1	OVERVIEW OF METHODS USED IN HISTOLOGY / 1 TISSUE PREPARATION / 2 Hematoxylin and Eosin Staining With Formalin Fixation / 2 Other Fixatives / 2 Other Staining Procedures / 3 HISTOCHEMISTRY AND CYTOCHEMISTRY / 3 Chemical Composition of Histologic Samples / 3 Chemical Basis of Staining / 5 Enzyme Digestion / 7 Enzyme Histochemistry / 7 Immunocytochemistry / 7 Hybridization Techniques / 10 Autoradiography / 12 MICROSCOPY / 13 Light Microscopy / 13 Examination of a Histologic Slide Preparation in the Light Microscope / 14 Other Optical Systems / 15 Electron Microscopy / 18 Atomic Force Microscopy / 20 Folder 1.1 Clinical Correlation: Frozen Sections / 4 Folder 1.2 Functional Considerations: Feulgen Microspectrophotometry / 7 Folder 1.3 Clinical Correlation: Monoclonal Antibodies in Medicine / 9 Folder 1.4 Proper Use of the Light Microscope / 11

1	The objective of a histology course is to lead the student to understand the microanatomy of cells, tissues, and organs and to correlate structure with function.

1	The methods used by histologists are extremely diverse. Much of the histology course content can be framed in terms of light microscopy. Today, students in histology laboratories use either light microscopes or, with increasing frequency, virtual microscopy, which represents a method of viewing a digitized microscopic specimen on a computer screen. In the past, more detailed interpretation of microanatomy was with the electron microscope (EM)—both the transmission electron microscope (TEM) and the scanning electron microscope (SEM). Now the atomic force microscope (AFM) can also provide high-resolution images, which are comparable in resolution to those obtained from TEM. Both EM and AFM, because of their greater resolution and useful magnification, are often the last step in data acquisition from many auxiliary techniques of cell and molecular biology.

1	These auxiliary techniques include:  histochemistry and cytochemistry,  immunocytochemistry and hybridization techniques,  autoradiography,  organ and tissue culture,  cell and organelle separation by differential centrifugation, and  specialized microscopic techniques and microscopes. The student may feel removed from such techniques and experimental procedures because direct experience with them is usually not available in current curricula. Nevertheless, it is important to know something about specialized procedures and the data they yield. This chapter provides a survey of methods and offers an explanation of how the data provided by these methods can help the student acquire a better understanding of cells, tissues, and organ function.

1	One problem students in histology face is understanding the nature of the two-dimensional image of a histologic slide or an electron micrograph and how the image relates to the three-dimensional structure from which it came. To bridge this conceptual gap, we must first present a brief description of the methods by which slides and electron microscopic specimens are produced. The routinely prepared hematoxylin and eosin–stained section is the specimen most commonly studied.

1	The routinely prepared hematoxylin and eosin–stained section is the specimen most commonly studied. The slide set given each student to study with the light microscope consists mostly of formalin-fixed, paraffin-embedded, hematoxylin and eosin (H&E)–stained specimens. Nearly all of the light micrographs in the Atlas section of this book are of slides from actual student sets. Also, most photomicrographs used to illustrate tissues and organs in histology lectures and conferences are taken from such slides. Other staining techniques are sometimes used to demonstrate specific cell and tissue components; several of these methods are discussed below. The first step in preparation of a tissue or organ sample is fixation to preserve structure.

1	The first step in preparation of a tissue or organ sample is fixation to preserve structure. Fixation, usually by a chemical or mixture of chemicals, permanently preserves the tissue structure for subsequent treatments. Specimens should be immersed in fixative immediately after they are removed from the body. Fixation is used to:  terminate cell metabolism, prevent enzymatic degradation of cells and tissues by autolysis (self-digestion), kill pathogenic microorganisms such as bacteria, fungi, and viruses, and  harden the tissue as a result of either cross-linking or dena turing protein molecules.

1	Formalin, a 37% aqueous solution of formaldehyde, at various dilutions and in combination with other chemicals and buffers, is the most commonly used fixative. Formaldehyde preserves the general structure of the cell and extracellular components by reacting with the amino groups of proteins (most often cross-linked lysine residues). Because formaldehyde does not significantly alter their three-dimensional structure, proteins maintain their ability to react with specific antibodies. This property is important in immunocytochemical staining methods (see page 7). The standard commercial solution of formaldehyde buffered with phosphates (pH 7) acts relatively slowly but penetrates the tissue well. However, because it does not react with lipids, it is a poor fixative of cell membranes. In the second step, the specimen is prepared for embedding in paraffin to permit sectioning.

1	In the second step, the specimen is prepared for embedding in paraffin to permit sectioning. Preparing a specimen for examination requires its infiltration with an embedding medium that allows it to be thinly sliced, typically in the range of 5 to 15 m (1 micrometer [ m] equals 1/1,000 of a millimeter [mm]; see Table 1.1). The specimen is washed after fixation and dehydrated in a series of alcohol solutions of ascending concentration as high as 100% alcohol to remove water. In the next step, clearing, organic solvents such as xylol or toluol, which are miscible in both alcohol and paraffin, are used to remove the alcohol before infiltration of the specimen with melted paraffin. TABLE Commonly Used Linear Equivalents1.1 1 picometer (pm) 0.01 angstrom (Å) 1 angstrom 0.1 nanometer (nm) 10 angstroms 1.0 nanometer 1 nanometer 1,000 picometers 1,000 nanometers 1.0 micrometer ( m) 1,000 micrometers 1.0 millimeter (mm)

1	When the melted paraffin is cool and hardened, it is trimmed into an appropriately sized block. The block is then mounted in a specially designed slicing machine—a microtome—and cut with a steel knife. The resulting sections are then mounted on glass slides using mounting medium (pinene or acrylic resins) as an adhesive. In the third step, the specimen is stained to permit examination.

1	In the third step, the specimen is stained to permit examination. Because paraffin sections are colorless, the specimen is not yet suitable for light microscopic examination. To color or stain the tissue sections, the paraffin must be dissolved out, again with xylol or toluol, and the slide must then be rehydrated through a series of solutions of descending alcohol concentration. The tissue on the slides is then stained with hematoxylin in water. Because the counterstain, eosin, is more soluble in alcohol than in water, the specimen is again dehydrated through a series of alcohol solutions of ascending concentration and stained with eosin in alcohol. Figure 1.1 shows the results of staining with hematoxylin alone, eosin alone, and hematoxylin with counterstain eosin. After staining, the specimen is then passed through xylol or toluol to a nonaqueous mounting medium and covered with a coverslip to obtain a permanent preparation.

1	Formalin does not preserve all cell and tissue components. Although H&E–stained sections of formalin-fixed specimens are convenient to use because they adequately display general structural features, they cannot elucidate the specific chemical composition of cell components. Also, many components are lost in the preparation of the specimen. To retain these components and structures, other fixation methods must be used. These methods are generally based on a clear understanding of the chemistry involved. For instance, the use of alcohols and organic solvents in routine preparations removes neutral lipids. To retain neutral lipids, such as those in adipose cells, frozen sections of formalin-fixed tissue and dyes that dissolve in fats must be used; to retain membrane structures, special fixatives

1	FIGURE 1.1 • Hematoxylin and eosin (H&E) staining. This series of specimens from the pancreas are serial (adjacent) sections that demonstrate the effect of hematoxylin and eosin used alone and hematoxylin and eosin used in combination. a. This photomicrograph reveals the staining with hematoxylin only. Although there is a general overall staining of the specimen, those components and structures that have a high affinity for the dye are most heavily stained−for example, the nuclear DNA and areas of the cell containing cytoplasmic RNA. b. In this photomicrograph, eosin, the counterstain, likewise has an overall staining effect when used alone. Note, however, that the nuclei are less conspicuous than in the specimen stained with hematoxylin alone. After the specimen is stained with hematoxylin and then prepared for staining with eosin in alcohol solution, the hematoxylin that is not tightly bound is lost, and the eosin then stains those components to which it has a high affinity. c. This

1	and then prepared for staining with eosin in alcohol solution, the hematoxylin that is not tightly bound is lost, and the eosin then stains those components to which it has a high affinity. c. This photomicrograph reveals the combined staining effect of H&E. 480.

1	containing heavy metals that bind to the phospholipids, such as permanganate and osmium, are used (Folder 1.1). The routine use of osmium tetroxide as a fixative for electron microscopy is the primary reason for the excellent preservation of membranes in electron micrographs. Hematoxylin and eosin are used in histology primarily to display structural features.

1	Hematoxylin and eosin are used in histology primarily to display structural features. Despite the merits of H&E staining, the procedure does not adequately reveal certain structural components of histologic sections such as elastic material, reticular fibers, basement membranes, and lipids. When it is desirable to display these components, other staining procedures, most of them selective, can be used. These procedures include the use of orcein and resorcin-fuchsin for elastic material and silver impregnation for reticular fibers and basement membrane material. Although the chemical bases of many staining methods are not always understood, they work. Knowing the components that a procedure reveals is more important than knowing precisely how the procedure works. Specific chemical procedures can provide information about the function of cells and the extracellular components of tissues.

1	Specific chemical procedures can provide information about the function of cells and the extracellular components of tissues. Histochemical and cytochemical procedures may be based on specific binding of a dye, use of a ﬂuorescent dye–labeled antibody with a particular cell component, or the inherent enzymatic activity of a cell component. In addition, many large molecules found in cells can be localized by the process of autoradiography, in which radioactively tagged precursors of the molecule are incorporated by cells and tissues before fixation. Many of these procedures can be used with both light microscopic and electron microscopic preparations. Before discussing the chemistry of routine staining and histochemical and cytochemical methods, it is useful to examine briefly the nature of a routinely fixed and embedded section of a specimen. Chemical Composition of Histologic Samples The chemical composition of a tissue ready for routine staining differs from living tissue.

1	Chemical Composition of Histologic Samples The chemical composition of a tissue ready for routine staining differs from living tissue. The components that remain after fixation consist mostly of large molecules that do not readily dissolve, especially after treatment with the fixative. These large molecules, particularly those that react with other large molecules to form macromolecular complexes, are usually preserved in a tissue section. Examples of such large macromolecular complexes include:  nucleoproteins formed from nucleic acids bound to protein,  intracellular cytoskeletal proteins complexed with as sociated proteins,  extracellular proteins in large insoluble aggregates, bound to similar molecules by cross-linking of neighbor ing molecules, as in collagen fiber formation, and  FOLDER 1.1 Clinical Correlation: Frozen Sections

1	Sometimes, the pathologist may be asked to immediately evaluate tissue obtained during surgery, especially when in-stant pathologic diagnosis may determine how the surgery will proceed. There are several indications to perform such an evaluation, routinely known as a frozen section. Most commonly, a surgeon in the operating room requests a frozen section when no preoperative diagnosis was available or when unexpected intraoperative findings must be identi-fied. In addition, the surgeon may want to know whether all of a pathologic mass within the healthy tissue limit has been re-moved and whether the margin of the surgical resection is free of diseased tissue. Frozen sections are also done in combination with other procedures such as endoscopy or thin-needle biopsy to confirm whether the obtained biopsy material will be usable in further pathologic examinations. Three main steps are involved in frozen section prepa-ration:  Freezing the tissue sample. Small tissue samples are frozen

1	obtained biopsy material will be usable in further pathologic examinations. Three main steps are involved in frozen section prepa-ration:  Freezing the tissue sample. Small tissue samples are frozen either by using compressed carbon dioxide or by immersion in a cold fluid (isopentane) at a temperature of 50 C. Freezing can be achieved in a special high-efficiency refrigerator. Freezing makes the tissue solid and allows sectioning with a microtome.  Sectioning the frozen tissue. Sectioning is usually per-formed inside a cryostat, a refrigerated compartment containing a microtome. Because the tissue is frozen solid, it can be cut into extremely thin (5 to 10 m) sec-tions. The sections are then mounted on glass slides.  Staining the cut sections. Staining is done to differen-tiate cell nuclei from the rest of the tissue. The most common stains used for frozen sections are H&E, methylene blue (Fig. F1.1.1), and PAS stains. The entire process of preparation and evaluation of frozen

1	nuclei from the rest of the tissue. The most common stains used for frozen sections are H&E, methylene blue (Fig. F1.1.1), and PAS stains. The entire process of preparation and evaluation of frozen sections may take as little as 10 minutes to complete. The total time to obtain results largely depends on the transport time of the tissue from the operating room to the pathology laboratory, on the pathologic technique used, and the expe-rience of the pathologist. The findings are then directly com-municated to the surgeon waiting in the operating room. aba b FIGURE F1.1.1 • Evaluation of a specimen obtained during surgery by frozen-section technique. a. This photomicrograph shows a specimen obtained from the large intestine that was prepared by frozen-section technique and stained with methylene blue. 160 b. Part of the specimen was fixed in formalin and processed as a routine H&E preparation. Examination of the frozen section revealed it to be normal. This diagnosis was later confirmed

1	blue. 160 b. Part of the specimen was fixed in formalin and processed as a routine H&E preparation. Examination of the frozen section revealed it to be normal. This diagnosis was later confirmed by examining the routinely prepared H&E specimen. 180. (Courtesy of Dr. Daniel W. Visscher.)  membrane phospholipid–protein (or carbohydrate) complexes.

1	These molecules constitute the structure of cells and tissues— that is, they make up the formed elements of the tissue. They are the basis for the organization that is seen in tissue with the microscope. In many cases, a structural element is also a functional unit. For example, in the case of proteins that make up the contractile filaments of muscle cells, the filaments are the visible structural components and the actual participants in the contractile process. The RNA of the cytoplasm is visualized as part of a structural component (e.g., ergastoplasm of secretory cells, Nissl bodies of nerve cells) and is also the actual participant in the synthesis of protein. Many tissue components are lost during the routine preparation of H&E–stained sections.

1	Many tissue components are lost during the routine preparation of H&E–stained sections. Despite the fact that nucleic acids, proteins, and phospholipids are mostly retained in tissue sections, many are also lost. Small proteins and small nucleic acids, such as transfer RNA, are generally lost during the preparation of the tissue. As previously described, neutral lipids are usually dissolved by the organic solvents used in tissue preparation. Other large molecules also may be lost, for example, by being hydrolyzed because of the unfavorable pH of the fixative solutions. Examples of large molecules lost during routine fixation in aqueous fixatives are:  glycogen (an intracellular storage carbohydrate common in liver and muscle cells), and  proteoglycans and glycosaminoglycans (extracellular complex carbohydrates found in connective tissue).

1	These molecules can be preserved, however, by using a non-aqueous fixative for glycogen or by adding specific binding agents to the fixative solution that preserve extracellular carbohydrate-containing molecules. Soluble components, ions, and small molecules are also lost during the preparation of paraffin sections. chapter 1 TABLE Some Basic and Acidic Dyes1.2 Dye Color Basic dyes Methyl green Green Methylene blue Blue Pyronin G Red Toluidine blue Blue Acidic dyes Acid fuchsin Red Aniline blue Blue Eosin Red Orange G Orange

1	Intermediary metabolites, glucose, sodium, chloride, and similar substances are lost during preparation of routine H&E paraffin sections. Many of these substances can be studied in special preparations, sometimes with considerable loss of structural integrity. These small soluble ions and molecules do not make up the formed elements of a tissue; they participate in synthetic processes or cellular reactions. When they can be preserved and demonstrated by specific  At a slightly acidic to neutral pH (5 to 7), sulfate and phos phate groups are ionized and available for reaction with the basic dye by electrostatic linkages.  At low pH (below 4), only sulfate groups remain ionized methods, they provide invaluable information about cell metabolism, active transport, and other vital cellular pro cesses. Water, a highly versatile molecule, participates in these reactions and processes and contributes to the stabilization of macromolecular structure through hydrogen bonding.

1	Chemical Basis of Staining Hematoxylin and eosin are the most commonly used dyes in histology. An acidic dye, such as eosin, carries a net negative charge on its colored portion and is described by the general formula [Na dye ]. A basic dye carries a net positive charge on its colored portion and is described by the general formula [dye Cl ]. Hematoxylin does not meet the definition of a strict basic dye but has properties that closely resemble those of a basic dye. The color of a dye is not related to whether it is basic or acidic, as can be noted by the examples of basic and acidic dyes listed in Table 1.2. Basic dyes react with anionic components of cells and tissue (components that carry a net negative charge).

1	Basic dyes react with anionic components of cells and tissue (components that carry a net negative charge). Anionic components include the phosphate groups of nucleic acids, the sulfate groups of glycosaminoglycans, and the carboxyl groups of proteins. The ability of such anionic groups to react with a basic dye is called basophilia [Gr., base-loving]. Tissue components that stain with hematoxylin also exhibit basophilia. The reaction of the anionic groups varies with pH. Thus:  At a high pH (about 10), all three groups are ionized and avail able for reaction by electrostatic linkages with the basic dye. and react with basic dyes. Therefore, staining with basic dyes at a specific pH can be used to focus on specific anionic groups; because the specific anionic groups are found predominantly on certain macromolecules, the staining serves as an indicator of these macromolecules.

1	As mentioned, hematoxylin is not, strictly speaking, a basic dye. It is used with a mordant (i.e., an intermediate link between the tissue component and the dye). The mordant causes the stain to resemble a basic dye. The linkage in the tissue–mordant–hematoxylin complex is not a simple electrostatic linkage; when sections are placed in water, hematoxylin does not dissociate from the tissue. Hematoxylin lends itself to those staining sequences in which it is followed by aqueous solutions of acidic dyes. True basic dyes, as distinguished from hematoxylin, are not generally used in sequences in which the basic dye is followed by an acidic dye. The basic dye then tends to dissociate from the tissue during the aqueous solution washes between the two dye solutions. Acidic dyes react with cationic groups in cells and tissues, particularly with the ionized amino groups of proteins.

1	Acidic dyes react with cationic groups in cells and tissues, particularly with the ionized amino groups of proteins. The reaction of cationic groups with an acidic dye is called acidophilia [Gr., acid-loving]. Reactions of cell and tissue components with acidic dyes are neither as specific nor as precise as reactions with basic dyes. Although electrostatic linkage is the major factor in the primary binding of an acidic dye to the tissue, it is not the only one; because of this, acidic dyes are sometimes used in combinations to color different tissue constituents selectively. For example, three acidic dyes are used in the Mallory staining technique: aniline blue, acid fuchsin, and orange G. These dyes selectively stain collagen, ordinary cytoplasm, and red blood cells, respectively. Acid fuchsin also stains nuclei.

1	In other multiple acidic dye techniques, hematoxylin is used to stain nuclei first, and then acidic dyes are used to stain cytoplasm and extracellular fibers selectively. The selective staining of tissue components by acidic dyes is attributable to relative factors such as the size and degree of aggregation of the dye molecules and the permeability and “compactness” of the tissue. Basic dyes can also be used in combination or sequentially (e.g., methyl green and pyronin to study protein synthesis and secretion), but these combinations are not as widely used as acidic dye combinations. A limited number of substances within cells and the extracellular matrix display basophilia.

1	A limited number of substances within cells and the extracellular matrix display basophilia. These substances include:  heterochromatin and nucleoli of the nucleus (chiefly because of ionized phosphate groups in nucleic acids of both), cytoplasmic components such as the ergastoplasm (also because of ionized phosphate groups in ribosomal RNA), and  extracellular materials such as the complex carbohydrates of the matrix of cartilage (because of ionized sulfate groups). Staining with acidic dyes is less specific, but more substances within cells and the extracellular matrix exhibit acidophilia. These substances include:  most cytoplasmic filaments, especially those of muscle cells, most intracellular membranous components and much of the otherwise unspecialized cytoplasm, and most extracellular fibers (primarily because of ionized amino groups).

1	Certain basic dyes react with tissue components that shift their normal color from blue to red or purple; this absorbance change is called metachromasia. The underlying mechanism for metachromasia is the presence of polyanions within the tissue. When these tissues are stained with a concentrated basic dye solution, such as toluidine blue, the dye molecules are close enough to form dimeric and polymeric aggregates. The absorption properties of these aggregations differ from those of the individual nonaggregated dye molecules. Cell and tissue structures that have high concentrations of ionized sulfate and phosphate groups—such as the ground substance of cartilage, heparin-containing granules of mast cells, and rough endoplasmic reticulum of plasma cells—exhibit metachromasia. Therefore, toluidine blue will appear purple to red when it stains these components. Aldehyde Groups and the Schiff Reagent

1	Aldehyde Groups and the Schiff Reagent The ability of bleached basic fuchsin (Schiff reagent) to react with aldehyde groups results in a distinctive red color and is the basis of the periodic acid–Schiff and Feulgen reactions. The periodic acid–Schiff (PAS) reaction stains carbohydrates and carbohydrate-rich macromolecules. It is used to demonstrate glycogen in cells, mucus in various cells and tissues, the basement membrane that underlies epithelia, and reticular fibers in connective tissue. The Feulgen reaction, which relies on a mild hydrochloric acid hydrolysis, is used to stain DNA.

1	The PAS reaction is based on the following facts:  Hexose rings of carbohydrates contain adjacent carbons, each of which bears a hydroxyl (–OH) group.  Hexosamines of glycosaminoglycans contain adjacent carbons, one of which bears an –OH group, whereas the other bears an amino (–NH2) group.  Periodic acid cleaves the bond between these adjacent carbon atoms and forms aldehyde groups. These aldehyde groups react with the Schiff reagent to give a distinctive magenta color. The PAS staining of basement membrane (Fig. 1.2) and reticular fibers is based on the content or association of proteoglycans (complex carbohydrates associated with a protein core). PAS staining is an alternative to silver-impregnation methods, which are also based on reaction with the sugar molecules in the proteoglycans.

1	The Feulgen reaction is based on the cleavage of purines from the deoxyribose of DNA by mild acid hydrolysis; the sugar ring then opens with the formation of aldehyde groups. Again, the newly formed aldehyde groups react with the FIGURE 1.2 • Photomicrograph of kidney tissue stained by the PAS method. This histochemical method demonstrates and localizes carbohydrates and carbohydrate-rich macromolecules. The basement membranes are PAS positive as evidenced by the magenta staining of these sites. The kidney tubules (T ) are sharply delineated by the stained basement membrane surrounding the tubules. The glomerular capillaries (C) and the epithelium of Bowman’s capsule (BC) also show PAS-positive basement membranes. 360.

1	 FOLDER 1.2 Functional Considerations: Feulgen Microspectrophotometry is a technique devel-sion. Currently, Feulgen microspectrophotometry is used to study changes in the DNA content in dividing cells undergoing differentiation. It is also used clinically to analyze abnormal chromosomal number (i.e., ploidy patterns) in malignant cells. Some malignant cells that have a largely diploid pattern are said to be well differentiated; tumors with these types of cells have a better prognosis than tumors with aneuploid (nonintegral multiples of the haploid amount of DNA) and tetraploid cells. Feulgen microspectrophotometry has been particularly useful in studies of specific adenocarcinomas (epithelial cancers), breast cancer, kidney cancer, colon and other gastrointestinal cancers, endometrial (uterine epithelium) cancer, and ovarian cancer. It is one of the most valuable tools for pathologists in evaluating the metastatic potential of these tumors and in making prognostic and treatment

1	(uterine epithelium) cancer, and ovarian cancer. It is one of the most valuable tools for pathologists in evaluating the metastatic potential of these tumors and in making prognostic and treatment decisions.

1	of reaction. In a typical reaction to display a hydrolytic enzyme, the tissue section is placed in a solution containing a substrate (AB) and a trapping agent (T) that precipitates one of the products as follows: where AT is the trapped end product and B is the hydrolyzed substrate. By using such methods, the lysosome, first identified in differential centrifugation studies of cells, was equated with a vacuolar component seen in electron micrographs. In lightly fixed tissues, the acid hydrolases and esterases contained in lysosomes react with an appropriate substrate. The reaction mixture also contains lead ions to precipitate (e.g., lead phosphate derived from the action of acid phosphatase). The precipitated reaction product can then be observed with both light and electron microscopy.

1	Similar light and electron microscopy histochemical procedures have been developed to demonstrate alkaline phosphatase, adenosine triphosphatases (ATPases) of many varieties (including the Na /K -ATPase that is the enzymatic basis of the sodium pump in cells and tissues), various esterases, and many respiratory enzymes (Fig. 1.3). The specificity of a reaction between an antigen and an antibody is the underlying basis of immunocytochemistry.

1	Antibodies, also known as immunoglobulins, are glyco proteins that are produced by specific cells of the immune system in response to a foreign protein, or antigen. In the laboratory, antibodies can be purified from the blood and conjugated (attached) to a fluorescent dye. In general, ﬂuorescent dyes (ﬂuorochromes) are chemicals that absorb oped to study DNA increases in developing cells and to analyze ploidy–that is, the number of times the normal DNA content of a cell is multiplied (a normal, nondividing cell is said to be diploid; a sperm or egg cell is haploid). Two techniques, static cytometry for tissue sections and ﬂow cytometry for isolated cells, are used to quantify the amount of nuclear DNA. The technique of static cytometry of Feulgen-stained sections of tumors uses microspectrophotometry coupled with a digitizing imaging system to measure the absorption of light emitted by cells and cell clusters at 560-nm wavelength. In contrast, the flow cytometry technique uses

1	coupled with a digitizing imaging system to measure the absorption of light emitted by cells and cell clusters at 560-nm wavelength. In contrast, the flow cytometry technique uses instrumentation able to scan only single cells flowing past a sensor in a liquid medium. This technique provides rapid, quantitative analysis of a single cell based on the measurement of fluorescent light emis-

1	Schiff reagent to give the distinctive magenta color. The reaction of the Schiff reagent with DNA is stoichiometric, meaning that the product of this reaction is measurable and proportional to the amount of DNA. It can be used, therefore, in spectrophotometric methods to quantify the amount of DNA in the nucleus of a cell. RNA does not stain with the Schiff reagent because it lacks deoxyribose. Enzyme digestion of a section adjacent to one stained for a specific component—such as glycogen, DNA, or RNA— can be used to confirm the identity of the stained material. Intracellular material that stains with the PAS reaction may be identified as glycogen by pretreatment of sections with diastase or amylase. Abolition of the staining after these treatments positively identifies the stained material as glycogen.

1	Similarly, pretreatment of tissue sections with deoxyribonuclease (DNAse) will abolish the Feulgen staining in those sections, and treatment of sections of protein secretory epithelia with ribonuclease (RNAse) will abolish the staining of the ergastoplasm with basic dyes. Histochemical methods are also used to identify and localize enzymes in cells and tissues. To localize enzymes in tissue sections, special care must be taken in fixation to preserve the enzyme activity. Usually, mild aldehyde fixation is the preferred method. In these procedures, the reaction product of the enzyme activity, rather than the enzyme itself, is visualized. In general, a capture reagent, either a dye or a heavy metal, is used to trap or bind the reaction product of the enzyme by precipitation at the site FIGURE 1.3 • Electron histochemical procedure for localization of membrane ATPase in epithelial cells of rabbit gallbladder.

1	FIGURE 1.3 • Electron histochemical procedure for localization of membrane ATPase in epithelial cells of rabbit gallbladder. Dark areas visible on the electron micrograph show the location of the enzyme ATPase. This enzyme is detected in the plasma membrane at the lateral domains of epithelial cells, which correspond to the location of sodium pumps. These epithelial cells are involved in active transport of molecules across the plasma membrane. 26,000.

1	light of different wavelengths (e.g., ultraviolet light) and then emit visible light of a specific wavelength (e.g., green, yellow, red). Fluorescein, the most commonly used dye, absorbs ultraviolet light and emits green light. Antibodies conjugated with fluorescein can be applied to sections of lightly fixed or frozen tissues on glass slides to localize an antigen in cells and tissues. The reaction of antibody with antigen can then be examined and photographed with a fluorescence microscope or confocal microscope that produces a three-dimensional reconstruction of the examined tissue (Fig. 1.4). Two types of antibodies are used in immunocytochemistry: polyclonal antibodies that are produced by immunized animals and monoclonal antibodies that are produced by immortalized (continuously replicating) antibody-producing cell lines.

1	In a typical procedure, a specific protein, such as actin, is isolated from a muscle cell of one species, such as a rat, and injected into the circulation of another species, such as a rabbit. In the immunized rabbit, the rat’s actin molecules are recognized by the rabbit immune system as a foreign antigen. This recognition triggers a cascade of immunologic reactions involving multiple groups (clones) of immune cells called B lymphocytes. The cloning of B lymphocytes eventually leads to the production of anti-actin antibodies. Collectively, these polyclonal antibodies represent mixtures of different antibodies produced by many clones of B lymphocytes that

1	FIGURE 1.4 • Confocal microscopy image of a rat cardiac muscle cell. This image was obtained from the confocal microscope using the indirect immunofluorescence method. Two primary antibodies were used. The first primary antibody recognizes a specific lactate transporter (MCT1) and is detected with a secondary antibody conjugated with rhodamine (red). The second primary antibody is directed against the transmembrane protein CD147, which is tightly associated with MCT1. This antibody was detected by a secondary antibody labeled with fluorescein (green). The yellow color is visible at the point at which the two labeled secondary antibodies exactly co-localize within the cardiac muscle cell. This three-dimensional image shows that both proteins are distributed on the surface of the muscle cell, whereas the lactate transporter alone is visible deep to the plasma membrane. (Courtesy of Drs. Andrew P. Halestrap and Catherine Heddle.) each recognize different regions of the actin molecule.

1	whereas the lactate transporter alone is visible deep to the plasma membrane. (Courtesy of Drs. Andrew P. Halestrap and Catherine Heddle.) each recognize different regions of the actin molecule. The antibodies are then removed from the blood, purified, and conjugated with a fluorescent dye. They can now be used to locate actin molecules in rat tissues or cells. If actin is present in a cell or tissue, such as a fibroblast in connective tissue, then the fluorescein-labeled antibody binds to it and the reaction is visualized by fluorescence microscopy.

1	Monoclonal antibodies (Folder 1.3) are those produced by an antibody-producing cell line consisting of a single group (clone) of identical B lymphocytes. The single clone that becomes a cell line is obtained from an individual with multiple myeloma, a tumor derived from a single antibody-producing plasma cell. Individuals with multiple myelomas produce a large population of identical, homogeneous antibodies with an identical specificity against an antigen. To produce monoclonal antibodies against a specific antigen, a mouse or rat is immunized with that antigen. The activated B lymphocytes are then isolated from the lymphatic tissue (spleen or lymph nodes) of the animal and fused with the myeloma cell line. This fusion produces a hybridoma, an immortalized individual antibody-secreting cell line. To obtain monoclonal antibodies against rat actin molecules, for example, the B lymphocytes from the lymphatic organs of immunized rabbits must be fused with myeloma cells.

1	FOLDER 1.3 Clinical Correlation: Monoclonal Antibodies in Medicine Monoclonal antibodies are now widely used in im-munocytochemical techniques and also have many clini-cal applications. Monoclonal antibodies conjugated with radioactive compounds are used to detect and diagnose tumor metastasis in pathology, differentiate subtypes of tumors and stages of their differentiation, and in infec-tious disease diagnosis to identify microorganisms in blood and tissue fluids. In recent clinical studies, mono-clonal antibodies conjugated with immunotoxins, chemotherapy agents, or radioisotopes have been used to deliver therapeutic agents to specific tumor cells in the body. Both direct and indirect immunocytochemical methods are used to locate a target antigen in cells and tissues.

1	Both direct and indirect immunocytochemical methods are used to locate a target antigen in cells and tissues. The oldest immunocytochemistry technique used for identifying the distribution of an antigen within cells and tissues is known as direct immunoﬂuorescence. This technique uses a fluorochrome-labeled primary antibody (either polyclonal or monoclonal) that reacts with the antigen within the sample (Fig. 1.5a). As a one-step procedure, this method involves only a single labeled antibody. Visualization of structures is not ideal because of the low intensity of the signal emission. Direct immunofluorescence methods are now being replaced by the indirect method because of suboptimal sensitivity.

1	Indirect immunoﬂuorescence provides much greater sensitivity than direct methods and is often referred to as the “sandwich” or “double-layer technique.” Instead of conjugating a fluorochrome with a specific (primary) antibody directed against the antigen of interest (e.g., a rat actin molecule), the fluorochrome is conjugated with a secondary antibody directed against rat primary antibody (i.e., goat anti-rat antibody; Fig. 1.5b). Therefore, when the fluorescein is conjugated directly with the specific primary antibody, the method is direct; when fluorescein is conjugated with a secondary antibody, the method is indirect. The indirect method considerably enhances the fluorescence signal emission from the tissue. An additional advantage of the indirect labeling method is that a single secondary antibody can be used to localize the tissue-specific binding of several different primary antibodies (Fig. 1.6). For microscopic studies, the secondary antibody can be conjugated with different

1	antibody can be used to localize the tissue-specific binding of several different primary antibodies (Fig. 1.6). For microscopic studies, the secondary antibody can be conjugated with different fluorescent dyes so that multiple labels can be shown in the same tissue section (see Fig. 1.4). Drawbacks of indirect immunofluorescence are that it is expensive, labor intensive, and not easily adapted to automated procedures.

1	It is also possible to conjugate polyclonal or monoclonal antibodies with other substances, such as enzymes FIGURE 1.5 • Direct and indirect immunoﬂuorescence. a. In direct immunofluorescence, a fluorochrome-labeled primary antibody reacts with a specific antigen within the tissue sample. Labeled structures are then observed in the fluorescence microscope in which an excitation wavelength (usually ultraviolet light) triggers the emission of another wavelength. The length of this wavelength depends on the nature of the fluorochrome used for antibody labeling. b. The indirect method involves two processes. First, the specific primary antibodies react with the antigen of interest. Second, the secondary antibodies, which are fluorochrome labeled, react with the primary antibodies. The visualization of labeled structures within the tissue is the same in both methods and requires the fluorescence microscope.

1	FIGURE 1.6 • Microtubules visualized by immunocytochemical methods. The behavior of microtubules (elements of the cell cytoskeleton) obtained from human breast tumor cells can be studied in vitro by measuring their nucleation activity, which is initiated by the centrosome. This image was photographed in the fluorescence microscope. By use of indirect immunofluorescence techniques, microtubules were labeled with a mixture of anti– -tubulin and anti– -tubulin monoclonal antibodies (primary antibodies) and visualized by secondary antibodies conjugated with fluorescein dye (fluorescein isothiocyanate–goat anti-mouse immunoglobulin G). The antigen–antibody reaction, performed directly on the glass coverslip, results in visualization of tubulin molecules responsible for the formation of more than 120 microtubules visible on this image. They originate from the centriole and extend outward approximately 20 to 25 m in a uniform radial array. 1,400. (Photomicrograph courtesy of Drs. Wilma L.

1	than 120 microtubules visible on this image. They originate from the centriole and extend outward approximately 20 to 25 m in a uniform radial array. 1,400. (Photomicrograph courtesy of Drs. Wilma L. Lingle and Vivian A. Negron.) (e.g., horseradish peroxidase), that convert colorless substrates into an insoluble product of a specific color that precipitates at the site of the enzymatic reaction. The staining that results from this immunoperoxidase method can be observed in the light microscope (Folder 1.4) with either direct or indirect immunocytochemical methods. In another variation, colloidal gold or ferritin (an iron-containing molecule) can be attached to the antibody molecule. These electron-dense markers can be visualized directly with the electron microscope.

1	Hybridization is a method of localizing messenger RNA (mRNA) or DNA by hybridizing the sequence of interest to a complementary strand of a nucleotide probe. In general, the term hybridization describes the ability of single-stranded RNA or DNA molecules to interact (hybridize) with complementary sequences. In the laboratory, hybridization requires the isolation of DNA or RNA, which is then mixed with a complementary nucleotide sequence (called a nucleotide probe). Hybrids are detected most often using a radioactive label attached to one component of the hybrid. Binding of the probe and sequence can take place in a solution or on a nitrocellulose membrane. In in situ hybridization, the binding of the nucleotide probe to the DNA or RNA sequence of interest is performed within cells or tissues, such as cultured cells or whole embryos. This technique allows the localization of specific nucleotide sequences as small as 10 to 20 copies of mRNA or DNA per cell.

1	Several nucleotide probes are used in in situ hybridization. Oligonucleotide probes can be as small as 20 to 40 base pairs. Single-or double-stranded DNA probes are much longer and can contain as many as 1,000 base pairs. For specific localization of mRNA, complementary RNA probes are used. These probes are labeled with radioactive isotopes (e.g., 32P, 35S, 3H), a specifically modified nucleotide (digoxigenin), or biotin (a commonly used covalent multipurpose label). Radioactive probes can be detected and visualized by autoradiography. Digoxigenin and biotin are detected by immunocytochemical and cytochemical methods, respectively.

1	The strength of the bonds between the probe and the complementary sequence depends on the type of nucleic acid in the two strands. The strongest bond is formed between a DNA probe and a complementary DNA strand and the weakest between an RNA probe and a complementary RNA strand. If a tissue specimen is expected to contain a minute amount of mRNA or a viral transcript, then polymerase chain reaction (PCR) amplification for DNA or reverse transcriptase-PCR (RT-PCR) for RNA can be used. The amplified transcripts obtained during these procedures are usually detected using labeled complementary nucleotide probes in standard in situ hybridization techniques. Recently, fluorescent dyes have been combined with nucleotide probes, making it possible to visualize multiple probes at the same time (Fig. 1.7). This technique, called

1	Recently, fluorescent dyes have been combined with nucleotide probes, making it possible to visualize multiple probes at the same time (Fig. 1.7). This technique, called FIGURE 1.7 • Example of the FISH technique used in a prenatal screening test. Interphase nuclei of cells obtained from amniotic fluid specimens were hybridized with two specific DNA probes. The orange probe (LSI 21) is locus specific for chromosome 21, and the green probe (LSI 13) is locus specific for chromosome 13. The right nucleus is from a normal amniotic fluid specimen and exhibits two green and two orange signals, which indicates two copies of chromosomes 13 and 21, respectively. The nucleus on the left has three orange signals, which indicate trisomy 21 (Down syndrome). DNA has been counterstained with a nonspecific blue stain (DAPI stain) to make the nucleus visible. 1,250. (Courtesy of Dr. Robert B. Jenkins.)

1	FOLDER 1.4 Proper Use of the Light Microscope continued next page This brief introduction to the proper use of the light micro-scope is directed to those students who will use the micro-scope for the routine examination of tissues. If the following comments appear elementary, it is only because most users of the microscope fail to use it to its fullest advan-tage. Despite the availability of today’s fine equipment, rel-atively little formal instruction is given on the correct use of the light microscope. Expensive and highly corrected optics perform optimally only when the illumination and observation beam paths are centered and properly adjusted. The use of proper settings and proper alignment of the optic pathway will contribute substantially to the recognition of minute details in the specimen and to the faithful display of color for the visual image and for photomicrography. Köhler illumination is one key to good microscopy and is incorporated in the design of practically all

1	the specimen and to the faithful display of color for the visual image and for photomicrography. Köhler illumination is one key to good microscopy and is incorporated in the design of practically all modern laboratory and research microscopes. Figure F1.4.1 shows the two light paths and all the controls for alignment on a modern laboratory microscope; it should be referred to in following the instructions given below to provide appropri-ate illumination in your microscope. The alignment steps necessary to achieve good Kler illumination are few and simple:  Focus the specimen.  Close the field diaphragm.  Focus the condenser by moving it up or down until the outline of its field diaphragm appears in sharp focus.  Center the field diaphragm with the centering controls on the (condenser) substage. Then open the field diaphragm until the light beam covers the full field observed.  Remove the eyepiece (or use a centering telescope or a phase telescope accessory if available) and

1	substage. Then open the field diaphragm until the light beam covers the full field observed.  Remove the eyepiece (or use a centering telescope or a phase telescope accessory if available) and observe the exit pupil of the objective. You will see an illuminated cir-cular field that has a radius directly proportional to the numeric aperture of the objective. As you close the con-denser diaphragm, its outline will appear in this circular field. For most stained materials, set the condenser di-aphragm to cover approximately two thirds of the objec-tive aperture. This setting results in the best compromise between resolution and contrast (contrast simply being the intensity difference between dark and light areas in the specimen). FIGURE F1.4.1 • Diagram of a typical light microscope. This drawing shows a cross-sectional view of the microscope, its operating components, and light path. (Courtesy of Carl Zeiss, Inc., Thornwood, NY.) eyepiece final image exit pupil (eyepoint) real

1	This drawing shows a cross-sectional view of the microscope, its operating components, and light path. (Courtesy of Carl Zeiss, Inc., Thornwood, NY.) eyepiece final image exit pupil (eyepoint) real interme-diate image exit pupil of objective specimen condenser diaphragm field diaphragm light source focusing control light source tube objective auxiliary condenser lens stage condenser diaphragm condenser stage control field diaphragm KÖHLER ILLUMINATION THROUGH THE MICROSCOPE IMAGING BEAM PATH ILLUMINATING BEAM PATH the ﬂuorescence in situ hybridization (FISH) procedure, is extensively used in the clinic for genetic testing. For example, a probe hybridized to metaphase chromosomes can be used to identify the chromosomal position of a gene.

1	The FISH procedure is used to simultaneously examine chromosomes, gene expression, and the distribution of gene products such as pathologic or abnormal proteins. Many specific ﬂuorescent probes are now commercially available and are used clinically in screening procedures for cervical cancer or for the detection of HIV-infected cells. The FISH procedure can also be used to examine chromosomes from the lymphocytes of astronauts to estimate the radiation dose absorbed by them during their stay in space. The frequency of chromosome translocations in lymphocytes is proportional to the absorbed radiation dose. Autoradiography makes use of a photographic emulsion placed over a tissue section to localize radioactive material within tissues.

1	Autoradiography makes use of a photographic emulsion placed over a tissue section to localize radioactive material within tissues. Many small molecular precursors of larger molecules, such as the amino acids that make up proteins and the nucleotides that make up nucleic acids, may be tagged by incorporating a radioactive atom or atoms into their molecular structure. The radioactivity is then traced to localize the larger molecules in cells and tissues. Labeled precursor molecules can be injected into animals or introduced into cell or organ cultures. In this way, synthesis of DNA and subsequent cell division, synthesis and secretion of proteins by cells, and localization of synthetic products within cells and in the extracellular matrix have been studied.

1	FIGURE 1.8 • Examples of autoradiography used in light and electron microscopy. a. Photomicrograph of a lymph node section from an animal injected with tritiated [3H]thymidine. Some of the cells exhibit aggregates of metallic silver grains, which appear as small black particles (arrows). These cells synthesized DNA in preparation for cell division and have incorporated the [3H]thymidine into newly formed DNA. Over time, the low-energy radioactive particles emitted from the [3H]thymidine strike silver halide crystals in a photographic emulsion covering the specimen (exposure) and create a latent image (much like light striking photographic film in a camera). During photographic development of the slide with its covering emulsion, the latent image, actually the activated silver halide in the emulsion, is reduced to the metallic silver, which then appears as black grains in the microscope. 1,200. (Original slide specimen courtesy of Dr. Ernst Kallenbach.) b. Electron microscopic

1	in the emulsion, is reduced to the metallic silver, which then appears as black grains in the microscope. 1,200. (Original slide specimen courtesy of Dr. Ernst Kallenbach.) b. Electron microscopic autoradiograph of the apical region of an intestinal absorptive cell. In this specimen, 125I bound to nerve growth factor (NGF) was injected into the animal, and the tissue was removed 1 hour later. The specimen was prepared in a manner similar to that for light microscopy. The relatively small size of the silver grains aids precise localization of the 125I–NGF complexes. Note that the silver grains are concentrated over apical invaginations (inv) and early endosomal tubular profiles (tub). 32,000. (Electron micrograph courtesy of Dr. Marian R. Neutra.)

1	Sections of specimens that have incorporated radioactive material are mounted on slides. In the dark, the slide is usually dipped in a melted photographic emulsion, thus producing a thin photographic film on the surface of the slide. After appropriate exposure in a light-tight box, usually for days to weeks, the exposed emulsion on the slide is developed by standard photographic techniques and permanently mounted with a coverslip. The slides may be stained either before or after exposure and development. The silver grains in the emulsion over the radioactively labeled molecules are exposed and developed by this procedure and appear as dark grains overlying the site of the radioactive emission when examined with the light microscope (Fig. 1.8a).

1	These grains may be used simply to indicate the location of a substance, or they may be counted to provide semiquantitative information about the amount of a given substance in a specific location. For instance, after injection of an animal with tritiated thymidine, cells that have incorporated this nucleotide into their DNA before they divide will have approximately twice as many silver grains overlying their nuclei as will cells that have divided after incorporating the labeled nucleotide. Autoradiography can also be carried out by using thin plastic sections for examination with the EM. Essentially the same procedures are used, but as with all TEM preparation techniques, the processes are much more delicate and difficult; however, they also yield much greater resolution and more precise localization (Fig. 1.8b).

1	A microscope, whether simple (one lens) or compound (multiple lenses), is an instrument that magnifies an image and allows visualization of greater detail than is possible with the unaided eye. The simplest microscope is a magnifying glass or a pair of reading glasses. TABLE Eye Versus Instrument Resolution1.3 Distance Between Resolvable Points Human eye 0.2 mm Bright-field microscope 0.2 m SEM 2.5 nm TEM Theoretical 0.05 nm Tissue section 1.0 nm Atomic force microscopy 50.0 pm The resolving power of the human eye—that is, the distance by which two objects must be separated to be seen as two objects (0.2 mm)—is determined by the spacing of the photoreceptor cells in the retina. The role of a microscope is to magnify an image to a level at which the retina can resolve the information that would otherwise be below its limit of resolution. Table 1.3 compares the resolution of the eye with that of various instruments.

1	Resolving power is the ability of a microscope lens or optical system to produce separate images of closely positioned objects. Resolution depends not only on the optical system but also on the wavelength of the light source and other factors such as specimen thickness, quality of fixation, and staining intensity. With light of wavelength 540 nm (see Table 1.1), a green-filtered light to which the eye is extremely sensitive, and with appropriate objective and condenser lenses, the greatest attainable resolving power of a bright-field microscope would be about 0.2 m (see Folder 1.4, page 12 for method of calculation). This is the theoretical resolution and, as mentioned, depends on all conditions being optimal. The ocular or eyepiece lens magnifies the image produced by the objective lens, but it cannot increase resolution.

1	Various light microscopes are available for general and specialized use in modern biologic research. Their differences are based largely on such factors as the wavelength of specimen illumination, physical alteration of the light coming to or leaving the specimen, and specific analytic processes that can be applied to the final image. These instruments and their applications are described briefly in this section. The microscope used by most students and researchers is the bright-field microscope.

1	The microscope used by most students and researchers is the bright-field microscope. The bright-field microscope is the direct descendant of the microscopes that became widely available in the 1800s and opened the first major era of histologic research. The bright-field microscope (Fig. 1.9) essentially consists of:  a light source for illumination of the specimen (e.g., a substage lamp), a condenser lens to focus the beam of light at the level of the specimen, a stage on which the slide or other specimen is placed, an objective lens to gather the light that has passed through the specimen, and an ocular lens (or a pair of ocular lenses in the more commonly used binocular microscopes) through which the image formed by the objective lens may be examined directly. cussed earlier are used. Examination of a Histologic Slide Preparation in the Light Microscope Organs are three-dimensional, whereas histologic sections are only two-dimensional.

1	cussed earlier are used. Examination of a Histologic Slide Preparation in the Light Microscope Organs are three-dimensional, whereas histologic sections are only two-dimensional. As discussed in the earlier “Tissue Preparation” section, every tissue sample prepared for light microscopic examination must be sliced into thin sections. Thus, two-dimensional sections are obtained from an original three-dimensional sample of tissue. One of the most challenging aspects for students using the microscope to study histology is the ability to mentally reconstruct the “missing” third dimension.

1	For example, slices in different planes through an orange are shown in Figure 1.10. Note that each cut surface (indicated by the dotted line) of the whole orange reveals different sizes and surface patterns, depending on the orientation of the cut. Thus, it is important when observing a given section cut through the orange to be able to mentally reconstruct the organization of the structure and its component parts. An example of a histologic structure—in this case, a kidney renal corpuscle—is shown as it would appear in different sectional planes (Fig. 1.10). Note the marked difference in each section of the renal corpuscle. By examining a number of such two-dimensional sections, it is possible to create the three-dimensional configuration of the examined structure. Artifacts in histologic slides can be generated in all stages of tissue preparation.

1	Artifacts in histologic slides can be generated in all stages of tissue preparation. The preparation of a histologic slide requires a series of steps beginning with the collection of the specimen and ending with the placement of the coverslip. During each step, an artifact (an error in the preparation process) may be introduced. In general, artifacts that appear on the finished glass slide are linked to methodology, equipment, or reagents used during preparation. The inferior purity of chemicals and reagents used in the process (fixatives, reagents, and stains), imperfections in the execution of the methodology (too short or too long intervals of fixation, dehydration, embedding, staining, or careless mounting and placement of the coverslip), or improper equipment (e.g., a microtome with a

1	FIGURE 1.9 • Diagram comparing the optical paths in different types of microscopes. For better comparison between all three types of microscopes, the light microscope (left) is shown as if it were turned upside down; the TEM (middle); and the SEM (right). Note that in both the TEM and the SEM, specimens need to be inserted into the high vacuum (10 4 to 10 7 Pa) environment. image in electron eye detector with CCD camera defective blade) can produce artifacts in the final preparation. It is important for students to recognize that not every slide in their slide collection is perfect and that they should be familiar with the most common artifacts found on their slides.

1	Besides bright-field microscopy, which is commonly used for routine examination of histologic slides, other optical systems (described below) are used in clinical and research laboratories. Some of them are used to enhance the contrast without staining (such as phase contrast microscope), whereas others are designed to visualize structures using specific techniques such as immunofluorescence (fluorescence and confocal microscopes). The phase contrast microscope enables examination of unstained cells and tissues and is especially useful for living cells.

1	The phase contrast microscope takes advantage of small differences in the refractive index in different parts of a cell or tissue sample. Light passing through areas of relatively high refractive index (denser areas) is deflected and becomes out of phase with the rest of the beam of light that has passed through the specimen. The phase contrast microscope adds other induced, out-of-phase wavelengths through a series of optical rings in the condenser and objective lenses, essentially abolishing the amplitude of the initially deflected portion of the beam and producing contrast in the image. Dark portions of the image correspond to dense portions of the specimen; light portions of the image correspond to less dense portions of the specimen. The phase contrast microscope is therefore used to examine living cells and tissues (such as cells in tissue culture) and is used extensively to examine unstained semithin (approximately 0.5-m) sections of plastic-embedded tissue.

1	Two modifications of the phase contrast microscope are the interference microscope, which also allows quantification of tissue mass, and the differential interference microscope (using Nomarski optics), which is especially useful for assessing surface properties of cells and other biologic objects. In dark-field microscopy, no direct light from the light source is gathered by the objective lens. In dark-field microscopy, only light that has been scattered or diffracted by structures in the specimen reaches the objective. The dark-field microscope is equipped with a special condenser that illuminates the specimen with strong, oblique

1	FIGURE 1.10 • Example of sections from an orange and a kidney renal corpuscle. The dotted lines drawn on the intact orange indicate the plane of section that correlates with each cut surface. Similarly, different sections through a kidney renal corpuscle, which is also a spherical structure, show differences in appearance. The size and internal structural appearance are reflected in the plane of section. light. Thus, the field of view appears as a dark background on which small particles in the specimen that reflect some light into the objective appear bright. The effect is similar to that of dust particles seen in the light beam emanating from a slide projector in a darkened room. The light reflected off the dust particles reaches the retina of the eye, thus making the particles visible.

1	The resolution of the dark-field microscope cannot be better than that of the bright-field microscope, using, as it does, the same wavelength source. Smaller individual particles can be detected in dark-field images, however, because of the enhanced contrast that is created. The dark-field microscope is useful in examining autoradiographs, in which the developed silver grains appear white in a dark background. Clinically, dark-field microscopy is useful in examining urine for crystals, such as those of uric acid and oxalate, and in demonstrating specific bacteria such as spirochetes, particularly Treponema pallidum, the microorganism that causes syphilis, a sexually transmitted disease. The fluorescence microscope makes use of the ability of certain molecules to fluoresce under ultraviolet light.

1	The fluorescence microscope makes use of the ability of certain molecules to fluoresce under ultraviolet light. A molecule that fluoresces emits light of wavelengths in the visible range when exposed to an ultraviolet (UV) source. The ﬂuorescence microscope is used to display naturally occurring fluorescent (autofluorescent) molecules such as vitamin A and some neurotransmitters. Because autofluorescent molecules are not numerous, however, the microscope’s most widespread application is the display of introduced fluores cence, as in the detection of antigens or antibodies in immunocytochemical staining procedures (see Fig. 1.6). Specific fluorescent molecules can also be injected into an animal or directly into cells and used as tracers. Such methods have been useful in studying intercellular (gap) junctions, in tracing the pathway of nerve fibers in neurobiology, and in detecting fluorescent growth markers of mineralized tissues.

1	Various filters are inserted between the UV light source and the specimen to produce monochromatic or near-monochromatic (single-wavelength or narrow-band–wavelength) light. A second set of filters inserted between the specimen and the objective allows only the narrow band of wavelength of the fluorescence to reach the eye or to reach a photographic emulsion or other analytic processor. The confocal scanning microscope combines components of a light optical microscope with a scanning system to dissect a specimen optically.

1	The confocal scanning microscope combines components of a light optical microscope with a scanning system to dissect a specimen optically. The confocal scanning microscope allows visualization of a biologic specimen in three dimensions. The two lenses in the confocal microscope (objective and phototube lens) are perfectly aligned to focus light from the focal point of one lens to the focal point of the other lens. The major difference between a conventional and a confocal microscope is the addition of a detector aperture (pinhole) that is conjugate with the focal point of the lens; therefore, it is confocal. This precisely positioned pinhole allows only “in-focus” light to pass into a photomultiplier (detector) device, whereas the “out-of-focus” light is blocked from entering the detector (Fig. 1.11). This system

1	FIGURE 1.11 • Diagram of the in-focus and out-of-focus emitted light in the confocal microscope. a. This diagram shows the path of the laser beam and emitted light when the imaging structure is directly at the focus of the lens. The screen with a pinhole at the other side of the optical system of the confocal microscope allows the light from the structure in focus to pass through the pinhole. The light is then translated into an image by computer software. Because the focal point of the objective lens of the microscope forms a sharp image at the level at which the pinhole is located, these two points are referred to as confocal points. b. This diagram shows the path of the laser beam and the emitted light, which is out of focus in relation to the pinhole. Thus, the light from the specimen that gets blocked by the pinhole is never detected.

1	FIGURE 1.12 • Structure of the confocal microscope and diagram of the beam path. The light source for the confocal microscope comes from a laser. The laser beam (red line) travels to the tissue sample via a dichroic beam splitter and then to two movable scanning mirrors; these mirrors scan the laser beam across the sample in both x and y directions. Finally, the laser beam enters the fluorescence microscope and travels through its optical system to illuminate an examined tissue sample. The emitted light by the illuminated tissue sample (blue line) travels back through the optical system of the microscope, through both scanning mirrors, passes through the beam splitter, and is focused onto the pinhole. The light that passes through the pinhole is received and registered by the detector attached to a computer that builds the image one pixel at a time.

1	has the capability to obtain exceptional resolution (0.2 to 0.5 m) and clarity from a thin section of a biologic sample simply by rejecting out-of-focus light. The confocal microscope uses an illuminating laser light system that is strongly convergent and therefore produces a high-intensity excitation light in the form of a shallow scanning spot. A mirror system is used to move the laser beam across the specimen, illuminating a single spot at a time (Fig. 1.12). Many single spots in the same focal plane are scanned, and a computer software program reconstructs the image from the data recorded during scanning. In this aspect, confocal microscopy resembles the imaging process in a computed axial tomography (CAT) scan.

1	Furthermore, by using only the narrow depth of the in-focus image, it is possible to create multiple images at varying depths within the specimen. Thus, one can literally dissect layer by layer through the thickness of the specimen. It is also possible to use the computer to make three-dimensional reconstructions of a series of these images. Because each individual image located at a specific depth within the specimen is extremely sharp, the resulting assembled three-dimensional image is equally sharp. Moreover, once the computer has assembled each sectioned image, the reconstructed three-dimensional image can be rotated and viewed from any orientation desired (see Fig. 1.4). The ultraviolet microscope uses quartz lenses with an ultraviolet light source.

1	The ultraviolet microscope uses quartz lenses with an ultraviolet light source. The image in the ultraviolet (UV) microscope depends on the absorption of UV light by molecules in the specimen. The UV source has a wavelength of approximately 200 nm. Thus, the UV microscope may achieve a resolution of 0.1 m. In principle, UV microscopy resembles the workings of a spectrophotometer; the results are usually recorded photographically. The specimen cannot be inspected directly through an ocular because the UV light is not visible and is injurious to the eye.

1	The method is useful in detecting nucleic acids, specifically the purine and pyrimidine bases of the nucleotides. It is also useful for detecting proteins that contain certain amino acids. Using specific illuminating wavelengths, UV spectrophotometric measurements are commonly made through the UV microscope to determine quantitatively the amount of DNA and RNA in individual cells. As described in the Folder 1.2 on page 7, Feulgen microspectrophoto metry is used clinically to evaluate the degree of ploidy (multiples of normal DNA quantity) in sections of tumors. The polarizing microscope uses the fact that highly ordered molecules or arrays of molecules can rotate the angle of the plane of polarized light. The polarizing microscope is a simple modification of the light microscope in which a polarizing filter (the polarizer) is located between the light source and the specimen, and a second polarizer (the analyzer) is located between the objective lens and the viewer.

1	Both the polarizer and the analyzer can be rotated; the difference between their angles of rotation is used to determine the degree by which a structure affects the beam of polarized light. The ability of a crystal or paracrystalline array to rotate the plane of polarized light is called birefringence (double refraction). Striated muscle and the crystalloid inclusions in the testicular interstitial cells (Leydig cells), among other common structures, exhibit birefringence. Two kinds of EMs can provide morphologic and analytic data on cells and tissues: the transmission electron microscope and the scanning electron microscope. The primary improvement in the EM versus the light microscope is that the wavelength of the EM beam is approximately 1/2,000 that of the light microscope beam, thereby increasing resolution by a factor of 103. The TEM uses the interaction of a beam of electrons with a specimen to produce an image.

1	The TEM uses the interaction of a beam of electrons with a specimen to produce an image. The optics of the TEM are, in principle, similar to those of the light microscope (see Fig. 1.9), except that the TEM uses a beam of electrons rather than a beam of light. The principle of the microscope is as follows:  An electron source (cathode, electron gun), such as a heated tungsten filament, emits electrons.  The electrons are attracted toward an anode.  An electrical difference between the cathode cover and the anode imparts an accelerating voltage of between 20,000 and 200,000 volts to the electrons, creating the electron beam.  The beam then passes through a series of electromag netic lenses that serve the same function as the glass lenses of a light microscope.

1	The condenser lens shapes and changes the diameter of the electron beam that reaches the specimen plane. The beam that has passed through the specimen is then focused and magnified by an objective lens and then further magnified by one or more projector lenses. The final image is viewed on a phosphor-coated ﬂuorescent screen or captured on a photographic plate. Portions of the specimen through which electrons have passed appear bright; dark portions of the specimen have absorbed or scattered electrons because of their inherent density or because of heavy metals added during specimen preparation. Often, an electron detector with a light-sensitive sensor such as a charge-coupled device (CCD) are placed above or below the viewing screen to observe the image in real time on a monitor. This allows for uncomplicated procedures of archiving images or videos in digital format on computers.

1	Specimen preparation for transmission electron microscopy is similar to that for light microscopy except that it requires finer methods. The principles used in the preparation of sections for viewing with the TEM are essentially the same as those used in light microscopy, with the added constraint that at every step one must work with specimens three to four orders of magnitude smaller or thinner than those used for light microscopy. The TEM, which has an electron beam wavelength of approximately 0.1 nm, has a theoretical resolution of 0.05 nm. Because of the exceptional resolution of the TEM, the quality of fixation—that is, the degree of preservation of sub-cellular structure—must be the best achievable. Routine preparation of specimens for transmission electron microscopy begins with glutaraldehyde fixation followed by a buffer rinse and fixation with osmium tetroxide.

1	Routine preparation of specimens for transmission electron microscopy begins with glutaraldehyde fixation followed by a buffer rinse and fixation with osmium tetroxide. Glutaraldehyde, a dialdehyde, preserves protein constituents by cross-linking them; the osmium tetroxide reacts with lipids, particularly phospholipids. The osmium also imparts electron density to cell and tissue structures because it is a heavy metal, thus enhancing subsequent image formation in the TEM. Ideally, tissues should be perfused with buffered glutaraldehyde before excision from the animal. More commonly, tissue pieces no more than 1 mm3 are fixed for the TEM (compared with light microscope specimens, which may be measured in centimeters). The dehydration process is identical to that used in light microscopy, and the tissue is infiltrated with a monomeric resin, usually an epoxy resin, that is subsequently polymerized.

1	The plastic-embedded tissue is sectioned on specially designed microtomes using diamond knives. Because of the limited penetrating power of electrons, sections for routine transmission electron microscopy range from 50 nm to no more than 150 nm. Also, for the reason that abrasives used to sharpen steel knives leave unacceptable scratches on sections viewed in the TEM, diamond knives with a nearly perfect cutting edge are used. Sections cut by the diamond knife are much too thin to handle; they are floated away from the knife edge on the surface of a fluid-filled trough and picked up from the surface onto plastic-coated copper mesh grids. The grids have 50 to 400 holes/inch or special slots for viewing serial sections. The beam passes through the holes in the copper grid and then through the specimen, and the image is then focused on the viewing screen, CCD, or photographic film.

1	Routine staining of transmission electron microscopy sections is necessary to increase the inherent contrast so that the details of cell structure are readily visible and photographable. In general, transmission electron microscopy sections are stained by adding materials of great density, such as ions of heavy metals, to the specimen. Heavy-metal ions may be bound to the tissues during fixation or dehydration or by soaking the sections in solutions of such ions after sectioning. Osmium tetroxide, routinely used in the fixative, binds to the phospholipid components of membranes, imparting additional density to the membranes. Uranyl nitrate is often added to the alcohol solutions used in dehydration to increase the density of components of cell junctions and other sites. Sequential soaking in solutions of uranyl acetate and lead citrate is routinely used to stain sections before viewing with the TEM to provide high-resolution, high-contrast electron micrographs.

1	Sometimes, special staining is required to visualize results of histocytochemical or immunocytochemical reactions with the TEM. The phosphatase and esterase procedures are used for this purpose (see Fig. 1.3). Substitution of a heavy metal–containing compound for the fluorescent dye that has been conjugated with an antibody allows the adaptation of immunocytochemical methods to transmission electron microscopy. Similarly, routine EM autoradiography techniques have been refined for use with transmission electron microscopy (see Fig. 1.8b). These methods have been particularly useful in elucidating the cellular sources and intracellular pathways of certain secretory products, the location on the cell surface of specific receptors, and the intracellular location of ingested drugs and substrates. Freeze fracture is a special method of sample preparation for transmission electron microscopy; it is especially important in the study of membranes.

1	Freeze fracture is a special method of sample preparation for transmission electron microscopy; it is especially important in the study of membranes. The tissue to be examined may be fixed or unfixed; if it has been fixed, then the fixative is washed out of the tissue before proceeding. A cryoprotectant such as glycerol is allowed to infiltrate the tissue, and the tissue is then rapidly frozen to about 160 C. Ice crystal formation is prevented by the use of cryoprotectants, rapid freezing, and extremely small tissue samples. The frozen tissue is then placed in a vacuum in the freeze fracture apparatus and struck with a knife edge or razor blade. The fracture plane passes preferentially through the hydrophobic portion of the plasma membrane, exposing the interior of the plasma membrane.

1	The fracture plane passes preferentially through the hydrophobic portion of the plasma membrane, exposing the interior of the plasma membrane. The resulting fracture of the plasma membrane produces two new surfaces. The surface of the membrane that is backed by extracellular space is called the E-face; the face backed by the protoplasm (cytoplasm) is called the P-face. The specimen is then coated, typically with evaporated platinum, to create a replica of the fracture surface. The tissue is then dissolved, and the surface replica, not the tissue itself, is picked up on grids to be examined with the TEM. Such a replica displays details at the macromolecular level (see Fig. 2.5, page 30). In scanning electron microscopy, the electron beam does not pass through the specimen but is scanned across its surface.

1	In scanning electron microscopy, the electron beam does not pass through the specimen but is scanned across its surface. In many ways, the images obtained from SEM more closely resemble those seen on a television screen than on the TEM monitor. They are three-dimensional in appearance and portray the surface structure of an examined sample. For the examination of most tissues, the sample is fixed, dehydrated by critical point drying, coated with an evaporated gold–carbon film, mounted on an aluminum stub, and placed in the specimen chamber of the SEM. For mineralized tissues, it is possible to remove all the soft tissues with bleach and then examine the structural features of the mineral.

1	Scanning is accomplished by the same type of raster that scans the electron beam across the face of a television tube. Electrons reflected from the surface (backscattered electrons) and electrons forced out of the surface (secondary electrons) are collected by one or more detectors and reprocessed to form a high-resolution three-dimensional image of a sample surface. In earlier models of microscopes, images were captured on high-resolution cathode ray tube (CRT) or photographic plate; modern instruments, however, capture digital images using sensitive detectors and CCD for display on a high-resolution computer monitor. Other detectors can be used to measure X-rays emitted from the surface, cathodoluminescence of molecules in the tissue below the surface, and Auger electrons emitted at the surface. The scanning-transmission electron microscope (STEM) combines features of the TEM and SEM to allow electron-probe X-ray microanalysis.

1	The scanning-transmission electron microscope (STEM) combines features of the TEM and SEM to allow electron-probe X-ray microanalysis. The SEM configuration can be used to produce a transmission image by inserting a grid holder at the specimen level, collecting the transmitted electrons with a detector, and reconstructing the image on a CRT. This latter configuration of an SEM or scanning-transmission electron microscope (STEM) facilitates the use of the instrument for electron-probe X-ray microanalysis.

1	Detectors can be fitted to the microscope to collect the X-rays emitted as the beam bombards the section; with appropriate analyzers, a map can be constructed that shows the distribution in the sections of elements with an atomic number above 12 and a concentration sufficient to produce enough X-rays to analyze. Semiquantitative data can also be derived for elements in sufficient concentration. Thus, both the TEM and the SEM can be converted into sophisticated analytical tools in addition to being used as “optical” instruments. The atomic force microscope has emerged as one of the most powerful tools for studying the surface topography at molecular and atomic resolution.

1	The atomic force microscope has emerged as one of the most powerful tools for studying the surface topography at molecular and atomic resolution. One newer microscope that has proved most useful for biologic studies is the atomic force microscope (AFM). It is a nonoptical microscope that works in the same way as a fingertip, which touches and feels the skin of our face when we cannot see it. The sensation from the fingertip is processed by our brain, which is able to deduce surface topography of the face while touching it.

1	In the AFM, an ultrasharp, pointed probe, approaching the size of a single atom at the tip, scans the specimen following parallel lines along the x-axis, repeating the scan at small intervals along the y-axis. The sharp tip is mounted at the end of a highly flexible cantilever so that the tip deflects the cantilever as it encounters the “atomic force” on the surface of the specimen (Fig. 1.13). The upper surface of the cantilever is reflective, and a laser beam is directed off the cantilever to a diode. This arrangement acts as an “optical lever” because extremely small deflections of the cantilever are greatly magnified on the diode. The AFM can work with the tip of the cantilever touching the sample (contact mode), or the tip can tap across the surface (tapping mode) much like the cane of a blind person (Fig. 1.13 insets).

1	As the tip moves up and down in the z-axis as it traverses the specimen, the movements are recorded on the diode as movements of the reflected laser beam. A piezoelectric device under the specimen is activated in a sensitive feedback loop with the diode to move the specimen up and down so that the laser beam is centered on the diode. As the tip dips down into a depression, the piezoelectric device moves the specimen up to compensate, and when the tip moves up over an elevation, the device compensates by lowering the specimen. The current to the piezoelectric device is interpreted as the z-axis, which along with the x-and y-axes renders the topography of the specimen at a molecular, and sometimes an atomic, resolution (Fig. 1.14).

1	A major advantage of the AFM for examining biologic specimens is that, unlike high-resolution optical instruments (i.e., TEM or SEM), the specimen does not have to be in a vacuum; it can even be in water. Thus, it is feasible to image living cells and their surrounding environments.

1	FIGURE 1.13 • Diagram of the atomic force microscope (AFM). An extremely sharp tip on a cantilever is moved over the surface of a biologic specimen. The feedback mechanism provided by the piezoelectric scanners enables the tip to be maintained at a constant force above the sample surface. The tip extends down from the end of a laser-reflective cantilever. A laser beam is focused onto the cantilever. As the tip scans the surface of the sample, moving up and down with the contour of the surface, the laser beam is deflected off the cantilever into a photodiode. The photodiode measures the changes in laser beam intensities and then converts this information into electrical current. Feedback from the photodiode is processed by a computer as a surface image and also regulates the piezoelectric scanner. In contact mode (left inset), the electrostatic or surface tension forces drag the scanning tip over the surface of the sample. In the tapping mode (right inset), the tip of the cantilever

1	scanner. In contact mode (left inset), the electrostatic or surface tension forces drag the scanning tip over the surface of the sample. In the tapping mode (right inset), the tip of the cantilever oscillates. The latter mode allows visualization of soft and fragile samples while achieving a high resolution.

1	FIGURE 1.14 • Atomic force microscopic image of a single DNA molecule. This image was obtained in the contact mode in which the sharp scanning tip “bumps” up and down as it is moved back and forth over the surface of the sample. The sample lies on an ultrasmooth mica surface. An individual molecule of DNA easily produces enough of a bump to be detected. Thickenings along the DNA molecule are produced by proteins bound to the molecule, and these thickenings produce an even larger movement of the scanning tip. The scan field measures 540 nm by 540 nm. The length of the DNA molecule ranges from 0 to 40 nm. 185,000. (Courtesy of Dr. Gabriela Bagordo, JPK Instruments AG, Berlin, Germany.)

1	OVERVIEW OF THE CELL AND CYTOPLASM / 22 MEMBRANOUS ORGANELLES / 25 Plasma Membrane / 25 Endosomes / 35 Lysosomes / 38 Autophagy / 41 Proteasome-Mediated Degradation / 44 Rough-Surfaced Endoplasmic Reticulum / 45 Smooth-Surfaced Endoplasmic Reticulum / 49 Golgi Apparatus / 50 Mitochondria / 53 Peroxisomes (Microbodies) / 56 NONMEMBRANOUS ORGANELLES / 56 Microtubules / 56 Actin Filaments / 59 Intermediate Filaments / 62 Centrioles and Microtubule-Organizing Centers / 65 Basal Bodies / 71 INCLUSIONS / 71 CYTOPLASMIC MATRIX / 73 Folder 2.1 Clinical Correlation: Lysosomal Storage Diseases / 42 Folder 2.2 Clinical Correlation: Abnormalities in Microtubules and Filaments / 68 Folder 2.3 Clinical Correlation: Abnormal Duplication of Centrioles and Cancer / 72 Cells are the basic structural and functional units of all multicellular organisms.

1	Cells are the basic structural and functional units of all multicellular organisms. The processes we normally associate with the daily activities of organisms—protection, ingestion, digestion, absorption of metabolites, elimination of wastes, movement, reproduction, and even death—are all reﬂections of similar processes occurring within each of the billions of cells that constitute the human body. To a very large extent, cells of different types use similar mechanisms to synthesize protein, transform energy, and move essential substances into the cell. They use the same kinds of molecules to engage in contraction, and they duplicate their genetic material in the same manner. Specific functions are identified with specific structural components and domains within the cell.

1	Specific functions are identified with specific structural components and domains within the cell. Some cells develop one or more of these functions to such a degree of specialization that they are identified by the function and the cell structures associated with them. For example, although all cells contain contractile filamentous proteins, some cells such as muscle cells, contain large amounts of these proteins in specific arrays. This allows them to carry out their specialized function of contraction at both the cellular and tissue level. The specialized activity or function of a cell may be reﬂected not only by the presence of a larger amount of the specific structural component performing the activity but also by the shape of the cell, its organization with respect to other similar cells, and its products (Fig. 2.1). Cells can be divided into two major compartments: the cytoplasm and the nucleus.

1	Cells can be divided into two major compartments: the cytoplasm and the nucleus. In general, the cytoplasm is the part of the cell located outside the nucleus. The cytoplasm contains organelles (“little organs”) and inclusions in an aqueous gel called the cytoplasmic matrix. The matrix consists of a variety of solutes, including inorganic ions (Na , K , Ca2 ) and organic molecules such as intermediate metabolites, carbohydrates, lipids, proteins, and RNAs. The cell controls the concentration

1	FIGURE 2.1 • Histologic features of different cell types. These three photomicrographs show different types of cells in three different organs of the body. The distinguishing features include size, shape, orientation, and cytoplasmic contents that can be related to each cell’s specialized activity or function. a. Epithelial cells in the kidney. Note several shapes of epithelial cells: columnar cells with well-defned borders in the collecting duct (CD), squamous cells in the thin segment (TS ) of the nephron, and even more-fattened cells lining blood vessels, the vasa recta in the kidney (VR). 380. b. Dorsal root ganglion cells. Note the large size of these nerve cell bodies and the large, pale (euchromatic) nuclei (N) with distinct nucleoli. Each ganglion cell is surrounded by fattened satellite cells (S ). The size of the ganglion cell and the presence of a euchromatic nucleus, prominent nucleolus, and Nissl bodies (rough-surfaced endoplasmic reticulum visible as darker granules

1	satellite cells (S ). The size of the ganglion cell and the presence of a euchromatic nucleus, prominent nucleolus, and Nissl bodies (rough-surfaced endoplasmic reticulum visible as darker granules within the cytoplasm) refect the extensive synthetic activity required to maintain the exceedingly long processes (axons) of these cells. 380. C. Smooth muscle cells of the small intestine. Note that these cells are typically elongated, fusiform shaped, and organized in a parallel array. The nuclei are also elongated to conform to the general shape of the cell. 380.

1	of solutes within the matrix, which inﬂuences the rate of metabolic activity within the cytoplasmic compartment. The nucleus is the largest organelle within the cell and contains the genome along with the enzymes necessary for DNA replication and RNA transcription. The cytoplasm and nucleus play distinct functional roles but also work in concert to maintain the cell’s viability. The structure and function of the nucleus is discussed in Chapter 3. Organelles are described as membranous (membranelimited) or nonmembranous.

1	Organelles are described as membranous (membranelimited) or nonmembranous. Organelles include the membrane systems of the cell and the membrane-limited compartments that perform the metabolic, synthetic, energy-requiring, and energy-generating functions of the cell, as well as nonmembranous structural components. All cells have the same basic set of intracellular organelles, which can be classified into two groups: (1) membranous organelles with plasma membranes that separate the internal environment of the organelle from the cytoplasm, and (2) nonmembranous organelles without plasma membranes.

1	The membranes of membranous organelles form vesicular, tubular, and other structural patterns within the cytoplasm that may be convoluted (as in smooth-surfaced endoplasmic reticulum) or plicated (as in the inner mitochondrial membrane). These membrane configurations greatly increase the surface area on which essential physiologic and biochemical reactions take place. The spaces enclosed by the organelles’ membranes constitute the intracellular microcompartments in which substrates, products, and other substances are segregated or concentrated. In addition, each type of organelle contains a set of unique proteins; in membranous organelles, these proteins are either incorporated into their membranes or sequestered within their lumens. For example, the enzymes of lysosomes are separated by a specific enzyme-resistant membrane from the cytoplasmic matrix because their hydrolytic activity would be detrimental to the cell. In nonmembranous organelles, the unique proteins usually

1	by a specific enzyme-resistant membrane from the cytoplasmic matrix because their hydrolytic activity would be detrimental to the cell. In nonmembranous organelles, the unique proteins usually self-assemble into polymers that form the structural elements of the cytoskeleton.

1	Besides organelles, the cytoplasm contains inclusions, structures that are not usually surrounded by a plasma membrane. They consist of such diverse materials as crystals, pigment granules, lipids, glycogen, and other stored waste products (for details, see page 71).

1	The membranous organelles include:  the plasma (cell) membrane, a lipid bilayer that forms the cell boundary as well as the boundaries of many organelles within the cell; rough-surfaced endoplasmic reticulum (rER), a region of endoplasmic reticulum associated with ribosomes and the site of protein synthesis and modification of newly synthesized proteins;  smooth-surfaced endoplasmic reticulum (sER), a region of endoplasmic reticulum involved in lipid and steroid synthesis but not associated with ribosomes; Golgi apparatus, a membranous organelle composed of multiple ﬂattened cisternae responsible for modifying, sorting, and packaging proteins and lipids for intracellular or extracellular transport;  endosomes, membrane-bounded compartments interposed within endocytotic pathways that have the major function of sorting proteins delivered to them via endocytotic vesicles and redirecting them to different cellular compartments for their final destination;  lysosomes, small

1	that have the major function of sorting proteins delivered to them via endocytotic vesicles and redirecting them to different cellular compartments for their final destination;  lysosomes, small organelles containing digestive enzymes that are formed from endosomes by targeted delivery of unique lysosomal membrane proteins and lysosomal enzymes;  transport vesicles—including pinocytotic vesicles, endocytotic vesicles, and coated vesicles—that are involved in both endocytosis and exocytosis and vary in shape and the material that they transport;  mitochondria, organelles that provide most of the energy to the cell by producing adenosine triphosphate (ATP) in the process of oxidative phosphorylation; and  peroxisomes, small organelles involved in the production and degradation of H2O2 and degradation of fatty acids.

1	The nonmembranous organelles include:  microtubules, which together with actin and intermedi ate filaments form elements of the cytoskeleton and continuously elongate (by adding tubulin dimers) and shorten (by removing tubulin dimers), a property referred to as dynamic instability;  flaments, which are also part of the cytoskeleton and can be classified into two groups—actin flaments, which are ﬂexible chains of actin molecules, and intermediate flaments, which are ropelike fibers formed from a variety of proteins—both groups providing tensile strength to withstand tension and confer resistance to shearing forces;  centrioles, or short, paired cylindrical structures found in the center of the microtubule-organizing center (MTOC) or centrosome and whose derivatives give rise to basal bodies of cilia; and  ribosomes, structures essential for protein synthesis and composed of ribosomal RNA (rRNA) and ribosomal proteins (including proteins attached to membranes of the rER and proteins

1	of cilia; and  ribosomes, structures essential for protein synthesis and composed of ribosomal RNA (rRNA) and ribosomal proteins (including proteins attached to membranes of the rER and proteins free in the cytoplasm).

1	An outline of the key features of cellular organelles and inclusions is provided in Table 2.1. The normal function and related pathologies of the organelles are summarized in Table 2.2.

1	Nucleus 3–10 Largest organelle within the cell Surrounded by two membranes (nuclear with distinct boundary envelope) containing nuclear pore complexes and perinuclear cisternal space Often visible nucleoli and Regions with condensed and diffused chromatin pattern regions chromatin pattern (heterochromatin and euchromatin) Nucleolus 1–2 Roughly circular, basophilic Dense, nonmembranous structure region within the nucleus containing fbrillar and granular material Visible in living cells throughout interphase with interference microscopy Plasma membrane 0.008–0.01 Not visible External membrane and membranes surrounding membranous organelles of cell; two inner and outer electron-dense layers separated by intermediate electron-lucent layer rER Area 5–10 Often observed as basophilic Flattened sheets, sacs, and tubes of region of cytoplasm referred membranes with attached ribosomes to as ergastoplasm sER Throughout Not visible Flattened sheets, sacs, and tubes of cytoplasm Cytoplasm in

1	sheets, sacs, and tubes of region of cytoplasm referred membranes with attached ribosomes to as ergastoplasm sER Throughout Not visible Flattened sheets, sacs, and tubes of cytoplasm Cytoplasm in region of sER may membranes without attached ribosomes exhibit distinct eosinophilia Golgi apparatus Area 5–10 Sometimes observed as Stack of fattened membrane sheets, “negative staining” region often adjacent to one side of nucleus Appears as network in heavy metal–stained preparations Visible in living cells with interference microscopy TABLE Review of Organelles and Cytoplasmic Inclusions: A Key to Light Microscopic and Electron Microscopic Identification2.1 Organelle or Inclusion Size (m) Light Microscopic Features Electron Microscopic Features continued next page chapter 2 Cell Cytoplasm MEMBRANOUS ORGANELLES TABLE Review of Organelles and Cytoplasmic Inclusions: A Key to Light Microscopic and Electron Microscopic Identification (Cont.)2.1 Organelle or Inclusion Size (m) Light

1	MEMBRANOUS ORGANELLES TABLE Review of Organelles and Cytoplasmic Inclusions: A Key to Light Microscopic and Electron Microscopic Identification (Cont.)2.1 Organelle or Inclusion Size (m) Light Microscopic Features Electron Microscopic Features Secretory vesicles 0.050–1.0 Observed only when vesicles Many relatively small, membrane-bounded are very large (e.g., zymogen vesicles of uniform diameter, often granules in pancreas) polarized on one side of cell Mitochondria 0.2–7 Sometimes observed in Two-membrane system: Outer membrane favorable situations (e.g., liver and inner membrane arranged in numerous or nerve cells) as miniscule, dark folds (cristae) dots; visible in living cells stained In steroid-producing cells, inner membrane with vital dyes (e.g., Janus green) arranged in tubular cristae Endosomes 0.02–0.5 Not visible Tubulovesicular structures with subdivided lumen containing electron-lucent material or other smaller vesicles Lysosomes 0.2–0.5 Visible only after special enzyme

1	Endosomes 0.02–0.5 Not visible Tubulovesicular structures with subdivided lumen containing electron-lucent material or other smaller vesicles Lysosomes 0.2–0.5 Visible only after special enzyme Membrane-bounded vesicles, often electron histochemical staining dense Peroxisomes 0.2–0.5 Visible only after special enzyme Membrane-bounded vesicles, often with histochemical staining electron-dense crystalloid inclusions Cytoskeletal elements 0.006–0.025 Only observed when organized Long, linear staining pattern with width into large structures (e.g., muscle and features characteristic of each fbrils) flament type Ribosomes 0.025 Not visible Minute dark dots, often associated with the rER Glycogen 0.010–0.040 Observed as a “purple haze” Nonmembranous, extremely dense grapelike region of cytoplasm inclusions metachromasia with toluidine blue–stained specimen Lipid droplets 0.2–5, up Readily visible when extremely Nonmembranous inclusions to 80 large (e.g., in adipocytes) Generally appear as a

1	inclusions metachromasia with toluidine blue–stained specimen Lipid droplets 0.2–5, up Readily visible when extremely Nonmembranous inclusions to 80 large (e.g., in adipocytes) Generally appear as a void in the section Appear as large empty holes in section (lipid itself is usually removed by embedding solvents)

1	The plasma membrane is a lipid-bilayered structure visible with transmission electron microscopy. The plasma membrane (cell membrane) is a dynamic structure that actively participates in many physiologic and biochemical activities essential to cell function and survival. When the plasma membrane is properly fixed, sectioned, stained, and viewed on edge with the transmission electron microscope (TEM), it appears as two electron-dense layers separated by an intermediate, electron-lucent (nonstaining) layer (Fig. 2.2). The total thickness of the plasma membrane is about 8 to 10 nm. The plasma membrane is composed of an amphipathic lipid layer containing embedded integral membrane proteins with peripheral membrane proteins attached to its surfaces.

1	The current interpretation of the molecular organization of the plasma membrane is referred to as the modifed fuid–mosaic model (Fig. 2.3). The membrane consists primarily of phospholipid, cholesterol, and protein molecules. The lipid molecules form a lipid bilayer with an amphipathic character (it is both hydrophobic and hydrophilic). The fatty-acid chains of the lipid molecules face each other, making the inner portion of the membrane hydrophobic (i.e., having no affinity for water). The surfaces of the membrane are formed by the polar head groups of the lipid molecules, thereby making the surfaces hydrophilic (i.e., they have an affinity for water). Lipids are distributed asymmetrically between the inner and outer leaﬂets of the lipid bilayer, and their composition varies considerably among different biologic membranes.

1	In most plasma membranes, protein molecules constitute approximately half of the total membrane mass. Most of the proteins are embedded within the lipid bilayer or pass through the lipid bilayer completely. These proteins are called integral membrane proteins. The other types of protein— peripheral membrane proteins—are not embedded within the lipid bilayer. They are associated with the plasma

1	TABLE Organelles and Cytoplasmic Inclusions: Functions and Pathologies 2.2 Organelle or Inclusion Function Examples of Associated Pathologies Nucleus Storage and use of genome Inherited genetic diseases; environmentally induced mutations Nucleolus Synthesis of rRNA and partial Werner syndrome (premature aging assembly of ribosomal subunits disease) Involved in regulation of cell cycle Malfunctions of cell cycle leading to cancerogenesis Plasmamembrane Ion and nutrient transport Cystic fbrosis Recognition of environmental signal Intestinal malabsorption syndromes Cell-to-cell and cell-to-extracellular Lactose intolerance matrix adhesions rER Binds ribosomes engaged in translating Pseudoachondroplasia mRNA for proteins destined for secretion Calcium phosphate dihydrate crystal or for membrane insertion deposition disease Also involved in chemical modifcations of proteins and membrane lipid synthesis sER Involved in lipid and steroid metabolism Hepatic endoplasmic reticular storage

1	insertion deposition disease Also involved in chemical modifcations of proteins and membrane lipid synthesis sER Involved in lipid and steroid metabolism Hepatic endoplasmic reticular storage disease Golgi apparatus Chemical modifcation of proteins I-cell disease Sorting and packaging of molecules Polycystic kidney disease for secretion or transport to other organelles Secretory vesicles Transport and storage of secreted proteins Lewy bodies of Parkinson’s disease to plasma membrane Proinsulin diabetes Mitochondria Aerobic energy supply (oxidative Mitochondrial myopathies such as MERRFa , phosphorylation, ATP) MELASb, Kearns-Sayre syndromes, and Initiation of apoptosis Leber’s hereditary optic atrophy Endosomes Transport of endocytosed material M-6-P receptor defciency Biogenesis of lysosomes Lysosomes Digestion of macromolecules Lysosomal storage diseases (see Folder 2.1, Clinical Correlation: Lysosomal Storage Diseases) Peroxisomes Oxidative digestion (e.g., fatty acids)

1	of lysosomes Lysosomes Digestion of macromolecules Lysosomal storage diseases (see Folder 2.1, Clinical Correlation: Lysosomal Storage Diseases) Peroxisomes Oxidative digestion (e.g., fatty acids) Zellweger’s syndrome Cytoskeletal elements Various functions, including cell motility, Immotile cilia syndrome, Alzheimer’s cell adhesions, intracellular and disease, epidermolysis bullosa extracellular transport Maintenance of cellular skeleton Ribosomes Synthesis of protein by translating Ribosomal dysfunction in Alzheimer’s protein-coding sequence from disease; Diamond-Blackfan anemia mRNA Many antibiotics act selectively on bacterial ribosomes: e.g., tetracyclines, aminoglycosides (gentamicin, streptomycin) Glycogen Short-term storage of glucose in Several known glycogen-storage diseases, the form of branched polymer including major groups of hepatic-Found in liver, skeletal muscle, hypoglycemic and muscle-energy and adipose tissue pathophysiologies Lipiddroplets Storage of esterified

1	the form of branched polymer including major groups of hepatic-Found in liver, skeletal muscle, hypoglycemic and muscle-energy and adipose tissue pathophysiologies Lipiddroplets Storage of esterified forms of fatty acids Lipid storage diseases such as Gaucher’s as high-energy storage molecules and Niemann-Pick disease, liver cirrhosis aMyoclonic epilepsy and ragged red fibers syndrome. bMitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes syndrome.

1	FIGURE 2.2 • Electron micrograph of microvilli on the apical surface of an absorptive cell. This electron micrograph shows the apical portion of absorptive cells with microvilli. Note that at this magnifcation the plasma membrane displays its characteristic appearance, showing two electron-dense lines separated by an electron-lucent intermediate layer. The glycoproteins of the glycocalyx can be seen extending from the tips of the microvilli into the lumen. The relationship between the outer plasma-membrane leafet and the glycocalyx is particularly well demonstrated. Glycoproteins of the glycocalyx include terminal digestive enzymes such as dipeptidases and disaccharidases.

1	100,000. (Courtesy of Dr. Ray C. Henrikson.) membrane by strong ionic interactions, mainly with integral proteins on both the extracellular and intracellular surfaces of the membrane (see Fig. 2.3). In addition, on the extracellular surface of the plasma membrane, carbohydrates may be attached to proteins, thereby forming glycoproteins; or to lipids of the bilayer, thereby forming glycolipids. These surface molecules constitute a layer at the surface of the cell, referred to as the cell coat or glycocalyx (see Fig. 2.2). They help establish extracellular microenvironments at the membrane surface that have specific functions in metabolism, cell recognition, and cell association and serve as receptor sites for hormones. Microdomains of the plasma membrane, known as lipid rafts, control the movement and distribution of proteins within the lipid bilayer.

1	The ﬂuidity of the plasma membrane is not revealed in static electron micrographs. Experiments reveal that the membrane behaves as though it were a two-dimensional lipid ﬂuid. For many years it was thought that integral membrane proteins moved freely within the plane of the membrane; this movement was compared to the movement of icebergs ﬂoating in the ocean (see Fig. 2.3). However, recent evidence shows that the distribution and movement of proteins within the lipid bilayer is not as random as once thought. Localized regions within the plasma membrane contain high concentrations of cholesterol and glycosphingolipids. These regions are called lipid rafts. Owing to the high concentration of cholesterol and the presence of longer, highly saturated fatty-acid chains, the lipid raft area is thicker and exhibits less ﬂuidity than the surrounding plasma membrane (Fig. 2.4). Lipid rafts contain a variety of integral and peripheral membrane proteins involved in cell signaling. They can be

1	thicker and exhibits less ﬂuidity than the surrounding plasma membrane (Fig. 2.4). Lipid rafts contain a variety of integral and peripheral membrane proteins involved in cell signaling. They can be viewed as “signaling platforms” ﬂoating in the ocean of lipids. Each individual raft is equipped with all of the necessary elements (receptors, coupling factors, effector enzymes, and substrates) to receive and convey specific signals. Signal transduction in lipid rafts occurs more rapidly and efficiently because of the close proximity of interacting proteins. In addition, different signaling rafts allow for the separation of specific signaling molecules from each other.

1	Integral membrane proteins can be visualized with the special tissue preparation technique of freeze fracture. The existence of protein within the substance of the plasma membrane (i.e., integral proteins) was confirmed by a technique called freeze fracture. When tissue is prepared for electron microscopy by the freeze fracture process (Fig. 2.5a), membranes typically split or cleave along the hydrophobic plane (i.e., between the two lipid layers) to expose two interior faces of the membrane, an E-face and a P-face (see Fig. 2.5b). The E-face is backed by extracellular space, whereas the P-face is backed by cytoplasm ( protoplasm). The numerous particles seen on the Eand P-faces with the TEM represent the integral proteins of the membrane. Usually, the P-face displays more particles, thus more protein, than the E-face (see Fig. 2.5c). Integral membrane proteins have important functions in cell metabolism, regulation, and integration.

1	Integral membrane proteins have important functions in cell metabolism, regulation, and integration. Six broad categories of membrane proteins have been defined in terms of their function: pumps, channels, receptors, linkers,

1	FIGURE 2.3 • Diagram of a plasma membrane showing the modifed fuid–mosaic model. The plasma membrane is a lipid bilayer consisting primarily of phospholipid molecules, cholesterol, and protein molecules. The hydrophobic fatty-acid chains of phospholipids face each other to form the inner portion of the membrane, whereas the hydrophilic polar heads of the phospholipids form the extracellular and intracellular surfaces of the membrane. Cholesterol molecules are incorporated within the gaps between phospholipids equally on both sides of the membrane. Note the elevated area of the lipid raft that is characterized by the high concentration of glycosphingolipids and cholesterol. It contains large numbers of integral and peripheral membrane proteins. The raft protrudes above the level of asymmetrically distributed phospholipids in the membrane bilayer (indicated by the different colors of the phospholipid heads). Carbohydrate chains attach to both integral and peripheral membrane proteins to

1	distributed phospholipids in the membrane bilayer (indicated by the different colors of the phospholipid heads). Carbohydrate chains attach to both integral and peripheral membrane proteins to form glycoproteins, as well as to polar phospholipid heads to form glycolipids.

1	enzymes, and structural proteins (Fig. 2.6). The categories are  Pumps serve to transport certain ions, such as Na , actively not mutually exclusive (e.g., a structural membrane protein across membranes. Pumps also transport metabolic precursors may simultaneously serve as a receptor, an enzyme, a pump, or of macromolecules, such as amino acids and sugars, across any combination of these functions). membranes, either by themselves or linked to the Na pump.

1	FIGURE 2.4 • An image of lipid rafts obtained with tapping-mode atomic force microscopy (AFM). This image shows a 5-nm-thick lipid bilayer spread on the mica support. The bilayer is composed of dioleoylphosphatidylcholine (dioleoyl-PC), sphingomyelin, and cholesterol. Sphingomyelin and cholesterol together form lipid rafts represented on the image by the pink areas; the blue-purple areas are the nonraft background of the bilayer. Because the sphingomyelin molecules are longer than the dioleoyl-PC molecules, the rafts protrude from the nonraft background by about 0.8 nm, and the AFM is sensitive enough to detect this protrusion. The black regions represent the mica support. The image also shows molecules of the Helicobacter pylori toxin VacA (white particles), which preferentially bind to protein receptors on the raft domains. The area depicted in this image is 800 nm square. (Courtesy of Drs. Nicholas A. Geisse, Timothy L. Cover, Robert M. Henderson, and J. Michael Edwardson.) inner

1	protein receptors on the raft domains. The area depicted in this image is 800 nm square. (Courtesy of Drs. Nicholas A. Geisse, Timothy L. Cover, Robert M. Henderson, and J. Michael Edwardson.) inner leaflet of lipid layer

1	FIGURE 2.5 • Freeze fracture examination of the plasma membrane. a. View of the plasma membrane seen on edge, with arrow indicating the preferential plane of splitting of the lipid bilayer through the hydrophobic portion of the membrane. When the membrane splits, some proteins are carried with the outer leaflet, though most are retained within the inner leaflet. b. View of the plasma membrane with the leaflets separating along the cleavage plane. The surfaces of the cleaved membrane are coated, forming replicas; the replicas are separated from the tissue and examined with the TEM. Proteins appear as bumps. The replica of the inner leaflet is called the P-face; it is backed by cytoplasm (protoplasm). A view of the outer leaflet is called the E-face; it is backed by extracellular space. c. Electron micrograph of a freeze fracture replica shows the E-face of the membrane of one epithelial cell and the P-face of the membrane of the adjoining cell. The cleavage plane has jumped from the

1	Electron micrograph of a freeze fracture replica shows the E-face of the membrane of one epithelial cell and the P-face of the membrane of the adjoining cell. The cleavage plane has jumped from the membrane of one cell to the membrane of the other cell, as indicated by the clear space (intercellular space) across the middle of the figure. Note the paucity of particles in the E-face compared with the P-face, from which the majority of the integral membrane proteins project. (Courtesy of Dr. Giuseppina d’Elia Raviola.)  Channels allow the passage of small ions, molecules, and include the family of integrins that link cytoplasmic actin water across the plasma membrane in either direction (i.e., filaments to an extracellular matrix protein (fibronectin). passive diffusion). Gap junctions formed by aligned chan- Enzymes have a variety of roles. ATPases have specific nels in the membranes of adjacent cells permit passage of roles in ion pumping: ATP synthase is the major protein of ions

1	by aligned chan- Enzymes have a variety of roles. ATPases have specific nels in the membranes of adjacent cells permit passage of roles in ion pumping: ATP synthase is the major protein of ions and small molecules from the cytoplasm of one cell to the inner mitochondrial membrane, and digestive enzymes the cytoplasm of the adjacent cells. such as disaccharidases and dipeptidases are integral mem  Receptor proteins allow recognition and localized brane proteins. binding of ligands (molecules that bind to the extracellu- Structural proteins are visualized by the freeze fracture lar surface of the plasma membrane) in processes such as method, especially where they form junctions with neigh-hormonal stimulation, coated-vesicle endocytosis, and boring cells. Often, certain proteins and lipids are concenantibody reactions. trated in localized regions of the plasma membrane to  Linker proteins anchor the intracellular cytoskeleton to carry out specific functions. Examples of such regions

1	are concenantibody reactions. trated in localized regions of the plasma membrane to  Linker proteins anchor the intracellular cytoskeleton to carry out specific functions. Examples of such regions can the extracellular matrix. Examples of linker proteins be recognized in polarized cells such as epithelial cells.

1	FIGURE 2.6 • Different functions of integral membrane proteins. The six major categories of integral membrane proteins are shown in this diagram: pumps, channels, receptors, linkers, enzymes, and structural proteins. These categories are not mutually exclu-sive. A structural membrane protein involved in cell-to-cell junctions might simultaneously serve as a receptor, enzyme, linker, or a com-bination of these functions. pumps channels receptors linkers enzymes structural proteins actin NaHK Integral membrane proteins move within the lipid bilayer of the membrane. Particles bound to the membrane can move on the surface of a cell; even integral membrane proteins, such as enzymes, may move from one cell surface to another (e.g., from apical to lateral) when barriers to ﬂow, such as cell junctions, are disrupted. The ﬂuidity of the membrane is a function of the types of phospholipids in the membrane and variations in their local concentrations.

1	As previously mentioned, lipid rafts containing integral membrane proteins may move to a different region of the plasma membrane. The movement of an integral protein anchored on a lipid raft makes signaling more precise and prevents nonspecific interactions. The lateral migration of proteins is often limited by physical connections between membrane proteins and intracellular or extracellular structures. Such connections may exist between:  proteins associated with cytoskeletal elements and portions of the membrane proteins that extend into the adjacent cytoplasm, the cytoplasmic domains of membrane proteins, and  peripheral proteins associated with the extracellular matrix and the integral membrane proteins that extend from the cell surface (i.e., the extracellular domain).

1	Through these connections, proteins can be localized or restricted to specialized regions of the plasma membrane or act as transmembrane linkers between intracellular and extracellular filaments (see the next section). Cell injury often manifests as morphologic changes in the cell’s plasma membrane, which results in the formation of plasma-membrane blebs. These are dynamic cell protrusions of the plasma membrane that are commonly observed in acute cell injury, in dividing and dying cells, and during cell movement. Blebbing is caused by the detachment of the plasma membrane from underlying actin flaments of the cell cytoskeleton. Cytoskeletal poisons that act on actin flaments such as phalloidin and cytochalasin-B cause extensive membrane blebbing. Substances that enter or leave the cell must traverse the plasma membrane.

1	Substances that enter or leave the cell must traverse the plasma membrane. Some substances (fat-soluble and small, uncharged molecules) cross the plasma membrane by simple diffusion down their concentration gradient (Fig. 2.7a). All other molecules require membrane transport proteins to provide them with indi vidual passage across the plasma membrane. There are generally two classes of transport proteins:  Carrier proteins transfer small, water-soluble molecules. They are highly selective, often transporting only one type of molecule. After binding to a molecule designated for transport, the carrier protein undergoes a series of conformational changes and releases the molecule on the other side of the membrane (Fig. 2.7b). Some carrier proteins, such as the Na /K pump or H pump, require energy for active transport of molecules against their concentration gradient. Other carrier proteins, such as glucose carriers, do not require energy and participate in passive transport.

1	 Channel proteins also transfer small, water-soluble molecules. In general, channels are made of transmembrane proteins with several membrane-spanning domains that create hydrophilic channels through the plasma membrane. Usually, channel proteins contain a pore domain that partially penetrates the membrane bilayer and serves as the ionselectivity filter. The pore domain is responsible for exquisite ion selectivity, which is achieved by regulation of its three-dimensional structure (Fig. 2.7c). Channels are ion selective and are regulated on the basis of the cell’s needs. Channel protein transport can be regulated by membrane potentials (e.g., voltage-gated ion channels in neurons), neurotransmitters (e.g., ligand-gated ion channels such as acetylcholine receptors in

1	FIGURE 2.7 • Movement of molecules through the plasma membrane. Fat-soluble and other small, uncharged molecules (in green) cross the plasma membrane by simple diffusion down their concentration gradient. Other molecules require membrane transport proteins to provide them with individual passage across the plasma membrane. Small water-soluble molecules (in blue) require highly selective carrier proteins to transfer them across the plasma membrane. After binding with a molecule, the carrier protein undergoes a series of conformational changes and releases the molecule on the other side of the membrane. If the process requires energy, it is called active transport (e.g., transport of H ions against their concentration gradient). The process is called passive transport when energy is not required (e.g., glucose transport). Ions and other small charged molecules (in red) are transported through the plasma membrane by ion-selective channel proteins. In neurons, for instance, ion transport

1	(e.g., glucose transport). Ions and other small charged molecules (in red) are transported through the plasma membrane by ion-selective channel proteins. In neurons, for instance, ion transport is regulated by membrane potentials (voltage-gated ion channels); in skeletal muscle cells, neuromuscular junctions possess ligand-gated ion channels.

1	muscle cells), or mechanical stress (e.g., mechanically gated ion channels in the internal ear). Vesicular transport maintains the integrity of the plasma membrane and also provides for the transfer of molecules between different cellular compartments. Some substances enter and leave cells by vesicular transport, a process that involves configurational changes in the plasma membrane at localized sites and subsequent formation of vesicles from the membrane or fusion of vesicles with the membrane (Fig. 2.8). The major mechanism by which large molecules enter, leave, and move within the cell is called vesicle budding. Vesicles formed by budding from the plasma membrane of one compartment fuse with the plasma membrane of another compartment. Within the cell, this process ensures intercompartmental transfer of the vesicle contents.

1	Vesicular transport involving the cell membrane may also be described in more specific terms:  Endocytosis is the general term for processes of vesicular transport in which substances enter the cell.  Exocytosis is the general term for processes of vesicular transport in which substances leave the cell. Both processes can be visualized with the electron microscope. FIGURE 2.8 • Endocytosis and exocytosis are two major forms of vesicular transport. Endocytosis brings molecules and other substances into the cell. In exocytosis, synthesized molecules and other substances leave the cell. Endocytosis is associated with the formation and budding of vesicles from the plasma membrane; exocytosis is associated with the fusion of vesicles originating from intracellular organelles with the plasma membrane, and it is a primary secretory modality. Uptake of ﬂuid and macromolecules during endocytosis depends on three different mechanisms.

1	Some of the endocytotic mechanisms require special proteins during vesicle formation. The best known protein that interacts with the plasma membrane in vesicle formation is clathrin. Therefore, endocytosis can also be classified as either clathrin dependent or clathrin independent. In general, three mechanisms of endocytosis are recognized in the cell:  Pinocytosis [Gr., cell drinking] is the nonspecific ingestion of ﬂuid and small protein molecules via small vesicles, usually smaller than 150 nm in diameter. Pinocytosis is performed by virtually every cell in the organism, and it is constitutive (i.e., it involves a continuous dynamic formation of small vesicles at the cell surface (Fig. 2.9a). Recent studies indicate that mechanoenzymes such as GTPase (dynamin) are involved in pinocytotic vesicle scission (the process of pinching off from the plasma membrane). Pinocytotic vesicles are visible with the TEM, and they have a smooth surface. These smooth pinocytotic vesicles are

1	vesicle scission (the process of pinching off from the plasma membrane). Pinocytotic vesicles are visible with the TEM, and they have a smooth surface. These smooth pinocytotic vesicles are especially numerous in the endothelium of blood vessels (Fig. 2.9b) and in smooth muscle cells. Pinocytosis does not require clathrin and

1	FIGURE 2.9 • Pinocytosis. a. Pinocytosis involves the dynamic formation of small vesicles at the cell surface. First, substances to be pinocytosed (e.g., small soluble proteins, colloidal tracers) make contact with the extracellular surface of the plasma membrane; next, the surface becomes indented; and fnally, the invaginated portion of the membrane pinches off from the membrane to become a pinocytotic vesicle within the cell. b. This electron micrograph shows numerous smooth-surfaced pinocytotic vesicles (arrows) within the cytoplasm of endothelial cells of a blood vessel. 60,000. therefore may be referred to as clathrin-independent endocytosis.

1	 Phagocytosis [Gr., cell eating ] is the ingestion of large particles such as cell debris, bacteria, and other foreign materials. In this nonselective process, plasma membrane sends out pseudopodia to engulf phagocytosed particles into large vesicles (larger than approximately 250 nm in diameter) called phagosomes. Phagocytosis is performed mainly by a specialized group of cells belonging to the mononuclear phagocytotic system (MPS). Phagocytosis is generally a receptor-mediated process in which receptors on the cell surface recognize non–antigen-binding domains (Fc fragments) of antibodies coating the surface of an invading microorganism or cell (Fig. 2.10a). Phagocytosis is also triggered by recognition of pathogen-associated molecular patterns (PAMPs) that are commonly expressed on pathogen surfaces by Toll-like receptors (page 278). This recognition of PAMPs leads to activation of nuclear factor kappa B (NF-B) transcription factor, which regulates genes that control cell

1	on pathogen surfaces by Toll-like receptors (page 278). This recognition of PAMPs leads to activation of nuclear factor kappa B (NF-B) transcription factor, which regulates genes that control cell responses in phagocytosis. However, nonbiologic materials such as inhaled carbon particles, inorganic dusts, and asbestos fibers, as well as biologic debris from inﬂammation, wound healing, and dead cells, are sequestered by cells of the MPS without involvement of Fc receptors (Fig. 2.10b). This process does not require clathrin for phagosome formation. However, because of initial pseudopodial extensions of plasma membrane that contribute to the formation of phagosome, the actin cytoskeleton must be rearranged in a process that requires depolymerization and repolymerization of the actin filaments. Thus, phagocytosis is referred to as clathrin-independent but actin-dependent endocytosis.

1	 Receptor-mediated endocytosis allows entry of specific molecules into the cell. In this mechanism, receptors for specific molecules, called cargo receptors, accumulate in well-defined regions of the cell membrane. These regions, which are represented by the lipid rafts in the plasma membrane, eventually become coated pits (Fig. 2.11a). The name coated pit is derived from these

1	FIGURE 2.10 • Phagocytosis. a. This drawing shows the steps in the phagocytosis of a large particle, such as a bacterium that has been killed as a result of an immune response. The bacterium is surrounded by antibodies attached to the bacterial surface antigens. Fc receptors on the surface of the plasma membrane of the phagocytotic cells recognize the Fc portion of the antibodies. This interaction triggers rearrangement of actin cytoskeleton. Depolymerizations and repolymerizations of actin flaments produce temporary projections of the plasma membrane called pseudopodia. They surround phagocytosed particle and lead to the formation of phagosomes. By targeted delivery of lysosomal enzymes, a phagosome matures into a lysosome that digests its phagocytosed content. b. Nonbiologic materials such as inhaled carbon particles, inorganic dusts, and asbestos fbers, as well as cellular debris resulting from infammation, are internalized without involvement of antibodies and Fc receptors. These

1	as inhaled carbon particles, inorganic dusts, and asbestos fbers, as well as cellular debris resulting from infammation, are internalized without involvement of antibodies and Fc receptors. These particles are bound to multiple receptors on the plasma membrane.

1	regions’ appearance in the electron microscope (EM) as an accumulation of electron-dense material that represents aggregation of clathrin molecules on the cytoplasmic surface of the plasma membrane. Cargo receptors recognize and bind to specific molecules that come in contact with the plasma membrane. Clathrin molecules then assemble into a basketlike cage that helps change the shape of the plasma membrane into a vesiclelike invagination (Fig. 2.11b). Clathrin interacts with the cargo receptor via another coating-protein complex, adaptin, which is instrumental in selecting appropriate cargo molecules for transport into the cells. Thus, selected cargo proteins and their receptors are pulled from the extracellular space into the lumen of a forming vesicle. The large (100kilodalton) mechanoenzyme GTPase called dynamin mediates the liberation of forming clathrin-coated vesicles from the plasma membrane during receptor-mediated endocytosis. The type of vesicle formed as a result of

1	GTPase called dynamin mediates the liberation of forming clathrin-coated vesicles from the plasma membrane during receptor-mediated endocytosis. The type of vesicle formed as a result of receptor-mediated endocytosis is referred to as a coated vesicle, and the process itself is known as clathrindependent endocytosis. Clathrin-coated vesicles are also involved in the movement of the cargo material from the plasma membrane to early endosomes and from the Golgi apparatus to the early and late endosomes.

1	Exocytosis is the process by which a vesicle moves from the cytoplasm to the plasma membrane, where it discharges its contents to the extracellular space.

1	A variety of molecules produced by the cell for export are initially delivered from the site of their formation to the Golgi apparatus. The next step involves sorting and packaging the secretory product into transport vesicles that are destined to fuse with the plasma membrane in a process known as exocytosis. Intracellular traffic of these vesicles is achieved by the presence of specific proteins on their surface (coatomers such as COP-I and COP-II) that mediate their movements (see page 48). The molecules that travel this route are often chemically modified (e.g., glycosylated, sulfated) as they pass through different cellular compartments. The membrane that is added to the plasma membrane by exocytosis is recovered into the cytoplasmic compartment by an endocytotic process. There are two general pathways of exocytosis:  In the constitutive pathway, substances designated for export are continuously delivered in transport vesicles to the plasma membrane. Proteins that leave the

1	two general pathways of exocytosis:  In the constitutive pathway, substances designated for export are continuously delivered in transport vesicles to the plasma membrane. Proteins that leave the cell by this process are secreted immediately after their synthesis and exit from the Golgi apparatus, as seen in the secretion of

1	FIGURE 2.11 • Receptor-mediated endocytosis. a. This diagram shows the steps in receptor-mediated endocytosis, a transport mechanism that allows selected molecules to enter the cell. Cargo receptors recognize and bind specifc molecules that come in contact with the plasma membrane. Cargo receptor–molecule complexes are recognized by adaptin, a protein that helps select and gather appropriate complexes in specifc areas of the plasma membrane for transport into cells. Clathrin molecules then bind to the adaptin–cargo receptor–molecule complex to assemble into a shallow basketlike cage and form a coated pit. Clathrin interactions then assist the plasma membrane to change shape to form a deep depression, a fully formed coated pit that becomes pinched off from the plasma membrane by the protein complex dynamin as a coated vesicle (i.e., budding from the membrane). Selected cargo proteins and their receptors are thus pulled from the extracellular space into the lumen of a forming coated

1	complex dynamin as a coated vesicle (i.e., budding from the membrane). Selected cargo proteins and their receptors are thus pulled from the extracellular space into the lumen of a forming coated vesicle. After budding and internalization of the vesicle, the coat proteins are removed and recycled for further use. The uncoated vesicle travels to its destination to fuse with a cytoplasmic organelle. b. Electron micrograph of the cytoplasmic surface of the plasma membrane of A431 cells prepared by the quick-freeze deep-etch technique. This image shows coated pits and clathrin-coated vesicles in different stages of their formation. Note that the coated pits and clathrin-coated vesicles are formed in areas devoid of actin flaments. The small uniform pinocytotic vesicles do not have a clathrin coat and are located in close proximity to actin flaments. 200,000. (Courtesy of Dr. John E. Heuser,

1	Washington University School of Medicine.) immunoglobulins by plasma cells and of procollagen by fibroblasts. This pathway is present to some degree in all cells. The TEM reveals that these cells lack secretory granules.  In the regulated secretory pathway, specialized cells, such as endocrine and exocrine cells and neurons, concentrate secretory proteins and transiently store them in secretory vesicles within the cytoplasm (Fig. 2.12). In this case, a regulatory event (hormonal or neural stimulus) must be activated for secretion to occur, as in the release of secretory vesicles by chief cells of the gastric mucosa and by acinar cells of the pancreas. The signaling stimulus causes a transient inﬂux of Ca2 into the cytoplasm, which in turn stimulates secretory vesicles to fuse with the plasma membrane and discharge their contents (Fig. 2.13). In the past, secretory vesicle containing inactive precursor (zymogen) were called zymogen granules.

1	In addition to excretory pathways, proteins can be transported between the Golgi apparatus and other organelles along endosomal pathways. These pathways are used for delivery of organelle-specific proteins, such as lysosomal structural proteins, into the appropriate organelles. The precise targeting of vesicles to the appropriate cellular compartment is initially controlled by docking proteins, and specificity is ensured by interactions between soluble NSF attachment receptor (SNARE) proteins.

1	As discussed previously, newly formed vesicles that bud off from the donor membrane (such as cell membrane or Golgi cisternae) can fuse with a number of possible target membranes within the cell. Shortly after budding and shedding its clathrin coat, a vesicle must be targeted to the appropriate cellular compartment. A targeting mechanism can be likened to a taxi driver in a large city who successfully delivers a passenger to the proper street address. In the cell, the street address is recognized by Rab-GTPase bound to the membrane of the traveling vesicle. Rab-GTPase interacts with tethering proteins located on the target membrane. This initial interaction provides recognition of the vesicle and recruits the necessary number of tethering proteins to dock the incoming vesicle. The docking complex between Rab-GT-Pase and its receptor immobilizes the vesicle near the target membrane (Fig. 2.14). To ensure accurate targeting, each vesicle contains a vesicle-specifc membrane protein

1	complex between Rab-GT-Pase and its receptor immobilizes the vesicle near the target membrane (Fig. 2.14). To ensure accurate targeting, each vesicle contains a vesicle-specifc membrane protein called a v-SNARE. The target membrane also contains a specific membrane protein, t-SNARE, that interacts with v-SNARE to form the cis-SNARE complex. SNAREs are a family of transmembrane proteins that were originally grouped according to their location within the vesicle (v-SNARE) or target membrane (t-SNARE). They guarantee the specificity of interaction between a particular vesicle and its target membrane and also promote membrane fusion that follows immediately after the cis-SNARE complexes are formed. After fusion, the SNARE complexes are dismantled with the help of the NSF/ -SNAP protein complex and recycled for use in another round of vesicle fusion.

1	FIGURE 2.12 • Photomicrograph of secretory cells of the pancreas. Note that secretory vesicles containing protein ready for secretion fll the apical portion of the cells. This process re-quires an external signaling mechanism for the cell to discharge the accumulated granules. 860. The TEM reveals the presence in the cytoplasm of membrane-enclosed compartments associated with all the endocytotic pathways described above (Fig. 2.15). These compartments, called early endosomes, are restricted to a portion of the

1	FIGURE 2.13 • Diagram showing two pathways for exocytosis. Newly synthesized proteins are synthesized in the rough endoplasmic reticulum (rER). After their initial post-translational modifcation, they are delivered in COP-II–coated vesicles to the Golgi apparatus. After additional modifcation in the Golgi apparatus, sorting, and packaging, the fnal secretory product is transported to the plasma membrane in vesicles that form from the trans-Golgi network (TGN). Note that retrograde transport is present between Golgi cisternae and is mediated by the COP-I–coated vesicle. Two distinct pathways are recognized. Blue arrows indicate the constitutive pathway in which proteins leave the cell immediately after their synthesis. In cells using this pathway, almost no secretory product accumulates, and thus few secretory vesicles are present in the cytoplasm. Red arrows indicate the regulated secretory pathway in which protein secretion is regulated by hormonal or neural stimuli. In cells using

1	and thus few secretory vesicles are present in the cytoplasm. Red arrows indicate the regulated secretory pathway in which protein secretion is regulated by hormonal or neural stimuli. In cells using this pathway, such as the pancreatic acinar cells in Figure 2.12, secretory proteins are concentrated and transiently stored in secretory vesicles within the cytoplasm. After appropriate stimulation, the secretory vesicles fuse with the plasma membrane and discharge their contents.

1	cytoplasm near the cell membrane where vesicles originating from the cell membrane fuse. From here, many vesicles return to the plasma membrane. However, large numbers of vesicles originating in early endosomes travel to deeper structures in the cytoplasm called late endosomes. The latter typically mature into lysosomes.

1	FIGURE 2.14 • Steps in formation, targeting, docking, and fusion of transport vesicles with the target membrane. (1) Lipid raft with cargo receptors ready to interact with cargo protein. Note the presence of the specifc targeting protein v-SNARE. (2) Initial step in vesicle formation: The binding of the adaptin complex and clathrin forms a coated pit. (3) Formation (budding) of fully assembled coated vesicle. (5) Disassembly of clathrin coat. Note the expression of Rab-GTPase activity. (6) Tethering of the vesicle to the target membrane by the interaction between Rab-GTPase and tethering proteins. (7) Beginning of the docking process (recruitment of tethering proteins). (8) Formation of the docking complex between Rab-GTPase and its protein in the target membrane: v-SNAREs on the immobilized vesicle interact with t-SNAREs on the target membrane to form the cis-SNARE complex. (9) Fusion of the vesicle to the target membrane. (10) Discharge of the cargo protein into the early endosomal

1	vesicle interact with t-SNAREs on the target membrane to form the cis-SNARE complex. (9) Fusion of the vesicle to the target membrane. (10) Discharge of the cargo protein into the early endosomal compartment and disassembly of the cis complex by the interaction of the NSF/ -SNAP protein complex. (11) Recycling of v-SNAREs in the transport vesicles for use in another round of vesicle targeting and fusion.

1	FIGURE 2.15 • Electron micrograph of an early endosome. This deep-etch electron micrograph shows the structure of an early endosome in Dictyostelium. Early endosomes are located near the plasma membrane and, as in many other sorting compartments, have a typical tubulovesicle structure. The tubular portions contain the majority of integral membrane proteins destined for membrane recycling, whereas the luminal portions collect secretory cargo proteins. The lumen of the endosome is subdivided into multiple compartments, or cisternae, by the invagination of its membrane and undergoes frequent changes in shape. 15,000. (Courtesy of Dr. John E. Heuser, Washington University School of Medicine.) Endosomes can be viewed either as stable cytoplasmic organelles or as transient structures formed as the result of endocytosis.

1	Endosomes can be viewed either as stable cytoplasmic organelles or as transient structures formed as the result of endocytosis. Recent experimental observations of endocytotic pathways conducted in vitro and in vivo suggest two different models that explain the origin and formation of the endosomal compartments in the cell:  The stable compartment model describes early and late endosomes as stable cellular organelles connected by vesicular transport with the external environment of the cell and with the Golgi apparatus. Coated vesicles formed at the plasma membrane fuse only with early endosomes because of their expression of specific surface receptors. The receptor remains a resident component of the early endosomal membrane.

1	 In the maturation model, early endosomes are formed de novo from endocytotic vesicles originating from the plasma membrane. Therefore, the composition of the early endosomal membrane changes progressively as some components are recycled between the cell surface and the Golgi apparatus. This maturation process leads to formation of late endosomes and then to lysosomes. Specific receptors present on early endosomes (e.g., for coated vesicles) are removed by recycling, degradation, or inactivation as this compartment matures. Both models actually complement rather than contradict each other in describing, identifying, and studying the pathways of internalized molecules. Endosomes destined to become lysosomes receive newly synthesized lysosomal enzymes that are targeted via the mannose-6-phosphate receptor.

1	Some endosomes also communicate with the vesicular transport system of the rER. This pathway provides constant delivery of newly synthesized lysosomal enzymes, or hydrolases. A hydrolase is synthesized in the rER as an enzymatically inactive precursor called a prohydrolase. This heavily glycosylated protein then folds in a specific way so that a signal patch is formed and exposed on its surface. This recognition signal is created when specific amino acids are brought into close proximity by the three-dimensional folding of the protein. The signal patch on a protein destined for a lysosome is then modified by several enzymes that attach mannose-6phosphate (M-6-P) to the prohydrolase surface. M-6-P acts as a target for proteins possessing an M-6-P receptor. M-6-P receptors are present in early and late endosomes, lysosomes, and the Golgi apparatus, which is involved in sorting and retrieving secreted prohydrolases destined for transport to endosomes (Fig. 2.16). The acidic environment

1	and late endosomes, lysosomes, and the Golgi apparatus, which is involved in sorting and retrieving secreted prohydrolases destined for transport to endosomes (Fig. 2.16). The acidic environment of late endosomes causes the release of prohydrolases from the M-6-P receptors. Prohydrolases are next activated by cleavage and by removing phosphate groups from the mannose residues.

1	Early and late endosomes differ in their cellular localization, morphology, and state of acidification and function. Early and late endosomes are localized in different areas of the cell. Early endosomes can be found in the more peripheral cytoplasm, whereas late endosomes are often positioned near FIGURE 2.16 • Pathways for delivery of newly synthesized lysosomal enzymes. Lysosomal enzymes (such as lysosomal hydrolases) are synthesized and glycosylated within the rough endoplasmic reticulum (rER). The enzymes then fold in a specifc way so that a signal patch is formed, which allows for further modifcation by the addition of M-6-P, which allows the enzyme to be targeted to specifc proteins that possess M-6-P receptor activity. M-6-P receptors are present in the TGN of the Golgi apparatus, where the lysosomal enzymes are sorted and packaged into vesicles later transported to the early or late endosomes.

1	the Golgi apparatus and the nucleus. An early endosome has a tubulovesicular structure: The lumen is subdivided into cisternae that are separated by invagination of its membrane. It exhibits only a slightly more acidic environment (pH 6.2 to 6.5) than the cytoplasm of the cell. In contrast, late endosomes have a more complex structure and often exhibit onionlike internal membranes. Their pH is more acidic, averaging 5.5. TEM studies reveal specific vesicles that transport substances between early and late endosomes. These vesicles, called multivesicular bodies (MVBs), are highly selective transporters. Within early endosomes, proteins destined to be transported to late endosomes are sorted and separated from proteins destined for recycling and packaging into MVBs (Fig. 2.17). In general, substances transported to late endosomes are eventually degraded in lysosomes in a default process that does not require any additional signals. Because late endosomes mature into lysosomes, they are

1	substances transported to late endosomes are eventually degraded in lysosomes in a default process that does not require any additional signals. Because late endosomes mature into lysosomes, they are also called prelysosomes. Advances in videomicroscopy now allow researchers to observe the complex behavior of these organelles; late lysosomes may fuse with each other or with mature lysosomes.

1	The major function of early endosomes is to sort and recycle proteins internalized by endocytotic pathways. MVBHpH 6.2 pH 5.5 pH 4.7

1	MVBHpH 6.2 pH 5.5 pH 4.7 FIGURE 2.17 • Schematic diagram of endosomal compartments of the cell. This diagram shows the fate of protein (red circles) endocytosed from the cell surface and destined for lysosomal destruction. Proteins are frst found in endocytotic (coated) vesicles that deliver them to early endosomes, which are located in the peripheral part of cytoplasm. Because of the sorting capability of the early endosomes, receptors are usually recycled to the plasma membrane, and endocytosed proteins are transported via multi-vesicular bodies (MVB) to late endosomes positioned near the Golgi apparatus and the nucleus. The proteins transported to late endosomes eventually will be degraded in lysosomes. Note the acidifcation scale (left) that illustrates changes of pH from early endosomes to lysosomes. The acidifcation is accomplished by the active transport of protons into endosomal compartments.

1	Early endosomes sort proteins that have been internalized by endocytotic processes. The morphologic shape and geometry of the tubules and vesicles emerging from the early endosome create an environment in which localized changes in pH constitute the basis of the sorting mechanism. This mechanism includes dissociation of ligands from their receptor protein; thus, in the past, early endosomes were referred to as compartments of uncoupling receptors and ligands (CURLs). In addition, the narrow diameter of the tubules and vesicles may also aid in the sorting of large molecules, which can be mechanically prevented from entering specific sorting compartments. After sorting, most of the protein is rapidly recycled, and the excess membrane is returned to the plasma membrane. The fate of the internalized ligand–receptor complex depends on the sorting and recycling ability of the early endosome.

1	The following pathways for processing internalized ligand–receptor complexes are present in the cell:  The receptor is recycled and the ligand is degraded. Surface receptors allow the cell to bring in substances selectively through the process of endocytosis. This pathway occurs most often in the cell; it is important because it allows surface receptors to be recycled. Most ligand–receptor complexes dissociate in the acidic pH of the early endosome. The receptor, most likely an integral membrane protein (see page 29), is recycled to the surface via vesicles that bud off the ends of narrow-diameter tubules of the early endosome. Ligands are usually sequestered in the spherical vacuolar part of the endosome that will later form MVBs, which will transport the ligand to late endosomes for further degradation in the lysosome (Fig. 2.18a). This pathway is described for the low-density lipoprotein (LDL)–receptor complex, insulin–glucose transporter (GLUT) receptor complex, and a variety

1	further degradation in the lysosome (Fig. 2.18a). This pathway is described for the low-density lipoprotein (LDL)–receptor complex, insulin–glucose transporter (GLUT) receptor complex, and a variety of peptide hormones and their receptors.

1	 Both receptor and ligand are recycled. Ligand–receptor complex dissociation does not always accompany receptor recycling. For example, the low pH of the endosome dissociates iron from the iron-carrier protein transferrin, but transferrin remains associated with its receptor. Once the transferrin–receptor complex returns to the cell surface, however, transferrin is released. At neutral extracellular pH, transferrin must again bind iron to be recognized by and bound to its receptor. A similar pathway is recognized for major histocompatibility complex (MHC) I and II molecules, which are recycled to the cell surface with a foreign antigen protein attached to them (Fig. 2.18b).

1	 Both receptor and ligand are degraded. This pathway has been identified for epidermal growth factor (EGF) and its receptor. Like many other proteins, EGF binds to its receptor on the cell surface. The complex is internalized and carried to the early endosomes. Here EGF dissociates from its receptor, and both are sorted, packaged in separate MVBs, and transferred to the late endosome. From there, both ligand and receptor are transferred to lysosomes, where they are degraded (Fig. 2.18c).  Both receptor and ligand are transported through the cell. This pathway is used for secretion of immunoglobulins (secretory IgA) into the saliva and human milk. During this process, commonly referred to as transcytosis, substances can be altered as they are transported across the epithelial cell (Fig. 2.18d). Transport of maternal IgG across the placental barrier into the fetus also follows a similar pathway.

1	Lysosomes are digestive organelles that were recognized only after histochemical procedures were used to demonstrate lysosomal enzymes. Lysosomes are organelles rich in hydrolytic enzymes such as proteases, nucleases, glycosidases, lipases, and phospholipases.

1	FIGURE 2.18 • Fate of receptor and ligand in receptor-mediated endocytosis. This diagram shows four major pathways along which the fate of internalized ligand–receptor complexes is determined. a. The internalized ligand–receptor complex dissociates, the receptor is recycled to the cell surface, and the ligand is directed to late endosomes and eventually degraded within lysosomes. This processing pathway is used by the LDL–receptor complex, insulin–GLUT receptor complex, and a variety of peptide hormone– receptor complexes. MVB, multivesicular bodies. b. Both internalized receptor and ligand are recycled. Ligand–receptor complex dissociation does not occur, and the entire complex is recycled to the surface. An example is the iron–transferrin–transferrin receptor complex that uses this processing pathway. Once iron (Fe) is released in the endosome, the transferrin–transferrin receptor complex returns to the cell surface, where transferrin is released. c. The internalized ligand–receptor

1	pathway. Once iron (Fe) is released in the endosome, the transferrin–transferrin receptor complex returns to the cell surface, where transferrin is released. c. The internalized ligand–receptor complex dissociates in the early endosome. The free ligand and the receptor are directed to the late endosomal compartment for further degradation. This pathway is used by many growth factors (i.e., the EGF–receptor complex). d. The internalized ligand–receptor complex is transported through the cell. Dissociation does not occur, and the entire complex undergoes transcytosis and release at a different site of the cell surface. This pathway is used during secretion of immunoglobulins (secretory IgA) into saliva. The antibody IgA–receptor complex is internalized at the basal surface of the secretory cells in the salivary gland and released at the apical surface.

1	A lysosome represents a major digestive compartment in the cell that degrades macromolecules derived from endocytotic pathways as well as from the cell itself in a process known as autophagy (removal of cytoplasmic components, particularly membrane-bounded organelles, by digesting them within lysosomes). For more information about autophagy, see page 41.

1	The first hypothesis for lysosomal biogenesis, formulated almost a half century ago, postulated that lysosomes arise as complete and functional organelles budding from the Golgi apparatus. These newly formed lysosomes were termed primary lysosomes in contrast to secondary lysosomes, which had already fused with incoming endosomes. However, the primary and secondary lysosome hypothesis has proved to have little validity as new research data allow a better understanding of the details of protein secretory pathways and the fate of endocytotic vesicles. It is now widely accepted that lysosomes are formed in a complex series of pathways that converge at the late endosomes, transforming them into lysosomes. These pathways are responsible for a targeted delivery of newly synthesized lysosomal enzymes and structural lysosomal membrane proteins into the late endosomes. As stated earlier, lysosomal enzymes are synthesized in the rER and sorted in the Golgi apparatus based on their binding

1	enzymes and structural lysosomal membrane proteins into the late endosomes. As stated earlier, lysosomal enzymes are synthesized in the rER and sorted in the Golgi apparatus based on their binding ability to M-6-P receptors (see page 37).

1	Lysosomes have a unique membrane that is resistant to the hydrolytic digestion occurring in their lumen.

1	Lysosomes contain a collection of hydrolytic enzymes and are surrounded by a unique membrane that resists hydrolysis by their own enzymes (Fig. 2.19). The lysosomal membrane has an unusual phospholipid structure that contains cholesterol and a unique lipid called lysobisphosphatidic acid. Most of the structural lysosomal membrane proteins are classified into lysosome-associated membrane proteins (lamps), lysosomal membrane glycoproteins (lgps), and lysosomal integral membrane proteins (limps). The lamps, lgps, and limps represent more than 50% of the total membrane proteins in lysosomes and are highly glycosylated on the luminal surface. Sugar molecules cover almost the entire luminal surface of these proteins, thus protecting them from digestion by hydrolytic enzymes. Lysobisphosphatidic acids within the lysosomal membrane may play an important role in restricting the activity of hydrolytic enzymes directed against the membrane. The same family of membrane proteins is also detected

1	acids within the lysosomal membrane may play an important role in restricting the activity of hydrolytic enzymes directed against the membrane. The same family of membrane proteins is also detected in late

1	FIGURE 2.19 • Schematic diagram of a lysosome. This diagram shows a few selected lysosomal enzymes residing inside the lysosome and their respective substrates. The major lysosomal membrane-specifc proteins, as well as a few other proteins associated H membrane impermeable to enzymes; contains specific lysosomal proteins, lamps, limps, and lgps with membrane transport, are also shown. endosomes. In addition, lysosomes and late endosomes contain proton (H ) pumps that transport H ions into the lysosomal lumen, maintaining a low pH (4.7). The lysosomal membrane also contains transport proteins that transport the final products of digestion (amino acids, sugars, nucleotides) to the cytoplasm, where they are used in the synthetic processes of the cell or are exocytosed.

1	Certain drugs can affect lysosomal function. For example, chloroquine, an agent used in the treatment and prevention of malaria, is a lysosomotropic agent that accumulates in the lysosomes. It raises the pH of the lysosomal content, thereby inactivating many lysosomal enzymes. The action of chloroquine on lysosomes accounts for its antimalarial activity; the drug concentrates in the acidic food vacuole of the malaria parasite (Plasmodium falciparum) and interferes with its digestive processes, eventually killing the parasite. Lysosomal membrane proteins are synthesized in the rER and have specific lysosomal targeting signal.

1	Lysosomal membrane proteins are synthesized in the rER and have specific lysosomal targeting signal. As mentioned previously, the intracellular trafficking leading to the delivery of many soluble lysosomal enzymes to late endosomes and lysosomes involves the M-6-P signal and its receptor. All membrane proteins destined for lysosomes (and late endosomes) are synthesized in the rER and transported to and sorted in the Golgi apparatus. However, they do not contain the M-6-P signals and must be targeted to lysosomes by a different mechanism. The targeting signal for integral membrane proteins is represented by a short cytoplasmic C-terminus domain, which is recognized by adaptin protein complexes and packaged into clathrin-coated vesicles. These proteins reach their destination by one of two pathways:  In the constitutive secretory pathway, limps exit the

1	Golgi apparatus in coated vesicles and are delivered to the cell surface. From here, they are endocytosed and, via the early and late endosomal compartments, finally reach lysosomes (Fig. 2.20).  In the Golgi-derived coated vesicle secretory pathway, limps, after sorting and packaging, exit the Golgi apparatus in clathrin-coated vesicles (see Fig. 2.20). These transport vesicles travel and fuse with late endosomes as a result of interaction between endosome-specific components of v-SNARE and t-SNARE docking proteins (see page 34). Three different pathways deliver material for intracellular digestion in lysosomes.

1	Three different pathways deliver material for intracellular digestion in lysosomes. Depending on the nature of the digested material, different pathways deliver material for digestion within the lysosomes (Fig. 2.21). In the digestion process, most of the digested material comes from endocytotic processes; however, the cell also uses lysosomes to digest its own obsolete parts, nonfunctional organelles, and unnecessary molecules. Three pathways for digestion exist:  Extracellular large particles such as bacteria, cell debris, and other foreign materials are engulfed in the process of phagocytosis. A phagosome, formed as the material is internalized within the cytoplasm, subsequently receives hydrolytic enzymes to become a late endosome, which matures into a lysosome.

1	 Extracellular small particles such as extracellular proteins, plasma-membrane proteins, and ligand–receptor complexes are internalized by pinocytosis and receptor-mediated endocytosis. These particles follow the endocytotic pathway through early and late endosomal compartments and are finally degraded in lysosomes.  Intracellular particles such as entire organelles, cytoplasmic proteins, and other cellular components are isolated from the cytoplasmic matrix by endoplasmic reticulum membranes, transported to lysosomes, and degraded. This process is called autophagy (see page 41).

1	FIGURE 2.20 • Lysosome biogenesis. This diagram shows regulated and constitutive pathways for delivery of lysosomal specifc membrane proteins into early and late endosomes. The lysosomal membrane possesses highly glycosylated specifc membrane proteins that protect the membrane from digestion by lysosomal enzymes. These lysosome-specifc proteins are synthesized in the rough endoplasmic reticulum, transported to the Golgi apparatus, and reach their destination by two pathways. Blue arrows indicate the constitutive secretory pathway in which certain lysosomal membrane proteins exit the Golgi apparatus and are delivered to the cell surface. From there they are endocytosed and, via the early and late endosomal compartments, fnally reach lysosomes. Green arrows indicate the endosomal Golgiderived coated vesicle secretory pathway. Here, other lysosomal proteins, after sorting and packaging, exit the Golgi apparatus in clathrin-coated vesicles to fuse with early and late endosomes.

1	In addition, some cells (e.g., osteoclasts involved in bone resorption and neutrophils involved in acute infammation) may release lysosomal enzymes directly into the extracellular space to digest components of the extracellular matrix. Lysosomes in some cells are recognizable in the light microscope because of their number, size, or contents. The numerous azurophilic granules of neutrophils (white blood cells) represent lysosomes and are recognized in aggregate by their specific staining. Lysosomes that contain phagocytized bacteria and fragments of damaged cells are often recognized in macrophages. Hydrolytic breakdown of the contents of lysosomes often produces a debris-filled vacuole called a residual body that may remain for the entire life of the cell. For example, in neurons, residual bodies are called age pig-

1	FIGURE 2.21 • Pathways of delivery of materials for digestion in lysosomes. Most of the small extracellular particles are internalized by both receptor-mediated endocytosis and pinocytosis. These two endocytic patways are labeled with red arrows. Large extracellular particles such as bacteria and cellular debris are delivered for cellular digestion via the phagocytotic pathway (blue arrows). The cell also uses lysosomes to digest its own organelles and other intracellular proteins via the autophagic pathway (green arrows). Intracellular particles are isolated from the cytoplasmic matrix by the isolation membrane of the sER, transported to lysosomes, and subsequently degraded. ment or lipofuscin granules. Residual bodies are a normal feature of cell aging. The absence of certain lysosomal enzymes can cause the pathologic accumulation of undigested substrate in residual bodies. This can lead to several disorders collectively termed lysosomal storage diseases (see Folder 2.1).

1	Autophagy represents the major cellular pathway in which a number of cytoplasmic proteins, organelles, and other cellular structures are degraded in the lysosomal compartment (Fig. 2.22). This important process maintains a well-controlled balance between anabolic and catabolic cell functions and permits the cell to eliminate unwanted or unnecessary organelles. Digested components of organelles are recycled and reused for normal cell growth and development.

1	Many genetic disorders have been identifed in individuals who have mutations in a gene that encodes lysosomal pro-teins. These diseases are termed lysosomal storage diseases (LSDs) and are characterized by dysfunctional lysosomes. The defective protein in most cases is a hy-drolytic enzyme or its cofactor; less commonly, lysosomal membrane proteins or proteins that are involved in sorting, targeting, and transporting lysosomal proteins are defec-tive. The result is an accumulation in cells of the specifc products that lysosomal enzymes normally use as sub-strates in their reactions. These undigested, accumulated products disrupt the normal function of the cell and lead to its death. Currently, 49 disorders are known LSDs with a collec-tive incidence of about 1 in 7,000 live births. The life ex-pectancy across the entire group of people with these disorders is 15 years. The frst LSD was described in 1881 by British ophthalmologist Warren Tay, who reported symp-toms of retinal

1	The life ex-pectancy across the entire group of people with these disorders is 15 years. The frst LSD was described in 1881 by British ophthalmologist Warren Tay, who reported symp-toms of retinal abnormalities in a 12-month-old infant with severe neuromuscular symptoms. In 1896, U.S. neurologist Bernard Sachs described a patient with similar eye symp-toms found earlier by Tay. This disease is now known as Tay-Sachs disease. It is caused by the absence of one enzyme, a lysosomal galactosidase ( -hexosaminidase) that catalyzes a step in lysosomal breakdown of ganglio-sides in neurons. The resulting accumulation of the GM2 ganglioside that is found within concentric lamellated struc-tures in residual bodies of neurons interferes with normal cell function. Children born with LSDs usually appear normal at birth; however, they soon show clinical signs of the disease. They often experience slower growth, show changes in fa-cial features, and develop bone and joint deformities that lead to

1	normal at birth; however, they soon show clinical signs of the disease. They often experience slower growth, show changes in fa-cial features, and develop bone and joint deformities that lead to signifcant restrictions of limb movement. They may lose already attained skills such as speech and learning ability. Behavioral problems may occur, as well as mental retardation. They are prone to frequent lung infections and heart disease. Some children have enlarged internal or-gans such as the liver and spleen (hepatosplenomegaly). The most common LSDs in children are Gaucher disease, Hurler syndrome (MPS I), Hunter syndrome (MPS II), and Pompe disease. Not long ago, LSDs were seen as neurodegenerative disorders without any potential treatment. In the last two decades, there has been limited success in treating the symptoms of LSDs. Considerable effort has been devoted to genetic research and fnding methods to replace the missing enzymes that cause various forms of LSD. Enzyme-replacement

1	in treating the symptoms of LSDs. Considerable effort has been devoted to genetic research and fnding methods to replace the missing enzymes that cause various forms of LSD. Enzyme-replacement therapy, which requires the cellu-lar delivery of a manufactured recombinant enzyme, is avail-able for some LSDs such as cystinosis and Gaucher disease. Enzymes have also been supplied by transplanta-tion of bone marrow containing normal genes from an un-affected person. Success of the enzyme-replacement therapy is often limited by insuffcient biodistribution of re-combinant enzymes and high costs. Recently emerging strategies for the treatment of LSDs include pharmaco-logical chaperone therapy in which chaperone molecules are delivered to affected cells. In some cases, synthetic chaparones can assist in folding of mutated en-zymes to improve their stability and advance their lysoso-mal delivery. In the future, the combination of different therapies such as enzyme replacement, pharmacological

1	in folding of mutated en-zymes to improve their stability and advance their lysoso-mal delivery. In the future, the combination of different therapies such as enzyme replacement, pharmacological chaperone, and gene-transfer therapies with the devel-opment of newborn screening tests will enable early detec-tion and improve clinical outcome of patients with LSDs. 

1	FOLDER 2.1 Clinical Correlation: Lysosomal Storage Diseases

1	Summary of Common Lysosomal Storage Diseases (LSDs) Accumulating Product Disease Protein Deficiency (or Defective Process) Gaucher disease Glucocerebrosidase Glucosylceramide Tay-Sachs disease -hexosaminidase, -subunit GM2 ganglioside Sandhoff disease -hexosaminidase, -subunit GM2 ganglioside, oligosaccharides Krabbe disease Galactosylceramidase Gal-ceramide, gal-sphingosine Niemann-Pick disease A,B Sphingomyelinase Sphingomyelin Disorders of Glycoprotein Degradation Aspartylglycosaminuria Aspartylglycosaminidase N-linked oligosaccharides -Mannosidosis -Mannosidase -Mannosides Disorders of Sphingolipid Degradation chapter 2 Disorders of Glycosaminoglycan Degradation Hurler syndrome (mucopolysaccharidosis I, -L-iduronidase Dermatan sulfate, heparan sulfate MPS I) Hunter syndrome (MPS II) L-Iduronate sulfatase Dermatan sulfate, heparan sulfate Maroteaux-Lamy syndrome (MPS IV) GalNAc 4-sulfatase/arylsulfatase B Dermatan sulfate Other Disorders of Single Enzyme Deficiency Pompe disease

1	L-Iduronate sulfatase Dermatan sulfate, heparan sulfate Maroteaux-Lamy syndrome (MPS IV) GalNAc 4-sulfatase/arylsulfatase B Dermatan sulfate Other Disorders of Single Enzyme Deficiency Pompe disease (glycogenosis II) -1,4-Glucosidase Glycogen Wolman disease (familial xanthomatosis) Acid lipase Cholesterol esters, triglycerides Canavan disease (aspartoacylase Aspartoacylase N-acetylaspartic acid deficiency) Disorders of Lysosomal Biogenesis Inclusion-cell (I-cell) disease, GlcNAc-1-phosphotransferase Lysosomal hydrolyses are not mucolipidosis II (GlcNAcPTase) present in lysosomes Leads to defective sorting of most soluble hydrolytic lysosomal enzymes Disorders of the Lysosomal Membrane Danon disease lamp2 Presence of autophagic vacuoles Cystinosis Cystinosin (cystine transporter) Cystine Summary of Common Lysosomal Storage Diseases (LSDs) (Cont.) Accumulating Product Disease Protein Deficiency (or Defective Process)

1	Cytoplasmic proteins and organelles are substrates for lysosomal degradation in the process of autophagy.

1	Autophagy plays an essential role during starvation, cellular differentiation, cell death, and cell aging. In the last few years, applying genetic-screening tests originally developed for yeasts, researchers uncovered several autophagy-related genes (Atg genes) in mammalian cell genome. The presence of adequate nutrients and growth factors stimulates enzymatic activity of a serine/threonine kinase known as mammalian target of rapamycin (mTOR). High mTOR activity exerts an inhibitory effect on autophagy. The opposite is found in nutrient starvation, hypoxia, and high temperature, where lack of mTOR activity causes activation of Atg genes. This results in formation of an Atg1 proteinkinase autophagy–regulatory complex that initiates process of autophagy. Generally, autophagy can be divided into three well-characterized pathways:  Macroautophagy, or simply autophagy, is a nonspecific process in which a portion of the cytoplasm or an entire organelle is first surrounded by a double or

1	into three well-characterized pathways:  Macroautophagy, or simply autophagy, is a nonspecific process in which a portion of the cytoplasm or an entire organelle is first surrounded by a double or multilamellar intracellular membrane of endoplasmic reticulum, called isolation membrane, to form a vacuole called an autophagosome. This process is aided by proteins encoded by several Atg genes. At first, the complex containing Atg12–Atg5–Atg16L proteins attaches to a part of endoplastic reticulum and localizes the isolation membrane. Subsequently, Atg8 is recruited and bound to the membrane. Together they change the shape of the isolation membrane, which bends to enclose and seal an organelle destined for digestion within the lumen of the autophagosome. Once the autophagosome is completed, the Atg12–Atg5–Atg16L complex and Atg8 dissociate from this structure. After targeted delivery of lysosomal enzymes autophagosome matures into a lysosome. The isolation membrane disintegrates within

1	the Atg12–Atg5–Atg16L complex and Atg8 dissociate from this structure. After targeted delivery of lysosomal enzymes autophagosome matures into a lysosome. The isolation membrane disintegrates within the hydrolytic compartment of a lysosome. Macroautophagy occurs in the liver during the first stages of starvation (Fig. 2.23).

1	 Microautophagy is also a nonspecific process in which cytoplasmic proteins are degraded in a slow, continuous process under normal physiologic conditions. In microautophagy, small cytoplasmic soluble proteins are internalized into the lysosomes by invagination of the lysosomal membrane.  Chaperone-mediated autophagy is the only selective process of protein degradation and requires assistance from specific cytosolic chaperones such as heat-shock chaperone protein called hsc73. This process is activated during nutrient deprivation and requires the presence of targeting signals on the degraded proteins and a specific receptor on

1	FIGURE 2.22 • Three autophagic pathways for degradation of cytoplasmic constituents. In macroautophagy, a portion of the cytoplasm or an entire organelle is surrounded by an intracellular membrane of the endoplasmic reticulum to form a double-membraned autophagosome vacuole. After fusion with a lysosome, the inner membrane and the contents of the vacuole are degraded. In microautophagy, cytoplasmic proteins are internalized into lysosomes by invagination of the lysosomal membrane. Chaperone-mediated autophagy to lysosomes is the most selective process for degradation of specifc cytoplasmic proteins. It requires assistance of proteins called chaperones. The chaperone protein—that is, hsc73—binds to the protein and helps transport it into the lysosomal lumen, where it is fnally degraded.

1	the lysosomal membrane. Chaperone-mediated direct transport resembles the process of protein import to various other cellular organelles: hsc73 binds to the protein and assists in its transport through the lysosomal membrane into the lumen, where it is finally degraded. Chaperone-mediated autophagy is responsible for the degradation of approximately 30% of cytoplasmic proteins in organs such as the liver and kidney. In addition to the lysosomal pathway of protein degradation, cells have an ability to destroy proteins without involvement of lysosomes. Such process occurs within large cytoplasmic or nuclear protein complexes called proteasomes. They represent FIGURE 2.23 • Electron micrograph of autophagosomes in a hepatocyte. This electron micrograph shows several autophagosomes containing degenerating mitochondria. Note the surrounding lysosomes that have been stained with acid phosphatase. 12,600. (Courtesy of Dr. William A. Dunn, Jr.)

1	12,600. (Courtesy of Dr. William A. Dunn, Jr.) ATP-dependent protease complexes that destroy proteins that have been specifically tagged for this pathway. Proteasomemediated degradation is used by cells to destroy abnormal proteins that are misfolded, denaturated, or contain abnormal amino acids. This pathway also degrades normal short-lived regulatory proteins that need to be rapidly inactivated and degraded such as mitotic cyclins that regulate cell-cycle progression, transcriptional factors, tumor suppressors, or tumor promoters. Proteins destined for proteasome-mediated degradation need to be recognized and specifically tagged by the polyubiquitin chain.

1	Proteins destined for proteasome-mediated degradation need to be recognized and specifically tagged by the polyubiquitin chain. Degradation of a protein in the proteasome-mediated pathway involves two successive steps:  Polyubiquitination, in which proteins targeted for destruction are repeatedly tagged by covalent attachments of a small (8.5-kilodalton) protein called ubiquitin. The tagging reaction is catalyzed by three ubiquitin ligases called ubiquitin-activating enzymes E1, E2, and E3. In a cascade of enzymatic reactions, the targeted protein is first marked by a single ubiquitin molecule. This creates a signal for consecutive attachment of several other ubiquitin molecules, resulting in a linear chain of ubiquitin conjugates. A protein target for destruction within the proteasome must be labeled with at least four ubiquitin molecules in the form of a polyubiquitin chain that serves as a degradation signal for proteasome complex.

1	 Degradation of the tagged protein by the 26S proteasome complex. Each proteasome consists of a hollow cylinder, shaped like a barrel, containing a 20S core particle (CP) that facilitates the multicatalytic protease activity in which polyubiquitinated proteins are degraded into small polypeptides and amino acids. On both ends of the CP cylinder are two 19S regulatory particles (RPs); one RP that forms the lid of the barrel recognizes polyubiquitin tags, unfolds the protein, and regulates its entry into the destruction chamber. The RP on the opposite side (on the base) of the barrel releases short peptides and amino acids after degradation of the protein is completed. Free ubiquitin molecules are released by de-ubiquitinating (DUB) enzymes and recycled. (Fig. 2.24).

1	Two groups of pathologic conditions are associated with the malfunction of proteasome-mediated degradation. The frst group of diseases results from a loss of proteasome function because of mutations in the system of ubiquitinactivating enzymes. This leads to a decrease in protein degradation and their subsequent accumulation in the cell cytoplasm (e.g., in Angelman syndrome and Alzheimer’s disease). The second group of diseases results from an accelerated degradation of proteins by overexpressed proteins involved in this system (e.g., infections with human papilloma virus). The recent discovery of specifc proteasome inhibitors holds promise for treatment of cancers and certain viral infections. The protein synthetic system of the cell consists of the rough endoplasmic reticulum and ribosomes.

1	The protein synthetic system of the cell consists of the rough endoplasmic reticulum and ribosomes. The cytoplasm of a variety of cells engaged chieﬂy in protein synthesis stains intensely with basic dyes. The basophilic staining is caused by the presence of RNA. That portion of the cytoplasm that stains with the basic dye is called ergastoplasm. The ergastoplasm in secretory cells (e.g., pancreatic acinar cells) is the light microscopic image of the organelle called the rough endoplasmic reticulum (rER).

1	With the TEM, the rER appears as a series of interconnected, membrane-limited, ﬂattened sacs called cisternae, with particles studding the exterior surface of the membrane (Fig. 2.25). These particles, called ribosomes , are attached to the membrane of the rER by ribosomal docking proteins. Ribosomes measure 15 to 20 nm in diameter and consist of a small and large subunit. Each subunit contains ribosomal RNA (rRNA) of different length as well as numerous different proteins. In many instances, the rER is continuous with the outer membrane of the nuclear envelope (see the next section). Groups of ribosomes form short spiral arrays called polyribosomes or polysomes (Fig. 2.26) in which many ribosomes are attached to a thread of messenger RNA (mRNA). Protein synthesis involves transcription and translation.

1	Protein synthesis involves transcription and translation. The production of proteins by the cell begins within the nucleus with transcription, in which the genetic code for a protein is transcribed from DNA to pre-mRNA. After posttranscriptional modifications of the pre-mRNA molecule— which includes RNA cleavage, excision of introns, rejoining of exons, and capping by addition of poly(A) tracks at the

1	FIGURE 2.24 • Proteasome-mediated degradation. This degradation pathway involves tagging proteins destined for destruction by a polyubiquitin chain and its subsequent degradation in proteasome complex with the release of free reusable ubiquitin molecules. Ubiquitin in the presence of ATP is activated by a complex of three ubiquitin-activating enzymes (E1, E2, and E3) to form a single polyubiquitin chain that serves as the degradation signal for the 26S proteasome complex. The regulatory particle (19S RP) that forms the lid of the main protein destruction chamber (20S core particle) recognizes polyubiquitin tags, unfolds the protein, and inserts and regulates its entry into the destruction chamber. The regulatory particle on the opposite side of the chamber releases short peptides and amino acids after degradation of the protein is completed. Free ubiquitin molecules are released by de-ubiquitinating enzymes (DUBs) and recycled.

1	3 end, and a methylguanosine cap [M(7) GPPP] at the 5 end—the resulting mRNA molecule leaves the nucleus and migrates into the cytoplasm (Fig. 2.27). Transcription is followed by translation in which the coded message contained in the mRNA is read by ribosomal complexes to form a polypeptide. A typical single cytoplasmic mRNA molecule binds to many ribosomes spaced as close as 80 nucleotides apart, thus forming a polyribosome complex, or FIGURE 2.25 • Electron micrograph of the rER. This image of the rER in a chief cell of the stomach shows the membranous cisternae (C) closely packed in parallel arrays. Polyribosomes are present on the cytoplasmic surface of the membrane surrounding the cisternae. The image of a ribosome-studded membrane is the origin of the term rough endoplasmic reticulum. A few ribosomes are free in the cytoplasm. M, mitochondrion. 50,000.

1	polysome. A polysome attached to the cytoplasmic surface of the rER can translate a single mRNA molecule and simultaneously produce many copies of a particular protein. In contrast, free ribosomes reside within the cytoplasm. They are not associated with any intracellular membranes and are structurally and functionally identical to polysomes of the rER. The differences between the structure of prokaryotic (bacterial) and eukaryotic ribosomes were exploited by researchers, who discovered chemical compounds (antibiotics) that bind to bacterial ribosomes, thereby destroying a bacterial infection without harming the cells of the infected individual. Several types of antibiotics, such as aminoglycosides (streptomycin), macrolides (erythromycin), lincosamides (clindamycin), tetracyclines, and chloramphenicol inhibit protein synthesis by binding to different portions of bacterial ribosomes. Signal peptides direct the post-translational transport of a protein.

1	Signal peptides direct the post-translational transport of a protein. FIGURE 2.26 • Electron micrograph of the rER and polyribosome complexes. This image shows a small section of the rER adjacent to the nucleus sectioned in two planes. The reticulum has turned within the section. Thus, in the upper right and left, the membranes of the reticulum have been cut at a right angle to their surface. In the center, the reticulum has twisted and is shown as in an aerial view (from above the membrane). The large spiral cytoplasmic assemblies (arrows) are chains of ribosomes that form polyribosomes that are actively engaged in translation of the mRNA molecule. 38,000.

1	Most proteins that are synthesized for export or to become a part of specific organelles (such as the plasma membrane, mitochondrial matrix, endoplasmic reticulum, or nucleus) require sorting signals that direct proteins to their correct destinations. These signal sequences (signal peptides) are often found in the sequence of the first group of 15 to 60 amino acids on the amino-terminus of a newly synthesized protein. For instance, almost all proteins that are transported to the endoplasmic reticulum have a signal sequence consisting of five to ten hydrophobic amino acids on their amino-termini. The signal sequence of the nascent peptide interacts with a signal-recognition particle (SRP), which arrests further growth of the polypeptide chain. The complex containing the SRP–polyribosome complex with arrested polypeptide synthesis is then relocated toward the rER excision of introns release of translational arrest

1	FIGURE 2.27 • Summary of events during protein synthesis. Proteins synthesis begins within the nucleus with transcription, during which the genetic code for a protein is transcribed from DNA to mRNA precursors. After post-transcriptional modifcations of the premRNA molecule—which include RNA cleavage, excision of introns, rejoining of exons, and capping by addition of poly(A) tracks at the 3 end and methylguanosine cap at the 5 end— the resulting mRNA molecule leaves the nucleus into the cytoplasm. In the cytoplasm, the mRNA sequence is read by the ribosomal complex in the process of translation to form a polypeptide chain. The frst group of 15 to 60 amino acids on the amino-terminus of a newly synthesized polypeptide forms a signal sequence (signal peptide) that directs protein to its destination (i.e., lumen of rER). The signal peptide interacts with a signal-recognition particle (SRP), which arrests further growth of the polypeptide chain until its relocation toward the rER

1	its destination (i.e., lumen of rER). The signal peptide interacts with a signal-recognition particle (SRP), which arrests further growth of the polypeptide chain until its relocation toward the rER membrane. Binding of THE SRP to a docking protein on the cytoplasmic surface of the rER aligns ribosome with the translocator protein. Binding of the ribosome to the translocator causes dissociation of the SRP–docking protein complex away from the ribosome, and protein synthesis is resumed. The translocator protein guides the polypeptide chain into the lumen of the rER cisterna. The signal sequence is cleaved from the polypeptide by signal peptidase and is subsequently digested by signal peptide peptidases. On completion of protein synthesis, the ribosome detaches from the translocator protein.

1	membrane. Binding of SRP to a docking protein on the cytoplasmic surface of rER aligns the ribosome with the translocator, an integral membrane protein of the rER. Binding of the ribosome to the protein translocator causes dissociation of the SRP–docking protein complex away from the ribosome and rER membrane, releasing the translational block and allowing the ribosome to resume protein synthesis (see Fig. 2.27). The translocator protein inserts the polypeptide chain into its aqueous pore, allowing newly formed protein to be discharged into the lumen of the rER cisterna. For simple secretory proteins, the polypeptide continues to be inserted by the translocator into the lumen as it is synthesized. The signal sequence is cleaved from the polypeptide by signal peptidase residing on the cisternal face of the rER membrane, even before the synthesis of the entire chain is completed. For integral membrane proteins, sequences along the polypeptide may instruct the forming protein to pass

1	face of the rER membrane, even before the synthesis of the entire chain is completed. For integral membrane proteins, sequences along the polypeptide may instruct the forming protein to pass back and forth through the membrane, creating the functional domains that the protein will exhibit at its final membrane. On completion of protein synthesis, the ribosome detaches from the translocator protein and is again free in the cytoplasm.

1	The post-translational modification and sequestration of proteins within the rER is the first step in the exportation of proteins destined to leave the cell. As polypeptide chains are synthesized by the membrane-bound polysomes, the protein is injected into the lumen of the rER cisterna, where it is further modified post-translationally by enzymes. These modifications include core glycosylation, disulfide-bond and internal hydrogen-bond formation, folding of the newly synthesized protein with the help of molecular chaperones, and partial subunit assembly. Proteins are then concentrated within a lumen of neighboring cisternae of rER, or they are carried to another part of the cell in the continuous channels of the rER.

1	Except for the few proteins that remain permanent residents of the rER membranes and those proteins secreted by the constitutive pathway, the newly synthesized proteins are normally delivered to the Golgi apparatus within minutes. A few diseases are characterized by an inability of the rER to export a mutated protein to Golgi. For example, in 1antitrypsin defciency, a single amino acid substitution renders the rER unable to export 1-antitrypsin (A1AT). This leads to decreased activity of A1AT in the blood and lungs and abnormal deposition of defective A1AT within the rER of liver hepatocytes, resulting in emphysema (chronic obstructive pulmonary disease) and impaired liver function. In cells in which the constitutive pathway is dominant— namely, plasma cells and activated fibroblasts—newly synthesized proteins may accumulate in the rER cisternae, causing their engorgement and distension.

1	The rER also serves as a quality checkpoint in the process of protein production. If the newly synthesized protein is not properly post-translationally modified or misfolded, it is then exported from the rER back to the cytoplasm via the mechanism of retrotranslocation. Defective proteins are here deglycosylated, polyubiquitylated, and degraded within proteasomes (see page 44). The rER is most highly developed in active secretory cells.

1	The rER is most highly developed in active secretory cells. The rER is particularly well developed in those cells that synthesize proteins destined to leave the cell (secretory cells) as well as in cells with large amounts of plasma membrane, such as neurons. Secretory cells include glandular cells, activated fibroblasts, plasma cells, odontoblasts, ameloblasts, and osteoblasts. The rER is not limited, however, to secretory cells and neurons. Virtually every cell of the body contains profiles of rER. However, they may be few in number, a reﬂection of the amount of protein secretion, and dispersed so that in the light microscope they are not evident as areas of basophilia.

1	The rER is most highly developed in active secretory cells because secretory proteins are synthesized exclusively by the ribosomes of the rER. In all cells, however, the ribosomes of the rER also synthesize proteins that are to become permanent components of the lysosomes, Golgi apparatus, rER, or nuclear envelope (these structures are discussed in the next sections) or integral components of the plasma membrane. Coatomers mediate bidirectional traffic between the rER and Golgi apparatus.

1	Coatomers mediate bidirectional traffic between the rER and Golgi apparatus. Experimental data indicate that two classes of coated vesicles are involved in the transport of protein from and to the rER. A protein coat similar to clathrin surrounds vesicles transporting proteins between the rER and the Golgi apparatus (page 35). However, unlike clathrins, which mediate bidirectional transport from and to the plasma membrane, one class of proteins is involved only in anterograde transport from the rER to the cis-Golgi network (CGN), the Golgi cisternae closest to the rER. Another class of proteins mediates retrograde transport from the CGN back to the rER (Fig. 2.28). These two classes of proteins are called coatomers or COPs.  COP-I mediates transport vesicles originating in the CGN back to the rER (Fig. 2.29a). This retrograde proteins mistakenly transferred to the CGN during nor mal anterograde transport. In addition, COP-I is also re the Golgi cisternae.

1	CGN back to the rER (Fig. 2.29a). This retrograde proteins mistakenly transferred to the CGN during nor mal anterograde transport. In addition, COP-I is also re the Golgi cisternae. FIGURE 2.28 • Anterograde and retrograde transport between the rER and cis-Golgi network. Two classes of coated vesicles are involved in protein transport to and from the rER. These vesicles are surrounded by COP-I and COP-II protein coat complexes, respectively. COP-II is involved in anterograde transport from the rER to the cis-Golgi network (CGN), and COP-I is involved in retrograde transport from the CGN back to the rER. After a vesicle is formed, the coat components dissociate from the vesicle and are recycled to their site of origin. The COP-I protein coat is also involved in retrograde transport between cisternae within the Golgi apparatus (see Fig. 2.13).

1	 COP-II is responsible for anterograde transport, forming rER transport vesicles destined for the CGN (Fig. 2.29b). COP-II assists in the physical deformation of rER membranes into sharply curved buds and further separation of vesicles from the rER membrane. Most proteins produced in the rER use COP-II–coated vesicles to reach the CGN. Shortly after formation of COP-I– or COP-II–coated vesicles, the coats dissociate from the newly formed vesicles, allowing the vesicle to fuse with its target. The coat components then recycle to their site of origin. “Free” ribosomes synthesize proteins that will remain in the cell as cytoplasmic structural or functional elements.

1	“Free” ribosomes synthesize proteins that will remain in the cell as cytoplasmic structural or functional elements. Proteins targeted to the nucleus, mitochondria, or peroxisomes are synthesized on free ribosomes and then released into the cytosol. In the absence of a signal sequence, proteins that are synthesized on free ribosomes remain in the cytosol. Cytoplasmic basophilia is associated with cells that produce large amounts of protein that will remain in the cell. Such cells and their products include developing red blood cells (hemoglobin), developing muscle cells (the contractile proteins actin and myosin), nerve cells (neurofilaments), and keratinocytes of the skin (keratin). In addition, most enzymes of the mitochondrion are synthesized by free polysomes and transferred into that organelle.

1	FIGURE 2.29 • Electron micrograph of COP-I– and COP-II–coated vesicles. a. This image shows COP-I–coated vesicles that initiate retrograde transport from the cis-Golgi network to the rER. In this image, taken with a quick-freeze deep-edge microscope, note the structure of the CGN and emerging vesicles. 27,000. b. Image of COP-II–coated vesicles that are responsible for anterograde transport. Note that the surface coat of these vesicles is different from that of clathrin-coated vesicles. 50,000. (Courtesy of Dr. John E. Heuser, Washington University School of Medicine.)

1	50,000. (Courtesy of Dr. John E. Heuser, Washington University School of Medicine.) Basophilia in these cells was formerly called ergastoplasm and is caused by the presence of large amounts of RNA. In this case, the ribosomes and polysomes are free in the cytoplasm (i.e., they are not attached to membranes of the endoplasmic reticulum). The large basophilic bodies of nerve cells, which are called Nissl bodies, consist of both rER and large numbers of free ribosomes (Fig. 2.30). All ribosomes contain RNA; it is the phosphate groups of the RNA of the ribosomes, not the membranous component of the endoplasmic reticulum, that accounts for basophilic staining of the cytoplasm.

1	FIGURE 2.30 • Electron micrograph of a nerve cell body showing the rER. This image shows rER profles as well as numerous free ribosomes located between the membranes of the rER. Collectively, the free ribosomes and membrane-attached ribosomes are responsible for the characteristic cytoplasmic basophilia (Nissl bodies) observed in the light microscope in the perinuclear cytoplasm of neurons. 45,000. The sER consists of short anastomosing tubules that are not associated with ribosomes.

1	The sER consists of short anastomosing tubules that are not associated with ribosomes. Cells with large amounts of smooth-surfaced endoplasmic reticulum may exhibit distinct cytoplasmic eosinophilia (acidophilia) when viewed in the light microscope. The sER is structurally similar to the rER but lacks the ribosome-docking proteins. It tends to be tubular rather than sheetlike, and it may be separate from the rER or an extension of it. The sER is abundant in cells that function in lipid metabolism (i.e., cells that synthesize fatty acids and phospholipids), and it proliferates in hepatocytes when animals are challenged with lipophilic drugs. The sER is well developed in cells that synthesize and secrete steroids such as adrenocortical cells and testicular Leydig (interstitial) cells (Fig. 2.31). In skeletal and cardiac muscle, the sER is also called the sarcoplasmic

1	FIGURE 2.31 • Electron micrograph of the sER. This image shows numerous profles of sER in an interstitial (Leydig) cell of the testis, a cell that produces steroid hormones. The sER seen here is a complex system of anastomosing tubules. The small, dense objects are glycogen particles. 60,000. reticulum. It sequesters Ca2 , which is essential for the contractile process and is closely apposed to the plasma-membrane invaginations that conduct the contractile impulses to the interior of the cell. The sER is the principal organelle involved in detoxification and conjugation of noxious substances.

1	The sER is the principal organelle involved in detoxification and conjugation of noxious substances. The sER is particularly well developed in the liver and contains a variety of detoxifying enzymes related to cytochrome P450 that are anchored directly into sER plasma membranes. They modify and detoxify hydrophobic compounds such as pesticides and carcinogens by chemically converting them into water-soluble conjugated products that can be eliminated from the body. The degree to which the liver is involved in detoxification at any given time may be estimated by the amount of sER present in liver cells. The sER is also involved in:  lipid and steroid metabolism,  glycogen metabolism, and  membrane formation and recycling. Because of these widely disparate functions, numerous other enzymes—including hydrolases, methylases, glucose-6phosphatase, ATPases, and lipid oxidases—are associated with the sER, depending on its functional role.

1	The Golgi apparatus is well developed in secretory cells and does not stain with hematoxylin or eosin. The Golgi apparatus was described more than 100 years ago by histologist Camillo Golgi. In studies of osmium-impregnated nerve cells, he discovered an organelle that formed networks around the nucleus. It was also described as well developed in secretory cells. Changes in the shape and FIGURE 2.32 • Photomicrograph of plasma cells. This photomicrograph of a plastic-embedded specimen showing the lamina propria of the small intestine is stained with toluidine blue. The plasma cells, where appropriately oriented, exhibit a clear area in the cytoplasm near the nucleus. These negatively stained regions (arrows) represent extensive accumulation of membranous cisternae that belong to the Golgi apparatus. The surrounding cytoplasm is deeply metachromatically stained because of the presence of ribosomes associated with the extensive rER. 1,200.

1	location of the Golgi apparatus relative to its secretory state were described even before it was viewed with the electron microscope and before its functional relationship to the rER was established. It is active both in cells that secrete protein by exocytosis and in cells that synthesize large amounts of membrane and membrane-associated proteins such as nerve cells. In the light microscope, secretory cells that have a large Golgi apparatus (e.g., plasma cells, osteoblasts, and cells of the epididymis) typically exhibit a clear area partially surrounded by ergastoplasm (Fig. 2.32). In EM, the Golgi apparatus appears as a series of stacked, ﬂattened, membrane-limited sacs or cisternae and tubular extensions embedded in a network of microtubules near the microtubule-organizing center (see page 65). Small vesicles involved in vesicular transport are seen in association with the cisternae. The Golgi apparatus is polarized both morphologically and functionally. The ﬂattened cisternae

1	page 65). Small vesicles involved in vesicular transport are seen in association with the cisternae. The Golgi apparatus is polarized both morphologically and functionally. The ﬂattened cisternae located closest to the rER represent the forming face, or the cis-Golgi network (CGN); the cisternae located away from the rER represent the maturing face, or the trans-Golgi network (TGN); (Figs. 2.33 and 2.34). The cisternae located between the TGN and CGN are commonly referred as the medial-Golgi network.

1	The Golgi apparatus functions in the post-translational modification, sorting, and packaging of proteins. Small COP-II coated transport vesicle s carry newly synthesized proteins (both secretory and membrane) from the rER to the CGN. From there, they travel within the transport vesicles from one cisterna to the next. The vesicles bud from one cisterna and fuse with the adjacent cisternae (Fig. 2.35). As proteins and lipids travel through the Golgi stacks, they undergo a series of post-translational modifcations that involve remodeling of N-linked oligosaccharides previously added in the rER.

1	FIGURE 2.33 • Electron micrograph of the Golgi apparatus. This electron micrograph shows the extensive Golgi apparatus in an islet cell of the pancreas. The fattened membrane sacs of the Golgi apparatus are arranged in layers. The CGN is represented by the fattened vesicles on the outer convex surface, whereas the fattened vesicles of the inner convex region constitute the TGN. Budding off the TGN are several vesicles (1). These vesicles are released (2) and eventually become secretory vesicles (3). 55,000.

1	In general, glycoproteins and glycolipids have their oligosaccharides trimmed and translocated. Glycosylation of proteins and lipids uses several carbohydrate-processing enzymes that add, remove, and modify sugar moieties of oligosaccharide chains. M-6-P is added to those proteins destined to travel to late endosomes and lysosomes (see page 37). In addition, glycoproteins are phosphorylated and sulfated. The proteolytic cleavage of certain proteins is also initiated within the cisternae. Four major pathways of protein secretion from the Golgi apparatus disperse proteins to various cell destinations. As noted, proteins exit the Golgi apparatus from the TGN. This network and the associated tubulovesicular array serve as

1	As noted, proteins exit the Golgi apparatus from the TGN. This network and the associated tubulovesicular array serve as FIGURE 2.34 • Electron micrograph of Golgi cisternae. a. This transmission electron micrograph shows a quick-frozen isolated Golgi apparatus replica from a cultured Chinese hamster ovary (CHO) cell line. The trans-Golgi cisternae are in the process of coated vesicle formation. b. Incubation of the trans-Golgi cisternae with the coatomer-depleted cytosol shows a decrease in vesicle formation activity. Note the lack of vesicles and the fenestrated shape of the trans-Golgi cisternae. 85,000. (Courtesy of Dr. John E. Heuser, Washington University School of Medicine.)

1	FIGURE 2.35 • The Golgi apparatus and vesicular traffcking. The Golgi apparatus contains several stacks of fattened cisternae with dilated edges. The Golgi cisternae form separate functional compartments. The closest compartment to the rER represents the CGN, to which COP-II–coated transport vesicles originating from the rER fuse and deliver newly synthesized pro teins. Retrograde transport from CGN to rER, as well retrograde transport between Golgi cisternae, is mediated by COP-I–coated vesicles. Once proteins have been modifed within the CGN, the transport vesicles bud off dilated ends of this compartment, and proteins are trans ferred into medial-Golgi cisternae. The process continues; in the same fashion, proteins are translocated into the trans-Golgi cisternae and the sorting station for shuttling vesicles that deliver proteins to the following locations (see Fig. 2.36).

1	 Apical plasma membrane. Many extracellular and membrane proteins are delivered to this site. This constitutive pathway most likely uses non–clathrin-coated vesicles. In most cells, secretory proteins destined for the apical plasma membrane have specific sorting signals that guide their sorting process in the TGN. Proteins are then delivered to the apical cell surface.

1	 Basolateral plasma membrane. Proteins targeted to the basolateral domain also have a specific sorting signal attached to them by the TGN. This constitutive pathway uses vesicles coated with an as yet unidentified protein associated with an epithelium-specific adaptor protein. The transported membrane proteins are continuously incorporated into the basolateral cell surface. This type of targeting is present in most polarized epithelial cells. In liver hepatocytes, however, the process of protein sorting into the basolateral and apical domains is quite different. All integral plasma-membrane proteins that are destined for both apical and basolateral domains are first transported from the TGN to the basolateral plasma membrane. From here, both proteins are endocytosed and sorted into early endo somal compartments. Basolateral proteins are recycled back into the basolateral membrane, whereas apical pro teins are transported across the cytoplasm to the apical cell membrane via

1	into early endo somal compartments. Basolateral proteins are recycled back into the basolateral membrane, whereas apical pro teins are transported across the cytoplasm to the apical cell membrane via transcytosis.

1	 Endosomes or lysosomes. Most proteins destined for organelles bear specific signal sequences. They are sorted in the TGN and delivered to specific organelles. However, TGN sorting mechanisms are never completely accurate. For instance, about 10% of lysosomal integral membrane proteins (limps) instead travel directly into early or late endosomes, take an extended route traveling via the apical plasma membrane (see Fig. 2.20), and from there move back into the endosomal pathways. Enzymes destined for lysosomes using M-6-P markers (see page 37) are delivered into early or late endosomes as they develop into mature lysosomes.

1	 Apical cytoplasm. Proteins that were aggregated or crystallized in the TGN as a result of changes in pH and Ca2 concentration are stored in large secretory vesicles. These vesicles undergo a maturation process in which secretory proteins are retained within the vesicle. All other nonsecretory proteins are recycled into the endosomal compartment or TGN in clathrin-coated vesicles

1	FIGURE 2.36 • Summary of events in protein traffcking from the TGN. The tubulovesicular array of the TGN serves as the sorting station for transporting vesicles that deliver proteins to the following destinations: (1) apical plasma membrane (i.e., epithelial cells); (2) apical region of the cell cytoplasm where proteins are stored in secretory vesicles (i.e., secretory cells); (3) early or late endosomal compartment; (4) selected proteins containing lysosomal signals, which are targeted to lysosomes; (5) lateral plasma membrane (i.e., epithelial cells); (6) basal plasma membrane (i.e., epithelial cells); (7) proteins destined for apical, basal, and lateral surfaces of plasma membrane, which are delivered to the basal plasma membrane (i.e., in hepatocytes); (8) all proteins endocytosed and sorted in early endosomes; (9) apical plasma membrane from early endosomes; (10) lateral plasma membrane; and (11) basal plasma membrane. Note two targeting mechanisms of proteins to different

1	and sorted in early endosomes; (9) apical plasma membrane from early endosomes; (10) lateral plasma membrane; and (11) basal plasma membrane. Note two targeting mechanisms of proteins to different surfaces of plasma membrane. In epithelial cells, proteins are directly targeted from the TGN into the appropriate cell surface as shown in steps (1), (5), and (6). In hepatocytes, all proteins are secreted frst to the basal cell surface, and then they are distributed to the appropriate cell surface via the endosomal compartment as shown in steps (7) to (11).

1	(see Fig. 2.35). Mature secretory vesicles eventually fuse with the plasma membrane to release the secretory product by exocytosis. This type of secretion is characteristic of highly specialized secretory cells found in exocrine glands. Sorting and packaging of proteins into transport vesicles occurs in the trans-Golgi network. Proteins that arrive in the TGN are distributed to different intercellular locations within transport vesicles. The intercellular destination of each protein depends on the sorting signals that are incorporated within the polypeptide chain of the protein.

1	The actual sorting and packaging of proteins in TGN is primarily based on sorting signals and physical properties.  Sorting signals are represented by the linear array of amino acid or associated carbohydrate molecules. This type of signal is recognized by the sorting machinery, which directs the protein into the appropriately coated transport vesicle.  Physical properties are important for packaging func tionally associated protein complexes. These groups of proteins are first partitioned into separate lipid rafts that targeted organelle. Mitochondria are abundant in cells that generate and expend large amounts of energy.

1	Mitochondria are abundant in cells that generate and expend large amounts of energy. Mitochondria were also known to early cytologists who observed them in cells vitally stained with Janus green B. It is now evident that mitochondria increase in number by division throughout interphase, and their divisions are not synchronized with the cell cycle. Videomicroscopy confirms that mitochondria can both change their location and undergo transient changes in shape. They may therefore be compared to mobile power generators as they migrate from one area of the cell to another to supply needed energy.

1	Because mitochondria generate ATP, they are more numerous in cells that use large amounts of energy such as striated muscle cells and cells engaged in ﬂuid and electrolyte transport. Mitochondria also localize at sites in the cell where energy is needed, as in the middle piece of the sperm, the intermyofibrillar spaces in striated muscle cells, and adjacent to the basolateral plasma-membrane infoldings in the cells of the proximal convoluted tubule of the kidney. Mitochondria evolved from aerobic bacteria that were engulfed by eukaryotic cells.

1	Mitochondria evolved from aerobic bacteria that were engulfed by eukaryotic cells. Mitochondria are believed to have evolved from an aerobic prokaryote (Eubacterium) that lived symbiotically within primitive eukaryotic cells. This hypothesis received support with the demonstration that mitochondria possess their own genome, increase their numbers by division, and synthesize some of their structural (constituent) proteins. Mitochondrial DNA is a closed circular molecule that encodes 13 enzymes involved in the oxidative phosphorylation pathway, two rRNAs, and 22 transfer RNAs (tRNAs) used in the translation of the mitochondrial mRNA.

1	Mitochondria possess a complete system for protein synthesis, including the synthesis of their own ribosomes. The remainder of the mitochondrial proteins is encoded by nuclear DNA; new polypeptides are synthesized by free ribosomes in the cytoplasm and then imported into mitochondria with the help of two protein complexes. These include translocase of the outer mitochondrial membrane (TOM complexes) and translocase of the inner mitochondrial membrane (TIM complexes). Translocation of proteins through mitochondrial membranes requires energy and assistance from several specialized chaperone proteins. Mitochondria are present in all cells except red blood cells and terminal keratinocytes.

1	Mitochondria are present in all cells except red blood cells and terminal keratinocytes. The number, shape, and internal structure of mitochondria are often characteristic for specific cell types. When present in large numbers, mitochondria contribute to the acidophilia of the cytoplasm because of the large amount of membrane they contain. Mitochondria may be stained specifically by histochemical procedures that demonstrate some of their constituent enzymes, such as those involved in ATP synthesis and electron transport. Mitochondria possess two membranes that delineate distinct compartments.

1	Mitochondria possess two membranes that delineate distinct compartments. Mitochondria display a variety of shapes, including spheres, rods, elongated filaments, and even coiled structures. All mitochondria, unlike other organelles described above, possess two membranes (Fig. 2.37). The inner mitochondrial membrane surrounds a space called the matrix. The outer mitochondrial membrane is in close contact with the cytoplasm. The space between the two membranes is called the intermembrane space. The following structural components of mitochondria possess specific characteristics related to their functions.

1	 Outer mitochondrial membrane. This 6to 7-nm-thick smooth membrane contains many voltage-dependent anion channels (also called mitochondrial porins). These large channels (approximately 3 nm in diameter) are permeable to uncharged molecules as large as 5,000 daltons. Thus, small molecules, ions, and metabolites can enter the intermembrane space but cannot penetrate the inner membrane. The environment of the intermembrane space is therefore similar to that of cytoplasm with respect to ions and small molecules. The outer membrane possesses receptors for proteins and polypeptides that translocate into the intermembrane space. It also contains several enzymes, including phospholipase A2, monoamine oxidase, and acetyl coenzyme A (CoA) synthase.

1	 Inner mitochondrial membrane. The TEM reveals that this membrane is thinner than the outer mitochondrial membrane. It is arranged into numerous cristae (folds) that significantly increase the inner membrane surface area (see Fig. 2.37). These folds project into the matrix that constitutes the inner compartment of the organelle. In some cells involved in steroid metabolism, the inner membrane may form tubular or vesicular projections into the matrix. The inner membrane is rich in the phospholipid cardiolipin, FIGURE 2.37 • Structure of the mitochondrion. a. This electron micrograph shows a mitochondrion in a pancreatic acinar cell. Note that the inner mitochondrial membrane forms the cristae (C) through a series of infoldings, as is evident in the region of the arrow. The outer mitochondrial membrane is a smooth continuous envelope that is separate and distinct from the inner membrane. 200,000.

1	b. Schematic diagram showing the components of a mitochondrion. Note the location of the elementary particles (inset), the shape of which refects the three-dimensional structure of ATP synthase. which makes the membrane impermeable to ions. The membrane forming the cristae contains proteins that have three major functions: (1) performing the oxidation reactions of the respiratory electron-transport chain, (2) synthesizing ATP, and (3) regulating transport of metabolites into and out of the matrix. The enzymes of the respiratory chain are attached to the inner membrane and project their heads into the matrix (Fig. 2.37, rectangle). With the TEM, these enzymes appear as tennis racquet–shaped structures called elementary particles. Their heads measure about 10 nm in diameter and contain enzymes that carry out oxidative phosphorylation, which generates ATP.

1	 Intermembrane space. This space is located between the inner and outer membranes and contains specific enzymes that use the ATP generated in the inner membrane. These enzymes include creatine kinase, adenylate kinase, and cytochrome c. The latter is an important factor in initiating apoptosis (see page 94).  Matrix. The mitochondrial matrix is surrounded by the inner mitochondrial membrane and contains the soluble enzymes of the citric acid cycle (Krebs cycle) and the enzymes involved in fatty-acid -oxidation. The major products of the matrix are CO2 and reduced NADH, which is the source of electrons for the electron-transport chain.

1	Mitochondria contain dense matrix granules that store Ca2 and other divalent and trivalent cations. These granules increase in number and size when the concentration of divalent (and trivalent) cations increases in the cytoplasm. Mitochondria can accumulate cations against a concentration gradient. Thus, in addition to ATP production, mitochondria also regulate the concentration of certain ions of the cytoplasmic matrix, a role they share with the sER. The matrix also contains mitochondrial DNA, ribosomes, and tRNAs. Mitochondria contain the enzyme system that generates ATP by means of the citric acid cycle and oxidative phosphorylation.

1	Mitochondria contain the enzyme system that generates ATP by means of the citric acid cycle and oxidative phosphorylation. Mitochondria generate ATP in a variety of metabolic pathways, including oxidative phosphorylation, the citric acid cycle, and -oxidation of fatty acids. The energy generated from these reactions, which take place in the mitochondrial matrix, is represented by hydrogen ions (H ) derived from reduced NADH. These ions drive a series of proton pumps located within the inner mitochondrial membrane that transfer H from the matrix to the intermembrane space (Fig. 2.38). These pumps constitute the electron-transport chain of respiratory enzymes (see Fig. 2.37). The transfer of H across the inner mitochondrial membrane establishes an

1	FIGURE 2.38 • Schematic diagram illustrating how mitochondria generate energy. The diagram indicates the ATP synthase complex and the electron-transport chain of proteins located in the inner mitochondrial membrane. The electron-transport chain generates a proton gradient between the matrix and intermembrane space that is used to produce ATP. Numbers represent sequential proteins involved in the electron-transport chain and ATP production: 1, NADH dehydrogenase complex; 2, ubiquinone; 3, cytochrome b-c1 complex; 4, cytochrome c; 5, cytochrome oxidase complex; and 6, ATP synthase complex.

1	electrochemical proton gradient. This gradient creates a large proton motive force that causes the movement of H to occur down its electrochemical gradient through a large, membrane-bound enzyme called ATP synthase. ATP synthase provides a pathway across the inner mitochondrial membrane in which H ions are used to drive the energetically unfavorable reactions leading to synthesis of ATP. This movement of protons back to the mitochondrial matrix is referred to as chemiosmotic coupling. The newly produced ATP is transported from the matrix to the intermembrane space by the voltage gradient–driven ATP/ADP exchange protein located in the inner mitochondrial membrane. From here, ATP leaves the mitochondria via voltage-dependent anion channels in the outer membrane to enter the cytoplasm. At the same time, ADP produced in the cytoplasm rapidly enters the mitochondria for recharging.

1	Several mitochondrial defects are related to defects in enzymes that produce ATP. Metabolically active tissues that use large amounts of ATP such as muscle cells and neurons are most affected. For example, myoclonic epilepsy with ragged red fbers (MERRF) is characterized by muscle weakness, ataxia, seizures, and cardiac and respiratory failure. Microscopic examination of muscle tissue from affected patients shows aggregates of abnormal mitochondria, providing a ragged appearance of red muscle fbers. MERRF is caused by mutation of the mitochondrial DNA gene encoding tRNA for lysine. This defect produces two abnormal complexes in the electron-transport chain of respiratory enzymes affecting ATP production. Mitochondria undergo morphologic changes related to their functional state.

1	Mitochondria undergo morphologic changes related to their functional state. TEM studies show mitochondria in two distinct configurations. In the orthodox confguration, the cristae are prominent, and the matrix compartment occupies a large part of the total mitochondrial volume. This configuration corresponds to a low level of oxidative phosphorylation. In the condensed confguration, cristae are not easily recognized, the matrix is concentrated and reduced in volume, and the intermembrane space increases to as much as 50% of the total volume. This configuration corresponds to a high level of oxidative phosphorylation. Mitochondria decide whether the cell lives or dies.

1	Mitochondria decide whether the cell lives or dies. Experimental studies indicate that mitochondria sense cellular stress and are capable of deciding whether the cell lives or dies by initiating apoptosis (programmed cell death). The major cell death event generated by the mitochondria is the release of cytochrome c from the mitochondrial intermembranous space into the cell cytoplasm. This event, regulated by the Bcl-2 protein family (see page 94), initiates the cascade of proteolytic enzymatic reactions that leads to apoptosis. Peroxisomes are single membrane-bounded organelles containing oxidative enzymes.

1	Peroxisomes are single membrane-bounded organelles containing oxidative enzymes. Peroxisomes (microbodies) are small (0.5 m in diameter), membrane-limited spherical organelles that contain oxidative enzymes, particularly catalase and other peroxidases. Virtually all oxidative enzymes produce hydrogen peroxide (H2O2) as a product of the oxidation reaction. Hydrogen peroxide is a toxic substance. The catalase universally present in peroxisomes carefully regulates the cellular hydrogen peroxide content by breaking down hydrogen peroxide, thus protecting the cell. In addition, peroxisomes contain D-amino acid oxidases, -oxidation enzymes, and numerous other enzymes.

1	Oxidative enzymes are particularly important in liver cells (hepatocytes), where they perform a variety of detoxification processes. Peroxisomes in hepatocytes are responsible for detoxification of ingested alcohol by converting it to acetaldehyde. The -oxidation of fatty acids is also a major function of peroxisomes. In some cells, peroxisomal fatty-acid oxidation may equal that of mitochondria. The proteins contained in the peroxisome lumen and membrane are synthesized on cytoplasmic ribosomes and imported into the peroxisome. A protein destined for peroxisomes must have a peroxisomal targeting signal attached to its carboxy-terminus.

1	Although abundant in liver and kidney cells, peroxisomes are also found in most other cells. The number of peroxisomes present in a cell increases in response to diet, drugs, and hormonal stimulation. In most animals, but not humans, peroxisomes also contain urate oxidase (uricase), which often appears as a characteristic crystalloid inclusion (nucleoid).

1	Various human metabolic disorders are caused by the inability to import peroxisomal proteins into the organelle because of a faulty peroxisomal targeting signal or its receptor. Several severe disorders are associated with nonfunctional peroxisomes. In the most common inherited disease related to nonfunctional peroxisomes, Zellweger syndrome, which leads to early death, peroxisomes lose their ability to function because of a lack of necessary enzymes. The disorder is caused by a mutation in the gene encoding the receptor for the peroxisome targeting signal that does not recognize the signal Ser-Lys-Leu at the carboxyterminus of enzymes directed to peroxisomes. Therapies for peroxisomal disorders have been unsatisfactory to date.

1	Microtubules are nonbranching and rigid hollow tubes of protein that can rapidly disassemble in one location and reassemble in another. In general, they grow from the microtubule-organizing center located near the nucleus and extend toward the cell periphery. Microtubules create a system of connections within the cell, frequently compared with railroad tracks, along which vesicular movement occurs. Microtubules are elongated polymeric structures composed of equal parts of -tubulin and -tubulin. Microtubules measure 20 to 25 nm in diameter (Fig. 2.39). The wall of the microtubule is approximately 5 nm thick and consists of 13 circularly arrayed globular dimeric tubulin molecules. The tubulin dimer has a molecular weight of 110 kilodaltons (kDa) and is formed from an -tubulin and a -tubulin molecule, each with a molecular weight of 55 kDa (Fig. 2.40). The dimers polymerize in an end-to-end fashion, head to tail, with the molecule of one dimer bound to the

1	FIGURE 2.39 • Electron micrographs of microtubules. a. Micrograph showing microtubules (arrows) of the mitotic spindle in a dividing cell. On the right, the microtubules are attached to chromosomes. 30,000. b. Micrograph of microtubules (arrows) in the axon of a nerve cell. In both cells, the microtubules are seen in longitudinal profle. 30,000. molecule of the next dimer in a repeating pattern. Longitudinal contacts between dimers link them into a linear structure called a protoflament. Axial periodicity seen along the 5-nm-diameter dimers corresponds to the length of the protein molecules. A small, 1-m segment of microtubule contains approximately 16,000 tubulin dimers. The arrangement of -tubulin and -tubulin molecules within the microtubule is visible in Figure 2.41. Microtubules grow from -tubulin rings within the MTOC that serve as nucleation sites for each microtubule.

1	Microtubule formation can be traced to hundreds of tubulin rings that form an integral part of the MTOC (Fig. 2.42). The and -tubulin dimers are added to a -tubulin ring in an end-to-end fashion (see Fig. 2.40). Polymerization of tubulin dimers requires the presence of guanosine triphosphate (GTP) and Mg2. Each tubulin molecule binds GTP before it is incorporated into the forming microtubule. The GTP–tubulin complex is then polymerized, and at some point GTP is hydrolyzed to guanosine diphosphate (GDP). As a result of this polymerization pattern, microtubules are polar because all of the dimers have the same orientation. Each microtubule possesses a minus (nongrowing) end that corresponds to -tubulin; in the cell, it is usually embedded in the MTOC. The plus (growing) end of microtubules corresponds to -tubulin and extends the cell periphery. Tubulin dimers dissociate from microtubules in the steady state, which adds a pool of free tubulin dimers to the cytoplasm. This pool is in

1	corresponds to -tubulin and extends the cell periphery. Tubulin dimers dissociate from microtubules in the steady state, which adds a pool of free tubulin dimers to the cytoplasm. This pool is in equilibrium with the polymerized tubulin in the micro-tubules; therefore, polymerization and depolymerization are in equilibrium. The equilibrium can be shifted in the direction of depolymerization by exposing the cell or isolated microtubules to low temperatures or high pressure. Repeated exposure to alternating low and high temperature is the basis of the purification technique for tubulin and microtubules. The speed of polymerization or depolymerization can also be modified by interaction with specific microtubule-associated proteins (MAPs). These proteins, such as MAP-1, 2, 3, and 4, MAP-, and TOG , regulate microtubule assembly and anchor the microtubules to specific organelles. MAPs are also responsible for the existence of stable populations of nondepolymerizing microtubules in the

1	and TOG , regulate microtubule assembly and anchor the microtubules to specific organelles. MAPs are also responsible for the existence of stable populations of nondepolymerizing microtubules in the cell, such as those found in cilia and ﬂagella.

1	The length of microtubules changes dynamically as tubulin dimers are added or removed in a process of dynamic instability.

1	Microtubules observed in cultured cells with real-time video microscopy appear to be constantly growing toward the cell periphery (by addition of tubulin dimers) and then suddenly shrink in the direction of the MTOC (by removal of tubulin dimers). This constant remodeling process, known as dynamic instability, is linked to a pattern of GTP hydrolysis during the microtubule assembly and disassembly process. The MTOC can be compared to a feeding chameleon, which fires its long, projectile tongue to make contact with potential food. The chameleon then retracts its tongue back into its mouth and repeats this process until it is successful in obtaining food. The same strategy of “firing” microtubules from the MTOC toward the cell periphery and subsequently retracting them enables the cell to establish an organized system of mi- ( end) ( end) tubulin dimer bound to GTP tubulin dimer bound to GDP 24 nm Pi Pi -tubulin -tubulin -tubulin capping proteins

1	FIGURE 2.40 • Polymerization of microtubules. On the left, the diagram depicts the process of polymerization and depolymerization of tubulin dimers during the process of microtubule assembly. Each tubulin dimer consists of an -tubulin and a -tubulin subunit. On the right is a diagram showing that each microtubule contains 13 tubulin dimers within its cross section. The minus ( ) end of the microtubule contains a ring of -tubulin, which is necessary for microtubule nucleation. This end is usually embedded within the MTOC and possesses numerous capping proteins. The plus ( ) end of the microtubule is the growing end to which tubulin dimers bound to guanosine triphosphate (GTP) molecules are incorporated. Incorporated tubulin dimers hydrolyze GTP, which releases the phosphate groups to form polymers with guanosine diphosphate (GDP)–tubulin molecules.

1	crotubules linking peripheral structures and organelles with the MTOC. As mentioned earlier, the association of a micro-tubule with MAPs, such as occurs within the axoneme of a cilium or ﬂagellum, effectively blocks this dynamic instability and stabilizes the microtubules. FIGURE 2.41 • Three-dimensional reconstruction of an intact microtubule. This image was obtained using cryo-electron microscopy. Tomographic (sectional) images of a frozen hydrated microtubule were collected and digitally reconstructed at a resolution of 8 angstroms (Å). The helical structure of the -tubulin molecules is recognizable at this magnifcation. 3,250,000. (Courtesy of Dr. Kenneth Downing.) The structure and function of microtubules in mitosis and in cilia and ﬂagella are discussed later in this chapter and in Chapter 5. Microtubules can be visualized in the light microscope and are involved in intracellular transport and cell motility.

1	Microtubules can be visualized in the light microscope and are involved in intracellular transport and cell motility. Microtubules may be seen in the light microscope by using special stains, polarization, or phase-contrast optics. Because of the limited resolution of the light microscope, in the past microtubules were erroneously called fibers, such as the “fibers” of the mitotic spindle. Microtubules may now be distinguished from filamentous and fibrillar cytoplasmic components even at the light microscopic level by using antibodies to tubulin, the primary protein component of microtubules, conjugated with ﬂuorescent dyes (Fig. 2.42). In general, microtubules are found in the cytoplasm, where they originate from the MTOC; in cilia and ﬂagella, where they form the axoneme and its anchoring basal body; in centrioles and the mitotic spindle; and in elongating processes of the cell, such as those in growing axons.

1	Microtubules are involved in numerous essential cellular functions:  Intracellular vesicular transport (e.g., movement of secre tory vesicles, endosomes, and lysosomes),  Movement of cilia and ﬂagella,  Attachment of chromosomes to the mitotic spindle and their movement during mitosis and meiosis,  Cell elongation and movement (migration), and  Maintenance of cell shape, particularly its asymmetry. FIGURE 2.42 • Staining of microtubules with fuorescent dye.

1	FIGURE 2.42 • Staining of microtubules with fuorescent dye. This confocal immunofuorescent image shows the organization of the microtubules within an epithelial cell in tissue culture. In this example, the specimen was immunostained with three primary antibodies against tubulin (green), centrin (red), and kinetochores (light blue) and then incubated in a mixture of three different fuorescently tagged secondary antibodies that recognized the primary antibodies. Nuclei were stained (dark blue) with a fuorescent molecule that intercalates into the DNA double helix. Note that the microtubules are focused at the MTOC or centrosome (red) located adjacent to the nucleus. The cell is in the S phase of the cell cycle, as indicated by the presence of both large unduplicated kinetochores and smaller pairs of duplicated kinetochores. 3,000. (Courtesy of Dr. Wilma L. Lingle and Ms. Vivian A. Negron.)

1	Movement of intracellular organelles is generated by molecular motor proteins associated with microtubules. In cellular activities that involve movement of organelles and other cytoplasmic structures—such as transport vesicles, mitochondria, and lysosomes—microtubules serve as guides to the appropriate destinations. Molecular motor proteins attach to these organelles or structures and ratchet along the microtubule track (Fig. 2.43). The energy required for the ratcheting movement is derived from ATP hydrolysis. Two families of molecular motor proteins have been identified that allow for unidirectional movement:  Dyneins constitute one family of molecular motors. They move along the microtubules toward the minus end of the tubule. Therefore, cytoplasmic dyneins are capable of transporting organelles from the cell periphery toward the

1	MTOC. One member of the dynein family, axonemal dynein, is present in cilia and ﬂagella. It is responsible for the sliding of one microtubule against an adjacent micro- tubule of the axoneme that effects their movement.  Kinesins, members of the other family, move along the microtubules toward the plus end; therefore, they are ca FIGURE 2.43 • The molecular motor proteins associated with microtubules. Microtubules serve as guides for molecular motor proteins. These ATP-driven microtubule-associated motor proteins are attached to moving structures (such as organelles) that ratchet them along a tubular track. Two types of molecular motors have been identifed: dyneins that move along microtubules toward their minus ( ) end (i.e., toward the center of the cell) and kinesins that move toward their plus ( ) end (i.e., toward the cell periphery). pable of moving organelles from the cell center toward the cell periphery.

1	pable of moving organelles from the cell center toward the cell periphery. Both dyneins and kinesins are involved in mitosis and meiosis. In these activities, dyneins move the chromosomes along the microtubules of the mitotic spindle. Kinesins are simultaneously involved in movement of polar microtubules. These microtubules extend from one spindle pole past the metaphase plate and overlap with microtubules extending from the opposite spindle pole. Kinesins located between these microtubules generate a sliding movement that reduces the overlap, thereby pushing the two spindle poles apart toward each daughter cell (Fig. 2.44). Actin filaments are present in virtually all cell types.

1	Actin molecules (42 kDa) are abundant and may constitute as much as 20% of the total protein of some nonmuscle cells (Fig. 2.45). Similar to the tubulin in microtubules, actin molecules also assemble spontaneously by polymerization into a linear helical array to form filaments 6 to 8 nm in diameter. They are thinner, shorter, and more ﬂexible than microtubules. Free actin molecules in the cytoplasm are referred to as G-actin (globular actin), in contrast to the polymerized actin of the filament, which is called F-actin (flamentous actin). An actin filament is a polarized structure; its fast-growing end is referred to as the plus (barbed) end, and its slow-growing end is referred to as the minus (pointed) end. The dynamic process of actin polymerization requires the presence of K , Mg2 , and ATP, which is hydrolyzed to ADP after each G-actin molecule is incorporated into the filament (Fig. 2.46). The control and regulation of the polymerization process depends on the local

1	K , Mg2 , and ATP, which is hydrolyzed to ADP after each G-actin molecule is incorporated into the filament (Fig. 2.46). The control and regulation of the polymerization process depends on the local concentration of G-actin and the interaction of actinbinding proteins (ABPs), which can prevent or enhance polymerization.

1	In addition to controlling the rate of polymerization of actin filaments, ABPs are responsible for the filaments’ organization. For example, a number of proteins can modify or act on actin filaments to give them various specific characteristics:

1	FIGURE 2.44 • Distribution of kinesinlike motor protein within the mitotic spindle. This confocal immunofuorescent image shows a mammary gland epithelial cell in anaphase of mitosis. Each mitotic spindle pole contains two centrioles (green). A mitosis-specifc kinesinlike molecule called Eg5 (red ) is associated with the subset of the mitotic spindle microtubules that connect the kinetochores (white) to the spindle poles. The motor action of Eg5 is required to separate the sister chromatids (blue) into the daughter cells. This cell was frst immunostained with three primary antibodies against Eg5 (red), centrin (green), and kinetochores (white) and then incubated in three different fuorescently tagged secondary antibodies that recognize the primary antibodies. Chromosomes were stained with a fuorescent molecule that intercalates into the DNA double helix. 3,500. (Courtesy of Dr. Wilma L. Lingle and Ms. Vivian A. Negron.)  Actin-bundling proteins cross-link actin filaments into parallel

1	fuorescent molecule that intercalates into the DNA double helix. 3,500. (Courtesy of Dr. Wilma L. Lingle and Ms. Vivian A. Negron.)  Actin-bundling proteins cross-link actin filaments into parallel arrays, creating actin filament bundles. An example of this modification occurs inside the microvillus, where actin filaments are cross-linked by the actin-bundling proteins fascin and fmbrin. This cross-linkage provides support and imparts rigidity to the microvilli.

1	FIGURE 2.45 • Distribution of actin flaments in pulmonary artery endothelial cells in culture. Cells were fxed and stained with NDB phallacidin stain conjugated with fuorescein dye. Phallacidin binds and stabilizes actin flaments, preventing their depolymerization. Note the accumulation of actin flaments at the periphery of the cell just beneath the plasma membrane. These cells were also stained with two additional dyes: a mitochondria-selective dye (i.e., MitoTracker Red) that allows the visualization of mitochondria (red ) in the middle of the cell and DAPI stain that reacts with nuclear DNA and exhibits blue fuorescence over the nucleus. 3,000. (Courtesy of Molecular Probes, Inc., Eugene, OR.) proteins they contain. There are two types of filaments (myoflaments) present in muscle cells: 6to 8-nm actin filaments (called thin flaments; Fig. 2.47) and 15-nm filaments (called thick flaments) of myosin II, which is the predominant protein in muscle cells. Myosin II is a double-headed

1	6to 8-nm actin filaments (called thin flaments; Fig. 2.47) and 15-nm filaments (called thick flaments) of myosin II, which is the predominant protein in muscle cells. Myosin II is a double-headed molecule with an elongated rodlike tail. The specific structural and functional relationships among actin, myosin, and other ABPs in muscle contraction are

1	FIGURE 2.46 • Polymerization of actin flaments. Actin flaments are polarized structures. Their fast-growing end is referred to as the plus ( ) or barbed end; the slow-growing end is referred to as the minus ( ) or pointed end. The dynamic process of actin polymerization requires energy in the form of an ATP molecule that is hydrolyzed to ADP after a G-actin molecule is incorporated into the flament.  Actin flament–severing proteins cut long actin filaments into short fragments. An example of such a protein is gelsolin, a 90-kDa ABP that normally initiates actin polymerization but at high Ca2 concentrations causes severing of the actin filaments, converting an actin gel into a ﬂuid state.

1	 Actin-capping proteins block further addition of actin molecules by binding to the free end of an actin filament. An example is tropomodulin, which can be isolated from skeletal and cardiac muscle cells. Tropomodulin binds to the free end of actin myofilaments, regulating the length of the filaments in a sarcomere.  Actin cross-linking proteins are responsible for cross-linking actin filaments with each other. An example of such proteins can be found in the cytoskeleton of erythrocytes. Several proteins—such as spectrin, adductin, protein 4.1,and protein 4.9—are involved in cross-linking actin filaments.  Actin motor proteins belong to the myosin family, which hydrolyzes ATP to provide the energy for movement along the actin filament from the minus end to the plus end. Some cells, such as muscle cells, are characterized by the size, amount, and nature of the filaments and actin-motor discussed in Chapter 11 (Muscle Tissue).

1	In addition to myosin II, nonmuscle cells contain myosin I, a protein with a single globular domain and short tail that attaches to other molecules or organelles. Extensive studies have revealed the presence of a variety of other nonmuscle myosin isoforms that are responsible for motor functions in many specialized cells, such as melanocytes, kidney and intestinal absorptive cells, nerve growth cones, and inner ear hair cells. Actin filaments participate in a variety of cell functions. Actin filaments are often grouped in bundles close to the plasma membrane. Functions of these membrane-associated actin filaments include the following.  Anchorage and movement of membrane protein. Actin filaments are distributed in three-dimensional networks throughout the cell and are used as anchors within specialized cell junctions such as focal adhesions.

1	 Formation of the structural core of microvilli on absorptive epithelial cells. Actin filaments may also help maintain the shape of the apical cell surface (e.g., the apical terminal web of actin filaments serves as a set of tension cables under the cell surface).  Locomotion of cells. Locomotion is achieved by the force exerted by actin filaments by polymerization at their growing ends. This mechanism is used in many migrating cells—in particular, on transformed cells of invasive tumors. As a result of actin polymerization at their leading edge, cells extend processes from their surface by pushing the plasma membrane ahead of the growing actin filaments. The leading-edge extensions of a crawling cell are called lamellipodia; they contain elongating organized

1	FIGURE 2.47 • Thin flament organization and structure in cardiac cells. a. Immunofuorescence micrograph of a chick cardiac myocyte stained for actin (green) to show the thin flaments and for tropomodulin (red ) to show the location of the slow-growing ( ) ends of the thin flaments. Tropomodulin appears as regular striations because of the uniform lengths and alignment of the thin flaments in sarcomeres. 320. (Courtesy of Drs. Velia F. Fowler and Ryan Littlefeld.) b. Diagram of a thin flament. The polarity of the thin flament is indicated by the fast-growing ( ) end and the slow-growing ( ) end. Only a portion of the entire thin flament is shown for clarity. Tropomodulin is bound to actin and tropomyosin at the slow-growing ( ) end. The troponin complex binds to each tropomyosin molecule every seven actin monomers along the length of the thin flament. (Courtesy of Drs. Velia F. Fowler and Ryan Littlefeld.) bundles of actin filaments with their plus ends directed to ward the plasma

1	every seven actin monomers along the length of the thin flament. (Courtesy of Drs. Velia F. Fowler and Ryan Littlefeld.) bundles of actin filaments with their plus ends directed to ward the plasma membrane. Extension of cell processes. These processes can be ob called flopodia, located around their surface. As in lamel lipodia, these protrusions contain loose aggregations of 10 to 20 actin filaments organized in the same direction, again with their plus ends directed toward the plasma mem brane. Actin filaments are also essential in cytoplasmic streaming (i.e., the streamlike movement of cytoplasm that can be observed in cultured cells).

1	Intermediate flaments play a supporting or general structural role. These ropelike filaments are called intermediate because their diameter of 8 to 10 nm is between those of actin filaments and microtubules. Nearly all intermediate filaments consist of subunits with a molecular weight of about 50 kDa. Some evidence suggests that many of the stable structural proteins in intermediate filaments evolved from highly conserved enzymes, with only minor genetic modification. Intermediate filaments are formed from nonpolar and highly variable intermediate filament subunits.

1	Intermediate filaments are formed from nonpolar and highly variable intermediate filament subunits. Unlike those of microfilaments and microtubules, the protein subunits of intermediate filaments show considerable diversity and tissue specificity. In addition, they do not posses enzymatic activity and form nonpolar filaments. Intermediate filaments also do not typically disappear and re-form in the continuous manner characteristic of most microtubules and actin filaments. For these reasons, intermediate filaments are believed to play a primarily structural role within the cell and to compose the cytoplasmic link of a tissuewide continuum of cytoplasmic, nuclear, and extracellular filaments (Fig. 2.48).

1	Intermediate flament proteins are characterized by a highly variable central rod-shaped domain with strictly conserved globular domains at either end (Fig. 2.49). Although the various classes of intermediate filaments differ in the amino acid sequence of the rod-shaped domain and show some variation in molecular weight, they all share a homologous region that is important in filament self-assembly. Intermediate filaments are assembled from a pair of helical monomers that twist around each other to form coiled-coil dimers. Then, two coiled-coil dimers twist around each other in antiparallel fashion (parallel but pointing in opposite directions) to generate a staggered tetramer of two coiled-coil dimers, thus forming the nonpolarized unit of the intermediate filaments (see Fig. 2.49). Each tetramer, acting as an individual unit, is aligned along the axis of the filament. The ends of the tetramers are bound together to form the free ends of the filament. This assembly process provides a

1	tetramer, acting as an individual unit, is aligned along the axis of the filament. The ends of the tetramers are bound together to form the free ends of the filament. This assembly process provides a stable, staggered, helical array in which filaments are packed together and additionally stabilized by lateral binding interactions between adjacent tetramers.

1	Intermediate filaments are a heterogeneous group of cytoskeletal elements found in various cell types. FIGURE 2.48 • Electron micrograph of the apical part of an epithelial cell demonstrating intermediate flaments. This electron micrograph, obtained using the quick-freeze deep-etch technique, shows the terminal web (TW ) of an epithelial cell and underlying intermediate flaments (IF ). The long, straight actin flament cores or rootlets (R) extending from the microvilli are cross-linked by a dense network of actin flaments containing numerous actin-binding proteins. The network of intermediate flaments can be seen beneath the terminal web anchoring the actin flaments of the microvilli. 47,000. (Reprinted with permission from Hirokawa N, Keller TC 3rd, Chasan R, Mooseker MS. Mechanism of brush border contractility studied by the quick-freeze, deep-etch method. J Cell Biol 1983;96:1325–1336.)

1	Intermediate filaments are organized into six major classes on the basis of gene structure, protein composition, and cellular distribution (Table 2.3).  Classes 1 and 2. These are the most diverse groups of intermediate filaments and are called keratins (cytokeratins). These classes contain more than 50 different isoforms and account for most of the intermediate filaments (about 54 genes out of the total of 70 human intermediate filament genes are linked to keratin molecules). Keratins only assemble as heteropolymers; an acid cytokeratin (class 1) and a basic cytokeratin (class 2) molecule form a heterodimer. Each keratin pair is characteristic of a particular type of epithelium; however, some epithelial cells may express more than one pair. Keratin filaments are found in different cells of epithelial origin. According to new nomenclature, keratins are divided into three expression groups: keratins of simple epithelia,

1	FIGURE 2.49 • Polymerization and structure of intermediate flaments. Intermediate flaments are self-assembled from a pair of monomers that twist around each other in parallel fashion to form a stable dimer. Two coiled-coil dimers then twist around each other in antiparallel fashion to generate a staggered tetramer of two coiled-coil dimers. This tetramer forms the nonpolarized unit of the intermediate flaments. Each tetramer, acting as an individual unit, aligns along the axis of the flament and binds to the free end of the elongating structure. This staggered helical array is additionally stabilized by lateral binding interactions between adjacent tetramers.

1	keratins of stratifed epithelia, and structural keratins, also called hard keratins. The latest are found in skin appendages such as hair and nails. Keratin filaments span the cytoplasm of epithelial cells and, via desmosomes, connect with keratin filaments in neighboring cells. Keratin subunits do not co-assemble with other classes of intermediate filaments; therefore, they form a distinct cell-specific and tissuespecific recognition system.

1	 Class 3. This group contains four proteins: vimentin, the most widely distributed intermediate filament protein in the body, and vimentin-like proteins such as desmin, glial fbrillary acidic protein (GFAP), and peripherin. They represent a diverse family of cytoplasmic filaments found in many cell types. In contrast to keratins, class 3 proteins (with the exception of desmin) preferentially form homopolymeric filaments containing only one type of intermediate protein. Vimentin is the most abundant intermediate filament found in all mesoderm-derived cells, including fibroblasts (Fig. 2.50); desmin is characteristic of muscle cells; GFAP is found in glial cells (highly specific for astrocytes), and peripherin is found in many peripheral nerve cells.

1	 Class 4. Historically this group has been called neuroflaments; they contain intermediate filament proteins that are expressed mostly in axons of nerve cells. The three types of neurofilament proteins are of different molecular weights: NF-L (a low-weight protein), NF-M (a medium-weight protein), and NF-H (a high-weight protein). They co-assemble to form a heterodimer that contains one NF-L molecule and one of the others. All three proteins form neurofilaments that extend from the cell body into the ends of axons and dendrites, providing structural support. However, genes for class 4 proteins also encode several other intermediate filament proteins. These include nestin and -internexin in nerve cells as well as synemin, syncoilin, and paranemin in muscle cells. Members of this group preferentially co-assemble in tissues as heteropolymers.

1	 Class 5. Lamins, specifically nuclear lamins, form a network-like structure that is associated with the nuclear envelope. Lamins are represented by two types of proteins, lamin A and lamin B. In contrast to other types of intermediate filaments found in the cytoplasm, lamins are located within the nucleoplasm of almost all differentiated cells in the body. A description of their structure and function can be found on page 82.  Class 6. This is a lens-specific group of intermediate filament, or “beaded flaments” containing two proteins, phakinin and flensin. The periodic beadlike surface appearance of these filaments is attributed to the globular structure of the carboxy-terminus of the filensin molecule, which projects out from the assembled filament core. Intermediate filament–associated proteins are essential for the integrity of cell-to-cell and cell-to-extracellular matrix junctions.

1	Intermediate filament–associated proteins are essential for the integrity of cell-to-cell and cell-to-extracellular matrix junctions. A variety of intermediate flament–associated proteins function within the cytoskeleton as integral parts of the molecular architecture of cells. Some proteins, such as those of the plectin family, possess binding sites for actin filaments,

1	TABLE Classes of Intermediate Filaments Their Location and Associated Diseases 2.3 Molecular Weight Examples of Type of Protein (kDa) Where Found Associated Diseases Class 1 and 2: Keratins Acid cytokeratins 40–64 All epithelial cells Epidermolysis bullosa simplex Basic cytokeratins 52–68 All epithelial cells Keratoderma disorders caused by keratin mutations Meesman corneal dystrophy Class 3: Vimentin and Vimentin-like Vimentin 55 Cells of mesenchymal origin Desmin-related myopathy (including endothelial cells, (DRM) myofbroblasts, some smooth Dilated cardiomyopathy muscle cells) and some cells of Alexander disease neuroectodermal origin Amyotrophic lateral sclerosis Desmin 53 Muscle cells; co-assembles with (ALS) nestin, synemin and paranemin Glial fbrillary acidic protein 50–52 Neuroglial cells (mainly astrocytes; (GFAP) to lesser degree, ependymal cells), Schwann cells, enteric glial cells, satellite cells of sensory ganglia, and pituicytes Peripherin 54 Peripheral neurons Class 4:

1	cells (mainly astrocytes; (GFAP) to lesser degree, ependymal cells), Schwann cells, enteric glial cells, satellite cells of sensory ganglia, and pituicytes Peripherin 54 Peripheral neurons Class 4: Neurofilaments Neuroflament L (NF-L) 68 Neurons Charcot-Marie-tooth disease Co-assembles with NF-M or NF-H Parkinson disease Neuroflament M (NF-M) 110 Neurons Co-assembles with NF-L Neuroflament H (NF-H) 130 Neurons Co-assembles with NF-L Nestin 240 Neural stem cells, some cells of neuroectodermal origin, muscle cells Co-assembles with desmin Α-Internexin 68 Neurons Synemin Α/ΒA 182 Muscle cells Co-assembles with desmin Syncoilin 64 Muscle cells Paranemin 178 Muscle cells Co-assembles with desmin Class 5: Lamins Lamin A/CB 62–72 Nucleus of all nucleated cells Emery-Dreyfuss muscular dystrophy Lamin B 65–68 Nucleus of all nucleated cells Limb girdle muscular dystrophy Class 6: Beaded Filaments Phakinin (CP49)C 49 Eye lens fber cells Juvenile-onset cataracts Co-assembles with flesin

1	Lamin B 65–68 Nucleus of all nucleated cells Limb girdle muscular dystrophy Class 6: Beaded Filaments Phakinin (CP49)C 49 Eye lens fber cells Juvenile-onset cataracts Co-assembles with flesin Congenital cataracts Filesin (CP115) 115 Eye lens fber cells Co-assembles with phakinin A Synemin and synemin represent two alternative transcripts of the DMN gene. BLamin C is a splice product of lamin A. C The molecular weight of filensin/phakinin heterodimer is 131 kilodaltons.

1	microtubules, and intermediate filaments and are thus important in the proper assembly of the cytoskeleton. Lamins, the intermediate filaments in the nucleus, are associated with numerous proteins in the inner nuclear membrane, including emerin, lamin B receptor (LBR), nurim, and several lamina-associated polypeptides. Some of these proteins have multiple binding sites to intermediate filaments, actin, chromatin, and signaling proteins; thus, they function in chromatin organization, gene expression, nuclear architecture, and cell signaling and provide an essential link between the nucleoskeleton and cytoskeleton of the cell. Another important family of intermediate filament–associated proteins consists of desmoplakins, desmoplakinlike proteins, and plakoglobins. These proteins form the attachment plaques for intermediate filaments, an essential part of desmosomes and hemidesmosomes. The interaction of intermediate filaments with cell-to-cell and cell-toextracellular matrix junctions

1	plaques for intermediate filaments, an essential part of desmosomes and hemidesmosomes. The interaction of intermediate filaments with cell-to-cell and cell-toextracellular matrix junctions provides mechanical stren gth and resistance to extracellular forces. Table 2.4 summarizes the characteristics of the three types of cytoskeletal filaments.

1	Centrioles represent the focal point around which the MTOC assembles. Centrioles, visible in the light microscope, are paired, short, rodlike cytoplasmic cylinders built from nine microtubule triplets. In resting cells, centrioles have an orthogonal

1	FIGURE 2.50 • Distribution of intermediate flaments in human fetal lung fbroblasts. Distribution of vimentin (red) and actin flaments (green) is shown in cultured fbroblasts from human fetal lung. Vimentin is an intermediate flament protein expressed in all cells of mesenchymal origin. In cultured fbroblasts, vimentin flaments are visible centrally within the cell cytoplasm, whereas the actin flaments are aggregated primary near the cell surface. This immunofuorescent image was obtained using the indirect immunofuorescence techniques in which vimentin flaments were treated with mouse anti-vimentin primary antibodies followed by goat anti-mouse secondary antibodies conjugated with Texas red fuorescent dye. Actin flaments were counter-stained with phalloidin conjugated with a green fuorescent dye. Nuclei were stained blue with Hoechst fuorescent stain. 3,500. (Reprinted with permission from Michael W. Davidson, Florida State University.) orientation: One centriole in the pair is arrayed

1	dye. Nuclei were stained blue with Hoechst fuorescent stain. 3,500. (Reprinted with permission from Michael W. Davidson, Florida State University.) orientation: One centriole in the pair is arrayed at a right angle to the other. Centrioles are usually found close to the nucleus, often partially surrounded by the Golgi apparatus, and associated with a zone of amorphous, dense pericentriolar material. The region of the cell containing the centrioles and pericentriolar material is called the microtubule-organizing center or centrosome (Fig. 2.51). The MTOC is the region where most microtubules are formed and from which they are then directed to specific destinations within the cell. Therefore, the MTOC controls the number, polarity, direction, orientation, and organization of microtubules formed during the interphase of the cell cycle. During mitosis, duplicated MTOCs serve as mitotic spindle poles. Development of the MTOC itself depends solely on the presence of centrioles. When

1	formed during the interphase of the cell cycle. During mitosis, duplicated MTOCs serve as mitotic spindle poles. Development of the MTOC itself depends solely on the presence of centrioles. When centrioles are missing, the MTOCs disappear, and formation of microtubules is severely impaired.

1	The pericentriolar matrix of MTOC contains nume rous ring-shaped structures that initiate microtubule formation. The MTOC contains centrioles and an amorphous pericentriolar matrix of more than 200 proteins, including tubulin that is organized in ring-shaped structures. Each -tubulin ring serves as the starting point (nucleation site) for the growth of one microtubule that is assembled from tubulin dimers; and -tubulin dimers are added with specific orientation to the -tubulin ring. The minus end of the micro-tubule remains attached to the MTOC, and the plus end

1	TABLE Summary Characteristics of Three Types of Cytoskeletal Elements 2.4 Shape Double-stranded linear Ropelike fibers Nonbranching long, hollow helical array cylinders Diameter 6–8 8–10 20–25 (nm) Basic Monomer of G-actin Various intermediate filament Dimers of and -tubulin protein (MW 42 kDa) proteins (MW ~50 kDa) (MW 54 kDa) -tubulin subunit found in MTOC is necessary for nucleation of microtubules -, -, -, -tubulins are associated with MTOC and basal bodies Enzymatic ATP hydrolytic activity None GTP hydrolytic activity activity Polarity Yes Nonpolar structures Yes Minus ( ) or pointed Minus ( ) end is nongrowing end is slow-growing end end embedded in MTOC Plus ( ) or barbed end Plus ( ) end is the growing end is faster-growing end Assembly Monomers of G-actin are Two pairs of monomers At the nucleation site, and process added to growing flament form two coiled-coil -tubulin dimers are added Polymerization requires dimers; then two coiled-to -tubulin ring in an presence of K , Mg2

1	At the nucleation site, and process added to growing flament form two coiled-coil -tubulin dimers are added Polymerization requires dimers; then two coiled-to -tubulin ring in an presence of K , Mg2 , and coil dimers twist around end-to-end fashion ATP, which is hydrolyzed to each other to generate Each tubulin dimer ADP after each G-actin a staggered tetramer, molecule binds GTP before it molecule is incorporated which aligns along the becomes incorporated into into the flament axis of the flament and the microtubule binds to the free end of Polymerization also requires the elongating structure presence of Mg2 GTP–tubulin complex is polymerized; after incorporation, GTP is hydrolyzed to GDP Source of ATP N/A GTP energy required for assembly Characteristics Thin, fexible flaments Strong, stable structures Exhibit dynamic instability Associated Variety of ABPs with different Intermediate flament– Microtubule-associated proteins functions: fascin bundling; associated proteins: plectins

1	stable structures Exhibit dynamic instability Associated Variety of ABPs with different Intermediate flament– Microtubule-associated proteins functions: fascin bundling; associated proteins: plectins proteins: MAP-1, 2, 3, gelsolin flament severing; bind micro tubules, actin, and 4, MAP-, and TOG CP protein capping; and intermediate flaments; regulate assembly, stabilize, spectrin cross-linking; desmoplakins and plakoglobins and anchor microtubules to myosin I and II motor attach intermediate flaments specifc organelles; motor functions to desmosomes and proteins—dyneins and kinesins— hemidesmosomes required for organelle movement Actin Filaments (Microfilaments) Intermediate Filaments Microtubules chapter 2 Location Core of microvilli Extend across cytoplasm Core of cilia in cell Terminal web connecting desmosomes Emerge from Concentrated beneath and hemidesmosomes MTOC and spread plasma membrane In nucleus just beneath into periphery of Contractile elements inner nuclear membrane

1	web connecting desmosomes Emerge from Concentrated beneath and hemidesmosomes MTOC and spread plasma membrane In nucleus just beneath into periphery of Contractile elements inner nuclear membrane cell of muscles Mitotic spindle, Contractile ring Centrosome in dividing cells Major Provide essential components Provide mechanical strength Provide network functions to contractile elements and resistance to shearing (“railroad tracks”) for of muscle cells (sarcomeres) forces movement of organelles within cell Provide movement for cilia and chromosomes during cell division TABLE Summary Characteristics of Three Types of Cytoskeletal Elements, Cont.2.4 Actin Filaments (Microfilaments) Intermediate Filaments Microtubules

1	FIGURE 2.51 • Structure of the MTOC. This diagram shows the represents the growing end directed toward the plasma membrane (see Fig. 2.51). Centrioles provide basal bodies for cilia and flagella and align the mitotic spindle during cell division. Although centrioles were discovered more than a century ago, their precise functions, replication, and assembly are still under intense investigation. The known functions of centrioles can be organized into two categories:  Basal body formation. One of the important functions of the centriole is to provide basal bodies, which are necessary for the assembly of cilia and flagella (Fig. 2.52).

1	Basal bodies are formed either by de novo formation without contact with the pre-existing centrioles (the acentriolar pathway) or by duplication of existing centrioles (the centriolar pathway). About 95% of the centrioles are generated through the acentriolar pathway. Both pathways give rise to multiple immediate precursors of centrioles, known as procentrioles, which mature as they migrate to the appropriate site near the apical cell membrane, where they become basal bodies (Fig. 2.53). The basal body acts as the organizing center for a cilium. Microtubules grow upward from the basal body, pushing the cell membrane outward, and elongate to form the mature cilium. The process of centriole duplication is described later on page 70.

1	 Mitotic spindle formation. During mitosis, the position location of the MTOC in relation to the nucleus and the Golgi apparatus. In some species, the MTOC is tethered to the nuclear envelope by a contractile protein, the nucleus–basal body connector (NBBC). The MTOC contains the centrioles and an amorphous protein matrix with an abundance of -tubulin rings. Each -tubulin ring serves as the nucleation site for the growth of a single microtubule. Note that the minus (–) end of the microtubule remains attached to the MTOC, and the plus ( ) end represents the growing end directed toward the plasma membrane.

1	of centrioles determines the location of mitotic spindle poles. Centrioles are also necessary for the formation of a fully functional MTOC, which nucleates mitotic spindle– associated microtubules. For instance, astral microtubules are formed around each individual centriole in a starlike fashion. They are crucial in establishing the axis of the developing mitotic spindle. In some animal cells, the mitotic spindle itself (mainly kinetochore microtubules) is  FOLDER 2.2 Clinical Correlation: Abnormalities in Microtubules and Filaments Abnormalities related to the organization and structure of microtubules, actin, and intermediate flaments underlie a variety of pathologic disorders. These abnormalities lead to defects in the cytoskeleton and can produce a variety of defects related to intracellular vesicular transport, intracellular accumulations of pathologic proteins, and impairment of cell mobility.

1	Defects in the organization of microtubules and microtubule-associated proteins can immobilize the cilia of respiratory epithelium, interfering with the ability of the respiratory system to clear accumulated secretions. This condition, known as Kartagener’s syndrome (see page 120), also causes dysfunction of microtubules, which affects sperm motility and leads to male sterility. It may also cause infertility in women because of impaired ciliary transport of the ovum through the oviduct.

1	Microtubules are essential for vesicular transport (endocytosis and exocytosis) as well as cell motility. Certain drugs, such as colchicine, bind to tubulin molecules and prevent their polymerization; this drug is useful in the treatment of acute attacks of gout, to prevent neutrophil migration, and to lower their ability to respond to urate crystal deposits in the tissues. Vinblastine and vincristine (Oncovin) represent another family of drugs that bind to microtubules and inhibit the formation of the mitotic spindle essential for cell division. FIGURE F.2.2.1 • Photomicrograph of Mallory bodies. Ac-These drugs are used as antimitotic and antiproliferative cumulation of keratin intermediate flaments forming intercelluagents in cancer therapy. Another drug, paclitaxel (Taxol), lar inclusions is frequently associated with specifc cell injuries. is used in chemotherapy for breast cancer. It stabilizes mi-In alcoholic liver cirrhosis, hepatocytes exhibit such inclusions crotubules,

1	is frequently associated with specifc cell injuries. is used in chemotherapy for breast cancer. It stabilizes mi-In alcoholic liver cirrhosis, hepatocytes exhibit such inclusions crotubules, preventing them from depolymerizing (an action (arrows), which are known as Mallory bodies. Lymphocytes and opposite to that of colchicine), and thus arrests cancer cells macrophages responsible for an intense infammatory reaction in various stages of cell division. surround cells containing Mallory bodies. 900.

1	Actin flaments are essential for various stages of leukocyte in neuroflaments within brain tissue are characteristic of migration as well as for the phagocytotic functions of vari Alzheimer’s disease, which produces neurofbrillary ous cells. Some chemical substances isolated from fungi, tangles containing neuroflaments and other microtubule such as cytochalasin B and cytochalasin D, prevent associated proteins.

1	actin polymerization by binding to the plus end of the actin Another disorder of the central nervous system,flament inhibiting lymphocyte migration, phagocytosis, and Alexander disease is associated with mutations in the cell division (cytokinesis). Several toxins of poisonous coding region of the GFAP gene. The pathologic feature of mushrooms, such as phalloidin, also bind to actin flathis disease is the presence of cytoplasmic inclusionsments, stabilizing them and preventing their depolymeriza tion. Conjugated with fuorescein dyes, derivatives of the tion of intermediate flament protein GFAP. Altered GFAP phallotoxin family (i.e., NDB-phallacidin) are frequently prevents the assembly not only of intermediate flamentsused in the laboratory to stain actin flaments (see Figs.

1	but also of other proteins that contribute to the structural2.45 and 2.50). Prolonged exposure of the cell to these integrity and function of astrocytes. Infants with Alexander substances can disrupt the dynamic equilibrium between disease develop leukoencephalopathy (infection of theF-actin and G-actin, causing cell death.

1	brain) with macrocephaly (abnormally large head), seizures, and psychomotor retardation, leading to death As noted, the molecular structure of intermediate flaments usually within the frst decade of life. is tissue specifc and consists of many different types of A prominent feature of alcoholic liver cirrhosis is the proteins. Several diseases are caused by defects in the presence of eosinophilic intracytoplasmic inclusions com-proper assembly of intermediate flaments. These defects posed predominantly of keratin intermediate flaments. have also been induced experimentally by mutations in in-These inclusions, called Mallory bodies, are visible in light termediate flament genes in laboratory animals. Changes microscopy within the hepatocyte cytoplasm (Fig. F.2.2.1).

1	FIGURE 2.52 • Basal bodies and cilia. This electron micro-graph shows the basal bodies and cilia in cross-sectional profle as seen in an oblique section through the apical part of a ciliated cell in the respiratory tract. Note the 9 2 microtubule arrangement of the cilia in which nine microtubules at the periphery of the cilia surround two central microtubules. The basal bodies lack the central tubule pair. On several cross sections, the basal foot is visible as it projects laterally from the basal body (asterisks).

1	28,000. (Courtesy of Patrice C. Abell-Aleff.) formed by MTOC-independent mechanisms and consists of microtubules that originate from the chromosomes. Recent experimental data indicate that in the absence of centrioles, astral microtubules fail to develop, causing errors in mitotic spindle orientation (Fig. 2.54). Thus, the primary role of centrioles in mitosis is to position the mitotic spindle properly by recruiting the MTOC from which astral microtubules can grow and establish the axis for the developing spindle. The dominant feature of centrioles is the cylindrical array of triplet microtubules with associated proteins. The TEM reveals that each rod-shaped centriole is about 0.2 m long and consists of nine triplets of microtubules that are oriented parallel to the long axis of the organelle and run in slightly twisted bundles (Fig. 2.55). The three micro-

1	FIGURE 2.53 • Two pathways of basal body formation. In the centriolar pathway, a pair of existing centriole serves as an organizing center for the duplication of new centrioles. Utilizing this pathway, ciliated cells have the ability to assemble large number of centrioles in the vicinity of an old mature centriole. In the acentriolar pathway, which plays a major role in the formation of basal bodies in ciliated cells, new centrioles are formed de novo from fbrous granules located in close proximity of nonmicrotubular structures called deuteresomes. Both pathways give rise to procentrioles, which mature as they migrate to the appropriate site near the apical cell membrane, where they become basal bodies. Fibrous granules contribute to the formation of the striated rootlet. (Based on Hagiwara H, Ohwada N, Takata K. Cell biology of normal and abnormal ciliogenesis in the ciliated epithelium. Int Rev Cytol 2004;234:101–139.) tubules of the triplet are fused, with adjacent microtubules

1	H, Ohwada N, Takata K. Cell biology of normal and abnormal ciliogenesis in the ciliated epithelium. Int Rev Cytol 2004;234:101–139.) tubules of the triplet are fused, with adjacent microtubules sharing a common wall. The innermost or A microtubule is a complete ring of 13 protofilaments containing and tubulin dimers; the middle and outer B and C micro-tubules, respectively, appear C-shaped because they share tubulin dimers with each other and with the A microtubule. The microtubules of the triplets are not equal in length. The C microtubule of the triplet is usually shorter than A and B.

1	The microtubule triplets of the centriole surround an internal lumen. The distal part of the lumen (away from the nucleus) contains a 20-kDa Ca2 -binding protein, centrin (Fig. 2.56). The proximal part of the lumen (close to the nucleus) is lined by -tubulin, which provides the template for the arrangement of the triplet microtubules. In addition, a family of newly discovered -, -, -, and -tubulin molecules as well as pericentrin protein complexes have also been localized with the centrioles. Other proteins, such a protein p210, form a ring of molecules that appears to link the distal end of the centriole to the plasma membrane. Filamentous connections between the centriole pair have been identified in human lymphocytes. In other organisms, two protein bridges, the proximal and distal connecting fbers, connect each centriole in a pair (see Fig. 2.56). In dividing cells, these connections participate in segregating the centrioles to each daughter cell. In some organisms, the proximal

1	fbers, connect each centriole in a pair (see Fig. 2.56). In dividing cells, these connections participate in segregating the centrioles to each daughter cell. In some organisms, the proximal end of

1	FIGURE 2.54 • Mitotic spindle during normal cell division and in cells lacking centrioles. a. This schematic drawing shows the orientation of the mitotic spindle in a normal cell undergoing mitosis. Note the positions of the centrioles and the distribution of the spindle microtubules. b. In a cell that lacks centrioles, mitosis occurs and a mitotic spindle containing only kinetochore micro-tubules is formed. However, both poles of the mitotic spindle lack astral microtubules, which position the spindle in proper plane during the mitosis. Such a misoriented spindle is referred to as an anastral bipolar spindle. (Based on Marshall WF, Rosenbaum JL. How centrioles work: lessons from green yeast. Curr Opin Cell Biol 2000;12:119–125.) each centriole is attached to the nuclear envelope by contractile proteins called nucleus–basal body connectors (NBBCs). Their function is to link the centriole to the mitotic spindle poles during mitosis. In human cells, the centrosome–nucleus connection

1	proteins called nucleus–basal body connectors (NBBCs). Their function is to link the centriole to the mitotic spindle poles during mitosis. In human cells, the centrosome–nucleus connection appears to be maintained by filamentous structures of cytoskeleton. A distinctive feature of mammalian centrioles is the difference between individual centrioles in the pair. One centriole (termed the mature centriole) contains stalklike satellite processes and sheetlike appendages whose function is not known (see Fig. 2.56). The other centriole (termed the immature centriole) does not possess satellites or appendages.

1	Centrosome duplication is synchronized with the cell-cycle events and linked to the process of ciliogenesis. Centrosome dynamics such as duplication or formation of basal bodies for ciliogenesis are synchronized with the cell- FIGURE 2.55 • Electron micrograph showing parent and daughter centrioles in a fbroblast. Note that the transverse-sectioned centriole in each of the pairs reveals the triplet confguration of microtubules. The lower right centriole represents a mid-longitudinal section, whereas the upper left centriole has also been longitudinally sectioned, but along the plane of its wall.

1	90,000. (Courtesy of Drs. Manley McGill, D. P. Highfeld, T. M. Monahan, and Bill R. Brinkley.) cycle progresion. Cilia are assembled during the G1 phase; they are most abundant in GO, and are disassembled before cell enters M phase of the cell cycle. These events are depicted in Figure 2.57, which shows an association between centrosome duplication, primary cilium formation, and progression through the cell cycle. Since each daughter cell receives only one pair of centrioles after cell division, the daughter cells must duplicate existing centrioles prior to cell division. In most somatic cells, duplication of centrioles begins near the transition between the G1 and S phases of the cell cycle. This event is closely associated with the activation of the cyclin E-Cdk2 complex during the S phase of the cell cycle (see Fig. 3.11). This

1	FIGURE 2.56  Schematic structure of centrioles. In nondividing cells, centrioles are arranged in pairs in which one centriole is aligned at a right angle to the other. One centriole is also more mature (generated at least two cell cycles earlier) than the other centriole, which was generated in the previous cell cycle. The mature centriole is characterized by the presence of satellites and appendages. Centrioles are located in close proximity to the nucleus. The basic components of each centriole are microtubule triplets that form the cylindrical structure surrounding an internal lumen. The proximal part of the lumen is lined by -tubulin, which provides the template for nucleation and arrangement of the microtubule triplets. The distal part of each lumen contains the protein centrin. In some species, two protein bridges, the proximal and distal connecting fbers, connect each centriole in a pair. In some species, but not in humans, the proximal end of each centriole is attached to the

1	species, two protein bridges, the proximal and distal connecting fbers, connect each centriole in a pair. In some species, but not in humans, the proximal end of each centriole is attached to the nuclear envelope by a contractile protein known as the nucleus–basal body connector.

1	complex directly phosphorylates the nucleus-chaperoning protein nucleophosmin/B23, which is responsible for initiating the duplication of centrioles.

1	In most cells, duplication begins with the splitting of a centriole pair, followed by the appearance of a small mass of fibrillar and granular material at the proximal lateral end of each original centrioles. Because the existing pair of centrioles serves as a core for new organelle formation, this process of centriole duplication is referred as the centriolar pathway (see Fig. 2.53). The fbrous granules coalesce into dense spherical structures called deuterosomes, and they give rise to the procentriole (or bud), which gradually enlarges to form a right-angle appendage to the parent (see Fig. 2.53). Microtubules begin to develop in the mass of fibrous granules as it grows (usually during the S to late G2 phases of the cell cycle), appearing first as a ring of nine single tubules, then as doublets, and finally as triplets. As procentrioles mature during the S and G2 phases of the cell cycle, each parent–daughter pair migrates around the nucleus. Before the onset of mitosis,

1	then as doublets, and finally as triplets. As procentrioles mature during the S and G2 phases of the cell cycle, each parent–daughter pair migrates around the nucleus. Before the onset of mitosis, centrioles with surrounding amorphous pericentriolar material position themselves on opposite sides of the nucleus and produce astral microtubules. In doing so, they define the poles between which the bipolar mitotic spindle develops.

1	The important difference between duplication of centrioles during mitosis and during ciliogenesis is the fact that during mitosis only one daughter centriole buds from the lateral side of parent organelle, whereas during ciliogenesis as many as ten centrioles may develop around parent centriole. Development of cilia on the cell surface requires the presence of basal bodies, structures derived from centrioles.

1	Each cilium requires a basal body. The generation of centrioles, which occurs during the process of ciliogenesis, is responsible for the production of basal bodies. The newly formed centrioles migrate to the apical surface of the cell and serve as organizing centers for the assembly of the microtubules of the cilium. The core structure (axoneme) of a motile cilium is composed of a complex set of microtubules consisting of two central microtubules surrounded by nine microtubule doublets (9 2 configuration). The organizing role of the basal body differs from that of the MTOC. The axonemal microtubule doublets are continuous with the A and B microtubules of the basal body from which they develop by addition of and -tubulin dimers at the growing plus end. A detail description of the structure of cilia, basal bodies, and the process of ciliogenesis can be found in Chapter 5, Epithelial Tissue.

1	Inclusions contain products of metabolic activity of the cell and consist largely of pigment granules, lipid droplets, and glycogen. Inclusions are cytoplasmic or nuclear structures with characteristic staining properties that are formed from the metabolic products of cell. They are considered nonmoving and nonliving components of the cell. Some of them, such as pigment granules, are surrounded by a plasma membrane; others (e.g., lipid droplets or glycogen) instead reside within the cytoplasmic or nuclear matrix.  Lipofuscin is a brownish-gold pigment visible in routine

1	H&E preparation. It is easily seen in nondividing cells such as neurons and skeletal and cardiac muscle cells. Lipofuscin accumulates during the years in most eukaryotic cells as a result of cellular senescence (aging); thus, it is often called the “wear-and-tear” pigment. Lipofuscin is a conglom erate of oxidized lipids, phospholipids, metals, and organic molecules that accumulate within the cells as a result of oxidative degradation of mitochondria and lysosomal digestion. Phagocytotic cells such as macrophages may also

1	One of the critical components of normal cell division is the precise redistribution of chromosomes and other cell or-ganelles during mitosis. Following replication of chromoso-mal DNA in the S phase of the cell cycle, centrioles undergo a single round of duplication that is closely coor-dinated with cell-cycle progression. During mitosis, centri-oles are responsible for forming the bipolar mitotic spindle, which is essential for equal segregation of chromosomes between daughter cells. Alterations of mechanisms regu-lating centriole duplication may lead to multiplication and abnormalities of centrioles and surrounding centrosomes (MTOCs). Such changes may distort the mitotic spindle (i.e., the presence of multipolar or misoriented spindles) (Fig. F.2.3.1), leading to abnormal sorting of chromosomes during cell divisions. The resulting changes in chromoso-mal number (aneuploidy) may increase the activity of onco-genes or decrease protection from tumor-suppressor genes. These changes

1	chromosomes during cell divisions. The resulting changes in chromoso-mal number (aneuploidy) may increase the activity of onco-genes or decrease protection from tumor-suppressor genes. These changes are known to promote malignant cell transformation. Increased numbers of centrioles are frequently observed in tumor cells. 

1	FOLDER 2.3 Clinical Correlation: Abnormal Duplication of Centrioles and Cancer ababFIGURE F2.3.1 • Multipolar mitotic spindle in a tumor cell. a. Electron micrograph of the invasive breast tumor cell showing abnormal symmetrical tripolar mitotic spindle in the metaphase of cell division. 16,000. b. This drawing composed by color tracings of microtubules (red), mitotic spindle poles (green), and metaphase chromosomes (blue) (obtained from six nonadja-cent serial sections of dividing tumor cell) shows more clearly the organization of this abnormal mitotic spindle. Detailed analy-sis and three-dimensional reconstruction of the spindle revealed that each spindle pole had at least two centrioles and that one spindle pole was composed of two distinct but adjacent foci of microtubules. (Reprinted with permission from Lingle WL. Salisbury JL. Altered centrosome structure is associated with abnormal mitoses in human breast tumors. Am J Path 1999; 155:1941–1951.) contain lipofuscin, which

1	with permission from Lingle WL. Salisbury JL. Altered centrosome structure is associated with abnormal mitoses in human breast tumors. Am J Path 1999; 155:1941–1951.) contain lipofuscin, which accumulates from the digestion of bacteria, foreign particles, dead cells, and their own organelles. Recent experiments indicate that lipofuscin accumulation may be an accurate indicator of cellular stress.

1	 Hemosiderin is an iron-storage complex found within the cytoplasm of many cells. It is most likely formed by the indigestible residues of hemoglobin, and its presence is related to phagocytosis of red blood cells. Hemosiderin is most easily demonstrated in the spleen, where aged erythrocytes are phagocytosed, but it can also be found in alveolar macrophages in the lung tissue, especially after pulmonary infection accompanied by small hemorrhage into the alveoli. It is visible in light microscopy as a deep brown granule, more or less indistinguishable from lipofuscin. Hemosiderin granules can be differentially stained using histochemical methods for iron detection.

1	 Glycogen is a highly branched polymer used as a storage material for glucose. It is not stained in the routine H&E preparation. However, it may be seen in the light microscope with special fixation and staining procedures (such as toluidine blue or the PAS method). Liver and striated muscle cells, which usually contain large amounts of glycogen, may display unstained regions where glycogen is located. Glycogen appears in EM as granules 25 to 30 nm in diameter or as clusters of granules that often occupy significant portions of the cytoplasm (Fig. 2.58).  Lipid inclusions (fat droplets) are usually nutritive inclusions that provide energy for cellular metabolism. The lipid droplets may appear in a cell for a brief time (e.g., in intestinal absorptive cells) or may reside for a long period (e.g., in adipocytes). In adipocytes, lipid inclusions often constitute most of the cytoplasmic vol

1	FIGURE 2.57 • Association of the centrosome duplication and primary cilium formation with the cell cycle. After a cell emerges from mitosis, it possesses a single centrosome (MTOC) surrounded by amorphous pericentriolar material. The primary cilium formation frst occurs during G1 phase in which the centrosome migrates toward the cell membrane and initiates the process of ciliogenesis. Necessary structural and transport proteins are acquired and activated to build primary cilium axoneme (9 0) directly on the top of the mature centriole. During the end of G1 phase, as well as in GO, the primary cilium functions as an external receiver antenna sensing and interpreting signals from the extracellular environment. Duplication of centrioles begins near the transition between the G1 and S phases of the cell cycle, and the two centrioles are visible in S phase. During the late G2 phase, centrioles reach their full maturity, whereas the primary cilium is disassembled. This allows centrioles to

1	of the cell cycle, and the two centrioles are visible in S phase. During the late G2 phase, centrioles reach their full maturity, whereas the primary cilium is disassembled. This allows centrioles to migrate away from the cell membrane and participate in the mitotic spindle formation. Once cell division is complete, the centrioles can proceed to ciliary reassembly in G1 phase. (Based on Santos N, Reiter JF. Building it up and taking it down: the regulation of vertebrate ciliogenesis. Dev Dyn 2008;237:1972–1981.) ume, compressing the other formed organelles into a thin rim at the margin of the cell. Lipid droplets are usually extracted by the organic solvents used to prepare tissues for both light and electron microscopy. What is seen as a fat droplet in light microscopy is actually a hole in the cytoplasm that represents the site from which the lipid was extracted. In individuals with genetic defects of enzymes involved in lipid metabolism, lipid droplets may accumulate in abnormal

1	in the cytoplasm that represents the site from which the lipid was extracted. In individuals with genetic defects of enzymes involved in lipid metabolism, lipid droplets may accumulate in abnormal locations or in abnormal amounts. Such diseases are classified as lipid storage diseases.

1	 Crystalline inclusions contained in certain cells are recognized in the light microscope. In humans, such inclusions are found in the Sertoli (sustentacular) and Leydig (interstitial) cells of the testis. With the TEM, crystalline inclusions have been found in many cell types and in virtually all parts of the cell, including the nucleus and most cytoplasmic organelles. Although some of these inclusions contain viral proteins, storage material, or cellular metabolites, the significance of others is not clear. The cytoplasmic matrix is a concentrated aqueous gel consisting of molecules of different sizes and shapes.

1	The cytoplasmic matrix (ground substance or cytosol) shows little specific structure by light microscopy or conventional TEM and has traditionally been described as a concentrated aqueous solution containing molecules of different size and shape (e.g., electrolytes, metabolites, RNA, and synthesized proteins). In most cells, it is the largest single compartment. The cytoplasmic matrix is the site of physiologic processes that are fundamental to the cell’s existence (protein synthesis, breakdown of nutrients). Studies with high-voltage EM (HVEM) of 0.25to 0.5-m sections reveal a complex three-dimensional structural network of thin microtrabecular strands and cross-linkers. This network provides a structural substratum on which cytoplasmic reactions occur, such as those involving free ribosomes, and along which regulated and directed cytoplasmic transport and movement of organelles occur.

1	FIGURE 2.58 • Electron micrographs of a liver cell with glycogen inclusions. a. Low-magnifcation electron micrograph showing a portion of a hepatocyte with part of the nucleus (N, upper left). Glycogen (G) appears as irregular electron-dense masses. Profles of rough endoplasmic reticulum (rER) and mitochondria (M) are also evident. 10,000. b. This higher-magnifcation EM reveals glycogen (G) as aggregates of small particles. Even the smallest aggregates (arrows) appear to be composed of several smaller glycogen particles. The density of the glycogen is considerably greater than that of the ribosomes (lower left). 52,000. The Cell Nucleus

1	The Cell Nucleus OVERVIEW OF THE NUCLEUS / 75 NUCLEAR COMPONENTS / 75 Chromatin / 75 Nucleolus / 79 Nuclear Envelope / 81 Nucleoplasm / 84 CELL RENEWAL / 84 CELL CYCLE / 86 Phases and Checkpoints Within the Cell Cycle / 86 Regulation of the Cell Cycle / 87 Mitosis / 89 Meiosis / 89 CELL DEATH / 93 Apoptosis / 94 Other Forms of Programmed Cell Death / 95 Folder 3.1 Clinical Correlation: Cytogenetic Testing / 80 Folder 3.2 Clinical Correlation: Regulation of Cell Cycle and Cancer Treatment / 81 The nucleus is a membrane-limited compartment that contains the genome (genetic information) in eukaryotic cells. The nucleus contains genetic information, together with the machinery for DNA replication and RNA transcription and processing. The nucleus of a nondividing cell, also called an interphase cell, consists of the following components.

1	 Chromatin, is nuclear material organized as euchromatin or heterochromatin. It contains DNA associated with roughly an equal mass of various nuclear proteins (e.g., histones) that are necessary for DNA to function.  The nucleolus (pl., nucleoli) is a small area within the nucleus that contains DNA in the form of transcription-ally active ribosomal RNA (rRNA) genes, RNA, and proteins. The nucleolus is the site of rRNA synthesis and contains regulatory cell-cycle proteins.  The nuclear envelope is the membrane system that surrounds the nucleus of the cell. It consists of an inner and an outer membrane separated by a perinuclear cisternal space and perforated by nuclear pores. The outer membrane of the nuclear envelope is continuous with that of the rough-surfaced endoplasmic reticulum (rER) and is often studded with ribosomes.  The nucleoplasm is nuclear content other than the chromatin and nucleolus.

1	 The nucleoplasm is nuclear content other than the chromatin and nucleolus. A simple microscopic evaluation of the nucleus provides a great deal of information about cell well-being. Evaluation of nuclear size, shape, and structure plays an important role in tumor diagnosis. For instance, dying cells have visible nuclear alterations. These include karyolysis, or the disappearance of nuclei due to complete dissolution of DNA by increased activity of DNAase, pyknosis, or condensation of chromatin leading to shrinkage of the nuclei (they appears as dense basophilic masses), and karyorrhexis, or fragmentations of nuclei (these changes usually are proceeded by pyknosis). Chromatin, a complex of DNA and proteins, is responsible for the characteristic basophilia of the nucleus.

1	Chromatin, a complex of DNA and proteins, is responsible for the characteristic basophilia of the nucleus. Each eukaryotic cell contains about 6 billion bits of information encoded in DNA structure, which has a total length of about 1.8 meter. The length of the DNA molecule is 100,000 times longer than the nuclear diameter. Therefore, the DNA must be highly folded and tightly packed in the cell nucleus. This is accomplished by the formation of a unique nucleoprotein complex called chromatin. The chromatin complex consists of DNA and structural proteins. Further folding of chromatin, such as that which occurs during mitosis, produces structures called chromosomes. Each human cell contains 46 chromosomes. Chromatin proteins include five basic proteins called histones along with other nonhistone proteins. A unique feature of chromatin packaging is that it permits the transcriptional machinery to access those regions of the chromosomes that are required for gene expression.

1	Sequencing human genome was successfully completed in 2003.

1	The human genome encompasses the entire length of human DNA that contains the genetic information packaged in all 46 chromosomes. Sequencing of the human genome took about 13 years and was successfully completed in 2003 by the Human Genome Project. The human genome contains a 2.85-billion base pair consensus sequence of nucleotides, which are arranged in about 23,000 protein-coding genes. For years it was thought that genes were usually present in two copies in a genome. However, recent discoveries have revealed that large segments of DNA can vary in numbers of copies. Such copy number variations (CNVs) are widespread in the human genome and most likely lead to genetic imbalances. For instance, genes that were thought to always occur in two copies per genome have sometimes one, three, or more copies. A previous definition of a gene as a segment of DNA involved in producing a polypeptide chain has been recently updated to now read as being a union of genomic sequences encoding a

1	or more copies. A previous definition of a gene as a segment of DNA involved in producing a polypeptide chain has been recently updated to now read as being a union of genomic sequences encoding a coherent set of potentially overlapping functional products.

1	In general, two forms of chromatin are found in the nucleus: A condensed form called heterochromatin and a dispersed form called euchromatin.

1	In most cells, chromatin does not have a homogeneous appearance; rather, clumps of densely staining chromatin are embedded in a more lightly staining background. The densely staining material is highly condensed chromatin called heterochromatin, and the lightly staining material (where most transcribed genes are located) is a dispersed form called euchromatin. It is the phosphate groups of the chromatin DNA that are responsible for the characteristic basophilia of chromatin (page 6). Heterochromatin is disposed in three locations (Fig. 3.1):  Marginal chromatin is found at the periphery of the nu cleus (the structure light microscopists formerly referred to as the nuclear membrane actually consists largely of marginal chromatin).  Karyosomes are discrete bodies of chromatin irregular in size and shape that are found throughout the nucleus.  Nucleolar-associated chromatin is chromatin found in association with the nucleolus.

1	Heterochromatin stains with hematoxylin and basic dyes; it is also readily displayed with the Feulgen procedure (a specific histochemical reaction for the deoxyribose of DNA, page 6) and fluorescent vital dyes such as Hoechst dyes and propidium iodide. It is the heterochromatin that accounts for the conspicuous staining of the nucleus in hematoxylin and eosin (H&E) preparations. Euchromatin is not evident in the light microscope. It is present within the nucleoplasm in the “clear” areas between and around the heterochromatin. In routine electron micro-graphs, there is no sharp delineation between euchromatin and heterochromatin; both have a granular, filamentous appearance, but the euchromatin is less tightly packed.

1	Euchromatin indicates active chromatin—that is, chromatin that is stretched out so that the genetic information in the DNA can be read and transcribed. It is prominent in metabolically active cells such as neurons and liver cells. Heterochromatin predominates in metabolically inactive cells such as small circulating lymphocytes and sperm or in cells that produce one major product such as plasma cells. The smallest units of chromatin structure are macromolecular complexes of DNA and histones called nucleosomes.

1	The smallest units of chromatin structure are macromolecular complexes of DNA and histones called nucleosomes. Nucleosomes are found in both euchromatin and heterochromatin and in chromosomes. These 10-nm-diameter particles represent the first level of chromatin folding and are formed by the coiling of the DNA molecule around a protein core. This step shortens the DNA molecule by approximately sevenfold relative to the unfolded DNA molecule. The core of the nucleosome consists of eight histone molecules (called an octamer). Two loops of DNA (approximately 146 nucleotide pairs) are wrapped around the core octomer. The DNA extends between each particle as a 2-nm filament that joins adjacent nucleosomes. When chromatin is extracted from the nucleus, the nucleosomal substructure of chromatin is visible in transmission electron microscopy (TEM) and is often described as “beads on a string” (Fig. 3.2a).

1	In the next step, a long strand of nucleosomes is coiled to produce a 30-nm chromatin fbril. Six nucleosomes form one turn in the coil of the chromatin fibril, which is approximately 40-fold shorter than unfolded DNA. Long stretches of 30-nm chromatin fibrils are further organized into loop domains (containing 15,000 to 100,000 base pairs), which are anchored into a chromosome scaffold or nuclear matrix composed of nonhistone proteins. In heterochromatin, the chromatin fibers are tightly packed and folded on each other; in euchromatin, the chromatin fibrils are more loosely arranged. In dividing cells, chromatin is condensed and organized into discrete bodies called chromosomes.

1	In dividing cells, chromatin is condensed and organized into discrete bodies called chromosomes. During mitotic division, chromatin fbers formed from chromatin loop domains attached to a flexible protein scaffold undergo condensation to form chromosomes [Gr., colored bodies]. Each chromosome is formed by two chromatids that are joined together at a point called the centromere (Fig. 3.2b). The double nature of the chromosome is produced in the preceding synthetic (S) phase of the cell cycle that maintains telomere length. For example, in cells that (see page 87), during which DNA is replicated in anticipation have been transformed into malignant cells, an enzyme of the next mitotic division. called telomerase is present that adds repeated nucleotide

1	FIGURE 3.1 • Electron micrographs of nuclei from two different cell types. The large electron micrograph shows the nucleus of a nerve cell. Two nucleoli are included in the plane of section. The nucleus of this active cell, exclusive of the nucleoli, comprises almost entirely extended chromatin or euchromatin. 10,000. Inset. The smaller nucleus belongs to a circulating lymphocyte (the entire cell is shown in the micrograph). It is a relatively inactive cell. Note the paucity of cytoplasm and cytoplasmic organelles. The chromatin in the nucleus is largely condensed (heterochromatin). The lighter areas represent euchromatin. 13,000.

1	The area located at each end of the chromosome is called sequences to the telomere ends. Recently, expression of this the telomere. Telomeres shorten with each cell division. Re-enzyme has been shown to extend the lifespan of cells. cent studies indicate that telomere length is an important in-With the exception of the mature gametes, the egg and dicator of the lifespan of the cell. To survive indefinitely sperm, human cells contain 46 chromosomes organized as 23 (become “immortalized”), cells must activate a mechanism homologous pairs (each chromosome in the pair has the chromatin fiber with loops of chromatin fibril

1	FIGURE 3.2 • Packaging of chromatin into the chromosomal structure. a. Sequential steps in the packaging of nuclear chromatin are shown in this diagram, beginning with the DNA double helix and ending with the highly condensed form found in chromosomes. b. Structure of human metaphase chromosome 2 as visible in atomic force microscopic image. 20,000. (Courtesy of Dr. Tatsuo Ushiki.) same shape and size). Twenty-two pairs have identical chromosomes (i.e., each chromosome of the pair contains the same portion of the genome) and are called autosomes. The twenty-third pair of chromosomes are the sex chromosomes, designated X and Y. Females contain two X chromosomes; males contain one X and one Y chromosome. The chromosomal number, 46, is found in most of the somatic cells of the body and is called the diploid (2n) number. To simplify the description of chromosomal number and DNA changes during mitosis and meiosis, we use the lower case letter (n) for chromosome number and lower case letter

1	the diploid (2n) number. To simplify the description of chromosomal number and DNA changes during mitosis and meiosis, we use the lower case letter (n) for chromosome number and lower case letter (d) for DNA content. Diploid chromosomes have the (2d) amount of DNA immediately after cell division but have twice that amount—that is, the (4d) amount of DNA—after the S phase (see page 89).

1	As a result of meiosis, eggs and sperm have only 23 chromosomes, the haploid (1n) number, as well as the haploid (1d) amount of DNA. The somatic chromosome number (2n) and the diploid (2d) amount of DNA are reestablished at fertilization by the fusion of the sperm nucleus with the egg nucleus. In a karyotype, chromosome pairs are sorted according to their size, shape, and emitted fluorescent color.

1	A preparation of chromosomes derived from mechanically ruptured, dividing cells that are then fixed, plated on a microscope slide, and stained is called a metaphase spread. In the past, chromosomes were routinely stained with Giemsa stain; however, with the recent development of in situ hybridization techniques, the fluorescent in situ hybridization (FISH) procedure is now more often used to visualize a chromosomal spread. These spreads are observed with fluorescence microscopes, and computer-controlled cameras are then used to capture images of the chromosome pairs. Image-processing software is used to sort the chromosome pairs according to their morphology to form a karyotype (see Fig. F3.1.1a). A variety of molecular probes that are now commercially available are used in cytogenetic testing to diagnose disorders caused by chromosomal abnormalities such as nondisjunctions, transpositions (see Fig. F3.1.1a), deletions (see Fig. F3.1.1b), and duplications of specifc gene sites.

1	testing to diagnose disorders caused by chromosomal abnormalities such as nondisjunctions, transpositions (see Fig. F3.1.1a), deletions (see Fig. F3.1.1b), and duplications of specifc gene sites. Karyotypes are also used for prenatal determination of sex in fetuses and for prenatal screening for certain genetic diseases (see Fig. 1.7).

1	The Barr body can be used to identify the sex of a fetus. Some chromosomes are repressed in the interphase nucleus and exist only in the tightly packed heterochromatic form. One X chromosome of the female is an example of such a chromosome. This fact can be used to identify the sex of a fetus. This chromosome was discovered in 1949 by Barr and Bartram in nerve cells of female cats, where it appears as a well-stained round body, now called the Barr body, adjacent to the nucleolus.

1	Although the Barr body was originally found in sectioned tissue, it was subsequently shown that any relatively large number of cells prepared as a smear (e.g., scrapings of the oral mucous membrane from the inside of the cheeks or neutrophils from a blood smear) can be used to search for the Barr body. In cells of the oral mucous membrane, the Barr body is located adjacent to the nuclear envelope. In neutrophils, the Barr body forms a drumstick-shaped appendage on one of the nuclear lobes (Fig. 3.3). In both sections and smears, many cells must be examined to find those whose orientation is suitable for the display of the Barr body. FIGURE 3.3 • Photomicrograph of a neutrophil from a female patient’s blood smear. The second X chromosome of the female patient is repressed in the interphase nucleus and can be demonstrated in the neutrophil as a drumstick-appearing appendage (arrow) on a nuclear lobe. 250.

1	The nucleolus is the site of ribosomal RNA (rRNA) synthesis and initial ribosomal assembly. The nucleolus is a nonmembranous region of the nucleus that surrounds transcriptionally active rRNA genes. It is the primary site of ribosomal production and assembly. The nucleolus varies in size but is particularly well developed in cells active in protein synthesis. Some cells contain more than one nucleolus (Fig. 3.4). The nucleolus has three morphologically distinct regions:  Fibrillar centers contain DNA loops of five different chromosomes (13, 14, 15, 21, and 22) that contain rRNA genes, RNA polymerase I, and transcription factors.  Fibrillar material (pars fbrosa) contain ribosomal genes that are actively undergoing transcription and large amounts of rRNA.  Granular material (pars granulosa) represents the site of initial ribosomal assembly and contains densely packed preribosomal particles.

1	 Granular material (pars granulosa) represents the site of initial ribosomal assembly and contains densely packed preribosomal particles. The network formed by the granular and the fibrillar materials is called the nucleolonema. rRNA is present in both granular and fibrillar material and is organized, respectively, as both granules and extremely fine filaments packed tightly together. Genes for the ribosomal subunits are localized in FIGURE 3.4 • Electron micrograph of the nucleolus. This nucleolus from a nerve cell shows fibrillar centers (FC) surrounded by the fibrillar (F) and granular (G) materials. Such a network of both materials is referred to as the nucleolonema. The rRNA, DNA-containing genes for the rRNA, and specific proteins are localized in the interstices of the nucleolonema. 15,000.

1	the interstices of this network and are transcribed by RNA polymerase I. After further processing and modification of rRNA by small nucleolar RNAs (snoRNAs), the subunits of rRNA are assembled using ribosomal proteins imported from the cytoplasm. The partially assembled ribosomal subunits (preribosomes) are exported from the nucleus via nuclear pores for full assembly into mature ribosomes in the cytoplasm. The nucleolus is involved in regulation of the cell cycle.

1	The nucleolus is involved in regulation of the cell cycle. Nucleostemin is a newly identified protein that has been found within the nucleolus. Nucleostemin is a p53 binding protein that regulates the cell cycle and influences cell differentiation (page 88). As cellular differentiation progresses, the level of this protein decreases. The presence of nucleostemin in malignant cells suggests that it could play a role in their uncontrolled proliferation (Folder 3.2). In addition, DNA, RNA, and retroviruses and their viral proteins interact with the nucleolus and cause redistribution of fbrillar and granular materials during the course of viral infection. These viruses can use components of the nucleolus as part of their own replication process. Evidence suggests that viruses may target the nucleolus and its components to favor viral transcription and translation and perhaps alter the cell cycle to promote viral replication.

1	The nucleolus stains intensely with hematoxylin and basic dyes and metachromatically with thionine dyes. That the basophilia and metachromasia of the nucleolus are related to the phosphate groups of the nucleolar RNA is confirmed by predigestion of specimens with ribonuclease (RNAse), which abolishes the staining. As mentioned above, DNA is present in the nucleolus; however, its concentration is below the detection capability of the Feulgen reaction. Thus, when examined in the light microscope, nucleoli

1	Cytogenetic testing is an important component in the di-agnosis and evaluation of genetic disorders and refers to the analysis of chromosomes. Chromosome abnormalities occur in approximately 0.5% of all live births and are de-tected in about 50% of the first trimester miscarriages (spontaneous abortions) and about 95% of various tumor cells. Chromosome analysis can be performed on periph-eral blood, bone marrow, tissues (such as skin or chorionic villi obtained from biopsies), and cells obtained from amni-otic fluid during amniocentesis. Studies of chromosomes begin with the extraction of whole chromosomes from the nuclei of dividing cells. These chromosomes are then placed on glass slides, hybridized with special fluorescence probes (FISH tech-nique), and examined under a microscope. A single fluo-rescent DNA probe produces a bright microscopic signal when the probe is hybridized to a specific part of a partic-ular chromosome. To obtain an image of all of the chromo-somes, a mixture

1	fluo-rescent DNA probe produces a bright microscopic signal when the probe is hybridized to a specific part of a partic-ular chromosome. To obtain an image of all of the chromo-somes, a mixture of different probes is used to produce dif-ferent colors in each chromosome. Karyotypes labeled by this method allow cytogeneticists to perform a comprehen-sive analysis of changes in the number of chromosomes and chromosomal abnormalities such as additions or dele-tions. The paired chromosomes are numbered in the kary-otype, and the male sex is indicated by the presence of chromosomes X and Y (see Fig. F3.1.1a). The white box inset in Figure F3.1.1a shows the XX chromosome pair as it appears in the female. Sometimes, part of a chromosome will break off and at-tach to another chromosome. When this happens, it is re-ferred to as a translocation. Note that the red box inset in Figure F3.1.1a shows a translocation between chromo-some 8 and 14 (t8;14). It is clearly visible on this color image that

1	it is re-ferred to as a translocation. Note that the red box inset in Figure F3.1.1a shows a translocation between chromo-some 8 and 14 (t8;14). It is clearly visible on this color image that a part of the original chromosome 8 (aqua blue region) is now attached to chromosome 14, and a small portion of chromosome 14 (red region) is now part of 

1	FOLDER 3.1 Clinical Correlation: Cytogenetic Testing FIGURE F3.1.1 • Karyotypes obtained with the FISH technique. a. Karyotype of a normal male. The white box inset shows the XX chromosome pair of a normal female. The red box inset reveals an abnormality in chromosomes 14 and 8. (Courtesy of the Applied Imaging International Ltd., Newcastle upon Tyne, UK.) b. A metaphase spread from a patient with Prader-Willi/Angelman syndrome. The yellow box inset shows the enlarged pair of chromosome 15. (Courtesy of Dr. Robert B. Jenkins.) 1 6 7 8 8 9 10 11 12 13 19 20 21 22 X XX Y 14 14 15 16 17 18 2 3 4 5 15 baba appear Feulgen-negative with Feulgen-positive nucleolus-associated chromatin that often rims the nucleolus. The nuclear envelope, formed by two membranes with a perinuclear cisternal space between them, separates the nucleoplasm from the cytoplasm.

1	The nuclear envelope provides a selectively permeable membranous barrier between the nuclear compartment and the cytoplasm, and it encloses the chromatin. The nuclear envelope is assembled from two (outer and inner) nuclear membranes with a perinuclear cisternal space between them. The peri nuclear clear cisternal space is continuous with the cisternal space of the rER (Fig. 3.5). The two membranes of the envelope are perforated at intervals by nuclear pores that mediate the active transport of proteins, ribonucleoproteins, and RNAs between the nucleus and cytoplasm. The membranes of the nuclear envelope differ in structure and functions:  The outer nuclear membrane closely resembles the membrane of the endoplasmic reticulum and in fact is continuous with rER membrane (see Fig 3.5). Polyribosomes are often attached to ribosomal docking proteins present on the cytoplasmic side of the outer nuclear membrane.

1	 The inner nuclear membrane is supported by a rigid network of intermediate protein filaments attached to its inner surface called the nuclear (fbrous) lamina. In addition, the inner nuclear membrane contains specific lamin receptors and several lamina-associated proteins that bind to chromosomes and secure the attachment of the nuclear lamina. The nuclear lamina is formed by intermediate filaments and lies adjacent to the inner nuclear membrane. The nuclear lamina, a thin, electron-dense intermediate filament networklike layer, resides underneath the nuclear FOLDER 3.2 Clinical Correlation: Regulation of Cell Cycle and Cancer Treatment

1	Understanding the details of cell-cycle regulation has had an impact on cancer research and has contributed to the development of new treatments. For instance, inactiva-tion of tumor-suppressor genes has been shown to play a role in the growth and division of cancer cells. The pro-teins encoded by these genes are used by the cell throughout several DNA-damage checkpoints. For in-stance, mutations in the breast cancer susceptibility gene 1 (BRCA-1) and breast cancer susceptibility gene 2 (BRCA-2) are associated with an increased risk for bilateral breast cancer. Both protein products of these tumor-suppressor genes–namely, BRCA-1 and BRCA-2 proteins–are directly involved in multiple cellular pro-cesses in response to DNA damage, including checkpoint activation, gene transcription, and repair of DNA double-strand breaks. Together with RAD-51 protein, which is involved in the homologous recombination and repair of DNA, they maintain stability of the human genome. The defective BRCA

1	repair of DNA double-strand breaks. Together with RAD-51 protein, which is involved in the homologous recombination and repair of DNA, they maintain stability of the human genome. The defective BRCA proteins are unable to interact with RAD-51. By screening patients for mutations in these genes, much earlier detection of cancer can be accomplished. It is also now known why in some individuals p53 mu-tations make their tumors resistant to radiotherapy. DNA damage caused by therapeutic radiation procedures is de-tected by DNA-damage checkpoints, which cause cancer cells to be arrested in the cell cycle. However, these cells will not die because of the absence of functional p53, which triggers apoptosis.

1	The Cell Nucleus NUCLEAR COMPONENTS

1	FIGURE 3.5 • Structure of the nuclear envelope and its relationship to the rER. a. The nuclear wall consists of a double membrane envelope that surrounds the nucleus. The outer membrane is continuous with the membranes of the rER; thus, the perinuclear space communicates with the rER lumen. The inner membrane is adjacent to nuclear intermediate filaments that form the nuclear lamina. b. This electron micrograph, prepared by the quick-freeze deep-etch technique, shows the nucleus, the large spherical object, surrounded by the nuclear envelope. Note that the outer membrane possesses ribosomes and is continuous with the rER. 12,000. (Courtesy of Dr. John E. Heuser, Washington University School of Medicine.) membrane. It addition to its supporting or “nucleoskeletal” function, nuclear lamina is essential in many nuclear activities such as DNA replication, transcription, and gene regulation. If the membranous component of the nuclear envelope is disrupted by exposure to detergent, the

1	is essential in many nuclear activities such as DNA replication, transcription, and gene regulation. If the membranous component of the nuclear envelope is disrupted by exposure to detergent, the nuclear lamina remains, and the nucleus retains its shape.

1	The major components of the lamina, as determined by biochemical isolation, are nuclear lamins, a specialized type of nuclear intermediate filament (see page 63), and laminassociated proteins. Nuclear lamina is essentially composed of lamin A and lamin C proteins that form intermediate filaments. These filaments are cross-linked into an orthogonal lattice (Fig. 3.6), which is attached mainly via lamin B protein to the inner nuclear membrane through its interactions with lamin receptors. The family of lamin receptors includes emerin (34 kilodaltons) that binds both lamin A and B, nurim (29 kilodaltons) that binds lamin A, and a 58-kilodalton lamin B receptor (LBR) that, as its name suggests, binds lamin B.

1	Unlike other cytoplasmic intermediate filaments, lamins disassemble during mitosis and reassemble when mitosis ends. The nuclear lamina appears to serve as scaffolding for chromatin, chromatin-associated proteins, nuclear pores, and the membranes of the nuclear envelope. In addition, it is involved in nuclear organization, cell-cycle regulation, differentiation, and gene expression.

1	Impairment in nuclear lamina architecture or function is associated with certain genetic diseases (laminopathies) and apoptosis. Mutations in lamin A/C cause tissue-specifc diseases that affect striated muscle, adipose tissue, peripheral nerve or skeletal development, and premature aging. Recently, two hereditary forms of Emery-Dreifuss muscular dystrophy (EDMD) have been associated with mutations in either lamins or lamin receptors. The X-linked recessive form of EDMD is caused by mutations of emerin, while the autosomal dominant form of EDMD is caused by mutations in lamin A/C. In general, EDMD is characterized by an early-onset contractures of major tendons, very slow progressive muscle weakness, muscle wasting in the upper and lower limbs, and cardiomyopathy (weakening of the heart muscle). The nuclear envelope has an array of openings called nuclear pores.

1	The nuclear envelope has an array of openings called nuclear pores. At numerous sites, the paired membranes of the nuclear envelope are punctuated by 70to 80-nm “openings” through the envelope. These nuclear pores are formed from the merging of the inner and outer membranes of the nuclear envelope. With an ordinary TEM, a diaphragmlike structure appears to cross the pore opening (Fig. 3.7). Often a small dense body is observed in the center of the opening (Fig. 3.8). Since such profiles are thought to represent either ribosomes or other protein complexes (transporters) captured during their passage through the pore at the time of fixation, the term central plug/transporter is commonly used to describe this feature. With special techniques—such as negative staining and high-voltage transmission electron microscopy, or recently,

1	FIGURE 3.6 • Structure of the nuclear lamina. a. This schematic drawing shows the structure of the nuclear lamina adjacent to the inner nuclear membrane. The cut window in the nuclear lamina shows the DNA within the nucleus. Note that the nuclear envelope is pierced by nuclear pore complexes, which allow for selective bidirectional transport of molecules between nucleus and cytoplasm. b. Electron micrograph of a portion of the nuclear lamina from a Xenopus oocyte. It is formed by intermediate filaments (lamins) that are arranged in a square lattice. 43,000. (Adapted from Aebi U, Cohn J, Buhle L, Gerace L. The nuclear lamina is a meshwork of intermediate-type filaments. Nature 1986;323:560–564.) nuclear lamina DNAnucleus reticulum perinuclear space nuclear pore complex a bb cryoelectron tomography—the nuclear pore exhibits additional structural detail (see Fig. 3.8). Eight multidomain protein subunits arranged in an octagonal central framework at the periphery of each pore form a

1	tomography—the nuclear pore exhibits additional structural detail (see Fig. 3.8). Eight multidomain protein subunits arranged in an octagonal central framework at the periphery of each pore form a cylinderlike structure known as the nuclear pore complex (NPC). The NPC, which has an estimated total mass of 125 106 daltons, is composed of about 50 different nuclear pore complex proteins collectively referred to as nucleoporins (Nup proteins). This central framework is inserted between the cytoplasmic ring and the nuclear ring (Fig. 3.9). From the cytoplasmic ring, eight short protein fbrils protrude into the cytoplasm and point toward the center of the structure. The nucleoplasmic ring complex anchors a nuclear basket (or nuclear “cage” that resembles a fish trap) assembled from eight thin 50-nm-long filaments joined distally by an adjustable terminal ring 30 to 50 nm in diameter (see Fig. 3.9). The cylinder-shaped central framework encircles the central pore of the NPC, which acts as

1	50-nm-long filaments joined distally by an adjustable terminal ring 30 to 50 nm in diameter (see Fig. 3.9). The cylinder-shaped central framework encircles the central pore of the NPC, which acts as a close-fitting diaphragm or gated channel. In addition, each NPC contains one or more water-filled channels for transport of small molecules.

1	The NPC mediates bidirectional nucleocytoplasmic transport.

1	Various experiments have shown that the NPC regulates the passage of proteins between the nucleus and the cytoplasm. The significance of the NPC can be readily appreciated, as the nucleus does not carry out protein synthesis. Ribosomal proteins are partially assembled into ribosomal subunits in the nucleolus and are transported through nuclear pores to the cytoplasm. Conversely, nuclear proteins, such as histones and lamins, are produced in the cytoplasm and are transported through nuclear pores into the nucleus. Transport through the NPC largely depends on the size of the molecules:  Large molecules (such as large proteins and macromolecular complexes) depend on the presence of an attached signal sequence called the nuclear localization signal (NLS) for passage through the pores. Labeled NLS proteins destined for the nucleus then bind to a soluble cytosolic receptor called a nuclear import receptor (importin) that directs them from the cytoplasm to an appropriate NPC.

1	FIGURE 3.7 • Electron micrograph of the nuclear envelope. Note the visible nuclear pore complexes (arrows) and the two membranes that constitute the nuclear envelope. At the periphery of each pore, the outer and inner membranes of the nuclear envelope appear continuous. 30,000. They are then actively transported through the pore by a GTP energy-dependent mechanism. An export of proteins and RNA from the nucleus is similar to the import mechanism into the nucleus. Proteins that possess the nuclear export sequence (NES) bind in the nucleus to exportin (a protein that moves molecules from nucleus into cytoplasm) and to a GTP molecule. Protein–exportin–GTP complexes pass through NPC into the cytoplasm where GTP is hydrolyzed and the NES protein is released. The NPC transports proteins, all forms of RNA, as well as ribosomal subunits in their fully folded configurations.

1	 Ions and smaller water-soluble molecules (less than 9 daltons) may cross the water-flled channels of the NPC by simple diffusion. This process is nonspecific and does not require nuclear signal proteins. The effective size of the pore is about 9 nm for substances that cross by diffusion rather than the 70to 80-nm measurement of the pore boundary. However, even the smaller nuclear proteins that are capable of diffusion are selectively transported, presumably because the rate is faster than simple diffusion. During cell division, the nuclear envelope is disassembled to allow chromosome separation and is later reassembled as the daughter cells form.

1	During cell division, the nuclear envelope is disassembled to allow chromosome separation and is later reassembled as the daughter cells form. In late prophase of cell division, enzymes (kinases) are activated that cause phosphorylation of the nuclear lamins and other lamina-associated proteins of the nuclear envelope. After phosphorylation, the proteins become soluble, and the nuclear envelope disassembles. The lipid component of the nuclear membranes then disassociates from the proteins and is retained in small cytoplasmic vesicles. The replicated chromosomes then attach to the microtubules of the mitotic spindle and undergo active movement.

1	Reassembly of the nuclear envelope begins in late anaphase, when phosphatases are activated to remove the phosphate residues from the nuclear lamins. During telophase, the nuclear lamins begin to repolymerize and form the nuclear lamina material around each set of daughter chromosomes. At the same time, vesicles containing the lipid components of the nuclear membranes and structural membrane protein components fuse, and an envelope is formed on the surface of the already-reassembled nuclear lamina. By the end of telophase, formation of a nuclear envelope in each daughter cell is complete. Nucleoplasm is the material enclosed by the nuclear envelope exclusive of the chromatin and the nucleolus.

1	Nucleoplasm is the material enclosed by the nuclear envelope exclusive of the chromatin and the nucleolus. Although crystalline, viral, and other inclusions are sometimes found in the nucleoplasm, until recently, morphologic techniques showed it to be amorphous. It must be assumed, however, that many proteins and other metabolites reside in or pass through the nucleus in relation to the synthetic and metabolic activity of the chromatin and nucleolus. New structures have recently been identified within the nucleoplasm, including intranuclear lamin-based arrays, the protein filaments emanating inward from the nuclear pore complexes, and the active gene-tethered RNA transcription and processing machinery itself. Somatic cells in the adult organism may be classified according to their mitotic activity.

1	Somatic cells in the adult organism may be classified according to their mitotic activity. The level of mitotic activity in a cell can be assessed by the number of mitotic metaphases visible in a single high-magnification light microscopic field or by autoradiographic studies of the incorporation of tritiated thymidine into the newly synthesized DNA before mitosis. Using these methods, cell populations may be classified as static, stable, or renewing. FIGURE 3.8 • Cryoelectron tomography of the nuclear pore complex. These surface renderings of electron tomograms obtained from the frozen-hydrated Dictyostelium nuclei show detailed structure of the nuclear pore complex (NPC). 320,000.

1	a. Cytoplasmic face of the NPC shows eight protein fibrils arranged around the central channel. They protrude from the cytoplasmic ring subunits and point toward the center of the structure. Note a presence of the central plug or transporter within the central pore, which represents either ribosomes or other protein transporters captured during their passage through the NPC. b. Nuclear face of the NPC shows the nucleoplasmic ring subunits connected by nuclear filaments with the basket indicated in brown color. (Adapted from Beck M, Fster F, Ecke M, Plitzko JM, Melchior F, Gerisch G, Baumeister W, Medalia, O. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 2004;306:1387–1390.)

1	FIGURE 3.9 • Sagittal section of the nuclear pore complex. Cryoelectron tomographic view of a sagittal section of the nuclear pore complex shown in Figure 3.8 is compared with a schematic drawing of the complex. Note that the central plug/transporter has been removed from the central pore. 320,000. Each pore contains eight protein subunits arranged in an octagonal central framework at the periphery of the pore. These subunits form a nuclear pore complex that is inserted between two cytoplasmic and nucleoplasmic rings. Eight short protein fibrils protrude from the cytoplasmic rings into the cytoplasm. The nuclear ring anchors a basket assembled from eight thin filaments joined distally into terminal ring which diameter can be adjusted to meet nuclear pore transport requirements. The cylindrical central framework encircles the central pore, which acts as a close-fitting diaphragm. (Adapted from Beck M, Frster F, Ecke M, Plitzko JM, Melchior F, Gerisch G, Baumeister W, Medalia, O.

1	cylindrical central framework encircles the central pore, which acts as a close-fitting diaphragm. (Adapted from Beck M, Frster F, Ecke M, Plitzko JM, Melchior F, Gerisch G, Baumeister W, Medalia, O. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 2004;306:1387–1390.)  Static cell populations consist of cells that no longer divide (postmitotic cells), such as cells of the central nervous system and skeletal or cardiac muscle cells. Under certain circumstances some of these cells (i.e., cardiac myocytes) may enter mitotic division.

1	 Stable cell populations consist of cells that divide episodically and slowly to maintain normal tissue or organ structure. These cells may be stimulated by injury to become more mitotically active. Periosteal and perichondrial cells, smooth muscle cells, endothelial cells of blood vessels, and fibroblasts of the connective tissue may be included in this category.  Renewing cell populations may be slowly or rapidly renewing but display regular mitotic activity. Division of such cells usually results in two daughter cells that differentiate both morphologically and functionally or two cells that remain as stem cells. Daughter cells may divide one or more times before their mature state is reached. The differentiated cell may ultimately be lost from the body.

1	 Slowly renewing populations include smooth muscle cells of most hollow organs, fibroblasts of the uterine wall, and epithelial cells of the lens of the eye. Slowly renewing populations may actually slowly increase in size during life, as do the smooth muscle cells of the gastrointestinal tract and the epithelial cells of the lens.  Rapidly renewing populations include blood cells, epithelial cells and dermal fibroblasts of the skin, and the epithelial cells and subepithelial fibroblasts of the mucosal lining of the alimentary tract. Phases and Checkpoints Within the Cell Cycle The cell cycle represents a self-regulated sequence of events that controls cell growth and cell division.

1	For renewing cell populations and growing cell populations, including embryonic cells, and cells in tissue culture, the goal of the cell cycle is to produce two daughter cells, each containing chromosomes identical to those of the parental cell. The cell cycle incorporates two principal phases: the interphase, representing continuous growth of the cell, and the M phase (mitosis), characterized by the partition of the genome. Three other phases, G1 (gap1) phase, S (synthesis) phase, and G2 (gap2) phase, further subdivide interphase (Fig. 3.10). Rapidly renewing populations of human cells progress through the full cell cycle in about 24 hours. Throughout the cycle, several internal quality-control mechanisms or checkpoints represented by biochemical pathways control transition between cell-cycle stages. The cell cycle stops at several checkpoints and can only proceed if certain conditions are met—for example, if the cell has reached a certain size. Checkpoints monitor and modulate the

1	stages. The cell cycle stops at several checkpoints and can only proceed if certain conditions are met—for example, if the cell has reached a certain size. Checkpoints monitor and modulate the progression of cells through the cell cycle in response to intracellular or environmental signals.

1	The G1 phase is usually the longest and the most variable phase of the cell cycle, and it begins at the end of M phase. During the G1 phase, the cell gathers nutrients and synthesizes RNA and proteins necessary for DNA synthesis and 912 h 7.510 h 3.54.5 h 1 h G1 GO GTD S G2 M G1 DNA-damage checkpoint G2 DNA-damage checkpoint S DNA-damage checkpoint unreplicated DNA checkpoint checkpoint checkpoint restriction checkpoint

1	FIGURE 3.10 • Cell cycle and checkpoints. This diagram illustrates the cell cycle of rapidly dividing cells in relation to DNA synthesis. After mitosis, the cell is in interphase. G1 represents the period during which a gap occurs in DNA synthesis. S represents the period during which DNA synthesis occurs. G2 represents a second gap in DNA synthesis. GO represents the path of a cell that has stopped dividing; however, such a cell may reenter the cell cycle after an appropriate stimulus. The cell residing in GO may undergo terminal differentiation, GTD, and produce a population of permanent nondividing cells (e.g., mature fat cells). The average timing of each phase of the cell cycle is indicated on the diagram. Each phase contains several checkpoints that ensure that the system only proceeds to the next stage when the previous stage has been completed and no damage to the DNA is detected.

1	chromosome replication. The cell’s progress through this phase is monitored by two checkpoints: (1) the restriction point, which is sensitive to the size of the cell, the state of the cell’s physiologic processes, and its interactions with extracellular matrix; and (2) the G1 DNA-damage checkpoint, which monitors the integrity of newly replicated DNA. For instance, if the DNA has irreparable damage, then the G1 DNA-damage checkpoint detects the high levels of tumor-suppressing protein p53 and it does not allow the cell to enter the S phase. The cell will then most likely undergo programmed cell death (apoptosis).

1	The restriction point (or “point of no return”) is the most important checkpoint in the cell cycle. At this checkpoint, the cell self-evaluates its own replicative potential before deciding to either enter the S phase and the next round of cell division or to retire and leave the cell cycle. A cell that leaves the cycle in the G1 phase usually begins terminal differentiation by entering the GO phase (“O” stands for “outside” the cycle). Thus, the G1 phase may last for only a few hours (average 9 to 12 hours) in a rapidly dividing cell, or it may last a lifetime in a nondividing cell. This checkpoint is mediated by interactions between the retinoblastoma susceptibility protein (pRb) and a family of essential transcription factors (E2F) with target promoters. In normal cells, proper interaction between pRb and E2F turns off many genes and blocks cell-cycle progression. In the S phase, DNA is replicated.

1	In the S phase, DNA is replicated. Initiation of DNA synthesis marks the beginning of the S phase, which is about 7.5 to 10 hours in duration. The DNA of the cell is doubled during the S phase, and new chromatids are formed that will become obvious at prophase or metaphase of the mitotic division. Chromosome replication is initiated at many different sites called replicons along the chromosomal DNA. Each replicon has a specifically assigned time frame for replication during S phase. Presence of the S DNA-damage checkpoint in this phase monitors quality of replicating DNA. In the G2 phase, the cell prepares for cell division.

1	In the G2 phase, the cell prepares for cell division. During this phase, the cell examines its replicated DNA in preparation for cell division. This is a period of cell growth and reorganization of cytoplasmic organelles before entering the mitotic cycle. The G2 phase may be as short as 1 hour in rapidly dividing cells or of nearly indefinite duration in some polyploid cells and in cells such as the primary oocyte that are arrested in G2 for extended periods. Two checkpoints monitor DNA quality: the G2 DNA-damage checkpoint and the unreplicated-DNA checkpoint. The latter checkpoint prevents the progression of the cell into the M phase before DNA synthesis is complete. Mitosis occurs in the M phase.

1	Mitosis occurs in the M phase. Mitosis nearly always includes both karyokinesis (division of the nucleus) and cytokinesis (division of the cell) and lasts about 1 hour. Mitosis takes place in several stages described in more detail below. Separation of two identical daughter cells concludes the M phase. The M phase possesses two checkpoints: the spindle-assembly checkpoint, which prevents premature entry into anaphase, and the chromosome-segregation checkpoint, which prevents the process of cytokinesis until all of the chromosomes have been correctly separated. The mitotic catastrophe caused by malfunction of cell-cycle checkpoints may lead to cell death and tumor cell development.

1	The mitotic catastrophe caused by malfunction of cell-cycle checkpoints may lead to cell death and tumor cell development. Malfunction of any of the three DNA-damage checkpoints at the G1, S, and G2 phases of the cell cycle and the spindle-assembly checkpoint at M phase may lead to a mitotic catastrophe. Mitotic catastrophe is defined as the failure to arrest the cell cycle before or at mitosis, resulting in aberrant chromosome segregation. Under normal conditions, death in these cells will occur by activation of the apoptotic cycle. Cells that fail to execute the apoptotic cycle in response to DNA or mitotic spindle damage are likely to divide asymmetrically in the next round of cell division. This leads to the generation of aneuploid cells (cells containing abnormal chromosome numbers). Thus, a mitotic catastrophe may be regarded as one of the mechanisms contributing to oncogenesis (tumor cell development).

1	Malfunction of the restriction checkpoint at the G1 phase may also result in malignant transformation of cells. Malignant cells lose contact inhibition, a normal process in which cells inhibit their division when they contact other cells. Malignant cells in culture continue to divide and may grow on top of one another rather then discontinuing growth when the plate is fully covered in a monolayer of cells. The malfunction of the restriction checkpoint may be facilitated by the viral proteins of several cancer-causing viruses, such as the T-antigen of simian virus (SV40) that binds to pRb. This binding alters the configuration of the pRb–T-antigen complex and renders the restriction checkpoint inoperable, thus facilitating the cell’s progression from the G1 to S phase of the cell cycle. This mechanism of carcinogenesis occurs in mesothelioma (cancer of the lining epithelium of the pleural cavities in the thorax), osteosarcoma (a type of bone cancer), and ependymoma (a type of childhood

1	mechanism of carcinogenesis occurs in mesothelioma (cancer of the lining epithelium of the pleural cavities in the thorax), osteosarcoma (a type of bone cancer), and ependymoma (a type of childhood brain tumor).

1	The reserve stem cell population may become activated and reenter the cell cycle. Cells identified as reserve stem cells may be thought of as GO cells that may be induced to reenter the cell cycle in response to injury of cells within the tissues of the body. Activation of these cells may occur in normal wound healing and in repopulation of the seminiferous epithelium after intense acute exposure of the testis to X-irradiation or during regeneration of an organ, such as the liver, after removal of a major portion. If damage is too severe, even the reserve stem cells die, and there is no potential for regeneration. Regulation of the Cell Cycle Passage through the cell cycle is driven by proteins that are cyclically synthesized and degraded during each cycle. A number of cytoplasmic protein complexes regulate and control the cell cycle. Some of these proteins function as

1	TABLE Functional Summary of Cyclin–Cdk Complexes Used in Regulating the Human Cell Cycle3.1 Associated Cyclin-Cyclin Type Dependent Protein Kinase Targeted Phase of Cell Cycle Targeted Effector Proteins Cyclin D Cdk4/6 G1 phase progression Tumor-suppressing protein p53, retinoblastoma susceptibility protein (pRb) Cyclin E Cdk2 S phase entry ATMA or ATRB protein kinases, tumor-suppressing protein p53 Cyclin A Cdk2 S phase progression Replication protein A, DNA polymerase, minichromosome maintenance (Mcm) protein Cyclin A Cdk1 S phase through G2 phase Cdc25 phosphatase, cyclin B and M phase entry Cyclin E Cdk1 M phase progression Chromatin-associated proteins, histone H1, nuclear lamins, myosin regulatory proteins, centrosomal proteins, transcription factors c-fos/jun, c-myb, oct-1, SWI5; p60src protein kinases, casein kinase II, c-mos protein kinases A Ataxia–telangiectasia mutated protein kinase B ATM—and Rad3—related kinase biochemical oscillators, whose synthesis and degradation are

1	protein kinases, casein kinase II, c-mos protein kinases A Ataxia–telangiectasia mutated protein kinase B ATM—and Rad3—related kinase biochemical oscillators, whose synthesis and degradation are coordinated with specific phases of the cycle. Cellular and molecular events induced during the increase and decrease of different protein levels are the basis of the cell-cycle “engine.” Other proteins actively monitor the quality of the molecular processes at the different checkpoints distributed throughout the cycle (described above). The protein complexes at the checkpoints may drive the cell into and out of the cell cycle, stimulating growth and division when conditions are favorable and, conversely, stopping or reducing the rate of cell division when conditions are not favorable.

1	A two-protein complex consisting of cyclin and a cyclindependent kinase (Cdk) helps power the cells through the checkpoints of cell-cycle division. The first milestone in understanding the regulation of the cell cycle was the discovery in the early 1970s of a protein called maturation promoting factor (MPF). MPF appeared to control the initiation of mitosis. When injected into the nuclei of immature frog oocytes, which are normally arrested in G2, the cells immediately proceeded through mitosis. MPF was eventually found to consist of two proteins:  Cdc2 (also known as Cdk-1), a 32-kilodalton member of the Cdk family of proteins; and  Cyclin B, a 45-kilodalton member of the cyclin family, which are key regulators of the cell cycle. Cyclins are synthe sized as constitutive proteins; however, their levels during the cell cycle are controlled by ubiquitin-mediated degradation.

1	It is now known that the cyclin–Cdk complex acts at different phases of the cell cycle and targets different proteins to control cell-cycle–dependent functions. Table 3.1 shows the combination of the different types of cyclins with different types of Cdks and how interactions between these two proteins affect cells progressing through the cell cycle. Passage through the cell cycle requires an increase in cyclin–Cdk activity in some phases followed by the decline of that activity in other phases (Fig. 3.11). The increased activity of FIGURE 3.11 • Regulation of the cell cycle by cyclin–Cdk complexes. This diagram shows the changing pattern of cyclin–Cdk activities during different phases of the cell cycle. cyclin–Cdk is achieved by the stimulatory action of cyclins and is counterbalanced by the inhibitory action of proteins such as Inks (inhibitors of kinase), Cips (Cdk inhibitory proteins), and Kips (kinase inhibitory proteins).

1	Cell division is a crucial process that increases the number of cells, permits renewal of cell populations, and allows wound repair. Mitosis is a process of chromosome segregation and nuclear division followed by cell division that produces two daughter cells with the same chromosome number and DNA content as the parent cell. The term mitosis is used to describe the equal partitioning of replicated chromosomes and their genes into two identical groups. The process of cell division includes division of both the nucleus (karyokinesis) and the cytoplasm (cytokinesis). The process of cytokinesis results in distribution of nonnuclear organelles into two daughter cells. Before entering mitosis, cells duplicate their DNA. This phase of the cell cycle is called the S or synthesis phase. At the beginning of this phase, the chromosome number is (2n), and the DNA content is also (2d); at the end, the chromosome number remains the same (2n), and the DNA content doubles to (4d).

1	Mitosis follows the S phase of the cell cycle and is described in four phases.

1	Mitosis consists of four phases (Fig. 3.12):  Prophase begins as the replicated chromosomes condense and become visible. As the chromosomes continue to condense, each of the four chromosomes derived from each homologous pair can be seen to consist of two chromatids. The sister chromatids are held together by the ring of proteins called cohesins and the centromere. In late prophase or prometaphase (sometimes identified as a separate phase of mitosis), the nuclear envelope begins to disintegrate into small transport vesicles and resembles the sER. The nucleolus, which may still be present in some cells, also completely disappears in prometaphase. In addition, a highly specialized protein complex called a kinetochore appears on each chromatid opposite to the centromere (Fig. 3.13). The protein complexes that form kinetochores in the centromere region of chromatid are attached to specific repetitive DNA sequences known as satellite DNA that are similar in each chromosome. Microtubules of

1	complexes that form kinetochores in the centromere region of chromatid are attached to specific repetitive DNA sequences known as satellite DNA that are similar in each chromosome. Microtubules of the developing mitotic spindle attach to the kinetochores and thus to the chromosomes.  Metaphase (Fig. 3.14) begins as the mitotic spindle, consisting of three types of microtubules, becomes organized around the microtubule-organization centers (MTOCs) located at opposite poles of the cell. The first type, the astral microtubules, are nucleated from the -tubulin rings in a starlike fashion around each MTOC (see Fig. 2.54). The second type, the polar micro-tubules, also originates from the MTOC; however, these microtubules grow away from the MTOC. The third type, the kinetochore microtubules, emanates from the MTOC to probe the cytoplasm in search of kinetochores. When a kinetochore is finally captured by a kinetochore microtubule, it is pulled toward the MTOC, where additional microtubules

1	from the MTOC to probe the cytoplasm in search of kinetochores. When a kinetochore is finally captured by a kinetochore microtubule, it is pulled toward the MTOC, where additional microtubules will attach. The kinetochore is capable of binding between 30 and 40 microtubules to each chromatid. In some species, kinetochore microtubules are formed by MTOCindependent mechanisms that involve kinetochores. Kinetochore microtubules and their associated motor proteins direct the movement of the chromosomes to a plane in the middle of the cell, the equatorial or metaphase plate.

1	 Anaphase (Fig. 3.15) begins at the initial separation of sister chromatids. This separation occurs when the cohesins that have been holding the chromatids together break down. The chromatids then begin to separate and are pulled to opposite poles of the cell by the molecular motors (dyneins) sliding along the kinetochore micro-tubules toward the MTOC.

1	 Telophase (Fig. 3.16) is marked by the reconstitution of a nuclear envelope around the chromosomes at each pole. The chromosomes uncoil and become indistinct except at regions that will remain condensed in the interphase nucleus. The nucleoli reappear, and the cytoplasm divides (cytokinesis) to form two daughter cells. Cytokinesis begins with the furrowing of the plasma membrane midway between the poles of the mitotic spindle. The separation at the cleavage furrow is achieved by a contractile ring consisting of a very thin array of actin filaments positioned around the perimeter of the cell. Within the ring, myosin II molecules are assembled into small filaments that interact with the actin filaments, causing the ring to contract. As the ring tightens, the cell is pinched into two daughter cells. Because the chromosomes in the daughter cells contain identical copies of the duplicated DNA, the daughter cells are genetically identical and contain the same kind and number of

1	two daughter cells. Because the chromosomes in the daughter cells contain identical copies of the duplicated DNA, the daughter cells are genetically identical and contain the same kind and number of chromosomes. The daughter cells are (2d) in DNA content and (2n) in chromosome number.

1	Meiosis involves two sequential nuclear divisions followed by cell divisions that produce gametes containing half the number of chromosomes and half the DNA found in somatic cells.

1	The zygote (the cell resulting from the fusion of an ovum and a sperm) and all the somatic cells derived from it are diploid (2n) in chromosome number; thus, their cells have two copies of every chromosome and every gene encoded on this chromosome. These chromosomes are called homologous chromosomes because they are similar but not identical; one set of chromosomes is of maternal origin, the other is from the male parent. The gametes, having only one member of each chromosome pair, are described as haploid (1n). During gametogenesis, reduction in chromosome number to the haploid state (23 chromosomes in humans) occurs through metaphase I continuation of continuation of meiosis as seen meiosis as seen metaphase in spermatogenesis in oogenesis (undergo apoptosis) spermatozoa *note: prophase II, anaphase II, and telophase II are not shown ovum

1	FIGURE 3.12 • Comparison of mitosis and meiosis in an idealized cell with two pairs of chromosomes (2n). The chromosomes of maternal and paternal origin are depicted in red and blue, respectively. The mitotic division produces daughter cells that are genetically identical to the parental cell (2n). The meiotic division, which has two components, a reductional division and an equatorial division, produces a cell that has only two chromosomes (1n). In addition, during the chromosome pairing in prophase I of meiosis, chromosome segments are exchanged, leading to further genetic diversity. It should be noted that in humans the first polar body does not divide. Division of the first polar body does occur in some species. FIGURE 3.13 • Atomic force microscopic image of the centromeric region of a human metaphase chromosome.

1	FIGURE 3.13 • Atomic force microscopic image of the centromeric region of a human metaphase chromosome. The facing surfaces of two sister chromatids visible on this image form the centromere, a point of junction of both chromatids. On the opposite side from the centromere, each chromatid possesses a specialized protein complex, the kinetochore, which serves as an attachment point for kinetochore microtubules of the mitotic spindle. Note that the surface of the chromosome has several protruding loop domains formed by chromatin fibrils anchored into the chromosome scaffold. 40,000. (Courtesy of Dr. Tatsuo Ushiki.) meiosis, a process that involves two successive divisions, the second of which is not preceded by an S phase. This reduction is necessary to maintain a constant number of chromosomes in a given species. Reduction in chromosome number to (1n) in the first meiotic division is followed by reduction in DNA content to the haploid (1d) amount in the second meiotic division.

1	FIGURE 3.15 • Mitotic spindle in anaphase. This immunofluorescent image comes from the same cell type and identical preparation as in Figure 3.13. Connections that hold the sister chromatids together break at this stage. The chromatids are then moved to opposite poles of the cell by microtubule-associated molecular motors (dyneins and kinesins) that slide along the kinetochore microtubules toward the centriole and are also pushed by the polar microtubules (visible between the separated chromosomes) away from each other, thus moving opposite poles of the mitotic spindle into the separate cells. 1,400. (Courtesy of Dr. Thomas U. Mayer.)

1	FIGURE 3.14 • Mitotic spindle in metaphase. Using indirect immunofluorescence techniques, the mitotic spindle in a Xenopus XL-177 cell was labeled with an antibody against -tubulin conjugated with fluorescein (green). DNA was stained blue with fluorescent DAPI stain. In metaphase, the nuclear membrane disassembles, DNA is condensed into chromosomes, and microtubules form a mitotic spindle. The action of microtubuleassociated motor proteins on the microtubules of the mitotic spindle creates the metaphase plate along which the chromosomes align in the center of the cell. 1,400. (Courtesy of Dr. Thomas U. Mayer.) During meiosis, the chromosome pair may exchange chromosome segments, thus altering the genetic composition of the chromosomes. This genetic exchange, called crossing-over, and the random assortment of each member of the chromosome pairs into haploid gametes give rise to infinite genetic diversity.

1	FIGURE 3.16 • Mitotic spindle in telophase. In this phase, DNA is segregated and a nuclear envelope is reconstituted around the chromosomes at each pole of the mitotic spindle. The cell divides into two during cytokinesis. In the middle of the cell, actin, septins, myosins, microtubules, and other proteins gather as the cell establishes a ring of proteins that will constrict, forming a bridge between the two sides of what was once one cell. The chromosomes uncoil and become indistinct except at regions that remain condensed in interphase. The cell types and preparation are the same as those in Figures 3.13 and 3.14. 1,400. (Courtesy of Dr. Thomas U. Mayer.) The cytoplasmic events associated with meiosis differ in the male and female.

1	The cytoplasmic events associated with meiosis differ in the male and female. The nuclear events of meiosis are the same in males and females, but the cytoplasmic events are markedly different. Figure 3.12 illustrates the key nuclear and cytoplasmic events of meiosis as they occur in spermatogenesis and oogenesis. The events of meiosis through metaphase I are the same in both sexes. Therefore, the figure illustrates the differences in the process as they diverge after metaphase I. In males, the two meiotic divisions of a primary spermatocyte yield four structurally identical, although genetically unique, haploid spermatids. Each spermatid has the capacity to differentiate into a spermatozoon. In contrast, in females, the two meiotic divisions of a primary oocyte yield one haploid ovum and three haploid polar bodies. The ovum receives most of the cytoplasm and becomes the functional gamete. The polar bodies receive very little cytoplasm and degenerate.

1	The nuclear events of meiosis are similar in males and females. Meiosis consists of two successive mitotic divisions without the additional S phase between the two divisions. During the S phase that precedes meiosis, DNA is replicated forming sister chromatids (two parallel strands of DNA) joined together by the centromere. The DNA content becomes (4d), but the chromosome number remains the same (2n). The cells then undergo a reductional division (meiosis I) and an equatorial division (meiosis II).

1	During meiosis I, as the name reductional division implies, the chromosome number is reduced from diploid (2n) to haploid (1n), and the amount of DNA is reduced from the (4d) to (2d). During prophase I, double-stranded chromosomes condense, and homologous chromosomes (normally one inherited from the mother and one from the father) are paired at centromeres. At this point, recombination of genetic material between the maternal and paternal chromosome pairs may occur. In metaphase I, the homologous chromosomes with their centromeres line up along the equator of the mitotic spindle and in anaphase I are separated and distributed to each daughter cell. This results in reduction of both the chromosome number (1n) and the DNA to the (2d) amount.

1	No DNA replication precedes meiosis II. The division during meiosis II is always equatorial because the number of chromosomes does not change. It remains at (1n), although the amount of DNA represented by the number of chromatids is reduced to (1d). During metaphase II, each chromosome aligns along the equator of the mitotic spindle, and at anaphase II sister chromatids are separated from each other. Thus, each chromosome splits into two single-stranded chromosomes that are then distributed to each haploid daughter cell. Phases in the process of meiosis are similar to the phases of mitosis. The prophase of meiosis I is an extended phase in which pairing of homologous chromosomes, synapsis (close association of homologous chromosomes), and recombination of genetic material on homologous chromosomes is observed. Prophase I is subdivided into the following five stages (see Fig. 3.12).

1	 Leptotene. This stage is characterized by the condensation of chromatin and by the appearance of chromosomes. Sister chromatids also condense and become connected with each other by meiosisspecifc cohesion complexes (Rec8p). At this phase, pairing of homologous chromosomes of maternal and paternal origin is initiated. Homologous pairing can be described as a process in which chromosomes actively search for each other. After finding their mates, they align themselves side by side with a slight space separating them.

1	 Zygotene. Synapsis, the close association of homologous chromosomes, begins at this stage and continues throughout pachytene. This process involves the formation of a synaptonemal complex, a tripartite structure that binds the chromosomes together. The synaptonemal complex is often compared to railroad tracks with an additional third rail positioned in the middle between two others. The cross ties in this track are represented by the transverse filaments that bind the scaffold material of both homologous chromosomes together.  Pachytene. At this stage, synapsis is complete. Crossing-over occurs early in this phase and involves transposition of DNA strands between two different chromosomes.

1	 Pachytene. At this stage, synapsis is complete. Crossing-over occurs early in this phase and involves transposition of DNA strands between two different chromosomes.  Diplotene. Early in this stage, the synaptonemal complex dissolves, and the chromosomes condense further. Homologous chromosomes begin to separate from each other and appear to be connected by newly formed junctions between chromosomes called chiasmata (sing., chiasma). Sister chromatids still remain closely associated with each other. Chiasmata indicate that crossing-over may have occurred.  Diakinesis. The homologous chromosomes condense and shorten to reach their maximum thickness, the nucleolus disappears, and the nuclear envelope disintegrates.

1	 Diakinesis. The homologous chromosomes condense and shorten to reach their maximum thickness, the nucleolus disappears, and the nuclear envelope disintegrates. Metaphase I is similar to the metaphase of mitosis except that the paired chromosomes are aligned at the equatorial plate with one member on either side. The homologous chromosomes are still held together by chiasmata. At late metaphase, chiasmata are cleaved and the chromosomes separate. Once the nuclear envelope has broken down, the spindle micro-tubules begin to interact with the chromosomes through the multilayered protein structure, the kinetochore, which is usually positioned near the centromere (see Fig. 3.13). The chromosomes undergo movement to ultimately align their centromeres along the equator of the spindle.

1	Anaphase I and telophase I are similar to the same phases in mitosis except that the centromeres do not split. The sister chromatids, held together by cohesin complexes and by the centromere, remain together. A maternal or paternal member of each homologous pair, now containing exchanged segments, moves to each pole. Segregation or random assortment occurs because the maternal and paternal chromosomes of each pair are randomly aligned on one side or the other of the metaphase plate, thus contributing to genetic diversity. At the completion of meiosis I, the cytoplasm divides. Each resulting daughter cell (a secondary spermatocyte or oocyte) is haploid in chromosome number (1n) and contains one member of each homologous chromosome pair. The cell is still diploid in DNA content (2d).

1	After meiosis I, the cells quickly enter meiosis II without passing through an S phase. Meiosis II is an equatorial division and resembles mitosis. During this phase, the proteinase enzyme separase cleaves the cohesion complexes between the sister chromatids. Cleavage of the cohesin complexes in the region of the centromere releases the bond between both centromeres. This cleavage allows the sister chromatids to separate at anaphase II and move to opposite poles of the cell. During meiosis II, the cells pass through prophase II, metaphase II, anaphase II, and telophase II. These stages are essentially the same as those in mitosis except that they involve a haploid set of chromosomes (1n) and produce daughter cells that have only haploid DNA content (1d). Unlike the cells produced by mitosis, which are genetically identical to the parent cell, the cells produced by meiosis are genetically unique.

1	In humans, as in all other multicellular organisms, the rates of cell proliferation and cell death determine the net cell production. An abnormality in any of these rates can cause disorders of cell accumulation (e.g., hyperplasia, cancer, autoimmune diseases) or disorders of cell loss (atrophy, degenerative diseases, AIDS, ischemic injury). Therefore, the balance (homeostasis) between cell production and cell death must be carefully maintained (Fig. 3.17). Cell death may occur as a result of acute cell injury or an internally encoded suicide program. Cell death may result from accidental cell injury or mechanisms that cause cells to self-destruct. The major two different mechanisms of cell death are necrosis and apoptosis.

1	Cell death may result from accidental cell injury or mechanisms that cause cells to self-destruct. The major two different mechanisms of cell death are necrosis and apoptosis.  Necrosis, or accidental cell death, is a pathologic process. It occurs when cells are exposed to an unfavorable physical or chemical environment (e.g., hypothermia, hypoxia, radiation, low pH, cell trauma) that causes acute cellular injury and damage to the plasma membrane. Under physiologic conditions, damage to the plasma membrane may also be initiated by viruses, or proteins called perforins. Rapid cell swelling and lysis are two characteristic features of this process.

1	 Apoptosis [Gr., falling off, as petals from flowers] was referred to in the past as programmed cell death. Today the term programmed cell death is applied more broadly to any kind of cell death mediated by an intracellular death program, irrespective of the trigger mechanism. Apoptosis represents a physiologic process. During apoptosis, cells that are no longer needed are eliminated from the organism. This process may occur during normal embryologic development or other normal physiologic processes, such as follicular atresia in the ovaries. Cells can initiate their own death through activation of an internally encoded suicide program. Apoptosis is characterized by controlled autodigestion, which maintains cell membrane integrity; thus, the cell “dies with dignity” without spilling its contents and damaging its neighbors.

1	FIGURE 3.17 • Schematic diagram showing the relationship between cell death and cell division. Under normal physiologic conditions (homeostasis), the rates of cell division and cell death are similar. If the rate of cell death is higher than that of cell division, then a net loss of cell number will occur. Such conditions are categorized as cell loss disorders. When the situation is reversed and the rate of cell division is higher than the rate of cell death, then the net gain in cell number will be prominent, leading to a variety of disorders of cell accumulation. TABLE Overview of Characteristic Features Distinguishing Necrosis from Apoptosis 3.2 Features of Dying Cells Necrosis Apoptosis Cell swelling —Cell shrinkage —Damage to the plasma membrane —Plasma membrane blebbing —Aggregation of chromatin —Fragmentation of the nucleus —Oligonucleosomal DNA fragmentation —Random DNA degradation —Caspase cascade activation —

1	In addition, certain cells or their secretions found in the immune system are toxic to other cells (e.g., cytotoxic T lymphocytes, natural killer [NK] cells); they initiate processes that destroy designated cells (e.g., cancer-transformed or virus-infected cells). In contrast to necrosis and apoptosis, cytotoxic death does not involve one specifc mechanism. For example, cell death mediated by cytotoxic T lymphocytes combines some aspects of both necrosis and apoptosis. For an overview of the apoptosis and necrosis, see Table 3.2. Necrosis begins with impairment of the cell’s ability to maintain homeostasis.

1	Necrosis begins with impairment of the cell’s ability to maintain homeostasis. As a result of cell injury, damage to the cell membrane leads to an influx of water and extracellular ions. Intracellular organelles such as the mitochondria, rER, and nucleus undergo irreversible changes that are caused by cell swelling and cell membrane rupture (cell lysis). As a result of the ultimate breakdown of the plasma membrane, the cytoplasmic contents, including lysosomal enzymes, are released into the extracellular space. Therefore, necrotic cell death is often associated with extensive surrounding tissue damage and an intense infammatory response (Fig. 3.18). Apoptosis is a mode of cell death that occurs under normal physiologic conditions.

1	Apoptosis is a mode of cell death that occurs under normal physiologic conditions. In apoptosis, the cell is an active participant in its own demise (“cellular suicide”). This process is activated by a variety of extrinsic and intrinsic signals. Cells undergoing apoptosis show the following characteristic morphologic and biochemical features (see Fig. 3.18);  DNA fragmentation occurs in the nucleus and is an irre versible event that commits the cell to die. DNA fragmen tation is a result of Ca2-dependent and Mg2-dependent activation of nuclear endonucleases. These enzymes selec tively cleave DNA, generating small oligonucleosomal fragments. Nuclear chromatin then aggregates, and the nucleus may divide into several discrete fragments bounded by the nuclear envelope.

1	 Decrease in cell volume is achieved by shrinking of the cytoplasm. The cytoskeletal elements become reorganized in bundles parallel to the cell surface. Ribosomes become clumped within the cytoplasm, the rER forms a series of concentric whorls, and most of the endocytotic vesicles fuse with the plasma membrane.

1	 Loss of mitochondrial function is caused by changes in the permeability of the mitochondrial membrane channels. The integrity of the mitochondrion is breached, the mitochondrial transmembrane potential drops, and the electron-transport chain is disrupted. Proteins from the mitochondrial intermembrane space, such as cytochrome c, are released into the cytoplasm to activate a cascade of proteolytic enzymes called caspases that are responsible for dismantling the cell. The regulated release of cytochrome c suggests that mitochondria, under the influence of Bcl-2 proteins (see page 95), are the decision makers for initiating apoptosis. Thus, many researchers view mitochondria either as the “headquarters for the leader of a crack suicide squad” or as a “high-security prison for the leaders of a military coup.”  Membrane blebbing results from cell membrane alterations. One alteration is related to translocation of certain molecules (e.g., phosphatidylserine) from the cytoplasmic surface

1	a military coup.”  Membrane blebbing results from cell membrane alterations. One alteration is related to translocation of certain molecules (e.g., phosphatidylserine) from the cytoplasmic surface to the outer surface of the plasma membrane. These changes cause the plasma membrane to change its physical and chemical properties and lead to blebbing without loss of membrane integrity (see Fig. 3.18).

1	 Formation of apoptotic bodies, the final step of apoptosis, results in cell breakage (Fig. 3.19a, b, and c). These membrane-bounded vesicles originate from the cytoplasmic bleb containing organelles and nuclear material. They are rapidly removed without a trace by phagocytotic cells. The removal of apoptotic bodies is so efficient that no inflammatory response is elicited. Apoptosis occurs more than 20 times faster than mitosis; therefore, it is challenging to find apoptotic cells in a routine H&E preparation (Fig. 3.19d). Apoptosis is regulated by external and internal stimuli.

1	Apoptotic processes can be activated by a variety of external and internal stimuli. Some factors, such as tumor necrosis factor (TNF), acting on cell membrane receptors, trigger apoptosis by recruiting and activating the caspase cascade. Consequently, the TNF receptor is known as the “death receptor.” Other external activators of apoptosis include transforming growth factor (TGF-), certain neurotransmitters, free radicals, oxidants, and UV and ionizing radiation. Internal activators of apoptosis include oncogenes (e.g., myc and rel ), tumor suppressors such as p53, and nutrient-deprivation antimetabolites (Fig. 3.20). Apoptotic pathways are also activated by the events leading to mitotic catastrophe—namely, malfunction of specific DNA-damage checkpoints in the cell cycle (see page 87). Mitotic catastrophe is accompanied by chromatin condensation, mitochondrial release of cytochrome c, activation of the caspase cascade, and DNA fragmentation.

1	Apoptosis can also be inhibited by signals from other cells and the surrounding environment via so-called survival factors. These include growth factors, hormones such as estrogen and androgens, neutral amino acids, zinc, and interactions with extracellular matrix proteins. Several cellular and viral proteins act as caspase inhibitors; for instance, nerve cells contain neuronal apoptosis inhibitory protein (NAIP) to protect them from premature apoptosis. However, the most important regulatory function in apoptosis is ascribed to internal signals from the Bcl-2 family of proteins. Members of this family consist of antiapoptotic and proapoptotic members that determine the life or death of a cell. These proteins interact with each other to suppress or propagate their own activity by acting on the downstream activation of various executional steps of apoptosis. They also act independently on mitochondria to regulate the release of cytochrome c, the most potent apoptosis-inducing agent.

1	Other Forms of Programmed Cell Death Several forms of programmed cell death were recently identified that differ from apoptosis or necrosis. There are several identified different forms of programmed cell death that do not fit into the classical apoptosis or necrosis scheme. They include the following.

1	There are several identified different forms of programmed cell death that do not fit into the classical apoptosis or necrosis scheme. They include the following. FIGURE 3.18 • Schematic diagram of changes occurring in necrosis and apoptosis. This diagram shows the major steps in necrosis and apoptosis. In necrosis (left side), breakdown of the cell membrane results in an influx of water and extracellular ions, causing the organelles to undergo irreversible changes. Lysosomal enzymes are released into the extracellular space, causing damage to neighboring tissue and an intense inflammatory response. In apoptosis (right side), the cell shows characteristic morphologic and biochemical features such as DNA fragmentation, decrease in cell volume, membrane blebbing without loss of membrane integrity, and formation of apoptotic bodies, causing cell breakage. Apoptotic bodies are later removed by phagocytotic cells without inflammatory reactions.

1	 Autophagy is a regulated cellular process that enables cells to turn over their contents by lysosomal degradation of their own components. It starts when an intracellular membrane (often part of sER cistern) wraps around an or-ganelle or portion of cytoplasm, forming a closed double membrane–bound vacuole. This vacuole, called an

1	FIGURE 3.19 • Electron micrographs of apoptotic cells. a. This electron micrograph shows an early stage of apoptosis in a lymphocyte. The nucleus is already fragmented, and the irreversible process of DNA fragmentation is turned on. Note the regions containing condensed heterochromatin adjacent to the nuclear envelope. 5,200. b. Further fragmentation of DNA. The heterochromatin in one of the nuclear fragments (left) begins to bud outward through the envelope, initiating a new round of nuclear fragmentation. Note the reorganization of the cytoplasm and budding of the cytoplasm to produce apoptotic bodies. 5,200. c. Apoptotic bodies containing fragments of the nucleus, organelles, and cytoplasm. These bodies will eventually be phagocytosed by cells from the mononuclear phagocytotic system. 5,200. (Courtesy of Dr. Scott H. Kaufmann, Mayo Clinic.) d. This photomicrograph taken with light microscopy of intestinal epithelium from the human colon shows apoptotic bodies (AB) within a single

1	5,200. (Courtesy of Dr. Scott H. Kaufmann, Mayo Clinic.) d. This photomicrograph taken with light microscopy of intestinal epithelium from the human colon shows apoptotic bodies (AB) within a single layer of absorptive cells. BM, basement membrane. 750.

1	autophagosome, initially devoid of any lysosomal enzymes, fuses with lysosomes and initiates digestion. For a detailed description of three pathways utilized in autophagy, see pages 43 to 44.  Mitotic catastrophe is a type of cell death that occurs during mitosis. It results from a combination of cellular damage and malfunction of several cell-cycle checkpoints such as the G1, S, and G2 DNA-damage checkpoints or the spindle-assembly checkpoint (page 87). Failure to arrest the cell cycle before mitosis occurs causes problems with chromosome separation, which triggers the apoptotic pathway and cell death.

1	 Paraptosis is an alternative, nonapoptotic cell death that may be induced by growth factor receptors (i.e., insulin growth factor [IGF-1] receptor). In contrast to apoptosis, cell death is not mediated by caspases but by mitogenactivated protein kinases (MAPKs). On a cellular level, paraptosis is characterized by the formation of multiple large vacuoles within the cell cytoplasm along with mitochondrial swellings.  Pyroptosis is a form of cell death induced by infection with certain microorganisms that generate intense inflammatory reactions. This pathway is uniquely dependent on the caspase-1 enzyme, which is not involved in caspase cascade in apoptotic cell death. Caspase-1 activates the inflammatory cytokines such as IL-1 and IL-18 that mediate intense inflammatory reactions in surrounding tissue.

1	 Necroptosis is a regulated caspase-independent cell death mechanism that can be induced in different cell types. It is initiated by the activation of the tumor necrosis factor receptors (TNFRs or death receptors) and Fas signaling pathway. Although it occurs under regulated conditions, necroptotic cell death is characterized by the same morphologic features as unregulated necrotic death. Necrostatin-1 is a specific inhibitor of necroptosis that significantly reduces ischemic damage in affected tissues. FIGURE 3.20 • Schematic drawing of mechanisms leading to apoptosis. Both external and internal stimuli can trigger apoptosis by activating the enzymatic caspase cascade. Many external activators act on the cell to initiate signals leading to apoptosis; note that TNF and TGFact through a “death receptor.” Controlled release of cytochrome c from mitochondria is an important internal step in the activation of apoptosis.

1	Microscopic studies of dying cells in the tissue reveals that different forms of cell death can occur simultaneously and that dying cells can share features of different types of cell death. Tissues: Concept and Classification OVERVIEW OF TISSUES / 98 EPITHELIUM / 99 CONNECTIVE TISSUE / 99 MUSCLE TISSUE / 100 NERVE TISSUE / 101 HISTOGENESIS OF TISSUES / 102 Ectodermal Derivatives / 102 Mesodermal Derivatives / 102 Endodermal Derivatives / 102 IDENTIFYING TISSUES / 102 Folder 4.1 Clinical Correlation: Ovarian Teratomas / 103 Tissues are aggregates or groups of cells organized to perform one or more specific functions.

1	Tissues are aggregates or groups of cells organized to perform one or more specific functions. At the light microscope level, the cells and extracellular components of the various organs of the body exhibit a recognizable and often distinctive pattern of organization. This organized arrangement reflects the cooperative effort of cells performing a particular function. Therefore, an organized aggregation of cells that function in a collective manner is called a tissue [Fr. tissu, woven; L. texo, to weave].

1	Although it is frequently said that the cell is the basic functional unit of the body, it is really the tissues, through the collaborative efforts of their individual cells, that are responsible for maintaining body functions. Cells within tissues communicate through specialized intercellular junctions (gap junctions, page 131), thus facilitating this collaborative effort and allowing the cells to operate as a functional unit. Other mechanisms that permit the cells of a given tissue to function in a unified manner include specific membrane receptors and anchoring junctions between cells. Despite their disparate structure and physiologic properties, all organs are made up of only four basic tissue types.

1	Despite their disparate structure and physiologic properties, all organs are made up of only four basic tissue types. The tissue concept provides a basis for understanding and recognizing the many cell types within the body and how they interrelate. Despite the variations in general appearance, structural organization, and physiologic properties of the various body organs, the tissues that compose them are classified into four basic types.  Epithelium (epithelial tissue) covers body surfaces, lines body cavities, and forms glands. Connective tissue underlies or supports the other three basic tissues, both structurally and functionally.  Muscle tissue is made up of contractile cells and is responsible for movement.  Nerve tissue receives, transmits, and integrates infor mation from outside and inside the body to control the activities of the body.

1	 Nerve tissue receives, transmits, and integrates infor mation from outside and inside the body to control the activities of the body. Each basic tissue is defined by a set of general morphologic characteristics or functional properties. Each type may be further subdivided according to specific characteristics of its various cell populations and any special extracellular substances that may be present.

1	In classifying the basic tissues, two different definitional parameters are used. The basis for definition of epithelium and connective tissue is primarily morphologic; for muscle and nerve tissue, it is primarily functional. Moreover, the same parameters exist in designating the tissue subclasses. For example, whereas muscle tissue itself is defined by its function, it is subclassified into smooth and striated categories, a purely morphologic distinction, not a functional one. Another kind of contractile tissue, myoepithelium, functions as muscle tissue but is typically designated epithelium because of its location. For these reasons, tissue classification cannot be reduced to a simple formula. Rather, students are advised to learn the features or characteristics of the different cell aggregations that define the four basic tissues and their subclasses. Epithelium is characterized by close cell apposition and presence at a free surface.

1	Epithelium is characterized by close cell apposition and presence at a free surface. Epithelial cells, whether arranged in a single layer or in multiple layers, are always contiguous with one another. In addition, they are usually joined by specialized cell-to-cell junctions that create a barrier between the free surface and the adjacent connective tissue. The intercellular space between epithelial cells is minimal and devoid of any structure except where junctional attachments are present. Free surfaces are characteristic of the exterior of the body, the outer surface of many internal organs, and the lining of the body cavities, tubes, and ducts, both those that ultimately communicate with the exterior of the body and those that are enclosed. The enclosed body cavities and tubes include the pleural, pericardial, and peritoneal cavities as well as the cardiovascular system. All of these are lined by epithelium.

1	Subclassifications of epithelium are usually based on the shape of the cells and the number of cell layers rather than on function. Cell shapes include squamous (flattened), cuboidal, and columnar. Layers are described as simple (single layer) or stratified (multiple layers). Figure 4.1 shows epithelia from two sites. Both are simple epithelia (i.e., one cell layer thick). The major distinction between the two examples is the shape of the cells, cuboidal versus columnar. In both epithelia, however, the cells occupy a surface position. Connective tissue is characterized on the basis of its extracellular matrix.

1	Connective tissue is characterized on the basis of its extracellular matrix. Unlike epithelial cells, connective tissue cells are conspicuously separated from one another. The intervening spaces are occupied by material produced by the cells. This extracellular material is called the extracellular matrix. The nature of the cells and matrix varies according to the function of the tissue. Thus, subclassification of connective tissue takes into account not only the cells but also the composition and organization of the extracellular matrix.

1	A type of connective tissue found in close association with most epithelia is loose connective tissue (Fig. 4.2a). In fact, it is the connective tissue that most epithelia rest on. The extracellular matrix of loose connective tissue contains loosely arranged collagen fibers and numerous cells. Some of these cells, the fibroblasts, form and maintain the extracellular matrix. However, most of the cells are migrants from the vascular system and have roles associated with the immune system. In contrast, where only strength is required, collagen fibers are more numerous and densely packed. Also, the cells are relatively sparse and limited to the fiber-forming cell, the fibroblast (Fig. 4.2b). This type of connective tissue is described as dense connective tissue.

1	Bone and cartilage are two other types of connective tissue characterized by the material associated with collagen (i.e., calcium [bones] and hyaluronan [cartilage]). Again, in both of these tissues, it is the extracellular material that characterizes the tissue, not the cells. FIGURE 4.1  Simple epithelia. a. An H&E–stained section showing a pancreatic duct lined by a single layer of contiguous cuboidal epithelial cells. The free surface of the cells faces the lumen; the basal surface is in apposition to the connective tissue. 540. b. An H&E–stained section showing a single layer of tall columnar epithelial cells lining the gallbladder. Note that the cells are much taller than the lining cells of the pancreatic duct. The free surface of the epithelial cells is exposed to the lumen of the gallbladder, and the basal surface is in apposition to the adjacent connective tissue. 540.

1	FIGURE 4.2  Loose and dense connective tissue. a. Mallory-Azan–stained specimen of a section through the epiglottis, showing the lower part of its stratified epithelium (Ep), subjacent loose connective tissue (LCT ), and dense connective tissue (DCT ) below. Loose connective tissue typically contains many cells of several types. Their nuclei vary in size and shape. The elongated nuclei most likely belong to fibroblasts. Because dense connective tissue contains thick collagen bundles, it stains more intensely with the blue dye. Also, note the relatively fewer nuclei. 540. b. A Mallory-stained specimen of dense connective tissue, showing a region composed of numerous, densely packed collagen fibers. The few nuclei (N) that are present belong to fibroblasts. The combination of densely packed fibers and the paucity of cells characterizes dense connective tissue. Relatively few small blood vessels (BV ) are shown on this section. 540.

1	To function efficiently to effect movement, most muscle cells are aggregated into distinct bundles that are easily Muscle tissue is categorized on the basis of a functional distinguished from the surrounding tissue. Muscle cells property, the ability of its cells to contract. are typically elongated and oriented with their long axes Muscle cells are characterized by large amounts of the in the same direction (Fig. 4.3). The arrangement of contractile proteins actin and myosin in their cytoplasm nuclei is also consistent with the parallel orientation of and by their particular cellular arrangement in the tissue. muscle cells.

1	FIGURE 4.3  Muscle tissue. a. An H&E–stained specimen showing a portion of three longitudinally sectioned skeletal muscle fibers (cells). Two striking features of these large, long cells are their characteristic cross-striations and the many nuclei located along the periphery of the cell. 420. b. A Mallory-stained specimen showing cardiac muscle fibers that also exhibit striations. These fibers are composed of individual cells that are much smaller than those of skeletal muscle and are arranged end to end to form long fibers. Most of the fibers are seen in longitudinal array. The organized aggregation–that is, the parallel array of the fibers in the case of muscle tissue, allows for collective effort in performing their function. Intercalated disks (arrows) mark the junction of adjoining cells. 420.

1	Although the shape and arrangement of cells in specific muscle types (e.g., smooth muscle, skeletal muscle, and cardiac muscle) are quite different, all muscle types share a common characteristic: The bulk of the cytoplasm consists of the contractile proteins actin and myosin. Although these proteins are ubiquitous in all cells, only in muscle cells are they present in such large amounts and organized in such highly ordered arrays that their contractile activity can produce movement in an entire organ or organism. Nerve tissue consists of nerve cells (neurons) and associated supporting cells of several types.

1	Although all cells exhibit electrical properties, nerve cells or neurons are highly specialized to transmit electrical impulses from one site in the body to another; they are also specialized to integrate those impulses. Nerve cells receive and process information from the external and internal environment and may have specific sensory receptors and sensory organs to accomplish this function. Neurons are characterized by two different types of processes through which they interact with other nerve cells and with cells of epithelia and muscle. A single, long axon (sometimes longer than a meter) carries impulses away from the cell body, which contains the neuron’s nucleus. Multiple dendrites receive impulses and carry them toward the cell body. (In histologic sections, it is usually impossible to differentiate axons and dendrites because they have the same structural appearance.) The axon terminates at a neuronal junction called a synapse at which electrical impulses are transferred

1	to differentiate axons and dendrites because they have the same structural appearance.) The axon terminates at a neuronal junction called a synapse at which electrical impulses are transferred from one cell to the next by secretion of neuromediators. These chemical substances are released at synapses to generate electrical impulses in the adjacent communicating neuron.

1	In the central nervous system (CNS), which comprises the brain and spinal cord, the supporting cells are called neuroglial cells. In the peripheral nervous system (PNS), which comprises the nerves in all other parts of the body, the supporting cells are called Schwann (neurilemmal) cells and satellite cells. Supporting cells are responsible for several important functions. They separate neurons from one another, produce the myelin sheath that insulates and speeds conduction in certain types of neurons, provide active phagocytosis to remove cellular debris, and contribute to the blood–brain barrier in the CNS.

1	In an ordinary hematoxylin and eosin (H&E)–stained section, nerve tissue may be observed in the form of a nerve, which consists of varying numbers of neuronal processes along with their supporting cells (Fig. 4.4a). Nerves are most commonly seen in longitudinal or cross sections in loose connective tissue. Nerve cell bodies in the PNS, including the autonomic nervous system (ANS), are seen in aggregations called ganglia, where they are surrounded by satellite cells (Fig. 4.4b). Neurons and supporting cells are derived from neuroectoderm, which forms the neural tube in the embryo. Neuroectoderm originates by invagination of an epithelial layer, the dorsal ectoderm of the embryo. Some nervous system cells, such as ependymal cells and cells of the choroid plexus in the CNS, retain the absorptive and secretory functions characteristic of epithelial cells.

1	FIGURE 4.4  Nerve tissue. a. A Mallory-stained section of a peripheral nerve. Nerve tissue consists of a vast number of threadlike myelinated axons held together by connective tissue. The axons have been cross-sectioned and appear as small, red, dotlike structures. The clear space surrounding the axons previously contained myelin that was dissolved and lost during preparation of the specimen. The connective tissue is stained blue. It forms a delicate network around the myelinated axons and ensheathes the bundle, thus forming a structural unit, the nerve. 270. b. An Azan-stained section of a nerve ganglion, showing the large, spherical nerve cell bodies and the nuclei of the small satellite cells that surround the nerve cell bodies. The axons associated with the nerve cell bodies are unmyelinated. They are seen as nerve fiber bundles (NFB) between clusters of the cell bodies. 270.

1	In the early developing embryo during the gastrulation phase, a trilaminar embryo (trilaminar germ disk) is being formed. The three germ layers include the ectoderm, mesoderm, and endoderm, which give rise to all the tissues and organs. The ectoderm is the outermost of the three germ layers. The derivatives of the ectoderm may be divided into two major classes: surface ectoderm and neuroectoderm. Surface ectoderm gives rise to:  epidermis and its derivatives (hair, nails, sweat glands, sebaceous glands, and the parenchyma and ducts of the mammary glands),  cornea and lens epithelia of the eye,  enamel organ and enamel of the teeth,  components of the internal ear,  adenohypophysis (anterior lobe of pituitary gland), and  mucosa of the oral cavity and lower part of the anal canal.

1	Neuroectoderm gives rise to:  the neural tube and its derivatives, including components of the central nervous system, ependyma (epithelium lining the cavities of the brain and spinal cord), pineal body, posterior lobe of pituitary gland (neurohypophysis), and the sensory epithelium of the eye, ear, and nose;  the neural crest and its derivatives, including components of the peripheral nervous system (cranial, spinal, and autonomic ganglia, peripheral nerves, and Schwann cells); glial cells (oligodendrocytes and astrocytes); chromaffin (medullary) cells of the adrenal gland; enteroendocrine (APUD) cells of the diffuse neuroendocrine system; melanoblasts, the precursors of melanocytes; the mesenchyme of the head and its derivatives (such as pharyngeal arches that contain muscles, connective tissue, nerves, and vessels); odontoblasts; and corneal and vascular endothelium.

1	Mesoderm is the middle of the three primary germ layers of an embryo. It gives rise to:  connective tissue, including embryonic connective tissue (mesenchyme), connective tissue proper (loose and dense connective tissue), and specialized connective tissues (cartilage, bone, adipose tissue, blood and hemopoietic tissue), and lymphatic tissue;  heart, blood vessels, and lymphatic vessels, including their endothelial lining;  kidneys and the gonads (ovaries and testes) with genital ducts and their derivatives (ureters, uterine tubes, uterus, ductus deferens);  mesothelium, the epithelium lining the pericardial, pleural, and peritoneal cavities; and the adrenal cortex.

1	Endoderm is the innermost layer of the three germ layers. In the early embryo it forms the wall of the primitive gut and gives rise to epithelial portions or linings of the organs arising from the primitive gut tube. Derivatives of the endoderm include:  alimentary canal epithelium (excluding the epithelium of the oral cavity and lower part of the anal canal, which are of ectodermal origin);  extramural digestive gland epithelium (e.g., the liver, pancreas, and gallbladder);  lining epithelium of the urinary bladder and most of the urethra; respiratory system epithelium; thyroid, parathyroid, and thymus gland epithelial components; parenchyma of the tonsils; lining epithelium of the tympanic cavity and auditory (Eustachian) tubes.

1	Thyroid and parathyroid glands develop as epithelial outgrowths from the floor and walls of the pharynx; they then lose their attachments from these sites of original outgrowth. As an epithelial outgrowth of the pharyngeal wall, the thymus grows into the mediastinum and also loses its original connection. Figure 4.5 summarizes the derivatives of the three germ layers. Recognition of tissues is based on the presence of specific components within cells and on specific cellular relationships. Keeping these few basic facts and concepts about the fundamental four tissues in mind can facilitate the task of examining and interpreting histologic slide material. The first goal is to recognize aggregates of cells as tissues and determine the special characteristics that they present. Are the cells present at a surface? Are they in contact with their neighbors, or are they separated by definable intervening material? Do they belong to a group with special properties such as muscle or nerve?

1	The structure and the function of each fundamental tissue are examined in subsequent chapters. In focusing on a single specific tissue we are, in a sense, artificially separating the constituent tissues of organs. However, this separation is necessary to understand and appreciate the histology of the various organs of the body and the means by which they operate as functional units and integrated systems.

1	chapter 4 Tissues: Concept and Classific ation I DE NTI FYI N G TISSU ES 103 FIGURE 4.5  Derivatives of the three germ layers. Schematic drawing illustrates the derivatives of the three germ layers: ectoderm, endoderm, and mesoderm. GI, gastrointestinal. (Based on Moore KL, Persaud TVN. The Developing Human, Clinically Oriented Embryology. Philadelphia: WB Saunders, 1998.) cranial and sensory ganglia and nerves adrenal medulla melanocytes pharyngeal arch cartilages head mesenchyme and connective tissue Schwann cells odontoblasts surface ectoderm epidermis, hair, nails, cutaneous, and mammary glands anterior pituitary gland enamel of theeth internal ear corneal epithelium and lens of eye neuroectoderm (neural tube) central nervous system retina pineal body posterior pituitary gland head mesoderm cranium (skull) connective tissue of head dentin paraxial mesoderm skeletal muscle of trunk and limbs except cranium muscles of head dermis of skin connective tissue intermediate mesoderm

1	mesoderm cranium (skull) connective tissue of head dentin paraxial mesoderm skeletal muscle of trunk and limbs except cranium muscles of head dermis of skin connective tissue intermediate mesoderm urogenital system including gonads, ducts and accessory glands lateral mesoderm connective tissue and muscle of viscera serous membranes of pleura, pericardium, and peritoneum blood and lymph cells cardiovascular and lymphatic systems spleen adrenal cortex endoderm respiratory tract (trachea, bronchi, lungs) GI tract (pharynx, esophagus, stomach, small and large intestines) urinary bladder and urachus thyroid gland tympanic cavity auditory tube tonsils parathyroid glands liver pancreas epithelial lining of: epithelial parts of: It is of clinical interest that, under certain conditions, abnor-mal differentiation may occur. Most of the tumors derive from the cells that originate from a single germ cell layer. However, if the tumor cells arise from the pluripotential stem cells, their mass may

1	differentiation may occur. Most of the tumors derive from the cells that originate from a single germ cell layer. However, if the tumor cells arise from the pluripotential stem cells, their mass may contain cells that differentiate and re-semble cells originating from all three germ layers. The result is formation of a tumor that contains a variety of mature tis-sues arranged in an unorganized fashion. Such masses are referred to as teratomas. Since pluripotential stem cells are primarily encountered in gonads, teratomas almost al-ways occur in the gonads. In the ovary, these tumors usually develop into solid masses that contain characteristics of the 

1	FOLDER 4.1 Clinical Correlation: Ovarian Teratomas

1	FOLDER 4.1 Clinical Correlation: Ovarian Teratomas (Cont.) mature basic tissues. Although the tissues fail to form functional structures, frequently organlike structures may be seen (i.e., teeth, hair, epidermis, bowel segments, and so forth). Teratomas may also develop in the testis, but they are rare. Moreover, ovarian teratomas are usually benign, whereas teratomas in the testis are composed of less-differentiated tissues that usually lead to malignancy. An example of a solid-mass ovarian teratoma containing fully differentiated tissue is shown in the center micrograph of Figure F4.1.1. The low power reveals the lack of organized structures but does not allow identification of the specific tissues present. However, with higher magnification, as shown in the insets (a–f) mature differentiated tissues are evident. This tumor represents a mature teratoma of the ovary often called a demoid cyst. This benign tumor has a normal female kariotype 46XX; based on genetic studies, these

1	tissues are evident. This tumor represents a mature teratoma of the ovary often called a demoid cyst. This benign tumor has a normal female kariotype 46XX; based on genetic studies, these tissues are thought to arise through parthenogenic oocyte development. Mature teratomas are common ovarian tumors in childhood and in early reproductive age.

1	The example given in Figure F4.1.1 shows that one can readily identify tissue characteristics, even in an unorganized structure. Again, the important point is the ability to recognize aggregates of cells and to determine the special characteristics that they exhibit. FIGURE F4.1.1  Ovarian teratoma. In the center is an H&E–stained section of an ovarian teratoma seen at low magnification. This mass is composed of various basic tissues that are well differentiated and easy to identify at higher magnification. The abnormal feature is the lack of organization of the tissues to form functional organs. The tissues within the boxed areas are seen at higher magnification in micrographs a–f. The higher magnification allows identification of some of the basic tissues that are present within this tumor. 10. a.

1	a. Simple columnar epithelium lining a cavity of a small cyst. 170. Inset. Higher magnification of the epithelium and the underlying connective tissue. 320. b. Dense regular connective tissue forming a tendon like structure. 170. c. Area showing hyaline cartilage (C) and developing bone spicules (B). 170. d. Brain tissue with glial cells. 170. e. Cardiac muscle fibers. 220. Inset. Higher magnification showing intercalated disks (arrows). 320. f. Skeletal muscle fibers cut in cross section. 220.

1	OVERVIEW OF EPITHELIAL STRUCTURE AND FUNCTION / 105 CLASSIFICATION OF EPITHELIUM / 106 CELL POLARITY / 107 THE APICAL DOMAIN AND ITS MODIFICATIONS / 109 Microvilli / 109 Stereocilia / 110 Cilia / 113 THE LATERAL DOMAIN AND ITS SPECIALIZATIONS IN CELL-TO-CELL ADHESION / 121 Occluding Junctions / 124 Anchoring Junctions / 127 Communicating Junctions / 131 Morphologic Specializations of the Lateral Cell Surface / 133 THE BASAL DOMAIN AND ITS SPECIALIZATIONS IN CELL-TO– EXTRACELLULAR MATRIX ADHESION / 134 Basement Membrane Structure and Function / 134 Cell-to–Extracellular Matrix Junctions / 144 Morphologic Modifications of the Basal Cell Surface / 146 GLANDS / 146 EPITHELIAL CELL RENEWAL / 150 Folder 5.1 Clinical Correlation: Epithelial Metaplasia / 109 Folder 5.2 Clinical Correlation: Primary Ciliary Dyskinesia / 120 Folder 5.3 Clinical Correlation: Junctional Complexes as a Target of Pathogenic Agents / 128 Folder 5.4 Functional Considerations: Basement Membrane and Basal Lamina

1	Primary Ciliary Dyskinesia / 120 Folder 5.3 Clinical Correlation: Junctional Complexes as a Target of Pathogenic Agents / 128 Folder 5.4 Functional Considerations: Basement Membrane and Basal Lamina Terminology / 138 Folder 5.5 Functional Considerations: Mucus and Serous Membranes / 150

1	Epithelium covers body surfaces, lines body cavities, and constitutes glands. Epithelium is an avascular tissue composed of cells that cover the exterior body surfaces and line internal closed cavities (including the vascular system) and body tubes that communicate with the exterior (the alimentary, respiratory, and genitourinary tracts). Epithelium also forms the secretory portion (parenchyma) of glands and their ducts. In addition, specialized epithelial cells function as receptors for the special senses (smell, taste, hearing, and vision). The cells that make up epithelium have three principal characteristics:  They are closely apposed and adhere to one another by means of specific cell-to-cell adhesion molecules that form specialized cell junctions (Fig. 5.1).

1	 They exhibit functional and morphologic polarity. In other words, different functions are associated with three distinct morphologic surface domains: a free surface or apical domain, a lateral domain, and a basal domain. The properties of each domain are determined by specific lipids and integral membrane proteins.  Their basal surface is attached to an underlying basement membrane, a noncellular, protein–polysaccharide-rich layer demonstrable at the light microscopic level by histochemical methods (see Fig. 1.2, page 6).

1	FIGURE 5.1 • Diagram of small intestine absorptive epithelial cells. a. All three cellular domains of a typical epithelial cell are indicated on the diagram. The junctional complex provides adhesion between adjoining cells and separates the luminal space from the intercellular space, limiting the movement of fluid between the lumen and the underlying connective tissue. The intracellular pathway of fluid movement during absorption (arrows) is from the intestinal lumen into the cell, then across the lateral cell membrane into the intercellular space, and, finally, across the basement membrane to the connective tissue. b. This photomicrograph of a plastic-embedded, thin section of intestinal epithelium, stained with toluidine blue, shows cells actively engaged in fluid transport. Like the adjacent diagram, the intercellular spaces are prominent, reflecting fluid passing into this space before entering the underlying connective tissue. 1,250.

1	In special situations, epithelial cells lack a free surface (epithelioid tissues).

1	In some locations, cells are closely apposed to one another but lack a free surface. Although the close apposition of these cells and the presence of a basement membrane would classify them as epithelium, the absence of a free surface more appropriately classifies such cell aggregates as epithelioid tissues. The epithelioid cells are derived from progenitor mesenchymal cells (nondifferentiated cells of embryonic origin found in connective tissue). Although the progenitor cells of these epithelioid tissues may have arisen from a free surface or the immature cells may have had a free surface at some time during development, the mature cells lack a surface location or surface connection. Epithelioid organization is typical of most endocrine glands; examples of such tissue include the interstitial cells of Leydig in the testis (Plate 3, page 156), the lutein cells of the ovary, the islets of Langerhans in the pancreas, the parenchyma of the adrenal gland, and the anterior lobe of the

1	cells of Leydig in the testis (Plate 3, page 156), the lutein cells of the ovary, the islets of Langerhans in the pancreas, the parenchyma of the adrenal gland, and the anterior lobe of the pituitary gland. Epithelioreticular cells of the thymus also may be included in this category. Epithelioid patterns are also formed by accumulations of connective tissue macrophages in response to certain types of injury and infections, as well as by many tumors derived from epithelium.

1	Epithelium creates a selective barrier between the external environment and the underlying connective tissue. Covering and lining epithelium forms a sheetlike cellular investment that separates underlying or adjacent connective tissue from the external environment, internal cavities, or fluid connective tissue such as the blood and lymph. Among other roles, this epithelial investment functions as a selective barrier that facilitates or inhibits the passage of specific substances between the exterior (including the body cavities) environment and the underlying connective tissue compartment. The traditional classification of epithelium is descriptive and based on two factors: the number of cell layers and the shape of the surface cells. The terminology, therefore, reflects only structure, not function. Thus, epithelium is described as:  simple when it is one cell layer thick and  stratifed when it has two or more cell layers.

1	The individual cells that compose an epithelium are described as:  squamous when the width of the cell is greater than its  cuboidal when the width, depth, and height are approximately the same; and  columnar when the height of the cell appreciably exceeds the width (the term low columnar is often used when a cell’s height only slightly exceeds its other dimensions). Thus, by describing the number of cell layers (i.e., simple or stratified) and the surface cell shape, the various configurations of epithelia are easily classified. The cells in some exocrine glands are more or less pyramidal, with their apices directed toward a lumen. However, these cells are still classified as either cuboidal or columnar, depending on their height relative to their width at the base of the cell.

1	In a stratifed epithelium, the shape and height of the cells usually vary from layer to layer, but only the shape of the cells that form the surface layer is used in classifying the epithelium. For example, stratified squamous epithelium consists of more than one layer of cells, and the surface layer consists of flat or squamous cells. In some instances, a third factor—specialization of the apical cell surface domain—can be added to this classification system. For example, some simple columnar epithelia are classified as simple columnar ciliated when the apical surface domain possesses cilia. The same principle applies to stratified squamous epithelium, in which the surface cells may be keratinized or nonkeratinized. Thus, epidermis would be designated as stratified squamous keratinized epithelium because of the keratinized cells at the surface. Pseudostratified epithelium and transitional epithelium are special classifications of epithelium.

1	Pseudostratified epithelium and transitional epithelium are special classifications of epithelium. Two special categories of epithelium are pseudostratified and transitional.  Pseudostratifed epithelium appears stratified, although some of the cells do not reach the free surface; all rest on the basement membrane (Plate 2, page 154). Thus, it is actually a simple epithelium. The distribution of pseudostratified epithelium is limited in the body. Also, it is often difficult to discern whether all of the cells contact the basement membrane. For these reasons, identification of pseudostratified epithelium usually depends on knowing where it is normally found.

1	 Transitional epithelium (urothelium) is a term applied to the epithelium lining the lower urinary tract, extending from the minor calyces of the kidney down to the proximal part of the urethra. Urothelium is a stratifed epithelium with specific morphologic characteristics that allow it to distend (Plate 3, page 156). This epithelium is described in Chapter 20. The cellular configurations of the various types of epithelia and their appropriate nomenclature are illustrated in Table 5.1. Endothelium and mesothelium are the simple squamous epithelia lining the vascular system and body cavities. Specific names are given to epithelium in certain locations:  Endothelium is the epithelial lining of the blood and lymphatic vessels.  Endocardium is the epithelial lining of ventricles and atria of the heart.

1	 Endocardium is the epithelial lining of ventricles and atria of the heart.  Mesothelium is the epithelium that lines the walls and covers the contents of the closed cavities of the body (i.e., the abdominal, pericardial, and pleural cavities; Plate 1, page 152). Both endothelium and endocardium, as well as mesothelium, are almost always simple squamous epithelia. An exception is found in postcapillary venules of certain lymphatic tissues in which the endothelium is cuboidal. These venules are called high endothelial venules (HEV). Another exception is found in the spleen in which endothelial cells of the venous sinuses are rod-shaped and arranged like the staves of a barrel. Diverse epithelial functions can be found in different organs of the body.

1	Diverse epithelial functions can be found in different organs of the body. A given epithelium may serve one or more functions, depending on the activity of the cell types that are present:  secretion, as in the columnar epithelium of the stomach and the gastric glands; absorption, as in the columnar epithelium of the intestines and proximal convoluted tubules in the kidney;  transportation, as in the transport of materials or cells along the surface of an epithelium by motile cilia or in the transport of materials across an epithelium to and from the connective tissue;  protection, as in the stratified squamous epithelium of the skin (epidermis) and the transitional epithelium of the urinary bladder; and  receptor function to receive and transduce external stimuli, as in the taste buds of the tongue, olfactory epithelium of the nasal mucosa, and the retina of the eye.

1	Epithelia involved in secretion or absorption are typically simple or, in a few cases, pseudostratified. The height of the cells often reflects the level of secretory or absorptive activity. Simple squamous epithelia are compatible with a high rate of transepithelial transport. Stratification of the epithelium usually correlates with transepithelial impermeability. Finally, in some pseudostratified epithelia, basal cells are the stem cells that give rise to the mature functional cells of the epithelium, thus balancing cell turnover. Epithelial cells exhibit distinct polarity. They have an apical domain, a lateral domain, and a basal domain. Specific biochemical characteristics are associated with each cell surface. These characteristics and the geometric arrangements of the cells in the epithelium determine the functional polarity of all three cell domains.

1	The free or apical domain is always directed toward the exterior surface or the lumen of an enclosed cavity or tube. The lateral domain communicates with adjacent cells and is characterized by specialized attachment areas. The basal domain rests on the basal lamina anchoring the cell to underlying connective tissue.

1	TABLE Types of Epithelium 5.1 Classification Some Typical Locations Major Function Simple squamous Vascular system (endothelium) Exchange, barrier in Body cavities (mesothelium) central nervous system Bowman’s capsule (kidney) Exchange and lubrication Respiratory spaces in lung Barrier Exchange Simple cuboidal Small ducts of exocrine glands Absorption, conduit Surface of ovary Barrier (germinal epithelium) Absorption and secretion Kidney tubules Thyroid follicles Simple columnar Small intestine and colon Absorption and secretion Stomach lining and gastric Secretion glands AbsorptionGallbladder Pseudostratified Trachea and bronchial tree Secretion, conduitDuctus deferens Efferent ductules of epididymis Absorption, conduit Stratified squamous Epidermis Barrier, protection Oral cavity and esophagus Vagina Stratified cuboidal Sweat gland ducts Large ducts of exocrine Barrier, conduit glands Anorectal junction Stratified columnar Largest ducts of exocrine Barrier, conduit glands Anorectal

1	Vagina Stratified cuboidal Sweat gland ducts Large ducts of exocrine Barrier, conduit glands Anorectal junction Stratified columnar Largest ducts of exocrine Barrier, conduit glands Anorectal junction Transitional Renal calyces (urothelium) Ureters Barrier, distensible property Bladder Urethra tttttttt  FOLDER 5.1

1	Clinical Correlation: Epithelial Metaplasia

1	Epithelial metaplasia is a reversible conversion of one mature epithelial cell type to another mature epithelial cell type. Metaplasia is generally an adaptive response to stress, chronic inflammation, or other abnormal stimuli. The original cells are substituted by cells that are better suited to the new environment and more resistant to the effects of abnor-mal stimuli. Metaplasia results from reprogramming of ep-ithelial stem cells that changes the patterns of their gene expression. The most common epithelial metaplasia is columnar-to-squamous and occurs in the glandular ep-ithelium, where the columnar cells become replaced by the stratified squamous epithelium. For example, squamous metaplasia frequently occurs in the pseudostratified respi-ratory epithelium of the trachea and bronchi in response to prolonged exposure to cigarette smoke. It also occurs in the cervical canal in women with chronic infections. In this exam-ple, simple columnar epithelium of the cervical canal is

1	in response to prolonged exposure to cigarette smoke. It also occurs in the cervical canal in women with chronic infections. In this exam-ple, simple columnar epithelium of the cervical canal is re-placed by the stratified squamous nonkeratinized epithelium (Fig F5.1.1). In addition, squamous metaplasia is noticeable in the urothelium (transitional epithelium) and is associated with chronic parasitic infections such as schistosomiasis. Squamous-to-columnar epithelial metaplasia may also occur. For example, as a result of gastroesophageal reflux (Barrett’s esophagus), the stratified squamous nonkeratinized epithelium of the lower part of the esopha-gus can undergo metaplastic transformation into an intestinal-like simple columnar epithelium containing gob-let cells. Metaplasia is usually a reversible phenomenon, and if the stimulus that caused metaplasia is removed, tissues re-turn to their normal pattern of differentiation. If abnormal stimuli persist for a long time, squamous

1	a reversible phenomenon, and if the stimulus that caused metaplasia is removed, tissues re-turn to their normal pattern of differentiation. If abnormal stimuli persist for a long time, squamous metaplastic cells may transform into squamous cell carcinoma. Cancers of the lung, cervix, and bladder often originate from squamous metaplastic epithelium. Squamous columnar epithelium may give rise to glandular adenocarcinomas. When metaplasia is diagnosed, all efforts should be di-rected toward removing the pathogenic stimulus (i.e., ces-sation of smoking, eradication of infectious agents, etc.) and monitoring the metaplastic site to ensure that cancer-ous changes do not begin to develop. FIGURE F5.1.1 • Squamous metaplasia of the uterine cervix. Photomicrograph of a cervical canal lined by simple columnar epithelium. Note that the center of the image is occupied by an island containing squamous stratified epithelium. This metaplastic epithelium is surrounded on both sides by simple columnar

1	columnar epithelium. Note that the center of the image is occupied by an island containing squamous stratified epithelium. This metaplastic epithelium is surrounded on both sides by simple columnar epithelium. Since metaplasia is triggered by reprogramming of stem cells, metaplastic squamous cells have the same characteristics as normal stratified squamous epithelium. 240. (Courtesy of Dr. Fabiola Medeiros.)

1	The molecular mechanism responsible for establishing polarity in epithelial cells is required to first create a fully functional barrier between adjucent cells. Junctional complexes (which will be discussed later in this chapter) are being formed in the apical parts of the epithelial cells. These specialized attachment sites not only are responsible for tight cell adhesions but also allow epithelium to regulate paracellular movements of solutes down their electroosmotic gradients. In addition, junctional complexes separate the apical plasma membrane domain from basal and lateral domains and allow them to specialize and recognize different molecular signals.

1	In many epithelial cells, the apical domain exhibits special structural surface modifications to carry out specific functions. In addition, the apical domain may contain specific enzymes (e.g., hydrolases), ion channels, and carrier proteins (e.g., glucose transporters). The structural surface modifications include:  microvilli, cytoplasmic processes containing a core of actin filaments;  stereocilia (stereovilli), microvilli of unusual length; and  cilia, cytoplasmic processes containing bundle of micro- tubules. Microvilli are fingerlike cytoplasmic projections on the apical surface of most epithelial cells.

1	Microvilli are fingerlike cytoplasmic projections on the apical surface of most epithelial cells. As observed with the electron microscope (EM), microvilli vary widely in appearance. In some cell types, microvilli are short, irregular, bleblike projections. In other cell types, they are tall, closely packed, uniform projections that greatly increase the free cell surface area. In general, the number and shape of the microvilli of a given cell type correlate with the cell’s absorptive capacity. Thus, cells that principally transport fluid and absorb metabolites have many closely packed, tall FIGURE 5.2 • Electron micrographs showing variation in microvilli of different cell types. a. Epithelial cell of uterine gland; small projections. b. Syncytiotrophoblast of placenta; irregular, branching microvilli. c. Intestinal absorptive cell; uniform, numerous, and regularly arranged microvilli. All figures 20,000.

1	microvilli. Cells in which transepithelial transport is less active have smaller, more irregularly shaped microvilli.

1	Among the fluid-transporting epithelia (e.g., those of the intestine and kidney tubules), a distinctive border of vertical striations at the apical surface of the cell, representing an astonishing number of 15,000 close packed microvilli, is easily seen in the light microscope. In intestinal absorptive cells, this surface structure was originally called the striated border; in the kidney tubule cells, it is called the brush border. Where there is no apparent surface modification based on light microscope observations, any microvilli present are usually short and not numerous, which explains why they may escape detection in the light microscope. The variations seen in microvilli of various types of epithelia are shown in Figure 5.2. The microvilli of the intestinal epithelium (striated border) are the most highly ordered and are even more uniform in appearance than those that constitute the brush border of kidney cells.

1	The internal structure of microvilli contains a core of actin filaments that are cross-linked by several actinbundling proteins.

1	Microvilli contain a conspicuous core of about 20 to 30 actin flaments. Their barbed (plus) ends are anchored to villin, a 95-kilodalton actin-bundling protein located at the tip of the microvillus. The actin bundle extends down into the apical cytoplasm. Here it interacts with a horizontal network of actin filaments, the terminal web, that lies just below the base of the microvilli (Fig. 5.3a). The actin filaments inside the microvillus are cross-linked at 10-nm intervals by other actin-bundling proteins such as fascin (57 kilodaltons), espin (30 kilodaltons), and fmbrin (68 kilodaltons). This cross-linkage provides support and gives rigidity to the microvilli. In addition, the core of actin filaments is associated with myosin I, a molecule that binds the actin filaments to the plasma membrane of the micro-villus. The addition of villin to epithelial cells growing in cultures induces formation of microvilli on the free apical surface.

1	The terminal web is composed of actin flaments stabilized by spectrin (468 kilodaltons), which also anchors the terminal web to the apical cell membrane (Fig. 5.3b). The presence of myosin II and tropomyosin in the terminal web explains its contractile ability; these proteins decrease the diameter of the apex of the cell, causing the microvilli, whose stiff actin cores are anchored into the terminal web, to spread apart and increase the intermicrovillous space. The functional and structural features of microvilli are summarized in Table 5.2. Stereocilia are unusually long, immotile microvilli. Stereocilia are not widely distributed among epithelia. They are, in fact, limited to the epididymis, the proximal part of the ductus deferens of the male reproductive system, and the sensory (hair) cells of the inner ear. They are included in this section because this unusual surface modification is traditionally treated as a separate structural entity.

1	FIGURE 5.3 • Molecular structure of microvilli. a. High magnification of microvilli from Figure 5.2c. Note the presence of the actin filaments in the microvilli (arrows), which extend into terminal web in the apical cytoplasm. 80,000. b. Schematic diagram showing molecular structure of microvilli and the location of specific actin filament–bundling proteins (fimbrin, espin, and fascin). Note the distribution of myosin I within the microvilli and myosin II within the terminal web. The spectrin molecules stabilize the actin filaments within the terminal web and anchor them into the apical plasma membrane.

1	Stereocilia of the genital ducts are extremely long processes that extend from the apical surface of the cell and facilitate absorption. Unique features include an apical cell protrusion from which they arise and thick stem portions that are interconnected by cytoplasmic bridges. Because electron microscopy reveals their internal structure to be that of unusually long microvilli, some histologists now use the term stereovilli (Fig. 5.4a). Seen in the light microscope, these processes frequently resemble the hairs of a paint brush because of the way they aggregate into pointed bundles. Like microvilli, stereocilia are supported by internal bundles of actin flaments that are cross-linked by fmbrin. The actin filaments’ barbed (plus) ends are oriented toward the tips of the stereocilia and the pointed (minus) ends at the base. This organization of actin core shares many construction principles with the microvilli, yet it can be as long as 120 m.

1	Stereocilia develop from microvilli by the lateral addition of actin filaments to the actin bundle as well as elongation of the actin filaments. Unlike microvilli, an 80kilodalton actin-binding protein, ezrin, closely associated with the plasma membrane of stereocilia, anchors the actin filaments to the plasma membrane. The stem portion of the stereocilium and the apical cell protrusion contain the crossbridge–forming molecule -actinin (Fig. 5.4b). A striking difference between microvilli and stereocilia, other than size and the presence of ezrin, is the absence of villin from the tip of the stereocilium. Stereocilia of the sensory epithelium of the ear have some unique characteristics.

1	Stereocilia of the sensory epithelium of the ear have some unique characteristics. Stereocilia of the sensory epithelium of the ear also derive from microvilli. They are exquisitely sensitive to mechanical vibration and serve as sensory mechanoreceptors rather than absorptive structures. They are uniform in diameter and organized into ridged bundles of increasing heights, forming characteristic staircase patterns (Fig. 5.5a). Their internal structure is characterized by the high density of actin flaments extensively cross-linked by espin, which is critical to normal structure and function of stereocilia. Stereocilia of sensory epithelia lack both ezrin and -actinin. Since stereocilia can be easily damaged by overstimulation, they have a molecular mechanism to continuously renew their structure, which needs to be maintained in proper working condition for a lifetime. Using fluorescent-labeled actin molecules, researchers found that actin

1	TABLE Summary of Apical Domain Modifications in the Epithelial Cells 5.2 FIGURE 5.4 • Molecular structure of stereocilia. a. Electron micrograph of stereocilia from the epididymis. The cytoplasmic projections are similar to microvilli, but they are extremely long. 20,000. b. Schematic diagram showing the molecular structure of stereocilia. They arise from the apical cell protrusions, having thick stem portions that are interconnected by cytoplasmic bridges. Note the distribution of actin filaments within the core of the stereocilium and the actin-associated proteins, fimbrin, and espin, in the elongated portion (enlarged box); and -actinin in the terminal web, apical cell protrusion, and occasional cytoplasmic bridges between neighboring stereocilia.

1	monomers are being constantly added at the tips and removed at the base of the stereocilia while the entire bundle of actin filaments moves toward the base of the stereocilium (Fig. 5.5b,c). This treadmilling effect of the actin core structure is highly regulated and depends on the length of the stereocilium. The functional and structural features of stereocilia in comparison to microvilli and cilia are summarized in Table 5.2.

1	The functional and structural features of stereocilia in comparison to microvilli and cilia are summarized in Table 5.2. Cilia are common surface modifications present on nearly every cell in the body. They are hairlike extensions of the apical plasma membrane containing an axoneme, the microtubulebased internal structure. The axoneme extends from the basal body, a centriole-derived, microtubuleorganizing center (MTOC) located in the apical region of a ciliated cell. The basal bodies are associated with several accessory structures that assist them with anchoring into cell cytoplasm. Cilia, including basal bodies and basal body–associated structures, form the ciliary apparatus of the cell. In general, cilia are classified as motile, primary, or nodal.

1	In general, cilia are classified as motile, primary, or nodal. Based on their functional characteristics cilia are classified into three basic categories:  Motile cilia have been historically the most studied of all cilia. They are found in large numbers on the apical domain of many epithelial cells. Motile cilia and their counterparts, fagella, possess a typical 9 2 axonemal organization with microtubule-associated motor proteins that are necessary for the generation of forces needed to induce motility.  Primary cilia (monocilia) are solitary projections found on almost all eukaryotic cells. The term monocilia imp lies that only a single cilium per cell is usually present. Primary cilia are immotile because of different arrangements

1	FIGURE 5.5 • Dynamic turnover of an internal architecture of sterocilia. a. This scanning electron micrograph shows stereocilia of sensory epithelium of the inner ear. They are uniform in diameter and organized into ridged bundles of increasing heights. 47,000. b. Confocal microscopy image shows incorporation of the -actin green fluorescent protein (GFP) and espin-GFP to the tip of the stereocilia (green). Actin filaments in the core of the stereocilia are counterstained with rhodamine/phalloidin (red). 35,000 c.

1	Diagram illustrates the mechanism by which the core of actin filaments is remodeled. Actin polymerization and espin cross-linking into the barbed (plus) end of actin filaments occurs at the tip of the stereocilia. Disassembly and actin filaments depolymerization occurs at the pointed (minus) end of actin filament near the base of the stereocilium. When the rate of assembly at the tip is equivalent to the rate of disassembly at the base, the actin molecules undergo an internal rearward flow or treadmilling, thus maintaining the constant length of the stereocilium. (Reprinted with permission from Rzadzinska AK, Schneider ME, Davies C, Riordan GP, Kachar B. An actin molecular treadmill and myosins maintain stereocilia functional architecture and self-renewal. J Cell Biol 2004;164:887–897.) of microtubules in the axoneme and lack of microtubule-concentrated in the area that surrounds the primitive associated motor proteins. They function as chemosen-node, hence their name nodal cilia.

1	of microtubules in the axoneme and lack of microtubule-concentrated in the area that surrounds the primitive associated motor proteins. They function as chemosen-node, hence their name nodal cilia. They have a similar axsors, osmosensors, and mechanosensors, and they onemal internal architecture as primary cilia; however, mediate light sensation, odorant, and sound perception in they are distinct in their ability to perform rotational multiple organs in the body. It is now widely accepted movement. They play an important role in early embrythat primary cilia of cells in developing tissues are essen-onic development. tial for normal tissue morphogenesis.

1	 Nodal cilia are found in the embryo on the bilaminar The functional and structural features of all three types of embryonic disc at the time of gastrulation. They are cilia are summarized in Table 5.2. Motile cilia are capable of moving fluid and particles along epithelial surfaces. Motile cilia possess an internal structure that allows them to move. In most ciliated epithelia, such as the trachea, bronchi, or oviducts, cells may have as many as several hundred cilia arranged in orderly rows. In the tracheobronchial tree, the cilia sweep mucus and trapped particulate material toward the oropharynx where it is swallowed with saliva and eliminated from the body. In the oviducts, cilia help transport ova and fluid toward the uterus. Cilia give a “crew-cut” appearance to the epithelial surface.

1	Cilia give a “crew-cut” appearance to the epithelial surface. In the light microscope, motile cilia appear as short, fine, hairlike structures, approximately 0.25 m in diameter and 5 to10 m in length, that emanate from the free surface of the cell (Fig. 5.6). A thin, dark-staining band is usually seen extending across the cell at the base of the cilia. This dark-staining band represents structures known as basal bodies. These structures take up stain and appear as a continuous band when viewed in the light microscope. When viewed with the EM, however, the basal body of each cilium appears as a distinct individual structure. Motile cilia contain an axoneme, which represents an organized core of microtubules arranged in a 9 2 pattern.

1	Motile cilia contain an axoneme, which represents an organized core of microtubules arranged in a 9 2 pattern. Electron microscopy of a cilium in longitudinal profile reveals an internal core of microtubules, called axoneme (Fig. 5.7a). A cross-sectional view reveals a characteristic configuration of nine pairs or doublets of circularly arranged microtubules surrounding two central microtubules (Fig. 5.7b). FIGURE 5.6 • Ciliated epithelium. Photomicrograph of an H&E–stained specimen of tracheal pseudostratified ciliated epithelium. The cilia (C) appear as hairlike processes extending from the apical surface of the cells. The dark line immediately below the ciliary processes is produced by the basal bodies (BB) associated with the cilia. 750.

1	The microtubules composing each doublet are constructed so that the wall of one microtubule, designated the B micro-tubule, is actually incomplete; it shares a portion of the wall of the other microtubule of the doublet, the A microtubule. The A microtubule is composed of 13 tubulin protoflaments, arranged in side-by-side configuration, whereas the B micro-tubule is composed of 10 tubulin protoflaments. Tubulin molecules incorporated into ciliary microtubules are tightly bound together and post-translationally modified in the process of acetylation and polyglutamylation. Such modifications ensure that microtubules of ciliary axoneme are highly stable and resist depolymerization.

1	When seen in cross-section at high resolution, each doublet exhibits a pair of “arms” that contain ciliary dynein, a microtubule-associated motor protein. This motor protein uses the energy of adenosine triphosphate (ATP) hydrolysis to move along the surface of the adjacent microtubule (see Fig. 5.7). The dynein arms occur at 24-nm intervals along the length of the A microtubule and extend out to form temporary cross-bridges with the B microtubule of the adjacent doublet. A passive elastic component formed by nexin (165 kilodaltons) permanently links the A microtubule with the B microtubule of adjacent doublets at 86-nm intervals. The two central microtubules are separate but partially enclosed by a central sheath projection at 14-nm intervals along the length of the cilium (see Fig. 5.7). Radial spokes extend from each of the nine doublets toward the two central microtubules at 29-nm intervals. The proteins forming the radial spokes and the nexin connections between the outer

1	5.7). Radial spokes extend from each of the nine doublets toward the two central microtubules at 29-nm intervals. The proteins forming the radial spokes and the nexin connections between the outer doublets make large-amplitude oscillations of the cilia possible.

1	Basal bodies and basal body–associated structures firmly anchor cilia in the apical cell cytoplasm. The 9 2 microtubule array courses from the tip of the cilium to its base, whereas the outer paired microtubules join the basal body. The basal body is a modified centriole. It functions as an MTOC consisting of nine short microtubule triplets arranged in a ring. Each of the paired microtubules of the ciliary axoneme (A and B microtubules) is continuous with two of the triplet microtubules of the basal body. The third incomplete C microtubule in the triplet extends from the bottom to the transitional zone at the top of the basal body near the transition between the basal body and the axoneme. The two central micro-tubules of the cilium originate at the transitional zone and extend to the top of axoneme (see Fig. 5.7b). Therefore, a cross-section of the basal body would reveal nine circularly arranged microtubule triplets but not the two central single microtubules of the cilium.

1	Basal bodies are associated with several basal body– associated structures such as alar sheets (transitional fibers), basal feet, and striated rootlets (see Fig. 5.7 and Fig 5.8).  The alar sheet (transitional fiber) is a collarlike extension between the transitional zone of basal body and plasma membrane. It originates near the top end of the basal body C microtubule and inserts into the cytoplasmic domain of the plasma membrane. It tethers the basal body to the apical plasma membrane (see Fig 5.7).

1	FIGURE 5.7 • Molecular structure of cilia. This figure shows a three-dimensional arrangement of microtubules within the cilium and the basal body. Cross section of the cilium (right) illustrates the pair of central microtubules and the nine surrounding microtubule doublets (9 2 configuration). The molecular structure of the microtubule doublet is shown below the cross section. Note that the A microtubule of the doublet is composed of 13 tubulin dimers arranged in a side-by-side configuration (lower right), whereas the B microtubule is composed of 10 tubulin dimers and shares the remaining dimers with those of the A microtubule. The dynein arms extend from the A microtubule and make temporary cross-bridges with the B microtubule of the adjacent doublet. The basal body is anchored by the striated rootlet within the cell cytoplasm. Note the presence of the basal foot in the midsection of the basal body. The cross section of the basal body (lower left) shows the arrangement of nine

1	the striated rootlet within the cell cytoplasm. Note the presence of the basal foot in the midsection of the basal body. The cross section of the basal body (lower left) shows the arrangement of nine microtubule triplets. These structures form a ring connected by nexin molecules. Each microtubule doublet of the cilium is an extension of two inner A and B microtubules of the corresponding triplet. The C microtubule is shorter and extends only to the transitional zone. Inset a. Electron micrograph of longitudinally sectioned cilia from the oviduct. The internal structures within the cilia are microtubules. The basal bodies appear empty because of the absence of the central pair of microtubules in this portion of the cilium. 20,000. Inset b. Electron micrograph of cross section of the cilium showing corresponding structures with drawing below. 180,000.

1	FIGURE 5.8 • Ciliated surface of the respiratory mucosa. Electron micrograph shows a longitudinally sectioned cilia from a respiratory epithelium of the nasal cavity. At this magnification, most of the basal bodies (BB) appear empty because of the absence of the central pair of microtubules in this portion of the cilium. Structural details of the basal body and basal body–associated structures are well visible on this section as well as on the higher magnification insert. Note that almost all basal bodies on this section possess striated rootlets (SR). They anchor the basal bodies deep within the apical cell cytoplasm. Each basal body has a single asymmetric basal foot (BF) projecting laterally; several are well visible on this section. The transitional zone (TZ) extends from the upper end of the basal body into the axomene (Ax) which is formed by a 9 2 microtubular arrangement. A central pair of microtubules is present on most of these sections. In addition, an alar sheath

1	upper end of the basal body into the axomene (Ax) which is formed by a 9 2 microtubular arrangement. A central pair of microtubules is present on most of these sections. In addition, an alar sheath (arrowheads) provides a winglike extension between the transitional zone and plasma membrane. The first and second basal bodies from the right have well preserved alar sheaths. 15,000. Inset 25,000. (Courtesy of Dr. Jeffrey L. Salisbury.)  The basal foot is an accessory structure that is usually Localization of myosin molecules associated with basal feet found in the midregion of the basal body (see Fig 5.8). supports this hypothesis. Since in the typical epithelial ciliated cells all basal feet are  The striated rootlet is composed of longitudinally aligned oriented in the same direction (Fig. 5.9), it has been hy-protofilaments, containing rootletin (a 220-kilodalton pothesized that they function in coordinating ciliary protein). Striated rootlet projects deep into cytoplasm and

1	(Fig. 5.9), it has been hy-protofilaments, containing rootletin (a 220-kilodalton pothesized that they function in coordinating ciliary protein). Striated rootlet projects deep into cytoplasm and movement. They are most likely involved in adjusting firmly anchors the basal body within the apical cell cytobasal bodies by rotating them to the desired position. plasm (see Fig 5.8).

1	FIGURE 5.9 • Basal bodies and cilia. This diagnostic electron micrograph obtained during biopsy of the nasal mucosa from a child undergoing evaluation for primary ciliary dyskinesia shows a normal appearance of basal bodies (BB) and cilia (C). It is an oblique section through the apical part of ciliated cells. Basal bodies seen in cross section appear as more dense structures than sectioned oblique and longitudinal profiles of the cilia above. Several profiles of microvilli (Mv) are visible at the apical cell surface. 11,000. Inset. Three basal bodies sectioned at the level of basal feet (BF). Note that all basal feet are oriented in the same direction. They most likely rotate the basal body to a desired angle in an effort to coordinate ciliary movement. 24,000. (Courtesy of Patrice C. Abell Aleff.) Cilia movement originates from the sliding of microtubule doublets, which is generated by the ATPase activity of the dynein arms.

1	Ciliary activity is based on the movement of the doublet microtubules in relation to one another. Ciliary movement is initiated by the dynein arms (see Fig. 5.7b). The ciliary dynein located in the arms of the A microtubule forms temporary cross-bridges with the B microtubule of the adjacent doublet. Hydrolysis of ATP produces a sliding movement of the bridge along the B microtubule. The dynein molecules produce a continuous shear force during this sliding directed toward the ciliary tip. Because of this ATP-dependent phase, a cilium that remains rigid exhibits a rapid forward movement called the effective stroke. At the same time, the passive elastic connections provided by the protein nexin and the radial spokes accumulate the energy necessary to bring the cilium back to the straight position. Cilia then become flexible and bend toward the lateral side on the slower return movement, the recovery stroke.

1	However, if all dynein arms along the length of the A microtubules in all nine doublets attempted to form temporary cross-bridges simultaneously, no effective stroke of the cilium would result. Thus, regulation of the active shear force is required. Current evidence suggests that the central pair of microtubules in 9 2 cilia undergo rotation with respect to the nine outer doublets. This rotation may be driven by another motor protein, kinesin, which is associated with the central pair of microtubules. The central microtubule pair can act as a “distributor” that progressively regulates the sequence of interactions of the dynein arms to produce the effective stroke. Cilia beat in a synchronous pattern.

1	Cilia beat in a synchronous pattern. Motile cilia with a 9 2 pattern display a precise and synchronous undulating movement. Cilia in successive rows start their beat so that each row is slightly more advanced in its cycle than the following row, thus creating a wave that sweeps across the epithelium. As previously discussed, basal feet of basal bodies are most likely responsible for synchronization of ciliary movement. During the process of cilia formation, all basal feet became oriented in the same direction of effective stroke by rotating basal bodies. This orientation allows cilia to achieve a metachronal rhythm that is responsible for moving mucus over epithelial surfaces or facilitating the flow of fluid and other substances through tubular organs and ducts. Primary cilia are nonmotile and contain a 9 0 pattern of microtubules.

1	Primary cilia are nonmotile and contain a 9 0 pattern of microtubules. Differing from motile cilia with the 9 2 pattern of microtubules is another type of cilia that display a 9 0 microtubule arrangement. Cilia with this pattern have the following characteristics:  They are nonmotile and passively bend by a flow of the fluid. to generate motile force.  The central pair of microtubules is missing.  The axoneme originates from a basal body that resembles a mature centriole positioned orthogonally in relation to its immature counterpart.  Primary cilium formation is synchronized with cell cycle progression and centrosome duplication events.

1	These cilia are present in a variety of cells and are called primary cilia or monocilia because each cell usually possesses only one such cilium (Fig. 5.10). They are also found in some epithelial cells (e.g., the epithelial cells of the rete testis in the male reproductive tract, epithelial cells lining the biliary tract, epithelial cells of kidney tubules, epithelial-like ependymal cells lining the fluid-filled cavities of the central nervous system, the connecting stalk of photoreceptor cells in the retina, and the vestibular hair cells of the ear. Primary cilia were formerly classified as nonfunctional vestigial developmental abnormalities of 9 2 motile cilia. Experimental studies of the last decade elevated the status of primary cilia to the level of important cellular-signaling devices functioning comparably to an antenna on a global positioning system (GPS) receiver. Similar to an antenna that takes information from satellites and allows the GPS receiver to calculate the

1	devices functioning comparably to an antenna on a global positioning system (GPS) receiver. Similar to an antenna that takes information from satellites and allows the GPS receiver to calculate the user’s exact location, primary cilia receive chemical, osmotic, light, and mechanical stimuli from the extracellular environment. In response to these stimuli, primary cilia generate signals that are transmitted into the cell to modify cellular processes in response to changes in the external environment. In many mammalian cells, signaling through the primary cilia seems to be essential for controlled cell division and subsequent gene expression.

1	Primary cilia containing a 9 0 pattern of microtubules function as signal receptors sensing a flow of fluid in developing organs.

1	Primary cilia function in secretory organs such as the kidneys, liver, or pancreas as sensors of fluid flow. They extend from the surface of epithelial cells lining secretory ducts into the extracellular lumen (Fig. 5.11). For instance, primary cilia found in the glomerulus and tubular cells of the kidney function as mechanoreceptors; fluid flow through the renal corpuscle and tubules causes them to bend, which initiates an influx of calcium into the cell (Fig. 5.11). In humans, mutations in two genes, ADPKD1 and ADPKD2, appear to affect development of these primary cilia, leading to polycystic kidney disease (PKD). The proteins encoded by these genes, polycystin-1 and polycystin-2, respectively, are essential in the formation of the calcium channels associated with primary cilia (see Fig 5.11b). This autosomal recessive disorder is characterized by multiple expanding cysts in both kidneys, which ultimately destroy the renal cortex and lead to renal failure. However, individuals with

1	5.11b). This autosomal recessive disorder is characterized by multiple expanding cysts in both kidneys, which ultimately destroy the renal cortex and lead to renal failure. However, individuals with PKD often exhibit other pathologies not associated with the kidney that are now attributed to ciliary

1	FIGURE 5.10 • Primary cilia in the connective tissue and the kidney tubule. a. Electron micrograph shows a fibroblast surrounded by the extracellular matrix from the uterine connective tissue containing a primary cilium. The primary cilium is characterized by a (9 0) pattern of the microtubule arrangement. 45,000. Inset shows higher magnification of the cilium. Note the visible basal body and doublets of microtubules emerging from the basal body. 90,000. b. This scanning electron micrograph shows a single primary cilium projecting into the lumen of the collecting tubule of the kidney. Primary cilia are prominent on the free surface of the collecting tubule cells and function as mechanoreceptors that become activated by fluid flow through the tubules. Passive bending of cilia opens calcium channels and initiates signaling cascades by the influx of calcium into the cell cytoplasm. 65,000. (Courtesy of Dr. Tetyana V. Masyuk.)

1	FIGURE 5.11 • Primary cilium in the kidney tubule is a primary sensor for the fuid fow. Primary cilia in kidney function as sensors for the flow of fluid through the tubules. Deflection of the primary cilium opens the mechanoreceptor calcium channels, which are formed by cystic kidney disease– associated proteins (polycystin-1 and policystin-2). This subsequently initiates the influx of calcium into the cell, releasing additional intracellular calcium from the endoplasmic reticulum. Scanning electron micrograph inset shows primary cilia projecting into the lumen of abnormalities. These include cysts in the pancreas and liver that are accompanied by an enlargement and dilatation of the biliary tree system. Other changes include retinitis pigmentosa (abnormalities of the photoreceptors cells of the retina that cause progressive vision loss), sensorineural hearing loss, diabetes, and learning disabilities. The knowledge of the distribution of primary cilia in the body may help to explain

1	of the retina that cause progressive vision loss), sensorineural hearing loss, diabetes, and learning disabilities. The knowledge of the distribution of primary cilia in the body may help to explain the crucial role of these once-forgotten cellular projections in the normal function of many vital internal organs.

1	During early embryonic development, nodal cilia containing a 9 0 pattern of microtubules establish the left–right asymmetry of internal organs. Recent studies suggest that specific primary cilia observed in embryos, despite their 9 0 architectural pattern, are motile and play an important role in early embryonic development by generating the left–right asymmetry of internal organs. During gastrulation, a clockwise rotation of these cilia was observed on the ventral surface of the bilaminar embryonic disc in the area near the primitive node, hence the name nodal cilia. These cilia contain motor proteins (dyneins or kinesins) and are capable of rotational movement in a counterclockwise direction as previously described. Most likely, the absence of the central pairs of microtubules is responsible for such movement, the trajectory of which resembles a full cone in contrast to a half-cone trajectory traceable in the motile 9 2 cilia (Table 5.2).

1	Movement of nodal cilia in the region known as the the collecting tubule. 27,000. (Courtesy of Dr. C. Craig Tisher.) primitive node generates a leftward, or “nodal” flow. This flow is detected by sensory receptors on the left side of the body, which then initiate signaling mechanisms that differ from those on the right side of the embryo. When nodal cilia are immotile or absent, nodal ﬂow does not occur, leading to random placement of internal body organs. Therefore, primary ciliary dyskinesia (immotile cilia syndrome) often results in situs inversus, a condition in which the position of the heart and abdominal organs are reversed. The first stage of ciliogenesis includes generation of centrioles.

1	The first stage of ciliary apparatus formation (ciliogenesis) in differentiating cells involves a generation of multiple centrioles. This process occurs either in the centriolar pathway (by duplication of pairs of existing centrioles, see page 69 in Chapter 2) or more commonly in the acentriolar pathway in which centrioles are formed de novo without involvement of existing centrioles. Both pathways give rise to multiple procentrioles, the immediate precursors of centrioles. Procentrioles mature (elongate) to form centrioles, one for each cilium, and migrate to the apical surface of the cell. After perpendicularly aligning themselves and securing to the apical cell membrane by alar sheets (transitional fibers), centrioles assume the function of basal bodies. The next stage of ciliary apparatus formation involves formation of the remaining basal body– associated structures that include basal feet and striated rootlets. From each of the nine triplets that make up the basal body, a

1	apparatus formation involves formation of the remaining basal body– associated structures that include basal feet and striated rootlets. From each of the nine triplets that make up the basal body, a microtubule doublet grows upward by poly

1	FOLDER 5.2 Clinical Correlation: Primary Ciliary Dyskinesia (Immotile Cilia Syndrome)

1	Cilia are present in almost all organs and play a significant role in the human body. There is increasing evidence that cilial dysfunction is involved in many human disorders. Sev-eral hereditary disorders grouped under the general name of primary ciliary dyskinesia (PCD), also known as immotile cilia syndrome, affect the function of cilia. PCD represents a group of autosomal recessive hereditary disorders affecting 1 in 20,000 individuals at birth. The clinical features of PCD reflect the distribution of motile cilia. For instance, the mucociliary transport that occurs in the respiratory epithelium is one of the important mechanisms protecting the body against invading bacteria and other pathogens. Motile cilia covering the epithelium of the respiratory tract are responsible for the clearance of the airway. Failure of the mucociliary transport system occurs in Kartagener’s syndrome, which is caused by a structural abnormality that results in absence of dynein arms (Fig. F5.2.1). In

1	of the airway. Failure of the mucociliary transport system occurs in Kartagener’s syndrome, which is caused by a structural abnormality that results in absence of dynein arms (Fig. F5.2.1). In addition, EM examination of basal bodies from individuals with the Kartagener’s syndrome often reveals misoriented basal feet pointing in different di-rections. Young’s syndrome, which is characterized by malformation of the radial spokes and dynein arms, also af-fects ciliary function in the respiratory tract. The most prominent symptoms of PCD are chronic respiratory dis -tress (including bronchitis and sinusitis), otitis media (in-flammation of the middle ear cavity), persistent cough, and asthma. Respiratory problems are caused by severely impaired or absent ciliary motility that results in reduced or absent mucociliary transport in the tracheobronchial tree. Flagellum of the sperm, cilia of the efferent ductules in the testis, and cilia of the female reproductive system share the same

1	reduced or absent mucociliary transport in the tracheobronchial tree. Flagellum of the sperm, cilia of the efferent ductules in the testis, and cilia of the female reproductive system share the same organization (9 2) pattern with the cilia of the respiratory tract. Therefore, males with PCD are sterile be-cause of immotile flagella. In contrast, some females with the syndrome may be fertile; however, there is an increased incidence of ectopic pregnancy. In such individuals, the FIGURE F5.2.1 • Electron micrograph of the cilium from an individual with primary cilary dyskinesia (PCD). Note the absence of dynein arms on microtubule doublets. 180,000. (Courtesy of Patrice Abell-Aleff.) ciliary movement may be sufficient, though impaired, to permit transport of the ovum through the oviduct to the uterus. Some individuals with PCD may also develop symp-toms of hydrocephalus internus (accumulation of fluid in the brain) or transient dilatation of inner brain ventricles. The ependymal cells

1	the uterus. Some individuals with PCD may also develop symp-toms of hydrocephalus internus (accumulation of fluid in the brain) or transient dilatation of inner brain ventricles. The ependymal cells lining the cerebrospinal fluid–filled spaced in the brain possess motile cilia with a 9 2 pat-tern. These cilia may be important for the circulation of cerebrospinal fluid through the narrow spaces between the brain ventricles. About 50% of patients with diagnosed PCD have situs inversus (a condition in which the organs of the viscera are transposed through the sagittal plane), providing a link be-tween left–right asymmetry and nodal cilia. Diagnosis of PCD in individuals with clinical syn-dromes compatible with PCD can be established by EM (see Fig. F5.2.1).

1	merization of and -tubulin molecules. A growing projection of the apical cell membrane become visible and contains the nine doublets found in the mature cilium. During the elongation stage of motile cilia, the assembly of two single, central microtubules starts in the transitional zone from -tubulin rings. The subsequent polymerization of tubulin molecules occurs within the ring of doublet microtubules, thus yielding the characteristic axonemal 9 2 arrangement. Subsequently, the axoneme grows upward from the basal body, pushing the cell membrane outward to form the mature cilium. Ciliogenesis dependents on the bidirectional intraflagellar transport mechanism that supplies precursor molecules to the growing cilium.

1	During growth and elongation of the cilium, precursor molecules are delivered from the cell body to the most distal end of the elongating axoneme by intrafagellar transport (IFT). Since cilia have no molecular machinery for protein synthesis, the IFT is the only mechanism for delivering proteins required for cilia assembly and growth. In some ways, the IFT can be compared to the vertical lift assembly used at a construction site to move building materials and tools up and down a building. As the building increases in height, the track of the lift extends as well. Similarly, the IFT utilizes raftlike platforms assembled from about 17 different intraflagellar transport proteins that move up and down the growing axoneme between the outer doublets of microtubules and plasma membrane of the elongating cilium (Fig. 5.12). Cargo molecules (including inactive cytoplasmic dynein molecules) are loaded onto the IFT platform while it is docked near the base of the cilium. Utilizing kinesin II as

1	cilium (Fig. 5.12). Cargo molecules (including inactive cytoplasmic dynein molecules) are loaded onto the IFT platform while it is docked near the base of the cilium. Utilizing kinesin II as a motor protein, the fully loaded platform is moved upward toward the tip of the cilium (anterograde transport). The “building materials” are then unloaded at the tip of the cilium (the site of axoneme assembly). Here particles turn around, and the platform heads back to the base of the cilium (retrograde transport) after picking up turnover products (including inactivated kinesin II). During this process, cytoplasmic dynein is activated and utilized as a motor protein to bring the platform back to the base of the cilium (see Fig. 5.12). Several proteins, including IFT raft proteins (kinesis, cytoplasmic dynein, polaris, IFT20, etc.), are important to ciliogenesis and subsequent maintenance of the functional cilium. Mutations in genes encoding these proteins result in loss of cilia or ciliary

1	dynein, polaris, IFT20, etc.), are important to ciliogenesis and subsequent maintenance of the functional cilium. Mutations in genes encoding these proteins result in loss of cilia or ciliary dysfunctions.

1	The lateral domain of epithelial cells is in close contact with the opposed lateral domains of neighboring cells. Like the other domains, the lateral domain is characterized by the presence of unique proteins, in this case the cell adhesion molecules (CAMs) that are part of junctional specializations. The molecular composition of the lipids and proteins that form the lateral cell membrane differ significantly from the composition of those that form the apical cell membrane. In addition, the lateral cell surface membrane in some epithelia may form folds and processes, invaginations and evaginations that create interdigitating and interleaving tongue-and-groove margins between neighboring cells. Viewed with the light microscope, terminal bars represent epithelial cell-to-cell attachment sites.

1	Viewed with the light microscope, terminal bars represent epithelial cell-to-cell attachment sites. Before the advent of EM, the close apposition of epithelial cells was attributed to the presence of a viscous adhesive substance referred to as intercellular cement. This cement stained deeply at the apicolateral margin of most cuboidal and columnar epithelial cells. When viewed in a plane perpendi cular to the epithelial surface, the stained material appears as a dotlike structure. When the plane of section passes parallel to and includes the epithelial surface, however, the dotlike component is seen as a dense bar or line between the apposing cells (Fig. 5.13). The bars, in fact, form a polygonal structure (or band) that encircles each cell to bind them together. Arrangement of this band can be compared to the plastic rings that hold together a six-pack of canned beverages.

1	Because of its location in the terminal or apical portion of the cell and its barlike configuration, the stainable material visible in light microscopy was called the terminal bar. It is now evident that intercellular cement as such does not exist. The terminal bar, however, does represent a significant structural complex. Electron microscopy has shown that it includes a specialized site that joins epithelial cells (Fig. 5.14a). It is also the site of a considerable barrier to the passage (diffusion) of substances between adjacent epithelial cells. The specific structural components that make up the barrier and the attachment device are readily identified with the EM and are collectively referred to as a junctional complex (see Table 5.4, page 135). These complexes are responsible for joining individual cells together. There are three types of junctional complexes (Fig. 5.14b):  Occluding junctions are impermeable and allow epithelial cells to function as a barrier. Also called tight

1	individual cells together. There are three types of junctional complexes (Fig. 5.14b):  Occluding junctions are impermeable and allow epithelial cells to function as a barrier. Also called tight junctions, occluding junctions form the primary intercellular diffusion barrier between adjacent cells.

1	By limiting the movement of water and other molecules through the intercellular space, they maintain physico chemical separation of tissue compartments. Because they are located at the most apical point between adjoin ing epithelial cells, occluding junctions prevent the mi gration of lipids and specialized membrane proteins between the apical and lateral surfaces, thus maintaining the integrity of these two domains. In addition, occlud ing junctions recruit various signaling molecules to the cell surface and link them to the actin flaments of the cell cytoskeleton.

1	FIGURE 5.12 • Intrafagellar transport mechanism within the cilium. Assembly and maintenance of cilia depends on the intraflagellar transport mechanism (IFT ) that utilizes raftlike platforms. They move up and down between the outer doublets of microtubules and plasma membrane of the elongating cilium. Cargo molecules (including inactive cytoplasmic dynein) are loaded onto the IFT platform while it is docked near the base of the cilium. Using kinesin II as a motor protein, the fully loaded platform is moved upward toward the plus end of microtubules at the tip of the cilium (anterograde transport). The cargo is then unloaded at the tip of the cilium (the site of axoneme assembly). Here particles turn around, and the platform powered by cytoplasmic dynein heads back to the base of the cilium (retrograde transport) after picking up turnover products (including inactivated kinesin II). Inset. Electron micrograph of a longitudinal section of a Chlamydomonas flagellum with two groups of IFT

1	(retrograde transport) after picking up turnover products (including inactivated kinesin II). Inset. Electron micrograph of a longitudinal section of a Chlamydomonas flagellum with two groups of IFT platforms. 55,000. (Reprinted with permission from Pedersen LB, Veland IR, Schrer JM, and Christensen ST. Assembly of primary cilia. Dev Dyn. 2008; 237:1993–2006.)

1	FIGURE 5.13 • Terminal bars in pseudostratifed epithelium. Photomicrograph of an H&E–stained specimen showing the terminal bars in a pseudostratified epithelium. The bar appears as a dot (arrowheads) when seen on its cut edge. When the bar is coursing parallel to the cut surface and lying within the thickness of the section, it is seen as a linear or barlike profile (arrows). 550. FIGURE 5.14 • Junctional complex. a. Electron micrograph of the apical portion of two adjoining epithelial cells of the gastric mucosa, showing the junctional complex. It consists of the zonula occludens (ZO), zonula adherens (ZA), and macula adherens (MA). 30,000. b. Diagram showing the distribution of cell junctions in the three cellular domains of columnar epithelial cells. The apical domain with microvilli has been lifted to better illustrate spatial arrangements of junctional complexes within the cell.

1	 Anchoring junctions provide mechanical stability to epithelial cells by linking the cytoskeleton of one cell to the cytoskeleton of an adjacent cell. These junctions are important in creating and maintaining the structural unity of the epithelium. Anchoring junctions interact with both actin and intermediate flaments and can be found not only on the lateral cell surface but also on the basal domain of the epithelial cell. Through their signal transduction capability, anchoring junctions also play important roles in cell-to-cell recognition, morphogenesis, and differentiation.  Communicating junctions allow direct communication between adjacent cells by diffusion of small (1,200 daltons) molecules (e.g., ions, amino acids, sugars, nucleotides, second messengers, metabolites). This type of intercellular communication permits the coordinated cellular activity that is important for maintaining organ homeostasis.

1	The zonula occludens (pl., zonulae occludentes) represents the most apical component in the junctional complex between epithelial cells. The zonula occludens is created by localized sealing of the plasma membrane of adjacent cells.

1	Examination of the zonula occludens or tight junction with the transmission electron microscope (TEM) reveals a narrow region in which the plasma membranes of adjoining cells come in close contact to seal off the intercellular space (Fig. 5.15a). At high resolution, the zonula occludens appears not as a continuous seal but as a series of focal fusions between the cells. These focal fusions are created by transmembrane proteins of adjoining cells that join in the intercellular space (Fig. 5.15b). The arrangement of these proteins in forming the seal is best visualized by the freeze fracture technique (Fig. 5.15c). When the plasma membrane is fractured at the site of the zonula occludens, the junctional proteins are observed on the P-face of the membrane, where they appear as ridgelike structures. The opposing surface of the fractured membrane, the E-face, reveals complementary grooves resulting from detachment of the protein particles from the opposing surface. The ridges and grooves

1	The opposing surface of the fractured membrane, the E-face, reveals complementary grooves resulting from detachment of the protein particles from the opposing surface. The ridges and grooves are arranged as a network of anastomosing particle strands, thus creating a functional seal within the intercellular space. The number of strands as well as the degree of anastomosing varies in different cells.

1	FIGURE 5.15 • Zonula occludens. a. Electron micrograph of the zonula occludens showing the close approximation of the outer lamellae of adjoining plasma membranes. The extracellular domains of proteins involved in the formation of this junction (occludins) appear as single, electron-dense lines (arrows). 100,000. b. Diagram showing the organization and pattern of distribution of the transmembrane protein occludin within the occluding junction. Compare the linear pattern of grooves with the ridges detected in the freeze fracture preparation on the right side. c. Freeze fracture preparation of zonula occludens shown here reveals an anastomosing network of ridges (arrows) located on the fracture membrane surface near the apical part of the cell [note microvilli (Mv) present at the cell surface]. This is the P-face of the membrane. (The E-face of the fractured membrane would show a complementary pattern of grooves.) The ridges represent linear arrays of transmembrane proteins (most likely

1	This is the P-face of the membrane. (The E-face of the fractured membrane would show a complementary pattern of grooves.) The ridges represent linear arrays of transmembrane proteins (most likely occludins) involved in the formation of the zonula occludens. The membrane of the opposing cell contains a similar network of proteins, which is in register with the first cell. The actual sites of protein interaction between the cells form the anastomosing network. 100,000. (Reprinted with permission from Hull BE, Staehelin LA. Functional significance of the variations in the geometrical organization of tight junction networks. J Cell Biol 1976;68:688–704.)

1	FIGURE 5.16 • Molecular structure of zonula occludens. Diagram showing three transmembrane proteins involved in the formation of zonula occludens: occludin, claudin, and junctional adhesion molecule (JAM). Occludin and claudin have four transmembrane domains with two extracellular loops, but JAM has only a single transmembrane domain, and its extracellular portion possesses two immunoglobulinlike loops. Several major associated proteins of the occluding junction and their interactions with each other are visible. Note that one of the associated proteins, ZO-1, interacts with the cell cytoskeleton binding actin filaments. Several proteins are involved in the formation of zonula occludens strands.

1	Several proteins are involved in the formation of zonula occludens strands. Zonula occludens strands correspond to the location of the rows of transmembrane proteins. Three major groups of transmembrane proteins are found in the zonula occludens (Fig. 5.16; Table 5.3):  Occludin, a 60-kilodalton protein, was the first protein identified in the zonula occludens. It participates in maintaining the barrier between adjacent cells as well as the barrier between the apical and lateral domains. Occludin is present in most occluding junctions. However, several types of epithelial cells do not have occludin within their strands, but they still possess well-developed and fully functional zonulae occludentes.

1	 Claudins constitute a family of proteins (20 to 27 kilodaltons) that have recently been identified as integral components of zonula occludens strands. Claudins form the backbone of each strand. In addition, claudins (especially claudin-2 and claudin-16) are able to form extracellular aqueous channels for the paracellular passage of ions and other small molecules. About 24 different members of the claudin family have been characterized to date. Mutations in the gene encoding claudin-14 have been recently linked to human hereditary deafness. A mutated form of claudin-14 causes an increased permeability of zonula occludens in the organ of Corti (receptor of hearing), affecting generation of action potentals.

1	 Junctional adhesion molecule (JAM) is a 40kilodalton protein that belongs to the immunoglobulin superfamily (IgSF). JAM does not itself form a zonula occludens strand but is instead associated with claudins. It is involved in the formation of occluding junctions in endothelial cells as well as between endothelial cells and monocytes migrating from the vascular space to the connective tissue.

1	The extracellular portions of these transmembrane proteins function as a zipper and seal the intercellular space between two adjacent cells, thus creating a barrier against paracellular diffusion. The cytoplasmic portions of all three proteins contain a unique amino acid sequence that attracts regulatory and signaling proteins called PDZ-domain proteins. These proteins include the zonula occludens proteins ZO-1, ZO-2, and ZO-3 (see Fig. 5.16). Occludin and claudins interact with the actin cytoskeleton through ZO-1 and ZO-3. Regulatory functions during the formation of the zonula occludens have been suggested for all ZO proteins. In addition, ZO-1 is a tumor suppressor, and ZO-2 is required in the epidermal growth factor–receptor signaling mechanism. The ZO-3 protein interacts with ZO-1 and the cytoplasmic domain of occludins. The proteins localized in the region of the zonula occludens are summarized in Table 5.3. Many pathogenic agents, such as cytomegalovirus and cholera toxins, act

1	the cytoplasmic domain of occludins. The proteins localized in the region of the zonula occludens are summarized in Table 5.3. Many pathogenic agents, such as cytomegalovirus and cholera toxins, act on ZO-1 and ZO-2, causing the junction to become permeable.

1	The zonula occludens separates the luminal space from the intercellular space and connective tissue compartment. It is now evident that the zonula occludens plays an essential role in the selective passage of substances from one side of an epithelium to the other. The ability of epithelia to create a diffusion barrier is controlled by two distinct pathways for transport of substances across the epithelia (Fig. 5.17a):  The transcellular pathway occurs across the plasma membrane of the epithelial cell. In most of these pathways, transport is active and requires specialized energy-dependent membrane transport proteins and channels. These proteins and channels move selected substances across the apical plasma membrane into the cytoplasm and then across the lateral membrane below the level of the occluding junction into the intercellular compartment.

1	 The paracellular pathway occurs across the zonula occludens between two epithelial cells. The amount of water, electrolytes, and other small molecules transported through this pathway is contingent on the tightness of the zonula occludens. The permeability of an occluding junction depends on the molecular composition of the zonula occludens strands and thus the number of active aqueous channels in the seal (see the following section). Under physiologic conditions, substances transported through this pathway may be regulated or coupled to transcellular transport. Permeability of the zonula occludens depends not only on the complexity and number of strands but also on the presence of functional aqueous channels formed by various claudin molecules.

1	TABLE Major Proteins Localized Within the Zonula Occludens Junction 5.3 Zona Occludens Protein Associated Protein Partners Function Occludin Occludin, ZO-1, ZO-2, ZO-3, Is present in most occluding junctions; maintains barrier Vap33, actin between apical and lateral cell surface Claudin Claudin, ZO-1, JAM Forms backbone of zonula occludens strands; forms and regulates aqueous channels used for paracellular diffusion JAM JAM, ZO-1, claudin Present in endothelial cells; mediates interactions between endothelial cells and monocyte adhesions ZO-1 ZO-2, ZO-3, occludin, claudin, Important link in transduction of signals from all JAM, cingulin, actin, ZONAB, transmembrane proteins; interacts with actin flaments; ASIP, AF-6 has tumor-suppressor actin ZO-2 ZO-1, occluding, cingulin, 4.1R Required in the epidermal growth factor–receptor signaling mechanism ZO-3 ZO-1, occludin, actin Interacts with ZO-1, occludin, and actin flaments of cell cytoskeleton AF-6 RAS, ZO-1 Small protein involved in

1	in the epidermal growth factor–receptor signaling mechanism ZO-3 ZO-1, occludin, actin Interacts with ZO-1, occludin, and actin flaments of cell cytoskeleton AF-6 RAS, ZO-1 Small protein involved in molecular transport system and signal transduction Cingulin ZO-1, ZO-2, ZO-3, cingulin, Acidic, heat-stable protein that cross-links myosin II actin flaments into sedimentable complexes Symplekin CPSF-100 Dual-location protein: localized in zonula occludens and in the interchromatin particles of the karyoplasm ASIP/Par3 PKC Controls relocation of asymmetrically distributed proteins Rab3b GTPase Rab13 -PDE Rab8 G/C kinase, Sec4 Sec4 Rab8 GTPase required for polarized delivery of cargo vesicles to plasma membrane Sec6 Sec8 Participates in fusion of Golgi vesicle with the plasma membrane Sec8 Sec6 Inhibits basolateral translocation of LDLP receptors after formation of zonula occludens Members of the RAS oncogene family of proteins; control the assembly of protein complexes for docking of

1	Sec6 Inhibits basolateral translocation of LDLP receptors after formation of zonula occludens Members of the RAS oncogene family of proteins; control the assembly of protein complexes for docking of transport vesicles

1	Observations of different kinds of epithelia reveal that the complexity and number of strands forming the zonulae occludentes varies. In epithelia in which anastomosing strands or fusion sites are sparse, such as certain kidney tubules, the intercellular pathway is partially permeable to water and solutes. In contrast, in epithelia in which the strands are numerous and extensively intertwined—for example, intestinal and urinary bladder epithelia—the intercellular region is highly impermeable.

1	However, in some epithelial cells, the number of strands does not directly correlate to the tightness of the seal. Differences in tightness between different zonulae occludentes could be explained by the presence of aqueous pores within individual zonula occludens strands (Fig. 5.17b). Recent experiments indicate that claudin-16 functions as an aqueous Mg2 channel between specific kidney epithelial cells. Similarly, claudin-2 is responsible for the presence of high-conductance aqueous pores in other kidney epithelia. Claudins not only form the backbone of the individual zonula occludens strand but also are responsible for the formation of extracellular aqueous channels. Thus, the combination and mixing ratios of claudins to occludins and other proteins found within individual paired zonula occludens strands determine tightness and selectivity of the seal between cells. PDZ domains of associated intracellular attachment proteins

1	FIGURE 5.17 • Two transcellular and paracellular pathways for transport of substances across the epithelia. a. The transcellular pathway occurs across the plasma membrane of the epithelial cell and represents an active transport system that requires specialized energy-dependent membrane transport proteins and channels. The paracellular pathway occurs across the zonula occludens between two epithelial cells. The amount of water, electrolytes, and other small molecules transported through this pathway is contingent on the tightness of the zonula occludens. b. Structure of the extracellular and cytoplasmic portions of tight-junction strands. Two zonula occludens strands from neighboring cells fuse together in a zipperlike fashion and create a barrier to movement between the cells. Aqueous pores allow water to move between the cells. The permeability of the barrier depends on the mixture of claudins and occludins in the zipper seal. The cytoplasmic portion of the strand attracts

1	pores allow water to move between the cells. The permeability of the barrier depends on the mixture of claudins and occludins in the zipper seal. The cytoplasmic portion of the strand attracts PDZ-domain proteins that function in cell signaling.

1	The zonula occludens establishes functional domains in the plasma membrane. As a junction, the zonula occludens controls not only the passage of substances across the epithelial layer but also the movement of lipid rafts containing specific proteins within the plasma membrane itself. The cell is able to segregate certain internal membrane proteins on the apical (free) surface and restrict others to the lateral or basal surfaces. In the intestine, for instance, the enzymes for terminal digestion of peptides and saccharides (dipeptidases and disaccharidases) are localized in the membrane of the microvilli of the apical surface. The NaK-ATPase that drives salt and transcellular water transport, as well as amino acid and sugar transport, is restricted to the lateral plasma membrane below the zonula occludens.

1	Anchoring junctions provide lateral adhesions between epithelial cells, using proteins that link into the cytoskeleton of adjacent cells. Two types of anchoring cell-to-cell junctions can be identified on the lateral cell surface:  zonula adherens (pl., zonulae adherentes), which inter acts with the network of actin filaments inside the cell; and  macula adherens (pl., maculae adherentes) or desmo some, which interacts with intermediate filaments In addition, two other types of anchoring junctions can be found where epithelial cells rest on the connective tissue matrix. These focal adhesions (focal contacts) and hemidesmosomes are discussed in the section on the basal domain (see pages 144 to 146). Cell adhesion molecules play important roles in cell-tocell and cell-to–extracellular matrix adhesions.

1	Cell adhesion molecules play important roles in cell-tocell and cell-to–extracellular matrix adhesions. Transmembrane proteins known as cell adhesion mole cules (CAMs) form an essential part of every anchoring junction on both lateral and basal cell surfaces. The extracellular domains of CAMs interact with similar domains belonging to CAMs of neighboring cells. If the binding occurs between different types of CAMs it is described as heterotypic binding; homotypic binding occurs between CAMs of the same type (Fig. 5.18). CAMs have a selective adhesiveness of relatively low strength, which allows cells to easily join and dissociate.  FOLDER 5.3 Clinical Correlation: Junctional Complexes as a Target of Pathogenic Agents

1	Epithelia form a physical barrier that allows the body to main-tain internal homeostasis while protecting the organism from harmful pathogenic agents from the external environment. The easiest way for many viruses, bacteria, and parasites to suc-cessfully compromise the protective functions of the epithelial layer is to destroy the junctional complexes between epithelial cells. Several proteins found in junctional specializations of the cell membrane are affected by molecules produced or ex-pressed by these pathogenic agents. Bacteria. A common bacterium that causes food poi-soning, Clostridium perfringens, attacks the zonula occlu-dens junction. This microorganism is widely distributed in the external environment and is found within the intestinal flora of humans and many domestic animals. Food poison-ing symptoms are characterized by intense abdominal pain and diarrhea that begins 8 to 22 hours after eating foods contaminated by these bacteria. Symptoms usually abate within 24

1	Food poison-ing symptoms are characterized by intense abdominal pain and diarrhea that begins 8 to 22 hours after eating foods contaminated by these bacteria. Symptoms usually abate within 24 hours. The enterotoxin produced by C. perfrin-gens is a small 35-kilodalton protein whose carboxy termi-nus binds specifically to the claudin molecules of the zonula occludens. Its amino terminus forms pores within the apical domain of the plasma membrane. Binding to claudins prevents their incorporation into the zonula occlu-dens strands and leads to malfunction and breakdown of the junction. Dehydration that occurs with this type of food poisoning is a result of a massive movement of fluids via paracellular pathways into the lumen of the intestines. Helicobacter pylori, another bacterium, resides within the stomach and binds to the extracellular domains of zonula occludens proteins. During this process, the CagA surface-exposed 128-kilodalton protein produced by the bacteria is translocated

1	within the stomach and binds to the extracellular domains of zonula occludens proteins. During this process, the CagA surface-exposed 128-kilodalton protein produced by the bacteria is translocated from the microorganism into the cy-toplasm, where it targets both ZO-1 and JAM proteins. As a result, the zonula occludens barrier becomes disrupted, and its capacity for tyrosine kinase signaling diminishes, causing cytoskeletal rearrangements. H. pylori cause injury to the protective barrier of the stomach that may lead to the development of gastric ulcers and gastric carcinomas. Viruses. The one specific group of RNA viruses responsible for infant enteritis (inflammation of the intestines) uses the intracellular JAM signaling pathway. The attachment and endocytosis of the reovirus is initiated by the interaction of its viral attachment protein with a JAM molecule. This interaction activates nuclear factor-B pro-tein (NF B), which migrates into the nucleus and triggers a cascade of

1	by the interaction of its viral attachment protein with a JAM molecule. This interaction activates nuclear factor-B pro-tein (NF B), which migrates into the nucleus and triggers a cascade of cellular events leading to apoptosis. This is ev-idence that JAMs are being used as signal transduction molecules to convey impulses from the external environ-ment to the cell nucleus. Zonula occludens–associated proteins that contain the PDZ-expressed sequence are targets of oncogenic aden-ovirus and papillomavirus. The viral oncoproteins produced by these viruses bind via their PDZ binding domains to ZO-2 and multi-PDZ–containing protein-1 (MUPP-1). The oncogenic effect of these interactions is attributed, in part, to the sequestration and degradation of the zonula occludens and the tumor-suppressor proteins associated with the viruses. Parasites. The common house dust mite, Der-matophagoides pteronyssinus, also destroys zonula occludens junctions. It belongs to the arachnid family, which

1	proteins associated with the viruses. Parasites. The common house dust mite, Der-matophagoides pteronyssinus, also destroys zonula occludens junctions. It belongs to the arachnid family, which includes spiders, scorpions, and ticks. When its fecal pellets are inhaled with dust particles, serine and cysteine peptidases present in the pellets cleave occluding and ZO-1 protein, resulting in the breakdown of zonula occludens junctions in the respiratory epithelium. The loss of the pro-tective epithelial barrier in the lung exposes the lung to in-haled allergens and initiates an immune response that can lead to severe asthma attacks.

1	The cytoplasmic domains are linked through a variety of intracellular proteins to components of the cell cytoskeleton. Through the cytoskeleton connection, CAMs are able to control and regulate diverse intracellular processes associated with cell adhesion, cell proliferation, and cell migration. In addition, CAMs are implicated in many other cellular functions such as intercellular and intracellular communications, cell recognition, regulation of intercellular diffusion barrier, generation of immune responses, and apoptosis. From early embryonic development, every type of tissue at every stage of differentiation is defined by the expression of specific CAMs. Changes in the expression pattern of one or several CAMs may lead to pathologic changes during tissue differentiation or maturation. To date, about 50 CAMs have been identified, and they are classified on the bases of their molecular structure into four major families: cadherins, integrins, selectins, and the immunoglobin

1	To date, about 50 CAMs have been identified, and they are classified on the bases of their molecular structure into four major families: cadherins, integrins, selectins, and the immunoglobin superfamily (see Fig. 5.18).

1	 Cadherins are represented by transmembrane Ca2dependent CAMs localized mainly within the zonula adherens. At these sites, cadherins maintain homotypic interactions with similar proteins from the neighboring cell. They are associated with a group of intracellular proteins (catenins) that link cadherin molecules to actin flaments of the cell cytoskeleton. Through this interaction, cadherins convey signals that regulate mechanisms of growth and cell differentiation. Cadherins control cell-tocell interactions and participate in cell recognition and embryonic cell migration. E-cadherin, the most studied member of this family, maintains the zonula adherens junction between epithelial cells. It also acts as an important suppressor of epithelial tumor cells.  Integrins are represented by two transmembrane glycoprotein subunits consisting of 15 and 9 chains. This composition allows for the formation of different combinations of integrin molecules that are able to interact with

1	FIGURE 5.18 • Cell adhesion molecules (CAMs). Cadherin and immunoglobin superfamily (IgSF) CAMs exhibit homotypic binding in which two identical molecules from the neighboring cells interact. Binding that occurs between different types of CAMs (e.g., selectins and integrins) is considered heterotypic binding (no identical pair of molecules reacts with each other). various proteins (heterotypic interactions). Integrins interact with extracellular matrix molecules (such as collagens, laminin, and fibronectin) and with actin and intermediate flaments of the cell cytoskeleton. Through these interactions, integrins regulate cell adhesion, control cell movement and shape, and participate in cell growth and differentiation.

1	 Selectins are expressed on white blood cells (leukocytes) and endothelial cells and mediate neutrophil– endothelial cell recognition. This heterotypic binding initiates neutrophil migration through the endothelium of blood vessels into the extracellular matrix. Selectins are also involved in directing lymphocytes into accumulations of lymphatic tissue (homing procedure).

1	 Immunoglobulin superfamily (IgSF). Many molecules involved in immune reactions share a common precursor element in their structure. However, several other molecules with no known immunologic function also share this same repeat element. Together, the genes encoding these related molecules have been defined as the immunoglobulin gene superfamily. It is one of the largest gene families in the human genome, and its glycoproteins perform a wide variety of important biologic functions. IgSF members mediate homotypic cell-to-cell adhesions and are represented by the intercellular cell adhesion molecule (ICAM), cell–cell adhesion mole cule (C-CAM), vascular cell adhesion molecule (VCAM), Down syndrome cell adhesion molecule (DSCAM), platelet endothelial cell adhesion molecules (PECAM), junctional adhesion molecules (JAM), and many others. These proteins play key roles in cell adhesion and differentiation, cancer and tumor metastasis, angiogenesis (new vessel formation), inflammation,

1	adhesion molecules (JAM), and many others. These proteins play key roles in cell adhesion and differentiation, cancer and tumor metastasis, angiogenesis (new vessel formation), inflammation, immune responses, and microbial attachment, as well as many other functions.

1	The zonula adherens provides lateral adhesion between epithelial cells.

1	The integrity of epithelial surfaces depends in large part on the lateral adhesion of the cells with one another and their ability to resist separation. Although the zonula occludens involves a fusion of adjoining cell membranes, their resistance to mechanical stress is limited. Reinforcement of this region depends on a strong bonding site below the zonula occludens. Like the zonula occludens, this lateral adhesion device occurs in a continuous band or beltlike configuration around the cell; thus, the adhering junction is referred to as a zonula adherens. The zonula adherens is composed of the transmembrane cell adhesion molecule E-cadherin. On the cytoplasmic side, the tail of E-cadherin is bound to catenin (Fig. 5.19a). The resulting E-cadherin–catenin complex binds to vinculin and -actinin and is required for the interaction of cadherins with the actin filaments of the cytoskeleton. The extracellular components of the E-cadherin molecules from adjacent cells are linked by Ca2 ions

1	and is required for the interaction of cadherins with the actin filaments of the cytoskeleton. The extracellular components of the E-cadherin molecules from adjacent cells are linked by Ca2 ions or an additional extracellular link protein. Therefore, the morphologic and functional integrity of the zonula adherens is calcium dependent. Removal of Ca2 leads to dissociation of E-cadherin molecules and disruption of the junction. Recent studies indicate that the E-cadherin–catenin complex functions as a master molecule in regulating not only cell adhesion but also polarity, differentiation, migration, proliferation, and survival of epithelial cells.

1	When examined with the TEM, the zonula adherens is characterized by a uniform 15to 20-nm space between the opposing cell membranes (Fig. 5.19b). The intercellular space is of low electron density, appearing almost clear, but it is evidently occupied by extracellular components of adjacent E-cadherin molecules and Ca2 ions. Within the confines of the zonula adherens, a moderately electron-dense material called fuzzy plaque is found along the cytoplasmic side of the membrane of each cell. This material corresponds to the location of the cytoplasmic component of the Ecadherin–catenin complex and the associated proteins (-actinin and vinculin) into which actin filaments attach. Evidence also suggests that the fuzzy plaque represents the stainable substance in light microscopy, the terminal bar. Associated with the electron-dense material is an array of 6-nm actin flaments that stretch across the apical cytoplasm of the epithelial cell, the terminal web.

1	FIGURE 5.19 • Zonula adherens. a. Molecular organization of zonula adherens. Actin filaments of adjacent cells are attached to the E-cadherin–catenin complex by -actinin and vinculin. The E-cadherin–catenin complex interacts with identical molecules embedded in the plasma membrane of the adjacent cell. Interactions between transmembrane proteins are mediated by calcium ions. b. Electron micrograph of the zonula adherens from Figure 5.14a at higher magnification. The plasma membranes are separated here by a relatively uniform intercellular space. This space appears clear, showing only a sparse amount of diffuse electron-dense substance, which represents extracellular domains of E-cadherin. The cytoplasmic side of the plasma membrane exhibits a moderately electron-dense material containing actin filaments. 100,000. The fascia adherens is a sheetlike junction that stabilizes nonepithelial tissues.

1	The fascia adherens is a sheetlike junction that stabilizes nonepithelial tissues. Physical attachments that occur between cells in tissues other than epithelia are usually not prominent, but there is at least one notable exception. Cardiac muscle cells are arranged end to end, forming threadlike contractile units. The cells are attached to each other by a combination of typical desmosomes, or maculae adherentes, and broad adhesion plates that morphologically resemble the zonula adherens of epithelial cells. Because the attachment is not ringlike but rather has a broad face, it is called the fascia adherens (Fig. 5.20). At the molecular level, the structure of the fascia adherens is similar to that of the zonula adherens; it also contains the zonula occludens ZO-1 protein found in the tight junctions of epithelial cells. The macula adherens (desmosome) provides a localized spotlike junction between epithelial cells.

1	The macula adherens (desmosome) provides a localized spotlike junction between epithelial cells. The macula adherens [L. macula, spot] represents a major anchoring cell-to-cell junction that provides a particularly strong attachment, as shown by microdissection studies. The macula adherens was originally described in epidermal cells and was called a desmosome [Gr. desmo, bond soma, body]. These junctions are localized on the lateral domain of the cell, much like a series of spot welds (see Fig. 5.14a), and they mediate direct cell-to-cell contact by providing anchoring sites for intermediate filaments. Increasing evidence suggests that the macula adherens, in addition to its structural function, participates in tissue morphogenesis and differentiation.

1	In simple epithelium formed by cuboidal or columnar cells, the macula adherens is found in conjunction with occluding (zonula occludens) and adhering (zonula adherens) junctions. Because the macula adherens occupies small, localized sites on the lateral cell surface, it is not a continuous structure around the cell, as is the zonula adherens. Thus, a section perpendicular to the surface of a cell that cuts through the entire lateral surface will often not include a macula adherens. The section will always, however, include the zonula adherens. In the area of the macula adherens, desmogleins and desmocollins provide the linkage between the plasma membranes of adjacent cells.

1	In the area of the macula adherens, desmogleins and desmocollins provide the linkage between the plasma membranes of adjacent cells. FIGURE 5.20 • Fascia adherens. Electron micrograph showing the end-to-end apposition of two cardiac muscle cells. The intercellular space appears as a clear undulating area. On the cytoplasmic side of the plasma membrane of each cell, there is a dense material similar to that seen in a zonula adherens containing actin filaments. Because the attachment site here involves a portion of the end face of the two cells, it is called a fascia adherens. 38,000.

1	Electron microscopy reveals that the macula adherens has a complex structure. On the cytoplasmic side of the plasma membrane of each of the adjoining cells is a disc-shaped structure consisting of very dense material called the desmosomal attachment plaque. This structure measures about 400 250 10 nm and anchors intermediate flaments (Fig. 5.21a). The filaments appear to loop through the attachment plaques and extend back out into the cytoplasm. They are thought to play a role in dissipating physical forces throughout the cell from the attachment site. At the molecular level, each attachment plaque is composed of several constitutive proteins, mainly desmoplakins and plakoglobins, which are capable of anchoring the intermediate filaments (Fig. 5.21b).

1	The intercellular space of the macula adherens is conspicuously wider (up to 30 nm) than that of the zonula adherens and is occupied by a dense medial band, the intermediate line. This line represents extracellular portions of transmembrane glycoproteins, the desmogleins and desmocollins, which are members of the cadherin family of Ca2-dependent cell adhesion molecules. In the presence of Ca2, extracellular portions of desmogleins and desmocollins bind adjacent identical molecules of neighboring cells (homotypic binding). X-ray crystallographic studies suggest that the extracellular binding domain of proteins from one cell interacts with two adjacent cadherin domains in an antiparallel orientation, thus forming a continuous cadherin zipper in the area of the desmosome (see Fig. 5.21b). The cytoplasmic portions of desmogleins and desmocollins are integral components of the desmosomal attachment plaque. They interact with the plakoglobins and desmoplakins that are involved in desmosome

1	cytoplasmic portions of desmogleins and desmocollins are integral components of the desmosomal attachment plaque. They interact with the plakoglobins and desmoplakins that are involved in desmosome assembly and the anchoring of intermediate flaments.

1	The cells of different epithelia require different types of attachments. In epithelia that serve as physiologic barriers, the junctional complex is particularly significant because it serves to create a long-term barrier, allowing the cells to compartmentalize and restrict the free passage of substances across the epithelium. Although it is the zonula occludens of the junctional complex that principally affects this function, it is the adhesive properties of the zonulae and maculae adherentes that guard against physical disruption of the barrier. In other epithelia, there is need for substantially stronger attachment between cells in several planes. In the stratified epithelial cells of the epidermis, for example, numerous maculae adherentes maintain adhesion between adjacent cells. In cardiac muscle, where there is a similar need for strong adhesion, a combination of the macula adherens and the fascia adherens serves this function.

1	Communicating junctions, also called gap junctions or nexuses, are the only known cellular structures that permit the direct passage of signaling molecules from one cell to another. They are present in a wide variety of tissues, including epithelia, smooth and cardiac muscle, and nerves. Gap junctions are important in tissues in which activity of adjacent cells must be coordinated, such as epithelia engaged in fluid and electrolyte transport, vascular and intestinal smooth muscle, and heart muscle. A gap junction consists of an accumulation of transmembrane channels or pores in a tightly packed array. It allows cells to exchange ions, regulatory molecules, and small metabolites through the pores. The number of pores in a gap junction can vary widely, as can the number of gap junctions between adjacent cells.

1	FIGURE 5.21 • Molecular structure of the macula adherens (desmosome). a. Electron micrograph of a macula adherens, showing the intermediate filaments (arrows) attaching into a dense, intracellular attachment plaque located on the cytoplasmic side of the plasma membrane. The intercellular space is also occupied by electron-dense material (arrowheads) containing desmocollins and desmogleins. The intercellular space above and below the macula adherens is not well defined because of extraction of the plasma membrane to show components of this structure. 40,000. (Courtesy of Dr. Ernst Kallenbach.) b. Schematic diagram showing the structure of a macula adherens. Note the intracellular attachment plaque with anchored intermediate filaments. The extracellular portions of desmocollins and desmogleins from opposing cells interact with each other in the localized area of the desmosome, forming the cadherin “zipper.”

1	A variety of methods are used to study structure and function of gap junctions. Various procedures have been used to study gap junctions, including the injection of dyes and fluorescent or radiolabeled compounds and measuring the flow of an electric current between cells. In dye studies, a fluorescent dye is injected with a micropipette into one cell. After a short period, the dye can be readily visualized in immediately adjacent cells. Electrical conductance studies show that neighboring cells joined by gap junctions exhibit a low electrical resistance between them and current flow is high; therefore, gap junctions are also called low-resistance junctions.

1	Current molecular biology techniques allow for isolation of cDNA clones encoding a family of gap junction proteins (connexins) and expressing them in tissue culture cells. Connexins expressed in transfected cells produce gap junctions, which can be isolated and studied by molecular and biochemical methods as well as by the improved imaging techniques of electron crystallography and atomic force microscopy. Gap junctions are formed by 12 subunits of the connexin protein family.

1	Gap junctions are formed by 12 subunits of the connexin protein family. When viewed with the TEM, the gap junction appears as an area of contact between the plasma membranes of adjacent cells (Fig. 5.22a). High-resolution imaging techniques such as cryo-electron microscopy have been used to examine the structure of gap junctions. These studies reveal groups of tightly packed channels, each formed by two half-channels called connexons embedded in the facing membranes. These channels are represented by pairs of connexons that bridge the extracellular space between adjacent cells. The connexon in one cell membrane is precisely aligned to dock with a corresponding connexon on the membrane of an adjacent cell, thus, as the name implies, allowing communication between the cells.

1	Each connexon contains six symmetrical subunits of an integral membrane protein called connexin (Cx) that is paired with a similar structure from the adjacent membrane. Therefore, the entire channel consists of 12 subunits. The subunits are configured in a circular arrangement to surround a 10-nm-long cylindrical transmembrane channel with a diameter of 2.8 nm (Fig. 5.22b). About 21 members of the connexin family of proteins have been identified. All traverse the lipid bilayer four times (i.e., they have four transmembrane domains). Most connexons pair with identical connexons (homotypic interaction) on the adjacent plasma membrane. These channels allow molecules to pass evenly in both directions; however, heterotypic channels can be asymmetrical in function, passing certain molecules faster in one direction than in another.

1	FIGURE 5.22 • Structure of a gap junction. a. Electron micrograph showing the plasma membranes of two adjoining cells forming a gap junction. The unit membranes (arrows) approach one another, narrowing the intercellular space to produce a 2-nm-wide gap. 76,000. b. Drawing of a gap junction showing the membranes of adjoining cells and the structural components of the membrane that form channels or passageways between the two cells. Each passageway is formed by a circular array of six subunits, dumbbell-shaped transmembrane proteins that span the plasma membrane of each cell. These complexes, called connexons, have a central opening of about 2-nm in diameter. The channels formed by the registration of the adjacent complementary pairs of connexons permit the flow of small molecules through the channel but not into the intercellular space. Conversely, substances in the intercellular space can permeate the area of a gap junction by flowing around the connexon complexes, but they cannot

1	the channel but not into the intercellular space. Conversely, substances in the intercellular space can permeate the area of a gap junction by flowing around the connexon complexes, but they cannot enter the channels. c. The diameter of the channel in an individual connexon is regulated by reversible changes in the conformation of the individual connexins.

1	Conformational changes in connexins leading to opening or closing gap junction channels have been observed with atomic force microscopy. Earlier electron microscopy studies of isolated gap junctions suggested that the gap junction channels are opened and closed by twisting of the connexin subunits (Fig. 5.22c). Recent atomic force microscopy (AFM) studies provide a dynamic view of the conformational changes that take place in connexons. Channels in gap junctions can fluctuate rapidly between an open and a closed state through reversible changes in the conformation of individual connexins. The conformational change in connexin molecules that triggers closure of gap junction channels at their extracellular surface appears to be induced by Ca2 ions (Fig. 5.23). However, other calcium-independent gating mechanisms responsible for closing and opening of the cytoplasmic domains of gap junction channels have also been identified.

1	Mutations in connexin genes are major pathogenic factors in several diseases. For instance, a mutation in the gene encoding connexin-26 (Cx26) is associated with congenital deafness. The gap junctions formed by Cx26 are found in the inner ear and are responsible for recirculating K in the cochlear sensory epithelium. Other mutations affecting Cx46 and Cx50 genes have been identified in patients with inherited cataracts. Both proteins are localized within the lens of the eye and form extensive gap junctions between the epithelial cells and lens fibers. These gap junctions play a crucial role in delivering nutrients to and removing metabolites from the avascular environment of the lens. A summary of the features of all of the junctions discussed in this chapter is found in Table 5.4. Morphologic Specializations of the Lateral Cell Surface Lateral cell surface folds (plicae) create interdigitating cytoplasmic processes of adjoining cells.

1	Morphologic Specializations of the Lateral Cell Surface Lateral cell surface folds (plicae) create interdigitating cytoplasmic processes of adjoining cells. The lateral surfaces of certain epithelial cells show a tortuous boundary as a result of infoldings or plicae along the border of each cell with its neighbor (Fig. 5.24). These in-foldings increase the lateral surface area of the cell and are particularly prominent in epithelia that are engaged in fluid and electrolyte transport, such as the intestinal and

1	FIGURE 5.23 • Atomic force microscopic (AFM) image of a gap junction. This image shows the extracellular surface of a plasma membrane preparation from the HeLa cell line. Multiple copies of the connexin-26 gene were incorporated into the HeLa cell genome to achieve overexpression of the connexin protein. Connexin-26 proteins self-assemble into functional gap junctions, and they were observed with AFM in two different buffer solutions. a. Gap junction containing individual connexons in a calcium-free buffer solution. 500,000. Inset shows a single connexon at higher magnification. Note the clear profiles of individual connexin molecules assembled into the connexon. The open profile of the channel is also visible. 2,000,000. b. The same preparation of connexons in a buffer containing Ca2 . 500,000. Inset: Note that the conformational change of the connexin molecules has caused the channel to close and has reduced the height of the connexon. 2,000,000. (Courtesy of Dr. Gina E. Sosinsky.)

1	Inset: Note that the conformational change of the connexin molecules has caused the channel to close and has reduced the height of the connexon. 2,000,000. (Courtesy of Dr. Gina E. Sosinsky.) gallbladder epithelium. In active fluid transport, sodium ions are pumped out of the cytoplasm at the lateral plasma membrane by NaK-ATPase localized in that membrane. Anions then diffuse across the membrane to maintain electrical neutrality, and water diffuses from the cytoplasm into the intercellular space, driven by the osmotic gradient between the salt concentration in the intercellular space and the concentration in the cytoplasm. The intercellular space distends because of the accumulating fluid moving across the epithelium, but it can distend only to a limited degree because of junctional attachments in the apical and basal portions of the cell. Hydrostatic pressure gradually builds up in the intercellular space and drives an essentially isotonic fluid from the space into the underlying

1	in the apical and basal portions of the cell. Hydrostatic pressure gradually builds up in the intercellular space and drives an essentially isotonic fluid from the space into the underlying connective tissue. The occluding junction at the apical end of the inter-cellular space prevents fluid from moving in the opposite direction. As the action of the sodium pump depletes the cytoplasm of salt and water, these are replaced by diffusion across the apical plasma membrane, whose surface area is greatly increased by the presence of microvilli, thus allowing the continuous movement of fluid from the lumen to the connective tissue as long as the Na/K-ATPase is active.

1	The basal domain of epithelial cells is characterized by several features:  The basement membrane is a specialized structure lo cated next to the basal domain of epithelial cells and the underlying connective tissue stroma.  Cell-to-extracellular matrix junctions anchor the cell to the extracellular matrix; they are represented by focal adhesions and hemidesmosomes.  Basal cell membrane infoldings increase the cell sur adjacent cells and extracellular matrix proteins.

1	The term basement membrane was originally given to an amorphous, dense layer of variable thickness at the basal surfaces of epithelia. Although a prominent structure referred to as basement membrane is observed with hematoxylin and eosin (H&E) stain in a few locations such as the trachea (Fig. 5.25) and, occasionally, the urinary bladder and ureters, basement membrane requires special staining to be seen in the light microscope. This requirement is caused, in part, by its thinness and by the effect of the eosin stain, which makes it indistinguishable from the immediately adjacent connective tissue. In the trachea, the structure that is often described as basement membrane includes not only the true basement membrane but also an additional layer of closely spaced and aligned collagen fibrils that belong to the connective tissue.

1	In contrast to H&E (Fig. 5.26a), the periodic acid–Schiff (PAS) staining technique (Fig. 5.26b) results in a positive reaction at the site of the basement membrane. It appears as a thin, well-defined magenta layer between the epithelium and the connective tissue. The stain reacts with the sugar moieties of proteoglycans, accumulating in sufficient amounts and density to make the basement membrane visible in the light microscope. Techniques involving the chapter 5 Epithelial Tissue TH E BASAL DOMAI N AN D ITS SPECIALIZATIONS TABLE Summary of Junctional Features 5.4 Associated Major Intracellular Link Extracellular Cytoskeleton Attachment Classification Proteins Ligands Components Proteins Functions Zonula Occludins, Occludins, Actin ZO-1, ZO-2, Seals adjacent occludens claudins, claudins, flaments ZO-3, AF6, cells together, (tight junction) JAMs JAMs in cingulin controls adjacent cell symplectin passage of ASIP/Povr 3 molecules Rab 36, 13, 8 between them Sec 4, 6, 8 (permeability),

1	flaments ZO-3, AF6, cells together, (tight junction) JAMs JAMs in cingulin controls adjacent cell symplectin passage of ASIP/Povr 3 molecules Rab 36, 13, 8 between them Sec 4, 6, 8 (permeability), defnes apical domain of plasma membrane, involved in cell signaling Zonula E-cadherin-E-cadherin-Actin -Actinin, Couples the adherens catenin catenin flaments vinculin actin cytoske-complex complex in leton to the adjacent cell plasma mem-brane at regi-ons of cell-cell adhesion Macula Cadherins (e.g., Desmogleins, Intermediate Desmoplakins, Couples the adherens desmogleins, desmocollins flaments plakoglobins intermediate (desmosome) desmocollins) in adjacent flaments to cell the plasma membrane at regions of cell-cell adhesion Focal Integrins Extracellular Actin Vinculin, talin, Anchors the adhesion matrix flaments -actinin, actin cytoske-proteins (e.g., paxillin leton to the fbronectin) extracellular matrix, detects and transdu-ces signals from outside the cell Hemides-Integrins

1	the adhesion matrix flaments -actinin, actin cytoske-proteins (e.g., paxillin leton to the fbronectin) extracellular matrix, detects and transdu-ces signals from outside the cell Hemides-Integrins Extracellular Intermediate Desmoplakin-Anchors the mosome (64 matrix protein flaments like proteins, intermediate integrin), (e.g., (possible microtu-BP 230 flaments to collagen laminin-5, bules and actin plectin, the extrace-XVII collagen-IV) flaments via inter-erbin llular matrix action with plectin) Gap junction Connexin Connexin in None Not Known Creates a (nexus) adjacent cell conduct between two adjacent cells for passage of small ions and informational micro-molecules Occluding Junction(cell-to-cell) Anchoring Junctions(cell-to-cell) Communicating Junction (cell-to-cell) Anchoring Junctions(cell-to–extracellular matrix) hhhh

1	FIGURE 5.24 • Lateral interdigitations. This electron micrograph shows infoldings or interdigitations at the lateral surfaces of two adjoining intestinal absorptive cells. 25,000. reduction of silver salts by the sugars blacken the basement membrane and are also used to demonstrate this structure. Although the basement membrane is classically described as exclusively associated with epithelia, similar PAS-positive and silver-reactive sites can be demonstrated surrounding peripheral nerve supporting cells, adipocytes, and muscle cells (Fig. 5.27); this helps to delineate them from the surrounding connective tissue in histologic sections. Connective tissue cells other than adipocytes do not show a similar PAS-positive or silver reaction. That most connective tissue cells are not surrounded by basement membrane material is consistent with their lack of adhesion to the connective tissue fibers. In fact, they must migrate within the tissue under appropriate stimuli to function.

1	The basal lamina is the structural attachment site for overlying epithelial cells and underlying connective tissue. Former descriptions of basal lamina were based on the investigation of specimens routinely prepared for electron microscopy. The examination of the site of epithelial base- FIGURE 5.25 • Tracheal basement membrane. Photomicrograph of an H&E–stained section of the pseudostratified ciliated epithelium of the trachea. The basement membrane appears as a thick homogeneous layer immediately below the epithelium. It is actually a part of the connective tissue and is composed largely of densely packed collagen fibrils. 450.

1	ment membranes with the EM reveals a discrete layer of electron-dense matrix material 40to 60-nm thick between the epithelium and the adjacent connective tissue (Fig. 5.28) called the basal lamina or, sometimes, lamina densa. When observed at high resolution, this layer exhibits a network of fine, 3to 4-nm filaments composed of laminins, a type IV collagen molecule, and various associated proteoglycans and glycoproteins. Between the basal lamina and the cell is a relatively clear or electron-lucent area, the lamina lucida (also about 40 nm wide). The area outlined by the lamina lucida contains extracellular portions of CAMs, mainly fbronectin and laminin receptors. These receptors are members of the integrin family of transmembrane proteins. With the development of new EM preparation techniques, the lamina lucida appears to be an artifact of fixation; in the living state, the basal lamina is composed of a single layer of the lamina densa.

1	If the tissue specimen for EM is fixed using low-temperature, high-pressure freezing (HPF) methods (without chemical fixatives), it retains much more of the tissue than specimens routinely fixed with glutaraldehyde. EM examination of such specimens reveals that the basal lamina is composed only of the lamina densa. No lamina lucida is detected. The lamina lucida may thus be an artifact of chemical fixation that appears as the epithelial cells shrink away from a high concentration of macromolecules deposited next to the basal domain of the epithelial cells. It probably results from the rapid dehydration that occurs during tissue processing for electron microscopy. Other structures visible with traditional electron microscopy are also not visible when tissues are prepared by the HPF method (Fig. 5.29).

1	FIGURE 5.26 • Photomicrographs showing serial sections of intestinal glands of the colon. The glands in this specimen have been cross-sectioned and appear as round profiles. a. This specimen was stained with H&E. Note that neither the basement membrane nor the mucin that is located within the goblet cells is stained. 550. b. This section was stained by the PAS method. It reveals the basement membrane as a thin, magenta layer (arrows) between the base of the epithelial cells of the glands and the adjacent connective tissue. The mucin within the goblet cells is also PAS positive. 550.

1	FIGURE 5.27 • Smooth muscle external lamina. This photomicrograph is stained by the PAS method and counterstained with hematoxylin (pale nuclei). The muscle cells have been cut in cross section and appear as polygonal profiles because of the presence of PAS-positive basement membrane material surrounding each cell. The cytoplasm is not stained. As the plane of section passes through each smooth muscle cell, it may or may not pass through the portion of the cell that includes the nucleus. Therefore, in some of the polygonal profiles, nuclei can be seen; in other profiles, no nuclei are seen. 850. The basal lamina in nonepithelial cells is referred to as the external lamina.

1	The basal lamina in nonepithelial cells is referred to as the external lamina. Muscle cells, adipocytes, and peripheral nerve supporting cells exhibit an extracellular electron-dense material that resembles the basal lamina of epithelium. This material also corresponds to a PAS-positive staining reaction, as described earlier (see Fig. 5.27). Although the term basement membrane is not ordinarily applied to the extracellular stainable material of these nonepithelial cells in light microscopy, the terms basal lamina or external lamina are typically used at the EM level. The basal lamina contains molecules that come together to form a sheetlike structure.

1	The basal lamina contains molecules that come together to form a sheetlike structure. Analyses of basal laminae derived from epithelia in many locations (kidney glomeruli, lung, cornea, lens of the eye) indicate that they consist of approximately 50 proteins that can be classified into four groups: collagens, laminins, glycoproteins, and proteoglycans. These proteins are synthesized and secreted by the epithelial cells and other cell types that possess an external lamina.  Collagens. At least three types of collagen species are present in the basal lamina; they represent a fraction of the approximately 28 types of collagen found in the body.

1	The major component, comprising 50% of all basal used inconsistently in the literature. Some authors use basement membrane when referring to both light and electron microscopic images. Others dispense with the term basement membrane altogether and use basal lam-ina in both light and electron microscopy. Because the term basement membrane originated with light mi-croscopy, it is used in this book only in the context of light microscopic descriptions and only in relation to epithelia. tural content to denote the layer present at the interface of connective tissue with epithelial cells. In this context, the light microscopy term basement membrane actually de-scribes basal lamina and the underlying reticular lamina combined. The term external lamina is used to identify basal lamina when it forms a peripheral cellular invest-ment, as in muscle cells and peripheral nerve supporting cells.  FOLDER 5.4 Functional Considerations: Basement Membrane and Basal Lamina Terminology

1	 FOLDER 5.4 Functional Considerations: Basement Membrane and Basal Lamina Terminology The terms basement membrane and basal lamina are The EM term basal lamina is reserved for the ultrastruc- FIGURE 5.28 • Electron micrograph of two adjoining epithelial cells with their basal lamina. The micrograph shows only the basal portions of the two cells and parts of their nuclei (N). The intercellular space is partially obscured by lateral interdigitations between the two cells (arrows). The basal lamina (BL) appears as a thin layer that follows the contours of the basal domain of the overlying cell. Below the basal lamina are numerous cross-sectioned collagen (reticular) fibrils. 30,000.

1	lamina proteins, is type IV collagen. The molecular characteristics and function of type IV collagen in forming a scaffold of basal lamina is described in the next section. The presence of different type IV collagen isoforms provides specificity to the basal lamina associated with different tissues. Two nonfibrillar types of collagens, type XV collagen and type XVIII collagen, are also found in the basal lamina. Type XV collagen plays an important role in stabilizing the structure of the external lamina in skeletal and cardiac muscle cells, whereas type XVIII collagen is mainly present in vascular and epithelial basal laminae and is believed to function in angiogenesis. In addition, type VII collagen forms anchoring fibrils that link the basal lamina to the underlying reticular lamina (described below).

1	 Laminins. These cross-shaped glycoprotein molecules (140 to 400 kilodaltons) are composed of three polypeptide chains. They are essential in initiating the assembly of the basal lamina. Laminins possess binding sites for different integrin receptors in the basal domain of the overlying epithelial cells. They are involved in many cellto–extracellular matrix interactions. They also play roles in the development, differentiation, and remodeling of epithelium. There are approximately 15 different variations of laminin molecules.  Entactin/nidogen. This small, rodlike sulfated glycoprotein (150 kilodaltons) serves as a link between laminin and the type IV collagen network in almost all basal laminae. Each entactin molecule is organized into distinct domains that bind calcium, support cell adhesion, promote neu

1	FIGURE 5.29 • Electron micrograph of epithelial cells preserved by low-temperature, high-pressure freezing. This electron micrograph shows basal domain of an epithelial cell obtained from human skin. The specimen was prepared by low-temperature, high-pressure freezing, which retains more tissue components than does chemical fixation. Note that a separate lamina densa or lamina lucida is not seen in this preparation. The lamina lucida is most likely an artifact that appears as the epithelial cell shrinks away from a high concentration of macromolecules just basal of the epithelial cell. This region of highly concentrated macromolecules precipitates into the artifact known as the lamina densa. BL, basal lamina; HD, hemidesmosome; CF, collagen fibrils. 55,000. (Courtesy of Douglas R. Keene.) trophil chemotaxis and phagocytosis, and interact with laminin, perlecan, fibronectin, and type IV collagen.

1	 Proteoglycans. Most of the volume of the basal lamina is probably attributable to its proteoglycan content. Proteoglycans consist of a protein core to which heparan sulfate (e.g., perlecan, agrin), chondroitin sulfate (e.g., bamacan), or dermatan sulfate side chains are attached. Because of their highly anionic character, these molecules are extensively hydrated. They also carry a high negative charge; this quality suggests that proteoglycans play a role in regulating the passage of ions across the basal lamina. The most common heparan sulfate proteoglycan found in all basal laminae is the large multidomain proteoglycan perlecan (400 kilodaltons). It provides additional cross-links to the basal lamina by binding to laminin, type IV collagen, and entactin/nidogen. Agrin (500 kilodaltons) is another important molecule found almost exclusively in the glomerular basement membrane of the kidney. It plays a major role in renal filtration as well as in cell-to–extracellular matrix

1	is another important molecule found almost exclusively in the glomerular basement membrane of the kidney. It plays a major role in renal filtration as well as in cell-to–extracellular matrix interactions.

1	The molecular structure of type IV collagen determines its role in the formation of the basal lamina network suprastructure. The type IV collagen molecule is similar to other collagens in that it contains three polypeptide chains. Each chain has a short amino-terminus domain (7S domain), a long middle collagenous helical domain (which interacts with the remaining two chains in the fully assembled molecule), and a carboxy-terminus globular noncollagenous domain (NC1 domain). The six known chains of type IV collagen molecules (1 to 6) form three sets of triple helical molecules known as collagen protomers. They are designated as [1(IV)]22(IV); 3(IV)4(IV)5(IV), and [5(IV)]26(IV) protomers (see Table 6.2).

1	Protomer assembly begins when the three NC1 domains assemble to form an NC1 trimer (Fig. 5.30). The next step in the assembly of the basal lamina structure is the formation of type IV collagen dimer molecules. This is achieved when two NC1 trimers interact to form an NC1 hexamer. Next, four dimers join in the region of the 7S domain to form a tetramer. The 7S domain of the tetramer (called the 7S box) determines the geometry of the tetramer. Finally, the type IV collagen scaffold is formed when other collagen tetramers interact end to end with each other. This scaffold forms the suprastructure of the basal lamina. Assembly of this suprastructure is genetically determined. Those containing [1(IV)]22(IV) protomers are found in all basal laminae. Those containing 3(IV)4 (IV)5(IV) protomers occur mainly in the kidney and lungs, and those containing [5(IV)]26(IV) protomers are restricted to the skin, esophagus, and Bowman capsule in the kidney.

1	Basal lamina self-assembly is initiated by the polymerization of laminins on the basal cell domain and interaction with the type IV collagen suprastructure.

1	The constituents of the basal lamina come together in a process of self-assembly to form a sheetlike structure. This process is initiated by both type IV collagen and laminins. The primary sequence of these molecules contains information for their self-assembly (other molecules of the basal lamina are incapable of forming sheetlike structures by themselves). Studies using cell lines have shown that the first step in self-assembly of the basal lamina is calcium-dependent polymerization of laminin molecules on the basal cell surface domain (Fig. 5.31). This process is aided by CAMs (integrins). At the same time, the type IV collagen suprastructure becomes associated with laminin polymers. These two structures are joined together primarily by entactin/nidogen bridges and are additionally secured by other proteins (perlecan, agrin, fibronectin, and so forth). The scaffold of type IV collagen and laminins provides the site for other basal lamina molecules to interact and form the fully

1	secured by other proteins (perlecan, agrin, fibronectin, and so forth). The scaffold of type IV collagen and laminins provides the site for other basal lamina molecules to interact and form the fully functional basal lamina.

1	FIGURE 5.30 • Formation of the type IV collagen suprastructure. Each type IV collagen molecule has three domains: an amino-terminus (7S domain), a middle collagenous helical domain, and a carboxy-terminus (NC1 domain). The NC1 domain initiates assembly of the type IV collagen protomer, which consists of three molecules. Protomer formation proceeds like a zipper from the NC1 domain toward the 7S domain, resulting in a fully assembled protomer. The next step in assembly is the dimerization of type IV collagen protomers. Two type IV collagen protomers become connected via their NC1 domains, and their two NC1 trimers join together to form an NC1 hexamer. Next, four dimers join together at their 7S domains to form tetramers connected by the 7S box. These tetramers interact to form the type IV collagen suprastructure via their interactions with the 7S domains of other tetramers and also by lateral associations between type IV collagen protomers.

1	A layer of reticular fibers underlies the basal lamina.

1	There is still lack of agreement about the extent to which the basal lamina seen with the EM corresponds to the structure described as the basement membrane in the light microscope. Some investigators contend that the basement membrane includes not only the basal lamina but also a secondary layer of small-unit fibrils of type III collagen (reticular fbers) that forms the reticular lamina. The reticular lamina, as such, belongs to the connective tissue and is not a product of the epithelium. The reticular lamina was once regarded as the component that reacted with silver, whereas the polysaccharides of the basal lamina and the ground substance associated with the reticular fibers were thought to be the components stained with the PAS reaction. However, convincing arguments can be made for the basal lamina reacting with both PAS and silver in several sites. In normal kidney glomeruli, for example, no collagen (reticular) fibers are associated with the basal lamina of the epithelial

1	for the basal lamina reacting with both PAS and silver in several sites. In normal kidney glomeruli, for example, no collagen (reticular) fibers are associated with the basal lamina of the epithelial cells (Fig. 5.32), although a positive reaction occurs with both PAS staining and silver impregnation. Also, in the spleen, where the basal lamina of the venous sinuses forms a unique pattern of ringlike bands rather than a thin, sheathlike layer around the vessel, exactly corresponding images are seen with the PAS and silver techniques as well as with the EM (Fig. 5.33).

1	Several structures are responsible for attachment of the basal lamina to the underlying connective tissue. On the opposite side of the basal lamina, the connective tissue side, several mechanisms provide attachment of the basal lamina to the underlying connective tissue:  Anchoring fbrils (type VII collagen) are usually found in close association with hemidesmosomes. They extend from the basal lamina either to the structures called anchoring plaques in the connective tissue matrix or loop back to the basal lamina (Fig. 5.34). The anchoring fibrils entrap type III collagen (reticular) fibers in the underlying connective tissue, which ensures sound epithelial anchorage. Anchoring fibrils are critical to the function of the anchoring junctions; mutations in the collagen VII gene result in dystrophic epidermolysis bullosa, an inherited blistering skin disease in which the epithelium is detached below the basement membrane.

1	 Fibrillin microfbrils are 10 to 12 nm in diameter and attach the lamina densa to elastic fibers. Fibrillin microfibrils are known to have elastic properties. A mutation in the fibrillin gene (FBN1) causes Marfan’s syndrome and other related connective tissue disorders.  Discrete projections of the lamina densa on its connective tissue side interact directly with the reticular lamina to form an additional binding site with type III collagen. An interwoven network of proteins provides the bases for a variety of basal lamina functions. In recent years, the basal lamina has been recognized as an important regulator of cell behavior rather than just a structural feature of the epithelial tissue. Organ-specific molecules have been identified in the basal lamina. Although morphologically single molecule of laminin integrin receptors

1	FIGURE 5.31 • Molecular components of the basal lamina. To produce a basal lamina, each epithelial cell must first synthesize and secrete its molecular components. The assembly of the basal lamina occurs outside the cell at its basal domain. The calcium-dependent polymerization of laminin molecules that occurs at the basal cell surface initiates basal lamina formation. Laminin polymers are next anchored to the cell surface by integrin receptors. At the same time, the type IV collagen suprastructure is assembled (see Fig. 5.30) in close proximity to laminin polymers. These two structures are connected by entactin or nidogen bridges and are additionally secured by other proteins (i.e., perlecan). The primary scaffold of type IV collagen connected to laminin polymers provides the site for other basal lamina molecules to interact and form the fully functional basal lamina.

1	LEnPEnBLPPNLL En P En BL P P N L FIGURE 5.32 • Basal lamina in the kidney glomerulus. Electron micrograph of a kidney glomerular capillary showing the basal lamina (BL) interposed between the capillary endothelial cell (En) and the cytoplasmic processes (P; podocytes) of epithelial cells. The epithelial cell is located on the outer (abluminal) surface of the endothelial cell. 12,000. Inset. Relationship at higher magnification. Note that the endothelial cells and epithelial cells are separated by the shared basal lamina and that no collagen fibrils are present. N, nucleus of epithelial cell; L, lumen of capillary. 40,000.

1	FIGURE 5.33 • Demonstration of basement membrane material in splenic vessels. a. Photomicrograph of a silver preparation revealing two longitudinally sectioned venous sinuses in the spleen. These blood vessels are surrounded by a modified basement membrane, which takes the form of a ringlike structure, much like the hoops of a barrel, rather than a continuous layer or lamina. The rings are blackened by the silver and appear as bands where the walls of the vessel have been tangentially sectioned (arrows). To the right, the cut has penetrated deeper into the vessel and shows the lumen (L). Here the cut edges of the rings are seen on both sides of the vessel. In the lower vessel, the cut rings have been sectioned in a virtually perpendicular plane, and the rings appear as a series of dots. 400. b. Electron micrograph of the wall of a venous sinus, showing a longitudinally sectioned endothelial cell (EnC). The nucleus (N) of the cell is protruding into the lumen (L). The basal lamina

1	400. b. Electron micrograph of the wall of a venous sinus, showing a longitudinally sectioned endothelial cell (EnC). The nucleus (N) of the cell is protruding into the lumen (L). The basal lamina material (asterisks) has the same homogeneous appearance as seen by electron microscopy in other sites except that it is aggregated into ringlike structures rather than into a flat layer or lamina. Moreover, its location and plane of section correspond to the silver-reactive, dotlike material in the panel above. 25,000.

1	all basal laminae appear similar, their molecular composition including all of its specialized tissues, such as bone and car-and functions are unique to each tissue. The following are tilage (with the exception of adipose tissue, in that its cells various functions now attributed to the basal lamina. possess an external lamina)—can be viewed as a single,  Structural attachment. As noted, the basal lamina serves continuous compartment. In contrast, epithelia, muscles, and nerves are separated from adjacent connective tissue as an intermediary structure in the attachment of cells to by intervening basal or external laminae. For any substance the adjacent connective tissue. Epithelial cells are anchored to move from one tissue to another (e.g., from one com- into the basal lamina by cell-to–extracellular matrix juncpartment to another), it must cross such a lamina.

1	tions, and the basal lamina is attached to underlying con nective tissue by anchoring fibrils and fibrillin microfibrils.  Filtration. The movement of substances to and from the  Compartmentalization. Structurally, basal and external connective tissue is regulated in part by the basal lamina, largely through ionic charges and integral spaces. Filtration laminae separate or isolate the connective tissue from epis well characterized in the kidney, in which the plasma ithelia, nerve, and muscle tissues. Connective tissue— projections of lamina densa

1	FIGURE 5.34 • Schematic diagram and electron micrograph of the basal portion of epithelial cell. a. This diagram shows the cellular and extracellular components that provide attachment between epithelial cells and the underlying connective tissue. On the connective tissue side of the basal lamina, anchoring fibrils extend from the basal lamina to the collagen (reticular) fibrils of the connective tissue, providing structural attachment at this site. On the epithelial side, laminin (green), collagen XVII (red), and integrins (yellow) are present in the lamina lucida and lamina densa and provide adhesion between the basal lamina and the intracellular attachment plaques of hemidesmosomes. b. This high-magnification electron micrograph of human skin shows the basal portion of human epithelial cells with underlying basal lamina. The electron-lucent space, the lamina lucida located just below the basal cell membrane, is occupied by anchoring filaments formed by the laminin-5 and type XVII

1	cells with underlying basal lamina. The electron-lucent space, the lamina lucida located just below the basal cell membrane, is occupied by anchoring filaments formed by the laminin-5 and type XVII collagen molecules. Anchoring filaments are responsible for attaching the basal cell membrane to the basal lamina. The looplike fibers originating from the basal lamina represent anchoring fibrils of type VII collagen that link the basal lamina with the reticular fibers (type III collagen) and with anchoring plaques located within the extracellular matrix. 200,000. (Courtesy of Douglas R. Keene.) filtrate must cross the compound basal laminae of capillar-processes of a cell use the basal lamina that remains after cell ies and adjacent epithelial cells to reach the urinary space loss, thus helping to maintain the original tissue architec within a renal corpuscle. ture. For example, when nerves are damaged, new neuro Tissue scaffolding. The basal lamina serves as a guide or muscular

1	helping to maintain the original tissue architec within a renal corpuscle. ture. For example, when nerves are damaged, new neuro Tissue scaffolding. The basal lamina serves as a guide or muscular junctions from a growing axon will be established scaffold during regeneration. Newly formed cells or growing only if the external lamina remains intact after injury. The basal laminae also allow cells to migrate under physiologic Focal adhesions create a dynamic link between the actin conditions but act as barriers against tumor cell invasion.

1	 Regulation and signaling. Many molecules that reside in the basal lamina interact with cell surface receptors, influencing epithelial cell behavior during morphogenesis, fetal development, and wound healing by regulating cell shape, proliferation, differentiation, and motility as well as gene expression and apoptosis. For instance, the basal lamina of endothelial cells has recently been found to be involved in the regulation of tumor angiogenesis. The organization of cells in epithelium depends on the support provided by the extracellular matrix on which the basal surface of each cell rests. Anchoring junctions maintain the morphologic integrity of the epithelium–connective tissue interface. The two major anchoring junctions are:  focal adhesions, which anchor actin filaments of the cytoskeleton into the basement membrane; and hemidesmosomes, which anchor the intermediate filaments of the cytoskeleton into the basement membrane.

1	In addition, transmembrane proteins located in the basal cell domain (mainly related to the integrin family of adhesion molecules) interact with the basal lamina. cytoskeleton and extracellular matrix proteins. Focal adhesions form a structural link between the actin cytoskeleton and extracellular matrix proteins. They are responsible for attaching long bundles of actin filaments (stress fibers) into the basal lamina (Fig. 5.35a). Focal adhesions play a prominent role during dynamic changes that occur in epithelial cells (e.g., migration of epithelial cells in wound repair). Coordinated remodeling of the actin cytoskeleton and the controlled formation and dismantling of focal adhesions provide the molecular bases for cell migration. Focal adhesions are also found in other nonepithelial cells such as fibroblasts and smooth muscle cells.

1	In general, focal adhesions consist of a cytoplasmic face to which actin flaments are bound, a transmembrane connecting region, and an extracellular face that binds to the proteins of the extracellular matrix. The main family of transmembrane proteins involved in focal adhesions are integrins, which are concentrated in clusters within the areas where the junctions can be detected. On the cytoplasmic face, integrins interact with actin-binding proteins (-actinin, vinculin, talin, paxillin) as well as many regulatory proteins such as focal adhesion kinase or tyrosine kinase (Fig. 5.35b). On the extracellular side, integrins bind to extracellular matrix glycoproteins, usually laminin and fibronectin.

1	FIGURE 5.35 • Molecular structure of focal adhesions. a. Diagram showing the molecular organization of focal adhesions. On the cytoplasmic side, note the arrangement of different actin-binding proteins. These proteins interact with integrins, the transmembrane proteins, the extracellular domains of which bind to proteins of the extracellular matrix (e.g., fibronectin). b. This image was obtained from the fluorescence microscope and shows cells cultured on the fibronectin-coated surface stained with fluoresceinlabeled phalloidin to visualize actin filaments (stress fibers) in green. Next, using indirect immunofluorescence techniques, focal adhesions were labeled with primary monoclonal antibody against phosphotyrosines and visualized with secondary rhodamine-labeled antibody (red). The phosphotyrosine is a product of the tyrosine kinase reaction in which tyrosine molecules of the associated proteins are phosphorylated by this enzyme. Tyrosine kinase is closely associated with focal

1	phosphotyrosine is a product of the tyrosine kinase reaction in which tyrosine molecules of the associated proteins are phosphorylated by this enzyme. Tyrosine kinase is closely associated with focal adhesion molecules, so the area where focal adhesions are formed is labeled red. Note the relationship of focal adhesions and actin filaments at the periphery of the cell. 3,000. (Courtesy of Dr. Keith Burridge.)

1	Focal adhesions play an important role in sensing and transmitting signals from the extracellular environment into the interior of the cell. Focal adhesions are also important sites of signal detection and transduction. They are able to detect contractile forces or mechanical changes in the extracellular matrix and convert them into biochemical signals. This phenomenon, known as mechanosensitivity, allows cells to alter their adhesion-mediated functions in response to external mechanical stimuli. Integrins transmit these signals to the interior of the cell, where they affect cell migration, differentiation, and growth. Recent studies indicate that focal adhesion proteins also serve as a common point of entry for signals resulting from stimulation of various classes of growth factor receptors. Hemidesmosomes occur in epithelia that require strong, stable adhesion to the connective tissue.

1	Hemidesmosomes occur in epithelia that require strong, stable adhesion to the connective tissue. A variant of the anchoring junction similar to the desmosome is found in certain epithelia subject to abrasion and mechanical shearing forces that would tend to separate the epithelium from the underlying connective tissue. Typically, it occurs in the cornea, the skin, and the mucosa of the oral cavity, esophagus, and vagina. In these locations, it appears as if half the desmosome is present, hence the name hemidesmosome. Hemidesmosomes are found on the basal cell surface, where they provide increased adhesion to the basal lamina (Fig. 5.36a).

1	When observed with the EM, the hemidesmosome exhibits an intracellular attachment plaque on the cytoplasmic side of the basal plasma membrane. The protein composition of this structure is similar to that of the desmosomal plaque, as it contains a desmoplakin-like family of proteins capable of anchoring intermediate filaments of the cytoskeleton. Three major proteins have been identified in the plaque:  Plectin (450 kilodaltons) functions as a cross-linker of the intermediate filaments that bind them to the hemidesmosomal attachment plaque. Recent studies indicate that plectin also interacts with microtubules, actin filaments, and myosin II. Thus, plectin cross-links and integrates all elements of the cytoskeleton.

1	 BP 230 (230 kilodaltons) attaches intermediate filaments to the intercellular attachment plaque. The absence of functional BP 230 causes bullous pemphigoid, a disease characterized clinically by blister formation. High levels of antibodies directed against components of the hemidesmosome, including antibodies against BP 230 and type XVII collagen, are detected in people with this disease. For this reason, BP 230 is called bullous pemphigoid antigen 1 (BPAG1), and the collagen XVII molecule is called bullous pemphigoid antigen 2 (BPAG2) or BP 180.  Erbin (180 kilodaltons) mediates association of BP 230 with integrins.

1	FIGURE 5.36 • Molecular structure of hemidesmosome. a. Electron micrograph of the basal aspect of a gingival epithelial cell. Below the nucleus (N), intermediate filaments are seen converging on the intracellular attachment plaques (arrows) of the hemidesmosome. Below the plasma membrane are the basal lamina (BL) and collagen (reticular) fibrils (most of which are cut in cross section) of the connective tissue. 40,000. b. Diagram showing the molecular organization of a hemidesmosome. The intracellular attachment plaque is associated with transmembrane adhesion molecules, such as the family of integrins and transmembrane type XVII collagen, and contains plectin, BP 230, and erbin. Note that the intermediate filaments seem to originate or terminate in the intracellular attachment plaque. Extracellular portions of integrins bind to laminin-5 and type IV collagen. With the help of anchoring fibrils (type VII collagen), laminin, and integrin, the attachment plaque is secured to the

1	Extracellular portions of integrins bind to laminin-5 and type IV collagen. With the help of anchoring fibrils (type VII collagen), laminin, and integrin, the attachment plaque is secured to the reticular fibers (type III collagen) of the extracellular matrix.

1	In contrast to the desmosome, whose transmembrane proteins belong to the cadherin family of calcium-dependent molecules, the majority of transmembrane proteins found in the hemidesmosome belong to the integrin class of cell matrix receptors. These include:  46 integrin, a heterodimer molecule containing two polypeptide chains. Its extracellular domain enters the basal lamina and interacts with type IV collagen supra structure containing laminins (laminin-5), entactin/nidogen or the perlecan. On the extracellular surface of the hemidesmosome, laminin-5 molecules form threadlike anchoring flaments that extend from the integrin molecules to the structure of the basement membrane (Fig. 5.36b). Interaction between laminin-5 and 64 integrin is essential for hemidesmosome formation and for the maintenance of epithelial adhesion. Mutation of the genes encoding laminin-5 chains results in junctional epidermolysis bullosa, another hereditary blistering skin disease.

1	 Type XVII collagen (BPAG2, BP 180), a transmembrane molecule (180 kilodaltons), that regulates expression and function of laminin-5. In experimental models, type XVII collagen inhibits migration of endothelial cells during angiogenesis and regulates keratinocyte migration in the skin (see Fig. 5.36b).  CD151 (32 kilodaltons), a glycoprotein that participates in the clustering of integrin receptors to facilitate cellto–extracellular matrix interactions. Despite their similarity in names, the terms anchoring filaments and anchoring fibrils do not describe the same structure. Anchoring flaments are formed mainly by laminin-5 and type XVII collagen molecules. They attach the basal cell membrane of epithelial cells into the underlying basal lamina. Anchoring fbrils are formed by type VII collagen and attach the basal lamina to the underlying reticular fibers (see page 140). Morphologic Modifications of the Basal Cell Surface

1	Morphologic Modifications of the Basal Cell Surface Many cells that transport fluid have infoldings at the basal cell surface. They significantly increase the surface area of the basal cell domain, allowing for more transport proteins and channels to be present. These basal surface modifications are prominent in cells that participate in active transport of molecules (e.g., in proximal and distal tubules of the kidney; Fig. 5.37) and in certain ducts of the salivary glands. Mitochondria are typically concentrated at this basal site to provide the energy requirements for active transport. The mitochondria are usually oriented vertically within the folds. The orientation of the mitochondria, combined with the basal membrane infoldings, results in a striated appearance along the basal aspect of the cell when observed in the light microscope. Because of this phenomenon, the salivary gland ducts that possess these cells are referred to as striated ducts.

1	FIGURE 5.37 • Basal infoldings. Electron micrograph of the basal portion of a kidney tubule cell showing the infolding of the plasma membrane. Note the aligned mitochondria. The infoldings of adjoining cells result in the interdigitations of cytoplasm between the two cells. 25,000. Typically, glands are classified into two major groups according to how their products are released (Table 5.5):  Exocrine glands secrete their products onto a surface directly or through epithelial ducts or tubes that are connected to a surface. Ducts may convey the secreted material in an unaltered form or may modify the secretion by concentrating it or adding or reabsorbing constituent substances.  Endocrine glands lack a duct system. They secrete their products into the connective tissue, from which they enter the bloodstream to reach their target cells. The products of endocrine glands are called hormones.

1	In some epithelia, individual cells secrete a substance that does not reach the bloodstream but rather affects other cells within the same epithelium. Such secretory activity is referred to as paracrine. The secretory material reaches the target cells by diffusion through the extracellular space or immediately subjacent connective tissue. TABLE Types of Glands 5.5 Exocrine Glands Endocrine Paracrine Merocine Apocrine Holocrine Glands Glands Cells of exocrine glands exhibit different mechanisms of secretion. The cells of exocrine glands have three basic release mechanisms for secretory products (see Table 5.5):  Merocrine secretion. This secretory product is delivered in membrane-bounded vesicles to the apical surface of the cell. Here vesicles fuse with the plasma membrane and extrude their contents by exocytosis. This is the most common mechanism of secretion and is found, for example, in pancreatic acinar cells.

1	 Apocrine secretion. The secretory product is released in the apical portion of the cell, surrounded by a thin layer of cytoplasm within an envelope of plasma membrane. This mechanism of secretion is found in the lactating mammary gland, where it is responsible for releasing large lipid droplets into the milk. It also occurs in the apocrine glands of skin, ciliary (Moll’s) glands of the eyelid, and the ceruminous glands of the external auditory meatus.  Holocrine secretion. The secretory product accumulates within the maturing cell, which simultaneously undergoes programmed cell death. Both secretory products and cell debris are discharged into the lumen of the gland. This mechanism is found in sebaceous glands of skin and the tarsal (Meibomian) glands of the eyelid. Exocrine glands are classified as either unicellular or multicellular.

1	Exocrine glands are classified as either unicellular or multicellular. Unicellular glands are the simplest in structure. In unicellular exocrine glands, the secretory component consists of single cells distributed among other nonsecretory cells. A typical example is the goblet cell, a mucus-secreting cell positioned among other columnar cells (Fig. 5.38). Goblet cells are located in the surface lining and glands of the intestines and in certain passages of the respiratory tract. Multicellular glands are composed of more than one cell. They exhibit varying degrees of complexity. Their structural organization allows subclassification according to the arrangement of the secretory cells (parenchyma) and the presence or absence of branching of the duct elements. The simplest arrangement of a multicellular gland is a cellular sheet in which each surface cell is a secretory cell. For example, the lining of the stomach and its gastric pits is a sheet of mucus-secreting cells (Fig. 5.39).

1	FIGURE 5.38 • Unicellular glands. Photomicrograph of intestinal epithelium showing single goblet cells (arrows) dispersed among absorptive cells. Each goblet cell may be regarded as a unicellular gland—the simplest exocrine type gland. 350. FIGURE 5.39 • Mucus-secreting surface cells of stomach. Photomicrograph of stomach surface. The epithelial cells lining the surface are all mucus-secreting cells, as are the cells lining the gastric pits (P). The cells of the gastric pit form simple tubular glands. 260.

1	Other multicellular glands typically form tubular invaginations from the surface. The end pieces of the gland contain the secretory cells; the portion of the gland connecting the secretory cells to the surface serves as a duct. If the duct is unbranched, the gland is called simple; if the duct is branched, it is called compound. If the secretory portion is shaped like a tube, the gland is tubular; if it is shaped like a flask, the gland is alveolar or acinar; if the tube ends in a saclike dilation, the gland is tubuloalveolar. Tubular secretory portions may be straight, branched, or coiled; alveolar portions may be single or branched. Various combinations of duct and secretory portion shapes are found in the body. Classification and description of exocrine glands may be found in Table 5.6. Mucus and serous glands are so named because of the type of secretion produced.

1	Mucus and serous glands are so named because of the type of secretion produced. The secretory cells of exocrine glands associated with the various body tubes (e.g., the alimentary canal, respiratory passages, and urogenital system) are often described as being mucus, serous, or both.

1	Mucus secretions are viscous and slimy, whereas serous secretions are watery. Goblet cells, secretory cells of the sublingual salivary glands, and surface cells of the stomach are examples of mucus-secreting cells. The mucus nature of the secretion results from extensive glycosylation of the constituent proteins with anionic oligosaccharides. The mucinogen granules, the secretory product within the cell, are therefore PAS positive (see Fig. 5.26a). However, they are water soluble and lost during routine tissue preparation. For this reason, the cytoplasm of mucus cells appears to be empty in H&E–stained paraffin sections. Another characteristic feature of a mucus cell is that its nucleus is usually flattened against the base of the cell by accumulated secretory product (Fig. 5.40).

1	In contrast to mucus-secreting cells, serous cells produce poorly glycosylated or nonglycosylated protein secretions. The nucleus is typically round or oval (Fig. 5.41). The apical cytoplasm is often intensely stained with eosin if its secretory granules are well preserved. The perinuclear cytoplasm often appears basophilic because of an extensive rough endoplasmic reticulum, a characteristic of protein-synthesizing cells. Serous cell–containing acini (sing., acinus) are found in the parotid gland and pancreas. Acini of some glands, such as the submandibular gland, contain both mucus and serous cells. In routine tissue preparation, the serous cells are more

1	FIGURE 5.40 • Mucus-secreting compound gland. Photomicrograph showing two small lobes of a mucus-secreting gland associated with the larynx. Each displays the beginning of a duct (D) into which mucin is secreted (arrows). The individual secretory cells that form the acinus (A) are difficult to define. Their nuclei (arrowheads) are flattened and located in the very basal portion of the cell, a feature typical of mucus-secreting glands. The cytoplasm is filled with mucin that has been retained during preparation of the tissue and appears stained. 350.

1	chapter 5 Epithelial Tissue G LAN D S TABLE Classification of Multicellular Glands5.6 Classification Typical Location Features Simple tubular Large intestine: intestinal Secretory portion of the gland glands of the colon is a straight tube formed by the secretory cells (goblet cells) Simple coiled Skin: eccrine sweat gland Coiled tubular structure is tubular composed of the secretory portion located deep in the dermis Simple branched Stomach: mucus-secreting Branched tubular glands with tubular glands of the pylorus wide secretory portion are formed by the secretory cells and produce a viscous mucous secretion Simple acinar Urethra: paraurethral Simple acinar glands develop and periurethral glands as an outpouching of the transitional epithelium and are formed by a single layer of secretory cells Branched acinar Stomach: mucus-secreting Branched acinar glands with glands of cardia secretory portions are formed by mucus-secreting cells; the short, single-duct portion opens directly

1	cells Branched acinar Stomach: mucus-secreting Branched acinar glands with glands of cardia secretory portions are formed by mucus-secreting cells; the short, single-duct portion opens directly into the lumen Compound Duodenum: submucosal Compound tubular glands with tubular glands of Brunner coiled secretory portions are located deep in the submucosa of the duodenum Compound Pancreas: excretory portion Compound acinar glands with acinar alveolar-shaped secretory units are formed by pyramid-shaped serous-secreting cells Compound Submandibular salivary Compound tubuloacinar glands tubuloacinar gland, mammary gland, can have both mucous branched lacrimal gland tubular and serous branched acinar secretory units; they have serous end-caps (demilunes) Simple GlandsCompound Glands hh

1	FIGURE 5.41 • Serous-secreting compound gland. Photomicro-graph of pancreatic acinus (A; outlined by the dotted line) with its duct (D). The small round objects within the acinar cells represent the zymogen granules, the stored secretory precursor material. 320. removed from the lumen of the acinus and are shaped as crescents or demilunes (half-moons) at the periphery of the mucus acinus. Most epithelial cells have a finite life span less than that of the whole organism.

1	Surface epithelia and epithelia of many simple glands belong to the category of continuously renewing cell populations. The rate of cell turnover (i.e., the replacement rate) is characteristic of a specific epithelium. For example, the cells lining the small intestine are renewed every 4 to 6 days in humans. The replacement cells are produced by mitotic activity of self-maintaining adult stem cells. They are located in sites called niches. In the small intestine, niches of adult stem cells are located in the lower portion of the intestinal glands (crypts; Fig. 5.42). They then migrate and differentiate into four principal cell types. Enterocytes (columnar absorptive cells), goblet cells (mucus-secreting), and enteroendocrine cells (regulatory and hormone-secreting) continue to differentiate and mature while they migrate up along the villi to the surface of the intestinal lumen. The migration of these new cells continues until they reach the tips of the villi, where they undergo

1	and mature while they migrate up along the villi to the surface of the intestinal lumen. The migration of these new cells continues until they reach the tips of the villi, where they undergo apoptosis and slough off into the lumen. The fourth cell type, Paneth cells, migrate downward and reside at the bottom of the crypt. The transcription factor Math1 expressed in the intestinal epithelium determines the fate of the cell. The cells committed to the secretory lineage (i.e., they will differentiate into goblet, enteroendocrine, and Paneth cells) have increased expression of Math1. Inhibition of Math1 expression characterizes the default developmental pathway into absorptive intestinal cells (enterocytes).

1	Similarly, the stratifed squamous epithelium of skin is replaced in most sites during a period of approximately 28 days. Cells in the basal layer of the epidermis, appropriately named the stratum basale (germinativum), undergo mitosis to provide for cell renewal. As these cells differentiate, they are pushed toward the surface by new cells in the basal layer. Ultimately, the cells become keratinized and slough off. In both of the above examples, a steady state is maintained within the epithelium, with new cells normally replacing ex-foliated cells at the same rate. In other epithelia, particularly in more complex glands, individual cells may live for a long time, and cell division is rare after the mature state is reached. These epithelial cells are characteristic of stable cell populations in which relatively little mitotic activity occurs such as in the liver. However, loss of significant amounts of liver tissue through physical

1	In two general locations, surface epithelium and its un-derlying connective tissue are regarded as a functional unit called a membrane. The two types of membrane are mucus membrane and serous membrane. The term membrane as used here should not be confused with the biologic membranes of cells, nor should the designations mucus and serous be confused with the nature of the gland secretion as discussed above. Mucus membrane, also called mucosa, lines those cavities that connect with the outside of the body, namely, the alimentary canal, the respiratory tract, and the geni-tourinary tract. It consists of surface epithelium (with or without glands), a supporting connective tissue called the lamina propria, a basement membrane separating the epithelium from the lamina propria, and sometimes a layer of smooth muscle called the muscularis mucosae as the deepest layer. Serous membrane, also called serosa, lines the peri-toneal, pericardial, and pleural cavities. These cavities are usually

1	a layer of smooth muscle called the muscularis mucosae as the deepest layer. Serous membrane, also called serosa, lines the peri-toneal, pericardial, and pleural cavities. These cavities are usually described as closed cavities of the body, although in the female the peritoneal cavity communicates with the exterior via the genital tract. Structurally, the serosa con-sists of a lining epithelium, the mesothelium, a support-ing connective tissue, and a basement membrane between the two. Serous membranes do not contain glands, but the fluid on their surface is watery.

1	 FOLDER 5.5 Functional Considerations: Mucus and Serous Membranes FIGURE 5.42 • Autoradiograph of intestinal gland (crypt). Autoradiograph of crypts in the jejunum of a rabbit that had been injected with tritiated thymidine 8 hours before death and fixation. Nearly all of the epithelial cells in this replicative zone of the intestinal mucosa are labeled, indicating that they were synthesizing DNA at the time the animal was injected. 600. (Reprinted with permission from Parker FG, Barnes EN, Kaye GI. The pericryptal fibroblast sheath. IV. Replication, migration, and differentiation of the subepithelial fibroblasts of the crypt and villus of the rabbit jejunum. Gastroenterology 1974;67: 607–621.) trauma or acute toxic destruction is accommodated by active proliferation of undamaged liver cells. The liver tissue is essentially restored by the stimulated mitotic activity of healthy liver tissue.

1	Epithelium consists of a diverse group of cell types, each of which possesses specific functional characteristics. The cells that make up a given epithelium are arranged in close apposition with one another and typically are located at what may be described as the free surfaces of the body. Such free surfaces include the exterior of the body, the outer surface of many internal organs, and the lining of body cavities, tubes, and ducts. Epithelium is classified on the basis of the arrangement of the cells that it contains and their shape. If the cells are present in a single layer, they constitute a simple epithelium. If they are present in multiple layers, they constitute a stratified epithelium. The shape of the cells is typically described as squamous, if the cell is wider than it is tall; cuboidal, if its height and width are approximately the same; or columnar, where the cell is taller than it is wide.

1	Simple squamous epithelium, mesovarium, human, body. The mesothelial cells (MC) are recognized by their nuclei at this low H&E, x350; inset x875. magnification. Beneath the mesothelial cells is a thin layer of connective tissue (CT) and adipose cells (A). The inset reveals at higher magnification the This micrograph shows the surface epithelium of the mesovarnuclei (N) of the mesothelial cells. ium covered by mesothelium, a name given to the simple squamous epithelium that lines the internal cavities of the Simple squamous epithelium, mesentery, rat, silver impregnation, x350; inset x700.

1	ium covered by mesothelium, a name given to the simple squamous epithelium that lines the internal cavities of the Simple squamous epithelium, mesentery, rat, silver impregnation, x350; inset x700. This is an intermediate magnification of a whole mount of a piece of mesentery. The mesentery was placed on the slide and prepared for microscopic examination. The microscope was focused at the surface of the mesentery. By this method, the boundaries of the surface mesothelial cells are delineated as black lines by the precipitated silver. Note that the cells are in close apposition to one another and that they have a polygonal shape. The inset reveals several mesothelial cells, each of which exhibits a nucleus (N) that has a round, or oval profile. Because of the squamous shape of the mesothelial cells, the nuclei are not spherical, but rather are disc-like. Simple squamous epithelium, kidney, human, H&E, x350.

1	Simple squamous epithelium, kidney, human, H&E, x350. This micrograph shows a kidney renal corpuscle. The wall of the renal corpuscle, known as the parietal layer of Bowman’s capsule, is a spherical structure that consists of a simple squaous epithelium (SSE). The interior of the corpuscle Simple cuboidal epithelium, pancreas, human, H&E, x700. This photomicrograph shows two pancreatic ducts (PD) that are lined by a simple cuboidal epithelium. The duct cell nuclei (N) tend to be spherical, a feature consistent with the Simple cuboidal epithelium, lung, human, H&E, x175; inset x525.

1	Simple cuboidal epithelium, lung, human, H&E, x175; inset x525. This photomicrograph shows the epithelium of the smallest conducting bronchioles of the lung. The simple cuboidal epithelium consists of cuboidal cells (CC). The inset shows a higher contains a capillary network from which fluid is filtered to enter the urinary space (US) and then into the proximal convoluted tubule (PCT). Nuclei (N) of the squamous cells of the parietal layer of Bowman’s capsule are ovoid and appear to protrude slightly into the urinary space. The free surface of this simple squamous epithelium faces the urinary space, whereas the basal surface of the epithelial cells rests on a layer of connective tissue (CT). cuboidal shape of the cell. The free surface of the epithelial cells face the lumen of the duct and the basal surface rests on connective tissue (CT). Careful examination of the free surface of the epithelial cells reveals some of the terminal bars (TB) between adjacent cells.

1	magnification of the cuboidal cells (CC). Note the spherical nuclei. These are small cells with relatively little cytoplasm, thus the nuclei appear close to one another. The free surface of the epithelial cells face the airway (AW), whereas the basal surface of these cells rests on its basement membrane and underlying dense connective tissue (CT). Simple cuboidal epithelium, liver, human, H&E, x450; inset x950. This micrograph reveals cords of hepatocytes (H), simple cuboidal cells that make up the liver parenchyma. The hepatic epithelial cell cords are mostly separated from one another by blood sinusoids (S). The inset shows a higher magnification of a hepatic cell and reveals an unusual feature in that several surfaces of these cells posses a groove representing the free surface of the cell. Where the groove of one cell faces a groove of the adjacent cell, a small canal-like structure, the canaliculus (C), is formed. Bile is secreted from the cell into the canaliculus.

1	PLATE 1 KEY A, adipose tissue AW, airway C, canaliculus CC, cuboidal cells CT, connective tissue H, hepatocytes MC, mesothelial cells N, nucleus PCT, proximal convoluted tubule PD, pancreatic duct S, sinusoid SSE, simple squamous epithelium TB, terminal bar US, urinary space Simple epithelia are only one cell layer thick. They are characteristic of organ systems primarily concerned with transport, absorption, and secretion, such as the intestine, the vascular system, the digestive glands and other exocrine glands, and the kidney. Stratified epithelia have more than one layer and are typical of surfaces that are subject to frictional stress, such as skin, oral mucosa and esophagus, and vagina. Simple epithelium, exocrine pancreas, monkey, H&E ×450.

1	This shows three epithelial forms. In the circle is a well-oriented acinus, a functional group of secretory cells, each of which is pyramidal in shape. The secretory cells form a spherical or tubular structure. The free surface of the cells and the lumen are located in the center of the circle. The lumen is not evident here but is evident in a similar cell arrangement in the middle right image below (see circle). Because the height of the cells (the distance from the edge of the circle to the lumen) is greater than the width, the epithelium is simple columnar. The second epithelial type is represented by a small, longitudinally sectioned duct (arrows) extending across the field. It is composed of flattened cells (note the nuclear shape), and on this basis, the epithelium is simple squamous. Finally, there is a larger cross-sectioned duct (asterisk) into which the smaller duct enters. The nuclei of this larger duct tend to be round, and the cells tend to be square in profile. Thus,

1	Finally, there is a larger cross-sectioned duct (asterisk) into which the smaller duct enters. The nuclei of this larger duct tend to be round, and the cells tend to be square in profile. Thus, these duct cells are a simple cuboidal epithelium.

1	Simple cuboidal epithelium, kidney, human, of a simple cuboidal epithelium. The arrows point to the lateral cell H&E ×450. boundaries; note that cell width approximates cell height. The cross-sectioned structures marked with asterisks are another type of tubule; they are This section shows cross-sectioned tubules of several types. smaller in diameter but are also composed of a simple cuboidal epithelium. Those that are labeled with the arrows provide another example Simple columnar epithelium, colon, human, H&E ×350. The simple columnar epithelium of the colon shown here consists of a single layer of absorptive cells and mucus-secreting cells (goblet cells). The latter can be recognized by the light Pseudostratifed epithelium, trachea, monkey, H&E ×450.

1	Pseudostratifed epithelium, trachea, monkey, H&E ×450. In addition to the tall columnar cells (CC) in this columnar epithelium, there is a definite layer of basal cells (BC). The columnar cells, which contain elongate nuclei and possess cilia (C), extend from the surface to the basement membrane (clearly visible in the trachea as a thick, acellular, homogeneous region that is part of the connective tissue (CT )). The basal cells are interspersed Pseudostratifed epithelium, epididymis, human, H&E ×450.

1	Pseudostratifed epithelium, epididymis, human, H&E ×450. This is another example of pseudostratified columnar epithelium. Again, two layers of nuclei are evident, those of basal cells (BC) and those of columnar cells (CC). As in the previous example, however, although not evident, the columnar cells staining “goblet” (arrows) that contains the cell’s secretory product. The epithelium lines the lumen of the colon and extends down into the connective tissue to form the intestinal glands (GL). Both cell types are tall with their nuclei located at the base of the cell. The connective tissue (CT) contains numerous cells, many of which are lymphocytes and plasma cells.

1	between the columnar cells. Because all of the cells rest on the basement membrane, they are regarded as a single layer, as opposed to two discrete layers, one over the other. Because the epithelium appears to be stratified but is not, it is called pseudostratified columnar epithelium. The circle in the micrograph delineates a tracheal gland similar to the acinus in exocrine pancreas (circle). Note that the lumen of the gland is clearly visible and the cell boundaries are also evident. The gland epithelium is simple columnar. rest on the basement membrane; thus, the epithelium is pseudostratified. Note that where the epithelium is vertically oriented, on the right of the micrograph, there appear to be more nuclei, and the epithelium is thicker. This is a result of a tangential plane of section. As a rule, always examine the thinnest area of an epithelium to visualize its true organization.

1	Stratifed squamous epithelium, vagina, human, small, with little cytoplasm, and thus the nuclei appear closely packed. As H&E ×225. the cells become larger, they tend to flatten out, forming disc-like squames. Because the surface cells retain this shape, the epithelium is This is the stratified squamous epithelium of the vaginal called stratified squamous. wall. The deeper cells, particularly those of the basal layer, are KEY BC, basal cell C, cilia CC, columnar cell CT, connective tissue GL, intestinal gland arrows: upper left, duct composed of simple squamous epithelium; upper right, lateral boundaries of cuboidal tubule cells; middle left, mucus cups of goblet cells asterisk, duct or tubule of simple cuboidal epithelium

1	Tissues that resemble epithelia but lack the characteristic free surface are designated epithelioid tissues. This is the characteristic structure of the endocrine organs, which develop from typical epithelia but lose their connection to a surface during development. Stratifed epithelia, esophagus, monkey, H&E ×250. This part of the wall of the esophagus reveals two different epithelia. On the left is the lining epithelium of the esophagus. It is multilayered with squamous surface cells; therefore, it is a stratified squamous epithelium (SS). On the right is the duct of an esophageal gland cut in several planes. By examining a region where the plane of section is at a right angle to the surface, the true character of the epithelium becomes apparent. In this case, the epithelium consists of two cell layers with cuboidal surface cells; thus, it is stratified cuboidal epithelium (StCu).

1	Stratifed epithelia, skin, human, H&E ×450. (StCu) in two layers; the cells of the inner layer (the surface cells) appear more or less square. Because the epidermal surface cells are not included in This shows a portion of the duct of a sweat gland just before the field, the designation stratified squamous cannot be derived from the in- the duct enters the stratified squamous epithelium (SS) of formation offered by the micrograph. the skin. The dashed line traces the duct within the epidermis. This duct also consists of a stratified cuboidal epithelium PLATE 3 • STRATI FI E D E PITH E LIA AN D E PITH E LIOI D TI SS U E S KEY C, capillary CT, connective tissue En, endocrine cells Ex, exocrine cells IC, interstitial (Leydig) cells SCol, simple columnar epithelium SS, stratified squamous epithelium StCu, stratified cuboidal epithelium arrowhead, transition site of simple stratified epithelium to stratified cuboidal arrows, surface cuboidal cells asterisks, dome-shaped cells

1	Epithelial transition, anorectal junction, human, H&E ×300. The area shown here is the terminal part of the large intestine. The luminal epithelium on the left is typical simple columnar epithelium (SCol) of the colon. This epithelium undergoes an abrupt transition (arrowhead) to a stratified cuboidal epithelium (StCu) at the anal canal. Note the general cuboidal Transitional epithelium (urothelium), urinary bladder, monkey, H&E ×400. The epithelium of the urinary bladder is called transitional epithelium, a stratified epithelium that changes in appearance according to the degree of distension of the bladder. In the nondistended state, as here, it is about four or five cells deep. The surface cells are large and dome shaped (asterisks). The cells Epithelioid tissues, testis, monkey, H&E ×350.

1	Epithelioid tissues, testis, monkey, H&E ×350. This shows the intestinal (Leydig) cells of the testis (IC). These cells possess certain epithelial characteristics. They do not possess a free surface, however, nor do they develop from a surface; shape of most of the surface cells (arrows) and the underlying layers of cells. The simple columnar epithelium on the left is part of an intestinal gland that is continuous with the simple columnar epithelium at the intestinal luminal surface. The connective tissue (CT) at this site is heavily infiltrated with lymphocytes, giving it an appearance unlike the connective tissue of other specimens on this page.

1	immediately under the surface cells are pear shaped and slightly smaller. The deepest cells are the smallest, and their nuclei appear more crowded. When the bladder is distended, the superficial cells are stretched into squamous cells, and the epithelium is reduced in thickness to about three cells deep. The bladder wall usually contracts when it is removed, unless special steps are taken to preserve it in a distended state. Thus, its appearance is usually like that in Figure 4. instead, they develop from mesenchymal cells. They are referred to as epithelioid cells because they contact similar neighboring cells much the same as epithelial cells contact each other. Leydig cells are endocrine in nature. Epithelioid tissues, endocrine pancreas, human, H&E ×450.

1	Epithelioid tissues, endocrine pancreas, human, H&E ×450. Cells of the endocrine islet (of Langerhans) (En) of the pancreas also have an epithelioid arrangement. The cells are in contact but lack a free surface, although they have developed from an epithelial surface by invagination. In contrast, the surrounding alveoli of the exocrine pancreas (Ex), which developed from the same epithelial surface, are made up of cells with a free surface onto which the secretory product is discharged. Capillaries (C) are prominent in endocrine tissues. Similar examples of epithelioid tissue are seen in the adrenal and the parathyroid and pituitary glands, all of which are endocrine glands.

1	GENERAL STRUCTURE AND FUNCTION OF CONNECTIVE TISSUE / 158 EMBRYONIC CONNECTIVE TISSUE / 159 CONNECTIVE TISSUE PROPER / 160 CONNECTIVE TISSUE FIBERS / 161 Collagen Fibers and Fibrils / 161 Biosynthesis and Degradation of Collagen Fibers / 164 Reticular Fibers / 171 Elastic Fibers / 171 EXTRACELLULAR MATRIX / 173 CONNECTIVE TISSUE CELLS / 178 Fibroblasts and Myofibroblasts / 178 Macrophages / 181 Mast Cells / 182 Basophils / 187 Adipocytes / 187 Adult Stem Cells and Pericytes / 187 Lymphocytes, Plasma Cells, and Other Cells of the Immune System / 189 Folder 6.1 Clinical Correlation: Collagenopathies / 170 Folder 6.2 Clinical Correlation: Sun Exposure and Molecular Changes in Photoaged Skin / 173 Folder 6.3 Clinical Correlation: Role of Myofibroblasts in Wound Repair / 183 Folder 6.4 Functional Considerations: The Mononuclear Phagocytotic System / 185 Folder 6.5 Clinical Correlation: The Role of Mast Cells and Basophils in Allergic Reactions / 188

1	Connective tissue comprises a diverse group of cells within a tissue-specific extracellular matrix. In general, connective tissue consists of cells and an extracellular matrix (ECM). ECM includes structural (fibers) and specialized proteins that constitute the ground substance. Connective tissue forms a vast and continuous compartment throughout the body, bounded by the basal laminae of the various epithelia and by the basal or external laminae of muscle cells and nerve-supporting cells. Different types of connective tissue are responsible for a variety of functions.

1	The functions of the various connective tissues are reflected in the types of cells and fibers present within the tissue and the composition of the ground substance in the ECM. For example, in loose connective tissue, many cell types are present (Fig. 6.1). One type, the fibroblast, produces the extracellular fibers that serve a structural role in the tissue. Fibroblasts also produce and maintain the ground substance. Other cell types, such as lymphocytes, plasma cells, macrophages, and eosinophils, are associated with the body’s defense system; they function within the ECM of the tissue. In contrast, bone tissue, another form of connective tissue, contains only a single cell type, the osteocyte. This cell produces the fibers that make up the bulk of bone tissue. A unique feature of bone is that its fibers are organized in a specific pattern and become calcified to create the hardness associated with this tissue. Similarly, in tendons and ligaments, fibers are the prominent feature of

1	that its fibers are organized in a specific pattern and become calcified to create the hardness associated with this tissue. Similarly, in tendons and ligaments, fibers are the prominent feature of the tissue. These fibers are arranged in parallel array and are densely packed to impart maximum strength.

1	Classification of connective tissue is based on the composition and organization of its cellular and extracellular components and on its functions. Connective tissue encompasses a variety of tissues with differing functional properties but with certain common characteristics that allow them to be grouped together. For

1	FIGURE 6.1 • Loose connective tissue. a. Photomicrograph of a mesentery spread stained with Verhoeff’s hematoxylin to show nuclei and elastic fibers; it has been counterstained with safranin for identification of mast cell granules and with orange G for identification of other proteins (mainly collagen fibers). The elastic fibers appear as blue-black, thin, long, and branching threads without discernible beginnings or endings. Collagen fibers appear as orange-stained, long, straight profiles, and are considerably thicker than the elastic fibers. Most of the visible nuclei are presumed to be those of fibroblasts. Nuclei of other cell types (e.g., lymphocytes, plasma cells, and macrophages) are also present but are not identifiable. Mast cells are identified by the bright reddish granules within their cytoplasm. Note the presence of the small blood vessel filled with red blood cells. 150. b. Schematic diagram illustrating the components of loose connective tissue. Note the association

1	within their cytoplasm. Note the presence of the small blood vessel filled with red blood cells. 150. b. Schematic diagram illustrating the components of loose connective tissue. Note the association of different cell types with the surrounding extracellular matrix, which contains blood vessels and different types of fibers.

1	TABLE Classification of Connective Tissue6.1 Embryonic connective tissue Mesenchyme Mucous connective tissue Connective tissue proper Loose connective tissue Dense connective tissue Regular Irregular Specialized connective tissuea Cartilage (Chapter 7) Blood (Chapter 10) Bone (Chapter 8) Hemopoietic tissue (Chapter 10) Adipose tissue (Chapter 9) Lymphatic tissue (Chapter 14) aIn the past, the designations elastic tissue and reticular tissue have been listed as separate categories of specialized connective tissue. The tissues usually cited as examples of elastic tissue are certain ligaments associated with the spinal column and the tunica media of elastic arteries. The identifying feature of reticular tissue is the presence of reticular fibers and reticular cells together forming a three-dimensional stroma. Reticular tissue serves as the stroma for hemopoietic tissue (specifically the red bone marrow) and lymphatic tissue organs (lymph nodes and spleen, but not the thymus).

1	tissues, including subtypes. , the middle embryonic germ layer, gives rise to alall of the connective tissues of the body. An exception is head region, where specific progenitor cells are derived a primitive connective tissue referred to as mes(in the head region, it is sometimes called ectomes) is established in the early embryo. Maturation and tissue is classified into two subtypes: Mesenchyme is primarily found in the embryo. It contains small, spindle-shaped cells of relatively uniform appearance (Fig. 6.2a). Processes extend from these cells and contact similar processes of neighboring cells, forming a three-dimensional cellular network. Gap junctions are present where the processes make contact. The extracellular space is occupied by a viscous ground substance. Collagen (reticular) fibers are present; they are very fine and relatively sparse. The paucity of collagen fibers is consistent with the limited physical stress on the growing fetus.

1	FIGURE 6.2 • Embryonic connective tissue. a. Photomicrograph of mesenchymal tissue from a developing fetus stained with H&E. Although morphologically the mesenchymal cells appear as a homogeneous population, they give rise to cells that will differentiate into various cell types. Their cytoplasmic processes often give the cell a tapering or spindle appearance. The extracellular component of the tissue contains a sparse arrangement of reticular fibers and abundant ground substance. 480. b. Photomicrograph of Wharton’s jelly from the umbilical cord stained with H&E. Wharton’s jelly consists of a specialized, almost gelatinlike ground substance that occupies large intercellular spaces located between the spindle-shaped mesenchymal cells. 480.

1	 Mucous connective tissue is present in the umbilical cord. It consists of a specialized, almost gelatinlike ECM; its ground substance is frequently referred to as Wharton’s jelly. The spindle-shaped cells are widely separated and appear much like fibroblasts in the near-term umbilical cord (e.g., the cytoplasmic processes are thin and difficult to visualize in routine hematoxylin and eosin [H&E] preparation). Wharton’s jelly occupies large inter-cellular spaces located between thin, wispy collagen fibers (Fig. 6.2b). Connective tissues that belong to this category are divided into two general subtypes:  Loose connective tissue, sometimes called areolar tissue, and  dense connective tissue, which can be further subcategorized into two basic types based on the organization of its collagen fibers: dense irregular connective tissue and dense regular connective tissue. Loose connective tissue is characterized by loosely arranged fibers and abundant cells of various types.

1	Loose connective tissue is characterized by loosely arranged fibers and abundant cells of various types. Loose connective tissue is a cellular connective tissue with thin and relatively sparse collagen fibers (Fig. 6.3). The FIGURE 6.3 • Loose and dense irregular connective tissue. Photomicrograph comparing loose and dense irregular connective tissue from the mammary gland stained with Masson’s trichrome. In the center, loose connective tissue surrounds the glandular epithelium. The loose connective tissue is composed of a wispy arrangement of collagen fibers with many cells. Note the large number of nuclei visible at this low magnification. On the upper left and lower right of the figure is dense irregular connective tissue. In contrast, few nuclei are revealed in the dense connective tissue. However, collagen is considerably more abundant and is composed of very thick fibers. 100.

1	ground substance, however, is abundant; in fact, it occupies more volume than the fibers do. It has a viscous to gel-like consistency and plays an important role in the diffusion of oxygen and nutrients from the small vessels that course through this connective tissue as well as in the diffusion of carbon dioxide and metabolic wastes back to the vessels.

1	Loose connective tissue is primarily located beneath the epithelia that cover the body surfaces and line the internal surfaces of the body. It is also associated with the epithelium of glands and surrounds the smallest blood vessels (Plate 4, page 192). This tissue is thus the initial site where pathogenic agents such as bacteria that have breached an epithelial surface are challenged and destroyed by cells of the immune system. Most cell types in loose connective tissue are transient wandering cells that migrate from local blood vessels in response to specific stimuli. Loose connective tissue is, therefore, the site of inﬂammatory and immune reactions. During these reactions, loose connective tissue can swell considerably. In areas of the body where foreign substances are continually present, large populations of immune cells are maintained. For example, the lamina propria, the loose connective tissue of mucous membranes, such as those of the respiratory and alimentary systems,

1	present, large populations of immune cells are maintained. For example, the lamina propria, the loose connective tissue of mucous membranes, such as those of the respiratory and alimentary systems, contains large numbers of these cells.

1	Dense irregular connective tissue is characterized by abundant fibers and few cells.

1	Dense irregular connective tissue contains mostly collagen fibers. Cells are sparse and are typically of a single type, the fibroblast. This tissue also contains relatively little ground substance (Plate 4, page 192). Because of its high pro portion of collagen fibers, dense irregular connective tissue provides significant strength. Typically, the fibers are arranged in bundles oriented in various directions (thus, the term irregular) that can withstand stresses on organs or structures. Hollow organs (e.g., the intestinal tract) possess a distinct layer of dense irregular connective tissue called the submucosa in which the fiber bundles course in varying planes. This arrangement allows the organ to resist excessive stretching and distension. Similarly, skin contains a relatively thick layer of dense irregular connective tissue called the reticular layer (or deep layer) of the dermis. The reticular layer provides resistance to tearing as a consequence of stretching forces from

1	layer of dense irregular connective tissue called the reticular layer (or deep layer) of the dermis. The reticular layer provides resistance to tearing as a consequence of stretching forces from different directions.

1	Dense regular connective tissue is characterized by ordered and densely packed arrays of fibers and cells. Dense regular connective tissue is the main functional component of tendons, ligaments, and aponeuroses. As in dense irregular connective tissue, the fibers of dense regular connective tissue are the prominent feature, and there is little ECM. However, in dense regular connective tissue, the fibers are arranged in parallel array and are densely packed to provide maximum strength. The cells that produce and maintain the fibers are packed and aligned between fiber bundles.  Tendons are cordlike structures that attach muscle to bone. They consist of parallel bundles of collagen fibers.

1	Situated between these bundles are rows of fibroblasts called tendinocytes (Fig. 6.4 and Plate 5, page 194). Tendinocytes are surrounded by a specialized ECM that separates them from the load-bearing collagen fibrils. In H&E–stained cross sections of tendon, the tendinocytes appear stellate. In transmission electron micrograph (TEM) sections parallel to the long axis of tendons, the cytoplasmic projections of the cell are seen to lie be tween the fibers and appear as thin cytoplasmic sheets. In most H&E–stained longitudinal sections, however, tendinocytes appear only as rows of typically flattened basophilic nuclei. The cytoplasmic sheets that extend from the body of the tendinocytes are not usually evident in longitudinal H&E–stained sections because they blend in with the collagen fibers. The substance of the tendon is surrounded by a thin connective tissue capsule, the epitendineum, in which the collagen fibers are not nearly as orderly (Plate 5, page 194). Typically, the tendon is

1	The substance of the tendon is surrounded by a thin connective tissue capsule, the epitendineum, in which the collagen fibers are not nearly as orderly (Plate 5, page 194). Typically, the tendon is subdivided into fascicles by endotendineum, a connective tissue extension of the epitendineum. It contains the small blood vessels and nerves of the tendon.

1	 Ligaments, like tendons, consist of fibers and fibroblasts arranged in parallel. The fibers of ligaments, however, are less regularly arranged than those of tendons. Ligaments join bone to bone, which in some locations, such as in the spinal column, requires some elasticity. Although collagen is the major extracellular fiber of most ligaments, some of the ligaments associated with the spinal column (e.g., ligamenta flava) contain many more elastic fibers and fewer collagen fibers. These ligaments are called elastic ligaments.

1	 Aponeuroses resemble broad, flattened tendons. Instead of fibers lying in parallel arrays, the fibers of aponeuroses are arranged in multiple layers. The bundles of collagen fibers in one layer tend to be arranged at a 90 angle to those in the neighboring layers. The fibers within each of the layers are arranged in regular arrays; thus, aponeurosis is a dense regular connective tissue. This orthogonal array is also found in the cornea of the eye and is responsible for its transparency. Connective tissue fibers are of three principal types. Connective tissue fibers are present in varying amounts, depending on the structural needs or function of the connective tissue. Each type of fiber is produced by fibroblasts and is composed of protein consisting of long peptide chains. The types of connective tissue fibers are Collagen fibers are the most abundant type of connective tissue fiber.

1	Collagen fibers are the most abundant type of connective tissue fiber. Collagen fbers are the most abundant structural components of the connective tissue. They are flexible and have a

1	FIGURE 6.4 • Dense regular connective tissue—tendon. a. Electron micrograph of a tendon at low magnification, showing tendinocytes (fibroblasts) and their thin processes (arrows) lying between the collagen bundles. 1,600. b. A tendinocyte with prominent profiles of rough endoplasmic reticulum (rER) is shown at higher magnification. The collagen fibers (C) can be resolved as consisting of very tightly packed collagen fibrils. The arrows indicate processes of tendinocytes. 9,500. Inset. Photomicrograph of a tendon. Note the orderly and regular alignment of the bundles of collagen fibers. Tendinocytes are aligned in rows between the collagen fibers. 200. (Electron micrographs modified from Rhodin J. Histology. New York: Oxford University Press, 1974.) remarkably high tensile strength. In the light microscope, collagen fibers typically appear as wavy structures of variable width and indeterminate length. They stain readily with eosin and other acidic dyes. They can also be colored with

1	light microscope, collagen fibers typically appear as wavy structures of variable width and indeterminate length. They stain readily with eosin and other acidic dyes. They can also be colored with the dye aniline blue used in Mallory’s connective tissue stain or with the dye light green used in Masson’s stain.

1	When examined with the TEM, collagen fibers appear as bundles of fine, threadlike subunits. These subunits are collagen fbrils (Fig. 6.5). Within an individual fiber, the collagen fibrils are relatively uniform in diameter. In different locations and at different stages of development, however, the fibrils differ in size. In developing or immature tissues, the fibrils may be as small as 15 or 20 nm in diameter. In dense, regular connective tissue of tendons or other tissues that are subject to considerable stress, they may measure up to 300 nm in diameter. Collagen fibrils have a 68-nm banding pattern.

1	When collagen fibrils stained with osmium or other heavy metals are examined with the TEM, they exhibit a sequence of closely spaced transverse bands that repeat every 68 nm along the length of the fibril (Fig. 6.5, inset). This regular banding pattern can also be observed on the surface of the collagen fibrils when they are examined with the atomic force microscope (AFM; Fig. 6.6). This banding pattern reflects the fibril’s subunit structure, specifically the size and shape of the collagen molecule and the arrangement of the molecules that form the fibril (Fig. 6.7). The collagen molecule (formerly called tropocollagen) measures about 300 nm long by 1.5 nm thick and has a head and a tail. Within each fibril, the collagen molecules align head to tail in overlapping rows with a gap between the molecules in each row and a onequarter-molecule stagger between adjacent rows. These gaps are clearly visible with the AFM (see Fig. 6.6). The strength of the fibril is created by the covalent

1	the molecules in each row and a onequarter-molecule stagger between adjacent rows. These gaps are clearly visible with the AFM (see Fig. 6.6). The strength of the fibril is created by the covalent bonds between the collagen molecules of adjacent rows, not the head-to-tail attachment of the molecules in a row. The banding pattern observed with the TEM (see Fig. 6.5, inset) is caused largely by osmium deposition in the space between the heads and tails of the molecules in each row.

1	FIGURE 6.5 • Collagen fbrils in dense irregular connective tissue. Electron micrograph of dense irregular connective tissue from the capsule of the testis of a young male. The threadlike collagen fibrils are aggregated in some areas (X ) to form relatively thick bundles; in other areas, the fibrils are more dispersed. 9,500. Inset. A longitudinal array of collagen fibrils from the same specimen seen at higher magnification. Note the banding pattern. The spacing of the arrows indicates the 68-nm repeat pattern. 75,000. Each collagen molecule is a triple helix composed of three intertwined polypeptide chains. A single collagen molecule consists of three polypeptides known as chains. The chains intertwine, forming a right-handed triple helix (see Fig. 6.7d). Every third amino acid in the chain is a glycine molecule, except at the ends of the chains. A hydroxyproline or hydroxylysine frequently precedes each glycine in the chain, and a proline frequently

1	FIGURE 6.6 • Collagen fbrils in dense irregular connective tissue. This atomic force microscopic image of type I collagen fibrils in the connective tissue shows the banding pattern on the surface of collagen fibrils. Note the random orientation of collagen fibrils that overlie and crisscross each other in the connective tissue matrix. 65,000. (Courtesy of Dr. Gabriela Bagordo, JPK Instruments AG, Berlin, Germany.) follows each glycine in the chain. Along with proline and hydroxyproline, the glycine is essential for the triple-helix conformation (see Fig. 6.7e). Associated with the helix are sugar groups that are joined to hydroxylysyl residues. Because of these sugar groups, collagen is properly described as a glycoprotein.

1	The chains that constitute the helix are not all alike. They vary in size from 600 to 3,000 amino acids. To date, a. Fibril D 68 b. Packing of (0.6 D) 0.4 D c. Collagen molecule 300 nm (4.4 D) 1.5 nm diameter d. Triple helix 10.4 nm (0.15 D) e. Typical sequence 1 of 1 and 2 1.74 nm .87 nm FIGURE 6.7 • Diagram showing the molecular character of a type 1 collagen fbril in increasing order of structure. a. A collagen fibril displays periodic banding with a distance (D) of 68 nm between repeating bands. b. Each fibril is composed of staggered collagen molecules. c. Each molecule is about 300 nm long and 1.5 nm in diameter. d. The collagen molecule is a triple helix. e. The triple helix consists of three chains. Every third amino acid of the chain is a glycine. The X position following glycine is frequently a proline, and the Y position preceding the glycine is frequently a hydroxyproline.

1	at least 42 types of chains encoded by different genes have been identified and mapped to loci on several different chromosomes. As many as 28 different types of collagens have been categorized on the basis of the combinations of chains they contain. These various collagens are classified by Roman numerals I to XXVIII according to the chronology of their discovery. A collagen molecule may be homotrimeric (consisting of three identical chains) or heterotrimeric (consisting of two or even three genetically distinct chains).

1	For example, type I collagen found in loose and dense connective tissue is heterotrimeric. Two of the chains, identified as 1, are identical, and one, identified as 2, is different. Thus, in collagen nomenclature it is designated [ 1(I)]2 2(I) (Table 6.2). Type II collagen is homotrimeric and present in hyaline and elastic cartilage, where it occurs as very fine fibrils. The collagen molecules of type II collagen are composed of three identical chains. Because these chains differ from those of other collagens, type II collagen is designated [ 1(II)]3. Several classes of collagens are identified on the basis of their polymerization pattern. Most of the collagen molecules polymerize into supramolecular aggregates such as fibrils or networks, and they are divided into several subgroups on the basis of their structural or amino acid sequence similarities.

1	 Fibrillar collagens include types I, II, III, V, and XI collagen molecules. These types are characterized by uninterrupted glycine–proline–hydroxyproline repeats and aggregate to form 68-nm-banded fibrils (as diagramed in Fig. 6.7a).  Fibril-associated collagens with interrupted triple helixes (FACITs) have interruptions in their triple helixes that provide flexibility to the molecule. They are located on the surface of different fibrils and are represented by types IX, XII, XIV, XVI, XIX, XX, XXI, and XXII collagens. For instance, type IX collagen molecule binds and interacts with type II collagen in the cartilage at the intersections of the fibrils. It serves to stabilize this tissue by binding type II collagen fibrils with proteoglycans of the ECM.  Hexagonal network–forming collagens are represented by collagen types VIII and X.

1	 Hexagonal network–forming collagens are represented by collagen types VIII and X.  Transmembrane collagens are represented by types: XIII (found in the focal adhesions), XVII (found within the hemidesmosomes), XXIII (found in metastatic cancer cells), and XXV (a brain-specific collagen).  Multiplexins (collagens with multiple triple-helix domains and interruptions) comprise collagen types XV and XVIII, which reside in the basement membrane zones.  Basement membrane–forming collagens include type IV collagen, which is responsible for the collagen suprastructure in the basement membrane of epithelial cells (page 139), type VI collagen, which forms beaded filaments, and type VII collagen, which forms anchoring fibrils that attach the basement membrane to the ECM.

1	Table 6.2 lists the collagens that have been characterized to date (I to XXV), including their structural variations and some of the roles presently ascribed to them. Recently identified collagen types (XXVI to XXVIII) have not been fully characterized and are not included in the table. Biosynthesis and Degradation of Collagen Fibers Collagen fiber formation involves events that occur both within and outside the fibroblast. The production of fbrillar collagen (I, II, III, V, and XI) involves a series of events within the fibroblast that leads to production of procollagen, the precursor of the collagen molecule. These events take place in membrane-bounded organelles within the cell. Production of the actual fibril occurs outside the cell and involves enzymatic activity at the plasma membrane to produce the collagen molecule, followed by assembly of the molecules into fibrils in the ECM under guidance by the cell (Fig. 6.8).

1	TABLE Types of Collagen, Composition, Location, and Function 6.2 Type Compositiona Location Functions I [ 1(I)]2 2(I) Connective tissue of skin, bone, tendon, Provides resistance to force, ligaments, dentin, sclera, fascia, and tension, and stretch organ capsules (accounts for 90% of body collagen) II [ 1(II)]3 Cartilage (hyaline and elastic), Provides resistance to notochord, and intervertebral disk intermittent pressure III [ 1(III)]3 Prominent in loose connective Forms reticular fibers, arranged tissue and organs (uterus, liver, spleen, as a loose meshwork of thin kidney, lung, etc.); smooth muscle; fibers, provides a supportive endoneurium; blood vessels; and scaffolding for the specialized fetal skin cells of various organs and blood vessels. IV [ 1(IV)]2 2(IV) or Basal laminae of epithelia, kidney Provides support and filtration 3(IV) 4(IV) 5(IV) or glomeruli, and lens capsule barrier [ 5(IV)]2 6(IV) V [ 1(V)]2 2(V) or Distributed uniformly throughout Localized at the surface of

1	kidney Provides support and filtration 3(IV) 4(IV) 5(IV) or glomeruli, and lens capsule barrier [ 5(IV)]2 6(IV) V [ 1(V)]2 2(V) or Distributed uniformly throughout Localized at the surface of type I 1(V) 2(V) 3(V) connective tissue stroma; may be collagen fibrils along with type XII related to reticular network and XIV collagen to modulate biomechanical properties of the fibril VI [ 1(VI)]2 2(VI) or Forms part of the cartilage matrix Attaches the chondrocyte to the 1(VI) 2(VI) 3(VI) immediately surrounding the matrix; covalently bound to type I chondrocytes collagen fibrils VII [ 1(VII)]3 Present in anchoring fibrils of skin, Secures basal lamina to connective eye, uterus, and esophagus tissue fibers VIII [ 1(VIII)]2 2(VIII) Product of endothelial cells Facilitates movement of endothelial cells during angiogenesis IX 1(IX) 2(IX) 3(IX) Found in cartilage associated with Stabilizes network of cartilage type II collagen fibrils type II collagen fibers by interaction with proteoglycan

1	cells during angiogenesis IX 1(IX) 2(IX) 3(IX) Found in cartilage associated with Stabilizes network of cartilage type II collagen fibrils type II collagen fibers by interaction with proteoglycan molecules at their intersections X [ 1(X)]3 Produced by chondrocytes in the Contributes to the bone mineral-zone of hypertrophy of normal ization process by forming growth plate hexagonal lattices necessary to arrange types II, IX, and XI collagen within cartilage XI [ 1(XI)]2 2(XI) or Produced by chondrocytes; Regulates size of type II collagen 1(XI) 2(XI) 3(XI) associated with type II collagen fibrils, fibrils; it is essential for cohesive forms core of type I collagen fibrils properties of cartilage matrix XII [ 1(XII)]3 Isolated from skin and placenta; Localized at the surface of type I abundant in tissues in which collagen fibrils along with type V mechanical strain is high and XIV collagen to modulate biomechanical properties of the fibril XIII [ 1(XIII)]3 An unusual transmembrane

1	abundant in tissues in which collagen fibrils along with type V mechanical strain is high and XIV collagen to modulate biomechanical properties of the fibril XIII [ 1(XIII)]3 An unusual transmembrane collagen Associated with the basal lamina detected in bone, cartilage, intestine, along with type VII collagen skin, placenta, and striated muscles continued next page

1	TABLE Types of Collagen, Composition, Location, and Function (Cont.)6.2 XIV [ 1(XIV)]3 Isolated from placenta; also Localized at the surface of type I detected in the bone marrow collagen fibrils along with type V and XII collagen to modulate biomechanical properties of the fibril; has a strong cell–cell binding property XV [ 1(XV)]3 Present in tissues derived from Involved in adhesion of basal lamina mesenchyme; expressed in heart to the underlying connective tissue and skeletal muscles XVI [ 1(XVI)]3 Broad tissue distribution; associated Contributes to structural with fibroblasts and arterial smooth integrity of connective tissue muscle cells, but not associated with type I collagen fibrils XVII [ 1(XVII)]3 Another unusual transmembrane collagen Interacts with integrins to stabilize found in epithelial cell membranes hemidesmosome structure XVIII [ 1(XVIII)]3 Found in epithelial and vascular Represents a basement membrane basement membrane heparan sulfate proteoglycan thought to

1	found in epithelial cell membranes hemidesmosome structure XVIII [ 1(XVIII)]3 Found in epithelial and vascular Represents a basement membrane basement membrane heparan sulfate proteoglycan thought to inhibit endothelial cell proliferation and angiogenesis XIX [ 1(XIX)]3 Discovered from the sequence of Pronounced vascular and stromal human rhabdomyosarcoma cDNA; interaction suggests involvement in present in fibroblasts and liver angiogenesis XX [ 1(XX)]3 Discovered from chick embryonic Binds to the surface of other tissue; also in corneal epithelium, collagen fibrils sternal cartilage, and tendons XXI [ 1(XXI)]3 Found in human gingiva, heart and Plays a role in maintaining three-skeletal muscle, and other tissues dimensional architecture of dense containing type I collagen fibrils connective tissues XXII [α1(XXII)]3 Found in myotendinous junction, Belongs to FACIT family skeletal and heart muscle, articular Expressed at tissue junctions cartilage–synovial fluid junction, at In skin,

1	tissues XXII [α1(XXII)]3 Found in myotendinous junction, Belongs to FACIT family skeletal and heart muscle, articular Expressed at tissue junctions cartilage–synovial fluid junction, at In skin, influences epithelial-the border between hair follicle mesenchymal interactions during hair and dermis follicle morphogenesis and cycling XXIII [ 3(XXIII)]3 Discovered in metastatic tumor cells Transmembrane collagen Also expressed in heart, retina, Interacts with ECM proteins (collagen and metastatic prostate cancer cells XIII and XXV, fibronectin, heparin) Increased expression in patient with metastatic prostate cancer XXIV [ 1(XXIV)]3 Found co-expressed with type I Fibrillarlike collagen collagen in the developing bone Regarded as an ancient molecule that and eye regulates type I collagen fibrillogenesis in bone and eye during fetal development XXV [ 1(XXV)]3 A brain-specific transmembrane collagen Binds to fibrillized β-amyloid peptide Discovered in amyloid of amyloid plaques in

1	fibrillogenesis in bone and eye during fetal development XXV [ 1(XXV)]3 A brain-specific transmembrane collagen Binds to fibrillized β-amyloid peptide Discovered in amyloid of amyloid plaques in Alzheimer’s plaques in brains of patients disease with Alzheimer’s disease Overexpressed in neurons aEach collagen molecule is composed of three polypeptide chains intertwined in a helical configuration. The Roman numerals in the parentheses in the Composition column indicate that the chains have a distinctive structure that differs from the chains with different numerals. Thus, collagen type I has two identical 1 chains and one 2 chain; collagen type II has three identical 1 chains. fibrillar collagen; FACITs; basement membrane–forming collagen; hexagonal network–forming collagen; transmembrane collagens; multiplexins Type Compositiona Location Functions

1	Formation of mRNA in the nucleus Initiation of synthesis of pro–α chains with signal sequences by ribosomes Synthesis of pro–α chains on the rER Hydroxylation of proline and lysine residues (vitamin C required) and cleavage of signal sequence from pro-α-chain Glycosylation of specific hydroxylysyl residues in the rER Formation of procollagen triple helix molecules from a C terminus toward the N terminus in a zipper-like manner 1. 2. 3. 4. 5. 6. Stabalization of the triple helix by formation of intraand interchain hydrogen and disulfide bounds and chaparone proteins (e.g., hsp-47) Transport of procollagan molecules to Golgi apparatus Packaging of procollagen molecules by Golgi into secretory vesicles Movement of vesicles to plasma membrane, assisted by molecular motor proteins associated with microtubules 7. 8. 9. 10. 11. Exocytosis of procollagen molecules 12. Cleavage of trimeric globular Cand helical N-procollagen domains by procollagen Nand C-proteinases 13. Polymerization

1	with microtubules 7. 8. 9. 10. 11. Exocytosis of procollagen molecules 12. Cleavage of trimeric globular Cand helical N-procollagen domains by procollagen Nand C-proteinases 13. Polymerization (self-assembly) of collagen molecules into collagen fibrils (in cove of fibroblast) with development of covalent cross-linking 14. Incorporation of other collagens (e.g, type V, FACITs, etc.) into collagen fibrils

1	FIGURE 6.8  Collagen biosynthesis. Schematic representation of the biosynthetic events and organelles participating in collagen synthesis. Bold numbers correspond to the events numbered in collagen biosynthesis listed at the bottom. Collagen molecule biosynthesis involves a number of intracellular events.

1	Collagen molecule biosynthesis involves a number of intracellular events. The steps in biosynthesis of almost all fibrillar collagens are similar, but type I collagen has been studied in most detail. In general, the synthetic pathway for collagen molecules is similar to other constitutive secretory pathways used by the cell. The unique features of collagen biosynthesis are expressed in multiple posttranslational processing steps that are required to prepare the molecule for the extracellular assembly process. Thus, we see the following:  Collagen chains are synthesized in the rER as long precursors containing large globular aminoand carboxyterminus propeptides called pro– chains (preprocollagen molecules). The newly synthesized polypeptides are simultaneously discharged into the cisternae of the rER, where intracellular processing begins.  Within the cisternae of the rER a number of posttranslational modifications of the preprocollagen molecules occur, including the following: 1.

1	 Within the cisternae of the rER a number of posttranslational modifications of the preprocollagen molecules occur, including the following: 1. The amino-terminus signal sequence is cleaved. 2. Proline and lysine residues are hydroxylated while the polypeptides are still in the nonhelical conformation. Ascorbic acid (vitamin C) is a required cofactor for the addition of hydroxyl groups to proline and lysine residues in pro– chains by the enzymes prolylhydroxylase and lysylhydroxylase; without hydroxylation of proline and lysine residues, the hydrogen bonds essential to the final structure of the collagen molecule cannot form. This explains why wounds fail to heal and bone formation is impaired in scurvy (vitamin C deficiency). 3. O-linked sugar groups are added to some hydroxy -lysine residues (glycosylation) and N-linked sugars are added to the two terminal positions. 4.

1	3. O-linked sugar groups are added to some hydroxy -lysine residues (glycosylation) and N-linked sugars are added to the two terminal positions. 4. The globular structure is formed at the carboxyterminus, which is stabilized by disulfide bonds. Formation of this structure ensures the correct alignment of the three chains during the formation of the triple helix. 5. A triple helix (beginning from the carboxy-terminus) is formed by three chains, except at the terminals where the polypeptide chains remain uncoiled. 6. Intrachain and interchain hydrogen and disulfide bonds form that influence the shape of the molecule. 7. The triple-helix molecule is stabilized by the binding of the chaperone protein hsp47, which also prevents the premature aggregation of the trimers within the cell. The resultant molecule is procollagen.

1	 The folded procollagen molecules pass to the Golgi apparatus and begin to associate into small bundles. This bundling is achieved by the lateral associations between uncoiled terminals of the procollagen molecules. Free and small aggregates of procollagen molecules are packaged into secretory vesicles and transported to the cell surface. Formation of collagen fibrils (fibrillogenesis) involves extracellular events.  As procollagen is secreted from the cell, it is converted into a mature collagen molecule by procollagen peptidase associated with the cell membrane, which cleaves the uncoiled ends of the procollagen (Fig. 6.9).

1	 The aggregated collagen molecules then align together to form the final collagen fbrils in a process known as fbrillogenesis. The cell controls the orderly array of the newly formed fibrils by directing the secretory vesicles to a localized surface site for discharge. The cell simultaneously creates specialized collagen assembly sites called coves. These invaginations of the cell surface allow molecules to concentrate where assembly will SS FIGURE 6.9 • Cleavage of the pro-collagen molecule. Illustration showing the procollagen molecule with Nand C termini. Small curved arrows in the upper part of the illustration show C-terminal where terminals are split from the propeptide procollagen molecule to form the collagen (tropocollagen) molecule. On the C terminus of the molecule, the sugar subunit is GlcNac (N acetylglucosamine) attached to mannose (Mann). (Adapted with permission from Prockop DJ, Kivirikko

1	On the C terminus of the molecule, the sugar subunit is GlcNac (N acetylglucosamine) attached to mannose (Mann). (Adapted with permission from Prockop DJ, Kivirikko KI, Tuderman L, Guzman NA. The (1.5 nm) biosynthesis of collagen and its disorders (first of two parts). N Engl J Med 1979;301:13–23. Copyright © 1979. Massachusetts Medical Society. All rights reserved.) FIGURE 6.10 • Type I collagen fbril. The type I collagen fibril contains small amounts of other collagen types such as types II, III, V, and XI. Note that the core of the fibril contains collagen types V and XI, which help initiate the assembly of the type I fibril.

1	occur (see Fig. 6.8). Within the cove, the collagen molecules align in rows and self-assemble longitudinally in head-to-tail fashion. They also aggregate laterally in a quarter-staggered pattern (see Fig. 6.7). The collagen molecules are then cross-linked by covalent bonds that are formed between the lysine and hydroxylysine aldehyde groups. Collagen biogenesis results in the formation of highly organized polymers called fbrils. Collagen fibrils often consist of more than one type of collagen.

1	Collagen fibrils often consist of more than one type of collagen. Usually different types of fibrillar collagens assemble into fibrils composed of more than one type of collagen molecule. For example, type I collagen fbrils often contain small amounts of types II, III, V, and XI. Current studies indicate that assembly of type I collagen fibrils is proceeded by formation of a fibrillar core containing type V and type XI molecules. Subsequently, type I collagen molecules are deposited and polymerized on the surface of the fibrillar core (Fig. 6.10). In addition, small amounts of type II and III collagen molecules are incorporated into type I collagen fibrils. Collagen types V and XI are important regulators of fibrillogenesis. They control the thickness of type I fibrils by limiting the deposition of collagen molecules after the fibril has reached the desired diameter.

1	Fully mature collagen fibers are usually associated with the FACIT family of collagen molecules that reside on their surfaces. For example, type I fibrils are associated with type XII and type XIV collagens. These collagens contribute to the three-dimensional organization of fibers within the ECM. Type II collagen fibrils, which are abundant within the cartilage, are usually smaller in diameter than type I fibrils. However, these fibrils are also associated with type IX collagen (another member of the FACIT subgroup). Collagen type IX resides on the surface of the type II fibril and anchors it to proteoglycans and other components of the cartilaginous ECM (Fig. 6.11). Collagen molecules are synthesized by various types of connective tissue and epithelial cells.

1	Collagen molecules are synthesized by various types of connective tissue and epithelial cells. Collagen molecules are largely synthesized by connective tissue cells. These cells include fibroblasts in a variety of tissues (e.g., chondrocytes in cartilage, osteoblasts in bone, and pericytes in blood vessels). In addition, the collagen molecules of basement membrane (see page 139) are produced by epithelial cells. The synthesis of collagen is regulated by complex interactions among growth factors, hormones, and cytokines. For example, transforming growth factor (TGF-) and platelet-derived growth factor (PDGF) stimulate collagen synthesis by fibroblasts, whereas steroid hormones (glucocorticoids) inhibit its synthesis.

1	FIGURE 6.11 • Type II collagen fbril. This diagram illustrates the interaction between type II collagen fibrils and type IX collagen molecules in the cartilaginous matrix. Collagen type IX provides the link between the collagen fibrils and GAG molecules, which stabilizes the network of cartilage fibers. Collagen fibers are degraded either by proteolytic or phagocytic pathways.

1	All proteins in the body are being continually degraded and resynthesized. These processes allow tissues to grow and to undergo remodeling. Initial fragmentation of insoluble collagen molecules occurs through mechanical wear, the action of free radicals, or proteinase cleavage. Further degradation is continued by specific enzymes called proteinases. The resulting colla gen fragments are then phagocytosed by cells and degraded by their lysosomal enzymes. Excessive collagen degradation is observed in several diseases (e.g., degradation of cartilage collagen in rheumatoid arthritis or bone collagen in osteoporosis). Secreted collagen molecules are degraded mainly by two different pathways:  Proteolytic degradation occurs outside the cells through the activity of enzymes called matrix metalloproteinases (MMPs). These enzymes are synthesized and secreted into the ECM by a variety of connective tissue cells (fibroblasts, chondrocytes, monocytes, neutrophils, and macrophages), some

1	metalloproteinases (MMPs). These enzymes are synthesized and secreted into the ECM by a variety of connective tissue cells (fibroblasts, chondrocytes, monocytes, neutrophils, and macrophages), some epithelial cells (keratinocytes in the epidermis), and cancer cells. The MMPs include collagenases (which degrade type I, II, III, and X collagens); gelatinases (which degrade most types of denatured collagens, laminin, fibronectin, and elastin); stromelysins (which degrade proteoglycans, fibronectin, and denatured collagens); matrilysins (which degrade type IV collagen and proteoglycans); membrane-type MMPs (which are produced by cancer cells and have a potent pericellular fibrinolytic activity); and macrophage metalloelastases (which degrade elastin, type IV collagen, and laminin).

1	In general, triple-helical undenatured forms of collagen molecules are resistant to degradation by MMPs. In contrast, damaged or denatured collagen (gelatin) is degraded by many MMPs, with gelatinases playing the prominent role. MMP activity can be specifically inhibited by tissue inhibitors of metalloproteinases (TIMPs). Because MMPs are secreted by invasive (migrating) cancer cells, researchers are investigating synthetic therapeutic agents that inhibit the activity of MMPs to control the spread of cancer cells.  Phagocytotic degradation occurs intracellularly and involves macrophages to remove components of the ECM. Fibroblasts are also capable of phagocytosing and degrading collagen fibrils within the lysosomes of the cell. FOLDER 6.1 Clinical Correlation: Collagenopathies

1	FOLDER 6.1 Clinical Correlation: Collagenopathies The important role of collagens in the body can be illus-trated by collagenopathies (collagen diseases), which are caused by a deficit or abnormality in the production of collagens. In the future, gene therapy could potentially be used either to control deposition of faulty collagen or to reverse the disease process caused by the mutated genes. specific collagens. Most collagenopathies are attributed to The following table lists the most common collagenopathies mutations in genes encoding the chains in the various that occur in humans.

1	The Most Common Collagenopathies in Humans Type of Collagen Disease Symptoms I Osteogenesis imperfecta Repeated fractures after minor trauma, brittle bones, abnormal teeth, thin skin, weak tendons, blue sclerae, progressive hearing loss II Kniest dysplasia; Short stature, restricted joint mobility, ocular changes Achondrogenesis, type 2 leading to blindness, wide metaphyses, and joint abnormality seen in radiographs III Ehlers-Danlos type IV Hypermobility of joints of digits, pale thin skin, severe bruisability, early morbidity and mortality, resulting from rupture of vessels and internal organs IV Alport’s syndrome Hematuria resulting from structural changes in the glomerular basement membrane of the kidney, progressive hearing loss, and ocular lesions VII Kindler’s syndrome Severe blistering and scarring of the skin after minor trauma, resulting from absence of anchoring fibrils IX Multiple ephiphyseal dysplasia Skeletal deformations resulting from impaired (MED) endochondral

1	blistering and scarring of the skin after minor trauma, resulting from absence of anchoring fibrils IX Multiple ephiphyseal dysplasia Skeletal deformations resulting from impaired (MED) endochondral ossification and dysplasia (MED), premature degenerative joint disease X Schmid metaphysal Skeletal deformations characterized by modifications chondrodysplasia of the vertebral bodies and chondrodysplasia metaphyses of the long bone XI Weissenbacher-Zweymuller Similar clinical features to type II collagenopathies syndrome Stickler’s syndrome in addition to craniofacial and skeletal deformations, (includes also additional mutations severe myopia, retinal detachment, and progressive of type II collagen gene) hearing loss XVII Generalized atrophic benign Blistering skin disease with mechanically induced epidermolysis bullosa (GABEB) dermal–epidermal separation, epidermolysis bullosa resulting from faulty hemidesmosomes, skin atrophy, nail dystrophy, and alopecia

1	Reticular fibers provide a supporting framework for the cellular constituents of various tissues and organs. Reticular fbers and collagen type I fibers share a prominent feature. They both consist of collagen fibrils. Unlike collagen fibers, however, reticular fibers are composed of type III collagen. The individual fibrils that constitute a reticular fiber exhibit a 68-nm banding pattern (the same as the fibrils of type I collagen). The fibrils have a narrow diameter (about 20 nm), exhibit a branching pattern, and typically do not bundle to form thick fibers.

1	In routinely stained H&E preparations, reticular fibers cannot be identified positively. When visualized in the light microscope with special techniques, reticular fibers have a threadlike appearance. Because they contain a greater relative number of sugar groups than collagen fibers, reticular fibers are readily displayed by means of the periodic acid–Schiff (PAS) reaction. They are also revealed with special silver-staining procedures such as the Gomori and Wilder methods. After silver treatment, the fibers appear black; thus, they are said to be argyrophilic (Fig. 6.12). The thicker collagen fibers in such preparations are colored brown. FIGURE 6.12 • Reticular fbers in the lymph node. Photo-micrograph of a lymph node silver preparation showing the connective tissue capsule at the top and a trabecula extending from it at the left. The reticular fibers (arrows) form an irregular anastomosing network. 650.

1	Reticular fibers are named for their arrangement in a mesh-like pattern or network.

1	In loose connective tissue, networks of reticular fbers are found at the boundary of connective tissue and epithelium, as well as surrounding adipocytes, small blood vessels, nerves, and muscle cells. They are also found in embryonic tissues. The prevalence of reticular fibers is an indicator of tissue maturity. They are prominent in the initial stages of wound healing and scar tissue formation, where they provide early mechanical strength to the newly synthesized ECM. As embryonic development or wound healing progresses, reticular fibers are gradually replaced by the stronger type I collagen fibers. Reticular fibers also function as a supporting stroma in hemopoietic and lymphatic tissues (but not in the thymus). In these tissues, a special cell type, the reticular cell, produces the collagen of the reticular fiber. This cell maintains a unique relationship to the fiber. It surrounds the fiber with its cytoplasm, thus isolating the fiber from other tissue components.

1	In most other locations, reticular fibers are produced by fibroblasts. Important exceptions to this general rule include the endoneurium of peripheral nerves, where Schwann cells secrete reticular fibers, tunica media of blood vessels, and muscularis of the alimentary canal, where smooth muscle cells secrete reticular and other collagen fibers. Elastic fibers allow tissues to respond to stretch and distension. Elastic fbers are typically thinner than collagen fibers and are arranged in a branching pattern to form a three-dimensional network. The fibers are interwoven with collagen fibers to limit the distensibility of the tissue and prevent tearing from excessive stretching (Plate 6, page 196).

1	Elastic fibers stain with eosin but not well, so they cannot always be distinguished from collagen fibers in routine H&E preparations. Because elastic fibers become somewhat refractile with certain fixatives, they may be distinguished from collagen fibers in specimens stained with H&E when they display this characteristic. Elastic fibers can also be selectively stained with special dyes such as orcein or resorcin-fuchsin, as shown in Figure 6.13. The elastic property of the elastin molecule is related to its unusual polypeptide backbone, which causes random coiling.

1	The elastic property of the elastin molecule is related to its unusual polypeptide backbone, which causes random coiling. Elastic fibers are produced by many of the same cells that produce collagen and reticular fibers, particularly fibroblasts and smooth muscle cells. In contrast to collagen fibers, however, elastic fibers are composed of two structural components: A central core of elastin and a surrounding network of fbrillin microfbrils.  Elastin (72 kilodaltons) is a protein that, like collagen, is rich in proline and glycine. Unlike collagen, it is poor in hydroxyproline and completely lacks hydroxylysine. The random distribution of glycines makes the elastin molecule

1	FIGURE 6.13 • Collagen and elastic fbers. Photomicrograph of a mesentery spread stained with resorcin-fuchsin. The mesentery is very thin, and the microscope can be focused through the entire thickness of the tissue. The delicate threadlike branching strands are the elastic fibers (E). Collagen fibers (C) are also evident. They are much thicker; although they cross one another, they do not branch. 200.

1	hydrophobic and allows for random coiling of its fibers. This permits elastic fibers to “slide” over one another or to be stretched and then recoil to their original state. Elastin also contains desmosine and isodesmosine, two large amino acids unique to elastin, which are responsible for the covalent bonding of elastin molecules to one another. These covalent bonds link four elastin molecules into either desmosine or isodesmosine cross-links (Fig. 6.14). Elastin forms fibers of variable thicknesses, or lamellar layers (as in elastic arteries). Elastin is encoded by one of the largest genes in the human genome. The elastin gene consists of 28 kilobases, but less than 10% of the kilobases carry the sequence that encodes elastin.

1	 Fibrillin-1 (350 kilodaltons) is a glycoprotein that forms fine microfibrils measuring 10 to 12 nm in diameter. During the early stages of elastogenesis, fibrillin-1 microfibrils are used as substrates for the assembly of elastic fibers. The microfibrils are formed first; elastin material is then deposited on the surface of the microfibrils. Elastin-associated fbrillin microfbrils play a major role in organizing elastin into fibers. The absence of fibrillin

1	FIGURE 6.14 • Diagram of elastin molecules and their interaction. a. Elastin molecules are shown joined by covalent bonding between desmosine and isodesmosine (purple) to form a cross-linked network. Inset shows enlargement of the elastin molecule in its individual and random-coiled conformation with the covalent bond formed by desmosine. b. The effect of stretching is shown. When the force is withdrawn, the network reverts to the relaxed state as in panel a. (Modified with permission from Alberts B, et al. Essential Cell Biology, p. 153. Copyright 1997. Routledge, Inc., part of The Taylor & Francis Group.) microfibrils during elastogenesis results in the formation of elastin sheets or lamellae, as found in blood vessels. Abnormal expression of the fbrillin gene (FBN1) is linked to Marfan’s syndrome, a complex, autosomal dominant, connective tissue disorder. Immunoﬂuorescence of a skin biopsy specimen from a person with this syndrome shows an absence of elastin-associated fibrillin

1	syndrome, a complex, autosomal dominant, connective tissue disorder. Immunoﬂuorescence of a skin biopsy specimen from a person with this syndrome shows an absence of elastin-associated fibrillin microfibrils. One of the consequences of the disease is abnormal elastic tissue.

1	With both the TEM and SEM, elastin appears as an amorphous structure of low electron density. In contrast, the fibrillin microfibrils are electron dense and are readily apparent even within the elastin matrix (Fig. 6.15). In mature fibers, the fibrillin microfibrils are located within the elastic fiber and at its periphery. The presence of microfibrils within the fiber is associated with the growth process; thus, as the fiber is formed and thickens, the microfibrils become entrapped within the newly deposited elastin.  FOLDER 6.2 Clinical Correlation: Sun Exposure and Molecular Changes in Photoaged Skin

1	Chronological aging of the skin is a complex process that is associated with functional and structural changes within the stratified squamous epithelium (epidermis) as well as the underlying connective tissue of the dermis. When these changes are intensified by prolonged exposure to solar or ultraviolet (UV) radiation, the process is referred to as photoaging. Chronic sun exposure ages the skin at an accelerated rate, especially in exposed areas of the body such as the face, neck, dorsal surface of the hands, and fore-arms. Clinical signs associated with photoaging include dyspigmentation, freckles, deep wrinkles, increased laxity, and increased risk for cutaneous cancers. The most prominent changes in the dermis of pho-toaged skin are associated with connective tissue fibers. Decreased production of type I and type III collagen fibers is observed in normal aged skin; however, these changes are more pronounced in sun-exposed regions. Sunlight exposure affects collagen biogenesis by

1	production of type I and type III collagen fibers is observed in normal aged skin; however, these changes are more pronounced in sun-exposed regions. Sunlight exposure affects collagen biogenesis by altering the cross-linking that occurs between collagen molecules during fibrillogenesis (page 168). These alterations result in for-mation of collagen fibers with abnormal stability and decreased resistance to enzymatic degradation. The overall number of elastic fibers also decreases with age; however, in photoaged skin, the number of abnormally thick and nonfunctional elastic fibers increases. Recent studies of fbrillin microfbrils from photoaged skin reveal that the microfibrillar network is affected by solar radiation. Excessive sun exposure causes the fibrillin microfibrils to undergo extensive changes. They became sparse and trun-cated, leading to the formation of aberrant nonfunctional elastic fibers ECM that finally degenerate into homogenous and amorphous elastin-containing

1	extensive changes. They became sparse and trun-cated, leading to the formation of aberrant nonfunctional elastic fibers ECM that finally degenerate into homogenous and amorphous elastin-containing masses. Photoaging is also characterized by abnormal degrada-tion of the connective tissue matrix associated with accu-mulation of nonfunctional matrix components. Fibroblasts and neutrophils residing in radiation-damaged areas of the skin secrete matrix metalloproteinases (MMP-1 and -9), elastases, and other proteases (cathepsin G). These enzymes are modulated by tissue inhibitors of metallopro-teinases (TIMPs) that protect extracellular proteins from endogenous degradation. In photoaged skin, TIMP levels are significantly reduced, which further contributes to photodamage of the skin. The best strategies to prevent photodamage caused by solar and UV radiation is the use of physical and chemical sunscreens to prevent UV penetration into skin. Other methods are also used in treating damaged

1	strategies to prevent photodamage caused by solar and UV radiation is the use of physical and chemical sunscreens to prevent UV penetration into skin. Other methods are also used in treating damaged skin. These include reducing skin inflammatory reactions with anti-inflammatory medications, inhibiting activities of elastase and other MMPs to prevent degradation of ECM, and stim-ulating natural or applying synthetic inhibitors of MMPs activities to control destruction of connective tissue ECM.

1	Elastic material is a major extracellular substance in vertebral ligaments, larynx, and elastic arteries. In elastic ligaments, the elastic material consists of thick fibers interspersed with collagen fibers. Examples of this material are found in the ligamenta flava of the vertebral column and the ligamentum nuchae of the neck. Finer fibers are present in elastic ligaments of the vocal folds of the larynx. In elastic arteries, the elastic material is in the form of fenestrated lamellae, sheets of elastin with gaps or openings. The lamellae are arranged in concentric layers between layers of smooth muscle cells. Like the collagen fibers in the tunica media of blood vessel walls, the elastic material of arteries is produced by smooth muscle cells, not by fibroblasts. In contrast to elastic fibers, microfibrils are not found in the lamellae. Only the amorphous elastin component is seen in electron micrographs. Elastin is synthesized by fibroblasts and vascular smooth muscle cells.

1	Elastin is synthesized by fibroblasts and vascular smooth muscle cells. As noted, elastic fibers are produced by fibroblasts or smooth muscle cells within the walls of the vessels. Elastin synthesis parallels collagen production; in fact, both processes can occur simultaneously in a cell. The orderly modification and assembly of procollagen and proelastin, as well as the synthesis of other connective tissue components, are controlled by signal sequences that are incorporated into the beginning of the polypeptide chains of each of the molecules.

1	Signal sequences can be compared to airline tags on luggage. Just as the tags ensure that baggage moves correctly from one aircraft to another at airports, so signal peptides ensure that the components of procollagen and proelastin remain separate and properly identified as they pass through the organelles of the cell. During this transit, a series of synthetic events and posttranslational modifications occur before the polypeptides ultimately arrive at their proper destination.

1	The extracellular matrix (ECM) is a complex and intricate structural network that surrounds and supports cells within the connective tissue. As previously mentioned, it contains a variety of fibers such as collagen and elastic fbers that are formed from different types of structural proteins. In addition, the ECM contains a variety of proteoglycans (e.g., aggrecan, syndecan); multiadhesive glycoproteins (such as fibronectin and laminin); and glycosaminoglycans (e.g., dermatan sulfate, keratan sulfate, hyaluronan). The last three groups of molecules constitute the ground substance.

1	FIGURE 6.15 • a. Electron micrograph of an elastic fber. The elastin (E) of the fiber has a relatively amorphous appearance. The fibrillin microfibrils (arrows) are present at the periphery and within the substance of the fiber. A number of collagen fibrils (C) are also present in this electron micrograph. 40,000. b. Scanning electron micrograph of an elastic fber. This scanning electron micrograph of the human dens irregular connective tissue from the dermis shows structure of elastic fiber (E) and illustrate it’s relative size in comparison to surrounding collagen fibrils (C). Note the presence of small fibrillin microfibrils (arrows) at the surface of elastic fiber 40,000. (Courtesy of Douglas R. Keene)

1	R. Keene) All molecules found in the ECM share common domains, and the function of the ECM relies largely on the interactions between these molecules. Each connective tissue cell secretes a different ratio of ECM molecules that contribute to the formation of many different architectural arrangements; therefore, the ECM possesses specific mechanical and biochemical properties characteristic for the tissue in which it is present. For instance, the properties of the ECM in loose connective tissue are different from those of the ECM in cartilage or bone. The extracellular matrix not only provides mechanical and structural support for tissue but also influences extracellular communication.

1	The ECM provides mechanical and structural support as well as tensile strength for the tissue. It also functions as a biochemical barrier and plays a role in regulating metabolic functions of the cells surrounded by the matrix. The ECM anchors cells within tissues through cell-to-ECM adhesion molecules and provides pathways for cell migration (e.g., during wound repair). Recent studies indicate that the ECM exerts a regulatory effect on embryonic development and cell differentiation. The matrix is capable of binding and retaining growth factors, which in turn modulate cell growth. With the aid of cell adhesion molecules, the ECM also influences the transmission of information across the plasma membrane of the connective tissue cells. Thus, the current view of ECM components (fibers and ground substance molecules) is that they form a dynamic and interactive system that informs cells about the biochemical and mechanical changes in their extracellular environment.

1	Ground substance is the part of the extracellular matrix that occupies the spaces between the cells and fibers; it consists of glycosaminoglycans (GAGs), proteoglycans, and multiadhesive glycoprotein.

1	Ground substance is a viscous, clear substance with a slippery feel and high water content. In the light microscope, ground substance appears amorphous in sections of tissue preserved by freeze-drying or in frozen sections stained with basic dyes or by the PAS method. In routine H&E preparations, ground substance is always lost because of its extraction during fixation and dehydration of the tissue. The result is an empty background; only cells and fibers are evident. Thus, in most histologic preparations, the appearance of ground substance—or its lack of appearance—belies its functional importance. Ground substance consists predominately of three groups of molecules: Proteoglycans, very large macromolecules composed of a core protein; glycosaminoglycan molecules (GAGs), which are covalently bound to the proteoglycans; and multiadhesive glycoproteins. The size and structure of the three groups of molecules vary enormously.

1	GAGs are responsible for the physical properties of ground substance. The GAGs are the most abundant heteropolysaccharide components of ground substance. These molecules represent long-chain unbranched polysaccharides composed of repeating disaccharide units. The disaccharide units contain either of two modified sugars—N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc)—and a uronic acid such as glucuronate or iduronate. GAGs (except hyaluronan) are synthesized by connective tissue cells as a covalent, posttranslational modification of proteins called proteoglycans. For example, heparin is formed by enzymatic cleavage of heparan sulfate; dermatan sulfate is similarly modified from chondroitin sulfate.

1	GAGs are highly negatively charged because of the sulfate and carboxyl groups located on many of the sugars, hence their propensity for staining with basic dyes. The high density of the negative charge (polyanions) also attracts water, forming a hydrated gel. The gel-like composition of ground substance permits rapid diffusion of water-soluble molecules. At the same time, the rigidity of the GAGs provides a structural framework for the cells. GAGs are located primarily within the ground substance as well as on the surface of cells within the ECM. On the basis of differences in specific sugar residues, the nature of their linkages, and the degree of their sulfation, a family of seven distinct GAGs is recognized. They are listed and partially characterized in Table 6.3. Hyaluronan is always present in the extracellular matrix as a free carbohydrate chain.

1	Hyaluronan is always present in the extracellular matrix as a free carbohydrate chain. The GAG hyaluronan (hyaluronic acid) deserves special note because it differs from the other GAGs in several respects. It is an exceedingly long, rigid molecule composed of a carbohydrate chain of thousands of sugars rather than the several hundred or fewer sugars found in other GAGs. Hyaluronan polymers are very large (100 to 10,000 kilodaltons) and can displace a large volume of water. They are synthesized by enzymes on the cell surface; therefore, they are not posttranslationally modified like all other GAGs. Hyaluronan is also unique among the GAGs in that it does not contain any sulfate.

1	Each hyaluronan molecule is always present in the form of a free carbohydrate chain; in other words, it is not covalently bound to protein, so it does not form proteoglycans. By means of special link proteins, however, proteoglycans indirectly bind to hyaluronan, forming giant macromolecules called proteoglycan aggregates (Fig. 6.16). These molecules are abundant in the ground substance of cartilage. The pressure, or turgor, that occurs in these giant hydrophilic proteoglycan aggregates accounts for the ability of cartilage to resist compression without inhibiting ﬂexibility, making them excellent shock absorbers.

1	Another important function of hyaluronan is to immobilize certain molecules in the desired location of the ECM. For instance, ECM contains binding sites for several growth factors, such as TGF-. The binding of growth factors to proteoglycans may cause either their local aggregation or dispersion, which in turn either inhibits or enhances the movement of migrating macromolecules, microorganisms, or metastatic cancer cells in the extracellular environment. In addition, hyaluronan molecules act as efficient insulators,

1	TABLE Glycosaminoglycans6.3 Molecular Disaccharide Name Weight (kDa) Composition Localization Function Hyaluronan 100–10,000 D-Glucuronic acid Synovial fluid, vitreous Large polymers of N-acetylglucosamine humor, ECM of connective hyaluronan can displace tissues a large volume of water. Thus this polymer is excellent lubricant and shock absorber Chondroitin 25 D-Glucuronic acid 4-sulfate N-acetylgalactosamine Chondroitin sulfates and 4-sulfate hyaluronan are fundamental components of aggrecan found in articular cartilage. Aggrecan confers on articular cartilage shock-Chondroitin 25 D-Glucuronic acid absorbing properties 6-sulfate N-acetylgalactosamine 6-sulfate Dermatan 35 L-Iduronic acid Skin, blood vessels, Dermatan sulfate proteogly-sulfate N-acetylgalactosamine heart valves cans have been implicated 4-sulfate in cardiovascular disease, tumorigenesis, infection, wound repair, fibrosis, and as a modulator in cell behavior Keratan 10 Galactose or galactose Bone, cartilage, cornea

1	been implicated 4-sulfate in cardiovascular disease, tumorigenesis, infection, wound repair, fibrosis, and as a modulator in cell behavior Keratan 10 Galactose or galactose Bone, cartilage, cornea Keratan sulfate proteoglycans sulfate 6-sulfate N-acetylgluco-function in cellular recognition samine 6-sulfate of protein ligands, axonal guidance, cell motility, corneal transparency, and embryo implantation Heparan 15 Glucuronic acid Basal lamina, normal Facilitates interactions with sulfate or L-iduronic acid component of cell fibroblastic growth factor 2-sulfate N-sulfamyl-surface (FGF) and its receptor glucosamine or N-acetyl-glucosamine Heparin 40 Glucuronic acid Limited to granules of Functions as an anticoagulant, or L-iduronic acid mast cells and facilitates interactions with 2-sulfate N-sulfamyl-basophiles FGF and its receptor glucosamine or N-acetylglucosamine 6-sulfate Cartilage, bone, heart valves kDa, kilodaltons.

1	because other macromolecules have difficulty diffusing through the dense hyaluronan network. With this property, hyaluronan (and other polysaccharides) regulates the distribution and transport of plasma proteins within the connective tissue. Proteoglycans are composed of GAGs covalently attached to core proteins.

1	Proteoglycans are composed of GAGs covalently attached to core proteins. The majority of GAGs in the connective tissue are linked to core proteins, forming proteoglycans. The GAGs extend perpendicularly from the core in a brushlike structure. The linkage of GAGs to the protein core involves a specific trisaccharide composed of two galactose residues and a xylulose residue. The trisaccharide linker is coupled through an O-glycosidic bond to the protein core that is rich in serine and threonine residues, allowing multiple GAG attachments. Proteoglycans are remarkable for their diversity (Fig. 6.17). The number of GAGs attached to the protein core varies from 1 (i.e., decorin) to more than 200 (i.e., aggrecan). A core protein may have identical GAGs attached to it (as in the

1	FIGURE 6.16 • Proteoglycan structure. This schematic drawing shows, on the right, a proteoglycan monomer and its relationship to the hyaluronan molecule as represented in the ground substance of cartilage. The proteoglycan monomer is composed of a core protein to which GAGs are covalently bound. The proteoglycan monomer consists of different numbers of GAGs joined to the core protein. The end of the core protein of the proteoglycan monomer interacts with a link protein, which attaches the monomer into the hyaluronan forming the proteoglycan aggregate. On the left, hyaluronan molecules forming linear aggregates, each with many proteoglycan monomers, are interwoven with a network of collagen fibrils. case of fibroglycan or versican) or different GAG molecules (as in the case of aggrecan or syndecan).

1	case of fibroglycan or versican) or different GAG molecules (as in the case of aggrecan or syndecan). Proteoglycans are found in the ground substance of all connective tissues and also as membrane-bound molecules on the surface of many cell types. Transmembrane proteoglycans such as syndecan link cells to ECM molecules (Fig. 6.17). For instance, syndecan is expressed two different times on the surface of B lymphocytes. Syndecan molecules are first expressed during early development when lymphocytes are attached to the matrix protein of the bone marrow as they undergo differentiation. The loss of expression of this proteoglycan coincides with the release of the B lymphocyte into the circulation. The second time the B lymphocyte expresses syndecan is during its differentiation into a plasma cell within the connective tissue. Syndecan anchors the plasma cell to the ECM proteins of the connective tissue.

1	Aggrecan is another important extracellular proteoglycan. Its molecules are noncovalently bound to the long molecule of hyaluronan (like bristles to the backbone in a bottle brush); this binding is facilitated by linking proteins. To each aggrecan core protein multiple chains of chondroitin sulfate and keratan sulfate are covalently attached through the trisaccharide linker. The most common proteoglycans are summarized in Table 6.4. Multiadhesive glycoproteins play an important role in stabilizing the ECM and linking it to cell surfaces.

1	Multiadhesive glycoproteins play an important role in stabilizing the ECM and linking it to cell surfaces. Multiadhesive glycoproteins represent a small but important group of proteins residing in the ECM. They are multidomain and multifunctional molecules that play an important role in stabilizing the ECM and linking it to the cell surface. They possess binding sites for a variety of ECM proteins such as collagens, proteoglycans, and GAGs; they also interact with cell-surface receptors such as integrin and laminin receptors (Fig. 6.18). Multiadhesive glycoproteins regulate and modulate functions of the ECM related to cell movement and cell migration as well as stimulate cell proliferation and differentiation. Among the best characterized multiadhesive glycoproteins are the following:  Fibronectin (250 to 280 kilodaltons) is the most abun dant glycoprotein in connective tissue. Fibronectins are

1	FIGURE 6.17 • Common proteoglycan monomers of the connective tissue matrix. Note the diversity of proteoglycan molecules; the number of GAGs attached to the protein core varies from one in decorin to more than 200 in aggrecan. Note also that versican has identical GAG molecules (chondroitin sulfate) attached to the core molecule whereas aggrecan has a mixture of chondroitin sulfate and keratan sulfate attached to the core protein. Syndecan is a transmembrane proteoglycan that attaches the cell membrane to the extracellular matrix.

1	by disulfide bonds at a carboxy terminus to form 50-nmlong arms (see Fig. 6.18). Each molecule contains several binding domains that interact with different ECM molecules (e.g., heparan sulfate; collagen types I, II, and III; fibrin; hyaluronan; and fibronectin) and integrin, a cell-surface receptor. Binding to a cell-surface receptor activates fibronectin, which then assembles into fibrils. Fibronectin plays an important role in cell attachment to the ECM. At least 20 different fibronectin molecules have been identified to date.  Laminin (140 to 400 kilodaltons) is present in basal and external laminae. It possesses binding sites for collagen type IV molecules, heparan sulfate, heparin, entactin, laminin, and the laminin receptor on the cell surface. The process of basal lamina assembly and the role of the laminin in this process are described in Chapter 5 (see page 138).

1	 Tenascin (280 kilodaltons/monomer) appears during embryogenesis, but its synthesis is switched off in mature tissues. It reappears during wound healing and is also found within musculotendinous junctions and malignant tumors. Tenascin is a disulfide-linked dimer molecule that consists of six chains joined at their amino-terminus (see Fig. 6.18). It has binding sites for fibrinogen, heparin, and EGF-like growth factors; thus, it participates in cell attachment to the ECM.  Osteopontin (44 kilodaltons) is present in the ECM of bone. It binds to osteoclasts and attaches them to the underlying bone surface. Osteopontin plays an important role in sequestering calcium and promoting calcification of the ECM. Important multiadhesive glycoproteins found in the ECM of connective tissue are summarized in Table 6.5. Connective tissue cells can be resident or wandering.

1	Important multiadhesive glycoproteins found in the ECM of connective tissue are summarized in Table 6.5. Connective tissue cells can be resident or wandering. The cells that make up the resident cell population are relatively stable; they typically exhibit little movement and can be regarded as permanent residents of the tissue. These resident cells include  fbroblasts and a closely related cell type, the myof broblast, macrophages, adipocytes, mast cells,and  adult stem cells. The wandering cell population or transient cell population consists primarily of cells that have migrated into the tissue from the blood in response to specific stimuli. These include  lymphocytes, plasma cells, neutrophils, eosinophils, basophils,and  monocytes. The fibroblast is the principal cell of connective tissue.

1	The fibroblast is the principal cell of connective tissue. Fibroblasts are responsible for the synthesis of collagen, elastic and reticular fibers, and the complex carbohydrates of the ground substance. Research suggests that a single fibro blast is capable of producing all of the ECM components.

1	Fibroblasts reside in close proximity to collagen fibers. In routine H&E preparations, however, often only the nucleus is visible. It appears as an elongated or disclike structure, sometimes with a nucleolus evident. The thin, pale-staining, flattened processes that form the bulk of the cytoplasm are usually not visible, largely because they blend with the collagen fibers. In some specially prepared specimens, it is possible to distinguish the cytoplasm of the cell from the fibrous components (Fig. 6.19a). When ECM material is produced during active growth or in wound repair (in activated fbroblasts), the cytoplasm of the fibroblast is more extensive and may display basophilia as a result of increased amounts of rER associated with protein synthesis (Fig. 6.19b). When examined with the TEM, the fibroblast cytoplasm exhibits profiles of rER and a prominent Golgi apparatus (Fig. 6.20).

1	TABLE Proteoglycans 6.4 kDa, kilodaltons. Molecular Molecular Name Weight (kDa) Composition Localization Function Aggrecan 250 Linear molecule; binds via a Cartilage, Responsible for hydration of link protein to hyaluronan; Chondrocytes extracellular matrix of contains 100 to 150 molecules cartilage of keratan sulfate and chondroitin sulfate chains Decorin 38 Small protein that contains Connective tissue, Functions in collagen only one chondroitin sulfate fibroblasts, cartilage, fibrillogenesis; by attaching or dermatan sulfate chain and bone to neighboring collagen molecules, helps to orient fibers. Regulates the thickness of the fibril and interacts with transforming growth factor (TGF-) Versican 260 Associated with a link protein; Fibroblasts, skin, Possesses EGF-like domains contains main and 12–15 smooth muscle, on the core protein; chains of chondroitin sulfate brain, and mesangial participates in cell-to-cell and attached to core protein cells of the kidney

1	domains contains main and 12–15 smooth muscle, on the core protein; chains of chondroitin sulfate brain, and mesangial participates in cell-to-cell and attached to core protein cells of the kidney cell-to-extracellular matrix interactions; binds to fibulin-1 Syndecan 33 Family of at least four different Embryonic epithelia, The extracellular domain types of transmembrane mesenchymal cells, binds collagens, heparin, proteoglycans, containing developing lymphatic tenascin, and fibronectin, varying amounts of both tissue cells, intracellular domain binds heparan sulfate and lymphocytes, and to cytoskeleton via actin chondroitin sulfate molecules plasma cells

1	The myofibroblast displays properties of both fibrmyosinoblasts and smooth muscle cells.

1	The myofbroblast is an elongated, spindly connective tissue cell not readily identifiable in routine H&E preparations. It is characterized by the presence of bundles of actin filament with associated actin motor proteins such as nonmuscle myosin (page 61). Expression of the -smooth muscle actin ( -SMA; actin isoform found in the vascular smooth muscles) in myo fibroblasts is regulated by TGF-1. The actin bundles transverse the cell cytoplasm originating and terminating on the opposite sites of the plasma membrane. The site of actin fibers attachment to the plasma membrane also serves as a cell-to-ECM anchoring junction and is called fbronexus. It resembles focal adhesion found in the epithelial cells (page 144). This arrangement is of the basis of a mechanotransduction system in which force that is generated by the contraction of intracellular actin bundles is transmitted to the ECM. With the TEM, the myofibroblast displays typical characteristics of the fibroblast along with

1	force that is generated by the contraction of intracellular actin bundles is transmitted to the ECM. With the TEM, the myofibroblast displays typical characteristics of the fibroblast along with characteristics of smooth muscle cells. In addition to rER and Golgi profiles, the myofibroblast contains bundles of longitudinally disposed actin filaments and dense bodies similar to those observed in smooth muscle cells (Fig. 6.21). As in the smooth muscle cell, the nucleus often shows an undulating surface profile, a phenomenon associated with cell contraction. The myofibroblast differs from the smooth muscle cell in that it lacks a surrounding basal lamina (smooth muscle cells are surrounded by a basal or external lamina). Also, it usually exists as an isolated cell, although its processes may contact the processes of other myofibroblasts. Such points of contact exhibit gap junctions, indicating intercellular communication.

1	FIGURE 6.18 • Common multiadhesive glycoproteins. These proteins reside in the extracellular matrix and are important in stabilizing the matrix and linking it to the cell surface. They are multifunctional molecules of different shapes and possess multiple binding sites for a variety of extracellular matrix proteins such as collagens, proteoglycans, and GAGs. Note that multiadhesive proteins interact with basal membrane receptors such as integrin and laminin receptors.

1	TABLE Multiadhesive Glycoproteins6.5 CAM, cell adhesion molecule; ECM, extracellular matrix; EGF, epithelial growth factor; kDa, kilodaltons. Molecular Name Weight (kDa) Molecular Composition Localization Function Fibronectin 250–280 Dimer molecule formed Present in the ECM of Responsible for cell adhesion from two similar peptides many tissues and mediate migration; possesses linked by a disulfide bond binding sites for integrins, type IV collagen, heparin, and fibrin Laminin 140–400 Cross-shaped molecule Present in basal laminae Anchors cell surfaces to the basal formed from three of all epithelial cells and lamina. It possesses binding polypeptides ( chain and external laminae of muscle sites for collagen type IV, heparan two chains) cells, adipocytes, and sulfate, heparin, entactin, laminin, Schwann cells and integrin receptors on the cell surface Tenascin 1,680 Giant protein formed Embryonic mesenchyme, Modulates cell attachments to the from six chains connected perichondrium,

1	laminin, Schwann cells and integrin receptors on the cell surface Tenascin 1,680 Giant protein formed Embryonic mesenchyme, Modulates cell attachments to the from six chains connected perichondrium, perio-ECM; possesses binding sites for by disulfide bonds steum, musculotendi-fibronectin, heparin, EGF-like nous junctions, wounds, growth factors, integrins, and CAMs tumors Osteopontin 44 Single-chain glycosylated Bone Binds to osteoclasts; possesses polypeptide binding sites for calcium, hydroxyapatite, and integrin receptor on the osteoclast membrane Entactin/ 150 Single-chain rodlike Basal lamina–specific Links laminin and type IV collagen; Nidogen sulfated glycoprotein protein has binding sites for perlecan and fibronectin

1	FIGURE 6.19 • Fibroblasts in connective tissue. a. Photomicrograph of a connective tissue specimen in a routine H&E-stained, paraffin-embedded preparation shows nuclei of fibroblasts (F ). 600. b. During the repair process of a wound, the activated fibroblasts (F ) exhibit more basophilic cytoplasm, which is readily observed with the light microscope. 500. Macrophages are phagocytotic cells derived from monocytes. Connective tissue macrophages, also known as tissue histiocytes, are derived from blood cells called monocytes. Monocytes migrate from the bloodstream into the connective tissue, where they differentiate into macrophages.

1	In the light microscope and with conventional stains, tissue macrophages are difficult to identify unless they display obvious evidence of phagocytotic activity—for example, visible ingested material within their cytoplasm. Another feature that assists in identifying macrophages is an indented or kidney-shaped nucleus (Fig. 6.22a). Lysosomes are abundant in the cytoplasm and can be revealed by staining for acid phosphatase activity (both in the light microscope and with the TEM); a positive reaction is a further aid in identification of the macrophage. With the TEM, the surface of the macrophage exhibits numerous folds and fingerlike projections (Fig. 6.22b). The surface folds engulf the substances to be phagocytosed.

1	FIGURE 6.20 • Electron micrograph of fbroblasts. The processes of several fibroblasts are shown. The nucleus of one fibroblast is in the upper right of the micrograph. The cytoplasm contains conspicuous profiles of rER. The cisternae of the reticulum are distended, indicating active synthesis. The membranes of the Golgi apparatus (G) are seen in proximity to the rER. Surrounding the cells are collagen fibrils (CF), almost all of which have been cut in cross section and thus appear as small dots at this magnification. 11,000. The macrophage contains a large Golgi apparatus, rER and sER, mitochondria, secretory vesicles, and lysosomes. The lysosomes of the macrophage, along with the surface cytoplasmic projections, are the structures most indicative of the specialized phagocytotic capability of the cell. The

1	The lysosomes of the macrophage, along with the surface cytoplasmic projections, are the structures most indicative of the specialized phagocytotic capability of the cell. The FIGURE 6.21 • Electron micrograph of a myofbroblast. The cell exhibits some features of a fibroblast, such as areas with a moderate amount of rER. Compare with Figure 6.20. Other areas, however, contain aggregates of thin filaments and cytoplasmic densities (arrows), features that are characteristic of smooth muscle cells. The arrowheads indicate longitudinal profiles of collagen fibrils. 11,000.

1	macrophage may also contain endocytotic vesicles, phagolysosomes, and other evidence of phagocytosis (i.e., residual bodies). The rER, sER, and Golgi apparatus support the synthesis of proteins involved in the cell’s phagocytotic and digestive functions, as well as in its secretory functions. The secretory products leave the cell by both the constitutive and regulated exocytotic pathways. Regulated secretion can be activated by phagocytosis, immune complexes, complement, and signals from lymphocytes (including the release of lymphokines, biologically active molecules that influence the activity of other cells). The secretory products released by the macrophage include a wide variety of substances related to the immune response, anaphylaxis, and inflammation. The release of neutral proteases and GAGases (enzymes that break down GAGs) facilitates the migration of the macrophages through the connective tissue.

1	Although the main function of the macrophage is phagocytosis, either as a defense activity (e.g., phagocytosis of bacteria) or as a cleanup operation (e.g., phagocytosis of cell debris), it also plays an important role in immune response reactions. Macrophages have specific proteins on their surfaces known as major histocompatibility complex II (MHC II) molecules that allow them to interact with helper CD4 T lymphocytes. When macrophages engulf a foreign cell, antigens—short polypeptides, 7 to 10 amino acids long, from the foreign cell—are displayed on the surface of MHC II molecules. If a CD4 T lymphocyte recognizes the displayed antigen, it becomes activated, triggering an immune response (see Chapter 14). Because macrophages “present” antigen to helper CD4 T lymphocytes, they are called antigen-presenting cells (APCs).

1	When macrophages encounter large foreign bodies, they may fuse to form a large cell with as many as 100 nuclei that engulfs the foreign body. These multinucleated cells are called foreign body giant cells (Langhans cells). Mast cells develop in bone marrow and differentiate in connective tissue. Mast cells are large, ovoid, connective tissue cells (20 to 30 m in diameter) with a spherical nucleus and cytoplasm filled with large, intensely basophilic granules. They are not easily identified in human tissue sections unless special fixatives are used to preserve the granules. After glutaraldehyde fixation, mast cell granules can be displayed with basic dyes such as toluidine blue. It stains the granules intensely and metachromatically because they contain heparin, a highly sulfated proteoglycan (Fig. 6.23a). The cytoplasm displays small amounts of rER, mitochondria, and a Golgi apparatus. The cell surface contains numerous microvilli and folds.

1	The mast cell is related, but not identical, to the basophil, a white blood cell that contains similar granules (Table 6.6). They both arise from a pluripotential hemopoietic stem cell (HSC) in the bone marrow. Mast cells initially circulate in the peripheral blood as agranular cells of monocytic appearance. After migrating into the connective tissue, immature mast cells differentiate and produce their characteristic granules (Fig. 6.23b). In contrast, basophils differentiate and remain within the circulatory system. The surface of mature mast cells express a large number of high-affinity Fc receptors (Fc RI) to which immunoglobulin E (IgE) antibodies are attached. Binding of specific antigen to exposed IgE antibody molecules on the mast cell surface leads to an aggregation of Fc receptors.

1	 FOLDER 6.3 Clinical Correlations: Role of Myofibroblasts in Wound Repair cess of wound healing. A clean surgical skin incision begins the healing process when a blood clot containing fibrin and blood cells fill the narrow space between the edges of the incision. The infammatory process, which begins as early as 24 hours after initial injury, contains the damage to a small area, aids in the removal of injured and dead tissues, and initiates deposition of new ECM proteins. During the initial phases of inflammation, neutrophils and monocytes infiltrate the injury (maximum infiltration by neutrophils occurs in the first 1 to 2 days after injury). Monocytes transform into macrophages (they usually replace neutrophils by day 3 after injury) (page 181). At the same time, in response to local growth factors, fibroblasts and vascular endothelial cells begin to proliferate and migrate into the delicate fibrin matrix of the blood clot, forming the granulation tissue, a specialized type of

1	growth factors, fibroblasts and vascular endothelial cells begin to proliferate and migrate into the delicate fibrin matrix of the blood clot, forming the granulation tissue, a specialized type of tissue characteristic of the repair process. Usually by day 5 after injury, the fully developed granulation tissue bridges the incision gap. It is composed mainly of large numbers of small vessels, fibroblasts, and myofibroblasts, and variable numbers of other inflammatory cells. Migra ting fibroblasts exert tractional forces on the ECM, reorganizing it along lines of stress. Under the influence of growth factors such as TGF-1 and mecha nical forces, fibro blasts undergo differentiation into myofbroblasts.This -SMA. This type of actin is not present in the cytoplasm of fibroblasts (Fig. 6.3.1). The myofibroblasts generate and maintain steady contractile force (similar to that of smooth muscle cells) that cause shortening of the connective tissue fibers and wound closure. At the same time,

1	The myofibroblasts generate and maintain steady contractile force (similar to that of smooth muscle cells) that cause shortening of the connective tissue fibers and wound closure. At the same time, myofibroblasts synthesize and lay down collagen fibers and other ECM components that are responsible for tissue remodeling. During the second week of wound healing, the amount of cells in tissue undergoing repair decreases; most of the myofibroblasts undergo apoptosis and disappear, resulting in a connective scar that has very few cellular elements. In some pathologic conditions, myofibroblasts persist and continue the process of remodeling. This continued remodeling causes hypertrophic scar formation, resulting in excessive connective tissue contracture. Extensive numbers of myofibroblasts are found in most contractive diseases of connective tissue (fibromatoses). For example, palmar fbromatosis (Dupuytren’s disease) is characterized by the thickening of palmar aponeurosis, which leads to

1	found in most contractive diseases of connective tissue (fibromatoses). For example, palmar fbromatosis (Dupuytren’s disease) is characterized by the thickening of palmar aponeurosis, which leads to progressive flex-ion contracture of the fourth and fifth digits of the hand (Fig. F 6.3.2) If scar tissue grows beyond boundaries of the original wound and does not regress, it is called a keloid. Its formation is more common among African Americans than other ethnic groups.

1	FIGURE F6.3.1 • Fibroblasts and myofbroblasts in the culture. This immunofluorescence image shows wild-type 3T3 fibroblasts cultured on the collagen lattice. Under the stimulation of certain growth factors such as TGF-1, some fibroblast differentiate to myofibroblasts, expressing -SMA, the marker of myofibroblasts differentiation. Cells were stained with fluorescein-labeled phalloidin to visualize F-actin filaments (green), and -SMA were labeled with primary antibodies against -SMA and visualized with secondary goat anti-mouse antibodies conjugated with FITC (red). Co-localization of -SMA with F-actin is indicated by yellow color. Note that some cells have completed their differentiation, and others are in the early stages. 1,000. (Courtesy of Dr. Boris Hinz.)

1	FIGURE 6.22 • Photomicrograph and electron micrograph of a macrophage. a. This photomicrograph shows several macrophages (M) in the connective tissue from the area of wound healing. They can be distinguished from other cells by the presence of an indented or kidney-shaped nucleus. Note several mature neutrophils (N) with segmented nuclei located in the connective tissue that surround blood vessel filled with red and white blood cells in the center of the image. 480. b. The most distinctive EM feature of the macrophage is its population of endocytotic vesicles, early and late endosomes, lysosomes, and phagolysosomes. The surface of the cell reveals a number of fingerlike projections, some of which may be sections of surface folds. 10,000.  FOLDER 6.4 Functional Considerations: The Mononuclear Phagocytotic System

1	The cells that are included in the mononuclear phagocy-totic system (MPS) are derived from monocytes and denote a population of antigen-presenting cells involved in the processing of foreign substances. These cells are able to phagocytose avidly vital dyes such as trypan blue and India ink, which makes them visible and easy to identify in the light microscope. The common origin of MPS cells from monocytes serves as the major distinguishing feature of the system as it is currently perceived and is the basis for the system’s name. In addition, cells of the MPS display receptors for complement and Fc fragments of im-munoglobulins. The various cells of the MPS are listed in the following table. Most cells of the MPS become fixed in specific tissues and may adopt a variety of morphologic appearances as they differentiate. The main functions of MPS cells are phagocytosis, secretion (lymphokines), antigen process-ing, and antigen presentation to other cells of the immune system. Some

1	appearances as they differentiate. The main functions of MPS cells are phagocytosis, secretion (lymphokines), antigen process-ing, and antigen presentation to other cells of the immune system. Some functionally important phagocytotic cells are not derived from monocytes. For example, microglia are small, stellate cells located primarily along capillaries of the central nervous system that function as phagocy-totic cells. They are generally thought to arise from the me-sectoderm of the neural crest and not from monocytes; nevertheless, they are included in the MPS. Similarly, fibroblasts of the subepithelial sheath of the lamina propria of the intestine and uterine endometrium have been shown to differentiate into cells with morphologic, enzy-matic, and functional characteristics of connective tissue macrophages. Cells of the Mononuclear Phagocytotic System Name of Cell Location Macrophage (histiocyte) Connective tissue Perisinusoidal macrophage (Kupffer cell) Liver Alveolar macrophage

1	tissue macrophages. Cells of the Mononuclear Phagocytotic System Name of Cell Location Macrophage (histiocyte) Connective tissue Perisinusoidal macrophage (Kupffer cell) Liver Alveolar macrophage Lungs Fetal placental antigen-presenting cell (Hofbauer cell) Placenta Macrophage Spleen, lymph nodes, bone marrow, and thymus Pleural and peritoneal macrophage Serous cavities Osteoclast Bone Microglia Central nervous system Langerhans’ cell Epidermis Fibroblast-derived macrophage Lamina propria of intestine, endometrium of uterus Dendritic cell Lymph nodes, spleen

1	This triggers mast cell activation, which results in granule exocytosis (degranulation) and the release of granule content into the ECM. Mast cells can also be activated by the IgEindependent mechanism during complement protein activation. Two types of human mast cells have been identified based on morphologic and biochemical properties. Most mast cells in the connective tissue of the skin and intestinal submucosa and breast and axillary lymph nodes contain cytoplasmic granules with a latticelike internal structure. These cells contain granule-associated tryptase and chymase and are referred to as MCTC mast cells. In contrast, mast cells in the lungs and intestinal mucosa have granules with a scroll-like internal structure. These cells produce only tryptase and are termed MCT mast cells. Nearly equivalent concentrations of each type are found in nasal mucosa.

1	Mast cells are especially numerous in the connective tissues of skin and mucous membranes, but are not present in the brain and spinal cord. Mast cells are distributed chiefly in the connective tissue of skin (MCTC mast cells) in the vicinity of small blood vessels, hair follicles, sebaceous glands, and sweat glands. Mast cells are also present in the capsules of organs and the connective tissue that surrounds the blood vessels of internal organs. A notable exception is the central nervous system. Although the meninges (sheets of connective tissue that surround the FIGURE 6.23 • The mast cell. a. Photomicrograph of a mast cell stained with H&E. The granules stain intensely and, because of their numbers, tend to appear as a solid mass in some areas. The nucleus of the cell is represented by the pale-staining area.

1	1,250. b. This electron micrograph shows the cytoplasm of a mast cell that is virtually filled with granules. Note a small lymphocyte present in the upper left of the figure. 6,000. brain and spinal cord) contain mast cells, the connective tissue around the small blood vessels within the brain and spinal cord is devoid of mast cells. The absence of mast cells protects the brain and spinal cord from the potentially disruptive effects of the edema of allergic reactions. Mast cells are also numerous in the thymus and, to a lesser degree, in other lymphatic organs, but they are not present in the spleen. Most mast cell secretory products (mediators of inflammation) are stored in granules and are released at the time of mast cell activation. chemical substances known as mediators of infammation.

1	chemical substances known as mediators of infammation. Mediators produced by mast cells are divided into two cate gories: Preformed mediators that are stored in secretory thesized mediators (mostly lipids and cytokines) that are often absent in the resting cells, although they are produced and secreted by activated mast cells. Preformed mediators found inside mast cell granules are the following:  Histamine is a biogenic amine that increases the permeability of small blood vessels, causing edema in the surrounding tissue and a skin reaction demonstrated by an itching sensation. In addition, it increases mucus production in the bronchial tree and prompts contraction of smooth muscle in the pulmonary airways. Histamine’s effects can be blocked by antihistaminic agents. These competitive inhibitors have a similar chemical structure and bind to histamine receptors without initiating histamine’s effects.

1	 Heparin is a sulfated GAG that is an anticoagulant. Its expression is limited essentially to the granules of mast cells and basophils. When heparin unites with antithrombin III and platelet factor IV, it can block numerous coagulation factors. On the basis of its anticoagulant properties, heparin is useful for treatment of thrombosis. It also interacts with FGF and its receptor to induce signal transduction in the cells.  Serine proteases (tryptase and chymase). Tryptase is selectively concentrated in the secretory granules of human mast cells (but not basophils). It is released by mast cells together with histamine and serves as a marker of mast cell activation. Chymase plays an important role in generating angiotensin II in response to vascular tissue injury. Mast cell chymase also induces apoptosis of vascular smooth muscle cells, particularly in the area of atherosclerotic lesions.

1	 Eosinophil chemotactic factor (ECF) and neutrophil chemotactic factor (NCF), which attract eosinophils and neutrophils, respectively, to the site of inflammation. The secretions of eosinophils counteract the effects of the histamine and leukotriens. Newly synthesized mediators include the following:  Leukotriene C (LTC4) is released from the must cell and then cleaved in the ECM, yelding two active leukotrienes— D (LTD4) and E (LTE4). They represent a family of modified lipids conjugated to glutathione (LTC4) or cysteine (LTD4 and LTE4). Leukotrienes are released by mast cells during anaphylaxis (see Folder 6.5 for a description of anaphylaxis). Similar to histamine, leukotrienes trigger prolonged constriction of smooth muscle in the pulmonary airways, causing bronchospasm. This constriction,

1	TABLE Comparison of Features Characteristic of Mast Cells and Basophils 6.6 Characteristic Features Mast Cells Basophils Origin Hemopoietic stem cell Hemopoietic stem cell Site of differentiation Connective tissue Bone marrow Cell divisions Yes (occasionally) No Cells in circulation No Yes Life span Weeks to months Days Size 20–30 m 7–10 m Shape of nucleus Round Segmented (usually bilobar) Granules Many, large, metachromatic Few, small, basophilic High-affinity surface receptors Present Present for IgE antibodies (Fc RI) Marker of cellular activity Tryptase Not yet established however, cannot be reversed by treatment with antihistaminic agents.  Tumor necrosis factor (TNF-) is a major cytokine produced by mast cells. It increases expression of adhesion molecules in endothelial cells and has antitumor effects.

1	 Tumor necrosis factor (TNF-) is a major cytokine produced by mast cells. It increases expression of adhesion molecules in endothelial cells and has antitumor effects.  Several interleukins (IL-4,-3 -5, -6, -8 and -16), growth factors (GM-CSF), and prostaglandin D2 (PGD2) are also released during mast cell activation. These mediators are not stored in granules but are synthesized by the cell and released immediately into the ECM. Mediators released during mast cell activation, as a result of interactions with allergens, are responsible for variety of symptoms and signs that are characteristic for allergic reactions. Basophils that develop and differentiate in bone marrow share many features with mast cells.

1	Basophils are granulocytes that circulate in the bloodstream and represent less than 1% of peripheral white blood cells (leukocytes). Developmentally, they represent a separate lineage from mast cells, despite sharing a common precursor cell in the bone marrow. Basophils develop and mature in the bone marrow and are released to the circulation as mature cells. They have also many other common features with mast cells such as basophilic secretory granules, an ability to secrete similar mediators, and an abundance of high-affinity Fc receptors for IgE antibodies on their cell membrane. They participate in allergic reactions (see Folder 6.5) and together with mast cells release histamine, heparin, heparan sulfate, ECF, NCF, and other mediators of inflammation. In contrast to mast cells, basophils do not produce prostaglandin D2 (PGD2) and interleukin-5 (IL-5). Basophils and their features are discussed in more details in Chapter 10.

1	The adipocyte is a connective tissue cell specialized to store neutral fat and produce a variety of hormones. Adipocytes differentiate from mesenchymal stem cells and gradually accumulate fat in their cytoplasm. They are located throughout loose connective tissue as individual cells and groups of cells. When they accumulate in large numbers, they are called adipose tissue. Adipocytes are also involved in the synthesis of a variety of hormones, inflammatory mediators, and growth factors. This specialized connective tissue is discussed in Chapter 9. Niches of adult stem cells are located in various tissues and organs.

1	Many tissues in mature individuals contain reservoirs of steam cells called adult stem cells. Compared with embryonic stem cells, adult stem cells cannot differentiate into multiple lineages. They usually are capable of differentiating only into lineage-specific cells. Adult stem cells are found in many tissues and organs, residing in specific sites referred to as niches. Niches of stem cells residing in tissues and organs (excluding bone marrow) are called tissue stem cells. They been identified in the gastrointestinal tract—for instance, in the stomach (isthmus of the gastric gland), small and large intestines (base of the intestinal gland), and many other areas. Bone marrow represents a unique reservoir of stem cells. In addition to containing HSCs (see Chapter 10), bone marrow also contains at least two other populations of stem cells: A heterogenous population of multipotent adult progenitor cells (MAPCs) that appear to have broad developmental capabilities and

1	FOLDER 6.5 Clinical Correlation: The Role of Mast Cells and Basophils in Allergic Reactions

1	When an individual is exposed to a specific antigen (aller-gen) that reacts with IgE antibodies bound to the surface of mast cells or basophils via their high-affinity receptors (FcεRI), it initiates mast cell activation. This type of IgE-dependent activation initiates a cascade of events, result-ing in allergic reactions. These reactions can occur as immediate hypersensitiviy reactions, (usually within sec-onds to minutes after exposure to an allegen), late-phase reactions, or chronic allergic inflammations. The immediate hypersensitivity reaction involves IgE-mediated release of histamine and other mediators from mast cells and also from basophils. The clinical symp-toms caused by the above mediators vary, depending on which organ system is affected. The release of mediators in the superficial layers of the skin can manifest as erythema (redness), swelling and itch-ing, or pain sensations. Respiratory system symptoms in-clude sneezing, rhinorrhea (runny nose), increased production

1	layers of the skin can manifest as erythema (redness), swelling and itch-ing, or pain sensations. Respiratory system symptoms in-clude sneezing, rhinorrhea (runny nose), increased production of mucus, coughing, bronchospasm (constric-tion of bronchi), and pulmonary edema. Individuals with these symptoms often complain of tightness in the chest, shortness of breath, and wheezing. The gastrointestinal tract can also be affected with symptoms of nausea, vomit-ing, diarrhea, and abdominal cramping. In highly sensitive individuals, the antigen injected by an insect can trigger a massive discharge of mast cells and ba-sophil granules that affect more than one system. This con-dition is known as anaphylaxis. Dilation and increased permeability of systemic blood vessels can cause anaphy-lactic shock. This often-explosive, life-threatening reaction is characterized by significant hypotension (decreased blood pressure), decreased circulating blood volume (leaky vessels), and smooth muscle cells

1	This often-explosive, life-threatening reaction is characterized by significant hypotension (decreased blood pressure), decreased circulating blood volume (leaky vessels), and smooth muscle cells constriction in the bronchial tree. The individual has difficulty breathing and may exhibit a rash, as well as have nausea and vomiting. Symptoms of anaphylactic shock usually develop within 1 to 3 minutes, and immediate treatment with vasoconstric-tors such as epinephrine is required. The assessment of the activation of basophils in systemic anaphylactic reac-tions is still problematic because an assay for a specific cellular marker released by basophils (and not by other cells such as mast cells) has not yet been developed. After the signs or symptoms of the immediate hyper-sensitiviy reaction have been resolved, an affected indi-vidual may develop late-phase allergic reactions 6 to 24 hours later. The symptoms of these reactions may in-clude redness, persistent swelling of the skin, nasal

1	have been resolved, an affected indi-vidual may develop late-phase allergic reactions 6 to 24 hours later. The symptoms of these reactions may in-clude redness, persistent swelling of the skin, nasal dis-charge, sneezing, and coughing, usually accompanied by an elevated white blood cell count. These symptoms usu-ally last a few hours and then disappear within 1 to 2 days of the initial allergen exposure. In the respiratory system, the late-phase reaction is believed to be responsible for the development of persistent asthma. If the exposure to an allergen is persistent (for instance by a dog-owning patient who is allergic to dogs), it can result in chronic allergic infammation. Tissues in such individuals accumulate variety of immune cells, such as eosinophils and T lymphocytes that cause more tissue dam-age and prolong inflammation. This can lead to permanent structural and functional changes in the affected tissue.

1	bone marrow stromal cells (BMSCs) that can generate chondrocytes, osteoblasts, adipocytes, muscle cells, and endothelial cells. The MAPCs are adult counterparts of embryonic stem cells. Niches of adult stem cells called mesenchymal stem cells are found in loose connective tissue of the adult. These cells give rise to differentiated cells that function in the repair and formation of new tissue such as in wound healing and in the development of new blood vessels (neovascularization). The vascular pericytes found around capillaries and venules are mesenchymal stem cells.

1	The vascular pericytes found around capillaries and venules are mesenchymal stem cells. Pericytes, also called adventitial cells or perivascular cells, are found around capillaries and venules (Fig. 6.24). Several observations support the interpretation that vascular pericytes are indeed mesenchymal stem cells. Experimental studies show that in response to external stimuli, pericytes express a cohort of proteins similar to those of stem cells in the bone marrow. Pericytes are surrounded by basal lamina material that is continuous with the basal lamina of the capillary endothelium; thus, they are not truly located in the connective tissue compartment. The pericyte is typically wrapped, at least partially, around the capillary, and its nucleus takes on a shape similar to that of endothelial cells (i.e., flattened but curved to conform to the tubular shape of the vessel).

1	TEM studies demonstrate that pericytes surrounding the smallest venules have cytoplasmic characteristics almost identical with those of the endothelial cells of the same vessel. Peri cytes associated with larger venules have characteristics of smooth muscle cells of the tunica media of small veins. In fortuitous sections cut parallel to the long axis of venules, the distal portion and proximal portion of the same pericyte exhibit characteristics of endothelial cells and smooth muscle cells, respectively. These studies suggest that during the development of new vessels, cells with characteristics of pericytes may differentiate into smooth muscle of the vessel wall. The role of pericytes as mesenchymal stem cells was confirmed experimentally in studies in which cultured pericytes from retinal capillaries were able to differentiate into a variety of cells, including osteoblasts, adipocytes, chondrocytes, and fibroblasts. FIGURE 6.24 • Electron micrograph of a small blood vessel.

1	FIGURE 6.24 • Electron micrograph of a small blood vessel. The nucleus at the upper left belongs to the endothelial cell that forms the wall of the vessel. At the right is another cell, a pericyte, that is in intimate relation to the endothelium. Note that the basal lamina (BL) covering the endothelial cell divides (arrows) to surround the pericyte. 11,000. The fibroblasts and blood vessels within healing wounds develop from mesenchymal stem cells associated with the tunica adventitia of venules. Autoradiographic studies of wound healing using parabiotic (crossed-circulation) pairs of animals have established that mesenchymal stem cells located in the tunica adventitia of venules and small veins are the primary source of new cells in healing wounds. In addition, fibroblasts, pericytes, and endothelial cells in portions of the connective tissue adjacent to the wound divide and give rise to additional cells that form new connective tissue and blood vessels.

1	Lymphocytes, Plasma Cells, and Other Cells of the Immune System Lymphocytes are principally involved in immune responses. Connective tissue lymphocytes are the smallest of the wandering cells in the connective tissue (see Fig. 6.23b). They have a thin rim of cytoplasm surrounding a deeply staining, heterochromatic nucleus. Often, the cytoplasm of connective tissue lymphocytes may not be visible. Normally, small numbers of lymphocytes are found in the connective tissue throughout the body. The number increases dramatically, however, at sites of tissue infammation caused by pathogenic agents. Lymphocytes are most numerous in the lamina propria of the respiratory and gastrointestinal tracts, where they are involved in immunosurveillance against pathogens and foreign substances that enter the body by crossing the epithelial lining of these systems. Lymphocytes are a heterogeneous population of at least three major functional cell types: T cells, B cells, and natural killer (NK) cells.

1	Lymphocytes are a heterogeneous population of at least three major functional cell types: T cells, B cells, and natural killer (NK) cells. At the molecular level, lymphocytes are characterized by the expression of specific molecules on the plasma membrane known as cluster of differentiation (CD) proteins. CD proteins recognize specific ligands on target cells. Because some CD proteins are present only on specific types of lymphocytes, they are considered specific marker proteins. On the basis of these specific markers, lymphocytes can be classified into three functional cell types.  T lymphocytes are characterized by the presence of the CD2, CD3, CD5, and CD7 marker proteins and the T-cell receptors (TCRs). These cells have a long life span and are effectors in cell-mediated immunity.

1	 B lymphocytes are characterized by the presence of CD9, CD19, and CD20 proteins and attached immunoglobulins IgM and IgD. These cells recognize antigen, have a variable life span, and are effectors in antibody-mediated (humoral) immunity.  NK lymphocytes are non-T, non-B lymphocytes that express the CD16, CD56, and CD94 proteins, not found on other lymphocytes. These cells neither produce immunoglobulins nor express TCR on their surface. Thus, NK lymphocytes are not antigen specific. Similar in action to T lymphocytes, however, they destroy virus-infected cells and some tumor cells by a cytotoxic mechanism. In response to the presence of antigens, lymphocytes become activated and may divide several times, producing clones of themselves. In addition, clones of B lymphocytes mature into plasma cells. A description of B and T lymphocytes and their functions during immune response reactions is presented in Chapter 14.

1	Plasma cells are antibody-producing cells derived from B lymphocytes. Plasma cells are a prominent constituent of loose connective tissue where antigens tend to enter the body (e.g., the gastrointestinal and respiratory tracts). They are also a normal component of salivary glands, lymph nodes, and hematopoietic tissue. Once derived from its precursor, the B lymphocyte, a plasma cell has only limited migratory ability and a somewhat short life span of 10 to 30 days. The plasma cell is a relatively large, ovoid cell (20 m) with a considerable amount of cytoplasm. The cytoplasm displays strong basophilia because of an extensive rER (Fig. 6.25a). The Golgi apparatus is usually prominent because of its relatively large size and lack of staining. It appears in light microscope preparations as a clear area in contrast to the basophilic cytoplasm.

1	The nucleus is spherical and typically offset or eccentrically positioned. It is small, not much larger than the nucleus of the lymphocyte. It exhibits large clumps of peripheral heterochromatin alternating with clear areas of euchromatin. This arrangement has traditionally been described as resembling a cartwheel or analog clock face, with the heterochromatin resembling the spokes of the wheel or the numbers on a clock (Fig. 6.25b). The heterochromatic nucleus of the plasma cell is somewhat surprising given the cell’s function in synthesizing large amounts of protein. However, because the cells produce large amounts of only one type of protein—a specific antibody—only a small segment of the genome is exposed for transcription. Eosinophils, monocytes, and neutrophils are also observed in connective tissue.

1	Eosinophils, monocytes, and neutrophils are also observed in connective tissue. As a result of immune responses and tissue injury, certain cells rapidly migrate from the blood to enter the connective tissue, particularly neutrophils and monocytes. Their presence generally indicates an acute inflammatory reaction. In these reactions, neutrophils migrate into the connective tissue in substantial numbers, followed by large numbers of monocytes. As noted, the monocytes then differentiate into macrophages. A description of these cells and their roles is found in Chapter 10. The eosinophil, which functions in allergic reactions and parasitic infections, is also presented in that chapter. Eosinophils may be observed in normal connective tissue, particularly the lamina propria of the intestine, as a result of chronic immunologic responses that occur in these tissues.

1	FIGURE 6.25 • The plasma cell. a. This photomicrograph shows the typical features of a plasma cell as seen in a routine H&E preparation. Note clumps of peripheral heterochromatin alternating with clear areas of euchromatin in the nucleus. Also note the negative Golgi (arrows) and basophilic cytoplasm. 5,000. b. Electron micrograph shows that an extensive rER occupies most of the cytoplasm of the plasma cell. The Golgi apparatus (G) is also relatively large, a further reflection of the cell’s secretory activity. 15,000. This page intentionally left blank.

1	This page intentionally left blank. Loose and dense connective tissue represents one of the several types of connective tissue. The others are namely cartilage, bone, blood, adipose tissue, and reticular tissue. Loose connective tissue is characterized by a relatively high proportion of cells within a matrix of thin and sparse collagen fibers. In contrast, dense irregular connective tissue contains few cells, almost all of which are fibroblasts that are re-sponsible for the formation and maintenance of the abundant collagen fibers that form the matrix of this tissue. The cells that are typically as-sociated with loose connective tissue are fibroblasts, the collagen-forming cells, and those cells that function in the immune system and those of the body’s general defense system. Thus, in loose connective tissue, there are, to varying degrees, lymphocytes, macrophages, eosinophils, plasma cells, and mast cells.

1	Loose and dense irregular connective tissue, mammary gland, human, H&E, ×175; insets ×350.

1	This micrograph shows at low magnification both loose connective tissue (LCT ) and dense irregular connective tissue (DICT ) for comparative purpose. The loose connective tissue surrounds the glandular epithelium (GE). The dense ir192 regular connective tissue consists mainly of thick bundles of collagen fibers with few cells present, whereas the loose connective tissue has a rela tive paucity of fibers and a considerable number of cells. The upper inset is a higher magnification of the dense connective tissue. Note that only a few cell nuclei are present relative to the larger expanse of collagen fibers. The lower inset, revealing the glandular epithelium and surrounding loose connective tissue, shows very few fibers, but large numbers of cells. Typically, the cellular component of loose connective tissue contains a relatively small proportion of fibroblasts, but large numbers of lymphocytes, plasma cells, and other connective tissue cell types.

1	Loose connective tissue, colon, monkey, Mallory, ×250. This micrograph reveals an extremely cellular loose connective tissue (LCT), also called lamina propria, which is located between the intestinal glands of the colon. The simple columnar mucous secreting epithelial cells seen here represent the glandular tissue. The Mallory stain colors cell nuclei red and collagen blue. Note how the cells are surrounded by a framework of the blue stained collagen fibers. Also shown in this micrograph is a band of smooth muscle, the muscularis mucosa (MM) of the colon and below that, seen in part, is dense irregular connective tissue (DICT) that forms the submucosa of the colon. Typically, the collagen fibers (C) that lie just below the epithelial cells (Ep) at the luminal surface are more concentrated and thus appear prominently in the micrograph.

1	Loose connective tissue, colon, monkey, threads that form a stroma surrounding the cells. The majority of cells that Mallory, ×700. are present here consists of lymphocytes and plasma cells (P). Other cells that are present within the stromal framework consist of fibroblasts, Shown at higher magnification is the boxed area in the adjasmooth muscle cells, macrophages, and occasional mast cells. cent figure. The base of the epithelial cells is seen on each side of the micrograph. The collagen fibers (C) appear as thin KEY CF, collagen fibers DICT, dense irregular connective tissue Ep, epithelial cells GE, glandular epithelium LCT, loose connective tissue MM, muscularis mucosa P, plasma cells

1	PLATE 5 Dense Regular Connective Tissue, Tendons,and Ligaments 194 Dense regular connective tissue is distinctive in that its fibers are very densely packed and are organized in parallel array into fascicles. The collagen fibrils that make up the fibers are also arranged in an ordered parallel array. Tendons, which attach muscle to bone, and ligaments, which attach bone to bone, are examples of this type of tissue. Ligaments are similar to tendons in most respects, but their fibers and the or-ganization of the fascicles tend to be less ordered. In tendons as well as ligaments, the fascicles are separated from one another by dense irregular connective tissue, the endotendineum, through which travel vessels and nerves. Also, a fascicle may be partially divided by connective tissue septa that extend from the endo-tendineum and contain the smallest vessels and nerves. Some of the fascicles may be grouped into larger functional units by a thicker, sur-rounding connective tissue, the

1	that extend from the endo-tendineum and contain the smallest vessels and nerves. Some of the fascicles may be grouped into larger functional units by a thicker, sur-rounding connective tissue, the peritendineum. Finally, the fascicles and groups of fascicles are surrounded by dense irregular connective tissue, the epitendineum. The fibroblasts, also called tendinocytes in tendons, are elongated cells that possess exceedingly thin, sheet-like cytoplasmic processes that reside between and embrace adjacent fibers. The margins of the cytoplasmic processes contact those of neighboring tendon cells, thus forming a syncytium-like cytoplasmic network. The most regular dense connective tissue is that of the stroma of the cornea of the eye (see Chapter 24). In this tissue, the collagen fibrils are arranged in parallel in lamellae that are separated by large, flattened fibroblasts. Adjacent lamellae are arranged at approximately right an-gles to one another, thus forming an orthogonal array. The

1	in parallel in lamellae that are separated by large, flattened fibroblasts. Adjacent lamellae are arranged at approximately right an-gles to one another, thus forming an orthogonal array. The extreme regularity of fibril size and fibril spacing in each lamella, in conjunction with the orthogonal array of the lamellae, is believed to be the basis of corneal transparency.

1	PLATE 5 • DENSE REGULAR CONNECTIVE TISSUE, TENDONS, AND LIGAMENTS Dense regular connective tissue, tendon, longitudinal section, human, H&E ×100. This specimen includes the surrounding dense irregular connective tissue of the tendon, the epitendineum (Ept). The tendon fascicles (TF) that make up the tendon are surrounded by a less dense connective tissue than that associated with the epitendineum. In longitudinal sections such as this, the connective tissue that surrounds the individual fascicles, the endotendineum (Ent), Dense regular connective tissue, tendon, longitudinal section, human, H&E ×400. This higher magnification micrograph shows the ordered single-file array of the tendinocyte nuclei (TC) along with the intervening collagen. The latter has a homogeneous appearance.

1	This higher magnification micrograph shows the ordered single-file array of the tendinocyte nuclei (TC) along with the intervening collagen. The latter has a homogeneous appearance. seems to disappear at certain points, with the result that one fascicle appears to merge with a neighboring fascicle. This is due to an obliqueness in the plane of section rather than an actual merging of fascicles. The collagen that makes up the bulk of the tendon fascicle has a homogeneous appearance as a result of the orderly packing of the individual collagen fibrils. The nuclei of the tendinocytes appear as elongate profiles arranged in linear rows. The cytoplasm of these cells blends in with the collagen, leaving only the nuclei as the representative feature of the cell.

1	The cytoplasm of the cells is indistinguishable from the collagen, as is typical in H&E paraffin specimens. The variation in nuclear appearance is due to the plane of section and the position of the nuclei within the thickness of the section. A small blood vessel (BV) coursing within the endotendineum is also present in the specimen. Dense regular connective tissue, tendon, cross section, human, H&E ×400.

1	Dense regular connective tissue, tendon, cross section, human, H&E ×400. This specimen is well preserved, and the densely packed collagenous fibers appear as a homogeneous field, even though the fibers are viewed on their cut ends. The nuclei appear irregularly scattered, as opposed to their more uniform pattern in the longitudinal plane. This is explained by examining the dashed line in lower left figure which is meant to represent an arbitrary cross-sectional cut of the tendon. Note the irregular spacing of the nuclei that are in the plane of the cut. Lastly, several small blood vessels (BV) are present within the endotendineum (Ent) within a fascicle. KEY BV, blood vessel Ent, endotendineum Ept, epitendineum TC, tendinocytes nuclei TF, fascicle of tendon dashed line, arbitrary cross-sectional cut of tendon PLATE 5 • DENSE REGULAR CONNECTIVE TISSUE, TENDONS, AND LIGAMENTS

1	Elastic fibers are present in loose and dense connective tissue throughout the body, but in lesser amounts than collagenous fibers. Elastic fibers are not conspicuous in routine H&E sections but are visualized readily with special staining methods. (The following selectively color elastic material: Weigert’s elastic tissue stain, purple-violet; Gomori’s aldehyde fuchsin stain, blue-black; Verhoeff’s hematoxylin elastic tissue stain, black; and modified Taenzer-Unna orcein stain, red-brown.) By using a combination of the special elastic stains and counterstains, such as H&E, not only the elastic fibers but also the other tissue components may be revealed, thus allowing study of the relationships between the elastic material and other connective tissue components. Elastic material occurs in both fibrous and lamellar forms. In loose and dense connective tissue and in elastic cartilage (see Plate 9, page 221), the elastic material is in fibrous form. Similarly, the elastic ligaments that

1	both fibrous and lamellar forms. In loose and dense connective tissue and in elastic cartilage (see Plate 9, page 221), the elastic material is in fibrous form. Similarly, the elastic ligaments that connect the cervical vertebrae and that are particularly prominent in grazing animals have a mixture of elastic and collagenous fibers in a tightly packed array. In the major, largest diameter arteries (e.g., aorta, pulmonary, common carotid, and other primary branches of the aorta), the tunica media consists of fenestrated layers of elastic tissue alternating with layers containing smooth muscle cells and collagenous tissue. This allows stretching and elastic rebound to assist in the propulsion of the blood. All arteries and most large arterioles have an internal elastic membrane that supports the delicate endothelium and its immediately subjacent connective tissue. It should be noted that both the collagen and elastic components of the tunica media are produced by the smooth muscle cells

1	the delicate endothelium and its immediately subjacent connective tissue. It should be noted that both the collagen and elastic components of the tunica media are produced by the smooth muscle cells of this layer.

1	Elastic fbers, dermis, monkey, Welgert’s ×160. This shows the connective tissue of the skin, referred to as the dermis, stained to show the nature and distribution of the elastic fibers (E), which appear purple. The collagen fibers (C) have been stained by eosin, and the two fiber types are easily differentiated. The connective tissue at the top of the figure, close to the epithelium (the papillary layer of the dermis), contains thin elastic fibers (see upper left of figure) as well as less coarse collagen fibers. The KEY BV, blood vessel C, collagen fibers D, duct of sweat gland E, elastic fibers PLATE 6 • E LASTIC FIBERS AND E LASTIC LAM E LLAE Elastic fbers, mesentery, rat, Weigert’s ×160. beginnings or endings and with a somewhat irregular course. Again, the collagen fibers (C) are contrasted by their eosin staining and appear as long, This is a whole mount specimen of mesentery prepared to straight profiles that are considerably thicker than the elastic fibers.

1	This is a whole mount specimen of mesentery prepared to straight profiles that are considerably thicker than the elastic fibers. show the connective tissue elements and differentially stained to reveal elastic fibers. The elastic fibers (E) appear as thin, long, crisscrossing and branching threads without discernible Elastic lamellae, elastic artery, monkey, Weigert’s ×80. Elastic material also occurs in sheets or lamellae rather than string-like fibers. This figure shows the wall of an elastic artery (pulmonary artery) that was stained to show the elastic material. Each of the wavy lines is a lamella of elastic material that is organized in the form of a fenestrated sheet or membrane. The plane of section is such that the elastic membranes are seen on edge. This specimen was not subsequently stained with H&E.

1	lower portion of the figure shows considerably heavier elastic and collagen fibers. Also note that many of the elastic fibers appear as short rectangular profiles. These profiles simply represent fibers traveling through the thickness of the section at an oblique angle to the path of the knife. Careful examination will also reveal a few fibers that appear as dot-like profiles. They represent cross-sectioned elastic fibers. Overall, the elastic fibers of the dermis have a three-dimensional interlacing configuration, thus the variety of forms. The empty-appearing spaces between elastic layers contain collagen fibers and smooth muscle cells, but they remain essentially unstained. In the muscular layer of blood vessel, both elastin and collagen are secreted by the smooth muscle cells. Tissues of the body containing large amounts of elastic material are limited in distribution to the walls of elastic arteries and some ligaments that are associated with the spinal column.

1	Tissues of the body containing large amounts of elastic material are limited in distribution to the walls of elastic arteries and some ligaments that are associated with the spinal column. OVERVIEW OF CARTILAGE / 198 HYALINE CARTILAGE / 199 ELASTIC CARTILAGE / 204 FIBROCARTILAGE / 204 CHONDROGENESIS AND CARTILAGE GROWTH / 206 REPAIR OF HYALINE CARTILAGE / 207 Folder 7.1 Clinical Correlation: Osteoarthritis / 199 Folder 7.2 Clinical Correlation: Malignant Tumors of the Cartilage; Chondrosarcomas / 208 Cartilage is a form of connective tissue composed of cells called chondrocytes and a highly specialized extracellular matrix. Cartilage is an avascular tissue that consists of chondrocytes and an extensive extracellular matrix. More than 95% of cartilage volume consists of extracellular matrix, which is a functional element of this tissue. The chondrocytes are sparse but essential participants in producing and maintaining the matrix (Fig. 7.1).

1	The extracellular matrix in cartilage is solid and firm but also somewhat pliable, which accounts for its resilience. Because there is no vascular network within cartilage, the composition of the extracellular matrix is crucial to the survival of the chondrocytes. The large ratio of glycosaminoglycans (GAGs) to type II collagen fibers in the cartilage matrix permits diffusion of substances between blood vessels in the surrounding connective tissue and the chondrocytes dispersed within the matrix, thus maintaining the viability of the tissue. Close interactions are seen between two classes of structural molecules that possess contrasting biophysical characteristics: the meshwork of tension-resisting collagen fibrils and the large amounts of heavily hydrated proteoglycan aggregates, the latter being extremely weak in shear, make the cartilage well adapted to bear weight, especially at points of movement, as in synovial joints. Because it maintains this property even while growing,

1	latter being extremely weak in shear, make the cartilage well adapted to bear weight, especially at points of movement, as in synovial joints. Because it maintains this property even while growing, cartilage is a key tissue in the development of the fetal skeleton and in most growing bones.

1	FIGURE 7.1 • General structure of hyaline cartilage. This photomicrograph of a routine H&E preparation of hyaline cartilage shows its general features. Note the extensive extracellular matrix that separates a sparse population of chondrocytes. 450. Three types of cartilage that differ in appearance and mechanical properties are distinguished on the basis of characteristics of their matrix:  Hyaline cartilage is characterized by matrix containing type II collagen fibers, GAGs, proteoglycans, and multiad hesive glycoproteins.  Elastic cartilage is characterized by elastic fibers and elas tic lamellae in addition to the matrix material of hyaline cartilage.  Fibrocartilage is characterized by abundant type I colla gen fibers as well as the matrix material of hyaline cartilage. Table 7.1 lists the locations, functions, and features of each type of cartilage. Hyaline cartilage is distinguished by a homogeneous, amorphous matrix.

1	The matrix of hyaline cartilage appears glassy in the living state: hence, the name hyaline [Gr. hyalos, glassy]. Throughout the cartilage matrix are spaces called lacunae. Located within these lacunae are the chondrocytes. Hyaline cartilage is not a simple, inert, homogeneous substance but a complex living tissue. It provides a low-friction surface, participates in lubricating synovial joints, and distributes applied forces to the underlying bone. Although its capacity for repair is limited, under normal circumstances it shows no evidence of abrasive wear over a lifetime. An exception is articular cartilage, which, in many individuals, breaks down with age (Folder 7.1). The macromolecules of hyaline cartilage matrix consist of collagen (predominantly type II fibrils and other cartilage-specific collagen molecules), proteoglycan aggregates containing GAGs, and multiadhesive glycoproteins (noncollagenous proteins). Figure 7.2 illustrates the relative distribution of the various

1	collagen molecules), proteoglycan aggregates containing GAGs, and multiadhesive glycoproteins (noncollagenous proteins). Figure 7.2 illustrates the relative distribution of the various components that constitute cartilage matrix.

1	Hyaline cartilage matrix is produced by chondrocytes and contains three major classes of molecules. Three classes of molecules exist in hyaline cartilage matrix.  Collagen molecules. Collagen is the major matrix pro tein. Four types of collagen participate in the formation of three-dimensional meshwork of the relatively thin (20-nm diameter) and short matrix fibrils. Type II collagen cells 3–5% multiadhesive glycoproteins 5% proteoglycans (aggrecan) 9% collagens 15% intercellular water 60–80% 5% III, VI, X, XII, XIV 15% IX, XI 80% II FIGURE 7.2 • Molecular composition of hyaline cartilage. Cartilage contains 60–80% of the wet weight of intercellular water, which is bound by proteoglycan aggregates. About 15% of the total weight is attributed to collagen molecules, of which type II collagen is the most abundant. Chondrocytes occupy only 3–5% of the total cartilage mass.

1	Osteoarthritis, a degenerative joint disease, is one of the most common types of joint diseases. The pathogenesis of osteoarthritis is unknown, but it is related to aging and injury of articular cartilage. Most individuals show some evidence of this disease by age 65. The disease is charac-terized by chronic joint pain with various degrees of joint deformity and destruction of the articular cartilage. Os-teoarthritis commonly affects weight-bearing joints: hips, knees, lower lumbar vertebra, and joints of the hand and foot. There is a decrease in proteoglycan content, which re-sults in reduction in intercellular water content in the carti-lage matrix. Chondrocytes also play an important role in the pathogenesis of osteoarthritis. By producing interleukin-1 (IL-1) and tumor necrosis factor (TNF-), the production of metalloproteinases is stimulated, whereas synthesis of type II collagen and proteoglycans by the chondrocyte is in-hibited. In the early stages of the disease, the

1	factor (TNF-), the production of metalloproteinases is stimulated, whereas synthesis of type II collagen and proteoglycans by the chondrocyte is in-hibited. In the early stages of the disease, the superficial layer of the articular cartilage is disrupted. Eventually, de-struction of the cartilage extends to the bone, where the ex-posed subchondral bone becomes a new articular surface. These changes result in progressive reduction of mobility and increased pain with joint movement. Osteoarthritis has no cure, and treatment focuses on relieving pain and stiff-ness to allow a greater range of joint movement. Os-teoarthritis may stabilize with age, but more often it slowly progresses with eventual long-term disability.

1	 FOLDER 7.1

1	Clinical Correlation: Osteoarthritis constitutes the bulk of the fibrils (see Fig. 7.2); type IX collagen facilitates fibril interaction with the matrix proteoglycan molecules; type XI collagen regulates the fibril size; and type X collagen organizes the collagen fibrils into a threedimensional hexagonal lattice that is crucial to its successful mechanical function. In addition, type VI collagen is also found in the matrix, mainly at the periphery of the chondrocytes where it helps to attach these cells to the matrix framework. Because types II, VI, IX, X, and XI are found in significant amounts only in the cartilage matrix, they are referred to as cartilage-specific collagen molecules. (Review the types of collagen in Table 6.2.)  Proteoglycans. The ground substance of hyaline cartilage contains three kinds of glycosaminoglycans: hyaluronan, chondroitin sulfate, and keratan sulfate. As in loose connective tissue matrix, the chondroitin and keratan sulfate of the cartilage matrix

1	contains three kinds of glycosaminoglycans: hyaluronan, chondroitin sulfate, and keratan sulfate. As in loose connective tissue matrix, the chondroitin and keratan sulfate of the cartilage matrix are joined to a core protein to form a proteoglycan monomer. The most important proteoglycan monomer in hyaline cartilage is aggrecan. It has a molecular weight of 250 kilodaltons. Each molecule contains about 100 chondroitin sulfate chains and as many as 60 keratan sulfate molecules. Because of the presence of the sulfate groups, aggrecan molecules have a large negative charge with an affinity for water molecules. Each linear hyaluronan molecule is associated with a large number of aggrecan molecules (more than 300), which are bound to the hyaluronan by link proteins at the N terminus of the molecule to form large proteoglycan aggregates. These highly charged proteoglycan aggregates are bound to the collagen matrix fibrils by electrostatic interactions and multiadhesive glycoproteins (Fig.

1	to form large proteoglycan aggregates. These highly charged proteoglycan aggregates are bound to the collagen matrix fibrils by electrostatic interactions and multiadhesive glycoproteins (Fig. 7.3). The entrapment of these aggregates within the intricate matrix of collagen fibrils is responsible for the unique biomechanical properties of hyaline cartilage.

1	Cartilage matrix also contains other proteoglycans (i.e., decorin, biglycan, and fibromodulin). These proteoglycans do not form aggregates but bind to other molecules and help stabilize the matrix.  Multiadhesive glycoproteins, also referred to as noncollagenous and nonproteoglycan-linked glycoproteins, influence interactions between the chondrocytes and the matrix molecules. Multiadhesive glycoproteins have clinical value as markers of cartilage turnover and degeneration. Examples of such proteins are anchorin CII (cartilage annexin V), a small 34-kilodalton molecule that functions as a collagen receptor on chondrocytes, tenascin, and fibronectin (see Table 6.5, page 180), which also help anchor chondrocytes to the matrix.

1	FIGURE 7.3 • Molecular structure of hyaline cartilage matrix. This schematic diagram shows the relationship of proteoglycan aggregates to type II collagen fibrils and chondrocytes in the matrix of hyaline cartilage. A hyaluronan molecule forming a linear aggregate with many proteoglycan monomers is interwoven with a network of collagen fibrils. The proteoglycan monomer (such as aggrecan) consists of approximately 180 glycosaminoglycans joined to a core protein. The end of the core protein contains a hyaluronan-binding region that is joined to the hyaluronan by a link protein. Hyaline cartilage matrix is highly hydrated to provide resilience and diffusion of small metabolites.

1	Hyaline cartilage matrix is highly hydrated to provide resilience and diffusion of small metabolites. Like other connective tissue matrices, cartilage matrix is highly hydrated. From 60% to 80% of the net weight of hyaline cartilage is intercellular water (see Fig. 7.2). Much of this water is bound tightly to the aggrecan– hyaluronan aggregates, imparting resilience to the cartilage. Some of the water is bound loosely enough, however, to allow diffusion of small metabolites to and from the chondrocytes.

1	In articular cartilage, both transient and regional changes occur in water content during joint movement and when the joint is subjected to pressure. The high degree of hydration and the movement of water in the matrix allow the cartilage matrix to respond to varying pressure loads and contribute to cartilage’s weight-bearing capacity. Throughout life, cartilage undergoes continuous internal remodeling as the cells replace matrix molecules lost through degradation. Normal matrix turnover depends on the ability of the chondrocytes to detect changes in matrix composition. The chondrocytes then respond by synthesizing appropriate types of new molecules. In addition, the matrix acts as a signal transducer for the embedded chondrocytes. Thus, pressure loads applied to the cartilage, as in synovial joints, create mechanical, electrical, and chemical signals that help direct the synthetic activity of the chondrocytes. As the body ages, however, the composition of the matrix changes, and the

1	joints, create mechanical, electrical, and chemical signals that help direct the synthetic activity of the chondrocytes. As the body ages, however, the composition of the matrix changes, and the chondrocytes lose their ability to respond to these stimuli.

1	Chondrocytes are specialized cells that produce and maintain the extracellular matrix. In hyaline cartilage, chondrocytes are distributed either singularly or in clusters called isogenous groups (Fig. 7.4). When the chondrocytes are present in isogenous groups, they represent cells that have recently divided. As the newly divided chondrocytes produce the matrix material that surrounds them, they are dispersed. They also secrete metalloproteinases, enzymes that degrade cartilage matrix, allowing the cells to expand and reposition themselves within the growing isogenous group.

1	The appearance of chondrocyte cytoplasm varies according to chondrocyte activity. Chondrocytes that are active in matrix production display areas of cytoplasmic basophilia, which are indicative of protein synthesis, and clear areas, which indicate their large Golgi apparatus (Fig. 7.5). Chondrocytes not only secrete the collagen present in the matrix but also all of the glycosaminoglycans and proteoglycans. In older, less active cells, the Golgi apparatus is smaller; clear areas of cytoplasm, when evident, usually indicate sites of extracted lipid droplets and glycogen stores. In such specimens, chondrocytes also display considerable distortion resulting from shrinkage after the glycogen and lipid are lost during preparation of the tissue. In the transmission electron microscope (TEM), the active chondrocyte displays numerous profiles of rough-surfaced endoplasmic reticulum (rER), a large Golgi apparatus, secretory granules, vesicles, intermediate filaments, microtubules, and actin

1	the active chondrocyte displays numerous profiles of rough-surfaced endoplasmic reticulum (rER), a large Golgi apparatus, secretory granules, vesicles, intermediate filaments, microtubules, and actin microfilaments (Fig. 7.6).

1	Components of the hyaline cartilage matrix are not uniformly distributed. Because the proteoglycans of hyaline cartilage contain a high concentration of bound sulfate groups, ground substance stains with basic dyes and hematoxylin (Plate 7, page 210). Thus, the basophilia and metachromasia seen in stained sections of cartilage provide information about the distribution and relative concentration of sulfated proteoglycans.

1	FIGURE 7.4 • Photomicrograph of a typical hyaline cartilage specimen stained with H&E. The upper portion of the micrograph shows the dense connective tissue (DCT ) overlying the perichondrium (P), from which new cartilage cells are derived. A slightly basophilic layer of growing cartilage (GC) underlying the perichondrium contains chondroblasts and immature chondrocytes that display little more than the nucleus residing in an empty-appearing lacuna. This layer represents deposition of new cartilage (appositional growth) on the surface of the existing hyaline cartilage. Mature chondrocytes with clearly visible nuclei (N) reside in the lacunae and are well preserved in this specimen. They produce the cartilage matrix that shows the dark-staining capsule or territorial matrix (TM) immediately surrounding the lacunae. The interterritorial matrix (IM) is more removed from the immediate vicinity of the chondrocytes and is less intensely stained. Growth from within the cartilage

1	immediately surrounding the lacunae. The interterritorial matrix (IM) is more removed from the immediate vicinity of the chondrocytes and is less intensely stained. Growth from within the cartilage (interstitial growth) is reflected by the chondrocyte pairs and clusters that are responsible for the formation of isogenous groups (rectangles). 480.

1	FIGURE 7.5 • Photomicrograph of young, growing cartilage. This specimen was preserved in glutaraldehyde, embedded in plastic, and stained with H&E. The chondrocytes, especially those in the upper part of the photomicrograph, are well preserved. The cytoplasm is deeply stained, exhibiting a distinct and relatively homogeneous basophilia. The clear areas (arrows) represent sites of the Golgi apparatus. 520. However, the matrix does not stain homogeneously. Rather, three different regions are described based on the staining property of the matrix (Fig. 7.7).

1	However, the matrix does not stain homogeneously. Rather, three different regions are described based on the staining property of the matrix (Fig. 7.7).  The capsular (pericellular) matrix is a ring of more densely staining matrix located immediately around the chondrocyte (see Fig. 7.4). It contains the highest concentration of sulfated proteoglycans, hyaluronan, bigly-cans, and several multiadhesive glycoproteins (e.g., fibronectin, decorin, and laminin). The capsular matrix contains almost exclusively type VI collagen fibrils that form a tightly woven enclosure around each chondrocyte. Type VI collagen binds to integrin receptors on the cell surface and anchors the chondrocytes to the matrix. A higher concentration of type IX collagen is also present in the capsular matrix.

1	 The territorial matrix is a region that is more removed from the immediate vicinity of the chondrocytes. It surrounds the isogenous group and contains a randomly arranged network of type II collagen fibrils with smaller quantities of type IX collagen. It also has a lower concentra-

1	FIGURE 7.6 • Electron micrograph of a young, active chondrocyte and surrounding matrix. The nucleus (N) of the chondrocyte is eccentrically located, like those in Figure 7.5, and the cytoplasm displays numerous and somewhat dilated profiles of rER, Golgi apparatus (G), and mitochondria (M). The large amount of rER and the extensive Golgi apparatus indicate that the cell is actively engaged in the production of cartilage matrix. The numerous dark particles in the matrix contain proteoglycans. The particularly large particles adjacent to the cell are located in the region of the matrix that is identified as the capsule or territorial matrix. 15,000. (Courtesy of Dr. H. Clarke Anderson.) tion of sulfated proteoglycans and stains less intensely than the capsular matrix. The interterritorial matrix is a region that surrounds the territorial matrix and occupies the space between groups of chondrocytes.

1	In addition to these regional differences in the concentration of sulfated proteoglycans and distribution of collagen fibrils, there is a decrease in proteoglycan content that occurs as cartilage ages, which is also reflected by staining differences. Hyaline cartilage provides a model for the developing skeleton of the fetus. FIGURE 7.7 • Diagram of cartilage matrices. Note the areas of capsular, territorial, and interterritorial matrices. The characteristics of each are described in the text above.

1	In early fetal development, hyaline cartilage is the precursor of bones that develop by the process of endochondral ossification (Fig. 7.8). Initially, most long bones are represented by cartilage models that resemble the shape of the mature bone (Plate 8, page 212). During the developmental process, in which much of the cartilage is replaced by bone, the remaining cartilage serves as a growth site called the epiphyseal growth plate (epiphyseal disc). This cartilage remains functional as long as the bone grows in length (Fig. 7.9). In a fully grown individual, the cartilage that remains from the developing skeleton is found on the articular surfaces of joints (articular cartilage) and within the rib cage (costal cartilages). Hyaline cartilage also exists in the adult as the skeletal unit in the trachea, bronchi, larynx, and nose. A firmly attached connective tissue, the perichondrium, surrounds hyaline cartilage.

1	A firmly attached connective tissue, the perichondrium, surrounds hyaline cartilage. The perichondrium is a dense connective tissue composed of cells that are indistinguishable from fibroblasts. In many respects, the perichondrium resembles the capsule that surrounds glands and many organs. It also serves as the source of new cartilage cells. When actively growing, the perichondrium appears divided into an inner cellular layer, which gives rise to new cartilage cells, and an outer fibrous layer. This division is not always evident, especially in perichondrium that is not actively producing new cartilage or in very slow-growing cartilage. The changes that occur during the differentiation of new chondrocytes in growing cartilage are illustrated in Figure 7.4. Hyaline cartilage of articular joint surfaces do not possess a perichondrium. Hyaline cartilage that covers the articular surfaces of movable joints is termed articular cartilage. In general, the

1	Hyaline cartilage of articular joint surfaces do not possess a perichondrium. Hyaline cartilage that covers the articular surfaces of movable joints is termed articular cartilage. In general, the FIGURE 7.8 • Photomicrograph of several cartilages that form the initial skeleton of the foot. The hyaline cartilage of developing tarsal bones will be replaced by bone as endochondral ossification proceeds. In this early stage of development, synovial joints are being formed between developing tarsal bones. Note that nonarticulating surfaces of the hyaline cartilage models of tarsal bones are covered by perichondrium, which also contributes to the development of joint capsules. Also, a developing tendon (T ) is evident in the indentation of the cartilage seen on the left side of the micrograph. 85.

1	structure of articular cartilage is similar to that of hyaline cartilage. However, the free, or articular, surface has no perichondrium. Also, on the opposite surface, the cartilage contacts the bone, and there is no perichondrium. Articular cartilage is a remnant of the original hyaline cartilage template of the developing bone, and it persists throughout adult life. In adults, the articular cartilage is 2 to 5 mm thick and is divided into four zones (Fig. 7.10).  The superficial (tangential) zone is a pressure-resistant region closest to the articular surface. It contains numerous

1	 The superficial (tangential) zone is a pressure-resistant region closest to the articular surface. It contains numerous FIGURE 7.9 • Photomicrograph of the proximal end of a growing long bone. A disc of hyaline cartilage−the epiphyseal plate−separates the more proximally located epiphysis from the funnel-shaped diaphysis located distal to the plate. The articular cartilage on the surface of the epiphysis contributes to the synovial joint and is also composed of hyaline cartilage. The cartilage of the epiphyseal plate disappears when lengthwise growth of the bone is completed, but the articular cartilage remains throughout life. The spaces within the bone are occupied by marrow. 85. condensation of type II collagen fibrils that are arranged in fascicles parallel to the free surface.

1	condensation of type II collagen fibrils that are arranged in fascicles parallel to the free surface.  The intermediate (transitional) zone lies below the superficial zone and contains round chondrocytes randomly distributed within the matrix. Collagen fibrils are less organized and are arranged in a somewhat oblique orientation to the surface.  The deep (radial) zone is characterized by small, round chondrocytes that are arranged in short columns perpendicular to the free surface of the cartilage. The collagen fibrils are positioned between columns parallel to the long axis of the bone.

1	 The calcified zone is characterized by a calcified matrix with the presence of small chondrocytes. This zone is separated from the deep (radial) zone by a smooth, undulating, heavily calcified line called the tidemark. Above this line, proliferation of chondrocytes within the cartilage lacunae provides the new cells for interstitial growth. In articular cartilage renewal, chondrocytes migrate from this region toward the joint surface. The renewal process of mature articular cartilage is very slow. This slow growth is a reflection of the highly stable type II collagen network and the long half-life of its proteoglycan molecules. Also, in healthy articular cartilage, metalloproteinase (MMP-1 and MMP-13) activity is low. Elastic cartilage is distinguished by the presence of elastin in the cartilage matrix.

1	Elastic cartilage is distinguished by the presence of elastin in the cartilage matrix. In addition to containing the normal components of hyaline cartilage matrix, elastic cartilage matrix also contains a dense network of branching and anastomosing elastic fibers and interconnecting sheets of elastic material (Fig. 7.11 and Plate 9, page 214). These fibers and lamellae are best demonstrated in paraffin sections with special stains such as resorcinfuchsin and orcein. The elastic material gives the cartilage elastic properties in addition to the resilience and pliability that are characteristic of hyaline cartilage.

1	Elastic cartilage is found in the external ear, the walls of the external acoustic meatus, the auditory (Eustachian) tube, and the epiglottis of the larynx. The cartilage in all of these locations is surrounded by a perichondrium similar to that found around most hyaline cartilage. Unlike hyaline cartilage, which calcifies with aging, the matrix of elastic cartilage does not calcify during the aging process. Fibrocartilage consists of chondrocytes and their matrix material in combination with dense connective tissue.

1	Fibrocartilage consists of chondrocytes and their matrix material in combination with dense connective tissue. Fibrocartilage is a combination of dense regular connective tissue and hyaline cartilage. The chondrocytes are dispersed among the collagen fibers singularly, in rows, and in isogenous groups (Fig. 7.12 and Plate 10, page 216). These chondrocytes appear similar to the chondrocytes of hyaline cartilage, but they have considerably less cartilage matrix material. There is also no surrounding perichondrium as in hyaline and elastic cartilage. In a section containing fibrocartilage, a population of cells with rounded nuclei and a small amount of surrounding amorphous matrix material can typically be seen. These nuclei belong to the chondrocytes. Within the fibrous areas are nuclei that are flattened or elongated. These are fibroblast nuclei.

1	Fibrocartilage is typically present in intervertebral discs, the symphysis pubis, articular discs of the sternoclavicular and temporomandibular joints, menisci of the knee joint, the triangular fibrocartilage complex of the wrist, and certain places where tendons attach to bones. The presence of fibrocartilage in these sites indicates that resistance to both compression and shearing forces is required of the tissue. The cartilage serves much like a shock absorber. The degree to which such forces occur is reflected in the amount of cartilage matrix material present. Extracellular matrix of fibrocartilage is characterized by the presence of both type I and type II collagen fibrils.

1	The cells in fibrocartilage synthesize a wide variety of extracellular matrix molecules not only during its development stage but also during its mature, fully differentiated state. This allows the fibrocartilage to respond to changes in the external environment (such as mechanical forces, nutritional changes, and changing levels of hormones and growth factors). The extracellular matrix of fibrocartilage contains significant quantities of both type I collagen (characteristic of connective tissue matrix) and type II collagen (characteristic of hyaline cartilage). The relative proportions of these collagens can vary. For example, menisci of the knee joint contain only a small quantity of the type II collagen, whereas the intervertebral disc contains equal amounts of type I and type II collagen fibers. The ratio between type I and type II collagen in fibrocartilage changes with age. In older individuals, there is more type II collagen because of the metabolic activity of chondrocytes,

1	fibers. The ratio between type I and type II collagen in fibrocartilage changes with age. In older individuals, there is more type II collagen because of the metabolic activity of chondrocytes, which constantly produce and discharge type II collagen fibrils into the surrounding matrix. In addition, the extracellular matrix of fibrocartilage contains larger amounts of versican (a proteoglycan monomer secreted by fibroblasts) than aggrecan (produced by chondrocytes). Versican can also bind hyaluronan to form highly hydrated proteoglycan aggregates (see Table 6.4, page 179).

1	(tangential) zone intermediate (transitional) zone deep (radial) zone tidemark calcified zone subchondral bone cancellous bone ababarticularcartilageSZIZDZCZbonearticularcartilageSZIZDZCZboneFIGURE 7.10 • Diagram and photomicrograph of articular cartilage. a. This diagram shows the organization of the collagen network and chondrocytes in the various zones of articular cartilage. b. Photomicrograph of normal articular cartilage from an adult. The superficial zone (SZ ) exhibits elongated and flattened chondrocytes. The intermediate zone (IZ ) contains round chondrocytes. The deep zone (DZ ) contains chondrocytes arranged in short columns. The calcified zone (CZ ), which borders the bone, exhibits a calcified matrix and lacks chondrocytes. Also, this zone is lighter staining than the matrix of the more superficial zones. The tidemark is indicated by the dashed line. 160.

1	FIGURE 7.11 • Photomicrograph of elastic cartilage from the epiglottis. This specimen was stained with orcein and reveals the elastic fibers, stained brown, within the cartilage matrix. The elastic fibers are of various sizes and constitute a significant part of the cartilage. Chondrocyte nuclei are evident in many of the lacunae. The perichondrium is visible at the top of the photomicrograph. 180.

1	TABLE Summary of Cartilage Features 7.1 Features Hyaline Cartilage Elastic Cartilage Fibrocartilage Location Fetal skeletal tissue, epiphyseal Pinna of external ear, external Intervertebral discs, symphysis plates, articular surface of acoustic meatus, auditory publis, articular discs synovial joints, costal cartilages (Eustachian) tube, cartilages of (sternoclavicular and of rib cage, cartilages of nasal larynx (epiglottis, corniculate, temporomandibular joints), cavity, larynx (thyroid, cricoid, and cuneiform cartilages) menisci (knee joint), triangular and arytenoids), rings of trachea fibrocartilage complex (wrist joint), and plates in bronchi insertion of tendons Function Resists compression Provides flexible support Resists deformation under stress Provides cushioning, smooth, and low-friction surface for joints Provides structural support in respiratory system (larynx, trachea, and bronchi) Forms foundation for development of fetal skeleton and further endochondral bone

1	low-friction surface for joints Provides structural support in respiratory system (larynx, trachea, and bronchi) Forms foundation for development of fetal skeleton and further endochondral bone formation and bone growth Presence of Yes (except articular cartilage Yes No perichondrium and epiphyseal plates) Undergoes Yes (i.e., during endochondral No Yes (i.e., calcification of calcification bone formation, during aging fibrocartilaginous callus during process) bone repair) Main cell Chondroblasts and Chondroblasts and Chondrocytes and fibroblasts types present chondrocytes chondrocytes Characteristic Type II collagen fibrils and Type II collagen fibrils, elastic Types I and II collagen fibers and features of aggrecan (the most important fibers, and aggrecan versican (a proteoglycan secreted extracellular proteoglycan) by fibroblasts) matrix Growth Interstitially and appositionally, very limited in adults Repair Very limited capability, commonly forms scar, resulting in

1	secreted extracellular proteoglycan) by fibroblasts) matrix Growth Interstitially and appositionally, very limited in adults Repair Very limited capability, commonly forms scar, resulting in fibrocartilage formation

1	Most cartilage arises from mesenchyme during chondrogenesis. Chondrogenesis, the process of cartilage development, begins with the aggregation of chondroprogenitor mesen chymal cells to form a mass of rounded, closely apposed cells. In the head, most of the cartilage arises from aggregates of ectomesenchyme derived from neural crest cells. The site of hyaline cartilage formation is recognized initially by an aggregate of mesenchymal or ectomesenchymal cells known as a chondrogenic nodule. Expression of transcription factor SOX-9 triggers differentiation of these cells into chondro blasts, which then secrete cartilage matrix (expression of SOX-9 coincides with secretion of type II collagen). The chondroblasts progressively move apart as they deposit matrix. When they are completely surrounded by matrix material, the cells are called chondrocytes. The mesenchymal tissue immediately surrounding the chondrogenic nodule gives rise to the perichondrium.

1	Chondrogenesis is regulated by many molecules, including extracellular ligands, nuclear receptors, transcription factors, adhesion molecules, and matrix proteins. Furthermore, FIGURE 7.12 • Photomicrograph of fibrocartilage from an intervertebral disc. The collagen fibers are stained green in this Gomori trichrome preparation. The tissue has a fibrous appearance and contains a relatively small number of fibroblasts with elongated nuclei (arrows) as well as more numerous chondrocytes with dark round nuclei. The chondrocytes exhibit close spatial groupings and are arranged either in rows among the collagen fibers or in isogenous groups. 60. Inset. Higher magnification of an isogenous group. Chondrocytes are contained within lacunae. Typically, there is little cartilage matrix surrounding the chondrocytes. 700.

1	the growth and development of the cartilage skeleton is influenced by biomechanical forces. These forces not only regulate the shape, regeneration, and aging of cartilage but also modify cell–to–extracellular matrix interactions within the cartilage. Cartilage is capable of two kinds of growth, appositional and interstitial. With the onset of matrix secretion, cartilage growth continues via a combination of two processes:  Appositional growth, the process that forms new carti lage at the surface of an existing cartilage; and  interstitial growth, the process that forms new cartilage

1	New cartilage cells produced during appositional growth are derived from the inner portion of the surrounding perichondrium. The cells resemble fibroblasts in form and function, producing the collagen component of the perichondrium (type I collagen). When cartilage growth is initiated, however, the cells undergo a differentiation process guided by an expression of the transcription factor SOX-9. The cytoplasmic processes disappear, the nucleus becomes rounded, and the cytoplasm increases in amount and prominence. These changes result in the cell becoming a chondro blast. Chondroblasts function in cartilage matrix production, including secretion of type II collagen. The new matrix increases the cartilage mass, while new fibroblasts are produced simultaneously to maintain the cell population of the perichondrium.

1	New cartilage cells produced during interstitial growth arise from the division of chondrocytes within their lacunae (see Fig. 7.4). This is possible only because the chondrocytes retain the ability to divide and the surrounding matrix is distensible, thus permitting further secretory activity. Initially, the daughter cells of the dividing chondrocytes occupy the same lacuna. As new matrix is secreted, a partition is formed between the daughter cells; at this point each cell occupies its own lacuna. With continued secretion of matrix, the cells move even farther apart from each other. The overall growth of cartilage thus results from the interstitial secretion of new matrix material by chondrocytes and by the appositional secretion of matrix material by newly differentiated chondro blasts (Folder 7.2). Cartilage has limited ability for repair.

1	Cartilage has limited ability for repair. Cartilage can tolerate considerable intense and repetitive stress. However, when damaged, cartilage manifests a striking inability to heal, even in the most minor injuries. This lack of response to injury is attributable to the avascularity of cartilage, the immobility of the chondrocytes, and the limited ability of mature chondrocytes to proliferate. Some repair can occur but only if the defect involves the perichondrium. In these injuries, repair results from activity of the pluripotential progenitor cells located in the perichondrium. Even then, however, few cartilage cells, if any, are produced. Repair mostly involves the production of dense connective tissue.

1	At the molecular level, cartilage repair is a tentative balance between deposition of type I collagen in the form of scar tissue and repair by expression of the cartilagespecific collagens. However, in adults, new blood vessels commonly develop at the site of the healing wound that stimulate the growth of bone rather than actual cartilage repair. The limited ability of cartilage to repair itself can cause significant problems in cardiothoracic surgery, such as coronary artery bypass graft surgery, when costal cartilage must be cut to enter the chest cavity. A variety of treatments may improve the healing of articular cartilage, including perichondral grafts, cell transplantation, insertion of artificial matrices, and application of growth factors. When hyaline cartilage calcifies, it is replaced by bone.

1	Hyaline cartilage is prone to calcification, a process in which calcium phosphate crystals become embedded in the tumors characterized by secretion of cartilage matrix. Ap-proximately 3.6% of primary bone tumors that are diagnosed in the United States each year are chondrosarcomas. These tumors are the second most common matrix-producing tu-mors of bone after osteosarcomas (malignant bone-forming tumors). They occur more commonly in men than women and affect individuals that are usually age 45 and older. Chondrosarcomas originate predominantly in the axial skele-ton (and most commonly involve vertebrae, pelvic bones, ribs, scapulae, and the sternum), and in metaphyses of prox-imal ends of long bones (most often the femur and humerus). The most common symptom reported by patients is a deep pain, often present for months and typically dull in character. Since cartilaginous tissue is compressed inside the bone, in most cases initial growth of a tumor cannot be palpated. Radiographs, CT,

1	pain, often present for months and typically dull in character. Since cartilaginous tissue is compressed inside the bone, in most cases initial growth of a tumor cannot be palpated. Radiographs, CT, and MRI scans are essential for the initial diagnosis and later for the evaluation of the extent of deep intramedullary tumors. Chondrosarcomas are classified by grades that strongly correlate with a patient’s prognosis. Micro-scopically, grade 1 represents the least aggressive and grade 3 represents the most aggressive tumor. Most chondrosarcomas (90%) are pathologically classified as conventional (grades 1 and 2); they rarely metastasize and are composed of hyaline cartilage that infiltrates bone marrow cavity and surrounds existing bony trabe-culae (Fig. F7.2.1). Multiple chondroblasts that are often binucleated with pleomorphic and hyperchromatic nu-clear patterns are frequently seen in a single lacuna. Cartilaginous matrix may also undergo mineralization and subsequent endochondral

1	are often binucleated with pleomorphic and hyperchromatic nu-clear patterns are frequently seen in a single lacuna. Cartilaginous matrix may also undergo mineralization and subsequent endochondral ossification. Metastatic spread to lungs and lymph nodes is more frequently asso-ciated with grade 3 lesions. Recently, immunohistochemical localization of collagen types has been used to determine stage of tissue differenti-ation, which in fact correlates with a patient’s prognosis. The presence of collagen types II and X and the proteogly-can aggrecan in biopsies indicates mature tumors associ-ated with good prognosis. On the other hand, the presence of collagen type I indicates changes in the extracellular ma-trix toward dedifferentiated (fibrous) types of tumor with poorer prognosis. In addition, transcription factor SOX9, which is essential for differentiation of mesenchymal cells into chondroblasts during normal fetal development, is expressed in chondrosarcomas. Treatment of

1	In addition, transcription factor SOX9, which is essential for differentiation of mesenchymal cells into chondroblasts during normal fetal development, is expressed in chondrosarcomas. Treatment of chondrosarcoma is primary surgical: The tumor is widely excised. Chemotherapy and radiation play limited roles in treatment. Patients with adequately re-sected low-grade tumors have an excellent survival rate. FIGURE F7.2.1 • Photomicrograph of a chondrosarcoma (grade 1) from the epiphysis of the long bone, stained with H&E. This photomicrograph shows a tissue mass of chondrosarcoma infiltrating intertrabecular spaces of the bone marrow. Note the presence of malignant chondrocytes in various stages of maturity. Small area of active bone marrow is visible in the upper left corner of the image. 240. (Courtesy of Dr. Fabiola Medeiros.) marrowmarrowmarrowbonebonebonechondrosarcomachondrosarcomachondrosarcoma  FOLDER 7.2 Clinical Correlation: Malignant Tumors of the Cartilage; Chondrosarcomas

1	Chondrosarcomas are generally slow-growing malignant cartilage matrix. The matrix of hyaline cartilage undergoes calcification as a regular occurrence in three well-defined situations:  The portion of articular cartilage that is in contact with bone tissue in growing and adult bones, but not the surface portion, is calcified.  Calcification always occurs in cartilage that is about to be replaced by bone (endochondral ossification) during an individual’s growth period.  Hyaline cartilage in the adult calcifies with time as part of the aging process.

1	 Hyaline cartilage in the adult calcifies with time as part of the aging process. In most of these situations, given sufficient time, cartilage that calcifies is replaced by bone. For example, in older individuals, portions of the cartilage rings in the trachea are often replaced by bone tissue (Fig. 7.13). Chondrocytes normally derive all of their nutrients and dispose of wastes by diffusion of materials through the matrix. When the matrix becomes heavily calcified, diffusion is impeded and the chondrocytes swell and die. The ultimate consequence of this event is removal of the calcified matrix and its replacement by bone. A number of investigators believe the process of cartilage removal involves a specific cell type designated as a chondroclast. This cell is described as resembling an osteoclast in both

1	A number of investigators believe the process of cartilage removal involves a specific cell type designated as a chondroclast. This cell is described as resembling an osteoclast in both FIGURE 7.13 • Photomicrograph of a tracheal ring from an elderly individual, stained with H&E. The darker, somewhat basophilic areas on the left side of the micrograph represent normal cartilage matrix (C). The lighter and more eosinophilic areas represent bone tissue (B) that has replaced the original cartilage matrix. A large marrow cavity has formed within the cartilage structure and is visible in the center of the micrograph. 75.

1	morphology and lytic function. It is thought that these cells enter cartilage along newly sprouting blood vessels and may, in fact, be derived from perivascular or bone marrow stem cells. However, the precise origin of these cells is unknown. Early studies of chondroclast structure and function were carried out on the developing mandible, in which the resorption of Meckel’s cartilage is not followed by bone replacement (endochondral ossification). It is likely that chondroclasts are cells that occur wherever cartilage is being removed. Their role is discussed in regard to endochondral bone formation.

1	Cartilage is an avascular form of connective tissue composed of cells called chondrocytes and a highly specialized extracellular matrix. Three kinds of cartilage are described on the basis of characteristics of the matrix: hyaline cartilage (described here), elastic cartilage (described in Plate 9), and fibrocartilage (described in Plate 10). Hyaline cartilage has a homogeneous-appearing amorphous matrix. It contains type II collagen. Type II collagen appears with the transmission electron microscope (TEM) as thin fibrils, ~20 nm in diameter, in which the character-istic 68-nm banding may not be obvious. The fibrils are arranged in a three-dimensional felt-like pattern. The matrix also contains large amounts of glycosaminoglycans, most of which form proteoglycans and proteoglycan aggregates. Hyaline cartilage is found in the adult as the structural framework for the larynx, trachea, and bronchi; it is found on the articular ends of the ribs and on the surfaces of synovial joints. In

1	Hyaline cartilage is found in the adult as the structural framework for the larynx, trachea, and bronchi; it is found on the articular ends of the ribs and on the surfaces of synovial joints. In addition, hyaline cartilage constitutes much of the fetal skeleton and plays an important role in the growth of most bones. Hyaline cartilage displays both appositional growth, the addition of new cartilage at its surface, and interstitial growth, the division and differentiation of chondrocytes within its substance.

1	Hyaline cartilage, trachea, human, H&E ×450. This micrograph reveals hyaline cartilage from the trachea as seen in a routinely prepared specimen. The cartilage appears as an avascular expanse of matrix material and a population of cells called chondrocytes (Ch). The chondrocytes produce the matrix; the space each chondrocyte occupies is called a lacuna (L). Surrounding the cartilage and in immediate apposition to it is a cover of connective tissue, the perichondrium (P). The perichondrium serves as a source of new chondrocytes during appositional growth of the cartilage. Often, the perichondrium reveals two distinctive layers: an outer, more fibrous layer and an inner, more cellular layer. The inner, more cellular layer is chondrogenic and provides for external growth. Cartilage matrix contains collagenous fibrils masked by ground substance in which they are embedded; thus, the fibrils are not evident. The Hyaline cartilage, trachea, human, H&E ×850.

1	This higher magnification micrograph reveals the area within the rectangle in lower left figure. The chondrocytes (Ch) in the upper part of the micrograph represent an isogenous group and are producing matrix material for interstitial growth. A prominent capsule is not yet evident. The lightly stained ba sophilic area reveals immature chondrocytes (arrows) within the perichondrium (P). Closest to the cartilage matrix, within the perichondrium (P), matrix also contains, among other components, sulfated glycosaminoglycans that exhibit basophilia with hematoxylin or other basic dyes. Also, the matrix material immediately surrounding a lacuna tends to stain more intensely with basic dyes. This region is referred to as a capsule (Cap). Not uncommonly, the matrix may appear to stain more intensely in localized areas (asterisks) that look much like the capsule matrix. This results from inclusion of a capsule within the thickness of the section, but not the lacuna it surrounds.

1	Frequently, two or more chondrocytes are located extremely close to one another, separated by only a thin partition of matrix. These are isogenous cell clusters that arise from a single predecessor cell. The proliferation of new chondrocytes by this means with the consequent addition of matrix results in interstitial growth of the cartilage. Hyaline cartilage, trachea, human, H&E ×160.

1	Hyaline cartilage, trachea, human, H&E ×160. The hyaline cartilage in this micrograph is from a specimen obtained shortly after death and kept cool during fixation. The procedure reduces the loss of its negatively charged sulfate groups; thus, the matrix is stained more heavily with hematoxylin. Also, note the very distinct and deeply stained capsules (arrows) surrounding the chondrocytes. The capsule represents the site where the sulfated glycosaminoglycans are most concentrated. In contrast to the basophilia of the cartilage matrix, the perichondrium (P) is stained with eosin. The lightly stained region between the perichondrium and the deeply stained matrix is matrix that has not yet matured. It has fewer sulfate groups.

1	are several chondrocytes that exhibit just barely detectable cytoplasm and elongate nuclei (FCh). These cells are formative chondrocytes that are just beginning to, or will shortly, produce matrix material. In contrast, the nuclei near the bottom edge of the micrograph are fibroblast nuclei (Fib); they belong to the outer layer of the perichondrium. Note how attenuated their nuclei are compared with the formative chondroblast nuclei of the inner perichondrial layer. KEY Cap, capsule Ch, chondrocytes FCh, formative chondrocytes Fib, fibroblasts L, lacuna P, perichondrium arrows, immature chondrocytes asterisk, capsule of a lacuna, but with lacuna and contained chondrocyte not included within the thickness of the section PLATE 8 Cartilage and the Developing Skeleton

1	PLATE 8 Cartilage and the Developing Skeleton Hyaline cartilage is present as a precursor to bones in the fetus. This cartilage is replaced by bone tissue except where one bone contacts another, as in a movable joint. In these locations, cartilage persists and covers the end of each bone as articular cartilage, providing a smooth, well-lubricated surface against which the end of one bone moves on the other in the joint. In addition, cartilage, being capable of interstitial growth, persists in weight-supporting bones and other long bones as a growth plate as long as growth in length occurs. The role of hyaline cartilage in bone growth is considered briefly below and in more detail in Plates 13 and 14. Developing skeleton, fetal foot, rat, H&E ×85.

1	This section shows the cartilages that will ultimately become the bones of the foot. In several places, developing ligaments (L) can be seen where they join the cartilages. The nuclei of the fibroblasts within the ligaments are just barely perceptible. They are aligned in rows and are separated from other rows of fibroblasts by collagenous material. The hue and intensity of color of the cartilage matrix, except at the periphery, are due to the combined uptake of 212 the H&E. The collagen of the matrix stains with eosin; however, the presence of sulfated glycosaminoglycans results in staining by hematoxylin. The matrix of cartilage that is about to be replaced by bone, such as that shown here, becomes impregnated with calcium salts, and the calcium is also receptive to staining with hematoxylin. The many enlarged lacunae (seen as light spaces within the matrix where the chondrocytes have fallen out of the lacunae) are due to hypertrophy of the chondrocytes, an event associated with

1	The many enlarged lacunae (seen as light spaces within the matrix where the chondrocytes have fallen out of the lacunae) are due to hypertrophy of the chondrocytes, an event associated with calcification of the matrix. Thus, where these large lacunae are present, i.e., in the center region of the cartilage, the matrix is heavily stained.

1	This figure also shows that the cartilage is surrounded by perichondrium, except where it faces a joint cavity (JC). Here, the bare cartilage forms a surface. Note that the joint cavity is a space between the cartilages whose boundaries are completed by connective tissue (CT). The connective tissue at the surface of the cavity is special. It will constitute the synovial membrane in the adult and contribute to the formation of a lubricating fluid (synovial fluid) that is present in the joint cavity. Therefore, all the surfaces that will enclose the adult joint cavity are derived originally from the mesenchyme. Synovial fluid is a viscous substance containing, among other things, glycosaminoglycans; it can be considered an exudate of interstitial fluid. The synovial fluid could be considered an extension of the extracellular matrix, as the joint cavity is not lined by an epithelium.

1	CARTI LAG E AN D TH E DEVE LOPI NG S KE LETON KEY B, bone C, cartilage CT, connective tissue JC, joint cavity L, ligament MC, marrow cavity arrowhead, calcified cartilage Developing skeleton, fetal fnger, human, thionine-picric acid ×30.

1	This figure shows a developing long bone of the finger and its articulation with the distal and proximal bones. Before the stage shown here, each bone consisted entirely of a hyaline cartilaginous structure similar to the cartilages seen in figure above but shaped like the long bones into which they would develop. Here, only the ends, or epiphyses, of the bone remain as cartilage, the epiphyseal cartilage (C). The shaft, or diaphysis, has become a cylinder of bone tissue (B) surrounding the marrow cavity (MC). The dark region at the ends of the marrow cavity is calcified cartilage (arrowhead) that is being replaced by bone. The bone at the ends of the marrow cavity constitutes the metaphysis. With this staining method, the calcified cartilage appears dark brown. The newly formed metaphyseal bone, which is admixed with this degenerating calcified cartilage and is difficult to define at this low magnification, has the same yellow-brown color as the diaphyseal bone. By the continued

1	bone, which is admixed with this degenerating calcified cartilage and is difficult to define at this low magnification, has the same yellow-brown color as the diaphyseal bone. By the continued proliferation of cartilage, the bone grows in length. Later, the cartilage becomes calcified; bone is then produced and occupies the site of the resorbed cartilage. With the cessation of cartilage proliferation and its replacement by bone, growth of the bone stops, and only the cartilage at the articular surface remains. The details of this process are explained under endochondral bone formation (Plates 13 and 14).

1	Elastic cartilage has a matrix containing elastic fibers and elastic lamellae in addition to type II collagen. It is found in the auricle of the exter-nal ear, in the auditory tube, in the epiglottis, and in part of the larynx. The elastic material imparts properties of elasticity, as distinguished from resiliency, which are not shared by hyaline cartilage. Elastic cartilage is surrounded by perichondrium, and it, too, increases in size by both ap-positional and interstitial growth. Unlike hyaline cartilage, however, elastic cartilage does not normally calcify. Elastic cartilage, epiglottis, human, H&E and orcein stains ×80. This section of the epiglottis contains elastic cartilage (EC) as the centrally located structure. The essential components of the cartilage, namely, the matrix that stains deep blue and the light, unstained lacunae surrounded by matrix, are evident in this low-magnification micrograph. The perimeter of the cartilage is covered

1	PLATE 9 • E LASTIC CARTI LAG E KEY AT, adipose tissue E, elastic fiber EC, elastic cartilage MG, mucous gland PC, perichondrium SE, stratified squamous epithelium Elastic cartilage, epiglottis, human, H&E and orcein stains ×250; inset ×400. This shows an area of the elastic cartilage at higher magnification. The elastic fibers appear as the blue, elongate profiles within the matrix. They are most evident at the edges of the cartilage, but they are obscured in some deeper parts of the matrix, where they blend with the elastic material that forms a honeycomb about the lacunae. Elastic fibers (E) are also apparent in the adipose tissue (AT), between the adipocytes.

1	Some of the lacunae in the cartilage are arranged in pairs separated by a thin plate of matrix. The plate of matrix appears as a bar between the adjacent lacunae. This is a reflection of interstitial growth by the cartilage, in that the adjacent cartilage cells are derived from the same parent cell. They have moved away from each other and secreted a plate of cartilage matrix by perichondrium (PC); its fibrous character is just barely visible in this figure. Also note the adipose tissue (AT) within the boundaries of the elastic cartilage. Both above and below the elastic cartilage is connective tissue, and each surface of the epiglottis is formed by stratified squamous epithelium (SE). Mucous glands (MG) are in the connective tissue in the bottom of this figure.

1	between them to form two lacunae. Most chondrocytes shown in this figure occupy only part of the lacuna. This is, in part, due to shrinkage, but it is also due to the fact that older chondrocytes contain lipid in large droplets that is lost during the processing of the tissue. The shrinkage of chondrocytes within the lacunae or their loss due to dropping out of the section during preparation causes the lacunae to stand out as light, unstained areas against the darkly stained matrix. The inset shows the elastic cartilage at still higher magnification. Here, the elastic fibers (E) are again evident as elongate profiles, chiefly at the edges of the cartilage. Most chondrocytes in this part of the specimen show little shrinkage. Many of the cells display a typically rounded nucleus, and the cytoplasm is evident. Note, again, that some lacunae contain two chondrocytes, indicating interstitial growth.

1	Fibrocartilage is a combination of dense connective tissue and cartilage. It has a matrix with large bundles of type I collagen in addition to type II collagen. The amount of cartilage varies, but in most locations the cartilage cells and their matrix occupy a lesser portion of the tissue mass. Fibrocartilage is found at the intervertebral discs, the symphysis pubis, the knee joint, the mandibular joint, the sternoclavicular joint, and the shoulder joint. It may also be present along the grooves or insertions for tendons and ligaments. Its presence is associated with sites where resilience is required in dense connective tissue to help absorb sudden physical impact, i.e., where resistance to both compressive and shearing forces is required in the tissue. Histologically, fibrocartilage appears as small fields of cartilage blending almost imperceptibly with regions of dense fibrous connective tissue. It is usually identified by the presence of aggregates of rounded chondrocytes

1	appears as small fields of cartilage blending almost imperceptibly with regions of dense fibrous connective tissue. It is usually identified by the presence of aggregates of rounded chondrocytes (isogenous groups) among bundles of collagen fibers and by the basophilic staining of the capsular matrix material and territorial matrix secreted by these cells. No perichondrium is present.

1	Fibrocartilage, intervertebral disc, human, Mallory’s trichrome ×160. This is a low-magnification view of fibrocartilage. The Mallory method stains collagen light blue. The tissue has a fibrous appearance, and at this low magnification the nuclei of the fibroblasts (F) appear as small, elongate, or spindle-shaped bodies. There are relatively few fibroblasts present, as is characteristic of dense connective tissue. The chondrocytes (C) are more numerous and exhibit close spatial groupings, i.e., isogenous groups. Some of the chondrocytes appear as elongate clusters of cells, whereas others appear in single-file rows. The matrix material immediately surrounding the chondrocytes has a homogeneous appearance and is, thereby, distinguishable from the fibrous connective tissue. PLATE 10 • FIBROCARTILAGE KEY C, chondrocytes F, fibroblast arrow, lacuna Fibrocartilage, intervertebral disc, human, Mallory’s trichrome ×700.

1	PLATE 10 • FIBROCARTILAGE KEY C, chondrocytes F, fibroblast arrow, lacuna Fibrocartilage, intervertebral disc, human, Mallory’s trichrome ×700. This figure shows the area circumscribed by the rectangle in figure above at higher magnification. The chondrocytes are contained within lacunae (arrows), and their cytoplasm stains deeply. The surrounding cartilage matrix material is scant and blends into the dense connective tissue. Cartilage matrix material can be detected best by observing the larger group of chondrocytes at the left of this figure and then observing this same area in figure above. Note the light homogeneous area around the cell nest in the lower-power view. This is the region of cartilage matrix. At the greater magnification of this figure, it is possible to see that some of the collagen fibers are incorporated in the matrix, where they appear as wispy bundles.

1	OVERVIEW OF BONE / 218 BONES AND BONE TISSUE / 219 GENERAL STRUCTURE OF BONES / 220 Outer Surface of Bones / 220 Bone Cavities / 221 Mature Bone / 221 Immature Bone / 223 CELLS OF BONE TISSUE / 223 Osteoprogenitor Cells / 225 Osteoblasts / 225 Osteocytes / 227 Bone-Lining Cells / 227 Osteoclasts / 227 BONE FORMATION / 232 Intramembranous Ossification / 234 Endochondral Ossification / 235 Growth of Endochondral Bone / 237 Development of the Osteonal (Haversian) System / 240 BIOLOGIC MINERALIZATION AND MATRIX VESICLES / 241 PHYSIOLOGIC ASPECTS OF BONE / 242 Folder 8.1 Clinical Correlation: Joint Diseases / 221 Folder 8.2 Clinical Correlation: Osteoporosis / 233 Folder 8.3 Clinical Correlation: Nutritional Factors in Bone Formation / 234 Folder 8.4 Functional Considerations: Hormonal Regulation of Bone Growth / 242 Bone is a connective tissue characterized by a mineralized extracellular matrix.

1	Bone is a connective tissue characterized by a mineralized extracellular matrix. Bone is a specialized form of connective tissue that, like other connective tissues, consists of cells and extracellular matrix. The feature that distinguishes bone from other connective tissues is the mineralization of its matrix, which produces an extremely hard tissue capable of providing support and protection. The mineral is calcium phosphate in the form of hydroxyapatite crystals [Ca10(PO4)6(OH)2]. By virtue of its mineral content, bone also serves as a storage site for calcium and phosphate. Both calcium and phosphate can be mobilized from the bone matrix and taken up by the blood as needed to maintain appropriate levels throughout the body. Thus, in addition to support and protection, bone plays an important secondary role in the homeostatic regulation of blood calcium levels. Bone matrix contains mainly type I collagen along with other matrix (noncollagenous) proteins.

1	Bone matrix contains mainly type I collagen along with other matrix (noncollagenous) proteins. The major structural component of bone matrix is type I collagen and, to a lesser extent, type V collagen. Trace amounts of other types such as type III, XI, and XIII collagens have also been found in the matrix. All collagen molecules constitute about 90% of the total weight of the bone matrix proteins.

1	The matrix also contains other matrix (noncollagenous) proteins that constitute the ground substance of bone. As a minor component of bone, constituting only 10% of the total weight of bone matrix proteins, they are essential to bone development, growth, remodeling, and repair. Both the collagen and the ground substance become mineralized to form bone tissue. The four main groups of noncollagenous proteins found in the bone matrix are the following:  Proteoglycan macromolecules contain a core protein with various numbers of covalently attached side chains of glycosaminoglycans (hyaluronan, chondroitin sulfate, and keratan sulfate). They contribute to the compressive strength of bone. They are also responsible for binding growth factors and may inhibit mineralization. Proteoglycans are described in detail in Chapter 6 (Table 6.3, page 176).

1	 Multiadhesive glycoproteins are responsible for attachment of bone cells and collagen fibers to the mineralized ground substance. Some of the more important glycoproteins are osteonectin (which serves as a glue between the collagen and hydroxyapatite crystals) and sialoproteins such as osteopontin (which mediates attachment of cells to bone matrix) and sialoprotein I and II (which mediate cell attachment and initiate calcium phosphate formation during the mineralization process).  Bone-specific, vitamin K–dependent proteins, which include osteocalcin (which captures calcium from the circulation and attracts and stimulates osteoclasts in bone remodeling), protein S (which assists in the removal of cells undergoing apoptosis), and matrix Gla-protein (MGP) (which participates in the development of vascular calcifications).

1	 Growth factors and cytokines, which are small regulatory proteins including insulinlike growth factors (IGFs), tumor necrosis factor (TNF-), transforming growth factor (TGF-), platelet-derived growth factors (PDGFs), bone morphogenic proteins (BMPs), and interleukins (IL-1, IL-6). The most unique members of this group are BMPs, because they induce the differentiation of mesenchymal cells into osteoblasts, the bone-producing cells. Recombinant human BMP-7, also known as osteogenic protein-1 (OP-1), is now used clinically to induce bone growth after bone surgery involving large bone defects, spinal fusions, or implantation of graft materials. Bone matrix contains lacunae connected by a network of canaliculi. Within the bone matrix are spaces called lacunae (sing., lacuna), each of which contains a bone cell, or osteocyte.

1	Bone matrix contains lacunae connected by a network of canaliculi. Within the bone matrix are spaces called lacunae (sing., lacuna), each of which contains a bone cell, or osteocyte. The osteocyte extends numerous processes into small tunnels called canaliculi. Canaliculi course through the mineralized matrix, connecting adjacent lacunae and allowing contact be tween the cell processes of neighboring osteocytes (Plate 11, page 244). In this manner, a continuous network of canaliculi and lacunae-containing cells and their processes is formed throughout the entire mass of mineralized tissue. Electron gap junctions. Bone tissue depends on the osteocytes to maintain viability. In addition to osteocytes, four other cell types are associ ated with bone.  Osteoprogenitor cells are cells derived from mesenchymal stem cells; they give rise to osteoblasts.

1	 Osteoprogenitor cells are cells derived from mesenchymal stem cells; they give rise to osteoblasts.  Osteoblasts are cells that secrete the extracellular matrix of bone; once the cell is surrounded with its secreted matrix, it is referred to as an osteocyte.  Bone-lining cells are cells that remain on the bone surface when there is no active growth. They are derived from those osteoblasts that remain after bone deposition ceases.  Osteoclasts are bone-resorbing cells present on bone surfaces where bone is being removed or remodeled (reorganized) or where bone has been damaged. Osteoprogenitor cells and osteoblasts are developmental precursors of the osteocyte. Osteoclasts are phagocytotic cells derived from fusion of hemopoietic progenitor cells in bone marrow that give rise to the neutrophilic granulocyte and monocyte lineages. Each of these cells is described in more detail below.

1	Bones are the organs of the skeletal system; bone tissue is the structural component of bones. Typically, a bone consists of bone tissue and other connective tissues, including hemopoietic tissue, fat tissue, blood vessels, and nerves. If the bone forms a freely movable or synovial joint, hyaline cartilage is present. The ability of the bone to perform its skeletal function is attributable to the bone tissue and, where present, the hyaline or articular cartilage. Bone tissue is classified as either compact (dense) or spongy (cancellous). If a bone is cut, two distinct structural arrangements of bone tissue can be recognized (Fig. 8.1 and Plate 12, page 246). A compact, dense layer forms the outside of the bone (compact bone); a spongelike meshwork consisting of trabe culae (thin, anastomosing spicules of bone tissue) forms the interior of the bone (spongy bone). The spaces within the meshwork are continuous and, in a living bone, are occupied by marrow and blood vessels.

1	Bones are classified according to shape; the location of spongy and compact bone varies with bone shape. Spongy and compact bone tissues are located in specific parts of bones. It is useful, then, to outline briefly the kinds of bones and survey where the two kinds of bone tissue are located. On the basis of shape, bones can be classified into four groups:  Long bones are longer in one dimension than other bones and consist of a shaft and two ends (e.g., the tibia and the metacarpals). A schematic diagram of a long bone sectioned longitudinally through the shaft is shown in Figure 8.2.  Short bones are nearly equal in length and diameter (e.g., the carpal bones of the hand).  Flat bones are thin and platelike (e.g., the bones of the calvaria [skull cap] and the sternum). They consist of two layers of relatively thick compact bone with an intervening layer of spongy bone.

1	 Irregular bones have a shape that does not fit into any one of the three groups just described; the shape may be complex (e.g., a vertebra), or the bone may contain air spaces or sinuses (e.g., the ethmoid bone). FIGURE 8.1 • Epiphysis of an adult long bone. This photo shows a longitudinally sectioned epiphysis of a long bone. The outer portion of the bone has a solid structure (arrows) and represents compact (dense) bone. The interior of the bone exhibits a spongy configuration and represents spongy (cancellous) bone. It consists of numerous interconnecting bony trabeculae separated by a labyrinth of interconnecting marrow spaces.

1	Long bones have a shaft, called the diaphysis, and two expanded ends, each called an epiphysis (see Fig. 8.2). The articular surface of the epiphysis is covered with hyaline cartilage. The flared portion of the bone between the diaphysis and the epiphysis is called the metaphysis. It extends from the diaphysis to the epiphyseal line. A large cavity filled with bone marrow, called the marrow or medullary cavity, forms the inner portion of the bone. In the shaft, almost the entire thickness of the bone tissue is compact; at most, only a small amount of spongy bone faces the marrow cavity. At the ends of the bone, the reverse is true. Here the spongy bone is extensive, and the compact bone consists of little more than a thin outer shell (see Fig. 8.1). Short bones possess a shell of compact bone and have spongy bone and a marrow space on the inside. Short bones usually form movable joints with their neighbors; like long

1	Short bones possess a shell of compact bone and have spongy bone and a marrow space on the inside. Short bones usually form movable joints with their neighbors; like long FIGURE 8.2 • Structure of a typical long bone. The diaphysis (shaft) of a long bone contains a large marrow cavity surrounded by a thick-walled tube of compact bone. A small amount of spongy bone may line the inner surface of the compact bone. The proximal and distal ends, or epiphyses, of the long bone consist chiefly of spongy bone with a thin outer shell of compact bone. The expanded or flared part of the diaphysis nearest the epiphysis is referred to as the metaphysis. Except for the articular surfaces that are covered by hyaline (articular) cartilage, indicated in blue, the outer surface of the bone is covered by a fibrous layer of connective tissue called the periosteum, indicated in pink.

1	bones, their articular surfaces are covered with hyaline cartilage. Elsewhere, periosteum, a fibrous connective tissue capsule covers the outer surface of the bone. Outer Surface of Bones Bones are covered by periosteum, a sheath of dense fibrous connective tissue containing osteoprogenitor cells. Bones are covered by a periosteum except in areas where they articulate with another bone. In the latter case, the articulating surface is covered by cartilage. The periosteum that covers an actively growing bone consists of an outer fibrous layer that resembles other dense connective tissues and an inner, more cellular layer that contains the osteoprogenitor cells. If active bone formation is not in progress on the bone surface, the fibrous layer is the main component of the periosteum, and the inner layer is not well defined. The relatively few cells that are present, the periosteal cells, are, however, capable of undergoing division and becoming osteoblasts under appropriate stimulus.

1	In general, the collagen fibers of the periosteum are arranged parallel to the surface of the bone in the form of a capsule. The character of the periosteum is different where ligaments and tendons attach to the bone. Collagen fibers from these structures extend directly, but at an angle, into the bone tissue, where they are continuous with the collagen fibers of the extracellular matrix of the bone tissue. These fibers are called Sharpey’s fbers. Bones that articulate with neighboring bones possess movable (synovial) joints.

1	Bones that articulate with neighboring bones possess movable (synovial) joints. Where a bone articulates with a neighboring bone, as in synovial joints, the contact areas of the two bones are referred to as articular surfaces. The articular surfaces are covered by hyaline cartilage, also called articular cartilage because of its location and function; articular cartilage is exposed to the joint cavity. This cartilage is not covered with perichondrium. The details of articular cartilage are discussed in Chapter 7 (page 203 and in Folder 8.1 (Clinical Correlation: Joint Diseases). Bone cavities are lined by endosteum, a layer of connective tissue cells that contains osteoprogenitor cells.

1	Bone cavities are lined by endosteum, a layer of connective tissue cells that contains osteoprogenitor cells. The lining tissue of both the compact bone facing the marrow cavity and the trabeculae of spongy bone within the cavity is referred to as endosteum. The endosteum is often only one cell layer thick and consists of osteoprogenitor cells that can differentiate into bone matrix–secreting cells, the osteoblasts, and bone-lining cells. Osteoprogenitor cells and bone-lining cells are difficult to distinguish at the microscopic level. They are both flattened in shape with elongated nuclei and indistinguishable cytoplasmic features. Because of their location within the bone cavities they are frequently called endosteal cells. The marrow cavity and the spaces in spongy bone contain bone marrow.

1	The marrow cavity and the spaces in spongy bone contain bone marrow. Red bone marrow consists of blood cells in different stages of development and a network of reticular cells and fibers that serve as a supporting framework for the developing blood cells and vessels. As an individual grows, the amount of red marrow does not increase proportionately with bone growth. In later stages of growth and in adults, when the rate of blood cell formation has diminished, the tissue in the marrow cavity consists mostly of fat cells; it is then called yellow marrow. In response to appropriate stimuli, such as extreme blood loss, yellow marrow can revert to red marrow. In the adult, red marrow is normally restricted to the spaces of spongy bone in a few locations such as the sternum and the iliac crest. Diagnostic bone marrow samples and marrow for transplantation are obtained from these sites. Mature bone is composed of structural units called osteons (Haversian systems).

1	Mature bone is composed of structural units called osteons (Haversian systems). Mature bone is largely composed of cylindrical units called osteons or Haversian systems (Fig. 8.3). The osteons consist of concentric lamellae (sing., lamella) of bone matrix surrounding a central canal, the osteonal (Haversian) canal, which contains the vascular and nerve supply of the osteon. Canaliculi containing the processes of osteocytes are generally arranged in a radial pattern with respect to the canal (Plate 11, page 244). The system of canaliculi that opens to the osteonal canal also serves for the passage of substances between the osteocytes and blood vessels. Between the osteons are remnants of previous concentric lamellae  FOLDER 8.1 Clinical Correlation: Joint Diseases

1	Inflammation of the joints or arthritis can be caused by many factors and can produce varying degrees of pain and disability, from the pathologic response of articular carti-lage to injury. Simple trauma to a joint by a single incident or by re-peated insult can so damage the articular cartilage that it calcifies and begins to be replaced by bone. This process can lead to ankylosis (i.e., bony fusion in the joint and subsequent loss of motion). The foot and knee joints of run-ners and football players and hand and finger joints of stringed instrument players are especially vulnerable to this condition. Immune responses or infectious processes that localize in joints, as in rheumatoid arthritis or tuberculosis, can also damage the articular cartilages, producing both severe joint pain and gradual ankylosis. Surgery that replaces the damaged joint with a prosthetic joint can often relieve the pain and restore joint motion in seriously debilitated individ-uals. Another common cause of

1	gradual ankylosis. Surgery that replaces the damaged joint with a prosthetic joint can often relieve the pain and restore joint motion in seriously debilitated individ-uals. Another common cause of damage to articular carti-lages is the deposition of crystals of uric acid in the joints, particularly those of the toes and fingers. This condition is known as gouty arthritis or, more simply, gout. Gout has become more common because of the widespread use of thiazide diuretics in the treatment of hypertension. In genet-ically predisposed individuals, gout is the most common side effect of these drugs. Gout causes severe, unbear-able pain because of the sharp crystals in the joint. The irritation also causes the formation of calcareous deposits that deform the joint and limit its motion.

1	lamellae of bone

1	FIGURE 8.3 • Diagram of a section of compact bone removed from the shaft of a long bone. The concentric lamellae and the Haversian canal that they surround constitute an osteon (Haversian system). One of the Haversian systems in this diagram is drawn as an elongated cylindrical structure rising above the plane of the bone section. It consists of several concentric lamellae that have been partially removed to show the perpendicular orientation of collagen fibers in adjacent layers. Interstitial lamellae result from bone remodeling and formation of new Haversian systems. The inner and outer surfaces of the compact bone in this diagram show additional lamellae—the outer and inner circumferential lamellae—arranged in broad layers. The inner circumferential lamella is covered by a thin layer of endosteum that faces the marrow cavity, similar to the outer surface of the bone, which is covered by periosteum. Branches of nutritional arteries accompanied by small veins are shown within the

1	of endosteum that faces the marrow cavity, similar to the outer surface of the bone, which is covered by periosteum. Branches of nutritional arteries accompanied by small veins are shown within the Haversian and Volkmann’s canals. These arteries also supply the periosteum, endosteum, and bone marrow.

1	called interstitial lamellae (see Fig. 8.3). Because of this organization, mature bone is also called lamellar bone. The long axis of an osteon is usually parallel to the long axis of the bone. The collagen fibers in the concentric lamellae in an osteon are laid down parallel to one another in any given lamella but in different directions in adjacent lamellae. This arrangement gives the cut surface of lamellar bone the appearance of plywood and imparts great strength to the osteon.

1	Lamellar bone is also found at sites other than the osteon. Circumferential lamellae follow the entire inner and outer circumferences of the shaft of a long bone, appearing much like the growth rings of a tree (see Fig. 8.3). Perforating canals (Volkmann’s canals) are channels in lamellar bone through which blood vessels and nerves travel from the periosteal and endosteal surfaces to reach the osteonal canal; they also connect osteonal canals to one another (Plate 11, page 244). They usually run at approximately right angles to the long axis of the osteons and of the bone (see Fig. 8.3). Volkmann’s canals are not surrounded by concentric lamellae, a key feature in their histologic identification. Mature spongy bone is structurally similar to mature compact bone.

1	Mature spongy bone is structurally similar to mature compact bone. Mature spongy bone is similar in structure to mature compact bone except that the tissue is arranged as trabeculae or spicules; numerous interconnecting marrow spaces of various sizes are present between the bone tissue. The matrix of the bone is lamellated. Arteries that enter the marrow cavity through the nutrient foramina supply blood to the shaft of long bones. Nutrient foramina are openings in the bone through which blood vessels pass to reach the marrow. The greatest numbers of nutrient foramina are found in the diaphysis and epiphysis (Fig. 8.4). Metaphyseal arteries supplement the blood supply to the bone. Veins that exit through the nutrient foramina

1	FIGURE 8.4 • Diagram showing the blood supply of an adult long bone. The nutrient artery and the epiphyseal arteries enter the bone through nutrient foramina. These openings in the bone arise developmentally as the pathways of the principal vessels of periosteal buds. Metaphyseal arteries arise from periosteal vessels that become incorporated into the metaphysis as the bone grows in diameter. or through the bone tissue of the shaft and continue out through the periosteum drain the blood from bone. The nutrient arteries that supply the diaphysis and epiphysis arise developmentally as the principal vessels of the periosteal buds. The metaphyseal arteries, in contrast, arise developmentally from periosteal vessels that become incorporated into the metaphysis during the growth process (i.e., through the widening of the bone). The blood supply to bone tissue is essentially centrifugal.

1	The blood supply to bone tissue is essentially centrifugal. The blood that nourishes bone tissue moves from the marrow cavity into and through the bone tissue and out via periosteal veins; thus, its flow is in a centrifugal direction. With respect to nourishment of the bone tissue itself, Volkmann’s canals provide the major route of entry for vessels to pass through the compact bone. The smaller blood vessels enter the Haversian canals, which contain a single arteriole and a venule or a single capillary. A lesser blood supply to the outermost portions of the compact bone is provided by the branches of periosteal arteries (see Fig. 8.4). Bone tissue lacks lymphatic vessels; lymphatic drainage occurs only from the periosteum.

1	Bone tissue initially formed in the skeleton of a developing fetus is called immature bone. It differs from mature bone in several respects (Fig. 8.5):  Immature bone does not display an organized lamellated appearance. On the basis of its collagen fiber arrangement, such bone is designated nonlamellar. Nonlamellar bone is also referred to as bundle bone or woven bone because of the interlacing arrangement of the collagen fibers.

1	osteoclast resorption canal osteocyte osteocyte interstitial lamellae MATURE BONE IMMATURE BONE osteon concentric lamella a b FIGURE 8.5 • Diagram of immature and mature bone. Immature bone does not display an organized lamellar appearance because of the interlacing arrangement of the collagen fibers. The cells tend to be randomly arranged, whereas the cells in mature bone are organized in a circular fashion that reflects the lamellar structure of the Haversian system. Resorption canals in mature bone have their long axes in the same direction as the Haversian canals. than does mature bone.  The cells in immature bone tend to be randomly arranged, whereas cells in mature bone are usually arranged with their long axes in the same direction as the lamellae.  The matrix of immature bone has more ground substance than does the matrix of mature bone. The matrix in im mature bone stains more intensely with hematoxylin, whereas the matrix of mature bone stains more intensely with eosin.

1	Although not evident in typical histologic sections (Fig. 8.6), immature bone is not heavily mineralized when it is initially formed, whereas mature bone undergoes prolonged secondary mineralization. The secondary mineralization of mature bone is evident in microradiographs of ground sections that show younger Haversian systems to be less mineralized than older Haversian systems (see Fig. 8.22). Immature bone forms more rapidly than mature bone. Although mature bone is clearly the major bone type in the adult and immature bone is the major bone type in the developing fetus, areas of immature bone are present in adults, especially where bone is being remodeled. Areas of immature bone are common in the alveolar sockets of the adult oral cavity and where tendons insert into bones. It is this immature bone in the alveolar sockets that makes it possible to make orthodontic corrections even in adults.

1	As noted previously, five designated cell types are associated with bone tissue: osteoprogenitor cells, osteoblasts, osteocytes, bone-lining cells, and osteoclasts. With the exception of the osteoclast, each of these cells may be regarded as a bone). In contrast, the osteoclast originates from a different differentiated form of the same basic cell type (Fig. 8.7). Each cell line and is responsible for bone resorption, an activity as-undergoes transformation from a less mature form to a more sociated with bone remodeling. mature form in relation to functional activity (growth of

1	FIGURE 8.6 • Photomicrographs of decalcified immature and mature bone. a. Decalcified immature bone, stained with H&E, showing the relationship of cells to the extracellular matrix. The immature bone has more cells, and the matrix is not layered in osteonal arrays. 130. b. This cross section of decalcified mature compact bone stained with H&E shows several osteons (O) with concentric lamellae. The Haversian canals contain blood vessels and connective tissue. Osteocytes undergo considerable shrinkage during routine slide preparation, revealing empty lacunae with a small nucleus attached to their walls. Mature bone has fewer osteocytes per unit area than immature bone. Note the presence of interstitial lamellae between neighboring osteons. 160. active inactive granulocyte/monocyte osteoclasts osteoclast progenitor (GMP, CFU-GM) endosteal cells

1	active inactive granulocyte/monocyte osteoclasts osteoclast progenitor (GMP, CFU-GM) endosteal cells FIGURE 8.7 • Schematic drawing of cells associated with bone. All cells except osteoclasts originate from the mesenchymal stem cells, which differentiate into osteoprogenitor cells, osteoblasts, and finally osteocytes and bone-lining cells. Bone-lining cells on external bone surfaces are part of the periosteum, hence the term periosteal cells. Bone-lining cells on internal bone surfaces are frequently called endosteal cells. Note that osteoprogenitor cells and bone-lining cells have a similar microscopic appearance and are often difficult to distinguish from each other. Osteoclasts originate from hemopoietic progenitor cells, which differentiate into boneresorbing cells. The specific details of osteoclast differentiation are illustrated in Figure 8.13. The osteoprogenitor cell is derived from mesenchymal stem cells.

1	The osteoprogenitor cell is derived from mesenchymal stem cells. Osteogenesis, the process of new bone formation, is essential to normal bone function. It requires a population of renewable osteoprogenitor cells (osteoblast precursor cells) that are responsive to molecular stimuli that transform them into bone-forming cells. Osteoprogenitor cells are derived from mesenchymal stem cells in the bone marrow that have the potential to differentiate into many different cell types including fibroblasts, osteoblasts, adipocytes, chondrocytes, and muscle cells. The key factor that triggers differentiation of osteoprogenitor cells is a transcription factor called core binding factor alpha-1 (CBFA1). This protein prompts the expression of genes that are characteristic of the phenotype of the osteoblast. As noted on page 219, bone morphogenic proteins also play a role in the differentiation of osteoblasts.

1	The osteoprogenitor cell is a resting cell that can differentiate into an osteoblast and secrete bone matrix.

1	Osteoprogenitor cells are found on the external and internal surfaces of bones and may also reside in the microvasculature supplying bone. Morphologically, they comprise the periosteal cells that form the innermost layer of the periosteum and the endosteal cells that line the marrow cavities, the osteonal (Haversian) canals, and the perforating (Volkmann’s) canals. In growing bones, osteoprogenitor cells appear as flattened or squamous cells with lightly staining, elongate, or ovoid nuclei and inconspicuous acidophilic or slightly basophilic cytoplasm. Electron micrographs reveal profiles of rough-surfaced endoplasmic reticulum (rER) and free ribosomes as well as a small Golgi apparatus and other organelles. The morphology of the osteoprogenitor cell is consistent with the finding that its stimulation leads to differentiation into a more active secretory cell, the osteoblast. The osteoblast is the differentiated bone-forming cell that secretes bone matrix.

1	The osteoblast is the differentiated bone-forming cell that secretes bone matrix. Like its close relatives, the fibroblast and the chondroblast, the osteoblast is a versatile secretory cell that retains the ability to divide. It secretes both type I collagen (which constitutes 90% of the protein in bone) and bone matrix proteins (BMPs), which constitute the initial unmineralized bone, or osteoid. The bone matrix proteins produced by the osteoblast include calcium-binding proteins such as osteocalcin and osteonectin; multiadhesive glycoproteins such as bone sialoproteins I and II, osteopontin, and thrombospondin, various proteoglycans and their aggregates, and alkaline phosphatase (ALP). Circulating levels of ALP and osteocalcin are used clinically as markers of osteoblast activity.

1	The osteoblast is also responsible for the calcification of bone matrix. The calcification process appears to be initiated by the osteoblast through the secretion into the matrix of small, 50to 250-nm, membrane-limited matrix vesicles. The vesicles are rich in ALP and are actively secreted only during the period in which the cell produces the bone matrix. The role of these vesicles is discussed later in this chapter (page 241). Osteoblasts are recognized in the light microscope by their cuboidal or polygonal shape and their aggregation into a single layer of cells lying in apposition to the forming bone (Fig. 8.8). The newly deposited matrix is not immediately calcified. It stains lightly or not at all compared with the mature mineralized matrix, which stains heavily with eosin. Because of this staining property of the newly formed matrix, osteoblasts appear to be separated from the bone by a light band. This band represents the osteoid, the nonmineralized matrix.

1	FIGURE 8.8 • Photomicrograph of a growing bone spicule stained with Mallory-Azan. Osteocytes are embedded within the bone matrix of the spicule, which is stained dark blue. These cells are metabolically active, laying down the unmineralized bone matrix (osteoid). A number of osteoblasts are aligned on the right side of the spicule. Between these cells and the calcified bone spicule is a thin, light-blue–stained layer of osteoid. This is the uncalcified matrix material produced by the osteoblasts. One of the cells (arrow) has virtually surrounded itself by its osteoid product; thus it can now be called an osteocyte. On the left side of the spicule, the nongrowing part, are inactive osteoblasts. The cells exhibit flattened nuclei and attenuated cytoplasm. 550.

1	The cytoplasm of the osteoblast is markedly basophilic, and the Golgi apparatus, because of its size, is sometimes observed as a clear area adjacent to the nucleus. Small, periodic acid–Schiff (PAS)-positive granules are observed in the cytoplasm, and a strong ALP reaction associated with the cell membrane can be detected by appropriate histochemical staining. In contrast to the secreting osteoblasts found in active matrix deposition, inactive osteoblasts are flat or attenuated cells that cover the bone surface. These cells resemble osteoprogenitor cells. Osteoblasts respond to mechanical stimuli to mediate the changes in bone growth and bone remodeling. As osteoid deposition occurs, the osteoblast is eventually surrounded by osteoid matrix and then becomes an osteocyte. Osteoblast processes communicate with other osteoblasts and with osteocytes by gap junctions.

1	Osteoblast processes communicate with other osteoblasts and with osteocytes by gap junctions. At the electron microscope level, osteoblasts exhibit thin cytoplasmic processes that penetrate the adjacent osteoid produced by the cell and are joined to similar processes of adjacent osteocytes by gap junctions. This early establishment of junctions between an osteoblast and adjacent osteocytes (as well as between adjacent osteoblasts) allows neighboring cells within the bone tissue to communicate. The osteoblast cytoplasm is characterized by abundant rER and free ribosomes (Fig. 8.9). These features are consistent with its basophilia observed in the light microscope as well as with its role in the production of collagen and proteoglycans

1	FIGURE 8.9 • Electron micrograph showing active bone formation. This electron micrograph is similar to the growing surface of the bone spicule in the preceding light micrograph (Fig. 8.8). The marrow cavity (M) with its developing blood cells is seen in the lower right corner. Osteoprogenitor cells (Opc) are evident between the marrow and the osteoblasts (Ob). They exhibit elongated or ovoid nuclei. The osteoblasts are aligned along the growing portion of the bone, which is covered by a layer of osteoid (Os). In this same region, one of the cells (upper right corner) embedded within the osteoid exhibits a small process (arrow). This cell, because of its location within the osteoid, can now be called an osteocyte (Oc). The remainder of the micrograph (upper left) is composed of calcified bone matrix (CB). Within the matrix are canaliculi (C) containing osteocyte processes. The boundary between two adjacent lamellae (L) of previously formed bone is evident as an irregular dark line.

1	bone matrix (CB). Within the matrix are canaliculi (C) containing osteocyte processes. The boundary between two adjacent lamellae (L) of previously formed bone is evident as an irregular dark line. 9,000.

1	for the extracellular matrix. The Golgi apparatus and surrounding regions of the cytoplasm contain numerous vesicles with a flocculent content that is presumed to consist of matrix precursors. These vesicles are the PAS-staining granules seen in light microscopy. The matrix vesicles, also produced by the osteoblast, appear to arise by a different pathway, originating as spherelike outgrowths that pinch off from the plasma membrane to become free in the matrix. Other cell organelles include numerous rod-shaped mitochondria and occasional dense bodies and lysosomes. The osteocyte is the mature bone cell enclosed by bone matrix that it previously secreted as an osteoblast.

1	The osteocyte is the mature bone cell enclosed by bone matrix that it previously secreted as an osteoblast. When completely surrounded by osteoid or bone matrix, the osteoblast is referred to as an osteocyte (see Fig. 8.8). Osteocytes are the cells responsible for maintaining the bone matrix. One of the roles of osteocytes is mechanotransduction, the process by which the osteocyte responds to mechanical forces applied to the bone. Different mechanical stimuli (e.g., weightlessness or increased mechanical loading) alter not only gene expression but also the cell’s apoptotic mechanism. Osteocytes can synthesize new matrix, as well as participate in matrix degradation. Such activities help to maintain calcium homeostasis. The death of osteocytes through trauma (e.g., a fracture), cell senescence, or apoptosis results in resorption of the bone matrix by osteoclast activity, followed by repair or remodeling of the bone tissue by osteoblast activity.

1	Each osteocyte occupies a space, or lacuna, that conforms to the shape of the cell. Osteocytes extend cytoplasmic processes through the canaliculi in the matrix to contact processes of neighboring osteocytes and bone-lining cells by means of gap junctions. Osteocytes can also communicate indirectly with distant osteoblasts, pericytes of blood vessels, and other bone cells through the expression of various signaling molecules such as nitric oxide and glutamate transporters. In hematoxylin and eosin (H&E)–stained sections, the canaliculi and the processes they contain are not discernible. In ground sections, the canaliculi are readily evident (Plate 11, page 244). Osteocytes are typically smaller than their precursors because of their reduced perinuclear cytoplasm. Often, in routinely prepared microscopic specimens, the cell is highly distorted by shrinkage and other artifacts that result from decalcifying the matrix before sectioning the bone. In such instances, the nucleus may be the

1	microscopic specimens, the cell is highly distorted by shrinkage and other artifacts that result from decalcifying the matrix before sectioning the bone. In such instances, the nucleus may be the only prominent feature. In wellpreserved specimens, osteocytes exhibit less cytoplasmic basophilia than osteoblasts, but little additional cytoplasmic detail can be seen (Plate 12, page 246).

1	Electron microscopy has revealed osteocytes in various functional states. Indeed, there is histologic and microradiologic evidence (i.e., enlarged lacunae and reduced radiodensity) that the osteocyte can modify the surrounding bone matrix. Three functional states, each with a characteristic morphology, have been described:  Quiescent osteocytes exhibit a paucity of rER and a markedly diminished Golgi apparatus (Fig. 8.10a). An osmiophilic lamina representing mature calcified matrix is seen in close apposition to the cell membrane. Formative osteocytes show evidence of matrix deposi tion and exhibit certain characteristics similar to those of osteoblasts. Thus, the rER and Golgi apparatus are more abundant, and there is evidence of osteoid in the pericel lular space within the lacuna (Fig. 8.10b).  Resorptive osteocytes, like formative osteocytes, con tain numerous profiles of endoplasmic reticulum and a well-developed Golgi apparatus. Moreover, lysosomes are conspicuous (Fig.

1	(Fig. 8.10b).  Resorptive osteocytes, like formative osteocytes, con tain numerous profiles of endoplasmic reticulum and a well-developed Golgi apparatus. Moreover, lysosomes are conspicuous (Fig. 8.10c).

1	The “resorptive” function of the osteocyte is not precisely defined and is supported mainly by the observation that the pericellular space is devoid of collagen fibrils and contains a flocculent material suggestive of a breakdown product. These observed changes could be explained by enzymatic degradation of collagen by osteocyte-secreted matrix metalloproteinases (MMPs). It has been shown under experimental conditions that a reduced load on bone initiates expression of MMP mRNA in the osteocyte. Degradation of bone by MMPs is called osteocytic osteolysis. The current concept of osteocytic osteolysis is that the lytic role of osteocytes is not related to bone matrix remodeling but functions to maintain blood calcium level. Bone-lining cells are derived from osteoblasts and cover bone that is not remodeling.

1	In sites where remodeling is not occurring, the bone surface is covered by a layer of flat cells with attenuated cytoplasm and a paucity of organelles beyond the perinuclear region (see Fig. 8.11a). These cells are designated simply as bone-lining cells. Bone-lining cells on external bone surfaces are called periosteal cells, and those lining internal bone surfaces are often called endosteal cells (see Fig. 8.7) Gap junctions are present where the bone-lining cell processes contact one another (Fig. 8.11b). Bone-lining cells represent a population of cells that are derived from osteoblasts. They are thought to function in the maintenance and nutritional support of the osteocytes embedded in the underlying bone matrix and regulate the movement of calcium and phosphate into and out of the bone. These suggested roles are based on the observation that the cell processes of bone-lining cells extend into the canalicular channels of the adjacent bone (see Fig. 8.11b) and communicate by means

1	These suggested roles are based on the observation that the cell processes of bone-lining cells extend into the canalicular channels of the adjacent bone (see Fig. 8.11b) and communicate by means of gap junctions with osteocytic processes. In these respects, bone-lining cells are somewhat comparable to osteocytes.

1	The osteoclast is responsible for bone resorption. Osteoclasts are large, multinucleated cells found at sites where bone is being removed. They rest directly on the bone tissue where resorption is taking place (Fig. 8.12). As a result of osteoclast activity, a shallow bay called a resorption bay FIGURE 8.10 • Electron micrographs of three different functional stages of an osteocyte. a. Relatively quiescent osteocyte that contains only a few profiles of rER and a few mitochondria (M). The cell virtually fills the lacuna that it occupies; the arrows indicate where cytoplasmic processes extend into canaliculi. Hydroxyapatite crystals have been lost from the matrix, which is ordinarily mineralized (MM), but some hydroxyapatite crystals fill the pericellular space. The hydroxyapatite crystals obscure the other substances within the pericellular space. The dark band marking the boundary of the lacuna is the osmiophilic lamina (OL). 25,000.

1	b. A formative osteocyte containing larger amounts of rER and a large Golgi apparatus (G). Of equal importance is the presence of a small amount of osteoid in the pericellular space within the lacuna. The osteoid shows profiles of collagen fibrils (arrows) not yet mineralized. The lacuna of a formative osteocyte is not bounded by an osmiophilic lamina. 25,000. c. A resorptive osteocyte containing a substantial amount of rER, a large Golgi apparatus, mitochondria (M), and lysosomes (L). The pericellular space is devoid of collagen fibrils and may contain some flocculent material. The lacuna containing a resorptive osteocyte is bounded by a less conspicuous osmiophilic lamina (OL). 25,000.

1	(Howship’s lacuna) can be observed in the bone directly under the osteoclast. The cell is conspicuous not only because of its large size but also because of its marked acidophilia. It also exhibits a strong histochemical reaction for acid phosphatase because of the numerous lysosomes that it contains. One of these enzymes, the 35-kilodalton iron-containing tartrate-resistant acid phosphatase (TRAP), is used clinically as a marker of osteoclast activity and differentiation. Osteoclasts are derived from the fusion of mononuclear hemopoietic progenitor cells under the influence of multiple cytokines.

1	Contrary to what was once thought, osteoclasts are not related to osteoblasts. They are derived from the fusion of mononuclear hemopoietic cells, namely, granulocyte/ macrophage progenitor cells (GMP, CFU-GM) that give rise to granulocyte and monocyte cell lineages (see Fig. 10.16). Osteoclast formation occurs in close association with stromal cells in bone marrow. These cells secrete essential cytokines for differentiation of both osteoclasts and macrophages from GMP progenitor cells, including monocyte colony-stimulating factor (M-CSF), TNF, and several interleukins. Initially, cells committed to become osteoclasts (osteoclast precursors) express two important transcription factors, c-fos and NFB; later, a receptor molecule called receptor activator of nuclear factor B (RANK) is expressed on their surface. The RANK receptor interacts with RANK ligand molecule (RANKL) produced and expressed on the stromal cell surface (Fig. 8.13). The RANK–RANKL signaling mechanism is essential for

1	on their surface. The RANK receptor interacts with RANK ligand molecule (RANKL) produced and expressed on the stromal cell surface (Fig. 8.13). The RANK–RANKL signaling mechanism is essential for osteoclast differentiation and maturation. Alternatively, during inﬂammation, activated T lymphocytes can produce both membrane-bound and soluble RANKL molecules. Therefore, inﬂammatory processes can stimulate osteoclast-mediated bone resorption. This pathway can be blocked by osteoprotegerin (OPG), which serves as a “decoy” receptor for RANKL. Lack of available ligand affects the RANK–RANKL signaling pathway and acts as a potent inhibitor of osteoclast formation. OPG is produced mainly by osteoblasts and is regulated by many bone metabolic regulators, such as IL-1, TNF, TGF, vitamin D, and prostaglandin E2. Recent studies indicate

1	FIGURE 8.11 • Electron micrograph of bone-lining cells. a. The cytoplasm of a bone-lining cell located on the surface of a spicule of mature bone is very attenuated and contains small amounts of rER and free ribosomes. A gap junction is seen between the two adjacent bone-lining cells. In addition, cytoplasmic processes are clearly seen where they pass through the matrix of unmineralized bone (osteoid). A fat cell of the marrow is also present. 8,900. (Reprinted with permission from Miller SC, Bowman BM, Smith JM, Jee WS. Characterization of endosteal bone-lining cells from fatty marrow bone sites in adult beagles. Anat Rec 1980;198:163–173.) Inset. High-magnification photomicrograph of a similar bone spicule stained with H&E, included for orientation purposes. The bone-lining cells (endosteal cells) on the surface of the spicule are indicated by the arrows. 350. b. Electron micrograph of the cytoplasm of two bone-lining cells observed at higher magnification. The gap junction is

1	cells) on the surface of the spicule are indicated by the arrows. 350. b. Electron micrograph of the cytoplasm of two bone-lining cells observed at higher magnification. The gap junction is clearly seen where the two cells are in apposition. The edge of a fat cell is seen at the top of the electron micrograph; its lipid, thin rim of cytoplasm, plasma membrane, and external lamina are also evident. 27,000.

1	that substances that promote osteoclast differentiation and bone resorption act through the OPG/RANKL system in the bone marrow. Both OPG and RANKL are detected in a free form in the blood, and their concentrations can be measured for diagnostic purposes and to monitor therapy of many bone diseases. Newly formed osteoclasts undergo an activation process to become bone-resorbing cells.

1	Newly formed osteoclasts undergo an activation process to become bone-resorbing cells. The newly formed osteoclast must be activated to become a bone-resorbing cell. During this process, it becomes highly polarized. When actively resorbing bone, osteoclasts exhibit three specialized regions:  The ruffled border is the part of the cell in direct contact with bone. It contains numerous deep plasma membrane infoldings forming microvillous-type structures responsible for increasing surface area for the exocytosis of hydrolytic enzymes and secretion of protons by ATP–dependent proton pumps, as well as endocytosis of degradation products and bone debris. The ruffled border stains less intensely than the remainder of the cell and often appears as a light band adjacent to the bone at

1	FIGURE 8.12 • Photomicrograph of an osteoclast on a bone spicule. This Mallory-stained specimen shows a spicule made of calcified cartilage (stained light blue) and a covering of bone tissue (stained dark blue). An osteoclast on the left side of the spicule has resorbed bone tissue and lies in a depression (Howship’s lacuna) in the spicule. The light band between the osteoclast and the bone spicule corresponds to the ruffled border of the osteoclast. The arrows on the nongrowing surface indicate cytoplasm of inactive bone-lining cells (osteoprogenitor cells). In contrast, bone is being deposited on the opposite side of the spicule, as evidenced by the presence of osteoblasts on this surface and newly formed osteocytes just below the surface of the spicule. 550. common myeloid progenitor (CMP, CFU-GEMM) granulocyte/ monocyte progenitors (GMP, CFU-GM)

1	FIGURE 8.13 • The origin of osteoclasts. Osteoclasts are derived from fusion of granulocyte/monocyte progenitor cells (GMP, CFUGM), which originate from multipotential common myeloid progenitor cells (CMP, CFU-GEMM). GMP cells also give rise to the granulocyte and monocyte cell lineages such as neutrophil progenitor (NoP, CFU-G) and monocyte progenitor (MoP, CFU-M) cells. Osteoclast formation occurs in close association with stromal cells in bone marrow, which secrete monocyte colony-stimulating factor (M-CSF ), tumor necrosis factor (TNF ), and several interleukins (ILs). Osteoclast precursors express c-fos and NFB, and receptor molecules called RANK (receptor activator of nuclear factor B). The signal generated by the interaction of the RANK receptor with the RANK ligand (RANKL) molecule is essential for osteoclast differentiation and maturation. During inflammation, T lymphocytes produce both soluble and membrane-bound RANKL molecules, which increase bone resorption. These

1	molecule is essential for osteoclast differentiation and maturation. During inflammation, T lymphocytes produce both soluble and membrane-bound RANKL molecules, which increase bone resorption. These pathways can be blocked by osteoprotegerin (OPG). Note that activated T lymphocytes can stimulate formation of osteoclasts by producing both membrane-bound and soluble RANKL molecules.

1	FIGURE 8.14 • Electron micrograph of an osteoclast. This micrograph shows a segment of bone surface (B) and a portion of an osteoclast that is in apposition to the partially digested bone. The resorption front (RF) of the osteoclast possesses numerous infoldings of the plasma membrane. When viewed in the light microscope, these infoldings are evident as the ruffled border. When the plane of section is parallel to the infoldings (asterisks), a broad, nonspecialized expanse of cytoplasm is seen. The cytoplasm of the osteoclast contains numerous mitochondria (M), lysosomes, and Golgi apparatus, all of which are functionally linked with the resorption and degradation of the bone matrix. In the upper part of the figure, some collagen fibrils are evident; the arrows indicate where 68-nm cross-banding is visible. 10,000.

1	the resorption site (see Fig. 8.12). At the electron microscopic level, hydroxyapatite crystals from the bone substance are observed between the processes of the ruffled border (Fig. 8.14). Internal to the ruffled border and in close proximity are numerous mitochondria and lysosomes. The nuclei are typically located in the part of the cell more removed from the bone surface. In this same region are profiles of rER, multiple stacks of Golgi apparatus, and many vesicles.

1	 The clear zone (sealing zone) is a ringlike perimeter of cytoplasm adjacent to the ruffled border that demarcates the bone area being resorbed. Essentially, the clear zone is a compartment at the site of the ruffled border where resorption and degradation of the matrix occurs. It contains abundant actin filaments but essentially lacks other organelles. The actin filaments are arranged in a ringlike structure surrounded on both sides by actin-binding proteins such as vinculin and talin (Fig. 8.15). The plasma membrane at the site of the clear zone contains cell and extracellular matrix adhesion molecules that are responsible for providing a tight seal between the plasma membrane and mineralized matrix of the bone. Several classes of integrin extracellular receptors (i.e., v 3 vitronectin receptor, 2 1 collagen receptor, or v 1 receptor) help maintain the seal.

1	 The basolateral region functions in the exocytosis of digested material (see Fig. 8.15). Transport vesicles containing degraded bone material endocytosed at the ruffled border fuse here with the cell membrane to release their contents. TRAP has been found within these vesicles, suggesting its role in the fragmentation of endocytosed material. Osteoclasts resorb bone tissue by releasing protons and lysosomal hydrolases into the constricted microenvironment of the extracellular space. Some, if not most, of the vesicles in the osteoclast are lysosomes. Their contents are released into the extracellular space in the clefts between the cytoplasmic processes of the ruffled border, a clear example of lysosomal enzymes functioning outside the cell. Once liberated, these hydrolytic enzymes, which include cathepsin K (a cysteine protease) and matrix metalloproteinases, degrade collagen and other proteins of the bone matrix.

1	Before digestion can occur, however, the bone matrix must be decalcified through acidification of the bony surface, which initiates dissolution of the mineral matrix. The cytoplasm of the osteoclast contains carbonic anhydrase II, which produces carbonic acid (H2CO3) from carbon dioxide and water. Subsequently, the carbonic acid dissociates to bicarbonate (HCO3) and a proton (H ). With the help of ATP-dependent proton pumps, protons are transported through the ruffled border, generating a low pH (4 to 5) in the microenvironment of the resorption bay. This local acidic environment created in the extracellular space between the bone and the osteoclast is protected by the clear zone. Chloride channels coupled with proton pumps facilitate the electroneutrality of the ruffled border membrane (see Fig. 8.15). Excess bicarbonate is removed by passive exchange with chloride ions via chloride–carbonate protein exchangers located at the basolateral membrane.

1	The acidic environment initiates the degradation of the mineral component of bone (composed primarily of hydroxyapatites) to calcium ions, soluble inorganic phosphates, and water. When resorption of designated bone tissue is completed, osteoclasts undergo apoptosis. Recent studies indicate that many drugs used to inhibit bone exocytosis of digested material lysosomes actin filaments vinculin, talin v 3 integrin receptors carbonic anhydrase chloride channel clear zone ruffled border ATP dependent proton pump Cl HCO3 HCO3 Cl H H CO2 H2O H2CO3 K cathepsin matrixmetalloproteinases

1	FIGURE 8.15 • Schematic drawing of an osteoclast. This drawing shows the structure of the osteoclasts and its three regions: the ruffled border, clear zone, and basolateral region. Note that the clear zone contains abundant actin filaments arranged in a ringlike structure surrounded on both sides by actin-binding proteins such as vinculin and talin. The plasma membrane at the site of the clear zone contains cell-to–extracellular matrix adhesion molecules (integrin receptors) that provide a tight seal between the plasma membrane and mineralized matrix of the bone. The pathways for proton and chloride transport are described in the text. resorption in osteoporosis (i.e., bisphosphonates and estrogens) promote osteoclast apoptosis (Folder 8.2). The phagocytotic function of osteoclasts is regulated by many factors.

1	resorption in osteoporosis (i.e., bisphosphonates and estrogens) promote osteoclast apoptosis (Folder 8.2). The phagocytotic function of osteoclasts is regulated by many factors. Numerous coated pits and coated vesicles are also present at the ruffled border, suggesting endocytotic activity. Osteoclasts are observed at sites where bone remodeling is in progress. (The process of remodeling is described in more detail shortly.) Thus, in sites where osteons are being altered or where a bone is undergoing change during the growth process, osteoclasts are relatively numerous.

1	An increase in parathyroid hormone (PTH) level promotes bone resorption and has a demonstrable effect on osteoclast activity, in addition to its effects on osteocytes, described previously. In contrast, calcitonin, secreted by parafollicular cells of the thyroid gland, has a counterbalancing effect, reducing osteoclast activity. PTH has also anabolic effect on bone either through a direct stimulatory effect on osteoblasts or indirectly through a mechanism that requires it to increase activity of osteoclasts. For example, PTH might stimulate bone formation directly by increasing the local production of IGF I or other bone-stimulating growth factors.

1	Other molecules that play an important role in regulating osteoclast activity include cathepsin K, carbonic anhydrase II, and proteins encoding the proton pump (TCIRG1). Deficiency of these proteins causes osteopetrosis, a congenital disease characterized by increased bone density and defective osteoclast function. In individuals with osteopetrosis, osteoclasts do not function properly, which causes bones to appear dense on X-ray; however, they are actually very fragile and break easily. The development of a bone is traditionally classified as endochondral or intramembranous.

1	The development of a bone is traditionally classified as endochondral or intramembranous. The distinction between endochondral and intramembranous formation rests on whether a cartilage model serves as the precursor of the bone (endochondral ossification) or whether the bone is formed by a simpler method, without the intervention of a cartilage precursor (intramembranous ossification). The bones of the extremities and those parts of the axial skeleton that bear weight (e.g., vertebrae) develop by endochondral ossification. The flat bones of the skull and face, the mandible, and the clavicle develop by intramembranous ossification.

1	The existence of two distinct types of ossification does not imply that existing bone is either membrane bone or endochondral bone. These names refer only to the mechanism by which a bone is initially formed. Because of the remodeling that occurs later, the initial bone tissue laid down by endochondral formation or by intramembranous formation is soon replaced. The replacement bone is established on the preexisting bone by appositional growth and is identical in both cases. Although the long bones are classified as being formed by endochondral formation, their continued growth involves the histogenesis of both endochondral and  FOLDER 8.2 Clinical Correlation: Osteoporosis

1	Osteoporosis, which literally means porous bone, is the most commonly occurring bone disease characterized by progressive loss of normal bone density accompanied by the deterioration of its microarchitecture. It is caused by an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone deposition, resulting in de-creased bone mass, enhanced bone fragility, and in-creased risk of fracture. In healthy individuals, osteoclast activity is primarily regulated by PTH and to a lesser degree by IL-1 and TNF. In addition, differentiation of osteoclast precursors is under the influence of M-CSF and IL-6. Fe-male hormones known as estrogens (especially estradiol) inhibit formation of these cytokines, therefore limiting the activity of osteoclasts. In postmenopausal women in whom estrogen levels are reduced, secretion of these cytokines is increased, resulting in enhanced activity of osteoclasts leading to intensified bone resorption. Osteoporosis is a disease that affects

1	estrogen levels are reduced, secretion of these cytokines is increased, resulting in enhanced activity of osteoclasts leading to intensified bone resorption. Osteoporosis is a disease that affects an estimated 75 million people in the United States, Europe, and Japan, including one-third of postmenopausal women and most of the elderly popula-tion. It results in more than 1.3 million fractures annually in the United States There are three general types of osteoporosis. 1. Type I primary osteoporosis occurs in post-menopausal women. Since this type appears at an earlier stage of life than type II, its long-term effect is usually more severe than osteoporosis that de-velops in the later years of life. 2. Type II primary osteoporosis occurs in elderly individuals in their seventh or eighth decade of life and is the leading cause of serious morbidity and functional loss in this age group. 3. Secondary osteoporosis develops as a result of drug therapy (i.e., corticosteroids) or disease

1	decade of life and is the leading cause of serious morbidity and functional loss in this age group. 3. Secondary osteoporosis develops as a result of drug therapy (i.e., corticosteroids) or disease pro-cesses that may affect bone remodeling, including malnutrition, prolonged immobilization, weightless-ness (i.e., with space travel), and metabolic bone dis-eases (i.e., hyperparathyroidism, metastatic cancers). Osteoporotic bone has normal histologic structure; however, there is less tissue mass (Fig. F8.2.1). This results in weakened bones that are more prone to fractures follow-ing even minor trauma. Femoral head and neck fractures (commonly known as hip fractures), wrist fractures, and compressed vertebrae fractures are common injuries that frequently disable and confine an elderly person to a wheelchair. Individuals suffering from fractures are at greater risk for death, not directly from the fracture, but from the complications of hospitalization because of immo-bilization and

1	to a wheelchair. Individuals suffering from fractures are at greater risk for death, not directly from the fracture, but from the complications of hospitalization because of immo-bilization and increased risk of pneumonia, pulmonary thrombosis, and embolism. Traditional treatment of individuals with osteoporosis in-cludes an improved diet with vitamin D and calcium sup-plementation and moderate exercise to help slow further bone loss. In addition to diet and exercise, pharmacologic therapy directed toward slowing down bone resorption is employed. Until recently, the treatment of choice in post-menopausal women with osteoporosis was hormone re-placement therapy with estrogen and progesterone. Estrogen is known to retard bone resorption, thereby di-minishing bone loss. The results of the Women’s Health Initiative have shown that hormone replacement therapy can indeed reduce the risk of fractures; however, it causes greater risk of adverse cardiovascular diseases as FIGURE F8.2.1 •

1	Women’s Health Initiative have shown that hormone replacement therapy can indeed reduce the risk of fractures; however, it causes greater risk of adverse cardiovascular diseases as FIGURE F8.2.1 • Scanning electron micrograph of trabecular bone. a. This image shows section from the trabecular bone obtained from a vertebral body of a healthy individual. b. This specimen was obtained from a vertebral body of elderly women showing extensive signs of osteoporosis. Compare the pattern of trabecular architecture in osteoporosis with normal vertebral bone. (Courtesy of Dr. Alan Boyd). aba b continued next page chapter 8 Bone BONE FORMATION 233 intramembranous bone, with the latter occurring through the activity of the periosteal (membrane) tissue.

1	In intramembranous ossification, bone is formed by differentiation of mesenchymal cells into osteoblasts.

1	The first evidence of intramembranous ossification is seen around the eighth week of gestation in humans. Some of the pale-staining, elongated mesenchymal cells within the mesenchyme migrate and aggregate in specific areas, the sites where bone is destined to form. This condensation of cells within the mesenchymal tissue initiates the process of intramembranous ossification (Fig. 8.16 and Plate 15, page 252). Mesenchymal cells then differentiate into osteoprogenitor cells expressing Cbfa1 transcription factor. This transcription factor is essential for osteoblast differentiation and expression of genes necessary for both intramembranous and endochondral ossification. As the process continues, the newly organized tissue at the presumptive bone site becomes more vascularized, and the aggregated mesenchymal cells become larger and rounded. The cytoplasm of osteoprogenitor cells changes from eosinophilic to basophilic, and a clear Golgi area becomes evident. These cytologic changes result

1	mesenchymal cells become larger and rounded. The cytoplasm of osteoprogenitor cells changes from eosinophilic to basophilic, and a clear Golgi area becomes evident. These cytologic changes result in the differentiated osteoblast, which then secretes the collagens (mainly type I collagen molecules), bone sialoproteins, osteocalcin, and other components of the bone matrix (osteoid). The osteoblasts within the bone matrix become increasingly separated from one another as the matrix is produced, but they remain attached by thin cytoplasmic processes. Because of the abundant collagen content, the bone matrix appears denser than the surrounding mesenchyme, in which the intercellular spaces reveal only delicate connective tissue fibers.

1	 FOLDER 8.3 Clinical Correlation: Nutritional Factors in Bone Formation

1	Both nutritional and hormonal factors affect the degree of bone mineralization. Calcium deficiency during growth causes rickets, a condition in which the bone matrix does not calcify normally. Rickets may be caused by insufficient amounts of dietary calcium or insufficient vitamin D (a steroid prohormone), which is needed for absorption of calcium by the intestines. An X-ray of the child with ad-vanced rickets presents classic radiological symptoms: bowed lower limbs (outward curve of long bones of the leg and thighs) and a deformed chest and skull (often having a distinctive “square” appearance). If rickets is not treated while children are still growing, then skeletal deformities and short stature may be permanent. In adults, the same nutritional or vitamin deficiency leads to osteomalacia. Although rickets and osteomalacia are no longer major health problems in populations where nutrition is adequate, it is among the most frequent childhood diseases in many developing countries. In

1	Although rickets and osteomalacia are no longer major health problems in populations where nutrition is adequate, it is among the most frequent childhood diseases in many developing countries. In addition to its influence on intestinal absorption of calcium, vitamin D is also needed for normal calcification. Other vitamins known to affect bone are vitamins A and C. Vitamin A deficiency suppresses endochondral growth of bone; vitamin A excess leads to fragility and subsequent frac-tures of long bones. Vitamin C is essential for synthesis of collagen, and its deficiency leads to scurvy. The matrix pro-duced in scurvy is not calcifiable. Another form of insufficient bone mineralization often seen in postmenopausal women is the condition known as osteoporosis (see Folder 8.2).

1	FIGURE 8.16 • Section of mandible developing by the process of intramembranous ossification. This photomicrograph shows a section from a developing mandible stained with H&E. In this relatively early stage of development, the mandible consists of bone spicules of various sizes and shapes. The bone spicules interconnect with each other and form trabeculae, providing the general shape of the developing bone (no cartilage model is present). The numerous osteoblasts responsible for this growing region of spicules are seen at the surface of the newly deposited bone. The older, calcified portion of spicules contains osteocytes surrounded by bone matrix. In the right portion of the figure, adjacent to the bone spicules, the connective tissue is very cellular and is developing into the early periosteum. 250. Newly formed bone matrix appears in histologic sections as small, irregularly shaped spicules and trabeculae.

1	Newly formed bone matrix appears in histologic sections as small, irregularly shaped spicules and trabeculae. With time, the matrix becomes calcified, and the interconnecting cytoplasmic processes of the bone-forming cells, now termed osteocytes, are contained within canaliculi. Concomitantly, more of the surrounding mesenchymal cells in the membrane proliferate, giving rise to a population of osteoprogenitor cells. Some of the osteoprogenitor cells come into apposition with the initially formed spicules, become osteoblasts, and add more matrix. By this process, called appositional growth, the spicules enlarge and become joined in a trabecular network with the general shape of the developing bone.

1	Through continued mitotic activity, the osteoprogenitor cells maintain their numbers and thus provide a constant source of osteoblasts for growth of the bone spicules. The new osteoblasts, in turn, lay down bone matrix in successive layers, giving rise to woven bone. This immature bone, discussed on page 223, is characterized internally by interconnecting spaces occupied by connective tissue and blood vessels. Bone tissue formed by the process just described is referred to as membrane bone or intramembranous bone. Endochondral ossification also begins with the proliferation and aggregation of mesenchymal cells at the site of the future bone. Under the influence of different fibroblastic growth factors (FGFs) and bone morphogenic proteins (BMPs) (see page 219), the mesenchymal cells initially express type II collagen and differentiate into chondroblasts that, in turn, produce cartilage matrix. Initially, a hyaline cartilage model with the general shape of the bone is formed.

1	Initially, a hyaline cartilage model with the general shape of the bone is formed. Once established, the cartilage model (a miniature version of the future definitive bone) grows by interstitial and appositional growth (Plate 13, page 248). The increase in the length of the cartilage model is attributed to interstitial growth. The increase in its width is largely the result of the addition of cartilage matrix produced by new chondrocytes that differentiate from the chondrogenic layer of the perichondrium surrounding the cartilage mass. Illustrations 1 of Figure 8.17 show an early cartilage model. The first sign of ossification is the appearance of a cuff of bone around the cartilage model.

1	At this stage, the perichondrial cells in the midregion of the cartilage model no longer give rise to chondrocytes. Instead, bone-forming cells or osteoblasts are produced. Thus, the connective tissue surrounding this portion of the cartilage is no longer functionally a perichondrium; rather, because of its altered role, it is now called periosteum. Moreover, because the cells within this layer are differentiating into osteoblasts, an osteogenic layer can now be identified within the periosteum. Because of these changes, a layer of bone is formed around the cartilage model (Plate 13, page 248). This bone can be classified as either periosteal bone, because of its location, or intramembranous bone, because of its method of development. In the case of a long bone, a distinctive cuff of periosteal bone, the bony collar, is established around the cartilage model in the diaphyseal portion of the developing bone. The bony collar is shown in illustration 2 of Figure 8.17.

1	With the establishment of the periosteal bony collar, the chondrocytes in the midregion of the cartilage model become hypertrophic. As the chondrocytes enlarge, their surrounding cartilage matrix is resorbed, forming thin irregular cartilage plates between the hypertrophic cells. The hypertrophic cells begin to synthesize alkaline phosphatase; concomitantly, the surrounding cartilage matrix undergoes calcification (see illustration 3 of Fig. 8.17). The calcification of the cartilage matrix should not be confused with mineralization that occurs in bone tissue.

1	FIGURE 8.17 • Schematic diagram of developing long bone. Illustrations 1 to 10 depict longitudinal sections through the long bone. The process begins with the formation of a cartilage model (1); next, a periosteal (perichondrial) collar of bone forms around the diaphysis (shaft) of the cartilage model (2); then, the cartilaginous matrix in the shaft begins to calcify (3). Blood vessels and connective tissue cells then erode and invade the calcified cartilage (4), creating a primitive marrow cavity in which remnant spicules of calcified cartilage remain at the two ends of the cavity. As a primary center of ossification develops, the endochondral bone is formed on spicules of calcified cartilage. The bone at the ends of the developing marrow cavity constitutes the metaphysis. Periosteal bone continues to form (5); the periosteal bone is formed as the result of intramembranous ossification. It can be recognized histologically because it is not accompanied by local cartilage erosion, nor

1	continues to form (5); the periosteal bone is formed as the result of intramembranous ossification. It can be recognized histologically because it is not accompanied by local cartilage erosion, nor is the bone deposited on spicules of calcified cartilage. Blood vessels and perivascular cells invade the proximal epiphyseal cartilage (6), and a secondary center of ossification is established in the proximal epiphysis (7). A similar epiphyseal (secondary) ossification center forms at the distal end of the bone (8), and an epiphyseal cartilage is thus formed between each epiphysis and the diaphysis. With continued growth of the long bone, the distal epiphyseal cartilage disappears (9), and finally, with cessation of growth, the proximal epiphyseal cartilage disappears (10). The metaphysis then becomes continuous with the epiphysis. Epiphyseal lines remain where the epiphyseal plate last existed.

1	The calcified cartilage matrix inhibits diffusion of nutrients, causing the death of the chondrocytes in the cartilage model. With the death of the chondrocytes, much of the matrix breaks down, and neighboring lacunae become confluent, producing an increasingly large cavity. While these events are occurring, one or several blood vessels grow through the thin diaphyseal bony collar to vascularize the cavity (see illustration 4 of Fig. 8.17). Mesenchymal stem cells migrate into the cavity along the growing blood vessels.

1	Mesenchymal stem cells residing in the developing periosteum migrate along the penetrating blood vessels and differentiate into osteoprogenitor cells in the bone marrow cavity. Hemopoietic stem cells (HSCs) also gain access to the cavity via the new vasculature, leaving the circulation to give rise to the marrow including all the blood cell lineages. As the calcified cartilage breaks down and is partially removed, some remains as irregular spicules. When the osteoprogenitor cells come in apposition to the remaining calcified cartilage spicules, they become osteoblasts and begin to lay down bone matrix (osteoid) on the spicule framework. Thus, the bone formed in this manner may be described as endochondral bone. This first site where bone begins to form in the diaphysis of a long bone is called the primary ossification center (see illustration 5 of Fig. 8.17). The combination of bone, which is initially only a thin layer, and the underlying calcified cartilage is described as a mixed

1	called the primary ossification center (see illustration 5 of Fig. 8.17). The combination of bone, which is initially only a thin layer, and the underlying calcified cartilage is described as a mixed spicule.

1	Histologically, mixed spicules can be recognized by their staining characteristics. Calcified cartilage tends to be basophilic, whereas bone is distinctly eosinophilic. With the Mallory stain, bone stains a deep blue, and calcified cartilage stains light blue (Fig. 8.18). Also, calcified cartilage no longer contains cells, whereas the newly produced bone may reveal osteocytes in the bone matrix. Such spicules persist for a short time before the calcified cartilage component is removed. The remaining bone component of the spicule may continue to grow by appositional growth, thus becoming larger and stronger, or it may undergo resorption as new spicules are formed. Growth of Endochondral Bone Endochondral bone growth begins in the second trimester of fetal life and continues into early adulthood.

1	Growth of Endochondral Bone Endochondral bone growth begins in the second trimester of fetal life and continues into early adulthood. The events described previously represent the early stage of endochondral bone formation that occurs in the fetus, beginning at about the twelfth week of gestation. The continuing growth process that lasts into early adulthood is described in the following section. Growth in length of long bones depends on the presence of epiphyseal cartilage. As the diaphyseal marrow cavity enlarges (see illustration 6 of Fig. 8.17), a distinct zonation can be recognized in the cartilage at both ends of the cavity. This remaining cartilage, referred to as epiphyseal cartilage, exhibits distinct

1	FIGURE 8.18 • Photomicrograph of a mixed bone spicule formed during endochondral bone formation. In this MalloryAzan–stained section, bone has been deposited on calcified cartilage spicules. In the center of the photomicrograph, the spicules have already grown to create an anastomosing trabecula. The initial trabecula still contains remnants of calcified cartilage, as shown by the light-blue staining of the calcified matrix compared with the dark-blue staining of the bone. In the upper part of the spicule, note a lone osteoclast (arrow) aligned near the surface of the spicule, where remodeling is about to be initiated. 275.

1	zones as illustrated in Figure 8.19 and Plate 14, page 250. During endochondral bone formation, the avascular cartilage is gradually replaced by vascularized bone tissue. This replacement is initiated by vascular endothelial growth factor (VEGF) and is accompanied by expression of genes responsible for production of type X collagen and matrix metalloproteases (enzymes responsible for degradation of cartilage matrix). The zones in the epiphyseal cartilage, beginning with the zone most distal to the diaphyseal center of ossification and proceeding toward that center, are the following.  The zone of reserve cartilage exhibits no cellular prolif eration or active matrix production.  The zone of proliferation is adjacent to the zone of re serve cartilage in the direction of the diaphysis. In this zone of reserve cartilage zone of proliferation zone of hypertrophy zone of calcified cartilage zone of resorption bone osteoclast blood vessel osteoblasts

1	FIGURE 8.19 • Longitudinal section through the distal end of a metatarsal bone of a 2-month-old infant. The epiphyseal (secondary) ossification center is well formed. Bone formation is taking place at both the epiphyseal and the diaphyseal surface of the epiphyseal plate. The zonation is apparent on the diaphyseal side because the growth rate there is so much greater than the epiphyseal ossification center. Because both centers are active, the zone of reserve cartilage is relatively narrow. H&E 280. (Reprinted with permission from Kelly DE, Wood RL, Enders AC. Bailey’s Textbook of Microscopic Anatomy. Baltimore: Williams & Wilkins, 1978.) zone, the cartilage cells undergo division and organize into distinct columns. These cells are larger than those in the reserve zone and actively produce collagen (mainly types II and XI) and other cartilage matrix proteins.

1	 The zone of hypertrophy contains greatly enlarged (hypertrophic) cartilage cells. The cytoplasm of these cells is clear, a reflection of the glycogen that they normally accumulate (which is lost during tissue preparation). Chondrocytes in this zone remain metabolically active; they continue to secrete type I collagen while increasing their secretion of type X collagen. Hypertrophic chondrocytes also secrete VEGF, which initiates vascular invasion. The cartilage matrix is compressed to form linear bands between the columns of hypertrophied cartilage cells.  In the zone of calcified cartilage, the hypertrophied cells begin to degenerate and the cartilage matrix becomes calcified. The calcified cartilage then serves as an initial scaffold for deposition of new bone. Chondrocytes positioned in the more proximal part of this zone undergo apoptosis.

1	 The zone of resorption is the zone nearest the diaphysis. The calcified cartilage here is in direct contact with the connective tissue of the marrow cavity. In this zone, small blood vessels and accompanying connective tissue invade the region previously occupied by the dying chondrocytes. They form a series of spearheads, leaving the calcified cartilage as longitudinal spicules. In a cross section, the calcified cartilage appears as a honeycomb because of the absence of the cartilage cells. The invading blood vessels are the source of osteoprogenitor cells, which will differentiate into bone-producing cells. Bone deposition occurs on the cartilage spicules in the same manner as described for the formation of the initial ossification center.

1	Bone deposition occurs on the cartilage spicules in the same manner as described for the formation of the initial ossification center. As bone is laid down on the calcified spicules, the cartilage is resorbed, ultimately leaving a primary spongy bone. This spongy bone undergoes reorganization through osteoclastic activity and addition of new bone tissue, thus accommodating the continued growth and physical stresses placed on the bone.

1	Shortly after birth, a secondary ossification center develops in the proximal epiphysis. The cartilage cells undergo hypertrophy and degenerate. As in the diaphysis, calcification of the matrix occurs, and blood vessels and osteogenic cells from the perichondrium invade the region, creating a new marrow cavity (see illustration 7 of Fig. 8.17). Later, a similar epiphyseal ossification center forms at the distal end of the bone (see illustration 8 of Fig. 8.17). This center is also regarded as a secondary ossification center, although it develops later. With the development of the secondary ossification centers, the only cartilage that remains from the original model is the articular cartilage at the ends of the bone and a transverse disc of cartilage, known as the epiphyseal growth plate, which separates the epiphyseal and diaphyseal cavities (Plate 13, page 248). Cartilage of the epiphyseal growth plate is responsible for maintaining the growth process.

1	Cartilage of the epiphyseal growth plate is responsible for maintaining the growth process. For a bone to retain proper proportions and its unique shape, both external and internal remodeling must occur as the bone grows in length. The proliferative zone of the epiphyseal plate gives rise to the cartilage on which bone is later laid down. In reviewing the growth process, it is important to realize the following:  The thickness of the epiphyseal plate remains relatively constant during growth.  The amount of new cartilage produced (zone of prolifera tion) equals the amount resorbed (zone of resorption).  The resorbed cartilage is, of course, replaced by spongy bone.

1	Actual lengthening of the bone occurs when new cartilage matrix is produced at the epiphyseal plate. Production of new cartilage matrix pushes the epiphysis away from the diaphysis, elongating the bone. The events that follow this incremental growth—namely, hypertrophy, calcification, resorption, and ossification—simply involve the mechanism by which the newly formed cartilage is replaced by bone tissue during development. Bone increases in width or diameter when appositional growth of new bone occurs between the cortical lamellae and the periosteum. The marrow cavity then enlarges by resorption of bone on the endosteal surface of the cortex of the bone. As bones elongate, remodeling is required. It consists of preferential epiphysis enlarges by growth of epiphyseal

1	FIGURE 8.20 • Diagram of external remodeling of a long bone. This diagram shows two periods during the growth of the bone. The younger bone profile (before remodeling) is shown on the right; the older (after remodeling), on the left. Superimposed on the left side of the figure is the shape of the bone (left half only) as it appeared at an earlier time. The bone is now longer, but it has retained its general shape. To grow in length and retain the general shape of the particular bone, bone resorption occurs on some surfaces, and bone deposition occurs on other surfaces, as indicated in the diagram. (Based on Ham AW. J Bone Joint Surg Am 1952;34:701.) resorption of bone in some areas and deposition of bone in other areas, as described previously and as outlined in Figure 8.20. When an individual achieves maximal growth, proliferation of new cartilage within the epiphyseal plate terminates.

1	When an individual achieves maximal growth, proliferation of new cartilage within the epiphyseal plate terminates. When the proliferation of new cartilage ceases, the cartilage that has already been produced in the epiphyseal plate continues to undergo the changes that lead to the deposition of new bone until, finally, there is no remaining cartilage. At this point, the epiphyseal and diaphyseal marrow cavities become confluent. The elimination of the epiphyseal plate is referred to as epiphyseal closure. In illustration 9 of Figure 8.17, the lower epiphyseal cartilage is no longer present; in illustration 10, both epiphyseal cartilages are gone. Growth is now complete, and the only remaining cartilage is found on the articular surfaces of the bone. Vestigial evidence of the site of the epiphyseal plate is reflected by an epiphyseal line consisting of bone tissue (see Fig. 8.2). Development of the Osteonal (Haversian) System Osteons typically develop in preexisting compact bone.

1	Development of the Osteonal (Haversian) System Osteons typically develop in preexisting compact bone. Compact bone can take several different forms. Compact bone may be formed from fetal spongy bone by continued deposition of bone on the spongy bone spicules; it may be deposited directly as adult compact bone (e.g., the circumferential lamellae of an adult bone); or it might be older compact bone consisting of osteons and interstitial lamellae. The process in which new osteons are formed is referred to as internal remodeling. During the development of new osteons, osteoclasts bore a tunnel, the resorption cavity, through compact bone.

1	During the development of new osteons, osteoclasts bore a tunnel, the resorption cavity, through compact bone. Formation of a new osteons in compact bone initially involves the creation of a tunnel-like space, the resorption cavity, by osteoclast activity. This resorption cavity will have the dimensions of the new osteon. When osteoclasts have produced an appropriately sized cylindrical tunnel by resorption of compact bone, blood vessels and their surrounding connective tissue occupy the tunnel. As the tunnel is occupied, new bone deposition on its wall begins almost immediately. These two aspects of cellular activity—namely, osteoclast resorption and osteoblast synthesis—constitute a bone-remodeling unit. A bone-remodeling unit consists of two distinct parts: an advancing cutting cone (also called a resorption canal) and a closing cone (Fig. 8.21). The tip of the cutting cone consists of advancing osteoclasts closely followed by an advancing capillary loop and pericytes. It

1	FIGURE 8.21 • Diagram of a bone-remodeling unit. A bone-remodeling unit consists of an advancing cutting cone and a closing cone. The cutting cone formed by osteoclasts is responsible for boring the tunnel or resorption cavity through the compact bone. Its action is initiated within the Haversian canal at the left of the diagram (in the area corresponding to section a). The cutting cone moves along the Haversian canal, in the direction indicated by the arrow, to the area corresponding to section d. Section d shows the cross section through the cutting cone. The resorption cavity is the site where the future osteon is formed by the action of the closing cone, which consists of osteoblasts. These cells begin to deposit the osteoid on the walls of the canal in successive lamellae. Gradual formation of the new bone fills the resorption cavity. Note the deposition of the osteoid deep to the osteoblasts seen in sections b and c. As successive lamellae of bone are deposited, the canal

1	formation of the new bone fills the resorption cavity. Note the deposition of the osteoid deep to the osteoblasts seen in sections b and c. As successive lamellae of bone are deposited, the canal ultimately attains the relatively narrow diameter of the mature Haversian canal, like that shown in section a. The growth-reversal line that appears at the outer limits of a newly formed osteon represents a border between the resorption activity of the cutting cone and the bony matrix not remodeled by this activity.

1	also contains numerous dividing cells that give rise to osteoblasts, additional pericytes, and endothelial cells. (Recall that osteoclasts are derived from mononuclear hemopoietic progenitor cells.) The osteoclasts drill a canal about 200 min diameter. This canal establishes the diameter of the future osteonal (Haversian) system. The cutting cone constitutes only a small fraction of the length of the bone-remodeling unit; thus, it is seen much less frequently than the closing cone. After the diameter of the future Haversian system is established, osteoblasts begin to fill the canal by depositing the organic matrix of bone (osteoid) on its walls in successive lamellae. With time, the bone matrix in each of the lamellae becomes mineralized. As the successive lamellae of bone are deposited, from the periphery inward, the canal ultimately attains the relatively narrow diameter of the adult osteonal canal. Compact adult bone contains Haversian systems of varying age and size.

1	Compact adult bone contains Haversian systems of varying age and size. Microradiographic examination of a ground section of bone reveals that younger Haversian systems are less completely mineralized than older systems (Fig. 8.22). They undergo a progressive secondary mineralization that continues (up to a point) even after the osteon has been fully formed. Figure 8.22 also illustrates the dynamic internal remodeling of compact bone. In the adult, deposition balances resorption. In the aged, resorption often exceeds deposition. If this imbalance becomes excessive, then osteoporosis develops (see Folder 8.2). Biologic mineralization is a cell-regulated extracellular event.

1	Biologic mineralization is a cell-regulated extracellular event. Mineralization occurs in the extracellular matrix of bone, cartilage and in the dentin, cementum, and enamel of teeth. The matrices of all of these structures except enamel contain collagen fibrils and ground substance. Mineralization is initiated in the same time within the collagen fibrils and in the ground substance surrounding them. In enamel, mineralization occurs within the extracellular matrix secreted by the enamel organ. Despite the extracellular location of biologic mineralization and the fact that physicochemical factors are basic to the process, biologic mineralization is a cell-regulated event. Mineralization involves the secretion of matrix vesicles into the bony matrix.

1	Mineralization involves the secretion of matrix vesicles into the bony matrix. In places where the mineralization of bone, cartilage, dentin, and cementum is initiated, the local concentration of Ca2 and PO4 ions in the matrix must exceed the normal threshold level. Several events are responsible for this mineralization:  The binding of extracellular Ca2 by osteocalcin and other sialoproteins creates a high local concentration of this ion.  The high Ca2 concentration stimulates the osteoblasts to secrete alkaline phosphatase (ALP), which increases the local concentration of PO4 ions. The high PO4 concentra where mineralization will be initiated.

1	FIGURE 8.22 • Microradiograph of the cross section of a bone. This 200-m-thick cross section of bone from a healthy 19-year-old male shows various degrees of mineralization in different osteons. Mature compact bone is actively replacing immature bone, which is seen on the periosteal (upper) surface. The degree of mineralization is reflected by the shade of light and dark in the microradiograph. Thus, very light areas represent the highly mineralized tissue that deflects the X-rays and prevents them from striking the photographic film. Conversely, dark areas contain less mineral and, thus, are less effective in deflecting the X-rays. Note that the interstitial lamellae (the older bone) are very light, whereas some of the osteons are very dark (these are the most newly formed). The Haversian canals appear black, as they represent only soft tissue. 157. (Courtesy of Dr. Jenifer Jowsey.)  At this stage of high extracellular Ca2 and PO4 concentration, the osteoblasts release small (50to

1	canals appear black, as they represent only soft tissue. 157. (Courtesy of Dr. Jenifer Jowsey.)  At this stage of high extracellular Ca2 and PO4 concentration, the osteoblasts release small (50to 200-nm) matrix vesicles into the bony matrix by exocytosis. The matrix vesicles contain ALP and pyrophosphatase that cleave PO 4 ions from other molecules of the matrix.

1	 The matrix vesicles that accumulate Ca2 and cleave PO4 ions cause the local isoelectric point to increase, which results in crystallization of CaPO4 in the surrounding matrix vesicles.  The CaPO4 crystals initiate matrix mineralization by the formation and deposition of hydroxyapatite crystals [Ca10(PO4)6(OH)2] in the matrix surrounding the osteoblasts. The osteoblast-derived matrix vesicles are the essential factors in controlling the initial site of mineral deposition in osteoid. Once the initial crystals of hydroxyapatite have precipitated, they grow rapidly by accretion until they join neighboring crystals produced around other matrix vesicles. In this way, a wave of mineralization sweeps through the osteoid. Other cells that produce osteoid are the ameloblasts and odontoblasts of developing teeth. Bone serves as a reservoir for body calcium.

1	Bone serves as a reservoir for body calcium. The maintenance of normal blood calcium levels is critical to health and life. Calcium may be delivered from the bone matrix to the blood if the circulating blood levels of calcium fall below a critical point (physiologic calcium concentration in the human ranges from 8.9 to 10.1 mg/dL). Conversely, excess blood calcium may be removed from the blood and stored in bone. These processes are regulated by parathyroid hormone (PTH), secreted by the parathyroid gland, and calcitonin, secreted by the parafollicular cells of the thyroid gland (see Folder 8.4).  PTH acts on the bone to raise low blood calcium levels to normal.  Calcitonin acts to lower elevated blood calcium levels to normal.

1	 PTH acts on the bone to raise low blood calcium levels to normal.  Calcitonin acts to lower elevated blood calcium levels to normal. PTH acts by stimulating both osteocytes and osteoclasts to resorb bone, allowing the release of calcium into the blood. As described previously (see page 227), resorption of bone by osteocytes constitutes osteocytic osteolysis. PTH also reduces excretion of calcium by the kidney and stimulates absorption of calcium by the small intestine. PTH further acts to maintain homeostasis by stimulating the kidney to excrete the excess phosphate produced by bone resorption. Calcitonin inhibits bone resorption, specifically inhibiting the effects of PTH on osteoclasts.

1	The classical concept of PTH action related to the regulation of serum calcium levels and bone resorption is more complex. For some time now, it has been known that PTH can also stimulate bone formation. In other words, it has an anabolic action (increases bone formation) in contrast to its catabolic action to cause bone resorption. In fact, clinical trials in which PTH hormone was administered to post-menopausal women with osteoporosis have shown significant increases in bone formation and bone mineral density. Increases in the amount of cancellous (trabecular) bone due to PTH treatment were shown in the ilium, vertebral bodies, and the shafts of radial and femoral bones (see Folder 8.2). The possible mechanisms behind this counterintuitive anabolic action of PTH are largely unknown. It is speculated that activation of different PTH-regulated genes might be responsible for each of the hormone’s contrasting effects. Bone can repair itself after injury.

1	Bone can repair itself after injury. The initial response to a fracture is similar to the response to any injury that produces tissue destruction and hemorrhage. Neutrophils are the first cells to arrive on the scene, followed by macrophages that begin to clean up the site of injury. Fibroblasts and capillaries then proliferate and grow into the site of injury. New loose connective tissue, granulation tissue, is formed, and as this tissue becomes denser, cartilage forms in parts of it. Both fibroblasts and periosteal cells participate in this phase of the healing process. The dense connective tissue and newly formed cartilage grow, covering the bone at the fracture site and producing a soft callus (Fig. 8.23). A callus will form whether or not the fractured parts of the bone are in immediate apposition to each other. The callus helps stabilize and bind together the fractured bone.  FOLDER 8.4 Functional Considerations: Hormonal Regulation of Bone Growth

1	Hormones other than PTH and calcitonin have major ef-fects on bone growth. One such hormone is pituitary growth hormone (GH, somatotropin). This hormone stimulates growth in general and, especially, growth of epiphyseal cartilage and bone. It acts directly on osteopro-genitor cells, stimulating them to divide and differentiate. Chondrocytes in epiphyseal growth plates are regulated by insulinlike growth factor I (IGF-I), which is primarily pro-duced by the liver in response to GH. In addition to IGF-I, insulin and thyroid hormones also stimulate chondrocyte activity. Oversecretion in childhood, caused by a defect in the mechanism regulating GH secretion or a GH-secreting tumor in the pituitary gland, leads to gigantism, an abnormal increase in the length of bones. Absence or hy-posecretion of GH in childhood leads to failure of growth of the long bones, resulting in pituitary dwarfism. Absence or severe hyposecretion of thyroid hormone dur-ing development and infancy leads to failure

1	of GH in childhood leads to failure of growth of the long bones, resulting in pituitary dwarfism. Absence or severe hyposecretion of thyroid hormone dur-ing development and infancy leads to failure of bone growth and dwarfism, a condition known as congenital hypothyroidism. When oversecretion of GH occurs in an adult, bones do not grow in length as a result of epiphy-seal closure. Instead, abnormal thickening and selective overgrowth of hands, feet, mandible, nose, and intramem-branous bones of the skull occurs. This condition, known as acromegaly, is caused by increased activity of osteoblasts on bone surfaces.

1	FIGURE 8.23 • Photomicrograph of fractured long bone undergoing repair. a. This low-magnification photomicrograph of a 3-week-old bone fracture, stained with H&E, shows parts of the bone separated from each other by the fibrocartilaginous callus. At this stage, the cartilage undergoes endochondral ossification. In addition, the osteoblasts of the periosteum are involved in secretion of new bony matrix on the outer surface of the callus. On the right of the microphotograph, the fibrocartilaginous callus is covered by periosteum, which also serves as the attachment site for the skeletal muscle. 35. b. Higher magnification of the callus from the area indicated by the upper rectangle in panel a shows osteoblasts lining bone trabeculae. Most of the original fibrous and cartilaginous matrix at this site has been replaced by bone. The early bone is deposited as an immature bone, which is later replaced by mature compact bone. 300. c. Higher magnification of the callus from the area indicated

1	site has been replaced by bone. The early bone is deposited as an immature bone, which is later replaced by mature compact bone. 300. c. Higher magnification of the callus from the area indicated by the lower rectangle in panel a. A fragment of old bone pulled away from the fracture site by the periosteum is now adjacent to the cartilage. It will be removed by osteoclast activity. The cartilage will calcify and be replaced by new bone spicules as seen in panel b. 300.

1	While the callus is forming, osteoprogenitor cells of the periosteum divide and differentiate into osteoblasts. The newly formed osteoblasts begin to deposit new bone on the outer surface of the bone at some distance from the fracture. This new formation of bone progresses toward the fracture site until new bone forms a bony sheath over the fibrocartilaginous callus. Osteogenic buds from the new bone invade the callus and begin to deposit new bone within the callus, gradually replacing the original fibrous and cartilaginous callus with a bony callus. The cartilage in the original callus calcifies and is replaced by bone as in endochondral ossification.

1	Endosteal proliferation and differentiation also occur in the marrow cavity, and medullary bone grows from both ends of the fracture toward the center. When this bone unites, the bony union of the fractured bone produced by the osteoblasts derived from both the periosteum and endosteum consists of spongy bone. As in normal bone formation, the spongy bone is gradually replaced by compact bone. While compact bone is being formed, the bony callus is removed by the action of osteoclasts, and gradual remodeling restores the bone to its original shape.

1	In healthy individuals, this process usually takes from 6 to 12 weeks, depending on the severity of the break and the particular bone that is broken. Setting the bone (i.e., reapproximating the normal structure) and holding the parts in place by internal fixation (by pins, screws, or plates) or by external fixation (by casts or by pins and screws) speeds the healing process and usually results in superior structural and functional restoration. PLATE 11 Bone, Ground Section

1	Bone is a specialized connective tissue characterized by a mineralized extracellular matrix. Calcium phosphate, in the form of hydroxyapatite crystals (Ca10(PO4)6OH2), is deposited along the collagen fibrils and in the proteoglycan ground substance. Bone serves as a storage site for calcium and phosphate, which can be released to the blood to maintain homeostatic levels. Osteocytes reside in lacunae in the bone ma-trix and extend fine cellular processes into canaliculi that connect the lacunae, thus forming a continuous network of cells within the mineral-ized tissue. Bones are organs of the skeletal system; bone tissue is the structural component of bones. Ground sections of bone are prepared from bone that has not been fixed but merely allowed to dry. Thin slices of the dried bone are then cut with a saw and further ground to a thinness that allows viewing in a light microscope. Slices may be treated with India ink to fill spaces that were formerly occupied by organic matter, e.g.,

1	cut with a saw and further ground to a thinness that allows viewing in a light microscope. Slices may be treated with India ink to fill spaces that were formerly occupied by organic matter, e.g., cells, blood vessels, and unmineralized matrix. A simpler method is to mount the ground specimen on a slide with a viscous medium that traps air in some of the spaces, as in the specimen in this plate. Here, some of the osteonal canals and a perforating canal are filled with the mounting medium, making them translucent instead of black. Specimens prepared in this manner are of value chiefly to display the architecture of the compact bone.

1	Ground bone, long bone, human, ×80. This figure reveals a cross-sectioned area of a long bone at low magnification and includes the outer or peripheral aspect of the bone, identified by the presence of circumferential lamellae (CL). (The exterior or periosteal surface of the bone is not included in the micrograph.) To their right are the osteons (O) or Haversian systems that appear as circular profiles. Between the osteons are interstitial lamellae (IL), the remnants of previously existing osteons.

1	Osteons are essentially cylindrical structures. In the shaft of a long bone, the long axes of the osteons are oriented parallel to the long axis of the bone. Thus, a cross section through the shaft of a long bone would reveal the osteons in cross section, as in this figure. At the center of each osteon is an osteonal (Haversian) canal (HC) that contains blood vessels, connective tissue, and cells lining the surface of the bone material. Because the organic material is not retained in ground sections, the Haversian canals and other spaces will appear black, as-they do here, if filled with India ink or air. Concentric layers of mineralized substance, the concentric lamellae, PLATE 11 • BONE, GROUND SECTION Ground bone-osteon, long bone, human, ×300.

1	PLATE 11 • BONE, GROUND SECTION Ground bone-osteon, long bone, human, ×300. This figure shows a higher-magnification micrograph of the labeled osteon from the upper figure. It includes some of the interstitial lamellae (IL) that are now seen at the bottom of the micrograph (the micrograph has been reoriented). Note the lacunae (L) and the fine thread-like profiles emanating from surround the Haversian canal and appear much the same as growth rings of a tree. The canal is also surrounded by concentric arrangements of lacunae. These appear as the small, dark, elongate structures.

1	During the period of bone growth and during adult life, there is constant internal remodeling of bone. This involves the destruction of osteons and formation of new ones. The breakdown of an osteon is usually not complete; however, part of the osteon may remain intact. Moreover, portions of adjacent osteons may also be partially destroyed. The space created by the breakdown process is reoccupied by a new osteon. The remnants of the previously existing osteons become the interstitial lamellae. Blood vessels reach the Haversian canals from the marrow through other tunnels called perforating (Volkmann’s) canals (VC). In some instances, as here, Volkmann’s canals travel from one Haversian canal to another. Volkmann’s canals can be distinguished from Haversian canals in that they pass through lamellae, whereas Haversian canals are surrounded by concentric rings of lamellae.

1	the lacunae. These thread-like profiles represent the canaliculi, spaces within the bone matrix that contained cytoplasmic processes of the osteocyte. The canaliculi of each lacuna communicate with canaliculi of neighboring lacunae to form a three-dimensional channel system throughout the bone. Ground bone, long bone, human, ×400.

1	Ground bone, long bone, human, ×400. In a still higher magnification, the circumferential lamellae are found around the shaft of the long bone at the outer as well as the inner surface of the bone. The osteoblasts that contribute to the formation of circumferential lamellae at these sites come from the periosteum and endosteum, respectively, whereas the osteons are constructed from osteoblasts in the canal of the developing Haversian system. This figure reveals not only the canaliculi but also the lamellae of the bone. The latter are just barely defined by the faint lines (arrows) that extend across the micrograph. Collagenous fibers in neighboring lamellae are oriented in different directions. This change in orientation accounts for the faint line or interface between adjacent lamellae. KEY CL, circumferential lamellae HC, Haversian canal IL, interstitial lamellae L, lacuna VC, Volkmann’s canal arrow, lamellar boundary

1	KEY CL, circumferential lamellae HC, Haversian canal IL, interstitial lamellae L, lacuna VC, Volkmann’s canal arrow, lamellar boundary PLATE 11 • BONE, GROUND SECTION VCVCILILHCHCCLCLOO CL HC IL ILILIL VC LL LL LL LL HCHCHC HCHCHC

1	Bone represents one of the specialized connective tissues. It is characterized by a mineralized extracellular matrix. It is the mineralization of the matrix that sets bone tissue apart from the other connective tissues and results in an extremely hard tissue that is capable of providing support and protection to the body. The mineral is calcium phosphate in the form of hydroxyapatite crystals. In addition to its supporting role, bone also provides a storage site for calcium and phosphate. Both can be mobilized from the bone matrix and taken up by the blood as needed to maintain normal levels. Bone matrix contains type I collagen and, in small amounts, a number of other types of collagen, i.e., types V, III, XI, and XIII. Other matrix proteins that constitute the ground substance of bone such as proteoglycan macromolecules, multiadhesive glycoproteins, growth factors, and cytokines are also present. Bone is typically studied in histological prepara-tions by removing the calcium content

1	as proteoglycan macromolecules, multiadhesive glycoproteins, growth factors, and cytokines are also present. Bone is typically studied in histological prepara-tions by removing the calcium content of the bone (decalcified bone), thus allowing it to be sectioned like other soft tissues. ORIENTATION MICROGRAPH: The orientation micrograph shows the upper end of a decal-cified humerus from an infant. The interior of the head of the bone, the epiphysis (E), consists of spongy cancellous bone made up of an anastomosing network of trabeculae (T) in the form of spicules of bone tissue. The outer portion consists of a dense layer of bone tissue known as compact bone (CB). Its thickness varies in different parts of the bone. The shaft of this bone, the diaphysis (D), is also made up of compact bone (CB) and in its interior, spongy bone (SB). Also within the shaft of the bone is bone marrow (BM), which at this stage of life is in the form of hemopoetic tissue. Lastly, cartilage is also a

1	bone (CB) and in its interior, spongy bone (SB). Also within the shaft of the bone is bone marrow (BM), which at this stage of life is in the form of hemopoetic tissue. Lastly, cartilage is also a component of the bone where it is present as an articular surface (AS) and as a growth plate (GP). The latter is described in a later plate. ASECBDCBCBSBBMCBTGPAS E CB D CB CB SB BM CB T GP

1	Compact bone, long bone, human, H&E, ×178. The area in the rectangle in the orientation micrograph containing compact bone in the epiphysis is shown here at higher magnification. The lighter staining area is cartilage (C). It serves as the articular surface of the epiphysis. Note the presence of isogenous groups of chondrocytes (Ch), a charac teristic feature of a growing cartilage. Below this is bone tissue (BT). It can Compact bone, long bone, human, H&E, ×135.

1	Compact bone, long bone, human, H&E, ×135. Bone from the diaphysis within the far right rectangle of the oritentation miocrograph is shown here at higher magnification. The outer surface of the bone is covered by dense connective tissue known as periosteum (P). The remaining tissue in be distinguished from the cartilage by the arrangement of its cells, the osteocytes (Oc). The osteocytes lie within the bone matrix, but are typically recognized only by their nuclei. Because bone matrix is laid down in layers (lamellae), bone characteristically shows linear or circular patterns that appear as striations. The irregular spaces seen within the bone tissue are vascular channels (VC) that contain, in addition to vessels, bone-forming tissue.

1	the micrograph is compact bone. The osteocytes (Oc) are again recognized by their nuclei within the bone matrix. Another feature worth noting in this growing bone is the presence of bone resorbing cells known as osteoclasts (Ocl). They are large multinucleated cells found at sites in bone where remodeling is taking place (see Plate 14). Spongy bone, long bone, human, H&E, ×135.

1	Spongy bone, long bone, human, H&E, ×135. The area in the rectangle in the orientation micrograph containing spongy bone in the epiphysis is shown here at higher magnification. Although the bone tissue at this site forms a three dimensional structure consisting of branching trabeculae, its structural organization and components are the same as that seen in compact bone. Note the osteocyte nuclei (N). As bone matures, the bone tissue becomes reorganized and forms osteons (O), which consist of a central vascular channel and surrounding layers (lamellae) of bone matrix. The two circular spaces are sites in which bone tissue has been removed and will be replaced by new tissue in the form of osteons. The spaces surrounding the spongy bone contain bone marrow consisting mainly of adipocytes. Other cells that have the capacity to form bone or hemopoetic tissue are also present.

1	KEY AS, articular surface BM, bone marrow BT, bone tissue C, cartilage CB, compact bone Ch, chondrocytes E, epiphysis GP, growth plate N, nuclei O, osteons Oc, osteocytes Ocl, osteoclasts P, periosteum SB, spongy bone T, trabeculae VC, vascular channels

1	Endochondral bone formation involves the continuing growth of a cartilage precursor, which serves as a fetal skeleton, and the simultane-ous removal of the cartilage and its replacement with bone tissue. In addition, as a bone grows, some of the bone tissue is removed while newer bone tissue is being laid down, a process called remodeling. Remodeling that alters the shape of the bone is called external remodel-ing; that which does not alter the shape of the bone, as in the formation of Haversian systems, is called internal remodeling. Two specialized cell types are identified with the process of bone growth and remodeling. The osteoblast is engaged in the formation of bone. Although the removal of bone is not as well described as its formation, it has been established that multinucleated cells, called osteo-clasts, are engaged in the removal of bone. Osteocytes, also, can alter and resorb bone in their immediate vicinity. The process is called os-teocytic osteolysis. It is important

1	cells, called osteo-clasts, are engaged in the removal of bone. Osteocytes, also, can alter and resorb bone in their immediate vicinity. The process is called os-teocytic osteolysis. It is important in calcium homeostasis, i.e., the maintenance of normal blood calcium concentrations.

1	Developing bone, short bone, monkey, H&E ×240. The early steps of endochondral bone formation are shown in this figure. The structure seen here is the cartilage model of the bone about to be formed. The steps of bone formation are 1. The cartilage (C) cells in the center of the cartilage model become hypertrophic (HC). 2. The matrix of the cartilage becomes calcified (CM). (The calcified matrix stains intensely with hematoxylin and appears as the darker condensed matrix material between the enlarged cartilage cells.) Developing bone, fetal fnger, human, H&E ×60.

1	Developing bone, fetal fnger, human, H&E ×60. The bone in this figure shows later events and a continuation of the earlier ones just described. A vascular bud (not shown) and accompanying perivascular cells from the periosteum have invaded the shaft of the cartilage model, resulting in the formation of a cavity (Cav). Examination at higher magnification would reveal that the cavity contains fat cells, hematopoietic tissue (the dark-blue–staining component), and other connective tissue elements. While the new steps of bone formation occur, the earlier steps continue: 1. Cartilage (C) cells proliferate at the epiphyses. They are responsible for production of new matrix material. It is this process that creates lengthening of the bone. 2. Periosteal bone (PB) continues to form. Developing bone, long bone, human, H&E ×60; inset ×200.

1	2. Periosteal bone (PB) continues to form. Developing bone, long bone, human, H&E ×60; inset ×200. This shows an early stage after the invasion of the epiphysis. A secondary ossification center (Os) has formed, and along with this event, the head of the long bone will develop a marrow cavity similar in its content to that of the diaphysis. The 3. A collar of bone forms around the circumference of the center of the cartilage model. This bone is called periosteal bone (PB) because the osteoblasts that have produced the bone material develop from the periosteum. (Note that the periosteal bone is, in fact, intramembranous bone [see Plate 15], because it develops within the connective tissue membrane that immediately surrounds the developing bone and not on a spicule of calcified cartilage.)

1	PLATE 13 • E N DOCHON DRAL BON E FOR MATION I KEY C, cartilage Cav, marrow cavity CC, calcified cartilage CM, calcified matrix EB, endochondral bone EP, epiphyseal plate HC, hypertrophic cartilage cell JC, joint cavity Os, secondary ossification center PB, periosteal bone 3. Cartilage cells facing the cavity become hypertrophic. 4. Cartilage matrix becomes calcified. 5. Erosion of cartilage occurs, creating spicules of cartilage. 6. Bone forms on the spicules of the calcified cartilage at the erosion front; this bone is endochondral bone (EB).

1	5. Erosion of cartilage occurs, creating spicules of cartilage. 6. Bone forms on the spicules of the calcified cartilage at the erosion front; this bone is endochondral bone (EB). As these processes continue in the shaft of the bone, one end of the cartilage model (the epiphysis) is invaded by blood vessels and connective tissue from the periosteum (periosteal bud), and it undergoes the same changes that occurred earlier in the shaft (except that no periosteal bone forms). This same process then occurs at the other end of the bone. Consequently, at each end of the developing long bone, a cartilaginous plate (epiphyseal plate) is created that lies between two sites of bone formation.

1	cartilage separating the two cavities is the epiphyseal plate (EP). At the early stage shown in this figure, the plate is not well defined. Despite the enlargement of the epiphyseal cavity, the remaining cartilage between the two cavities persists as a disc or plate until growth ceases. The inset shows some calcified cartilage as well as the deposition of endochondral bone (EB) within the secondary ossification center.

1	Endochondral bone formation is the principal process by which the long bones, e.g., the bones of the axial skeleton and the appendages and digits, increase in length to achieve their adult dimensions. So long as epiphyseal cartilage exists between the diaphyseal and epiphy-seal ossification centers, the bone will continue to grow. Cessation of bone growth is the result of the cessation of interstitial growth of the epiphyseal cartilages. X-ray examination of the bones of late adolescents can determine whether there is still an epiphyseal cartilage plate and, therefore, determine the potential for further growth in bone length and body height. Endochondral bone formation, epiphysis of long bone, human, H&E ×80; inset ×380.

1	This is a photomicrograph of an epiphysis at higher magnification than that seen in Plate 13. Different zones of the cartilage of the epiphyseal plate reflect the progressive changes that occur in active growth of endochondral bone. These zones are not sharply delineated, and the boundaries between them are somewhat arbitrary. They lead toward the marrow cavity (M), so that the first zone is fur 250 thest from the cavity. There are five zones:  Zone of reserve cartilage (RC). The cartilage cells of this zone have not yet begun to participate in the growth of the bone; thus, they are reserve cells. These cells are small, usually only one to a lacuna, and not grouped. At some time, some of these cells will proliferate and undergo the changes outlined for the next zone.  Zone of proliferating cartilage (PC). The cells of this zone are increasing in number; they are slightly larger than the reserve cells and close to their neighbors; they begin to form rows.

1	 Zone of hypertrophic cartilage (HC). The cells of this zone are aligned in rows and are significantly larger than the cells in the preceding zone.  Zone of calcified matrix (CM). In this zone the cartilage matrix is impregnated with calcium salts.  Zone of resorption (R). This zone is represented by eroded cartilage that is in direct contact with the connective tissue of the marrow cavity. Spicules (actually a honeycomb at the level of the advancing blood vessels) of cartilage are formed because the pericapillary cells invade and resorb in spearheads rather than along a straight front. Specifically, the pericapillary cells break into the rows of hypertrophied chondrocytes, temporarily leaving the calcified cartilage (C) between the rows of cells. In this manner, spicules of calcified cartilage are formed. Endochondral bone (EB) is then deposited on the surfaces of these calcified cartilage spicules by osteoblasts (Ob), thus forming mixed spicules as seen in the inset.

1	Endochondral bone formation, epiphysis of long bone, human, H&E ×150; inset ×380. This is a higher magnification of the lower middle area of upper figure. It shows calcified cartilage spicules on which bone has been deposited. In the lower portion of the figure, the spicules have already grown to create anastomosing bone trabeculae. These initial trabeculae still contain remnants of calcified cartilage, as shown by the bluish color of the cartilage matrix (compared with the red staining of the bone). Osteoblasts (Ob) are aligned on the surface of the spicules, where bone formation is active. The upper inset shows the surface of several spicules from the left circle in lower figure, at higher magnification. Note the osteoblasts (Ob), some of which are just beginning to produce bone in apposition to the calcified cartilage (C). The lower right of the inset shows bone (EB) with an osteocyte (Oc) already embedded in the bone matrix.

1	The lower inset, an enlargement of the right circle in lower figure, reveals several osteoclasts (Ocl). They are in apposition to the spicule, which is mostly cartilage. A small amount of bone is evident, based on the red-staining material in this inset. Note the light area (arrow) representing the ruffled border of the osteoclast. Examination of lower figure reveals a number of other osteoclasts (Ocl). • KEY CM, zone of calcified matrix C, calcified cartilage EB, endochondral bone HC, zone of hypertrophic cartilage M, marrow Ob, osteoblast Oc, osteocyte Ocl, osteoclast PC, zone of proliferating cartilage RC, zone of reserve cartilage arrow, ruffled border of osteoclast R, zone of resorption

1	Intramembranous bone formation is limited to those bones that are not required to perform an early supporting function, e.g., the flat bones of the skull. This process requires the proliferation and differentiation of cells of the mesenchyme to become osteoblasts, the bone-forming cells. They produce ground substance and collagen. This initial matrix, called osteoid, calcifies to form bone. As the osteoblasts continue to secrete their product, some are entrapped within their matrix and are then known as osteocytes. They are responsible for maintenance of the newly formed bone tissue. The remaining osteoblasts continue the bone deposition process at the bone surface. They are capable of reproducing to maintain an adequate population for continued growth. This newly formed bone appears first as spicules that enlarge and interconnect as growth proceeds, creating a three-dimensional trabecu-lar structure similar in shape to the future mature bone. The interstices contain blood vessels and

1	as spicules that enlarge and interconnect as growth proceeds, creating a three-dimensional trabecu-lar structure similar in shape to the future mature bone. The interstices contain blood vessels and connective tissue (mesenchyme). As the bone continues to grow, remodeling occurs. This involves resorption of localized areas of bone tissue by osteoclasts in order to maintain ap-propriate shape in relation to size and to permit vascular nourishment during the growth process.

1	Intramembranous bone formation, fetal head, human, Mallory trichrome ×45. A cross section of the developing mandible, as seen at this relatively early stage of development, consists of bone spicules (BS) of various sizes and shapes. The bone spicules interconnect and, in three dimensions, have the general shape of the mandible. Other structures present that will assist in orientation include Intramembranous bone formation, fetal head, human, Mallory trichrome ×350.

1	This higher-magnification micrograph of a portion of the field in lower left figure shows to advantage the distinction between newly deposited osteoid, which stains blue, and mineralized bone, which stains red. Osteoblasts are seen in two different levels of activity. Those that are relatively inactive and are in apposition to well-formed osteoid (Ob1) exhibit elongate nuclear profiles and appear to be flattened on the surface of the osteoid. Those osteoblasts developing teeth (DT), the tip of Meckel’s cartilage (MC), also referred to as the mandibular process, seen on the left side, and the oral cavity (OC). The bottom surface of the specimen shows the epidermis (Ep) of the underside of the chin. A large portion of the developing tongue is seen in the upper half of the figure. The tongue consists largely of developing striated visceral muscle fibers arranged in a three-dimensional orthogonal array that is characteristic of this organ.

1	Intramembranous bone formation, fetal head, human, Mallory trichrome ×175. This higher-magnification view of the boxed area in upper figure shows the interconnections of the bone spicules (BS) of the developing mandible. Within and around the spaces enclosed by the developing spicules is mesenchymal tissue. These mesenchymal cells will give rise to new osteoblasts as well as to the cells that will form the vascular components of the bone. The more dense connective tissue (CT) will differentiate into the periosteum on one side of the developing mandible. Other structures shown in the field include numerous blood vessels (BV) and the enamel organ of a developing tooth (DT).

1	(Ob2) that are actively secreting new osteoid appear as tall, columnar-like cells adjacent to osteoid. One of the spicules shows a cell completely surrounded by bone matrix; this is an osteoblast that has become trapped in its own secretions and is now an osteocyte (OC). At this magnification, the very loose connective tissue characteristics of the mesenchyme and the sparseness of the mesenchymal cells (MC) are well demonstrated. The highly cellular connective tissue (CT) on the right margin of the figure is the developing perichondrium. Some of its cells will also develop into osteoblasts to allow growth of the bone at its surface. • KEY BS, bone spicules BV, blood vessels CT, connective tissue DT, developing tooth EO, enamel organ Ep, epithelium MC, Meckel’s cartilage (Fig. 1.) MC, mesenchymal cells (Fig. 3.) Ob1 , inactive osteoblast Ob2 , active osteoblast OC, oral cavity (upper figure) OC, osteocyte (lower right figure)

1	OVERVIEW OF ADIPOSE TISSUE / 254 WHITE ADIPOSE TISSUE / 254 Function of White Adipose Tissue / 254 Differentiation of Adipocytes / 255 Structure of Adipocytes and Adipose Tissue / 256 Regulation of Adipose Tissue / 257 BROWN ADIPOSE TISSUE / 259 Folder 9.1 Clinical Correlation: Obesity / 261 Folder 9.2 Clinical Correlation: Adipose Tissue Tumors / 262 Folder 9.3 Clinical Correlation: PET Scanning and Brown Adipose Tissue Interference / 264 Adipose tissue is a specialized connective tissue that plays an important role in energy homeostasis. Individual fat cells, or adipocytes, and groups of adipocytes are found throughout loose connective tissue. Tissues in which adipocytes are the primary cell type are designated adipose tissue. Adipocytes play a key role in energy homeostasis.

1	For its survival, the body needs to ensure a continuous delivery of energy despite highly variable supplies of nutrients from the external environment. To meet the body’s energy demands when nutrient supplies are low, adipose tissue efficiently stores excess energy. The body has a limited capacity to store carbohydrate and protein, therefore energy reserves are stored within lipid droplets of adipocytes in the form of triglycerides. Triglycerides represent a dynamic form of energy storage that is added to when food intake is greater than energy expenditure and is tapped when energy expenditure is greater than food intake. The energy stored in adipocytes can be rapidly released for use at other sites in the body.

1	Triglycerides are the most concentrated form of metabolic energy storage available to humans. Because triglycerides lack water, they have about twice the energy density of carbohydrates and proteins. The energy density of triglycerides is approximately 37.7 kJ/g (9 cal/g), whereas the density of carbohydrates and proteins is 16.8 kJ/g (4 cal/g). In the event of food deprivation, triglycerides are an essential source of water and energy. Some animals can rely solely on metabolic water obtained from fatty-acid oxidation for maintenance of their water balance. For instance, the hump of a camel consists largely of adipose tissue and is a source of water and energy for this desert animal.

1	Adipocytes perform other functions in addition to their role as fat-storage containers. They also regulate energy metabolism by secreting paracrine and endocrine substances. The newly discovered secretory functions of adipocytes have shifted views on adipose tissue, which is now considered a major endocrine organ. Considerable evidence already exists to link increased endocrine activity of adipocytes to the metabolic and cardiovascular complications associated with obesity. There are two types of adipose tissue: white (unilocular) and brown (multilocular). The two types of adipose tissue, white adipose tissue and brown adipose tissue, are so named because of their color in the living state.  White adipose tissue is the predominant type in adult humans.  Brown adipose tissue is present in humans during fetal life but diminishes during the first decade after birth. Function of White Adipose Tissue

1	Function of White Adipose Tissue Functions of white adipose tissue include energy storage, insulation, cushioning of vital organs, and secretion of hormones.

1	White (unilocular) adipose tissue forms a layer called the panniculus adiposus [Lat. panniculus, a little garment; adipatus, fatty] or hypodermis in the connective tissue under the skin. Since the thermal conductivity of adipose tissue is only about half that of skeletal muscle, the subcutaneous layer of connective tissue provides a significant thermal insulation against cold by reducing the rate of heat loss. Concentrations of adipose tissue are found in the connective tissue under the skin of the abdomen, buttocks, axilla, and thigh. Sex differences in the thickness of this fatty layer in the skin of different parts of the body account, in part, for the differences in body contour between females and males. In both sexes, the mammary fat pad is a preferential site for accumulation of adipose tissue; the nonlactating female breast is composed primarily of this tissue. In the lactating female, mammary fat pad plays an important role in supporting breast function. It provides lipids

1	adipose tissue; the nonlactating female breast is composed primarily of this tissue. In the lactating female, mammary fat pad plays an important role in supporting breast function. It provides lipids and energy for milk production, but it is also a site for the synthesis of different growth factors that modulate responses to different steroid and proteins and hormones acting on mammary gland function.

1	Internally, adipose tissue is preferentially located in the greater omentum, mesentery, and retroperitoneal space and is usually abundant around the kidneys. It is also found in bone marrow and between other tissues, where it fills in spaces. In the palms of the hands and the soles of the feet, beneath the visceral pericardium (around the outside of the heart), and in the orbits around the eyeballs, adipose tissue functions as a cushion. It retains this structural function even during reduced caloric intake; when adipose tissue elsewhere becomes depleted of lipid, this structural adipose tissue remains undiminished. White adipose tissue produces a variety of hormones, growth factors, and cytokines.

1	White adipose tissue produces a variety of hormones, growth factors, and cytokines. Adipocytes actively synthesize and secrete hormones, growth factors, and cytokines. Leptin [Gr. leptos, thin], a 16-kilodalton peptide hormone involved in the regulation of energy homeostasis, is exclusively secreted by adipocytes. Leptin inhibits food intake and loss of body weight and stimulates metabolic rate. Thus, leptin fulfills the criteria for a circulating satiety factor that controls food intake when the body’s store of energy is sufficient. Leptin also participates in an endocrine signaling pathway that communicates the energy state of adipose tissue to brain centers that regulate food uptake. It acts on the central nervous system by binding to specific receptors, mainly in the hypothalamus. In addition, leptin communicates the fuel state of adipocytes from fat-storage sites to other metabolically active tissues (i.e., from adipose tissue to muscle at a different site).

1	In addition to leptin, adipose tissue secretes other hormones, including angiotensinogen (AGE), adiponectin, and resistin, and produces steroid hormones (testosterone, estrogens, and glucocorticoids). AGE is synthesized in other tissues, including the liver; increased production of this peptide contributes to hypertension (elevation of blood pressure), which is a frequent complication of obesity. Sex hormones and glucocorticoids are not synthesized de novo; instead they are converted from inactive forms by specific enzymes expressed in adipocytes. These enzymes can therefore inﬂuence the sex steroid profiles of obese individuals. Obesity-increased secretion of growth factors (tumor necrosis factor a [TNF-a], transforming growth factor b [TGF-b], and insulinlike growth factor I [IGF-I]) and cytokines (interleukin 6 and prostaglandins) may be linked to metabolic abnormalities and development of diabetes. Table 9.1 presents a summary of the molecules produced by adipocytes and their

1	and cytokines (interleukin 6 and prostaglandins) may be linked to metabolic abnormalities and development of diabetes. Table 9.1 presents a summary of the molecules produced by adipocytes and their functions.

1	Differentiation of Adipocytes White adipocytes differentiate from mesenchymal stem cells under the control of PPAR/RXR transcription factors.

1	Early histologists debated whether adipose tissue was a specific tissue, distinct from connective tissue, or ordinary connective tissue in which fibroblasts store fat globules. The current consensus is that adipocytes are a specific cell type derived from undifferentiated mesenchymal stem cells associated with the adventitia of small venules (Fig. 9.1). Current evidence suggests that a transcription factor called peroxisome proliferator–activated receptor gamma (PPAR) in complex with the retinoid X receptor (RXR) play a critical role in adipocyte differentiation and initiation of lipid metabolism. It induces the maturation of early lipoblasts (adipoblasts) or preadipocytes into fat cells of white adipose tissue. Most of the PPAR target genes in adipose tissue influence lipogenic pathways and initiate the storage of triglycerides. Therefore, PPAR/RXR is regarded as the “master switch” regulator in the white adipocytes’ differentiation.

1	White adipose tissue begins to form midway through fetal development. Lipoblasts initially develop from stromal-vascular cell along the small blood vessels in the fetus and are free of lipids. These cells are committed to becoming adipocytes at this early stage by expressing PPAR/RXR transcription factors. Collections of such cells are sometimes called primitive fat organs. They are characterized by proliferating early lipoblasts and proliferating capillaries. Lipid accumulation in lipoblasts produces the typical morphology of adipocytes. Early lipoblasts look like fibroblasts but develop small lipid inclusions and a thin external lamina.

1	Early lipoblasts look like fibroblasts but develop small lipid inclusions and a thin external lamina. Transmission electron microscopy (TEM) studies reveal that early lipoblasts have an elongated configuration, multiple cytoplasmic processes, and abundant endoplasmic reticulum and Golgi apparatus. As lipoblastic differentiation begins, vesicles increase in number, with a corresponding decrease in rough-surfaced endoplasmic reticulum (rER). Small lipid inclusions appear at one pole of the cytoplasm. Pinocytotic vesicles and an external lamina also appear. The presence of an external lamina is a feature that further distinguishes adipocytes from proper connective tissue cells. Midstage lipoblasts become ovoid as lipid accumulation changes the cell dimensions.

1	Midstage lipoblasts become ovoid as lipid accumulation changes the cell dimensions. With continued development, the early lipoblasts assume an oval configuration. The most characteristic feature at this stage is an extensive concentration of vesicles and small lipid droplets around the nucleus and extending toward both poles of the cell. Glycogen particles appear at the periphery of the lipid droplets, and pinocytotic vesicles and basal lamina become more apparent. These cells are designated midstage lipoblasts.

1	TABLE Summary of Molecules Synthesized and Secreted by Adipose Tissue and Their Functions 9.1 Molecule Major Function or Effect Acylation-stimulating Influences the rate of triglyceride synthesis in adipose tissue protein (ASP) Adiponectin, adipocyte Stimulates fatty-acid oxidation complement–related Decreases plasma triglycerides and glucose concentrations and increases insulin protein (ACRP30) sensitivity in cells Plays a role in the pathogenesis of familial combined hyperlipidemia Correlated with insulin resistance and hyperinsulinemia Adipophilin Serves as a specific marker for lipid accumulation in cells Adipsin Serine proteinase that regulates adipose tissue metabolism by facilitating fatty-acid storage and stimulating triglyceride synthesis Angiotensinogen (AGE) ACG is the precursor of vasoactive angiotensin II (AngII) that regulates blood pressure and Angiotensin II and electrolyte levels in the serum and is also involved in the metabolism and (AngII) differentiation of

1	of vasoactive angiotensin II (AngII) that regulates blood pressure and Angiotensin II and electrolyte levels in the serum and is also involved in the metabolism and (AngII) differentiation of adipose tissue During development, AngII inhibits differentiation of lipoblasts; in mature adipocytes it regulates lipid storage Insulinlike growth factor I Stimulates proliferation of a wide variety of cells and mediates many of the effects (IGF-I) of growth hormone Interleukin 6 (IL-6) Interacts with cells of immune system and regulates glucose and lipid metabolism Decreases activity of adipose tissue in cancer and other wasting disorders Leptin Regulates appetite and body energy expenditure Signals to the brain about body fat stores Increases formation of new vessels (angiogenesis) Involved in blood pressure control by regulating vascular tone Potent inhibitor of bone formation Plasminogen activator Inhibits the fibrinolytic system inhibitor-1 (PAI-1) Elevated levels are associated with

1	in blood pressure control by regulating vascular tone Potent inhibitor of bone formation Plasminogen activator Inhibits the fibrinolytic system inhibitor-1 (PAI-1) Elevated levels are associated with increased formation of blood clots Prostaglandins I2 and Helps regulate inflammation, blood clotting, ovulation, menstruation, F2, (PGI2 and PGF2) and acid secretion Resistin Increases insulin resistance Linked to obesity and to type 2 diabetes Transforming growth Regulates a wide variety of biologic responses, including proliferation, differentiation, factor (TGF-) apoptosis, and development Tumor necrosis factor Interferes with insulin receptor signaling and is a possible cause of development (TNF-) of insulin resistance in obesity (Modified from Frbeck G, Gomez-Ambrosi J, Muruzabal FJ, Burrell MA. The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation. Am J Physiol Endocrinol Metab 2001;280:E827–E847.)

1	The mature adipocyte is characterized by a single, large lipid inclusion surrounded by a thin rim of cytoplasm. In the late stage of differentiation, the cells increase in size and become more spherical. Small lipid droplets coalesce to form a single large lipid droplet that occupies the central portion of the cytoplasm. Smooth-surfaced endoplasmic reticulum (sER) is abundant, whereas rER is less prominent. These cells are designated late lipoblasts. Eventually, the lipid mass compresses the nucleus to an eccentric position, producing a signet-ring appearance in hematoxylin-and-eosin (H&E) preparations. Because these cells have a single lipid droplet, they are designated unilocular [Lat. unus, single; loculus, a little place] adipocytes or mature lipocytes. Structure of Adipocytes and Adipose Tissue Unilocular adipocytes are large cells, sometimes 100 m or more in diameter.

1	Structure of Adipocytes and Adipose Tissue Unilocular adipocytes are large cells, sometimes 100 m or more in diameter. When isolated, white adipocytes are spherical, but they may appear polyhedral or oval when crowded together in adipose are surrounded by reticular fibers (type III collagen), which are mesechymal stem cell secreted by the adipocytes. Special stains also reveal the pres ence of unmyelinated nerve fibers and numerous mast cells. A summary of white adipose tissue features is listed in Table 9.2.

1	FIGURE 9.1 • Development of adipose tissue cells. Like all connective tissue cells, adipocytes are derived from undifferentiated mesenchymal stem cells. By expressing PPAR-/ RXR transcription factors, they become committed to becoming early lipoblasts (preadipocytes) committed to white adipocyte lineage development. By expressing PRDM16/PGC-1 transcription factors, these cells will differentiate into early lipoblasts committed to brown adipocyte lineage development. Lipoblasts develop an external (basal) lamina and begin to accumulate numerous lipid droplets in their cytoplasm. In white adipose tissue, these droplets fuse to form a single large lipid droplet that ultimately fills the mature cell, compressing the nucleus, cytoplasm, and cytoplasmic organelles into a thin rim around the droplet. In brown adipose tissue, the individual lipid droplets remain separate.

1	tissue. Their large size is due to the accumulated lipid in the cell. The nucleus is flattened and displaced to one side of the lipid mass; the cytoplasm forms a thin rim around the lipid. In routine histologic sections, the lipid is lost through extraction by organic solvents such as xylene; consequently, adipose tissue appears as a delicate meshwork of polygonal profiles (Fig. 9.2). The thin strand of meshwork that separates adjacent adipocytes represents the cytoplasm of both cells and the extracellular matrix. The strand is usually so thin, however, that it is not possible to resolve its component parts in the light microscope. Adipose tissue is richly supplied with blood vessels, and capillaries are found at the angles of the meshwork where adjacent adipocytes meet. Silver stains show that adipocytes The lipid mass in the adipocyte is not membrane bounded.

1	The lipid mass in the adipocyte is not membrane bounded. The TEM reveals that the interface between the contained lipid and surrounding cytoplasm of the adipocyte is composed of a 5-nm-thick condensed layer of lipid reinforced by parallel vimentin flaments measuring 5 to 10 nm in diameter. This layer separates the hydrophobic contents of the lipid droplet from the hydrophilic cytoplasmic matrix. The perinuclear cytoplasm of the adipocyte contains a small Golgi apparatus, free ribosomes, short profiles of rER, microfilaments, and intermediate filaments. Filamentous mitochondria and multiple profiles of sER are also found in the thin rim of cytoplasm surrounding the lipid droplet (Fig. 9.3). Regulation of Adipose Tissue

1	Regulation of Adipose Tissue It is almost impossible to separate regulation of adipose tissue from digestive processes and functions of the central nervous system. These interconected hormonal and neural signals emanating from the adipose tissue, alimentary tract, and central nervous system form the brain–gut–adipose axis that regulates appetite, hunger, satiety, and energy homeostasis (Fig 9. 4). The amount of an individual’s adipose tissue is determined by two physiologic systems: One associated with short-term weight regulation, the other with long-term weight regulation.

1	The amount of an individual’s adipose tissue is determined by two physiologic systems: One associated with short-term weight regulation, the other with long-term weight regulation. The amount of adipose tissue in an individual is regulated by two physiologic systems. The first system, which is associated with short-term weight regulation, controls appetite and metabolism on a daily basis. Recently, two small peptide hormones produced in the gastrointestinal tract— ghrelin, an appetite stimulant, and peptide YY (PYY), an appetite suppressant—have been linked to this system. The second system, which is associated with long-term weight regulation, controls appetite and metabolism on a continual basis (over months and years). Two major hormones influence this system, leptin and insulin, along with other hormones, including thyroid hormone, glucocorticoids, and hormones of the pituitary gland (see Fig. 9.4).

1	Ghrelin and peptide YY control appetite as part of the short-term weight control system. The recently discovered potent appetite stimulant ghrelin is a small, 28-amino-acid polypeptide produced by gastric epithelial cells. In addition to its appetite stimulatory role, it acts on the anterior lobe of the pituitary gland to release growth hormone. In humans, ghrelin functions through receptors located in the hypothalamus, increasing the sense of hunger. As such it is considered a “meal initiator” factor. A genetic mutation in chromosome 15 causes Prader-Willi syndrome, in which an overproduction of ghrelin leads to morbid obesity. In individuals with this syndrome, compulsive eating and an obsession with food usually arise at an early age. The urge to eat

1	FIGURE 9.2 • White adipose tissue. a. Photomicrograph of white adipose tissue, showing its characteristic meshwork in an H&E–stained paraffin preparation. Each space represents a single large drop of lipid before its dissolution from the cell during tissue preparation. The surrounding eosin-stained material represents the cytoplasm of the adjoining cells and some intervening connective tissue. 320. b. High-power photomicrograph of a glutaraldehyde-preserved, plastic-embedded specimen of white adipose tissue. The cytoplasm of the individual adipose cells is recognizable in some areas, and part of the nucleus of one of the cells is included in the plane of section. A second nucleus (arrow), which appears intimately related to one of the adipose cells, may actually belong to a fibroblast; it is difficult to tell with assurance. Because of the large size of adipose cells, the nucleus is infrequently observed in a given cell. A capillary and a small venule are also evident in the

1	it is difficult to tell with assurance. Because of the large size of adipose cells, the nucleus is infrequently observed in a given cell. A capillary and a small venule are also evident in the photomicrograph. 950.

1	in these individuals is physiologic, overwhelming, and very difficult to control. If not treated, these individuals often die before age 30 of complications attributable to obesity. The small, 36-amino-acid long gastrointestinal hormone peptide YY is produced by the small intestine and plays an important role in promoting and maintaining weight loss by inducing a greater sense of fullness soon after a meal. It also acts through receptors in the hypothalamus that suppress appetite. It decreases food intake in individuals by inducing satiety or a sense of fullness and the desire to stop eating. In experimental clinical studies, the infusion of PYY into humans has been shown to reduce food intake by 33% over a period of 24 hours. Two hormones, leptin and insulin, are responsible for long-term regulation of body weight.

1	The discovery of the leptin (ob) gene, which encodes a fat-specific messenger RNA (mRNA) for leptin, has given some insight into the mechanism of energy homeostasis. In experimental animal models, the addition of recombinant leptin to obese, leptin-deficient ob/ob mice causes them to reduce their food intake and lose about 30% of their total body weight after 2 weeks of treatment. Unlike mutant mice, in most obese humans, levels of leptin mRNA in adipose tissue as well as serum levels of leptin are elevated. This was observed in all types of obesity, regardless of whether it is caused by genetic factors, hypothalamic lesions, or increased efficiency of food utilization. For unknown reasons, adipocytes in these obese individuals are resistant to leptin’s action, and administration of leptin does not reduce the amount of adipose tissue. Conversely, studies of individuals who have lost weight and those with anorexia nervosa show that leptin mRNA levels in their adipose tissue and serum

1	does not reduce the amount of adipose tissue. Conversely, studies of individuals who have lost weight and those with anorexia nervosa show that leptin mRNA levels in their adipose tissue and serum levels of leptin are significantly reduced. Recent clinical findings indicate that leptin most likely protects the body against weight loss in times of food deprivation.

1	Insulin, the pancreatic hormone that regulates blood glucose levels, is also involved in regulation of adipose tissue metabolism. It enhances the conversion of glucose into FIGURE 9.3 • Electron micrograph showing portions of two adjacent adipose cells. The cytoplasm of the adipose cells reveals mitochondria (M) and glycogen (the latter appears as the very dark particles). 15,000. Upper inset. Attenuated cytoplasm (Cy) of two adjoining adipose cells. Each cell is separated by a narrow space containing external (basal) lamina and an extremely attenuated process of a fibroblast. 65,000. Lower inset. The external (basal) lamina (BL) of the adipose cells appears as a discrete layer by which the cells are adequately separated from one another. F, fibroblast processes. 30,000.

1	the triglycerides of the lipid droplet by the adipocyte. Like leptin, insulin regulates weight by acting on brain centers in the hypothalamus. In contrast to leptin, insulin is required for the accumulation of adipose tissue. Antiobesity drug research is currently focusing on substances that can inhibit insulin and leptin signaling in the hypothalamus. Deposition and mobilization of lipid are influenced by neural and hormonal factors.

1	Deposition and mobilization of lipid are influenced by neural and hormonal factors. One of the major metabolic functions of adipose tissue involves the uptake of fatty acids from the blood and their conversion to triglyceride within the adipocyte. Triglyceride is then stored within the cell’s lipid droplet. When adipose tissue is stimulated by neural or hormonal mechanisms, triglycerides are broken down into glycerol and fatty acids, a process called mobilization. The fatty acids pass through the adipocyte cell membrane to enter a capillary. Here they are bound to the carrier protein albumin and transported to other cells, which use fatty acids as metabolic fuel.

1	Neural mobilization is particularly important during periods of fasting and exposure to severe cold. During the early stages of experimental starvation in rodents, adipose cells in a denervated fat pad continue to deposit fat. Adipose cells in the intact contralateral fat pad mobilize fat. It is now known that norepinephrine (which is liberated by the endings of nerve cells of the sympathetic nervous system) initiates a series of metabolic steps that lead to the activation of lipase. This enzyme splits triglycerides, which constitute more than 90% of the lipids stored in the adipocyte. This enzymatic activity is an early step in the mobilization of lipids.

1	Hormonal mobilization involves a complex system of hormones and enzymes that controls fatty-acid release from adipocytes. These include insulin, thyroid hormones, and adrenal steroids. Insulin is an important hormone that promotes lipid synthesis by stimulating lipid synthesis enzymes (fatty-acid synthase, acetyl-CoA carboxylase) and suppresses lipid degradation by inhibiting the action of hormone-sensitive lipase and thus blocking the release of fatty acids. Glucagon, another pancreatic hormone, and growth hormone from the pituitary gland both increase lipid utilization (lipolysis). In addition, elevated levels of tumor necrosis factor (TNF-) have been implicated as a causative factor in the development of insulin resistance associated with obesity and diabetes. Adipocytes of brown, multilocular adipose tissue contain numerous fat droplets. The cells of brown (multilocular) adipose tissue are smaller than those of white adipose tissue. The nucleus of a muscle, other tissues

1	The cells of brown (multilocular) adipose tissue are smaller than those of white adipose tissue. The nucleus of a muscle, other tissues FIGURE 9.4 • Regulation of energy homeostasis. This schematic diagram shows the relationship of adipose tissue to the central nervous system and gastrointestinal system within the brain– gut–adipose axis that is responsible for regulating energy homeostasis.

1	mature multilocular adipocyte is typically in an eccentric position within the cell, but it is not flattened as is the nucleus of a unilocular adipocyte. In routine H&E–stained sections, the cytoplasm of the multilocular adipocyte consists largely of empty vacuoles because the lipid that ordinarily occupies the vacuolated spaces is lost during preparation (Fig. 9.5). Multilocular adipocytes depleted of their lipid bear a closer resemblance to epithelial cells than to connective tissue cells. The multilocular adipocyte contains numerous mitochondria, a small Golgi apparatus, and only small amounts of rER and sER. The mitochondria contain large amounts of cytochrome oxidase, which imparts the brown color to the cells. Brown adipose tissue features are listed in Table 9.2. Brown adipose tissue, abundant in newborns, is markedly reduced in adults.

1	Brown adipose tissue, abundant in newborns, is markedly reduced in adults. Brown adipose tissue is present in large amounts in the newborn, which helps offset the extensive heat loss that results from the newborn’s high surface-to-mass ratio and avoid lethal hypothermia (a major risk of death for premature babies). In newborns, brown adipose tissue makes up about 5% of the total body mass and is located on the back, along the upper half of the spine, and toward the shoulders. The amount of brown adipose tissue gradually decreases as the body grows, but it remains widely distributed throughout the first decade of life in the cervical, axillary, paravertebral, mediastinal, sternal, and abdominal regions of the body. It then disappears from most sites except for regions around the kidney, adrenal glands, large vessels (i.e., aorta), and regions of the neck (deep cervical and supraclavicular), regions of the back (interscapular and paravertebral), and thorax (mediastinum).

1	Brown adipose tissue is subdivided into lobules by partitions of connective tissue, but the connective tissue stroma between individual cells within the lobules is sparse. The tissue has a rich supply of capillaries that enhance its color. Numerous unmyelinated nerve fibers are present among the fat cells. Brown adipocytes differentiate from mesenchymal stem cells under the control of PRDM16/PGC-1 transcription factors in the presence of catecholamines.

1	Brown adipocytes are derived from undifferentiated mesenchymal stem cells. In contrast to white adipocytes, differentiation of brown adipocytes is under the influence of a different pair of transcription factors. When the zinc-finger protein known as PR domain containing 16 (PRDM16) is activated, mesenchymal stem cells synthesize several members of the PPAR coactivator-1 (PGC-1) family of transcription factors. Therefore, PRDM16/PGC-1 is regarded as a “master switch” regulator in the brown adipocytes’ differentiation. These factors in turn regulate expression of genes (i.e., UPC-1) that control brown fat differentiation. The UPC-1 gene encodes a specific mitochondrial protein called uncoupling protein (UCP-1) or thermogenin (a 33-kilodalton inner mitochondrial membrane protein) is essential for brown adipocytes metabolism (thermogenesis). Clinical observations confirm that under normal conditions brown adipose tissue can expand in response to increased blood levels of norepinephrine.

1	for brown adipocytes metabolism (thermogenesis). Clinical observations confirm that under normal conditions brown adipose tissue can expand in response to increased blood levels of norepinephrine. This becomes evident in patients with pheochromocytoma, an endocrine tumor of adrenal medulla secreting excessive amounts of epinephrine and norepinephrine. In these individuals, the  FOLDER 9.1

1	Clinical Correlation: Obesity

1	Obesity is epidemic in the United States. According to current estimates by the National Institutes of Health (NIH), about two-thirds of Americans are considered to be obese, and 300,000 die annually from obesity-related metabolic diseases (i.e., diabetes, hypertension, cardiovascular dis-eases, and cancer). An individual is considered obese when the percentage of body fat exceeds the average per-centage for the individual’s age and sex. The prevalence of obesity has increased in the last decade from 12% to 18%. The increases are seen in both sexes and at all so-cioeconomic levels, with the greatest increase reported in the 18to 29-year-old age group. The body mass index (BMI), expressed as weight/ height2, is closely correlated with the total amount of body fat and is commonly used to classify overweight and obe-sity among adults. A BMI of approximately 25 kg/m2 is considered normal. A BMI greater than 27 kg/m2, which correlates to an excess body weight of approximately 20%, is

1	to classify overweight and obe-sity among adults. A BMI of approximately 25 kg/m2 is considered normal. A BMI greater than 27 kg/m2, which correlates to an excess body weight of approximately 20%, is considered a health risk. Obesity is associated with an increased risk of mortality as well as with many diseases such as hypertension, car-diovascular diseases, diabetes, and cancer. It is a chronic condition that develops as a result of an interaction be-tween a person’s genetic makeup and his or her environ-ment. Obesity genes encode the molecular components of the short-term and long-term weight regulation systems, which include leptin, ghrelin, and other factors that regulate energy balance. In addition, several of these factors modu-late glucose metabolism by adipose tissue and contribute to the development of insulin resistance, which is associ-ated with type 2 diabetes. Intensive research directed to-ward adipocyte-derived proteins may in the future provide potential drugs for

1	to the development of insulin resistance, which is associ-ated with type 2 diabetes. Intensive research directed to-ward adipocyte-derived proteins may in the future provide potential drugs for reducing obesity and overcoming insulin resistance. blood vesselblood vessel connectiveconnective tissue septatissue septa

1	FIGURE 9.5 • Brown adipose tissue. a. Photomicrograph of brown adipose tissue from a newborn in an H&E–stained paraffin preparation. The cells contain fat droplets of varying size. 150. b. This photomicrograph, obtained at a higher magnification, shows the brown adipose cells with round and often centrally located nuclei. Most of the cells are polygonal and closely packed, with numerous lipid droplets. In some cells, large lipid droplets displace nuclei toward the cell periphery. A network of collagen fibers and capillaries surrounds the brown adipose cells. 320. UCP-1 gene is activated by norepinephrine stimulation, which also protects brown adipocytes by inhibiting apoptosis.

1	UCP-1 gene is activated by norepinephrine stimulation, which also protects brown adipocytes by inhibiting apoptosis. In the past, it was thought that uncoupling proteins were expressed only in brown adipose tissue. Recently, several similar uncoupling proteins have been discovered in other tissues. UCP-2 is linked to hyperinsulimenia and obesity and may be involved in regulation of body weight. UPC-3 is expressed in skeletal muscles and may account for the thermogenic effects of thyroid hormone. UPC-4 is a brain-specific molecule. Metabolism of lipid in brown adipose tissue generates heat in process known as thermogenesis. Hibernating animals have large amounts of brown adipose tissue. The tissue serves as a ready source of lipid. When oxidized, it produces heat to warm the blood flowing through the brown fat on arousal from hibernation and in the maintenance of body temperature in the cold. This type of heat production is known as nonshivering thermogenesis.

1	Brown adipose tissue is also present in nonhibernating animals and humans and again serves as a source of heat. As in the mobilization of lipid in white adipose tissue, lipid is mobilized, and heat is generated by brown adipocytes when they are stimulated by the sympathetic nervous system. Therefore, normally present brown adipose tissue can most likely be induced and function in the context of human adaptive thermogenesis. Future research is being directed toward finding mechanisms for increased brown fat differentiation, which may potentially be an attractive treatment in both diet-induced and genetically acquired obesity.

1	The study of the numerous varieties of benign and malig-nant adipose tissue tumors provides further insight into, and confirmation of, the sequence of adipose tissue differ-entiation described above. As with epithelial tumors and tu-mors of fibroblast origin, the variety of adipose tissue tumors reflects the normal pattern of adipose tissue differ-entiation; that is, discrete tumor types can be described that consist primarily of cells resembling a given stage in normal adipose tissue differentiation. The most common benign tumor of adipose tissue of adulthood is the lipoma. It is more common than all other soft tissue tumors combined. Lipomas are subclassified by the morphology of the predominant cell in the tumor. For in-stance, the conventional lipoma consists of mature white adipocytes, whereas a fbrolipoma has adipocytes sur-rounded by an excess of fibrous tissue and an angio lipoma contains adipocytes separated by an unusually large num-ber of vascular channels. The majority of

1	whereas a fbrolipoma has adipocytes sur-rounded by an excess of fibrous tissue and an angio lipoma contains adipocytes separated by an unusually large num-ber of vascular channels. The majority of lipomas show structural chromosome aberrations that include balanced re-arrangements, often involving chromosome 12. Lipomas are usually found in subcutaneous tissues in middle-aged and elderly individuals. They are characterized as well-defined, soft, and painless masses of mature adipocytes usually found in the subcutaneous fascia of the back, thorax, and proximal parts of the upper and lower limbs. Treatment of lipomas usually involves a simple surgical excision. Malignant tumors of adipose tissue, called liposarco-mas, are rare. They are typically detected in older individu-als and are mainly found in the deep adipose tissues of the lower limbs, abdomen, and the shoulder area. Liposarco-mas may contain both well-differentiated, mature adipocytes and early, undifferentiated cells (Fig.

1	found in the deep adipose tissues of the lower limbs, abdomen, and the shoulder area. Liposarco-mas may contain both well-differentiated, mature adipocytes and early, undifferentiated cells (Fig. F9.2.1). Tumors containing more cells in earlier stages of differenti-ation are more aggressive and more frequently metasta-size. Typically, liposarcomas are surgically removed, but if a tumor has already metastasized, then both chemotherapy and radiation therapy can be utilized as a preor postsurgi-cal treatment. Although the term lipoma relates primarily to white adi-pose tissue tumors, tumors of brown adipose tissue are also found. Not surprisingly, these are called hibernomas. They are rare, benign, and slow-growing soft tissue tumors of brown fat most commonly arising in the periscapular region, axillary fossa, neck, or mediastinum. Most hibernomas contain a mixture of white and brown adipose tissue; pure hibernomas are very rare.

1	 FOLDER 9.2 Clinical Correlation: Adipose Tissue Tumors FIGURE F9.2.1 • Well-differentiated liposarcoma. This photomicrograph was obtained from a tumor surgically removed from the retroperitoneal space of the abdomen. Well-differentiated liposarcoma is characterized by a predominance of mature adipocytes that vary in size and shape. They are interspersed between broad fibrous septa of connective tissue containing cells (majority of them are fibroblasts) with atypical hyperchromatic nuclei. A relatively few scattered spindle cells with hyperchromatic and pleomorphic nuclei are found within connective tissue. 340. (Courtesy of Dr. Fabiola Medeiros.) Thermogenic activity of brown adipose tissue is facilitated by UCP-1 that is found at the inner mitochondrial membrane.

1	Thermogenic activity of brown adipose tissue is facilitated by UCP-1 that is found at the inner mitochondrial membrane. The mitochondria in eukaryotic cells produce and store energy as an electrochemical proton gradient across the inner mitochondrial membrane. As described earlier (see page 55), this energy is used to synthesize ATP when the protons return to the mitochondrial matrix through the ATP synthase enzyme located at the inner mitochondrial membrane.

1	The mitochondria in the cytoplasm of brown adipose tissue cells contain uncoupling protein (UCP-1), which uncouples the oxidation of fatty acids from the production of ATP. Therefore, protons are allowed to travel from the inter-membrane space back to the mitochondrial matrix along the gradient without passing through ATP synthase and thus without producing ATP. This can occur since an alternative pathway for the protons return is available through an UCP-1 that facilitates proton transport across the inner mitochondrial membrane. The movement of protons from the inner mitochondrial compartment dissipates the mitochondrial proton gradient, thus uncoupling respiration from ATP synthesis. The energy produced by the mitochondria is then dissipated as heat in process known as thermogenesis. The metabolic activity of brown adipose tissue is regulated by the sympathetic nerve system and is related to ambient outdoor temperature.

1	The metabolic activity of brown adipose tissue is largely regulated by norepinephrine released from the sympathetic nerve terminals, which stimulates lipolysis and hydrolysis of triglycerydes as well as increases mitochondrial expression and activity of UCP-1 molecules. In experimental animals, chapter 9 Adipose Tissue B ROWN AD I POSE TISS U E TABLE Summary of Adipose Tissue Features 9.2 Features White Adipose Tissue Brown Adipose Tissue Location Subcutaneous layer, mammary gland, Large amounts in newborn greater omentum, mesenteries, Remnants in adults at the retroperitoneal retroperitoneal space, visceral space, deep cervical and supraclavicular pericardium, orbits (eye sockets), regions of the neck, interscapular, bone marrow cavity paravertebral regions of the back, mediastinum Function Metabolic energy storage, insulation, Heat production (thermogenesis) cushioning, hormone production, source of metabolic water Adipocyte morphology Unilocular, spherical, flatten nucleus,

1	Function Metabolic energy storage, insulation, Heat production (thermogenesis) cushioning, hormone production, source of metabolic water Adipocyte morphology Unilocular, spherical, flatten nucleus, Multilocular, spherical, round rim of cytoplasm eccentric nucleus Large diameter (15–150 m) Smaller diameter (10–25 m) Transcription factors “master PPAR-/RXR PRDM16/PGC-1 switch” in differentiation UCP-1 genes expression No Yes (unique to brown fat) Mitochondria Few, poorly developed Many, well developed Innervation Few sympathetic nerve fibers High density of sympathetic nerve fibers Vascularization Few blood vessels Highly vascularized tissue Response to environmental Decreased lipogenesis Increased lipogenesis stress (cold exposure) Increased lipoprotein lipase activity Decreased lipoprotein lipase activity Growth and differentiation Throughout entire life from Only during fetal period stromal-vascular cells Decreases in adult life (exception: individuals with pheochromocytoma and

1	lipase activity Growth and differentiation Throughout entire life from Only during fetal period stromal-vascular cells Decreases in adult life (exception: individuals with pheochromocytoma and hibernoma)  FOLDER 9.3 Clinical Correlation: PET Scanning and Brown Adipose Tissue Interference

1	Positron emission tomography, also called a “PET scan,” is a diagnostic tool that can locate malignant cells in the body. PET is based on the detection of high-energy gamma rays created when positrons (subatomic particles of antimatter), produced during decay of radioactive mate-rials, are encountered by electrons. The procedure re-quires the injection of a radioactive tracer, most commonly 18-fuorine-2-fuoro-2-deoxy-D-glucose (18F-FDG). This radioactive glucose isotope is used in PET imaging because malignant cells metabolize glucose at a greater rate than normal cells. After injection of the isotope, a de-tector scans the entire body and records radiation emitted by the 18F-FDG tracer as it becomes incorporated within the body’s cells. A computer reassembles the signals into images, which are, in effect, biological maps of 18F-FDG distribution in the body. Recently, due to greater diagnostic accuracy and improved biopsy methods, combined positron emission tomography and computed

1	which are, in effect, biological maps of 18F-FDG distribution in the body. Recently, due to greater diagnostic accuracy and improved biopsy methods, combined positron emission tomography and computed tomography (PET/CT) scanners are utilized more frequently. One drawback to PET imaging is that many normal tissues and benign lesions also show increased glucose metabolism and can thus be misinterpreted as malignant. For example, brown adipose tissue, with its increased glucose uptake me-diated by increased activity of glucose transporters, can be a potential source of false-positive interpretation of PET scans. Since brown adipose tissue is present in the neck, supraclavicular regions, and mediastinum (see page 260) it is commonly observed on PET scans, especially in underweight patients and during winter months, when brown adipose tis-sue is more predominant. This 18F-FDG uptake most likely represents activated brown adipose tissue during increased sympathetic nerve activity related to

1	during winter months, when brown adipose tis-sue is more predominant. This 18F-FDG uptake most likely represents activated brown adipose tissue during increased sympathetic nerve activity related to cold stress. A typical PET image of brown fat is usually bilateral and symmetric; however, in the mediastinum, the image may be asymmetric or focal and can mimic malignancy. False-positive results from brown fat 18F-FDG uptake in these areas have been reported in young women undergoing scans for diagnosis and staging of breast cancer. Therefore, under-standing that brown fat can show that increased radioac-tive tracer uptake is crucial for establishing an accurate diagnosis and avoiding false-positive results (Fig. F9.3.1). FIGURE F9.3.1 • Coronal positron emission tomography/ computed tomography (PET/CT) image of a healthy young woman. This upper part of the coronal section of this whole-body PET/CT scan shows extensive bilateral increased 18F-FDG uptake (red color) in the neck,

1	tomography (PET/CT) image of a healthy young woman. This upper part of the coronal section of this whole-body PET/CT scan shows extensive bilateral increased 18F-FDG uptake (red color) in the neck, supraclavicular, and upper axillary regions. Note that moderate increased radioactive tracer uptake is also detectable in the myocardium (yellow color). Regions of extensive metabolic activity correlate with the distribution pattern of low-density brown adipose tissue. PET/CT imaging allows for precise localization of increased 18F-FDG uptake areas and differentiation between brown adipose tissue tracer uptake and malignant tumor findings. (Courtesy of Dr. Jolanta Durski.)

1	UCP-1 activity has been shown to increase during cold stress. In addition, cold stimulates glucose utilization in brown adipocytes by overexpression of glucose transporters (Glut-4). Recent clinical studies using PET scans in adults have shown a direct relationship between outdoor temperature and the amount of brown fat accumulated in the body. An increase in the amount of brown adipose tissue has been reported on the neck and supraclavicular regions during the winter months especially in lean individuals. This is supported by autopsy findings of larger amounts of brown fat in outdoor workers exposed to cold. Modern molecular imaging techniques now allow clinicians to precisely locate where brown fat is distributed in the body, which is essential for proper diagnosis of cancerous lesions (see Folder 9.3). This page intentionally left blank.

1	ADI POS E TI SS U E 266 Adipose tissue is widely distributed throughout the body and in varying amounts in different individuals. It is a specialized connective tissue consisting of fat-storing cells, adipocytes, and has a rich blood supply. Two types of adipose tissue are recognized. The more common and abundant is referred to as white adipose tissue. Its adipocytes are very large cells whose cytoplasm contains a single large vacuole in which the fat is stored in the form of triglycerides. When observed in a typical H&E section, white adi-pose tissue appears as a mesh-like structure (see orientation micrograph). The second type is brown adipose tissue. It consists of smaller cells. Their cytoplasm is characterized by numerous vesicles that occupy much of the cells’ volume. It also is very richly vascu-larized. Brown adipose tissue is found in human newborns where it assists in maintaining body temperature. ORIENTATION MICROGRAPH: Shown here is white adipose tissue from the

1	is very richly vascu-larized. Brown adipose tissue is found in human newborns where it assists in maintaining body temperature. ORIENTATION MICROGRAPH: Shown here is white adipose tissue from the hypo-dermis of skin. It consists of numerous adipocytes closely packed in lobules. Dense con-nective tissue (DICT) surrounds the adipose tissue. The loss of the fat within the cell gives the adipose tissue a mesh-like appearance. Note the small blood vessels (BV) ob-served at the periphery of the tissue. They provide a rich capillary network within the adi-pose tissue. Several sweat gland ducts (SGD) are also present in the dense connective tissue. DICTBVSGDDICT BV SGD

1	White adipose tissue, human, H&E, ×363; inset ×700.

1	This is a higher magnification micrograph of white adipose tissue from the specimen shown in the orientation micro-graph. It reveals portions of several lobules of adipose cells. Dense connective tissue (DICT) separates the lobules from surrounding structures. In well preserved specimens, the adipocytes (A) have a spherical profile in which they exhibit a very thin rim of cytoplasm surrounding a single, large fat-containing vacuole. Because the fat is lost during tissue preparation, one only sees the rim of cytoplasm and an almost clear space. Between the cells, there is an extremely thin, delicate connective tissue stroma holding the adipocytes together and within this stroma are small blood vessels (BV), mostly capillaries and venules. The majority of nuclei that are observed within the adipose tissue belong to fibroblasts, adipocytes, or cells of small blood vessels. However, distinguishing between fibroblast nuclei and adipocyte nuclei is often difficult. The inset shows an

1	the adipose tissue belong to fibroblasts, adipocytes, or cells of small blood vessels. However, distinguishing between fibroblast nuclei and adipocyte nuclei is often difficult. The inset shows an adipocyte whose nucleus (N) is relatively easy to identify. It appears to reside within the rim of cytoplasm (Cy), giving the adipocyte the classic “signet ring” appearance. A second nucleus (N’), partially out of the plane of section, appears to reside between the cytoplasmic rim of two adjacent cells. This is probably the nucleus of a fibroblast. Because of the relatively large size of the adipocyte, it is very infrequent that the nucleus of the cell is included in the plane of section of a given cell. Other cells that may be seen within the delicate connective tissue stroma are mast cells (MC).

1	Brown adipose tissue, human, H&E, ×450; inset ×1100.

1	The brown adipose tissue shown here consists of small fat cells that are very closely packed with minimal intercellular space. Because of this arrangement, it is hard to define individual cells at this magnification. At higher magnification (not shown), it is possible to identify some individual cells. One cell, whose boundaries could be identified at higher magnification is circumscribed by a dotted line. Each cell contains many small, fat-containing vacuoles surrounded by cytoplasm. Included in this cell is its nucleus (N). As noted, brown adipose tissue is highly vascularized, and in this specimen, one can see numerous blood vessels (BV) as evidenced by the red blood cells that they contain. It is even more difficult to distinguish fibroblasts within the lobule from nuclei of the fat cells. Even at higher magnification (inset), it is difficult to determine which nuclei belong to which cells. A capillary (C) can be identified in the inset. Again, it is recognized by the presence of

1	Even at higher magnification (inset), it is difficult to determine which nuclei belong to which cells. A capillary (C) can be identified in the inset. Again, it is recognized by the presence of red blood cells. Where the lobules are slightly separated from one another (arrows), small elongate nuclei can be recognized. These belong to fibroblasts in the connective tissue forming the septa.

1	KEY DICT, dense irregular connective tissue A, adipocytes BV, blood vessels N, nucleus Cy, cytoplasm MC, mast cells C, capillary SGD, sweat gland ducts

1	OVERVIEW OF BLOOD / 268 PLASMA / 269 ERYTHROCYTES / 270 LEUKOCYTES / 275 Neutrophils / 275 Eosinophils / 280 Basophils / 282 Lymphocytes / 283 Monocytes / 286 THROMBOCYTES / 286 FORMATION OF BLOOD CELLS (HEMOPOIESIS) / 289 Monophyletic Theory of Hemopoiesis / 289 Development of Erythrocytes (Erythropoiesis) / 293 Kinetics of Erythropoiesis / 295 Development of Thrombocytes (Thrombopoiesis) / 295 Development of Granulocytes (Granulopoiesis) / 295 Kinetics of Granulopoiesis / 296 Development of Monocytes / 298 Development of Lymphocytes (Lymphopoiesis) / 298 BONE MARROW / 298 Folder 10.1 Clinical Correlation: ABO and Rh Blood Group Systems / 273 Folder 10.2 Clinical Correlation: Hemoglobin in Patients with Diabetes / 274 Folder 10.3 Clinical Correlation: Hemoglobin Disorders / 276 Folder 10.4 Clinical Correlation: Inherited Disorders of Neutrophils; Chronic Granulomatous Disease (CGD) / 281 Folder 10.5 Clinical Correlation: Hemoglobin Breakdown and Jaundice / 281 Folder 10.6 Clinical

1	10.4 Clinical Correlation: Inherited Disorders of Neutrophils; Chronic Granulomatous Disease (CGD) / 281 Folder 10.5 Clinical Correlation: Hemoglobin Breakdown and Jaundice / 281 Folder 10.6 Clinical Correlation: Cellularity of the Bone Marrow / 300

1	Blood is a fluid connective tissue that circulates through the cardiovascular system. Like the other connective tissues, blood consists of cells and an extracellular component. Total blood volume in the average adult is about 6 L or 7% to 8% of total body weight. The heart’s pumping action propels blood through the cardiovascular system to the body tissues. Blood’s many functions include:  delivery of nutrients and oxygen directly or indirectly to cells, transport of wastes and carbon dioxide away from cells, delivery of hormones and other regulatory substances to and from cells and tissues,  maintenance of homeostasis by acting as a buffer and participating in coagulation and thermoregulation, and  transport of humoral agents and cells of the immune system that protect the body from pathogenic agents, foreign proteins, and transformed cells (i.e., cancer cells). Blood consists of cells and their derivatives and a protein-rich fluid called plasma.

1	Blood consists of cells and their derivatives and a protein-rich fluid called plasma. Blood cells and their derivatives include:  erythrocytes, also called red blood cells (RBCs), leukocytes, also known as white blood cells (WBCs), and thrombocytes, also termed platelets. Plasma is the liquid extracellular material that imparts fluid properties to blood. The relative volume of cells and plasma in whole blood is approximately 45% and 55%, respectively. The TABLE Formed Elements of the Blood 10.1 Cells/L Formed Elements Male Female % Erythrocytes 4.3– 5.7 1012 3.9–5.0 1012 Leukocytes 3.5–10.5 109 3.5–10.5 109 100 Agranulocytes Lymphocytes 0.9–2.9 109 0.9–2.9 109 25.7–27.6a Monocytes 0.3–0.9 109 0.3–0.9 109 8.6a Granulocytes Neutrophils 1.7–7.0 109 1.7–7.0 109 48.6–66.7a Eosinophils 0.05–0.5 109 0.05–0.5 109 1.4–4.8a Basophils 0–0.03 109 0–0.03 109 0–0.3a Thrombocytes (platelets) 150–450 109 150–450 109 aPercentage of leukocytes.

1	volume of packed erythrocytes in a sample of blood is called the hematocrit. The hematocrit is measured by centrifuging a blood sample to which anticoagulants have been added, and then calculating the percentage of the centrifuge tube volume occupied by the erythrocytes compared with that of the whole blood. A normal hematocrit reading is about 39% to 50% in men and 35% to 45% in women; thus, 39% to 50% and 35% to 45% of the blood volume for men and women, respectively, consists of erythrocytes. Low hematocrit values often reﬂect reduced numbers of circulating erythrocytes (a condition called anemia) and may indicate significant blood loss caused by internal or external bleeding.

1	Leukocytes and platelets constitute only 1% of the blood volume. In a blood sample that has been centrifuged, the cell fraction (the part of the sample that contains the cells) consists mainly of packed erythrocytes (99%). The leukocytes and platelets are contained in a narrow layer at the upper part of the cell fraction called the buffy coat. As Table 10.1 indicates, there are nearly 1,000 times more erythrocytes (5 1012 cells/L of blood) than leukocytes (7 109/L of blood). Although the blood cells are the major objects of interest in histology, a brief examination of plasma is also useful. The composition of plasma is summarized in Table 10.2. More than 90% of plasma by weight is water, which serves as the solvent for a variety of solutes, including proteins, dissolved gases, electrolytes, nutrients, regulatory substances, and waste materials. The solutes in the plasma help maintain homeostasis, a steady state that provides optimal pH and osmolarity for cellular metabolism.

1	Plasma proteins consist primarily of albumin, globulins, and fibrinogen. Albumin is the main protein constituent of the plasma, accounting for approximately half of the total plasma proteins. It is the smallest plasma protein (about 70 kilodaltons) and is made in the liver. Albumin is responsible for exerting the concentration gradient between blood and extracellular tissue fluid. This major osmotic pressure on the blood vessel wall, called the colloid osmotic pressure, maintains the correct proportion of blood to tissue fluid volume. If a significant amount of albumin leaks out of the blood vessels into the loose connective tissue or is lost from the blood to urine in the kidneys, then the colloid osmotic pressure of the blood decreases, and ﬂuid accumulates in the tissues. (This increase in tissue ﬂuid is most

1	TABLE Composition of Blood Plasma10.2 Component % Water 91–92 Protein (albumin, globulins, fibrinogen) 7–8 Other solutes: 1–2 • Electrolytes (Na, K, Ca2, Mg2, Cl, HCO3 , PO4 3, SO4 2) • Nonprotein nitrogen substances (urea, uric acid, creatine, creatinine, ammonium salts) • Nutrients (glucose, lipids, amino acids) • Blood gases (oxygen, carbon dioxide, nitrogen) • Regulatory substances (hormones, enzymes) readily noted by swelling of the ankles at the end of a day.) Albumin also acts as a carrier protein; it binds and transports hormones (thyroxine), metabolites (bilirubin), and drugs (barbiturates). Globulins include the immunoglobulins (-globulins), the largest component of the globulin fraction, and nonimmune globulins (-globulin and -globulin). The immunoglobulins are antibodies, a class of functional immune-system molecules secreted by plasma cells. (Antibodies are discussed in Chapter 14, Lymphatic System.)

1	Nonimmune globulins are secreted by the liver. They help maintain the osmotic pressure within the vascular system and also serve as carrier proteins for various substances such as copper (by ceruloplasmin), iron (by transferrin), and the protein hemoglobin (by haptoglobin). Nonimmune globulins also include fibronectin, lipoproteins, coagulation factors, and other molecules that may exchange between the blood and the extravascular connective tissue. Fibrinogen, the largest plasma protein (340 kilodaltons), is made in the liver. In a series of cascade reactions with other coagulation factors, soluble fibrinogen is transformed into the insoluble protein fibrin (323 kilodaltons). During conversion of fibrinogen to fibrin, fibrinogen chains are broken to produce fibrin monomers that rapidly polymerize to form long fibers. These fibers become cross-linked to form an impermeable net at the site of damaged blood vessels, thereby preventing further blood loss.

1	With the exception of these large plasma proteins and regulatory substances, which are small proteins or polypeptides, most plasma constituents are small enough to pass through the blood vessel wall into the extracellular spaces of the adjacent connective tissue. In general, plasma proteins react with common fixatives; they are often retained within the blood vessels in tissue sections. Plasma proteins do not possess a structural form above the molecular level; thus, when they are retained in blood vessels in the tissue block, they appear as a homogeneous substance that stains evenly with eosin in hematoxylin and eosin (H&E)–stained sections. Serum is the same as blood plasma except that clotting factors have been removed.

1	For laboratory purposes, samples of blood are often drawn from a vein (the procedure called venipuncture). When blood is removed from the circulation, it immediately clots. A blood clot consists mostly of erythrocytes entangled in a network of fine fibers composed of fibrin. To prevent clotting of a blood sample, an anticoagulant such as citrate or heparin is added to the blood specimen as it is obtained. Citrate binds calcium ions, which are essential for triggering the cascade of coagulation reactions; heparin deactivates the clotting factors in the plasma. Plasma that lacks coagulation factors is called serum. For many biochemical laboratory tests, plasma and blood serum can be used interchangeably. Serum is preferred for several specific tests because the anticoagulants in plasma can interfere with the results. However, tests of clotting ability require that all coagulation factors be preserved; therefore, serum is inappropriate for these tests.

1	The interstitial fluid of connective tissues is derived from blood plasma. The fluid that surrounds tissue cells, called interstitial ﬂuid, not surprisingly, has an electrolyte composition that reflects that of blood plasma, from which it is derived. The composition of interstitial fluid in nonconnective tissues, however, is subject to considerable modification by the absorptive and secretory activities of epithelia. Epithelia may create special microenvironments conducive to their function. For example, a blood–brain barrier exists between the blood and nerve tissue. Barriers also exist between the blood and the parenchymal tissue in the testis, thymus gland, eye, and other epithelial compartments. Fluids, barriers, and their functions are discussed in subsequent chapters that describe these particular organs. Examination of blood cells requires special preparation and staining.

1	Examination of blood cells requires special preparation and staining. The preparation method that best displays the cell types of peripheral blood is the blood smear. This method differs from the usual preparation seen in the histology laboratory in that the specimen is not embedded in paraffin and sectioned. Rather, a drop of blood is placed directly on a slide and spread thinly over the surface of the slide (i.e., “pulled” with the edge of another slide) to produce a monolayer of cells (Fig. 10.1a). The preparation is then air dried and stained. Another difference in the preparation of a blood smear is that instead of H&E, special mixtures of dyes are used to stain the blood cells. The resulting preparation may then be examined with a high-power oil-immersion lens, with or without a coverslip (Fig. 10.1b and Plate 17, page 302).

1	The modified Romanovsky-type stain commonly used for blood smears consists of a mixture of methylene blue (a basic dye), related azures (also basic dyes), and eosin (an acidic dye). On the basis of their appearance after staining, leukocytes are traditionally divided into granulocytes (neutrophils, eosinophils, and basophils) and agranulocytes (lymphocytes and monocytes). Although both cell types may contain granules, the granulocytes possess obvious, specifically stained granules in their cytoplasm. In general, the basic dyes stain nuclei, granules of basophils, and the RNA of the cytoplasm, whereas the acidic dye stains the erythrocytes and the granules of eosinophils. Scientists originally thought that the fine neutrophil granules were stained by a “neutral dye” that formed when methylene blue and its related azures were combined with eosin. The mechanism by which the specific neutrophil granules are stained is still not clearly understood. Some of the basic dyes (the azures) are

1	blue and its related azures were combined with eosin. The mechanism by which the specific neutrophil granules are stained is still not clearly understood. Some of the basic dyes (the azures) are metachromatic and may impart a violet to red color to the material they stain.

1	Erythrocytes are anucleate, biconcave discs. Erythrocytes or red blood cells (RBCs), are anucleate cells devoid of typical organelles. They function only within the bloodstream to bind oxygen for delivery to the FIGURE 10.1  Blood smear: Preparation technique and overview photomicrograph. a. Photograph showing the method of producing a blood smear. A drop of blood is placed directly on a glass slide and spread over its surface with the edge of another slide. b. Photomicrograph of smear from peripheral blood stained with Wright’s stain, showing the cells evenly distributed. The cells are mainly erythrocytes. Three leukocytes are present. Platelets are indicated by arrows. 350. tissues and, in exchange, bind carbon dioxide for removal from the tissues. Their shape is that of a biconcave discs with a diameter of 7.8 m, an edge thickness of 2.6 m, and a central thickness of 0.8 m. This shape maximizes the cell’s surface area (140 m2), an important attribute in gas exchange.

1	7.8 m 0.8 m 2.6 m The life span of erythrocytes is approximately 120 days, after which most (90%) of them are phagocytosed by macrophages in the spleen, bone marrow, and liver. The remaining aged erythrocytes (10%) break down intravascularly, releasing insignificant amounts of hemoglobin into the blood. In H&E–stained sections, erythrocytes are usually 7 to 8 m in diameter. Because their size is relatively consistent in fixed tissue, they can be used to estimate the size of other cells and structures in tissue sections; in this role, erythrocytes are appropriately referred to as the “histologic ruler.”

1	Because both living and preserved erythrocytes usually appear as biconcave discs, they can give the impression that their form is rigid and inelastic (Fig. 10.2). They are, in fact, extremely deformable. They pass easily through the narrowest capillaries by folding over on themselves. They stain uniformly with eosin. In thin sections viewed with the transmission electron microscope (TEM), the contents of an erythrocyte appear as a dense, finely granular material. The shape of the erythrocyte is maintained by membrane proteins. The cell membrane of an erythrocyte is composed of a typical lipid bilayer and contains two functionally significant groups of proteins.

1	The cell membrane of an erythrocyte is composed of a typical lipid bilayer and contains two functionally significant groups of proteins.  Integral membrane proteins represent most of the proteins in the lipid bilayer. They consist of two major families: glycophorins and band 3 proteins. The extracellular domains of these integral membrane proteins are glycosylated and express specific blood group antigens. Glycophorin C, a member of the glycophorin family of transmembrane proteins, plays an important role in attaching the underlying cytoskeletal protein network to the cell membrane. Band 3 protein binds hemoglobin and acts as an additional anchoring site for the cytoskeletal proteins (Fig. 10.3).

1	 Peripheral membrane proteins reside on the inner surface of the cell membrane. They are organized into a two-dimensional hexagonal lattice network that laminates the inner layer of the membrane. The lattice itself, which is positioned parallel to the membrane, is composed mainly of cytoskeletal proteins including spectrin tetramers, actin, band 4.1 protein, adducin, band 4.9 protein, and tropomyosin (see Fig. 10.3) that form a network or mesh. The lattice is anchored to the lipid bilayer by the globular protein ankyrin, which interacts with band 4.2 protein as well as with band 3 integral membrane protein. glycophorin C adductin spectrin ankyrin actin tropomyosin band 3 protein 4.2

1	glycophorin C adductin spectrin ankyrin actin tropomyosin band 3 protein 4.2 FIGURE 10.2  Erythrocyte morphology. a. Photomicrograph of three capillaries (Cap) joining to form a venule (V ), as observed in adipose tissue within a full-thickness mesentery spread. The erythrocytes appear in single file in one of the capillaries (the other two are empty). The light center area of some of the erythrocytes results from their biconcave shape. Erythrocytes are highly plastic and can fold on themselves when passing through very narrow capillaries. The large round structures are adipose cells (A). 470. b. Scanning electron micrograph of erythrocytes collected in a blood tube. Note the concave shape of the cells. The stacks of erythrocytes in these preparations are not unusual and are referred to as rouleau. Such formations in vivo indicate an increased level of plasma immunoglobulin. 2,800.

1	This unique cytoskeletal arrangement contributes to the shape of the erythrocyte and imparts elastic properties and stability to the membrane. The cytoskeleton is not static; it undergoes continuous rearrangement in response to various protein 4.9 protein 4.1 FIGURE 10.3  Erythrocyte membrane organization. The rectangle in the sectioned erythrocyte (upper left) represents the area of the membrane in the larger diagram. The large diagram shows the arrangement of the peripheral and integral membrane proteins. The peripheral proteins form a cytoskeletal lattice on the interior surface of the plasma membrane; the predominant protein is spectrin. The lattice is anchored to the plasma membrane by a number of protein complexes.

1	physical factors and chemical stimuli as the cell moves through the vascular network. Any defect in the expression of genes that encode these cytoskeleton proteins can result in abnormally shaped and fragile erythrocytes. For instance, hereditary spherocytosis is caused by a primary defect in spectrin gene expression that results in spherical erythrocytes. Hereditary elliptocytosis is caused by a deficiency in band 4.1 proteins that results in elliptical erythrocytes. In both conditions, erythrocytes are unable to adapt to changes in their environment (e.g., osmotic pressure and mechanical deformations), which results in premature destruction of the cells, or hemolysis. Erythrocytes contain hemoglobin, a protein specialized for the transport of oxygen and carbon dioxide.

1	Erythrocytes contain hemoglobin, a protein specialized for the transport of oxygen and carbon dioxide. Erythrocytes transport oxygen and carbon dioxide bound to the protein hemoglobin (68 kilodaltons). A monomer of hemoglobin is similar in composition and structure to myoglobin, the oxygen-binding protein found in striated muscle. A high concentration of hemoglobin is present within erythrocytes and is responsible for their uniform staining with eosin and the cytoplasmic granularity seen with the TEM. The disc shape of the erythrocyte facilitates gas exchange because more hemoglobin molecules are closer to the plasma membrane than they would be in a spherical cell. Thus, gases have less distance to diffuse within the cell to reach a binding site on the hemoglobin.  FOLDER 10.1 Clinical Correlation: ABO and Rh Blood a life-threatening complication, blood for transfusion must

1	An important factor in blood transfusion is the ABO blood group system, which essentially involves three antigens called A, B, and O (Table F10.1.1). These antigens are glycoproteins and glycolipids and differ only slightly in their composition. They are present on the surface of erythrocytes and are attached to the extracellular domains of integral membrane proteins called glycophorins. The presence of A, B, or O antigens determines the four primary blood groups: A, B, AB, and O. All humans have enzymes that catalyze the synthesis of the O antigen. Individuals with A blood group have an additional enzyme (N-acetylgalactosamine transferase or Aglycosyltransferase) that adds N-acetylgalactosamine to the O antigen. Individuals with B blood group have an enzyme (galactose transferase or B-glycosyltransferase) that adds galactose to the O antigen (Fig. F10.1.1). Individuals with the AB blood group express both enzymes, whereas individuals with type O blood group lack both enzymes. In

1	B-glycosyltransferase) that adds galactose to the O antigen (Fig. F10.1.1). Individuals with the AB blood group express both enzymes, whereas individuals with type O blood group lack both enzymes. In humans, ABO genes consist of at least seven exons, and they are located on chromosome 9. The O allele is recessive, whereas A and B alleles are codominant.

1	The differences in the carbohydrate molecules of these antigens are detected by specific antibodies against either A or B antigens. Individuals with A antigens possess serum anti-B antibodies that are directed against the B antigen. Individuals with B antigens possess serum anti-A antibodies that are directed against A antigen. Individuals with blood group AB do not have antibodies directed against A or B antigens. Thus, they are universal acceptors of any blood type. Group O individuals have both anti-A and anti-B antibodies in their serum and neither A nor B antigens on their erythrocytes. Thus, these individuals are universal blood donors.

1	If an individual is transfused with blood of an incompatible type, the recipient’s antibodies will attack the donor erythrocytes, causing a hemolytic transfusion reaction, or destruction of the transfused erythrocytes. To prevent such be always cross-matched to the blood of a recipient. In this procedure, serum from the recipient is tested against the donor’s erythrocytes. If there is no reaction to this cross-match test, then the donors’ blood can be used for the transfusion.

1	Rh blood group system The other important blood group system, the Rh system, is based on the Rhesus (Rh) antigen. In humans, this system is represented by a 40-kilodalton transmembrane nonglycosylated Rh30 polypeptide that shares antigenic sites with rhesus monkey erythrocytes. Rh30 polypeptide is a component of a larger (90-kilodalton) erythrocyte integral membrane protein complex that includes Rh50 glycoprotein. Although the Rh30 polypeptide expresses many antigen sites on its extracellular domain, only three of them—D, C, and E antigens—have clinical significance. Interactions between Rh30 and Rh50 molecules are essential for the expression of D, C, and E antigens. An individual who possesses only one of these three antigens is referred to as Rh positive (Rh). All three antigens stimulate production of anti-Rh antibodies in individuals without the same antigens.

1	Rh incompatibility may induce a hemolytic transfusion reaction and in newborns causes the hemolytic disease erythroblastosis fetalis. Erythroblastosis fetalis occurs in Rh(D) newborns delivered by Rh(D ) mothers and results from an immune reaction of anti-D immunoglobulins passed across the placenta from the mother. The anti-D antibodies are produced by the mother in response to the D antigen expressed on the fetal erythrocytes that leak into her circulation during pregnancy. Administration of anti-D antibodies (RhoGAM) to the mother during pregnancy and after parturition destroys any circulating Rh(D) fetal erythrocytes that persist in the mother’s blood, thus preventing Rh-incompatibility reactions in future pregnancies.

1	Blood Type Erythrocyte Surface Antigen Serum Antibody Can Give Blood to Can Receive Blood From A A antigen Anti-B A and AB A and O B B antigen Anti-A B and AB B and O AB A and B antigens No antibodies Only AB A, B, AB, and O (universal blood recipient) O O antigen (no A or B antigens) Anti-A and anti-B A, B, AB, and O (universal blood donor) Only O TABLE ABO Blood Group System F10.1.1 Hemoglobin consists of four polypeptide chains of globin , , ,and , each complexed to an iron-containing heme group (Fig. 10.4). The structure of the polypeptide chains varies. Depending on the particular polypeptides present, the following types of hemoglobin can be distinguished:  Hemoglobin HbA is most prevalent in adults, accounting for about 96% of total hemoglobin. It is a tetramer with two and two chains (2 2).  Hemoglobin HbA2 accounts for 1.5% to 3% of total hemoglobin in adults. It consists of two and two chains (22).

1	 Hemoglobin HbA2 accounts for 1.5% to 3% of total hemoglobin in adults. It consists of two and two chains (22).  Hemoglobin HbF accounts for less than 1% of total hemoglobin in adults. It contains two and two chains (22) and is the principal form of hemoglobin in the fetus. HbF production falls dramatically after birth; however, in some individuals HbF is produced throughout their entire lives. Although HbF persists in slightly higher percentages than normal in those with sickle cell disease and thalassemia, it does not appear to have a pathologic role. Mutations in the genes encoding the globin chains can cause disorders in hemoglobin production. An example of a mutation in the gene encoding hemoglobin is discussed in the Folder 10.3. Interestingly, more than 550 types of abnormal hemoglobin molecules have been identified, but the majority of them have no clinical significance.  FOLDER 10.2 Clinical Correlation: Hemoglobin in Patients with Diabetes

1	As mentioned in the text, about 96% of total hemoglobin in adults is represented by hemoglobin type HbA. Approx-imately 8% of HbA consists of several subtypes that dis-play slight chemical differences. These subtypes are hemoglobins HbA1a1, HbA1a2, HbA1b, and HbA1c. Of these subtypes, hemoglobin type A1c is of clinical sig-nificance because it binds irreversibly to glucose. It is re-ferred to as glycated or glycosylated hemoglobin. Levels of this hemoglobin subtype are used to monitor an individual’s blood glucose levels over the previous 2 to 3 months (clinically referred to as an A1c test). Individuals with diabetes have increased levels of glycated hemoglobin HbA1c in the blood because of their elevated blood glu-cose. Since the normal life span of erythrocytes is about 120 days (see page 295), glycated hemoglobin can only be eliminated when the RBCs containing it are de-stroyed. Thus, HbA1c values are directly proportional to the concentration of glucose in the blood over the

1	page 295), glycated hemoglobin can only be eliminated when the RBCs containing it are de-stroyed. Thus, HbA1c values are directly proportional to the concentration of glucose in the blood over the entire life span of the erythrocyte. In healthy individuals and in those with diabetes that is being effectively controlled, HbA1c levels should not be higher than 7% of the total hemoglobin. Since HbA1c values are not subject to the short-term fluctuations in blood glucose levels that are seen, for example, after meals or during fasting, blood for HbA1c testing can be obtained without regard to when food is consumed.

1	FIGURE 10.4  Structural diagram of the hemoglobin molecule. Each hemoglobin molecule is composed of four subunits. Each subunit contains a heme, the iron-containing portion of hemoglobin, embedded in a hydrophobic cleft of a globin chain. The folding of the globin chain places the heme near the surface of the molecule, where it is readily accessible to oxygen. There are four different types of globin chains: , , , and occurring in pairs. The types of globin chains present in the molecules determine the type of hemoglobin. The figure illustrates hemoglobin A (HbA), which is composed of two and two chains.

1	Leukocytes are subclassified into two general groups. The basis for this division is the presence or absence of prominent specific granules in the cytoplasm. As previously noted, cells containing specific granules are classified as granulocytes (neutrophils, eosinophils, and basophils) (Plate 17, page 302), and cells that lack specific granules are classified as agranulocytes (lymphocytes and monocytes) (Plate 18, page 304). However, both agranulocytes and granulocytes possess small number of nonspecific azurophilic granules, which are lysosomes. The relative number of the various leukocytes is given in Table 10.1. Neutrophils are the most numerous WBCs as well as the most common granulocytes.

1	Neutrophils are the most numerous WBCs as well as the most common granulocytes. Neutrophils measure 10 to 12 m in diameter in blood smears and are obviously larger than erythrocytes. Although named for their lack of characteristic cytoplasmic staining, they are also readily identified by their multilobal nucleus; thus, they are also called polymorphonuclear neutrophils or polymorphs. Mature neutrophils possess two to four lobes of nuclear material joined by thinner nuclear strands (Plate 17, page 302). The arrangement is not static; rather, in living neutrophils the lobes and connecting strands change their shape, position, and even number.

1	The chromatin of the neutrophil has a characteristic arrangement. Wide regions of heterochromatin are located chiefly at the periphery of the nucleus in contact with the nuclear envelope. Regions of euchromatin are located primarily at the center of the nucleus with relatively smaller regions contacting the nuclear envelope (Fig. 10.5). In women, the Barr body (the condensed, single, inactive X chromosome) forms a drumstick-shaped appendage on one of the nuclear lobes. Neutrophils contain three types of granules. The cytoplasm of a neutrophil contains three kinds of granules. The different types of granules reflect the various phagocytotic functions of the cell.

1	Neutrophils contain three types of granules. The cytoplasm of a neutrophil contains three kinds of granules. The different types of granules reflect the various phagocytotic functions of the cell.  Specific granules (secondary granules) are the smallest granules and are at least twice as numerous as azurophilic granules. They are barely visible in the light microscope; in electron micrographs, they are ellipsoidal (see Fig. 10.5). Specific granules contain various enzymes (i.e., type IV collagenase, phospholipase) as well as complement activators and other antimicrobial peptides (i.e., lysozymes, lactoferrins).

1	 Azurophilic granules (primary granules) are larger and less numerous than specific granules. They arise early in granulopoiesis and occur in all granulocytes, as well as in monocytes and lymphocytes. The azurophilic granules are the lysosomes of the neutrophil and contain myeloperoxidase (MPO) (a peroxidase enzyme), which appears as a finely stippled material with the TEM. Myeloperoxidase helps to generate highly reactive bactericidal hypochlorite and chloramines. In addition to containing a variety of the typical acid hydrolases, azurophilic granules also contain cationic proteins called defensins, which function analogously to antibodies and the antimicrobial peptide cathelicidin to kill pathogens.

1	 Tertiary granules in neutrophils are of two types. One type contains phosphatases (enzymes that remove a phosphate group from a substrate) and is sometimes called a phosphasome. The other type contains metalloproteinases, such as gelatinases and collagenases, which are thought to facilitate the migration of the neutrophil through the connective tissue. Aside from these granules, membrane-bounded organelles are sparse. A small Golgi apparatus is evident in the center of the cell, and mitochondria are relatively few in number (see Fig. 10.5). Neutrophils are motile cells; they leave the circulation and migrate to their site of action in the connective tissue.

1	Neutrophils are motile cells; they leave the circulation and migrate to their site of action in the connective tissue. An important property of neutrophils and other leukocytes is their motility. Neutrophils are the most numerous of the first wave of cells to enter an area of tissue damage. Their migration is controlled by the expression of adhesion molecules on the neutrophil surface that interact with corresponding ligands on endothelial cells (Fig. 10.6) and are often involved in cell binding. The initial phase of neutrophil migration occurs in the post-capillary venules and is regulated by a mechanism involving neutrophil–endothelial cell recognition. Selectins (a type of cell adhesion molecule) on the surface of the circulating neutrophil (CD62L) interact with receptors (GlyCAM-1) on the surface of the endothelial cells. The neutrophil becomes partially tethered to the endothelial cell as a result of this  FOLDER 10.3 Clinical Correlation: Hemoglobin Disorders

1	Anemia Anemia is defined clinically as a decrease in the concentration of hemoglobin in the blood for the age and sex of an individual. Although in certain anemias this decreased concentration of hemoglobin is caused by a decrease in the amount of hemoglobin in each cell, most anemias are caused by a reduction in the number of erythrocytes. Causes of anemia include loss of blood (hemorrhage), insufficient production of erythrocytes, or accelerated destruction of erythrocytes in the circulation. Insufficient dietary iron or deficiencies of vitamins such as vitamin B12 or folic acid can lead to decreased production of erythrocytes. Gastric atrophy, as a result of autoimmune disease, with concomitant destruction of the parietal cells that secrete intrinsic factor, a molecule essential for absorption of vitamin B12 by cells in the ileum, is the cause of a form of anemia called pernicious anemia. The clinical symptoms of anemia vary, depending on the type of anemia, the underlying cause,

1	of vitamin B12 by cells in the ileum, is the cause of a form of anemia called pernicious anemia. The clinical symptoms of anemia vary, depending on the type of anemia, the underlying cause, and other associated medical conditions. The common symptoms of even mild anemia include weakness, fatigue, and loss of energy. The other symptoms associated with anemia are shortness of breath, frequent headaches, difficulty concentrating, mental confusion, loss of sexual drive, dizziness, leg cramps, insomnia, and pale skin.

1	interaction, which slows the neutrophil and causes it to roll on the surface of the endothelium. In the second phase, another group of adhesion molecules on the neutrophil surface, called integrins (VLA-5), are activated by chemokine signals from the endothelial cells. In the third phase, integrins and other adhesion molecules from the immunoglobulin superfamily (e.g., intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]) expressed on the neutrophil surface engage with their specific receptors on the endothelial cells,

1	Sickle Cell Disease Sickle cell disease is caused by a single-point mutation in the gene that encodes the -globin chain of hemoglobin A. The result of this mutation is an abnormal -globin chain in which the amino acid valine is substituted for glutamic acid in position 6. Hemoglobin containing this abnormal -globin chain is designated sickle hemoglobin (HbS). The substitution of the hydrophobic valine for the hydrophilic glutamic acid causes HbS to aggregate under conditions of reduced oxygen tension. Instead of the normal biconcave disc shape, many of the erythrocytes become sickle-shaped at low oxygen tension, hence the name of this disease (Fig. F10.3.1). Sickled erythrocytes are more rigid than normal cells and adhere more readily to the endothelial surface. Thus, sickled erythrocytes may pile up in the smallest capillaries, depriving portions of tissues and organs of oxygen and nutrients. Large-vessel obstruction may also occur, which in children frequently leads to stroke.

1	may pile up in the smallest capillaries, depriving portions of tissues and organs of oxygen and nutrients. Large-vessel obstruction may also occur, which in children frequently leads to stroke. Sickled erythrocytes are also more fragile and break down or are destroyed more quickly (after 20 days) than normal erythrocytes.

1	Sickle cell disease is a homozygous recessive genetic disorder. However, heterozygous individuals with sickled cell trait may occasionally have clinical consequences at high altitude or when under extreme physical stress. FIGURE F10.3.1 • Photomicrograph of a sickle cell anemia blood smear. Blood smear stained with Wright’s stain shows abnormal “boat”and “sickle”-shaped cells from an individual with sickle cell anemia. 400. attaching the neutrophil to the endothelial cell. The neutrophil then extends a pseudopod to an intercellular junction. Histamine and heparin released at the injury site by perivascular mast cells open the intercellular junction, allowing the neutrophil to migrate into the connective tissue. With the TEM, the cytoplasmic contents of a neutrophil pseudopod appear as an expanse of finely granular cytoplasmic matrix with no membranous organelles (see Fig. 10.5). The finely granular appearance is attributable to the presence of actin filaments, some

1	FIGURE 10.5 • Electron micrograph of a human mature neutrophil. The nucleus shows the typical multilobed configuration with the heterochromatin at the periphery and the euchromatin more centrally located. A small Golgi apparatus (G) is present; other organelles are sparse. The punctate appearance of the cytoplasm adjacent to the convex aspect of the nuclear profile is caused by glycogen particles. Adjacent to the concave aspect of the nuclear profile are numerous granules. Specific granules appear less dense and more rounded than azurophilic granules. The latter are fewer in number and are extremely electron dense. 22,000. (Courtesy of Dr. Dorothea Zucker-Franklin.) For comparison, the inset shows a neutrophil from a blood smear observed in the light microscope. 1,800. FIGURE 10.6 • Diagram of events in the migration of a neutrophil from a postcapillary venule into the connective tissue. a.

1	Circulating neutrophils are slowed down by the interaction of their surface adhesion molecules, selectins (CD62L), with the endothelium of the venule (b). c. As the result of this interaction, the cell rolls on the surface of the endothelium. The neutrophil then adheres to the endothelium and responds to chemokines secreted by the endothelial cells. d. Their secretion induces the expression of other adhesion molecules on the surface of the neutrophil such as integrins (VLA-5) and the immunoglobulin superfamily of adhesion molecules (e.g., intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]). e. These adhesion molecules allow the neutrophil to bind to adhesion molecule receptors on the endothelial cells. f. The neutrophil then extends a pseudopod to an intercellular junction previously opened by histamine and heparin released from the mast cells in the connective tissue, allowing the neutrophil to migrate through the vessel wall (g).

1	microtubules, and glycogen, which are involved in the extension of the cytoplasm to form the pseudopod and the subsequent contraction that pulls the cell forward. Once the neutrophil enters the connective tissue, further migration to the injury site is directed by a process known as chemotaxis, the binding of chemoattractant molecules and extracellular matrix proteins to specific receptors on the surface of the neutrophil. Neutrophils are active phagocytes that utilize a variety of surface receptors to recognize bacteria and other infectious agents at the site of inflammation.

1	Once at the site of tissue injury, the neutrophil must first recognize any foreign substances before phagocytosis can occur. Like most phagocytic cells, neutrophils have a variety of receptors on their cell membrane that can recognize and bind to bacteria, foreign organisms, and other infectious agents (Fig 10.7). Some of these organisms and agents bind directly to neutrophils (no modifications of their surfaces are required), whereas others must be opsonized (coated with antibodies or complement) to make them more attractive to the neutrophil. The most common receptors used by neutrophils during phagocytosis include the following.  Fc receptors on the neutrophil surface bind to the ex posed Fc region of IgG antibodies that coat bacterial surfaces (see Fig 10.7). Binding of IgG-coated bacteria activates the neutropil’s phagocitic activity and causes a rapid surge in intracellular metabolism.

1	 Complement receptors (CRs) facilitate binding and uptake of immune complexes that are opsonized by active C3 complement protein, namely, C3b. Binding of bacteria or other C3b-coated antigens to CRs triggers phagocytosis, resulting in activation of a neutrophil’s lytic pathways and respiratory burst reactions.  Scavenger receptors (SRs) are a structurally diverse group of transmembrane glycoproteins that bind to modified (acetylated or oxidized) forms of low-density lipoproteins (LDLs), polyanionic molecules that are often on the surface of both Gram-positive and Gram-negative bacteria and apoptotic bodies. Binding of these receptors increases the phagocitic activity of neutrophils.

1	 Toll-like receptors, also known as pattern recognition receptors (PRRs), are neutrophil receptors that recognize pathogenic molecules such as endotoxins, lipopolysaccharides, peptidoglycanes, and lipoteichoic acids that are arranged in predictable pathogen-associated molecular patterns (PAMPs) and are commonly expressed on bacterial surfaces and other infectious agents. Like other phagocytic cells, neutrophils possess a variety of toll-like receptors that recognize PAMPs. Binding of bacterial

1	FIGURE 10.7 • Neutrophil phagocytosis. a. Phagocytosis begins with recognition and attachment of foreign material (antigen), mainly by Fc receptors that interact with the Fc region of antibodies bound to the antigen. b. The antigen is then engulfed by pseudopods of the neutrophil. c. As the pseudopods come together and fuse, the antigen is internalized. d. Once the phagosome is formed, digestion is initiated by activation of membrane-bounded oxidases of the phagosome. e. Next, both specific and azurophilic granules fuse with the phagosome and release their contents, forming a phagolysosome. This fusion and release of granules is called degranulation. f. The enzymatic contents of the granules are responsible for killing and digesting the microorganism. The entire digestive process occurs within the phagolysosome, which protects the cell from self-injury. g. The digested material is either exocytosed into the extracellular space or stored as residual bodies within the neutrophil.

1	antigens to these receptor causes phagocytosis and the release of cytokines such as interleukin-1 (IL-1), interleukin-3 (IL-3), and tumor necrosis factor (TNF-) from the neutrophil. IL-1, historically known as a pyrogen (fever-causing agent), induces synthesis of prostaglandins, which in turn act on the thermoregulatory center of the hypothalamus to produce fever. Therefore, fever is a consequence of acute reaction to invading pathogens that cause a massive neutrophilic response. Phagocytosed bacteria are killed within phagolysosomes by the toxic reactive oxygen intermediates produced during respiratory burst.

1	Phagocytosis begins when the neutrophil recognizes and attaches to the antigen. Extended pseudopods of the neutrophil engulf the antigen and internalize it to form a phagosome (see Fig. 10.7). Specific and azurophilic granules fuse with the phagosome membrane, and the lysosomal hydrolases of the azurophilic granules digest the foreign material. During phagocytosis, the neutrophil’s glucose and oxygen utilization increases noticeably and is referred to as the respiratory burst. It results in synthesis of several oxygen-containing compounds called reactive oxygen intermediates (ROIs). They include free radicals such as oxygen and hydroxyl radicals that are used in immobilizing and killing live bacteria within the phagolysosomes. By definition, free radicals possess an unpaired electron within their chemical structure, which makes them highly reactive and therefore capable of damaging intracellular molecules, including lipids, proteins, and nucleic acids. The process by which

1	within their chemical structure, which makes them highly reactive and therefore capable of damaging intracellular molecules, including lipids, proteins, and nucleic acids. The process by which microorganisms are killed within neutrophils is termed oxygen-dependent intracellular killing. In general, two biochemical pathways are involved in this process: the first is the phagocyte oxidase (phox) system that utilizes the phagocyte’s nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in the phagolysosome membrane; the second is associated with the lysosomal enzyme myeloperoxidase (MPO) found in the azurophilic granules of neutrophils (Fig 10.8).

1	Within the phagocyte oxidase pathway, or phox system, phagocytosis proceeds by signaling the cell to produce sufficient amounts of NADPH needed to generate superoxide anions. Increased glucose uptake and shunting of NADPH metabolism is achieved via the pentose phosphate pathway (also known as pentose shunt). The cytosolic NADPH becomes an electron donor: The NADPH oxidase enzyme complex transports electrons across the membrane to molecular O2 inside the phagolysosome to generate the free radical superoxide anions (O–2). These superoxide anions are converted into ROIs. The superoxide dismutase converts superoxide anions to singlet oxygen (1O2) and hydrogen peroxide (H2O2), which further reacts with superoxide anions to produce bactericidal hydroxyl radicals (OH–) (the neutral form of the hydroxyl ion) and more singlet oxygen molecules (see Fig 10.8).

1	Oxygen-dependent killing with MPO involvement occurs when azurophilic granules containing MPO fuse with phagosomes containing phagocytosed bacteria. During the neutrophil’s respiratory burst, MPO, using heme as a cofactor, catalyzes a reaction that produces hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and a chloride anion (Cl–). Hypochlorous acid, which is about 1,000 times more effective in bacterial killing than hydrogen peroxide, is further metabolized to a highly toxic hypochlorite OCl– (bleach) and chlorine (Cl2). Some of the hypochloride may spontaneously break down to yield toxic singlet oxygen (1O2) and chloride ions (Cl–) (see Fig. 10.8).

1	In addition, nitric oxide (NO) and other reactive nitrogen intermediates (RNIs) have also been implicated in the intracellular microbial killing mechanisms. NO has been found in neutrophils; however, it is believed that RNI-mediated killing mechanisms do not appear to have a critical role in humans. The main role of neutrophil-derived NO is to induce vasodilatation, which in turn facilitates the migration of neutrophils from blood vessels to surrounding connective tissue. Phagocytosed bacteria can also be killed by a diverse arsenal of oxygen-independent killing mechanisms utilizing bacteriolytic enzymes and antimicrobial peptides.

1	In addition to the oxygen-dependent respiratory burst reactions, microorganisms can be killed by bacteriolytic enzymes and cationic antimicrobial peptides that are stored within the granules of the neutrophil’s cytoplasm. These oxygen-independent killing mechanisms are directed toward the bacterial cell membrane, causing its breakdown and leakage. Neutrophils contain particularly large amounts of cationic antimicrobial proteins such as defensins and antimicrobial peptides called cathelicidins. Similar to lysozymes and cathepsins stored in the specific granules, these cationic antimicrobial proteins break down the bacterial wall. In addition, lysosomal hydrolytic enzymes that digest bacterial proteins and lactoferrins that chelate iron from nutritional bacterial pathways contribute to the destruction of the invading bacteria. These mechanisms are not as efficient as oxygendependent killing pathways. Neutrophils from patients with defects in oxygen-dependent pathways, such as those with

1	of the invading bacteria. These mechanisms are not as efficient as oxygendependent killing pathways. Neutrophils from patients with defects in oxygen-dependent pathways, such as those with chronic granulomatous disease (see Folder 10.4), are still able to destroy phagocytosed bacteria to some degree. However, because of the low efficiency of these processes, individuals with these defects are more susceptible to serious infections.

1	After intracellular digestion by the neutrophil, the remnants of degraded material are stored in residual bodies or exocytosed. Most neutrophils die in this process; the accumulation of dead bacteria and dead neutrophils constitutes the thick exudate called pus. The yellow–green color of the pus and of mucus secretions (e.g., from infected lungs) comes from the heme pigment of MPO enzyme in azurophilic granules of neutrophils. Inflammation and wound healing also involve monocytes, lymphocytes, eosinophils, basophils, and fibroblasts. Monocytes also enter the connective tissue as a secondary response to tissue injury. At the site of tissue injury, they transform into macrophages that phagocytose cell and tissue debris, fibrin, remaining bacteria, and dead neutrophils. Normal wound healing depends on the participation of FIGURE 10.8 • Pathways leading to synthesis of reactive oxygen intermediates during neutrophil’s respiratory burst reactions.

1	This schematic diagram shows a phagolysosome that contains already pagocytosed bacterium. Two oxygen-dependent killing mechanisms are depicted in this drawing. The first mechanism depends on a phagocyte oxidase (phox) system that utilizes the NADPH oxidase complex (contains five subunits). This complex transports excess electrons across the membrane of the phagolysosome, where they interact with molecular oxygen to generate free superoxide anions. These anions are converted into reactive oxygen intermediates. Another enzyme superoxide dismutase converts superoxide anions to singlet oxygen and hydrogen peroxide, which further reacts with superoxide anions to produce bactericidal hydroxyl radicals and more singlet oxygen molecules. The second mechanism involves lysosomal enzyme myeloperoxidase (MPO) found in the azurophilic granules of neutrophils. MPO catalizes the production of hypochlorous acids from hydrogen peroxide and chloride anions. Hypochlorous acid is further metabolized to a

1	(MPO) found in the azurophilic granules of neutrophils. MPO catalizes the production of hypochlorous acids from hydrogen peroxide and chloride anions. Hypochlorous acid is further metabolized to a highly toxic hypochlorite (bleach) and chlorine. Some of the hypochloride may spontaneously break down to yield toxic singlet oxygen and chloride ions. All molecules produced during oxygen bursts in neutrophils (associated with red arrows) are highly effective in killing ingested bacteria.

1	macrophages in the inflammatory response; they become the major cell type in the inflammatory site after the neutrophils are spent. At the same time that the macrophages become active at the site of inflammation, fibroblasts near the site and undifferentiated mesenchymal cells in the adventitia of small vessels at the site begin to divide and differentiate into fibroblasts and myofibroblasts that then secrete the fibers and ground substance of the healing wound. Like neutrophils, monocytes are attracted to the inflammatory site by chemotaxis. Lymphocytes, eosinophils, and basophils also play a role in inﬂammation, but they are more involved in the immunologic aspects of the process (see Chapter 14, Lymphatic System). Eosinophils and lymphocytes are more commonly found at sites of chronic inﬂammation.

1	Eosinophils are about the same size as neutrophils, and their nuclei are typically bilobed (Fig. 10.9; Plate 17, page 302). As in neutrophils, the compact heterochromatin of eosinophils is chiefly adjacent to the nuclear envelope, whereas the euchromatin is located in the center of the nucleus. Eosinophils are named for the large, eosinophilic, refractile granules in their cytoplasm. The cytoplasm of eosinophils contains two types of granules: numerous, large, elongated specific granules and azurophilic granules (otherwise, the eosinophil contains only a sparse representation of membranous organelles).  FOLDER 10.4 Clinical Correlation: Inherited Disorders of Neutrophils; Chronic Granulomatous Disease (CGD)

1	A primary example of genetic immunodeficiency that af-fects oxygen-dependent killing mechanisms is chronic granulomatous disease (CGD). In this inherited disor-der of neutrophils and other phagocytic cells, one of the components of the NADPH oxidase complex (phox system) has mutated or is absent. As a result, neutrophils cannot produce reactive oxygen intermediates (ROIs). The NADPH oxidase complex consists of five molecules. Two of them, glycoprotein 91 (gp91) and protein 22 (p22), are part of a membrane-bound cytochrome called cy-tochrom B558 (see Fig. 10.8). Three other cytosolic com-ponents—protein 47 (p47), protein 67 (p67), and protein 40 (p40)—are components of Rac-2 GTPase, which is required for oxidase activity. Neutrophil activation and stimulation from phagocytosis cause cytosolic pro-teins to translocate to the plasma membrane of the phagolysosome to assemble the active NADPH oxidase complex. The assembled enzyme transports electrons from cytosolic NADPH across the

1	cytosolic pro-teins to translocate to the plasma membrane of the phagolysosome to assemble the active NADPH oxidase complex. The assembled enzyme transports electrons from cytosolic NADPH across the membrane to molecular O2 residing inside the phagolysosome, generating bactericidal superoxide anions (O2 –) and other ROIs. Approximately 50% to 70% of all CGD cases are caused by a mutation in the CYBB (cytochrome B, b subunit) gene located on the X chromosome. This gene encodes glycoprotein 91 (gp91), which is necessary for proper function of the NADPH oxidase complex. Since gp91 deficiency is an X-linked disease, CGD caused by this mutation is often re-ferred as X91 disease. Another 20% to 40% of those with CGD have mutations in the NCF1 gene on chromo-some 7 that codes for protein 47. Mutations in gene NCF2 (which encodes protein 67) and CYBA (which encodes protein 22) are rare, accounting for fewer than 10% of all cases of CGD. Mutations in the NCF1, NCF2, and CYBA genes produce

1	Mutations in gene NCF2 (which encodes protein 67) and CYBA (which encodes protein 22) are rare, accounting for fewer than 10% of all cases of CGD. Mutations in the NCF1, NCF2, and CYBA genes produce autosomal recessive forms of CGD. CGD decreases the ability of neutrophils to kill certain types of bacteria and fungi. Individuals with this disease are frequently affected by recurrent life-threatening bacte-rial and fungal infections and chronic inflammatory condi-tions. The most common pathologic changes occur in tissues and organs that form barriers against the entry of microorganisms from the external environment. They in-clude skin (skin infections), gingiva (swollen inflamed gums), lungs (pneumonia), lymph nodes (lymphoadenitis), gastrointestinal tract (enteritis, diarrhea), liver, and spleen. Another characteristic feature of CGD is the development of enlarged, tumorlike masses called granulomas. The presence of granulomas may cause serious problems in the gastrointestinal tract

1	spleen. Another characteristic feature of CGD is the development of enlarged, tumorlike masses called granulomas. The presence of granulomas may cause serious problems in the gastrointestinal tract by obstructing the passage of food and in the genitourinary tract by blocking the flow of urine from the kidneys and bladder.

1	 Specific granules of eosinophils contain a crystalloid body that is readily seen with the TEM, surrounded by a less electron-dense matrix. These crystalloid bodies are responsible for the refractivity of the granules in the light microscope. They contain four major proteins: an arginine-rich protein called major basic protein (MBP), which accounts for the intense acidophilia of the granule; eosinophil cationic protein (ECP); eosinophil peroxidase (EPO); and eosinophil-derived neurotoxin (EDN). MBP is localized in the crystalloid body; the other three proteins are found in the granule matrix. MBP, ECP, and EPO have a strong cytotoxic effect on protozoans and helminthic parasites; EDN causes nervous system dysfunction in parasitic organisms; histaminase neutralizes the activity of histamine; and arylsulfatase neutralizes leukotrienes secreted by basophils and mast cells (see Chapter 6, Connective Tissue,  FOLDER 10.5 Clinical Correlation: Hemoglobin Breakdown and Jaundice

1	If the conjugation of bilirubin or its excretion into bile by the liver cells is inhibited, or if blockage of the bile duct system occurs, bilirubin may reenter the blood, causing a yellow appearance of the sclera of the eye and the skin. This con-dition is called jaundice. Jaundice can be caused by the destruction of circulating erythrocytes. An example of such a condition is a hemolytic transfusion reaction when ABO-incompatible blood is administered to a patient, usu-ally because of a clerical error. Massive hemolysis of the transfused erythrocytes may be associated with severe systemic complications such as hypotension (decreased blood pressure), renal failure, and even death. Jaundice is also characteristic in a variety of hemolytic anemias that result from either inherited defects in the erythrocyte (e.g., hereditary spherocytosis) or external fac-tors such as pathogenic microorganisms, animal venoms, chemicals, and drugs. Some jaundice is common in new-born infants (physiologic

1	the erythrocyte (e.g., hereditary spherocytosis) or external fac-tors such as pathogenic microorganisms, animal venoms, chemicals, and drugs. Some jaundice is common in new-born infants (physiologic jaundice) because of ineffi-ciency of the bilirubin-conjugating system in the newborn liver.

1	page 186). Specific granules also contain histaminase, arylsulfatase, collagenase, and cathepsins.  Azurophilic granules are lysosomes. They contain a variety of the usual lysosomal acid hydrolases and other hydrolytic enzymes that function in destruction of parasites and hydrolysis of antigen–antibody complexes internalized by the eosinophil. Eosinophils are associated with allergic reactions, parasitic infections, and chronic inflammation. Eosinophils develop and mature in the bone marrow. Once released from the bone marrow, they circulate in peripheral blood and then migrate to the connective tissue. Eosinophils are activated by interactions with IgG, IgA, or secretory IgA antibodies.

1	The release of arylsulfatase and histaminase by eosinophils at sites of allergic reaction moderates the potentially deleterious effects of inﬂammatory vasoactive mediators. The eosinophil also participates in other immunologic responses and phagocytoses antigen–antibody complexes. Thus, the count of eosinophils in blood samples of individuals with allergies and parasitic infections is usually high. Eosinophils play a major role in host defense against helminthic parasites. They are also found in large numbers in the lamina propria of the intestinal

1	FIGURE 10.9 • Electron micrograph of a human eosinophil. The nucleus is bilobed, but the connecting segment is not within the plane of section. The granules are of moderate size, compared with those of the basophil, and show a crystalline body (Cr) within the less electron-dense matrix of the granule. M, mitochondria. 26,000. (Courtesy of Dr. Dorothea Zucker-Franklin.) Inset. Light microscopic image of an eosinophil from a blood smear. 1,800. tract and at other sites of potential chronic inﬂammation (i.e., lung tissues in patients with asthma). Basophils are about the same size as neutrophils and are so named because the numerous large granules in their cytoplasm stain with basic dyes (Plate 17, page 302). Basophils are the least numerous of the WBCs, accounting for less than 0.5% of total leukocytes.

1	Basophils are the least numerous of the WBCs, accounting for less than 0.5% of total leukocytes. Often, several hundred WBCs must be examined in a blood smear before one basophil is found. The lobed basophil nucleus is usually obscured by the granules in stained blood smears, but its characteristics are evident in electron micro-graphs (Fig. 10.10). Heterochromatin is chiefly in a peripheral location, and euchromatin is chiefly centrally located; typical cytoplasmic organelles are sparse. The basophil plasma membrane possesses numerous high-affinity Fc receptors for IgE antibodies. In addition, a specific 39-kilodalton protein called CD40L is expressed on the basophil’s surface. CD40L interacts with a complementary receptor (CD40) on B lymphocytes, which results in increased synthesis of IgE.

1	FIGURE 10.10 • Electron micrograph of a human basophil. The nucleus appears as three separate bodies; the connecting strands are not in the plane of section. The basophil granules (B) are very large and irregularly shaped. Some granules reveal myelin figures (MF ). M, mitochondria. 26,000. (Courtesy of Dr. Dorothea Zucker-Franklin.) Inset. Light microscopic appearance of a basophil from a blood smear. 1,800. The basophil cytoplasm contains two types of granules: specific granules, which are larger than the specific granules of the neutrophil, and nonspecific azurophilic granules.

1	The basophil cytoplasm contains two types of granules: specific granules, which are larger than the specific granules of the neutrophil, and nonspecific azurophilic granules.  Specific granules exhibit a grainy texture and myelin figures when viewed with the TEM. These granules contain a variety of substances, namely, heparin, histamine, heparan sulfate, leukotrienes, IL-4, and IL-13. Heparin, a sulfated glycosaminoglycan, is an anticoagulant. Histamine and heparan sulfate are vasoactive agents that among other actions cause dilation of small blood vessels. Leukotrienes are modified lipids that trigger prolonged constriction of smooth muscles in the pulmonary airways (see page 186). Interleukin-4 (IL-4) and interleukin-13 (IL-13) promote synthesis of IgE antibodies. The intense basophilia of these specific granules correlates with the high concentration of sulfates within the glycosaminoglycan molecules of heparin and heparan sulfate.

1	 Azurophilic granules are the lysosomes of basophils and contain a variety of the usual lysosomal acid hydrolases that are similar to those in other leukocytes. The function of basophils is closely related to that of mast cells.

1	Basophils are functionally related to, but not identical with, mast cells of the connective tissue (see Table 6.6, page 187). Both mast cells and basophils bind an antibody secreted by plasma cells, IgE, through high-affinity Fc receptors expressed on their cell surface. The subsequent exposure to, and reaction with, the antigen (allergen) specific for IgE triggers the activation of basophils and mast cells and the release of vasoactive agents from cell granules. These substances are responsible for the severe vascular disturbances associated with hypersensitivity reactions and anaphylaxis. Furthermore, both basophils and mast cells are derived from the same basophil–mast cell progenitor (BMCP) cell. If a specific BMCP expresses the granulocyte-related transcription factor CCAAT/enhancer-binding protein (C/EBP), the cell becomes committed to differentiate into a basophil progenitor (BaP) cell. Basophils develop and differentiate in the bone marrow and are released to the peripheral

1	protein (C/EBP), the cell becomes committed to differentiate into a basophil progenitor (BaP) cell. Basophils develop and differentiate in the bone marrow and are released to the peripheral blood as mature cells. In the absence of C/EBP transcription factor, a BMCP cell migrates to the spleen and after further differentiation travels as a mast cell precursor (MPC) to the intestine, where it becomes a mature mast cell.

1	Lymphocytes are the main functional cells of the lymphatic or immune system.

1	Lymphocytes are the most common agranulocytes and account for about 30% of the total blood leukocytes. In understanding the function of the lymphocytes, one must realize that most lymphocytes found in blood or lymph represent recirculating immunocompetent cells (i.e., cells that have developed the capacity to recognize and respond to antigens and are in transit from one lymphatic tissue to another). In the tissues associated with the immune system (see Chapter 14, Lymphatic System), three groups of lymphocytes can be identified according to size: small, medium, and large lymphocytes, ranging in diameter from 6 to 30 m. The large lymphocytes are either activated lymphocytes, which possess surface receptors that interact with a specific antigen, or natural killer (NK) lymphocytes. In the bloodstream, most lymphocytes are small or medium sized, 6 to 15 m in diameter. The majority—more than 90%—are small lymphocytes.

1	In blood smears, the mature lymphocyte approximates the size of an erythrocyte. When observed in the light microscope in a blood smear, small lymphocytes have an intensely staining, slightly indented, spherical nucleus (Plate 17, page 302). The cytoplasm appears as a very thin, pale blue rim surrounding the nucleus. In general, there are no recognizable cytoplasmic organelles other than an occasional fine azurophilic granule. The TEM reveals that the cytoplasm primarily contains free ribosomes and a few mitochondria. Other organelles are so sparse that they are usually not seen in a thin section. Small, dense lysosomes that correspond to the azurophilic granules seen in the light microscope are occasionally observed; a pair of centrioles and a small Golgi apparatus are located in the cell center, the area of the indentation of the nucleus.

1	In the medium lymphocyte, the cytoplasm is more abundant, the nucleus is larger and less heterochromatic, and the Golgi apparatus is somewhat more developed (Fig. 10.11). Greater numbers of mitochondria and polysomes and small profiles of rough endoplasmic reticulum are also seen in these medium-sized cells. The ribosomes are the basis for the slight basophilia displayed by lymphocytes in stained blood smears. Three functionally distinct types of lymphocytes are present in the body: T lymphocytes, B lymphocytes, and NK cells. The characterization of lymphocyte types is based on their function, not on their size or morphology. T lymphocytes (T cells) are so named because they undergo differentiation in the thymus. B lymphocytes (B cells) are so named because they were first recognized as a separate population in the bursa of Fabricius in birds or bursa-equivalent organs (e.g., bone marrow) in mammals. Natural killer (NK) cells

1	FIGURE 10.11 • Electron micrograph of a medium-sized lymphocyte. The punctate appearance of the cytoplasm is caused by the presence of numerous free ribosomes. Several mitochondria (M) are evident. The cell center or centrosphere region of the cell (the area of the nuclear indentation) also shows a small Golgi apparatus (G) and a centriole (C). 26,000. (Courtesy of Dr. Dorothea Zucker-Franklin.) Inset. Light microscopic appearance of a medium-sized lymphocyte from a blood smear. 1,800. develop from the same precursor cell as B and T cells and are so named because they are programmed to kill certain types of transformed cells.

1	develop from the same precursor cell as B and T cells and are so named because they are programmed to kill certain types of transformed cells.  T cells have a long life span and are involved in cell-mediated immunity. T cells are characterized by the presence of cell-surface recognition proteins called T-cell receptors (TCRs), which in a majority of T cells comprise two glycoprotein chains called and -TCR chains. They express CD2, CD3, CD5, and CD7 marker proteins on their surface; however, they are subclassified on the basis of the presence or absence of CD4 and CD8 proteins. CD4 T lymphocytes possess the CD4 marker and recognize antigens bound to major histocompatability complex II (MHC II) molecules. CD8 T lymphocytes possess the CD8 marker and recognize antigen bound to MHC I molecules.

1	 B cells have variable life spans and are involved in the production of circulating antibodies. Mature B cells in blood express IgM and IgD and MHC II molecules on their surface. Their specific markers are CD9, CD19, CD20, and CD24.  NK cells are programmed during their development to kill certain virus-infected cells and some types of tumor cells. They also secrete an antiviral agent, interferon (IFN-). NK cells are larger than B and T cells (15 m in diameter) and have a kidney-shaped nucleus. Because NK cells have several large cytoplasmic granules easily seen by light microscopy, they are also called large granu lar lymphocytes (LGLs). Their specific markers include CD16, CD56, and CD94.

1	T and B cells are indistinguishable in blood smears and tissue sections; immunocytochemical staining for different types of markers and receptors on their cell surface must be used to identify them. NK lymphocytes can be identified in the light microscope by size, nuclear shape, and presence of cytoplasmic granules; however, immunocytochemical staining for their specific markers is used to confirm microscopic identification. T and B lymphocytes express different surface molecules.

1	T and B lymphocytes express different surface molecules. Although the T and B cells cannot be distinguished on the basis of their morphology, their distinctive surface proteins (CD proteins) can be used to identify the cells with immunolabeling techniques. In addition, immunoglobulins are expressed on the surface of B cells that function as antigen receptors. In contrast, T cells do not have antibodies but express TCRs. These recognition proteins appear during discrete stages in the maturation of the cells within the thymus. In general, the surface molecules mediate or augment specific T-cell functions and are required for the recognition or binding of T cells to antigens displayed on the surface of target cells.

1	In human blood, 60% to 80% of lymphocytes are mature T cells, and 20% to 30% are mature B cells. Approximately 5% to 10% of the cells do not demonstrate the surface markers associated with either T or B cells. These are NK cells and the rare circulating hemopoietic stem cells (see below). The size differences described above may have functional significance; some of the large lymphocytes may be cells that have been stimulated to divide, whereas others may be plasma cell precursors that are undergoing differentiation in response to the presence of antigen. Several different types of T lymphocytes have been identified: cytotoxic, helper, suppressor, and gamma/delta (). The activities of cytotoxic, helper, suppressor, and gamma/delta T lymphocytes are mediated by molecules located on their surface. Immunolabeling techniques have made it possible to identify specific types of T cells and study their function.

1	 Cytotoxic CD8 T cells serve as the primary effector cells in cell-mediated immunity. CD8 cells are specifically sensitized T lymphocytes that recognize antigens through the TCRs on viral or neoplastic host cells. Cytotoxic CD8 T lymphocytes only recognize antigens bound to MHC I molecules. After the TCR binds the antigen–MHC I complex, the cytotoxic CD8 T cells secrete lymphokines and perforins that produce ion channels in the membrane of the infected or neoplastic cell, leading to its lysis (see Chapter 14, Lymphatic System). Cytotoxic CD8 T lymphocytes play a significant role in rejection of allografts and in tumor immunology.

1	 Helper CD4 T cells are critical for induction of an immune response to a foreign antigen. Antigen bound to MHC II molecules is presented by antigen-presenting cells such as macrophages to a helper CD4 T lymphocyte. Binding of the TCR to the antigen–MHC II complex activates the helper CD4 T cells. The activated helper CD4 T lymphocytes then produce interleukins (mainly IL-2), which act in an autocrine mode to simulate the proliferation and differentiation of more helper CD4 T lymphocytes. Newly differentiated cells synthesize and secrete lymphokines that affect function as well as differentiation of B cells, T cells, and NK cells. B cells differentiate into plasma cells and synthesize antibody.

1	 Regulatory (suppressor) T-cells represent a phenotypically diverse population of T lymphocytes that can functionally suppress an immune response to foreign and self-antigen by influencing the activity of other cells in the immune system. The CD4CD25FOXP3 regulator T cells represent a classical example of cells that can down-regulate the ability of T lymphocytes to initiate immune responses. The FOXP3 marker indicates an expression of forkhead family transcription factors that are characteristic of many T cells. Furthermore, tumor-associated CD8CD45RO T suppressor cells secrete IL-10 and also suppress T-cell activation. The suppressor T cells may also function in suppressing B-cell differentiation and in regulating erythroid cell maturation in the bone marrow.

1	 Gamma/delta () T cells represent a small population of T cells that possess a distinct TCR on their surface. As discussed previously, most T cells have a TCR receptor composed of two glycoprotein chains called and TCR chains. In contrast, T cells possess TCR receptors made up of one -chain and one -chain. These cells develop in the thymus and migrate into various epithelial tissues (e.g., skin, oral mucosa, intestine, and vagina). Once they colonize an epithelial tissue, they do not recirculate between blood and lymphatic organs. They are also known as intraepithelial lymphocytes. Their location within the skin and mucosa of internal organs allows them to function in the first line of defense against invading organisms. Monocytes are the precursors of the cells of the mononuclear phagocytotic system.

1	Monocytes are the precursors of the cells of the mononuclear phagocytotic system. Monocytes are the largest of the WBCs in a blood smear (average diameter, 18 m). They travel from the bone marrow to the body tissues, where they differentiate into the various phagocytes of the mononuclear phagocytotic system— that is, connective tissue macrophages, osteoclasts, alveolar macrophages, perisinusoidal macrophages in the liver (Kupffer cells), and macrophages of lymph nodes, spleen, and bone marrow among others (see Chapter 6, Connective Tissue). Monocytes remain in the blood for only about 3 days.

1	The nucleus of the monocyte is typically more indented than that of the lymphocyte (Fig. 10.12 and Plate 18, page 304). The indentation is the site of the cell center where the well-developed Golgi apparatus and centrioles are located. Monocytes also contain smooth endoplasmic reticulum, rough endoplasmic reticulum, and small mitochondria. Although these cells are classified as agranular, they contain small, dense, azurophilic granules. These granules contain typical lysosomal enzymes similar to those found in the azurophilic granules of neutrophils. Monocytes transform into macrophages, which function as antigen-presenting cells in the immune system.

1	Monocytes transform into macrophages, which function as antigen-presenting cells in the immune system. During inﬂammation, the monocyte leaves the blood vessel at the site of inﬂammation, transforms into a tissue macrophage, and phagocytoses bacteria, other cells, and tissue debris. The monocyte–macrophage is an antigen-presenting cell and plays an important role in immune responses by partially degrading antigens and presenting their fragments on the MHC II molecules located on the macrophage surface of helper CD4 T lymphocytes for recognition. Thrombocytes are small, membrane-bounded, anucleate cytoplasmic fragments derived from megakaryocytes.

1	Thrombocytes are small, membrane-bounded, anucleate cytoplasmic fragments derived from megakaryocytes. Thrombocytes (platelets) are derived from large polyploid cells (cells whose nuclei contain multiple sets of chromosomes) in the bone marrow called megakaryocytes (Fig. 10.13). In platelet formation, small bits of cytoplasm are separated from the peripheral regions of the megakaryocyte by extensive platelet demarcation channels. The membrane that lines these channels arises by invagination of the plasma membrane; therefore, the channels are in continuity with the extracellular space. The continued development and fusion of the platelet demarcation membranes result in the complete partitioning of cytoplasmic fragments to form individual platelets. After entry into the vascular system from the bone marrow, the platelets circulate as discoid structures about 2 to 3 m in diameter. Their life span is about 10 days.

1	Structurally, platelets may be divided into four zones based on organization and function. The TEM reveals a structural organization of the thrombocyte cytoplasm that can be categorized into the following four zones (Fig. 10.14).  The peripheral zone consists of the cell membrane covered by a thick surface coat of glycocalyx. The glyco calyx consists of glycoproteins, glycosaminoglycans, and FIGURE 10.12 • Electron micrograph of a human mature monocyte. The nucleus is markedly indented, and adjacent to this site, a centriole (C) and several Golgi profiles (G) are evident. The small dark granules are azurophilic granules, the lysosomes (L) of the cell. The slightly larger and less dense profiles are mitochondria (M). 22,000. (Courtesy of Dr. Dorothea Zucker-Franklin.) Inset. Light microscopic appearance of a monocyte from a blood smear. 1,800.

1	FIGURE 10.13 • Electron and light micrographs of a megakaryocyte. This electron micrograph shows a portion of a megakaryocyte from a bone marrow section. Two lobes of the nucleus and the surrounding cytoplasm are visible. The cell border is indicated by the dotted line (upper right). The cytoplasm reveals evidence of platelet formation as indicated by the extensive platelet demarcation channels. 13,000. Left inset. Light micrograph showing an entire megakaryocyte from a marrow smear. Its nucleus is multilobed and folded on itself, giving an irregular outline. The “foamy” peripheral cytoplasm of the megakaryocyte represents areas in which segmentation to form platelets is occurring. The smaller surrounding cells are developing blood cells. 1,000. Right inset. Higher-power electron micrograph showing a section of cytoplasm that is almost fully partitioned by platelet demarcation channels (arrows). It also shows mitochondria (M), a very dense granule, and glycogen particles. For

1	showing a section of cytoplasm that is almost fully partitioned by platelet demarcation channels (arrows). It also shows mitochondria (M), a very dense granule, and glycogen particles. For comparison, Figure 10.14a shows a mature circulating platelet. 30,000.

1	several coagulation factors adsorbed from the plasma. The integral membrane glycoproteins function as receptors in platelet function.  The structural zone comprises microtubules, actin filaments, myosin, and actin-binding proteins that form a network supporting the plasma membrane. From 8 to 24 microtubules reside as a bundle immediately below the actin filament network. They are circumferentially arranged and are responsible for maintaining the platelet’s disc shape.

1	 The organelle zone occupies the center of the platelet. It consists of mitochondria, peroxisomes, glycogen particles, and at least three types of granules dispersed within the cytoplasm. The most numerous granules are granules (300 to 500 nm in diameter) that contain mainly fibrinogen, coagulation factors, plasminogen, plasminogen activator inhibitor, and platelet-derived growth factor. The contents of these granules play an important role in the initial phase of vessel repair, blood coagulation, and platelet aggregation. The smaller, denser, and less numerous granules mainly contain adenosine diphosphate (ADP), adenosine triphosphate (ATP), serotonin, and histamine, which facilitate platelet adhesion and vasoconstriction in the area of the injured vessel. The granules are similar to lysosomes found in other cells and contain several hydrolytic enzymes. The contents of granules function in clot resorption during the later stages of vessel repair.

1	 The membrane zone consists of two types of membrane channels. The open canalicular system (OCS) is the first type of membrane channel. The OCS is a developmental remnant of the platelet demarcation channels and is simply a membrane that did not participate in subdividing the megakaryocyte cytoplasm. In effect, open canaliculi are invaginations into the cytoplasm from the plasma membrane. The dense tubular system (DTS) is the

1	FIGURE 10.14 • Platelet electron micrograph and diagram. a. High-magnification electron micrograph of a platelet situated between an erythrocyte on the left and an endothelial cell on the right. Visible organelles include a mitochondrion, microtubules, a single profile of the surface-connected open canalicular system, profiles of the dense tubular system, the moderately dense granules, a single very dense granule, and glycogen particles. The microfilaments are not evident against the background matrix of the platelet. b. Diagram of a platelet showing the components of the four structural zones.

1	second type of membrane channel. The DTS contains an electron-dense material originating from the rough endoplasmic reticulum of the megakaryocyte, which serves as a storage site for calcium ions. DTS channels do not connect with the surface of the platelet; however, both the OCS and DTS fuse in various areas of the platelet to form membrane complexes that are important in regulation of the intraplatelet calcium concentration. Platelets function in continuous surveillance of blood vessels, blood clot formation, and repair of injured tissue. Platelets are involved in several aspects of hemostasis (control of bleeding). They continuously survey the endothelial lining of blood vessels for gaps and breaks. When a blood vessel wall is injured or broken, the exposed connective tissue at the damaged site promotes platelet adhesion. Adhesion of the platelets at the damaged site triggers their degranulation and release of serotonin, ADP, and thromboxane A2.

1	Serotonin is a potent vasoconstrictor that causes the vascular smooth muscle cells to contract, thereby reducing local blood flow at the site of injury. Adenosine diphosphate (ADP), a nucleotide, and the signaling molecule thromboxane A2, are responsible for further aggregation of platelets into a primary hemostatic plug. The mass of aggregated platelets stop extravasation of blood. At the same time, the activated platelets release their and granules, which contain among other substances coagulation factors such as platelet thromboplastic factor (PF3) and additional serotonin. The glycocalyx of the platelets provides a reaction surface for the conversion of soluble fibrinogen into fibrin. Fibrin then forms a loose mesh over the initial plug and is further FIGURE 10.15 • Scanning electron micrograph of blood clot.

1	FIGURE 10.15 • Scanning electron micrograph of blood clot. High-magnification scanning electron micrograph shows initial stage of blood clot formation. Red blood cells are entrapped in a loose mesh of fibrin fibers that are extensively cross-linked to form impermmable hemostatic plug that prevents movement of cells and fluids from the lumen of the injured vessel. 1,600. (Copyright Dennis Kunkel Microscopy, Inc.) stabilized by covalent cross-links that produce a dense aggregation of fibers (Fig 10.15). Platelets and red blood cells become trapped in this mesh. The initial platelet plug is transformed into a definitive clot known as a secondary hemostatic plug by additional tissue factors secreted by the damaged blood vessel.

1	After the definitive clot is formed, platelets cause clot retraction, probably as a function of the actin and myosin found in the structural zone of the platelet. Contraction of the clot permits the return of normal blood flow through the vessel. Finally, after the clot has served its function, it is lysed by plasmin, a fibrinolytic enzyme that circulates in the plasma in an inactive form known as plasminogen. The hydrolytic enzymes released from the granules assist in this process. The activator for plasminogen conversion, tissue plasminogen activator (TPA), is derived principally from endothelial cells. A synthetic form of TPA is currently used as an emergency treatment to minimize the damage caused by clots that lead to strokes. An additional role of platelets is to help repair the injured tissues beyond the vessel itself. Platelet-derived growth factor released from the granules stimulates smooth muscle cells and fibroblasts to divide and allow tissue repair.

1	Hemopoiesis (hematopoiesis) includes both erythropoiesis and leukopoiesis (development of red and white blood cells, respectively), as well as thrombopoiesis (development of platelets; Fig. 10.16). Blood cells have a limited life span; they are continuously produced and destroyed. The ultimate objective of hemopoiesis is to maintain a constant level of the different cell types found in the peripheral blood. Both the human erythrocyte (life span of 120 days) and the platelet (life span of 10 days) spend their entire life in the circulating blood. Leukocytes, however, migrate out of the circulation shortly after entering it from the bone marrow and spend most of their variable life spans (and perform all of their functions) in the tissues.

1	In the adult, erythrocytes, granulocytes, monocytes, and platelets are formed in the red bone marrow; lymphocytes are also formed in the red bone marrow and in the lymphatic tissues. To study the stages of blood cell formation, a sample of bone marrow is prepared as a stained smear in a manner similar to that described on page 270 for the preparation of a smear of blood. Hemopoiesis is initiated in early embryonic development.

1	During fetal life, both erythrocytes and leukocytes are formed in several organs before the differentiation of the bone marrow. The first or yolk-sac phase of hemopoiesis begins in the third week of gestation and is characterized by the formation of “blood islands” in the wall of the yolk sac of the embryo. In the second, or hepatic phase, early in fetal development, hemopoietic centers appear in the liver (Fig. 10.17). Blood cell formation in these sites is largely limited to erythroid cells although some leukopoiesis occurs in the liver. The liver is the major blood-forming organ in the fetus during the second trimester. The third or bone marrow phase of fetal hemopoiesis and leukopoiesis involves the bone marrow (and other lymphatic tissues) and begins during the second trimester of pregnancy. After birth, hemopoiesis takes place only in the red bone marrow and lymphatic tissues, as in the adult (Fig. 10.18). The precursors of both the blood cells and germ cells arise in the yolk

1	After birth, hemopoiesis takes place only in the red bone marrow and lymphatic tissues, as in the adult (Fig. 10.18). The precursors of both the blood cells and germ cells arise in the yolk sac.

1	Monophyletic Theory of Hemopoiesis According to the monophyletic theory of hemopoiesis, blood cells are derived from a common hemopoietic stem cell. Considerable circumstantial evidence has for many years supported the monophyletic theory of hemopoiesis in which all blood cells arise from a common stem cell. Decisive evidence for the validity of the monophyletic theory has come with the isolation and demonstration of the hemopoietic stem cell (HSC). The hemopoietic stem cell, also known as pluripotential stem cell (PPSC), is capable not only of differentiating into all the blood cell lineages but also of self-renewal ( i.e., the pool progenitor (CLP, CFU-L)

1	Hemopoietic stem cell (HSC, PPSC) Common myeloid progenitor (CMP, CFU-GEMM) Granulocyte/ monocyte progenitor (GMP, CFU-GM) Pro-dendritic cella (pro-DC) Dendritic cell Neutrophil progenitor (NoP, CFU-G) Neutrophil Basophil/mast cell progenitorb (BMCP) Mast cell progenitor (MCP) Mast cellBasophil progenitor (BaP, CFU-Ba) Basophil Eosinophil cell progenitor (EoP, CFU-Eo) Eosinophil Neutrophil Eosinophil Monocyte progenitor (MoP, CFU-M) Monocyte Macrophage Megakaryocyte progenitorc (MKP, CFU-Meg) Megakaryocyte/ erythrocyte progenitor (MEP) Megakaryocyte Megakaryoblast Platelets Erythrocyte progenitor (ErP, CFU-E) Proerythroblast Orthochromatophilic erythroblast Erythrocyte

1	FIGURE 10.16 • Hemopoiesis. This chart is based on the most recent concepts in hemopoiesis. It shows blood cells’ development from hemopoietic stem cells in the bone marrow to mature cells and their distribution in the blood and connective tissue compartments. In all lineages, extensive proliferation occurs during differentiation. Cytokines (including hemopoietic growth factors) may and do act individually and severally at any point in the process from the first stem cell to the mature blood or connective tissue cell. aProdendritic cells are possible to differentiate from common lymphoid progenitor. bIf committed to enter the mast cell lineage, the basophil/mast cell progenitor cell migrates to the spleen where it differentiates into a mast cell progenitor cell. After further differentiation in the spleen, it migrates to the intestine to become a mast cell precursor. cA megakariocyte progenitor cell may also differentiate directly from a common myeloid progenitor cell.

1	FIGURE 10.17 • Hepatic stage of hemopoiesis. Photomicro -graph of the fetal liver stained with H&E shows active hemopoiesis. The small round bodies (arrows) are mostly nuclei of developing erythrocytes. Although it is difficult to discern, these cells are located between developing liver cells and the wall of the vascular sinus. 350.

1	of stem cells is self-sustaining). Recent studies indicate that HSCs also have the potential to differentiate into multiple non–blood cell lineages and contribute to the cellular regeneration of various tissues and multiple organs. During embryonic development, HSCs are present in the circulation and undergo tissue-specific differentiation in different organs. Human HSCs have been isolated from umbilical cord blood, fetal liver, and fetal and adult bone marrow. In the adult, HSCs have the potential to repair tissues under pathologic conditions (e.g., is-chemic injury, organ failure). Human HSCs express specific molecular marker proteins such as CD34 and CD90 and at the same time do not express lineage-specific markers (Lin–) that are found on lymphocytes, granulocytes, monocytes, megakaryocytes, and erythroid cells. It is now believed that human HSC can be identified by the Lin , CD34, CD90 , and CD38 cellsurface markers. HSCs are not identifiable in the routine preparation; however,

1	and erythroid cells. It is now believed that human HSC can be identified by the Lin , CD34, CD90 , and CD38 cellsurface markers. HSCs are not identifiable in the routine preparation; however, they can be identified and isolated using immunocytochemical methods.

1	A hemopoietic stem cell (HSC) in the bone marrow gives rise to multiple colonies of progenitor stem cells. In the bone marrow, descendants of the HSC differentiate into two major colonies of multipotential progenitor cells: The common myeloid progenitor (CMP) cells and the common lymphoid progenitor (CLP) cells. Ultimately, common myeloid progenitor (CMP) cells which were previously called colony-forming units–granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM), differentiate into specific lineage-restricted progenitors (Table 10.3). These include the following.

1	FIGURE 10.18 • Dynamics of hemopoiesis in embryonic and fetal life. During embryonic and fetal life, erythrocytes are formed in several organs. Essentially, three major organs involved in hemopoiesis can be sequentially identified: The yolk sac in the early developmental stages of the embryo; the liver during the second trimester of pregnancy; and the bone marrow during the third trimester. The spleen participates to a very limited degree during the second trimester of pregnancy. At birth, most hemopoiesis occurs in the red bone marrow, as it does in the adult.  Megakaryocyte/erythrocyte progenitor (MEP) cells: These bipotential stem cells give rise to monopotent megakaryocyte-committed progenitor cells (MKP or CFU-Meg) and other monopotent erythrocyte-committed progenitor cells (ErP or CFU-E) that give rise to the erythrocyte lineage.

1	 Granulocyte/monocyte progenitor (GMP or CFUGM) cells: Development of the GMP (CFU-GM) cells requires high-level expression of PU.1 transcription factor. These cells then give rise to the neutrophil progenitors (NoP or CFU-G) which differentiate into the neutrophilic lineage; eosinophil progenitors (EoP or CFU-Eo), cells that give rise to eosinophils; basophil/mast cell progenitors (BMCP) that give rise either to basophil progenitor cells (BaP or CFU-Ba) in the bone marrow or MCPs in the gastrointestinal mucosa; and finally monocyte progenitors (MoP or CFU-M) that develop toward monocyte lineages. In addition to the specific lineage progenitors, GMP cells can give rise to dendritic cells (DCs), which are professional antigen-presenting cells. Dendritic cells are discuss in Chapter 14, Lymphatic System.

1	The common lymphoid progenitor (CLP) cells are capable of diferentiating into T cells, B cells, and natural killer (NK) cells. These multipotential CLP cells have been previously called colony-forming units–lymphoid (CFU-L) The NK cells are thought to be the prototype of T cells; they both possess similar capability to destroy other cells. Lymphocytes are discussed in Chapter 14, Lymphatic System. Dendritic cells can also developed from CLP cells. Perhaps the easiest way to begin the histologic study of blood cell development is to refer to Figure 10.16 and TABLE Summary of Features During Maturation of Common Myeloid Progenitor (CMP) Cell10.3 • Large, round nucleuswith 1–2 nucleoli Smaller, •moreheterochromatic nucleus • Last cell capable of mitosis Small, deeply stained nucleus Begins to acquire acidophilia Acidophilic cytoplasm •with trace of earlier gray Life span:in blood, 1–120 days Slightly basophilic •cytoblasm with multiplenucleoli

1	Small, deeply stained nucleus Begins to acquire acidophilia Acidophilic cytoplasm •with trace of earlier gray Life span:in blood, 1–120 days Slightly basophilic •cytoblasm with multiplenucleoli Round, invaginated •nucleus; may bebinucleate • Enlarges by endomitosis• Polyploid (8–64n) Life span: in marrow, unknowm Platelets (constantly produced in marrow)Life span:in blood, 7–10 days • Large, euchromatic spherical nucleus; 3–5 nucleoli • Increased size, 18–24 m • Last cell capable of mitosis • Nucleus is elongate andacquires U shape • Nucleus, 3–5 segmentsor lobes Life span:in blood, 8–12 hrin CT, 1–2 days • Difficult to Identify 55 hr • Nucleus large, slightly Large, kidney-shaped • Life span:in blood, 16 hr Life span:in CT, unknown Large,•euchromatc spherical nucleus;3–5 nucleoli Increased size, Life span:in blood, 8–12 hrin CT, unknown Large,•euchromatic spherical nucleus; 3–5nucleoli • Increased size, 18–24 m • Last cell capable of mitosis

1	Increased size, Life span:in blood, 8–12 hrin CT, unknown Large,•euchromatic spherical nucleus; 3–5nucleoli • Increased size, 18–24 m • Last cell capable of mitosis Life span:in blood, 8 hrin CT, unknown This table summarizes the maturation of blood cells with histologic characteristics at the various stages, maturation time, and life span after leaving the marrow. Times indicated along vertical lines are the approximate time between recognizable stages. ↑M-1 wk indicates increase in number by mitosis for 1 week before differentiation begins; MEP, megakaryocyte/erythrocyte progenitor cell; MKP, megakaryocyte progenitor cell; BMCP, basophil/mast cell progenitor cell; ErP, erythrocyte progenitor cell; NoP, neutrophil progenitor cell; MoP, monocyte progenitor cell; BaP, basophil progenitor cell; CT, connective issue.

1	Figure 10.19. Figure 10.19 shows the stages of blood cell development in which characteristic cell types can be identified in the light microscope in a tissue section or bone marrow smear. Hemopoiesis is initiated in an apparently random manner when individual HSCs begin to differentiate into one of the lineage-restricted progenitor cells. Progenitor cells have surface receptors for specific cytokines and growth factors, including colony-stimulating factors FIGURE 10.19 • Stages of erythrocytic and granular leukocytic differentiation with Romanovskytype stain. Shown here are normal human bone marrow cells as they basophil would typically appear in a smear. (CSFs), that influence their proliferation and maturation into a specific lineage. Development of Erythrocytes (Erythropoiesis)

1	(CSFs), that influence their proliferation and maturation into a specific lineage. Development of Erythrocytes (Erythropoiesis) Erythrocyte development starts from CMP cells that, under the influence of erythropoietin, IL-3, and IL-4, differentiate to MEP cells. Expression of transcription factor GATA-1is required for the terminal differentiation of MEP cells to definitive erythroid cell lineage. Under GATA-1 influence, MEP cells transform into erythropoietin-sensitive erythrocyte-committed progenitors (ErPs or CFU-E) that give rise to the proerythroblast. The first microscopically recognizable precursor cell in erythropoiesis is called the proerythroblast.

1	The first microscopically recognizable precursor cell in erythropoiesis is called the proerythroblast. The proerythroblast is a relatively large cell measuring 12 to 20 m in diameter. It contains a large spherical nucleus with one or two visible nucleoli. The cytoplasm shows mild basophilia because of the presence of free ribosomes. Although recognizable, the proerythroblast is not easily identified in routine bone marrow smears. The basophilic erythroblast is smaller than the proerythroblast, from which it arises by mitotic division.

1	The basophilic erythroblast is smaller than the proerythroblast, from which it arises by mitotic division. The nucleus of the basophilic erythroblast is smaller (10 to 16 m in diameter) and progressively more heterochromatic with repeated mitoses. The cytoplasm shows strong basophilia because of the large number of free ribosomes (polyribosomes) that synthesize hemoglobin. The accumulation of hemoglobin in the cell gradually changes the staining reaction of the cytoplasm so that it begins to stain with eosin. At the stage when the cytoplasm displays both acidophilia, because of the staining of hemoglobin, and basophilia, because of the staining of the ribosomes, the cell is called a polychromatophilic erythroblast. The polychromatophilic erythroblast shows both acidophilic and basophilic staining of cytoplasm.

1	The polychromatophilic erythroblast shows both acidophilic and basophilic staining of cytoplasm. The staining reactions of the polychromatophilic erythroblast may blend to give an overall gray or lilac color to the cytoplasm, or distinct pink (acidophilic) and purple (basophilic) regions may be resolved in the cytoplasm. The nucleus of the cell is smaller than that of the basophilic erythroblast, and coarse heterochromatin granules form a checkerboard pattern that helps identify this cell type. The orthochromatophilic erythroblast is recognized by its increased acidophilic cytoplasm and dense nucleus. The next named stage in erythropoiesis is the orthochromatophilic erythroblast (normoblast). This cell has a small, compact, densely stained nucleus. The cytoplasm is eosinophilic because of the large amount of hemoglobin (Fig. 10.20). It is only slightly larger than a mature erythrocyte. At this stage, the orthochromatophilic erythroblast is no longer capable of division.

1	The polychromatophilic erythrocyte has extruded its nucleus. The orthochromatic erythroblast loses its nucleus by extruding it from the cell; it is then ready to pass into the blood sinusoids of the red bone marrow. Some polyribosomes that can still synthesize hemoglobin are retained in the cell. These polyribosomes impart a slight basophilia to the otherwise eosinophilic cells; for this reason, these new cells are called polychromatophilic erythrocytes (Fig. 10.21). The polyribosomes of FIGURE 10.20 • Electron micrograph of an orthochromatophilic erythroblast (normoblast). The cell is shown just before extrusion of the nucleus. The cytoplasm contains a group of mitochondria located below the nucleus and small cytoplasmic vacuoles. The cytoplasm is relatively dense because of its hemoglobin content. The fine, dense particles scattered in the cytoplasm are ribosomes. 10,000. (Courtesy of Dr. Dorothea Zucker-Franklin.)

1	FIGURE 10.21 • Electron micrograph of a polychromatophilic erythrocyte (reticulocyte). The nucleus is no longer present, and the cytoplasm shows the characteristic fimbriated processes that occur just after nuclear extrusion. Mitochondria are still present, as are early and late endosomes and ribosomes. 16,500. (Courtesy of Dr. Dorothea Zucker-Franklin.) the new erythrocytes can also be demonstrated with special stains that cause the polyribosomes to clump and form a reticular network. Consequently, polychromatophilic erythrocytes are also (and more commonly) called reticulocytes. In normal blood, reticulocytes constitute about 1% to 2% of the total erythrocyte count. However, if increased numbers of erythrocytes enter the bloodstream (as during increased erythropoiesis to compensate for blood loss), the number of reticulocytes increases. Kinetics of Erythropoiesis Mitoses occur in proerythroblasts, basophilic erythroblasts, and polychromatophilic erythroblasts.

1	Kinetics of Erythropoiesis Mitoses occur in proerythroblasts, basophilic erythroblasts, and polychromatophilic erythroblasts. At each of these stages of development, the erythroblast divides several times. It takes about a week for the progeny of a newly formed basophilic erythroblast to reach the circulation. Nearly all erythrocytes are released into the circulation as soon as they are formed; bone marrow is not a storage site for erythrocytes. Erythrocyte formation and release are regulated by erythropoietin, a 34-kilodalton glycoprotein hormone synthesized and secreted by the kidney in response to decreased blood oxygen concentration. Erythropoietin acts on the specific receptors expressed on the surface of ErP. Erythrocytes have a life span of about 120 days in humans.

1	Erythrocytes have a life span of about 120 days in humans. When erythrocytes are about 4 months old, they become senescent. The macrophage system of the spleen, bone marrow, and liver phagocytoses and degrades the senescent erythrocytes. The heme and globin dissociate, and the globin is hydrolyzed to amino acids, which enter the metabolic pool for reuse. The iron on the heme is released, enters the iron-storage pool in the spleen in the form of hemosiderin or ferritin, and is stored for reuse in hemoglobin synthesis. The rest of the heme moiety of the hemoglobin molecule is partially degraded to bilirubin, bound to albumin, released into the bloodstream, and transported to the liver, where it is conjugated and excreted via the gallbladder as the bilirubin glucuronide of bile. Development of Thrombocytes (Thrombopoiesis)

1	Development of Thrombocytes (Thrombopoiesis) Each day bone marrow of a healthy adult produces about 1 1011 platelets, a number that can increase 10-fold in time of increased demand. The thrombocytopoiesis from the bone marrow progenitors is a complex process of cell divisions and differentiation that requires the support of interleukins, colony-stimulating factors, and hormones. Thrombocytes (platelets) develop from a bipotent megakaryocyte/erythrocyte progenitor (MEP) cell that differentiates into megakariocyte-committed progenitor (MKP) cell and finally into a megakaryocyte. Platelets are produced in the bone marrow from the same common myeloid progenitor (CMP) cells as the erythroid and myeloid series. Under the influence of granucolyte-macrophage colony-stimulating factor (GMCSF) and IL-3, CMP stem cell differentiates into a bipotent megakaryocyte/erythrocyte progenitor (MEP) cell.

1	Further development proceeds toward a unipotent megakariocytecommitted progenitor (MKP) cell (or CFU-Meg), which further develops into the megakaryoblast. The megakaryoblast that develops from this MKP is a large cell (about 30 m in diameter) with a nonlobed nucleus. No evidence of platelet formation is seen at this stage. Successive endomitoses occur in the megakaryoblast (i.e., chromosomes replicate), but neither karyokinesis nor cytokinesis occurs. Under stimulation by thrombopoietin, a 30-kilodalton glycoprotein hormone produced by liver and kidney, ploidy increases from 8n to 64n before chromosomal replication ceases. The cell then becomes a platelet-producing megakaryocyte, a cell measuring 50 to 70 m in diameter with a complex multilobed nucleus and scattered azurophilic granules. Both the nucleus and the cell increase in size in proportion to the ploidy of the cell. With the TEM, multiple centrioles and multiple Golgi apparatuses are also seen in these cells.

1	When bone marrow is examined in a smear, platelet fields are seen to fill much of the peripheral cytoplasm of the megakaryocyte. When examined with the TEM, the peripheral cytoplasm of the megakaryocyte appears to be divided into small compartments by invagination of the plasma membrane. As described above, these invaginations are the platelet demarcation channels (see Fig. 10.13). Thrombocytopenia (a low blood platelet count) is an important clinical problem in the management of patients with immune-system disorders and cancer (i.e., leukemia). It increases the risk of bleeding and in cancer patients often limits the dose of chemotherapeutic agents. Development of Granulocytes (Granulopoiesis)

1	Granulocytes originate from the multipotential common myeloid progenitor (CMP) stem cell, which differentiates into granulocyte/monocyte progenitors (GMPs) under the influence of cytokines such as GM-CSF, granulocyte colony-stimulating factor (G-CSF), and IL-3. GM-CSF is a cytokine secreted by endothelial cells, T cells, macrophages, mast cells, and fibroblasts. It stimulates GMP cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes. The neutrophil progenitor (NoP) undergoes six morphologically identifiable stages in the process of maturation: myeloblast, promyelocyte, myelocyte, metamyelocyte, band cell, and mature neutrophil. Eosinophils and basophils undergo a morphologic maturation similar to that of neutrophils. GMP cells, when induced by GM-CSF, IL-3, and IL-5, differentiate to eosinophil progenitors (EoPs), and eventually mature to eosinophils. Lack of IL-5 causes the GMP cells to differentiate into basophil progenitors (BaPs), which produce

1	and IL-5, differentiate to eosinophil progenitors (EoPs), and eventually mature to eosinophils. Lack of IL-5 causes the GMP cells to differentiate into basophil progenitors (BaPs), which produce basophils. One cannot differentiate eosinophilic or basophilic precursors from neutrophilic precursors morphologically in the light microscope until the cells reach the myelocytic stage when the specific granules appear.

1	Myeloblasts are the first recognizable cells that begin the process of granulopoiesis. The myeloblast is the earliest microscopically recognizable neutrophil precursor cell in the bone marrow. It has a large, euchromatic, spherical nucleus with three to five nucleoli. It measures 14 to 20 m in diameter and has a large nuclear-tocytoplasmic volume. The small amount of agranular cytoplasm stains intensely basophilic. A Golgi area is often seen where the cytoplasm is unstained. The myeloblast matures into a promyelocyte. Promyelocytes are the only cells to produce azurophilic granules.

1	Promyelocytes are the only cells to produce azurophilic granules. The promyelocyte has a large spherical nucleus with azurophilic (primary) granules in the cytoplasm. Azurophilic granules are produced only in promyelocytes; cells in subsequent stages of granulopoiesis do not make azurophilic granules. For this reason, the number of azurophilic granules is reduced with each division of the promyelocyte and its progeny. Promyelocytes do not exhibit subtypes. Recognition of the neutrophil, eosinophil, and basophil lines is possible only in the next stage—the myelocyte—when specific (secondary) and tertiary granules begin to form. Myelocytes first exhibit specific granules.

1	Myelocytes first exhibit specific granules. Myelocytes begin with a more or less spherical nucleus that becomes increasingly heterochromatic and acquires a distinct indentation during subsequent divisions. Specific granules begin to emerge from the convex surface of the Golgi apparatus, whereas azurophilic granules are seen at the concave side. The significance of this separation is unclear. Myelocytes continue to divide and give rise to metamyelocytes. The metamyelocyte is the stage at which neutrophil, eosinophil, and basophil lines can be clearly identified by the presence of numerous specific granules.

1	The metamyelocyte is the stage at which neutrophil, eosinophil, and basophil lines can be clearly identified by the presence of numerous specific granules. A few hundred granules are present in the cytoplasm of each metamyelocyte, and the specific granules of each variety outnumber the azurophilic granules. In the neutrophil, this ratio of specific to azurophilic granules is about 2 to 1. The nucleus becomes more heterochromatic, and the indentation deepens to form a kidney bean–shaped structure. Theoretically, the metamyelocyte stage in granulopoiesis is followed by the band stage and then the segmented stage. Although these stages are obvious in the neutrophil line, they are rarely if ever observed in the eosinophil and basophil lines in which the next easily recognized stages of development are the mature eosinophil and mature basophil, respectively. In the neutrophil line, the band (stab) cell precedes development of the first distinct nuclear lobes.

1	In the neutrophil line, the band (stab) cell precedes development of the first distinct nuclear lobes. The nucleus of the band (stab) cell is elongated and of nearly uniform width, giving it a horseshoelike appearance. Nuclear constrictions then develop in the band neutrophil and become more prominent until two to four nuclear lobes are recognized; the cell is then considered a mature neutrophil, also called a polymorphonuclear neutrophil or segmented neutrophil. Although the percentage of band cells in the circulation is almost always low (0% to 3%), it may increase in acute or chronic inflammation and infection. Kinetics of Granulopoiesis Granulopoesis in the bone marrow takes about two weeks.

1	Kinetics of Granulopoiesis Granulopoesis in the bone marrow takes about two weeks. The mitotic (proliferative) phase in granulopoiesis lasts about a week and stops at the late myelocyte stage. The post-mitotic phase, characterized by cell differentiation—from metamyelocyte to mature granulocyte—also lasts about a week. The time it takes for half of the circulating segmented neutrophils to leave the peripheral blood is about 6 to 8 hours. Neutrophils leave the blood randomly—that is, a given neutrophil may circulate for only a few minutes or as long as 16 hours before entering the perivascular connective tissue (a measured half-life of circulating human neutrophils is only 8 to 12 hours). Neutrophils live for 1 to 2 days in the connective tissue, after which they are destroyed by apoptosis and are subsequently engulfed by macrophages. Also, large numbers of neutrophils are lost by migration into the lumen of the gastrointestinal tract from which they are discharged with the feces.

1	Bone marrow maintains a large reserve of fully functional neutrophils ready to replace or supplement circulating neutrophils at times of increased demand. In normal conditions, the bone marrow produces more than 1011 neutrophils each day. As a result of the release of neutrophils from the bone marrow, approximately 5 to 30 times as many mature and near-mature neutrophils are normally present in the bone marrow as are present in the circulation. This bone marrow reserve pool constantly releases neutrophils into the circulation and is replenished by maturing cells. The reserve neutrophils can be released abruptly in response to inﬂammation, infection, or strenuous exercise.

1	A reservoir of neutrophils is also present in the vascular compartment. This reserve consists of a freely circulating pool and a marginated pool, with the latter contained in small blood vessels. The neutrophils adhere to the endothelium much as they do before leaving the vasculature at sites of injury or infection (see page 275). The normally marginated neutrophils, however, loosely adhere to the endothelium through the action of selectin and can be recruited very quickly. They are in dynamic equilibrium with the circulating pool, which is approximately equal to the size of the marginated pool. The size of the reserve pool in the bone marrow and in the vascular compartment depends on the rate of granulopoiesis, the life span of the neutrophils, and the rates of migration into the bloodstream and connective tissue. The entire hemopoietic process is summarized in Table 10.3.

1	Transcription factors control the fate of hemopoietic cells, whereas cytokines and local mediators regulate all stages of hemopoiesis.

1	Intimate interactions between HSCs and their bone marrow microenvironment work toward redefining the identity and the differentiation pathways of these multipotential stem cells. Signaling molecules from a variety of bone marrow cells initiate intracellular pathways that ultimately target a select group of synergistic and inhibitory proteins known as transcription factors. They specifically bind to promoter or enhancer regions on DNA in the affected cell. By controlling transcription of the specific genes downstream, these transcription factors trigger a cascade of genetic changes that ultimately determines the course of the cells during differentiation. In addition to identifying the various intracellular transcription factors, recent studies have identified and begun to characterize numerous signaling molecules found in the bone marrow. These include glycoproteins that act as both circulating hormones and local mediators to regulate the progress of hemopoiesis and the rate of

1	numerous signaling molecules found in the bone marrow. These include glycoproteins that act as both circulating hormones and local mediators to regulate the progress of hemopoiesis and the rate of differentiation of other cell types (Table 10.4). Specific hormones such as erythropoietin or thrombopoietin, discussed in a previous section, regulate erythrocyte and thrombocyte development, respectively. Other factors, collectively called colony-stimulating factors (CSFs), are subclassified according to the specific cell or group of cells that they affect. Among the recently isolated and most completely characterized factors are several that stimulate granulocyte and monocyte formation, GM-CSF, G-CSF, and macrophage colony-stimulating factor (M-CSF). Interleukins, produced by lymphocytes, act on other leukocytes and their progenitors. IL-3 is a cytokine that appears to affect most progenitor cells and even terminally differentiated cells. Any particular cytokine may act at one or more

1	act on other leukocytes and their progenitors. IL-3 is a cytokine that appears to affect most progenitor cells and even terminally differentiated cells. Any particular cytokine may act at one or more stages in hemopoiesis, affecting cell division, differentiation, or cell function. These factors are synthesized by many different cell types, including kidney cells (erythropoietin), liver hepatocytes (thrombopoietin), T lymphocytes (IL-3), endothelial cells (IL-6), adventitial cells in the bone marrow (IL-7), and macrophages (the CSFs that affect granulocyte and macrophage development).

1	Blood FOR MATION OF B LOOD CE LLS (H E MOPOI ESIS) TABLE Hemopoietic Cytokines, Their Sources, and Target Cells 10.4 CytokineaSymbol Source Target Granulocyte-macrophage colony-stimulating factor GM-CSF T cells, endothelial cells, fibroblasts CMP, ErP, GMP, EoP, BaP, MKP, all granulocytes, erythrocytes Granulocyte colony-stimulating factor G-CSF Endothelial cells, monocytes ErP, GMP, EoP, BaP, MKP Monocyte colony-stimulating factor M-CSF Monocytes, macrophages, endothelial and adventitial cells GMP, MoP, monocytes, macrophages, osteoclasts Erythropoietin EPO Kidney, liver CMP, MEP, ErP Thrombopoietin TPO Bone marrow MKP, megakaryocytes Interferon-IFN-CD4T cells, NK cells B cells, T cells, NK cells, neutrophils, monocytes Interleukin 1 IL-1 Neutrophils, monocytes, macrophages, endothelial cells CD4T cells, B cells Interleukin 2 IL-2 CD4T cells T cells, B cells, NK cells Interleukin 3 IL-3 CD4T cells CMP, ErP, GMP, EoP, BaP, MKP, all granulocytes, erythroid cells Interleukin 4 IL-4 CD4T

1	CD4T cells, B cells Interleukin 2 IL-2 CD4T cells T cells, B cells, NK cells Interleukin 3 IL-3 CD4T cells CMP, ErP, GMP, EoP, BaP, MKP, all granulocytes, erythroid cells Interleukin 4 IL-4 CD4T cells, mast cells B cells, T cells, mast cells Interleukin 5 IL-5 CD4T cells EoP, eosinophils, B cells Interleukin 6 IL-6 Endothelial cells, neutrophils, macrophages, T cells CMP, ErP, GMP, B cells, T cells, macrophages, hepatocytes Interleukin 7 IL-7 Adventitial cells of bone marrow Early pre-B, pre-T cells Interleukin 8 IL-8 Macrophages, endothelial cells T cells, neutrophils Interleukin 9 IL-9 CD4T cells CD4T cells, CMP, ErP Interleukin 10 IL-10 Macrophages, T cells T cells, B cells, NK cells Interleukin 11 IL-11 Macrophages CMP, ErP, GMP, T cells, B cells, macrophages, megakaryocytes aHemopoietic cytokines include colony-stimulating factors (CSFs), interleukins, and inhibitory factors. They are almost all glycoproteins with a basic polypeptide chain of about 20 kilodaltons. Nearly all of

1	cytokines include colony-stimulating factors (CSFs), interleukins, and inhibitory factors. They are almost all glycoproteins with a basic polypeptide chain of about 20 kilodaltons. Nearly all of them act on progenitor stem cells, lineage-restricted progenitor cells, committed cells, and maturing and mature cells. Therefore, the targets listed above are target lines rather than individual target cells.

1	The isolation, characterization, manufacture, and clinical testing of cytokines (proteins and peptides that are signaling compounds) in the treatment of human disease is a major activity of the rapidly growing biotechnology industry. Several hemopoietic and lymphopoietic cytokines have been manufactured by recombinant DNA technology and are already used in clinical settings. These include recombinant erythropoietin, G-CSF, GM-CSF, and IL-3; others are under active development. GM-CSF (sargramostim, Leukine) is used clinically to stimulate production of WBCs following chemotherapy and to accelerate WBC recovery following bone marrow transplantation. Development of Monocytes The multipotential CMP stem cell also gives rise to the cells that develop along the monocyte–macrophage pathway.

1	The multipotential CMP stem cell also gives rise to the cells that develop along the monocyte–macrophage pathway. Monocytes are produced in the bone marrow from a GMP stem cell that can mature into a monocyte or another of the three granulocytic cell lines. In addition, GMP gives rise to dendritic cells. The proliferation and differentiation of CMP into committed GMP is controlled by IL-3. Further progression of monocyte progenitor (MoP) cell lineage depends on the continued presence of PU.1 and Egr-1 transcription factors and is stimulated by IL-3 and GM-CSF. The GMCSF also controls further differentiation into mature cells, which are then released into circulation. The transformation of MoPs to monocytes takes about 55 hours, and the monocytes remain in the circulation for only about 16 hours before emigrating to the tissues where they differentiate under influence of both GM-CSF and M-CSF into tissue macrophages. Their subsequent life span is not yet fully understood.

1	Development of Lymphocytes (Lymphopoiesis) Development and lineage commitment of CLP cells depend on the expression of variety of transcription factors.

1	Although lymphocytes continuously proliferate in the peripheral lymphatic organs, the bone marrow remains the primary site of lymphopoiesis in humans. Members of the Ikaros family of transcription factors play major roles in the differentiation of pluripotent HSCs toward common lymphoid progenitor (CLP) cells. Progeny of the CLP cells that express GATA-3 transcription factor are destined to become T lymphocytes. These cells that express GATA-3 leave the bone marrow as pre–T lymphocytes and travel to the thymus, where they complete their differentiation and thymic cell education (see Chapter 14, Lymphatic System). They then enter the circulation as long-lived, small T lymphocytes. Another transcription factor, Pax5, activates B-cell–specific genes in CLP cells destined to become B lymphocytes. In mammals, these cells originate in bursa-equivalent organs such as the bone marrow, gut-associated lymphatic tissue, and spleen. Although a number of transcription factors have been identified

1	In mammals, these cells originate in bursa-equivalent organs such as the bone marrow, gut-associated lymphatic tissue, and spleen. Although a number of transcription factors have been identified in the development of lymphoid cell lineages, little is known about factors that may influence development and lineage commitment of NK cells. NK cells most likely differentiate under the influence of IL-2 and IL-15 into immature pre–NK cells, and, after acquisition of NK–cell effector functions (ability to secrete interferon and cytotoxicity), become mature NK cells. The bone marrow is the main NK cell–producing organ. However, recent studies suggest that lymph nodes or fetal thymus may also contain NK-progenitor cells. Lymphocytes constitute as much as 30% of all nucleated cells in the bone marrow. The production and differentiation of lymphocytes are discussed in more detail in Chapter 14, Lymphatic System.

1	Red bone marrow lies entirely within the spaces of bone, medullary cavity of young long bones, and spaces of spongy bone. Bone marrow consists of blood vessels, specialized units of blood vessels called sinusoids, and a spongelike network of hemopoietic cells (Fig. 10.22). The bone marrow sinusoids provide the barrier between the hemopoietic compartment and the peripheral circulation. In sections, the cells in hemopoietic compartment appear to lie in “cords” between sinusoids or between sinusoids and bone.

1	The sinusoid of red bone marrow is a unique vascular unit. It occupies the position normally occupied by a capillary; that is, it is interposed between arteries and veins. It is believed to be derived from vessels that have just nourished the cortical bone tissue. The sinusoids arise from these vessels at the corticomedullary junction. The sinusoid wall consists of an endothelial lining, a basal lamina, and an incomplete covering of adventitial cells. The endothelium is a simple squamous epithelium.

1	The adventitial cell, also called a reticular cell, sends sheetlike extensions into the substance of the hemopoietic cords, which provide some support for the developing blood cells. In addition, adventitial cells produce reticular fibers. They also play a role in stimulating the differentiation of developing progenitor cells into blood cells by secreting several cytokines (e.g., CSFs, IL-5, IL-7). When blood cell formation and the passage of mature blood cells into the sinusoids are active, adventitial cells and the basal lamina become displaced by mature blood cells as they approach the endothelium to enter the sinusoid from the bone marrow cavity. The bone marrow sinusoidal system is a closed circulation system; newly formed blood cells must penetrate the endothelium to enter the circulation.

1	The bone marrow sinusoidal system is a closed circulation system; newly formed blood cells must penetrate the endothelium to enter the circulation. As a maturing blood cell or a megakaryocyte process pushes against an endothelial cell, the abluminal plasma membrane is pressed against the luminal plasma membrane until they fuse, thus forming a transitory opening, or aperture. The migrating cell or the megakaryocyte process literally pierces the endothelial cell. Thus, migration across the bone marrow

1	FIGURE 10.22 • Bone marrow with active hemopoiesis. a. Schematic drawing of bone marrow shows the erythroblastic islets engaged in the formation of erythrocytes, megakaryocytes discharging platelets into the sinusoids, endothelial cells adjacent to a basal lamina that is sparse in places and absent where blood cells are entering the sinusoids, and adventitial or reticular cells extending from the basal lamina into the hemopoietic compartment. (Modified from Weiss L, ed. Cell and Tissue Biology: A Textbook of Histology, 6th ed. Baltimore: Urban & Schwarzenberg, 1988.) b. Photomicrograph of bone marrow stained with H&E shows active hemopoietic centers in a close proximity to bone marrow sinusoids. 420.

1	endothelium is a transcellular and not an intercellular event. Each blood cell must squeeze through an aperture to enter the lumen of a sinusoid. Similarly, a megakaryocyte process must protrude through an aperture so that the platelets can be released directly into the sinusoid lumen. The aperture is lined by the fused plasma membrane, thus maintaining the integrity of the endothelial cell during the transcellular passage. As the blood cell completes its passage through the aperture or the megakaryocyte that has extruded its platelets withdraws its process, the endothelial cell “repairs itself,” and the aperture disappears.

1	In active red bone marrow, the cords of hemopoietic cells contain predominately developing blood cells and megakaryocytes. The cords also contain macrophages, mast cells, and some adipose cells. Although the cords of hemopoietic tissue appear to be unorganized, specific types of blood cells develop in nests or clusters. Each nest in which erythrocytes develop contains a macrophage. These nests are located near the sinusoid wall. Megakaryocytes are also located adjacent to the sinusoid wall, and they discharge their platelets directly into the sinusoid through apertures in the endothelium. Granulocytes develop in cell nests farther from the sinusoid wall. When mature, the granulocyte migrates to the sinusoid and enters the bloodstream. Bone marrow not active in blood cell formation contains predominately adipose cells, giving it the appearance of adipose tissue.

1	Bone marrow not active in blood cell formation contains predominately adipose cells, giving it the appearance of adipose tissue. Inactive bone marrow is called yellow bone marrow. It is the chief form of bone marrow in the medullary cavity of bones in the adult that are no longer hemopoietically active, such as the long bones of the arms, legs, fingers, and toes. In these bones, the red bone marrow has been replaced completely by fat. Even in hemopoietically active bone marrow in adult humans—such as that in the ribs, vertebrae, pelvis, and shoulder girdle—about half of the bone marrow space is occupied by adipose tissue and half by hemopoietic tissue. The yellow bone marrow retains its hemopoietic potential, however, and when necessary, as after severe loss of blood, it can revert to red bone marrow, both by extension of the hemopoietic tissue into the yellow bone marrow and by repopulation of the yellow bone marrow by circulating stem cells.

1	 FOLDER 10.6 Clinical Correlation: Cellularity of the Bone Marrow Bone marrow cellularity is one of the most important factors in evaluating the function of the bone marrow. The assessment of bone marrow cellularity is semiquantita-tive and represents the ratio of hemopoietic cells to adipocytes. The most reliable evaluation of cellularity is ob-tained from the microscopic examination of a bone marrow biopsy that preserves the organization of the marrow. Smear preparations are not accurate preparations with which to assess bone marrow cellularity. Bone cellularity changes with age. Normal bone marrow cellularity for a specifc age can be calculated by subtract-ing an individual’s age from 100 and adding 10%. Thus, a 30-year-old individual’s bone marrow contains between 60% and 80% of active bone-producing cells (100 – 30 70 10%); in contrast, a 70-year-old individual’s marrow is in the range of 20% to 40% (100 70 30 10%).

1	As can be seen from this calculation, the number of hemopoietic cells decreases with age. Bone marrow with a normal age–specifc index is called normocellular bone marrow. Deviation from age-specifc normal indices indicates a pathologic change in the marrow. In hypocellular bone marrow, which occurs in aplastic anemia or after chemotherapy, only a small number of blood-forming cells can be found in a marrow biopsy (Fig. F10.6.1a). Thus, a 50-year-old individual with this condition might have a bone cellularity index of 10% to 20%. In the same-aged individual with acute myelogenous leukemia, the bone cellularity index might be 80% to 90%. Hypercellular bone marrow is characteristic of bone marrow affected by tumors originating from hemopoietic cells (Fig. F10.6.1b).

1	FIGURE F10.6.1 • Cellularity of the bone marrow. a. This is an example of hypocellular bone marrow from an individual with aplastic anemia. The bone marrow consists largely of adipose cells and lacks normal hemopoietic activity. 120. b. This photomicrograph of bone marrow section from an individual with acute myelogenous leukemia shows hypercellular bone marrow. Note that the entire field of view next to the bony trabecula is filled with tightly packed myeloblasts. Only a few adipose cells are visible on this image. 280. (Reprinted with permission from Rubin E, Gorstein F, Schwarting R, Strayer DS. Rubin’s Pathology, 4th ed. Baltimore: Lippincott Williams & Wilkins, 2004, Fig. 20-12, Fig. 20-54.) This page intentionally left blank.

1	Blood is regarded as a connective tissue, fluid in character, and consists of formed elements and plasma. Red blood cells (erythrocytes), white blood cells (leukocytes), and thrombocytes (platelets) constitute the formed elements. Collectively, they make up 45% of the blood volume. Red blood cells transport and exchange oxygen and carbon dioxide. They constitute 99% of the blood cells. White blood cells are categorized as agranulocytes and granulocytes. The agranulocytes are further classified as lymphocytes and monocytes. The granulocytes, so named for the character of the granules that they contain in their cytoplasm, consist of neutrophils, eosinophils, and basophils. Each type of white cell has a specific role in immune and protective responses in the body. They typically leave the circulation and enter the connective tissue to perform their specific role. In contrast, red blood cells function only within the vascular system. Blood platelets are responsible for blood clotting and

1	and enter the connective tissue to perform their specific role. In contrast, red blood cells function only within the vascular system. Blood platelets are responsible for blood clotting and consequently have an essential role in incidents of small vessel damage. Blood smears are utilized for microscope examination and identification of relative numbers of white cells in circulating blood. The blood smear is prepared by placing a small drop of blood on a microscope slide and then smearing it across the slide with the edge of another slide. When properly executed, this method provides a uniform, single layer of blood cells that is allowed to air dry and then stained. Wright’s stain, a modified Romanovsky stain, is generally utilized. In examining the specimen under the microscope, it is useful to use a low magnifica-tion to find areas in which the blood cells have a uniform distribution like that seen in the smear on the adjacent page. Once this is accom-plished, by switching to a

1	useful to use a low magnifica-tion to find areas in which the blood cells have a uniform distribution like that seen in the smear on the adjacent page. Once this is accom-plished, by switching to a higher magnification, one can identify the various types of white blood cells and, in fact, determine the relative number of each cell type. A normal cell count is as follows: neutrophils, 48.6–66.7%; eosinophils, 1.4–4.8%; basophils, 0–0.3%; lympho-cytes, 25.7–27.6%; monocytes, 8.6–9.0%.

1	Blood smear, human, Wright’s stain, ×200. This low magnification photomicrograph shows part of a blood smear in which the blood cells are uniformly distributed. The greater majority of cells are red blood cells. Because of their biconcave shape, most of the red blood cells Neutrophils, blood smear, human, Wright’s stain, ×2,200. Neutrophils exhibit variation in size and nuclear morphology that is associated with age of the cell. The nucleus seen on the left is that of a neutrophil that has just passed the band stage and has recently entered the blood stream. The cell is relatively small; its cytoplasm exhibits distinctive, fine granules. The middle neutrophil is considerably larger and its cytoplasm contains more Eosinophils, blood smear, human, Wright’s stain, ×2,200.

1	Eosinophils, blood smear, human, Wright’s stain, ×2,200. The eosinophils seen in these micrographs similarly represent different stages of maturity. The eosinophil at the left is relatively small and is just beginning to show lobulation. The cytoplasm is almost entirely filled with eosinophilic granules that characterize this cell type. The lighter stained area, devoid of granules, probably represents the site of the Golgi apparatus (arrow). The Basophils, blood smear, human, Wright’s stain, ×2,200.

1	Basophils, blood smear, human, Wright’s stain, ×2,200. The cells shown here are basophils and also represent different stages of maturation. The basophil at the left is relatively young and small. The basophilic granules are variable in size and tend to obscure the morphology of the nucleus. Also, they are less plentiful than the granules seen in the eosinophil. The appear donut-shaped. Two white cells, both granulocytes, are evident. One cell is a neutrophil (N), the other granulocyte is an eosinophil (E). However, at this magnification, the major distinction is in the staining of their cytoplasm. Higher magnification, as in the figures below, would allow for a more precise characterization of the cell type.

1	fine granules. The nucleus still exhibits a U shape, but lobulation (arrows) is becoming apparent with the constriction of the nucleus at several points. The neutrophil to the right shows greater maturity by virtue of its very distinctive lobulation. Here the lobules are connected by a very thin nuclear “bridge.” A very distinctive feature associated with the nucleus of this cell is the presence of a the Barr body (arrow), indicative of blood that has been drawn from a female. eosinophil shown in the middle is larger and its nucleus is now distinctively bilobed. At one site, three distinct granules (arrows) are evident. Note their spherical shape and their relative uniform size. The eosinophil at the right is more mature in that it displays at least three lobes. By going through focus, the eosinophil granules often appear to “light up” due to their crystalline structure.

1	nucleus of the middle basophil appears to be bilobed, but the granules that lie over the nucleus, again tend to obscure the precise shape. The basophil at the right is probably more mature. The granules almost entirely obscure the nuclear shape. A few blood platelets (arrowheads) are seen in several of the micrographs. Typically they appear as small, irregular shaped bodies. PLATE 17 KEY E, eosinophil N, neutrophil Lymphocytes, blood smear, human, Wright’s stain, 7–9m. A large lymphocyte is seen in the right panel. These cells may be as ×2,150. large as 16m. The lymphocyte in the middle panel is intermediate in size. The difference in lymphocyte size is attributable mostly to the amount of

1	The lymphocytes shown here vary in size, but each represents cytoplasm present. However, the nucleus also contributes to the size of the a mature cell. Circulating lymphocytes are usually described as cell, but to a lesser degree. In differential counts, lymphocyte size is small, medium, and large. A small lymphocyte is shown in the disregarded. Two platelets (arrows) are evident in the left panel. left panel. Lymphocytes in this category range in size from Monocytes, blood smear, human, Wright’s stain, ×2,150.

1	left panel. Lymphocytes in this category range in size from Monocytes, blood smear, human, Wright’s stain, ×2,150. The white cells in these panels are mature monocytes. Their size ranges from approximately 13–20m, with the majority falling in the upper size range. The nucleus exhibits the most characteristic features of the monocyte, namely an indentation, which is sometimes so prominent that it exhibits a U shape as is evident in the right hand panel. The cytoplasm is very weakly basophilic. Small, azurophilic granules (lysosomes) are also characteristic of the cytoplasm and are similar to those seen in neutrophils. Platelets (arrows) are present in the left and middle panels. Bone marrow smear, human, Giemsa, ×180.

1	This low magnification photomicrograph shows a bone marrow smear. This type of preparation allows for the examination of developing red and white cells. A marrow smear is made in a manner similar to that of a peripheral blood smear. A sample of bone marrow is aspirated from a bone and simply placed on a slide and spread into a thin monolayer of cells. A wide variety of cell types are present in the marrow smear. Most of the cells are developing granulocytes and developing erythrocytes. Mature erythrocytes (Ey) are also present in large numbers. They are readily identified by their lack of a nucleus and eosinophilic staining. Often intermixed with these red cells are small groups of reticulocytes. These are very young erythrocytes that contain residual ribosomes in their cytoplasm. The presence of the ribosomes slightly alters the color of the reticulocyte giving it a just perceptible blue coloration in comparison to the mature eosinophilic erythrocyte. The reticulocytes are best

1	presence of the ribosomes slightly alters the color of the reticulocyte giving it a just perceptible blue coloration in comparison to the mature eosinophilic erythrocyte. The reticulocytes are best distinguished at higher magnifications. In addition, adipocytes (A) are found in variable numbers. In preparations such as this, the lipid content is lost during preparation and recognition of the cell is based on a clear or unstained round space. Another large cell that is typically present is the megakaryocyte (M). The megakaryocyte is a polyploid cell that exhibits a large and irregular nuclear profile. It is the platelet-producing cell.

1	At this low magnification, it is difficult to distinguish the earlier stages of the developing cell types. However, examples of each stage of development in both cell lines are presented in the following plates. In contrast, many cells in their late stage of development, particularly in the granulocyte series, can be identified with some degree of assuredness at low magnification. For example, some band neutrophils (BN) and young eosinophils (E) can be identified by their morphology and staining characteristics.

1	AG RAN U LOCYTE S AN D R E D MAR ROW KEY A, adipocytes BN, band neutrophil E, eosinophils Ey, erythrocytes M, megakaryocyte 306 Erythropoiesis is a process by which the concentration of erythrocytes in the peripheral blood stream is maintained under normal condi-tions in a steady state. Stimulation of erythroid stem cells (ErP or CFU-E) by hormonal action results in a proliferation of precursor cells that undergo differentiation and maturation in the bone marrow. The earliest recognizable precursor of the red blood cell is the proerythroblast. These cells lack hemoglobin. Their cytoplasm is basophilic and the nucleus exhibits a dense chromatin structure and several nucleoli. The Golgi apparatus, when evident, appears as a light staining area. The basophilic erythroblast is smaller than the proerythroblast, from which it arises by mitotic division. Its nucleus is smaller. The cytoplasm shows strong basophilia due to the increasing number of ribosomes involved in hemoglobin synthesis.

1	proerythroblast, from which it arises by mitotic division. Its nucleus is smaller. The cytoplasm shows strong basophilia due to the increasing number of ribosomes involved in hemoglobin synthesis. The accumulation of hemoglobin in the cell gradually changes the staining reaction of the cytoplasm so that it begins to stain with eosin. The recognizable presence of hemoglobin in the cell by virtue of its staining signifies its transition to the polychro-matophilic erythroblast. The cytoplasm in the earlier part of this stage may exhibit a blue-grey color. With time, increasing amounts of hemoglobin are synthesized and concomitantly, decreasing numbers of ribosomes are present. The nucleus of the cell is smaller than that of the basophilic erythroblast and the heterochromatin is much coarser. At the end of this stage, the nucleus has become much smaller and the cytoplasm more eosinophilic. This is the last stage in which mitosis occurs. The next definable stage is the orthochromatophilic

1	At the end of this stage, the nucleus has become much smaller and the cytoplasm more eosinophilic. This is the last stage in which mitosis occurs. The next definable stage is the orthochromatophilic erythrob-last, also called normoblast. Its nucleus is smaller than earlier stages and is extremely condensed. The cytoplasm is considerably less blue leaning more to a pink or eosinophilic coloration. It is slightly larger than a mature erythrocyte. At this stage, it is no longer capable of division. In the next stage, the polychromatophilic erythrocyte, also more commonly called a reticulocyte, has lost its nucleus and is ready to pass into the blood sinusoids of the red bone marrow. Some ribosomes that can still synthesize hemoglobin are present in the cell. These ribosomes create a very slight basophilia to the cell. Comparison of this cell to typical mature erythrocytes in the marrow smear reveals a slight difference in coloration.

1	Polychromatophilic erythroblast, bone marrow smear, human, Giemsa, ×2,200. Two polychromatophilic erythroblasts are seen in this micrograph. The larger and less mature cell exhibits a pronounced clumping of its chromatin. The cytoplasm is basophilic, but is considerably lighter in color than that of the basophilic erythroblast. The cytoplasm also exhibits some eosinophilia, which is indicative of hemoglobin production. The smaller cell represents a later stage of a polychromatophilic erythroblast. Note how much more dense the chromatin appears as well as how much smaller the nucleus has become. Also, the cytoplasm now favors an eosinophilia. However, some basophilia is still evident. Orthochromatophilic erythrocyte, bone marrow predominantly eosinophilhic, but still possesses a degree of basophilia. Over-smear, human, Giemsa, ×2,200. all the cell is only slightly larger than a mature erythrocyte. At this stage, the cell is no longer capable of division.

1	Two orthrochromatophilic erythrocytes are seen in this micrograph. Their nuclei have become even smaller and the nucleus exhibits a compact, dense staining. The cytoplasm is Polychromatophilic erythrocyte, bone marrow smear, human, Giemsa, ×2,200. A polychromatophilic erythrocyte (PE) is seen in this micro-graph. Its nucleus has been extruded and the cytoplasm exhibits a slight basophilia. In proximity are a number of mature erythrocytes (E). Compare the coloration of the polychromatophilic erythrocyte with that of the mature red blood cells. Polychromatophilic erythrocytes can also be readily demonstrated with special stains that cause the remaining ribosomes in the cytoplasm to clump and form a visible reticular network, hence the polychromatophilic erythrocyte is also commonly called a reticulocyte.

1	KEY E, erythrocytes N, nucleoli PE, polychromatophilic erythrocyte 308 Granulopoiesis is the process by which the granulocyte blood cells (neutrophils, eosinophils, and basophils) differentiate and mature in the bone marrow. The earliest recognizable stage is the myeloblast, which is followed consecutively by the promyelocyte, myelocyte, metamyelo-cyte, band cell, and finally, the mature granulocyte. It is not possible to differentiate eosinophil, basophil, or neutrophil precursors morphologi-cally until the myelocyte stage is reached – when specific granules characteristic of each cell type appear. The cells of the basophil lineage are extremely difficult to locate in a marrow smear because of the minimal number of these cells in the marrow. The myeloblast is characterized by a large euchromatic, spherical nucleus with three to five nucleoli. The cell measures 14 to 20 m in di-ameter. The cytoplasm stains deeply basophilic. The presence of a light or poorly staining area indicates a

1	spherical nucleus with three to five nucleoli. The cell measures 14 to 20 m in di-ameter. The cytoplasm stains deeply basophilic. The presence of a light or poorly staining area indicates a Golgi apparatus. The promyelocyte exhibits a similar size range, 15 to 21 m; nucleoli are present. Promyelocyte cytoplasm stains similarly to that of the myoblast, but it is distin-guished by the presence of large, blue/black, primary azurophilic granules, also called nonspecific granules. The myelocyte ranges from 16 to 24 m. Its chromatin is more condensed than its precursor and nucleoli are absent. The cytoplasm of the neutrophilic myelocyte is character-ized by small, pink-to-red specific granules with some azurophilic granules present. The eosinophilic lineage has a similar appearing nucleus, but its specific granules are large. The metamyelocyte ranges from 12 to 18 m. The nuclear-cytoplasmic ratio is further decreased and the nu-cleus assumes a kidney shape. There are few azurophilic

1	but its specific granules are large. The metamyelocyte ranges from 12 to 18 m. The nuclear-cytoplasmic ratio is further decreased and the nu-cleus assumes a kidney shape. There are few azurophilic granules at this stage in cells, and there is a predominance of small, pink-to-red spe-cific granules. The eosinophilic metamyelocyte shows an increased number of specific granules compared to the neutrophilic metamyelocyte. The band cells are further reduced in size, 9 to 15 m. The chromatin of the nucleus exhibits further condensation and has a horseshoe shape. In the neutrophilic band cell, the small, pink-to-red specific granules are the only granule type present. The eosinophilic band cell shows little or no change relative to the specific granules, but the nucleus exhibits a kidney shape. Mature granulocytes are shown on Plate 17.

1	Myeloblast, bone marrow smear, human, Giemsa, ×2,200. The myeloblast shown here exhibits a deep blue cytoplasm with a lighter region that represents the Golgi area (G). The nucleus is-round. Several nucleoli (N) are evident. Promyelocyte, bone marrow smear, human, Giemsa, ×2,200. The promyelocyte exhibits a round nucleus with one or more nucleoli (N) present. The cytoplasm is basophilic and exhibits relatively large blue/black azurophilic granules (AG). Eosinophilic myelocyte, bone marrow smear, human, Giemsa, ×2,200. The eosinophilic myelocyte exhibits a nucleus the same as that described for the neutrophilic myelocyte. The cytoplasm, however, contains the large specific granules characteristic of eosinophils, but they are fewer in number. Neutrophilic myelocyte, bone marrow smear, human, Giemsa, ×2,200. The neutrophilic myelocyte retains the round nucleus, but nucleoli are now absent. The cytoplasm exhibits small pink-to-red specific granules.

1	The neutrophilic myelocyte retains the round nucleus, but nucleoli are now absent. The cytoplasm exhibits small pink-to-red specific granules. Eosinophilic metamyelocyte, bone marrow smear, human, Giemsa, ×2,200. The eosinophilic metamyelocyte exhibits a kidney or bean sha ped nucleus. The cytoplasm exhibits numerous the characteristic eosinophilic granules that are present throughout the cytoplasm. Neutrophilic metamyelocyte, bone marrow smear, human, Giemsa, ×2,200. The neutrophilic metamyelocyte differs from its precursor by the presence of a kidney or bean shaped nucleus. The small, pink-to-red specific granules are now seen in the cytoplasm and few or no azurophilic granules are present. Eosinophilic band cell, bone marrow smear, human, Neutrophilic band cell, bone marrow smear, human, Giemsa, ×2,200. Giemsa, ×2,200.

1	Eosinophilic band cell, bone marrow smear, human, Neutrophilic band cell, bone marrow smear, human, Giemsa, ×2,200. Giemsa, ×2,200. The eosinophilic band cell exhibits a horseshoe-shaped nu-The band or non-segmented neutrophil exhibits a horseshoe cleus. Its cytoplasm is filled with the eosinophilic granules. shaped nucleus with abundant small, pink-to-red specific granules in the cytoplasm. KEY AG, azurophilic granules G, Golgi apparatus N, nucleoli

1	KEY AG, azurophilic granules G, Golgi apparatus N, nucleoli OVERVIEW AND CLASSIFICATION OF MUSCLE / 310 SKELETAL MUSCLE / 311 Myofibrils and Myofilaments / 314 The Contraction Cycle / 317 Motor Innervation / 322 Sensory Innervation / 324 Development, Repair, Healing, and Renewal / 325 CARDIAC MUSCLE / 327 Structure of Cardiac Muscle / 328 Injury and Repair / 331 SMOOTH MUSCLE / 331 Structure of Smooth Muscle / 331 Functional Aspects of Smooth Muscle / 335 Renewal, Repair, and Differentiation / 336 Folder 11.1 Functional Considerations: Muscle Metabolism and Ischemia / 316 Folder 11.2 Clinical Correlation: Muscular Dystrophies—Dystrophin and Dystrophin-Associated Proteins / 319 Folder 11.3 Functional Considerations: The Sliding Filament Model / 323 Folder 11.4 Clinical Correlation: Myasthenia Gravis / 325 Folder 11.5 Functional Considerations: Comparison of the Three Muscle Types / 337

1	Muscle tissue is responsible for movement of the body and its parts and for changes in the size and shape of internal organs. This tissue is characterized by aggregates of specialized, elongated cells arranged in parallel array that have the primary role of contraction (Fig. 11.1). Myofilament interaction is responsible for muscle cell contraction. Two types of myofilaments are associated with cell contraction.  Thin filaments (6 to 8 nm in diameter, 1.0 m long) are composed primarily of the protein actin. Each thin filament of fibrous actin (F-actin) is a polymer formed from globular actin molecules (G-actin). Thick filaments (15 nm in diameter, 1.5 m long) are composed of the protein myosin II. Each thick filament consists of 200 to 300 myosin II molecules. The long, rod-shaped tail portion of each molecule aggregates in a regular parallel but staggered array, whereas the head portions project out in a regular helical pattern.

1	The two types of myofilaments occupy the bulk of the cytoplasm, which in muscle cells is also called sarcoplasm [Gr. sarcos, flesh; plasma, thing ]. Actin and myosin are also present in most other cell types (although in considerably smaller amounts), where they play a role in cellular activities such as cytokinesis, exocytosis, and cell migration. In contrast, muscle cells contain a large number of aligned contractile filaments that the cells use for the single purpose of producing mechanical work. Muscle is classified according to the appearance of the contractile cells. Two principal types of muscle are recognized:  Striated muscle, in which the cells exhibit cross-striations at the light microscope level, and  Smooth muscle, in which the cells do not exhibit cross-striations.

1	FIGURE 11.1 • Photomicrograph of a skeletal muscle. a. This low-magnification photomicrograph shows skeletal muscle in longitudinal section. Muscle fibers (cells) are arranged in parallel; they are vertically oriented, and the length of each fiber extends beyond the upper and lower edge of the micrograph. The fibers appear to be of different thicknesses. This is largely a reflection of the plane of section through the muscle fibers. Note on the left the epimysium, the sheath of dense connective tissue surrounding the muscle. 160. b. At higher magnification, cross-striations of the muscle fibers are readily seen. The nuclei of skeletal muscle fibers are located in the cytoplasm immediately beneath the plasma membrane. 360.

1	Striated muscle tissue is further subclassified on the basis of its location:  Skeletal muscle is attached to bone and is responsible for movement of the axial and appendicular skeleton and for maintenance of body position and posture. In addition, skeletal muscles of the eye (extraocular muscles) provide precise eye movement.  Visceral striated muscle is morphologically identical to skeletal muscle but is restricted to the soft tissues, namely, the tongue, pharynx, lumbar part of the diaphragm, and upper part of the esophagus. These muscles play essential roles in speech, breathing, and swallowing.  Cardiac muscle is a type of striated muscle found in the wall of the heart and in the base of the large veins that empty into the heart.

1	 Cardiac muscle is a type of striated muscle found in the wall of the heart and in the base of the large veins that empty into the heart. The cross-striations in striated muscle are produced largely by the specific cytoarchitectural arrangement of both thin and thick myofilaments. This arrangement is the same in all types of striated muscle cells. The main differences between skeletal muscle cells and cardiac muscle cells are in their size, shape, and organization relative to one another. Smooth muscle cells do not exhibit cross-striations because the myofilaments do not achieve the same degree of order in their arrangement. In addition, the myosin-containing myofilaments in smooth muscle are highly labile. Smooth muscle is restricted to the viscera and vascular system, the arrector pili muscles of the skin, and the intrinsic muscles of the eye. A skeletal muscle cell is a multinucleated syncytium.

1	A skeletal muscle cell is a multinucleated syncytium. In skeletal muscle, each muscle cell, more commonly called a muscle fiber, is actually a multinucleated syncytium. A muscle fiber is formed during development by the fusion of small, individual muscle cells called myoblasts (see page 326). When viewed in cross section, the mature multinucleated muscle fiber reveals a polygonal shape with a diameter of 10 to 100 m (Plate 21, page 340). Their length varies from almost a meter, as in the sartorius muscle of the lower limb, to as little as a few millimeters, as in the strapedius muscle of the middle ear. (Note: A muscle fiber should not be confused with a connective tissue fiber; muscle fibers are skeletal muscle cells, whereas connective tissue fibers are extracellular products of connective tissue cells.)

1	The nuclei of a skeletal muscle fiber are located in the cy-branches are present within the endomysium, running paral toplasm immediately beneath the plasma membrane, also called the sarcolemma. In the past, the term sarcolemma was used to describe a thick “membrane” that was thought to be the cytoplasmic boundary of the muscle cell. It is now known that the thick sarcolemma actually represents the plasma membrane of the cell, its external lamina, and the surrounding reticular lamina. A skeletal muscle consists of striated muscle fibers held together by connective tissue. The connective tissue that surrounds both individual muscle fibers and bundles of muscle fibers is essential for force transduction (Fig. 11.2). At the end of the muscle, the connective tissue continues as a tendon or some other arrangement of collagen fibers that attaches the muscle, usually to bone. A rich supply of blood vessels and nerves travels in the connective tissue.

1	The connective tissue associated with muscle is named according to its relationship with the muscle fibers:  Endomysium is the delicate layer of reticular fibers that im mediately surrounds individual muscle fibers (see Fig. 11.2a). Only small-diameter blood vessels and the finest neuronal lel to the muscle fibers.  Perimysium is a thicker connective tissue layer that surrounds a group of fibers to form a bundle or fascicle. Fascicles are functional units of muscle fibers that tend to work together to perform a specific function. Larger blood vessels and nerves travel in the perimysium.  Epimysium is the sheath of dense connective tissue that surrounds a collection of fascicles that constitutes the muscle (see Fig. 11.1a). The major vascular and nerve supply of the muscle penetrates the epimysium. Three types of skeletal muscle fibers—red, white, and intermediate—can be identified by color in vivo.

1	It has long been known that skeletal muscle fibers differ in diameter and in their natural color in vivo. The color differences are not apparent in hematoxylin and eosin (H&E)–stained sections. However, histochemical reactions based on oxidative enzyme activity, specifically the succinic dehydrogenase and nicotinamide adenine dinucleotide– tetrazolium (NADH-TR) reactions, confirm the observations seen in fresh tissue and reveal several types of skeletal muscle fibers (Fig. 11.3). The most obvious nomenclature muscle fiber muscle fascicle myofibril epimysium perimysium endomysium aa b FIGURE 11.2 • General organization of skeletal muscle. a. This freeze fracture scanning electron micrograph of an intramuscular connective tissue was obtained from the bovine semitendinous muscle. The specimen was routinely fixed for SEM and subsequently treated according to the cell maceration method with sodium hydroxide to remove muscle cells. Note a delicate honeycomb structure of the endomysium

1	was routinely fixed for SEM and subsequently treated according to the cell maceration method with sodium hydroxide to remove muscle cells. Note a delicate honeycomb structure of the endomysium surrounding individual muscle cells. 480. (Reprinted with permission from Nishimura T, Hattori A, Takahashi K. Structural changes in intramuscular connective tissue during the fattening of Japanese Black Cattle: effect of marbling on beef tenderization. J Anim Sci 1999; 77:93–104.) b. This schematic diagram shows the general organization of skeletal muscle and its relation to the surrounding connective tissue. Note the organization of the endomysium that surrounds individual muscle cells (fibers), the perimysium that surrounds a muscle bundle, and the epimysium that surrounds the entire muscle.

1	FIGURE 11.3 • Cross section of skeletal muscle fibers. This cross section of muscle fibers stained with the NADH-TR reaction demonstrates two fiber types. The deeply stained, smaller muscle fibers exhibit strong oxidative enzyme activity and correspond to the type I slow oxidative fibers. The lighter-staining, larger fibers correspond to the type IIb fast glycolytic fibers. 280. Inset. Portions of the two fiber types at higher magnification. The reaction also reveals the mitochondria that contain the oxidative enzymes. The contractile components, the myofibrils, are unstained. 550. (Original slide specimen courtesy of Dr. Scott W. Ballinger.) to describe these differences is division into red, white, and intermediate fibers. Skeletal muscle fibers are characterized by speed of contraction, enzymatic velocity, and metabolic activity.

1	The current classification of skeletal muscle fibers is based on contractile speed, enzymatic velocity of the fiber’s myosin ATPase reaction, and metabolic profile. The contractile speed determines how fast the fiber can contract and relax. Velocity of the myosin ATPase reaction determines the rate at which this enzyme is capable of breaking down ATP molecules during the contraction cycle. The metabolic profile indicates the capacity for ATP production by oxidative phosphorylation or glycolysis. Fibers characterized by oxidative metabolism contain large amounts of myoglobin and an increased number of mitochondria, with their constituent cytochrome electron transport complexes. Myoglobin is an oxygen-binding protein that closely resembles hemoglobin found in erythrocytes and occurs in varying amounts in muscle fibers. It provides a ready source of oxygen for muscle metabolism.

1	The three types of skeletal muscle fibers are type I (slow oxidative), type IIa (fast oxidative glycolytic), and type IIb (fast glycolytic) fibers. Three types of fiber are typically found in any given skeletal muscle; the proportion of each type varies according to the functional role of the muscle.

1	 Type I fibers or slow oxidative fibers are small fibers that appear red in fresh specimens and contain many mitochondria and large amounts of myoglobin and cytochrome complexes. Their high levels of mitochondrial oxidative enzymes are demonstrated by their strong succinic dehydrogenase and NADH-TR histochemical staining reactions as described previously (see Fig. 11.3). Type I fibers are slow-twitch, fatigue-resistant motor units (a twitch is a single, brief contraction of the muscle). These fibers have great resistance to fatigue but generate less tension than other fibers. Their myosin ATPase reaction velocity is the slowest of all of the fiber types. Type I fibers are typically found in the limb muscles of mammals and in the breast muscle of migrating birds. More importantly, they are the principal fibers of the long muscles of the back in humans, where they are particularly adapted to the long, slow contraction needed to maintain erect posture. A high percentage of these fibers

1	the principal fibers of the long muscles of the back in humans, where they are particularly adapted to the long, slow contraction needed to maintain erect posture. A high percentage of these fibers make up the muscles of high-endurance athletes such as marathon runners.

1	 Type IIa fibers or fast oxidative glycolytic fibers are the intermediate fibers seen in fresh tissue. They are of medium size with many mitochondria and a high myoglobin content. In contrast to type I fibers, type IIa fibers contain large amounts of glycogen and are capable of anaerobic glycolysis. They make up fast-twitch, fatigue-resistant motor units that generate high peak muscle tension. Athletes who have a high percentage of these fast oxidative glycolytic fibers include 400and 800-m sprinters, middledistance swimmers, and hockey players.

1	 Type IIb fibers or fast glycolytic fibers are large fibers that appear light pink in fresh specimens and contain less myoglobin and fewer mitochondria than type I and type IIa fibers. They have a low level of oxidative enzymes but exhibit high anaerobic enzyme activity and store a considerable amount of glycogen. These fibers are fast-twitch, fatigue-prone motor units and generate high peak muscle tension. Their myosin ATPase velocity is the fastest of all the fiber types. They also fatigue rapidly as a result of production of lactic acid. Thus, type IIb fibers are adapted for rapid contraction and precise, fine movements. They constitute most fibers of the extraocular muscles and the muscles that control the movements of the digits. These muscles have a greater number of neuromuscular junctions than do type I fibers, thus allowing more precise neuronal control of movements in these muscles. Short-distance sprinters, weight lifters, and other field athletes have a high percentage of

1	than do type I fibers, thus allowing more precise neuronal control of movements in these muscles. Short-distance sprinters, weight lifters, and other field athletes have a high percentage of type IIb fibers.

1	The structural and functional subunit of the muscle fiber is the myofibril. A muscle fiber is filled with longitudinally arrayed structural subunits called myofibrils (Fig. 11.4). Myofibrils are visible in favorable histologic preparations and are best seen in cross-sections of muscle fibers. In these sections they give the fiber a stippled appearance. Myofibrils extend the entire length of the muscle cell. Myofibrils are composed of bundles of myofilaments.

1	Myofibrils are composed of bundles of myofilaments. Myofilaments are the individual filamentous polymers of myosin II (thick filaments) and actin and its associated proteins (thin filaments). Myofilaments are the actual contractile elements of striated muscle. The bundles of myofilaments that make up the myofibril are surrounded by a welldeveloped smooth-surfaced endoplasmic reticulum (sER), also called the sarcoplasmic reticulum. This reticulum forms a highly organized tubular network around the contractile elements in all striated muscle cells. Mitochondria and glycogen deposits are located between the myofibrils in association with the sER. Cross-striations are the principal histologic feature of striated muscle.

1	Cross-striations are the principal histologic feature of striated muscle. Cross-striations are evident in H&E–stained preparations of longitudinal sections of muscle fibers. They may also be seen in unstained preparations of living muscle fibers examined with a phase contrast or polarizing microscope, in which they appear as alternating light and dark bands. These bands are termed the A band and the I band (see Fig. 11.4). In polarizing microscopy, the dark bands are birefringent (i.e., they alter the polarized light in two planes). Therefore, the dark bands, being doubly refractive, are anisotropic and are given the name A band. The light bands are monorefringent (i.e., they do not alter the plane of polarized light). Therefore, they are isotropic and are given the name I band.

1	Both the A and I bands are bisected by narrow regions of contrasting density (see Fig. 11.4). The light I band is bisected by a dense line, the Z line, also called the Z disc [Ger. Zwischenscheibe, between discs]. The dark A band is bisected by a less dense, or light, region called the H band [Ger. Hell, light]. Furthermore, bisecting the light H band is a narrow dense line called the M line [Ger. Mitte, middle]. The M line is best demonstrated in electron micrographs (Fig. 11.5), although in ideal H&E preparations it can be detected in the light microscope. As noted above, the cross-banding pattern of striated muscle is caused by the arrangement of the two kinds of myofilaments. To understand the mechanism of contraction, this banding pattern must be considered in functional terms. muscle fascicle (composed of muscle fibers) myofibril (composed of myofilaments) muscle fiber (composed of myofibrils) sarcomere Z lineZ line

1	FIGURE 11.4 • Organization of a skeletal muscle. A skeletal muscle consists of bundles of muscle fibers called fascicles. In turn, each fascicle consists of a bundle of elongate muscle fibers (cells). The muscle fiber represents a collection of longitudinal units, the myofibrils, which in turn are composed of myofilaments of two types: thick (myosin) filaments and thin (actin) filaments. The myofilaments are organized in a specific manner that imparts a cross-striated appearance to the myofibril and to the fiber. The functional unit of the myofibril is the sarcomere; it extends in both directions from one Z line to the next Z line. The A band marks the extent of the myosin filaments. Actin filaments extend from the Z line into the region of the A band, where they interdigitate with the myosin filaments as shown. The cross sections through different regions of the sarcomere are also shown (from left to right): Through thin filaments of the I band; through thick filaments of the H band;

1	filaments as shown. The cross sections through different regions of the sarcomere are also shown (from left to right): Through thin filaments of the I band; through thick filaments of the H band; through the center of the A band, where adjacent thick filaments are linked to form the M line; and through the A band, where thin and thick filaments overlap. Note that each thick filament is within the center of a hexagonal array of thin filaments.

1	FIGURE 11.5 • Electron micrograph of skeletal muscle fiber. This low-magnification electron micrograph shows the general organization of skeletal muscle fibers. Small portions of three muscle fibers in longitudinal profile are included in this micrograph. The muscle fiber on the right reveals a nucleus at its periphery. Two fibers—one in the middle and another on the left—exhibit regular profiles of myofibrils separated by a thin layer of surrounding sarcoplasm (Sr). Each repeating part of the myofibril between adjacent Z lines is a sarcomere (S). The cross-banded pattern visible on this micrograph reflects the arrangement, in register, of the individual myofibrils (M); a similar pattern found in the myofibril reflects the arrangement of myofilaments. The detailed features of a sarcomere are shown at higher magnification in Figure 11.7a. The presence of the connective tissue in the extracellular space between the fibers constitutes the endomysium of the muscle. 6,500.

1	The functional unit of the myofibril is the sarcomere, the segment of the myofibril between two adjacent Z lines. The sarcomere is the basic contractile unit of striated muscle. It is the portion of a myofibril between two adjacent Z lines. A sarcomere measures 2 to 3 m in relaxed mammalian muscle. It may be stretched to more than 4 m and, during extreme contraction, may be reduced to as little as 1 m (Fig. 11.6). The entire muscle cell exhibits cross-striations because sarcomeres in adjacent myofibrils are in register. The arrangement of thick and thin filaments gives rise to the density differences that produce the cross-striations of the myofibril.

1	The arrangement of thick and thin filaments gives rise to the density differences that produce the cross-striations of the myofibril. The myosin-containing thick filaments are about 1.5 m long and are restricted to the central portion of the sarcomere (i.e., the A band). The thin filaments attach to the Z line and extend into the A band to the edge of the H band. Portions of two sarcomeres, on either side of a Z line, constitute the I band and contain only thin filaments. In a longitudinal section of a sarcomere, the Z line appears as a zigzag structure, with matrix material, the Z matrix, bisecting the zigzag. The Z line and its matrix material anchor the thin filaments from adjacent sarcomeres to the angles of the zigzag by the actin-binding protein -actinin. These features are illustrated in Figures 11.4 and 11.6. F-actin, troponin, and tropomyosin in thin filaments and myosin II in thick filaments are the primary proteins in the contractile apparatus.

1	F-actin, troponin, and tropomyosin in thin filaments and myosin II in thick filaments are the primary proteins in the contractile apparatus. Thin filaments contain F-actin, tropomyosin, and troponin. Thick filaments contain only myosin II. G-actin is a small, 42-kilodalton molecule that polymerizes to form a double-stranded helix, the F-actin filament. These actin filaments are polar; all G-actin molecules are oriented in the same direction. The plus end of each filament is bound to the Z line by -actinin; the minus end extends toward the M line and is protected by an actin-capping protein. Each G-actin molecule of the thin filament has a binding site for myosin. Tropomyosin is a 64-kilodalton protein that also consists of a double helix of two polypeptides. It forms filaments that run in the groove between the F-actin molecules in the thin filament. In resting muscle, tropomyosin and its regulatory protein, the troponin complex, mask the myosin-binding site on the actin molecule.

1	Troponin consists of a complex of three globular subunits. Each tropomyosin molecule contains one troponin complex. Troponin-C (TnC) is the smallest subunit of the troponin complex (18 kilodaltons). It binds Ca2, the essential step in the initiation of contraction (see illustration that follows). Troponin-T (TnT), a 30-kilodalton subunit, binds to tropomyosin, anchoring the troponin complex.

1	FIGURE 11.6 • Sarcomeres in different functional stages. In the resting state (middle), interdigitation of thin (actin) and thick (myosin) filaments is not complete; the H and I bands are relatively wide. In the contracted state (bottom), the interdigitation of the thin and thick filaments is increased according to the degree of contraction. In the stretched state (top), the thin and thick filaments do not interact; the H and I bands are very wide. The length of the A band always remains the same and corresponds to the length of the thick filaments; the lengths of the H and I bands change, again in proportion to the degree of sarcomere relaxation or contraction. Troponin-I (TnI), also a 30-kilodalton subunit, binds to actin, thus inhibiting actin–myosin interaction.  FOLDER 11.1 Functional Considerations: Muscle Metabolism and Ischemia

1	Like all cells, muscle cells depend on the energy source contained in the high-energy phosphate bonds of ATP and phosphocreatine. The energy stored in these high-energy phosphate bonds comes from the metabolism of fatty acids and glucose. Glucose is the primary metabolic sub-strate in actively contracting muscle. It is derived from the general circulation as well as from the breakdown of glyco-gen, which is normally stored in the muscle fiber cyto-plasm. As much as 1% of the dry weight of skeletal and cardiac muscle may be glycogen. In rapidly contracting muscles, such as the leg muscles in running or the extraocular muscles, most of the energy for contraction is supplied by anaerobic glycolysis of stored glycogen. The buildup of intermediary metabolites from this pathway, particularly lactic acid, can produce an oxygen deficit that causes ischemic pain (cramps) in cases of extreme muscular exertion. Most of the energy used by muscle recovering from contraction or by resting muscle is

1	acid, can produce an oxygen deficit that causes ischemic pain (cramps) in cases of extreme muscular exertion. Most of the energy used by muscle recovering from contraction or by resting muscle is derived from oxidative phosphorylation. This process closely follows the -oxidation of fatty acids in mitochondria that liberates two carbon fragments. The oxygen needed for oxidative phosphorylation and other terminal metabolic reactions is derived from hemoglobin in circulating erythrocytes and from oxygen bound to myoglobin stored in the mus-cle cells.

1	Myosin II, a 510-kilodalton protein, is composed of two polypeptide heavy chains (222 kilodaltons each) and four light chains. Light chains are of two types (essential light chains [18 kilodaltons] and regulatory light chains [22 kilodaltons]), and one molecule of each type is present in association with each myosin head. The phosphorylation by myosin light chain kinase of the regulatory light chain initiates contraction in smooth muscles. Each heavy chain has a small globular head that projects at an approximately right angle at one end of the long rod-shaped molecule. This globular head has two specific binding sites, one for ATP and one for actin. It also demonstrates ATPase and motor activity. Myosin molecules in striated muscles aggregate tail to tail to form bipolar thick myosin filaments; the rod-shaped segments overlap so that the globular heads project from the thick filament. The “bare” zone in the middle of the filament (i.e., the portion of the filament that does not have

1	the rod-shaped segments overlap so that the globular heads project from the thick filament. The “bare” zone in the middle of the filament (i.e., the portion of the filament that does not have globular projections) is the H band. The projecting globular heads of the myosin molecules form cross-bridges between the thick and thin filaments on either side of the H band (see Fig. 11.6).

1	Accessory proteins maintain precise alignment of thin and thick filaments. To maintain efficiency and speed of muscle contraction, both thin and thick filaments in each myofibril must be aligned precisely and kept at an optimal distance from one another. Proteins known as accessory proteins are essential in regulating the spacing, attachment, and alignment of the myofilaments. These structural protein components of skeletal muscle fibrils constitute less than 25% of the total protein of the muscle fiber. They include the following (see also Fig. 11.7):  Titin, a large (2,500-kilodalton) protein, forms an elastic lattice that anchors thick filaments in the Z lines. Two springlike portions of the protein adjacent to the thin filaments help stabilize the centering of the myosincontaining thick filament, preventing excessive stretching of the sarcomere.

1	 -Actinin, a short, bipolar, rod-shaped, 190-kilodalton actin-binding protein, bundles thin filaments into parallel arrays and anchors them at the Z line.  Nebulin, an elongated, inelastic, 600-kilodalton protein, is attached to the Z lines and runs parallel to the thin filaments. It helps -actinin anchor thin filaments to Z lines and is thought to regulate the length of thin filaments during muscle development.  Tropomodulin, a small, 40-kilodalton actin-binding protein, is attached to the free portion of the thin filament. This actin-capping protein maintains and regulates the length of the sarcomeric actin filament. Variations in thin filament length (such as those in type I and type IIb muscle fibers) affect the length–tension relationship during muscle contraction and therefore influence the physiologic properties of the muscle.

1	 Desmin, a type of 53-kilodalton intermediate filament, forms a lattice that surrounds the sarcomere at the level of the Z lines, attaching them to one another and to the plasma membrane, thus forming stabilizing cross-links between neighboring myofibrils.  Myomesin, a 185-kilodalton myosin-binding protein, holds thick filaments in register at the M line.  C protein, one of possibly several myosin-binding proteins (140 to 150 kilodaltons), serves the same function as myomesin and forms several distinct transverse stripes on either side of the M line.

1	 Dystrophin, a large 427-kilodalton protein, is thought to link laminin, which resides in the external lamina of the muscle cell, to actin filaments. Absence of this protein is associated with progressive muscular weakness, a genetic condition called Duchenne’s muscular dystrophy. Dystrophin is encoded on the X chromosome, which explains why only boys suffer from Duchenne’s muscular dystrophy. Recently, characterization of the dystrophin gene and its product has been clinically important (Folder 11.2). When a muscle contracts, each sarcomere shortens and becomes thicker, but the myofilaments remain the same length.

1	When a muscle contracts, each sarcomere shortens and becomes thicker, but the myofilaments remain the same length. During contraction the sarcomere and I band shorten, whereas the A band remains the same length. To maintain the myofilaments at a constant length, the shortening of the sarcomere must be caused by an increase in the overlap of the thick and thin filaments. This overlap can readily be seen by comparing electron micrographs of resting and contracted muscle. The H band narrows, and the thin filaments penetrate the H band during contraction. These observations indicate that the thin filaments slide past the thick filaments during contraction. The Contraction Cycle Shortening of a muscle involves rapid contraction cycles that move the thin filaments along the thick filament. Each contraction cycle consists of five stages: attachment, release, bending, force generation, and reattachment.

1	Attachment is the initial stage of the contraction cycle; the myosin head is tightly bound to the actin molecule of the thin filament. At the beginning of the contraction cycle, the myosin head is tightly bound to the actin molecule of the thin filament, and

1	FIGURE 11.7 • Electron micrograph of skeletal muscle and corresponding molecular structure of a sarcomere. a. This high-magnification electron micrograph shows a longitudinal section of the myofibrils. The I band, which is bisected by the Z line, is composed of barely visible, thin (actin) filaments. They are attached to the Z line and extend across the I band into the A band. The thick filaments, composed of myosin, account for the full width of the A band. Note that in the A band there are additional bands and lines. One of these, the M line, is seen at the middle of the A band; another, the less electron-dense H band, consists only of thick filaments. The lateral parts of the A band are more electron dense and represent areas where the thin filaments interdigitate with the thick filaments. 35,000. b. Diagram illustrating the distribution of myofilaments and accessory proteins within a sarcomere. The accessory proteins are titin, a large elastic molecule that anchors the thick

1	35,000. b. Diagram illustrating the distribution of myofilaments and accessory proteins within a sarcomere. The accessory proteins are titin, a large elastic molecule that anchors the thick (myosin) filaments to the Z line; -actinin, which bundles thin (actin) filaments into parallel arrays and anchors them at the Z line; nebulin, an elongated inelastic protein attached to the Z lines that wraps around the thin filaments and assists -actinin in anchoring the thin filament to Z lines; tropomodulin, an actin-capping protein that maintains and regulates the length of the thin filaments; tropomyosin, which stabilizes thin filaments and, in association with troponin, regulates binding of calcium ions; and myomesin and C proteins, myosin-binding proteins that hold thick filaments in register at the M line. The interactions of these various proteins maintain the precise alignment of the thin and thick filaments in the sarcomere.

1	 FOLDER 11.2 Clinical Correlation: Muscular Dystrophies— Dystrophin and Dystrophin-Associated Proteins Dystrophin is a rod-shaped cytoskeletal protein with a short head and a long tail that is located just beneath the skeletal muscle cell membrane. F-actin is bound at the end portion of the tail. Two groups of transmembrane proteins— and -dystroglycans and -, -, -, and -sarcoglycans— participate in a dystrophin–glycoprotein complex that links dystrophin to the extracellular matrix proteins laminin and agrin. Dystroglycans form the actual link between dystrophin and laminin; sarcoglycans are merely associated with the dystroglycans in the membrane. Distribution of dystrophin in healthy individuals is visualized using immunostaining methods (Fig F11.2.1).

1	Several forms of muscular dystrophy are attributed to mutations of single genes encoding several proteins of the dystrophin–glycoprotein complex. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are associated with mutations that affect dystrophin expression (Fig F11.2. 2); different forms of limb FIGURE F11.2.2 • Distribution of dystrophin in a patient girdle muscular dystrophy (LGMD) are caused by muta-with Duchenne muscular dystrophy (DMD). This crosstions in the genes found on the short arm of the X chromo-section of skeletal muscle was obtain from a patient diagnosed some encoding the four different sarcoglycans, and with DMD. Slide preparation similar to Fig F11.2.1. Compare another form of congenital muscular dystrophy (CMD) is the pattern and intensity of the dystrophin distribution within caused by a mutation in the gene encoding the 2 chain of affected muscle fibers to the normal individual. This muscle shows signs of hypertrophy. Some fibers do not have

1	distribution within caused by a mutation in the gene encoding the 2 chain of affected muscle fibers to the normal individual. This muscle shows signs of hypertrophy. Some fibers do not have any muscle laminin. Recent research has successfully characexpression of dystrophin; others still express variable levels of terized the dystrophin gene and its products. Most cases dystrophin. 480. (Courtesy of Dr. Andrew G. Engel.) of DMD are caused by a high frequency of gene deletions that create frame shifts, resulting in the absence of dystrophin in affected muscle fibers. This finding in affected individuals opened the way to direct genetic testing and prenatal diagnosis.

1	Because of its inheritance as an X-linked recessive trait, DMD primarily affect boys (an estimated 1 in 3,500 boys worldwide). Onset of DMD is between 3 and 5 years of age and progresses rapidly. Most boys become unable to walk by age 12 and by age 20 must use a respirator to breathe. BMD is similar to DMD except that it progresses at a much slower rate. Symptoms usually appear at about age 12, and the ability to walk is lost at an average age of 25 to 30. At the current time, there is no known cure for muscular dystrophies, and available treatment is aimed at controlling symptoms to maximize quality of life. Intensive research efforts are directed to implement gene therapy

1	FIGURE F11.2.1 • Distribution of dystrophin in human into treatment of affected patients. One method may lead skeletal muscle. This cross section of skeletal muscle fibers from a healthy individual was immunostained with cells. To achieve that objective, specially engineered forms goat polyclonal antibody against dystrophin using of viruses need to be develop that would carry “normal” immunoperoxydase method. Since dystrophin and associated dystrophin-glycoprotein complexes connect the muscle genes, infect muscle cells, and induce cells to express dyscytoskeleton to the surrounding extracellular matrix through trophin. The other method that might be tried is transplanthe cell membrane, the localization of dystrophin outlines cell tation of “healthy” satellite (muscle stem) cells that can membrane. Note a regular shape of skeletal muscle cells and divide and differentiate into normal muscle cells. Stem cell pattern of dystrophin distribution. 480. (Courtesy of therapy has been tested

1	membrane. Note a regular shape of skeletal muscle cells and divide and differentiate into normal muscle cells. Stem cell pattern of dystrophin distribution. 480. (Courtesy of therapy has been tested in laboratory animals and yielded Dr. Andrew G. Engel.) encouraging results.

1	ATP is absent. This arrangement is known as the rigor configuration. The muscular stiffening and rigidity that begins at the moment of death is caused by lack of ATP and is known as rigor mortis. In an actively contracting muscle, this step ends with the binding of ATP to the myosin head. Release is the second stage of the cycle; the myosin head is uncoupled from the thin filament. In this stage of the contraction cycle, ATP binds to the myosin head and induces conformational changes of the actin-binding site. This change reduces the affinity of the myosin head for the actin molecule of the thin filament, causing the myosin head to uncouple from the thin filament. STAGE 2: RELEASE Bending is the third stage of the cycle; the myosin head, as a result of hydrolysis of ATP, advances a short distance in relation to the thin filament.

1	STAGE 2: RELEASE Bending is the third stage of the cycle; the myosin head, as a result of hydrolysis of ATP, advances a short distance in relation to the thin filament. The ATP-binding site on the myosin head undergoes further conformational changes, causing the myosin head to bend. This movement is initiated by the breakdown of ATP into adenosine diphosphate (ADP) and inorganic phosphate; both products, however, remain bound to the myosin head. In this stage of the cycle, the linear displacement of the myosin head relative to the thin filament is approximately 5 nm. STAGE 3: BENDING Force generation is the fourth stage of the cycle; the myosin head releases inorganic phosphate and the power stroke occurs.

1	STAGE 3: BENDING Force generation is the fourth stage of the cycle; the myosin head releases inorganic phosphate and the power stroke occurs. The myosin head binds weakly to its new binding site on the neighboring actin molecule of the thin filament, causing release of the inorganic phosphate. This release has two effects. First, the binding affinity between the myosin head and its new attachment site increases. Second, the myosin head generates a force as it returns to its original unbent position. Thus, as the myosin head straightens, it forces movement of the thin filament along the thick filament. This is the “power stroke” of the cycle. During this stage, ADP is lost from the myosin head. STAGE 4: Reattachment is the fifth and last stage of the cycle; the myosin head binds tightly to a new actin molecule. The myosin head is again tightly bound to a new actin molecule of the thin filament (rigor configuration), and the cycle can repeat.

1	The myosin head is again tightly bound to a new actin molecule of the thin filament (rigor configuration), and the cycle can repeat. Although an individual myosin head may detach from the thin filament during the cycle, other myosin heads in the same thick filament will attach to actin molecules, thereby resulting in movement. Because the myosin heads are arranged as mirror images on either side of the H band (antiparallel arrangement), this action pulls the thin filaments into the A band, thus shortening the sarcomere. Regulation of contraction involves Ca2, sarcoplasmic reticulum, and the transverse tubular system. Ca2 must be available for the reaction between actin and myosin. After contraction, Ca2 must be removed. This rapid delivery and removal of Ca2 is accomplished by the combined work of the sarcoplasmic reticulum and the transverse tubular system.

1	The sarcoplasmic reticulum is arranged as a repeating series of networks around the myofibrils. Each network of the reticulum extends from one A–I junction to the next A–I junction within a sarcomere. The adjacent network of sarcoplasmic reticulum continues from the A–I junction to the next A–I junction of the neighboring sarcomere. Therefore, one network of sarcoplasmic reticulum surrounds the A band, and the adjacent network surrounds the I band (Fig. 11.8). Where the two networks meet, at the junction between A and I bands, the sarcoplasmic reticulum forms a slightly more regular ringlike channel called the terminal cisterna. The terminal cisternae serve as reservoirs for Ca2. To release Ca2 into the sarcoplasm, the plasma membrane of the terminal cisternae contains an abundance of gated Ca2-release channels. Also located around the myofibrils in association with the sarcoplasmic reticulum are large numbers of mitochondria and glycogen granules, both of which are involved in

1	of gated Ca2-release channels. Also located around the myofibrils in association with the sarcoplasmic reticulum are large numbers of mitochondria and glycogen granules, both of which are involved in providing the energy necessary for the reactions involved in contraction.

1	The transverse tubular system, or T system, consists of numerous tubular invaginations of the plasma membrane; each one is called a T tubule. T tubules penetrate to all levels of the muscle fiber and are located between adjacent terminal cisternae at the A–I junctions (see Fig. 11.8). They contain voltage-sensor proteins, depolarization-sensitive trans-membrane channels that are activated when the plasma

1	FIGURE 11.8 • Diagram of the organization of striated muscle fiber. This diagram illustrates the organization of the sarcoplasmic reticulum and its relationship to the myofibrils. Note that in striated muscle fibers, two transverse (T) tubules supply a sarcomere. Each T tubule is located at an A–I band junction and is formed as an invagination of the sarcolemma of striated muscle. It is associated with two terminal cisternae of the sarcoplasmic reticulum that surrounds each myofibril, one cisterna on either side of the T tubule. The triple structure as seen in cross section, where the two terminal cisternae flank a transverse tubule at the A–I band junction, is called a triad. Depolarization of the T tubule membrane initiates the release of calcium ions from the sarcoplasmic reticulum and eventually triggers muscle contraction.

1	membrane depolarizes. Conformational changes of these proteins directly affect gated Ca2-release channels located in the adjacent plasma membrane of the terminal cisternae. The complex of T tubule and the two adjacent terminal cisternae is called a triad. The depolarization of the T-tubule membrane triggers the release of Ca from the terminal cisternae to initiate muscle contraction.

1	When a nerve impulse arrives at the neuromuscular junction, the release of neurotransmitter (acetylcholine) from the nerve ending triggers a localized plasma-membrane depolarization of the muscle cell. The depolarization, in turn, causes voltage-gated Na channels in the plasma membrane to open, allowing an influx of Na from the extracellular space into the muscle cell. The influx of Na results in general depolarization, which spreads rapidly over the entire plasma membrane of the muscle fiber. When the depolarization encounters the opening of the T tubule, it is transmitted along the membranes of the T system into the depths of the cell. Electrical charges activate voltage-sensor proteins located in the membrane of the T tubule. These proteins have the structural and functional properties of Ca2 channels. During skeletal muscle depolarization, short activation of these sensors is not sufficient to open Ca2 channels. Thus, Ca2 transport from the lumen of the T tubule into the

1	of Ca2 channels. During skeletal muscle depolarization, short activation of these sensors is not sufficient to open Ca2 channels. Thus, Ca2 transport from the lumen of the T tubule into the sarcoplasm does not occur and is not essential to trigger the contraction cycle. Instead, activation of these sensors opens gated Ca2-release channels in adjacent terminal sacs of the sarcoplasmic reticulum, causing the rapid release of Ca2 into the sarcoplasm. The increased concentration of Ca2 in the sarcoplasm initiates contraction of the myofibril by binding to the TnC portion of the troponin complex on the thin filaments (see page 316). The change in molecular conformation of TnC causes the TnI to dissociate from the actin molecules, allowing the troponin complex to uncover myosin-binding sites on the actin molecules. The myosin heads are now free to interact with actin molecules to initiate the muscle contraction cycle.

1	Simultaneously, a Ca2-activated ATPase pump in the membrane of the sarcoplasmic reticulum transports Ca2 back into the terminal cisternae. The resting concentration of Ca2 is restored in the cytosol in less than 30 milliseconds. This restoration of resting Ca2 concentration near the myo filaments normally causes contraction to stop. Contraction will continue, however, as long as nerve impulses continue to depolarize the plasma membrane of the T tubules. Skeletal muscle fibers are richly innervated by motor neurons that originate in the spinal cord or brainstem. The axons of the neurons branch as they near the muscle, giving rise to twigs or terminal branches that end on individual muscle fibers (Fig. 11.9). FIGURE 11.9 • Photomicrograph of neuromuscular junction.

1	FIGURE 11.9 • Photomicrograph of neuromuscular junction. This silver preparation shows a motor nerve and its final branches that lead to the neuromuscular junctions (motor end plates). The skeletal muscle fibers are oriented horizontally in the field and are crossed perpendicularly by the motor nerve fibers. Note that these fibers distally lose their myelin sheath and divide extensively into small swellings, forming a cluster of neuromuscular junctions. 620. The neuromuscular junction is the contact made by the terminal branches of the axon with the muscle fiber.

1	The neuromuscular junction is the contact made by the terminal branches of the axon with the muscle fiber. At the neuromuscular junction (motor end plate), the myelin sheath of the axon ends, and the terminal portion of the axon is covered by only a thin portion of the neurilemmal (Schwann) cell with its external lamina. The end of the axon ramifies into a number of end branches, each of which lies in a shallow depression on the surface of the muscle fiber, the receptor region (Fig. 11.10). The axon ending is a typical presynaptic structure and contains numerous mitochondria and synaptic vesicles that contain the neurotransmitter acetylcholine (ACh). Release of acetylcholine into the synaptic cleft initiates depolarization of the plasma membrane, which leads to muscle cell contraction.

1	Release of acetylcholine into the synaptic cleft initiates depolarization of the plasma membrane, which leads to muscle cell contraction. The muscle fiber plasma membrane that underlies the synaptic cleft has many deep junctional folds (subneural folds). Specific cholinergic receptors for ACh are limited to the plasma membrane immediately bordering the cleft and at the  FOLDER 11.3 Functional Considerations: The Sliding

1	The sliding filament model postulates that the ratchetlike movements of the myosin heads bound to actin produce the movement of the thin filaments relative to the thick fil-aments, which in turn causes the sarcomere to shorten. Although the sliding filament model can explain contrac-tion in a single sarcomere, it cannot adequately explain the shortening of a myofibril of a muscle fiber. Obviously, if the activity just described were to occur simultaneously in adjacent sarcomeres, no contraction could occur. Equal and opposite forces would be exerted on either side of the Z line, and the contraction of any given sar-comere would be prevented by the contraction of its two immediate serial neighbors. Recent studies with ultrahigh-speed photography have demonstrated that an extremely small temporal delay occurs between the contraction of adjacent sarcomeres, so that a wavelike contraction actu-ally occurs in each muscle fibril and, consequently, in each muscle fiber.

1	FIGURE 11.10 • Neuromuscular junction. a. Diagram of a neuromuscular junction. An axon is shown making contact with a muscle cell. Note how the junctional folds of the muscle cell augment the surface area within the synaptic cleft. The external lamina extends throughout the cleft area. The cytoplasm of the Schwann cell is shown covering the axon terminal. (Modified from Kelly DE, Wood RL, Enders AC, eds. Bailey’s Textbook of Microscopic Anatomy. Baltimore: Williams & Wilkins, 1984.) b. Electron micrograph of a neuromuscular junction shows the axon ending within the synaptic cleft of a skeletal muscle fiber. An aggregation of mitochondria (M) and numerous synaptic vesicles (SV ) is visible. The portion of the motor axon ending that is not in apposition to the muscle fiber is covered by Schwann cell cytoplasm (S), but no myelin is present. The muscle fiber shows the junctional folds (JF ) and the subneural clefts (SnC) between them. The external lamina of the muscle fiber is barely

1	Schwann cell cytoplasm (S), but no myelin is present. The muscle fiber shows the junctional folds (JF ) and the subneural clefts (SnC) between them. The external lamina of the muscle fiber is barely evident within the subneural clefts. Other structures present are the aggregated mitochondria of the muscle fiber (M) in the region of the neuromuscular junction, the nucleus (N) of the muscle fiber, and some myofibrils (MF ). 32,000. (Courtesy of Dr. George D. Pappas.) top of the folds. The external lamina extends into the subneural folds (see Fig. 11.10). The synaptic vesicles of the axon terminal release ACh into the cleft, which then binds to nicotinic ACh receptors on the sarcolemma of the striated muscle. The nicotinic ACh receptor in striated muscles is a transmitter-gated Na channel. Binding of ACh opens

1	ACh Na channels, causing an influx of Na into striated muscle Na receptor cell. This influx results in a localized membrane depolariza tion, which in turn leads to the events described above. An enzyme called acetylcholinesterase (AChE) quickly breaks down the acetylcholine to prevent continued stimulation. For more detail description of ACh function, see Chapter 12. The muscle fiber cytoplasm that underlies the junctional folds contains nuclei, many mitochondria, rough-surfaced endoplasmic reticulum (rER), free ribosomes, and glycogen. These cytoplasmic organelles are believed to be involved in the synthesis of specific acetylcholine receptors in the membrane of the cleft, as well as acetylcholinesterase. A neuron along with the specific muscle fibers that it in- nervates is called a motor unit. before depolarization depolarization

1	A neuron along with the specific muscle fibers that it in- nervates is called a motor unit. before depolarization depolarization A single neuron may innervate several to a hundred or more muscle fibers. Muscles capable of the most delicate movements have the fewest muscle fibers per motor neuron in their motor units. For example, in eye muscles, the innervation ratio is about one neuron to three muscle fibers; in the postural muscles of the back, a single neuron may innervate hundreds of muscle fibers. The nature of muscle contraction is determined by the number of motor neuron endings as well as by the number of specific types of muscle fibers that are depolarized. Al-voltage sensor though depolarization of a muscle fiber at a single neuromuscular junction is characterized as an “all-or-none” phenomenon, not all nerve terminals discharge at once, FIGURE 11.11 • Summary of events leading to contraction of which allows a graded response to the contractile stimulus.

1	FIGURE 11.11 • Summary of events leading to contraction of which allows a graded response to the contractile stimulus. skeletal muscle. See the text for a description of the events Innervation is necessary for muscle cells to maintain their structural integrity. The motor nerve cell not only instructs the muscle cells to contract but also exerts a trophic influence on the muscle cells. If the nerve supply to a muscle is disrupted, the muscle cell undergoes regressive changes known as tissue atrophy. The most conspicuous indication of this atrophy is thinning of the muscle and its cells. If innervation is reestablished surgically or by the slower process of natural regeneration of the nerve, the muscle can regain normal shape and strength. The events leading to contraction of skeletal muscle can be summarized as a series of steps. The events involved in contraction can be summarized as follows (the numbers refer to the numbers in Fig. 11.11): 1.

1	The events involved in contraction can be summarized as follows (the numbers refer to the numbers in Fig. 11.11): 1. The contraction of a skeletal muscle fiber is initiated when a nerve impulse traveling along the axon of a motor neuron arrives at the neuromuscular junction. 2. The nerve impulse prompts the release of acetylcholine into the synaptic cleft that binds into ACh-gated Na channels causing local depolarization of sarcolemma. 3. Voltage-gated Na channels open, and Na enters the cell. indicated by the numerals. ACh, acetylcholine. 4. General depolarization spreads over the plasma membrane of the muscle cell and continues via membranes of the T tubules. 5. Voltage sensor proteins in the plasma membrane of T tubules change their conformation. 6. At the muscle cell triads, the T tubules are in close contact with the lateral enlargements of the sarcoplasmic reticulum, where gated Ca2-release channels are activated by conformational changes of voltage-sensor proteins.

1	7. Ca2 is rapidly released from the sarcoplasmic reticulum into the sarcoplasm. 8. Ca2 binds to the TnC portion of the troponin complex. 9. The contraction cycle is initiated, and Ca2 is returned to the terminal cisternae of the sarcoplasmic reticulum. Encapsulated sensory receptors in muscles and tendons are examples of proprioreceptors. These receptors are part of  FOLDER 11.4 Clinical Correlation: Myasthenia Gravis

1	During normal function, acetylcholine (ACh) molecules re-leased to the synaptic cleft at the neuromuscular junction bind to the nicotinic ACh receptors on the sarcolemma of the skeletal muscle cell. As discussed earlier in the text, these re-ceptors represent transmitter-gated Nachannels that con-trol the influx of Nanecessary for generating an action potential leading to initiation of muscle contraction. After stimulating their own receptors, ACh molecules are quickly degraded by the enzyme acetylcholinesterase (AChE) into acetic acid and choline, which is taken up by the axon termi-nal and reused for ACh synthesis (see page 363). In a clinical condition called myasthenia gravis, the ACh nicotinic receptors are blocked by antibodies directed to the body’s own receptor protein. Thus, myasthenia gravis is an autoimmune disease caused by the reduced number of functional ACh receptor sites. In addition, other abnormalities within the synaptic cleft (e.g., widening of the synaptic cleft,

1	gravis is an autoimmune disease caused by the reduced number of functional ACh receptor sites. In addition, other abnormalities within the synaptic cleft (e.g., widening of the synaptic cleft, disappearance of junctional folds) also occur, further reducing the effectiveness of the muscle fibers. Myasthenia gravis is characterized by noticeable weakening of the muscle fiber response to the nerve stim-ulus. Initially, weakness begins with extraocular muscles, drooping eyelids, double vision, and generalized muscular weakness. Other somatic musculature might be affected, including respiratory muscles. As the disease progresses, the number of neuromuscular junctions is reduced. An effective pharmacologic treatment for myasthenia gravis is administration of AChE inhibitors. These substances rein-force neuromuscular transmission by extending the life of released ACh within the synaptic cleft. In addition to AChE inhibitors, the immunosuppressive treatment and resection of the enlarged

1	rein-force neuromuscular transmission by extending the life of released ACh within the synaptic cleft. In addition to AChE inhibitors, the immunosuppressive treatment and resection of the enlarged thymus (if present) are used to slow down the activity of the immune system and rate of production of antibodies against ACh receptors.

1	the somatic sensory system that provides information about the degree of stretching and tension in a muscle. Proprioreceptors inform the central nerve system about the body’s position and movement in space. The muscle spindle is the specialized stretch receptor located within the skeletal muscle.

1	The muscle spindle is a specialized stretch receptor in muscle; it consists of two types of modified muscle fibers called spindle cells and neuron terminals (Fig. 11.12). Both types of modified muscle fibers are surrounded by an internal capsule. A fluid-filled space separates the internal capsule from an outer external capsule. One type of spindle cell, the nuclear bag fiber, contains an aggregation of nuclei in an expanded midregion; the other type, called a nuclear chain fiber, has many nuclei arranged in a chain. The muscle spindle transmits information about the degree of stretching in a muscle. The sensory (afferent, Ia) nerve fibers that carry information from the muscle spindle have endings that are spirally arranged around the midregion of both types of spindle cells. In addition, spindle cells receive motor (efferent) innervation from the spinal cord and brain via motor (efferent) nerve fibers, which are thought to regulate the sensitivity of the stretch receptor. When

1	spindle cells receive motor (efferent) innervation from the spinal cord and brain via motor (efferent) nerve fibers, which are thought to regulate the sensitivity of the stretch receptor. When skeletal muscle is stretched, nerve endings of sensory nerves become activated. They convey their impulses to the central nervous system, which in turn modulates the activity of motor neurons innervating that particular muscle.

1	Recent real-time studies with computed tomography (CT) scans of living muscle in different states of contraction suggest that muscle spindles may also represent the axes of functional units within large skeletal muscles. Such functional units precisely regulate contractions of portions of the muscle by creating “fixation points” within the muscle substance. Similar encapsulated receptors, Golgi tendon organs, are found in the tendons of muscle and respond to increased tension on the muscle. These receptors contain only sensory (afferent, Ib) nerve fibers, and they monitor muscle tension (or the force of contraction) within an optimal range. Development, Repair, Healing, and Renewal Development of myogenic stem cell linage depends on expression of various myogenic regulatory factors.

1	Myoblasts are derived from a self-renewing population of multipotential myogenic stem cells that originate in the embryo from unsegmented paraxial mesoderm (cranial muscle progenitors) or segmented mesoderm of somites (epaxial and hypaxial muscle progenitors). Early in embryonic development, these cells express MyoD transcription factor, which, along with other myogenic regulatory factors (MRFs), plays a key role in activation of muscle-specific gene expressions and differentiation of all skeletal muscle lineages. A balancing effect on skeletal-muscle development is achieved by the expression of negative regulatory myostatin gene, which leads to synthesis of myostatin, a 26-kilodalton protein belonging to the bone morphogenetic protein/transforming growth factor(BMP/TGF-) protein superfamily. Myostatin exerts an inhibitory effect on muscle growth and differentiation. It is thought that MyoD preferentially upregulates myostatin gene expression and controls myogenesis during not only

1	Myostatin exerts an inhibitory effect on muscle growth and differentiation. It is thought that MyoD preferentially upregulates myostatin gene expression and controls myogenesis during not only the embryonic and fetal periods but also postnatal stages of the development. The hypermuscular phenotypes observed on inactivation of the myostatin gene in animals and humans have confirmed the role of myostatin as a negative regulator of skeletal-muscle development. Experimental studies have demonstrated that muscle mass increases through myostatin inhibition, and the myostatin signaling

1	FIGURE 11.12 • Muscle spindle. a. Schematic diagram of a muscle spindle. The diameter of the spindle is expanded to illustrate structural details. Each spindle contains approximately two to four nuclear bag fibers and six to eight nuclear chain fibers. In the nuclear bag fibers, the muscle fiber nuclei are clumped in the expanded central portion of the fiber, hence the name bag. In contrast, the nuclei concentrated in the central portion of the nuclear chain fibers are arranged in a chain. Both afferent Ia (sensory) and efferent (motor) nerve fibers supply muscle spindle cells. The afferent nerve fibers respond to excessive stretching of the muscle, which in turn inhibits the somatic motor stimulation of the muscle. The efferent nerve fibers regulate the sensitivity of the afferent endings in the muscle spindle. b. Photomicrograph of a cross section of a muscle spindle, showing two bundles of spindle cells in the encapsulated, fluid-filled receptor. In one bundle, several of the

1	in the muscle spindle. b. Photomicrograph of a cross section of a muscle spindle, showing two bundles of spindle cells in the encapsulated, fluid-filled receptor. In one bundle, several of the spindle cells are cut at the level that reveals their nuclei. An internal capsule surrounds the spindle cells. The external capsule of the muscle spindle and the adjacent perimysium can be seen as a faint double-layer boundary of the receptor. Immediately above and outside of the muscle spindle is a nerve that may be supplying the spindle. The several types of nerves associated with the spindle cells as well as the type of spindle cells cannot be distinguished in this H&E–stained section. Near one of the bundles of spindle cells is a small blood vessel. The flocculent material within the capsule consists of precipitated proteoglycans and glycoproteins from the fluid that filled the spindle before fixation. 550.

1	pathway may be a potent therapeutic intervention point in the treatment of muscle-wasting diseases, such as muscular dystrophy, amyotrophic lateral sclerosis (ALS), AIDS, and cancer. Pharmacologic manipulation of myostatin expression could also lead to the development of new therapeutic approaches in a variety of musculoskeletal pathologies. Skeletal muscle progenitors differentiate into early and late myoblasts. Developing muscle contains two types of myoblasts:  Early myoblasts are responsible for the formation of primary myotubes, chainlike structures that extend between tendons of the developing muscle. Primary myotubes are formed by nearly synchronous fusion of early myoblasts. Myotubes undergo further differentiation into mature skeletal muscle fibers. Primary myotubes observed in the light microscope exhibit a chain of multiple central nuclei surrounded by myofilaments.

1	 Late myoblasts give rise to secondary myotubes, which are formed in the innervated zone of developing muscle where the myotubes have direct contact with nerve terminals. Secondary myotubes continue to be formed by sequential fusion of myoblasts into the already-formed secondary myotubes at random positions along their length. Secondary myotubes are characterized by a smaller diameter, more widely spaced nuclei, and an increased number of myofilaments (Fig. 11.13). In the mature multinucleated muscle fiber, the nuclei are all in the peripheral sarcoplasm, just inside the plasma membrane. Some nuclei that appear to belong to the skeletal muscle fiber are nuclei of satellite cells.

1	Some nuclei that appear to belong to the skeletal muscle fiber are nuclei of satellite cells. FIGURE 11.13 • Photomicrograph of developing skeletal muscle myotubes. This photomicrograph shows a cross section (on the left) and a longitudinal section (on the right) of developing skeletal muscle fibers in the stage of secondary myotubes. These myotubes are formed by sequential fusion of myoblasts, forming elongated tubular structures. Note that the myotubes have a small diameter and widely spaced, centrally positioned nuclei that gradually become displaced into the cell periphery by the increased number of newly synthesized myofilaments. In the mature multi-nucleated muscle fiber (upper left), all nuclei are positioned in the peripheral sarcoplasm, just inside the plasma cell membrane. 220.

1	Satellite cells are interposed between the plasma membrane of the muscle fiber and its external lamina. They are small cells with scant cytoplasm. The cytoplasm typically blends in with the muscle cell sarcoplasm when viewed in the light microscope, thus making them difficult to identify. Each satellite cell has a single nucleus with a chromatin network denser and coarser than that of muscle cell nuclei. Satellite cells are responsible for the skeletal muscle’s ability to regenerate, but their regenerative capacity is limited. These myogenic precursors of muscle cells are normally quiescent and do not express myogenic regulatory factors. However, after injury of muscle tissue, some satellite cells become activated, reenter the cell cycle, and begin to express MRFs. They proliferate and give rise to new myoblasts. As long as the external lamina remains intact, the myoblasts fuse within the external lamina to form myotubes, which then mature into a new fiber. In contrast, if the

1	and give rise to new myoblasts. As long as the external lamina remains intact, the myoblasts fuse within the external lamina to form myotubes, which then mature into a new fiber. In contrast, if the external lamina is disrupted, fibroblasts repair the injured site, with subsequent scar tissue formation.

1	Muscular dystrophies are characterized by progressive degeneration of skeletal muscle fibers, which places a constant demand on the satellite cells to replace the degenerated fibers. Ultimately, the satellite cell pool is exhausted. New experimental data indicate that, during this process, additional myogenic cells are recruited from the bone marrow and supplement the available satellite cells. The rate of degeneration exceeds the rate of regeneration, however, resulting in loss of muscle function. A future treatment strategy for muscular dystrophies may include the trans-plantation of satellite cells or their myogenic bone marrow counterparts into damaged muscle. FIGURE 11.14 • Photomicrograph of longitudinally sectioned cardiac muscle. The arrows point to the intercalated discs. The disc represent specialized cell-to-cell attachments of the cardiac muscle cells. Also note the apparent branching of the muscle fibers. 360.

1	Cardiac muscle has the same types and arrangement of contractile filaments as skeletal muscle. Therefore, cardiac muscle cells and the fibers they form exhibit cross-striations evident in routine histologic sections. In addition, cardiac muscle fibers exhibit densely staining cross-bands, called intercalated discs, that cross the fibers in a linear fashion or frequently in a way that resembles the risers of a stairway (Fig. 11.14 and Plate 24, page 346). The intercalated discs represent highly specialized attachment sites between adjacent cells. This linear cell-to-cell attachment of the cardiac muscle cells results in “fibers” of variable length. Thus, unlike skeletal and visceral striated muscle fibers that represent multinucleated single cells, cardiac muscle fibers consist of numerous cylindrical cells arranged end to end. Furthermore, some cardiac muscle cells in a fiber may join with two or more cells through intercalated discs, thus creating a branched fiber.

1	terminal cisterna of sarcoplasmic reticulum FIGURE 11.15 • Diagram of the organization of cardiac muscle fiber. The T tubules of cardiac muscle are much larger than the T tubules of skeletal muscle and carry an investment of external lamina material into the cell. They also differ in that they are located at the level of the Z disc. The portion of the sarcoplasmic reticulum adjacent to the T tubule is not in the form of an expanded cisterna but rather is organized as an anastomosing network. Structure of Cardiac Muscle The cardiac muscle nucleus lies in the center of the cell.

1	The central location of the nucleus in cardiac muscle cells is one feature that helps distinguish them from multinucleated skeletal muscle fibers, whose nuclei lie immediately under the plasma membrane. The transmission electron microscope (TEM) reveals that the myofibrils of cardiac muscle separate to pass around the nucleus, thus outlining a biconical juxtanuclear region in which the cell organelles are concentrated. This region is rich in mitochondria and contains the Golgi apparatus, lipofuscin pigment granules, and glycogen. In the atria of the heart, atrial granules measuring 0.3 to 0.4 m in diameter are also concentrated in the juxtanuclear cytoplasm. These granules contain two polypeptide hormones: atrial natriuretic factor (ANF) [L. natrium, sodium] and brain natriuretic factor (BNF). Both hormones are diuretics, affecting urinary excretion of sodium. They inhibit renin secretion by the kidney and aldosterone secretion by the adrenal gland. They also inhibit contractions of

1	(BNF). Both hormones are diuretics, affecting urinary excretion of sodium. They inhibit renin secretion by the kidney and aldosterone secretion by the adrenal gland. They also inhibit contractions of vascular smooth muscle. In congestive heart failure, levels of circulating BNF increase.

1	Numerous large mitochondria and glycogen stores are adjacent to each myofibril. In addition to the juxtanuclear mitochondria, cardiac muscle cells are characterized by large mitochondria that are densely packed between the myofibrils. These large mitochondria often extend the full length of a sarcomere and contain numerous, closely packed cristae (Fig. 11.15). Concentrations of glycogen granules are also located between the myofibrils. Thus, the structures that store energy (glycogen granules) and the structures that release and recapture energy (mitochondria) are located adjacent to the structures (myofibrils) that use the energy to drive contraction. The intercalated discs represent junctions between cardiac muscle cells.

1	As previously noted, the intercalated disc represents the attachment site between cardiac muscle cells. In the light microscope, the disc appears as a densely staining linear structure that is oriented transversely to the muscle fiber. Often it consists of short segments arranged in a steplike fashion (Fig. 11.16). When the site of the intercalated disc is examined with the TEM, the densely staining structure seen in the light microscope can be attributed to the presence of a transverse component that crosses the fibers at a right angle to the myofibrils. The transverse component is analogous to the risers of the stairway. A lateral component (not visible in the light microscope) occupies a series of surfaces perpendicular to the transverse component and lies parallel to the myofibrils. The lateral component is analogous to the steps of the stairway. Both components of the intercalated disc contain specialized cell-to-cell junctions between adjoining cardiac muscle cells:  Fascia

1	The lateral component is analogous to the steps of the stairway. Both components of the intercalated disc contain specialized cell-to-cell junctions between adjoining cardiac muscle cells:  Fascia adherens (adhering junction) is the major constituent of the transverse component of the intercalated disc and is responsible for its staining in routine H&E

1	FIGURE 11.16 • Structure of cardiac muscle fiber. a. This scanning electron micrograph shows the heart muscle tissue preparation obtained from the monkey right ventricle. The sample was ultrasonicated within the sodium hydroxide that resulted in the digestion of collagen fibers and separation of cardiac myocytes at the intercalated discs. Note the branching pattern of myocytes and clearly visible transverse and lateral components of intercalated disc. 32,000. b. Three-dimensional drawing of an intercalated disc, which represents a highly specialized attachment site between adjacent cardiac muscle cells. The intercalated disc is composed of the transverse component (blue area) that crosses the fibers at a right angle to the myofibrils (analogous to the risers of a stairway) and a lateral component (pink area) that occupies a series of surfaces perpendicular to the transverse component and parallel to the myofibrils (analogous to the steps of a stairway). The fascia adherens is the

1	component (pink area) that occupies a series of surfaces perpendicular to the transverse component and parallel to the myofibrils (analogous to the steps of a stairway). The fascia adherens is the major constituent of the transverse component. It holds the cardiac muscle cells at their ends and serves as the attachment site for thin filaments. The maculae adherentes reinforce the fascia adherens and are also found in the lateral components. The gap junctions are found only in the lateral component of the intercalated disc. c. This electron micrograph reveals portions of two cardiac muscle cells joined by an intercalated disc. The line of junction between the two cells takes an irregular, steplike course, making a number of nearly right-angle turns. In its course, different parts of the intercalated disc are evident. These include the transverse components (fascia adherens and maculae adherentes) and lateral components (gap junctions and maculae adherentes). The macula adherens (MA) is

1	disc are evident. These include the transverse components (fascia adherens and maculae adherentes) and lateral components (gap junctions and maculae adherentes). The macula adherens (MA) is enlarged in inset 1 (62,000). The fascia adherens (FA) is more extensive than the macula adherens, being disposed in a larger area of irregular outline. The fascia adherens is enlarged in inset 3 (62,000). The fascia adherens of the intercalated disc corresponds to the zonula adherens of other tissues. The gap junction (GJ) is enlarged in inset 2 (62,000). Other features typical of cardiac muscle are also present: mitochondria (Mi), sarcoplasmic reticulum (SR), and components of the sarcomere, including Z lines (Z), M line (M), and myofilaments. This particular specimen is in a highly contracted state, and consequently, the I band is practically obscured. 30,000. (Part A reprinted with permission from Zhang L, Ina K, Kitamura H, Campbell GR, Shimada T. The intercalated discs of monkey myocardial

1	and consequently, the I band is practically obscured. 30,000. (Part A reprinted with permission from Zhang L, Ina K, Kitamura H, Campbell GR, Shimada T. The intercalated discs of monkey myocardial cells and Purkinje fibers as revealed by scanning electron microscopy. Arch Histol Cytol 1996;59:453–465.) preparations. It holds the cardiac muscle cells at their ends to form the functional cardiac muscle fiber (see Fig. 5.20, page 131). It always appears as a transverse boundary between the cardiac muscle cells. The TEM reveals an intercellular space between the adjacent cells that is filled with electron-dense material resembling the material found in the zonula adherens of epithelia. The fascia adherens serves as the site at which the thin filaments in the terminal sarcomere anchor onto the plasma membrane. In this way, the fascia adherens is functionally similar to the epithelial zonula adherens, where actin filaments of the terminal web are also anchored.

1	 Maculae adherentes (desmosomes) bind the individual muscle cells to one another. Maculae adherentes help prevent the cells from pulling apart under the strain of regular repetitive contractions. They reinforce the fascia adherens and are found in both the transverse and lateral components of the intercalated discs.  Gap junctions (communicating junctions) constitute the major structural element of the lateral component of the intercalated disc. Gap junctions provide ionic continuity between adjacent cardiac muscle cells, thus allowing informational macromolecules to pass from cell to cell. This exchange permits cardiac muscle fibers to behave as a syncytium while retaining cellular integrity and individuality. The position of the gap junctions on the lateral surfaces of the intercalated disc protects them from the forces generated during contraction. The sER in cardiac muscle cells is organized into a single network along the sarcomere, extending from Z line to Z line.

1	The sER in cardiac muscle cells is organized into a single network along the sarcomere, extending from Z line to Z line. The sER of cardiac muscle is not as well organized as that of skeletal muscle. It does not separate bundles of myofilaments into discrete myofibrils. The T tubules in cardiac muscle penetrate into the myofilament bundles at the level of the Z line, between the ends of the sER network. Thus, there is only one T tubule per sarcomere in cardiac muscle. Small terminal cisternae of the sER are in close proximity to the T tubules to form a diad at the level of the Z line (see Fig. 11.15). The external lamina adheres to the invaginated plasma membrane of the T tubule as it penetrates into the cytoplasm of the muscle cell. The T tubules are larger and more numerous in cardiac ventricular muscle than in skeletal muscle. They are less numerous, however, in cardiac atrial muscle.

1	Passage of Ca2 from the lumen of the T tubule to the sarcoplasm of a cardiac muscle cell is essential to initiate the contraction cycle. As discussed in the section on skeletal muscle, depolarization of the T tubule membrane activates voltage-sensor proteins, which are similar in structure and function to Ca2 channels. In contrast to skeletal muscle, long-lasting depolarization in cardiac muscle activates these sensors and prompts their slow conformation change into functional Ca2 channels (Fig. 11.17). Thus, in the first stage of the cardiac muscle contraction cycle, Ca2 from the lumen of the T tubule is transported to the sarcoplasm of cardiac muscle, which opens gated Ca2-release channels in adjacent terminal sacs of the FIGURE 11.17 • Movement of the calcium ions after depola rization of the plasma membrane in cardiac muscle.

1	FIGURE 11.17 • Movement of the calcium ions after depola rization of the plasma membrane in cardiac muscle. Depolarization of the T tubule membrane activates voltage-sensor proteins that function as Ca2 channels. Initially, Ca2 is transported from the lumen of the T tubule through channels in voltage-sensor proteins into the sarcoplasm of the cardiac muscle (illustrated next to the upper terminal sac of the sER). Next, Ca2 activates gated Ca2-release channels in adjacent terminal sacs of the sarcoplasmic reticulum. This causes the massive release of sequestrated Ca2 from the sER into the sarcoplasm and initiates the contraction cycle.

1	sarcoplasmic reticulum. This calcium-triggered calcium release mechanism causes a massive and rapid release of additional Ca2 that initiates subsequent steps of the contraction cycle, which are identical to those in skeletal muscle. The differences between initiation of cardiac and skeletal muscle contractions—the longer-lasting membrane depolarization and activation of voltage-sensitive Ca2 channels in the wall of the T tubule—account for an approximately 200-millisecond delay from the start of a depolarization in a cardiac muscle twitch (see Fig. 11.11). Cardiac muscle cells exhibit a spontaneous rhythmic contraction.

1	The intrinsic spontaneous contraction or beat of cardiac muscle is evident in embryonic cardiac muscle cells as well as in cardiac muscle cells in tissue culture. The heartbeat is initiated, locally regulated, and coordinated by specialized, modified cardiac muscle cells called cardiac conducting cells (Plate 25, page 348). These cells are organized into nodes and highly specialized conducting fibers called Purkinje fibers that generate and rapidly transmit the contractile impulse to various parts of the myocardium in a precise sequence. Both parasympathetic and sympathetic nerve fibers terminate in the nodes. Sympathetic stimulation accelerates the heartbeat by increasing the frequency of impulses to the cardiac conducting cells. Parasympathetic stimulation slows down the heartbeat by decreasing the frequency of the impulses. The impulses carried by these nerves do not initiate contraction but only modify the rate of intrinsic cardiac muscle contraction by their effect at the nodes.

1	the frequency of the impulses. The impulses carried by these nerves do not initiate contraction but only modify the rate of intrinsic cardiac muscle contraction by their effect at the nodes. The structure and functions of the conducting system of the heart are described in Chapter 13, Cardiovascular System.

1	A localized injury to cardiac muscle tissue that results in the death of cells is repaired by replacement with fibrous connective tissue. Consequently, cardiac function is lost at the site of injury. This pattern of injury and repair is seen in nonfatal myocardial infarction (MI). Confirmation of suspected MI in the individual can be made through the detection of specific markers in the blood. These markers are the structural subunits TnI and TnT of the cardiac troponin complex. They are usually released into the bloodstream within 3 to 12 hours after an MI. TnI levels remain elevated for up to 2 weeks from the time of the initial injury; thus, it is regarded as an excellent marker for diagnosing MI that has recently occurred. Mature cardiac muscle cells are able to divide.

1	Mature cardiac muscle cells are able to divide. In the past, it was thought that once cardiac muscle cells are destroyed, they cannot be replaced by new muscle cells. Recent studies of hearts removed from individuals who had received transplants reveal nuclei undergoing mitosis. Although the number of dividing nuclei in these hearts is low (0.1%), it suggests that damaged cells can potentially be replaced. Perhaps in the future, a method might be developed that could induce human cardiac muscle to regenerate into healthy tissue.

1	Smooth muscle generally occurs as bundles or sheets of elongated fusiform cells with finely tapered ends (Fig. 11.18 and Plate 26, page 350). The cells, also called fibers, range in length from 20 m in the walls of small blood vessels to about 200 m in the wall of the intestine; they may be as large as 500 m in the wall of the uterus during pregnancy. Smooth muscle cells are interconnected by gap junctions, the specialized communication junctions between the cells (Fig. 11.19). Small molecules or ions can pass from cell to cell via these junctions and provide communication links that regulate contraction of the entire bundle or sheet of smooth muscle.

1	Smooth muscle cytoplasm stains rather evenly with eosin in routine H&E preparations because of the concentrations of actin and myosin that these cells contain. The nuclei of smooth muscle cells are located in the center of the cell and often have a corkscrew appearance in longitudinal section. This characteristic is a result of contraction of the cell during fixation and is often useful in distinguishing smooth muscle cells from fibroblasts in routine histologic sections. In the noncontracted cell, the nucleus appears as an elongated structure with tapering ends, lying in the center axis of the cell. When the nucleus is included in a cross section of a smooth muscle fiber, it appears as a round or circular profile whether the cell is contracted or relaxed. The TEM shows that most of

1	FIGURE 11.18 • Photomicrograph of smooth muscle from a human colon. The smooth muscle shown in this micrograph is arranged in two layers. On the left, the muscle cells are cut in longitudinal section; on the right, they are cut in cross section. Smooth muscle cells are elongate and have tapering ends. Note that the nuclei in the longitudinally sectioned muscle cells appear elongate and also exhibit tapering ends, thus matching the shape of the cell. In contrast, the nuclei in the cross-sectioned muscle cells are circular in profile. Also, some of the cross-sectioned cells appear to lack a nucleus, a reflection that the section passed through one of the ends of the cell. Also note that the longitudinally sectioned muscle cells are not easily delineated from one another, which is on account of the way they lie over one another within the thickness of the section. 400.

1	the cytoplasmic organelles are concentrated at each end of the nucleus. These include numerous mitochondria, some cisternae of the rER, free ribosomes, glycogen granules, and a small Golgi apparatus. Structure of Smooth Muscle Smooth muscle cells possess a contractile apparatus of thin and thick filaments and a cytoskeleton of desmin and vimentin intermediate filaments. The remaining sarcoplasm is filled with thin filaments that form a part of the contractile apparatus. Thick myosin filaments are scattered throughout the sarcoplasm of a smooth muscle cell. They are extremely labile and tend to be lost during tissue preparation. Special techniques can be used, however, to retain the structural integrity of the thick filaments and thus demonstrate them with the TEM. The thin FIGURE 11.19 • Electron micrograph of smooth muscle cells.

1	FIGURE 11.19 • Electron micrograph of smooth muscle cells. This electron micrograph shows parts of three smooth muscle cells. The nucleus of one cell is in the lower part of the micrograph. The bulk of the cytoplasm is occupied by thin (actin) filaments, which are just recognizable at this magnification. The -actinin–containing cytoplasmic densities, or dense bodies, are visible among the myofilaments (arrows). Elements of the sarcoplasmic reticulum (SR) and the pinocytotic vesicles (PV ) are also indicated. The other two cells in the middle and upper part of the micrograph possess visible gap junctions (GJ) that allow communication between adjacent cells. The small dark particles are glycogen. 25,000. Inset. Enlargement of the gap junction. Note the presence of pinocytotic vesicles. 35,000.

1	filaments in a smooth muscle cell are attached to cytoplasmic densities or dense bodies that are visible among the filaments (Fig. 11.20). These structures are distributed throughout the sarcoplasm in a network of intermediate filaments containing the protein desmin. Intermediate filaments are part of the cytoskeleton of the cell. Note that vascular smooth muscle contains vimentin filaments in addition to desmin filaments.

1	The components of the contractile apparatus in smooth muscle cells are the following.  Thin filaments contain actin, the smooth muscle isoform of tropomyosin, and two smooth muscle–specific proteins, caldesmon and calponin. No troponin is associ ated with smooth muscle tropomyosin. Actin is involved in the force-generating interaction with myosin II molecules. Research suggests that the tropomyosin position on the actin filament is regulated by phosphorylation of myosin heads. Caldesmon (120 to 150 kilodaltons) and calponin (34 kilodaltons) are actin-binding proteins that block the myosin-binding site. The action of these proteins is Ca2-dependent and is also controlled by the phosphorylation of myosin heads.

1	 Thick filaments containing myosin II differ slightly from those found in skeletal muscle. They, too, are composed of two polypeptide heavy chains and four light chains. However, the structure of thick filaments in smooth muscle is different than in skeletal muscle. Rather than a bipolar arrangement, myosin II molecules are oriented in one direction on one side of the filament and in an opposite direction on the other side of the filament. In this arrangement, myosin molecules are staggered in parallel between two immediate neighbors and are also bound to an antiparallel partner via a short overlap at the very tip of their tails (Fig. 11.21). The polarity of the myosin heads is the same along the entire length of one side of the filament and the opposite on the opposite side. This side-polar myosin filament also has no central “bare zone,” but instead has asymmetrically tapered bare ends. This organization maximizes the interaction between thick and thin filaments, allowing the

1	myosin filament also has no central “bare zone,” but instead has asymmetrically tapered bare ends. This organization maximizes the interaction between thick and thin filaments, allowing the overlapped thin filaments to be pulled over the entire length of the thick filaments.

1	Several more proteins are associated with the contractile apparatus and are essential to initiation or regulation of the smooth muscle contractions.  Myosin light chain kinase (MLCK) is a 130to 150kilodalton enzyme that is important in the mechanism of contraction in smooth muscle. It initiates the contraction cycle after its activation by Ca2–calmodulin complex. Active MLCK phosphorylates one of the myosin regulatory light chains, enabling it to form a cross-bridge with actin filaments.  Calmodulin, a 17-kilodalton Ca2-binding protein, is related to the TnC found in skeletal muscle, which regulates the intracellular concentration of Ca2. A Ca2 calmodulin complex binds to MLCK to activate this enzyme. It may also, with caldesmon, regulate its phosphorylation and release from F-actin.  -actinin, a 31-kilodalton protein, provides structural component to dense bodies. Dense bodies provide an attachment site for thin filaments and intermediate filaments.

1	 -actinin, a 31-kilodalton protein, provides structural component to dense bodies. Dense bodies provide an attachment site for thin filaments and intermediate filaments. Dense bodies contain a variety of attachment plaque proteins, including -actinin, which anchors both thin filaments and intermediate filaments either directly or indirectly to the sarcolemma. They play an important role in transmitting contractile forces generated inside the cell to the cell surface, altering the cell’s shape (Fig. 11.22). Dense bodies are intracellular analogs of the striated muscle Z lines. In support of this concept is the finding that dense bodies, although frequently appearing as small, isolated, irregular, electron-dense bodies,

1	FIGURE 11.20 • Electron micrographs showing the cytoplasmic densities in vascular smooth muscle cells. Upper inset. The plane of section includes only the smooth muscle cells in the vascular wall. The rectangle in the inset shows portions of three smooth muscle cells that appear at higher magnification in the large electron micrograph. The -actinin–containing cytoplasmic densities (single arrows) usually appear as irregular masses, some of which are in contact with, and attached to, the plasma membrane. The cell in the center of the micrograph has been cut in a plane closer to the cell surface and reveals these same densities as a branching structure (double arrows). A three-dimensional model of the cytoplasmic densities would reveal an anastomosing network. BL, basal (external) lamina; PV, pinocytotic vesicles. 27,000. Lower inset. Higher magnification of cytoplasmic densities attached to the plasma membrane from the area indicated by the rectangle. Note that each cell possesses a

1	PV, pinocytotic vesicles. 27,000. Lower inset. Higher magnification of cytoplasmic densities attached to the plasma membrane from the area indicated by the rectangle. Note that each cell possesses a basal (external) lamina. In addition, the pinocytotic vesicles can be observed in different stages of their formation. 49,500.

1	FIGURE 11.21 • Comparison of myosin filaments of skeletal muscle and smooth muscle. This drawing shows the different arrangements of myosin thick filaments. a. Bipolar thick filaments are present in skeletal and cardiac muscle. They have a helical parallel–antiparallel arrangement of myosin molecules with their globular heads projecting from both ends of the filament. This filament has a “bare zone” in the middle of the filaments that does not have globular heads. b. Side-polar nonhelical thick filaments are present in smooth muscle. In these filaments, myosin molecules are staggered in parallel by two immediate neighbors and are also bound to an antiparallel partner via a short overlap at the very tip of their tails. The polarity of the myosin heads is the same along the entire length of one side of the filament and the opposite on the opposite side. There is no central “bare zone”; instead, the filament b has asymmetrically tapered bare ends.

1	-actinin-containing cytoplasmic densities (dense bodies) actin-myosin filaments nucleus actin filament actin tropomyosin myosin filament intermediate filament (desmin, vimentin) dense body -actinin FIGURE 11.22 • A suggested model for smooth muscle cell contraction. Bundles of myofilaments containing thin and thick filaments, shown in dark brown, are anchored on cytoplasmic densities, shown in beige. These densities, in turn, are anchored on the sarcolemma. Cytoplasmic densities are intracellular analogs of striated muscle Z lines. They contain the actin-binding protein -actinin. Because the contractile filament bundles are oriented obliquely to the long axis of the cell, their contraction shortens the cell and produces the “corkscrew” shape of the nucleus.

1	may also appear as irregular linear structures. In fortuitous sections, they exhibit a branching configuration consistent with a three-dimensional anastomosing network that extends from the sarcolemma into the interior of the cell (see Fig. 11.20). Contraction in smooth muscles is initiated by a variety of impulses, including mechanical, electrical, and chemical stimuli. The mechanisms that cause contraction of smooth muscle cells are very different from those of striated muscle. Smooth muscle has diverse signal transduction pathways that initiate and modulate smooth muscle contraction. They all lead to elevation of the intracellular concentration of Ca2, which is directly responsible for muscle contraction. Thus muscle contraction can be triggered by the following.  Mechanical impulses, such as passive stretching of vascular smooth muscle, activate mechanosensitive ion channels, leading to initiation of spontaneous muscle contraction (myogenic reflex).

1	 Mechanical impulses, such as passive stretching of vascular smooth muscle, activate mechanosensitive ion channels, leading to initiation of spontaneous muscle contraction (myogenic reflex).  Electrical depolarizations can occur, such as those during neural stimulation of smooth muscle. The release of the neurotransmitters acetylcholine and norepinephrine from their synaptic nerve endings stimulates receptors located in the neuronal plasma membrane and changes the membrane potential. This causes opening of voltage-sensitive Ca2 channels (see below).

1	 Chemical stimuli, such as those elicited by angiotensin II, vasopressin, or thromboxane A2, act on specific cell membrane receptors, leading to muscle contraction. These substances use second-messenger pathways that do not require the generation of an action potential and cell depolarization to trigger contraction. The most common second-messenger pathways used by smooth muscle are inositol 1,4,5-trisphosphate (IP3), G-protein-coupled, and nitric oxide (NO)-cGMP pathways. Smooth muscle cells lack a T system.

1	Smooth muscle cells lack a T system. A characteristic feature of smooth muscle cells is the presence of large numbers of invaginations of the cell membrane that resemble caveolae (see Fig. 11.19). Beneath the plasma membrane, and often in proximity to the sparse profiles of the sER, are cytoplasmic vesicles. It is thought that the invaginations of the cell membrane and the underlying vesicles along with the sER function in a manner analogous to the T system of striated muscle to deliver Ca2 to the cytoplasm. Intracellular Ca2 concentrations are very important in regulating smooth muscle contraction.

1	An elevation of intracellular Ca2 levels in smooth muscle is achieved either by depolarization of the cell membrane with subsequent activation of voltage-sensor proteins or by direct activation of gated Ca2-release channels in the sER by a second-messenger molecule, most commonly IP3. The IP3 receptor is located in the sER membrane and has properties similar to those of gated Ca2-release channels. The amount of Ca2 entering the cell after activation of the voltage-sensor protein is usually insufficient to initiate smooth muscle contraction and needs to be supplemented by release of Ca2 from the sER. The Ca2 then binds to calmodulin, which activates phosphorylation of the myosin light chain kinase to initiate contraction. After the contraction cycle commences, Ca2 is removed from the sarcoplasm by ATP-dependent calcium pumps and resequestered in the sER or delivered to the extracellular environment.

1	Contraction of smooth muscle is regulated by the Ca2–calmodulin–myosin light chain kinase system.

1	A modified version of the sliding filament model described on page 323 can explain contraction in both striated and smooth muscle (see Fig. 11.22). As in striated muscle, contraction is initiated by an increase in the Ca2 concentration in the cytosol, but the contraction does not act through a troponin–tropomyosin complex on the thin filament. Rather, in smooth muscle, an increase in Ca2 concentration stimulates a myosin light chain kinase (MLCK) to phosphorylate one of the two regulatory light chains of the myosin molecule. The Ca2 binds to calmodulin to form the Ca2–calmodulin complex, which in turn binds to MLCK to activate the phosphorylation reaction of the regulatory light chain of myosin (Fig. 11.23). When the light chain is phosphorylated, the actin-binding site of the myosin head is activated and attaches to actin. In the presence of ATP, the myosin head bends, producing contraction. When it is dephosphorylated, the myosin head dissociates from actin. This phosphorylation

1	is activated and attaches to actin. In the presence of ATP, the myosin head bends, producing contraction. When it is dephosphorylated, the myosin head dissociates from actin. This phosphorylation occurs slowly, with maximum contraction often taking up to a second to achieve.

1	Smooth muscle cell myosin hydrolyzes ATP at about 10% of the rate of skeletal muscle, producing a slow cross-bridging cycle that results in slow contraction of these cells. Thus, smooth muscle cells, and nonmuscle cells that contract by this same mechanism, are capable of sustained contractions over long periods of time while using only 10% of the ATP that would be used by a striated muscle cell performing the same work. The force of smooth muscle contraction may be maintained for long periods in a “latch state.”

1	The force of smooth muscle contraction may be maintained for long periods in a “latch state.” In addition to normal phosphorylation of the regulatory light chains of myosin, smooth muscle cells possess a secondary mechanism that allows them to maintain long-term contraction with minimal expenditure of ATP. This mechanism is detected in vascular smooth muscles, for example, and is used to maintain the force of contraction (tone of blood vessels) for an extended time. This so-called latch state of smooth muscle contraction occurs after the initial Ca2-dependent myosin phosphorylation. The myosin head attached to the actin molecule becomes dephosphorylated, causing its ATPase activity to decrease. As a result of the decrease in ATP activity, the myosin head is unable to detach from the actin flament, which maintains the contracted state. The latch state is comparable in many ways to rigor mortis in striated muscle. Functional Aspects of Smooth Muscle

1	Functional Aspects of Smooth Muscle Smooth muscle is specialized for slow, prolonged contraction. As noted previously, smooth muscle cells may enter the latch state and remain contracted for long periods of time without fatiguing. They may contract in a wavelike manner, producing peristaltic movements such as those in the gastrointestinal tract and the male genital tract, or contraction may occur along the entire muscle, producing extrusive movements (e.g., those in the urinary bladder, gallbladder, and uterus). Smooth muscle exhibits a spontaneous contractile activity in the absence of nerve stimuli. Contraction of smooth muscle is usually regulated by post-synaptic neurons of the autonomic nervous system (ANS); most smooth muscle is directly innervated by both sympathetic and parasympathetic nerves. In the gastrointestinal tract, the third component of the ANS, the enteric division, is the primary source of nerves to the muscular layers.

1	FIGURE 11.23 • Schematic diagram illustrating steps leading to initiation of smooth muscle contraction. An increase in the Ca2 level concentration within the cytosol is necessary to initiate smooth muscle contraction. This increase is achieved either by initial depolarization of the cell membrane or hormonal stimulation of cell surface receptors. The intracellular Ca2 binds to calmodulin (4 Ca2 per 1 molecule of calmodulin) to form the Ca2–calmodulin complex. This complex then binds to myosin light chain kinase (MLCK) to phosphorylate one of the two regulatory light chains of the myosin molecule. When phosphorylated, the myosin changes its conformation and the actin-binding site on the myosin head is activated, allowing it to attach to actin. In the presence of ATP, the myosin head bends, producing contraction. sER, smooth endoplasmic reticulum.

1	Although Ca2 enters the cytoplasm during depolarization by voltage-gated Ca2 channels, some Ca2 channels, called ligand-gated Ca2 channels, are activated by hormones via their second-messenger pathways (see Fig. 11.23). Thus, smooth muscle contraction may also be initiated by certain hormones secreted from the posterior pituitary gland (e.g., oxytocin and, to a lesser extent, antidiuretic hormone [ADH]). In addition, smooth muscle cells may be stimulated or inhibited by hormones secreted by the adrenal medulla (e.g., epinephrine and norepinephrine). Also oxytocin is a potent stimulator of smooth muscle contraction, and its release by the posterior pituitary plays an essential role in uterine contraction during parturition. It is often used to induce or enhance labor. Many peptide secretions of enteroendocrine cells also stimulate or inhibit smooth muscle contraction, particularly in the alimentary canal and its associated organs.

1	Nerve terminals in smooth muscle are observed only in the connective tissue adjacent to the muscle cells. Nerve fibers pass through the connective tissue within the bundles of smooth muscle cells; enlargements in the passing nerve fiber, or bouton en passant (see page 359), occur adjacent to the muscle cells to be innervated. The enlargements contain synaptic vesicles with neuromuscular transmitters. However, the neuromuscular site is not comparable to the neuromuscular junction of striated muscle. Rather, a considerable distance, usually 10 to 20 m (in some locations, up to 200 m), may separate the nerve terminal and the smooth muscle. The neurotransmitter released by the nerve terminal must diffuse across this distance to reach the muscle.

1	Not all smooth muscle cells are exposed directly to the neurotransmitter, however. As discussed above, smooth muscle cells make contact with neighboring cells by gap junctions. As in cardiac muscle, contraction is propagated from cell to cell via gap junctions, thus producing coordinated activity within a smooth muscle bundle or layer. The gap junction between two smooth muscle cells was originally designated a nexus, a term still in use. Smooth muscle cells also secrete connective tissue matrix.

1	Smooth muscle cells also secrete connective tissue matrix. Smooth muscle cells have organelles typical of secretory cells. A well-developed rER and Golgi apparatus are found in the perinuclear zone. Smooth muscle cells synthesize both type IV (basal lamina) collagen and type III (reticular) collagen as well as elastin, proteoglycans, and multiadhesive glycoproteins. Except at the gap junctions, smooth muscle cells are surrounded by an external lamina. In some locations, such as the walls of blood vessels and the uterus, smooth muscle cells secrete large amounts of both type I collagen and elastin. Renewal, Repair, and Differentiation Smooth muscle cells are capable of dividing to maintain or increase their number.

1	Smooth muscle cells may respond to injury by undergoing mitosis. In addition, smooth muscle contains regularly replicating populations of cells. Smooth muscle in the uterus proliferates  FOLDER 11.5 Functional Considerations: Comparison of the Three Muscle Types 337 Cardiac muscle shares structural and functional charac-teristics with skeletal muscle and smooth muscle. In both cardiac and skeletal muscle, the contractile elements— thick and thin filaments—are organized into sarcomeres surrounded by sER and mitochondria. Both cardiac and smooth muscle cells retain their individuality, although both are in functional communication with their neighbors through gap junctions. In addition, cardiac and smooth muscle cells have a spontaneous beat that is regulated but not initiated by autonomic or hormonal stimuli. Both have centrally located nuclei and perinuclear organelles. These common characteristics suggest that cardiac mus-cle may have evolved in the direction of skeletal muscle from

1	or hormonal stimuli. Both have centrally located nuclei and perinuclear organelles. These common characteristics suggest that cardiac mus-cle may have evolved in the direction of skeletal muscle from the smooth muscle of primitive circulatory systems. A summary of major characteristics of all three muscle types is provided in the table below. Comparison of the Three Muscle Types Skeletal Cardiac Smooth Structural features Muscle cell Large, elongate cell, 10–100 m in diameter, up to 100 cm in length (sartorius m.) Short, narrow cell, 10–15 m in diameter, 80–100 m in length Short, elongate, fusiform cell, 0.2–2 m in diameter, 20–200 m in length Location Muscles of skeleton visceral striated (e.g., tongue, esophagus, diaphragm) Heart, superior and inferior vena cava, pulmonary veins Vessels, organs, and viscera Connective tissue components Epimysium, perimysium, endomysium Endomysium (subendocardial and subpericardial connective tissue) Endomysium, sheaths, and bundles Fiber Single

1	organs, and viscera Connective tissue components Epimysium, perimysium, endomysium Endomysium (subendocardial and subpericardial connective tissue) Endomysium, sheaths, and bundles Fiber Single skeletal muscle cell Linear branched arrangement of several cardiac muscle cells Single smooth muscle cell Striation Present Present None Nucleus Many peripheral Single central, surrounded by juxtanuclear region Single central T tubules Present at A-I junction (triad: with two terminal cisternae), two T tubules/sarcomere Present at Z lines (diad: with small terminal cisternae), one T tubule/sarcomere None, well-developed sER, many invaginations and vesicles similar to caveolae Cell-to-cell junctions None Intercalated discs containing 1. Fasciae adherentes 2. Macula adherens (desmosome) 3. Gap junctions Gap junctions (nexus) Special features Well-developed sER and T tubules Intercalated discs Dense bodies, caveolae, and cytoplasmic vesicles Functions Type of innervation Voluntary Involuntary

1	Gap junctions (nexus) Special features Well-developed sER and T tubules Intercalated discs Dense bodies, caveolae, and cytoplasmic vesicles Functions Type of innervation Voluntary Involuntary Involuntary Efferent innervation Somatic Autonomic Autonomic Type of contraction “AlI or none” (type I and type II fibers) “All or none” rhythmic (pacemakers, conductive system of the heart) Slow, partial, rhythmic, spontaneous contractions (pacemakers of stomach) Regulation of contraction By binding of Ca2to TnC, causes tropomyosin movement and exposes myosin-binding sites on actin filaments By binding of Ca2to TnC, causes tropomyosin movement and exposes myosin-binding sites on actin filaments By phosphorylation of myosin light chain by myosin light chain kinase in the presence of Ca2-calmodulin complex Growth and regeneration Mitosis None None (in normal condition) Present Response to demand Hypertrophy Hypertrophy Hypertrophy and hyperplasia Regeneration Limited (satellite cells and myogenic

1	Growth and regeneration Mitosis None None (in normal condition) Present Response to demand Hypertrophy Hypertrophy Hypertrophy and hyperplasia Regeneration Limited (satellite cells and myogenic cells from bone marrow) None (in normal condition) Present chapter 11 Muscle Tissue SMOOTH M USCLE 337 during the normal menstrual cycle and during pregnancy; both activities are under hormonal control. The smooth muscle cells of blood vessels also divide regularly in the adult, presumably to replace damaged or senile cells; the smooth muscle of the muscularis externa of the stomach and colon regularly replicates and may even slowly thicken during life.

1	New smooth muscle cells have been shown to differentiate from undifferentiated mesenchymal stem cells in the adventitia of blood vessels. Differentiation of smooth muscle progenitor cells is regulated by a variety of intracellular and environmental stimuli, and developing muscles exhibit a wide range of different phenotypes at different stages of their development. To date, no transcription factors have been identified that are characteristic for the smooth muscle cell lineage. However, serum response factor (RF), a member of the MADS–box transcription factor family, has been shown to regulate most smooth muscle differentiation marker genes. Smooth muscle cells have also been shown to develop from the division and differentiation of endothelial cells and pericytes during the repair process after vascular injury.

1	Vascular pericytes are located within the basal lamina of capillaries and postcapillary venules. They function as multi-potential mesenchymal progenitor cells. In capillaries, their cytoplasmic morphology is difficult to distinguish from that of the endothelial cell. In postcapillary venules and pericytic venules, they may form a nearly complete investment of the vessel with cells that resemble smooth muscle cells (see Chapter 13, Cardiovascular System).

1	Fibroblasts in healing wounds may develop morphologic and functional characteristics of smooth muscle cells (myofibroblasts; see page 179). Epithelial cells in numerous locations, particularly sweat glands, mammary glands, salivary glands, and the iris of the eye, may acquire the characteristics of smooth muscle cells (myoepithelial cells). Myoid cells of the testis have a contractile function in the seminiferous tubules, and cells of the perineurium, a concentric layer of connective tissue that surrounds groups of nerve fibers and partitions peripheral nerves into distinct fascicles, function as contractile cells as well as transport barrier cells. This page intentionally left blank.

1	340 Muscle tissue is classified on the basis of the appearance of its contractile cells. Two major types are recognized: striated muscle, in which the cells exhibit a cross-striation pattern when observed at the light microscope level; and smooth muscle, in which the cells lack striations. Striated muscle is further subclassified based on location namely skeletal muscle, visceral striated muscle, and cardiac muscle. Skeletal mus-cle is attached to bone and is responsible for movement of the axial and apendicular skeleton, and for maintenance of body position and pos-ture. Visceral striated muscle is morphologically identical, but is restricted to soft tissues including the tongue, pharynx, upper part of the esophagus, and the diaphragm. Cardiac muscle is a type of striated muscle found in the heart and the base of the large veins that empty into the heart. The cross-striations in striated muscle are due to the organization of the contractile elements that occur in the muscle cell,

1	in the heart and the base of the large veins that empty into the heart. The cross-striations in striated muscle are due to the organization of the contractile elements that occur in the muscle cell, namely thin fila-ments composed largely of the protein actin and thick filaments composed of the protein myosin II. The two types of myofilaments occupy bulk of the cytoplasm. The skeletal and visceral striated muscle cells, more commonly called fibers, are a multinucleated syncytium formed during development by the fusion of individual small muscle cells called myoblasts. Surrounding each fiber is a delicate mesh of collagen fibrils referred to as endomysium. In turn, bundles of muscle fibers that form func-tional units within a muscle are surrounded by a thicker connective tissue layer. This connective tissue is referred to as perimysium. Lastly, a sheath of dense connective tissue that surrounds the muscle, is referred to as epimysium. The force generated by individual muscle fibers is

1	connective tissue is referred to as perimysium. Lastly, a sheath of dense connective tissue that surrounds the muscle, is referred to as epimysium. The force generated by individual muscle fibers is transferred to the collagenous elements of each of these connective tissue elements to end in a tendon.

1	Skeletal muscle, human, H&E, ×33. but are not individually discernable. However, the small blue dot-like structures are nuclei of the fibers. Between the fascicles, though difficult to see at This low power micrograph shows a longitudinal section of this magnification, is connective tissue, the perimysium (P). Also evidentstriated muscle. The muscle tissue within the muscle is arin the micrograph is a nerve (Nv). ranged in a series of fascicles (F). The individual muscle fibers within a fascicle are in close proximity to one another, Skeletal muscle, human, H&E, ×33.

1	ranged in a series of fascicles (F). The individual muscle fibers within a fascicle are in close proximity to one another, Skeletal muscle, human, H&E, ×33. This micrograph reveals part of a muscle that has been cut in cross section. Again, individual bundles of muscle fibers or fascicles (F) can be readily identified. In contrast to the previous micrograph, even at this low magnification, upon careful examination, individual muscle fibers (MF) can be identified in many of the fascicles. Each is bounded by connective tissue, which constitutes the perimysium (P). Also identifiable in this micrograph is a dense connective tissue surrounding the muscle, namely epimysium (E). Skeletal muscle, human, H&E, ×256; inset ×700.

1	This higher magnification of a longitudinal section of a muscle reveals two muscle fibers (MF). At this magnification, the cross-banding pattern is just perceptible. With few exceptions, the nuclei (N), which tend to run in linear arrays, belong to the muscle fibers. Also evident in this micrograph is a small blood vessel (BV). The inset, taken from a gluteraldehyde fixed plastic embedded specimen, is a much higher magnification of a portion of two muscle fibers. The major bands are readily identifiable at this magnification and degree of specimen preservation. The thick, dark stained band is the A-band. Between A-bands is a lightly stained area, the I-band, which is bisected by the Z-line. The two elongate nuclei (N) belong to the muscle fibers. Below them are a capillary (C) and a portion of an endothelial cell nucleus (End). At this higher magnification, the endothelial nuclei, as well as the nuclei of the fibroblasts, can be distinguished from the muscle cell nuclei by their

1	portion of an endothelial cell nucleus (End). At this higher magnification, the endothelial nuclei, as well as the nuclei of the fibroblasts, can be distinguished from the muscle cell nuclei by their smaller size and heterochromatin giving them a dark stain. The muscle cell nuclei (N) exhibit more euchromatin with a speckling of heterochromatin, thus given them a lighter staining appearance.

1	Skeletal muscle, human, H&E, ×256. In this cross section, individual muscle fibers (MF) are readily discernable in contrast to identifying individual muscle fibers in longitudinal sections. For example, if one imagines a cut crossing a number of cells (see dashed line), the close proximity of the muscle cells can mask the boundary between individual cells within a fascicle when observed in the opposite or longitudinal plane. The connective tissue (CT) that is readily apparent here belongs to the perimysium, which separates fascicles. The nuclei of the individual fibers are located at the periphery of the cell. At this magnification, it is difficult to distinguish between occasional fibroblasts that belong to the endomysium from the nuclei of the muscle cells. KEY BV, blood vessel C, capillary CT, connective tissue E, epimysium End, endothelial cell nucleus F, fascicles MF, muscle fibers N, nuclei Nv, nerve P, perimysium

1	The myofibril is the structural and functional subunit of a muscle fiber. They are best seen at higher magnification in the light microscope in a cross section of the cell where they appear as dot-like structures. The overall effect is a stippled appearance of the cytoplasm. Each myofibril is composed of a bundle of myofilaments that are of two types. One type is the myosin II thick filament. The other is actin and its associated proteins that make up the thin filaments. It is the arrangement of the thick and thin filaments that produce density differences that in turn cre-ate the cross-striations of the myofibril when viewed in longitudinal section (see diagram). As evident in the diagram, the site of overlap of thin and thick filaments produces the dark A-band. The light appearing I-band contains the thin filaments. Careful examination of the A-band in the light microscope reveals a light staining area in the middle of the A-band. This is referred to as the H-band. It is an area

1	contains the thin filaments. Careful examination of the A-band in the light microscope reveals a light staining area in the middle of the A-band. This is referred to as the H-band. It is an area that is occupied by thin filaments and is devoid of thick filaments. At the middle of each I-band is the thin dense Z-line to which the thin filaments are attached. The distance between Z-lines is referred to as a sarcomere. When a muscle contracts, the sarcomere and I-band shorten. The filaments, however, maintain a constant length, thus the contraction is produced by an increase in the overlap between the two filament types.

1	Skeletal muscle, human, H&E, ×512; inset ×985. This micrograph reveals a cross section of a muscle fascicle. The individual muscle fibers (MF) exhibit a polygonal shape, but vary only slightly in width. Of the many nuclei that can be observed in this plane of section, only some belong to the muscle fibers. The muscle fiber nuclei (MFN) appear to be embedded within the extreme periphery of the fiber. In contrast, fibroblast nuclei (FN) that belong to the endomysium lie clearly outside of the muscle fiber, are typically smaller and exhibit greater density than the nuclei of Skeletal muscle, human, H&E, ×512; inset ×985.

1	Skeletal muscle, human, H&E, ×512; inset ×985. This micrograph, a longitudinal section of a gluteraldehyde fixed, plastic embedded specimen reveals four myofibers (M). Although they appear to be markedly different in width, the difference is due mainly to the plane of section through each of the fibers. Because the nuclei of the myofibers are located at the periphery of the cell, their location is variable when observed in a longitudinal section. For example, three nuclei (N) are seen in what appears to be a central location of a fiber. This is due to the section grazing the periphery of this fiber. The clear space at either end of two of these nuclei represents the cytoplasmic portion of the cell that contains organelles and is devoid of Skeletal muscle, human, electron micrograph, ×5,000.

1	The low power electron micrograph shown here should be compared to the inset of the longitudinally sectioned myofibers above. It reveals portions of three myofibers (M), two of which exhibit a nucleus (N). Between cells, various amounts of collagenous fibers are present representing the endomy sium (E). The micrograph illustrates the banding pattern of the myofibrils to advantage. In contrast to the longitudinally sectioned muscle in the inset above, individual myofibrils (My) can be identified in this electron the muscle fibers. Also present between the muscle fibers are capillaries (C). The endothelial cell nuclei (ECN) are also relatively dense. Other nuclei that may be present, but are very difficult to identify belong to satellite cells. The inset which shows the boxed area reveals several nuclei, two of which belong to the muscle fibers (MF). The small, very dense nucleus (FN) probably belongs to a fibroblast of the endomysium. Also clearly evident here is a cross-sectioned

1	several nuclei, two of which belong to the muscle fibers (MF). The small, very dense nucleus (FN) probably belongs to a fibroblast of the endomysium. Also clearly evident here is a cross-sectioned capillary (C). The more striking feature at this magnification is the appearance of the muscle cells’ myofibrils which appear as the punctate or dot-like structures.

1	myofibrils. Other myofiber nuclei (MFN) can be seen at the periphery of the myofibers. Note that they exhibit a similar chromatin pattern as the three nuclei previously described. Also present in this micrograph is a capillary (C) coursing along the center of the micrograph. In this plane of section, it is difficult to clearly distinguish between the endothelial cell nuclei and nuclei of fibroblasts in the endomysium. Perhaps the most significant feature of a longitudinal section of a muscle fiber is the striations that they exhibit. The inset shows at higher magnification the banding pattern of the myofiber. The dark staining lines represent the A-band. The light staining area is the I-band which is bisected by the dark staining Z-line.

1	micrograph. They correspond to the dot-like structures seen in the inset of the cross-sectioned myofibers above. Note that adjadcent myofibrils are aligned with one another with respect to their banding pattern and also that they exhibit different widths. Each myofibril is essentially a cylindrical structure much like a dowel, thus when sectioned in a longitudinal plane, the width of each myofibril will vary depending on what portion of the cylindrical structure has been cut. The sarcoplasmic reticulum, a membrane system that is present between myofibrils and the nature of the bands in a myofibril are shown to advantage in the next plate. • KEY C, capillaries E, endomysium ECN, endothelial cell nuclei FN, fibroblast nuclei MF, muscle fibers MFN, muscle fiber nuclei M, myofibers My, myofibrils MyN, myofiber nuclei N, nucleus

1	The work effected by skeletal muscle to allow body movement is through tendons to which the muscle fibers are attached. The site of attach-ment between a muscle fiber and the collagen of the tendon is referred to as the myotendinal junction. The muscle fibers at the junctional site end in numerous fingerlike cytoplasmic projections. At the ends of each projection and between these projections, the collagen fibrils of the tendon attach to the cell at its basal lamina (see electron micrograph in adjacent plate). In the light microscope, these fingerlike projections appear to merge into the tendon. The detailed relationship is seen at the electron microscope level. The last sarcomeres in the muscle fiber end where the fingerlike projections begin. At this point, the ending sarcomere lacks its Z-line and the actin filaments from the A-band con-tinue into the cytoplasmic fingers ending at the sarcolemma.

1	Myotendinal junction, monkey, H&E, ×365. collagenous bundles of the tendon. Several of the muscle fibers (MF’) are seen at the point where they terminate and are attached to the tendon This light micrograph reveals a tendon (T) and adjacent to it fibers. The area in the rectangle is seen in higher magnification in the mi-several muscle fibers (MF). The tendon contains dispersed crograph below. tendinocytes whose nuclei (N) are compressed between the Myotendinal junction, monkey, H&E, ×1,560. end of the muscle fiber are clearly seen. Between the fingerlike structures are the collagen fibers of the tendon. The nuclei of the tendinocytes (T) are seen The muscle fiber (MF) in this micrograph is seen at the point in the tendon where it continues from the muscle fiber.

1	where it ends. Note the banding pattern of the muscle fiber. At this magnification, the fingerlike projections (arrows) at the • MYOTE N DI NAL J U NCTION KEY MF, muscle fibers MF’, terminating muscle fibers N, nuclei S, sarcomere T, tendon Tc, tendinocytes Myotendinal junction, monkey, electron micrograph, ×24,000. This electron micrograph shows the end of part of a muscle. Note that the last sarcomere (S) lacks a Z-line. The actin filaments appear to extend from the A-band and continue along the length of the finger projections and seemingly attach to the sarcolemma. Between the finger projec-tions are the collagen fibrils (arrows) that make up the tendon. (Micrograph courtesy of Dr. Douglas Kelly.)

1	Cardiac muscle consists of fibers that possess the same arrangement of contractile filaments and thus the same cross-banding patterns that are present in striated skeletal and visceral muscle. Although cardiac muscle is, therefore, also striated, it differs in many significant respects from skeletal and striated visceral muscle. Cardiac muscle consists of individual cells that are joined by complex junctions to form a functional unit (fiber). The histologically obvious differences between cardiac and the other striated muscle fibers are the presence in cardiac muscle of intercalated discs (the light microscopic representation of the complex junctions), the location of cardiac muscle cell nuclei in the center of the fiber, and the branching of the cardiac muscle fibers. All of these characteristics are evident in a well-prepared longitudinal section of the muscle. Cardiac muscle, heart, human, H&E ×160.

1	Cardiac muscle, heart, human, H&E ×160. This figure shows a longitudinal section of cardiac muscle. The muscle fibers are disposed horizontally in the illustration and show cross-striations. In addition to the regular cross-striations (those of greater frequency), however, there is another group of very pronounced cross-bands, namely, the interca346 lated discs (ID). Intercalated discs most often appear as a straight band, but sometimes they are arranged in a stepwise manner (see also figure on right). These discs are not always displayed in routine H&E sections; therefore, one Cardiac muscle, heart, human, H&E ×400.

1	Cardiac muscle, heart, human, H&E ×400. Like skeletal muscle, the cardiac muscle is composed of linear contractile units, the myofibrils. These are evident in this figure as the longitudinally disposed linear structures that extend through the length of the cell. The myofibrils separate to bypass the nuclei, and in doing so, they delineate a perinuclear region of cytoplasm that is free of myofibrils and their cross-striations. These perinuclear cytoplasmic areas (asterisks) contain the cytoplasmic organelles that are not directly involved in the contractile process. Many cardiac muscle cells are binucleate; both nuclei typically occupy the myofibrilfree region of cytoplasm, as shown in the cell marked by the asterisks. The third nucleus in this region appears to belong to the connective tissue either Cardiac muscle, heart, human, H&E ×160.

1	This figure shows cross-sectioned cardiac muscle fibers. Many have rounded or smooth-contoured polygonal profiles. Some fibers, however, are generally more irregular and elongate in profile. These probably reflect a profile of both a fiber and a branch of the fiber. The more lightly stained region in the center of many fibers represents the myofibril-free region of the cell already re- may not be able to depend on these structures for identifying cardiac muscle. Intercalated discs are opposing cell-to-cell contacts. Thus, cardiac muscle fibers differ in a very fundamental respect from fibers of skeletal muscle. The cardiac muscle fiber consists of an end-to-end alignment of individual cells; in contrast, the skeletal muscle fiber is a single multinucleated protoplasmic unit. In examining a longitudinal section of cardiac muscle, it is useful to scan specific fibers along their long axes. By doing so, one can find places where the fibers obviously branch. Two such branchings are

1	a longitudinal section of cardiac muscle, it is useful to scan specific fibers along their long axes. By doing so, one can find places where the fibers obviously branch. Two such branchings are indicated by the arrows in this figure.

1	above or below the “in-focus” plane of section. Often, the staining of muscle cell nuclei in a specific specimen is very characteristic, especially when seen in face view as here. Notice, in the nucleus between the asterisks, the well-stained nucleolus and the delicate pattern of the remainder of the nucleus. Once such features have been characterized for a particular specimen, it becomes easy to identify nuclei with similar staining characteristics throughout the specimen. For example, survey the field in figure on left for nuclei with similar features. Having done this, it is substantially easier to identify nuclei of connective tissue cells (CT), which display different staining properties and are not positioned in the same relationship to the muscle cells.

1	ferred to above and indicated by the asterisks in top right figure. Delicate connective tissue surrounds the individual muscle fibers. This contains capillaries and sometimes larger vessels, such as the venule (V) in the center of the bundle of muscle fibers. Larger amounts of connective tissue (CT) surround bundles of fibers, and this tissue contains larger blood vessels, such as the arteriole (A) marked in the figure. Cardiac muscle, heart, human, H&E ×400. At higher magnification, it is possible to see the cut ends of the myofibrils. These appear as the numerous red areas that give the cut face of the muscle cell a stippled appearance. The nuclei (N) occupy a central position surrounded by myofibrils.

1	Remember, in contrast, that nuclei of skeletal muscle fibers are located at the periphery of the cell. Note, also, that as mentioned, the nucleus-free central area of the cell, devoid of myofibrils, shows areas of perinuclear cytoplasm similar to that marked with asterisks in figure directly above. KEY A, arteriole C, capillaries CT, connective tissue ID, intercalated discs N, nuclei of cardiac muscle cells V, venule arrows, sites where fibers branch asterisks, perinuclear cytoplasmic areas PLATE 25 Cardiac Muscle, Purkinje Fibers

1	Cardiac muscle cells possess the ability for spontaneous rhythmic contractions. The contraction or beat of the heart is regulated and coordinated by specialized and modified cardiac muscle cells that are found in nodes and muscle bundles. The beat of the heart is initiated at the sinoatrial (SA) node which consists of a group of specialized cardiac muscle cells located at the junction of the superior vena cava in the right atrium. The impulse spreads from this node along the cardiac mus-cle fibers of the atria. The impulse is then received at the atrioventricular (AV) node which is lo-cated on the inner or medial wall of the right ventricle adjacent to the tricuspid valve. Specialized cardiac muscle cells then conduct impulses from the AV node along the ventricular septum and into the ventricular walls. Within the ventricular septum, the specialized cells are grouped into a bundle, the AV bundle (of His). This bundle then divides into two main branches, a left and right bundle branch,

1	walls. Within the ventricular septum, the specialized cells are grouped into a bundle, the AV bundle (of His). This bundle then divides into two main branches, a left and right bundle branch, the former going to the left ventricle and the latter to the right ventricle. The specialized con-ducting fibers carry the impulse at a rate that is approximately four times faster than the cardiac muscle fibers. They are responsible for the final distribution of the electrical stimulus to the my-ocardium. While the sinoatrial node on its own exhibits a constant or inherent rhythm, it is modu-lated by the autonomic nervous system. Thus, the rate of the heartbeat can be decreased by parasympathetic fibers from the vagus nerve or increased by fibers from sympathetic ganglia. The specialized conducting cells within the ventricles are referred to as Purkinje fibers. The cells that make up the Purkinje fibers differ from cardiac muscle cells in that they are larger and have their my-ofibrils located

1	cells within the ventricles are referred to as Purkinje fibers. The cells that make up the Purkinje fibers differ from cardiac muscle cells in that they are larger and have their my-ofibrils located mostly at the periphery of the cell. Their nuclei are also larger. The cytoplasm be-tween the nucleus and the peripherally located myofibrils stains poorly, a reflection, in part, of the large amount of glycogen present in this part of the cell. ORIENTATION MICROGRAPH: The specimen shown here is a saggital section revealing part of the atrial wall (A) and the ventricular wall (V). Between these two portions of the heart is the atri-oventricular septum (AS). The clear space is the interior of the atrium. AAVVASASAVAS

1	PLATE 25 • CARDIAC MUSCLE, PURKINJE FIBERS KEY A, atrial wall AS, atrioventricular septum DICT, dense irregular connective tissue Ec, endocardium Ec’, deep endocardium Et, endothelium EtC, endothelial cells ID, intercalated discs M, myofibrils My, myocardium PF, Purkinje fibers SM, smooth muscle cells V, ventricular wall Purkinje fibers, heart, human, Masson, ×180. This micrograph shows the area in the rectangle of the orientation figure. At this site, the endocardium (Ec) has been separated by bundles of Purkinje fibers (bundle of His) (PF) coursing along the ventricle wall. Normally, the endocardium consists of three layers. The endothelium (Et) lining the ventricle is the most superficial, but is barely detectable at this magnification. Purkinje fibers, heart, human, Masson, ×365; inset ×600.

1	Purkinje fibers, heart, human, Masson, ×365; inset ×600. This higher magnification is the boxed area in the above photomicrograph. It reveals the endothelial cells of the endocardium (EtC) and the underlying connective tissue containing smooth muscle cells (SM). Where the Purkinje fibers are cut in cross section or obliquely sectioned, the myofibrils (M) are seen at the periphery of the Beneath the endothelium is a middle layer consisting of dense connective tissue (DICT), in which elastic fibers are present as well as some smooth muscle cells. The third layer, the deepest part of the endocardium (Ec’), consists of more irregularly arranged connective tissue with blood vessels and occasional fat cells. At the bottom of the micrograph is myocardium (My). Note how darkly stained the cardiac muscle fibers are compared to those of the Purkinje fibers.

1	cell. The cytoplasm in the inner portion of the cell appears unstained. Where the nuclei are included in the section of the cell, they are surrounded by the clear cytoplasm. In the lower portion of the figure, several longitudinally sectioned Purkinje fibers can be seen. Note the intercalated discs (ID) when seen in this profile. The inset reveals to advantage the intercalated discs and the myofibrils with their cross-banding. Note the clear area or unstained cytoplasm surrounding the nuclei. PLATE 25 • CARDIAC MUSCLE, PURKINJE FIBERS EtEtDICTDICTPFPFEc’Ec’EcEcDICTDICTMyMyEtDICTPFEc’EcDICTMyEtCEtCSMSMSMSMIDIDMMMMMMEtCSMSMIDMMM

1	Smooth muscle is the intrinsic muscle of the alimentary canal, blood vessels, the genitourinary and respiratory tracts, and other hollow and tubular organs. It is also a component of the nipple, scrotum, skin (arrector pili muscle) and parts of the eye (iris). In most locations, smooth muscle consists of bundles or layers of elongate fusiform cells. They may range in length from 20 m in the walls of small blood vessels to about 200 m in the intestinal wall. In the case of the uterus, they may become as large as 500 m during pregnancy. The smooth muscle cells are joined by gap junctions that allow small molecules or ions to pass from cell to cell and allow regulation of contraction of the entire bundle or sheet of smooth muscle. The cytoplasm of smooth muscle cells stains uniformly with eosin in routine H&E preparations because of the concentration of actin and myosin that these cells contain. The nucleus of the cell is located in its center and is elongate with tapering ends, matching

1	in routine H&E preparations because of the concentration of actin and myosin that these cells contain. The nucleus of the cell is located in its center and is elongate with tapering ends, matching the shape of the cell. When the cell is maximally contracted, the nucleus displays a corkscrew shape. During lesser degrees of contraction, the nucleus may appear to have a slight spiral shape. Often in H&E preparations, smooth muscle stains much the same as dense connective tissue. A distinguishing feature relative to smooth muscle is that nuclei are considerably more numerous and they tend to look the same, appearing as elongate profiles when smooth muscle is longitudinally sectioned and as circular profiles when smooth muscle is cross-sectioned. In contrast, the nuclei of dense connective tissue, though fewer in number per unit area, may appear in varying profiles in a given section.

1	Smooth muscle, small intestine, human, H&E, ×256. This low power micrograph reveals part of the wall of the small intestine, the muscularis externa. The left side of the micrograph shows two bundles, both are longitudinally sectioned (LS), whereas on the right side, smooth muscle bundles are seen in cross section (CS). Note that the nuclei of Smooth muscle, small intestine, human, H&E, ×512.

1	Smooth muscle, small intestine, human, H&E, ×512. This higher magnification photomicrograph shows a bundle of smooth muscle cells (SMC). Note how the nuclei exhibit an undulating or wavy form indicating that the cells are partially contracted. The nuclei seen in the dense connective tissue the smooth muscle cells in the longitudinally sectioned bundles are all elongate; in contrast the nuclei in the cross-sectioned smooth muscle bundles appear as circular profiles. Intermixed between the bundles is dense irregular connective tissue (DICT). While both the smooth muscle cells and the dense connective tissue stain with eosin, the dense connective tissue exhibits a paucity of nuclei compared to the smooth muscle cell bundles.

1	(DCT) in contrast show a variety of shapes. The collagen fibers in this case, as in the previous micrograph, have a brighter red coloration than the cytoplasm of the smooth muscle cells, which provides further distinction between the two types of tissue. However, this is not always the case and the two may appear similarly stained. Smooth muscle, small intestine, human, H&E, ×256. dense connective tissue (DCT) and the numerous circular profiles of the smooth muscle cell nuclei. This micrograph shows at low magnification several bundles of cross-sectioned smooth muscle (SM). Again, note how the smooth muscle bundles are separated from one another by Smooth muscle, small intestine, human, H&E, ×512; inset ×1,185.

1	Smooth muscle, small intestine, human, H&E, ×512; inset ×1,185. At this higher magnification, the smooth muscle is again seen in cross section. As is typically the case, the distribution of the smooth muscle cell nuclei is not uniform, thus in some areas there appears to be a crowding of nuclei (lower box), whereas in other areas, there appears to be a paucity of nuclei (upper box). This is a reflection of the side-by-side orientation of the smooth muscle cells; thus, in this area, the cells are aligned in a manner that the nucleus has not been included in the thickness of the section. The inset is a higher magnification of this area and shows the cross-sectioned smooth muscle cells as circular profiles of varying size. Where the nuclei appear more numerous, the cells simply are aligned where the section has included the nucleus.

1	KEY CS, cross-sectioned bundles DCT, dense connective tissue DICT, dense irregular connective tissue LS, longitudinally sectioned bundles SM, smooth muscle SMC, smooth muscle cells

1	OVERVIEW OF THE NERVOUS SYSTEM / 352 COMPOSITION OF NERVE TISSUE / 353 THE NEURON / 353 Cell Body / 355 Dendrites and Axons / 357 Synapses / 358 Axonal Transport Systems / 363 SUPPORTING CELLS OF THE NERVOUS SYSTEM: THE NEUROGLIA / 363 Peripheral Neuroglia / 363 Schwann Cells and the Myelin Sheath / 364 Satellite Cells / 366 Central Neuroglia / 367 Impulse Conduction / 371 ORIGIN OF NERVE TISSUE CELLS / 373 ORGANIZATION OF THE PERIPHERAL NERVOUS SYSTEM / 375 Peripheral Nerves / 375 Connective Tissue Components of a Peripheral Nerve / 375 Afferent (Sensory) Receptors / 377 ORGANIZATION OF THE AUTONOMIC NERVOUS SYSTEM / 378 Sympathetic and Parasympathetic Divisions of the Autonomic Nervous System / 378 Enteric Division of the Autonomic Nervous System / 378 A Summarized View of Autonomic Distribution / 381 ORGANIZATION OF THE CENTRAL NERVOUS SYSTEM / 381 Cells of the Gray Matter / 382 Organization of the Spinal Cord / 382 Connective Tissue of the Central Nervous System / 383 Blood–Brain

1	/ 381 ORGANIZATION OF THE CENTRAL NERVOUS SYSTEM / 381 Cells of the Gray Matter / 382 Organization of the Spinal Cord / 382 Connective Tissue of the Central Nervous System / 383 Blood–Brain Barrier / 385 RESPONSE OF NEURONS TO INJURY / 386 Degeneration / 386 Regeneration / 387 Folder 12.1 Clinical Correlation: Parkinson’s Disease / 358 Folder 12.2 Clinical Correlation: Demyelinating Diseases / 366 Folder 12.3 Clinical Correlation: Gliosis: Scar formation in the CNS / 389

1	The nervous system enables the body to respond to continuous changes in its external and internal environment. It controls and integrates the functional activities of the organs and organ systems. Anatomically, the nervous system is divided into the following:  The central nervous system (CNS) consists of the brain and the spinal cord, which are located in the cranial cavity and spinal canal, respectively.

1	 The peripheral nervous system (PNS) consists of cranial, spinal, and peripheral nerves that conduct impulses from (efferent or motor nerves) and to (the afferent or sensory nerves of ) the CNS, collections of nerve cell bodies outside the CNS called ganglia, and specialized nerve endings (both motor and sensory). Interactions between sensory (afferent) nerves that receive stimuli, the CNS that interprets them, and motor (efferent) nerves that initiate responses create neural pathways. These pathways mediate reflex actions called reflex arcs. In humans, most sensory neurons do not pass directly into the brain but instead communicate by specialized terminals (synapses) with motor neurons in the spinal cord.

1	Functionally, the nervous system is divided into the following:  The somatic nervous system (SNS) consists of somatic [Gr. soma, body] parts of the CNS and PNS. The SNS controls functions that are under conscious voluntary control with the exception of reflex arcs. It provides sensory and motor innervation to all parts of the body except viscera, smooth and cardiac muscle, and glands.  The autonomic nervous system (ANS) consists of autonomic parts of the CNS and PNS. The ANS provides efferent involuntary motor innervation to smooth muscle, the conducting system of the heart, and glands. It also provides afferent sensory innervation from the viscera (pain and autonomic reflexes). The ANS is further subdivided into a sympathetic division and a parasympathetic division. A third division of ANS, the enteric division, serves the alimentary canal. It communicates with the CNS through the parasympathetic and sympathetic nerve fibers; however, it can also function independently of the other

1	ANS, the enteric division, serves the alimentary canal. It communicates with the CNS through the parasympathetic and sympathetic nerve fibers; however, it can also function independently of the other two divisions of the ANS (see page 378).

1	Nerve tissue consists of two principal types of cells: neurons and supporting cells. The neuron or nerve cell is the functional unit of the nervous system. It consists of a cell body, containing the nucleus, and several processes of varying length. Nerve cells are specialized to receive stimuli from other cells and to conduct electrical impulses to other parts of the system via their processes. Several neurons are typically involved in sending impulses from one part of the system to another. These neurons are arranged in a chainlike fashion as an integrated communications network. Specialized contacts between neurons that provide for transmission of information from one neuron to the next are called synapses.

1	Supporting cells are nonconducting cells that are located close to the neurons. They are referred to as neuroglial cells or simply glia. The CNS contains four types of glial cells: oligodendrocytes, astrocytes, microglia, and ependymal cells (see page 367). Collectively, these cells are called the central neuroglia. In the PNS, supporting cells are called peripheral neuroglia and include Schwann cells, satellite cells, and a variety of other cells associated with specific structures. Schwann cells surround the processes of nerve cells and isolate them from adjacent cells and extracellular matrix. Within the ganglia of the PNS, peripheral neuroglial cells are called satellite cells. They surround the nerve cell bodies, the part of the cell that contains the nucleus, and are analogous to Schwann cells. The supporting cells of the ganglia in the wall of the alimentary canal are called enteric neuroglial cells. They are morphologically and functionally similar to central neuroglia (see

1	Schwann cells. The supporting cells of the ganglia in the wall of the alimentary canal are called enteric neuroglial cells. They are morphologically and functionally similar to central neuroglia (see page 367).

1	Functions of the various neuroglial cell types include:  physical support (protection) for neurons,  insulation for nerve cell bodies and processes that facilitates rapid transmission of nerve impulses,  repair of neuronal injury,  regulation of the internal fluid environment of the CNS,  clearance of neurotransmitters from synaptic clefts, and  metabolic exchange between the vascular system and the neurons of the nervous system In addition to neurons and supporting cells, an extensive vasculature is present in both the CNS and the PNS. The blood vessels are separated from the nerve tissue by the basal laminae and variable amounts of connective tissue, depending on vessel size. The boundary between blood vessels and nerve tissue in the CNS excludes many substances that normally leave blood vessels to enter other tissues. This selective restriction of blood-borne substances in the CNS is called the blood–brain barrier, which is discussed on page 385.

1	The nervous system allows rapid response to external stimuli. The nervous system evolved from the simple neuroeffector system of invertebrate animals. In primitive nervous systems, only simple receptor–effector reflex loops exist to respond to external stimuli. In higher animals and humans, the SNS retains the ability to respond to stimuli from the external environment through the action of effector cells (such as skeletal muscle), but the neuronal responses are infinitely more varied. They range from simple reflexes that require only the spinal cord to complex operations of the brain, including memory and learning. The autonomic part of the nervous system regulates the function of internal organs.

1	The autonomic part of the nervous system regulates the function of internal organs. The specific effectors in the internal organs that respond to the information carried by autonomic neurons include the following:  Smooth muscle. Contraction of smooth muscle modifies the diameter or shape of tubular or hollow viscera such as the blood vessels, gut, gallbladder, and urinary bladder.  Cardiac conducting cells (Purkinje fbers) located within the conductive system of the heart. The inherent frequency of Purkinje fiber depolarization regulates the rate of cardiac muscle contraction and can be modified by autonomic impulses. Glandular epithelium. The autonomic nervous system reg ulates the synthesis, composition, and release of secretions.

1	The regulation of the function of internal organs involves close cooperation between the nervous system and the endocrine system. Neurons in several parts of the brain and other sites behave as secretory cells and are referred to as neuroendocrine tissue. The varied roles of neurosecretions in regulating the functions of the endocrine, digestive, respiratory, urinary, and reproductive systems are described in subsequent chapters. The neuron is the structural and functional unit of the nervous system. The human nervous system contains more than 10 billion neurons. Although neurons show the greatest variation in size and shape of any group of cells in the body, they can be grouped into three general categories.

1	 Sensory neurons convey impulses from receptors to the CNS. Processes of these neurons are included in somatic afferent and visceral afferent nerve fibers. Somatic afferent fbers convey sensations of pain, temperature, touch, and pressure from the body surface. In addition, these fibers convey pain and proprioception (nonconscious sensation) from organs within the body (e.g., muscles, tendons, and joints) to provide the brain with information related to the orientation of the body and limbs. Visceral afferent fbers transmit pain impulses and other sensations from internal organs, mucous membranes, glands, and blood vessels.

1	 Motor neurons convey impulses from the CNS or ganglia to effector cells. Processes of these neurons are included in somatic efferent and visceral efferent nerve fibers. Somatic efferent neurons send voluntary impulses to skeletal muscles. Visceral efferent neurons transmit involuntary impulses to smooth muscle, cardiac conducting cells (Purkinje fibers), and glands (Fig. 12.1).  Interneurons, also called intercalated neurons, form a communicating and integrating network between the node of Ranvier FIGURE 12.1 • Diagram of a motor neuron. The nerve cell body, dendrites, and proximal part of the axon are within the CNS. The axon leaves the CNS and, while in the PNS, is part of a nerve (not shown) as it courses to its effectors (striated muscle). In the CNS, the myelin for the axon is produced by, and is part of, an oligodendrocyte; in the PNS, the myelin is produced by, and is part of, a Schwann cell.

1	sensory and motor neurons. It is estimated that more than 99.9% of all neurons belong to this integrating network. The functional components of a neuron include the cell body, axon, dendrites, and synaptic junctions. The cell body (perikaryon) of a neuron contains the nucleus and those organelles that maintain the cell. The processes extending from the cell body constitute the single common structural characteristic of all neurons. Most neurons have only one axon, usually the longest process extending from the cell, which transmits impulses away from the cell body to a specialized terminal (synapse). The synapse makes contact with another neuron or an effector cell (e.g., a muscle cell or glandular epithelial cell). A neuron usually has many dendrites, shorter processes that transmit impulses from the periphery (i.e., other neurons) toward the cell body. Neurons are classified on the basis of the number of processes extending from the cell body.

1	Neurons are classified on the basis of the number of processes extending from the cell body. Most neurons can be anatomically characterized as the following:  Multipolar neurons have one axon and two or more dendrites (Fig. 12.2). The direction of impulses is from dendrite to cell body to axon or from cell body to axon. Functionally, the dendrites and cell body of multipolar neurons are the receptor portions of the cell, and their plasma membrane is specialized for impulse generation. The axon is the conducting portion of the cell, and its plasma membrane is specialized for impulse conduction. The terminal portion of the axon, the synaptic ending, contains various neurotransmitters—that is, small molecules released at the synapse that affect other neurons as well as muscle cells and glandular epithelium. Motor neurons and interneurons constitute most of the multipolar neurons in the nervous system.

1	 Bipolar neurons have one axon and one dendrite (see Fig. 12.2). Bipolar neurons are rare. They are most often associated with the receptors for the special senses (taste, smell, hearing, sight, and equilibrium). They are generally found within the retina of the eye and the ganglia of the vestibulocochlear nerve (cranial nerve VIII) of the ear. Some neurons in this group do not fit the above generalizations. For example, amacrine cells of the retina have no axons, and olfactory receptors resemble neurons of primitive neural systems, in that they retain a surface location and regenerate at a much slower rate than other neurons.

1	 Pseudounipolar (unipolar) neurons have one process, the axon, that divides close to the cell body into two long axonal branches. One branch extends to the periphery, and the other extends to the CNS (see Fig. 12.2). The two axonal branches are the conducting units. Impulses are generated in the peripheral arborizations (branches) of the neuron that are the receptor portion of the cell. Each pseudounipolar neuron develops from a bipolar neuron, as its axon and dendrite migrate around the cell body and fuse into a single process. The majority of pseudounipolar neurons are sensory neurons located close to the CNS (Fig. 12.3). Cell bodies of sensory neurons are situated in the dorsal root ganglia and cranial nerve ganglia.

1	FIGURE 12.2 • Diagram illustrating different types of neurons. The cell bodies of pseudounipolar (unipolar), bipolar, and postsynaptic autonomic neurons are located outside the CNS. Purkinje and pyramidal cells are restricted to the CNS; many of them have elaborate dendritic arborizations that facilitate their identification. Central axonal branch and all axons in remaining cells are indicated in green. Cell Body surfaced endoplasmic reticulum (rER) and free ribosomes when observed with the transmission electron microscope The cell body of a neuron has characteristics of a protein (TEM), a feature consistent with its protein synthetic activity. producing cell.

1	The cell body of a neuron has characteristics of a protein (TEM), a feature consistent with its protein synthetic activity. producing cell. In the light microscope, the ribosomal content appears as small The cell body is the dilated region of the neuron that contains bodies called Nissl bodies that stain intensely with basic dyes a large, euchromatic nucleus with a prominent nucleolus and and metachromatically with thionine dyes (see Fig. 12.4a). surrounding perinuclear cytoplasm (Fig.12.4a, Plate 27, Each Nissl body corresponds to a stack of rER. The perinupage 390). The perinuclear cytoplasm reveals abundant rough- clear cytoplasm also contains numerous mitochondria, a large node of Ranvier nucleus of Schwann cell spinal nerve Pacinian corpuscle cell body of motor neuron cell body of sympathetic neuron smooth muscle and striated enteroceptors of ANS muscle

1	FIGURE 12.3 • Schematic diagram showing arrangement of motor and sensory neurons. The cell body of a motor neuron is located in the ventral (anterior) horn of the gray matter of the spinal cord. Its axon, surrounded by myelin, leaves the spinal cord via a ventral (anterior) root and becomes part of a spinal nerve that carries it to its destination on striated (skeletal) muscle fibers. The sensory neuron originates in the skin within a receptor (here, a Pacinian corpuscle) and continues as a component of a spinal nerve, entering the spinal cord via the dorsal (posterior) root. Note the location of its cell body in the dorsal root ganglion (sensory ganglion). A segment of the spinal nerve is enlarged to show the relationship of the nerve fibers to the surrounding connective tissue (endoneurium, perineurium, and epineurium). In addition, segments of the sensory, motor and autonomic unmyelinated neurons have been enlarged to show the relationship of the axons to the Schwann cells.

1	perinuclear Golgi apparatus, lysosomes, microtubules, neurofilaments (intermediate filaments), transport vesicles, and inclusions (Fig. 12.4b). Nissl bodies, free ribosomes, and occasionally the Golgi apparatus extend into the dendrites but not into the axon. This area of the cell body, called the axon hillock, lacks large cytoplasmic organelles and serves as a landmark to distinguish between axons and dendrites in both light microscope and TEM preparations. The euchromatic nucleus, large nucleolus, prominent Golgi apparatus, and Nissl bodies indicate the high level of anabolic activity needed to maintain these large cells. Neurons do not divide; however, in some areas of the brain, neural stem cells are present and are able to differentiate and replace damaged nerve cells.

1	Neurons do not divide; however, in some areas of the brain, neural stem cells are present and are able to differentiate and replace damaged nerve cells. Although neurons do not replicate, the subcellular components of the neurons turn over regularly and have life spans measured in hours, days, and weeks. The constant need to replace enzymes, neurotransmitter substances, membrane components, and other complex molecules is consistent with the morphologic features characteristic of a high level of synthetic activity. Newly synthesized protein molecules are transported to distant locations within a neuron in a process referred to as axonal transport (page 363).

1	It is generally accepted that nerve cells do not divide. However, recently it has been shown that the adult brain retains some cells that exhibit the potential to regenerate. In certain regions of the brain such as olfactory bulb and dentate gyrus of the hippocampus, these neural stem cells are able to divide and generate new neurons. They are characterized by prolonged expression of a 240-kDa intermediate filament protein nestin, which is used to identify these cells by histochemical methods. Neural stem cells are also able to migrate to sites of injury and differentiate into new nerve

1	FIGURE 12.4 • Nerve cell bodies. a. This photomicrograph shows a region of the ventral (anterior) horn of a human spinal cord stained with toluidine blue. Typical features of the nerve cell bodies visible in this image include large, spherical, pale-stained nuclei with a single prominent nucleolus and abundant Nissl bodies within the cytoplasm of the nerve cell body. Most of the small nuclei belong to neuroglial cells. The remainder of the field consists of nerve fibers and cytoplasm of cental neuroglial cells. 640. b. Electron micrograph of a nerve cell body. The cytoplasm is occupied by aggregates of free ribosomes and profiles of rough-surfaced endoplasmic reticulum (rER) that constitute the Nissl bodies of light microscopy. The Golgi apparatus (G) appears as isolated areas containing profiles of flattened sacs and vesicles. Other characteristic organelles include mitochondria (M) and lysosomes (L). The neurofilaments and neurotubules are difficult to discern at this relatively low

1	of flattened sacs and vesicles. Other characteristic organelles include mitochondria (M) and lysosomes (L). The neurofilaments and neurotubules are difficult to discern at this relatively low magnification. 15,000.

1	cells. Research studies on the animal model demonstrate that newly generated cells mature into functional neurons in the adult mammalian brain. These findings may lead to therapeutic strategies that use neural cells to replace nerve cells lost or damaged by neurodegenerative disorders such as Alzheimer and Parkinson diseases. Dendrites are receptor processes that receive stimuli from other neurons or from the external environment.

1	Dendrites are receptor processes that receive stimuli from other neurons or from the external environment. The main function of dendrites is to receive information from other neurons or from the external environment and carry that information to the cell body. Generally, dendrites are located in the vicinity of the cell body. They have a greater diameter than axons, are unmyelinated, are usually tapered, and form extensive arborizations called dendritic trees. Dendritic trees significantly increase the receptor surface area of a neuron. Many neuron types are characterized by the extent and shape of their dendritic trees (see Fig. 12.2). In general, the contents of the perinuclear cytoplasm of the cell body and cytoplasm of dendrites are similar, with the exception of the Golgi apparatus. Other organelles characteristic of the cell body, including ribosomes and rER, are found in the dendrites, especially in the base of the dendrites.

1	Axons are effector processes that transmit stimuli to other neurons or effector cells. The main function of the axon is to convey information away from the cell body to another neuron or to an effector cell, such as a muscle cell. Each neuron has only one axon, and it may be extremely long. Axons that originate from neurons in the motor nuclei of the CNS (Golgi type I neurons) may travel more than a meter to reach their effector targets, skeletal muscle. In contrast, interneurons of the CNS (Golgi type II neurons) have very short axons. Although an axon may give rise to a recurrent branch near the cell body (i.e., one that turns back toward the cell body) and to other collateral branches, the branching of the axon is most extensive in the vicinity of its targets. The axon originates from the axon hillock. The axon hillock usually lacks large cytoplasmic organelles such as Nissl  FOLDER 12.1 Clinical Correlation: Parkinson’s Disease

1	Parkinson’s disease is a slowly progressive neurologic disorder caused by the loss of dopamine (DA)-secreting cells in the substantia nigra and basal ganglia of the brain. DA is a neurotransmitter responsible for synaptic trans -mission in the nerve pathways coordinating smooth and focused activity of skeletal muscles. Loss of DA-secreting cells is associated with a classic pattern of symptoms, including the following:  Resting tremor in the limb, especially of the hand when in a relaxed position; tremor usually increases during stress and is often more severe on one side of the body  Rigidity or increased tone (stiffness) in all muscles  Slowness of movement (bradykinesia) and inability to initiate movement (akinesia)  Lack of spontaneous movements  Loss of postural reflexes, which leads to poor balance and abnormal walking (festinating gait)  Slurred speech, slowness of thought, and small, cramped handwriting The cause of idiopathic Parkinson’s disease, in which DA-secreting

1	leads to poor balance and abnormal walking (festinating gait)  Slurred speech, slowness of thought, and small, cramped handwriting The cause of idiopathic Parkinson’s disease, in which DA-secreting neurons in the substantia nigra are damaged and lost by degeneration or apoptosis, is not known. However, some evidence suggests a hereditary predisposition; about 20% of Parkinson’s patients have a family member with similar symptoms. Symptoms that resemble idiopathic Parkinson’s dis-ease may also result from infections (e.g., encephalitis), toxins (e.g., MPTP), drugs used in the treatment of neuro-logic disorders (e.g., neuroleptics used to treat schizophre-nia), and repetitive trauma. Symptoms with these causes are called secondary parkinsonism. On the microscopic level, degeneration of neurons in the substantia nigra is very evident. This region loses its typical pigmentation, and an increase in the number of glial cells is noticeable (gliosis). In addition, nerve cells in this region

1	in the substantia nigra is very evident. This region loses its typical pigmentation, and an increase in the number of glial cells is noticeable (gliosis). In addition, nerve cells in this region display characteristic intracellular inclusions called Lewy bodies, which represent accumulation of intermedi-ate neurofilaments in association with proteins -synuclein and ubiquitin. Treatment of Parkinson’s disease is primarily symp-tomatic and must strike a balance between relieving symp-toms and minimizing psychotic side effects. L-Dopa is a precursor of DA that can cross the blood–brain barrier and is then converted to DA. It is often the primary agent used to treat Parkinson’s disease. Other drugs that are used in-clude a group of cholinergic receptor blockers and aman-tadine, a drug that stimulates release of DA from neurons. If drug therapies are not effective, several surgical op-tions can be considered. Stereotactic surgery, in which nuclei in selective areas of the brain (globus

1	stimulates release of DA from neurons. If drug therapies are not effective, several surgical op-tions can be considered. Stereotactic surgery, in which nuclei in selective areas of the brain (globus pallidus, thalamus) are destroyed by a thermocoagulative probe in-serted into the brain, can be effective in some cases. Sev-eral new surgical procedures are being developed and are still in experimental stages. These include transplantation of DA-secreting neurons into the substantia nigra to replace lost neurons.

1	bodies and Golgi cisternae. Microtubules, neurofilaments, mitochondria, and vesicles, however, pass through the axon hillock into the axon. The region of the axon between the apex of the axon hillock and the beginning of the myelin sheath (see below) is called the initial segment. The initial segment is the site at which an action potential is generated in the axon. The action potential (described in more detail below) is stimulated by impulses carried to the axon hillock on the membrane of the cell body after other impulses are received on the dendrites or the cell body itself. Some large axon terminals are capable of local protein synthesis, which may be involved in memory processes.

1	Some large axon terminals are capable of local protein synthesis, which may be involved in memory processes. Almost all of the structural and functional protein molecules are synthesized in the nerve cell body. These molecules are distributed to the axons and dendrites via axonal transport systems (described on page 363). However, contrary to the common view that the nerve cell body is the only site of protein synthesis, recent studies indicate that local synthesis of axonal proteins takes place in some large nerve terminals. Some vertebral axon terminals (i.e., from the retina) contain polyribosomes with complete translational machinery for protein synthesis. These discrete areas within the axon terminals, called periaxoplasmic plaques, possess biochemical and molecular characteristics of active protein synthesis. Protein synthesis within the periaxoplasmic plaques is modulated by neuronal activity. These proteins may be involved in the processes of neuronal cell memory.

1	Neurons communicate with other neurons and with effector cells by synapses. Synapses are specialized junctions between neurons that facilitate the transmission of impulses from one (presynaptic) neuron to another (postsynaptic) neuron. Synapses also occur between axons and effector (target) cells, such as muscle and gland cells. Synapses between neurons may be classified morphologically as:  axodendritic, occurring between axons and dendrites;  axosomatic, occurring between axons and the cell body; or  axoaxonic, occurring between axons and axons (Fig. 12.5).

1	Synapses are not resolvable in routine hematoxylin and eosin (H&E) preparations. However, silver precipitation staining methods (e.g., Golgi method) not only demonstrate the overall shape of some neurons but also show synapses as oval bodies on the surface of the receptor neuron. Typically, an axon makes several of these buttonlike contacts with the receptor portion of the neuron. Often, the incoming neuron travels along the surface of the neuron, making several synaptic contacts called boutons en passant (Fr., buttons in passing). The axon then continues, ending finally as a terminal branch with an enlarged tip, a bouton terminal (Fr., terminal button), or end bulb. The number of synapses on a neuron or its processes, which may vary from a few to tens of thousands per neuron (Fig. 12.6), appears to be directly related to the number of impulses that a neuron is receiving and processing.

1	FIGURE 12.5 • Schematic diagram of different types of synapses. a. Axodendritic or axosomatic. b. Axodendritic, in which an axon terminal synapses with a dendritic spine. c. Axoaxonic. The axoaxonic synapse may enhance or inhibit the axodendritic (or axosomatic) synapse. (Modified from Barr ML. The Human Nervous System. New York: Harper & Row, 1979.) Synapses are classified as chemical or electrical. Classification depends on the mechanism of conduction of the nerve impulses and the way the action potential is gener-ated in the target cells. Thus, synapses may also be classified as the following.

1	FIGURE 12.6 • Scanning electron micrograph of the nerve cell body. This micrograph shows the cell body of a neuron. Axon endings forming synapses are visible as are numerous oval bodies with tail-like appendages. Each oval body represents axon terminal from different neuron making contact with the cell body. 76,000. (Courtesy of Dr. George Johnson.)  Chemical synapses: Conduction of impulses is achieved by the release of chemical substances (neurotransmitters) from the presynaptic neuron. Neurotransmitters then diffuse across the narrow intercellular space that separates the presynaptic neuron from the postsynaptic neuron or target cell.

1	 Electrical synapses: Common in invertebrates, these synapses contain gap junctions that permit movement of ions between cells and consequently permit the direct spread of electrical current from one cell to another. These synapses do not require neurotransmitters for their function. Mammalian equivalents of electrical synapses include gap junctions in smooth muscle and cardiac muscle cells. A typical chemical synapse contains a presynaptic element, synaptic cleft, and postsynaptic membrane. Components of a typical chemical synapse include the following.

1	Components of a typical chemical synapse include the following.  A presynaptic element (presynaptic knob, presynaptic component, or synaptic bouton) is the end of the neuron process from which neurotransmitters are released. The presynaptic element is characterized by the presence of synaptic vesicles, membrane-bound structures that range from 30 to 100 nm in diameter and contain neurotransmitters (Fig. 12.7). The binding and fusion of synaptic vesicles to the presynaptic plasma membrane is mediated by a family of transmembrane proteins called SNAREs (which stands for “soluble NSF attachment receptors”), (see page 34). The specific SNARE proteins involved in this activity are known as v-SNARE (vesicle–bound) proteins and t-SNARE (targetmembrane–bound found in specialized areas of the presynaptic membrane). Another vesicle-bound protein called synaptotagmin 1 then replaces the SNARE complex, which is subsequently dismantled and recycled by

1	FIGURE 12.7 • Diagram of a chemical axodendritic synapse. This diagram illustrates three components of a typical synapse. The presynaptic knob is located at the distal end of the axon from which neurotransmitters are released. The presynaptic element of the axon is characterized by the presence of numerous neurotransmitter-containing synaptic vesicles. The plasma membrane of the presynaptic knob is recycled by the formation of clathrin-coated endocytotic vesicles. The synaptic cleft separates the presynaptic knob of the axon from the postsynaptic membrane of the dendrite. The postsynaptic membrane of the dendrite is frequently characterized by a postsynaptic density and contains receptors with an affinity for the neurotransmitters. Note two types of receptors: Green-colored molecules represent transmitter-gated channels, and the purple-colored structure represents a G-protein–coupled receptor that, when bound to a neurotransmitter, may act on G-protein–gated ion channels or on enzymes

1	transmitter-gated channels, and the purple-colored structure represents a G-protein–coupled receptor that, when bound to a neurotransmitter, may act on G-protein–gated ion channels or on enzymes producing a second messenger. a. Diagram showing the current view of neurotransmitter release from a presynaptic knob by a fusion of the synaptic vesicles with presynaptic membrane. b. Diagram showing a new proposed model of the neurotransmitter release via porocytosis. In this model, the synaptic vesicle is anchored and juxtaposed to calcium-selective channels in the presynaptic membrane. In the presence of Ca2, the bilayers of the vesicle and presynaptic membranes are reorganized to create a 1-nm transient pore connecting the lumen of the vesicle, with the synaptic cleft allowing the release of a neurotransmitter. Note the presence of the SNARE complex and the synaptotagmin that anchor the vesicle to the active zones within plasma membrane of the presynaptic element.

1	presynaptic element of axon postsynaptic membrane of dendrite NSF/SNAP25 protein complexes. Dense accumulations of proteins are present on the cytoplasmic side of the presynaptic plasma membrane. These presynaptic densities represent specialized areas called active zones where synaptic vesicles are docked and where neurotransmitters are released. Active zones are rich in Rab-GTPase docking complexes (see page 35), t-SNAREs and synaptotagmin binding proteins. The vesicle membrane that is added to the presynaptic membrane is retrieved by endocytosis and reprocessed into synaptic vesicles by the smooth-surfaced endoplasmic reticulum (sER) located in the nerve ending. Numerous small mitochondria are also present in the presynaptic element.  The synaptic cleft is the 20to 30-nm space that separates the presynaptic neuron from the postsynaptic neuron or target cell, which the neurotransmitter must cross.

1	 The synaptic cleft is the 20to 30-nm space that separates the presynaptic neuron from the postsynaptic neuron or target cell, which the neurotransmitter must cross.  The postsynaptic membrane (postsynaptic component) contains receptor sites with which the neurotransmitter interacts. This component is formed from a portion of the plasma membrane of the postsynaptic neuron (Fig. 12.8) and is characterized by an underlying layer of dense material. This postsynaptic density represents an elaborate complex of interlinked proteins that serve numerous functions such as translation of the neurotransmitter–receptor interaction into an intracellular signal, anchoring of and trafficking neurotransmitter receptors to the plasma membrane, and anchoring various proteins that modulate receptor activity. Voltage-gated Ca2 channels in the presynaptic membrane regulate transmitter release.

1	Voltage-gated Ca2 channels in the presynaptic membrane regulate transmitter release. When a nerve impulse reaches the synaptic bouton, the voltage reversal across the membrane produced by the impulse (called depolarization) causes voltage-gated Ca2

1	FIGURE 12.8 • Electron micrograph of nerve processes in the cerebral cortex. A synapse can be seen in the center of the micrograph, where an axon ending is apposed to a dendrite. The ending of the axon exhibits numerous neurotransmitter-containing synaptic vesicles that appear as circular profiles. The postsynaptic membrane of the dendrite shows a postsynaptic density. A substance of similar density is also present in the synaptic cleft (intercellular space) at the synapse. 76,000. (Courtesy of Drs. George D. Pappas and Virginia Kriho.) channels to open in the plasma membrane of the bouton. The influx of Ca2 from the extracellular space causes the synaptic vesicles to migrate, anchor, and fuse with the presynaptic membrane, thereby releasing the neurotransmitter into the synaptic cleft by exocytosis. Vesicle docking and fusion is mainly driven by the actions of SNARE and synaptotagmin proteins. Alternative to the massive release of neurotransmitter following vesicle fusion is the

1	by exocytosis. Vesicle docking and fusion is mainly driven by the actions of SNARE and synaptotagmin proteins. Alternative to the massive release of neurotransmitter following vesicle fusion is the process of porocytosis, in which vesicles anchored at the active zones release neurotransmitters through a transient pore connecting the lumen of the vesicle with the synaptic cleft. The neurotransmitter then diffuses across the synaptic cleft. At the same time, the presynaptic membrane of the synaptic bouton that released the neurotransmitter quickly forms endocytotic vesicles that return to the endosomal compartment of the bouton for recycling or reloading with neurotransmitter.

1	The neurotransmitter binds to either transmitter-gated channels or G-protein–coupled receptors on the postsynaptic membrane. The released neurotransmitter molecules bind to the extracellular part of postsynaptic membrane receptors called transmitter-gated channels. Binding of neurotransmitter induces a conformational change in these channel proteins that causes its pore to open. The response that is ultimately generated depends on the identity of the ion that enters the cell. For instance, influx of Na causes local depolarization in the postsynaptic membrane, which under favorable conditions (sufficient amount and duration of neurotransmitter release) prompts the opening of voltage-gated Na channels, thereby generating a nerve impulse.

1	Some amino acid and amine neurotransmitters may bind to G-protein–coupled receptors to produce longer-lasting and more diverse postsynaptic responses. The neurotransmitter binds to a transmembrane receptor protein on the postsynaptic membrane. Receptor binding activates G-proteins, which move along the intracellular surface of the postsynaptic membrane and eventually activate effector proteins. These effector proteins may include transmembrane G-protein–gated ion channels or enzymes that synthesize second-messenger molecules (page 335). Several neurotransmitters (i.e., acetylcholine) can generate different postsynaptic actions, depending on which receptor system they act (see below). Porocytosis describes the secretion of neurotransmitter that does not involve the fusion of synaptic vesicles with the presynaptic membrane.

1	Porocytosis describes the secretion of neurotransmitter that does not involve the fusion of synaptic vesicles with the presynaptic membrane. Based on evaluation of physiologic data and the structural organization of nerve synapses, an alternate model of neurotransmitter secretion called porocytosis has recently been proposed to explain the regulated release of neurotransmitters. In this model, secretion from the vesicles occurs without fusion of the vesicle membrane with the presynaptic membrane. Instead, the synaptic vesicle is anchored to the presynaptic membrane next to Ca2-selective channels by SNARE and synaptotagmin proteins. In the presence of Ca2, the vesicle and presynaptic membranes are reorganized to create a 1-nm transient pore connecting the lumen of the vesicle with the synaptic cleft. Neurotransmitters can then be released in a controlled fashion through these transient membrane pores (see Fig. 12.7).

1	The chemical nature of the neurotransmitter determines the type of response at that synapse in the generation of neuronal impulses. The release of neurotransmitter by the presynaptic component can cause either excitation or inhibition at the postsynaptic membrane.  In excitatory synapses, release of neurotransmitters such as acetylcholine, glutamine, or serotonin opens channels), prompting an influx of Na that causes local reversal of voltage of the postsynaptic membrane to a threshold level (depolarization). This leads to initiation of an action potential and generation of a nerve impulse.  In inhibitory synapses, release of neurotransmitters such as -aminobutyric acid (GABA) or glycine opens transmitter-gated Cl channels (or other anion channels), causing Cl– to enter the cell and hyperpolarize the postsynaptic membrane, making it even more negative. In these synapses, the generation of an action potential then becomes more difficult.

1	The ultimate generation of a nerve impulse in a postsynaptic neuron (firing) depends on the summation of excitatory and inhibitory impulses reaching that neuron. This allows precise regulation of the reaction of a postsynaptic neuron (or muscle fiber or gland cell). The function of synapses is not simply to transmit impulses in an unchanged manner from one neuron to another. Rather, synapses allow for the processing of neuronal input. Typically, the impulse passing from the presynaptic to the postsynaptic neuron is modified at the synapse by other neurons that, although not in the direct pathway, nevertheless have access to the synapse (see Fig. 12.5). These other neurons may influence the membrane of the presynaptic neuron or the postsynaptic neuron and facilitate or inhibit the transmission of impulses. The firing of impulses in the postsynaptic neuron is caused by the summation of the actions of hundreds of synapses.

1	A number of molecules that serve as neurotransmitters have been identified in various parts of the nervous system. The most common neurotransmitters are the following.

1	 Acetylcholine (ACh). ACh is the neurotransmitter between axons and striated muscle at the neuromuscular junction (see page 322) and serves as a neurotransmitter in the ANS. ACh is released by the presynaptic sympathetic and parasympathetic neurons and their effectors. ACh is also secreted by postsynaptic parasympathetic neurons, as well as by a specific type of postsynaptic sympathetic neuron that innervates sweat glands. Neurons that use ACh as their neurotransmitter are called cholinergic neurons. The receptors for ACh in the postsynaptic membrane are known as cholinergic receptors and are divided into two classes on the basis of their interactions with muscarine, a substance isolated from poisonous mushrooms (muscarinic ACh receptor), and nicotine, isolated from tobacco plants (nicotinic ACh receptor). The muscarinic ACh receptor in the heart is an example of a G-protein–coupled receptor that is linked to K channels. ACh release stimulated by parasympathetic stimulation of the

1	ACh receptor). The muscarinic ACh receptor in the heart is an example of a G-protein–coupled receptor that is linked to K channels. ACh release stimulated by parasympathetic stimulation of the heart opens K channels, causing hyperpolarization of cardiac muscle fibers. This hyperpolarization slows rhythmic contraction of the heart. In contrast, the nicotinic ACh receptor in skeletal muscles is a transmitter-gated Na channel. Opening of this channel causes rapid depolarization of skeletal muscle fibers and initiation of contraction. Various drugs affect the release of ACh into the synaptic cleft as well as its binding to its receptors. For instance, curare, the South American arrow-tip poison—binds to Na channels and blocks the action of nicotinic ACh receptors, causing muscle paralysis. Atropin, an alkaloid extracted from the belladonna plant, blocks the action of muscarinic ACh receptors. The botulinum toxin produced by Clostridium botulinum that grows in improperly canned meat and

1	Atropin, an alkaloid extracted from the belladonna plant, blocks the action of muscarinic ACh receptors. The botulinum toxin produced by Clostridium botulinum that grows in improperly canned meat and vegetable products inhibits ACh release. Inhibition of ACh release decreases receptor stimulation and leads to paralysis of skeletal muscles, including respiratory muscles.

1	 Catecholamines such as norepinephrine (NE), epinephrine (EPI, adrenaline), and dopamine (DA). These neurotransmitters are synthesized in a series of enzymatic reactions from the amino acid tyrosine. Neurons that use catecholamines as their neurotransmitter are called catecholaminergic neurons. Catecholamines are secreted by cells in the CNS that are involved in the regulation of movement, mood, and attention. Neurons that utilize epinephrine (adrenaline) as their neurotransmitter are called adrenergic neurons. They all contain an enzyme that converts NE to adrenaline (EPI), which serves as a transmitter between postsynaptic sympathetic axons and effectors in the ANS. EPI is also released into the bloodstream by the endocrine cells (chromaffin cells) of the adrenal medulla during the fight-or-ﬂight response.

1	 Serotonin or 5-hydroxytryptamine (5-HT). Serotonin is formed by the hydroxylation and decarboxylation of tryptophan. It functions as a neurotransmitter in neurons of the CNS and enteric nervous system. Neurons that use serotonin as their neurotransmitter are called serotonergic. After the release of serotonin, a portion is recycled by reuptake into presynaptic serotonergic neurons.  Amino acids such as -aminobutyrate (GABA), glutamate (GLU), aspartate (ASP), and glycine (GLY) also act as neurotransmitters, mainly in the CNS.

1	 Amino acids such as -aminobutyrate (GABA), glutamate (GLU), aspartate (ASP), and glycine (GLY) also act as neurotransmitters, mainly in the CNS.  Nitric oxide (NO), a simple gas with free radical properties, also has been identified as a neurotransmitter. At low concentrations, NO carries nerve impulses from one neuron to another. Unlike other neurotransmitters, which are synthesized in the nerve cell body and stored in synaptic vesicles, NO is synthesized within the synapse and used immediately. It is postulated that excitatory neurotransmitter GLU induces a chain reaction in which NO synthase is activated to produce NO, which in turn diffuses from the presynaptic knob via the synaptic cleft and post-synaptic membrane to the adjacent cell, resulting ultimately in generation of an action potential.

1	 Small peptides have also been shown to act as synaptic transmitters. Among these are substance P (so named because it was originally found in a powder of acetone extracts of brain and intestinal tissue), hypothalamic releasing hormones, enkephalins, vasoactive intestinal peptide (VIP), cholecystokinin (CCK), and neurotensin. Many of these same substances are synthesized and released by enteroendocrine cells of the intestinal tract. They may act immediately on neighboring cells (paracrine secretion) or be carried in the bloodstream as hormones to act on distant target cells (endocrine secretion). They are also synthesized and released by endocrine organs and by the neurosecretory neurons of the hypothalamus. Neurotransmitters released into the synaptic cleft may be degraded or recaptured.

1	The degradation or recapture of neurotransmitters is necessary to limit the duration of stimulation or inhibition of the post-synaptic membrane. The most common process of neurotransmitter removal after its release into the synaptic cleft is called high-affnity reuptake. About 80% of released neurotransmitters are removed by this mechanism, in which they are bound into specifc neurotransmitter transport proteins located in the presynaptic membrane. Neurotransmitters that were transported into the cytoplasm of the presynaptic bouton are either enzymatically destroyed or reloaded into empty synaptic vesicles. For example, the action of catecholamines on postsynaptic receptors is terminated by the reuptake of neurotransmitters into the presynaptic bouton utilizing Nadependent transporters. The efficiency of this uptake can be regulated by several pharmacologic agents such as amphetamine and cocaine, which block catecholamine reuptake and prolong the actions of neurotransmitters on the

1	The efficiency of this uptake can be regulated by several pharmacologic agents such as amphetamine and cocaine, which block catecholamine reuptake and prolong the actions of neurotransmitters on the postsynaptic neurons. Once inside the presynaptic bouton, catecholamines are reloaded into synaptic vesicles for future use. The excess of catecholamines is inactivated by the enzyme catechol O-methyltransferase (COMT) or is destroyed by another enzyme found on the outer mitochondrial membrane, monoamine oxidase (MAO). Therapeutic substances that inhibit the action of MAO are frequently used in the treatment of clinical depression; selective COMT inhibitors have been also developed.

1	Enzymes associated with the postsynaptic membrane degrade the remaining 20% of neurotransmitters. For example, acetylcholinesterase (AChE), which is secreted by the muscle cell into the synaptic cleft, rapidly degrades ACh into acetic acid and choline. Choline is then taken up by the cholinergic presynaptic bouton and reused for ACh synthesis. The AChE action at the neuromuscular junction can be inhibited by various pharmacological compounds, nerve agents, and pesticides resulting in prolonged muscle contraction. Clinically, AChE inhibitors have been used clinically in the treatment of myasthenia gravis (see Folder 11.4 in Chapter 11), a degenerative neuromuscular disorder, glaucoma, and more recently Alzheimer’s disease. Substances needed in the axon and dendrites are synthesized in the cell body and require transport to those sites.

1	Substances needed in the axon and dendrites are synthesized in the cell body and require transport to those sites. Most neurons possess elaborate axonal and dendritic processes. Because the synthetic activity of the neuron is concentrated in the nerve cell body, axonal transport is required to convey newly synthesized material to the processes. Axonal transport is a bidirectional mechanism. It serves as a mode of intracellular communication, carrying molecules and information along the microtubules and intermediate filaments from the axon terminal to the nerve cell body and from the nerve cell body to the axon terminal. Axonal transport is described as the following:  Anterograde transport carries material from the nerve cell body to the periphery. Kinesin, a microtubule-associated motor protein that uses ATP, is involved in anterograde transport (see page 59).

1	 Retrograde transport carries material from the axon terminal and the dendrites to the nerve cell body. This transport is mediated by another microtubule-associated motor protein, dynein (see page 59). The transport systems may also be distinguished by the rate at which substances are transported:  A slow transport system conveys substances from the cell body to the terminal bouton at the speed of 0.2 to 4 mm/day. It is only an anterograde transport system. Structural elements such as tubulin molecules (microtubule precursors), actin molecules, and the proteins that form neurofilaments are carried from the nerve cell body by the slow transport system. So, too, are cytoplasmic matrix proteins such as actin, calmodulin, and various metabolic enzymes.

1	 A fast transport system conveys substances in both directions at a rate of 20 to 400 mm/day. Thus, it is both an anterograde and a retrograde system. The fast anterograde transport system carries to the axon terminal different membrane-limited organelles, such as sER components, synaptic vesicles, and mitochondria, and low-molecular-weight materials such as sugars, amino acids, nucleotides, some neurotransmitters, and calcium. The fast retrograde transport system carries to the nerve cell body many of the same materials as well as proteins and other molecules endocytosed at the axon terminal. Fast transport in either direction requires ATP, which is used by microtubuleassociated motor proteins, and depends on the microtubule arrangement that extends from the nerve cell body to the termination of the axon. Retrograde transport is the pathway followed by toxins and viruses that enter the CNS at nerve endings. Retrograde transport of exogenous enzymes, such as horseradish peroxidase,

1	of the axon. Retrograde transport is the pathway followed by toxins and viruses that enter the CNS at nerve endings. Retrograde transport of exogenous enzymes, such as horseradish peroxidase, and of radiolabeled or immunolabeled tracer materials is now used to trace neuronal pathways and to identify the nerve cell bodies related to specific nerve endings.

1	Dendritic transport appears to have the same characteris tics and to serve the same functions for the dendrite as axonal transport does for the axon. SUPPORTING CELLS OF THE NERVOUS SYSTEM: THE NEUROGLIA In the PNS, supporting cells are called peripheral neuroglia; in the CNS, they are called central neuroglia. Peripheral neuroglia include Schwann cells, satellite cells, and a variety of other cells associated with specific organs or tissues. Examples of the latter include terminal neuroglia (teloglia), which are associated with the motor end plate; enteric neuroglia associated with the ganglia located in the wall of the alimentary canal; and Mler’s cells in the retina. Schwann Cells and the Myelin Sheath In the PNS, Schwann cells produce the myelin sheath.

1	Schwann Cells and the Myelin Sheath In the PNS, Schwann cells produce the myelin sheath. The main function of Schwann cells is to support myelinated and unmyelinated nerve cell fibers. Schwann cells develop from neural crest cells and differentiate by expressing transcription factor Sox-10. In the PNS, Schwann cells produce a lipid-rich layer called the myelin sheath that surrounds the axons (Fig. 12.9). The myelin sheath isolates the axon from the surrounding extracellular compartment of endoneurium. Its presence ensures the rapid conduction of nerve impulses. The axon hillock and the terminal arborizations where the axon synapses with its target cells are not covered by myelin. Unmyelinated fibers are also enveloped and nurtured by Schwann cell cytoplasm. In addition, Schwann cells aid in cleaning up PNS debris and guide the regrowth of PNS axons. Myelination begins when a Schwann cell surrounds the axon and its cell membrane becomes polarized.

1	Myelination begins when a Schwann cell surrounds the axon and its cell membrane becomes polarized. During formation of the myelin sheath (also called myelination), the axon initially lies in a groove on the surface of the Schwann cell (Fig. 12.10a). A 0.08to 0.1-mm segment of the axon then becomes enclosed within each Schwann cell that lies along the axon. The Schwann cell surface becomes polarized into two functionally distinct membrane domains. The part of the Schwann cell membrane that is exposed to the external environment or endoneurium, the abaxonal plasma membrane, represents one domain. The other domain is represented by the adaxonal or periaxonal plasma membrane, which is in direct contact with the axon. When the axon is completely enclosed by the Schwann cell membrane, a third domain, the mesaxon, is created (Fig. 12.10b). This third domain is a double membrane that connects the abaxonal and adaxonal membranes and encloses the narrow extracellular space.

1	The myelin sheath develops from compacted layers of Schwann cell mesaxon wrapped concentrically around the axon. Myelin sheath formation is initiated when the Schwann cell mesaxon surrounds the axon. A sheetlike extension of the mesaxon then wraps around the axon in a spiraling motion. The first few layers or lamellae of the spiral are not compactly

1	FIGURE 12.9 • Photomicrographs of a peripheral nerve in cross and longitudinal sections. a. Photomicrograph of an osmium-fixed, toluidine blue–stained peripheral nerve cut in cross-section. The axons (A) appear clear. The myelin is represented by the dark ring surrounding the axons. Note the variation in diameter of the individual axons. In some of the nerves, the myelin appears to consist of two separate rings (asterisks). This is caused by the section passing through a Schmidt-Lanterman cleft. Epi, epineurium. 640. b. Photomicrograph showing longitudinally sectioned myelinated nerve axons (A) in the same preparation as above. A node of Ranvier (NR) is seen near the center of the micrograph. In the same axon, a Schmidt-Lanterman cleft (SL) is seen on each side of the node. In addition, a number of Schmidt-Lanterman clefts can be seen in the adjacent axons. The perinodal cytoplasm of the Schwann cell at the node of Ranvier and the Schwann cell cytoplasm at the Schmidt-Lanterman cleft

1	number of Schmidt-Lanterman clefts can be seen in the adjacent axons. The perinodal cytoplasm of the Schwann cell at the node of Ranvier and the Schwann cell cytoplasm at the Schmidt-Lanterman cleft appear virtually unstained. 640.

1	FIGURE 12.10 • Diagram showing successive stages in the formation of myelin by a Schwann cell. a. The axon initially lies in a groove on the surface of the Schwann cell. b. The axon is surrounded by a Schwann cell. Note the two domains of the Schwann cell, the adaxonal plasma-membrane domain and abaxonal plasma-membrane domain. The mesaxon plasma membrane links these domains. The mesaxon membrane initiates myelination by surrounding the embedded axon. c. A sheetlike extension of the mesaxon membrane then wraps around the axon, forming multiple membrane layers. d. During the wrapping process, the cytoplasm is extruded from between the two apposing plasma membranes of the Schwann cell, which then become compacted to form myelin. The outer mesaxon represents invaginated plasma membrane extending from the abaxonal surface of the Schwann cell to the myelin. The inner mesaxon extends from the adaxonal surface of the Schwann cell (the part facing the axon) to the myelin. The inset shows the

1	from the abaxonal surface of the Schwann cell to the myelin. The inner mesaxon extends from the adaxonal surface of the Schwann cell (the part facing the axon) to the myelin. The inset shows the major proteins responsible for compaction of the myelin sheath. MBP, myelin basic protein; Nrq1, neurorequlin; P0, protein 0; PMP22, peripheral myelin protein of 22 kilodaltons.

1	arranged—that is, some cytoplasm is left in the first few concentric layers (Fig. 12.10c). The TEM reveals the presence of a 12to 14-nm gap between the outer (extracellular) leaflets and the Schwann cell cytoplasm that separates the inner (cytoplasmic) leaflets. As the wrapping progresses, cytoplasm is squeezed out from between the membrane of the concentric layers of the Schwann cell.

1	External to, and contiguous with, the developing myelin sheath is a thin outer collar of perinuclear cytoplasm called the sheath of Schwann. This part of the cell is enclosed by an abaxonal plasma membrane and contains the nucleus and most of the organelles of the Schwann cell. Surrounding the Schwann cell is a basal or external lamina. The apposition of the mesaxon of the last layer to itself as it closes the ring of the spiral produces the outer mesaxon, the narrow intercellular space adjacent to the external lamina. Internal to the concentric layers of the developing myelin sheath is a narrow inner collar of Schwann cell cytoplasm surrounded by the adaxonal plasma membrane. The narrow intercellular space between mesaxon membranes communicates with the adaxonal plasma membrane to produce the inner mesaxon (Fig. 12.10d).

1	Once the mesaxon spirals on itself, the 12to 14-nm gaps disappear and the membranes form the compact myelin sheath. Compaction of the sheath corresponds with the expression of transmembrane myelin-specifc proteins such as protein 0 (P0), a peripheral myelin protein of 22 kilodaltons (PMP22),and myelin basic protein (MBP). The inner (cytoplasmic) leaflets of the plasma membrane come close together as a result of the positively charged cytoplasmic domains of P0 and MBP. With the TEM, these closely aligned inner leaflets are electron opaque, appearing as the major dense lines in the TEM image of myelin (Fig. 12.10d). The concentric dense lamellae alternate with the slightly less dense intraperiod lines that are formed by closely apposed, but not fused, outer (extracellular) membrane leaflets. The narrow 2.5-nm gap corresponds to the remaining extracellular space containing the extracellular domains of P0 protein (Fig. 12.10d). P0 is a 30-kilodalton cell adhesion molecule expressed within

1	The narrow 2.5-nm gap corresponds to the remaining extracellular space containing the extracellular domains of P0 protein (Fig. 12.10d). P0 is a 30-kilodalton cell adhesion molecule expressed within the mesoaxial plasma membrane during myelination. This transmembrane glycoprotein mediates strong adhesions between the two opposite membrane layers and represents a key structural component of peripheral nerve myelin. Structural and genetic studies indicate that mutations in human genes encoding P0 produce unstable myelin and may contribute to the development of demyelinating diseases (see Folder 12.2).

1	In general, demyelinating diseases are characterized by preferential damage to the myelin sheath. Clinical symp-toms of these diseases are related to decreased or lost ability to transmit electrical impulses along nerve fibers. Several immune-mediated diseases affect the myelin sheath. Guillain-Barré syndrome, known also as acute in-fammatory demyelinating polyradiculoneuropathy, is one of the most common life-threatening diseases of the PNS. Microscopic examination of nerve fibers obtained from patients affected by this disease shows a large accu-mulation of lymphocytes, macrophages, and plasma cells around nerve fibers within nerve fascicles. Large segments of the myelin sheath are damaged, leaving the axons ex-posed to the extracellular matrix. These findings are consis-tent with a T cell–mediated immune response directed against myelin, which causes its destruction and slows or blocks nerve conduction. Patients exhibit symptoms of as-cending muscle paralysis, loss of muscle

1	a T cell–mediated immune response directed against myelin, which causes its destruction and slows or blocks nerve conduction. Patients exhibit symptoms of as-cending muscle paralysis, loss of muscle coordination, and loss of cutaneous sensation. Multiple sclerosis (MS) is a disease that attacks myelin in the CNS. MS is also characterized by preferential damage to myelin, which becomes detached from the axon and is eventually destroyed. In addition, destruction of oligodendroglia, which are responsible for the synthesis and maintenance of myelin, occurs. The myelin basic protein appears to be the major autoimmune target in this disease. Chemical changes in the lipid and protein con-stituents of myelin produce irregular, multiple plaques throughout the white matter of the brain. Symptoms of MS depend on the area in the CNS in which myelin is dam-aged. MS is usually characterized by distinct episodes of neurologic deficits such as unilateral vision impairment, loss of cutaneous

1	of MS depend on the area in the CNS in which myelin is dam-aged. MS is usually characterized by distinct episodes of neurologic deficits such as unilateral vision impairment, loss of cutaneous sensation, lack of muscle coordination and movement, and loss of bladder and bowel control. Treatment of both diseases is related to diminishing the causative immune response by immunomodulatory therapy with interferon as well as by administrating adrenal steroids. For more severe, progressive forms, immunosup-pressive drugs may be used.

1	 FOLDER 12.2 Clinical Correlation: Demyelinating Diseases The thickness of the myelin sheath at myelination is determined by axon diameter and not by the Schwann cell. Myelination is an example of cell-to-cell communication in which the axon interacts with the Schwann cell. Experimental studies show that the number of layers of myelin is determined by the axon and not by the Schwann cell. Myelin sheath thickness is regulated by a growth factor called neuregulin (Ngr1) that acts on Schwann cells. Ngr1 is a transmembrane protein expressed on the axolemma (cell membrane) of the axon. The node of Ranvier represents the junction between two adjacent Schwann cells.

1	The node of Ranvier represents the junction between two adjacent Schwann cells. The myelin sheath is segmented because it is formed by numerous Schwann cells arrayed sequentially along the axon. The junction where two adjacent Schwann cells meet is devoid of myelin. This site is called the node of Ranvier. Therefore, the myelin between two sequential nodes of Ranvier is called an internodal segment (Plate 28, page 392).

1	Myelin is composed of about 80% lipids because, as the Schwann cell membrane winds around the axon, the cytoplasm of the Schwann cell, as noted, is extruded from between the opposing layers of the plasma membranes. Electron micrographs, however, typically show small amounts of cytoplasm in several locations (Figs. 12.11 and 12.12): The inner collar of Schwann cell cytoplasm, between the axon and the myelin; the Schmidt-Lanterman clefts, small islands within successive lamellae of the myelin; perinodal cytoplasm, at the node of Ranvier; and the outer collar of perinuclear cytoplasm, around the myelin (Fig. 12.13). These areas of cytoplasm are what light microscopists identified as the Schwann sheath. If one conceptually unrolls the Schwann cell process, as shown in Figure 12.14, its full extent can be appreciated, and the inner collar of Schwann cell cytoplasm can be seen to be continuous with the body of the Schwann cell through the Schmidt-Lanterman clefts and through the perinodal

1	extent can be appreciated, and the inner collar of Schwann cell cytoplasm can be seen to be continuous with the body of the Schwann cell through the Schmidt-Lanterman clefts and through the perinodal cytoplasm. Cytoplasm of the clefts contains lysosomes and occasional mitochondria and microtubules, as well as cytoplasmic inclusions, or dense bodies. The number of Schmidt-Lanterman clefts correlates with the diameter of the axon; larger axons have more clefts.

1	Unmyelinated axons in the peripheral nervous system are enveloped by Schwann cells and their external lamina. The nerves in the PNS that are described as unmyelinated are nevertheless enveloped by Schwann cell cytoplasm as shown in Figure 12.15. The Schwann cells are elongated in parallel to the long axis of the axons, and the axons fit into grooves in the surface of the cell. The lips of the groove may be open, exposing a portion of the axolemma of the axon to the adjacent external lamina of the Schwann cell, or the lips may be closed, forming a mesaxon. A single axon or a group of axons may be enclosed in a single invagination of the Schwann cell surface. Large Schwann cells in the PNS may have 20 or more grooves, each containing one or more axons. In the ANS, it is common for bundles of unmyelinated axons to occupy a single groove.

1	The neuronal cell bodies of ganglia are surrounded by a layer of small cuboidal cells called satellite cells. Although they form a complete layer around the cell body, only their nuclei are typically visible in routine H&E preparations (Fig. 12.16, a and b). In paravertebral and peripheral ganglia, neural cell

1	FIGURE 12.11 • Electron micrograph of an axon in the process of myelination. At this stage of development, the myelin (M) consists of about six membrane layers. The inner mesaxon (IM) and outer mesaxon (OM) of the Schwann cell (SC) represent parts of the mesaxon membrane. Another axon (see upper left A) is present that has not yet been embedded within a Schwann cell mesaxon. Other notable features include the Schwann cell basal (external) lamina (BL) and the considerable amount of Schwann cell cytoplasm associated with the myelination process. 50,000. (Courtesy of Dr. Stephen G. Waxman.) processes must penetrate between the satellite cells to establish a synapse (there are no synapses in sensory ganglia). They help to establish and maintain a controlled microenvironment around the neuronal body in the ganglion, providing electrical insulation as well as a pathway for metabolic exchanges. Thus, the functional role of the satellite cell is analogous to that of the Schwann cell except

1	body in the ganglion, providing electrical insulation as well as a pathway for metabolic exchanges. Thus, the functional role of the satellite cell is analogous to that of the Schwann cell except that it does not make myelin.

1	Neurons and their processes located within ganglia of the enteric division of the ANS are associated with enteric neuroglial cells. These cells are morphologically and functionally similar to astrocytes in the CNS (see below). Enteric neuroglial cells share common functions with astrocytes, such as structural, metabolic, and protective support of neurons. However, recent studies indicate that enteric glial cells may also participate in enteric neurotransmission and help coordinate activities of the nervous and immune systems of the gut.

1	There are four types of central neuroglia:  Astrocytes are morphologically heterogeneous cells that provide physical and metabolic support for neurons of the CNS.  Oligodendrocytes are small cells that are active in the formation and maintenance of myelin in the CNS.  Microglia are inconspicuous cells with small, dark, elongated nuclei that possess phagocytotic properties.  Ependymal cells are columnar cells that line the ventricles of the brain and the central canal of the spinal cord. Only the nuclei of glial cells are seen in routine histologic preparations of the CNS. Heavy-metal staining or immunocytochemical methods are necessary to demonstrate the shape of the entire glial cell.

1	Although glial cells have long been described as supporting cells of nerve tissue in the purely physical sense, current concepts emphasize the functional interdependence of neuroglial cells and neurons. The most obvious example of physical support occurs during development. The brain and spinal cord develop from the embryonic neural tube. In the head region, the neural tube undergoes remarkable thickening and folding, leading ultimately to the final structure, the brain. During the early stages of the process, embryonic glial cells extend through the entire thickness of the neural tube in a radial manner. These radial glial cells serve as the physical scaffolding that directs the migration of neurons to their appropriate position in the brain. Astrocytes are closely associated with neurons to support and modulate their activities.

1	Astrocytes are closely associated with neurons to support and modulate their activities. Astrocytes are the largest of the neuroglial cells. They form a network of cells within the CNS and communicate with neurons to support and modulate many of their activities. Some astrocytes span the entire thickness of the brain, providing a scaffold for migrating neurons during brain development. Other astrocytes stretch their processes from blood vessels to neurons. The ends of the processes expand, forming end feet that cover large areas of the outer surface of the vessel or axolemma. Astrocytes do not form myelin. Two kinds of astrocytes are identified:  Protoplasmic astrocytes are more prevalent in the outermost covering of brain called gray matter. These astrocytes have numerous, short, branching cytoplasmic processes (Fig. 12.17).

1	 Fibrous astrocytes are more common in the inner core of the brain called white matter. These astrocytes have fewer processes, and they are relatively straight (Fig. 12.18). Both types of astrocytes contain prominent bundles of intermediate filaments composed of glial fbrillary acidic protein (GFAP). The filaments are much more numerous in

1	FIGURE 12.12 • Electron micrograph of a mature myelinated axon. The myelin sheath (M) shown here consists of 19 paired layers of Schwann cell membrane. The pairing of membranes in each layer is caused by the extrusion of the Schwann cell cytoplasm. The axon displays an abundance of neurofilaments, most of which have been cross-sectioned, giving the axon a stippled appearance. Also evident in the axon are microtubules (MT ) and several mitochondria (Mit). The outer collar of Schwann cell cytoplasm (OCS) is relatively abundant compared with the inner collar of Schwann cell cytoplasm (ICS). The collagen fibrils (C) constitute the fibrillar component of the endoneurium. BL, basal (external) lamina. 70,000. Inset. Higher magnification of the myelin. The arrow points to cytoplasm within the myelin that would contribute to the appearance of the Schmidt-Lanterman cleft as seen in the light microscope. It appears as an isolated region here because of the thinness of the section. The

1	the myelin that would contribute to the appearance of the Schmidt-Lanterman cleft as seen in the light microscope. It appears as an isolated region here because of the thinness of the section. The intercellular space between axon and Schwann cell is indicated by the arrowhead. A coated vesicle (CV) in an early stage of formation appears in the outer collar of the Schwann cell cytoplasm. 130,000. (Courtesy of Dr. George D. Pappas.) inner collar of Schwann cell cytoplasm

1	FIGURE 12.14 • Three-dimensional diagrams conceptualizing the relationship of myelin and cytoplasm of a Schwann cell. This diagram shows a hypothetically uncoiled Schwann cell. Note how the inner collar of the Schwann cell cytoplasm is continuous with the outer collar of Schwann cell cytoplasm via Schmidt-Lanterman clefts. the fibrous astrocytes, however, hence the name. Antibodies to GFAP are used as specific stains to identify astrocytes in sections and tissue cultures (see Fig. 12.18b). Tumors arising from fbrous astrocytes, fibrous astrocytomas, account for about 80% of adult primary brain tumors. They can be identified microscopically and by their GFAP specificity.

1	Astrocytes play important roles in the movement of metabolites and wastes to and from neurons. They help maintain the tight junctions of the capillaries that form the blood–brain barrier (see page 385). In addition, astrocytes provide a covering for the “bare areas” of myelinated axons— for example, at the nodes of Ranvier and at synapses. They may confine neurotransmitters to the synaptic cleft and remove excess neurotransmitters by pinocytosis. Protoplasmic astrocytes on the brain and spinal cord surfaces extend their processes (subpial feet) to the basal lamina of the pia mater to form the glia limitans, a relatively impermeable barrier surrounding the CNS (Fig. 12.19). Astrocytes modulate neuronal activities by buffering the K concentration in the extracellular space of the brain.

1	Astrocytes modulate neuronal activities by buffering the K concentration in the extracellular space of the brain. It is now generally accepted that astrocytes regulate K concentrations in the brain’s extracellular compartment, thus maintaining the microenvironment and modulating activities of the neurons. The astrocyte plasma membrane contains an abundance of K pumps and K channels that mediate the transfer K ions from areas of high to low concentration. Accumulation of large amounts of intracellular K in astrocytes decreases local extracellular K gradients. The astrocyte membrane becomes depolarized, and the charge is dissipated over a large area by the extensive network of astrocyte processes. The maintenance of the K concentration in the brain’s extracellular space by astrocytes is called potassium spatial buffering. Oligodendrocytes produce and maintain the myelin sheath in the CNS.

1	Oligodendrocytes produce and maintain the myelin sheath in the CNS. The oligodendrocyte is the cell responsible for producing CNS myelin. The myelin sheath in the CNS is formed by concentric layers of oligodendrocyte plasma membrane. The formation of the sheath in the CNS is more complex, however, than the simple wrapping of Schwann cell mesaxon membranes that occurs in the PNS (page 364).

1	Oligodendrocytes appear in specially stained light microscopic preparations as small cells with relatively few processes compared with astrocytes. They are often aligned in rows between axons. Each oligodendrocyte gives off several tonguelike processes that find their way to the axons, where each process wraps itself around a portion of an axon, forming an internodal segment of myelin. The multiple processes of a single oligodendrocyte may myelinate one axon or several nearby axons (Fig. 12.20). The nucleus-containing region of the oligodendrocyte may be at some distance from the axons it myelinates. Because a single oligodendrocyte may myelinate several nearby axons simultaneously, the cell cannot embed multiple axons in its cytoplasm and allow the mesaxon membrane to spiral around each axon. Instead, each tonguelike process appears to spiral around the axon, always staying in proximity to it, until the myelin sheath is formed.

1	The myelin sheath in the CNS differs from that in the PNS. There are several other important differences between the myelin sheaths in the CNS and those in the PNS. Oligodendrocytes in the CNS express different myelin-specific proteins during myelination than those expressed by Schwann cells in the PNS. Instead of P0 and PMP-22, which are expressed only in myelin of the PNS, other proteins, including proteolipid protein (PLP), myelin oligodendrocyte glyco protein (MOG), and oligodendrocyte myelin glycoprotein (OMgp), perform similar functions in CNS myelin. Deficiencies in the expression of these proteins appear to be important in the pathogenesis of several autoimmune demyelinating diseases of the CNS.

1	Deficiencies in the expression of these proteins appear to be important in the pathogenesis of several autoimmune demyelinating diseases of the CNS. On the microscopic level, myelin in the CNS exhibits fewer Schmidt-Lanterman clefts because the astrocytes provide metabolic support for CNS neurons. Unlike Schwann cells of the PNS, oligodendrocytes do not have an external lamina. Furthermore, because of the manner in which oligodendrocytes form CNS myelin, little or no cytoplasm may be present in the outermost layer of the myelin sheath, and with the absence of external lamina, the myelin of adjacent axons may come into contact. Thus, where myelin sheaths of adjacent axons touch, they may share an intraperiod line. Finally, the nodes of Ranvier in the CNS are larger than those in the PNS. The larger areas of exposed

1	FIGURE 12.15 • Electron micrograph of unmyelinated nerve fbers. The individual fibers or axons (A) are engulfed by the cytoplasm of a Schwann cell. The arrows indicate the site of mesaxons. In effect, each axon is enclosed by the Schwann cell cytoplasm, except for the intercellular space of the mesaxon. Other features evident in the Schwann cell are its nucleus (N), the Golgi apparatus (G), and the surrounding basal (external) lamina (BL). In the upper part of the micrograph, myelin (M) of two myelinated nerves is also evident. 27,000. Inset. Schematic diagram showing the relationship of axons engulfed by the Schwann cell. (Reprinted with permission from Barr ML, Kiernan JA. The Human Nervous System. New York: Harper & Row, 1983.) axolemma thus make saltatory conduction (see below) even more efficient in the CNS.

1	Another difference between the CNS and the PNS in regard to the relationships between supporting cells and neurons is that unmyelinated neurons in the CNS are often found to be bare—that is, they are not embedded in glial cell processes. The lack of supporting cells around unmyelinated axons as well as the absence of basal lamina material and connective tissue within the substance of the CNS helps to distinguish the CNS from the PNS in histologic sections and in TEM specimens. Microglia possess phagocytotic properties.

1	Microglia possess phagocytotic properties. Microglia are phagocytotic cells. They normally account for about 5% of all glial cells in the adult CNS but proliferate and become actively phagocytotic (reactive microglial cells) in regions of injury and disease. Microglial cells are considered part of the mononuclear phagocytotic system (see Folder 6.4, page 185) and originate from granulocyte/monocyte progenitor (GMP) cells. Microglia precursor cells enter the CNS parenchyma from the vascular system. Recent evidence suggests that microglia play a critical role in defense against invading microorganisms and neoplastic cells. They remove bacteria, injured cells, and the debris of cells that undergo apoptosis. They also mediate neuroimmune reactions, such as those occurring in chronic pain conditions.

1	Microglia are the smallest of the neuroglial cells and have relatively small, elongated nuclei (Fig. 12.21). When stained with heavy metals, microglia exhibit short, twisted processes. Both the processes and the cell body are covered with numerous spikes. The spikes may be the equivalent of the ruffled border seen on other phagocytotic cells. The TEM reveals numerous lysosomes, inclusions, and vesicles. However, microglia contain little rER and few microtubules or actin filaments. Ependymal cells form the epithelial-like lining of the ventricles of the brain and spinal canal.

1	Ependymal cells form the epithelial-like lining of the ventricles of the brain and spinal canal. Ependymal cells form the epithelium-like lining of the fluid-filled cavities of the CNS. They form a single layer of cuboidalto-columnar cells that have the morphologic and physiologic characteristics of fluid-transporting cells (Fig. 12.22). They are tightly bound by junctional complexes located at the apical surfaces. Unlike a typical epithelium, ependymal cells lack an external lamina. At the TEM level, the basal cell surface exhibits numerous infoldings that interdigitate with adjacent astrocyte processes. The apical surface of the cell possesses cilia and microvilli. The latter are involved in absorbing cerebrospinal fluid. Within the system of the brain ventricles, this epithelium-like lining is further modified to produce the cerebrospinal fluid by transport and secretion of materials

1	Within the system of the brain ventricles, this epithelium-like lining is further modified to produce the cerebrospinal fluid by transport and secretion of materials FIGURE 12.16 • Photomicrograph of a nerve ganglion. a. Photomicrograph showing a ganglion stained by the Mallory-Azan method. Note the large nerve cell bodies (arrows) and nerve fibers (NF ) in the ganglion. Satellite cells are represented by the very small nuclei at the periphery of the neuronal cell bodies. The ganglion is surrounded by a dense irregular connective tissue capsule (CT ) that is comparable to, and continuous with, the epineurium of the nerve. 200. b. Higher magnification of the ganglion, showing individual axons and a few neuronal cell bodies with their satellite cells (arrows). The nuclei in the region of the axons are mostly Schwann cell nuclei. 640. derived from adjacent capillary loops. The modified ependymal cells and associated capillaries are called the choroid plexus.

1	derived from adjacent capillary loops. The modified ependymal cells and associated capillaries are called the choroid plexus. An action potential is an electrochemical process triggered by impulses carried to the axon hillock after other impulses are received on the dendrites or the cell body itself.

1	A nerve impulse is conducted along an axon much as a flame travels along the fuse of a firecracker. This electrochemical process involves the generation of an action potential, a wave of membrane depolarization that is initiated at the initial segment of the axon hillock. Its membrane contains a large number of voltage-gated Na and K channels. In response to a stimulus, voltage-gated Na channels in the initial segment of the axon membrane open, causing an influx of Na into the axoplasm. This influx of Na briefly reverses (depolarizes) the negative membrane potential of the resting membrane (70 mV) to positive (30 mV). After depolarization, the voltage-gated Na channels close and voltage-gated K channels open. K rapidly exits the axon, returning the membrane to its resting potential. Depolarization of one part of the membrane sends electrical current to neighboring portions of unstimulated membrane, which is still positively charged. This local current stimulates adjacent portions of

1	of one part of the membrane sends electrical current to neighboring portions of unstimulated membrane, which is still positively charged. This local current stimulates adjacent portions of the axon’s membrane and repeats depolarization along the membrane. The entire process takes less than one thousandth of a second. After a very brief (refractory) period, the neuron can repeat the process of generating an action potential once again.

1	Rapid conduction of the action potential is attributable to the nodes of Ranvier. Myelinated axons conduct impulses more rapidly than unmyelinated axons. Physiologists describe the nerve impulse as “jumping” from node to node along the myelinated axon. This process is called saltatory (L. saltus, to jump) or discontinuous conduction. In myelinated nerves, the myelin sheath around the nerve does not conduct an electric current and forms an insulating layer around the axon. However, the voltage reversal can only occur at the nodes of Ranvier, where the axolemma lacks a myelin sheath. Here, the axolemma is exposed to extracellular fluids and possesses a high concentration of voltage-gated Na and K channels (see Figs. 12.13 and 12.20). Because of this, the voltage reversal (and, thus, the impulse) jumps as current flows from one node of Ranvier to the

1	FIGURE 12.17 • Protoplasmic astrocyte in the gray matter of the brain. a. This schematic drawing shows the foot processes of the protoplasmic astrocyte terminating on a blood vessel and the axonal process of a nerve cell. The foot processes terminating on the blood vessel contribute to the blood–brain barrier. The bare regions of the vessel as shown in the drawing would be covered by processes of neighboring astrocytes, thus forming the overall barrier. b. This laser-scanning confocal image of protoplasmic astrocyte in the gray matter of the dentate gyrus was visualized by intracellular labeling method. In lightly fixed tissue slices, selected astrocytes were impaled and iontophoretically injected with fluorescent dye (AlexaFluor 568) using pulses of negative current. Note the density and spatial distribution of cell processes. 480. (Reprinted with permission from Bushong EA, Martone ME, Ellisman MH. Examination of the relationship between astrocyte morphology and laminar boundaries

1	spatial distribution of cell processes. 480. (Reprinted with permission from Bushong EA, Martone ME, Ellisman MH. Examination of the relationship between astrocyte morphology and laminar boundaries in the molecular layer of adult dentate gyrus. J Comp Neurol 2003;462:241–251.)

1	FIGURE 12.18 • Fibrous astrocytes in the white matter of the brain. a. Schematic drawing of a fibrous astrocyte in the white mater a bb of the brain. b. Photomicrograph of the white matter of the brain, showing the extensive radiating cytoplasmic processes for which astrocytes are named. They are best visualized, as shown here, with immunostaining methods that use antibodies against GFAP. 220. (Reprinted with permission from Fuller GN, Burger PC. Central nervous system. In: Sternberg SS, ed. Histology for Pathologists. Philadelphia: Lippincott-Raven, 1997.)

1	FIGURE 12.19 • Distribution of glial cells in the brain. This diagram shows the three types of glial cells—astrocytes, oligodendrocytes, and microglial cells—interacting with several structures and cells found in the brain tissue. Note that the astrocytes and their processes interact with the blood vessels as well as with axons and dendrites. Note that astrocytes also send their processes toward the brain surface, where they contact the basement membrane of the pia mater, forming the glia limitans. In addition, processes of astrocytes extend toward the fluid-filled spaces in the CNS, where they contact the ependymal lining cells. Oligodendrocytes are involved in myelination of the nerve fibers in the CNS. Microglia exhibit phagocytotic functions. pia mater myelin oligo-dendrocyte ependyma astrocyte microglial cell pericyte neuron nerve fibers myelin axons node of Ranvier (showing axon in contact with extracellular space) oligodendrocyte

1	FIGURE 12.20 • Three-dimensional view of an oligodendrocyte as it relates to several axons. Cytoplasmic processes from the oligodendrocyte cell body form flattened cytoplasmic sheaths that wrap around each of the axons. The relationship of cytoplasm and myelin is essentially the same as that of Schwann cells. next. The speed of saltatory conduction is related not only to the thickness of the myelin but also to the diameter of the axon. Conduction is more rapid along axons of greater diameter. In unmyelinated axons, Na and K channels are distributed uniformly along the length of the fiber. The nerve impulse is conducted more slowly and moves as a continuous wave of voltage reversal along the axon. CNS neurons and central glia, except microglial cells, are derived from neuroectodermal cells of the neural tube.

1	CNS neurons and central glia, except microglial cells, are derived from neuroectodermal cells of the neural tube. Neurons, oligodendrocytes, astrocytes, and ependymal cells are derived from cells of the neural tube. After developing neurons have migrated to their predestined locations in the neural tube and have differentiated into mature neurons, they no longer divide. However, in the adult mammalian brain, a very small number of cells left from development called neural stem cells retain the ability to divide. These cells migrate into sites of injury and differentiate into fully functional nerve cells. Oligodendrocyte precursors are highly migratory cells. They appear to share a developmental lineage with motor neurons migrating from their site of origin to developing axonal projections (tracts) in the white matter of the brain or

1	FIGURE 12.21 • Microglial cell in the gray matter of the brain. a. This diagram shows the shape and characteristics of a microglial cell. Note the elongated nucleus and relatively few processes emanating from the body. b. Photomicrograph of microglial cells (arrows) showing their characteristic elongated nuclei. The specimen was obtained from an individual with diffuse microgliosis. In this condition, the microglial cells are present in large numbers and are readily visible in a routine H&E preparation. 420. (Reprinted with permission from Fuller GN, Burger PC. Central nervous system. In: Sternberg SS, ed. Histology for Pathologists. Philadelphia: Lippincott-Raven, 1997.) spinal cord. The precursors then proliferate in response to the local expression of mitogenic signals. The matching of oligodendrocytes to axons is accomplished through a combination of local regulation of cell proliferation, differentiation, and apoptosis.

1	Astrocytes are also derived from cells of the neural tube. During the embryonic and early postnatal stages, immature astrocytes migrate into the cortex, where they differentiate and become mature astrocytes. Ependymal cells are derived from the proliferation of neuroepithelial cells that immediately surround the canal of the developing neural tube. In contrast to other central neuroglia, microglia cells are derived from mesodermal macrophage precursors, specifically from Granulocyte/monocyte progenitor (GMP) cells in bone marrow. They infiltrate the neural tube in the early stages of its development and under the influence of growth factors such as colony stimulating factor-1 (CSF-1) produced by developing neural cells undergo proliferation and differentiation into motile ameboid cells. These motile cells are commonly ob-

1	FIGURE 12.22 • Ependymal lining of the spinal canal. a. Photomicrograph of the cental region of the spinal cord stained with toluidine blue. The arrow points to the central canal. 20. b. At higher magnification, ependymal cells, which line the central canal, can be seen to consist of a single layer of columnar cells. 340. (Courtesy of Dr. George D. Pappas.) c. Transmission electron micrograph showing a portion of the apical region of two columnar ependymal cells. They are joined by a junctional complex (JC) that separates the lumen of the canal from the lateral intercellular space. The apical surface of the ependymal cells has both cilia (C) and microvilli (M). Basal bodies (BB) and a Golgi apparatus (G) within the apical cytoplasm are also visible. 20,000. (Courtesy of Dr. Paul Reier.) served in the developing brain. As the only glial cells of mesenchymal origin, microglia possess the vimentin class of intermediate flaments, which can be useful in identifying these cells with

1	served in the developing brain. As the only glial cells of mesenchymal origin, microglia possess the vimentin class of intermediate flaments, which can be useful in identifying these cells with immunocytochemical methods.

1	PNS ganglion cells and peripheral glia are derived from the neural crest. The development of the ganglion cells of the PNS involves the proliferation and migration of ganglion precursor cells from the neural crest to their future ganglionic sites, where they undergo further proliferation. There, the cells develop processes that reach the cells’ target tissues (e.g., glandular tissue or smooth muscle cells) and sensory territories. Initially, more cells are produced than are needed. Those that do not make functional contact with a target tissue undergo apoptosis.

1	Schwann cells also arise from migrating neural crest cells that become associated with the axons of early embryonic nerves. Several genes have been implicated in Schwann cell development. Sex-determining region Y (SRY) box 10 (Sox10) is required for the generation of all peripheral glia from neural crest cells. Axon-derived neuregulin 1 (Nrg-1) sustains the Schwann cell precursors that undergo differentiation and divide along the growing nerve processes. The fate of all immature Schwann cells is determined by the nerve processes with which they have immediate contact. Immature Schwann cells that associate with large-diameter axons mature into myelinating Schwann cells, while those that associate with small-diameter axons mature into nonmyelinating cells. The peripheral nervous system (PNS) consists of peripheral nerves with specialized nerve endings and ganglia containing nerve cell bodies that reside outside the central nervous system.

1	The peripheral nervous system (PNS) consists of peripheral nerves with specialized nerve endings and ganglia containing nerve cell bodies that reside outside the central nervous system. A peripheral nerve is a bundle of nerve fibers held together by connective tissue. The nerves of the PNS are made up of many nerve fibers that carry sensory and motor (effector) information between the organs and tissues of the body and the brain and spinal cord. The term nerve fber is used in different ways that can be confusing. It can connote the axon with all of its coverings (myelin and Schwann cell), as used above, or it can connote the axon alone. It is also used to refer to any process of a nerve cell, either dendrite or axon, especially if insufficient information is available to identify the process as either an axon or a dendrite.

1	The cell bodies of peripheral nerves may be located within the CNS or outside the CNS in peripheral ganglia. Ganglia contain clusters of neuronal cell bodies and the nerve fibers leading to and from them (see Fig. 12.16). The cell bodies in dorsal root ganglia as well as ganglia of cranial nerves belong to sensory neurons (somatic afferents and visceral afferents that belong to the autonomic nervous system [discussed below]), whose distribution is restricted to specific locations (Table 12.1 and Fig. 12.3). The cell bodies in the paravertebral, prevertebral, and terminal ganglia belong to postsynaptic “motor” neurons (visceral efferents) of the autonomic nervous system (see Table 12.1 and Fig. 12.16). To understand the PNS, it is also necessary to describe some parts of the CNS. Motor neuron cell bodies of the PNS lie in the CNS.

1	To understand the PNS, it is also necessary to describe some parts of the CNS. Motor neuron cell bodies of the PNS lie in the CNS. The cell bodies of motor neurons that innervate skeletal muscle (somatic efferents) are located in the brain, brain stem, and spinal cord. The axons leave the CNS and travel in peripheral nerves to the skeletal muscles that they innervate. A single neuron conveys impulses from the CNS to the effector organ. Sensory neuron cell bodies are located in ganglia outside of, but close to, the CNS. In the sensory system (both the somatic afferent and the visceral afferent components), a single neuron connects the receptor, through a sensory ganglion, to the spinal cord or brain stem. Sensory ganglia are located in the dorsal roots of the spinal nerves and in association with sensory components of cranial nerves V, VII, VIII, IX, and X (see Table 12.1). Connective Tissue Components of a Peripheral Nerve

1	Connective Tissue Components of a Peripheral Nerve The bulk of a peripheral nerve consists of nerve fibers and their supporting Schwann cells. The individual nerve fibers and their associated Schwann cells are held together by connective tissue organized into three distinctive components, each with specific morphologic and functional characteristics (Fig. 12.23; also, see Fig. 12.3).  The endoneurium includes loose connective tissue sur rounding each individual nerve fiber.  The perineurium includes specialized connective tissue surrounding each nerve fascicle.  The epineurium includes dense irregular connective tis sue that surrounds a peripheral nerve and fills the spaces between nerve fascicles. Endoneurium constitutes the loose connective tissue associated with individual nerve fibers.

1	Endoneurium constitutes the loose connective tissue associated with individual nerve fibers. The endoneurium is not conspicuous in routine light microscope preparations, but special connective tissue stains permit its demonstration. At the electron microscope level, collagen fibrils that constitute the endoneurium are readily apparent (see Figs. 12.11 and 12.12). The fibrils run both parallel to, and around, the nerve fibers, binding them together into a fascicle, or bundle. Because fbroblasts are relatively sparse in the interstices of the nerve fibers, it is likely that most of the collagen fibrils are secreted by the Schwann cells. This conclusion is supported by tissue culture studies in which collagen fibrils are formed in pure cultures of Schwann cells and dorsal root neurons.

1	Other than occasional fibroblasts, the only other connective tissue cells normally found within the endoneurium are mast cells and macrophages. Macrophages mediate immunologic surveillance and also participate in nerve tissue re-

1	TABLE Peripheral Gangliaa12.1 aPractical note: Neuron cell bodies seen in tissue sections such as tongue, pancreas, urinary bladder, and heart are invariably terminal ganglia or “ganglion cells” of the parasympathetic nervous system. Ganglia that contain cell bodies of sensory neurons; these are not synaptic stations  Dorsal root ganglia of all spinal nerves  Sensory ganglia of cranial nerves  Trigeminal (semilunar, Gasserian) ganglion of the trigeminal (V) nerve  Geniculate ganglion of the facial (VII) nerve  Spiral ganglion (contains bipolar neurons) of the cochlear division of the vestibulocochlear (VIII) nerve  Vestibular ganglion (contains bipolar neurons) of the vestibular division of the vestibulocochlear (VIII) nerve  Superior and inferior ganglia of the glossopharyngeal (IX) nerve  Superior and inferior ganglia of the vagus (X) nerve Ganglia that contain cell bodies of autonomic (postsynaptic) neurons; these are synaptic stations  Sympathetic ganglia  Sympathetic

1	(IX) nerve  Superior and inferior ganglia of the vagus (X) nerve Ganglia that contain cell bodies of autonomic (postsynaptic) neurons; these are synaptic stations  Sympathetic ganglia  Sympathetic trunk (paravertebral) ganglia (the highest of these is the superior cervical ganglion)  Prevertebral ganglia (adjacent to origins of large unpaired branches of abdominal aorta), including celiac, superior mesenteric, inferior mesenteric, and aorticorenal ganglia  Adrenal medulla, which may be considered a modified sympathetic ganglion (each of the secretory cells of the medulla, as well as the recognizable ganglion cells, is innervated by cholinergic presynaptic sympathetic nerve fibers)  Parasympathetic ganglia  Head ganglia  Ciliary ganglion associated with the oculomotor (III) nerve  Submandibular ganglion associated with the facial (VII) nerve  Pterygopalatine (sphenopalatine) ganglion of the facial (VII) nerve  Otic ganglion associated with the glossopharyngeal (IX) nerve 

1	 Submandibular ganglion associated with the facial (VII) nerve  Pterygopalatine (sphenopalatine) ganglion of the facial (VII) nerve  Otic ganglion associated with the glossopharyngeal (IX) nerve  Terminal ganglia (near or in wall of organs), including ganglia of the submucosal (Meissner’s) and myenteric (Auerbach’s) plexuses of the gastrointestinal tract (these are also ganglia of the enteric division of the ANS) and isolated ganglion cells in a variety of organs pair. Following nerve injury, they proliferate and actively phagocytose myelin debris. In general, most of the nuclei (90%) found in cross-sections of peripheral nerves belong to Schwann cells; the remaining 10% is equally distributed between the occasional fibroblasts and other cells such as endothelial cells of capillaries, macrophages, and mast cells.

1	Perineurium is the specialized connective tissue surrounding a nerve fascicle that contributes to the formation of the blood–nerve barrier.

1	Surrounding the nerve bundle is a sheath of unique connective tissue cells that constitutes the perineurium. The perineurium serves as a metabolically active diffusion barrier that contributes to the formation of a blood–nerve barrier. This barrier maintains the ionic milieu of the ensheathed nerve fibers. In a manner similar to the properties exhibited by the endothelial cells of brain capillaries forming the blood–brain barrier (see page 385), perineurial cells possess receptors, transporters, and enzymes that provide for the active transport of substances across perineurial cells. The perineurium may be one or more cell layers thick, depending on the nerve diameter. The cells that compose this layer are squamous; each layer exhibits an external (basal) lamina on both surfaces (Fig. 12.23b and Plate 27, page 390). The cells are contractile and contain an appreciable number of actin filaments, a characteristic of smooth muscle cells and other contractile cells. Moreover, when there

1	and Plate 27, page 390). The cells are contractile and contain an appreciable number of actin filaments, a characteristic of smooth muscle cells and other contractile cells. Moreover, when there are two or more perineurial cell layers (as many as five of six layers may be present in larger nerves), collagen fibrils are present between the perineurial cell layers, but fibroblasts are absent. Tight junctions provide the basis for the blood–nerve barrier and are present between the cells located within the same layer of the perineurium. In effect, the arrangement of these cells as a barrier—the presence of tight junctions and external (basal) lamina material—liken them to an epithelioid tissue. On the other hand, their contractile nature and their apparent ability to produce collagen fibrils also liken them to smooth muscle cells and fibroblasts.

1	The limited number of connective tissue cell types within the endoneurium (page 375) undoubtedly reflects the protective role that the perineurium plays. Typical immune system cells (i.e., lymphocytes, plasma cells) are not found within the endoneurial and perineurial compartments. This absence of immune cells (other than the mast cells and macrophages) is accounted for by the protective barrier created by the perineurial cells. Typically only fibroblasts, a small number of resident macrophages, and occasional mast cells are present within the nerve compartment. Epine.urium consists of dense irregular connective tissue that surrounds and binds nerve fascicles into a common bundle.

1	FIGURE 12.23 • Electron micrograph of a peripheral nerve and its surrounding perineurium. a. Electron micrograph of unmyelinated nerve fibers and a single myelinated fiber (MF). The perineurium (P), consisting of several cell layers, is seen at the left of the micrograph. Perineurial cell processes (arrowheads) have also extended into the nerve to surround a group of axons (A) and their Schwann cell as well as a small blood vessel (BV ). The enclosure of this group of axons represents the root of a small nerve branch that is joining or leaving the larger fascicle. 10,000. The area within the circle encompassing the endothelium of the vessel and the adjacent perineurial cytoplasm is shown in the inset at higher magnification. Note the basal (external) laminae of the vessel and the perineurial cell (arrows). The junction between endothelial cells of the blood vessel is also apparent (arrowheads). 46,000. b. Electron micrograph showing the perineurium of a nerve. Four cellular layers of

1	cell (arrows). The junction between endothelial cells of the blood vessel is also apparent (arrowheads). 46,000. b. Electron micrograph showing the perineurium of a nerve. Four cellular layers of the perineurium are present. Each layer has a basal (external) lamina (BL) associated with it on both surfaces. Other features of the perineurial cell include an extensive population of actin microfilaments (MF), pinocytotic vesicles (arrows), and cytoplasmic densities (CD). These features are characteristic of smooth muscle cells. The innermost perineurial cell layer (right) exhibits tight junctions (asterisks) where one cell is overlapping a second cell in forming the sheath. Other features seen in the cytoplasm are mitochondria (M), rough-surfaced endoplasmic reticulum (rER), and free ribosomes (R). 27,000.

1	The epineurium forms the outermost tissue of the peripheral nerve. It is a typical dense connective tissue that surrounds the fascicles formed by the perineurium (Plate 28, page 392). Adipose tissue is often associated with the epineurium in larger nerves. The blood vessels that supply the nerves travel in the epineurium, and their branches penetrate into the nerve and travel within the perineurium. Tissue at the level of the endoneurium is poorly vascularized; metabolic exchange of substrates and wastes in this tissue depends on diffusion from and to the blood vessels through the perineurial sheath (see Fig. 12.23). Afferent receptors are specialized structures located at the distal tips of the peripheral processes of sensory neurons. Although receptors may have many different structures, they have one basic characteristic in common: They can initiate a nerve impulse in response to a stimulus. Receptors may be classified as the following.

1	 Exteroceptors react to stimuli from the external environment—for example, temperature, touch, smell, sound, and vision.  Enteroceptors react to stimuli from within the body— for example, the degree of filling or stretch of the alimen tary canal, bladder, and blood vessels.  Proprioceptors, which also react to stimuli from within the body, provide sensation of body position and muscle tone and movement. The simplest receptor is a bare axon called a nonencapsulated (free) nerve ending. This ending is found in epithelia, in connective tissue, and in close association with hair follicles. Most sensory nerve endings acquire connective tissue capsules or sheaths of varying complexity.

1	Most sensory nerve endings acquire connective tissue capsules or sheaths of varying complexity. Sensory nerve endings with connective tissue sheaths are called encapsulated endings. Many encapsulated endings are mechanoreceptors located in the skin and joint capsules (Krause’s end bulb, Ruffini’s corpuscles, Meissner’s corpuscles, and Pacinian corpuscles) and are described in Chapter 15, Integumentary System (page 501). Muscle spindles are encapsulated sensory endings located in skeletal muscle; they are described in Chapter 11, Muscle Tissue (page 325). Functionally related Golgi tendon organs are encapsulated tension receptors found at musculotendinous junctions. Although the ANS was introduced early in this chapter, it is useful here to describe some of the salient features of its organization and distribution. The ANS is classified into three divisions: The ANS controls and regulates the body’s internal environment.

1	The ANS controls and regulates the body’s internal environment. The ANS is the portion of the PNS that conducts involuntary impulses to smooth muscle, cardiac muscle, and glandular epithelium. These effectors are the functional units in the organs that respond to regulation by nerve tissue. The term visceral is sometimes used to characterize the ANS and its neurons, which are referred to as visceral motor (efferent) neurons. However, visceral motor neurons are frequently accompanied by visceral sensory (afferent) neurons that transmit pain and reflexes from visceral effectors (i.e., blood vessels, mucous membrane, and glands) to the CNS. These pseudounipolar neurons have the same arrangement as other sensory neurons—that is, their cell bodies are located in sensory ganglia, and they possess long peripheral and central axons, as described above.

1	The main organizational difference between the efferent flow of impulses to skeletal muscle (somatic effectors) and the efferent flow to smooth muscle, cardiac muscle, and glandular epithelium (visceral effectors) is that one neuron conveys the impulses from the CNS to the somatic effector (Fig. 12.24a), whereas a chain of two neurons conveys the impulses from the CNS to the visceral effectors (Fig. 12.24b). Thus, there is a synaptic station in an autonomic ganglion outside the CNS, where a presynaptic neuron makes contact with postsynaptic neurons. Each presynaptic neuron synapses with several post-synaptic neurons. Sympathetic and Parasympathetic Divisions of the Autonomic Nervous System The presynaptic neurons of the sympathetic division are located in the thoracic and upper lumbar portions of the spinal cord.

1	The presynaptic neurons of the sympathetic division are located in the thoracic and upper lumbar portions of the spinal cord. The presynaptic neurons send axons from the thoracic and upper lumbar spinal cord to the vertebral and paravertebral ganglia. The paravertebral ganglia in the sympathetic trunk contain the cell bodies of the postsynaptic effector neurons of the sympathetic division (Figs. 12.24b and 12.25). The presynaptic neurons of the parasympathetic division are located in the brain stem and sacral spinal cord. The presynaptic parasympathetic neurons send axons from the brain stem—that is, the midbrain, pons, and medulla, and the sacral segments of the spinal cord (S2 through S4)— to visceral ganglia. The ganglia in or near the wall of abdominal and pelvic organs and the visceral motor ganglia of cranial nerves III, VII, IX, and X contain cell bodies of the postsynaptic effector neurons of the parasympathetic division (Figs. 12.24c and 12.25).

1	The sympathetic and parasympathetic divisions of the ANS often supply the same organs. In these cases, the actions of the two are usually antagonistic. For example, sympathetic stimulation increases the rate of cardiac muscle contractions, whereas parasympathetic stimulation reduces the rate.

1	Many functions of the SNS are similar to those of the adrenal medulla, an endocrine gland. This functional similarity is partly explained by the developmental relationships between the cells of the adrenal medulla and postsynaptic sympathetic neurons. Both are derived from the neural crest, are innervated by presynaptic sympathetic neurons, and produce closely related physiologically active agents, EPI and NE. A major difference is that the sympathetic neurons deliver the agent directly to the effector, whereas the cells of the adrenal medulla deliver the agent indirectly through the bloodstream. The innervation of the adrenal medulla may constitute an exception to the rule that autonomic innervation consists of a two-neuron chain from the CNS to an effector unless the adrenal medullary cell is considered the functional equivalent of the second neuron (in effect, a neurosecretory neuron). Enteric Division of the Autonomic Nervous System

1	Enteric Division of the Autonomic Nervous System The enteric division of the ANS consists of the ganglia and their processes that innervate the alimentary canal. The enteric division of the ANS represents a collection of neurons and their processes within the walls of the alimentary canal. It controls motility (contractions of the gut wall), exocrine and endocrine secretions, and blood flow through the gastrointestinal tract; it also regulates immunologic and inflammatory processes.

1	The enteric nervous system can function independently from the CNS and is regarded as the “brain of the gut.” However, digestion requires communication between enteric neurons and the CNS, which is provided by parasympathetic and sympathetic nerve fibers. Enteroceptors located in the alimentary tract provide sensory information to the CNS regarding the state of digestive functions. The CNS then coordinates sympathetic stimulation that inhibits gastrointestinal secretion, motor activity, and contraction of gastrointestinal sphincters and blood vessels and parasympathetic stimuli that produce opposite actions. Interneurons integrate information from sensory neurons and relay this information to enteric motor neurons in the form of reflexes. For instance, the gastrocolic reflex is elicited when distention of the stomach stimulates contraction of musculature of the colon, triggering defecation.

1	Ganglia and postsynaptic neurons of the enteric division are located in the lamina propria, muscularis mucosae, submucosa, muscularis externa, and subserosa of the alimentary canal from the esophagus to the anus (Fig. 12.26). Because the enteric division does not require presynaptic input from the vagus nerve and sacral outflow, sympathetic trunk with paravertebral ganglion splanchnic nerve containing axons of presynaptic neurons

1	FIGURE 12.24 • Schematic diagram comparing somatic efferent and visceral efferent neurons. a. In the somatic efferent (motor) system, one neuron conducts the impulses from the CNS to the effector (skeletal muscle). b. In the visceral efferent system (repre sented in this diagram by the sympathetic division of the ANS), a chain of two neurons conducts the impulses: A presy naptic neuron located within the CNS and a postsynaptic neuron located in the paravertebral or prevertebral ganglia. Moreover, each presynaptic neuron makes synaptic contact with more than one postsynaptic neuron. Postsy naptic sympathetic fibers supply smooth muscles (as in blood vessels) or glandular epithelium (as in sweat glands). c. Neurons of the ANS that supply organs of the abdomen reach these organs by way of the splanchnic nerves. In this example, the splanchnic nerve joins with the celiac ganglion, where most of the synapses of the two-neuron chain occur. Note that one presynaptic neuron makes contact with

1	splanchnic nerves. In this example, the splanchnic nerve joins with the celiac ganglion, where most of the synapses of the two-neuron chain occur. Note that one presynaptic neuron makes contact with several postsynaptic neurons. (Redrawn with permission from Reith EJ, Breidenbach B, Lorenc

1	M. Textbook of Anatomy and Physiology. St. Louis: CV Mosby, 1978.) blood vessels of visceral structures, blood vessels, sweat glands, and arrector muscles of hairs eye (iris) cephalic arterial ramus carotid periarterial plexus cardiopulmonary splanchnic nerves heart larynx trachea bronchi lungs diaphragm stomach pancreas spleen liver gallbladder bladder penis (clitoris) gonad inferior mesenteric ganglion superior mesenteric ganglion aorticorenal ganglion celiac ganglion abdominopelvic splanchnic nerves large intestine small intestine kidney suprarenal (adrenal) gland rectum internal anal sphincter presynaptic postsynaptic sympathetic fibers lacrimal gland nasal, palatine, and pharyngeal glands eye (iris, ciliary mm.) sacral parasympathetic outflow (via pelvic splanchnic nerves) parotid gland sublingual and submandibular glands heart larynx trachea bronchi lungs liver gallbladder left colic (splenic) flexure dividing sacral para-sympathetic supply from cranial supply large intestine

1	gland sublingual and submandibular glands heart larynx trachea bronchi lungs liver gallbladder left colic (splenic) flexure dividing sacral para-sympathetic supply from cranial supply large intestine small intestine rectum innervation via sacral outflow innervation via cranial outflow ciliary ganglion bladder penis (clitoris) stomach pancreas pterygopalatine ganglion otic ganglion submandibular ganglion cranial parasympathetic outflow (via 4 cranial nerves) III VII IX X S2 S3 S4

1	FIGURE 12.25 • Schematic diagram showing the general arrangement of sympathetic and parasympathetic neurons of the ANS. The sympathetic outflow is shown on the right; the parasympathetic, on the left. The sympathetic (thoracolumbar) outflow leaves the CNS from the thoracic and upper lumbar segments (T1–L2 or L3) of the spinal cord. These presynaptic fibers communicate with postsynaptic neurons in two locations, the paravertebral and prevertebral ganglia. Paravertebral ganglia are linked together and form two sympathetic trunks (yellow columns on each side of the spinal cord). Prevertebral ganglia are associated with the main branches of the abdominal aorta (yellow circles). Note the distribution of postsynaptic sympathetic nerve fibers to the viscera. The parasympathetic (craniosacral) outflow leaves the CNS from the gray matter of the brain stem within cranial nerves III, VII, IX, and X and the gray matter of sacral segments (S2–S4) of the spinal cord and is distributed to the

1	outflow leaves the CNS from the gray matter of the brain stem within cranial nerves III, VII, IX, and X and the gray matter of sacral segments (S2–S4) of the spinal cord and is distributed to the viscera. The presynaptic fibers traveling with cranial nerves III, VII, and IX communicate with postsynaptic neurons in various ganglia located in the head and neck region (yellow circles). The presynaptic fibers traveling with cranial nerve X and with pelvic splanchnic nerves have their synapses with postsynaptic neurons in the wall of visceral organs (terminal ganglia). The viscera thus contain both sympathetic and parasympathetic innervation. Note that a two-neuron chain carries impulses to all viscera except the adrenal medulla. (Modified from Moore KL, Dalley AF. Clinically Oriented Anatomy. Baltimore: Lippincott Williams & Wilkins, 1999:48–50.) the intestine will continue peristaltic movements even by the same pathologic changes that can occur in neuafter the vagus nerve or pelvic

1	Baltimore: Lippincott Williams & Wilkins, 1999:48–50.) the intestine will continue peristaltic movements even by the same pathologic changes that can occur in neuafter the vagus nerve or pelvic splanchnic nerves are rons of the brain. Lewy bodies associated with Parkin severed. son’s disease (see Folder 12.1) as well as amyloid plaques

1	Neurons of the enteric division are not supported by and neurofibrillary tangles associated with Alzheimer’s Schwann or satellite cells; instead, they are supported by disease have been found in the walls of the large intesenteric neuroglial cells that resemble astrocytes (see tine. This finding may lead to development of routine page 367). Cells of the enteric division are also affected rectal biopsies for early diagnosis of these conditions

1	FIGURE 12.26 • Enteric nervous system. This diagram shows the organization of the enteric system in the wall of the small intestine. Note the location of two nerve plexuses containing ganglion cells. The more superficial plexus, the myenteric plexus (Auerbach’s plexus), lies between two muscle layers. Deeper in the region of submucosa is a network of unmyelinated nerve fibers and ganglion cells, forming the submucosal plexus (Meissner’s plexus). Parasympathetic fibers originating from the vagus nerve enter the mesentery of the small intestine and synapse with the ganglion cells of both plexuses. Postsynaptic sympathetic nerve fibers also contribute to the enteric nervous system. rather than the more complex and risk-associated biopsy of the brain. A Summarized View of Autonomic Distribution

1	rather than the more complex and risk-associated biopsy of the brain. A Summarized View of Autonomic Distribution Figures 12.24 and 12.25 summarize the origins and distribution of the ANS. Refer to these figures as you read the descriptive sections. Note that the diagrams indicate both the paired innervation (parasympathetic and sympathetic) common to the ANS as well as the important exceptions to this general characteristic.

1	Head  Parasympathetic presynaptic outfow to the head leaves the brain with the cranial nerves, as indicated in Figure 12.25, but the routes are quite complex. Cell bodies may also be found in structures other than head ganglia listed in Table 12.1 and Figure 12.25 (e.g., in the tongue). These are “terminal ganglia” that contain nerve cell bodies of the parasympathetic system. Sympathetic presynaptic outfow to the head comes from the thoracic region of the spinal cord. The postsynaptic neurons have their cell bodies in the superior cervical ganglion; the axons leave the ganglion in a nerve network that hugs the wall of the internal and external carotid arteries to form the periarterial plexus of nerves. The inter nal carotid plexus and external carotid plexus follow the branches of the carotid arteries to reach their destination.

1	 Parasympathetic presynaptic outfow to the thoracic viscera is via the vagus nerve (X). The postsynaptic neurons have their cell bodies in the walls or in the parenchyma of the organs of the thorax.  Sympathetic presynaptic outfow to the thoracic organs is from the upper thoracic segments of the spinal cord. Sympathetic postsynaptic neurons for the heart are mostly in the cervical ganglia; their axons make up the cardiac nerves. Postsynaptic neurons for the other thoracic viscera are in ganglia of the thoracic part of the sympathetic trunk. The axons travel via small splanchnic nerves from the sympathetic trunk to organs within the thorax and form the pulmonary and esophageal plexuses.

1	 Parasympathetic presynaptic outfow to the abdominal viscera is via the vagus (X) and pelvic splanchnic nerves. Postsynaptic neurons of the parasympathetic system to abdominopelvic organs are in terminal ganglia that generally are in the walls of the organs, such as the ganglia of the submucosal (Meissner’s) plexus and the myenteric (Auerbach’s) plexus in the alimentary canal. These ganglia are part of the enteric division of the ANS.

1	 Sympathetic presynaptic outfow to the abdominopelvic organs is from the lower thoracic and upper lumbar segments of the spinal cord. These fibers travel to the prevertebral ganglia through abdominopelvic splanchnic nerves consisting of the greater, lesser, and least thoracic splanchnic and lumbar splanchnic nerves. Postsynaptic neurons have their cell bodies mostly in the prevertebral ganglia (see Fig. 12.24c). Only presynaptic fibers terminating on cells in the medulla of the suprarenal (adrenal) gland originate from paravertebral ganglia of the sympathetic trunk. The adrenal medullary cells function as a special type of postsynaptic neuron that release neurotrans mitter directly into the bloodstream instead of into the synaptic cleft.

1	 There is no parasympathetic outflow to the body wall and extremities. Anatomically, the autonomic innervation in the body wall is only sympathetic (see Fig. 12.24b). Each spinal nerve contains postsynaptic sympathetic fibers— that is, unmyelinated visceral efferents of neurons whose cell bodies are in paravertebral ganglia of the sympathetic trunk. For sweat glands, the neurotransmitter released by the “sympathetic” neurons is ACh instead of the usual NE. The central nervous system consists of the brain located in the cranial cavity and the spinal cord located in the vertebral canal. The CNS is protected by the skull and vertebrae and is surrounded by three connective tissue membranes called meninges. The brain and spinal cord essentially float in the cerebrospinal fluid that occupies the space between the two inner meningeal layers. The brain is further subdivided into the cerebrum, cerebellum, and brain stem that connects with the spinal cord.

1	In the brain, the gray matter forms an outer covering or cortex; the white matter forms an inner core or medulla. The cerebral cortex that forms the outermost layer of the brain contains nerve cell bodies, axons, dendrites, and central glial cells, and it is the site of synapses. In a freshly dissected brains, the cerebral cortex has a gray color, hence the name gray matter. In addition to the cortex, islands of gray matter called nuclei are found in the deep portions of the cerebrum and cerebellum.

1	The white matter contains only axons of nerve cells plus the associated glial cells and blood vessels (axons in fresh preparations appear white). These axons travel from one part of the nervous system to another. Whereas many of the axons going to, or coming from, a specific location are grouped into functionally related bundles called tracts, the tracts themselves do not stand out as delineated bundles. The demonstration of a tract in white matter of the CNS requires a special procedure, such as the destruction of cell bodies that contribute fibers to the tract. The damaged fibers can be displayed by the use of appropriate staining or labeling methods and then traced. Even in the spinal cord, where the grouping of tracts is most pronounced, there are no sharp boundaries between adjacent tracts. Cells of the Gray Matter The types of cell bodies found in the gray matter vary according to which part of the brain or spinal cord is being examined.

1	Cells of the Gray Matter The types of cell bodies found in the gray matter vary according to which part of the brain or spinal cord is being examined. Each functional region of the gray matter has a characteristic variety of cell bodies associated with a meshwork of axonal, dendritic, and glial processes. The meshwork of axonal, dendritic, and glial processes associated with the gray matter is called the neuropil. The organization of the neuropil is not demonstrable in H&E–stained sections. It is necessary to use methods other than H&E histology to decipher the cytoarchitecture of the gray matter (Plate 29, page 394). Although general histology programs usually do not deal with the actual arrangements of the neurons in the CNS, the presentation of two examples will add to the appreciation of H&E sections that students usually examine. These examples present a region of the cerebral cortex (Fig. 12.27) and the cerebellar cortex (Fig. 12.28), respectively.

1	The brain stem is not clearly separated into regions of gray matter and white matter. The nuclei of the cranial nerves located in the brain stem, however, appear as islands surrounded by more or less distinct tracts of white matter. The nuclei contain the cell bodies of the motor neurons of the cranial nerves and are both the morphologic and functional counterparts of the anterior horns of the spinal cord. FIGURE 12.27 • Nerve cells in intracortical cerebral circuits. This simple diagram shows the organization and connections between cells in different layers of the cortex contributing to cortical afferent fibers (arrows pointing up) and cortical efferent fibers (arrows pointing down). The small interneurons are indicated in yellow. In other sites in the brain stem, as in the reticular formation, the distinction between white matter and gray matter is even less evident. Organization of the Spinal Cord

1	In other sites in the brain stem, as in the reticular formation, the distinction between white matter and gray matter is even less evident. Organization of the Spinal Cord The spinal cord is a flattened cylindrical structure that is directly continuous with the brain stem. It is divided into 31 segments (8 cervical, 12 thoracic, 5 lumbar, 5 sacral, and 1 coccygeal), and each segment is connected to a pair of spinal nerves. Each spinal nerve is joined to its segment of the cord by a number of rootlets grouped as dorsal (posterior) or ventral (anterior) roots (Fig. 12.29; see also Fig. 12.3). In cross-section, the spinal cord exhibits a butterfly-shaped grayish-tan inner substance surrounding the central canal, the gray matter, and a whitish peripheral substance, the white matter (Fig. 12.30). The white matter (see Fig. 12.3) contains only tracks of myelinated and unmyelinated axons traveling to and from other parts of the spinal cord and to and from the brain.

1	The gray matter contains neuronal cell bodies and their dendrites, along with axons and central neuroglia (Plate 31, ventral ramus of spinal nerve FIGURE 12.28 • Cytoarchitecture of the cerebellar cortex. a.

1	This diagram shows a section of the folium, a narrow, leaflike gyrus of the cerebellar cortex. Note that the cerebellar cortex contains white matter and gray matter. Three distinct layers of gray matter are identified on this diagram: The superficially located molecular layer, the middle Purkinje cell layer, and the granule cell layer adjacent to the white matter. b. Purkinje cell layer from rat cerebellum visualized using double-fluorescent–labeling methods. Red DNA staining indicates the nuclei of cells in molecular and granular cell layer thin section. Note that each Purkije cell exhibits an abundance of dendrites. 380. (Coutesy of Thomas J. Deerinck.) page 398). Functionally related groups of nerve cell bodies in the gray matter are called nuclei. In this context, the term nucleus means a cluster or group of neuronal cell bodies plus fibers and neuroglia. Nuclei of the CNS are the morphologic and functional equivalents of the ganglia of the PNS. Synapses occur only in the gray

1	means a cluster or group of neuronal cell bodies plus fibers and neuroglia. Nuclei of the CNS are the morphologic and functional equivalents of the ganglia of the PNS. Synapses occur only in the gray matter.

1	The cell bodies of motor neurons that innervate striated muscle are located in the ventral (anterior) horn of the gray matter. Ventral motor neurons, also called anterior horn cells, are large basophilic cells easily recognized in routine histologic preparations (see Fig. 12.30 and Plate 31, page 398). Because the motor neuron conducts impulses away from the CNS, it is an efferent neuron. The axon of a motor neuron leaves the spinal cord, passes through the ventral (anterior) root, becomes a component of

1	The axon of a motor neuron leaves the spinal cord, passes through the ventral (anterior) root, becomes a component of FIGURE 12.29 • Posterior view of the spinal cord with surrounding meninges. Each spinal nerve arises from the spinal cord by rootlets, which merge together to form dorsal (posterior) and ventral (anterior) nerve roots. These roots unite to form a spinal nerve that, after short course, divides into larger ventral (anterior) and smaller dorsal (posterior) primary rami. Note the dura mater (the outer layer of the meninges) surrounds the spinal cord and emerging spinal nerves. The denticulate ligament of the pia mater that anchors the spinal cord to the wall of the spinal canal is also visible. the spinal nerve of that segment, and as such is conveyed to the muscle. The axon is myelinated except at its origin and termination. Near the muscle cell, the axon divides into numerous terminal branches that form neuromuscular junctions with the muscle cell (see page 322).

1	The cell bodies of sensory neurons are located in ganglia that lie on the dorsal root of the spinal nerve. Sensory neurons in the dorsal root ganglia are pseudounipolar (Plate 27, page 390). They have a single process that divides into a peripheral segment that brings information from the periphery to the cell body and a central segment that carries information from the cell body into the gray matter of the spinal cord. Because the sensory neuron conducts impulses to the CNS, it is an afferent neuron. Impulses are generated in the terminal receptor arborization of the peripheral segment. Connective Tissue of the Central Nervous System Three sequential connective tissue membranes, the meninges, cover the brain and spinal cord.  The dura mater is the outermost layer.  The arachnoid layer lies beneath the dura.  The pia mater is a delicate layer resting directly on the surface of the brain and spinal cord.

1	FIGURE 12.30 • Cross-section of the human spinal cord. The photomicrograph shows a cross-section through the lower lumbar (most likely L4–L5) level of the spinal cord stained by the Bielschowsky silver method. The spinal cord is organized into an outer part, the white matter, and an inner part, the gray matter that contains nerve cell bodies and associated nerve fibers. The gray matter of the spinal cord appears roughly in the form of a butterfly. The anterior and posterior prongs are referred to as ventral horns (VH) and dorsal horns (DH), respectively. They are connected by the gray commissure (GC). The white matter contains nerve fibers that form ascending and descending tracts. The outer surface of the spinal cord is surrounded by the pia mater. Blood vessels of the pia mater, the ventral fissure (VF), and some dorsal roots of the spinal nerves are visible in the section. 5.

1	Because arachnoid and pia mater develop from the single layer of mesenchyme surrounding the developing brain, they are commonly referred as the pia-arachnoid. In adults, pia mater represents the visceral portion, and arachnoid represents the parietal portion of the same layer. This common origin of pia-arachnoid is evident in adult meninges in which numerous strands of connective tissue (arachnoid trabeculae) pass between pia mater and arachnoid. The dura mater is a relatively thick sheet of dense connective tissue.

1	The dura mater is a relatively thick sheet of dense connective tissue. In the cranial cavity, the thick layer of connective tissue that forms the dura mater (L., tough mother) is continuous at its outer surface with the periosteum of the skull. Within the dura mater are spaces lined by endothelium (and backed by periosteum and dura mater, respectively) that serve as the principal channels for blood returning from the brain. These venous (dural) sinuses receive blood from the principal cerebral veins and carry it to the internal jugular veins. Sheetlike extensions of the inner surface of the dura mater form partitions between parts of the brain, supporting those parts within the cranial cavity and carrying the arachnoid to some of the deeper parts of the brain. In the spinal canal, the vertebrae have their own periosteum, and the dura mater forms a separate tube surrounding the spinal cord (see Fig. 12.29).

1	The arachnoid is a delicate sheet of connective tissue adjacent to the inner surface of the dura. The arachnoid abuts on the inner surface of the dura and extends delicate arachnoid trabeculae to the pia mater on the surface of the brain and spinal cord. The weblike trabeculae of the arachnoid give this tissue its name (Gr., resembling a spider’s web). Trabeculae are composed of loose connective tissue fibers containing elongated fibroblasts. The space bridged by these trabeculae is the subarachnoid space; it contains the cerebrospinal fuid (Fig. 12.31). The pia matter lies directly on the surface of brain and spinal cord. The pia mater (L., tender mother) is also a delicate connective tissue layer. It lies directly on the surface of the brain and spinal cord and is continuous with the perivascular connective tissue sheath of the blood vessels of the brain and spinal cord. Both surfaces of the arachnoid, the inner FIGURE 12.31 • Schematic diagram of the cerebral meninges.

1	FIGURE 12.31 • Schematic diagram of the cerebral meninges. The outer layer, the dura mater, is joined to adjacent bone of the cranial cavity (not shown). The inner layer, the pia mater, adheres to the brain surface and follows all its contours. Note that the pia mater follows the branches of the cerebral arteries as they enter the cerebral cortex. The intervening layer, the arachnoid, is adjacent but not attached to the dura mater. The arachnoid sends numerous, weblike arachnoid trabeculae to the pia mater. Located between the arachnoid and the pia mater is the subarachnoid space; it contains cerebrospinal fluid. The space also contains the larger blood vessels (cerebral arteries) that send branches into the substance of the brain. surface of the pia mater, and the trabeculae are covered with a thin squamous epithelial layer. Both the arachnoid and pia mater fuse around the opening for the cranial and spinal nerves as they exit the dura mater.

1	The blood–brain barrier protects the CNS from fluctuating levels of electrolytes, hormones, and tissue metabolites circulating in the blood vessels. The observation more than 100 years ago that vital dyes injected into the bloodstream can penetrate and stain nearly all organs except the brain provided the first description of the blood–brain barrier. More recently, advances in microscopy and molecular biology techniques have revealed the precise location of this unique barrier and the role of endothelial cells in transporting essential substances to the brain tissue.

1	The blood–brain barrier develops early in the embryo through an interaction between glial astrocytes and capillary endothelial cells. The barrier is created largely by the elaborate tight junctions between the endothelial cells, which form continuous-type capillaries. Studies with the TEM using electron-opaque tracers show complex tight junctions between the endothelial cells. Morphologically, these junctions more closely resemble epithelial tight junctions than tight junctions present between other endothelial cells. In addition, TEM studies reveal a close association of astrocytes and their end foot processes with the endothelial basal lamina (Fig. 12.32). The tight junctions eliminate gaps between endothelial cells and prevent simple diffusion of solutes and fluid into the neural tissue. Evidence suggests that the integrity of blood–brain barrier tight junctions depends on normal functioning of the associated astrocytes. In several brain diseases, the blood–brain barrier loses

1	Evidence suggests that the integrity of blood–brain barrier tight junctions depends on normal functioning of the associated astrocytes. In several brain diseases, the blood–brain barrier loses effectiveness. Examination of brain tissue in these conditions by TEM reveals loss of the tight junctions as well as alterations in the morphology of astrocytes. Other experimental evidence has revealed that astrocytes release soluble factors that increase barrier properties and tight junction protein content.

1	The blood–brain barrier restricts passage of certain ions and substances from the bloodstream to tissues of the CNS. The presence of only a few small vesicles indicates that pinocytosis across the brain endothelial cells is severely restricted. Substances with a molecular weight greater than 500 daltons generally cannot cross the blood–brain barrier. Many molecules that are required for neuronal integrity leave and enter the blood capillaries through the endothelial cells. Thus, O2 and CO2 as well as certain lipid-soluble molecules (e.g., ethanol and steroid hormones) easily penetrate the endothelial cells and pass freely between the blood and extracellular fluid of the CNS. Due to the high K permeability of the neuronal membrane, neurons are particularly sensitive to changes in the concentration of extracellular K. As previously discussed, astrocytes are foot processes of astrocytes FIGURE 12.32 • Schematic drawing of blood–brain barrier.

1	FIGURE 12.32 • Schematic drawing of blood–brain barrier. This drawing shows the blood–brain barrier, which consists of endothelial cells joined together by elaborate, complex tight junctions, endothelial basal lamina, and the end foot processes of astrocytes. responsible for buffering the concentration of K in the brain extracellular fluid (page 369). They are assisted by endothelial cells of the blood–brain barrier that effectively limit movement of K into the extracellular fluid of the CNS. Substances that do cross the capillary wall are actively transported by specific receptor-mediated endocytosis. For instance, glucose (which the neuron depends on almost exclusively for energy), amino acids, nucleosides, and vitamins are actively transported by specific transmembrane carrier proteins. The permeability of the blood–brain barrier to these macromolecules is attributable to the level of expression of specific carrier proteins on the endothelial cell surface.

1	Several other proteins that reside within the plasma membrane of endothelial cells protect the brain by metabolizing certain molecules, such as drugs and foreign proteins, thus preventing them from crossing the barrier. For example, L-dopa (levodopa), the precursor of the neuromediators dopamine and noradrenaline, easily crosses the blood–brain barrier. However, the dopamine formed from the decarboxylation of L-dopa in endothelial cells cannot cross the barrier and is restricted from the CNS. In this case, the blood–brain barrier regulates the concentration of L-dopa in the brain. Clinically, this restriction explains why L-dopa is administered for the treatment of dopamine deficiency (e.g., Parkinson’s disease) rather than dopamine.

1	Recent studies indicate that the end feet of astrocytes also play an important role in maintaining water homeostasis in brain tissue. Water channels (aquaporin AQP4) are found in end foot processes in which water crosses the blood–brain barrier. In pathologic conditions such as brain edema, these channels play a key role in reestablishing osmotic equilibrium in the brain. The midline structures bordering the third and fourth ventricles are unique areas of the brain that are outside the blood–brain barrier.

1	Some parts of the CNS, however, are not isolated from substances carried in the bloodstream. The barrier is ineffective or absent in the sites located along the third and fourth ventricles of the brain, which are collectively called circumventricular organs. Circumventricular organs include the pineal gland, median eminence, subfornical organ, area postrema, subcommissural organ, organum vasculosum of the lamina terminalis, and posterior lobe of the pituitary gland. These barrierdeficient areas are most likely involved in sampling of materials circulating in the blood that are normally excluded by the blood–brain barrier and then conveying information about these substances to the CNS. Circumventricular organs are important in regulating body fluid homeostasis and controlling neurosecretory activity of the nervous system. Some researchers describe them as “windows of the brain” within central neurohumoral system.

1	Neuronal injury induces a complex sequence of events termed axonal degeneration and neural regeneration. Neurons, Schwann cells, oligodendrocytes, macrophages, and microglia are involved in these responses. In contrast to the PNS, in which injured axons rapidly regenerate, axons severed in the CNS usually cannot regenerate. This striking difference is most likely related to the inability of oligodendrocytes and microglia cells to phagocytose myelin debris quickly and the restriction of large numbers of migrating macrophages by the blood–brain barrier. Because myelin debris contains several inhibitors of axon regeneration, its removal is essential to the regeneration progress. The portion of a nerve fiber distal to a site of injury degenerates because of interrupted axonal transport.

1	Degeneration of an axon distal to a site of injury is called anterograde (Wallerian) degeneration (Fig. 12.33, a and b). The first sign of injury, which occurs 8–24 hours after the axon is damaged, is axonal swelling followed by its disintegration. This leads to breakdown of the axonal cytoskeleton. Microtubules, neurofilaments, and other cytoskeleton components are disassembled, resulting in the fragmentation of the axon. This process is known as granular disintegration of the axonal cytoskeleton. In the PNS, loss of axon contact causes dedifferentiation of Schwann cells and breakdown of the myelin sheath that enclosed the axon. Schwann cells downregulate expression of myelin-specific proteins (see page 365) and at the same time upregulate and secrete several glial growth factors (GGFs), members of a family of axon-associated neuregulins and potent stimulators of proliferation. Under the influence of GGFs, Schwann cells divide and arrange themselves in a line along their external

1	members of a family of axon-associated neuregulins and potent stimulators of proliferation. Under the influence of GGFs, Schwann cells divide and arrange themselves in a line along their external laminae. Since axonal processes distal to the site of injury have been by phagocytosis, the linear arrangement of the Schwann cells’ external laminae resembles a long tube with an empty lumen (Fig. 12.33b). In the CNS, oligodendrocyte survival is dependent on signals from axons. In contrast to Schwann cells, if oligodendrocytes lose contact with axons, they respond by initiating apoptotic programmed cell death.

1	The most important cells in clearing myelin debris from the site of nerve injury are monocyte-derived macrophages. In the PNS, even before the arrival of phagocytotic cells at the site of nerve injury, Schwann cells initiate removal of myelin debris. Recent studies demonstrate that resident macrophages (normally present in small numbers in the peripheral nerves) become activated after nerve injury. They migrate to the site of nerve injury, proliferate, and then phagocytize myelin debris.

1	The efficient clearance of myelin debris in the PNS is attributed to the massive recruitment of monocyte-derived macrophages that migrate from blood vessels and infiltrate the vicinity of the nerve injury (Fig 12. 34). When an axon is injured, the blood–nerve barrier (see page 376) is disrupted along the entire length of the injured axon, which allows for the influx of these cells into the site of injury. The presence of large numbers of monocyte-derived macrophages speeds up the process of myelin removal, which in peripheral nerves is usually completed within 2 weeks. In the CNS, inefficient clearance of myelin debris due to limited access of monocyte-derived macrophages, the inefficient phagocytic activity of microglia, and the formation of an astrocyte-derived scar severely restrict nerve regeneration.

1	A key difference in the CNS response to axonal injury relates to the fact that the blood–brain barrier (see page 385) is disrupted only at the site of injury and not along entire length of the injured axon (see Fig 12.34). This limits infiltration of monocyte-derived macrophages to the CNS and dramatically slows the process of myelin removal, which can take months or even years. Although the number of microglial cells increases at sites of CNS injury, these reactive microglia cells do not possess the full phagocytotic capabilities of migrating macrophages. The inefficient clearance of myelin debris is a major factor in the failure of nerve regeneration in the CNS. Another factor that affects nerve regeneration is the formation of a glial (astrocyte-derived) scar that fills the empty space left by degenerated axons. Scar formation is discussed in Folder 12.3. Traumatic degeneration occurs in the proximal part of the injured nerve.

1	Traumatic degeneration occurs in the proximal part of the injured nerve. FIGURE 12.33 • Response of a nerve fber to injury. a. A normal nerve fiber at the time of injury, with its nerve cell body and the effector cell (striated skeletal muscle). Note the position of the neuron nucleus and the number and distribution of Nissl bodies.

1	b. When the fiber is injured, the neuronal nucleus moves to the cell periphery, and the number of Nissl bodies is greatly reduced. The nerve fiber distal to the injury degenerates along with its myelin sheath. Schwann cells dedifferentiate and proliferate; myelin debris are phagocytosed by macrophages. c. Proliferated Schwann cells form cellular cords of Bunger that are penetrated by the growing axonal sprout. The axon grows at a rate of 0.5 to 3 mm/day. Note that the muscle fiber show a pronounced atrophy. d. A confocal immunofluorescent image showing reinnervated skeletal muscle of the mouse. Regenerating motor axons are stained green for neurofilaments reestablished connections with two neuromuscular junctions visualized in pink color, which reflects specific staining for postsynaptic acetylcholine receptors; Schwann cells are stained blue for S100, which represents a Schwann cell–specific calcium-binding protein. Regenerating axons extended along Schwann cells, which led them to

1	acetylcholine receptors; Schwann cells are stained blue for S100, which represents a Schwann cell–specific calcium-binding protein. Regenerating axons extended along Schwann cells, which led them to the original synaptic sites of the muscle fibers. 640. (Courtesy of Dr. Young-Jin Son.)

1	Some retrograde degeneration also occurs in the proximal axon and is called traumatic degeneration. This process appears to be histologically similar to antegrade (Wallerian) degeneration. The coverage of traumatic degeneration depends on the severity of the injury and usually extends for only one or a few internodal segments. Sometimes, traumatic degeneration extends more proximally than one or a few nodes of Ranvier and may result in death of the cell body. When a motor fiber is cut, the muscle innervated by that fiber undergoes atrophy (Fig. 12.33c). Retrograde signaling to the cell body of an injured nerve causes a change in gene expression that initiates reorganization of the perinuclear cytoplasm.

1	Retrograde signaling to the cell body of an injured nerve causes a change in gene expression that initiates reorganization of the perinuclear cytoplasm. Axonal injury also initiates retrograde signaling to the nerve cell body leading to the upregulation of a gene called c-jun. C-jun transcription factor is involved in early as well as later stages of nerve regeneration. Reorganization of the perinuclear cytoplasm and organelles starts within a few days. The cell body of the injured nerve swells, and its nucleus moves peripherally. Initially, Nissl bodies disappear from the center of the neuron and move to the periphery of the neuron in a process called chromatolysis. Chromatolysis is first observed within 1 to 2 days after injury and reaches a peak at about 2 weeks (see Fig. 12.33b). The changes in the cell body are proportional to the amount of axoplasm destroyed by the injury; extensive loss of axoplasm can lead to death of the cell.

1	Before the development of modern dyes and radioisotope tracer techniques, Wallerian degeneration and chromatolysis were used as research tools. These tools allowed researchers to trace the pathways and destination of axons and the localization of the cell bodies of experimentally injured nerves. In the PNS, Schwann cells divide and develop cellular bands that bridge a newly formed scar and direct growth of new nerve processes. As mentioned above, division of dedifferentiated Schwann cells is the first step in the regeneration of a severed or crushed peripheral nerve. Initially, these cells arrange themselves in a series of cylinders called endoneurial tubes. Removal of myelin failure of axon regeneration

1	FIGURE 12.34 • Schematic diagram of response to neuronal injury within peripheral and central nervous systems. Injuries of nerve processes (axons and dendrites) both in PNS and CNS induce axonal degeneration and neural regeneration. These processes involve not only neurons but also supportive cells such as Schwann cells and oligodendrocytes, as well as phagocytic cells such as macrophages and microglia. Injuries to axons in PNS lead to their degeneration, which accompanies divisions and dedifferentiation of Schwann cells and disruption of the blood–nerve barrier along entire length of the injured axon. This allows massive infiltration of monocyte-derived macrophages, which are responsible for the process of myelin removal. Rapid clearance of myelin debris allows for axon regeneration and subsequential restoration of blood–nerve barrier. In the CNS, limited disruption of the blood–brain barrier restricts infiltration of monocyte-derived macrophages and dramatically slows the process of

1	restoration of blood–nerve barrier. In the CNS, limited disruption of the blood–brain barrier restricts infiltration of monocyte-derived macrophages and dramatically slows the process of myelin removal. In addition, appoptosis of oligodendrocytes, an inefficient phagocytic activity of microglia, and the formation of an astrocyte-derived scar lead to failure in nerve regeneration in the CNS.

1	and axonal debris from inside the tubes causes them to eventu-They preferentially interact with proteins of the extracellular ally collapse. Proliferating Schwann cells organize themselves matrix such as fibronectin and laminin found within the into cellular bands resembling longitudinal columns called external lamina of the Schwann cell. Thus, if a sprout associates bands of Bungner. Cellular bands guide the growth of new itself with a band of Bungner, it regenerates between the layers nerve processes (neurites or sprouts) of regenerating axons. of external lamina of the Schwann cell. This sprout will grow Once the bands are in place, large numbers of sprouts begin to along the band at a rate of about 3 mm per day. Although many grow from the proximal stump (see Fig. 12.33c). A growth new sprouts do not make a contact with cellular bands and decone develops in the distal portion of each sprout that consists generate, their large number increases the probability of of filopodia rich

1	sprouts do not make a contact with cellular bands and decone develops in the distal portion of each sprout that consists generate, their large number increases the probability of of filopodia rich in actin filaments. The tips of the filopodia reestablishing sensory and motor connections. After crossing establish a direction for the advancement of the growth cone. the site of injury, sprouts enter the surviving cellular bands in  FOLDER 12.3 Clinical Correlation: Reactive Gliosis: Scar Formation in the CNS

1	When a region of the CNS is injured, astrocytes near the leasion become activated. They divide and undergo marked hypertrophy with a visible increase in the number of their cytoplasmic processes. In time, the processes be-come densly packed with GFAP intermediate fla-ments. Eventually, scar tissue is formed. This process is referred to as reactive gliosis, while the resulting per-manent scar is most often called a plaque. Reactive glio-sis varies widely in duration, degree of hyperplasia, and time course of expression of GFAP immunostaining. Se-veal biological mechanisms for induction and mainte-nance of reactive gliosis have been proposed. The type of glial cell that responds during reactive gliosis depends on the brain structure that is damagd. In addition, activation of microglial cell population occurs almost immedietaly after any kind of injury to the CNS. These reactive mi-croglial cells migrate toward the site of injury and exhibit marked phagocitic activity. However, their

1	population occurs almost immedietaly after any kind of injury to the CNS. These reactive mi-croglial cells migrate toward the site of injury and exhibit marked phagocitic activity. However, their phagocitic ac-tivity and ability to remove myelin debris is much less than that of monocyte-derived macrophages. Gliosis is a prominent feature of many diseases of the CNS, includ-ing stroke, neurotoxic damage, genetic diseases, inflam-matory demyelination, and neurodegenerative disorders such as multiple sclerosis. Much of the research in CNS regeneration is focused on preventing or inhibiting glial scar formation.

1	the distal stump. These bands then guide the neurites to their destination as well as provide a suitable microenvironment for continued growth (Fig. 12.33d). Axonal regeneration leads to Schwann cell redifferentiation, which occurs in a proximal-todistal direction. Redifferentiated Schwann cells upregulate genes for myelin-specific proteins and downregulate c-jun. If physical contact is reestablished between a motor neuron and its muscle, function is usually reestablished.

1	If physical contact is reestablished between a motor neuron and its muscle, function is usually reestablished. Microsurgical techniques that rapidly reestablish intimate apposition of severed nerve and vessel ends have made reat tachment of severed limbs and digits, with subsequent reestablishment of function, a relatively common procedure. If the axonal sprouts do not reestablish contact with the appropriate Schwann cells, then the sprouts grow in a disorganized manner, resulting in the mass of tangled axonal processes known as traumatic neuroma or amputation neuroma. Clinically, traumatic neuroma usually appears as a freely movable nodule at the site of nerve injury and is characterized by pain, particularly on palpation. Traumatic neuroma of the injured motor nerve prevents reinnervation of the affected muscle.

1	Ganglia are clusters of neuronal cell bodies located outside the central nervous system (CNS); nerve fibers lead to and from them. Sensory ganglia lie just outside the CNS and contain the cell bodies of sensory nerves that carry impulses into the CNS. Autonomic ganglia are pe-ripheral motor ganglia of the autonomic nervous system (ANS) and contain the cell bodies of postsynaptic neurons that conduct nerve im-pulses to smooth muscle, cardiac muscle, and glands. Synapses between presynaptic neurons (all of which have their cell bodies in the CNS) and postsynaptic neurons occur in autonomic ganglia. Sympathetic ganglia constitute a major subclass of autonomic ganglia; parasym-pathetic ganglia and enteric ganglia constitute the other subclasses. Sympathetic ganglia are located in the sympathetic chain (paravertebral ganglia) and on the anterior surface of the aorta (prevertebral ganglia). They send long postsynaptic axons to the viscera. Parasympathetic ganglia (terminal ganglia) are

1	chain (paravertebral ganglia) and on the anterior surface of the aorta (prevertebral ganglia). They send long postsynaptic axons to the viscera. Parasympathetic ganglia (terminal ganglia) are located in, or close to, the or-gans innervated by their postsynaptic neurons. The enteric ganglia are located in the submucosal plexus and the myenteric plexus of the ali-mentary canal. They receive parasympathetic presynaptic input as well as intrinsic input from other enteric ganglia and innervate smooth muscle of the gut wall.

1	Sympathetic ganglion, human, silver and H&E stains ×160. A sympathetic ganglion stained with silver and counter-stained with H&E is illustrated here. Shown to advantage are several discrete bundles of nerve fibers (NF) and numerous large circular structures, namely, the cell bodies (CB) of the postsynaptic neurons. Random patterns of nerve fibers are also seen. Moreover, careful examination of the cell bodies shows that some display several processes joined to them. Thus, these are multipolar neurons (one contained within the rectangle is shown at higher magnification). Generally, the connective tissue is not conspicuous in a silver preparation, although it can be identified by virtue of its location about the larger blood vessels (BV), particularly in the upper part of this figure.

1	PLATE 27 • SYM PATH ETIC AN D DORSAL ROOT GANG LIA KEY A, axon BV, blood vessels CB, cell body of neuron CT, connective tissue L, lipofuscin N, nucleus of nerve cell NF, nerve fibers NL, nucleolus P, processes of nerve cell body Sat C, satellite cells arrowheads, neurilemma asterisks, clusters of satellite cells Sympathetic ganglion, human, silver and H&E stains ×500.

1	Sympathetic ganglion, human, silver and H&E stains ×500. The cell bodies of the sympathetic ganglion are typically large, and the one labeled here shows several processes (P). In addition, the cell body contains a large, pale-staining spherical nucleus (N); this, in turn, contains a spherical, intensely staining nucleolus (NL). These features, namely, a large pale-staining nucleus (indicating much-extended chromatin) and a large nucleolus, reflect a cell active in protein synthesis. Also shown in the cell body are accumulations of lipofuscin (L), a yellow pigment that is darkened by the silver. Because of the large size of the cell body, the nucleus is not always included in the section; in that case, the cell body appears as a rounded cytoplasmic mass. Dorsal root ganglion, cat, H&E ×160.

1	Dorsal root ganglion, cat, H&E ×160. Dorsal root ganglia differ from autonomic ganglia in a number of ways. Whereas the latter contain multipolar neurons and have synaptic connections, dorsal root ganglia contain pseudounipolar sensory neurons and have no synaptic connections in the ganglion. Part of a dorsal root ganglion stained with H&E is shown in this figure. The specimen includes the edge of the ganglion, where it is covered with connective tissue (CT). The dorsal root ganglion contains large cell bodies (CB) that are typically arranged as closely packed clusters. Also, between and around the cell clusters, there are bundles of nerve fibers (NF). Most of the fiber bundles indicated by the label have been sectioned longitudinally. Dorsal root ganglion, cat, H&E ×350.

1	Dorsal root ganglion, cat, H&E ×350. At higher magnification of the same ganglion, the constituents of the nerve fiber show their characteristic structure, namely, a centrally located axon (A) surrounded by a myelin space (not labeled), which, in turn, is bounded on its outer border by the thin cytoplasmic strand of the neurilemma (arrowheads). The cell bodies of the sensory neurons display large, pale-staining spherical nuclei (N) and intensely staining nucleoli (NI). Also seen in this H&E preparation are the nuclei of satellite cells (Sat C) that completely surround the cell body and are continuous with the Schwann cells that invest the axon. Note how much smaller these cells are than the neurons. Clusters of cells (asterisks) within the ganglion that have an epithelioid appearance are en face views of satellite cells where the section tangentially includes the satellite cells but barely grazes the adjacent cell body.

1	Peripheral nerves are composed of bundles of nerve fibers held together by connective tissue and a specialized layer (or layers) of cells, the perineurium. The connective tissue consists of an outer layer, the epineurium, surrounding the whole nerve; the perineurium, surrounding bundles of nerve fibers; and the endoneurium, associated with individual neurons. Each nerve fiber consists of an axon that is surrounded by a cellular investment called the neurilemma, or the sheath of Schwann. The fiber may be myelinated or unmyelinated. The myelin, if present, is immediately around the axon and is formed by the concentric wrapping of the Schwann cell around the axon. This, in turn, is surrounded by the major portion of the cytoplasm of the Schwann cell, forming the neurilemma. Unmyelinated axons rest in grooves in the Schwann cell. Peripheral nerve, cross-section, femoral nerve, H&E. ×200 and 640

1	Peripheral nerve, cross-section, femoral nerve, H&E. ×200 and 640 This cross-section shows several bundles of nerve fibers (BNF). The external cover for the entire nerve is the epineurium (Epn), the layer of dense connective tissue that one touches when a nerve has been exposed during a dissection. The epineurium may also serve as part of the outermost cover of individual bundles. It contains blood vessels (BV) and may contain some fat cells. Typically, adipose tissue (AT) is found about the nerve. Figure on right shows, at higher magnification, the perineurial septum (marked with arrows on left image, which is now rotated and vertically disposed (arrows)).

1	Figure on right shows, at higher magnification, the perineurial septum (marked with arrows on left image, which is now rotated and vertically disposed (arrows)). The layer under the epineurium that directly surrounds the bundle of nerve fibers is the perineurium (Pn). As seen in the cross-section through the nerve, the nuclei of the perineurial cells appear flat and elongate; they are actually being viewed on edge and belong to flat cells that are also being viewed on edge. Again, as noted by the distribution of nuclei, it can be ascertained that the perineurium is only a few cells thick. The perineurium is a specialized layer of cells and extracellular material whose arrangement is not evident in H&E sections. The perineurium (Pn) and epineurium (Epn) are readily distinguished in the triangular area formed by the diverging perineurium of the two adjacent nerve bundles.

1	The nerve fibers included in Figure on right are mostly myelinated, and because the nerve is cross-sectioned, the nerve fibers are also seen in this plane. They have a characteristic cross-sectional profile. Each nerve fiber shows a centrally placed axon (A); this is surrounded by a myelin space (M) in which some radially disposed precipitate may be retained, as in this specimen. External to the myelin space is a thin cytoplasmic rim representing the neurilemma. On occasion, a Schwann cell nucleus (SS ) appears to be perched on the neurilemma. As shown in the illustration, the upper edge of the nuclear crescent appears to occupy the same plane as that occupied by the neurilemma (N). These features enable one to identify the nucleus as belonging to a Schwann (neurilemma) cell. Other nuclei are not related to the neurilemma but, rather, appear to be between the nerve fibers. Such nuclei belong to the rare fibroblasts (F) of the endoneurium. The latter is the delicate connective tissue

1	are not related to the neurilemma but, rather, appear to be between the nerve fibers. Such nuclei belong to the rare fibroblasts (F) of the endoneurium. The latter is the delicate connective tissue between the individual nerve fibers; it is extremely sparse and contains the capillaries (C) of the nerve bundle.

1	Peripheral nerve, longitudinal section, femoral nerve, H&E. ×200 and 640

1	The edge of a longitudinally sectioned nerve bundle is shown on left; a portion of the same nerve bundle is shown at higher magnification on right image. The boundary between the epineurium (Epn) and perineurium is ill-defined. Within the nerve bundle, the nerve fibers show a characteristic wavy pattern. Included among the wavy nerve fibers are nuclei belonging to Schwann cells and to cells within the endoneurium. Higher magnification allows one to identify certain specific components of the nerve. Note that the nerve fibers (NF) are now shown in longitudinal profile. Moreover, each myelinated nerve fiber shows a centrally positioned axon (A) surrounded by a myelin space (M), which, in turn, is bordered on its outer edge by the thin cytoplasmic band of the neurilemma cell (NI). Another diagnostic feature of myelinated nerve fibers is also seen in longitudinal section, namely, the node of Ranvier (NR). This is where the ends of the two Schwann cells meet. Histologically, the node

1	diagnostic feature of myelinated nerve fibers is also seen in longitudinal section, namely, the node of Ranvier (NR). This is where the ends of the two Schwann cells meet. Histologically, the node appears as a constriction of the neurilemma, and sometimes, the constriction is marked by a cross-band, as in figure on right. It is difficult to determine whether the nuclei (N) shown here belong to Schwann cells or to endoneurial fibroblasts.

1	KEY A, axon AT, adipose tissue BNF, bundle of nerve fibers BV, blood vessels C, capillary Epn, epineurium F, fibroblast M, myelin N, nucleus of Schwann cell NF, nerve fiber Nl, neurilemma NR, node of Ranvier Pn, perineurium SS, Schwann cell nucleus arrows, septum formed by perineurium The cerebrum is the principal portion of the brain and contains the cell bodies of nerves that receive and store sensory information, nerves that control voluntary motor activity, and nerves that integrate and coordinate the activity of other nerves, as well as the nerves and neural pathways that constitute memory. Cerebral cortex, brain, human, Luxol fast blue-PAS ×65.

1	This micrograph shows a low-magnification view of the cerebral cortex (CC). It includes the full thickness of the gray matter and a small amount of white matter at the bottom of the micrograph (WM). The white matter contains considerably fewer cells per unit area; these are neuroglial cells rather than nerve cell bodies that are present in the cortex. Covering the cortex is the pia mater (PM). A vein (V) can be seen enclosed by the pia mater. Also, a smaller blood vessel (BV) can be seen entering the substance of the cortex. The six layers of the cortex are marked by dashed lines, which represent only an approximation of the 394 boundaries. Each layer is distinguished on the basis of predominant cell types and fiber (axon and dendrite) arrangement. Unless the fibers are specifically stained, they cannot be utilized to further aid in identification of the layers. Rather, the delineation of the layers, as they are identified here, is based on cell types, and more specifically, the shape

1	they cannot be utilized to further aid in identification of the layers. Rather, the delineation of the layers, as they are identified here, is based on cell types, and more specifically, the shape and appearance of the cells. The six layers of the cortex are named and described as follows:

1	I: The plexiform layer (or molecular layer) consists largely of fibers, most of which travel parallel to the surface, and relatively few cells, mostly neuroglial cells and occasional horizontal cells of Cajal. II: The small pyramidal cell layer (or outer granular layer) consists mainly of small pyramidal cells, and granule cells, also called stellate cells. III: The layer of medium pyramidal cells (or layer of outer pyramidal cells) is not sharply demarcated from layer II. However, the pyramidal cells are somewhat larger and posses a typical pyramidal shape. IV: The granular layer (or inner granular layer) is characterized by the presence of many small granule cells (stellate cells). V: The layer of large pyramidal cells (or inner layer of pyramidal cells) contains pyramidal cells that, in many parts of the cerebrum, are smaller than the pyramidal cells of layer III but in the motor area are extremely large and are called Betz cells.

1	VI: The layer of polymorphic cells contains cells with diverse shapes, many of which have a spindle of fusiform shape. These cells are called fusiform cells. In addition to pyramidal cells, granule cells, and fusiform cells, two other cell types are also present in the cerebral cortex but are not recognizable in this preparation: the horizontal cells of Cajal, which are present only in layer I and send their processes laterally, and the cells of Martinotti, which send their axons toward the surface (opposite to that of pyramidal cells). Layer I of cerebral cortex, brain, human, Luxol fast cells appear as naked nuclei, with the cytoplasm being indistinguishable blue—PAS ×350. from the nerve fibers that make up the bulk of this layer. Also present is a small capillary (Cap). The pink outline of the vessel is due to the PAS stain-This micrograph is a higher power of layer I, the plexiform ing reaction of its basement membrane.

1	layer. It consists of nerve fibers, numerous neuroglial cells (NN) and occasional horizontal cells of Cajal. The neuroglial Layer II of cerebral cortex, brain, human, Luxol fast blue—PAS ×350. This micrograph shows layer II, the small pyramidal cell layer. Many small pyramidal cells (PC) are present. Granule cells (GC) are also numerous, though difficult to identify here. Layer IV of cerebral cortex, brain, human, Luxol fast prominent. The micrograph also reveals a number of capillaries. Note how blue—PAS ×350. they travel in various directions. This micrograph shows layer IV, the granular layer. Many of the cells here are granule cells, but neuroglial cells are also

1	Layer VI of cerebral cortex, brain, human, Luxol fast region. Pyramidal cells (PC) are readily recognized. Other cell types present blue—PAS ×350. include fusiform cells (FC), granule cells, and Martinotti cells. This micrograph shows layer VI, the layer of polymorphic cells, so named because of the diverse shape of the cells in this White matter, brain, human, Luxol fast blue—PAS ×350. the cortex, the cytoplasm of the cell is not distinguishable. Thus, they appear as naked nuclei in the bed of nerve processes. The neuropil is essentially a This micrograph shows the outer portion of the white matter. densely packed aggregation of nerve fibers and neuroglial cells. The small round nuclei (NN) belong to neuroglial cells. As in KEY BV, blood vessel Cap, capillary CC, cerebral cortex FC, fusiform cells GC, granule cells NN, neuroglial nuclei PC, pyramidal cells PM, pia mater V, vein WM, white matter

1	KEY BV, blood vessel Cap, capillary CC, cerebral cortex FC, fusiform cells GC, granule cells NN, neuroglial nuclei PC, pyramidal cells PM, pia mater V, vein WM, white matter The cerebellum is a portion of the brain lying behind and below the cerebrum; it serves to coordinate both voluntary movements and muscle function in the maintenance of normal posture. Cerebellum, brain, human, H&E ×40. The cerebellar cortex has the same appearance regardless of which region is examined. In this low-magnification view of the cerebellum, the outermost layer, the molecular layer (Mol), is lightly stained with eosin. Under this is the granular layer (Gr), which stains intensely with hematoxylin. Together, these two layers constitute the cortex of the cerebellum. Deep in the granular layer is Cerebellum, brain, human, H&E ×400.

1	At the junction between the molecular and granular layers are the extremely large flask-shaped cell bodies of the Purkinje cells (Pkj ). These cells are characteristic of the cerebellum. Each possesses numerous dendrites (D) that arborize in the molecular layer. The Purkinje cell has a single axon that is not usually evident in H&E sections. This nerve fiber represents the beginning of the outflow from the cerebellum. The figure shows relatively few neuron cell bodies, those of the basket cells (BC ), in the molecular layer; they are widely removed from each other and, at another region that stains lightly with H&E and, except for location, shows no distinctive histologic features. This is the white matter (WM ). As in the cerebrum, it contains nerve fibers, supporting neuroglial cells, and small blood vessels, but no neuronal cell bodies. The fibrous cover on the cerebellar surface is the pia mater (Pia). Cerebellar blood vessels (BV ) travel in this layer. (Shrinkage artifact has

1	and small blood vessels, but no neuronal cell bodies. The fibrous cover on the cerebellar surface is the pia mater (Pia). Cerebellar blood vessels (BV ) travel in this layer. (Shrinkage artifact has separated the pia mater from the cerebellar surface.) The rectangular area is shown at higher magnification in Figure on right.

1	best, show only a small amount of cytoplasm surrounding the nucleus. In contrast, the granular layer presents an overall spotted-blue appearance due to the staining of numerous small nuclei with hematoxylin. These small neurons, called granule cells, receive incoming impulses from other parts of the CNS and send axons into the molecular layer, where they branch in the form of a T, so that the axons contact the dendrites of several Purkinje cells and basket cells. Incoming (mossy) fibers contract granule cells in the lightly stained areas called glomeruli (arrows). Careful examination of the granular layer where it meets the molecular layer will reveal a group of nuclei (G ) that are larger than the nuclei of granule cells. These belong to Golgi type II cells. Cerebellum, brain, human, silver stain ×40.

1	Cerebellum, brain, human, silver stain ×40. The specimen in this figure has been stained with a silver procedure. Such procedures do not always color the specimen evenly, as do H&E. Note that the part of the molecular layer on the right is much darker than that on the left. A rectangular area on the left has been selected for examination at higher magnification in Figure 4. Even at the relatively low magnification shown here, however, the Purkinje cells can be recognized in the silver preparation because of their large size, characteristic shape, and location between an outer molecular layer (Mol ) and an inner granular layer (Gr). The main advantage of this silver preparation is that the white matter (WM ) can be recognized as being composed of fibers; they have been blackened by the silver-staining procedure. The pia mater (Pia) and cerebellar blood vessels are also evident in the preparation. Cerebellum, brain, human, silver stain ×400.

1	Cerebellum, brain, human, silver stain ×400. At higher magnification, the Purkinje cell bodies (Pkj ) stand out as the most distinctive and conspicuous neuronal cell type of the cerebellum, and numerous dendritic branches (D) can be seen. Note, also, the blackened fibers within the granular layer (Gr), about the Purkinje cell bodies, and in the molecular layer (Mol) disposed in a horizontal direction (relative to the cerebellar surface). The arrow indicates a T turn characteristic of the turn made by axons of granule cells. As these axonal branches travel horizontally, they make synaptic contact with numerous Purkinje cells. KEY BC, basket cells BV, blood vessels D, dendrites G, Golgi type II cells Gr, granular layer Mol, molecular layer Pia, pia mater Pkj, Purkinje cells WM, white matter arrows: Upper right figure, glomeruli; Lower right figure, T branching of axon in molecular layer rectangular area, areas shown at higher magnification

1	The spinal cord is organized into two discrete parts. The outer part, called the white matter of the cord because of its appearance in unfixed specimens, contains ascending and descending nerve fibers. Some of the fibers go to and from the brain, whereas others connect different lev-els of the spinal cord. The inner part of the spinal cord, called the gray matter because of its appearance in unfixed specimens, contains the cell bodies of neurons as well as nerve fibers. The gray matter forms an Hor butterfly-shaped pattern surrounding the central canal. The gray matter is described as having dorsal (posterior) horns and ventral (anterior) horns. The ventral horns contain the large cell bodies of motor neu-rons, whereas the dorsal horns contain neurons that receive, process, and retransmit information from the sensory neurons whose cell bodies are located in the dorsal root ganglia. The size of the gray matter (and, therefore, the size of the spinal cord) is different at different

1	information from the sensory neurons whose cell bodies are located in the dorsal root ganglia. The size of the gray matter (and, therefore, the size of the spinal cord) is different at different levels. Where the gray matter contains many large motor nerve cells that control the movement of the upper and lower limbs, the gray matter and the spinal cord are considerably larger than where the gray matter contains only the motor neurons for the muscle of the torso.

1	Spinal cord, human, silver stain ×16. A cross-section through the lower lumbar region of the spinal cord is shown here. The preparation is designed to stain the gray matter that is surrounded by the ascending and descending nerve fibers. Although the fibers that have common origins and destinations in the physiologic sense are arranged in tracts, these tracts cannot be distinguished unless they have been marked by special techniques, such as causing injury to the cell bodies from which they arise or by using special dyes or radioisotopes to label the axons.

1	The gray matter of the spinal cord appears roughly in the form of a butterfly. The anterior and posterior prongs are referred to as ventral horns (VH ) and dorsal horns (DH ), respectively. The connecting bar is called the gray commissure (GC ). The neuron cell bodies that are within the ventral horns (ventral horn cells) are so large that they can be seen even at this extremely low magnification (arrows). The pale-staining fibrous material that surrounds the spinal cord is the pia mater (Pia). It follows the surface of the spinal cord intimately and dips into the large ventral fissure (VF ) and into the shallower sulci. Blood vessels (BV ) are present in the pia mater. Some dorsal roots (DR ) of the spinal nerves are included in the section.

1	KEY BV, blood vessels DH, dorsal horn DR, dorsal root GC, gray commissure N, nucleus of ventral horn cell NB, Nissl bodies NN, nucleus of neuroglial cell Np, neuropil Pia, pia mater VF, ventral fissure VH, ventral horn arrows, cell bodies of ventral horn cell

1	OVERVIEW OF THE CARDIOVASCULAR SYSTEM / 400 HEART / 402 Intrinsic Regulation of Heart Rate / 405 Systemic Regulation of Heart Function / 407 GENERAL FEATURES OF ARTERIES AND VEINS / 408 Layers of Vascular Wall / 408 Vascular Endothelium / 409 ARTERIES / 414 Large Arteries (Elastic Arteries) / 415 Medium Arteries (Muscular Arteries) / 417 Small Arteries and Arterioles / 420 CAPILLARIES / 421 Classification of Capillaries / 421 Functional Aspects of Capillaries / 422 ARTERIOVENOUS SHUNTS / 423 VEINS / 424 Venules and Small Veins / 425 Medium Veins / 425 Large Veins / 425 ATYPICAL BLOOD VESSELS / 426 LYMPHATIC VESSELS / 427 Folder 13.1 Clinical Correlation: Atherosclerosis / 411 Folder 13.2 Clinical Correlation: Hypertension / 416 Folder 13.3 Clinical Correlation: Ischemic Heart Disease / 429

1	The cardiovascular system is a transport system that carries blood and lymph to and from the tissues of the body. The constitutive elements of these fluids include cells, nutrients, waste products, hormones, and antibodies. The cardiovascular system includes the heart, blood vessels, and lymphatic vessels. The cardiovascular system consists of a pump represented by the heart and blood vessels, which provide the route by which blood circulates to and from all parts of the body (Fig. 13.1). The heart pumps the blood through the arterial system under significant pressure; blood is returned to the heart under low pressure with the assistance of negative pressure in the thoracic cavity during inspiration and compression of the veins by skeletal muscle. The blood vessels are arranged so that blood delivered from the heart quickly reaches a network of narrow, thin-walled vessels—the blood capillaries—within or in proximity to the tissues in every part of the body.

1	In the capillaries, a two-directional exchange of fluid occurs between the blood and tissues. The fluid, called blood filtrate, carries oxygen and metabolites and passes through the capillary wall. In the tissues, these molecules are exchanged for carbon dioxide and waste products. Most of the fluid reenters the distal or venous end of the blood capillaries. The remaining fluid enters lymphatic capillaries as lymph and is ultimately returned to the bloodstream through a system of lymphatic vessels that join the blood system at the junction of the internal jugular veins with the subclavian veins. Normally, many of the white blood cells conveyed in the blood leave the blood vessels to enter the tissues. This occurs at the level of the postcapillary venules. When pathologic changes occur in the body, as in the inﬂammatory reaction, large numbers of white blood cells emigrate from these venules.

1	Arteries are the vessels that deliver blood to the capillaries. The smallest arteries, called arterioles, are functionally associated with networks of capillaries into which they deliver blood. The arterioles regulate the amount of blood that enters these capillary networks. Together, the arterioles, associated capillary network, and postcapillary venules form a functional unit called the microcirculatory or microvascular bed of that tissue. Veins, beginning with the postcapillary venule, collect blood from the microvascular bed and carry it away. FIGURE 13.1 • Photograph of the human heart. This specimen was sectioned in the oblique plane to visualize all of the chambers of the heart. The posterior part of the heart is on the left; the anterior part has been removed and is shown on the right. Note the thickness of the ventricular walls and the intermuscular septum. The interatrial septum, which separates the atria, is also visible.

1	Two circuits distribute blood in the body: the systemic and the pulmonary circulations. Two pathways of circulation are formed by the blood vessels and the heart:  Pulmonary circulation conveys blood from the heart to the lungs and from the lungs to the heart (Fig. 13.2). arch of aorta  Systemic circulation conveys blood from the heart to other tissues of the body and from other tissues of the body to the heart. Although the general arrangement of blood vessels in both circulations is from arteries to capillaries to veins, in some parts of the systemic circulation it is modified so that a vein or an arteriole is interposed between two capillary networks; these vessels constitute a portal system. Venous portal systems occur in vessels carrying blood to the liver, namely, the hepatic portal system (portal vein), and in vessels leading to the pituitary, the hypothalamic–hypophyseal portal system.

1	FIGURE 13.2 • Diagram depicting circulation of blood through the heart. Blood returns from the tissues of the body via the superior vena cava and inferior vena cava. These two major venous vessels carry the blood to the right atrium. Blood then passes into the right ventricle and is pumped into the pulmonary trunk before flowing into the pulmonary arteries, which convey the blood to the lungs. The blood is oxygenated in the lungs and is then returned to the left atrium via the pulmonary veins. Blood then passes to the left ventricle and is pumped into the aorta, which conveys the blood to the tissues of the body. From the heart to the lungs and from the lungs to the heart constitutes the pulmonary circulation; from the heart to the tissues and from the tissues to the heart constitutes the systemic circulation.

1	The heart lies obliquely, about two-thirds on the left side of the thoracic cavity, in the middle mediastinum—the space enclosed between sternum, vertebral column, diaphragm, and lungs. It is surrounded by a tough fibrous sac, the pericardium, which also contains the beginnings and ends of the great vessels entering and leaving the heart. Through the pericardium, the heart is strongly attached to the diaphragm and neighboring organs that lie in the thoracic cavity. The heart is a muscular pump that maintains unidirectional flow of blood.

1	The heart contains four chambers—the right and left atria and right and left ventricles—through which blood is pumped (see Fig. 13.1). Valves guard the exits of the chambers, preventing backflow of blood. An interatrial septum and an interventricular septum separate the right and left sides of the heart. The right side of the heart pumps blood through pulmonary circulation. The right atrium receives blood returning from the body via the inferior and superior venae cavae, the two largest veins of the body (Fig. 13.3). The right ventricle receives blood from the right atrium and pumps it to the lungs for oxygenation via the pulmonary arteries. The left side of the heart pumps blood through systemic circulation. The left atrium receives the oxygenated blood returning from the lungs via the four pulmonary veins. The left ventricle receives blood from the left atrium and pumps it into the aorta for distribution into the remainder of the body.

1	FIGURE 13.3 • Diagram of the blood circulation. This diagram shows the right and left side of the heart artificially separated. The right side of the heart pumps blood through the low-pressure pulmonary circulation. The right atrium receives deoxygenated blood returning from the body via the inferior and superior venae cavae. The right ventricle receives blood from the right atrium and pumps it to the lungs for oxygenation via the pulmonary arteries. The left side of the heart pumps blood through the high-pressure systemic circulation. The left atrium receives the oxygenated blood returning from the lungs via the four pulmonary veins. The left ventricle receives blood from the left atrium and pumps it into the aorta for systemic distribution. The heart contains the following:  A musculature of cardiac muscle for contraction to propel the blood.

1	 A fibrous skeleton that consists of four fibrous rings surrounding the valve orifices, two fibrous trigones connecting the rings, and the membranous part of the inter-ventricular and interatrial septa. The fibrous rings are composed of dense irregular connective tissue. They encircle the base of the two arteries, leaving the heart (aorta and pulmonary trunk) and the openings between the atria and the ventricles (right and left atrioventricular [AV] orifices) (Fig. 13.4). These rings provide the attachment site for the leaflets of all four valves of the heart that allow blood flow in only one direction through the openings. The membranous part of the interventricular septum is devoid of cardiac muscle; it consists of dense connective tissue that contains a short length of the atrioventricular bundle of the conducting system of the heart. The fibrous skeleton provides independent attachments for the atrial and ventricular myocardium. It also acts as an electrical insulator by

1	bundle of the conducting system of the heart. The fibrous skeleton provides independent attachments for the atrial and ventricular myocardium. It also acts as an electrical insulator by preventing the free flow of electrical impulses between atria and ventricles.

1	 A conducting system for initiation and propagation of rhythmic depolarizations, which results in rhythmic cardiac muscle contractions (Fig. 13.5). This system is formed by modified cardiac muscle cells (Purkinje fibers), which generate and conduct electrical impulses rapidly through the heart. In the sudden cessation of normal heart rhythm leading to abrupt cessation of blood circulation called cardiac arrest, the conducting system of the heart fails to produce or conduct electrical fibrous ring of pulmonary conus trunk ligament left fibrous fibrous ring trigone of tricuspid fibrous ring of mitral valve

1	FIGURE 13.4 • Fibrous skeleton of the heart as seen with the two atria removed. This fibrous network (indicated in blue) serves for the attachment of cardiac muscle; it also serves for the attachment of the cuspid valves between the atria and ventricles and for the semilunar valves of the aorta and the pulmonary artery. The atrioventricular bundle passes from the right atrium to the ventricular septum via the membranous septum of the fibrous skeleton. FIGURE 13.5 • Chambers of the heart and the impulse-conducting system. The heart has been cut open in the coronal plane to expose its interior and the main parts of its impulse-conducting system (indicated in yellow). Impulses are generated in the sinuatrial (SA) node, transmitted through the atrial wall to the atrioventricular (AV) node and then sent along the AV bundle to the Purkinje fibers.

1	impulses that cause the heart to contract and supply blood to the body. Sudden cardiac arrest is a medical emergency; first-aid treatment such as cardiopulmonary resuscitation (CPR) and defibrillation (delivering a therapeutic dose of electrical energy to the heart) can improve the chances of survival. If not treated, cardiac arrest leads to sudden cardiac death. Heart rhythm pathologies associated with cardiac arrest include tachycardia (accelerated heart rhythm), fibrillation (rapid, irregular, and ineffective contractions), bradycardia (decelerated heart rhythm), and asystole (total absence of heart rhythm).

1	 A coronary vasculature that consists of two coronary arteries and cardiac veins. The right and left coronary arteries provide the arterial blood supply to the heart. They originate from the initial part of the ascending aorta near the aortic valves and circle the base of the heart, with branches converging toward the apex of the heart. Venous drainage of the heart occurs via several cardiac veins, most of which drain into the coronary sinus located on the posterior surface of the heart. The coronary sinus drains into right atrium. The wall of the heart is composed of three layers: epicardium, myocardium, and endocardium. The structural organization of the wall of the heart is continuous within the atria and ventricles. The wall of the heart is composed of three layers. From the outside to the inside, they are as follow.

1	 The epicardium, also known as the visceral layer of serous pericardium, adheres to the outer surface of the heart (Fig 13.6). It consists of a single layer of mesothelial cells and underlying connective and adipose tissue. The blood vessels and nerves that supply the heart lie in the epicardium and are surrounded by adipose tissue that cushions the heart in the pericardial cavity. The epicardium is reflected back at the great vessels entering and leaving the heart as the parietal layer of serous pericardium, which lines the inner surface of the pericardium that surrounds the heart and roots of great vessels. Thus, there is a potential space containing a minimal amount (15–50 ml) of serous (pericardial) fluid between the visceral and parietal layers of the serous pericardium. This space is known as the pericardial cavity; its lining consists of mesothelial cells (see Fig. 13.6). The condition in which excess ﬂuid (blood or pericardial effusion) rapidly accumulates in the pericardial

1	is known as the pericardial cavity; its lining consists of mesothelial cells (see Fig. 13.6). The condition in which excess ﬂuid (blood or pericardial effusion) rapidly accumulates in the pericardial cavity is called cardiac tamponade. It is commonly caused by both blunt and penetrating chest injuries and by myocardial rupture or pericarditis (inﬂammation of pericardium). This is a life-threatening condition in which accumulating ﬂuid compresses the heart, preventing the heart’s chambers from filling properly with the blood. Relieving the pressure is usually accomplished with pericardiocentesis (a procedure to drain the ﬂuid from the pericardial cavity).

1	 The myocardium, consisting of cardiac muscle, is the principal component of the heart. The detailed histologic structure and function of cardiac muscle is discussed in Chapter 11, Muscle Tissue. The myocardium of the atria is substantially thinner than that of the ventricles. The atria receive blood from the large veins and deliver it to adjacent ventricles, a process that requires relatively low pressure. The myocardium of the ventricles is substantially thicker because of the higher pressure required to pump the blood through the pulmonary and systemic circulations (Fig. 13.7).

1	 The endocardium consists of an inner layer of endothelium and subendothelial connective tissue, a middle layer of connective tissue and smooth muscle cells, and a deeper layer of connective tissue, which is also called the subendocardial layer. The latter is continuous with the connective tissue of the myocardium. The conducting system of the heart (see the following section called “Intrinsic Regulation of Heart Rate”) is located in the subendocardial layer of the endocardium. The interventricular septum is the wall between the right and left ventricles. It contains cardiac muscle in all but the membranous portion. Endocardium lines each surface of the interventricular septum. The interatrial septum is much thinner than the interventricular septum. Except in certain localized areas that contain fibrous tissue, it has a center layer of cardiac muscle and a lining of endocardium facing each chamber.

1	FIGURE 13.6 • Layers of the heart and pericardium. This schematic diagram shows the anatomical relationship between the layers of the heart. In the middle mediastinum, the heart and roots of the great vessels are surrounded by the pericardium, which is often covered by highly variable amounts of adipose tissue. The pericardium has two layers: a tough external fibrous layer called the fibrous pericardium and a parietal layer of serous pericardium that lines its inner surface. The parietal layer of the serous pericardium is reflected back at the great vessels entering and leaving the heart as the visceral layer of the serous pericardium or epicardium. The epicardium lines the outer surface of the heart. The pericardial cavity is a space between the visceral and parietal layers of the serous pericardium, and it is lined by the mesothelial cells. Deep to the epicardium is the myocardium consisting of cardiac muscle. Note a small amount of adipose tissue of the epicardium, which contains

1	pericardium, and it is lined by the mesothelial cells. Deep to the epicardium is the myocardium consisting of cardiac muscle. Note a small amount of adipose tissue of the epicardium, which contains the coronary arteries and cardiac veins. The inner layer of the myocardium is called the endocardium, which is lined by the mesothelium with an underlying thin layer of connective tissue.

1	Heart valves are composed of connective tissue with overlying endocardium. The heart valves attach to the complex framework of dense irregular connective tissue that forms the fibrous rings and surrounds the orifices containing the valves (Fig. 13.8). Each valve is composed of three layers.  The fibrosa forms the core of the valve and contains fibrous extensions from the dense irregular connective tissue of the skeletal rings of the heart.  The spongiosa is loose connective tissue located on the atrial or blood vessel side of each valve. It is composed of loosely arranged collagen and elastic fibers infiltrated with

1	 The spongiosa is loose connective tissue located on the atrial or blood vessel side of each valve. It is composed of loosely arranged collagen and elastic fibers infiltrated with FIGURE 13.7 • Horizontal section through the ventricles of the heart. This photograph shows a cross section of the human heart at the level of the ventricles. Cusps of both the tricuspid valve in the right ventricle and the mitral valve in left ventricle are visible with their attachments to the chordae tendineae. Cross sections of the papillary muscles in both ventricles are visible. Note the differences in the thickness between the wall of the right and left ventricles. Adipose tissue of the epicardium contains branches of the coronary arteries and tributaries of the coronary veins. RV, right ventricle; LV, left ventricle. (Courtesy of Dr. William D. Edwards, Mayo Clinic, Rochester, MN.)

1	FIGURE 13.8 • Photomicrograph of the left atrial and left ventricular walls. a. This photomicrograph shows a sagittal section of the posterior wall of the left atrium and left ventricle. The line of section crosses the coronary (AV) groove containing the coronary sinus and circumflex branch of the left coronary artery. Note that the section has cut through the fibrous AV ring of the mitral valve, which provides the attachment site for the muscle of the left atrium and the left ventricle and the cusp of the mitral valve. The ventricular wall consists of three layers: (1) endocardium (arrowheads), (2) myocardium, and (3) epicardium. The visible blood vessels lie in the epicardium and are surrounded by adipose tissue. The layers of the mitral valve are shown at higher magnification in Figure 13.9b. 35.

1	b. This high magnification of the area indicated by the rectangle in a shows the characteristic features of the inner surface of the heart. Note that the endocardium consists of a squamous inner layer of endothelium (End), a middle layer of subendothelial dense connective tissue (DCT ) containing smooth muscle cells (SMC), and a deeper subendocardial layer containing Purkinje fibers (PF). The myocardium contains cardiac muscle fibers (CMF) and is seen on the left. 120. large numbers of proteoglycans. The spongiosa acts as a shock absorber to dampen vibrations associated with the closing of the valve. It also confers flexibility and plasticity to the valve cusps. In the aortic and pulmonary valves, spongiosa located on the blood vessel side is called arterialis. It corresponds to the loose connective tissue located on the atrial side of the AV (tricuspid and mitral) valves, which is called the auricularis.

1	 The ventricularis is immediately adjacent to the ventricular or atrial surface of each valve and is covered with endothelium. It contains dense connective tissue with many layers of elastic fibers. In the AV valves, the ventricularis continues into the chordae tendineae, which are fibrous, threadlike cords also covered with endothelium (Fig. 13.9). They extend from the free edge of the AV valves to muscular projections from the wall of the ventricles, which are called papillary muscles. Valve cusps are normally avascular. Small blood vessels and smooth muscle can be found only in the base of the cusp. The surfaces of the valve are exposed to blood, and the cusps are thin enough to allow nutrients and oxygen to diffuse from the blood.

1	Several diseases affect the valves of the heart, causing their degeneration (e.g., calcification, fibrosis) and resulting in heart malfunction because of insufficiency or stenosis of valvular orifices. These conditions, known collectively as valvular heart disease, include rheumatic heart disease, vegetative endocarditis, degenerative calcific aortic valve stenosis, and mitral annular calcification. For example, rheumatic fever causes inﬂammation of the heart valves (valvulitis). Inﬂammation induces angiogenesis in the valve and vascularization in the normally avascular layers of the valve. These changes most commonly affect the mitral valve (65% to 70%) and aortic valve (20% to 25%). This inﬂammation can lead to progressive replacement of elastic tissue by irregular masses of collagen fibers, causing the valve to thicken. The valves become rigid and inﬂexible, which affects their ability to open and close. Intrinsic Regulation of Heart Rate

1	Intrinsic Regulation of Heart Rate Contraction of the heart is synchronized by specialized cardiac conducting cells. Cardiac muscle can contract in a rhythmic manner without any direct stimulus from the nervous system. For the heart to be an effective pump, it is necessary for the atria and ventricles to contract in a coordinated rhythmic manner. The electrical activity (impulses) that results in the rhythmic pulsations of the heart is initiated and propagated by the conducting system of the heart. The rate of depolarization of cardiac muscle

1	FIGURE 13.9 • Mitral valve in the human heart. a. This photograph shows a sagittal section of the posterior wall of the left ventricle and the posterior cusp of the mitral valve. The chordae tendineae extend from the papillary muscle to the ventricular side of the mitral valve cusp. Note the thickness of the myocardium in the left ventricle. Glistening inner surface of the heart represents the endocardium, the outer surface of the myocardium is covered by the epicardium. 2. (Courtesy of Dr. William D. Edwards, Mayo Clinic, Rochester, MN.) b. Photomicrograph of a mitral valve. This photomicrograph shows a section through one of the two cusps of the mitral valve. Both sides of the cusp are covered by the endothelium. Note that the valve exhibits a layered architecture. Beginning at the atrial side (top of the image), the first layer underlying the endothelium is the spongiosa—not well developed in this part of the cusp. The second layer is the fibrosa, which forms the majority of the

1	side (top of the image), the first layer underlying the endothelium is the spongiosa—not well developed in this part of the cusp. The second layer is the fibrosa, which forms the majority of the dense connective tissue in the core of the valve. The third layer, the ventricularis, is formed by dense connective tissue containing layers of elastic and collagen fibers. 125.

1	varies in different parts of the conducting system; the fastest is in the atria, the slowest in the ventricles. The contraction cycle of the heart is initiated in the atria, forcing blood into the ventricles. A wave of contraction in the ventricles then begins at the apex of the heart, forcing blood from the heart into the aorta and pulmonary trunk.

1	The conducting system of the heart consists of two nodes—the sinoatrial node and the atrioventricular node—and a series of conduction fibers or bundles (tracts). Electrical impulses are generated at the sinoatrial (SA) node, a group of specialized nodal cardiac muscle cells located near the junction of the superior vena cava and the right atrium (see Fig. 13.5). Since the SA node has the fastest rate of depolarizations, it is referred to as the pacemaker of the heart. The pacemaker rate of the SA node is about 60 to 100 beats per minute. The SA node initiates an impulse that spreads along the cardiac muscle fibers of the atria and along internodal tracts composed of modified cardiac muscle fibers. The impulse is then picked up at the atrioventricular (AV) node and carried across the fibrous skeleton to the ventricles by the AV bundle (of His). The bundle divides into smaller right and left bundle branches and then into subendothelial branches, commonly called Purkinje fibers. The

1	skeleton to the ventricles by the AV bundle (of His). The bundle divides into smaller right and left bundle branches and then into subendothelial branches, commonly called Purkinje fibers. The components of the conducting system convey impulses at a rate approximately four times faster than the cardiac muscle fibers and are the only elements that can convey impulses across the fibrous skeleton.

1	If the SA node fails to function (e.g., because of insufficient blood supply), then the area with the next fastest intrinsic rate of depolarization will take over. In this situation, the AV node will drive the heart contractions at a rate of about 50 beats per minute. In complete heart block when the conduction of electric impulses to the ventricles is interrupted, the ventricles will beat at their own rate of about 30 to 40 beats per minute, driven by depolarization of Purkinje fibers. Purkinje fibers have the slowest rate of intrinsic depolarization of the entire conducting system. The spread of electrical impulses through the myocardium can be monitored and recorded by the electrocardiogram (ECG). The ECG is obtained by placing electrodes at different points on the skin at specific distances from the heart. Electrodes record electrical activity of the heart by measuring voltage differences between different points. The coordinated spread of the electrical activity through the heart

1	from the heart. Electrodes record electrical activity of the heart by measuring voltage differences between different points. The coordinated spread of the electrical activity through the heart is responsible for the shape of the ECG waveform, careful analysis of which can provide information about heart rate, cardiac rhythm, conduction times through various parts of the heart, effects of electrolyte concentration, effects of cardiac medication, and location of pathologic (ischemic) damages in the heart.

1	The nodal cardiac muscle cells in both the SA and AV nodes are modified cardiac muscle fibers that are smaller than the surrounding atrial cardiac muscle cells. They contain fewer myofibrils and lack typical intercalated discs. The AV bundle, the bundle branches, and the Purkinje fibers are also composed of modified cardiac muscle cells, but they are larger than the surrounding ventricular muscle cells (Fig. 13.10 and Plate 32, page 432). The terminal ramifications of the conducting system consist of Purkinje fibers. Cardiac conducting cells that make up the bundle of His originate at the AV node, pass through the fibrous skeleton of

1	FIGURE 13.10 • Photomicrograph of the ventricular wall containing the conducting system. This photomicrograph shows a Mallory-Azan–stained section of the ventricular wall of a human heart. The upper two thirds of the micrograph is occupied by the endocardium (E) containing a thick layer of Purkinje fibers. The free luminal surface of the ventricle (top) is covered by endothelium and an underlying layer of subendothelial connective tissue (stained blue). The deep layer of endocardium contains the Purkinje fibers. Note the intercalated discs in the fibers (arrows). The Purkinje fibers contain large amounts of glycogen, which appear as homogeneous, pale-staining regions that occupy the center portion of the cell surrounded by the myofibrils. The nuclei (N) are round and are larger than the nuclei of the cardiac muscle cells in the myocardium (M). They are frequently surrounded by the lighter-stained cytoplasm, which represents the juxtanuclear region of the cell. Because of the

1	the nuclei of the cardiac muscle cells in the myocardium (M). They are frequently surrounded by the lighter-stained cytoplasm, which represents the juxtanuclear region of the cell. Because of the considerable size of the Purkinje cells, the nuclei are often not included in the section. Among the Purkinje fibers course nerves (NF ) that belong to the autonomic nervous system. 320.

1	the heart, course along both sides of the interventricular septum (see Fig. 13.5), and terminate as Purkinje fibers in the myocardium of the ventricles. The cells that form the Purkinje fibers are larger than ventricular muscle cells. Their myofibrils are located at the periphery of the cell. The nuclei are round and are larger than the nuclei of the cardiac muscle cells in the myocardium. Because of the considerable size of the cells, the nuclei are often not included in the section. Intercalated disks are present in Purkinje fibers, but they are variable in appearance and number depending on their location. They are positive for periodic acid–Schiff (PAS) staining because of the large amount of glycogen they contain. With hematoxylin and eosin (H&E) and most other stains, the glycogen-rich center portion of the cell appears homogeneous and stains pale (see Fig 13.10). Because of the stored glycogen, Purkinje fiber cells are more resistant to hypoxia than are ventricular muscle

1	center portion of the cell appears homogeneous and stains pale (see Fig 13.10). Because of the stored glycogen, Purkinje fiber cells are more resistant to hypoxia than are ventricular muscle cells.

1	Systemic Regulation of Heart Function As mentioned above, the heart beats independently of any nervous stimulation. This spontaneous rhythm of the heart can be altered by nerve impulses from both sympathetic and parasympathetic divisions of the autonomic nervous system. The autonomic nerves do not initiate contraction of the cardiac muscle but rather regulate the heart rate (a chronotropic effect) according to the body’s immediate needs. Stimulation of the parasympathetic nerves decreases the heart rate.

1	Stimulation of the parasympathetic nerves decreases the heart rate. The parasympathetic nerve supply to the heart originates in the vagus nerve (cranial nerve X). Presynaptic parasympathetic fibers synapse with postsynaptic neurons within the heart. Their short postsynaptic fibers terminate chiefly at the SA and AV nodes but also extend into the coronary arteries that supply the heart. The release of the neurotransmitter acetylcholine from the terminals of these fibers slows the heart rate (an effect known as bradycardia), reduces the force of the heartbeat, and constricts the coronary arteries of the heart. Stimulation of the sympathetic nerves increases the heart rate.

1	Stimulation of the sympathetic nerves increases the heart rate. The sympathetic presynaptic fibers that supply the heart originate in the lateral horns at the level of the T1 to T6 segments of the spinal cord. They conduct electrical signals to the cell bodies of postsynaptic neurons located in the cervical and thoracic paravertebral ganglia of sympathetic trunks (see Fig. 12.25, page 380). The postsynaptic fibers terminate at the SA and AV nodes, extend into the myocardium, and also pass through the epicardium to reach the coronary arteries. The autonomic fibers secrete norepinephrine that regulates the rate of impulses emanating from the SA node. The sympathetic component causes the rate of contraction to increase (an effect known as tachycardia) and increases the force of muscle contraction. Sympathetic stimulation produces dilation of the coronary arteries by inhibiting their constriction.

1	The heart rate and the force of contraction can be regulated by circulating hormones and other substances. Changes in the force and rate of cardiac muscle contractions are regulated by hormones secreted from the adrenal medulla. These hormones include epinephrine and norepinephrine that reach the heart muscle cells via the coronary circulation.

1	Activation of adrenergic receptors (mainly 1 type) by epinephrine and less efficiently by norepinephrine increases the force of contraction (a positive inotropic effect) and the heart rate (a positive chronotropic effect). Other substances that have positive inotropic and chronotropic effects on the heart include Ca2, thyroid hormones, caffeine, theophylline, and the cardiac glycoside digoxin. These substances all increase intracellular Ca2 levels in cardiac myocytes. Substances that have negative inotropic and chronotropic actions on the heart muscle include adrenergic-receptor antagonists such as propranolol or Ca2 channel blockers. These substances decrease the heart rate and the force of cardiac muscle contraction. The central nervous system monitors arterial pressure and heart function through specialized receptors located within the cardiovascular system.

1	The central nervous system monitors arterial pressure and heart function through specialized receptors located within the cardiovascular system. The activity of the cardiovascular system is monitored by specialized centers in the central nervous system (CNS). Specialized sensory nerve receptors that supply afferent information about blood pressure are located in the walls of large blood vessels near the heart and within the heart itself. The information received from all types of cardiovascular receptors initiates the appropriate physiologic reflexes. The receptors function as follow.

1	blood pressure. These receptors are located in the carotid sinus and aortic arch.  Volume receptors (low-pressure receptors) located within the walls of the atria and ventricles sense central ve nous pressure and provide the CNS with information about cardiac distention.  Chemoreceptors detect alterations in oxygen, carbon dioxide tension, and pH. These receptors are the carotid and aortic bodies located at the bifurcation of the com mon carotid arteries and in the aortic arch, respectively. The carotid bodies consist of cords and irregular groups of epithelioid cells. A rich supply of nerve fibers is associated with these cells. The neural elements are both afferent and efferent. The structure of the aortic bodies is essentially similar to that of the carotid bodies. Both receptors function in neural reflexes that adjust cardiac output and respiratory rate. Layers of Vascular Wall The walls of arteries and veins are composed of three layers called tunics.

1	Layers of Vascular Wall The walls of arteries and veins are composed of three layers called tunics. The three layers of the vascular wall, from the lumen outward (Fig. 13.11 and Plate 33, page 434), are the following. FIGURE 13.11 • Schematic diagram of the major structural features of blood vessels. The layers or tunics of the blood vessel walls are labeled in the upper two panels. The arrangement of the microcirculatory bed in certain parts of the body is shown in the lowest panel. Note the location of pericytes and their relationship to the basal lamina. Also, an arteriovenous (AV) anastomosis is shown within the microcirculatory bed.

1	 The tunica intima, the innermost layer of the vessel, consists of three components: (a) a single layer of squamous epithelial cells, the endothelium; (b) the basal lamina of the endothelial cells (a thin extracellular layer composed chiefly of collagen, proteoglycans, and glycoproteins); and (c) the subendothelial layer, consisting of loose connective tissue. Occasional smooth muscle cells are found in the loose connective tissue. The subendothelial layer of the intima in arteries and arterioles contains a sheetlike layer or lamella of fenestrated elastic material called the internal elastic membrane. Fenestrations enable substances to diffuse readily through the layer and reach cells deep within the wall of the vessel.

1	 The tunica media, or middle layer, consists primarily of circumferentially arranged layers of smooth muscle cells. In arteries, this layer is relatively thick and extends from the internal elastic membrane to the external elastic membrane. The external elastic membrane is a layer of elastin that separates the tunica media from the tunica adventitia. Variable amounts of elastin, reticular fibers, and proteoglycans are interposed between the smooth muscle cells of the tunica media. The sheets or lamellae of elastin are fenestrated and arranged in circular concentric layers. All of the extracellular components of the tunica media are produced by the smooth muscle cells.

1	 The tunica adventitia, or outermost connective tissue layer, is composed primarily of longitudinally arranged collagenous tissue and a few elastic fibers. These connective tissue elements gradually merge with the loose connective tissue surrounding the vessels. The tunica adventitia ranges from relatively thin in most of the arterial system to quite thick in the venules and veins, where it is the major component of the vessel wall. In addition, the tunica adventitia of large arteries and veins contains a system of vessels called the vasa vasorum that supplies blood to the vascular walls themselves, as well as a network of autonomic nerves called nervi vascularis that control contraction of the smooth muscle in the vessel walls. Histologically, the various types of arteries and veins are distinguished from each other by the thickness of the vascular wall and differences in the composition of the layers. Table 13.1 summarizes the features of the various types of blood vessels.

1	In the adult human body, a circulatory system consists of about 60,000 miles of different-sized vessels that are lined by a simple squamous epithelium called endothelium. The endothelium is formed by a continuous layer of flattened, elongated, and polygonally shaped endothelial cells that are aligned with their long axes in the direction of the blood flow. At the luminal surface, they express a variety of surface adhesion molecules and receptors (i.e., low-density lipoprotein [LDL], insulin, and histamine receptors). Endothelial cells play an important role in blood homeostasis. The functional properties of these cells change in response to various stimuli. This process, known as endothelial activation, is also responsible for the pathogenesis of many vascular diseases (e.g., atherosclerosis; Folder 13.1). Inducers of endothelial activation include bacterial and viral antigens, cytotoxins, complement products, lipid products, and hypoxia. Activated endothelial cells exhibit new

1	Folder 13.1). Inducers of endothelial activation include bacterial and viral antigens, cytotoxins, complement products, lipid products, and hypoxia. Activated endothelial cells exhibit new surface adhesion molecules and produce different classes of cytokines, lymphokines, growth factors, and vasoconstrictor and vasodilator molecules, as well as molecules that control blood coagulation.

1	Endothelial cells participate in the structural and functional integrity of the vascular wall. Endothelial cells are active participants in a variety of interactions between the blood and underlying connective tissue and are responsible for many properties of the vessels (Table 13.2). These properties include the following.

1	 The maintenance of a selective permeability barrier allows selective movement of small and large molecules from the blood to the tissues and from the tissues to the blood. This movement is related to the size and charge of the molecules. The endothelium is permeable to small hydrophobic (lipid-soluble) molecules (e.g., oxygen, carbon dioxide) that readily pass through the lipid bilayer of the endothelial cell membrane (a process called simple diffusion). However, water and hydrophilic (water-soluble) molecules (e.g., glucose, amino acids, electrolytes) cannot diffuse across the endothelial cell membrane. These molecules and solutes must be either actively transported across the plasma membrane and released into the extracellular space (transcellular pathways) or transported across the zonula occludens between two epithelial cells (paracellular pathway; see Chapter 5, Epithelial Tissue, page 125). The transcellular pathway uses numerous small pinocytotic vesicles (a

1	across the zonula occludens between two epithelial cells (paracellular pathway; see Chapter 5, Epithelial Tissue, page 125). The transcellular pathway uses numerous small pinocytotic vesicles (a clathrin-independent form of endocytosis) to transport bulk material from the blood into the cell. In addition, some specific molecules (e.g., LDL, cholesterol, transferrin) are transported via receptor-mediated endocytosis (a clathrin-dependent process), which uses endothelial-specific surface receptors. In some blood vessels, larger molecules are transported through fenestrations within the endothelial cells visible in transmission electron microscope (TEM) preparations.

1	 The maintenance of a nonthrombogenic barrier between blood platelets and subendothelial tissue is done by producing anticoagulants (agents that prevent coagulation such as thrombomodulin and others) and antithrombogenic substances (agents that prevent or interfere with platelet aggregation and release of factors that cause formation of clots, or thrombi, such as prostacyclin [PGI2] and tissue plasminogen activator). Normal endothelium does not support the adherence of platelets or the formation of thrombi on its surface. Damage to endothelial cells causes them to release prothrombogenic agents (agents that promote thombi formation) such as von Willebrand factor or plasminogen-activator inhibitor.

1	 The modulation of blood ﬂow and vascular resistance is achieved by the secretion of vasoconstrictors (endothelins, angiotensin-converting enzyme [ACE], prostaglandin H2, thromboxane A2) and vasodilators (nitrous oxide [NO], prostacyclin). This subject is discussed in more depth in the next section.  The regulation and modulation of immune responses is done by controlling the interaction of lymphocytes with the endothelial surface, which is mainly achieved through the expression of adhesion molecules and their receptors on the endothelial-free surface as well as by secretion of three classes of interleukins (IL-1, IL-6, and IL-8).

1	 Hormonal synthesis and other metabolic activities are done by the synthesis and secretion of various growth factors—for example, hemopoietic colony-stimulating factors (CSFs) such as granulocyte–macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), and macrophage CSF (M-CSF); fibroblast growth factor (FGF); and platelet-derived growth factor (PDGF). Endothelial cells also synthesize growth inhibitors such as heparin and

1	TABLE Characteristics of Blood Vessels 13.1 Arteries Vessel Diameter Tunica intima (inner layer) Tunica media (middle layer) Tunica adventitia (outer layer) Large artery (elastic artery) >10 mm Endothelium Connective tissue Smooth muscle Smooth muscle Elastic lamellae Connective tissue Elastic fibers Thinner than tunica media Medium artery (muscular artery) 2–10 mm Endothelium Connective tissue Smooth muscle Prominent internal elastic membrane Smooth muscle Collagen fibers Relatively little elastic tissue Connective tissue Some elastic fibers Thinner than tunica media Small artery 0.1–2 mm Endothelium Connective tissue Smooth muscle Internal elastic membrane Smooth muscle (8–10 cell layers) Collagen fibers Connective tissue Some elastic fibers Thinner than tunica media Arteriole 10–100 µm Endothelium Connective tissue Smooth muscle Smooth muscle (1–2 cell layers) Thin, ill-defined sheath of connective tissue Capillary 4–10 µm Endothelium None None Veins Vessel Diameter Tunica intima

1	Endothelium Connective tissue Smooth muscle Smooth muscle (1–2 cell layers) Thin, ill-defined sheath of connective tissue Capillary 4–10 µm Endothelium None None Veins Vessel Diameter Tunica intima (inner layer) Tunica media (middle layer) Tunica adventitia (outer layer) Postcapillary venule 10–50 µm Endothelium Pericytes None None Muscular venule 50–100 µm Endothelium Smooth muscle (1–2 cell layers) Connective tissue Some elastic fibers Thicker than tunica media Small vein 0.1–1 mm Endothelium Connective tissue Smooth muscle (2–3 layers) Smooth muscle (2–3 layers continuous with tunica intima) Connective tissue Some elastic fibers Thicker than tunica media Medium vein 1–10 mm Endothelium Connective tissue Smooth muscle Internal elastic membrane in some cases Smooth muscle Collagen fibers Connective tissue Some elastic fibers Thicker than tunica media Large vein 10 mm Endothelium Connective tissue Smooth muscle Smooth muscle (2–15 layers) Cardiac muscle near heart Collagen fibers

1	Connective tissue Some elastic fibers Thicker than tunica media Large vein 10 mm Endothelium Connective tissue Smooth muscle Smooth muscle (2–15 layers) Cardiac muscle near heart Collagen fibers Connective tissue Some elastic fibers, longitudinal smooth muscles Much thicker than tunica media transforming growth factor (TGF-). Endothelial cells function in the conversion of angiotensin I to angiotensin II in the renin–angiotensin system that controls blood pressure, as well as in the inactivation or conversion of a several compounds conveyed in the blood (norepinephrine, thrombin, prostaglandins, bradykinin, and serotonin) to inactive forms.

1	 Modification of the lipoproteins occurs by oxidation. Lipoproteins, mainly LDLs with a high cholesterol content and very low-density lipoproteins (VLDLs), are oxidized by chapter 13 Cardiovascular System GENERAL FEATURES OF ARTERIES AND VEINS 411 Atherosclerotic lesions are the most common acquired abnormality of blood vessels. More than half of the annual deaths in the United States are related to complications of atherosclerotic disease, which includes ischemic heart disease (see Folder 13.3), myocardial infarction, stroke, and gangrene of the limbs. Lesions develop pri-marily in the tunica intima of large elastic arteries follow-ing endothelial injury, which leads to endothelial dysfunction. Factors that predispose to endothelial in-juries include low-LDL cholesterol hyperlipidemia, hyper-glycemia (in diabetes), hypertension, increased toxin levels associated with cigarette smoking, and certain viral and bacterial infections caused by cytomegalovirus (CMV) or Chlamydia

1	hyper-glycemia (in diabetes), hypertension, increased toxin levels associated with cigarette smoking, and certain viral and bacterial infections caused by cytomegalovirus (CMV) or Chlamydia pneumoniae, respectively. Altered function of vascular endothelium leads to increased per-meability to LDL cholesterol and increased adherence of white blood cells to the endothelium. Endothelial injury in-creases the production of reactive oxygen species such as O2 , H2O2, OH, and ONOO, which in turn oxidize LDL in the tunica intima of the artery. In response to this injury, monocytes from the bloodstream enter the tunica intima and differentiate into macrophages. Macrophages phagocytize oxidized LDL, slowly transforming themselves into foam cells with a characteristic spongy appearance of the cytoplasm loaded with lipid-containing vesicles. Foam cells and infiltrated T lymphocytes form the initial atherosclerotic lesion, or fatty streak. This lesion under-goes further remodeling and growth into

1	loaded with lipid-containing vesicles. Foam cells and infiltrated T lymphocytes form the initial atherosclerotic lesion, or fatty streak. This lesion under-goes further remodeling and growth into fibrofatty plaque as smooth muscle cells migrate from the media and fibrob-lasts form a protective capsule of connective tissue (Fig. F13.1.1). A thick layer of fibrous connective tissue con-taining scattered smooth muscle cells, macrophages, foam cells, T lymphocytes, cholesterol crystals, and cell debris is known as an atheromatous plaque. Progression of the plaque is marked by accumulation of lipid and loss of integrity of the endothelium. In advanced lesions, blood stasis and clotting (thrombosis) may lead to occlusion of the vessel. Other changes seen in advanced lesions in-clude thinning of the tunica media, calcification of accu-mulated extracellular lipids, and necrosis within the lesion (Fig. F13.1.2ab). Progression from simple to complicated lesions can be found in some people as

1	of the tunica media, calcification of accu-mulated extracellular lipids, and necrosis within the lesion (Fig. F13.1.2ab). Progression from simple to complicated lesions can be found in some people as early as their 20s and in most individuals by age 50 or 60 years.

1	 FOLDER 13.1 Clinical Correlation: Atherosclerosis

1	FIGURE F13.1.1 • Schematic diagram of cellular interactions in the formation of an atheromatous plaque. Endothelial cells express cell adhesion molecules that initiate monocyte migration through the endothelium. Platelet-derived growth factor (PDGF) and other growth factors (blue arrow) released from endothelial cells stimulate migration of the smooth muscle cells from the tunica media to the tunica intima. In the tunica intima, smooth muscle cells produce large amounts of extracellular matrix (proteoglycans, collagen) that increase the thickness of the tunica intima. Foam cells derived from both macrophages and smooth muscle cells accumulate LDLs, which cross the endothelial barrier (yellow arrows) and are oxidized by free radicals produced by the endothelial cells. lumen lipid uptake cell adhesion molecules endothelium tunicaintimainternal elastic membrane tunicamediamonocyte smooth muscle cells cytokines oxidized LDL foam cells LDL LDL PDGF continued next page free radicals

1	cell adhesion molecules endothelium tunicaintimainternal elastic membrane tunicamediamonocyte smooth muscle cells cytokines oxidized LDL foam cells LDL LDL PDGF continued next page free radicals produced by endothelial cells. Modified LDLs, in turn, are rapidly endocytosed by macrophages to form foam cells (see Fig. F13.1.1). Foam cells are a characteristic feature in the formation of atheromatous plaques.

1	Endothelium of blood vessels controls contraction and relaxation of smooth muscle cells in the tunica media influencing local blood flow and pressure. The endothelial-derived relaxing factor (EDRF) was historically one of the early compounds discovered in the endothelial cells that was causing relaxation of blood vessels. For years, researchers had difficulty characterizing EDRF chemically. It is now known that most of the vascular effects of EDRF can be attributed to nitric oxide (NO) and its related compounds, which are released by endothelial cells in arteries, blood capillaries, and even lymphatic capillaries. As a chemical compound, NO is a gas with a very short physiologic half-life measured in seconds; hence, the difficulty with its discovery. Shear stress produced during the interaction of blood flow with vascular endothelial cells initiates nitric oxide (NO)-derived relaxation of blood vessels.

1	Vasodilation (the relaxation of smooth muscle cells) increases the luminal diameter of the vessels, decreasing vascular resistance and systemic blood pressure. Endotheliumderived nitric oxide (NO) is one of several critical regulators of cardiovascular homeostasis. It regulates the blood vessel diameter, inhibits monocyte adhesion to dysfunctional endothelial cells, and maintains an antiproliferative and anti-apoptotic environment in the vessel wall. NO is an endogenous vasodilatory gas continuously synthesized in endothelial cells by endothelial nitric oxide synthase (eNOS). This Ca2-dependent enzyme catalyzes oxidation of L-arginine and acts through the G-protein–signaling cascade. Endothelial cells are constantly subjected to shear stress, the dragging force generated by the blood flow. Shear stress increases synthesis of a potent eNOS stimulator, the vascular endothelial growth factor (VEGF), and triggers a variety of other molecular and physical changes in endothelial cell

1	flow. Shear stress increases synthesis of a potent eNOS stimulator, the vascular endothelial growth factor (VEGF), and triggers a variety of other molecular and physical changes in endothelial cell structure and function. Once NO is produced by endothelial cells, it diffuses out through the cell and basement membrane to the underlying tunica media and binds to guanylate cyclase in smooth muscle cytoplasm. This enzyme increases production of cGMP, which activates smooth muscle protein kinase G (PKG). Activation of protein kinase G has a negative effect on intracellular concentration of Ca2, causing smooth muscle relaxation (Fig 13.12). Note that NO is also a signaling molecule in many pathologic and physiologic processes.

1	TABLE Summary of Endothelial Cell Properties and Functions 13.2 Major Properties Associated Functions Active Molecules Involved Maintenance of selective permeability barrier Simple diffusion Active transport Pinocytosis Receptor-mediated endocytosis Oxygen, carbon dioxide Glucose, amino acids, electrolytes Water, small molecules, soluble proteins LDL, cholesterol, transferrin, growth factors, antibodies, MHC complexes Maintenance of nonthrombogenic barrier Secretion of anticoagulants Secretion of antithrombogenic agents Secretion of prothrombogenic agents Thrombomodulin Prostacyclin (PGI2), tissue plasminogen activator (TPA), antithrombin III, heparin Tissue thromboplastin, von Willebrand factor, plasminogenactivator inhibitor Modulation of blood flow and vascular resistance Secretion of vasoconstrictors Secretion of vasodilators Endothelin, angiotensin-converting enzyme (ACE) Endothelial-derived relaxation factor (EDRF)/nitric oxide (NO), prostacyclin Regulation of cell growth

1	of vasoconstrictors Secretion of vasodilators Endothelin, angiotensin-converting enzyme (ACE) Endothelial-derived relaxation factor (EDRF)/nitric oxide (NO), prostacyclin Regulation of cell growth Secretion of growth-stimulating factors Secretion of growth-inhibiting factors Platelet-derived growth factor (PDGF), hemopoietic colony-stimulating factors (GM-CSF, G-CSF, M-CSF) Heparin, transforming growth factor (TGF-) Regulation of immune responses Regulation of leukocyte migration by expression of adhesion molecules Regulation of immune functions Selectins, integrins, CD marker molecules Interleukin molecules (IL-1, IL-6, IL-8), MHC molecules Maintenance of extracellular matrix Synthesis of basal lamina Synthesis of glycocalyx Type IV collagen, laminin Proteoglycans Involvement in lipoprotein, cholesterol, metabolism Production of free radicals Oxidation of LDL Reactive oxygen species (ROS), LDL, VLDL Modified from Cotran S, Kumar V, Collins T, Robbins SL, eds. Robbins Pathologic

1	cholesterol, metabolism Production of free radicals Oxidation of LDL Reactive oxygen species (ROS), LDL, VLDL Modified from Cotran S, Kumar V, Collins T, Robbins SL, eds. Robbins Pathologic Basis of Disease. Philadelphia: WB Saunders, 1999.

1	It acts as an anti-inflammatory agent under normal physiologic conditions, although its overproduction induces inflammation. NO is also involved in immune reactions (it stimulates macrophages to release high concentrations of NO), is a potent neurotransmitter in the nervous system, and contributes to the regulation of apoptosis. The pathogenesis of inflammatory disorders of the joint, gut, and lungs is linked to local overproduction of NO. Recently, NO inhibitors have been used to manage inflammatory diseases.

1	Metabolic stress in endothelial cells also contributes to smooth muscle relaxation. Endothelium-derived relaxing factors include prostacyclin (PGI2), which in addition to relaxing smooth muscles is a potent inhibitor of platelet aggregation. PGI2 binds to receptors on the smooth muscles; stimulates cAMP-activated protein kinase A (PKA), which in turn phosphylates myosin light chain kinase (MLCK); and prevents activation of the calcium– calmodulin complex. This type of relaxation occurs without change in the intracellular Ca2 concentration. represents another endothelium-derived relaxing factor that acts on Ca2-dependent potassium channels causing hyperpolarization of smooth muscle cells and their relaxation (see Fig 13.12). Endothelins produced by vascular endothelial cells play an important role in both physiologic and pathologic mechanisms of the circulatory system.

1	Endothelins produced by vascular endothelial cells play an important role in both physiologic and pathologic mechanisms of the circulatory system. Vasoconstriction (contraction of smooth muscle) in the tunica media of small arteries and arterioles reduces the luminal diameter of these vessels and increases vascular resistance. Vasoconstriction increases systemic blood pressure. In the past, vasoconstriction was thought to be mainly induced by nerve impulses or circulating hormones. Today it is known that endothelium-derived factors play an important role in both physiologic and pathologic mechanisms of the circulatory system. Members of the endothelin

1	FIGURE 13.12 • Molecular mechanism of blood vessel vasodilatation. Relaxation of smooth muscle cells in the wall of the blood vessel causes an increase of its diameter and decreases in vascular resistance and systemic blood pressure. Nitric oxide (NO) produced by the endothelial nitric oxide synthase (eNOS) in endothelial cells is an important molecule regulating relaxation of vascular smooth muscles. Other molecules include ADP, vascular endothelial growth factor (VEGF ), bradykinin, prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF ). Shear stress produced between erythrocytes and endothelial cells as well as VEGF activate eNOS, increasing the production of NO. Once NO is produced, it diffuses to the underlying smooth muscles and activates guanylate cyclase production of cGMP, which in turn activates cGMP-dependent protein kinase G (PKG) metabolic pathways, causing smooth muscle relaxation. Metabolic stress of endothelial cells caused by increased levels of

1	of cGMP, which in turn activates cGMP-dependent protein kinase G (PKG) metabolic pathways, causing smooth muscle relaxation. Metabolic stress of endothelial cells caused by increased levels of ADP or PGI2 stimulates cAMP-activated protein kinase A (PKA) metabolic pathways in smooth muscles, causing their relaxation. In addition, EDHF opens the potassium channels, causing hyperpolarization of smooth muscle cell membranes, further leading to their relaxation. (Based on Noble A, Johnson R, Thomas A, Bass P. The Cardiovascular System. London, New York: Churchill Livingstone, 2005.) family of 21 amino acid peptides produced by vascular endothelial cells are the most potent vasoconstrictors. The family consists of three members: endothelin-1 (ET-1), endothelin-2 (ET-2), and endothelin-3 (ET-3). Endothelins act mainly as paracrine and autocrine agents and bind to their own receptors on the epithelial cells and vascular smooth muscles (Fig 13.13). The ET-1 is the most potent naturally

1	(ET-3). Endothelins act mainly as paracrine and autocrine agents and bind to their own receptors on the epithelial cells and vascular smooth muscles (Fig 13.13). The ET-1 is the most potent naturally occurring vasoconstricting agent that interacts with its ETA receptor on vascular smooth muscles.

1	High levels of ET-1 gene expression are associated with many diseases that are caused in part by sustained en-dothelium-induced vasoconstriction. These include systemic hypertension (see Folder 13.2), pulmonary hypertension, atherosclerosis, congestive heart failure, idiopathic cardiomyopathy, and renal failure. It is interesting to mention that snake venom from the Israeli burrowing asp (Atractaspis engaddensis) contains sarafotoxin, a highly toxic protein that exhibits a very high degree of sequence homology with ET-1. After it enters the circulation, it binds to ETA receptors and causes life-threatening, intense coronary vasoconstriction. This is remarkable because endothelin is a natural compound of the human vascular system, whereas sarafotoxin is a toxin in the snake venom. The other endothelium-derived vasoconstrictors include thromboxane A2 and prostaglandin

1	H2. Thromboxane A2 is synthesized from prostaglandin H2. In addition, decreased rate of NO production or inactivation of NO by the superoxide anion (O2–) has a stimulating effect on smooth muscle contraction (see Fig. 13.13). Traditionally, arteries are classified into three types on the basis of size and the characteristics of the tunica media.  Large arteries or elastic arteries such as the aorta and pulmonary arteries convey blood from the heart to the systemic and pulmonary circulations, respectively (see Fig. 13.2). Their main branches—the brachiocephalic trunk, common carotid, subclavian, and common iliac arteries—are also classified as elastic arteries.  Medium arteries or muscular arteries (most of the “named” arteries of the body) cannot be sharply distinguished from elastic arteries. Some of these arteries are difficult to classify because they have features that are intermediate between the two types.

1	 Small arteries and arterioles are distinguished from one another by the number of smooth muscle layers in the tunica media. By definition, arterioles have only one or two

1	FIGURE 13.13 • Molecular mechanism of blood vessel vasoconstriction. Contraction of vascular smooth muscle in a blood vessel (vasoconstriction) decreases its diameter and increases vascular resistance, leading to increased systemic blood pressure. Binding of angiotensin II and thrombin to vascular endothelial cells stimulate synthesis of endothelium-derived factors that regulate smooth muscle contraction. These include endothelins (the most potent family of vasoconstrictors), prostaglandin H2 (PGH2), and its derivative thromboxane A2. They bind to their own receptors on the smooth muscle cell membrane, causing an influx of Ca2 and an increase in the release of intracellular-stored Ca2 from the sarcoplasmic reticulum. The reduced rate of nitric oxide (NO) production, which is a potent vasodilatator, or inactivation of NO by the superoxide anion (O2–) has a stimulating effect on smooth muscle contraction. (Based on Noble A, Johnson R, Thomas A, Bass P. The Cardiovascular System. London,

1	or inactivation of NO by the superoxide anion (O2–) has a stimulating effect on smooth muscle contraction. (Based on Noble A, Johnson R, Thomas A, Bass P. The Cardiovascular System. London, New York: Churchill Livingstone, 2005.) layers, and small arteries may have as many as eight layers of smooth muscle in their tunica media.

1	Elastic arteries have multiple sheets of elastic lamellae in their walls.

1	From a functional standpoint, elastic arteries serve primarily as conduction tubes; however, they also facilitate the continuous and uniform movement of blood along the tube. Blood flow occurs as follows: The ventricles of the heart pump blood into the elastic arteries during systole (the contraction phase of the cardiac cycle). The pressure generated by contraction of the ventricles moves the blood through the elastic arteries and along the arterial tree. Simultaneously, it also causes the wall of the large elastic arteries to distend. The distension is limited by the network of collagenous fibers in the tunica media and tunica adventitia (Fig. 13.14). During diastole (the relaxation phase of the cardiac cycle), when no pressure is generated by the heart, the recoil of the distended elastic arteries serves to maintain arterial blood pressure and the flow of blood within the vessels. Initial elastic recoil forces blood both away from and back toward the heart. The flow of blood toward

1	arteries serves to maintain arterial blood pressure and the flow of blood within the vessels. Initial elastic recoil forces blood both away from and back toward the heart. The flow of blood toward the heart causes the aortic and pulmonary valves to close. Continued elastic recoil then maintains continuous flow of blood away from the heart.

1	The tunica intima of the elastic artery consists of endothelium, subendothelial connective tissue, and an inconspicuous internal elastic membrane. The tunica intima of elastic arteries is relatively thick and consists of the following.

1	 In the endothelial lining with its basal lamina, the cells are typically flat and elongated, with their long axes oriented parallel to the direction of blood flow in the artery (Fig. 13.15). In forming the epithelial sheet, the cells are joined by tight junctions (zonulae occludentes) and gap junctions (Fig. 13.16). Endothelial cells possess rodlike inclusions called Weibel-Palade bodies that are present in the cytoplasm. These specific endothelial organelles are electron-dense structures and contain von Willebrand factor and P-selectin. Von Willebrand factor is a glycoprotein synthesized by arterial endothelial cells. When secreted into the blood, it binds coagulating factor VIII) and plays an important role in platelets’ adhesion to the site of endothelial injury. The antibody to von Willebrand factor is commonly used as an immunohistochemical marker for identification of en-dothelium-derived tumors. P-selectin is a cell adhesion molecule involved in the mechanism of neutrophil–

1	Willebrand factor is commonly used as an immunohistochemical marker for identification of en-dothelium-derived tumors. P-selectin is a cell adhesion molecule involved in the mechanism of neutrophil– endothelial cell recognition. It initiates neutrophil migration from the blood to the site of action in the connective tissue.

1	 FOLDER 13.2 Clinical Correlation: Hypertension

1	Hypertension, or high blood pressure, occurs in about 25% of the population and is defined by a sustained dias-tolic pressure greater than 90 mmHg or a sustained systolic pressure greater than 140 mmHg. Hypertension is often as-sociated with atherosclerotic vascular disease and with an increased risk of cardiovascular disorders such as stroke and angina pectoris. In most cases of hypertension, the luminal diameter of small muscular arteries and arterioles is reduced, which leads to increased vascular resistance. Re-striction in the luminal size may also result from active con-traction of smooth muscle in the vessel wall, an increase in the amount of smooth muscle in the wall, or both. In individuals with hypertension, smooth muscle cells multiply. The additional smooth muscle then adds to the thickness of the tunica media. Concomitantly, some of the smooth muscle cells accumulate lipid. This is one reason why hypertension is a major risk factor for atherosclerosis. In fat-fed animals,

1	the thickness of the tunica media. Concomitantly, some of the smooth muscle cells accumulate lipid. This is one reason why hypertension is a major risk factor for atherosclerosis. In fat-fed animals, hypertension accelerates the rate of lipid accumulation in vessel walls. In the absence of a fatty diet, hypertension increases the rate of intimal thickening that occurs naturally with age. Cardiac muscle is also affected by chronic hypertension that leads to pressure overload, resulting in compensatory left ventricular hypertrophy. Ventricular hypertrophy in this condition is caused by an increased diameter (not length) of cardiac muscle cells with characteristic enlarged and rectangular nuclei. Left ventricular hypertrophy is a com-mon manifestation of the hypertensive heart disease. Ventricular hypertrophy makes the wall of the left ventricle uniformly much thicker and less elastic, and the heart must then work harder to pump blood (Fig. F13.2.1). Untreated hypertensive heart disease

1	hypertrophy makes the wall of the left ventricle uniformly much thicker and less elastic, and the heart must then work harder to pump blood (Fig. F13.2.1). Untreated hypertensive heart disease would lead to cardiac failure. Recent studies have shown that prolonged reduction of blood pressure in patients with ventricular hypertrophy as a result of chronic hypertension can reduce the degree of hypertrophy. normal wall of right ventricle hypertrophied wall of left ventricle FIGURE F13.2.1 • Horizontal section of the heart with left ventricular hypertrophy. This photograph shows a cross section of the ventricles of the heart from a patient with chronic hypertension. The walls of the left ventricle are concentrically thickened, resulting in decreases of the cavity diameter. Note the wall of the right ventricle, which has normal dimensions. (Reprinted with permission from Rubin R, Strayer DS. Rubin’s Pathology. 5th ed. Baltimore: Lippincott Williams & Wilkins, 2008.)  The subendothelial

1	the right ventricle, which has normal dimensions. (Reprinted with permission from Rubin R, Strayer DS. Rubin’s Pathology. 5th ed. Baltimore: Lippincott Williams & Wilkins, 2008.)  The subendothelial layer of connective tissue in larger elastic arteries consists of connective tissue with both collagen and elastic fibers. The main cell type in this layer is the smooth muscle cell. It is contractile and secretes extracellular ground substance as well as collagen and elastic fibers. Occasional macrophages may also be present.

1	 The internal elastic membrane in elastic arteries is not conspicuous because it is one of many elastic layers in the wall of the vessel. It is usually identified only because it is the innermost elastic layer of the arterial wall. Endothelial cells participate in the structural and functional integrity of the vascular wall. Endothelial cells not only provide a physical barrier between the circulating blood and the subendothelial tissues but also produce vasoactive agents that cause constriction and relaxation of underlying vascular smooth muscles. Multiple roles and functions of the endothelium lining of blood vessels are described in detail at the beginning of this chapter (see page 409). The tunica media of elastic arteries consists of multiple layers of smooth muscle cells separated by elastic lamellae. The tunica media is the thickest of the three layers of elastic arteries and consists of the following.

1	The tunica media is the thickest of the three layers of elastic arteries and consists of the following.  Elastin in the form of fenestrated sheets or lamellae between the muscle cell layers. These lamellae are arranged in concentric layers (Figs. 13.17a, 13.14, and Plate 33, page 434). As noted, fenestrations in the lamellae facilitate the diffusion of substances within the arterial wall. The number and thickness of these lamellae are related to blood pressure and age. At birth, the aorta is almost devoid of lamellae; in the adult, the aorta has 40 to 70 lamellae. In individuals with hypertension, both the number and the thickness of the lamellae are increased.

1	FIGURE 13.14 • Diagram and photomicrograph of an elastic artery. a. This schematic diagram of a typical elastic artery shows its cellular and extracellular components. Note the organization of smooth muscle cells in the tunica media and the distribution of elastic lamellae. The internal elastic membrane is not well defined and is represented by the innermost elastic lamellae of the arterial wall. b. This low magnification photomicrograph shows the section of the wall of the human aorta stained with Weigert’s resorcin-fuchsin elastic stain to visualize elastic lamellae interspersed with the smooth muscle cells of the tunica media. Only the tunica media, which is the thickest of the three layers of the elastic arteries, is labeled on this image. Note that elastic lamellae, collagen fibrils, and blood vessels are present in the tunica adventitia. 48.

1	 Smooth muscle cells are arranged in layers. The smooth muscle cells are arranged in a low-pitch spiral relative to the long axis of the vessel; thus, in cross sections of the artery they appear in a circular array. The smooth muscle cells are spindle-shaped with an elongated nucleus. They are invested with an external (basal) lamina except where they are joined by gap junctions. Fibroblasts are not present in the tunica media. Smooth muscle cells synthesize the collagen, elastin, and other molecules of the extracellular matrix. In addition, in response to growth factors (i.e., PDGF, FGF) produced by endothelial cells, smooth muscle cells may proliferate and migrate to the adjacent intima. This characteristic is important in normal repair of the vascular wall and in pathologic processes similar to those occurring in atherosclerosis.  Collagen fibers and ground substance (proteoglycans) are synthesized and secreted by the smooth muscle cells.

1	 Collagen fibers and ground substance (proteoglycans) are synthesized and secreted by the smooth muscle cells. The tunica adventitia in the elastic artery is a relatively thin connective tissue layer. In elastic arteries, the tunica adventitia is usually less than half the thickness of the tunica media. It consists of the following.  Collagen fibers and elastic fibers form a loose network of elastic fibers (not lamellae) that are less organized than those in the tunica media. The collagen fibers help prevent the expansion of the arterial wall beyond physiologic limits during systole of the cardiac cycle.  Fibroblasts and macrophages are the principal cells of the tunica adventitia.

1	 Fibroblasts and macrophages are the principal cells of the tunica adventitia.  The vasa vasorum (blood vessels) and nervi vascularis (nerves) include arterial branches and veins similar to that of vasculature in general and provide nutrients and remove waste products from the outer portion of the arterial wall. They may partially enter the outer part of the tunica media. The inner part of the wall is supplied by nutrients from the lumen of the vessel. The hemodynamic impact (i.e., increased blood pressure) on the function of the vasa vasorum may play a role in the pathogenesis of atheromatous plaques. Muscular arteries have more smooth muscle and less elastin in the tunica media than do elastic arteries.

1	Muscular arteries have more smooth muscle and less elastin in the tunica media than do elastic arteries. Generally, in the region of transition between elastic arteries and large muscular arteries, the amount of elastic material decreases, and smooth muscle cells become the predominant endothelial cells cytoplasm of endothelial cells basal lamina junctional complex ba FIGURE 13.15 • Diagram and scanning electron micrograph of the endothelium. a. This schematic drawing shows the luminal surface of the endothelium. The cells are elongated with their long axis parallel to the direction of blood flow. Nuclei of endothelial cells are also elongated in the direction of blood flow. b. Scanning electron micrograph of a small vein, showing the cells of the endothelial lining. Note the spindle shape with the long axis of the cells running parallel to the vessel. 1,100.

1	FIGURE 13.16 • Diagram depicting segments of two adjacent endothelial cells. This diagram shows cell-to-cell and cellto-extracellular matrix junctions in the endothelial cells. Junctional complex near the luminal surface (dashed line box) comprises the zonula occludens and the zonula adherens, and a desmosome represents a group of cell-to-cell junctions that maintain selective permeability barrier. Cell-to-cell communication junctions are represented by an area of gap junctions (green). Anchoring junctions (cell-to-extracellular matrix) are represented here by hemidesmosomes and focal adhesions. Observe the organization of the cytoplasm and cytoplasmic inclusions, the Weibel-Palade bodies, that are characteristic of endothelial cells. Pinocytotic vesicles in the cell on the left have been positioned to suggest the pathway of vesicles from the lumen of the blood vessel to the basal cell membrane or to the lateral cell membrane as indicated by the dashed arrows. Various markers have

1	positioned to suggest the pathway of vesicles from the lumen of the blood vessel to the basal cell membrane or to the lateral cell membrane as indicated by the dashed arrows. Various markers have been traced through pinocytotic pathways across the endothelial cell. Luminal surface of the endothelial cells expresses variety of endothelial cell-surface receptors.

1	FIGURE 13.17 • Photomicrographs of the wall of elastic and muscular types of arteries. a. This photomicrograph shows a cross section of the human aorta stained with resorcin-fuchsin to demonstrate elastic material. Three layers can be recognized: the tunica intima, the tunica media, and the tunica adventitia. The tunica intima consists of a lining of endothelial cells that rest on a thin layer of connective tissue containing smooth muscle cells, occasional macrophages, and collagen and elastic fibers. The boundary between it and the tissue below, the tunica media, is not sharply defined. The tunica media contains an abundance of smooth muscle cells (note the blue-staining nuclei) and numerous elastic fenestrated membranes (the red, wavy lamellae). The tunica adventitia, the outermost part, lacks elastic laminae, consists mainly of connective tissue, and contains the blood vessels and nerves that supply the aortic wall. 300. b. Photomicrograph of a cross section through a muscular

1	part, lacks elastic laminae, consists mainly of connective tissue, and contains the blood vessels and nerves that supply the aortic wall. 300. b. Photomicrograph of a cross section through a muscular artery in a routine H&E preparation shows that the wall of the muscular artery is also divided into the same three layers as in the elastic artery. The tunica intima consists of an endothelial lining, a small amount of connective tissue, and the internal elastic membrane. This structure has a scalloped appearance when the vessel is constricted and is highly refractile. Constriction also causes the endothelial cell nuclei to appear rounded. The tunica media consists mainly of circularly arranged smooth muscle cells and collagen and elastic fibers. The nuclei of the smooth muscle cells, when contracted, have a corkscrew appearance. The tunica adventitia consists mostly of connective tissue. A well-defined external elastic membrane is not apparent in this vessel, but profiles of elastic

1	contracted, have a corkscrew appearance. The tunica adventitia consists mostly of connective tissue. A well-defined external elastic membrane is not apparent in this vessel, but profiles of elastic material (arrows) are present. 360.

1	constituent of the tunica media (Fig. 13.18 and Plate 34, page 436). Also, a prominent internal elastic membrane becomes apparent, helping to distinguish muscular arteries from elastic arteries. In many instances, a recognizable external elastic membrane is also evident. The tunica intima is thinner in muscular arteries and contains a prominent internal elastic membrane. The tunica intima is relatively thinner in muscular arteries than in elastic arteries and consists of an endothelial lining with its basal lamina, a sparse subendothelial layer of connective tissue, and a prominent internal elastic membrane. In some muscular arteries, the subendothelial layer is so scanty that the basal lamina of the endothelium appears to make contact with the internal elastic membrane. In histologic sections, the internal elastic membrane usually appears as a well-defined, undulating or wavy structure because of contraction of the smooth muscle (Fig. 13.17b).

1	The thickness of the tunica intima varies with age and other factors. In young children, it is very thin. In muscular arteries of young adults, the tunica intima accounts for about one-sixth of the total wall thickness. In older adults, the tunica intima may be expanded by lipid deposits, often in the form of irregular “fatty streaks.” The tunica media of muscular arteries is composed almost entirely of smooth muscle, with little elastic material. The tunica media of muscular arteries consists of smooth muscle cells amid collagen fibers and relatively little elastic material. The smooth muscle cells are arranged in a spiral fashion in the arterial wall. Their contraction helps maintain blood pressure. As in elastic arteries, there are no fibroblasts in this layer. The smooth muscle cells possess an external (basal) lamina except at the sites of gap junctions and produce extracellular collagen, elastin, and ground substance.

1	FIGURE 13.18 • Diagram and photomicrograph of a muscular artery. a. In this schematic diagram of a muscular artery, the cellular and extracellular components are labeled. Note the distribution of cellular components in all three tunics and the locations of external and internal elastic membranes. b. In this photomicrograph of a cross section through a muscular artery in Weigert’s resorcin-fuchsin elastic stain preparation, note two distinct layers of elastic tissue: a wavy-appearing inner layer of the internal elastic membrane and a well-defined outer layer of the external elastic membrane. The relatively thick tunica media enclosed by the internal and external elastic membranes consists mainly of circularly arranged smooth muscle cells, collagen, and fine elastic fibers. The tunica intima in this preparation is indiscernible; the tunica adventitia is well defined, consisting mostly of connective tissue with collagen and elastic fibers. 175.

1	The tunica adventitia of muscular arteries is relatively thick and is often separated from the tunica media by a recognizable external elastic membrane. The tunica adventitia of muscular arteries consists of fibroblasts, collagen fibers, elastic fibers, and, in some vessels, scattered adipose cells. Compared with elastic arteries, the tunica adventitia of muscular arteries is relatively thick—about the same thickness as the tunica media. Collagen fibers are the principal extracellular component. However, a concentration of elastic material immediately adjacent to the tunica media is often present and as such constitutes the external elastic membrane. Nerves and small vessels travel in the adventitia and give off branches that penetrate into the tunica media in the large muscular arteries as the vasa vasorum. Small arteries and arterioles are distinguished from one another by the number of smooth muscle cell layers in the tunica media.

1	Small arteries and arterioles are distinguished from one another by the number of smooth muscle cell layers in the tunica media. As mentioned previously, arterioles have only one or two layers, and a small artery may have as many as eight layers of smooth muscle in the tunica media (Fig. 13.19) and (Plate 35, page 438). Typically, the tunica intima of a small artery has an internal elastic membrane, whereas this layer may or may not be present in the arteriole. The endothelium in both is essentially similar to endothelium in other arteries, except that at the electron microscope level, gap junctions may be found between endothelial cells and the smooth muscle cells of the tunica media. Last, the tunica adventitia is a thin, ill-defined sheath of connective tissue that blends with the connective tissue in which these vessels travel. Arterioles control blood flow to capillary networks by contraction of the smooth muscle cells.

1	Arterioles control blood flow to capillary networks by contraction of the smooth muscle cells. Arterioles serve as flow regulators for the capillary beds. In the normal relationship between an arteriole and a capillary network, contraction of the smooth muscle in the wall of an arteriole increases the vascular resistance and reduces or shuts off the blood going to the capillaries. The slight thickening of the smooth muscle at the origin of a capillary bed from an arteriole is called the precapillary sphincter. Most arterioles can dilate 60% to 100% from their resting diameter, and they can maintain as much as 40% constriction for a long time. Therefore, a large decrease or increase in vascular resistance has a direct effect on distribution of blood flow and systemic arterial pressure. This regulation directs blood to where it may be most needed. For instance, during strenuous physical exertion such as running, blood

1	FIGURE 13.19 • Electron micrograph and photomicrograph of arterioles. a. This electron micrograph shows a cross section of an arteriole. The tunica intima of the vessel is composed of an endothelium and a very thin layer of subendothelial connective tissue (collagen fibrils and ground substance). The arrows indicate the site of junction between adjoining endothelial cells. The tunica media consists of a single layer of smooth muscle cells (SM). The tunica adventitia is composed of collagen fibrils and several layers of fibroblasts (F) with extremely attenuated processes. Red blood cells are visible in the lumen. 6,000. b. Photomicrograph of arteriole and venule in the dermis. One arteriole is seen in longitudinal section, and another is seen in cross section. The round and ovoid nuclei in the wall of the longitudinally sectioned arteriole belong to the smooth muscle cells of the tunica media. Their round to ovoid shape indicates that these cells have been cut in cross section. The

1	in the wall of the longitudinally sectioned arteriole belong to the smooth muscle cells of the tunica media. Their round to ovoid shape indicates that these cells have been cut in cross section. The elongated nuclei (arrows) belong to endothelial cells. 320. Inset. The cross-sectioned arteriole is shown here at higher magnification and reveals the endothelial cell nuclei bulging into the lumen (arrows). They reflect a cross-sectional cut. The nuclei of the smooth muscle cells in the tunica media appear as elongate profiles reflecting their circular pattern around the vessel. 600.

1	ﬂow to skeletal muscle is increased by dilation of arterioles, and blood ﬂow to the intestine is reduced by arteriolar constriction. After ingestion of a large meal, however, the reverse is true. Capillaries are the smallest diameter blood vessels, often smaller than the diameter of an erythrocyte.

1	Capillaries form blood vascular networks that allow fluids containing gases, metabolites, and waste products to move through their thin walls. The human body contains approximately 50,000 miles of capillaries. Each consists of a single layer of endothelial cells and their basal lamina. The endothelial cells form a tube just large enough to allow the passage of red blood cells one at a time. In many capillaries the lumen is so narrow that the red cells literally fold on themselves to pass through the vessel (Fig. 13.20). The passing red blood cells fill virtually the entire capillary lumen, minimizing the diffusion path for gases and nutrients between the capillary and the extravascular tissue. In cross sections and with the TEM, the tube appears to be formed by only one cell or portions of several cells. Because of their thin walls and close physical association with metabolically active cells and tissues, capillaries are particularly well suited for the exchange of gases and

1	of several cells. Because of their thin walls and close physical association with metabolically active cells and tissues, capillaries are particularly well suited for the exchange of gases and metabolites between cells and the bloodstream. The ratios of capillary volume to endothelial surface area and thickness also favor movement of substances across the vessel wall.

1	Classification of Capillaries Capillary structure varies in different tissues and organs. On the basis of their morphology, three types of capillaries are described: continuous capillaries, fenestrated capillaries, and discontinuous capillaries. Continuous capillaries are typically found in muscle, lung, and the CNS. With the TEM, they appear in cross sections as two plasma membranes enclosing a ribbon of cytoplasm that may include the nucleus (Fig. 13.21). Occluding junctions can be seen in the typical cross section of a continuous capillary. They only allow the passage of relatively small molecules (less than 10,000 daltons) between adjacent endothelial cells.

1	FIGURE 13.20 • Photomicrograph of the capillary network in the retina. This is a whole-mount preparation of retinal capillaries. After mild enzymatic digestion, retina was spread on a glass slide, stained with the periodic acid–Schiff (PAS) procedure, and counterstained with hematoxylin. Vertically crossing the image is an arterial (A) with a clearly visible layer of circularly arranged smooth muscle cells (SM). The arterial is crossed perpendicularly by a venule (V). Note the extensive network of capillaries connecting both vessels. Nuclei of endothelial cells (E) are clearly visible within capillaries. At this magnification, pericytes are difficult to discern. 560. (Courtesy Mr. Denifield W. Player.) Numerous pinocytotic vesicles underlie both the luminal and basal plasma membrane surfaces. The vesicles are approximately 70 nm in diameter and function in the transport of larger molecules between the lumen and the connective tissue and vice versa.

1	In some continuous capillaries and postcapillary venules, pericytes (historically known as Rouget cells) may be associated with the endothelium (see Figs. 13.20 and 13.21). The pericyte, when present, intimately surrounds the capillary, with branching cytoplasmic processes, and is enclosed by a basal lamina that is continuous with that of the endothelium. Pericytes are contractile and are controlled by NO produced by endothelial cells. They provide vascular support and promote stability of capillaries and postcapillary venules through physical and chemical signaling with vascular endothelial cells. Histologically, pericytes display features of undifferentiated mesenchymal stem cells with large nuclei rich in heterochromatin. During embryonic development or angiogenesis (i.e., wound healing), pericytes give rise to both endothelial cells and smooth muscle cells. Pericytes are directly involved in the pathogenesis of vascular driven diseases (i.e., diabetic retinopathy and tumor

1	healing), pericytes give rise to both endothelial cells and smooth muscle cells. Pericytes are directly involved in the pathogenesis of vascular driven diseases (i.e., diabetic retinopathy and tumor angiogenesis). In addition, uncontrolled divisions of pericytes give rise to the hemangiopericytoma, a rare vascular tumor that can originate in the body anywhere there are capillaries.

1	Fenestrated capillaries are typically found in endocrine glands and sites of fluid and metabolite absorption such as the gallbladder, kidney, and intestinal tract. They are characterized by fenestrations, 80 to 100 nm in diameter, that provide channels across the capillary wall (Fig. 13.22). Fenestrated capillaries also have pinocytotic vesicles. Some research suggests that fenestrations are formed when a forming pinocytotic vesicle spans the narrow cytoplasmic layer and simultaneously opens on the opposite surface. A fenestration may have a thin, nonmembranous diaphragm across its opening. When viewed from the luminal surface, this diaphragm has a cartwheel-like shape with a central thickening and 14 wedge-shaped gaps. It is derived from the glycocalyx formerly enclosed in the pinocytotic vesicle from which the fenestration may have formed.

1	Fenestrated capillaries in the gastrointestinal tract and gallbladder have fewer fenestrations and a thicker wall when no absorption is occurring. When absorption takes place, the walls thin, and the number of pinocytotic vesicles and fenestrations increases rapidly. The ionic changes in the perivascular connective tissue, caused by the absorbed solutes, stimulate pinocytosis. These observations support the suggested mode of formation of the fenestrations described above.

1	Discontinuous capillaries (also called sinusoidal capillaries or sinusoids) are typically found in the liver, spleen, and bone marrow. They are larger in diameter and more irregularly shaped than other capillaries. Structural features of these capillaries vary from organ to organ and include specialized cells. Kupffer cells (stellate sinusoidal macrophages) and vitamin A–storing Ito cells (hepatic stellate cells) in the liver occur in association with the endothelial cells. In the spleen, endothelial cells exhibit a unique spindle shape with gaps between the neighboring cells; the basal lamina underlying the endothelium may be partially or even completely absent. Functional Aspects of Capillaries To understand capillary function, two important points— vasomotion (i.e., capillary blood flow) and the extent or density of the capillary network—should be considered.

1	Blood flow is controlled through local and systemic signals. In response to vasodilating agents (e.g., NO, low O2 tension), the smooth muscle in the walls of the arterioles relaxes, resulting in vasodilation and increased blood ﬂow through the capillary system. Pressure within the capillaries increases, and much of the plasma ﬂuid is driven into the tissue. This process occurs in peripheral edema. Local endothelial-derived factors, systemic signals carried by the autonomic nervous system, and norepinephrine released by the adrenal gland cause the smooth muscle of the arterioles to contract (vasoconstriction), resulting in decreased blood ﬂow through the capillary bed. In this condition, capillary pressure can decrease and greatly increase absorption of tissue ﬂuid. This situation occurs during loss of blood volume and can add considerable amount of ﬂuid into the blood, preventing hypovolemic shock.

1	FIGURE 13.21 • Electron micrograph and diagram of a continuous capillary. The endothelial cells that make up the wall of a continuous capillary contain numerous pinocytotic vesicles. The cell junctions are frequently marked by cytoplasmic (marginal) folds that protrude into the lumen. The endothelial cell nuclei are not included within the plane of section in the micrograph, but an endothelial cell with its nucleus is shown in the diagram. Similarly, the electron micrograph shows only a small amount of pericyte cytoplasm; a nucleus is not seen but is shown in the diagram (see the upper right and lower left of the micrograph). Note that the pericyte cytoplasm is enclosed by basal lamina. 30,000.

1	The density of the capillary network determines the total surface area available for exchange between the blood and tissue. It is related to the metabolic activity of the tissue. The liver, kidney, cardiac muscle, and skeletal muscle have rich capillary networks. Dense connective tissue is less metabolically active and has less extensive capillary networks. Arteriovenous shunts allow blood to bypass capillaries by providing direct routes between arteries and veins.

1	Generally, in a microvascular bed, arteries convey blood to the capillaries, and veins convey blood away from the capillaries. However, all the blood does not necessarily pass from arteries to capillaries and thence to veins. In many tissues, there are direct routes between the arteries and veins that divert blood from the capillaries. These routes are called arteriovenous (AV) anastomoses or AV shunts (see Fig. 13.11). AV shunts are commonly found in the skin of the fingertips, nose, and lips and in the erectile tissue of the penis and clitoris. The arteriole of AV shunts is often coiled, has a relatively thick smooth muscle layer, is enclosed in a connective tissue capsule, and is richly innervated. Contrary to the ordinary precapillary sphincter, contraction of the arteriole smooth muscle of the AV shunt sends blood to a capillary bed; relaxation of the smooth muscle sends blood to a venule, bypassing the capillary bed. AV shunts serve in thermoregulation at the body surface.

1	muscle of the AV shunt sends blood to a capillary bed; relaxation of the smooth muscle sends blood to a venule, bypassing the capillary bed. AV shunts serve in thermoregulation at the body surface. Closing an AV shunt in the skin causes blood to ﬂow through the capillary bed, enhancing heat loss. Opening an AV shunt in the skin reduces the blood ﬂow to the skin capillaries, conserving body heat. In erectile tissue such as the penis, closing the AV shunt directs blood ﬂow into the corpora cavernosa, initiating the erectile response.

1	FIGURE 13.22 • Electron micrograph and schematic diagram of a fenestrated capillary. The cytoplasm of the endothelial cells contains numerous fenestrations (small arrows). In some of the thicker regions of the endothelial cells where the fenestrations are absent, pinocytotic vesicles are present. Part of a pericyte is seen on the left side of the electron micrograph, including its nucleus in the upper left corner of the micrograph. 21,500. The inset shows to advantage the fenestrations and the diaphragm that spans the openings (large arrows). 55,000.

1	Preferential thoroughfares, whose proximal segment is called a metarteriole (Fig. 13.23), also allow some blood to pass more directly from artery to vein. Capillaries arise from both arterioles and metarterioles. Although capillaries themselves have no smooth muscle in their walls, a sphincter of smooth muscle called the precapillary sphincter is located at their origin from either an arteriole or a metarteriole. These sphincters control the amount of blood passing through the capillary bed. The tunics of veins are not as distinct or well defined as the tunics of arteries. Traditionally, veins are divided into four types on the basis of size.  Venules are further subclassified as postcapillary and muscular venules. They receive blood from capillaries and have a diameter as small as 0.1 mm.  Small veins are less then 1 mm in diameter and are continuous with muscular venules.

1	 Small veins are less then 1 mm in diameter and are continuous with muscular venules.  Medium veins represent most of the named veins in this category. They usually are accompanied by arteries and have a diameter of as much as 10 mm.  Large veins usually have a diameter greater than 10 mm. Examples of such veins include the superior and inferior vena cava and hepatic portal vein. Although large and medium veins have three layers— also designated tunica intima, tunica media, and tunica adventitia—these layers are not as distinct as they are in arteries. Largeand medium-sized veins usually travel with largeand medium-sized arteries; arterioles and muscular venules also sometimes travel together, thus allowing comparison in histologic sections. Typically, veins have thinner walls than their accompanying arteries, and the lumen of the vein is larger than that of the artery. The arteriole lumen

1	FIGURE 13.23 • Diagram of microcirculation. This schematic diagram shows a metarteriole (initial segment of a thoroughfare channel) giving rise to capillaries. The precapillary sphincters of the arteriole and metarteriole control the entry of blood into the capillaries. The distal segment of the thoroughfare channel receives capillaries from the microcirculatory bed, but no sphincters are present where the afferent capillaries enter the thoroughfare channels. Blind-ended lymphatic vessels are shown in association with the capillary bed. Note the presence of anchoring filaments and the valve system within the lymphatic capillaries. is usually patent; that of the vein is often collapsed. Many veins, especially those that convey blood against gravity, such as those of the limbs, contain valves that allow blood to flow in only one direction, back toward the heart. The valves are semilunar flaps consisting of a thin connective tissue core covered by endothelial cells.

1	Postcapillary venules collect blood from the capillary network and are characterized by the presence of pericytes. Postcapillary venules possess an endothelial lining with its basal lamina and pericytes (Plate 35, page 438). The endothelium of postcapillary venules is the principal site of action of vasoactive agents such as histamine and serotonin.

1	Response to these agents results in extravasation of ﬂuid and emigration of white blood cells from the vessel during inﬂammation and allergic reactions. Postcapillary venules of lymph nodes also participate in the transmural migration of lymphocytes from the vascular lumen into the lymphatic tissue. Pericytes form umbrella-like connections with the endothelial cells. The relationship between endothelial cells and pericytes promote their mutual proliferation and survival. Both synthesize and share the basal lamina (see Fig. 13.21), synthesize growth factors, and communicate with each other through tight and gap junctions. Pericyte coverage is more extensive in the postcapillary venules than capillaries. The postcapillary venules in the lymph nodes are also called high endothelial venules (HEVs) because of the prominent cuboidal appearance of their endothelial cells and their ovoid nuclei.

1	The postcapillary venules in the lymph nodes are also called high endothelial venules (HEVs) because of the prominent cuboidal appearance of their endothelial cells and their ovoid nuclei. Muscular venules are distinguished from postcapillary venules by the presence of a tunica media. Muscular venules are located distal to the postcapillary venules in the returning venous network and have a diameter of as much as 0.1 mm. Whereas postcapillary venules have no true tunica media, the muscular venules have one or two layers of smooth muscle that constitute a tunica media. These vessels also have a thin tunica adventitia. Usually, pericytes are not found in muscular venules.

1	Medium veins have a diameter of as much as 10 mm. Most deep veins that accompany arteries are in this category (e.g., radial vein, tibial vein, popliteal vein). Valves are a characteristic feature of these vessels and are most numerous in the inferior portion of the body, particularly the lower limbs, to prevent retrograde movement of blood because of gravity. Often deep veins of lower limbs are the site of thrombus (blood clot) formation, a condition known as deep venous thrombosis (DVT). DVT is associated with immobilization of the lower limbs due to prolonged bed rest (after surgery or hospitalization), orthopedic casts, or restricted movement during long-haul ﬂights. DVT can be a life-threatening condition because of the potential for development of pulmonary embolism (blockage of the pulmonary arteries) by a dislodged blood clot originating from deep veins. The three tunics of the venous wall are most evident in medium-sized veins (Fig. 13.24).

1	The three tunics of the venous wall are most evident in medium-sized veins (Fig. 13.24).  The tunica intima consists of an endothelium with its basal lamina, a thin subendothelial layer with occasional smooth muscle cells scattered in the connective tissue elements, and, in some cases, a thin internal elastic membrane.  The tunica media of medium-sized veins is much thinner than the same layer in medium-sized arteries. It contains several layers of circularly arranged smooth muscle cells with interspersed collagen and elastic fibers. In addition, longitudinally arranged smooth muscle cells may be present just beneath the tunica adventitia.  The tunica adventitia is typically thicker than the tunica media and consists of collagen fibers and networks of elastic fibers (Fig. 13.24b). In large veins, the tunica media is relatively thin, and the tunica adventitia is relatively thick. Veins with a diameter greater than 10 mm are classified as large veins.

1	In large veins, the tunica media is relatively thin, and the tunica adventitia is relatively thick. Veins with a diameter greater than 10 mm are classified as large veins.  The tunica intima of these veins (Fig. 13.25 and Plate 34, page 436) consists of an endothelial lining with its basal lamina, a small amount of subendothelial connective tissue, and some smooth muscle cells. Often the boundary between the tunica intima and tunica media is not clear, and it is not always easy to decide whether the smooth muscle cells close to the intimal endothelium belong to the tunica intima or to the tunica media.

1	FIGURE 13.24 • Schematic diagram and photomicrograph of a medium-sized vein. a. The cellular and extracellular components are labeled. Note that the tunica media contains a few layers of circularly arranged smooth muscle cells with interspersed collagen and elastic fibers. Also, longitudinally arranged smooth muscle cells are present at the junction with the tunica adventitia. b. This photomicrograph shows a section through the wall of a medium-sized vein in routine H&E preparation. The tunica intima consists of endothelium and a very thin subendothelial layer of connective tissue containing some smooth muscle cells. The tunica media contains a few layers of circularly and spirally arranged smooth muscle cells with collagen and elastic fibers. Note that the thickest layer is the tunica adventitia, which contains an abundance of collagen and some elastic fibers. The few nuclei seen in this layer belong to fibroblasts. 360.

1	 The tunica media is relatively thin and contains circumferentially arranged smooth muscle cells, collagen fibers, and some fibroblasts. In some animals, but not in humans, cardiac muscle cells extend into the tunica media of the both superior and inferior vena cava and the pulmonary veins, near their junction with the heart.  The tunica adventitia of large veins (e.g., the subclavian veins, portal vein, and the venae cavae) is the thickest layer of the vessel wall. Along with the usual collagen and elastic fibers and fibroblasts, the tunica adventitia also contains longitudinally disposed smooth muscle cells (Fig. 13.26). In several locations in the body, blood vessels—both arteries and veins with an atypical structure are present. These include the following.

1	 Coronary arteries, considered to be medium-sized muscular arteries, originate from the proximal part of the ascending aorta and lie on the surface of the heart in the epicardium surrounded by adipose tissue. The walls of coronary arteries are usually thicker than those of comparable arteries on the upper or lower limb because of the large amounts of circular smooth muscle layers in the tunica media. In routine H&E preparation, the subendothelial layer of the tunica intima of younger people is inconspicuous, but it progressively thickens by increasing amounts of smooth muscle cell and fibroelastic tissue with aging (Fig. 13.27). The internal elastic membrane is well developed, although it may be fragmented, duplicated, or focally lost in older individuals. The relatively “loose” consistency of the tunica adventitia is reinforced by the longitudinal bundles of collagen fibers that allow for continuous changes of the vascular diameter.

1	Atherosclerotic changes in coronary arteries that restrict blood ﬂow and oxygen supply to cardiac muscle lead to ischemic heart disease (see Folder 13.3).  Dural venous sinuses represent venous channels in the cranial cavity. They are essentially broad spaces within the dura mater that are lined with endothelial cells and devoid of smooth muscles.  The great saphenous vein represents a long subcutaneous vein of the lower limb that originates in the foot and

1	FIGURE 13.25 • Schematic diagram and photomicrograph of a large vein. a. The cellular and extracellular components are labeled. Note a thin layer of circumferentially arranged smooth muscles of tunica media and the tunica adventitia with a large amount of longitudinally arranged smooth muscle bundles. b. This photomicrograph shows a section through the wall of a human portal vein in a routine H&E preparation. The tunica intima is indiscernible at this magnification. The tunica media contains a layer of circumferentially arranged smooth muscle cells with collagen and elastic fibers. Note the thickest layer of this wall is the tunica adventitia. In addition to an extensive collagen and elastic fiber network, the tunica adventitia contains a broad layer of smooth muscle cells arranged in longitudinal bundles. These bundles are variable in size and separated from each other by connective tissue fibers. 125. (Courtesy of Dr. Donald J. Lowrie Jr., University of Cincinnati College of

1	in longitudinal bundles. These bundles are variable in size and separated from each other by connective tissue fibers. 125. (Courtesy of Dr. Donald J. Lowrie Jr., University of Cincinnati College of Medicine.) drains into the femoral vein just below the inguinal ligament. This vein is often described as a muscular vein because of the presence of an unusual amount of smooth muscle (Fig. 13.28). In addition to the thick circular arrangement of smooth muscle in its tunica media, the great saphenous vein possesses numerous longitudinal smooth muscle bundles in the intima and in the well-developed adventitia. A thin, poorly developed internal elastic membrane separates the tunica intima from the media. The great saphenous vein is frequently harvested from the lower limb and used for autotransplantation in coronary artery bypass graft (CABG) surgery when arterial grafts (usually taken from internal thoracic artery) are not available or many grafts are required for multiple-bypass

1	autotransplantation in coronary artery bypass graft (CABG) surgery when arterial grafts (usually taken from internal thoracic artery) are not available or many grafts are required for multiple-bypass anastomoses. CABG is one of the most commonly performed major surgical operations in the United States.

1	 The central adrenomedullary vein that passes through the adrenal medulla and its tributaries have an unusual tunica media. It contains several longitudinally oriented bundles of smooth muscle cells that vary in size and apperance (Fig 13.29). These irregularly arranged smooth muscle bundles (also called muscle cushions) extend into larger tributaries of the central adrenomedullary vein. This unique eccentric arrangement of smooth muscle bundles results in the irregularity in the thickness of the vascular wall. In areas where muscle bundles are absent, cells of the adrenal medulla or sometimes adrenal cortex are separated from the lumen of the vein only by a thin layer of the tunica intima (see Fig. 13.29). Contraction of the longitudinally arranged smooth muscles in the tunica media enhances the efflux of hormones from the adrenal medulla into the circulation.

1	Veins in certain other locations (e.g., retina, placenta, trabeculae of the spleen) also have atypical walls and are discussed in the chapters that describe these organs. Lymphatic vessels convey fluids from the tissues to the bloodstream. In addition to blood vessels, another set of vessels circulates fluid called lymph through most parts of the body. These lymph-carrying vessels serve as adjuncts to the blood vessels. Unlike the blood vessels, which convey blood to and from tissues, the lymphatic vessels are unidirectional, conveying fluid

1	FIGURE 13.26 • Photomicrograph of a large vein. This photomicrograph shows the three tunics in a section through the wall of the portal vein stained with H&E. The tunica intima consists of endothelium and a thin subendothelial layer of connective tissue containing a few smooth muscle cells. The tunica media contains relatively thin layer of circularly arranged smooth muscle cells. Tunica adventitia is the thickest layer of this vessel. It contains a thick layer of longitudinally arranged smooth muscle bundles (seen here in cross section) separated by collagen and elastic fibers. Note a layer of connective tissue containing coarse collagen and elastic fibers that separates longitudinal bundles of smooth muscle in the tunica adventitia from a layer of smooth muscles of the tunica media. 240. (Courtesy of Dr. Donald

1	J. Lowrie Jr., University of Cincinnati College of Medicine.) only from tissues. The smallest lymphatic vessels are called lymphatic capillaries. They are especially numerous in the loose connective tissues under the epithelium of the skin and mucous membranes. The lymphatic capillaries begin as “blind-ended” tubes in the microcapillary beds (see Fig. 13.23). Lymphatic capillaries converge into increasingly larger vessels called lymphatic vessels. They ultimately unite to form two main channels that empty into the blood vascular system by draining into the large veins in the base of the neck. Lymph enter the vascular system at the junctions of the internal jugular and subclavian veins. The largest lymphatic vessel, draining most of the body and emptying into the veins on the left side, is the thoracic duct. The other main channel is the right lymphatic trunk. Lymphatic capillaries are more permeable than blood capillaries and collect excess protein-rich tissue fluid.

1	Lymphatic capillaries are more permeable than blood capillaries and collect excess protein-rich tissue fluid. FIGURE 13.27 • Photomicrograph of the coronary artery. This photomicrograph of a cross section of the coronary artery obtained from adult human shows all three vascular tunics similar to those in muscular arteries. The subendothelial layer of the tunica intima (TI) is considerably thicker because of the aging process than a comparable muscular artery. The internal elastic membrane (IEM) is visible at the border with the tunica media (TM), which is also thicker than in other muscular-type arteries. Connective tissue of the tunica adventitia (TA) is loosely arranged and contains peripherally positioned longitudinal bundles of collagen fibers. There is an artificial separation between cardiac muscle (CM) and tunica adventitia. 175.

1	Lymphatic capillaries are a unique part of the circulatory system, forming a network of small vessels within the tissues. Because of their greater permeability, lymphatic capillaries are more effective than blood capillaries in removing protein-rich fluid from the intercellular spaces. Once the collected fluid enters the lymphatic vessel, it is called lymph. Lymphatic vessels also serve to convey proteins and lipids that are too large to cross the fenestrations of the absorptive capillaries in the small intestine. Before lymph is returned to the blood, it passes through lymph nodes, where it is exposed to the cells of the immune system. Thus, the lymphatic vessels serve not only as an adjunct to the blood vascular system but also as an integral component of the immune system.

1	Lymphatic capillaries are essentially tubes of endothelium that, unlike the typical blood capillary, lack a continuous basal lamina. This incomplete basal lamina can be correlated with their high permeability. Anchoring filaments extend between the incomplete basal lamina and the perivascular collagen. These filaments may help maintain the patency of the vessels during times of increased tissue pressure such as in inflammation.

1	FIGURE 13.28 • Photomicrograph of the great saphenous vein. This photomicrograph shows a section through the wall of the greater saphenous vein. Tunica intima is usually thicker than in the other medium size veins and is characterized by a presence of numerous longitudinal smooth muscle bundles (SM) separated by connective tissue fibers. Tunica media contains relatively thick layer of circularly arranged smooth muscles. Tunica adventitia is well developed and contains additional layers of smooth muscle fibers arranged in spiral, oblique, and longitudinal bundles. 380. (Courtesy of Dr. Joseph J. Maleszewski, Mayo Clinic, Rochester, MN.)

1	As lymphatic vessels become larger, the wall becomes thicker. The increasing thickness is because of connective tissue and bundles of smooth muscle. Lymphatic vessels possess valves that prevent backflow of the lymph, thus aiding unidirectional flow (Plate 35, page 438). There is no central pump in the lymphatic system. Lymph moves sluggishly, driven primarily by compression of the lymphatic vessels by adjacent skeletal muscles.

1	FIGURE 13.29 • Photomicrograph of the central adrenomedullary vein. This photomicrograph of the human adrenal gland shows a large central adrenomedullary vein with its tributary stained with H&E. The wall of the vein is highly irregular, containing several longitudinally oriented bundles of smooth muscles (SM) that extend into the wall of a tributary. This unique eccentric arrangement of smooth muscles, sometimes called muscle cushions, results in the irregularity in the thickness of the vascular wall. Note that in the cleft between two smooth muscle bundles (asterisk), the lumen of the vein is separated from the chromaffin cells of the adrenal medulla only by the tunica intima. On the opposite side of the wall, muscle bundles are absent (arrowheads) and the cells of the adrenal cortex are in direct contact with the tunica intima. 120. (Courtesy of Dr. Donald J. Lowrie Jr., University of Cincinnati College of Medicine.)  FOLDER 13.3 Clinical Correlation: Ischemic Heart Disease

1	Clinical Correlation: Ischemic Heart Disease Ischemic heart disease or ischemic cardiomyopathy is defined as the imbalance between supply and demand of the heart for oxygenated blood. Ischemic heart disease is the most common type of heart disease in the United States and affects approximately 1 in every 100 people. The most common cause of ischemic heart disease is atherosclerosis. The risk of developing atherosclerosis in-creases with age, family history, hypertension, cigarette smoking, hypercholesterolemia, and diabetes. In atheroscle-rosis, the lumina of the coronary arteries progressively narrow because of the accumulation of lipids, extracellular matrix, and cells, leading to the development of atheroma-tous plaques (Fig. F13.3.1). Plaques are formed by intra-cellular and extracellular lipid deposition, smooth muscle proliferation, and increased synthesis of proteoglycans and collagen within the intima of the vessel wall. Blood flow continued next page

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1	The cardiovascular system is a transport system that carries blood and lymph to and from the tissues of the body. The cardiovascular system includes the heart, blood vessels, and lymphatic vessels. Blood vessels provide the route by which blood circulates to and from all parts of the body. The heart pumps the blood. Lymphatic vessels carry tissue-derived fluid, called lymph, back to the blood vascular system. The heart is a four-chambered organ consisting of a right and left atrium and a right and left ventricle. Blood from the body is returned to the right atrium from which it enters the right ventricle. Blood is pumped from the right ventricle to the lungs for oxygenation and returns to the left atrium. Blood from the left atrium enters the left ventricle from which it is pumped to the rest of the body, i.e., the systemic circulation. The heart, which differentiates from a straight vascular tube in the embryo, has the same basic three-layered structure in its wall as do the blood

1	rest of the body, i.e., the systemic circulation. The heart, which differentiates from a straight vascular tube in the embryo, has the same basic three-layered structure in its wall as do the blood vessels above the level of capillaries and postcapillary venules. In the blood vessels, the three layers are called the tunica intima, includ-ing the vascular endothelium and its underlying connective tissue; the tunica media, a muscular layer that varies in thickness in arteries and veins; and the tunica adventitia, the outermost layer of relatively dense connective tissue. In the heart, these layers are called the endo-cardium, the myocardium, and the epicardium, respectively.

1	Atrioventricular septum, heart, human, H&E ×45; inset ×125.

1	This micrograph of the field shows portions of the atrial (A) and ventricular (V) walls at the level of the atrioventricular septum and the root of the mitral valve (MV). Both chambers and the valve are lined with the squamous endothelium of the endocardium (En). Purkinje fibers (PF) of the cardiac conduction system are seen in the atrial wall between the relatively thin subendocardial connective tissue (CT) and the underlying modified cardiac muscle cells (CM) of the atrioventricular node (AVN). Dense fibrous connective tissue (DCT) that is continuous with that of the septum and the subendocardial layers of the atrium and ventricle extends from the root of the valve into the leaflet. Thin cardiac muscle fibers can also be seen extending from the wall of the atrium into the upper portion of the valve. Inset. This higher-magnification view of the field outlined by the rectangle (turned ~90°) shows more clearly the endothelial layer of the endocardium (En) and the dense fibrous

1	portion of the valve. Inset. This higher-magnification view of the field outlined by the rectangle (turned ~90°) shows more clearly the endothelial layer of the endocardium (En) and the dense fibrous connective tissue of the endocardium (DCT) and subendocardial layer. A thin layer of smooth muscle (SM) appears between the more densely packed fibrous tissue immediately subjacent to the endothelium and the more loosely packed dense fibrous tissue of the subendocardium. Particularly evident are the longitudinally sectioned Purkinje fibers (PF) of the cardiac conduction system. These modified cardiac muscle cells contain the same fibrillar contractile system as their smaller counterparts in the myocardium, but the fibrils are fewer, are more loosely packed, and often surround what appear to be vacuolated areas. Intercalated discs (ID), typical of cardiac muscle cell organization, are evident in some areas.

1	Coronary artery and cardiac vein, heart, human, H&E ×30. This micrograph shows cross sections of a coronary artery and cardiac vein in the coronary sulcus. The surrounding adipose tissue (AT) serves to cushion the blood vessels that run in the coronary sulcus. The coronary artery (CA) in the lower left of this micrograph is surrounded by small bundles of small cardiac muscle cells (CM) that are part of the atrioventricular node (AVN). A loop of the conduction bundle (CB) containing Purkinje fibers is evident to the right of the artery. The darkly stained tunica intima (TI) is delimited by an internal elastic membrane (IEM) that is easily distinguished even at this relatively low magnification. The thick muscular tunica media (TM) is also easily distinguished from the thinner, fibrous tunica adventitia (TA). A smaller arterial vessel (A ).

1	KEY A, atrium A, small artery AT, adipose tissue AVN, atrioventricular node B, blood CA, coronary artery CB, conduction bundle CM, cardiac muscle CT, connective tissue CV, cardiac vein DCT, dense connective tissue En, endothelium ID, intercalated disc IEM, internal elastic membrane LN, lymph node MV, mitral valve PF, Purkinje fibers SM, smooth muscle TA, tunica adventitia TM, tunica media TI, tunica intima V, ventricle

1	PLATE 33 • AORTA The aorta, the main systemic artery of the body, is an elastic artery. The presence of numerous fenestrated elastic lamellae allows it to resist the pressure variations caused by rhythmic contraction of the left ventricle. The intima is comparatively much thicker than that seen in muscular arteries. The subendothelial layer of the in-tima consists of connective tissue with both collagen and elastic fibers. The cellular component consists of smooth muscle cells and fibroblasts. The external border of the intima is bounded by an internal elastic membrane that represents the first layer of the many concentric fenestrated laminae in the media of the vessel. The media constitutes the bulk of the wall. Between the elastic laminae are collagen fibers and smooth muscle cells. The latter are responsible for the synthesis of collagen and elastic fibers. With age, the number and thickness of elastic laminae in the wall in-creases. By 35 years as many as 60 laminae are found in

1	latter are responsible for the synthesis of collagen and elastic fibers. With age, the number and thickness of elastic laminae in the wall in-creases. By 35 years as many as 60 laminae are found in the thoracic aorta. At ap-proximately 50 years, individual laminae begin to show signs of degeneration and gradually become replaced by collagen leading to a gradual loss of elasticity of the aortic wall. The adventitia consists of irregular dense connective tissue with intermixed elas-tic fibers that tend to be organized in a circumferential pattern. It also contains small blood vessels that supply the outer portion of the media. They are the vasa vasorum of the aorta. Also present in the adventitia are lymphatic capillaries. ORIENTATION MICROGRAPHS: The upper micrograph shows a cross section of an H&E stained human aorta from a child. The intima (I) stains considerably lighter than the adjacent media (M) The adventitia (A) contains an abundance of collagenous fibers and stains more

1	of an H&E stained human aorta from a child. The intima (I) stains considerably lighter than the adjacent media (M) The adventitia (A) contains an abundance of collagenous fibers and stains more densely than that of either the media or intima. The lower micro-graph is from an adult and has been stained to reveal the elastic component of the vessel wall. The intima (I) is very lightly stained, in this case, due to the paucity of elastic material. The media (M) is heavily stained due to the presence of large amounts of elastic laminae. The adventitia (A) contains in addition to the dense connective tissue a moderate amount of elastic fibers. MMMMAAAAIIIIMMAAII

1	Aorta, human, H&E, ×365; inset ×700. This micrograph shows the layers of the aortic wall. The intima consists of an endothelium (En) overlying loose connective tissue (LCT). The thickest portion of the vessel wall is the media (M). The wavy eosinophilic material is the collagenous fibers. The eosin stain does not reveal the elastic laminae. The nuclei are those of smooth muscle cells. Fibroblasts are absent. The outer Aorta, human, iron hematoxylin and aniline blue, ×255; inset ×350.

1	Aorta, human, iron hematoxylin and aniline blue, ×255; inset ×350. The specimen shown here has been stained to distinguish collagen from elastic material. The intima (I) consists mostly of collagenous fibers. The endothelium (En) represented by several nuclei is just barely evident. The media (M) contains nu merous elastic lamellae that appear as the black wavy lines. The intervening blue stained material consists of collagen fibers. Careful examination of the layer of the vessel wall is the adventitia (A). The eosinophilic material here consists of dense connective tissue. The nuclei that are evident belong to fibroblasts. Also note the small blood vessel (BV) in the adventitia. The inset shows the intima at higher magnification and includes part of the media. Note the endothelium (En). The eosinophilic material in the intima consists of collagenous fibers (CF). The main cell type here is the smooth muscle cell (SMC).

1	media reveals nuclei of smooth muscle cells dispersed between the elastic lamellae. The inset shows the intima at higher magnification. Note the nuclei of the endothelial cells (EnC) at the luminal surface. The remainder of the intima consists mostly of collagenous fibers (stained blue) with occasional elastic fibers (EF) identified by their darker coloration. The nuclei of the fibroblasts and occasional smooth muscle cells (SMC) appear randomly arranged. KEY A, adventitia BV, blood vessel CF, collagenous fibers EF, elastic fibers En, endothelium EnC, endothelial cells I, intima LCT, loose connective tissue M, media SMC, smooth muscle cells

1	Muscular arteries have more smooth muscle and less elastin in the tunica media than do elastic arteries. Thus, as the arterial tree is traced fur-ther from the heart, the elastic tissue is considerably reduced and smooth muscle becomes the predominant component of the tunica media. The muscular arteries are characterized, however, by a refractile internal elastic membrane separating the tunica intima from the tunica media and, usually, by an external elastic membrane separating the tunica media from the tunica adventitia. Muscular arteries, or arteries of medium caliber, constitute the majority of the named arteries in the body. Veins usually accompany arteries as they travel in the loose connective tissue. The veins have the same three layers in their walls, but the tunica media is thinner than in the accompanying artery, and the tunica adventitia is the predominant layer in the wall. The veins usually have the same name as the artery they accompany.

1	Muscular artery and medium vein, monkey, H&E ×365. In this photomicrograph, the lumen of the artery is at the left, the lumen of the vein is at the right. The arterial endothelium (AEn) is clearly seen on the corrugated surface of the tunica intima, whereas the venous endothelium (VEn) is somewhat harder to distinguish. The internal elastic membrane (IEM) is seen as a thin clear zone immediately beneath the endothelial layer, separating the tunica intima from the underlying smooth muscle (SM) of the tunica media (TM). It is evident here that the tunica media is almost twice as thick as the tunica adventitia (TA ). Muscular artery, monkey, H&E ×545. elastic membrane (IEM), which rests directly on the most luminal layer of smooth muscle cells (SM) of the thick tunica media (TM). The thinner tu- This is a higher-magnification micrograph of the portion of nica adventitia (TA ).

1	This is a higher-magnification micrograph of the portion of nica adventitia (TA ). the figure above outlined by the rectangle turned 90°. At this magnification, it is evident that the flattened endothelial cells (EN) follow the contours of the refractile, corrugated internal M USCU LAR ARTERIES AND MEDIUM VEINS KEY AEn, arterial endothelium C, collagen bundles EF, elastic fibers EN, endothelial cells IEM, internal elastic membrane N, nuclei SM, smooth muscle SSm, small smooth muscle TA' tunica adventitia of artery TA, tunica adventitia of accompanying vein TI, tunica intima TM, tunica media VEn, venous endothelium Medium vein, monkey, H&E ×600.

1	Medium vein, monkey, H&E ×600. In this higher-magnification view of a portion of the wall of the vein in the figure above, the endothelial cells (EN) are more easily recognized and are seen to be plumper than those of the arterial endothelium. The margin between the tunica intima (TI) and the thin tunica media (TM) is difficult to discern, but the smooth muscle cells (SM) in the thin media are more easily recognized than in the figure above because of the shape of their nuclei and the slight basophilia of their cytoplasm. The tunica adventitia (TA) is about twice as thick as the tunica media and appears to contain only bundles of collagen fibers and fibroblasts, with the latter recognizable by their nuclei (N). The collagen bundles of the loose connective tissue beneath the tunica adventitia are larger than those of the adventitia, and there are fewer cells in this portion of the specimen. PLATE 35 Arterioles, Venules, and Lymphatic Vessels

1	PLATE 35 Arterioles, Venules, and Lymphatic Vessels The terminal components of the arterial tree just before a capillary bed or an arteriovenous shunt are the arterioles. Arterioles have an en-dothelial lining and smooth muscle in the wall but the smooth muscle is limited in thickness to one or two cells. There may or may not be an internal elastic membrane, according to the size of the vessel. Arterioles control blood flow into capillary networks. In the normal relationship between an arteriole and a capillary network, contraction of the smooth muscle of the arteriole wall reduces or shuts off the blood going to the capillaries. A precapillary sphincter is formed by a slight thickening of the smooth muscle at the origin of a capillary bed from an arteriole. Nerve impulses and hormonal stimulation can cause the muscle cells to contract, directing blood into capillary beds where it is most needed. Arteriole, venule, and small nerve, fngertip, human, H&E ×600.

1	Arteriole, venule, and small nerve, fngertip, human, H&E ×600. This micrograph shows two cross-sectioned arterioles (A) and a venule (V). The arteriole on the left is identified as a large arteriole, based on the presence of two discrete layers of smooth muscle cells that form the tunica media of the vessel. The nu clei of the muscle cells appear in longitudinal profile as a result of the cir 438 cumferential arrangement of the cells. The endothelial cell nuclei of the vessel appear as small round profiles surrounding the lumen. These cells are elongate and oriented with their long axis in the direction of flow. Thus, Arteriole, fngertip, human, H&E ×350.

1	Arteriole, fngertip, human, H&E ×350. This micrograph shows a longitudinal section of an arteriole. Because of its twisting path through the section, its wall has been cut such that the single layer of muscle cells of the tunica media is seen in different planes along its length. In the segment numbered 1, at the left, the vessel wall has been cut tan gentially. Thus, the vessel lumen is not included in the plane of section, but the smooth muscle cell nuclei of the tunica media are seen in longitudinal Lymphatic vessel, fngertip, human, H&E ×175.

1	The lymphatic vessel shown in this figure shows a region where the vessel is making a U-shaped turn in the plane of the section, thus disappearing at the top and bottom of the micro-graph. The wall of the vessel consists of an endothelial lining and a small amount of connective tissue, with one being indis tinguishable from the other. A valve (Val), which is characteristic of their nuclei are seen here as cross-sectioned profiles. The arteriole on the right is a very small arteriole, having only a single layer of smooth muscle. Again, the muscle cell nuclei are seen in longitudinal profile. The endothelial cell nuclei appear as the small round profiles at the luminal surface. A venule is seen in proximity to the larger arteriole, and a cross section of peripheral nerve (N) is seen in proximity to the smaller arteriole. Compare the wall of the venule, consisting only of endothelium and a thin layer of connective tissue, with the arterioles. Also, note the relatively large lumen of the

1	to the smaller arteriole. Compare the wall of the venule, consisting only of endothelium and a thin layer of connective tissue, with the arterioles. Also, note the relatively large lumen of the venule.

1	PLATE 35 • ARTERIOLES, VENULES, AND LYMPHATIC VESSELS profile. After the arteriole makes an acute turn (segment numbered 2), the vessel wall is cut to reveal the lumen. Here, the smooth muscle nuclei appear as round profiles and the nuclei of the endothelial cells lining the lumen appear in longitudinal profile. In the segment numbered 3, the vessel wall is again only grazed. In the segment numbered 4, the cut is deeper, again showing the lumen and some of the endothelial cells in face view (arrowheads). The structure below the vessel is a Pacinian corpuscle (P).

1	lymphatic vessels, is seen within the vessel. It is formed of a miniscule layer of connective tissue that is covered on both sides by endothelium. The arrows indicate nuclei that are just barely visible at this magnification; most of them belong to endothelial cells. Typically, the lumen contains precipitated lymph material (L); sometimes, lymphocytes may be present. Adjacent to the vessel, on the right, is adipose tissue (AT) and on the upper left is dense irregular connective tissue (DCT). Lymphatic vessel, fngertip, human, Mallory ×375.

1	Lymphatic vessel, fngertip, human, Mallory ×375. The lymphatic vessel shown here is contained within dense irregular connective tissue (DCT). The lumen is irregular, appearing relatively narrow below the valve (Val). A few endothelial cell nuclei are evident (arrows). A thin layer of connective tissue that is present outside of the endothelium blends with the dense connective tissue beyond the wall of the vessel. A venule (V) is also present; it can readily be distinguished from the lymphatic vessel by the presence of red blood cells in the lumen. KEY A, arteriole Ad, adipocyte AT, adipose tissue DCT, dense irregular connective tissue L, lymph material N, nerve P, Pacinian corpuscle V, venule Val, valve arrowheads, endothelial cells arrows, endothelial cell nuclei PLATE 35 • ARTERIOLES, VENULES, AND LYMPHATIC VESSELS VVV NNN AAA ATATDCTDCTPP11223344AT DCT P 1 2 3 4 ValValVal DCTDCTATAT LLDCT AT L AdAdDCTDCTDCTDCTVVAALLLLAd DCT DCTV ValValVal A L L

1	PLATE 35 • ARTERIOLES, VENULES, AND LYMPHATIC VESSELS VVV NNN AAA ATATDCTDCTPP11223344AT DCT P 1 2 3 4 ValValVal DCTDCTATAT LLDCT AT L AdAdDCTDCTDCTDCTVVAALLLLAd DCT DCTV ValValVal A L L OVERVIEW OF THE LYMPHATIC SYSTEM / 440 CELLS OF THE LYMPHATIC SYSTEM / 441 Overview / 441 Lymphocytes / 444 Antigen-Presenting Cells / 453 LYMPHATIC TISSUES AND ORGANS / 453 Lymphatic Vessels / 453 Diffuse Lymphatic Tissue and Lymphatic Nodules / 456 Lymph Nodes / 460 Cells of the Reticular Meshwork / 461 Thymus / 466 Spleen / 471 Folder 14.1 Functional Considerations: Origin of the Names T Lymphocyte and B Lymphocyte / 447 Folder 14.2 Clinical Correlation: Hypersensitivity Reactions / 447 Folder 14.3 Clinical Correlation: Human Immunodeficiency Virus (HIV) and Acquired Immunodeficiency Syndrome (AIDS) / 455 Folder 14.4 Clinical Correlation: Reactive (Inflammatory) Lymphadenitis / 466

1	Throughout history, it has been noted that people who recover from certain diseases such as chickenpox, measles, and mumps become resistant (i.e., immune) to the same disease. Another longstanding observation is that immunity is specific—that is, immunity to chickenpox does not prevent infection with measles. We also now recognize that the immune system can react against itself, causing autoimmune diseases such as lupus erythematosus, autoimmune hemolytic anemia, some forms of diabetes mellitus, and autoimmune thyroiditis (Hashimoto’s thyroiditis).

1	The lymphatic system consists of groups of cells, tissues, and organs that monitor body surfaces and internal fluid compartments and react to the presence of potentially harmful substances. Lymphocytes are the definitive cell type of the lymphatic system and the effector cells in the response of the immune system to harmful substances. Included in this system are the diffuse lymphatic tissue, lymphatic nodules, lymph nodes, spleen, bone marrow, and thymus (Fig. 14.1). The various lymphatic organs and lymphatic tissues are often collectively referred to as the immune system. Lymphatic vessels connect parts of the system to the blood vascular system.

1	Lymphatic vessels connect parts of the system to the blood vascular system. Lymphatic tissues serve as sites where lymphocytes proliferate, differentiate, and mature. In addition, in the thymus, bone marrow, and gut-associated lymphatic tissue (GALT), lymphocytes are “educated” to recognize and destroy specific antigens. These are now immunocompetent cells that can distinguish between “self” (molecules normally present within an organism) and “nonself” (foreign molecules—i.e., those not normally present). An antigen is any substance that can induce a specific immune response.

1	An antigen is any substance that can induce a specific immune response. The body is constantly exposed to pathogenic (diseasecausing) organisms and hazardous substances from the external environment (infectious microorganisms, toxins, and foreign cells and tissues). In addition, changes may occur in cells (such as transformation of normal cells into cancerous cells) that give them characteristics of foreign cells. An immune response is generated against a specific antigen, which can be a soluble substance (e.g., a foreign protein, polysaccharide, or toxin) or an infectious organism, foreign tissue, or transformed tissue. Most antigens must be “processed” by cells of the immune system before other cells can mount the immune response.

1	FIGURE 14.1  Overview of the structures constituting the lymphatic system. Because lymphatic tissue is the main component of some organs, they are regarded as organs of the lymphatic system (spleen, thymus, lymph nodes). Lymphatic tissue is present as part of other organs such as red bone marrow, lymphatic nodules of the alimentary canal: tonsils, vermiform appendix, gut-associated lymphatic tissue (GALT) and of the respiratory system (bronchus-associated lymphatic tissue, or BALT ), and, not shown in the illustration, diffuse lymphatic tissue of mucous membranes (mucosa-associated lymphatic tissue, or MALT). The lymph nodes are interspersed along the superficial lymphatic vessels (associated with the skin and superficial fascia) and deep lymphatic vessels (associated with main arteries). Ultimately, the lymphatic vessels empty into the bloodstream by joining the large veins at the base of the neck. The thoracic duct is the largest lymphatic vessel.

1	The immune responses can be divided into nonspecific (innate) and specific (adaptive) defenses. The body has two lines of immune defenses against foreign invaders and transformed cells: nonspecific immunity and specific immunity.  In nonspecific (innate) immunity, preexisting nonspecific defenses constitute the innate immune response. In all living organisms, the innate immunity represents the frst line of defense against microbial aggression. It consists of (1) physical barriers (e.g., the skin and mucous membranes) that prevent foreign organisms from invading the tissues, (2) chemical defenses (e.g., low pH ) that destroy many invading microorganisms, (3) various secretory substances (e.g., thiocyanate in saliva, lysozymes, interferons, fbronectin, and complement in serum) that neutralize foreign cells, and (4) phagocytic cells (e.g., macrophages, neutrophils, and monocytes) and natural killer (NK) cells.

1	 With specific (adaptive) immunity, if nonspecific defenses fail, the immune system provides specific, or adaptive, defenses that target specific invaders. The initial contact with a specific antigen or foreign agent initiates a chain of reactions that involve effector cells of the immune system and frequently leads to a state of immune “memory.” Adaptive immunity induces acquired resistance against microbial aggression through random somatic rearrangements of genes that encode immunoglobulins and specifc receptors on T lymphocytes (i.e., T-cell receptors, or TCRs). During adaptive immune responses, specifc B and T lymphocytes become activated to destroy invading organisms. Two types of specific defenses have been identified: Humoral response results in the production of proteins called antibodies that mark invaders for destruction by other immune cells, and the cellular immune response targets transformed and virus-infected cells for destruction by specific “killer” cells.

1	Therefore, shortly after invasion by bacteria or other pathogenic agents, the immune system becomes activated (inflammatory response) to destroy infectious agents and generate long-term memory against pathogens. Cells of the immune system include lymphocytes and various supporting cells.

1	Cells of the immune system include lymphocytes and various supporting cells. Lymphocytes and a variety of supporting cells make up the cells of the immune system. Three major types of lym phocytes are recognized: B cells, T cells, and NK cells. Supporting cells interact with lymphocytes and play important roles in the presentation of antigen to lymphocytes and the regulation of immune responses. These cells include monocytes, macrophages, neutrophils, basophils, eosinophils, reticular cells, dendritic cells, follicular dendritic cells, Langerhans’ cells, and epithelioreticular cells. In addition, a series of specialized epithelial and stromal cells provide the environment for many immune reactions to occur by secreting specific substances that regulate growth, migration, and the activation of effector and supporting cells. Supporting cells in the lymphatic organs are organized into loose meshworks.

1	Supporting cells in the lymphatic organs are organized into loose meshworks. In lymph nodules, lymph nodes, and the spleen, reticular cells and the reticular fibers produced by these cells form elaborate meshworks. Lymphocytes, macrophages, dendritic cells, follicular dendritic cells, and other cells of the immune system reside in these meshworks and in the loose connective tissue of the body; Langerhans’ cells are found only in the middle layers of epidermis. At these sites, they carry out their mission of surveillance and defense. In the thymus, epithelioreticular cells form the structural meshwork within the tissue. Despite their name, these cells neither produce nor are related to reticular fibers. Different types of cells in lymphatic tissue are identified by specific cluster of differentiation (CD) markers on their surface.

1	Different types of cells in lymphatic tissue are identified by specific cluster of differentiation (CD) markers on their surface. Different lymphatic and hematopoietic tissue cells possess unique cell surface molecules. These specific markers, called cluster of differentiation (CD) molecules, are designated by numbers according to an international system that relates them to antigens expressed at different stages of their differentiation. CD molecules can be visualized by immunohistochemical methods using monoclonal antibodies and are useful in identifying specific subtypes of lymphatic or hematopoietic cells. Some CD markers are expressed by cells throughout their entire life; others are expressed only during one phase of differentiation or during cell activation. Table 14.1 lists the most clinically useful markers.

1	TABLE Most Common CD Markers Used in Clinical Practice 14.1 Marker Main Cellular Expression Function/Identity Molecular Weight (kDa) CD1 T cells in the midstage of development Interact with MHC I molecules Developmental markers for T cells and Langerhans’ cells of the skin 49 CD2 T cells Adhesion molecules Used as clinical markers for T cells 50 CD3 T cells Form complex with T-cell receptor (TCR) 100 CD4 Helper T cells, monocytes, macrophages Members of immunoglobulin superfamily Interact with MHC II molecules Bind viral protein gp120 of HIV-1 and HIV-2 56 CD5 T cells, some B cells Costimulatory molecules High levels in chronic lymphocytic leukemia 67 CD7 T cells Members of immunoglobulin superfamily Bind the PI-3 kinase Useful clinical markers for T-cell leukemia stem cells 40 CD8 Cytotoxic T cells Members of immunoglobulin superfamily Interact with MHC I molecules 34 CD9 B cells, T cells, monocytes, eosinophils, basophils, platelets, endothelial cells Facilitate aggregation of

1	T cells Members of immunoglobulin superfamily Interact with MHC I molecules 34 CD9 B cells, T cells, monocytes, eosinophils, basophils, platelets, endothelial cells Facilitate aggregation of platelets, cell adhesion, and cell migration 24 CD10 Pre-B cells, pre-T cells Zinc metalloproteas Common markers for acute lymphoblastic leukemia 100 CD16a NK cells, granulocytes, monocytes Clinical markers for NK cells Function as Fc receptors for aggregated IgG Mediate phagocytosis and antibody-dependent cell-mediated cytotoxicity 27 CD19 B cells, dendritic cells Coreceptors with CD21 Clinical markers for all stages of B-cell development 90 continued next page chapter 14 Lymphatic System CE LLS OF TH E LYM PHATIC SYSTE M CD20 B cells Form Ca2channels Markers for late stage of B-cell development 37 CD21 B cells, follicular dendritic cells Receptors for C3d complement protein and for Epstein-Barr virus 145 CD22 B cells B-lymphocyte cell adhesion molecules Mediate B-cell to T-cell adhesion 140

1	37 CD21 B cells, follicular dendritic cells Receptors for C3d complement protein and for Epstein-Barr virus 145 CD22 B cells B-lymphocyte cell adhesion molecules Mediate B-cell to T-cell adhesion 140 CD24 B cells, granulocytes, epithelial cells Expressed in late stage of B-cell differentiation 41 CD28 T cells T-cell costimulatory molecule interacts with CD80 (B7.1) and CD86 (B7.2); the costimulatory signal induces T-cell activation and IL-2 production 44 CD34 Hemopoietic stem cells (HSCs) Clinical markers for HSCs and ligand for CD62L Mediate attachment of stem cells to bone marrow extracellular matrix 120 CD35 T cells, B cells, monocytes, dendritic cells, granulocytes, erythrocytes Complement receptor 1 Promote phagocytosis of complement-coated particles Bind C3b and C4b complement protein 250 CD38 Activated T cells NAD glycohydrolase Used as markers for T-cell activation and proliferation 45 CD40 B cells, macrophages, dendritic cells Active in proliferating B cells Costimulatory

1	250 CD38 Activated T cells NAD glycohydrolase Used as markers for T-cell activation and proliferation 45 CD40 B cells, macrophages, dendritic cells Active in proliferating B cells Costimulatory molecules for CD40L (CD154) Facilitate cytokine production in macrophages and dendritic cells 48 CD40L Activated CD4T cells; known as CD154 Facilitate interaction between T and B cells Regulate B-cell function Costimulatory molecules for CD40 39 CD45 All human leukocytes Thyrosine phosphatase Leukocyte common antigen 220 CD56 NK cells Clinical markers for NK cells Isoforms of neural adhesion molecules (N-CAM) 135 CD62L Leukocytes Bind CD34 Represent L-selectins, leukocyte adhesion molecules that allow lymphocytes to roll along the endothelial surface 150 CD80 B cells, macrophages, dendritic cells, monocytes, macrophages APC costimulatory molecule interacts with CD28 45 CD86 Activated B cells, macrophages, monocytes, dendritic cells, endothelial cells APC costimulatory molecule interacts with

1	monocytes, macrophages APC costimulatory molecule interacts with CD28 45 CD86 Activated B cells, macrophages, monocytes, dendritic cells, endothelial cells APC costimulatory molecule interacts with CD28 70 CD94 NK cells Clinical markers for NK cells 43 TABLE Most Common CD Markers Used in Clinical Practice (Cont.)14.1 Marker Main Cellular Expression Function/Identity Molecular Weight (kDa) APC, antigen-presenting cell; NK, natural killer.

1	Circulating lymphocytes are the chief cellular constituents of lymphatic tissue. To understand the function of lymphocytes, one must realize that most lymphocytes (approximately 70%) in blood or lymph represent a circulating pool of immunocompetent cells. These cells participate in a cycle during which they exit the systemic circulation to enter the lymphatic tissue. While there, they are responsible for immunologic surveillance of surrounding tissues. The cells then return to the systemic circulation. This population of cells is represented mainly by long-lived, mature lymphocytes (mainly T cells) that have developed the capacity to recognize and respond to foreign antigens and are in transit from one site of lymphatic tissue to another.

1	The remaining 30% of lymphocytes in the blood vessels do not circulate between the lymphatic tissues and the systemic circulation. This population comprises mainly short-lived, immature cells or activated cells destined for a specific tissue. These cells leave the capillaries and migrate directly to the tissues, especially into the connective tissue that underlies the lining epithelium of the respiratory, gastrointestinal, and urogenital tracts as well as into the intercellular spaces of these epithelia. Functionally, three major types of lymphocytes are present in the body: T lymphocytes, B lymphocytes, and NK cells. The functional classifcation of lymphocytes is independent of their morphologic (size) characteristics. T lymphocytes differentiate in the thymus and account for the majority of circulating lymphocytes.

1	T lymphocytes differentiate in the thymus and account for the majority of circulating lymphocytes. T lymphocytes (T cells) are named for the thymus, where they differentiate. They have a long lifespan and are involved in cell-mediated immunity. They account for 60% to 80% of circulating lymphocytes. T cells express CD2, CD3, CD5, and CD7 markers and T-cell receptors (TCRs); however, they are subclassified according to the presence or absence of two other important surface markers: CD4 and CD8.  Helper CD4 T lymphocytes are T cells that also express CD4 markers. These cells are further subdivided by their ability to secrete cytokines (see page 454). Helper T cells that synthesize interleukin 2 (IL-2), interferon (IFN-), and tumor necrosis factor (TNF-) are called TH1 cells.

1	These cells interact with cytotoxic CD8 T lymphocytes (CTLs), NK cells, and macrophages in cell-mediated im mune responses and are essential for controlling intracellu lar pathogens such as viruses and certain microorganisms. The other group of helper T cells synthesize IL-4, IL-5, IL 10, and IL-13 and are called TH2 cells. They interact with B lymphocytes and are essential for initiating antibody-me pathogens.  Cytotoxic CD8 T lymphocytes (CTLs) are T cells that also express CD8 markers. They kill other target cells such as virus-infected cells, cancer-transformed cells, cells infected with intracellular microorganisms, parasites, and transplanted cells.

1	 Regulatory (suppressor) T lymphocytes represent a phenotypically diverse population of T lymphocytes that can functionally suppress an immune response to foreign and self-antigen by influencing the activity of other cells in the immune system. For example, T lymphocyte with CD4 CD25FOXP3 markers represent a classical example of the regulatory cells that can diminish the ability of T lymphocytes to initiate immune responses. The FOXP3 marker indicates an expression of forkhead family transcription factors that are characteristic of many T cells. Another tumor-associated T lymphocyte with CD8CD45RO markers is able to suppress T-cell activation. Other suppressor T cells may also function in suppressing B-cell differentiation and in regulating erythroid cell maturation in the bone marrow.

1	 Gamma/delta (/) T lymphocytes represent a small population of T cells that possess a distinct TCR on their surface made of one -chain and one -chain. Most other TCRs are composed of two glycoprotein chains called -and -TCR chains. These cells develop in the thymus and migrate into various epithelial tissues (e.g., the skin, oral mucosa, intestines, and vagina). Once they colonize an epithelial tissue, they do not recirculate between blood and lymphatic organs. Gamma/delta (/) T cells are strategically positioned at the interfaces of the external and internal environments and function as the first line of defense against invading organisms. They encounter antigen on the surface of the epithelial cells even before it enters the body. B lymphocytes differentiate in the bursa-equivalent organs and participate in humoral immunity.

1	B lymphocytes (B cells) are so named because they were first recognized as a separate population in the bursa of Fabricius in birds (page 447) or bursa-equivalent organs such as bone marrow and GALT in mammals. They have variable life spans and are involved in the production and secretion of the various circulating antibodies, also called immunoglobulins (Ig), the immune proteins associated with humoral immunity (Fig. 14.2 and Table 14.2). B cells account for 20% to 30% of the circulating lymphocytes. In addition to secreting circulating immunoglobulins, B cells express membrane-bound forms of immunoglobulin called B-cell receptors (BCRs) that serve as the antigen-specific binding site. During diff erentiation, the BCR isotope switches from immunoglobulin M (IgM) in immature B cells to immu noglobulin D (IgD) in mature B cells. B cells also express the major histocompatibility complex II (MHC II) molecules on the cell surface. Their CD markers are CD9, CD19, and CD20.

1	tion in the primary lymphatic organs. In humans and other mammals, the bone marrow and GALT region (together called the bursa-equivalent organ) and the thy mus have been identified as primary (central) lymphatic organs. Lymphocytes differentiate into immunocompetent cells in these organs. Initially, lymphocytes are genetically programmed to recognize a single antigen out of virtually an infinite number of possible antigens, a process called These immunocompetent cells then enter the blood or lymph antigen-independent proliferation and differentiation. region of IgG, IgM carboxy terminus FIGURE 14.2 • Schematic diagram of an antibody molecule.

1	Antibodies are Y-shaped molecules produced by plasma cells. They consist of two heavy (H) and two light (L) polypeptide chains connected by disulfide bonds (SOS). Both H and L chains are composed of domains of amino acids that are constant (at the carboxy terminus) or variable (at the amino terminus) in their sequence. The five different immunoglobulin (Ig) isotypes (see Table 14.2) are determined by the type of heavy chain present. An antibody molecule binds an antigen (Ag) at the two sites of the amino terminus, where the heavy and light chains are associated with each other. Digestion of an antibody molecule by the proteolytic enzyme papain cleaves the antibody into two Fab fragments and one crystallizable Fc fragment. The Fab fragments impart the specific antigen binding, whereas the Fc fragment, which is composed of two carboxy-terminus heavy-chain segments (CH2 and CH3), fulfills the effector functions (e.g., in complement activation). Many cells express Fc receptors on their

1	Fc fragment, which is composed of two carboxy-terminus heavy-chain segments (CH2 and CH3), fulfills the effector functions (e.g., in complement activation). Many cells express Fc receptors on their surfaces, which anchor antibodies at the Fc fragment.

1	Natural killer lymphocytes (NK cells) are neither T nor B cells and are specialized to kill certain types of target cells. Natural killer (NK) cells, which develop from the same precursor cell as B and T cells, are named for their ability to kill certain types of target cells. They constitute about 5% to 10% of circulating lymphocytes. They do not mature in the thymus; however, during their development, they are genetically programmed to recognize transformed cells (i.e., cells infected with a virus or tumor cells). NK cells kill target cells in a similar fashion to that of cytotoxic CD8 T lymphocytes. After recognition of a transformed cell, they release perforins and granzymes (fragmentins), substances that create channels in the cell’s plasma membrane, which induces them to self-destruct (a process known as apoptosis). Their specific markers include CD16a, CD56, and CD94. and are transported throughout the body, where they are dispersed in the connective tissue.

1	and are transported throughout the body, where they are dispersed in the connective tissue. Lymphocytes undergo antigen-dependent activation in the secondary lymphatic organs. Immunocompetent lymphocytes (together with plasma cells derived from B lymphocytes and with macrophages) organize around reticular cells and their reticular fibers to form the adult effector lymphatic tissues and organs (i.e., lymphatic nodules, lymph nodes, tonsils, and spleen). Within these secondary (peripheral) lymphatic organs, T and B lymphocytes undergo antigen-dependent activation into effector lymphocytes and memory cells. Immune Responses to Antigens Inflammation is the initial response to an antigen. The initial reaction of the body to invasion by an antigen, either a foreign molecule or a pathogenic organism, is the nonspecific defense known as the inﬂammatory response.

1	The initial reaction of the body to invasion by an antigen, either a foreign molecule or a pathogenic organism, is the nonspecific defense known as the inﬂammatory response. The infammatory response may either sequester the antigen, physically digest it with enzymes secreted by neutrophils, or phagocytose and degrade the antigen in the cytoplasm of macrophages. Degradation of antigens by macrophages may lead to subsequent presentation of a portion of the antigen to immunocompetent lymphocytes to elicit a specifc immune response. Specific immune responses are either primary or secondary. When immunocompetent cells encounter a foreign antigen (e.g., antigen associated with pathogenic microorganisms, tissue transplants, or toxins), a specific immune response to the antigen is generated.

1	A primary immune response refers to the body’s first encounter with an antigen. This response is characterized by a lag period of several days before antibodies (mostly IgM) or specific lymphocytes directed against the invading antigen can be detected in the blood. The initial response to an antigen is initiated by only one or a few B lymphocytes that have been genetically programmed to respond to that specifc antigen. After this initial immune response, a few antigen-specifc B lymphocytes remain in circulation as memory cells. The secondary immune response is usually more rapid and more intense (characterized by higher levels of secreted antibodies, usually of the IgG class) than the primary

1	TABLE Characteristics of Human Immunoglobulins14.2 Isotype Molecular Weight (kDa) Serum Level (mg/mL) Percentage of all Ig in Adult Blood Cells to Which Bind via Fc Region Major Functions IgG 145 12.0 85 Macrophages, B cells, NK cells, neutrophils, eosinophils Principal Ig in secondary immune response Longest half-life (23 days) of all five Igs Activates complement Stimulates chemotaxis Crosses placenta, providing newborn with passive immunity IgM 190 (950)a1.5 5–10 B cells Principal Ig produced during primary immune response Most efficient Ig in fixing complement Activates macrophages Serves as Ag receptor of B lymphocytes IgA 160 (385)b2.0 5–15 B cells Ig present in body secretions, including tears, colostrum, saliva, and vaginal fluid, and in secretions of nasal cavity, bronchi, intestine, and prostate Provides protection against proliferation of microorganisms in these fluids and aids in defense against microbes and foreign molecules penetrating body via cell linings of these

1	and prostate Provides protection against proliferation of microorganisms in these fluids and aids in defense against microbes and foreign molecules penetrating body via cell linings of these cavities IgD 185 0.03 1 B cells Acts as an antigen receptor (together with IgM) on surface of mature B lymphocytes (only traces in serum) IgE 190 3 × 10 5 1 Mast cells, basophils Stimulates mast cells to release histamine, heparin, leukotrienes, and eosinophil chemotactic factor of anaphylaxis Responsible for anaphylactic hypersensitivity reactions Increased levels in parasitic infections a IgM found in serum as a pentameric molecule. b IgA found in serum as dimeric molecule. Ag, antigen; Ig, immunoglobulin; NK, natural killer.

1	response because of the presence of specific memory B lymphocytes already programmed to respond to that specific antigen. The secondary response is the basis of most immunizations for common bacterial and viral diseases. Some antigens, such as penicillin and insect venoms, may trigger an intense secondary immune response that produces a hypersensitivity reaction or even anaphylaxis (see Folder 14.2). However, antibodies themselves do not kill or destroy invading antigens; they simply mark them for destruction by cells of the immune system. The two types of specific immune responses are the humoral and cell-mediated responses. In general, an encounter with a given antigen triggers a response characterized as either a humoral immune response (antibody production) or a cell-mediated immune response.  FOLDER 14.2 Clinical Correlation: Hypersensitivity Reactions

1	 FOLDER 14.2 Clinical Correlation: Hypersensitivity Reactions When an individual has been immunologically sensitized by exposure to antigen, subsequent exposure may lead not only to secondary response but also to tissue-damaging reactions called hypersensitivity reactions. Such reac-tions are observed in sensitized humans after insect bites or injections of penicillin. One common type of hypersen-sitivity reaction is the allergic reaction. Certain aspects of a hypersensitivity reaction are caused by the antibody-induced discharge of mast cell granules. These granules contain histamine, which accounts for the distressing fea-tures of hypersensitivity reactions. Eosinophils are  FOLDER 14.1 Functional Considerations: Origin of the Names T Lymphocyte and B Lymphocyte

1	In the early 1960s, investigators using chicken embryos demonstrated that the bursa of Fabricius, a mass of lym-phatic tissue associated with the cloaca of birds, was one of the anatomic sites of lymphocyte differentiation. When this tissue was destroyed in the chicken embryos (by either surgical removal or administration of high doses of testos-terone), the adult chickens were unable to produce antibod-ies, leading to impaired humoral immunity. The chickens also demonstrated a marked reduction in the number of lymphocytes found in specific bursa-dependent areas of the spleen and lymph nodes. These affected lymphocytes were therefore named B lymphocytes or B cells. The bursa-equivalent organs in mammals (including humans) are the GALT and the bone marrow, where B lymphocytes differentiate into immunocompetent cells. Thus, the “B” refers to the bursa of Fabricius or the bursa-equivalent organs of mammals. Investigators studying newborn mice found that removal of the thymus results in

1	into immunocompetent cells. Thus, the “B” refers to the bursa of Fabricius or the bursa-equivalent organs of mammals. Investigators studying newborn mice found that removal of the thymus results in profound deficiencies in cell-mediated immune responses. The rejection of transplanted skin from a heterologous donor is an example of cell-medi-ated immune response. Thymectomized mice demonstrate a marked reduction in the number of lymphocytes found in specific regions of the spleen and the lymph nodes (thymus-dependent areas). The areas of depletion differ from those identified after removal of the bursa of Fabricius in the chicken. These affected lymphocytes were therefore named T lymphocytes or T cells; T refers to thymus.

1	attracted to the site of mast cell degranulation, where they neutralize the effects of histamine. Thus, eosinophils are frequently seen in connective tissue at allergic or other hypersensitivity reaction sites. Typically, however, both cellular and humoral immune systems are involved, although one system generally predominates, depending on the stimulus.  Humoral (antibody-mediated) immunity is mediated by antibodies that act directly on an invading agent. These antibodies are produced by B lymphocytes and by plasma cells derived from B lymphocytes. In some diseases (e.g., tetanus), a nonimmune person can be rendered immune by receiving an injection of antibody purifed from the blood of an immune person or animal. The effectiveness of this passive transfer proves that it is the antibody that is responsible for the protection.

1	 Cell-mediated immunity is mediated by specific T lymphocytes that attack and destroy virus-infected host cells or foreign cells. Cell-mediated immunity is important in the defense against viral, fungal, and mycobacterial infections, as well as tumor cells. Cell-mediated immunity is also responsible for transplant rejection. Helper T and cytotoxic T lymphocytes ( CTLs) recognize and bind to antigens that are bound to MHC molecules.

1	Helper T and cytotoxic T lymphocytes ( CTLs) recognize and bind to antigens that are bound to MHC molecules. To understand how the specific immune responses (hu-moral and cell-mediated responses) are initiated, one must grasp the central role played by the helper and cytotoxic T lymphocytes. Helper T and cytotoxic lymphocytes act as the immune system “patrols.” Both kinds of lymphocytes have a T-cell receptor (TCR), a transmembrane protein whose exposed portion is on the T-cell membrane in close proximity to the CD3 marker (Fig. 14.3). The TCR recognizes antigen only when it is attached to “identification molecules,” the MHC molecules. In addition, helper T lymphocytes can only recognize an antigen when it is “presented” to them by cells called antigen-presenting cells (APCs). Cytotoxic T lymphocytes can only recognize antigen on other body cells such as those transformed by cancer or infected with a virus. The two classes of MHC molecules display peptides on the surface of cells.

1	The two classes of MHC molecules display peptides on the surface of cells. MHC molecules display short fragments of digested foreign proteins on the surface of cells. These proteins bind to MHC molecules inside the cell and are then transported to the cell FIGURE 14.3 • Schematic diagram of the molecular structure of the CD3– TCR complex. The CD3 molecule consists of five different polypeptide chains with molecular weights ranging from 16 to 28 kilodaltons. This molecule is closely associated with the T-cell receptor (TCR), which has two polypeptide chains ( and ). The T cell may be activated after the interaction of the TCR with antigen (Ag) displayed on the surface of a major histocompatability complex (MHC) molecule. This interaction transmits the signals to the interior of the cell through the CD3 molecule. This signal stimulates the T cell to secrete interleukins, which in turn stimulate T cells to divide and differentiate.

1	surface. MHC I and MHC II molecules are products of a “supergene” located on chromosome 6 in humans known as the major histocompatibility gene complex. The expression of this gene complex produces molecules that are specific not only to the individual cell that produces them but also to the tissue type and degree of cellular differentiation. MHC I is expressed on the surface of all nucleated cells and platelets. MHC I molecules act as a target to allow the elimination of abnormal host cells (e.g., virus-infected or transformed cancer cells). MHC I molecules perform this function by displaying on their surface short fragments of all peptides that are actively synthesized by the cell. Therefore, all endogenous “self ” peptides are displayed on the surface of every cell in the body, but viral or cancer-specific peptides are displayed only on the surface of infected or transformed cells (Fig. 14.4). The MHC I molecules present peptides fragments to cytotoxic CD8 T lymphocytes.

1	MHC II is limited in its distribution (see Fig. 14.4). It is expressed on the surface of all APCs and is critical in immune interactions. The MHC II molecules present partially digested, endocytosed foreign peptides to helper CD4 T lymphocytes. Activation of T and B Cells Activation of T cells requires the presence of costimulatory signals. become fully activated and to subsequently differentiate and proliferate. The interaction of the TCR and the CD4 or CD8 molecules with the antigen–MHC complex is referred to as a first signal. The second signal, which is called the costimulatory signal, is delivered by the interaction of membrane molecules on T cells and molecules on the APC. The most important interactions are between the CD28 molecule expressed on the T-cell membrane and the B7 (CD86) molecule expressed on the APC membrane. Another pair of costimulatory signals is generated by the interaction of CD40 (on APC cells) with CD40L (CD154), on T cells.

1	When a helper (CD4) T lymphocyte recognizes an antigen bound to an MHC molecule, the TCR attaches to the antigen–MHC II complex. The binding of the TCR to the antigen–MHC II complex in the presence of a costimulatory signal (derived from the CD28–B7 interaction) activates the helper T lymphocyte to release immune chemicals, or cytokines. Cytokines are immune substances (proteins) that are biologic modulators of immune responses. The specific cytokines secreted by helper CD4 T lymphocytes are called interleukins (ILs). Interleukins stimulate other T cells, B cells, and NK cells to differentiate and proliferate. When a cytotoxic CD8 T lymphocyte (CTL) recognizes an antigen–MHC I complex, the TCR attaches to it. If a costimulatory signal is present (derived from the interaction of CD40 and CD40L), the CTL is activated. Once activated,

1	FIGURE 14.4 • Schematic diagram of the molecular structure of MHC I and MHC II molecules. The MHC I molecule is a glycoprotein that is expressed on the surface of all nucleated cells of the body and on platelets. MHC I molecules present endogenously synthesized peptides for recognition by cytotoxic CD8 T lymphocytes. Therefore, the MHC I molecule acts as the target for the elimination of abnormal host cells producing abnormal proteins (e.g., cells infected by an intracellular agent such as a virus, or cells that have been transformed such as cancer cells). MHC I consists of an heavy chain (45 kilodaltons) and a smaller, noncovalently attached 2 microglobulin polypeptide (12 kilodaltons). The 2 microglobulin promotes maturation of T cells and acts as a chemotactic factor. The MHC II molecule is also a glycoprotein but is expressed only on a restricted population of cells known as antigen-presenting cells (APCs). MHC II molecules present exogenous (foreign) peptides to helper CD4 T

1	is also a glycoprotein but is expressed only on a restricted population of cells known as antigen-presenting cells (APCs). MHC II molecules present exogenous (foreign) peptides to helper CD4 T lymphocytes. They consist of two chains–an chain (33 kilodaltons) and a chain (29 kilodaltons)—each of which possesses oligosaccharide groups.

1	the CTL also releases cytokines that stimulate cells to proliferate and destroy the abnormal host cells. CD8 T lymphocytes are MHC I restricted, and CD4 T lymphocytes are MHC II restricted. MHC molecules are recognized by helper CD4 T lymphocytes or CTLs, depending on the class of the MHC molecule engaged. This restricted presentation of foreign antigens by MHC molecules to either cytotoxic or helper T lymphocytes is a key component of immune surveillance. The MHC I molecule with the peptide antigen displayed on its surface interacts only with the TCR and CD8 molecule expressed on cytotoxic CD8 T lymphocytes; these cells are therefore described as MHC I restricted. This interaction allows cytotoxic T lymphocytes to recognize infected or transformed target cells (Fig. 14.5a).

1	In contrast, the MHC II molecule with the peptide antigen displayed on its surface interacts only with the TCR and CD4 molecule expressed on helper CD4 T lymphocytes (Fig. 14.5b); these cells are therefore described as MHC II restricted. MHC II molecules are found on APCs, such as macrophages, whose main function is to present antigen to T lymphocytes. For B cells to become activated and differentiate into plasma cells, they require interactions with helper T lymphocytes.

1	Each B lymphocyte reacts only with a single antigen or type of antigenic site that it has been genetically programmed to recognize. Activation of B cells requires two signals. One is derived from interaction between BCRs and antigen. The bound antigen molecules are engulfed into B cells by receptor-mediated endocytosis, and fragments of the antigen are then displayed at the cell surface with the help of MHC II molecules. Helper T cells with complementary TCRs bind B cells and provide the second costimulatory signal. The binding usually involves a reaction between CD40 molecules on a B-cell surface with their ligands (CD40L or CD154) residing at the surface of a helper T cell. These interactions complete the activation process of a B lymphocyte and induce an involved T cell to secrete specific cytokines that stimulate divisions and differentiation of B cells. Details of B-cell activation are illustrated in Figure 14.6.

1	FIGURE 14.5 • Schematic diagram of the molecular interactions that occur during antigen presentation. To become activated, both cytotoxic and helper T lymphocytes need to identify presented antigens as “nonself” as well as recognize the appropriate class of MHC molecules. Note that each interaction between an antigen–MHC complex with its specific T-cell receptor (TCR) requires a costimulatory signal from the interaction of CD28 with B7 molecules. Without a costimulatory signal, the T cell cannot be fully activated. a. In all nucleated cells of the body, viral antigen or cancer (tumor-specific) proteins are displayed in the context of MHC I molecules to interact with cytotoxic CD8 T lymphocytes. b. On antigen-presenting cells (e.g., macrophages), the foreign antigen is displayed in the context of MHC II molecules to interact with a helper CD4 T lymphocyte. FIGURE 14.6 • Schematic diagram of B lymphocyte activation leading to plasma cell and B memory cell formation.

1	FIGURE 14.6 • Schematic diagram of B lymphocyte activation leading to plasma cell and B memory cell formation. B cells are activated by the binding of antigen (Ag) to B-cell receptors (BCRs; membrane-bound antibodies) expressed on their surface. As an antigen-presenting cell, a B cell internalizes the BCR–antigen complex, partially digests the antigen, and then displays parts of it on the surface of its own MHC II molecules. The T-cell receptor (TCR) on a helper CD4 T lymphocyte (TH2 cell) recognizes both the antigen and the MHC II molecule, activating the helper CD4 T lymphocyte. The activated helper CD4 T lymphocyte releases interleukins IL-2, IL-4, IL-5, IL-10, and IL-13, which promote division and differentiation of the B lymphocyte into plasma cells and memory B cells. Note the presence of a costimulatory molecule complex between the B and T cells. Ab, antibody.

1	FIGURE 14.7 • Schematic diagram of activation of natural killer cells leading to destruction of a transformed tumor cell by antibody-dependent, cell-mediated cytotoxicity (ADCC). The ADCC reaction involves (1) activation of natural killer (NK) cells by the binding of interferon (IFN-), the powerful NK cell activator, to its cell surface receptor (IFNreceptor) and (2) the binding of an antibodyor an antibodyand complement-coated target cell to an NK cell bearing Fc receptors. These reactions induce apoptosis, or lysis, of the target cell, usually through the action of tumor-specific antibodies or the action of perforins and granzymes (fragmentins) secreted by activated NK cells. Activated B lymphocytes differentiate into plasma cells and memory B cells.  Plasma cells synthesize and secrete a specific antibody.

1	Activated B lymphocytes differentiate into plasma cells and memory B cells.  Plasma cells synthesize and secrete a specific antibody. During this process, activated B cells switch from synthe sizing their BCRs as integral membrane proteins to form ing a soluble version, which are called antibodies.  Memory B cells respond more quickly to the next encounter with the same antigen. The specific antibody produced by the plasma cell binds to the stimulating antigen, forming an antigen–antibody complex. These complexes are eliminated in a variety of ways, including destruction by NK cells and phagocytosis by macrophages and eosinophils. In antibody-dependent cell-mediated cytotoxicity (ADCC), IgG molecules direct NK cells to their target.

1	In antibody-dependent cell-mediated cytotoxicity (ADCC), IgG molecules direct NK cells to their target. The membranes of a number of cells, including NK cells, macrophages, neutrophils, and eosinophils, possess immunoglobulin Fc receptors and can kill certain target cells. NK cells recognize the Fc region of antibodies and preferentially attack and destroy target cells, usually those coated with IgG antibodies (Fig. 14.7). The recognition and subsequent destruction of antibody-coated target cells by NK cells is called antibody-dependent cell-mediated cytotoxicity (ADCC). The antibodies in ADCC that coat the target cells often include tumor-specifc antibodies. This binding (through the Fc region) results in the apoptosis and lysis of the target cell.

1	If the antigen is a bacterium, the antigen–antibody complex may also activate a system of plasma proteins called the complement system and cause one of its components, usually C3, to bind to the bacterium and act as a ligand for its phagocytosis by macrophages. Complement-bound foreign cells are also targets of ADCC. The cell-mediated immune response: Cytotoxic CD8 T lymphocytes (CTLs) target and destroy transformed and virus-infected cells.

1	When the TCR of a CTL recognizes and binds to an anti-gen–MHC I complex on the surface of a transformed or virusinfected cell, the activation process is triggered. First, CTLs undergo “clonal expansion” by entering the cell cycle and proceeding with cell divisions followed by differentiation into effector (“killer”) cells. During differentiation, a large number of secretory vesicles are formed containing specific proteins that include perforins and granzymes (fragmentins). As a result of interaction with antigen, CTLs secrete these proteins. Perforins are pore-forming proteins that enter the target cell by forming ringlike transmembrane channels in their cell membranes. These channels cause an increase in the permeability of the membrane that contributes to cell death. Granzymes are exogenous serine proteinases that are released from cytoplasmic granules and pass into the target cells through the pores created by perforins. Once inside the cell, granzymes activate caspases that induce

1	serine proteinases that are released from cytoplasmic granules and pass into the target cells through the pores created by perforins. Once inside the cell, granzymes activate caspases that induce the cell to undergo apoptosis (Fig. 14.8). After killing the target cell, the majority of activated CTLs will die (of apoptosis), but some of them that interacted with helper T cells will become memory cells.

1	CD4CD25FOXP3 suppressor T lymphocytes suppress the immune responses of other lymphocytes. Once the immune reactions are initiated by contact with antigen, the immune system is capable of controlling the

1	FIGURE 14.8 • Schematic diagram of T-cell activation leading to elimination of a virus-infected host cell. The TCR–CD3 complex on a helper CD4 T lymphocyte recognizes foreign antigen displayed on an MHC II molecule on the surface of a macrophage. This recognition triggers a rapid response from B lymphocytes and the release of interleukin 2 (IL-2). The same macrophage also expresses MHC I molecules (like every other cell in the body) that interact with the appropriate TCR on the surface of a cytotoxic CD8 T lymphocyte. The cytotoxic CD8 T lymphocyte also possesses IL-2 receptors. IL-2 binding to these receptors stimulates the cell to divide and differentiate. The newly formed cytotoxic CD8 T lymphocytes migrate to the site of viral infection. There the TCRs recognize the viral antigens displayed on the surface of MHC I molecules of infected cells. After successfully recognizing these “nonself” proteins, the cytotoxic CD8 T lymphocytes secrete perforins and granzymes, killing the

1	displayed on the surface of MHC I molecules of infected cells. After successfully recognizing these “nonself” proteins, the cytotoxic CD8 T lymphocytes secrete perforins and granzymes, killing the infected cells.

1	magnitude of this response and terminating it over time. Certain T lymphocytes called suppressor T cells diminish or suppress responses of the other lymphocytes to antigen. Characterization of these cells has proved to be difficult, but recent studies have convincingly shown that these cells belong to the population of CD4 T lymphocytes that coexpress the CD25 and FOXP3 marker proteins. CD4CD25FOXP3 suppressor T cells originate in the thymus and account for about 5% of the total population of T cells. They secrete cytokines such as IL-10 and transforming growth factor (TGF-), the latter a potent suppressor of proliferation of specific classes of T and B effector cells.

1	Suppressor T cells diminish or suppress antibody formation by B cells, as well as lower the ability of CTLs to provide a cell-mediated immune response. They play important roles in delayed hypersensitivity reactions (allergic reactions) by downregulating responses to antigen that enter the body through the skin or mucosa. They are also important in the prevention of graft rejection. Suppressor T lymphocytes may also function in the regulation of erythroid cell maturation in bone marrow. Activated T lymphocytes synthesize a variety of cytokines.

1	Cytokines are soluble polypeptide substances, synthesized mainly by activated T lymphocytes, which affect the function of immune system effector cells (T and B cells), monocytes, macrophages, and other APCs. In general, cytokines and growth factors are similar in nature; the distinction between them is related to their effects on their target cell populations. Cytokines are defined as substances that are involved in immune defense mechanisms and act on lymphocytes, whereas growth factors act on other somatic cells. Included among these substances are chemotactic and mitogenic agents, migration inhibitory factors, interferon, and interleukins. Cytokines serve as chemical messengers between cells of the immune system and act locally on the same cell that secreted them (autocrine control) or on neighboring cells (paracrine control). In a way similar to hormones, they may also communicate the state of the immune system to cells in other systems (e.g., central nervous system, endocrine

1	or on neighboring cells (paracrine control). In a way similar to hormones, they may also communicate the state of the immune system to cells in other systems (e.g., central nervous system, endocrine system, and hemopoietic system). Cytokines function through specific receptors. Therefore, cells regulated by cytokines possess cytokine receptors.

1	Interleukins are synthesized mainly by helper CD4 T lymphocytes and to a lesser extent by monocytes, macrophages, and endothelial cells. Interleukins promote growth and differentiation of T cells, B cells, and hematopoietic cells. Currently, more than 29 interleukins have been identified. Interleukin 2 was the first cytokine to be discovered and characterized. Mutations in the genes encoding several cytokine receptors have been identified in several immunodeficiency disorders, bacterial sepsis, certain lymphoid cancers, and diseases of autoimmunity. For instance, individuals with a mutation in the IL-12 receptor gene cannot mount an effective immune response against mycobacterial (fungal) infections. Cytokines have been used with promising results to prevent transplant rejection, reverse cellular deficiencies after chemotherapy and radiation therapy, and treat certain cancers. The major functions of selected interleukins are summarized in Table 14.3.

1	APCs interact with helper CD4 T lymphocytes to facilitate immune responses. The interaction between most antigens and antibodies is insufficient to stimulate immune responses. The antigen must be broken into small peptides and presented in conjunction with MHC II molecules by specialized APCs to the appropriate helper CD4 T lymphocytes. Antigen can also be processed as a part of the B-cell activation pathway. Most APCs belong to the mononuclear phagocytotic system (MPS; described in Chapter 6, Connective Tissue, page 185). APCs include macrophages, perisinusoidal macrophages (Kupffer cells) of the liver, Langerhans’ cells in the epidermis, and dendritic cells of spleen and lymph nodes. Two APCs that do not belong to the MPS are B lymphocytes and type II and type III epithelioreticular cells of the thymus.

1	To present an antigen to a helper T cell, the APC first processes the antigen intracellularly and then displays antigen peptides on its surface. Antigen processing begins when the APC endocytoses the antigen and breaks it down into 8 to 10 amino acid peptides. In the endosomal compartment of the APC, the peptides bind to MHC II molecules. The anti-gen–MHC II complex is then translocated to the plasma membrane of the APC and displayed on the cell surface (Fig. 14.9). In addition to acting as APCs, macrophages perform other crucial functions in the immune response. In addition to presenting antigens to both T and B lymphocytes, macrophages have other important, although nonspecific, functions in the immune response: conjunction with MHC II molecules to helper CD4 T lymphocytes.  They digest pathogenic microorganisms through lyso somal action in combination with the helper CD4

1	T lymphocytes.  They digest pathogenic microorganisms through lyso somal action in combination with the helper CD4 T lymphocytes. They secrete multiple cytokines including lymphokines, complement components, and interleukins, as well as acid hydrolases, proteases, and lipases. After contact with an antigen, macrophages undergo an activation process characterized by multiple functional and morphologic changes. The macrophage increases in size, as do the number of lysosomes and cytoplasmic vacuoles. The activated macrophage becomes avidly phagocytotic with a greater ability to lyse ingested pathogenic microorganisms (Fig. 14.10). Activated macrophages destroy phagocytosed bacteria and foreign antigens. Macrophages also play a vital role in sequestering and removing foreign materials and organisms that either do not provoke an immune response or are ingested but not digested.

1	Macrophages also play a vital role in sequestering and removing foreign materials and organisms that either do not provoke an immune response or are ingested but not digested. These include both organic and inorganic particulate materials (e.g., carbon particles), pigment (e.g., from tattoos), cellulose, and asbestos, as well as tuberculosis and leprosy bacilli and the organisms that cause malaria and other diseases. In these instances, macrophages often fuse to form multinucleate, foreign body giant cells called Langerhans’ giant cells that isolate these pathogens from the body. Lymphatic vessels are the route by which cells and large molecules pass from the tissue spaces back to the blood.

1	Lymphatic vessels are the route by which cells and large molecules pass from the tissue spaces back to the blood. Lymphatic vessels begin as networks of blind capillaries in loose connective tissue. They are most numerous beneath the epithelium of skin and mucous membranes. These vessels remove substances and fluid from the extracellular spaces of the connective tissues, thus producing lymph. Because the walls of the lymphatic capillaries are more permeable than the walls of blood capillaries, large molecules, including antigens and cells, gain entry more readily into the lymphatic capillaries than into blood capillaries. As lymph circulates through the lymphatic vessels, it passes through lymph nodes. Within the lymph nodes, foreign substances (antigens) conveyed in the lymph are trapped by the follicular dendritic cells. The antigen exposed on the surface of follicular dendritic cells can be processed by APCs present within the lymph node.

1	Lymphocytes circulate through both lymphatic and blood vessels. The circulation of lymphocytes through the lymphatic vessels and the bloodstream enables them to move from one part of

1	TABLE Characteristics of Interleukins14.3 Interleukin 12 IL-12 T cells Stimulates growth of NK cells, CD4T cells, and CD8T cells Interleukin 13 IL-13 T cells Modulates B-cell responses and promotes IgE synthesis Interleukin 14 IL-14 T cells, follicular dendritic cells Induces production of memory B cells Interleukin 15 IL-15 T cells, monocytes Induces proliferation and differentiation of CD8T cells Interleukin 16 IL-16 T cells Activates migration of CD8T cells, mono-cytes, and eosinophils Interleukin 17 IL-17 Memory CD4T cells Stimulates endothelial cells and fibroblasts to secrete cytokines Name Symbol Source Major Functions Interleukin 1 IL-1 Neutrophils, monocytes, macrophages, endothelial cells Stimulates various cells in inflammatory response Induces fever Facilitates proliferation of CD4T cells and proliferation and differentiation of B cells Interleukin 2 IL-2 CD4T cells Induces proliferation and differentiation of CD4T cells and to a lesser degree CD8T cells, B cells, and NK

1	of CD4T cells and proliferation and differentiation of B cells Interleukin 2 IL-2 CD4T cells Induces proliferation and differentiation of CD4T cells and to a lesser degree CD8T cells, B cells, and NK cells Interleukin 3 IL-3 CD4T cells Induces proliferation of hematopoietic stem cells Interleukin 4 IL-4 CD4T cells, mast cells Induces proliferation and differentiation of B cells, CD4T cells Activates macrophages Promotes synthesis of IgE and IgG Interleukin 5 IL-5 CD4T cells Induces proliferation and differentiation of eosinophils Stimulates B cells to secrete IgA Interleukin 6 IL-6 Endothelial cells, neutrophils, macrophages, T cells Stimulates differentiation of hematopoietic cells Induces growth of activated B cells Interleukin 7 IL-7 Adventitial cells of bone marrow Stimulates growth and differentiation of progenitor B and T cells Interleukin 8 IL-8 Macrophages, endothelial cells Acts as chemotactic factor on T lymphocytes and neutrophils Interleukin 9 IL-9 CD4T cells Facilitates

1	and differentiation of progenitor B and T cells Interleukin 8 IL-8 Macrophages, endothelial cells Acts as chemotactic factor on T lymphocytes and neutrophils Interleukin 9 IL-9 CD4T cells Facilitates growth of CD4T cell (but not CD8T cells) Stimulates growth of hematopoietic cells Activates mast cells Interleukin 10 IL-10 Macrophages, T cells Acts on T cells as cytokine synthesis inhibitory factor Inhibits macrophage functions Interleukin 11 IL-11 Macrophages Facilitates growth of hematopoietic cells, mainly megakaryocytes  FOLDER 14.3 Clinical Correlation: Human Immunodeficiency Virus (HIV) and Acquired Immunodeficiency

1	Human immunodeficiency virus (HIV) is an RNA retro-helper CD4T cells, reducing the number of helper T cells virus; it contains an enzyme called reverse transcriptase. HIV is the virus that causes acquired immunodeficiency syndrome (AIDS). It has an incubation period that may be as long as 11 years before symptoms of clinical AIDS occur. The great majority of HIV-infected individuals eventually develop AIDS. HIV gains entry to helper T cells by binding to CD4 molecules. The virus then injects its own genetic information into the cell cytoplasm (Fig. F14.3.1). This injected genetic information consists of single-stranded RNA. The viral RNA is incorporated into the infected T-cell genome through reverse transcription of the RNA into DNA. The transcribed DNA is then incorporated into the host DNA. The T cell then makes copies of the virus, which are extruded from the T cell through exocytosis. These HIV particles then infect other helper T cells. The immune system responds to this

1	host DNA. The T cell then makes copies of the virus, which are extruded from the T cell through exocytosis. These HIV particles then infect other helper T cells. The immune system responds to this condition by generating cytotoxic CD8 T cells and antibodies directed against the virus particles. Cytotoxic CD8 T cells kill HIV-infected (the helper T-cell count is actually used as a clinical indicator of the progress of HIV infection). As the helper CD4 T-cell population becomes depleted, infected individuals eventually become incapable of generating an immune response against bacterial or viral infections. They usually die of secondary infections caused by opportunistic microorganisms or cancer.

1	Anti-HIV treatment is the major strategy against HIV infection and AIDS. Azidothymidine (AZT), an inhibitor of reverse transcriptase, was the first promising drug used to treat HIV infection. Currently, the most effective treatment is multiple drug therapy known as highly active antiretroviral therapy (HAART), which uses a combination of several chemotherapeutic agents. These include nucleoside and nonnucleoside reverse transcriptase inhibitors and HIV protease inhibitors. HAART offers several advantages over monotherapy such as synergistic dosage effects and reduced side effects as well as reduced drug resistance.

1	FIGURE F14.3.1 • Schematic diagram of the interaction between HIV and the helper CD4T cell. Human immu -nodeficiency virus (HIV) is the RNA virus that causes AIDS. It contains reverse transcriptase. HIV gains entry into the helper CD4T lymphocyte by binding to the CD4 molecule and injecting its genetic information into the cell cytoplasm. Accessory cell surface molecules such as gp 120 assist in viral entry into the cell. These proteins interact with CD4 molecules. The injected genetic information is incorporated into the host cell genome through reverse transcription of RNA into DNA. This DNA containing viral information is then incorporated into host DNA. HIV HIV gp 120 gp 120 CD4 CD4 chemokine receptor chemokine receptor gp 41 gp 41 RNA RNACD4T lymphocyte

1	FIGURE 14.9 • Schematic diagram of processing pathways for MHC I and MHC II synthesis and antigen presentation. During the processing and presentation of cytoplasmic antigen (Ag) for MHC I molecules (red pathway), cytoplasmic protein antigens are degraded by protease into 8 to 10 amino acid fragments that then enter the rough-surfaced endoplasmic reticulum (rER). In the rER, newly synthesized chains of MHC I molecules interact with both the processed antigen and 2 microglobulin (2M) and form a stable complex. This complex leaves the rER via the typical secretory pathway through the Golgi apparatus. The antigen–MHC I complex is displayed on the cell surface, where it is available for recognition by cytotoxic CD8 T lymphocytes. MHC II molecules are assembled in the rER and then bind to an invariant chain, which blocks the antigen-binding site. At this point, the MHC II molecule and the invariant chain are secreted to the cell surface (blue pathway). After a brief stay on the cell

1	an invariant chain, which blocks the antigen-binding site. At this point, the MHC II molecule and the invariant chain are secreted to the cell surface (blue pathway). After a brief stay on the cell surface, the MHC II molecule and invariant chain are endocytosed, and in an early endosome, the invariant chain is degraded. The foreign (exogenous) antigen is endocytosed and partially digested by proteolytic degradation in endosomes (white pathway). The MHC II molecule can now bind the processed foreign antigen and return with it to the cell surface. On the cell surface, the antigen–MHC II complex is recognized by helper CD4 T lymphocytes, which initiates the immune response. If the MHC II molecule fails to capture the antigen, it will be degraded in the lysosomal compartment (green pathway).

1	the lymphatic system to another at different stages in their development and to reach sites within the body where they are needed. Lymphocytes conveyed in the lymph enter lymph nodes via afferent lymphatic vessels, whereas lymphocytes conveyed in the blood enter the node through the walls of postcapillary venules (high endothelial venules [HEVs]; Fig. 14.11). B and T cells migrate to and populate different regions within the lymph node. Some lymphocytes pass through the substance of the node and leave via the efferent lymphatic vessels, which lead to the right lymphatic trunk or to the thoracic duct. In turn, both of these channels empty into the blood circulation at the junctions of the internal jugular and subclavian veins at the base of the neck. The lymphocytes are conveyed to and from the various lymphatic tissues via the blood vessels. Diffuse lymphatic tissue and lymphatic nodules guard the body against pathogenic substances and are the site of the initial immune response.

1	Diffuse lymphatic tissue and lymphatic nodules guard the body against pathogenic substances and are the site of the initial immune response. The alimentary canal, respiratory passages, and genitourinary tract are guarded by accumulations of lymphatic

1	The alimentary canal, respiratory passages, and genitourinary tract are guarded by accumulations of lymphatic FIGURE 14.11 • Diagram depicting circulation of lymphocytes in the body. Lymphocytes enter lymph nodes by two routes: afferent lymphatic vessels and through the wall of high endothelial venules (HEVs) in the deep cortex. Some lymph node lymphocytes move to the T and B domains of the lymph node; others efferent pass through the parenchyma of the lymphatic vessel. Ultimately, the lym phocytes enter a major lymphatic vessel—in this case, the right lymphatic trunk—that opens into the junction of the right internal jugular and right subclavian vein. The lymphocytes continue to the arterial side of the circulation and, via the arteries, to the lymphatic tissues of the body or to tissues where they participate in immune reactions. From the lymphatic tissues, lymphocytes again return to the lymph nodes to gain entry via the HEV.

1	FIGURE 14.10 • Macrophage activation by a helper CD4 T cell. Helper CD4 T lymphocytes recognize the bacterial antigen expressed in the context of MHC II molecules on the surface of a macrophage that has phagocytosed the bacteria. The recognition of MHC II molecules activates the T cell, which in turn secretes IL-2. IL-2 acts as an autocrine hormone to stimulate T-cell division and differentiation. Newly formed helper CD4 T lymphocytes also interact with MHC II molecules and release interferon (IFN-). This cytokine stimulates the macrophage to destroy the bacteria inside its phagosomes. CD4 molecules on the surface of the T cell also potentiate antibacterial reactions.

1	tissue that are not enclosed by a capsule. Lymphocytes and other free cells of this tissue are found in the lamina propria (subepithelial tissue) of these tracts. This form of lymphatic tissue is called diffuse lymphatic tissue or mucosa-associated lymphatic tissue (MALT) because of its association with mucous membranes (Fig. 14.12). These cells are strategically located to intercept antigens and initiate an immune response. After contact with antigen, they travel to regional lymph nodes, where they undergo proliferation and differentiation. Progeny of these cells then return to the lamina propria as effector B and T lymphocytes. The importance of diffuse lymphatic tissue in protecting the body from antigens is indicated by two factors:  The regular presence of large numbers of plasma cells, especially in the lamina propria of the gastrointestinal tract, a morphologic indication of local antibody secretion.

1	 The presence of large numbers of eosinophils, also frequently observed in the lamina propria of the intestinal and respiratory tracts, an indication of chronic inflammation and hypersensitivity reactions. Lymphatic nodules are discrete concentrations of lymphocytes contained in a meshwork of reticular cells.

1	Lymphatic nodules are discrete concentrations of lymphocytes contained in a meshwork of reticular cells. In addition to diffuse lymphatic tissue, localized concentrations of lymphocytes are commonly found in the walls of the alimentary canal, respiratory passages, and genitourinary tract. These concentrations, called lymphatic nodules or lymphatic follicles, are sharply defined but not encapsulated (Fig. 14.13). A lymphatic nodule consisting chiefly of small lymphocytes is called a primary nodule. However, most nodules are secondary nodules and have distinctive features that include the following:  A germinal center is located in the central region of the nodule (Fig. 14.14) and appears lightly stained in histologic sections. The germinal center develops when a lymphocyte that has recognized an antigen returns to a primary nodule and undergoes proliferation. The lighter FIGURE 14.12 • Photomicrograph of diffuse lymphatic tissue.

1	FIGURE 14.12 • Photomicrograph of diffuse lymphatic tissue. This photomicrograph shows the diffuse lymphatic tissue in the lamina propria (LP) of the large intestine. The lower portion of two intestinal glands (Gl) is also evident. The highly cellular, diffuse lymphatic tissue includes fibroblasts, plasma cells, and eosinophils. However, the most abundant cell component, whose presence characterizes diffuse lymphatic tissue, is the lymphocyte, which can be identified by its small, round, dark-staining nucleus. 320.

1	320. FIGURE 14.13 • Photomicrograph of a lymphatic nodule. This photomicrograph shows a section of the wall of the small intestine (duodenum). Short villi and intestinal glands are present in the upper part of the micrograph. A lymphatic nodule (LN) occupies most of the remainder of the micrograph. The lighter central region of the nodule is the germinal center. The lymphocytes in the germinal center are larger than those in the denser region of the nodule. They have more cytoplasm, so, their nuclei are more dispersed, giving the appearance of a less compact cellular mass. 120.

1	staining is attributable to the large immature lymphocytes (lymphoblasts and plasmablasts) that it contains. These lymphocytes have large amounts of dispersed euchromatin in their nuclei rather than the dense heterochromatin of small lymphocytes. Follicular dendritic cells (FDCs) are also present in germinal centers interdispersed between populations of B lymphocytes. The germinal center is a morphologic indication of lymphatic tissue response to antigen. The presence of a germinal center represents a cascade of events that includes activation and proliferation of lymphocytes, differentiation of plasma cells, and antibody production. Mitotic figures are frequently observed in the germinal center, reflecting the proliferation of new lymphocytes at this site. The number of FDCs and macrophages in the germinal center often increases dramatically after a period of intense response to an antigen.

1	 A mantle zone or corona is present that represents an outer ring of small lymphocytes that encircles the germinal center. Lymphatic nodules are usually found in structures associated with the alimentary canal such as the tonsils, ileum, and vermiform appendix.

1	Generally, nodules are dispersed singly in a random manner. In the alimentary canal, however, some aggregations of nodules are found in specific locations. These include the following:  Tonsils form a ring of lymphatic tissue at the entrance of the oropharynx. The pharyngeal tonsils (adenoids) (located in the roof of the pharynx), the palatine tonsils (or simply the tonsils, located on either side of the pharynx and between the palatopharyngeal and palatoglossal arches), and the lingual tonsils at the base of the tongue all contain aggregates of lymphatic nodules. The palatine tonsils consist of dense accumulations of lymphatic tissue located in the mucous membrane. The squamous epithelium that forms the surface of the tonsil dips into the underlying connective tissue in numerous places, forming tonsillar crypts (Fig. 14.15). The walls of these crypts usually possess numerous lymphatic nodules. Like other aggregations of lymph nodules, tonsils do not possess afferent lymphatic

1	forming tonsillar crypts (Fig. 14.15). The walls of these crypts usually possess numerous lymphatic nodules. Like other aggregations of lymph nodules, tonsils do not possess afferent lymphatic vessels; however, lymph drains from the lymphatic tissue of the tonsil via efferent lymphatic vessels.

1	 Peyer’s patches are located in the ileum (distal portion of the small intestine) and consist of numerous aggregations of lymphatic nodules containing T and B lymphocytes (Fig. 14.16). In addition, numerous isolated single (solitary) lymph nodules are located along both large and small intestines.  The vermiform appendix arises from the cecum. The lamina propria is heavily infiltrated with lymphocytes and contains numerous lymphatic nodules. Although the appendix is often described as a vestigial organ, the abundant lymphatic tissue that it contains during early life suggests that it is functionally associated with bursa-equivalent organs.

1	FIGURE 14.14 • Photomicrograph of a lymph node. This photomicrograph shows the superficial cortex (SC), deep cortex (DC), and medulla (M) of the lymph node in a routine H&E preparation. The capsule (Cap) is composed of dense connective tissue from which trabeculae (T) penetrate into the organ. Below the capsule is the subcapsular sinus (SCS). It receives lymph from afferent lymphatic vessels that penetrate the capsule. The subcapsular sinus is continuous with the trabecular sinuses that course along the trabeculae. The superficial cortex contains the lymphatic nodules (LN). The deep cortex is nodule free. It consists of densely packed lymphocytes and contains the unique high endothelial venules (not visible at this magnification). The medulla consists of narrow strands of anastomosing lymphatic tissue called medullary cords (MC), which are separated by light-appearing spaces, the medullary sinuses (MS). The medullary sinuses receive lymph from the trabecular sinuses as well as lymph

1	tissue called medullary cords (MC), which are separated by light-appearing spaces, the medullary sinuses (MS). The medullary sinuses receive lymph from the trabecular sinuses as well as lymph that has filtered through the cortical tissue. 140.

1	FIGURE 14.15 • Photomicrograph of a palatine tonsil. a. This low-magnification photomicrograph shows an H&E–stained palatine tonsil. The stratified squamous epithelium that forms the surface of the tonsil dips into the underlying connective tissue in numerous places, forming tonsillar crypts. 25. b. This higher-magnification photomicrograph of the rectangular area in a shows the stratified squamous epithelium (SSE) lining the tonsillar crypt. In the portion of the photomicrograph below the lumen of the crypt, SSE is well defined and separated by a connective tissue layer (CT) from the lymphatic nodule (LN). In the upper portion of the photomicrograph, the SSE is just barely recognized because of the heavy infiltration of lymphocytes; the epithelial cells are present, however, although they are difficult to identify. In effect, the lymphatic nodule has literally grown into the epithelium, distorting it and resulting in the disappearance of the more typical, well-defined

1	although they are difficult to identify. In effect, the lymphatic nodule has literally grown into the epithelium, distorting it and resulting in the disappearance of the more typical, well-defined epithelial–connective tissue boundary. 450.

1	With age, the amount of lymphatic tissue within the organ regresses and is diffcult to recognize. As noted, diffuse lymphatic tissue and lymphatic nodules are named according to the region or organ in which they appear. In the alimentary canal, they are collectively referred to as gut-associated lymphatic tissue (GALT); in the bronchial tree, they are known as bronchus-associated lymphatic tissue (BALT). The term mucosa-associated lymphatic tissue (MALT) includes GALT and BALT. Diffuse lymphatic tissue and lymphatic nodules of MALT are present in many other regions of the body (e.g., female reproductive tract) where the mucosa is exposed to the external environment. All lymphatic nodules become enlarged as a consequence of encounters with antigen. Lymph nodes are small encapsulated organs located along the pathway of lymphatic vessels.

1	Lymph nodes are small encapsulated organs located along the pathway of lymphatic vessels. Lymph nodes are small, bean-shaped, encapsulated lymphatic organs. They range in size from about 1 mm (barely visible with the unaided eye) to about 1 to 2 cm in their longest dimension. Lymph nodes are interposed along lymphatic vessels (Fig. 14.17) and serve as filters through which lymph percolates on its way to the blood vascular system. Although widely distributed throughout the body, they are concentrated in certain regions such as the axilla, groin, and mesenteries.

1	Two types of lymphatic vessels serve the lymph node:  Afferent lymphatic vessels convey lymph toward the node and enter it at various points on the convex surface of the capsule. Efferent lymphatic vessels convey lymph away from the node and leave at the hilum, a depression on the concave surface of the node that also serves as the entrance and exit for blood vessels and nerves. Note that activated lymphocytes, which remain in the lymph node to proliferate and differentiate, are carried to the node primarily by blood vessels. The supporting elements of the lymph node are:  capsule, composed of dense connective tissue that surrounds the node;

1	FIGURE 14.16 • Photomicrograph of aggregated nodules in the wall of the ileum. This low-magnification photomicrograph provides an example of aggregated nodules. The multiple lymphatic nodules (indicated by a dashed line) with visible germinal centers are typically found in the ileum. This accumulation of lymphatic tissue is known as a Peyer’s patch. The nodules originate in the lamina propria and extend into the submucosa of the ileum. 5.  trabeculae, also composed of dense connective tissue, which extend from the capsule into the substance of the node, forming a gross framework; and  reticular tissue composed of reticular cells and reticular fibers that form a fine supporting meshwork throughout the remainder of the organ (Fig. 14.18). The reticular meshwork of lymphatic tissues and organs (except the thymus) consists of cells of mesenchymal origin and reticular fibers and ground substance produced by those cells. Cells of the Reticular Meshwork

1	Cells of the Reticular Meshwork The reticular meshwork of the lymph node contains several types of cells that perform different functions in generating immune responses. The cells of the reticular meshwork appear as stellate or elongated cells with an oval euchromatic nucleus and a small amount of acidophilic cytoplasm. These cells can take up dyes and colloidal materials. Using immunocytochemistry and transmission electron microscopy, several populations of cells have been identified.

1	 Reticular cells are indistinguishable from typical fibroblasts. These cells synthesize and secrete type III collagen (reticular fibers) and the associated ground substance that forms the stroma observed with the light microscope (Plate 38, page 481). Elongated cytoplasmic processes of these cells wrap around the bundles of reticular fibers, effectively isolating these structural components from the parenchyma of the lymphatic tissue and organs (Fig. 14.19). Besides their supporting role, they express surface molecules and produce substances that attract T cells, B cells, and dendritic cells.

1	 Dendritic cells (DCs) are unique bone marrow–derived APCs. DCs monitor the local environment for foreign substances that they then process and present to antigen-specific T cells. They are much more efficient in antigen presentation than other APCs and can present virtually any form of protein antigen on both MHC I and MHC II molecules. They express an exceptionally high level of MHC II and costimulatory molecules necessary for activation of T cells. In the lymph node, DCs are usually localized in T lymphocyte–rich areas.

1	 Macrophages are both phagocytic and antigen-presenting cells that express MHC I, MHC II, and costimulatory molecules. However, the expression levels of MHC II and costimulatory molecules are much lower than those of the dendritic cells, making them less efficient APCs. Instead, they have an immense capacity for endocytosis and digestion of internalized materials. The structure, microscopic characteristics, and functions of macrophages are described in Chapter 6, Connective Tissue.

1	 Follicular dendritic cells (FDCs) have multiple, thin, hairlike branching cytoplasmic processes that interdigitate between B lymphocytes in the germinal centers (Fig. 14.20). Antigen–antibody complexes adhere to the dendritic cytoplasmic processes by means of the antibody’s Fc receptors, and the cell can retain antigen on its surface for weeks, months, or years. Although this mechanism is similar to the adhesion of antigen–antibody complexes to macrophages, the antigen is not generally endocytosed, as it is by the macrophage. FDCs are thus not APCs because they lack MHC II molecules. General Architecture of the Lymph Node

1	General Architecture of the Lymph Node The parenchyma of the lymph node is divided into a cortex and medulla (Fig. 14.21). The cortex forms the outer portion of the node except at the hilum. It consists of a dense mass of lymphatic tissue (reticular framework, dendritic cells, follicular dendritic cells, lymphocytes, macrophages, and plasma cells) and lymphatic sinuses, the lymph channels. The medulla is the inner part of the lymph node.

1	FIGURE 14.17 • Structure of a lymph node. a. This diagram depicts the general features of a lymph node as seen in a section. The substance of the lymph node is divided into a cortex, including a deep cortex, and a medulla. The cortex, the outermost portion, contains spherical or oval aggregates of lymphocytes called lymphatic nodules. In an active lymph node, nodules contain a lighter center called the germinal center. The medulla, the innermost region of the lymph node, consists of lymphatic tissue that appears as irregular cords separated by lymphatic medullary sinuses. The dense population of lymphocytes between the superficial cortex and the medulla constitutes the deep cortex. It contains the high endothelial venules. Surrounding the lymph node is a capsule of dense connective tissue from which trabeculae extend into the substance of the node. Under the capsule and adjacent to the trabeculae are, respectively, the subcapsular sinus and the trabecular lymphatic sinuses. Afferent

1	from which trabeculae extend into the substance of the node. Under the capsule and adjacent to the trabeculae are, respectively, the subcapsular sinus and the trabecular lymphatic sinuses. Afferent lymphatic vessels (arrows) penetrate the capsule and empty into the subcapsular sinus. The subcapsular sinus and trabecular sinuses communicate with the medullary sinuses. The upper portion of the lymph node shows an artery and a vein and the location of the high endothelial venules of the lymph node. b. Photomicrograph of a lymph node in a routine H&E preparation. The dense outer portion of the lymph node is the cortex. It consists of aggregations of lymphocytes organized as nodules and a nodule-free deep cortex. The innermost portion of the lymph node, the medulla, extends to the surface at the hilum, where blood vessels enter or leave and where efferent lymphatic vessels leave the node. Surrounding the lymph node is the capsule, and immediately beneath it is the subcapsular sinus. 18.

1	FIGURE 14.18 • Photomicrograph of a lymph node. This silver preparation shows the connective tissue capsule (at the top), subcapsular sinus, and the superficial cortex of the lymph node (at the bottom). The reticular fibers (arrows) form an irregular anastomosing network throughout the stroma of the lymph node. Note elongated oval nuclei of reticular cells (arrowheads), which are in intimate contact with reticular fibers in the sinus. 640. FIGURE 14.19 • Electron micrograph of a reticular cell. The body of a reticular cell and its processes (arrows) are evident. The arrangement of the reticular cells contains and isolates the collagen fibrils from exposure to the lymphocytes. Note the adjacent lymphocytes on the right. In the light microscope and using a silver staining method, these collagen fibrils are recognized as a reticular fiber. 12,600. Lymphocytes in the superficial cortex are organized into nodules.

1	Lymphocytes in the superficial cortex are organized into nodules. As elsewhere, the lymphatic nodules of the cortex are designated primary nodules if they consist chiefly of small lymphocytes and secondary nodules if they possess a germinal center. Lymphatic nodules are found in the outer part of the cortex, called the superficial (nodular) cortex (Plate 37, page 479). The portion of the cortex between the medulla and superficial cortex is free of nodules; it is called the deep cortex (paracortex). This region contains most of the T cells in the lymph node (Fig. 14.22a). Because of its dependence on the thymus, perinatal thymectomy in animals results in a poorly developed deep cortex. On the basis of this

1	FIGURE 14.20 • Diagram of a follicular dendritic cell. This cell, usually found in germinal centers, has multiple, thin, hairlike cytoplasmic processes that interdigitate between B lymphocytes. Antigen–antibody complexes adhere to the dendritic cytoplasmic processes by means of Fc receptors. Follicular dendritic cells are not antigen-presenting cells because they lack MHC II molecules. observation, the deep cortex is also called the thymus-dependent cortex. The medulla of the lymph node consists of the medullary cords and medullary sinuses.

1	observation, the deep cortex is also called the thymus-dependent cortex. The medulla of the lymph node consists of the medullary cords and medullary sinuses. The medulla, the inner part of the lymph node, consists of cords of lymphatic tissue separated by lymphatic sinuses called medullary sinuses. As described above, a network of reticular cells and fibers traverses the medullary cords and medullary sinuses and serves as the framework of the parenchyma. In addition to reticular cells, the medullary cords contain lymphocytes (mostly B lymphocytes), macrophages, dendritic, and plasma cells (Fig. 14.22b). The medullary sinuses converge near the hilum, where they drain into efferent lymphatic vessels. Filtration of lymph in the lymph node occurs within a network of interconnected lymphatic channels called sinuses.

1	Filtration of lymph in the lymph node occurs within a network of interconnected lymphatic channels called sinuses. There are three types of lymphatic channels called sinuses in the lymph node. Just beneath the capsule of the lymph node is a sinus interposed between the capsule and the cortical lymphocytes called the subcapsular (cortical) sinus

1	FIGURE 14.21 • Schematic diagram of lymphocyte circulation within a lymph node. The green arrows indicate the circulation pathway of lymphocytes that enter the lymph node with the flow of lymph. Afferent lymphatic vessels carry lymph from the surrounding tissues and neighboring lymph nodes into the elaborate network of lymphatic sinuses. The wall of the sinuses allows lymph to percolate freely into the superficial and deep cortex, allowing lymphocytes to engage in immunosurveillance. The lymphocytes that enter the tissue next migrate back to the sinuses and leave the lymph node with the flow of the lymph. Lymphocytes that migrate to the lymph node from the blood (blue arrows) enter the deep cortex via high endothelial venules (HEVs) and also migrate to the superficial cortex. Here lymphocytes perform the same functions as lymphocytes that enter via lymphatic vessels. They also leave the lymph node by the efferent lymph vessels.

1	(Plate 38, page 481). Afferent lymphatic vessels drain lymph into this sinus. Trabecular sinuses that originate from the subcapsular sinuses extend through the cortex along the trabeculae and drain into medullary sinuses. Lymphocytes and macrophages or their processes readily pass back and forth between the lymphatic sinuses and the parenchyma of the node. The sinuses have a lining of endothelium that is continuous where it is directly adjacent to the connective tissue of the capsule or trabeculae but discontinuous where it faces the lymphatic parenchyma. Although a macrophage may reside in the lymphatic parenchyma, it often sends pseudopods (long cytoplasmic processes) into the sinus through these endothelial discontinuities. These pseu

1	FIGURE 14.22 • Distribution of T and B lymphocytes in the superficial cortex of the lymph node. a. Distribution of T lymphocytes in the lymph node of a marmoset monkey was visualized using an immunocytochemical method employing antibodies against CD3 protein, a specific marker for T lymphocytes. Tissue sections were initially treated with primary rabbit antihuman antibodies against a CD3 marker and later exposed to biotinylated secondary swine antirabbit antibodies. After incubation with avidin-biotin-peroxidase complex, the positive response was then visualized with diaminobenzidine (DAB) solution (brown-colored reaction). Cell nuclei were counterstained with hematoxylin. Note that the majority of T cells are distributed within the deep cortex (DP), a small number of T cells is present in the superficial cortex (SC), mainly around germinal centers (GC). b. Using the same immunoperoxidase DAB reaction described above, B cells were localized using primary monoclonal antibodies against

1	the superficial cortex (SC), mainly around germinal centers (GC). b. Using the same immunoperoxidase DAB reaction described above, B cells were localized using primary monoclonal antibodies against human CD20 protein (specific marker for B lymphocytes). Subsequently, secondary rabbit antimouse antibodies were used to visualize location of B cells, which the accumulations are found within germinal centers (GC) of the superficial cortex (SC). Cap, capsule. 200. (Courtesy of Dr. Douglas F. Paulsen.) dopods monitor the lymph as it percolates through the sinus.

1	Lymphatic sinuses are not open spaces, as are blood sinuses. Particularly in the medulla, macrophage processes, along with the reticular fbers surrounded by reticular cell processes, span the lumen of the sinus and form a crisscrossing meshwork that retards the free fow of lymph and enhances its fltration. Antigenic material and transformed cells of metastatic cancer are trapped by this mechanical flter and then phagocytosed by macrophages. In metastatic cancer, the system can be overwhelmed by an excessive number of cancer cells fowing through the lymphatic sinuses; as a result, the cells may establish a new metastatic site in the lymph node. Specialized high endothelial venules (HEVs) are the site of fluid absorption and the entrance for circulating lymphocytes into the lymph node.

1	In addition to lymph, lymphocytes also circulate through the lymph nodes. Although some lymphocytes enter nodes through afferent lymphatic vessels as components of lymph, most (about 90%) enter the node through the walls of postcapillary venules located in the deep cortex (see Fig. 14.21 and Plate 38, page 481). Because the postcapillary venules are lined by cuboidal or columnar endothelial cells, they are referred to as high endothelial venules (HEVs); Fig. 14.23). Cells of HEVs play an important role in circulating and concentrating lymph by transporting approximately 35% of fluid and electrolytes entering via afferent lymph vessels directly into the bloodstream. The cells of HEVs express a high concentration of water channels (aquaporin-1 [AQP-1] molecules). The rapid resorption of the interstitial fluid via water channels into the bloodstream causes lymph entering through the afferent lymph vessels to be drawn into the deep cortex by solvent drag.

1	These specialized endothelial cells also possess receptors for antigen-primed lymphocytes. They signal lymphocytes to leave the circulation and migrate into the lymph node. Both B and T cells leave the bloodstream through HEVs, crossing the endothelium by diapedesis—that is, by migrating between the endothelial cells in a manner similar to that described for neutrophils (see Fig. 10.6, page 277). The T cells remain in the thymus-dependent deep cortex; the B cells migrate to the nodular cortex (see Fig. 14.22). Most lymphocytes leave the lymph node by entering lymphatic sinuses from which they flow to an efferent lymphatic vessel. The lymph node is an important site for phagocytosis and initiation of immune responses.

1	The lymph node is an important site for phagocytosis and initiation of immune responses. Phagocytosis of particulate material by phagocytotic cells within the lymph nodes is an important step in initiating an immune response. The physical accumulation of microorganisms and particulate substances conveyed in the lymph and phagocytosis of the particulate material help to concentrate antigen, thus enhancing its presentation to lymphocytes. Antigens conveyed in the lymph percolate through the sinuses and penetrate the lymph nodules to initiate an immune response. Some antigens become trapped on the surface of the follicular dendritic cells, whereas others are processed by macrophages, dendritic cells, and B cells, leading to activation and differentiation of B cells into antibody-producing plasma cells and memory B cells.

1	The plasma cells then migrate to the medullary cords where they synthesize and release specific antibodies into the lymph flowing through the sinuses. Plasma cells account for 1% to 3% of the cells in resting lymph nodules. Their number increases dramatically during an immune response, thereby increasing the amount of circulating immunoglobulins. Memory B cells may leave the lymph nodes and circulate to various regions throughout the body, where they can proliferate in response to subsequent exposure to their specific antigen. The presence of memory cells in various sites

1	FIGURE 14.23 • Photomicrograph of the deep cortex of a lymph node. This photomicrograph shows several longitudinally sectioned high endothelial venules (HEVs) as well as several that are seen in cross section (arrows). These vessels are lined by cuboidal endothelial cells. In some preparations, the walls of an HEV may be infiltrated with migrating lymphocytes, making it difficult to recognize. 400. Inset. The cross section of an HEV shown here at higher magnification reveals several lymphocytes (arrowheads) in the process of migrating from the HEV into the parenchyma of the lymph node. 640. throughout the body ensures a more rapid response to an antigen, the secondary response.

1	throughout the body ensures a more rapid response to an antigen, the secondary response. Lymph nodes in which lymphocytes are responding to antigens often enlarge, refecting formation of germinal centers and proliferation of lymphocytes. This phenomenon is often seen in the lymph nodes of the neck in response to nasal or oropharyngeal infection, and in axillary and inguinal regions because of infection in extremities. Lymphadenitis, a reactive (infammatory) lymph node enlargement, is a common complication of microbial infections. These enlarged lymph nodes are commonly referred to as swollen glands (see Folder 14.4).  FOLDER 14.4 Clinical Correlation: Reactive (Inflammatory) Lymphadenitis

1	 FOLDER 14.4 Clinical Correlation: Reactive (Inflammatory) Lymphadenitis Reactive (inﬂammatory) lymphadenitis refers to enlargement of the lymph nodes that is often secondary to bacterial and other microbial infections. Lymph nodes enlarge because of edema and hyperplasia of lymphatic nodules and their cellular components (Fig. F14.4.1). These include B lymphocytes, T lymphocytes, macrophages, and other antigen-presenting cells. In addition, infiltration of lymphatic sinuses by neutrophils is also prominent. In severe bacterial infections, lymphadenitis may be accompanied by lymphangitis, an inflammation of afferent lymphatic vessels that carry infected lymph into regional lymph nodes. Inflamed lymph vessels may be visible as red streaks under the skin in the affected area of lymphatic drainage. Common symptoms of acute lymphadenitis are swollen lymph nodes that are tender to palpation, fever, chills, loss of appetite, tachycardia, and general weakness. Lymph

1	Common symptoms of acute lymphadenitis are swollen lymph nodes that are tender to palpation, fever, chills, loss of appetite, tachycardia, and general weakness. Lymph FIGURE F14.4.1 • Photomicrograph of a lymph node with nodes are usually palpable and tender, with red discol reactive lymphadenitis. Section though a superficial cortex oration on the overlying skin. In severe cases of suppura-of the lymph node shows a hyperplastic germinal center (GC) tive necrosis (necrosis with pus formation), a fistula (false projecting toward the connective tissue capsule. The majority opening) may develop that allows pus to drain from the of pale staining cells within the germinal center are enlarged lymph node to the surface. represented by B lymphocytes and macrophages;

1	The most common microbial organisms that cause lym-accumulation of T lymphocytes forms a distinct mantle zone phadenitis are streptococcal and staphylococcal bacteria. that encircles the germinal center. 120. (Reproduced from Other less common organisms are viruses (as in mononu-Schwarting R, McKenzie S, Rubin R. Hematopathology. cleosis or rubella), protozoa, rickettsiae, fungi, and the In: Rubin R, Strayer DS [eds]: Rubin’s Pathology: Clinicotuberculosis bacilli. Tonsillitis, infections originating from pathologic Foundations of Medicine, 5th ed. Baltimore: Lippincott Williams & Wilkins, 2008.) teeth, and bacterial pharyngitis (sore throats) are the most common causes of lymphadenitis in the neck area. Generalized lymphadenopathy is typical for rheumatoid arthritis and is detected as an early sign of HIV infection. In chronic lymphadenitis, lymph nodes are enlarged, but they are usuxally not tender. The thymus is a lymphoepithelial organ located in the superior mediastinum.

1	The thymus is a lymphoepithelial organ located in the superior mediastinum. The thymus is a bilobed organ located in the superior mediastinum, anterior to the heart and great vessels. It develops bilaterally from the third (and sometimes also the fourth) branchial (oropharyngeal) pouch. During development, the epithelium invaginates, and the thymic rudiment grows caudally as a tubular projection of the endodermal epithelium into the mediastinum of the chest. The advancing tip proliferates and ultimately becomes disconnected from the branchial epithelium. Multipotential lymphoid stem cells (CFU-Ls) from the bone marrow are destined to develop into immunocompetent T cells that invade the epithelial rudiment and occupy spaces between the epithelial cells so that the thymus develops into a lymphoepithelial organ.

1	The thymus is fully formed and functional at birth. It persists as a large organ until about the time of puberty, when T-cell differentiation and proliferation are reduced and most of the lymphatic tissue is replaced by adipose tissue (involution). The organ can be restimulated under conditions that demand rapid T-cell proliferation. General Architecture of the Thymus Connective tissue surrounds the thymus and subdivides it into thymic lobules. The thymus possesses a thin connective tissue capsule from which trabeculae extend into the parenchyma of the organ. The capsule and trabeculae contain blood vessels, efferent (but not afferent) lymphatic vessels, and nerves. In addition to collagen fibers and fibroblasts, the connective tissue of the thymus contains variable numbers of plasma cells, granulocytes, lymphocytes, mast cells, adipose cells, and macrophages.

1	The trabeculae establish domains in the thymus called thymic lobules. They are not true lobules, but cortical caps over portions of the highly convoluted but continuous inner FIGURE 14.24 • Photomicrograph of an infant human thymus. This H&E preparation reveals multiple lobules separated by connective tissue trabeculae that extend into the organ from the surrounding capsule. Each lobule is composed of a dark-staining basophilic cortex and a lighter-staining and relatively eosinophilic medulla. The medulla is actually a continuous branching mass surrounded by the cortex. The cortex contains numerous densely packed lymphocytes, whereas the medulla contains fewer lymphocytes. Note that in some instances the medulla may bear a resemblance to germinal centers of lymphatic nodules (upper right and center left). Such isolated medullary profiles are continuous with the overall medullary tissue, but this continuity may not be seen within the plane of section. 25.

1	medullary tissue (Fig. 14.24 and Plate 41, page 487). In some planes of section, the “lobular” arrangement of the cortical cap and medullary tissue superficially resembles a lymphatic nodule with a germinal center, which often confuses students. Other morphologic characteristics (described below) allow positive identification of the thymus in histologic sections. The thymic parenchyma contains developing T cells in an extensive meshwork formed by epithelioreticular cells.

1	The thymic parenchyma contains developing T cells in an extensive meshwork formed by epithelioreticular cells. The outer portion of the parenchyma, the thymic cortex, is markedly basophilic in hematoxylin and eosin (H&E) preparations because of the closely packed developing T lymphocytes with their intensely staining nuclei. These T lymphocytes, also called thymocytes, occupy spaces within an extensive mesh-work of epithelioreticular cells (Fig. 14.25). Macrophages are also dispersed among the cortical cells. The developing T cells arise from CFU-Ls, which originate in bone marrow. As development proceeds in the thymus, the cells derived from CFU-Ls pass through a series of developmental stages that are reflected by their expression of different CD molecules.

1	As their name implies, epithelioreticular cells have features of both epithelial and reticular cells. They provide a framework for the developing T cells; thus, they correspond to the reticular cells and their associated reticular fibers in other lymphatic tissues and organs. Reticular connective tissue cells and their fibers, however, are not present in the thymic parenchyma. Epithelioreticular cells exhibit certain features characteristic of epithelium such as intercellular junctions and intermediate filaments. Six types of epithelioreticular cells are recognized on the basis of function: Three types in the cortex and three types in the medulla. Each type is designated by roman numerals. In the cortex, the following cell types are recognized.

1	 Type I epithelioreticular cells are located at the boundary of the cortex and the connective tissue capsule as well as between the cortical parenchyma and the trabeculae. They also surround the adventitia of the cortical blood vessels. In essence, type I epithelioreticular cells serve to separate the thymic parenchyma from the connective tissue of the organ. The occluding junctions between these cells reflect their function as a barrier that isolates developing T cells from the connective tissue of the organ—that is, capsule, trabeculae, and perivascular connective tissue.

1	 Type II epithelioreticular cells are located within the cortex. The transmission electron microscope (TEM) reveals maculae adherents (desmosomes) that join long cytoplasmic processes of adjacent cells. The cell body and cytoplasmic processes contain abundant intermediate filaments. Because of their processes, these cells are stellate. They have a large nucleus that stains lightly with H&E because of its abundant euchromatin. This nuclear feature allows the cell to be easily identified in the light microscope. Type II cells compartmentalize the cortex into isolated areas for the developing T cells. Unlike type I cells, type II cells express MHC I and MHC II molecules, which are involved in thymic cell education.

1	 Type III epithelioreticular cells are located at the boundary of the cortex and medulla. The TEM reveals occluding junctions between sheetlike cytoplasmic processes of adjacent cells. Like type I cells, type III epithelioreticular cells create a functional barrier—in this case, between the cortex and medulla. Like type II cells, they possess MHC I and MHC II molecules.  Macrophages reside within the thymic cortex and are responsible for phagocytosis of T cells that do not fulfill thymic education requirements. These T cells are programmed to die before leaving the cortex. Approximately 98% of the T cells undergo this apoptosis and are then phagocytosed by the macrophages. The macrophages in the cortex are difficult to identify in H&E preparations. However, the periodic acid– Schiff (PAS) reaction readily defines them because of the

1	FIGURE 14.25 • Photomicrograph of a human thymus. a. The cortex contains a dense population of small, maturing T cells that creates the dark staining of this region of the thymus. The medulla, in contrast, appears lighter. The medulla also contains the thymic corpuscles that stain with eosin and give it a further distinction. 120. b. This higher-magnification photomicrograph shows the medulla with a thymic corpuscle (left) and surrounding cells. Thymic corpuscles are isolated masses of closely packed, concentrically arranged type VI epithelioreticular cells; these cells exhibit flattened nuclei. The more central mass of the corpuscle contains fully keratinized cells. In addition to numerous lymphocytes, the micrograph also shows type V epithelioreticular cells (arrows), with their eosinophilic cytoplasm and large, pale-staining nuclei. 600. staining of their numerous large lysosomes. Accordingly, these macrophages are called PAS cells.

1	staining of their numerous large lysosomes. Accordingly, these macrophages are called PAS cells. Although the epithelioreticular cells of the thymic cortex play an important role in the development of immunocompetent T cells, recent evidence shows that T cells at the different stages of differentiation control the microarchitecture of the thymic epithelioreticular cells, a phenomenon called crosstalk. The developing lymphocytes and epithelioreticular cells thus influence each other during T-cell development. Thymic or Hassall’s corpuscles (derived from type VI epithelioreticular cells) are a distinguishing feature of the thymic medulla.

1	Thymic or Hassall’s corpuscles (derived from type VI epithelioreticular cells) are a distinguishing feature of the thymic medulla. The thymic medulla, the inner portion of the parenchyma, contains a large number of epithelioreticular cells and loosely packed T cells (Fig. 14.25). The medulla stains less intensely than the cortex because, like the germinal centers of lymph nodules, it contains mostly large lymphocytes. These lymphocytes have pale-staining nuclei and quantitatively more cytoplasm than small lymphocytes. Like the cortex, the medulla also contains three types of epithelioreticular cells:  Type IV epithelioreticular cells are located between the cortex and the medulla close to type III cells. They possess sheetlike processes with occluding junctions between adjacent cells as well as between them and type III cells. In cooperation with type III cells, they create the barrier at the corticomedullary junction.

1	 Type V epithelioreticular cells are located throughout the medulla. Like the type II cells located in the cortex, processes of adjacent cells are joined by desmosomes to provide the cellular framework of the medulla and to compartmentalize groups of lymphocytes. These nuclei contrast markedly with the densely staining lymphocyte nuclei.  Type VI epithelioreticular cells form the most characteristic feature of the thymic medulla, the thymic (Hassall’s) corpuscles (Fig. 14.26 and Plate 41, page 487). Thymic corpuscles are isolated masses of closely packed, concentrically arranged type VI epithelioreticular cells that exhibit flattened nuclei. TEM studies of these cells reveal keratohyalin granules, bundles of cytoplasmic intermediate filaments, and lipid droplets. The cells are joined by desmosomes. The center of a thymic corpuscle

1	FIGURE 14.26 • Electron micrograph of a thymic (Hassall’s) corpuscle. This relatively low-magnification electron micrograph shows some of the nuclei (N) and cytoplasm of the concentrically arranged epithelioreticular cells of a thymic (Hassall’s) corpuscle. Bundles of intermediate filaments, keratohyalin granules, and lipid droplets are also evident within the cytoplasm of the epithelioreticular cells. Fully keratinized cells (black layer) are present in the center of the thymic corpuscle. 5,000. (Courtesy of Dr. Johannes A. G. Rhodin.) may display evidence of keratinization, not a surprising feature for cells developed from oropharyngeal epithelium. Thymic corpuscles are unique, antigenically distinct, and functionally active multicellular components of the medulla. Although the function of thymic corpuscles is not fully understood, it is thought that thymic corpuscles produce interleukins (IL-4 and IL-7) that function in thymic differentiation and education of T lymphocytes.

1	Blood vessels pass from the trabeculae to enter the parenchyma of the thymus. Typically, the blood vessels enter the medulla from the deeper parts of the trabeculae and carry a sheath of connective tissue along with them. This perivascular connective tissue sheath varies in thickness. It is thicker around larger vessels and gradually becomes thinner around smaller vessels. Where it is thick, it contains reticular fibers, fibroblasts, macrophages, plasma cells, and other cells found in loose connective tissue; where it is thin, it may contain only reticular fibers and occasional fibroblasts. The blood–thymus barrier protects developing lymphocytes in the thymus from exposure to antigens.

1	The blood–thymus barrier protects developing lymphocytes in the thymus from exposure to antigens. Lymphocytes reaching the thymic cortex are prevented from contact with antigen by a physical barrier called the blood– thymus barrier (Fig. 14.27). The following components constitute the blood–thymus barrier between the T cells and the lumen of cortical blood vessels, from the lumen outward:  The endothelium lining the capillary wall is of the continuous type with occluding junctions. It is highly impermeable to macromolecules and is considered a major structural component of the barrier within the cortical parenchyma. The underlying basal lamina of endothelial cells and occasional pericytes are also part of the capillary wall.

1	FIGURE 14.27 • Schematic diagram of the blood–thymus barrier. The blood–thymus barrier consists of three major elements: (1) capillary endothelium and its basal lamina, (2) perivascular connective tissue space occupied by macrophages, and (3) type I epithelioreticular cells with their basal lamina. The perivascular connective tissue is enclosed between the basal lamina of the epithelioreticular cells and the endothelial cell basal lamina. These layers provide the necessary protection to the developing immature T cells and separate them from mature immunocompetent lymphocytes circulating in the bloodstream.  Macrophages residing in the surrounding perivascular connective tissue may phagocytose antigenic molecules that escape from the capillary lumen into the cortical parenchyma.

1	 Macrophages residing in the surrounding perivascular connective tissue may phagocytose antigenic molecules that escape from the capillary lumen into the cortical parenchyma.  Type I epithelioreticular cells with their occluding junctions provide further protection to the developing T cells. The epithelioreticular cells surround the capillary wall in the cortex; with their basal lamina, they represent another major structural component of the blood–thymus barrier. The thymus is the site of T-cell education. During fetal life, the thymus is populated by multipotential lymphoid stem cells that originate from the bone marrow and are destined to develop into immunocompetent T cells. Stem cell maturation and differentiation into immunocompetent T cells is called thymic cell education (Fig. 14.28). This process is characterized by the expression and deletion of specific surface CD antigens.

1	immunocompetent T cells is accomplished by the expression and deletion of specific surface CD DO NOT selection antigens. The CFU-L stem cells enter the medulla PASS of the thymus via a postcapillary venule and then type migrate to the periphery of the thymic lobule. The II erc presence of CD2 and CD7 molecules on the cell surface indicates an early stage of differentiation. This is followed by expression of the CD1

1	The expression of CD2 and CD7 molecules on the T-cell surface indicates an early stage of differentiation (doublenegative stage). The double-negative term refers to lack of both CD4 and CD8 molecules. This early stage is followed by the expression of the CD1 molecule, which indicates the middle stage of T-cell differentiation. As maturation progresses, the T-cells express TCRs, CD3, and both CD4 and CD8 molecules. This is the double-positive stage of T-cells differentiation. These cells are then presented with selfand foreign antigens by type II and III epithelioreticular cells. If the lymphocyte recognizes self-MHC molecules and selfor foreign antigen, it will survive, a process called positive selection. If not, the cell will die. Cells that pass the positive-selection test leave the cortex and enter the medulla. Here, they undergo another selection process in which cells that recognize self-antigen displayed by self-MHC are eliminated, a process called negative selection. The cells

1	and enter the medulla. Here, they undergo another selection process in which cells that recognize self-antigen displayed by self-MHC are eliminated, a process called negative selection. The cells that survive become either

1	FIGURE 14.28 • Schematic drawing of the major steps in thymic education. The process of multipotential lymphatic stem cell (CFU-L) molecule, indicating the middle stage of T-cell differentiation. As maturation progresses, the cells express TCRs, CD3, CD4, and CD8 molecules. These cells are then presented with epithelioreticular (erc) cells. If the lymphocyte recognizes self MHC and self or foreign antigen, then it will survive the selection (positive TCR selection); if not, death of the cell will occur. Cells that pass the positive selection test leave the cortex and enter the medulla. Here they undergo to self-antigen displayed by self MHC are typeV eliminated (negative selection). Cells that survive erc that selection then become either cytotoxic CD8 T lymphocytes or helper CD4 T lymphocytes.

1	CD8 These cells are now ready for the immune response; they leave the thymus from the medulla and enter the blood circulation. Hormonal substances secreted by type VI epithelioreticular cells within the thymic (Hassall’s) corpuscle promote the process of thymic cell education. Note the distribution of all six types of epithelioreticular cells. cytotoxic CD8 T lymphocytes (by losing CD4 and retaining CD8) or helper CD4 T lymphocytes (by losing CD8 and retaining CD4). This stage is called the single-positive stage of T-cells differentiation. Now the cells leave the thymus by passing from the medulla into the blood circulation. The process of thymic cell education is promoted by substances secreted by the epithelioreticular cells, including interleukins (IL-4 and IL-7), colony-stimulating factors, and interferon .

1	The spleen is about the size of a clenched fist and is the largest lymphatic organ. It is located in the upper left quadrant of the abdominal cavity and has a rich blood supply. The spleen filters blood and reacts immunologically to blood-borne antigens. The spleen has both morphologic and immunologic filtering functions. In addition to large numbers of lymphocytes, it contains specialized vascular spaces or channels, a meshwork of reticular cells and reticular fibers, and a rich supply of macrophages and dendritic cells. These contents allow the spleen to monitor the blood immunologically, much as the macrophages and dendritic cells of the lymph nodes monitor the lymph. The spleen is enclosed by a dense connective tissue capsule from which trabeculae extend into the parenchyma of the organ (Fig. 14.29). The connective tissue of the capsule

1	FIGURE 14.29 • Schematic diagram and photomicrograph of splenic structure. a. The substance of the spleen is divided into white pulp and red pulp. White pulp consists of a cylindrical mass of lymphocytes arranged around a central artery that constitutes the periarterial lymphatic sheath (PALS). Splenic nodules occur along the length of the PALS. When observed in cross section through part of the sheath that contains a nodule, the central artery appears eccentrically located with respect to the lymphatic mass. The red pulp consists of splenic sinuses surrounded by splenic cords (cords of Billroth). A capsule surrounds the spleen; trabeculae project from it into the substance of the spleen. Both capsule and trabeculae give the appearance of dense connective tissue infiltrated by numerous myofibroblasts. Blood vessels traverse the capsule and trabeculae before and after passage within the substance of the spleen. Lymphatic vessels originate in the white pulp near the trabeculae. b. This

1	Blood vessels traverse the capsule and trabeculae before and after passage within the substance of the spleen. Lymphatic vessels originate in the white pulp near the trabeculae. b. This low-magnification photomicrograph of the spleen reveals the same components shown in the previous drawing. Note the capsule with several trabeculae projecting into the substance of the spleen. In the center, there is a trabecula containing a trabecular vein through which blood leaves the organ. The red pulp constitutes the greater bulk of the splenic tissue. The white pulp contains lymphatic tissue that follows and ensheathes the central artery. Expansion of the white pulp creates the splenic nodules. 45.

1	and trabeculae contains myofibroblasts. These contractile cells also produce extracellular connective tissue fibers. In many mammals, the spleen holds large volumes of red blood cells in reserve. In these species, contraction in the capsule and trabeculae helps discharge stored red blood cells into the systemic circulation. The human spleen normally retains relatively little blood, but it has the capacity for contraction by means of the contractile cells in the capsule and trabeculae. The hilum, located on the medial surface of the spleen, is the site for the passage of the splenic artery and vein, nerves, and lymphatic vessels. The lymphatic vessels originate in the white pulp near the trabeculae and constitute a route for lymphocytes leaving the spleen.

1	Most of the spleen consists of splenic pulp. Splenic pulp, in turn, is divided into two functionally and morphologically different regions: white pulp and red pulp, based on the color of fresh sections. White pulp appears as circular or elongated whitish gray areas surrounded by red pulp. White pulp consists of a thick accumulation of lymphocytes surrounding an artery.

1	The white pulp consists of lymphatic tissue, mostly lymphocytes. In H&E–stained sections, white pulp appears basophilic because of the dense heterochromatin in the nuclei of the numerous lymphocytes (Plate 39, page 483). Branches of the splenic artery course through the capsule and trabeculae of the spleen and then enter the white pulp. Within the white pulp, the branch of the splenic artery is called the central artery. Lymphocytes that aggregate around the central artery constitute the periarterial lymphatic sheath (PALS). The PALS has a roughly cylindrical configuration that conforms to the course of the central artery. In cross sections, the PALS appears circular and may resemble a lymphatic nodule. The presence of the central artery, however, distinguishes the PALS from typical lymphatic nodules found in other sites. Nodules appear as localized expansions of the PALS and displace the central artery so that it occupies an eccentric rather than a central position.

1	The nodules are the territory of B lymphocytes; other lymphocytes of the PALS are chiefly T lymphocytes that surround the nodules. Thus, the PALS may be considered a thymus-dependent zone similar to the deep cortex of a lymph node. The nodules usually contain germinal centers, which, as in other lymphatic tissues, develop as B cells proliferate after their activation. In humans, germinal centers develop within 24 hours after antigen exposure and may become extremely large and visible with the naked eye. These enlarged nodules are called splenic nodules or Malpighian corpuscles (not to be confused with the renal corpuscles that have the same name). Red pulp contains large numbers of red blood cells that it filters and degrades.

1	Red pulp contains large numbers of red blood cells that it filters and degrades. Red pulp has a red appearance in the fresh state as well as in histologic sections because it contains large numbers of red blood cells (Plate 40, page 485). Essentially, red pulp consists of splenic sinuses separated by splenic cords (cords of Billroth). Splenic cords consist of the now-familiar loose meshwork of reticular cells and reticular fibers that contain large numbers of erythrocytes, macrophages, lymphocytes, dendritic cells, plasma cells, and granulocytes. Splenic macrophages phagocytose damaged red blood cells. The iron from destroyed red blood cells is used in the formation of new red blood cells; splenic macrophages begin the process of hemoglobin breakdown and iron reclamation. Megakaryocytes are also present in certain species, such as rodents and the cat, but not in humans except during fetal life.

1	The splenic or venous sinuses are special sinusoidal vessels lined by rod-shaped endothelial cells. The endothelial cells that line the splenic sinuses are extremely long. Their longitudinal axis runs parallel to the direction of the vessel (Fig. 14.30). There are few contact points between adjacent cells, thus producing prominent intercellular spaces. These spaces allow blood cells to pass readily into and out of the sinuses. Processes of macrophages extend between the endothelial cells and into the lumen of the sinuses to monitor the passing blood for foreign antigens.

1	The sinuses do not possess a continuous basal lamina. Strands of basal lamina loop around the outside of the sinus much like the hoops that loop around the staves of a barrel. These strands are at right angles to the long axes of the endothelial cells. This material stains with silver-containing reagents or with the PAS reaction (Plate 40, page 485). Neither smooth muscle nor pericytes are present in the wall of splenic sinuses. Reticular cell processes may extend to the basal side of the endothelial cells and are probably associated with the reticular fibers that appear to merge with the perisinusoidal loops of basal lamina. Blood fills both the sinuses and cords of the red pulp, often obscuring the underlying structures and making it difficult to distinguish between the cords and the sinuses in histologic sections. Circulation within red pulp allows macrophages to screen antigens in the blood.

1	Branches of the splenic artery enter the white pulp from the trabeculae. The central artery sends branches to the white pulp itself and to the sinuses at the perimeter of the white pulp called marginal sinuses (see Fig. 14.29). The central artery continues into the red pulp, where it branches into several relatively straight arterioles called penicillar arterioles. The penicillar arterioles then continue as arterial capillaries. Some arterial capillaries are surrounded by aggregations of macrophages and are thus called sheathed capillaries. Sheathed capillaries then empty directly into the reticular meshwork of the splenic cords rather than connecting to the endothelium-lined splenic sinuses. Blood entering the red pulp in this manner percolates through the cords and is exposed to the macrophages of the cords before returning to the circulation by squeezing through the walls of the splenic sinuses (Fig. 14.31). This type of circulation is referred to as open circulation, and it is the

1	of the cords before returning to the circulation by squeezing through the walls of the splenic sinuses (Fig. 14.31). This type of circulation is referred to as open circulation, and it is the only route by which blood returns to the venous circulation in humans. In other species such as the rat and dog, some of the blood from the sheathed capillaries passes directly to the splenic sinuses of the red pulp. This type of circulation is referred to as closed circulation.

1	FIGURE 14.30 • Splenic sinus and splenic cord structure. a. This scanning electron micrograph shows a cross section of a splenic sinus (SS), revealing the lattice structure of its wall. Through the multiple openings in the wall, processes of macrophages (arrows) are inserted into the sinus lumen. The remainder of the micrograph shows characteristically smooth-surfaced processes of reticular cells (RC). The spaces of the reticular cell framework contain neutrophils (N), macrophages (M), and blood platelets (P). 4,400. b. Schematic diagram of the reconstructed structure of splenic sinus. Note the direction of blood flow in open and closed circulations. c.

1	b. Schematic diagram of the reconstructed structure of splenic sinus. Note the direction of blood flow in open and closed circulations. c. Scanning electron micrograph of the splenic sinus, showing the architecture of the sinus wall as seen from its luminal side. Rodlike endothelial cells run in parallel and are intermittently connected to each other by side processes. A nuclear swelling is shown at lower right. The tapered ends of a few of the rod cells are seen. The macrophage, neutrophil, and lymphocyte are outside the sinus. 5,300. (Reprinted with permission from Fujita T, Tanaka K, Tokunga J. SEM Atlas of Cells and Tissues. Tokyo: Igaku-Shoin, 1981.)

1	5,300. (Reprinted with permission from Fujita T, Tanaka K, Tokunga J. SEM Atlas of Cells and Tissues. Tokyo: Igaku-Shoin, 1981.) FIGURE 14.31 • Schematic diagram of open and closed splenic circulations. In the open circulation, which occurs in humans, penicillar arterioles empty directly into the reticular meshwork of the cords rather than connect to the endotheliumlined splenic sinuses. Blood entering the red pulp then percolates through the cords and is exposed to the macrophages residing there. In the closed circulation, which occurs in other species, the penicillar arterioles empty directly into the splenic sinuses of the red pulp. PALS, periarterial lymphatic sheath.

1	Open circulation exposes the blood more efficiently to the macrophages of the red pulp. Both transmission and scanning electron micrographs often show blood cells in transit across the endothelium of the sinus, presumably reentering the vascular system from the red pulp cords. The blood collected in the sinuses drains to tributaries of the trabecular veins that converge into larger veins and eventually leaves the spleen by the splenic vein. The splenic vein in turn joins the drainage from the intestine in the hepatic portal vein. The spleen performs both immune and hemopoietic functions.

1	The spleen performs both immune and hemopoietic functions. Because the spleen filters blood as the lymph nodes filter lymph, it functions in both the immune and the hemopoietic systems. Immune system functions of the spleen include  antigen presentation by APCs (mostly dendritic cells and macrophages) and initiation of immune response;  activation and proliferation of B and T lymphocytes;  production of antibodies against antigen present in circu lating blood; and removal of macromolecular antigens from the blood. Activation and proliferation of T cells and differentiation of B cells and plasma cells, as well as secretion of antibodies, occur in the white pulp of the spleen; in this regard, the white pulp is the equivalent of other lymphatic organs.

1	Hemopoietic functions of the spleen include:  removal and destruction of senescent, damaged, and abnormal erythrocytes and platelets;  retrieval of iron from erythrocyte hemoglobin;  formation of erythrocytes during early fetal life; and  storage of blood, especially red blood cells, in some species.

1	The role of the red pulp is primarily blood filtration (i.e., removal of particulate material, macromolecular antigens, and aged, abnormal, or damaged blood cells and platelets from the circulating blood). These functions are accomplished by the macrophages embedded in the reticular mesh-work of the red pulp. Senescent, damaged, or abnormal red cells are broken down by the lysosomes of the macrophages; the iron of the hemoglobin is retrieved and stored as ferritin or hemosiderin for future recycling. The heme portion of the molecule is broken down to bilirubin, which is transported to the liver via the portal system and there conjugated to glucuronic acid. Conjugated bilirubin is secreted into the bile, giving it a characteristic color. Macrophages recognize senescent or abnormal blood cells by several different mechanisms: become more rigid and are thus more easily trapped in the mesh of the red pulp.

1	Macrophages recognize senescent or abnormal blood cells by several different mechanisms: become more rigid and are thus more easily trapped in the mesh of the red pulp. Specific mechanisms include opsonization of the cell membrane with anti-band 3 IgG antibodies, which trigger Fc receptor–dependent phagocytosis of erythrocytes. In addition, specific changes in glycosylation of glycophorins (see page 274) in aging erythrocytes act as a recognition signal that triggers the elimination of senescent erythrocytes by macrophages. Despite these important functions, the spleen is not essential for human life. It can be removed surgically, which is often done after trauma that causes intractable bleeding from the spleen. The removal and destruction of aging red blood cells then occurs in the bone marrow and liver. This page intentionally left blank.

1	The palatine tonsils are paired structures consisting of masses of lymphatic tissue lo-cated on either side of the pharynx. They, along with the pharyngeal tonsils (adenoids) and lingual tonsils, form a ring at the entrance to the oropharynx (Waldeyer’s ring). Structurally, the tonsils contain numerous lymphatic nodules located in the mucosa. The stratified squamous epithelium that covers the surface of the palatine tonsil (and pharyngeal) dips into the underlying connective tissue forming many crypts, the tonsi-lar crypts. The walls of these crypts contain lymphatic nodules. The epithelial lining of the crypts is typically infiltrated with lymphocytes and often to such a degree that the epithelium may be difficult to discern. While the nodules principally occupy the con-nective tissue, the infiltration of lymphocytes into the epithelium tends to mask the ep-ithelial connective tissue boundary. The tonsils guard the opening of the pharynx, the common entry to the respiratory and

1	the infiltration of lymphocytes into the epithelium tends to mask the ep-ithelial connective tissue boundary. The tonsils guard the opening of the pharynx, the common entry to the respiratory and digestive tracts. The palatine and pharyngeal tonsils can become enflamed due to repeated infection in the oropharynx and na-sopharynx and can harbor bacteria that cause repeated infections if they are over-whelmed. When this occurs, the enflamed tonsils are removed surgically (tonsillectomy and adenoidectomy). Tonsils, like other aggregations of lymphatic nodules, do not possess afferent lymphatic vessels. Lymph, however, does drain from the tonsular lymphatic tissue through efferent lymphatic vessels. ORIENTATION MICROGRAPH: This low magnification micrograph is a section through a palatine tonsil. The hematoxylin staining areas represent the lymphatic tis-sue (L). The tonsil is surfaced by stratified squamous epithelium (SSE) which dips into the underlying connective tissue forming the

1	tonsil. The hematoxylin staining areas represent the lymphatic tis-sue (L). The tonsil is surfaced by stratified squamous epithelium (SSE) which dips into the underlying connective tissue forming the tonsilar crypts (TC). At the base of one of the crypts are a number of mucous secreting glands (MG). LLLLLLMGMGTCTCTCTCSSESSELLLMGTCTCSSE

1	Tonsil, human, H&E, ×47. This micrograph is from the area in the rectangle of the orientation micrograph. At this higher magnification, part of the surface epithelium (SE) of the tonsil can be readily identified. In other sites, the lymphocytes (Ly) have infiltrated the epithelium to such an extent that the epithelium is difficult to identify. The body of the nodules (N) lie within the mucosa and because of Tonsil, human, H&E, ×365.

1	Tonsil, human, H&E, ×365. At the higher magnification of this micrograph, the characteristic invasiveness of the lymphocytes into the overlying epithelium is readily evident. Note on the lower left side of the micrograph a clear boundary between the epithelium and the underlying lamina propria. The basal cells (BC) of the stratified squamous epithelium can be recognized. The underlying lamina propria is occupied by numerous lymphocytes; only a few have entered the epithelial compartment. Also note the thin band of collagen fibers (CF) that their close proximity, they tend to merge. Several of the nodules have been cut in a plane that includes their germinal center (GC). Note the eosinophilic staining in these areas. Beneath the nodules is the submucosa (S) consisting of dense connective tissue, which is continuous with the dense connective tissue beyond the tonsilar tissue.

1	can be seen at the boundary between the epithelium and lamina propria. In contrast, the lower right side of the micrograph displays numerous lymphocytes that have invaded the epithelium. More striking is the presence of what appear as isolated islands of epithelial cells (Ep) within the periphery. The thin band of collagen (C) lying at the interface of the epithelium is so disrupted in this area that it appears as small fragments. In effect, the small portion of the nodule seen in the right side of the micrograph has literally grown into the epithelium with the consequent disappearance of the well-defined epithelial-connective tissue boundary. KEY BC, basal cells C, collagen CF, collagen fibers Ep, islands of epithelial cells GC, germinal center L, lymphatic tissue Ly, lymphocytes MG, mucous secreting glands N, nodule S, submucosa SE, surface epithelium SSE, stratified squamous epithelium TC, tonsilar crypts

1	PLATE 37 • LYM PH NODE I Lymph nodes are small, encapsulated lymphatic organs that are located in the path of the lymph ves-sels. They serve as filters of the lymph and as the principal site in which T and B lymphocytes undergo antigen-dependent proliferation and differentiation into effector lymphocytes (plasma cells and T cells) and memory B cells and T cells. A low-magnification (×14) micrograph of a section through a human lymph node is shown on this page for orientation. The capsule appears as a thin connective tissue covering. The parenchyma of the node is composed of a mass of lymphatic tissue, arranged as a cortex (C) that surrounds a less dense area, the medulla (M). The cortex is interrupted at the hilum of the organ (H), where there is a recognizable concavity. It is at this site that blood vessels enter and leave the lymph node; the efferent lymphatic vessels also leave the node at the hilum. Afferent lymphatic vessels penetrate the capsule at multiple sites to empty into

1	that blood vessels enter and leave the lymph node; the efferent lymphatic vessels also leave the node at the hilum. Afferent lymphatic vessels penetrate the capsule at multiple sites to empty into an endothelium-lined space, the cortical or subcapsular sinus. This sinus drains into the trabecular sinuses that extend through the cortex alongside the trabeculae and then supply the medullary sinuses. These, in turn, drain to the efferent lymphatics that leave the node at the hilum. MM CC H

1	Cortex of lymph node, human, H&E ×120. An area from the cortex is shown here at higher magnification. The capsule (Cap) is composed of dense connective tissue from which trabeculae (T) penetrate into the organ. Immediately below the capsule is the cortical or subcapsular sinus (CS), which receives lymph from the afferent lymphatic vessels after they penetrate the capsule. The cortical sinus is continuous with the trabecular sinuses (TS) that course along the trabeculae. The cortex contains the lymphatic nodules (LN) and a deeper component that lacks nodules, known as the deep cortex. Whereas lymph nodules Lymphatic nodule, lymph node, human, H&E ×400; inset ×640.

1	This higher-magnification micrograph of a lymphatic nodule from figure above illustrates the germinal center (GC) containing medium and large lymphocytes. Germinal centers also contain plasma cells. Dividing lymphocytes are shown at slightly higher magnification in the inset (arrows), which corresponds to the area in the circle in this figure. The inset also reveals nuclei of the reticular cells (RC) that form the connective tissue stroma throughout the organ. The and their lighter-staining germinal centers characterize the outer cortex, a more dense mass of lymphocytes, which impart a distinct basophilia, characterize the deep cortex. In contrast to these areas, the medulla is characterized by narrow strands of anastomosing lymphatic tissue containing numerous lymphocytes, the medullary cords (MC), separated by light-appearing areas known as the medullary sinuses (MS). The medullary sinuses receive lymph from the trabecular sinuses and lymph filtered through the cortical tissue.

1	ovoid reticular cell has a large pale-staining nucleus, and its cytoplasm forms long processes that surround the reticular fibers. In H&E preparations, the reticular fibers and the surrounding cytoplasm are difficult to identify. Reticular cells are best seen in the sinuses, where they extend across the lymphatic space and are relatively unobscured by other cells. A unique vessel, the high endothelial venule (HEV), is found in relation to the lymphatic nodules, particularly in the deep cortex. These vessels have an endothelium composed of tall cells between which lymphocytes migrate from the vessel lumen into the parenchyma. KEY C, cortex Cap, capsule CS, cortical or subcapsular sinus GC, germinal center H, hilum LN, lymphatic nodule M, medulla MC, medullary cords MS, medullary sinus HEV, high endothelial venule RC, reticular cells T, trabecula TS, trabecular sinus arrows, dividing lymphocytes

1	Immunocompetent B cells that have been exposed to an antigen that they can recognize and bind migrate to a lymph node, where they undergo activation and begin a series of mitotic divisions that produce large numbers of immature lymphocytes. They proliferate further in superficial cortex into a clone of lymphocytes that differentiate into antibody-secreting plasma cells and memory cells. B cell proliferation and differentiation take place in germinal centers in the superficial cortex of the lymph node. T cell activation and differentiation take place in the deep cortex. Newly differentiated plasma cells migrate to the medulla, where they release antibodies into the lymph leaving the node. They may also leave the node, enter the blood vascular system at the thoracic duct, and travel to localized sites in the connective tissue where they may continue to produce antibodies. Deep cortex, lymph node, human, H&E ×365.

1	Deep cortex, lymph node, human, H&E ×365. This micrograph shows the lymph node deep cortex. As noted in the previous plate, it lies below the region containing the lymph nodules and consists of closely packed lymphocytes. A number of blood vessels can be seen in this region. Whereas typical small blood vessels such as capillaries (Cap) and480 venules are present, the more unusual postcapillary venule called high endothelial venule (HEV) is also found in this region. A small vessel that can be identified as a venule (Ven), based on lumen size and wall thickness, is seen at a point of transition to become a high endothelial venule (arrowheads). The endothelial cell nuclei at this point of juncture have become Hilar region, lymph node, human, H&E ×250.

1	The area shown here, near the hilar region of the node, shows part of a lymph nodule (LN), the cortical sinus (CS) just below the capsule (Caps), and some of the medullary sinus (MS). Both the cortical sinus and the medullary sinus are spanned by reticular cells (RC). These cells wrap around the collagen cuboidal. The high endothelial venule is identified by its endothelium, which is composed of cells that are cuboidal. A cross-sectioned profile of a postcapillary venule is shown in the inset at higher magnification (×700). The endothelial cell nuclei are round and are lightly stained, in contrast to the nuclei of the surrounding lymphocytes, which are of similar size and shape but are densely stained. This vessel also shows three lymphocytes (arrows) that are in the process of migrating through the wall of the vessel. The lower right corner of this figure reveals a region where there is a considerably lesser concentration of lymphocytes. This area, part of the medulla, contains

1	through the wall of the vessel. The lower right corner of this figure reveals a region where there is a considerably lesser concentration of lymphocytes. This area, part of the medulla, contains spaces that represent medullary sinuses (MS).

1	bundles that form the supporting trabecular framework of the node. The inset reveals the boxed area at higher magnification (×530). The nuclei of the reticular cells (RC) are larger and less densely staining than the lymphocyte nuclei, which are round and densely stained. In H&E preparations, these characteristics allow for the distinction between the reticular cell and the lymphocyte. KEY A, artery Cap, capillary Caps, capsule CS, cortical sinus LN, lymph nodule MS, medullary sinus HEV, high endothelial venule RC, reticular cells V, vein Val, valve Ven, venule arrowheads, endothelial cells of postcapillary venule arrows, Top image endothelial cells of HEV; Bottom image opening of medullary sinus to lymph vessel

1	The spleen is the largest lymphatic organ; it is surrounded by a capsule and located in the path of the bloodstream (splenic artery and vein). The spleen filters the blood and reacts immunologically to blood-borne antigens. It has both morphologic and immunologic filtering functions. The substance of the spleen, the splenic pulp, consists of red pulp and white pulp, so named because of their appearance in fresh tissue. The white pulp is rich in lymphocytes that form a periarterial lymphatic sheath (PALS) around branches of the splenic artery that penetrate the white pulp. The red pulp contains large numbers of red blood cells that it filters and degrades. Aged, damaged, or abnormal red blood cells are trapped by macrophages associated with unusual vascular sinuses in the red pulp. These macrophages break down the red cells, begin the metabolic breakdown of hemoglobin, and retrieve and store the iron from the heme for reutilization in the formation of new red blood cells in the bone

1	macrophages break down the red cells, begin the metabolic breakdown of hemoglobin, and retrieve and store the iron from the heme for reutilization in the formation of new red blood cells in the bone marrow.

1	Spleen, human, H&E ×65. This low-magnification micrograph of the spleen reveals its two major components, the red pulp (RP) and white pulp (WP). In the center of the figure, there is a trabecula containing a blood vessel, a trabecular vein (TV) through which blood leaves the organ. The red pulp constitutes the greater bulk of the splenic tissue. In life, the red pulp has pulp-like texture; it is red as a result of the natural coloration of the numerous red blood cells present, hence its name. The white pulp, on the other hand, is so named because its content of lymphocytes appears in life as whitish areas. In tissue sections, however, the nuclei of the closely packed lymphocytes impart an overall blue-staining Red pulp, spleen, human, H&E ×160.

1	Red pulp, spleen, human, H&E ×160. This figure reveals, at a higher magnification, the red pulp and a portion of the trabecular vein from the area enclosed in the uppermost rectangle in top figure. The red pulp is composed of two elements: venous sinuses (VS) and the splenic cords (of Billroth), the tissue that lies between the sinuses. In this speci men, the venous sinuses can be seen to advantage because the red blood cells in the sinuses have lysed and appear as unstained “ghosts”; only the nuclei of the white cells are readily seen. (This is better shown in Plate 40.) The paler, unstained areas thus represent the sinus lumina. response. The lymphatic tissue that constitutes the white pulp differs from nodules seen elsewhere in that it follows and ensheathes a blood vessel, the central artery. The lymphatic tissue surrounding the artery exhibits periodic expansion, thus forming the nodules. When this occurs, the central artery (CA) is displaced peripherally within the nodule.

1	In those regions where the lymphatic tissue is not in nodular form, it is present as a thin cuff around the central artery and is referred to as the periarterial lymphatic sheath. If the plane of section does not include the artery, the sheath may appear only as a localized and irregular aggregation of lymphocytes. Near the top of the micrograph, two venous sinuses (arrows) empty into the trabecular vein (TV), thus showing the continuity between venous sinuses and the trabecular veins. The wall of the vein is thin, but the trabecula (T) containing the vessel gives the appearance of being part of the vessel wall. In humans as well as in other mammals, the capsule and the trabeculae that extend from the capsule contain myofibroblasts. Under conditions of increasing physical stress, contraction of these cells will occur and cause rapid expulsion of blood from the venous sinuses into the trabecular veins and, thus, into the general circulation. White pulp, spleen, human, H&E ×240.

1	White pulp, spleen, human, H&E ×240. This figure reveals, at higher magnification, the splenic nodule in the rectangle in the right portion of figure above. Present are a germinal center (GC) and a cross section through the thick-walled central artery (CA). As noted above, the central artery is eccentrically placed in the nodule. The marginal zone (MZ) is the area that separates white pulp and red pulp (RP). Small arterial vessels and capillaries, branches of the central artery, supply the white pulp, and some pass into the reticular network of the marginal zone, terminating in a funnel-shaped orifice. Venous sinuses are also found in the marginal zone, and occasionally, arterial vessels may open into the sinuses. The details of the vascular supply are, at best, difficult to resolve in typical H&E preparations. The penicillar arterioles, the terminal branches of the central artery, supply the red pulp but are likewise difficult to resolve.

1	KEY CA, central artery GC, germinal center MZ, marginal zone RP, red pulp T, trabecula TV, trabecular vein VS, venous sinus WP, white pulp arrows, venous sinuses emptying into the trabecular vein Red pulp, spleen, human, H&E, ×360. As previously noted, the red pulp consists of the venous sinuses (VS) and the area between the venous sinuses, the splenic cords (of Bilroth) (SC). In this specimen, the red blood cells have been lysed leaving only a clear outline of the individual cells. Thus, the relatively clear spaces with scattered nuclei represent the lumen of the venous sinus; the nuclei are those of white blood cells. When the wall of a venous sinus is tangentially sectioned (VW), as in this figure, the endothelial cells, which are rod-like in shape, appear as a series of thin, linear bodies. Red pulp, spleen, human, H&E, ×1,200.

1	This micrograph is a high magnification of the area in the rectangle of the previous micrograph. The venous sinus in the center of the micrograph has been cut in cross section. Other than the lysed red blood cells, which appear as empty circular profiles, a number of lymphocytes (Ly) are present in the lumen. The wall of the sinus as seen here consists of rod-like endothelial cells (EC) that have been cut in cross section. A narrow, but clearly visible intercellular space is present between adjacent cells. These spaces allow blood cells to pass readily into and out of the sinuses. Also, processes of macrophages located outside of the sinuses in the splenic cords extend between the endothelial cells and into the lumen of the sinuses to monitor the passing blood for foreign antigens. The endothelial cell nuclei (ECN) project into the lumen of the vessel and appear to be sitting on top of the cell. A macrophage (M), identified by residual bodies in its cytoplasm, is seen just outside of

1	cell nuclei (ECN) project into the lumen of the vessel and appear to be sitting on top of the cell. A macrophage (M), identified by residual bodies in its cytoplasm, is seen just outside of the sinus.

1	Spleen, human, silver preparation, ×128. This micrograph shows a splenic nodule (SN) occupying the upper portion of the micrograph and below red pulp (RP). The components that can be identified are a germinal center (GC), a central artery (CA), and venus sinuses (VS) in the red pulp. The structural elements that are stained by the silver in the nodule consist of reticular fibers. Note their paucity within the germinal center. The fine, thread-like stained material that encircles the venous sinuses is a usual modification of basement membrane. Venous sinuses, spleen, human, silver preparation, vessel has been cut deeper along its long axis, the basement membrane ap×515. pears as dot-like structures (arrowheads). A three-dimensional reconstruc tion of the basement membrane would reveal it as a series of ring-like This micrograph reveals several venous sinuses (VS). Where structures.

1	the vessel wall has been tangentially sectioned the basement membrane appears as a ladder-like structure (BM). Where the KEY BM, basement membrane CA, central artery EC, rod-like endothelial cells ECN, endothelial cell nuclei GC, germinal center Ly, lymphocytes M, macrophage RP, red pulp SC, splentic cords SN, splenic nodule TV, trabecular vein VS, venous sinuses VW, venous sinus wall

1	The thymus is a lymphatic organ that exhibits certain unique structural features. The supporting reticular stroma arises from endodermal ep-ithelium and produces a cellular reticulum. There are no reticular fibers associated with these cells; instead, the cells, designated epithe-lioreticular cells, serve as the stroma. Lymphocytes come to lie in the interstices of the cellular reticulum, and these two cellular elements, the lymphocytes and the epithelioreticular cells, comprise the bulk of the organ. The stem lymphocytes that migrate into the endodermal rudiment in the embryo derive from the yolk sac and, later, from the red bone marrow. These lymphocytes proliferate and become immunologically com-petent in the thymus, differentiating into the thymus-dependent lymphocytes (i.e., T lymphocytes). Some of these lymphocytes migrate to other tissues to populate the thymus-dependent portions of lymph nodes and spleen as well as to reside in the loose connective tissue. Many lymphocytes die

1	Some of these lymphocytes migrate to other tissues to populate the thymus-dependent portions of lymph nodes and spleen as well as to reside in the loose connective tissue. Many lymphocytes die or are destroyed in the thymus because in the random process by which they acquire the ability to recognize and react to antigens they become programmed against “self” antigens. Numerous macrophages are present to phagocytize these destroyed lympho-cytes. A blood-thymus barrier is formed by the sheathing of the perivascular connective tissue of the thymus by the epithelioreticular cells. In addition, there are no afferent lymphatic vessels to the thymus. Thus, it cannot react to circulating antigens. The thymus involutes during ado-lescence and is often difficult to recognize in the adult. A connective tissue capsule (Cap) surrounds each lobe of the two lobes of the thymus and sends trabeculae (T) into the parenchyma to form lobules. The lobules are not completely separate units; rather, they

1	tissue capsule (Cap) surrounds each lobe of the two lobes of the thymus and sends trabeculae (T) into the parenchyma to form lobules. The lobules are not completely separate units; rather, they interconnect because of the discontinuous nature of the trabeculae.

1	Thymus, human, H&E ×40. whereas the medulla contains fewer lymphocytes and is consequently less densely packed. Examination of the thymus at low magnification reveals the lobules (L) composed of a dark-staining basophilic cortex (C) and a lighter-staining and relatively eosinophilic medulla (M). The cortex contains numerous densely packed lymphocytes, Thymus, human, H&E ×140.

1	Thymus, human, H&E ×140. It is the relative difference in the lymphocyte population (per unit area) and, in particular, the staining of their nuclei with hematoxylin that creates the difference in appearance between cortex (C) and medulla (M). Note that some of the medullary areas bear a resemblance to germinal centers of other lymphatic organs because of the medulla appearing as isolated circular areas (upper left of top figure). The medullary component, however, is actually a continuous branching mass surrounded by cortical tissue. Thus, the “isolated” medullary profiles are actually united with one another, although not within the plane of section. A suggestion of such continuity can be seen on the right in top figure where the medulla appears to extend across several lobules.

1	The main cellular constituents of the thymus are lymphocytes (thymocytes), with characteristic small, round, dark-staining nuclei, and epithelioreticular supporting cells, with large pale-staining nuclei. Both of the cell types can be distinguished in figure on right, which provides a high-magnification view of the medulla. Because there are fewer lymphocytes in the medulla, it is the area of choice to examine the epithelioreticular cells. The thymus also contains macrophages; however, they are difficult to distinguish from the epithelioreticular cells. Medulla, thymus, human, H&E ×600.

1	Medulla, thymus, human, H&E ×600. The medulla usually possesses varying numbers of circular bodies called Hassall’s, or thymic, corpuscles (HC). The corpuscles are large concentric layers of flattened type VI epithelioreticular cells (Ep). They stain readily with eosin and can be distinguished easily with low magnification, as in top figure and lower left (arrows). The center of a corpuscle, particularly a large one, may show evidence of keratinization and appear somewhat amorphous.

1	The thymus gland remains as a large structure until the time of puberty. At that time, regressive changes occur that result in a significant reduction in the amount of thymic tissue. The young thymus is highly cellular and contains a minimum of adipose tissue. On the other hand, in the older thymus, much adipose tissue is present between the lobules. With continued involution, adipose cells are found even within the thymic tissue itself. Occasional plasma cells may be present in the periphery of the thymic cortex of the involuting thymus gland. KEY BV, blood vessels C, cortex Cap, capsule Ep, epithelioreticular cells HC, Hassall’s corpuscles L, lobule M, medulla T, trabeculae arrowheads, nuclei of type VI epithelioreticular cells of Hassall’s corpuscles arrows, Hassall’s corpuscles

1	OVERVIEW OF THE INTEGUMENTARY SYSTEM / 488 LAYERS OF THE SKIN / 489 Epidermis / 489 Dermis / 491 CELLS OF THE EPIDERMIS / 493 Keratinocytes / 494 Melanocytes / 496 Langerhans’ Cells / 499 Merkel’s Cells / 500 STRUCTURES OF SKIN / 501 Nerve Supply / 501 Epidermal Skin Appendages / 503 Folder 15.1 Clinical Correlation: Cancers of Epidermal Origin / 492 Folder 15.2 Functional Considerations: Skin Color / 499 Folder 15.3 Functional Considerations: Hair Growth and Hair Characteristics / 504 Folder 15.4 Functional Considerations: The Role of Sebum / 505 Folder 15.5 Clinical Correlation: Sweating and Disease / 507 Folder 15.6 Clinical Correlation: Skin Repair / 512

1	The skin (cutis, integument) and its derivatives constitute the integumentary system. The skin forms the external covering of the body and is its largest organ, constituting 15% to 20% of its total mass. The skin consists of two main layers:  The epidermis is composed of a keratinized stratified squamous epithelium that grows continuously but maintains its normal thickness by the process of desquamation. Epidermis is derived from ectoderm.  The dermis is composed of a dense connective tissue that imparts mechanical support, strength, and thickness to the skin. Dermis is derived from mesoderm. The hypodermis contains variable amounts of adipose tissue arranged into lobules separated by connective tissue septa. It lies deep to the dermis and is equivalent to the subcutaneous fascia described in gross anatomy. In well-nourished individuals and in individuals living in cold climates, the adipose tissue can be quite thick.

1	The epidermal derivatives of the skin (epithelial skin appendages) include the following structures and integumentary products: The integumentary system performs essential functions related to its external surface location. Skin and its derivatives constitute a complex organ composed of many different cell types. The diversity of these cells and their ability to work together provide many functions that allow the individual to cope with the external environment. Major functions of the skin include the following:  It acts as a barrier that protects against physical, chemical, and biologic agents in the external environment (i.e., mechanical barrier, permeability barrier, ultraviolet barrier).  It provides immunologic information obtained during antigen processing to the appropriate effector cells in the lymphatic tissue.

1	 It provides immunologic information obtained during antigen processing to the appropriate effector cells in the lymphatic tissue.  It participates in homeostasis by regulating body temperature and water loss.  It conveys sensory information about the external environment to the nervous system.  It performs endocrine functions by secreting hormones, cytokines, and growth factors and converting precursor molecules into hormonally active molecules (vitamin D). It functions in excretion through the exocrine secretion of sweat, sebaceous, and apocrine glands.

1	In addition, certain lipid-soluble substances may be absorbed through the skin. Although not a function of skin, this property is frequently used to deliver therapeutic agents. For example, nicotine, steroid hormones, and seasickness medications are frequently delivered through the skin in the form of small sticking plasters or patches. To reduce nicotine withdrawal symptoms during smoking cessation, nicotine patches are often used to provide a small constant dose of nicotine without the dangerous effects of tobacco smoke. Skin is categorized as thick or thin, a reflection of thickness and location.

1	Skin is categorized as thick or thin, a reflection of thickness and location. The thickness of the skin varies over the surface of the body, from less than 1 mm to more than 5 mm. However, the skin is obviously both grossly and histologically different at two locations: The palms of the hands and the soles of the feet. These areas are subject to the most abrasion, are hairless, and have a much thicker epidermal layer than skin in any other location. This hairless skin is referred to as thick skin. Elsewhere, the skin possesses a much thinner epidermis and is called thin skin. It contains hair follicles in all but a few locations.

1	The terms thick skin and thin skin, as used in histologic description, are misnomers and refer only to the thickness of the epidermal layer. Anatomically, the thickest skin is found on the upper portion of the back where the dermis is exceedingly thick. The epidermis of the upper back, however, is comparable to that of thin skin found elsewhere on the body. In contrast, in certain other sites such as the eyelid, the skin is extremely thin.

1	The epidermis is composed of stratified squamous epithelium in which four distinct layers can be identified. In the case of thick skin, a fifth layer is observed (Figs. 15.1 and 15.2). Beginning with the deepest layer, these are as follow:  the stratum basale, also called the stratum germina tivum because of the presence of mitotically active cells, the stem cells of the epidermis; the stratum spinosum, also called the spinous layer or prickle cell layer because of the characteristic light microscopic appearance of short processes extending from cell to cell; the stratum granulosum, which contains numerous in tensely staining granules; the stratum lucidum, which is limited to thick skin and considered a subdivision of the stratum corneum; and the stratum corneum, which is composed of keratinized cells.

1	FIGURE 15.1 • Photomicrograph showing the layers of thin skin. This hematoxylin and eosin (H&E)–stained specimen from human skin shows the two chief layers of the skin—the epidermis (Epi) and dermis (Derm). The epidermis forms the surface; it consists of stratified squamous epithelium that is keratinized. The dermis consists of two layers: The papillary layer, which is the most superficial layer and is adjacent to the epidermis, and the more deeply positioned reticular layer. The boundary between these two layers is not conspicuous; the papillary layer is, however, more cellular than the reticular layer. In addition, the collagen fibers of the reticular layer are thick (clearly visible in the lower part of the figure); those of the papillary layer are thin. 45. Differentiation of epithelial cells constitutes a specialized form of apoptosis.

1	Differentiation of epithelial cells constitutes a specialized form of apoptosis. Terminal differentiation of the epidermal cells, which begins with the cell divisions in the stratum basale, is considered a specialized form of apoptosis. Cells in the stratum granulosum exhibit typical apoptotic nuclear morphology, including fragmentation of their DNA. However, the cellular fragmentation associated with normal apoptosis does not occur; instead, the cells become filled with filaments of the intracellular protein keratin and are later sloughed from the skin surface. The stratum basale provides for epidermal cell renewal. The stratum basale is represented by a single layer of cells that rests on the basal lamina (Plate 42, page 515). It contains the stem cells from which new cells, the keratinocytes, arise by mitotic division. For this reason, the stratum basale is also called the stratum germinativum. The cells are small and cuboidal to low columnar. They have less cytoplasm than the

1	FIGURE 15.2 • Photomicrograph showing the layers of thick skin. This specimen obtained from the skin of the sole of the foot (human) shows epidermis (Epi) containing the extremely thick stratum corneum (SC). Remaining layers of the epidermis (except for the stratum lucidum, which is not present on this slide)—that is, the stratum basale (SB), the stratum spinosum (SS), and the stratum granulosum (SGr)—are clearly visible in this routine H&E preparation. The duct of a sweat gland (D) can be seen on the left as it traverses the dermis (Derm) and further spirals through the epidermis. At the sites where the ducts of the sweat gland enter the epidermis, note the epidermal downgrowths known as interpapillary pegs. The dermis contains papillae, protrusions of connective tissue that lie between the interpapillary pegs. Note also the greater cellularity of the papillary layer (PL) and that the collagen fibers of the reticular layer (RL) are thicker than those of the papillary layer. 65.

1	cells in the layer above; consequently, their nuclei are more closely spaced. The closely spaced nuclei, in combination with the basophilic cytoplasm of these cells, impart a noticeable basophilia to the stratum basale. The basal cells also contain various amounts of melanin (described later) in their cytoplasm that is transferred from neighboring melanocytes interspersed in this layer. Basal cells exhibit extensive cell junctions; they are connected to each other and to keratinocytes by desmosomes and to the underlying basal lamina by hemidesmosomes. As new keratinocytes arise in this layer by mitotic division, they move into the next layer, thus beginning their process of upward migration. This process terminates when the cell becomes a mature keratinized cell, which is eventually sloughed off at the skin surface.

1	FIGURE 15.3 • Photomicrograph of the stratum spinosum and stratum basale. The epidermis of thin skin is shown here at higher magnification. The one-cell-deep layer at the base of the epidermis just above the connective tissue (CT ) of the dermis is the stratum basale (SB). The cells of this layer rest on the basement membrane. A layer referred to as the stratum spinosum (SS) is located just above the stratum basale. It consists of cells with spinous processes on their surfaces. These processes are attached to spinous processes of neighboring cells by desmosomes and together appear as intercellular bridges. 640. The cells of the stratum spinosum characteristically exhibit spinous processes.

1	The stratum spinosum is at least several cells thick. Keratinocytes in this layer are larger than those of the stratum basale. They exhibit numerous cytoplasmic processes or spines, which gives this layer its name (Fig. 15.3 and Plate 42, page 515). The processes are attached to similar processes of adjacent cells by desmosomes. In the light microscope, the site of the desmosome appears as a slight thickening called the node of Bizzozero. The processes are usually conspicuous, in part because the cells shrink during preparation and a resultant expanded intercellular space develops between the spines. Because of their appearance, the cells that constitute this layer are often referred to as prickle cells. As the cells mature and move to the surface, they increase in size and become flattened in a plane parallel to the surface. This arrangement is particularly notable in the most superficial spinous cells, where the nuclei also become elongate instead of ovoid, matching the acquired

1	in a plane parallel to the surface. This arrangement is particularly notable in the most superficial spinous cells, where the nuclei also become elongate instead of ovoid, matching the acquired squamous shape of the cells.

1	The cells of the stratum granulosum contain conspicuous keratohyalin granules. The stratum granulosum is the most superficial layer of the nonkeratinized portion of the epidermis. This layer varies from one to three cells thick. Keratinocytes in this layer contain numerous keratohyalin granules, hence the name of the layer. These granules contain cystine-rich and histidine-rich proteins, which are the precursors of the protein flaggrin, which aggregates the keratin flaments present within the cornified cells of the stratum corneum. Keratohyalin granules are irregular in shape and variable in size. Because of their intense basophilic staining, they are readily seen in routine histologic sections. The stratum corneum consists of anucleate squamous cells largely filled with keratin filaments.

1	The stratum corneum consists of anucleate squamous cells largely filled with keratin filaments. Usually, an abrupt transition occurs between the nucleated cells of the stratum granulosum and the flattened, desiccated, anucleate cells of the stratum corneum. The cells in the stratum corneum are the most differentiated cells in the skin. They lose their nucleus and cytoplasmic organelles and become filled almost entirely with keratin filaments. The thick plasma membrane of these cornified, keratinized cells is coated from the outside, in the deeper portion of this layer, with an extracellular layer of lipids that form the major constituent of the water barrier in the epidermis.

1	The stratum corneum is the layer that varies most in thickness, being thickest in thick skin. The thickness of this layer constitutes the principal difference between the epidermis of thick and thin skin. This cornified layer will become even thicker at sites subjected to unusual amounts of friction, as in the formation of calluses on the palms of the hand and on the fingertips. The stratum lucidum, considered a subdivision of the stratum corneum by some histologists, is normally only well seen in thick skin. In the light microscope, it often has a refractile appearance and may stain poorly. This highly refractile layer contains eosinophilic cells in which the process of keratinization is well advanced. The nucleus and cytoplasmic organelles become disrupted and disappear as the cell gradually fills with keratin. Attachment of epidermis to dermis is enhanced by an increased interface between the two tissues.

1	The junction between the dermis and epidermis is seen in the light microscope as an uneven boundary except in the thinnest skin. Sections of skin cut perpendicular to the surface reveal numerous fingerlike connective tissue protrusions, dermal papillae, that project into the undersurface of the epidermis (see Figs. 15.1 and 15.2). The papillae are complemented by what appear to be similar epidermal protrusions, called epidermal ridges or rete ridges, that project into the dermis. If the plane of section is parallel to the surface of the epidermis and passes at a level that includes the dermal papillae, however, the epidermal tissue appears as a continuous sheet of epithelium, containing circular islands of connective tissue within it. The islands are cross sections of true fingerlike dermal papillae that project into the epidermis. At sites where increased mechanical stress is placed on the skin, the epidermal ridges are much deeper (the epithelium is thicker), and the dermal papillae

1	papillae that project into the epidermis. At sites where increased mechanical stress is placed on the skin, the epidermal ridges are much deeper (the epithelium is thicker), and the dermal papillae are much longer and more closely spaced, creating a more extensive interface between the dermis and epidermis. This phenomenon is particularly well demonstrated in histologic sections that show both palmar and dorsal surfaces of the hand, as in a section of a finger.

1	True dermal ridges are present in thick skin in addition to dermal papillae. Dermal ridges tend to have a parallel arrangement, with the dermal papillae located between them. These ridges form a distinctive pattern that is genetically unique to each individual and is reflected in the appearance of epidermal grooves and ridges on the surface of the skin. These patterns are the basis of the science of dermatoglyphics, or fingerprint and footprint identification. The dermal ridges and papillae are most prominent in the thick skin of the palmar and plantar surfaces. Here, the basal surface of the epidermis greatly exceeds its free surface. The germinal layer is thus spread over a large area; assuming a near-constant rate of mitosis in the stratum germinativum, more cells per unit time enter the stratum corneum in thick skin than in thin skin. These additional cells are thought to account for the greater thickness of the cornified layer in thick skin.

1	Hemidesmosomes strengthen the attachment of the epidermis to the underlying connective tissue. When studied with the transmission electron microscope (TEM), the basal surface of the basal epidermal cells exhibits a pattern of irregular cytoplasmic protrusions that increase the attachment surface between the epithelial cell and its subjacent basal lamina. A series of hemidesmosomes link the intermediate filaments of the cytoskeleton into the basal lamina. In addition, focal adhesions that anchor actin filaments into the basal lamina are also present. These specialized anchoring junctions are discussed on page 144. The dermis is composed of two layers: The papillary layer and the reticular layer.

1	The dermis is composed of two layers: The papillary layer and the reticular layer. Examination of the full thickness of the dermis at the light microscope level reveals two structurally distinct layers.  The papillary layer, the more superficial layer, consists of loose connective tissue immediately beneath the epidermis (Plate 43, page 517). The collagen fibers located in this part of the dermis are not as thick as those in the deeper portion. I and type III collagen molecules. Similarly, the elastic fibers are threadlike and form an irregular network. The papillary

1	Three major types of skin cancer originate from cells in the epidermis. In general, skin cancer is caused by unpro-tected, long-term exposure to the sun’s ultraviolet radiation. The most common type is the basal cell carcinoma, which microscopically, as its name implies, resembles cells from the stratum basale of the epidermis. Basal cell carcinoma is a slow-growing tumor that usually does not metastasize. Typically, the cancer cells arise from the follicular bulge of the outer root sheath of the hair follicle. Almost in all cases of basal cell carcinoma, the recommended treatment is surgical removal of the tumor. The second most common skin cancer is the squa-mous cell carcinoma with more than 200,000 cases each year. Individuals with this form of cancer usually develop a small painless nodule or patch that is surrounded by an area of inflammation. Squamous cell carcinoma is charac-terized by highly atypical cells at all levels of the epidermis (carcinoma in situ). Disruption of the

1	nodule or patch that is surrounded by an area of inflammation. Squamous cell carcinoma is charac-terized by highly atypical cells at all levels of the epidermis (carcinoma in situ). Disruption of the basement membrane results in spread (metastasis) of tumor cells to the lymph nodes. Squamous cell carcinoma is known for variable dif-ferentiation patterns ranging from polygonal squamous cells arranged in orderly lobules and zones of keratinization to rounded cells with foci of necrosis and occasional single keratinized cells. Treatment for squamous cell carcinoma depends on histological type, size, and location. It may in-clude surgical excision, curettage and electrodesiccation, cryotherapy (freezing with liquid nitrogen), or chemoor ra-diotherapy. Moh’s micrographic surgical procedure is being used for locally recurrent skin cancers. This proce-dure involves shaving away one-by-one thin layers of epi-dermis and examining them under a microscope for the presence of malignant cells.

1	is being used for locally recurrent skin cancers. This proce-dure involves shaving away one-by-one thin layers of epi-dermis and examining them under a microscope for the presence of malignant cells. When the shaving is cancer free, the surgery is complete. This method preserves as many unaffected skin layers as possible while making cer-tain that all cancer cells are removed. Malignant melanoma is the most serious form of skin cancer if not recognized at an early stage and surgically removed. Individual melanoma cells, which originate from melanocytes, contain large nuclei with irregular contours and prominent eosinophilic nucleoli. These cells either aggregate in nests or are scattered through the entire thick-ness of the epidermis (Fig F15.1.1). They may reside only in the epidermis (melanoma in situ) or extend into the underly-ing papillary layer of the dermis. With time, the melanoma undergoes a radial growth phase. The melanocytes grow in all directions, upward in the epidermis,

1	in situ) or extend into the underly-ing papillary layer of the dermis. With time, the melanoma undergoes a radial growth phase. The melanocytes grow in all directions, upward in the epidermis, downward into the dermis, and peripherally in the epidermis. At this early stage, the melanoma tends not to metastasize. On the skin surface, it presents itself as an irregularly pigmented multicolor lesion, appearing black with parts brown to light brown, and a mixture of pink to red or shades of blue (Fig. F15.1.2). In time (approximately 1 to 2 years), melanocytes exhibit mitotic activity and form round nodules growing perpendicularly to the surface of the skin. In this vertical growth phase, the melanocytes display little or no pigment and usually metastasize into regional lymph nodes.

1	 FOLDER 15.1 Clinical Correlation: Cancers of Epidermal Origin FIGURE F15.1.1 • Photomicrograph of a malignant melanoma lesion in the early stage of radial growth phase. This section of the skin shows a layer of the epidermis containing atypical (hyperplasic) cells loaded with dark-brown pigment granules containing melanin. These cells represent atypical melanocytes that normally should reside in the stratum basale of the epidermis. At this stage of disease, these abnormal melanocytes migrate to the upper layers of the epidermis (melanocytic hyperplasia). There are scattered small nests of atypical cells in the dermis. Note the accumulation of lymphocytes in the superficial dermis. 320. Inset shows enlarged nest containing melanocytes with clearly visible processes containing melanin granules. 640. continued next page FOLDER 15.1 Clinical Correlation: Cancers of Epidermal Origin (Cont.)

1	FOLDER 15.1 Clinical Correlation: Cancers of Epidermal Origin (Cont.) FIGURE F15.1.2 • Photograph of the skin with malignant melanoma during radial growth phase. In this individual, malignant melanoma is represented by the relatively flat, irregularly pigmented multicolor lesion. The largest nodule appears ebony black. It is adjacent to a slightly elevated region that is colored in shades from dark brown to light brown with two reddish smaller nodules in pink coloration. At this early stage, melanocytes grow in all directions, upward in the epidermis, downward into the dermis, and peripherally in the epidermis. (Reproduced from Storm CA, Elder DE. The Skin. In: Rubin R, Strayer DS (eds): Rubin’s Pathology: Clinicopathologic Foundations of Medicine, 5th ed. Baltimore: Lippincott Williams & Wilkins, 2008.)

1	The ABCD rule is helpful for remembering the signs and symptoms of melanoma (see Fig F15.1.2): shape of skin lesion•Border of the lesion is irregular•Color variations; melanomas usually have multiple colors of skin lesion; moles greater than 6 mm are more likely to be suspicious layer is relatively thin and includes the substance of the dermal papillae and dermal ridges. It contains blood vessels that serve but do not enter the epidermis. It also contains nerve processes that either terminate in the dermis or penetrate the basal lamina to enter the epithelial compartment. Because the blood vessels and sensory nerve endings are concentrated in this layer, they are particularly apparent in the dermal papillae.

1	 The reticular layer lies deep to the papillary layer. Although its thickness varies in different parts of the body, it is always considerably thicker and less cellular than the papillary layer. It is characterized by thick, irregular bundles of mostly type I collagen and by coarser elastic fibers. The collagen and elastic fibers are not randomly oriented but form regular lines of tension in the skin called Langer’s lines. Skin incisions made parallel to Langer’s lines heal with the least scarring. In the skin of the areolae, penis, scrotum, and perineum, smooth muscle cells form a loose plexus in the deepest parts of the reticular layer. This arrangement accounts for the puckering of the skin at these sites, particularly in erectile organs. Layers of adipose tissue, smooth muscle, and, in some sites, striated muscle may be found just beneath the reticular layer.

1	Layers of adipose tissue, smooth muscle, and, in some sites, striated muscle may be found just beneath the reticular layer. Deep to the reticular layer is a layer of adipose tissue, the panniculus adiposus, which varies in thickness. This layer Surgery is a choice of treatment for localized malignant melanoma of the skin. A multidisciplinary approach is used for advanced malignant melanoma, including surgery combined with chemotherapy or immunotherapy with adjuvant treatment. serves as a major energy storage site and also provides insulation. It is particularly thick in individuals who live in cold climates. This layer and its associated loose connective tissue constitute the hypodermis or subcutaneous fascia.

1	Individual smooth muscle cells or small bundles of smooth muscle cells that originate in this layer form the arrector pili muscles that connect the deep part of hair follicles to the more superficial dermis. Contraction of these muscles in humans produces the erection of hairs and puckering of skin called “goose flesh.” In other animals, the erection of hairs serves in both thermal regulation and fright reactions. A thin layer of striated muscle, the panniculus carnosus, lies deep to the subcutaneous fascia in many animals. Although largely vestigial in humans, it remains well defined in the skin of the neck, face, and scalp, where it constitutes the platysma muscle and the other muscles of facial expression.

1	The cells of the epidermis consist of four different cell types:  Keratinocytes are highly specialized epithelial cells de signed to perform a very specific function: Separation of the organism from its external environment.  Melanocytes are the pigment-producing cells of the epi dermis.  Langerhans’ cells are involved in signaling in the im mune system. Merkel’s cells are associated with sensory nerve endings. The keratinocyte is the predominate cell type of the epidermis. These cells originate in the stratum basale of the epidermis. On leaving this layer, keratinocytes assume two essential activities:  They produce keratins (cytokeratins), major heteropoly meric structural proteins of the epidermis (see Table 2.3, page 64). Keratins form intermediate filaments; they con stitute almost 85% of fully differentiated keratinocytes.  They participate in the formation of the epidermal water barrier.

1	The keratinocytes in the basal layer contain numerous free ribosomes, scattered 7to 9-nm intermediate (keratin) filaments, a small Golgi apparatus, mitochondria, and rough-surfaced endoplasmic reticulum (rER). The cytoplasm of immature keratinocytes appears basophilic in histologic sections because of the large number of free ribosomes, most of which are engaged in the synthesis of keratins, which will later be assembled into keratin flaments. These filaments are classified as intermediate filaments, although they are more commonly called tonoflaments. As the cells enter and are moved through the stratum spinosum, the synthesis of keratin filaments continues, and the filaments become grouped into bundles sufficiently thick to be visualized in the light microscope. These bundles are called tonofbrils. The cytoplasm becomes eosinophilic because of the staining reaction of the tonofibrils that fill more and more of the cytoplasm.

1	Keratohyalin granules contain intermediate filament– associated proteins that aid in the aggregation of keratin filaments.

1	In the upper part of the stratum spinosum (Fig. 15.4), the free ribosomes within the keratinocytes begin to synthesize keratohyalin granules that become the distinctive feature of the cells in the stratum granulosum (Plate 42, page 515). Keratohyalin granules contain the two major intermediate filament–associated proteins, filaggrin and trichohyalin. The appearance of the granules and expression of filaggrin in the keratinocytes are often used as a clinical marker for the initiation of the final stage of apoptosis. As the number of granules increases, the contents of the granules are released into the keratinocyte cytoplasm. Filaggrin and trichohyalin function as promoters in the aggregation of keratin filaments into tonofibrils, thus initiating the conversion of granular cells into cornified cells. This process is called keratinization and occurs in 2 to 6 hours, the time it takes for the cells to leave the stratum granulosum and enter the stratum corneum. The keratin fibril formed

1	cells. This process is called keratinization and occurs in 2 to 6 hours, the time it takes for the cells to leave the stratum granulosum and enter the stratum corneum. The keratin fibril formed in this process is called soft keratin in contrast to the hard keratin of hair and nails (see below).

1	The transformation of a granular cell into a keratinized cell also involves breakdown of the nucleus and other organelles and thickening of the plasma membrane. This is accompanied by a change in pH, which decreases from approximately neutral (pH 7.17) in the stratum granulosum to acidic at the surface of the stratum corneum, ranging between pH 4.5 and 6.0. Desquamation of surface keratinocytes from the stratum corneum is regulated by proteolytic degradation of the cells’ desmosomes.

1	Cells are regularly exfoliated or desquamated from the surface of the stratum corneum. The continuous exfoliation of surface keratinocytes is a regulated proteolytic process that involves degradation of the cells’ desmosomes. The human kallikrein-related serine peptidases such as KLK5, KLK7, and KLK14 cause cleavage of desmosomes in a pH-dependent manner. A physiological serine protease inhibitor, lymphoepithelial Kazal-type inhibitor (LEKTI), through its interactions with KLKs in neutral pH, prevents desmosomal cleavage. However, as pH decreases in more superficial portions of the stratum corneum as described, LEKTI progressively releases KLKs at the lower pH, thus allowing KLKs to degrade the desmosomes and cause keratinocyte release (see Fig 15.4). Under normal condition, the process allows a controlled renewal of the epidermis by means of its pH gradient. Recently, pathogenic mutations were identified in the gene called serine protease inhibitor Kazal-type 5 (SPINK5), which

1	allows a controlled renewal of the epidermis by means of its pH gradient. Recently, pathogenic mutations were identified in the gene called serine protease inhibitor Kazal-type 5 (SPINK5), which encodes LEKTI. Netherton syndrome, a rare genetic disorder associated with a defective SPINK5 gene, is characterized by decreased skin barrier function, generalized redness of the skin (erythroderma), and scaling.

1	Lamellar bodies contribute to the formation of the inter-cellular epidermal water barrier. An epidermal water barrier is essential for mammalian “dry” epithelia and is responsible for maintaining body homeostasis. The barrier is established primarily by two factors in terminally differentiating keratinocytes: (1) deposition of insoluble proteins on the inner surface of the plasma membrane and (2) a lipid layer that is attached to the outer surface of the plasma membrane.

1	As the keratinocytes in the stratum spinosum begin to produce keratohyalin granules, they also produce membrane-bounded lamellar bodies (membrane-coating granules). They are tubular or ovoid-shaped membrane-bound organelles that are unique to mammalian epidermis. Spinous and granular cells synthesize a heterogenous mixture of pro-barrier lipids and their respective lipid-processing enzymes such as glycosphingolipids, phospholipids, ceramides, acidic sphingomyelinase, and secretory phospholipase A2 (Fig. 15.5); this mixture is assembled into lamellar bodies in the Golgi apparatus. In addition, lamellar bodies contain proteases (i.e., SC chymotryptic enzyme, cathepsin D, acid phosphatase, glycosidases, protease inhibitors). The contents of the granules are then secreted by exocytosis into the intercellular spaces between the stratum granulosum and stratum corneum. The organization of these intercellular lipid lamellae is responsible for the formation of the epidermal water barrier

1	the intercellular spaces between the stratum granulosum and stratum corneum. The organization of these intercellular lipid lamellae is responsible for the formation of the epidermal water barrier (Fig. 15.6). In addition to their major role in the formation of barrier homeostasis, lamellar bodies are also pH 4.5–5.3pH 6.8–7.5

1	FIGURE 15.4 • Schematic diagram of keratinocytes in the epidermis. The keratinocytes in this figure reflect different stages in the life cycle of the cell as it passes from the basal layer to the skin surface, where it becomes desquamated. The basal cell begins to synthesize intermediate (keratin) filaments; these are grouped into bundles and are seen in the light microscope as tonofibrils. The cell enters the spinous layer, where the synthesis of intermediate filaments continues. In the upper part of the spinous layer, the cells begin to produce keratohyalin granules containing intermediate filament–associated proteins and glycolipid-containing lamellar bodies. Within the granular layer, the cell discharges lamellar bodies that contribute to formation of the water barrier of the epidermis; the remainder of the cell cytoplasm contains numerous keratohyalin granules that, in close association with tonofilaments, form the cell envelope. The surface cells are keratinized; they contain a

1	the remainder of the cell cytoplasm contains numerous keratohyalin granules that, in close association with tonofilaments, form the cell envelope. The surface cells are keratinized; they contain a thick cell envelope and bundles of tonofilaments in a specialized matrix. Desquamation of keratinized cells is controlled by pH that monitors KLK activity and its interaction with LEKTI. Keratinocytes positioned near the granular layer exhibit neutral pH, which maintains desmosomal interactions and allows a strong interaction in the extracellular matrix between LEKTI and its KLK targets. As the pH acidifies toward the skin surface, LEKTI and KLK dissociate, allowing proteinase to activate and search for other protein targets in the extracellular space. In the most superficial layers of keratinocytes, pH is low enough for active KLK molecules to digest desmosomal proteins. In concert with other proteinase activities, this action leads to a complete degradation of desmosomal junctions,

1	pH is low enough for active KLK molecules to digest desmosomal proteins. In concert with other proteinase activities, this action leads to a complete degradation of desmosomal junctions, resulting in the detachment of the most superficial layer of keratinocytes. rER, rough-surfaced endoplasmic reticulum.

1	FIGURE 15.5 • Schematic diagram of the epidermal water barrier. The heterogeneous mixture of glycosphingolipids, phospholipids, and ceramides makes up the lamellae of the lamellar bodies. The lamellar bodies, produced within the Golgi apparatus, are secreted by exocytosis into the intercellular spaces between the stratum granulosum and stratum corneum, where they form the lipid envelope. The lamellar arrangement of lipid molecules is depicted in the intercellular space just below the thickened plasma membrane and forms the cell envelope of the keratinized keratinocyte. The innermost part of the cell envelope consists primarily of loricrin molecules (pink spheres) that are cross-linked by small proline-rich (SPR) proteins and elafin. The layer adjacent to the cytoplasmic surface of the plasma membrane consists of the two tightly packed proteins involucrin and cystatin . Keratin filaments (tonofilaments) bound by filaggrin are anchored into the cell envelope.

1	involved in formation of the cornified envelope, desquamation of cornified cells, and antimicrobial defenses in the skin. The epidermal water barrier thus consists of two structural elements:  The cell envelope (CE) is a 15-nm-thick layer of insoluble proteins deposited on the inner surface of the plasma membrane that contributes to the strong mechanical properties of the barrier. The thickness of the CE increases in epithelia that are subject to considerable mechanical stress (e.g., lip, palm of the hand, sole of the foot). The CE is formed by cross-linking small prolinerich (SPR) proteins and larger structural proteins. The structural proteins include cystatin, desmosomal proteins (desmoplakin), elafn, envoplakin, flaggrin, involucrin, five different keratin chains, and loricrin. Loricrin is the major structural protein and accounts for almost 80% of the total CE protein mass. This 26-kilodalton insoluble protein has the highest glycine content of any known protein in the body.

1	 The lipid envelope is a 5-nm-thick layer of lipids attached to the cell surface by ester bonds. The major lipid components of the lipid envelope are ceramides, which belong to the class of sphingolipids; cholesterol; and free fatty acids. However, the most important component is the monomolecular layer of acylglucosylceramide, which provides a “Teflon-like” coating on the cell surface. Ceramides also play an important role in cell signaling and are partially responsible for inducing cell differentiation, triggering apoptosis, and reducing cell proliferation. As the cells continue to move toward the free surface, the barrier is constantly maintained by keratinocytes entering the process of terminal differentiation. Lamellae may remain as recognizable discs in the intercellular space or may fuse into broad sheets or layers.

1	Experiments have shown that the epidermis of animals with induced essential fatty acid defciency (EFAD) is more permeable than normal to water. The membrane-coating granules also have fewer lamellae than normal. Destruction of the epidermal barrier over large areas, as in severe burns, can lead to life-threatening loss of ﬂuid from the body. Neural crest–derived melanocytes are scattered among the basal cells of the stratum basale.

1	Neural crest–derived melanocytes are scattered among the basal cells of the stratum basale. During embryonic life, melanocyte precursor cells migrate from the neural crest and enter the developing epidermis. A specific functional association is then established—the epidermal-melanin unit—in which one melanocyte maintains an association with a given number of keratinocytes. This ratio varies in different parts of the body. In adults, a pool of undifferentiated melanocyte stem cells resides in the area of the hair follicle called the follicular bulge. Differentiation of the melanocyte stem cell is regulated by the expression of the Pax3 gene that belongs to the paired box (PAX) family of transcription factors. Pax3 activates expression of the microphthalmia transcription factor (MITF), which is critical for the development and differentiation of melanocytes (melanogenesis).

1	The epidermal melanocyte is a dendritic cell that is scattered among the basal cells of the stratum basale (Fig. 15.7). They are called dendritic cells because the rounded cell body resides in the basal layer and extends long processes between the keratinocytes of the stratum spinosum. Neither the processes nor the cell body forms desmosomal attachments with neighboring keratinocytes. However, melanocytes that reside close to the basal lamina have structures that resemble hemidesmosomes. The ratio of melanocytes to keratinocytes or their precursors in the basal layer ranges from 1:4 to 1:10 in different parts of the body and is constant in all races. In routine hematoxylin and eosin (H&E) preparations, melanocytes are seen in the stratum basale with elongated nuclei surrounded by a clear cytoplasm. With the TEM, however, they are readily identified by the developing and mature melanin granules in the cytoplasm (see Fig. 15.7). Melanocytes maintain the capacity to replicate throughout

1	cytoplasm. With the TEM, however, they are readily identified by the developing and mature melanin granules in the cytoplasm (see Fig. 15.7). Melanocytes maintain the capacity to replicate throughout their life, although at a much slower rate than keratinocytes, thus maintaining the epidermal-melanin unit.

1	FIGURE 15.6 • Electron micrographs of keratinocytes. a. Much of the keratinocyte cytoplasm is filled with tonofilaments. One keratinocyte exhibits a keratohyalin granule (KG). Near the plasma membrane closest to the surface (upper left), two keratinocytes display lamellar bodies (arrowheads). 8,500. b. A lamellar body at higher magnification. 135,000. c. Part of a keratinized cell and the underlying keratinocyte. Located between the cells are the contents of the lamellar bodies, which have been discharged into the intercellular space (arrow) to form the lipid envelope. 90,000. (Courtesy of Dr. Albert I. Farbman.) Melanocytes produce and distribute melanin into keratinocytes.

1	The epidermal melanocytes produce and secrete the pigment melanin. The most important function of melanin is to protect the organism against the damaging effects of non-ionizing ultraviolet irradiation. Melanin is produced by the oxidation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) by tyrosinase and the subsequent transformation of DOPA into melanin. These reactions initially occur in membrane-bounded structures called premelanosomes, which are derived from the Golgi apparatus (Fig. 15.8). Premelanosomes and the early melanosomes, which have a low melanin content, exhibit a finely ordered internal structure with the TEM, reflecting their content of tyrosinase molecules. As more melanin is produced by the oxidation of tyrosine, the internal structure of the premelanosome becomes obscured until the mature melanin granule, the melanosome, is formed and then appears as an electron-opaque granule. Premelanosomes are concentrated near the Golgi apparatus; nearly mature melanosomes at

1	until the mature melanin granule, the melanosome, is formed and then appears as an electron-opaque granule. Premelanosomes are concentrated near the Golgi apparatus; nearly mature melanosomes at the bases of the cell processes; and mature melanosomes most commonly in and at the ends of the processes (see Fig. 15.8). Developing melanosomes and their melanin contents are transferred to neighboring keratinocytes by pigment donation. This process, which involves the phagocytosis of the tips of the melanocyte processes by keratinocytes, is a type of cytocrine

1	FIGURE 15.7 • Diagram of the epidermis and electron micrograph of a melanocyte. a. This diagram shows a melanocyte interacting with several cells of the stratum basale and the stratum spinosum. The melanocyte has long dendritic processes that contain accumulated melanosomes and extend between the cells of the epidermis, which are also visible on the electron micrograph. The Langerhans’ cell is a dendritic cell often confused with a melanocyte but is actually part of the mononuclear phagocytotic system and functions as an antigen-presenting cell of the immune system in the initiation of cutaneous hypersensitivity reactions (contact allergic dermatitis). b. The melanocyte reveals several processes extending between neighboring keratinocytes. The small dark bodies are melanosomes. 8,500. (Courtesy of Dr. Bryce L. Munger.)

1	FIGURE 15.8 • Formation of melanin pigment and secretion of pigment granules into keratinocytes. Melanocytes produce membrane-bounded structures that originate in the Golgi apparatus as premelanosomes. a. Within the early melanosomes, as maturation proceeds, melanin is produced from tyrosine by a series of enzymatic reactions. b. Mature melanosomes and their melanin contents are transferred to neighboring keratinocytes by pigment donation, which involves the phagocytosis of the tips of the melanocyte. c. In darker skin (on the right), the melanin is degraded slowly, and melanosomes remain discrete; in lighter skin (on the left), the melanin is degraded more rapidly d. through the process of macroautophagy. (Based on Weiss L, Greep RO. Histology. New York: McGraw-Hill, 1977.) tors, including major genetic determinants, several modifier genes, and environmental influences such as exposure to ultraviolet radiation and gender effects. The most signifi-cant is melanin content. Although the

1	major genetic determinants, several modifier genes, and environmental influences such as exposure to ultraviolet radiation and gender effects. The most signifi-cant is melanin content. Although the number of melanocytes is essentially the same in all races, the fate of the melanin that is produced by the melanocytes differs. For example, because of the lysosomal activity of the ker-atinocytes, melanin is degraded more rapidly in individuals with light skin than in individuals with dark skin. In the former, melanosomes are more concentrated in the ker-atinocytes nearest the basal layer and are relatively sparse in the midregion of the stratum granulosum. In contrast, dark skin may exhibit melanosomes throughout the epider-mis, including the stratum corneum. In addition, melanin pigment comprises two distinct forms. One form, eumelanin, is a brownish black pigment. The other form, pheomelanin, is a reddish yellow pigment. Each is genetically determined. Coloration is most appar-ent in

1	two distinct forms. One form, eumelanin, is a brownish black pigment. The other form, pheomelanin, is a reddish yellow pigment. Each is genetically determined. Coloration is most appar-ent in hair because of the concentration of melanin pig-ment granules, but it is also reflected in skin coloration. Exposure to ultraviolet radiation, particularly the sun’s rays is called tanning. It increases the number of melanocytes and accelerates the rate of melanin produc-tion, thus protecting against further radiation effects. Response to ultraviolet radiation is genetically determined and is more pronounced in individuals with darker color of skin. Increased pigmentation of the skin may also result from hormonal imbalance—for example, in Addison’s disease. Lack of pigmentation occurs in a condition known as albinism. In this hereditary condition, premelanosomes are produced by melanocytes, but because of the absence of dihydroxyphenylalanine (DOPA) and the subsequent trans formation of DOPA

1	known as albinism. In this hereditary condition, premelanosomes are produced by melanocytes, but because of the absence of dihydroxyphenylalanine (DOPA) and the subsequent trans formation of DOPA into melanin fail to occur. Thus, there is no pigmentation in the skin or hair of these individuals. Two genes—Bcl2 and Mitf—appear to be responsible for the process of graying. The expression of Bcl2 in melanocyte stem cells is essential to maintaining their population within the niche of the follicular bulge. Defi-ciency in Bcl2 expression causes apoptosis of melanocyte stem cells and a consequent decrease in the number of melanocytes. Melanocyte depletion occurs with age, resulting in a decreased rate of pigment donation to keratinocytes. Therefore, the skin becomes lighter with increased age, and the incidence of skin cancer also increases. Melanocyte depletion caused by defective self-maintenance of melanocyte stem cells is also linked to hair graying, the most obvious sign of aging in

1	and the incidence of skin cancer also increases. Melanocyte depletion caused by defective self-maintenance of melanocyte stem cells is also linked to hair graying, the most obvious sign of aging in humans. Individ-uals with a mutation in the Bcl2 gene may become prema-turely gray. Other normal factors that affect skin coloration include the presence of oxyhemoglobin in the dermal vascular bed, which imparts a red hue; the presence of carotenes, an ex-ogenous orange pigment taken up from foods and concen-trated in tissues containing fat; and the presence of certain endogenous pigments. The latter include degradation products of hemoglobin, iron-containing hemosiderin, and iron-free bilirubin, all of which impart color to the skin. Hemosiderin is a golden brown pigment, whereas bilirubin is a yellowish brown pigment. Bilirubin is normally removed from the bloodstream by the liver and eliminated via the bile. A yellowish skin color as a result of abnormal accumu-lation of bilirubin

1	is a yellowish brown pigment. Bilirubin is normally removed from the bloodstream by the liver and eliminated via the bile. A yellowish skin color as a result of abnormal accumu-lation of bilirubin reflects liver dysfunction and is evidenced as jaundice.

1	 FOLDER 15.2 Functional Considerations: Skin Color The color of an individual’s skin is caused by several fac-tyrosinase, the transformation of tyrosine into 3,4 secretion because a small amount of cytoplasm surrounding the melanosome is also phagocytosed. Taking into consideration the complexity of melanine biogenesis, protein trafficking, organelle movement, and cell-to-cell interactions within the epidermal-melanin unit, it is explainable that even minor changes in the cellular environment can affect melanosome structure and pigment donation process. Numerous intrinsic and extrinsic factors are also responsible for skin pigmentation, including age, ethnicity and gender differences, variable hormone levels and affinities of their receptors, genetic defects, ultraviolet radiation, climate and season changes, and chemical exposure to toxins and pollutants. Langerhans’ cells are antigen-presenting cells in the epidermis.

1	Langerhans’ cells are antigen-presenting cells in the epidermis. Langerhans’ cells are dendritic-appearing, antigen-presenting cells in the epidermis. They originate from common lymphoid progenitor (CLP) cells in bone marrow, migrate via the bloodstream, and ultimately enter the epidermis where they differentiate into immunocompetent cells. Langerhans’ cells encounter and process antigens entering through the skin. Therefore, they constitute part of the mononuclear phagocytotic system (MPS; page 185). Once antigen is phagocytized, processed, and displayed on the surface of the Langerhans’ cell, the cell migrates from the epidermis to a regional lymph node where it interacts with T lymphocytes.

1	Langerhans’ cells cannot be distinguished with certainty in routine H&E–stained paraffin sections. Like melanocytes, Langerhans’ cells do not form desmosomes with neighboring keratinocytes. The nucleus stains heavily with hematoxylin, and the cytoplasm is clear. With special techniques, such as gold chloride impregnation or immunostaining with antibody against CD1a molecules, Langerhans’ cells can be readily seen in the stratum spinosum. They possess dendritic processes resembling those of the melanocyte. The TEM reveals several distinctive features of a Langerhans’ cell (Fig. 15.9). Its nucleus is characteristically indented in many

1	FIGURE 15.9 • Electron micrograph of a Langerhans’ cell. The nucleus (N) of a Langerhans’ cell is characteristically indented in many places, and the cytoplasm contains distinctive rod-shaped bodies (arrows). Note the presence of tonofilaments (T ) in adjacent keratinocytes (K) but the absence of these filaments in the Langerhans’ cell. 19,000. Inset. Photomicrograph of the epidermis shows the distribution and dendritic nature of the Langerhans’ cells that were stained via immunostaining techniques with antibodies against CD1a surface antigen. 300. (Reprinted with permission from Urmacher CD. In: Sternberg SS, ed. Histology for Pathologists. Philadelphia: Lippincott-Raven, 1997.) places, so the nuclear profile is uneven. Also, it possesses characteristic, tennis racquet–shaped Birbeck granules. They represent relatively small vesicles, which appear as rods with a bulbous expansion at their end.

1	Like macrophages, Langerhans’ cells express both MHC I and MHC II molecules, as well as Fc receptors for immunoglobulin G (IgG). Langerhans’ cells also express complement C3b receptors as well as fluctuating quantities of CD1a molecules. As an antigen-presenting cell, the Langerhans’ cell is involved in delayed-type hypersensitivity reactions (e.g., contact allergic dermatitis and other cell-mediated immune responses in the skin) through the uptake of antigen in the skin and its transport to the lymph nodes. Skin biopsy specimens from individuals with AIDS or AIDS-related complex reveal that Langerhans’ cells contain HIV in their cytoplasm. Langerhans’ cells appear to be more resistant than T cells to the deadly effects of the HIV and may, therefore, serve as a reservoir for the virus.

1	In addition, a malignant transformation of Langerhans’ cells is responsible for histiocytosis X (Langerhans’ cell histiocytosis), a group of immune diseases that are characterized by abnormal increase and spread of Langerhans’ cells. Accumulation of these abnormal cells may form tumors, which can affect various parts of the body, including the bones, lungs, skull, and other areas and organs. Merkel’s cells are epidermal cells that function in cutaneous sensation.

1	Merkel’s cells are dendritic cells located in the stratum basale. The origin of Merkel’s cells is unknown; they possess antigenic markers of both epidermal and neural type. They are most abundant in skin where sensory perception is acute such as the fingertips. Merkel’s cells are bound to adjoining keratinocytes by desmosomes and contain intermediate (keratin) filaments in their cytoplasm. The nucleus is lobed, and the cytoplasm is somewhat denser than that of melanocytes and Langerhans’ cells. They may contain some melanosomes in their cytoplasm, but they are best characterized by the presence of 80-nm dense-cored neurosecretory granules that resemble those found in the adrenal medulla and carotid body (Fig. 15.10). Merkel’s cells are closely associated with the expanded terminal bulb of afferent myelinated nerve fibers. The neuron terminal loses its Schwann cell covering and immediately penetrates the basal lamina, where it expands into a disc or platelike ending that lies in close

1	afferent myelinated nerve fibers. The neuron terminal loses its Schwann cell covering and immediately penetrates the basal lamina, where it expands into a disc or platelike ending that lies in close apposition to the base of the Merkel’s cell. The combination of the neuron and epidermal cell, called a Merkel’s corpuscle, is a sensitive mechanoreceptor.

1	FIGURE 15.10 • Electron micrograph of a Merkel’s cell. The cell has small neurosecretory granules in the cytoplasm and makes contact with a peripheral terminal (NT ) of a neuron. The dermis (D) is in the lower part of the micrograph. 14,450. (Courtesy of Dr. Bryce L. Munger.) Merkel cell carcinoma (MCC) is a rare but highly aggressive type of skin cancer that develops when Merkel cells undergo uncontrolled proliferation. It starts most often in areas of skin exposed to the sun, such as the head, neck, and upper and lower limbs. MCC tends to grow quickly and to metastasize via lymph vessels at an early stage. The skin is endowed with sensory receptors of various types that are peripheral terminals of sensory nerves (Fig. 15.11). It is also well supplied with motor nerve endings to the blood vessels, arrector pili muscles, and sweat glands. Free nerve endings are the most numerous neuronal receptors in the epidermis.

1	Free nerve endings are the most numerous neuronal receptors in the epidermis. Free nerve endings in the epidermis terminate in the stratum granulosum. The endings are “free” in that they lack a connective tissue or Schwann cell investment. Such neuronal endings subserve multiple sensory modalities including fine touch, heat, and cold, without apparent morphologic distinction. Networks of free dermal endings surround most hair follicles and attach to their outer root sheath (Fig. 15.12). In this position, they are particularly sensitive to hair movement and serve as mechanoreceptors. This relationship imparts a sophisticated degree of specialization in the receptors that surround tactile hairs (vibrissae), such as the whiskers of a cat or rodent, in which each vibrissa has a specific representation in the cerebral cortex.

1	Other nerve endings in the skin are enclosed in a connective tissue capsule. Encapsulated nerve endings include the following: tions applied on the skin surface.  Meissner’s corpuscles are responsible for sensitivity to light touch. Ruffni’s corpuscles that sensitive to skin stretch and torque. Pacinian corpuscles are deep pressure receptors for mechanical and vibratory pressure.

1	Pacinian corpuscles are large ovoid structures found in the deeper dermis and hypodermis (especially in the fingertips), in connective tissue in general, and in association with joints, periosteum, and internal organs. Pacinian corpuscles usually have macroscopic dimensions, measuring more than 1 mm along their long axis. They are composed of a myelinated nerve ending surrounded by a capsule structure (see Figs. 15.11 and 15.12a). The nerve enters the capsule at one pole with its myelin sheath intact. The myelin is retained for one or two nodes and is then lost. The unmyelinated portion of the axon extends toward the opposite pole from which it entered, and its length is covered by a series of tightly packed, flattened Schwann cell lamellae that form the inner core of the corpuscle. The remainder or bulk of the capsule, the outer core, is formed by a series of concentric lamellae; each lamella is separated

1	Merkel’s cell free endings of afferent axon terminal disk of afferent axon terminal of afferent axon b terminal branches of afferent axon dermal papilla terminal branches of afferent axon spiral terminals of afferent axon FIGURE 15.11 • Diagram of the sensory receptors in skin. from its neighbor by a narrow space containing lymphlike fluid (Plate 46, page 523). The appearance of the concentric lamellae as observed in the light microscope is reminiscent of the cut surface of a hemisected onion. Each lamella is composed of flattened cells that correspond to the cells of the endoneurium outside the capsule. In addition to fluid between the lamellae, collagen fibrils are present, although sparse, as are occasional capillaries. Pacinian corpuscles respond to pressure and vibration through the displacement of the capsule lamellae. This displacement effectively causes depolarization of the axon. Meissner’s corpuscles are localized within dermal papillae and serve as touch receptors.

1	Meissner’s corpuscles (see Figs. 15.11 and 15.12b) are touch receptors that are particularly responsive to low-frequency stimuli in the papillary layer of hairless skin (e.g., the lips and the palmar and volar surfaces, particularly those of the fingers and toes). Generally, they are tapered cylinders that measure about 150 m along their long axis and are oriented perpendicular to the skin surface. Meissner’s corpuscles are present in the dermal papillae just beneath the epidermal basal lamina (Plate 46, page 523). Within these receptors, one or two unmyelinated endings of myelinated nerve fibers follow spiral paths in the corpuscle. The cellular component consists of flattened Schwann cells that form several irregular lamellae through which the axons course to the pole of the corpuscle. In H&E–stained slides of sagittal sections, this structure resembles a loose, twisted skein of wool. It is the Schwann cells that give this impression.

1	Ruffini’s corpuscles respond to mechanical displacement of adjacent collagen fibers. Ruffni’s corpuscles are the simplest encapsulated mechanoreceptors. They have an elongated fusiform shape and

1	FIGURE 15.12 • Pacinian and Meissner’s corpuscles in H&E preparations. a. In this photomicrograph, the concentric cellular lamellae of the Pacinian corpuscle are visible because of flat, fibroblastlike supportive cells. Although not evident within the tissue section, these cells are continuous with the endoneurium of the nerve fiber. The spaces between lamellae contain mostly fluid. The neural portion of the Pacinian corpuscle travels longitudinally through the center of the structure (arrow). Several nerves (N) are present adjacent to the corpuscle. 85. b. Three Meissner’s corpuscles (MC) are shown residing within the dermal papillae. Note the direct proximity of the corpuscle to the undersurface of the epidermis. 150. Inset. A higher magnification of a Meissner’s corpuscle. The nerve fiber terminates at the superficial pole of the corpuscle. Note that supporting cells are oriented approximately at right angles to the long axis of the corpuscle. 320.

1	measure 1 to 2 m in length (see Fig. 15.11f ). Structurally, they consist of a thin connective tissue capsule that encloses a fluid-filled space. Collagen fibers from the surrounding connective tissue pass through the capsule. The neural element consists of a single myelinated fiber that enters the capsule, where it loses its myelin sheath and branches to form a dense arborization of fine axonal endings, each terminating in a small knoblike bulb. The axonal endings are dispersed and intertwined inside the capsule. The axonal endings respond to displacement of the collagen fibers induced by sustained or continuous mechanical stress, thus they respond to stretch and torque. Ruffini’s corpuscles functionally belong to the family of rapidly adapting receptors (phasic receptors) that generate short action potentials at the beginning and end of a stimulus.

1	Skin appendages are derived from downgrowths of epidermal epithelium during development. They include the following:  Hair follicles and their product, hair  Sebaceous glands and their product, sebum  Eccrine sweat glands and their product, sweat taining a form of sweat with a high concentration of carbo hydrates, lipids, and proteins Both hairs and sweat glands play specific roles in the regulation of body temperature. Sebaceous glands secrete an oily substance that may have protective functions. Apocrine glands produce a serous secretion containing pheromones that act as a sex attractant in other animals and possibly in humans. The epithelium of the skin appendages (especially hair follicles) can serve as a source of new epithelial stem cells for skin wound repair. Each hair follicle represents an invagination of the epidermis in which a hair is formed.

1	Each hair follicle represents an invagination of the epidermis in which a hair is formed. Hair follicles and hairs are present over almost the entire body; they are absent only from the sides and palmar surfaces of the hands, sides, and plantar surfaces of the feet, the lips, and the region around the urogenital orifices. Hair distribution is influenced to a considerable degree by sex hormones;  FOLDER 15.3 Functional Considerations: Hair Growth and Hair Characteristics

1	Unlike the renewal of the surface epidermis, hair growth is not continuous, but a cyclic process. A period of growth (anagen) in which a new hair develops is followed by a brief period in which growth stops (catagen). Catagen is followed by a long rest period (telogen) in which the follicle atrophies, and the hair is eventually lost. Epidermal stem cells found in the follicular bulge are capable of pro-viding stem cells that give rise to mature anagen follicles. During the hair growth cycle, mature anagen hairs period-ically undergo apoptosis and regress to the catagen stage. In this phase, entire follicles retract toward the epidermal layer. As the base of the retracted follicle approximates the follicular bulge, the hair shaft is no longer supported by the nutrient-rich anagen bulb and eventually is ejected from the resting telogen follicle. This makes room for a new shaft that will grow during anagen regeneration. More than 80% of the hair present in the normal scalp is in the

1	and eventually is ejected from the resting telogen follicle. This makes room for a new shaft that will grow during anagen regeneration. More than 80% of the hair present in the normal scalp is in the anagen phase. In catagen, the germinative zone is reduced to an epithelial strand still attached to a remnant of the dermal papilla. In the telogen phase, the atrophied follicle may contract to one half or less of its original length. The hair may remain attached to the follicle for several months during this stage and is called a club hair because of the shape of its proximal end. Hairs vary in size from long, coarse terminal hairs that may reach a meter or more in length (scalp hair and beard hair in males) to short, fine vellus hairs that may be visible only with the aid of a magnifying lens (vellus hairs of the forehead and anterior surface of the forearm). Terminal hairs are produced by large-diameter, long follicles; vellus hairs are produced by relatively small follicles. Terminal

1	(vellus hairs of the forehead and anterior surface of the forearm). Terminal hairs are produced by large-diameter, long follicles; vellus hairs are produced by relatively small follicles. Terminal hair follicles may spend up to several years in anagen and only a few months in telogen. In the balding individual, large ter-minal follicles are gradually converted into small vellus folli-cles after several growth cycles. The ratio of vellus follicles to terminal follicles increases as baldness progresses. The “completely bald” scalp is not hairless but is populated by vellus follicles that produce fine hairs and remain in telogen for relatively long periods.

1	these include, in the male, the thick, pigmented facial hairs that begin to grow at puberty and the pubic and axillary hair that develops at puberty in both genders. In the male, the hairline tends to recede with age; in both genders, the scalp hair thins with age because of reduced secretion of estrogen and estrogenlike hormones. The hair follicle is responsible for the production and growth of a hair. Coloration of the hair is attributable to the content and type of melanin that the hair contains. The follicle varies in histologic appearance, depending on whether it is in a growing or a resting phase. The growing follicle shows the most elaborate structure; thus, it is described here.

1	The hair follicle is divided into three segments:  The infundibulum extends from the surface opening of the follicle to the level of the opening of its sebaceous gland. The infundibulum is a part of the pilosebaceous canal, which is used as a route for the discharge of the oily substance sebum.  The isthmus extends from the infundibulum to the level of insertion of the arrector pili muscle.  The inferior segment in the growing follicle (Fig. 15.13) is of nearly uniform diameter except at its base, where it expands to form the bulb. The base of the bulb is invaginated by a tuft of vascularized loose connective tissue called, not surprisingly, a dermal papilla (Plate 47, page 525). Other cells forming the bulb, including those that surround the connective tissue papilla, are collectively referred to as the matrix, which consists simply of matrix cells.

1	Matrix cells immediately adjacent to the dermal papilla represent the germinative layer of the follicle. Division and proliferation of these cells accounts for the growth of the hair. Scattered melanocytes are also present in this germinative layer. They contribute melanosomes to the developing hair cells in a manner analogous to that in the stratum germinativum of the epidermis. The dividing matrix cells in the germinative layer differentiate into the keratin-producing cells of the hair and the internal root sheath. The internal root sheath is a multilayered cellular covering that surrounds the deep part of the hair. The internal root sheath has three layers:  The cuticle consists of squamous cells whose outer free surface faces the hair shaft.  Huxley’s layer consists of a single or double layer of flat tened cells that form the middle plate of the internal root sheath.

1	 Henle’s layer consists of an outer single layer of cuboidal cells. These cells are in direct contact with the outermost part of the hair follicle, which represents a downgrowth of the epidermis and is designated the external root sheath. A niche of epidermal stem cells that resides within the follicular bulge of the external root sheath provides stem cells for hair growth. The progression of the external root sheath of the hair follicle upward toward the epidermal surface reveals the insertion site of the arrector pili muscle and the origin of the sebaceous

1	The progression of the external root sheath of the hair follicle upward toward the epidermal surface reveals the insertion site of the arrector pili muscle and the origin of the sebaceous The role of sebum is not clearly defined. Various investiga-tors have ascribed bacteriostatic, emollient, barrier, and pheromone functions to sebum. Sebum does appear to play a critical role in the development of acne. The amount of sebum secreted increases significantly at puberty in both males and females. Triglycerides contained in sebum are broken down to fatty acids by bacteria on the skin surface, and the free fatty acids liberated may be an irritant in the formation of acne lesions. On histologic examina-tion, acne is characterized by retention of the sebum in the isthmus of the hair follicle, with variable lymphocytic infiltra-tion. In severe cases, dermal abscesses may form in asso-ciation with inflamed hair follicles.

1	FIGURE 15.13 • Hair follicle and other skin appendages. a. Diagram showing a hair follicle. Note the cell layers that form the hair shaft and the surrounding external and internal root sheaths. The sebaceous gland consists of the secretory portion and a short duct that empties into the infundibulum. The arrector pili muscle accompanies the sebaceous gland; its contraction assists in gland secretion and discharge into the infundibulum. The apocrine gland also empties into the infundibulum of the hair follicle. Note that eccrine sweat glands are independent structures and are not associated directly with the hair follicle. b. Photomicrograph of H&E–stained section of thin skin from human scalp. The growing end of a hair follicle consists of an expanded hair bulb (HB) of epithelial cells that is invaginated by a papilla of connective tissue. The epithelial cells form the unspecialized matrix surrounding the papilla; as the cells leave the matrix, they form cell layers that differentiate

1	is invaginated by a papilla of connective tissue. The epithelial cells form the unspecialized matrix surrounding the papilla; as the cells leave the matrix, they form cell layers that differentiate into the shaft of the hair and the inner and outer root sheaths of the hair follicle (HF ). Note that several oblique and longitudinal sections of the hair follicles are embedded in the adipose tissue (AT ) of the hypodermis. Some of them reveal a section of the hair. Sebaceous glands (SG) are visible in conjunction with the upper part of the hair follicle. 60.

1	duct and gland from the wall of the follicular canal (see reside in this area indefinitely and undergo self-renewal or Fig 15.13). In this general region resides an aggregate of rela-differentiation into specific cell lineages. Under normal contively undifferentiated epithelial cells called the follicular ditions, ES cells are responsible for providing stem cells for bulge. Recent studies identified the follicular bulge as a the growth of hair follicles (the internal root sheath, cortex, niche of epidermal stem (ES) cells. The ES cells can and medulla) as well as sebaceous glands. The ES cells that  FOLDER 15.4

1	Functional Considerations: The Role of Sebum normally reside in the follicular bulge do not contribute to the population of the basal stem cells of the epidermis. However, when the epidermis is injured or lost (such as in extensive skin burns and superficial skin wounds), the ES cells become reprogrammed, migrate toward the wound surface from their follicular niches, and participate in the initial resurfacing of the wound. Hairs are composed of keratinized cells that develop from hair follicles.

1	Hairs are composed of keratinized cells that develop from hair follicles. Keratinization of the hair and internal root sheath occurs shortly after the cells leave the matrix in a region called the keratogenous zone. By the time the hair emerges from the follicle, it is entirely keratinized as hard keratin. The internal root sheath, consisting of soft keratin, does not emerge from the follicle with the hair but is broken down at about the isthmus level where sebaceous secretions enter the follicle. A thick basal lamina, called the glassy membrane, separates the hair follicle from the dermis. Surrounding the follicle is a dense irregular connective tissue sheath containing follicular bulge. The arrector pili muscle is attached to follicular bulge which, as was indicated above, also serves as a epidermal stem cell niche.

1	Hairs are elongated filamentous structures that project from the hair follicles. They also consist of three layers (see Fig. 15.13):  The medulla forms the central part of the shaft and con tains large vacuolated cells. The medulla is present only in thick hairs.  The cortex is located peripherally to the medulla and con tains cuboidal cells. These cells undergo differentiation into keratin-filled cells.  The cuticle of the hair shaft contains squamous cells that form the outermost layer of the hair. In addition, the hair shaft contains melanin pigment produced by melanocytes present in the germinative layer of the hair bulb. Sebaceous glands secrete sebum that coats the hair and skin surface.

1	Sebaceous glands secrete sebum that coats the hair and skin surface. Sebaceous glands develop as outgrowths of the external root sheath of the hair follicle, usually producing several glands per follicle (Fig. 15.14 and Plate 45, page 521). The oily substance produced in the gland, sebum, is the product of holocrine secretion. The entire cell produces and becomes filled with the fatty product while it simultaneously undergoes programmed cell death (apoptosis) as the product fills the cell. Ultimately, both the secretory product and cell debris are discharged from the gland as sebum into the infundibulum of a hair follicle, which forms the pilosebaceous canal with the short duct of the sebaceous gland. New cells are produced by mitosis of the basal cells at the periphery of the gland, and the cells of the gland remain linked to one another by desmosomes. The basal lamina of these cells is continuous with that of the FIGURE 15.14 • Photomicrograph of a sebaceous gland. a.

1	FIGURE 15.14 • Photomicrograph of a sebaceous gland. a. This micrograph shows the secretory lobules and their pilosebaceous canal of two sebaceous glands. The canal of the gland on the left is about to enter the hair follicle seen at the top of the micrograph. The canal of the sebaceous gland on the right has been sectioned in a manner that shows mostly the wall of the canal. 60. b. The secretory component of the lobule in the lower box of Figure a is shown here at higher magnification. Note the light staining of the secretory cells due to the lack of staining of the sebum that they contain. These cells are actively producing sebum. The basal cells at the periphery of the lobule proliferate producing new sebum-producing cells. 120. c. The secretory component of the lobule in the upper box of Figure a is shown here at higher magnification. The sebum-filled cells are now within the canal. Note their pyknotic nuclei, signifying the death of the cell. 120.

1	Although many neural and emotional factors can alter the composition of sweat, altered sweat composition can also be a sign of disease. For example, elevated sodium and chlo-ride levels in sweat can serve as an indicator of cystic fbro-sis. Individuals with cystic fibrosis have two to five times higher than normal amounts of sodium and chloride in their sweat. In pronounced uremia, when the kidneys are unable to rid the body of urea, the concentration of urea in sweat increases. In this condition, after the water evaporates, crystals may be discerned on the skin, especially on the upper lip. These include urea crystals and are called urea frost.  FOLDER 15.5 Clinical Correlation: Sweating and Disease epidermis and the hair follicle. The process of sebum production from the time of basal cell mitosis to the secretion of the sebum takes about 8 days.

1	The basal cells of the sebaceous gland contain smooth-surface endoplasmic reticulum (sER), rER, free ribosomes, mitochondria, glycogen, and a well-developed Golgi apparatus. As the cells move away from the basal layer and begin to produce the lipid secretory product, the amount of sER increases, reflecting the role of the sER in lipid synthesis and secretion. The cells gradually become filled with numerous lipid droplets separated by thin strands of cytoplasm.

1	Sweat glands are classified on the bases of their structure and the nature of their secretion. Two types of sweat glands are recognized:  Eccrine sweat glands are distributed over the entire body surface except for the lips and part of the external genitalia. Apocrine sweat glands are limited to the axilla, areola, and nipple of the mammary gland; skin around the anus; and the external genitalia. The ceruminous glands of the external acoustic meatus canal and the apocrine glands of eyelashes (glands of Moll) are also apocrine-type glands. Eccrine sweat glands are simple coiled glands that regulate body temperature.

1	Eccrine sweat glands are simple coiled glands that regulate body temperature. Eccrine sweat glands are independent structures, not associated with the hair follicle that arises as a downgrowth from the fetal epidermis. Each eccrine gland is arranged as a blind-ended, simple, coiled tubular structure. It consists of two segments: A secretory segment located deep in the dermis or in the upper part of the hypodermis and a directly continuous, less coiled duct segment that leads to the epidermal surface (Fig. 15.15 and Plate 44, page 519). Eccrine sweat glands play a major role in temperature regulation through the cooling that results from the evaporation of water from sweat on the body surface. The secretory portion of the glands produces a secretion similar in composition to an ultrafiltrate of blood. Resorption of some of the sodium and water in the duct results in the FIGURE 15.15 • Photomicrograph of an eccrine sweat gland.

1	FIGURE 15.15 • Photomicrograph of an eccrine sweat gland. This photomicrograph of a H&E–stained section of human skin shows profiles of both the secretory component and the duct of an eccrine sweat gland. The secretory component appears as a double layer of cuboidal epithelial cells and peripherally, within the basal lamina, a layer of myoepithelial cells. The duct portion of the gland has a narrower outside diameter and lumen than the secretory portion of the gland. It consists of a double layer of small cuboidal cells without the myoepithelial cells. 320. release of a hypotonic sweat at the skin surface. This hypo-tonic watery solution is low in protein and contains varying amounts of sodium chloride, urea, uric acid, and ammonia. Thus, the eccrine sweat gland also serves, in part, as an excretory organ.

1	Excessive sweating can lead to the loss of other electrolytes such as potassium and magnesium, as well as significant water loss. Normally, the body loses about 600 ml of water a day through evaporation from the lungs and skin. Under conditions of high ambient temperature, water loss can be increased in a regulated manner by an increased rate of sweating. This thermoregulatory sweating first occurs on the forehead and scalp, extends to the face and to the rest of the body, and occurs last on the palms and soles. Under conditions of emotional stress, however, the palms, soles, and axillae are the first surfaces to sweat. Control of thermoregulatory sweating is cholinergic, whereas emotional sweating may be stimulated by adrenergic portions of the sympathetic division of the autonomic nervous system. The secretory segment of the eccrine sweat gland contains three cell types.

1	The secretory segment of the eccrine sweat gland contains three cell types. Three cell types are present in the secretory segment of the gland: clear cells and dark cells, both of which are secretory epithelial cells, and myoepithelial cells, which are contractile epithelial cells (Fig. 15.16 and Plate 45, page 521). All of the cells rest on the basal lamina; their arrangement is that of a pseudostratified epithelium.

1	 Clear cells are characterized by abundant glycogen. The glycogen is conspicuous in Figure 15.16a because of its amount; it would stain intensely with the periodic acid–Schiff (PAS) method. In routine H&E preparations, the cytoplasm of clear cells stains poorly. Membranous organelles include numerous mitochondria, profiles of sER, and a relatively small Golgi apparatus. The plasma membrane is remarkably amplified at the lateral and apical surfaces by extensive cytoplasmic folds. In addition, the basal surface of the cell possesses infoldings, although they are considerably less complex than the cytoplasmic folds. The morphology of these cells indicates that they produce the watery component of sweat.

1	 Dark cells are characterized by abundant rER and se cretory granules (see Fig. 15.16). The Golgi apparatus is relatively large, a feature consistent with the glyco protein secretion of these cells. The apical cytoplasm contains mature secretory granules and occupies most of the luminal surface (see Fig. 15.16a). Clear cells have considerably less cytoplasmic exposure to the lumen; their secretion is largely via the lateral surfaces of the cell, which are in contact with intercellular canaliculi that allow the watery secretion to reach the lumen. Here it mixes with the proteinaceous secretion of the dark cells.  Myoepithelial cells are limited to the basal aspect of the secretory segment. They lie between the secretory cells, with their processes oriented transversally to the tubule.

1	 Myoepithelial cells are limited to the basal aspect of the secretory segment. They lie between the secretory cells, with their processes oriented transversally to the tubule. The cytoplasm contains numerous contractile filaments (actin) that stain deeply with eosin, thus making them readily identifiable in routine H&E specimens. Contraction of these cells is responsible for rapid expression of sweat from the gland. The duct segment of eccrine glands is lined by stratified cuboidal epithelium and lacks myoepithelial cells.

1	The duct segment of the gland continues from the secretory portion with coiling. In histologic sections, multiple duct profiles typically appear among the secretory profiles. As the duct passes upward through the dermis, it takes a gentle spiral course until it reaches the epidermis, where it then continues in a tighter spiral to the surface. When the duct enters the epidermis, however, the duct cells end and the epidermal cells form the wall of the duct. The duct is composed of stratifed cuboidal epithelium, consisting of a basal cell layer and a luminal cell layer. The duct cells are smaller and appear darker than the cells of the secretory portion of the gland. Also, the duct has a smaller diameter than the secretory portion. In contrast to the secretory portion of the eccrine gland, the duct portion does not possess myoepithelial cells. These features are useful in distinguishing the duct from the secretory portion in a histologic section (see Fig. 15.15).

1	The basal or peripheral cells of the duct have a rounded or ovoid nucleus and contain a prominent nucleolus. The cytoplasm is filled with mitochondria and ribosomes. The apical or luminal cells are smaller than the basal cells, but their nuclei are similar in appearance. The most conspicuous feature of the luminal cells is the deeply stained, glassy (hyalinized) appearance of the apical cytoplasm. The glassy appearance is attributable to the presence of large numbers of aggregated tonofilaments in the apical cytoplasm. Apocrine glands are large-lumen tubular glands associated with hair follicles. Apocrine sweat glands develop from the same down-growths of epidermis that give rise to hair follicles. The connection to the follicle is retained, allowing the secretion of the gland to enter the follicle, typically at a level just above the entry of the sebaceous duct. From here, the secretion makes its way to the surface.

1	Like eccrine glands, apocrine glands are coiled tubular glands. They are sometimes branched. The secretory portion of the gland is located deep in the dermis or, more commonly, in the upper region of the hypodermis. The secretory portion of apocrine glands has a wider lumen than that of eccrine glands and is composed of a single cell type. The secretory portion of apocrine glands differs in several respects from that of eccrine glands. The most obvious difference, readily noted in the light microscope, is its very wide lumen (Fig. 15.17 and Plate 44, page 519). Unlike eccrine glands, apocrine glands store their secretory product in the lumen. The secretory portion of the gland is composed of simple

1	FIGURE 15.16 • Electron micrographs of an eccrine sweat gland. a. This micrograph shows myoepithelial cells (My) and two distinctive gland cell types, dark cells (D) and clear cells (C). The apical portion of the dark cell is broad; it faces the lumen (L) of the gland and contains numerous secretory granules. The dashed line marks the boundary of one dark cell. The clear cell is more removed from the lumen of the gland. Its base rests on the myoepithelial cells or directly on the basal lamina. Most of the free surface of the clear cell faces an intercellular canaliculus (IC). Clear cells contain numerous mitochondria, extensive infoldings of the plasma membrane, and large numbers of electron-dense glycogen inclusions. 5,600. (Courtesy of Dr. John A. Terzakis.) b. At higher magnification, dark cells display rER (arrow) and a Golgi apparatus (G) in addition to secretory granules. Clear cells show large amounts of folded membrane, mitochondria, and glycogen. The myoepithelial cells (My)

1	dark cells display rER (arrow) and a Golgi apparatus (G) in addition to secretory granules. Clear cells show large amounts of folded membrane, mitochondria, and glycogen. The myoepithelial cells (My) contain large numbers of contractile actin filaments. Short stubby arrows (upper right) mark the boundary of a clear cell. 17,500. (Courtesy of Dr. John A. Terzakis.)

1	FIGURE 15.17 • Photomicrograph of an apocrine sweat gland. This section of adult skin from the area around the anus shows several apocrine (anal) sweat glands, which are easily identified by the large lumen of their secretory components. This apocrine sweat gland is close to a hair follicle (center of photomicrograph) and deep to the dense, irregular connective tissue of the dermis. 45. Inset. Higher magnification of secretory component shows the cell types of the apocrine gland. The gland consists of a simple epithelium whose cells are either cuboidal or columnar and myoepithelial cells located in the basal portion of the epithelial cell layer. 230.

1	epithelium. Only one cell type is present, and the cytoplasm of the cell is eosinophilic. The apical part of the cell often exhibits a bleblike protrusion. It was once thought that this part of the cell pinched off and was discharged into the lumen as an apocrine secretion, thus the name of the gland. However, TEM studies confirm that the secretion is a merocrine type. The apical cytoplasm contains numerous small granules, the secretory component within the cell, which are discharged by exocytosis. Other features of the cell include numerous lysosomes and lipofuscin pigment granules. The latter represent secondary and tertiary lysosomes. Mitochondria are also numerous. During the refractory phase, after expulsion of the secretion, the Golgi apparatus enlarges in preparation for a new secretory phase.

1	Myoepithelial cells are also present in the secretory portion of the gland and are situated between the secretory cells and the adjacent basal lamina. As in eccrine glands, contraction of the processes of myoid cells facilitates expulsion of the secretory product from the gland. The duct portion of apocrine glands is lined by stratified cuboidal epithelium and lacks myoepithelial cells. The duct of the apocrine gland is similar to that of the eccrine duct; it has a narrow lumen. However, it continues from the secretory portion of the gland in a relatively straight path to empty into the follicle canal. Because of its straight course, the probability of viewing both the duct and the secretory portion of an apocrine gland in the same histologic section is reduced. Also in contrast to the eccrine duct, resorption does not take place in the apocrine duct. The secretory product is not altered in its passage through the duct.

1	The duct epithelium is stratified cuboidal, usually two but sometimes three cell layers thick. The apical cytoplasm of the luminal cells appears hyalinized, a consequence of the aggregated tonofilaments in the apical cytoplasm. In this aspect, they resemble the luminal cells of the eccrine duct. Apocrine glands produce a protein-rich secretion containing pheromones. Apocrine glands produce a secretion that contains protein, carbohydrate, ammonia, lipid, and certain organic compounds that may color the secretion. However, the secretions vary with the anatomic location. In the axilla, the secretion is milky and slightly viscous. When secreted, the fluid is odorless; through bacterial action on the skin surface, it develops an acrid odor.

1	Apocrine glands become functional at puberty; as with axillary and pubic hair, their development depends on sex hormones. In the female, both axillary and areolar apocrine glands undergo morphologic and secretory changes that parallel the menstrual cycle. In many mammals, similar glands secrete pheromones, chemical signals used in marking territory, in courtship behavior, and in certain maternal and social behaviors. It is generally believed that apocrine secretions may function as pheromones in humans. Male pheromones (androstenol and androstenone) in the secretion of apocrine glands have a direct effect on the female menstruation cycle. Furthermore, female pheromones (copulins) inﬂuence male perception of females and may also induce hormonal changes in males. Innervation of sweat glands

1	Innervation of sweat glands Both eccrine and apocrine sweat glands are innervated by the sympathetic portion of the autonomic nervous system. Eccrine sweat glands are stimulated by cholinergic transmitters (usually identified with the parasympathetic component of the autonomic system), whereas apocrine glands are stimulated by adrenergic transmitters. As described above, eccrine glands respond to heat and stress. Apocrine glands respond to emotional and sensory stimuli but not to heat. Nails are plates of keratinized cells containing hard keratin. The slightly arched fingernails and toenails, more properly referred to as nail plates, rest on nail beds. The nail bed consists of epithelial cells that are continuous with the stratum basale and stratum spinosum of the epidermis (Fig. 15.18 and Plate 47, page 525).

1	The proximal part of the nail, the nail root, is buried in a fold of epidermis and covers the cells of the germinative zone, or matrix. The matrix contains a variety of cells, including stem cells, epithelial cells, melanocytes, Merkel’s cells, and Langerhans’ cells. The stem cells of the matrix regularly divide, migrate toward the root of the nail, and there differentiate and produce the keratin of the nail. Nail keratin is a hard keratin, like that of the hair cortex. Unlike the soft keratin of the epidermis, it does not desquamate. It consists of densely packed keratin filaments embedded in a matrix of amorphous keratin with a high sulfur content, which is responsible for the hardness of the nail. The process of hard keratin formation, as with the hair cortex, does not involve keratohyalin granules. In addition, a cornified cell envelope contains proteins similar to those found in the epidermis.

1	FIGURE 15.18 • Photomicrograph of a sagittal section of distal phalanx with a nail. A nail is a keratinized plate located on the dorsal aspect of the distal phalanges. Under the free edge of the nail is a boundary layer, the hyponychium, which is continuous with the stratum corneum of the adjacent epidermis. The proximal end, the root of the nail, is overlapped by skin, the eponychium, which is also continuous with the stratum corneum of the adjacent epidermis. Deep to the nail is a layer of epithelium with underlying dermis. The proximal portion of this epithelium is referred to as the nail matrix. The bone in this section represents a distal phalanx. Numerous Pacinian corpuscles are present in the connective tissue of the palmar side of the finger. Note that even at this low magnification, the stratum lucidum is visible in the epidermis of the fingertip. 10.

1	The constant addition of new cells at the root and their keratinization account for nail growth. As the nail plate grows, it moves over the nail bed. On the microscopic level, the nail plate contains closely packed interdigitating corneocytes lacking nuclei and organelles. The crescent-shaped white area near the root of the nail, the lunula, derives its color from the thick, opaque layer of partially keratinized matrix cells in this region. When the nail plate becomes fully keratinized, it is more transparent and takes on the coloring of the underlying vascular bed. The edge of the skin fold covering the root of the nail is the eponychium, or cuticle. The cuticle is also composed of hard keratin; therefore, it does not desquamate. Because of its thinness, it tends to break off or, as with many individuals, it is trimmed and pushed back. A thickened epidermal layer, the hyponychium, secures the free edge of the nail plate at the fingertip.

1	 FOLDER 15.6 Clinical Correlation: Skin Repair

1	The process of wound healing of the skin is classically described as either a primary or secondary union. Healing by primary union (frst intention) occurs after surgical in-cisions in which wounds that are usually clean and unin-fected have their edges approximated by surgical sutures. The healing by secondary union (secondary intention) occurs in traumatic wounds with separated edges, which are characterized by more extensive loss of cells and tis-sues. Wound healing in such cases involves generating a large amount of granulation tissue, which represents a specialized type of tissue formed during the repair process. The repair of an incision or laceration of the skin requires stimulated growth of both the dermis and the epidermis. Der-mal repair involves (1) blood clot formation, (2) removal of damaged collagen fibers, primarily through the effort of macrophage activity that is associated with inflammation, (3) formation of granulation tissue, (4) reepithelialization of the exposed

1	of damaged collagen fibers, primarily through the effort of macrophage activity that is associated with inflammation, (3) formation of granulation tissue, (4) reepithelialization of the exposed surface, (5) proliferation and migration of fibroblasts and differentiation of myofibroblasts involved in wound contraction, and (6) deposition and remodeling of the extracellular matrix of underlying connective tissue. Healing by primary union following application of sutures reduces the extent of the repair area through maximal closure of a wound, minimizing scar formation. Surgical incisions are typically made along cleavage lines; the cut tends to parallel the collagen fibers, thus minimizing the need for excess collagen production and the inherent scarring that may occur. Repair of the epidermis involves the proliferation of the basal keratinocytes in the stratum basale in the undamaged site surrounding the wound (Fig. F15.6.1). Mitotic activity is markedly increased within the first 24

1	involves the proliferation of the basal keratinocytes in the stratum basale in the undamaged site surrounding the wound (Fig. F15.6.1). Mitotic activity is markedly increased within the first 24 hours. In a short time, the wound site is covered by a scab that represents dehydrated blood clot. The proliferating basal cells of the stratum basale begin migrating beneath the scab and across the wound surface. The migration rate may be as much as 0.5 mm/day, starting within 8 to 18 hours after wounding. Further proliferation and differentiation occur behind the migration front, leading to restoration of the mul-tilayered epidermis. As new cells ultimately keratinize and desquamate, the overlying scab is freed with the desqua-mating cells, which explains why a scab detaches from its periphery inward. In cases in which the full thickness of the epidermal layer is removed either by trauma or in surgery, the parts of hair follicles, the follicular bulge that contains niche of the epidernal

1	inward. In cases in which the full thickness of the epidermal layer is removed either by trauma or in surgery, the parts of hair follicles, the follicular bulge that contains niche of the epidernal stem cells, will produce cells that migrate over the exposed surface to reestablish a complete epithelial (epidermal) layer. Massive destruction of all of the epithelial structures of the skin, as in a third-degree burn or extensive full-thickness abrasion, prevents reepithelialization. Such wounds can be healed only by grafting epidermis to cover the wounded area. In the absence of a graft, the wound would, at best, reepithelialize slowly and imperfectly by ingrowth of cells from the margins of the wound. FIGURE F15.6.1 • Photomicrograph showing a late stage in the epidermal repair of a skin wound. The initial injury was caused by an incision through the full thickness of the skin and partially into the hypodermis, which contains adipose cells (A). The epidermis has re-formed beneath the

1	wound. The initial injury was caused by an incision through the full thickness of the skin and partially into the hypodermis, which contains adipose cells (A). The epidermis has re-formed beneath the scab. The asterisk marks an artifact where epithelium separated during specimen preparation. The scab, which contains numerous dead neutrophils in its inferior aspect, is close to the point of release. The dermis at this stage shows little change during the repair process but will ultimately reestablish itself to form a continuous layer. 110. LLLL L*scabL*scabL*scabL *scabA A * scab

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1	The skin, or integument, consists of two main layers: the epidermis, composed of stratified squamous epithelium that is keratinized, and the dermis, composed of connective tissue. Under the dermis is a layer of loose connective tissue called the hypodermis, which is also generally referred to as the subcutaneous tissue or, by gross anatomists, as the superficial fascia. Typically, the hypodermis contains large amounts of adipose tissue, particularly in an adequately nourished individual. The epidermis gives rise to nails, hairs, sebaceous glands, and sweat glands. On the palms of the hands and soles of the feet, the epider-mis has an outer keratinized layer that is substantially thicker than that over the other parts of the body. Accordingly, the skin over the palms and soles is referred to as thick skin, in contrast to the skin over other parts of the body, which is referred to as thin skin. There are no hairs in thick skin. In addition, the interface between the epidermis and the

1	to as thick skin, in contrast to the skin over other parts of the body, which is referred to as thin skin. There are no hairs in thick skin. In addition, the interface between the epidermis and the dermis is more complex in thick skin than in thin skin. The fingerlike projections of the dermis into the base of the epidermis, the dermal papillae, are much longer and more closely spaced in thick skin. This provides greater resistance to frictional forces acting on this skin.

1	Thick skin, human, H&E ×45. In this sample of thick skin, the epidermis (Ep) is at the top; the remainder of the field consists of dermis, in which a large number of sweat glands (SW) can be observed. Although the layers of the epidermis are examined more advantageously at higher magnification, it is easy to see, even at this relatively low magnification, that about half of the thickness of the epidermis consists of a distinctive surface layer that stains more lightly than the remainder of the epidermis. This is the keratinized layer. The dome-shaped surface contours represent a cross section through the minute ridges on the surface of thick skin that produce the characteristic fingerprints of an individual.

1	In addition to sweat glands, the dermis displays blood vessels (BV) and adipose tissue (AT). The ducts of the sweat glands (D) extend from the glands to the epidermis. One of the ducts is shown as it enters the epidermis at the bottom of an epithelial ridge. It will pass through the epidermis in a spiral course to open onto the skin surface. Thin skin, human, H&E ×60. Often, as in this tissue sample, the hair follicles and the glands, both sebaceous and sweat, extend beyond the dermis (De) and into the hypodermis. A sample of thin skin is shown here to compare with the thick Note the blood vessels (BV) and adipose tissue (AT) in the hypodermis. skin in the above figure. In addition to sweat glands, thin skin contains hair follicles (HF) and their associated sebaceous glands (SGI). Each sebaceous gland opens into a hair follicle. Epidermis, skin, human, H&E ×320; inset ×640.

1	The layers of the epidermis of thin skin are shown here at higher magnification. The cell layer that occupies the deepest location is the stratum basale (SB). This is one cell deep. Just above this is a layer several cells in thickness, the stratum spinosum (SS). It consists of cells that have spinous processes on their surface. These processes meet with spinous processes of neighboring cells and, together, appear as intercellular bridges (arrows, inset). The next layer is the stratum granulosum (SGr), whose cells contain keratohyalin granules (arrowhead, inset). On the surface is the stratum corneum (SC). This consists of keratinized cells, i.e., cells that no longer possess nuclei. The keratinized cells are flat and generally adhere to other cells above and below without evidence of cell boundaries. In thick skin, a fifth layer, the stratum lucidum, is seen between the stratum granulosum and the stratum corneum. The pigment in the cells of the stratum basale is melanin; some of this

1	boundaries. In thick skin, a fifth layer, the stratum lucidum, is seen between the stratum granulosum and the stratum corneum. The pigment in the cells of the stratum basale is melanin; some of this pigment (P) is also present in connective tissue cells of the dermis.

1	KEY AT, adipose tissue BV, blood vessels D, duct of sweat glands De, dermis Ep, epidermis HF, hair follicle P, pigment SB, stratum basale SC, stratum corneum SGl, sebaceous gland SGr, stratum granulosum SS, stratum spinosum SW, sweat gland arrowhead, granules in cell of stratum granulosum arrows, intercellular bridges

1	The epidermis contains four distinctive cell types: keratinocytes, melanocytes, Langerhans’ cells, and Merkel’s cells. Keratinocytes are the most numerous of these cells; they are generated in the stratum basale and move toward the surface. As they do so, they produce the intracellular protein keratin and the special extracellular lipid that serves as a water barrier in the upper layers of the epidermis. Histologically, the keratinocytes are the cells that show spinous processes in the stratum spinosum. The other three cell types are not readily identified in H&E–stained paraffin sections. The product of the melanocyte is, however, evident in H&E sections, and this is considered in the first two figures of this plate. The skin contains a pigment, melanin, which protects the tissue against the harmful effects of ultraviolet light. It is formed by the melanocytes that then pass the pigment to the keratinocytes. More pigment is present in dark skin than in light skin; this can be seen by

1	the harmful effects of ultraviolet light. It is formed by the melanocytes that then pass the pigment to the keratinocytes. More pigment is present in dark skin than in light skin; this can be seen by com-paring light skin (top figure) and dark skin (middle figure). The epidermis and a small amount of the dermis are shown in each figure. Whereas the deep part of the dark skin contains considerable pigment, the amount of pigment in light skin is insufficient to be noticeable at this magni-fication. Cells for producing the pigment are present in both skin types and in approximately equal numbers. The difference is due to more rapid digestion of the pigment by lysosomes of keratinocytes in light skin. After prolonged exposure to sunlight, pigment is also produced in sufficient amounts to be seen in light skin.

1	Light skin, human, H&E ×300. may also appear as clear cells, but they are located more superficially in the stratum spinosum. Merkel’s cells may also appear as clear cells, thus mak-In routine H&E–stained paraffin sections of light skin, such ing it difficult to identify these three cell types with certainty. as this sample, the melanocytes are among the cells that appear as small, rounded, clear cells (CC) mixed with the other cells of the stratum basale. However, not all clear cells of the epidermis are melanocytes. For example, Langerhans’ cells Dark skin, human, H&E ×300. corneum. In light skin, the melanin is broken down before it leaves the upper part of the stratum spinosum. Thus, pigment is not seen in the upper In dark skin, most of the pigment is in the basal portion of the layers of the epidermis.

1	In dark skin, most of the pigment is in the basal portion of the layers of the epidermis. epidermis, but it is also present in cells progressing toward the surface and within the nonnucleated cells of the keratinized layer. The arrows indicate the melanin pigment in keratinocytes of the stratum spinosum and in the stratum Dermis, skin, human, H&E and elastin stain ×200; inset ×450.

1	Dermis, skin, human, H&E and elastin stain ×200; inset ×450. This figure is included because it shows certain features of the dermis, the connective tissue layer of the skin. The dermis is divided into two layers: the papillary layer (PL) of loose connective tissue and the reticular layer (RL) of more dense connective tissue. The papillary layer is immediately under the epidermis. It includes the connective tissue papillae that project into the undersurface of the epidermis. The reticular layer is deep to the papillary layer. The boundary between these two layers is not demarcated by any specific structural feature except for the change in the histologic composition of the two layers.

1	This specimen was stained with H&E and also with a procedure to display elastic fibers (EF). They are relatively thick and conspicuous in the reticular layer (see also inset), where they appear as the dark-blue profiles, some of which are elongate, whereas others are short. In the papillary layer, the elastic fibers are thinner and relatively sparse (arrows). The inset shows the typical eosinophilic staining of the thick collagenous fibers in the reticular layer. Although the collagenous fibers at the lower magnification of this figure are not as prominent, it is nevertheless possible to note that they are thicker in the reticular layer than in the papillary layer. The papillary layer is evidently more cellular than the reticular layer. Many of the small dark-blue profiles in the reticular layer represent oblique and cross sections through elastic fibers (see inset) and not nuclei of cells.

1	KEY CC, clear cells EF, elastic fibers PL, papillary layer RL, reticular layer arrows, middle figure pigment in different layers of epidermis; Lower figure delicate elastic fibers

1	Skin possesses three types of glands: eccrine, apocrine and sebaceous. Eccrine sweat glands are distributed over the entire body surface except for the lips, glans penis, prepuce, clitoris, and labia minora. They are especially numerous in the thick skin of the hands and feet. Evaporation of the secreted sweat on the skin surface cools the body. Apocrine sweat glands are localized in the axilla, areolae, perineal and perianal area, prepuce, scrotum, mons pubis, and labia majora. Many of the epithelial cells in the secretory segment of these glands exhibit an apical bleb-like protrusion that was earlier thought to represent their mode of secretion, i.e., pinching off of the bleb as the secretory product, thus the name apocrine. It is now known that secretion occurs as a merocrine process. The secretion is a clear, viscous product that becomes odife-rous through the action of resident microbes on the skin surface. In the human, its role is unclear, but it is generally believed that the

1	The secretion is a clear, viscous product that becomes odife-rous through the action of resident microbes on the skin surface. In the human, its role is unclear, but it is generally believed that the secretion may act as a sex attractant (pheromone). Apocrine glands are present at birth but do not fully develop and become functional until puberty. In the female, these glands undergo changes that parallel the menstrual cycle. ORIENTATION MICROGRAPH: The adjacent orientation micrograph of the skin of the axilla shows both the large, branched tubular apocrine glands (A) and the smaller, simple tubular eccrine glands (E) in the hypodermis (H). Also evident is a tangentially cut hair follicle (HF). The overlying dermis (D) consists of dense connec-tive tissue and includes part of a sebaceous gland (SG). AAEESGDHFHA A E E SG D HF H

1	Apocrine sweat gland, skin, human, H&E, ×33. image are two sweat glands (SwG) also surrounded by dense connective tissue. Note the considerable difference in diameter and lumen size of the Low-power micrograph showing the secretory segment of an two types of glands. apocrine sweat gland. The sectioned profiles seen here represent several coiled branches of a single gland surrounded by dense connective tissue (DCT). In the upper part of this Apocrine sweat gland, skin, human, H&E, ×256. The epithelium (Ep) of the apocrine sweat gland from the boxed area to the left is simple columnar. The individual cells vary in height, and some show the bleb-like protrusions (B). At the base of the epithelium are the spindle-shaped myoepithelial cells. In some regions of the tubule, these cells have been cut longitudinally and thus appear as a deeply stained eosin band (EB). At other sites, the cells have been sectioned tangentially and appear as a series of parallel linear profiles (MyC).

1	Eccrine sweat gland, skin, human, H&E, ×512. At this very high magnification, two cross-sectioned profiles of the secretory segment (SS) and one profile of the duct segment (DS) from the boxed area to the left are shown. When the tubule wall of the secretory segment is cut in a perpendicular plane, the simple columnar nature of the epithelium (Ep) is evident. Because the tubule is so tortuous, more often the epithelium appears to be multilayered. The myoepithelial cells of the secretory segment appear here both as a circumferential band (CB) and in a cross-sectioned array (CA) in which they resemble the teeth of a saw blade. Occasionally, myoepithelial cell nuclei (MyN) are present. Such profiles give the appearance of a pseudostratified epithelium. The duct segment (DS) lacks the myoepithelium and also differs in that it is stratified cuboidal. See next plate.

1	KEY A, apocrine gland B, bleb-like protrusions CA, cross-sectioned array CB, circumferential band D, dermis DCT, dense connective tissue DS, duct segment E, eccrine gland EB, eosin band Ep, epithelium H, hypodermis HF, hair follicle MyC, linear profiles of myoepithelial cells MyN, myoepithelial cell nuclei SG, sebaceous gland SS, secretory segment SwG, sweat glands

1	Normally, the body loses ~600 mL of water a day through evaporation from the lungs and skin. Under conditions of high ambient temperature, water loss is increased by an increased rate of sweating. This thermoregulatory sweating first occurs on the forehead and scalp, extends to the face and the rest of the body, and occurs last on the palms and soles. Emotional sweating, however, occurs first on the palms and soles and in the axillae. Sweating is under both nervous control through the autonomic nervous system and hormonal control. Sebaceous glands secrete sebum, an oily substance that coats the hair and skin surface. Sebaceous secretion is a holocrine secretion; the entire cell produces, and becomes filled with, the fatty secretory product while it simultaneously undergoes progressive disruption, fol-lowed by necrosis, as the product fills the cell. Both secretory product and cell debris are discharged into the pilosebaceous canal. Sweat gland, skin, human, H&E ×1000.

1	Sweat gland, skin, human, H&E ×1000. This section through a sweat gland shows five profiles of the ductal portion (D) and two profiles of the secretory portion (SG). The larger secretory segment is through a region either just below or above where a U turn was made; therefore, it shows two luminal profiles. The lumina of both the ductal 520 and the secretory units are marked by asterisks. The glandular unit of the eccrine sweat gland contains two epithelial cell types and myoepithelial cells (M). Arrowheads show small cross sections of myoepithelial cell cytoplasm; large arrows show where more elongate profiles of myoepithelial cytoplasm are evident. The epithelial cells are of Sebaceous gland, skin, human, H&E ×160.

1	Sebaceous gland, skin, human, H&E ×160. Sebaceous glands develop from the epithelial cells of the hair follicle and discharge their secretion into the follicle, from where it reaches the skin surface. The sebaceous secretion is rich in lipid, and this is reflected in the cells of the sebaceous gland. A section of a sebaceous gland and its related hair folli cle is shown in this figure. At this level, the hair follicle consists of the ex- Sebaceous gland, skin, human, H&E ×320.

1	The same sebaceous gland as in figure above is shown here at higher magnification. Numbers 1 to 4 show a series of cells filled with an increasingly greater amount of lipid and progressively closer to the opening of the gland into the hair follicle. The sebaceous secretion includes the entire cell, and two types, designated dark cells and clear cells. Unfortunately, the characteristic dark cytoplasmic staining of the dark cells is not evident unless special precautions are taken to preserve the secretory granules in their apical cytoplasm. Nevertheless, note that the dark cells are closer to the lumen, whereas the clear cells are closer to the base of the epithelial layer, making contact with either the basal lamina or, more frequently, the myoepithelial cells. In addition, the clear cells are in contact with intercellular canaliculi. Several such intercellular canaliculi are shown in the secretory units (small arrows). This figure also shows that the duct consists of two layers of

1	are in contact with intercellular canaliculi. Several such intercellular canaliculi are shown in the secretory units (small arrows). This figure also shows that the duct consists of two layers of small cuboidal cells.

1	PLATE 45 • SW EAT AN D S E BACEOUS G LAN DS KEY BC, basal cells CT, connective tissue D, duct of eccrine sweat gland eRS, junction between sebaceous gland and external root sheath M, myoepithelial cell RS, external root sheath of hair follicle Seb, sebaceous gland SG, secretory component of eccrine sweat gland arrowheads, myoepithelial cell cytoplasm (cross section) asterisks, lumina of glands and ducts large arrows, myoepithelial cell cytoplasm (longitudinal section) numbers 1 to 4 (lower right image), see text small arrows, intercellular canaliculi ternal root sheath (RS) surrounding the hair shaft. The sebaceous gland (Seb) appears as a cluster of cells, most of which display a washed-out or finely reticulated cytoplasm. This is because these cells contain numerous lipid droplets and the lipid is lost by dissolution in fat solvents during the routine preparation of the H&E–stained paraffin section. The opening of the sebaceous gland through the external root sheath (eRS) and into

1	the lipid is lost by dissolution in fat solvents during the routine preparation of the H&E–stained paraffin section. The opening of the sebaceous gland through the external root sheath (eRS) and into the hair follicle is shown in the lower right.

1	therefore, cells need to be replaced constantly in the functional gland. Cells at the periphery of the gland are basal cells (BC). Dividing cells in the basal layer replace those that are lost with the secretion.

1	The skin is endowed with numerous sensory receptors of various types. These are the peripheral terminals of sensory nerves whose cell bodies are in the dorsal root ganglia. The receptors in the skin are described as free nerve endings and encapsulated nerve endings. Free nerve endings are the most numerous. They subserve fine touch, heat, and cold and are found in the basal layers of the epidermis and as a network around the root sheath of hair follicles. Encapsulated nerve endings include Pacinian corpuscles (deep pressure), Meissner’s corpuscles (touch, especially in the lips and thick skin of fingers and toes), and Ruffini endings (sustained mechanical stress on the dermis). Motor endings of the autonomic nervous system supply the blood vessels, the arrector pili muscles, and the apocrine and eccrine sweat glands. Skin, fngertip, human, H&E ×20.

1	Skin, fngertip, human, H&E ×20. This specimen is a section of thick skin from the finger tip, showing the epidermis (Ep) and the dermis (De) and, under the skin, a portion of the hypodermis (Hy). The thickness of the epidermis is largely due to the thickness of the stratum corneum. This layer is more lightly stained than the deeper 522 portions of the epidermis. Note, even at this low magnification, the thick collagenous fibers in the reticular layer of the dermis. Sweat glands (SG) are present in the upper part of the hypodermis, and several sweat ducts (D) are seen passing through the epidermis. A feature of this specimen is that it depicts those sensory receptors that can be recognized in a routine low-power H&E–stained paraffin section. Meissner's corpuscle, skin, human, H&E ×190.

1	Meissner's corpuscle, skin, human, H&E ×190. This high-magnification micrograph shows portions of the upper left field of figure above in which two Meissner’s corpuscles (MC) are in direct proximity to the undersurface of the epidermis in adjacent dermal papillae. The section shows the long axis of the corpuscles. A Meissner’s corpuscle consists of an axon (sometimes two) taking a zigzag or flat spiral course They are Meissner’s corpuscles and Pacinian corpuscles (PC). Several nerve bundles (N) are seen in proximity to the Pacinian corpuscles. Meissner’s corpuscles are in the upper part of the dermis, in the dermal papillae immediately under the epidermis. These corpuscles are small and difficult to identify at this low magnification; however, their location is characteristic. Knowing where they are located is a major step in finding Meissner’s corpuscles in a tissue section; they are shown at higher magnification in figure below.

1	Pacinian corpuscles are seen in the lower part of the hypodermis. These corpuscles are large, slightly oval structures, and even at low magnification, a layered or lamellated pattern can be discerned. Pacinian corpuscle, skin, human, H&E ×320. At this higher magnification, the concentric layers or lamellae of the Pacinian corpuscle can be seen to be due to flat cells. These are fibroblast-like cells, and although not evident within the tissue section, these cells are continuous with the perineurium of the nerve fiber. The space between the cellular lamellae contains mostly fluid. The neural portion of the Pacinian corpuscle travels longitudinally through the center of the corpuscle. In this specimen, the corpuscle has been cross-sectioned; an arrowhead points to the centrally located nerve fiber.

1	from one pole of the corpuscle to the other. The nerve fiber terminates at the superficial pole of the corpuscle. Consequently, as seen here, the nerve fibers and supporting cells are oriented approximately at right angles to the long axis of the corpuscle. Meissner’s corpuscles are particularly numerous near the tips of the fingers and toes. Meissner's corpuscle, skin, human, H&E ×550. seen) and its supporting cells is evident here, as is the fibrous capsule (FC) that surrounds the ending. At the even higher magnification of this figure, the close apposition of Meissner’s corpuscle to the undersurface of the epidermis is well demonstrated throughout the entire area of the dermal papilla. The flat spiral path of the neuron (not PLATE 46KEY D, ducts of sweat glands De, dermis Ep, epidermis FC, fibrous capsule Hy, hypodermis MC, Meissner’s corpuscles. N, nerve bundles PC, Pacinian corpuscles SG, sweat glands arrowhead, nerve fiber in center of Pacinian corpuscle

1	Hairs are composed of keratinized cells that develop from hair follicles. Hairs are present over almost the entire body, being conspicuously absent only from the sides and palmar surfaces of the hands, from the sides and plantar surfaces of the feet, from the lips, and from the skin around the urogenital orifices. Coloration of the hair is due to the content and type of melanin that it contains. The follicle varies in appear-ance, depending on whether it is in a growing or a resting phase; the growing follicle is the more elaborate. The skin appendages (adnexa), especially hair follicles and sweat glands, are particularly important in healing of skin wounds. They serve as the source of new epithelial cells when there is extensive loss of epidermis, as in deep abrasions and second-degree burns. Hair follicle, skin, human, H&E ×300; inset ×440.

1	The growing end of a hair follicle consists of an expanded bulb of epithelial cells that is invaginated by a papilla (HP) of connective tissue. The epithelial cells surrounding the papilla at the very tip of the follicle are not yet specialized; they constitute the matrix, the region of the hair follicle where cell division occurs. As the cells leave the matrix, they form cell layers that will 524 become the shaft of the hair and the inner and outer root sheaths of the hair follicle. The cells that will develop into the shaft of the hair are seen just to the right of the expanded bulb. They constitute the cortex (C), medulla (M), and cuticle (asterisks) of the hair. The cells of the cortex become keratinized. This layer will come to constitute most of the hair shaft as a thick cylinder. The medulla forms the centrally located axis of the hair shaft; it does not always extend through the entire length of the hair and is absent from some hairs. The cuticle consists of overlapping cells

1	medulla forms the centrally located axis of the hair shaft; it does not always extend through the entire length of the hair and is absent from some hairs. The cuticle consists of overlapping cells that ultimately lose their nuclei and become filled with keratin. The cuticle covers the hair shaft like a layer of overlapping shingles.

1	The root sheath (RS) has two parts: the outer root sheath, which is continuous with the epidermis of the skin, and the inner root sheath, which extends only as far as the level at which sebaceous glands enter the hair follicle. The inner root sheath is further divided into three layers: Henle’s layer, Huxley’s layer, and the cuticle of the inner root sheath. These layers are seen in the growing hair follicle and are shown at higher magnification in the inset with numbers 1 to 5: 1, cells of the outer root sheath; 2, Henle’s layer; 3, Huxley’s layer; 4, cuticle of the inner root sheath; and 5, future cuticle of the hair. Many of the cells of the growing hair follicle contain pigment that contributes to the color of the hair. Most of this pigment is inside the cell (inset); however, in very dark hair some pigment is also extracellular. The connective tissue surrounding the hair follicle forms a distinct layer referred to as the sheath, or dermal sheath (DS), of the hair follicle.

1	The connective tissue surrounding the hair follicle forms a distinct layer referred to as the sheath, or dermal sheath (DS), of the hair follicle. Nail, skin, human, H&E ×12. A nail is a keratinized plate located on the dorsal aspect of the distal phalanges. A section through a nail plate is shown here. The nail itself (N) is difficult to stain. Under the free edge of the nail is a boundary layer, the hyponychium (Hypon), which is continuous with the stratum corneum of the adjacent epidermis. The proximal end of the nail is overlapped by skin; here, the junctional region is called the eponychium (Epon) and is also continuous with the stratum corneum of the adjacent epidermis. Under the nail is a layer of epithelium, the proximal portion of which is referred to as the nail matrix (NM). The cells of the nail matrix function in the growth of the nail.

1	Together, the epithelium under the nail and the underlying dermis (D) constitute the nail bed. The proximal portion of the nail, covered by the fold of the skin, is the root of the nail (NR). The relationship of the nail to other structures in the fingertip is also shown in this figure. The bone (B) in the specimen represents a distal phalanx. Note that in this bone there is an epiphyseal growth plate (EP) at the proximal extremity of the bone but not at the distal extremity. Numerous Pacinian corpuscles (PC) are present in the connective tissue of the palmar side of the finger. Also seen to advantage in this section is the stratum lucidum (SL) in the epidermis of the thick skin of the fingertip.

1	PLATE 47 KEY B, bone C, cortex D, dermis DS, dermal sheath EP, epiphyseal plate Epon, eponychium HP, dermal papilla of hair follicle Hypon, hyponychium M, medulla N, nail or nail plate NM, nail matrix NR, nail root PC, Pacinian corpuscles RS, root sheath SL, stratum lucidum asterisks, cuticle of hair numbers: 1, external root sheath; 2, Henle’s layer; 3, Huxley’s layer; 4, cuticle of inner root sheath; 5, future cuticle of the hair Digestive System I: Oral Cavity and Associated Structures

1	Digestive System I: Oral Cavity and Associated Structures OVERVIEW OF THE DIGESTIVE SYSTEM / 526 ORAL CAVITY / 527 TONGUE / 529 TEETH AND SUPPORTING TISSUES / 534 Enamel / 536 Cementum / 539 Dentin / 539 Dental Pulp and Central Pulp Cavity (Pulp Chamber) / 543 Supporting Tissues of the Teeth / 544 SALIVARY GLANDS / 545 Secretory Gland Acini / 546 Salivary Ducts / 549 Major Salivary Glands / 550 Parotid Gland / 550 Submandibular Gland / 551 Sublingual Gland / 551 Saliva / 551 Folder 16.1 Clinical Correlation: The Genetic Basis of Taste / 533 Folder 16.2 Clinical Correlation: Classification of Permanent (Secondary) and Deciduous (Primary) Dentition / 534 Folder 16.3 Clinical Correlation: Dental Caries / 547 Folder 16.4 Clinical Correlation: Salivary Gland Tumors / 555 The digestive system consists of the alimentary canal and its principal associated organs, namely, the tongue, teeth, salivary glands, pancreas, liver, and gallbladder.

1	The digestive system consists of the alimentary canal and its principal associated organs, namely, the tongue, teeth, salivary glands, pancreas, liver, and gallbladder. The lumen of the alimentary canal is physically and functionally external to the body. As it passes through the alimentary canal, food is broken down physically and chemically so that the degraded products can be absorbed into the body. The various segments of the alimentary canal are morphologically specialized for specific aspects of digestion and absorption.

1	After preliminary maceration, moistening, and formation into a bolus by the actions of the structures of the oral cavity and salivary glands, food passes rapidly through the pharynx to the esophagus. The rapid passage of food through the pharynx keeps it clear for the passage of air. The food passes more slowly through the gastrointestinal tract, and during its transit through the stomach and small intestine, the major alterations associated with digestion, solubilization, and absorption occur. Absorption occurs chiefly through the wall of the small intestine. Undigested food and other substances within the alimentary canal, such as mucus, bacteria, desquamated cells, and bile pigments, are excreted as feces. The alimentary mucosa is the surface across which most substances enter the body.

1	The alimentary mucosa is the surface across which most substances enter the body. The alimentary mucosa performs numerous functions in its role as an interface between the body and the environment. These functions include the following:  Secretion. The lining of the alimentary canal secretes, at specific sites, digestive enzymes, hydrochloric acid, mucin, and antibodies.  Absorption. The epithelium of the mucosa absorbs metabolic substrates (e.g., the breakdown products of digestion) as well as vitamins, water, electrolytes, recyclable materials such as bile components and cholesterol, and other substances essential to the functions of the body.  Barrier. The mucosa serves as a barrier to prevent the entry of noxious substances, antigens, and pathogenic organisms. Immunologic protection. Lymphatic tissue within the mucosa serves as the body’s first line of immune defense.

1	The functions listed above are discussed at the beginning of the next chapter. The digestive system is considered in three chapters that deal, respectively, with the oral cavity and pharynx (this chapter); the esophagus and gastrointestinal tract (Chapter 17); and the liver, gallbladder, and pancreas (Chapter 18). The oral cavity consists of the mouth and its structures, which include the tongue, teeth and their supporting structures (periodontium), major and minor salivary glands, and tonsils. The oral cavity is divided into a vestibule and the oral cavity proper. The vestibule is the space between the lips, cheeks, and teeth. The oral cavity proper lies behind the teeth and is bounded by the hard and soft palates superiorly, the tongue and the floor of the mouth inferiorly, and the entrance to the oropharynx posteriorly.

1	Each of the three major salivary glands are paired structures; they include the following:  Parotid gland, the largest of the three glands, located in the infratemporal region of the head. Its excretory duct, the parotid (Stensen’s) duct, opens at the parotid papilla, a small elevation on the mucosal surface of the cheek opposite the second upper molar tooth.  Submandibular gland, located in the submandibular triangle of the neck. Its excretory duct, the submandibular (Wharton’s) duct, opens at a small fleshy prominence (the sublingual caruncle) on each side of the lingual frenulum on the floor of the oral cavity.  Sublingual gland, lying inferior to the tongue within the sublingual folds at the floor of the oral cavity. It has a number of small excretory ducts; some enter the submandibular duct, and others enter individually into the oral cavity.

1	The parotid and submandibular glands have relatively long ducts that extend from the secretory portion of the gland to the oral cavity. The sublingual ducts are relatively short. The minor salivary glands are located in the submucosa of the oral cavity. They empty directly into the cavity via short ducts and are named for their location (i.e., buccal, labial, lingual, and palatine). The tonsils consist of aggregations of lymphatic nodules that are clustered around the posterior opening of the oral and nasal cavities.

1	The tonsils consist of aggregations of lymphatic nodules that are clustered around the posterior opening of the oral and nasal cavities. Lymphatic tissue is organized into a tonsillar (Waldeyer’s) ring of immunologic protection located at the shared entrance to the digestive and respiratory tracts. This lymphatic tissue surrounds the posterior orifice of the oral and nasal cavities and contains aggregates of lymphatic nodules that include the following:  Palatine tonsils, or simply the tonsils, which are located at either side of the entrance to the oropharynx between the palatopharyngeal and palatoglossal arches  Tubal tonsils, which are located in the lateral walls of the nasopharynx posterior to the opening of the auditory tube  Pharyngeal tonsil, or adenoid, which is located in the roof of the nasopharynx  Lingual tonsil, which is located at the base of the tongue

1	The oral cavity is lined by the oral mucosa that consists of masticatory mucosa, lining mucosa, and specialized mucosa. The masticatory mucosa is found on the gingiva (gums) and the hard palate (Fig. 16.1). It has a keratinized and, in some areas, a parakeratinized stratifed squamous epithelium (Fig. 16.2). Parakeratinized epithelium is similar to keratinized FIGURE 16.1 • Roof of oral cavity. The hard palate, which contains bone, is bisected into right and left halves by a raphe. Anteriorly, in the fatty zone, the submucosa of the hard palate contains adipose tissue; posteriorly, in the glandular zone, there are mucous glands within the submucosa. Neither the raphe nor the gingiva contains a submucosa; instead, the mucosa is attached directly to the bone. The soft palate has muscle instead of bone, and its glands are continuous with those of the hard palate in the submucosa. (Based on Bhaskar SN, ed. Orban’s Oral Histology and Embryology. St. Louis: CV Mosby, 1991.)

1	FIGURE 16.2 • Stratified squamous epithelium of the hard palate. This photomicrograph shows a transition in the oral mucosa from a stratified squamous epithelium (on the right) to a stratified squamous parakeratinized epithelium (on the left). The flattened surface cells of the keratinized epithelium are devoid of nuclei. The layer of keratohyalin granule–containing cells is clearly visible in this type of epithelium. The flattened surface cells of the parakeratinized epithelium display the same characteristics as the keratinized cells, except they retain their nuclei, i.e., they are parakeratinized. In addition, note the paucity of keratohyalin granules present in the subsurface cells. 380.

1	epithelium except that the superficial cells do not lose their nuclei and their cytoplasm does not stain intensely with eosin (Plate 48, page 557). The nuclei of the parakeratinized cells are pyknotic (highly condensed) and remain until the cell is exfoliated (see Fig. 16.2). The keratinized epithelium of the masticatory mucosa resembles that of the skin but lacks a stratum lucidum. The underlying lamina propria consists of a thick papillary layer of loose connective tissue that contains blood vessels and nerves, some of which send bare axon endings into the epithelium as sensory receptors, and some of which end in Meissner’s corpuscles. Deep to the lamina propria is a reticular layer of more-dense connective tissue.

1	As in the skin, the depth and number of connective tissue papillae contribute to the relative immobility of the masticatory mucosa, thus protecting it from frictional and shearing stress. At the midline of the hard palate, in the palatine raphe, the mucosa adheres firmly to the underlying bone. The reticular layer of the lamina propria blends with the periosteum, and thus there is no submucosa. The same is true of the gingiva. Where there is a submucosa underlying the lamina propria on the hard palate (see Fig. 16.1), it contains adipose tissue anteriorly (fatty zone) and mucous glands posteriorly (glandular zone) that are continuous with those of the soft palate. In the submucosal regions, thick collagenous bands extend from the mucosa to the bone.

1	Lining mucosa is found on the lips, cheeks, alveolar mucosal surface, floor of the mouth, inferior surfaces of the tongue, and soft palate. At these sites it covers striated muscle (lips, cheeks, and tongue), bone (alveolar mucosa), and glands (soft palate, cheeks, inferior surface of the tongue). The lining mucosa has fewer and shorter papillae so that it can adjust to the movement of its underlying muscles.

1	Generally, the epithelium of the lining mucosa is nonkeratinized, although in some places it may be parakeratinized. The epithelium of the vermilion border of the lip (the reddish portion between the moist inner surface and the facial skin) is keratinized. The nonkeratinized lining epithelium is thicker than keratinized epithelium. It consists of only three layers:  Stratum basale, a single layer of cells resting on the basal lamina  Stratum spinosum, which is several cells thick  Stratum superfciale, the most superficial layer of cells, also referred as the surface layer of the mucosa The cells of the mucosal epithelium are similar to those of the epidermis of the skin and include keratinocytes, Langerhans’ cells, melanocytes, and Merkel’s cells.

1	The cells of the mucosal epithelium are similar to those of the epidermis of the skin and include keratinocytes, Langerhans’ cells, melanocytes, and Merkel’s cells. The lamina propria contains blood vessels, nerves that send bare axon endings into the basal layers of the epithelium, and encapsulated sensory endings in some papillae. The sharp contrast between the numerous deep papillae of the alveolar mucosa and the shallow papillae in the rest of the lining mucosa allows easy identification of the two different regions in a histologic section.

1	A distinct submucosa underlies the lining mucosa except on the inferior surface of the tongue. This layer contains large bands of collagen and elastic fibers that bind the mucosa to the underlying muscle; it also contains the many minor salivary glands of the lips, tongue, and cheeks. Occasionally, sebaceous glands not associated with a hair follicle are found in the submucosa just lateral to the corner of the mouth and in the cheeks opposite the molar teeth. They are visible to the eye and are called Fordyce spots. The submucosa contains the larger blood vessels, nerves, and lymphatic vessels that supply the subepithelial neurovascular networks in the lamina propria throughout the oral cavity. Specialized mucosa is associated with the sensation of taste and is restricted to the dorsal surface of the tongue. It contains papillae and taste buds responsible for generating the chemical sensation of taste.

1	Oral mucosa forms an important protective barrier between the external environment of the oral cavity and internal environments of the surrounding tissues. It is resistant to the pathogenic organisms that enter the oral cavity and to indigenous microorganisms residing there as microbial ﬂora. Epithelial cells, migratory neutrophils, and saliva all contribute to maintaining the health of the oral cavity and protecting the oral mucosa from bacterial, fungal, and viral infections. The protective mechanisms include several salivary antimicrobial peptides, the -defensins expressed in the epithelium, the -defensins expressed in neutrophils, and the secretory immunoglobin A (sIgA). However, in individuals with immunodeficiency or undergoing antibiotic therapy, in which the balance between microorganisms and protective mechanisms is disrupted, oral infections are rather common.

1	The tongue is a muscular organ projecting into the oral cavity from its inferior surface. Lingual muscles (i.e., muscles of the tongue) are both extrinsic (having one attachment outside of the tongue) and intrinsic (confined entirely to the tongue, without external attachment). The striated muscle of the tongue is arranged in bundles that generally run in three planes, with each arranged at right angles to the other two. This arrangement of muscle fibers allows enormous flexibility and precision in the movements of the tongue, which are essential to human speech as well as to its role in digestion and swallowing. This form of muscle organization is found only in the tongue, which allows easy identification of this tissue as lingual muscle. Variable amounts of adipose tissue are found among the muscle fiber groups.

1	Grossly, the dorsal surface of the tongue is divided into an anterior two thirds and a posterior one third by a V-shaped depression, the sulcus terminalis (Fig. 16.3). The apex of the V points posteriorly and is the location of the foramen cecum, the remnant of the site from which an evagination of the floor of the embryonic pharynx occurred to form the thyroid gland. Papillae cover the dorsal surface of the tongue. Numerous mucosal irregularities and elevations called lingual papillae cover the dorsal surface of the tongue anterior to the sulcus terminalis. The lingual papillae and their associated taste buds constitute the specialized mucosa of the oral cavity. Four types of papillae are described: filiform, fungiform, circumvallate, and foliate.

1	 Filiform papillae are the smallest and most numerous in humans. They are conical, elongated projections of connective tissue that are covered with highly keratinized stratified squamous epithelium (Fig. 16.4a and Plate 49, page 559). This epithelium does not contain taste buds. The papillae serve only a mechanical role. Filiform papillae are distributed over the entire anterior dorsal surface of the tongue, with their tips pointing backward. They appear to form rows that diverge to the left and right from the midline and that parallel the arms of the sulcus terminalis.

1	 Fungiform papillae, as the name implies, are mushroom-shaped projections located on the dorsal surface of the tongue (Fig. 16.4b). They project above the filiform papillae, among which they are scattered, and are just visible to the unaided eye as small spots (see Fig. 16.3 and Plate 50, page 561). They tend to be more numerous near the tip of the tongue. Taste buds are present in the stratified squamous epithelium on the dorsal surface of these papillae.  Circumvallate papillae are the large, dome-shaped structures that reside in the mucosa just anterior to the sulcus terminalis (see Fig. 16.3). The human tongue has 8 to 12 of these papillae. Each papilla is surrounded by a moatlike invagination lined with stratified squamous epithelium that contains numerous taste buds (Fig. 16.4d). Ducts of lingual salivary (von Ebner’s) glands empty their

1	FIGURE 16.3 • Human tongue. Circumvallate papillae are positioned in a V configuration, separating the anterior two thirds of the tongue from the posterior third. Fungiform and filiform papillae are on the anterior portion of the dorsal tongue surface. The uneven contour of the posterior tongue surface is attributable to the lingual tonsils. The palatine tonsil is at the junction between the oral cavity and the pharynx. serous secretion into the base of the moats. This secretion presumably flushes material from the moat to enable the taste buds to respond rapidly to changing stimuli.

1	serous secretion into the base of the moats. This secretion presumably flushes material from the moat to enable the taste buds to respond rapidly to changing stimuli.  Foliate papillae consist of parallel low ridges separated by deep mucosal clefts (see Fig. 16.4c and Plate 50, page 561), which are aligned at right angles to the long axis of the tongue. They occur on the lateral edge of the tongue. In aged individuals, the foliate papillae may not be recognized; in younger individuals, they are easily found on the posterior lateral surface of the tongue and contain many taste buds in the epithelium of the facing walls of neighboring papillae (Fig. 16.3e). Small serous glands empty into the clefts. In some animals, such as the rabbit, foliate papillae constitute the principal site of aggregation of taste buds. The dorsal surface of the base of the tongue exhibits smooth bulges that reflect the presence of the lingual tonsil in the lamina propria (see Fig. 16.3).

1	FIGURE 16.4 • Lingual papillae. a. Structurally, the filiform papillae are posteriorly bent conical projections of the epithelium. These papillae do not possess taste buds and are composed of stratified squamous keratinized epithelium. 45. b. Fungiform papillae are slightly rounded, elevated structures situated among the filiform papillae. A highly vascularized connective tissue core forms the center of the fungiform papilla and projects into the base of the surface epithelium. Because of the deep penetration of connective tissue into the epithelium (arrows), combined with a very thin keratinized surface, the fungiform papillae appear as small red dots when the dorsal surface of the tongue is examined by gross inspection. 45. c. In a section, foliate papillae can be distinguished from fungiform papillae because they appear in rows separated by deep clefts (arrows). The foliate papillae are covered by stratified squamous nonkeratinized epithelium containing numerous taste buds on their

1	papillae because they appear in rows separated by deep clefts (arrows). The foliate papillae are covered by stratified squamous nonkeratinized epithelium containing numerous taste buds on their lateral surfaces. The free surface epithelium of each papilla is thick and has a number of secondary connective tissue papillae projecting into its undersurface. The connective tissue within and under the foliate papillae contains serous glands (von Ebner’s glands) that open via ducts into the cleft between neighboring papillae. 45. d. Circumvallate papillae are covered by stratified squamous epithelium that may be slightly keratinized. Each circumvallate papilla is surrounded by a trench or cleft. Numerous taste buds are on the lateral walls of the papillae. The dorsal surface of the papilla is smooth. The deep trench surrounding the circumvallate papillae and the presence of taste buds on the sides rather than on the free surface are features that distinguish circumvallate from fungiform

1	smooth. The deep trench surrounding the circumvallate papillae and the presence of taste buds on the sides rather than on the free surface are features that distinguish circumvallate from fungiform papillae. The connective tissue near the circumvallate papillae also contains many serous-type glands that open via ducts into the bottom of the trench. 25.

1	Taste buds are present on fungiform, foliate, and circumvallate papillae. In histologic sections, taste buds appear as oval, pale-staining bodies that extend through the thickness of the epithelium (Fig. 16.5). A small opening onto the epithelial surface at the apex of the taste bud is called the taste pore. Three principal cell types are found in taste buds:  Neuroepithelial (sensory) cells are the most numerous cells in the taste bud. These elongated cells extend from the basal lamina of the epithelium to the taste pore, through which the tapered apical surface of each cell extends microvilli (see Fig. 16.5). Near their apical surface they are connected to neighboring neuroepithelial or supporting cells by tight junctions. At their base they form a synapse with the processes of afferent sensory neurons of the facial (cranial nerve VII), glossopharyngeal (cranial nerve IX), or vagus (cranial nerve X) nerves. The turnover time of neuroepithelial cells is about 10 days.

1	 Supporting cells are less numerous. They are also elongated cells that extend from the basal lamina to the taste pore. Like neuroepithelial cells, they contain microvilli on FIGURE 16.5 • Diagram and photomicrograph of a taste bud. a. This diagram of a taste bud shows the neuroepithelial (sensory), supporting, and basal cells. One of the basal cells is shown in the process of dividing. Nerve fibers have synapses with the neuroepithelial cells. (Based on Warwick R, Williams PL, eds. Gray’s Anatomy, 35th ed. Edinburgh: Churchill Livingstone, 1973.) b. This high-magnification photomicrograph shows the organization of the cells within the taste bud. The sensory and supporting cells extend through the full length of the taste bud. The apical surface of these cells contains microvilli. The basal cells are located at the bottom of the taste bud. Note that the taste bud opens at the surface by means of a taste pore. 640.

1	their apical surface and possess tight junctions, but they do not synapse with the nerve cells. The turnover time of sup porting cells is also about 10 days. Basal cells are small cells located in the basal portion of the taste bud, near the basal lamina. They are the stem cells for the two other cell types. In addition to those associated with the papillae, taste buds are also present on the glossopalatine arch, the soft palate, the posterior surface of the epiglottis, and the posterior wall of the pharynx down to the level of the cricoid cartilage. Taste is a chemical sensation in which various chemicals elicit stimuli from neuroepithelial cells of taste buds.

1	Taste is a chemical sensation in which various chemicals elicit stimuli from neuroepithelial cells of taste buds. Taste is characterized as a chemical sensation in which various tastants (taste-stimulating substances) contained in food or beverages interact with taste receptors located at the apical surface of the neuroepithelial cells. These cells react to five basic stimuli: sweet, salty, bitter, sour, and umami [Jap. delicious]. The molecular action of tastants can involve opening and passing through ion channels (i.e., salt and sour), closing ion channels (sour), or acting on a specific taste G protein–coupled receptors (i.e., bitter, sweet, and umami). Stimulation of bitter, sweet, and umami receptors activates G protein–coupled taste receptors that belong to T1R and T2R chemosensory receptors families.

1	Stimulation of bitter, sweet, and umami receptors activates G protein–coupled taste receptors that belong to T1R and T2R chemosensory receptors families. Bitter, sweet, and umami tastes are detected by a variety of receptor proteins encoded by the two taste receptor genes (T1R and T2R). Their products are all characterized as being G protein–coupled taste receptors.

1	 Bitter taste is detected by about 30 different types of T2R chemosensory receptors. Each receptor represents a single transmembrane protein coupled to its own G protein. After receptor activation by the tastant, the G protein stimulates the enzyme phospholipase C, leading to increased intracellular production of inositol 1,4,5-trisphosphate (IP3), a second messenger molecule. IP3 in turn activates taste-specifc Na channels causing influx of Na ions, thus depolarizing the neuroepithelial cell. Depolarization of the plasma membrane causes voltage-gated Ca2 channels in neuroepithelial cells to open. Increasing the concentration of intracellular Ca2 levels either by influx of extracellular Ca2 into the cell (the effect of depolarization) or by its release from intracellular stores (direct IP3 stimulation) results in the release of neurotransmitter molecules,

1	FIGURE 16.6 • Diagram of taste receptors and their signaling mechanism. a. This diagram shows the signaling mechanism of bitter, sweet and umami receptors in the neuroepithelial cells. These cells selectively express only one class of receptor proteins; for simplicity all three taste receptors are depicted in the apical cell membrane. See text for details. PLC – phospholipase C, IP2–inositol-1,4-diphosphate, IP3 – inositol 1,4,5-trisphosphate (IP3). b. Signaling mechanism in sour sensation is generated by H protons that primary blocks K channels. The H protons enter the cell via amiloride-sensitive Na channels and through taste-specific H channels (PKD1L3 and PKD2L1) exclusively expressed in cells involved in sour taste transduction. c. Salty sensation derives from Na ions that enter the neuroepithelial cells through the amiloride-sensitive Na channels. Intracellular Na causes a depolarization of membrane and activation of additional voltage-sensitive Na and Ca2 channels. Calcium

1	the neuroepithelial cells through the amiloride-sensitive Na channels. Intracellular Na causes a depolarization of membrane and activation of additional voltage-sensitive Na and Ca2 channels. Calcium mediated release of neurotransmitters from synaptic vesicles results in stimulating gustatory nerve fiber.

1	which generate nerve impulses along the gustatory afferent nerve fiber (Fig 16.6a).  Sweet taste receptors are also G protein–coupled receptors. In contrast to the bitter taste receptors, they have two protein subunits, T1R2 and T1R3. The sweet tastants bound to these receptors activate the same second messanger system cascade of reactions that the bitter receptors do (see Fig 16.6a).

1	 Umami taste is linked to certain amino acids (e.g., L-glutamate, aspartate, and related compounds) and is common to asparagus, tomatoes, cheese, and meat. Umami taste receptors are very similar to sweet receptors; they are also comprised of two subunits. One subunit, T1R3, is identical to that in the sweet receptor, but the second subunit formed by the T1R1 protein is unique for umami receptors (see Fig 16.6a). The transduction process is identical to that described previously for bitter taste pathways. Monosodium glutamate, added to many foods to enhance their taste (and the main ingredient of soy sauce), stimulates the umami receptors. The mechanism of transtuction can be similar to several tastes (i.e., bitter or sweet); however, it is important to remember that neuroepithelial cells selectively express only one class of receptor proteins. Therefore, the messages about bitterness or sweetness from eating food are transferred to the CNS along different nerve fibers.

1	Sodium ions and hydrogen protons, which are responsible for salty and sour taste respectively, act directly on ion channels. Signaling mechanisms, in the case of sour and salty tastes, are similar to other signaling mechanisms found in synapses and neuromuscular junctions.  Sour taste is generated by H protons that are formed by hydrolysis of acidic compounds. The H primary blocks K channels that are responsible for generating the cell membrane potential that causes depolarization of the cell membrane. In addition, H protons enter the cell through amiloride-sensitive Na channels and through specification channels, called PKD1L3 and PKD2L1, found in neuroepithelial cells exclusively involved in sour taste transduction. The entry of H into receptor cell activates the voltage-sensitive Ca2 channels. Influx of Ca2 triggers migration of synaptic vesicles, their fusion, and transmitter release, which results in generating action potentials in apposed sensory nerve fiber (Fig 16.6b).

1	 Salty taste that is stimulated by table salt (NaCl) is essentualy derived from the taste of the sodium ions. The Na enter the neuroepithelial cells through the specific amiloride-sensitive Na channels (the same that are involved in sour taste transmission). These channels are different from voltage-sensitive Na channels that generate action potentials in nerve or muscle cells. The entry of Na into receptor cell causes a depolarization of its membrane and activation of additional voltage-sensitive Na channels and voltage-sensitive Ca2 channels. As previously described, influx of Ca2 triggers migration and release of neurotransmitter from synaptic vesicles, which results in stimulating gustatory nerve fiber (Fig 16.6c). Some areas of the tongue are more responsive to certain tastes than others.

1	Some areas of the tongue are more responsive to certain tastes than others. In general, taste buds at the tip of the tongue detect sweet stimuli, those immediately posterolateral to the tip detect salty stimuli, and those more posterolateral detect sour-tasting stimuli. Taste buds on the circumvallate papillae detect bitter and umami stimuli. However, studies with thermal stimulation of the tongue have shown that the classic taste maps as described above represent an oversimplified view of the distribution of taste receptors. Sensitivity to all tastes is distributed across the entire tongue, but some areas are indeed more responsive to certain tastes than others. The lingual tonsil consists of accumulations of lymphatic tissue at the base of the tongue.

1	The lingual tonsil consists of accumulations of lymphatic tissue at the base of the tongue. The lingual tonsil is located in the lamina propria of the root or base of the tongue. It is found posterior to the sulcus terminalis (see Fig. 16.3). The lingual tonsil contains diffuse lymphatic tissue with lymphatic nodules containing germinal centers. These structures are discussed in Chapter 14, Lymphatic Tissues and Organs. Epithelial crypts usually invaginate into the lingual tonsil. However, the structure of the epithelium may be difficult to distinguish because of the extremely large number of lymphocytes that normally invade it. Between nodules, the lingual epithelium has the characteristics of lining epithelium. Mucous lingual salivary glands may be seen within the lingual tonsil and may extend into the muscle of the base of the tongue. The complex nerve supply of the tongue is provided by cranial nerves and the autonomic nervous system.

1	The complex nerve supply of the tongue is provided by cranial nerves and the autonomic nervous system.  General sensation for the anterior two thirds of the tongue (anterior to the sulcus terminalis) is carried in the mandibular division of the trigeminal nerve (cranial nerve V). General sensation for the posterior one third of the tongue is carried in the glossopharyngeal nerve (cranial nerve IX) and the vagus nerve (cranial nerve X).  Taste sensation is carried by the chorda tympani, a branch of the facial nerve (cranial nerve VII) anterior to the sulcus terminalis, and by the glossopharyngeal  FOLDER 16.1 Clinical Correlation: The Genetic Basis of Taste

1	The general ability to taste as well as the ability to sense specific tastes is genetically determined. Studies in large populations demonstrate that taste variation is common. About 25% of the population, referred to as “super-tasters,” have more than the normal number of lingual papillae and a high density of taste buds. Rare individuals in this group, such as wine, brandy, coffee, or tea tasters, have prodigious taste discrimination and taste memory. These individuals are characterized by their extreme sensi-tivity to the chemical phenylthiocarbamide (PTC) and its derivative 6-N-propylthiouracil (PROP); they typically re-port an intensely bitter taste after a drop of PTC/PROP solution is placed on the tip of their tongue. On the other side of the spectrum (about 25% of the population) are in-dividuals known as “nontasters,” with a smaller than normal number of lingual papillae and a lower density of taste buds. When tested with PTC/PROP solution, these individuals are unaware of

1	are in-dividuals known as “nontasters,” with a smaller than normal number of lingual papillae and a lower density of taste buds. When tested with PTC/PROP solution, these individuals are unaware of its bitter taste. Many clinical conditions can affect taste perception. They include lesions in the nerves that transmit the taste sensa-tion to the central nervous system; inflammations of the oral cavity; mucosal disorders including radiation-induced in-flammation of the lingual mucosa; nutritional deficiencies; endocrine disorders such as diabetes mellitus, hypogo-nadism, and pseudohypoparathyroidism; and hormonal fluctuations during menstruation and pregnancy. Some rare genetic disorders also affect taste sensation. Type I familial dysautonomia (Riley-Day syndrome) causes se-vere hypogeusia (decreased ability to detect taste) be-cause of the developmental absence of taste buds and fungiform papillae. This sensory and autonomic neuropathy is an autosomal recessive disorder caused by a

1	(decreased ability to detect taste) be-cause of the developmental absence of taste buds and fungiform papillae. This sensory and autonomic neuropathy is an autosomal recessive disorder caused by a mutation in the DYS gene (also referred to as the IKBKAP gene) lo-cated on chromosome 9. In addition to hypogeusia, these individuals experience other symptoms related to develop-mental defects in the peripheral and autonomic nervous systems, including diminished lacrimation, defective ther-moregulation, orthostatic hypotension, excessive sweating, loss of pain and temperature sensation, and absent re-flexes. A test that detects the causative mutation in the DYS gene has recently been developed to confirm the di-agnosis of familial dysautonomia.

1	nerve (cranial nerve IX) and vagus nerve (cranial nerve X) posterior to the sulcus.  Motor innervation for the musculature of the tongue is supplied by the hypoglossal nerve (cranial nerve XII).  Vascular and glandular innervation is provided by the sympathetic and parasympathetic nerves. They supply blood vessels and small salivary glands of the tongue. Ganglion cells are often seen within the tongue. These cells belong to postsynaptic parasympathetic neurons and are destined for the minor salivary glands within the tongue. The cell bodies of sympathetic postsynaptic neurons are located in the superior cervical ganglion.

1	Teeth are a major component of the oral cavity and are essential for the beginning of the digestive process. Teeth are embedded in and attached to the alveolar processes of the maxilla and mandible. Children have 10 deciduous (primary, milk) teeth in each jaw, on each side:  A medial (central) incisor, the first tooth to erupt (usually in the mandible) at approximately 6 months of age (in some infants, the first teeth may not erupt until 12 to 13 months of age)  A lateral incisor, which erupts at approximately 8 months  A canine tooth, which erupts at approximately 15 months  Two molar teeth, the first of which erupts at 10 to 19 months and the second of which erupts at 20 to 31 months

1	During a period of years, usually beginning at about age 6 and ending at about age 12 to 13, deciduous teeth are gradually replaced by 16 permanent (secondary) teeth in each jaw (Folder 16.2). Each side of both upper and lower jaws consists of the following:  A medial (central) incisor, which erupts at age 7 to 8  A lateral incisor, which erupts at age 8 to 9  A canine tooth, which erupts at age 10 to 12  Two premolar teeth, which erupt between ages 10 and 12  Three molar teeth, which erupt at different times; the first molar usually erupts at age 6, the second molar in the early teens, and the third molar (wisdom teeth) during the late teens or early twenties Incisors, canines, and premolars have one root each, except for the first premolar of the maxilla, which has two roots. Molars have either two roots (lower jaw) or three (upper jaw) and, on rare occasions, four roots. All teeth have the same basic structure, however.

1	 FOLDER 16.2 Clinical Correlation: Classification of Permanent (Secondary) and Deciduous (Primary) Dentition

1	Three systems are currently used to classify permanent and deciduous teeth (Fig. F16.2.1):  Palmer system, which was the most commonly used notation worldwide. In this system, uppercase letters are used for the deciduous teeth, and arabic numerals are used for the permanent teeth. Each quadrant in this sys-tem is designated by angled lines: for upper right (UR), for upper left (UL), for lower right (LR), and for lower left (LL). For example, permanent canines are called number 3 in each quadrant, and the quadrant is desig-nated by its angled line.  International system, which uses two arabic numer-als to designate the individual tooth. In this system, the first numeral indicates the location of the tooth in a spe-cific quadrant. The permanent quadrants are designated UR 1, UL 2, LL 3, and LR 4; the deciduous quadrants are designated UR 5, UL 6, LL 7, and LR 8. The second numeral designates the individual tooth, which is numbered beginning from the dental mid-line. For example, in

1	and LR 4; the deciduous quadrants are designated UR 5, UL 6, LL 7, and LR 8. The second numeral designates the individual tooth, which is numbered beginning from the dental mid-line. For example, in this system, the permanent canines are named 13, 23, 33, and 43, and the deciduous ca-nines would be 53, 63, 73, and 83.  American (Universal) system, which is the most commonly used notation in North America. In this system, the permanent dentition is designated by arabic numer-als, and the deciduous dentition is designated with up-percase letters. For permanent dentition, numbering begins in the UR quadrant, with the UR third molar des-ignated number 1. Numbering continues across the maxillary arch to the UL third molar, designated tooth number 16. Tooth number 17 is the third molar located in the LL quadrant inferior and opposite to tooth number 16. Then, the numbering progresses across the mandibular arch and terminates with tooth number 32, the LR third molar. In this system, the

1	in the LL quadrant inferior and opposite to tooth number 16. Then, the numbering progresses across the mandibular arch and terminates with tooth number 32, the LR third molar. In this system, the sum of the num-bers of opposing teeth adds up to 33. For the decidu-ous dentition, the same pattern is followed, but the letters A to T are used to designate the individual teeth. Thus, in this system, the permanent canines are desig-nated 6, 11, 22, and 27, and the deciduous canines, C, H, M, and R. Also note that in Figure F16.2.1 the color outline demonstrates the relationship of the deciduous and per-manent dentitions. Examination of the table reveals that de-ciduous molars are replaced with permanent premolars after exfoliation and that the permanent molars have no de-ciduous precursors. continued next page

1	FOLDER 16.2 Clinical Correlation: Classification of Permanent (Secondary) and Deciduous (Primary) Dentition (Cont.)

1	FIGURE F16.2.1 • Classification of permanent and deciduous teeth. Three systems of tooth classification are used. The central panel of the diagram shows the permanent teeth, whereas the upper and lower panels show the deciduous teeth. Dentition is divided into four quadrants: upper left (UL), upper right (UR), lower left (LL), and lower right (LR). Each quadrant includes 8 permanent teeth or 5 deciduous teeth. In the American (Universal) system (blue), permanent teeth are designated with Arabic numerals. The numbering begins from the wisdom tooth in the upper right quadrant designated as tooth number 1 and continues along all the teeth in the maxilla to tooth number 16, which designates the third upper left molar. The numbering progresses to the mandible, beginning at the third left lower molar designated as number 17 to the third lower right molar designated as number 32. In the American system, deciduous teeth are marked with capital letters designated for each tooth. The pattern is

1	molar designated as number 17 to the third lower right molar designated as number 32. In the American system, deciduous teeth are marked with capital letters designated for each tooth. The pattern is the same as that for permanent teeth, so the numbering begins from the second upper right molar and finishes with the second lower right molar. In the International system (red), also referred to as the Two-Digit System, each tooth is designated with two numbers: The first number indicates the dentition quadrant, which is marked from 1 to 4 and from 5 to 8 in clockwise direction beginning from the upper right quadrant for permanent and deciduous teeth, respectively. The second number specifies individual teeth in each quadrant beginning from the midline where the medial incisors are designated as number 1 and third molars are designated as number 8. In the Palmer system (yellow), the dentition is divided into four quadrants with a right-angle bracket. The vertical line of the bracket

1	as number 1 and third molars are designated as number 8. In the Palmer system (yellow), the dentition is divided into four quadrants with a right-angle bracket. The vertical line of the bracket divides the dentition into a right and a left side beginning at the midline. The horizontal line of the bracket divides the dentition into the upper and lower parts to designate teeth in the maxilla and mandible. In the Palmer system, permanent teeth are numbered with Arabic numerals beginning from the midline. The deciduous teeth are marked with capital letters also starting from the midline. To mark a particular tooth with the Palmer system, two lines (vertical and horizontal) and the correct number or letter of the tooth are needed. (Table design courtesy of Dr. Wade T. Schultz.)

1	Teeth consist of several layers of specialized tissues. Teeth are made up of three specialized tissues:  Enamel, a hard, thin, translucent layer of acellular mineralized tissue that covers the crown of the tooth.  Dentin, the most abundant dental tissue; it lies deep to the enamel in the crown and cementum in the root. Its unique tubular structure and biochemical composition support the more rigid enamel and cementum overlying the surface of the tooth.  Cementum, a thin, pale-yellowish layer of bone like calcified tissue covering the dentin of the root of the teeth. Cementum is softer and more permeable than dentin and is easily removed by abrasion when the root surface is exposed to the oral environment. Enamel is the hardest substance in the body; it consists of 96 to 98% calcium hydroxyapatite. Enamel is an acellular mineralized tissue that covers the crown of the tooth. Once formed it cannot be replaced. lines of Retzius

1	Enamel is an acellular mineralized tissue that covers the crown of the tooth. Once formed it cannot be replaced. lines of Retzius Enamel is a unique tissue because, unlike bone, which is formed from connective tissue, it is a mineralized material derived from epithelium. Enamel is more highly mineralized and harder than any other mineralized tissue in the body; it consists of 96 to 98% of calcium hydroxyapatite. The enamel that is exposed and visible above the gum line is called the clinical crown; the anatomic crown describes all of the tooth that is covered by enamel, some of which is below the gum line. Enamel varies in thickness over the crown and may be as thick as 2.5 mm on the cusps (biting and grinding surfaces) of some teeth. The enamel layer ends at the neck, or cervix, of the tooth at the cementoenamel junction (Fig. 16.7); the root of the tooth is then covered by cementum, a bonelike material.

1	Enamel is composed of enamel rods that span the entire thickness of the enamel layer. The nonstoichiometric carbonated calcium hydroxyapatite enamel crystals that form the enamel are arranged as rods that measure 4 m wide and 8 m high. Each enamel rod spans the full thickness of the enamel layer from the dentin showing dentinal tubules interglobular spaces odontoblasts gingival sulcus epithelium of gingiva pulp chamber granular layer of Tomes fibers of periodontal membrane alveolar bone with marrow pulp canal cellular cementum apical foramen

1	FIGURE 16.7 • Diagram of a section of an incisor tooth and surrounding bony and mucosal structures. The three mineralized components of the tooth are dentin, enamel, and cementum. The central soft core of the tooth is the pulp. The periodontal ligament (membrane) contains bundles of collagenous fibers that bind the tooth to the surrounding alveolar bone. The clinical crown of the tooth is the portion that projects into the oral cavity. The anatomic crown is the entire portion of the tooth covered by enamel.

1	dentinoenamel junction to the enamel surface. When examined in cross section at higher magnification, the rods reveal a keyhole shape (Fig. 16.8); the ballooned part, or head, is oriented superiorly, and the tail is directed inferiorly toward the root of the tooth. The enamel crystals are primarily oriented parallel to the long axis of the rod within the head, and in the tail they are oriented more obliquely (Figs. 16.8 and 16.9). The limited spaces between the rods are also filled with enamel crystals. Striations observed on enamel rods (contour lines of Retzius) may represent evidence of rhythmic growth of the enamel in the developing tooth. A wider line of hypomineralization is observed in the enamel of the deciduous teeth. This line, called the neonatal line, marks the nutritional changes that take place between prenatal and postnatal life.

1	Although the enamel of an erupted tooth lacks cells and cell processes, it is not a static tissue. It is influenced by the secretion of the salivary glands, which are essential to its maintenance. The substances in saliva that affect teeth include digestive enzymes, secreted antibodies, and a variety of inor FIGURE 16.8 • Diagram showing the basic organization and structure of enamel rods. The enamel rod is a thin structure extending from the dentinoenamel junction to the surface of the enamel. Where the enamel is thickest, at the tip of the crown, the rods are longest, measuring up to 2,000 m. On cross section, the rods reveal a keyhole shape. The upper ballooned part of the rod, called the head, is oriented superiorly, and the lower part of the rod, called the tail, is directed inferiorly. Within the head, most of the enamel crystals are oriented parallel to the long axis of each rod. Within the tail, the crystals are oriented more obliquely. ganic (mineral) components.

1	ganic (mineral) components. Mature enamel contains very little organic material. Despite its hardness, enamel can be decalcified by acid-producing bacteria acting on food products trapped on the enamel surface. This is the basis of the initiation of dental caries. Fluoride added to the hydroxyapatite complex makes the enamel more resistant to acid demineralization. The widespread use of fluoride in drinking water, toothpaste, pediatric vitamin supplements, and mouthwashes significantly reduces the incidence of dental caries. Enamel is produced by ameloblasts of the enamel organ, and dentin is produced by neural crest–derived odontoblasts of the adjacent mesenchyme.

1	The enamel organ is an epithelial formation that is derived from ectodermal epithelial cells of the oral cavity. The onset of tooth development is marked by proliferation of oral epithelium to form a horseshoe-shaped cellular band of tissue, the dental lamina, in the adjacent mesenchyme where the upper and lower jaws will develop. At the site of each future tooth, there is a further proliferation of cells that arise from the dental lamina, resulting in a rounded, cellular, budlike outgrowth, one for each tooth, that projects into the underlying mesenchymal tissue. This outgrowth, referred to as the bud stage, represents the early enamel organ (Fig. 16.10a). Gradually, the rounded cell mass enlarges and then develops a concavity at the site opposite where it arose from the dental lamina. The enamel organ is now referred to as being in the cap stage (Fig. 16.10b). Further growth and development of the enamel organ results in the bell stage (Fig. 16.10, c and d). At this stage the enamel

1	enamel organ is now referred to as being in the cap stage (Fig. 16.10b). Further growth and development of the enamel organ results in the bell stage (Fig. 16.10, c and d). At this stage the enamel organ consists of four recognizable cellular components:  Outer enamel epithelium, made up of a cell layer that forms the convex surface  Inner enamel epithelium, made up of a cell layer that forms the concave surface  Stratum intermedium, a cell layer that develops internal to the inner enamel epithelium Stellate reticulum, made up of cells that have a stellate ap pearance and occupy the inner portion of the enamel organ

1	The neural crest–derived preodontoblasts lined up within the “bell” adjacent to the inner enamel epithelial cells become columnar and have an epithelial-type appearance. They will become odontoblasts and form the dentin of the tooth. The inner enamel epithelial cells of the enamel organ will become ameloblasts. Along with the cells of the stratum intermedium, they will be responsible for enamel production. At the early stage, just before dentinogenesis and amelogenesis, the dental lamina degenerates, leaving the developing tooth primordium detached from its site of origin. Dental enamel is formed by a matrix-mediated biomineralization process known as amelogenesis. These are the major stages of amelogenesis:

1	Dental enamel is formed by a matrix-mediated biomineralization process known as amelogenesis. These are the major stages of amelogenesis: FIGURE 16.9 • Structure of young enamel. a. This electron micrograph shows enamel rods cut obliquely. Arrows indicate the boundaries between adjacent rods. 14,700. b. Parts of two adjacent rods are seen at higher magnification. Arrows mark the boundary between the two rods. The dark needlelike objects are young hydroxyapatite crystals; the substance between the hydroxyapatite crystals is the organic matrix of the developing enamel. As the enamel matures, the hydroxyapatite crystals grow, and the bulk of the organic matrix is removed. 60,000.

1	 Matrix production, or secretory stage. In the formation of mineralized tissues of the tooth, dentin is produced first. Then, partially mineralized enamel matrix (Fig. 16.11) is deposited directly on the surface of the previously formed dentin. The cells producing this partially mineralized organic matrix are called secretory-stage ameloblasts. As do osteoblasts in bone, these cells produce an organic proteinaceous matrix by activity of the rough endoplasmic reticulum (rER), Golgi apparatus, and secretory granules. The secretory-stage ameloblasts continue to produce enamel matrix until the full thickness of the future enamel is achieved.

1	 Matrix maturation. Maturation of the partially mineralized enamel matrix involves the removal of organic material as well as continued influx of calcium and phosphate into the maturing enamel. Cells involved in this second stage of enamel formation are called maturation-stage ameloblasts. Maturation-stage ameloblasts differentiate from secretory-stage ameloblasts and function primarily as a transport epithelium, moving substances into and out of the maturing enamel. Maturation-stage ameloblasts undergo cyclical alterations in their morphology that correspond to cyclical entry of calcium into the enamel. Secretory-stage ameloblasts are polarized columnar cells that produce enamel.

1	The secretory-stage ameloblast lies directly adjacent to the developing enamel. At the apical pole of each ameloblast is a process, Tomes’s process, which is surrounded by the developing enamel (Fig. 16.12). A cluster of mitochondria in the base of the cell accounts for the eosinophilic staining of this region in hematoxylin and eosin (H&E)– stained paraffin sections (Fig. 16.13). Adjacent to the mitochondria is the nucleus; in the main column of cytoplasm are the rER, Golgi, secretory granules, and other cell elements. Junctional complexes are present at both apical and basal parts of the cell. They maintain the integrity and orientation of the ameloblasts as they move away from the dentoenamel junction. Actin filaments joined to these junctional complexes are involved in moving the secretory-stage ameloblast over the developing enamel. The rod produced by the ameloblast follows in the wake of the cell. Thus, in mature enamel, the direction of the enamel rod is a record of the path

1	ameloblast over the developing enamel. The rod produced by the ameloblast follows in the wake of the cell. Thus, in mature enamel, the direction of the enamel rod is a record of the path taken earlier by the secretory-stage ameloblast.

1	At their base, the secretory-stage ameloblasts are adjacent to a layer of enamel organ cells called the stratum intermedium (see Fig. 16.10, b, c, and g). The plasma membrane of these cells, especially at the base of the ameloblasts, contains alkaline phosphatase, an enzyme active in calcification. Stellate enamel organ cells are external to the stratum intermedium and are separated from the adjacent blood vessels by a basal lamina. Maturation-stage ameloblasts transport substances needed for enamel maturation. The histologic feature that marks the cycles of maturation-stage ameloblasts is a striated or ruffled border (Fig. 16.14) Maturation-stage ameloblasts with a striated border occupy approximately 70% of a specific cycle, and those that are smooth-ended, approximately 30% of a specific cycle. There is no stratum intermedium in the enamel organ during enamel maturation; stellate papillary cells are adjacent to the maturation-stage ameloblasts.

1	The maturation-stage ameloblasts and the adjacent papillary cells are characterized by numerous mitochondria. Their presence indicates cellular activity that requires large amounts of energy and reflects the function of maturation-stage ameloblasts and adjacent papillary cells as a transporting epithelium. Recent advances in the molecular biology of ameloblast gene products have revealed the enamel matrix to be highly heterogeneous. It contains proteins encoded by a number of different genes. Listed here are the principal proteins in the extracellular matrix of the developing enamel:  Amelogenins, important proteins in establishing and maintaining the spacing between enamel rods in early stages of enamel development.

1	 Ameloblastins, signaling proteins produced by ameloblasts from the early secretory to late maturation stages. Their function is not well understood; however, their developmental pattern suggests that ameloblastins play a much broader role in amelogenesis than the other proteins. Ameloblastins are believed to guide the enamel mineralization process by controlling elongation of the enamel crystals and to form junctional complexes between individual enamel crystals.  Enamelins, proteins distributed throughout the enamel layer. These proteins undergo proteolytic cleavage as the enamel matures. Low-molecular-weight products of this cleavage are retained in the mature enamel, often localized on the surface of enamel crystals.

1	 Tuftelins, the earliest detected proteins located near the dentinoenamel junction. Their acidic and insoluble nature aids in the nucleation of enamel crystals. Tuftelins are present in enamel tufts and account for hypomineralization, i.e., enamel tufts have a higher percentage of organic material than the remainder of the mature enamel. The maturation of the developing enamel results in its continued mineralization so that it becomes the hardest substance in the body. Amelogenins and ameloblastins are removed during enamel maturation. Thus, mature enamel contains only enamelins and tuftelins. The ameloblasts degenerate after the enamel is fully formed, at about the time of tooth eruption through the gum. Cementum covers the root of the tooth.

1	Cementum covers the root of the tooth. The root is the part of the tooth that fits into its alveolus, or socket in the maxilla or mandible. Cementum is a thin layer of bonelike material that is secreted by cementocytes, cells that closely resemble osteocytes. Like bone, cementum is 65% mineral. The lacunae and canaliculi in the cementum contain the cementocytes and their processes, respectively. They resemble those structures in bone that contain osteocytes and osteocyte processes. Unlike bone, cementum is avascular. Also, the canaliculi in cementum do not form an interconnecting network. A layer of cementoblasts (cells that resemble the osteoblasts of the surface of growing bone) is seen on the outer surface of the cementum, adjacent to the periodontal ligament.

1	Collagen fbers that project out of the matrix of the cementum and embed in the bony matrix of the socket wall form the bulk of the periodontal ligament. These fibers are another example of Sharpey’s fbers (Fig. 16.15). In addition, elastic fibers are also a component of the periodontal ligament. This mode of attachment of the tooth in its socket allows slight movement of the tooth to occur naturally. It also forms the basis of orthodontic procedures used to straighten teeth and reduce malocclusion of the biting and grinding surfaces of the maxillary and mandibular teeth. During corrective tooth movements, the alveolar bone of the socket is resorbed and resynthesized, but the cementum is not. Dentin is a calcified material that forms most of the tooth substance.

1	Dentin is a calcified material that forms most of the tooth substance. Dentin lies deep to the enamel and cementum. It contains less hydroxyapatite than enamel, about 70%, but more than is found in bone and cementum. Dentin is secreted by odontoblasts that form an epithelial layer over the inner surface of the dentin, i.e., the surface that is in contact with the pulp (Fig. 16.16). Like ameloblasts, odontoblasts are columnar cells that contain a well-developed rER, a large Golgi apparatus, and other organelles associated with the synthesis and secretion of large amounts of protein (Fig. 16.17). The apical surface of the odontoblast is in contact with the forming dentin; junctional complexes between the odontoblasts at that level separate the dentinal compartment from the pulp compartment. primordium of enamel primordium of pulp dental papilla dental papilla dental pulp

1	primordium of enamel primordium of pulp dental papilla dental papilla dental pulp FIGURE 16.11 • Diagram showing the cellular relationships during enamel formation. In the initial secretory stage, dentin is produced first by odontoblasts. Enamel matrix is then deposited directly on the surface of the previously formed dentin by secretory-stage ameloblasts. The secretory-stage ameloblasts continue to produce enamel matrix until the full thickness of the future enamel is achieved. (Adapted with permission from Schour I. The neonatal line in the enamel and dentin of the human deciduous teeth and first permanent molar. JADA 1936;23:1946. Copyright (c) 1936 American Dental Association. All rights reserved.)

1	The layer of odontoblasts retreats as the dentin is laid down, leaving odontoblast processes embedded in the dentin in narrow channels called dentinal tubules (see Fig. 16.15). The tubules and processes continue to elongate as the dentin continues to thicken by rhythmic growth. The rhythmic growth of dentin produces certain “growth lines” in the dentin (incremental lines of von Ebner and thicker lines of Owen) that mark significant developmental times such as birth (neonatal line) and when unusual substances such as lead are incorporated into the growing tooth. Study of growth lines has proved useful in forensic medicine.

1	Predentin is the newly secreted organic matrix, closest to the cell body of the odontoblast, which has yet to be mineralized. Although most of the proteins in the organic matrix are similar to those of bone, predentin contains two unique proteins:  Dentin phosphoprotein (DPP), a 45-kilodalton highly acidic phosphorylated protein, which is rich in aspartic acid and phosphoserine and binds large amounts of calcium. DPP is involved in the initiation of mineralization and in control of mineral size and shape.  Dentin sialoprotein (DSP), a 100-kilodalton proteogly can which is rich in aspartic and glutamic acids, serine, glycine, and chondroitin 6-sulfate. DSP is also involved in the mineralization process.

1	An unusual feature of the secretion of collagen and hydroxy-apatite by odontoblasts is the presence, in Golgi vesicles, of arrays of a formed filamentous collagen precursor. Granules believed to contain calcium attach to these precursors, giving rise to structures called abacus bodies (Figs. 16.17 and 16.18). Abacus bodies become more condensed as they mature into secretory granules. Dentin is produced by odontoblasts. Dentin is the first mineralized component of the tooth to be deposited. The outermost dentin, which is referred to as mantle dentin, is formed by subodontoblastic cells that produce small bundles of collagen fibers (von Korff ’s fibers). The odontoblasts differentiate from cells at the

1	FIGURE 16.10 • Diagrams and photomicrographs of a developing tooth. a. In this bud stage, the oral epithelium invaginates into the underlying mesenchyme, giving origin to the enamel organ (primordium of enamel). Mesenchymal cells adjacent to the tooth bud begin to differentiate, forming the dental papilla that protrudes into the tooth bud. b. Tooth bud in cap stage. In this stage, cells located in the concavity of the cap differentiate into tall, columnar cells (ameloblasts) forming the inner enamel epithelium. The condensed mesenchyme invaginates into the inner enamel epithelium, forming the dental papilla, which gives rise to the dentin and the pulp. c. In this bell stage, the connection with the oral epithelium is almost cut off. The enamel organ consists of a narrow line of outer enamel epithelium, an inner enamel epithelium formed by ameloblasts, several condensed layers of cells that form the stratum intermedium, and the widely spaced stellate reticulum. The dental papilla is

1	epithelium, an inner enamel epithelium formed by ameloblasts, several condensed layers of cells that form the stratum intermedium, and the widely spaced stellate reticulum. The dental papilla is deeply invaginated against the enamel organ. d. In this appositional dentin and enamel stage, the tooth bud is completely differentiated and independent from the oral epithelium. The relationship of the two mineralized tissues of the dental crown, enamel and dentin, is clearly visible. The surrounding mesenchyme has developed into bony tissue. e. In this stage of tooth eruption, the apex of the tooth emerges through the surface of the oral epithelium. The odontoblastic layer lines the pulp cavity. Note the developed periodontal ligaments that fasten the root of the tooth to the surrounding bone. The apex of the root is still open, but after eruption occurs, it becomes narrower. f. Functional tooth stage. Note the distribution of enamel and dentin. The tooth is embedded in surrounding bone and

1	apex of the root is still open, but after eruption occurs, it becomes narrower. f. Functional tooth stage. Note the distribution of enamel and dentin. The tooth is embedded in surrounding bone and gingiva. g. This photomicrograph of the developing tooth in the cap stage (comparable to b) shows its connection with the oral epithelium. The enamel organ consists of a single layer of cuboidal cells forming the outer enamel epithelium, the inner enamel epithelium has differentiated into columnar ameloblasts, and the layer of cells adjacent to the inner enamel epithelium has formed the stratum intermedium. The remainder of the structure is occupied by the stellate reticulum. The mesenchyme of the dental papilla has proliferated and pushed into the enamel organ. At this stage, the forming tooth is surrounded by condensed mesenchyme, called the dental sac, which gives rise to periodontal structures. 300. h. This photomicrograph shows the developing crown of an incisor, which is surrounded by

1	is surrounded by condensed mesenchyme, called the dental sac, which gives rise to periodontal structures. 300. h. This photomicrograph shows the developing crown of an incisor, which is surrounded by the outer enamel epithelium and remnants of the stellate reticulum. It is comparable to d. The underlying lighter-stained layer of dentin is a product of the odontoblasts. These tall columnar odontoblasts have differentiated from cells of the dental papilla. The pulp cavity is filled with dental pulp, and blood vessels permeate the pulp tissue. 40.

1	FIGURE 16.12 • Schematic diagrams of a partially formed tooth showing details of amelogenesis. a. The enamel is drawn to show the enamel rods extending from the dentinoenamel junction to the surface of the tooth. Although the full thickness of the enamel is formed, the full thickness of the dentin has not yet been established. The contour lines within the dentin show the extent to which the dentin has developed at a particular time, as labeled in the illustration. Note that the pulp cavity in the center of the tooth becomes smaller as the dentin develops. (Based on Schour I, Massler M. The neonatal line in the enamel and dentin of the human deciduous teeth and first permanent molar. J Am Dent Assoc 1936;23:1948.) b. During amelogenesis, enamel formation is influenced by the path of the ameloblasts. The rod produced by the ameloblast forms in the wake of the cell. Thus, in mature enamel, the direction of the enamel rod is a record of the path taken earlier by the secretory-stage

1	the ameloblasts. The rod produced by the ameloblast forms in the wake of the cell. Thus, in mature enamel, the direction of the enamel rod is a record of the path taken earlier by the secretory-stage ameloblast. c. At the apical pole of the secretory-stage ameloblasts are Tomes’ processes, surrounded by the developing enamel. Junctional complexes at the apical pole are also shown. Note the numerous matrix-containing secretory vesicles in the cytoplasm of the processes.

1	periphery of the dental papilla. The progenitor cells have the appearance of typical mesenchymal cells, i.e., they contain little cytoplasm. During their differentiation into odontoblasts, the cytoplasmic volume and organelles characteristic of collagen-producing cells increase. The cells form a layer at the periphery of the dental papilla, and they secrete the organic matrix of dentin, or predentin, at their apical end (away from the dental papilla; Fig. 16.19). As the predentin thickens, the odontoblasts move or are displaced centrally (see Fig. 16.12). A wave of mineralization follows the receding odontoblasts; this mineralized product is the dentin. As the cells move centrally, the odontoblastic

1	FIGURE 16.13 • Enamel organ cells and odontoblasts in a developing tooth. This photomicrograph of an unstained plastic thick section viewed with the phase contrast microscope shows enamel organ cells and odontoblasts as they begin to produce enamel (E) and dentin (D), respectively. Young enamel is deposited by secretory-stage ameloblasts (SA) onto the previously formed dentin. The enamel appears dark in the illustration. At the top, the enamel surface displays a characteristic picket-fence pattern because of the sharp contrast between the lightly stained Tomes’ processes (TP) of the secretory-stage ameloblasts and the darkly stained young enamel product that partly surrounds the cell processes. The nuclei (N) at the right belong to cells of the stratum intermedium. The nuclei (N) on the left belong to odontoblasts located in the basal part of the cells. The odontoblast cytoplasm extends to the dashed line. At this point, cytoplasmic processes (OP) extend into the dentin. 85.

1	CTPLMABVbCT PL MA BV ccbb CTPLMABVaCT PL MA BV a BVCTPLPLMAEBV CT PL PL MA E FIGURE 16.14 • Ameloblasts in different stages of maturation. a. This black and white photomicrograph of an H&E–stained specimen shows maturation-stage ameloblasts (MA) in demineralized tissue. The maturing enamel has been lost during slide preparation, and the space below the ameloblasts previously occupied by the enamel appears empty. Maturation-stage ameloblasts with a striated border account for 80% of the cell population in the maturation zone. BV, blood vessels; CT, connective tissue; PL, papillary layer. 650. b. This photomicrograph shows smooth-ended maturation-stage ameloblasts (MA), which account for 20% of the cell population in the mature zone. At the basal pole of the ameloblasts are the cells of the papillary layer (PL). A layer of stratum intermedium is no longer present during this stage of ameloblast maturation. 650. c. Colorized scanning electron micrograph of freeze fracture section of the

1	papillary layer (PL). A layer of stratum intermedium is no longer present during this stage of ameloblast maturation. 650. c. Colorized scanning electron micrograph of freeze fracture section of the tooth shows layer of smooth-ended maturation-stage ameloblasts (MA, green) on the enamel surface (orange). During slide preparation apical surfaces of ameloblasts were detached from the enamel. Basal surface of ameloblast is attached to connective tissue (CT) containing blood vessels 1,300. (Part C from SPL / Photo Researchers, Inc, with permission.) processes elongate; the longest are surrounded by the mineralized dentin. In newly formed dentin, the wall of the dentinal tubule is simply the edge of the mineralized dentin. With time, the dentin immediately surrounding the dentinal tubule becomes more highly mineralized; this more mineralized sheath of dentin is referred to as the peritubular dentin. The remainder of the dentin is called the intertubular dentin.

1	FIGURE 16.15 • Electron micrograph of Sharpey’s fibers. Sharpey’s fibers extend from the periodontal ligament (right) into the cementum. They consist of collagen fibrils. Sharpey’s fibers within the cementum are mineralized; those within the periodontal ligament are not mineralized. 13,000. The dental pulp cavity is a connective tissue compartment bounded by the tooth dentin. The central pulp cavity is the space within a tooth that is occupied by dental pulp, a loose connective tissue that is richly vascularized and supplied by abundant nerves. The pulp cavity takes the general shape of the tooth. The blood vessels and nerves enter the pulp cavity at the tip (apex) of the root, at a site called the apical foramen. (The designations apex and apical in this context refer only to the narrowed tip of the root of the tooth rather than to a luminal (apical) surface, as used in describing secretory and absorptive epithelia.)

1	The blood vessels and nerves extend to the crown of the tooth, where they form vascular and neural networks beneath and within the layer of odontoblasts. Some bare nerve fibers also enter the proximal portions of the dentinal tubules and contact odontoblast processes. The odontoblast processes are believed to serve a transducer function in transmitting stimuli from the tooth surface to the nerves in the dental pulp. In teeth with more than one cusp, pulpal horns extend into the cusps and contain large numbers of nerve fibers. More of these fibers extend into the dentinal tubules than at other sites. Because dentin continues to be secreted throughout life, the pulp cavity decreases in volume with age.

1	FIGURE 16.16 • Dental pulp and structure of dentin. This photomicrograph of a decalcified tooth shows the centrally located dental pulp, surrounded by dentin on both sides. The dental pulp is a soft tissue core of the tooth that resembles embryonic connective tissue, even in the adult. It contains blood vessels and nerves. Dentin contains the cytoplasmic processes of the odontoblasts within dentinal tubules. They extend into the dentinoenamel junction. The cell bodies of the odontoblasts are adjacent to the unmineralized dentin called the predentin. 120. Left inset. Longitudinal profiles of the dentinal tubules. 240. Right inset. Cross-sectional profiles of dentinal tubules. The dark outline of the dentinal tubules, as seen in both insets, represents the peritubular dentin, which is the more mineralized part of the dentin. 240. Supporting Tissues of the Teeth

1	Supporting Tissues of the Teeth Supporting tissues of the teeth include the alveolar bone of the alveolar processes of the maxilla and mandible, periodontal ligaments, and gingiva. The alveolar processes of the maxilla and mandible contain the sockets or alveoli for the roots of the teeth. The alveolar bone proper, a thin layer of compact bone, forms the wall of the alveolus (see Fig. 16.7) and is the bone to which the periodontal ligament is attached. The rest of the alveolar process consists of supporting bone. The surface of the alveolar bone proper usually shows regions of bone resorption and bone deposition, particularly when a tooth is being moved (Fig. 16.20). Periodontal disease usually leads to loss of alveolar bone, as does the absence of functional occlusion of a tooth with its normal opposing tooth.

1	The periodontal ligament is the fibrous connective tissue joining the tooth to its surrounding bone. This ligament is also called the periodontal membrane, but neither term describes its structure and function adequately. The periodontal ligament provides for the following:  Tooth attachment (fixation) Tooth support  Bone remodeling (during movement of a tooth)

1	A histologic section of the periodontal ligament shows that it contains areas of both dense and loose connective tissue. The dense connective tissue contains collagen fibers and fibroblasts that are elongated parallel to the long axis of the collagen fibers. The fibroblasts are believed to move back and forth, leaving behind a trail of collagen fibers. Periodontal fibroblasts also contain internalized collagen fibrils that are digested by the hydrolytic enzymes of the cytoplasmic lysosomes. These observations indicate that the fibroblasts not only produce collagen fibrils but also resorb collagen fibrils, thereby adjusting continuously to the demands of tooth stress and movement.

1	The loose connective tissue in the periodontal ligament contains blood vessels and nerve endings. In addition to fibroblasts and thin collagenous fibers, the periodontal ligament also contains thin, longitudinally disposed oxytalan fbers. They are attached to bone or cementum at each end. Some appear to be associated with the adventitia of blood vessels. The gingiva is the part of the mucous membrane commonly called the gums. The gingiva is a specialized part of the oral mucosa located around the neck of the tooth. It is firmly attached to the teeth and to underlying alveolar bony tissue. An idealized diagram

1	The gingiva is a specialized part of the oral mucosa located around the neck of the tooth. It is firmly attached to the teeth and to underlying alveolar bony tissue. An idealized diagram FIGURE 16.17 • Electron micrograph of odontoblasts. The plasma membrane of one odontoblast has been marked with arrows. The cell contains a large amount of rough endoplasmic reticulum and a large Golgi apparatus. The odontoblast processes are not included in this image; one process would extend from the apical pole of each cell (top). The black objects in the Golgi region are abacus bodies. The tissue has been treated with pyroantimonate, which forms a black precipitate with calcium. 12,000.

1	of the gingiva is presented in Figure 16.20. The gingiva is composed of two parts:  Gingival mucosa, which is synonymous with the masticatory mucosa described above  Junctional epithelium, or attachment epithelium, which adheres firmly to the tooth. A basal lamina-like material is secreted by the junctional epithelium and adheres firmly to the tooth surface. The cells then attach to this material via hemidesmosomes. The basal lamina and the hemidesmosomes are together referred to as the epithelial attachment. In young individuals, this attachment is to the enamel; in older individuals, where passive tooth eruption and gingival recession expose the roots, the attachment is to the cementum. FIGURE 16.18 • Golgi apparatus in an odontoblast. This electron micrograph shows a region of the Golgi apparatus containing numerous large vesicles. Note the abacus bodies (arrows) that contain parallel arrays of filaments studded with granules. 52,000.

1	Above the attachment of the epithelium to the tooth, a shallow crevice called the gingival sulcus is lined with crevicular epithelium that is continuous with the attachment epithelium. The term periodontium refers to all the tissues involved in the attachment of a tooth to the mandible and maxilla. These include the crevicular and junctional epithelium, the cementum, the periodontal ligament, and the alveolar bone. The major salivary glands are paired glands with long ducts that empty into the oral cavity.

1	The major salivary glands are paired glands with long ducts that empty into the oral cavity. The major salivary glands, as noted above, consist of the paired parotid, submandibular, and sublingual glands. The parotid and the submandibular glands are actually located outside the oral cavity; their secretions reach the cavity by ducts. The parotid gland is located subcutaneously, below and in front of the ear in the space between the ramus of the mandible and the styloid process of the temporal bone. The submandibular gland is located under the floor of the mouth, in the submandibular triangle of the neck. The sub-lingual gland is located in the floor of the mouth anterior to the submandibular gland. The minor salivary glands are located in the submucosa of different parts of the oral cavity. They include the lingual, labial, buccal, molar, and palatine glands.

1	The minor salivary glands are located in the submucosa of different parts of the oral cavity. They include the lingual, labial, buccal, molar, and palatine glands. Each salivary gland arises from the developing oral cavity epithelium. Initially, the gland takes the form of a solid cord of cells that enters the mesenchyme. The proliferation of epithelial cells eventually produces highly branched epithelial cords with bulbous ends. Degeneration of the innermost FIGURE 16.19 • Odontoblast process of a young odontoblast. This electron micrograph shows a process of the odontoblast entering a dentinal tubule. The process extends into the predentin and, after passing the mineralization front (arrows), lies within the dentin. The collagen fibrils in the predentin are finer than the more mature, coarser fibrils of the mineralization front and beyond. 34,000.

1	FIGURE 16.20 • Schematic diagram of gingiva. This schematic diagram of gingiva corresponds to the rectangular area of the orientation diagram. The gingival epithelium is attached to the enamel of the tooth. Here, the junction between epithelium and connective tissue is smooth. Elsewhere, the gingival epithelium is deeply indented by connective tissue papillae, and the junction between the two is irregular. The black lines represent collagen fibers from the cementum of the tooth and from the crest of the alveolar bone that extend toward the gingival epithelium. Note the shallow papillae in the lining mucosa (alveolar mucosa) that contrast sharply with those of the gingiva. cells of the cords and bulbous ends leads to their canalization. The cords become ducts, and the bulbous ends become secretory acini. Secretory acini are organized into lobules.

1	cells of the cords and bulbous ends leads to their canalization. The cords become ducts, and the bulbous ends become secretory acini. Secretory acini are organized into lobules. The major salivary glands are surrounded by a capsule of moderately dense connective tissue from which septa divide the secretory portions of the gland into lobes and lobules. The septa contain the larger blood vessels and excretory ducts. The connective tissue associated with the groups of secretory acini blends imperceptibly into the surrounding loose connective tissue. The minor salivary glands do not have a capsule. Numerous lymphocytes and plasma cells populate the connective tissue surrounding the acini in both the major and minor salivary glands. Their significance in the secretion of salivary antibodies is described below. Acini are of three types: serous, mucous, or mixed.

1	Acini are of three types: serous, mucous, or mixed. The basic secretory unit of salivary glands, the salivon, consists of the acinus, intercalated duct, and excretory duct (Fig. 16.21). The acinus is a blind sac composed of secretory cells. The term acinus [L., berry or grape] refers to the secretory unit of the salivary glands. The acini of salivary glands contain either serous cells (protein secreting), mucous cells (mucin secreting), or both. The relative frequencies of the three types of acini are a prime characteristic by which the

1	Dental caries is an infectious microbial disease of teeth that results in the destruction of affected calcified tissues, i.e., enamel, dentin, and cementum. Carious lesions gener-ally occur under masses of bacterial colonies referred to as “dental plaque.” The onset of dental caries is primarily as-sociated with bacterial colonies of Streptococcus mutans, whereas lactobacilli are associated with active progression of the disease. These bacterial colonies metabolize carbo-hydrates, producing an acidic environment that demineral-izes the underlying tooth structure. Frequent sucrose ingestion is strongly associated with the development of these acidogenic bacterial colonies. Trace amounts of fluoride, from sources such as water supplies (0.5 to 1.0 ppm is optimal), toothpaste, and even diet, can improve resistance to the effects of cariogenic bacteria. Fluoride improves the acid resistance of the tooth structure, acts as an antimicrobial agent, and promotes remineralization of small

1	can improve resistance to the effects of cariogenic bacteria. Fluoride improves the acid resistance of the tooth structure, acts as an antimicrobial agent, and promotes remineralization of small carious lesions. Resis-tance to acid breakdown of enamel is facilitated by the substitution of fluoride ion for the hydroxyl ion in the hydroxyapatite crystal. This decreases enamel crystal solubility in acid. Treatment of cavitated lesions, or “tooth cavities” (Fig. F16.3.1), includes excavation of the infected tooth tis-sue and replacement with dental materials such as amal-gam, composite, and glass ionomer cements. Microbial invasion of tooth structure can reach the “pulp” of the tooth and elicit an inflammatory response. In this case, endodon-tic treatment, or a “root canal,” is generally recommended, with subsequent placement of a crown to add strength to the compromised coronal tooth structure.

1	 FOLDER 16.3 Clinical Correlation: Dental Caries aabba b EEEEDDDDEE CLCLCL D D DDD FIGURE F16.3.1 • Photomicrographs of carious lesions. a. Photomicrograph of an unstained ground section of a tooth showing a carious lesion (CL) that has penetrated the entire thickness of the enamel (E) and spread laterally at the amelodentinal junction. D, dentin. b. The lesion here is more advanced. The enamel (E) has been undermined and weakened, causing fracture and a resulting cavity. At this point, bacteria can invade and penetrate down the exposed dental tubules, resulting in destructive liquefaction foci in the dentin (D) and, ultimately, exposure of the pulp. 16. (From Eveson JW, Scully C. Color Atlas of Oral Pathology. London: Times Mirror International Publishers, 1995.) major salivary glands are distinguished. Thus, three types of acini are described:  Serous acini, which contain only serous cells and are generally spherical Mucous acini, which contain only mucous cells and are usually

1	are distinguished. Thus, three types of acini are described:  Serous acini, which contain only serous cells and are generally spherical Mucous acini, which contain only mucous cells and are usually more tubular  Mixed acini, which contain both serous and mucous cells. In routine H&E preparations, mucous acini have a cap of serous cells that are thought to secrete into the highly convoluted intercellular space between the mucous cells. Because of their appearance in histologic sections, such caps are called serous demilunes [Fr., half-moon].

1	Serous demilunes are artifacts of the traditional fixation method.

1	As noted above, each mixed acinus, such as those found in the sublingual and submandibular glands, contains serous and mucus-producing cells. In routine preparation for both light and electron microscopy, serous cells have traditionally been regarded as the structures that make up the demilune. Recent electron microscopic studies now challenge this classic interpretation of the demilune. Rapid freezing of the tissue in liquid nitrogen, followed by rapid freeze substitution with osmium tetroxide in cold acetone, reveals that both mucous and serous cells are aligned in the same row to surround the lumen of the secretory acinus. No serous demilune is found. Sections prepared from the same specimen by conventional methods show swollen mucous cells with enlarged secretory granules. The serous cells form typical demilunes and are positioned in the peripheral region of the acinus with slender cytoplasmic processes interposed between the mucous cells. These findings indicate that the demilune

1	form typical demilunes and are positioned in the peripheral region of the acinus with slender cytoplasmic processes interposed between the mucous cells. These findings indicate that the demilune observed in light or electron microscopy is an artifact of the routine fxation method (Fig. 16.22). The process of demilune formation can be explained by the expansion of mucinogen, a major component of secretory granules, during routine fixation. This expansion increases the volume of the mucous cells and displaces the serous cells from their original position, thus creating the demilune effect. A similar phenomenon is sometimes seen in the intestinal mucosa, in which swollen goblet cells displace adjacent absorptive cells.

1	FIGURE 16.21 • Diagram comparing the components of the salivon in the three major salivary glands. The four major parts of the salivon–the acinus, intercalated duct, striated duct, and excretory duct—are color-coded. The three columns on the right of the salivon compare the length of the different ducts in the three salivary glands. The red-colored cells of the acinus represent serous-secreting cells, and the yellow-colored cells represent mucus-secreting cells. The ratio of serous-secreting cells to mucus-secreting cells is depicted in the acini of the various glands. Serous cells are protein-secreting cells. Serous cells have a pyramidal shape, with a relatively wide basal surface facing the basal lamina and a small apical surface facing the lumen of the acinus. They contain large amounts of rER, free ribosomes, a prominent Golgi appara-

1	FIGURE 16.22 • Relationship of serous-secreting cells and mucus-secreting cells in the mixed acinus. a. This drawing indicates the relationship of the mucous and serous cells as observed in the electron microscope after the rapid-freezing method. The serous cells extend from the basal lamina to the lumen of the acinus. b. In this drawing, serous cells are shown occupying the periphery of the acinus to form the so-called serous demilune. This feature is visible in routine preparations using immersion fixation. The swollen mucous cells have forced out the serous cells, leaving small remnants of the cytoplasm between the mucous cells.

1	tus, and numerous spherical secretory granules (Fig. 16.23). As in most protein-secreting cells that store their secretions in zymogen granules, the granules are located in the apical cytoplasm. Most other organelles are located in the basal or perinuclear cytoplasm. In H&E sections, the basal cytoplasm of the serous cell stains with hematoxylin because of the rER and the free ribosomes, whereas the apical region stains with eosin, in large part because of the secretory granules. When examined with the transmission electron microscope (TEM), the base of the serous cell may display infoldings of the plasma membrane and basolateral folds in the form of processes that interdigitate with similar processes of adjacent cells. The serous cells are joined near their apical surface by junctional complexes to neighboring cells of the acinus (see Fig. 16.23). Mucous cells are mucin-secreting cells.

1	As in other mucus-secreting epithelia, the mucous cells of the mucous salivary acini undergo cyclic activity. During part of the cycle, mucus is synthesized and stored within the cell as mucinogen granules. When the product is discharged after hormonal or neural stimulation, the cell begins to resynthesize mucus. After discharge of most or all of the mucinogen granules, the cell is difficult to distinguish from an inactive serous cell. However, most mucous cells contain large numbers of mucinogen granules in their apical cytoplasm, and because the mucinogen is lost in H&E–stained paraffin sections, the apical portion of the cell usually appears empty. In TEM preparation, the rER, mitochondria, and other components are seen chiefly in the basal portion of the cell; this part of the cell also contains the nucleus, which is typically flattened against the base of the cell (Fig. 16.24). In rapid-freeze preparations (Fig. 16.25), cells are rounded and clearly isolated from each other. The

1	contains the nucleus, which is typically flattened against the base of the cell (Fig. 16.24). In rapid-freeze preparations (Fig. 16.25), cells are rounded and clearly isolated from each other. The nuclei are round and centrally located. The apical portion of the mucous cell contains numerous mucinogen granules and a large Golgi apparatus, in which large amounts of carbohydrate are added to a protein base to synthesize the glycoprotein of the mucin. Mucous cells possess apical junctional complexes, the same as those seen between serous cells.

1	Myoepithelial cells are contractile cells that embrace the basal aspect of the acinar secretory cells. Myoepithelial cells are contractile cells with numerous processes. They lie between the basal plasma membrane of the epithelial cells and the basal lamina of the epithelium (Fig. 16.26). Myoepithelial cells also underlie the cells of the proximal portion of the duct system. In both locations, the myoepithelial cells are instrumental in moving secretory products toward the excretory duct. Myoepithelial cells are sometimes difficult to identify in H&E sections. The nucleus of the cell is often seen as a small round profile near the basement membrane. The contractile filaments stain with eosin and are sometimes recognized as a thin eosinophilic band adjacent to the basement membrane.

1	FIGURE 16.23 • Electron micrograph of the apical portion of parotid gland serous cells. The cells are polarized, with their product package within the secretory vesicles (SV ) near the lumen (L) of the acinus. The cells display rough endoplasmic reticulum (rER) and several profiles of the Golgi apparatus (G). Immature secretory vesicles (IV ) are present close to the Golgi apparatus. At the apical pole of the cells are junctional complexes (JC). The intercellular space (IC) is dilated, and profiles of sectioned lateral plications are seen. M, mitochondria. 15,000. The lumen of the salivary acinus is continuous with that of a duct system that may have as many as three sequential segments:  Intercalated duct, which leads from the acinus  Striated duct, so-called because of the presence of “stria tions,” the infoldings of the basal plasma membrane of the columnar cells that form the duct  Excretory ducts, which are the larger ducts that empty into the oral cavity

1	The degree of development of the intercalated ducts and striated ducts varies, depending on the nature of the acinar secretion (see Fig. 16.21). Serous glands have well-developed intercalated ducts and striated ducts that modify the serous secretion by both absorption of specific components from the secretion and secretion of additional components to form the final product. Mucous glands, in which the secretion is not modified, have very poorly developed intercalated ducts that may not be recognizable in H&E sections. Moreover, they do not display striated ducts. Intercalated ducts are located between a secretory acinus and a larger duct.

1	Intercalated ducts are located between a secretory acinus and a larger duct. Intercalated ducts are lined by low cuboidal epithelial cells that usually lack any distinctive feature to suggest a function other than that of a conduit. However, the cells of intercalated ducts possess carbonic anhydrase activity. In serous-secreting glands and mixed glands, they have been shown to  secrete HCO3 into the acinar product.  absorb Cl from the acinar product. As noted above, intercalated ducts are most prominent in those salivary glands that produce a watery serous secretion. In mucus-secreting salivary glands, the intercalated ducts, when present, are short and difficult to identify. Striated duct cells have numerous infoldings of the basal plasma membrane.

1	Striated duct cells have numerous infoldings of the basal plasma membrane. FIGURE 16.24 • Low-magnification electron micrograph of a mucous acinus. The mucous cells contain numerous mucinogen granules. Many of the granules have coalesced to form larger irregular masses that will ultimately discharge into the lumen (L) of the acinus. Myoepithelial cell processes (MyC) are evident at the periphery of the acinus. 5,000.

1	Striated ducts are lined by a simple cuboidal epithelium that gradually becomes columnar as it approaches the excretory duct. The infoldings of the basal plasma membrane are seen in histologic sections as “striations.” Longitudinally oriented, elongated mitochondria are enclosed in the infoldings. Basal infoldings associated with elongated mitochondria are a morphologic specialization associated with reabsorption of fluid and electrolytes. The striated duct cells also have numerous basolateral folds that are interdigitated with those of adjacent cells. The nucleus typically occupies a central (rather than basal) location in the cell. Striated ducts are the sites of  reabsorption of Na from the primary secretion.  secretion of K and HCO3 into the secretion.

1	More Na is resorbed than K is secreted, so the secretion becomes hypotonic. When secretion is very rapid, more Na and less K appear in the final saliva because the reabsorption and secondary secretion systems cannot keep up with the rate of primary secretion. Thus, the saliva may become isotonic to hypertonic. The diameter of striated ducts often exceeds that of the secretory acinus. Striated ducts are located in the parenchyma of the glands (they are intralobular ducts) but may be surrounded by small amounts of connective tissue in which blood vessels and nerves can be seen running in parallel with the duct. Excretory ducts travel in the interlobular and interlobar connective tissue.

1	Excretory ducts travel in the interlobular and interlobar connective tissue. Excretory ducts constitute the principal ducts of each of the major glands. They ultimately connect to the oral cavity. The epithelium of small excretory ducts is simple cuboidal. It gradually changes to pseudostratified columnar or stratified cuboidal. As the diameter of the duct increases, stratified columnar epithelium is often seen, and as the ducts approach the oral epithelium, stratified squamous epithelium may be present. The parotid duct (Stensen’s duct) and the submandibular duct (Wharton’s duct) travel in the connective tissue of the face and neck, respectively, for some distance from the gland before penetrating the oral mucosa. The parotid glands are completely serous. The paired serous parotid glands are the largest of the major salivary glands. The parotid duct travels from the gland,

1	FIGURE 16.25 • Electron micrographs of mixed acini. a. Low-magnification electron micrograph of the sublingual gland, prepared by the rapid freezing and freeze-substitution method, shows the arrangement of the cells within a single acinus. The mucous cells have well-preserved round mucinogen granules. The mucous and serous cells are aligned to surround the acinus lumen. Serous demilunes are not evident. 6,000. b. Electron micrograph of the sublingual gland prepared by traditional fixation in formaldehyde. Note the considerable expansion and coalescence of the mucinogen granules and the formation of a serous demilune. 15,000. (Courtesy of Dr. Shohei Yamashina.) which is located below and in front of the ear, to enter the oral cavity opposite the second upper molar tooth. The secretory units in the parotid are serous and surround numerous, long, narrow intercalated ducts. Striated ducts are large and conspicuous (Fig. 16.27a).

1	Large amounts of adipose tissue often occur in the parotid gland; this is one of its distinguishing features (Plate 52, page 565). The facial nerve (cranial nerve VII) passes through the parotid gland; large cross sections of this nerve may be encountered in routine H&E sections of the gland and are useful in identifying the parotid. Mumps, a viral infection of the parotid gland, can damage the facial nerve. The submandibular glands are mixed glands that are mostly serous in humans. The large, paired, mixed submandibular glands are located under either side of the floor of the mouth, close to the mandible. A duct from each of the two glands runs forward and medially to a papilla located on the floor of the mouth just lateral to the frenulum of the tongue. Some mucous acini capped by serous demilunes are generally found among the predominant serous acini. Intercalated ducts are less extensive than in the parotid gland (Fig. 16.27b and Plate 51, page 563).

1	The small sublingual glands are mixed glands that are mostly mucous secreting in humans. The sublingual glands, the smallest of the paired major salivary glands, are located in the floor of the mouth anterior to the submandibular glands. Their multiple small sublingual ducts empty into the submandibular duct as well as directly onto the floor of the mouth. Some of the predominant mucous acini exhibit serous demilunes, but purely serous acini are rarely present (Fig. 16.27c and Plate 53, page 567). Intercalated ducts and striated ducts are short, difficult to locate, or sometimes absent. The mucous secretory units may be more tubular than purely acinar. Saliva includes the combined secretions of all the major and minor salivary glands. Most saliva is produced by the salivary glands. A smaller amount is derived from the gingival sulcus, tonsillar crypts, and general transudation from the epithelial lining of the oral

1	Most saliva is produced by the salivary glands. A smaller amount is derived from the gingival sulcus, tonsillar crypts, and general transudation from the epithelial lining of the oral FIGURE 16.26 • Electron micrograph of the basal portion of an acinus. This electron micrograph shows the basal portion of two secretory cells from a submandibular gland. A myoepithelial cell process is also evident. Note the location of the myoepithelial cell process on the epithelial side of the basal lamina. The cytoplasm of the myoepithelial cell contains contractile filaments and densities (arrows) similar to those seen in smooth muscle cells. The cell on the left with the small nucleus is a lymphocyte. Having migrated through the basal lamina, it is also within the epithelial compartment. Arrowheads, cell boundaries; asterisks, basolateral folds. 15,000.

1	cavity. One of the unique features of saliva is the large and variable volume produced. The volume (per weight of gland tissue) of saliva exceeds that of other digestive secretions by as much as 40 times. The large volume of saliva produced is undoubtedly related to its many functions, only some of which are concerned with digestion. Saliva performs both protective and digestive functions.

1	Saliva performs both protective and digestive functions. The salivary glands produce about 1,200 mL of saliva a day. Saliva has numerous functions relating to metabolic and nonmetabolic activities, including these:  Moistening the oral mucosa Moistening dry foods to aid swallowing Providing a medium for dissolved and suspended food ma terials that chemically stimulate taste buds Buffering the contents of the oral cavity, because of its high concentration of bicarbonate ions  Digesting carbohydrates with the digestive enzyme -amylase, which breaks one to four glycosidic bonds and continues to act in the esophagus and stomach  Controlling the bacterial flora of the oral cavity by use of lysozyme (muramidase), an enzyme that lyses the muramic acid in certain bacteria (e.g., staphylococci) The unique composition of saliva is summarized in Table 16.1. Saliva is a source of calcium and phosphate ions essential for normal tooth development and maintenance.

1	The unique composition of saliva is summarized in Table 16.1. Saliva is a source of calcium and phosphate ions essential for normal tooth development and maintenance. Calcium and phosphate in the saliva are essential for the mineralization of newly erupted teeth and for repair of precarious lesions of the enamel in erupted teeth. In addition, saliva serves several other roles in protecting the teeth. Proteins in saliva cover the teeth with a protective coat called the acquired pellicle. Antibodies and other antibacterial agents retard bacterial action that would otherwise lead to tooth decay. Patients whose salivary glands are irradiated, as in the treatment of salivary gland tumors, fail to produce normal amounts of saliva; these patients typically develop rampant caries. Anticholinergic drugs used to treat some forms of heart disease also significantly reduce salivary secretion, leading to dental caries. Saliva performs immunologic functions.

1	Saliva performs immunologic functions. As noted, saliva contains antibodies, salivary immunoglobulin A (IgA). IgA is synthesized by plasma cells in the connective tissue surrounding the secretory acini of the salivary glands, and both dimeric and monomeric forms are released into the connective tissue matrix (Fig. 16.28).

1	FIGURE 16.27 • Photomicrographs of the three major salivary glands. a. The parotid gland in the human is composed entirely of serous acini and their ducts. Typically, adipose cells are also distributed throughout the gland. The lower portion of the figure reveals an excretory duct within a connective tissue septum. 120. Inset. Higher magnification of the serous acinar cells. 320. b. The submandibular glands contain both serous and mucous acini. In humans, the serous components predominate. The mucus-secreting acini are readily discernible at this low magnification because of their light staining. The remainder of the field is composed largely of serous acini. Various ducts—excretory, striated, and intercalated—are evident in the field. 120. Left inset. Higher magnification of an acinus revealing a serous demilune surrounding mucus-secreting cells. 360. Right inset. Higher magnification of a striated duct. These ducts have columnar epithelium with visible basal striations. 320. c. The

1	a serous demilune surrounding mucus-secreting cells. 360. Right inset. Higher magnification of a striated duct. These ducts have columnar epithelium with visible basal striations. 320. c. The sublingual gland also contains both serous and mucous elements. Here, the mucous acini predominate. The mucous acini are conspicuous because of their light staining. Critical examination of the mucous acini at this relatively low magnification reveals that they are not spherical structures but, rather, elongate or tubular structures with branching outpockets. Thus, the acinus is rather large, and much of it is usually not seen within the plane of a single section. The ducts of the sublingual gland that are observed with the greatest frequency in a section are the interlobular ducts. 120. Inset. The serous component of the gland is composed largely of demilunes (asterisks), artifacts of conventional fixation. 320.

1	TABLE Composition of Unstimulated Saliva16.1 Organic constituents Mean (mg/mL) Protein 220.0 Amylase 38.0 Mucin 2.7 Muramidase (lysozyme) 22.0 Lactoferrin 0.03 ABO group markers 0.005 EGF 3.4 sIgA 19.0 IgG 1.4 IgM 0.2 Glucose 1.0 Urea 20.0 Uric acid 1.5 Creatinine 0.1 Cholesterol 8.0 cAMP 7.0 Inorganic constituents Sodium 15.0 Potassium 80.0 Thiocyanate Smokers 9.0 Nonsmokers 2.0 Calcium 5.8 Phosphate 16.8 Chloride 50.0 Fluoride Traces (according to intake) Modified from Jenkins GN. The Physiology and Biochemistry of the Mouth, 4th ed. Oxford: Blackwell Scientific Publications, 1978. sIgA, secretory IgA; EGF, epithelial growth factor. A polymeric immunoglobulin receptor (pIgR) protein is synthesized by the salivary gland cells and inserted into the basal plasma membrane, where it serves as a receptor for dimeric IgA.

1	A polymeric immunoglobulin receptor (pIgR) protein is synthesized by the salivary gland cells and inserted into the basal plasma membrane, where it serves as a receptor for dimeric IgA. When the dimeric IgA binds to the receptor, the pIgR-dIgA complex is internalized by receptor-mediated endocytosis and carried through the acinar cell to the apical plasma membrane. FIGURE 16.28 • Diagram of different forms of immunoglobulin A (IgA). This drawing shows the monomer of IgA (top). The dimer of IgA is a product of the plasma cell and contains a J chain (J ) connecting two monomers (middle). The secretory component (SC), a product of proteolytically cleaved pIgR is added to the dimer to form secretory IgA (sIgA) (sIgA, bottom).

1	Here, pIgR is proteolytically cleaved and the extracellular part of the receptor that is bound to dIgA is released into the lumen as secretory IgA (sIgA). This process of synthesis and secretion of IgA is essentially identical to that which occurs in the more distal parts of the gastrointestinal tract, where sIgA is transported across the absorptive columnar epithelium of the small intestine and colon (see page 596). Saliva contains water, various proteins, and electrolytes. Saliva contains chiefly water, proteins and glycoproteins (enzymes and antibodies), and electrolytes. It has a high potassium concentration that is approximately seven times that of blood, a sodium concentration approximately one tenth that of blood, a bicarbonate concentration approximately three times that of blood, and significant amounts of calcium, phosphorus, chloride, thiocyanate, and urea. Lysozyme and -amylase are the principal enzymes present (see Table 16.1).

1	 FOLDER 16.4 Clinical Correlation: Salivary Gland Tumors chapter 16 Digestive System I SALIVARY G LAN DS 555 Tumors of salivary glands usually occur in the major salivary glands (parotid, submandibular, and sublingual); however, a small percentage occur in the minor glands located within the oral mucosa, palate, uvula, floor of mouth, tongue, pharynx, larynx, and paranasal sinuses. Approxi-mately 80% of salivary gland tumors are benign. Most orig-inate in the parotid gland (Fig. F16.4.1a). The palate is the most common site of minor salivary gland tumors. The most common benign tumor is the pleomorphic adenoma, which accounts for 65% of all salivary gland tu-mors. It is characterized by epithelial tissue containing duc-tal and myoepithelial cells intermingled with areas resembling ground substance of connective tissues (e.g., in cartilage). These connective-like tissues are produced by myoepithelial cells (Fig. F16.4.1b). Most patients with benign tumors present with painless

1	ground substance of connective tissues (e.g., in cartilage). These connective-like tissues are produced by myoepithelial cells (Fig. F16.4.1b). Most patients with benign tumors present with painless swelling of the involved gland. Because of nerve involvement, signs such as numbness or weakness of innervated muscle are also reported. For instance, paralysis of facial muscles or persistent facial pain may be present in some individuals with parotid tumors. The most common treatment is surgical removal of the tumor. For parotid gland tumors, a total parotidectomy (ex-cision of parotid gland) is often necessary. Postoperative radiation therapy is also used when the tumor is cancer-ous. Complications of surgical treatment of parotid gland tumors include facial nerve dysfunction and Frey’s syn-drome (also known as gustatory sweating). a bb FIGURE F16.4.1 • Pleomorphic adenoma of the parotid gland. a. This photograph shows a patient with a parotid mass located near the angle of the

1	syn-drome (also known as gustatory sweating). a bb FIGURE F16.4.1 • Pleomorphic adenoma of the parotid gland. a. This photograph shows a patient with a parotid mass located near the angle of the mandible. b. This low magnification photomicrograph shows the features of a pleomorphic adenoma. Note that normal parotid tissue (basophilic-stained areas in upper right) is infiltrated by nodules containing connective-like tissue that resembles the extracellular matrix of cartilage (lighter eosinophilic-stained regions). 120. (From Rubin E, Gorstein F, Schwarting R, Strayer DS. Rubin’s Pathology, 4th ed. Baltimore: Lippincott Williams & Wilkins, 2004, Figs 25–20 and 25–22.)

1	The lips are the entry point of the alimentary canal. Here, the thin keratinized epithelium of face skin changes to the thick parakeratinized epithelium of the oral mucosa. At the mucocutaneous junction, the red portion of the lips, is characterized by deep pene-tration of connective tissue papillae into the base of the stratified squamous kera-tinized epithelium. The blood vessels and nerve endings in these papillae are responsible for both the color and the exquisite touch sensitivity of the lips. ORIENTATION MICROGRAPH: An H&E–stained sagittal section through the upper lip in this low-power orientation photomicrograph to the right (×8) reveals the skin of the face, the red margin of the lip, and the transition to the oral mucosa (OM). The marked rectangles indicate representative areas of each of these sites, shown at higher magnifications in upper, middle and lower rows of figures, on the adjacent plate. Note the change in thickness of the epithelium from the exterior or facial

1	of each of these sites, shown at higher magnifications in upper, middle and lower rows of figures, on the adjacent plate. Note the change in thickness of the epithelium from the exterior or facial portion of the lip (the vertical surface on the right) to the interior surface of the oral cavity (the surface beginning with rectangle marked lower and continuing down the left surface of the lip) in this micrograph. lower OM skin middle top

1	Keratinized epithelium, lip, human, H&E ×120. The keratinized epithelium (EP) of the face is relatively thin and has the general features of thin skin found in other sites. Associated with it are hair follicles (HF) and sebaceous glands (SGl). Red margin, lip, human, H&E ×120. The epithelium of the red margin of the lip is much thicker than that of the face. The stratum granulosum is still present; thus, the epithelium is keratinized. The feature that accounts for the coloration of the red margin is the deep penetration of the connective tissue papillae into the epithelium (arrowheads). The thinness of the epithelium combined with the extensive vascularity of the underlying connective tissue, particularly the extensive venous blood vessels (BV), allows the color of the blood to show through. Keratinized epithelium, lip, human, H&E ×380.

1	Keratinized epithelium, lip, human, H&E ×380. The circled area in figure on left is shown at higher magnification here. The reddish brown material in the basal cells is the pigment melanin (M), and the dark blue near the surface is the stratum granulosum (SG) with its deep-blue-stained keratohyalin granules. Red margin, lip, human, H&E ×380. The sensitivity of the red margin to stimuli such as light touch is due to the presence of an increased number of sensory receptors. In fact, each of the two deep papillae seen in figure on left contains a Meissner’s corpuscle, one of which (MC) is more clearly seen in this figure. Mucocutaneous junction, lip, human, H&E ×120. The transition from the keratinized red margin to the fairly thick stratified squamous parakeratinized epithelium of the oral mucosa is evident in this figure. Note how the stratum granulosum suddenly ends. This is more clearly shown at higher magnification in figure on right.

1	Mucocutaneous junction, lip, human, H&E ×380. Beyond the site where the stratum granulosum cells disappear, nuclei are seen in the superficial cells up to the surface (arrows). The epithelium is also much thicker at this point and remains so throughout the oral cavity. PLATE 48 KEY BV, venous blood vessels EP, epithelium HF, hair follicle M, melanin pigment MC, Meissner’s corpuscle OM, oral mucosa SG, stratum granulosum SGl, sebaceous gland arrowheads, connective tissue papillae arrows, nuclei of superficial cells up to surface

1	The tongue is a muscular organ projecting into the oral cavity from its inferior surface. It is covered with a mucous membrane that consists of stratified squamous epithelium, keratinized in parts, resting on a loose connective tissue. The undersurface of the tongue is relatively uncomplicated. The mucosa of the dorsal surface, however, is modified to form three types of papillae: filiform, fungiform, and circumvallate. The circumvallate papillae form a V-shaped row that divides the tongue into a body and a root; the dorsal surface of the body, i.e., the portion anterior to the circumvallate papillae, contains filiform and fungiform papillae. Parallel ridges bearing taste buds are found on the sides of the tongue and are particularly evident in infants. When sectioned at right angles to their long axis, they appear as papillae and, although not true papillae, are called foliate papillae. The tongue contains both intrinsic and extrinsic voluntary striated muscle. The striated muscles

1	their long axis, they appear as papillae and, although not true papillae, are called foliate papillae. The tongue contains both intrinsic and extrinsic voluntary striated muscle. The striated muscles of the tongue are arranged in three inter-weaving planes, with each arrayed at right angles to the other two. This arrangement is unique to the tongue. It provides enormous flexibility and precision in the movements of the tongue that are essential to human speech as well as to its role in digestion and swallowing. The ar-rangement also allows easy identification as lingual muscle.

1	Dorsal surface, tongue, monkey, H&E ×65; inset ×130.

1	This figure shows the dorsal surface of the tongue with the filiform papillae (Fil P). They are the most numerous of the three types of papillae. Structurally, they are bent, conical projections of the epithelium, with the point of the projection directed posteriorly. These papillae do not possess taste buds and are composed of stratified squamous keratinized epithelium. The fungiform papillae are scattered about as isolated, slightly rounded, elevated structures situated among the filiform papillae. A fungiform papilla is shown in the inset. A large connective tissue core (primary connective tissue papilla) forms the center of the fungiform papilla, and smaller connective tissue papillae (secondary connective tissue papillae) project into the base of the surface epithelium (arrowhead). The connective tissue of the papillae is highly vascularized. Because of the deep penetration of connective tissue into the epithelium, combined with a very thin keratinized surface, the fungiform

1	The connective tissue of the papillae is highly vascularized. Because of the deep penetration of connective tissue into the epithelium, combined with a very thin keratinized surface, the fungiform papillae appear as small red dots when the dorsal surface of the tongue is examined by gross inspection.

1	Ventral surface, tongue, monkey, H&E ×65. The ventral surface of the tongue is shown in this figure. The smooth surface of the stratified squamous epithelium (Ep) contrasts with the irregular surface of the dorsum of the tongue. Moreover, the epithelial surface on the ventral surface of the tongue is usually not keratinized. The connective tissue (CT) is immediately deep to the epithelium; deeper still is the striated muscle (M). The numerous connective tissue papillae that project into the base of the epithelium of both ventral and dorsal surfaces give the epithelial– connective tissue junction an irregular profile. Often, these connective tissue papillae are cut obliquely and then appear as small islands of connective tissue within the epithelial layer (see figure above).

1	The connective tissue extends as far as the muscle without changing character, and no submucosa is recognized. The muscle (M) is striated and is unique in its organization; i.e., the fibers travel in three planes. Therefore, most sections will show bundles of muscle fibers cut longitudinally, at right angles to each other, and in cross section. Nerves (N) that innervate the muscle are also frequently observed in the connective tissue septa between the muscle bundles. The surface of the tongue behind the vallate papillae (the root of the tongue) contains lingual tonsils (not shown). These are similar in structure and appearance to the palatine tonsils illustrated in Plate 36. KEY CT, connective tissue Ep, epithelium Fil P, filiform papillae M, striated muscle bundles N, nerves arrowhead (inset), secondary connective tissue papilla

1	The papillae and their associated taste buds constitute the specialized mucosa of the oral cavity. Although filiform papillae do not have taste buds, the other three types, viz., foliate, fungiform, and circumvallate, contain taste buds in their epithelium. The fungiform (i.e., mushroom-shaped) papillae (see inset on Plate 49) are most numerous near the tip of the tongue. Taste buds are present in the epithelium on their dor-sal surface. The taste buds in the epithelium covering the circumvallate and foliate papillae are located in deep clefts that separate the papillae from adjacent mucosa or from each other, respectively. Ducts of lingual salivary glands (von Ebner’s glands; a component of the minor salivary glands) empty their serous secretions into the moat surrounding each circumvallate papilla. The secretions flush material from the moat to allow the taste buds to respond to new stimuli. Similarly, ducts of small serous glands empty into the clefts between foliate papillae.

1	papilla. The secretions flush material from the moat to allow the taste buds to respond to new stimuli. Similarly, ducts of small serous glands empty into the clefts between foliate papillae. Taste buds in section appear as oval, pale-staining bodies that extend through the thickness of the epithelium. A small opening at the epithe-lial surface is called the taste pore. Taste buds react to only five stimuli: sweet, salty, bitter, sour, and umami. These modalities appear to be more concentrated in the specific areas of the tongue; taste buds at the tip of the tongue detect sweet stimuli, those immediately posterolat-eral to the tip detect salty stimuli, those on the circumvallate papillae detect bitter and umami stimuli.

1	Foliate papillae, tongue, human, H&E, ×50.

1	Foliate papillae consist of a series of parallel ridges that are separated by narrow, deep mucosal clefts (see orientation photograph, pg 75). They are aligned at right angles to the long axis of the tongue on its posterior lateral edge. In younger individuals, they are readily observed by gross inspection. However, with age, foliate papillae may not be recognized. This micrograph shows three papillae, each is separated from its neighbor by a narrow cleft (C). The surface of these papillae is covered by a thick stratified non keratinized epithelium (SE). The basal surface of the epithelium is extremely uneven due to the presence of deep, penetrating connective tissue papillae (CTP). In contrast, the epithelium lining the clefts (Ep) is relatively thin and uniform. It contains numerous taste buds. These are the light-staining objects seen in the cleft epithelium. Underlying the epithelium is a layer of loose connective tissue (LCT) and a central core of dense connective tissue. Within

1	buds. These are the light-staining objects seen in the cleft epithelium. Underlying the epithelium is a layer of loose connective tissue (LCT) and a central core of dense connective tissue. Within this core and between bundles of muscle fibers beneath the papillae are lingual serous glands (LSG). These glands, like the serous glands associated with the circumvalate papillae, have ducts (D) that empty into the base of the clefts between papillae.

1	Taste bud, tongue, human, H&E, ×1100. This micrograph shows to advantage the taste pore (TP), the cells of the taste bud, and its associated nerve fibers (NF). The cells with the large, round nuclei are neuroepithelial sensory cells (NSC). They are the most numerous cells of the taste bud. At their apical surface, they possess microvilli that extend into the taste pore. At their basal surface, they form a synapse with the afferent sensory fibers that make up the underlying nerve. Among the sensory cells are supporting cells (SC). These cells contain microvilli on their apical surface. Also present in the taste bud at its base are small cells simply referred to as basal cells (BC), one of which is identified here. They are the stem cells for the supporting and neuroepithelial cells which have a turnover life of about 10 days.

1	KEY BC, basal cells C, cleft CTP, connective tissue papillae D, ducts Ep, epithelium lining the clefts LCT, loose connective tissue LSG, lingual serous glands NF, nerve fibers NSC, neuroepithelial sensory cells SC, supporting cells SE, stratified nonkeratinized epithelium TP, taste pore

1	Like the parotid glands, the submandibular glands are located outside of the oral cav-ity. They are located under either side of the floor of the mouth near the mandible. A duct runs forward and medially from each of the two glands to a papilla located on the floor of the mouth just lateral to the frenulum of the tongue. The secretory component of the submandibular glands are the acini which are of three types, namely serous acini that are protein secreting like those of the parotid gland, mucous acini that secrete mucin, and third, acini containing both serous and mucous secreting cells. In the case of the mixed acini, the mucous cells are capped by serous cells, which are typically described as demilumes. Recent studies suggest that the demilume is an artifact of tissue preparation and that all of the cells are aligned to secrete into the acinus lumen. Traditional fixation in formaldehyde appears to expand the mucous cells with the consequent squeezing of the serous cells to form

1	that all of the cells are aligned to secrete into the acinus lumen. Traditional fixation in formaldehyde appears to expand the mucous cells with the consequent squeezing of the serous cells to form their cap like position. ORIENTATION MICROGRAPH: This micrograph reveals a portion of the sub-mandibular gland. A single well-defined lobe (L) is seen in the upper part of the micro-graph. Within the central portion of the gland, there is a dense connective tissue core (DCT) containing the larger arteries (A), veins (V), and excretory ducts (ED) of the gland. The submandibular gland is a mixed gland; those regions containing serous acini (SA) are darkly stained whereas, regions containing mucous acini (MA) are lighter in appearance. SASASASAMAMASASADCTDCTVVEDEDEDEDAASASAMAMALLSASAMASADCTVEDEDASAMAL

1	Submandibular gland, human, H&E, ×175. This micrograph reveals the various components of the sub-mandibular gland. The serous acini (SA) are darkly stained in contrast to the lighter staining mucous acini (MA). Furthermore, the serous acini are generally spherical in shape. The mucous acini are more tubular or elongate and sometimes can be seen to branch. The secretion from the acini enters an intercalated duct. They are the smallest ducts and are of relatively short length. They reside within the lobule, but are often difficult to find because of their shortness. Submandibular gland, human, H&E, ×725.

1	The boxed area in the above micrograph is shown here at higher magnification. It includes several mucous acini (MA) on the left side of the micrograph, a number of serous acini (SA) on the right side of the micrograph, and in the center, two mixed acini (MxA) consisting of mucous-secreting cells and serous-secreting cells. Characteristically, the mucous-secreting cells have a pale-staining cytoplasm with their nuclei flattened at the base of the cell. In contrast, the serous-secreting cells are deeply stained and exhibit round nuclei. In addition, the lumen (Lu) of the acini associated with the mucous se-These ducts, in turn, empty into the larger striated duct (SD). This type of duct is better demonstrated in the micrograph below. Their contents then empty into an excretory duct (ED) which is recognized by a stratified or pseudostratified epithelium. Other features of note in this micrograph are arteries (A) and veins (V) which are found coursing in the connective tissue with the

1	which is recognized by a stratified or pseudostratified epithelium. Other features of note in this micrograph are arteries (A) and veins (V) which are found coursing in the connective tissue with the larger ducts. Also evident in this micrograph is an area containing an accumulation of lymphocytes and plasma cells (LP).

1	creting cells is relatively wide; the lumen of the serous acini is relatively narrow and difficult to find. An additional point that should be made is that the serous cells of the mixed acini generally appear as a cap in relation to the mucous cells. These cells are referred to as demilumes. In evaluating some of those acini that appear to be serous in nature, it is possible that they simply represent a tangential section of a demilume. A striated duct (SD) is also included in the micrograph. It is so named because of the faint striations that can be seen in the basal cytoplasm. These ducts, as noted, receive secretion from the intercalated ducts and empty into the larger excretory ducts. KEY A, arteries DCT, dense connective tissue core ED, excretory ducts L, lobe LP, lymphocytes and plasma cells Lu, lumen MA, mucous acini MxA, mixed acini SA, serous acini SD, striated duct V, veins

1	KEY A, arteries DCT, dense connective tissue core ED, excretory ducts L, lobe LP, lymphocytes and plasma cells Lu, lumen MA, mucous acini MxA, mixed acini SA, serous acini SD, striated duct V, veins The parotid glands are the largest of the major salivary glands. They are composed of alveoli containing only serous secretory cells. Adipose tissue often occurs in the parotid gland and may be one of its distinguishing features. The facial nerve (cranial nerve VII) passes through the parotid gland; large cross sections of this nerve, often found in routine H&E sections of the gland, may also be of help in identifying the parotid. Mumps, a viral infection of the parotid gland, can damage the facial nerve. Parotid gland, human, H&E ×160.

1	Parotid gland, human, H&E ×160. The parotid gland in the human is composed entirely of serous acini (A) and their ducts. However, numerous adipose cells (AC) are usually distributed throughout the gland. Both the serous acini and their duct system in the parotid gland are comparable in structure and arrangement to the same components in the submandibular gland. Within the lobule, the striated ducts Parotid gland, monkey, glutaraldehyde-osmium tetroxide fxed, H&E ×640.

1	Parotid gland, monkey, glutaraldehyde-osmium tetroxide fxed, H&E ×640. The serous cells are optimally preserved in this specimen and reveal their secretory (zymogen) granules. The granules appear as fine dot-like objects within the cytoplasm. The acinus in the upper right of the figure has been cut in cross section and reveals the acinar lumen (AL). The small rectangle drawn in the acinus represents an area comparable to the electron micrograph shown as Figure 16.23. The large acinar profile to the left of the striated duct (StD) shows that the acini are not simple spheres but, rather, irregular elongate structures. Because of the small size of the acinar lumen and the variability in sectioning an acinus, the lumen is seen infrequently.

1	(StD) are readily observed. They exhibit a simple columnar epithelium. The intercalated ducts are smaller; at the low magnification of this figure, they are difficult to recognize. A few intercalated ducts (ID) are indicated. The lower portion of the figure reveals an excretory duct (ED) within a connective tissue septum (CT). The epithelium of this excretory duct exhibits two layers of nuclei and is either pseudostratified or, possibly, already true stratified epithelium.

1	A cross-sectional profile of an intercalated duct (ID) appears on the left of the micrograph; note its simple cuboidal epithelium. A single flattened nucleus is present at the top of the duct and may represent one of the myoepithelial cells that are associated with the beginning of the duct system as well as with the acini (A). The large duct occupying the center of the micrograph is a striated duct (StD). It is composed of columnar epithelium. The striations (S) that give the duct its name are evident. Also of significance is the presence of plasma cells (PC) within the connective tissue surrounding the duct. These cells produce the immunoglobulins taken up and resecreted by the acinar cells, particularly secretory IgA (sIgA). PLATE 52 • PAROTI D G LAN D KEY A, acinus AC, adipose cell AL, acinar lumen CT, connective tissue ED, excretory duct ID, intercalated duct PC, plasma cells S, striations of duct cells StD, striated duct

1	The sublingual glands are the smallest of the paired major salivary glands. Their multiple small ducts empty into the submandibular ducts as well as directly onto the floor of the mouth. The sublingual gland resembles the submandibular gland, in that it contains both serous and mu-cous elements. In the sublingual gland, however, the mucous acini predominate. Some of the predominant mucous acini have serous de-milunes, but purely serous acini are rarely present. Saliva includes the combined secretions of all the major and minor salivary glands. The functions of saliva include moistening dry foods to aid swallowing, dissolving and suspending food materials that chemically stimulate taste buds, buffering the contents of the oral cavity through its high concentration of bicarbonate ion, digestion of carbohydrates by the digestive enzyme -amylase (which breaks the 1–4 gly-coside bonds and continues to act in the esophagus and stomach), and controlling the bacterial flora of the oral cavity

1	of carbohydrates by the digestive enzyme -amylase (which breaks the 1–4 gly-coside bonds and continues to act in the esophagus and stomach), and controlling the bacterial flora of the oral cavity because of the pres-ence of the antibacterial enzyme lysozyme. Saliva is a source of calcium and phosphate ions essential for normal tooth development and maintenance. It also contains antibodies, no-tably salivary sIgA. Salivation is part of a reflex arc that is normally stimulated by the ingestion of food, although sight, smell, or even thoughts of food can also stimulate salivation.

1	Sublingual gland, human, H&E ×160. This figure shows a sublingual gland at low power. The mucous acini (MA) are conspicuous because of their light staining. Critical examination of the mucous acini at this relatively low magnification reveals that they are not spherical structures but, rather, elongate or tubular structures with branch ing outpockets. Thus, the acinus is rather large, and much of it is usually not seen within the plane of a single section. The serous component of the gland is composed largely of demilunes, but occasional serous acini are present. As noted earlier, some of the serous Sublingual gland, human, H&E ×400.

1	Sublingual gland, human, H&E ×400. Note that through a fortuitous plane of section the lumen of a mucous acinus (MA) (upper right) is seen joining an intercalated duct (ID). The juncture between the acinus and the beginning of the intercalated duct is marked by an arrowhead. The intercalated duct is composed of a flattened or low columnar epithelium similar to that seen in the other salivary glands. The intercalated ducts of the sublingual gland are extremely short, however, and thus are usually difficult to find. The intercalated duct seen in this micrograph joins with one or more other intercalated ducts to become the intralobular duct (InD), which is identified by its columnar epithelium and relatively large lumen. The point of transition from intercalated to intralobular duct is not recognizable in the micrograph, however, because the duct wall has only been grazed and the shape of the cells cannot be determined.

1	demilunes may be sectioned in a plane that does not include the mucous component of the acinus, thus giving the appearance of a serous acinus. The ducts of the sublingual gland that are observed most frequently in a section are the intralobular ducts. They are the equivalent of the striated duct of the submandibular and parotid glands but lack the extensive basal infoldings and mitochondrial array that creates the striations. One of the intralobular ducts (InD) is evident in this figure (upper right). The area within the rectangle includes part of this duct and is shown at higher magnification in figure below.

1	Examination of the acini at this higher magnification also reveals the serous demilunes (SD). Note how they form a caplike addition to the mucous end pieces. The cytologic appearance of the mucous cells (MC) and serous cells is essentially the same as that described for the submandibular gland. The area selected for this higher magnification also reveals isolated cell clusters that bear some resemblance to serous acini. It is likely, however, that these cells are actually mucous cells that either have been cut in a plane parallel to their base and do not include the mucinogen-containing portions of the cell or are in a state of activity in which, after depletion of their granules, the production of new mucinogen granules does not yet suffice to give the characteristic “empty” mucous cell appearance.

1	An additional important feature of the connective tissue stroma is the presence of numerous lymphocytes and plasma cells. Some of the plasma cells are indicated by the arrows. The plasma cells are associated with the production of salivary IgA and are also present in the other salivary glands. KEY MA, mucous acinus MC, mucous cells ID, intercalated duct InD, intralobular duct SD, serous demilune arrowhead, mucous acinus joining intercalated duct arrows, plasma cells Digestive System II: Esophagus and Gastrointestinal Tract

1	OVERVIEW OF THE ESOPHAGUS AND GASTROINTESTINAL TRACT / 569 Mucosa / 569 Submucosa / 571 Muscularis Externa / 571 Serosa and Adventitia / 571 ESOPHAGUS / 572 STOMACH / 574 Gastric Mucosa / 574 Fundic Glands of the Gastric Mucosa / 577 Cardiac Glands of the Gastric Mucosa / 583 Pyloric Glands of the Gastric Mucosa / 583 Epithelial Cell Renewal in the Stomach / 584 Lamina Propria and Muscularis Mucosae / 584 Gastric Submucosa / 585 Gastric Muscularis Externa / 585 Gastric Serosa / 586 SMALL INTESTINE / 586 Submucosa / 596 Muscularis Externa / 597 Serosa / 597 Epithelial Cell Renewal in the Small Intestine / 597 LARGE INTESTINE / 597 Mucosa / 599 Epithelial Cell Renewal in the Large Intestine / 600 Lamina Propria / 600 Muscularis Externa / 601 Submucosa and Serosa / 601 Cecum and Appendix / 601 Rectum and Anal Canal / 603 Folder 17.1 Clinical Correlation: Pernicious Anemia and Peptic Ulcer Disease / 578 Folder 17.2 Clinical Correlation: Zollinger-Ellison Syndrome / 580 Folder 17.3

1	/ 601 Rectum and Anal Canal / 603 Folder 17.1 Clinical Correlation: Pernicious Anemia and Peptic Ulcer Disease / 578 Folder 17.2 Clinical Correlation: Zollinger-Ellison Syndrome / 580 Folder 17.3 Functional Considerations: The Gastrointestinal Endocrine System / 581 Folder 17.4 Functional Considerations: Digestive and Absorptive Functions of Enterocytes / 587 Folder 17.5 Functional Considerations: Immune Functions of the Alimentary Canal / 595 Folder 17.6 Clinical Correlation: The Pattern of Lymph Vessel Distribution and Diseases of the Large Intestine / 602  Serosa, a serous membrane consisting of a simple squa-GASTROINTESTINAL TRACT mous epithelium, the mesothelium, and a small amount of

1	The portion of the alimentary canal that extends from the proximal part of the esophagus to the distal part of the anal canal is a hollow tube of varying diameter. This tube has the same basic structural organization throughout its length. Its wall is formed by four distinctive layers. From the lumen outward (Fig. 17.1), they are as follows:  Mucosa, consisting of a lining epithelium, an underlying connective tissue called the lamina propria, and the muscularis mucosae, composed of smooth muscle  Submucosa, consisting of dense irregular connective tissue  Muscularis externa, consisting in most parts of two layers of smooth muscle underlying connective tissue. An adventitia consisting only of connective tissue is found where the wall of the tube is directly attached or fixed to adjoining structures (i.e., body wall and certain retroperitoneal organs).

1	The structure of the esophagus and gastrointestinal tract varies considerably from region to region; most of the variation occurs within the mucosa. The epithelium differs throughout the alimentary canal and is adapted to the specific function of each part of the tube. The mucosa has three principal functions: protection, absorption, and secretion.

1	FIGURE 17.1 • Diagram of general organization of the alimentary canal. This composite diagram shows the wall structure of the alimentary canal in four representative organs: esophagus, stomach, small intestine, and large intestine. Note that villi, a characteristic feature of the small intestine, are not present in other parts of the alimentary canal. Mucosal glands are present throughout the length of the alimentary canal but sparingly in the esophagus and oral cavity. Submucosal glands are present in the esophagus and duodenum. The extramural glands (liver and pancreas) empty into the duodenum. Diffuse lymphatic tissues and nodules are found in the lamina propria throughout the entire length of the alimentary canal (shown here only in the large intestine). Nerves, blood vessels, and lymphatic vessels reach the alimentary canal via the mesenteries or via the adjacent connective tissue (as in the retroperitoneal organs).

1	The histologic characteristics of these layers are described below in relation to specific regions of the digestive tube. The epithelium of the mucosa serves as a barrier that separates the lumen of the alimentary canal from the rest of the organism. The epithelial barrier separates the external luminal environment of the tube from the tissues and organs of the body. The barrier aids in protection of the individual from the entry of antigens, pathogens, and other noxious substances. In the esophagus, a stratified squamous epithelium provides protection from physical abrasion by ingested food. In the gastrointestinal portion of the alimentary tract, tight junctions between the simple columnar epithelial cells of the mucosa serve as a selectively permeable barrier. Most epithelial cells transport products of digestion and other essential substances such as water through the cell and into the extracellular space beneath the tight junctions.

1	The absorptive function of the mucosa allows the movement of digested nutrients, water, and electrolytes into the blood and lymph vessels. The absorption of digested nutrients, water, and electrolytes is possible because of projections of the mucosa and submucosa into the lumen of the digestive tract. These surface projections greatly increase the surface area available for absorption, and vary in size and orientation. They consist of the following structural specializations (see Fig. 17.1):  Plicae circulares are circumferentially oriented submu cosal folds present along most of the length of the small intestine.  Villi are mucosal projections that cover the entire surface of the small intestine, the principal site of absorption of the products of digestion.  Microvilli are tightly packed, microscopic projections of the apical surface of intestinal absorptive cells. They fur ther increase the surface available for absorption.

1	In addition, the glycocalyx consists of glycoproteins that project from the apical plasma membrane of epithelial absorptive cells. It provides additional surface for adsorption and includes enzymes secreted by the absorptive cells that are essential for the final steps of digestion of proteins and sugars. The epithelium selectively absorbs the products of digestion both for its own cells and for transport into the vascular system for distribution to other tissues. The secretory function of the mucosa provides lubrication and delivers digestive enzymes, hormones, and antibodies into the lumen of the alimentary tube.

1	The secretory function of the mucosa provides lubrication and delivers digestive enzymes, hormones, and antibodies into the lumen of the alimentary tube. Secretion is carried out largely by glands distributed throughout the length of the digestive tube. The various secretory products provide mucus for protective lubrication, as well as buffering of the tract lining and substances that assist in digestion, including enzymes, hydrochloric acid, peptide hormones, and water (see Fig. 17.1). The mucosal epithelium also secretes antibodies that it receives from the underlying connective tissue.

1	The glands of the alimentary tract (see Fig. 17.1) develop from invaginations of the luminal epithelium and include  mucosal glands that extend into the lamina propria.  submucosal glands that either deliver their secretions directly to the lumen of mucosal glands or via ducts that pass through the mucosa to the luminal surface.  extramural glands that lie outside the digestive tract and deliver their secretions via ducts that pass through the wall of the intestine to enter the lumen. The liver and the pan creas are extramural digestive glands (see Chapter 18) that greatly increase the secretory capacity of the digestive sys tem. They deliver their secretions into the duodenum, the first part of the small intestine. The lamina propria contains glands, vessels that transport absorbed substances, and components of the immune system.

1	The lamina propria contains glands, vessels that transport absorbed substances, and components of the immune system. As noted, the mucosal glands extend into the lamina propria throughout the length of the alimentary canal. In addition, in several parts of the alimentary canal (e.g., the esophagus and anal canal), the lamina propria contains aggregations of mucus-secreting glands. In general, they lubricate the epithelial surface to protect the mucosa from mechanical and chemical injury. These glands are described below in relation to specific regions of the digestive tube.

1	In segments of the digestive tract in which absorption occurs, principally the small and large intestines, the absorbed products of digestion diffuse into the blood and lymphatic vessels of the lamina propria for distribution. Typically, the blood capillaries are of the fenestrated type and collect most of the absorbed metabolites. In the small intestine, lymphatic capillaries are numerous and receive some absorbed lipids and proteins.

1	The lymphatic tissues in the lamina propria function as an integrated immunologic barrier that protects against pathogens and other antigenic substances that could potentially enter through the mucosa from the lumen of the alimentary canal. The lymphatic tissue is represented by  diffuse lymphatic tissue consisting of numerous lym phocytes and plasma cells, located in the lamina propria, and lymphocytes transiently residing in the intercellular spaces of the epithelium. lymphatic nodules with well-developed germinal centers.  eosinophils, macrophages, and sometimes neutrophils.

1	The diffuse lymphatic tissue and the lymphatic nodules are referred to as gut-associated lymphatic tissue (GALT). In the distal small intestine, the ileum, extensive aggregates of nodules, called Peyer’s patches, occupy much of the lamina propria and submucosa. They tend to be located on the side of the tube opposite the attachment of the mesentery. Aggregated lymphatic nodules are also present in the appendix. The muscularis mucosae forms the boundary between mucosa and submucosa. The muscularis mucosae, the deepest portion of the mucosa, consists of smooth muscle cells arranged in an inner circular and outer longitudinal layer. Contraction of this muscle produces movement of the mucosa, forming ridges and valleys that facilitate absorption and secretion. This localized movement of the mucosa is independent of the peristaltic movement of the entire wall of the digestive tract.

1	The submucosa consists of a dense, irregular connective tissue layer containing blood and lymphatic vessels, a nerve plexus, and occasional glands.

1	The submucosa contains the larger blood vessels that send branches to the mucosa, muscularis externa, and serosa. The submucosa also contains lymphatic vessels and a nerve plexus. The extensive nerve network in the submucosa contains visceral sensory fibers mainly of sympathetic origin, parasympathetic (terminal) ganglia, and preganglionic and postganglionic parasympathetic nerve fibers. The nerve cell bodies of parasympathetic ganglia and their postganglionic nerve fibers represent the enteric nervous system, the third division of the autonomic nervous system. This system is primarily responsible for innervating the smooth muscle layers of the alimentary canal and can function totally independently of the central nervous system. In the submucosa, the network of unmyelinated nerve fibers and ganglion cells constitute the submucosal plexus (also called Meissner’s plexus).

1	As noted, glands occur occasionally in the submucosa in certain locations. For example, they are present in the esophagus and the initial portion of the duodenum. In histologic sections, the presence of these glands often aids in identifying the specific segment or region of the tract. In most parts of the digestive tract, the muscularis externa consists of two concentric and relatively thick layers of smooth muscle. The cells in the inner layer form a tight spiral, described as a circularly oriented layer; those in the outer layer form a loose spiral, described as a longitudinally oriented layer. Located between the two muscle layers is a thin connective tissue layer. Within this connective tissue lies the myenteric plexus (also called Auerbach’s plexus), containing nerve cell bodies (ganglion cells) of postganglionic parasympathetic neurons and neurons of the enteric nervous system, as well as blood vessels and lymphatic vessels.

1	Contractions of the muscularis externa mix and propel the contents of the digestive tract. Contraction of the inner circular layer of the muscularis externa compresses and mixes the contents by constricting the lumen; contraction of the outer, longitudinal layer propels the contents by shortening the tube. The slow, rhythmic contraction of these muscle layers under the control of the enteric nervous system produces peristalsis (i.e., waves of contraction). Peristalsis is marked by constriction and shortening of the tube, which moves the contents through the intestinal tract.

1	A few sites along the digestive tube exhibit variations in the muscularis externa. For example, in the wall of the proximal portion of the esophagus (pharyngoesophageal sphincter) and around the anal canal (external anal sphincter), striated muscle forms part of the muscularis externa. In the stomach, a third, obliquely oriented layer of smooth muscle is present deep into the circular layer. Finally, in the large intestine, part of the longitudinal smooth muscle layer is thickened to form three distinct, equally spaced longitudinal bands called teniae coli. During contraction, the teniae facilitate shortening of the tube to move its contents. The circular smooth muscle layer forms sphincters at specific locations along the digestive tract.

1	The circular smooth muscle layer forms sphincters at specific locations along the digestive tract. At several points along the digestive tract, the circular muscle layer is thickened to form sphincters or valves. From the oropharynx distally, these structures include the following:  Pharyngoesophageal sphincter. Actually, the lowest part of the cricopharyngeus muscle is physiologically referred to as the superior (upper) esophageal sphincter. It prevents the entry of air into the esophagus.

1	 Inferior (lower) esophageal sphincter. As the name implies, this sphincter is located at the lower end of the esophagus; its action is reinforced by the diaphragm that surrounds this part of the esophagus as it passes into the abdominal cavity. It creates a pressure difference between the esophagus and stomach that prevents reflux of gastric contents into the esophagus. Abnormal relaxation of this sphincter allows acidic content of the stomach to return (reﬂux) into the esophagus. If not treated, this condition may progress into gastroesophageal refux disease (GERD), characterized by inﬂammation of the esophageal mucosa (reﬂux esophagitis), strictures, and difficulty in swallowing (dysphagia) with accompanying chest pain.  Pyloric sphincter. Located at the junction of the pylorus of the stomach and duodenum (gastroduodenal sphincter), it controls the release of chyme, the partially digested contents of the stomach, into the duodenum.

1	 Ileocecal valve. Located at the junction of the small and large intestines, it prevents reflux of the contents of the colon with its high bacterial count into the distal ileum, which normally has a low bacterial count.  Internal anal sphincter. This, the most distally located sphincter, surrounds the anal canal and prevents passage of the feces into the anal canal from the undistended rectum. Serosa or adventitia constitutes the outermost layer of the alimentary canal. The serosa is a serous membrane consisting of a layer of simple squamous epithelium, called the mesothelium, and a small amount of underlying connective tissue. It is equivalent to the visceral peritoneum described in gross anatomy. The serosa is the most superficial layer of those parts of the digestive tract that are suspended in the peritoneal cavity. As such, the serosa is continuous with both the mesentery and the lining of the abdominal cavity.

1	Large blood and lymphatic vessels and nerve trunks travel through the serosa (from and to the mesentery) to reach the wall of the digestive tract. Large amounts of adipose tissue can develop in the connective tissue of the serosa (and in the mesentery). Parts of the digestive tract do not possess a serosa. These include the thoracic part of the esophagus and portions of structures in the abdominal and pelvic cavities that are fixed to the cavity wall—the duodenum, ascending and descending colon, rectum, and anal canal. These structures are attached to the abdominal and pelvic wall by connective tissue, the adventitia, which blends with the connective tissue of the wall. The esophagus is a fixed muscular tube that delivers food and liquid from the pharynx to the stomach.

1	The esophagus is a fixed muscular tube that delivers food and liquid from the pharynx to the stomach. The esophagus courses through the neck and mediastinum, where it is attached to adjacent structures by connective tissue. As it enters the abdominal cavity, it is free for a short distance, approximately 1 to 2 cm. The overall length of the esophagus is about 25 cm. On cross section (Fig. 17.2), the lumen in its normally collapsed state has a branched appearance because of longitudinal folds. When a bolus of food passes through the esophagus, the lumen expands without mucosal injury.

1	The mucosa that lines the length of the esophagus has a nonkeratinized stratified squamous epithelium (Fig. 17.3 and Plate 54, page 606). In many animals, however, the epithelium is keratinized, reflecting a coarse food diet. In humans, the surface cells may exhibit some keratohyalin granules, but keratinization does not normally occur. The underlying lamina propria is similar to the lamina propria throughout the alimentary tract; diffuse lymphatic tissue is scattered throughout, and lymphatic nodules are present, often in proximity to ducts of the esophageal mucous glands (see page 573). The deep layer of the mucosa, the muscularis mucosae, is composed of longitudinally organized smooth muscle that begins near the level of the cricoid cartilage. It is unusually thick in the proximal portion of the esophagus and presumably functions as an aid in swallowing.

1	The submucosa consists of dense irregular connective tissue that contains the larger blood and lymphatic vessels, nerve fibers, and ganglion cells. The nerve fibers and ganglion cells make up the submucosal plexus (Meissner’s plexus). Glands are also present (see page 571). In addition, diffuse lymphatic tissue and lymphatic nodules are present mostly in the upper and lower parts of the esophagus where submucosal glands are more prevalent.

1	The muscularis externa consists of two muscle layers, an inner circular layer and an outer longitudinal layer (Plate 54, page 606). It differs from the muscularis externa found in the rest of the digestive tract in that the upper one third is striated muscle, a continuation of the muscle of the pharynx. Striated muscle and smooth muscle bundles are mixed and interwoven in the muscularis externa of the middle third of the esophagus; the muscularis externa of the distal third consists only of smooth muscle, as in the rest of the digestive tract. A nerve

1	FIGURE 17.2 • Photomicrograph of the esophagus. This low-magnification photomicrograph shows an H&E–stained section of the esophagus with its characteristically folded wall, giving the lumen an irregular appearance. The mucosa consists of a relatively thick stratified squamous epithelium, a thin layer of lamina propria containing occasional lymphatic nodules, and muscularis mucosae. Mucous glands are present in the submucosa; their ducts, which empty into the lumen of the esophagus, are not evident in this section. External to the submucosa in this part of the esophagus is a thick muscularis externa made up of an inner layer of circularly arranged smooth muscle and an outer layer of longitudinally arranged smooth muscle. The adventitia is seen just external to the muscularis externa. 8. FIGURE 17.3 • Photomicrograph of the esophageal mucosa.

1	FIGURE 17.3 • Photomicrograph of the esophageal mucosa. This higher-magnification photomicrograph shows the mucosa of the wall of the esophagus in an H&E preparation. It consists of a stratified squamous epithelium, lamina propria, and muscularis mucosae. The boundary between the epithelium and lamina propria is distinct, although uneven, because of the connective tissue papillae. The basal layer of the epithelium stains intensely, appearing as a dark band because the basal cells are smaller and have a high nucleus-to-cytoplasm ratio. Note that the loose connective tissue of the lamina propria is very cellular, containing many lymphocytes. The deepest part of the mucosa is the muscularis mucosae that is arranged in two layers (inner circular and outer longitudinal) similar in orientation to the muscularis externa. 240.

1	plexus, the myenteric plexus (Auerbach’s plexus), is present between the outer and inner muscle layers. As in the submucosal plexus (Meissner’s plexus), nerves and ganglion cells are present here. This plexus innervates the muscularis externa and produces peristaltic activity. As noted, the esophagus is fixed to adjoining structures throughout most of its length; thus, its outer layer is composed of adventitia. After entering the abdominal cavity, the short remainder of the tube is covered by serosa, the visceral peritoneum. Mucosal and submucosal glands of the esophagus secrete mucus to lubricate and protect the luminal wall. Glands are present in the wall of the esophagus and are of two types. Both secrete mucus, but their locations differ.

1	Glands are present in the wall of the esophagus and are of two types. Both secrete mucus, but their locations differ. FIGURE 17.4 • Photomicrograph of an esophageal submucosal gland. This photomicrograph shows a mucicarmine-stained section of the esophagus. An esophageal gland, deeply stained red by the carmine, and an adjacent excretory duct are seen in the submucosa. These small, compound, tubuloalveolar glands produce mucus that lubricates the epithelial surface of the esophagus. Note the stained mucus within the excretory duct. The remaining submucosa consists of irregular dense connective tissue. The inner layer of the muscularis externa (bottom) is composed of circularly arranged smooth muscle. 110.

1	 Esophageal glands proper lie in the submucosa. These glands are scattered along the length of the esophagus but are somewhat more concentrated in the upper half. They are small, compound, tubuloalveolar glands (Fig. 17.4). The excretory duct is composed of stratified squamous epithelium and is usually conspicuous when present in a section, because of its dilated appearance.  Esophageal cardiac glands are named for their similarity to the cardiac glands of the stomach and are found in the lamina propria of the mucosa. They are present in the terminal part of the esophagus and frequently, though not consistently, in the beginning portion of the esophagus.

1	The mucus produced by the esophageal glands proper is slightly acidic and serves to lubricate the luminal wall. Because the secretion is relatively viscous, transient cysts often occur in the ducts. The esophageal cardiac glands produce neutral mucus. Those glands near the stomach tend to protect the esophagus from regurgitated gastric contents. Under certain conditions, however, they are not fully effective, and excessive reﬂux results in pyrosis, a condition more commonly known as heartburn. This condition may progress to fully developed gastroesophageal refux disease (GERD). The muscle of the esophageal wall is innervated by both autonomic and somatic nervous systems.

1	The muscle of the esophageal wall is innervated by both autonomic and somatic nervous systems. The striated musculature in the upper part of the esophagus is innervated by somatic motor neurons of the vagus nerve, cranial nerve X (from the nucleus ambiguus). The smooth muscle of the lower part of the esophagus is innervated by visceral motor neurons of the vagus (from the dorsal motor nucleus). These motor neurons synapse with postsynaptic neurons whose cell bodies are located in the wall of the esophagus. The stomach is an expanded part of the digestive tube that lies beneath the diaphragm. It receives the bolus of macerated food from the esophagus. Mixing and partial digestion of the food in the stomach by its gastric secretions produce a pulpy fluid mix called chyme. The chyme then passes into the small intestine for further digestion and absorption. The stomach is divided histologically into three regions based on the type of gland that each contains.

1	Gross anatomists subdivide the stomach into four regions. The cardia surrounds the esophageal orifice; the fundus lies above the level of a horizontal line drawn through the esophageal (cardiac) orifice; the body lies below this line; and the pyloric part is the funnel-shaped region that leads into the pylorus, the distal, narrow sphincteric region between the stomach and duodenum. Histologists also subdivide the stomach, but into only three regions (Fig. 17.5). These subdivisions are based not on location but on the types of glands that occur in the gastric mucosa. The histologic regions are as follows:  Cardiac region (cardia), the part near the esophageal ori fice, which contains the cardiac glands (Fig. 17.6 and Plate 55, page 608) Pyloric region (pylorus), the part proximal to the pyloric sphincter, which contains the pyloric glands  Fundic region (fundus), the largest part of the stomach, which is situated between the cardia and pylorus and con tains the fundic or gastric

1	pyloric sphincter, which contains the pyloric glands  Fundic region (fundus), the largest part of the stomach, which is situated between the cardia and pylorus and con tains the fundic or gastric glands (see Fig. 17.6)

1	Longitudinal submucosal folds, rugae, allow the stomach to distend when filled. The stomach has the same general structural plan throughout, consisting of a mucosa, submucosa, muscularis externa, and serosa. Examination of the inner surface of the empty stomach reveals a number of longitudinal folds or ridges called rugae. They are prominent in the narrower regions of the stomach but poorly developed in the upper portion (see Fig. 17.5). When the stomach is fully distended, the rugae, composed of the mucosa and underlying submucosa, virtually disappear. The rugae do not alter total surface area; rather, they serve to accommodate expansion and filling of the stomach. A view of the stomach’s surface with a hand lens shows that smaller regions of the mucosa are formed by grooves or FIGURE 17.5 • Photograph of a hemisected human stomach.

1	FIGURE 17.5 • Photograph of a hemisected human stomach. This photograph shows the mucosal surface of the posterior wall of the stomach. Numerous longitudinal gastric folds are evident. These folds or rugae allow the stomach to distend as it fills. The histologic divisions of the stomach differ from the anatomic division. The former is based on the types of glands found in the mucosa. Histologically, the portion of the stomach adjacent to the entrance of the esophagus is the cardiac region (cardia) in which cardiac glands are located. A dashed line approximates its boundary. A slightly larger region leading toward the pyloric sphincter, the pyloric region (pylorus), contains the pyloric glands. Another dashed line approximates its boundary. The remainder of the stomach, the fundic region (fundus), is located between the two dashed lines and contains the fundic (gastric) glands.

1	shallow trenches that divide the stomach surface into bulging irregular areas called mamillated areas. These grooves provide a slightly increased surface area for secretion. At higher magnification, numerous openings can be observed in the mucosal surface. These are the gastric pits, or foveolae. They can be readily demonstrated with the scanning electron microscope (Fig. 17.7). The gastric glands open into the bottom of the gastric pits. Surface mucous cells line the inner surface of the stomach and the gastric pits. The epithelium that lines the surface and the gastric pits of the stomach is simple columnar. The columnar cells are designated surface mucous cells. Each cell possesses a large, apical cup of mucinogen granules, creating a glandular sheet of cells (Fig. 17.8). The mucous cup occupies

1	FIGURE 17.6 • Photomicrograph of esophagogastric junction. This low-magnification photomicrograph shows the junction between the esophagus and stomach. At the esophagogastric junction, the stratified squamous epithelium of the esophagus ends abruptly, and the simple columnar epithelium of the stomach mucosa begins. The surface of the stomach contains numerous and relatively deep depressions called gastric pits that are formed by the surface epithelium. The glands in the vicinity of the esophagus, the cardiac glands, extend from the bottom of these pits. The fundic (gastric) glands similarly arise at the base of the gastric pits and are evident in the remaining part of the mucosa. Note the relatively thick muscularis externa. 40.

1	FIGURE 17.7 • Mucosal surface of the stomach. a. Scanning electron micrograph showing the mucosal surface of the stomach. The gastric pits contain secretory material, mostly mucus (arrows). The surface mucus has been washed away to reveal the surface mucous cells. 1,000. b. Higher magnification showing the apical surface of the surface mucous cells that line the stomach and gastric pits. Note the elongate polygonal shape of the cells. 3,000.

1	FIGURE 17.8 • Gastric glands. a. This photomicrograph shows the fundic mucosa from an Alcian blue/PAS preparation to visualize mucus. Note that the surface epithelium invaginates to form the gastric pits. The surface mucous cells and the cells lining the gastric pits are readily identified in this preparation because the neutral mucus within these cells is stained intensely. One of the gastric pits and its associated fundic gland are depicted by the dashed lines. This gland represents a simple branched tubular gland (arrows indicate the branching pattern). It extends from the bottom of the gastric pit to the muscularis mucosae. Note the segments of the gland: the short isthmus, the site of cell divisions; the relatively long neck; and a shorter and wider fundus. The mucous secretion of mucous neck cells is different from that produced by the surface mucous cells as evidenced by the lighter magenta staining in this region of the gland. 320. b. Schematic diagram of a gastric gland,

1	mucous neck cells is different from that produced by the surface mucous cells as evidenced by the lighter magenta staining in this region of the gland. 320. b. Schematic diagram of a gastric gland, illustrating the relationship of the gland to the gastric pit. Note that the isthmus region contains dividing cells and undifferentiated cells; the neck region contains mucous neck cells, parietal cells, and enteroendocrine cells, including amine precursor uptake and decarboxylation (APUD) cells. Parietal cells are large, pear-shaped acidophilic cells found throughout the gland. The fundus of the gland contains mainly chief cells, some parietal cells, and several types of enteroendocrine cells.

1	most of the volume of the cell. It typically appears empty in routine hematoxylin and eosin (H&E) sections because the mucinogen is lost in fixation and dehydration. When the mucinogen is preserved by appropriate fixation, however, the granules stain intensely with toluidine blue and with the periodic acid–Schiff (PAS) procedure. The toluidine blue staining reflects the presence of many strongly anionic groups in the glycoprotein of the mucin, among which is bicarbonate. The nucleus and Golgi apparatus of the surface mucous cells are located below the mucous cup. The basal part of the cell contains small amounts of rough endoplasmic reticulum (rER) that may impart a light basophilia to the cytoplasm when observed in well-preserved specimens.

1	The mucous secretion from the surface mucous cells is described as visible mucus because of its cloudy appearance. It forms a thick, viscous, gel-like coat that adheres to the epithelial surface; thus, it protects against abrasion from rougher components of the chyme. Additionally, its high bicarbonate and potassium concentration protects the epithelium from the acidic content of the gastric juice. The bicarbonate that makes the mucus alkaline is secreted by the surface cells but is prevented from mixing rapidly with the contents of the gastric lumen by its containment within the mucous coat. Finally, prostaglandins (PGE2) appear to play an important role in protecting gastric mucosa. They stimulate secretion of bicarbonates and increase thickness of the mucus layer with accompanied vasodilatation in the lamina propria. This action improves supply of nutrients to any damaged area of gastric mucosa, thus optimizing conditions for tissue repair.

1	The lining of the stomach does not function in an absorptive capacity. However, some water, salts, and lipid-soluble drugs may be absorbed. For instance, alcohol and certain drugs such as aspirin or nonsteroidal anti-inﬂammatory drugs (NSAIDs) enter the lamina propria by damaging the surface epithelium. Even small doses of aspirin suppress the production of protective prostaglandins by the gastric mucosa. In addition, aspirin’s direct contact with the wall of the stomach interferes with the hydrophobic properties of the gastric mucosa. Fundic Glands of the Gastric Mucosa The fundic glands produce the gastric juice of the stomach.

1	The fundic glands, also called gastric glands, are present throughout the entire gastric mucosa except for the relatively small regions occupied by cardiac and pyloric glands. The fundic glands are simple, branched, tubular glands that extend from the bottom of the gastric pits to the muscularis mucosae (see Fig. 17.8). Located between the gastric pit and the gland below is a short segment known as the isthmus. Isthmus of the fundic gland is a site of stem cells location (stem cell niche) in which stem cells replicate and differentiate. Cells destined to become mucous surface cells migrate upward in the gastric pits to the stomach surface. Other cells migrate downward, maintaining the population of the fundic gland epithelium. Typically, several glands open into a single gastric pit. Each gland has a narrow, relatively long neck segment and a shorter and wider base or fundic segment. The base of the gland usually divides into two and sometimes three branches that become slightly

1	Each gland has a narrow, relatively long neck segment and a shorter and wider base or fundic segment. The base of the gland usually divides into two and sometimes three branches that become slightly coiled near the muscularis mucosae. The cells of the gastric glands produce gastric juice (about 2 L/day), which contains a variety of substances. In addition to water and electrolytes, gastric juice contains four major components:  Hydrochloric acid (HCl) in a concentration ranging from 150 to 160 mmol/L, which gives the gastric juice a low pH (1.0 to 2.0). It is produced by parietal cells and initiates digestion of dietary protein (it promotes acid hydrolysis of substrates). It also converts inactive pepsinogen into the active enzyme pepsin. Because HCl is bacteriostatic, most of the bacteria entering the stomach with the ingested food are destroyed. However, some bacteria can adapt to the low pH of the gastric contents. Helicobacter pylori contains large amounts of urease, the enzyme

1	entering the stomach with the ingested food are destroyed. However, some bacteria can adapt to the low pH of the gastric contents. Helicobacter pylori contains large amounts of urease, the enzyme that hydrolyzes urea, in its cytoplasm and on its plasma membrane. This highly active enzyme creates a protective basic “ammonia cloud” around the bacterium, allowing it to survive in the acidic environment of the stomach (Folder 17.1).

1	 Pepsin, a potent proteolytic enzyme. It is converted from pepsinogen produced by the chief cells by HCl at a pH lower than 5. Pepsin hydrolyzes proteins into small peptides by splitting interior peptide bonds. Peptides are further digested into amino acids by enzymes in the small intestine.  Mucus, an acid-protective coating for the stomach secreted by several types of mucus-producing cells. The mucus and bicarbonates trapped within the mucous layer maintain a neutral pH and contribute to the so-called physiologic gastric mucosa barrier. In addition, mucus serves as a physical barrier between the cells of the gastric mucosa and the ingested material in the lumen of the stomach.  Intrinsic factor, a glycoprotein secreted by parietal cells that binds to vitamin B12. It is essential for its absorption, which occurs in the distal part of the ileum. Lack of intrinsic factor leads to pernicious anemia and vitamin B-12 defciency (see Folder 17.1).

1	In addition, gastrin and other hormones and hormone-like secretions are produced by enteroendocrine cells in the fundic glands and secreted into the lamina propria, where they enter the circulation or act locally on other gastric epithelial cells. Fundic glands are composed of four functionally different cell types. The cells that constitute the fundic glands are of four functional types. Each has a distinctive appearance. In addition, undifferentiated cells that give rise to these cells are also present. These are the various cells that constitute the gland:  Mucous neck cells  Chief cells  Parietal cells, also called oxyntic cells  Enteroendocrine cells  Undifferentiated adult stem cells  FOLDER 17.1 Clinical Correlation: Pernicious Anemia and Peptic Ulcer Disease

1	Achlorhydria is a chronic autoimmune disease character-Recently, new proton pump inhibitors (e.g., omeprazole ized by the destruction of the gastric mucosa. Conse-and lansoprazole) have been designed that inhibit the quently, in the absence of parietal cells, the intrinsic factor H/K-ATPase. They suppress acid production in the pariis not secreted, thereby leading to pernicious anemia. etal cells and do not affect intrinsic factor secretion. Lack of intrinsic factor is the most common cause of vita-Although it was generally thought that the parietal cells min B12 deficiency. However, other factors such as gram-are the direct target of the H2 receptor–antagonist drugs, negative anaerobic bacterial overgrowth in the small recent evidence from a combination of in situ hybridization intestine are associated with B12 deficiency. These bacte-histochemistry and antibody staining has unexpectedly reria bind to the vitamin B12–intrinsic factor complex, pre-vealed that the immunoglobulin A

1	are associated with B12 deficiency. These bacte-histochemistry and antibody staining has unexpectedly reria bind to the vitamin B12–intrinsic factor complex, pre-vealed that the immunoglobulin A (IgA)–secreting plasma venting its absorption. Parasitic tapeworm infections also cells and some of the macrophages in the lamina propria produce clinical symptoms of pernicious anemia. Because display a positive reaction for gastrin receptor mRNA, not the liver has extensive reserve stores of vitamin B12, the the parietal cells. These findings indicate that the agents disease is often not recognized until long after significant used to treat peptic ulcers may act directly on plasma cells changes in the gastric mucosa have taken place. or macrophages and that these cells then transmit their ef-

1	Another cause of reduced secretion of intrinsic factor fects to the parietal cells to inhibit HCl secretion. The facand subsequent pernicious anemia is the loss of gastric tor that mediates the interaction between the connective epithelium in partial or total gastrectomy. Loss of functional tissue cells and the epithelial cells has not been elucidated. gastric epithelium also occurs in chronic or recurrent pep-Recent evidence, however, suggests that most common tic ulcer disease (PUD). Often, even healed ulcerated re-peptic ulcers (95%) are actually caused by a chronic infecgions produce insufficient intrinsic factor. Repeated loss of tion of the gastric mucosa by the bacterium Helicobacter epithelium and consequent scarring of the gastric mucosa pylori. Lipopolysaccharide antigens are expressed on its can significantly reduce the amount of functional mucosa. surface that mimic those on human gastric epithelial cells.

1	Histamine H2 receptor–antagonist drugs such as The mimicry appears to cause an initial immunologic tolerranitidine (Zantac) and cimetidine (Tagamet), which block ance to the pathogen by the host immune system, thus help-attachment of histamine to its receptors in the gastric mu-ing to enhance the infection and ultimately causing the cosa, suppress both acid and intrinsic factor production production of antibodies. These antibodies against H. pylori and have been used extensively in the treatment of peptic bind to the gastric mucosa and cause damage to the mu-ulcers. These drugs prevent further mucosal erosion and cosal cells. Treatment includes antibiotic eradication of the promote healing of the previously eroded surface. How-bacteria. These treatments for ulcerative disease have made ever, long-term use can cause vitamin B12 deficiency. the common surgical interventions of the past infrequent.

1	Mucous neck cells are localized in the neck region of the gland and are interspersed with parietal cells. As the name implies, the mucous neck cells are located in the neck region of the fundic gland. Parietal cells are usually interspersed between groups of these cells. The mucous neck cell is much shorter than the surface mucous cell and contains considerably less mucinogen in the apical cytoplasm. Consequently, these cells do not exhibit a prominent mucous cup. Also, the nucleus tends to be spherical compared with the more prominent, elongated nucleus of the surface mucous cell. The mucous neck cells secrete a soluble mucus compared with the insoluble or cloudy mucus produced by the surface mucous cell. Release of mucinogen granules is induced by vagal stimulation; thus, secretion from these cells does not occur in the resting stomach. Chief cells are located in the deeper part of the fundic glands.

1	Chief cells are located in the deeper part of the fundic glands. Chief cells are typical protein-secreting cells (Fig. 17.9 and Plate 57, page 612). The abundant rER in the basal cytoplasm gives this part of the cell a basophilic appearance, whereas the apical cytoplasm is eosinophilic owing to the presence of the secretory vesicles, also called zymogen granules because they contain enzyme precursors. The basophilia, in particular, allows easy identification of these cells in H&E sections. The eosinophilia may be faint or absent when the secretory vesicles are not adequately preserved. Chief cells secrete pepsinogen and a weak lipase. On contact with the acid gastric juice, pepsinogen is converted to pepsin, a proteolytic enzyme. Parietal cells secrete HCl and intrinsic factor.

1	Parietal cells secrete HCl and intrinsic factor. Parietal (oxyntic) cells are found in the neck of the fundic glands, among the mucous neck cells, and in the deeper part of the gland. They tend to be most numerous in the upper and middle portions of the neck. They are large cells, sometimes binucleate, and appear somewhat triangular in sections, with the apex directed toward the lumen of the gland and the base resting on the basal lamina. The nucleus is spherical, and the cytoplasm stains with eosin and other acidic dyes. Their size and distinctive staining characteristics

1	FIGURE 17.9 • Diagram of a chief cell. The large amount of rER in the basal portion of the cell accounts for the intense basophilic staining seen in this region. Secretory vesicles (zymogen granules) containing pepsinogen and a weak lipase are not always adequately preserved, and thus the staining in the apical region of the cell is somewhat variable. This cell produces and secretes the precursor enzyme of the gastric secretion. allow them to be easily distinguished from other cells in the fundic glands.

1	When examined with the transmission electron microscope (TEM), parietal cells (Fig. 17.10) are seen to have an extensive intracellular canalicular system that communicates with the lumen of the gland. Numerous microvilli project from the surface of the canaliculi, and an elaborate tubulovesicular membrane system is present in the cytoplasm adjacent to the canaliculi. In an actively secreting cell, the number of microvilli in the canaliculi increases, and the tubulovesicular system is reduced significantly or disappears. The membranes of the tubulovesicular system serve as a reservoir of plasma membrane containing active proton pumps. This membrane material can be inserted into the plasma membrane of the canaliculi to increase their surface area and the number of proton pumps available for acid production. Numerous mitochondria with complex cristae and many matrix granules supply the high levels of energy necessary for acid secretion.

1	HCl is produced in the lumen of the intracellular canaliculi. Parietal cells have three different types of membrane receptors for substances that activate HCl secretion: gastrin receptors, histamine H2 receptors, and acetylcholine M3 receptor. Activation of the gastrin receptor by gastrin, a gastrointestinal peptide hormone, is the major path for parietal cell stimulation (Folder 17.2). After stimulation, several steps occur in the production of HCl (Fig. 17.11): FIGURE 17.10 • Diagram of a parietal cell. The cytoplasm of the parietal cell stains with eosin largely because of the extensive amount of membrane comprising the intracellular canaliculus, tubulovesicular system, mitochondria, and the relatively small number of ribosomes. This cell produces HCl and intrinsic factor.

1	 Production of H ions in the parietal cell cytoplasm by the enzyme carbonic anhydrase. This enzyme hydrolyzes carbonic acid (H2CO3) to H and HCO3 . Carbon dioxide (CO2), necessary for synthesis of carbonic acid, diffuses across the basement membrane into the cell from the blood capillaries in the lamina propria.  Transport of H ions from the cytoplasm, across the membrane to the lumen of the canaliculus by the H/ K-ATPase proton pump. Simultaneously, K from the canaliculus is transported into the cell cytoplasm in exchange for the H ions.  Transport of K and Cl ions from the parietal cell cytoplasm into the lumen of the canaliculus through activation of K and Cl channels (uniporters) in the plasma membrane.  Formation of HCl from the H and Cl that were transported into the lumen of the canaliculus.

1	 Formation of HCl from the H and Cl that were transported into the lumen of the canaliculus. In humans, intrinsic factor is secreted by the parietal cells (chief cells do so in some other species). Its secretion is stimulated by the same receptors that stimulate gastric acid secretion. Intrinsic factor is a 44-kilodalton glycoprotein that complexes with vitamin B12 in the stomach and duodenum, a step necessary for subsequent absorption of the vitamin in the ileum. Autoantibodies directed against intrinsic factor or parietal cells themselves lead to an intrinsic factor deficiency, resulting in malabsorption of vitamin B12 and pernicious anemia (see Folder 17.1).  FOLDER 17.2 Clinical Correlation: Zollinger-Ellison Syndrome

1	Excessive secretion of gastrin usually has its origin in a tumor of the gastrin-producing enteroendocrine cells located in the duodenum or in the pancreatic islet. This condition, known as the Zollinger-Ellison syndrome or gastrinomas, is characterized by excessive secretion of hydrochloric acid by continuously stimulated parietal cells. The excess acid cannot be adequately neutralized in the duodenum, thereby leading to gastric and duodenal ulcers. Gastric ulcers are present in 95% of patients with this syndrome and are six times more prevalent than the duodenal ulcers. Patients with Zollinger-Ellison syndrome may experience intermittent abdominal pain, diarrhea, and steatorrhea (excretion of stool containing a large amount of fat). Patients without symptoms who have severe ulceration of the stomach and small intestine, especially if they fail to respond to conventional treatment, should be also suspected of having a tumor that is producing excess gastrin. Treatment of

1	ulceration of the stomach and small intestine, especially if they fail to respond to conventional treatment, should be also suspected of having a tumor that is producing excess gastrin. Treatment of Zollinger-Ellison syndrome in the past involved blockage of the parietal cell membrane receptors that stimulate HCl production. Recently, proton pump inhibitors have become the treatment of choice in managing HCl hypersecretion. In addition, surgical excision of the tumor, when possible, removes the source of gastrin production and alleviates symptoms.

1	FIGURE 17.11 • Diagram of parietal cell HCl synthesis. After parietal cell stimulation, several steps occur leading to the production of HCl. Carbon dioxide (CO2) from the blood diffuses across the basement membrane into the cell to form H2CO3. The H2CO3 dissociates into H and HCO3 . The reaction is catalyzed by carbonic anhydrase, which leads to the production of H ions in the cytoplasm, which are then transported across the membrane to the lumen of the intracellular canaliculus by a H/K-ATPase proton pump. Simultaneously, K within the canaliculus is transported into the cell in exchange for the H ions. Cl ions are also transported from the cytoplasm of the parietal cell into the lumen of the canaliculus by Cl channels in the membrane. HCl is then formed from H and Cl. The HCO3 / Cl anion channels maintain the normal concentration of both ions in the cell, as well as Na/K-ATPase on the basolateral cell membrane.

1	Enteroendocrine cells secrete their products into either Most of them represent small cells that rest on the basal lamina the lamina propria or underlying blood vessels. and do not always reach the lumen; they are known as enteroendocrine “closed” cells (Fig. 17.12ab and Plate 57, Enteroendocrine cells are found at every level of the fundic page 612). Some, however, have a thin cytoplasmic extension gland, although they tend to be somewhat more prevalent in the bearing microvilli that are exposed to the gland lumen base (Folder 17.3). In general, two types of enteroendocrine (Fig 17.12c); these are referred as enteroendocrine cells can be distinguished throughout the gastrointestinal tract.  FOLDER 17.3 Functional Considerations: The Gastrointestinal Endocrine System

1	Enteroendocrine cells are specialized cells present in the mucosa of the digestive tract. They account for less than 1% of all epithelial cells in the gastrointestinal tract, but as a whole, they collectively constitute the largest endocrine “organ” in the body. Enteroendocrine cells are also found in the ducts of the pancreas, the liver, and the respiratory sys-tem, another endodermal derivative that originates by in-vagination of the epithelium of the embryonic foregut. Because enteroendocrine cells closely resemble neurose-cretory cells of the central nervous system (CNS) that se-crete many of the same hormones, signaling molecules, and regulatory agents, they are also called neuroendocrine cells. Most of these cells are not grouped as clusters in any specific part of the gastrointestinal tract. Rather, enteroen-docrine cells are distributed singly throughout the gastroin-testinal epithelium. For that reason, they are described as constituting part of a diffuse neuroendocrine

1	tract. Rather, enteroen-docrine cells are distributed singly throughout the gastroin-testinal epithelium. For that reason, they are described as constituting part of a diffuse neuroendocrine system (DNES). Figure 17.13 shows the parts of the gastrointesti-nal tract from which the gastrointestinal peptides are pro-duced. One notable exception to this distribution pattern is found in the pancreas. Here, enteroendocrine cells, derived from pancreatic buds that also arise from the em-bryonic foregut, form specialized accumulations called en-docrine islets of Langerhans (see page 649). In the current view, the DNES includes both neurons and endocrine cells that share common characteristics, including the expression of specific markers (e.g., neu-ropeptides, chromogranins, and neuropeptide processing enzymes) and the presence of dense-core secretory gran-ules. Secretory products of enteroendocrine cells derive from a variety of genes; they are expressed in different forms because of

1	processing enzymes) and the presence of dense-core secretory gran-ules. Secretory products of enteroendocrine cells derive from a variety of genes; they are expressed in different forms because of alternative splicing and differential pro-cessing. Secretion of enteroendocrine cells is regulated by G protein–coupled receptors and tyrosine-kinase activity. There is evidence that chromogranin-A regulates biosyn-thesis of dense-core secretory granules, whereas chromo-granin-B controls sorting and packaging of produced peptides into secretory vesicles. Table 17.1 lists important gastrointestinal hormones, their sites of origin, and their major functions. Neoplastic transformations of DNES cells are responsi-ble for development of gastroenteropancreatic (GEP) neuroendocrine tumors. These tumors represent rare neoplasms of the gastrointestinal tract and pancreas that often secrete hormonally active agents, causing distinct clinical syndromes. The appendix is the most common gastrointestinal

1	represent rare neoplasms of the gastrointestinal tract and pancreas that often secrete hormonally active agents, causing distinct clinical syndromes. The appendix is the most common gastrointestinal site of origin for neuroendocrine tumors. The classical example is the carcinoid syndrome caused by the release of a variety of hormonally active substances by tumor cells. Symptoms include diarrhea (case by sero-tonin), episodic flushing, bronchoconstriction, and right-sided cardiac valve disease. Some enteroendocrine cells may be classifiable func-tionally as amine precursor uptake and decarboxylation (APUD) cells. They should not, however, be confused with the APUD cells that are derived from the embryonic neural crest and migrate to other sites in the body. APUD cells se-crete a variety of regulator substances in tissues and or-gans including the respiratory epithelium, adrenal medulla, islets of Langerhans, thyroid gland (parafollicular cells), and pituitary gland. The enteroendocrine

1	regulator substances in tissues and or-gans including the respiratory epithelium, adrenal medulla, islets of Langerhans, thyroid gland (parafollicular cells), and pituitary gland. The enteroendocrine cells differentiate from the progeny of the same stem cells as all of the other epithelial cells of the digestive tract. The fact that two dif-ferent cells may produce similar products should not imply that they have the same origin. Enteroendocrine cells produce not only gastrointestinal hormones such as gastrin, ghrelin, secretin, cholecys-tokinin (CCK), gastric inhibitory peptide (GIP), and motilin, but also paracrine hormones. A paracrine hormone dif-fers from an endocrine hormone in that it diffuses locally to its target cell instead of being carried by the bloodstream to a target cell. A well-known substance that appears to act as a paracrine hormone within the gastrointestinal tract and pancreas is somatostatin, which inhibits other gas-trointestinal and pancreatic islet endocrine

1	A well-known substance that appears to act as a paracrine hormone within the gastrointestinal tract and pancreas is somatostatin, which inhibits other gas-trointestinal and pancreatic islet endocrine cells. In addition to the established gastrointestinal hormones, several gastrointestinal peptides have not been definitely classified as hormones or paracrine hormones. These pep-tides are designated candidate or putative hormones. Other locally active agents isolated from the gastroin-testinal mucosa are neurotransmitters. These agents are released from nerve endings close to the target cell, usu-ally the smooth muscle of the muscularis mucosae, the muscularis externa, or the tunica media of a blood vessel. Enteroendocrine cells can also secrete neurotransmitters that activate afferent neurons, sending signals to the CNS and enteric division of autonomic nervous system. In addi-tion to acetylcholine (not a peptide), peptides found in nerve fibers of the gastrointestinal tract are

1	neurons, sending signals to the CNS and enteric division of autonomic nervous system. In addi-tion to acetylcholine (not a peptide), peptides found in nerve fibers of the gastrointestinal tract are vasoactive in-testinal peptide (VIP), bombesin, and enkephalins. Thus, a particular peptide may be produced by endocrine and paracrine cells and also be localized in nerve fibers.

1	FIGURE 17.12 • Electron micrograph and diagrams of enteroendocrine cells. a. This electron micrograph shows an example of the “closed” enteroendocrine cell. Arrowheads mark the boundary between the enteroendocrine cell and the adjacent epithelial cells. At its base, the enteroendocrine cell rests on the basal lamina (BL). This cell does not extend to the epithelial or luminal surface. Numerous secretory vesicle (G) in the base of the cell are secreted in the direction of the arrows across the basal lamina and into the connective tissue (CT ). En, endothelium of capillary; M, mitochondria; rER, rough endoplasmic reticulum; sER, smooth endoplasmic reticulum. b. This diagram of an enteroendocrine “closed” cell is drawn to show that it does not reach the epithelial surface. The secretory vesicles are regularly lost during routine preparation. Because of the absence of other distinctive organelles, the nucleus appears to be surrounded by a small amount of clear cytoplasm in H&E–stained

1	are regularly lost during routine preparation. Because of the absence of other distinctive organelles, the nucleus appears to be surrounded by a small amount of clear cytoplasm in H&E–stained sections. c. The enteroendocrine “open” extend to the epithelial surface. Microvilli on the apical surface of these cells possess taste receptors and are able to detect sweet, bitter, and umami sensations. These cells serve as chemoreceptor cells, which monitor an environment on the surface of the epithelium. They are involved in a regulation of gastrointestinal hormones secretion.

1	“open” cells. It is now known that open cells serve as primary chemoreceptors that sample the contents of the gland lumen and release hormones based on the information obtained from those samples. The taste receptors, similar to those found in taste buds of the specialized oral mucosa (page 530), detect sweet, bitter, and umami sensations and have now been characterized on the free surface of the open enteroendocrine cells. They belong to the T1R and T2R families of G protein–coupled receptors described in Chapter 16 (page 531). Secretion from closed cells, however, is regulated by luminal content indirectly through neural and paracrine mechanisms.

1	Electron micrographs reveal small membrane-bound secretory vesicles throughout the cytoplasm; however, the vesicles are typically lost in H&E preparations, and the cytoplasm appears clear because of the lack of sufficient stainable material. Although these cells are often difficult to identify because of their small size and lack of distinctive staining, the clear cytoplasm of the cell sometimes stands out in contrast to adjacent chief or parietal cells, thus allowing their easy recognition.

1	The names given to the enteroendocrine cells in the older literature were based on their staining with salts of silver and chromium (i.e., enterochromaffin cells, argentaffin cells, and argyrophil cells). Such cells are currently identified and characterized by immunochemical staining for the more than 20 peptide and polypeptide hormones and hormone-like regulating agents that they secrete (a list of many of these agents and their actions is given in Fig. 17.13 and in Tables 17.1 and 17.2). With the aid of the TEM, at least 17 different types of enteroendocrine cells have been described on the basis of size, shape, and density of their secretory vesicles. Cardiac Glands of the Gastric Mucosa Cardiac glands are composed of mucus-secreting cells.

1	Cardiac glands are limited to a narrow region of the stomach (the cardia) that surrounds the esophageal orifice. Their secretion, in combination with that of the esophageal cardiac glands, contributes to the gastric juice and helps protect the esophageal epithelium against gastric reflux. The glands are tubular, somewhat tortuous, and occasionally branched (Fig. 17.14 and Plate 56, page 610). They are composed mainly of mucus-secreting cells, with occasional interspersed enteroendocrine cells. The mucus-secreting cells are similar in appearance to the cells of the esophageal cardiac glands. They have a flattened basal nucleus, and the apical cytoplasm is typically filled with mucin granules. A short duct segment containing columnar cells with elongate nuclei is interposed between the secretory portion of the gland and the shallow pits into which the glands secrete. The duct segment is the site at which the surface mucous cells and the gland cells are produced.

1	FIGURE 17.13 • Gastrointestinal hormones. This schematic diagram shows the distribution of gastrointestinal peptide hormones produced by enteroendocrine cells in the alimentary canal. CCK, cholecystokinin; VIP, vasoactive intestinal peptide; GIP, gastric inhibitory peptide. (Modified from Johnson LR, ed. Gastrointestinal Physiology. St. Louis: Mosby-Year Book, 1997.) Pyloric Glands of the Gastric Mucosa Pyloric gland cells are similar to surface mucous cells and help protect the pyloric mucosa. Pyloric glands are located in the pyloric antrum (the part of the stomach between the fundus and the pylorus). They are branched, coiled, tubular glands (Plate 58, page 614).

1	583 TABLE Physiologic Actions of Gastrointestinal Hormones 17.1 Major Action Hormone Site of Synthesis Stimulates Inhibits Gastrin G cells in stomach Gastric acid secretion Ghrelin Gr cells in stomach GH secretion Appetite and perception of hunger Lipid metabolism Fat utilization in adipose tissue Cholecystokinin (CCK) I cells in duodenum and jejunum Gallbladder contraction Pancreatic enzyme secretion Pancreatic bicarbonate ion secretion Pancreatic growth Gastric emptying Secretin S cells in duodenum Pancreatic enzyme secretion Pancreatic bicarbonate ion secretion Pancreatic growth Gastric acid secretion Gastric inhibitory peptide (GIP) K cells in duodenum and jejunum Insulin release Gastric acid secretion Motilin Mo cells in duodenum and jejunum Gastric motility Intestinal motility Modified from Johnson LR, ed. Essential Medical Physiology. Philadelphia: Lippincott-Raven, 1998. GH, growth hormone.

1	TABLE Physiologic Actions of Other Hormones in the Gastrointestinal Tract 17.2 Modified from Johnson LR, ed. Essential Medical Physiology. Philadelphia: Lippincott-Raven, 1998. GI, gastrointestinal. Major Action Hormone Site of Synthesis Stimulates Inhibits Candidate hormones Pancreatic polypeptide PP cells in pancreas Gastric emptying and gut motility Pancreatic enzyme secretion Pancreatic bicarbonate secretion Peptide YY L cells in ileum and colon Electrolyte and water absorption in the colon Gastric acid secretion Gastric emptying Food intake Glucagon-like peptide-1 (GLP-1) L cells in ileum and colon Insulin release Gastric acid secretion Gastric emptying Paracrine hormones Somatostatin D cells in mucosa throughout GI tract Gastrin release Gastric acid secretion Release of other GI hormones Histamine Mucosa throughout GI tract Gastric acid secretion Neurocrine hormones Bombesin Stomach Gastrin release Enkephalins Mucosa and smooth muscle throughout GI tract Smooth muscle

1	GI hormones Histamine Mucosa throughout GI tract Gastric acid secretion Neurocrine hormones Bombesin Stomach Gastrin release Enkephalins Mucosa and smooth muscle throughout GI tract Smooth muscle contraction Intestinal secretion Vasoactive inhibitory peptide (VIP) Mucosa and smooth muscle throughout GI tract Pancreatic enzyme secretion Intestinal secretion Smooth muscle contraction Sphincter contraction

1	The lumen is relatively wide, and the secretory cells are similar in appearance to the surface mucous cells, suggesting a relatively viscous secretion. Enteroendocrine cells are found interspersed within the gland epithelium along with occasional parietal cells. The glands empty into deep gastric pits that occupy about half the thickness of the mucosa (Fig. 17.15). Epithelial Cell Renewal in the Stomach Surface mucous cells are renewed approximately every 3 to 5 days.

1	Epithelial Cell Renewal in the Stomach Surface mucous cells are renewed approximately every 3 to 5 days. The relatively short lifespan of the surface mucous cells, 3 to 5 days, is accommodated by mitotic activity in the isthmus, the narrow segment that lies between the gastric pit and the fundic gland (Fig. 17.16). The isthmus of the fundic gland contains a reservoir of tissue stem cells that undergo mitotic activity, providing for continuous cell renewal. Most of the newly produced cells at this site become surface mucous cells. They migrate upward along the wall of the pit to the luminal surface of the stomach and are ultimately shed into the stomach lumen. The cells of the fundic glands have a relatively long lifespan.

1	Other cells from the isthmus migrate down into the gastric glands to give rise to the parietal cells, chief cells, mucous gland cells, and enteroendocrine cells that constitute the gland epithelium. These cells have a relatively long lifespan. The parietal cells have the longest lifespan, approximately 150 to 200 days. Although parietal cells develop from the same undifferentiated stem cells, their lifespan is distinctly different. Recently, it has been hypothesized that parietal cells may have originated from a bacterium called Neurospora crassa that previously existed in a symbiotic relationship with the cells of the human stomach. The basis for this hypothesis is that the human proton pump (H/K-ATPase) found in parietal cells bears a strong genetic similarity to proton pumps found in this bacterium. The bacterial DNA is thought to have been translocated and subsequently incorporated into the nucleus of the stem cells, probably with the help of a virus.

1	The chief and enteroendocrine cells are estimated to live for about 60 to 90 days before they are replaced by new cells migrating downward from the isthmus. The mucous neck cell, in contrast, has a much shorter lifespan, approximately 6 days. The lamina propria of the stomach is relatively scant and restricted to the limited spaces surrounding the gastric pits and glands. The stroma is composed largely of reticular fibers with associated fibroblasts and smooth muscle cells. Other components include cells of the immune system, namely, lymphocytes, plasma cells, macrophages, and some eosinophils. When

1	FIGURE 17.14 • Photomicrograph of cardiac glands. This photomicrograph shows the esophagogastric junction. Note the presence of the stratified squamous epithelium of the esophagus in the upper right corner of the micrograph. The cardiac glands are tubular, somewhat tortuous, and occasionally branched. They are composed mainly of mucus-secreting cells similar in appearance to the cells of the esophageal glands. Mucous secretion reaches the lumen of the gastric pit via a short duct segment containing columnar cells. 240. inflammation occurs, as is often the case, neutrophils may also be prominent. Occasional lymphatic nodules are also present, usually intruding partially into the muscularis mucosae.

1	inflammation occurs, as is often the case, neutrophils may also be prominent. Occasional lymphatic nodules are also present, usually intruding partially into the muscularis mucosae. The muscularis mucosae is composed of two relatively thin layers, usually arranged as an inner circular and outer longitudinal layer. In some regions, a third layer may be present; its orientation tends to be in a circular pattern. Thin strands of smooth muscle cells extend toward the surface in the lamina propria from the inner layer of the muscularis mucosae. These smooth muscle cells in the lamina propria are thought to help outflow of the gastric gland secretions. The submucosa is composed of a dense connective tissue containing variable amounts of adipose tissue and blood vessels, as well as the nerve fibers and ganglion cells that compose the submucosal (Meissner’s) plexus. The latter innervates the vessels of the submucosa and the smooth muscle of the muscularis mucosae.

1	FIGURE 17.15 • Photomicrograph of pyloric glands. This photomicrograph shows a section of the wall of the pylorus. The pyloric glands are relatively straight for most of their length but are slightly coiled near the muscularis mucosae. The lumen is relatively wide, and the secretory cells are similar in appearance to the surface mucous cells, suggesting a relatively viscous secretion. They are restricted to the mucosa and empty into the gastric pits. The boundary between the pits and glands is, however, hard to ascertain in routine H&E preparations. 120.

1	The muscularis externa of the stomach is traditionally described as consisting of an outer longitudinal layer, a middle circular layer, and an inner oblique layer. This description is somewhat misleading, as distinct layers may be difficult to discern. As with other hollow, spheroidal organs (e.g., gallbladder, urinary bladder, and uterus), the smooth muscle of the muscularis externa of the stomach is somewhat more randomly oriented than the term layer implies. Moreover, the longitudinal layer is absent from much of the anterior and posterior stomach surfaces, and the circular layer is poorly developed in the periesophageal region. The arrangement of the muscle layers is functionally important, as it relates to its role in mixing chyme during the digestive process as well as to its ability to force the partially digested contents into the small intestine. Groups of ganglion cells and bundles of unmyelinated nerve fibers are present between the muscle layers. Collectively, they

1	ability to force the partially digested contents into the small intestine. Groups of ganglion cells and bundles of unmyelinated nerve fibers are present between the muscle layers. Collectively, they represent the myenteric (Auerbach’s) plexus, which provides innervation of the muscle layers.

1	FIGURE 17.16 • Photomicrograph of a dividing cell in the isthmus of a pyloric gland. The gastric pits in this photomicrograph were sectioned in a plane that is oblique to the axis of the pit. Note that on this section, gastric pits (arrows) can be recognized as invaginations of surface epithelium that are surrounded by lamina propria. The lamina propria is highly cellular because of the presence of large numbers of lymphocytes. 240. Inset. This high magnification of the area indicated by the rectangle shows a dividing cell in the isthmus. 580. The serosa of the stomach is as described above for the alimentary canal in general. It is continuous with the parietal peritoneum of the abdominal cavity via the greater omentum and with visceral peritoneum of the liver at the lesser omen-tum. Otherwise, it exhibits no special features.

1	The small intestine is the longest component of the digestive tract, measuring over 6 m, and is divided into three anatomic portions:  Duodenum (25 cm long) is the first, shortest, and widest part of the small intestine. It begins at the pylorus of the stomach and ends at the duodenojejunal junction (Plate 59, page 616).  Jejunum (2.5 m long) begins at the duodenojejunal junction and constitutes the upper two fifths of the small intestine. It gradually changes its morphologic characteristics to become the ileum (Plate 60, page 618).  Ileum (3.5 m long) is a continuation of the jejunum and constitutes the lower three fifths of the small intestine. It ends at the ileocecal junction, the union of the distal ileum and cecum (Plate 61, page 620). The small intestine is the principal site for the digestion of food and absorption of the products of digestion.

1	The small intestine is the principal site for the digestion of food and absorption of the products of digestion. Chyme from the stomach enters the duodenum, where enzymes from the pancreas and bile from the liver are also delivered to continue the solubilization and digestion process. Enzymes, particularly disaccharidases and dipeptidases, are also located in the glycocalyx of the microvilli of the enterocytes, the intestinal absorptive cells. These enzymes contribute to the digestive process by completing the breakdown of most sugars and proteins to monosaccharides and amino acids, which are then absorbed (Folder 17.4). Water and electrolytes that reach the small intestine with the chyme and pancreatic and hepatic secretions are also reabsorbed in the small intestine, particularly in the distal portion. Plicae circulares, villi, and microvilli increase the absorptive surface area of the small intestine.

1	Plicae circulares, villi, and microvilli increase the absorptive surface area of the small intestine. The absorptive surface area of the small intestine is amplified by tissue and cell specializations of the submucosa and mucosa.  Plicae circulares (circular folds), also known as the valves of Kerckring, are permanent transverse folds that contain a core of submucosa. Each circular fold is circularly arranged and extends about one half to two thirds of the way around the circumference of the lumen (Fig. 17.17). The folds begin to appear about 5 to 6 cm beyond the pylorus. They are most numerous in the distal part of the duodenum and the beginning of the jejunum and become reduced in size and frequency in the middle of the ileum.

1	 Villi are unique, fingerlike and leaflike projections of the mucosa that extend from the theoretical mucosal surface for 0.5 to 1.5 mm into the lumen (Fig. 17.18). They completely cover the surface of the small intestine, giving it a velvety appearance when viewed with the unaided eye.  Microvilli of the enterocytes provide the major amplification of the luminal surface. Each cell possesses several thousand closely packed microvilli. They are visible in the light microscope and give the apical region of the cell a striated appearance, the so-called striated border. Enterocytes and their microvilli are described below. The villi and intestinal glands, along with the lamina propria, associated GALT, and muscularis mucosae, constitute the essential features of the small intestinal mucosa.

1	The villi and intestinal glands, along with the lamina propria, associated GALT, and muscularis mucosae, constitute the essential features of the small intestinal mucosa. Villi, as noted, are projections of the mucosa. They consist of a core of loose connective tissue covered by a simple columnar epithelium. The core of the villus is an extension of the lamina propria, which contains numerous fibroblasts, smooth muscle cells, lymphocytes, plasma cells,  FOLDER 17.4 Functional Considerations: Digestive and Absorptive Functions of Enterocytes

1	Carbohydrate final digestion is brought about by enzymes bound to the microvilli of the enterocytes (Fig. 17.4.2). Galactose, glucose, and fructose are absorbed directly into venous capillaries and conveyed to the liver by the vessels of the hepatic portal system. Some infants and a larger percentage of adults cannot tolerate milk and unfermented milk products because of the absence of lactase, the disaccharidase that splits lactose into galactose and

1	The plasma membrane of the microvilli of the enterocyte plays a role in digestion as well as absorption. Digestive enzymes are anchored in the plasma membrane, and their functional groups extend outward to become part of the glycocalyx. This arrangement brings the end products of digestion close to their site of absorption. Included among the enzymes are peptidases and disaccharidases. The plasma membrane of the apical microvilli also contains the enzyme enteropeptidase (enterokinase), which is particularly important in the duodenum, where it converts trypsinogen into trypsin. Trypsin can then continue to convert additional trypsinogen into trypsin, and trypsin converts several other pancreatic zymogens into active enzymes (Fig. F17.4.1). A summary of digestion and absorption of the three major nutrients is outlined in the following paragraphs.

1	FIGURE F17.4.1 • Diagram showing events in the activation of the proteolytic enzymes of the pancreas. The majority of pancreatic enzymes (proteases) are secreted as inactive proenzymes. Their activation is triggered by the arrival of chyme into the duodenum. This stimulates the mucosal cells to release and to activate the enterokinase (blue box) within the glycocalyx. The enterokinase activates trypsinogen, converting it into its active form, trypsin (green box). In turn, trypsin activates other pancreatic proenzymes (red box) into their active forms (purple box). The active proteases hydrolyze peptide bonds of proteins or polypeptides and reduce them to small peptides and amino acids. FIGURE F17.4.2 • Diagram showing the digestion and absorption of carbohydrates by an enterocyte.

1	Carbohydrates are delivered to the alimentary canal as monosaccharides (e.g., glucose, fructose, and galactose), disaccharides (e.g., sucrose, lactose, and maltose), and polysaccharides (e.g., glycogen and starch). Enzymes involved in digestion of carbohydrates are classified as salivary and pancreatic amylases. Further digestion is performed at the striated border of the enterocytes by enzymes breaking down oligosaccharides and polysaccharides into three basic monosaccharides (glucose, galactose, and fructose). Glucose and galactose are absorbed by the enterocyte via an active transport using Na-dependent glucose transporters (SGLT1). These transporters are localized at the apical cell membrane (brown circles with G and Na labels). Fructose enters the cell via facilitated Na-independent transport using GLUT5 (gray circle with F label) and GLUT2 glucose transporters (orange octagon with G2 label). The three absorbed monosaccharides then pass through the basal membrane of the

1	transport using GLUT5 (gray circle with F label) and GLUT2 glucose transporters (orange octagon with G2 label). The three absorbed monosaccharides then pass through the basal membrane of the enterocyte, using GLUT2 glucose transporters, into the underlying capillaries of the portal circulation to reach their final destination in the liver.

1	eosinophils, macrophages, and a network of fenestrated blood capillaries located just beneath the epithelial basal lamina. In addition, the lamina propria of the villus contains a central, blind-ending lymphatic capillary, the lacteal (Fig. 17.19 and Plate 60, page 618). Smooth muscle cells derived from the muscularis mucosae extend into the villus and accompany the lacteal. These smooth muscle cells may account for reports that villi contract and shorten intermittently, an action that may force lymph from the lacteal into the lymphatic vessel network that surrounds the muscularis mucosae. The intestinal glands, or crypts of Lieberkn, are simple tubular structures that extend from the muscularis mucosae through the thickness of the lamina propria, where they open onto the luminal surface of the intestine at the base of the villi (see Fig. 17.18). The glands are composed of a simple columnar epithelium that is continuous with the epithelium of the villi.

1	FIGURE 17.17 • Photograph of the mucosal surface of the small intestine. This photograph of a segment of a human jejunum shows the mucosal surface. The circular folds (plicae circulares) appear as a series of transversely oriented ridges that extend partially around the lumen. Consequently, some of the circular folds appear to end (or begin) at various sites along the luminal surface (arrows). The entire mucosa has a velvety appearance because of the presence of villi.

1	As in the stomach, the lamina propria surrounds the intestinal glands and contains numerous cells of the immune system (lymphocytes, plasma cells, mast cells, macrophages, and eosinophils), particularly in the villi. The lamina propria also contains numerous nodules of lymphatic tissue that represent a major component of the GALT. The nodules are particularly large and numerous in the ileum, where they are preferentially located on the side of the intestine opposite the mesenteric attachment (Fig. 17.20). These nodular aggregations are known as aggregated nodules or Peyer’s patches. In gross specimens, they appear as aggregates of white specks. The muscularis mucosae consists of two thin layers of smooth muscle cells, an inner circular and an outer longitudinal layer. As noted above, strands of smooth muscle cells extend from the muscularis mucosae into the lamina propria of the villi. At least five types of cells are found in intestinal mucosal epithelium.

1	At least five types of cells are found in intestinal mucosal epithelium. The mature cells of the intestinal epithelium are found both in the intestinal glands and on the surface of the villi. They include the following:  Enterocytes, whose primary function is absorption  Goblet cells, unicellular mucin-secreting glands  Paneth cells, whose primary function is to maintain substances  Enteroendocrine cells, which produce various paracrine and endocrine hormones  M cells (microfold cells), modified enterocytes that cover enlarged lymphatic nodules in the lamina propria Enterocytes are absorptive cells specialized for the transport of substances from the lumen of the intestine to the circulatory system. Enterocytes are tall columnar cells with a basally positioned nucleus (see Figs. 17.18 and 17.21). Microvilli increase the apical surface area as much as 600 times; they are recognized in the light microscope as forming a striated border on the luminal surface.

1	Each microvillus has a core of vertically oriented actin microfilaments that are anchored to villin located in the tip of the microvillus and that also attach to the microvillus plasma membrane by myosin I molecules. The actin microfilaments extend into the apical cytoplasm and insert into the terminal web, a network of horizontally oriented contractile microfilaments that form a layer in the most apical cytoplasm and attach to the intracellular density associated with the zonula adherens. Contraction of the terminal web causes the microvilli to spread apart, thus increasing the space between them to allow more surface area exposure for absorption to take place. In addition, contraction of the terminal web may aid in “closing” the holes left in the epithelial sheet by exfoliation of aging cells. Enterocytes are bound to one another and to the goblet, enteroendocrine, and other cells of the epithelium by junctional complexes.

1	Tight junctions establish a barrier between the intestinal lumen and the epithelial intercellular compartment. The tight junctions between the intestinal lumen and the connective tissue compartment of the body allow selective retention of substances absorbed by the enterocytes. As noted in the section on occluding junctions, the “tightness” of these junctions can vary. In relatively impermeable tight junctions, as in the ileum and colon, active transport is required to move solutes across the barrier. In simplest terms, active transport systems, e.g., sodium pumps (Na/K-ATPase), located in the lateral plasma membrane transiently reduce the cytoplasmic concentration of Na by transporting it across the lateral plasma membrane into the extracellular space below the tight junction. This transport of Na creates a high intercellular Na concentration, causing water from the cell to enter the intercellular space, reducing both the water and Na concentrations in the

1	FIGURE 17.18 • Intestinal villi in the small intestine. a. Scanning electron micrograph of the intestinal mucosa showing its villi. Note the openings (arrows) located between the bases of the villi that lead into the intestinal glands (crypts of Lieberkn). 800. b. This three-dimensional diagram of the intestinal villi shows the continuity of the epithelium covering the villi with the epithelium lining the intestinal glands. Note blood vessels and the blind-ending lymphatic capillary, called a lacteal, in the core of the villus. Between the bases of the villi, the openings of the intestinal glands can be seen (arrows). Also, the small openings on the surface of the villi indicate the location of discharged goblet cells.

1	cell. Consequently, water and Na enter the cell at its apical surface, passing through the cell, and exiting at the lateral plasma membrane as long as the sodium pump continues to function. Increased osmolarity in the intercellular space draws water into this space, establishing a hydrostatic pressure that drives Na and water across the basal lamina into the connective tissue. In epithelia with more permeable tight junctions, such as those in the duodenum and jejunum, a sodium pump also creates low intracellular Na concentration. When the contents that pass into the duodenum and jejunum are hypotonic, however, considerable absorption of water, along with additional Na and other small solutes, takes place directly across the tight junctions of the enterocytes into the intercellular spaces. This mechanism of absorption is referred to as solvent drag.

1	Other transport mechanisms also increase the concentrations of specific substances, such as sugars, amino acids, and other solutes in the intercellular space. These substances then diffuse or flow down their concentration gradients within the intercellular space to cross the epithelial basal lamina and enter the fenestrated capillaries in the lamina propria located immediately beneath the epithelium. Substances that are too large to enter the blood vessels, such as lipoprotein particles, enter the lymphatic lacteal.

1	The lateral cell surface of the enterocytes exhibits elaborate, flattened cytoplasmic processes (plicae) that interdigitate with those of adjacent cells (see Fig. 5.000). These folds increase the lateral surface area of the cell, thus increasing the amount of plasma membrane containing transport enzymes. During active absorption, especially of solutes, water, and lipids, these lateral plications separate, enlarging the intercellular compartment. The increased hydrostatic pressure from the accumulated solutes and solvents causes a directional flow through the basal lamina into the lamina propria (see Fig. 5.1).

1	FIGURE 17.19 • Photomicrograph of an intestinal villus. The surface of the villus consists of columnar epithelial cells, chiefly enterocytes with a striated border. Also evident are goblet cells that can be readily identified by the presence of the apical mucous cup. Located beneath the epithelium is the highly cellular loose connective tissue, the lamina propria. The lamina propria contains large numbers of round cells, mostly lymphocytes. In addition, smooth muscle cells can be identified. A lymphatic capillary called a lacteal occupies the center of the villus. When the lacteal is dilated, as it is in this specimen, it is easily identified. 160.

1	In addition to the membrane specializations associated with absorption and transport, the enterocyte cytoplasm is also specialized for these functions. Elongated mitochondria that provide energy for transport are concentrated in the apical cytoplasm between the terminal web and the nucleus. Tubules and cisternae of the smooth endoplasmic reticulum (sER), which are involved in the absorption of fatty acids and glycerol and in the resynthesis of neutral fat, are found in the apical cytoplasm beneath the terminal web. Enterocytes are also secretory cells, producing enzymes needed for terminal digestion and absorption as well as secretion of water and electrolytes.

1	Enterocytes are also secretory cells, producing enzymes needed for terminal digestion and absorption as well as secretion of water and electrolytes. The secretory function of enterocytes, primarily the synthesis of glycoprotein enzymes that will be inserted into the apical plasma membrane, is represented morphologically by aligned stacks of Golgi cisternae in the immediate supranuclear region and by the presence of free ribosomes and rER lateral to the Golgi apparatus (see Fig. 17.21). Small secretory vesicles con-

1	FIGURE 17.20 • Photomicrograph of Peyer’s patches. This photomicrograph shows a longitudinal section through the wall of a human ileum. Note the extensive lymphatic nodules located in the mucosa and the section of a circular fold projecting into the lumen of the ileum. Lymphatic nodules within the Peyer’s patch are primarily located within the lamina propria, although many extend into the submucosa. They are covered by the intestinal epithelium, which contains enterocytes, occasional goblet cells, and specialized antigen-presenting M cells. 40. taining glycoproteins destined for the cell surface are located in the apical cytoplasm, just below the terminal web, and along the lateral plasma membrane. Histochemical or autoradiographic methods are needed, however, to distinguish these secretory vesicles from endocytotic vesicles or small lysosomes. The small intestine also secretes water and electrolytes. This activity occurs mainly in the cells within the intestinal

1	FIGURE 17.21 • Diagrams of an enterocyte in different phases of absorption. a. This cell has a striated border on its apical surface and junctional complexes that seal the lumen of the intestine from the lateral intercellular space. The characteristic complement of major organelles is depicted in the diagram. b. This cell shows the distribution of lipid during fat absorption as seen with the TEM. Initially, lipids are seen in association with the microvilli of the striated border. Lipids are internalized and seen in vesicles of the smooth endoplasmic reticulum (sER) in the apical portion of the cell. The membrane-bounded lipid can be traced to the center of the cell, where many of the lipid-containing vesicles fuse. The lipid is then discharged into the intercellular space. The extracellular lipids, recognized as chylomicrons, pass beyond the basal lamina for further transport into lymphatic (green) and/or blood vessels (red).

1	glands. The secretion that occurs in these glands is thought ination with the TEM reveals a large accumulation of mu-to assist the process of digestion and absorption by maintain-cinogen granules in the apical cytoplasm that distends the ing an appropriate liquid state of the intestinal chyme. apex of the cell and distorts the shape of neighboring cells Under normal conditions, the absorption of fluid by the vil-(Fig. 17.22). With the apex of the cell containing a large lus enterocyte is balanced by the secretion of fluid by the accumulation of mucinogen granules, the basal portion of the gland enterocyte. cell resembles a narrow stem. This basal portion is intensely basophilic in histologic preparations because it is occupied by Goblet cells represent unicellular glands that are inter- a heterochromatic nucleus, extensive rER, and free ribo spersed among the other cells of the intestinal epithelium.

1	Goblet cells represent unicellular glands that are inter- a heterochromatic nucleus, extensive rER, and free ribo spersed among the other cells of the intestinal epithelium. somes. Mitochondria are also concentrated in the basal cyto-As in other epithelia, goblet cells produce mucus. In the plasm. The characteristic shape, with the apical accumulation small intestine, goblet cells increase in number from the duo-of granules and the narrow basal stem, is responsible for the denum to the terminal part of the ileum. Also, as in other name of the cell, as in a glass “goblet.” An extensive array of epithelia, because water-soluble mucinogen is lost during flattened Golgi cisternae forms a wide cup around the newly preparation of routine H&E sections, the part of the cell that formed mucinogen granules adjacent to the basal part of the normally contains mucinogen granules appears empty. Exam-cell (see Fig. 17.22a). The microvilli of goblet cells are

1	FIGURE 17.22 • Electron micrograph and the diagram of a goblet cell. a. This electron micrograph shows the basal portion of a goblet cell depicted on the adjacent diagram. The cell rests on the basal lamina. The basal portion of the cell contains the nucleus, rough endoplasmic reticulum, and mitochondria. Just apical to the nucleus are extensive profiles of Golgi apparatus. As the mucous product accumulates in the Golgi cisternae, they become enlarged (asterisks). The large mucinogen granules fill most of the apical portion of the cell and collectively constitute the “mucous cup” seen in the light microscope. 15,000. b. This diagram shows the entire goblet cell. The boxed region on this diagram represents an area from which the adjacent electron micrograph was most likely obtained. The nucleus is located at the basal portion of the cell. The major portion of the cell is filled with mucinogen granules forming the mucous cup that is evident in the light microscope. At the base and lower

1	is located at the basal portion of the cell. The major portion of the cell is filled with mucinogen granules forming the mucous cup that is evident in the light microscope. At the base and lower sides of the mucous cup are flattened saccules of the large Golgi apparatus. Other organelles are distributed throughout the remaining cytoplasm, especially in the perinuclear cytoplasm in the base of the cell.

1	restricted to a thin rim of cytoplasm (the theca) that surrounds the apical-lateral portion of the mucinogen granules. Microvilli are more obvious on the immature goblet cells in the deep one half of the intestinal gland. Paneth cells play a role in regulation of normal bacterial flora of the small intestine. Paneth cells are found in the bases of the intestinal glands. (They are also occasionally found in the normal colon in small numbers; their number may increase in certain pathologic conditions.) They have a basophilic basal cytoplasm, a supranuclear Golgi apparatus, and large, intensely acidophilic, refractile apical secretory vesicles. These vesicles allow their easy identification in routine histologic sections (Fig. 17.23).

1	The secretory vesicles contain the antibacterial enzyme lysozyme, -defensins, other glycoproteins, an arginine-rich protein (probably responsible for the intense acidophilia), and zinc. Lysozyme digests the cell walls of certain groups of bacteria. -defensins are homologs of peptides that function as mediators in cytotoxic CD8 T lymphocytes. Their antibacterial action and ability to phagocytose certain bacteria and protozoa suggest that Paneth cells play a role in regulating the normal bacterial ﬂora of the small intestine. Enteroendocrine cells in the small intestine produce nearly all of the same peptide hormones as they do in the stomach.

1	Enteroendocrine cells in the small intestine produce nearly all of the same peptide hormones as they do in the stomach. Enteroendocrine cells in the small intestine resemble those that reside in the stomach (see Fig. 17.12). The “closed cells” are concentrated in the lower portion of the intestinal gland, whereas the “open cells” can be found at all levels of each villus. Activation of taste receptors found on the apical cell membrane of “open cells” activates G protein–signaling cascade, resulting in releasing of peptides that regulate a variety of gastrointestinal functions. These include regulating pancreatic secretion, inducing digestion and absorption, and controlling energy homeostasis by acting on neural pathways

1	FIGURE 17.23 • Photomicrograph of intestinal glands showing Paneth cells. This photomicrograph shows the base of intestinal (jejunal) glands in an H&E preparation. The gland on the right is sectioned longitudinally; the circular cross-sectional profile of another gland is seen on the left. Paneth cells are typically located in the base of the intestinal glands and are readily seen in the light microscope because of the intensive eosin staining of their vesicles. The lamina propria contains an abundance of plasma cells, lymphocytes, and other connective tissue cells. Note several lymphocytes in the epithelium of the gland (arrows). 240. Inset. This high magnification of the area indicated by the rectangle shows the characteristic basophilic cytoplasm in the basal portion of the cell and large accumulations of intensely staining, eosinophilic, refractile secretory vesicles in the apical portion of the cell. An arginine-rich protein found in the vesicles is probably responsible for the

1	accumulations of intensely staining, eosinophilic, refractile secretory vesicles in the apical portion of the cell. An arginine-rich protein found in the vesicles is probably responsible for the intense eosinophilic reaction. 680.

1	of brain-gut-adipose axis. Nearly all of the same peptide hormones identified in this cell type in the stomach can be demonstrated in the enteroendocrine cells of the intestine (see Table 17.1). Cholecystokinin (CCK), secretin, gastric inhibitory polypeptide (GIP), and motilin are the most active regulators of gastrointestinal physiology that are released in this portion of the gut (see Fig. 17.13). CCK and secretin increase pancreatic and gallbladder activity and inhibit gastric secretory function and motility. GIP stimulates insulin release in the pancreas, and motilin initiates gastric and intestinal motility. Although other peptides produced by enteroendocrine cells have been isolated, they are not considered hormones and are therefore called candidate hormones (page 584). Enteroendocrine cells also produce at least two hormones, somatostatin and histamine, which act as paracrine hormones (see page 584) (i.e., hormones that have a local effect and do not circulate in the

1	cells also produce at least two hormones, somatostatin and histamine, which act as paracrine hormones (see page 584) (i.e., hormones that have a local effect and do not circulate in the bloodstream). In addition, several peptides are secreted by the nerve cells located in the submucosa and muscularis externa. These peptides, called neurocrine hormones, are represented by VIP, bombesin, and the enkephalins. The functions of these peptides are listed in Table 17.2.

1	M cells convey microorganisms and other macromolecules from the intestinal lumen to Peyer’s patches. M cells are epithelial cells that overlie Peyer’s patches and other large lymphatic nodules; they differ significantly from the surrounding intestinal epithelial cells (Folder 17.5). M cells have microfolds rather than microvilli on their apical surface, and they take up microorganisms and macromolecules from the lumen in endocytotic vesicles. The M cell is an antigen-transporting cell. The vesicles are transported to the basolateral membrane where they discharge their contents into the epithelial intercellular space in the vicinity of CD4 T lymphocytes. Thus, substances that gain access to the body from the intestinal lumen via M cells come into contact with cells of the immune system as they reach the basolateral surface. Antigens that reach lymphocytes in this manner stimulate a response in the GALT that is described below.

1	Intermediate cells constitute the amplifying compartment of the intestinal stem cell niche.

1	Intermediate cells constitute most of the cells found within the intestinal stem cell niche that is located in the lower half of the intestinal gland. These cells constitute the amplifying compartment of the cells that are still capable of cell division and usually undergo one or two divisions before they become committed to differentiation into either absorptive or goblet cells. These cells have short, irregular microvilli with long core filaments extending deep into the apical cytoplasm and numerous macular (desmosomal) junctions with adjacent cells. Small mucin-like secretory granules form a column in the center of the supranuclear cytoplasm. Intermediate cells that are committed to becoming goblet cells develop a small, rounded collection of secretory granules just beneath the apical plasma membrane; those that are committed to becoming absorptive cells lose the secretory granules and begin to show concentrations of mitochondria, rER, and ribosomes in the apical cytoplasm.

1	GALT is prominent in the lamina propria of the small intestine. As noted above, the lamina propria of the digestive tract is heavily populated with elements of the immune system; approximately one-fourth of the mucosa consists of a loosely organized layer of lymphatic nodules, lymphocytes, macrophages, plasma cells, and eosinophils in the lamina propria (Plate 55, page 608). Lymphocytes are also located between epithelial cells. This GALT serves as an immunologic barrier throughout the length of the gastrointestinal tract. In cooperation with the overlying epithelial cells, particularly M cells, the lymphatic  FOLDER 17.5 Functional Considerations: Immune Functions of the Alimentary Canal

1	Immunologists have shown that the GALT not only re-sponds to antigenic stimuli but also functions in a monitor-ing capacity. This function has been partially clarified for the lymphatic nodules of the intestinal tract. The M cells that cover Peyer’s patches and lymphatic nodules have a distinctive surface that might be misinterpreted in sections as thick microvilli. The cells are readily identified with the scanning electron microscope because microfolds con-trast sharply with the microvilli that constitute the striated border of the adjacent enterocytes. It has been shown with horseradish peroxidase (an enzyme used as an experimental marker) that the M cells pinocytose protein, from the intestinal lumen, transport the pinocytotic vesicles through the cell and discharge the protein by exocytosis into deep recesses of the adjacent extracellular space (Fig. F17.5.1). Lymphocytes within the deeply recessed extracellular space sample the luminal protein, including antigens, and thus have

1	into deep recesses of the adjacent extracellular space (Fig. F17.5.1). Lymphocytes within the deeply recessed extracellular space sample the luminal protein, including antigens, and thus have the opportunity to stimulate development of specific antibodies against the antigens. The destination of these exposed lympho-cytes has not yet been fully determined. Some remain within the local lymphatic tissue, but others may be des-tined for other sites in the body, such as the salivary and mammary glands. Recall that in the salivary glands, cells of the immune system (plasma cells) secrete IgA, which the glandular epithelium then converts into sIgA. Some exper-imental observations suggest that antigen contact neces-sary for the production of IgA by plasma cells occurs in the lymphatic nodules of the intestines. bb M cells lymphocytes absorptive cells lamina propria a FIGURE F17.5.1 • Diagram of M cells in a lymphatic nodule of the intestine. a. This diagram shows the relationship of the M

1	of the intestines. bb M cells lymphocytes absorptive cells lamina propria a FIGURE F17.5.1 • Diagram of M cells in a lymphatic nodule of the intestine. a. This diagram shows the relationship of the M cells (microfold cells) and absorptive cells to the lymphatic nodule. The M cell is an epithelial cell that displays microfolds rather than microvilli on its apical surface. It has deep recesses within which lymphocytes come close to the lumen of the small intestine. M cells have MHC II molecules on their surface and are therefore considered antigen-presenting cells. Antigen from the intestinal lumen is presented to T lymphocytes residing within the recesses of the M cell. (Based on Owen RL, Nemanic PC, eds. Scanning Electron Microscopy. Vol II. O’Hare, IL: AMF, 1978.) b. Scanning electron micrograph of a Peyer’s patch lymphatic nodule bulging into the lumen of the ileum. Note that the area of the follicle covered by M cells is surrounded by the fingerlike projections of the intestinal

1	micrograph of a Peyer’s patch lymphatic nodule bulging into the lumen of the ileum. Note that the area of the follicle covered by M cells is surrounded by the fingerlike projections of the intestinal villi. The surface of the M cells has a smooth appearance. The absence of absorptive cells and mucus-producing goblet cells in the area covered by M cells facilitates immunoreactions to antigens. 80. (Reprinted with permission from Owen RL, Johns AL. Epithelial cell specialization within human Peyer’s patches: an ultrastructural study of intestinal lymphoid follicles. Gastroenterology 1974;66:189–203.) tissue samples the antigens in the epithelial intercellular spaces. Lymphocytes and other antigen-presenting cells process the antigens and migrate to lymphatic nodules in the lamina propria where they undergo activation (see page 444), leading to antibody secretion by newly differentiated plasma cells.

1	Mucosal surface is protected by immunoglobulinmediated responses.

1	Mucosal surface of the gut tube is constantly challenged by the presence of ingested microorganisms (i.e., viruses, bacteria, parasites) and toxins, which after compromising the epithelial barrier may cause infections or diseases. An example of a specific defense mechanism is the immunoglobulinmediated response using IgA, IgM and IgE antibodies. Most of the plasma cells in the lamina propria of the intestine secrete dimeric dIgA antibodies rather than the more common IgG; other plasma cells produce pentameric IgM and IgE (see page 554). Dimeric dIgA is composed of two monomeric IgA subunits and a polypeptide J chain (see Fig. 16.28). Secreted dIgA molecules bind to the polymeric immunoglobulin receptor (pIgR) located at the basal domain of the epithelial cells (Fig. 17.24). The pIgR receptor is a transmembrane glycoprotein (75-kilodalton) synthesized by enterocytes and expressed on the basal plasma membrane. The pIgR–dIgA complex is then endocytosed and transported across the

1	receptor is a transmembrane glycoprotein (75-kilodalton) synthesized by enterocytes and expressed on the basal plasma membrane. The pIgR–dIgA complex is then endocytosed and transported across the epithelium to the apical surface of the enterocyte (this type of transport refers as transcytosis). After the pIgR–dIgA complex reach the apical surface, pIgR is proteolytically cleaved and the extracellular part of the receptor that is bound to dIgA is released into the gut lumen (see Fig. 17.24). This cleaved extracellular binding domain of the receptor is known as the secretory component (SC); secreted dIgA in association with the SC is known as secretory IgA (sIgA). The release of sIgA immunoglobulins is critical for proper immunological surveillance by the mucosal immune system. In the lumen, sIgA binds to antigens, toxins, and microorganisms. Secretory IgA prevents the attachment and invasion of viruses and bacteria into the mucosa by either inhibiting their motility, causing microbial

1	sIgA binds to antigens, toxins, and microorganisms. Secretory IgA prevents the attachment and invasion of viruses and bacteria into the mucosa by either inhibiting their motility, causing microbial aggregation or masking pathogen adhesion sites on the epithelial surface. For example, sIgA binds to a glycoprotein on the viral envelope of HIV virus preventing its attachment, internalization, and subsequent replication in the cell.

1	Secretory IgA is the principal molecule of mucosal immunity. However, IgM molecules utilize similar pathway of the receptor-mediated transcytosis to reach the mucosal surface. Some of the IgE binds to the plasma membranes of mast cells in the lamina propria (see page 182), selectively sensitizing them to specific antigens derived from the lumen. A distinguishing characteristic of the duodenum is the presence of submucosal glands. The submucosa consists of a dense connective tissue and localized sites that contain aggregates of adipose cells. A conspicuous feature in the duodenum is the presence of submucosal glands, also called Brunner’s glands. The branched, tubular submucosal glands of the duodenum have secretory cells with characteristics of both zymogensecreting and mucus-secreting cells (Fig. 17.25).

1	The branched, tubular submucosal glands of the duodenum have secretory cells with characteristics of both zymogensecreting and mucus-secreting cells (Fig. 17.25). The secretion of these glands has a pH of 8.1 to 9.3 and contains neutral and alkaline glycoproteins and bicarbonate ions. This highly alkaline secretion probably serves to protect the proximal small intestine by neutralizing the acid-containing chyme delivered to it. It also brings the intestinal contents close to the optimal pH for the pancreatic enzymes that are also delivered to the duodenum.

1	FIGURE 17.24 • Diagram of immunoglobulin A (IgA) secretion and transport. A monomeric form of immunoglobulin A (IgA) is synthesized by the plasma cell. IgAs are secreted into the lamina propria in a dimeric form as dIgA. Dimeric dIgA is composed of two monomeric IgA subunits and a polypeptide J chain also produced by the plasma cell. In the lamina propria dIgA binds to the polymeric immunoglobulin receptor (pIgR) on the basal cell membrane of the enterocyte. The pIgR-IgA complex enters the cell by endocytosis and is carried out within the endocytotic vesicles to the eary endosomal compartment and then to the apical surface (a process called transcytosis). Endocytic vesicles fuse with the apical plasma membrane, the pIgR is proteolytically cleaved and dIgA is released with the extracellular portion of the pIgR receptor. This portion of the pIgR remains with the IgA dimer and become the secretory component (SC) of the secretory IgA (sIgA).

1	FIGURE 17.25 • Photomicrograph of Brunner’s glands in the duodenum. This photomicrograph shows part of the duodenal wall in an H&E preparation. A distinctive feature of the duodenum is the presence of Brunner’s glands. The dashed line marks the boundary between the villi and the typical intestinal glands (crypts of Lieberkn). The latter extend to the muscularis mucosae. Under the mucosa is the submucosa, which contains Brunner’s glands. These are branched tubular glands whose secretory component consists of columnar cells. The duct of the Brunner’s gland opens into the lumen of the intestinal gland (arrow). 120.

1	The muscularis externa consists of an inner layer of circularly arranged smooth muscle cells and an outer layer of longitudinally arranged smooth muscle cells. The main components of the myenteric plexus (Auerbach’s plexus) are located between these two muscle layers (Fig. 17.26). Two kinds of muscular contraction occur in the small intestine. Local contractions displace intestinal contents both proximally and distally; this type of contraction is called segmentation. These contractions primarily involve the circular muscle layer. They serve to circulate the chyme locally, mixing it with digestive juices and moving it into contact with the mucosa for absorption. Peristalsis, the second type of contraction, involves coordinated action of both circular and longitudinal muscle layers and moves the intestinal contents distally.

1	The serosa of the parts of the small intestine that are located intraperitoneally in the abdominal cavity corresponds to the general description at the beginning of the chapter. Epithelial Cell Renewal in the Small Intestine All of the mature cells of the intestinal epithelium are derived from a single stem cell population.

1	Epithelial Cell Renewal in the Small Intestine All of the mature cells of the intestinal epithelium are derived from a single stem cell population. Stem cells are located in the base of the intestinal gland. This intestinal stem cell niche (zone of cell replication) is restricted to the lower one half of the gland and contains highly proliferative intermediate cells (as previously explained) and cells at various stages of differentiation. A cell destined to become a goblet cell or absorptive cell usually undergoes several additional divisions after it leaves the pool of stem cells. The epithelial cells migrate upward in the intestinal gland onto the villus where they undergo apoptosis and slough off into the lumen. Autoradiographic studies have shown that the renewal time for absorptive and goblet cells in the human small intestine is 4 to 6 days.

1	Enteroendocrine cells and Paneth cells are also derived from the stem cells at the base of the intestinal gland. Enteroendocrine cells appear to divide only once before differentiating. They migrate with the absorptive and goblet cells but at a slower rate. Paneth cells migrate downward and reside at the bottom of the intestinal gland. They live for approximately 4 weeks and are then replaced by differentiation of a nearby “committed” cell in the intestinal gland. Cells that are recognizable as Paneth cells no longer divide. As mentioned in the chapter on epithelial tissue (page 150) expression of the transcription factor Math1 appears to determine the fate of differentiating cells in the intestinal stem cell niche. The cells committed to the secretory lineage (i.e., they will differentiate into goblet, enteroendocrine, and Paneth cells) have increased expression of Math1. Inhibition of Math1 expression characterizes the default developmental pathway into absorptive intestinal cells

1	into goblet, enteroendocrine, and Paneth cells) have increased expression of Math1. Inhibition of Math1 expression characterizes the default developmental pathway into absorptive intestinal cells (enterocytes).

1	The large intestine comprises the cecum with its projecting vermiform appendix, the colon, the rectum, and the anal canal. The colon is further subdivided on the basis of its anatomic location into ascending colon, transverse colon, descending colon, and sigmoid colon. The four layers characteristic of the alimentary canal are present throughout. However, several distinctive features exist at the gross level (Fig. 17.27):  Teniae coli that represent three narrowed, thickened, equally spaced bands of the outer longitudinal layer of the muscularis externa. They are primarily visible in the cecum

1	FIGURE 17.26 • Electron micrograph of the myenteric (Auerbach’s) plexus. The plexus is located between the two smooth muscle (SM) layers of the muscularis externa. It consists of nerve cell bodies (CB) and an extensive network of nerve fibers (N). A satellite cell (SC), also referred to as an enteric glial cell, is seen in proximity to the neuron cell bodies. These cells have structural and chemical features in common with glial cells of the CNS. BV, blood vessel. 3,800.

1	FIGURE 17.27 • Photograph of the large intestine. This photograph shows the outer (serosal) surface (left) and internal (mucosal) surface (right) of the transverse colon. On the outer surface, note the characteristic features of the large intestine: a distinctive smooth muscle band representing one of the three teniae coli (TC); haustra coli (HC), the sacculations of the colon located between the teniae; and omental appendices (OA), small peritoneal projections filled with fat. The smooth mucosal surface shows semilunar folds (arrows) formed in response to contractions of the muscularis externa. Compare the mucosal surface as shown here with that of the small intestine (Fig. 17.17).

1	and colon and they are absent in the rectum, anal canal, and vermiform appendix. Haustra coli that are visible sacculations between the teniae coli on the external surface of the cecum and colon.  Omental appendices that are small fatty projections of the serosa, observed on the outer surface of the colon. The mucosa of the large intestine has a “smooth” surface; neither plicae circulares nor villi are present. It contains numerous straight tubular intestinal glands (crypts of Lieberkn) that extend through the full thickness of the mucosa (Fig. 17.28a). The glands consist of simple columnar epithelium, as does the intestinal surface from which they invaginate. Examination of the luminal surface of the large intestine at the microscopic level reveals the openings of the glands, which are arranged in an orderly pattern (Fig. 17.28b). The principal functions of the large intestine are reabsorption of electrolytes and water and elimination of undigested food and waste.

1	The principal functions of the large intestine are reabsorption of electrolytes and water and elimination of undigested food and waste. The primary function of the columnar absorptive cells is reabsorption of water and electrolytes. The morphology of absorptive cells is essentially identical to that of the enterocytes of the small intestine. Reabsorption is accomplished by the same Na/K-activated ATPase-driven transport system as described for the small intestine. Elimination of semisolid to solid waste materials is facilitated by the large amounts of mucus secreted by the numerous goblet cells of the intestinal glands. Goblet cells are more numerous in the large intestine than in the small intestine (see Fig. 17.28a and Plate 62, page 622). They produce mucin that is secreted continuously to lubricate the bowel, facilitating the passage of the increasingly solid contents.

1	The mucosal epithelium of the large intestine contains the same cell types as the small intestine except Paneth cells, which are normally absent in humans. Columnar absorptive cells predominate (4:1) over goblet cells in most of the colon, although this is not always apparent in histologic sections (see Fig. 17.28a). The ratio decreases, however, approaching 1:1, near the rectum, where the number of goblet cells increases. Although the absorptive cells secrete glycocalyx at a rapid rate (turnover time is 16 to 24 hours in humans), this layer has not been shown to contain digestive enzymes in the colon. As in the small intestine, however, Na/K-ATPase is abundant and is localized in the lateral plasma membranes of the absorptive cells. The intercellular space is often dilated, indicating active transport of fluid.

1	FIGURE 17.28 • Mucosa of the large intestine. a. This photomicrograph of an H&E preparation shows the mucosa and part of the submucosa. The surface epithelium is continuous with the straight, unbranched, tubular intestinal glands (crypts of Lieberkn). The openings of the glands at the intestinal surface are identified (arrows). The epithelial cells consist principally of absorptive and goblet cells. As the absorptive cells are followed into the glands, they become fewer in number, whereas the goblet cells increase in number. The highly cellular lamina propria contains numerous lymphocytes and other cells of the immune system. b. Scanning electron micrograph of the human mucosal surface of the large intestine. The surface is divided into territories by clefts (arrows). Each territory contains 25 to 100 gland openings. 140. (Reprinted with permission from Fenoglio CM, Richart RM, Kaye GI. Comparative electron-microscopic features of normal, hyperplastic, and adenomatous human colonic

1	25 to 100 gland openings. 140. (Reprinted with permission from Fenoglio CM, Richart RM, Kaye GI. Comparative electron-microscopic features of normal, hyperplastic, and adenomatous human colonic epithelium. II. Variations in surface architecture found by scanning electron microscopy. Gastroenterology 1975;69:100–109.)

1	Goblet cells may mature deep in the intestinal gland, even in the replicative zone (Fig. 17.29). They secrete mucus continuously, even to the point where they reach the luminal surface. Here, at the surface, the secretion rate exceeds the synthesis rate, and “exhausted” goblet cells appear in the epithelium. These cells are tall and thin and have a small number of mucinogen granules in the central apical cytoplasm. An infrequently observed cell type, the caveolated “tuft” cell, has also been described in the colonic epithelium; however, this cell may be a form of exhausted goblet cell. Epithelial Cell Renewal in the Large Intestine All intestinal epithelial cells in the large intestine derive from a single stem cell population.

1	Epithelial Cell Renewal in the Large Intestine All intestinal epithelial cells in the large intestine derive from a single stem cell population. As in the small intestine, all of the mucosal epithelial cells of the large intestine arise from stem cells located at the bottom of the intestinal gland. The lower third of the gland constitutes the intestinal stem cell niche, where newly generated cells undergo two to three more divisions as they begin their migration up to the luminal surface, where they are shed about 5 days later. The intermediate cell types found in the lower third of the intestinal gland are identical to those seen in the small intestine.

1	The turnover times of the epithelial cells of the large intestine are similar to those of the small intestine (i.e., about 6 days for absorptive cells and goblet cells and up to 4 weeks for enteroendocrine cells). Senile epithelial cells that reach the mucosal surface undergo apoptosis and are shed into the lumen at the midpoint between two adjacent intestinal glands.

1	Although the lamina propria of the large intestine contains the same basic components as the rest of the digestive tract, it demonstrates some additional structural features and greater development of some others. These include the following:  Collagen table, which represents a thick layer of collagen and proteoglycans that lies between the basal lamina of the epithelium and that of the fenestrated absorptive venous capillaries. This layer is as much as 5 m thick in the normal human colon and can be up to three times that thickness in human hyperplastic colonic polyps. The collagen table participates in regulation of water and electrolyte transport from the intercellular compartment of the epithelium to the vascular compartment.

1	 Pericryptal fbroblast sheath, which constitutes a well-developed fibroblast population of regularly replicating cells. They divide immediately beneath the base of the intestinal gland, adjacent to the stem cells found in the epithelium (in both the large and small intestines). The fibroblasts then differentiate and migrate upward in parallel and synchrony with the epithelial cells. Although the ultimate fate of the pericryptal fibroblast is unknown, most FIGURE 17.29 • Electron micrograph of dividing goblet cells.

1	FIGURE 17.29 • Electron micrograph of dividing goblet cells. This electron micrograph demonstrates that certain cells of the intestine continue to divide even after they have differentiated. Here, two goblet cells (GC) are shown in division. Typically, the dividing cells move away from the basal lamina toward the lumen. One of the goblet cells shows mucinogen granules (M) in its apical cytoplasm. The chromosomes (C) of the dividing cells are not surrounded by a nuclear membrane. Compare with the nuclei (N) of the nondividing intestinal epithelial cells. The lumen of the gland (L) is on the right. CT, connective tissue; E, eosinophil. 5,000.

1	of these cells, after they reach the level of the luminal surface, take on the morphologic and histochemical characteristics of macrophages. Some evidence suggests that the macrophages of the core of the lamina propria in the large intestine may arise as a terminal differentiation of the pericryptal fibroblasts:  GALT, which is continuous with that of the terminal ileum. In the large intestine, GALT is more extensively developed; large lymphatic nodules distort the regular spacing of the intestinal glands and extend into the submucosa. The extensive development of the immune system in the colon probably reflects the large number and variety of microorganisms and noxious end products of metabolism normally present in the lumen.

1	 Lymphatic vessels. In general, there are no lymphatic vessels in the core of lamina propria between the intestinal glands and none that extend toward the luminal surface of the large intestine. Only recently, using new very selective markers for lymphatic epithelium, researchers have found occasional small lymphatic vessels at the bases of the intestinal glands. These lymphatic vessels drain into the lymphatic network within the muscularis mucosae. The next step in lymph drainage occurs in the lymphatic plexuses in the submucosa and muscularis externa before lymph leaves the wall of the large intestine and drains into the regional lymph nodes. To understand the clinical significance of the lymphatic pattern in the large intestine, see Folder 17.6.

1	As noted, in the cecum and colon (the ascending, transverse, descending, and sigmoid colons), the outer layer of the muscularis externa is, in part, condensed into prominent longitudinal bands of muscle, called teniae coli, which may be seen at the gross level (see Fig. 17.27). Between these bands, the longitudinal layer forms an extremely thin sheet. In the rectum, anal canal, and vermiform appendix, the outer longitudinal layer of smooth muscle is a uniformly thick layer, as in the small intestine. Bundles of muscle from the teniae coli penetrate the inner, circular layer of muscle at irregular intervals along the length and circumference of the colon. These apparent discontinuities in the muscularis externa allow segments of the colon to contract independently, leading to the formation of haustra colli, sacculations of the colon wall.

1	The muscularis externa of the large intestine produces two major types of contraction: segmentation and peristalsis. Segmentation is local and does not result in the propulsion of contents. Peristalsis results in the distal mass movement of the colonic contents. Mass peristaltic movements occur infrequently; in healthy persons, they usually occur once a day to empty the distal colon. The submucosa of the large intestine corresponds to the general description already given. Where the large intestine is directly in contact with other structures (as on much of its posterior surface), its outer layer is an adventitia; elsewhere, the outer layer is a typical serosa.

1	The cecum forms a blind pouch just distal to the ileocecal valve; the appendix is a thin, fingerlike extension of this pouch. The histology of the cecum closely resembles that of the rest of the colon; the appendix differs from it in having a uniform layer of longitudinal muscle in the muscularis externa (Fig. 17.30 and Plate 63, page 624). The most conspicuous feature of the appendix is the large number of lymphatic nodules that extend into the submucosa. In many adults, the normal structure of the appendix is lost, and the appendage is filled with fibrous scar tissue. Blockage of the opening between the appendix and the cecum, usually due to scarring, buildup of thick mucus, or stool that enters the lumen of the appendix from the cecum, may cause appendicitis

1	FIGURE 17.30 • Photomicrograph of a cross section through the vermiform appendix. The vermiform appendix displays the same four layers as those of the large intestine except that its diameter is smaller. Typically, lymphatic nodules are seen within the entire mucosa and usually extend into the submucosa. Note the distinct germinal centers within the lymphatic nodules. The muscularis externa is composed of a relatively thick circular layer and a much thinner outer longitudinal layer. The appendix is covered by a serosa that is continuous with the mesentery of the appendix (lower right). 10.  FOLDER 17.6 Clinical Correlation: The Pattern of Lymph Vessel Distribution and Diseases of the Large Intestine

1	The absence of lymphatic drainage from the lamina pro-pria of the large intestine was initially discovered using stan-dard techniques of analyzing tissue samples obtained from biopsies with the light and electron microscopy. Recently, specific monoclonal antibodies D2-40 that react with a 40-kilodalton O-linked sialoglycoprotein expressed on the lym-phatic endothelium became available to study distribution of lymphatic vessels. This examination becomes important to monitor the morphologic integrity of the lamina propria in the large intestine that is associated with the absence of lymphatic vessels. For instance in a chronic superficial in-flammation of the colon and rectum known as ulcerative colitis, the formation of granulation tissue is associated with proliferation of blood and lymphatic vessels within the lamina propria. The lymphangiogenesis (the growth of lym-phatic vessels) in this disease is linked to the expression of vascular endothelial growth factors (VEGFs). The

1	lymphatic vessels within the lamina propria. The lymphangiogenesis (the growth of lym-phatic vessels) in this disease is linked to the expression of vascular endothelial growth factors (VEGFs). The progress of treatment in ulcerative colitis can be monitored by biop-sies, which show the disappearance of lymphatic vessels from the lamina propria. On the other hand, the increased number of lymphatic vessels in the lamina propria signals the presence of active inflammation. Discovery of the distribution of lymphatic vessels in the large intestine established the basis for the current man-agement of adenomas (adenomatous polyps of the large intestine). These are intraepithelial neoplasms located on the mass of tissue that protrudes into the lumen of the large intestine (Fig F17.6.1). The absence of lymphatic vessels from the lamina propria was important in under-standing the slow rate of metastasis from certain colon cancers. Cancers that develop in large adenomatous colonic polyps may

1	of lymphatic vessels from the lamina propria was important in under-standing the slow rate of metastasis from certain colon cancers. Cancers that develop in large adenomatous colonic polyps may grow extensively within the epithelium and lamina propria before they even have access to the lymphatic vessels at the level of the muscularis mucosae. Because almost 50% of all adenomatous polyps of the large intestine are located in the rectum and sigmoid colon, they can be detected with rectosigmoidoscopy. As long as the lesion is confined to the mucosa, the endo-scopic removal of such polyps is regarded as an adequate clinical treatment. However, the final therapeutic decision must be confirmed after careful microscopic examination of the resected specimen. aa b FIGURE F17.6.1 • Adenomatous polyp of the large intestine. a. This image shows a macroscopic view of the polyp (about 2 cm in diameter) that was surgically removed from the large intestine during endoscopic colonoscopy. It has

1	polyp of the large intestine. a. This image shows a macroscopic view of the polyp (about 2 cm in diameter) that was surgically removed from the large intestine during endoscopic colonoscopy. It has characteristic bosselated surface (with rounded swellings) and a stalk by which attaches to the wall of the colon. b. This photomicrograph was obtained from the center of the polyp. At the tip of the polyp, note a repetitive pattern of tubules covered with the neoplastic epithelial cells that have migrated and accumulated on the intestinal surface. Stalk in the center is continuous with the submucosa of the colon. Note also the presence of a normal simple columnar epithelium of the large intestine at the base of the stalk. (Reproduced from Mitros FA, Rubin E. The Gastrointestinal Tract. In: Rubin R, Strayer DS (eds): Rubin’s Pathology: Clinicopathologic Foundations of Medicine, 5th ed. Baltimore: Lippincott Williams & Wilkins, 2008.) skin internal anal sphincter lymphatic nodules anal

1	Rubin R, Strayer DS (eds): Rubin’s Pathology: Clinicopathologic Foundations of Medicine, 5th ed. Baltimore: Lippincott Williams & Wilkins, 2008.) skin internal anal sphincter lymphatic nodules anal columns external anal sphincter transverse rectal folds ampulla of rectum mucosal folds rectum anal canal perianal skin colorectal zone anal transitional zone squamous zone anal sinuses

1	FIGURE 17.31 • Drawing of the rectum and anal canal. The rectum and anal canal are the terminal portions of the large intestine. They are lined by the colorectal mucosa that possesses a simple columnar epithelium containing mostly goblet cells and numerous anal glands. In the anal canal the simple columnar epithelium undergoes transition into a stratified columnar (or cuboidal) epithelium and then to a stratified squamous epithelium. This transition occurs in the area referred to as the anal transitional zone, which occupies the middle third of the anal canal between the colorectal zone and the squamous zone of the perianal skin. (inﬂammation of the appendix). The appendix is also a common site for carcinoid, a type of tumor originating from enteroendocrine cells of lining mucosa (see Folder 17.3).

1	(inﬂammation of the appendix). The appendix is also a common site for carcinoid, a type of tumor originating from enteroendocrine cells of lining mucosa (see Folder 17.3). The rectum is the dilated distal portion of the alimentary canal. Its upper part is distinguished from the rest of the large intestine by the presence of folds called transverse rectal folds. The mucosa of the rectum is similar to that of the rest of the distal colon, having straight, tubular intestinal glands with many goblet cells.

1	The most distal portion of the alimentary canal is the anal canal. It has an average length of 4 cm and extends from the upper aspect of the pelvic diaphragm to the anus (Fig. 17.31). The upper part of the anal canal has longitudinal folds called anal columns. Depressions between the anal columns are called anal sinuses. The anal canal is divided into three zones according to the character of the epithelial lining:  Colorectal zone, which is found in the upper third of the anal canal and contains simple columnar epithelium with characteristics identical to that in the rectum.

1	 Anal transitional zone (ATZ), which occupies the middle third of the anal canal. It represents a transition between the simple columnar epithelium of the rectal mucosa and the stratified squamous epithelium of the perianal skin. The ATZ possesses a stratified columnar epithelium interposed between the simple columnar epithelium and the stratified squamous epithelium, which extends to the cutaneous zone of the anal canal (Fig. 17.32 and Plate 64, page 626).  Squamous zone, which is found in the lower third of the anal canal. This zone is lined with stratified squamous epithelium that is continuous with that of the perineal skin. In the anal canal, anal glands extend into the submucosa and even into the muscularis externa. These branched, straight tubular glands secrete mucus onto the anal surface through ducts lined with stratified columnar epithelium.

1	Sometimes the anal glands are surrounded by diffuse lymphatic tissue. They often lead to the formation of pathologic fistulas (a false opening between the anal canal and the perianal skin). Large apocrine glands, the circumanal glands, are found in the skin surrounding the anal orifice. In some animals, the

1	FIGURE 17.32 • Photomicrographs of the anal canal. a. This photomicrograph shows a longitudinal section through the wall of the anal canal. Note the three zones in the anal canal: the squamous zone (SQZ) containing stratified squamous epithelium; the anal transitional zone (ATZ) containing stratified squamous, stratified cuboidal, or columnar epithelium and simple columnar epithelium of the rectal mucosa; and the colorectal zone (CRZ) containing only simple columnar epithelium like the rest of the colon. Note the anal valve that demarcates the transition between the ATZ and SQZ. The internal anal sphincter is derived from the thickening of the circular layer of the muscularis externa. A small portion of the external anal sphincter is seen subcutaneously. 10. b. This high magnification of the area indicated by the rectangle in a shows the area of the anal transitional zone. Note the abrupt transition between stratified cuboidal and simple columnar epithelium. The simple columnar

1	of the area indicated by the rectangle in a shows the area of the anal transitional zone. Note the abrupt transition between stratified cuboidal and simple columnar epithelium. The simple columnar epithelium of anal glands extends into the submucosa. These straight, mucus-secreting tubular glands are surrounded by diffuse lymphatic tissue. 200.

1	secretion of these glands acts as a sex attractant. Hair follicles sion). There are no teniae coli at the level of the rectum; the and sebaceous glands are also found at this site. longitudinal layer of the muscularis externa forms a uniform The submucosa of the anal columns contains the terminal sheet. The muscularis mucosae disappears at about the level ramifications of the superior rectal artery and the rectal ve-of the ATZ, where the circular layer of the muscularis ex-nous plexus. Enlargements of these submucosal veins consti-terna thickens to form the internal anal sphincter. The extute internal hemorrhoids, which are related to elevated ternal anal sphincter is formed by striated muscle of the venous pressure in the portal circulation (portal hyperten-pelvic floor. This page intentionally left blank.

1	The esophagus is a muscular tube that conveys food and other substances from the pharynx to the stomach. The mucosa that lines the length of the esophagus has a nonkeratinized stratified squamous epithelium. The underlying lamina propria is similar to the lamina propria throughout the alimentary tract; diffuse lymphatic tissue is scattered throughout, and lymphatic nodules are present. The deep layer of the mucosa, the muscularis mucosae, is composed of longitudinally organized bundles of smooth muscle fibers. The submucosa consists of dense irregular connective tissue that contains the larger blood and lymphatic vessels, nerve fibers, and ganglion cells. The nerve fibers and ganglion cells make up the submucosal plexus (Meissner’s plexus). The muscularis externa consists of two muscle layers, an inner circular layer and an outer longitudinal layer. The upper one third of muscularis externa consists of striated muscle, a continuation of the muscle of the pharynx. Striated muscle and

1	an inner circular layer and an outer longitudinal layer. The upper one third of muscularis externa consists of striated muscle, a continuation of the muscle of the pharynx. Striated muscle and smooth muscle bundles are mixed and interwoven in the muscularis externa of the middle third of the esophagus. The muscularis externa of the distal one third consists only of smooth muscle, as in the rest of the digestive tract.

1	Esophagus, monkey, H&E ×60; inset ×400. A cross section of the wall of the esophagus is shown here. The mucosa (Muc) consists of stratified squamous epithelium (Ep), a lamina propria (LP), and muscularis mucosae (MM ). The boundary between the epithelium and lamina propria is distinct, although uneven, as a result of the presence of numerous deep connective tissue papillae. The basal layer of the epithelium stains intensely, appearing as a dark band that is relatively conspicuous at low magnification. This is, in part, due to the cytoplasmic basophilia of the basal cells. That the basal cells are small results in a high nuclear-cytoplasmic ratio, which further intensifies the hematoxylin staining of this layer.

1	The submucosa consists of irregular dense connective tissue that contains the larger blood vessels and nerves. No glands are seen in the submucosa in this figure, but they are regularly present throughout this layer and are likely to be included in a section of the wall. Whereas the boundary between the epithelium and lamina propria is striking, the boundary between the mucosa (Muc) and submucosa (SubM ) is less well marked, although it is readily discernable. The muscularis externa (ME ) shown here is composed largely of smooth muscle, but it also contains areas of striated muscle. Although the striations are not evident at this low magnification, the more densely stained eosinophilic areas (asterisks) prove to be striated muscle when observed at higher magnification. Reference to the inset, which is from an area in the lower half of the figure, substantiates this identification.

1	The inset shows circularly oriented striated and smooth muscle. The striated muscle stains more intensely with eosin, but of greater significance are the distribution and number of nuclei. In the center of the inset, numerous elongated and uniformly oriented nuclei are present; this is smooth muscle (SM ). Above and below, few elongated nuclei are present; moreover, they are largely at the periphery of the bundles. This is striated muscle (StM ); the cross-striations are just perceptible in some areas. The specimen shown here is from the middle of the esophagus, where both smooth and striated muscle are present. The muscularis externa of the distal third of the esophagus would contain only smooth muscle, whereas that of the proximal third would consist of striated muscle. External to the muscularis externa is the adventitia (Adv) consisting of dense connective tissue. Mucosa, esophagus, monkey, H&E ×300.

1	External to the muscularis externa is the adventitia (Adv) consisting of dense connective tissue. Mucosa, esophagus, monkey, H&E ×300. As in other stratified squamous epithelia, new cells are produced in the basal layer, from which they move to the surface. During this migration, the shape and orientation of the cells change. This change in cell shape and orientation is also reflected in the appearance of the nuclei. In the deeper layers, the nuclei are spherical; in the more superficial layers, the nuclei are elongated and oriented parallel to the surface. That nuclei can be seen throughout the epithelial layer, particularly the surface cells, indicates that the epithelium is not keratinized. In some instances, the epithelium of the upper regions of the esophagus may be parakeratinized or, more rarely, keratinized.

1	In some instances, the epithelium of the upper regions of the esophagus may be parakeratinized or, more rarely, keratinized. As shown in this figure, the lamina propria (LP) is a very cellular, loose connective tissue containing many lymphocytes (Lym), small blood vessels, and lymphatic vessels (LV ). The deepest part of the mucosa is the muscularis mucosae (MM ). That layer of smooth muscle defines the boundary between mucosa and submucosa. The nuclei of the smooth muscle cells of the muscularis mucosae appear spherical because the cells have been cut in cross section. KEY Adv, adventitia Ep, stratified squamous epithelium L, longitudinal layer of muscularis externa LP, lamina propria LV, lymphatic vessels Lym, lymphocytes ME, muscularis externa MM, muscularis mucosae Muc, mucosa SM, smooth muscle StM, striated muscle SubM, submucosa arrows (upper figure), lymphocytes in epithelium asterisks (lower figure), areas containing striated muscle in the muscularis externa

1	PLATE 55 Esophagus And Stomach, Cardiac Region The esophagogastric junction marks a change in function from that of a conduit (esophagus) to that of a digestive organ (stomach). The epithelium of the mucosa changes from stratified squamous (protective) to a simple columnar secretory epithelium that forms mucosal glands that secrete mucinogen, digestive enzymes, and hydrochloric acid. The very cellular lamina propria is rich in diffuse lymphatic tis-sue, emphasizing the role of this layer in the immune system. Esophagogastric junction, esophagus-stomach, human, H&E ×100.

1	Esophagogastric junction, esophagus-stomach, human, H&E ×100. The junction between the esophagus and stomach is shown here. The esophagus is on the right, and the cardiac region of the stomach is on the left. The large rectangle marks a representative area of the cardiac mucosa seen at higher magnification in figure below; the smaller rectangle shows part of the junction examined at higher magnification in figure on right. As noted in Plate 54, the esophagus is lined by stratifed squamous 608 epithelium (Ep) that is indented on its undersurface by deep connective tissue papillae. When these are sectioned obliquely (as five of them have been), they appear as islands of connective tissue within the thick epithelium. Under the epithelium are the lamina propria and the muscularis mucosae Cardiac region, stomach, human, H&E ×260.

1	Cardiac region, stomach, human, H&E ×260. The cardiac glands and pits seen in top figure are surrounded by a very cellular lamina propria. At this higher magnification, it can be seen that many cells of the lamina propria are lymphocytes and other cells of the immune system. Large numbers of lymphocytes (L) may be localized between the smooth muscle cells of the muscularis mucosae (MM ), and thus, the muscularis mu cosae in these locations appears to be disrupted. Cardiac region, stomach, human, H&E ×440.

1	Cardiac region, stomach, human, H&E ×440. The epithelium of the cardiac glands (GL ) also consists of mucous gland cells (MGC ). As seen in the photomicrograph, the nucleus of the gland cell is typically flattened; one side is adjacent to the base of the cell, while the other side is adjacent to the pale-staining cytoplasm. Again, mucus is lost during processing of the tissue, and this accounts for the pale-staining appearance of the cytoplasm. Although the cardiac glands are mostly unbranched, some branching (MM ). At the junction between the esophagus and the stomach (see also middle right figure ), the stratified squamous epithelium of the esophagus ends abruptly, and the simple columnar epithelium of the stomach surface begins.

1	The surface of the stomach contains numerous and relatively deep depressions called gastric pits (P ), or foveolae, that are formed by epithelium similar to, and continuous with, that of the surface. Glands (GL ) open into the bottom of the pits; they are cardiac glands. The entire gastric mucosa contains glands. There are three types of gastric glands: cardiac, fundic, and pyloric. Cardiac glands are in the immediate vicinity of the opening of the esophagus; pyloric glands are in the funnel-shaped portion of the stomach that leads to the duodenum; and fundic glands are throughout the remainder of the stomach. PLATE 55 • E SOPHAG US AN D STOMACH, CAR DIAC R EG ION KEY D, ducts of cardiac glands Ep, epithelium GL cardiac glands L, lymphocytes LP, lamina propria MGC, mucous gland cells MM, muscularis mucosae P, gastric pits SMC, surface mucous cells arrows, intraepithelial lymphocytes

1	The cardiac glands (GL) are limited to a narrow region around the cardiac orifice. They are not sharply delineated from the fundic region of the stomach that contains parietal and chief cells. Thus, at the boundary, occasional parietal cells are seen in the cardiac glands. In certain animals (e.g., ruminants and pigs), the anatomy and histology of the stomach are different. In these, at least one part of the stomach is lined with stratified squamous epithelium. Esophagogastric junction, esophagus-stomach, of cells named surface mucous cells (SMC). The content of the mucous human, H&E ×440. cup is usually lost during the preparation of the tissue, and thus, the apical cup portion of the cells appears empty in routine H&E paraffin sections The columnar cells of the stomach surface and gastric pits (P ) such as the ones shown in this plate.

1	produce mucus. Each surface and pit cell contains a mucous cup in its apical cytoplasm, thereby forming a glandular sheet is occasionally seen. The glands empty their secretions via ducts (D) into the bottom of the gastric pits. The cells forming the ducts are columnar, and the cytoplasm stains well with eosin. This makes it easy to distinguish the duct cells from mucous gland cells. Among the cells forming the duct portion of the gland are those that undergo mitotic division to replace both surface mucous and gland cells. Cardiac glands also contain enteroendocrine cells, but they are difficult to identify in routine H&E paraffin sections. PLATE 55 • ESOPHAGUS AND STOMACH, CARDIAC REGIONMGCMGCGLGLLPLPMMMMGLGLDD PLEpEpMMMMGLGLEp P GL MM Ep L P P PP PP PP PP DD DD DD DD DD DD GL MM SMC LP MMMMMM GL GLGLGL MGC GLGLGL LL

1	PLATE 55 • ESOPHAGUS AND STOMACH, CARDIAC REGIONMGCMGCGLGLLPLPMMMMGLGLDD PLEpEpMMMMGLGLEp P GL MM Ep L P P PP PP PP PP DD DD DD DD DD DD GL MM SMC LP MMMMMM GL GLGLGL MGC GLGLGL LL The stomach is divided into three regions: the cardia, nearest the esophagus, contains cardiac glands that secrete primarily mucinogen; the pylorus, proximal to the gastrointestinal (pyloric) sphincter contains pyloric glands that secrete a mucinogen that resembles that of the sur-face mucous cells; and the fundus, the body or largest part of the stomach that contains the fundic (gastric) glands. Fundic glands contain parietal (oxyntic) cells, acidophilic cells that secrete 0.16 N HCl; and chief cells, basophilic cells that contain acidophilic secretory granules in the apical cytoplasm. The granules contain, principally, pepsinogen. The glands in all parts of the stomach contain enteroendocrine cells. Stomach, human, H&E.

1	Stomach, human, H&E. As with other parts of the gastrointestinal tract, the wall of the stomach consists of four layers: a mucosa (Muc), a submucosa (SubM ), a muscularis externa (ME ), and a serosa. The mucosa is the innermost layer and reveals three distinctive regions (arrows). The most superficial region contains the gastric pits; the middle region contains the necks of the glands, which tend to stain with eosin; and the deepest part of the mucosa stains most heavily with hema 610 toxylin. The cell types of the deep (hematoxylin-staining) portion of the fundic mucosa are considered in bottom figure. The cells of all three regions and their staining characteristics are considered in Plate 57. The inner surface of the empty stomach is thrown into long folds referred to as rugae. One such cross-sectioned fold is shown here. It consists Fundocardiac junction, stomach, human, H&E.

1	Fundocardiac junction, stomach, human, H&E. This figure and figure below show the fundocardiac junction between the cardiac and fundic regions of the stomach. This junction can be identified histologically on the basis of the structure of the mucosa. The gastric pits (P ), some of which are seen opening at the surface (arrows), are similar in both regions, but the glands are different. They are composed mostly of mucus- Fundocardiac junction, stomach, human, H&E.

1	Fundocardiac junction, stomach, human, H&E. This figure provides a comparison between the cardiac and fundic glands at higher magnification. The cardiac glands (CG ) consist of mucous gland cells arranged as a simple columnar epithelium; the nucleus is in the most basal part of the cell and is somewhat flattened. The cytoplasm appears as a faint network of lightly stained material. The lumina (L) of the cardiac glands are relatively wide. On the other hand, the fundic glands (FG ) (left of the dashed line) are small, and a lumen is seen readily only in certain fortuitously sectioned glands. As a consequence, most of the glands of mucosa and submucosa (asterisks). The rugae are not permanent folds; they disappear when the stomach wall is stretched, as in the distended stomach. Also evident are mamillated areas (M ), which are slight elevations of the mucosa that resemble cobblestones. The mamillated areas consist only of mucosa without submucosa.

1	The submucosa and muscularis externa stain predominantly with eosin; the muscularis externa appears darker. The smooth muscle of the muscularis externa gives an appearance of being homogeneous and uniformly solid. In contrast, the submucosa, being connective tissue, may contain areas with adipocytes and contains numerous profiles of blood vessels (BV ). The serosa is so thin that it is not evident as a discrete layer at this low magnification. secreting cells and occasional enteroendocrine cells. The boundary between cardiac glands (CG ) and fundic glands (FG ) is marked by the dashed line in each figure. The full thickness of the gastric mucosa is shown here, as indicated by the presence of the muscularis mucosae (MM ) deep to the fundic glands. The muscularis mucosae under the cardiac glands is obscured by a large infiltration of lymphocytes forming a lymphatic nodule (LN ).

1	appear to be cords of cells. Because this is a deep region of the fundic mucosa, most of the cells are chief cells. The basal portion of the chief cell contains the nucleus and extensive ergastoplasm, thus, its basophilia. The apical cytoplasm, normally occupied by secretory granules that were lost during the preparation of the tissue, stains poorly. Interspersed among the chief cells are parietal cells (PC). These cells typically have a round nucleus that is surrounded by eosinophilic cytoplasm. Among the cells of the lamina propria are some with pale elongate nuclei. These are smooth muscle cells (SM ) that extend into the lamina propria from the muscularis mucosae.

1	KEY BV, blood vessels CG, cardiac glands FG, fundic glands L, lumen LN, lymphatic nodule M, mamillated areas ME, muscularis externa MM, muscularis mucosae Muc, mucosa P, gastric pits PC, parietal cells SM, smooth muscle cells SubM, submucosa arrows: top left figure, three differently stained regions of fundic mucosa; Top right figure, opening of gastric pits asterisks, submucosa in ruga dashed line, boundary between cardiac and fundic glands The epithelial lining of the alimentary canal is a regularly renewing epithelium; each portion has a characteristic turnover time and stem cell location. In the stomach, stem cells are located in the mucous neck. Cells that migrate upward to form the mucous cells of the gastric pit and surface have a turnover time of 3 to 5 days; cells that migrate downward to form the parietal cells, chief cells, and enteroendocrine cells of the glands have a turnover time of about 1 year. Fundic glands, stomach, monkey, H&E ×320.

1	Fundic glands, stomach, monkey, H&E ×320. This figure shows an area of the fundic mucosa that includes the bottom of the gastric pits and the neck and deeper part of the fundic glands. It includes the areas marked by the arrows in top left figure of Plate 56. The surface mucous cells (SMC) of the gastric pits are readily identified because the mucous cup in the apical pole of each cell has an empty, washed-out appearance. Just below the gastric pits are the necks (N ) of the fundic glands, in which one can identify mucous neck cells (MNC ) and parietal cells (PC ). The mu612 cous neck cells produce a mucous secretion that differs from that produced by the surface mucous cells. As seen here, the mucous neck cells display a cytoplasm that is lightly stained; there are no cytoplasmic areas that stain intensely, nor is there a characteristic local absence of staining as in the mucous Submucosa, stomach, monkey, H&E ×320.

1	Submucosa, stomach, monkey, H&E ×320. This figure shows the bottom of the stomach mucosa, the sub-mucosa (SubM ), and part of the muscularis externa (ME ). The muscularis mucosae (MM ) is the deepest part of the mucosa. It consists of smooth muscle cells arranged in at least two layers. As seen in the photomicrograph, the smooth muscle cells immediately adjacent to the submucosa have been sectioned longitudinally and display elongate nuclear profiles. Just above this layer, the smooth muscle cells have been cut in cross section and display rounded nuclear profiles. Gastric glands, stomach, silver stain ×160. Enteroendocrine cells constitute a class of cells that can be displayed with special histochemical or silver-staining methods but that are not readily evident in H&E sections. The distribution of cells demonstrable with special silver-staining procedures is shown here (arrows). Because of the staining procedure, Gastric glands, stomach, silver stain ×640.

1	Gastric glands, stomach, silver stain ×640. At higher magnification, the argentaffn cells (arrows) are almost totally blackened by the silver staining, although a faint nucleus can be seen in some cells. The silver stains the secretory product lost during the preparation of routine sections, cup of the surface mucous cells. The mucous neck cells are also the stem cells that divide to give rise to the surface mucous cells and the gland cells. Parietal cells are distinctive primarily because of the pronounced eosinophilic staining of their cytoplasm. Their nucleus is round, like that of the chief cell, but tends to be located closer to the basal lamina of the epithelium than to the lumen of the gland because of the pearlike shape of the parietal cell.

1	This figure also reveals the significant characteristics of chief cells (CC ), namely, the round nucleus in a basal location; the ergastoplasm, deeply stained with hematoxylin (particularly evident in some of the chief cells where the nucleus has not been included in the plane of the section); and the apical, slightly eosinophilic cytoplasm (normally occupied by the secretory granules). The submucosa consists of connective tissue of moderate density. Present in the submucosa are adipocytes (A), blood vessels (BV), and a group of ganglion cells (GC ). These particular cells belong to the submucosal plexus (Meissner’s plexus [MP] ). The inset shows some of the ganglion cells (GC ) at higher magnification. These are the large cell bodies of the enteric neurons. Each cell body is surrounded by satellite cells intimately apposed to the neuron cell body. The arrowheads point to the nuclei of the satellite cells.

1	these cells are properly designated as argentaffn cells. The surface mucous cells (SMC) in the section mark the bottom of the gastric pits and establish the fact that the necks of the fundic glands are represented in the section. The argentaffin cells appear black in this specimen. The relatively low magnification permits the viewer to assess the frequency of distribution of these cells. and accordingly, in H&E–stained paraffin sections the argentaffin cell appears as a clear cell. The special silver staining in this figure and in figure on left shows that many of the argentaffin cells tend to be near the basal lamina and away from the lumen of the gland. KEY A, adipocytes BV, blood vessels CC, chief cells GC, ganglion cells ME, muscularis externa MM, muscularis mucosae MNC, mucous neck cells MP, Meissner’s plexus N, neck of fundic glands PC, parietal cells SMC, surface mucous cells SubM, submucosa arrows, argentaffin cells arrowheads, nuclei of satellite cells

1	The gastroduodenal junction marks the entry into the absorptive portion of the alimentary canal. Thickening of the circular layer of muscularis externa at this site forms the pyloric sphincter that regulates passage of chyme from stomach to intestine. The mucous secretion of the pyloric glands helps to neutralize the chyme as it enters the intestine. Gastroduodenal junction, stomach-duodenum, monkey, H&E ×40. The gastroduodenal junction between the stomach and the duodenum is shown here. Most of the mucosa in the micro-graph belongs to the stomach; it is the pyloric mucosa (PMuc). The pyloric sphincter appears as a thickened region of smooth muscle below the pyloric mucosa. On the far right is the duodenal mucosa, the first part of the intestinal mucosa (IMuc). The area marked by the rectangle is shown at higher magnification in figure below. It provides a comparison of the two mucosal regions and also shows the submucosal 614 glands (Brunner’s glands).

1	Gastroduodenal junction, stomach-duodenum, monkey ×120. Examination of this region at higher magnification reveals that in addition to intestinal glands (IGl ) within the mucosa, there are glands within the duodenal submucosa. These are submucosal (Brunner’s) glands (BGl ). Some of the glandular elements (arrows) can be seen to pass from the submucosa to the mucosa, thereby interrupting the muscularis mucosae (MM ). The submucosal glands empty their secretions into the duodenal lumen by means of ducts (D). In contrast, the pyloric glands (PGl ) are relatively straight for most of their length but are coiled in the deepest part of the mucosa and are sometimes branched. They are restricted to the mucosa and empty into deep Gastroduodenal junction, stomach-duodenum, monkey ×640.

1	Gastroduodenal junction, stomach-duodenum, monkey ×640. The rectangular area in figure below is considered at higher magnification here. It shows how the epithelium of the stomach differs from that of the intestine. In both cases, the epithelium is simple columnar, and the underlying lamina propria (LP ) is highly cellular because of the presence of large numbers of lymphocytes. The boundary between gastric and duodenal epithelium is marked by The submucosa of the duodenum contains submucosal (Brunner’s) glands. These are below the muscularis mucosae; therefore, this structure serves as a useful landmark in identifying the glands. In the stomach, the muscularis mucosae is readily identified as narrow bands of muscular tissue (MM ). It can be followed toward the right into the duodenum but is then interrupted in the region between the two asterisks.

1	This figure also shows the thickened region of the gastric muscularis externa, where the stomach ends. This is the pyloric sphincter (PS ). Its thickness, mostly due to the amplification of the circular layer of smooth muscle of the muscularis externa, can be appreciated by comparison with the muscularis externa in the duodenum (ME ). gastric pits. The boundary between the pits and glands is, however, hard to ascertain in H&E sections.

1	gastric pits. The boundary between the pits and glands is, however, hard to ascertain in H&E sections. With respect to the mucosal aspects of gastroduodenal histology, as mentioned above, the glands of the stomach empty into gastric pits. These are depressions; accordingly, when the pits are sectioned in a plane that is oblique or at right angles to the long axis of the pit, as in this figure, the pits can be recognized as being depressions because they are surrounded by lamina propria. In contrast, the inner surface of the small intestine has villi (V). These are projections into the lumen of slightly varying height. When the villus is cross-sectioned or obliquely sectioned, it is surrounded by space of the lumen, as is one of the villi shown here. In addition, the villi have lamina propria (LP ) in their core.

1	the arrow. On the stomach side of the arrow, the epithelium consists of surface mucous cells (SMC). The surface cells contain an apical cup of mucous material that typically appears empty in an H&E–stained paraffin section. In contrast, the absorptive cells (AC ) of the intestine do not possess mucus in their cytoplasm. Although goblet cells are found in the intestinal epithelium and are scattered among the absorptive cells, they do not form a complete mucous sheet. The intestinal absorptive cells also possess a striated border, which is shown in Plate 60.

1	PLATE 58KEY AC, absorptive cells BGl, Brunner’s glands D, ducts IGl, intestinal glands IMuc, intestinal mucosa LP, lamina propria ME, muscularis externa MM, muscularis mucosae PGl, pyloric glands PMuc, pyloric mucosa PS, pyloric sphincter SMC, surface mucous cells V, villi arrows: bottom figure, Brunner’s gland element that passes from the submucosa to the mucosa; upper right figure, boundary between gastric and duodenal epithelium asterisks, interruption in muscularis mucosae

1	The small intestine is the principal site for the digestion of food and absorption of the products of digestion. It is the longest component of the alimentary canal, measuring over 6 m, and is divided into three segments: duodenum (~25 cm); jejunum (~2.5 m); and ileum (~3.5 m). The first portion, the duodenum, receives a partially digested bolus of food (chyme) from the stomach, as well as secretions from the stomach, pancreas, liver, and gallbladder that contain digestive enzymes, enzyme precursors, and other products that aid digestion and absorption. The small intestine is characterized by plicae circulares, permanent transverse folds that contain a core of submucosa, and villi, fingerlike and leaflike projections of the mucosa that extend into the lumen. Microvilli, multiple finger like extensions of the apical surface of each intesti-nal epithelial cell (enterocyte), further increase the surface for absorption of metabolites. Mucosal glands extend into the lamina propria. They

1	like extensions of the apical surface of each intesti-nal epithelial cell (enterocyte), further increase the surface for absorption of metabolites. Mucosal glands extend into the lamina propria. They contain the stem cells and developing cells that will ultimately migrate to the surface of the villi. In the duodenum, submucosal glands (Brunner’s glands) secrete an alkaline mucus that helps to neutralize the acidic chyme. Enterocytes not only absorb metabolites digested in the intestinal lumen but also synthesize enzymes inserted into the membrane of the mi-crovilli for terminal digestion of disaccharides and dipeptides.

1	Duodenum, monkey, H&E ×120. This figure shows a segment of the duodenal wall. As in the stomach, the layers of the wall, in order from the lumen, are the mucosa (Muc), the submucosa (SubM ), the muscularis externa (ME ), and the serosa (S ). Both longitudinal (L) and circular (C ) layers of the muscularis externa can be distinguished. Although plicae circulares are found in the wall of the small intestine, including the duodenum, none is included in this photomicrograph. A distinctive feature of the intestinal mucosa is the presence of fingerlike and leaflike projections into the intestinal lumen, called villi. Most of the villi (V ) shown here display profiles that correspond to their description as fingerlike. One villus, however, displays the form of a leaflike villus (asterisk). The dashed line marks the boundary between the villi and the intestinal glands (also called crypts of Lieberkn). The latter extend as far as the muscularis mucosae (MM ).

1	Under the mucosa is the submucosa, containing the Brunner’s glands (BGl ). These are branched tubular or branched tubuloalveolar glands whose secretory components, shown at higher magnification in figure below, consist of columnar epithelium. A duct (D) through which the glands open into the lumen of the duodenum is shown here and, at higher magnification, in figure below, where it is marked by an arrow. Mucosa, duodenum, monkey, H&E ×240.

1	Mucosa, duodenum, monkey, H&E ×240. The histologic features of the duodenal mucosa are shown at higher magnification here. Two kinds of cells can be recognized in the epithelial layer that forms the surface of the villus: enterocytes (absorptive cells) and goblet cells (GC ). Most of the cells are absorptive cells. They have a striated border that will be seen at higher magnification in Plate 60; their elongate nuclei are located in the basal half of the cell. Goblet cells are readily identified by the presence of the apical mucous cup, which appears empty. Most of the dark round nuclei also seen in the epithelial layer covering the villi belong to lymphocytes.

1	The lamina propria (LP ) makes up the core of the villus. It contains large numbers of round cells whose individual identity cannot be ascertained at this magnification. Note, however, that these are mostly lymphocytes (and other cells of the immune system), which accounts for the designation of the lamina propria as diffuse lymphatic tissue. The lamina propria surrounding the intestinal glands (IGl ) similarly consists largely of lymphocytes and related cells. The lamina propria also contains components of loose connective tissue and isolated smooth muscle cells.

1	The intestinal glands (IGl ) are relatively straight and tend to be dilated at their base. The bases of the intestinal crypts contain the stem cells from which all of the other cells of the intestinal epithelium arise. They also contain Paneth cells. These cells possess eosinophilic granules in their apical cytoplasm. The granules contain lysozyme, a bacteriolytic enzyme thought to play a role in regulating intestinal microbial flora. The main cell type in the intestinal crypt is a relatively undifferentiated columnar cell. These cells are shorter than the enterocytes of the villus surface; they usually undergo two mitoses before they differentiate into absorptive cells or goblet cells. Also present in the intestinal crypts are some mature goblet cells and enteroendocrine cells.

1	KEY BGl, Brunner’s glands C, circular (inner) layer of muscularis externa D, duct of Brunner’s gland GC, goblet cells IGl, intestinal glands (crypts) L, longitudinal (outer) layer of muscularis externa LP, lamina propria ME, muscularis externa MM, muscularis mucosae Muc, mucosa S, serosa SubM, submucosa V, villi arrow, duct of Brunner’s gland asterisk, leaflike villus dashed line (top figure), boundary between base of villi and intestinal glands The jejunum is the principal site of absorption of nutrients in the small intestine. The villi are more finger like than leaf like and are covered largely with absorptive columnar epithelial cells (enterocytes), although goblet cells and enteroendocrine cells are also present. The stem cells for all of these cells and the Paneth cells that secrete the antibacterial enzyme lysozyme are found deep in the intestinal gland. Replicat-ing cells line the lower half of the gland. Jejunum, monkey, H&E ×22.

1	Jejunum, monkey, H&E ×22. This is a longitudinal section of the jejunum, showing the permanent circular folds of the small intestine, the plicae circulares (PC ). These folds or ridges are mostly arranged with their long axis at roughly right angles to the longitudinal axis of the intestine; therefore, the plicae circulares shown here are cut in cross section. The plicae circulares consist of mucosa (Muc) as well as sub-mucosa (SubM ). The broad band of tissue external to the submucosa is the Plica circularis, jejunum, monkey, H&E ×60.

1	Plica circularis, jejunum, monkey, H&E ×60. Part of the plica circularis marked by the bracket in figure above is shown at higher magnification. Note the muscularis mucosae (MM ), the intestinal glands (GI ), and the villi (V ). The boundary between the glands and villi is marked by the dashed line. Some of the glands are cut longitudinally; some are cut in cross section; most of the villi have been cut longitudinally. In conceptualizing the mucosal structure of the small intestine, it is important to recognize that the glands are epithelial depressions that project into the wall Intestinal villi, jejunum, monkey, H&E ×500.

1	This figure shows portions of two adjacent villi at higher magnification. The epithelium consists chiefly of enterocytes. These are columnar absorptive cells that typically exhibit a striated border (SB), the light microscopic representation of the microvilli on the apical surface of each enterocyte. The dark band at the base of the striated border is due to the terminal web of the cell, a layer of actin filaments that extends across the apex of the cell to which the actin filaments of the cores of the microvilli attach. The nuclei of the enterocytes have essentially the same shape, orientation, and staining characteristics. Even if the cytoplasmic boundaries were not evident, the nuclei would be an indication of the columnar shape and orientation of the cells. The enterocytes rest on a basal lamina not evident in H&E–stained paraffin sections. The eosinophilic band (arrow) at the base of the cell layer, muscularis externa (ME ) and is not included in the plicae. (The serosa cannot be

1	lamina not evident in H&E–stained paraffin sections. The eosinophilic band (arrow) at the base of the cell layer, muscularis externa (ME ) and is not included in the plicae. (The serosa cannot be distinguished at this magnification.) Most of the villi (V ) in this specimen have been cut longitudinally, thereby revealing their full length as well as the fact that some are slightly shorter than others. The shortening is considered to be due to the contraction of smooth muscle cells in the villi. Also seen here are the lacteals (L), which in most of the villi are dilated. Lacteals are lymphatic capillaries that begin in the villi and carry certain absorbed dietary lipids and proteins from the villi to the larger lymphatic vessels of the submucosa.

1	of the intestine, whereas the villi are projections that extend into the lumen. The glands are surrounded by cells of the lamina propria; the villi are surrounded by space of the intestinal lumen. The lamina propria with its lacteal occupies a central position in the villus; the lumen occupies the central position of the gland. Also note that the lumen of the gland tends to be dilated at its base. Studies of enzymatically isolated preparations of mucosa show that the bases of the glands are often divided into two to three fingerlike extensions resting on the muscularis mucosae. where one would expect a basement membrane, actually consists of flat lateral cytoplasmic processes from the enterocytes. These processes partially delimit the basal-lateral intercellular spaces (asterisks) that are dilated, as can be seen here, during active transport of absorbed substrates.

1	The epithelial cells with an expanded apical cytoplasm in the form of a cup are goblet cells (GC ). In this specimen, the nucleus of almost every goblet cell is just at the base of the cup, and a thin cytoplasmic strand (not always evident) extends to the level of the basement membrane. The scattered round nuclei within the epithelium belong to lymphocytes (Ly). The lamina propria (LP ) and the lacteal (L) are located beneath the intestinal epithelium. The cells forming the lacteal are simple squamous epithelium (endothelium). Two nuclei of these cells (EC ) appear to be exposed to the lumen of the lacteal; another elongate nucleus slightly removed from the lumen belongs to a smooth muscle cell (M) accompanying the lacteals.

1	KEY EC, endothelial cell GC, goblet cell GI, intestinal glands (crypts) L, lacteal LP, lamina propria Ly, lymphocytes M, smooth muscle cell ME, muscularis externa MM, muscularis mucosae Muc, mucosa PC, plicae circulares S, serosa SB, striated border SubM, submucosa V, villi arrow, basal processes of enterocytes asterisks, basal-lateral intercellular spaces dashed line, boundary between villi and intestinal glands

1	The ileum is the principal site of water and electrolyte reabsorption in the small intestine. It has essentially the same histologic features as the jejunum. Some, however, are emphasized; namely, villi in the ileum are more frequently leaflike, and lymphatic tissue in the lamina propria is organized into small and large nodes that are found in great number on the antimesenteric side of the ileum. The nodes fuse to form large ac-cumulations of lymphatic tissue called Peyer’s patches. The surface epithelium of the small intestine renews itself every 5 to 6 days. The stem cells are restricted to the bottoms of the mucosal glands, and the zone of cell replication is restricted to the lower half of the gland. The cells migrate onto the villus and are lost from its tip. All of the epithelial cells, absorptive cells, and goblet cells, as well as enteroendocrine cells and Paneth cells, derive from the same stem cell population, but enteroendocrine cells migrate only slowly, and Paneth cells

1	absorptive cells, and goblet cells, as well as enteroendocrine cells and Paneth cells, derive from the same stem cell population, but enteroendocrine cells migrate only slowly, and Paneth cells do not migrate.

1	Ileum, monkey, H&E ×20. For purposes of orientation, the submucosa (SM ) and muscularis externa (ME ) have been marked in the cross section through the ileum shown here. Just internal to the submucosa is the mucosa; external to the muscularis externa is the serosa. The mucosa reveals several longitudinally sectioned villi (V ), which have been labeled, and other unlabeled villi, which can be identified easily on the basis of their appearance as islands of tissue completely surrounded by the space of the lumen. They are, of course, not islands because this appearance is due to the plane of section that slices completely through some of the villi obliquely or in cross section, thereby isolating them from their base. Below the villi are the intestinal glands, many of which are obliquely or transversely sectioned and can be readily identified, as was done in the preceding plates, because they are totally surrounded by lamina propria.

1	There are about 8 to 10 projections of tissue into the intestinal lumen that are substantially larger than the villi. These are the plicae circulares. As Aggregated lymph nodule, ileum, monkey, H&E ×100; inset ×200.

1	Part of an aggregated lymph nodule and part of the overlying epithelium are shown here at higher magnification. The lymphocytes and related cells are so numerous that they virtually obscure the cells of the muscularis mucosae. Their location, however, can be estimated as being near the presumptive label (MM?? ), inasmuch as the muscularis mucosae is ordinarily adjacent to the base of the intestinal glands (Gl ). Moreover, on examination of this area at higher magnification (inset), groups of smooth muscle cells (MM ) can be seen noted above, plicae generally have circular orientation, but they may travel in a longitudinal direction for short distances and may branch. In addition, even if all the plicae are arranged in a circular manner, if the section is somewhat oblique, the plicae will be cut at an angle, as appears to be the case with several plicae in this figure. One of the distinctive features of the small intestine is the presence of single and aggregated lymph nodules in the

1	be cut at an angle, as appears to be the case with several plicae in this figure. One of the distinctive features of the small intestine is the presence of single and aggregated lymph nodules in the intestinal wall. Isolated nodules of lymphatic tissue are common in the proximal end of the intestinal canal. As one proceeds distally through the intestines, however, the lymph nodules occur in increasingly larger numbers. In the ileum, large aggregates of lymph nodules are regularly seen; they are referred to as Peyer’s patches. Several lymphatic nodules (LN ) forming a Peyer’s patch are shown in this figure. The nodules are partly within the mucosa of the ileum and extend into the submucosa. Although not evident in the figure, the nodules are characteristically located opposite where the mesentery connects to the intestinal tube.

1	Plica circularis, ileum, monkey, H&E ×40. Sometimes, in a cross section through the intestine, a plica displays a clear cross-sectional profile such as that shown here. Note, again, that the submucosa (SM ) constitutes the core of the plica. Although many of the villi in this figure present profiles (V ) that would be expected if the villus were a fingerlike projection, others clearly do not. In particular, one villus (marked with three asterisks) shows the broad profile of a longitudinally sectioned leaf like villus. If this same villus were cut at a right angle to the plane shown here, it would appear as a finger like villus. separated by numerous lymphocytes close to the intestinal glands (Gl ). Clearly, the lymphocytes of the nodule are on both sides of the muscularis mucosae and, thus, within both the mucosa and the submucosa.

1	In places, the lymph nodule is covered by the intestinal epithelium. Whereas the nature of the epithelium cannot be appreciated fully in the light microscope, electron micrographs (both scanning and transmission) have shown that among the epithelial cells are special cells, designated M cells, that sample the intestinal content (for antigen) and present this antigen to the lymphocytes in the epithelial layer. KEY Gl, intestinal glands LN, lymphatic nodules ME, muscularis externa MM, muscularis mucosae MM??, presumptive location of muscularis mucosae SM, submucosa V, villi asterisks, leaf like villus PLATE 61 • ILEUM MMMMMM??MM??GIGIMEMESMSMLNLNV LN SM ME V MEMEME SMSMSM V GI MM?? MM GIGIGI ** ** **

1	PLATE 61 • ILEUM MMMMMM??MM??GIGIMEMESMSMLNLNV LN SM ME V MEMEME SMSMSM V GI MM?? MM GIGIGI ** ** ** The principal functions of the colon are reabsorption of electrolytes and water and elimination of undigested food and other waste. The mu-cosa has a smooth surface; neither plicae circulares nor villi are present. Numerous simple glands (crypts of Lieberkn) extend through the full thickness of the mucosa. The glands, as well as the surface, are lined with a simple columnar epithelium that contains goblet cells, absorp-tive cells, and enteroendocrine cells but does not normally contain Paneth cells. Here, too, stem cells are restricted to the bottoms of the glands (crypts), and the normal zone of replication extends about one third of the height of the crypt. Colon, monkey, H&E ×30.

1	Colon, monkey, H&E ×30. A cross section through the large intestine is shown at low magnification. It shows the four layers that make up the wall of the colon: the mucosa (Muc), the submucosa (SubM ), the muscularis externa (ME ), and the serosa (S ). Although these layers are the same as those in the small intestine, several differences should be noted. The large intestine has no villi, nor does it have plicae circulares. On the other hand, the muscularis externa is arranged in a 622 distinctive manner, and this is evident in the photomicrograph. The longi- Mucosa, colon, monkey, H&E ×140. The mucosa, shown at higher magnification, contains straight, unbranched, tubular glands (crypts of Lieberkn) that extend to the muscularis mucosae (MM). The arrows identify the openings of some of the glands at the intestinal surface. Gener- Lamina propria, colon, monkey, H&E ×525.

1	Lamina propria, colon, monkey, H&E ×525. This figure reveals the muscularis mucosae (MM) and the cells in the lamina propria (LP), many of which can be recognized as lymphocytes and plasma cells. The smooth muscle cells of the muscularis mucosae are arranged in two layers. Note that the smooth muscle cells marked by the arrowheads Intestinal glands, colon, monkey, H&E ×525.

1	Intestinal glands, colon, monkey, H&E ×525. The cells that line the surface of the colon and the glands are principally absorptive cells (AC) and goblet cells (GC). The absorptive cells have a thin striated border that is evident where the arrows show the opening of the glands. Interspersed among the absorptive cells are the goblet cells (GC). As the absorptive cells are followed into the glands, they become fewer, whereas the tudinal layer (ME[l] ) is substantially thinner than the circular layer (ME[c] ) except in three locations where the longitudinal layer of smooth muscle is present as a thick band. One of these thick bands, called a tenia coli (TC ), is shown in this figure. Because the colon is cross-sectioned, the tenia coli is also cross-sectioned. The three teniae coli extend along the length of the large intestine as far as, but not into, the rectum.

1	The submucosa consists of a rather dense irregular connective tissue. It contains the larger blood vessels (BV ) and areas of adipose tissue (see A in figure below). ally, the lumen of the glands is narrow except in the deepest part of the gland, where it is often slightly dilated (asterisks, lower left figure). Between the glands (Gl) is a lamina propria (LP) that contains considerable numbers of lymphocytes and other cells of the immune system. Two rectangles mark areas of the mucosa that are examined at higher magnification in figures below. show rounded nuclei; however, other smooth muscle cells appear as more or less rounded eosinophilic areas. These smooth muscle cells have been cut in cross section. Just above these cross-sectioned smooth muscle cells are others that have been cut longitudinally; they display elongate nuclei and elongate strands of eosinophilic cytoplasm.

1	goblet cells increase in number. Other cells in the gland are enteroendocrine cells, not easily identified in routine H&E–stained paraffin sections, and, in the deep part of the gland, undifferentiated cells of the replicative zone, derived from the stem cells in the base of the crypt. The undifferentiated cells are readily identified if they are undergoing division, by virtue of the mitotic figures (M) they display (see figure on left). KEY A, adipose tissue AC, absorptive cells BV, blood vessels GC, goblet cells GI, intestinal glands LP, lamina propria M, mitotic figures ME, muscularis externa ME(c), circular layer of muscularis externa ME(l), longitudinal layer of muscularis externa MM, muscularis mucosae Muc, mucosa S, serosa SubM, submucosa TC, tenia coli arrowheads, smooth muscle cells showing rounded nuclei arrows, opening of intestinal glands asterisks, lumen of intestinal glands

1	The appendix (vermiform appendix) is typically described as a wormor fingerlike structure [L. vermis, worm; forma, form]. It arises from the cecum (the first segment of the large intestine; the others, in order, are the ascending, transverse, and descending colon; the sigmoid colon; the rectum; and the anal canal) and forms a blind-ending tube ranging from 2.5 cm to as much as 13 cm in length (average length of ~8 cm). Because it is a blind-ended pouch, intestinal contents may be trapped or sequestered in the appendix, often leading to inflammation and in-fection. In infants and children it is both relatively and absolutely longer than in adults and contains numerous lymphatic nodules, suggesting that it has an immunologic role. Recent evidence indicates that it (and the cecum and terminal ileum) may be the “bursa equivalent” in mam-mals, i.e., the portion of the immature immune system in which potential B lymphocytes achieve immunocompetence (equivalent to the bursa of Fabricius in

1	ileum) may be the “bursa equivalent” in mam-mals, i.e., the portion of the immature immune system in which potential B lymphocytes achieve immunocompetence (equivalent to the bursa of Fabricius in birds). The wall of the appendix is much like that of the small intestine, having a complete longitudinal layer of muscularis externa, but it lacks both plicae circulares and villi. Thus, the mucosa is similar to that of the colon, having simple glands. Even this resemblance is often obliterated, however, by the large number and size of the lymphatic nodules that usually fuse and extend into the submucosa. In later life, the amount of lymphatic tissue in the appendix regresses, and there is a consequent reduction in size. In many adults, the normal structure is lost, and the appendage is filled with fibrous scar tissue.

1	Appendix, human, H&E ×25. mucosa (Muc), submucosa (Subm), muscularis externa (ME ), and serosa (S ) are identified. Cross section of an appendix from a preadolescent, showing the various structures composing its wall. The lumen (L), Appendix, human, H&E ×80; inset ×200.

1	Appendix, human, H&E ×80; inset ×200. This micrograph is a higher magnification of the boxed area in figure above. It reveals the straight tubular glands (Gl ) that extend to the muscularis mucosae. Below is the submucosa (Subm) in which the lymphatic nodules (LN ) and considerable diffuse lymphatic tissue are present. Note the distinct germinal centers (GC ) of the lymph nodules and the cap region (Cap) that faces the lumen. The more superficial part of the submucosa blends and merges with the mucosal lamina propria because of the numerous lymphocytes in these two sites. The deeper part of the submucosa is relatively de void of lymphocyte infiltration and contains the large blood vessels (BV ) and nerves. The muscularis externa (ME ) is composed of a relatively thick circular layer and a much thinner outer longitudinal layer. The serosa (S ) is only partially included in this micrograph.

1	The inset is a higher magnification of the rectangular area in lower figure. Note that the epithelium of the glands in the appendix is similar to that of the large intestine. Most of the epithelial cells contain mucin; hence, the light appearance of the apical cytoplasm. The lamina propria, as noted, is heavily infiltrated with lymphocytes, and the muscularis mucosae at the base of the glands is difficult to recognize (arrows). KEY BV, blood vessel Cap, cap of lymphatic nodule GC, germinal center Cl, gland L, lumen LN, lymphatic nodule ME, muscularis externa Muc, mucosa S, serosa Subm, submucosa arrows, muscularis mucosae at base of glands

1	At the anal canal, there is a transition from the simple columnar epithelium of the intestinal mucosa to the keratinized stratified squamous ep-ithelium of the skin. Between these two distinctly different epithelia there is a narrow region (anal transitional zone) where the epithelium is first stratified columnar (or stratified cuboidal) and then nonkeratinized stratified squamous. At the level of the anal canal, the muscularis mucosae disappears. At the same level, the circular layer of the muscularis externa thickens to become the internal anal sphincter. The external anal sphincter is formed by the striated muscles of the pelvic floor. Anal canal, human, H&E ×40.

1	Anal canal, human, H&E ×40. A view of the anal canal is shown at low magnification. Mucosa characteristic of the large intestine (colorectal zone) is seen on the upper left of the micrograph. This region is the upper part of the anal canal, and the intestinal glands are the same as those present in the colon. The muscularis mucosae (MM ) is readily identified as the narrow band of tissue under the glands. Both the intestinal glands and the muscularis mucosae terminate within626 the left rectangular area of the field, and here, at the diamond, there is the first major change in the epithelium. This area called the anal transitional zone is examined at higher magnification in bottom left figure. The right rectangular area includes the stratified squamous epithelium (StS ) of the Anal transitional zone, anal canal, human, H&E ×160; inset ×300.

1	Anal transitional zone, anal canal, human, H&E ×160; inset ×300. The junction between the simple columnar (SC ) and the stratified (ST ) epithelium called the anal transitional zone is marked with the diamond. The simple columnar epithelium of the upper part of the anal canal contains numerous goblet cells, and as in the mucosa of the colon, this epithelium is continuous with skin in the squamous zone of the anal canal and is examined at higher magnification in bottom right figure. Between the two diamonds in the rectangular areas shown is epithelium of the lower part of the anal canal. Under this epithelium, there is a lymphatic nodule that has a well-formed germinal center. Isolated lymphatic nodules under mucous membranes should not be construed to have fixed locations. Rather, they may or may not be present, according to local demands.

1	Also, at this low magnification, note the internal anal sphincter muscle (IAS ), i.e., the thickened, most distal portion of the circular layer of smooth muscle of the muscularis externa. Under the skin on the right is the subcutaneous part of the external anal sphincter muscle (EAS ). It is composed of striated muscle fibers, which are seen in cross section. the epithelium of the intestinal glands (IG ). These glands continue to about the same point as the muscularis mucosae (MM ). Characteristically, the lamina propria contains large numbers of lymphocytes (Lym), particularly so in the region marked. A higher magnification of the stratified columnar epithelium (StCol ) and stratified cuboidal epithelium (StC ) found in the transition zone is shown in the inset. Squamous zone, anal canal, human, H&E ×160.

1	Squamous zone, anal canal, human, H&E ×160. The final change in epithelial type that occurs at the squamous zone of the anal canal is shown here. On the right is the stratified squamous epithelium of skin (StS(k)). The keratinized nature of the surface is apparent. On the other hand, the stratified squamous epithelium (StS ) below the level of the diamond is not keratinized, and nucleated cells can be seen all the way to the surface. Again, numerous lymphocytes (Lym) are in the underlying connective tissue, and many have migrated into the epithelium in the nonkeratinized area.

1	KEY EAS, external anal sphincter IAS, internal anal sphincter IG, intestinal glands LN, lymphatic nodules Lym, lymphocytes MM, muscularis mucosae SC, simple columnar epithelium ST, stratified epithelium StC, stratified cuboidal epithelium StCol, stratified columnar epithelium StS, stratified squamous epithelium StS(k), stratified squamous epithelium (keratinized) arrow, termination of muscularis mucosae diamonds, junctions between epithelial types Digestive System III: Liver, Gallbladder, and Pancreas

1	Digestive System III: Liver, Gallbladder, and Pancreas LIVER / 628 Overview / 628 Liver Physiology / 629 Blood Supply to the Liver / 631 Structural Organization of the Liver / 632 Liver Lobules / 633 Blood Vessels of the Parenchyma / 636 Perisinusoidal Space (Space of Disse) / 636 Lymphatic Pathway / 638 Hepatocytes / 639 Biliary Tree / 641 GALLBLADDER / 643 PANCREAS / 647 Overview / 647 Exocrine Pancreas / 647 Duct System of the Exocrine Pancreas / 649 Endocrine Pancreas / 649 Functions of Pancreatic Hormones / 651 Regulation of Islet Activity / 653 Folder 18.1 Clinical Correlation: Lipoproteins / 630 Folder 18.2 Clinical Correlation: Congestive Heart Failure and Liver Necrosis / 635 Folder 18.3 Insulin Production and Alzheimer’s Disease / 655 Folder 18.4 Functional Considerations: Insulin Synthesis, an Example of Posttranslational Processing / 655

1	The liver is the largest mass of glandular tissue in the body and the largest internal organ, weighing approximately 1,500 g and accounting for nearly 2.5% of adult body weight. It is located in the upper right and partially in the upper left quadrants of the abdominal cavity, protected by the ribcage. The liver is enclosed in a capsule of fibrous connective tissue (Glisson’s capsule); a serous covering (visceral peritoneum) surrounds the capsule, except where the liver adheres directly to the diaphragm or the other organs. The liver is anatomically divided by deep grooves into two large lobes (the right and left lobes) and two smaller lobes (the quadrate and caudate lobes; Fig. 18.1). This anatomic division has only topographic importance because it relates lobes of the liver to other abdominal organs. Division into functional or surgical segments that correspond to the blood supply and bile drainage is more clinically important.

1	In the embryo, the liver develops as an endodermal evagination from the wall of the foregut (specifically the site that will become the duodenum) to form the hepatic diverticulum. The diverticulum proliferates, giving rise to the hepatocytes, which become arranged in cellular (liver) cords, FIGURE 18.1 • Anatomic structure of the liver. This diagram shows the gross view of the diaphragmatic and visceral surfaces of the liver, with labeled anatomic landmarks found on both surfaces. The enlarged cross-sectional area of the liver (bottom) shows the general microscopic organization of the liver into lobules. Note the presence of hepatic portal triads at the periphery of each lobule, with the terminal hepatic venule (central vein) in the center of the lobule.

1	thus forming the parenchyma of the liver. The original stalk of the hepatic diverticulum becomes the common bile duct. An outgrowth from the common bile duct forms the cystic diverticulum that gives rise to the gallbladder and cystic duct. Many circulating plasma proteins are produced and secreted by the liver. The liver plays an important role in the uptake, storage, and distribution of both nutrients and vitamins from the bloodstream. It also maintains the blood glucose level and regulates circulating levels of very low-density lipoproteins (VLDLs). In addition, the liver degrades or conjugates numerous toxic substances and drugs, but it can be overwhelmed by such substances and damaged. The liver is also an exocrine organ; it produces a bile secretion that contains bile salts, phospholipids, and cholesterol. Finally, the liver performs important endocrine-like functions. The liver produces most of the body’s circulating plasma proteins.

1	The circulating plasma proteins produced by the liver include  albumins, which are involved in regulating plasma volume and tissue fluid balance by maintaining the plasma colloid osmotic pressure.  lipoproteins, in particular, VLDLs. The liver synthesizes most VLDLs, which participate in the transport of triglycerides from the liver to other organs. The liver also produces small amounts of other plasma lipoproteins, such as low-density lipoproteins (LDLs) and high-density lipoproteins (HDLs). LDLs transport cholesterol esters from the liver to other tissues. HDLs remove cholesterol from the peripheral tissues and transport it to the liver (see Folder 18.1).  glycoproteins, which include proteins involved in iron transport such as haptoglobin, transferrin, and hemopexin.  prothrombin and fbrinogen, important components of the blood-clotting cascade. nonimmune -globulins and -globulins, which also help maintain plasma colloid osmotic pressure and serve as carrier proteins for

1	and fbrinogen, important components of the blood-clotting cascade. nonimmune -globulins and -globulins, which also help maintain plasma colloid osmotic pressure and serve as carrier proteins for various substances (see Chapter 10, page 270).

1	The liver stores and converts several vitamins and iron. Several vitamins are taken up from the bloodstream and are then stored or biochemically modified by the liver. They include  vitamin A (retinol), an important vitamin in vision. Vitamin A is the precursor of retinal, which is required for the synthesis of rhodopsin in the eye. The liver plays a major role in the uptake, storage, and maintenance of circulating levels of vitamin A. When the vitamin A levels in the blood decrease, the liver mobilizes its storage sites in the hepatic stellate cells (see page 637). Vitamin A is then released into the circulation in the form of retinol bound to retinolbinding protein (RBP). The liver also synthesizes RBP; RBP synthesis is regulated by plasma levels of vitamin A. Night blindness and multiple skin disorders are related to vitamin A deficiency.

1	 vitamin D (cholecalciferol), an important vitamin in calcium and phosphate metabolism. Vitamin D is acquired from dietary vitamin D3 and is also produced in the skin during exposure to ultraviolet light by conversion of 7-dehydrocholesterol. Unlike vitamin A, vitamin D is not stored in the liver but is distributed to skeletal muscles and adipose tissue. The liver plays an important role in vitamin D metabolism by converting vitamin D3 to 25-hydroxycholecalciferol, the predominant form of circulating vitamin D. Further chapter 18 Digestive System III: Liver, Gallbladder, and Pancreas LIVE R 629

1	Lipoproteins are multicomponent complexes of proteins and lipids that are involved in the transport of cholesterol and triglycerides in the blood. Cholesterol and triglyc-erides do not circulate freely in the plasma because lipids, on their own, would be unable to remain in suspension. The association of the protein with the lipid-containing core makes the complex sufficiently hydrophilic to remain suspended in the plasma. Lipoproteins serve a variety of functions in cellular mem-branes and in the transport and metabolism of lipids. Lipoprotein precursors are produced in the liver. The lipid component is produced in the sER; the protein component is produced in the rER of the hepatocytes. The lipoprotein complexes pass to the Golgi, where secretory vesicles containing electron-dense lipoprotein particles bud off and are then released at the cell surface bordering the perisinu-soidal space to reach the bloodstream. Several hormones, such as estrogen and thyroid hormones, regulate the

1	particles bud off and are then released at the cell surface bordering the perisinu-soidal space to reach the bloodstream. Several hormones, such as estrogen and thyroid hormones, regulate the se-cretion of lipoproteins. In general, four classes of lipoproteins have been de-fined by their characteristic density, molecular weight, size, and chemical composition: chylomicrons, VLDLs, LDLs, and HDLs. These lipoproteins differ in chemical composi-tion and can be isolated from plasma according to their flotation properties, from largest and least dense to small-est and most dense. Chylomicrons, the lightest of all lipoproteins, are made only in the small intestine. Their main function is to transport the large amount of absorbed fat to the bloodstream. VLDLs are denser and smaller than chylomicrons; they are synthesized predominately in the liver and to a lesser extent in the small intestine. VLDLs are rich in triglycerides. Their function is to transport most of the triglycerides from the

1	they are synthesized predominately in the liver and to a lesser extent in the small intestine. VLDLs are rich in triglycerides. Their function is to transport most of the triglycerides from the liver to other organs. Liver VLDLs are associated with circulating apolipoprotein B-100, also synthesized in the liver, which aids in secretion of VLDLs. In congenital liver disease, such as abetalipoproteinemia, and to a lesser degree in acute and chronic disorders, the liver is unable to produce apolipoprotein B-100, leading to blockage in the secretion of VLDLs. In liver biopsy specimens from these individuals, large lipid droplets occupy most of the hepato-cyte cytoplasm. LDLs and HDLs are produced in the plasma; however, small amounts of these fractions are produced by the liver. LDLs are denser than VLDLs, and HDLs are denser than LDLs. The function of LDLs is to transport cholesterol es-ters from the liver to the peripheral organs. The HDLs are involved in the transport of cholesterol

1	than VLDLs, and HDLs are denser than LDLs. The function of LDLs is to transport cholesterol es-ters from the liver to the peripheral organs. The HDLs are involved in the transport of cholesterol from the peripheral tissues to the liver. High levels of LDL are directly corre-lated with increased risk of cardiovascular disease; high levels of HDL or low levels of LDL are associated with decreased risk.

1	 FOLDER 18.1 Clinical Correlation: Lipoproteins conversion takes place in the kidney to 1,25-hydroxycholecalciferol, which is 10 times more active than vitamin D3. Vitamin D is essential for development and growth of the skeletal system and teeth. Deficiency of vitamin D is associated with rickets and disorders of bone mineralization.  vitamin K, which is important in hepatic synthesis of prothrombin and several other clotting factors. Like vitamin D, it is derived from two sources: dietary vitamin K and synthesis in the small intestine by intestinal bacterial flora. Vitamin K is transported to the liver with chylomicrons, where it is rapidly absorbed, partially used, and then partially secreted with the VLDL fraction. Vitamin K deficiency is associated with hypoprothrombinemia and bleeding disorders.

1	In addition, the liver functions in the storage, meta bolism, and homeostasis of iron. It synthesizes almost all of the proteins involved in iron transport and metabolism, including transferrin, haptoglobin, and hemopexin. Transferrin is a plasma iron-transport protein. Haptoglobin binds to free hemoglobin in the plasma, from which the entire complex is removed by the liver to preserve iron. Hemopexin is involved in the transport of free heme in the blood. Iron is stored within the hepatocyte cytoplasm in the form of ferritin or may be converted to hemosiderin granules. Recent studies indicate that hepatocytes are the major sites of long-term storage of iron. Iron overload (as in multiple blood transfusions) may lead to hemochromatosis, a form of liver damage characterized by excessive amounts of hemosiderin in hepatocytes. The liver degrades drugs and toxins. Hepatocytes are involved in degradation of drugs, toxins, and other proteins foreign to the body (xenobiotics).

1	The liver degrades drugs and toxins. Hepatocytes are involved in degradation of drugs, toxins, and other proteins foreign to the body (xenobiotics). Many drugs and toxins are not hydrophilic; therefore, they cannot be eliminated effectively from the circulation by the kidneys. The liver converts these substances into more soluble forms. This process is performed by the hepatocytes in two phases:  Phase I (oxidation) includes hydroxylation (adding an OOH group) and carboxylation (adding a OCOOH group) to a foreign compound. This phase is performed in the hepatocyte smooth endoplasmic reticulum (sER) and mitochondria. It involves a series of biochemical reactions with proteins collectively named cytochrome P450.  Phase II (conjugation) includes conjugation with glucuronic acid, glycine, or taurine. This process makes the product of phase I even more water soluble so that it can be easily removed by the kidney. The liver is involved in many other important metabolic pathways.

1	The liver is important in carbohydrate metabolism as it maintains an adequate supply of nutrients for cell processes. In glucose metabolism, the liver phosphorylates absorbed glucose from the gastrointestinal tract to glucose-6phosphate. Depending on energy requirements, glucose-6-phosphate is either stored in the liver in the form of glycogen or used in the glycolytic pathways. During fasting, glycogen is broken down by the process of glycogenolysis, and glucose is released into the bloodstream. In addition, the liver functions in lipid metabolism. Fatty acids derived from plasma are consumed by hepatocytes using -oxidation to provide energy. The liver also produces ketone bodies that are used as a fuel by other organs (the liver cannot use them as an energy source). The involvement in cholesterol metabolism (synthesis and uptake from the blood) is also an important function of the liver. Cholesterol is used in the formation of bile salts, synthesis of VLDLs, and biosynthesis of

1	in cholesterol metabolism (synthesis and uptake from the blood) is also an important function of the liver. Cholesterol is used in the formation of bile salts, synthesis of VLDLs, and biosynthesis of organelles. The liver synthesizes most of the urea in the body from ammonium ions derived from protein and nucleic acid degradation. Finally, the liver is involved in the synthesis and conversion of nonessential amino acids.

1	Bile production is an exocrine function of the liver. The liver is engaged in numerous metabolic conversions involving substrates delivered by blood from the digestive tract, pancreas, and spleen. Some of these products are involved in the production of bile, an exocrine secretion of the liver. Bile contains conjugated and degraded waste products that are returned to the intestine for disposal, as well as substances that bind to metabolites in the intestine to aid in absorption (Table 18.1). Bile is carried from the parenchyma of the liver by bile ducts that fuse to form the hepatic duct. The cystic duct then carries the bile into the gallbladder, where it is concentrated. Bile is returned, via the cystic duct, to the common bile duct, which delivers bile from the liver and gallbladder to the duodenum (see Fig. 18.15). The endocrine-like functions of the liver are represented by its ability to modify the structure and function of many hormones.

1	The endocrine-like functions of the liver are represented by its ability to modify the structure and function of many hormones. The liver modifes the action of hormones released by other organs. The liver’s endocrine-like actions involve  vitamin D, which is converted by the liver to 25hydroxycholecalciferol, the predominant form of circulating vitamin D (page 629).  thyroxine, a hormone secreted by the thyroid gland as tetraiodothyronine (T4), which is converted in the liver to the biologically active form, triiodothyronine (T3), by deiodination.  growth hormone (GH), a hormone secreted by the pituitary gland. The action of GH is modified by liver-produced growth hormone–releasing hormone (GHRH) and inhibited by somatostatin, which is secreted by enteroendocrine cells of the gastrointestinal tract.  insulin and glucagon, both pancreatic hormones. These hormones are degraded in many organs, but the liver and kidney are the most important sites of their degradation.

1	 insulin and glucagon, both pancreatic hormones. These hormones are degraded in many organs, but the liver and kidney are the most important sites of their degradation. Blood Supply to the Liver To appreciate the myriad functions of the liver introduced previously, one must first understand its unique blood supply and how blood is distributed to the hepatocytes. The liver has a dual blood supply consisting of a venous (portal) supply via the hepatic portal vein and an arterial supply via the hepatic artery. Both vessels enter the liver at a hilum or porta hepatis, the same site at which the common bile duct, carrying the bile secreted by the liver, and the lymphatic vessels leave the liver. Therefore, bile flows in a direction opposite to that of the blood. The liver receives the blood that initially supplied the intestines, pancreas, and spleen.

1	The liver receives the blood that initially supplied the intestines, pancreas, and spleen. The liver is unique among organs because it receives its major blood supply (about 75%) from the hepatic portal vein, which carries venous blood that is largely depleted of oxygen.

1	631 TABLE Composition of Bile18.1 Component Function Water Serves as solute in which other components are carried Phospholipids (i.e., lecithin) and cholesterol Are metabolic substrates for other cells in the body; act as precursors of membrane components and steroids; largely reabsorbed in the gut and recycled Bile salts (also called bile acids): primary (secreted by liver): cholic acid, chenodeoxycholic acid; secondary (converted by bacterial flora in the intestine): deoxycholic acid, lithocholic acid Act as emulsifying agents that aid in the digestion and absorption of lipids from the gut and help to keep the cholesterol and phospholipids of the bile in solution, largely recycled, going back and forth between the liver and gut Bile pigments, principally the glucuronide of the bilirubin produced in the spleen, bone marrow, and liver by the breakdown of hemoglobin Detoxify bilirubin, the end product of hemoglobin degradation, and carry it to the gut for disposal Electrolytes: Na,

1	produced in the spleen, bone marrow, and liver by the breakdown of hemoglobin Detoxify bilirubin, the end product of hemoglobin degradation, and carry it to the gut for disposal Electrolytes: Na, K, Ca2, Mg2, Cl, and HCO3 Establish and maintain bile as an isotonic fluid; also largely reabsorbed in the gut

1	Digestive System III: Liver, Gallbladder, and Pancreas LIVER The blood delivered to the liver by the hepatic portal vein comes from the digestive tract and the major abdominal organs, such as the pancreas and spleen. The portal blood carried to the liver contains  nutrients and toxic materials absorbed in the intestine.  blood cells and breakdown products of blood cells from the spleen. endocrine secretions of the pancreas and enteroendocrine cells of the gastrointestinal tract. Thus, the liver stands directly in the pathway of blood vessels that convey substances absorbed from the digestive tract. Although the liver is the first organ to receive metabolic substrates and nutrients, it is also the first exposed to toxic substances that have been absorbed.

1	The hepatic artery, a branch of the celiac trunk, carries oxygenated blood to the liver, providing the remaining 25% of its blood supply. Because blood from the two sources mixes just before it perfuses the hepatocytes of the liver parenchyma, the liver cells are never exposed to fully oxygenated blood. Within the liver, the distributing branches of the portal vein and hepatic artery, which supply the sinusoidal capillaries (sinusoids) that bathe the hepatocytes, and the draining branches of the bile duct system, which lead to the common hepatic duct, course together in a relationship termed the portal triad. Although a convenient term, it is a misnomer because one or more vessels of the lymphatic drainage system of the liver always travel with the vein, artery, and bile duct (Fig. 18.2).

1	The sinusoids are in intimate contact with the hepatocytes and provide for the exchange of substances between the blood and liver cells. The sinusoids lead to a terminal hepatic venule (central vein) that in turn empties into the sublobular veins. Blood leaves the liver through the hepatic veins, which empty into the inferior vena cava. Structural Organization of the Liver As introduced previously, the structural components of the liver include  parenchyma, consisting of organized plates of hepatocytes, which in the adult are normally one cell thick and are separated by sinusoidal capillaries. In young individuals up to 6 years of age, the liver cells are arranged in plates two cells thick.  connective tissue stroma that is continuous with the fibrous capsule of Glisson. Blood vessels, nerves, lymphatic vessels, and bile ducts travel within the connective tissue stroma.

1	 connective tissue stroma that is continuous with the fibrous capsule of Glisson. Blood vessels, nerves, lymphatic vessels, and bile ducts travel within the connective tissue stroma.  sinusoidal capillaries (sinusoids), the vascular channels between the plates of hepatocytes.  perisinusoidal spaces (spaces of Disse), which lie between the sinusoidal endothelium and the hepatocytes. With this information as background, one can now consider several ways to describe the organization of these structural elements to understand the major functions of the liver. periportal space (space of Mall) capillary terminal branch of hepatic artery terminal branch of portal vein portal canal

1	FIGURE 18.2 • Blood supply to the liver: the portal triad. The portal triad is composed of the branches of the hepatic artery, portal vein, and bile duct. Blood from the terminal branches of the hepatic artery and portal vein enters the hepatic sinusoids. The mixture of venous and arterial blood is transported by the sinusoids toward the terminal hepatic venule (central vein). From here, blood drains into the sublobular veins, the tributaries of the hepatic vein. Note the small vessels and capillary network in the perivascular connective tissue surrounding each hepatic triad within the portal canal. Also note the periportal space of Mall, located between the portal canal and the outermost hepatocytes. This space is also filled with a small amount of connective tissue in which lymph drainage begins. From here, blind-ended lymphatic capillaries form larger lymphatic vessels that accompany branches of the hepatic artery.

1	There are three ways to describe the structure of the liver in terms of a functional unit: the classic lobule, the portal lobule, and the liver acinus. The classic lobule is the traditional way to describe the organization of the liver parenchyma, and it is relatively easy to visualize. It is based on the distribution of the branches of the portal vein and hepatic artery within the organ and the pathway that blood from them follows as it ultimately perfuses the liver cells. The classic hepatic lobule is a roughly hexagonal mass of tissue.

1	The classic hepatic lobule is a roughly hexagonal mass of tissue. The classic lobule (Fig. 18.3 and Plate 65, page 656) consists of stacks of anastomosing plates of hepatocytes, one cell thick, separated by the anastomosing system of sinusoids that perfuse the cells with the mixed portal and arterial blood. Each lobule measures about 2.0 0.7 mm. At the center of the lobule is a relatively large venule, the terminal hepatic venule (central vein), into which the sinusoids drain. The plates of cells radiate from the central vein to the periphery of the lobule, as do the sinusoids. At the angles of the hexagon are the portal areas (portal canals), loose stromal connective tissue characterized by the presence of the portal triads. This connective tissue is ultimately continuous with the fibrous capsule of the liver. The portal canal is bordered by the outermost hepatocytes of the

1	FIGURE 18.3 • Diagram of a classic liver lobule. A classic liver lobule can be schematically diagramed as a six-sided polyhedral prism with portal triads (hepatic artery, portal vein, and bile duct) at each of the corners. The blood vessels of the portal triads send distributing branches along the sides of the lobule, and these branches open into the hepatic sinusoids. The long axis of the lobule is traversed by the terminal hepatic venule (central vein), which receives blood from the hepatic sinusoids. Note that a wedge of the tissue has been removed from the lobule for better visualization of the terminal hepatic venule. Interconnecting sheets or plates of hepatocytes are disposed in a radial pattern from the terminal hepatic venule to the periphery of the lobule.

1	lobule. At the edges of the portal canal, between the connective tissue stroma and the hepatocytes, is a small space called the periportal space (space of Mall). This space is thought to be one of the sites where lymph originates in the liver. In some species (e.g., the pig; Fig. 18.4a), the classic lobule is easily recognized because the portal areas are connected by relatively thick layers of connective tissue. In humans, however, there is normally very little interlobular connective tissue, and it is necessary, when examining histologic sections of liver, to draw imaginary lines between portal areas surrounding a central vein to get some sense of the size of the classic lobule (Fig. 18.4b). The portal lobule emphasizes the exocrine functions of the liver.

1	The portal lobule emphasizes the exocrine functions of the liver. The major exocrine function of the liver is bile secretion. Thus, the morphologic axis of the portal lobule is the interlobular bile duct of the portal triad of the classic lobule. Its outer margins are imaginary lines drawn between the three central veins that are closest to that portal triad (Fig. 18.5). These lines define a roughly triangular block of tissue that includes those portions of three classic lobules that secrete the bile that drains into its axial bile duct. This concept allows a description of hepatic parenchymal structure comparable to that of other exocrine glands. The liver acinus is the structural unit that provides the best correlation between blood perfusion, metabolic activity, and liver pathology.

1	The liver acinus is the structural unit that provides the best correlation between blood perfusion, metabolic activity, and liver pathology. The liver acinus is lozenge shaped and represents the smallest functional unit of the hepatic parenchyma. The short axis of the acinus is defined by the terminal branches of the portal triad that lie along the border between two classic lobules. The long axis of the acinus is a line drawn between the two central veins closest to the short axis. Therefore, in a two-dimensional view (Fig. 18.6) the liver acinus occupies parts of adjacent classic lobules. This concept allows a description of the exocrine secretory function of the liver comparable to that of the portal lobule.

1	The hepatocytes in each liver acinus are described as being arranged in three concentric elliptical zones surrounding the short axis (see Fig. 18.6).  Zone 1 is closest to the short axis and the blood supply from penetrating branches of the portal vein and hepatic artery. This zone corresponds to the periphery of the clas sic lobules.  Zone 3 is farthest from the short axis and closest to the ter minal hepatic vein (central vein). This zone corresponds to the most central part of the classic lobule that surrounds the terminal hepatic vein.  Zone 2 lies between zones 1 and 3 but has no sharp boundaries.

1	The zonation is important in the description and interpretation of patterns of degeneration, regeneration, and specific toxic effects in the liver parenchyma relative to the degree or quality of vascular perfusion of the hepatic cells. As a result of the sinusoidal blood ﬂow, the oxygen gradient, metabolic activity of the hepatocytes, and distribution of hepatic enzymes vary chapter 18 Digestive System III: Liver, Gallbladder, and Pancreas LIVE R 633

1	FIGURE 18.4 • Photomicrographs of pig and human livers. a. This photomicrograph shows a cross section of a pig liver lobule stained by the Mallory-Azan method to visualize connective tissue components. Note the relatively thick interlobular connective tissue (stained blue) surrounding the lobules. The terminal hepatic venule (central vein) is visible in the center of the lobule. 65. b. Photomicrograph of a human liver from a routine H&E preparation. Note that in contrast to the pig liver, the lobules of the human liver lack connective tissue septa. The plates of hepatocytes of one lobule merge with those of adjacent lobules. The boundaries of a lobule can be approximated, however, by drawing a line (dashed line) from one portal canal to the next, thus circumscribing the lobule. 65.

1	across the three zones. The distribution of liver damage result-are the first to show ischemic necrosis (centrilobular necrosis) ing from ischemia and exposure to toxic substances can be ex-in situations of reduced perfusion and the first to show fat ac-plained using this zonal interpretation. cumulation. They are the last to respond to toxic substances Cells in zone 1 are the first to receive oxygen, nutrients, and bile stasis. Normal variations in enzyme activity, the and toxins from the sinusoidal blood and the first to show number and size of cytoplasmic organelles, and the size of cymorphologic changes after bile duct occlusion (bile stasis). toplasmic glycogen deposits are also seen between zones 1 These cells are also the last to die if circulation is impaired and 3. Cells in zone 2 have functional and morphologic char-and the first to regenerate. On the other hand, cells in zone 3 acteristics and responses intermediate to those of zones 1 and 3.

1	FIGURE 18.5 • Comparison of the classic liver lobule, portal lobule, and liver acinus. The area indicated in blue shows the territory of each of the three units relating to liver organization and function. The classic lobule has the terminal hepatic venule (central vein) at the center of the lobule and the portal canals containing portal triads at the peripheral angles of the lobule. The portal lobule has a portal canal at the center of the lobule and terminal hepatic venules (central veins) at the peripheral angles of the lobule. The liver acinus has distributing vessels at the equator and terminal hepatic venules (central veins) at each pole.

1	FIGURE 18.6 • The liver acinus. The liver acinus is a functional interpretation of liver organization. It consists of adjacent sectors of neighboring hexagonal fields of classic lobules partially separated by distributing blood vessels. The zones, marked 1, 2, and 3, are supplied with blood that is most oxygenated and richest in nutrients in zone 1 and least so in zone 3. The terminal hepatic venules (central veins) in this interpretation are at the edges of the acinus instead of in the center, as in the classic lobule. The vessels of the portal canals, namely, terminal branches of the portal vein and hepatic artery that, along with the smallest bile ducts, make up the portal triad, are shown at the corners of the hexagon that outlines the cross-sectioned profile of the classic lobule.  FOLDER 18.2 Clinical Correlation: Congestive Heart Failure and Liver Necrosis

1	Liver injury may be triggered by hemodynamic changes in the circulatory system. In congestive heart failure, the heart is unable to provide sufficient oxygenated blood to meet the metabolic requirements of many tissues and organs, including the liver, which is readily affected by hypoperfusion and hypoxia (low blood oxygen content). Zone 3 of the liver acinus is the first to be affected by this condition. The hepatocytes in this zone are the last to receive blood as it passes along the sinusoids; as a result, these cells receive a blood supply already depleted in oxygen. Examination of a liver biopsy specimen from an individual with congestive heart failure shows a distinct pattern of liver necrosis. Hepatocytes in zone 3, which is located around the central vein, undergo ischemic necrosis. Typically, no noticeable changes are seen in zones 1 and 2, representing the periphery of a classic lobule. Necrosis of this type is referred to as centrilobular necrosis. Figure F18.2.1 shows the

1	Typically, no noticeable changes are seen in zones 1 and 2, representing the periphery of a classic lobule. Necrosis of this type is referred to as centrilobular necrosis. Figure F18.2.1 shows the centrilobular portion of a classic lobule. The multiple round vacuoles indicate lipid accumulation, and the atrophic changes are the result of dying hepatocytes undergoing autophagocytosis. Centrilobular necrosis as a result of hypoxia is referred to as cardiac cirrhosis; however, unlike true cirrhosis, nodular regeneration of hepatocytes is minimal.

1	FIGURE F18.2.1 • Photomicrograph of centrilobular necrosis in human liver. This photomicrograph shows a routine H&E liver biopsy specimen from an individual with congestive heart failure. Pathologic changes (referred to as ischemic necrosis) are most severe in hepatocytes in zone 3. This zone surrounds the terminal hepatic venule (central vein). This type of necrosis is referred to as centrilobular necrosis. Note the presence of multiple round vacuoles, which indicates extensive lipid accumulation. No noticeable changes are seen in the periphery of the lobule, that is zone 1 and much of zone 2. 320. Digestive System III: Liver, Gallbladder, and Pancreas LIVER Blood Vessels of the Parenchyma

1	Digestive System III: Liver, Gallbladder, and Pancreas LIVER Blood Vessels of the Parenchyma The blood vessels that occupy the portal canals are called interlobular vessels. Only the interlobular vessels that form the smallest portal triads send blood into the sinusoids. The larger interlobular vessels branch into distributing vessels that are located at the periphery of the lobule. These distributing vessels send inlet vessels to the sinusoids (Fig. 18.7). In the sinusoids, the blood flows centripetally toward the central vein. The central vein courses through the central axis of the classic liver lobule, becoming larger as it progresses through the lobule and empties into a sublobular vein. Several sublobular veins converge to form larger hepatic veins that empty into the inferior vena cava.

1	The structure of the portal vein and its branches within the liver is typical of veins in general. The lumen is much larger than that of the artery associated with it. The structure of the hepatic artery is like that of other arteries (i.e., it has a thick muscular wall). In addition to providing arterial blood directly to the sinusoids, the hepatic artery provides arterial blood to the connective tissue and other structures in the larger portal canals. Capillaries in these larger portal canals return the blood to the interlobular veins before they empty into the sinusoid.

1	The central vein is a thin-walled vessel receiving blood from the hepatic sinusoids. The endothelial lining of the central vein is surrounded by small amounts of spirally arranged connective tissue fibers. The central vein, so named because of its central position in the classic lobule, is actually the terminal venule of the system of hepatic veins and, thus, is more properly called the terminal hepatic venule. The sublobular FIGURE 18.7 • Diagram of the fow of blood and bile in the liver. This schematic diagram of a part of a classic lobule shows the components of the portal triads, hepatic sinuses, terminal hepatic venule (central vein), and associated plates of hepatocytes. Red arrows indicate the direction of the blood flow in the sinusoids. Note that the direction of bile flow (green arrows) is opposite that of the blood flow.

1	vein, the vessel that receives blood from the terminal hepatic venules, has a distinct layer of connective tissue fibers, both collagenous and elastic, just external to the endothelium. The sublobular veins and the hepatic veins, into which they drain, travel alone. Because they are solitary vessels, they can be readily distinguished in a histologic section from the portal veins that are members of a triad. There are no valves in hepatic veins. Hepatic sinusoids are lined with a thin discontinuous endothelium. The discontinuous sinusoidal endothelium has a discontinuous basal lamina that is absent over large areas. The discontinuity of the endothelium is evident in two ways:  Large fenestrae, without diaphragms, are present within the endothelial cells.  Large gaps are present between neighboring endothelial cells.

1	Hepatic sinusoids differ from other sinusoids in that a second cell type, the stellate sinusoidal macrophage, or Kupffer cell (Fig. 18.8 and Plate 66, page 658), is a regular part of the vessel lining. Kupffer cells belong to the mononuclear phagocytotic system. Like other members of the mononuclear phagocytotic system, Kupffer cells are derived from monocytes. The scanning electron microscope (SEM) and transmission electron microscope (TEM) clearly show that the Kupffer cells form part of the lining of the sinusoid. Previously, they had been described as lying on the luminal surface of the endothelial cells. This older histologic description was probably based on the fact that processes of the Kupffer cells occasionally overlap endothelial processes on the luminal side. Kupffer cells do not form junctions with neighboring endothelial cells.

1	Processes of Kupffer cells often seem to span the sinusoidal lumen and may even partially occlude it. The presence of red cell fragments and iron in the form of ferritin in the cytoplasm of Kupffer cells suggests that they may be involved in the final breakdown of some damaged or senile red blood cells that reach the liver from the spleen. Some of the ferritin iron may be converted to hemosiderin granules and stored in the cells. This function is greatly increased after splenectomy when it is then essential for red blood cell disposal. Perisinusoidal Space (Space of Disse) The perisinusoidal space is the site of exchange of materials between blood and liver cells. The perisinusoidal space (space of Disse) lies between the basal surfaces of hepatocytes and the basal surfaces of endothelial cells and Kupffer cells that line the sinusoids. Small, irregular microvilli project into this space from the basal surface of the hepatocytes (Fig. 18.9).

1	The microvilli increase the surface area available for exchange of materials between hepatocytes and plasma by as much as six times. Because of the large gaps in the endothelial layer and the absence of a continuous basal lamina, no FIGURE 18.8 • Electron micrograph of two hepatic sinusoids of the liver. One hepatic sinusoid (top) displays a stellate sinusoidal macrophage (Kupffer cell). The remainder of the sinusoid as well as the other sinusoid is lined by thin endothelial cell cytoplasm. Surrounding each sinusoid is the perisinusoidal space (space of Disse), which contains numerous hepatocyte microvilli. Also present in the perisinusoidal space is a hepatic stellate cell (Ito cell) with a large lipid droplet and several smaller droplets. Its nucleus conforms to the curve of the lipid droplet. 6,600.

1	significant barrier exists between the blood plasma in the sinusoid and the hepatocyte plasma membrane. Proteins and lipoproteins synthesized by the hepatocyte are transferred into the blood in the perisinusoidal space; this pathway is for liver secretions other than bile. In the fetal liver, the space between blood vessels and hepatocytes contains islands of blood-forming cells. In cases of chronic anemia in the adult, blood-forming cells may again appear in the perisinusoidal space.

1	FIGURE 18.9 • Electron micrograph showing the perisinusoidal space (of Disse). The perisinusoidal space (D) is located between the hepatocytes (H) and the sinusoid. A gap (large arrow) separates the endothelial cells (En) that line the sinusoid. Such gaps allow easy passage of small substances between the sinusoid and the perisinusoidal space. Numerous microvilli extend from the hepatocytes into the perisinusoidal space. These processes are long and frequently branch (small arrow). A red blood cell (RBC) is within the sinusoid. 18,000. The hepatic stellate cells (Ito cells) store vitamin A; however, in pathologic conditions, they differentiate into myofibroblasts and synthesize collagen.

1	The hepatic stellate cells (Ito cells) store vitamin A; however, in pathologic conditions, they differentiate into myofibroblasts and synthesize collagen. The other cell type found in the perisinusoidal space is the hepatic stellate cell (commonly called an Ito cell). These cells of mesenchymal origin are the primary storage site for hepatic vitamin A in the form of retinyl esters within cytoplasmic lipid droplets. Vitamin A is released from the hepatic stellate cell as retinol (alcohol form) bound to retinol chapter 18 Digestive System III: Liver, Gallbladder, and Pancreas LIVE R 637 binding protein (RBP). It is then transported from the liver to the retina, where its stereoisomer 11-cis retinal binds to the protein opsin to form rhodopsin, the visual pigment of rods and cones of the retina. For many years, fish liver oils (e.g., cod liver oil) were medically and economically important nutritional sources of vitamin A.

1	In certain pathologic conditions, such as chronic inﬂammation or liver cirrhosis, hepatic stellate cells lose their lipid and vitamin A storage capability and differentiate into cells with characteristics of myofibroblasts. These cells appear to play a significant role in hepatic fibrogenesis; they synthesize and deposit type I and type III collagen within the perisinusoidal space, resulting in liver fibrosis. This collagen is continuous with the connective tissue of the portal space and the connective tissue surrounding the central vein. An increased amount of perisinusoidal fibrous stroma is an early sign of liver response to toxic substances. The cytoplasm of hepatic stellate cells contains contractile elements, such as desmin and smooth muscle -actin filaments. During cell contraction, they increase the vascular resistance within the sinusoids by constricting the vascular channels, leading to portal hypertension. In addition, hepatic stellate cells play a role in remodeling the

1	they increase the vascular resistance within the sinusoids by constricting the vascular channels, leading to portal hypertension. In addition, hepatic stellate cells play a role in remodeling the extracellular matrix during recovery from liver injury.

1	Hepatic lymph originates in the perisinusoidal space. Plasma that remains in the perisinusoidal space drains into the periportal connective tissue, where a small space, the periportal space (space of Mall) (see Fig. 18.10b), is described between the stroma of the portal canal and the outermost hepatocytes. From this collecting site, the fluid then enters lymphatic capillaries that travel with the other components of the portal triad. The lymph moves in progressively larger vessels, in the same direction as the bile (i.e., from the level of the hepatocytes, toward the portal canals and eventually to the

1	FIGURE 18.10 • Canals of Hering and the intrahepatic ductile. a. Photomicrograph showing an area near a portal canal. Arrows indicate regions where bile canaliculi are draining into canals of Hering. Note that the canal of Hering is partially lined by hepatocytes and partially by cholangiocytes. It drains into intrahepatic bile ductule surrounded by hepatocytes, in contrast to the interlobular bile duct, which is embedded in the connective tissue of the portal canal. The terminal branch of a portal vein (lower right) accompanied by a small bile ductule are evident.800. b. Electron micrograph showing an intrahepatic bile ductule. The ductule collects bile from the canals of Hering. It is close to the hepatocytes, but the actual connection between bile canaliculi and the intrahepatic ductule is not evident in this plane of section. The ductule is composed of cholangiocytes (CH) surrounded by a complete basal lamina (BL). The narrow space (asterisks) into which microvilli of hepatocytes

1	is not evident in this plane of section. The ductule is composed of cholangiocytes (CH) surrounded by a complete basal lamina (BL). The narrow space (asterisks) into which microvilli of hepatocytes project is the periportal space (of Mall), not the perisinusoidal space (of Disse). 6,000.

1	chapter 18 Digestive System III: Liver, Gallbladder, and Pancreas LIVE R 639 hilum of the liver). About 80% of the hepatic lymph follows this pathway and drains into the thoracic duct, forming the major portion of the thoracic duct lymph. Hepatocytes make up the anastomosing cell plates of the liver lobule. Hepatocytes are large, polygonal cells measuring between 20 and 30 m in each dimension. They constitute about 80% of the cell population of the liver. Hepatocyte nuclei are large and spherical and occupy the center of the cell. Many cells in the adult liver are binucleate; most cells in the adult liver are tetraploid (i.e., they contain the 4d amount of DNA). Heterochromatin is present as scattered clumps in the nucleoplasm and as a distinct band under the nuclear envelope. Two or more well-developed nucleoli are present in each nucleus.

1	Hepatocytes are relatively long-lived for cells associated with the digestive system; their average lifespan is about 5 months. In addition, liver cells are capable of considerable regeneration when liver substance is lost to hepatotoxic processes, disease, or surgery. The hepatocyte cytoplasm is generally acidophilic. Specific cytoplasmic components may be identified by routine and special staining procedures, including  basophilic regions that represent rough endoplasmic retic ulum (rER) and free ribosomes.  numerous mitochondria; as many as 800 to 1,000 mito enzyme histochemistry.  multiple small Golgi complexes seen in each cell after specific staining. large numbers of peroxisomes demonstrated by immunocytochemistry.  deposits of glycogen stained by means of the periodic acid–Schiff (PAS) procedure. However, in a well-preserved hematoxylin and eosin (H&E) preparation, glycogen is also visible as irregular spaces, usually giving a fine foamy appearance to the cytoplasm.

1	 lipid droplets of various sizes seen after appropriate fixation and Sudan or toluidine blue staining (Plate 66, page 658). In routinely prepared histologic sections, round spaces are sometimes seen that represent dissolved lipid droplets. The number of lipid droplets increases after injection or ingestion of certain hepatotoxins, including ethanol.  lipofuscin pigment within lysosomes seen with routine H&E staining in various amounts. Well-delineated brown granules can also be visualized by the PAS method.

1	 lipofuscin pigment within lysosomes seen with routine H&E staining in various amounts. Well-delineated brown granules can also be visualized by the PAS method. As noted previously, the hepatocyte is polyhedral; for convenience, it is described as having six surfaces, although there may be more. A schematic section of a cuboidal hepatocyte is shown in Figure 18.11. Two of its surfaces face the perisinusoidal space. The plasma membrane of two surfaces faces a neighbo ring hepatocyte and a bile canaliculus. Assuming that the cell is cuboidal, the remaining two surfaces, which cannot be seen in the diagram, would also face neighboring cells and bile canaliculi. The surfaces that face the perisinusoidal space correspond to the basal surface of other epithelial cells; the surfaces that face neighboring cells and bile canaliculi correspond to the late ral and apical surfaces, respectively, of other epithelial cells.

1	FIGURE 18.11 • Schematic diagram of a plate of hepatocytes interposed between hepatic sinusoids. This diagram shows a one-cell-thick plate of hepatocytes interposed between two sinusoids. If it is assumed that the cell is cuboidal, two sides of each cell (shown) would face hepatic sinusoids, two sides of each cell (shown) would face bile canaliculi, and the additional two sides (not shown) would face bile canaliculi. Note the location and features of a hepatic stellate cell (Ito cell) filled with cytoplasmic vacuoles containing vitamin A. The sparse collagen fibers found in the perisinusoidal space (of Disse) are produced by the hepatic stellate cells (Ito cells). In certain pathologic conditions, these cells lose their storage vacuoles and differentiate into myofibroblasts that produce collagen fibers, leading to liver fibrosis. Observe that the stellate sinusoidal macrophage (Kupffer cell) forms an integral part of the sinusoidal lining.

1	FIGURE 18.12 • Electron micrographs of a hepatocyte. a. This electron micrograph shows organelles and other cytoplasmic structures near the nucleus (N). These include a peroxisome (P), mitochondrion (M), glycogen inclusions (Gl), smooth endoplasmic reticulum (sER), and rough endoplasmic reticulum (rER). In the lower left, the membranes of the rER have been cut in a tangential plane showing the ribosomes (encircled by a dashed line) on the cytoplasmic face of the membrane. 12,000. b. This micrograph shows a region of cytoplasm near a bile canaliculus (C). It includes a lysosome (L), mitochondria (M), and both sER and rER. Note the microvilli in the bile canaliculus. 18,000. Peroxisomes are numerous in hepatocytes.

1	Peroxisomes are numerous in hepatocytes. Hepatocytes have as many as 200 to 300 peroxisomes per cell. They are relatively large and vary in diameter from 0.2 to 1.0 m (see Fig. 18.12a). Peroxisomes are a major site of oxygen use and in this way perform a function similar to that of mitochondria. They contain a large amount of oxidase that generates toxic hydrogen peroxide, H2O2. The enzyme catalase, also residing within peroxisomes, degrades hydrogen peroxide to oxygen and water. These types of reactions are involved in many detoxification processes occurring in the liver (e.g., detoxification of alcohol). In fact, about one half of the ethanol that is ingested is converted to acetaldehyde by enzymes contained in liver peroxisomes. In humans, catalase and D-amino acid oxidase, as well as alcohol dehydrogenase, are found in peroxisomes. In addition, peroxisomes are also involved in breakdown of fatty acids ( oxidation) as well as gluconeogenesis and metabolism of purines.

1	sER can be extensive in hepatocytes. The sER in hepatocytes may be extensive but varies with metabolic activity (see Fig. 18.12b). The sER contains enzymes involved in degradation and conjugation of toxins and drugs as well as enzymes responsible for synthesizing cholesterol and the lipid portion of lipoproteins. Under conditions of hepatocyte challenged by drugs, toxins, or metabolic stimulants, the sER may become the predominant organelle in the cell. In addition to stimulating sER activity, certain drugs and hormones induce synthesis of new sER membranes and their associated enzymes. The sER undergoes hypertrophy after administration of alcohol, drugs (i.e., phenobarbital, anabolic steroids, and progesterone), and certain chemotherapeutic agents used to treat cancer.

1	Stimulation of the sER by ethanol enhances its ability to detoxify other drugs, certain carcinogens, and some pesticides. On the other hand, metabolism by the sER can actually increase the hepatocyte-damaging effects of some toxic compounds, such as carbon tetrachloride (CCl4) and 3, 4-benzpyrene. The large Golgi apparatus in hepatocytes consists of as many as 50 Golgi units.

1	Examination of hepatocytes with the TEM shows the Golgi apparatus to be much more elaborate than those seen in routine histologic specimens. Heavy-metal preparations (Golgi stains) of thick sections of liver give an indication of the extent of the Golgi network. As many as 50 Golgi units, each consisting of three to five closely stacked cisternae, plus many large and small vesicles, are found in hepatocytes. These “units” are actually branches of the tortuous Golgi apparatus seen in heavy-metal preparations. Elements of the Golgi apparatus concentrated near the bile canaliculus are believed to be associated with the exocrine secretion of bile. Golgi cisternae and vesicles near the sinusoidal surfaces of the cell, however, contain electron-dense granules 25 to 80 nm in diameter that are believed to be VLDL and other lipoprotein precursors. These substances are subsequently released into the circulation as part of the endocrine secretory function of the hepatocytes. Similar dense

1	are believed to be VLDL and other lipoprotein precursors. These substances are subsequently released into the circulation as part of the endocrine secretory function of the hepatocytes. Similar dense globules are seen in dilated portions of the sER and, occasionally, in the dilated ends of rER cisternae, where they are synthesized.

1	Lysosomes concentrated near the bile canaliculus correspond to the peribiliary dense bodies seen in histologic sections. Hepatocyte lysosomes are so heterogeneous that they can only be positively identified, even at the TEM level, by histochemical means. In addition to normal lysosomal enzymes, TEM reveals other components: Hepatocyte lysosomes may also be a normal storage site for iron (as a ferritin complex) and a site of iron accumulation in certain storage diseases. FIGURE 18.13 • Scanning electron micrograph of the luminal surface of the bile duct. Bile duct is lined by epithelial lining cells called cholangiocytes. Their apical surfaces exhibit numerous short microvili projecting into the lumen of the bile duct. Each cholangiocyte possesses a long primary cilium which sense changes in luminal flow of the bile. Note that all cilia are bent in the same direction of a bile flow. 3,600. (Courtesy of Dr. Tetyana V. Masyuk.)

1	The number of lysosomes increases in a variety of pathologic conditions, ranging from simple obstructive bile stasis to viral hepatitis and anemia. However, although the range of normal liver function—particularly the rate of bile secretion—is quite wide, no statistically significant morphologic changes take place in the Golgi apparatus or lysosomes of the peribiliary cytoplasm to correlate with the rate of bile secretion. The biliary tree is the three-dimensional system of channels of increasing diameter that bile flows through from the hepatocytes to the gallbladder and then to the intestine. In the adult human liver, there are more than 2 kilometers of interconnecting bile ductules and ducts of different sizes and shapes. These structures are not only passive conduits, but they are also capable of modifying bile flow and changing its composition in response to hormonal and neural stimulation.

1	The biliary tree is lined by cholangiocytes which monitor bile flow and regulate its content. Cholangiocytes are epithelial cells that line the biliary tree. When examined in TEM, cholangiocytes are identified by their organelle-scant cytoplasm, presence of tight junctions between adjacent cells, and presence of complete basal lamina. An apical domain of cholangiocytes appears similar to hepatocytes with microvilli projecting into the lumen. In addition, each cholangiocyte contains primary cilium that sense changes in lumenal flow resulting in alterations of cholangiocytes secretion (Fig. 18.13). Small bile ductules are lined by small cholangiocytes, mainly cuboidal in shape, but as the diameter of the bile duct increases, they become progressively larger and more columnar in shape. The bile canaliculus is a small canal formed by apposed grooves in the surface of adjacent hepatocytes.

1	The smallest branches of the biliary tree are the bile canaliculi into which the hepatocytes secrete bile. They form a complete loop around four sides of the idealized six-sided hepatocytes (Fig. 18.14 and Plate 66, page 658). They are approximately 0.5 m in luminal diameter and are isolated from the rest of the intercellular compartment by tight junctions, which are part of junctional complexes that also include zonulae adherentes and desmosomes. Microvilli of the two adjacent hepatocytes extend into the canalicular lumen. Adenosine triphosphatase (ATPase) and other alkaline phosphatases can be localized on the plasma membranes of the canaliculi, suggesting that bile secretion into this space is an active process. Bile flow is centrifugal, that is from the region of the terminal hepatic venule (central vein) toward the portal canal (a direction opposite to the blood flow). Near the portal canal but still within the lobule, bile canaliculi transform into the short canals of Hering.

1	A characteristic feature of the canal of Hering is its lining made of two types of cells, hepatocytes and cholangiocytes. chapter 18 Digestive System III: Liver, Gallbladder, and Pancreas LIVE R 641 FIGURE 18.14 • Photomicrograph of bile canaliculi. This high-magnification photomicrograph shows several one-cell-thick plates of hepatocytes separated by hepatic sinusoids. The plane of section in certain areas is parallel to the bile canaliculi. In this plane, the canaliculi reveal their arrangement on four sides of the hepatocytes (arrows). Arrowheads indicate those bile canaliculi that appear only in cross-sectional profile. 1,240.

1	The canal of Hering is a channel partially lined by hepatocytes and partially by cuboidal shaped cholangiocytes. Similar to other cholangiocytes, hepatocytes possess microvilli at their apical surface and tight junctions, and their basal domain is rested on basal lamina, as is the rest of the distal biliary epithelium. Functionally, as demonstrated by video-microscopy, the canal of Hering exhibits contractile activity that assists with unidirectional bile flow toward the portal canal. Because the canal of Hering represents the smallest and most proximal tributary of the biliary tree containing cholangiocytes, it often is involved in the same diseases that affect small bile ducts. Functional disturbance in contractile activity as well as injury or destruction of the canals of Hering may contribute to intrahepatic cholestasis (obstruction of the bile ﬂow). Canal of Hering serves as a reservoir of liver progenitor cells.

1	Due to their location at the crucial interface between hepatocytes and cholangiocytes, it has been proposed that the hepatic stem cells’ niche is present either in the canals of Hering or in their vicinity. This hypothesis was supported by the appearance of liver cell precursors near the canals of Hering in most of pathologic conditions characterized by extensive damage to hepatocytes. These cells could migrate and differentiate into either hepatocytes or bile duct cells. Recently, the three-dimensional reconstruction of ductular reactions in liver necrosis suggests that small cholangiocytes lining the canals of Hering proliferate extensively and migrate into the parenchyma of the liver. In immunocytochemical staining, these cells express dual markers of both biliary and hepatocyte antigens and appear to be involved in the repair of liver tissue damaged by chronic pathologic processes. Therefore, it has been concluded that the canal of Hering consists of or harbors specific hepatic

1	and appear to be involved in the repair of liver tissue damaged by chronic pathologic processes. Therefore, it has been concluded that the canal of Hering consists of or harbors specific hepatic stem cells. Laboratory studies suggest that in the future, hepatic stem cells may ultimately have therapeutic use in the treatment of liver diseases.

1	The bile ductule represents the part of the biliary tree that is lined entirely by cholangiocytes. The bile from the canal of Hering continues to flow into the intrahepatic bile ductule, which is lined entirely by cholangiocytes. Three-dimensional analysis of immunocytochemical-stained serial sections of the liver reveals that the canal of Hering often crosses the boundary of the lobule and becomes bile ductule in the periportal space (of Mall). The main distinction between the canal of Hering and the bile ductule is not its location within the lobule, but whether the structure is partially or completely lined by cholangiocytes. Intrahepatic bile ductules carry bile to hepatic ducts.

1	Intrahepatic bile ductules carry bile to hepatic ducts. The ductules have a diameter of about 1.0 to 1.5 m and carry bile to the interlobular bile ducts that form part of the portal triad (see Fig. 18.10b). These ducts range from 15 to 40 m in diameter and are lined by cholangiocytes that are cuboidal near the lobules and gradually become columnar as the ducts near the porta hepatis. The columnar cells have well-developed microvilli, as do those of the extrahepatic bile ducts and gallbladder. As the bile ducts get larger, they gradually acquire a dense connective tissue investment containing numerous elastic fibers. Smooth muscle cells appear in this connective tissue as the ducts approach the hilum. Interlobular ducts join to form the right and left hepatic ducts, which in turn join at the hilum to form the common hepatic duct (Fig. 18.15).

1	In some individuals, the ducts of Luschka are located in the connective tissue between the liver and the gallbladder, near the neck of the gallbladder. These ducts connect with the cystic duct, not with the lumen of the gallbladder. They are histologically similar to the intrahepatic bile ducts and may be remnants of aberrant embryonic bile ducts. Extrahepatic bile ducts carry the bile to the gallbladder and duodenum. The common hepatic duct is about 3 cm long and is lined with tall columnar epithelial cells that closely resemble those of the gallbladder. All of the layers of the alimentary canal (see page 569) are represented in the duct, except the muscularis mucosae. The cystic duct connects the common hepatic duct to the gallbladder and carries bile both main of common sphincter of main pancreatic duct major duodenal hepatopancreatic papilla ampulla (of Vater) sphincter of hepatopancreatic ampulla (of Oddi)

1	FIGURE 18.15 • Diagram showing the relationship of hepatic, pancreatic, and gallbladder ducts. The gallbladder is a blind pouch joined to a single cystic duct in which numerous mucosal folds form the spiral valve (of Heister). The cystic duct joins with the common hepatic duct, and together they form the common bile duct that leads into the duodenum. At the entry to the duodenum, the common bile duct is joined by the main pancreatic duct to form the hepatopancreatic ampulla (of Vater), and together they enter the second part of the duodenum. Sphincters can be found at the distal part of these ducts. The sphincters of the common bile duct (of Boyden), the main pancreatic duct, and the hepatopancreatic ampulla (of Oddi) control the flow of bile and pancreatic secretion into the duodenum. When the common bile duct sphincter contracts, bile cannot enter the duodenum; it backs up and flows into the gallbladder, where it is concentrated and stored.

1	into and out of the gallbladder. Distal to the junction with the cystic duct, the fused duct is called the common bile duct and extends for about 7 cm to the wall of the duodenum at the ampulla of Vater. A thickening of the muscularis externa of the duodenum at the ampulla constitutes the sphincter of Oddi, which surrounds the openings of both the common bile duct and the pancreatic duct and acts as a valve to regulate the flow of bile and pancreatic juice into the duodenum. The adult human liver secretes, on average, about 1 L of bile a day. The bile fulfills two major functions. It is involved in the absorption of fat and is used by the liver as a vehicle for excretion of cholesterol, bilirubin, iron, and copper. The composition of bile and the specific functions of most of its components are listed in Table 18.1. As noted in the table, many components of the bile are recycled via the portal circulation.

1	 About 90% of the bile salts, a component of bile, is reab sorbed by the gut and transported back to the liver in the portal blood. The bile salts are then reabsorbed and rese creted by hepatocytes. Hepatocytes also synthesize new bile salts to replace those that are lost.  Cholesterol and the phospholipid lecithin, as well as most of the electrolytes and water delivered to the gut with the bile, are also reabsorbed and recycled. Bilirubin glucuronide, the detoxified end product of hemoglobin breakdown, is not recycled. It is excreted with the feces and gives them their color. Failure to absorb bilirubin or failure to conjugate it or secrete glucuronide can produce jaundice.

1	Bile fow from the liver is regulated by hormonal and neural control. The rate of blood flow to the liver and the concentration of bile salts in the blood exert regulatory effects on the bile flow. Bile flow is increased when hormones such as cholecystokinin (CCK), gastrin, and motilin are released by enteroendocrine cells during digestion. Steroid hormones (i.e., estrogen during pregnancy) decrease bile secretion from the liver. In addition, parasympathetic stimulation increases bile flow by prompting contraction of the gallbladder and relaxation of the sphincter of Oddi. Bile that leaves the liver via the common hepatic duct flows through the cystic duct to the gallbladder. Following stimulation, the gallbladder contracts steadily and delivers the bile to the duodenum via the common bile duct. The liver has both sympathetic and parasympathetic innervation.

1	The liver has both sympathetic and parasympathetic innervation. The liver (and gallbladder) receives nerves from both sympathetic and parasympathetic divisions of the autonomic nervous system. The nerves enter the liver at the porta hepatis and ramify through the liver in the portal canals along with the members of the portal triad. Sympathetic fbers innervate blood vessels, and increased stimulation in this system causes an increase of vascular resistance, decreased hepatic blood volume, and a rapid increase of serum levels of glucose. The parasympathetic fbers are assumed to innervate the large ducts (those that contain smooth muscle in their walls) and possibly blood vessels; their stimulation promotes glucose uptake and utilization. The cell bodies of parasympathetic neurons are often present near the porta hepatis.

1	The gallbladder is a pear-shaped, distensible sac with a volume of about 50 mL in humans (see Fig. 18.15). It is attached to the visceral surface of the liver. The gallbladder is a secondary derivative of the embryonic foregut, arising as an evagination of the primitive bile duct that connects the embryonic liver to the developing intestine. The gallbladder concentrates and stores bile.

1	The gallbladder concentrates and stores bile. The gallbladder is a blind pouch that leads, via a neck, to the cystic duct. Through this duct it receives dilute bile from the chapter 18 Digestive System III: Liver, Gallbladder, and Pancreas GALLB LADDE R 643 hepatic duct. The gallbladder can store and remove about  Localized concentrations of mitochondria in the apical 90% of the water from the incoming bile, which results in an and basal cytoplasm increase of bile salts, cholesterol, and bilirubin concentrations  Complex lateral plications up to 10-fold. Hormones secreted by the enteroendocrine cells of the small intestine, in response to the presence of fat in the proximal duodenum, stimulate contractions of the smooth muscle of the gallbladder. Because of these contractions, concentrated bile is discharged into the common bile duct, which carries it to the duodenum. Mucosa of the gallbladder has several characteristic features.

1	Mucosa of the gallbladder has several characteristic features. The empty or partially filled gallbladder has numerous deep mucosal folds (Fig. 18.16). The mucosal surface consists of simple columnar epithelium (Fig. 18.17). The tall epithelial cells exhibit the following features:  Numerous, but short and not well-developed apical microvilli  Apical junctional complexes that join adjacent cells and form a barrier between the lumen and the intercellular compartment These cells closely resemble the absorptive cells of the intestine. Both cells share the above characteristics, as well as localization of Na/K–activated ATPase on their lateral plasma membranes and secretory vesicles filled with glycoproteins in their apical cytoplasm.

1	The lamina propria of the mucosa is particularly rich in fenestrated capillaries and small venules, but there are no lymphatic vessels in this layer. The lamina propria is also very cellular, containing large numbers of lymphocytes and plasma cells. The characteristics of the lamina propria resemble those of the colon, another organ specialized for the absorption of electrolytes and water. Mucin-secreting glands are sometimes present in the lamina propria in the normal human gallbladder, especially near the neck of the organ, but they are more commonly found in

1	Mucin-secreting glands are sometimes present in the lamina propria in the normal human gallbladder, especially near the neck of the organ, but they are more commonly found in FIGURE 18.16 • Photomicrograph of the wall of the gallbladder. The mucosa of the gallbladder consists of a lining of simple columnar epithelial cells and a lamina propria of loose connective tissue, which typically exhibits numerous deep folds in the mucosa. Beneath this layer is a relatively thick layer, the muscularis externa. There is no muscularis mucosae or submucosa. The smooth muscle bundles of the muscularis externa are randomly oriented. External to the muscle is an adventitia containing adipose tissue and blood vessels. The portion of the gallbladder not attached to the liver displays a typical serosa instead of an adventitia. 175.

1	FIGURE 18.17 • Electron micrographs of gallbladder epithelium. a. The tall columnar cells display features typical of absorptive cells, with microvilli on their apical surface, an apical junctional complex separating the lumen of the gallbladder from the lateral intercellular space, and numerous mitochondria in the apical portion of the cell. 3,000. b. During active fluid transport, salt is pumped from the cytoplasm into the intercellular space, and water follows the salt. Both salt and water then diffuse into the cell from the lumen. As this process continues, the intercellular space becomes greatly distended (arrows). Fluid moves from the engorged intercellular space (arrows) across the basal lamina into the underlying connective tissue (CT) and then into blood vessels. The increase in size of the lateral intercellular space during active fluid transport is evident with the light microscope. 3,000.

1	chapter 18 Digestive System III: Liver, Gallbladder, and Pancreas GALLB LADDE R 645 inflamed gallbladders. Cells that appear identical to enteroendocrine cells of the intestine are also found in these glands. The wall of the gallbladder lacks a muscularis mucosae and submucosa. External to the lamina propria is a muscularis externa that has numerous collagen and elastic fibers among the bundles of smooth muscle cells. Despite its origin from a foregutderived tube, the gallbladder does not have a muscularis mucosae or submucosa. The smooth muscle bundles are somewhat randomly oriented, unlike the layered organization of the intestine. Contraction of the smooth muscle reduces the volume of the bladder, forcing its contents out through the cystic duct.

1	External to the muscularis externa is a thick layer of dense connective tissue (see Fig. 18.16). This layer contains large blood vessels, an extensive lymphatic network, and the autonomic nerves that innervate the muscularis externa and the blood vessels (cell bodies of parasympathetic neurons are found in the wall of the cystic duct). The connective tissue is also rich in elastic fibers and adipose tissue. The layer of tissue where the gallbladder attaches to the liver surface is referred to as the adventitia. The unattached surface is covered by a serosa or visceral peritoneum consisting of a layer of mesothelium and a thin layer of loose connective tissue.

1	In addition, deep diverticula of the mucosa, called Rokitansky-Aschoff sinuses, sometimes extend through the muscularis externa (Fig. 18.18 and Plate 67, page 660). They are thought to presage pathologic changes and develop as the result of hyperplasia (excessive growth of cells) and herniation of epithelial cells through the muscularis externa. Also, bacteria may accumulate in these sinuses, causing chronic inﬂammation that is a risk factor for the formation of gallstones. Concentration of the bile requires the coupled transport of salt and water.

1	Concentration of the bile requires the coupled transport of salt and water. The epithelial cells of the gallbladder actively transport both Na, Cl and HCO3 from the cytoplasm into the intercellular compartment of the epithelium. ATPase is located in the lateral plasma membranes of the epithelial cells. This active transport mechanism is essentially identical to that described in Chapter 17 for the enterocytes of the small intestine and the absorptive cells of the colon. The epithelial cells of the gallbladder also express two types of aquaporins water channels (AQP1 and AQP8), integral membrane channel proteins that facilitate rapid passive movement of water (see Chapter 20, Folder 20.XX). The presence of water channels on apical and basolateral plasma membranes of the gallbladder epithelial cells suggests that they may be involved in both water absorption and secretion.

1	Active transport of Na, Cl, and HCO3 across the lateral plasma membrane into the intercellular (paracellular) compartment causes the concentration of electrolytes in the intercellular space to increase. The increased electrolyte concentration creates an osmotic gradient between the intercellular space and the cytoplasm and between the intercellular space and the lumen. Water moves from the cytoplasm and from the lumen into the intercellular space because of the osmotic gradient (i.e., it moves down its concentration gradient; see Fig. 18.17b). Although the intercellular space can distend to a degree often visible with the light microscope, this ability is limited. The movement of electrolytes and water into the space creates hydrostatic pressure that forces a nearly isotonic fluid out of the inter-cellular compartment into the subepithelial connective tissue (the lamina propria). The fluid that enters the lamina propria quickly passes into the numerous fenestrated capillaries and the

1	the inter-cellular compartment into the subepithelial connective tissue (the lamina propria). The fluid that enters the lamina propria quickly passes into the numerous fenestrated capillaries and the venules that closely underlie the epithelium. Studies of fluid transport in the gallbladder first demonstrated the essential role of the intercellular compartment in transepithelial transport of an isotonic fluid from the lumen to the vasculature. Therefore, the final modification of bile is mainly the result of the active transport of Na , Cl, and HCO3 , and the passive, aquaporin-mediated transport of water across the plasma membrane of epithelial cells of the gallbladder.

1	FIGURE 18.18 • Photomicrograph of the Rokitansky-Aschoff sinuses in the wall of the gallbladder. This photomicrograph shows deep invaginations of the mucosa extending into the muscularis externa. These invaginations are referred to as Rokitansky-Aschoff sinuses. 120. FIGURE 18.19 • Diagram of pancreas, duodenum, and associated excretory ducts. The main pancreatic duct (of Wirsung) traverses the length of the pancreas and enters the duodenum after joining with the common bile duct. An accessory pancreatic duct (of Santorini) is commonly present, as shown, and empties into the duodenum at a separate minor duodenal papilla. The site of entry of the common bile duct and main pancreatic duct into the duodenum is typically marked by a major duodenal papilla visible on the inner surface of the duodenum.

1	The pancreas is an elongate gland described as having a head, body, and tail. The head is an expanded portion that lies in the C-shaped curve of the duodenum (Fig. 18.19). It is joined to the duodenum by connective tissue. The centrally located body of the pancreas crosses the midline of the human body, and the tail extends toward the hilum of the spleen. The pancreatic duct (of Wirsung) extends through the length of the gland and empties into the duodenum at the hepatopan creatic ampulla (of Vater), through which the common bile duct from the liver and gallbladder also enters the duodenum.

1	The hepatopancreatic sphincter (of Oddi) surrounds the ampulla and not only regulates the flow of bile and pancreatic juice into the duodenum but also prevents reflux of intestinal contents into the pancreatic duct. In some individuals, an ac cessory pancreatic duct (of Santorini) is present, a vestige of the pancreas’s origin from two embryonic endodermal pri mordia that evaginate from the foregut. A thin layer of loose connective tissue forms a capsule around the gland. From this capsule, septa extend into the gland, dividing it into ill-defined lobules. Within the lob ules, a stroma of loose connective tissue surrounds the parenchymal units. Between the lobules, larger amounts of connective tissue surround the larger ducts, blood vessels, and nerves. Moreover, in the connective tissue surrounding the pancreatic duct, there are small mucous glands that empty into the duct. The pancreas is an exocrine and endocrine gland.

1	The pancreas is an exocrine and endocrine gland. Unlike the liver, in which the exocrine and secretory (endocrine) functions reside in the same cell, the dual functions of the pancreas are relegated to two structurally distinct components.  The exocrine component synthesizes and secretes en zymes into the duodenum that are essential for digestion in the intestine.  The endocrine component synthesizes and secretes the hormones insulin and glucagon into the blood. These hormones regulate glucose, lipid, and protein metabolism in the body. The exocrine pancreas is found throughout the organ; within the exocrine pancreas, distinct cell masses called islets of Langerhans are dispersed and constitute the endocrine pancreas. The exocrine pancreas is a serous gland.

1	The exocrine pancreas is a serous gland. The exocrine pancreas closely resembles the parotid gland, with which it can be confused. The secretory units are acinar or tubuloacinar in shape and are formed by a simple epithelium of pyramidal serous cells (Fig. 18.20a and Plate 68, page 662). The cells have a narrow free (luminal) surface and a broad basal surface. Periacinar connective tissue is minimal. The serous secretory cells of the acinus produce the digestive enzyme precursors secreted by the pancreas. Pancreatic acini are unique among glandular acini; the initial duct that leads from the acinus, the intercalated duct, actually begins within the acinus (Fig. 18.20b; Fig. 18.21). The duct cells located inside the acinus are referred to as centroacinar cells.

1	The acinar cells are characterized by distinct basophilia in the basal cytoplasm and by acidophilic zymogen granules in the apical cytoplasm (see Figs. 18.20a and 18.21). Zymogen granules are most numerous in the pancreas of fasting individuals. The squamous centroacinar cells lack both ergastoplasm and secretory granules (see Fig. 18.21); thus, they stain very lightly with eosin. This weak staining helps identify them in routine histologic sections. Zymogen granules contain a variety of digestive enzymes in an inactive form. Pancreatic enzymes can digest most food substances. The inactive enzymes, or proenzymes, contained in pancreatic zymogen granules are listed here along with the specific substances they digest when activated.  Proteolytic endopeptidases (trypsinogen, chy carboxypeptidase, proaminopeptidase) digest proteins by by cleaving amino acids from the carboxyl or amino end of the peptide. chapter 18 Digestive System III: Liver, Gallbladder, and Pancreas PANCR EAS 647

1	chapter 18 Digestive System III: Liver, Gallbladder, and Pancreas PANCR EAS 647 FIGURE 18.20 • Pancreatic acinus and its duct system. a. In this photomicrograph of a thin, H&E–stained plastic section, an intercalated duct can be seen beginning within a pancreatic acinus. The cells forming the duct within the acinus are the centroacinar cells. The eosinophilic zymogen granules are clearly seen in the apical cytoplasm of the parenchymal cells. 860. b. In this schematic diagram, observe the beginning of the intercalated duct. Note the location and shapes of the centroacinar cells within the acinus. They represent the initial lining of the intercalated duct, which drains into an intralobular collecting duct.

1	 Amylolytic enzymes (-amylase) digest carbohydrates by cleaving the glycosidic linkages of glucose polymers. Lipases digest lipids by cleaving ester bonds of triglycerides, producing free fatty acids. Nucleolytic enzymes (deoxyribonuclease and ribonuclease) digest nucleic acids, producing mononucleotides. The pancreatic digestive enzymes are activated only after they reach the lumen of the small intestine. Initially, the proteolytic activity of enzymes enterokinases in the glycocalyx of the microvilli of the intestinal absorptive cells converts trypsinogen to trypsin, a potent proteolytic enzyme. Trypsin then catalyzes the conversion of the other inactive enzymes as well as the digestion of proteins in the chyme.

1	The cytoplasmic basophilia of the pancreatic acinar cells when observed with the TEM appears as an extensive array of rER and free ribosomes. The presence of these numerous organelles correlates with the high level of protein synthetic activity of the acinar cells (Fig. 18.22). A well-developed Golgi apparatus is present in the apical cytoplasm and is involved in concentration and packaging of the secretory products. Mitochondria are small and, although found throughout the cell, are concentrated among the rER cisternae. Acinar cells are joined to one another by junctional complexes at their apical poles, thus forming an isolated lumen into which small microvilli extend from the apical surfaces of the acinar cells and into which the zymogen granules are released by exocytosis.

1	FIGURE 18.21 • Electron micrograph of the pancreatic acinus and intercalated duct. Note, that pancreatic acinus is formed from a pyramidal in shape acinar cells. Their basal area contains nucleus surrounded by extensive rER and Golgi apparatus. The apical portion exhibit well preserved zymogen granules. The origin of the intercalated duct lined by the centroacinar cells is visible on this electron micrograph. 5,800. (Courtesy of Dr. Holger Jastrow.) Duct System of the Exocrine Pancreas The centroacinar cells (see Figs. 18.20a and 18.21) are the beginning of the duct system of the exocrine pancreas. They have a centrally placed, flattened nucleus and attenuated cytoplasm, which is typical of a squamous cell. Centroacinar cells are intercalated duct cells located in the acinus.

1	Centroacinar cells are intercalated duct cells located in the acinus. Centroacinar cells are continuous with the cells of the short intercalated duct that lies outside the acinus. The structural unit of the acinus and centroacinar cells resembles a small balloon (the acinus) into which a drinking straw (the intercalated duct) has been pushed. The intercalated ducts are short and drain into intralobular collecting ducts. There are no striated (secretory) ducts in the pancreas.

1	The complex, branching network of intralobular ducts drains into the larger interlobular ducts, which are lined with a low columnar epithelium in which enteroendocrine cells and occasional goblet cells may be found. The interlobular ducts, in turn, drain directly into the main pancreatic duct, which runs the length of the gland parallel to its long axis, giving this portion of the duct system a herringbone-like appearance (see Fig. 18.19). A second large duct, the accessory pancreatic duct, arises in the head of the pancreas. The intercalated ducts add bicarbonate and water to the exocrine secretion.

1	The intercalated ducts add bicarbonate and water to the exocrine secretion. The pancreas secretes about 1 L of fluid per day, about equal to the initial volume of the hepatic bile secretion. Whereas bile is concentrated in the gallbladder, the entire volume of the pancreatic secretion is delivered to the duodenum. Although the acini secrete a small volume of protein-rich fluid, the intercalated duct cells secrete a large volume of fluid rich in sodium and bicarbonate. The bicarbonate serves to neutralize the acidity of the chyme that enters the duodenum from the stomach and to establish the optimal pH for the activity of the major pancreatic enzymes. Pancreatic exocrine secretion is under hormonal and neural control.

1	Pancreatic exocrine secretion is under hormonal and neural control. Two hormones secreted by the enteroendocrine cells of the duodenum, secretin and cholecystokinin (CCK), are the principal regulators of the exocrine pancreas (see Tables 17.1 page 583). The entry of the acidic chyme into the duodenum stimulates the release of these hormones into the blood:  Secretin is a polypeptide hormone (27 amino acid residues) that stimulates the duct cells to secrete a large vol ume of fluid with a high HCO3 concentration but little or no enzyme content. CCK is a polypeptide hormone (33 amino acid residues) that causes the acinar cells to secrete their proenzymes.

1	The coordinated action of the two hormones results in the secretion of a large volume of enzyme-rich, alkaline fluid into the duodenum. In addition to hormonal influences, the pancreas also receives autonomic innervation. Sympathetic nerve fibers are involved in regulation of pancreatic blood flow. Parasympathetic fibers stimulate activity of acinar as well as centroacinar cells. Cell bodies of neurons occasionally seen in the pancreas belong to parasympathetic postganglionic neurons. The endocrine pancreas is a diffuse organ that secretes hormones that regulate blood glucose levels.

1	The endocrine pancreas is a diffuse organ that secretes hormones that regulate blood glucose levels. The islets of Langerhans, the endocrine component of the pancreas, are scattered throughout the organ in cell groupings of varying size (Fig. 18.23). It is estimated that 1 million to 3 million islets constitute about 1% to 2% of the volume of the pancreas but are most numerous in the tail. Individual islets may contain only a few cells or many hundreds of cells (Plate 68, page 662). Their polygonal cells are arranged in short, chapter 18 Digestive System III: Liver, Gallbladder, and Pancreas PANCR EAS 649

1	FIGURE 18.22 • Electron micrograph of the apical cytoplasm of several pancreatic acinar cells. One pancreatic acinar cell is outlined by the dashed line. Nuclei (N) of adjoining cells are evident at the bottom left and right of the electron micrograph. The apical cytoplasm contains extensive rough endoplasmic reticulum (rER), mitochondria (M), zymogen-containing secretory granules (Z), and Golgi profiles (G). At the apices of these cells, a lumen (L) is present, into which the zymogen granules are discharged. A junctional complex (JC) is indicated near the lumen. 20,000. irregular cords that are profusely invested with a network of fenestrated capillaries. The definitive endocrine cells of the islets develop between 9 and 12 weeks of gestation.

1	irregular cords that are profusely invested with a network of fenestrated capillaries. The definitive endocrine cells of the islets develop between 9 and 12 weeks of gestation. In H&E–stained sections, the islets of Langerhans appear as clusters of pale-staining cells surrounded by more intensely staining pancreatic acini. It is not practical to attempt to identify the several cell types found in the islets in routinely prepared specimens (Fig. 18.24). After Zenkerformol fixation and staining by the Mallory-Azan method, however, it is possible to identify three principal cell types designated A (alpha), B (beta), and D (delta) cells (Table 18.2 and Fig. 18.25). With this method, the A cells stain red, the B cells stain brownish orange, and the D cells stain blue. About 5% of the cells appear to be

1	FIGURE 18.23 • Photomicrograph of the pancreas. This H&E–stained specimen shows a number of pancreatic lobules separated by connective tissue septa that are continuous with the thin surrounding capsule of the gland. The pancreatic lobules consist largely of the exocrine acini and their intralobular duct system. Most of the lobules exhibit small, round, lighter-staining profiles, which are the islets of Langerhans (arrows). Adjacent to the lobules, at the lower left, is a large interlobular duct that serves the exocrine pancreas. 25. unstained after this procedure. TEM allows identification of the principal cell types by the size and density of their secretory granules. Islet cells, other than B cells, are counterparts of the enteroendocrine cells of the gastrointestinal mucosa.

1	Islet cells, other than B cells, are counterparts of the enteroendocrine cells of the gastrointestinal mucosa. In addition to the three principal islet cells, three minor islet cell types have also been identified by using a combination of the TEM and immunocytochemistry (Table 18.3). Each cell type can be correlated with a specific hormone, and each has a specific location in the islet. B cells constitute about 70% of the total islet cells in humans and are generally located in its central portion. They secrete insulin (see Table 18.2). B cells contain numerous secretory granules about 300 nm in diameter with a dense polyhedral core and a pale matrix. The polyhedral core is believed to be crystallized insulin.

1	A cells constitute about 15% to 20% of the human islet population and are generally located peripherally in the islets. They secrete glucagon (see Table 18.2). A cells contain secretory granules about 250 nm in diameter that are more uniform in size and more densely packed in the cytoplasm than the granules of B cells. The granule is the site of stored glucagon (Fig. 18.26). D cells constitute about 5% to 10% of the total pancreatic endocrine tissue and are also located peripherally in the islets. D cells secrete somatostatin, which is contained in secretory granules that are larger than those of the A and B cells (300 to 350 nm) and contain material of low to medium electron density (see Fig. 18.26). The minor islet cells constitute about 5% of the islet tissue and may be equivalent to the pale cells seen after Mallory-Azan staining. Their characteristics and functions are summarized in Table 18.3.

1	Evidence suggests that some cells may secrete more than one hormone. Immunocytochemical staining has localized several hormones in addition to glucagon in the A-cell cytoplasm. These include gastric inhibitory peptide (GIP), CCK, and adrenocorticotropic hormone (ACTH)endorphin. Although there is no clear morphologic evidence for the presence of G cells (gastrin cells) in the islets, gastrin may also be secreted by one or more of the islet cells. Certain pancreatic islet cell tumors secrete large amounts of gastrin, thereby producing excessive acid secretion in the stomach (Zollinger-Ellison syndrome). Functions of Pancreatic Hormones All of the hormones secreted by the endocrine pancreas regulate metabolic functions either systemically, regionally (in the gastrointestinal tract), or locally (in the islet itself). Insulin, the major hormone secreted by the islet tissue, decreases blood glucose levels.

1	Insulin, the major hormone secreted by the islet tissue, decreases blood glucose levels. Insulin is the most abundant endocrine secretion. Its principal effects are on the liver, skeletal muscle, and adipose tissue. Insulin has multiple individual actions in each of these tissues. In general, insulin stimulates  uptake of glucose from the circulation. Specific cell membrane glucose transporters are involved in this process.  storage of glucose by activation of glycogen synthase and subsequent glycogen synthesis.  phosphorylation and use of glucose by promoting its gly colysis within cells. Absence or inadequate amounts of insulin lead to elevated blood glucose levels and the presence of glucose in the urine, a condition known as diabetes mellitus. Reduced expression of insulin and insulin growth factors in the central nervous system (CNS) has recently been linked to Alzheimer’s disease (Folder 18.3).

1	In addition to its effects on glucose metabolism, insulin stimulates glycerol synthesis and inhibits lipase activity in adipose cells. Circulating insulin also increases the amount of amino acids taken up by cells (which may involve cotransport with glucose) and inhibits protein catabolism. Glucagon, secreted in amounts second only to insulin, increases blood glucose levels. chapter 18 Digestive System III: Liver, Gallbladder, and Pancreas PANCR EAS 651 FIGURE 18.24 • Photomicrographs of islets of Langerhans. a. In this routine H&E preparation, it is difficult to identify specific islet cell types without special stains. At best, one can identify small cells (arrows) at the periphery of the islet that are probably A cells. 360. b. This photo micrograph shows an islet of Langerhans stained with a special Grimelius silver stain that reacts with glucagonsecreting cells. The silver-impregnated A cells are arranged around the periphery of the islet. 360.

1	The actions of glucagon are essentially reciprocal to those gluconeogenesis, mobilizes fats from adipose cells, and of insulin. Glucagon stimulates release of glucose into the stimulates hepatic lipase. bloodstream and stimulates gluconeogenesis (synthesis Somatostatin inhibits insulin and glucagon secretion. of glucose from metabolites of amino acids) and glycogenolysis (breakdown of glycogen) in the liver. Somatostatin is secreted by the D cells of the islets. It is Glucagon also stimulates proteolysis to promote identical to the hormone secreted by the hypothalamus that regulates somatotropin (growth hormone) release from the anterior pituitary gland. Although the precise role of somatostatin in the islets is unclear, it has been shown to inhibit both insulin and glucagon secretion.

1	TABLE Principal Cell Types in Pancreatic Islets18.2 Cell Type % Cytoplasmic Staining with Mallory-Azan Product Granules (TEM) A 15–20 Red Glucagon About 250 nm; dense, eccentric core surrounded by light substance B 60–70 Brownish orange Insulin About 300 nm; many with dense, crystalline (angular) core surrounded by light substance D 5–10 Blue Somatostatin About 325 nm; homogeneous matrix FIGURE 18.25 • Diagram of an islet of Langerhans stained by the Mallory-Azan method. A cells display red cytoplasmic staining, B cells (comprising most of the islet cells) display brownish-orange staining, and D cells show a blue cytoplasm. The molecular characteristics of the major and some minor islet hormones are summarized in Table 18.4. Regulation of Islet Activity

1	The molecular characteristics of the major and some minor islet hormones are summarized in Table 18.4. Regulation of Islet Activity A blood glucose level above the normal 70 mg/100 mL (70 mg/dL) stimulates release of insulin from beta cells, leading to uptake and storage of glucose by liver and muscle. The resultant decrease in the blood glucose level stops insulin secretion. Some amino acids also stimulate insulin secretion, either alone or in concert with elevated blood glucose levels. Increased blood fatty acid levels also stimulate insulin release, as do circulating gastrin, CCK, and secretin. CCK and glucagon, released in the islet by the A cells, act as paracrine secretions to stimulate B cell secretion of insulin.

1	Blood glucose levels below 70 mg/100 mL stimulate release of glucagon; blood glucose levels significantly above 70 mg/100 mL inhibit glucagon secretion. Glucagon is also released in response to low levels of fatty acids in the blood. Insulin inhibits release of glucagon by A cells, but because of the cascading circulation in the islet (explained in a following paragraph), this inhibition is effected by a hormonal action of insulin carried in the general circulation. The islets have both sympathetic and parasympathetic innervation. About 10% of the islet cells have nerve endings directly on their plasma membrane. Well-developed gap junctions are located between islet cells. Ionic events triggered by synaptic transmitters at the nerve endings are carried from cell to cell across these junctions. Autonomic nerves may have direct effects on hormone secretion by A and B cells.

1	Parasympathetic (cholinergic) stimulation increases secretion of both insulin and glucagon; sympathetic (adrenergic) stimulation increases glucagon release but inhibits insulin release. This neural control of insulin and glucagon may

1	TABLE Minor Cell Types in Pancreatic Islets 18.3 Cell Type Secretion Location (in Addition to Islet) Actions PP cell (F cell)a Pancreatic polypeptide Stimulates gastric chief cells, inhibits bile secretion and intestinal motility, inhibits pancreatic enzymes and HCO3 secretion D 1 cell Vasoactive intestinal peptide (VIP) Also in exocrine acini and duct epitheliumb Similar to those of glucagon (hyperglycemic and glycogenolytic); also affects secretory activity and motility in gut; stimulates pan-creatic exocrine secretion EC cella Secretin, motilin, substance P Also in exocrine acini and duct epitheliumb Secretin: acts locally to stimulate HCO3 secretion in pancreatic juice and pancreatic enzyme secretion Motilin: increases gastric and intestinal motility Substance P: has neurotransmitter properties Epsilon cell Ghrelin Epithelium lining the fundus of the stomachc Stimulate appetite aPP, protein polypeptide; EC, enterochromaffin cell. bThis localization further emphasizes the ontogeny

1	properties Epsilon cell Ghrelin Epithelium lining the fundus of the stomachc Stimulate appetite aPP, protein polypeptide; EC, enterochromaffin cell. bThis localization further emphasizes the ontogeny of the pancreas from the embryonic gut. cGhrelin is produced in the stomach by P/D1 cells.

1	Digestive System III: Liver, Gallbladder, and Pancreas PANCR EAS FIGURE 18.26 • Electron micrograph of pancreatic islet cells. The portion of the cell in the upper part of the illustration is an A cell. It contains characteristic granules (arrows) showing a dense spherical core surrounded by a clear area and then a membrane. This cell also displays a characteristically well-developed Golgi apparatus. The cell in the bottom of the illustration is a D cell. It contains numerous membrane-bounded granules of moderately low density (arrowheads). 15,000.

1	TABLE Characteristics of Pancreatic Hormones18.4 Hormone Molecular Weight (daltons) Structure Insulin 5,700–6,000 Two protein chains linked by disulfide bridges: chain, 21 amino acids; chain, 30 amino acids Glucagon 3,500 Linear polypeptide: 29 amino acids Somatostatin 1,638 Cyclic polypeptide: 14 amino acids VIP 3,300 Linear polypeptide: 28 amino acids Pancreatic 4,200 Linear polypeptide polypeptide: 36 amino acids contribute to the availability of circulating glucose in stress reactions. The blood supply to the pancreas provides a cascading perfusion of the islets and acini.

1	The blood supply to the pancreas provides a cascading perfusion of the islets and acini. Several arterioles enter the periphery of the islets and branch into fenestrated capillaries. In humans, the capillaries first perfuse the A and D cells, peripherally, before the blood reaches the B cells, centrally. Larger vessels that travel in septa that penetrate the central portion of the islet are also accompanied by A and D cells, so that blood reaching the B cells has always first perfused the A and D cells. Large efferent capillaries leave the islet and branch into the capillary networks that surround the acini of the exocrine pancreas. This cascading flow resembles the portal systems of other endocrine glands (pituitary, adrenal). Secretions of the islet cells have regulatory effects on the acinar cells:  Insulin, the vasoactive intestinal peptide (VIP), and CCK stimulate exocrine secretion.  Glucagon, pancreatic polypeptide (PP), and somatostatin inhibit exocrine secretion.

1	 FOLDER 18.3  FOLDER 18.4 Functional Considerations: Insulin Synthesis, an Example of Posttranslational Processing chapter 18 Digestive System III: Liver, Gallbladder, and Pancreas PANCR EAS 655 655 Recently, researchers identified expression of insulin and insulin growth factors (IGF I and IGF II) in nerve cells in several regions of the brain. It is known that insulin resis-tance characteristic of diabetes mellitus is linked to neural degeneration, cognitive dysfunction, and dementia. The reduced rate of insulin and IGF production in the brain contributes to the degeneration of brain cells, an early symptom of Alzheimer’s disease (AD). Postmortem ex-amination of the brain tissue from individuals diagnosed with AD confirmed that levels of insulin and IGFs were sig-nificantly reduced in the hippocampus (the part of the brain responsible for memory), frontal lobes, and hypothalamus. In contrast, in the cerebellum (which is generally not af-fected by AD), researchers noted normal

1	in the hippocampus (the part of the brain responsible for memory), frontal lobes, and hypothalamus. In contrast, in the cerebellum (which is generally not af-fected by AD), researchers noted normal levels of these hormones. Abnormalities in insulin production in the brain do not present symptoms typical to type 1 or type 2 dia-betes mellitus, but they may indicate that AD could be a manifestation of type 3 diabetes. If these new observations are confirmed in the future, it might be possible to develop a targeted treatment for AD that is not available today. Insulin is produced within B cells in the pancreas. It is a small protein consisting of two polypeptide chains joined by disulfide bridges. Its biosynthesis presents a clear ex-ample of the importance of posttranslational processing in the achievement of the final, active structure of a protein. Insulin is originally synthesized as a single 110–amino acid polypeptide chain with a molecular weight of about 12,000 daltons. This

1	in the achievement of the final, active structure of a protein. Insulin is originally synthesized as a single 110–amino acid polypeptide chain with a molecular weight of about 12,000 daltons. This polypeptide is called preproinsulin. Preproinsulin contains an amino-terminus signal sequence (24 amino acids in length) that is required for the precursor hormone to enter rER. As the molecule is inserted into the cisternae of the rER, the preproinsulin signal sequence is proteolytically cleaved to form proinsulin. Post translational processing reduces the preproinsulin to a polypeptide with a molecular weight of about 9,000 daltons. Proinsulin is a single polypeptide chain of 81 to 86 amino acids that has the approximate shape of the letter G (Fig. F18.4.1). Two disulfide bonds connect the bar of the G to the top loop. During packaging and storage of proinsulin in the Golgi apparatus, a cathepsin-like enzyme cleaves most of the side of the loop, leaving the bar of the G as an A chain of 21

1	of the G to the top loop. During packaging and storage of proinsulin in the Golgi apparatus, a cathepsin-like enzyme cleaves most of the side of the loop, leaving the bar of the G as an A chain of 21 amino acids cross-linked by the disulfide bridges to the top of the loop, which becomes the B chain of 30 amino acids. The 35–amino acid peptide removed from the loop is called a C peptide (connecting peptide). It is stored in the secretory vesicles and released with the insulin in equimolar amounts. No physiologic function has been identified for the C peptide. Because C peptide has a longer half-life than insulin, higher concentrations of C peptides are detected in the peripheral blood. For these reasons, measurement of cir-culating levels of C peptides provides important clinical information about the secretory activity of B cells. Be-cause C peptide is cleared from the body by the kidney, measurement of its urinary excretion provides a useful in-formation about B cell insulin

1	about the secretory activity of B cells. Be-cause C peptide is cleared from the body by the kidney, measurement of its urinary excretion provides a useful in-formation about B cell insulin secretion. C peptide mea-surements are frequently used to assess the residual B cell function in patients treated with insulin, to distinguish between types 1 and 2 diabetes and in the diagnosis and monitoring therapy of insulinoma (tumor of B cells). C peptide may also be used to monitor the progress of pan-creas or islet cell transplantation. FIGURE F18.4.1 • Posttranslational processing of insulin. Insulin is synthesized as the preproinsulin, a single polypeptide chain that undergoes posttranslational modifications. Initially, the signal sequence is removed within cysternae of the rER. Resulted shorter polypeptide chain know as proinsulin is transported to Golgi apparatus where is further modified by formation of internal disulfide bonds and removal of C chain that produces biologically active

1	polypeptide chain know as proinsulin is transported to Golgi apparatus where is further modified by formation of internal disulfide bonds and removal of C chain that produces biologically active insulin. signal sequence chain B chain A chain C H2N C peptide preproinsulin proinsulin insulin COOH S H2N H2N H2N COOH COOH COOH S S S S S chain B chain A chain C S S S S S S chain B chain A S S S S S S

1	The liver is the largest mass of glandular tissue in the body and the largest internal organ. It is unique because it receives its major blood sup-ply from the hepatic portal vein, which carries venous blood from the small intestine, pancreas, and spleen. Thus the liver is directly in the pathway that conveys materials absorbed in the intestine. This gives the liver the first exposure to metabolic substrates and nutrients; it also makes the liver the first organ exposed to noxious and toxic substances absorbed from the intestine. One of the major roles of the liver is to degrade or conjugate toxic substances to render them harmless. It can, however, be seriously damaged by an excess of such substances. Each liver cell has both exocrine and endocrine functions. The exocrine secretion of the liver, called bile, contains conjugated and de-graded waste products that are delivered back to the intestine for disposal. It also contains substances that bind to metabolites in the intes-tine to

1	liver, called bile, contains conjugated and de-graded waste products that are delivered back to the intestine for disposal. It also contains substances that bind to metabolites in the intes-tine to aid absorption. A series of ducts of increasing diameter and complexity, beginning with bile canaliculi between individual hepatocytes and ending with the common bile duct, deliver bile from the liver and gallbladder to the duodenum. The endocrine secretions of the liver are released directly into the blood that supplies the liver cells; these secretions include albumin, nonimmune and -globulins, prothrombin, and glycoproteins, including fibronectin. Glucose, released from stored glycogen, and triiodothy-ronine (T3), the more active deiodination product of thyroxine, are also released directly into the blood. Functional units of the liver, described as lobules or acini, are made up of irregular interconnecting sheets of hepatocytes separated from one another by the blood sinusoids.

1	Liver, human, H&E ×65; inset ×65. At the low magnification shown here, large numbers of hepatic cells appear to be uniformly disposed throughout the specimen. The hepatocytes are arranged in one-cell-thick plates, but when sectioned, they appear as interconnecting cords one or more cells thick, depending on the plane of section. The sinusoids appear as light areas between the cords of cells; they are more clearly shown in figure below (asterisks). Also present in this figure is a portal canal. It is a connective tissue septum that carries the branches of the hepatic artery (HA) and portal vein (PV), bile ducts (BD), and lymphatic vessels and nerves. The artery and vein, along with the bile duct, are collectively referred to as a portal triad. The hepatic artery and the portal vein are easy to identify because they are found in relation to one another within the surrounding connective tis- Liver lobule, liver, human, H&E ×160.

1	The hepatic artery and the portal vein are easy to identify because they are found in relation to one another within the surrounding connective tis- Liver lobule, liver, human, H&E ×160. The terminal hepatic venules or central veins (CV) are the most distal radicals of the hepatic vein, and like the hepatic vein, they also travel alone. Their distinguishing features are the sinusoids that penetrate the wall of the vein and the paucity of surrounding connective tissue. These characteristics are shown to advantage in Plate 66.

1	It is best to examine low-magnification views of the liver to define the boundaries of a lobule. A lobule is best identifed when it is cut in cross section. The central vein then appears as a circular profle, and the hepatic sue of the portal canal. The vein is typically thin walled; the artery is smaller in diameter and has a thicker wall. The bile ducts are composed of a simple cuboidal or columnar epithelium, depending on the size of the duct. Multiple profiles of the blood vessels and bile ducts may be evident in the canal because of either branching or their passage out of the plane of section and then back in again.

1	The vessel through which blood leaves the liver is the hepatic vein. It is readily identified because it travels alone (inset) and is surrounded by an appreciable amount of connective tissue (CT). If more than one profile of a vein is present within this connective tissue, but no arteries or bile ducts are present, the second vessel will also be a hepatic vein. Such is the case in the inset, where a profile of a small hepatic vein is seen just above the larger hepatic vein (HV). cells appear as cords radiating from the central vein. Such a lobule is outlined by the dashed line in fgure above.

1	cells appear as cords radiating from the central vein. Such a lobule is outlined by the dashed line in fgure above. The limits of the lobule are defned, in part, by the portal canal. In other directions, the plates of the lobule do not appear to have a limit; that is they have become contiguous with plates of an adjacent lobule. One can estimate the dimensions of the lobule, however, by approximating a circle with the central vein as its center and incorporating those plates that exhibit a radial arrangement up to the point where a portal canal is present. If the lobule has been cross-sectioned, the radial limit is set by the location of one or more of the portal canals as indicated by the bile ducts (BD) in this fgure. KEY BD, bile duct CT, connective tissue CV, central vein (terminal hepatic venule) HA, hepatic artery HV, hepatic vein L, lymphatic nodule PV, portal vein asterisks (bottom fgure), blood sinusoids dotted line (top fgure), approximates the limits of a lobule

1	There are three ways of describing the liver parenchyma in terms of a functional unit, “classic” lobules, portal lobules, or acini. The classic lobule is a roughly hexagonal block of tissue that has at its center the terminal hepatic venule (central vein) and at its six corners the portal canals (portal triads) containing in each a branch of the portal vein, hepatic artery, and bile duct. The portal lobule is a triangular construct that emphasizes the exocrine secretory function. It has as its axis the bile duct of the portal triad of the classic lobule, and its outer margins are imaginary lines drawn between the central veins closest to that portal triad. The liver acinus provides the best correlation among blood perfusion, metabolic activity, and liver pathology. The acinus is a small diamondor lozenge-shaped mass of tissue that has as its short axis the fine branches of the portal triad that lie along the border of two classic lobules and as its long axis a line drawn between the

1	diamondor lozenge-shaped mass of tissue that has as its short axis the fine branches of the portal triad that lie along the border of two classic lobules and as its long axis a line drawn between the two central veins closest to the short axis. The hepatocytes in each acinus are described as arranged in three concentric elliptical zones around the short axis; zone 1 is closest to, and zone 3 is farthest from, the axis.

1	Central vein, liver, human, H&E ×500; inset ×800. The central vein and surrounding hepatocytes from lower figure of Plate 65 are shown here at higher magnification. The cytoplasm of the hepatocytes in this specimen has a foamy appearance because of extraction of glycogen and lipid during tissue preparation. The boundaries between individual hepatocytes are discernable in some locations but not between those cells where the knife has cut across the boundary in an oblique plane. Frequently, when cell boundaries are observed at still higher magnification (inset), a very small circular or oval profile is observed midway along the boundary. These profiles represent the bile canaliculi (BC).

1	The cells that line the sinusoids (S) show little, if any, cytoplasmic detail in routine preparations. Perisinusoidal macrophages (Kupffer cells [KC]) are generally recognized by their ovoid nuclei and the projection of the cell into the lumen. The endothelial cell, in contrast, is a squamous cell that has a smaller, attenuated or elongated nucleus. Some nuclei of this description are evident in the micrograph. The termination of two of the sinusoids and their union with the central vein (CV) is indicated by the curved arrows. Note that the wall of the vein is strengthened by connective tissue, mostly collagen, which appears as homogeneous eosin-stained material (asterisks). Fibroblasts (F) within this connective tissue can be identified and distinguished from the endothelial cell (EN) lining of the vein. Hepatic sinusoids, liver, rat, glutaraldehyde–osmium fxation, toluidine blue ×900.

1	This figure shows a plastic-embedded liver specimen fixed by the method normally used for electron microscopy. In contrast to the H&E–stained preparation, it demonstrates to advantage the cytologic detail of the hepatocytes and the sinusoids (S). The hepatocytes are deeply colored with toluidine blue. Note that the cytoplasm exhibits irregular magenta masses (arrows). This is glycogen that has been retained by the glutaraldehyde fixation and stained metachromatically by toluidine blue. Also evident are lipid droplets (L) of varying size that have been preserved and stained black by the osmium used as the secondary fixative. The quantities of lipid and glycogen are variable and, under normal conditions, reflect dietary intake. Examination of the hepatocyte cytoplasm also reveals small, punctate, dark-blue bodies contrasted against the lighter-blue background of the cell. These are the mitochondria. Another feature of this specimen is the clear representation of the bile canaliculi (BC)

1	dark-blue bodies contrasted against the lighter-blue background of the cell. These are the mitochondria. Another feature of this specimen is the clear representation of the bile canaliculi (BC) between liver cells. They appear as empty circular profiles when cross-sectioned and as elongate channels (lower right) when longitudinally sectioned.

1	The sinusoidal lining cells are of two distinct types. The Kupffer cells (KC) are the more prominent cells. They exhibit a large nucleus and a substantial amount of cytoplasm. They protrude into the lumen and may give the appearance of occluding it. However, they do not block the channel. The surface of the Kupffer cell exhibits a very irregular or jagged contour because of the numerous processes that provide the cell with an extensive surface area. The endothelial cell (EN) has a smaller nucleus, attenuated cytoplasm, and a smooth surface contour. A third cell type, the less frequently observed perisinusoidal lipocyte (Ito cell), is not seen in this micrograph. This cell would appear as a light cell containing numerous lipid droplets. The lipid droplets contain stored vitamin A.

1	KEY BC, bile canaliculus CV, central vein EN, endothelial cell F, fibroblast KC, Kupffer cell L, lipid droplet S, sinusoid arrows, glycogen asterisks, connective tissue of central vein curved arrows, opening of sinusoid into central vein The gallbladder concentrates and stores bile for delivery to the duodenum. The bile is concentrated by the active transport of salt from the bile and the passive movement of water in response to the salt transport. The mucosa is characterized by a tall columnar absorptive epithe-lium that closely resembles that of the intestine and the colon in both its morphology and function. The epithelial cells are characterized by nu-merous short apical microvilli, apical junctional complexes, concentrations of mitochondria in the apical and basal cytoplasm, and complex lateral plications. In addition, Na+-K+–activated ATPase is localized on the lateral plasma membrane of the epithelial cell. Gallbladder, human, H&E ×45.

1	Gallbladder, human, H&E ×45. The gallbladder is a hollow, pear-shaped organ that concentrates and stores the bile. The full thickness of its wall is shown here. It is composed of a mucosa (Muc), muscularis (Mus), and adventitia (Adv) and, on its free surface (not shown), a serosa. The mucosa is considered at higher magni fication in figure below. The muscularis consists of interlacing bundles of smooth muscle (SM). The adventitia (Adv) consists of irregular dense con660 nective tissue through which the larger blood vessels (BV) travel and, more peripherally, of varying amounts of adipose tissue (AT). Mucosa, gallbladder, human, H&E ×325.

1	Mucosa, gallbladder, human, H&E ×325. The mucosa consists of a tall simple columnar absorptive epithelium (Ep) resting on a lamina propria of loose irregular connective tissue (CT). The epithelium has characteristics that distinguish it from the absorptive epithelium of other organs, such as the intestines. Only one cell type, tall columnar cells, is present in the epithelial layer (see upper right figure). The nuclei are in the basal portion of the cell. The cells possess a thin apical striated bor- Mucosa, gallbladder, human, H&E ×550. The smaller of the two gland-like structures is composed of mucous cells (MC) and represents a section through a mucous gland. This specimen was taken from a site near the neck of the gallbladder where mucous glands are often present. Note the characteristic flattened nuclei at the base of the cell and the lightly stained appearance of the cytoplasm, features characteristic

1	The mucosa is thrown into numerous folds that are particularly pronounced when the muscularis is highly contracted. This is the usual histologic appearance of the gallbladder unless, of course, steps are taken to fix and preserve it in a distended state. Occasionally, the section cuts through a recess in a fold, and the recess may then resemble a gland (arrows). The mucosa, however, does not possess glands, except in the neck region, where some mucous glands are present (see lower right figure).

1	der. However, this is not always evident in routine H&E–stained sections. The cytoplasm stains rather uniformly with eosin. This is related to its absorptive function and is in contrast to the staining of cells that are engaged in the production of protein. Lastly, with respect to its absorptive function, the epithelial cells frequently exhibit distended intercellular spaces at their basal aspect (see upper right figure arrows). This is a feature associated with the transport of fluid across the epithelium and, as noted above, commonly seen in intestinal absorptive cells. Mucosa, gallbladder, human, H&E ×550. The lamina propria underlying the epithelium is usually very cellular. In this specimen, in addition to lymphocytes (L), a relatively common finding, a large number of plasma cells (PC) is also present within the lamina propria. (The high concentration of plasma cells suggests chronic inflammation.)

1	Another feature of note in the lamina propria is the presence of several out-pouching of the mucosa, called Rokitansky-Aschoff sinuses (RAS), other than those seen in the mucosa and noted above. These are readily apparent in figure on right and two of these structures, marked by RAS are shown at higher magnification in figure below. of mucin-secreting cells. In contrast, the epithelium of the large gland-like profile that is only partially included in the micrograph has rounded or ovoid nuclei. This epithelial-lined structure is not a true gland but represents an invagination of the mucous membrane that extends into and often through the thickness of the muscularis. These invaginations are known as Rokitansky-Aschoff sinuses.

1	KEY Adv, adventitia AT, adipose tissue BV, blood vessel CT, connective tissue, lamina propria Ep, epithelium RAS, Rokitansky-Aschoff sinus L, lymphocytes MC, mucous cells Muc, mucosa Mus, muscularis PC, plasma cells SM, smooth muscle arrows: Top left fgure, recess in luminal surface; Top right figure, intercellular spaces

1	The pancreas is an elongated extramural digestive gland with a head nestled in the C-shaped bend of the duodenum, a body that crosses the midline of the abdomen, and a tail extending across the back of the abdomen. It is a mixed gland containing both an exocrine component and an endocrine component that have distinctive characteristics. The exocrine component is a compound tubuloacinar gland with a branching network of ducts that convey the exocrine secretions to the duodenum. These secretions consist primarily of inactive forms of potent prote-olytic enzymes, as well as amylase, lipase, nucleases, and electrolytes, particularly HCO3 . The endocrine component is isolated as highly vascularized islets of epithelioid tissue (islets of Langerhans). The islet cells secrete a va-riety of polypeptide and protein hormones, most notably insulin and glucagon, which regulate glucose metabolism throughout the other tis-sues of the body. Other hormones secreted by islet cells include

1	of polypeptide and protein hormones, most notably insulin and glucagon, which regulate glucose metabolism throughout the other tis-sues of the body. Other hormones secreted by islet cells include somatostatin, pancreatic polypeptide, vasoactive intestinal peptide, secretin, motilin, and substance P. All of these substances, with the exception of insulin, are also secreted by the population of enteroendocrine cells in the intestine, the organ from which the pancreas is derived during embryonic development. While insulin and glucagon act primarily in en-docrine regulation of distant cells, the other hormones (and glucagon) have significant roles in the paracrine regulation of the insulin-secreting B cells of the pancreatic islet.

1	Pancreas, human, H&E ×160; inset ×360. The pancreas is surrounded by a delicate capsule of moderately dense connective tissue. Septa from the capsule divide the pancreas into lobules, one of which is shown here, bounded by connective tissue (CT). Larger blood vessels (BV ) travel in the connective tissue septa; nerves also travel in the septa, but they are seen infrequently. Within the lobule are the numerous acini of the exocrine component, an intralobular duct (InD), intercalated ducts (not readily evident at this low magnification), and islets of Langerhans (IL). Also within the lobule are the small blood vessels and the connective tissue serving as a stroma for the parenchymal elements of the gland. Pancreas, human, H&E ×600.

1	Pancreas, human, H&E ×600. Acini of the pancreas consist of serous cells. In sections, the acini present circular and irregular profiles. The lumen of the acinus is small, and only in fortuitous sections through an acinus is the lumen included (asterisks). The nucleus is characteristically in the base of the acinar cell. There is a region of intense basophilia adjacent to the nucleus. This is the ergastoplasm (Er), and it reflects the presence of rER that is active in the synthesis of pancreatic enzymes. Some acini reveal a centrally positioned cell with cytoplasm that shows no special staining characteristics in H&E–stained paraffin sections. These are centroacinar cells (CC). They are the beginning of the intercalated ducts. This figure demonstrates particularly well the morphology and relationships of the intercalated ducts. Note, first, the cross-sectioned intralobular duct (InD) consisting of cuboidal epithelium. (There are no striated

1	This figure shows an islet of Langerhans (IL) among the far more numerous acini. (Islets are most numerous in the tail of the pancreas and least numerous in the head). Cells within the islets are arranged as irregular cords. In routine preparations, it is not possible to identify the various cell types within the islets. Note, however, that B cells are the most numerous; these produce insulin. The next most numerous are A cells; these produce glucagon. The inset also shows numerous capillaries (arrows). The labels A and B are not intended to identify specific cells but rather to show those parts of the islets where A and B cells are found in greatest number.

1	ducts in the pancreas.) Leading to the intralobular duct is an intercalated duct (ID), which is seen in cross section at the furthest distance from the intralobular duct and then, in longitudinal section, in the center of the illustration as it travels toward the intralobular duct. The lumen is evident where the intercalated duct is seen in cross section but is not evident where it is seen in longitudinal section. This is because the plane of section cuts chiefly through the cells rather than the lumen. As a consequence, this figure provides a good view of the nuclei of the duct cells. They are elongate, with their long axis oriented in the direction of the duct. In addition, they display a staining pattern similar to that of centroacinar cells and different from that of nuclei of the parenchymal cells.

1	Once the cells of the intercalated duct have been identified in one part of the section, their staining characteristics and location can be used to identify the intercalated ducts in other parts of the lobule, several of which are marked (ID). KEY A, region with most A cells B, region with most B cells BV, blood vessels CC, centroacinar cells CT, connective tissue Er, ergastoplasm ID, intercalated ducts IL, islets of Langerhans InD, intralobular duct arrows, capillaries asterisks, lumen of acini

1	OVERVIEW OF THE RESPIRATORY SYSTEM / 664 NASAL CAVITIES / 665 Vestibule of the Nasal Cavity / 665 Respiratory Region of the Nasal Cavity / 666 Olfactory Region of the Nasal Cavity / 667 Paranasal Sinuses / 670 PHARYNX / 670 LARYNX / 670 TRACHEA / 670 Tracheal Epithelium / 672 Basement Membrane and Lamina Propria / 673 BRONCHI / 676 BRONCHIOLES / 677 Bronchiolar Structure / 677 Bronchiolar Function / 678 ALVEOLI / 678 BLOOD SUPPLY / 687 LYMPHATIC VESSELS / 687 NERVES / 687 Folder 19.1 Clinical Correlations: Squamous Metaplasia in the Respiratory Tract / 672 Folder 19.2 Clinical Correlations: Cystic Fibrosis / 685 Folder 19.3 Clinical Correlations: Emphysema and Pneumonia / 686 The respiratory system consists of the paired lungs and a series of air passages that lead to and from the lungs. Within the lung, the air passages branch into increasingly smaller tubes until the very smallest air spaces, called alveoli, are reached (Fig. 19.1).

1	Three principal functions are performed by this system: air conduction, air filtration, and gas exchange (respiration). The latter occurs in the alveoli. In addition, air passing through the larynx is used to produce speech, and air passing over the olfactory mucosa in the nasal cavities carries stimuli for the sense of smell. The respiratory system also participates to a lesser degree in endocrine functions (hormone production and secretion), as well as regulation of immune responses to inhaled antigens. The lungs develop in the embryo as a ventral evagination of the foregut; thus, the epithelium of the respiratory system is of endodermal origin. This initial respiratory diverticulum grows into the thoracic mesenchyme. The bronchial cartilages, smooth muscle, and the other connective tissue elements are derived from the thoracic mesenchyme. The air passages of the respiratory system consist of a conducting portion and a respiratory portion.

1	The air passages of the respiratory system consist of a conducting portion and a respiratory portion. The conducting portion of the respiratory system consists of those air passages that lead to the sites of respiration within the lung where gas exchange takes place. The conducting passages include those located outside as well as within the lungs. The passages external to the lungs consist of the following:  Nasal cavities that represent two large air-filled spaces in the uppermost part of the respiratory system (and, during forced breathing, the oral cavity residing inferior to the nasal cavities).  Nasopharynx that lies behind the nasal cavities and above the level of the soft palate and communicate inferiorly with oropharynx that is posterior to the oral cavity.  Larynx that is a hollow tubular organ containing cartilaginous framework responsible for producing sounds.

1	 Larynx that is a hollow tubular organ containing cartilaginous framework responsible for producing sounds.  Trachea that is a flexible air tube that extend from the larynx to the thorax. It serves as a conduit for air and in the mediastinum it birurcates into paired main bronchi.  Paired main (primary) bronchi that enter root of the right or left lung. FIGURE 19.1 • Diagram of respiratory passages. The nasal cavities, nasopharynx, oropharynx, larynx, trachea, bronchi, and bronchioles constitute the conducting portion of the respiratory system. The respiratory portion of the system, where gas exchange occurs, is composed of respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli. (Based on Boileau G. A Method of Anatomy. Baltimore: Williams & Wilkins, 1980.)

1	Within the lungs, the main bronchi undergo extensive branching to give rise ultimately to the distributing bronchioles. The bronchioles represent the terminal part of the conducting passages. Collectively, the internal bronchi and the bronchioles constitute the bronchial tree. The respiratory portion is that part of the respiratory tract in which gas exchange occurs. Sequentially, it includes these:  Respiratory bronchioles that are involve in both air conduction and gas exchange.  Alveolar ducts that are elongated airways formed from the confluence openings to alveoli.  Alveolar sacs that respresnt spaces surrounded by clusters of alveoli.  Alveoli that are the primary sites of gas exchange.

1	Blood vessels enter the lung with the bronchi. The arteries branch into smaller vessels as they follow the bronchial tree into the substance of the lung. Capillaries come into intimate contact with the terminal respiratory units, the alveoli. This intimate relationship between the alveolar air spaces and the pulmonary capillaries is the structural basis for gas exchange within the lung parenchyma. The essential features of the lung blood supply are described on page 687. Air passing through the respiratory passages must be conditioned before reaching the terminal respiratory units.

1	Air passing through the respiratory passages must be conditioned before reaching the terminal respiratory units. Conditioning of the air occurs in the conducting portion of the respiratory system and includes warming, moistening, and removal of particulate materials. Mucous and serous secretions play a major role in the conditioning process. These secretions moisten the air and also trap particles that have managed to slip past the special short thick hairs, called vibrissae, in the nasal cavities. Mucus, augmented by these serous secretions, also prevents the dehydration of the underlying epithelium by the moving air. Mucus covers almost the entire luminal surface of the conducting passages and is continuously produced by goblet cells and mucus-secreting glands in the walls of the passages. The mucus and other secretions are moved toward the pharynx by means of coordinated sweeping movements of cilia and are then normally swallowed.

1	The nasal cavities are paired chambers separated by a bony and cartilaginous septum. They are elongated spaces with a wide base that rests on the hard and soft palate and a narrow apex that points toward the anterior cranial fossa. The skeletal framework of the nasal cavities is formed by bones and cartilages; most are located centrally in the skull except for the small anterior region that is enclosed within the external nose. Each cavity or chamber communicates anteriorly with the external environment through the anterior nares (nostrils), posteriorly with the nasopharynx through the choanae, and laterally with the paranasal sinuses and nasolacrimal duct, which drains tears from the eye into the nasal cavity (Fig. 19.2). The chambers are divided into three regions:  Nasal vestibule, which is a dilated space of the nasal cav ity just inside nostrils and lined by skin  Respiratory region, which is the largest part (inferior two-thirds) of the nasal cavities and lined by respiratory

1	is a dilated space of the nasal cav ity just inside nostrils and lined by skin  Respiratory region, which is the largest part (inferior two-thirds) of the nasal cavities and lined by respiratory mucosa  Olfactory region, which is located at the apex (upper one- third) of each nasal cavity and lined by specialized olfac

1	Vestibule of the Nasal Cavity The nasal vestibule forms a part of the external nose and communicates anteriorly with the external environment. It is lined with stratified squamous epithelium, a continuation of the skin of the face, and contains a variable number of vibrissae that entrap large particulate matter before it is carried in the air stream to the rest of the cavity. Sebaceous glands are also present, and their secretions assist in the entrapment of particulate matter. Posteriorly, where the vestibule ends, the stratified squamous epithelium becomes thinner and undergoes a transition to the pseudostratified epithelium that characterizes the respiratory region. At this site, sebaceous glands are absent.

1	trachea cricoid cartilage thyroid cartilage larynx epiglottis hyoid bone oral cavity soft palate hard palate vestibule nasal mucosa conchae olfactory mucosa choanae orifice of auditory tube nasopharynx oropharynx ventricular fold vocal fold esophagus laryngopharynx FIGURE 19.2 • Diagram of the relationship of the pharynx to the respiratory and digestive systems. The pharynx is divided into three parts: nasopharynx, oropharynx, and laryngopharynx. It is located posterior to the nasal and oral cavities and extends inferiorly past the larynx. The pharynx serves both respiratory and digestive systems. This midsagittal section also transects the cartilages forming the skeleton of the larynx (i.e., epiglottis, thyroid cartilage, and cricoid cartilage). Note the ventricular and vocal folds in the middle of the larynx, approximately at the level of the thyroid cartilage. This part of the larynx represents the narrowest portion of the respiratory system and is responsible for producing sound

1	middle of the larynx, approximately at the level of the thyroid cartilage. This part of the larynx represents the narrowest portion of the respiratory system and is responsible for producing sound by audible vibration of the vocal folds.

1	Respiratory Region of the Nasal Cavity The respiratory region constitutes most of the volume of the nasal cavities. It is lined by the respiratory mucosa that contains a ciliated, pseudostratified columnar epithelium on its surface. The underlying lamina propria is firmly attached to the periosteum and perichondrium of the adjacent bone or cartilage. The medial wall of the respiratory region, the nasal septum, is smooth, but the lateral walls are thrown into folds by the presence of three shelf-like, bony projections called conchae or turbinates. The conchae divide each nasal cavity into separate air chambers and play a dual role. They increase surface area and cause turbulence in airflow to allow more efficient conditioning of inspired air.

1	The ciliated, pseudostratified columnar epithelium of the respiratory mucosa is composed of five cell types:  Ciliated cells, tall columnar cells with cilia that project into the mucus covering the surface of the epithelium  Goblet cells that synthesize and secrete mucus  Brush cells, a general name for those cells in the respira tory tract that bear short, blunt microvilli  Small granule cells (Kulchitsky cells) that resemble basal cells but contain secretory granules. These are enteroendocrine cells of the APUD system  Basal cells, stem cells from which the other cell types arise The epithelium of the respiratory region of the nasal cavity is essentially the same as the epithelium lining most of the parts that follow in the conducting system. Because the respiratory epithelium of the trachea is studied and examined in preference to that of the nasal cavity, the above cell types are discussed in the section on the trachea (page 672).

1	The mucosa of the respiratory region warms, moistens, and filters inspired air. The lamina propria of the respiratory mucosa has a rich, vascular network that includes a complex set of capillary loops. The arrangement of the vessels allows the inhaled air to be warmed by blood flowing through the part of the loop closest to the surface. The capillaries that reside near the surface are arranged in rows; the blood flows perpendicular to the airflow, much as one would find in a mechanical heat exchange system. These same vessels may become engorged and leaky during allergic reactions or viral infections such as the common cold. The lamina propria then becomes distended with ﬂuid, resulting in marked swelling of the mucous membrane with consequent restriction of the air passage, making breathing difficult. The lamina propria also contains mucous glands, many exhibiting serous demilunes. Their secretions supplement that of the goblet cells in the respiratory epithelium.

1	By increasing surface area, the conchae (turbinates) increase the efficiency with which the inspired air is warmed. The turbinates also increase the efficiency of filtration of inspired air through the process of turbulent precipitation. The air stream is broken into eddies by the turbinates. Particulate matter suspended in the air stream is thrown out of the stream and adheres to the mucus-covered wall of the nasal cavity. Particles trapped in this layer of mucus are transported to the pharynx by means of coordinated sweeping movements of cilia and are then swallowed. Olfactory Region of the Nasal Cavity

1	Olfactory Region of the Nasal Cavity The olfactory region is located on part of the dome of each nasal cavity and, to a variable extent, the contiguous lateral and medial nasal walls. It is lined with a specialized olfactory mucosa. In living tissue, this mucosa is distinguished by its slight yellowish brown color caused by pigment in the olfactory epithelium and the associated olfactory glands. In humans, the total surface area of the olfactory mucosa is only about 10 cm2; in animals with an acute sense of smell, the total surface area of the olfactory mucosa is considerably more extensive. For instance, certain dogs species have more than 150 cm2. The lamina propria of the olfactory mucosa is directly contiguous with the periosteum of the underlying bone (Plate 69, page 688). This connective tissue contains numerous blood and lymphatic vessels, unmyelinated olfactory nerves, myelinated nerves, and olfactory glands.

1	The olfactory epithelium, like the epithelium of the respiratory region, is also pseudostratified, but it contains very different cell types. Also, it lacks goblet cells (Fig. 19.3 and Plate 69, page 688). The olfactory epithelium is composed of the following cell types:  Olfactory receptor cells are bipolar neurons that span the thickness of the epithelium and enter the central nerve system.

1	FIGURE 19.3 • Olfactory mucosa of the nasal cavity. a. This diagram shows the three major cell types located within the olfactory epithelium: the olfactory cell, supporting (sustentacular) cell, and basal cell. The olfactory cell is the receptor cell; it has an apical expansion, the olfactory vesicle, from which long, nonmotile cilia extend. At its basal surface it extends an axon into the connective tissue that joins with axons of other olfactory cells to form an olfactory nerve. The basal cells are small and cuboidal. They are restricted to the basal part of the epithelium. The supporting cells, in contrast, are columnar and extend the full thickness of the epithelium; their nuclei are located in the upper portion of the cell. Note the olfactory (Bowman’s) gland and its duct that empties on the surface of the mucosa. b. Photomicrograph of the olfactory mucosa. The olfactory epithelium exhibits nuclei through much of its thickness, but the individual cell types to which they belong

1	on the surface of the mucosa. b. Photomicrograph of the olfactory mucosa. The olfactory epithelium exhibits nuclei through much of its thickness, but the individual cell types to which they belong are not discernible. The underlying connective tissue is largely occupied by numerous olfactory (Bowman’s) glands, olfactory nerves, and blood vessels. Note that the ducts of the olfactory glands extend from the secretory portion of the gland to the epithelial surface. 240.

1	 Supporting or sustentacular cells are columnar cells that are similar to neuroglia cells and provide mechanical and metabolic support to the olfactory receptor cells. They synthesize and secrete odorant-binding proteins.  Basal cells are stem cells from which new olfactory receptor cells and supporting cells differentiate.  Brush cells are the same cell type that occurs in the respi ratory epithelium. Olfactory receptor cells are bipolar neurons that possess an apical projection bearing cilia.

1	The apical domain of each olfactory receptor cell has a single dendritic process that projects above the epithelial surface as a knoblike structure called the olfactory vesicle. A number of long, thin cilia (10 to 23) with typical basal bodies arise from the olfactory vesicle and extend radially in a plane parallel to the epithelial surface (see Fig. 19.3). The cilia are usually up to 200 m long and may overlap with cilia of adjacent olfactory receptor cells. The cilia are regarded as nonmotile, although some research suggests that they may have limited motility. The basal domain of the cell gives rise to an unmyelinated axonal process that leaves the epithelial compartment. Collections of axons from olfactory receptor cells do not come together as a single nerve, but instead they are grouped into bundles that pass through a thin cribriform plate of the ethmoid bone, course through the dura and arachnoid matters, and finally are surrounded by pia matter, entering the olfactory bulb

1	into bundles that pass through a thin cribriform plate of the ethmoid bone, course through the dura and arachnoid matters, and finally are surrounded by pia matter, entering the olfactory bulb of the brain. The collections of axons from olfactory receptor cells form the olfactory nerve (cranial nerve I). The olfactory axons are very fragile, and can be harmed during traumatic head injury. They can be permanently severed, resulting in anosmia (loss of the sense of smell).

1	Autoradiographic studies show that olfactory receptor cells have a lifespan of about 1 month. If injured, they are quickly replaced. Olfactory receptor cells (and some neurons of the enteric division of the autonomic nervous system) appear to be the only neurons in the nervous system that are readily replaced during postnatal life. Entire olfactory transduction pathways occurs within the cilia of the olfactory receptor cells.

1	All the molecules that are involved in olfactory transduction are located within long cilia that arise from the olfactory bulb. The chemical signals (the odorants) are detected and selectively bind to the odorant-binding proteins (OBPs) that are concentrated in the olfactory mucus (Fig 19.4). The OBPs are small (10 to 30 kilodalton), water-soluble proteins that are synthesized and secreted by supporting cells. First, incoming odorant molecules are solubilized in the olfactory mucus, and then OBPs act as molecular carriers that transport odorants and deliver them to the olfactory receptors (ORs) located in the plasma membrane of the cilia. Olfactory receptors are specific for the olfactory receptor cells and belong to the family of G protein–coupled receptors (known as Golf ). When stimulated by odorant molecules, Golf ORs activate enzyme adenylyl cyclase and initiate the cyclic adenosine monophosphate (cAMP) cascade of evens (see Fig 19.4). These include binding of cAMP to specific Na

1	by odorant molecules, Golf ORs activate enzyme adenylyl cyclase and initiate the cyclic adenosine monophosphate (cAMP) cascade of evens (see Fig 19.4). These include binding of cAMP to specific Na and Ca2 channel proteins and influx of Na and Ca2, which is responsible for plasma membrane depolarization that generates action potential. To accurately detect several thousands of known odor molecules by only 350 known different ORs in humans requires a special coding system for different impulses. This is achieved by a population coding scheme, in which each OR protein binds to different odorants with different sensitivity. Thus, the olfactory system must decode olfactory impulses not from only one cell, but from the entire population of cells within the olfactory epithelium.

1	Supporting cells provide mechanical and metabolic support for the olfactory receptor cells.

1	Supporting cells are the most numerous cells in the olfactory epithelium. The nuclei of these tall columnar or sustentacular cells occupy a more apical position in the epithelium than do those of the other cell types, thus aiding in their identification in the light microscope (see Fig. 19.3 and Plate 69, page 688). They have numerous microvilli on their apical surface, and abundant mitochondria. Numerous profiles of smooth endoplasmic reticulum (sER) and, to a more limited extent, rough endoplasmic reticulum (rER) are observed in the cytoplasm. They also possess lipofuscin granules. Adhering junctions are present between these cells and the olfactory receptor cells, but gap and tight junctions are absent. The supporting cells function in a manner comparable to that of neuroglial cells, providing both metabolic support (secretion of OBP molecules) and physical support to the olfactory receptor cells. Brush cells are columnar cells specialized for transduction of general sensation.

1	Brush cells are columnar cells specialized for transduction of general sensation. The olfactory epithelium also contains cells present in much smaller numbers, called brush cells. As noted, these cells are present in the epithelium of other parts of the conducting air passages. With the electron microscope (EM), brush cells exhibit large, blunt microvilli at their apical surface, a feature that gives them their name. The basal surface of a brush cell is in synaptic contact with nerve fibers that penetrate the basal lamina. The nerve fibers are terminal branches of the trigeminal nerve (cranial nerve V) that function in general sensation rather than olfaction. Brush cells appear to be involved in transduction of general sensory stimulation of the mucosa. In addition, presence of a microvillous border, vesicles near the apical cell membrane, and a well-defined Golgi apparatus suggest that brush cells might be involved in an absorptive as well as a secretory functions.

1	Basal cells are progenitors of the other mature cell types. Basal cells are small, rounded cells located close to the basal lamina. Their nuclei are frequently invaginated and lie at a level below those of the olfactory receptor cell nuclei. The cytoplasm contains few organelles, a feature consistent with their role as a reserve or stem cell. A feature consistent with their differentiation into supporting cells is the observation of

1	FIGURE 19.4 • Diagram of olfactory transduction pathway. This diagram shows interactions of the odorant molecules with proteins associated with olfactory receptor cell. Incoming in inhaled air odorant molecules are solubilized in the olfactory mucus and bind to olfactory binding proteins, which deliver them to the olfactory receptors. Note that different odorant molecules bind with different affinity to the olfactory receptors. Strong signal (see green G protein–coupled olfactory receptor) is produced by high affinity binding where odorant molecule (green) matches perfectly the binging site on the receptor. Other olfactory receptors (yellow and pink) show less affinity binding, thus producing weaker signals. Stimulated by odorant molecules olfactory receptors activate enzyme adenylyl cyclase and initiate the cAMP cascade of evens leading to opening of specific Na and Ca2channels. Influx of Na and Ca2 is responsible for cell depolarization. Generated action potential travels on axons

1	and initiate the cAMP cascade of evens leading to opening of specific Na and Ca2channels. Influx of Na and Ca2 is responsible for cell depolarization. Generated action potential travels on axons of olfactory receptor cells from nasal cavity passing through the ethmoid bone and surrounding brain coverings to the olfactory bulb of the brain.

1	processes in some basal cells that partially ensheathe the first portion of the olfactory receptor cell axon. They thus maintain a relationship to the olfactory receptor cell even in their undifferentiated state. Olfactory glands are a characteristic feature of the olfactory mucosa. The olfactory glands (Bowman’s glands), a characteristic feature of the mucosa, are branched tubuloalveolar serous glands that deliver their proteinaceous secretions via ducts onto the olfactory surface (see Fig. 19.3 and Plate 69, page 688). Lipofuscin granules are prevalent in the gland cells, and in combination with the lipofuscin granules in the supporting cells of the olfactory epithelium, they give the mucosa its natural yellow-brown coloration. Short ducts composed of cuboidal cells lead from the glands and pass through the basal lamina into the olfactory epithelium, where they continue to the epithelial surface to discharge their contents.

1	The serous secretion of the olfactory glands serves as a trap and solvent for odoriferous substances. Constant flow from the glands rids the mucosa of remnants of detected odoriferous substances so that new scents can be continuously detected as they arise. The identifying feature of the olfactory region of the nasal mucosa in a histologic preparation is the presence of the olfactory nerves in combination with olfactory glands in the lamina propria. The nerves are particularly conspicuous because of the relatively large diameter of the individual unmyelinated fibers that they contain (see Figs. 19.3 and 19.4). Paranasal sinuses are air-filled spaces in the bones of the walls of the nasal cavity.

1	Paranasal sinuses are air-filled spaces in the bones of the walls of the nasal cavity. The paranasal sinuses are extensions of the respiratory region of the nasal cavity and are lined by respiratory epithelium. The sinuses are named for the bone in which they are found (i.e., the ethmoid, frontal, sphenoid, and maxillary bones). The sinuses communicate with the nasal cavities via narrow openings onto the respiratory mucosa. The mucosal surface of the sinuses is a thin, ciliated, pseudostratified columnar epithelium with numerous goblet cells. Mucus produced in the sinuses is swept into the nasal cavities by coordinated ciliary movements. The sinuses are often subject to acute infection after viral infection of the upper respiratory tract. Severe infections may require physical drainage.

1	The pharynx connects the nasal and oral cavities to the larynx and esophagus. It serves as a passageway for air and food and acts as a resonating chamber for speech. The pharynx is located posterior to the nasal and oral cavities and is divided regionally into the nasopharynx and oropharynx, respectively (see Fig. 19.2). The auditory (Eustachian) tubes connect the nasopharynx to each middle ear. Diffuse lymphatic tissue and lymphatic nodules are present in the wall of the nasopharynx. The concentration of lymphatic nodules at the junction between the superior and posterior walls of the pharynx is called the pharyngeal tonsil.

1	The passageway for air between the oropharynx and trachea is the larynx (see Fig. 19.2). This complex tubular region of the respiratory system is formed by irregularly shaped plates of hyaline and elastic cartilage (the epiglottis and the vocal processes of the arytenoid cartilages). In addition to serving as a conduit for air, the larynx serves as the organ for producing sounds. Vocal folds control the flow of air through the larynx and vibrate to produce sound.

1	Vocal folds control the flow of air through the larynx and vibrate to produce sound. The vocal folds, also referred to as vocal cords, are two folds of mucosa that project into the lumen of the larynx (Fig. 19.5 and Plate 70, page 690). They are oriented in an anteroposterior direction and define the lateral boundaries of the opening of the larynx, the rima glottidis. A supporting ligament and skeletal muscle, the vocalis muscle, is contained within each vocal fold. Ligaments and the intrinsic laryngeal muscles join the adjacent cartilaginous plates and are responsible for generating tension in the vocal folds and for opening and closing the glottis. The extrinsic laryngeal muscles insert on cartilages of the larynx but originate in extralaryngeal structures. These muscles move the larynx during deglutition (swallowing).

1	Expelled air from lungs passing through a narrow space of rima glottidis causes the vocal folds to vibrate. The vibrations are altered by modulating the tension on the vocal folds and by changing the degree of glottal opening. This alteration of the vibrations produces sounds of different pitch. Sounds created in the larynx during the process of phonation are modified in upper parts of the respiratory system (nasopharynx, nasal cavities, and paranasal sinuses) and the oral cavity (oropharynx, soft and hard palate, tongue, teeth, lips, etc.) to produce the individual speech sounds (different vowels and consonants). The ventricular folds located above the vocal folds are the “false vocal cords.”

1	The ventricular folds located above the vocal folds are the “false vocal cords.” Above the vocal folds is an elongated recess in the larynx called the ventricle. Immediately above the ventricle is another pair of mucosal folds, the ventricular folds, or false vocal cords (see Fig. 19.5 and Plate 70, page 690). These folds do not have the intrinsic muscular investment of the true vocal cords and therefore, do not modulate in phonation. They and the ventricle, however, are important in creating sound resonance. Inﬂammation and swelling of the larynx caused by viruses (such as in common cold virus) and other microbial agents is called acute laryngitis. Symptoms of acute laryngitis may include hoarseness or, in more severe cases, the total loss of voice, coughing, and difficulty with swallowing and breathing. Chronic laryngitis is usually caused by prolonged exposure of irritating agents such as tobacco smoke, dust, and/or polluted air.

1	Stratified squamous and ciliated pseudostratified columnar epithelium line the larynx. The luminal surface of the vocal cords is covered with stratified squamous epithelium, as is most of the epiglottis (Plate 70, page 690). The epithelium serves to protect the mucosa from abrasion caused by the rapidly moving air stream. The rest of the larynx is lined with the ciliated, pseudostratified columnar epithelium that characterizes the respiratory tract (see Fig. 19.5 and Plate 70, page 690). The connective tissue of the larynx contains mixed mucoserous glands that secrete through ducts onto the laryngeal surface.

1	The trachea is a short, flexible, air tube about 2.5 cm in diameter and about 10 cm long. It serves as a conduit for air; additionally, its wall assists in conditioning inspired air. The trachea extends from the larynx to about the middle of the thorax, where it divides into the two main (primary) bronchi. The lumen of the trachea stays open because of the arrangement of the series of cartilaginous rings. vestibule of the larynx stratified squamous epithelium

1	FIGURE 19.5 • Photomicrograph of a frontal section of the larynx. a. This photomicrograph shows three parts of the larynx: the vestibule above the ventricular folds, the ventricles between the vestibular folds and superior to the vocal folds, and the infraglottic cavity that extends from the vocal folds to the cricoid cartilage. Note that mucous glands are prominent in the ventricular folds and are covered by the typical pseudostratified ciliated epithelium. The vocal fold is composed of the epithelium, vocal ligament, and underlying vocalis muscle. Numerous lymph nodules are also present within the mucosa of the larynx (arrows). 10. b. High magnification of the area of the ventricular fold indicated by the upper rectangle in a shows on the left the pseudostratified ciliated epithelium that lines most of the larynx. Many nonsmoking adults and virtually all smokers exhibit patches of stratified squamous epithelium, as seen on the right of the micrograph. 240. c. High magnification of

1	lines most of the larynx. Many nonsmoking adults and virtually all smokers exhibit patches of stratified squamous epithelium, as seen on the right of the micrograph. 240. c. High magnification of the area of the vocal fold indicated by the lower rectangle in a reveals normal stratified squamous epithelium at this site. Just beneath the epithelium is the connective tissue known as Reinke’s space. This clinically important site lacks lymphatic vessels and is poorly vascularized. The vocal ligament, inscribed by the dashed line, is seen at the bottom of the micrograph. 240.

1	The wall of the trachea consists of four definable layers:  Mucosa, composed of a ciliated, pseudostratified epithelium and an elastic, fiber-rich lamina propria  Submucosa, composed of a slightly denser connective tissue than the lamina propria  Cartilaginous layer, composed of C-shaped hyaline cartilages  Adventitia, composed of connective tissue that binds the trachea to adjacent structures A unique feature of the trachea is the presence of a series of C-shaped hyaline cartilages that are stacked one on top of each other to form a supporting structure (Fig. 19.6). These cartilages, which might be described as a skeletal framework, prevent collapse of the tracheal lumen, particularly during  FOLDER 19.1 Clinical Correlation: Squamous Metaplasia in the Respiratory Tract

1	In human respiratory mucosa, ciliated pseudostratified in certain regions to a stratified squamous form. The alcolumnar epithelium may change to stratified squamous ep-tered epithelium is more resistant to physical stress and inithelium. This change from columnar to squamous epithelium sult, but it is less effective functionally. In smokers, a similar epithelium is referred as columnar-to-squamous metapla-epithelial change occurs. Initially, the cilia on ciliated cells sia or simply the squamous metaplasia. Epithelial alter-lose their synchronous beating pattern as a result of noxations of this kind are reversible and are characterized by ious elements in smoke. As a result, removal of mucus is change from one type of fully differentiated adult cell to a dif-impaired. To compensate, the individual begins to cough, ferent type of adult cell. A given mature cell does not change thereby facilitating the expulsion of accumulated mucus in to another type of mature cell; rather, basal cell

1	begins to cough, ferent type of adult cell. A given mature cell does not change thereby facilitating the expulsion of accumulated mucus in to another type of mature cell; rather, basal cell proliferation the airway, particularly in the trachea. With time, the numgives rise to the new differentiated cell type. These cellular ber of ciliated cells decreases because of chronic cough-changes are considered to be controlled and adaptive. ing. This reduction in ciliated cells further impairs the

1	Squamous metaplasia is a normal occurrence on the normal epithelium and results in its replacement with stratrounded, more exposed portions of the turbinates, on the ified squamous epithelium at affected sites in the airway. If vocal folds, and in certain other regions. factors (i.e., tobacco smoking) that predispose to squa- Changes in the character of the respiratory epithelium mous metaplasia are not eliminated, the metaplastic epmay, however, occur in other ciliated epithelial sites when ithelium may undergo malignant transformation. Therefore, the pattern of airflow is altered or when forceful airflow oc-one of the two most common forms of cancer in the respicurs, as in chronic coughing. Typically, in chronic bronchi-ratory tract, the squamous cell carcinoma, has its origin tis and bronchiectasis, the respiratory epithelium changes from the squamous metaplastic cells.

1	expiration. Fibroelastic tissue and smooth muscle, the trachealis muscle, bridge the gap between the free ends of the C-shaped cartilages at the posterior border of the trachea, adjacent to the esophagus. Tracheal epithelium is similar to respiratory epithelium in other parts of the conducting airway. Ciliated columnar cells, mucous (goblet) cells, and basal cells are the principal cell types in the tracheal epithelium (Figs. 19.7 and 19.8). Brush cells are also present, but in small numbers, as are small granule cells.

1	19.7 and 19.8). Brush cells are also present, but in small numbers, as are small granule cells.  Ciliated cells, the most numerous of the tracheal cell types, extend through the full thickness of the epithelium. Cilia appear in histologic sections as short, hair-like profiles projecting from the apical surface (Plate 71, page 692). Each cell has approximately 250 cilia. Immediately below the cilia is a dark line formed by the aggregated ciliary basal bodies (Fig. 19.9). The cilia provide a coordinated sweeping motion of the mucous coat from the farthest reaches of the air passages toward the pharynx. In effect, the ciliated cells function as a “mucociliary escalator” that serves as an important protective mechanism for removing small inhaled particles from the lungs.

1	 Mucous cells are similar in appearance to intestinal goblet cells and are thus often referred to by the same name. They are interspersed among the ciliated cells and also extend through the full thickness of the epithelium (see Fig. 19.9). They are readily seen in the light microscope after they have accumulated mucinogen granules in their cytoplasm. Although the mucinogen is typically washed out in hematoxylin and eosin (H&E) preparations, the identity of the cell is made apparent by the remaining clear area in the cytoplasm and the lack of cilia at the apical surface. In contrast to ciliated cells, the number of mucous cells increases during chronic irritation of the air passages.

1	 Brush cells have the same general features as those described for the respiratory epithelium of the nasal cavity (Fig. 19.10). They are columnar cells that bear blunt microvilli. The basal surface of the cells is in synaptic contact with an afferent nerve ending (epitheliodendritic synapse). Thus, the brush cell is regarded as a receptor cell.

1	 Small granule cells (Kulchitsky cells) are respiratory representatives of the general class of enteroendocrine cells of the gut and gut derivatives (see Fig. 19.10). Their presence is explained by the development of the respiratory tract and lungs from an evagination of the primitive foregut. Small granule cells usually occur singly in the trachea and are sparsely dispersed among the other cell types. They are difficult to distinguish from basal cells in the light microscope without special techniques such as silver staining, which reacts with the granules. The nucleus is located near the basement membrane; the cytoplasm is somewhat more extensive than that of the smaller basal cells. With the transmission electron microscope (TEM), a thin, tapering cytoplasmic process is sometimes observed extending to the lumen. Also, with the TEM, the cytoplasm exhibits numerous, membrane-bounded, dense-core granules. In one type of small granule cell, the secretion is a catecholamine. A second

1	extending to the lumen. Also, with the TEM, the cytoplasm exhibits numerous, membrane-bounded, dense-core granules. In one type of small granule cell, the secretion is a catecholamine. A second cell type produces polypeptide hormones such as serotonin, calcitonin, and gastrin-releasing peptide

1	FIGURE 19.6 • Photomicrograph of a section of the trachea and esophagus. a. This specimen, obtained from an elderly individual, shows the relationship between the trachea and the esophagus at the base of the neck. The cartilaginous tracheal rings, which keep the trachea patent, have a C-shaped appearance. The cartilage gap, where the trachea is adjacent to the esophageal wall, is spanned by a fibroelastic membrane. It contains the trachealis muscle and numerous seromucous glands. In this specimen, the tracheal ring has been transformed, in part, to bone, a process that occurs in aging. The darker-staining material represents cartilage, whereas the lighter-staining material has been replaced by bone tissue. The very light areas (arrows) are marrow spaces. 3.25. b. This high-magnification photomicrograph shows an area of the tracheal ring that has partially transformed into bone. The top of the micrograph shows the tracheal mucosa and submucosa. Below is part of the tracheal ring. In

1	photomicrograph shows an area of the tracheal ring that has partially transformed into bone. The top of the micrograph shows the tracheal mucosa and submucosa. Below is part of the tracheal ring. In this particular region, however, a substantial portion of the cartilage has been replaced by bone tissue and marrow. The bone tissue exhibits typical lamellae and osteocytes. The cartilage tissue, in contrast, exhibits nests of chondrocytes. 100.

1	(bombesin). Some small granule cells appear to be innervated. The function of these cells is not well understood. Some are present in groups in association with nerve fibers, forming neuroepithelial bodies, which are thought to function in reflexes regulating the airway or vascular caliber.  Basal cells serve as a reserve cell population that maintains individual cell replacement in the epithelium. Basal cells tend to be prominent because their nuclei form a row in close proximity to the basal lamina. Although nuclei of other cells reside at this same general level within the epithelium, they are relatively sparse. Thus, most of the nuclei near the basement membrane belong to basal cells. A thick “basement membrane” is characteristic of tracheal epithelium.

1	A thick “basement membrane” is characteristic of tracheal epithelium. Located beneath the tracheal epithelium is a distinctive layer typically referred to as a basement membrane (see Fig. 19.9). It usually appears as a glassy or homogeneous light-staining layer approximately 25 to 40 m thick. Electron microscopy reveals that it consists of densely packed collagenous fibers that lie immediately under the epithelial basal lamina. Structurally, it can be regarded as an unusually thick and dense reticular lamina and, as such, is part of the lamina propria. In smokers, particularly those who experience chronic coughing, this layer may be considerably thicker, a response to mucosal irritation.

1	The lamina propria, excluding that part just designated as basement membrane, appears as a typical loose connective tissue. It is very cellular, containing numerous lymphocytes, many of which infiltrate the epithelium. Plasma cells, mast cells, eosinophils, and fibroblasts are the other cell types readily observed in this layer. Lymphatic tissue, in both diffuse and nodular forms, is consistently present in the lamina propria and submucosa of the tracheal wall. It is also present in other parts of the respiratory system involved primarily with air conduction. This lymphatic tissue is the developmental and functional equivalent of the bronchus-associated lymphatic tissue (BALT).

1	FIGURE 19.7 • Electron micrograph of human trachea. This electron micrograph shows the three main cell types of this respiratory epithelium. They are represented by ciliated epithelial cells extending to the surface, where they possess cilia; goblet cells with mucinogen granules; and basal cells, which are confined to the basal portion of the epithelial layer near the connective tissue. 1,800. (Courtesy of Dr. Johannes A. G. Rhodin.) The boundary between mucosa and submucosa is defined by an elastic membrane. Interspersed among the collagenous fibers are numerous elastic fibers. Where the lamina propria ends, the elastic material is more extensive, and in specimens stained for these fibers, a distinct band of elastic material is seen. This band or elastic membrane marks the boundary between the lamina propria and submucosa. In H&E preparations, however, the boundary is not obvious.

1	The submucosa is unlike that of most other organs, in which this connective tissue typically has a dense character. In the trachea, the submucosa is a relatively loose connective tissue similar in appearance to the lamina propria, which makes it difficult to determine where it begins. Diffuse lymphatic tissue and lymphatic nodules characteristically extend into this layer from the lamina propria. The submucosa contains the larger distributing vessels and lymphatics of the tracheal wall. Submucosal glands composed of mucus-secreting acini with serous demilunes are also present in the submucosa. Their ducts consist of a simple cuboidal epithelium and extend through the lamina propria to deliver their product, largely glycoproteins, on the epithelial surface. The glands are

1	FIGURE 19.8 • Scanning electron micrograph of the luminal surface of a bronchus. The nonciliated cells are the goblet cells (G). Their surface is characterized by small blunt microvilli that give a stippled appearance to the cell at this low magnification. The cilia of the many ciliated cells occupy the remainder of the micrograph. Note how all are “synchronously” arrayed (i.e., uniformly leaning in the same direction) appearing just as they were when fixed at a specific moment during their wavelike movement. 1,200. especially numerous in the cartilage-free gap on the posterior portion of the trachea. Some penetrate the muscle layer at this site and, therefore, also lie in the adventitia. The submucosal layer ends where its connective tissue fibers blend with the perichondrium of the cartilage layer. The tracheal cartilages and trachealis muscle separate submucosa from adventitia.

1	The tracheal cartilages and trachealis muscle separate submucosa from adventitia. The tracheal cartilages, which number about 16 to 20 in humans, represent the next layer of the tracheal wall. As noted, the cartilages are C shaped. They sometimes anastomose with

1	FIGURE 19.9 • Photomicrograph of tracheal epithelium. Three major cell types are evident in the tracheal epithelium (Ep): ciliated columnar cells; mucus-secreting goblet cells (G) interspersed between the ciliated cells; and basal cells, which are close to the basement membrane (BM ). The ciliated columnar cells extend from the basement membrane to the surface. At their free surface they contain numerous cilia that, together, give the surface a brushlike appearance. At the base of the cilia is a dense eosinophilic line. This is owing to the linear aggregation of structures referred to as basal bodies, located at the proximal end of each cilium. Although basement membranes are not ordinarily seen in H&E preparations, a structure identified as such is seen regularly under the epithelium in the human trachea. The underlying lamina propria (LP ) consists of loose connective tissue. The more deeply located submucosa (SM) contains dense irregular connective tissue with blood and lymphatic

1	the human trachea. The underlying lamina propria (LP ) consists of loose connective tissue. The more deeply located submucosa (SM) contains dense irregular connective tissue with blood and lymphatic vessels, nerves, and numerous mucus-secreting tracheal glands. 400.

1	adjacent cartilages, but their arrangement provides flexibility to the tracheal pipe and also maintains patency of the lumen. With age, the hyaline cartilage may be partially replaced by bone tissue (see Fig. 19.6), causing it to lose much of its flexibility. The adventitia, the outer layer, lies peripheral to the cartilage rings and trachealis muscle. It binds the trachea to adjacent

1	The adventitia, the outer layer, lies peripheral to the cartilage rings and trachealis muscle. It binds the trachea to adjacent FIGURE 19.10 • Diagram of a brush cell and small granule cell. a. The brush cell, as illustrated here, is interposed between type I and type II alveolar cells of an alveolus. Blunt microvilli are distinctive features of the brush cell. The cytoplasm typically shows a Golgi apparatus, lysosomes, mitochondria, and glycogen inclusions. b. This small granule cell is shown located between two Clara cells, as in a terminal or respiratory bronchiole. The cell contains small secretory vesicles, most of which are in the basal portion of the cell. In addition to the vesicles, the most conspicuous organelles of the cell are rough-surfaced endoplasmic reticulum (rER), a Golgi apparatus, and mitochondria. A nerve terminal is shown within the epithelium adjacent to the cell.

1	structures in the neck and mediastinum and contains the largest blood vessels and nerves that supply the tracheal wall, as well as the larger lymphatics that drain the wall.

1	The trachea divides into two branches forming the main (primary) bronchi. Anatomically, these divisions are more frequently described as simply the right and left main bronchi, terms that are more useful because of the physical difference between the two. The right bronchus is wider and significantly shorter than the left. On entering the hilum of the lung, each main bronchus divides into the lobar bronchi (secondary bronchi). The left lung is divided into two lobes; the right lung is divided into three lobes. Thus, the right bronchus divides into three lobar bronchial branches, and the left into two lobar bronchial branches, with each branch supplying one lobe. The left lung is further divided into 8 bronchopulmonary segments and the right lung into 10 such segments. Thus, in the right lung the lobar bronchi give rise to 10 segmental bronchi (tertiary bronchi); the lobar bronchi of the left lung give rise to 8 segmental bronchi.

1	A segmental bronchus and the lung parenchyma that it supplies constitute a bronchopulmonary segment. The significance of the bronchopulmonary segment in the human lung becomes apparent when considering the need for surgical resection, which may be required in certain disease states. The segments, each with their own blood supply and connective tissue septa, are convenient subunits that facilitate surgical resection.

1	Initially, the bronchi have the same general histologic structure as the trachea. At the point where the bronchi enter the lungs to become intrapulmonary bronchi, the structure of the bronchial wall changes. The cartilage rings are replaced by cartilage plates of irregular shape. The plates are distributed in a linear array around the entire circumference of the wall, giving the bronchi a circular or cylindrical shape in contrast to the ovoid shape with a flattened posterior wall of the trachea. As the bronchi decrease in size because of branching, the cartilage plates become smaller and less numerous. The plates ultimately disappear at the point where the airway reaches a diameter of about 1 mm, whereupon the branch is designated a bronchiole. Bronchi can be identified by their cartilage plates and a circular layer of smooth muscle.

1	Bronchi can be identified by their cartilage plates and a circular layer of smooth muscle. The second change observed in the wall of the intrapulmonary bronchus is the addition of smooth muscle to form a complete circumferential layer. The smooth muscle becomes an increasingly conspicuous layer as the amount of cartilage diminishes. Initially, the smooth muscle is arranged in interlacing bundles forming a continuous layer. In the smaller bronchi, the smooth muscle may appear discontinuous.

1	Because the smooth muscle forms a separate layer, namely, a muscularis, the wall of the bronchus can be regarded as having five layers:  Mucosa, composed of a pseudostratified epithelium with the same cellular composition as the trachea. The height of the cells decreases as the bronchi decrease in diameter. In H&E specimens the “basement membrane” is conspicuous in the primary bronchi but quickly diminishes in thickness and disappears as a discrete structure in the secondary bronchi. The lamina propria is similar to that of the trachea but is reduced in amount in proportion to the diameter of the bronchi.  Muscularis, a continuous layer of smooth muscle in the larger bronchi. It is more attenuated and loosely organized in smaller bronchi, where it may appear discontinuous because of its spiral course. Contraction of the muscle regulates the appropriate diameter of the airway.

1	 Submucosa remains as a relatively loose connective tissue. Glands are present as well as adipose tissue in the larger bronchi.  Cartilage layer consists of discontinuous cartilage plates that become smaller as the bronchial diameter diminishes.  Adventitia is moderately dense connective tissue that is continuous with that of adjacent structures, such as pulmonary artery and lung parenchyma.

1	 Adventitia is moderately dense connective tissue that is continuous with that of adjacent structures, such as pulmonary artery and lung parenchyma. The bronchopulmonary segments are further subdivided into pulmonary lobules; each lobule is supplied by a bronchiole. Delicate connective tissue septa that partially separate adjacent lobules may be represented on the surface of the lung as faintly outlined polygonal areas. Pulmonary acini are smaller units of structure that make up the lobules. Each acinus consists of a terminal bronchiole and the respiratory bronchioles and alveoli that it aerates (Fig. 19.11). The smallest functional unit of pulmonary structure is thus the respiratory bronchiolar unit. It consists of a single respiratory bronchiole and the alveoli that it supplies.

1	Bronchioles are air-conducting ducts that measure 1 mm or less in diameter. The larger bronchioles represent branches of the segmental bronchi. These ducts branch repeatedly, giving rise to the smaller terminal bronchioles that also branch. The terminal bronchioles finally give rise to the respiratory bronchioles. Cartilage plates and glands are not present in bronchioles. The larger-diameter bronchioles initially have a ciliated, pseudostratified columnar epithelium that gradually transforms into a simple ciliated columnar epithelium as the duct narrows. Goblet cells are still present in the largest bronchioles but are not present in the terminal bronchioles that follow. An exception is in smokers and others exposed to irritants in the air. There are no subepithelial glands in

1	FIGURE 19.11 • Photomicrograph showing the respiratory portion of the bronchial tree. In this photomicrograph a terminal bronchiole (TB) is shown longitudinally sectioned as it branches into two respiratory bronchioles (RB). The terminal bronchiole is the most distal part of the conducting portion of the respiratory system and is not engaged in gas exchange. The respiratory bronchiole engages in gas exchange and is the beginning of the respiratory portion of the bronchial tree. Respiratory bronchioles give rise to alveolar ducts (AD), which are elongate airways that have almost no walls, only alveoli surrounding the duct space. Alveolar sacs (AS) are spaces at the termination of the alveolar ducts that, likewise, are surrounded by alveoli. 120.

1	bronchioles. Cartilage plates, characteristic of bronchi, are absent in bronchioles. Instead, small elements of cartilage may be present, particularly at branching points. A relatively thick layer of smooth muscle is present in the wall of all bronchioles. Small bronchioles have a simple cuboidal epithelium. The smallest conducting bronchioles, the terminal bronchioles, are lined with a simple cuboidal epithelium in which Clara cells are interspersed among the ciliated cells (Fig. 19.12). Clara cells increase in number as the ciliated cells decrease along the length of the bronchiole. Occasional brush cells and small granule cells are also present. A small amount of connective tissue underlies the epithelium, and a circumferential layer of smooth muscle underlies the connective tissue in the conducting portions. Clara cells are nonciliated cells that have a characteristic rounded or dome-shaped apical surface projection. They display TEM characteristics of protein-secreting cells

1	Clara cells are nonciliated cells that have a characteristic rounded or dome-shaped apical surface projection. They display TEM characteristics of protein-secreting cells FIGURE 19.12 • Scanning electron micrograph of a terminal bronchiole. This scanning photomicrograph shows a longitudinal section throughout the terminal bronchiole and surrounding alveoli (A). Note that the apical surfaces of the Clara cells possess no cilia and have a characteristic dome-shaped appearance. 150. The inset shows some of the Clara cells at a higher magnification and the cilia of a neighboring ciliated cell, which are present in very small numbers at this level. Note the relatively few cilia present on these small cells. 1,200.

1	(Fig. 19.13). They have a well-developed basal rER, a lateral or supranuclear Golgi apparatus, secretory granules that stain for protein, and numerous cisternae of sER in the apical cytoplasm. Clara cells secrete a surface-active agent, a lipoprotein that prevents luminal adhesion should the wall of the airway collapse on itself, particularly during expiration. In addition, Clara cells produce a 16-kilodalton protein known as Clara cell secretory protein (CC16), which is an abundant component of the airway secretion. Chronic lung diseases such as chronic obstructive pulmonary disease (COPD) and asthma are associated with changes in the abundance of CC16 in airway ﬂuid and serum. CC16 is used as a measurable pulmonary marker in bronchoalveolar lavage ﬂuid and serum. Secretion of CC16 into the bronchial tree decreases during lung injury (because of damage to the Clara cells), whereas serum levels of CC16 may increase because of leakage across the air–blood barrier.

1	FIGURE 19.13 • Diagram of a Clara cell between bronchiolar ciliated epithelial cells. The nucleus is in a basal location. Rough-surfaced endoplasmic reticulum (rER), a Golgi apparatus, and mitochondria are chiefly in basal and paranuclear locations of the cell. Smooth-surfaced endoplasmic reticulum (sER) and secretory vesicles are chiefly in the apical cytoplasm. One of the secretory vesicles is shown discharging its contents onto the surface of the cell. Respiratory bronchioles are the first part of the bronchial tree that allows gas exchange.

1	Respiratory bronchioles are the first part of the bronchial tree that allows gas exchange. Respiratory bronchioles constitute a transitional zone in the respiratory system; they are involved in both air conduction and gas exchange. They have a narrow diameter and are lined by cuboidal epithelium. The epithelium of the initial segments of the respiratory bronchioles contains both ciliated cells and Clara cells (see Fig. 19.12). Distally, Clara cells predominate. Occasional brush cells and dense-core granule cells are also present along the length of the respiratory bronchiole. Scattered, thin-walled outpocketings, alveoli, extend from the lumen of the respiratory bronchioles (see Fig. 19.11). Alveoli are the sites at which air leaves and enters the bron chiole to allow gas exchange. Alveoli are the site of gas exchange.

1	Alveoli are the site of gas exchange. The surface area available for gas exchange is increased by the lung alveoli. Alveoli are the terminal air spaces of the respiratory system and are the actual sites of gas exchange between the air and the blood. Each alveolus is surrounded by a network of capillaries that brings blood into close proximity to inhaled air inside the alveolus. About 150 to 250 million alveoli are found in each adult lung; their combined internal surface area is approximately 75 m2, roughly the size of a tennis court. Each alveolus is a thin-walled polyhedral

1	FIGURE 19.14 • Photomicrograph showing an alveolar sac with adjacent alveoli. This photomicrograph shows the terminal components of the respiratory system, namely, the alveolar sac (AS) and the surrounding alveoli (A). The alveoli are surrounded and separated from one another by a thin connective tissue layer, the interalveolar septa, containing blood capillaries. On the right is the lung surface, which is covered by visceral pleura containing simple squamous epithelium and an underlying layer of connective tissue. 360. chamber approximately 0.2 mm in diameter that is confluent with an alveolar sac (Fig. 19.14).  Alveolar ducts are elongated airways that have almost no walls, only alveoli, as their peripheral boundary. Rings of smooth muscle are present in the knoblike interalveolar septa (see the next paragraph). Alveolar sacs are spaces surrounded by clusters of alveoli. The surrounding alveoli open into these spaces.

1	The surrounding alveoli open into these spaces. Alveolar sacs usually occur at the termination of an alveolar duct but may occur anywhere along its length. Alveoli are surrounded and separated from one another by an exceedingly thin connective tissue layer that contains blood capillaries. The tissue between adjacent alveolar air spaces is called the alveolar septum or septal wall (Fig. 19.15). Alveolar epithelium is composed of type I and II alveolar cells and occasional brush cells.

1	Alveolar epithelium is composed of type I and II alveolar cells and occasional brush cells. The alveolar surface forms a vulnerable biologic interface that is subject to many destabilizing surface forces and to continuous exposure to inhaled particles, pathogens, and toxins. The alveolar epithelium is composed of several specialized cells and their products, some of which play defensive and protective roles:  Type I alveolar cells, also known as type I pneumocytes, comprise only 40% of the entire alveolar lining cells. They are extremely thin squamous cells; they line most (95%) of the surface of the alveoli (see Fig. 19.15). These cells are joined to one another and to the other cells of the alveolar epithelium by occluding junctions (Fig. 19.16). The junctions form an effective barrier between the air space and the components of the septal wall. Type I alveolar cells are not capable of cell division.

1	 Type II alveolar cells, also called type II pneumocytes or septal cells, are secretory cells. These cuboidal cells are interspersed among the type I cells but tend to congregate at septal junctions. Type II cells account for 60% of the alveolar lining cells, but because of their different shape they cover only about 5% of the alveolar air surface. Like Clara cells, type II cells tend to bulge into the air space (see Fig. 19.16). Their apical cytoplasm is filled with granules that are resolved with the TEM (Fig. 19.17) as stacks of parallel membrane lamellae, the lamellar bodies. They are rich in a mixture of phospholipids, neutral lipids, and proteins, that is secreted by exocytosis to form an alveolar lining, surface-active agent called surfactant. In addition to secretion of surfactant, type II alveolar cells are progenitor cells for type I alveolar cells. After lung injury, they proliferate and restore both types of alveolar cells within the alveolus. Hyperplasia of type II

1	type II alveolar cells are progenitor cells for type I alveolar cells. After lung injury, they proliferate and restore both types of alveolar cells within the alveolus. Hyperplasia of type II alveolar cells is an important marker of alveolar injury and repair of alveoli.

1	 Brush cells are also present in the alveolar wall, but they are few in number. They may serve as receptors that monitor air quality in the lung. Surfactant decreases the alveolar surface tension and actively participates in the clearance of foreign materials.

1	The surfactant layer produced by type II alveolar cells reduces the surface tension at the air–epithelium interface. The most critical agent for air space stability is a specific phospholipid called dipalmitoylphosphatidylcholine (DPPC), which accounts for almost all surface tension–reducing properties of surfactant. Surfactant synthesis in the fetus occurs after the 35th week of gestation and is modulated by a variety of hormones, including cortisol, insulin, prolactin, and thyroxine. Without adequate secretion of surfactant, the alveoli would collapse on each successive exhalation. Such collapse occurs in premature infants whose lungs have not developed sufficiently to produce surfactant, causing neonatal respiratory distress syndrome (RDS). Prophylactic administration of exogenous surfactant at birth to extremely premature infants and administration to symptomatic newborns reduces the risk of RDS. In addition, administration of cortisol to mothers with threatened premature delivery

1	at birth to extremely premature infants and administration to symptomatic newborns reduces the risk of RDS. In addition, administration of cortisol to mothers with threatened premature delivery decreases neonatal mortality.

1	Surfactant proteins help organize the surfactant layer and modulate alveolar immune responses. In addition to phospholipids, hydrophobic proteins are necessary for the structure and function of surfactant. These proteins are listed here:  Surfactant protein A (SP-A), the most abundant surfactant protein. SP-A is responsible for surfactant homeostasis (regulating synthesis and secretion of surfactant by type II alveolar cells). It also modulates immune responses to viruses, bacteria, and fungi.

1	FIGURE 19.15 • Electron micrograph of lung alveoli. This electron micrograph shows two alveolar spaces separated by an alveolar septum containing capillaries, some of which contain red blood cells. Note the areas of thin and thick portions of the alveolar septum. These are shown at a higher magnification in Figure 19.19. 5,800. Inset. Photomicrograph of an alveolus for comparison with the alveolar wall as seen in an electron micrograph. Arrows indicate alveolar capillaries containing red blood cells. 480.  Surfactant protein B (SP-B), an important protein for the transformation of the lamellar body into the thin surface film of surfactant. SP-B is a critical surfactant-organizing protein responsible for adsorption and spreading of surfactant onto the surface of the alveolar epithelium.

1	 Surfactant protein C (SP-C), which represents only 1% of the total mass of surfactant protein. Along with SP-B, SP-C aids in orientation of DPPC within the surfactant and maintenance of the thin film layer within the alveoli.  Surfactant protein D (SP-D), a primary protein involved in host defense. It binds to various microorganisms (e.g., Gram-negative bacteria) and to lymphocytes. SP-D participates in a local inﬂammatory response to acute lung injury and with SP-A modulates an allergic response to various inhaled antigens. The alveolar septum is the site of the air–blood barrier.

1	The alveolar septum is the site of the air–blood barrier. The air–blood barrier refers to the cells and cell products across which gases must diffuse between the alveolar and capillary compartments. The thinnest air–blood barrier consists of a thin layer of surfactant, a type I epithelial cell and its basal lamina, and a capillary endothelial cell and its basal lamina. Often, these two basal laminae are fused (Fig. 19.18). Connective tissue cells and fibers that may be present between the two basal laminae widen the air–blood barrier. These two arrangements produce a thin portion and a thick portion of the barrier (Fig. 19.19). It is thought that most gas exchange occurs across the thin portion of the barrier. The thick portion is thought to be a site in which tissue fluid can accumulate and even cross into the alveolus. Lymphatic vessels in the connective tissue of the terminal bronchioles drain fluid that accumulates in the thick portion of the septum.

1	Alveolar macrophages remove inhaled particulate matter from the air spaces and red blood cells from the septum. Alveolar macrophages are unusual in that they function both in the connective tissue of the septum and in the air FIGURE 19.16 • Electron micrograph of a type II alveolar cell. The type II alveolar cell has a dome-shaped apical surface with a number of short microvilli at its periphery and a relatively smooth-contoured apical center. The lateral cell margins are overlain to a variable degree by the type I alveolar cells that are joined to the type II cell by occluding junctions. Both cell types rest on the basal lamina (BL). The secretory vesicles (G) in this specimen are largely dissolved, but their lamellar character is shown to advantage in Figure 19.17b. 24,000.

1	space of the alveolus (Fig. 19.20). In air spaces, they scavenge the surface to remove inhaled particulate matter (e.g., dust and pollen), thus giving them one of their alternative names, dust cells. Alveolar macrophages are derived from blood monocytes and belong to the mononuclear phagocytotic system (see page 185). They phagocytize red blood cells that may enter the alveoli in heart failure (see Fig. 19.20). Some engorged macrophages pass up the bronchial tree in the mucus and are disposed of by swallowing or expectoration when they reach the pharynx. Other macrophages return to or remain in the septal connective tissue, where, filled with accumulated phagocytized material, they may remain for much of an individual’s life. Thus, at autopsy, the lungs of urban dwellers and smokers usually show many alveolar and septal macrophages filled with carbon particles, anthracotic pigment, and birefringent needle-like particles of silica. Alveolar macrophages also phagocytose infectious

1	usually show many alveolar and septal macrophages filled with carbon particles, anthracotic pigment, and birefringent needle-like particles of silica. Alveolar macrophages also phagocytose infectious organisms such as Mycobacterium tuberculosis, which can be recognized in the cells in appropriately stained specimens. These bacilli are not digested by macrophages, however, other infections or conditions that damage alveolar macrophages can cause release of the bacteria and recurrent tuberculosis.

1	Collateral air circulation through alveolar pores allows air to pass between alveoli. Scanning electron microscopic studies of alveolar structure show openings in the interalveolar septa that allow circulation of air from one alveolus to another. These alveolar pores (of Kohn) can be of great significance in some pathologic conditions in which obstructive lung disease blocks the normal pathway of air to the alveoli. The alveoli distal to the blockage may continue to be aerated, via the pores, from an adjacent lobule or acinus. A basic summary of information related to the respiratory system is included in Figure 19.21. surfactant discharged into lumen of alveolus

1	surfactant discharged into lumen of alveolus FIGURE 19.17 • Diagram of a type II alveolar cell and electron micrograph of lamellar bodies. a. Surfactant is an oily mixture of proteins, phospholipids, and neutral lipids that are synthesized in the rER from precursors in the blood. These precursors are glucose, fatty acids, choline, and amino acids. The protein constituents of surfactant are produced in the rER and stored in the cytoplasm within lamellar bodies, which are discharged into the lumen of the alveolus. With the aid of surfactant protein, surfactant is distributed, on the surface of epithelial cells lining the alveolus, as a thin film that reduces the surface tension. b. Higher-magnification electron micrograph showing the typical lamellar pattern of the secretory vesicles of type II alveolar cells. These vesicles contain the pulmonary surfactant precursor proteins. 38,000. (Courtesy of Dr. A. Mercuri.)

1	FIGURE 19.18 • Diagram of the interalveolar septum. This diagram shows the thick and thin portions of the interalveolar septum. The thin portion forms the air–blood barrier and is responsible for most of the gas exchange that occurs in the lung. Arrows indicate the direction of CO2 and O2 exchange between the alveolar air space and the blood. The thick portion of the interalveolar septum plays an important role in fluid distribution and its dynamics. It contains connective tissue cells. Note the macrophage in the thick portion that extends its processes into the lumen of the alveolus.

1	FIGURE 19.19 • Electron micrograph of the alveolar septum. This high-magnification micrograph shows the thin portion of the air–blood barrier where it consists of type I alveolar cells, capillary endothelium, and the fused basal lamina shared by both cells. In the thick portion, the type I alveolar cell (arrows) rests on a basal lamina, and on the opposite side is connective tissue in which collagen fibrils and elastic fibers are evident. 33,000. FIGURE 19.20 • Photomicrograph of alveolar macrophages.

1	FIGURE 19.20 • Photomicrograph of alveolar macrophages. This high-magnification photomicrograph shows the structure of the alveolar septum and the lumen of an alveolus containing alveolar macrophages and red blood cells. The cytoplasm of the alveolar macrophages, when they are present in significant numbers, often contains the brown pigment hemosiderin from phagocytosed red blood cells. These hemosiderin-laden macrophages (often called “heart failure cells”) are typically found in heart disease, mostly left ventricular failures that cause pulmonary congestion and edema. This results in enlargement of the alveolar capillaries and small hemorrhages into the alveoli. 560. trachea main bronchus lobar bronchus segmental bronchus terminal bronchiole respiratory bronchioles alveolar duct alveolar sacs alveoli RESPIRATORY CONDUCTING FIGURE 19.21 • Divisions of the bronchial tree and summary of its histological features. 684  FOLDER 19.2 Clinical Correlation: Cystic Fibrosis

1	Cystic fibrosis (CF, mucoviscidosis) is a chronic obstructive pulmonary disease of children and young adults. It is an autosomal recessive disorder caused by a mutation in a gene called the cystic fibrosis transmembrane conductance regulator (CFTR) located on chromosome 7. The product of this gene the Cl channel protein is involved in final alteration of mucus and digestive secretions, sweat, and tears. All mutations of CFTR gene results in abnormal epithelial transport of Cl that affects the viscosity of the secretion of the exocrine glands. Almost all exocrine glands secrete abnormally viscid mucus that obstructs the glands and their excretory ducts. The course of the disease is largely determined by the the CFTR gene could lead to the use of gene therapy in degree of pulmonary involvement. At birth, the lungs are the near future.

1	The course of the disease is largely determined by the the CFTR gene could lead to the use of gene therapy in degree of pulmonary involvement. At birth, the lungs are the near future. FIGURE F19.2.1 • Schematic diagram of pathology in cystic fibrosis. In cystic fibrosis, secretion of Cl anions into the lumen of the bronchial tree is markedly decreased because of a defective or nonexistent chloride channel protein. Na resorption from the lumen of the bronchial tree is then increased, causing movement of water into the cell. As a result, the mucous layer within the bronchial tree becomes dehydrated and viscous. This thick mucus is difficult to move by the mucociliary escalator mechanism, and it clogs the lumen of the bronchial tree, obstructing airflow.

1	normal. However, the defective Cl channel protein in the bronchial epithelium causes decreased Cl secretion and increased Na and water reabsorption from the lumen (Fig. F19.2.1). As a result, the “mucociliary escalator” malfunctions, with consequent accumulation of an unusually thick, viscous mucous secretion. The pulmonary lesion is probably initiated by obstruction of the bronchioles. Bronchiolar obstruction blocks the airways and leads to thickening of the bronchiole walls and to other degenerative changes in the alveoli. Because fluids remain trapped in the lungs, individuals with cystic fibrosis have frequent respiratory tract infections. The cloning of

1	Emphysema is a condition of the lung characterized by permanent enlargement of the air spaces distal to the ter-minal bronchiole. This enlargement is caused by chronic obstruction of airflow, most often because of narrowing of the bronchioles, and is accompanied by destruction of the alveolar wall (Fig. F19.3.1). Thus, significant area for gas exchange is lost in this disease. Emphysema is relatively common; it is seen in about half of all autopsies and is eas-ily recognized. Pathologists identify several types of em-physema. The severity of the disease is clinically more important, however, than recognition of the specific type. Emphysema is often caused by chronic inhalations of foreign particulate material such as coal dust, textile fibers, and construction dust. The most common cause, however, is cigarette smoking. The destruction of the alveolar wall may be associated with excess lysis of elastin and other structural proteins in the alveolar septa. Elastase and other proteases

1	however, is cigarette smoking. The destruction of the alveolar wall may be associated with excess lysis of elastin and other structural proteins in the alveolar septa. Elastase and other proteases are derived from lung neutrophils, macrophages, and monocytes. A specific genetic disease, 1-antitrypsin deficiency, causes a particularly severe form of emphy-sema in both heterozygous and homozygous individuals and/or chronic obstructive pulmonary disease (COPD). It is usually fatal in homozygotes if untreated, but its sever-ity can be reduced by supplying the enzyme inhibitor ex-ogenously. FIGURE F19.3.1 • Photomicrographs of emphysema and pneumonia. a. This photomicrograph from the lung of an individual with emphysema shows the partial destruction of interalveolar septa, resulting in permanent enlargement of the air spaces. Note that the changes in the lung parenchyma are accompanied by thickening of the wall of the pulmonary vessels (arrows) and the presence of numerous cells within the

1	enlargement of the air spaces. Note that the changes in the lung parenchyma are accompanied by thickening of the wall of the pulmonary vessels (arrows) and the presence of numerous cells within the air spaces. These cells are the alveolar macrophages and are shown at higher magnification in Figure 19.20. 240. b. This photomicrograph is from the lung of an individual in the early stages of acute pneumonia (inflammation of the lung). Note that the air spaces are filled with exudate containing white blood cells (mainly neutrophils), red blood cells, and fibrin. The capillaries in the alveolar septum are enlarged and congested with red blood cells. Pathologists recognize this stage as the red hepatization stage of the pneumonia. At this stage, the affected portion of the lung on gross examination appears red (because of enlarged capillaries), firm (because of the lack of air spaces), and heavy (because of the presence of exudate within the alveoli); the term hepatization stems from the

1	appears red (because of enlarged capillaries), firm (because of the lack of air spaces), and heavy (because of the presence of exudate within the alveoli); the term hepatization stems from the tissue’s resemblance to the liver. 240. Inset. Part of an alveolus at a higher magnification. Note the enlarged, congested capillary within the alveolar septum. The air space is filled with neutrophils and red blood cells. The lower right corner shows early organization of the intra-alveolar exudate; observe that the developing fibrin network contains entrapped neutrophils and several red blood cells. 420. aaaaa bbbbb  FOLDER 19.3 Clinical Correlation: Emphysema and Pneumonia

1	The lung has both pulmonary and bronchial circulations. The pulmonary circulation supplies the capillaries of the alveolar septum and is derived from the pulmonary artery that leaves the right ventricle of the heart. The branches of the pulmonary artery travel with those of the bronchi and bronchioles and carry blood down to the capillary level at the alveoli. This blood is oxygenated and collected by pulmonary venous capillaries that join to form venules. They ultimately form the four pulmonary veins that return blood to the left atrium of the heart. The pulmonary venous system is located at a distance from the respiratory passages at the periphery of the bronchopulmonary segments.

1	The bronchial circulation, via bronchial arteries that branch from the aorta, supplies all of the lung tissue other than the alveoli (i.e., the walls of the bronchi and bronchioles and the connective tissue of the lung other than that of the alveolar septum). The finest branches of the bronchial arterial tree also open into the pulmonary capillaries. Therefore, the bronchial and pulmonary circulations anastomose at about the level of the junction between the conducting and respiratory passages. Bronchial veins drain only the connective tissue of the hilar region of the lungs. Most of the blood reaching the lungs via the bronchial arteries leaves the lungs via the pulmonary veins.

1	A dual lymphatic drainage of the lungs parallels the dual blood supply. One set of lymphatic vessels drains the parenchyma of the lung and follows the air passages to the hilum. Lymph nodes are found along the route of the larger lymphatic vessels. A second set of lymphatic vessels drains the surface of the lung and travels in the connective tissue of the visceral pleura, a serous membrane consisting of a surface mesothelium and the underlying connective tissue. Most of the nerves that serve the lung are not visible at the level of the light microscope. They are components of the sympathetic and parasympathetic divisions of the autonomic nervous system and mediate reflexes that modify the dimensions of the air passages (and blood vessels) by contraction of the smooth muscle in their walls.

1	Olfactory mucosa is located in the roof and part of the walls of the nasal cavity. Its pseudostratified epithelium is thicker than that of nonsen-sory epithelium, and it serves as the receptor for smell. Olfactory epithelium consists of olfactory cells, supporting (sustentacular) cells, basal cells, and brush cells. Olfactory cells are bipolar neurons. The apex of the cell is expanded into the olfactory vesicle from which nonmotile cilia, the actual recep-tors, extend into surface secretions. The base of the cell tapers into an axonal process that enters the lamina propria and joins axons from other receptor cells to form the olfactory nerve. Large, cuboidal Schwann cells are a prominent feature of these axons, giving the nerve an un-usual appearance. Supporting cells are columnar cells with apical microvilli. They attach to the receptor cells through adhering junctions and provide me-chanical and metabolic support to the olfactory cells. Basal cells are stem cells from which

1	cells with apical microvilli. They attach to the receptor cells through adhering junctions and provide me-chanical and metabolic support to the olfactory cells. Basal cells are stem cells from which olfactory and supporting cells differentiate. Brush cells are the same cell type that occurs in nonsensory respiratory epithelium. The lamina propria is directly contiguous with periosteum. It contains numerous blood and lymphatic vessels, unmyelinated and myelinated nerves, and olfactory (Bowman’s) glands. These are tubuloalveolar serous glands whose watery secretion serves as a trap and solvent for odorant substances and continuously washes the olfactory surface.

1	Olfactory mucosa, nasal cavity, human, Azan ×75. This low-magnification orientation micrograph shows part of the wall of the nasal cavity. The olfactory mucosa (OM ) and adjacent ethmoid bone (EB ) are indicated. The olfactory mucosa is directly attached to the bone tissue; no submucosa is present. In this specimen, however, the mucosa is separated from the bone tissue because of shrinkage, a frequently encountered artifact. The olfactory epithelium (OEp) is pseudostratified, like respiratory epithelium; however, it is typically thicker. Note the respiratory epithelium (REp) included in the lower right of the micrograph. The feature that is most useful in identifying olfactory mucosa is the presence of numerous large, unmyelinated nerves (N ) and extensive olfactory (Bowman’s) glands (BG) in the connective tissue of the mucosa. Note that the adjacent respiratory mucosa lacks the nerves and exhibits a relative paucity of glands. Olfactory mucosa, nasal cavity, human, Azan ×375.

1	At this higher magnification, it is possible to distinguish in a general way the three principal cell types of the olfactory epithelium on the basis of nuclear location and appearance, as well as by certain cytoplasmic characteristics. For example, the nuclei of the supporting cells (SC ) are relatively dense and are located closest to the epithelial surface. They are arranged in an almost discrete single layer. The supporting cell has a cylindrical shape and extends from the basement membrane through the full thickness of the epithelium. Immediately beneath this layer are the cell bodies of the olfactory receptor cells (OC ). They lie at different levels within the thickness of the epithelium. Careful examination of the nuclei of these bipolar neuronal cells reveals that they contain more euchromatin than the nuclei of the supporting cells and often exhibit several nucleoli. In this preparation, the nucleoli appear as small round red bodies. In some cases, particularly when there is

1	euchromatin than the nuclei of the supporting cells and often exhibit several nucleoli. In this preparation, the nucleoli appear as small round red bodies. In some cases, particularly when there is shrinkage, the thin tapering dendritic process that extends to the olfactory surface may be observed. Similarly, an axonal process may sometimes be observed extending basally. The basal cells (BC ), the least numerous of the principal cell types, are characterized by their small round nuclei and scant cytoplasm. They are irregularly spaced and lie in proximity to the basement membrane. Note that the olfactory mucosa in contrast to respiratory mucosa lacks goblet cells.

1	The lamina propria contains numerous blood vessels (capillaries [C ], veins [V ]), lymphatics, olfactory nerves (N ), and olfactory (Bowman’s) glands (BG ). The Bowman’s glands are branched tubuloalveolar structures. They exhibit a very small lumen (arrows). The duct elements extend from the secretory portion of the gland beginning in close proximity to the overlying epithelium (arrowhead) and pass directly through the epithelium to deliver their secretions at the surface. The ducts are very short, making it difficult to identify them. The very thin axonal processes (AP ) of the olfactory cells are sometimes evident within the lamina propria prior to being ensheathed by Schwann cells to form the prominent olfactory nerves. The nuclei present within the olfactory nerves represent Schwann cell nuclei (ScC ).

1	KEY A, artery AP, axonal process BC, basal cells BG, Bowman’s glands C, capillary EB, ethmoid bone ES, ethmoid sinus N, olfactory nerves OC, olfactory cells OEp, olfactory epithelium OM, olfactory mucosa REp, respiratory epithelium SC, supporting cell nuclei ScC, Schwann cell nuclei V, vein arrows, lumina of Bowman’s glands arrowhead, duct of a Bowman’s gland entering epithelium

1	The larynx is the passageway for air between the oropharynx and the trachea that functions in the production of sound. It consists of a carti-laginous framework to which both extrinsic and intrinsic muscles are attached and a mucosal surface that varies in character from pseudos-tratified to stratified squamous in regions subject to abrasion by the air stream. The muscles move certain cartilages with respect to others, thus increasing or decreasing the opening of the rima glottis and increasing or decreasing the tension on the vocal folds (cords). In this way, vibrations of different wavelengths are generated in the passing air, and sound is produced. Larynx, monkey, H&E ×15.

1	Larynx, monkey, H&E ×15. The vocal folds are ridge-like structures that are oriented in an anteroposterior (ventral-dorsal) direction. In frontal sections, the vocal folds (VF ) are cross-sectioned, giving the appearance seen here. The two vocal folds and the space between them constitute the glottis. Just above each vocal fold is an elongated recess called the ventricle (V ), and above the ventricle is another ridge called the ventricular fold (VnF ) or, sometimes, the false vocal fold. Below and lateral to the vocal folds are the vocalis muscles (VM ). Within the vocal fold is a considerable amount of elastic material, although it is usually not evident in routine H&E preparations. This elastic material is part of the vocal ligament. It lies in an anteroposterior direction within the substance of the vocal fold and plays an important role in phonation.

1	Ventricular and vocal folds, larynx, monkey, H&E ×160. the contact between surfaces is considerable. Laterally, the surfaces consist of stratified columnar epithelium (SCE). The contact between these surfaces The surfaces of a vocal fold and the facing ventricular fold is less wearing. Small glands (Gl) are in the lamina propria of the laryngeal within rectangle 1 in top figure are turned 90° clockwise and mucosa. shown at higher magnification in this figure. Medially, both are lined by stratified squamous epithelium (SSE). Here, Infraglottic cavity, larynx, monkey, H&E ×160. infraglottic cavity. It shows the junction between the stratified squamous epithelium (SSE ), with its flat surface cells, and the stratified columnar

1	Infraglottic cavity, larynx, monkey, H&E ×160. infraglottic cavity. It shows the junction between the stratified squamous epithelium (SSE ), with its flat surface cells, and the stratified columnar Rectangle 2 in top figure is shown at higher magnification in epithelium (SCE ), with its columnar surface cells. The lamina propria this figure. This area of the larynx below the ventricles and consists of loose connective tissue in which glands (Gl ) are present. rima glottidis communicates with trachea and is called the Infraglottic cavity, larynx, monkey, H&E ×160.

1	Epithelium of the infraglottic cavity of the larynx just below the portion shown in top figure changes again, giving way, below, to the ciliated pseudostratified columnar epithelium (PSE) shown here. Note the cylinders of cytoplasm that clearly indicate the columnar nature of the surface cells. In the upper part of the figure, the epithelium is stratified columnar; in the lower part of the figure, it is pseudostratified columnar. This distinction is difficult to make from the examination of a single sample such as that shown here, and other information is needed to make the assessment. The additional information is the presence of cilia on the pseudostratified columnar epithelium; this epithelium is typically ciliated. Although not evident in the photomicrographs, note that stratified columnar epithelium has a very limited distribution, usually occurring between stratified squamous epithelium and some other epithelial types (e.g., pseudostratified columnar here or simple columnar at

1	epithelium has a very limited distribution, usually occurring between stratified squamous epithelium and some other epithelial types (e.g., pseudostratified columnar here or simple columnar at the anorectal junction, Plate 64). The lamina propria is a loose cellular connective tissue, and it also shows some glands (Gl ).

1	KEY Gl, glands PSE, pseudostratified columnar epithelium SCE, stratified columnar epithelium SSE, stratified squamous epithelium V, ventricles VF, vocal folds VM, vocalis muscles VnF, ventricular folds

1	The trachea is a short tube about 2.5 cm in diameter and about 10 cm long. It extends from the larynx to about the middle of the thorax, where it divides into the two main bronchi (primary bronchi). Its major function is to serve as a conduit for air. The lumen of the trachea is held open by a series of C-shaped hyaline cartilages that are stacked on one another to form a supporting structure. Fibroelastic tissue and smooth muscle (the trachealis muscle) bridge the gap between the free ends of the cartilages at the posterior border of the trachea, adja-cent to the esophagus. Typical respiratory (ciliated pseudostratified columnar) epithelium lines the trachea and primary bronchi. On entering the lungs, the primary bronchi branch immediately to give rise to the lobar bronchi (secondary bronchi) that supply the two lobes of the left lung and the three lobes of the right lung. Within the lung, the C-shaped cartilages are replaced by an investment of (some-times overlapping) cartilaginous

1	that supply the two lobes of the left lung and the three lobes of the right lung. Within the lung, the C-shaped cartilages are replaced by an investment of (some-times overlapping) cartilaginous plates that completely surround the bronchi.

1	Trachea, human, H&E ×90. This low-magnification micrograph of the posterior wall of the human trachea shows the pseudostratified ciliated columnar epithelium (EP ) subtended by a well-developed basement membrane (Bm). The basement membrane, which consists of tightly packed, fine collagen fibers, is actually an unusually thick and dense reticular layer and is, thus, part of the lamina propria. It is particularly distinct in the human trachea and may thicken with Trachea, human H&E ×65. This micrograph shows the wall of the trachea at the level of one end of the C-shaped tracheal cartilage (TC ). The portion of the pseudostratified ciliated columnar epithelium (EP ) does not exhibit as many goblet cells as are seen in the figure above. However, the basement membrane (Bm) is clear, as are the cellular lamina propria (LP ) and the submucosa (SM ) of the tra- Trachea, human, H&E ×250; inset ×500.

1	In this higher-magnification micrograph of the tracheal wall and in the inset, the cilia of the pseudostratified ciliated columnar epithelium (EP ) are particularly well demonstrated, as is the dense line (BB) formed by the basal bodies of the cilia in the apical cytoplasm of the epithelial cells. Goblet cells (GC ) are easily recognized, and the displacement of the flattened nucleus (N ) toward the base of the cell is well demonstrated. The thickness chronic irritation, as in smokers. Numerous goblet cells (GC ) are evident as clear ovoid spaces in the respiratory epithelium. A thin lamina propria (LP ) and a dense thick submucosa (SM ) underlie the respiratory epithelium. Seromucous glands (Gl ) are seen on both sides of the trachealis muscle (TM), a band of smooth muscle that fills the gap between the posterior ends of the C-shaped tracheal cartilages (not shown) and serves to separate the trachea from the esophagus. Adipose tissue (Ad ) is also present in the submucosa between

1	the gap between the posterior ends of the C-shaped tracheal cartilages (not shown) and serves to separate the trachea from the esophagus. Adipose tissue (Ad ) is also present in the submucosa between the esophagus and trachea.

1	chea. Again, seromucous glands (Gl ) are evident beneath the submucosa. The ends of the bundles of the trachealis muscle (TM ) are located toward the posterior midline from the glands. A small lymphatic nodule (LN ) is located adjacent to the end of one of the bundles. A significant amount of adipose tissue (Ad ) is found in the connective tissue between the trachealis muscle and the wall of the esophagus (not shown in this figure). and the density of the basement membrane (Bm) are more easily seen here than in the lower-magnification views in the other figures. A venule (V ) containing red cell ghosts is seen in the middle of the submucosa, and some inflammatory cells (IC ), probably lymphocytes, are seen adjacent to the vein as well as distributed lightly through the submucosa and more densely in the lamina propria. Portions of the seromucous glands (Gl ) are just visible at the bottom edge of the figure.

1	KEY Ad, adipose tissue BB, brush border Bm, basement membrane C, cilia EP, epithelium GC, goblet cells Gl, glands IC, inflammatory cells LN, lymphatic nodule LP, lamina propria N, nuclei of goblet cells SM, submucosa TC, tracheal cartilage TM, trachealis muscle V, vein

1	The primary bronchus that enters each lung divides into smaller secondary and tertiary bronchi. As the bronchi become smaller, some compo-nents of the wall are lost or reduced in amount. Ultimately, the respiratory passage has distinctly different features than those of a bronchus, and it is called a bronchiole. The features that characterize the bronchiole are the absence of cartilage, loss of submucosal glands, and grad-ual disappearance of goblet cells. The epithelium changes from pseudostratified columnar to simple ciliated columnar, and some columnar cells even lack cilia. Smooth muscle occupies a relatively larger portion of the bronchiolar wall than of the bronchial wall. The smallest diameter conducting bronchioles, the terminal bronchioles, are lined with simple ciliated cuboidal epithelium in which Clara cells, cells that secrete a surface-active agent that prevents luminal adhesion of bronchiolar walls during expiration, are found among the cili-ated cells. Respiratory

1	epithelium in which Clara cells, cells that secrete a surface-active agent that prevents luminal adhesion of bronchiolar walls during expiration, are found among the cili-ated cells. Respiratory bronchioles are the first part of the bronchial tree that allows gas exchange to occur. Respiratory bronchioles consti-tute a transition zone in which both air conduction and gas exchange occur. Scattered, thin-walled evaginations of the lumen of the respiratory bronchiole are called alveoli; these are the structures in which gas exchange between the air passages and the blood capillaries occurs.

1	Bronchiole, lungs, human, H&E ×75. A typical bronchiole is shown here. Characteristically, blood vessels (BV ) are adjacent to the bronchiole. The main features of the bronchiolar wall that are evident in the figure are bundles of smooth muscle (SM ) and the lining epithelium (shown at higher magnification in Plate 73). Higher magnification would reveal that the epithelium is ciliated. The connective tissue is mini mal and, at this low magnification, not conspicuous. Nevertheless, it is present and separates the muscle into bundles (i.e., the muscle layer is not a single continuous layer). The connective tissue contains collagenous and some elastic fibers. Glands are not present in the wall of the bronchiole. Surrounding the bronchiole, comprising most of the lung substance, are the air spaces or alveoli of the lung. Bronchiole and respiratory bronchioles, lungs, human, H&E ×75.

1	Bronchiole and respiratory bronchioles, lungs, human, H&E ×75. In this figure, a short length of a bronchiole (B) is shown longitudinally sectioned as it branches into two respiratory bronchioles (RB ). The last portion of a bronchiole that leads into respiratory bronchioles is called a terminal bronchiole. It is not engaged in exchange of air with the blood; the respiratory bronchiole does engage in air exchange. Arrows mark the place where the terminal bronchiole ends. Not uncommonly, as shown here, cartilage (C ) is found in the bronchiolar wall where branching occurs. Blood vessels (BV ) and a nodule of lymphocytes (L) are adjacent to the bronchiole.

1	The respiratory bronchiole has a wall composed of two components: One consists of recesses that have a wall similar to that of the alveoli and are thus capable of gas exchange; the other has a wall formed by small cuboidal cells that appear to rest on a small bundle of eosinophilic material. This is smooth muscle surrounded by a thin investment of connective tissue. Both of these components are shown at higher magnification in Plate 73. Alveoli, lungs, human, H&E ×75. The outer surface of lung tissue is the serosa (S); it consists of a lining of mesothelial cells resting on a small amount of connective tissue. This is the The most distal component of the respiratory passage is the layer that gross anatomists refer to as the visceral pleura. alveolus. Groups of alveoli clustered together and sharing a common opening are referred to as an alveolar sac (AS). Alveoli that form a tube are referred to as alveolar ducts (AD).

1	alveolus. Groups of alveoli clustered together and sharing a common opening are referred to as an alveolar sac (AS). Alveoli that form a tube are referred to as alveolar ducts (AD). KEY AD, alveolar ducts AS, alveolar sacs B, bronchiole BV, blood vessels C, cartilage L, nodule of lymphocytes RB, respiratory bronchiole S, serosa SM, smooth muscle arrows, end of terminal bronchiole PLATE 73 Terminal Bronchiole, Respiratory Bronchiole, and Alveolus

1	Respiratory bronchioles continue to divide to form alveolar ducts, passages lined solely with rows of alveoli that have rings of smooth muscle in knob-like interalveolar septa. The alveolar ducts terminate in alveolar sacs, enlarged spaces surrounded by clusters of alveoli that open into the spaces. The alveoli are lined with type I alveolar cells, extremely thin squamous cells that cover about 95% of the alveolar surface, and with type II alveolar cells, cuboidal cells that secrete surfactant, a surface-active agent that reduces surface tension at the air-epithelium surface. The tissue between adjacent alveoli is called the alveolar septum. This consists of the alveolar epithelial cells and their basal lamina, the basal lamina of the underlying capillary endothelium and the endothelial cells, themselves, and any other connective tissue elements that may lie between the two basal laminae. The alveolar septum is the site of the air-blood barrier.

1	Terminal bronchiole, lungs, human, H&E ×550. The histologic features of the terminal bronchiolar wall are shown here. Ciliated epithelium extends from the top of the figure to the diamond. This is ciliated pseudostratified columnar epithelium (PsEp). Some basal cells are still present and, therefore, the designation pseudostratified columnar. Elsewhere, the epithelium might be ciliated simple columnar, and just be Respiratory bronchiole, lungs, human, H&E ×550.

1	Respiratory bronchiole, lungs, human, H&E ×550. The wall of a respiratory bronchiole is shown here and in lower left figure. The alveoli (A) are terminal air spaces on the left in each of the two figures. The lumen of the respiratory bronchiole is on the right. Characteristically, the wall of the respiratory bronchiole consists of alternating thick and fore it becomes a respiratory bronchiole, the epithelium may include cuboidal or low columnar nonciliated cells. These nonciliated cells are Clara cells (CC, beyond the diamond). Clara cells produce a surface-active agent that is instrumental in expansion of the lungs. The smooth muscle (SM) in the bronchiolar wall is organized in bundles; other cells under the epithelium and around the smooth muscle belong to the connective tissue.

1	thin regions. The thick regions are similar to the wall of the bronchiole except that cuboidal Clara cells instead of columnar epithelium form the surface. Thus, as seen here, Clara cells (CC) are the surface-lining cells of the thick regions, and smooth muscle bundles (SM) are under the Clara cells, with a small amount of intervening connective tissue. The thin regions have a wall similar to the alveolar wall; this is considered below. Respiratory bronchiole, lungs, human, H&E ×550. Structurally, it shows essentially the same features as those seen in upper right figure except that there are fewer Clara cells and the smooth muscle is The respiratory bronchiole shown in lower left figure is somewhat thinner. slightly more distal than the area seen in top right figure. Alveolus, lungs, human, H&E ×800.

1	The respiratory bronchiole shown in lower left figure is somewhat thinner. slightly more distal than the area seen in top right figure. Alveolus, lungs, human, H&E ×800. The central component of the alveolar wall is the capillary (C) and, in certain locations, associated connective tissue. On each side, where it faces the alveolus (A), a flat squamous cell is interposed between the capillary and the air spaces. This is a pneumocyte type I cell. In some places, the type I cell is sep arated from the capillary endothelial cell by a single basal lamina shared by the two cells. This is the thin portion of the alveolar-capillary complex, readily seen in the upper part of the figure (arrows). Gas exchange occurs through the thin portion of the alveolar-capillary complex. Elsewhere, connective tissue is interposed between the pneumocyte type I cell and the endothelial cell of the capillary; each of these epithelial cells retains its own basal lamina.

1	A second cell type, the pneumocyte type II cell or septal cell (SC), also lines the alveolar air space. This cell typically displays a rounded (rather than flattened) shape, and the nucleus is surrounded by a noticeable amount of cytoplasm, some of which may appear clear. The septal cell produces a surface-active agent different from that of the Clara cell, which also acts in permitting the lung to expand. KEY A, alveolus C, capillary CC, Clara cells PsEp, pseudostratified squamous epithelium SC, septal cell SM, smooth muscle arrows, thin portion of alveolar-capillary complex diamond, junction between pseudostratified columnar epithelium and Clara cells PLATE 73

1	OVERVIEW OF THE URINARY SYSTEM / 698 GENERAL STRUCTURE OF THE KIDNEY / 699 Capsule / 699 Cortex and Medulla / 700 Kidney Lobes and Lobules / 701 The Nephron / 702 General Organization of the Nephron / 702 Tubes of the Nephron / 703 Types of Nephrons / 703 Collecting Tubules and Ducts / 703 Filtration Apparatus of the Kidney / 705 Mesangium / 710 Juxtaglomerular Apparatus / 711 KIDNEY TUBULE FUNCTION / 714 Proximal Convoluted Tubule / 715 Proximal Straight Tubule / 716 Thin Segment of Loop of Henle / 717 Distal Straight Tubule / 718 Distal Convoluted Tubule / 718 Collecting Tubules and Collecting Ducts / 719 INTERSTITIAL CELLS / 720 HISTOPHYSIOLOGY OF THE KIDNEY / 720 BLOOD SUPPLY / 721 LYMPHATIC VESSELS / 723 NERVE SUPPLY / 723 URETER, URINARY BLADDER, AND URETHRA / 723 Ureters / 725 Urinary Bladder / 726 Urethra / 726 Folder 20.1 Functional Considerations: Kidney and Vitamin D / 699 Folder 20.2 Clinical Correlation: Antiglomerular Basement Membrane Antibody-Induced

1	Ureters / 725 Urinary Bladder / 726 Urethra / 726 Folder 20.1 Functional Considerations: Kidney and Vitamin D / 699 Folder 20.2 Clinical Correlation: Antiglomerular Basement Membrane Antibody-Induced Glomerulonephritis; Goodpasture Syndrome / 712 Folder 20.3 Clinical Correlation: Examination of the Urine—Urinalysis / 714 Folder 20.4 Clinical Correlation: Renin–Angiotensin–Aldosterone System and Hypertension / 714 Folder 20.5 Functional Considerations: Structure and Function of Aquaporin Water Channels / 717 Folder 20.6 Functional Considerations: Hormonal Regulation of Collecting Duct Function / 721

1	The urinary system consists of the paired kidneys; paired ureters, which lead from the kidneys to the urinary bladder; and the urethra, which leads from the bladder to the exterior of the body. The kidneys conserve body fluid and electrolytes and remove metabolic waste. Like the lungs and liver, the kidneys retrieve essential materials and dispose of wastes. They conserve water, essential electrolytes, and metabolites, and they remove certain waste products of metabolism from the body. The kidneys play an important role in regulating and maintaining the composition and volume of extracellular fluid. They also are essential in maintaining acid–base balance by excreting hydrogen ions when bodily fluids become too acidic or excreting bicarbonates when bodily fluids become too basic.

1	The kidneys are highly vascular organs; they receive approximately 25% of the cardiac output. The kidneys produce urine, initially a glomerular ultrafiltrate of the blood or primary urine, which is then modified by selective resorption  FOLDER 20.1 Functional Considerations: Kidney and Vitamin D

1	Despite its name, vitamin D is actually an inactive precursor that undergoes a series of transformations to become the fully active hormone that regulates plasma calcium levels. In the human body vitamin D is derived from two sources:  Skin, in which vitamin D3 (cholecalciferol) is rapidly produced by the action of ultraviolet light on the precur-sor 7-dehydrocholesterol. The skin is the major source of vitamin D3, especially in regions where food is not supple-mented with vitamin D. Typically, 30 minutes to 2 hours of sunlight exposure per day can provide enough vitamin D to fulfill daily body requirements for this vitamin.  Diet, from which vitamin D3 is absorbed by the small intestine in association with chylomicrons. In the blood vitamin D3 is bound to vitamin D–binding protein and transported to the liver. The first transforma-tion occurs in the liver and involves hydroxylation of vitamin D3 to form 25-OH vitamin D3. This compound is released into the bloodstream and undergoes

1	transported to the liver. The first transforma-tion occurs in the liver and involves hydroxylation of vitamin D3 to form 25-OH vitamin D3. This compound is released into the bloodstream and undergoes a second hydroxyla-tion in the proximal tubules of the kidney to produce the highly active 1,25-(OH)2 vitamin D3 (calcitriol). The pro-cess is regulated indirectly by an increase in plasma Ca2concentration, which triggers secretion of PTH, or directly by a decrease in circulating phosphates, which in turn stim-ulates activity of 1 -hydroxylase responsible for conver-sion of 25-OH vitamin D3 into active 1,25-(OH)2 vitamin D3. Active 1,25-(OH)2 vitamin D3 stimulates intestinal absorption of Ca2and phosphate and mobilization of Ca2from bones. It is therefore necessary for normal development and growth of bones and teeth. The related compound vitamin D2 (ergocalciferol) undergoes the same conversion steps as vitamin D3 and produces the same biologic effects. Patients with end-stage chronic

1	growth of bones and teeth. The related compound vitamin D2 (ergocalciferol) undergoes the same conversion steps as vitamin D3 and produces the same biologic effects. Patients with end-stage chronic kidney diseases have inadequate conversion of vitamin D into active metabolites resulting in vitamin D3 deficiency. In adults, vitamin D3 defi-ciency is manifested by impaired bone mineralization and reduced bone density. Therefore, patients with chronic kidney diseases, especially those on prolonged renal hemodialysis are often supplemented with vitamin D3 and calcium to avoid severe disturbance of calcium homeostasis because of sec-ondary hyperparathyroidism, a condition prevalent in these patients. Vitamin D3 deficiency in childhood results in rickets, a disease that causes abnormal bone ossification.

1	and specific secretion by the cells of the kidney. The final urine is conveyed by the ureters to the urinary bladder, where it is stored until discharged via the urethra. The final urine contains water and electrolytes as well as waste products, such as urea, uric acid, and creatinine, and breakdown products of various substances. The kidney also functions as an endocrine organ. Endocrine activities of the kidneys include  Synthesis and secretion of the glycoprotein hormone erythropoietin (EPO), which acts on the bone marrow and regulates red blood cell formation in response to decreased blood oxygen concentration. EPO is synthesized by endothelial cells of the peritubular capillaries in the renal cortex and acts on specific receptors expressed on the surface of erythrocyte progenitor (Er-P) cells in the bone marrow.

1	The recombinant form of erythropoietin (RhEPO) is used for the treatment of anemia in patients with end-stage renal disease. It is also used to treat anemia resulting from bone marrow suppression that develops in AIDS patients undergoing treatment with antiretroviral drugs, such as azidothymidine (AZT).  Synthesis and secretion of the acid protease renin, an enzyme involved in control of blood pressure and blood volume. Renin is produced by juxtaglomerular cells and cleaves circulating angiotensinogen to release angiotensin I (see page 713).  Hydroxylation of 25-OH vitamin D3, a steroid precursor produced in the liver, to hormonally active 1,25-(OH)2 vitamin D3. This step is regulated primarily by parathyroid hormone (PTH), which stimulates activity of the enzyme 1-hydroxylase and increases the production of the active hormone (see Folder 20.1).

1	The kidneys are large, reddish, bean-shaped organs located on either side of the spinal column in the retroperitoneal space of the posterior abdominal cavity. They extend from the 12th thoracic to the 3rd lumbar vertebrae, with the right kidney positioned slightly lower. Each kidney measures approximately 10 cm long 6.5 cm wide (from concave to convex border) 3 cm thick. On the upper pole of each kidney, embedded within the renal fascia and a thick protective layer of perirenal adipose tissue, lies an adrenal gland. The medial border of the kidney is concave and contains a deep vertical fissure, called the hilum, through which the renal vessels and nerves pass and the expanded, funnel-shaped origin of the ureter, called the renal pelvis, exits. A section through the kidney shows the relationship of these structures as they lie just within the hilum of the kidney in a space called the renal sinus (Fig. 20.1). Although not shown in the illustration, the space between and around these

1	of these structures as they lie just within the hilum of the kidney in a space called the renal sinus (Fig. 20.1). Although not shown in the illustration, the space between and around these structures is filled largely with loose connective tissue and adipose tissue.

1	The kidney surface is covered by a connective tissue capsule. The capsule consists of two distinct layers: an outer layer of fibroblasts and collagen fibers, and an inner layer with a cellular component of myofibroblasts (Fig. 20.2). The contractility of FIGURE 20.1 • Diagram of kidney structure. The diagram represents a hemisection of a kidney, revealing its structural organization. the myofibroblasts may aid in resisting volume and pressure variations that can accompany variations in kidney function. Its specific role, however, is unknown. The capsule passes inward at the hilum, where it forms the connective tissue covering of the sinus and becomes continuous with the connective tissue forming the walls of the calyces and renal pelvis (see Fig. 20.1). Examination with the naked eye of the cut face of a fresh, hemisected kidney reveals that its substance can be divided into two distinct regions:  Cortex, the outer reddish brown part  Medulla, the much lighter-colored inner part

1	The color seen in the cut surface of the unfixed kidney reflects the distribution of blood in the organ. Approximately 90% to 95% of the blood passing through the kidney is in the cortex; 5% to 10% is in the medulla. The cortex is characterized by renal corpuscles and their associated tubules. The cortex consists of renal corpuscles along with the convoluted tubules and straight tubules of the nephron, the collecting tubules, collecting ducts, and an extensive vascular supply. The nephron is the basic functional unit of the kidney and is described in a following section. The renal corpuscles are spherical structures, barely visible with the naked eye. They constitute the beginning segment of the nephron and contain a unique capillary network called a glomerulus. Examination of a section cut through the cortex at an angle perpendicular to the surface of the kidney reveals a series of FIGURE 20.2 • Photomicrograph of human kidney capsule.

1	Examination of a section cut through the cortex at an angle perpendicular to the surface of the kidney reveals a series of FIGURE 20.2 • Photomicrograph of human kidney capsule. This photomicrograph of a Mallory-Azan–stained section shows the capsule (cap) and part of the underlying cortex. The outer layer of the capsule (OLC) is composed of dense connective tissue. The fibroblasts in this part of the capsule are relatively few in number; their nuclei appear as narrow, elongate, red-staining profiles against a blue background representing the stained collagen fibers. The inner layer of the capsule (ILC) consists of large numbers of myofibroblasts whose nuclei appear as round or elongate, red-staining profiles, depending on their orientation within the section. Note that the collagen fibers in this layer are relatively sparse and that the myofibroblast nuclei are more abundant than those of the fibroblasts in the outer layer of the capsule. 180.

1	vertical striations that appear to emanate from the medulla (see Fig 20.1). These striations are the medullary rays (of Ferrein). Their name reflects their appearance, as the striations seem to radiate from the medulla. Approximately 400 to 500 medullary rays project into the cortex from the medulla. Each medullary ray is an aggregation of straight tubules and collecting ducts. Each medullary ray contains straight tubules of the nephrons and collecting ducts. The regions between medullary rays contain the renal corpuscles, the convoluted tubules of the nephrons, and the collecting tubules. These areas are referred to as cortical labyrinths. Each nephron and its collecting tubule (which connects to a collecting duct in the medullary ray) form the uriniferous tubule. The medulla is characterized by straight tubules, collecting ducts, and a special capillary network, the vasa recta.

1	The medulla is characterized by straight tubules, collecting ducts, and a special capillary network, the vasa recta. The straight tubules of the nephrons and the collecting ducts continue from the cortex into the medulla. They are accompanied by a capillary network, the vasa recta, that runs in parallel with the various tubules. These vessels represent the vascular part of the countercurrent exchange system that regulates the concentration of the urine.

1	The tubules in the medulla, because of their arrangement and differences in length, collectively form a number of conical structures called pyramids. Usually 8 to 12 but as many as 18 pyramids may be present in the human kidney. The bases of the pyramids face the cortex, and the apices face the renal sinus. Each pyramid is divided into an outer medulla (adjacent to the cortex) and an inner medulla. The outer medulla is further subdivided into an inner stripe and an outer stripe. The zonation and stripes are readily recognized in a sagittal section through the pyramid of a fresh specimen. They reflect the location of distinct parts of the nephron at specific levels within the pyramid (Fig. 20.3). The renal columns represent cortical tissue contained within the medulla.

1	The renal columns represent cortical tissue contained within the medulla. The caps of cortical tissue that lie over the pyramids are sufficiently extensive that they extend peripherally around the lateral portion of the pyramid, forming the renal columns (of Bertin). Although renal columns contain the same components as the rest of the cortical tissue, they are regarded as part of the medulla. In effect, the amount of cortical tissue is so extensive that it “spills” over the side of the pyramid much as a large scoop of ice cream extends beyond and overlaps the sides of an ice cream cone.

1	The apical portion of each pyramid, which is known as the papilla, projects into a minor calyx, a cup-shaped structure that represents an extension of the renal pelvis. The tip of the papilla, also known as the area cribrosa, is perforated by the openings of the collecting ducts (Fig. 20.4). The minor calyces are branches of the two or three major calyces that in turn are major divisions of the renal pelvis (see Fig. 20.1). The number of lobes in a kidney equals the number of medullary pyramids.

1	The number of lobes in a kidney equals the number of medullary pyramids. Each medullary pyramid and the associated cortical tissue at its base and sides (one half of each adjacent renal column) constitutes a lobe of the kidney. The lobar organization of the kidney is conspicuous in the developing fetal kidney (Fig. 20.5). Each lobe is reflected as a convexity on the outer surface of the organ, but they usually disappear after birth. The surface convexities typical of the fetal kidney may persist, however, until the teenage years and, in some cases, into adulthood. Each human kidney contains 8 to 18 lobes. Kidneys of some animals possess only one pyramid; these kidneys are classified as unilobar, in contrast to the multilobar kidney of the human. A lobule consists of a collecting duct and all the nephrons that it drains.

1	A lobule consists of a collecting duct and all the nephrons that it drains. The lobes of the kidney are further subdivided into lobules consisting of a central medullary ray and surrounding cortical material (Fig. 20.6 and Plate 75, page 730). Although the center or axis of a lobule is readily identifiable, the

1	FIGURE 20.3 • Diagram of two types of nephrons in the kidney and their associated collecting duct systems. A long-looped nephron is shown on the left, and a short-looped nephron is shown on the right. The relative position of the cortex, medulla, papilla, and capsule are indicated. The inverted cone-shaped area in the cortex represents a medullary ray. The parts of the nephron are indicated by number: 1, renal corpuscle including the glomerulus and Bowman’s capsule; 2, proximal convoluted tubule; 3, proximal straight tubule; 4, descending thin limb; 5, ascending thin limb; 6, thick ascending limb (distal straight tubule); 7, macula densa located in the final portion of the thick ascending limb; 8, distal convoluted tubule; 9, connecting tubule; 9*, collecting tubule that forms an arch (arched collecting tubule); 10, cortical collecting duct; 11, outer medullary collecting duct; and 12, inner medullary collecting duct. (Modified from Kriz W, Bankir

1	L. A standard nomenclature for structures of the kidney. The Renal Commission of the International Union of Physiological Sciences (IUPS). Kidney Int 1988;33:1–7.) boundaries between adjacent lobules are not obviously demarcated from one another by connective tissue septa. The concept of the lobule has an important physiologic basis; the medullary ray containing the collecting duct for a group of nephrons that drain into that duct constitutes the renal secretory unit. It is the equivalent of a glandular secretory unit or lobule.

1	FIGURE 20.4 • Renal papilla and calyx. a. This scanning electron micrograph shows the conical structure that represents the renal papilla, projecting into the renal calyx. The apex of the papilla contains openings (arrows) of the collecting ducts (of Bellini). These ducts deliver urine from the pyramids to the minor calyx. The surface of the papilla containing the openings is designated the area cribrosa. (Courtesy of Dr. C. Craig Tisher.) b. Photomicrograph of an H&E–stained specimen of the papilla, showing the distal portion of the collecting ducts opening into the minor calyx. 120. The Nephron The nephron is the structural and functional unit of the kidney.

1	The Nephron The nephron is the structural and functional unit of the kidney. The nephron is the fundamental structural and functional unit of the kidney (see Fig. 20.3). Each human kidney contains approximately 2 million nephrons. Nephrons are responsible for the production of urine and correspond to the secretory part of other glands. The collecting ducts are responsible for the final concentration of the urine and are analogous to the ducts of exocrine glands that modify the concentration of the secretory product. Unlike the typical exocrine gland in which the secretory and duct portions arise from a single epithelial outgrowth, nephrons and their collecting tubules arise from separate primordia and only later become connected. General Organization of the Nephron The nephron consists of the renal corpuscle and a tubule system.

1	General Organization of the Nephron The nephron consists of the renal corpuscle and a tubule system. As stated previously, the renal corpuscle represents the beginning of the nephron. It consists of the glomerulus, a tuft of capillaries composed of 10 to 20 capillary loops, surrounded by a double-layered epithelial cup, the renal or Bowman’s capsule. Bowman’s capsule is the initial portion of the nephron, where blood flowing through the glomerular capillaries undergoes filtration to produce the glomerular ultrafiltrate. The glomerular capillaries are supplied by an afferent arteriole and are drained by an efferent arteriole that then branches, forming a new capillary network to supply the kidney tubules. The site where the afferent and efferent arterioles penetrate and exit from the parietal layer of Bowman’s capsule is called the vascular pole. Opposite this site is the urinary pole of the renal corpuscle, where the proximal convoluted tubule begins (see Fig. 20.7).

1	Continuing from Bowman’s capsule, the remaining parts of the nephron (the tubular parts) are as follows:  Proximal thick segment, consisting of the proximal con voluted tubule (pars convoluta) and the proximal straight tubule (pars recta) Thin segment, which constitutes the thin part of the loop of Henle  Distal thick segment, consisting of the distal straight tubule (pars recta) and the distal convoluted tubule (pars The distal convoluted tubule connects to the collecting tubule, often through a connecting tubule, thus forming the uriniferous tubule (i.e., the nephron plus collecting tubule; see Fig. 20.3). FIGURE 20.5 • Photomicrograph of fetal kidney. This photo-micrograph of an H&E–stained human fetal kidney shows the cortex, the medulla, and two associated pyramids. Note each surface convexity corresponds to a kidney lobe. During postnatal life the lobar convexities disappear and the kidney then exhibits a smooth surface. 30. Tubes of the Nephron

1	Tubes of the Nephron The tubular segments of the nephron are named according to the course that they take (convoluted or straight), location (proximal or distal), and wall thickness (thick or thin). Beginning from Bowman’s capsule, the sequential parts of the nephron consist of the following tubules:  Proximal convoluted tubule originates from the urinary pole of Bowman’s capsule. It follows a very tortuous or convoluted course and then enters the medullary ray to continue as the proximal straight tubule.  Proximal straight tubule, commonly referred to as the thick descending limb of the loop of Henle, descends into the medulla.  Thin descending limb is the continuation of the proximal straight tubule within the medulla. It makes a hairpin turn and returns toward the cortex.  Thin ascending limb is the continuation of the thin descending limb after its hairpin turn.

1	 Thin ascending limb is the continuation of the thin descending limb after its hairpin turn.  Distal straight tubule, which is also referred to as the thick ascending limb of the loop of Henle, is the continuation of the thin ascending limb. The distal straight tubule ascends through the medulla and enters the cortex in the medullary ray to reach the vicinity of its renal corpuscle of origin. The distal straight tubule then leaves the medullary ray and makes contact with the vascular pole of its parent renal corpuscle. At this point, the epithelial cells of the tubule adjacent to the afferent arteriole of the glomerulus are modified to form the macula densa. The distal tubule then leaves the region of the corpuscle and becomes the distal convoluted tubule.

1	 Distal convoluted tubule is less tortuous than the proximal convoluted tubule; thus, in a section showing the cortical labyrinth, there are fewer distal tubule profiles than proximal tubule profiles. At its termination, the distal convoluted tubule empties into a collecting duct that lies in the medullary ray via either an arched collecting tubule or a shorter tubule simply called the connecting tubule. The loop of Henle forms the entire U-shaped portion of a nephron. The proximal straight tubule, the thin descending limb with its hairpin turn, the thin ascending limb, and the distal straight tubule are collectively called the loop of Henle. In some nephrons, the thin descending and ascending segments are extremely short; therefore, the hairpin turn may be made by the distal straight tubule. Types of Nephrons

1	Types of Nephrons Several types of nephrons are identified, based on the location of their renal corpuscles in the cortex (see Fig. 20.3):  Subcapsular nephrons or cortical nephrons have their renal corpuscles located in the outer part of the cortex. They have short loops of Henle, extending only into the outer medulla. They are typical of the nephrons described previously, wherein the hairpin turn occurs in the distal straight tubule.  Juxtamedullary nephrons make up about one eighth of the total nephron count. Their renal corpuscles occur in proximity to the base of a medullary pyramid. They have long loops of Henle and long ascending thin segments that extend well into the inner region of the pyramid. These structural features are essential to the urine-concentrating mechanism, which is described in a further section.  Intermediate nephrons or midcortical nephrons have their renal corpuscles in the midregion of the cortex. Their loops of Henle are of intermediate length.

1	 Intermediate nephrons or midcortical nephrons have their renal corpuscles in the midregion of the cortex. Their loops of Henle are of intermediate length. The collecting tubules begin in the cortical labyrinth, as either connecting tubules or arched collecting tubules, and proceed to the medullary ray, where they join the collecting ducts. The collecting ducts within the cortex are referred to as cortical collecting ducts. When cortical collecting ducts reach the medulla, they are referred to as medullary collecting ducts. These ducts travel to the apex of the pyramid, where they merge into larger collecting ducts (up to 200 m), the papillary ducts (ducts of Bellini) that open into the minor calyx (see Fig. 20.4). The area on the papilla that contains the openings of these collecting ducts is called the area cribrosa.

1	FIGURE 20.6 • Diagrams and photomicrograph of an adult human kidney. The diagram in the upper left is a hemisection of the adult human kidney included for orientation. The diagram on the right represents an enlarged portion emphasizing the relationship of two nephrons and their collecting tubules and ducts ( yellow) to the cortex and medulla. The upper nephron, a midcortical nephron, extends only a short distance into the medulla and possesses a short thin segment in the loop of Henle. The lower nephron, a juxtamedullary nephron, has a long loop of Henle that extends deep into the medulla. Both nephrons drain into the collecting tubules in the medullary ray. The photomicrograph shows a section of the cortex. It is organized into a series of medullary rays containing straight tubules and collecting tubules and between them the cortical labyrinths containing the renal corpuscles and their associated proximal and distal convoluted tubules. A kidney lobule consists of a medullary ray at

1	collecting tubules and between them the cortical labyrinths containing the renal corpuscles and their associated proximal and distal convoluted tubules. A kidney lobule consists of a medullary ray at its center and half of the adjacent cortical labyrinth on either side. 60.

1	In summary, the gross appearance of the kidney parenchyma reflects the structure of the nephron. The renal corpuscle and the proximal and the distal convoluted tubules are all located in and make up the substance of the cortical labyrinths. The portions of the straight proximal and straight distal tubules and the descending thin and ascending thin limbs of the loop of Henle in the cortex are located in and make up the major portion of the medullary rays. The thin descending and thin ascending limbs of the loop of Henle are always located in the medulla. Thus, the arrangement of the nephrons (and the collecting tubules and ducts) accounts for the characteristic appearance of the cut surface of the kidney, as can be seen in Figure 20.6. foot processes (pedicels) of podocytes parietal layer of basal lamina

1	foot processes (pedicels) of podocytes parietal layer of basal lamina FIGURE 20.7 • Structure of the renal corpuscle. a. This schematic diagram shows the organization of the renal corpuscle and the structures associated with it at the vascular and urinary poles. Mesangial cells are associated with the capillary endothelium of the glomerulus and the glomerular basement membrane. The macula densa cells of the distal tubule are shown intimately associated with the juxtaglomerular cells of the afferent arteriole and the extraglomerular mesangial cells. (Modified from Kriz W, Sakai T. Morphological aspects of glomerular function. In: Nephrology: Proceedings of the Tenth International Congress of Nephrology. London: Bailliere-Tindall, 1987.) b. Photomicrograph of an H&E–stained specimen showing a renal corpuscle. The macula densa is seen in close proximity to the vascular pole. 160. Filtration Apparatus of the Kidney

1	Filtration Apparatus of the Kidney The renal corpuscle contains the filtration apparatus of the kidney, which consists of the glomerular endothelium, underlying glomerular basement membrane, and the visceral layer of Bowman’s capsule.

1	The renal corpuscle is spherical and has an average diameter of 200 m. It consists of the glomerular capillary tuft and the surrounding visceral and parietal epithelial layers of Bowman’s capsule (Fig. 20.8). The filtration apparatus, also called glomerular fltration barrier, enclosed by the parietal layer of Bowman’s capsule, consists of three different components:  Endothelium of the glomerular capillaries, which possesses numerous fenestrations (Fig. 20.9). These fenestrations are larger (70 to 90 nm in diameter), more numerous, and more irregular in outline than fenestrations in other capillaries. Moreover, the diaphragm that spans the fenestrations in other capillaries is absent in the glomerular capillaries. Endothelial cells of glomerular capillaries possess a large number of aquaporin-1 (AQP-1) water channels that allow the fast movement of water through the epithelium. Secretory products of endothelial cells, such as nitric oxide (NO) or prostaglandins (PGE2), play an

1	aquaporin-1 (AQP-1) water channels that allow the fast movement of water through the epithelium. Secretory products of endothelial cells, such as nitric oxide (NO) or prostaglandins (PGE2), play an important role in the pathogenesis of several thrombotic glomerular diseases.

1	 Glomerular basement membrane (GBM), a thick (300 to 370 nm) basal lamina that is the joint product of the endothelium and the podocytes, the cells of the visceral layer of Bowman’s capsule. Because of its thickness, it is prominent in histologic sections stained with the periodic acid–Schiff (PAS) procedure (see Fig. 1.2, page 6). The GMB is composed of a network consisting of type IV collagen (mainly 3, 4, and 5 chains), laminin, nidogen, entactin, together with proteoglycans such as agrin and perlecan, as well as multiadhvesive glycoproteins (see page 138). GBM can also be visualized employing immunofluorescence techniques using antibodies directed to a specific chain of type IV collagen (Fig 20.10). Mutation in the gene encoding for the 5 chain of type IV collagen gives rise to the Alport’s syndrome of Bowman’s capsule glomerular basement membrane endothelial cells with fenestrations foot process of podocytepodocyte subpodocyte spaceendothelial blood

1	FIGURE 20.8 • Schematic dia gram of fltration barrier. The arrow indicates movement of plasma fluid across the glomerular filtration barrier, forming the glomerular ultrafiltrate (primary urine) that accumulates in the urinary space of the Bowman’s capsule. Note the layers of the filtration barrier that include, fenestrated glomerular endothelial cells, glomerular basement membrane, and podocytes with filtration slit diaphragms spanned between their foot processes. In addition, the endothelial surface layer of glycoproteins and subpodocyte spaces are shown on this diagram. (hereditary glomerulonephritis), manifesting by hematuria (presence of the red blood cells in the urine); proteinuria (presence of significant amount of protein in the urine); and progressive renal failure. In Alport’s syndrome, the GBM becomes irregularly thickened with laminated lamina densa and fails to serve as an effective filtration barrier.

1	FIGURE 20.9 • Scanning electron micrograph of the interior surface of a glomerular capillary. The wall of the capillary shows horizontal ridges formed by the cytoplasm of the endothelial cell. Elsewhere, fenestrations are seen as numerous dark oval and circular profiles. 5,600. (Courtesy of Dr. C. Craig Tisher.)  Visceral layer of Bowman’s capsule, which contains specialized cells called podocytes or visceral epithelial cells. These cells extend processes around the glomerular capillaries (Fig. 20.11 and Plate 76, page 732). The podocytes arise during embryonic development from one of the blind ends of the developing nephron through invagination of the end of the tubule to form a double-layered epithelial cup. The inner cell layer (i.e., the visceral cell layer) lies in apposition to a capillary network, the glomerulus, which forms at this site. The outer layer of these cells, the parietal layer, forms the squamous cells of Bowman’s capsule. The cup eventually closes to form the

1	a capillary network, the glomerulus, which forms at this site. The outer layer of these cells, the parietal layer, forms the squamous cells of Bowman’s capsule. The cup eventually closes to form the spherical structure containing the glomerulus. As they differentiate, the podocytes extend processes around the capillaries and develop numerous secondary processes called pedicels or foot processes. The foot processes interdigitate with foot processes of neighboring podocytes, a feature that can be clearly seen with the scanning electron microscope (SEM; Fig. 20.12). The elongated spaces between the interdigitating foot processes, called fltration slits, are about 40 nm wide and covered by an ultrathin fltration slit diaphragm that spans the filtration slit slightly above the GBM (Fig. 20.13, inset).

1	Nephrin is an important structural protein of the filtration slit diaphragm. Recent studies of the fltration slit diaphragm revealed its complex protein structure as a zipper-like sheet configuration with a central density. A transmembrane protein, nephrin is

1	FIGURE 20.10 • Immunofuorescent-stained glomerular basement membrane in human kidney. Glomerular basement membrane (GBM) is composed of five (1 to 5) of the six chains of type IV collagen. This high magnification micrograph of the GBM within the kidney glomerulus was obtained using primary monoclonal antibodies against 1 chain of type IV collagen molecules that were visualized by secondary antibody conjugated with fluorescein dye. 1,200. (Courtesy of Dr. L. Barisoni.) a key structural and functional component of the slit diaphragm. Nephrin molecules emerging from opposite foot processes interact in the center of the slit (homophilic interactions), forming a central density with pores on both sides (Fig. 20.14). This intercellular protein sheet also contains other adhesion molecules, such as Neph-1, Neph-2, P-cadherin, FAT1, and FAT 2. The filtration slit diaphragm is firmly anchored to numerous actin filaments within the foot processes of podocytes. Regulation and maintenance of the

1	as Neph-1, Neph-2, P-cadherin, FAT1, and FAT 2. The filtration slit diaphragm is firmly anchored to numerous actin filaments within the foot processes of podocytes. Regulation and maintenance of the actin cytoskeleton of podocytes has emerged as a critical process for regulating size, patency, and selectivity of the filtration slits. Mutations in the nephrin gene (NPHS1) are associated with congenital nephrotic syndrome, a disease characterized by massive proteinuria and edema.

1	Endothelial surface layer of glomerular capillaries and subpodocyte space also make an important contribution to overall glomerular function. The filtration apparatus is a very complex semipermeable barrier, with properties that allow for high filtration rate of water, nonrestricted passage of small and middle-sized molecules, and almost total exclusion of serum albumins and other larger proteins. The filtration apparatus may thus be described as a barrier having two discontinuous cellular layers, the endothelium of glomerular capillaries and visceral layer of Bowman’s capsule applied to either side of a continuous extracellular layer of the glomerular basement membrane. These three layers have traditionally been considered as the glomerular filtration barrier. However, recently two additional physiologically important layers, the endothelial surface layer of glomerular capillaries and subpodocyte space are included as part of the filtration apparatus.

1	 Endothelial surface layer of the glomerular capillaries consists of a thick carbohydrate–rich meshwork (200–400 nm) attached to the luminal surface of glomerular endothelial cells. It contains glycocalyx, which refers to plasma membrane-bound negatively charged proteoglycans (such as perlecan, syndecan, and versican) associated with glycosaminoglycan side-chains (such as heparan sulphate and chondroitin sulphate) and peripheral membrane proteins. Plasma proteins (e.g., albumins) adsorbed from the blood coat the luminal surface of glycocalyx.

1	 Subpodocyte space represents a narrow space between the foot processes with their filtration slit diaphragms on one side and a cell body of the podocyte on the other side (see Fig. 20.13). Recent three-dimensional reconstruction of these spaces revealed their interconnected but structurally restrictive character. They cover approximately 60% of the entire surface area of the glomerular filtration barrier and may function in regulating glomerular fluid flux across the filtration apparatus. The glomerular basement membrane (GBM) acts as a physical barrier and an ion-selective filter.

1	The glomerular basement membrane (GBM) acts as a physical barrier and an ion-selective filter. As discussed earlier, the GBM contains type IV and XVIII collagens, sialoglycoproteins, and other noncollagenous glycoproteins (e.g., laminin, fibronectin, entactin), as well as proteoglycans (e.g., perlecan, agrin) and glycosaminoglycans, particularly heparan sulfate (Fig. 20.15). These components are localized in particular portions of the GBM:  The lamina rara externa, adjacent to the podocyte processes. It is particularly rich in polyanions, such as heparan sulfate, that specifically impede the passage of negatively charged molecules.  The lamina rara interna, adjacent to the capillary endothelium. Its molecular features are similar to those of the lamina rara externa.

1	 The lamina rara interna, adjacent to the capillary endothelium. Its molecular features are similar to those of the lamina rara externa.  The lamina densa, the overlapping portion of the two basal laminae, sandwiched between the laminae rarae. It contains type IV collagen, which is organized into a network that acts as a physical filter. Type XVIII collagen, perlecan, and agrin are responsible for the bulk of anionic charges found in glomerular basement membrane. The laminin and other proteins present in the laminae rara interna and externa are involved in the attachment of the endothelial cells and podocytes to the GBM.

1	The GBM restricts the movement of particles, usually proteins, larger than approximately 70,000 daltons or 3.6 nm radius (e.g., albumin or hemoglobin). Although albumin is not a usual constituent, it may sometimes be found in urine, indicating that the size of albumin is close to the effective pore size of the filtration barrier. The polyanionic glycosaminoglycans of the laminae rarae have strong negative charges and restrict the movement of anionic particles and molecules across the GBM, even those smaller than 70,000 daltons. Despite the ability of the filtration barrier to restrict protein, several grams of protein do pass through the barrier each day. This

1	FIGURE 20.11 • Transmission electron micrograph of a glomerulus in the region of the urinary pole. The nuclear and perinuclear regions of the endothelial cells (EC) that line the glomerular capillaries (C) bulge into the vascular lumen. On the outer surface of the capillaries are the processes of the podocytes (P). External to the podocytes is the urinary space (U). Bowman’s capsule (BC) is shown on the left; it is continuous at the dashed line (marked by arrowheads) with the tubule cells of the proximal tubule (PTC). Note the numerous mitochondria (M) in the base of these cells and the brush border (BB) at the apex, projecting into the urinary space. The nuclei of three adjacent mesangial cells (MC) can be seen in the upper right of the micrograph. 4,700.

1	protein is reabsorbed by endocytosis in the proximal convoluted tubule. Albuminuria (presence of significant amounts of albumin in the urine) or hematuria (presence of significant amounts of red blood cells in the urine) indicate physical or functional damage to the GBM. In such cases (e.g., diabetic nephropathy), the number of anionic sites, especially in the lamina rara externa, is significantly reduced. The filtration slit diaphragm acts as a size-selective filter.

1	The filtration slit diaphragm acts as a size-selective filter. The narrow slit pores formed by the foot processes of podocytes and the filtration slit diaphragms act as physical barriers to restrict the movement of solutes and solvents across the filtration barrier. The discovery of specific proteins that form the slit diaphragm has led to new insights into the function of the filtration apparatus in the kidney. Most of the proteins found in the diaphragm are crucial for normal development and function of the kidney. The slit diaphragm architecture accounts for a true size-selective filter properties, which determine the molecular sieving characteristics of the glomerulus. Several mechanisms prevent clogging of the filtration slit diaphragms. These include, the negative charges of glycosaminoglycans in the GBM, negative charges of the podocyte cell membrane, and the phagocytic function of mesangial cells in the renal corpuscle.

1	FIGURE 20.12 • Scanning electron micrograph of a glomerulus. a. Low-magnification image revealing the tortuous course of the podocyte-covered glomerular capillaries. 700. b. A higher magnification of the area in the rectangle in a. Note the podocyte and its processes embracing the capillary wall. The primary processes (1 ) of the podocyte give rise to secondary processes (2 ), which in turn give rise to the pedicels. The space between the interdigitating pedicels creates the slit pores. 14,000. Inset. This higher magnification of the area in the rectangle reveals the slit pores and clearly shows that alternating pedicels belong to the secondary process of one cell; the intervening pedicels belong to the adjacent cell. 6,000.

1	FIGURE 20.13 • Transmission electron micrograph of a glomerular capillary and adjacent podocyte. The pedicels of the podocytes rest on the basal lamina adjacent to the capillary endothelium, and together, the three components—capillary endothelium, basal lamina, and podocyte—form a filtration apparatus. 5,600. Inset. The large arrows point to the fenestrations in the endothelium. On the other side of the basal lamina are the pedicels of the podocytes. Note the slit diaphragm (small arrows) spanning the gap between adjacent pedicels. 12,000. Changes in different components of the filtration apparatus influence the functions of one another.

1	Changes in different components of the filtration apparatus influence the functions of one another. The molecular structure and composition of each component of the glomerular filtration barrier has important consequences for adjacent components of the barrier. For instance, molecular changes in the GBM not only modify the contribution of this layer but also modify the rate at which solutes and solvents pass through the endothelium of glomerular capillaries on one side and the visceral layer of the Bowman’s capsule on the other. In addition, it is important to understand that glomerular filtration barrier is not a passive but an active structure that can remodel itself and modify its own permeability. Simple squamous epithelium constitutes the parietal layer of Bowman’s capsule.

1	Simple squamous epithelium constitutes the parietal layer of Bowman’s capsule. The parietal layer of Bowman’s capsule contains parietal epithelial cells and forms a simple squamous epithelium. At the urinary pole of the renal corpuscle, the parietal layer is continuous with the cuboidal epithelium of the proximal convoluted tubule (see Figs. 20.7 and 20.11). Proliferation of parietal epithelial cells is a typical diagnostic feature in certain types of glomerulonephritis (inﬂammation of the glomerulus). For an example of such a disease, see Folder 20.2. The space between the visceral and parietal layers of Bowman’s capsule is called the urinary space or Bowman’s space (see Fig. 20.11). It is the receptacle for the glomerular ultrafiltrate (primary urine) produced by the filtration apparatus of the renal corpuscle. At the urinary pole of the renal corpuscle, the urinary space is continuous with the lumen of the proximal convoluted tubule.

1	The renal corpuscle contains an additional group of cells called mesangial cells. These cells and their extracellular matrix constitute the mesangium. It is most obvious at the vascular stalk of the glomerulus and at the interstices of adjoining glomerular capillaries. Mesangial cells are positioned much the same as pericytes, in that they are enclosed by the GBM (Fig. 20.16). The mesangial cells are not confined entirely to the renal corpuscle; some are located outside the corpuscle along the vascular pole, where they are also designated as lacis cells and form part of what is called the juxtaglomerular apparatus (see Fig. 20.7).

1	Important functions of the mesangial cells follow:  Phagocytosis and endocytosis. Mesangial cells remove trapped residues and aggregated proteins from the GBM and filtration slit diaphragm, thus keeping the glomerular filter free of debris. They also endocytose and process a variety of plasma proteins including immune complexes. Maintaining the structure and function of glomerular barrier is the primary function of the mesangial cells. Structural support. Mesangial cells produce components of mesengial matrix, which provide support for the FIGURE 20.14 • Diagram of the fltration slit diaphragm.

1	Filtration slit diaphragm is a complex zipper-like sheet structure formed by a transmembrane protein nephrin. The extracellular domains of nephrins emerge from the opposite foot processes of neighboring podocytes and interdigitate in the center of the slit forming a central density with pores on both sides. The intracellular domains of nephrins interact with actin cytoskeleton within the cytoplasm of foot processes. The sheet of nephrin molecules is reinforced near its attachment to the foot processes by Neph1 and Neph2 proteins that interact with each other and with nephrin. The other adhesion molecules such as P-cadherin, FAT1, and FAT2 are also found in this region. Note that foot processes of podocytes are separated by the glomerular basement membrane (GBM) from fenestrated endothelial cells lining glomerular capillaries. (Redrawn from Tryggvason K, Patrakka J, Wartiovaara J. Hereditary proteinuria syndromes and mechanisms of proteinuria. N Engl J Med 2006;354:1387–401.) podocytes

1	cells lining glomerular capillaries. (Redrawn from Tryggvason K, Patrakka J, Wartiovaara J. Hereditary proteinuria syndromes and mechanisms of proteinuria. N Engl J Med 2006;354:1387–401.) podocytes in the areas where the epithelial basement membrane is absent or incomplete (see Fig 20.16).

1	 Secretion. Mesangial cells synthesize and secrete a variety of molecules such as interleukin 1 (IL-1), PGE2, and platelet-derived growth factor (PDGF), which play a central role in response to glomerular injury.  Modulation of glomerular distension. Mesangial cells have contractile properties. In the past, it was suggested that contraction of mesangial cells could increase the intraglomerular blood volume and filtration pressure. Recent studies revealed that mesangial contribution to glomerular filtration rate is minimal, and the mesangial cells may function in regulating glomerular distension in response to increased blood pressure.

1	 Clinically, it has been observed that mesangial cells proliferate in certain kidney diseases, in which abnormal amounts of protein and protein complexes are trapped in the GBM. Proliferation of mesangial cells is a prominent feature in the immunoglobulin A (IgA) nephropathy (Berger disease), membranoproliferative glomerulonephritis, lupus nephritis, and diabetic nephropathy. FIGURE 20.15 • Immunofuorescent-stained glomerulus.

1	FIGURE 20.15 • Immunofuorescent-stained glomerulus. This triple-exposure micrograph of a normal adult rat glomerulus is immunostained with two different antibodies. One antibody recognizes specific extracellular components, namely, basement membrane heparan sulfate proteoglycan (BM-HSPG, rhodamine label). The other antibody recognizes basement membrane chondroitin sulfate proteoglycan (BM-CSPG, fluorescein label). Because it is a triple-exposure micrograph, a yellow color occurs where the two fluorescent labels exactly codistribute. The blue fluorescence is nuclear counterstaining with Hoechst nuclear stain. The micrograph shows that compartmentalization occurs with respect to glomerular proteoglycan populations. The glomerular capillary basement membrane is composed exclu sively of BM-HSPG, whereas the mesangial matrix (yellow) contains both BM-HSPG and BM-CSPG. Bowman’s capsule appears to be strongly stained by only BM-CSPG antibodies. 360. (Courtesy of Dr. Kevin J. McCarthy.)

1	Embryologically, mesangial, and juxtaglomerular cells (discussed in a following paragraph) are derived from smooth muscle cell precursors. Although mesangial cells are clearly phagocytotic, they are unusual in the sense that they are not derived from the usual precursor cells of the mononuclear phagocytotic system, the blood-borne monocytes. The juxtaglomerular apparatus includes the macula densa, the juxtaglomerular cells, and the extraglomerular mesangial cells. Lying directly adjacent to the afferent and efferent arterioles and adjacent to some extraglomerular mesangial cells at the  FOLDER 20.2 Clinical Correlation: Antiglomerular Basement Membrane Antibody-Induced Glomerulonephritis; Goodpasture Syndrome

1	As discussed earlier in the section on the basal lamina assembly (see Chapter 5, page 139), the major building block of any basement membrane, including glomerular basement membrane (GBM), is the type IV collagen molecule. Its core structure is composed of three -chain monomers, each representing one or more of six types of -chains known for type IV collagen (see Table 6.2; page 165). Each molecule has three domains: an amino-terminus 7S domain, a middle collagenous helical domain, and a carboxy-terminus noncallagenous NC1 domain. Molecular architecture of type IV collagen is a key to understanding pathophysiology of glomerular kidney diseases. For instance, an autoimmune response to the noncollagenous NC1 domain of the 3-chain of type IV collagen [3(IV)] in the GBM is responsible for the development of anti-GBM antibody-induced glomerulonephritis. This condition is characterized by a linear deposition of immunoglobulin G (IgG) antibodies in the GBM. In some individuals, anti-GBM

1	the development of anti-GBM antibody-induced glomerulonephritis. This condition is characterized by a linear deposition of immunoglobulin G (IgG) antibodies in the GBM. In some individuals, anti-GBM antibodies may cross-react with alveolar basement membrane in the lungs, producing Goodpasture syndrome.

1	The clinical feature of Goodpasture syndrome is rapidly progressive glomerulonephritis (inflammation in the glomeruli) and pulmonary hemorrhage due to disruption of the air–blood barrier. In response to deposition of IgG in the glomerulus, the complement system is activated and circulating leukocytes elaborate a variety of proteases, leading to disruption of the GBM and deposition of fibrin. Fibrin, in turn, stimulates the proliferation of parietal cells lining Bowman’s capsule and cause influx of monocytes from the circulation. The product of these reactions is often seen within the glomerulus as a crescent, a characteristic microscopic feature of glomerulonephritis (Fig. F20.2.1). Most patients affected by Goodpasture syndrome have a severe crescentic glomerulonephritis with transiently elevated levels of circulating anti-GBM antibodies. Formation of anti-GBM antibodies is most likely triggered by viruses, cancers, pharmacologic agents, and chemical compounds found in a variety of

1	elevated levels of circulating anti-GBM antibodies. Formation of anti-GBM antibodies is most likely triggered by viruses, cancers, pharmacologic agents, and chemical compounds found in a variety of paints, solvents, and dyes.

1	Individuals with Goodpasture syndrome present with both respiratory and urinary symptoms. These include shortness of breath, cough, and bloody sputum, as well as hematuria (blood in urine), proteinuria (proteins in the urine), and other symptoms of progressing kidney failure. The main therapeutic goal in treating Goodpasture syndrome is to remove the circulating pathogenic antibodies from the blood. This is achieved by plasmapheresis, in which blood plasma is removed from the circulation and replaced by fluid, protein, or donated plasma. In addition, treatment with immunosuppressive drugs and corticosteroids is beneficial to keep the immune system from producing pathogenic autoantibodies.

1	FIGURE F20.2.1 • Photomicrograph of a glomerulus in Goodpasture syndrome. a. In this Mallory trichome-stained specimen obtained from a kidney biopsy, the collagen molecules in the mesangial matrix and glomerular capillaries are stained dark blue. The bright red stain within the renal corpuscle represents fibrin, which leaked out of the glomerular capillary loops into the urinary space. A cellular crescent (outlined in dashed line) is formed by deposition of fibrin infiltrated by macrophages and proliferated parietal cells of the Bowman capsule. The light blue color surrounding the glomerulus is reflective of edematous reaction containing cells mediating inflammatory reactions. Note the basal lamina of a parietal layer of the Bowman capsule. 320. b. This immunofluorescence image of the renal corpuscle shows the glomerular basement membrane labeled with antibodies directed against human IgG and visualized with secondary antibodies conjugated with fluorescent dye. In Goodpasture

1	the renal corpuscle shows the glomerular basement membrane labeled with antibodies directed against human IgG and visualized with secondary antibodies conjugated with fluorescent dye. In Goodpasture syndrome, IgGs bind to the NC1 domain of type IV collagen (3 chain) found in the GMB. Note the irregular thickness pattern of the GBM surrounding capillary loops. The remaining space is occupied by the cellular crescent. 360. (Courtesy of Dr. Joseph P. Grande.) endothelial cell foot processes of podocytes glomerular basement membrane endothelial cell fenestrae of endothelial cell mesangial cell mesangial matrix foot processes of podocytes

1	FIGURE 20.16 • Schematic diagram showing the relationship between the intraglomerular mesangial cells and the glomerular capillaries. The mesangial cell and its surrounding matrix are enclosed by the basal lamina of the glomerular capillaries. Note that the mesangial cells are in the same compartment as the endothelium and that they can be intimately associated with the basal lamina as well as with the endothelial cells. (Modified from Sakai T, Kriz W. The structural relationship between mesangial cells and basement membrane of the renal glomerulus. Anat Embryol 1987; 176:373–386.) vascular pole of the renal corpuscle is the terminal portion of the distal straight tubule of the nephron. At this site, the wall of the tubule contains cells that are referred to collectively as the macula densa. When viewed in the light microscope, the cells of the macula densa are distinctive, in that they are narrower and usually taller than other distal tubule cells (see Fig. 20.7). The nuclei of these

1	When viewed in the light microscope, the cells of the macula densa are distinctive, in that they are narrower and usually taller than other distal tubule cells (see Fig. 20.7). The nuclei of these cells appear crowded, even to the extent that they appear partially superimposed over one another, thus the name “macula densa.”

1	In this same region, the smooth muscle cells of the adjacent afferent arteriole (and, sometimes, the efferent arteriole) are modified. They contain secretory granules, and their nuclei are spherical, as opposed to the typical elongate smooth muscle cell nucleus. These juxtaglomerular cells (see Fig. 20.7) require spe cial stains to reveal the secretory vesicles in the light microscope. The juxtaglomerular apparatus regulates blood pressure by activating the renin–angiotensin–aldosterone system.

1	In certain physiologic (low sodium intake) or pathologic conditions (decrease in volume of circulating blood because of hemorrhage or reduction in renal perfusion owing to compression of the renal arteries), juxtaglomerular cells are responsible for activating the renin–angiotensin–aldosterone system (RAAS). This system plays an important role in maintaining sodium homeostasis and renal hemodynamics. The granules of the juxtaglomerular cells contain an aspartyl protease, called renin, which is synthesized, stored, and released into the bloodstream from the modified smooth muscle cells. In the blood, renin catalyzes the hydrolysis of a circulating 2-globulin, angiotensinogen, to produce the decapeptide angiotensin I. Then,  Angiotensin I is converted to the active octapeptide angiotensin II by angiotensin-converting enzyme (ACE) present on the endothelial cells of lung capillaries.

1	 Angiotensin II stimulates the synthesis and release of the hormone aldosterone from the zona glomerulosa of the adrenal gland (see page 766).  Aldosterone, in turn, acts on collecting ducts to increase reabsorption of sodium and concomitant reabsorption of water, thereby raising blood volume and pressure.

1	with suspected renal disease. It typically includes several measurement of physical, biochemical, and microscopic urine characteristics such as pH, specific gravity (indirect measurement of ion concentration), bilirubin, concentration of intermediate compounds derived from the fatty acid metabolism known as ketone bodies, hemoglobin, and con-centration of the proteins. Important part of this analysis in-cludes the determination of the amount of protein excreted in the urine. The excretion of excessive amounts of protein (i.e., proteinuria [albuminuria]) is an important diagnostic sign of renal disease. Normally, less than 150 mg of protein is of protein almost always indicates renal disease, extreme exercise, such as jogging, or severe dehydration may pro-duce increased proteinuria in individuals without renal dis-ease. Microscopic examination of the urine may reveal presence of red and white blood cells, mineral crystals, and pathogenic agents such as bacteria or fungi. Often these

1	without renal dis-ease. Microscopic examination of the urine may reveal presence of red and white blood cells, mineral crystals, and pathogenic agents such as bacteria or fungi. Often these elements are enclosed within the cylindrical structures called urinary casts. The matrix of urinary cast is formed by an 85kilodalton protein, uromodulin (Tamm-Horsfall protein) that precipitate in the lumen of distal convoluted tubes and collecting ducts during a disease process.

1	 FOLDER 20.3 Clinical Correlation: Examination of the Urine—Urinalysis Urinalysis is an important part of the examination of patients excreted in the urine each day. Although excessive excretion

1	For years, cardiologists and nephrologists believed that chronic essential hypertension, the most common form of hypertension, was somehow related to an abnor-mality in the RAAS. However, 24-hour urine renin levels in such patients were usually normal. Not until a factor in the venom of a South American snake was shown to be a potent inhibitor of angiotensin-converting enzyme (ACE) in the lung did investigators have both a clue to the cause of chronic essential hypertension and a new series of drugs with which to treat this common disease. The “lesion” in chronic essential hypertension is now be-lieved to be excessive production of angiotensin II in the lung. Development of the so-called ACE inhibitors—captopril, enalapril, and related derivatives of the original snake venom factor—has revolutionized the treatment of chronic essential hypertension. These antihypertensive drugs do not cause the often-dangerous side effects of the diuretics and -blockers that were previously the most

1	revolutionized the treatment of chronic essential hypertension. These antihypertensive drugs do not cause the often-dangerous side effects of the diuretics and -blockers that were previously the most commonly used drugs for control of this condition.

1	 FOLDER 20.4 Clinical Correlation: Renin–Angiotensin– Aldosterone System (RAAS) and Hypertension Angiotensin II is also a potent vasoconstrictor that has a regulatory role in the control of renal and systemic vascu lar resistance.

1	The juxtaglomerular apparatus functions not only as an endocrine organ that secretes renin but also as a sensor of blood volume and tubular fluid composition. The cells of the macula densa monitor the Na concentration in the tubular fluid and regulate both the glomerular filtration rate and the release of renin from the juxtaglomerular cells. The decreased Na concentration in the distal convoluted tubule is believed to be a stimulus for unique ion-transporting molecules expressed on the apical membrane of macula densa cells. These molecules include Na/2Cl /K cotransporters, Na/H exchangers, and pHand calcium-regulated K channels. Activation of membrane transport pathways changes the intracellular ion concentration within the macula densa cells and initiates signaling mechanisms by releasing various mediators such as ATP, adenosine, nitric oxide (NO), and prostaglandins (PGE2). These molecules act in a paracrine manner and signal both the underlying juxtaglomerular cells of the

1	releasing various mediators such as ATP, adenosine, nitric oxide (NO), and prostaglandins (PGE2). These molecules act in a paracrine manner and signal both the underlying juxtaglomerular cells of the afferent arteriole to secrete renin and the vascular smooth muscle cells to contract. An increase in blood volume sufficient to cause stretching of the juxtaglomerular cells in the afferent arteriole may be the stimulus that closes the feedback loop and stops secretion of renin.

1	As the glomerular ultrafltrate passes through the uriniferous and collecting tubules of the kidney, it undergoes changes that involve both active and passive absorption, as well as secretion. Certain substances within the ultrafiltrate are reabsorbed, some partially (e.g., water, sodium, and bicarbonate) and some completely (e.g., glucose). Other substances (e.g., creatinine and organic acids and bases) are added to the ultrafiltrate (i.e., the primary urine) by secretory activity of the tubule cells. Thus, the volume of the ultrafiltrate is reduced substantially, and the urine is made hyperosmotic. The long loop of Henle and the collecting tubules that pass parallel to similarly arranged blood vessels, the vasa recta, serve as the basis for the countercurrent multiplier mechanism that is instrumental in concentrating the urine, thereby making it hyperosmotic. The proximal convoluted tubule is the initial and major site of reabsorption.

1	The proximal convoluted tubule receives the ultrafiltrate from the urinary space of Bowman’s capsule. The cuboidal cells of the proximal convoluted tubule have the elaborate surface specializations associated with cells engaged in absorption and fluid transport. They exhibit the following features:  A brush border, composed of relatively long, closely packed, and straight microvilli (Fig. 20.17)  A junctional complex, consisting of a narrow, tight junction that seals off the intercellular space from the lumen of the tubule and a zonula adherens that maintains the adhesion between neighboring cells  Plicae or folds located on the lateral surfaces of the cells, which are large flattened processes, alternating with similar processes of adjacent cells (see Fig. 20.16)  Extensive interdigitation of basal processes of adjacent cells (Figs. 20.18 and 20.19)  Basal striations, consisting of elongate mitochondria concentrated in the basal processes and oriented vertically to the basal

1	of basal processes of adjacent cells (Figs. 20.18 and 20.19)  Basal striations, consisting of elongate mitochondria concentrated in the basal processes and oriented vertically to the basal surface (see Fig. 20.18)

1	In well-fixed histologic preparations, the basal striations and the apical brush border help to distinguish the cells of the proximal convoluted tubule from those of the other tubules. At the very base of the proximal convoluted tubule cell, in the interdigitating processes, bundles of 6-nm microfilaments FIGURE 20.17 • Drawing of proximal convoluted tubule cells.

1	The drawing, at the electron microscopic level, shows the sectioned face on the right and a three-dimensional view of the basolateral surface of a cell with a partial cut face on the left. Here the interdigitating parts of the adjoining cell have been removed to show the basolateral interdigitations. Some of the interdigitating processes extend the full height of the cell. The processes are long in the basal region and create an elaborate extracellular compartment adjacent to the basal lamina. Apically, the microvilli (M) constitute the brush border. In some locations, the microvilli have been omitted, thereby revealing the convoluted character of the apical cell boundary (CB). (Based on Bulger RE. The shape of rat kidney tubular cells. Am J Anat 1965;116:253.) are present (see arrows, Figs. 20.18 and 20.19). These actin filaments may play a role in regulating the movement of fluid from the basolateral extracellular space across the tubule basal lamina toward the adjacent peritubular

1	20.18 and 20.19). These actin filaments may play a role in regulating the movement of fluid from the basolateral extracellular space across the tubule basal lamina toward the adjacent peritubular capillary.

1	Of the 180 L/day of ultrafiltrate entering the nephrons, approximately 120 L/day, or 65% of the ultrafiltrate, is reabsorbed by the proximal convoluted tubule. Two major proteins are responsible for fluid reabsorption in the proximal convoluted tubules:

1	FIGURE 20.18 • Electron micrograph of a proximal tubule cell. The apical surface of the cell shows the closely packed microvilli (Mv) that collectively are recognized as the brush border in the light microscope. Many vesicles (V) are evident in the apical cytoplasm. Also present in the apical region of the cell are lysosomes (L). The nucleus has not been included in the plane of section. Extensive numbers of longitudinally oriented mitochondria (M) are present in the cell within the interdigitating processes. The mitochondria are responsible for the appearance of the basal striations seen in the light microscope, particularly if the extracellular space is enlarged. The electron micrograph also reveals a basal lamina and a small amount of connective tissue and the fenestrated endothelium (En) of an adjacent peritubular capillary. 15,000. Upper inset. This higher magnification of the microvilli shows the small endocytotic vesicles that have pinched off from the plasma membrane at the

1	(En) of an adjacent peritubular capillary. 15,000. Upper inset. This higher magnification of the microvilli shows the small endocytotic vesicles that have pinched off from the plasma membrane at the base of the microvilli. 32,000. Lower inset. A higher magnification of the basal portion of the interdigitating processes (IP) below the reach of the mitochondria. The extreme basal aspect of these processes reveals a dense material (arrows) that represents bundles of actin filaments (see Fig. 20.16). 30,000.

1	FIGURE 20.19 • Electron micrograph of a proximal convoluted tubule cell. This section is almost tangential and slightly oblique to the base of a proximal convoluted tubule cell and the subjacent basal lamina and capillary. In the left part of the micrograph is the capillary endothelium (En). Characteristically, the endothelium possesses numerous fenestrations (EnF), and in this plane of section, the fenestrations are seen en face, displaying circular profiles. The plane of section also makes the basal lamina (BL) appear as a broad band of homogenous material. To the right of the basal lamina are the interdigitating basal processes of the proximal tubule cells. The long, straight processes contain longitudinally oriented actin filaments (arrows). In this plane of section, the basal extracellular space appears as a maze between the cellular processes. 32,000.

1	 Na/K-ATPase pumps, transmembrane proteins that are localized in the lateral folds of the plasma membrane. They are responsible for the reabsorption of Na, which is the major driving force for reabsorption of water in the proximal convoluted tubule. As in the intestinal and gallbladder epithelia, this process is driven by active transport of Na into the lateral intercellular space. Active transport of Na is followed by passive diffusion of Cl to maintain electrochemical neutrality. The accumulation of NaCl in the lateral intercellular spaces creates an osmotic gradient that draws water from the lumen into the intercellular compartment. This compartment distends as the amount of fluid in it increases; the lateral folds separate to allow this distension.

1	 AQP-1, a small (30 kilodaltons) transmembrane protein that functions as a molecular water channel in the cell membrane of proximal convoluted tubules. Movement of water through these membrane channels does not require the high energy of Na/K-ATPase pumps. Immunocytochemical methods can be used to demonstrate the presence of these proteins.  The hydrostatic pressure that builds up in the distended intercellular compartment, presumably aided by contractile activity of the actin filaments in the base of the tubule cells, drives an essentially isosmotic fluid across the tubule basement membrane into the renal connective tissue. Here, the fluid is reabsorbed into the vessels of the peritubular capillary network. The proximal convoluted tubule also reabsorbs amino acids, sugars, and polypeptides.

1	The proximal convoluted tubule also reabsorbs amino acids, sugars, and polypeptides. As in the intestine, the microvilli of proximal convoluted tubule cells are covered with a well-developed glycocalyx that contains several ATPases, peptidases, and high concentrations of disaccharidases. In addition to amino acids and monosaccharides, the ultrafiltrate also contains small peptides and disaccharides. The latter adsorb on the glycocalyx for further digestion before internalization of the resulting amino acids and monosaccharides (including glucose). Also, as in the gut, amino acid and glucose resorption in the proximal convoluted tubule depends on active Na transport. Proteins and large peptides are endocytosed in the proximal convoluted tubule.

1	Proteins and large peptides are endocytosed in the proximal convoluted tubule. Deep tubular invaginations are present between the microvilli of the proximal convoluted tubule cells. Proteins in the ultra-filtrate, on reaching the tubule lumen, bind to the glycocalyx that covers the plasma membrane of the invaginations. Then endocytotic vesicles containing the bound protein bud from the invaginations and fuse in the apical cytoplasm to form large protein-containing early endosomes (see Fig. 20.18). These early endosomes are destined to become lysosomes, and the endocytosed proteins are degraded by acid hydro-lases. The amino acids produced in the lysosomal degradation are recycled into the circulation via the intercellular compartment and the interstitial connective tissue.

1	Also, the pH of the ultrafiltrate is modified in the proximal convoluted tubule by the reabsorption of bicarbonate and by the specific secretion into the lumen of exogenous organic acids and organic bases derived from the peritubular capillary circulation. The cells of the proximal straight tubule (i.e., the thick descending limb of the loop of Henle) are not as specialized for absorption as are those of the proximal convoluted tubule.  FOLDER 20.5 Functional Considerations: Structure and Function of Aquaporin Water Channels

1	Aquaporins (AQPs) are a recently recognized family of nodes, endothelial cells lining lymphatic sinuses, and on small, hydrophobic, transmembrane proteins that mediate the vascular endothelium of high endothelial venules as water transport in the kidney and other organs (i.e., liver, well as in the endothelial cells of intestinal lacteals. gallbladder). To date, 13 proteins have been characterized  AQP-2, present in the terminal portion of the distal con-and cloned. The molecular size of AQPs ranges from 26 to voluted tubules and in the epithelium of collecting34 kilodaltons. Each protein consists of six transmembrane tubules and ducts. AQP-2 is under the regulation of an-domains arranged to form a distinct pore. The sites where tidiuretic hormone (ADH) and is thus known as an ADH-AQPs are expressed implicate their role in water transport, regulated water channel. Mutation of the AQP-2 gene such as renal tubules (water reabsorption), brain and spinal has been linked to congenital

1	are expressed implicate their role in water transport, regulated water channel. Mutation of the AQP-2 gene such as renal tubules (water reabsorption), brain and spinal has been linked to congenital nephrogenic diabetes cord (cerebrospinal fluid reabsorption), pancreatic acinar insipidus.

1	cells (secretion of pancreatic fluids), lacrimal apparatus (secretion and resorption of tears), and eye (aqueous  AQP-3 and AQP-4 have also been detected in the bahumor secretion and reabsorption). Most AQPs are selec-solateral cell surface of the light cells of kidney collecttive for the passage of water (AQP-1, AQP-2, AQP-4, ing ducts as well in the gastrointestinal epithelium AQP-5, AQP-6, and AQP-8), whereas others, such as (AQP-3), pancreatic acinar cells (AQP-12), and the AQP-3, AQP-7, and AQP-9, called aquaglyceroporins, also brain and spinal cord (AQP-4). transport glycerol and other larger molecules in addition to

1	AQP-3, AQP-7, and AQP-9, called aquaglyceroporins, also brain and spinal cord (AQP-4). transport glycerol and other larger molecules in addition to Current research into the function and structure of the water. Prominent members of the AQP family include AQP proteins may lead to the development of water chan  AQP-1, expressed in kidney (proximal convoluted nel blockers that could be used to treat hypertension, con-tubules) and other cell types such as hepatocytes and gestive heart failure, and brain swelling and to regulate red blood cells. AQP-1 is also expressed in the lymph intracranial or intraocular pressure. They are shorter, with a less well-developed brush border and with fewer and less complex lateral and basolateral processes. The mitochondria are smaller than those of the cells of the convoluted segment and are randomly distributed in the cytoplasm. There are fewer apical invaginations and endocytotic vesicles, as well as fewer lysosomes. Thin Segment of Loop of Henle

1	Thin Segment of Loop of Henle As noted above, the length of the thin segment varies with the location of the nephron in the cortex. Juxtamedullary nephrons have the longest limbs; cortical nephrons have the shortest. Furthermore, various cell types are present in the thin segment. In the light microscope, it is possible to detect at least two kinds of thin segment tubules, one with a more squamous epithelium than the other. Electron microscopic examination of the thin segments of various nephrons reveals further differences, namely, the existence of four types of epithelial cells (Fig. 20.20):  Type I epithelium is found in the thin descending and ascending limbs of the loop of Henle of short-looped nephrons. It consists of a thin, simple epithelium. The cells have almost no interdigitations with neighboring cells and few organelles.

1	 Type II epithelium, found in the thin descending limb of long-looped nephrons in the cortical labyrinth, consists of taller epithelium. These cells possess abundant organelles and have many small, blunt microvilli. The extent of lateral interdigitation with neighboring cells varies by species.  Type III epithelium, found in the thin descending limb in the inner medulla, consists of a thinner epithelium. The cells have a simpler structure and fewer microvilli than type II epithelial cells. Lateral interdigitations are absent.  Type IV epithelium, found at the bend of long-looped nephrons and through the entire thin ascending limb, consists of a low, flattened epithelium without microvilli. The cells possess few organelles.

1	The specific functional roles of the four cell types in the thin segment are not yet clear, although this segment is part of the countercurrent exchange system that functions in concentrating urine. Morphologic differences, such as microvilli, mitochondria, and degree of cellular interdigitation, probably reflect specific active or passive roles in this process. The thin descending and ascending limbs of the loop of Henle differ in structural and functional properties.

1	Studies of ultrafiltrate that enters the thin descending limb and leaves the thin ascending limb of the loop of Henle reveal dramatic changes in ultrafiltrate osmolality. The ultrafiltrate that enters the thin descending limb is isosmotic, whereas the ultrafiltrate leaving the thin ascending limb is hyposmotic to plasma. This change is achieved by reabsorbing more salts than water. The two limbs of the loop of Henle have different permeabilities and thus different functions:  The thin descending limb of the loop of Henle is highly permeable to water and much less permeable to solutes like NaCl and urea. Because the interstitial fluid in the medulla is hyper-osmotic, water diffuses out of this nephron segment. In addition, a small amount of NaCl and urea enters the nephron at this site. The cells of this limb do not actively transport ions; thus the increased tubular fluid osmolality that occurs in this nephron segment is caused in large part by the passive movement of water into

1	The cells of this limb do not actively transport ions; thus the increased tubular fluid osmolality that occurs in this nephron segment is caused in large part by the passive movement of water into the peritubular connective tissue.

1	FIGURE 20.20 • Schematic diagram of loop of Henle thin-limb epithelial cells. Roman numerals (I–IV) identify the various segments of the epithelium and the region where they are found in the thin limb of the short and long loops of Henle. The diagrams of the epithelium do not include nuclear regions of the epithelial cells. (Modified from Madsen KM, Tisher CC. Physiologic anatomy of the kidney. In: Fisher JW (Ed). Kidney Hormones, London, UK: Academic Press 1986;3:45–100.)  The thin ascending limb of the loop of Henle does not actively transport ions, but it is highly permeable to NaCl and thus allows passive diffusion of NaCl into the interstitium. Cl diffuses into the interstitium following its concentration gradient through Cl conducting channels. Although the energy from ATP is required to open these channels, the movement of Cl is not an example of active transport and does not require Cl -stimulated ATPase activity. Counter ions, in this case Na (the majority) and K, follow

1	to open these channels, the movement of Cl is not an example of active transport and does not require Cl -stimulated ATPase activity. Counter ions, in this case Na (the majority) and K, follow passively to maintain electrochemical neutrality. The hyperosmolarity of the interstitium is directly related to the transport activity of the cells in this limb of the loop of Henle.

1	Further, the thin ascending limb is largely impermeable to water, so that at this site, as the salt concentration increases in the interstitium, the interstitium becomes hyperosmotic and the fluid in the lumen of the nephron becomes hyposmotic. In addition, epithelial cells lining the thick ascending limb produce an 85-kilodalton protein called uromodulin (Tamm-Horsfall protein) that influences NaCl reabsorption and urinary concentration ability. Uromodulin also modulates cell adhesion and signal transduction by interacting with various cytokines, as well as it inhibits the aggregation of calcium oxalate crystals (preventing kidney stones formation) and provides a defense against urinary tract infection. In individuals with inﬂammatory kidney diseases, a precipitated uromodulin is detected in urine in the form of urinary casts (see Folder 20.3). The distal straight tubule is a part of the ascending limb of the loop of Henle.

1	The distal straight tubule (thick ascending limb), as previously noted, is a part of the ascending limb of the loop of Henleand includes both medullary and cortical portions, with the latter located in the medullary rays. The distal straight tubule, like the ascending thin limb, transports ions from the tubular lumen to the interstitium. The apical cell membrane in this segment has electroneutral transporters (synporters) that allow Cl , Na, and K to enter the cell from the lumen. Na is actively transported across the extensive basolateral plications by the Na/K-ATPase pumps; Cl and K diffuse out from the intracellular space by the Cl and K channels. Some K ions leak back into the tubular fluid through K channels, causing the tubular lumen to be positively charged with respect to the interstitium. This positive gradient provides the driving force for the reabsorption of many other ions such as Ca2 and Mg2. Note that this significant movement of ions occurs without the movement of

1	interstitium. This positive gradient provides the driving force for the reabsorption of many other ions such as Ca2 and Mg2. Note that this significant movement of ions occurs without the movement of water through the wall of the distal straight tubule, resulting in separation of water from its solutes.

1	In routine histologic preparations, the large cuboidal cells of the distal straight tubule stain lightly with eosin, and the lateral margins of the cells are indistinct (Plate 77, page 734). The nucleus is located in the apical portion of the cell and sometimes, especially in the straight segment, causes the cell to bulge into the lumen. As noted above, these cells have extensive basolateral plications, and there are numerous mitochondria associated with these basal folds (Fig. 20.21). They also have considerably fewer and less well-developed microvilli than proximal straight tubule cells (compare Figs. 20.18 and 20.19). The distal convoluted tubule exchanges Na for K under aldosterone regulation. The distal convoluted tubule, located in the cortical labyrinth, is only about one third as long (5 mm) as the proximal convoluted tubule. This short tubule is responsible for  reabsorption of Naand secretion of K into the ultrafiltrate to conserve Na .

1	FIGURE 20.21 • Electron micrograph of a distal convoluted tubule cell. The apical surface of the cell displays some microvilli (Mv), but they are not sufficiently long or numerous to give the appearance of a brush border (compare with Fig. 20.15). The nucleus and Golgi apparatus (G) are in the upper portion of the cell. Mitochondria (M) are chiefly in the basal region of the cell within the interdigitating processes (IP). As in the proximal tubule cell, the mitochondria account for the appearance of basal striations in the light microscope. A basal lamina (BL) is seen adjacent to the basal surface of the cell. 12,000.  reabsorption of bicarbonate ions, with concomitant secretion of hydrogen ions, leading to further acidification of the urine.  secretion of ammonium in response to the kidneys’ need to excrete acid and generate bicarbonate.

1	Aldosterone, secreted by the adrenal gland and released under stimulation by angiotensin II, increases the reabsorption of Na and secretion of K . These effects increase blood volume and blood pressure in response to increased blood Na concentration.

1	The collecting tubules as well as the cortical collecting ducts and medullary collecting ducts are composed of simple epithelium. The collecting tubules and cortical col lecting ducts have flattened cells, somewhat squamous to cuboidal in shape. The medullary collecting ducts have cuboidal cells, with a transition to columnar cells as the ducts increase in size. The collecting tubules and ducts are readily distinguished from proximal and distal tubules by virtue of the cell boundaries that can be seen in the light microscope (Plate 77, page 734). Two distinct types of cells are present in the collecting tubules and collecting ducts:  Light cells, also called collecting duct cells or CD cells, are the principal cells of the system. They are pale-staining cells with true basal infoldings rather than processes that interdigitate with those of adjacent cells. They possess a single primary cilium and relatively few short microvilli (Fig. 20.22). They contain small, spherical mitochondria.

1	than processes that interdigitate with those of adjacent cells. They possess a single primary cilium and relatively few short microvilli (Fig. 20.22). They contain small, spherical mitochondria. These cells possess an abundance of antidiuretic hormone (ADH)–regulated water channels, aquaporin-2 (AQP-2), which are responsible for water permeability of the collecting ducts. In addition, aquaporins AQP-3 and AQP-4 are present within the basolateral membrane of these cells.

1	 Dark cells, also called intercalated (IC) cells, occur in considerably smaller numbers. They have many mitochondria, and their cytoplasm appears denser. Microplicae, cytoplasmic folds, are present on their apical surface, as well as microvilli. The microplicae are readily observed with the SEM but may be mistaken for microvilli with the TEM (see Fig. 20.22). They do not show basal infoldings but have basally located interdigitations with neighboring cells. Numerous vesicles are present in the apical cytoplasm. The intercalated cells are involved in the secretion of H (-intercalated cells) or bicarbonate (-intercalated cells), depending on the whether the kidneys need to

1	FIGURE 20.22 • Scanning electron micrograph of a collecting tubule. This micrograph shows dark cells (asterisks), with numerous short lamellipodia or microridges on their surface, and light cells, each with a primary cilium on its free surface along with small microvilli. The terms light and dark refer to the staining character of sectioned cells and not to the density differences reflecting charge characteristics of the coated surface of the specimen. (Courtesy of Dr. C. Craig Tisher.) excrete acid or alkali. The -intercalated cell actively secretes H into the collecting duct lumen via ATP-depen dent pumps and releases HCO3 via Cl /HCO3 exchangers located in their basolateral cell membrane. The -intercalated cells have opposite polarity and secrete bicarbonate ions into the lumen of the collecting duct. Because of the nature of the diet and thus the need to excrete acid, the epithelium of collecting ducts contains more than -intercalated cells.

1	The cells of the collecting ducts gradually become taller as the ducts pass from the outer to the inner medulla and become columnar in the region of the renal papilla. The number of dark cells gradually decreases until there are none in the ducts as they approach the papilla. The connective tissue of the kidney parenchyma, called interstitial tissue, surrounds the nephrons, ducts, and blood and lymphatic vessels. This tissue increases considerably in amount from the cortex (where it constitutes approximately 7% of the volume) to the inner region of the medulla and papilla (where it may constitute more than 20% of the volume).

1	In the cortex, two types of interstitial cells are recognized: cells that resemble fbroblasts, found between the basement membrane of the tubules and the adjacent peritubular capillaries, and occasional macrophages. In their intimate relationship with the base of the tubular epithelial cells, the fibroblasts resemble the subepithelial fibroblasts of the intestine. These cells synthesize and secrete the collagen and glycosaminoglycans of the extracellular matrix of the interstitium. In the medulla, the principal interstitial cells resemble myofbroblasts. They are oriented to the long axes of the tubular structures and may have a role in compressing these structures. The cells contain prominent bundles of actin filaments, abundant rough endoplasmic reticulum (rER), a well-developed Golgi complex, and lysosomes. Prominent lipid droplets in the cytoplasm appear to increase and decrease in relation to the diuretic state.

1	Most fibroblasts originate within the interstitial tissue through a mechanism called epithelial–mesenchymal transition. The conversion of tubular epithelial cells into a mesenchymal phenotype is initiated by an alteration in the balance of local cytokine concentrations. During persistent injury and chronic inflammation of the kidney parenchyma, fibroblasts increase their numbers and, by secreting excess extracellular matrix, destroy normal interstitial architecture of the kidney. Research studies suggest that in renal fbrosis, more than one third of all disease-related fibroblasts originate from tubular epithelial cells at the site of injury. Proliferation of fibroblasts in response to local mitogens usually leads to irreversible renal failure characterized by tubulointerstitial nephritis. Recent therapeutic interventions in renal fibrosis are directed toward inhibiting fibroblast formation by shifting local cytokine balance in favor of reversal mesenchymal– epithelial transition.

1	The countercurrent multiplier system creates hyperosmotic urine. The term countercurrent indicates a flow of fluid in adjacent structures in opposite directions. The ability to excrete hyper-osmotic urine depends on the countercurrent multiplier system that involves three structures:  Loop of Henle, which acts as a countercurrent multiplier. The ultrafiltrate moves within the descending limb of the thin segment of the loop toward the renal papilla and moves back toward the corticomedullary junction within the ascending limb of the thin segment. The osmotic gradients of the medulla are established along the axis of the loop of Henle.  Vasa recta, form loops parallel to the loop of Henle. They act as countercurrent exchangers of water and solutes between the descending part (arteriolae rectae) and ascending part (venulae rectae) of the vasa recta. The vasa recta help to maintain the osmotic gradient of the medulla.

1	 Collecting duct in the medulla acts as an osmotic equilibrating device. Modified ultrafiltrate in the collecting ducts can be further equilibrated with the hyperosmotic medullary interstitium. The level of equilibration depends on activation of ADH-dependent water channels (AQP-2). A standing gradient of ion concentration produces hyper-osmotic urine by a countercurrent multiplier effect. The loop of Henle creates and maintains a gradient of ion concentration in the medullary interstitium that increases from the corticomedullary junction to the renal papilla. As noted above, the thin descending limb of the loop of Henle is freely permeable to water, whereas the ascending limb of the loop of Henle is impermeable to water. Further, the thin ascending limb cells add Na and Cl to the interstitium.

1	Because water cannot leave the thin ascending limb, the interstitium becomes hyperosmotic relative to the luminal contents. Although some of the Cl and Na of the interstitium diffuses back into the nephron at the thin descending limb, the ions are transported out again in the thin ascending limb and distal straight tubule (thick ascending limb). This produces the countercurrent multiplier effect. Thus, the concentration of NaCl in the interstitium gradually increases down the length of the loop of Henle and, consequently, through the thickness of the medulla from the corticomedullary junction to the papilla. Vasa recta containing descending arterioles and ascending venules act as countercurrent exchangers. For an understanding of the countercurrent exchange mechanism, it is necessary to resume the description of the renal circulation at the point at which the efferent arteriole leaves the renal corpuscle.

1	The efferent arterioles of the renal corpuscles of most of the cortex branch to form the capillary network that surrounds the tubular portions of the nephron in the cortex, the peritubular capillary network. The efferent arterioles of the juxtamedullary renal corpuscles form several unbranched arterioles that descend into the medullary pyramid. These arteriolae rectae make a hairpin turn deep in the medullary regulated by antidiuretic hormone (ADH, vasopressin), a hormone produced in the hypothalamus and released from the posterior lobe of the pituitary gland. ADH increases the permeability of the collecting duct to water, thereby producing more-concentrated urine. At the molecu-lar level, ADH acts on ADH-regulated water channels, aqua-porin 2 (AQP-2), located in the epithelium of the terminal portion of the distal convoluted tubule and in the epithelium of the collecting tubules and ducts. However, the action of ADH is more significant in the collecting tubules and collect-ing ducts.

1	portion of the distal convoluted tubule and in the epithelium of the collecting tubules and ducts. However, the action of ADH is more significant in the collecting tubules and collect-ing ducts. ADH binds to receptors on the cells of these tubules and triggers the following actions:  Translocation of the AQP-2–containing intracyto-plasmic vesicles into the apical cell surface—a short-term effect. This results in an increased number of available AQP-2 channels at the cell surface, thus increas-ing water permeability of the epithelium.  Synthesis of AQPs-2 and their insertion into the apical cell membrane—a long-term effect volume stimulates release of ADH, as does nicotine. In the absence of ADH, copious, dilute urine is produced. This condition is called central diabetes insipidus (CDI). Recent studies indicate that mutation of two genes encoding AQP-2 and ADH receptors is responsible for a form of CDI called nephrogenic diabetes insipidus. In this disease, the kidney does not

1	(CDI). Recent studies indicate that mutation of two genes encoding AQP-2 and ADH receptors is responsible for a form of CDI called nephrogenic diabetes insipidus. In this disease, the kidney does not respond to ADH because of defective AQP-2 and ADH receptor proteins synthesized by the col-lecting tubule and duct epithelial cells. Excess water con-sumption can also inhibit ADH release, thereby promoting the production of a large volume of hyposmotic urine. Increased secretion of ADH can produce an extremely hyperosmotic urine, thereby conserving water in the body. Inadequate consumption of water or loss of water because of sweating, vomiting, or diarrhea stimulates release of ADH. This leads to an increase in the permeability of the epithelium of the distal and collecting tubules and pro-motes the production of a small volume of hyperosmotic urine.

1	 FOLDER 20.6 Functional Considerations: Hormonal Regulation of Collecting Duct Function Water permeability of the epithelium of the collecting ducts is An increase in plasma osmolality or a decrease in blood pyramid and ascend as the venulae rectae. Together, the descending arterioles and the ascending venules are called the vasa recta. The arteriolae rectae form capillary plexuses lined by fenestrated endothelium that supply the tubular structures at the various levels of the medullary pyramid. Interaction between collecting ducts, loops of Henle, and vasa recta is required for concentrating urine by the countercurrent exchange mechanism.

1	Interaction between collecting ducts, loops of Henle, and vasa recta is required for concentrating urine by the countercurrent exchange mechanism. Because the thick ascending limb of the loop of Henle has a high level of transport activity and because it is impermeable to water, the modified ultrafiltrate that ultimately reaches the distal convoluted tubule is hyposmotic. When ADH is present, the distal convoluted tubules, the collecting tubules, and the collecting ducts are highly permeable to water. Therefore, within the cortex, in which the interstitium is isosmotic with blood, the modified ultrafiltrate within the distal convoluted tubule equilibrates and becomes isosmotic, partly by loss of water to the interstitium and partly by addition of ions other than Na and Cl to the ultrafiltrate. In the medulla, increasing amounts of water leave the ultrafiltrate as the collecting ducts pass through the increasingly hyperosmotic interstitium on their course to the papillae.

1	As noted previously, the vasa rectae also form loops in the medulla that parallel the loop of Henle. This arrangement ensures that the vessels provide circulation to the medulla without disturbing the osmotic gradient established by transport of Cl in the epithelium of the ascending limb of the loop of Henle. The vasa recta form a countercurrent exchange system in the following manner: Both the arterial and venous sides of the loop are thin-walled vessels that form plexuses of fenestrated capillaries at all levels in the medulla. As the arterial vessels descend through the medulla, the blood loses water to the interstitium and gains salt from the interstitium so that at the tip of the loop, deep in the medulla, the blood is essentially in equilibrium with the hyperosmotic interstitial fluid.

1	As the venous vessels ascend toward the corticomedullary junction, the process is reversed (i.e., the hyperosmotic blood loses salt to the interstitium and gains water from the interstitium). This passive countercurrent exchange of water and salt between the blood and the interstitium occurs without expenditure of energy by the endothelial cells. The energy that drives this system is the same energy that drives the multiplier system, namely, the movement of Na and Cl out of the cells of the water-impermeable ascending limb of the loop of Henle. The counter-current exchange system and other movements of molecules in different parts of the nephron are shown in Figure 20.23. Some aspects of the blood supply of the kidney have been described in relation to specific functions (i.e., glomerular filtration, control of blood pressure, and countercurrent exchange). It remains, however, to provide an overall description of the blood supply of the kidney.

1	Each kidney receives a large branch from the abdominal aorta, called the renal artery. The renal artery branches within the renal sinus and sends interlobar arteries into the substance of the kidney (Fig. 20.24). These arteries travel between the pyramids as far as the cortex and then turn to follow an arched course along the base of the pyramid between the medulla and the cortex. Thus, these interlobar arteries are designated arcuate arteries.

1	Na+ H2O H2O H2O H2O H2O H2O H2O H2O H2O Cl-Cl-K+ K+ K+ H+ H+ H+glomerulus glucose amino acids polypeptides distal convoluted tubule collecting tubule proximal convoluted tubule loop of Henleurea thin descending limb collecting duct MEDULLA CORTEX urea distal straight tubule H2O Na+ organic acids Ca2+ Mg2+ Na+ Na+ Na+ Cl-thin ascending limb proximal straight tubule Active transport Active exchange Active cotransport Passive diffusion ADH-dependent H2O movement Aquaporin-dependent H2O movement (AQP)2 2 2 3 3 H+ bicarbonate anion bicarbonate anion Na+ Na+ K+ 1 1 K+K+ Cl- FIGURE 20.23 • Diagram showing movement of substances into and out of the nephron and collecting system. The symbols indicate the mode of transport as noted in the key.

1	FIGURE 20.23 • Diagram showing movement of substances into and out of the nephron and collecting system. The symbols indicate the mode of transport as noted in the key. Interlobular arteries branch from the arcuate arteries and ascend through the cortex toward the capsule. Although the boundaries between lobules are not distinct, the interlobular arteries, when included in a section cut perpendicular to the vessel, are located midway between adjacent medullary rays, traveling in the cortical labyrinth. As they traverse the cortex toward the capsule, the interlobular arteries give off branches, the afferent arterioles, one to each glomerulus. A single afferent arteriole may spring directly from the interlobular artery, or a common stem from the interlobular artery may branch to form several afferent arterioles. Some interlobular arteries terminate near the periphery of the cortex, whereas others enter the kidney capsule to provide its arterial supply.

1	Afferent arterioles give rise to the capillaries that form the glomerulus. The glomerular capillaries reunite to form an efferent arteriole that, in turn, gives rise to a second network of capillaries, the peritubular capillaries. The arrangement of these capillaries differs according to whether they originate from cortical or juxtamedullary glomeruli.  Efferent arterioles from cortical glomeruli lead into a peritubular capillary network that surrounds the local uriniferous tubules (G1 and G2, Fig. 20.24).  Efferent arterioles from juxtamedullary glomeruli descend into the medulla alongside the loop of Henle; they break up into smaller vessels that continue toward the apex of the pyramid but make hairpin turns at various levels to

1	FIGURE 20.24 • Schematic diagram of the renal blood supply. The renal artery gives rise to interlobar arteries that branch into arcuate arteries at the border between the medulla and cortex. Interlobular arteries (IL) branch from the arcuate arteries and travel toward the renal capsule, giving off afferent arterioles to the glomeruli (G). Glomeruli in the outer part of the cortex (G1, G2) send efferent arterioles to the peritubular capillaries (PC) that surround the tubules in the cortex; glomeruli near the medulla (G3), the juxtamedullary glomeruli, send efferent arterioles almost entirely into the medullary plexus (MP) of capillaries via the arteriolae rectae spuriae (ARS). Blood returns from the capillaries via veins that enter the arcuate veins. Stellate veins (SV) near the capsule drain both the capsular (CC) and the peritubular capillaries.

1	return as straight vessels toward the base of the pyramid (see G3, Fig. 20.24). Thus, the efferent arterioles from the juxtamedullary glomeruli give rise to vasa recta involved in the countercurrent exchange system and their peritubular capillary network. These vessels are described in the explanation of the countercurrent exchange system (page 721). Generally, venous flow in the kidney follows a reverse course to arterial flow, with the veins running in parallel with the corresponding arteries (see Fig. 20.24). Thus,  Peritubular cortical capillaries drain into interlobular veins, which in turn drain into arcuate veins, interlobar veins, and the renal vein.  The medullary vascular network drains into arcuate veins and so forth.  Peritubular capillaries near the kidney surface and capillaries of the capsule drain into stellate veins (so called for their pattern of distribution when viewed from the kidney surface), which drain into interlobular veins , and so forth.

1	The kidneys contain two major networks of lymphatic vessels. These networks are not usually visible in routine histologic sections but can be demonstrated by experimental methods. One network is located in the outer regions of the cortex and drains into larger lymphatic vessels in the capsule. The other network is located more deeply in the substance of the kidney and drains into large lymphatic vessels in the renal sinus. There are numerous anastomoses between the two lymphatic networks.

1	The fibers that form the renal plexus are derived mostly from the sympathetic division of the autonomic nervous system. They cause contraction of vascular smooth muscle and consequent vasoconstriction.  Constriction of the afferent arterioles to the glomeruli reduces the filtration rate and decreases the production of urine.  Constriction of the efferent arterioles from the glomeruli increases the filtration rate and increases the production of urine.  Loss of sympathetic innervation leads to increased urinary output. It is evident, however, that the extrinsic nerve supply is not necessary for normal renal function. Although the nerve fibers to the kidney are cut during renal transplantation, transplanted kidneys subsequently function normally. URETER, URINARY BLADDER, AND URETHRA All excretory passages, except the urethra, have the same general organization.

1	URETER, URINARY BLADDER, AND URETHRA All excretory passages, except the urethra, have the same general organization. On leaving the collecting ducts at the area cribrosa, the urine enters a series of structures that do not modify it but are specialized to store and pass the urine to the exterior of the body. The urine flows sequentially to a minor calyx, a major calyx, and the renal pelvis, and leaves each kidney through the ureter to the urinary bladder, where it is stored. The urine is finally voided through the urethra. All of these excretory passages, except the urethra, have the same general structures, namely, a mucosa (lined by transitional epithelium), muscularis, and adventitia (or, in some regions, a serosa). Transitional epithelium lines the calyces, ureters, bladder, and the initial segment of the urethra.

1	Transitional epithelium lines the calyces, ureters, bladder, and the initial segment of the urethra. Transitional epithelium (urothelium) lines the excretory passages leading from the kidney. This stratified epithelium is essentially impermeable to salts and water. The epithelium begins in the minor calyces as two cell layers and increases to an apparent four to five layers in the ureter (Fig. 20.25) and as many as six or more layers in the empty bladder. However, chapter 20 Urinary System U R ETE R, U R I NARY B LADD E R, AN D U R ETH RA 723 FIGURE 20.25 • Photomicrograph of transitional epithelium (urothelium). This H&E–stained specimen shows the 4to 5-celllayer thickness of the epithelium in the relaxed ureter. The surface cells exhibit a rounded or dome-shaped profile. The connective tissue below the epithelium (Ep) is relatively cellular and contains a number of lymphocytes. Blood vessels (BV) are also abundant in this area. 450.

1	when the bladder is distended, as few as three layers are seen. This change reflects the ability of the cells to accommodate to distension. The cells in the distended bladder, particularly the large surface cells and those in the layers below, flatten and unfold to accommodate the increasing surface area. As the individual cells unfold and flatten, the resulting appearance is the “true” three layers.

1	In routine histologic sections obtained from the empty bladder, the surface epithelial cells are usually cuboidal and bulge into the lumen. They are frequently described as “dome shaped” or “umbrella” cells because of the curvature of the apical surface (see Fig. 20.25). When examined with the TEM, the plasma membrane exhibits an unusual feature (e.g., modified areas of the plasma membrane called plaques) are seen (Fig. 20.26). These plaques appear to be more rigid and thicker (up to 12 nm) than the rest of the apical plasma membrane. Actin filaments are observed stretching from the inner surface of the plaques into the cytoplasm. In the undistended urinary bladder, the plaques give the luminal surface of cells an irregular scalloped contour (Fig. 20.27). Each cell appears to fold inward on itself. Because of this folding, the plaques appear as a series of fusiform vesicles. Their lumina, however, are in continuity with the cell’s exterior. As the bladder distends, the fusiform

1	on itself. Because of this folding, the plaques appear as a series of fusiform vesicles. Their lumina, however, are in continuity with the cell’s exterior. As the bladder distends, the fusiform vesicles unfold and become part of the surface as the cell stretches and flattens (Fig. 20.28).

1	FIGURE 20.26 • Transmission electron micrograph of urinary bladder epithelium. The mucous membrane of the urinary bladder consists of transitional epithelium (Ep) with an underlying lamina propria (LP). The epithelial cells contain unique fusiform vesicles, which are evident here at this relatively low magnification. These are seen at higher magnification in Figure 20.24. 5,000. Smooth muscle of the urinary passages is arranged in bundles. A dense collagenous lamina propria underlies the urothelium throughout the excretory passages. Neither a muscularis mucosae nor a submucosal layer is present in their walls. In the tubular portions (ureters and urethra), usually two layers of smooth muscle lie beneath the lamina propria:  Longitudinal layer, the inner layer that is arranged in a loose spiral pattern Circular layer, the outer layer that is arranged in a tight

1	Note that this arrangement of the smooth muscle is opposite that of the muscularis externa of the intestinal tract. The smooth muscle of the urinary passages is mixed with connective tissue, so that it forms parallel bundles rather than pure FIGURE 20.27 • Transmission electron micrograph of the apical portion of a transitional epithelial cell. a. The cytoplasm displays small vesicles, filaments, and mitochondria, but the most distinctive feature of the cell is its fusiform vesicles (FV). 27,000. b. The higher magnification shows that the membrane forming the vesicles appears to be similar to the plasma membrane of the cell surface (arrows). Both membranes are thickened and give the impression of possessing a degree of rigidity greater than that of plasma membrane in other locations. The thickened plasma membrane represents a sectioned view of a surface plaque. The fusiform vesicles are formed by the infolding of the plaques in the cells of the relaxed urinary bladder. 60,000.

1	muscular sheets. Peristaltic contractions of the smooth muscle move the urine from the minor calyces through the ureter to the bladder. Each ureter conducts urine from the renal pelvis to the urinary bladder and is approximately 24 to 34 cm long. The distal part of the ureter enters the urinary bladder and follows an oblique path through the wall of the bladder. Transitional epithelium (urothelium) lines the luminal surface of the wall of the ureter. The remainder of the wall is composed of smooth muscle and connective tissue. The smooth

1	FIGURE 20.28 • Diagrams of the luminal surface of transitional epithelial cells. The upper drawing depicts part of a surface cell in a distended bladder; the lower drawing depicts the same cell as it would appear in a relaxed bladder. The plasma membrane is thickened in regions to form plaques. The interplaque regions consist of membrane that is not thickened. In the relaxed bladder, the plaques are invaginated into the cell, and although they retain their continuity with the surface, the invaginated plaques typically appear as isolated fusiform vesicles in electron micrographs. Filaments attached to the undersurface of the plaques may prevent undue stretching in the distended bladder. (Modified from Staehelin LA, Chlapowski FJ, Bonneville MA. Lumenal plasma membrane of the urinary bladder. I. Three-dimensional reconstruction from freeze-etch images. J Cell Biol 1972;53:73–91.) muscle is arranged in three layers: an inner longitudinal layer, a middle circular layer, and an outer

1	I. Three-dimensional reconstruction from freeze-etch images. J Cell Biol 1972;53:73–91.) muscle is arranged in three layers: an inner longitudinal layer, a middle circular layer, and an outer longitudinal layer (Plate 78, page 736). However, the outer longitudinal layer is present only at the distal end of the ureter. Usually, the ureter is embedded in the retroperitoneal adipose tissue. The adipose tissue, vessels, and nerves form the adventitia of the ureter.

1	As the bladder distends with urine, the openings of the ureters are compressed, reducing the possibility of reflux of urine into the ureters. Contraction of the smooth muscle of the bladder wall also compresses the openings of the ureters into the bladder. This action helps prevent the spread of infection from the bladder and urethra, frequent sites of chronic infection (particularly in females), to the kidney. In the terminal portion of the ureters, a thick outer layer of longitudinal muscle is present in addition to the two listed above, particularly in the portion of the ureter that passes through the bladder wall. Most descriptions of the bladder musculature indicate that this longitudinal layer continues into the wall of the bladder to form a principal component of its wall. The smooth muscle of the bladder, however, is not as clearly separated into distinctive layers. chapter 20 Urinary System U R ETE R, U R I NARY B LADD E R, AN D U R ETH RA 725

1	chapter 20 Urinary System U R ETE R, U R I NARY B LADD E R, AN D U R ETH RA 725 The urinary bladder is a distensible reservoir for urine, located in the pelvis, posterior to the pubic symphysis; its size and shape change as it fills. It contains three openings, two for the ureters (ureteric orifces) and one for the urethra (internal urethral orifce). The triangular region defined by these three openings, the trigone, is relatively smooth and constant in thickness, whereas the rest of the bladder wall is thick and folded when the bladder is empty and thin and smooth when the bladder is distended. These differences reflect the embryologic origins of the trigone and the rest of the bladder wall: the trigone is derived from the embryonic mesonephric ducts, and the major portion of the wall originates from the cloaca.

1	The smooth muscle of the bladder wall forms the detrusor muscle. Toward the opening of the urethra, the muscle fibers form the involuntary internal urethral sphincter, a ringlike arrangement of muscle around the opening of the urethra. The smooth muscle bundles of the detrusor muscle are less regularly arranged than that of the tubular portions of the excretory passages, and thus the muscle and collagen bundles are randomly mixed (Plate 79, page 738). Contraction of the detrusor muscle of the bladder compresses the entire organ and forces the urine into the urethra.

1	The bladder is innervated by both sympathetic and parasympathetic divisions of the autonomic nervous system:  Sympathetic fbers form a plexus in the adventitia of the bladder wall. These fibers probably innervate blood vessels in the wall.  Parasympathetic fbers originate from S2 to S4 seg ments of the spinal cord and travel with pelvic splanchnic nerves into the bladder. They end in terminal ganglia in the muscle bundles and the adventitia and are the efferent fibers of the micturition refex.  Sensory fbers from the bladder to the sacral portion of the spinal cord are the afferent fibers of the micturition reflex. The urethra is the fibromuscular tube that conveys urine from the urinary bladder to the exterior through the external urethral orifce. The size, structure, and functions of the urethra differ in males and females.

1	In the male, the urethra serves as the terminal duct for both the urinary and genital systems. It is about 20 cm long and has three distinct segments:  Prostatic urethra extends for 3 to 4 cm from the neck of the bladder through the prostate gland (see page 785). It is lined with transitional epithelium (urothelium). The ejaculatory ducts of the genital system enter the posterior wall of this segment, and many small prostatic ducts also empty into this segment.

1	 Membranous urethra extends for about 1 cm from the apex of the prostate gland to the bulb of the penis. It passes through the deep perineal pouch of the pelvic floor as it enters the perineum. Skeletal muscle of the deep perineal pouch surrounding the membranous urethra forms the external (voluntary) sphincter of the urethra. Transitional epithelium ends in the membranous urethra. This segment is lined with a stratified or pseudostratified columnar epithelium that resembles the epithelium of the genital duct system more than it resembles the epithelium of the more proximal portions of the urinary duct system.

1	 Penile (spongy) urethra extends for about 15 cm through the length of the penis and opens on the body surface at the glans penis. The penile urethra is surrounded by the corpus spongiosum as it passes through the length of the penis. It is lined with pseudostratified columnar epithelium except at its distal end, where it is lined with stratified squamous epithelium continuous with that of the skin of the penis. Ducts of the bulbourethral glands (Cowper’s glands) and of the mucus-secreting urethral glands (glands of Littré) empty into the penile urethra.

1	In the female, the urethra is short, measuring 3 to 5 cm in length from the bladder to the vestibule of the vagina, where it normally terminates just posterior to the clitoris. The mucosa is traditionally described as having longitudinal folds. As in the male urethra, the lining is initially transitional epithelium, a continuation of the bladder epithelium, but changes to stratified squamous epithelium before its termination. Some investigators have reported the presence of stratified columnar and pseudostratified columnar epithelium in the midportion of the female urethra.

1	Numerous small urethral glands, particularly in the proximal part of the urethra, open into the urethral lumen. Other glands, the paraurethral glands, which are homologous to the prostate gland in the male, secrete into the common paraurethral ducts. These ducts open on each side of the external urethral orifice. They produce an alkaline secretion. The lamina propria is a highly vascularized layer of connective tissue that resembles the corpus spongiosum in the male. Where the urethra penetrates the urogenital diaphragm (membranous part of the urethra), the striated muscle of this structure forms the external (voluntary) urethral sphincter. This page intentionally left blank.

1	The urinary system consists of the paired kidneys, the paired ureters, which lead from the kidneys to the urinary bladder, and the urethra, which leads from the bladder to the exterior of the body. The kidneys conserve body fluid and electrolytes and remove metabolic wastes such as urea, uric acid, creatinine, and breakdown products of various substances. They produce urine, initially an ultrafiltrate of blood that is modi-fied by selective resorption and specific secretion by kidney tubule cells. The kidneys also function as endocrine organs, producing erythropoi-etin, a growth factor that regulates red blood cell formation, and renin, a hormone involved in blood pressure and blood volume control. They also hydroxylate vitamin D, a steroid prohormone, to produce its active form. Each kidney is a flattened, bean-shaped structure approximately 10 cm long, 6.5 cm wide (from convex to concave border), and 3 cm thick. The concave medial border of each kidney contains a hilum, an indented

1	is a flattened, bean-shaped structure approximately 10 cm long, 6.5 cm wide (from convex to concave border), and 3 cm thick. The concave medial border of each kidney contains a hilum, an indented region through which blood vessels, nerves, and lymphatic vessels enter and leave the kidney. The funnel-shaped origin of the ureter, the renal pelvis, also leaves the kidney at the hilum. A cut, hemi-sected fresh kidney reveals two distinct regions: a cortex, the reddish-brown outer region, and a medulla, a much lighter inner part continu-ous with the renal pelvis. The cortex is characterized by renal corpuscles and their tubules, including the convoluted and straight tubules of the nephron, the collecting tubules, and an extensive vascular supply.

1	Kidney, human, fresh specimen ×3.

1	A frontal section through the cortex and medulla of an unembalmed kidney obtained from autopsy is shown here. The visible hilar region consists of minor calyces (grey/white) surrounded by yellow in appearance adipose tissue. The outer part of the kidney has a reddish-brown appearance; this is the cortex. It is easily distinguished from the inner portion, the medulla, which is further divided into an outer portion (OM), identified here by the presence of straight blood vessels, the vasa recta (VR), and an inner portion (IM), which has a lighter and more homogenous appearance. The medulla consists of renal pyramids, which have their base facing the cortex and their apex in the form of a papilla (P) is directed towards the hilum. The pyramids are separated, sometimes only partially as in this figure, by cortical material that is designated the renal columns (RC). The majority of the outer part of the pyramid on the left, has not been included in the plane of

1	Cortex and medulla, kidney, human, H&E ×20. A histologic section including the cortex and part of the medulla is shown here. Located at the boundary between the two (partly marked by the dashed line) are numerous profiles of arcuate arteries (AA) and arcuate veins (AV ). The most distinctive feature of the renal cortex, regardless of the plane of section, is the presence of the renal corpuscles (RC). These are spherical structures composed of a glomerulus (glomerular vascular tuft) surrounded by the visceral the section. The papillae are free tips of the pyramids that project into the first of a series of large urine collecting spaces referred to as the minor calyces (MC); the inner surface of the calyx is white. The minor calyces drain into major calyces, and in turn, these open into the renal pelvis, which funnels urine into the ureter.

1	An interesting feature in this specimen is that the blood has been retained in many of the vessels, thereby allowing for visualization of several renal vessels in their geographic location. Among the vessels that can be identified in the cut face of the kidney shown here are the interlobular vessels (IV) within the cortex; the arcuate veins (AV) and the arcuate arteries (AA) at the base of the pyramids; the interlobar arteries (ILA) and veins (ILV) between renal pyramids and, in the medulla, the vessels going to and from the capillary network of the pyramid. The latter vessels, both arterioles and venules, are relatively straight and are designated collectively as the vasa recta (VR). (Specimen courtesy of Dr. Eric A. Pfeifer, Mayo Clinic, Rochester, MN).

1	and parietal epithelium of Bowman’s capsule. Also seen in the cortex are groups of tubules that are more or less straight and disposed in a radial direction from the base of the medulla (arrows); these are the medullary rays. In contrast, the medulla presents profiles of tubular structures that are arranged as gentle curves in the outer part of the medulla, turning slightly to become straight in the inner part of the medulla. The disposition of the tubules (and blood vessels) gives the cut face of the pyramid a slightly striated appearance that is also evident in the gross specimen (see figure above). KEY AA, arcuate arteries AV, arcuate veins ILA, Interlobar artery ILV, Interlobar vein IM, inner medulla IV, interlobular vessels MC, minor calyx OM, outer medulla P, papilla RCol, renal column RC, renal corpuscles VR, vasa recta arrows, medullary rays dashed line, boundary between cortex and medulla

1	The nephron is the functional unit of the kidney. There are about 2 million nephrons in each human kidney. They are responsible for the production of urine and correspond to the secretory part of other glands. The collecting tubules, responsible for the final concentration of the urine, are analogous to the ducts of exocrine glands. The nephron is made up of the renal corpuscle and the renal tubule. The renal corpuscle consists of the glomerulus, a tuft of 10 to 20 capillary loops, surrounded by a double-layered epithelial cup, the renal or Bowman’s capsule. The glomeru-lar capillaries are supplied at the vascular pole of Bowman’s capsule by an afferent arteriole and drained by an efferent arteriole that leaves Bow-man’s capsule at the vascular pole and then branches to form a new capillary network to supply the kidney tubules. The opposite pole of Bowman’s capsule, the urinary pole, is where the filtrate leaves the renal capsule. The tubular parts of the nephron are the proximal

1	network to supply the kidney tubules. The opposite pole of Bowman’s capsule, the urinary pole, is where the filtrate leaves the renal capsule. The tubular parts of the nephron are the proximal thick segment (consisting of the proximal convoluted tubule and the proximal straight tubule), the thin segment, which constitutes the thin limb of the loop of Henle, and the distal thick segment, consisting of the distal straight tubule and the distal convoluted tubule. The loop of Henle is the U-shaped portion of the nephron consisting of the thick straight portions of the proximal and distal tubules and the thin segment between them. The distal convoluted tubule joins the collecting tubule. The nephron and the collecting tubule constitute the uriniferous tubule.

1	Cortex, kidney, human, H&E ×60.

1	The renal cortex can be divided into regions referred to as the cortical labyrinth (CL) and the medullary rays (MR). The cortical labyrinth contains the renal corpuscles (RC ), which appear as relatively large spherical structures. Surrounding each renal corpuscle are the proximal and distal convoluted tubules. They are also part of the cortical labyrinth. The convoluted tubules, particularly the proximal, because of their tortuosity, present a variety of profiles, most of which are oval or circular; others, more elongate, are in the shape of a letter J, a C, or even an S. The medullary rays are composed of groups of straight tubules oriented in the same direction and appear to radiate from the base of the pyramid. When the medullary rays are cut longitudinally, as they are in this figure, the tubules present elongated profiles. The medullary rays contain proximal straight tubules (thick segments; descending limb of Henle’s loop), distal straight tubules (thick segments; ascending

1	the tubules present elongated profiles. The medullary rays contain proximal straight tubules (thick segments; descending limb of Henle’s loop), distal straight tubules (thick segments; ascending limbs of Henle’s loop), and collecting tubules.

1	Cortex, kidney, human, H&E ×120. This figure presents another profile of the renal cortex, at a somewhat higher magnification, cut in a plane at a right angle to the section in figure above. The peripheral part of the micrograph shows the cortical labyrinth in which the tubules display chiefly round and oval profiles but also some that are more elongate and curved. The appearance is the same as the cortical labyrinth areas of figure above. A renal corpuscle (RC) is also present in the cortical labyrinth. In contrast, the profiles presented by the tubules of the medullary ray in this figure are quite different from those seen in figure above. All of the tubules bounded by the dashed line belong to the medullary ray (MR), and all are cut in cross section.

1	A general survey of the tubules within the medullary ray reveals that several distinct types can be recognized on the basis of the size of the tubule, shape of the lumen, and size of the tubule cells. These features as well as those of the cortical labyrinth are considered in Plate 76. PLATE 75 KEY CL, cortical labyrinth MR, medullary ray RC, renal corpuscle dashed line, approximate boundary of the medullary ray

1	Proximal and distal convoluted tubules display features that aid in their identification in H&E-stained paraffin sections. Proximal convoluted tubules generally have a larger diameter than distal tubules have; cross sections of the lumen often appear stellate. A brush border (apical microvilli) is often visible on the proximal tubule cells. Also, the proximal convoluted tubule is more than twice as long as the distal convo-luted tubule; thus, the majority of tubular profiles in the cortical labyrinth will be of proximal tubules. Mesangial cells and their extracellular matrix constitute the mesangium of the renal corpuscle. They underlie the endothelium of the cap-illaries of the glomerular tuft and extend to the vascular pole, where they become part of the juxtaglomerular apparatus. The terminal portion of the distal thick segment of the nephron lies close to the afferent arteriole. Tubule epithelial cells closest to the arteriole are thinner, taller, and more closely packed than

1	terminal portion of the distal thick segment of the nephron lies close to the afferent arteriole. Tubule epithelial cells closest to the arteriole are thinner, taller, and more closely packed than other tubule cells and constitute the macula densa. Arterial smooth muscle cells opposite the macula densa are modified into juxtaglomerular cells that secrete renin in response to decreased blood NaCl concentration.

1	Proximal and distal convoluted tubules, kidney, human, H&E ×240. In this figure, an area of cortical labyrinth, there are six distal convoluted tubule (DC) profiles. The proximal convoluted tubules (unlabeled) have a slightly larger outside diameter than the distal tubules have. The proximal tubules have a brush border, whereas the distal tubules have a cleaner, sharper luminal Renal corpuscles, kidney, human, H&E ×360. The renal corpuscle appears as a spherical structure whose periphery is composed of a thin capsule that encloses a narrow clear-appearing space, the urinary space (asterisks), and a capillary tuft or glomerulus that appears as a large cellular mass. The capsule of the renal corpuscle, known as the renal or

1	Bowman’s capsule, actually has two parts: a parietal layer, which is marked (BC ), and a visceral layer. The parietal layer consists of simple squamous epithelial cells. The visceral layer consists of cells, called podocytes (Pod ), that lie on the outer surface of the glomerular capillary. Except where they clearly line the urinary space, as the labeled cells do in figure on left, podocytes may be difficult to distinguish from the capillary endothelial cells. To complicate matters, the mesangial cells are also a component of the glomerulus. In general, nuclei of podocytes are larger and stain less intensely than do the endothelial and mesangial cell nuclei. surface. The lumen of the proximal tubules is often star shaped; this is not the case with distal tubules. Typically, fewer nuclei appear in a cross section of a proximal tubule than in an equivalent segment of a distal tubule.

1	Most of the above points can also be utilized in distinguishing the straight portions of the proximal and distal thick segments in the medullary rays, as shown in figure on right. Proximal and distal straight tubules, kidney, human, H&E ×240. In this figure, all of the tubular profiles within the medullary ray are rounded except for a proximal convoluted tubule (PC) included in the lower right corner of the figure (it belongs to the adjacent cortical labyrinth). Second, the number of proximal straight (P) and distal straight (D) tubular profiles are about equal in the medullary ray, as is shown by the labeling of each tubule in this figure. Note that, in contrast to the distal straight tubules, the proximal straight tubules display a brush border and have a larger outside diameter, with many displaying a star-shaped lumen. The medullary ray also contains collecting tubules (CT ). They are considered in Plate 77.

1	A distal (DC) and two proximal (PC) convoluted tubules are marked in figure on left. The cells of the distal tubule are more crowded on one side. These crowded cells constitute the macula densa (MD) that lies adjacent to the afferent arteriole. In figure on right, both the vascular pole and the urinary pole of the renal corpuscle are evident. The vascular pole is characterized by the presence of arterioles (A), one of which is entering or leaving (double-headed arrow) the corpuscle. The afferent arteriole possesses modified smooth muscle cells with granules, the juxtaglomerular cells (not evident in this figure). At the urinary pole, the parietal layer of Bowman’s capsule is continuous with the beginning of the proximal convoluted tubule (PC ). Here, the urinary space of the renal corpuscle continues into the lumen of the proximal tubule, and the lining cells change from simple squamous to simple cuboidal or low columnar with a brush border.

1	KEY A, arteriole BC, Bowman’s capsule (parietal layer) CT, collecting tubule D, distal straight tubule DC, distal convoluted tubule MD, macula densa P, proximal (straight tubule) PC, proximal convoluted tubule Pod, podocyte (visceral layer of Bowman’s capsule) asterisks, urinary space double-headed arrow, blood vessel at vascular pole of renal corpuscle Renal corpuscles are restricted to the cortical labyrinth. The medulla contains the thick straight segments of proximal and distal tubules, along with their thin segments, the collecting tubules and ducts, and the blood vessels that run in parallel with them. These structures function as the countercurrent multiplier and countercurrent exchange systems that, ultimately, produce hypertonic urine. The final urine drains from the papillary ducts (of Bellini) into calyces that then empty into the renal pelvis. Medulla, kidney, human, H&E ×240.

1	A section through the outer portion of the medulla is shown in this figure. This region contains proximal and distal thick segments, thin segments, and collecting tubules. All of the tubules are parallel, and all are cut in cross section; thus, they present circular profiles. The proximal straight tubules (P) display typical star-shaped lumina and a brush border (or the fragmented apical cell surface from which the brush border has been partially broken). These tubules have outside diameters that are generally larger than those of 734 the distal straight tubules (D). As mentioned previously and as shown here, the distal straight tubules display a larger number of nuclei than do comparable segments of proximal straight tubule cells. Note, also, that the lumen of the distal tubule is more rounded and the apical surface of the cells is sharper. The collecting tubules (CT ) have outer diameters that are about the same as those of the proximal tubules and larger than those of distal

1	more rounded and the apical surface of the cells is sharper. The collecting tubules (CT ) have outer diameters that are about the same as those of the proximal tubules and larger than those of distal tubules. The cells forming the collecting tubules are cuboidal and smaller than those of proximal tubules; thus, they also display a relatively larger number of nuclei than do comparable segments of proximal tubule cells. Count them! Finally, boundaries between the cells that constitute the collecting tubules are usually evident (asterisks); this serves as one of the most dependable features for the identification of collecting tubules.

1	The thin segments (T ) have the thinnest walls of all renal tubules seen in the medulla. They are formed by a low cuboidal or simple squamous epithelium, as seen here, and the lumina are relatively large. Occasionally, a section includes the region of transition from a thick to a thin segment and can be recognized even in a cross section through the tubule. One such junction is evident in this figure (the tubule with two arrows in the lumen). On one side, the tubule cell (left-pointing arrow) is characteristic of the proximal segment; it possesses a distinctive brush border. The other side of the tubule (right-pointing arrow) is composed of low cuboidal cells that resemble the cells forming the thin segments. In addition to the renal and collecting tubules, there are many other small tubular structures in this figure. Thin-walled and lined by endothelium, they are small blood vessels. Renal pyramid, kidney, human, H&E ×20.

1	Renal pyramid, kidney, human, H&E ×20. This figure shows a renal pyramid at low magnification. The pyramid is a conical structure composed principally of medullary straight tubules, ducts, and the straight blood vessels (vasa recta). The dashed line at the left of the micrograph is placed at the junction between cortex and medulla; thus, it marks the base of the pyramid. Note the arcuate vessels (AV ) that lie at the boundary of cortex and medulla. They define the boundary line. The few renal corpuscles (RC), upper left, belong to the renal column of the medulla. They are referred to as juxtamedullary corpuscles. The pyramid is somewhat distorted in this specimen, as evidenced by regions of longitudinally sectioned tubules, lower left, and cross-sectioned and obliquely sectioned tubules in other regions. In effect, part of the pyramid was bent, thus the change in the plane of section of the tubules.

1	The apical portion of the pyramid (arrowhead), known as the renal papilla, is lodged in a cupor funnel-like structure referred to as the calyx. It collects the urine that leaves the tip of the papilla from the papillary ducts (of Bellini). (The actual tip of the papilla is not seen within the plane of section, nor are the openings of the ducts at this low magnification.) The surface of the papilla that faces the lumen of the minor calyx is simple columnar or cuboidal epithelium (SCEp). (In places, this epithelium has separated from the surface of the papilla and appears as a thin strand of tissue.) The calyx is lined by transitional epithelium (TEp). Although not evident at the low magnification shown here, the boundary between the columnar epithelium covering the papilla and the transitional epithelium covering the inner surface of the calyx is marked by the diamonds.

1	KEY AV, arcuate vessels CT, collecting tubules D, distal straight tubule P, proximal straight tubule RC, renal corpuscle SCEp, simple columnar epithelium T, thin segment TEp, transitional epithelium arrowhead, location of apex of pyramid asterisks, boundaries between cells of a collecting tubule diamonds, boundary between a transitional and a columnar epithelium left-pointing arrow, proximal tubule cell right-pointing arrow, thin segment cell

1	PLATE 78 • URETER The ureters are paired tubular structures that convey urine from the kid-neys to the urinary bladder. They are lined with transitional epithelium (urothelium), an impervious layer that lines the urinary excretory passa-ges from the renal calyces through the urethra. The ability of this epithe-lium to become thinner and flatter allows all of these passages to accommodate to distension by the urine. The epithelium rests on a dense collagenous lamina propria, which in turn, rests on an inner longitudinal and an outer circular layer of smooth muscle. Regular peristaltic contractions of this muscle contribute to the flow of urine from the kidney to the urinary bladder. ORIENTATION MICROGRAPH: As shown in this low-power orienta-tion micrograph, the wall of the ureter consists of a mucosa (Muc), a muscularis (Mus), and an adventitia (Adv). Note that the ureters are lo-cated behind the peritoneum of the abdominal cavity in their course to the bladder. Thus, a serosa (Ser)

1	of a mucosa (Muc), a muscularis (Mus), and an adventitia (Adv). Note that the ureters are lo-cated behind the peritoneum of the abdominal cavity in their course to the bladder. Thus, a serosa (Ser) may be found covering a portion of the circumference of the tube. Also, because of contraction of the smooth muscle of the muscularis, the luminal surface is characteristically folded, thus creating a star-shaped lumen. ATATAT BVBVBV AdvAdvAdv MucMucMuc MusMusMus Ser

1	Ureter, monkey, H&E ×160. The wall of the ureter from the rectangular area in the orientation micrograph is examined at higher magnification in this figure. One can immediately recognize the thick epithelial lining, which appears distinct and sharply delineated from the remainder of the wall. This is the transitional epithelium (urothelium), (Ep). The remainder of the wall is made up of connective tissue (CT ) and smooth muscle. The latter can be recognized as the darker-staining layer. The section also shows some adipose tissue (AT ), a component of the adventitia. The transitional epithelium and its supporting connective tissue constitute the mucosa (Muc). A distinct submucosa is not present, although the term is sometimes applied to the connective tissue that is closest to the muscle.

1	The muscularis (Mus) is arranged as an inner longitudinal layer (SM(l)), a middle circular layer (SMc), and an outer longitudinal layer (SM(l)). However, the outer longitudinal layer is present only at the lower end of the ureter. In a cross section through the ureter, the inner and outer smooth muscle layers are cut in cross section, whereas the middle circular layer of the muscle cells is cut longitudinally. This is as they appear in this figure. Transitional epithelium, ureter, monkey, H&E ×400.

1	Transitional epithelium, ureter, monkey, H&E ×400. This figure shows the inner longitudinal smooth muscle layer (SM(l)) at higher magnification. Note that the nuclei appear as round profiles, indicating that the muscle cells have been cross-sectioned. This figure also shows the transitional epithelium (Ep) to advantage. The surface cells of the transitional epithelium (urothelium) are characteristically the largest, and some are binucleate (arrow). The basal cells are the smallest, and typically, the nuclei appear crowded because of the minimal cytoplasm of each cell. The intermediate cells appear to consist of several layers and are composed of cells larger in size than the basal cells but smaller than the surface cells. KEY Adv, adventitia AT, adipose tissue BV, blood vessels CT, connective tissue Ep, transitional epithelium Muc, mucosa Mus, muscularis Ser, serosa SM(c), circular layer of smooth muscle SM(l), longitudinal layer of smooth muscle arrow, binucleate surface cell

1	The urinary bladder receives the urine from the two ureters and stores it until neural stimulation causes it to contract and expel the urine via the urethra. It, too, is lined with transitional epithelium (urothelium). Beneath the epithelium and its underlying con-nective tissue, the wall of the urinary bladder contains smooth muscle that is usually described as being arranged as an inner longitudinal layer, a middle circular layer, and an outer longitudinal layer. As in most distensible hollow viscera that empty their con-tents through a narrow aperture, the smooth muscle in the wall of the urinary bladder is less regularly arranged than the description indicates, allowing contraction to re-duce the volume relatively evenly throughout the bladder. ORIENTATION MICROGRAPH: This orientation micrograph of the urinary blad-der reveals the full thickness of the bladder wall. The luminal surface epithelium is at the top of the micrograph. One of the ureters can be seen as it passes through

1	micrograph of the urinary blad-der reveals the full thickness of the bladder wall. The luminal surface epithelium is at the top of the micrograph. One of the ureters can be seen as it passes through the bladder wall to empty its contents into the bladder lumen. Most of the tissue to the sides and below the ureteral profile is smooth muscle.

1	Urinary bladder, human, H&E ×60. This micrograph shows most of the entire thickness of the urinary bladder. An unusual feature is the presence of one of the ureters (U ) as it is passing through the bladder wall to empty its contents into the bladder lumen. The transitional epithelium (Ep) lining the bladder is seen on the right. Beneath the epithe lium is a relatively thick layer of connective tissue (CT ) containing blood vessels (BV ) of various sizes. Note that the connective tissue stains more eosinophilic than the smooth muscle of the underlying muscularis (M). The epithelium and connective tissue constitute the mucosa of the bladder. The muscularis consists of smooth muscle arranged in three indistinct layers. It should be noted that as the ureter passes through the bladder wall, it carries with it a layer of longitudinally oriented smooth muscle (SM(L)). Medium-size arteries (A) and veins (V ) are occasionally seen in the muscularis.

1	Transitional epithelium, urinary bladder, human, H&E ×250. This higher magnification of the left rectangle of figure above shows the transitional epithelium (Ep) and the underlying connective tissue (CT ) that represent the mucosa of the ureter. Adjacent to the mucosa are bundles of longitudinally sectioned smooth muscle (SM(L)) that belong to the ureter. A small lymphatic vessel (Lym) is present in the connective tissue adjacent to the smooth muscle. Note the lymphocytes, identified by their small round densely stained nuclei, within the lumen of the vessel. Transitional epithelium, urinary bladder, human, H&E ×250.

1	Transitional epithelium, urinary bladder, human, H&E ×250. This higher magnification of the right rectangle of figure above shows the bladder transitional epithelium (Ep) and the underlying connective tissue (CT ) of the bladder wall. The transitional epithelium is often characterized by the presence of surface cells that exhibit a “dome” shape. In addition, many of these cells are binucleate (arrows). The thickness of transitional epithelium is variable. When the bladder is fully distended, as few as three cell layers are seen. Here, in the contracted bladder, it appears that there are as many as ten cell layers, a result of the cells folding over one another as the smooth muscle contracts and the lining surface is reduced. The connective tissue consists of bundles of collagen fibers interspersed with varying numbers of lymphocytes identified by their densely stained round nuclei. A vein (V ) filled with red blood cells is also evident in the mucosal connective tissue.

1	KEY A, artery BV, blood vessel CT, connective tissue Ep, transitional epithelium Lym, lymphatic vessel M, muscularis SM(L), longitudinally cut smooth muscle U, ureter V, vein arrows, binucleate cells

1	OVERVIEW OF THE ENDOCRINE SYSTEM / 740 Hormones and Their Receptors / 740 Regulation of Hormone Secretion and Feedback Mechanism / 742 PITUITARY GLAND (HYPOPHYSIS) / 742 Gross Structure and Development / 743 Blood Supply / 743 Nerve Supply / 745 Structure and Function of the Pituitary Gland / 745 Anterior Lobe of the Pituitary Gland (Adenohypophysis) / 745 Posterior Lobe of the Pituitary Gland (Neurohypophysis) / 748 HYPOTHALAMUS / 751 PINEAL GLAND / 752 THYROID GLAND / 755 PARATHYROID GLANDS / 760 ADRENAL GLANDS / 762 Blood Supply / 762 Cells of the Adrenal Medulla / 764 Zonation of the Adrenal Cortex / 766 Zona Glomerulosa / 766 Zona Fasciculata / 767 Zona Reticularis / 768 Fetal Adrenal Gland / 768 Folder 21.1 Functional Considerations: Regulation of Pituitary Gland Secretion / 743 Folder 21.2 Clinical Correlation: Principles of Endocrine Diseases / 750 Folder 21.3 Clinical Correlation: Pathologies Associated with ADH Secretion / 753 Folder 21.4 Clinical Correlation: Abnormal

1	Folder 21.2 Clinical Correlation: Principles of Endocrine Diseases / 750 Folder 21.3 Clinical Correlation: Pathologies Associated with ADH Secretion / 753 Folder 21.4 Clinical Correlation: Abnormal Thyroid Function / 758 Folder 21.5 Clinical Correlation: Chromaffin Cells and Pheochromocytoma / 766 Folder 21.6 Functional Considerations: Biosynthesis of Adrenal Hormones / 769

1	The endocrine system produces various secretions called hormones [Gr. hormaein, to set in motion] that serve as effectors to regulate the activities of various cells, tissues, and organs in the body. Its functions are essential in maintaining homeostasis and coordinating body growth and development and are similar to that of the nervous system: Both communicate information to peripheral cells and organs. Communication in the nervous system is through transmission of neural impulses along nerve cell processes and the discharge of neurotransmitter. Communication in the endocrine system is through hormones, which are carried to their destination via connective tissue spaces and the vascular system. These two systems are functionally interrelated. The endocrine system produces a slower and more prolonged response than the nervous system. Both systems may act simultaneously on the same target cells and tissues, and some nerve cells secrete hormones.

1	In general, a hormone is described as a biologic substance acting on specific target cells. In the classic definition, a hormone is a secretory product of endocrine cells and organs that passes into the circulatory system (bloodstream) for transport to target cells. For years this endocrine control of target tissues became a central part of endocrinology. Recent research shows that a variety of hormones and hormonally active substances are not always FIGURE 21.1 • Hormonal control mechanisms. This schematic diagram shows three basic types of control mechanisms. a. In endocrine control, the hormone is discharged from a cell into the bloodstream and is transported to the effector cells. b. In paracrine control, the hormone is secreted from one cell and acts on adjacent cells that express specific receptors. c. In autocrine control, the hormone responds to the receptors located on the cell that produces it.

1	discharged into the bloodstream but are released into connective tissue spaces. They may act on adjacent cells or diffuse to nearby target cells that express specific receptors for that particular hormone. This type of hormonal action is referred to as paracrine control. In addition, some cells express receptors for hormones that they secrete. This type of hormonal action is referred to as autocrine control. These hormones regulate the cell’s own activity. Figure 21.1 summarizes various hormonal control mechanisms. Hormones include three classes of compounds.

1	Hormones include three classes of compounds. Cells of the endocrine system release more than 100 hormones and hormonally active substances that are chemically divided into three classes of compounds:  Steroids, cholesterol-derived compounds, are synthesized and secreted by cells of the ovaries, testes, and adrenal cortex. These hormones (gonadal and adrenocortical steroids) are released into the bloodstream and transported to target cells with the help of plasma proteins or specialized carrier proteins such as androgen-binding protein. Hormone-binding carrier proteins protect the hormone from degradation during transport to the target tissue. When needed, the hormone is released from the carrier protein to become active.

1	 Small peptides, polypeptides,and proteins are synthesized and secreted by cells of the hypothalamus, pituitary gland, thyroid gland, parathyroid gland, pancreas, and scattered enteroendocrine cells of the gastrointestinal tract and respiratory system. This group of hormones (e.g., insulin, glucagon, growth hormone [GH], adreno corticotropic hormone [ACTH], follicle-stimulating hormone [FSH], luteinizing hormone [LH], antidiuretic hormone [ADH], oxytocin, interleukins, and various growth factors), when released into the circulation, dissolve readily in the blood and generally do not require special transport proteins. However, most if not all polypeptides and proteins have specific carrier proteins (e.g., insulin growth factor–binding protein (IGFBP).

1	 Amino acids and arachidonic acid analogs, and their derivatives, including the catecholamines (norepinephrine and epinephrine–phenylalanine/tyrosine derivatives) and prostaglandins, prostacyclins, and leukotrienes (arachidonic acid derivatives). They are synthesized and secreted by many neurons as well as a variety of cells including cells of the adrenal medulla. Also included in this group of compounds are thyroid hormones, the iodinated derivatives of the amino acid tyrosine that are synthesized and secreted by the thyroid gland. When released into the circulation, catecholamines dissolve readily in the blood, in contrast to thyroid hormones that bind to the prealbumin fraction of serum proteins (transthyretin) and a specialized thyroxin-binding protein. Hormones interact with specific hormone receptors to alter biologic activity of the target cells.

1	The first step in hormone action on a target cell is its binding to a specifc hormone receptor. However, recent studies suggest that some hormones are involved in non–receptor-mediated responses. Hormones interact with their receptors exposed on the surface of the target cell or within its cytoplasm or nucleus. In general, two groups of hormone receptors have been identified:  Cell surface receptors interact with peptide hormones or catecholamines that are unable to penetrate the cell membrane. Activation of these receptors as a result of hormone binding rapidly generates large quantities of small intracellular molecules called second messengers. These molecules amplify the signal initiated by hormone–receptor interaction and are produced by activation of membrane-associated G proteins. Examples of such systems include the adenylate cyclase/cyclic adenosine monophosphate (cAMP) system (for most protein hormones and catecholamines), the guanylyl cyclase/cyclic guanosine monophosphate

1	Examples of such systems include the adenylate cyclase/cyclic adenosine monophosphate (cAMP) system (for most protein hormones and catecholamines), the guanylyl cyclase/cyclic guanosine monophosphate (cGMP) system (an antagonistic system for action of cAMP in some protein hormones), the tyrosine kinase system (for insulin and epidermal growth factor [EGF]), the phosphatidylinositol system (for certain hormones such as oxytocin, gonadotropinreleasing hormone [GnRH], angiotensin II, and neurotransmitters such as epinephrine), and activation of ion channels (as with most neurotransmitters). The majority of second messenger molecules exert a stimulatory function on cell metabolism. Examples of second messenger molecules include cAMP, 1,2-diacylglycerol (DAG), inositol 1,4,5-triphosphate (IP3), and Ca2. An inhibitory response is mainly achieved by cGMP, which interferes with the production of cAMP. The second-messenger molecules produced in the cascade reactions of these systems alter the

1	and Ca2. An inhibitory response is mainly achieved by cGMP, which interferes with the production of cAMP. The second-messenger molecules produced in the cascade reactions of these systems alter the cell’s metabolism and produce hormone-specific responses (Fig. 21.2).

1	 Intracellular receptors, which are localized within the cell (mainly within the nucleus), are used by steroids and thyroid hormones that can easily penetrate both plasma and nuclear membranes. Their receptors consist of large multi-protein complexes of chaperones containing three binding domains: a hormone-binding region, a DNA-binding

1	FIGURE 21.2 • General mechanisms of hormone action. a. This schematic diagram shows the basis for protein hormone action involving cell surface receptors. Hormone molecules bind to the receptor and initiate synthesis of second-messenger molecules. These molecules, in turn, activate a cascade of reactions that produce hormone-specific responses in the stimulated cell. b. This diagram shows the mechanism of action of steroid hormones, which use intracellular receptors. Binding of the hormone to this receptor causes allosteric transformation of the receptor into a form that binds to DNA. This binding leads to mRNA transcription and production of new proteins that produce hormone-specific responses in the stimulated cell.

1	region, and an amino-terminal region. Binding of the hormone to these receptors causes allosteric transformation of the receptor into a form that binds to chromosomal DNA and activates RNA polymerase activity. This in turn increases transcription of mRNA, resulting in the production of new proteins that regulate cell metabolism. Therefore, hormones acting on intracellular receptors infuence gene expression directly, without the help of a second messenger (see Fig. 21.2). Regulation of Hormone Secretion and Feedback Mechanism Regulation of hormonal function is controlled by feedback mechanisms.

1	Regulation of Hormone Secretion and Feedback Mechanism Regulation of hormonal function is controlled by feedback mechanisms. Hormonal production is often controlled through feedback mechanisms from the target organ. In general, feedback occurs when the response to a stimulus (action of a hormone) has an effect on the original stimulus (hormone-secreting cell). The nature of this response determines the type of feedback. Two types of feedback are recognized; a negative feedback occurs when the response diminishes the original stimulus. It is much more common than a positive feedback, which occurs when the response enhances the original stimulus.

1	To better understand the function of feedback mechanisms, one can point to an air-conditioning system, which also uses a simple negative feedback system. When the compressor produces enough cold air to lower the temperature below the set point of the thermostat, the thermostat is triggered and shuts off the compressor. In this negative feedback system, the lower temperature is then fed back to the compressor and diminishes its response (it shuts off its production of cold air). When the temperature rises back above the set point, the negative feedback is abolished and the compressor comes back on (for more information on negative feedback, see Folder 21.1, Functional Considerations: Regulation of Pituitary Gland Secretion).

1	Activities of hormones are constantly monitored on many levels beginning with molecular biosynthetic processes to the final end points of hormonal action. Several examples of feedback mechanisms are discussed in the sections on the pituitary, hypothalamus, and thyroid gland. Hormone-secreting cells are present in many organs to regulate their activity.

1	Hormone-secreting cells are present in many organs to regulate their activity. This chapter primarily describes the discrete endocrine glands that release their hormones for delivery to the bloodstream for transport to target cells and organs. In other chapters, the endocrine function of adipose tissue as well as individual cells within the gonads, liver, pancreas, kidney, and gastrointestinal system is discussed. The cells of the diffuse neuroendocrine (DNES) system (see page 581) constitute a collection of endocrine cells in the body. In addition to their endocrine function, cells of DNES system exercise autocrine and paracrine control of the activity of their own and adjacent epithelial cells by diffusion of peptide secretions through the extracellular spaces.

1	The pituitary gland and the hypothalamus, the portion of the brain to which the pituitary gland is attached, are morphologically and functionally linked in the endocrine and neuroendocrine control of other endocrine glands. Because they play central roles in a number of regulatory feedback systems, they are often called the “master organs” of the endocrine system. In the past, the control of pituitary hormone secretion by the hypothalamus was classically regarded as the major function of the neuroendocrine system. However, the field of neuroendocrinology today has expanded to encompass multiple reciprocal interactions between the central nervous system (CNS), autonomic nervous system (ANS), endocrine system, and immune system in the regulation of homeostasis and behavioral responses to environmental stimuli. For example, the neuroendocrine axes in maintaining energy homeostasis were discussed in Chapter 9 (Adipose Tissue).

1	 FOLDER 21.1 Functional Considerations: Regulation of Pituitary Gland Secretion

1	The release of hormones from the anterior lobe of the pituitary gland is carefully regulated by three tiers of control mechanisms that include the following:  Tier I: hypothalamic secretion of hypothalamic-regulating hormones. The pituitary gland is under signif-icant control by the hypothalamus, which regulates release of hypothalamic-regulating hormones into the hypophyseal portal veins. The hypothalamic-regulating hormones are produced by the cells of the hypothalamus in response to circulating levels of systemic hormones and impulses from the CNS. These hormones act directly on the highly spe-cific G protein–linked receptors on the plasma mem-branes of cells residing in the anterior lobe of the pituitary gland. Activation of receptors elicits positive or negative signals that affect gene transcription and lead to stimula-tion or inhibition of pituitary hormone secretion. Most of the tropic hormones produced by the anterior lobe of the pitu-itary gland are regulated by

1	affect gene transcription and lead to stimula-tion or inhibition of pituitary hormone secretion. Most of the tropic hormones produced by the anterior lobe of the pitu-itary gland are regulated by polypeptide-releasing hor-mones, with the notable exception of dopamine. Prolactin (PRL) production is primarily regulated by the inhibitory ef-fect of dopamine (i.e., PRL secretion is tonically inhibited by the release of dopamine from the hypothalamus).  Tier II: Paracrine and autocrine secretions of the pituitary cells. Release of hormones from the pituitary gland is also regulated by soluble growth factors and cy-tokines produced by the cells residing in the pituitary gland.  Tier III: Feedback effect of circulating hormones. The level of hormones in the systemic circulation regu-lates the secretion of cells in the anterior lobe of the pituitary gland. This is primary achieved by negative feedback regulation of hormones secreted by the pitu-itary gland by target hormones. For instance,

1	secretion of cells in the anterior lobe of the pituitary gland. This is primary achieved by negative feedback regulation of hormones secreted by the pitu-itary gland by target hormones. For instance, secretion of TSH is inhibited by thyroid hormones produced in the thyroid gland under TSH influence. To better understand the mechanism of negative regula-tion, consider a simple negative feedback system that controls the synthesis and discharge of T3 and T4 thyroid hormones (see Fig. 21.6). The secretion of thyroid hor-mones is controlled by the release of TSH from the anterior lobe of the pituitary gland into the bloodstream. If blood lev-els of T3 and T4 are high, TRH is not produced or released. If blood levels of T3 and T4 are low, the hypothalamus dis-charges TRH into the hypothalamohypophyseal portal sys-tem. Release of TRH stimulates specific cells within the anterior lobe of the pituitary gland to produce TSH, which in turn stimulates the thyroid to produce and release more

1	portal sys-tem. Release of TRH stimulates specific cells within the anterior lobe of the pituitary gland to produce TSH, which in turn stimulates the thyroid to produce and release more thyroid hormones. As the thyroid hormone levels rise, the negative feedback system stops the hypothalamus from discharging TRH. Using the same mechanism of negative feedback regulation, thyroid hormones also act on the thy-rotropes in the anterior lobe of the pituitary gland to inhibit their secretion of TSH.

1	The pituitary gland is composed of glandular epithelial tissue and neural (secretory) tissue. The pituitary gland [Lat. pituta, phlegm—reflecting its nasopharyngeal origin] is a pea-sized, compound endocrine gland that weighs 0.5 g in males and 1.5 g in multiparous women (i.e., a woman who has given birth two or more times). It is centrally located at the base of the brain, where it lies in a saddle-shaped depression of the sphenoid bone called the sella turcica. A short stalk, the infundibulum, and a vascular network connect the pituitary gland to the hypothalamus. The pituitary gland has two functional components (Fig. 21.3):  Anterior lobe (adenohypophysis), the glandular epithe lial tissue  Posterior lobe (neurohypophysis), the neural secretory

1	The pituitary gland has two functional components (Fig. 21.3):  Anterior lobe (adenohypophysis), the glandular epithe lial tissue  Posterior lobe (neurohypophysis), the neural secretory These two portions are of different embryologic origin. The anterior lobe of the pituitary gland is derived from an evagination of the ectoderm of the oropharynx toward the brain (Rathke’s pouch). The posterior lobe of the pituitary gland is derived from a downgrowth (the future infundibulum) of neuroectoderm of the foor of the third ventricle (the diencephalon) of the developing brain (Fig. 21.4).

1	The anterior lobe of the pituitary gland consists of three derivatives of Rathke’s pouch:  Pars distalis, which comprises the bulk of the anterior lobe of the pituitary gland and arises from the thickened anterior wall of the pouch Pars intermedia, a thin remnant of the posterior wall of the pouch that abuts the pars distalis  Pars tuberalis, which develops from the thickened lateral walls of the pouch and forms a collar or sheath around the infundibulum The embryonic infundibulum gives rise to the posterior lobe of the pituitary gland. The posterior lobe of the pituitary gland consists of the following:  Pars nervosa, which contains neurosecretory axons and their endings.  Infundibulum, which is continuous with the median eminence and contains the neurosecretory axons forming the hypothalamohypophyseal tracts Knowledge of the unusual blood supply of the pituitary gland is important to understanding its functions. The

1	Knowledge of the unusual blood supply of the pituitary gland is important to understanding its functions. The FIGURE 21.4 • Development of the pituitary gland. This diagram shows sequential stages (a to c) in the development of the pituitary gland. FIGURE 21.3 • Pituitary gland. a. Photomicrograph of a pituitary gland. The pituitary gland lobes can be identified on the basis of their appearance, location, and relation to each other. 7. b. Drawing of a pituitary and related regions of the hypothalamus. The anterior lobe of the pituitary gland consists of the pars distalis, pars tuberalis, and pars intermedia; the posterior lobe of the pituitary gland consists of the infundibulum and pars nervosa.

1	pituitary blood supply is derived from two sets of vessels The hypothalamohypophyseal portal system provides the (Fig. 21.5): crucial link between the hypothalamus and the pituitary  Superior hypophyseal arteries supply the pars tuber-gland. alis, median eminence, and infundibulum. These vessels The arteries that supply the pars tuberalis, median eminence, arise from the internal carotid arteries and posterior com-and infundibulum give rise to fenestrated capillaries (the primunicating artery of the circle of Willis. mary capillary plexus). These capillaries drain into portal  Inferior hypophyseal arteries primarily supply the pars veins, called the hypophyseal portal veins, which run nervosa. These vessels arise solely from the internal carotid along the pars tuberalis and give rise to a second fenestrated arteries. An important functional observation is that most sinusoidal capillary network (the secondary capillary plexus). of the anterior lobe of the pituitary gland has no

1	to a second fenestrated arteries. An important functional observation is that most sinusoidal capillary network (the secondary capillary plexus). of the anterior lobe of the pituitary gland has no direct This system of vessels carries the neuroendocrine secretions arterial supply. of hypothalamic nerves from their sites of release in the

1	FIGURE 21.5 • Diagram of the blood supply to the pituitary gland. The hypophyseal portal veins begin in the capillary beds of the median eminence and infundibulum and end in the capillaries of the pars distalis. median eminence and infundibulum directly to the cells of the pars distalis. Most of the blood from the pituitary gland drains into the cavernous sinus at the base of the diencephalon and then into the systemic circulation. Some evidence suggests, however, that blood can flow via short portal veins from the pars distalis to the pars nervosa and that blood from the pars nervosa may flow toward the hypothalamus. These short pathways provide a route by which the hormones of the anterior lobe of the pituitary gland could provide feedback directly to the brain without making the full circuit of the systemic circulation.

1	The nerves that enter the infundibulum and pars nervosa from the hypothalamic nuclei are components of the posterior lobe of the pituitary gland (see the section that follows on the posterior lobe). The nerves that enter the anterior lobe of the pituitary gland are postsynaptic fibers of the autonomic nervous system and have vasomotor function. Structure and Function of the Pituitary Gland Anterior Lobe of the Pituitary Gland (Adenohypophysis) The anterior lobe of the pituitary gland regulates other endocrine glands and some nonendocrine tissues. Most of the anterior lobe of the pituitary gland has the typical organization of endocrine tissue. The cells are organized in clumps and cords separated by fenestrated sinusoidal capillaries of relatively large diameter. These cells respond to signals from the hypothalamus and synthesize and secrete a number of pituitary hormones. Four hormones of the anterior

1	FIGURE 21.6 • Interaction of the hypothalamus, anterior lobe of the pituitary gland, and thyroid gland. Production of thyroid hormones is regulated through a negative feedback system. The thyroid hormone can feed back on the system and inhibit further release of thyroid hormones. Such inhibition occurs at the level of the anterior lobe and the hypothalamus. The system is activated in response to low thyroid hormone levels or in response to metabolic needs. TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone (thyrotropin).

1	lobe—adrenocorticotropic hormone (ACTH), thyroid-stimulating (thyrotropic) hormone (TSH, thyrotropin), follicle-stimulating hormone (FSH), and luteinizing hormone (LH)—are called tropic hormones because they regulate the activity of cells in other endocrine glands throughout the body (Fig. 21.6). The two remaining hormones of the anterior lobe, growth hormone (GH) and prolactin (PRL), are not considered tropic because they act directly on target organs that are not endocrine. The general character and effects of the pituitary hormones of the anterior lobe are summarized in Table 21.1.

1	Pars Distalis. The cells within the pars distalis vary in size, shape, and staining properties. The cells are arranged in cords and nests with interweaving capillaries. Early descriptions of the cells within the pars distalis were based solely on the staining properties of secretory vesicles within the cells. Using mixtures of acidic and basic dyes (Fig. 21.7), histologists identified three types of cells according to their staining reaction, namely, basophils (10%), acidophils (40%),and chromophobes (50%). However, this classification contains no information regarding the hormonal secretory activity or functional role of these cells.

1	TABLE Hormones of the Anterior Lobe of the Pituitary Gland 21.1 Hormone Composition MW (kDa) Major Functions Growth hormone (somatotropin, GH) Straight-chain protein (191 aa) 21,700 Stimulates liver and other organs to synthesize and secrete insulin-like growth factor I (IGF-I), which in turn stimulates division of progenitor cells located in growth plates and in skeletal muscles, resulting in body growth Prolactin (PRL) Straight-chain protein (198 aa) 22,500 Promotes mammary gland development; initiates milk formation; stimulates and maintains secretion of casein, lactalbumin, lipids, and carbohydrates into the milk Adrenocorticotropic hormone (ACTH) Small polypeptide (39 aa) 4,000 Maintains structure and stimulates secretion of gluco-corticoids and gonadocorticoids by the zona fasciculata and zona reticularis of the adrenal cortex Follicle-stimulating hormone (FSH) 2-chain glycoproteina (, 92 aa; , 111 aa) 28,000 Stimulates follicular development in the ovary and spermato-genesis

1	and zona reticularis of the adrenal cortex Follicle-stimulating hormone (FSH) 2-chain glycoproteina (, 92 aa; , 111 aa) 28,000 Stimulates follicular development in the ovary and spermato-genesis in the testis Luteinizing hormone (LH) 2-chain glycoproteina (, 92 aa; , 116 aa) 28,300 Regulates final maturation of ovarian follicle, ovulation, and corpus luteum formation; stimulates steroid secretion by follicle and corpus luteum; in males, essential for maintenance of and androgen secretion by the Leydig (interstitial) cells of the testis Thyrotropic hormone (TSH) 2-chain glycoproteina (, 92 aa; , 112 aa) 28,000 Stimulates growth of thyroid epithelial cells; stimulates production and release of thyroglobulin and thyroid hormones aThe chains of FSH, LH, and TSH are identical and encoded by a single gene; the chains are specific for each hormone. kDa, kilodaltons; aa, amino acids.

1	Five functional cell types are identified in the pars distalis on the basis of immunocytochemical reactions.

1	All known hormones of the anterior lobe of the pituitary gland are small proteins or glycoproteins. This important fact has led to definitive identification of specific cell types by immunocytochemistry (Table 21.2). These studies have classified cells of the anterior lobe of the pituitary gland into five cell types:  Somatotropes (GH cells) are most commonly found within the pars distalis and constitute approximately 50% of the parenchymal cells in the anterior lobe of the pituitary gland. These medium-sized, oval cells exhibit round, centrally located nuclei and produce growth hormone (GH; somatotropin). The presence of eosinophilic vesicles in their cytoplasm classifies them as acidophils. Three hormones regulate the release of GH from somatotropes. Two of these hormones are opposing hypothalamic-releasing hormones: growth hormone–releasing hormone (GHRH), which stimulates GH release from the somatotropes, and somatostatin, which inhibits GH release from the somatotropes.

1	hypothalamic-releasing hormones: growth hormone–releasing hormone (GHRH), which stimulates GH release from the somatotropes, and somatostatin, which inhibits GH release from the somatotropes. Recently, a third hormone, a 28-amino acid peptide ghrelin, was isolated from the stomach. It is a potent stimulator of GH secretion and appears to coordinate food intake with GH secretion. Hormonally active tumors that originate from somatotropes are associated with hypersecretion of GH and cause gigantism in children and acromegaly in adults.

1	 Lactotropes (PRL cells, mammotropes) constitute 15% to 20% of the parenchymal cells in the anterior lobe of the pituitary gland. These are large, polygonal cells with oval nuclei. They produce prolactin (PRL). In their storage phase, lactotropes exhibit numerous acidophilic vesicles (the histologic feature of an acidophil). When the content of these vesicles is released, the cytoplasm of the lactotrope does not stain (the histologic feature of a chromophobe). Secretion of PRL is under inhibitory control by dopamine, the catecholamine produced by the hypothalamus. However, thyrotropin-releasing hormone (TRH) and vasoactive inhibitory peptide (VIP) are known to stimulate synthesis and secretion of PRL. During pregnancy and lactation, these cells undergo hypertrophy and hyperplasia, causing the pituitary gland to increase in size. These processes account for the larger size of the pituitary gland in multiparous women.

1	 Corticotropes (ACTH cells) also constitute 15% to 20% of the parenchymal cells in the anterior lobe of the pituitary gland. These polygonal, medium-sized cells with round and eccentric nuclei produce a precursor molecule of adrenocorticotropic hormone (ACTH), known as proopiomelanocortin (POMC). Corticotropes stain as basophils and also exhibit a strong positive reaction with periodic acid–Schiff (PAS) reagent, because of the carbohydrate moieties associated with POMC. POMC is further cleaved by proteolytic enzymes within the corticotrope into several fragments, namely ACTH, -lipotrophic hormone (-LPH), melanocyte-stimulating hormone (MSH), -endorphin, and enkephalin. ACTH release is regulated

1	FIGURE 21.7 • Pars distalis. This specimen of the pars distalis is stained with brilliant crystal scarlet, aniline blue, and Martius yellow to distinguish the various cell types and connective tissue stroma. The cords of cells are surrounded by a delicate connective tissue stroma stained blue. The sinusoidal capillaries are seen in close association with the parenchyma and contain erythrocytes stained yellow. In the region shown here, the acidophils (Ac) are the most numerous cell type present. Their cytoplasm stains cherry red. The basophils (Bas) stain blue. The chromophobes (Ch), although few in number in this particular region, are virtually unstained. 640. by corticotropin-releasing hormone (CRH) produced by the hypothalamus.

1	by corticotropin-releasing hormone (CRH) produced by the hypothalamus.  Gonadotropes (FSH and LH cells) constitute about 10% of the parenchymal cells in the anterior lobe of the pituitary gland. These small, oval cells with round and eccentric nuclei produce both luteinizing hormone (LH) and follicle-stimulating hormone (FSH). They are scattered throughout the pars distalis and stain intensely with both basic stains (thus classifying them as the basophil cell type) and PAS reagent. Many gonadotropes are capable of producing both FSH and LH. However, immunocytochemical studies indicate that some gonadotropes may produce only one hormone or the other. The release of FSH and LH is regulated by gonadotropin-releasing hormone (GnRH) produced by the hypothalamus. Both FSH and LH play an important role in male and female reproduction, which is discussed in Chapters 22 and 23.

1	 Thyrotropes (TSH cells) constitute about 5% of the parenchymal cells in the anterior lobe of the pituitary gland. These large, polygonal cells with round and eccentric nuclei produce thyrotropic hormone called thyroid-stimulating hormone (TSH), which acts on the follicular cells of the thyroid gland to stimulate production of thyroglobulin and thyroid hormones. Thyreotropes exhibit cytoplasmic basophilia (basophils) and stain positively with PAS reagent. Release of TSH is under the hypothalamic control of thyrotropin-releasing hormone (TRH), which also stimulates secretion of PRL. Distinctive characteristics of the five cell types of the anterior lobe of the pituitary gland are readily seen with transmission electron microscopy (TEM). These characteristics are summarized in Table 21.3. In addition to the five types of hormone-producing cells, anterior lobe of the pituitary gland contains folliculostellate cells.

1	Folliculo-stellate cells present in the anterior lobe of the pituitary gland are characterized by a starlike appearance with their cytoplasmic processes encircling hormone-producing cells. They have the ability to make cell clusters or small follicles, and they do not produce hormones. Folliculostellate cells are interconnected by gap junctions containing connexin-43 protein. Based on immunocytochemical and electrophysiological studies, it is hypothesized that the network of folliculo-stellate cells interconnected by gap junctions transmits signals from the pars tuberalis to pars distalis. These signals may regulate hormone release throughout the anterior lobe of the pituitary gland. Thus, the folliculo-stellate network may appear to function in addition to the hypophyseal portal vein system. Recent in vitro findings of gap junctions interconnecting not only folliculo-stellate cells but also hormone-producing cells support this proposed signaling mechanism in the anterior lobe of the

1	Recent in vitro findings of gap junctions interconnecting not only folliculo-stellate cells but also hormone-producing cells support this proposed signaling mechanism in the anterior lobe of the pituitary gland.

1	Pars Intermedia. The pars intermedia surrounds a series of small cystic cavities that represent the residual lumen of Rathke’s pouch. The parenchymal cells of the pars intermedia surround colloid-filled follicles. The cells lining these follicles appear to be derived either from folliculo-stellate cells or various hormone-secreting cells. TEM reveals that these cells form apical junctional complexes and have vesicles larger than those found in the pars distalis. The nature of this follicular colloid is yet to be determined; however, often cell debris is found within it. The pars intermedia contains basophils and chromophobes (Fig. 21.8). Frequently, the basophils and cystic cavities extend into the pars nervosa.

1	The function of the pars intermedia cells in humans remains unclear. From studies of other species, however, it is known that basophils have scattered vesicles in their cytoplasm that contain either or -endorphin (a morphine-related compound). In frogs, the basophils produce MSH, which stimulates pigment production in melanocytes and pigment dispersion in melanophores. In humans, MSH is not a distinct, functional hormone but is a byproduct of LPH posttranslational processing. Because MSH is found in the human pars intermedia in small amounts, the basophils of the pars intermedia are assumed to be corticotropes.

1	TABLE Staining Characteristics of Cells Found in the Anterior Lobe of the Pituitary Gland 21.2 Cell Type Percentage of Total Cells General Staining Specific Staining Product Somatotrope (GH cell) 50 Acidophil Orange G (PAS ) Growth hormone (GH) Lactotrope (PRL cell) 15–20 Acidophil Orange G (PAS ) Herlant’s erythrosin Brooke’s carmosine Prolactin (PRL) Corticotrope (ACTH cell) 15–20 Basophil Lead hematoxylin (PAS ) Proopiomelanocortin (POMC), which is cleaved in human into adrenocorticotropic hormone (ACTH) and -lipotrophic hormone (-LPH) Gonadotrope (FSH and LH cells) 10 Basophil Aldehyde-fuchsin Aldehyde-thionine (PAS ) Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) Thyrotrope (TSH cell) 5 Basophil Aldehyde-fuchsin Aldehyde-thionine (PAS ) Thyrotropic hormone (TSH)

1	Pars Tuberalis. The pars tuberalis is an extension of the anterior lobe along the stalklike infundibulum. It is a highly vascular region containing veins of the hypothalamohypophyseal system. The parenchymal cells are arranged in small clusters or cords in association with the blood vessels. Nests of squamous cells and small follicles lined with cuboidal cells are scattered in this region. These cells often show immunoreactivity for ACTH, FSH, and LH. Posterior Lobe of the Pituitary Gland (Neurohypophysis) The posterior lobe of the pituitary gland is an extension of the central nervous system (CNS) that stores and releases secretory products from the hypothalamus. The posterior lobe of the pituitary gland, also known as the neurohypophysis, consists of the pars nervosa and the infundibulum that connects it to the hypothalamus.

1	The posterior lobe of the pituitary gland, also known as the neurohypophysis, consists of the pars nervosa and the infundibulum that connects it to the hypothalamus. TABLE Electron Microscopic Characteristics of Cells Found in the Anterior Lobe of the Pituitary Gland21.3 Cell Type Size/Shape Nucleus/Location Secretory Vesicle Size/ Characteristics Other Cytoplasmic Characteristics Somatotrope Medium/oval Round/central, with prominent nucleoli Dense: 350 nm, closely packed None Lactotrope Large/polygonal Oval/central Inactive: 200 nm, sparse Active: dense, pleomorphic, 600 nm, sparse Lysosomes increase after lactation Corticotrope Medium/polygonal Round/eccentric 100–300 nm Lipid droplets, large lysosomes, perinu-clear bundles of intermediate filaments Gonadotrope Small/oval Round/eccentric Dense: 200–250 nm Prominent Golgi apparatus, distended rER cisternae Thyrotrope Large/polygonal Round/eccentric Dense: 150 nm Prominent Golgi apparatus with numerous vesicles

1	FIGURE 21.8 • Photomicrograph of the pars intermedia of an adult human pituitary gland. This photomicrograph of a toluidine blue–stained specimen shows the pars intermedia located between the pars distalis (on the left) and pars nervosa (on the right). In humans, this portion of the gland is somewhat rudimentary. However, a characteristic feature of the pars intermedia is the presence of different-sized follicles filled with colloid (CF ) and small groups of cells consisting of chromophobes and basophils. 120.

1	The pars nervosa, the neural lobe of the pituitary, contains the nonmyelinated axons and their nerve endings of approximately 100,000 neurosecretory neurons whose cell bodies lie in the supraoptic nuclei and paraventricular nuclei of the hypothalamus. The axons form the hypothalamohypophyseal tract and are unique in two respects. First, they do not terminate on other neurons or target cells but end in close proximity to the fenestrated capillary network of the pars nervosa. Second, they contain secretory vesicles in all parts of the cells, i.e., the cell body, axon, and axon terminal. Because of their intense secretory activity, the neurons have well-developed Nissl bodies and in this respect resemble ventral horn and ganglion cells.

1	The posterior lobe of the pituitary gland is not an endocrine gland. Rather, it is a storage site for neurosecretions of the neurons of the supraoptic and paraventricular nuclei of the hypothalamus. The nonmyelinated axons convey neurosecretory products to the pars nervosa. Other neurons from the hypothalamic nuclei (described below) also release their secretory products into the fenestrated capillary network of the infundibulum, the first capillary bed of the hypothalamohypophyseal portal system. Electron microscopy reveals three morphologically distinct neurosecretory vesicles in the nerve endings of the pars nervosa.

1	Electron microscopy reveals three morphologically distinct neurosecretory vesicles in the nerve endings of the pars nervosa. Three sizes of membrane-bounded vesicles are present in the pars nervosa:  Neurosecretory vesicles with diameters ranging between 10 and 30 nm accumulate in the axon terminals. They also form accumulations that dilate portions of the axon near the terminals (Fig. 21.9). These dilations, called Herring bodies, are visible in the light microscope (Plate 81, page 774). In the electron microscope, Herring bodies in addition to abundant neurosecretory vesicles, contain mitochondria, few micro-tubules and profiles of SER (Fig. 21.10).  Nerve terminals also contain 30-nm vesicles that contain acetylcholine. These vesicles may play a specific role in the release of neurosecretory vesicles.

1	 Larger 50to 80-nm vesicles that resemble the dense core vesicles of the adrenal medulla and adrenergic nerve endings are present in the same terminal as the other membrane-bound vesicles. The membrane-bound neurosecretory vesicles that aggregate to form Herring bodies contain either oxytocin or antidiuretic hormone (ADH; also called vasopressin; Table 21.4). Each hormone is a small peptide of nine amino acid residues. The two hormones differ in only two of these residues. Each vesicle also contains ATP and a neurophysin, a protein that binds to the hormone by noncovalent bonds. Oxytocin and ADH are synthesized as part of a large molecule that includes the hormone and its specific neurophysin. The large molecule is proteolytically cleaved into the hormone and neurophysin as it travels from the nerve cell body to the axon terminal. Immunocytochemical staining demonstrates that oxytocin and ADH are secreted by different cells in the hypothalamic nuclei.

1	ADH facilitates resorption of water from the distal tubules and collecting ducts of the kidney by altering the permeability of the cells to water. ADH’s original name, vasopressin, was derived from the observation that large nonphysiologic doses increase blood pressure by promoting the contraction of smooth muscle in small arteries and arterioles. However, the physiologic levels of ADH have only minimal effects on blood pressure. ADH is the main hormone involved in regulation of water homeostasis and osmolarity of body fuids. The primary physiologic effect of ADH on the kidney is the insertion of water channels (aquaporins) into cells of the distal convoluted tubules and collecting ducts, which increases the permeability for water. Insertion of aquaporin-2 (AQP-2) into the apical domain and aquaporin-3 (AQP-3) into the basolateral domain of these cells is responsible for rapid resorption of water across the tubule epithelium.

1	ADH acts through its specific V2 receptor on the basolateral domain of cells lining the distal convoluted tubules and collecting ducts; mutation of this receptor is responsible for nephrogenic diabetes insipidus (Folder 21.3). Plasma osmolality and blood volume are monitored by specialized receptors of the cardiovascular system (e.g., carotid bodies and juxtaglomerular apparatus). An increase

1	Abnormalities in the signaling mechanisms that coordinate and control the function of multiple organs and biological processes are the bases of many endocrine diseases. Classical biochemistry, physiology, and advances in cell and molecular biology and genetics combined with clinical observations are able to explain the mechanisms of hor-monal action and endocrine diseases. Endocrine diseases can be classified into four major categories:  Hormone overproduction. The most common cause of hormone overproduction is an increase in the total num-ber of cells producing a specific hormone. An example of this mechanism is hyperthyroidism (Graves’ disease; see Folder 21.4). Briefly, the presence of abnormal antibodies that mimic the action of TSH stimulates a dramatic in-crease in the number of thyroid cells. In some instances, increased hormone secretion is related to genetic abnor-mality that affects regulation of hormone synthesis and re-lease. In addition, mutation in tumor suppressor

1	thyroid cells. In some instances, increased hormone secretion is related to genetic abnor-mality that affects regulation of hormone synthesis and re-lease. In addition, mutation in tumor suppressor genes and protooncogenes may lead to proliferation of mutant cells that produce the specific hormone. This commonly occurs in cells of the anterior lobe of the pituitary gland.  Hormone underproduction. Underproduction of hor-mones may result from destruction of an endocrine organ by a disease process (e.g., tuberculosis of the adrenal glands) or autoimmunity (e.g., Hashimoto’s disease in which abnormal antibodies target and destroy thyroid hormone–producing cells). Also, genetic abnormalities that lead to abnormal development of endocrine glands (e.g., hypogonadotropic hypogonadism), abnormal hormone synthesis (e.g., deletion of the GH gene), or abnormal reg-ulation of hormone secretion (e.g., hypoparathyroidism associated with mutation of the calcium-sensing receptor expressed on

1	hormone synthesis (e.g., deletion of the GH gene), or abnormal reg-ulation of hormone secretion (e.g., hypoparathyroidism associated with mutation of the calcium-sensing receptor expressed on parathyroid cells) can cause decreased serum levels or lack of active hormones. Iatrogenic injury to endocrine glands such as occurs when the parathyroid gland is removed during thyroidectomy (thyroid gland removal) may also be responsible.  Altered tissue responses to hormones. This cate-gory of endocrine disease is often cause by a variety of genetic mutation in hormone receptors (e.g., TSH, LH, and PTH). In diabetic patients, the resistance to insulin in muscles and the liver is mainly caused by signals origi-nating from adipose tissue (see Chapter 9).  Tumors of endocrine glands. Most of the tumors of endocrine gland are hormonally active and are responsi-ble for hormone overproduction. However, some tumors of endocrine glands do not produce hormones, but com-press neighboring organs or

1	tumors of endocrine gland are hormonally active and are responsi-ble for hormone overproduction. However, some tumors of endocrine glands do not produce hormones, but com-press neighboring organs or cause destructions of other organs due to metastasis. An example of such a tumor is thyroid cancer that can metastasize throughout the body without presenting signs of thyroid hormone overpro-duction (hyperthyroidism). Hormones are used to treat endocrine diseases. A com-mon use is as hormone replacement therapies when a specific endocrine gland is not developed or ceases to produce the required hormone. Hormones and their syn-thetic analogs can be used to suppress the effects of other hormones. In general, thyroid and steroid hormones can be administered orally, whereas protein hormones (e.g., insulin, GH) need to be injected. Recent technological innovations including computerized mini-pumps and depot intramuscular injections have made therapy more manage-able for patients.

1	 FOLDER 21.2 Clinical Correlation: Principles of Endocrine Diseases in osmolality or a decrease in blood volume stimulates ADH release. Additionally, the cell bodies of the hypothalamic secretory neurons may also serve as osmoreceptors, initiating ADH release. Pain, trauma, emotional stress, and drugs such as nicotine also stimulate release of ADH. Oxytocin promotes contraction of smooth muscle of the uterus and myoepithelial cells of the breast. Oxytocin is a more potent promoter of smooth muscle contraction than is ADH. Its primary effect includes promotion of contraction of  uterine smooth muscle during orgasm, menstruation, and parturition. As parturition approaches, uterine smooth muscle cells demonstrate about a 200-fold increase in the responsiveness to oxytocin. This is accompanied by increased formation of gap junctions between smooth muscle cells and increased density of oxytocin receptors.

1	 myoepithelial cells of the secretory alveoli and alveolar ducts of the mammary gland. Oxytocin secretion is triggered by neural stimuli that reach the hypothalamus. These stimuli initiate a neurohumoral reflex that resembles a simple sensorimotor reflex. In the uterus, the neurohumoral reﬂex is initiated by distension of the vagina and cervix. In the breast, the reﬂex is initiated by breastfeeding (suckling). Contraction of the myoepithelial cells that surround the base of the alveolar secretory cells and cells of the larger ducts cause milk to be released and pass through the ducts that open onto the nipple (i.e., milk ejection; see page 867). Synthetic analogs of oxytocin are often used in intravenous infusion pumps to initiate and strengthen uterine contractions during active labor and delivery. Nasal preparations of oxytocin are also used to promote lactation in breastfeeding women. The pituicyte is the only cell specific to the posterior lobe of the pituitary gland.

1	The pituicyte is the only cell specific to the posterior lobe of the pituitary gland. In addition to the numerous axons and terminals of the hypothalamic neurosecretory neurons, the posterior lobe of the pituitary gland contains fibroblasts, mast cells, and specialized glial cells called pituicytes associated with the fenestrated capillaries. These cells are irregular in shape, with many branches, and resemble astroglial cells. Their nuclei are round or oval, and pigment vesicles are present in the cytoplasm. Like astroglia, they possess specific intermediate filaments assembled from glial fibrillary acidic proteins (GFAP). Pituicytes often have processes that terminate in the perivascular space. Because of their many processes and relationships to the blood, the pituicyte serves a supporting role similar to that of astrocytes in the rest of the CNS (see page 367).

1	FIGURE 21.9 • Electron micrograph of Herring bodies of rat posterior lobe. Dilated portions of axons near their terminals called Herring bodies (HB) contain numerous neurosecretory vesicles filled with either oxytocin or ADH. They are surrounded by the specialized glial cells called pituicytes (P). Note that Herring bodies reside in a close proximity to blood vessels (BV), mainly fenestrated capillaries, lined by endothelial cells (En). 6,000. (Courtesy of Dr. Holger Jastrow). The hypothalamus regulates pituitary gland activity.

1	The hypothalamus regulates pituitary gland activity. The hypothalamus is located in the middle of the base of the brain, and it encapsulates the ventral portion of the third ventricle. It coordinates most endocrine functions of the body and serves as one of the major controlling centers of the autonomic nervous system. Some of the functions that it regulates include blood pressure, body temperature, fluid and electrolyte balance, body weight, and appetite. The hypothalamus produces numerous neurosecretory products. In addition to oxytocin and ADH, hypothalamic neurons secrete polypeptides that promote and inhibit the secretion and release of hormones from the anterior lobe of the pituitary gland (Table 21.5). These hypothalamic polypeptides also accumulate in nerve endings near the median eminence and infundibulum and are released into the capillary bed of the hypothalamohypophyseal portal system for transport to the pars distalis of the pituitary gland.

1	A feedback system regulates endocrine function at two levels: hormone production in the pituitary gland and hypothalamic releasing hormone production in the hypothalamus. The circulating level of a specific secretory product of a target organ, a hormone or its metabolite, may act directly on the cells of the anterior lobe of the pituitary gland or the hypothalamus to regulate the secretion of hypothalamic releasing hormones (see Fig. 21.6). The two levels of feedback FIGURE 21.10 • Electron micrograph of rat posterior lobe.

1	FIGURE 21.10 • Electron micrograph of rat posterior lobe. Neurosecretory granules and small vesicles are present in the terminal portions of the axonal processes of the hypothalamohypophyseal tract fibers. Capillaries with fenestrated endothelium are present in close proximity to the nerve endings. 20,000. (Courtesy of Drs. Sanford L. Palay and P. Orkland.) allow exquisite sensitivity in the control of secretory function. The hormone itself normally regulates the secretory activity of the cells in the hypothalamus and pituitary gland that regulate its secretion.

1	In addition, information from most physiologic and psychologic stimuli that reach the brain also reaches the hypothalamus. The hypothalamohypophyseal feedback loop provides a regulatory path whereby general information from the CNS contributes to the regulation of the anterior lobe of the pituitary gland and, consequently, to the regulation of the entire endocrine system. The secretion of hypothalamic regulatory peptides is the primary mechanism by which changes in emotional state are translated into changes in the physiologic homeostatic state.

1	The pineal gland (pineal body, epiphysis cerebri) is an endocrine or neuroendocrine gland that regulates daily body rhythm. It develops from neuroectoderm of the posterior portion of the roof of the diencephalon and remains attached to the brain by a short stalk. In humans, it is located at the posterior wall of the third ventricle near the center of the brain. The pineal gland is a flattened, pine cone–shaped structure, hence its name (Fig. 21.11). It measures 5 to 8 mm high and 3 to 5 mm in diameter and weighs between 100 and 200 mg. The pineal gland contains two types of parenchymal cells: pinealocytes and interstitial (glial) cells.

1	The pineal gland contains two types of parenchymal cells: pinealocytes and interstitial (glial) cells. Pinealocytes are the chief cells of the pineal gland. They are arranged in clumps or cords within lobules formed by connective tissue septa that extend into the gland from the pia mater that covers its surface. These cells have a large, deeply infolded nucleus with one or more prominent nucleoli and contain lipid droplets within their cytoplasm. When examined with the TEM, pinealocytes show typical cytoplasmic

1	TABLE Hormones of the Posterior Lobe of the Pituitary Gland21.4 Hormone Composition Source Major Functions Oxytocin Polypeptide containing 9 amino acids Cell bodies of neurons located in the supraoptic and paraventricular nuclei of the hypothalamusa Stimulates activity of the contractile cells around the ducts of the mammary glands to eject milk from the glands; stimulates contraction of smooth muscle cells in the pregnant uterus Antidiuretic hormone (ADH; vasopressin) Polypeptide containing 9 amino acids; two forms: arginine-ADH (most common in humans) and lysine-ADH Cell bodies of neurons located in the supraoptic and paraventricular nuclei of the hypothalamusa Decreases urine volume by increasing reabsorption of water by collecting ducts of the kidney; decreases the rate of perspiration in response to dehydration; increases blood pressure by stimulating contractions of smooth muscle cells in the wall of arterioles a Immunocytochemical studies indicate that oxytocin and ADH are

1	in response to dehydration; increases blood pressure by stimulating contractions of smooth muscle cells in the wall of arterioles a Immunocytochemical studies indicate that oxytocin and ADH are produced by separate sets of neurons within the supraoptic and paraventricular nuclei of the hypothalamus. Biochemical studies have demonstrated that the supraoptic nucleus contains equal amounts of both hormones, whereas the paraventricular nucleus contains more oxytocin than ADH, but less than the amount found in the supraoptic nucleus.

1	 FOLDER 21.3 Clinical Correlation: Pathologies Associated with ADH Secretion chapter 21 Endocrine Organs PI N EAL G LAN D 753 The absence or reduced production of ADH leads to a condition known as diabetes insipidus, which is char-acterized by polyuria (production of large volumes of di-luted urine—up to 20 L per day) with hypotonic and tasteless (insipid) urine. Individuals with this condition have extreme thirst, which allows them to counteract the loss of water by drinking large amounts of fluids. This dis-ease commonly results from head injuries, tumors, or other lesions that can damage the hypothalamus or pos-terior lobe of the pituitary gland. This form of the disease is classified as hypothalamic diabetes insipidus in contrast to nephrogenic diabetes insipidus, in which secretion of the ADH is normal or elevated, but there is a lack of renal response to circulating levels of ADH. Nephrogenic diabetes is usually a congenital disorder re-lated to the mutation of the aquaporins-2

1	the ADH is normal or elevated, but there is a lack of renal response to circulating levels of ADH. Nephrogenic diabetes is usually a congenital disorder re-lated to the mutation of the aquaporins-2 (AQP-2) water channels gene or different ADH V2 receptor mutations in kidney tubules. Hypothalamic diabetes insipidus is usu-ally treated by administration of synthetic analogs of ADH (desmopressin), whereas the treatment of the nephro-genic type of this disease is aimed at reducing the volume of urine output. Abnormally high levels of ADH are found in the syn-drome of inappropriate antidiuretic hormone secre-tion (SIADH), which is characterized by hyponatremia (low serum levels of sodium), decreased serum osmolality asso-ciated with excessive urine sodium excretion, and elevated urine osmolality. In SIADH, the elevated level of ADH in-creases the absorption of water, thereby leading to produc-tion of concentrated urine, inability to excrete water, and hyponatremia that results from excess

1	In SIADH, the elevated level of ADH in-creases the absorption of water, thereby leading to produc-tion of concentrated urine, inability to excrete water, and hyponatremia that results from excess water rather than sodium deficiency. The increase in ADH secretion may be related to CNS disorders (tumors, injuries, infections, or cerebrovascular accidents); pulmonary diseases (pneumo-nia, chronic obstructive pulmonary disease, a lung abscess, or tuberculosis); tumors that secrete ADH (small-cell carci-noma of the lung, tumors of the pancreas, thymoma, or lym-phomas); and certain drugs (anti-inflammatories, nicotine, diuretics, and many others). Treatment of SIADH depends on the underlying etiology and includes fluid restrictions as well as pharmacological treatment. An ADH V2–receptor antagonist (Conivaptan) is now available to improve hy-ponatremia and to increase the free water diuresis without loss of other ions in the urine of patients with SIADH. TABLE Hypothalamic Regulating

1	antagonist (Conivaptan) is now available to improve hy-ponatremia and to increase the free water diuresis without loss of other ions in the urine of patients with SIADH. TABLE Hypothalamic Regulating Hormones21.5 Hormone Composition Source Major Functions Growth hormone– releasing hormone (GHRH) Two forms in human: polypeptides containing 40 and 44 amino acids Cell bodies of neurons located in the arcuate nucleus of hypothalamus Stimulates secretion and gene expression of GH by somatotropes Somatostatin Two forms in human: polypeptides containing 14 and 28 amino acids Cell bodies of neurons located in the periventricular, paraven-tricular, and arcuate nuclei of the hypothalamus Inhibits secretion of GH by somatotropes, inhibits insulin secretion by B cells of pancreatic islets Dopamine Catecholamine (amino acid derivative) Cell bodies of neurons located in the arcuate nucleus of hypothalamus Inhibits secretion of PRL by lactotropes Corticotropin-releasing hormone (CRH) Polypeptide

1	Catecholamine (amino acid derivative) Cell bodies of neurons located in the arcuate nucleus of hypothalamus Inhibits secretion of PRL by lactotropes Corticotropin-releasing hormone (CRH) Polypeptide containing 41 amino acids Cell bodies of neurons located in the arcuate, periventricular, and medial paraventricular nuclei of hypothalamus Stimulates secretion of ACTH by corticotropes; stimulates gene expression for POMC in corticotropes Gonadotropin-releasing hormone (GnRH) Polypeptide containing 10 amino acids Cell bodies of neurons located in the arcuate, ventromedial, dorsal, and paraventricular nuclei of hypothalamus Stimulates secretion of LH and FSH by gonadotropes Thyrotropin-releasing hormone (TRH) Polypeptide containing 3 amino acids Cell bodies of neurons located by the ventromedial, dorsal, and paraventricular nuclei of hypothalamus Stimulates secretion and gene expression of TSH by thyrotropes; stimulates synthesis and secretion of PRL

1	FIGURE 21.11 • Photomicrograph of infant pineal gland. This H&E–stained section is from a median cut through the pine cone–shaped gland. The conical anterior end of the gland is at the top of the micrograph. The arrows indicate the part of the gland that connects with the posterior commissure. The gland is formed by an evagination of the posterior portion of the roof of the third ventricle (diencephalon). The dark areas indicated by asterisks are caused by bleeding within the gland. 25. organelles along with numerous, dense-core, membrane-bounded vesicles in their elaborate, elongated cytoplasmic processes. The processes also contain numerous parallel bundles of microtubules. The expanded, clublike endings of the processes are associated with the blood capillaries. This feature strongly suggests neuroendocrine activity.

1	The interstitial (glial) cells constitute about 5% of the cells in the gland. They have staining and ultrastructural features that closely resemble those of astrocytes and are reminiscent of the pituicytes of the posterior lobe of the pituitary gland. In addition to the two cell types, the human pineal gland is characterized by the presence of calcified concretions, called corpora arenacea or brain sand (Fig. 21.12). These concretions appear to be derived from precipitation of calcium phosphates and carbonates on carrier proteins that are released into the cytoplasm when the pineal secretions are

1	FIGURE 21.12 • Photomicrograph of human pineal gland. This higher-magnification photomicrograph shows the characteristic concretions called brain sand or corpora arenacea. Pinealocytes (chief cells of the pineal gland) account for the majority of the cells seen in the specimen. They are arranged in clumps or cords. Those blood vessels (BV ) that contain red blood cells are readily apparent; numerous other blood vessels are also present but are not recognized at this magnification without evidence of the blood cells. 250. exocytosed. The concretions are recognizable in childhood and increase in number with age. Because they are opaque to X-rays and located in the midline of the brain, they serve as convenient markers in radiographic and computed tomography (CT) studies. The human pineal gland relates light intensity and duration to endocrine activity.

1	The human pineal gland relates light intensity and duration to endocrine activity. The pineal gland is a photosensitive organ and an important timekeeper and regulator of the day/night cycle (circadian rhythm). It obtains information about light and dark cycles from the retina via the retinohypothalamic tract, which connects in the suprachiasmatic nucleus with sympathetic neural tracts traveling into the pineal gland. During the day, light impulses inhibit the production of the major pineal gland hormone, melatonin. Therefore, pineal activity, as measured by changes in the plasma level of melatonin, increases during darkness and decreases during light. In humans, these circadian changes of melatonin secretion play an important role in regulating daily body rhythms.

1	TABLE Hormones of the Pineal Gland21.6 Hormone Composition Source Major Functions Melatonin Indolamine (N-acetyl-5-methoxytryptamine) Pinealocytes Regulates daily body rhythms and day/night cycle (circadian rhythms); inhibits secretion of GnRH and regulates steroidogenic activity of the gonads particularly as related to the menstrual cycle; in animals, influences seasonal sexual activity

1	Melatonin is released in the dark and regulates reproductive function in mammals by inhibiting the steroidogenic activity of the gonads (Table 21.6). Production of gonadal steroids is decreased by the inhibitory action of melatonin on neurosecretory neurons located in the hypothalamus (arcuate nucleus) that produce GnRH. Inhibition of GnRH causes a decrease in the release of FSH and LH from the anterior lobe of the pituitary gland. In addition to melatonin, extracts of pineal glands from many animals contain numerous neurotransmitters, such as serotonin, norepinephrine, dopamine,and histamine, and hypothalamic-regulating hormones, such as somatostatin and TRH. Clinically, tumors that destroy the pineal gland are associated with precocious (early-onset) puberty.

1	Animal studies demonstrate that information relating to the length of daylight reaches the pineal gland from photoreceptors in the retina. The pineal gland thus influences seasonal sexual activity. Recent studies in humans suggest that the pineal gland has a role in adjusting to sudden changes in day length, such as those experienced by travelers who suffer from jet lag. In addition, the pineal gland may play a role in altering emotional responses to the reduced length of day during winter in temperate and subarctic zones known as seasonal affective disorder (SAD). The thyroid gland is located in the anterior neck region adjacent to the larynx and trachea.

1	The thyroid gland is located in the anterior neck region adjacent to the larynx and trachea. The thyroid gland is a bilobate endocrine gland located in the anterior neck region and consists of two large lateral lobes connected by an isthmus, a thin band of thyroid tissue. The two lobes, each approximately 5 cm in length, 2.5 cm in width, and 20 to 30 g in weight, lie on either side of the larynx and upper trachea. The isthmus crosses anterior to the upper part of the trachea. A pyramidal lobe often extends upward from the isthmus. A thin connective tissue capsule surrounds the gland. It sends trabeculae into the parenchyma that partially outline irregular lobes and lobules. Thyroid follicles constitute the functional units of the gland. The thyroid gland develops from the endodermal lining of the floor of the primitive pharynx.

1	The thyroid gland begins to develop during the fourth week of gestation from a primordium originating as an endodermal thickening of the floor of the primitive pharynx. The primordium grows caudally and forms a ductlike invagination known as the thyroglossal duct. The thyroglossal duct descends through the tissue of the neck to its final destination in front of the trachea, where it divides into two lobes. During this downward migration, the thyroglossal duct undergoes atrophy, leaving an embryologic remnant, the pyramidal lobe of the thyroid, which is present in about 40% of the population. About the ninth week of gestation, endodermal cells differentiate into plates of follicular cells that become arranged into follicles. By week 14, well-developed follicles lined by the follicular cells contain colloid in their lumen. During week 7, epithelial cells lining the invagination of the fourth branchial pouches (sometimes called the fifth branchial pouches), known as the ultimobranchial

1	contain colloid in their lumen. During week 7, epithelial cells lining the invagination of the fourth branchial pouches (sometimes called the fifth branchial pouches), known as the ultimobranchial bodies, start their migration toward the developing thyroid gland and become incorporated into the lateral lobes. After fusing with the thyroid, ultimobranchial body cells disperse among the follicles, giving rise to parafollicular cells that become incorporated into the follicular epithelium.

1	The thyroid follicle is the structural and functional unit of the thyroid gland. A thyroid follicle is a roughly spherical cystlike compartment with a wall formed by a simple cuboidal or low columnar epithelium, the follicular epithelium. Hundreds of thousands of follicles that vary in diameter from about 0.2 to 1.0 mm constitute nearly the entire mass of the human thyroid gland. The follicles contain a gel-like mass called colloid (Fig. 21.13). The apical surfaces of the follicular cells are in contact with the colloid, and the basal surfaces rest on a typical basal lamina. Follicular epithelium contains two types of cells: follicular and parafollicular cells.

1	Follicular epithelium contains two types of cells: follicular and parafollicular cells. The parenchyma of the thyroid gland is composed of epithelium containing two types of cells:  Follicular cells (principal cells) are responsible for pro duction of the thyroid hormones T4 and T3. These cells vary in shape and size according to the functional state of the gland. In routine hematoxylin and eosin (H&E) prepa rations, follicular cells exhibit a slightly basophilic basal cy toplasm with spherical nuclei containing one or more FIGURE 21.13 • Thyroid gland. This photomicrograph of a human thyroid is from a section stained with H&E. It shows the colloid-containing follicles of the gland. Each follicle consists of a single layer of epithelial cells surrounding a central mass of colloid. The arrows indicate some of the blood capillaries between the follicles. 500.

1	prominent nucleoli. The Golgi apparatus has a supranuclear position. Lipid droplets and PAS-positive droplets can be identified with appropriate staining. At the ultrastructural level, the follicle cells reveal organelles commonly associated with both secretory and absorptive cells (Fig. 21.14), including typical junctional complexes at the apical end of the cell and short microvilli on the apical cell surface. Numerous profiles of rough-surfaced endoplasmic reticulum (rER) are present in the basal region. Small vesicles present in the apical cytoplasm are morphologically similar to vesicles associated with the Golgi apparatus. Abundant endocytotic vesicles, identified as colloidal resorption droplets, and lysosomes are also present in the apical cytoplasm.

1	 Parafollicular cells (C cells) are located in the periphery of the follicular epithelium and lie within the follicle basal lamina. These cells have no exposure to the follicle lumen. They secrete calcitonin, a hormone that regulates calcium metabolism. In routine H&E preparations, C cells are pale staining and occur as solitary cells or small clusters of cells. Human parafollicular cells are difficult to identify with light microscopy. At the electron microscope level, the parafollicular cells reveal numerous small secretory vesicles, which range in diameter from 60 to 550 nm, and a prominent Golgi apparatus (Fig. 21.15).

1	FIGURE 21.14 • Electron micrograph of follicular cells in rat thyroid gland. This electron micrograph shows a single layer of epithelium containing low columnar follicular cells. The apical surfaces with visible microvilli (Mv) are in contact with the colloid, whereas basal surfaces of follicular cells rest on the basal lamina (FBL). A narrow extracellular connective tissue space separates the follicular cells from the lumen of the capillary. Note that the fenestrated endothelial cells (En) lining capillary lumen rest on the basal lamina (EBL). Accumulation of lysosomes (L) and colloid resorption droplets (CRD), extensive Golgi apparatus (G), rough endoplasmic reticulum (rER), and presence of enlarged intercellular spaces are indicative of intensive activity of follicular cells. N, nucleus; JC, junctional complex. 14,000. (Courtesy of Dr. Holger Jastrow).

1	An extensive network of fenestrated capillaries derived from the superior and inferior thyroid arteries surrounds the follicles. Blind-ended lymphatic capillaries are present in the interfollicular connective tissue and may also provide a second route for conveying the hormones from the gland. Thyroid gland function is essential to normal growth and development. The thyroid gland produces three hormones, each of which is essential to normal metabolism and homeostasis (Table 21.7):  Thyroxine (tetraiodothyronine, T4) and triiodothyronine (T3) are synthesized and secreted by the follicular cells. Both hormones regulate cell and tissue basal metabolism and heat production and influence body growth and development. Secretion of these hormones is regulated by TSH released from the anterior lobe of the pituitary gland.

1	 Calcitonin (thyrocalcitonin) is synthesized by the parafollicular cells (C cells) and is a physiologic antagonist to parathyroid hormone (PTH). Calcitonin has an important role in regulating serum calcium levels in lower animals; however, its physiological role in humans remains elusive. Calcitonin lowers blood calcium levels by suppressing the

1	FIGURE 21.15 • Electron micrograph of a parafollicular cell. Cytoplasmic processes of follicular cells (arrows) partially surround the parafollicular cell (PC), which contains numerous electron-dense granules and a prominent Golgi apparatus (G). A basal lamina (BL) is associated with the follicular cells (FC). A portion of the central mass of colloidal material (C) in two adjacent follicles can be seen in the left corners of the micrograph. 12,000. (Courtesy of Dr. Emmanuel-Adrien Nunez.) resorptive action of osteoclasts and promotes calcium deposition in bones by increasing the rate of osteoid calcification. Secretion of calcitonin is regulated directly by blood calcium levels. High levels of calcium stimulate secretion; low levels inhibit it. Secretion of calcitonin is unaffected by the hypothalamus and pituitary gland. Calcitonin is secreted by several endocrine tumors (e.g., a medullary carcinoma of the thyroid); therefore, it is used as a tumor marker to monitor progress of

1	the hypothalamus and pituitary gland. Calcitonin is secreted by several endocrine tumors (e.g., a medullary carcinoma of the thyroid); therefore, it is used as a tumor marker to monitor progress of recovery after surgical resection of the tumor. Although calcitonin is used to treat patients with several disorders associated with excess bone resorption (e.g., osteoporosis and Paget’s disease), no clinical disease has been associated with its deficiency or even its absence after total thyroidectomy.

1	The principal component of colloid is thyroglobulin, an inactive storage form of thyroid hormones. The principal component of colloid is a large (660 kilodalton) iodinated glycoprotein called thyroglobulin containing about 120 tyrosine residues. Colloid also contains several enzymes and other glycoproteins. It stains with both basic and acidic dyes and is strongly PAS positive. Thyroglobulin is not a hormone. It is an inactive storage form of the thyroid hormones. Active thyroid hormones are liberated from thyroglobulin and released into the fenestrated blood capillaries that surround the follicles only after further cellular processing. The thyroid is unique among endocrine glands because it stores large amounts of its secretory product extracellularly. Synthesis of thyroid hormone involves several steps. The synthesis of the two major thyroid hormones, thyroxine (T4) and T3 takes place in the thyroid follicle in a series of discrete steps (Fig. 21.16): 1.

1	The synthesis of the two major thyroid hormones, thyroxine (T4) and T3 takes place in the thyroid follicle in a series of discrete steps (Fig. 21.16): 1. Synthesis of thyroglobulin. The precursor of thyroglobulin is synthesized in the rER of the follicular epithelial cells. Thyroglobulin is posttranslationally glycosylated in the rER and the Golgi apparatus before it is packaged into vesicles and secreted by exocytosis into the lumen of the follicle. 2.

1	Resorption, diffusion, and oxidation of iodide. Follicular epithelial cells actively transport iodide from the 758 The most common symptom of thyroid disease is a goiter, the enlargement of the thyroid gland. It may indicate either hypothyroidism or hyperthyroidism. Hypothyroidism can be caused by insufficient dietary iodine (iodine-defciency goiter, endemic goiter) or by one of several inherited autoimmune diseases, such as autoimmune thyroiditis (Hashimoto’s thyroiditis). Autoimmune thyroiditis is characterized by the presence of abnormal autoimmunoglobulins directed against thyroglobu-lin (TgAb), thyroid peroxidase (TPOAb), and the TSH recep-tor (TSHAb). The results are thyroid cell apoptosis and follicular destruction. The low levels of circulating thyroid hormone stimulate release of excessive amounts of TSH, which cause hypertrophy of the thyroid through synthesis of more thyroglobulin. Adult hypothyroidism, formerly called myxedema (due to the puffy appearance of the skin) is

1	excessive amounts of TSH, which cause hypertrophy of the thyroid through synthesis of more thyroglobulin. Adult hypothyroidism, formerly called myxedema (due to the puffy appearance of the skin) is char-acterized by mental and physical sluggishness. The edema that occurs in the severe stages of hypothyroidism is caused by the accumulation of large amounts of hyaluronan in the extracellular matrix of the connective tissue of the dermis. In hyperthyroidism (toxic goiter or Graves’ dis-ease), excessive amounts of thyroid hormones are re-leased into the circulation. Individuals with Graves’ disease have detectable levels of autoantibodies. These abnormal immunoglobulins (IgG) bind to the TSH receptors on the follicular cells and stimulate adenylate cyclase activity. As a result, increased levels of cAMP in follicular cells lead to continuous stimulation of the cells and increased thyroid hormone secretion. Because of negative feedback, the lev-els of TSH in the circulation are usually

1	levels of cAMP in follicular cells lead to continuous stimulation of the cells and increased thyroid hormone secretion. Because of negative feedback, the lev-els of TSH in the circulation are usually normal. However, under such stimulation the thyroid gland undergoes hyper-trophy, and the thyroid hormone is secreted at abnormally high rates, causing increased metabolism. Most of the clin-ical futures are associated with increased metabolic rate and increased sympathetic nerve activities. These include weight loss, excessive sweating, tachycardia, and nervous-ness. Noticeable features include protrusion of the eyeballs and retraction of the eyelids, resulting from increased sym-pathetic activity and increased deposition of extracellular matrix in the adipose tissue located behind the eyeball (Fig. F21.4.1a). The thyroid gland is enlarged. Microscopic features include the presence of columnar follicular cells lin-ing the thyroid follicles. Because of the high utilization of col-loid,

1	(Fig. F21.4.1a). The thyroid gland is enlarged. Microscopic features include the presence of columnar follicular cells lin-ing the thyroid follicles. Because of the high utilization of col-loid, the follicle tends to be depleted in the areas of contact with the apical surface of follicular cells (Fig. F21.4.1b). The treatment for Graves’ disease is either surgical to remove the thyroid gland or radiotherapy by ingestion of radioactive iodine (131I), which destroys most active follicular cells.

1	 FOLDER 21.4 Clinical Correlation: Abnormal Thyroid Function aaabbbFIGURE F21.4.1 • Hyperthyroidism. a. A young woman with signs of hyperthyroidism. Note the enlarged mass on the neck and the typical ocular symptoms known as exophthalmos. b. Photomicrograph of a thyroid gland specimen from an individual with Graves’ disease. Due to the increased utilization of colloid, there is a lack of staining at the periphery of the colloid near the apical surface of the follicular cell. Note that the majority of the cells are columnar in shape. (Reprinted with permission from Rubin E, Gorstein F, Rubin R, Schwarting R, Strayer D. Rubin’s Pathology, Clinicopathologic Foundations of Medicine, 4th ed. Baltimore: Lippincott Williams & Wilkins, 2005.)

1	TABLE 21.7 Hormones of the Thyroid Gland carbohydrate use, protein synthesis and degradation, and fat synthesis and degradation); regulates heat production; influences body and tissue growth and development of the nervous system in the fetus and young childb; increases absorption of carbohydrates from the intestine Decreases blood calcium levels by inhibiting bone resorption and stimulating absorption of calcium by the bones Thyroxine (tetraiodothyronine, T4) and aThyroid gland secretes substantially more T4 than T3; however, about 40% of T4 is peripherally converted to T3, which acts more rapidly and is a more potent hormone. bDeficiency of T3 and T4 during development results in fewer and smaller neurons, defective myelination, and mental retardation.

1	blood into their cytoplasm using ATPase-dependent lishing an intracellular concentration of iodide that is 30 sodium/iodide symporters (NIS). The NIS is the to 40 times greater than that of the serum. Iodide ions 87-kilodalton transmembrane protein that mediates ac-then diffuse rapidly toward the apical cell membrane. tive iodide uptake in the basolateral membrane of the From here iodide ions are transported to the lumen of follicular epithelial cells. These cells are capable of estab-the follicle by the 86 kilodalton iodide/chloride receptor FIGURE 21.16 • Diagram of steps in thyroid hormone synthesis. This diagram 5L depicts two follicular cells: one in the process of thyroglobulin synthesis (on the left with red pathways) and the other in the process of thyroglobulin resorption (on the right with blue pathways). The numbers, which are described more fully in the text, indicate the sequential steps that occur: 1, synthesis and secretion of thyroglobulin; 2, uptake and concentration

1	right with blue pathways). The numbers, which are described more fully in the text, indicate the sequential steps that occur: 1, synthesis and secretion of thyroglobulin; 2, uptake and concentration of iodide from the blood by sodum/iodide symporters (NIS), release of iodide into the colloid via iodide/chloride (pendrin) 6 transporters, and oxidation of iodide to iodine by thyroid peroxidase; 3, iodination of thyroglobulin in the colloid; 4, formation of T3 and T4 hormones in the colloid by oxidative coupling reactions; 5L, resorption pathway); 5TE, resorption of colloid via pathway; and 6, release of T4 and T3 from synthesis resorption the cell into the circulation.

1	transporter called pendrin located in the apical cell membrane. Iodide is then immediately oxidized to iodine, the active form of iodide. This process occurs in the colloid and is catalyzed by membrane-bound thyroid peroxidase (TPO). 3. Iodination of thyroglobulin. One or two iodine atoms are then added to the specific tyrosine residues of thyroglobulin. This process occurs in the colloid at the microvillar surface of the follicular cells and is also catalyzed by thyroid peroxidase (TPO). Addition of one iodine atom to a single tyrosine residue forms monoiodotyrosine (MIT). Addition of a second iodine atom to the MIT residue forms a diiodotyrosine (DIT) residue. 4.

1	4. Formation of T3 and T4 by oxidative coupling reactions. The thyroid hormones are formed by oxidative coupling reactions of two iodinated tyrosine residues in close proximity. For example, when neighboring DIT and MIT residues undergo a coupling reaction, T3 is formed; when two DIT residues react with each other, T4 is formed. After iodination, T4 and T3 as well as the DIT and MIT residues that are still linked to a thyroglobulin molecule are stored as the colloid within the lumen of the follicle.

1	5. Resorption of colloid. In response to TSH, follicular cells take up thyroglobulin from the colloid by a process of receptor-mediated endocytosis. After endocytosis, thyroglobulin follows at least two different intracellular pathways. In the lysosomal pathway, thyreoglobulin is internalized and transported within endocytotic vesicles to early endosomes. They eventually mature into lysosomes or fuse with existing lysosomes. Resorption of thyroglobulin at this stage can be confirmed by the presence of large endocytic vesicles called colloidal resorption droplets in the apical region of the follicular cells. Thyroglobulin is then degraded by lysosomal proteases into constituent amino acids and carbohydrates, leaving free T4, T3, DIT, and MIT molecules (see 5L labeled pathway in Fig 21.16). Under physiological condition, this is a major pathway of colloid resorption.

1	 In the transepithelial pathway, thyroglobulin is transported intact from the apical to the basolateral surface of follicular cells. To enter this pathway thyroglobulin binds to its receptor megalin, a 330-kilodalton member of the LDL endocytic receptor family. Megalin is a transmembrane protein expressed at the apical surface of follicular epithelial cells directly facing colloid. Thyreoglobulin internalized by megalin avoids the lysosomal pathway and endocytic vesicles are delivered to the basolateral membrane of follicular cells (see 5TE labeled pathway in Fig 21.16). In pathologic conditions of high TSH or TSH-like stimulation, megalin expression is increased and large amounts of thyroglobulin follow the transepithelial pathway. This pathway may reduce the extent of T4 and T3 release by diverting thyroglobulin away from the lysosomal pathway.

1	Patients with Graves’ and other thyroid diseases have detectable amounts of circulating thyroglobulin that contains portion of megalin receptor. If the levels of TSH remain high, the amount of colloid in the follicle is reduced because it is synthesized, secreted, iodinated, and resorbed too rapidly to accumulate.

1	6. Release of T4 and T3 into the circulation and recycling processes. Majority of T4 and T3 are liberated from thyroglobulin in the lysosomal pathway in a T4 to T3 ratio of 20:1. They cross the basal membrane and enter the blood and lymphatic capillaries. Most of the released hormones are immediately bound to either a specific plasma protein (54 kilodalton), thyroxinbinding protein (70%), or a nonspecific prealbumin fraction of serum protein (25%), leaving only small amounts (~5%) of free circulating hormones that are metabolically active. Very small amounts of T4 to T3 are released bound to thyroglobulin. Only the follicular cells are capable of producing T4, whereas most T3, which is five times more active than T4, is produced through conversion from T4 by organs such as the kidney, liver, and heart. The free circulating hormones also function in the feedback system that regulates the secretory activity of the thyroid. Once uncoupled from thyroglobulin, DIT and MIT molecules are

1	liver, and heart. The free circulating hormones also function in the feedback system that regulates the secretory activity of the thyroid. Once uncoupled from thyroglobulin, DIT and MIT molecules are further deiodinated within the cytoplasm of the follicular cells to release the amino acid tyrosine and iodide, which are then available for recycling.

1	Thyroid hormones play an essential role in normal fetal development.

1	In humans, thyroid hormones are essential to normal growth and development. In normal pregnancy, both T3 and T4 cross the placental barrier and are critical in the early stages of brain development. In addition, the fetal thyroid gland begins to function during the 14th week of gestation and also contributes additional thyroid hormones. Thyroid hormone deficiency during fetal development results in irreversible damage to the central nervous system (CNS), causing reduced numbers of neurons, defective myelination, and mental retardation. If maternal thyroid deficiency is present before the development of the fetal thyroid gland, the mental retardation is severe. Recent studies reveal that thyroid hormones also stimulate gene expression for GH in the somatotropes. Therefore, in addition to neural abnormalities, a generalized stunted body growth is typical. The combination of these two abnormalities is called congenital hypothyroidism.

1	The parathyroid glands are small endocrine glands closely associated with the thyroid. They are ovoid, a few millimeters in diameter, and arranged in two pairs, constituting the superior and inferior parathyroid glands. They are usually located in the connective tissue on the posterior surface of the lateral lobes of the thyroid gland. However, the number and location may vary. In 2% to 10% of individuals, additional glands are associated with the thymus. Structurally, each parathyroid gland is surrounded by a thin connective tissue capsule that separates it from the thyroid. Septa extend from the capsule into the gland to divide it into poorly defined lobules and to separate the densely packed cords of cells. The connective tissue is more evident in the adult, with the development of fat cells that increase with age and ultimately constitute as much as 60% to 70% of the glandular mass.

1	The glands receive their blood supply from the inferior thyroid arteries or from anastomoses between the superior and inferior thyroid arteries. Typical of endocrine glands, rich networks of fenestrated blood capillaries and lymphatic capillaries surround the parenchyma of the parathyroids. Parathyroid glands develop from the endodermal cells derived from the third and fourth branchial pouches. Embryologically, the inferior parathyroid glands (and the thymus) are derived from the third branchial pouch; the superior glands, from the fourth branchial pouch. Normally, the inferior parathyroids separate from the thymus and come to lie below the superior parathyroids. Failure of these structures to separate results in the atypical association of the parathyroids with the thymus in the adult. The principal (chief) cells differentiate during embryonic development and are functionally active in regulating fetal calcium metabolism. The oxyphil cells differentiate later at puberty.

1	Principal cells and oxyphil cells constitute the epithelial cells of the parathyroid gland.  Principal (chief) cells, the more numerous of the parenchymal cells of the parathyroid (Fig. 21.17), are responsible for regulating the synthesis, storage, and secretion of large amounts of PTH. They are small, polygonal cells, with a diameter of 7 to 10 m and a centrally located nucleus. The pale-staining, slightly acidophilic cytoplasm contains lipofuscin-containing vesicles, large accumulations of glycogen, and lipid droplets. Small, dense, membrane-limited vesicles seen with the TEM or after using special stains with the light microscope are thought to be the storage form of PTH. Principal cells can replicate when they are chronically stimulated by changes in blood calcium levels.

1	 Oxyphil cells constitute a minor portion of the parenchymal cells and are not known to have a secretory role. They are found singly or in clusters; the cells are more rounded, considerably larger than the principal cells, and have a distinctly acidophilic cytoplasm (see Fig. 21.17). Mitochondria, often with bizarre shapes and sizes, almost fill the cytoplasm and are responsible for the strong acidophilia of these cells. No secretory vesicles and little if any rER are present. Cytoplasmic inclusion bodies consist of occasional lysosomes, lipid droplets, and glycogen distributed among the mitochondria. Parathyroid hormone regulates calcium and phosphate levels in the blood. The parathyroids function in the regulation of calcium and phosphate levels. Parathyroid hormone (PTH) is essential

1	Parathyroid hormone regulates calcium and phosphate levels in the blood. The parathyroids function in the regulation of calcium and phosphate levels. Parathyroid hormone (PTH) is essential FIGURE 21.17 • Photomicrograph of human parathyroid gland. This H&E–stained specimen shows the gland with part of its connective tissue capsule (Cap). The blood vessels (BV) are located in the connective tissue septum between lobes of the gland. The principal cells are arranged in two masses (top and bottom) and are separated by a large cluster of oxyphil cells (center). The oxyphil cells are the larger cell type with prominent eosinophilic cytoplasm. They may occur in small groups or in larger masses, as seen here. The principal cells are more numerous. They are smaller, having less cytoplasm, and consequently exhibit closer proximity of their nuclei. Adipose cells (AC) are present in variable, although limited, numbers. 175.

1	for life. Therefore, care must be taken during thyroidectomy to leave some functioning parathyroid tissue. If the glands are totally removed, death will ensue because muscles, including the laryngeal and other respiratory muscles, go into tetanic contraction as the blood calcium level falls. PTH is an 84–amino acid linear peptide (Table 21.8). It binds to a specific PTH receptor on target cells that interacts with G protein to activate a second-messenger system. PTH release causes the level of calcium in the blood to increase. Simultaneously, it reduces the concentration of serum phosphate. Secretion of PTH is regulated by the serum calcium level through a simple feedback system. When parathyroid calcium-sensing receptors on principal cells detect low serum

1	TABLE Parathyroid Hormone 21.8 Hormone Composition Source Major Functions Parathyroid hormone (PTH) Polypeptide containing 84 amino acids Principal (chief cells)a Increases blood calcium level in three ways: (1) promotes calcium release from bone (acting on osteoblasts via RANK-RANKL signaling system, it increases the relative number of osteoclasts); (2) acts on the kidney to stimulate calcium reabsorption by the distal tubule while inhibiting phos-phate reabsorption in the proximal tubule; and (3) increases formation of hormonally active 1,25-dihydroxycholecalciferol (1,25-(OH)2 vitamin D3) in the kidney, which promotes tubular reabsorption of calcium. aSome evidence suggests that oxyphil cells, which first appear in the parathyroid gland at about 4 to 7 years of age and increase in number after puberty, may also produce PTH.

1	calcium levels, they stimulate secretion of PTH; high levels of serum calcium inhibit its secretion. PTH functions at several sites:  Action on bone tissue. For years, the bone resorption has been considered the major effect of PTH action on bone. However, the actions of PTH on bone are more complicated. PTH acts directly and indirectly on several cell types. Receptors for PTH are found on osteoprogenitor cells, osteoblasts, osteocytes, and bone lining cells. Surprisingly, the bone-resorbing osteoclasts do not have PTH receptors; thus, they are indirectly activated by the RANKRANKL signaling mechanism of osteoblasts (page 228). The binding of PTH to its receptors on osteoblasts increases local RANK production and decreases osteoprotegerin (OPG) secretion. These changes then stimulate osteoclast differentiation, which leads to increased bone resorption and release of calcium and phosphates into the extracellular fluid. PTH also has an anabolic effect on bone that leads to increased

1	differentiation, which leads to increased bone resorption and release of calcium and phosphates into the extracellular fluid. PTH also has an anabolic effect on bone that leads to increased bone mass; therefore, it is utilized in the treatment of osteoporosis (see Folder 8.2 in Chapter 8 on page 233).

1	 Kidney excretion of calcium is decreased by PTH stimulation of tubular reabsorption, thus conserving calcium.  Urinary phosphate excretion is increased by PTH secretion, thus lowering phosphate concentration in the blood and extracellular fluids.  Kidney conversion of 25-OH vitamin D3 to hormonally active 1,25-(OH)2 vitamin D3 is regulated primarily by PTH, which stimulates activity of 1-hydroxylase and increases the production of active hormone.  Intestinal absorption of calcium is increased under the influence of PTH. Vitamin D3, however, has a greater effect than PTH on intestinal absorption of calcium. PTH and calcitonin have reciprocal effects in the regulation of blood calcium levels.

1	PTH and calcitonin have reciprocal effects in the regulation of blood calcium levels. Although PTH increases blood calcium levels, the peak increase after its release is not reached for several hours. PTH appears to have a rather slow, long-term homeostatic action. Calcitonin, however, rapidly lowers blood calcium levels and has its peak effect in about 1 hour; therefore, it has a rapid, acute homeostatic action. The adrenal (suprarenal) glands secrete both steroid hormones and catecholamines. They have a flattened triangular shape and are embedded in the perirenal fat at the superior poles of the kidneys.

1	The adrenal (suprarenal) glands secrete both steroid hormones and catecholamines. They have a flattened triangular shape and are embedded in the perirenal fat at the superior poles of the kidneys. The adrenal glands are covered with a thick connective tissue capsule from which trabeculae extend into the parenchyma, carrying blood vessels and nerves. The secretory parenchymal tissue is organized into two distinct regions (Fig. 21.18):  The cortex is the steroid-secreting portion. It lies beneath the capsule and constitutes nearly 90% of the gland by weight.  The medulla is the catecholamine-secreting portion. It lies deep to the cortex and forms the center of the gland. Parenchymal cells of the cortex and medulla are of different embryologic origin.

1	Parenchymal cells of the cortex and medulla are of different embryologic origin. Embryologically, the cortical cells originate from mesodermal mesenchyme, whereas the medulla originates from neural crest cells that migrate into the developing gland (Fig. 21.19). Although embryologically distinct, the two portions of the adrenal gland are functionally related (see below). The parenchymal cells of the adrenal cortex are controlled in part by the anterior lobe of the pituitary gland and function in regulating metabolism and maintaining normal electrolyte balance (Table 21.9).

1	The adrenal glands are supplied with blood by the superior, middle, and inferior suprarenal arteries. These vessels branch before entering the capsule, to produce many small arteries that penetrate the capsule. In the capsule, the arteries branch to give rise to three principal patterns of blood distribution (Figs. 21.20 and 21.21). The vessels form a system that consists of  capsular capillaries that supply the capsule. the cortex and then drain into the fenestrated medullary capillary sinusoids.  medullary arterioles that traverse the cortex, traveling within the trabeculae, and bring arterial blood to the medullary capillary sinusoids. sympathetic ganglion (from neural crest) cortical primordium aorta of fetal cortex chromaffin aorta cells of future medulla

1	sympathetic ganglion (from neural crest) cortical primordium aorta of fetal cortex chromaffin aorta cells of future medulla FIGURE21.18 • Photomicrograph of the adrenal gland. This low-power micrograph of an H&E–stained specimen shows the full thickness of the adrenal gland with the cortex seen on both surfaces and a central region containing the medulla. Within the medulla are profiles of the central vein. Note that the deeper portion of the cortex stains darker than the outer portion, a reflection of the washed-out lipid in the zona glomerulosa and outer region of the zona fasciculata. This section also includes a cross section of the adrenal vein, which is characterized by the longitudinally arranged bundles of smooth muscle in its wall. 20.

1	The medulla thus has a dual blood supply: arterial blood from the medullary arterioles and “venous” blood from the cortical sinusoidal capillaries that have already supplied the cortex. The venules that arise from the cortical and medullary sinusoids drain into the small adrenomedullary collecting veins that join to form the large central adrenomedullary vein, which then drains directly into the inferior vena cava on the right side and into the left renal vein on the left side. In humans, the central adrenomedullary vein and its tributaries are unusual in that they have a tunica media containing conspicuous, longitudinally oriented bundles of smooth muscle cells. Synchronous contraction of longitudinal smooth muscle bundles along the central adrenomedullary vein and its tributaries cause the volume of the adrenal gland to decrease. This volume decrease enhances the efflux of hormones from the adrenal medulla into the circulation, an action comparable to squeezing a wet sponge.

1	Lymphatic vessels are present in the capsule and the connective tissue around the larger blood vessels in the gland. FIGURE21.19 • Development of the adrenal gland. a. In this early stage, the cortex is shown developing from cells of the intermediate mesoderm, and the medulla is shown differentiating from cells in the neural crest and migrating from the neighboring sympathetic ganglion. Note that the gland develops between the root of the dorsal mesentery of the primitive gut and the developing urogenital ridges. b. Mesodermal cells from the fetal cortex surround the cells of the developing medulla. c. At this stage (about 7 months of development), the fetal cortex occupies about 70% of the cortex. The permanent cortex develops outside the fetal cortex. d. The fully developed adrenal cortex is visible at the age of 4 months. The permanent cortex replaces the fetal cortex, which at this age has completely disappeared. Note the fully developed zonation of the permanent cortex.

1	TABLE Hormones of the Adrenal Glands 21.9 Hormone Composition Source Major Functions Adrenal cortex Mineralocorticoids (95% of mineralocorticoid activity in aldosterone) Steriod hormones (cholesterol derivatives) Parenchymal cells of the zona glomerulosa Aid in controlling electrolyte homeostasis (act on distal tubule of kidney to increase sodium reabsorption and decrease potassium reabsorp-tion); function in maintaining the osmotic balance in the urine and in preventing serum acidosis Glucocorticoids (cortico -sterone, and cortisol; 95% of glucocorticoid activity in cortisol) Steriod hormones (cholesterol derivatives) Parenchymal cells of the zona fasciculata (and to a lesser extent of the zona reticularis) Promote normal metabolism, particularly carbohy-drate metabolism (increase rate of amino acid transport to live, promote removal of protein from skeletal muscle and its transport to liver, reduce rate of glucose metabolism by cells and stimulate glycogen synthesis by liver,

1	rate of amino acid transport to live, promote removal of protein from skeletal muscle and its transport to liver, reduce rate of glucose metabolism by cells and stimulate glycogen synthesis by liver, stimulate mobilization of fats from storage deposits for energy use); provide resistance to stress; suppress inflammatory response and some allergic reactions Gonadocorticoids (dehydroepiandrosterone [DHEA] is a major sex steroid produced in both men and women) Steriod hormones (cholesterol derivatives) Parenchymal cells of the zona reticularis (and to a lesser extent of the zona fasciculata) Induce weak masculinizing effect; at normal serum levels usually their function is insignificant Adrenal medulla Norepinephrine and epinephrine (in human, 80% epinephrine) Catecholamines (amino acid derivatives) Chromaffin cells Sympathomimetic (produce effects similar to those induced by the sympathetic division of the autonomic nervous system)a; increase heart rate, increase blood pressure, reduce

1	Chromaffin cells Sympathomimetic (produce effects similar to those induced by the sympathetic division of the autonomic nervous system)a; increase heart rate, increase blood pressure, reduce blood flow to viscera and skin; stimulate conversion of glycogen to glucose; increase sweating; induce dilation of bronchioles; increase rate of respiration; decrease digestion; decrease enzyme production by digestive system glands; decrease urine production a The catecholamines influence the activity of glandular epithelium, cardiac muscle, and smooth muscle located in the walls of blood vessels and viscera.

1	Lymphatic vessels have also been demonstrated in the parenchyma of the adrenal medulla. Recent physiologic studies indicate an important role of the lymph vessels in distributing high-molecular-weight secretory products of chromaffin cells, such as chromogranin A, into the circulation. Cells of the Adrenal Medulla Chromaffin cells located in the adrenal medulla are innervated by presynaptic sympathetic neurons.

1	The central portion of the adrenal gland, the medulla, is composed of a parenchyma of large, pale-staining epithelioid cells called chromaffn cells (medullary cells), connective tissue, numerous sinusoidal blood capillaries, and nerves. The chromaffin cells are, in effect, modified neurons (Folder 21.5). Numerous myelinated, presynaptic sympathetic nerve fibers pass directly to the chromaffin cells of the medulla (see Chapter 12, page 378). When nerve impulses carried by the sympathetic fibers reach the catecholamine-secreting chromaffin cells, they release their secretory products. Therefore, chromaffin cells are considered the equivalent of postsynaptic neurons. However, they lack axonal processes. Experimental studies reveal that when chromaffin cells are grown in culture, they extend axon-like processes. However, axonal growth can be inhibited by glucocorticoids— hormones secreted by the adrenal cortex. Thus, the hormones of the adrenal cortex exert control over the morphology of

1	axon-like processes. However, axonal growth can be inhibited by glucocorticoids— hormones secreted by the adrenal cortex. Thus, the hormones of the adrenal cortex exert control over the morphology of the chromaffin cells and prevent them from forming neural processes. Chromaffin cells therefore more closely resemble typical endocrine cells, in that their secretory product enters the bloodstream via the fenestrated capillaries.

1	Ganglion cells are also present in the medulla. Their axons extend peripherally to the parenchyma of the adrenal cortex to modulate its secretory activity and innervate blood vessels, and extend outside the gland to the splanchnic nerves innervating abdominal organs. FIGURE 21.20 • Diagram illustrating the blood supply to the human adrenal gland. The region of the capsule, the zones within the cortex, and the medulla are indicated. (Modified from Warwick R, Williams PL, eds. Gray’s Anatomy, 35th ed. Edinburgh: Churchill Livingstone, 1973.) Chromaffin cells of the adrenal medulla have a secretory function. Chromaffn cells are organized in ovoid clusters and short interconnecting cords. The blood capillaries are arranged in intimate relation to the parenchyma. They originate either from the cortical capillaries or, as branches, from the cortical arterioles.

1	Ultrastructurally, the chromaffin cells are characterized by numerous secretory vesicles with diameters of 100 to 300 nm, profiles of rER, and a well-developed Golgi apparatus. The secretory material in the vesicles can be stained specifically to demonstrate histochemically that the catecholamines epinephrine and norepinephrine secreted by the chromaffin cells are produced by different cell types (Fig. 21.22). The TEM also reveals two populations of chromaffin cells distinguished by the nature of their membrane-bounded vesicles:  One population of cells contains only large dense core vesicles. These cells secrete norepinephrine.  The other population of cells contains vesicles that are smaller, more homogeneous, and less dense. These cells secrete epinephrine. Exocytosis of the secretory vesicles is triggered by release of acetylcholine from presynaptic sympathetic axons that synapse with each chromaffin cell. secretes: secretes: secretes: secretes:

1	Exocytosis of the secretory vesicles is triggered by release of acetylcholine from presynaptic sympathetic axons that synapse with each chromaffin cell. secretes: secretes: secretes: secretes: FIGURE 21.21 • Diagram illustrating the organization of the cells within the adrenal gland and their relationship to the blood vessels. Refer to Figure 21.20 for identification of the blood vessels. The ultrastructural features of the basic cell types and their secretions are noted. (Modified from Warwick R, Williams PL, eds. Gray’s Anatomy, 35th ed. Edinburgh: Churchill Livingstone, 1973.)

1	Epinephrine and norepinephrine account for less than 20% of the contents of the medullary secretory vesicles. The vesicles also contain large amounts of soluble 48-kilodalton proteins, called chromogranins, that appear to impart the density to the vesicle contents. These proteins, along with ATP and Ca2 , may help to bind the low-molecular-weight catecholamines and are released with the hormones during exocytosis. The catecholamines, synthesized in the cytosol, are transported into the vesicles through the action of a magnesium-activated ATPase in the membrane of the vesicle. Drugs such as reserpine, which cause depletion of catecholamines from the vesicles, may act by inhibiting this transport mechanism. Glucocorticoids secreted in the cortex induce the conversion of norepinephrine to epinephrine in chromaffin cells.

1	Glucocorticoids secreted in the cortex induce the conversion of norepinephrine to epinephrine in chromaffin cells. Glucocorticoids produced in the adrenal cortex reach the medulla directly through the continuity of the cortical and medullary sinusoidal capillaries. They induce the enzyme that catalyzes the methylation of norepinephrine to produce epinephrine. The nature of the blood flow correlates with regional differences in distribution of norepinephrineand epinephrine-containing chromaffin cells. The epinephrine-containing cells are more numerous in areas of the medulla  FOLDER 21.5 Clinical Correlation: Chromaffin Cells and Pheochromocytoma

1	Chromaffn cells (so named because they react with cells that secrete norepinephrine in comparison with the chromate salts) of the adrenal medulla are part of the amine normal adrenal medulla that comprises about 85% precursor uptake and decarboxylation (APUD) system of epinephrine-secreting cells. Stimulation of -adrenergic re-cells. The chromaffin reaction is thought to involve oxidation ceptors results in elevated blood pressure, increased car-and polymerization of the catecholamines contained within diac contractility, glycogenolysis, gluconeogenesis, and the secretory vesicles of these cells. Classically, chromaffin intestinal relaxation. Stimulation of -adrenergic receptors cells have been defined as being derived from neuroecto-results in an increase in heart rate and contractility. Surgiderm, innervated by presynaptic sympathetic nerve fibers, cal resection of the tumor is the treatment of choice. Care-and capable of synthesizing and secreting catecholamines. ful monitoring

1	Surgiderm, innervated by presynaptic sympathetic nerve fibers, cal resection of the tumor is the treatment of choice. Care-and capable of synthesizing and secreting catecholamines. ful monitoring with and -blockers is required during

1	A rare tumor derived from chromaffin cells called surgery to prevent hypertensive crises. pheochromocytoma produces excessive amounts of To summarize, pheochromocytomas are often described catecholamines. Because chromaffin cells are also found according to the “rule of 10’s”: outside of the adrenal medulla in paravertebral and prever- 10% are extraadrenal (paragangliomas) and of those,tebral sympathetic ganglia and other locations, tumors may 10% reside outside the abdomen. arise from outside the adrenal gland. These extraadrenal  10% occurs in children. pheochromocytomas are called paragangliomas be 10% are multiple or bilateral. cause scattered groups of chromaffin cells located among or near the components of the autonomic nervous system  10% are not associated with hypertension. (ANS) are called paraganglia. Episodic symptoms may  10% are malignant. occur, related to the pharmacological effects of excessive  10% are familial.

1	occur, related to the pharmacological effects of excessive  10% are familial. catecholamine secretion. Therefore, pheochromocytomas  10% recur after surgical removal. may precipitate life-threatening hypertension, cardiac arrhythmias, anxiety, and fear of impending death. Most  10% are found incidentally during unrelated imaging pheochromocytomas contain predominantly chromaffin studies. supplied with blood that has passed through the cortical sinusoids and thus contains secreted glucocorticoids. In some species, the norepinephrine-containing cells are more numerous in those regions of the medulla supplied by capillaries derived from the cortical arterioles. The catecholamines, in concert with the glucocorticoids, prepare the body for the “fight-or-flight” response.

1	The catecholamines, in concert with the glucocorticoids, prepare the body for the “fight-or-flight” response. The sudden release of catecholamines establishes conditions for maximum use of energy and thus maximum physical effort. Both epinephrine and norepinephrine stimulate glycogenolysis (release glucose into the bloodstream) and mobilization of free fatty acids from adipose tissue. The release of catecholamines also causes an increase in blood pressure, dilation of the coronary blood vessels, vasodilation of vessels supplying skeletal muscle, vasoconstriction of vessels conveying blood to the skin and gut, an increase in heart rate and output, and an increase in the rate and depth of breathing. Zonation of the Adrenal Cortex

1	Zonation of the Adrenal Cortex The adrenal cortex is divided into three zones on the basis of the arrangement of its cells (Fig. 21.23):  Zona glomerulosa, the narrow outer zone that consti tutes up to 15% of the cortical volume  Zona fasciculata, the thick middle zone that constitutes nearly 80% of the cortical volume  Zona reticularis, the inner zone that constitutes only 5% to 7% of the cortical volume but is thicker than the glomerulosa because of its more central location

1	The cells of the zona glomerulosa are arranged in closely packed ovoid clusters and curved columns that are continuous with the cellular cords in the zona fasciculata. Cells of the zona glomerulosa are relatively small and columnar or pyramidal. Their spherical nuclei appear closely packed and stain densely. In humans, some areas of the cortex may lack a recognizable zona glomerulosa. A rich network of fenestrated sinusoidal capillaries surrounds each cell cluster. The cells have abundant smooth-surfaced endoplasmic reticulum (sER), multiple Golgi complexes, large mitochondria with shelf-like cristae, free ribosomes, and some rER. Lipid droplets are sparse. The zona glomerulosa secretes aldosterone, which functions in the control of blood pressure.

1	The zona glomerulosa secretes aldosterone, which functions in the control of blood pressure. The cells of the zona glomerulosa secrete mineralocorticoids, compounds that function in the regulation of sodium and potassium homeostasis and water balance. The principal secretion, aldosterone, acts on the distal tubules of the nephron in the kidney, the gastric mucosa, and the salivary and sweat glands to stimulate resorption of sodium at these sites, as well as to stimulate excretion of potassium by the kidney. FIGURE 21.22 • Electron micrograph of medullary cells. Two types of medullary cells are present. The norepinephrinesecreting cells (NE) are identified by their vesicles, which contain a very dense core. The epinephrine-secreting cells (E) possess vesicles with less intensely staining granules. 15,000. The renin–angiotensin–aldosterone system provides feedback control of the zona glomerulosa.

1	The renin–angiotensin–aldosterone system provides feedback control of the zona glomerulosa. The zona glomerulosa is under feedback control of the renin–angiotensin–aldosterone system. The juxtaglomerular cells in the kidney release renin in response to a decrease in blood pressure or a low blood sodium level. Circulating renin catalyzes the conversion of circulating angiotensinogen to angiotensin I, which in turn is converted by angiotensin-converting enzyme (ACE) in the lung to angiotensin II. Angiotensin II then stimulates the cells of the zona glomerulosa to secrete aldosterone. As the blood pressure, sodium concentration, and blood volume increase in response to aldosterone, the release of renin from the juxtaglomerular cells is inhibited. Drugs that inhibit ACE in the lung are effective in the treatment of chronic essential hypertension.

1	The cells of the zona fasciculata are large and polyhedral. They are arranged in long straight cords, one or two cells thick, that are separated by sinusoidal capillaries. The cells of the zona fasciculata have a lightly staining spherical nucleus. Binucleate cells are common in this zone. TEM studies reveal characteristics typical of steroid-secreting cells, i.e., a highly developed sER (more so than cells of the zona glomerulosa) and mitochondria with tubular cristae. They also have a well-developed Golgi apparatus and numerous profiles of rER that may give a slight basophilia to some parts of the cytoplasm (Fig. 21.24). In general, however, the cytoplasm is acidophilic and contains numerous lipid droplets, although it usually appears vacuolated in routine histologic sections because of the extraction of lipid during dehydration. The lipid droplets contain neutral fats, fatty acids, cholesterol, and phospholipids that are precursors for the steroid hormones secreted by these cells.

1	The principal secretion of the zona fasciculata is glucocorticoids that regulate glucose and fatty acid metabolism.

1	The zona fasciculata secretes glucocorticoids, so called because of their role in regulating gluconeogenesis (glucose synthesis) and glycogenesis (glycogen polymerization). One of the glucocorticoids secreted by the zona fasciculata, cortisol acts on many different cells and tissues to increase the metabolic availability of glucose and fatty acids, both of which are immediate sources of energy. Within this broad function, glucocorticoids may have different, even opposite effects in different tissues:  In the liver, glucocorticoids stimulate conversion of amino acids to glucose, stimulate the polymerization of glucose to glycogen, and promote the uptake of amino acids and fatty acids. In adipose tissue, glucocorticoids stimulate the break down of lipids to glycerol and free fatty acids. In other tissues, they reduce the rate of glucose use and promote the oxidation of fatty acids. In cells such as fibroblasts, they inhibit protein synthesis and even promote protein catabolism to

1	In other tissues, they reduce the rate of glucose use and promote the oxidation of fatty acids. In cells such as fibroblasts, they inhibit protein synthesis and even promote protein catabolism to provide amino acids for conversion to glucose in the liver.

1	Glucocorticoids also depress the immune and inﬂammatory responses and, as a result of the latter, inhibit wound healing. Hydrocortisone, a synthetic form of cortisol is used in treatment of allergies and inﬂammation. It depresses the inﬂammatory response by suppressing interleukin 1 (IL-1) and IL-2 production by lymphocytes and macrophages. Glucocorticoids also stimulate destruction of lymphocytes in lymph nodes and inhibit mitosis by transformed lymphoblasts. Cells of the zona fasciculata also secrete small amounts of gonadocorticoids, principally androgens. ACTH regulates secretion of the zona fasciculata.

1	ACTH regulates secretion of the zona fasciculata. The secretion and production of glucocorticoids and sex steroids by the zona fasciculata is under feedback control of the CRH–ACTH system. ACTH is necessary for cell growth and maintenance and also stimulates steroid synthesis and increases blood flow through the adrenal gland. Exogenous ACTH maintains the structure and function of the zona fasciculata after hypophysectomy. In animals, administration of ACTH causes hypertrophy of the zona fasciculata. Circulating glucocorticoids may act directly on the pituitary gland, but they most commonly exert their feedback

1	Circulating glucocorticoids may act directly on the pituitary gland, but they most commonly exert their feedback FIGURE 21.23 • Photomicrographs of the cortex and medulla of the human adrenal gland. a. This photomicrograph shows an H&E–stained specimen of the outer cortex. It includes the connective tissue capsule, the zona glomerulosa, and the zona fasciculata. Continuous with the zona glomerulosa are the straight cords of cells that characterize the zona fasciculata. Between the cords are the capillaries and the less numerous arterioles. The red linear stripes represent capillaries that are engorged with red blood cells. 120.

1	b. The deep parts of the zona fasciculata, zona reticularis, and medulla are shown here. Note that the linear arrays of the cords in the zona fasciculata give way to irregular groups of cells of the zona reticularis. The medulla, in contrast, consists of ovoid groups of cells and short interconnecting cords of cells. A central adrenomedullary vein is also seen here. Note the thick longitudinally sectioned smooth muscle in part of its wall. 120. control on neurons in the arcuate nucleus of the hypothalamus, causing the release of CRH into the hypothalamohypophyseal portal circulation. Evidence also suggests that circulating glucocorticoids and the physiologic effects that they produce stimulate higher brain centers that, in turn, cause the hypothalamic neurons to release CRH. The zona reticularis produces glucocorticoids and androgens.

1	The zona reticularis produces glucocorticoids and androgens. The cells of the zona reticularis are noticeably smaller than those of the zona fasciculata, and their nuclei are more deeply stained. They are arranged in anastomosing cords separated by fenestrated capillaries. The cells have relatively few lipid droplets. Both light and dark cells are seen. Dark cells have abundant large lipofuscin pigment granules, and deeply staining nuclei are evident. The cells in this zone are small because they have less cytoplasm than the cells in the zona fasciculata; thus the nuclei appear more closely packed. They exhibit features of steroid-secreting cells, namely, a well-developed sER and numerous elongated mitochondria with tubular cristae, but they have little rER. The principal secretions of the zona reticularis are weak androgens.

1	The principal secretions of the zona reticularis are weak androgens. The principal secretion of the cells in the zona reticularis consists of weak androgens, mostly dehydroepiandrosterone (DHEA). The cells also secrete some glucocorticoids, in much smaller amounts than those of the zona fasciculata. Here, too, the principal glucocorticoid secreted is cortisol. The zona reticularis is also regulated by the feedback control of the CRH–ACTH system and atrophies after hypophysectomy. Exogenous ACTH maintains the structure and function of the zona reticularis after hypophysectomy. The fetal adrenal gland consists of an outer narrow permanent cortex and an inner thick fetal cortex or fetal zone.

1	The fetal adrenal gland consists of an outer narrow permanent cortex and an inner thick fetal cortex or fetal zone. Once fully established, the fetal adrenal gland is unusual in terms of its organization and its large size relative to other developing organs. The gland arises from mesodermal cells located between the root of the mesentery and the developing gonad zone (see Fig. 21.19a). The mesodermal cells penetrate the underlying mesenchyme and give rise to a large

1	FIGURE 21.24 • Electron micrograph of cells in the zona fasciculata. The boundary between adjacent cells of the cord is indicated by the arrowheads. Lipid droplets (L) are numerous (the lipid has been partially extracted). 15,000. Inset. A higher magnification of an area in the cell at the top of the micrograph reveals the extensive sER that is characteristic of steroid-secreting cells. Portions of the Golgi apparatus are also evident. 40,000 eosinophilic cell mass that will become the functional fetal terms of body weight and is only slightly smaller than the cortex or zone (see Fig. 21.19b). Later, a second wave of adjacent kidney. At term, the adrenal glands are equivalent cells proliferates from the mesenchyme and surrounds the in size and weight to those of the adult and produce 100 to primary cell mass (see Fig. 21.19c). By the fourth fetal 200 mg of steroid compounds per day, about twice that of month, the adrenal gland reaches its maximum mass in the adult adrenals.

1	 FOLDER 21.6 Functional Considerations: Biosynthesis of Adrenal Hormones

1	Cholesterol is the basic precursor of several steroid hor-mones, namely, corticosteroids, sex hormones, bile acids, and vitamin D. About half of the cholesterol in the body comes from the diet and the other half derives from de novo biosynthesis. Cholesterol synthesis occurs in the cytoplasm and organelles from acetyl-CoA. Biosynthesis in the liver accounts for approximately 10%, and in the in-testines, approximately 15%, of the amount produced each day. In addition, a small portion of cholesterol is synthe-sized by the adrenal cortical cells. Both dietary cholesterol and that synthesized de novo are transported within low-density lipoproteins (LDLs). Cholesterol is stored in lipid droplets within the cytoplasm of adrenal cortical cells in the form of cholesterol esters. Steroid hormones in adrenal glands are synthesized from cholesterol esters by removal of part of the side chain and modifications at specific sites on the remainder of the molecule. The enzymes catalyzing these

1	in adrenal glands are synthesized from cholesterol esters by removal of part of the side chain and modifications at specific sites on the remainder of the molecule. The enzymes catalyzing these modifications are located in different zones of the cortex as well as in differ-ent cytoplasmic sites within the cells. For instance, cleav-age of the side chain is catalyzed by P450-linked side chain cleavage enzyme (P450ssc) or desmolase, which is found only in mitochondria of steroid-producing cells. The other enzymes necessary for steroid production are located within sER, cytosol, and mitochondria. Thus, a precursor molecule may move from the sER to a mitochon-drion and back again several times before the definitive molecular structure of a given corticosteroid is obtained. Cholesterol esters removed from cytoplasmic lipid droplets and used in steroid hormone synthesis are quickly replenished from the cholesterol esters contained within LDL carried in the bloodstream. These esters are the

1	removed from cytoplasmic lipid droplets and used in steroid hormone synthesis are quickly replenished from the cholesterol esters contained within LDL carried in the bloodstream. These esters are the pri-mary source of the cholesterol used in corticosteroid syn-thesis. Under conditions of short-term or prolonged ACTH stimulation, the lipid stores in adrenal cortical cells are re-cruited for corticosteroid synthesis.

1	FIGURE 21.25 • Photomicrographs of a human fetal adrenal gland. a. Low-power micrograph of an H&E–strained section of a fetal adrenal gland. The permanent cortex (PC) is indicated in the upper portion of the micrograph. Below is the fetal zone (FZ ) in which the cells are arranged in anastomosing linear cords. Some of the capillaries (C) are engorged with red blood cells, thereby making them more apparent. 100. b. Higher-power micrograph of the same specimen showing the capsule (Cap) and the underlying permanent cortex. The cells are arranged in arched groups that extend into short cords. Note the close proximity of the nuclei and the small amount of cytoplasm in these cells. 200. c. This micrograph shows the cells of the fetal zone at the same magnification as in b. Note the slightly larger size of the nuclei and the considerable amount of cytoplasm in each of the fetal zone cells. Also note the eosinophilia of the cytoplasm, compared with the more basophilic cytoplasm of the cells

1	size of the nuclei and the considerable amount of cytoplasm in each of the fetal zone cells. Also note the eosinophilia of the cytoplasm, compared with the more basophilic cytoplasm of the cells of the permanent cortex. 200. (Original specimen courtesy of Dr. William H. Donnelly.)

1	The histologic appearance of the fetal adrenal gland is superficially similar to that of the adult adrenal gland. During late fetal life, most of the gland consists of cords of large eosinophilic cells that constitute approximately 80% of its mass. This portion of the gland, referred to as the fetal cortex (also called fetal zone), arises from the initial mesodermal cell migration. The remainder of the gland is composed of the peripheral layer of small cells with scanty cytoplasm. This portion, referred to as the permanent cortex, arises from the secondary mesodermal cell migration. The narrow permanent cortex, when fully established in the embryo, appears similar to the adult zona glomerulosa. The cells are arranged in arched groups that extend into short cords. They, in turn, become continuous with the cords of the underlying fetal zone (Fig. 21.25). In H&E preparations, the cytoplasm of the cells in the permanent cortex exhibits some basophilia; in combination with the closely

1	continuous with the cords of the underlying fetal zone (Fig. 21.25). In H&E preparations, the cytoplasm of the cells in the permanent cortex exhibits some basophilia; in combination with the closely packed nuclei, this gives this part of the gland a blue appearance, in contrast to the eosinophilic staining of the fetal zone.

1	With the TEM, the cells of the permanent cortex exhibit small mitochondria with shelf-like cristae, abundant ribosomes, and small Golgi profiles. The cells of the fetal zone, in contrast, are considerably larger and are arranged in irregular cords of varying width. With the TEM, these cells exhibit spherical mitochondria with tubular cristae, small lipid droplets, an extensive sER that accounts for the eosinophilia of the cytoplasm, and multiple Golgi profiles. Collectively, these features are characteristic of steroid-secreting cells. The development of the fetal adrenal gland is a part of a complex process of maturation and preparation of the fetus for extrauterine life.

1	The development of the fetal adrenal gland is a part of a complex process of maturation and preparation of the fetus for extrauterine life. The fetal adrenal lacks a definitive medulla. Chromaffin cells are present but are scattered among the cells of the fetal zone and are difficult to recognize in H&E preparations. The chromaffin cells originate from the neural crest (see Fig. 21.19a) and invade the fetal zone at the time of its formation (see Fig. 21.19b). They remain in this location in small, scattered cell clusters during fetal life (see Fig. 21.19c). The blood supply to both the permanent cortex and the fetal zone is through sinusoidal capillaries that course between the cords and join to form larger venous channels in the center of the gland. Unlike the postnatal adrenal, arterioles are absent in the parenchyma of the fetal adrenal gland.

1	Functionally, the fetal adrenal gland is under the control of the CRH–ACTH feedback system through the fetal pituitary. It interacts with the placenta to function as a steroid-secreting organ because it lacks certain enzymes necessary for steroid synthesis that are present in the placenta. Similarly, the placenta lacks certain enzymes necessary for steroid synthesis that are present in the fetal adrenal gland. Thus, the fetal adrenal gland is part of a fetal–placental unit. Precursor molecules are transported back and forth between the two organs to enable synthesis of glucocorticoids, aldosterone, androgens, and estrogens.

1	At birth, the fetal cortex undergoes a rapid involution that reduces the gland within the first postnatal month to about a quarter of its previous size. The permanent cortex grows and matures to form the characteristic zonation of the adult cortex. With the involution and disappearance of the fetal zone cells, the chromaffin cells aggregate to form the medulla. If the adrenal glands fail to develop properly, congenital adrenal hyperplasia may result.

1	The pituitary gland is located in a bony fossa in the floor of the cranial cavity. It is connected by a stalk to the base of the brain. Although joined to the brain, only posterior lobe of the gland, the neurohypophysis, develops from the neural ectoderm. The larger anterior lobe of the pituitary, the adenohypophysis, develops from oral ectoderm as a diverticulum of the buccal epithelium, called Rathke’s pouch. The adenohypophysis regulates other endocrine glands. It is composed of clumps and cords of epithelioid cells, separated by large-diameter fenestrated capillaries. The neurohypophysis is a nerve tract whose terminals store and release secretory products synthesized by their cell bodies in the supraoptic and paraventricular nuclei. The secretions contain either oxytocin or vasopressin (antidiuretic hormone [ADH]). Other neurons from the hypothalamus release secretions into the fenestrated capillaries of the infundibulum, the first capillary bed of the hypophyseal portal system

1	(antidiuretic hormone [ADH]). Other neurons from the hypothalamus release secretions into the fenestrated capillaries of the infundibulum, the first capillary bed of the hypophyseal portal system that carries blood to the fenestrated capillaries of the adenohypophysis. These hypothalamic secretions regulate the activity of the adenohypophysis.

1	Pituitary, human, H&E ×50. This specimen is a sagittal section of the pituitary gland. The neurohypophysis, the posterior lobe of the gland, is delineated by the dashed line (indicated by arrows) that separates it from the adenohypophysis. The pars nervosa (PN ) is the expanded portion of the neurohypohysis that is continuous with the infundibulum. The pars tuberalis (PT ) of adenohypophysis is located around the infundibular stem but may cover the pars nervosa to a variable extent. The pars intermedia (PI ) is a narrow band of tissue that lies between Pars distalis, pituitary, human, H&E ×375. This photomicrograph shows a region of the pars distalis of adenohypophysis that is rich in acidophils (A). Basophils (B) are present in this area in lesser numbers. The acidophils are readily identified by the acidophilic staining of their cytoplasm, in contrast to the basophils whose cytoplasm is clearly Pars distalis, pituitary, human, H&E ×375.

1	Pars distalis, pituitary, human, H&E ×375. This photomicrograph shows a region of the pars distalis of adenohypophysis that is rich in basophils (B). At this particular site, there are no recognizable acidophils (at other sites, it is possible to find a more equal distribution of acidophils and Pars intermedia, pituitary, human, PAS/aniline blue-black ×80.

1	Pars intermedia, pituitary, human, PAS/aniline blue-black ×80. This photomicrograph shows a small portion of the pars distalis (PD); the remainder reveals the pars intermedia (Pl ) of adenohypophysis. The pars distalis shown here contains numerous capillaries filled with red blood cells, thus producing the pars distalis (PD) and the pars nervosa. It borders a small cleft (Cl ) that constitutes the remains of the lumen of Rathke’s pouch. The pars distalis, the anterior lobe of the gland, is its largest part. It contains a variety of cell types that are not uniformly distributed. This accounts for differences in staining (light and dark staining areas) that are seen throughout the pars distalis.

1	Each of the components of the adenohypophysis; i.e., the pars distalis, pars tuberalis, and pars intermedia, when examined at higher magnification, exhibit features at the cellular level that aid in their identification. These features are described in the following figures as well as those on Plate 81. basophilic. Chromophobes (C ) are also very numerous in this field. The cytoplasm stains poorly in contrast to that of the acidophils and basophils. The cells are arranged in cords and clumps, between which are capillaries (Cap), some of which can be recognized, but most are in a collapsed state and difficult to visualize at this magnification. basophils, though, typically, one cell type outnumbers the other in a given region). Chromophobes (C) are also relatively numerous at this site. In this particular region, the chromophobe nuclei are readily apparent, but the cytoplasm of the cells is difficult to discern.

1	the bright red appearance. The pars intermedia contains a number of small cysts (Cy). The cells that make up the pars intermedia, which is relatively small in humans, consist of small basophils and chromophobes. The basophils have taken up the blue stain, thus making them prominent. To the extreme right is a less cellular area, the pars nervosa (PN ). KEY A, acidophils B, basophils C, chromophobes Cap, capillaries Caps, capsule Cl, cleft Cy, cysts PD, pars distalis PI, pars intermedia PN, pars nervosa PT, pars tuberalis

1	The parenchyma of the pars distalis consists of two general cell types: chromophobes and chromophils. Chromophobes stain poorly; chromophils stain well. Chromophils are further subdivided into acidophils and basophils. Basophils stain with basic dyes or hematoxylin, whereas the cytoplasm of the acidophil stains with acid dyes such as eosin. The cytoplasm of basophils also stains with the periodic acid-Schiff (PAS) reaction because of the glycoprotein in its secretory granules. Acidophils can be further subdivided into two groups on the basis of special cytochemical and ultrastructural features. One group, called somatotropes, produces the growth hormone, somatotropic hormone (STH); the other group of acidophils, called lactotropes, produces the prolactin (PRL). The groups of basophils can also be distinguished with the electron microscope and with special cytochemical procedures. One group of thyrotropes produces the thyroid-stimulating hormone (TSH); another group of gonadotropes

1	also be distinguished with the electron microscope and with special cytochemical procedures. One group of thyrotropes produces the thyroid-stimulating hormone (TSH); another group of gonadotropes produces the gonadotropic hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH); and a third group of corticotropes produces adrenocorticotropic hormone (ACTH) and lipotropic hormone (LPH). Chromophobes are also a heterogeneous group of cells. Many are considered to be depleted acidophils or basophils.

1	Pars distalis, pituitary, human, Mallory ×360; inset ×1200. This photomicrograph of the pars distalis is from an area where there is an almost equal distribution of acidophils (A) and basophils (B). The clumps and cords of cells are delineated by strands of connective tissue (stained blue) that surround them. A number of engorged capillaries (Cap) containing red blood cells (stained yellow) are also seen. The acidophil cytoplasm in this preparation Pars nervosa, pituitary, human, H&E ×325.

1	Pars nervosa, pituitary, human, H&E ×325. The pars nervosa of neurohypophysis seen here contains cells called pituicytes, and unmyelinated nerve fibers form the supraoptic and paraventricular nuclei of the hypothalamus. The pituicytes (P) are comparable with neuroglial cells of the central nervous system. The nuclei are round to oval; the cyto plasm extends from the nuclear region of the cell as long processes. In H&E preparations such as this, the cytoplasm of the pituicyte cannot be stains a reddish or rust color. The basophils stain a reddish blue to deep blue, and the chromophobes (C ) exhibit a pale-blue color. The inset shows the three general cell types at higher magnification. The secretory granules of the acidophils (A) and basophils (B) are just discernable. It is the granules that stain and provide the overall coloration to the two cell types. In contrast, the chromophobe (C) lacks granules and simply reveals a pale-blue background color.

1	distinguished from the unmyelinated nerve fibers. The hormones of the neurohypophysis, oxytocin and antidiuretic hormone (ADH) (also called vasopressin), are formed in the hypothalamic nuclei and pass via the fibers of the hypothalamohypophyseal tract to the neurohypophysis, where they are stored in the expanded nerve terminal portion of the nerve fibers. The stored neurosecretory material appears as Herring bodies (HB). In H&E preparations, the Herring bodies simply appear as small islands of eosinstained substance. Interspersed among the nerve fibers are capillaries (Cap). Pars nervosa, pituitary, human, PAS/aniline blue-black ×250; inset ×700.

1	Pars nervosa, pituitary, human, PAS/aniline blue-black ×250; inset ×700. In this specimen from pars nervosa, the aniline blue has stained the nuclei of the pituicytes (P); the nerve fibers have taken up some of the stain to give a light-blue background. With this staining technique, the Herring bodies (HB) appear as the dark black islands. The inset shows the Herring body near the bottom of the micrograph at high magnification. The granular texture of the Herring body as seen here is a reflection of the accumulated secretory granules in the nerve terminals. Also of note in this specimen are the capillaries (Cap), which are prominent as a result of the contrasting red staining of the red blood cells within them. KEY A, acidophils B, basophils C, chromophobes Cap, capillaries HB, Herring bodies P, pituicytes

1	The pineal gland (pineal body, epiphysis cerebri) is located in the brain above the superior colliculi. It de-velops from neuroectoderm but, in the adult, bears little resemblance to nerve tissue. Two cell types have been described within the pineal gland: parenchymal cells and glial cells. The full extent of these cells cannot be appreciated without the application of special methods. Those would show that the glial cells and the parenchymal cells have processes and that the processes of the parenchymal cells are expanded at their periphery. The parenchymal cells are more numerous. In an H&E preparation, the nuclei of the parenchymal cells are pale staining. The nuclei of the glial cells, on the other hand, are smaller and stain more intensely. Although the physiology of the pineal gland is not well understood, the secretions of the gland evidently have an antigonadal effect. For example, hypogenitalism has been reported in pineal tumors that consist chiefly of parenchymal cells,

1	is not well understood, the secretions of the gland evidently have an antigonadal effect. For example, hypogenitalism has been reported in pineal tumors that consist chiefly of parenchymal cells, whereas sexual precocity is associated with glial cell tumors (presumably, the parenchymal cells have been destroyed). In addition, experiments with animals indicate that the pineal gland has a neuroendocrine function whereby the pineal gland serves as an intermediary that relates endocrine function (particularly gonadal function) to cycles of light and dark. The external photic stimuli reach the pineal gland via optical pathways that connect with the superior cervical ganglion. In turn, the superior cervical ganglion sends postganglionic nerve fibers to the pineal gland. The extent to which these findings with laboratory animals apply to humans is not yet clear. Recent studies in humans suggest that the pineal gland has a role in adjusting to sudden changes in day length, such as those

1	these findings with laboratory animals apply to humans is not yet clear. Recent studies in humans suggest that the pineal gland has a role in adjusting to sudden changes in day length, such as those experienced by travelers who suffer from jet lag, and a role in regulating emo-tional responses to reduced day length during winter in temperate and subarctic zones (seasonal affec-tive disorder [SAD]).PI N EAL G LAN D 776

1	Pineal gland, human, H&E ×180. The pineal gland is surrounded by a very thin capsule (Cap) that is formed by the pia mater. Connective tissue trabeculae (CT ) extend from the capsule into the substance of the gland dividing it into lobules. The lobules (L) appear often as indistinct groups of cells of varying size surrounded Pineal gland, human, H&E ×360; inset ×700.

1	This micrograph shows at higher magnification the parenchyma of the pineal gland as well as a component called brain sand (BS) or corpora arenacea. When viewed at even higher magnifications, the corpora arenacea are seen to have an indistinct lamellated structure. Typically, they stain heavily with hematoxylin. The presence of these structures is an identifying feature of the pineal gland. A careful examination of the cells within the gland at the light microscopic level reveals two specific cell types. One cell type represents the parenchymal cells. These are by far the most numerous and are referred to as by the connective tissue. Blood vessels, generally small arteries (A) and veins (V ), course through the connective tissue. The arteries give rise to capillaries that surround and penetrate the lobules to supply the parenchyma of the gland. In this specimen and even at this low magnification, the capillaries (C ) are prominent as a consequence of the red blood cells present in

1	penetrate the lobules to supply the parenchyma of the gland. In this specimen and even at this low magnification, the capillaries (C ) are prominent as a consequence of the red blood cells present in their lumina.

1	pinealocytes (or chief cells of the pineal gland). Pinealocytes are modified neurons. Their nuclei are spherical and are relatively lightly stained because of the amount of euchromatin that they contain. The second cell type is the interstitial cell or glial cell that constitutes a relatively small percentage of the cells in the gland. Their nuclei are smaller and more elongate than those of the pinealocytes. The inset reveals several glial cells (G) that can be identified by their more densely staining nuclei. The majority of the nuclei of the other cells seen here belong to pinealocytes. Also seen in the inset are several fibroblasts (F) that are present within a trabecula. KEY A, artery BS, brain sand C, capillary Cap, capsule CT, connective tissue F, fibroblast G, glial cell L, lobule V, vein

1	The parathyroid glands are usually four in number. Each is surrounded by a capsule and lies on or is partially embedded in the thyroid gland. Connective tissue trabeculae extend from the capsule into the substance of the gland. The parathyroid glands elaborate a hormone that influences calcium and bone metabolism. Injection of parathyroid hormone into laboratory animals results in the release of calcium from bone by the action of osteocytes (osteocytic osteolysis) and osteoclasts. Removal of parathyroid glands results in a rapid drop in blood calcium levels. The thyroid gland is located in the neck in close relation to the upper part of the trachea and the lower part of the larynx. It consists of two lateral lobes that are joined by a narrow isthmus. The follicle, which consists of a single layer of cuboidal or low columnar epithelium surround-ing a colloid-filled space, is the functional unit of the thyroid gland. A rich capillary network is present in the connective tissue that

1	single layer of cuboidal or low columnar epithelium surround-ing a colloid-filled space, is the functional unit of the thyroid gland. A rich capillary network is present in the connective tissue that separates the follicles. The connective tissue also contains lymphatic capillaries.

1	Parathyroid gland, human, H&E ×320. As seen here, the larger blood vessels are associated with the trabeculae (BV) and, occasionally, adipose cells (A). The parenchyma of the parathyroid glands appears as cords or sheets of cells separated by capillaries and delicate connective tissue septa.

1	Two parenchymal cell types can be distinguished in routine H&E sections: chief cells (principal cells) and oxyphil cells. The chief cells (CC ) are more numerous. They contain a spherical nucleus surrounded by a small amount of cytoplasm. Oxyphil cells (OC ) are less numerous. They are conspicuously larger than chief cells but have a slightly smaller and more intensely staining nucleus. Their cytoplasm stains with eosin, and the boundaries between the cells are usually well marked. Moreover, the oxyphils are arranged in groups of variable size that appear scattered about in a much larger field of chief cells. Even with low magnification it is often possible to identify clusters of oxyphil cells because a unit area contains fewer nuclei than a comparable unit area of chief cells, as is clearly evident in this figure. Oxyphil cells appear during the end of the first decade of life and become more numerous around puberty. A further increase may be seen in older individuals.

1	PARATHYROI D AN D THYROI D G LAN DS KEY A, adipose cells BV, blood vessels CC, chief cells CT, connective tissue F, follicles OC, oxyphil cells arrows, tangential section of follicle wall asterisks, shrinkage artifact Thyroid gland, human, H&E ×200. A histologic section of the thyroid gland is shown here. The thyroid follicles (F ) vary somewhat in size and shape and appear closely packed. The homogeneous mass in the center of each follicle is the colloid. The thyroid cells appear to form a ring around the colloid. Although the individual cells are difficult to distinguish at this magnification, the nuclei of the cells serve as an indication of their location and arrangement. Large groups of cells are seen in association with some follicles. Where the nuclei are of the same size and staining characteristics, one can conclude that in these sites, the section includes the wall of the follicle (arrows) in a tangential manner without including the lumen.

1	There are two adrenal glands, one at the upper pole of each kidney. The gland is a composite of two distinct structural and functional components: a cortex and a medulla. The cortex develops from mesoderm and secretes steroid hormones; the medulla develops from neuroectoderm of the neural crest and secretes catecholamines. The adrenal cortex is divided into three zones according to the type and arrangement of its parenchymal cells. These are designated zona glomerulosa, zona fasciculata, and zona reticularis. The zona glomerulosa constitutes 15% of the cortical volume. It secretes mineralocorti-coids (aldosterone and deoxycorticosterone). The zona fasciculata constitutes nearly 80% of the cortical volume. It secretes the glucocorti-coids (cortisol, cortisone, and corticosterone) and a small amount of adrenal androgens. The zona reticularis (5% to 7% of cortical volume) produces most of the adrenal androgens. The zona fasciculata and the zona reticularis are regulated by

1	and a small amount of adrenal androgens. The zona reticularis (5% to 7% of cortical volume) produces most of the adrenal androgens. The zona fasciculata and the zona reticularis are regulated by adrenocorticotropic hormone (ACTH) secreted by the adenohypophysis in response to corticotropin-releasing factor (CRF) produced by the hypothalamus. The zona glomerulosa is not regulated by ACTH but is under feedback control of the renin-angiotensin-aldosterone system that also regulates blood pressure.

1	Adrenal gland, human, H&E ×45. This low-magnification micrograph of a section through the partial thickness of an adrenal gland shows the outer capsule (Cap), the cortex (Cort) from one surface of the gland, the underlying medulla (Med ), and a very small portion of the cortex from the other surface of the gland (Cort, bottom center). The cortex has a distinctly different appearance in both structural organization Cortex, adrenal gland, human, H&E ×180. This is a higher magnification of a portion of the capsule and the full thickness of the cortex from an area in figure above. The capsule consists of dense connective tissue in which the larger arteries (A) travel to give rise to smaller vessels that will supply the cortex and medulla. The zona glomerulosa (ZG) is located at the outer part of the cortex, immediately under the capsule. The parenchyma of this zone consists of small cells that appear as arching cords or as oval groups of cells. Cortex, adrenal gland, human, H&E ×245.

1	Cortex, adrenal gland, human, H&E ×245. This is a higher magnification of the area inscribed by the left rectangle in figure above. It shows the zona glomerulosa (ZG) and the outer portion of the zona fasciculata (ZF). Note the smaller size of the cells in the zona glomerulosa than those in the zona fasciculata. In addition, cells of the zona glomerulosa contain fewer lipid droplets than those of the zona fasciculata. and staining characteristics. From the inner portion, the medulla, note the lighter appearance of the medullary tissue. A small amount of adipose tissue (AT ) in which the gland is partially embedded is seen at the upper center of the micrograph. The corticomedullary boundary (dashed lines) has a wavelike contour, a reflection of the irregular shape of the gland. Within the medulla are a number of relatively large blood vessels (BV ). These are the adrenomedullary collecting veins that drain both the cortex and the medulla.

1	The zona fasciculata (ZF ) consists of radially oriented cords and sheets of cells, usually two cells in width, that extend toward the medulla. The cells of the outer part of the zona fasciculata are generally larger than those of the inner portion of this zone and typically stain poorly because of the large number of lipid droplets that they contain. The cells of the zona reticularis (ZR) are relatively small and contain little or no lipid droplets and, consequently, stain prominently with eosin. Because of their small size, the nuclei are in close proximity to one another, much like the cells of the zona glomerulosa.

1	Typically, the cells in this part of the zona fasciculata are filled with lipid droplets, thus, the very poor staining characteristic of their cytoplasm. Delicate connective tissue trabeculae (arrows) extend from the capsule to surround the glomerular groups of cells and extend between the cords of cells in the zona fasciculata. Capillaries and arterioles are located within the connective tissue trabeculae. Usually, the capillaries are collapsed and, without the presence of red blood cells in their lumina, are thus difficult to identify.

1	Cortex, adrenal gland, human, H&E ×245. in cords and contain lipid droplets, though in lesser amounts. The cells of This is a higher magnification of the area inscribed by the right the zona reticularis (ZR) are arranged in irregular anastomosing cords and rectangle in figure above. This deep portion of the zona fascic-contain at best only a small amount of lipid and, consequently, their cyto ulata (ZF) reveals smaller cells, although they are still arranged plasm stains with eosin. KEY A, arteries AT, adipose tissue BV, blood vessels Cap, capsule Cort, cortex Med, medulla ZF, zona fasciculata ZG, zona glomerulosa ZR, zona reticularis arrows, connective tissue trabeculae dashed line, corticomedullary boundary

1	The cells of the adrenal medulla develop from the same source as the postganglionic cells of the sympathetic nervous system. They are directly innervated by preganglionic cells of the sympathetic system and may be regarded as modified postganglionic cells that are specialized to secrete. These cells produce the catecholamines epinephrine and norepinephrine. The adrenal medulla receives its blood supply via two routes: by arterioles that pass through the cortex and by capillaries that continue from the cortex, a type of portal circulation. Thus, some of the blood supplying the medulla contains cortical secretions that regulate medullary function. Blood leaves the medulla via the central adrenomedullary vein. Its structure is unusual in that the tunica media of the vessel contains prominent bundles of longitudinally oriented smooth muscle, the contraction of which facilitates rapid outflow of blood when medullary cate-cholamines are released.

1	Medulla, adrenal gland, human, H&E ×175; inset ×250. This moderately low power photomicrograph shows the cells of the adrenal medulla. The medullary cells are organized in ovoid groups and short interconnecting cords. The cytoplasm of the medullary cells may stain with different intensity. The cytoplasm of some cells is very poorly stained, appearing almost clear, whereas others show greater intensity of eosin Central adenomedullary vein, adrenal gland, human, H&E ×350.

1	Central adenomedullary vein, adrenal gland, human, H&E ×350. This higher-magnification view of the rectangle in the bottom left figure shows part of the lumen (L) of the central adrenomedullary vein (AMV) at the bottom of the field. The tunica intima (TI ) of the vessel is relatively thin but may con tain a variable amount of connective tissue. The smooth muscle (SM) of the staining. In this photomicrograph, a portion of the wall, namely, the tunica media (TM ) of the central adrenomedullary vein, can be seen. The nature of the central adrenomedullary vein is described in lower left figure. The inset shows the ovoid groups of medullary cells at a higher magnification. Between these groups of cells are capillaries (Cap) that, as in the cortex, can be identified when they contain red blood cells as shown here. Medulla, adrenal gland, human, H&E ×125.

1	Medulla, adrenal gland, human, H&E ×125. This micrograph shows a central adrenomedullary vein (AMV ) that drains the adrenal medulla. The tunica media (TM) is unusually thick. The smooth muscle that constitutes this part of the vessel wall is in the form of bundles that are arranged longitudinally, that is, in the same direction as the vessel. Thus, the muscle seen here is cut in cross section, as is the vein. While the central adrenomedulary vein occupies most of the micrograph, medullary cells (MC) can be seen in several locations surrounding the vein. The portion of the figure outlined by the rectangle is seen at higher magnification in the bottom right figure.

1	tunica media (TM) is readily seen here as being arranged in bundles and appears in cross section. There is no discrete tunica adventitia in this vein. Instead, its connective tissue blends in with surrounding structures. Ganglion cells (GC) are frequently found in proximity to the wall of the central adrenomedullary vein. They are large cells with a moderately basophilic cytoplasm. Because of the large size of the cell, the nucleus is often missed in the section, and only the cell cytoplasm is seen. PLATE 85KEY AMV, central adrenomedullary vein Cap, capillary GC, ganglion cells L, lumen of central adrenomedullary vein MC, medullary cells SM, smooth muscle TI, tunica intima TM, tunica media

1	OVERVIEW OF THE MALE REPRODUCTIVE SYSTEM / 784 TESTIS / 784 Gender Determination and Development of the Testis / 785 Structure of the Testis / 788 Leydig Cells / 789 SPERMATOGENESIS / 792 Spermatogonial Phase / 792 Spermatocyte Phase (Meiosis) / 793 Spermatid Phase (Spermiogenesis) / 794 Structure of the Mature Sperm / 796 SEMINIFEROUS TUBULES / 798 Cycle of the Seminiferous Epithelium / 798 Waves of the Seminiferous Epithelium / 798 Sertoli Cells / 800 INTRATESTICULAR DUCTS / 802 EXCURRENT DUCT SYSTEM / 803 Epididymis / 803 Ductus Deferens / 807 ACCESSORY SEX GLANDS / 808 PROSTATE GLAND / 808 Bulbourethral Glands / 812 SEMEN / 813 PENIS / 813 Folder 22.1 Functional Considerations: Hormonal Regulation of Spermatogenesis / 788 Folder 22.2 Clinical Correlation: Factors Affecting Spermatogenesis / 789 Folder 22.3 Clinical Correlation: Sperm-Specific Antigens and the Immune Response / 803 Folder 22.4 Clinical Correlation: Benign Prostatic Hypertrophy and Cancer of the Prostate / 811

1	/ 789 Folder 22.3 Clinical Correlation: Sperm-Specific Antigens and the Immune Response / 803 Folder 22.4 Clinical Correlation: Benign Prostatic Hypertrophy and Cancer of the Prostate / 811 Folder 22.5 Clinical Correlation: Mechanism of Erection and Erectile Dysfunction / 815

1	The male reproductive system consists of the testes, genital excurrent ducts, accessory sex glands, and penis (Fig. 22.1). The accessory sex glands include the seminal vesicles, the prostate, and bulbourethral glands. The two primary functions of the testis are spermatogenesis (the production of sperm, called male gametes), and steroidogenesis (synthesis of androgens, also called sex hormones). Androgens, mainly testosterone, are essential for spermatogenesis, play an important role in embryonic development of the male embryo into the phenotypic male fetus, and are responsible for sexual dimorphism (male physical and behavioral characteristics). The events of cell division that occur during production of male gametes, as well as those of the female, the ova, involve both normal division, mitosis, and reduction division, meiosis.

1	The events of cell division that occur during production of male gametes, as well as those of the female, the ova, involve both normal division, mitosis, and reduction division, meiosis. A brief description of mitosis and meiosis is included in Chapter 3. A basic understanding of these processes is essential to understanding the production of gametes in both men and women. The adult testes are paired ovoid organs that lie within the scrotum, located outside the body cavity. Each testis is suspended within the end of an elongated musculofacial pouch, which is continuous with layers of the anterior abdominal FIGURE 22.1 • Schematic diagram demonstrating the components of the male reproductive system. Midline structures are depicted in sagittal section; bilateral structures including the testis, epididymis, ductus deferens, and seminal vesicle are shown intact.

1	wall and projects into the scrotum. Testes are connected by the spermatic cords to the abdominal wall and tethered to the scrotum by scrotal ligaments, the remnants of the gubernaculum (see further in the chapter). Gender Determination and Development of the Testis Gender differentiation is accomplished through a cascade of gene activations. Genetic sex is determined at fertilization by the presence or absence of the Y chromosome. The testes, however, do not form until the seventh week of development. Gonadal sex is determined by the presence of the SRY gene located in the sex-determining region of the short arm of the Y chromosome. The SRY gene expression in early embryonic development is responsible for gender determination.

1	The genetic information encoded in the Y chromosome itself is not sufficient to guide a complex development of the male gonads. Instead, the SRY gene operates as a master switch that controls the cascade of several gene activations on autosomes 9, 11, 17, and 19 and the X chromosome. A transcription factor called the testis-determining factor (TDF), encoded by the SRY gene, has a molecular domain that binds into a specific region of DNA and alters it structure. The affected DNA forms a loop that permits binding of other transcription factors. They in turn cause the expression of other genes that initiate formation of not only the testes but also other male sex organs. Several other genes are expressed about the same time as the SRY gene, including these:  WT-1 gene, that is required for the development of the urogenital system and for regulation of the SRY transcrip tion. Mutations of the WT-1 gene are found in children with familial Wilms’s tumor and in children with accompanied

1	the development of the urogenital system and for regulation of the SRY transcrip tion. Mutations of the WT-1 gene are found in children with familial Wilms’s tumor and in children with accompanied genitourinary malformations.

1	 SOX-9 gene found in the genital ridges activates the AMH gene that is responsible for Mlerian-inhibiting factor synthesis. The mutation of the SOX9 gene is linked to a reversal of the gender of a male individual (46, XY).  SF-1 gene (steroidogenic factor-1 gene) that regulates the expression of a number of steroidogenic genes.  DAX-1 gene that encodes nuclear receptor DAX-1. Acti vation of this receptor suppresses the SRY gene during gonadal sex differentiation and its mutation is responsible for congenital adrenal hypoplasia. The testes develop on the posterior wall of the abdomen and later descend into the scrotum.

1	The testes develop on the posterior wall of the abdomen and later descend into the scrotum. The testes develop in close association with the urinary system retroperitoneally on the posterior wall of the abdominal cavity. Testes (like ovaries) are derived from three sources:  Intermediate mesoderm that forms the urogenital ridges on the posterior abdominal wall, giving rise to Leydig cells (interstitial cells) and myoid cells (peritubular contractile cells) that lines the urogenital ridges and gives rise to Sertoli cells  Primordial germ cells that migrate from the yolk sac into developing gonads, where they divide and differentiate into spermatogonia

1	Migration of the primordial germ cells into the genital ridges induces mesodermal cells of the urogenital ridges and cells of the coelomic mesothelium to proliferate and form the primary sex cords. At this stage, these cords are comprised of primordial germ cells, pre-Sertoli cells, and a surrounding layer of myoid cells. Later, primary sex cords duct of epididymis degenerating paramesonephric duct

1	FIGURE 22.2 • Schematic diagram of the stages of testicular development. a. This diagram shows the 5-week embryo in the stage of indifferent gonads. The gonadal ridges visible on the posterior abdominal wall are being infiltrated by primordial germ cells (green) that migrate from the yolk sac. Most of the developing gonad is formed by mesenchyme derived from the coelomic epithelium. The primordial germ cells become incorporated in the primary sex cords. b. At a later stage, under hormonal influence of testis-determining factor (TDF), the developing gonad initiates production of testosterone. This is followed by differentiation of the primary sex cords into seminiferous cords. At the same time, the developing gonad produces Mlerian-inhibiting factor (MIF), which causes regression of the paramesonephric duct and those structures derived from it. Note that the mesonephric tubules come in close contact with the developing rete testis. c. Final stages of testicular development. The tunica

1	duct and those structures derived from it. Note that the mesonephric tubules come in close contact with the developing rete testis. c. Final stages of testicular development. The tunica albuginea surrounding the testis contributes to development of the testicular septa. The rete testis connects with the seminiferous cords and with the excurrent duct system that develops from the mesonephric duct and tubules.

1	differentiate into the seminiferous cords, which give rise to the seminiferous tubules, straight tubules, and rete testis (Fig. 22.2).

1	In the first stage of development, the testes develop on the posterior abdominal wall from indifferent primordia of urogenital ridges that are identical in both sexes. During this indifferent stage an embryo has the potential to develop into either a male or female. However, expression of the SRY gene—exclusively in the pre-Sertoli cells—orchestrates male development of the embryo. Early in male development, mesenchyme separating the seminiferous cords gives rise to Leydig (interstitial) cells that produce testosterone to stimulate development of the indifferent primordium into a testis. Testosterone is also responsible for the growth and differentiation of the mesonephric (Wolffian) ducts that develop into the male genital excurrent ducts. Also in this early stage, the Sertoli (sustentacular) cells that develop within the seminiferous cords produce another important hormonal substance, called Mlerian-inhibiting factor (MIF). MIF’s molecular structure is similar to that of

1	(sustentacular) cells that develop within the seminiferous cords produce another important hormonal substance, called Mlerian-inhibiting factor (MIF). MIF’s molecular structure is similar to that of transforming growth factor(TGF-). It is a large glycoprotein that inhibits cell division of the paramesonephric (Mlerian) ducts, which in turn inhibits development of female reproductive organs (Fig. 22.3).

1	Development and differentiation of the external genitalia (also from the sexually indifferent stage) occur at the same time and result from the action of dihydrotestosterone (DHT), a product of the conversion of testosterone by 5reductase. Without DHT, regardless of the genetic or gonadal sex, the external genitalia will develop along the female template. The appearance of testosterone, MIF, and DHT in the developing male embryo determines its hormonal sex. (Folder 22.1)

1	At approximately the 26th week of gestation, the testes descend from the abdomen into the scrotum. This migration of testes is caused by differential growth of the abdominal cavity combined with the action of testosterone that causes shortening of the gubernaculum, the testosterone-sensitive ligament connecting the inferior pole of each testis with the developing scrotum. The testes descend into the scrotum by passing through the inguinal canal, the narrow passage between the abdominal cavity and the scrotum. During descent, the testes carry their blood vessels, lymphatic vessels, and nerves as well as their principal ex-current duct system, the ductus deferens with them. Descent of the testis is sometimes obstructed, resulting in cryptorchidism, or undescended testes. This condition is common (30%) in premature newborns and about 1% of fullterm newborns. Cryptorchidism can lead to irreversible histologic changes in the testis and increases the risk of testicular cancer. Therefore,

1	common (30%) in premature newborns and about 1% of fullterm newborns. Cryptorchidism can lead to irreversible histologic changes in the testis and increases the risk of testicular cancer. Therefore, an undescended testis requires surgical correction. Orchiopexy (placement in the scrotal sac) should be performed, preferably before histologic changes become irreversible at approximately 2 years of age.

1	Spermatogenesis requires that the testes be maintained below normal body temperature. As the testes descend from the abdominal cavity into the scrotum, they carry with them blood vessels, lymphatic vessels, autonomic nerves, and an extension of the abdominal peritoneum called the tunica vaginalis, which covers their anterolateral surface. Within the scrotum the temperature of the testes is 2C to 3C below body temperature. This lower temperature is essential for spermatogenesis, but is not required for hormone production (steroidogenesis), which can occur at normal body temperature. If the testes are maintained at higher temperatures (e.g., because of fever) or if they fail to descend into the scrotum, sperm are not produced.

1	Each testis receives blood through a testicular artery, a direct branch of the abdominal aorta. It is highly convoluted near the testis, where it is surrounded by the pampiniform venous plexus, which carries blood from the testis to the abdominal veins. This arrangement allows heat exchange between the blood vessels and helps maintain the testes at a lower temperature. The cooler venous blood returning from the testis cools the arterial blood before it enters the testis through a countercurrent heat exchange mechanism. In addition, the cremaster muscle, whose fibers originate from the internal abdominal oblique muscle of the anterior abdominal wall, responds to changes in ambient temperature. Its contraction moves the testes closer to the abdominal wall, and its relaxation lowers the testes within the scrotum. Cold temperatures also cause contraction of a thin sheet of smooth muscle (dartos muscle) in the superficial fascia of the scrotum. Contraction of dartos

1	FIGURE 22.3 • Schematic diagram of male sex development and hormonal infuence on developing reproductive organs. This diagram illustrates three levels on which the sex of the developing embryo is determined. The genetic sex is determined at the time of fertilization; gonadal sex is determined by activation of the SRY gene located on the short arm of chromosome Y; and hormonal sex is determined by a hormone secreted by the developing gonad. The diagram shows the influence of Mlerianinhibiting factor (MIF), testosterone, and dihydrotestosterone (DHT) on the developing structures.  FOLDER 22.1 Functional Considerations: Hormonal Regulation of Spermatogenesis

1	Normal function of testis is dependent upon hormones acting through endocrine and paracrine pathways. The endocrine function of the testis resides primarily in the Leydig cell pop-ulation that synthesizes and secretes the principal circulating androgen, testosterone. Nearly all of the testosterone is produced by the testis; less than 5% is produced by the adrenal glands. It is estimated in humans that the total Leydig cell population produces about 7 mg of testosterone per day. As testosterone leaves the Leydig cells, it passes into blood and lymphatic capillaries and across the peritubular tissue to reach the seminiferous epithelium. High local levels of testosterone within the testis (esti-mated to be as much as 200 times the circulating levels) are necessary for the proliferation and differentiation of sper-matogenic cells. This high testicular level of testosterone can be significantly decreased by negative feedback from exogenous testosterone. Intensive research in this area is

1	differentiation of sper-matogenic cells. This high testicular level of testosterone can be significantly decreased by negative feedback from exogenous testosterone. Intensive research in this area is being directed into development of a prototype of testos-terone-based contraceptive drugs for men. In early clinical studies, these drugs have been shown to cause a significant decrease in the testicular testosterone concentration and inhibition of spermatogenesis. Recovery of spermatogene-sis occurs after discontinuation of contraceptive use. How-ever, in some individuals, this type of contraceptive is not efficacious and does not cause spermatogenic suppression. Peripheral testosterone levels have the following effects:  Differentiation of the central nervous system (CNS) and the genital apparatus and genital excurrent duct system  Growth and maintenance of secondary sexual charac-teristics (such as the beard, male distribution of pubic hair, and low-pitched voice)  Growth and

1	apparatus and genital excurrent duct system  Growth and maintenance of secondary sexual charac-teristics (such as the beard, male distribution of pubic hair, and low-pitched voice)  Growth and maintenance of the accessory sex glands (seminal vesicles and prostate and bulbourethral glands), genital excurrent duct system, and the external genitalia (mainly byproducts of testosterone conversion to DHT)  Anabolic and general metabolic processes, including skeletal growth, skeletal muscle growth, distribution of subcutaneous fat, and kidney function  Behavior, including libido The steroidogenic and spermatogenic activities of the testis are regulated by hormonal interaction among the hy-pothalamus, anterior lobe of the pituitary gland, and gonadal cells (i.e., Sertoli, spermatogenic, and Leydig cells). The anterior lobe of the pituitary gland produces three hormones involved in this process: luteinizing hormone (LH), which in the male is sometimes referred to as interstitial

1	and Leydig cells). The anterior lobe of the pituitary gland produces three hormones involved in this process: luteinizing hormone (LH), which in the male is sometimes referred to as interstitial cell–stimulating hormone (ICSH); follicle-stimulating hormone (FSH); and prolactin (PRL). In response to LH release by the pituitary, Leydig cells pro-duce increasing amounts of testosterone. PRL acts in combination with LH to increase the steroidogenic activity of Leydig cells. Because FSH and testosterone receptors are found in Sertoli cells, these cells are the primary regu-lators of spermatogenesis.

1	muscles causes the scrotum to wrinkle when cold to help regulate heat loss (Folder 22.2). Structure of the Testis The testes have an unusually thick connective tissue capsule, the tunica albuginea. An unusually thick, dense connective tissue capsule, the tunica albuginea, covers each testis (Fig. 22.4). The inner part of this capsule, the tunica vasculosa, is a loose connective tissue layer that contains blood vessels. Each testis is divided into approximately 250 lobules by incomplete connective tissue septa that project from the capsule. Along the posterior surface of the testis, the tunica albuginea thickens and projects inward as the mediastinum testis. Blood vessels, lymphatic vessels, and the genital excurrent ducts pass through the mediastinum as they enter or leave the testis. Each lobule consists of several highly convoluted seminiferous tubules.

1	Each lobule consists of several highly convoluted seminiferous tubules. Each lobule of the testis consists of one to four seminiferous tubules, in which sperm are produced, and a connective tissue stroma, in which Leydig (interstitial) cells are contained (Fig. 22.5). Each tubule within the lobule forms a loop and, because of its considerable length, is highly convoluted, actually folding on itself within the lobule. The ends of the loop are located near the mediastinum of the testis, where they assume a short straight course. This part of the seminiferous tubule is called the straight tubule (tubulus rectus). It becomes continuous with the rete testis, an anastomosing channel system within the mediastinum. The seminiferous tubules consist of a seminiferous epithelium surrounded by a tunica propria.

1	The seminiferous tubules consist of a seminiferous epithelium surrounded by a tunica propria. Each seminiferous tubule is approximately 50 cm long (range, 30 to 80 cm) and 150 to 250 m in diameter. The seminiferous epithelium is an unusual and complex stratified epithelium composed of two basic cell populations:  Sertoli cells, also known as supporting, or sustentacular, cells. These cells do not replicate after puberty. Sertoli cells are columnar cells with extensive apical and lateral processes that surround the adjacent spermatogenic cells and occupy the spaces between them. However, this elaborate configuration of the Sertoli cells cannot be seen distinctly in routine hematoxylin and eosin (H&E) preparations.

1	Degenerative changes, such as apoptosis, premature sloughing of cells, or formation of multinucleated giant cells, are readily apparent after exposure to such agents. Factors that negatively affect spermatogenesis include these:  Dietary defciencies. Reduced dietary intake is known to impair spermatogenesis. Vitamins, coenzymes, and microelements such as vitamin A, B12, C, E, -carotenes, zinc, and selenium have been shown to affect sperm formation.  Environmental/lifestyle factors. A recent study con-ducted in Denmark compared the sperm count in two groups of young men from rural and urban populations. A higher median sperm count (24%) was found in the men from the rural group compared with those from the urban group.  Developmental disorders. Cryptorchidism, hypospa-dias, and factors such as low birth weight have been found to be important risk factors for testicular cancer associated with reduced semen quality and reduced fertility.  Systemic diseases or local infections.

1	such as low birth weight have been found to be important risk factors for testicular cancer associated with reduced semen quality and reduced fertility.  Systemic diseases or local infections. Infections involving the testis (orchitis) may have an effect on sper-matogenesis. Systemic diseases that can impair sper-matogenesis include fever, kidney diseases, HIV and other viral infections, and metabolic disorders.  Elevated testicular temperature. A sedentary lifestyle may impair the ability to maintain the lower temperature of the testis in the scrotum. A higher than average scrotal temperature has been linked to failure of spermatogenesis. sure to synthetic estrogen (diethylstilbestrol) and other sex steroids can exert negative feedback on FSH secre-tion, resulting in decreased spermatogenesis. Prenatal exposure to estrogens can potentially inhibit fetal go-nadotropin secretion and inhibit Sertoli cell proliferation.  Toxic agents. Mutagenic agents, antimetabolites, and some

1	Prenatal exposure to estrogens can potentially inhibit fetal go-nadotropin secretion and inhibit Sertoli cell proliferation.  Toxic agents. Mutagenic agents, antimetabolites, and some pesticides, e.g., dibromochloropropane (DBCP), can drastically affect spermatogenesis and production of normal sperm. DBCP is a nematocide pesticide that is still used in some developing countries. It has been shown to cause a major decrease in sperm count and infertility after human exposure. Other agents that may affect fertility include chemicals in plastics (e.g., phtha-lates), pesticides (e.g., DDT), products of combustion (e.g., dioxins), polychlorinated biphenyls (PCBs), and others. Most of these chemicals possess very weak estrogen properties and may affect fertility. Direct toxic-ity to the spermatogonia is linked to changes in sperm quality.  Ionizing radiation and alkylating agents. Nitrogen mustard gas and procarbazine have been found to have toxic effects on spermatogonia. Electromagnetic

1	is linked to changes in sperm quality.  Ionizing radiation and alkylating agents. Nitrogen mustard gas and procarbazine have been found to have toxic effects on spermatogonia. Electromagnetic and mi-crowave radiation also affect sperm count and motility. Proliferating cells are particularly sensitive to mutagenic agents and the absence of essential metabolites. There-fore, nondividing Sertoli cells, Leydig cells, and reserve stem cells, which demonstrate low mitotic activity, are much less vulnerable than actively dividing, differentiating spermatogenic cells.

1	 FOLDER 22.2 Clinical Correlation: Factors Affecting Spermatogenesis Spermatogenic cells are very sensitive to noxious agents.  Steroid hormones and related medications. Expo- Sertoli cells give structural organization to the tubules as they extend through the full thickness of the seminiferous epithelium.  Spermatogenic cells, which regularly replicate and differentiate into mature sperm. These cells are derived from primordial germ cells originating in the yolk sac that colonize the gonadal ridges during early development of the testis. Spermatogenic cells are organized in poorly defined layers of progressive development between adjacent Sertoli cells (Fig. 22.6). The most immature spermatogenic cells, called spermatogonia, rest on the basal lamina. The most mature cells, called spermatids, are attached to the apical portion of the Sertoli cell, where they border the lumen of the tubule.

1	The tunica (lamina) propria, also called peritubular tissue, is a multilayered connective tissue that lacks typical fibroblasts. In man, it consists of three to five layers of myoid cells (peritubular contractile cells) and collagen fibrils, external to the basal lamina of the seminiferous epithelium (see Fig. 22.6). At the ultrastructural level, myoid cells demonstrate features associated with smooth muscle cells, including a basal lamina and large numbers of actin filaments. They also exhibit a significant amount of rough endoplasmic reticulum (rER), a feature indicating their role in collagen synthesis in the absence of typical fibroblasts. Rhythmic contractions of the myoid cells create peristaltic waves that help move spermatozoa and testicular fluid through the seminiferous tubules to the excurrent duct system. Blood vessels and extensive lymphatic vasculature as well as Leydig cells are present external to the myoid layer.

1	As a normal consequence of aging, the tunica propria increases in thickness. This thickening is accompanied by a decreased rate of sperm production and an overall reduction in the size of the seminiferous tubules. Excessive thickening of the tunica propria earlier in life is associated with infertility. Leydig cells (interstitial cells) are large, polygonal, eosinophilic cells that typically contain lipid droplets (Fig. 22.7). head of

1	Leydig cells (interstitial cells) are large, polygonal, eosinophilic cells that typically contain lipid droplets (Fig. 22.7). head of FIGURE 22.4 • Sagittal section of the human testis. a. This schematic diagram shows a midsagittal section of the human testis. The genital duct system, which includes the tubuli recti, rete testis, efferent ducts, duct of the epididymis, and ductus deferens, is also shown. Note the thick connective tissue covering, the tunica albuginea, and the surrounding tunica vaginalis. (Modified from Dym M. In: Weiss L, ed. Cell and Tissue Biology: A Textbook of Histology, 6th ed. Baltimore: Urban & Schwarzenberg, 1988.) b. Sagittal section of an H&E–stained section of the testis and the head and body of the epididymis. Again note the surrounding tunica albuginea and tunica vaginalis. Only a small portion of the rete testis is visible in this section. Its connection with the excurrent duct system is not evident in the plane of this section. 3.

1	Lipofuscin pigment is also frequently present in these cells as well as distinctive, rod-shaped cytoplasmic crystals, the crystals of Reinke (Fig. 22.8). In routine histologic preparations, these crystals are refractile and measure approximately 3 20 m. Although their exact nature and function remain unknown, they probably represent a protein product of the cell. Like other steroid-secreting cells, Leydig cells have an elaborate smooth endoplasmic reticulum (sER), a feature that accounts for their eosinophilia (see Fig. 22.7). The enzymes necessary for the synthesis of testosterone from cholesterol are associated with the sER. Mitochondria with tubulovesicular cristae, another characteristic of steroid-secreting cells, are also present in Leydig cells.

1	Leydig cells differentiate and secrete testosterone during early fetal life. Secretion of testosterone is required during embryonic development, sexual maturation, and reproductive function:  In the embryo, secretion of testosterone and other andro gens is essential for the normal development of the gonads in the male fetus.  At puberty, secretion of testosterone is responsible for the initiation of sperm production, accessory sex gland secre tion, and development of secondary sex characteristics. In the adult, secretion of testosterone is essential for the maintenance of spermatogenesis and of secondary sex char acteristics, genital excurrent ducts, and accessory sex glands.

1	The Leydig cells are active in the early differentiation of the male fetus and then undergo a period of inactivity beginning at about 5 months of fetal life. Inactive Leydig cells are difficult to distinguish from fibroblasts. When Leydig cells are exposed to gonadotropic stimulation at puberty, they again become androgen-secreting cells and remain active throughout life. Leydig cell tumors represents predominately benign tumors, which occur during two distinct periods (in childhood and in adults between 20 and 60 years old). They are hormonally active and secrete androgens or combination of androgens and estrogens. Commonly, they are composed of uniform cells with all characteristics of steroid hormone-secreting cells containing Reinke crystals. The frst symptom of these benign tumors, besides testicular enlargement, usually is related to abnormal level of hormone production. In prepubertal boys,

1	FIGURE 22.5 • Photomicrographs of human testis. a. This low-magnification photomicrograph of an H&E–stained section of a human testis shows seminiferous tubules and the tunica albuginea. The larger blood vessels are present in the inner aspect of the tunica albuginea. The seminiferous tubules are highly convoluted; thus the profiles that they present in the section are variable in appearance. 30. b. A higher magnification of the previous specimen shows several seminiferous tubules. Note the population of Leydig (interstitial) cells that occur in small clusters in the space between adjoining tubules. 250.

1	FIGURE 22.6 • Schematic drawing of human seminiferous epithelium. This drawing shows the relationship of the Sertoli cells to the spermatogenic cells. The seminiferous epithelium rests on a basal lamina, and a layer of peritubular cells surrounds the seminiferous tubule. The spermatogonia—type A pale, type A dark, and type B pale— and preleptotene spermatocytes are located in the basal compartment of the seminiferous epithelium below the junctional complex, between adjacent Sertoli cells. Pachytene primary spermatocytes, early spermatids, and late spermatids, with partitioning residual cytoplasm that becomes the residual body, are seen above the junctional complex in the abluminal compartment. (Redrawn from Clermont Y. The cycle of the seminiferous epithelium in man. Am J Anat 1963;112:35.) this leads to sexual precocity (unexpected pubertal changes in early age), whereas in adults it may be observed as feminization (development of female sexual characteristics) and gynecomastia

1	this leads to sexual precocity (unexpected pubertal changes in early age), whereas in adults it may be observed as feminization (development of female sexual characteristics) and gynecomastia (development of breast in males).

1	Spermatogenesis is the process by which spermatogonia develop into sperm. Spermatogenesis, the process by which sperm are produced, involves a complex and unique series of events. It begins shortly before puberty, under the influence of rising levels of pituitary gonadotropins, and continues throughout life. For descriptive purposes, spermatogenesis is divided into three distinct phases:  Spermatogonial phase, in which spermatogonia divide by mitosis to replace themselves as well as provide a popu lation of committed spermatogonia that will eventually

1	FIGURE 22.7 • Electron micrograph of Leydig cells. This electron micrograph shows portions of several Leydig cells. The cytoplasm contains an abundance of sER, a characteristic of Leydig cells. Other features characteristic of the Leydig cell seen in the lower-power micrograph are the numerous lipid droplets (L), the segmented profiles of the Golgi apparatus (G), and the presence of variable numbers of lysosomes (Ly). Occasional profiles of rER are also seen. Note also the presence of microvilli along portions of the cell surface (arrows). M, cytoplasm of adjacent macrophage. 10,000. Inset. sER at higher magnification. The very dense particles are glycogen. 60,000.  Spermatocyte phase (meiosis), in which primary sper matocytes undergo two meiotic divisions to reduce both the chromosome number and amount of DNA to produce haploid cells called spermatids Spermatid phase (spermiogenesis), in which sper

1	At the end of spermatogenesis, spermatids undergo their final maturation and are released during a process called spermiation from the supporting Sertoli cells into the lumen of the seminiferous tubule. In the spermatogonial phase, stem cells divide to replace themselves and provide a population of committed spermatogonia. Spermatogonial stem cells undergo multiple divisions and produce spermatogonial progeny that display differences in nuclear appearance in routine H&E preparations. FIGURE 22.8 • Electron micrograph of a Reinke crystal. This electron micrograph shows the internal structure of a Reinke crystal in the cytoplasm of a human Leydig cell. Also note the sER (arrows) and a lipid droplet (L) in the cytoplasm. 16,000. (Courtesy of Dr. Don F. Cameron.)

1	Human spermatogonia are classified into three types based on the appearance of the nuclei in routine histologic preparations:  Type A dark (Ad) spermatogonia have ovoid nuclei with intensely basophilic, finely granular chromatin. These spermatogonia are thought to be the stem cells of the seminiferous epithelium. They divide at irregular intervals to give rise to either a pair of type Ad spermatogonia that remain as stem cells or to a pair of type Ap spermatogonia.  Type A pale (Ap) spermatogonia have ovoid nuclei with lightly staining, finely granular chromatin. Ap spermatogonia are committed to the differentiation process that produces the sperm. They undergo several successive mitotic divisions, thereby increasing their number.  Type B spermatogonia have generally spherical nuclei with chromatin that is condensed into large clumps along the nuclear envelope and around a central nucleolus (see Fig. 22.6).

1	 Type B spermatogonia have generally spherical nuclei with chromatin that is condensed into large clumps along the nuclear envelope and around a central nucleolus (see Fig. 22.6). An unusual feature of the division of an Ad spermatogonium into two type Ap spermatogonia is that the daughter cells remain connected by a thin cytoplasmic bridge. This same phenomenon occurs through each subsequent mitotic and meiotic division of the progeny of the original pair of Ap spermatogonia (Fig. 22.9). Thus, all of the progeny of an initial pair of Ap spermatogonia are connected, much like a strand of pearls. These cytoplasmic connections remain intact to the last stages of spermatid maturation and are essential for the synchronous development of each clone from an original pair of Ap cells. After several divisions, type A spermatogonia differentiate into type B spermatogonia. The appearance of type B spermatogonia represents the last event in the spermatogonial phase.

1	After several divisions, type A spermatogonia differentiate into type B spermatogonia. The appearance of type B spermatogonia represents the last event in the spermatogonial phase. In the spermatocyte phase, primary spermatocytes undergo meiosis to reduce both the chromosome number and the amount of DNA. The mitotic division of type B spermatogonia produces primary spermatocytes. They replicate their DNA shortly after they form and before meiosis begins, so that each primary spermatocyte contains the normal chromosomal number (2n) and double the amount of DNA (4d ). Each chromosome consists of two sister chromatids; hence, 4d amount of DNA.

1	Meiosis I results in reduction of both the number of chromosomes (from 2n to 1n) and the amount of DNA to the haploid condition (from 4d to 2d); thus, secondary spermatocyte is characterized by haploid number of chromosomes (1n) and 2d amount of DNA. Because no DNA replication precedes meiosis II, after this division each spermatid has the haploid (1n) number of chromosomes, each containing a single chromatid (1d). Meiosis is described in detail in Chapter 3 (see page 89); a brief description of spermatocyte meiosis follows. Prophase of the first meiotic division, during which the chromatin condenses into visible chromosomes, lasts up to 22 days in human primary spermatocytes. At the end of prophase, 44 autosomes and an X and a Y chromosome, each having two chromatin strands (chromatids), can be identified. Homologous chromosomes are paired as they line up on the metaphase plate.

1	The paired homologous chromosomes, called tetrads because they consist of four chromatids, exchange genetic material in a process called crossing-over. During this exchange, the four chromatids rearrange into a tripartite structure called a synaptonemal complex. This process ensures genetic diversity. Through genetic exchange, the four spermatids produced from each spermatocyte differ from each other and from every other spermatid. After crossing-over is complete, the homologous chromosomes separate and move to the opposite poles of the meiotic spindle. Thus, the tetrads, which have been modified by crossing-over, separate and become dyads again. The two chromatids of each original chromosome (although modified by crossing-over) remain together. This is just the opposite of what happens in mitosis, in which the paired chromatids—one representing the “template” and the other, newly synthesized DNA—separate.

1	FIGURE 22.9 • Schematic diagram illus trating the generations of spermato genic cells. This diagram shows the clonal nature of the successive generations of spermatogenic cells. Cytoplasmic division is complete only in the primitive type A dark spermatogonia that serve as stem cells. All other spermatogenic cells remain connected by intercellular bridges as they undergo mitotic and meiotic division and differentiation of the spermatids. The cells separate into individual spermatozoa as they are released from the seminiferous epithelium. The residual bodies remain connected and are phagocytosed by the Sertoli cells. (Reprinted with permission from Dym M, Fawcett DW. Further observations on the numbers of spermatogonia, spermatocytes, and spermatids connected by intercellular bridges in the mammalian testis. Biol Reprod 1971;4:195–215.)

1	The movement of a particular chromosome of a homologous pair to either pole of the spindle is random (i.e., maternally derived chromosomes and paternally derived chromosomes do not sort themselves out at the metaphase plate). This random sorting is another source of genetic diversity in the resulting sperm.

1	The cells derived from the first meiotic division are called secondary spermatocytes. These cells immediately enter the prophase of the second meiotic division without synthesizing new DNA (i.e., without passing through an S phase; see pages 89–93). Each secondary spermatocyte has a reduced number of chromosomes to (1n), which is represented by 22 autosomes and an X or a Y chromosome. Each of these chromosomes consists of two sister chromatids. The secondary spermatocyte has the (2d), diploid amount of DNA. During metaphase of the second meiotic division, the chromosomes line up at the metaphase plate, and the sister chromatids separate and move to opposite poles of the spindle. As the second meiotic division is completed and the nuclear membranes re-form, two haploid spermatids, each containing 23 single-stranded chromosomes (1n) and the (1d) amount of DNA, are formed from each secondary spermatocyte (Fig. 22.10).

1	In the spermatid phase, spermatids undergo extensive cell remodeling as they differentiate into mature sperm. Each spermatid that results from the second meiotic division is haploid in DNA content (1d) and chromosome number (1n) represented by 22 autosomes and an X or Y chromosome. No further division occurs. The haploid spermatids undergo a differentiation process that produces mature sperm, which are also haploid. The normal diploid condition is restored when a sperm fertilizes an oocyte. The extensive cell remodeling that occurs during differentiation of the spermatid population into mature sperm (spermiogenesis) consists of four phases. These phases occur while the spermatids are physically attached to the Sertoli cell plasma membrane by specialized junctions. The morphologic changes in all four phases that occur during spermiogenesis are described here and summarized in Figure 22.11.

1	 Golgi phase. This phase is characterized by the presence of in the multiple Golgi complexes of the spermatid. These two pairs of homologous chromosomes G1 phase G2 phase *note: prophase II, anaphase II, and telophase II are not shown ovum FIGURE 22.10 • Comparison of mitosis and meiosis in a spermatogonial cell. The two pairs of chromosomes (2n) of maternal and paternal origin are depicted in red and blue, respectively. The mitotic division produces daughter cells that are genetically identical to the parental (2n) cell. The meiotic division, which has two components, a reductional division and an equatorial division, produces a cell that has only half the number of chromosomes (n). In addition, during the chromosome pairing in prophase I of meiosis, chromosome segments are exchanged, crossing-over, creating genetic diversity. In humans, the first polar body does not divide, but it does so in other species. *Note that prophase II, anaphase II, and telophase II are not shown.

1	Golgi acrosomal vesicle acrosome manchette flagellum centriole sheath FIGURE 22.11 • Schematic diagram of spermiogenesis in the human. The basic changes in the structure of the key organelles of the spermatid are illustrated (see text for detailed explanation). (Modified from Dym M. In: Weiss L, ed. Cell and Tissue Biology: A Textbook of Histology, 6th ed. Baltimore: Urban & Schwarzenberg, 1988.) proacrosomal granules, rich in glycoproteins, coalesce into a membrane-bounded vesicle, the acrosomal vesicle, adjacent to the nuclear envelope. The vesicle enlarges and its contents increase during this phase. The position of the acrosomal vesicle determines the anterior pole of the developing sperm. Also during this phase, the centrioles migrate from the juxtanuclear region to the posterior pole of the spermatid, where the mature centriole aligns at right angles to the plasma membrane. The centriole initiates the assembly of the nine peripheral microtubule doublets and two central

1	pole of the spermatid, where the mature centriole aligns at right angles to the plasma membrane. The centriole initiates the assembly of the nine peripheral microtubule doublets and two central micro-tubules that constitute the axoneme of the sperm tail.

1	 Cap phase. In this phase, the acrosomal vesicle spreads over the anterior half of the nucleus. This reshaped structure is called the acrosomal cap. The portion of the nuclear envelope beneath the acrosomal cap loses its pores and becomes thicker. The nuclear contents also condense.  Acrosome phase. In this phase, the spermatid reorients itself so that the head becomes deeply embedded in the Sertoli cell and points toward the basal lamina. The developing flagellum extends into the lumen of the seminiferous tubule. The condensed nucleus of the spermatid flattens and elongates, the nucleus and its overlying acrosome also move to a position immediately adjacent to the anterior plasma membrane, and the cytoplasm is displaced posteriorly. The cytoplasmic microtubules become organized into a cylindrical sheath, the manchette, which extends from the posterior rim of the acrosome toward the posterior pole of the spermatid.

1	The centrioles, which had earlier initiated the development of the flagellum, now move back to the posterior surface of the nucleus where the immature centriole becomes attached to a shallow groove in the nucleus. They are then modified to form the connecting piece, or neck region, of the developing sperm. Nine coarse fibers develop from the centrioles attached to the nucleus and extend into the tail as the outer dense fibers peripheral to the microtubules of the axoneme. These fibers unite the nucleus with the flagellum; hence, the name connecting piece.

1	As the plasma membrane moves posteriorly to cover the growing fagellum, the manchette disappears, and the mitochondria migrate from the rest of the cytoplasm to form a tight, helically wrapped sheath around the coarse fibers in the neck region and its immediate posterior extension (Fig. 22.12). This region is the middle piece of the tail of the sperm. Distal to the middle piece, a fbrous sheath consisting of two longitudinal columns and numerous connecting ribs surrounds the nine longitudinal fibers of the principal piece and extends nearly to the end of the flagellum. This short segment of the tail distal to the fibrous sheath is called the end piece.  Maturation phase. This last phase of spermatid remodel ing reduces excess cytoplasm from around the flagella to form mature spermatozoon. The Sertoli cells then phagocytose this excess cytoplasm, also termed the resid ual body. The intercellular bridges that have characterized the developing gametes since the prespermatocyte stages

1	The Sertoli cells then phagocytose this excess cytoplasm, also termed the resid ual body. The intercellular bridges that have characterized the developing gametes since the prespermatocyte stages remain with the residual bodies. Spermatids are no longer attached to each other and are released from the Sertoli cells.

1	Spermatids are released into the lumen of the seminiferous tubules during the process called spermiation. Toward the end of maturation phase of spermatogenesis, elongated spermatids are released from Sertoli cells into the lumen of seminiferous tubule. This complex process, called spermiation involves progressive removal of specialized Sertoli-to-spermatid junctional complexes and disengagement of spermatids from the Sertoli cell. The presence of the 1-integrins in the Sertoli-to-spermatid junctions, as well as increased activity of the integrin-linked kinase at the time of spermiation, suggests an enzymatic control of spermatid release. The rate of spermiation in the testis determines the number of sperm cells in the ejaculate of semen. Various pharmacologic treatments, toxic agents, and gonadotropin suppression result in spermiation failure, in which spermatids are not released but instead are retained and phagocytosed by the Sertoli cell. Structure of the Mature Sperm

1	Structure of the Mature Sperm The events of spermiogenesis result in a structurally unique cell. The mature human sperm is about 60 m long. The sperm head is flattened and pointed and measures 4.5 m long by 3 m wide by 1 m thick (see Fig. 22.12). The acrosomal cap that covers the anterior two-thirds of the nucleus contains hyaluronidase, neuraminidase, acid phosphatase, and a trypsin-like protease called acrosin. These acrosomal enzymes are essential for penetration of the zona pellucida of the outer dense fibers 4, 5, 6 outer doublets of axonemal complex central pair of microtubules of axonemal complex FIGURE 22.12 • Diagram of a human spermatozoon. Regions of the spermatozoon are indicated on the left. Key structural features of the head (viewed in frontal and sagittal planes), the middle piece, and the principal piece of the spermatozoon are illustrated on the right. (Modified from Pederson PL, Fawcett DW. In: Hafez ESE, ed. Human Semen and Fertility Regulation in the

1	Male. St. Louis: CV Mosby, 1976.) ovum. The release of acrosomal enzymes as the sperm touches the egg is the first step in the acrosome reaction. This complex process facilitates sperm penetration and subsequent fertilization and prevents the entry of additional sperm into the ovum. The sperm tail is subdivided into the neck, the middle piece, the principal piece, and the end piece. The short neck contains the centrioles and the origin of the coarse fibers. The middle piece is approximately 7 m long and contains the mitochondria, helically wrapped around the coarse fibers and the axonemal complex. These mitochondria provide the energy for movement of the tail and thus are responsible for the motility of the sperm. The principal piece is approximately 40 m long and contains the fibrous sheath external to the coarse fibers and the axonemal complex. The end piece, approximately the last 5 m of the flagellum in the mature sperm, contains only the axonemal complex.

1	Newly released sperm cells are processed in the epididymidis where they acquire motility and undergo further maturation.

1	Newly released sperm cells are nonmotile and are carried from the seminiferous tubules in a fluid secreted by the Sertoli cells. The fluid and sperm flow through the seminiferous tubules, facilitated by peristaltic contractions of the peritubular contractile cells of the lamina propria. They then enter the straight tubules, a short segment of the seminiferous tubule where the epithelium consists only of Sertoli cells. At the mediastinum testis, the fluid and sperm enter the rete testis, an anastomosing system of ducts lined by simple cuboidal epithelium (Plate 87, page 820). From the rete testis, they move into the extratesticular portion of the efferent ductules (ductuli efferentes), the first part of the excurrent duct system, and then into the proximal portion of the duct of the epididymis (ductus epididymis). As the sperm cells move through the 4 to 5 m of the highly coiled duct of the epididymis, they acquire motility and undergo several maturational changes,. These changes

1	(ductus epididymis). As the sperm cells move through the 4 to 5 m of the highly coiled duct of the epididymis, they acquire motility and undergo several maturational changes,. These changes include  condensation of nuclear DNA. The head of the sperm decreases in size.  further reduction of cytoplasm. The sperm cells become more slender.  changes in plasma membrane lipids, proteins, and glycosy lation.  alterations in the outer acrosomal membrane (decapacita tion). The surface-associated decapacitation factor is added to inhibit the fertilizing ability of the sperm cells (page 803).

1	Initiation of sperm cell motility during cell transit through the epididymis is most likely related to changes in the intracellular levels of cyclic adenosine monophosphate (cAMP), calcium ions (Ca2 ), and intracellular pH. These factors regulate flagellar activity through changes in protein phosphorylation, resulting from activities of protein kinases and protein phosphatases. For instance, pharmacologic stimulation of protein kinase A activity increases motility of sperm cells, whereas inhibition of protein phosphatase activity may initiate or stimulate such motility. This suggests that phosphatases have an important role in the regulation of sperm kinetic activity. Contractions of the smooth muscle that surrounds the progressively distal and larger ducts continue to move the sperm by peristaltic action until they reach the distal portion of the duct of the epididymis, where they are stored before ejaculation.

1	Sperm can live for several weeks in the male excurrent duct system, but they will survive only 2 to 3 days in the female reproductive tract. They acquire the ability to fertilize the ovum only after some time in the female tract. This process, which involves removal and replacement of glycocalyx components (glycoconjugates) on the sperm membrane, is called capacitation. Capacitation of spermatozoa is described in detail in Chapter 23 (page 840). Cycle of the Seminiferous Epithelium

1	Cycle of the Seminiferous Epithelium Differentiating spermatogenic cells are not arranged at random in the seminiferous epithelium; specific cell types are grouped together. These groupings or associations occur because intercellular bridges are present between the progeny of each pair of type Ap spermatogonia and because the synchronized cells spend specific times in each stage of maturation. All phases of differentiation occur sequentially at any given site in a seminiferous tubule as the progeny of stem cells remain connected by cytoplasmic bridges and undergo synchronous mitotic and meiotic divisions and maturation (see Fig. 22.10).

1	Each recognizable grouping, or cell association, is considered a stage in a cyclic process. The series of stages that appears between two successive occurrences of the same cell association pattern at any given site in the seminiferous tubule constitutes a cycle of the seminiferous epithelium. The cycle of the seminiferous epithelium has been most thoroughly studied in rats, in which 14 successive stages occur in linear sequence along the tubule. In man, six stages or cell associations are defined in the cycle of the seminiferous epithelium (Fig. 22.13). These stages are not as clearly delineated as those in rodents because in man the cellular associations occur in irregular patches that form a mosaic pattern. Duration of spermatogenesis in humans is approximately 74 days.

1	After injecting a pulse of tritiated thymidine, a specific generation of cells can be followed by sequential biopsies of the seminiferous tubules. In this way, the time required for the labeled cells to go through the various stages can be determined. Several generations of developing cells may be present in the thickness of the seminiferous epithelium at any given site and at any given time, which produces the characteristic cell associations. Autoradiographic studies have revealed that the duration of the cycle of the seminiferous epithelium is constant, lasting about 16 days in humans. In humans it would require about 4.6 cycles (each 16 days long), or approximately 74 days, for a spermatogonium produced by a stem cell to complete the process of spermatogenesis. It would then require approximately 12 days for the spermatozoon to pass through the epididymis. Approximately 300 million sperm cells are produced daily in the human testis. The length of the cycle and the time required

1	approximately 12 days for the spermatozoon to pass through the epididymis. Approximately 300 million sperm cells are produced daily in the human testis. The length of the cycle and the time required for spermatogenesis are constant and specific in each species. Therefore, in any pharmacologic intervention (e.g., therapy for male infertility), if a drug is given that affects the initial phases of spermatogenesis, approximately 86 days are required to see the effect of that compound on sperm production.

1	Waves of the Seminiferous Epithelium

1	As indicated above, the cycle of the seminiferous epithelium describes changes that occur with time at any given site in the tubule. In addition, the wave of the seminiferous epithelium describes the distribution of patterns of cellular association (stages) along the length of the tubule. In rodents and other mammals that have been studied, including subhuman primates, each stage occupies a significant length of the seminiferous tubule, and the stages appear to occur sequentially along the length of the tubule. In the rat, there are approximately 12 waves in each tubule. A transverse section through the tubule usually reveals only one pattern of cell associations. There are no waves in the human seminiferous epithelium. Each pattern of cellular associations (stage of the cycle) has a patch-like distribution in the human seminiferous tubule (Fig. 22.14). Patches do not extend around the circumference of the tubule, nor are they in sequence. Therefore, a transverse section through a

1	distribution in the human seminiferous tubule (Fig. 22.14). Patches do not extend around the circumference of the tubule, nor are they in sequence. Therefore, a transverse section through a human seminiferous tubule may reveal as many as six different stages of the cycle arranged in a pie-wedge fashion around the circumference of the tubule.

1	FIGURE 22.13 • Schematic drawing of the stages of the human seminiferous epithelium. This diagram shows each of the six recognizable cell associations that occur in the cycle of the human seminiferous epithelium. Sa, Sb, Sc, and Sd indicate spermatids in various steps of differentiation. (Based on Clermont Y. The cycle of the seminiferous epithelium in man. Am J Anat 1963;112:50.) FIGURE 22.14 • Diagram of organization of seminiferous epithelium in humans and other species. a. In subhuman species, a particular cellular association occupies varying lengths along the tubule. Therefore, in a typical cross section only a single cellular association is observed. b. In humans, cellular associations occur in irregularly shaped areas along the tubule, and therefore, a cross section typically shows two or more cellular associations. (Modified from Dym M. In: Weiss L, ed. Cell and Tissue Biology: A Textbook of Histology, 6th ed. Baltimore: Urban & Schwarzenberg, 1988.)

1	Sertoli cells constitute the true epithelium of the seminiferous epithelium. Sertoli cells (sustentacular cells) are tall, columnar, non-replicating epithelial cells that rest on the thick, multilayered basal lamina of the seminiferous epithelium (Fig. 22.15). They are the supporting cells for the developing sperm that attach to their surface after meiosis. Sertoli cells contain an extensive sER, a well-developed rER, and stacks of annulate lamellae. They have numerous spherical and elongated mitochondria, a well-developed Golgi apparatus, and varying numbers of microtubules, lysosomes, lipid droplets, vesicles, glycogen granules, and filaments. A sheath of 7to 9-nm filaments surrounds the nucleus and separates it from other cytoplasmic organelles.

1	The euchromatic Sertoli cell nucleus, a reflection of this very active cell, is generally ovoid or triangular and may have one or more deep infoldings. Its shape and location vary. It may be flattened, lying in the basal portion of the cell near and parallel to the base of the cell, or it may be triangular or ovoid, lying near or some distance from the base of the cell. In some species, the Sertoli cell nucleus contains a unique tripartite structure that consists of an RNA-containing nucleolus flanked by a pair of DNA-containing bodies called karyosomes (Fig. 22.16).

1	In man, characteristic inclusion bodies (of Charcot-Btcher) are found in the basal cytoplasm. These slender fusiform crystalloids measure 10 to 25 m long by 1 m wide and are visible in routine histologic preparations. With transmission electron microscopy, they are resolved as bundles of poorly ordered, parallel or converging, straight, dense 15-nmdiameter filaments (see Fig. 22.15). Their chemical composition and function are unknown; however, recent studies detected an accumulation of lipoprotein receptor (CLA-1) proteins. This suggests that inclusion bodies might be involved in lipids transport and their utilization by the Sertoli cells. FIGURE 22.15 • Electron micrograph of a human Sertoli cell.

1	FIGURE 22.15 • Electron micrograph of a human Sertoli cell. This electron micrograph shows characteristic crystalloid inclusion bodies of Charcot-Btcher in the basal cytoplasm of the Sertoli cell. The basal lamina (BL) is indicated for orientation. 9,000. Inset. This higher magnification shows filaments of the crystalloid. 27,000. (Courtesy of Dr. Don F. Cameron.) The Sertoli cell–to–Sertoli cell junctional complex consists of a structurally unique combination of membrane and cytoplasmic specializations.

1	The Sertoli cell–to–Sertoli cell junctional complex consists of a structurally unique combination of membrane and cytoplasmic specializations. Sertoli cells are bound to one another by an unusual Sertoli cellto-Sertoli cell junctional complex (Fig. 22.17). This complex is characterized, in part, by an exceedingly tight junction (zonula occludens) that includes more than 50 parallel fusion lines in the adjacent membranes. In addition, two cytoplasmic components characterize this unique junctional complex:  A fattened cisterna of sER lies parallel to the plasma membrane in the region of the junction in each cell. Actin flament bundles, hexagonally packed, are inter posed between the sER cisternae and the plasma membranes.

1	FIGURE 22.16 • Schematic drawing of the Sertoli cell and its relationship to adjacent spermatogenic cells. This drawing shows the Sertoli–to–Sertoli junctional specialization between adjacent Sertoli cells and the Sertoli–to–spermatid junctional specialization between the Sertoli cell and late spermatids. The Sertoli–to–Sertoli junctional complex is an adhesion device that includes a tight junction that contributes to the blood–testis barrier. The junctional specialization between the Sertoli cell and late spermatids residing in deep recesses within the apical cytoplasm is an adhesion device only. Lateral processes of the Sertoli cells extend over the surface of the spermatocytes and spermatids. Note the ultrastructural features of the Sertoli cell, including the microtubule arrays and characteristic shape of the nucleus and its karyosome. (Reprinted with permission from Bloom W, Fawcett DW. A Textbook of Histology. Philadelphia: WB Saunders, 1975.)

1	Philadelphia: WB Saunders, 1975.) A similar-appearing junctional complex in the Sertoli cell is also present at the site where the spermatids are attached. However, no tight junction is present, and the spermatid lacks flattened cisternae of sER and actin filament bundles (see Figs. 22.16 and 22.17). Other junctional specializations of the Sertoli cells include gap junctions between Sertoli cells, desmosome-like junctions between Sertoli cells and early-stage spermatogenic cells, and hemidesmosomes at the Sertoli cell–basal lamina interface. The Sertoli cell–to–Sertoli cell junctional complex divides the seminiferous epithelium into basal and luminal compartments.

1	The Sertoli cell–to–Sertoli cell junctions establish two epithelial compartments, a basal epithelial compartment and a luminal compartment. Spermatogonia and early primary spermatocytes are restricted to the basal compartment (i.e., between the Sertoli cell–to–Sertoli cell junctions and the basal lamina). More mature spermatocytes and spermatids are restricted to the luminal side of the Sertoli cell–to–Sertoli cell junctions. Early spermatocytes produced by mitotic division of type B spermatogonia must pass through the junctional complex to move from the basal compartment to the luminal compartment. This movement occurs via the formation of a new junctional complex between Sertoli cell processes that extend beneath the newly formed spermatocytes, followed by the breakdown of the junction above them. Thus, in the differentiation of the spermatogenic cells, the processes of meiosis and spermiogenesis occur in the luminal compartment.

1	In both compartments, spermatogenic cells are surrounded by complex processes of the Sertoli cells. Because of the unusually close relationships between Sertoli cells and differentiating spermatogenic cells, it has been suggested that Sertoli cells serve as “nurse,” or supporting, cells (i.e., they function in the exchange of metabolic substrates and wastes between the developing spermatogenic cells and the circulatory system). In addition, Sertoli cells phagocytose and break down the residual bodies formed in the last stage of spermiogenesis. They also phagocytose any spermatogenic cells that fail to differentiate completely. The Sertoli cell–to–Sertoli cell junctional complex is the site of the blood–testis barrier. In addition to the physical compartmentalization described above, the Sertoli cell–to–Sertoli cell junctional complex also FIGURE 22.17 • Electron micrograph of Sertoli cell junctions.

1	This electron micrograph demonstrates a Sertoli–to–Sertoli junctional complex and, in close proximity, a Sertoli–to–spermatid junctional specialization. Condensation and shaping of the spermatid nucleus (N) are well advanced. The acrosome (A) of the spermatid appears as a V-shaped profile, and in close association with it is the Sertoli cell junctional specialization characterized by bundles of microfilaments that are cut in cross section (arrows). The associated profile of endoplasmic reticulum resides immediately adjacent to the microfilament bundles. The Sertoli–to–Sertoli junction lies below, joining one Sertoli cell (S1) to the adjacent Sertoli cell (S2). The arrowheads indicate the limits of the junction. Note that the junction here reveals the same elements, the microfilament bundles (arrows) and a profile of endoplasmic reticulum, as are seen in the Sertoli–to–spermatid junctional specialization. Not evident at this magnification is the tight junction associated with the

1	bundles (arrows) and a profile of endoplasmic reticulum, as are seen in the Sertoli–to–spermatid junctional specialization. Not evident at this magnification is the tight junction associated with the Sertoli–to–Sertoli junctional complex. 30,000.

1	creates a permeability barrier called the blood–testis barrier. This barrier is essential in creating a physiologic compartmentalization within the seminiferous epithelium with respect to ionic, amino acid, carbohydrate, and protein composition. Therefore, the composition of the fluid in the seminiferous tubules and excurrent ducts differs considerably from the composition of the blood plasma and testicular lymph. Plasma proteins and circulating antibodies are excluded from the lumen of the seminiferous tubules. The exocrine secretory products of the Sertoli cells (particularly the androgen-binding protein (ABP), which has a high binding affinity for testosterone and DHT) are highly concentrated in the lumen of the seminiferous tubules and maintain a high concentration of testosterone, which provides a favorable microenvironment for the differentiating spermatogenic cells.

1	Most important, the blood–testis barrier isolates the genetically different and therefore antigenic haploid germ cells (secondary spermatocytes, spermatids, and sperm) from the immune system of the adult male. Antigens produced by, or specific to, the sperm are prevented from reaching the systemic circulation. Conversely, -globulins and specific sperm antibodies found in some individuals are prevented from reaching the developing spermatogenic cells in the seminiferous tubule (Folder 22.3). Therefore, the blood–testis barrier serves an essential role in isolating the spermatogenic cells from the immune system. Sertoli cells have both exocrine and endocrine secretory functions.

1	Sertoli cells have both exocrine and endocrine secretory functions. In addition to secreting fluid that facilitates passage of the maturing sperm along the seminiferous tubules to the intratesticular ducts, Sertoli cells produce critical factors necessary for the successful progression of spermatogonia into spermatozoa. They secrete a 90-kilodalton androgen-binding protein (ABP). ABP concentrates testosterone in the luminal compartment of the seminiferous tubule, where high concentrations of testosterone are essential for normal maturation of the developing sperm.

1	FSH and testosterone receptors are present on Sertoli cells; therefore, their secretory function is regulated by both FSH and testosterone (Fig. 22.18). Sertoli cells secrete several endocrine substances, such as inhibin, a 32-kilodalton glycoprotein hormone involved in the feedback loop that inhibits follicle-stimulating hormone (FSH) release from the anterior pituitary gland. In addition, Sertoli cells synthesize plasminogen activator, which converts plasminogen to the active proteolytic hormone plasmin, transferrin (an iron-transporting protein), and ceruloplasmin (a copper-transporting protein). Furthermore, the Sertoli cells secrete other glycoproteins that function as growth factors or paracrine factors, such as the Mlerian-inhibiting factor (MIF), stem cell factor (SCF), and glial cell line-derived neurotrophic factor (GDNF).

1	At the end of each seminiferous tubule there is an abrupt transition to the straight tubules, or tubuli recti. This short terminal section of the seminiferous tubule is lined only by Sertoli cells (Plate 87, page 820). Near their termination, the straight tubules narrow, and their lining changes to a simple cuboidal epithelium. The straight tubules empty into the rete testis, a complex series of interconnecting channels within the highly vascular connective tissue of the mediastinum (Fig. 22.19). A simple cuboidal or low columnar epithelium lines the channels of the rete testis. These cells have a single apical cilium and relatively few short apical microvilli.

1	logic importance of the blood–testis barrier:  Spermatozoa and spermatogenic cells possess molecules that are unique to these cells and are recognized as “foreign” (not self) by the immune system.  Spermatozoa are first produced at puberty, long after the individual has become immunocompetent (i.e., capa-ble of recognizing foreign molecules and producing an-tibodies against them). Failure of the spermatogenic cells and spermatozoa to remain isolated results in the production of sperm-specific seen after vasectomy and in some cases of infertility. After vasectomy, sperm-specific antibodies are pro-duced as the cells of the immune system are exposed to the spermatozoa that may leak from the severed ductus deferens. Thus, sperm no longer remain isolated from the immune system within the reproductive tract. In some cases of infertility, sperm-specific antibodies have been found in the semen. These antibodies cause the sperm to agglutinate, preventing movement and interaction with the

1	reproductive tract. In some cases of infertility, sperm-specific antibodies have been found in the semen. These antibodies cause the sperm to agglutinate, preventing movement and interaction with the ovum.

1	 FOLDER 22.3 Clinical Correlation: Sperm-Specific Antigens and the Immune Response Two basic facts are well established about the immuno-antibodies. Such an immune response is sometimes The excurrent duct system develops from the mesonephric (Wolffian) duct and mesonephric tubules.

1	The excurrent duct system develops from the mesonephric (Wolffian) duct and mesonephric tubules. The initial development of Leydig cells and initiation of testosterone secretion stimulate the mesonephric (Wolffian) duct to differentiate into the excretory duct system for the developing testis (Fig. 22.20). The portion of the mesonephric duct adjacent to the developing testis becomes convoluted and differentiates into the duct of the epididymis. In addition, a number (about 20) of the remaining mesonephric tubules in this region make contact with the developing seminiferous cords and finally develop into the efferent ductules (Fig. 22.21 and Plate 88, page 822). They connect the developing rete testis with the duct of the epididymis. The distal part of the mesonephric duct acquires a thick, smooth muscle coat and becomes the ductus deferens. The end of the distal mesonephric duct gives rise to the ejaculatory duct and seminal vesicles.

1	The efferent ductules are lined with pseudostratified columnar epithelium. In man, approximately 20 efferent ductules connect the channels of the rete testis at the superior end of the mediastinum to the proximal portion of the duct of the epididymis. As the efferent ductules exit the testis, they become highly coiled and form 6 to 10 conical masses, the coni vasculosi, whose bases form part of the head of the epididymis. The coni vasculosi, each about 10 mm in length, contain the highly convoluted ducts that measure 15 to 20 cm in length. At the base of the cones, the efferent ducts open into a single channel, the duct of the epididymis (see Fig. 22.4).

1	The efferent ductules are lined with a pseudostratified columnar epithelium that contains clumps of tall and short cells, giving the luminal surface a sawtooth appearance (see Fig. 22.21). Interspersed among the columnar cells are occasional basal cells that serve as epithelial stem cells. The tall columnar cells are ciliated. The short nonciliated cells have numerous microvilli and canalicular invaginations of the apical surface as well as numerous pinocytotic vesicles, membrane-bounded dense bodies, lysosomes, and other cytoplasmic structures associated with endocytotic activity. Most of the fluid secreted in the seminiferous tubules is reabsorbed in the efferent ductules.

1	Most of the fluid secreted in the seminiferous tubules is reabsorbed in the efferent ductules. A smooth muscle layer in the excurrent ducts first appears at the beginning of the efferent ductules. The smooth muscle cells form a layer several cells thick in which the cells are arrayed as a circular sheath in the wall of the ductule. Interspersed among the muscle cells are elastic fibers. Transport of the sperm in the efferent ductules is affected largely by both ciliary action and contraction of this fibromuscular layer. The epididymis is an organ that contains the efferent ductules and the duct of the epididymis.

1	The epididymis is a crescent-shaped structure that lies along the superior and posterior surfaces of the testis. It measures about 7.5 cm in length and consists of the efferent ductules and the duct of the epididymis and associated vessels, smooth muscles, and connective tissue coverings (Fig. 22.22 and Plate 88, page 822). The duct of the epididymis is a highly coiled tube measuring 4 to 6 m in length. The epididymis is divided into a head, a body, and a tail (see Fig. 22.4). The efferent ductules occupy the head, and the duct of the epididymis occupies the body and tail. Newly produced sperm, which enter the epididymis from the testis, mature during their passage through the duct of the epididymis, acquiring motility and the ability to fertilize an oocyte. During this androgen-dependent maturation process, the head of the sperm is modified by the addition of surface-associated decapacitation factor containing epididymal fluid glycoconjugates. This process, called decapacitation,

1	maturation process, the head of the sperm is modified by the addition of surface-associated decapacitation factor containing epididymal fluid glycoconjugates. This process, called decapacitation, inhibits the fertilizing ability of the sperm in a reversible inhibitory adenohypophysis testosterone stimulatory GnRH-like peptides spermatogenic cells Leydig cells Sertoli cells ? hypothalamus other CNS centers GnRH FSH LH prolactin inhibin estradiol DHT

1	FIGURE 22.18 • Diagram depicting the hormonal regulation of male reproductive function. Blue arrows indicate stimulatory action on the system; red arrows indicate inhibitory feedback. See text for explanation.

1	FIGURE 22.19 • Photomicrograph of human testis. a. This H&E–stained specimen shows the site that includes the mediastinum of the testis. On the right are seminiferous tubules, and on the left are the anastomosing channels of the rete testis. The arrow indicates termination of a straight tubule that is lined only by Sertoli cells. It is at this site that the tubule content enters the rete testis and the channels are then lined by a simple cuboidal epithelium. 70. b. This higher magnification from a slightly deeper section of the same specimen shows the rete testis (left), a cross section of a seminiferous tubule (upper right), and a terminating straight tubule (asterisk) where it is entering the rete testis. Note the abrupt change in the epithelial lining at this site. As noted, the lining epithelium of the rete testis is simple cuboidal. 275.

1	extend from the luminal surface of the principal cells (Plate 88, page 822). The stereocilia vary in height from 25 m in the head to approximately 10 m in the tail.  Basal cells that represent small, round cells resting on the basal lamina. They are the stem cells of the duct epithelium. In addition, migrating lymphocytes called halo cells are often found within the epithelium. Under normal conditions, the epithelium of the epididymis represents the most proximal level of the excurrent duct system in which lymphocytes are present. Epididymal cells function in both absorption and secretion.

1	Epididymal cells function in both absorption and secretion. Most of the fluid that is not reabsorbed by the efferent ductules is reabsorbed in the proximal portion of the epididymis. The epithelial cells also phagocytose any residual bodies not removed by the Sertoli cells as well as sperm that degenerate in the duct. The apical cytoplasm of the principal cells contains numerous invaginations at the bases of the stereocilia, along with coated vesicles, multivesicular bodies, and lysosomes (Fig. 22.24). The principal cells secrete glycerophosphocholine, sialic acid, and glycoproteins, which, in addition to the glycocalyx and steroids, aid in the maturation of the sperm. They have numerous cisternae of rER surrounding the basally located nucleus and a remarkably large supranuclear Golgi apparatus. Profiles of sER and rER are also present in the apical cytoplasm. The smooth muscle coat of the duct of the epididymis gradually increases in thickness to become three-layered in the tail.

1	The smooth muscle coat of the duct of the epididymis gradually increases in thickness to become three-layered in the tail. In the head of the epididymis and most of the body, the smooth muscle coat consists of a thin layer of circular smooth muscle resembling that of the efferent ductules. In the tail, inner and outer longitudinal layers are added. These three layers are then continuous with the three smooth muscle layers of the ductus deferens, the next component of the excurrent duct system (Plate 89, page 824).

1	Differences in smooth muscle function parallel these morphologic differences. In the head and body of the epididymis, spontaneous, rhythmic peristaltic contractions serve to move the sperm along the duct. Few peristaltic contractions occur in the tail of the epididymis, which serves as the principal reservoir for mature sperm. These sperm are forced into the ductus deferens by intense contractions of the three smooth muscle layers after appropriate neural stimulation associated with ejaculation.

1	FIGURE 22.20 • Schematic diagram of development of intratesticular and excurrent duct systems. a. This diagram shows the testis in the seventh week of development before it descends into the scrotal sac. Note that the mesonephric duct and its tubules give rise to the excurrent duct system for the developing testis. b. Sagittal section of a fully developed testis positioned within the scrotum. Note that the seminal vesicles, ejaculatory ducts, ductus deferens, epididymis, and efferent ductules are all developed from the mesonephric duct and tubules. The seminiferous tubules, straight tubules, and rete testis develop from the indifferent gonads. The prostate gland develops from the prostatic primordium that originates from the pelvic urethra.

1	FIGURE 22.21 • Photomicrograph of efferent ductules. The specimen in this photomicrograph was stained with picric acid and hematoxylin to better visualize the epithelial components of the efferent ductules. The efferent ductules are lined by pseudostratified columnar epithelium. The luminal surface has an uneven or wavy appearance because of the presence of alternating groups of tall columnar cells and cuboidal cells. The ductules are surrounded by several layers of circularly arranged smooth muscle (SM). Within the ductule lumina are clumped spermatozoa (asterisks). Connective tissue (CT ) makes up the stroma of the organ and contains blood vessels (BV ) of various sizes. 120. Inset. This higher magnification of the pseudostratified epithelium shows columnar and cuboidal cells that contain sparse cilia. 500.

1	FIGURE 22.22 • Photomicrograph of human epididymis. This photomicrograph of an H&E–stained section shows the highly coiled ductus epididymis. Its coiled nature is reflected in the variously shaped profiles of the duct. Within the connective tissue are numerous profiles of blood vessels (BV ). The vessels tend to follow the duct; thus they too reflect multiple profiles of several vessels. The section of the duct within the rectangle is shown at higher magnification in Figure 22.23. 30. The ductus deferens is the longest part of the excurrent duct system.

1	The ductus deferens (vas deferens) is a direct continuation of the tail of the epididymis (see Fig. 22.1). It ascends along the posterior border of the testis, close to the testicular vessels and nerves. It then enters the abdomen as a component of the spermatic cord, by passing through the inguinal canal. The spermatic cord contains all of the structures that pass to and from the testis. In addition to the ductus deferens, the spermatic cord contains the testicular artery, small arteries to the ductus deferens and cremaster muscle, the pampiniform plexus, lymphatic vessels, sympathetic nerve fibers, and the genital branch of the genitofemoral nerve. All of these structures are surrounded by fascial coverings derived from the anterior abdominal wall. After leaving the spermatic cord, the ductus deferens descends in the pelvis to the level of the bladder, where its distal end enlarges to form the ampulla of ductus deferens. The ampulla is joined there by the duct of the seminal

1	FIGURE 22.23 • Photomicrograph of human ductus epididymis. This higher magnification of the rectangular area in Figure 22.22 reveals the two cell types of the epididymal epithelium, the principal cells and the basal cells. Stereocilia (arrows) extend from the apical surface of the principal cells. The nuclei of the basal cells are spherical and are located in close proximity to the basement membrane, whereas the nuclei of the principal cells are cylindrical and conform to the columnar shape of the cell. Surrounding the duct epithelium is a layer of circularly arranged smooth muscle cells. The duct lumen contains numerous sperm. 250. vesicle and continues through the prostate gland to the urethra as the ejaculatory duct.

1	vesicle and continues through the prostate gland to the urethra as the ejaculatory duct. The ductus deferens is lined with a pseudostratified columnar epithelium that closely resembles that of the epididymis (Plate 89, page 824). The tall columnar cells also have long microvilli that extend into the lumen. The rounded basal cells rest on the basal lamina. Unlike the epididymis, however, the lumen of the duct does not appear smooth. In histologic preparations (Fig. 22.25), it appears to be thrown into deep longitudinal folds throughout most of its length, probably because of contraction of the thick (1 to 1.5 mm) muscular coat of the duct during fixation.

1	The ampulla of ductus deferens has taller, branched mucosal folds that often show glandular diverticula. The muscle coat surrounding the ampulla is thinner than that of the rest of the ductus deferens, and the longitudinal layers disappear near the origin of the ejaculatory duct. The epithelium of the ampulla and ejaculatory duct appears to have a FIGURE 22.24 • Electron micrograph of epididymis. a. Electron micrograph of the epididymal epithelium, showing principal cells (PC) extending to the lumen and a basal cell (BC) limited to the basal portion of the epithelium. Profiles of sperm (S) are seen in the lumen. The apical cytoplasm of the principal cells exhibits numerous long microvilli (stereocilia). 3,000. b. Apical surface of the epithelial cell with its numerous long microvilli (stereocilia). The middle piece of a sperm (S) is evident in the lumen. The small, light circular profiles (arrowheads) are endocytotic vesicles. 13,000.

1	secretory function. The cells contain large numbers of yellow pigment granules. The wall of the ejaculatory duct does not have a muscularis layer; the fibromuscular tissue of the prostate substitutes for it. The paired seminal vesicles secrete a fluid rich in fructose.

1	The paired seminal vesicles secrete a fluid rich in fructose. The seminal vesicles are paired, elongate, and highly folded tubular glands located on the posterior wall of the urinary bladder, parallel to the ampulla of the ductus deferens. A short excretory duct from each seminal vesicle combines with the ampulla of the ductus deferens to form the ejaculatory duct. Seminal vesicles develop as evaginations of the mesonephric (Wolffian) ducts in the region of future ampullae. The wall of the seminal vesicles contains a mucosa, a thin layer of smooth muscle, and a fibrous coat (Fig. 22.26). The mucosa is thrown into numerous primary, secondary, and tertiary folds that increase the secretory surface area (Plate 91, page 828). All of the irregular chambers thus formed, however, communicate with the lumen.

1	The pseudostratified columnar epithelium contains tall, nonciliated columnar cells and short, round cells that rest on the basal lamina. The short cells appear identical to those of the rest of the excurrent duct system. They are the stem cells from which the columnar cells are derived. The columnar cells have the morphology of protein-secreting cells, with a well-developed rER and large secretory vacuoles in the apical cytoplasm.

1	The secretion of the seminal vesicles is a whitish yellow, viscous material. It contains fructose, which is the principal metabolic substrate for sperm, along with other simple sugars, amino acids, ascorbic acid, and prostaglandins. Although prostaglandins were first isolated from the prostate gland (hence the name), they are actually synthesized in large amounts in the seminal vesicles. Contraction of the smooth muscle coat of the seminal vesicles during ejaculation discharges their secretion into the ejaculatory ducts and helps to flush sperm out of the urethra. The secretory function and morphology of the seminal vesicles are under the control of testosterone. The prostate, the largest accessory sex gland, is divided into several morphologic and functional zones. The prostate is the largest accessory sex gland of the male reproductive system. Its size and shape are commonly compared to those of a walnut. The main function of the prostate

1	FIGURE 22.25 • Photomicrograph of human spermatic cord. a. This low-magnification photomicrograph shows a cross section of the spermatic cord containing several structures. These include the ductus deferens, the accompanying testicular artery and vein, and veins of the pampiniform plexus. 15. Inset. A higher magnification of a pampiniform vein. Note the bundles of longitudinal smooth muscles (cut in cross section) in the tunica adventitia and tunica intima. 55. b. This cross section of the ductus deferens shows the thick muscular wall organized in three distinct smooth muscle layers: an inner longitudinal (SM(L)), middle circular (SM(C)), and outer longitudinal (SM(L)). 100. Inset. A higher magnification shows the pseudostratified epithelium lining the ductus deferens. The tall principal cells possess long microvilli (stereocilia; arrows). The basal cells are in close proximity to the basement membrane and possess spherical nuclei. 215.

1	gland is to secrete a clear, slightly alkaline (pH 7.29) fluid that contributes to the formation of seminal fluid. The gland is located in the pelvis, inferior to the bladder, where it surrounds the prostatic part of the urethra. It consists of 30 to 50 tubuloalveolar glands arranged in three concentric layers: an inner mucosal layer, an intermediate submucosal layer, and a peripheral layer containing the main prostatic glands (Fig. 22.27). The glands of the mucosal layer secrete directly into the urethra; the other two layers have ducts that open into the prostatic sinuses located on either side of the urethral crest on the posterior wall of the urethra.

1	The adult prostatic parenchyma is divided into four anatomically and clinically distinct zones:  The central zone surrounds ejaculatory ducts as they pierce the prostate gland. It contains about 25% of the glandular tissue and is resistant to both carcinoma and inflammation. In comparison to the other zones, cells in the central zone have distinctive morphologic features (a more prominent and slightly basophilic cytoplasm and a larger nuclei displaced at different levels in adjacent cells). Recent findings suggest that this zone originates embryologically from the inclusion of mesonephric duct cells into the developing prostate.

1	 The peripheral zone comprises 70% of the glandular tissue of the prostate. It surrounds the central zone and occupies posterior and lateral parts of the gland. Most prostatic carcinomas arise from the peripheral zone of the prostate gland. The peripheral zone is palpable during digital examination of the rectum. This zone is also the most susceptible to infammation.  The transitional zone surrounds the prostatic urethra; it comprises about 5% of the prostatic glandular tissue and contains the mucosal glands. In older individuals, the parenchymal cells of this zone frequently undergo extensive division (hyperplasia) and form nodular masses of epithelial cells. Because this zone is proximate to the pro-static urethra, these nodules can compress the prostatic urethra, causing diffcult urination. This condition is known as benign prostatic hyperplasia (BPH) and is discussed in Folder 22.4 (page 811).

1	FIGURE 22.26 • Photomicrograph of human seminal vesicle a. This low-magnification photomicrograph shows part of an H&E–stained section of a human seminal vesicle. This gland is a tortuous tubular structure and in a section exhibits what appear to be a number of isolated lumina. In actuality, there is only one lumen. The mucosa is characterized by extensive folding (arrows). It rests on a thick smooth muscle (SM) investment that is organized in two layers: an inner circular layer and an outer longitudinal layer. 20. b. This higher magnification shows the mucosal folds surfaced by a pseudostratified epithelium. Arrows indicate the basal cells. 500. FIGURE 22.27 • Schematic drawing of the zones of the human prostate gland. This drawing illustrates the relative location, by color, of the four zones of the prostate gland and anterior fibromuscular stroma of the gland.

1	 The periurethral zone contains mucosal and submucosal glands. In later stages of BPH, this zone may undergo pathologic growth but mainly from the stromal components. Together with the glandular nodules of the transitional zone, this growth causes increased urethral compression and further retention of urine in the bladder. In addition, fbromuscular stroma occupies the anterior surface of the prostate gland anterior to the urethra and is composed of dense irregular connective tissue with large amount of smooth muscle fibers. The growth of the prostatic glandular epithelium is regulated by the hormone dihydrotestosterone.

1	Within each prostate zone, the glandular epithelium is generally simple columnar, but there may be patches that are simple cuboidal, squamous, or occasionally pseudostratified (Fig. 22.28). The alveoli of the prostatic glands, especially those in older men, often contain prostatic concretions (corpora amylacea) of varied shape and size, often up to 2 mm in diameter (see Fig. 22.28 and Plate 90, page 826). They appear in sections as concentric lamellated bodies  FOLDER 22.4 Clinical Correlation: Benign Prostatic Hypertrophy and Cancer of the Prostate chapter 22 Male Reproductive System PROSTATE G LAN D 811 Benign prostatic hypertrophy (nodular hyperplasia, BPH) occurs almost exclusively in the transitional and peri-urethral zones, leading to partial or total obstruction of the urethra (Fig. F22.4.1a). A widely accepted theory of the pathogenesis of BPH is related to the action of dihy-drotestosterone (DHT). DHT is synthesized in the stromal cells by conversion from circulating

1	(Fig. F22.4.1a). A widely accepted theory of the pathogenesis of BPH is related to the action of dihy-drotestosterone (DHT). DHT is synthesized in the stromal cells by conversion from circulating testosterone in the presence of 5-reductase. Once synthesized, DHT acts as an autocrine agent on the stromal cells and as a paracrine hormone on the glandular epithelial cells, causing them to proliferate (Fig. F22.4.1b). BHP is believed to occur to some extent in all men by age 80. Several options are available to treat BHP. Noninva-sive treatment includes medications (–receptor block-ers) to relax the prostate smooth muscles and relieve pressure on the compressed urethra. Clinical trials have shown that inhibitors of 5-reductase reduce the DHT concentration and thus decrease the size of the prostate and reduce urethral obstruction. Minimally invasive treatment options use laser, microwave, or radiofrequency energy to destroy the prostate tissue causing urethral obstruction. These include

1	and reduce urethral obstruction. Minimally invasive treatment options use laser, microwave, or radiofrequency energy to destroy the prostate tissue causing urethral obstruction. These include interstitial laser coagulation (ILC), microwave hyperthermia, and transurethral needle ablation (TUNA). Finally, a variety of surgical procedures are used to remove hypertrophied regions of the prostate gland. They include transurethral in-cision of the prostate (TUIP), a more extensive transurethral resection of the prostate (TURP), and most recently, a mod-ification of the TURP procedure that uses laser energy to vaporize the prostate tissue called Greenlight PVP. Cancer of the prostate is one of the most common cancers in the male: the lifetime risk of developing prostate cancer is 16.7% (1 in 6). The incidence of prostatic cancer increases with age, and it is estimated that 70% of men be-tween the ages of 70 and 80 will develop this disease. Tumors usually develop in the peripheral zone of

1	The incidence of prostatic cancer increases with age, and it is estimated that 70% of men be-tween the ages of 70 and 80 will develop this disease. Tumors usually develop in the peripheral zone of the gland. In the past, early detection was uncommon, because the ab-normal growth of the tumor did not impinge on the urethra to produce symptoms that demanded prompt attention. There-fore, prostatic cancer was often inoperable by the time it was discovered. However, beginning in the late 1980s, the introduction of prostate-specific antigen (PSA) testing for aabba b FIGURE F22.4.1 • Benign prostatic hyperplasia (BPH) a. This photograph shows a horizontal section through the prostate gland from an individual with BPH. The cut section shows numerous well-defined nodules that compress prostatic urethra (marked with paper clip). b. Photomicrograph of prostatic glands shows the hypertrophy of epithelium lining the glands. Note that the cells form folds that protrude into the lumen of the glands.

1	(marked with paper clip). b. Photomicrograph of prostatic glands shows the hypertrophy of epithelium lining the glands. Note that the cells form folds that protrude into the lumen of the glands. 200. (Reprinted with permission from Rubin E, Gorstein F, Schwarting R, Strayer DS. Rubin’s Pathology, 4th ed. Baltimore: Lippincott Williams & Wilkins, 2004; Fig. 17–40.) continued next page and are believed to be formed by precipitation of secretory material around cell fragments. They may become partially calcified.

1	The glandular epithelium is influenced by sex hormones, such as testosterone and adrenal androgens. These hormones enter the secretory cells of glandular epithelium and are converted to dihydrotestosterone (DHT) by the enzyme 5-reductase. DHT is approximately 30 times more potent than testosterone. The binding of DHT to the androgen receptor (AR) results in a conformational change of the receptor and its relocation from the cytosol to the cell nucleus. Here the phosphorylated dimers of AR complex bind to a specific sequence of DNA known as a hormone-response element residing in the promoter regions of target genes. The primary function for AR is direct upor down-regulation of specific gene transcription. DHT stimulates growth of normal prostatic epithelium and proliferation and growth of BPH and androgen-dependent prostate cancer. The prostate gland secretes prostatic acid phosphatase (PAP), fibrinolysin, citric acid, and prostate-specific antigen (PSA).

1	The prostate gland secretes prostatic acid phosphatase (PAP), fibrinolysin, citric acid, and prostate-specific antigen (PSA). The epithelial cells in prostate gland produce several enzymes, particularly prostate-specific antigen (PSA), pro-static acid phosphatase (PAP), fibrinolysin, and citric acid.

1	 Prostate-specifc antigen (PSA), a 33-kilodalton serine protease, is one of the most clinically important tumor markers. In normal conditions, PSA is secreted into prostatic gland alveoli and ultimately incorporated into seminal fluid. The alveolar secretion from the prostate gland is pumped into the prostatic urethra during ejaculation by contraction of the fibromuscular tissue of the prostate. Because PSA is predominately released into prostatic secretion, only a very small amount of PSA (usually below 4 ng/mL) is circulating in the blood of a healthy individual. However, in prostate cancer, serum concentration of PSA increases; large amounts of PSA are produced and misdirected into the circulation by the transformed prostatic epithelium. Therefore, the elevated levels of PSA are directly related to increased activity of the prostatic cancer cells. A PSA level between 4 and 10 ng/mL suggests about 25% percent cancer risk; levels above 10 ng/mL suggest a risk greater than 67%. An

1	related to increased activity of the prostatic cancer cells. A PSA level between 4 and 10 ng/mL suggests about 25% percent cancer risk; levels above 10 ng/mL suggest a risk greater than 67%. An increased PSA serum level is used as marker for the presence and progression of the disease. Recently, it has become widely accepted that small amounts of PSA are also present in many nonprostatic tissues, including breast, ovary, salivary gland, and liver tissue, and in various tumors. It is also important to mention that high circulating levels of PSA can be associated with benign (noncancerous) conditions such as prostatitis (infection of the prostate gland), interrupted blood fow to the prostate, or BPH.

1	 The prostatic acid phosphatase (PAP) (100-kilodalton) enzyme regulates cell growth and metabolism of prostate glandular epithelium. Because elevated serum levels of PAP are found in patients with metastatic prostate cancer, this enzyme is routinely used as an alternate marker to PSA for prostatic tumors. Measurements of PAP and PSA are useful in assessing the prognosis of prostate cancer.  Fibrinolysin, secreted from the prostate gland, liquefies semen. The bulbourethral glands secrete preseminal fluid. The paired bulbourethral glands (Cowper’s glands) are pea-sized structures located in the urogenital diaphragm (see Fig. 22.1). The duct of each gland passes through the inferior fascia of the urogenital diaphragm and joins the initial portion of the penile urethra. The glands are compound tubuloalveolar glands that structurally resemble mucus secretory glands (Fig. 22.29). The simple columnar epithelium, which varies considerably in height depending

1	FIGURE 22.28 • Photomicrograph of human prostate gland. a. This Mallory-azan–stained specimen shows the tubuloalveolar glands (Gl) and the fibromuscular tissue that forms the septa between glandular tissue. Within the lumina, various-sized prostatic concretions can be seen. The stain used for this specimen readily distinguishes the smooth muscle component (stained red) from the dense connective tissue component (stained blue) of the stroma. 60. b. This higher magnification shows an area where the glandular epithelium is pseudostratified. The round nuclei adjacent to the connective tissue (arrowheads) belong to the basal cells. Those nuclei that are more elongate and further removed from the base of the epithelium belong to the secretory cells. Note the terminal bars (arrows) that are evident at the apical region of these cells. The red-stained sites within the dense connective tissue represent smooth muscle cells. 635.

1	on the functional state of the gland, is under the control of testosterone. The clear, mucus-like glandular secretion contains considerable amounts of galactose and galactosamine, galacturonic acid, sialic acid, and methylpentose. Sexual stimulation causes release of this secretion, which constitutes the major portion of the preseminal fuid and probably serves to lubricate the penile urethra. Semen contains fluids and sperm from the testis and secretory products from the epididymis, ductus deferens, prostate, seminal vesicles, and bulbourethral glands. It is alkaline and may help to neutralize the acid environment of the urethra and the vagina. Semen also contains prostaglandins that may influence sperm transit in both the male and female reproductive ducts and that may have a role in implantation of a fertilized ovum.

1	The average ejaculate of semen has a volume of about 3 mL and normally contains up to 100 million sperm per milliliter. It is estimated that 20% of the sperm in any ejaculate are morphologically abnormal and nearly 25% are immotile. Erection of the penis involves the filling of the vascular spaces of the corpora cavernosa and corpus spongiosum. The penis consists principally of two dorsal masses of erectile tissue, the corpora cavernosa, and a ventral mass of erectile

1	The penis consists principally of two dorsal masses of erectile tissue, the corpora cavernosa, and a ventral mass of erectile FIGURE 22.29 • Photomicrograph of human bulbourethral gland. This photomicrograph shows an H&E–stained section of the compound tubuloalveolar bulbourethral gland. The epithelium consists of columnar mucus-secreting cells. The nuclei are displaced to the base of the cells by the accumulated secretory material that they contain. The cytoplasm has an appearance similar to typical mucus-secreting cells. Note several ducts (D) lined by a simple columnar epithelium. The ducts will merge to form a single excretory duct. In some sites the ducts contain mucus-secreting cells (arrows). 40.

1	tissue, the corpus spongiosum, in which the spongy part of the urethra is embedded. A dense, fibroelastic layer, the tunica albuginea, binds the three together and forms a capsule around each (Fig. 22.30). The corpora cavernosa contain numerous wide, irregularly shaped vascular spaces lined with vascular endothelium. These spaces are surrounded by a thin layer of smooth muscle that forms trabeculae within the tunica albuginea interconnecting and criss-crossing the corpus cavernosum. Irregular smooth muscle bundles are observed frequently as “subendothelial cushions” surrounding irregular vascular spaces (Fig. 22.31). The interstitial connective tissue contains many nerve endings and lymphatic vessels. The vascular spaces increase in size and rigidity by filling with

1	FIGURE 22.30 • Photomicrograph of a histologic section of the penis. This photomicrograph shows an H&E–stained specimen of a cross section of the penis near the base of the organ. Note the arrangement of the corpora cavernosa and corpus spongiosum; the latter contains the urethra. 3. blood, principally derived from the helicine arteries. These arteries dilate during erection (see Folder 22.5) to increase the blood flow to the penis. An arteriovenous (AV) anastomosis exists between the deep artery of the penis and the peripheral venous system (Folder 22.5).

1	The skin of the penis is thin and loosely attached to the underlying loose connective tissue except at the glans penis, where it is very thin and tightly attached. The skin of the glans is so thin that blood within its large, muscular anastomosing veins that drain the corpus spongiosum may give it a bluish color. There is no adipose tissue in the subcutaneous tissue. There is, however, a thin layer of smooth muscle that is continuous with the dartos layer of the scrotum. In uncircumcised males, the glans is covered with a fold of skin, the prepuce, which resembles a mucous membrane on its inner aspect. Numerous sebaceous glands are present in the skin of the penis just proximal to the glans.

1	The penis is innervated by somatic, sympathetic, and parasympathetic nerves. Many sensory nerve endings are distributed throughout the tissue of the penis, and sympathetic and parasympathetic visceral motor fibers innervate the smooth muscle of the trabeculae of the tunica albuginea and the blood vessels. Both sensory and visceral motor fibers play essential roles in erectile and ejaculatory responses.

1	CNS and maintained by complex interactions between vascular and neurologic events. The CNS responds to external or internal stimuli (sensory impulses, perception, desire, etc.) that involve the sympathetic and parasympa-thetic innervation of the penis. Parasympathetic stimulation initiates erection by relaxation of the trabecular smooth muscle cells and dila-tion of the helicine arteries. This leads to expansion of the corpora cavernosa and, to a lesser degree, the corpus spongiosum. Arterial blood accumulates in these erectile tissues by compression of the venules against the nondis-tensible tunica albuginea. This process is referred to as the corporal venoocclusive mechanism. The tunica albuginea also compresses the larger veins that drain blood from the corpora cavernosa so that venous outflow is also blocked, resulting in tumescence, and rigidity of the penis. Two neuromediators, acetylcholine and nitric oxide, are involved in the relaxation of smooth muscle during the initi-ation

1	outflow is also blocked, resulting in tumescence, and rigidity of the penis. Two neuromediators, acetylcholine and nitric oxide, are involved in the relaxation of smooth muscle during the initi-ation and maintenance of penile erection. nerve endings and acts primarily on the endothelial cells that line the vascular spaces of the corpora cavernosa. This causes the release of vasoactive intestinal peptide (VIP) and, more importantly, nitric oxide.  Nitric oxide (NO) activates guanylate cyclase in the trabecular smooth muscle cells to produce cyclic guanosine monophosphate (cGMP). cGMP causes the smooth muscle cells to relax. Sympathetic stimulation terminates penile erection by causing contraction of the trabecular smooth muscle cells of the helicine arteries. These events decrease the flow of blood to the corpora cavernosa, reducing blood pressure in the erectile tissue to normal venous pressure. The lower pressure within the corpus cavernosum allows the veins leading from the corpora

1	of blood to the corpora cavernosa, reducing blood pressure in the erectile tissue to normal venous pressure. The lower pressure within the corpus cavernosum allows the veins leading from the corpora cavernosa to open and drain the excess blood. Erectile dysfunction (ED) is an inability to achieve and maintain sufficient penile erection to complete satisfactory intercourse. Adequate arterial blood supply is critical for continued next page

1	FIGURE 22.31 • Photomicrograph of corpus spongiosum. a. This photomicrograph of an H&E–stained section shows the corpus spongiosum and urethra. 20. b. This higher magnification of the corpus spongiosum shows the numerous irregularly shaped vascular spaces. Note the surrounding layer of smooth muscle (SM) forming the “subendothelial cushions.” 135.  FOLDER 22.5 Clinical Correlation: Mechanism of Erection and Erectile Dysfunction Erection of the penis is a vascular event initiated by the  Acetylcholine is released by the parasympathetic This page intentionally left blank.

1	This page intentionally left blank. The male reproductive system consists of the paired testes, epididymides, and genital ducts, as well as accessory reproductive glands and the penis. The functions of the testis are the production of sperm and the synthesis and secretion of androgens, especially testosterone. The events of cell division that lead to the mature sperm involve both normal cell division, mitosis, and reduction division, meiosis, to yield a hap-loid chromosome number and haploid DNA content. Androgen secretion by the testis begins early in fetal development and is essential for continued normal development of the male fetus. At puberty, androgen secretion resumes and is responsible for initiation and maintenance of sperm production (spermatogenesis), secretion by accessory sex glands (e.g., prostate and seminal vesicles), and development of secondary sex characteristics. Testis, monkey, H&E 65.

1	Testis, monkey, H&E 65. This section of the testis shows the seminiferous tubules and the tunica albuginea (TA), the capsule of the organ. Extending from the very thick capsule are connective tissue septa (S) that divide the organ into compartments. Each compartment contains several seminiferous tubules and represents a lobule (L). Blood vessels (BV) are present within the inner portion of the capsule, the part referred to as the tunica vasculosa, and in the connective tissue septa. The seminiferous tubules are convoluted; thus, the profiles they present in a section are variable in appearance. Not infrequently, the wall of a tubule is sectioned tangentially, thus obscuring the lumen and revealing what appears to be a solid mass of cells (X). Seminiferous tubules, testis, monkey, H&E 400.

1	Seminiferous tubules, testis, monkey, H&E 400. Examination at higher magnification, as in this figure, reveals a population of interstitial cells that occur in small clusters and lie in the space between adjoining tubules. They consist mostly of Leydig cells (LC), the chief source of testosterone in the male. They are readily identified by virtue of their location and by their small round nucleus and eosinophilic cytoplasm. Macrophages are also found, in close association with the Leydig cells, but in lesser number. They are, however, difficult to identify in H&E sections.

1	A layer of closely apposed squamous cells forms a sheath-like investment around the tubule epithelium of each seminiferous tubule. In man, several layers of cells invest the tubule epithelium. The cells of this peritubular investment exhibit myoid features and account for the slow peristaltic activity of the tubules. Peripheral to the myoid layer is a broad lymphatic channel that occupies an extensive space between the tubules. In routine histologic sections, however, the lymphatic channels are usually collapsed and, thus, unrecognizable. The cellular elements that surround the tubule epithelium are generally referred to as a lamina propria (LP) or as a boundary tissue. As a lamina propria, it is atypical. It is not a loose connective tissue. Indeed, under normal circumstances, lymphocytes and other cell types related to the immune system are conspicuously absent.

1	Examination of the tubule epithelium reveals two kinds of cells: a proliferating population of spermatogenic cells and a nonproliferating population, the sustentacular, or Sertoli, cells. The Sertoli cells are considerably fewer and can be recognized by their elongate, pale-staining nuclei (Sn) and conspicuous nucleolus. The Sertoli cell cytoplasm extends from the periphery of the tubule to the lumen.

1	The spermatogenic cells consist of successive generations arranged in concentric layers. Thus, the spermatogonia (Sg ) are found at the periphery. The spermatocytes (Sc), most of which have large round nuclei with a distinctive chromatin pattern (because of their chromatin material being reorganized), come to lie above the spermatogonia. The spermatid population (Sp) consists of one or two generations and occupies the site closest to the lumen. The tubules in this figure have been identified according to their stage of development. The tubule at the upper right can be identified as stage VI. At this stage, the mature population of spermatids (identified by their dark-blue heads and eosinophilic threadlike flagella protruding into the lumen) are in the process of being released (spermiogenesis). The younger generation of spermatids is composed of round cells and exhibits round nuclei. Moving clockwise, the tubule indicated as stage VII is slightly more advanced. The mature spermatids

1	The younger generation of spermatids is composed of round cells and exhibits round nuclei. Moving clockwise, the tubule indicated as stage VII is slightly more advanced. The mature spermatids are now gone. Progressing to stage VIII, the tubule at the bottom of the micrograph reveals that the spermatid population is undergoing a change in nuclear shape. Note the tapered nuclei (arrows). Further maturation of the spermatids is reflected in the tubule at the top of the micrograph, stage XI. Finally, the tubule marked stage II, on the left, reveals slightly greater maturation of the luminal spermatids, and with the start of the new cycle (stage I), a newly formed spermatid population is now present. By examining the spermatid population and assessing the number of generations present (i.e., one or two) and the degree of maturation, it is possible with the aid of a chart to approximate the stage of a tubule.

1	KEY BV, blood vessels L, lobule LC, Leydig cells LP, lamina propria S, connective tissue septa Sc, spermatocytes Sg, spermatogonia Sn, Sertoli nuclei Sp, spermatids TA, tunica albuginea X, tangential section of tubule with lumen obscured arrows, spermatid nuclei displaying early shape change

1	While the mature testis is characterized by seminiferous tubules, the immature testis is characterized by cords of cells consisting of an epithelium of sustentacular (Sertoli) cells surrounding occasional gonocytes, precursors of spermatogonia that are derived from the primordial germ cells that invaded the developing gonad in the embryo. At puberty, these cords become canalized, and the gonocytes begin the multiple divisions that give rise to the spermatogonia that, in turn, will divide and differentiate into the mature sperm. The seminiferous tubules terminate as straight tubules (tubuli recti) that are lined only by Sertoli cells. The tubuli recti lead to the rete testis, a complex series of anastomosing channels in the mediastinum testis that is the termination of the intratesticular tubule system. Prepubertal testis, newborn human, H&E 180; inset 360.

1	Prepubertal testis, newborn human, H&E 180; inset 360. The various germ cell types representative of spermatogenesis in the mature seminiferous tubules are not present in the testis before puberty or in the postpubertal undescended testis. Instead, the “tubules” are represented by cords of cells in which a lumen is lacking. The seminiferous cords display the same tortuosity as in the adult; the tunica albuginea (TA) of the testis, though thinner, is of the same relative thickness.

1	The seminiferous cords are of considerably smaller diameter than the tubules of the adult and are composed of two cell types: the gonocyte, or first-generation spermatogonium, derived from the primordial germ cell that migrates from the yolk sac to the developing gonad in the embryo; and a cell that resembles the Sertoli cell of the adult. The latter cell type predominates and constitutes the bulk of the cord. The cells are columnar, and their nuclei are close to the basement membrane. The gonocytes (G) are the precursors of the definitive germ cells, or spermatogonia. They are round cells Mediastinum testis, testis, monkey, H&E 65.

1	Mediastinum testis, testis, monkey, H&E 65. In the posterior portion of the testis, the connective tissue of the tunica albuginea extends more deeply into the organ. This inward extension of connective tissue is called the mediastinum testis. It contains a network of anastomosing channels called the rete testis. Only a small portion of the mediastinum testis (MT) is evident in the figure. The area includes, however, a few seminiferous tubules (ST) in the upper portion of the micrograph Straight tubule, testis, monkey, H&E 400.

1	Straight tubule, testis, monkey, H&E 400. The straight tubule or tubulus rectus (TR) in this figure appears to end on one side before it ends on the other. This simply reflects the angle of section. When the straight tubule ends, however, the epithelial lining suddenly becomes cuboidal. This represents the rete testis, which constitutes an anastomosing system of channels that lead to the efferent ductules. The that have a centrally placed, spherical nucleus. The cytoplasm takes little stain and usually appears as a light ring around the nucleus. This gives the gonocyte a distinctive appearance in histologic sections (inset). Generally, the gonocytes are found at the periphery of the cord, but many are also found more centrally. The gonocytes give rise to spermatogonia that begin to proliferate in males between the ages of 10 and 13 years. The seminiferous epithelium then becomes populated with cells at various stages of spermatogenesis, as seen in the adult.

1	The seminiferous cords are surrounded by one or two layers of cells with long processes and flat nuclei. They resemble fibroblasts at the ultrastructural level and give rise to the myoid peritubular cells of the adult. The interstitial cells (of Leydig) are conspicuous in the newborn, a reflection of the residual effects of maternal hormones. Leydig cells, however, regress and do not become conspicuous again until puberty. In this preparation, the Leydig cells (LC) can be seen between the cords (inset). They are ovoid or polygonal and are closely grouped, so that adjacent cells are in contact with each other. Overall, they have the same appearance as the Leydig cells of the adult.

1	and, fortuitously, the site where one of the seminiferous tubules terminates and joins the rete testis (RT). This can be recognized in the area delineated by the rectangle, which is shown at higher magnification in figure below. As noted above, the seminiferous tubules are arranged in the form of a loop, with each end joining the rete testis. The seminiferous tubules open into the rete testis by way of a straight tubule. Straight tubules are very short and are lined by Sertoli-like cells; no germ cell component is present. epithelial lining cells of the rete are sometimes more squamous than cuboidal or, occasionally, may be low columnar in appearance. Typically, they possess a single cilium; however, this is difficult to see in routine H&E preparations.

1	The connective tissue of the mediastinum is very dense but exhibits no other special features, nor is smooth muscle present. Adipose cells (AC) and blood vessels (BV), particularly veins of varying size, are present within the connective tissue. KEY AC, adipose cells BV, blood vessels G, gonocytes LC, Leydig cells MT, mediastinum testis RT, rete testis ST, seminiferous tubules TA, tunica albuginea TR, tubulus rectus

1	The rete testis, in turn, is connected via 20 efferent ductules (ductuli efferentes; remnants of nephrons of the fetal mesonephric kidney) to the ductus epididymis. These are the first elements of the excurrent duct system of the male genital system. Most of the fluid secreted in the seminiferous tubules is reabsorbed in the efferent ductules. The muscular coat characteristic of the excurrent duct system first appears at the beginning of the efferent ductules. The ductus epididymis is a highly coiled tube, 4 to 6 m long; sperm mature during their passage along its length, acquiring motility as well as the ability to fertilize an egg. This maturation is also androgen dependent and involves changes in the sperm plasma membrane and addition to the glycocalyx of glycoproteins secreted by the epididymal epithelial cells. Efferent ductules, testis-epididymis, monkey, H&E 60; inset 360.

1	About 12 to 20 efferent ductules leave the testis and serve as channels from the rete testis to the ductus epididymis. Each of the efferent ductules undergoes numerous spiral windings and convolutions to form a group of conical structures; together they constitute the initial part of the head of the epididymis. When examined in a 822 tissue section, the ductules exhibit a variety of irregular profiles due to their twisting and turning. This is evident on the right side of this micrograph. The epithelium that lines the efferent ductules is distinctive in that groups of tall columnar cells alternate with groups of cuboidal cells, giving the luminal surface an unevenly contoured appearance. Thus, small cup-like depressions are created where the epithelium contains groups of cuboidal or low columnar cells. Typically, these shorter cells exhibit a brush border-like apical surface because of the microvilli that they possess (arrowhead, inset).

1	PLATE 88 • E FFE R E NT DUCTU LE S AN D E P I DI DYM I S KEY AT, adipose tissue BV, blood vessel C, cilia CT, connective tissue P, pigment SC, stereocilia SM, smooth muscle arrowhead (inset), brush border arrows, “islands” of epithelium in the lumen Epididymis, monkey, H&E 180. The epididymis, by virtue of its shape, is divided into a head, body, and tail. The initial part of the head contains the ductus epididymis, a single convoluted duct into which the efferent ductules open. The duct is, at first, highly convoluted but becomes less tortuous in the body and tail. A section through the head of the epididymis, as shown in figure above, cuts the ductus epididymis in numerous places, and as in the efferent ductules, different-shaped profiles are observed.

1	The epithelium contains two distinguishable cell types: tall columnar cells and basal cells similar to those of the efferent ductules. The epithelium is, thus, also pseudostratified columnar. The columnar cells are tallest in the head of the epididymis and diminish in height as the tail is reached. The free surface of the cell possesses stereocilia (SC). These are extremely long, branching microvilli. They evidently adhere to each other during the preparation of the tissue to form the fine tapering structures that are characteristically seen with the light microscope. The nuclei of the columnar cells are elongated and are located a moderate distance from the base of the cell.

1	The basal surface of the ductule, in contrast, has a smooth contour (see figure below and inset). Some of the cells, generally the tall columnar cells, possess cilia (C) (inset). Whereas the ciliated cells aid in moving the contents of the tubule toward the epididymis, the cells with the microvilli are largely responsible for absorbing fluid from the lumen. In addition to the columnar and cuboidal cells, basal cells are also present; thus, the epithelium is designated pseudostratified columnar. The basal cells possess little cytoplasm and presumably serve as stem cells.

1	The efferent ductules possess a thin layer of circularly arranged smooth muscle cells (SM, inset). The muscle is close to the basal surface of the epithelial cells, being separated from it by only a small amount of connective tissue (CT, inset). Because of this close association, the smooth muscle may be overlooked or misidentified as connective tissue. Smooth muscle facilitates movement of luminal contents of the ductule to the ductus epididymis. They are readily distinguished from the spherical nuclei of the basal cells that lie close to the basement membrane. Other conspicuous features of the columnar cells include a very large supranuclear Golgi apparatus (not seen at the magnification offered here), pigment accumulations (P), and numerous lysosomes, demonstrable with appropriate techniques.

1	Because of the unusual height of the columnar cells and, again, the tortuosity of the duct, an uneven lumen appears in some sites; indeed, even “islands” of epithelium can be encountered in the lumen (see arrows, figure above). Such profiles are accounted for by sharp turns in the duct where the epithelial wall on one side of the duct is partially cut. For example, a cut in the plane of the double-headed arrow indicated in this figure would create such an isolated epithelial island. A thin layer of smooth muscle circumscribes the duct and appears similar to that associated with the efferent ductules. In the terminal portion of the epididymis, however, the smooth muscle acquires a greater thickness, and longitudinal fibers are also present. Beyond the smooth muscle coat there is a small amount of connective tissue (CT) that binds the loops of the duct together and carries the blood vessels (BV) and nerves.

1	The ductus (vas) deferens continues from the duct of the epididymis as a thick-walled muscular tube that leaves the scrotum and passes through the inguinal canal as a component of the spermatic cord. At the deep inguinal ring, it continues into the pelvis and, behind the urinary bladder, joins with the excretory duct from the seminal vesicle to form the ejaculatory duct. The ejaculatory duct then pierces the prostate gland and opens into the urethra. Mature sperm are stored in the terminal portion (tail) of the ductus epididymis. These sperm are forced into the ductus deferens by intense contractions of the three smooth muscle layers of the ductus deferens following appropriate neural stimulation. Contraction of the smooth muscle of the ductus deferens continues the movement of the sperm through the ejaculatory duct into the urethra during the ejaculatory reflex. The seminal vesicles (see Plate 91) are not storage sites for sperm but, rather, secrete a fructose-rich fluid that becomes

1	the ejaculatory duct into the urethra during the ejaculatory reflex. The seminal vesicles (see Plate 91) are not storage sites for sperm but, rather, secrete a fructose-rich fluid that becomes part of the ejaculated semen. Fructose is the principal metabolic substrate for sperm.

1	Spermatic cord, human, H&E 80. A cross section through the ductus deferens and some of the vessels and nerves that accompany the duct in the spermatic cord are shown in this figure. The wall of the ductus deferens is extremely thick, mostly because of the presence of a large amount of smooth muscle. The muscle contracts when the tissue is removed, causing the mucosa to form longitudinal folds. For this reason, in histologic sections, the lumen (L) usually appears irregular in cross section.

1	The smooth muscle of the ductus deferens is arranged as a thick outer longitudinal layer (SM(L)), a thick middle circular layer (SM(C)), and a thinner inner longitudinal layer (SM(L)). Between the epithelium and the inner longitudinal smooth muscle layer there is a moderately thick cellular layer of loose connective tissue, the lamina propria (LP). The connective tissue immediately surrounding the ductus deferens contains nerves and some of the smaller blood vessels that supply the duct. In fact, some of these vessels can be seen penetrating the outer longitudinal smooth muscle layer (asterisks). SPERMATIC CORD AND DUCTUS DEFERENS KEY BV, blood vessels CT, connective tissue L, lumen of ductus deferens LP, lamina propria Lu, lumen of blood vessel N, nerve SM(C), circular layer of smooth muscle SM(L), longitudinal layer of smooth muscle arrowheads, stereocilia arrows, basal cell nucleus asterisks, small arteries supplying ductus deferens Ductus deferens, human, H&E 320; inset 250.

1	Ductus deferens, human, H&E 320; inset 250. The epithelial lining of the ductus deferens consists of pseudostratified columnar epithelium with stereocilia (arrowheads). It resembles the epithelium of the epididymis, but the cells are not as tall. The elongated nuclei of the columnar cells are read ily distinguished from the spherical nuclei of the basal cells (arrows). The epithelium rests on a loose connective tissue that extends to the smooth muscle; no submucosa is described. A unique feature of the spermatic cord is the presence of a plexus of atypical veins (pampiniform plexus) that arise from the spermatic veins.

1	A unique feature of the spermatic cord is the presence of a plexus of atypical veins (pampiniform plexus) that arise from the spermatic veins. These vessels receive the blood from the testis. (The pampiniform plexus also receives tributaries from the epididymis.) The plexus is an anastomosing vascular network that constitutes the bulk of the spermatic cord. Portions of several of these veins (BV) are evident in the upper right of figure above along with a number of nerves (N). The unusual feature of the veins is their thick muscular wall that, at a glance, gives the appearance of an artery rather than a vein. Careful examination of these vessels (inset) shows that the bulk of the vessel wall is composed of two layers of smooth muscle—an outer circular layer SM(C) and an inner longitudinal layer SM(L).

1	The prostate gland is the largest accessory sex gland. It consists of 30 to 50 tubuloalveolar glands that surround the proximal urethra. Because of this relationship, a common condition in later life, benign prostatic hyperplasia, can result in partial or total obstruction of the urethra. The prostatic glands are arranged in three concentric layers: a mucosal layer, a submucosal layer, and a peripheral layer containing the main prostatic glands. The mucosal glands secrete directly into the urethra; the other two sets of glands deliver their secretions through ducts that open into the prostatic sinuses on the posterior wall of the urethra. All of the glands are made up of a pseudostratified columnar epithe-lium that secretes several components of the semen, including acid phosphatase, citric acid (a nutrient for sperm), and fibrinolysin (which keeps the semen liquified). Aggregations of dead epithelial cells and precipitated secretory products form prostatic concretions in the alveoli

1	acid (a nutrient for sperm), and fibrinolysin (which keeps the semen liquified). Aggregations of dead epithelial cells and precipitated secretory products form prostatic concretions in the alveoli of the glands; these are a characteristic feature that aids in recognition of the prostate. The stroma is characterized by numerous small bundles of smooth muscle, so that it can also be described as a fbromuscular stroma. Contraction of this muscle occurs at ejaculation, forcing the secretion into the urethra. Surrounding the gland is a fibroelastic capsule that also contains small bundles of smooth muscle.

1	Prostate gland, human H&E 47. A portion of the prostate gland in shown in this low-magnification micrograph. A small section of the capsule (Cap) of the gland is seen in the upper left corner. The rest of the field is filled with the glandular and stromal components of the prostate. The secretory tubuloalveoli of the prostate gland vary greatly in form, as is evident in the figure. They may appear as tubes, as isolated alveoli, as alveoli with branches, or as tubes with branches. Tangential sections through alveoli may even produce the appearance of “epithelial islands” (arrowheads) in the lumen of the alveoli.

1	This is due to the extremely uneven contour of the epithelial surface. It should also be noted that many of the alveoli may appear rudimentary in structure (arrows). These are simply in an inactive state and are increasingly observed in older individuals. As noted above, aggregations of dead epithelial cells and precipitated secretions form prostatic concretions (C) in the lumina of the alveoli; these gradually increase in number and size with age. The concretions stain with eosin and may have a concentric lamellar appearance, as is clearly shown in the concretion in the lower right. With time, they may become impregnated with calcium salts and thus be easily recognized in X-rays of the lower abdomen. Glands and fbromuscular stroma, prostate, human, H&E 178; upper inset 350; lower inset 650.

1	In this higher magnification view of a portion of the prostate gland, the fbromuscular stroma is clearly seen both immediately subtending the secretory epithelium of the tubuloalveoli as well as in deeper, nonsecretory areas. In the upper inset, corresponding to the larger rectangle, the intensity of the staining of the smooth muscle (SM) clearly distinguishes it from the fibrous stromal connective tissue with which it is intimately intermingled. There are no clearly outlined bundles or layers of smooth muscle in the prostate; rather, it is randomly arrayed throughout the stroma. Prostatic concretions (C) are again evident in the lumina of alveoli, in one instance compressing the epithelium to a degree that makes it nearly unrecognizable. The lower inset, corresponding to the smaller rectangle, clearly demonstrates the pseudostratified columnar nature of the prostatic epithelium (Ep). Well-delineated basal cells (arrowheads) are seen along with the taller columnar secretory cells. A

1	clearly demonstrates the pseudostratified columnar nature of the prostatic epithelium (Ep). Well-delineated basal cells (arrowheads) are seen along with the taller columnar secretory cells. A small blood vessel immediately subtending the epithelium is recognizable by the red blood cells in its lumen. A lymphocytic infiltration appears to fill the stroma along the lower border of this image suggesting an inflammatory process occurring in the prostate gland.

1	PLATE 90 KEY BV, blood vessel C, prostatic concretion Cap, capsule Ep, epithelium L, lymphocytes SM, smooth muscle arrows, inactive alveoli arrowheads: top fgure, “epithelial islands”; bottom figure, basal cells

1	The seminal vesicles are evaginations from the end of each ductus deferens that form tightly coiled tubes. Although sections through this structure may show many lumina, they are all profiles of a single continuous tubular lumen. The seminal vesicles are lined with a pseudostratified columnar epithelium that closely resembles that of the prostate gland. The secretion of the seminal vesicles is a whitish yellow viscous material that contains fructose, other simple sugars, amino acids, ascorbic acid, and prostaglandins. Although prostaglandins were first isolated from the prostate gland (hence the name), they are actually synthesized in large amounts in the seminal vesicles. Fructose is the primary nutrient source for the sperm in the semen. The mucosa rests on a thick layer of smooth muscle that is directly continuous with that of the ductus deferens, from which the seminal vesicle evaginates. The smooth muscle consists of an indistinct inner circular layer and an outer longitudinal

1	that is directly continuous with that of the ductus deferens, from which the seminal vesicle evaginates. The smooth muscle consists of an indistinct inner circular layer and an outer longitudinal layer (compare with the three layers of the ductus epididymis and the ductus deferens, Plate 88), which are difficult to distinguish. Contraction of the smooth muscle coat during ejaculation forces the secretions of the seminal vesicles into the ejaculatory ducts. Beyond the smooth muscle is the connective tissue of the adventitia.

1	Seminal vesicle, human, H&E 30. This figure shows a cross section of a seminal vesicle. Because of the coiled nature of the vesicle, two almost distinct lumina, lying side by side, appear to be present. They are, however, connected so that, in effect, all of the internal spaces are continuous and what is seen here is actually a two-dimensional configuration reflecting coiling of the tube. The mucosa of the seminal vesicles is characterized by being extensively folded or ridged. The ridges vary in size and typically branch and interconnect with one another. The larger ridges may form recesses that contain smaller ridges, and when cut obliquely, these appear as mucosal arches that enclose the smaller folds (arrows). When the plane of section is normal to Mucosal folds, seminal vesicle, human, H&E 220.

1	Mucosal folds, seminal vesicle, human, H&E 220. This higher magnification of the mucosal folds reveals the epithelium (Ep) and the underlying loose connective tissue or lamina propria (LP). The epithelium is described as pseudostratified. It is composed of low columnar or cuboidal cells and small, round basal cells. The latter are randomly inter spersed between the larger principal cells, but they are relatively sparse. the surface, the mucosal ridges appear as “villi.” In some areas, particularly the peripheral region of the lumen, the interconnecting folds of the mucosa appear as alveoli. Each of these chambers is, however, simply a pocket-like structure that is open and continuous with the lumen. The mucosa is subtended by a very cellular loose connective tissue (CT) that, in turn, is surrounded by smooth muscle (SM).

1	The seminal vesicles are paired elongated sacs. Each vesicle consists of a single tube folded and coiled on itself with occasional diverticula in its wall. The upper extremity ends as a cul-de-sac; the lower extremity is constricted into a narrow straight excretory duct that joins and empties into its corresponding ductus deferens. For this reason, the epithelium may not be readily recognized as pseudostratified. In some areas, the epithelium appears thick (arrowhead) and, based on the disposition of the nuclei, would seem to be multilayered. This is due to a tangential section of the epithelium and is not a true stratification. The lamina propria of the mucosa is composed of a very cellular connective tissue containing some smooth muscle cells and is rich in elastic fibers. PLATE 91 KEY CT, connective tissue Ep, epithelium LP, lamina propria SM, smooth muscle arrowhead, oblique section of epithelium arrows, mucosal arches

1	OVERVIEW OF THE FEMALE REPRODUCTIVE SYSTEM / 830 OVARY / 831 Ovarian Structure / 832 Follicle Development / 833 Ovulation / 837 Capacitation and Fertilization / 840 Atresia / 843 Blood Supply and Lymphatics / 845 Innervation / 845 UTERINE TUBES / 845 UTERUS / 848 Cyclic Changes During the Menstrual Cycle / 850 Implantation / 852 Cervix / 853 PLACENTA / 854 VAGINA / 860 EXTERNAL GENITALIA / 861 MAMMARY GLANDS / 863 Hormonal Regulation of the Mammary Gland / 866 Involution of the Mammary Gland / 867 Blood Supply and Lymphatics / 870 Innervation / 870 Folder 23.1 Clinical Correlation: Polycystic Ovarian Disease / 839 Folder 23.2 Clinical Correlation: In Vitro Fertilization / 844 Folder 23.3 Functional Considerations: Summary of Hormonal Regulation of the Ovarian Cycle / 846 Folder 23.4 Clinical Correlation: Fate of the Mature Placenta at Birth / 860 Folder 23.5 Clinical Correlation: Cytologic Pap Smears / 862 Folder 23.6 Clinical Correlation: Cervix and Human Papillomavirus (HPV)

1	Clinical Correlation: Fate of the Mature Placenta at Birth / 860 Folder 23.5 Clinical Correlation: Cytologic Pap Smears / 862 Folder 23.6 Clinical Correlation: Cervix and Human Papillomavirus (HPV) Infections / 868 Folder 23.7 Functional Considerations: Lactation and Infertility / 870

1	The female reproductive system consists of internal sex organs and external genital structures. The internal female reproductive organs are located in the pelvis, and the external genital structures (external genitalia) are situated in the anterior part of the perineum known as the vulva.  The internal female reproductive organs are the ovaries, uterine tubes, uterus, and vagina (Fig. 23.1). They are contained mainly within the pelvic cavity and in the perineum.  The external genitalia include the mons pubis, labia majora and minora, clitoris, vestibule and opening of the vagina, hymen, and external urethral orifice. The mammary glands are included in this chapter because their development and functional state are directly fundus of uterus body of uterine portion isthmus of mesosalpinx ampulla of of uterine tube uterine tube uterine tube suspensory ligament of ovary ovary ligament of ovary mesometrium perimetrium

1	FIGURE 23.1 • Schematic drawing of female internal sex organs. This drawing shows the posterior view of the female internal sex organs. Part of the wall of the uterus, uterine tube, and vagina has been removed to reveal their internal structure. Note the three distinct layers of the uterine wall: the inner layer, the endometrium lining the uterine cavity; the middle and thickest layer, the myometrium; and the outer layer, the perimetrium, which is the peritoneal covering of the uterus. related to the hormonal activity of the female reproductive system. Similarly, the placenta is included because of its functional and physical relationship to the uterus in pregnancy. Female reproductive organs undergo regular cyclic changes from puberty to menopause.

1	The ovaries, uterine tubes, and uterus of the sexually mature female undergo marked structural and functional changes related to neural activity and changes in hormone levels during each menstrual cycle and during pregnancy. These mechanisms also regulate the early development of the female reproductive system. The initiation of the menstrual cycle, referred to as the menarche, occurs in females between 9 and 14 years of age (the mean age of menarche is 12.7 years) and marks the end of puberty and the beginning of the reproductive life span. During this phase of life, the menstrual cycle averages about 28 to 30 days in length. Between 45 and 55 years of age (the mean age is 51.4 years), the menstrual cycle becomes infrequent and eventually ceases. This change in reproductive function is referred to as the menopause or climacterium (commonly called the “change of life”). The ovaries cease their reproductive function of producing oocytes and their endocrine function of producing

1	is referred to as the menopause or climacterium (commonly called the “change of life”). The ovaries cease their reproductive function of producing oocytes and their endocrine function of producing hormones that regulate reproductive activity. Other organs (e.g., vagina and mammary glands) show varying degrees of reduced function, particularly secretory activity.

1	Production of gametes and steroid hormones are the two major functions of the ovary. The ovaries have two interrelated functions: gametogenesis (the production of gametes) and steroidogenesis (the production of steroids). In women, the production of gametes is called oogenesis. Developing gametes are called oocytes; mature gametes are called ova. Two major groups of steroid hormones—estrogens and progestogens—are secreted by the ovaries.  Estrogens promote the growth and maturation of internal and external sex organs and are responsible for the female sex characteristics that develop at puberty. Estrogens also act on mammary glands to promote breast development by stimulating ductal and stromal growth and accumulation of adipose tissue.

1	 Progestogens prepare the internal sex organs, mainly the uterus, for pregnancy by promoting secretory changes in the endometrium (discussed in the section on cyclic changes in the endometrium). Progestogens also prepare the mammary gland for lactation by promoting lobular proliferation. FIGURE 23.2 • Schematic drawing of a section through the ovary. This drawing shows stages of follicular development from the early primary follicle to the mature (Graafian) follicle. The maturation of the follicles occurs in the direction of the arrow. Changes in the follicle after ovulation lead to development of the corpus luteum and eventually the corpus albicans. Note the highly coiled blood vessels in the hilum and medulla of the ovary. Both hormones play an important role in the menstrual cycle by preparing the uterus for implantation of a fertilized ovum. If implantation does not occur, the endometrium of the uterus degenerates and menstruation follows.

1	In nulliparas (women who have not borne children), the ovaries are paired, almond-shaped, pinkish white structures measuring about 3 cm in length, 1.5 cm in width, and 1 cm in thickness. Each ovary is attached to the posterior surface of the broad ligament by a peritoneal fold, the mesovarium (see Fig. 23.1). The superior (or tubal) pole of the ovary is attached to the pelvic wall by the suspensory ligament of the ovary, which carries the ovarian vessels and nerves. The inferior (or uterine) pole is attached to the uterus by the ovarian ligament. This ligament is a remnant of the gubernaculum, the embryonic fibrous cord that attaches the developing gonad to the floor of the pelvis. Before puberty, the surface of the ovary is smooth, but during reproductive life it becomes progressively scarred and irregular because of repeated ovulations. In postmenopausal women, the ovaries are about one-fourth the size observed during the reproductive period.

1	The ovary is composed of a cortex and a medulla. A section through the ovary reveals two distinct regions:  The medulla or medullary region is located in the central portion of the ovary and contains loose connective tissue, a mass of relatively large contorted blood vessels, lymphatic vessels, and nerves (Fig. 23.2).  The cortex or cortical region is found in the peripheral portion of the ovary surrounding the medulla. The cortex contains the ovarian follicles embedded in a richly cellular connective tissue (Plate 92, page 872). Scattered smooth muscle fibers are present in the stroma around the follicles. The boundary between the medulla and cortex is indistinct. “Germinal epithelium” instead of mesothelium covers the ovary.

1	The surface of the ovary is covered by a single layer of cuboidal and, in some parts, almost squamous cells. This cellular layer, known as the germinal epithelium, is continuous with the mesothelium that covers the mesovarium. The term germinal epithelium is a carryover from the past when it was incorrectly thought to be the site of germ cell formation during embryonic development. It is now known that the primordial germ cells (both male and female) are of extragonadal origin and that they migrate from the embryonic yolk sac into the cortex of the embryonic gonad, where they differentiate and induce differentiation of the ovary. A dense connective tissue layer, the tunica albuginea, lies between the germinal epithelium and the underlying cortex (Plate 92, page 872). Tumors that arise from the epithelial surface of the ovary account for more than 70% of ovarian cancers. The origin of surface epithelial tumors may be related to repeated disruption and repair of the germinal

1	arise from the epithelial surface of the ovary account for more than 70% of ovarian cancers. The origin of surface epithelial tumors may be related to repeated disruption and repair of the germinal epithelium that occurs during ovulations.

1	Ovarian follicles provide the microenvironment for the developing oocyte.

1	Ovarian follicles of various sizes, each containing a single oocyte, are distributed in the stroma of the cortex. The size of a follicle indicates the developmental state of the oocyte. Early stages of oogenesis occur during fetal life when mitotic divisions massively increase the number of oogonia (see the section on oogenesis). The oocytes present at birth remain arrested in development at the first meiotic division (see page 92). During puberty, small groups of follicles undergo cyclic growth and maturation. The first ovulation generally does not take place for a year or more after menarche. A cyclic pattern of follicular maturation and ovulation is then established that continues in parallel with the menstrual cycle. Normally, only one oocyte reaches full maturity and is released from the ovary during each menstrual cycle. Obviously, the maturation and release of more than one egg at ovulation may lead to multiple zygotes. During the reproductive life span, a woman produces only

1	the ovary during each menstrual cycle. Obviously, the maturation and release of more than one egg at ovulation may lead to multiple zygotes. During the reproductive life span, a woman produces only about 400 mature ova. Most of the estimated 600,000 to 800,000 primary oocytes present at birth do not complete maturation and are gradually lost through atresia, the spontaneous death and subsequent resorption of immature oocytes. This process begins as early as the fifth month of fetal life and is mediated by apoptosis of cells surrounding the oocyte. Atresia reduces the number of primary oocytes in a logarithmic fashion throughout life from as many as 5 million in the fetus to less than 20% of that number at birth. The oocytes that remain at menopause degenerate within a few years.

1	Histologically, three basic types of ovarian follicles can be identified on the basis of developmental state:  primordial follicles; growing follicles which are further subcategorized as pri mary and secondary (or antral) follicles; and mature follicle or Graafan follicles. Some histologists and clinicians identify additional stages in the continuum of follicular development. In the cycling ovary, follicles are found at all stages of development, but primordial follicles predominate. The primordial follicle is the earliest stage of follicular development.

1	The primordial follicle is the earliest stage of follicular development. Primordial follicles first appear in the ovaries during the third month of fetal development. Early growth of the primordial follicles is independent of gonadotropin stimulation. In the mature ovary, primordial follicles are found in the stroma of the cortex just beneath the tunica albuginea. A single layer of squamous follicle cells surrounds the oocyte (Fig. 23.3 and Plate 92, page 872). The outer surface of the follicle cells is bounded by a basal lamina. At this stage, the oocyte and the

1	FIGURE 23.3 • Primordial follicle. a. Schematic drawing of a primordial follicle shows the oocyte arrested in prophase of the first meiotic division. The oocyte is closely surrounded by a single layer of squamous follicle cells. The outer surface of these cells is separated from the connective tissue by a basal lamina. The ooplasm contains characteristic organelles, as seen with the electron microscope, including a Balbiani body, annulate lamellae, and small spherical mitochondria. b. This photomicrograph of primordial follicles shows the oocytes surrounded by a single layer of flattened follicle cells (FC). Usually, the nucleus (N) of the oocyte is in an eccentric position. Two oocytes in which the nucleus is not included in the plane of section are indicated (X ). Similarly, there are two follicles (arrows) in which the follicle cells are revealed in face or tangential view and the enclosed oocytes are not included in the section. 640.

1	surrounding follicle cells are closely apposed to one another. The oocyte in the follicle measures about 30 m in diameter and has a large, eccentric nucleus that contains finely dispersed chromatin and one or more large nucleoli. The cytoplasm of the oocyte, referred to as ooplasm, contains a Balbiani body (Fig. 23.3a). At the ultrastructural level, the Balbiani body is revealed as a localized accumulation of Golgi membranes and vesicles, endoplasmic reticulum, numerous mitochondria, and lysosomes. In addition, human oocytes contain annulate lamellae, and numerous small vesicles are scattered throughout the cytoplasm along with small, spherical mitochondria. Annulate lamellae resemble a stack of nuclear envelope profiles. Each layer of the stack includes pore structures that are morphologically identical to nuclear pores. The primary follicle is the first stage in the development of the growing follicle.

1	As a primordial follicle develops into a growing follicle, changes occur in the oocyte, in the follicle cells, and in the adjacent stroma. Initially, the oocyte enlarges, and the surrounding flattened follicle cells proliferate and become cuboidal. At this stage—that is, when the follicle cells become cuboidal— the follicle is identified as a primary follicle. As the oocyte grows, it secretes specific proteins that are assembled into an extracellular coat called the zona pellucida. It appears between the oocyte and the adjacent follicle cells (Fig. 23.4). The zonal pellucida in humans is composed of three classes of sulfated acidic zona pellucida (ZP) glycoproteins termed ZP-1 (80 to 120 kilodaltons), ZP-2 (73 kilodaltons), and ZP-3 (59 to 65 kilodaltons). Of the three, the most important is the ZP-3, which functions as the spermatozoa-binding receptor and inducer of the acrosome reaction (see page 841); ZP-2 is believed to act as a secondary spermatozoa-binding protein, and ZP-1 has

1	is the ZP-3, which functions as the spermatozoa-binding receptor and inducer of the acrosome reaction (see page 841); ZP-2 is believed to act as a secondary spermatozoa-binding protein, and ZP-1 has not yet been functionally characterized. In the light microscope, zona pellucida is clearly visible as a homogeneous and refractile layer that stains deeply with acidophilic stains and with the periodic acid–Schiff (PAS) reagents (Plate 92, page 872). It is first apparent when the oocyte, surrounded by a single layer of cuboidal or columnar follicle cells, has grown to a diameter of 50 m to 80 m.

1	Follicle cells undergo stratification to form the granulosa layer of the primary follicle. Through rapid mitotic proliferation, the single layer of follicle cells gives rise to a stratified epithelium, the membrana granulosa (stratum granulosum), surrounding the oocyte. The follicle cells are now identified as granulosa cells. The basal lamina retains its position between the outermost layer of the follicle cells, which become columnar, and the connective tissue stroma. During follicular growth, extensive gap junctions develop between granulosa cells. Unlike Sertoli cells in the testis, however, the basal layer of the granulosa cells does not possess elaborate tight junctions (zonulae occludentes), indicating the absence of a blood–follicle barrier. Movement of nutrients and small informational macromolecules from the blood into the follicular fluid is essential for normal development of the ovum and follicle. Connective tissue cells form the theca layers of the primary follicle.

1	Connective tissue cells form the theca layers of the primary follicle. As the granulosa cells proliferate, stromal cells immediately surrounding the follicle form a sheath of connective tissue cells, known as the theca folliculi, just external to the basal PRIMARY FOLLICLE stromal cells forming zona pellucida oocyte a follicle cells bbbbb FCFCFCFCFC FIGURE 23.4 • Early primary follicle. a. Schematic drawing of a primary follicle in an early stage of development. Note the formation of the zona pellucida between the oocyte and the adjacent follicle cells. A single layer of cuboidal follicle cells surrounds the growing oocyte. b. Photomicrograph of a primary follicle. Note the distinct layer of follicle cells (FC) surrounding the oocyte. 640.

1	LATE PRIMARY FOLLICLE granulosa cells stratum granulosum cortical granules oocyte cytoplasm granulosa cells microvilli of oocyte gap junction basal lamina zona pellucida process of granulosa cell theca folliculi a bbbbb GCGCGCGCGC ZPZPZPZPZP FIGURE 23.5 • Late primary follicle. a. Schematic drawing of a late primary follicle shows a multilayered mass of granulosa cells (differentiated from follicle cells) surrounding the oocyte. Note that the innermost layer of granulosa cells is adjacent to the zona pellucida, and the outermost layer of these cells rests on the basal lamina, which is adjacent to the stromal cells now called the theca folliculi. The Balbiani body at this stage reorganizes into multiple Golgi units, and cortical granules appear in the cytoplasm. The wedge-shaped enlargement depicts the ultrastructure of an oocyte and adjacent follicle cells. Numerous microvilli from the oocyte and slender processes from the granulosa cells extend into the zona pellucida that surrounds

1	depicts the ultrastructure of an oocyte and adjacent follicle cells. Numerous microvilli from the oocyte and slender processes from the granulosa cells extend into the zona pellucida that surrounds the oocyte. Processes of the granulosa cells contact the plasma membrane of the oocyte. b. Photomicrograph of a late primary follicle (monkey). Multiple layers of granulosa cells (GC) can be seen surrounding the primary oocyte. The zona pellucida (ZP) is present between the oocyte and follicle cells. 160.

1	lamina (Fig. 23.5). The theca folliculi further differentiates into two layers:  The theca interna is the inner, highly vascularized layer of cuboidal secretory cells (Plate 93, page 874). The fully differentiated cells of the theca interna possess ultrastructural features characteristic of steroid-producing cells. Cells of the theca interna possess a large number of luteinizing hormone (LH) receptors. In response to LH stimulation, they synthesize and secrete the androgens that are the precursors of estrogen. In addition to secretory cells, the theca interna contains fibroblasts, collagen bundles, and a rich network of small vessels typical of endocrine organs.  The theca externa is the outer layer of connective tissue cells. It contains mainly smooth muscle cells and bundles of collagen fibers.

1	 The theca externa is the outer layer of connective tissue cells. It contains mainly smooth muscle cells and bundles of collagen fibers. Boundaries between the thecal layers and between the theca externa and surrounding stroma are not distinct. However, the basal lamina between the granulosa layer and the theca interna establishes a distinct boundary between these layers. It separates the rich capillary bed of the theca interna from the granulosa layer, which is avascular during the period of follicular growth. Maturation of the oocyte occurs in the primary follicle.

1	Maturation of the oocyte occurs in the primary follicle. The distribution of organelles changes as the oocyte matures. Multiple, dispersed Golgi elements derived from the single Balbiani body of the primordial oocyte become scattered in the cytoplasm. The number of free ribosomes, mitochondria, small vesicles, and multivesicular bodies and the amount of rough-surfaced endoplasmic reticulum (rER) increase. Occasional lipid droplets and masses of lipochrome pigment may also be seen. The oocytes of many species, including mammals, exhibit specialized secretory vesicles known as cortical granules (see Fig. 23.5a). They are located just beneath the oolemma (oocyte’s plasma membrane). The granules contain proteases that are released by exocytosis when the ovum is activated by the sperm (discussed in the section on fertilization).

1	Numerous irregular microvilli project from the oocyte into the perivitelline space between the oocyte and the surrounding granulosa cells as the zona pellucida is deposited (see Fig. 23.5). At the same time, slender processes from the granulosa cells develop and project toward the oocyte, intermingling with oocyte microvilli and, occasionally, invaginating into the oocyte plasma membrane. The processes may contact the plasma membrane but do not establish cytoplasmic continuity between the cells. The secondary follicle is characterized by a fluid-containing antrum. The primary follicle initially moves deeper into the cortical stroma as it increases in size, mostly through proliferation of the granulosa cells. Several factors are required for oocyte and follicular growth:  Follicle-stimulating hormone (FSH) Growth factors (e.g., epidermal growth factor [EGF],

1	When the stratum granulosum reaches a thickness of 6 to 12 cell layers, fuid-flled cavities appear among the granulosa cells (Fig. 23.6). As the hyaluronan-rich fluid called liquor folliculi continues to accumulate among the granulosa cells, the cavities begin to coalesce, eventually forming a single, crescent-shaped cavity called the antrum. The follicle is now identified as a secondary follicle or antral follicle (Plate 93, page 874). The eccentrically positioned oocyte, which has attained a diameter of about 125 m, undergoes no further growth. The inhibition of growth is achieved by the presence of a small, 1to 2-kilodalton peptide called oocyte maturation inhibitor (OMI), which is secreted by the granulosa cells into the antral fluid. A direct correlation is observed between the size of the secondary follicle and OMI concentration. The concentration is highest in small follicles and lowest in mature follicles. The follicle, which was 0.2 mm in diameter as an early secondary

1	size of the secondary follicle and OMI concentration. The concentration is highest in small follicles and lowest in mature follicles. The follicle, which was 0.2 mm in diameter as an early secondary follicle when the fluid first appeared, continues to grow and reaches 10 mm or more in diameter.

1	Cells of the cumulus oophorus form a corona radiata around the secretory follicle oocyte. As the secondary follicle increases in size, the antrum, lined by several layers of granulosa cells, also enlarges (Fig. 23.7). The stratum granulosum has a relatively uniform thickness except for the region associated with the oocyte. Here the granulosa cells form a thickened mound, the cumulus oophorus, which projects into the antrum. The cells of the cumulus oophorus that immediately surround the oocyte and remain with it at ovulation are referred to as the corona radiata. The corona radiata is composed of cumulus cells that send penetrating microvilli throughout the zona pellucida to communicate via gap junctions with microvilli of the oocyte. During follicular maturation, the number of surface microvilli of

1	FIGURE 23.6 • Secondary follicle. a. Schematic drawing of a secondary follicle showing the fluid-filled antrum, which arises by the coalescence of small fluid-filled cavities among the granulosa cells. Note that this actively growing follicle has many dividing granulosa cells. Call-Exner bodies appear at this stage. The wedge-shaped enlargement of the shadowed area depicts the relationship of the granulosa cells, basal lamina, and the theca interna and theca externa. The theca interna cells differentiate into highly vascularized, steroid-producing cells. The theca interna is surrounded by an outer layer of stromal cells called the theca externa. The basal lamina separates the granulosa cells from the theca interna. b. Photomicrograph of a secondary follicle. The antrum (A), filled with follicular fluid, is visible within the stratum granulosum (GC). Multiple layers of theca interna cells (TI) and theca externa cells (TE) can be seen outside the basal lamina of the secondary follicle.

1	fluid, is visible within the stratum granulosum (GC). Multiple layers of theca interna cells (TI) and theca externa cells (TE) can be seen outside the basal lamina of the secondary follicle. 85.

1	FIGURE 23.7 • Secondary follicle in a late stage of development. a. Schematic drawing of a mature (Graafian) follicle with a large antrum containing an oocyte embedded within the cumulus oophorus. The cells of the cumulus oophorus immediately surrounding the oocyte remain with it after ovulation and are referred to as the corona radiata. b. Photomicrograph of a mature secondary follicle. Note the large fluid-filled antrum (A) and the cumulus oophorus (CO) containing the oocyte. The remaining cells that surround the lumen of the antrum make up the membrana granulosa (stratum granulosum, SG). The surface of the ovary is visible on the right. Note the presence of two primary follicles (upper right). TI, theca interna. 45.

1	granulosa cells increases and is correlated with an increased number of LH receptors on the free antral surface. Extracellular, densely staining, PAS-positive material called Call-Exner bodies (see Fig. 23.6a) may be seen between the granulosa cells. These bodies are secreted by granulosa cells and contain hyaluronan and proteoglycans. The mature or Graafian follicle contains the mature secondary oocyte.

1	The mature or Graafian follicle contains the mature secondary oocyte. The mature follicle, also known as a Graafan follicle, has a diameter of 10 mm or more. Because of its large size, it extends through the full thickness of the ovarian cortex and causes a bulge on the surface of the ovary. As the follicle nears its maximum size, the mitotic activity of the granulosa cells decreases. The stratum granulosum appears to become thinner as the antrum increases in size. As the spaces between the granulosa cells continue to enlarge, the oocyte and cumulus cells are gradually loosened from the rest of the granulosa cells in preparation for ovulation. The cumulus cells immediately surrounding the oocyte now form a single layer of cells of the corona radiata. These cells and loosely attached cumulus cells remain with the oocyte at ovulation.

1	During this period of follicle maturation, the thecal layers become more prominent. Lipid droplets appear in the cytoplasm of the theca interna cells, and the cells demonstrate ultrastructural features associated with steroid-producing cells. In humans, LH stimulates the cells of the theca interna to secrete androgens, which serve as estrogen precursors. Some androgens are transported to the smooth-surfaced endoplasmic reticulum (sER) in the granulosa cells. In response to FSH, the granulosa cells catalyze the conversion of androgens to estrogens, which in turn stimulate the granulosa cells to proliferate and thereby increase the size of the follicle. Increased estrogen levels from both follicular and systemic sources are correlated with increased sensitization of gonadotropes to gonadotropin-releasing hormone. A surge in the release of FSH or LH is induced in the adenohypophysis approximately 24 hours before ovulation. In response to the LH surge, LH receptors on granulosa cells are

1	hormone. A surge in the release of FSH or LH is induced in the adenohypophysis approximately 24 hours before ovulation. In response to the LH surge, LH receptors on granulosa cells are downregulated (desensitized), and granulosa cells no longer produce estrogens in response to LH. Triggered by this surge, the first meiotic division of the primary oocyte resumes. This event occurs between 12 and 24 hours after the LH surge, resulting in the formation of the secondary oocyte and the first polar body. The granulosa and thecal cells then undergo luteinization and produce progesterone (see page 839, section on the corpus luteum).

1	Ovulation is a hormone-mediated process resulting in the release of the secondary oocyte. Ovulation is the process by which a secondary oocyte is released from the Graafian follicle. The follicle destined to ovulate in any menstrual cycle is recruited from a cohort of several primary follicles in the first few days of the cycle. During ovulation, the oocyte traverses the entire follicular wall, including the germinal epithelium.

1	A combination of hormonal changes and enzymatic effects is responsible for the actual release of the secondary oocyte in the middle of the menstrual cycle (i.e., on the 14th day of a 28-day cycle). These factors include:  Increase in the volume and pressure of the follicular fluid  Enzymatic proteolysis of the follicular wall by activated plasminogen Hormonally directed deposition of glycosaminoglycans between the oocyte–cumulus complex and the stratum granulosum Contraction of the smooth muscle fibers in the theca externa layer, triggered by prostaglandins

1	Just before ovulation, blood flow stops in a small area of the ovarian surface overlying the bulging follicle. This area of the germinal epithelium, known as the macula pellucida or follicular stigma, becomes elevated and then ruptures (Fig. 23.8a). The oocyte, surrounded by the corona radiata and cells of the cumulus oophorus, is released from the ruptured follicle. At the time of ovulation, the fimbriae of the uterine tube become closely apposed to the surface of the ovary, and the cumulus mass containing oocyte is then gently swept by the fimbriae into the abdominal ostium of the uterine tube. The cumulus mass firmly adheres to the fimbriae and is actively transported by the ciliated cells lining the uterine tube, preventing its passage into the peritoneal cavity. Recently, nonsurgical ultrasound technology is being used to monitor ovarian follicle development. Transvaginal ultrasound examination can provide detailed information about

1	FIGURE 23.8 • Endoscopic and ultrasound examination of the ovary. a. This photograph shows a view of the human ovary during the endoscopic oocyte harvest surgery. The ovary is in stage just before ovulation. Note the area of bulging follicle with follicular stigma that is clearly visible. The germinal epithelium covering the tunica albuginea is ruptured in the area of imminent ovulation. b. Development of nonsurgical techniques such as ultrasound imaging helps in monitoring of follicular growth and is useful as a method for determining the time for harvesting preovulatory oocytes. (Courtesy Dr. Charles C. Coddington, III. Mayo Clinic.) the number and size of developing follicles (Fig 23.8b). After ovulation, the secondary oocyte remains viable for approximately 24 hours. If fertilization does not occur during this period, the secondary oocyte degenerates as it passes through the uterine tube.

1	Oocytes that fail to enter the uterine tube usually degenerate in the peritoneal cavity. Occasionally, however, one may be fertilized and implant in the peritoneal cavity on the surface of the ovary or intestine or inside the rectouterine (Douglas) pouch. Such ectopic implantations usually do not develop beyond early fetal stages but may have to be removed surgically. Ectopic pregnancy that develops at any site other than the endometrium of the uterine cavity continues to be the most common cause of death in the first half of pregnancy.

1	Normally, only one follicle completes maturation in each cycle and ruptures to release its secondary oocyte. Rarely, oocytes are released from other follicles that have reached full maturity during the same cycle, leading to the possibility of multiple zygotes. Drugs such as clomiphene citrate (Serophene) or human menopausal gonadotropins that stimulate ovarian activity greatly increase the possibility of multiple births by causing simultaneous maturation of several follicles. The primary oocyte is arrested for 12 to 50 years in the diplotene stage of prophase of the first meiotic division.

1	The primary oocyte is arrested for 12 to 50 years in the diplotene stage of prophase of the first meiotic division. The primary oocytes within the primordial follicles begin the first meiotic division in the embryo, but the process is arrested at the diplotene stage of meiotic prophase (see the section on meiosis in Chapter 3). The first meiotic prophase is not completed until just before ovulation. Therefore, primary oocytes remain arrested in the first meiotic prophase for 12 to 50 years. This long period of meiotic arrest exposes the primary oocyte to adverse environmental inﬂuences and may contribute to errors in meiotic division such as nondisjunction. Such errors result in anomalies such as trisomy of chromosome 21 (Down syndrome).

1	As the first meiotic division (reduction division) is completed in the mature follicle (Fig. 23.9), each daughter cell of the primary oocyte receives an equal share of chromatin, but one daughter cell receives most of the cytoplasm and becomes the secondary oocyte. It measures 150 m in diameter. The other daughter cell receives a minimal amount of cytoplasm and becomes the frst polar body. The secondary oocyte is arrested at metaphase in the second meiotic division just before ovulation.

1	As soon as the first meiotic division is completed, the secondary oocyte begins the second meiotic division. As the secondary oocyte surrounded by the cells of the corona radiata leaves the follicle at ovulation, the second meiotic division (equatorial division) is in progress. This division is arrested at metaphase and completed only if the secondary oocyte is penetrated by a spermatozoon. If fertilization occurs, the secondary oocyte completes the second meiotic division and forms a mature ovum with the maternal pronucleus containing a set of 23 chromosomes. The other cell produced at this division is a second polar body. In humans, the first polar body persists for more than 20 hours after ovulation and does not divide; therefore, the fertilized egg can be recognized by the presence of two polar bodies (diploid first polar body and haploid second polar body). In some mammals, the first polar body can divide, so the final outcome of meiosis consists of one oocyte and three haploid

1	two polar bodies (diploid first polar body and haploid second polar body). In some mammals, the first polar body can divide, so the final outcome of meiosis consists of one oocyte and three haploid polar bodies (see Fig. 23.9). The polar bodies, which are not capable of further development, undergo apoptosis.

1	Polycystic ovarian disease is characterized by bilaterally enlarged ovaries with numerous follicular cysts. When asso-ciated with oligomenorrhea, scanty menstruation, the clinical term Stein-Leventhal syndrome is used. The individual is infertile because of lack of ovulation. Morphologically, the ovaries resemble a small, white balloon filled with tightly packed marbles. Affected ovaries, often called oyster ovaries, have a smooth, pearl-white surface but do not show surface scarring because no ovulations have occurred. The condition is attributable to the large number of fluid-filled fol-licular cysts and atrophic secondary follicles that lie beneath an unusually thick tunica albuginea. The pathogenesis is not clear but seems to be related to a defect in the regulation of androgen biosynthesis that causes production of excessive amounts of androgens that are converted to estrogens. The selection process of the follicles that undergo maturation also seems to be disturbed. The

1	biosynthesis that causes production of excessive amounts of androgens that are converted to estrogens. The selection process of the follicles that undergo maturation also seems to be disturbed. The individual has an anovula-tory cycle characterized by only estrogenic stimulation of the endometrium because of the inhibition of progesterone pro-duction. Progesterone inhibition is caused by failure of the Graafian follicle to transform into a progesterone-producing corpus luteum. The treatment of choice is hormonal to stabi-lize and reconstruct the estrogen-to-progesterone ratio, but in some cases, surgical intervention is necessary. A wedge-shaped incision is made into the ovary to expose the cortex, thus allowing the ova, after hormonal treatment, to leave the ovary without physical restrictions created by the preexisting thickened tunica albuginea (Fig. F23.1.1).

1	 FOLDER 23.1 Clinical Correlation: Polycystic Ovarian Disease TATATATATA FIGURE F23.1.1 • Polycystic ovarian disease. This photo-micrograph shows a section through the cortex of the ovary from an individual with polycystic ovarian disease. Note the unusually thick tunica albuginea (TA) that overlies numerous follicles. The thickness of the tunica albuginea prevents ovulation of the mature (Graafian) follicles. Note that one of the follicles has developed to the primary follicle stage. 45. The collapsed follicle undergoes reorganization into the corpus luteum after ovulation.

1	At ovulation, the follicular wall, composed of the remaining granulosa and thecal cells, is thrown into deep folds as the follicle collapses and is transformed into the corpus luteum (yellow body), or luteal gland (Fig. 23.10a and Plate 94, page 876). At first, bleeding from the capillaries in the theca interna into the follicular lumen leads to formation of the corpus hemorrhagicum with a central clot. Connective tissue from the stroma then invades the former follicular cavity. Cells of the granulosa and theca interna layers then differentiate into granulosa luteal and theca luteal cells in the process called luteinization. These luteal cells undergo dramatic morphologic changes, increasing in size and filling with lipid droplets (Fig. 23.10b). A lipid-soluble pigment, lipochrome, in the cytoplasm of the cells gives them a yellow appearance in fresh preparations. At the ultrastructural level, the cells demonstrate features associated with steroid-secreting cells, namely, abundant sER

1	cytoplasm of the cells gives them a yellow appearance in fresh preparations. At the ultrastructural level, the cells demonstrate features associated with steroid-secreting cells, namely, abundant sER and mitochondria with tubular cristae (Fig. 23.11).

1	Two types of luteal cells are identified:  Granulosa lutein cells are large (about 30 m in diame ter), centrally located cells derived from the granulosa cells. Theca lutein cells are smaller (about 15 m), more deeply staining, and peripherally located cells derived from the cells of the theca interna layer (Plate 94, page 876) As the corpus luteum begins to form, blood and lymphatic vessels from the theca interna rapidly grow into the granulosa layer. A rich vascular network is established within the corpus luteum. This highly vascularized structure located in the cortex of the ovary secretes progesterone and estrogens. These hormones stimulate the growth and secretory activity of the lining of the uterus, the endometrium, to prepare it for the implantation of the developing zygote in the event that fertilization occurs. The corpus luteum of menstruation is formed in the absence of fertilization.

1	The corpus luteum of menstruation is formed in the absence of fertilization. If fertilization and implantation do not occur, the corpus luteum remains active only for 14 days; in this case it is called the corpus luteum of menstruation. In the absence of formation of centrosome of spermatozoon unpacked nucleus of spermatozoon formation of second polar body human chorionic gonadotropin (hCG) and other luteotropins, the rate of secretion of progestogens and estrogens declines, and the corpus luteum begins to degenerate about 10 to 12 days after ovulation.

1	The corpus luteum degenerates and undergoes a slow involution after pregnancy or menstruation. The cells become loaded with lipid, decrease in size, and undergo autolysis. A white scar, the corpus albicans, is formed as intercellular hyaline material accumulates among the degenerating cells of the former corpus luteum (Fig. 23.12). The corpus albicans sinks deeper into the ovarian cortex as it slowly disappears over a period of several months. During capacitation, the mature spermatozoa acquire the ability to fertilize the oocyte.

1	During capacitation, the mature spermatozoa acquire the ability to fertilize the oocyte. Following their maturation in the epididymis, spermatozoa must be activated within the female reproductive tract. During this activation process, called capacitation, structural and functional changes take place in the spermatozoon that result in its increased affinity to bind to zona pellucida receptors. Successful capacitation is confirmed by hyperactivation of the spermatozoa, which manifests as a vigorous, whiplash-like beating pattern of their flagella.

1	Capacitation involves several biochemical changes and modifications to the spermatozoon and its plasma membrane. They include:  Increased activity of adenylyl cyclase leading to increasing levels of cAMP  Increased rate of tyrosine phosphorylation (measurement of tyrosine phosphorylation is used clinically as a biochemical marker of capacitation)  Activation of Ca2 channels resulting in increased intracellular Ca2 levels  Release of seminal fluid glycoconjugates from the surface of the head of the spermatozoon. These surface glycosides (also called decapacitation factors) added during sperm maturation in the epididymis inhibit binding to the zona pellucida receptors  Extensive modification of the plasma membrane by removal of cholesterol, the predominant inhibitor of capacitation, and redistribution of phospholipids and carbohydrate moieties

1	FIGURE 23.9 • Diagram illustrating changes that occur during growth, maturation, and fertilization of the oocyte. The primary oocyte remains arrested in prophase I of meiosis. The first meiotic or reductional division is completed only after the oocyte progresses to ovulation. The second meiotic or equatorial division is not completed unless the secondary oocyte is impregnated by a spermatozoon. Note the formation of first and second polar bodies. In some mammals, the first polar body divides (as shown on this drawing) so that there are four total meiotic products. However, in humans, the first polar body does not divide but persists for about 20 hours; therefore, the fertilized egg can be recognized by the presence of two polar bodies. migration of nucleus to cell surface hyperactivation of spermatozoa impregnation of oocyte and resumption of second meiotic division alignment of male and female pronuclei

1	migration of nucleus to cell surface hyperactivation of spermatozoa impregnation of oocyte and resumption of second meiotic division alignment of male and female pronuclei FIGURE 23.10 • Photomicrograph of human corpus luteum. a. The corpus luteum is formed from the collapsed follicle wall that contains the granulosa and theca cells. The granulosa lutein cells form a thick, folded layer around the former follicular cavity (Cav). Within the folds are cells of the theca interna (arrows). 12. b. This photomicrograph shows the wall of the corpus luteum at higher magnification. The main cell mass is composed of granulosa lutein cells (GLC). These cells have a large spherical nucleus and a large amount of cytoplasm. The theca lutein cells (TLC) also have a spherical nucleus, but the cells are considerably smaller than the granulosa lutein cells. 240. Fertilization normally occurs in the ampulla of the uterine tube.

1	Fertilization normally occurs in the ampulla of the uterine tube. Usually, only a few hundred of the millions of spermatozoa in an ejaculate reach the site of fertilization, typically the ampulla of the uterine tube. On arrival, spermatozoa are confronted with the secondary oocyte surrounded by the corona radiata. Spermatozoa must penetrate the corona radiata to gain access to the zona pellucida. Although several spermatozoa may penetrate the zona pellucida, only one spermatozoon completes the fertilization process. Capacitation is complete when spermatozoa are able to bind to the zona pellucida receptors. Binding to the ZP-3 receptors on the zona pellucida triggers the acrosome reaction in which enzymes (mainly hyaluronidases) released from the acrosome enable a single spermatozoon to penetrate the zona pellucida. Penetration is accomplished by limited proteolysis of the zona pellucida in front of the advancing hypermotile spermatozoon.

1	After penetrating the zona pellucida, the spermatozoon enters the perivitelline space between the zona pellucida and the oocyte plasma membrane (or oolemma). Here the spermatozoon plasma membrane fuses with the oolemma, and the nucleus of the sperm head is finally incorporated into the oocyte. It forms the male pronucleus containing 23 paternal chromosomes. After the alignment and dissolution of nuclear membranes of the two pronuclei, the resulting zygote, with its diploid (2n) complement of 46 chromosomes, undergoes a mitotic division or first cleavage. This two-cell stage marks the beginning of embryonic development. Before spermatozoa can fertilize the secondary oocyte, they must acquire more thrust to penetrate the corona radiata and zona pellucida.

1	Before spermatozoa can fertilize the secondary oocyte, they must acquire more thrust to penetrate the corona radiata and zona pellucida. When approaching a secondary oocyte, a spermatozoon becomes hyperactivated: It swims faster, and its tail movements become more forceful and erratic. Recent studies indicate that this hyperactivation reaction is caused by a sudden influx of Ca2 into the tails of spermatozoa. The plasma membrane of the sperm tail contains large numbers of transmembrane Ca2 FIGURE 23.12 • Photomicrograph of the corpus albicans of a human ovary. Large amounts of hyaline material can be seen among the degenerating cells of the former corpus luteum. The corpus albicans is surrounded by ovarian stroma. 125.

1	FIGURE 23.11 • Electron micrograph of theca lutein cells from the corpus luteum of a monkey. At this early implantation stage (day 10.5 of gestation), membrane-bounded dense bodies are clustered near the Golgi apparatus (G); most of the cytoplasm is packed with tubules of smooth endoplasmic reticulum (sER), lipid droplets (L), and mitochondria (M). Note the capillary (Cap) and the closely apposed cell membranes of the theca lutein cells (arrows). 10,000. (Courtesy of Dr. Carolynn B. Booher.) channel proteins called CatSpers (cation channels of sperm). CatSper proteins are expressed exclusively in membranes of the tail. Influx of Ca2 causes the tail to be more active and bend more forcefully, resulting in faster sperm movement through the viscous environment of the uterine tube. Together with limited proteolysis of the zona pellucida, hyperactivation is responsible for the physical penetration of the oocyte.

1	Sperm hyperactivity is necessary for breaking physical barriers that protect the secondary oocyte from fertilization. Thus, activation of CatSpers is required for male fertility. Impregnation of the oocyte allows structures lying inside the spermatozoon to enter the cytoplasm of the oocyte. After penetrating the zona pellucida, the spermatozoon enters the perivitelline space between the zona pellucida and the oolemma (oocyte’s plasma membrane). Here, after docking to the oolemma, the spermatozoon plasma membrane fuses with the oolemma. This process, called the impregnation of the oocyte, allows the nucleus of the sperm (containing highly concentrated DNA), the centrosome, the midpiece with the mitochondria, and the kinocilium to be incorporated into the cytoplasm of the oocyte. The tail plasma membrane remains as an appendage to the oolemma.

1	An impregnating spermatozoon generates a molecular signal for resumption and termination of the second meiotic division. This division transforms the secondary oocyte into a mature oocyte and triggers the expulsion of the second polar body into the perivitelline space.

1	The male genetic material lying within the nucleus of incorporated sperm head is unpacked and used for building the male pronucleus, which contains 23 paternal chromosomes. Nuclear membranes of both female and male pronuclei dissolve (without fusion), and the chromosomes align themselves within the common mitotic spindle. The resulting zygote contains a diploid (2n) complement of 46 chromosomes and later undergoes the first mitotic division or cleavage. The male centrosome is essential for the alignment of the mitotic spindle that divides chromosomes into the first two cells of the embryo. Only centrosomes from the father are used in building first and subsequent mitotic spindles. The incorporated kinocilium is finally dissolved, and all sperm mitochondria are eliminated from the cytoplasm of the oocyte. Note that all mitochondria in human cells normally derive from the mother, but all centrosomes originate from the father’s sperm cell.

1	Several spermatozoa may penetrate the zona pellucida, but only one spermatozoon completes the fertilization process. As the fertilizing spermatozoon penetrates the ooplasm, at least three types of postfusion reactions occur to prevent other spermatozoa from entering the secondary oocyte (polyspermy). These events include the following.  Fast block to polyspermy. A large and long-lasting (up to 1 minute) depolarization of the oolemma creates a transient electrical block to polyspermy.  Cortical reaction. Changes in the polarity of the oolemma then trigger release of Ca2 from the ooplasmic stores. The Ca2 propagates a cortical reaction wave in which cortical granules move to the surface and fuse with the oolemma, leading to a transient increase in surface area of the ovum and reorganization of the membrane. The contents of the cortical granules are released into the perivitelline space.

1	 Zona reaction. The released enzymes (proteases) of the cortical granules not only degrade the glycoprotein oocyte plasma membrane receptors for sperm binding but also form the perivitelline barrier by cross-linking proteins on the surface of the zona pellucida. This event creates the final and permanent block to polyspermy. The corpus luteum of pregnancy is formed after fertilization and implantation. If fertilization and implantation occur, the corpus luteum increases in size to form the corpus luteum of pregnancy. The existence and function of the corpus luteum depends on a combination of paracrine and endocrine secretions, collectively described as luteotropins. Paracrine luteotropins are locally produced by the ovary. They include:

1	Paracrine luteotropins are locally produced by the ovary. They include: Endocrine luteotropins are produced at a distance from their target organ, the corpus luteum. They include:  hCG, a 37-kilodalton glycoprotein secreted by the trophoblast of the chorion, which stimulates LH receptors on the corpus luteum and prevents its degeneration (page 840)  LH and prolactin, both secreted by the pituitary gland  Insulin, produced by the pancreas

1	High levels of progesterone, produced from cholesterol by the corpus luteum, block the cyclic development of ovarian follicles. In early pregnancy, the corpus luteum measures 2 cm to 3 cm, thus filling most of the ovary. Its function begins to decline gradually after 8 weeks of pregnancy, although it persists throughout pregnancy. Although the corpus luteum remains active, the placenta produces sufficient amounts of estrogens and progestogens from maternal and fetal precursors to take over the function of the corpus luteum after 6 weeks of pregnancy. Human chorionic gonadotropin (hCG) can be detected in the serum as early as 6 days after conception and in the urine as early as 10 to 14 days of pregnancy. Detection of hCG in the urine by specific antibodies forms the basis of most over-the-counter pregnancy tests. In addition, the rapid increase in circulating level of hGC in early pregnancy is responsible for “morning sickness,” a condition characterized by nausea and vomiting. These

1	pregnancy tests. In addition, the rapid increase in circulating level of hGC in early pregnancy is responsible for “morning sickness,” a condition characterized by nausea and vomiting. These symptoms usually occur in the early hours of the morning are often among the first signs of pregnancy.

1	Most ovarian follicles are lost by atresia mediated by apoptosis of granulosa cells. As stated, very few of the ovarian follicles that begin their differentiation in the embryonic ovary are destined to complete their maturation. Most of the follicles degenerate and disappear through a process called ovarian follicular atresia. Atresia is mediated by apoptosis of granulosa cells. Large numbers of follicles undergo atresia during fetal development, early postnatal life, and puberty. After puberty, groups of follicles begin to mature during each menstrual cycle; normally, only one follicle completes its maturation. Atresia is now thought to be a mechanism whereby a few follicles are stimulated to maintain their development through the programmed death of the other follicles. Thus, at any stage of its maturation a follicle may undergo atresia. The process becomes more complex as the follicle progresses toward maturation.

1	In atresia of primordial and small, growing follicles, the immature oocyte becomes smaller and degenerates; similar changes occur in the granulosa cells. Atretic follicles shrink and eventually disappear from the stroma of the ovary as a result of repeated apoptosis and phagocytosis by granulosa cells (Plate 93, page 874). As the cells are reabsorbed and disappear, the surrounding stromal cells migrate into the space previously occupied by the follicle, leaving no trace of its existence.

1	In atresia of large, growing follicles, the degeneration of the mature oocyte is delayed and appears to occur secondary to degenerative changes in the follicular wall (Plate 93, page 874). This delay indicates that once the oocyte has achieved its maturity and competence, it is no longer sensitive to the same stimuli that initiate the atresia in granulosa cells. The follicular changes include the following sequential events:  Initiation of apoptosis within the granulosa cells, indicated by cessation of mitosis and expression of endonucleases and other hydrolytic enzymes within the granulosa cells  Invasion of the granulosa layer by neutrophils and macrophages  Invasion of the granulosa layer by strands of vascularized connective tissue  Sloughing of the granulosa cells into the antrum of the follicle  Hypertrophy of the theca interna cells  Collapse of the follicle as degeneration continues Invasion of connective tissue into the cavity of the follicle

1	Recent studies indicate that several gene products regulate the process of follicular atresia. One of these products is the

1	There are several indications for in vitro fertilization (IVF), but the primary one is infertility as a result of surgically un-correctable damage to, or absence of, the uterine tubes. To induce multiple follicle development and maturation, women selected for an IVF procedure undergo controlled hyper-stimulation of the ovaries. Hyperstimulation is achieved by different hormonal therapies using human menopausal go-nadotropins and clomiphene citrate (Serophene), with or without FSH. Mature preovulatory oocytes are collected from the Graafian follicles by either laparoscopic or ultrasound-guided percutaneous aspiration or transvaginal aspiration. Before insemination, the oocytes are preincubated in a specialized medium with serum complements for a time determined by their stage of maturity. The collected semen is placed in a special medium. The oocytes are then added to the medium containing the col-lected semen for fertilization. Twelve to 16 hours later, the oocytes are examined with

1	The collected semen is placed in a special medium. The oocytes are then added to the medium containing the col-lected semen for fertilization. Twelve to 16 hours later, the oocytes are examined with the differential interference con-trast microscope to determine the presence of female and male pronuclei, the indication of successful fertilization (Fig. F23.2.1a). At this stage, the fertilized oocyte may be frozen for future IVF transfers. Generally, 80% of mature oocytes cultured in vitro are fertilized. At this point, the embryo is transferred to a special growth medium for 24 to 48 hours, where it is allowed to grow to the stage of four to six cells (Fig. F23.2.1b). Several embryos are then trans-ferred into the uterus via the vagina and cervical canal on the third or fourth day after the initial aspiration of the oocyte. Before embryo transfer, the uterus has been pre-pared to receive the embryo by administration of appropri-ate hormones. Embryos are therefore placed into a

1	after the initial aspiration of the oocyte. Before embryo transfer, the uterus has been pre-pared to receive the embryo by administration of appropri-ate hormones. Embryos are therefore placed into a hormonally prepared uterus under conditions equivalent to those in normal implantation (see page 852). Intensive pro-gesterone treatment is usually begun just after the transfer to mimic the function of the corpus luteum of pregnancy. In recent years, existing treatment protocols have been optimized to such an extent that success rates of pregnancy and delivery with IVF programs have reached greater than 30% per embryo transfer. Further improvements in preg-nancy rates may be achieved by the introduction of new drugs, such as recombinant FSH or gonadotropin-releasing hormone (GnRH) antagonists that provide individualized hormonal treatment. Additionally, the occurrence of multiple pregnancies, which is the main complication of IVF, may be limited by reducing the number of transferred

1	that provide individualized hormonal treatment. Additionally, the occurrence of multiple pregnancies, which is the main complication of IVF, may be limited by reducing the number of transferred embryos.

1	 FOLDER 23.2 Clinical Correlation: In Vitro Fertilization aaba b FIGURE F23.2.1 • Early developmental stages of the human embryo. a. This image, obtained with an interference contrast microscope equipped with Nomarski optics, shows a human fertilized oocyte with two pronuclei. The zygote develops after alignment and dissolution of nuclear membranes of both female and male pronuclei. The resulting cell will contain a diploid complement of 46 chromosomes. 400. b. This image shows a 48-hour-old human embryo growing in a special growth medium. At this stage, the embryo consists of four cells. In IVF procedures, it is at this stage that the embryo is usually transferred into the uterine cavity. 400. (Courtesy of Dr. Peter Fehr.) gonadotropin-induced neural apoptosis inhibitory protein (NAIP), which inhibits and delays apoptotic changes in the granulosa cell. NAIP gene expression is present in all stages of the growing follicle but absent in follicles undergoing atresia. A high level of

1	which inhibits and delays apoptotic changes in the granulosa cell. NAIP gene expression is present in all stages of the growing follicle but absent in follicles undergoing atresia. A high level of gonadotropins inhibits apoptosis in ovarian follicles by increasing the expression of NAIP in the ovaries.

1	The oocyte undergoes typical changes associated with degeneration and autolysis, and the remnants are phagocytosed by invading macrophages. The zona pellucida, which is resistant to the autolytic changes occurring in the cells associated with it, becomes folded and collapses as it is slowly broken down within the cavity of the follicle. Macrophages in the connective tissue are involved in the phagocytosis of the zona pellucida and the remnants of the degenerating cells. The basement membrane between the follicle cells from the theca interna may separate from the follicle cells and increase in thickness, forming a wavy hyaline layer called the glassy membrane. This structure is characteristic of follicles in late stages of atresia.

1	Enlargement of the cells of the theca interna occurs in some atretic follicles. These cells are similar to theca lutein cells and become organized into radially arranged strands separated by connective tissue. A rich capillary network develops in the connective tissue. These atretic follicles, which resemble an old corpus luteum, are called corpora lutea atretica. The interstitial gland arises from the theca interna of the atretic follicle.

1	The interstitial gland arises from the theca interna of the atretic follicle. As atretic follicles continue to degenerate, a scar with hyaline streaks develops in the center of the cell mass, giving it the appearance of a small corpus albicans. This structure eventually disappears as the ovarian stroma invades the degenerating follicle. In the ovaries of a number of mammals, the strands of luteal cells do not degenerate immediately but become broken up and scattered in the stroma. These cords of cells contribute to the interstitial gland of the ovary and produce steroid hormones. The development of the interstitial gland is most extensive in animal species that have large litters.

1	In the human ovary, there are relatively few interstitial cells. They occur in the largest numbers in the first year of life and during the early phases of puberty, corresponding to times of increased follicular atresia. At menarche, involution of the interstitial cells occurs; therefore, few are present during the reproductive life span and menopause. It has been suggested that in humans the interstitial cells are an important source of the estrogens that influence growth and development of the secondary sex organs during the early phases of puberty. In other species, the interstitial cells have been shown to produce progesterone.

1	In humans, cells called ovarian hilar cells are found in the hilum of the ovary in association with vascular spaces and nonmyelinated nerve fibers. These cells, which appear to be structurally related to the interstitial cells of the testis, contain Reinke crystalloids. The hilar cells appear to respond to hormonal changes during pregnancy and at the onset of menopause. Research suggests that the hilar cells secrete androgens; hyperplasia or tumors associated with these cells usually lead to masculinization. Blood supply to the ovaries comes from two different sources: Ovarian and uterine arteries.

1	Blood supply to the ovaries comes from two different sources: Ovarian and uterine arteries. The ovarian arteries are the branches of the abdominal aorta that pass to the ovaries through the suspensory ligaments and provide the principal arterial supply to the ovaries and uterine tubes. These arteries anastomose with the second blood source to the ovary, the ovarian branches of the uterine arteries, which arise from the internal iliac arteries. Relatively large vessels arising from this region of anastomosis pass through the mesovarium and enter the hilum of the ovary. These large arteries are called spiral arteries because they branch and become highly coiled as they pass into the ovarian medulla (see Fig. 23.2). Veins accompany the arteries and form a plexus called the pampiniform plexus as they emerge from the hilum. The ovarian vein is formed from the plexus.

1	Veins accompany the arteries and form a plexus called the pampiniform plexus as they emerge from the hilum. The ovarian vein is formed from the plexus. In the cortical region of the ovary, networks of lymphatic vessels in the thecal layers surround the large developing and atretic follicles and corpora lutea. The lymphatic vessels follow the course of the ovarian arteries as they ascend to paraaortic lymph nodes in the lumbar region. Ovaries are innervated by the autonomic ovarian plexus.

1	Ovaries are innervated by the autonomic ovarian plexus. Autonomic nerve fibers that supply the ovary are conveyed mainly by the ovarian plexus. Although it is clear that the ovary receives both sympathetic and parasympathetic fibers, little is known about their actual distribution. Groups of parasympathetic ganglion cells are scattered in the medulla. Nerve fibers follow the arteries, supplying the smooth muscle in the walls of these vessels, as they pass into the medulla and cortex of the ovary. Nerve fibers associated with the follicles do not penetrate the basal lamina. Sensory nerve endings are scattered in the stroma. The sensory fibers convey impulses via the ovarian plexus and reach the dorsal root ganglia of the first lumbar spinal nerves. Therefore, ovarian pain is referred over the cutaneous distribution of these spinal nerves.

1	At ovulation, about 45% of women experience midcycle pain (“mittelschmerz”). It is usually described as a sharp, lower abdominal pain that lasts from a few minutes to as long as 24 hours and is frequently accompanied by a small amount of bleeding from the uterus. It is believed that this pain is related to smooth muscle cell contraction in the ovary as well as in its ligaments. These contractions are in response to an increased level of prostaglandin F2 mediated by the surge of LH.

1	The uterine tubes are paired tubes that extend bilaterally from the uterus toward the ovaries (see Fig. 23.1). Also commonly referred to as the Fallopian tubes, the uterine tubes transport the ovum from the ovary to the uterus and provide the necessary environment for fertilization and initial development of the zygote to the morula stage. One end of 846 changes that involve two phases:  Follicular phase  Luteal phase Ovulation occurs between the two phases (Fig. F23.3.1). The follicular phase begins with the development of a small number of primary follicles (10 to 20) under the influ-ence of FSH and LH. Selection of dominant follicles occurs by days 5 to 7 of the menstrual cycle. During the first 8 to 10 days of the cycle, FSH is the principal hormone influenc-ing the growth of the follicles. It stimulates the granulosa and thecal cells, which begin to secrete steroid hormones, principally estrogens, into the follicular lumen. As estrogen production from the dominant follicle

1	follicles. It stimulates the granulosa and thecal cells, which begin to secrete steroid hormones, principally estrogens, into the follicular lumen. As estrogen production from the dominant follicle increases, FSH is in-hibited by a negative feedback loop from the pituitary gland. Estrogens continue to accumulate in the follicular lumen, fi-nally reaching a level that makes the follicle independent of FSH for its continued growth and development. Late in the follicular phase, before ovulation, progesterone levels begin to rise under the influence of LH. The amount of estrogens in the circulating blood inhibits further production of FSH by the adenohypophysis. Ovulation is induced by a surge in the in the FSH level. It occurs approximately 34 to 36 hours after the start of the LH surge or about 10 to 12 hours after the peak of the LH surge. The luteal phase begins immediately after ovulation as the granulosa and thecal cells of the ruptured follicle undergo rapid morphologic

1	surge or about 10 to 12 hours after the peak of the LH surge. The luteal phase begins immediately after ovulation as the granulosa and thecal cells of the ruptured follicle undergo rapid morphologic transformation to form the cor-pus luteum. Estrogens and large amounts of progesterone are secreted by the corpus luteum. Under the influence of both hormones, but primarily progesterone, the en-dometrium begins its secretory phase, which is essential for the preparation of the uterus for implantation in the event that the egg is fertilized. LH appears to be responsi-ble for the development and maintenance of the corpus lu-teum during the menstrual cycle. If fertilization does not occur, the corpus luteum degenerates within a few days as the hormonal levels drop. If fertilization does occur, the corpus luteum is maintained and continues to secrete pro-gesterone and estrogens. hCG, which is initially produced by the embryo and later by the placenta, stimulates the corpus luteum and is

1	the corpus luteum is maintained and continues to secrete pro-gesterone and estrogens. hCG, which is initially produced by the embryo and later by the placenta, stimulates the corpus luteum and is responsible for its maintenance dur-ing pregnancy. menstrual phase proliferative phase secretory phase 0 5 10 14 20 25 28 0 5 10 15 20 0 10 20 30 40 50 menstrual cycle ovarian hormones (in blood) estrogen progesterone ovarian cycle pituitary hormones (in blood) LH FSH FIGURE F23.3.1 • Relationship of morphologic and physiologic events that occur in the men -strual cycle. This diagram illustrates the relation of the morphologic changes in the endo metrium and ovary to the pituitary and ovarian blood hormone levels that occur during the menstrual cycle. The pituitary and ovarian hormones and their plasma con centrations are indicated in arbitrary units. LH, luteinizing hormone; FSH, follicle-stimulating hormone.

1	 FOLDER 23.3 Functional Considerations: Summary of Hormonal Regulation of the Ovarian Cycle During each menstrual cycle, the ovary undergoes cyclic LH level, which occurs concomitantly with a smaller increase the tube is adjacent to the ovary and opens into the peritoneal cavity; the other end communicates with the uterine cavity. Each uterine tube is approximately 10 cm to 12 cm long and can be divided into four segments by gross inspection:  The infundibulum is the funnel-shaped segment of the tube adjacent to the ovary. At the distal end, it opens into the peritoneal cavity. The proximal end communicates with the ampulla. Fringed extensions, or fmbriae, extend from the mouth of the infundibulum toward the ovary.  The ampulla is the longest segment of the tube, constituting about two thirds of the total length, and is the site of fertilization.  The isthmus is the narrow, medial segment of the uterine tube adjacent to the uterus.

1	 The isthmus is the narrow, medial segment of the uterine tube adjacent to the uterus.  The uterine or intramural part, measuring about 1 cm in length, lies within the uterine wall and opens into the cavity of the uterus. The wall of the uterine tube is composed of three layers. The uterine tube wall resembles the wall of other hollow viscera, consisting of an external serosal layer, an intermediate muscular layer, and an internal mucosal layer. However, there is no submucosa.  The serosa or peritoneum is the outermost layer of the uterine tube and is composed of mesothelium and a thin layer of connective tissue.  The muscularis, throughout most of its length, is organized into an inner, relatively thick circular layer and an outer, thinner longitudinal layer. The boundary between these layers is often indistinct.

1	 The mucosa, the inner lining of the uterine tube, exhibits relatively thin longitudinal folds that project into the lumen of the uterine tube throughout its length. The folds are most numerous and complex in the ampulla (Fig. 23.13 and Plate 95, page 878) and become smaller in the isthmus. The mucosal lining is simple columnar epithelium composed of two kinds of cells—ciliated and nonciliated (Fig. 23.13b). They represent different functional states of a single cell type.  Ciliated cells are most numerous in the infundibulum and ampulla. The wave of the cilia is directed toward the uterus.

1	 Ciliated cells are most numerous in the infundibulum and ampulla. The wave of the cilia is directed toward the uterus. FIGURE 23.13 • Photomicrograph of a human uterine tube. a. This cross section is near the ampulla region of the uterine tube. The mucosa is thrown into extensive folds that project into the lumen of the tube. The muscularis is composed of a thick inner layer of circularly arranged fibers and an outer layer of longitudinal fibers. Note several branches of the uterine and ovarian arteries (BV ) that travel along the uterine tube. 16. b. The lumen of the tube is lined by a simple columnar epithelium composed of ciliated cells (above the point of the arrow) and nonciliated cells (below the point of the arrow). 640.  Nonciliated, peg cells are secretory cells that produce the fluid that provides nutritive material for the ovum.

1	 Nonciliated, peg cells are secretory cells that produce the fluid that provides nutritive material for the ovum. The epithelial cells undergo cyclic hypertrophy during the follicular phase and atrophy during the luteal phase in response to changes in hormonal levels, particularly estrogens. Also, the ratio of ciliated to nonciliated cells changes during the hormonal cycle. Estrogen stimulates ciliogenesis, and progesterone increases the number of secretory cells. At about the time of ovulation, the epithelium reaches a height of about 30 m and is then reduced to about one-half that height just before the onset of menstruation. Bidirectional transport occurs in the uterine tube.

1	The uterine tube demonstrates active movements just before ovulation as the fimbriae become closely apposed to the ovary and localize over the region of the ovarian surface where rupture will occur. As the oocyte is released, the ciliated cells in the infundibulum sweep it toward the opening of the uterine tube and thus prevent it from entering the peritoneal cavity. The oocyte is transported along the uterine tube by peristaltic contractions. The mechanisms by which spermatozoa and the oocyte are transported from opposite ends of the uterine tube are not fully understood. Research suggests that both ciliary movements and peristaltic muscular activity are involved in the movements of the oocyte. The movement of the spermatozoa is much too rapid, however, to be accounted for by intrinsic motility. Fertilization usually occurs in the ampulla, near its junction with the isthmus. The ovum remains in the uterine tube for about 3 days before it enters the uterine cavity.

1	Several conditions that may alter the integrity of the tubal transport system (e.g., inﬂammation, use of intrauterine devices, surgical manipulation, tubal ligation) may cause ectopic pregnancy. The majority of ectopic pregnancies, 98% occur in the uterine tube (tubal pregnancies); remaining sites for the implantation of the blastocyst in ectopic pregnancies are the peritoneal cavity, ovaries, and cervix.

1	The uterus receives the rapidly developing morula from the uterine tube. All subsequent embryonic and fetal development occurs within the uterus, which undergoes dramatic increases in size and development. The human uterus is a hollow, pear-shaped organ located in the pelvis between the bladder and rectum. In a nulliparous woman, it weighs 30 g to 40 g and measures 7.5 cm in length, 5 cm in width at its superior aspect, and 2.5 cm in thickness. Its lumen, which is also flattened, is continuous with the uterine tubes and the vagina. Anatomically, the uterus is divided into two regions:  The body is the large upper portion of the uterus. The an terior surface is almost flat; the posterior surface is convex.

1	The upper, rounded part of the body that expands above the attachment of the uterine tubes is termed the fundus.  The cervix is the lower, barrel-shaped part of the uterus separated from the body by the isthmus (see Fig. 23.1). The lumen of the cervix, the cervical canal, has a constricted opening at each end. The internal os communicates with the cavity of the uterus; the external os with the vagina. FIGURE 23.14 • Photomicrograph of a sagittal section of a human uterus. This section shows the three layers of the uterine wall: the endometrium, the innermost layer that lines the uterine cavity; the myometrium, the middle layer of smooth muscle; and the perimetrium, the very thin layer of peritoneum that covers the exterior surface of the uterus. The deep portion of the myometrium contains the larger blood vessels (BV ) that supply the uterus. 8. The uterine wall is composed of three layers (Fig. 23.14).

1	The uterine wall is composed of three layers (Fig. 23.14). From the lumen outward they are as follows.  The endometrium is the mucosa of the uterus.  The myometrium is the thick muscular layer. It is contin uous with the muscle layer of the uterine tube and vagina. The smooth muscle fibers also extend into the ligaments connected to the uterus.  The perimetrium, the outer serous layer or visceral peritoneal covering of the uterus, is continuous with the pelvic and abdominal peritoneum and consists of a mesothelium and a thin layer of loose connective tissue. Beneath the mesothelium, a layer of elastic tissue is usually prominent. The perimetrium covers the entire posterior surface of the uterus but only part of the anterior surface. The remaining part of the anterior surface consists of connective tissue or adventitia.

1	The perimetrium covers the entire posterior surface of the uterus but only part of the anterior surface. The remaining part of the anterior surface consists of connective tissue or adventitia. Both myometrium and endometrium undergo cyclic changes each month to prepare the uterus for implantation of an embryo. These changes constitute the menstrual cycle. If an embryo implants, the cycle stops, and both layers undergo considerable growth and differentiation during pregnancy (described in the next section). The myometrium forms a structural and functional syncytium. The myometrium is the thickest layer of the uterine wall. It is composed of three indistinctly defined layers of smooth muscle:  The middle muscle layer contains numerous large blood vessels (venous plexuses) and lymphatics and is called the stratum vasculare. It is the thickest layer and has interlaced smooth muscle bundles oriented in a circular or spiral pattern.

1	 The smooth muscle bundles in the inner and outer layers are predominantly oriented parallel to the long axis of activity of the endometrium during the cycle are correlated with the maturation of the ovarian follicles (see Folder 23.3). The end of each cycle is characterized by the partial destruction and sloughing of the endometrium, accompanied by bleeding from the mucosal vessels. The discharge of tissue and blood from the vagina, which usually continues for 3 to 5 days, is referred to as menstruation or menstrual fow. The menstrual cycle is defined as beginning on the day when menstrual flow begins. During reproductive life, the endometrium consists of two layers or zones that differ in structure and function (Fig. 23.15 and Plate 96, page 880): contraction, all three layers of the myometrium work together as a functional syncytium expelling the contents of the lumen through a narrow orifice.

1	In the nonpregnant uterus, the smooth muscle cells are about 50 m long. During pregnancy, the uterus undergoes enormous enlargement. The growth is primarily owing to the hypertrophy of existing smooth muscle cells, which may reach more than 500 m in length, and secondarily at tributable to the development of new fibers through the division of existing muscle cells and the differentiation of undifferentiated mesenchymal cells. The amount of connective tissue also increases. As pregnancy proceeds, the uterine wall becomes progressively thinner as it stretches because of the growth of the fetus. After parturition, the uterus returns straight artery to almost its original size. Some muscle fibers degenerate, but most return to their original size. The collagen pro duced during pregnancy to strengthen the myometrium is then enzymatically degraded by the cells that secreted it. The uterine cavity remains larger and the muscular wall remains thicker than before pregnancy.

1	Compared with the body of the uterus, the cervix has more connective tissue and less smooth muscle. Elastic fibers are abundant in the cervix but are found in appreciable quantities only in the outer layer of the myometrium of the body of the uterus. The endometrium proliferates and then degenerates during a menstrual cycle. Throughout the reproductive lifespan, the endometrium undergoes cyclic changes each month that prepare it for the implantation of the embryo and the subsequent events of embryonic and fetal development. Changes in the secretory

1	FIGURE 23.15 • Schematic diagram illustrating arterial blood supply to the endometrium of the uterus. The two layers of the endometrium, the stratum basale and stratum functionale, are supplied by branches of the uterine artery. The spiral arteries located at the interface between these two layers degenerate and regenerate during the menstrual cycle under the influence of estrogens and progesterone. (Based on Weiss L, ed. Cell and Tissue Biology: A Textbook of Histology, 6th ed. Baltimore: Urban & Schwarzenberg, 1988.)  The stratum functionale or functional layer is the thick part of the endometrium, which is sloughed off at men struation.  The stratum basale or basal layer is retained during menstruation and serves as the source for the regeneration of the stratum functionale. The stratum functionale is the layer that proliferates and degenerates during the menstrual cycle.

1	The stratum functionale is the layer that proliferates and degenerates during the menstrual cycle. During the phases of the menstrual cycle, the endometrium varies from 1 mm to 6 mm in thickness. It is lined by a simple columnar epithelium with a mixture of secretory and ciliated cells. The surface epithelium invaginates into the underlying lamina propria, the endometrial stroma, forming the uterine glands. These simple tubular glands, containing fewer ciliated cells, occasionally branch in the deeper aspect of the endometrium. The endometrial stroma, which resembles mesenchyme, is highly cellular and contains abundant inter-cellular ground substance. As in the uterine tube, no submucosa separates the endometrium from the myometrium. The vasculature of the endometrium also proliferates and degenerates during each menstrual cycle.

1	The endometrium contains a unique system of blood vessels (see Fig. 23.15). The uterine artery gives off 6 to 10 arcuate arteries that anastomose in the myometrium. Branches from these arteries, the radial arteries, enter the basal layer of the endometrium where they give off small straight arteries that supply this region of the endometrium. The main branch of the radial artery continues upward and becomes highly coiled; it is therefore called the spiral artery. Spiral arteries give off numerous arterioles that often anastomose as they supply a rich capillary bed. The capillary bed includes thin-walled dilated segments called lacunae. Lacunae may also occur in the venous system that drains the endometrium. The straight arteries and the proximal part of the spiral arteries do not change during the menstrual cycle. The distal portion of the spiral arteries, under the influence of estrogens and progesterone, undergoes degeneration and regeneration with each menstrual cycle.

1	Cyclic Changes During the Menstrual Cycle Cyclic changes of the endometrium during the menstrual cycle are represented by the proliferative, secretory, and menstrual phases. The menstrual cycle is a continuum of developmental stages in the functional layer of the endometrium. It is ultimately controlled by gonadotropins secreted by the pars distalis of the pituitary gland that regulate the steroid secretions of the ovary. The cycle normally repeats every 28 days, during which the endometrium passes through a sequence of morphologic and functional changes. It is convenient to describe the cycle as having three successive phases:  The proliferative phase occurs concurrently with follicular maturation and is influenced by ovarian estrogen secretion.  The secretory phase coincides with the functional activ ity of the corpus luteum and is primarily influenced by progesterone secretion.

1	 The menstrual phase commences as hormone produc tion by the ovary declines with the degeneration of the cor pus luteum (see Folder 23.3). The phases are part of a continuous process; there is no abrupt change from one to the next. The proliferative phase of the menstrual cycle is regulated by estrogens.

1	The proliferative phase of the menstrual cycle is regulated by estrogens. At the end of the menstrual phase, the endometrium consists of a thin band of connective tissue, about 1 mm thick, containing the basal portions of the uterine glands and the lower portions of the spiral arteries (see Fig. 23.15). This layer is the stratum basale; the layer that was sloughed off was the stratum functionale. Under the infuence of estrogens, the proliferative phase is initiated. Stromal, endothelial, and epithelial cells in the stratum basale proliferate rapidly, and the following changes can be seen:  Epithelial cells in the basal portion of the glands reconstitute the glands and migrate to cover the denuded endometrial surface.  Stromal cells proliferate and secrete collagen and ground substance.  Spiral arteries lengthen as the endometrium is reestablished; these arteries are only slightly coiled and do not extend into the upper third of the endometrium.

1	The proliferative phase continues until 1 day after ovulation, which occurs at about day 14 of a 28-day cycle. At the end of this phase, the endometrium has reached a thickness of about 3 mm. The glands have narrow lumina and are relatively straight but have a slightly wavy appearance (Fig. 23.16a). Accumulations of glycogen are present in the basal portions of the epithelial cells. In routine histologic preparations, extraction of the glycogen gives an empty appearance to the basal cytoplasm. The secretory phase of the menstrual cycle is regulated by progesterone.

1	The secretory phase of the menstrual cycle is regulated by progesterone. Under the infuence of progesterone, dramatic changes occur in the stratum functionale, beginning a day or two after ovulation. The endometrium becomes edematous and may eventually reach a thickness of 5 mm to 6 mm. The glands enlarge and become corkscrew shaped, and their lumina become sacculated as they fill with secretory products (Fig. 23.16b). The mucoid fluid produced by the gland epithelium is rich in nutrients, particularly glycogen, required to support development if implantation occurs. Mitoses are now rare. The growth seen at this stage results from hypertrophy of the epithelial cells, an increase in vascularity, and edema of the endometrium. The spiral arteries, however, lengthen and become more coiled. They extend nearly to the surface of the endometrium (Plate 97, page 882).

1	The sequential influence of estrogens and progesterone on the stromal cells enables their transformation into decidual cells. The stimulus for transformation is the implantation of the blastocyst. Large, pale cells rich in glycogen result from this transformation. Although the precise function of these cells is not known, it is clear that they provide a favorable environment for the nourishment of the embryo and that they create a FIGURE 23.16 • Photomicrographs of the uterine lining in proliferative, secretory, and menstrual phases of the menstrual cycle.

1	a. The upper panel shows the endometrium at the proliferative phase of the cycle. During this phase, the stratum functionale (separated by the dashed line from the stratum basale) greatly thickens. 15. The lower panel shows at higher magnification the endometrial glands that extend from the stratum basale to the surface. 55. b. The upper panel shows the endometrium at the secretory phase of the cycle. The glands have acquired a corkscrew shape as the endometrium increases further in thickness. The stratum basale (below the dashed line) exhibits less dramatic changes in morphology. 20. The lower panel shows uterine glands that have been cut in a plane that is close to their long axes. Note the pronounced corkscrew shape of the glands and mucous secretion (arrows). 60. c. The upper panel shows the stratum functionale (above the dashed line). Much of the stratum functionale has degenerated and sloughed away. 15. The lower panel shows the extravasated blood and necrosis of the stratum

1	shows the stratum functionale (above the dashed line). Much of the stratum functionale has degenerated and sloughed away. 15. The lower panel shows the extravasated blood and necrosis of the stratum functionale. 55.

1	specialized layer that facilitates the separation of the placenta The corpus luteum actively produces hormones for about from the uterine wall at the termination of pregnancy. 10 days if fertilization does not occur. As hormone levels rapidly decline, changes occur in the blood supply to the stra-The menstrual phase results from a decline in the ovarian tum functionale. Initially, periodic contractions of the walls of secretion of progesterone and estrogen. the spiral arteries, lasting for several hours, cause the stratum functionale to become ischemic. The glands stop secreting, and the endometrium shrinks in height as the stroma becomes less edematous. After about 2 days, extended periods of arterial contraction, with only brief periods of blood flow, cause disruption of the surface epithelium and rupture of the blood vessels. When the spiral arteries close off, blood flows into the stratum basale but not into the stratum functionale. Blood, uterine fluid, and sloughing stromal and

1	and rupture of the blood vessels. When the spiral arteries close off, blood flows into the stratum basale but not into the stratum functionale. Blood, uterine fluid, and sloughing stromal and epithelial cells from the stratum functionale constitute the vaginal discharge. As patches of tissue separate from the endometrium, the torn ends of veins, arteries, and glands are exposed (Fig. 23.16c). The desquamation continues until only the stratum basale remains. Clotting of blood is inhibited during this period of menstrual flow. Arterial blood flow is restricted except for the brief periods of relaxation of the walls of the spiral arteries. Blood continually seeps from the open ends of the veins. The period of menstrual fow normally lasts about 5 days. The average blood loss in the menstrual phase is 35 ml to 50 ml. Blood flow through the straight arteries maintains the stratum basale.

1	As noted, this process is cyclic. Figure F23.2.1 in Folder 23.3 shows a single cycle of the endometrium and then demonstrates a gravid state as it is established at the end of a secretory phase. In the absence of fertilization, cessation of bleeding would accompany the growth and maturation of new ovarian follicles. The epithelial cells would rapidly proliferate and migrate to restore the surface epithelium as the proliferative phase of the next cycle begins. In the absence of ovulation (a cycle referred to as an anovulatory cycle), a corpus luteum does not form, and progesterone is not produced. In the absence of progesterone, the endometrium does not enter the secretory phase and continues in the proliferative phase until menstruation. In cases of infertility, biopsies of the endometrium can be used to diagnose such anovulatory cycles as well as other disorders of the ovary and endometrium.

1	If fertilization and implantation occur, a gravid phase replaces the menstrual phase of the cycle. If fertilization and subsequent implantation occur, decline of the endometrium is delayed until after parturition. As the blastocyst becomes embedded in the uterine mucosa in the early part of the second week, cells in the chorion of the developing placenta begin to secrete hCG and other luteotropins. These hormones maintain the corpus luteum and stimulate it to continue the production of progesterone and estrogens. Thus, the decline of the endometrium is prevented, and the endometrium undergoes further development during the first few weeks of pregnancy. Implantation is the process by which the blastocyst settles into the endometrium.

1	Implantation is the process by which the blastocyst settles into the endometrium. The fertilized human ovum undergoes a series of changes as it passes through the uterine tube into the uterine cavity in preparation for becoming embedded in the uterine mucosa. The zygote undergoes cleavage, followed by a series of mitotic divisions without cell growth, resulting in a rapid increase in the number of cells in the embryo. Initially, the embryo is under the control of maternal informational macromolecules that have accumulated in the cytoplasm of the ovum during oogenesis. Later development depends on activation of the embryonic genome, which encodes various growth factors, cell junction components, and other macromolecules required for normal progression to the blastocyst stage.

1	The cell mass resulting from the series of mitotic divisions is known as a morula (L. morum, mulberry), and the individual cells are known as blastomeres. During the third day after fertilization, the morula, which has reached a 12to 16cell stage and is still surrounded by the zona pellucida, enters the uterine cavity. The morula remains free in the uterus for about a day while continued cell division and development occur. The early embryo gives rise to a blastocyst, a hollow sphere of cells with a centrally located clump of cells. This inner cell mass will give rise to the tissues of the embryo proper; the surrounding layer of cells, the outer cell mass, will form the trophoblast and then the placenta (Fig. 23.17).

1	Fluid passes inward through the zona pellucida during this process, forming a fluid-filled cavity, the blastocyst cavity. This event defines the beginning of the blastocyst. As the blastocyst remains free in the uterine lumen for 1 or 2 days and undergoes further mitotic divisions, the zona pellucida disappears. The outer cell mass is now called the trophoblast, and the inner cell mass is referred to as the embryoblast. Implantation occurs during a short period known as the implantation window. The attachment of the blastocyst to the endometrial epithelium occurs during the implantation window, the period when the uterus is receptive for implantation of the blastocyst. This short period results from a series of programmed actions of progesterone and estrogens on the endometrium.

1	Antiprogesterone drugs such as mifepristone (RU-486) and its derivatives compete for the receptors in the endometrial epithelium, thus blocking hormone binding. The failure of progesterone to gain access to its receptors prevents implantation, thus effectively closing the window. In the human, the implantation window begins on day 6 after the LH surge and is completed by day 10. As contact is made with the uterine wall by the trophoblastic cells over the embryoblast pole, the trophoblast rapidly proliferates and begins to invade the endometrium. The invading trophoblast differentiates into the syncytiotrophoblast and the cytotrophoblast.  The cytotrophoblast is a mitotically active inner cell layer producing cells that fuse with the syncytiotrophoblast, the outer erosive layer.  The syncytiotrophoblast is not mitotically active and consists of a multinucleate cytoplasmic mass; it actively invades the epithelium and underlying stroma of the endometrium.

1	Through the activity of the trophoblast, the blastocyst is entirely embedded within the endometrium on about the 11th day of development. (Further development of the syncytiotrophoblast and cytotrophoblast is described in the section on the placenta.) FIGURE 23.17 • Schematic diagrams of sectioned blastocysts. a. A human blastocyst at about 4.5 days of development showing formation of the inner cell mass. b. A monkey blastocyst at about 9 days of development. The trophoblastic cells of the monkey blastocyst have begun to invade the epithelial cells of the endometrium. In humans, the blastocyst begins to invade the endometrium at about the fifth or sixth day of development. c. A human blastocyst at 14 days after implantation. At this stage, the trophoblast cells have differentiated into syncytiotrophoblasts and cytotrophoblasts.

1	The syncytiotrophoblast has well-developed Golgi complexes, abundant sER and rER, numerous mitochondria, and relatively large numbers of lipid droplets. These features are consistent with the secretion of progesterone, estrogens, hCG, and lactogens by this layer. Recent evidence indicates that cytotrophoblast cells may also be a source of steroid hormones and hCG. After implantation, the endometrium undergoes decidualization. During pregnancy, the portion of the endometrium that undergoes morphologic changes is called the decidua or decidua graviditas. As its name implies, this layer is shed with the placenta at parturition. The decidua includes all but the deepest layer of the endometrium. The stromal cells differentiate into large, rounded decidual cells (see page 850). The uterine glands enlarge and become more coiled during the early part of pregnancy and then become thin and flattened as the growing fetus fills the uterine lumen.

1	Three different regions of the decidua are identified by their relationship to the site of implantation (Fig. 23.18):  The decidua basalis is the portion of the endometrium that underlies the implantation site. The decidua capsularis is a thin portion of en dometrium that lies between the implantation site and the uterine lumen.  The decidua parietalis includes the remaining en dometrium of the uterus. By the end of the third month, the fetus grows to the point that the overlying decidua capsularis fuses with the decidua parietalis of the opposite wall, thereby obliterating the uterine cavity. By the 13th day of development, an extraembryonic space, the chorionic cavity, has been established (see Fig. 23.17c).

1	By the 13th day of development, an extraembryonic space, the chorionic cavity, has been established (see Fig. 23.17c). The cell layers that form the outer boundary of this cavity (i.e., the syncytiotrophoblast, cytotrophoblast, and extraembryonic somatic mesoderm) are collectively referred to as the chorion. The innermost membranes enveloping the embryo are called the amnion (see Fig. 23.18). The endometrium of the cervix differs from the rest of the uterus.

1	The cervical mucosa measures about 2 to 3 mm in thickness and differs dramatically from the rest of the uterine endometrium in that it contains large, branched glands (Fig. 23.19 and Plate 98, page 884). It also lacks spiral arteries. The cervical mucosa undergoes little change in thickness during the menstrual cycle and is not sloughed during the period of menstruation. During each menstrual cycle, however, the cervical glands undergo important functional changes that are related to the transport of spermatozoa within the cervical canal. The amount and properties of the mucus secreted by the gland cells vary during the menstrual cycle under the influence of the ovarian hormones. At midcycle, the amount of mucus produced increases 10-fold. This mucus is less viscous and appears to provide a more favorable environment for sperm migration. The cervical mucus at other times in the cycle restricts the passage of sperm into the uterus. Thus, hormonal mechanisms ensure that ovulation and

1	a more favorable environment for sperm migration. The cervical mucus at other times in the cycle restricts the passage of sperm into the uterus. Thus, hormonal mechanisms ensure that ovulation and changes in the cervical mucus are coordinated, thereby increasing the possibility that fertilization will occur if freshly ejaculated spermatozoa and the ovum arrive simultaneously at the site of fertilization in the uterine tube.

1	Blockage of the openings of the mucosal glands results in the retention of their secretions, leading to formation of dilated cysts within the cervix called Nabothian cysts. Nabothian FIGURE 23.18 • Development of the placenta. This schematic drawing shows growth of the uterus during human pregnancy and development of the placenta and its membranes. Note that there is a gradual obliteration of the uterine lumen and disappearance of the decidua capsularis as the definitive placenta is established. (Modified from Williams J. Am J Obstet Gynecol 1927;13:1.) cysts develop frequently but are clinically important only if numerous cysts produce marked enlargement of the cervix. The transformation zone is the site of transition between vaginal stratified squamous epithelium and cervical simple columnar epithelium.

1	The transformation zone is the site of transition between vaginal stratified squamous epithelium and cervical simple columnar epithelium. The portion of the cervix that projects into the vagina, the vaginal part or ectocervix, is covered with a stratified squamous epithelium (Fig. 23.20). An abrupt transition between this squamous epithelium and the mucus-secreting columnar epithelium of the cervical canal, the endocervix, occurs in the transformation zone that during the reproductive age of the woman is located just outside the external os (Plate 98, page 884). Before puberty and after menopause, the transformation zone resides in the cervical canal (Fig. 23.21). Metaplastic changes in this transformation zone constitute precancerous lesions of the cervix. Metaplasia (Gr.

1	Metaplastic changes in this transformation zone constitute precancerous lesions of the cervix. Metaplasia (Gr. í, change in form) represents an adoptive and reversible response to persistent injury of the epithelium caused by chronic infections. It results from a reprogramming of epithelial stem cells that begin to differentiate into new cell lineage. Within the cervical canal (endocervix) it is manifested as a replacement of the simple columnar epithelium with fully mature stratified squamous epithelium (Fig. 23.22). The cervical epithelial cells are constantly exfoliated into the vagina. Stained preparations of the cervical cells (Papanicolaou [Pap] smears) are used routinely for screening and diagnosis of precancerous and cancerous lesions of the cervix. The developing fetus is maintained by the placenta, which develops from fetal and maternal tissues.

1	The developing fetus is maintained by the placenta, which develops from fetal and maternal tissues. The placenta consists of a fetal portion, formed by the chorion, and a maternal portion, formed by the decidua basalis. The two parts are involved in physiologic exchange of substances between the maternal and fetal circulations. The uteroplacental circulatory system begins to develop around day 9, with development of vascular spaces called trophoblastic lacunae within the syncytiotrophoblast. Maternal sinusoids, which develop from capillaries of the maternal side, anastomose with the trophoblastic lacunae (Fig. 23.23). The differential pressure between the arterial and venous channels that communicate with the lacunae establishes directional flow from the arteries into the veins, thereby establishing a primitive uteroplacental circulation. Numerous pinocytotic vesicles present in the syncytiotrophoblast indicate the transfer of nutrients from the maternal vessels to the embryo.

1	Proliferation of the cytotrophoblast, growth of chorionic mesoderm, and blood vessel development successively give rise to the following.  Primary chorionic villi are formed by the rapidly proliferating cytotrophoblast. They send cords or masses of cells into the blood-filled trophoblastic lacunae in the syncytiotrophoblast (see Fig. 23.17b). The primary villi appear between days 11 and 13 of development.  Secondary chorionic villi are composed of a central core of mesenchyme surrounded by an inner layer of cytotrophoblast and an outer layer of syncytiotrophoblast. They develop at about day 16 when the primary chorionic villi become invaded by loose connective tissue from chorionic mesenchyme. The secondary villi cover the entire surface of the chorionic sac (Fig. 23.23a).

1	 Tertiary chorionic villi are formed by the end of the third week as the secondary villi become vascularized by blood vessels that have developed in their connective tissue cores (Fig. 23.23b and Plate 100, page 888). As the tertiary villi are forming, cytotrophoblastic cells in the villi continue to grow out through the syncytiotrophoblast.

1	FIGURE 23.19 • Photomicrograph of a human cervix. This H&E–stained specimen is from a postmenopausal woman. Its lower portion projects into the upper vagina where an opening, the external os, leads to the uterus through the cervical canal. The surface of the cervix is covered by stratified squamous epithelium (SSE) that is continuous with the epithelial lining of the vagina. An abrupt transition from stratified squamous epithelium to simple columnar epithelium (SCE) occurs at the entry to the cervical canal. In this specimen, the stratified epithelium has extended into the canal, an event that occurs with aging. Mucus-secreting cervical glands are seen along the cervical canal. These are simple branched tubular glands that arise as invaginations of the epithelium lining the canal. Frequently, the glands develop into nabothian cysts as a result of retention of mucous secretion by blockage of the gland opening. The material marked by the X is mucus secreted from the cervical glands. 10.

1	When they meet the maternal endometrium, they grow laterally and meet similar processes growing from neighboring villi. Thus, a thin layer of cytotrophoblastic cells called the trophoblastic shell is formed around the syncytiotrophoblast. The trophoblastic shell is interrupted only at sites where maternal vessels communicate with the intervillous spaces. Future growth of the placenta is accomplished by interstitial growth of the trophoblastic shell. FIGURE 23.20 • Stratified squamous epithelium of the ectocervix. The stratified squamous epithelium and underlying fibrous connective tissue within the lower rectangle in Figure 23.22 is shown here at higher magnification. The more mature epithelial cells have a clear cytoplasm (arrowheads), a reflection of their high glycogen content. Also, note the connective tissue papillae protruding into the epithelium (arrows). The bulk of the cervix is made up of dense, fibrous connective tissue with relatively little smooth muscle. 120.

1	FIGURE 23.21 • Transformation zone of the cervix. The site of the squamocolumnar junction from the upper rectangle in Figure 23.19 is shown here at higher magnification. Note the abrupt change from stratified squamous epithelium to simple columnar epithelium (arrow). Neoplastic changes leading to development of cervical cancer most frequently begin in this transformation zone. Within the connective tissue are the branched, mucus-secreting cervical glands (CG) composed of a simple columnar epithelium that is continuous with the lining epithelium of the cervical canal. 120. FIGURE 23.22 • Metaplastic stratified squamous epithelium of the cervical canal. This photomicrograph shows an island of the fully mature stratified squamous epithelium surrounded by the simple columnar epithelium normally found in the cervical canal. 450. (Courtesy of Dr. Fabiola Medeiros, Mayo Clinic.)

1	Several types of cells are recognized in the connective tissue stroma of the villi: mesenchymal cells, reticular cells, fibroblasts, myofibroblasts, smooth muscle cells, macrophages, and fetal placental antigen–presenting cells, historically also known as Hofbauer cells (Fig. 23.24 and Plate 100, page 888). Fetal placental antigen–presenting cells are the specific villous macrophages of fetal origin that participate in the placental innate immune reactions. In response to antigen, they proliferate and upregulate specific surface receptors that recognize and bind to a variety of pathogens. Like other antigen-presenting cells, if stimulated, they increase the number of MHC II (major histocompatibility complex) molecules on their surface.

1	They are more common in the early placenta. The vacuoles in these cells contain lipids, glycosaminoglycans, and glycoproteins. Recent studies of HIV-infected placentas indicate that HIV is primarily localized within the fetal placental antigen–presenting cells and in the syncytiotrophoblast. Early in development, the blood vessels of the villi become connected with vessels from the embryo. Blood begins to circulate through the embryonic cardiovascular system and the villi at about 21 days. The intervillous spaces provide the site of exchange of nutrients, metabolic products and intermediates, and wastes between the maternal and fetal circulatory systems.

1	During the first 8 weeks, villi cover the entire chorionic surface, but as growth continues, villi on the decidua capsularis begin to degenerate, producing a smooth, relatively avascular surface called the chorion laeve. The villi adjacent to the decidua basalis rapidly increase in size and number and become highly branched. This region of the chorion, which is the fetal component of the placenta, is called the chorion frondosum or villous chorion. The layer of the placenta from which the villi project is called the chorionic plate (Plate 99, page 886).

1	During the period of rapid growth of the chorion frondosum, at about the fourth to fifth month of gestation, the fetal part of the placenta is divided by the placental (decidual) septa into 15 to 25 areas called cotyledons. Wedge-like placental septa form the boundaries of the cotyledons, and because they do not fuse with the chorionic plate, maternal blood can circulate easily between them. Cotyledons are visible as the bulging areas on the maternal side of the basal plate. The decidua basalis forms a compact layer known as the basal plate that is the maternal component of the placenta. maternal sinusoid syncytiotrophoblast cytotrophoblast cytotrophoblastic shell tertiary villus developing vessels in wall of chorionic sac intervillus space

1	maternal sinusoid syncytiotrophoblast cytotrophoblast cytotrophoblastic shell tertiary villus developing vessels in wall of chorionic sac intervillus space FIGURE 23.23 • Schematic diagrams of sections through a developing human embryo. a. This drawing shows the chorionic sac and placenta at 16 days of development. b. The same embryo at 21 days of development. The diagrams illustrate the separation of the fetal and maternal blood vessels by the placental membrane, which is composed of the endothelium of the capillaries, mesenchyme, cytotrophoblast, and syncytiotrophoblast. Vessels within this part of the endometrium supply blood to the intervillous spaces. Except for relatively rare rupturing of capillary walls, which is more common at delivery, fetal blood and maternal blood do not mix. Fetal and maternal blood are separated by the placental barrier.

1	Fetal and maternal blood are separated by the placental barrier. Separation of the fetal and maternal blood, referred to as the placental barrier, is maintained primarily by the layers of fetal tissue (Fig. 23.25). Starting at the fourth month, these layers become very thin to facilitate the exchange of products across the placental barrier. The thinning of the wall of the villus is caused in part by the degeneration of the inner cytotrophoblast layer. At its thinnest, the placental barrier consists of the:  Syncytiotrophoblast Discontinuous inner cytotrophoblast layer  Basal lamina of the trophoblast Connective (mesenchymal) tissue of the villus Basal lamina of the endothelium  Endothelium of the fetal placental capillary in the tertiary

1	This barrier bears a strong resemblance to the air–blood barrier of the lung, with which it has an important parallel function, namely, the exchange of oxygen and carbon dioxide—in this case between the maternal blood and the fetal blood. It also resembles the air–blood barrier by having a particular type of macrophage in its connective tissue— in this instance, the fetal placental antigen–presenting cells (Hofbauer cell). FIGURE 23.24 • Photomicrographs of a human placenta. a.

1	This H&E–stained specimen shows the amniotic surface (A), the chorionic plate (CP), and, below, the various-sized profiles of the chorionic villi (CV ). These villi emerge from the chorionic plate as large stem villi and branch into the increasingly smaller villi. Blood vessels (BV ) are evident in the larger villi. The smallest villi contain capillaries where exchange takes place. 60. Upper inset. This higher magnification shows the simple cuboidal epithelium of the amnion and the underlying connective tissue. 200. Lower inset. This higher magnification shows a cross-sectioned villus containing several larger blood vessels and its thin surface syncytiotrophoblast layer. 200. b. This H&E–stained specimen shows the maternal side of the placenta. The stratum basale (SB), the part of the uterus to which some of the chorionic villi (CV ) anchor, is seen at the bottom of the micrograph. Also evident is a stromal connective tissue (CT ) component, part of the stratum basale, to which many

1	to which some of the chorionic villi (CV ) anchor, is seen at the bottom of the micrograph. Also evident is a stromal connective tissue (CT ) component, part of the stratum basale, to which many of the chorionic villi are also attached. Within the stratum basale and the connective tissue stroma are clusters of cells, the decidual cells (arrows), which arose from connective tissue cells. 60. Inset. Decidual cells seen at higher magnification. 200.

1	FIGURE 23.25 • Human placental barrier in a third trimester of the pregnancy. This high magnification electron micrograph shows the thinnest layer of a fully developed placental barrier (section does not include cytotrophoblast cells that form a discontinuous layer in the human placenta). The lumen of intervillous space containing maternal erythroblasts (ME) (to the left ) is separated from the fetal capillary space containing fetal erythroblasts (FE) (to the right ). The intervillous space is lined by the multinucleated syncytiotrophoblast (Syn). Its surface contains microvilli projecting into maternal blood space. The cytoplasm of syncytiotrophoblast contains multiple nuclei (N) and has an abundance of transport vesicles, rER, and sER, mitochondria, and occasional lipid droplets. The syncytiotrophoblast rests on the basal lamina (TBL), which is separated by the thin layer of the connective tissue (CT ) from the basal lamina (EBL) of the fetal endothelial cells (FEn). 11,000.

1	The syncytiotrophoblast rests on the basal lamina (TBL), which is separated by the thin layer of the connective tissue (CT ) from the basal lamina (EBL) of the fetal endothelial cells (FEn). 11,000. (Courtesy of Dr. Holger Jastrow.)

1	The placenta is the site of exchange of gases and metabolites between the maternal and fetal circulation. Fetal blood enters the placenta through a pair of umbilical arteries (Fig. 23.26). As they pass into the placenta, these arteries branch into several radially disposed vessels that give numerous branches in the chorionic plate. Branches from these vessels pass into the villi, forming extensive capillary networks in close association with the intervillous spaces. Gases and metabolic products are exchanged across the thin fetal layers that separate the two bloodstreams at this level. Antibodies can also cross this layer and enter the fetal circulation to provide passive immunity against a variety of infectious agents—for example, those of diphtheria, smallpox, and measles. Fetal blood returns through a system of veins that parallel the arteries except that they converge on a single umbilical vein.

1	Maternal blood is supplied to the placenta through 80 to 100 spiral endometrial arteries that penetrate the basal plate. Blood from these spiral arteries flows into the base of the intervillous spaces, which contain about 150 ml of maternal blood that is exchanged three to four times per minute. The blood pressure in the spiral arteries is much higher than that in the intervillous spaces. As blood is injected into these spaces at each pulse, it is directed deep into the spaces. As the pressure decreases, the blood flows back over the surfaces of the villi and eventually enters endometrial veins also located in the base of the spaces.

1	Exchange of gases and metabolic products occurs as the blood passes over the villi. Normally, water, carbon dioxide, metabolic waste products, and hormones are transferred from the fetal blood to the maternal blood; water, oxygen, metabolites, electrolytes, vitamins, hormones, and some antibodies pass in the opposite direction. The placental barrier does not exclude many potentially dangerous agents such as alcohol, nicotine, viruses, drugs, exogenous hormones, and heavy metals. Therefore, during pregnancy, exposure to or ingestion of such agents should be avoided to reduce the risk of injury to the embryo or fetus. Before the establishment of blood flow through the placenta, the growth of the embryo is supported in part by metabolic products that are synthesized by or transported through the trophoblast. The syncytiotrophoblast synthesizes glycogen, cholesterol, and fatty acids, as well as other nutrients used by the embryo.

1	The placenta is a major endocrine organ producing steroid and protein hormones. The placenta also functions as an endocrine organ, producing steroid and peptide hormones as well as prostaglandins that play an important role in the onset of labor. Immunocytochemical studies indicate that the syncytiotrophoblast is the site of synthesis of these hormones. The steroid hormones progesterone and estrogen have essential roles in the maintenance of pregnancy. As pregnancy proceeds, the placenta takes over the major role in the secretion of these steroids from the corpus luteum. The placenta produces enough progesterone by the end of the eighth week to maintain pregnancy if the corpus luteum is surgically

1	FIGURE 23.26 • Schematic diagram of mature human placenta. The sagittal section of the uterus (left) with the developing embryo shows the most common location of the placenta. The mature placenta (right) is divided into cotyledons by placental septa that are formed by outgrowths of the decidua basalis. Maternal blood enters the placenta through numerous endometrial spiral arteries that penetrate the basal plate. As the blood enters the cotyledon, it is directed deep into the intervillous spaces (red arrows). It then passes over the surface of the villi, where exchange of gases and metabolic products occurs. The maternal blood finally leaves the intervillous space (black arrows) through endometrial veins. The fetal blood enters the placenta through the umbilical arteries that divide into a series of radially disposed arteries within the chorionic plate. Branches from the vessels pass into the main stem villi and there form extensive capillary networks. The veins within the villi then

1	a series of radially disposed arteries within the chorionic plate. Branches from the vessels pass into the main stem villi and there form extensive capillary networks. The veins within the villi then carry the blood back through a system of veins that parallels the fetal arteries.

1	removed or fails to function. In the production of placental estrogen, the fetal adrenal cortex plays an essential role, providing the precursors needed for estrogen synthesis. Because the placenta lacks the enzymes needed for the production of estrogen precursors, a cooperative fetoplacental (endocrine) unit is established. Clinically, the monitoring of estrogen production during pregnancy can be used as an index of fetal development.

1	The following peptide hormones are secreted by the placenta:  Human chorionic gonadotropin (hCG) is required for implantation and maintenance of the pregnancy. Its synthesis begins around day 6, even before the syncytiotrophoblast is formed. HCG exhibits extensive (about 85%) sequence homology to LH, which is required for ovulation and maintenance of the corpus luteum during the menstrual cycle. Similar to the work of LH during the menstrual cycle, hCG maintains the corpus luteum during early pregnancy. HCG also possesses marked homology to pituitary thyroid-stimulating hormone (TSH), which may account for hyperthyroidism in pregnancy by stimulating the maternal thyroid gland to increase secretion of tetraiodothyronine (T4). Measurement of hCG is used to detect early pregnancy and assess pregnancy viability. Two other clinical conditions that increase the blood levels of hCG include trophoblastic diseases and ectopic pregnancies.

1	 Human chorionic somatomammotropin (hCS), also known as human placental lactogen (hPL), is closely related to human growth hormone. Synthesized in the syncytiotrophoblast, it promotes general growth, regulates glucose metabolism, and stimulates mammary duct proliferation in the maternal breast. The effects of hCS on maternal metabolism are significant, but the role of this hormone in fetal development remains unknown.  IGF-I and IGF-II are produced by and stimulate proliferation and differentiation of the cytotrophoblast.  Endothelial growth factor (EGF) exhibits an age-dependent dual action on the early placenta. In the 4to 5-week-old placenta, EGF is synthesized by the cytotrophoblast and stimulates proliferation of the trophoblast. In the 6to 12-week-old placenta, synthesis of EGF is shifted to the syncytiotrophoblast; it then stimulates and maintains the function of the differentiated trophoblast.

1	 Relaxin is synthesized by decidual cells and is involved in the “softening” of the cervix and the pelvic ligaments in preparation for parturition.  Leptin is synthesized by the syncytiotrophoblast, particularly during the last month of gestation. Leptin appears to  FOLDER 23.4 Clinical Correlation: Fate of the Mature Placenta at Birth

1	The mature placenta measures about 15 to 20 cm in when the placenta attaches too deeply into the uterine wall diameter and 2 to 3 cm in thickness, covers 25% to 30% but does not penetrate the myometrium. Placenta increta of the uterine surface, and weighs 500 g to 600 g at term. (about 15% of all cases) occurs when the placental villi The surface area of the villi of human placenta is estimated penetrate deep into the muscular layer of the myometrum. to be about 10 m2. The microvilli on the syncytiotrophoblast In the remaining 10% of all cases, placenta percreta pen-increase the effective area for metabolic exchange to more etrates through the entire uterine wall and attaches to an-than 90 m2. After birth, the uterus continues to contract, other organ such as the bladder, rectum, intestines, or reducing the luminal surface and inducing placental separa-large blood vessels. It is the most serious complication of tion from the uterine wall. The entire fetal portion of the

1	intestines, or reducing the luminal surface and inducing placental separa-large blood vessels. It is the most serious complication of tion from the uterine wall. The entire fetal portion of the pla-placentation and may cause rupture of the uterus and other centa, fetal membranes, and the intervening projections of complications related to its attachment. A retained abnordecidual tissue are released. During uncomplicated labor, mal placenta or placental fragments may cause massive the placenta separates from the uterine wall and is delivered postpartum bleeding and need to be manually removed. approximately 30 minutes after birth. Placenta increta and percreta are often treated by perform-

1	One of the most severe complications of labor results ing a hysterectomy. from abnormal placentation (abnormal attachment of After physiologic delivery of the placenta, the endomethe placenta on uterine wall). If decidual tissue during im-trial glands and stroma of the decidua basalis regenerate. plantation is disrupted, the placenta invades deep into the Endometrial regeneration is completed by the end of the uterine wall. This may cause one of the three clinical condi-third week postpartum except at the placental site, where tions, known as placenta accreta, placenta increta, or pla-regeneration usually extends for another three weeks. Durcenta percreta. Classification depends on the severity and ing the first week after delivery, remnants of the decidua deepness of the placental attachment. Placenta accreta, are shed and constitute the red-brown uterine discharge accounting for approximately 75% of all cases, occurs known as lochia rubra.

1	regulate maternal nutrient storage to the nutrient requirements of the fetus. It is also involved in transporting nutrients across the placental barrier from mother to the fetus.  Other growth factors stimulate cytotrophoblastic growth (e.g., fibroblast growth factor, colony-stimulating factor [CSF-1], platelet-derived growth factor, and interleukins [IL-1 and IL-3]) or inhibit trophoblast growth and proliferation (e.g., tumor necrosis factor). The vagina is a fibromuscular tube that joins internal reproductive organs to the external environment. The vagina is a fibromuscular sheath extending from the cervix to the vestibule, which is the area between the labia minora. In a virgin, the opening into the vagina may be surrounded by the hymen, folds of mucous membrane extending into the vaginal lumen. The hymen or its remnants are derived from the endodermal membrane that separated the developing vagina from the cavity of the definitive urogenital sinus in the embryo.

1	The vaginal wall (Fig. 23.27) consists of the following.  An inner mucosal layer has numerous transverse folds or rugae (see Fig. 23.1) and is lined with stratified squamous epithelium (Fig. 23.28). Connective tissue papillae from the underlying lamina propria project into the epithelial layer. In humans and other primates, keratohyalin granules may be present in the epithelial cells, but under normal conditions, keratinization does not occur. Therefore, nuclei can be seen in epithelial cells throughout the thickness of the epithelium.  An intermediate muscular layer is organized into two sometimes indistinct, intermingling smooth muscle layers, an outer longitudinal layer, and an inner circular layer. The outer layer is continuous with the corresponding layer in the uterus and is much thicker than the inner layer. Stria ted muscle fibers of the bulbospongiosus muscle are present at the vaginal opening (Plate 101, page 890).

1	 An outer adventitial layer is organized into an inner dense connective tissue layer adjacent to the muscularis and an outer loose connective tissue layer that blends with the adventitia of the surrounding structures. The inner layer contains numerous elastic fibers that contribute to the elasticity and strength of the vaginal wall. The outer layer contains numerous blood and lymphatic vessels and nerves. The vagina possesses a stratified, squamous nonkeratinized epithelium and lacks glands. The lumen of the vagina is lined by stratifed squamous, nonkeratinized epithelium. Its surface is lubricated mainly by mucus produced by the cervical glands. The greater and lesser vestibular glands located in the wall of the vaginal vestibule produce additional mucus that lubricates the vagina. Glands are not present in the wall of the vagina. The epithelium of the vagina undergoes cyclic changes during the menstrual cycle. Under the influence of estrogens, during the follicular

1	FIGURE 23.27 • Photomicrograph of a human vagina. This low-magnification H&E–stained specimen of the vaginal wall shows two of three layers of the vagina: the mucosal layer and the muscular layer (the outer layer, the adventitia, is not included). The mucosal layer consists of a stratified squamous epithelium and the underlying connective tissue. The epithelial connective tissue boundary is typically very irregular, with prominent papillae projecting into the undersurface of the epithelium. The muscular layer is seen only in part; it consists of irregularly arranged bundles of smooth muscle cells. Also, the deep region of the connective tissue contains a rich supply of blood vessels that supply the various layers of the vaginal wall. 40. phase, the epithelial cells synthesize and accumulate glycogen as they migrate toward the surface. Cells are continuously desquamated, but near or during the menstrual phase, the superficial layer of the vaginal epithelium may be shed.

1	The lamina propria exhibits two distinct regions. The outer region immediately below the epithelium is a highly cellular loose connective tissue. The deeper region, adjacent to the muscular layer, is denser and may be considered a submucosa. The deeper region contains many thin-walled veins that simulate erectile tissue during sexual arousal. Numerous elastic fibers are present immediately below the epithelium, and some of the fibers extend into the muscular layer. Many lymphocytes and leukocytes (particularly neutrophils) are found in the lamina propria and migrate into the epithelium. Solitary lymphatic nodules may also be present. The number of lymphocytes and leukocytes in the mucosa and vaginal lumen dramatically increases around the time of menstrual flow. The vagina has few general sensory nerve endings. The sensory nerve endings that FIGURE 23.28 • Photomicrograph of the vaginal mucosa.

1	FIGURE 23.28 • Photomicrograph of the vaginal mucosa. This micrograph, a higher magnification of Figure 23.27, shows the stratified squamous epithelium and mature cells with small pyknotic nuclei. Note a single layer of basal cells and two or three layers of cells undergoing differentiation (with eosinophilic cytoplasm). Projections of the connective tissue papillae into the epithelium give the connective tissue–epithelial junction an uneven appearance. The tips of these projections often appear as isolated structures surrounded by epithelium (arrows). 180. are more plentiful in the lower third of the vagina are probably associated primarily with pain and stretch sensations. The female external genitalia consist of the following parts, which are collectively referred to as the vulva and have a stratified squamous epithelium as follows.  The mons pubis is the rounded prominence over the pubic symphysis formed by subcutaneous adipose tissue.

1	 The mons pubis is the rounded prominence over the pubic symphysis formed by subcutaneous adipose tissue.  The labia majora are two large longitudinal folds of skin, homologous to the skin of the scrotum, that extend from the mons pubis and form the lateral boundaries of the urogenital cleft. They contain a thin layer of smooth muscle that resembles the dartos muscle of the scrotum and a large amount of subcutaneous adipose tissue. The outer surface, like that of the mons pubis, is covered with pubic hair. The inner surface is smooth and devoid of hair. Sebaceous and sweat glands are present on both surfaces (Fig. 23.29).  The labia minora are paired, hairless folds of skin that border the vestibule and are homologous to the skin of the

1	The examination of Pap smears is a valuable diagnostic tool in evaluating the vaginal and cervical mucosae (Fig. F23.5.1). The superficial epithelial cells are scraped from the mucosa, spread on a glass slide, fixed, and then stained with the Papanicolaou stain (a combination of hematoxylin, orange G, and eosin azure). Examination of the Pap smear provides valuable diagnostic information about the epithelium regard-ing pathologic changes, response to hormonal changes dur-ing the menstrual cycle, and the microbial environment of the vagina. The synthesis and release of glycogen by the epithelial cells of the uterus and vagina are directly related to changes in the pH of vaginal fluid. The pH of the fluid, which is normally low, around pH 4, becomes more acid near midcycle as Lactobacillus acidophilus, a lactic acid–forming bacterium in the vagina, metabolizes the se-creted glycogen. An alkaline environment can favor the growth of infectious agents such as Staphylococci, Corynebacterium

1	a lactic acid–forming bacterium in the vagina, metabolizes the se-creted glycogen. An alkaline environment can favor the growth of infectious agents such as Staphylococci, Corynebacterium vaginale, Trichomonas vaginalis, and Candida albicans, causing an abnormal increase in vaginal transexudates and inflammation of the vaginal mucosa and vulvar skin known as vulvovaginitis. These pathologic con-ditions are readily diagnosed with Pap smears. Specific antimicrobial agents (antibiotics, sulfonamides) are used to-gether with nonspecific therapy (acidified 0.1% hexetidine gel) to restore the normal low pH in the vagina and thus pre-vent the growth of these agents. In addition, cervicovaginal Pap smears are widely used for diagnosis of early cervical cancer as well as endome-trial carcinoma. Because cervical lesions may exist in a noninvasive stage for as long as 20 years, the abnormal cells shed from the epithelium are easily detected with a Pap smear examination. Microscopic examination

1	cervical lesions may exist in a noninvasive stage for as long as 20 years, the abnormal cells shed from the epithelium are easily detected with a Pap smear examination. Microscopic examination of these cells permits differentiation between normal and abnormal cells, determines their site of origin, and allows classifying cellular changes related to the spread of the disease. The Pap smear is an extremely effective and in-expensive screening method in preventing cervical can-cer. Most of the cell abnormalities detected by Pap smears are in the precancerous stage, which allows the clinician to implement appropriate therapy.

1	 FOLDER 23.5 Clinical Correlation: Cytologic Pap Smears aabba b FIGURE F23.5.1 • Photomicrographs of cervical smears. a. Negative cervical smear. The surface squamous cells reveal small pyknotic nuclei and abundant cytoplasm. Other cells in the micrograph include red blood cells and neutrophils. 600. b. Abnormal smear. Many of the cells in this specimen contain large nuclei with no evidence of pyknosis (arrows). The cytoplasm is relatively scant. Other cells exhibit a more normal appearance with pyknotic nuclei and more surrounding cytoplasm (arrowheads). Neutrophils are also present. 600. penis. Abundant melanin pigment is present in the deep cells of the epithelium. The core of connective tissue within each fold is devoid of fat but does contain numerous blood vessels and fine elastic fibers. Large sebaceous glands are present in the stroma.

1	 The clitoris is an erectile structure that is homologous to the penis. Its body is composed of two small erectile bodies, the corpora cavernosa; the glans clitoris is a small, rounded tubercle of erectile tissue. The skin over the glans is very thin, forms the prepuce of the clitoris, and contains numerous sensory nerve endings.  Vestibule. The vestibule is lined with stratified squamous epithelium. Numerous small mucous glands, the lesser vestibular glands (also called Skene’s glands), are present primarily near the clitoris and around the external urethral orifice. The large, paired greater vestibular glands (also called Bartholin’s glands) are homologous to the male bulbourethral glands. These tuboalveolar glands are

1	FIGURE 23.29 • Photomicrograph of the inner surface of the labia majora. This low-power H&E–stained specimen of the labia majora’s inner surface shows its nonkeratinized epithelium (Ep) and abundant sebaceous glands (SG). Two sebaceous ducts (SD) are also evident. Note the continuity of the duct epithelium with the epithelium of the skin and the sebaceous gland epithelium. At this magnification, several smooth muscle bundles can just barely be discerned (arrows).

1	about 1 cm in diameter and are located in the lateral wall of the vestibule posterior to the bulb of the vestibule. The greater vestibular glands secrete lubricating mucus. The ducts of these glands open into the vestibule near the vaginal opening. If the duct of the Bartholin’s gland becomes obstructed, it usually dilates and fills with a secretion produced by the gland. This condition, known as Bartholin’s cyst, may become infected within a few days, causing severe pain, redness, and enlargement of the involved labia majora. Purulent material in Bartholin’s abscess usually requires surgical incision with drainage or complete excision. Numerous sensory nerve endings are present in the external genitalia:  Meissner’s corpuscles are particularly abundant in the skin over the mons pubis and labia majora.

1	Numerous sensory nerve endings are present in the external genitalia:  Meissner’s corpuscles are particularly abundant in the skin over the mons pubis and labia majora.  Pacinian corpuscles are distributed in the deeper layers of the connective tissue and are found in the labia majora and in association with the erectile tissue. The sensory impulses from these nerve endings play an important role in the physiologic response during sexual arousal.  Free nerve endings are present in large numbers and are equally distributed in the skin of the external genitalia.

1	 Free nerve endings are present in large numbers and are equally distributed in the skin of the external genitalia. The mammary glands, or breasts, are a distinguishing feature of mammals. They are structurally dynamic organs, varying with age, menstrual cycle, and reproductive status of the female. During embryologic development, growth and development of breast tissue occur in both sexes. Multiple glands develop along paired epidermal thickenings called mammary ridges (milk lines) that extend from the developing axilla to the developing inguinal region. In humans, normally only one group of cells develops into a breast on each side. An extra breast (polymastia) or nipple (polythelia) may occur as an inheritable condition in about 1% of the female population. These relatively rare conditions may also occur in men. In females, mammary glands develop under the influence of sex hormones.

1	In females, mammary glands develop under the influence of sex hormones. Until puberty, both females and males mammary glands develop in similar fashion. At the onset of puberty in males, testosterone acts on the mesenchymal cells to inhibit further growth of the mammary gland. In the same time, the mammary glands in women undergo further development under hormonal influence of estrogen and progesterone. Estrogen stimulates mesenchymal cells further development. The mammary gland increases in size, mainly due to the growth of interlobular adipose tissue. The ducts extend and branch into the expanding connective tissue stroma. Proliferation of epithelial cells is controlled by interactions between the epithelium and the specialized intralobular hormone-sensitive loose connective tissue stroma. By adulthood, the complete ductal architecture of the gland has been established.

1	The mammary glands remain in inactive state until pregnancy, during which the mammary glands assume their complete morphologic and functional maturation. This occurs in response to estrogens and progesterone initially secreted from the corpus luteum and later from placenta, prolactin from pituitary gland, and gonadocorticoids produced by adrenal cortex. By the end of pregnancy, secretory vessicles are found in the epithelial cells, but milk production is inhibited by high levels of progesterone. The actual initiation of milk secretion occurs immediately after birth and is induced by prolactin (PRL) secreted by the adenohypophysis. The ejection of the milk from the breast is stimulated by oxytocin released from the neurohypophysis. With the change in the hormonal environment at menopause, the glandular component of the breast regresses or involutes and is replaced by fat and connective tissue. In men, some additional development of the mammary glands normally occurs after puberty, and

1	the glandular component of the breast regresses or involutes and is replaced by fat and connective tissue. In men, some additional development of the mammary glands normally occurs after puberty, and the glands remain rudimentary.

1	Constant hormonal exposure and genetic predisposition are the major risk factors for the development of breast cancer. It is the most common malignancy in women in the United States. Each year an estimated nearly FIGURE 23.30 • Schematic drawing of the human breast as seen during lactation. The breast is composed largely of terminal duct lobular units (TDLUs) containing branched tubuloalveolar glands. TDLUs are contained within an extensive connective tissue stroma and variable amounts of adipose tissue. (Modified from Warwick R, Williams PL, eds. Gray’s Anatomy, 35th ed. Edinburgh: Churchill Livingstone, 1973.) 200,000 women (and also 1,700 men) are diagnosed with breast cancer. Most breast cancers are linked to hormonal exposure (which increases with age, early menarche, late menopause, and with older age of a first full-term pregnancy). About 5% of all breast cancers are attributable to mutation in autosomal dominant breast cancer genes (BRCA1 and BRCA2).

1	Mammary glands are modified tubuloalveolar apocrine sweat glands. The tubuloalveolar mammary glands, derived from modified sweat glands in the epidermis, lie in the subcutaneous tissue. The inactive adult mammary gland is composed of 15 to 20 irregular lobes separated by fibrous bands of connective tissue. They radiate from the mammary papilla, or nipple, and are further subdivided into numerous lobules known as terminal duct lobular units (TDLUs) (Fig. 23.30). Some of the fibrous bands, called suspensory or Cooper’s ligaments, connect with the dermis. Abundant adipose tissue is present in the dense connective tissue of the interlobular spaces.

1	Each gland ends in a lactiferous duct that opens through a constricted orifice into the nipple. Beneath the areola, the pigmented area surrounding the nipple, each duct has a dilated portion, the lactiferous sinus. Near their openings, the lactiferous ducts are lined with stratified squamous keratinized epithelium. The epithelial lining of the duct shows a gradual transition from stratified squamous to two layers of cuboidal cells in the lactiferous sinus and finally to a single layer of columnar or cuboidal cells through the remainder of the duct system.

1	The epidermis of the adult nipple and areola is highly pigmented and somewhat wrinkled and has long dermal papillae invading into its deep surface (Fig. 23.31). It is covered by keratinized stratified squamous epithelium. The pigmentation of the nipple increases at puberty, and the nipple becomes more prominent. During pregnancy, the areola becomes larger and the degree of pigmentation increases further. Deep to the areola and nipple, bundles of smooth muscle fibers are arranged radially and circumferentially in the dense connective tissue and longitudinally along the lactiferous ducts. These muscle fibers allow the nipple to become erect in response to various stimuli.

1	The areola contains sebaceous glands, sweat glands, and modified mammary glands (glands of Montgomery). These glands have a structure intermediate between sweat glands and true mammary glands, and they produce small elevations on the surface of the areola. It is believed that Montgomery glands produce a lubricating and protective secretion that changes the skin’s pH and discourages microbial growth. Numerous sensory nerve endings are present in the nipple; the areola contains fewer sensory nerve endings. Terminal duct lobular unit (TDLU) of the mammary gland represents a cluster of small secretory alveoli (in lactating gland) or terminal ductules (in inactive gland) surrounded by intralobular stroma.

1	Successive branching of lactiferous ducts leads to the terminal duct lobular unit (TDLU). Each TDLU represents a grapelike cluster of small alveoli that forms a lobule (Fig. 23.32) and consists of the following.  Terminal ductules are present in the inactive gland. Dur ing pregnancy and after birth, epithelium of the terminal ductules, which is lined by secretory cells, differentiates into fully functional secretory alveoli producing milk.  The intralobular collecting duct carries alveolar secre tions into the lactiferous duct.  The intralobular stroma is specialized hormonally sensi tive loose connective tissue that surrounds the terminal ductules and alveoli. The intralobular connective tissue contains little adipose cells.

1	Glandular epithelial and myoepithelial cells are the most important cells associated with mammary ducts and lobules. Glandular epithelial cells line the duct system, whereas myoepithelial cells lie deep within the epithelium between the epithelial cells and the basal lamina. These cells, arranged in a basketlike network, are present in the secretory portions of the gland. In routine hematoxylin and eosin (H&E) preparation, the myoepithelial cells are more apparent in the larger ducts. However, in the immunocytochemical preparation, their discontinuous, basketlike arrangement is better visualized within the alveoli (Fig. 23.33). Contraction of myoepithelial cells assists in milk ejection during lactation. Recent immunoﬂuorescence studies have proven that breast progenitor cells found in the ductular epithelium give rise to both glandular cells of the alveoli and myoepithelial cells. The morphology of the secretory portion of the mammary gland varies with the menstrual cycle.

1	FIGURE 23.31 • Photomicrographs of a section through the female nipple. a. This low-magnification micrograph of an H&E–stained sagittal section through the nipple shows the wrinkled surface contour, a thin stratified squamous epithelium, and associated sebaceous glands (arrows). The core of the nipple consists of dense connective tissue, smooth muscle bundles, and the lactiferous ducts that open at the nipple surface. 6. b. The wall of one of the lactiferous ducts is shown here at higher magnification. Its epithelium is stratified cuboidal, consisting of two-cell layers. As it approaches the tip of the nipple, it changes to a stratified squamous epithelium and becomes continuous with the epidermis. 175. c. A higher magnification of the sebaceous gland from the rectangle in a. Note how the glandular epithelium is continuous with the epidermis (arrows), and the sebum is being secreted onto the epidermal surface. 90. d. A higher magnification showing bundles of smooth muscle in

1	how the glandular epithelium is continuous with the epidermis (arrows), and the sebum is being secreted onto the epidermal surface. 90. d. A higher magnification showing bundles of smooth muscle in longitudinal and cross-sectional profiles. 350.

1	In the inactive gland, the glandular component is sparse and ductules appear as cords formed by the cuboidal-shaped epconsists chiefly of duct elements (Fig. 23.34 and Plate 102, ithelial cells with little or no lumen. During the luteal phase, page 892). During the menstrual cycle, the inactive breast the epithelial cells increase in height, and lumina appear in undergoes slight cyclic changes. Early during the follicular the ducts as small amounts of secretions accumulate. Also, phase, the intralobular stroma is less dense, and terminal fluid accumulates in the connective tissue. This is followed by

1	FIGURE 23.32 • Terminal Duct Lobular Unit a. This schematic diagram shows components of the terminal duct lobular unit (TDLU). Terminal ductules and intralobular collecting duct are surrounded by a specialized hormonally sensitive loose connective tissue called intralobular stroma. TDLU are separated from each other by interlobular stroma containing variable amount of dense irregular connective tissue and adipose tissue. In active mammary glands, terminal ductules differentiate into milk-producing alveoli. b. This photomicrograph shows the TDLU from an inactive mammary gland. The clear area in the upper part of the image represents adipose cells. 120. FIGURE 23.33 • Myoepithelial cells in the mammary gland.

1	FIGURE 23.33 • Myoepithelial cells in the mammary gland. This imunofluorescence image is obtained from mammary gland of lactating mouse two days post-parturition. The mouse carried a transgene composed of the smooth muscle -actin promoter conjugated to enhance green fluorescent protein (GFP) reaction. Three-dimensional organization of myoepithelial cells is visualized in green color due to the expression of the promoter transgene in myoepithelial cells. The tissue was also stained red with antibody against smooth muscle -actin conjugated directly with CY3 fluorescent dye. The orange color staining results from overlapping of the green and red staining. The cells on the surface of the terminal duct lobular unit are stained orange, while those deeper in the tissue stained only green because the antibody did not penetrate deep into the tissue. Note a small intralobular duct that merges into the larger lactiferous duct. 600. (Courtesy Dr. James

1	J. Tomasek, University of Oklahoma Health Science Center.) abrupt involution and apoptosis during the last few days of menstrual cycle before onset of menstruation. Mammary glands undergo dramatic proliferation and development during pregnancy. The mammary glands exhibit a number of changes in preparation for lactation. These can be examined by the trimester of pregnancy.  First trimester is characterized by elongation and branching of the terminal ductules. The lining epithelial and myoepithelial cells proliferate and differentiate from breast progenitor cells found in epithelium of terminal ductules. Myoepithelial cells proliferate between the base of the epithelial cells and the basal lamina in both the alveolar and ductal portions of the gland.

1	 Second trimester is characterized by differentiation of alveoli from the growing ends of the terminal ductules. The development of the glandular tissue is not uniform, and variation in the degree of development is seen even within a single lobule. The cells vary in shape from flattened to low columnar. Plasma cells, lymphocytes, and eosinophils infiltrate the intralobular connective tissue stroma as the breast develops (Plate 103, page 894). At this stage, amount of glandular tissue and mass of the breast increases mainly due to the growth of the alveoli (Fig. 23.35).

1	 Third trimester commences maturation of the alveoli. The epithelial glandular cells become cuboidal with nuclei positioned at the basal cell surface. They develop an extensive rER; secretory vesicles and lipid droplets appear in the cytoplasm. The actual proliferation of the interlobular stromal cells declines and subsequent enlargement of the breast occurs through hypertrophy of the secretory cells and accumulation of secretory product in the alveoli. The changes in glandular tissue during pregnancy are accompanied by a decrease in the amount of connective tissue and adipose tissue. Both merocrine and apocrine secretion are involved in production of milk.

1	Both merocrine and apocrine secretion are involved in production of milk. The secreting cells contain abundant granular endoplasmic reticulum, a moderate number of large mitochondria, a supranuclear Golgi apparatus, and a number of dense lysosomes (Fig. 23.36). Depending on the secretory state, large lipid droplets and secretory vesicles may be present in the apical cytoplasm. The secretory cells produce two distinct products that are released by different mechanisms.  Merocrine secretion: The protein component of the milk is synthesized in the rER, packaged into membrane-limited secretory vesicles for transport in the Golgi apparatus, and released from the cell by fusion of the vesicle’s limiting membrane with the plasma membrane.

1	 Apocrine secretion: The fatty or lipid component of the milk arises as lipid droplets free in the cytoplasm. The lipid coalesces to form large droplets that pass to the apical region of the cell and project into the lumen of the acinus. The droplets are invested with an envelope of plasma membrane as they are released. A thin layer of cytoplasm is trapped between the plasma membrane and lipid droplet and is released with the lipid, but the cytoplasmic loss in this process is minimal.

1	The secretion released in the first few days after childbirth is known as colostrum. This premilk is an alkaline, yellowish secretion with a higher protein, vitamin A, sodium, and chloride content and a lower lipid, carbohydrate, and potassium content than milk. It contains considerable amounts of antibodies (mainly secretory IgA) that provide the newborn with some degree of passive immunity. The antibodies in the colostrum are believed to be produced by the lymphocytes and plasma cells that infiltrate the loose connective tissue of the breast during its proliferation and development and are secreted across the glandular cells as in salivary glands and intestine. As these wandering cells decrease in number after parturition, the production of colostrum stops, and lipid-rich milk is produced. Hormonal Regulation of the Mammary Gland

1	The initial growth and development of the mammary gland at puberty occur under the influence of estrogens and progesterone produced by the maturing ovary. Under hormonal influence, the TDLUs develop and differentiate into dynamic functional units. Subsequent to this initial development, slight changes in the morphology of the glandular tissue occur during each ovarian cycle. During the follicular phase of the menstrual cycle, estrogen in the circulation stimulates proliferation of the lactiferous duct components. After ovulation in the luteal phase, progesterone stimulates growth of alveoli; intralobular stroma become edematous. Clinically, during the luteal phase, women perceive tenderness and a progressive increase of breast tissue mass. During pregnancy, the corpus luteum and placenta continuously produce estrogens and progesterone, causing a massive increase in TDLUs. It is now believed that the growth of the mammary glands also depends on the presence of prolactin, which is

1	continuously produce estrogens and progesterone, causing a massive increase in TDLUs. It is now believed that the growth of the mammary glands also depends on the presence of prolactin, which is produced by the adenohypophysis; hCS, which is produced by the placenta; and adrenal glucocorticoids.

1	FIGURE 23.34 • Photomicrograph of an inactive mammary gland. a. This low-magnification H&E–stained specimen shows several lobules within the dense connective tissue of the breast. The epithelial component consists of a branching duct system that makes up the lobule. The clear areas (arrows) are adipose cells. 60. b. A higher magnification of the area in the rectangle of a. The epithelial cells of the ducts are columnar and exhibit interspersed lymphocytes (arrows) that have entered the epithelium. The surrounding stained material (arrowheads) represents the myoepithelial cells (MEp) and collagen bundles in the adjacent connective tissue. 700. Lactation is under the neurohormonal control of the adenohypophysis and hypothalamus.

1	Lactation is under the neurohormonal control of the adenohypophysis and hypothalamus. Although estrogen and progesterone are essential for the physical development of the breast during pregnancy, both of these hormones also suppress the effects of prolactin and hCS, the levels of which increase as pregnancy progresses. Immediately after birth, however, the sudden loss of estrogen and progesterone secretion from the placenta and corpus luteum allows prolactin to assume its lactogenic role. Production of milk also requires adequate secretion of growth hormone, adrenal glucocorticoids, and parathyroid hormones.

1	The act of suckling during breast-feeding initiates sensory impulses from receptors in the nipple to the hypothalamus. The impulses inhibit the release of prolactin-inhibiting factor, and prolactin is then released from the adenohypophysis. The sensory impulses also cause the release of oxytocin in the neurohypophysis. Oxytocin stimulates the myoepithelial cells that surround the base of the alveolar secretory cells and the base of the cells in the larger ducts, causing them to contract and eject the milk from the alveoli and the ducts. In the absence of suckling, secretion of milk ceases, and the mammary glands begin to regress and atrophy. The glandular tissue then returns to an inactive condition. Involution of the Mammary Gland

1	Involution of the Mammary Gland The mammary gland atrophies or its specialized stroma involutes after menopause. In the absence of ovarian hormone stimulation, the secretory cells of the TDLUs degenerate and disappear, leaving only ducts to create a histologic pattern that resembles that of male breast. The connective tissue also demonstrates degenerative changes, marked by a decrease in the number of fibroblasts and collagen fibers, and loss of elastic fibers.

1	FIGURE 23.35 • Photomicrograph of an active mammary gland during late pregnancy. a. This low-magnification H&E–stained specimen shows the marked proliferation of the duct system giving rise to the secretory alveoli that constitute the major portion of the lobules. The intralobular ducts are difficult to identify because their epithelium also secretes. Outside the lobules is a large excretory duct. 60. b. A higher magnification of an area in a. The secretory alveolar cells are mostly cuboidal here. A myoepithelial cell (mEp) as well as a number of plasma cells (arrows) can be identified in the adjacent loose connective tissue. 700.  FOLDER 23.6 Clinical Correlation: Cervix and Human Papillomavirus (HPV) Infections

1	Human papillomavirus (HPV) is the most common and cancer of the penis in men. Most HPV-associated sexually transmitted virus in the United States. More than lesions can be diagnosed by microscopic examination of 40 HPV types are known to infect the urogenital and anal smears or biopsies. In difficult cases, ancillary techniques regions of men and women, targeting the stratified squa-such as in situ hybridization can help to confirm the diagnosis mous epithelium of the perineal skin or mucous mem-(Fig. F23.6.1). Recently, two vaccines (Cervarix and Garbranes. Most women will be infected by HPV in their dasil) have become available to protect women against the lifetime, but only a small percentage (5%–10%) will de-types of HPV that cause most cervical cancers. Cervarix is velop a persistent infection and the associated risk of cer-designed to prevent infections from HPV types 16 and 18 vical cancer. Of the approximately 40 sexually transmitted and contains recombinant noninfectious

1	infection and the associated risk of cer-designed to prevent infections from HPV types 16 and 18 vical cancer. Of the approximately 40 sexually transmitted and contains recombinant noninfectious viruslike particles HPV types, most (90%) cause genital warts rather than (VLPs) from both virus types. Gardasil contains a mixture of cervical cancer and so are called low-risk HPV types (e.g., recombinant VLPs from HPV types 6, 11, 16, and 18. Nei-HPV types 6 and 11). Low-risk HPV types tend to infect ther vaccine is therapeutic (i.e., they do not clear prior in-mature epithelial cells and lead to genital warts or mild fection), but both lead to the development of specific cervical dysplasia. HPV types 16 and 18 are the most immunity against HPV infections. The vaccines are most efcommon high-risk HPV types and are associated with 70% fective for girls and young women between ages 9 and 26 of cervical cancers. High-risk HPV types usually infect di-who have had no prior HPV exposure and who

1	HPV types and are associated with 70% fective for girls and young women between ages 9 and 26 of cervical cancers. High-risk HPV types usually infect di-who have had no prior HPV exposure and who complete viding cells, cause moderate to severe cervical dysplasia or the three-injection immunization protocol before initiation carcinomas, and are linked to anal cancer, vulvar cancer, of sexual activity.

1	 FOLDER 23.6 Clinical Correlation: Cervix and Human Papillomavirus (HPV) Infections (Cont.) FIGURE F23.6.1 • Photomicrograph of in situ hybridization of a human cervical biopsy showing HPV infection. a. This low-magnification photomicrograph shows stratified squamous epithelium of the cervix hybridized with DNA probes to HPV types 6 and 11 and counterstained with nuclear fast red. Note that the majority of infected cells are mature cells located in the upper layers of the stratified squamous epithelium of the ectocervix. 120. b. This higher-magnification photomicrograph shows viral particles stained purple within the nuclei of infected cells. 225. (Courtesy of Dr. Fabiola Medeiros, Mayo Clinic.)

1	FIGURE 23.36 • Photomicrographs and diagram of a lactating mammary gland. a. Low-magnification micrograph of a fast greenosmium–stained section of a lactating mammary gland. Portions of several large lobules and an excretory duct are seen. Many of the alveoli exhibit a prominent lumen, even at this magnification. 60. b. A higher magnification of an area in a shows lipid droplets (black circular profiles) within the secretory cells of the alveoli as well as in the alveolar lumina. The arrows indicate plasma cells within the interstitial spaces. 480. c. Diagram of a lactating mammary gland epithelial cell. (Redrawn after Bloom W, Fawcett DW. A Textbook of Histology, 10th ed. Philadelphia: WB Saunders, 1975.) and infertility. This effect is caused by high levels of serum prolactin, which suppress secretion of LH. Ovulation usually for 2 to 3 years, lactational amenorrhea is the principal means of birth control.  FOLDER 23.7 Functional Considerations: Lactation and Infertility

1	 FOLDER 23.7 Functional Considerations: Lactation and Infertility Almost 50% of fully breast-feeding women exhibit lacta-resumes after 6 months or earlier with a decrease in suckling tional amenorrhea (lack of menstruation during lactation) frequency. In cultures in which breast-feeding may continue The arteries that supply the breast are derived from the thoracic branches of the axillary artery, the internal thoracic artery, and anterior intercostal arteries. Branches of the vessels pass primarily along the path of the alveolar ducts as they reach capillary beds surrounding the alveoli. Veins basically follow the path of the arteries as they return to the axillary and internal thoracic veins. Lymphatic capillaries are located in the connective tissue surrounding the alveoli. The larger lymphatic vessels drain into axillary, supraclavicular, or parasternal lymph nodes.

1	Lymphatic capillaries are located in the connective tissue surrounding the alveoli. The larger lymphatic vessels drain into axillary, supraclavicular, or parasternal lymph nodes. The nerves that supply the breast are anterior and lateral cutaneous branches from the second to sixth intercostal nerves. The nerves convey afferent and sympathetic fibers to and from the breast. The secretory function is primarily under hormonal control, but afferent impulses associated with suckling are involved in the reflex secretion of prolactin and oxytocin. This page intentionally left blank.

1	The ovaries are small, paired, ovoid structures that exhibit a cortex and medulla when sectioned. On one side is a hilum for the transit of neu-rovascular structures; on this same side is a mesovarium that joins the ovary to the broad ligament. The functions of the ovary are the produc-tion of ova and the synthesis and secretion of estrogen and progesterone. In the cortex are numerous primordial follicles that are present at the time of birth and that remain unchanged until sexual maturation. The oogonia in these follicles are arrested in prophase of the first meiotic division. At puberty, under the influence of pituitary gonadotropins, the ovaries begin to undergo the cyclical changes designated the ovarian cycle. During each cycle, the ovaries normally produce a single oocyte that is ready for fertilization. At the beginning of the ovarian cycle, under the influence of pituitary follicle-stimulating hormone (FSH), some of the primordial follicles begin to undergo changes that lead

1	for fertilization. At the beginning of the ovarian cycle, under the influence of pituitary follicle-stimulating hormone (FSH), some of the primordial follicles begin to undergo changes that lead to the development of a mature (Graafian) follicle. These changes include a proliferation of follicular cells and enlargement of the follicle. Although several primordial follicles begin these developmental changes, usually only one reaches maturity and yields an oocyte. Occasionally, two follicles will mature and ovulate, leading to the possibility of dizygotic twin development. The dis-charge of the oocyte and its adherent cells is called ovulation. At ovulation, the oocyte completes the first meiotic division. Only if fertilization occurs does the oocyte complete the second meiotic division. Whether or not fertilization occurs, the other follicles that began to proliferate in the same cycle degenerate, a process referred to as atresia.

1	Cortex, ovary, monkey, H&E ×120. The cortex of an ovary from a sexually mature individual is shown here. On the surface, there is a single layer of epithelial cells designated the germinal epithelium (GEp). This epithelium is continuous with the serosa (peritoneum) of the mesovarium. Contrary to its name, the epithelium does not give rise to the germ cells. The germinal epithelium covers a dense fibrous connective Early primary follicles, ovary, monkey, H&E ×450. When a primordial follicle begins the changes leading to the formation of a mature follicle, the layer of squamous follicular cells becomes cuboidal, as in this figure. In addition, the follicular cells proliferate and become multilayered. A follicle Primordial follicles, ovary, monkey, H&E ×450.

1	Primordial follicles, ovary, monkey, H&E ×450. This figure shows several primordial follicles at higher magnification. Each follicle consists of an oocyte surrounded by a single layer of squamous follicular cells (F). The nucleus (N) of the oocyte is typically large, but the oocyte itself is so Late primary follicle, ovary, monkey, H&E ×450. The primary follicle in this figure shows a multilayered mass of follicular cells (FC) surrounding the oocyte. The innermost layer of follicular cells is adjacent to a thick eosinophilic layer of extracellular homogeneous material called the zona pellucida (ZP). At this stage of development, the oocyte has also enlarged slightly. The entire structure surrounded by the zona pellucida is actually the oocyte.

1	tissue layer, the tunica albuginea (TA); under the tunica albuginea are the primordial follicles (PF ). It is not unusual to see follicles at various stages of development or atresia in the ovary. In this figure, along with the large number of primordial follicles, there are four growing follicles (SF ), an atretic follicle (AF ), and part of a large follicle on the right. The region of the large follicle shown in the figure includes the theca interna (TI ), granulosa cells (GC ), and part of the antrum (A). undergoing these early changes is called a primary follicle. Thus, an early primary follicle may still be unilaminar, but it is surrounded by cuboidal cells, and this distinguishes it from the more numerous unilaminar primordial follicles that are surrounded by squamous cells.

1	large that the nucleus is often not included in the plane of section, as in the oocyte marked X. The group of epithelioid-appearing cells (arrowhead) are follicular cells of a primordial follicle that has been sectioned in a plane that just grazes the follicular surface. In this case, the follicular cells are seen en face. Surrounding the follicles are elongate cells of the highly cellular connective tissue, referred to as stromal cells. The stromal cells surrounding a secondary follicle become disposed into two layers designated the theca interna and the theca externa. As seen in figure above stromal cells become epithelioid in the cell-rich theca interna (TI). A, antrum AF, atretic follicle F, follicle cells, primordial FC, follicle cells GEp, germinal epithelium N, nucleus of oocyte PF, primordial follicles SF, growing follicles TA, tunica albuginea TI, theca interna X, oocyte showing only cytoplasm ZP, zona pellucida arrowhead, follicle cells seen en face KEY

1	Atresia of follicles is a regular event in the ovary, beginning in embryonic life. In any section through the postpubertal ovary, follicles of various stages can be seen undergoing atresia. In atresia, the initial changes involve pyknosis of the nuclei of the follicular cells and dissolution of their cytoplasm. The follicle is then invaded by macrophages and other connective tissue cells. The oocyte degenerates, leaving behind the prominent zona pellucida. This may fold inward or collapse, but it usually retains its thickness and staining characteristics. When included in the plane of section, a distorted zona pellucida serves as a reliable diagnostic feature of an atretic follicle. In atresia of large, nearly mature follicles, cells of the theca interna remain to form clusters of epithelioid cells in the ovarian cortex. These are referred to collectively as interstitial glands and continue to secrete steroid hormones. Secondary follicles, ovary, monkey, H&E ×120.

1	Two follicles growing under the influence of FSH are shown in figure on left. The more advanced follicle is a secondary follicle. The oocyte in this follicle is surrounded by several layers of follicular cells (FC) that, at this stage, are identified as granulosa cells. At a slightly earlier time, small lakes of fluid formed 874 between the follicular cells, and these lakes have now fused into a well-defined larger cavity called the follicular antrum (FA), which is evident in the figure. The antrum is also filled with fluid and stains with the periodic acid-Schiff (PAS) reaction, although only lightly. The substance that stains with the PAS reaction has been retained as an eosinophilic precipitate in the antra of the secondary follicles shown here and in figure on right. Immediately above the obvious secondary follicle is a slightly smaller follicle. Because no antral spaces are evident between the follicular cells, it is appropriate to classify it as a primary follicle. In both

1	above the obvious secondary follicle is a slightly smaller follicle. Because no antral spaces are evident between the follicular cells, it is appropriate to classify it as a primary follicle. In both follicles, but particularly in the larger follicle with the antrum, the surrounding stromal cells have become altered to form two distinctive layers designated theca interna (TI) and theca externa (TE). The theca interna is a more cellular layer, and the cells are epithelioid. When seen with the electron microscope, they display the characteristics of endocrine cells, particularly steroid-secreting cells. In contrast, the theca externa is a connective tissue layer. Its cells are more or less spindle shaped.

1	In figure on right, a later stage in the growth of the secondary follicle is shown. The antrum (FA) is larger, and the oocyte is off to one side, surrounded by a mound of follicular cells called the cumulus oophorus. The remaining follicular cells that surround the antral cavity are referred to as the membrana granulosa (MG), or simply granulosa cells. Atretic follicle, ovary, monkey, H&E ×65. marked by arrows in adjacent figure on right. The two larger, more advanced follicles do not display the remains of a zona pellucida, but they do display Atretic follicles (AF) are shown here and at higher magnificaother features of follicular atresia. tion in adjacent figure on right. The two smaller atretic follicles can be identified by virtue of the retained zona pellucida (ZP) Atretic follicles, ovary, monkey, H&E ×120.

1	tion in adjacent figure on right. The two smaller atretic follicles can be identified by virtue of the retained zona pellucida (ZP) Atretic follicles, ovary, monkey, H&E ×120. In atresia of a more advanced follicle, the follicular cells tend to degenerate more rapidly than the cells of the theca interna, and the basement membrane separating the two becomes thickened to form a hyalinized membrane, the glassy membrane. Thus, the glassy membrane (arrows) separates an outer layer of remaining theca interna cells from the degenerating inner follicular cells. The remaining theca interna cells may show cytologic integrity (RTI); these intact theca cells remain temporarily functional in steroid secretion. AF, atretic follicle FA, antrum of follicle FC, follicle cells MG, membrana granulosa RTI, remaining theca interna cells TE, theca externa TI, theca interna ZP, zona pellucida arrowhead, persisting theca interna cells arrows, glassy membrane KEY

1	After the oocyte and its immediately surrounding cells (i.e., the cells of the cumulus oophorus) are discharged from the mature ovarian follicle (ovulation), the remaining follicle cells (membrana granulosa) and the adjacent theca interna cells differentiate into a new functional unit, the corpus luteum. The cells of the corpus luteum, luteal cells, rapidly increase in size and become filled with lipid droplets. A lipid-soluble pigment in the cytoplasm of the cells, lipochrome, gives them their yellow appearance in fresh tissue. Electron micrographs of the luteal cells demonstrate that they have features typical of steroid-secreting cells, namely, abundant smooth endoplasmic reticulum and mitochondria with tubular cristae. Two types of luteal cells are identified: Large, centrally located granulosa lutein cells are derived from the granulosa cells; smaller, peripherally located theca lutein cells are derived from the theca interna. A rich vascular network is established in the corpus

1	granulosa lutein cells are derived from the granulosa cells; smaller, peripherally located theca lutein cells are derived from the theca interna. A rich vascular network is established in the corpus luteum into which progesterone and estrogen are secreted by the lutein cells. These hormones stimulate growth and differentiation of the uterine endometrium to prepare it for implantation of a fertilized ovum.

1	Corpus luteum, ovary, human, H&E ×20. This figure shows ovarian cortex shortly after ovulation. The arrowhead points toward the surface of the ovary at the site of ovulation. The cavity (FC) of the former follicle has been invaded by connective tissue (CT). The membrana granulosa has become plicated, and the granulosa cells, now transforming into cells of the corpus luteum, are called granulosa lutein cells (TC). The plication of the membrana granulosa begins just before ovulation and Corpus luteum, ovary, human, H&E ×65 (on left) and ×240 (on right).

1	A segment of the plicated corpus luteum is shown in figure on left at higher magnification. As noted above, the main cell mass is composed of granulosa lutein cells (GLC). On one side of this cell mass is the connective tissue (CT) within the former follicular cavity; on the other side are the theca lutein cells. The granulosa lutein cells contain a large spherical nucleus (see, also, GLC, in figure on right) and a large amount of cytoplasm. The cytoplasm contains yellow pigment (usually not evident in routine H&E sections), hence the name, corpus luteum. Theca lutein cells (TLC) also contain a spherical nucleus, but the cells are smaller than the granulosa lutein cells. Thus, when identifying the two-cell types, aside from location, note that the nuclei of adjacent theca lutein cells generally appear to be closer to each continues as the corpus luteum develops. As the corpus luteum becomes more plicated, the former follicular cavity becomes reduced in size. At the same time, blood

1	appear to be closer to each continues as the corpus luteum develops. As the corpus luteum becomes more plicated, the former follicular cavity becomes reduced in size. At the same time, blood vessels (BV) from the theca of the follicle invade the former cavity and the transforming membrana granulosa cells. Cells of the theca interna follow the blood vessels into the outermost depressions of the plicated structure. These theca interna cells become transformed into cells of the corpus luteum called theca lutein cells.

1	Corpus luteum, ovary, human, H&E ×20. A portion of a fully formed corpus luteum is shown here. Most endocrine cells are the granulosa lutein cells (GLC). These form a folded cell mass that surrounds the remains of the former follicular cavity (FC). External to the corpus luteum is the connective tissue of the ovary (CT). Keep in mind that the theca interna was derived from the connective tissue stroma of the ovary. The location of theca lutein cells (TLC) reflects this origin, and these cells can be found in the deep outer recesses of the glandular mass, adjacent to the surrounding connective tissue. other than nuclei of adjacent granulosa lutein cells. The connective tissue (CT) and small blood vessels that invaded the mass of granulosa lutein cells can be identified as the flattened and elongated components between the granulosa lutein cells.

1	The changes whereby the ruptured ovarian follicle is transformed into a corpus luteum occur under the influence of pituitary luteinizing hormone. In turn, the corpus luteum itself secretes progesterone, which has a profound effect on the estrogen-primed uterus. If pregnancy occurs, the corpus luteum remains functional; if pregnancy does not occur, the corpus luteum regresses after having reached a point of peak development, roughly 2 weeks after ovulation. The regressing cellular components of the corpus luteum are replaced by fibrous connective tissue, and the structure is then called a corpus albicans.

1	BV, blood vessels CT, connective tissue FC, former follicular cavity GLC, granulosa lutein cells TC, granulosa cells transforming into corpus luteum cells TLC, theca lutein cells KEY 878 The uterine tubes (oviducts, Fallopian tubes) are joined to the uterus and extend to the ovaries, where they present an open flared end (abdominal ostium) for entry of the ovum at ovulation. The oviduct undergoes cyclical changes along with those of the uterus, but these are not nearly as pronounced. The epithelial cells increase in height during the middle of the cycle, just about the time the ovum will be passing through the tube, and become reduced during the premenstrual period. Some of the epithelial cells are ciliated. The epithelial cells depend on the ovaries for their viability. Not only does the number of ciliated cells increase during the follicular phase of the ovarian cycle, but also removal of the ovaries leads to atrophy of the epithelium and loss of ciliated cells. The uterine tube

1	the number of ciliated cells increase during the follicular phase of the ovarian cycle, but also removal of the ovaries leads to atrophy of the epithelium and loss of ciliated cells. The uterine tube varies in size and degree of mucosal folding along its length. The mucosal folds are evident in its distal portion, the infundibulum, as it nears the open end. Near the opening, the tube flares outward and is called the infundibulum. It has fringed folded edges called fimbria. The infundibulum leads proximally to the ampulla, which constitutes about two thirds of the length of the oviduct, has the most numerous and complex mucosal folds, and is the site of fertilization. Mucosal folds are least numerous at the proximal end of the oviduct, near the uterus, where the tube is narrow and referred to as the isthmus. A uterine or intramural portion measures about 1 cm in length and passes through the uterine wall to empty into the uterine cavity. Fertilization of the ovum usually occurs in the

1	to as the isthmus. A uterine or intramural portion measures about 1 cm in length and passes through the uterine wall to empty into the uterine cavity. Fertilization of the ovum usually occurs in the distal portion of the ampulla. For the first several days of development, as it navigates the complex pathway created by the mucosal folds, the embryo is transported proximally by the beating of the cilia of the ciliated epithelial cells and by peristaltic contractions of the well-developed muscularis layer that underlies the mucosa.

1	Uterine tube, human, H&E ×40. A cross section through the ampulla of the uterine tube is shown here. Many mucosal folds project into the lumen (L), and the complicated nature of the folds is evident by the variety of profiles that is seen. In addition to the mucosa (Muc), the remainder of the wall consists of a muscularis (Mus) and connective tissue. The muscularis consists of smooth muscle that forms a relatively thick layer of circular fibers and a thinner outer layer of longitudinal fibers. The layers are not clearly delineated, and no sharp boundary separates them. Mucosal fold, uterine tube, human, H&E ×160; inset ×320.

1	The area enclosed by the rectangle in figure above is shown here at higher magnification. The specimen shows a longitudinal section through a lymphatic vessel (Lym). In other planes of section, the lymphatic vessels are difficult to identify. The for tuitously sectioned lymphatic vessel is seen in the core of the mucosal fold, along with a highly cellular connective tissue (CT) and the blood vessels (BV) within the connective tissue. The epithelium lining the mucosa is shown in the inset. The ciliated cells are readily identified by the presence of well-formed cilia (C). Nonciliated cells, also called peg cells (PC), are readily identified by the absence of cilia; moreover, they have elongate nuclei and sometimes appear to be squeezed between the ciliated cells. The connective tissue (CT) contains cells whose nuclei are arranged typically in a random manner. They vary in shape, being elongated, oval, or round. Their cytoplasm cannot be distinguished from the intercellular material

1	contains cells whose nuclei are arranged typically in a random manner. They vary in shape, being elongated, oval, or round. Their cytoplasm cannot be distinguished from the intercellular material (inset). The character of the connective tissue is essentially the same from the epithelium to the muscularis, and for this reason, no submucosa is described.

1	PLATE 95 BV, blood vessels C, cilia CT, connective tissue Ep, epithelium L, lumen Lym, lymphatic vessel Muc, mucosa Mus, muscularis PC, peg cells KEY 880 The uterus is a hollow, pear-shaped organ with a thick wall and, in the nonpregnant state, a narrow cavity. The uterine wall is composed of a mucosa, referred to as the endometrium; a muscularis, referred to as a myometrium; and, externally, a serosal cover, the perimetrium. The myometrium consists of smooth muscle and connective tissue and contains the large blood vessels that give rise to the vessels that supply the endometrium. The uterus undergoes cyclical changes that are largely manifested by changes that occur in the endometrium. If implantation of an ovum does not occur after preparation for this event, the state of readiness is not maintained, and much of the endometrium degenerates and is sloughed off, constituting the menstrual flow. The part of the endometrium that is lost is referred to as the stratum functionale; the

1	not maintained, and much of the endometrium degenerates and is sloughed off, constituting the menstrual flow. The part of the endometrium that is lost is referred to as the stratum functionale; the part that is retained is called the stratum basale. The stratum basale is the deeper part of the endometrium and adjoins the myometrium. The myometrium also undergoes changes associated with implantation of a zygote. In the nonpregnant uterus, the smooth muscle cells are about 50 m in length; during pregnancy, they undergo enormous hypertrophy, often reaching more than 500 m in length. In addition, new muscle fibers develop after division of existing muscle cells and division and differentiation of undifferentiated mesenchymal cells. The connective tissue also increases to strengthen the uterine wall. Fibroblasts increase by division and secrete additional collagen and elastic fibers. After parturition, the uterus nearly returns to its normal size. Most muscle fibers return to their normal

1	wall. Fibroblasts increase by division and secrete additional collagen and elastic fibers. After parturition, the uterus nearly returns to its normal size. Most muscle fibers return to their normal size, and some degenerate. Collagen secreted during pregnancy is digested by the very cells that secreted it, the fibroblasts. Similar, but less pronounced, proliferation and degeneration of fibroblasts and collagen occur in each menstrual cycle.

1	Uterus, human, H&E ×25; inset ×120.

1	After the stratum functionale (SF) is sloughed off, resurfacing of the raw tissue occurs. The epithelial resurfacing comes from the glands that remain in the stratum basale (SB). The gland epithelium simply proliferates and grows over the surface. This figure shows the endometrium as it appears when resurfacing is complete. The area inscribed in the upper small rectangle is shown at higher magnification in the inset on the right. Note the simple columnar epithelium (SEp) that covers the endometrial surface and its similarity to the glandular epithelium (Gl). The endometrium is relatively thin at this phase and over half of it consists of the stratum basale. The area inscribed by the lower small rectangle, located in the region of the stratum basale, is shown at higher magnification in the inset in figure below. The glandular epithelium of the deep portion of the glands is similar to that of the endometrial surface. Below the endometrium is the myometrium (M), in which a number of

1	the inset in figure below. The glandular epithelium of the deep portion of the glands is similar to that of the endometrial surface. Below the endometrium is the myometrium (M), in which a number of large blood vessels (BV) are present.

1	Endometrium, proliferative phase, uterus, human, H&E ×25; inset ×120. Under the influence of estrogen, the various components of the endometrium proliferate (proliferative phase), so that the total thickness of the endometrium is increased. As shown in this figure, the endometrial glands (Gl) become rather long and follow a fairly straight course within the stratum functionale (SF) to reach the surface. The stratum basale (SB) remains essentially unaffected by the estrogen and appears much the same as in above figure. In this figure, the stratum functionale (SF), on the other hand, has increased in thickness and constitutes about four fifths of the endometrial thickness. BV, blood vessels Gl, endometrial glands M, myometrium SB, stratum basale SEp, surface epithelium SF, stratum functionale KEY

1	After estrogen brings about the uterine events designated the proliferative phase another hormone, progesterone, influences additional uterine changes that constitute the secretory phase of the uterine cycle. This hormone brings the endometrium to a state of readiness for implantation, and as a consequence of its actions, the thickness of the endometrium increases further. There are conspicuous changes in the glands, primarily in the stratum functionale, where the glands take on a more pronounced corkscrew shape and secrete mucus that accumulates in sacculations along their length. The vasculature of the endometrium also proliferates and degenerates in each menstrual cycle. Radial arteries enter the stratum basale of the endometrium from the myometrium and give rise to small, straight arteries that supply the stratum basale and continue into the endometrium to become the highly coiled spiral arteries. Arterioles derived from the spiral arteries supply the stratum functionale. The

1	arteries that supply the stratum basale and continue into the endometrium to become the highly coiled spiral arteries. Arterioles derived from the spiral arteries supply the stratum functionale. The distal portion of the spiral arteries and the arterioles are sloughed with the stratum functionale during menstruation. Alternating contraction and relaxation of the basal portions of the spiral arteries prevent excessive blood loss during menstruation.

1	Uterus, human, H&E ×25.

1	This view of the endometrium in the secretory phase shows the stratum functionale (SF), the stratum basale (SB), and, in the lower left of the photomicrograph, a small amount of the myometrium (M). The uterine glands have been cut in a plane that is close to their long axes, and one gland (arrow) is seen opening at the uterine surface. Except for a few glands near the center of the figure that resemble those of the proliferative phase, most of the glands (Gl) in this figure, including those that are labeled, show numerous shallow sacculations that give the profile of the glandular epithelium a serrated appearance. This is one of the distinctive features of the secretory phase. It is seen most advantageously in areas where the plane of section is close to the long axis of the gland. In contrast to the characteristic sinuous course of the glands in the stratum functionale, glands of the stratum basale more closely resemble those in the proliferative phase. They are not oriented in any

1	to the characteristic sinuous course of the glands in the stratum functionale, glands of the stratum basale more closely resemble those in the proliferative phase. They are not oriented in any noticeable relationship to the uterine surface, and many of their long profiles are even parallel to the plane of the surface.

1	Endometrium, secretory phase, uterus, human, H&E ×30; inset ×120. This slightly higher-magnification view of the stratum functionale shows essentially the same characteristics of the endometrial glands (Gl) described above; it also shows other modifications that occur during the secretory phase. One of these is that the endometrium becomes edematous. The increase in endometrial thickness because of edema is reflected by the presence of empty spaces between cells and other formed elements. Thus, many areas of this figure, especially the area within and near the rectangle, show histologic signs of edema. In addition, in this phase the glandular epithelial cells begin to secrete a mucoid fluid that is rich in glycogen. This product is secreted into the lumen of the glands, causing them to dilate. Typically, the glands of the secretory endometrium are more dilated than those of the proliferative endometrium.

1	The rectangle in this figure highlights two glands that are shown at higher magnification in the inset. Each of these glands contains some substance within the lumen. The mucoid character of the substance within one of the glands can be surmised from its blue staining. Although not evident in routine H&E paraffin sections, the epithelial cells also contain glycogen during the secretory phase and as mentioned above, this becomes part of the secretion. The arrowheads indicate stromal cells; some of these cells undergo enlargement late in the secretory phase. These modified stromal cells, called decidual cells, play a role in implantation. Gl, endometrial glands M, myometrium SB, stratum basale SF, stratum functionale arrow, glandular opening at uterine surface arrowheads, stromal cells KEY

1	The cervix is the narrow or constricted inferior portion of the uterus, part of which projects into the vagina. The cervical canal traverses the cervix and provides a channel connecting the vagina and the uterine cavity. The structure of the cervix resembles the rest of the uterus in that it consists of a mucosa (endometrium) and a myometrium. There are, however, some important differences in the mucosa. The endometrium of the cervix does not undergo the cyclical growth and loss of tissue that is characteristic of the body and fundus of the uterus. Rather, the amount and character of the mucous secretion of its simple columnar epithelium vary at different times in the uterine cycle under the influence of the ovarian hormones. At midcycle, there is a 10-fold increase in the amount of mucus produced; this mucus is less viscous and provides a favorable environment for sperm migration. At other times in the cycle, the mucus restricts the passage of sperm into the uterus. The myometrium

1	produced; this mucus is less viscous and provides a favorable environment for sperm migration. At other times in the cycle, the mucus restricts the passage of sperm into the uterus. The myometrium forms the major thickness of the cervix. It consists of interweaving bundles of smooth muscle cells in an extensive, continuous network of fibrous connective tissue.

1	Cervix, uterus, human, H&E ×15. The portion of the cervix that projects into the vagina, the vaginal part or ectocervix, is represented by the upper two thirds of top figure. The lower third of the micrograph reveals the portion of the cervical canal (CC). Lower figure shows the continuation of cervical canal (CC). The plane of section in both figures passes through the long axis of the cervical canal which is narrowed and cone shaped at its two ends. The upper end, the internal os, communicates with the uterine cavity, and the lower end, the external os (Os), communicates with the vagina. (For purposes of orientation, realize that only one side of the longitudinal section of the cervix is shown in these Ectocervix, uterus, human, H&E ×240.

1	Ectocervix, uterus, human, H&E ×240. The surface of the vaginal part of the cervix, ectocervix is covered by the stratified squamous epithelium (SSEp). The epithelium-connective tissue junction presents a relatively even contour in contrast to the irregular profile seen in the vagina. Transformation zone, cervix, uterus, human, H&E ×240. The mucosa of the cervical canal is covered with columnar epithelium. An abrupt change from stratified squamous epithelium (SSEp) to simple columnar epithelium (CEp) occurs within the transformation zone at the vaginal opening Cervical glands, cervix, uterus, human, H&E ×500.

1	Cervical glands, cervix, uterus, human, H&E ×500. This figure shows, at high magnification, portions of the cervical gland identified in the rectangle in Figure on left. Note the tall epithelial cells and the lightly staining supranuclear cytoplasm, a reflection of the mucin dissolved out of the cell during figures and that the actual specimen, as seen in a section, would present a similar image on the other side of the cervical canal.) The mucosa (Muc) of the cervix differs according to the cavity it faces. The two rectangles in upper figure delineate representative areas of the mucosa that are shown at higher magnification in upper right and middle right figures, respectively. Bottom figure emphasizes the nature of the cervical glands (Gl). The glands differ from those of the uterus in that they branch extensively. They secrete a mucous substance into the cervical canal that serves to lubricate the vagina.

1	In other respects, the epithelium has the same general features as the vaginal epithelium. Another similarity is that the epithelial surface of the ectocervix undergoes cyclical changes similar to those of the vagina in response to ovarian hormones. The mucosa of the ectocervix, like that of the vagina, is devoid of glands. of the cervical canal (external os). The lower rectangle in top left figure marks this site, known as the transition zone, which is shown at higher magnification here. Note the abrupt change in the epithelium at the point indicated by the diamond-shaped marker, as well as the large number of lymphocytes present in this region.

1	tissue preparation. The crowding and the change in shape of the nuclei (asterisk) seen at the upper part of one of the glands in this figure are due to a tangential cut through the wall of the gland as it passed out of the plane of section. (It is not uncommon for cervical glands to develop into cysts as a result of obstruction in the duct. Such cysts are referred to as Nabothian cysts.) BV, blood vessels CC, cervical canal CEp, columnar epithelium Gl, cervical glands Muc, mucosa Os, ostium of the uterus SSEp, stratified squamous epithelium asterisk, tangential cut of the epithelial surface KEY

1	The placenta is a disc-shaped organ that serves for the exchange of materials between the fetal and maternal circulations during pregnancy. It develops primarily from embryonic tissue, the chorion frondosum. One side of the placenta is embedded in the uterine wall at the basal plate. The other side faces the amniotic cavity that contains the fetus. After birth, the placenta separates from the wall of the uterus and is discharged along with the contiguous membranes of the amniotic cavity. The umbilical cord connects the fetus to the placenta. It contains two arteries that carry blood from the fetus to the placenta and a vein that returns blood from the placenta to the fetus. The umbilical arteries have thick muscular walls. These are arranged as two layers, an inner longitudinal layer and an outer circular layer. Elastic lamellae are poorly developed in these vessels and, indeed, may be absent. The umbilical vein is similar to the arteries, also having a thick muscular wall arranged as

1	outer circular layer. Elastic lamellae are poorly developed in these vessels and, indeed, may be absent. The umbilical vein is similar to the arteries, also having a thick muscular wall arranged as an inner longitudinal and an outer circular layer.

1	Placenta, human, H&E ×16. A section extending from the amniotic surface into the substance of the placenta is shown here. This includes the amnion (A), the chorionic plate (CP), and the chorionic villi (CV). The amnion consists of a layer of simple cuboidal epithelium and an underlying layer of connective tissue. The connective tissue of the amnion is continuous with the connective tissue of the chorionic plate as a result of their fusion at an earlier time. The plane of fusion, however, is not evident in H&E sections; the separation (asterisks) in parts of this figure in the vicinity of the fusion is an artifact.

1	The chorionic plate is a thick connective tissue mass that contains the ramifications of the umbilical arteries and vein. These vessels (BVp) do not have the distinct organizational features characteristic of arteries and veins; rather, they resemble the vessels of the umbilical cord. Although their identification as blood vessels is relatively simple, it is difficult to distinguish which vessels are branches of an umbilical artery and which are tributaries of the vein.

1	The main substance of the placenta consists of chorionic villi of different sizes (see Plate 100). These emerge from the chorionic plate as large stem villi that branch into increasingly smaller villi. Branches of the umbilical arteries and vein (BVv, in figure below) enter the stem villi and ramify through the branching villous network. Some villi extend from the chorionic plate to the maternal side of the placenta and make contact with the maternal tissue; these are called anchoring villi. Other villi, the free villi, simply arborize within the substance of the placenta without anchoring onto the maternal side. Placenta, human, H&E ×70; inset ×370.

1	Placenta, human, H&E ×70; inset ×370. The maternal side of the placenta is shown in this figure. The basal plate, or stratum basale (SB), is on the right side of the illustration. This is the part of the uterus to which the chorionic villi anchor. Along with the usual connective tissue elements, the basal plate contains specialized cells called decidual cells (DC). The same cells are shown at higher magnification in the inset. Decidual cells are usually found in clusters and have an epithelial appearance. Because of these features, they are easily identified. Septa from the basal plate extend into the portion of the placenta that contains the chorionic villi. The septa do not contain the branches of the umbilical vessels and, on this basis, can frequently be distinguished from stem villi or their branches.

1	PLATE 99 A, amnion BVp, blood vessels in chorionic plate BVv, blood vessels in chorionic villi CP, chorionic plate CV, chorionic villi DC, decidual cells SB, stratum basale asterisk, separation that is actually an artifact KEY

1	As the embryo develops, the invasive activity of the syncytiotrophoblast erodes the maternal capillaries and anastomoses them with the tro-phoblast lacunae, forming the maternal blood sinusoids. These communicate with each other and form a single blood compartment, lined by syn-cytiotrophoblasts, called the intervillous space. At the end of the second week of development, cytotrophoblast cells form primary chorionic villi. They project into the maternal blood space. In the third week of development, invasion of the extra embryonic mesenchyme into the primary chori-onic villi creates secondary chorionic villi. At the end of the third week, core mesenchyme differentiates into connective tissue and blood vessels that connect with the embryonic circulation. These tertiary chorionic villi constitute functional units for exchange of gases, nutrients, and waste products between maternal and fetal circulation without direct contact with each other. This separation of fetal and maternal blood

1	functional units for exchange of gases, nutrients, and waste products between maternal and fetal circulation without direct contact with each other. This separation of fetal and maternal blood is referred to as the placental barrier. Each tertiary villus consists of a connective tissue core surrounded by two distinct layers of trophoblast-derived cells. The outermost layer consists of the syncytiotrophoblast; immediately under it is a layer of cytotrophoblast cells. Starting at the fourth month, these lay-ers become very thin to facilitate the exchange of products across the placental barrier. The thinning of the wall of the villus is due to the loss of the inner, cytotrophoblastic layer. At this stage, the syncytiotrophoblast forms numerous trophoblastic buds that resemble the primary chorionic villi; however, the cytotrophoblast and the connective tissue grow very rapidly into these structures, transforming them into tertiary villi. At term, the placental barrier consists of the

1	chorionic villi; however, the cytotrophoblast and the connective tissue grow very rapidly into these structures, transforming them into tertiary villi. At term, the placental barrier consists of the syncytiotrophoblasts; a spare, discontinuous, inner cytotrophoblast layer; the basal lamina of the trophoblast; the connective tissue of the villus; the basal lamina of the endothelium; and the endothelium of the fetal placental capillary in the tertiary villus.

1	Tertiary chorionic villi, placenta, full-term, human, H&E ×280. This photomicrograph shows a section through the intervillous space of the placenta at term. It includes chorionic villi (CV) of different sizes and the surrounding intervillous space (IS). The connective tissue of the villi contains branches and tributaries of the umbilical vein (UV) and arteries. The intervillous space usually contains maternal blood (only a few maternal blood cells are seen here). The outermost layer of each chorionic villus derives from the fusion of cytotrophoblast cells. This layer, known as the syncytiotrophoblast (S), has no intercellular boundaries, and its nuclei are rather evenly distributed, giving this layer an appearance similar to that of cuboidal epithelium. In some areas, nuclei are gathered in clusters (arrowheads); in other regions the Tertiary chorionic villi, placenta, mid-term, human, H&E ×320.

1	This higher-magnification photomicrograph shows a cross section through immature chorionic tertiary villi surrounded by the intervillous space (IS). At this stage, chorionic villi are growing by proliferation of their core mesenchyme, syncy tiotrophoblast (S), and fetal endothelial cells. The syncytiotrophoblast syncytiotrophoblast layer appears relatively free of nuclei (arrows). These stretches of the syncytiotrophoblast may be so attenuated in places that the villous surface appears devoid of a covering. The syncytiotrophoblast contains microvilli that project into the intervillous space. In well-preserved specimens they may appear as a striated border (see inset below). The cytotrophoblast consists of an irregular layer of mono-nucleated cells that lies beneath the syncytiotrophoblast. In immature placentas, the cytotrophoblasts form an almost complete layer of cells. In this full-term placenta, only occasional cytotrophoblast cells (C) can be discerned. Most of the cells within

1	In immature placentas, the cytotrophoblasts form an almost complete layer of cells. In this full-term placenta, only occasional cytotrophoblast cells (C) can be discerned. Most of the cells within the core of the villus are typical connective tissue fibroblasts and endothelial cells. Other cells have a visible amount of cytoplasm that surrounds the nucleus. These are considered to be fetal placental antigen–presenting cells or placental macrophages (PM) historically known as the Hofbauer cells.

1	Secondary chorionic villi, placenta, human, H&E x320; inset ×640. This micrographs shows the secondary chorionic villi in the third week of embryonic development. These villi are composed of a mesenchymal core (MC) surrounded by two distinct layers of the trophoblast. Secondary villi have a much larger number of cytotrophoblast cells (C) than the mature tertiary villi and form an almost complete layer of cells immediately deep to the syncytiotrophoblast (S) (see inset). The syncytiotrophoblast covers not only the surface of the chorionic villi, but also extends into chorionic plate. Maternal red blood cells are present in the intervillous space.

1	surrounding the chorionic villus (center of the image) forms trophoblastic bud (TB), which will be invaded subsequently by cells of the cytotrophoblast (C), connective tissue, and rapidly developing new blood vessels. In addition to fibroblasts, several fetal placental antigen–presenting cells (placental macrophages) (PM) can be identified by the amount of cytoplasm surrounding their nuclei. C, cytotrophoblast cells CV, chorionic villiI S, intervillous space MC, mesenchymal core PM, placental macrophages S, syncytiotrophoblast TB, trophoblastic bud UV, umbilical vein KEY

1	The vagina is the fibromuscular tube of the female reproductive tract that leads to the exterior of the body. The wall of the vagina consists of three layers: a mucosa, a muscularis, and an adventitia. The epithelium of the mucosa is nonkeratinized stratified squamous. It undergoes changes that correspond to the ovarian cycle. The amount of glycogen stored in the epithelial cells increases under the influence of estrogen, whereas the rate of desquamation increases under the influence of progesterone. The glycogen liberated from the desquamated cells is fermented by lactobacilli vaginalis, producing lactic acid that acidifies the vaginal surface and inhibits colonization by yeasts and potentially harmful bacteria. The vagina has certain histologic similarities to the proximal portion of the alimentary canal but is distinguished by the following features: The epithelium does not keratinize, and except for the deepest layers, the cells appear to be empty in routine H&E sections; the

1	the alimentary canal but is distinguished by the following features: The epithelium does not keratinize, and except for the deepest layers, the cells appear to be empty in routine H&E sections; the mucosa contains neither glands nor a muscularis mucosae; the muscle is smooth and not well ordered. This should be contrasted with the oral cavity, pharynx, and upper part of the esophagus in which the muscle is striated. The more distal portion of the esophagus, which contains smooth muscle, can be distinguished easily from the vagina because it has a muscularis mucosae.

1	Vagina, human, H&E ×90. The mucosa of the vagina consists of a stratified squamous epithelium (Ep) and an underlying fibrous connective tissue (CT) that often appears more cellular than other fibrous connective tissue. The boundary between the two is readily identified because of the conspicuous staining of the closely packed small cells of the basal layer (B) of the epithelium. Connective tissue papillae project into the underside of the epithelium, giving the epithelial– connective tissue junction an uneven appearance. The papillae may be cut obliquely or in cross section and thus may appear as connective tissue islands (arrows) within the lower portion of the epithelium. The epithelium is characteristically thick and although keratohyaline granules may be found in the Mucosa, vagina, human H&E ×110.

1	Mucosa, vagina, human H&E ×110. This is a higher magnification of the epithelium that includes the area outlined by the rectangle in upper figure (turned 90 ). The obliquely cut and cross-sectioned portions of connective tissue papillae that appear as connective tissue islands in the Mucosa, vagina, human H&E ×225. This is a higher-magnification micrograph of the basal portion of the epithelium (Ep) between connective tissue papillae. Note the regularity and dense packing of the basal epithelial cells. They are the stem cells for the stratified squamous epithelium. Daughter cells of these cells migrate toward the surface Muscularis, vagina, human, H&E ×125. This higher-magnification micrograph of the smooth muscle of the vaginal wall emphasizes the irregularity of the arrangement of the muscle bundles. At the right edge of the figure is a bundle of smooth muscle cut in a longitudinal section (SML).

1	superficial cells, keratinization does not occur in human vaginal epithelium. Thus, nuclei can be observed throughout the entire thickness of the epithelium despite the fact that the cytoplasm of most of the cells above the basal layers appears empty. These cells are normally filled with large deposits of glycogen that is lost in the processes of fixation and embedding of the tissue. The rectangle outlines a portion of the epithelium and connective tissue papillae that is examined at higher magnification below. The muscular layer of the vaginal wall consists of smooth muscle arranged in two ill-defined layers. The outer layer is generally said to be longitudinally arranged (SML), and the inner layer is generally said to be circularly arranged (SMC), but the fibers are more usually organized as interlacing bundles surrounded by connective tissue. Many blood vessels (BV) are seen in the connective tissue.

1	epithelium are more clearly seen here (arrows), in some instances outlined by the surrounding closely packed cells of the basal epithelial cell layer. Note, again, that the epithelial cells even at the surface still retain their nuclei and there is no evidence of keratinization. and begin to accumulate glycogen and become less regularly arranged as they move toward the surface. The highly cellular connective (CT) tissue immediately beneath the basal layer (B) of the epithelium typically contains many lymphocytes (L). The number of lymphocytes varies with the stage of the ovarian cycle. Lymphocytes invade the epithelium around the time of menstruation and appear along with the epithelial cells in vaginal smears.

1	Adjacent to this is a bundle of smooth muscle cut in cross section (SMC). This bundle abuts on a longitudinally sectioned lymphatic vessel (LV). To the left of the lymphatic vessel is another longitudinal bundle of smooth muscle (SML). A valve (Va) is seen in the lymphatic vessel. A small vein (V) is present in the circular smooth muscle close to the lymphatic. B, basal layer of vaginal epithelium BV, blood vessels CT, connective tissue Ep, epithelium L, lymphocytes LV, lymphatic vessel SMC, smooth muscle, cross section SML, smooth muscle, longitudinal section V, vein Va, valve in lymphatic vessel arrows, connective tissue islands in epithelium KEY

1	The mammary glands are branched tubuloalveolar glands that develop from epidermis and come to lie in the subcutaneous tissue (superficial fascia). They begin to develop at puberty in the female but do not reach a fully functional state until after pregnancy. The glands also develop in the male at puberty; the development is limited, however, and the glands usually remain in a stabilized state. Mammary gland, inactive stage, human, H&E ×80. This figure is a section through an inactive gland. The parenchyma is sparse and consists mainly of duct elements. Several ducts (D) are shown in the center of the field. A small lumen can be seen in each. The ducts are surrounded by a loose Mammary gland, inactive stage, human, H&E ×200; inset ×400.

1	Additional details are evident at higher magnification. In distinguishing between the loose and dense connective tissue, recall that both extracellular and cellular features show differences that are evident in both the figure and the inset. Note the thicker collagenous fibers in the dense connective tissue in contrast to the much thinner fibers of the loose connective tissue. The loose connective tissue contains far more cells per unit area and a greater variety of cell types. This figure shows a cluster of lymphocytes (L) and, at still higher magnification (inset), plasma cells (P) and individual lymphocytes (L). Both plasma cells and lymphocytes are cells with a rounded shape, but plasma cells are larger and show more cytoplasm. In addition, regions of plasma cell cytoplasm display basophilia. Elongate nuclei in spindle-shaped cells belong to fibroblasts. In con- connective tissue (see CT(L), in figure below, and together, the ducts and surrounding connective tissue constitute a

1	Elongate nuclei in spindle-shaped cells belong to fibroblasts. In con- connective tissue (see CT(L), in figure below, and together, the ducts and surrounding connective tissue constitute a lobule. Two terminal duct lobular units (TDLU) are bracketed in this figure. Beyond the lobular unit, the connective tissue is more dense (CT(D)). The two types of connective tissues can be distinguished at the low magnification of this figure.

1	trast, although the cell types in the dense connective tissue may also be diverse, a simple examination of equal areas of loose and dense connective tissue will, by far, show fewer cells in the dense connective tissue. Characteristically, the dense connective tissue contains numerous aggregates of adipocytes (A).

1	The epithelial cells within the resting lobular units are regarded as being chiefly duct elements. Usually, alveoli are not found; their precursors, however, are represented as cellular thickenings of the duct wall. The epithelium of the resting lobule is cuboidal; in addition, myoepithelial cells are present. Reexamination of the inset shows a thickening of the epithelium in one location, presumably the precursor of an alveolus, and myoepithelial cells (M) at the base of the epithelium. As elsewhere, the myoepithelial cells are on the epithelial side of the basement membrane. During pregnancy, the glands begin to proliferate. This can be thought of as a dual process in which ducts proliferate and alveoli grow from the ducts. MAM MARY G LAN D I NACTIVE STAG E A, adipocytes CT(D), dense connective tissue CT(L), loose connective tissue D, ducts L, lymphocytes M, myoepithelial cells P, plasma cells TDLU, terminal duct lobular unit KEY

1	PLATE 103 Mammary Gland, Late Proliferative and Lactating Stages

1	Mammary glands exhibit a number of changes during pregnancy in preparation for lactation. Lymphocytes and plasma cells infiltrate the loose connective tissue as the glandular tissue develops. As the cells of the glandular portion proliferate by mitotic division, the ducts branch and alveoli begin to develop at their growing ends. Alveolar development becomes most prominent in the later stages of pregnancy, and accumulation of secretory product takes place in the alveoli. At the same time, lymphocytes and plasma cells become prominent in the loose connective tissue of the developing lobules. Myoepithelial cells proliferate between the base of the epithelial cells and the basal lamina in both the alveolar and the ductal portion of the glands. They are most prominent in the larger ducts. Both merocrine and apocrine secretion are involved in the production of milk. The protein component is synthesized, concentrated, and secreted by exocytosis in a manner typical for protein secretion. The

1	merocrine and apocrine secretion are involved in the production of milk. The protein component is synthesized, concentrated, and secreted by exocytosis in a manner typical for protein secretion. The lipid component begins as droplets in the cytoplasm that coalesce into large droplets in the apical cytoplasm of the alveolar cells and cause the apical plasma membrane to bulge into the alveolar lumen. The droplets are surrounded by a thin layer of cytoplasm and are enveloped in plasma membrane as they are released. The initial secretion in the first days after birth is called colostrum. This premilk is an alkaline secretion with a higher protein, vitamin A, sodium, and chloride content than milk and a lower lipid, carbohydrate, and potassium content. Considerable amounts of antibodies are contained in colostrum, and these provide the newborn with passive immunity to many antigens. The antibodies are produced by the plasma cells in the stroma of the breast and are carried across the

1	contained in colostrum, and these provide the newborn with passive immunity to many antigens. The antibodies are produced by the plasma cells in the stroma of the breast and are carried across the glandular cells in a manner similar to that for secretory IgA in the salivary glands and intestine. A few days after parturition, the secretion of colostrum stops and lipid-rich milk is produced.

1	PLATE 103 • MAMMARY GLAND, LATE PROLIFERATIVE AND LACTATING STAGES Mammary gland, late proliferative stage, human, H&E ×90; inset ×560. Whereas the development of the duct elements in the mammary gland occurs during the early proliferative stage, the development of the alveolar elements becomes conspicuous in the late proliferative stage. This figure shows the terminal duct lobular units (TDLU) at the late proliferative stage. Individual lobular units are separated by narrow dense connective tissue septa (S). The connective tissue within the lobular unit is a typical loose connective tissue that is now more cellular, containing mostly plasma cells and lymphocytes. The alveoli are well developed, and many exhibit precipitated secretory product. Each of the alveoli is joined to a duct, although that relationship can be difficult to identify. The epithelium of the intralobular ducts is similar in Mammary gland, lactating stage, human, methyl green-osmium ×90; inset ×700.

1	The specimen shown here is from a lactating mammary gland. It is similar in appearance to the gland at the late proliferative stage but differs mainly to the extent that the alveoli are more uniform in appearance and their lumina are larger. As in the late proliferative stage, several alveoli can be seen merging with one another (asterisks). The use of osmium in this specimen stains the lipid component of the secretion. The inset reveals the lipid droplets within the epithelial cell cytoplasm as well as lipid that has been secreted into the lumen appearance to the alveolar epithelium. The cells of both components are secretory. The alveoli as well as the intralobular ducts consist of a single layer of cuboidal epithelial cells subtended by myoepithelial cells. Often, what appear to be several alveoli are seen merging with one another (asterisks). Such profiles represent alveolar units opening into a duct. Interlobular ducts are easy to identify as they are surrounded by dense

1	be several alveoli are seen merging with one another (asterisks). Such profiles represent alveolar units opening into a duct. Interlobular ducts are easy to identify as they are surrounded by dense connective tissue. In one instance, an intralobular duct can be seen emptying into an interlobular duct (arrow). The inset shows the secretory epithelium at a much higher magnification. Note that it is a simple columnar epithelium. The nucleus of a myoepithelial cell (M) is seen at the base of the epithelium. Generally, these cells are difficult to recognize. Also, as noted above, numerous plasma cells (P) and lymphocytes (Ly) are present in the loose connective tissue of the lobule.

1	of the alveolus. The lipid first appears as small droplets within the epithelial cells. These droplets become larger and ultimately are secreted into the alveolar lumen along with milk proteins. The milk proteins are present in small vacuoles in the apical part of the cell but cannot be seen by light microscopic methods. They are secreted by exocytosis. The lipid droplets, in contrast, are large and surrounded by the apical cell membrane as they are pinched off to enter the lumen; thus it is an apocrine secretion. Several interlobular ducts are evident. One of these ducts reveals a small branch, an ending intralobular duct (arrows) joining the interlobular duct. D, interlobular duct Ly, lymphocyte M, myoepithelial cell P, plasma cell S, connective tissue septa TDLU, terminal duct lobular unit arrow, union of intralobular duct with interlobular duct asterisks, sites of merging alveoli KEY

1	PLATE 103 • MAMMARY GLAND, LATE PROLIFERATIVE AND LACTATING STAGES TDLUTDLUTDLU TDLUTDLUTDLU TDLUTDLUTDLU DDD MMM *** *** *** *** *** *** *** *** DDDDD DDDD LyLyPPLy P SSS SSS OVERVIEW OF THE EYE / 896 GENERAL STRUCTURE OF THE EYE / 896 Layers of the Eye / 896 Chambers of the Eye / 897 Development of the Eye / 898 MICROSCOPIC STRUCTURE OF THE EYE / 899 Corneoscleral Coat / 899 Vascular Coat (Uvea) / 902 Retina / 907 Crystalline Lens / 915 Vitreous Body / 915 Accessory Structures of the Eye / 916 Folder 24.1 Clinical Correlation: Glaucoma / 905 Folder 24.2 Clinical Correlation: Retinal Detachment / 908 Folder 24.3 Clinical Correlation: Age-Related Macular Degeneration (ARMD) / 909 Folder 24.4 Clinical Correlation: Conjunctivitis / 917

1	The eye is a complex sensory organ that provides the sense of sight. In many ways, the eye is similar to a digital camera. Like the optical system of a camera, the cornea and lens of the eye capture and automatically focus light. The iris also automatically adjusts the eye to differences in illumination of visual fields. In many aspects, the optical system of the eye is far more elaborate and complex than a camera. For example, the eye has the ability to track moving objects with coordinated eye movements. The eye can also protect, maintain, self-repair, and clean its transparent optical system. The light detector in a digital camera, the charge-coupled device (CCD), consists of closely spaced photodiodes that capture, collect, and convert the light image into a series of electrical impulses. Similarly, the photoreceptor cells in the retina of the eye detect light intensity and color (wavelengths of visible light that are reflected by different objects) and encode these parameters

1	Similarly, the photoreceptor cells in the retina of the eye detect light intensity and color (wavelengths of visible light that are reflected by different objects) and encode these parameters into electrical impulses for transmission to the brain via the optic nerve. The retina has other capabilities beyond those of a CCD: It can extract and modify specific impulses from the visual image before sending them to the central nervous system (CNS).

1	Because the eyes are paired, two somewhat different and overlapping images (visual fields) are sent to the brain. The brain can be compared to a computer, which processes the slightly different images from each eye, separates them into layers, and projects them onto the primary visual cortex located in the occipital lobes. Complex neural mechanisms coordinate eye movements, enabling us to perceive depth and distance to achieve a three-dimensional image. Therefore, the way in which we perceive the world around us largely depends on impulses processed within the retina and analysis and interpretation of these impulses by the CNS. The eye measures approximately 25 mm in diameter. It is suspended in the bony orbital socket by six extrinsic muscles that control its movement. A thick layer of adipose tissue partially surrounds and cushions the eye as it moves within the orbit. The extraocular muscles are coordinated so that the eyes move symmetrically around their own central axes.

1	Layers of the Eye The wall of the eye consists of three concentric layers or coats. The eyeball is composed of three structural layers (Fig. 24.1):  The corneoscleral coat, the outer or fibrous layer, includes the sclera, the white portion, and the cornea, the transparent portion. FIGURE 24.1 • Schematic diagram of the layers of the eye. The wall of the eyeball is organized in three separate concentric layers: (a) an outer supporting layer, the corneoscleral coat (colorless and blue); (b) a middle vascular coat or uvea (pink); and (c) an inner photosensitive layer, the retina (yellow).  The vascular coat, the middle layer, or uvea, includes the choroid and the stroma of the ciliary body and iris.  The retina, the inner layer, includes an outer pigment ep ithelium, the inner neural retina, and the epithelium of the ciliary body and iris. The neural retina is continuous with the central nervous system through the optic nerve.

1	The corneoscleral coat consists of the transparent cornea and the white opaque sclera. The cornea covers the anterior one-sixth of the eye (Fig. 24.1a). In this windowlike region, the surface of the eye has a prominence or convexity. The cornea is continuous with the sclera (Gr. skleros, hard). The sclera is composed of dense fibrous connective tissue that provides attachment for the extrinsic muscles of the eye. The sclera constitutes the “white” of the eye. In children, it has a slightly blue tint because of its thinness; in elderly people, it is yellowish because of the accumulation of lipofuscin in its stromal cells. The corneoscleral coat encloses the inner two layers except where it is penetrated by the optic nerve. The uvea consists principally of the choroid, the vascular layer that provides nutrients to the retina.

1	The uvea consists principally of the choroid, the vascular layer that provides nutrients to the retina. Blood vessels and melanin pigment give the choroid an intense dark brown color. The pigment absorbs scattered and reflected light to minimize glare within the eye. The choroid contains many venous plexuses and layers of capillaries and is firmly attached to the retina (Fig. 24.1b). The anterior rim of the uveal layer continues forward, where it forms the stroma of the ciliary body and iris. The ciliary body is a ringlike thickening that extends inward just posterior to the level of the corneoscleral junction. Within the ciliary body is the ciliary muscle, a smooth muscle that is responsible for lens accommodation. Contraction of the ciliary muscle changes the shape of the lens, which enables it to bring light rays from different distances to focus on the retina.

1	The iris is a contractile diaphragm that extends over the anterior surface of the lens. It also contains smooth muscle and melanin-containing pigment cells scattered in the connective tissue. The pupil is the central circular aperture of the iris. It appears black because one looks through the lens toward the heavily pigmented back of the eye. In the process of adaptation, the pupil changes in size to control the amount of light that passes through the lens to reach the retina. The retina consists of two components: The neural retina and pigment epithelium. The retina is a thin, delicate layer (Fig. 24.1c) consisting of two components:  The neural retina is an inner layer that contains light-sensitive receptors and complex neuronal networks.  The retinal pigment epithelium (RPE) is an outer layer composed of simple cuboidal melanin-containing cells.

1	Externally, the retina rests on the choroid; internally, it is associated with the vitreous body. The neural retina consists largely of photoreceptor cells, called retinal rods and cones, and interneurons. Visual information encoded by the rods and cones is sent to the brain via impulses conveyed along the optic nerve. Chambers of the Eye The layers of the eye and the lens serve as boundaries for three chambers within the eye. The chambers of the eye are the following.  The anterior chamber is the space between the cornea and the iris.  The posterior chamber is the space between the posterior surface of the iris and the anterior surface of the lens.  The vitreous chamber is the space between the posterior surface of the lens and the neural retina (Fig. 24.2). The cornea, the anterior and posterior chambers, and their contents constitute the anterior segment of the eye. The

1	FIGURE 24.2 • Schematic diagram canal of illustrating the internal structures of the human eye. The retina consists of that differ in their function. Note that the photosensitive region of the retina occupies the posterior part of the eye and terminates iris anteriorly along the ora serrata. The nonphotosensitive region of the retina is located anterior to the ora serrata and lines the inner aspect of the ciliary body and the posterior surface of the iris. The other layers of the eyeball as well as the attachment of two of the extraocular muscles to the sclera inferior rectus muscle are also shown. vitreous chamber, visual retina, RPE, posterior sclera, and Development of the Eye uvea constitute the posterior segment. To appreciate the unusual structural and functional relationships in the eye, it is helpful to understand how it forms in the The refractile media components of the eye alter the light embryo. path to focus it on the retina.

1	The refractile media components of the eye alter the light embryo. path to focus it on the retina. As light rays pass through the components of the eye, they are The tissues of the eye are derived from neuroectoderm, refracted. Refraction focuses the light rays on the photorecep surface ectoderm, and mesoderm. tor cells of the retina. Four transparent components of the By the 22nd day of development, the eyes are evident as eye, called the refractile (or dioptric) media, alter the path shallow grooves—the optic sulci or grooves—in the neural of the light rays: folds at the cranial end of the human embryo. As the neural  The cornea is the anterior window of the eye. tube closes, the paired grooves form outpocketings called optic The aqueous humor is the watery fluid located in the vesicles (Fig. 24.3a). As each optic vesicle grows laterally, the anterior and posterior chambers.

1	connection to the forebrain becomes constricted into an optic  The lens is a transparent, crystalline, biconcave structure stalk, and the overlying surface ectoderm thickens and forms a suspended from the inner surface of the ciliary body by a lens placode. These events are followed by concomitant in-ring of radially oriented fibers, the zonule of Zinn. vagination of the optic vesicles and the lens placodes. The in The vitreous body is composed of a transparent gel subvagination of the optic vesicle results in the formation of a stance that fills the vitreous chamber. It acts as a “shock double-layered optic cup (Fig. 24.3b). The inner layer becomes absorber” that protects the fragile retina during rapid eye the neural retina. The outer layer becomes the RPE. movement and helps to maintain the shape of the eye.

1	movement and helps to maintain the shape of the eye. Invagination of the central region of each lens placode re-The vitreous body is almost 99% water with soluble prosults in the formation of the lens vesicles. By the fifth week teins, hyaluronan, glycoproteins, widely dispersed collaof development, the lens vesicle loses contact with the surface gen fibrils, and traces of other insoluble proteins. The ectoderm and comes to lie in the mouth of the optic cup. fluid component of the vitreous body is called the vitre-After the lens vesicle detaches from the surface ectoderm, this ous humor.

1	same site again thickens to form the corneal epithelium. Mes-The cornea is the chief refractive element of the eye. It has a enchymal cells from the periphery then give rise to the refractive index of 1.376 (air has a refractive index of 1.0). corneal endothelium and the corneal stroma. The lens is second in importance to the cornea in the refrac-Grooves containing blood vessels derived from mesenchyme tion of light rays. Because of its elasticity, the shape of the lens develop along the inferior surface of each optic cup and stalk. can undergo slight changes in response to the tension of the Called the choroid fssures, the grooves enable the hyaloid ciliary muscle. These changes are important in accommoda-artery to reach the inner chamber of the eye. This artery and its tion for proper focusing on near objects. The aqueous humor branches supply the inner chamber of the optic cup, lens vesicle, and vitreous body have only minor roles in refraction. How-and mesenchyme within the optic

1	on near objects. The aqueous humor branches supply the inner chamber of the optic cup, lens vesicle, and vitreous body have only minor roles in refraction. How-and mesenchyme within the optic cup. The hyaloid vein returns ever, the aqueous humor plays an important role in providing blood from these structures. The distal portions of the hyaloid nutrients to two avascular structures, the lens and cornea. In vessels degenerate, but the proximal portions remain as the cenaddition to transmitting light, the vitreous body helps main-tral retinal artery and central retinal vein. By the end of the tain the position of the lens and helps keep the neural retina seventh week, the edges of the choroid fissure fuse, and a round in contact with the RPE. opening, the future pupil, forms over the lens vesicle.

1	forebrain of optic cup lumen of choroid inner layer of optic nerveoptic stalk FIGURE 24.3 • Schematic drawing illustrating the development of the eye. a. Forebrain and developing optic vesicles as seen in a 4-mm embryo. b. Bilayered optic cup and invaginating lens vesicle as seen in a 7.5-mm embryo. The optic stalk connects the developing eye to the brain. c. The eye as seen in a 15-week fetus. All the layers of the eye are established, and the hyaloid artery traverses the vitreous body from the optic disc to the posterior surface of the lens. (Modified from Mann IC. The Development of the Human Eye. New York: Grune & Stratton, 1974.)

1	Human Eye. New York: Grune & Stratton, 1974.) The outer layer of the optic cup forms a single layer of pigmented cells (Fig. 24.3c). Pigmentation begins at the end of the fifth week. The inner layer undergoes a complex differentiation into the nine layers of the neural retina. The photoreceptor cells (rods and cones), as well as the bipolar, amacrine, and ganglion cells and nerve fibers are present by the seventh month. The macular depression begins to develop during the eighth month and is not complete until about 6 months after birth.

1	During the third month, growth of the optic cup gives rise to the ciliary body and the future iris, which forms a double row of epithelium in front of the lens. The mesoderm located external to this region becomes the stroma of the ciliary body and iris. Both epithelial layers of the iris become pigmented. In the ciliary body, however, only the outer layer is pigmented. At birth, the iris is light blue in fair-skinned people because pigment is usually not present. The dilator and sphincter pupillary muscles develop during the sixth month as derivatives of the neuroectoderm of the outer layer of the optic cup. The embryonic origins of the individual eye structures are summarized in Table 24.1. The cornea consists of five layers: Three cellular layers and two noncellular layers.

1	The embryonic origins of the individual eye structures are summarized in Table 24.1. The cornea consists of five layers: Three cellular layers and two noncellular layers. The transparent cornea (see Figs. 24.1 and 24.2) is only 0.5 mm thick at its center and about 1 mm thick peripherally. It consists of three cellular layers that are distinct in both appearance and origin. These layers are separated by two important membranes that appear homogeneous when viewed in the light microscope. Thus, the fve layers of the cornea seen in a transverse section are the following:

1	TABLE Embryonic Origins of the Individual Structures of the Eye24.1 Source Derivative Surface ectoderm Lens Epithelium of the cornea, conjunctiva, and lacrimal gland and its drainage system Neural ectoderm Vitreous body (derived partly from neural ectoderm of the optic cup and partly from mesenchyme) Epithelium of the retina, iris, and ciliary body Sphincter pupillae and dilator papillae muscles Optic nerve Mesoderm Sclera Stroma of the cornea, ciliary body, iris, and choroid Extraocular muscles Eyelids (except epithelium and conjunctiva) Hyaloid system (most of which degenerates before birth) Coverings of the optic nerve Connective tissue and blood vessels of the eye, bony orbit, and vitreous body The corneal epithelium is a nonkeratinized stratified squamous epithelium.

1	The corneal epithelium is a nonkeratinized stratified squamous epithelium. The corneal epithelium (Fig. 24.4) consists of approximately five layers of nonkeratinized cells and measures about 50 m in average thickness. It is continuous with the conjunctival epithelium that overlies the adjacent sclera. The epithelial cells adhere to neighboring cells via desmosomes that are present on short interdigitating processes. Like other stratified epithelium, such as that of the skin, the cells proliferate from a basal layer and become squamous at the surface. The basal cells are low columnar with round, ovoid nuclei; the surface cells acquire a squamous or discoid shape, and their nuclei are flattened and pyknotic (see Fig. 24.4b). As the cells migrate to the surface, the cytoplasmic organelles gradually disappear, indicating a progressive decline in metabolic activity. The corneal epithelium has a remarkable regenerative capacity with a turnover time of approximately 7 days.

1	The actual stem cells for the corneal epithelium reside at the corneoscleral limbus, the junction of the cornea and sclera. The microenvironment of this stem cell niche is important in maintaining the population of stem cells that also act as a “barrier” to conjunctival epithelial cells and normally prevent their migration to the corneal surface. The corneal epithelial stem cells may be partially or totally depleted by disease or extensive injury, resulting in abnormalities of the corneal surface that lead to conjunctivalization of the cornea, which is characterized by vascularization, appearance of goblet cells, and an irregular and unstable epithelium. These changes cause ocular discomfort and reduced vision. Minor injuries of the corneal surface heal rapidly by inducing stem cell proliferation and migration of cells from the corneoscleral limbus to fll the defect. Numerous free nerve endings in the corneal epithelium provide it with extreme sensitivity to touch. Stimulation of

1	FIGURE 24.4 • Photomicrograph of the cornea. a. This photomicrograph of a section through the full thickness of the cornea shows the corneal stroma and the two corneal surfaces covered by different types of epithelia. The corneal stroma does not contain blood or lymphatic vessels. 140. b. A higher magnification of the anterior surface of the cornea showing the corneal stroma covered by a stratified squamous (corneal) epithelium. The basal cells that rest on Bowman’s membrane, which is a homogeneous condensed layer of corneal stroma, are low columnar in contrast to the squamous surface cells. Note that one of the surface cells is in the process of desquamation (arrow). 280. c. A higher-magnification photomicrograph of the posterior surface of the cornea covered by a thin layer of simple squamous epithelium (corneal endothelium). These cells are in direct contact with the aqueous humor of the anterior chamber of the eye. Note the very thick Descemet’s membrane (basal lamina) of the

1	squamous epithelium (corneal endothelium). These cells are in direct contact with the aqueous humor of the anterior chamber of the eye. Note the very thick Descemet’s membrane (basal lamina) of the corneal endothelial cells. 280.

1	these nerves (e.g., by small foreign bodies) elicits blinking of the eyelids, flow of tears, and, sometimes, severe pain. Microvilli present on the surface epithelial cells help retain the tear film over the entire corneal surface. Drying of the corneal surface may cause ulceration. DNA in corneal epithelial cells is protected from UV light damage by nuclear ferritin. Despite constant exposure of the corneal epithelium to UV light, cancer of this epithelium is extremely rare. Unlike the epidermis, which is also exposed to UV light, melanin is not present as a defense mechanism in corneal epithelium. The presence of melanin in the cornea would diminish light transmission. Instead, it has recently been shown that corneal epithelial cell nuclei contain ferritin, an iron-storage protein. Experimental studies with avian corneas have shown that nuclear ferritin protects the DNA in the corneal epithelial cells from free radical damage caused by UV light exposure.

1	Bowman’s membrane is a homogeneous-appearing layer on which the corneal epithelium rests. Bowman’s membrane (anterior basement membrane) is a homogeneous, faintly fibrillar lamina that is approximately 8 m to 10 m thick. It lies between the corneal epithelium and the underlying corneal stroma and ends abruptly at the corneoscleral limbus. The collagen fibrils of Bowman’s membrane have a diameter of about 18 nm and are randomly oriented. Bowman’s membrane imparts some strength to the cornea, but more signifcantly, it acts as a barrier to the spread of infections. It does not regenerate. Therefore, if damaged, an opaque scar forms that may impair vision. In addition, changes in Bowman’s membrane are associated with recurrent corneal erosions. The corneal stroma constitutes 90% of the corneal thickness.

1	The corneal stroma constitutes 90% of the corneal thickness. The corneal stroma, also called substantia propria, is composed of about 60 thin lamellae. Each lamella consists of parallel bundles of collagen fibrils. Located between lamellae are nearly complete sheets of slender, flattened fibroblasts. The fibrils measure approximately 23 nm in diameter and are as long as 1 cm. The collagen fibrils in each lamella are arranged at approximately right angles to those in the adjacent lamellae (Fig. 24.5). The ground substance contains corneal proteoglycans, which are sulfated glycosaminoglycans— chiefly, keratan sulfate (lumican) and chondroitin sulfate covalently bound to protein (decorin). Lumican regulates normal collagen fibril assembly in the cornea and is critical in the development of a highly organized collagenous matrix.

1	It is believed that the uniform spacing of collagen fibrils and lamellae, as well as the orthogonal array of the lamellae (alternating layers at right angles), is responsible for the transparency of the cornea. Proteoglycans (lumican), along with type V collagen, regulate the precise diameter and spacing of the collagen fibrils. Swelling of the cornea after injury to the epithelium or endothelium disrupts this precise array and leads to translucency or opacity of the cornea. Lumican is FIGURE 24.5 • Electron micrograph of the corneal stroma. This electron micrograph shows parts of three lamellae and a portion of a corneal fibroblast (CF) between two of the lamellae. Note that the collagen fibrils in adjacent lamellae are oriented at right angles to one another. 16,700. overexpressed during the wound-healing process following corneal injury.

1	overexpressed during the wound-healing process following corneal injury. Normally, the cornea contains no blood vessels or pigments. During an infammatory response involving the cornea, large numbers of neutrophilic leukocytes and lymphocytes migrate from blood vessels of the corneoscleral limbus and penetrate the stromal lamellae. Descemet’s membrane is an unusually thick basal lamina. Descemet’s membrane (posterior basement membrane) is the basal lamina of corneal endothelial cells. It is intensely positive to periodic acid–Schiff (PAS) and can be as thick as 10 m. Descemet’s membrane has a feltlike appearance and consists of an interwoven meshwork of fibers and pores. It separates the corneal endothelium from the adjacent corneal stroma. Unlike Bowman’s membrane, Descemet’s membrane readily regenerates after injury. It is produced continuously but slowly thickens with age.

1	Unlike Bowman’s membrane, Descemet’s membrane readily regenerates after injury. It is produced continuously but slowly thickens with age. Descemet’s membrane extends peripherally beneath the sclera as a trabecular meshwork forming the pectinate ligament. Strands from the pectinate ligament penetrate the ciliary muscle and sclera and may help to maintain the normal curvature of the cornea by exerting tension on Descemet’s membrane. The corneal endothelium provides for metabolic exchange between the cornea and aqueous humor.

1	The corneal endothelium provides for metabolic exchange between the cornea and aqueous humor. The corneal endothelium is a single layer of squamous cells covering the surface of the cornea that faces the anterior chamber (see Fig. 24.4). The cells are joined by well-developed zonulae adherentes, relatively leaky zonulae occludentes, and desmosomes. Virtually all of the metabolic exchanges of the cornea occur across the endothelium. The endothelial cells contain many mitochondria and vesicles and an extensive rough-surfaced endoplasmic reticulum (rER) and Golgi apparatus. They demonstrate endocytotic activity and are engaged in active transport. Na/K-activated ATPase is located on the lateral plasma membrane.

1	Transparency of the cornea requires precise regulation of the water content of the stroma. Physical or metabolic damage to the endothelium leads to rapid corneal swelling and, if the damage is severe, corneal opacity. Restoration of endothelial integrity is usually followed by deturgescence, although corneas can swell beyond their ability for self-repair. Such swelling can result in permanent focal opacities caused by aggregation of collagen fbrils in the swollen cornea. Essential sulfated glycosaminoglycans that normally separate the corneal collagen fbers are extracted from the swollen cornea.

1	Human corneal endothelium has a limited proliferative capacity. Severely damaged endothelium can only be repaired by transplantation of a donor cornea. Recent studies indicate that the periphery of the cornea represents a regenerative zone of the corneal endothelial cells. However, soon after corneal transplantation, endothelial cells exhibit contact inhibition when exposed to the extracellular matrix of Descemet’s membrane. This fnding that inhibitory factors released by Descemet’s membrane prevent proliferation of endothelial cells has focused some current corneal research on reversal or prevention of this inhibition with exogenous growth factors. The sclera is an opaque layer that consists predominantly of dense connective tissue.

1	The sclera is an opaque layer that consists predominantly of dense connective tissue. The sclera is a thick fibrous layer containing flat collagen bundles that pass in various directions and in planes parallel to its surface. Both the collagen bundles and fibrils that form them are irregular in diameter and arrangement. Interspersed between the collagen bundles are fine networks of elastic fibers and a moderate amount of ground substance. Fibroblasts are scattered among these fibers (Plate 107, page 926).

1	The opacity of the sclera, like that of other dense connective tissues, is primarily attributable to the irregularity of its structure. The sclera is pierced by blood vessels, nerves, and the optic nerve (see Fig. 24.2). It is 1 mm thick posteriorly, 0.3 to 0.4 mm thick at its equator, and 0.7 mm thick at the corneoscleral margin or “limbus.” The sclera is divided into three rather ill-defined layers:  The episcleral layer (episclera), the external layer, is the loose connective tissue adjacent to the periorbital fat.  The substantia propria (sclera proper, also called Tenon’s capsule), is the investing fascia of the eye and is composed of a dense network of thick collagen fibers.  The suprachoroid lamina (lamina fusca), the inner aspect of the sclera, is located adjacent to the choroid and contains thinner collagen fibers and elastic fibers as well as fibroblasts, melanocytes, macrophages, and other connective tissue cells.

1	In addition, the episcleral space (Tenon’s space) is located between the episcleral layer and substantia propria of the sclera. This space and the surrounding periorbital fat allow the eye to rotate freely within the orbit. The tendons of the extraocular muscles attach to the substantia propria of the sclera. The corneoscleral limbus is the transitional zone between the cornea and sclera. At the junction of the cornea and sclera (Fig. 24.6 and Plate 107, page 926), Bowman’s membrane ends abruptly. The overlying epithelium at this site thickens from the 5 cell layers of the cornea to the 10 to 12 cell layers of the conjunctiva. The surface of the limbus is composed of two distinct types of epithelial cells: one type constitutes the conjunctival cells and the other corneal epithelial cells.

1	At this junction, the corneal lamellae become less regular as they merge with the oblique bundles of collagen fibers of the sclera. An abrupt transition from the avascular cornea to the well-vascularized sclera also occurs here.

1	The limbus region, specifically, the iridocorneal angle, contains the apparatus for the outflow of aqueous humor (Fig. 24.7). In the stromal layer, endothelium-lined channels called the trabecular meshwork (or spaces of Fontana) merge to form the scleral venous sinus (canal of Schlemm). This sinus encircles the eye (see Figs. 24.6 and 24.7). The aqueous humor is produced by the ciliary processes that border the lens in the posterior chamber of the eye. The fluid passes from the posterior chamber into the anterior chamber through the valvelike potential opening between the iris and lens. The fluid then passes through the openings in the trabecular meshwork in the limbus region as it continues its course to enter the scleral venous sinus. Collecting vessels in the sclera, called aqueous veins because they convey aqueous humor instead of blood, transport the aqueous humor to (blood) veins located in the sclera. Changes in the iridocorneal angle may lead to blockage in the drainage of

1	veins because they convey aqueous humor instead of blood, transport the aqueous humor to (blood) veins located in the sclera. Changes in the iridocorneal angle may lead to blockage in the drainage of aqueous humor causing glaucoma (see Folder 24.1). The iridocorneal angle can be visualized during eye examination using a gonioscope, a specialized optical device that utilizes mirrors or prisms to refect the light from the iridocorneal angle into the direction of the observer. In conjunction with a slit lamp or operating microscope, the ophthalmologist can examine this region to monitor various eye conditions associated with glaucoma.

1	The iris, the most anterior part of the vascular coat, forms a contractile diaphragm in front of the lens. The iris arises from the anterior border of the ciliary body (Fig. 24.7) and is attached to the sclera about 2 mm posterior to the corneoscleral junction. The pupil is the central aperture of this thin disc. The iris is pushed slightly forward as it changes in size in response to light intensity. It consists of highly vascularized connective tissue stroma that is covered on its posterior surface by highly pigmented cells, the posterior pigment epithelium (Fig. 24.8). The basal lamina of sphincter m. pigment layer iris dilator m. canal of Schlemm ciliary muscle ciliary process zonular fibers

1	FIGURE 24.6 • Schematic diagram of the structure of the eye. This drawing shows a horizontal section of the eyeball with color-coded layers of its wall. Upper inset. Enlargement of the anterior and posterior chambers is shown in more detail. Note the location of the iridocorneal angle and canal of Schlemm (scleral venous sinus), which drains the aqueous humor from the anterior chamber of the eye. Lower inset. Typical organization of the cells and nerve fibers of the fovea.

1	these cells faces the posterior chamber of the eye. The degree of pigmentation is so great that neither the nucleus nor character of the cytoplasm can be seen in the light microscope. Located beneath this layer is a layer of myoepithelial cells, the anterior pigment myoepithelium. The apical (posterior) portions of these myoepithelial cells are laden with melanin granules, which effectively obscure their boundaries with the adjacent posterior pigment epithelial cells. The basal (anterior) portions of myoepithelial cells possess processes containing contractile elements that extend radially and collectively make up the dilator pupillae muscle of the iris. The contractile processes are enclosed by a basal lamina that separates them from the adjacent stroma. Constriction of the pupil is produced by smooth muscle cells located in the stroma of the iris near the pupillary margin of the iris. These circumferentially oriented cells collectively compose the sphincter pupillae muscle.

1	The anterior surface of the iris reveals numerous ridges and grooves that can be seen in clinical examination with the ophthalmoscope. When this surface is examined in the light microscope, it appears as a discontinuous layer of fibroblasts and melanocytes. The number of melanocytes in the stroma is responsible for variation in eye color. The function of these pigment-containing cells in the iris is to absorb light rays. If there are few melanocytes in the stroma, eye color is derived from light refected from the pigment present in the cells of the posterior surface of the iris, giving it a blue appearance. As the amount of pigment present in the stroma increases, the color changes from blue to shades of greenish blue, gray, and, fnally, brown. The sphincter pupillae is innervated by parasympathetic nerves; the dilator pupillae muscle is under sympathetic nerve control.

1	The sphincter pupillae is innervated by parasympathetic nerves; the dilator pupillae muscle is under sympathetic nerve control. The size of the pupil is controlled by contraction of the sphincter pupillae and dilator pupillae muscles. The process of adaptation (increasing or decreasing the size of the pupil) ensures that only the appropriate amount of light enters the eye. Two muscles are actively involved in adaptation:  The sphincter pupillae muscle, a circular band of smooth muscle cells (Plate 106, page 924), is innervated by parasympathetic nerves carried in the oculomotor nerve (cranial nerve III) and is responsible for reducing pupillary size in response to bright light. Failure of the

1	FIGURE 24.7 • Photomicrograph of the ciliary body and iridocorneal angle. This photomicrograph of the human eye shows the anterior portion of the ciliary body and parts of the iris and sclera. The inner surface of the ciliary body forms radially arranged, ridge-shaped elevations, the ciliary processes, to which the zonula fibers are anchored. The ciliary body contains the ciliary muscle, connective tissue with blood vessels of the vascular coat, and the ciliary epithelium, which is responsible for the production of aqueous humor. Anterior to the ciliary body, between the iris and the cornea, is the iridocorneal angle. The scleral venous sinus (canal of Schlemm) located in close proximity to this angle drains the aqueous humor to regulate intraocular pressure. 120. The inset shows that the ciliary epithelium consists of two layers, the outer pigmented layer and the inner nonpigmented layer. 480.

1	pupil to respond when light is shined into the eye— “pupil fxed and dilated”—is an important clinical sign showing lack of nerve or brain function.  The dilator pupillae muscle is a thin sheet of radially oriented contractile processes of pigmented myoepithelial cells constituting the anterior pigment epithelium of the iris. This muscle is innervated by sympathetic nerves from the superior cervical ganglion and is responsible for increasing pupillary size in response to dim light. Just before ophthalmoscopic examination, mydriatic agents such as atropine are given as eye drops to cause dilation of the pupil. Acetylcholine (ACh) is the neurotransmitter of the parasympathetic nervous system (it innervates the sphincter pupillae muscle); the addition of atropine blocks muscarinic acetylcholine receptors, temporally blocking the action of the sphincter muscle and leaving the pupil wide open and unreactive to light originating from ophthalmoscope.

1	The ciliary body is the thickened anterior portion of the vascular coat and is located between the iris and choroid. The ciliary body extends about 6 mm from the root of the iris posterolaterally to the ora serrata (see Fig. 24.2). As seen from behind, the lateral edge of the ora serrata bears 17 to 34 grooves or crenulations. These grooves mark the anterior limit of both the retina and the choroid. The anterior third of the ciliary body has about 75 radial ridges or ciliary processes (see Fig. 24.7). The fibers of the zonule arise from the grooves between the ciliary processes. The layers of the ciliary body are similar to those of the iris and consist of a stroma and an epithelium. The stroma is divided into two layers:  An outer layer of smooth muscle, the ciliary muscle, makes up the bulk of the ciliary body.  An inner vascular region extends into the ciliary processes.

1	The epithelial layer covering the internal surface of the ciliary body is a direct continuation of the two layers of the retinal epithelium (see Fig. 24.1). The ciliary muscle is organized into three functional portions or groups of smooth muscle fibers. The smooth muscle of the ciliary body has its origin in the scleral spur, a ridgelike projection on the inner surface of the sclera at the corneoscleral junction. The muscle fibers spread out in several directions and are classified into three functional groups on the basis of their direction and insertion:

1	Glaucoma is a clinical condition resulting from increased intraocular pressure over a sustained period of time. It can be caused by excessive secretion of aqueous humor or impedance of the drainage of aqueous humor from the an-terior chamber. The internal tissues of the eye, particularly the retina, are nourished by the diffusion of oxygen and nutrients from the intraocular vessels. Blood flows normally through these vessels (including the capillaries and veins) when the hydrostatic pressure within the vessels exceeds the intraocular pressure. If the drainage of the aqueous humor is impeded, the intraocular pressure increases be-cause the layers of the eye do not allow the wall to expand. This increased pressure interferes with normal retinal nour-ishment and function, causing the retinal nerve fiber layer to atrophy (Fig. F24.1.1). There are two major types of glaucoma:  Open-angle glaucoma is the most common type of glaucoma and the leading cause of blindness among adults. The

1	nerve fiber layer to atrophy (Fig. F24.1.1). There are two major types of glaucoma:  Open-angle glaucoma is the most common type of glaucoma and the leading cause of blindness among adults. The removal of aqueous humor is obstructed be-cause of reduced flow through the trabecular meshwork of the iridocorneal angle into the scleral venous sinus (canal of Schlemm).  Close-angle glaucoma (acute glaucoma) is much less common and is characterized by a narrowed irido-corneal angle that obstructs inflow of the aqueous humor into the scleral venous sinus. Usually it is associ-ated with a sudden, painful, complete blockage of the scleral venous sinus and can result in permanent blind-ness if not treated promptly. Visual deficits associated with glaucoma include blurring of vision and impaired dark adaptation (symptoms that indicate loss of normal retinal function) and halos around lights (a symptom indicating corneal endothelial damage). If the con-dition is not treated, the retina will be

1	adaptation (symptoms that indicate loss of normal retinal function) and halos around lights (a symptom indicating corneal endothelial damage). If the con-dition is not treated, the retina will be permanently damaged, and blindness will occur. Treatments are directed toward lowering the intraocular pressure by decreasing the rate of production of aqueous humor or eliminating the cause of the obstruction of normal drainage. Recently, carbonic anhydrase inhibitors that specifically inhibit carbonic an-hydrase isoenzyme CA-II, which plays an important role in the production of aqueous humor in humans, are used as the pharmacologic treatment of choice. Dorzolamide and brinzolamide are two carbonic anhydrase inhibitors that are currently available as eyedrops to treat glaucoma.

1	 FOLDER 24.1 Clinical Correlation: Glaucoma FIGURE F24.1.1 • Glaucoma. This image shows a view of the fundus of the left eye in a patient with advanced glaucoma. As a result of the increased intraocular pressure, retinal nerve fibers undergo atrophy and shrink in size. Note a pale optic disc in the center of the image with a less pronounced rim due to atrophy of nerve fibers. Enlargement of the optic nerve cup (central area of the optic disc) is also visible and a characteristic finding for glaucoma. Compare this image to a normal retina in Fig. 24.14. (Courtesy of Dr. Renzo A. Zaldivar.)  The meridional (or longitudinal) portion consists of the outer muscle fibers that pass posteriorly into the stroma of the choroid. These fibers function chiefly in stretching the choroid. It also may help open the iridocorneal angle and facilitate drainage of the aqueous humor.

1	 The radial (or oblique) portion consists of deeper muscle fiber bundles that radiate in a fanlike fashion to insert in the ciliary body. Its contraction causes the lens to flatten and thus focus for distant vision.  The circular (or sphincteric) portion consists of inner muscle fiber bundles oriented in a circular pattern that forms a sphincter. It reduces the tension on the lens, causing the lens to accommodate for near vision. Examination of a histologic preparation does not clearly reveal the arrangement of the muscle fibers. Rather, the organizational grouping is based on microdissection techniques. Ciliary processes are ridgelike extensions of the ciliary body from which zonular fibers emerge and extend to the lens.

1	Ciliary processes are ridgelike extensions of the ciliary body from which zonular fibers emerge and extend to the lens. Ciliary processes are thickenings of the inner vascular region of the ciliary body. They are continuous with the vascular layers of the choroid. Scattered macrophages containing melanin pigment granules and elastic fibers are present in these processes (Plate 106, page 924). The processes and the ciliary body are covered by a double layer of columnar epithelial cells, the ciliary epithelium, which was originally

1	FIGURE 24.8 • Structure of the iris. a. This schematic diagram shows the layers of the iris. Note that the pigmented epithelial cells are reflected as occurs at the pupillary margin of the iris. The two layers of pigmented epithelial cells are in contact with the dilator pupillae muscle. The incomplete layer of fibroblasts and stromal melanocytes is indicated on the anterior surface of the iris. b. Photomicrograph of the iris showing the histologic features of this structure. The lens, which lies posterior to the iris, is included for orientation. The iris is composed of a connective tissue stroma covered on its posterior surface by the posterior pigment epithelium. The basal lamina (not visible) faces the posterior chamber of the eye. Because of intense pigmentation, the histologic features of these cells are not discernible. Just anterior to these cells is the anterior pigment myoepithelium layer (the dashed line separates the two layers). Note that the posterior portion of the

1	of these cells are not discernible. Just anterior to these cells is the anterior pigment myoepithelium layer (the dashed line separates the two layers). Note that the posterior portion of the myoepithelial cells contains melanin, whereas the anterior portion contains contractile elements forming the dilator pupillae muscle of the iris. The sphincter pupillae muscle is evident in the stroma. The color of the iris depends on the number of stromal melanocytes scattered throughout the connective tissue stroma. At the bottom, note the presence of the lens. 570.

1	derived from the two layers of the optic cup. The ciliary epithelium has three principal functions:  Secretion of aqueous humor Participation in the blood–aqueous barrier (part of the blood–ocular barrier  Secretion and anchoring of the zonular fbers that form the suspensory ligament of the lens The inner cell layer of the ciliary epithelium has a basal lamina facing the posterior and vitreous chambers. The cells in this layer are nonpigmented. The cell layer that has its basal lamina facing the connective tissue stroma of the ciliary body is heavily pigmented and is directly continuous with the pigmented epithelial layer of the retina. The double-layered ciliary epithelium continues over the iris, where it becomes the posterior pigmented epithelium and anterior pigmented myoepithelium. The zonular fibers extend from the basal lamina of the nonpigmented epithelial cells of the ciliary processes and insert into the lens capsule (the thickened basal lamina of the lens).

1	The cells of the nonpigmented layer have all the characteristics of a fluid-transporting epithelium, including complex cell-to-cell junctions with a well-developed zonular occludens, extensive lateral and basal plications, and localization of Na/K-ATPase in the lateral plasma membrane. In addition, they have an elaborate rER and Golgi complex consistent with their role in secreting the zonular fibers. The cells of the pigmented layer have a less developed junctional zone and often exhibit large, irregular lateral intercellular spaces. Both desmosomes and gap junctions hold together the apical surfaces of the two cell layers, creating discontinuous “luminal” spaces called ciliary channels.

1	The aqueous humor is similar in ionic composition to plasma but contains less than 0.1% protein (compared with 7% protein in plasma). The aqueous humor passes from the ciliary body toward the lens, and then between the iris and lens, before it reaches the anterior chamber of the eye (see Fig. 24.6). In the anterior chamber of the eye, the aqueous humor passes laterally to the angle formed between the cornea and iris. Here it penetrates the tissues of the limbus as it enters the labyrinthine spaces of trabecular meshwork and finally reaches the canal of Schlemm, which communicates with the veins of the sclera (see Folder 24.1). The choroid is the portion of the vascular coat that lies deep to the retina. The choroid is a dark brown vascular sheet only 0.25 mm thick posteriorly and 0.1 mm thick anteriorly. It lies between the sclera and retina (see Fig. 24.1).

1	The choroid is a dark brown vascular sheet only 0.25 mm thick posteriorly and 0.1 mm thick anteriorly. It lies between the sclera and retina (see Fig. 24.1). Two layers can be identified in the choroid:  The choriocapillary layer, an inner vascular layer  Bruch’s membrane, a thin, amorphous hyaline membrane

1	The choroid is attached firmly to the sclera at the margin of the optic nerve. A potential space, the perichoroidal space (between the sclera and retina), is traversed by thin, ribbon-like, branching lamellae or strands that pass from the sclera to the choroid. These lamellae originate from the suprachoroid lamina (lamina fusca) and consist of large, flat melanocytes scattered between connective tissue elements including collagen and elastic fibers, fibroblasts, macrophages, lymphocytes, plasma cells, and mast cells. The lamellae pass inward to surround the vessels in the remainder of the choroid layer. Free smooth muscle cells, not associated with blood vessels, are present in this tissue. Lymphatic channels called epichoroid lymph spaces, long and short posterior ciliary vessels, and nerves on their way to the front of the eye are also present in the suprachoroid lamina.

1	Most of the blood vessels decrease in size as they approach the retina. The largest vessels continue forward beyond the ora serrata into the ciliary body. These vessels can be seen with an ophthalmoscope. The large vessels are mostly veins that course in whorls before passing obliquely through the sclera as vortex veins. The inner layer of vessels, arranged in a single plane, is called the choriocapillary layer. The vessels of this layer provide nutrients to the cells of the retina. The fenestrated capillaries have lumina that are large and irregular in shape. In the region of the fovea, the choriocapillary layer is thicker, and the capillary network is denser. This layer ends at the ora serrata.

1	Bruch’s membrane measures 1 to 4 m in thickness and lies between the choriocapillary layer and the pigment epithelium of the retina. It runs from the optic nerve to the ora serrata, where it undergoes modifications before continuing into the ciliary body. Bruch’s membrane is a thin, amorphous refractile layer, also called the lamina vitrea. The transmission electron microscope (TEM) reveals its structure as consisting of a multilaminar sheet containing a center layer of elastic and collagen fibers. Five different layers are identified in Bruch’s membrane:  The basal lamina of the endothelial cells of the choriocap illary layer  A layer of collagen fibers approximately 0.5 m thick  A layer of elastic fibers approximately 2 m thick  A second layer of collagen fibers (thus forming a “sandwich” around the intervening elastic tissue layer)  The basal lamina of the retinal epithelial cells

1	At the ora serrata, the collagenous and elastic layers disappear into the ciliary stroma, and Bruch’s membrane becomes continuous with the basal lamina of the RPE of the ciliary body. The retina represents the innermost layer of the eye. The retina, derived from the inner and outer layers of the optic cup, is the innermost of the three concentric layers of the eye (see Fig. 24.1c). It consists of two basic layers:  The neural retina or retina proper is the inner layer that contains the photoreceptor cells.  The RPE is the outer layer that rests on and is firmly attached through the Bruch’s membrane to the choriocapillary layer of the choroid. A potential space exists between the two layers of the retina. The two layers may be separated mechanically in the preparation of histologic specimens. Separation of the layers, “retinal detachment” (Folder 24.2), also occurs in the living state because of eye disease or trauma.

1	In the neural retina, two regions or portions that differ in function are recognized:  The nonphotosensitive region (nonvisual part), located anterior to the ora serrata, lines the inner aspect of the ciliary body and the posterior surface of the iris (this portion of the retina is described in the sections on the iris and ciliary body).  The photosensitive region (optic part) lines the inner surface of the eye posterior to the ora serrata except where it is pierced by the optic nerve (see Fig. 24.1).

1	 The photosensitive region (optic part) lines the inner surface of the eye posterior to the ora serrata except where it is pierced by the optic nerve (see Fig. 24.1). The site where the optic nerve joins the retina is called the optic disc or optic papilla. Because the optic disc is devoid of photoreceptor cells, it is a blind spot in the visual field. The fovea centralis is a shallow depression located about 2.5 mm lateral to the optic disc. It is the area of greatest visual acuity. The visual axis of the eye passes through the fovea. A yellow-pigmented zone called the macula lutea surrounds the fovea. In relative terms, the fovea is the region of the retina that contains the highest concentration and most precisely ordered arrangement of the visual elements. Layers of the Retina Ten layers of cells and their processes constitute the retina.

1	Layers of the Retina Ten layers of cells and their processes constitute the retina. Before discussing the ten layers of the retina, it is important to identify the types of cells found there. This identification will aid in understanding the functional relationships of the cells. Studies of the retina in primates have identified at least 15 types of neurons that form at least 38 different types of synapses. For convenience, neurons and supporting cells can be classified into four groups of cells (Fig. 24.9):  Photoreceptor cells—the retinal rods and cones  Conducting neurons—bipolar neurons and ganglion cells  Association neurons and others—horizontal, centrifugal, interplexiform, and amacrine neurons  Supporting (neuroglial) cells—Mler’s cells, microglial cells, and astrocytes

1	The specific arrangement and associations of the nuclei and processes of these cells result in the retina being organized in ten layers that are seen with the light microscope. The ten layers of the retina, from outside inward, are (Figs. 24.9 and 24.10). 1. Retinal pigment epithelium (RPE)—the outer layer of the retina, actually not part of the neural retina but intimately associated with it 2. Layer of rods and cones—contains the outer and inner segments of photoreceptor cells

1	A potential space exists in the retina as a vestige of the space between the apical surfaces of the two epithelial lay-ers of the optic cup. If this space expands, the neural retina separates from the retinal pigment epithelium (RPE), which remains attached to the choroid layer. This condition is called retinal detachment. As a result of retinal detach-ment, the photoreceptor cells are no longer supplied by nu-trients from the underlying vessels in the choriocapillary plexus of the choroid. Clinical symptoms of retinal detachment include visual sensations commonly described as a “shower of pepper” or floaters. These are caused by red blood cells extravasated from the capillary vessels that have been injured during the retinal tear or detachment. In addition, some individuals describe sudden flashes of light, as well as a “web” or “veil” in front of the eye in conjunction with the onset of floaters. A detached retina can be observed and diagnosed during ophthalmoscopic eye examination

1	flashes of light, as well as a “web” or “veil” in front of the eye in conjunction with the onset of floaters. A detached retina can be observed and diagnosed during ophthalmoscopic eye examination (Fig. F24.2.1). If not repositioned quickly, the detached area of the retina will undergo necrosis, resulting in blindness. More commonly, as the vitreous body ages (in the sixth and sev-enth decades of life), it tends to shrink and pull away from the neural retina, which causes single or multiple tears in the neural retina. An argon laser is often used to repair reti-nal detachment by photocoagulating the edges of the de-tachment and producing scar tissue. This method prevents the retina from further detachment and facilitates the repo-sitioning of photoreceptor cells.

1	 FOLDER 24.2 Clinical Correlation: Retinal Detachment fovea area ofretinalarea of detachmentretinal detachment optic disc FIGURE F24.2.1 • Retinal detachment. This image shows a view of the fundus of the right eye in a patient with retinal detachment. The central retinal vessels emerging from the optic disc are in focus, but in the area of the retinal detachment they appear to be out of focus. This is due to the fact that the area of retinal detachment is elevated (note multiple ridges and shadows) and is located anterior to the plane of focus of the ophtalmoscope. (Courtesy of Dr. Renzo A. Zaldivar.) 3. Outer limiting membrane—the apical boundary of Mler’s cells 4. Outer nuclear layer—contains the cell bodies (nuclei) of retinal rods and cones 5. Outer plexiform layer—contains the processes of retinal rods and cones and processes of the horizontal, amacrine, and bipolar cells that connect to them 6.

1	Outer plexiform layer—contains the processes of retinal rods and cones and processes of the horizontal, amacrine, and bipolar cells that connect to them 6. Inner nuclear layer—contains the cell bodies (nuclei) of horizontal, amacrine, bipolar, and Mler’s cells 7. Inner plexiform layer—contains the processes of horizontal, amacrine, bipolar, and ganglion cells that connect to each other 8. Ganglion cell layer—contains the cell bodies (nuclei) of ganglion cells 9. Layer of optic nerve fbers—contains processes of ganglion cells that lead from the retina to the brain 10. Inner limiting membrane—composed of the basal lamina of Mler’s cells Each of the layers is more fully described in the following sections (see corresponding numbers). The cells of the RPE (layer 1) have extensions that surround the processes of the rods and cones.

1	Each of the layers is more fully described in the following sections (see corresponding numbers). The cells of the RPE (layer 1) have extensions that surround the processes of the rods and cones. The RPE is a single layer of cuboidal cells about 14 m wide and 10 to 14 m tall. The cells rest on Bruch’s membrane of the choroid layer. The pigment cells are tallest in the fovea and adjacent regions, which accounts for the darker color of this region. Adjacent RPE cells are connected by a junctional complex consisting of gap junctions and elaborate zonulae occludentes and adherentes. This junctional complex is the site of the blood–retina barrier.

1	The pigment cells have cylindrical sheaths on their apical surface that are associated with, but do not directly contact, the tip of the photoreceptor processes of the adjacent rod and cone cells. Complex cytoplasmic processes project for a short distance between the photoreceptor cells of the rods and cones. Numerous elongated melanin granules, unlike those found elsewhere in the eye, are present in many of these processes. They aggregate on the side of the cell nearest the rods and cones and are the most prominent feature of the cells. The nucleus with its many convoluted infoldings is located near the basal plasma membrane adjacent to Bruch’s membrane. The cells also contain material phagocytosed from the processes of the photoreceptor cells in the form of lamellar debris contained in residual bodies or phagosomes. A supranuclear Golgi apparatus and an extensive network of smooth-surfaced  FOLDER 24.3 Clinical Correlation: Age-Related Macular Degeneration (ARMD)

1	Age-related macular degeneration (ARMD) is the most common cause of blindness in older individuals. Al-though the cause of this disease is still unknown, evi-dence suggests both genetic and environmental (UV irradiation, drugs) components. The disease causes loss of central vision, although peripheral vision remains unaffected. Two forms of ARMD are recognized: a dry (at-rophic, nonexudative) form and a wet (exudative, neovas-cular) form. The latter is considered a complication of the first. Dry ARMD is the most common form (90% of all cases) and involves degenerative lesions localized in the area of the macula lutea. The degenerative lesions in-clude a focal thickening of Bruch’s membrane called drusen, atrophy, and depigmentation of RPE, and obliter-ation of capillaries in the underlying choroid layer. These changes lead to deterioration of the overlying photosensi-tive retina, resulting in the formation of blind spots in the visual field (Fig. F24.3.1). Wet ARMD is a complication

1	choroid layer. These changes lead to deterioration of the overlying photosensi-tive retina, resulting in the formation of blind spots in the visual field (Fig. F24.3.1). Wet ARMD is a complication of dry ARMD caused by neovascularization of blind spots of the retina in the large drusen. These newly formed, thin, fragile vessels frequently leak and produce exudates and hemorrhages in the space just beneath the retina, result-ing in fibrosis and scarring. These changes are responsi-ble for the progressive loss of central vision over a short time. The treatment of wet ARMD includes conventional laser treatment therapy; however, new surgical methods such as macular translocation have been recently introduced. In this procedure, the retina is detached, translocated, and reattached in a new location, away from the choroid neovascular tissue. Conventional laser treat-ment is then applied to destroy pathologic vessels with-out destroying central vision. FIGURE F24.3.1 • Photograph depicting

1	away from the choroid neovascular tissue. Conventional laser treat-ment is then applied to destroy pathologic vessels with-out destroying central vision. FIGURE F24.3.1 • Photograph depicting the visual feld in individuals with age-related macular degeneration. Note that central vision is absent because of the changes in the macula region of the retina. To maximize their remaining vision, individuals with this condition are instructed to use eccentric fixation of their eyes.

1	endoplasmic reticulum (sER) surround the melanin granules and residual bodies that are present in the cytoplasm. The RPE serves several important functions, including the following:  It absorbs light passing through the neural retina to prevent reflection and resultant glare.  It isolates the retinal cells from blood-borne substances. It serves as a major component of the blood–retina barrier via tight junctions between RPE cells.  It participates in restoring photosensitivity to visual pigments that were dissociated in response to light. The metabolic apparatus for visual pigment resynthesis is present in the RPE cells.  It phagocytoses and disposes of membranous discs from the rods and cones of the retinal photoreceptor cells. The rods and cones of the photoreceptor cell (layer 2) extend from the outer layer of the neural retina to the pigment epithelium.

1	The rods and cones of the photoreceptor cell (layer 2) extend from the outer layer of the neural retina to the pigment epithelium. The rods and cones are the outer segments of photoreceptor cells whose nuclei form the outer nuclear layer of the retina (Figs. 24.10 and 24.11). The light that reaches the photoreceptor cells must first pass through all of the internal layers of the neural retina. The rods and cones are arranged in a palisade manner; therefore, in the light microscope, they appear as vertical striations. The retina contains approximately 120 million rods and 7 million cones. The rods are about 2 m thick and 50 m long (ranging from about 60 m at the fovea to 40 m peripherally). The cones vary in length from 85 m at the fovea to 25 m at the periphery of the retina.

1	Functionally, the rods are more sensitive to light and are the receptors used during periods of low light intensity (e.g., at dusk or at night). The rod pigments have a maximum absorption at 496 nm of visual spectrum, and the image provided is one composed of gray tones (a “black and white picture”). In contrast, the cones exist in three classes: L, M, and S (long-, middle-, and short-wavelength sensitive, respectively) that cannot be distinguished morphologically. They are less sensitive to low light but more sensitive to red, green, and blue regions of the visual spectrum. Each class of cones contains a different visual pigment molecule that is activated by the absorption of light at the blue (420 nm), green (531 nm), and red (588 nm) ranges in the color spectrum. Cones provide a visual image composed of color by mixing the appropriate proportion of red, green, and blue light. segment of cones outer membrane outer segment of cones and rods

1	segment of cones outer membrane outer segment of cones and rods FIGURE 24.9 • Schematic drawing of the layers of the retina. The interrelationship of the neurons is indicated. Light enters the retina and passes through the inner layers of the retina before reaching the photoreceptor cells of the rods and cones that are closely associated with the pigment epithelium.

1	The specificity of the cones provides a functional basis to explain color blindness. Trichromats, almost 90% of the population, are those people who can mix a given color from impulses generated in all three classes of cones. True color-blind individuals (almost all are male) are dichromats and are believed to have a defect in the red-, green-, or (much less commonly) blue-sensitive cones. They are able to distinguish different colors by matching the impulses generated by any of the two normal classes of cones. In addition, about 6% of the population of trichromats matches colors with an unusual proportion of red and green. These individuals are called anomalous trichromats. Each rod and cone photoreceptor consists of three parts:  The outer segment of the photoreceptor is roughly cylindrical or conical (hence, the descriptive name rod or cone). This portion of the photoreceptor is intimately related to microvilli projecting from the adjacent pigment epithelial cells.

1	 The connecting stalk contains a cilium composed of nine peripheral microtubule doublets extending from a basal body. The connecting stalk appears as the constricted region of the cell that joins the inner to the outer segment. In this layer of FIGURE 24.10 • Photomicrograph of a human retina. On the basis of histologic features that are evident in this micrograph, the retina can be divided into ten layers as indicated on this photomicrograph. Note that Bruch’s membrane (lamina vitrea) separates the inner layer of the vascular coat (choroid) from the pigment epithelium. 440. region, a thin, tapering process called the calyceal process extends from the distal end of the inner segment to surround the proximal portion of the outer segment (see Fig. 24.11).

1	region, a thin, tapering process called the calyceal process extends from the distal end of the inner segment to surround the proximal portion of the outer segment (see Fig. 24.11).  The inner segment is divided into an outer ellipsoid and an inner myoid portion. This segment contains a typical complement of organelles associated with a cell that actively synthesize proteins. A prominent Golgi apparatus, rER, and free ribosomes are concentrated in the myoid region. Mitochondria are most numerous in the ellipsoid region. Micro-tubules are distributed throughout the inner segment. In the outer ellipsoid portion, cross-striated fibrous rootlets may extend from the basal body among the mitochondria.

1	The outer segment is the site of photosensitivity, and the inner segment contains the metabolic machinery that supports the activity of the photoreceptor cells. The outer segment is considered a highly modified cilium because it is joined to the inner segment by a short connecting stalk containing a basal body (Fig. 24.12a). With the TEM, 600 to 1,000 regularly spaced horizontal discs are seen in the outer segment (Fig. 24.12). In rods, these FIGURE 24.11 • Schematic diagram of the ultrastructure of rod and cone cells. The outer segments of the rods and cones are closely associated with the adjacent pigment epithelium.

1	FIGURE 24.11 • Schematic diagram of the ultrastructure of rod and cone cells. The outer segments of the rods and cones are closely associated with the adjacent pigment epithelium. discs are membrane-bounded structures measuring about 2 m in diameter. They are enclosed within the plasma membrane of the outer segment (Fig. 24.12a). The parallel membranes of the discs are about 6 nm thick and are continuous at their ends. The central enclosed space is about 8 nm across. In both rods and cones, the membranous discs are formed from repetitive transverse infolding of the plasma membrane in the region of the outer segment near the cilium. Autoradiographic studies have demonstrated that rods form new discs by infolding of the plasma membrane throughout their life span. Discs are formed in cones in a similar manner but are not replaced on a regular basis.

1	Rod discs lose their continuity with the plasma membrane from which they are derived soon after they are formed. They then pass like a stack of plates, proximally to distally, along the length of the cylindrical portion of the outer segment until they are eventually shed and phagocytosed by the pigment epithelial cells. Thus, each rod disc is a membrane-enclosed compartment within the cytoplasm. Discs within the cones retain their continuity with the plasma membrane (Fig. 24.12b). Rod cells contain the visual pigment rhodopsin; cone cells contain the visual pigment iodopsin.

1	Rod cells contain the visual pigment rhodopsin; cone cells contain the visual pigment iodopsin. Rhodopsin (also called visual purple) in rod cells initiates the visual stimulus when it is bleached by light. Rhodopsin is present in globular form on the outer surface of the lipid bilayer (on the cytoplasmic side) of the membranous discs. In the cone cells, the visual pigment on the membranous discs is the photopigment iodopsin. Each cone cell is specialized to respond maximally to one of three colors: red, green, or blue. Both rhodopsin and iodopsin contain a membrane-bound subunit called an opsin and a second component called a chromophore. The opsin of rods is scotopsin; the opsins of cones are photopsins. The chromophore of rods is a vitamin A–derived carotenoid called retinal. Thus, an adequate intake of vitamin A is essential for normal vision. Prolonged dietary defciency of vitamin A leads to the inability to see in dim light (night blindness).

1	The interior of the discs of cones is continuous with the extracellular space. The basic difference in the structure of the rod and cone discs—that is, continuity with the plasma membrane—is correlated with the slightly different means by which the visual pigments are renewed in rods and cones. Newly synthesized rhodopsin is incorporated into the membrane of the rod disc as the disc is being formed at the base of the outer segment. It then takes several days for the disc to reach the tip of the outer segment. In contrast, although visual proteins are constantly produced in retinal cones, the proteins are incorporated into cone discs located anywhere in the outer segment. Vision is a process by which light striking the retina is converted into electrical impulses that are transmitted to the brain.

1	Vision is a process by which light striking the retina is converted into electrical impulses that are transmitted to the brain. The impulses produced by light reaching the photoreceptor cells are conveyed to the brain by an elaborate network of nerves. The conversion of the incident light into nerve impulses is called visual processing and involves two basic steps:  Step 1 is a photochemical reaction that occurs in the outer segment of the rod and cone receptors as absorbed light energy causes conformational changes in the chromophores. The activated opsin molecules interact with a G-protein called transducin. Transducin then activates phosphodiesterase, which breaks down cyclic GMP (cGMP). In the dark, high levels of cGMP in the photoreceptor cells are bound to the cytoplasmic surface of Na channels, causing them to stay open. As a result, photoreceptor cells have a low membrane potential.

1	 Step 2 consists of a decrease in the concentration of cGMP within the cytoplasm of the inner segment of the photoreceptor cells. These changes, which are activated by light energy, decrease the Na permeability of the plasma membrane. When fewer cGMP molecules are bound to Na channel proteins, the photoreceptor becomes hyperpolarized, resulting in a decrease of neurotransmitter (glutamate) secretion. This decrease in glutamate secretion is detected by bipolar cells of the retina, which initiate electrical impulses that are conveyed to the brain. In rods, absorbed light energy causes conformational changes in retinal, converting it to retinol. The conversion of retinal to retinol results in its release from scotopsin (a reaction called bleaching). The energy for this process is provided by the mitochondria located in the inner segment. Mler’s cells and pigment epithelial cells

1	FIGURE 24.12 • Electron micrographs of portions of the inner and outer segments of cones and rods. a. This electron micrograph shows the junction between the inner and outer segments of the rod cell. The outer segments contain the horizontally flattened discs. The plane of this section passes through the connecting stalk and cilium. A centriole, a cilium and its basal body, and a calyceal process are identified. 32,000. b. Another electron micrograph shows a similar section of a cone cell. The interior of the discs in the outer segment of the cone is continuous with the extracellular space (arrows). 32,000. (Courtesy of Dr. Toichiro Kuwabara.) also participate in the interconversion of retinal and retinol and the reactions necessary for the resynthesis of rhodopsin.

1	During normal functioning of the photoreceptor cells, the membranous discs of the outer segment are shed and phagocytosed by the pigment epithelial cells (Fig. 24.13). It is estimated that each of these cells is capable of phagocytosing and disposing of about 7,500 discs per day. The discs are constantly turning over, and the production of new discs must equal the rate of disc shedding. Discs are shed from both rods and cones. In rods, after a period of sleep, a burst of disc shedding occurs as light first enters the eye. The time of disc shedding in cones is more variable. The shedding of discs in cones also enables the receptors to eliminate superfluous membrane. Although not fully understood, the shedding process in cones also alters the size of the discs so that the conical form is maintained as discs are released from the distal end of the cone.

1	FIGURE 24.13 • Electron micrograph of the retinal pigment epithelium in association with the outer segments of rods and cones. Retinal pigment epithelial cells (RPE) contain numerous elongated melanin granules that are aggregated in the apical portion of the cell, where the microvilli extend from the surface toward the outer segments of the rod and cone cells. The retinal pigment epithelial cells contain numerous mitochondria and phagosomes. The arrow indicates the location of the junctional complex between two adjacent cells. 20,000. (Courtesy of Dr. Toichiro Kuwabara.) The outer limiting membrane (layer 3) is formed by a row of zonulae adherentes between Mler’s cells.

1	The outer limiting membrane (layer 3) is formed by a row of zonulae adherentes between Mler’s cells. The outer limiting membrane is not a true membrane. It is a row of zonulae adherentes that attaches the apical ends of Mler’s cells (i.e., the end that faces the pigment epithelium) to each other and to the rods and cones (see Fig. 24.10). Because Mler’s cells end at the base of the inner segments of the receptors, they mark the location of this layer. Thus, the supporting processes of Mler’s cells, on which the rods and cones rest, are pierced by the inner and outer segments of the photoreceptor cells. This layer is thought to be a metabolic barrier that restricts the passage of large molecules into the inner layers of the retina. The outer nuclear layer (4) contains the nuclei of the retinal rods and cones.

1	The outer nuclear layer (4) contains the nuclei of the retinal rods and cones. The region of the rod cytoplasm that contains the nucleus is separated from the inner segment by a tapering process of the cytoplasm. In cones, the nuclei are located close to the outer segments, and no tapering is seen. The cone nuclei stain lightly and are larger and more oval than rod nuclei. Rod nuclei are surrounded by only a thin rim of cytoplasm. In contrast, a relatively thick investment of cytoplasm surrounds the cone nuclei (see Fig. 24.11). The outer plexiform layer (5) is formed by the processes of the photoreceptor cells and neurons.

1	The outer plexiform layer is formed by the processes of retinal rods and cones and the processes of horizontal, interplexiform, amacrine, and bipolar cells. The processes allow the electrical coupling of photoreceptor cells to these specialized interneurons via synapses. A thin process extends from the region of the nucleus of each rod or cone to an inner expanded portion with several lateral processes. The expanded portion is called a spherule in a rod and a pedicle in a cone. Normally, many photoreceptor cells converge onto one bipolar cell and form interconnecting neural networks. Cones located in the fovea, however, synapse with a single bipolar cell. The fovea is also unique in that the compactness of the inner neural layers of the retina causes the photoreceptor cells to be oriented obliquely. Horizontal cell dendritic processes synapse with photoreceptor cells throughout the retina and further contribute to the elaborate neuronal connections in this layer.

1	The inner nuclear layer (6) consists of the nuclei of horizontal, amacrine, bipolar, interplexiform, and Mler’s cells. Mler’s cells form the scaffolding for the entire retina. Their processes invest the other cells of the retina so completely that they fill most of the extracellular space. The basal and apical ends of Mler’s cells form the inner and outer limiting membranes, respectively. Microvilli extending from their apical border lie between the photoreceptor cells of the rods and cones. Capillaries from the retinal vessels extend only to this layer. The rods and cones carry out their metabolic exchanges with extracellular fluids transported across the blood–retina barrier of the RPE. The four types of conducting cells—bipolar, horizontal, interplexiform, and amacrine—found in this layer have distinct orientations (see Fig. 24.9).

1	The four types of conducting cells—bipolar, horizontal, interplexiform, and amacrine—found in this layer have distinct orientations (see Fig. 24.9).  Bipolar cells and their processes extend to both the inner and outer plexiform layer. In the peripheral regions of the retina, the axons of bipolar cells pass to the inner plexiform layer where they synapse with several ganglion cells. Through these connections, the bipolar cells establish communication with multiple cells in each layer except in the fovea, where they may synapse only with a single ganglion cell to provide greater visual acuity in this region.  Horizontal cells and their processes extend to the outer plexiform layer where they intermingle with processes of bipolar cells. The cells have synaptic connections with rod spherules, cone pedicles, and bipolar cells. This electrical coupling of cells is thought to affect the functional threshold between rods and cones and bipolar cells.

1	 Amacrine cells processes pass inward, contributing to a complex interconnection of cells. Their processes branch extensively to provide sites of synaptic connections with axonal endings of bipolar cells and dendrites of ganglion cells. Besides bipolar and ganglion cells, the amacrine cells synapse in the inner plexiform layer with interplexiform and other amacrine cells (see Fig. 24.9).  Interplexiform cells and their processes have synapses in both inner and outer plexiform layers. These cells convey impulses from the inner plexiform to the outer plexiform layer. The inner plexiform layer (7) consists of a complex array of intermingled neuronal cell processes.

1	The inner plexiform layer (7) consists of a complex array of intermingled neuronal cell processes. The inner plexiform layer consists of synaptic connections between axons of the bipolar neurons and dendrites of ganglion cells. It also contains synapses between intermingling processes of amacrine cells and bipolar neurons, ganglion cells, and interplexiform neurons. The course of these processes is parallel to the inner limiting membrane, thus giving the appearance of horizontal striations to this layer (see Fig. 24.10). The ganglion cell layer (8) consists of the cell bodies of large multipolar neurons.

1	The cell bodies of large multipolar nerve cells, measuring as much as 30 m in diameter, constitute the ganglion cell layer. These nerve cells have lightly staining round nuclei with prominent nucleoli and Nissl bodies in their cytoplasm. An axonal process emerges from the rounded cell body, passes into the nerve fiber layer, and then goes into the optic nerve. The dendrites extend from the opposite end of the cell to ramify in the inner plexiform layer. In the peripheral regions of the retina, a single ganglion cell may synapse with a hundred bipolar cells. In marked contrast, in the macular region surrounding the fovea, the bipolar cells are smaller (some authors refer to them as “midget” bipolar cells), and they tend to make one-to-one connections with ganglion cells. Over most of the retina, the ganglion cells are only a single layer of cells. At the macula, however, they are piled as many as eight deep, although they are absent over the fovea itself. Scattered among the ganglion

1	retina, the ganglion cells are only a single layer of cells. At the macula, however, they are piled as many as eight deep, although they are absent over the fovea itself. Scattered among the ganglion cells are small neuroglial cells with densely staining nuclei (see Fig. 24.10).

1	The layer of optic nerve fibers (9) contains axons of the ganglion cells. The axonal processes of the ganglion cells form a flattened layer running parallel to the retinal surface. This layer increases in depth as the axons converge at the optic disc (Fig. 24.14). The axons are thin, nonmyelinated processes measuring as much as 5 m in diameter (see Fig. 24.10). The retinal vessels, including the superficial capillary network, are primarily located in this layer. The inner limiting membrane (layer 10) consists of a basal lamina separating the retina from the vitreous body. The inner limiting membrane forms the innermost boundary of the retina. It serves as the basal lamina of Mler’s cells (see Fig. 24.10). In younger individuals, refections from the internal limiting membrane produce a retinal sheen that is seen during ophthalmoscopic examination of the eye. Specialized Regions of the Retina

1	Specialized Regions of the Retina The fovea (fovea centralis) appears as a small (1.5 mm in diameter), shallow depression located at the posterior pole of the optical axis of the eye. Its central region, known as the foveola, is about 200 m in diameter (see Fig.24.14). Except for the photoreceptor layer, most of the layers of the retina are markedly reduced or absent in this region (see Fig. 24.6). Here the photoreceptor is composed entirely of cones (approximately 4,000) that are longer and more slender and rodlike than they are elsewhere. In this area, the retina is specialized for discrimination of details and color vision. The ratio between cones and ganglion cells is close to 1:1. Retinal vessels are absent in the fovea, allowing light to pass unobstructed into the cone’s outer segment. The adjacent pigment epithelial cells and choriocapillaris are also thickened in this region.

1	The macula lutea is the area surrounding the fovea, approximately 5.5 mm in diameter. It is yellowish because of the presence of yellow pigment (xanthophyll). The macula lutea contains approximately 17,000 cones and gains rods at its periphery. Retinal vessels are also absent in this region. Here the retinal cells and their processes, especially the ganglion cells, are heaped up on the sides of the fovea so that light may pass unimpeded to this most sensitive area of the retina. Vessels of the Retina The central retinal artery and central retinal vein, the vessels that can be seen and assessed with an ophthalmoscope, pass through the center of the optic nerve to enter the bulb of the eye at the optic disc (see Fig. 24.2 and page 898,

1	FIGURE 24.14 • Normal view of the fundus in ophthalmoscopic examination of the right eye. The site where the axons converge to form the optic nerve is called the optic disc. Because the optic disc is devoid of photoreceptor cells, it is a blind spot in the visual field. From the center of the optic nerve (clinically called the optic cup), central retinal vessels emerge. The artery divides into upper and lower branches, each of which further divides into nasal and temporal branches (note the nasal and temporal directions on the image). Veins have a similar pattern of tributaries. Approximately 17 degrees or 2.5 times optic disc diameters lateral to the disc, the slightly oval-shaped, blood-vessel-free, and pigmented area represents the macula lutea. The fovea centralis, a shallow depression in the center of the macula lutea, is also visible. (Courtesy of Dr. Renzo A. Zaldivar.) the section on the development of the eye). The central retinal artery provides nutrients to the inner

1	in the center of the macula lutea, is also visible. (Courtesy of Dr. Renzo A. Zaldivar.) the section on the development of the eye). The central retinal artery provides nutrients to the inner retinal layers. The artery branches immediately into upper and lower branches, each of which divides again into nasal and temporal branches (see Fig. 24.14). Veins undergo a similar pattern of branching. The vessels initially lie between the vitreous body and inner limiting membrane. As the vessels pass laterally, they also move deeper within the inner retinal layers. Branches from these vessels form a capillary plexus that reaches the inner nuclear layer and therefore provides nutrients to the inner retinal layers (layers 6–10; see pages 907–908). Nutrients to the remaining layers (layers 1–5) are provided by diffusion from the vascular choriocapillary layer of the choroid. The branches of the central retinal artery do not anastomose and therefore are classifed as anatomic end arteries.

1	are provided by diffusion from the vascular choriocapillary layer of the choroid. The branches of the central retinal artery do not anastomose and therefore are classifed as anatomic end arteries. Evaluation of the retinal vessels and appearance of the optic disc during the ophthalmoscopy not only provides important information on the state of the eye but also provides early clinical signs of a number of conditions, including elevated intracranial pressure, hypertension, glaucoma, and diabetes.

1	The lens is a transparent, avascular, biconvex structure. It is suspended between the edges of the ciliary body by the zonular fbers. The pull of the zonular fibers keeps the lens in a flattened condition. Release of tension causes the lens to fatten or accommodate to bend light rays originating close to the eye so that they focus on the retina. The lens has three principal components (Fig. 24.15):  The lens capsule is a thick basal lamina measuring ap proximately 10 m to 20 m produced by the anterior lens cells.  The Subcapsular epithelium is a cuboidal layer of cells present only on the anterior surface of the lens.  The lens fbers are structures derived from subcapsular epithelial cells. The lens capsule, composed primarily of type IV collagen and proteoglycans, is elastic. It is thickest at the equator where the fibers of the zonule attach to it.

1	The lens capsule, composed primarily of type IV collagen and proteoglycans, is elastic. It is thickest at the equator where the fibers of the zonule attach to it. Gap junctions connect the cuboidal cells of the subcapsular epithelium. They have few cytoplasmic organelles and stain faintly. The apical region of the cell is directed toward the internal aspect of the lens and the lens fbers, with which they form junctional complexes. The lens increases in size during normal growth and then continues to produce new lens fibers at an ever-decreasing rate throughout life. The new lens fibers develop from the subcapsular epithelial cells located near the equator (see Fig. 24.15). Cells in this region increase in height and then differentiate into lens fibers.

1	As the lens fibers develop, they become highly elongated and appear as thin, flattened structures. They lose their nuclei and other organelles as they become filled with proteins called crystallins. Mature lens fibers attain a length of 7 to 10 mm, a width of 8 to 10 m, and a thickness of 2 m. Near the center of the lens, in the nucleus, the fibers are compressed and condensed to such a degree that individual fibers are impossible to recognize. Despite its density and protein content, the lens is normally transparent (see Fig. 24.15). The high density of lens fibers makes it difficult to obtain routine histologic sections of the lens that are free from artifacts. Changes in the lens are associated with aging. With increasing age, the lens gradually loses its elasticity and ability to accommodate. This condition, called presbyopia, usually occurs in the fourth decade of life. It is easily corrected by wearing reading glasses or using a magnifying lens.

1	Loss of transparency of the lens or its capsule is also a relatively common condition associated with aging. This condition, called cataract, may be caused by conformational changes or cross-linking of proteins. The development of a cataract may also be related to disease processes, metabolic or hereditary conditions, trauma, or exposure to a deleterious agent (such as ultraviolet radiation). Cataracts that signifcantly impair vision can usually be corrected surgically by removing the lens and replacing it with a plastic lens implanted in the posterior chamber. The vitreous body is the transparent jellylike substance that fills the vitreous chamber in the posterior segment of the eye. The vitreous body is loosely attached to the surrounding structures, including the inner limiting membrane of the

1	The vitreous body is loosely attached to the surrounding structures, including the inner limiting membrane of the FIGURE 24.15 • Structure of the lens. a. This schematic drawing of the lens indicates its structural components. Note that the capsule of the lens is formed by the basal lamina of the lens fibers and the subcapsular epithelium located on the anterior surface of the lens. Also note the location of the germinal zone at the equatorial area of the lens. b. This high-magnification photomicrograph of the germinal zone of the lens (near its equator) shows the active process of lens fiber formation from the subcapsular epithelium. Note the thick lens capsule and the underlying layer of nuclei of lens fibers during their differentiation. The mature lens fibers do not possess nuclei. 570.

1	retina. The main portion of the vitreous body is a homogeneous gel containing approximately 99% water (the vitreous humor), collagen, glycosaminoglycans (principally hyaluronan), and a small population of cells called hyalocytes. These cells are believed to be responsible for synthesis of collagen fibrils and glycosaminoglycans. Hyalocytes in routine hematoxylin and eosin (H&E) preparation are difficult to visualize. Often, they exhibit a well-developed rER and Golgi apparatus. Fibroblasts and tissue macrophages are sometimes seen in the periphery of the vitreous body. The hyaloid canal (or Cloquet’s canal), which is not always visible, runs through the center of the vitreous body from the optic disc to the posterior lens capsule. It is the remnant of the pathway of the hyaloid artery of the developing eye. Accessory Structures of the Eye The conjunctiva lines the space between the inner surface of the eyelids and the anterior surface of the eye lateral to the cornea.

1	Accessory Structures of the Eye The conjunctiva lines the space between the inner surface of the eyelids and the anterior surface of the eye lateral to the cornea. The conjunctiva is a thin, transparent mucous membrane that extends from the corneoscleral limbus located on the peripheral margin of the cornea across the sclera (bulbar conjunctiva) and covers the internal surface of the eyelids (palpebral conjunctiva). It consists of a stratified columnar epithelium containing numerous goblet cells and rests on a lamina propria composed of loose connective tissue. The goblet cell secretion is a component of the tears that bathe the eye. Conjunctivitis, an infammation of the conjunctiva that is commonly called pinkeye, is characterized by redness, irritation, and watering of the eyes. For more clinical information, see Folder 24.4. The primary function of the eyelids is to protect the eye.

1	The primary function of the eyelids is to protect the eye. The skin of the eyelids is loose and elastic to accommodate their movement. Within each eyelid is a flexible support, the tarsal plate, consisting of dense fibrous and elastic tissue. Its lower free margin extends to the lid margin, and its superior border serves for the attachment of smooth muscle fibers of the superior tarsal muscle (of Mler). The undersurface of the tarsal plate is covered by the conjunctiva (Fig. 24.16). The orbicularis oculi muscle, a facial expression muscle, forms a thin oval sheet of circularly oriented skeletal muscle fibers overlying the tarsal plate. In addition, the connective tissue of the eyelid contains tendon fibers of the levator palpebrae superioris muscle that open the eyelid (see Fig. 24.16).

1	In addition to eccrine sweat glands, which discharge their secretions directly onto the skin, the eyelid contains four other major types of glands (see Fig. 24.16):  The tarsal glands (Meibomian glands), long seba ceous glands embedded in the tarsal plates, appear as ver tical yellow streaks in the tissue deep in the conjunctiva.  FOLDER 24.4 Clinical Correlation: Conjunctivitis

1	Conjunctivitis otherwise known as pinkeye, is an inflam-mation of the conjunctiva. It may be localized in either the palpebral conjunctiva or bulbar conjunctiva. Individuals may present with relatively nonspecific symptoms and signs that include redness, irritation, and watery discharge from the eye (Fig. F24.4.1). The symptoms can also mimic a foreign body sensation. Extended use of contact lenses can cause an allergic or bacterial conjunctivitis and may be the first sign of more serious ocular disease (i.e., corneal ulcer). In general, symptoms that last less than 4 weeks are classified as acute conjunctivitis, and those extending for a longer period are referred to as chronic conjunctivitis. Acute conjunctivitis is most commonly caused by bac-teria; a variety of viruses including HIV, varicella-zoster virus (VZV), and herpes simplex virus (HSV); or allergic reac-tions. Bacterial conjunctivitis often shows an opaque puru-lent discharge containing white cells and desquamated

1	HIV, varicella-zoster virus (VZV), and herpes simplex virus (HSV); or allergic reac-tions. Bacterial conjunctivitis often shows an opaque puru-lent discharge containing white cells and desquamated epithelial cells. On eye examination, the purulent discharge and conjunctival papillae help to differentiate between bac-terial and viral etiology. Viral conjunctivitis is most common in adults. Clinically, it presents as a diffuse pinkness of the conjunctiva with particularly numerous lymphoid follicles on the palpebral conjunctiva, often accompanied by en-larged preauricular lymph nodes. Viral conjunctivitis is very contagious and usually associated with a recent upper res-piratory infection. Patients need to be advised to avoid touching their eyes, to wash their hands frequently, and to avoid sharing towels and washcloths. Bacterial conjunctivitis is usually treated with antibiotic eye drops or ointments. For viral etiology, no antimicrobials therapy is needed. However, conservative

1	avoid sharing towels and washcloths. Bacterial conjunctivitis is usually treated with antibiotic eye drops or ointments. For viral etiology, no antimicrobials therapy is needed. However, conservative management with artificial tears to keep the eye lubricated may relieve symptoms. Although there is no cure for viral conjunctivitis, symp-tomatic relief may be achieved with warm compresses and artificial tears. For the worst cases, topical corticos-teroid drops may be prescribed to reduce the discomfort of inflammation. However, prolonged use of corticos-teroid drops increases the risk of side effects. Antibiotic drops may also be used for treatment of complementary infections. Viral conjunctivitis usually resolves within 3 weeks. However, in worst cases it may take more than a month. FIGURE F24.4.1 • Conjunctivitis. This photograph of the lower part of the eyeball with reflected lower eyelid shows an infected conjunctiva. The enlarged blood vessels of the conjunctiva are responsible

1	F24.4.1 • Conjunctivitis. This photograph of the lower part of the eyeball with reflected lower eyelid shows an infected conjunctiva. The enlarged blood vessels of the conjunctiva are responsible for moderate redness of the eye with conjunctival swelling. Frequently moderate, clear (in allergic conjunctivitis) or purulent (in bacterial conjunctivitis) discharge is visible. (Courtesy of Dr. Renzo A. Zaldivar.)

1	About 25 tarsal glands are present in the upper eyelid, and 20 are present in the lower eyelid. The sebaceous secretion of the tarsal glands produces an oily layer on the surface of the tear film that retards the evaporation of the normal tear layer. Blockage of the tarsal gland secretion leads to chalazion (tarsal gland lipogranuloma), an inflammation of the tarsal gland. It presents as a painless cyst usually on the upper eyelid that disappears after a few months without any therapeutic intervention.  Sebaceous glands of eyelashes (glands of Zeis) are small, modified sebaceous glands that are connected with and empty their secretion into the follicles of the eyelashes. Bacterial infection of these sebaceous glands causes a stye (also called hordeolum), a painful tenderness and redness of the affected area of the eyelid.  Apocrine glands of eyelashes (glands of Moll) are small sweat glands with unbranched sinuous tubules that begin as a simple spiral.

1	 Apocrine glands of eyelashes (glands of Moll) are small sweat glands with unbranched sinuous tubules that begin as a simple spiral.  Accessory lacrimal glands are compound serous tubuloalveolar glands that have distended lumina. They are located on the inner surface of the upper eyelids (glands of Wolfring) and in the fornix of the lacrimal sac (glands of Krause). All glands of the human eyelid are innervated by neurons of the autonomic nervous system, and their secretion is synchronized with the lacrimal glands by a common neurotransmitter, vasoactive intestinal polypeptide (VIP). The eyelashes emerge from the most anterior edge of the lid margin, in front of the openings of the Meibomian glands. The lashes are short, stiff, curved hairs and may occur in double or triple rows. The lashes on the same eyelid margin may have different lengths and diameters. The lacrimal gland produces tears that moisten the cornea and pass to the nasolacrimal duct.

1	The lacrimal gland produces tears that moisten the cornea and pass to the nasolacrimal duct. Tears are produced by the lacrimal glands and to a lesser degree by the accessory lacrimal glands. The lacrimal gland is duct of tarsalgland glands apocrine glands of eyelids (of Moll) eyelids (of Moll) eyelash sebaceous glands(of Zeis) of eyelashes (of Zeis) FIGURE 24.16 • Structure of the eyelid. a. This schematic drawing of the eyelid shows the skin, associated skin appendages, muscles, tendons, connective tissue, and conjunctiva. Note the distribution of multiple small glands associated with the eyelid and observe the reflection of the palpebral conjunctiva in the fornix of the lacrimal sac to become the bulbar conjunctiva.

1	b. Photomicrograph of a sagittal section of the eyelid stained with picric acid for better visualization of epithelial components of the skin and the numerous glands. In this preparation, muscle tissue (i.e., orbicularis oculi muscle) stains yellow, and the epithelial cells of skin, conjunctiva, and glandular epithelium are green. Note the presence of the numerous glands within the eyelid. The tarsal (Meibomian) gland is the largest gland, and it is located within the dense connective tissue of the tarsal plates. This sebaceous gland secretes into ducts opening onto the eyelids. 20. Inset. Higher magnification of a tarsal gland from the boxed area, showing the typical structure of a holocrine gland. 60.

1	located beneath the conjunctiva on the upper lateral side of the orbit (Fig. 24.17). The lacrimal gland consists of several separate lobules of tubuloacinar serous glands. The acini have large lumina lined with columnar cells. Myoepithelial cells, located below the epithelial cells within the basal lamina, aid in the release of tears. Approximately 12 ducts drain from the lacrimal gland into the reflection of conjunctiva just beneath the upper eyelid, known as the fornix of the lacrimal sac.

1	Tears drain from the eye through lacrimal puncta, the small openings of the lacrimal canaliculi, located at the medial angle. The upper and lower canaliculi join to form the common canaliculus, which opens into the lacrimal sac. The sac is continuous with the nasolacrimal duct, which opens into the nasal cavity below the inferior turbinate. A pseudostratified ciliated epithelium lines the lacrimal sac and the nasolacrimal duct. Dacryocystitis is an infammation of the lacrimal sac that is frequently caused by an obstruction of the nasolacrimal duct. It can be acute, chronic, or congenital. It usually affects older individuals and is most often secondary to stenosis of the lacrimal canaliculi. Tears protect the corneal epithelium and contain antibacterial and UV-protective agents.

1	Tears protect the corneal epithelium and contain antibacterial and UV-protective agents. Tears keep the conjunctiva and corneal epithelium moist and wash foreign material from the eye as they flow across the corneal surface toward the medial angle of the eye (see Fig. 24.17). The thin film of tears covering the corneal surface is not homogeneous but a mixture of products secreted by the lacrimal glands, the accessory lacrimal glands, the goblet cells of the conjunctiva, and the tarsal glands of the eyelid. The tear film contains proteins (tear albumins, lactoferrin), enzymes (lysozyme), lipids, metabolites, electrolytes, and drugs, the latter secreted during therapy. The tear cationic protein lactoferrin increases the activity of various antimicrobial agents such as lysozyme.

1	The tear cationic protein lactoferrin increases the activity of various antimicrobial agents such as lysozyme. FIGURE 24.17 • Schematic diagram of the eye and lacrimal apparatus. This drawing shows the location of the lacrimal gland and components of the lacrimal apparatus, which drains the lacrimal fluid into the nasal cavity. The eye is moved within the orbit by coordinated contraction of extraocular muscles.

1	The eye is moved within the orbit by coordinated contraction of extraocular muscles. Six muscles of the eyeball (also called extraocular or extrinsic muscles) attach to each eye. These are the medial, lateral, superior, and inferior rectus muscles and the superior and inferior oblique muscles. The superior oblique muscle is innervated by the trochlear nerve (cranial nerve IV). The lateral rectus muscle is innervated by the abducens nerve (cranial nerve VI). All of the remaining extraocular muscles are innervated by the oculomotor nerve (cranial nerve III). The combined, precisely controlled action of these muscles allows vertical, lateral, and rotational movement of the eye. Normally, the actions of the muscles of both eyes are coordinated so that the eyes move in parallel (called conjugate gaze).

1	The human eye is a complex sensory organ that provides sight. The wall of the eye consists of three concentric layers or coats: the retina, the inner layer; the uvea, the middle or vascular layer; and the corneosclera, the outer fibrous layer. The eye is often compared to a simple camera with a lens to capture and focus light, a diaphragm to regulate the amount of light, and film to record the image. In the eye, the cornea and lens concentrate and focus light on the retina. The iris, located between the cornea and lens, regulates the size of the pupil through which light enters the eye. Photoreceptor cells (rods and cones) in the retina detect the intensity (rods) and color (cones) of the light that reaches them and encode the various parameters for transmission to the brain via the optic nerve (cranial nerve II). The eye measures 25 mm in diameter. It is suspended in the bony orbit by six extrinsic striated muscles that control its movement. The extraocular muscles are coordinated so

1	nerve (cranial nerve II). The eye measures 25 mm in diameter. It is suspended in the bony orbit by six extrinsic striated muscles that control its movement. The extraocular muscles are coordinated so that both eyes move synchronously, with each moving symmetrically around its own central axis. A thick layer of adipose tissue partially surrounds and cushions the eye as it moves within the orbit.

1	Modifed drawing of human eye, meridional perspective by E. Sobotta. The innermost layer is the retina (R), which consists of several layers of cells. Among these are receptor cells (rods and cones), neurons (e.g., bipolar and ganglion cells), supporting cells, and a pigment epithelium (see Plate 105). The receptor components of the retina are situated in the posterior three fifths of the eyeball. At the anterior boundary of the receptor layer, the ora serrata (OS), the retina becomes reduced in thickness, and nonreceptor components of the retina continue forward to cover the posterior or inner surface of the ciliary body (CB) and the iris (I). This anterior nonreceptor extension of the inner layer is highly pigmented, and the pigment (melanin) is evident as the black inner border of these structures.

1	The uvea, the middle layer of the eyeball, consists of the choroid, the ciliary body, and the iris. The choroid is a vascular layer; it is relatively thin and difficult to distinguish in the accompanying figure except by location. On this basis, the choroid (Ch) is identified as being just external to the pigmented layer of the retina. It is also highly pigmented; the choroidal pigment is evident as a discrete layer in several parts of the section. Anterior to the ora serrata, the uvea is thickened; here, it is called the ciliary body (CB). This contains the ciliary muscle (see Plate 106), which brings about adjustments of the lens to focus light. The ciliary body also contains processes to which the zonular fibers are attached. These fibers function as suspensory ligaments of the lens (L). The iris (I ) is the most anterior component of the uvea and contains a central opening, the pupil.

1	The outermost layer of the eyeball, the fbrous layer, consists of the sclera (S) and the cornea (C). Both of these contain collagenous fibers as their main structural element; however, the cornea is transparent, and the sclera is opaque. The extrinsic muscles of the eye insert into the sclera and effect movements of the eyeball. These are not included in the preparation except for two small pieces of a muscle insertion (arrows) in the lower left and top center of the illustration. Posteriorly, the sclera is pierced by the emerging optic nerve (ON ). A deep depression in the neural retina lateral to the optic nerve (above the ON in this figure) is the fovea centralis (FC ), the thinnest and most sensitive portion of the neural retina.

1	The lens is considered in Plate 107. Just posterior to the lens is the large cavity of the eye, the vitreous cavity (V ), which is filled with a thick jellylike material, the vitreous humor or body. Anterior to the lens are two additional, fluid-filled chambers of the eye, the anterior chamber (AC ) and the posterior chamber (PC ), separated by the iris. KEY AC, anterior chamber C, cornea CB, ciliary body Ch, choroid FC, fovea centralis I, iris L, lens ON, optic nerve OS, ora serrata PC, posterior chamber R, retina S, sclera V, vitreous cavity arrows, muscle insertions PLATE 105 Eye II: Retina

1	The retina and optic nerve are projections of the forebrain. The fibrous cover of the optic nerve is an extension of the meninges of the brain. The neural retina is a multilayered structure consisting of photoreceptors (rods and cones), neurons, some of which are specialized as conducting and associating neurons, and supporting cells (Mler’s cells). External to the neural retina is a layer of simple columnar retinal pigment epithelium (RPE). The Mler’s cells are comparable to neuroglia in the rest of the central nervous system. Processes of Mler’s cells ramify virtually through the entire thickness of the retina. The internal limiting membrane is the basal lamina of these cells; the external limiting membrane is actually a line formed by the junctional complexes between processes of these cells and the photoreceptor cells. The neurons of the retina are arranged sequentially in three layers: (1) a deep layer of rods and cones; (2) an intermediate layer of bipolar, horizontal, and

1	these cells and the photoreceptor cells. The neurons of the retina are arranged sequentially in three layers: (1) a deep layer of rods and cones; (2) an intermediate layer of bipolar, horizontal, and amacrine cells; and (3) a superficial layer of ganglion cells. Nerve impulses originating in the rods and cones are transmitted to the intermediate layer and then to the ganglion cells. Synaptic connections occur in the outer plexiform layer (between the rods and cones and the intermediate neuronal layer) and the inner plexiform layer (between the intermediate layer and the ganglion cells), resulting in summation and neuronal integration. Finally, the ganglion cells send their axons to the brain as components of the optic nerve.

1	Optic disc and nerve, eye, human, H&E 65. The site where the optic nerve leaves the eyeball is called the optic disc (OD). It is characteristically marked by a depression, evident here. Receptor cells are not present at the optic disc, and because it is not sensitive to light stimulation, it is sometimes referred to as the blind spot. The fibers that give rise to the optic nerve originate in the retina, more specifically, in the ganglion cell layer (see below). They traverse the sclera through a number of openings (arrows) to form the optic nerve (ON ). The region of the sclera that contains these openings is called the lamina cribrosa (LC) or cribriform plate. The optic nerve contains a central artery and vein (not seen here) that also traverse the lamina cribrosa. Branches of these blood vessels (BV ) supply the inner portion of the retina. PLATE 105 • EYE II: RETINA Retina, eye, human, H&E 325.

1	PLATE 105 • EYE II: RETINA Retina, eye, human, H&E 325. On the basis of structural features that are evident in histologic sections, the retina is divided into ten layers, from posterior to anterior, as listed below and labeled in this figure: 1. Retinal pigment epithelium (RPE ), the outermost layer of the retina 2. Layer of rods and cones (R&C ), the photoreceptor layer of the retina 3. External limiting membrane (ELM ), a line formed by the junctional complexes of the photoreceptor cells 4. Outer nuclear layer (ONL), containing nuclei of rod and cone cells 5. Outer plexiform layer (OPL), containing neural processes and synapses of rod and cone cells with bipolar, amacrine, interplexiform and horizontal cells 6. Inner nuclear layer (INL), containing nuclei of bipolar, horizontal, interplexiform amacrine, and Mler’s cells 7. Inner plexiform layer (IPL), containing processes and synapses of bipolar, horizontal, interplexiform amacrine, and ganglion cells 8.

1	Inner plexiform layer (IPL), containing processes and synapses of bipolar, horizontal, interplexiform amacrine, and ganglion cells 8. Layer of ganglion cells (GC ), containing cell bodies and nuclei of ganglion cells 9. Nerve fiber layer (NFL), containing axons of ganglion cells 10. Internal limiting membrane (ILM ), consisting of the external (basal) lamina of Mler’s cells This figure also shows the innermost layer of the choroid (Ch), a cell-free membrane, the lamina vitrea (LV ), also called Bruch’s membrane. Electron micrographs reveal that it corresponds to the basement membrane of the pigment epithelium. Immediately external to the lamina vitrea is the capillary layer of the choroid (lamina choriocapillaris). These vessels supply the outer part of the retina.

1	KEY BV, blood vessels Ch, choroid ELM, external limiting membrane GC, layer of ganglion cells ILM, internal limiting membrane INL, inner nuclear layer (nuclei of bipolar, horizontal, amacrine, and Mler’s cells) IPL, inner plexiform layer LC, lamina cribrosa LV, lamina vitrea NFL, nerve fiber layer OD, optic disc ON, optic nerve ONL, outer nuclear layer (nuclei of rod and cone cells) OPL, outer plexiform layer RPE, retinal pigment epithelium R&C, layer of rods and cones arrows, openings in sclera (lamina cribrosa) PLATE 106 Eye III: Anterior Segment

1	PLATE 106 Eye III: Anterior Segment The anterior segment is that part of the eye anterior to the ora serrata, the most anterior extension of the neural retina, and includes the anterior and posterior chambers and the structures that define them. These include the cornea and sclera, the iris, the lens, the ciliary body, and the connections between the basal lamina of the ciliary processes and the lens capsule (thick basal lamina of the lens epithelium) that form the suspensory ligament of the lens, the zonular fbers. The posterior chamber is bounded posteriorly by the anterior surface of the lens and anteriorly by the posterior surface of the iris. The ciliary body forms the lateral boundary. Aqueous humor flows through the pupil into the anterior chamber, which occupies the space between the cornea and the iris, and drains into the canal of Schlemm. Anterior segment, eye, human, H&E 45; inset 75.

1	Anterior segment, eye, human, H&E 45; inset 75. A portion of the anterior segment of the eye, shown in this figure, includes parts of the cornea (C ), sclera (S), iris (I ), ciliary body (CB), anterior chamber (AC ), posterior chamber (PC ), lens (L), and zonular fibers (ZF ).

1	The relationship of the cornea to the sclera is illustrated to advantage here. The junction between the two (arrows) is marked by a 924 change in staining, with the substance of the cornea appearing lighter than that of the sclera. The corneal epithelium (CEp) is continuous with the conjunctival epithelium (CjEp) that covers the sclera. Note that the epithelium thickens considerably at the corneoscleral junction and resembles that of the oral mucosa. The conjunctival epithelium is separated from the dense fibrous component of the sclera by a loose vascular connective tissue. Together, this connective tissue and the epithelium constitute the conjunctiva (Cj). The epithelial-connective tissue junction of the conjunctiva is irregular; in contrast, the undersurface of the corneal epithelium presents an even profile.

1	Just lateral to the junction of the cornea and sclera is the canal of Schlemm (CS; see also figure below). This canal takes a circular route about the perimeter of the cornea. It communicates with the anterior chamber through a loose trabecular meshwork of tissue, the spaces of Fontana. The canal of Schlemm also communicates with episcleral veins. By means of its communications, the canal of Schlemm provides a route for the fluid in the anterior and posterior chambers to reach the bloodstream. The inset shows the tip of the iris. Note the heavy pigmentation on the posterior surface of the iris, which is covered by the same double-layered epithelium as the ciliary body and ciliary processes. In the ciliary epithelium, the outer layer is pigmented and the inner layer is nonpigmented. In the iris, both layers of the iridial epithelium (IEp) are heavily pigmented. A portion of the iridial constrictor muscle (M ) is seen beneath the epithelium. PLATE 106 • EYE III: ANTERIOR SEGMENT

1	PLATE 106 • EYE III: ANTERIOR SEGMENT Anterior segment, eye, human, H&E 90; inset 350. Immediately internal to the anterior margin of the sclera (S) is the ciliary body (CB). The inner surface of this forms radially arranged, ridge-shaped elevations, the ciliary processes (CP), to which the zonular fibers (ZF ) are anchored. From the outside in, the components of the ciliary body are the ciliary muscle (CM), the connective tissue (vascular) layer (VL) representing the choroid coat in the ciliary body, the lamina vitrea (LV, inset), and the ciliary epithelium (CiEp, inset). The ciliary epithelium consists of two layers (inset), the pigmented layer (PE ) and the nonpigmented layer (npE ). The lamina vitrea is a continuation of the same layer of the choroid; it is the basement membrane of the pigmented ciliary epithelial cells.

1	The ciliary muscle is arranged in three patterns. The outer layer is immediately deep to the sclera. These are the meridionally arranged fibers of Brke. The outermost of these continues more posteriorly into the choroid and is referred to as the tensor muscle of the choroid. The middle layer is the radial group. It radiates from the region of the sclerocorneal junction into the ciliary body. The innermost layer of muscle cells is circularly arranged. These are seen in cross section. The circular artery (CA; barely discernible) and vein (CV) for the iris, also cut in cross section, are just anterior to the circular group of muscle cells.

1	KEY A, artery AC, anterior chamber C, cornea CA, circular artery CB, ciliary body CEp, corneal epithelium Ch, choroid CiEp, ciliary epithelium Cj, conjunctiva CjEp, conjunctival epithelium CM, ciliary muscle CP, ciliary processes CS, canal of Schlemm CV, circular vein I, iris IEp, iridial epithelium L, lens LV, lamina vitrea M, iridial constrictor muscle npE, nonpigmented layer of ciliary epithelium PC, posterior chamber PE, pigmented layer of ciliary epithelium S, sclera V, vein VL, vascular layer (of ciliary body) ZF, zonular fibers arrows, junction between cornea and sclera PLATE 107 Eye IV: Sclera, Cornea, and Lens

1	The transparent cornea is the primary dioptric (refractive element) of the eye and is covered with nonkeratinized, stratified squamous epithelium. Its stroma consists of alternating lamellae of collagen fibrils and fibroblasts (keratocytes). The fibrils in each lamella are extremely uniform in diameter and uniformly spaced; fibrils in adjacent lamellae are arranged at approximately right angels to each other. This orthogonal array of highly regular fibrils is responsible for the transparency of the cornea. The posterior surface is covered with a single layer of low cuboidal cells, the corneal endothelium, which rest on a thickened basal lamina called Descemet’s membrane. Nearly all of the metabolic exchanges of the avascular cornea occur across the endothelium. Damage to this layer leads to corneal swelling and can produce temporary or permanent loss of transparency. The lens is a transparent, avascular, biconvex epithelial structure suspended by the zonular fibers. Tension on these

1	to corneal swelling and can produce temporary or permanent loss of transparency. The lens is a transparent, avascular, biconvex epithelial structure suspended by the zonular fibers. Tension on these fibers keeps the lens flattened; reduced tension allows it to fatten or accommodate to bend light rays originating close to the eye to focus them on the retina.

1	PLATE 107 • EYE IV: SCLERA, CORNEA, AND LENS Corneoscleral junction, eye, human, H&E 130. in thickness and rests on a loose vascular connective tissue. Together, this epithelium and underlying connective tissue represents the conjunctiva This low-magnification micrograph shows the full thickness of (Cj). The white opaque appearance of the sclera is due to the irregular dense the sclera just lateral to the corneoscleral junction or limbus. arrangement of the collagen fibers that make up the stroma (S ). The canal To the left of the arrow is sclera; to the right is a small amount of Schlemm (CS ) is seen at the left close to the inner surface of the sclera. of corneal tissue. The conjunctival epithelium (CjEp) is irregular

1	of corneal tissue. The conjunctival epithelium (CjEp) is irregular Corneoscleral junction and canal of Schlemm, eye, the sclera. Note that Bowman’s membrane (B), lying under the corneal human, H&E 360. epithelium, is just perceptible but disappears beneath the conjunctival epithelium. Figure below shows at higher magnification the canal of Schlemm Upper figure is a higher-magnification micrograph showing (CS ) than does top left figure. That the space shown here is not an artithe transition from the corneal epithelium (CEp) to the irfact is evidenced by the endothelial lining cells (En) that face the lumen. Cornea, eye, human, H&E 175. white spaces seen here and in figure on left are artifacts). Nuclei (N ) of the keratocytes of the stroma lie between lamellae. The corneal epithelium rests

1	Cornea, eye, human, H&E 175. white spaces seen here and in figure on left are artifacts). Nuclei (N ) of the keratocytes of the stroma lie between lamellae. The corneal epithelium rests This low-magnification micrograph shows the full thickness of on a thickened anterior basement membrane called Bowman’s membranethe cornea (C) and can be compared with the sclera shown in (B). The posterior surface of the cornea is lined by a simple squamous figure on left. The corneal epithelium (CEp) presents a uniepithelium called the corneal endothelium (CEn); its thick posterior base-form thickness and the underlying stroma (S ) has a more ment membrane is called Descemet’s membrane (D). homogeneous appearance than the stroma of the sclera (the

1	homogeneous appearance than the stroma of the sclera (the Corneal epithelium and endothelium, eye, human, underlying stroma (S ). Note that the stromal tissue has a homogeneous ap-H&E 360. pearance, a reflection of the dense packing of its collagen fibrils. The flattened nuclei belong to the keratocytes. Lower figure shows the posterior Upper is a higher magnification showing the corneal epithe surface of the cornea. Note the thick homogeneous Descemet’s memlium (CEp) with its squamous surface cells, the very thick brane (D) and the underlying corneal endothelium (CEn). homogeneous-appearing Bowman’s membrane (B), and the Lens, eye, human, H&E 360. are present on the anterior surface of the lens, but at the lateral margin they become extremely elongated and form layers that extend toward the center This micrograph shows a portion of the lens near its equator.

1	This micrograph shows a portion of the lens near its equator. of the lens. These elongated columns of epithelial cytoplasm are referred to The lens consists entirely of epithelial cells surrounded by a as lens fbers (LF ). New cells are produced at the margin of the lens and homogeneous-appearing lens capsule (LC ) to which the displace the older cells inwardly. Eventually, the older cells lose their nuclei, zonula fibers attach. The lens capsule is a very thick basal lamas evidenced by the deeper portion of the cornea in this micrograph. ina of the epithelial cells. Simple cuboidal lens epithelial cells KEY AC, anterior chamber B, Bowman’s membrane BV, blood vessels C, cornea CEn, corneal endothelium CEp, corneal epithelium Cj, conjunctiva CjEp, conjunctival epithelium CS, canal of Schlemm D, Descemet’s membrane En, endothelial lining cells LC, lens capsule LF, lens fibers N, nuclei S, stroma

1	PLATE 107 • EYE IV: SCLERA, CORNEA, AND LENSLFLFCEnCEnDCSCSBCjEpCjEpCEpCEpBCEpCEpCEnCEnDCSNBCEpCEpBVBVCSCSSCjEpCjEpCjCjCjCjEpSBVCSACCEpBSCCEnDACNCjEpCEpBCEpBDCEnEnCSLFLCSSSS OVERVIEW OF THE EAR / 928 EXTERNAL EAR / 928 MIDDLE EAR / 929 INTERNAL EAR / 932 Structures of the Bony Labyrinth / 932 Structures of the Membranous Labyrinth / 933 Sound Perception / 941 Innervation of the Internal Ear / 942 Blood Vessels of the Membranous Labyrinth / 945 Folder 25.1 Clinical Correlation: Otosclerosis / 933 Folder 25.2 Clinical Correlation: Hearing Loss— Vestibular Dysfunction / 934 Folder 25.3 Clinical Correlation: Vertigo / 937

1	The ear is a three-chambered sensory organ that functions as an auditory system for sound perception and as a vestibular system for balance. Each of the three divisions of the ear— the external ear, middle ear, and internal ear—is an essential part of the auditory and vestibular systems (Fig. 25.1). The external and middle ear collect and conduct sound energy to the internal ear, where auditory sensory receptors convert that energy into electrical impulses. The sensory receptors of the vestibular system respond to gravity and movement of the head. They are responsible for the sense of balance and equilibrium and help to coordinate movements of the head and eyes. The ear develops from surface ectoderm and components of the first and second pharyngeal arch.

1	Embryologically, the functions of the ear—hearing and balance—are elaborated from an invagination of surface ectoderm that appears on each side of the myelencephalon. This invagination forms the otic vesicle (otocyst), which sinks deep to the surface ectoderm into the underlying mesenchyme (Fig. 25.2). The otic vesicle serves as a primordium for development of the epithelia that line the membranous labyrinth of the internal ear. Later, development of the first and part of the second pharyngeal arch provides structures that augment hearing. The endodermal component of the first pouch gives rise to the tubotympanic recess, which ultimately develops into the auditory tube (Eustachian tube) and the middle ear and its epithelial lining. The corresponding ectodermal outgrowth of the first pharyngeal groove gives rise to the external acoustic meatus and its epithelial lining (see Fig. 25.2). The connective tissue part of the pharyngeal arches produces the ossicles. The malleus and incus

1	groove gives rise to the external acoustic meatus and its epithelial lining (see Fig. 25.2). The connective tissue part of the pharyngeal arches produces the ossicles. The malleus and incus develop from the first pharyngeal arch, and the stapes from the second pharyngeal arch. The sensory epithelia of the membranous labyrinth that originates from the otic vesicle link with cranial nerve VIII, which is an outgrowth of the central nervous system. The cartilaginous, bony, and muscular structures of the ear develop from the mesenchyme surrounding these early epithelia.

1	The auricle is the external component of the ear that collects and amplifies sound. The auricle (pinna) is the oval appendage that projects from the lateral surface of the head. The characteristic shape of the auricle is determined by an internal supporting structure of elastic cartilage. Thin skin with hair follicles, sweat glands, and sebaceous glands cover the auricle. The auricle is considered a nearly vestigial structure in humans, compared with its development and role in other animals. However, it is an essential component in sound localization and amplification. The external acoustic meatus conducts sounds to the tympanic membrane. The external acoustic meatus is an air-filled tubular space that follows a slightly S-shaped course for about 25 mm to the tympanic membrane (eardrum). The wall of the canal

1	The external acoustic meatus is an air-filled tubular space that follows a slightly S-shaped course for about 25 mm to the tympanic membrane (eardrum). The wall of the canal FIGURE 25.1 • Three divisions of the ear. The three divisions of the ear are represented by different colors and consist of the external ear (auricle and external acoustic meatus; pink tone), the middle ear (tympanic cavity, auditory ossicles, tympanic membrane, and auditory tube; green), and the internal ear containing the bony labyrinth (semicircular canals, vestibule, and cochlea; blue) and the membranous labyrinth (not visible). is continuous externally with the auricle. The wall of the lateral one third of the canal is cartilaginous and is continuous with the elastic cartilage of the auricle. The medial two thirds of the canal is contained within the temporal bone.

1	The lateral part of the canal is lined by skin that contains hair follicles, sebaceous glands, and ceruminous glands but no eccrine sweat glands. The coiled tubular ceruminous glands closely resemble the apocrine glands found in the axillary region. Their secretion mixes with that of the sebaceous glands and desquamated cells to form cerumen, or earwax. The cerumen lubricates the skin and coats the meatal hairs to impede the entry of foreign particles into the ear. Excessive accumulation of cerumen can plug the meatus, however, resulting in conductive hearing loss. The medial part of the canal located within the temporal bone has thinner skin and fewer hairs and glands. The middle ear is an air-filled space that contains three small bones, the ossicles.

1	The middle ear is an air-filled space that contains three small bones, the ossicles. The middle ear is located in an air-filled space, called the tympanic cavity, within the temporal bone (Fig. 25.3). It is spanned by three small bones, the auditory ossicles, which are connected by two movable joints. The middle ear also contains the auditory tube (Eustachian tube) as well as the muscles that attach to the ossicles. The middle ear is bounded anteriorly by the auditory tube and posteriorly by the spongy bone of the mastoid process, which contains the mastoid antrum and other, smaller air-filled spaces called mastoid cells. Laterally, the middle ear is bounded by the tympanic membrane and medially by the bony wall of the internal ear.

1	The middle ear works as a full-fledged mechanical energy transformer. Its primary function is to convert sound waves (air vibrations) arriving from the external acoustic meatus into mechanical vibrations that are transmitted to the internal ear. Two openings in the medial wall of the middle ear, the oval (vestibular) window and the round (cochlear) window, are essential components in this conversion process. The tympanic membrane separates the external acoustic meatus from the middle ear.

1	The tympanic membrane separates the external acoustic meatus from the middle ear. The tympanic membrane is in the shape of an irregular cone, the apex of which is located at the umbo that corresponds to the tip of the manubrium of the malleus. During otoscopic examination of the normal ear, tympanic membrane is in a light gray color and a cone of light (light reflex) should reflect off its surface (Fig. 25.4). The malleus is one of three small auditory ossicles residing in the middle ear and the only one that attaches to the tympanic membrane (see Fig. 25.1). Tympanic membrane forms the medial boundary of the external acoustic meatus and the lateral wall of the middle ear (Fig. 25.5). The layers of the tympanic membrane from outside to inside are  The skin of the external acoustic meatus  A core of radially and circularly arranged collagen fibers  The mucous membrane of the middle ear

1	Sound waves cause the tympanic membrane to vibrate, and these vibrations are transmitted through the ossicular chain of three small bones that link the external ear to the internal ear. petrous part of temporal bone

1	petrous part of temporal bone FIGURE 25.2 • Schematic drawings showing development of the ear. a. This drawing shows the relationship of the surface ectodermderived otic vesicle to the first pharyngeal arch during the fourth week of development. b. The otic vesicle sinks deep into the mesenchymal tissue and develops into the membranous labyrinth. Note the development of the tubotympanic recess lined by endoderm into the future middle-ear cavity and auditory tube. In addition, accumulation of mesenchyme from the first and second pharyngeal arches gives rise to the auditory ossicles. c. At this later stage of development, the first pharyngeal groove grows toward the developing tubotympanic recess. The auditory ossicles assume a location inside the tympanic cavity. d. This final stage of development shows how the tympanic membrane develops from all three germ layers: surface ectoderm, mesoderm, and endoderm. Note that the wall of the otic vesicle develops into the membranous labyrinth.

1	Perforation of the tympanic membrane may cause transient or permanent hearing impairment. The auditory ossicles connect the tympanic membrane to the oval window. The three auditory ossicles, or bones—the malleus, the incus, and the stapes—cross the space of the middle ear in series (Fig. 25.6) and connect the tympanic membrane to the oval window. These bones work like a lever system that increases the force transmitted from the vibrating tympanic membrane to the stapes by decreasing the ratio of their oscillation amplitudes. The ossicles help to convert sound waves to mechanical (hydraulic) vibrations in tissues and fluid-filled chambers. Movable synovial joints connect the bones, which are named according to their approximate shape:  The malleus (hammer) attaches to the tympanic mem brane and articulates with the incus.

1	 The incus (anvil), is the largest of the ossicles and links the malleus to the stapes. The stapes (stirrup), the footplate of which fits into the oval window, acts like a small piston on the cochlear fluid. Diseases that affect the external acoustic meatus, tympanic membrane, or ossicles are responsible for the conductive hearing loss (see Clinical Folders 25.1 and 25.2). Two muscles attach to the ossicles and affect their movement. The tensor tympani muscle lies in a bony canal above the auditory tube; its tendon inserts on the malleus. Contraction of this muscle increases tension on the tympanic membrane. The stapedius muscle lies in a bony eminence on the posterior wall of the middle ear; its tendon inserts on the stapes. Contraction of the stapedius tends to dampen the

1	FIGURE 25.3 • Horizontal section of a human temporal bone. The relationships of the three divisions of the ear within the temporal bone are shown. The tympanic membrane (TM) separates the external acoustic meatus from the tympanic cavity (TC). Within the tympanic cavity, sections of the malleus (M) and incus (I) can be seen. The posterior wall of the tympanic cavity is associated with the mastoid air cells (AC). The lateral wall of the cavity is formed principally by the tympanic membrane. The opening to the internal ear or oval window (arrowhead) is seen in the medial wall of the cavity (the stapes has been removed). The facial nerve (F) can be observed near the oval window. The cochlea (C), the vestibule (V ), and a portion of the lateral semicircular canal (LSC) of the bony labyrinth are identified. The cochlear and vestibular divisions of cranial nerve VIII (N) can also be observed within the internal acoustic meatus. 65. lateral process of malleus manubrium of malleus

1	lateral process of malleus manubrium of malleus FIGURE 25.4 • The tympanic membrane in otoscopic examination of the external ear. This photograph shows the left tympanic membrane seen with otoscope during examination of the external acoustic meatus. The landmarks of tympanic membrane include the manubrium of the malleus with its visible attachment to the tympanic membrane, umbo at the tip of the manubrium, and projecting lateral process of the malleus. Note the cone of light (light reflex) that is usually seen extending anteroinferiorly from the umbo of tympanic membrane. (Courtesy of Dr. Eric J. Moore, Mayo Clinic, Rochester, MN.) movement of the stapes at the oval window. The stapedius is only a few millimeters long and is the smallest skeletal muscle.

1	The two muscles of the middle ear are responsible for a protective reflex called the attenuation reﬂex. Contraction of the muscles makes the chain of ossicles more rigid, thus reducing the transmission of vibrations to the internal ear. This reflex protects the internal ear from the damaging effects of very loud sound. The auditory tube connects the middle ear to the nasopharynx. The auditory (Eustachian) tube is a narrow flattened channel approximately 3.5 cm long. This tube is lined with ciliated pseudostratified columnar epithelium, about one fifth of which is composed of goblet cells. It vents the middle ear, equalizing the pressure of the middle ear with atmospheric pressure. The walls of the tube are normally pressed together but separate during yawning and swallowing. It is common for infections to spread from the pharynx to the middle ear via the auditory tube (causing

1	FIGURE 25.5 • Cross section through a human tympanic membrane. This photomicrograph shows the tympanic membrane, external acoustic meatus, and tympanic cavity. 9. Inset. Higher magnification of the tympanic membrane. The outer epithelial layer of the membrane consists of stratified squamous epithelium (SSE), and the inner epithelial layer of the membrane consists of low simple cuboidal epithelium (SCE). A middle layer of connective tissue (CT ) lies between the two epithelial layers. 190. otitis media). A small mass of lymphatic tissue, the tubal tonsil, is often found at the pharyngeal orifice of the auditory tube. The mastoid air cells extend from the middle ear into the temporal bone.

1	The mastoid air cells extend from the middle ear into the temporal bone. A system of air cells projects into the mastoid portion of the temporal bone from the middle ear. The epithelial lining of these air cells is continuous with that of the tympanic cavity and rests on periosteum. This continuity allows infections in the middle ear to spread into these cells, causing mastoiditis. Before the development of antibiotics, repeated episodes of otitis media and mastoiditis usually led to deafness. The internal ear consists of two labyrinthine compartments, one contained within the other. The bony labyrinth is a complex system of interconnected cavities and canals in the petrous part of the temporal bone. The membranous labyrinth lies within the bony labyrinth and consists of a complex system of small sacs and tubules that also form a continuous space enclosed within a wall of epithelium and connective tissue.

1	There are three fluid-filled spaces in the internal ear:  Endolymphatic spaces are contained within the membranous labyrinth. The endolymph of the membranous labyrinth is similar in composition to intracellular ﬂuid (it has a high K concentration and a low Na concentration).  The perilymphatic space lies between the wall of the bony labyrinth and the wall of the membranous labyrinth. The perilymph is similar in composition to extracellular ﬂuid (it has a low K concentration and a high Na concentration).  The cortilymphatic space lies within the organ of Corti. It is a true intercellular space. The cells surrounding the space loosely resemble an absorptive epithelium. The cortilymphatic space is filled with cortilymph, which has a composition similar to that of extracellular ﬂuid. Structures of the Bony Labyrinth The bony labyrinth consists of three connected spaces within the temporal bone. The three spaces of the bony labyrinth, as illustrated in Figure 25.7, are the:

1	Structures of the Bony Labyrinth The bony labyrinth consists of three connected spaces within the temporal bone. The three spaces of the bony labyrinth, as illustrated in Figure 25.7, are the: FIGURE 25.6 • Photograph of the three articulated human auditory ossicles. The three ossicles are the malleus, the incus, and the stapes. 30. The vestibule is the central space that contains the utricle and saccule of the membranous labyrinth. The vestibule is the small oval chamber located in the center of the bony labyrinth. The utricle and saccule of the membranous labyrinth lie in elliptical and spherical recesses, respectively. The semicircular canals extend from the vestibule posteriorly, and the cochlea extends from the vestibule anteriorly. The oval window into which the footplate of the stapes inserts lies in the lateral wall of the vestibule. The semicircular canals are tubes within the temporal bone that lie at right angles to each other.

1	The semicircular canals are tubes within the temporal bone that lie at right angles to each other. Three semicircular canals, each forming about three quarters of a circle, extend from the wall of the vestibule and return to it. The semicircular canals are identified as anterior, posterior, and lateral and lie within the temporal bone at approximately right angles to each other. They occupy three planes in space— sagittal, frontal, and horizontal. The end of each semicircular canal closest to the vestibule is expanded to form the ampulla (Fig. 25.8a). The three canals open into the vestibule through five orifices; the anterior and posterior semicircular canals join at one end to form the common bony limb (see Fig. 25.8a). The cochlea is a cone-shaped helix connected to the vestibule.

1	The cochlea is a cone-shaped helix connected to the vestibule. The lumen of the cochlea, like that of the semicircular canals, is continuous with that of the vestibule. It connects to the vestibule on the side opposite the semicircular canals. Between its base and the apex, the cochlea makes about 2.75 turns around a central core of spongy bone called the modiolus (Plate 108, page 946). A sensory ganglion, the spiral ganglion, lies in the modiolus. A thin membrane (the secondary tympanic membrane) covers one opening of the canal, the round window on its inferior surface near the base. Structures of the Membranous Labyrinth The membranous labyrinth contains the endolymph and is suspended within the bony labyrinth. The membranous labyrinth consists of a series of communicating sacs and ducts containing endolymph. It is  FOLDER 25.1 Clinical Correlation: Otosclerosis

1	Otosclerosis is one of most common causes of acquired hearing loss. It is reported that about 13% of the U.S. population has nonclinical otosclerosis (histologic oto-sclerosis); however, incidence of clinical disease ranges from 0.5% to 1.0%. Individuals with otosclerosis complain about progressive hearing loss. Symptoms usually become apparent between ages 20 and 45. Otosclerosis is a metabolic bone disease that uniquely affects the temporal bone and ossicles and is characterized by abnormal bone remodeling. The stimulus to initiate bone remodeling in oto-sclerosis is still unknown, but recent studies associate this event with measles virus infection. Mature bone in the area of the oval window on the medial wall of the tympanic cav-ity, which separates the middle ear from the internal ear, is removed by osteoclasts and replaced with much thicker immature (woven) bone. Since the footplate of the stapes normally resides and freely vibrates within the oval window to allow the

1	ear, is removed by osteoclasts and replaced with much thicker immature (woven) bone. Since the footplate of the stapes normally resides and freely vibrates within the oval window to allow the transmission of sound into the internal ear, the bone remodeling in this area results in fixation of the stapes into the surrounding bone. The cemented (or frozen in place) stapes does not vibrate and prevents sound waves from reaching the perilymphatic fluid space of the internal ear, causing conductive hearing loss. The treatment of otosclerosis includes several options: phar-macologic treatment to suppress bone remodeling with fluorides and bisphosphonates, amplification of sounds with hearing aids, and surgical removal of the stapes (stapedectomy) with subsequent implantation of a pros-thesis between the incus and the oval window. Surgery is usually the most effective method of managing otosclero-sis; more than 90% of patients experience complete elim-ination of conductive hearing loss.

1	 FOLDER 25.2 Clinical Correlation: Hearing Loss—Vestibular Dysfunction

1	Several types of disorders can affect the auditory and vestibular system and result in deafness, dizziness (vertigo), or both. Auditory disorders are classified as either sen-sorineural or conductive. Conductive hearing loss results when sound waves are mechanically impeded from reaching the auditory sensory receptors within the internal ear. This type of hearing loss principally involves the external ear or structures of the middle ear. Conductive hearing loss is the second most common type of loss after sensorineural hear-ing loss, and it usually involves a reduction in sound level or the inability to hear faint sounds. A conductive hearing loss may be caused by otitis media (ear infection); in fact, this is the most common cause of temporary hearing loss in chil-dren. Fluid that collects in the tympanic cavity can also cause significant hearing problems in children. Other com-mon causes of conductive hearing loss include excess wax or foreign bodies in the external acoustic meatus

1	in the tympanic cavity can also cause significant hearing problems in children. Other com-mon causes of conductive hearing loss include excess wax or foreign bodies in the external acoustic meatus or dis-eases that affect the ossicles in the middle ear (otosclero-sis; see also Folder 25.1). In many cases, conductive hearing loss can be treated either medically or surgically and may not be permanent. Sensorineural hearing impairment may also occur after injury to the auditory sensory hair cells within the inter-nal ear, cochlear division of cranial nerve VIII, nerve path-ways in the CNS, or auditory cortex. Sensorineural hearing loss accounts for about 90% of all hearing loss. It may be congenital or acquired. Causes of acquired sensorineural hearing loss include infections of the membranous labyrinth (e.g., meningitis, chronic otitis media), fractures of the tem-poral bone, acoustic trauma (i.e., prolonged exposure to excessive noise), and administration of certain classes of

1	membranous labyrinth (e.g., meningitis, chronic otitis media), fractures of the tem-poral bone, acoustic trauma (i.e., prolonged exposure to excessive noise), and administration of certain classes of antibiotics and diuretics. Another example of sensorineural hearing loss often re-sults from aging. Sensorineural hearing loss not only in-volves a reduction in sound level; it also affects the ability to hear clearly or to distinguish speech. A loss of the sensory hair cells or associated nerve fibers begins in the basal turn of the cochlea and progresses apically over time. The char-acteristic impairment is a high-frequency hearing loss termed presbycusis (see presbyopia, page 915). In selected patients, the use of a cochlear implant can partially restore some hearing function. The cochlear implant is an electronic device consisting of an external microphone, amplifier, and speech processor linked to a receiver implanted under the skin of the mastoid region. The receiver is con-nected

1	implant is an electronic device consisting of an external microphone, amplifier, and speech processor linked to a receiver implanted under the skin of the mastoid region. The receiver is con-nected to the multielectrode intracochlear implant inserted along the wall of the cochlear canal. After considerable train-ing and tuning of the speech processor, the patient’s hearing can be partially restored to various degrees ranging from recognition of critical sounds to the ability to converse.

1	suspended within the bony labyrinth (Fig. 25.8b), and the remaining space is filled with perilymph. The membranous labyrinth is composed of two divisions: the cochlear labyrinth and the vestibular labyrinth (Fig. 25.8c). FIGURE 25.7 • Photograph of a cast of the bony labyrinth of the internal ear. The cochlear portion of the bony labyrinth appears blue-green; the vestibule and semicircular canals appear orange-red. (Courtesy of Dr. Merle Lawrence.) The vestibular labyrinth contains the following:  Three semicircular ducts lie within the semicircular canals and are continuous with the utricle.  The utricle and the saccule, which are contained in recesses in the vestibule, are connected by the membranous utriculosaccular duct. The cochlear labyrinth contains the cochlear duct, which is contained within the cochlea and is continuous with the saccule (see Fig. 25.8b and Fig. 25.8c). Sensory Cells of the Membranous Labyrinth

1	Sensory Cells of the Membranous Labyrinth Specialized sensory cells are located in six regions in the membranous labyrinth. Six sensory regions of membranous labyrinth are composed of sensory hair cells and accessory supporting cells. These regions project from the wall of the membranous labyrinth into the endolymphatic space in each internal ear (see Fig. 25.7c):  Three cristae ampullaris (ampullary crests) are located in the membranous ampullae of the semicircular ducts. They are sensitive to angular acceleration of the head (i.e., turning the head).  Two maculae, one in the utricle (macula of utricle) and the other in the saccule (macula of saccule), sense the position of the head and its linear movement.  The spiral organ of Corti projects into the endolymph of the cochlear duct. It functions as the sound receptor.

1	 The spiral organ of Corti projects into the endolymph of the cochlear duct. It functions as the sound receptor. FIGURE 25.8 • Diagrams of the human internal ear. a. This lateral view of the left bony labyrinth shows its divisions: the vestibule, cochlea, and three semicircular canals. The openings of the oval window and the round window can be observed.

1	b. Diagram of the membranous labyrinth of the internal ear lying within the bony labyrinth. The cochlear duct can be seen spiraling within the bony cochlea. The saccule and utricle are positioned within the vestibule, and the three semicircular ducts are lying within their respective canals. This view of the left membranous labyrinth allows the endolymphatic duct and sac to be observed. c. This view of the left membranous labyrinth shows the sensory regions of the internal ear for equilibrium and hearing. These regions are the macula of the saccule and macula of the utricle, the cristae ampullaris of the three semicircular ducts, and the spiral organ of Corti of the cochlear duct. macula of saccule organ of Corti macula of utricle crista ampullaris of anterior semicircular duct crista ampullaris of lateral semicircular duct crista ampullaris of posterior semicircular duct Hair cells are epithelial mechanoreceptors of the vestibular and cochlear labyrinth.

1	The hair cells of the vestibular and cochlear labyrinths function as mechanoelectric transducers; they convert mechanical energy into electrical energy that is then transmitted via the vestibulocochlear nerve to the brain. The hair cells derive their name from the organized bundle of rigid projections at their apical surface. This surface holds a hair bundle that is formed of rows of stereocilia called sensory hairs. The rows increase in height in one particular direction across the bundle (Fig. 25.9). In the vestibular system, each hair cell possesses a single true cilium called a kinocilium, which is located behind the row of longest stereocilia (Fig. 25.10). In the auditory system, the hair cells lose their cilium during development but retain the basal body. The position of the kinocilium (or basal body) behind the longest row of stereocilia defines the polarity of this asymmetric hair bundle. Therefore, movement of the stereocilia toward the kinocilium is perceived differently

1	(or basal body) behind the longest row of stereocilia defines the polarity of this asymmetric hair bundle. Therefore, movement of the stereocilia toward the kinocilium is perceived differently than movement in the opposite direction (see below).

1	Stereocilia of hair cells are rigid structures that contain mechanoelectric transducer channel proteins at their distal ends.

1	The stereocilia of hair cells have a molecular structure similar to those described on page 110. Tightly packed actin filaments cross-linked by fimbrin and espin (actin-bundling proteins) form their internal core structure. The high density of actin filaments and the extensive cross-linking pattern imparts rigidity and stiffness to the shaft of the stereocilium. The shaft tapers at its proximal end near the apical surface of the cell, where the core filaments of each stereocilium are anchored within the terminal web (cuticular plate). When stereocilia are deflected, they pivot at their proximal ends like stiff rods (see Fig. 25.10). Transmission electron microscope examination of the distal free end of the stereocilium reveals an electron-dense plaque at the cytoplasmic site of the plasma membrane. This plaque represents mechanoelectric transducer (MET) channel protein. A fibrillar cross-link called the tip link connects the tip of the stereocilium with the shaft of an adjacent

1	plasma membrane. This plaque represents mechanoelectric transducer (MET) channel protein. A fibrillar cross-link called the tip link connects the tip of the stereocilium with the shaft of an adjacent longer stereocilium (see Fig. 25.10). The tip link plays an important role in activating the MET channels at the tip of the stereocilia and opening additional transduction K channels at the site of its attachment to the shaft of neighboring stereocilium (see Fig. 25.10). The molecular structures of the transduction K channels and tip links are as yet unknown. Individual stereocilia are also connected by a variety of fibrillar extracellular cross-links.

1	A mutation that disrupts the gene that produces espin causes cochlear and vestibular symptoms in experimental mice. They lose their hearing early in life; these animals also spend most of their time walking or spinning in circles. The stereocilia of these animals do not maintain the rigidity necessary for proper functioning of the MET channels. All hair cells use mechanically gated ion channels to generate action potentials. All hair cells of the internal ear appear to function by moving (pivoting) their rigid stereocilia. Mechanoelectric transduction

1	FIGURE 25.9 • Electron micrographs of the kinocilium and stereocilia of a vestibular sensory hair cell. a. Scanning electron micrograph of the apical surface of a sensory hair cell from the macula of the utricle. Note the relationship of the kinocilium (K) to the stereocilia (S). 47,500. (Reprinted with permission from Rzadzinska AK, Schneider ME, Davies C, Riordan GP, Kachar B. An actin molecular treadmill and myosins maintain stereocilia functional architecture and self-renewal. J Cell Biol 2004;164:887-897.) b. Transmission electron micrograph of the kinocilium (K) and stereocilia (S) of a vestibular hair cell in cross section. The kinocilium has a larger diameter than the stereocilia. 47,500. (Reprinted with permission from Hunter-Duvar IM, Hinojosa R. Vestibule: sensory epithelia. In: Friedmann I, Ballantyne J, eds. Ultrastructural Atlas of the Inner Ear. London: Butterworth, 1984.) occurs in stereocilia that are deflected toward its tallest edge (toward the kinocilium if

1	In: Friedmann I, Ballantyne J, eds. Ultrastructural Atlas of the Inner Ear. London: Butterworth, 1984.) occurs in stereocilia that are deflected toward its tallest edge (toward the kinocilium if present). This movement exerts tension on the fibrillar tip links, and the generated force is used to open mechanically gated ion channels near the tip of the stereocilium. This allows for an influx of K, causing depolarization of the receptor cell. This depolarization results in the opening of voltage-gated Ca2 channels in the basolateral surface of the hair cells and the secretion of a neurotransmitter that generates an action potential in afferent nerve endings. Movement in the opposite direction (away from the kinocilium) closes the MET channels, causing hyperpolarization of the receptor cell. The means by which stereocilia are deflected varies from receptor to receptor; these are discussed in the sections describing each receptor area.

1	Two types of hair cells are present in the vestibular labyrinth. Both hair cell types are associated with afferent and efferent nerve endings (Fig. 25.10). Type I hair cells are flask-shaped, with a rounded base and thin neck, and are surrounded by an afferent nerve chalice and a few efferent nerve fibers. Type II hair cells are cylindrical and have afferent and efferent bouton nerve endings at the base of the cell (see Fig. 25.10). Sensory Receptors of the Membranous Labyrinth Cristae ampullaris are sensors of angular movements of the head. Each ampulla of the semicircular duct contains a crista ampullaris, which is a sensory receptor for angular movements of the head (see Figs. 25.8c and 25.11). The crista ampullaris is a thickened transverse epithelial ridge that is oriented perpendicularly to the long axis of the semicircular canal and consists of the epithelial hair cells and supporting cells (Plate 108, page 946).

1	A gelatinous protein–polysaccharide mass, known as the cupula, is attached to the hair cells of each crista (see Fig. 25.11). The cupula projects into the lumen and is surrounded by endolymph. During rotational movement of the head, the walls of the semicircular canal and the membranous semicircular ducts move, but the endolymph contained within the ducts tends to lag behind because of inertia. The cupula, projecting into the endolymph, is swayed by the

1	FIGURE 25.10 • Diagram of two types of sensory hair cells in the sensory areas of the membranous labyrinth. The type I hair cell has a flask-shaped structure with a rounded base. The base is enclosed in a chalicelike afferent nerve ending that has several synaptic boutons for efferent nerve endings. Note the apical surface specializations of this cell, which include a kinocilium and hair bundle. The apical cytoplasm of hair cells contains basal bodies for the attachment of the kinocilium and a terminal web for the attachment of stereocilia. The type II hair cell is cylindrical and possesses several nerve terminals at its base for both afferent and efferent nerve fibers. The apical surface specializations are identical to those of the type I cell. The molecular organization of the stereocilia is depicted in the enlarged rectangle. The top link connects the lateral plasma membrane of the stereocilium shaft (where K transduction channels are located) with the tip of the shorter

1	stereocilia is depicted in the enlarged rectangle. The top link connects the lateral plasma membrane of the stereocilium shaft (where K transduction channels are located) with the tip of the shorter stereocilium (where the mechanoelectric transduction [MET] channel protein is located). Note that the proximal end of each stereocilium is tapered and its narrow rootlets are anchored within the terminal web (cuticular plate) of the hair cell. Several other fibrillar connectors between neighboring stereocilia are also shown.

1	 FOLDER 25.3 Clinical Correlation: Vertigo

1	The sensation of rotation without equilibrium (dizziness, vertigo) signifies dysfunction of the vestibular system. Causes of vertigo include viral infections, certain drugs, and tumors such as acoustic neuroma. Acoustic neuro-mas develop in or near the internal acoustic meatus and exert pressure on the vestibular division of cranial nerve VIII or branches of the labyrinthine artery. Vertigo can also be produced normally in individuals by excessively stimulating the semicircular ducts. Similarly, excessive stimulation of the utricle can produce motion sickness (seasickness, car-sickness, or airsickness) in some individuals. Some diseases of the internal ear affect both hearing and equilibrium. For example, people with Ménière’s syn-drome initially complain of episodes of dizziness and tinni-tus (ringing in the ears) and later develop a low-frequency hearing loss. The causes of Ménière’s syndrome are related to blockage of the cochlear aqueduct, which drains excess endolymph from the

1	(ringing in the ears) and later develop a low-frequency hearing loss. The causes of Ménière’s syndrome are related to blockage of the cochlear aqueduct, which drains excess endolymph from the membranous labyrinth. Blockage of this duct causes an increase in endolymphatic pressure and distension of the membranous labyrinth (endolymphatic hydrops).

1	FIGURE 25.11 • Diagram of the crista ampullaris within a semicircular duct. The cellular organization of the crista ampullaris of a semicircular duct is shown in the large diagram and the enlarged rectangle. The crista ampullaris is composed of both type I and type II sensory hair cells and supporting cells. The stereocilia and kinocilium of each hair are embedded in the cupula that projects toward the nonsensory wall of the ampulla. movement differential between the crista fixed to the wall of the duct and the endolymph. Deflection of the stereocilia in the narrow space between the hair cells and the cupula generates nerve impulses in the associated nerve endings. The maculae of the saccule and utricle are sensors of gravity and linear acceleration.

1	The maculae of the saccule and utricle are sensors of gravity and linear acceleration. The maculae of the saccule and the utricle are innervated sensory thickenings of the epithelium that face the endolymph of the saccule and utricle (see Fig. 25.8c). As in the cristae, each macula consists of type I and type II hair cells, supporting cells, and nerve endings associated with the hair cells. The maculae of the utricle and saccule are oriented at right angles to each another. When a person is standing, the macula of the utricle is in a horizontal plane, and the macula of the saccule is in a vertical plane. The gelatinous polysaccharide material that overlies the maculae is called the otolithic membrane (Fig. 25.12). Its outer surface contains 3-m to 5-m crystalline bodies of calcium carbonate and a protein (Fig. 25.13). Otoliths are heavier than the endolymph. The outer surface of the otolithic membrane

1	FIGURE 25.12 • Diagram of a macula within the utricle. A more detailed diagram of the cellular organization of the macula of the utricle is shown in the enlarged rectangle. Supporting cells can be seen lying between the two principal types of sensory hair cells (types I and II). The stereocilia and kinocilium of each sensory hair cell are embedded in the otolithic membrane on which otoconia lie. FIGURE 25.13 • Scanning electron micrograph of human otoconia. Each otoconium has a long cylindrical body with a three-headed facet on each end. 5,000.

1	FIGURE 25.13 • Scanning electron micrograph of human otoconia. Each otoconium has a long cylindrical body with a three-headed facet on each end. 5,000. lies opposite the surface in which the stereocilia of the hair cells are embedded. The otolithic membrane moves on the macula in a manner analogous to that by which the cupula moves on the crista. Stereocilia of the hair cells are deflected by gravity in the stationary individual when the otolithic membrane and its otoliths pull on the stereocilia. They are also displaced during linear movement when the individual is moving in a straight line and the otolithic membrane drags on the stereocilia because of inertia. In both cases, movement of the otolithic membrane activates MET channels and depolarizes hair cells. The spiral organ of Corti is the sensor of sound vibrations.

1	The spiral organ of Corti is the sensor of sound vibrations. The cochlear duct divides the cochlear canal into three par allel compartments or scalae:  Scala media, the middle compartment in the cochlear canal  Scala vestibuli  Scala tympani The cochlear duct itself is the scala media (Figs. 25.14 and 25.15). The scala vestibuli and scala tympani are the spaces above and below, respectively, the scala media. The scala media is an endolymph-containing space that is continuous FIGURE 25.14 • Schematic diagram of the cochlea. a.

1	FIGURE 25.14 • Schematic diagram of the cochlea. a. Schematic diagram of a midmodiolar section of the cochlea that illustrates the position of the cochlear duct within the 2.75 turns of the bony cochlea. Observe that the scala vestibuli and scala tympani are continuous apically (helicotrema). b. Cross section of the basal turn of the cochlear duct. The cochlear duct and the osseous spiral lamina divide the cochlea into the scala vestibuli and the scala tympani, which contain perilymph. The scala media (the space within the cochlear duct) is filled with endolymph and contains the organ of Corti. (Modified from Goodhill V. Ear, Diseases, Deafness, and Dizziness. Hagerstown, MD: Harper & Row, 1979.)

1	FIGURE 25.15 • Photomicrograph of cochlear canal. This photomicrograph shows a section of the basal turn of the cochlear canal. The osseous spiral lamina (OSL) and its membranous continuation, the basilar membrane (BM), as well as the vestibular membrane (VM) divide the cochlear canal into three parallel compartments: The scala vestibuli, the cochlear duct (CD), and the scala tympani. Both the scala vestibuli and the scala tympani are filled with perilymph, whereas the cochlear canal is filled with endolymph. Note the three walls of the cochlear canal, which are formed by the basilar membrane inferiorly, the stria vascularis (SV ) and underlying spiral ligament (SL) laterally, and the vestibular membrane superiorly. The spiral organ of Corti resides on the inferior wall of the cochlear canal. Dendrites of the cochlear nerve (CN) fibers that originate in the spiral ganglion (SG) enter the spiral organ of Corti. The axons of the cochlear nerve form the cochlear part of the

1	cochlear canal. Dendrites of the cochlear nerve (CN) fibers that originate in the spiral ganglion (SG) enter the spiral organ of Corti. The axons of the cochlear nerve form the cochlear part of the vestibulocochlear nerve. 65.

1	with the lumen of the saccule and contains the spiral organ of Corti, which rests on its lower wall (see Fig. 25.15). The scala vestibuli and the scala tympani are perilymphcontaining spaces that communicate with each other at the apex of the cochlea through a small channel called the helicotrema (see Fig. 25.14). The scala vestibuli begins at the oval window, and the scala tympani ends at the round window. The scala media is a triangular space with its acute angle attached to the modiolus. In transverse section, the scala media appears as a triangular space with its most acute angle attached to a bony extension of the modiolus, the osseous spiral lamina (see Fig. 25.15). The upper wall of the scala media, which mesothelial cellperilymph of scala vestibuli endolymph of scala media epithelial cell

1	FIGURE 25.16 • Transmission electron micrograph of the vestibular (Reissner’s) membrane. Two cell types can be observed: a mesothelial cell, which faces the scala vestibuli and is bathed by perilymph, and an epithelial cell, which faces the scala media and is bathed by endolymph. 8,400. separates it from the scala vestibuli, is the vestibular (Reissner’s) membrane (Fig. 25.16). The lateral or outer wall of the scala media is bordered by a unique epithelium, the stria vascularis. It is responsible for production and maintenance of endolymph. The stria vascularis encloses a complex capillary network and contains three types of cells (Fig. 25.17). The marginal cells, primarily involved in K transport, line the endolymphatic space of the scala media.

1	FIGURE 25.17 • Transmission electron micrograph of the stria vascularis. The apical surfaces of the marginal cells (M) of the stria are bathed by endolymph (E) of the scala media. Intermediate cells (I) are positioned between the marginal cells and the basal cells (B). The basal cells separate the other cells of the stria vascularis from the spiral ligament (SpL). 4,700. Intermediate pigment-containing cells are scattered among capillaries. The basal cells separate stria vascularis from underlying spiral ligament. The lower wall or floor of the scala media is formed by a relatively flaccid basilar membrane that increases in width and decreases in stiffness as it coils from the base to apex of the cochlea. The spiral organ of Corti rests on the basilar membrane and is overlain by the tectorial membrane. The spiral organ of Corti is composed of hair cells, phalangeal cells, and pillar cells.

1	The spiral organ of Corti is composed of hair cells, phalangeal cells, and pillar cells. The spiral organ of Corti is a complex epithelial layer on the floor of the scala media (Fig. 25.18 and Plate 109, page 948). It is formed by the following:  Inner hair cells (close to the spiral lamina) and outer hair cells (farther from the spiral lamina) Inner phalangeal (supporting) cells and outer pha Several other named cell types of unknown function are also present in the spiral organ. The hair cells are arranged in inner and outer rows of cells. The inner hair cells form a single row of cells throughout all 2.75 turns of the cochlear duct. The number of cells forming the width of the continuous row of outer hair cells is variable. Three ranks of hair cells are found in the basal part of the coil (Fig. 25.19). The width of the row gradually increases to five ranks of cells at the apex of the cochlea. The phalangeal and pillar cells provide support for the hair cells.

1	The phalangeal and pillar cells provide support for the hair cells. Phalangeal cells are supporting cells for both rows of hair cells. The phalangeal cells associated with the inner hair cells surround the cells completely (Fig. 25.20a). The phalangeal cells associated with the outer hair cells surround only the basal portion of the hair cell completely and send apical processes toward the endolymphatic space (Fig. 25.20b). These processes flatten near the apical ends of the hair cells and collectively form a complete plate surrounding each hair cell (Fig. 25.21).

1	FIGURE 25.18 • Photomicrograph of the vestibular duct and spiral organ of Corti. This higher-magnification photomicrograph of the cochlear duct shows the structure of the spiral organ of Corti. Relate this structure to the inset, which labels the structural features of the spiral organ. 180. Inset. Diagram of the sensory and supporting cells of the spiral organ of Corti. The sensory cells are divided into an inner row of sensory hair cells and three rows of outer sensory hair cells. The supporting cells are the inner and outer pillar cells, inner and outer (Deiters’) phalangeal cells, outer border cells (Hensen’s cells), inner border cells, Claudius’ cells, and Btcher’s cells. (Modified from Goodhill V. Ear, Diseases, Deafness, and Dizziness. Hagerstown, MD: Harper & Row, 1979.)

1	The apical ends of the phalangeal cells are tightly bound to one another and to the hair cells by elaborate tight junctions. These junctions form the reticular lamina that seals the endolymphatic compartment from the true intercellular spaces of the organ of Corti (Figs. 25.18 and 25.20b). The extracellular fluid in this intercellular space is cortilymph. Its composition is similar to that of other extracellular fluids and to perilymph. Pillar cells have broad apical and basal surfaces that form plates and a narrowed cytoplasm. The inner pillar cells rest on the tympanic lip of the spiral lamina; the outer pillar cells rest on the basilar membrane. Between them, they form a triangular tunnel, the inner spiral tunnel (see Fig. 25.18). The tectorial membrane extends from the spiral limbus over the cells of the spiral organ of Corti.

1	The tectorial membrane extends from the spiral limbus over the cells of the spiral organ of Corti. The tectorial membrane is attached medially to the modiolus. Its lateral free edge projects over and attaches to the organ of Corti by the stereocilia of the hair cells. It is formed from the radially oriented bundles of collagen types II, V, and IX embedded in a dense amorphous ground substance. Glycoproteins unique to the internal ear, called otogelin and tectorin, are associated with the collagen bundles. These proteins are also present in the otolithic membranes overlying the maculae of the utricle and saccule, as well as in the cupulae of the cristae in the semicircular canals. As described on page 929, sound waves striking the tympanic membrane are translated into simple mechanical vibrations. The ossicles of the middle ear convey these vibrations to the cochlea. In the internal ear, the vibrations of the ossicles are transformed into waves in the perilymph.

1	In the internal ear, the vibrations of the ossicles are transformed into waves in the perilymph. Movement of the stapes in the oval window of the vestibule sets up vibrations or traveling waves in the perilymph of the scala vestibuli. The vibrations are transmitted through FIGURE 25.19 • Scanning electron micrograph of the spiral organ of Corti. This electron micrograph illustrates the configuration of stereocilia on the apical surfaces of the inner row and three outer rows of the cochlear sensory hair cells. 3,250. the vestibular membrane to the scala media (cochlear duct), which contains endolymph, and are also propagated to the perilymph of the scala tympani. Pressure changes in this closed perilymphatic–endolymphatic system are reflected in movements of the membrane that covers the round window in the base of the cochlea.

1	As a result of sound vibrations entering the internal ear, a traveling wave is set up in the basilar membrane (Fig. 25.22). A sound of specified frequency causes displacement of a relatively long segment of the basilar membrane, but the region of maximal displacement is narrow. The point of maximal displacement of the basilar membrane is specified for a given frequency of sound and is the morphologic basis of frequency discrimination. High-frequency sounds cause maximal vibration of the basilar membrane near the base of the cochlea; low-frequency sounds cause maximal displacement nearer the apex. Amplitude discrimination (i.e., perception of sound intensity or loudness) depends on the degree of displacement of the basilar membrane at any given frequency range. Thus, coding acoustic information into nerve impulses depends on the vibratory pattern of the basilar membrane. Movement of the stereocilia of the hair cells in the cochlea initiates neuronal transduction.

1	Movement of the stereocilia of the hair cells in the cochlea initiates neuronal transduction. Hair cells are attached through the phalangeal cells to the basilar membrane, which vibrates during sound reception. The stereocilia of these hair cells are, in turn, attached to the tectorial membrane, which also vibrates. However, the tectorial membrane and the basilar membrane are hinged at different points. Thus, a shearing effect occurs between the basilar membrane (and the cells attached to it) and the tectorial membrane when sound vibrations impinge on the internal ear.

1	Because they are inserted into the tectorial membrane, the stereocilia of the hair cells are the only structures that connect the basilar membrane and its complex epithelial layer to the tectorial membrane. The shearing effect between the basilar membrane and the tectorial membrane deflects the stereocilia and thus the apical portion of the hair cells. This deflection activates MET channels located at the tips of stereocilia and generates action potentials that are conveyed to the brain via the cochlear nerve (cochlear division of the vestibulocochlear nerve, cranial nerve VIII). Innervation of the Internal Ear The vestibular nerve originates from the sensory receptors associated with the vestibular labyrinth.

1	Innervation of the Internal Ear The vestibular nerve originates from the sensory receptors associated with the vestibular labyrinth. The vestibulocochlear nerve (cranial nerve VIII) is a special sensory nerve and is composed of two divisions: A vestibular division called the vestibular nerve and a cochlear division called the cochlear nerve. The vestibular nerve is associated with equilibrium and carries impulses from the sensory receptors located within the vestibular labyrinth. The cochlear nerve is associated with hearing and conveys impulses from the sensory receptors within the cochlear labyrinth (Fig. 25.23).

1	The cell bodies of the bipolar neurons of the vestibular nerve are located in the vestibular ganglion (of Scarpa) in the internal acoustic meatus. Dendritic processes of the vestibular ganglion cells originate in the cristae ampullaris of the three semicircular ducts, the macula of the utricle, and the macula of the saccule. They synapse at the base of the vestibular sensory hair cells, either as a chalice around a type I hair cell or as a bouton associated with a type II hair cell. The axons of the vestibular nerve originate from the vestibular ganglion, enter the brain stem and terminate in four vestibular nuclei. Some secondary neuronal fibers travel to the cerebellum and to the nuclei of cranial nerves III, IV, and VI, which innervate the muscles of the eye. The cochlear nerve originates from the sensory receptors of the spiral organ of Corti.

1	The cochlear nerve originates from the sensory receptors of the spiral organ of Corti. Neurons of the cochlear nerve are also bipolar, and their cell bodies are located in the spiral ganglion within the modiolus. Dendritic processes of spiral ganglion cells exit the

1	FIGURE 25.20 • Electron micrograph of an inner and outer hair cell. a. Observe the rounded base and constricted neck of the inner hair cell. Nerve endings (NE) from afferent nerve fibers (AF ) to the inner hair cells are seen basally. IP, inner pillar cell; IPH, inner phalangeal cell. 6,300. b. Afferent (AF ) and efferent (EF ) nerve fiber endings on the base of an outer sensory hair cell are evident. Outer phalangeal cells (OPH) surround the outer hair cells basally. Their apical projections form the apical cuticular plate (ACP). Note that the lateral domains in the middle third of the outer hair cells are not surrounded by supporting cells. 6,300. (Reprinted with permission from Kimura RS. Sensory and accessory epithelia of the cochlea. In: Friedmann I, Ballantyne J, eds. Ultrastructural Atlas of the Inner Ear. London: Butterworth, 1984.)

1	FIGURE 25.21 • Structure of the outer phalangeal cell. a. This scanning electron micrograph illustrates the architecture of the outer phalangeal (Deiters’) cells. Each phalangeal cell cups the basal surface of an outer hair cell and extends its phalangeal process apically to form an apical cuticular plate that supports the outer sensory hair cells. 2,400. b. Schematic drawing showing the relationship of an outer phalangeal cell to an outer hair cell.

1	FIGURE 25.22 • Schematic diagram illustrating the dynamics of the three divisions of the ear. The cochlear duct is shown here as if straightened. Sound waves are collected and transmitted from the external ear to the middle ear, where they are converted into mechanical vibrations. The mechanical vibrations are then converted at the oval window into fluid vibrations within the internal ear. Fluid vibrations cause displacement of the basilar membrane (traveling wave) on which rest the auditory sensory hair cells. Such displacement leads to stimulation of the hair cells and a discharge of neural impulses from them. Note that high-frequency sounds cause vibrations of the narrow, thick portion of the basilar membrane at the base of the cochlea, whereas low-frequency sounds displace basilar membrane toward the apex of the cochlea near its helicotrema.

1	modiolus through the small openings in the bony spiral lam-the spiral ganglion cells form the cochlear nerve, which enina and enter the spiral organ. About 90% of dendrites orig-ters the bony cochlea through the modiolus to appear in the inating from the spiral ganglion cells synapse with the inner internal acoustic meatus (see Fig. 25.23). From the internal hair cells; the remaining 10% of dendrites synapse with acoustic meatus, the cochlear nerve enters the brain stem and outer hair cells of the spiral ganglion of Corti. The axons of terminates in the cochlear nuclei of the medulla. Nerve fibers superior part of ventral vestibular nerve cochlear nuclei inferior part of vestibular nerve

1	FIGURE 25.23 • Diagram illustrating the innervation of the sensory regions of the membranous labyrinth. Note the two parts of the vestibulocochlear nerve. The cochlear nerve carries the hearing impulses from the cochlear duct; the vestibular nerve carries balance information from the semicircular canals. The cell bodies of these sensory fibers are located in the spiral ganglion (for hearing) and vestibular ganglion (for equilibrium). (Modified from Hawke M, Keene M, Alberti PW. Clinical Otoscopy: A Text and Colour Atlas. Edinburgh: Churchill Livingstone, 1984.) from these nuclei pass to the geniculate nucleus of the thalamus and then to the auditory cortex of the temporal lobe.

1	It is interesting that the organ of Corti also receives small amounts of efferent fibers conveying impulses from the brain pass parallel to the afferent nerve fibers of the vestibulocochlear nerve (olivocochlear tract, cochlear efferents of Rasmussen). Efferent nerve fibers from the brain stem pass through the vestibular nerve. They synapse either on afferent endings of the inner hair cell or on the basal aspect of an outer hair cell. Efferent fibers are thought to affect control of auditory and vestibular input to the central nervous system, presumably by enhancing some afferent signals while suppressing other signals. Damage to the organ of Corti, cochlear nerve, nerve pathways, or auditory cortex is responsible for permanent sensorineural hearing loss. Blood Vessels of the Membranous Labyrinth Arterial blood is supplied to the membranous labyrinth by the labyrinthine artery; venous blood drainage is to the venous dural sinuses.

1	Blood Vessels of the Membranous Labyrinth Arterial blood is supplied to the membranous labyrinth by the labyrinthine artery; venous blood drainage is to the venous dural sinuses. The blood supply to the external ear, middle ear, and bony labyrinth of the internal ear is derived from vessels associated with the external carotid arteries. The arterial blood supply to tissues of the membranous labyrinth of the internal ear is derived intracranially from the labyrinthine artery, a common branch of the anterior inferior cerebellar or basilar artery (Fig. 25.24). The labyrinthine artery is a terminal artery: It has no anastomoses with other surrounding arteries. Branches of this artery exactly parallel the distribution of the superior and inferior parts of the vestibular nerve.

1	FIGURE 25.24 • Diagram of the arterial supply of the membranous labyrinth of the internal ear. The blood supply to the membranous labyrinth of the internal ear is derived from the labyrinthine artery, a branch of the anterior inferior cerebellar or basilar artery. (Modified from Schuknecht HF. Pathology of the Ear. Cambridge, MA: Harvard University Press, 1974.) Venous drainage from the cochlear labyrinth is via the posterior and anterior spiral modiolar veins that form the common modiolar vein. The common modiolar vein and the vestibulocochlear vein form the vein of the cochlear aqueduct, which empties into the inferior petrosal sinus. Venous drainage from the vestibular labyrinth is via vestibular veins that join the vein of the cochlear aqueduct and by the vein of vestibular aqueduct, which drains into the sigmoid sinus.

1	The internal ear, located in the temporal bone, consists of a system of chambers and canals that contain a network of membranous channels. These are referred to, respectively, as the bony labyrinth and membranous labyrinth. In places, the membranous labyrinth forms the lining of the bony labyrinth; in other places, there is a separation of the two. Within the space lined by the membranous labyrinth is a watery fluid called endolymph. External to the membranous labyrinth, i.e., between the membranous and bony labyrinths, is additional fluid called perilymph. The bony labyrinth is divided into three parts: cochlea, semicircular canals, and vestibule. The cochlea and semicircular canals contain membranous counterparts of the same shape; however, the membranous components of the vestibule are more complex in form, being composed of ducts and two chambers, the utricle and saccule. The cochlea contains the receptors for hearing, i.e., the organ of Corti, the semicircular canals contain the

1	more complex in form, being composed of ducts and two chambers, the utricle and saccule. The cochlea contains the receptors for hearing, i.e., the organ of Corti, the semicircular canals contain the receptors for movement of the head, and the utricle and saccule contain receptors for position of the head.

1	Internal ear, ear, guinea pig, H&E 20. In this section through the internal ear, bone surrounds the entire internal ear cavity. Because of its labyrinthine character, sections of the internal ear appear as a number of separate chambers and ducts. These, however, are all interconnected (except that the perilymphatic and endolymphatic spaces remain separate). The largest chamber is the vestibule (V ). The left side of this chamber (black arrow) leads into the cochlea (C). Just below the black arrow and to the right is the oval ligament (OL) surrounding the base of the stapes (S). Both structures have been cut obliquely and are not seen in their entirety. The facial nerve (FN ) is in an osseous tunnel to the left of the oval ligament. The com munication of the vestibule with one of the semicircular canals is marked by the white arrow. At the upper right are cross sections of the membranous labyrinth passing through components of the semicircular duct system (DS).

1	The cochlea is a spiral structure having the general shape of a cone. The specimen illustrated here makes 31⁄2 turns (in humans, there are 23⁄4 turns). The section goes through the central axis of the cochlea. This consists of a bony stem called the modiolus (M). It contains the beginning of the cochlear nerve (CN ) and the spiral ganglion (SG). Because of the plane of section and the spiral arrangement of the cochlear tunnel, the tunnel is cut crosswise in seven places (note 31⁄2 turns). A more detailed examination of the cochlea and the organ of Corti is provided in Plate 109. Semicircular canal, ear, guinea pig, H&E 225.

1	A higher magnification of one of the semicircular canals and of the crista ampullaris (CA) within the canal seen in the lower right corner of figure above is provided here. The receptor for movement, the crista ampullaris (note its relationships in figure above), is present in each of the semicircular canals. The epithelial (EP) surface of the crista consists of two cell types, sustentacular (supporting) cells and hair (receptor) cells. (Two types of hair cells are distinguished with the electron microscope.) It is difficult to identify the hair and sustentacular cells on the basis of specific characteristics; they can, however, be distinguished on the basis of location (see inset), as the hair cells (HC) are situated in a more superficial location than the sustentacular cells (SC). A gelatinous mass, the cupula (Cu), surmounts the epithelium of the crista ampullaris. Each receptor cell sends a hair-like projection deep into the substance of the cupula.

1	The epithelium rests on a loose, cellular connective tissue (CT ) that also contains the nerve fibers associated with the receptor cells. The nerve fibers are difficult to identify because they are not organized into a discrete bundle. KEY C, cochlea CA, crista ampullaris CN, cochlear nerve CT, connective tissue Cu, cupula DS, duct system (of membranous labyrinth) EP, epithelium FN, facial nerve HC, hair cell M, modiolus OL, oval ligament S, stapes SC, sustentacular cell SG, spiral ganglion V, vestibule black arrow, entry to cochlea white arrow, entry to semicircular canal PLATE 109 Cochlear Canal and Organ of Corti

1	PLATE 109 Cochlear Canal and Organ of Corti The hair cell, a nonneuronal mechanoreceptor, is the common receptor cell of the vestibulocochlear system. Hair cells are epithelial cells that possess numerous stereocilia, modified microvilli also called sensory hairs. They convert mechanical energy to electrical energy that is transmitted via the vestibulocochlear nerve (cranial nerve VIII) to the brain. Hair cells are associated with afferent, as well as efferent, nerve endings. All hair cells have a common basis of receptor cell function that involves bending or flexing of their stereocilia. The specific means by which the stereocilia are bent varies from receptor to receptor, but in each case, stretching of the plasma membrane caused by the bending of the stereocilia generates transmembrane potential changes that are transmitted to the afferent nerve endings associated with each cell. Efferent nerve endings on the hair cells serve to regulate their sensitivity.

1	Cochlear canal, ear, guinea pig, H&E 65; inset 380.

1	A section through one of the turns of the cochlea is shown here. The most important functional component of the cochlea is the organ of Corti, enclosed by the rectangle and shown at higher magnification in figure below. Other structures are included in this figure. The spiral ligament (SL) is a948 thickening of the periosteum on the outer part of the tunnel. Two membranes, the basilar membrane (BM ) and the vestibular membrane (VM), join with the spiral ligament and divide the cochlear tunnel into three parallel canals, namely, the scala vestibuli (SV ), the scala tympani (ST ), and the cochlear duct (CD). Both the scala vestibuli and the scala tympani are perilymphatic spaces; these communicate at the apex of the cochlea. The cochlear duct, on the other hand, is the space of the membranous labyrinth and is filled with endolymph. It is thought that the endolymph is formed by the portion of the spiral ligament that faces the cochlear duct, the stria vascularis (StV ). This is highly

1	labyrinth and is filled with endolymph. It is thought that the endolymph is formed by the portion of the spiral ligament that faces the cochlear duct, the stria vascularis (StV ). This is highly vascularized and contains specialized “secretory” cells.

1	A shelf of bone, the osseous spiral lamina (OSL), extends from the modiolus to the basilar membrane. Branches of the cochlear nerve (CN) travel along the spiral lamina to the modiolus, where the main trunk of the nerve is formed. The components of the cochlear nerve are bipolar neurons whose cell bodies constitute the spiral ganglion (SG). These cell bodies are shown at higher magnification in the inset (upper right). The spiral lamina supports an elevation of cells, the limbus spiralis (LS). The surface of the limbus is composed of columnar cells. Organ of Corti, ear, guinea pig, H&E 180; inset 380.

1	Organ of Corti, ear, guinea pig, H&E 180; inset 380. The components of the organ of Corti, beginning at the limbus spiralis (LS), are as follows: inner border cells (IBC ); inner phalangeal and hair cells (IP&HC); inner pillar cells (IPC ); (the sequence continues, repeating itself in reverse) outer pillar cells (OPC ); hair cells (HC ) and outer phalangeal cells (OP); and outer border cells or cells of Hensen (CH ). Hair cells are receptor cells; the other cells are collectively referred to as supporting cells. The hair and outer phalangeal cells can be distinguished in this figure by their location (see inset) and because their nuclei are well aligned. Because the hair cells rest on the phalangeal cells, it can be concluded that the upper three nuclei belong to outer hair cells, whereas the lower three nuclei belong to outer phalangeal cells.

1	The supporting cells extend from the basilar membrane (BM) to the surface of the organ of Corti (this is not evident here but can be seen in the inset), where they form a reticular membrane (RM). The free surface of the receptor cells fits into openings in the reticular membrane, and the “hairs” of these cells project toward, and make contact with, the tectorial membrane (TM ). The latter is a cuticular extension from the columnar cells of the limbus spiralis. In ideal preparations, nerve fibers can be traced from the hair cells to the cochlear nerve (CN). In their course from the basilar membrane to the reticular membrane, groups of supporting cells are separated from other groups by spaces that form spiral tunnels. These tunnels are named the inner tunnel (IT ), the outer tunnel (OT ), and the internal spiral tunnel (IST ). Beyond the supporting cells are two additional groups of cells, the cells of Claudius (CC) and the cells of Btcher (CB).

1	KEY BM, basilar membrane CB, cells of Btcher CC, cells of Claudius CD, cochlear duct CH, cells of Hensen CN, cochlear nerve HC, hair cells IBC, inner border cells IPHC, inner phalangeal and hair cells IPC, inner pillar cells IST, internal spiral tunnel IT, inner tunnel LS, limbus spiralis OP, outer phalangeal cells OPC, outer pillar cells OSL, osseous spiral lamina OT, outer tunnel RM, reticular membrane SG, spiral ganglion SL, spiral ligament ST, scala tympani StV, stria vascularis SV, scala vestibuli TM, tectorial membrane VM, vestibular membrane

1	We are pleased that the following section authors have continued as members of the seventh edition team: Drs. Kalman Rubinson and Eric Lang (nervous system), Dr. James Watras (muscle), Dr. Achilles Pappano (cardiovascular system), Drs. Michelle Cloutier and Roger Thrall (respiratory system), Drs. Kim Barrett and Helen Raybould (gastrointestinal system), and Dr. Bruce White (endocrine and reproductive systems). We also welcome the following authors: Dr. Withrow Gil Wier (cardiovascular system), and Dr. John Harrison (endocrine and reproduction systems).

1	As in the previous editions of this textbook, we have attempted to emphasize broad concepts and to minimize the compilation of isolated facts. Each chapter has been written to make the text as lucid, accurate, and current as possible. We have included both clinical and molecular information in each section, as feedback on these features has indicated that this information serves to provide clinical context and new insights into physiologic phenomena at the cellular and molecular levels. New to this edition is a list of sources that the reader can consult for further information on the topics covered in each chapter. We hope that you find this a valuable addition to the book.

1	The human body consists of billions of cells that are organized into tissues (e.g., muscle, epithelia, and nervous tissue) and organ systems (e.g., nervous, cardiovascular, respiratory, renal, gastrointestinal, endocrine, and reproductive). For these tissues and organ systems to function properly and thus allow humans to live and carry out daily activities, several general conditions must be met. First and foremost, the cells within the body must survive. Survival requires adequate cellular energy supplies, maintenance of an appropriate intracellular milieu, and defense against a hostile external environment. Once cell survival is ensured, the cell can then perform its designated or specialized function (e.g., contraction by skeletal muscle cells). Ultimately, the function of cells, tissues, and organs must be coordinated and regulated. All of these functions are the essence of the discipline of physiology and are presented throughout this book. What follows is a brief introduction to

1	and organs must be coordinated and regulated. All of these functions are the essence of the discipline of physiology and are presented throughout this book. What follows is a brief introduction to these general concepts.

1	Cells need a constant supply of energy. This energy is derived from the hydrolysis of adenosine triphosphate (ATP). If not replenished, the cellular ATP supply would be depleted in most cells in less than 1 minute. Thus, ATP must be continuously synthesized. This in turn requires a steady supply of cellular fuels. However, the cellular fuels (e.g., glucose, fatty acids, and ketoacids) are present in the blood at levels that can support cellular metabolism only for a few minutes. The blood levels of these cellular fuels are maintained through the ingestion of precursors (i.e., carbohydrates, proteins, and fats). In addition, these fuels can be stored and then mobilized when ingestion of the precursors is not possible. The storage forms of these fuels are triglycerides (stored in adipose tissue), glycogen (stored in the liver and skeletal muscle), and protein. The maintenance of adequate levels of cellular fuels in the blood is a complex process involving the following tissues, organs,

1	glycogen (stored in the liver and skeletal muscle), and protein. The maintenance of adequate levels of cellular fuels in the blood is a complex process involving the following tissues, organs, and organ systems:

1	Liver: Converts precursors into fuel storage forms (e.g., glucose → glycogen) when food is ingested, and converts storage forms to cellular fuels during fasting (e.g., glycogen → glucose and amino acids → glucose). Skeletal muscle: Like the liver, stores fuel (glycogen and protein) and converts glycogen and protein to fuels (e.g., glucose) or fuel intermediates (e.g., protein → amino acids) during fasting. Gastrointestinal tract: Digests and absorbs fuel precursors. Adipose tissue: Stores fuel during feeding (e.g., fatty acids → triglycerides) and releases the fuels during fasting. Cardiovascular system: Delivers the fuels to the cells and to and from their storage sites. Endocrine system: Maintains the blood levels of the cellular fuels by controlling and regulating their storage and their release from storage (e.g., insulin and glucagons).

1	Endocrine system: Maintains the blood levels of the cellular fuels by controlling and regulating their storage and their release from storage (e.g., insulin and glucagons). Nervous system: Monitors oxygen levels and nutrient content in the blood and, in response, modulates the cardiovascular, pulmonary, and endocrine systems and induces feeding and drinking behaviors. In addition to energy metabolism, the cells of the body must maintain a relatively constant intracellular environment to survive. This includes the uptake of fuels needed to produce ATP, the export from the cell of cellular wastes, the maintenance of an appropriate intracellular ionic environment, the establishment of a resting membrane potential, and the maintenance of a constant cellular volume. All of these functions are carried out by specific membrane transport proteins.

1	The composition of the extracellular fluid (ECF) that bathes the cells must also be maintained relatively constant. In addition, the volume and temperature of the ECF must be regulated. Epithelial cells in the lungs, gastrointestinal tract, and kidneys are responsible for maintaining the volume and composition of the ECF, while the skin plays a major role in temperature regulation. On a daily basis, H2O and food are ingested, and essential components are absorbed across the epithelial cells of the gastrointestinal tract. This daily intake of solutes and water must be matched by excretion from the body, thus maintaining steady-state balance. The kidneys are critically involved in the maintenance of steady-state balance for water and many components of the ECF (e.g., Na+ , K+ , HCO3 − , pH, Ca++ , organic solutes). The lungs ensure an adequate supply of O2 to “burn” the cellular fuels for the production of ATP and excrete the major waste product of this process (i.e., CO2). Because CO2

1	Ca++ , organic solutes). The lungs ensure an adequate supply of O2 to “burn” the cellular fuels for the production of ATP and excrete the major waste product of this process (i.e., CO2). Because CO2 can affect the pH of the ECF, the lungs work with the kidneys to maintain ECF pH.

1	Because humans inhabit many different environments and often move between environments, the body must be able to rapidly adapt to the challenges imposed by changes in ambient temperature and availability of food and water. Such adaptation requires coordination of the function of cells in different tissues and organs as well as their regulation. The nervous and endocrine systems coordinate and regulate cell, tissue, and organ function. The regulation of function can occur rapidly (seconds to minutes), as is the case for levels of cellular fuels in the blood, or over much longer periods of time (days to weeks), as is the case for acclimatization when an individual moves from a cool to a hot environment or changes from a high-salt to a low-salt diet.

1	The function of the human body represents complex processes at multiple levels. This book explains what is currently known about these processes. Although the emphasis is on the normal function of the human body, discussion of disease and abnormal function is also appropriate, as these often illustrate physiologic processes and principles at the extremes. The authors for each section have presented what they believe to be the most likely mechanisms responsible for the phenomena under consideration. We have adopted this compromise to achieve brevity, clarity, and simplicity. Bruce M. Koeppen, MD, PhD Bruce A. Stanton, PhD 29TheGastricPhaseoftheIntegratedResponsetoaMeal,529 30TheSmallIntestinalPhaseoftheIntegratedResponsetoaMeal,541 31TheColonicPhaseoftheIntegratedResponsetoaMeal,559 32TransportandMetabolicFunctionsoftheLiver,568 Section 7: The Renal System, 580 37RoleoftheKidneysintheRegulationofAcid-BaseBalance,670 Section 8: The Endocrine and Reproductive Systems, 685

1	Section 7: The Renal System, 580 37RoleoftheKidneysintheRegulationofAcid-BaseBalance,670 Section 8: The Endocrine and Reproductive Systems, 685 SECTION 1Cellular Physiology BRUCE M. KOEPPEN AND BRUCE A. STANTON Upon completion of this chapter, the student should be able to answer the following questions: 1. What organelles are found in a typical eukaryotic cell, and what is their function? 2. What is the composition of the plasma membrane? 3. What are the major classes of membrane transport proteins, and how do they transport biologically important molecules and ions across the plasma membrane? 4. What is the electrochemical gradient, and how it is used to determine whether the transport of a molecule or ion across the plasma membrane is active or passive? 5. What are the driving forces for movement of water across cell membrane and the capillary wall?

1	5. What are the driving forces for movement of water across cell membrane and the capillary wall? In addition, the student should be able to define and understand the following properties of physiologically important solutions and fluids: he human body is composed of billions of cells. Although cells can perform different functions, they share certain common elements. This chapter provides an overview of these common elements and focuses on the important function of the transport of molecules and water into and out of the cell across its plasma membrane. Overview of Eukaryotic Cells

1	Overview of Eukaryotic Cells Eukaryotic cells are distinguished from prokaryotic cells by the presence of a membrane-delimited nucleus. With the exception of mature human red blood cells and cells within the lens of the eye, all cells within the human body contain a nucleus. The cell is therefore effectively divided into two compartments: the nucleus and the cytoplasm. The cytoplasm is an aqueous solution containing numerous organic molecules, ions, cytoskeletal elements, and a number of organelles. Many of the organelles are membrane-enclosed compartments that carry out specific cellular function. An idealized eukaryotic cell is depicted in Fig. 1.1 , and the primary function of some components and compartments of the cell are summarized in . Readers who desire a more in-depth presentation of this material are encouraged to consult one of the many textbooks on cell and molecular biology that are currently available. The Plasma Membrane

1	The Plasma Membrane The cells within the body are surrounded by a plasma membrane that separates the intracellular contents from the extracellular environment. Because of the properties of this membrane and, in particular, the presence of specific membrane proteins, the plasma membrane is involved in a number of important cellular functions, including the following: Selective transport of molecules into and out of the cell. A function carried out by membrane transport proteins. Cell recognition through the use of cell surface antigens. Cell communication through neurotransmitter and hormone receptors and through signal transduction pathways. Tissue organization, such as temporary and permanent cell junctions, and interaction with the extracellular matrix, with the use of a variety of cell adhesion molecules. Membrane-dependent enzymatic activity.

1	Membrane-dependent enzymatic activity. Determination of cell shape by linkage of the cytoskeleton to the plasma membrane. In this chapter, the structure and function of the plasma membrane of eukaryotic cells are considered. More specifically, the chapter focuses on the transport of molecules and water across the plasma membrane. Only the principles of membrane transport are presented here. Additional details that relate to specific cells are presented in the various sections and chapters of this book. The plasma membrane of eukaryotic cells consists of a 5-nm-thick lipid bilayer with associated proteins ( Fig. 1.2 ). Some of the membrane-associated proteins are integrated into the lipid bilayer; others are more loosely attached to the CHAPTER 1 Principles of Cell and Membrane Function 3 • Fig. 1.1 Schematic drawing of a eukaryotic cell. The top portion of the cell is omitted to illustrate the nucleus and various intracellular organelles. See text for details.

1	Primary Functions of Some Eukaryotic Cellular Components and Compartments 1.1 CytosolMetabolism, protein synthesis (free ribosomes)CytoskeletonCell shape and movement, intracellular transportNucleusGenome (22 autosomes and 2 sex chromosomes), DNA and RNA synthesisMitochondriaATP synthesis by oxidative phosphorylation, Ca2+ storageSmooth endoplasmic reticulumSynthesis of lipids, Ca2+ storageFree ribosomesTranslation of mRNA into cytosolic proteinsRough endoplasmic reticulumTranslation of mRNA into membrane associated proteins or for secretion out of the cellLysosomeIntracellular degradationEndosomeCellular uptake of cholesterol, removal of receptors from the plasma membrane, uptake of small molecules and water into the cell, internalization of large particles (e.g., bacteria, cell debris)Golgi apparatusModification, sorting, and packaging of proteins and lipids for delivery to other organelles within ATP, adenosine triphosphate; mRNA, messenger RNA.

1	ATP, adenosine triphosphate; mRNA, messenger RNA. inner or outer surfaces of the membrane, often by binding molecules that contain a charged (or polar) hydrophilic to the integral membrane proteins. head and two (nonpolar) hydrophobic fatty acyl chains ( Fig. 1.3 ). The amphipathic nature of the phospholipid molecule Membrane Lipids is critical for the formation of the bilayer: The hydrophobic The major lipids of the plasma membrane are phospholipids fatty acyl chains form the core of the bilayer, and the polar and phosphoglycerides. Phospholipids are amphipathic head groups are exposed on the surface. • Fig. 1.2 Schematic diagram of the cell plasma membrane. Not shown are lipid rafts. See text for details. (Modified from Cooper GM. The Cell—A Molecular Approach. 2nd ed. Washington, DC: Sinauer; 2000, Fig. 12.3.) Phospholipid Glycolipid (e.g., phosphatidylcholine) (e.g., galactosylceramide) Cholesterol

1	Phospholipid Glycolipid (e.g., phosphatidylcholine) (e.g., galactosylceramide) Cholesterol Sugar (e.g., galactose) • Fig. 1.3 Models of the major classes of plasma membrane lipids, depicting the hydrophilic and hydrophobic regions of the molecules. The molecules are arranged as they exist in one leaflet of the bilayer. The opposing leaflet is not shown. One of the fatty acyl chains in the phospholipid molecule is unsaturated. The presence of this double bond produces a “kink” in the fatty acyl chain, which prevents tight packing of membrane lipids and increases membrane fluidity. (Modified from Hansen JT, Koeppen BM: Netter’s Atlas of Human Physiology. Teterboro, NJ: Icon Learning Systems; 2002.)

1	The majority of membrane phospholipids have a glycerol carbons in length and may be saturated or unsaturated (i.e., “backbone” to which are attached the fatty acyl chains, contain one or more double bonds). and an alcohol is linked to glycerol via a phosphate group. The phospholipid composition of the membrane The common alcohols are choline, ethanolamine, serine, varies among different cell types and even between the inositol, and glycerol. Another important phospholipid, bilayer leaflets. For example, in the erythrocyte plasma sphingomyelin, has the amino alcohol sphingosine as its membrane, phosphatidylcholine and sphingomyelin are “backbone” instead of glycerol. lists these common found predominantly in the outer leaflet of the membrane, phospholipids. The fatty acyl chains are usually 14 to 20 whereas phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol are found in the inner leaflet.

1	, phosphatidylinositol plays an important role in signal transduction, and its location in the inner leaflet of the membrane facilitates this signaling role. The sterol molecule cholesterol is also a critical component of the bilayer (see Fig. 1.3 ). It is found in both leaflets and serves to stabilize the membrane at normal body temperature (37°C). As much as 50% of the lipids found in the membrane can be cholesterol. A minor lipid component of the plasma membrane is glycolipids. These lipids, as their name indicates, consist of two fatty acyl chains linked to polar head groups that consist of carbohydrates (see Fig.

1	Fig. 1.3 ). As discussed in the section on , one glycolipid, glycosylphosphatylinositol (GPI), plays an important role in anchoring proteins to the outer leaflet of the membrane. Both cholesterol and glycolipids, like the phospholipids, are amphipathic, and they are oriented with their polar groups on the outer surface of the leaflet in which they are located. Their hydrophobic portion is thus located within the interior of the bilayer. The lipid bilayer is not a static structure. The lipids and associated proteins can diffuse within the plane of the membrane. The fluidity of the membrane is determined by temperature and by its lipid composition. As temperature increases, the fluidity of the membrane increases. The presence of unsaturated fatty acyl chains in the phospholipids and glycolipids also increases membrane fluidity. If a fatty acyl chain is unsaturated, the presence of a double bond introduces a “kink” in the molecule (see Fig. 1.3).

1	Fig. 1.3). This kink prevents the molecule from associating closely with surrounding lipids, and, as a result, membrane fluidity is increased. Although the lipid bilayer is “fluid,” movement of proteins in the membrane can be constrained or limited. For example, membrane proteins can be anchored to components of the intracellular cytoskeleton, which limits their movement. Membrane domains can also be isolated from one another. An important example of this can be found in epithelial tissues. Junctional complexes (e.g., tight junctions) separate the plasma membrane of epithelial cells into two domains: apical and basolateral (see ). The targeted localization of membrane proteins into one or other of these domains allows epithelial cells to carry

1	CHAPTER 1 Principles of Cell and Membrane Function out vectorial transport of substances from one side of the epithelium to the opposite side. The ability to carry out vectorial transport is crucial for the functioning of several organ systems (e.g., the gastrointestinal tract and kidneys). In addition, some regions of the membrane contain lipids (e.g., sphingomyelin and cholesterol) that aggregate into what are called lipid rafts. These lipid rafts often have an association with specific proteins, which diffuse in the plane of the membrane as a discrete unit. Lipid rafts appear to serve a number of functions. One important function of these rafts is to segregate signaling molecules. As much as 50% of the plasma membrane is composed of proteins. These membrane proteins are classified as integral, lipid-anchored, or peripheral.

1	As much as 50% of the plasma membrane is composed of proteins. These membrane proteins are classified as integral, lipid-anchored, or peripheral. Integral membrane proteins are imbedded in the lipid bilayer, where hydrophobic amino acid residues are associated with the hydrophobic fatty acyl chains of the membrane lipids. Many integral membrane proteins span the bilayer; such proteins are termed transmembrane proteins. Transmembrane proteins have both hydrophobic and hydrophilic regions. The hydrophobic region, often in the form of an α helix, spans the membrane. Hydrophilic amino acid residues are then exposed to the aqueous environment on either side of the membrane. Transmembrane proteins may pass through the membrane multiple times.

1	Thereisasuperfamilyofmembraneproteinsthatserveasreceptorsformanyhormones,neurotransmitters,andnumerousdrugs.ThesereceptorsarecoupledtoheterotrimericGproteinsandaretermedG protein–coupled receptors (see ).Theseproteinsspanthemembranewithsevenα-helicaldomains.Thebindingsiteofeachligandiseitherontheextracellularportionoftheprotein(largeligands)orinthemembrane-spanningportion(smallligands),whereasthecytoplasmicportionbindstotheGprotein.Thissuperfamilyofmembraneproteinsmakesupthethirdlargestfamilyofgenesinhumans.NearlyhalfofallnonantibioticprescriptiondrugsaretargetedtowardGprotein–coupledreceptors.

1	A protein can also be attached to the membrane via lipid anchors. The protein is covalently attached to a lipid molecule, which is then embedded in one leaflet of the bilayer. Glycosylphosphatidylinositol (GPI) anchors proteins to the outer leaflet of the membrane. Proteins can be attached to the inner leaflet via their amino-terminus by fatty acids (e.g., myristate or palmitate) or via their carboxyl-terminus by prenyl anchors (e.g., farnesyl or geranylgeranyl). Peripheral proteins may be associated with the polar head groups of the membrane lipids, but they more commonly bind to integral or lipid-anchored proteins.

1	Peripheral proteins may be associated with the polar head groups of the membrane lipids, but they more commonly bind to integral or lipid-anchored proteins. In many cells, some of the outer leaflet lipids, as well as many of the proteins exposed on the outer surface of the membrane, are glycosylated (i.e., have short chains of sugars, called oligosaccharides, attached to them). Collectively, these glycolipids and glycoproteins form what is called the glycocalyx. Depending on the cell these glycolipids and glycoproteins may be involved in cell recognition (e.g., cell surface antigens) and formation of cell-cell interactions (e.g., attachment of neutrophils to vascular endothelial cells).

1	Although plasma membrane proteins perform many important cellular functions, as noted previously, the remainder of this chapter focuses on one group of plasma membrane proteins: the membrane transport proteins, or transporters. It has been estimated that approximately 10% of human genes (≈2000) code for transporters. They are also targets for numerous drugs. The normal function of cells requires the continuous movement of water and solutes into and out of the cell. The intracellular and extracellular fluids are composed primarily of H2O, in which solutes (e.g., ions, glucose, amino acids) are dissolved. The plasma membrane, with its hydrophobic core, is an effective barrier to the movement of virtually all of these biologically important solutes. It also restricts the movement of water across the membrane. The presence of specific membrane transporters in the membrane is responsible for the movement of these solutes and water across the membrane.

1	Membrane transporters have been classified in several different ways. In this chapter, the transporters are divided into four general groups: water channels, ion channels, solute carriers, and adenosine triphosphate (ATP)–dependent transporters. lists these groups of membrane transporters, their modes of transport, and estimates of the rates at which they transport molecules or ions across the membrane.

1	lists these groups of membrane transporters, their modes of transport, and estimates of the rates at which they transport molecules or ions across the membrane. Water channels, or aquaporins (AQPs), are the main routes for water movement into and out of the cell. They are widely distributed throughout the body (e.g., the brain, lungs, kidneys, salivary glands, gastrointestinal tract, and liver). Cells express different AQP isoforms, and some cells even express multiple isoforms. For example, cells in the collecting ducts of the kidneys express AQP3 and AQP4 in their basolateral membrane and AQP2 in their apical membrane. Moreover, the abundance of AQP2 in the apical membrane is regulated by antidiuretic hormone (also called arginine vasopressin), which is crucial for the ability of the kidneys to concentrate the urine (see

1	Although all AQP isoforms allow the passive movement of H2O across the membrane, some isoforms also provide a pathway for other molecules such as glycerol, urea, mannitol, purines, pyrimidines, CO2, and NH3 to cross the membrane. Because glycerol was one of the first molecules identified as crossing the membrane via some AQPs, this group of AQPs is collectively called aquaglyceroporins (see also ). Regulation of the amount of H2O that can enter or leave the cell via AQPs occurs primarily by altering the number of AQPs in the membrane. EachAQPmoleculeconsistsofsixmembrane-spanningdomainsandacentralwater-transportingpore.FourAQPmonomersassembletoformahomotetramerintheplasmamembrane,witheachmonomerfunctioningasawaterchannel.

1	Ion channels are found in all cells, and are especially important for the function of excitable cells (e.g., neurons and muscle cells). Ion channels are classified by their selectivity, conductance and mechanism of channel gating (i.e., opening and closing). Selectivity is defined as the nature of the ions that pass through the channel. At one extreme, ion channels can be highly selective, in that they allow only a specific ion through. At the other extreme, they may be nonselective, allowing all or a group of cations or anions through. Channel conductance refers to the number of ions that pass through the channel and is typically expressed in picosiemens (pS). The range of conductance is considerable: Some channels have a conductance of only 1 to 2 pS, whereas others have a conductance of more than 100 pS. For some channels, the conductance varies, depending on the direction in which the ion is moving. For example, if the channel has a larger conductance when ions are moving into the

1	more than 100 pS. For some channels, the conductance varies, depending on the direction in which the ion is moving. For example, if the channel has a larger conductance when ions are moving into the cell than when they are moving out of the cell, the channel is said to be an inward rectifier. Moreover, ion channels fluctuate between an open state or a closed state, a process called gating (

1	Fig. 1.4 ). Factors that can control gating include membrane voltage, extracellular agonists or antagonists (e.g., acetylcholine is an extracellular agonist mitochondria,andwaterchannels(i.e.,aquaporins)thatfunctionas that controls the gating of a cation-selective channel in the apore. ATP,adenosinetriphosphate.motor end plate of skeletal muscle cells; see Chapter 6), intracellular messengers (e.g., Ca++ , ATP, cyclic guanosine CHAPTER 1 Principles of Cell and Membrane Function •Fig. 1.4 Recordingofcurrentflowthroughasingleionchannel.Thechannelspontaneouslyfluctuatesbetweenanopenstateandaclosedstate.Theamplitudeofthecurrentisapproximately2pA(2× 10−12amps);thatis,12.5millionions/secondcrossthemembrane. monophosphate), and mechanical stretch of the plasma membrane. Ion channels can be regulated by a change in the number of channels in the membrane or by gating of the channels.

1	Solute carriers (denoted SLCs by the HUGO Gene Nomenclature Committee) represent a large group of membrane transporters categorized into more than 50 families; almost 400 specific transporters have been identified to date. These carriers can be divided into three groups according to their mode of transport. One group, uniporters (or facilitated transporters), transports a single molecule across the membrane. The transporter that brings glucose into the cell (glucose transporter 1 [GLUT-1], or SLC2A1) is an important member of this group. The second group, symporters (or cotransporters), couples the movement of two or more molecules/ions across the membrane. As the name implies, the molecules/ions are transported in the same direction. The Na+,K+,2Cl− (NKCC) symporter found in the kidney (NKCC2, or SLC12A1), which is crucial for diluting and concentrating the urine (see ), is a member of this group. The third group, antiporters (or exchange transporters), also couples the movement of

1	(NKCC2, or SLC12A1), which is crucial for diluting and concentrating the urine (see ), is a member of this group. The third group, antiporters (or exchange transporters), also couples the movement of two or more molecules/ions across the membrane; in this case, however, the molecules/ions are transported in opposite directions. The Na+-H+ antiporter is a member of this group of solute carriers. One isoform of this antiporter (NHE-1, or SLC9A1) is found in all cells and plays an important role in regulating intracellular pH.

1	The ATP-dependent transporters, as their name implies, use the energy in ATP to drive the movement of molecules/ ions across the membrane. There are two groups of ATP-dependent transporters: the ATPase ion transporters and the ATP-binding cassette (ABC) transporters. The ATPase ion transporters are subdivided into P-type ATPases and V-type ATPases. The P-type ATPases are phosphorylated during the transport cycle. Na+,K+-ATPase is an important example of a P-type ATPase. With the hydrolysis of each ATP molecule, it transports three Na+ ions out of the cell and two K+ ions into the cell. Na+,K+-ATPase is present in all cells and plays a critical role in establishing cellular ion and electrical gradients, as well as maintaining cell volume (see

1	V-type H+-ATPases are found in the membranes of several intracellular organelles (e.g., endosomes, lysosomes); as a result, they are also referred to as vacuolar H+-ATPases. The aAnother type of ATPases, F-type ATPases, is found in the mitochondria, and they are responsible for ATP synthesis. They are not considered in this chapter.

1	Na+,K+-ATPase(alsocalledtheNa+,K+-pump orjusttheNa+pump)isfoundinallcellsandisresponsibleforestablishingthegradientsofNa+ andK+ acrosstheplasmamembrane.Thesegradientsinturnprovideenergyforseveralessentialcellfunctions(see ).Na+,K+-ATPaseiscomposedofthreesubunits(α,β,andγ),andtheproteinexistsinthemembranewithastoichiometriccompositionof1α,1β,1γ.Theα subunitcontainsbindingsitesforNa+,K+ andATP.Itisalsothesubunitthatbindscardiacglycosides(e.g.,ouabain),whichspecificallyinhibittheenzyme.Ithasatransmembranedomainandthreeintracellulardomains:phosphorylation(P-domain),nucleotidebinding(N-domain),andactuator(A-domain).Althoughtheα subunitisthefunctionalsubunitoftheenzyme(i.e.,ithydrolyzesATP,bindsNa+ andK+ ,andtranslocatesthemacrossthemembrane),itcannotfunctionwithouttheβ subunit.Theβ subunitisresponsiblefortargetingtheα subunittothemembraneandalsoappearstomodulatetheaffinityoftheNa+,K+-ATPaseforNa+ andK+ .Theα andβ subunitscancarryoutNa+ andK+ transportintheabsenceoftheγ subunit.However,theγ

1	subunittothemembraneandalsoappearstomodulatetheaffinityoftheNa+,K+-ATPaseforNa+ andK+ .Theα andβ subunitscancarryoutNa+ andK+ transportintheabsenceoftheγ subunit.However,theγ subunitappearstoplayaregulatoryrole.Theγ subunitisamemberofafamilyofproteinscalledFXYD proteins (sonamedfortheFXYDaminoacidsequencefoundintheprotein).

1	H+-ATPase in the plasma membrane plays an important role in urinary acidification (see ABC transporters represent a large group of membrane transporters. They are found in both prokaryotic and eukaryotic cells, and they have amino acid domains that bind ATP (i.e., ABC domains). Seven subgroups of ABC transporters in humans and more than 40 specific transporters have been identified to date. They transport a diverse group of molecules/ions, including Cl−, cholesterol, bile acids, drugs, iron, and organic anions.

1	Because biologically important molecules enter and leave cells through membrane transporters, membrane transport is specific and regulated. Although some membrane transporters are ubiquitously expressed in all cells (e.g., Na+,K+-ATPase), the expression of many other transporters is limited to specific cell types. This specificity of expression tailors the function of the cell to the organ system in which it is located (e.g., the sodium-glucose–linked transporters SGLT-1 and SGLT-2 in the epithelial cells of the intestines and renal proximal tubules). In addition, the amount of a molecule being transported across the membrane can be regulated. Such regulation can take place through altering the number of transporters in the membrane or altering the rate or kinetics of individual transporters (e.g., the time an ion channel stays in the open versus closed state), or both.

1	Solute and water can be brought into the cell through a process of endocytosis and released from the cell through the process of exocytosis. Endocytosis is the process whereby a piece of the plasma membrane pinches off and

1	Cystic fibrosis isanautosomalrecessivediseasecharacterizedbychroniclunginfections,pancreaticinsufficiency,andinfertilityinboysandmen.Deathusuallyoccursbecauseofrespiratoryfailure.Itismostprevalentinwhitepeopleandisthemostcommonlethalgeneticdiseaseinthispopulation,occurringin1per3000livebirths.Itisaresultofmutationsinageneonchromosome7thatcodesforanABCtransporter.Todate,morethan1000mutationsinthegenehavebeenidentified.Themostcommonmutationisadeletionofaphenylalanineatposition508(F508del).Becauseofthisdeletion,degradationoftheproteinbytheendoplasmicreticuluminenhanced,and,asaresult,thetransporterdoesnotreachtheplasmamembrane.Thistransporter,calledcystic fibrosis transmembrane conductance regulator (CFTR), normallyfunctionsasaCl− channelandalsoregulatesothermembranetransporters(e.g.,theepithelialNa+

1	fibrosis transmembrane conductance regulator (CFTR), normallyfunctionsasaCl− channelandalsoregulatesothermembranetransporters(e.g.,theepithelialNa+ channel[ENaC]).Thusinindividualswithcysticfibrosis,epithelialtransportisdefective,whichisresponsibleforthepathophysiologicprocess.Forexample,inpatientsnotaffectedbycysticfibrosis,theepithelialcellsthatlinetheairwayofthelungarecoveredwithalayerofmucusthatentrapsinhaledparticulatesandbacteria.Ciliaontheepithelialcellsthentransporttheentrappedmaterialoutofthelung,aprocesstermedmucociliary transport (see formoredetails).Inpatientswithcysticfibrosis,theinabilitytosecreteCl− ,Na+ ,andH2Oresultsinanincreaseintheviscosityoftheairwaysurfacemucus;thustheciliacannottransporttheentrappedbacteriaandotherpathogensoutofthelung.Thisinturnleadstorecurrentandchroniclunginfections.Theinflammatoryprocessthataccompaniestheseinfectionsultimatelydestroysthelungtissue,causingrespiratoryfailureanddeath.In2015,the

1	U.S.FoodandDrugAdministrationapprovedlumacaftor/ivacaftor(Orkambi),adrugthatincreasestheamountofF508delCFTRintheplasmamembraneoflungepithelialcells. is internalized into the cell interior, and exocytosis is the process whereby vesicles inside the cell fuse with the plasma membrane. In both of these processes, the integrity of the plasma membrane is maintained, and the vesicles allow for the transfer of the contents among cellular compartments. In some cells (e.g., the epithelial cells lining the gastrointestinal tract), endocytosis across one membrane of the cell is followed by exocytosis across the opposite membrane. This allows the transport of substances inside the vesicles across the epithelium, a process termed transcytosis.

1	Endocytosis occurs in three mechanisms. The first is pinocytosis, which consists of the nonspecific uptake of small molecules and water into the cell. Pinocytosis is a prominent feature of the endothelial cells that line capillaries and is responsible for a portion of the fluid exchange that occurs across these vessels. The second form of endocytosis, phagocytosis, allows for the cellular internalization of large particles (e.g., bacteria, cell debris). This process is an important characteristic of cells in the immune system (e.g., neutrophils and macrophages). Often, but not always, phagocytosis is a receptor-mediated process. For example, CHAPTER 1 Principles of Cell and Membrane Function 9

1	Proteins within the plasma membrane of cells are constantly being removed and replaced with newly synthesized proteins. As a result, membrane proteins are constantly being replaced. One mechanism by which membrane proteins are “tagged” for replacement is by the attachment of ubiquitin to the cytoplasmic portion of the protein. Ubiquitin is a 76–amino acid protein that is covalently attached to the membrane protein (usually to lysine) by a class of enzymes called ubiquitin protein

1	protein ligases. One important group of these ligases is the developmentally downregulated protein 4 (Nedd4)/Nedd4-like family. Once a membrane protein is ubiquitinated, it undergoes endocytosis and is degraded either by lysosomes or by the proteosome. Cells also contain deubiquitinating enzymes (DUBs). Thus the amount of time a protein stays in the plasma membrane depends on the rate that ubiquitin groups are added by the ligases versus the rate that they are removed by the DUBs. For example, Na+ reabsorption by the collecting ducts of the kidneys is stimulated by the adrenal hormone aldosterone (see ). One of the actions of aldosterone is to inhibit Nedd4-2. This prevents ubiquitination of ENaC in the apical membrane of epithelial cells. Thus the channels are retained for a longer period of time in the membrane, and as a result, more Na+ enters the cell and is thereby reabsorbed.

1	macrophages have receptors on their surface that bind the Fc portion of immunoglobulins. When bacteria invade the body they are often coated with antibody, a process called opsonization. These bacteria then attach to the membrane of macrophages via the fragment crystallizable (Fc) portion of the immunoglobulin, undergo phagocytosis, and are destroyed inside the cell. The third mechanism of endocytosis is receptor-mediated endocytosis, which allows the uptake of specific molecules into the cell. In this form of endocytosis, molecules bind to receptors on the surface of the cell. Endocytosis involves a number of accessory proteins, including adaptin, clathrin, and the GTPase dynamin ( Fig. 1.5

1	Exocytosis can be either constitutive or regulated. Constitutive secretion occurs, for example, in plasma cells that are secreting immunoglobulin or in fibroblasts secreting collagen. Regulated secretion occurs in endocrine cells, neurons, and exocrine glandular cells (e.g., pancreatic acinar cells). In these cells, the secretory product (e.g., hormone, neurotransmitter, or digestive enzyme), after synthesis and processing in the rough endoplasmic reticulum and Golgi apparatus, is stored in the cytoplasm in secretory granules until an appropriate signal for secretion is received. These signals may be hormonal or neural. Once the cell receives the appropriate stimulus, the secretory vesicle fuses with the plasma membrane and releases its contents into the extracellular fluid. Fusion of the vesicle with the membrane is mediated by a number of accessory proteins. One important group is the SNARE (soluble N-ethylmaleimide sensitive fusion protein [NSF] attachment protein receptors)

1	of the vesicle with the membrane is mediated by a number of accessory proteins. One important group is the SNARE (soluble N-ethylmaleimide sensitive fusion protein [NSF] attachment protein receptors) proteins. These membrane proteins help target the secretory

1	Receptor Formation of Formation of coated pit coated vesicle Dynamin Uncoated vesicle ready to fuse Vesicle (e.g., lysosome) uncoating • Fig. 1.5 Receptor-mediated endocytosis. Receptors on the surface of the cell bind the ligand. A clathrin-coated pit is formed with adaptin linking the receptor molecules to clathrin. Dynamin, a guanosine triphosphatase (GTPase), assists in separation of the endocytic vesicle from the membrane. Once inside the cell, the clathrin and adaptin molecules dissociate and are recycled. The uncoated vesicle is then ready to fuse with other organelles in the cell (e.g., lysosomes). (Adapted from Ross MH, Pawlina W: Histology. 5th ed. Baltimore: Lippincott Williams & Wilkins; 2006.)

1	Cholesterol is an important component of cells (e.g., it is a key component of membranes). However, most cells are unable to synthesize cholesterol and therefore must obtain it from the blood. Normally, cholesterol is ingested in the diet, and it is transported through the blood in association with lipoproteins. Low-density lipoproteins (LDLs) in the blood carry cholesterol to cells, where they bind to LDL receptors in the plasma membrane. After the receptors bind LDL, they collect into “coated pits” and undergo endocytosis as clathrin-coated vesicles. Once inside the cell, the endosomes release LDL and then recycle the LDL receptors back to the cell surface. Inside the cell, LDL is then degraded in lysosomes, and the cholesterol is made available to the cell. Defects in the LDL receptor prevent cellular uptake of LDL. Individuals with this defect have elevated levels of blood LDL, often called “bad cholesterol,” because it is associated with the development of cholesterol-containing

1	prevent cellular uptake of LDL. Individuals with this defect have elevated levels of blood LDL, often called “bad cholesterol,” because it is associated with the development of cholesterol-containing plaques in the smooth muscle layer of arteries. This process, atherosclerosis, is associated with an increased risk for heart attacks as a result of occlusion of the coronary arteries.

1	vesicle to the plasma membrane. The process of secretion is usually triggered by an increase in the concentration of intracellular Ca++ ([Ca++]). However, two notable exceptions to this general rule exist: (1) Renin secretion by the juxtaglomerular cells of the kidney occurs with a decrease in intracellular Ca++ (see ), as does (2) the secretion of parathyroid hormone by the parathyroid gland (see Basic Principles of Solute and Water Transport

1	Basic Principles of Solute and Water Transport As already noted, the plasma membrane, with its hydrophobic core, is an effective barrier to the movement of virtually all biologically important molecules into or out of the cell. Thus membrane transport proteins provide the pathway that allows transport to occur into and out of cells. However, the presence of a pathway is not sufficient for transport to occur; an appropriate driving force is also required. In this section, the basic principles of diffusion, active and passive transport, and osmosis are presented. These topics are discussed in greater depth, as appropriate, in the other sections of the book.

1	Diffusion is the process by which molecules move spontaneously from an area of high concentration to one of low concentration. Thus wherever a concentration gradient exists, diffusion of molecules from the region of high concentration to the region of low concentration dissipates the gradient (as discussed later, the establishment of concentration gradients for molecules requires the expenditure of energy). Diffusion is a random process driven by the thermal motion of the molecules. Fick’s first law of diffusion quantifies the rate at which a molecule diffuses from point A to point B: Equation 1.1 where J = the flux or rate of diffusion per unit time D = the diffusion coefficient A = area across which the diffusion is occurring ΔC = the concentration difference between point A and B ΔX = the distance along which diffusion is occurring

1	The diffusion coefficient takes into account the thermal energy of the molecule, its size, and the viscosity of the medium through which diffusion is taking place. For spherical molecules, D is approximated by the Stokes-Einstein equation: Equation 1.2 where k = Boltzmann’s constant T = temperature in degrees Kelvin r = radius of the molecule η= viscosity of the medium According to eqs. 1.1 and 1.2 , the rate of diffusion will be faster for small molecules than for large molecules. In addition, diffusion rates are high at elevated temperatures, in the presence of large concentration gradients, and when diffusion occurs in a low-viscosity medium. With all other variables held constant, the rate of diffusion is linearly related to the concentration gradient.

1	Fick’s equation can also be applied to the diffusion of molecules across a barrier, such as a lipid bilayer. When applied to the diffusion of a molecule across a bilayer, the diffusion coefficient (D) incorporates the properties of the bilayer and especially the ability of the molecule to diffuse through the bilayer. To quantify the interaction of the molecule with the bilayer, the term partition coefficient (β) is used. For a molecule that “dissolves” equally in the fluid bathing the lipid bilayer (e.g., water) and in the lipid bilayer, β= 1. If the molecule dissolves more easily in the lipid bilayer, β> 1; and if it dissolves less easily in the lipid bilayer, β< 1. For a simple lipid bilayer, the more lipid soluble the molecule is, the larger the partition coefficient is, and thus the diffusion coefficient—therefore the rate of diffusion of the molecule across the bilayer—is greater. In this situation, ΔC represents the concentration difference across the membrane, A is the membrane

1	coefficient—therefore the rate of diffusion of the molecule across the bilayer—is greater. In this situation, ΔC represents the concentration difference across the membrane, A is the membrane area, and ΔX is the thickness of the membrane.

1	Another useful equation for quantitating the diffusion of molecules across the plasma membrane (or any membrane) is as follows: Equation 1.3 J =−PC( i − Co), where J = the flux or rate of diffusion across the membrane P = is the permeability coefficient Ci = concentration of the molecule inside the cell Co = the concentration of the molecule outside the cell This equation is derived from Fick’s equation ( eq. 1.1 ). P incorporates D, ΔX, A, and the partition coefficient (β). P is expressed in units of velocity (e.g., centimeters per second), and C the units of moles/cm3. Thus the units of flux are moles per square centimeter per second (mol/cm2/ sec). Values for P can be obtained experimentally for any molecule and bilayer.

1	As noted, the phospholipid portion of the plasma membrane represents an effective barrier to many biologically important molecules. Consequently, diffusion through the lipid phase of the plasma membrane is not an efficient process for movement of these molecules across the membrane. It has been estimated that for a cell 20 µm in diameter, with a plasma membrane composed only of phospholipids, dissipation of a urea gradient imposed across the membrane would take approximately 8 minutes. Similar gradients for glucose and amino acids would take approximately 14 hours to dissipate, whereas ion gradients would take years to dissipate.

1	As noted previously, the vast majority of biologically important molecules cross cell membranes via specific membrane transporters, rather than by diffusing through the lipid portion of the membrane. Nevertheless, eq. 1.3 can be and has been used to quantitate the diffusion of molecules across many biological membranes. When this is done, the value of the permeability coefficient (P) reflects the properties of the pathway (e.g., membrane transporter or, in some cases, multiple transporters) that the molecule uses to cross the membrane.

1	Despite the limitations of using diffusion to describe and understand the transport of molecules across cell membranes, it is also important for understanding gas exchange in the lungs (see ), the movement of molecules through the cytoplasm of the cell, and the movement of molecules between cells in the extracellular fluid. For example, one of the physiological responses of skeletal muscle to exercise is the recruitment or opening of capillaries that are not perfused at rest. This opening of previously closed capillaries increases capillary density and thereby reduces the diffusion distance between the capillary and the muscle fiber so that oxygen and cellular fuels (e.g., fatty acids and glucose) can be delivered more quickly to the contracting muscle fiber. In resting muscle, the average distance of a muscle fiber from a capillary is estimated to be 40 µm. However, with exercise, this distance decreases to 20 µm or less.

1	The electrochemical gradient (also called the electrochemical potential difference) is used to quantitate the driving force acting on a molecule to cause it to move across a membrane. The electrochemical gradient for any molecule (Δµx) is calculated as follows: Equation 1.4 ˜µx = RTln + zFV ,[X]o xm where R = the gas constant T = temperature in degrees Kelvin Ln = natural logarithm [X]i = the concentration of X inside the cell [X]o = the concentration of X outside the cell zx = the valence of charged molecules F = the Faraday constant Vm = the membrane potential (Vm = Vi − Vo)

1	The electrochemical gradient is a measure of the free energy available to carry out the useful work of transporting the molecule across the membrane. It has two components: One component represents the energy in the concentration gradient for X across the membrane (chemical potential bBy convention, membrane voltages are determined and reported with regard to the exterior of the cell. In a typical cell, the resting membrane potential (Vm) is negative. Positive Vm values can be observed in some excitable cells at the peak of an action potential.

1	CHAPTER 1 Principles of Cell and Membrane Function difference). The second component (electrical potential difference) represents the energy associated with moving charged molecules (e.g., ions) across the membrane when a membrane potential exits (i.e., Vm ≠ 0 mV). Thus for the movement of glucose across a membrane, only the concentrations of glucose inside and outside of the cell need to be considered (Fig. 1.6A ). However, the movement of K+ across the membrane, for example, would be determined both from the K+ concentrations inside and outside of the cell and from the membrane voltage (see Fig. 1.6B Eq. 1.4 can be used to derive the Nernst equation for the situation in which a molecule is at equilibrium across the membrane (i.e., Δµ = 0): Equation 1.5a Equation 1.5b

1	Fig. 1.6B Eq. 1.4 can be used to derive the Nernst equation for the situation in which a molecule is at equilibrium across the membrane (i.e., Δµ = 0): Equation 1.5a Equation 1.5b The value of Vm calculated with the Nernst equation represents the equilibrium condition and is referred to as the Nernst equilibrium potential (Ex, the Vm at which there is no net transport of the molecule across the membrane). It should be apparent that the Nernst equilibrium potential quantitates the energy in a concentration gradient and expresses that energy in millivolts. For example, for the cell depicted in

1	Fig. 1.6B , the energy in the K+ gradient (derived from the Nernst equilibrium potential for K+ [EK+]) is proportional to 90.8 mV (causing K+ to move out of the cell). This is opposite to, and of greater magnitude than, the energy in the membrane voltage (Vm =−60 mV), which causes K+ to enter the cell. As a result, the electrochemical gradient is such that the net movement of K+ across the membrane will be out of the cell. Another way to state this is that the net driving force for K+ (Vm − EK+) is 30.8 mV (driving K+ out of the cell). This is described in more detail in The Nernst equation, at 37° C, can be written as follows by replacing the natural logarithm function with the base 10 logarithm function: Equation 1.6a 61 5. mV [X]i

1	The Nernst equation, at 37° C, can be written as follows by replacing the natural logarithm function with the base 10 logarithm function: Equation 1.6a 61 5. mV [X]i Equation 1.6b 615. mV [X]E = log o x zx [X]i •Fig. 1.6 Electrochemicalgradientsandcellulartransportofmolecules.A, Becauseglucoseisuncharged,theelectrochemicalgradientisdeterminedsolelybytheconcentrationgradientforglucoseacrossthecellmembrane.Asshown,theglucoseconcentrationgradientwouldbeexpectedtodriveglucoseintothecell.B, BecauseK+ ischarged,theelectrochemicalgradientisdeterminedbyboththeconcentrationgradientandthemembranevoltage(Vm).TheNernstequilibriumpotentialforK+ (EK+ ),calculatedwith eq.1.5a ,is−90.8mV(EK+= Vm atequilibrium).Theenergyintheconcentrationgradient,whichdrivesK+ outofthecell,isthusproportionalto+90.8mV.Themembranevoltageof−60mVdrivesK+ intothecell.Thustheelectrochemicalgradient,ornetdrivingforce,is2.97kJ/mol(equivalentto30.8mV),whichdrivesK+ outofthecell.

1	These are the most common forms of the Nernst equation in use. In these equations, it is apparent that for a univalent ion (e.g., Na+ , K+ , Cl−), a 10-fold concentration gradient across the membrane is equivalent in energy to an electrical potential difference of 61.5 mV (at 37° C), and a 100-fold gradient is equivalent to an electrical potential difference of 123 mV. Similarly, for a divalent ion (e.g., Ca++), a 10-fold concentration gradient is equivalent to a 30.7-mV electrical potential difference, because z in eqs. 1.6a and 1.6b is equal to 2. When the net movement of a molecule across a membrane occurs in the direction predicted by the electrochemical gradient, that movement is termed passive transport. Thus for the examples given in

1	When the net movement of a molecule across a membrane occurs in the direction predicted by the electrochemical gradient, that movement is termed passive transport. Thus for the examples given in Fig. 1.6 , the movement of glucose into the cell and the movement of K+ out of the cell would be considered passive transport. Transport that is passive is sometimes referred to as either “downhill transport” or “transport with the electrochemical gradient.” In contrast, if the net movement of a molecule across the membrane is opposite to that predicted by the electrochemical gradient, that movement is termed active transport, a process that requires the input of energy (e.g., ATP). Active transport is sometimes referred to as either “uphill transport” or “transport against the electrochemical gradient.”

1	In the various classes of plasma membrane transport proteins, the movement of H2O through water channels is a passive process (see later discussion), as is the movement of ions through ion channels and the transport of molecules via uniporters (e.g., transport of glucose via GLUT-1). The ATPase-dependent transporters can use the energy in ATP to drive active transport of molecules (e.g., Na+,K+-ATPase, H+-ATPase, or ABC transporters). Because the transport is directly coupled to the hydrolysis of ATP, it is referred to as primary active transport. Solute carriers that couple movement of two or more molecules (e.g., 3Na+,Ca++ antiporter) often transport one or more molecules (one Ca++ molecule in this example) against their respective electrochemical gradient through the use of the energy in the electrochemical gradient of the other molecule or molecules (three Na+ in this example). When this occurs, the molecule or molecules transported against their electrochemical gradient are said

1	in the electrochemical gradient of the other molecule or molecules (three Na+ in this example). When this occurs, the molecule or molecules transported against their electrochemical gradient are said to be transported by secondary active transport mechanisms (

1	Fig. 1.7 The movement of water across cell membranes occurs by the process of osmosis. The movement of water is passive, with the driving force for this movement being the osmotic pressure difference across the cell membrane. Fig. 1.8 illustrates the concept of osmosis and the measurement of the osmotic pressure of a solution. Osmotic pressure is determined by the number of solute molecules dissolved in the solution. It is not dependent on such factors as the size of the molecules, their mass, or their chemical nature (e.g., valence). Osmotic pressure (π), measured in atmospheres (atm), is calculated by van’t Hoff’s law as follows: Equation 1.7 ˜= nCRT, CHAPTER 1 Principles of Cell and Membrane Function

1	Equation 1.7 ˜= nCRT, CHAPTER 1 Principles of Cell and Membrane Function K+ Na+ 3Na+ Na+, K+ -ATPase 3Na+-Ca++ antiporter IntracellularconcentrationsExtracellularconcentrations3Na+ Na+: 145 mEq/L K+: 4 mEq/L Glucose: 5 mmol/L Ca++: 2.5 mEq/L (ionized) Na+: 12 mEq/L K+: 120 mEq/L Glucose: 2 mmol/L Ca++: 0.001 mEq/L (ionized) Ca++ Secondary active transport of Ca++ Primary active transport of Na+ and K+ Passive transport Na+ channel K+ channel Glucose uniporter Glucose 2K+ ATP •Fig. 1.7 Examplesofseveralmembranetransporters,illustratingprimaryactive,passive,andsecondaryactivetransport.Seetextfordetails.ATP,adenosinetriphosphate.

1	Glucoseistransportedbytheepithelialcellsthatlinethegastrointestinaltract(smallintestine),andbycellsthatformtheproximaltubulesofthekidneys.Inthegastrointestinaltract,theglucoseisabsorbedfromingestedfood.Inthekidney,theproximaltubulereabsorbstheglucosethatwasfilteredacrosstheglomerularcapillariesandtherebypreventsitfrombeinglostintheurine.Theuptakeofglucoseintotheepithelialcellfromthelumenofthesmallintestineandfromthelumenoftheproximaltubuleisasecondaryactiveprocessinvolvingthesodium-glucose–linkedtransportersSGLT-1andSGLT-2.SGLT-2transportsoneglucosemoleculewithoneNa+ ion,andtheenergyintheelectrochemicalgradientforNa+ (intothecell)drivesthesecondaryactiveuptakeofglucose.Accordingtothefollowingequation,forcalculatingtheelectrochemicalgradient,andifthemembranepotential(Vm)is−60mVandthereisa10-fold[Na+]gradientacrossthemembrane,anapproximate100-foldglucosegradientcouldbegeneratedbySGLT-2:[Glucose]i [Na+ ]o V 615. mV

1	Thus,iftheintracellularglucoseconcentrationwas2mmol/L,thecellcouldlowertheextracellularglucoseconcentrationtoapproximately0.02mmol/L.However,byincreasingthenumberofNa+ ionstransportedwithglucosefromonetotwo,SGLT-1cangenerateanearly10,000-foldglucosegradient: 615. mV Again,iftheintracellularglucoseconcentrationis2mmol/L,SGLT-1couldremovevirtuallyallglucosefromeitherthelumenofthesmallintestineorthelumenoftheproximaltubule(i.e.,theluminalglucoseconcentration≅ 0.0002mmol/L). where molecules at 37°C can exert an osmotic pressure of 2.54 n = number of dissociable particles per molecule × 10−2 atm, as calculated with eq. 1.7 and the following C = total solute concentration values: R = gas constant n = 1 T = temperature in degrees Kelvin C = 0.001 mol/L

1	For a molecule that does not dissociate in water, such as R = 0.082 atm L/mol K glucose or urea, a solution containing 1 mmol/L of these T = 310 °K •Fig. 1.8

1	= 310 °K •Fig. 1.8 Schematicrepresentationofosmoticwatermovementandthegenerationofanosmoticpressure.CompartmentAandcompartmentBareseparatedbyasemipermeablemembrane(i.e.,themembraneishighlypermeablebywaterbutimpermeablebysolute).CompartmentAcontainsasolute,whereascompartmentBcontainsonlydistilledwater.Overtime,watermovesbyosmosisfromcompartmentBtocompartmentA.(Note:Thiswatermovementisdrivenbytheconcentrationgradientforwater.BecauseofthepresenceofsoluteparticlesincompartmentA,theconcentrationofwaterincompartmentAislessthanthatincompartmentB.Consequently,watermovesacrossthesemipermeablemembranefromcompartmentBtocompartmentAdownitsconcentrationgradient.)ThiscausestheleveloffluidtoberaisedincompartmentAandloweredincompartmentB.Atequilibrium,thehydrostaticpressureexertedbythecolumnofwater(h)stopsthenetmovementofwaterfromcompartmentBtoA.Thusatequilibrium,thehydrostaticpressureisequalandoppositetotheosmoticpressureexertedbythesoluteparticlesincompartmentA.(RedrawnfromKoeppenBM,StantonBA.Renal

1	Physiology. 4thed.St.Louis:Mosby;2006.)

1	Because 1 atm equals 760 mm Hg at sea level, π for this solution can also be expressed as 19.3 mm Hg. Alternatively, osmotic pressure is expressed in terms of osmolarity (see the following section). Regardless of the molecule, a solution containing 1 mmol/L of the molecule therefore exerts an osmotic pressure proportional to 1 mOsm/L. For molecules that dissociate in a solution, n of eq. 1.7 will have a value other than 1. For example, a 150-mmol/L solution of NaCl has an osmolarity of approximately 300 mOsm/L because each molecule of NaCl dissociates into a Na+ and a Cl− ion (i.e., n = 2). If dissociation of a molecule into its component ions is not complete, n will not be an integer. Accordingly, osmolarity for any solution can be calculated as follows: Equation 1.8 Osmolarity = concentration × number of dissociable particles mOsm/L = mmol/L × number of particles

1	Equation 1.8 Osmolarity = concentration × number of dissociable particles mOsm/L = mmol/L × number of particles The terms osmolarity and osmolality are frequently confused and incorrectly interchanged. Osmolarity refers to the osmotic pressure generated by the dissolved solute molecules in 1 L of solvent, whereas osmolality is the number of molecules dissolved in 1 kg of solvent. For a dilute solution, the difference between osmolarity and osmolality is cNaCl does not completely dissociate in water. The value for n is 1.88 rather than 2. However, for simplicity, the value of 2 is most often used.

1	insignificant. Measurements of osmolarity are temperature dependent because the volume of the solvent varies with temperature (i.e., the volume is larger at higher temperatures). In contrast, osmolality, which is based on the mass of the solvent, is temperature independent. For this reason, osmolality is the preferred term for biologic systems and is used throughout this book. Because the solvent in biological solutions and bodily fluids is water, and because of the dilute nature of biological solutions and bodily solutions, osmolalities are expressed as milliosmoles per kilogram of water (mOsm/kg H2O).

1	The tonicity of a solution is related to the effect of the solution on the volume of a cell. Solutions that do not change the volume of a cell are said to be isotonic. A hypotonic solution causes a cell to swell, whereas a hypertonic solution causes a cell to shrink. Although related to osmolality, tonicity also accounts for the ability of the molecules in solution to cross the cell membrane.

1	Consider two solutions: a 300-mmol/L solution of sucrose and a 300-mmol/L solution of urea. Both solutions have an osmolality of 300 mOsm/kg H2O and therefore are said to be isosmotic (i.e., they have the same osmolality). When red blood cells—which for the purpose of this illustration also have an intracellular fluid osmolality of 300 mOsm/kg H2O—are placed in the two solutions, those in the sucrose solution maintain their normal volume, whereas those placed in urea swell and eventually burst. Thus the sucrose solution is isotonic and the urea solution is hypotonic. The differential effect of these solutions on

1	CHAPTER 1 Principles of Cell and Membrane Function red blood cell volume is related to the permeability of the red blood cell plasma membrane to sucrose and urea. The red blood cell membrane contains uniporters for urea. Thus urea easily crosses the cell membrane (i.e., the cell is permeable by urea), driven by the concentration gradient (i.e., extracellular urea concentration > intracellular urea concentration). In contrast, the red blood cell membrane does not contain sucrose transporters, and sucrose cannot enter the cell (i.e., the cell is impermeable by sucrose).

1	To exert an osmotic pressure across a membrane, a molecule must not cross the membrane. Because the red blood cell membrane is impermeable by sucrose, it exerts an osmotic pressure equal and opposite to the osmotic pressure generated by the contents within the red blood cell (in this case, 300 mOsm/kg H2O). In contrast, urea is readily able to cross the red blood cell membrane, and it cannot exert an osmotic pressure to balance that generated by the intracellular solutes of the red blood cell. Consequently, sucrose is termed an effective osmole, whereas urea is an ineffective osmole. To take into account the effect of a molecule’s ability to permeate the membrane on osmotic pressure, it is necessary to rewrite eq. 1.7 as follows: Equation 1.9 ˜e = ˙(nCRT), where σ is the reflection coefficient (or osmotic coefficient) and is a measure of the relative ability of the molecule to cross the cell membrane, and Πe is the “effective osmotic pressure.”

1	For a molecule that can freely cross the cell membrane, such as urea in the preceding example, σ= 0, and no effective osmotic pressure is exerted (e.g., urea is an ineffective osmole for red blood cells). In contrast, σ= 1 for a solute that cannot cross the cell membrane (in the preceding example, sucrose). Such a substance is said to be an effective osmole. Many molecules are neither completely able nor completely unable to cross cell membranes (i.e., 0 <σ< 1) and generate an osmotic pressure that is only a fraction of what is expected from the molecules’ concentration in solution. Oncotic pressure is the osmotic pressure generated by large molecules (especially proteins) in solution. As illustrated in

1	Oncotic pressure is the osmotic pressure generated by large molecules (especially proteins) in solution. As illustrated in Fig. 1.9 , the magnitude of the osmotic pressure generated by a solution of protein does not conform to van’t Hoff’s law. The cause of this anomalous relationship between protein concentration and osmotic pressure is not completely understood, but it appears to be related to the size and shape of the protein molecule. For example, the correlation to van’t Hoff’s law is more precise with small, globular proteins than with larger protein molecules.

1	The oncotic pressure exerted by proteins in human plasma has a normal value of approximately 26 to 28 mm Hg. Although this pressure appears to be small in relation to osmotic pressure (28 mm Hg ≅ 1.4 mOsm/kg H2O), it is an important force involved in fluid movement across capillaries (see •Fig. 1.9 Relationshipbetweentheconcentrationofplasmapro-teinsinsolutionandtheosmoticpressure(oncoticpressure)theygenerate.Proteinconcentrationisexpressedingramsperdeciliter.Normalplasmaproteinconcentrationisindicated.Notehowtheactualpressuregeneratedexceedsthatpredictedbyvan’tHoff’slaw.80 60 40 20 0 2 6 10 14 Protein (g/dl) Predicted by van’t Hoff’s law Normal plasma ActualOsmotic pressure (g/dL)

1	The total concentration of all molecules in a solution can also be measured as specific gravity. Specific gravity is defined as the weight of a volume of solution divided by the weight of an equal volume of distilled water. Thus the specific gravity of distilled water is 1. Because biological fluids contain a number of different molecules, their specific gravities are greater than 1. For example, normal human plasma has a specific gravity in the range of 1.008 to 1.010.

1	Thespecificgravityofurineissometimesmeasuredinclinicalsettingsandusedtoassesstheurineconcentratingabilityofthekidneys.Thespecificgravityofurinevariesinproportiontoitsosmolality.However,becausespecificgravitydependsbothonthenumberofmoleculesandontheirweight,therelationshipbetweenspecificgravityandosmolalityisnotalwayspredictable.Forexample,inpatientswhohavereceivedaninjectionofradiocontrastdye(molecularweight> 500g/mole)forx-raystudies,valuesofurinespecificgravitycanbehigh(1.040to1.050),eventhoughtheurineosmolalityissimilartothatofplasma(e.g.,300mOsm/kgH2O). The plasma membrane is a lipid bilayer composed of phospholipids and cholesterol, into which are embedded a wide range of proteins. One class of these membrane proteins (membrane transport proteins or transporters) is involved in the selective and regulated transport of molecules into and out of the cell. These transporters include water channels (aquaporins), ion channels, solute carriers, and ATP-dependent transporters.

1	The movement of molecules across the plasma membrane through ion channels and solute carriers is driven by chemical concentration gradients and, chemical concentration gradients and electrical potential differences (charged molecules only). The electrochemical gradient is used to quantitate this driving force. ATP-dependent transporters use the energy in ATP to transport molecules across the membrane and often establish the chemical Alberts B, et al. Essential Cell Biology. 4th ed. New York: Garland Science; 2014. Altenberg GA, Ruess L. Mechanisms of water transport across cell membranes and epithelia. In: Alpern R, Moe O, Kaplan M, eds. Seldin and Giebisch’s The Kidney—Physiology and Pathophysiology. 5th ed. New York: Academic Press; 2013. Hediger MA, et al. The ABCs of membrane transporters in health and disease (SLC series): introduction. Mol Aspects Med. 2013; 34:95-107.

1	Hediger MA, et al. The ABCs of membrane transporters in health and disease (SLC series): introduction. Mol Aspects Med. 2013; 34:95-107. and electrical gradients that then drive the transport of other molecules through channels and by the solute carriers. Water movement through aquaporins is driven by an osmotic pressure difference across the membrane.

1	• Transport across the membrane is classified as passive or active. Passive transport is the movement of molecules as expected from the electrochemical gradient for that molecule. Active transport represents transport against the electrochemical gradient. Active transport is further divided into primary active and secondary active transport. Primary active transport is directly coupled to the hydrolysis of ATP (e.g., ATP-dependent transporters). Secondary active transport occurs with coupled solute carriers, for which passive movement of one or more molecules drives the active transport of other molecules (e.g., Na+-glucose symporter, Na+-H+ antiporter). Pawlina W. Histology: A Text and Atlas with Correlated Cell and Molecular Biology. 7th ed. Alphen aan den Rijn, The Netherlands: Wolters Kluwer; 2016. Rojeck A, et al. A current view of mammalian aquaglyceroporins. Annu Rev Physiol. 2008;70:301-327.

1	Rojeck A, et al. A current view of mammalian aquaglyceroporins. Annu Rev Physiol. 2008;70:301-327. Ruess L, Altenberg GA. Mechanisms of ion transport across cell membranes. In: Alpern R, Moe O, Kaplan M, eds. Seldin and Giebisch’s The Kidney—Physiology and Pathophysiology. 5th ed. New York: Academic Press; 2013. Upon completion of this chapter, the student should be able to answer the following questions: 1. What is steady-state balance, and, with water balance as an example, what are the elements needed to achieve steady-state balance? 2. What are the volumes of the body fluid compartments, and how do they change under various conditions? 3. How do the body fluid compartments differ with regard to their composition? 4. What determines the resting membrane potential of cells? 5. How do cells regulate their volume in isotonic, hypotonic, and hypertonic solutions? 6.

1	4. What determines the resting membrane potential of cells? 5. How do cells regulate their volume in isotonic, hypotonic, and hypertonic solutions? 6. What are the structural features of epithelial cells, how do they carry out vectorial transport, and what are the general mechanisms by which transport is regulated?

1	ormal cellular function requires that the intracellular composition—with regard to ions, small molecules, water, pH, and a host of other substances—be maintained within a narrow range. This is accomplished by the transport of many substances and water into and out of the cell via membrane transport proteins, as described in . In addition, each day, food and water are ingested, and waste products are excreted from the body. In a healthy individual, these processes occur without significant changes in either the volume of the body fluid compartments or their composition. The maintenance of constant volume and composition of the body fluid compartments (and their temperature in warm-blooded animals and humans) is termed homeostasis. The human body has multiple systems designed to achieve homeostasis, the details of which are explained in the various chapters of this book. In this chapter, the basic principles that underlie the maintenance of homeostasis are outlined. In addition, the

1	homeostasis, the details of which are explained in the various chapters of this book. In this chapter, the basic principles that underlie the maintenance of homeostasis are outlined. In addition, the volume and composition of the various body fluid compartments are defined.

1	Concept of Steady-State Balance The human body is an “open system,” which means that substances are added to the body each day and, similarly, substances are lost from the body each day. The amounts added to or lost from the body can vary widely, depending on the environment, access to food and water, disease processes, and even cultural norms. In such an open system, homeostasis occurs through the process of steady-state balance.

1	To illustrate the concept of steady-state balance, consider a river on which a dam is built to create a synthetic lake. Each day, water enters the lake from the various streams and rivers that feed it. In addition, water is added by underground springs, rain, and snow. At the same time, water is lost through the spillways of the dam and by the process of evaporation. For the level of the lake to remain constant (i.e., steady-state balance), the rate at which water is added, regardless of source, must be exactly matched by the amount of water lost, again regardless of route. Because the addition of water is not easily controlled and the loss by evaporation cannot be controlled, the only way to maintain a constant level of the lake is to regulate the amount that is lost through the spillways. To understand steady-state balance as it applies to the human body, the following key concepts are important. 1.

1	To understand steady-state balance as it applies to the human body, the following key concepts are important. 1. There must be a “set point” so that deviations from this baseline can be monitored (e.g., the level of the lake in the preceding example, or setting the temperature in a room by adjusting the thermostat). 2. The sensor or sensors that monitor deviations from the set point must generate “effector signals” that can lead to changes in either input or output, or both, to maintain the desired set point (e.g., electrical signals to adjust the spillway in the dam analogy, or electrical signals sent to either the furnace or air conditioner to maintain the proper room temperature). 3. “Effector organs” must respond in an appropriate way to the effector signals generated by the set point monitor (i.e., the spillway gates must operate, and the furnace or air conditioner must turn on). 4.

1	4. The sensitivity of the system (i.e., how much of a deviation from the set point is tolerated) depends on several factors, including the nature of the sensor (i.e., how much of a deviation from the set point is needed for the sensor to detect the deviation), the time necessary for generation of the effector signals, and how rapidly the effector organs respond to the effector signals. 1. 2. 1. 2. 3. 4. •Fig. 2.1 Whole-Body Steady-State Water Balance. Seetextfordetails.ADH,antidiuretichormone(alsocalledargininevasopressin);CNS,centralnervoussystem;GI,gastrointestinal. It is important to recognize that deviations from steady-state balance do occur. When input is greater than output, a state of positive balance exists. When input is less than output, a state of negative balance exists. Although transient periods of imbalance can be tolerated, prolonged states of positive or negative balance are generally incompatible with life.

1	Fig. 2.1 illustrates several important concepts for the maintenance of steady-state water balance (details related to the maintenance of steady-state water balance are presented in ). As depicted in Fig. 2.1 , there are multiple inputs and outputs of water, many of which can vary but nevertheless cannot be regulated. For example, the amount of water lost through the lungs depends on the humidity of the air and the rate of respiration (e.g., low humidity and rapid breathing increase water loss from the lungs). Similarly, the amount of water lost as sweat varies according to ambient temperature and physical activity. Finally, water loss via the gastrointestinal tract can increase from a normal level of 100 to 200 mL/day to many liters with acute diarrhea. Of these inputs and outputs, the only two that can be regulated are increased ingestion of water in response to thirst and alterations in urine output by the kidneys (see Chapter 35).

1	Chapter 35). Water balance determines the osmolality of the body fluids. Cells within the hypothalamus of the brain monitor body fluid osmolality for deviations from the set point (normal range: 280-295 mOsm/kg H2O). When deviations are sensed, two effector signals are generated. One is neural and relates to the individual’s sensation of thirst. The other is hormonal (antidiuretic hormone, also called arginine vasopressin), which regulates the amount of water excreted by the kidneys. With appropriate responses to these two signals, water input, water output, or both are adjusted to maintain balance and thereby keep body fluid osmolality at the set point. Volumes and Composition of Body Fluid Compartments

1	Unicellular organisms maintain their volume and composition through exchanges with the environment they inhabit (e.g., sea water). The billions of cells that constitute the human body must maintain their volume and composition as well, but their task is much more difficult. This challenge, as well as its solution, was first articulated by the French physiologist Claude Bernard (1813-1878). He recognized that although cells within the body cannot maintain their volume and composition through exchanges with the environment, they can do so through exchanges with the fluid environment that surrounds them (i.e., the extracellular fluid). Bernard referred to the extracellular fluid as the milieu intérieur (“the environment within”). He also recognized that the organ systems of the body are designed and function to maintain a constant milieu interieur or a “constant internal environment.” This in turn allows all cells to maintain their volume and composition through exchanges with the

1	designed and function to maintain a constant milieu interieur or a “constant internal environment.” This in turn allows all cells to maintain their volume and composition through exchanges with the extracellular fluid as a result of membrane transport (see

1	Transport by the epithelial cells of the gastrointestinal tract, kidneys, and lungs are the body’s interface with the CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments external environment and control both the intake and excretion of numerous substances, as well as water. The cardiovascular system delivers nutrients to and removes waste products from the cells and tissues and keeps the extracellular fluid well mixed. Finally, the nervous and endocrine systems provide regulation and integration of these important functions.

1	To provide background for the study of all organ systems, this chapter presents an overview of the normal volume and composition of the body fluid compartments and describes how cells maintain their intracellular composition and volume. Included is a presentation on how cells generate and maintain a membrane potential, which is fundamental for understanding the function of excitable cells (e.g., neurons and muscle cells). Finally, because epithelial cells are so central to the process of regulating the volume and composition of the body fluids, the principles of solute and water transport by epithelial cells are also reviewed. Definition and Volumes of Body Fluid Compartments

1	Definition and Volumes of Body Fluid Compartments Water makes up approximately 60% of the body’s weight; variability among individuals is a function of the amount of adipose tissue. Because the water content of adipose tissue is lower than that of other tissue, increased amounts of adipose tissue reduce the fraction of water in the total body weight. The percentage of body weight attributed to water also varies with age. In newborns, it is approximately 75%. This decreases to the adult value of 60% by the age of 1 year. Fig. 2.2, total body water is distributed between two major compartments, which are divided by the cell membrane. The intracellular fluid (ICF) compartment is the larger compartment, and contains approximately two thirds of the total body water. The remaining third is contained in the extracellular fluid (ECF) compartment. Expressed as percentages of body weight, the volumes of total body water, ICF, and ECF are as follows:

1	Total body water = 0 6. × (body weight) ICF = 0 4. × (body weight) ECF = 0 2. × (body weight) The ECF compartment is further subdivided into interstitial fluid and plasma. The ECF also includes fluid contained within bone and dense connective tissue, as well as the cerebrospinal fluid. The interstitial fluid surrounds the cells in the various tissues of the body and makes up three fourths of the ECF volume. Plasma is contained within the vascular compartment and represents the remaining fourth of the ECF. In some pathological conditions, additional fluid may accumulate in what is referred to as a third space. Third-space collections of fluid are part of the ECF; an aIn these and all subsequent calculations, it is assumed that 1 L of fluid (e.g., ICF and ECF) has a mass of 1 kg. Although, 1 L of the ICF and ECF has a mass of slightly more than 1 kg, this simplification allows conversion from measurements of body weight to volume of body fluids.

1	Total body water (TBW) 0.6 x body weight 42 L Extracellular fluid (ECF) 0.2 x body weight 14 L Intracellular fluid (ICF) 0.4 x body weight 28 L Cell membrane Interstitial fluid 3/4 of ECF 10.5 L Plasma 1/4 of ECF 3.5 L •Fig. 2.2 Relationship Between the Volumes of the Various Body Fluid Compartments. Theactualvaluesshownareforanindividualweighing70kg.(ModifiedfromLevyMN,KoeppenBM,StantonBA.Berne & Levy’s Principles of Physiology.4thed.St.Louis:Mosby;2006.) example is the accumulation of fluid in the peritoneal cavity (ascites) of individuals with liver disease. Movement of Water Between Body Fluid Compartments Fig. 2.2 , water moves between the ICF and ECF compartments across the plasma membranes of cells, and it moves between the vascular (plasma) and interstitial compartments across capillary walls. The pathways and driving forces for this water movement are different across cell membranes, in comparison to the capillary walls.

1	Movement of water between the ICF and ECF compartments, across cell membranes, occurs through aquaporins expressed in the plasma membrane (see Chapter 1).

1	The driving force for this water movement is an osmotic pressure difference. The osmotic pressure of both the ICF and ECF is determined by the molecules/ions present in these fluids. For simplicity, these can be divided into (1) molecules of low molecular weight (e.g., glucose) and ions (e.g., Na+) and (2) macromolecules (e.g., proteins). The osmotic pressures of both the ICF and ECF are in the range of 280 to 295 mOsm/kg H2O. For the ECF, the low-molecular-weight molecules and ions account for nearly all of this pressure because the osmotic pressure contributed by proteins is only 1 to 2 mOsm/kg H2O. The molecules/ions contributing to the osmotic pressure within the cell are less well understood, but they also include low-molecular-weight molecules (e.g., glucose), ions (e.g., Na+), and macromolecules (e.g., proteins). The fact that cell volume remains constant when ECF osmolality is constant means that the osmotic pressure inside the cells is equal to that of the ECF. If an osmotic

1	macromolecules (e.g., proteins). The fact that cell volume remains constant when ECF osmolality is constant means that the osmotic pressure inside the cells is equal to that of the ECF. If an osmotic pressure difference did exist, the cells would either swell or shrink, as described in the section “Nonisotonic Cell Volume Regulation.”

1	Movement of water between the vascular (plasma) compartment and the interstitial fluid compartment occurs across the capillary wall. The amount of water that moves across the capillary wall, and the mechanism of the water movement varies depending on the capillary. For example, in the capillary sinusoids of the liver, endothelial cells are often separated by large gaps (discontinuous capillary). As a result, water and all components of the plasma (and some cellular elements) can pass easily across the wall. Other capillaries are lined by endothelial cells that contain fenestrations that are up to 80 to 100 nm in diameter (e.g., in the kidneys). These fenestrations allow all components of the plasma (only cellular elements of blood cannot pass through the fenestrations) to move across the capillary wall. Some capillaries (e.g., in the brain) form a relatively tight barrier to water and small molecules and ions, and water movement occurs through small pores on the endothelial cell

1	the capillary wall. Some capillaries (e.g., in the brain) form a relatively tight barrier to water and small molecules and ions, and water movement occurs through small pores on the endothelial cell surface or through clefts between adjacent endothelial cells. These pores and clefts allow water and molecules smaller than 4 nm to pass. In addition, a small amount of water traverses the capillary wall via pinocytosis by endothelial cells.

1	The driving forces for fluid (water) movement across the capillary wall are hydrostatic pressure and oncotic pressure (i.e., osmotic pressure generated by proteins). Collectively, these are called the Starling forces. Capillary fluid movement is discussed in detail in in brief, hydrostatic pressure within the capillary (as a result of the pumping of the heart and the effect of gravity on the column of blood in the vessels feeding a capillary) is a force that causes fluid to move out of the capillary. Hydrostatic pressure in the surrounding interstitial tissue opposes the effect of the capillary hydrostatic pressure. The oncotic pressure of the plasma in the capillary tends to draw fluid from the interstitium into the capillary. The oncotic pressure of the interstitial fluid opposes this. Thus the amount of fluid moving across the wall of the capillary is determined as follows: Equation 2.1

1	Equation 2.1 Kf = filtration constant (measure of surface area + in πi = interstitial fluid oncotic pressure. Depending on the magnitude of these forces, fluid may move out of the capillary or into the capillary. The compositions of the various body fluid compartments differ; however, as described later, the osmolalities of the fluid within these compartments are essentially identical. Thus the compartments are in “osmotic equilibrium.” In addition, any change in the osmolality of one compartment quickly causes water to redistribute across all compartments, which brings them back into osmotic equilibrium. Because of this rapid redistribution of water, measuring the osmolality of plasma or serum, which is easy to do, reveals the osmolality of the other body fluid compartments (i.e., interstitial fluid and intracellular fluid).

1	As described later, Na+ is a major constituent of the ECF. Because of its high concentration in comparison with other molecules and ions, Na+ (and its attendant anions, primarily Cl− and HCO3 −) is the major determinant of the osmolality of this compartment. Accordingly, it is possible to obtain an approximate estimate of the ECF osmolality by simply doubling the sodium concentration [Na+]. For example, if a blood sample is obtained from an individual, and the [Na+] of the serum is 145 mEq/L, its osmolality can be estimated as follows: Equation 2.2 In contrast to water, the movement of ions across cell membranes is more variable from cell to cell and depends on the presence of specific membrane transport proteins (see the section “

1	In contrast to water, the movement of ions across cell membranes is more variable from cell to cell and depends on the presence of specific membrane transport proteins (see the section “ Composition of Body Fluid Compart ”). Consequently, in trying to understand the physiology of fluid shifts between body fluid compartments, it can be assumed that while water moves freely between the compartments, there is little net movement of solutes. For most situations, this is a reasonable assumption. To illustrate the physiologic characteristics of fluid shifts, consider what happens when solutions containing various amounts of NaCl are added to the ECF. Example 1: Addition of Isotonic Sodium Chloride to the Extracellular Fluid

1	Addition of an isotonic NaCl solution (e.g., intravenous infusion of 0.9% NaCl: osmolality ≈ 290 mOsm/kg H2O) bSome exceptions do exist. The cerebrospinal fluid is part of the ECF, but its osmolality is slightly higher than that of the ECF elsewhere in the body. Also, regions within the kidney can have osmolalities that are either less than or greater than that of the ECF. However, these volumes are small (≈150 mL) in comparison with the total volume of the ECF (≥12 L). cSerum is derived from clotted blood. Thus serum differs from plasma by the absence of clotting factors. With regard to osmolality and the concentrations of other molecules and ions, the osmolality and concentrations in plasma and serum are virtually identical. dFluids are usually administered intravenously. When electrolyte solutions are infused by this route, equilibration between plasma and interstitial fluid is rapid (i.e., minutes) because of the high permeability of many capillary walls for water and

1	electrolyte solutions are infused by this route, equilibration between plasma and interstitial fluid is rapid (i.e., minutes) because of the high permeability of many capillary walls for water and electrolytes. Thus these fluids are essentially added to the entire ECF. eA 0.9% NaCl solution (0.9 g NaCl/100 mL) contains 154 mmol/L of NaCl. Because NaCl does not dissociate completely in solution (i.e., 1.88 Osm/mol), the osmolality of this solution is 290 mOsm/kg H2O, which is very similar to that of normal ECF.

1	CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments Insomeclinicalsituations,itispossibletoobtainamoreaccurateestimateoftheserumosmolality,andthustheosmolalitiesoftheECFandICF,byalsoconsideringtheosmolescontributedbyglucoseandurea,asthesearethenextmostabundantsolutesintheECF(theothercomponentsoftheECFcontributeonlyafewadditionalmilliosmoles).Accordingly,serumosmolalitycanbeestimatedasfollows: 18 28. Theglucoseandureaconcentrationsareexpressedinunitsofmilligramsperdeciliter(dividingby18forglucoseand2.8for allowsconversionfromtheunitsofmilligramsperdecilitertomillimolesperliterandthustomilliosmolesperkilogramofH2O).Thisestimationofserumosmolalityisespeciallyusefulintreatingpatientswhohaveanelevatedserumglucoseconcentrationsecondarytodiabetesmellitus,andinpatientswithchronicrenalfailure,whoseserumureaconcentrationiselevatedbecauseofreducedrenalexcretion.

1	,theabilityofasubstancetocausewatertomoveacrosstheplasmamembraneofacelldependsonwhetherthesubstanceitselfcrossesthemembrane.Recall Eq.1.9 whereΠe= theeffectiveosmoticpressureandσ= thereflectioncoefficientforthesubstance.Formanycells,glucoseandureacrossthecellmembrane.Althoughtheycontributetoserumosmolality,asmeasuredbyalaboratoryosmometerwhereallmoleculesare“effectiveosmoles,”theyareineffectiveosmolesforwatermovementacrossmany,butnotall,cellmembranes.Incontrast,Na+ isan“effectiveosmole”forwatermovementacrosstheplasmamembraneofvirtuallyallcells. Eq.2.2 givesthebestestimateoftheeffectiveosmolalityoftheserum. *Theureaconcentrationinplasmaismeasuredasthenitrogenintheureamolecule,orbloodureanitrogen(BUN).

1	Eq.2.2 givesthebestestimateoftheeffectiveosmolalityoftheserum. *Theureaconcentrationinplasmaismeasuredasthenitrogenintheureamolecule,orbloodureanitrogen(BUN). to the ECF increases the volume of this compartment by the volume of fluid administered. Because this fluid has the same osmolality as does the ECF, and therefore the ICF, there is no driving force for fluid movement between these compartments, and the volume of the ICF remains unchanged. Although Na+ can cross cell membranes, it is effectively restricted to the ECF by the activity of the Na+,K+-ATPase, which is present in the plasma membrane of all cells (see the section “ Ionic Composition of Cells”). Therefore, there is no net movement of the infused isotonic NaCl solution into cells. Example 2: Addition of Hypotonic Sodium Chloride to the Extracellular Fluid

1	Ionic Composition of Cells”). Therefore, there is no net movement of the infused isotonic NaCl solution into cells. Example 2: Addition of Hypotonic Sodium Chloride to the Extracellular Fluid Addition of a hypotonic NaCl solution to the ECF (e.g., intravenous infusion of 0.45% NaCl; osmolality ≅ 145 mOsm/kg H2O) decreases the osmolality of this fluid compartment, which results in the movement of water into

1	Neurosurgicalproceduresandcerebrovascularaccidents(strokes)oftenresultintheaccumulationofinterstitialfluidinthebrain(i.e.,edema)andswellingoftheneurons.Becausethebrainisenclosedwithintheskull,edemacanraiseintracranialpressureandtherebydisruptneuronalfunction,whichleadstocomaanddeath.Theblood-brainbarrier,whichseparatesthecerebrospinalfluidandbraininterstitialfluidfromblood,canbepermeatedfreelybywaterbutnotbymostothersubstances.Asaresult,excessfluidinbraintissuecanberemovedbyimposinganosmoticgradientacrosstheblood-brainbarrier.Mannitolcanbeusedforthispurpose.Mannitolisasugar(molecularweight,182g/mol)thatdoesnotreadilycrosstheblood-brainbarrierandmembranesofcells(neuronsandothercellsinthebody).Therefore,mannitolisaneffectiveosmole,andintravenousinfusionresultsinthemovementofinterstitialfluidoutofthebrainbyosmosis.

1	the ICF. After osmotic equilibration, the osmolalities of the ICF and ECF are again equal but lower than before the infusion, and the volume of each compartment is increased. The increase in ECF volume is greater than the increase in ICF volume. Example 3: Addition of Hypertonic Sodium Chloride to the Extracellular Fluid Addition of a hypertonic NaCl solution to the ECF (e.g., intravenous infusion of 3% NaCl: osmolality ≅ 1000 mOsm/kg H2O) increases the osmolality of this compartment, which results in the movement of water out of cells. After osmotic equilibration, the osmolalities of the ECF and ICF are again equal but higher than before the infusion. The volume of the ECF is increased, whereas that of the ICF is decreased. Composition of Body Fluid Compartments

1	Composition of Body Fluid Compartments The compositions of the ECF and ICF differ considerably. The ICF has significantly more proteins and macromolecules than the ECF. There are also differences in the concentrations of many ions. The composition of the ICF is maintained by the action of a number of specific cell membrane transport proteins. Principal among these transporters is the Na+,K+adenosine triphosphatase (Na+,K+-ATPase), which converts the energy in ATP into ion and electrical gradients, which can in turn be used to drive the transport of other ions and molecules by means of ion channels and solute carriers (e.g., symporters and antiporters).

1	The compositions of the plasma and interstitial fluid compartments of the ECF are similar because those compartments are separated only by the capillary endothelium, a barrier that ions and small molecules can permeate. The major difference between the interstitial fluid and plasma is that the latter contains significantly more protein. Although this differential concentration of protein can affect the Fluidandelectrolytedisordersareobservedcommonlyinclinicalpractice(e.g.,inpatientswithvomitingordiarrhea,orboth).Inmostinstances,thesedisordersareself-limited,andcorrectionofthedisorderoccurswithoutneedforintervention.However,moresevereorprolongeddisordersmaynecessitatefluidreplacementtherapy.Suchtherapymaybeadministeredorally,withspecialelectrolytesolutions,orintravenously,withfluid.

1	Intravenoussolutionsareavailableinmanyformulations.Thetypeoffluidadministeredtoaparticularpatientisdictatedbythepatient’sneed.Forexample,ifanincreaseinthepatient’svascularvolumeisnecessary,asolutioncontainingsubstancesthatdonotreadilycrossthecapillarywallisinfused(e.g.,5%proteinordextransolutions).Theoncoticpressuregeneratedbythealbuminmoleculescausesfluidtoberetainedinthevascularcompartment,whichexpandsitsvolume.ExpansionoftheECFisaccomplishedmostoftenwithisotonicsalinesolutions(e.g.,0.9%NaClorlactatedRingersolution).Asalreadynoted,administrationofanisotonicNaClsolutiondoesnotresultinthedevelopmentofanosmoticpressuregradientacrosstheplasmamembraneofcells.Therefore,theentirevolumeoftheinfusedsolutionremainsintheECF.

1	Patientswhosebodyfluidsarehyperosmoticneedhypotonicsolutions.ThesesolutionsmaybehypotonicNaCl(e.g.,0.45%NaCl)or5%dextroseinwater(D5W).AdministrationoftheD5WsolutionisequivalenttotheinfusionofdistilledwaterbecausethedextroseismetabolizedtoCO2andwater.AdministrationofthesefluidsincreasesthevolumesofboththeICFandECF.Inaddition,patientswhosebodyfluidsarehypotonicneedhypertonicsolutions.ThesearetypicallyNaCl-containingsolutions(e.g.,3%or5%NaCl).ThesesolutionsexpandthevolumeoftheECFbutdecreasethevolumeoftheICF.Otherconstituents,suchaselectrolytes(e.g.,K+)ordrugs,canbeaddedtointravenoussolutionstotailorthetherapytothepatient’sfluid,electrolyte,andmetabolicneeds. distribution of cations and anions between these two compartments by the Gibbs-Donnan effect (see the section “ ” for details), this effect is small, and the ionic compositions of the interstitial fluid and plasma can be considered to be identical. Maintenance of Cellular Homeostasis

1	Maintenance of Cellular Homeostasis Normal cellular function requires that the ionic composition of the ICF be tightly controlled. For example, the activity of some enzymes is pH dependent; therefore, intracellular pH must be regulated. In addition, the intracellular composition of other electrolytes is similarly held within a narrow range. This is necessary for the establishment of the membrane potential, a cell property especially important for the normal function of excitable cells (e.g., neurons and muscle cells) and for intracellular signaling (e.g., intracellular [Ca++]; see for details). Finally, the volume of cells must be maintained because shrinking or swelling of cells can lead to cell damage or death. The regulation of intracellular K+ 3.5-5.0mEq/L120-150mEq/L Ca++ 2.1-2.8(total)mmol/L 1.1-1.4(ionized)≈10−7M(ionized)

1	K+ 3.5-5.0mEq/L120-150mEq/L Ca++ 2.1-2.8(total)mmol/L 1.1-1.4(ionized)≈10−7M(ionized) Pi1.0-1.4(total)mmol/L 0.5-0.7(ionized)0.5-0.7(ionized) *Ca++ andPi(H2PO4−/HPO4−2)areboundtoproteinsandotherorganicmolecules.Inaddition,largeamountsofCa++ canbesequesteredwithincells.LargeamountsofPiarepresentincellsaspartoforganicmolecules,suchasadenosinetriphosphate(ATP). composition and cell volume is accomplished through the activity of specific transporters in the plasma membrane of the cells. This section is a review of the mechanisms by which cells maintain their intracellular ionic environment and their membrane potential and by which they control their volume. Ionic Composition of Cells

1	The intracellular ionic composition of cells varies from tissue to tissue. For example, the intracellular composition of neurons is different from that of muscle cells, both of which differ from that of blood cells. Nevertheless, there are similar patterns, and these are presented in . In comparison with the ECF, the ICF is characterized by a low [Na+] and a high [K+]. This is the result of the activity of the Na+,K+-ATPase, which transports 3 Na+ ions out of the cell and 2 K+ ions into the cell for each ATP molecule hydrolyzed. As discussed later in this chapter, the activity of the Na+,K+-ATPase not only is important for establishing the cellular Na+ and K+ gradients but also is involved in determining, indirectly, the cellular gradients for many other ions and molecules. Of importance is that the cellular K+ gradient generated by the activity of the Na+,K+-ATPase is a major determinant of the membrane voltage because of the leak of K+ out of the cell through K+selective channels

1	is that the cellular K+ gradient generated by the activity of the Na+,K+-ATPase is a major determinant of the membrane voltage because of the leak of K+ out of the cell through K+selective channels (see the section “

1	Membrane Potential”). Thus the Na+,K+-ATPase converts the energy in ATP into ion gradients (i.e., Na+ and K+), and a voltage gradient (i.e., membrane voltage). The Na+,K+-ATPase–generated ion and electrical gradients are used to drive the transport of other ions and molecules into or out of the cell ( Fig. 2.3 ). For example, as described in , a number of solute carriers couple the transport of Na+ to that of other ions or molecules. The Na+-glucose and Na+–amino acid symporters use the energy in the Na+ electrochemical gradient, directed to bring Na+

1	CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments •Fig. 2.3 Cell Model Depicting How Cellular Gradients and the Membrane Potential (Vm) Are Established. (1)TheNa+,K+-ATPasedecreasestheintracellular[Na+]andincreasestheintracellular[K+].SomeK+ exitsthecellviaK+-selectivechannelsandgeneratestheVm(cell’sinterioriselectricallynegative).(2)TheenergyintheNa+ electrochemicalgradientdrivesthetransportofotherionsandmoleculesthroughtheuseofvarioussolutecarriers.(3)TheVmdrivesCl− outofthecellviaCl−-selectivechannels.(4)TheCa++-ATPaseandthe3Na+-Ca++ antiportersmaintainthelowintracellular[Ca++].

1	into the cell, to drive the secondary active cellular uptake of glucose and amino acids. Similarly, the inwardly directed Na+ gradient drives the secondary active extrusion of H+ from the cell and thus contributes to the maintenance of intracellular pH. The 3Na+-Ca++ antiporter, along with the plasma membrane Ca++-ATPase, extrudes Ca++ from the cell and thus contributes to the maintenance of a low intracellular [Ca++]. In addition, the membrane voltage drives Cl− out of the cell through Cl−-selective channels, thus lowering the intracellular concentration below that of the ECF. As described previously, the Na+,K+-ATPase and K+-selective channels in the plasma membrane are important determinants of the membrane potential (Vm) of the cell. For all cells within the body, the resting Vm is oriented with the interior of the cell electrically negative in relation to the ECF. However, the magnitude of the Vm can vary widely.

1	To understand what determines the magnitude of the Vm, it is important to recognize that any transporter that transfers charge across the membrane has the potential to influence the Vm. Such transporters are said to be fIn muscle cells, in which contraction is regulated by the intracellular [Ca++], the maintenance of a low intracellular [Ca++] during the relaxed state involves not only the activity of the plasma membrane 3Na+-Ca++ antiporter and the Ca++-ATPase but also a Ca++-ATPase molecule located in the smooth endoplasmic reticulum (see

1	Chapters 12 to 14 electrogenic. As might be expected, the contribution of various electrogenic transporters to the Vm is highly variable from cell to cell. For example, the Na+,K+-ATPase channel transports three Na+ and two K+ ions and thus transfers one net positive charge across the membrane. However, the direct contribution of the Na+,K+-ATPase to the Vm of most cells is only a few millivolts at the most. Similarly, the contribution of other electrogenic transporters, such as the 3Na+-Ca++ antiporter and the Na+-glucose symporter is minimal. The major determinants of the Vm are ion channels. The type (e.g., selectivity), number, and activity (e.g., gating) of these channels determine the magnitude of the Vm. As described in , rapid changes in ion channel activity underlies the action potential in neurons and other excitable cells, such as those of skeletal and cardiac muscle (see

1	As ions move across the membrane through a channel, they generate a current. As described in , this current can be measured, even at the level of a single channel. By convention, the current generated by the movement of cations into the cell, or the movement of anions out of the cell, is defined as negative current. Conversely, the movement of cations out of the cell, or the movement of anions into the cell, is defined as positive current. Also by convention, the magnitude of the Vm is expressed in relation to the outside of the cell; thus for a cell with a Vm of −80 mV, the interior of the cell is electrically negative in relation to the outside of the cell. The current carried by ions moving through a channel depends on the driving force for that ion and on the conductance of the channel. As described in , the driving force is determined by the energy in the concentration gradient for the ion across the membrane (Ei), as calculated by the Nernst equation (Eq. 1.5a ) and the Vm:

1	Equation 2.3 Driving force = Vm − Ei. Thus as defined by Ohm’s law, the ion current through the channel (Ii) is determined as follows: Equation 2.4 where gi is the conductance of the channel. For a cell, the conductance of the membrane to a particular ion (Gi) is determined by the number of ion channels in the membrane and by the amount of time each channel is in the open state. Fig. 2.4 , the Vm is the voltage at which there is no net ion flow into or out of the cell. Thus for a cell that has ion channels selective for Na+ , K+ , and Cl− , Equation 2.5 Equation 2.6 + [(Vm − E − )× G − ]= 0.

1	Equation 2.5 Equation 2.6 + [(Vm − E − )× G − ]= 0. •Fig. 2.4 Current-Voltage Relationship of a Hypothetical Cell Containing Na+-, K+-, and Cl−-Selective Channels. Membranecurrentsareplottedoverarangeofmembranevoltages(i.e.,current-voltagerelationships).EachioncurrentiscalculatedwiththeuseofOhm’slaw,theNernstequilibriumpotentialfortheion(ECl,EK,andENa),andthemembraneconductancefortheion.Thecurrent-voltagerelationshipforthewholecellisalsoshown.Totalcellcurrent(Icell)wascalculatedwiththechordconductanceequation(see Eq.2.7).Because80%of cellconductanceisduetoK+ ,therestingmembranevoltage(Vm)of−64.4mVisneartothatoftheNernstequilibriumpotentialforK+ . Equation 2.7 where ˜G = G ++ G ++ G −. Inspection of Eq. 2.7 , which is often called the chord conductance equation, reveals that the Vm will be near to the Nernst equilibrium potential of the ion to which the membrane has the highest conductance. In

1	Eq. 2.7 , which is often called the chord conductance equation, reveals that the Vm will be near to the Nernst equilibrium potential of the ion to which the membrane has the highest conductance. In Fig. 2.4 , 80% of the membrane conductance is attributable to K+; as a result, Vm is near to the Nernst equilibrium potential for K+ (EK+). For most cells at rest, the membrane has a high conductance to K+ , and thus the Vm approximates EK+. Moreover, the Vm is greatly influenced by the magnitude of EK+ , which in turn is greatly influenced by changes in the [K+] of the ECF. For example, if the intracellular [K+] is 120 mEq/L and the extracellular [K+] is 4 mEq/L, EK+ has a value of −90.8 mV. If the extracellular [K+] is increased to 7 mEq/L, EK+ would be −79.9 mV. This change in EK+ depolarizes the Vm (i.e., Vm is less negative). Conversely, if the extracellular [K+] is decreased to 2 mEq/L, EK+ becomes −109.4 mV, and the Vm hyperpolarizes (i.e., Vm is more negative).

1	•Fig. 2.5 Nerve Action Potential Showing the Changes in Na+ and K+ Conductances ( G and GK+ , Respectively) and the Membrane Potential (Vm). Atrest,themembranehasahighK+ conductance,andVmisneartheNernstequilibriumpotentialforK+ (EK+ ).Withtheinitiationoftheactionpotential,thereisalargeincreaseintheNa+ conductanceofthemembrane,andtheVmapproachestheNernstequilibriumpotentialforNa+ (ENa+ ).TheincreaseinNa+ conductanceistransient,andtheK+ conductancethenincreasesaboveitsvaluebeforetheactionpotential.ThishyperpolarizesthecellasVmapproachesEK+.AstheK+ conductancereturnstoitsbaselinevalue,Vmreturnstoitsrestingvalueof−70mV.(ModifiedfromLevyMN,KoeppenBM,StantonBA.Berne & Levy’s Principles of Physiology. 4thed.St.Louis:Mosby;2006.)

1	Changesintheextracellular[K+]canhaveimportanteffectsonexcitablecells,especiallythoseoftheheart.Adecreaseinextracellular[K+](hypokalemia) hyperpolarizestheVmofcardiacmyocytesand,insodoing,makesinitiatinganactionpotentialmoredifficult,becausealargerdepolarizingcurrentisneededtoreachthreshold(see ).Ifsevere,hypokalemiacanleadtocardiacarrhythmias,andeventuallytheheartcanstopcontracting(asystole). Anincreaseintheextracellular[K+](hyperkalemia) canbeequallydeleterioustocardiacfunction.Withhyperkalemia,theVmisdepolarized,anditiseasiertoinitiateanactionpotential.However,oncetheactionpotentialfiresthechannelsbecomeinactivated,andareunabletoinitiateanotheractionpotential,untiltheyarereactivatedbynormalrepolarizationoftheVm.BecausetheVmisdepolarizedinhyperkalemia,thechannelsstayinaninactivatedstate.ThusdepolarizationoftheVmwithhyperkalemiacanleadtocardiacarrhythmiasandlossofcardiacmusclecontraction. Eq. 2.7 also defines the limits for the membrane potential. In the example depicted in

1	Eq. 2.7 also defines the limits for the membrane potential. In the example depicted in Fig. 2.4, it is apparent that the Vm cannot be more negative than EK+ (−90.8 mV), as would be the case if the membrane were only conductive to K+ . Conversely, the Vm could not be more positive than ENa+ (66.6 mV); such a condition would be met if the membrane were conductive only to Na+ . The dependence of the Vm on the conductance of the membrane to specific ions is the basis by which action potentials in excitable cells are generated ( Fig. 2.5). As noted previously, in all excitable cells, the CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments membrane at rest is conductive predominantly to K+ , and thus Vm is near EK+. When an action potential is initiated, Na+-channels open and the membrane is now conductive predominantly to Na+ . As a result, Vm now approaches ENa+. The generation of action potentials is discussed in more detail in

1	TheestablishmentoftheVmrequirestheseparationofchargeacrosstheplasmamembrane.However,thenumberofionsthatmustmoveacrossthemembraneisatinyfractionofthetotalnumberofionsinthecell.Forexample,considerasphericalcellwithadiameterof20µmandaVmof−80mV.Furthermore,assumethatthisVmof−80mVistheresultofthediffusionofK+ outofthecellandthattheintracellular[K+]is120mmol/L.TheamountofK+ thatwouldhavetodiffuseoutofthecelltoestablishtheVmof−80mVisthencalculatedasfollows. Firstthechargeseparationacrossthemembraneneedstobecalculated.Thisisdonewiththeknowledgethattheplasmamembranebehaveselectricallylikeacapacitor,thecapacitance(C)ofwhichisapproximately1µF/cm2,and whereQ= chargeandisexpressedinunitsofcoulombs.Ifthesurfaceareaofthecellis4πr2or1.26× 10−5cm2,thecapacitanceofthecelliscalculatedasfollows: 1× 10 F/cm × . × 10 cm = . × 10 126 126 F. Thusthechargeseparationacrossthemembraneiscalculatedasfollows: 1 01× 10−12 coulombs.

1	Thusthechargeseparationacrossthemembraneiscalculatedasfollows: 1 01× 10−12 coulombs. Because1moleofK+ contains96,480coulombs,theamountofK+ thathadtodiffuseacrossthemembranetoestablishtheVmof−80mViscalculatedasfollows: . coul = 1 05. × 10 mole ofK , ombs/mol Withacellvolumeof4.19× 10−12L(volume= 4πr3/3)andanintracellular[K+]of120mmol/L,thetotalintracellularK+ contentis 4 19 × 10 × . mol/L = 5 03 . 012 . × 10 moles Therefore,thediffusionof1.05× 10−17molesofK+ outofthecellrepresentsonlya0.002%changeintheintracellularK+ content: . mol −13 ° 0 002. % 5 03 × 10 es . mol Thustheintracellular[K+]ofthecellisnotappreciablyalteredbythediffusionofK+ outofthecell. Regulation of Cell Volume As already noted, changes in cell volume can lead to cell damage and death. Cells have developed mechanisms to regulate their volume. Most cells are highly permeable by water because of the presence of aquaporins in their plasma membranes. As discussed in

1	Chapter 1, osmotic pressure gradients across the cell membrane that are generated by effective osmoles cause water to move either into or out of the cell, which result in changes in cell volume. Thus cells swell when placed in hypotonic solutions and shrink when placed in hypertonic solutions (see the section ”). However, even when a cell is placed in an isotonic solution, the maintenance of cell volume is an active process requiring the expenditure of ATP and specifically the activity of the Na+,K+-ATPase.

1	The importance of the Na+,K+-ATPase in isotonic cell volume regulation can be appreciated by the observation that red blood cells swell when chilled (i.e., reduced ATP synthesis) or when the Na+,K+-ATPase is inhibited by cardiac glycosides (e.g., ouabain, digoxin [Lanoxin]). The necessity for energy expenditure to maintain cell volume in an isotonic solution is the result of the effect of intracellular proteins on the distribution of ions across the plasma membrane: the so-called Gibbs-Donnan effect ( Fig. 2.6

1	Fig. 2.6 The Gibbs-Donnan effect occurs when a membrane separating two solutions can be permeated by some but not all of the molecules in solution. As noted previously, this effect accounts for the small differences in the ionic compositions of the plasma and the interstitial fluid. In this case, the capillary endothelium represents the membrane, and the plasma proteins are the molecules whose ability to permeate across the capillary is restricted. For cells, the membrane is the plasma membrane, and the impermeant molecules are the intracellular proteins and organic molecules.

1	Fig. 2.6 , the presence of impermeant molecules (e.g., protein) in one compartment results over time in the accumulation of permeant molecules/ions in the same compartment. This increases the number of osmotically active particles in the compartment containing the impermeant anions, which in turn increases the osmotic pressure, and water thereby enters that compartment. For cells, the Gibbs-Donnan effect would increase the number of osmotically active particles in the cell, and result in cell swelling. However, the activity of the Na+,K+-ATPase counteracts the Gibbs-Donnan effect by actively extruding cations (three Na+ ions are extruded, whereas two K+ ions are brought into the cell). In addition, the K+ gradient established by the Na+,K+-ATPase allows for the development of the Vm (in which the cell’s interior is electrically negative), that in turn drives Cl− and other anions out of the cell. Thus through the activity of the Na+,K+-ATPase, the number of intracellular osmotically

1	which the cell’s interior is electrically negative), that in turn drives Cl− and other anions out of the cell. Thus through the activity of the Na+,K+-ATPase, the number of intracellular osmotically active particles is

1	B the intracellular osmotic pressure and thereby restoring cell volume to normal. Conversely with cell shrinking a regulatory volume increase response transports osmolytes into the cell, raising the intracellular osmotic pressure and thereby restoring cell volume to normal. These osmolytes include ions and organic molecules such as polyols (sorbitol and myo-inositol), methylamines (glycerophosphorylcholine and betaine), and some amino acids (taurine, glutamate, and β-alanine). If the cell is exposed to the nonisotonic ECF for an extended period of time, the cell alters the intracellular levels of the organic osmolytes through metabolic processes.

1	•Fig. 2.6 The Gibbs-Donnan Effect. Top, TwosolutionsareseparatedbyamembranethatispermeablebyNa+ ,Cl− ,andH2Obutnotpermeablebyprotein(P−).TheosmolalityofsolutionAisidenticaltothatofsolutionB.Bottom, Cl− diffusesfromcompartmentBtocompartmentAdownitsconcentrationgradient.ThiscausescompartmentAtobecomeelectricallynegativewithregardtocompartmentB.ThemembranevoltagethendrivesthediffusionofNa+ fromcompartmentBtocompartmentA.TheaccumulationofadditionalNa+ andCl− incompartmentAincreasesitsosmolalityandcauseswatertoflowfromcompartmentBtocompartmentA(Note:theincreasevolumeofcompartmentAresultsinalower[P−]).IfthecontainercontainingthetwosolutionsweresealedatthetopsothatwatercouldnotmovefromcompartmentBtocompartmentA,thepressureincompartmentAwouldincreaseasthenumberofosmoticallyactiveparticlesincreasesinthatcompartment. reduced from what would be caused by the Gibbs-Donnan effect, and cell volume is maintained in isotonic solutions.

1	reduced from what would be caused by the Gibbs-Donnan effect, and cell volume is maintained in isotonic solutions. Most cells throughout the body are bathed with isotonic ECF, the composition of which is tightly regulated (see ). However, certain regions within the body are not isotonic (e.g., the medulla of the kidney), and with disorders of water balance, the ECF can become either hypotonic or hypertonic. When this occurs, cells either swell or shrink. Cell swelling or shrinkage can result in cell damage or death, but many cells have mechanisms that limit the degree to which the cell volume changes. These mechanisms are particularly important for neurons, in which swelling within the confined space of the skull can lead to serious neurological damage. In general, when a cell is exposed to nonisotonic ECF, volume-regulatory responses are activated within seconds to minutes to restore cell volume (

1	In general, when a cell is exposed to nonisotonic ECF, volume-regulatory responses are activated within seconds to minutes to restore cell volume ( Fig. 2.7 ). With cell swelling, a regulatory volume decrease response transports osmotically active particles (osmolytes) out of the cell, reducing

1	Fig. 2.7 ). With cell swelling, a regulatory volume decrease response transports osmotically active particles (osmolytes) out of the cell, reducing TheECFofindividualswithdisordersinwaterbalancemaybeeitherhypotonic(positivewaterbalance)orhypertonic(negativewaterbalance).WithadecreaseinECFosmolality,neuronsandglialcellsswellaswaterentersthecell.Tominimizethisswelling,theneuronsandglialcellsreduceintracellularosmolytes.IftheECFosmolalityiscorrected(i.e.,increased)tooquickly,theneuronsandglialcellsthenshrinkbecauseofthereducednumberofintracellularosmolytes.ThisresponsetoarapidcorrectionofECFosmolalitycanleadtocelldamage.Damagetotheglialcellsthatsynthesizemyelinwithinthebraincanresultindemyelinization.Thisdemyelinizationresponse,termedosmotic demyelinization syndrome, canaffectanyofthewhitematterofthebrain,butespeciallyregionsofthepons.Theseeffectsareoftenirreversible.Therefore,correctionofdisordersofwaterbalanceisusuallyaccomplishedslowlytoavoidthisseriousneurologicalcomplication.

1	The regulatory volume increase response results in the rapid uptake of NaCl and a number of organic osmolytes. To increase cell volume there is an activation of the Na+-H+ antiporter (NHE-1), the 1Na+,1K+,2Cl− symporter (NKCC1), and a number of cation-selective channels, which together bring NaCl into the cell. The Na+,K+-ATPase then extrudes the Na+ in exchange for K+ , so that ultimately the KCl content of the cell is increased. Several organic osmolyte transporters are also activated to increase cell volume. These include a 3Na+,1Cl−-taurine symporter, a 3Na+,2Cl−-betaine symporter, a 2Na+–myo-inositol symporter, and a Na+– amino acid symporter. These transporters use the energy in the Na+ and Cl− gradients to drive the secondary active uptake of these organic osmolytes into cells.

1	The regulatory volume decrease response results in the loss of KCl and organic osmolytes from the cell. The loss of KCl occurs through the activation of a wide range of K+selective, Cl−-selective, and anion-selective channels (the specific channels involved vary depending on the cell), as well as through activation of K+-Cl− symporters. Some of the organic osmolytes appear to leave the cell via anion channels (e.g., volume-sensitive organic osmolyte-anion channels). Several mechanisms are involved in activation of these various transporters during the volume regulatory

1	CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments 2Cl Na+, Cl– Na+ + •Fig. 2.7 Volume Regulation of Cells in Hypotonic and Hypertonic Media. Top, Whencellsareexposedtoahypotonicmedium,theyswellandthenundergoavolume-regulatorydecrease(RVD).TheRVDinvolveslossofKClandorganicosmolytesfromthecell.ThedecreaseincellularKClandorganicosmolytescausesintracellularosmoticpressuretodecrease,waterleavesthecell,andthecellreturnstonearlyitsoriginalvolume.Bottom, Whencellsareexposedtoahypertonicmedium,theyshrinkandthenundergoavolume-regulatoryincrease(RVI).DuringtheRVI,NaClandorganicosmolytesenterthecell.TheincreaseintheactivityofNa+,K+-ATPase(notdepicted)enhancestheexchangeNa+ forK+ sothattheK+ (andCl−)contentofthecellisincreased.TheincreaseincellularKCl,alongwithariseinintracellularorganicosmolytes,increasesintracellularosmoticpressure,whichbringswaterbackintothecell,andthecellvolumereturnstonearlyitsoriginalvolume.π,theoncoticpressureinsidethecell.

1	responses. Changes in cell volume appear to monitored by the cytoskeleton, by changes in macromolecular crowding and ionic strength of the cytoplasm, and by channels whose gating is influenced, either directly or indirectly, by stretch of the plasma membrane (e.g., stretch-activated cation channels). A number of second messenger systems may also be involved in these responses (e.g., intracellular [Ca++], calmodulin, protein kinase A, and protein kinase C), but the precise mechanisms have not been defined completely. Principles of Epithelial Transport

1	Principles of Epithelial Transport Epithelial cells are arranged in sheets and provide the interface between the external world and the internal environment (i.e., ECF) of the body. Depending on their location, epithelial cells serve many important functions, such as establishing a barrier to microorganisms (lungs, gastrointestinal tract, and skin), prevention of the loss of water from the body (skin), and maintenance of a constant internal environment (lungs, gastrointestinal tract, and kidneys). This latter function is a result of the ability of epithelial cells to carry out regulated vectorial transport (i.e., transport from one side of the epithelial cell sheet to the opposite side). In this section, the principles of epithelial transport are reviewed. The transport functions of specific epithelial cells are discussed in the appropriate chapters throughout this book.

1	Fig. 2.8 shows a schematic representation of an epithelial cell. The free surface of the epithelial layer is referred to as the apical membrane. It is in contact with the external environment (e.g., air within the alveoli and larger airways of the lungs and the contents of the gastrointestinal tract) or with extracellular fluids (e.g., glomerular filtrate in the nephrons of the kidneys and the secretions of the ducts of the pancreas or sweat glands). The basal side of the epithelium rests on a basal lamina, which is secreted by the epithelial cells, and this in turn is attached to the underlying connective tissue. Epithelial cells are connected to one another and to the underlying connective tissue by a number of specialized junctions (see

1	Epithelial cells are connected to one another and to the underlying connective tissue by a number of specialized junctions (see Fig. 2.8 ). The adhering junction, desmosomes, and hemidesmosomes provide mechanical adhesion by linking together the cytoskeleton of adjacent cells (adhering junction and desmosome) or to the underlying connective tissue (hemidesmosome). The gap junction and tight junction play important physiological roles. Gap junctions provide low-resistance connections between cells. The functional unit of the gap junction is the connexon. The connexon is composed of six integral gGap junctions are not limited to epithelial cells. A number of other cells also have gap junctions (e.g., cardiac myocytes and smooth muscle cells). •Fig. 2.8 Schematic of an Epithelial Cell, Illustrating the Various Adhering Junctions. Thetightjunctionseparatestheapicalmembranefrombasolateralmembrane(seetextfordetails).

1	•Fig. 2.8 Schematic of an Epithelial Cell, Illustrating the Various Adhering Junctions. Thetightjunctionseparatestheapicalmembranefrombasolateralmembrane(seetextfordetails). membrane protein subunits called connexins. A connexon in one cell is aligned with the connexon in the adjacent cell, forming a channel. The channel may be gated, and when it is open, it allows the movement of ions and small molecules between cells. Because of their low electrical resistance, they effectively couple electrically one cell to the adjacent cell.

1	The tight junction serves two main functions. It divides the cell into two membrane domains (apical and basolateral) and, in so doing, restricts the movement of membrane lipids and proteins between these two domains. This so-called fence function allows epithelial cells to carry out vectorial transport from one surface of the cell to the opposite surface by segregating membrane transporters to one or other of the membrane domains. They also serve as a pathway for the movement of water, ions, and small molecules across the epithelium. This pathway between the cells is referred to as the paracellular pathway, as opposed to the transcellular pathway through the cells. The apical surface of epithelial cells may have specific structural features. One such feature is microvilli (Fig.

1	The apical surface of epithelial cells may have specific structural features. One such feature is microvilli (Fig. 2.9A ). Microvilli are small (typically 1 to 3 µm in length), nonmotile projections of the apical plasma membrane that serve to increase surface area. They are commonly located on cells that must transport large quantities of ions, water, and molecules (e.g., epithelial cells lining the small intestine and cells of the renal proximal tubule). The core of the microvilli is composed of actin filaments and a number of accessory proteins. This actin core is connected to the cytoskeleton of the cell via the terminal web (a network of actin fibers at

1	Epithelialcelltightjunctions(alsocalledzonula occludens)arecomposedofseveralintegralmembraneproteins,includingoccludins, claudins, andseveralmembersoftheimmunoglobulinsuperfamily(e.g.,thejunctional adhesion molecule [JAM]).Occludinsandclaudinsaretransmembraneproteinsthatspanthemembraneofonecellandlinktotheextracellularportionofthesamemoleculeintheadjacentcell.Cytoplasmiclinkerproteins(e.g.,tightjunctionprotein[ZO-1,ZO-2,andZO-3])thenlinkthemembranespanningproteinstothecytoskeletonofthecell.

1	Ofthesejunctionalproteins,claudinsappeartobeimportantindeterminingthepermeabilitycharacteristicsofthetightjunction,especiallywithregardtocationsandanions.Todate,27mammalianclaudingeneshavebeenidentified,and26arefoundinthehumangenome(thegeneforclaudin13isnotfoundinhumans).Certainclaudinsserveasbarrierproteinsthatrestrictthemovementofionsthroughthetightjunction,whereasothersforma“pore”thatfacilitatesthemovementofionsthroughthejunction.Thusthepermeabilitycharacteristicsofthetightjunctionofanepitheliumaredeterminedbythecomplementofclaudinsexpressedbythecell.Forexample,theproximaltubuleofthekidneyistermeda“leaky”epithelium,inwhichwaterandsolutes(e.g.,Na+)movethroughthejunction.Claudin4andclaudin10areexpressedinthetightjunctionofproximaltubulecells.Incontrast,thecollectingductofthekidneyisconsidereda“tight”epithelium,withrestrictedmovementofionsthroughthetightjunction.Collectingductcellsexpressclaudins3,4,7,8,10,and18.

1	Thefunctionofclaudinscanberegulatedatseverallevels,includinggeneexpression,posttranslationalmodification,interactionswithcytoplasmicscaffoldingproteins,andinteractionswithotherclaudinsinthesamemembrane(cis-interaction),aswellaswithclaudinsofadjacentcells(trans-interaction).ThemineralocorticoidhormonealdosteronestimulatesNa+ reabsorptionbydistalsegmentsoftherenalnephron(see ).Inadditiontothehormone’seffectonNa+ transportersinthecell,aldosteronealsoupregulatesexpressionofclaudin8inthetightjunction.Theincreasedexpressionofclaudin8reducestheabilityofNa+ topermeatethetightjunction,whichthenreducesthebackwardsleakofNa+ fromtheinterstitiumintothetubulelumen,therebyallowingmoreefficientNa+ reabsorptionbytheepithelium.

1	Mutationsinthegenethatcodesforclaudin16resultintheautosomalrecessiveconditionknowasfamilial hypomagnesemia, hypercalcuria, and nephrocalcinosis (FHHNC).Claudin16isfoundinthetightjunctionofthethickascendingportionofHenle’sloopinthekidneysandservesasaroutefortheparacellularreabsorptionofCa++ andMg++ fromthetubularfluid.IndividualswithFHHNClackfunctionalcopiesofclaudin16,andreabsorptionofthesedivalentionsisthusreduced,whichleadstohypomagnesemia,hypercalcuria,andnephrocalcinosis.

1	CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments 29 • Fig. 2.9 Illustration of Apical Membrane Specializations of Epithelial Cells (Not Drawn to Scale). A, Microvilli 1 to 3 µm in length serve to increase the surface area of the apical membrane (e.g., those of the epithelial cells of the small intestine). B, Stereocilia can be up to 120 µm in length (e.g., those of the epididymis of the male reproductive tract). Both microvilli and stereocilia have a core structure composed primarily of actin, with a number of associated proteins. Both are nonmotile. (Redrawn from Pawlina, W. Histology: A Text and Atlas, with Correlated Cell and Molecular Biology. 7th ed. Philadelphia: Wolters Kluwer Health, 2016.) the base of the microvilli) and provides structural support for the microvilli. Another surface feature is stereocilia (see

1	Fig. 2.9B ). Stereocilia are long (up to 120 µm), nonmotile membrane projections that, like microvilli, increase the surface area of the apical membrane. They are found in the epididymis of the testis and in the “hair cells” of the inner ear. Their core also contains actin filaments and accessory proteins. A third apical membrane feature is cilia ( Fig. 2.10 ). Cilia may be either motile (called secondary cilia) or non-motile (called primary cilia). The motile cilia contain a microtubule core arranged in a characteristic “9+2” pattern (nine pairs of microtubules around the circumference of the cilium, and one pair of microtubules in the center). Dynein is the molecular motor that drives the movement of the cilium. Motile cilia are characteristic features of the epithelial cells that line the respiratory tract. They pulsate in a synchronized manner and serve to transport mucus and inhaled particulates out of the lung, a process termed mucociliary transport (see Chapter 26).

1	Chapter 26). Nonmotile cilia serve as mechanoreceptors and are involved in determining left-right asymmetry of organs during embryological development, as well as sensing the flow rate of fluid in the nephron of the kidneys (see ). Only a single nonmotile cilium is found in the apical membrane of cells. Nonmotile cilia have a microtubule core (“9+0” arrangement) and lack a motor protein. As noted previously, the tight junction effectively divides the plasma membrane of an epithelial cell into two domains: an apical surface and a basolateral surface. The basolateral membrane of many epithelial cells is folded or invaginated. This is especially so for epithelial cells that have high transport rates. These invaginations serve to increase the membrane surface area to accommodate the large number of membrane transporters (e.g., Na+,K+-ATPase) needed in the membrane.

1	Because the tight junction divides the plasma membrane into two domains (i.e., apical and basolateral), epithelial cells are capable of vectorial transport, whereby an ion or molecule can be transported from one side of the epithelial sheet to the opposite side ( Fig. 2.11 ). The accomplishment of vectorial transport requires that specific membrane transport proteins be targeted to and remain in one or the other of the membrane domains. In the example shown in

1	Fig. 2.11 , the Na+ channel is present only in the apical •Fig. 2.10 Ciliaareapicalmembranespecializationsofsomeepithelialcells.Ciliaare5to10µminlengthandcontainarraysofmicrotubules,asdepictedinthesecross-sectiondiagrams.Left, Theprimaryciliumhasnineperipheralmicrotubulearrays.Itisnonmotileandservesasamechanoreceptor(e.g.,cellsoftherenalcollectingduct).Cellsthathaveaprimaryciliumhaveonlyasinglecilium.Right, Thesecondaryciliumhasacentralpairofmicrotubulesinadditiontothenineperipheralmicrotubulearrays.Alsointhesecondarycilium,themotorproteindyneinisassociatedwiththemicrotubulearraysandthereforeismotile.Asinglecellcanhavethousandsofsecondaryciliaonitsapicalsurface(e.g.,epithelialcellsoftherespiratorytract).(RedrawnfromRodat-DespoixL,DelmasP.Ciliaryfunctionsinthe nephron.Pflugers Archiv.2009;458:179.) membrane, whereas the Na+,K+-ATPase and the K+ channels are confined to the basolateral membrane. The operation of the Na+,K+-ATPase channel and the leakage of K+ out of the cell across the

1	membrane, whereas the Na+,K+-ATPase and the K+ channels are confined to the basolateral membrane. The operation of the Na+,K+-ATPase channel and the leakage of K+ out of the cell across the basolateral membrane sets up a large electrochemical gradient for Na+ to enter the cell across the apical membrane through the Na+ channel (intracellular [Na+] < extracellular [Na+], and Vm which is oriented with the cell’s interior electrically negative with respect to the cell’s exterior). The Na+ is then pumped out of the cell by the Na+,K+-ATPase, and vectorial transport from the apical side of the epithelium to the basolateral side of the epithelium occurs. Transport from the apical side to the basolateral side of an epithelium is termed either absorption or reabsorption: For example, the uptake of nutrients from the lumen of the gastrointestinal tract is termed absorption, whereas the transport of NaCl and water from the lumen of the renal nephrons is termed reabsorption. Transport from the

1	of nutrients from the lumen of the gastrointestinal tract is termed absorption, whereas the transport of NaCl and water from the lumen of the renal nephrons is termed reabsorption. Transport from the basolateral side of the epithelium to the apical side is termed secretion.

1	As noted previously, the Na+,K+-ATPase and K+-selective channels play an important role in establishing cellular ion gradients for Na+ and K+ and in generating the Vm. In all epithelial cells except the choroid plexus and retinal pigment epithelium, the Na+,K+-ATPase channel is located in the basolateral membrane of the cell. Numerous K+selective channels are in epithelial cells and may be located in either membrane domain. Through the establishment of these chemical and voltage gradients, the transport of other ions and solutes can be driven (e.g., Na+-glucose symporter, Na+-H+ antiporter, 1Na+,1K+,2Cl− symporter, 1Na+-3HCO3 − symporter). The direction of transepithelial transport (reabsorption or secretion) depends simply on which membrane domain the transporters are located. Because of the dependence on the Na+,K+-ATPase, epithelial transport requires the expenditure of energy. Other ATP-dependent transporters, such as the H+-ATPase, H+,K+-ATPase, and a host of ABC

1	Because of the dependence on the Na+,K+-ATPase, epithelial transport requires the expenditure of energy. Other ATP-dependent transporters, such as the H+-ATPase, H+,K+-ATPase, and a host of ABC transporters—such as P-glycoprotein (PGP) and multidrug resistance-associated protein 2 (MRP2), which transport xenobiotics (drugs), and cystic fibrosis transmembrane conductance regulator (CFTR), which transports Cl−—are involved in epithelial transport.

1	hThe choroid plexus is located in the ventricles of the brain and secretes the cerebrospinal fluid. The Na+,K+-ATPase channel is located in the apical membrane of these cells. CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments •Fig. 2.11 Insymmetricalcells(A; e.g.,redbloodcells),membranetransportproteinsaredistributedovertheentiresurfaceofthecell.Epithelialcells(B), incontrast,areasymmetricalandtargetvariousmembranetransportproteinstoeithertheapicalorthebasolateralmembrane.Whenthetransportersareconfinedtoamembranedomain,vectorialtransportcanoccur.Inthecelldepicted,Na+ istransportedfromtheapicalsurfacetothebasolateralsurface.ATP,adenosinetriphosphate.

1	Solutes and water can be transported across an epithelium by traversing both the apical and basolateral membranes (transcellular transport) or by moving between the cells across the tight junction (paracellular transport). Solute transport via the transcellular route is a two-step process, in which the solute molecule is transported across both the apical and basolateral membrane. Uptake into the cell, or transport out of the cell, may be either a passive or an active process. Typically, one of the steps is passive, and the other is active. For the example shown in

1	Fig. 2.11B , the uptake of Na+ into the cell across the apical membrane through the Na+-selective channel is passive and driven by the electrochemical gradient for Na+ . The exit of Na+ from the cell across the basolateral membrane is primary active transport via the Na+,K+-ATPase channel. Because a transepithelial gradient for Na+ can be generated by this process (i.e., the [Na+] in the apical compartment can be reduced below that of the basolateral compartment, the overall process of transepithelial Na+ transport is said to be active). Any solute that is actively transported across an epithelium must be transported via the transcellular pathway.

1	Depending on the epithelium, the paracellular pathway is an important route for transepithelial transport of solute and water. As noted, the permeability characteristics of the paracellular pathway are determined, in large part, by the specific claudins that are expressed by the cell. Thus the tight junction can have low permeability for solutes, water, or both, or it can have a high permeability. For epithelia in which there are high rates of transepithelial transport, •Fig. 2.12 The Electrical Profile Across an Epithelial Cell. Themagnitudeofthemembranevoltages,andthetransepithelialvoltagearedeterminedbythevariousmembranetransportproteinsintheapicalandbasolateralmembranes.Thetransepithelialvoltageisequaltothesumoftheapicalandbasolateralmembranevoltages(seetextfordetails).

1	the tight junctions typically have a high permeability (i.e., are leaky). Examples of such epithelia include the proximal tubule of the renal nephron and the early segments of the small intestine (e.g., duodenum and jejunum). If the epithelium must establish large transepithelial gradients for solutes, water, or both, the tight junctions typically have low permeability (i.e., are tight). Examples of this type of epithelium include the collecting duct of the renal nephron, the urinary bladder, and the terminal portion of the colon. In addition, the tight junction may be selective for certain solutes (e.g., cation versus anion selective).

1	All solute transport that occurs through the paracellular pathway is passive in nature. The two driving forces for this transport are the transepithelial concentration gradient for the solute and, if the solute is charged, the transepithelial voltage (Fig. 2.12 ). The transepithelial voltage may be oriented with the apical surface electrically negative in relation to the basolateral surface as shown in Fig. 2.12 , or it may be oriented with the apical surface electrically positive in relation to the basolateral surface. The polarity and magnitude of the transepithelial voltage is determined by the specific membrane transporters in the apical and basolateral membranes, as well as by the permeability characteristics of the tight junction. It is important to recognize that transcellular transport processes set up the transepithelial chemical and voltage gradients, which in turn can drive paracellular transport. This is illustrated in

1	It is important to recognize that transcellular transport processes set up the transepithelial chemical and voltage gradients, which in turn can drive paracellular transport. This is illustrated in Fig. 2.13 for an epithelium that reabsorbs NaCl and for an epithelium that secretes NaCl. In both epithelia, the transepithelial voltage is oriented with the apical surface electrically negative in relation to the basolateral surface. For the NaCl-reabsorbing epithelium, the transepithelial voltage is generated by the active, transcellular reabsorption of Na+ . This voltage in turn drives Cl− reabsorption through the paracellular pathway. In contrast, for the NaCl-secreting epithelium, the transepithelial voltage is generated by the active transcellular secretion of Cl− . Na+ is then secreted passively via the paracellular pathway, driven by the negative transepithelial voltage.

1	Water movement across epithelia is passive and driven by transepithelial osmotic pressure gradients. Water movement can occur by a transcellular route involving aquaporins in both the apical and basolateral membranes. In addition, water may also move through the paracellular pathway. In the NaCl-reabsorbing epithelium depicted in Fig. 2.13A , the reabsorption of NaCl from the apical compartment lowers the osmotic pressure in that compartment, whereas the addition of NaCl to the basolateral compartment raises the osmotic pressure in that compartment. As a result, a transepithelial osmotic pressure gradient is established that drives the movement of water from the apical to the basolateral compartment (i.e., reabsorption). The opposite occurs with NaCl-secreting epithelia (see Fig. 2.13B ), in which the transepithelial secretion of NaCl establishes a transepithelial osmotic pressure gradient that drives water secretion.

1	Fig. 2.13B ), in which the transepithelial secretion of NaCl establishes a transepithelial osmotic pressure gradient that drives water secretion. In some epithelia (e.g., proximal tubule of the renal nephron), the movement of water across the epithelium via the paracellular pathway can drive the movement of additional solute. This process is termed solvent drag and reflects the fact that solutes dissolved in the water will traverse the tight junction with the water. As is the case with the establishment of transepithelial concentration and voltage gradients, the establishment of transepithelial osmotic pressure gradients requires transcellular transport of solutes by the epithelial cells. iDifferent aquaporin isoforms are often expressed in the apical and basolateral membrane. In addition, multiple isoforms may be expressed in one or more of the membrane domains. CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments

1	CHAPTER 2 Homeostasis: Volume and Composition of Body Fluid Compartments NaCl reabsorption •Fig. 2.13 The Role of the Paracellular Pathway in Epithelial Transport. A, Na+ transportthroughthecellgeneratesatransepithelial voltagethatthendrivesthepassivemovementofCl− throughthetightjunction.NaClreabsorptionresults.B, Cl− transportthroughthecellgeneratesatransepithelialvoltagethatthendrivesthepassivetransportofNa+ throughthetightjunction.NaClsecretionresults. Regulation of Epithelial Transport

1	Epithelial transport must be regulated to meet the homeostatic needs of the individual. Depending on the epithelium, this regulation involves neural or hormonal mechanisms, or both. For example, the enteric nervous system of the gastrointestinal tract regulates solute and water transport by the epithelial cells that line the intestine and colon. Similarly, the sympathetic nervous system regulates transport by the epithelial cells of the renal nephron. Aldosterone, a steroid hormone produced by the adrenal cortex (see ), is an example of a hormone that stimulates NaCl transport by the epithelial cells of the colon, renal nephron, and sweat ducts. Epithelial cell transport can also be regulated by locally produced and locally acting substances, a process termed paracrine regulation. The stimulation of HCl secretion in the stomach by histamine is an example of this process. Cells that are located near the epithelial cells of the stomach release histamine, which acts on the HCl-secreting

1	of HCl secretion in the stomach by histamine is an example of this process. Cells that are located near the epithelial cells of the stomach release histamine, which acts on the HCl-secreting cells of the stomach (parietal cells) and stimulates them to secrete HCl.

1	When acted upon by a regulatory signal, the epithelial cell may respond in several different ways, including: • Retrieval of transporters from the membrane, by endocytosis, or insertion of transporters into the membrane from an intracellular vesicular pool, by a process called Change in activity of membrane transporters (e.g., channel gating) Synthesis of specific transporters, and their insertion into the membrane Na+ The first two mechanisms can occur quite rapidly (seconds 2Cl– to minutes), but the synthesis of transporters takes addi-K+ tional time (minutes to days).

1	Na+ The first two mechanisms can occur quite rapidly (seconds 2Cl– to minutes), but the synthesis of transporters takes addi-K+ tional time (minutes to days). The body maintains steady-state balance for water and a number of important solutes. This occurs when input into the body equals output from the body. For each solute and water, there is a normal set point. Deviations from this set point are monitored (i.e., when input ≠ output), and effector mechanisms are activated that restore balance. This balance is achieved by adjustment of either intake or excretion of water and solutes. Thereafter, input and output are again equal to maintain balance. The Na+,K+-ATPase and K+-selective channels are critically important in establishing and maintaining the intracellular composition, the membrane potential (Vm), and cell volume. Na+,K+-ATPase converts the energy in

1	Altenberg GA, Ruess L. Mechanisms of water transport across cell membranes and epithelia. In: Alpern R, Moe O, Kaplan M, eds. Seldin and Giebisch’s The Kidney—Physiology and Pathophysiology. 5th ed. New York: Academic Press; 2013. Günzel D, Yu ASL. Claudins and the modulation of tight junction permeability. Physiol Rev. 2013;93:525-569. Hoffman EK, et al. Physiology of cell volume regulation in vertebrates. Physiol Rev. 2009;89:193-277. Lang F. Cell volume control. In: Alpern R, Moe O, Kaplan M, eds. Seldin and Giebisch’s The Kidney—Physiology and Pathophysiology. 5th ed. New York: Academic Press; 2013. ATP into potential energy of ion gradients and the membrane potential. The ion and electrical gradients created by this process are then used to drive the transport of other ions and other molecules, especially by solute carriers (i.e., symporters and antiporters).

1	• Epithelial cells constitute the interface between the external world and the internal environment of the body. Vectorial transport of solutes and water across epithelia helps maintain steady-state balance for water and a number of important solutes. Because the external environment constantly changes, and because dietary intake of food and water is highly variable, transport by epithelia is regulated to meet the homeostatic needs of the individual. Pawlina W. Histology: A Text and Atlas, with Correlated Cell and Molecular Biology. 7th ed. Philadelphia: Wolters Kluwer Health; 2016. Pedersen SF, Kapus A, Hoffmann EK. Osmosensory mechanisms in cellular and systemic volume regulation. J Am Soc Nephrol. 2011;22:1587-1597. Sackin H, Palmer LG. Electrophysiological analysis of transepithelial transport. In: Alpern R, Moe O, Kaplan M, eds. Seldin and Giebisch’s The Kidney—Physiology and Pathophysiology. 5th ed. New York: Academic Press; 2013.

1	3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression Upon completion of this chapter, the student should be able to answer the following questions: 1. How do cells communicate with each other? 2. What are the four classes of receptors, and what signal transduction pathways are associated with each class of receptors? 3. How do steroid and thyroid hormones, cyclic adenosine monophosphate, and receptor tyrosine kinases regulate gene expression?

1	he human body is composed of billions of cells, each with a distinct function. However, the function of cells is tightly coordinated and integrated by external chemical signals, including hormones, neurotransmitters, growth factors, odorants, and products of cellular metabolism that serve as chemical messengers and provide cell-to-cell communication. Mechanical and thermal stimuli and light are physical external signals that also coordinate cellular function. Chemical and physical messengers interact with receptors located in the plasma membrane, cytoplasm, and nucleus. Interaction of these messengers with receptors initiates a cascade of signaling events that mediate the response to each stimulus. These signaling pathways ensure that the cellular response to external messengers is specific, amplified, tightly regulated, and coordinated. This chapter provides an overview of how cells communicate via external messengers and a discussion of the signaling pathways that process external

1	amplified, tightly regulated, and coordinated. This chapter provides an overview of how cells communicate via external messengers and a discussion of the signaling pathways that process external information into a highly coordinated cellular response. In subsequent chapters, details on signaling pathways in the nervous system, muscular system, cardiovascular system, respiratory system, gastrointestinal system, renal system, and endocrine system are discussed in greater detail.

1	The significance of signaling pathways in medicine is illustrated by the following short list of popular drugs that act by regulating signaling pathways. Details on these pathways are presented later in this and other chapters. Aspirin, the first pharmaceutical (1899), inhibits cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX2) and therefore is antithrombotic (i.e., reduces the formation of blood clots). β-Adrenergic receptor agonists and antagonists are used to treat a variety of medical conditions. β1-Agonists increase cardiac contractility and heart rate in patients with low blood pressure. β2-Agonists dilate bronchi and are used to treat asthma and chronic obstructive lung disease. In contrast, β-adrenergic antagonists are used to treat hypertension, angina, cardiac arrhythmias, and congestive heart failure (see Chapter 18).

1	Chapter 18). Fluoxetine (Prozac) is an antidepressant medication that inhibits reuptake of the neurotransmitter serotonin into the presynaptic cell, which results in enhanced activation of serotonin receptors (see Several monoclonal antibodies are used to treat cancer caused by the activation of growth factor receptors in cancer cells. For example, trastuzumab (Herceptin) is a monoclonal antibody used to treat metastatic breast cancer in women who overexpress HER2/neu, a member of the family of epidermal growth factor (EGF) receptors, which stimulate cell growth and differentiation. Cetuximab (Erbitux) and bevacizumab (Avastin) are monoclonal antibodies that are used to treat metastatic colorectal cancer and cancers of the head and neck. These antibodies bind to and inhibit the EGF receptor and thereby inhibit EGF-induced cell growth in cancer cells.

1	Drugs that inhibit cyclic guanosine monophosphate (cGMP)–specific phosphodiesterase type 5, such as sildenafil (Viagra), tadalafil (Cialis), and vardenafil (Levitra), prolong the vasodilatory effects of nitric oxide and are used to treat erectile dysfunction and pulmonary arterial hypertension (see Chapter 17). An overview of how cells communicate with each other is presented in

1	Fig. 3.1 . Cells communicate by releasing extracellular signaling molecules (e.g., hormones and neurotransmitters) that bind to receptor proteins located in the plasma membrane, cytoplasm, or nucleus. This signal is transduced into the activation, or inactivation, of one or more intracellular messengers by interacting with receptors. Receptors interact with a variety of intracellular signaling proteins, including kinases, phosphatases, and guanosine triphosphate (GTP)–binding proteins (G proteins). These signaling proteins interact with and regulate the activity of target proteins and thereby modulate cellular function. Target proteins include, but are not limited to, ion channels and other transport proteins, metabolic enzymes, cytoskeletal proteins, gene regulatory proteins, and cell cycle proteins that regulate cell growth and division. Signaling pathways are characterized by (1) multiple, hierarchical steps; (2) amplification of the signal-receptor binding event, which magnifies

1	proteins that regulate cell growth and division. Signaling pathways are characterized by (1) multiple, hierarchical steps; (2) amplification of the signal-receptor binding event, which magnifies the response; (3) activation of multiple pathways and regulation of multiple cellular functions; mechanisms, which minimize the response and provide tight regulatory control over these signaling pathways. A brief description of how cells communicate follows. Readers who desire a more in-depth presentation of this material are encouraged to consult one of the many cellular and molecular biology textbooks currently available.

1	Cells in higher animals release into the extracellular space hundreds of chemicals, including (1) peptides and proteins (e.g., insulin); (2) amines (e.g., epinephrine and norepinephrine); (3) steroid hormones (e.g., aldosterone, estrogen); and (4) small molecules, including amino acids, nucleotides, ions (e.g., Ca++), and gases, such as nitric oxide and carbon dioxide. Secretion of signaling molecules is • Fig. 3.1 An Overview of How Cells Communicate. A signaling molecule (i.e., hormone or neurotransmitter) binds to a receptor, which may be in the plasma membrane, cytosol, or nucleus. Binding of ligand to a receptor activates intracellular signaling proteins, which interact with and regulate the activity of one or more target proteins to change cellular function. Signaling molecules regulate cell growth, division, and differentiation and influence cellular metabolism. In addition, they modulate the intracellular ionic composition by regulating the activity of ion channels and

1	regulate cell growth, division, and differentiation and influence cellular metabolism. In addition, they modulate the intracellular ionic composition by regulating the activity of ion channels and transport proteins. Signaling molecules also control cytoskeleton-associated events, including cell shape, division, and migration and cell-to-cell and cell-to-matrix adhesion. (Redrawn from Alberts B, et al: Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.)

1	CHAPTER 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression 37 cell-type specific. For example, beta cells in the pancreas release insulin, which stimulates glucose uptake into cells. The ability of a cell to respond to a specific signaling molecule depends on the expression of receptors that bind the signaling molecule with high affinity and specificity. Receptors are located in the plasma membrane, the cytosol, and the nucleus ( Fig. 3.2 Fig. 3.3). Contact-dependent signaling, in which a membrane-bound signaling molecule of one cell binds directly to a plasma membrane receptor of another cell, is important during development, in immune responses, and in cancer (see Fig. 3.3A ). Molecules that are released and act locally are called paracrine (see Fig. 3.3B

1	Fig. 3.3C ) hormones. Paracrine signals are released by one type of cell and act on another type; they are usually taken up by target cells or rapidly degraded (within • Fig. 3.2 Signaling molecules, especially ones that are hydrophilic and cannot cross the plasma membrane, bind directly to their cognate receptors in the plasma membrane (A). Other signaling molecules— including steroid hormones, triiodothyronines, retinoic acids, and vitamin D—bind to carrier proteins in blood and readily diffuse across the plasma membrane, where they bind to cognate nuclear receptors in the cytosol or nucleus (B). Still other signaling molecules, including nitric oxide, can diffuse without carrier proteins and cross the membrane to act on intracellular protein targets (B). Both classes of receptors, when ligand bound, regulate gene transcription. mRNA, messenger RNA. (Redrawn from Alberts B, et al: Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.) minutes) by enzymes. For

1	when ligand bound, regulate gene transcription. mRNA, messenger RNA. (Redrawn from Alberts B, et al: Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.) minutes) by enzymes. For example, enterochromaffin-like cells in the stomach secrete histamine, which stimulates the production of acid by neighboring parietal cells (see for details). Autocrine signaling involves the release of a molecule that affects the same cell or other cells of the same type (e.g., cancer cells). In synaptic signaling (see

1	Fig. 3.3D ), neurons transmit electrical signals along their axons and release neurotransmitters at synapses that affect the function of other neurons or cells that are distant from the neuron cell body. The close physical relationship between the nerve terminal and the target cell ensures that the neurotransmitter is delivered to a specific cell. Details on synaptic signaling are discussed in Chapter 6. Endocrine signals are hormones that are secreted into the blood and are widely dispersed in the body (see Fig. 3.3E ). Details on endocrine signaling are discussed in

1	Chapter 6. Endocrine signals are hormones that are secreted into the blood and are widely dispersed in the body (see Fig. 3.3E ). Details on endocrine signaling are discussed in In addition to paracrine, autocrine, endocrine, and synaptic signaling, cell-to-cell communication also occurs via gap junctions that form between adjacent cells (see ). Gap junctions are specialized junctions that allow intracellular signaling molecules, generally less than 1200 D in size, to diffuse from the cytoplasm of one cell to an adjacent cell. The permeability of gap junctions is regulated by cytosolic [Ca++], [H+], and cyclic adenosine monophosphate (cAMP) and by the membrane potential. Gap junctions also allow cells to be electrically coupled, which is vitally important for the coordinated activity of cardiac and smooth muscle cells (see

1	The speed of a response to an extracellular signal depends on the mechanism of delivery. Endocrine signals are relatively slow (seconds to minutes) because time is required for diffusion and blood flow to the target cell, whereas synaptic signaling is extremely fast (milliseconds). If the response involves changes in the activity of proteins in the cell, the response may occur in milliseconds to seconds. However, if the response involves changes in gene expression and the de novo synthesis of proteins, the response may take hours to occur, and a maximal response may take days. For example, the stimulatory effect of aldosterone on sodium transport by the kidneys requires days to develop fully (see

1	The response to a particular signaling molecule also depends on the ability of the molecule to reach a particular cell, on expression of the cognate receptor (i.e., receptors that recognize a particular signaling molecule or ligand with a high degree of specificity), and on the cytoplasmic signaling molecules that interact with the receptor. Thus signaling molecules frequently have many different effects that are dependent on the cell type. For example, the neurotransmitter acetylcholine stimulates contraction of skeletal muscle but decreases the force of contraction in heart muscle. This is because skeletal muscle and heart cells express different acetylcholine receptors. aThe acetylcholine receptor in skeletal muscle is termed nicotinic because nicotine can mimic this action of the neurotransmitter. In contrast, the acetylcholine receptor in cardiac muscle is termed muscarinic because this effect is mimicked by muscarine, an alkaloid derived from the mushroom Amanita muscaria.

1	Amanita muscaria. • Fig. 3.3 Cell-to-cell communication is mediated by five basic mechanisms: contact-dependent (A), paracrine (B), autocrine (C), synaptic (D), and endocrine signaling (E). These mechanisms are described in detail in the text. (Redrawn from Alberts B, et al: Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.) CHAPTER 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression Nuclear receptors Extracellular ligand: GABA ACh (muscle) ATP Glutamate: NMDA Intracellular ligand: Neurotransmitters (ACh) Peptides (PTH, oxytocin) Odorants Cytokines, lipids ANP TGF-β Insulin, EGF Interleukin-6, erythropoietin Steroid hormones: Miscellaneous hormones: Membrane currents: Cl− Na+ , K+ , Ca++ Ca++ , Na+ , K+ Na+ , K+ , Ca++ K+ Na+ , K+ Ca++ βγ Subunits activate ion channels α Subunit activates enzymes:

1	Miscellaneous hormones: Membrane currents: Cl− Na+ , K+ , Ca++ Ca++ , Na+ , K+ Na+ , K+ , Ca++ K+ Na+ , K+ Ca++ βγ Subunits activate ion channels α Subunit activates enzymes: Cyclases that generate cAMP, cGMP, phospholipases that generate InsP3 and diacylglycerol, and phospholipases that generate arachidonic acid and its metabolites. Monomeric G proteins Receptor guanylyl cyclase Receptor serine/threonine kinase Receptor tyrosine kinase Tyrosine kinase–associated receptor Bind to regulatory sequences in DNA and increase or Bind to regulatory sequences in DNA and increase or decrease gene transcription ACh, acetylcholine; ANP, atrial natriuretic peptide; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; EGF, epidermal growth factor; GABA, gamma-aminobutyric acid; InsP3, inositol 1,4,5-triphosphate; NMDA, N-methyl-D-aspartate; PTH, parathyroid hormone; TGF, transforming growth factor.

1	All signaling molecules bind to specific receptors that act as signal transducers, thereby converting a ligand-receptor binding event into intracellular signals that affect cellular function. Receptors can be divided into four basic classes on the basis of their structure and mechanism of action: (1) ligand-gated ion channels, (2) G protein–coupled receptors (GPCRs), (3) enzyme-linked receptors, and (4) nuclear receptors ( Table 3.1 Figs. 3.4 3.5). Fig. 3.4A). Neurotransmitters bind to receptors and either open or close ion channels, thereby changing the ionic permeability of the plasma membrane and altering the membrane potential. For examples and more details, see GPCRs regulate the activity of other proteins, such as enzymes and ion channels (see Fig. 3.4B ). In the example in

1	GPCRs regulate the activity of other proteins, such as enzymes and ion channels (see Fig. 3.4B ). In the example in Fig. 3.4B , the interaction between the receptor and the target protein is mediated by heterotrimeric G proteins, which are composed of α, β, and γ subunits. Stimulation of G proteins by ligand-bound receptors activates or inhibits downstream target proteins that regulate signaling pathways if the target protein is an enzyme or changes membrane ion permeability if the target protein is an ion channel. Enzyme-linked receptors either function as enzymes or are associated with and regulate enzymes (see Fig. 3.4C ). Most enzyme-linked receptors are protein kinases or are associated with protein kinases, and ligand binding causes the kinases to phosphorylate a specific subset of proteins on specific amino acids, which in turn activates or inhibits protein activity.

1	Nuclear receptors are small hydrophobic molecules, including steroid hormones, thyroid hormones, retinoids, and vitamin D, that have a long biological half-life (hours to days), diffuse across the plasma membrane, and bind to nuclear receptors or to cytoplasmic receptors that, once bound to their ligand, translocate to the nucleus (see Fig. 3.5 ). Some nuclear receptors, such as those that bind cortisol

1	Signal molecule as a dimer Signal molecule • Fig. 3.4 Three of the Four Classes of Plasma Membrane Receptors. See text for details. (Redrawn from Alberts B, et al: Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.) and aldosterone, are located in the cytosol and enter the nucleus after binding to hormone, whereas other receptors, including the thyroid hormone receptor, are located in the nucleus. In both cases, inactive receptors are bound to inhibitory proteins, and binding of hormone results in dissociation of the inhibitory complex. Hormone binding causes the receptor to bind coactivator proteins that activate gene transcription. Once activated, the hormone-receptor complex regulates the transcription of specific genes. Activation of specific genes usually occurs in two steps: an early primary response (≈30 minutes), which activates genes that stimulate other genes to produce a delayed (hours to days) secondary response (see Fig. 3.5).

1	Fig. 3.5). Each hormone elicits a specific response that is based on cellular expression of the cognate receptor, as well as on cell type–specific expression of gene regulatory proteins that interact with the activated receptor to regulate the transcription of a specific set of genes (see for more details). In addition to steroid receptors that regulate gene expression, evidence also suggests the existence of membrane and juxtamembrane steroid receptors that mediate the rapid, nongenomic effects of steroid hormones. Some membrane proteins do not fit the classic definition of receptors, but they subserve a receptor-like function in that they recognize extracellular signals and transduce the signals into an intracellular second messenger that has a biological effect. For example, on activation by a ligand, some membrane proteins undergo regulated intramembrane proteolysis (RIP), which elaborates a cytosolic peptide fragment that enters the nucleus and regulates gene expression (

1	Fig. 3.6 ). In this signaling pathway, binding of ligand to a plasma membrane receptor leads to ectodomain shedding, facilitated by members of the metalloproteinase-disintegrin family, and produces a carboxy-terminal fragment that is the substrate for γ-secretase. γ-Secretase induces RIP, thereby causing the release of an intracellular domain of CHAPTER 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression 41 • Fig. 3.5 Steroid Hormones Stimulate the Transcription of Early-Response Genes and Late-Response Genes. See text for details. (Redrawn from Alberts B, et al: Molecular Biology of Cell. 6th ed. New York: Garland Science; 2015.) Translocation to the nucleus • Fig. 3.6 Regulated Intramembrane Proteolysis. See text for details. (Redrawn from Alberts B, et al: Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.) the protein that enters the nucleus and regulates transcription (see

1	Fig. 3.6 ). The best characterized example of RIP is the sterol regulatory element–binding protein (SREBP), a transmembrane protein expressed in the membrane of the endoplasmic reticulum. When cellular cholesterol levels are low, SREBP undergoes RIP, and the proteolytically cleaved fragment is translocated into the nucleus, where it transcriptionally activates genes that promote cholesterol biosynthesis. When hormones bind to plasma membrane receptors, signals are relayed to effector proteins via intracellular signaling pathways. When hormones bind to nuclear or cytosolic receptors, they relay signals primarily through regulation of gene expression. Signaling pathways can amplify and integrate signals but can also downregulate and desensitize signals, reducing or terminating the response, even in the continued presence of hormone.

1	Alzheimer’s disease, a progressive neurodegenerative brain disease characterized by the formation of amyloid plaques, affects approximately 44 million people worldwide. In Alzheimer’s disease, regulated intramembrane proteolysis of amyloid β-protein precursor (APP) causes the accumulation of amyloid β-protein (Aβ), which forms amyloid plaques that contribute to the pathogenesis of Alzheimer’s disease. APP is a type I transmembrane protein (i.e., its spans the membrane only once). After ectodomain shedding, its sequential proteolysis by β-secretase and γ-secretase produces the Aβ40 and Aβ42 peptides that are normally produced throughout life but accumulate in individuals with Alzheimer’s disease. Missense mutations in presenilins, proteins that regulate γ-secretase protease activity, enhance the production of Aβ42, which is more hydrophobic and prone to aggregation into amyloid fibrils than is the more abundant Aβ40 protein.

1	Intracellular signaling molecules—so-called second messengers (the first messenger of the signal is the ligand that binds to the receptor)—include small molecules such as cAMP, cGMP, Ca++, and diacylglycerol. Signaling pathways often include dozens of small molecules that form complicated networks within the cell (Fig. 3.7 ). Some proteins in the intracellular signaling pathways relay the signal by passing the message directly to another protein (e.g., by phosphorylating a target, or by binding and causing an allosteric change). Such intracellular signaling proteins act as reversible molecular switches: When a signal is received, they switch from an inactive to an active form or vice versa, until another signaling molecule reverses the process. This principle of reversibility is central to many signaling pathways. In many cases, activation is achieved by reversing inhibition: For example, the thyroid hormone receptor is bound to an inhibitory protein in the absence of signal.

1	Signaling complexes, composed of multiple proteins that interact physically, enhance the speed, efficiency, and specificity of signaling. Many proteins, usually enzymes or ion channels, transduce the signal into a different chemical form and simultaneously amplify the signal either by producing large amounts of additional signaling molecules or by activating a large number of downstream signaling proteins. For example, adenylyl cyclase, the enzyme that makes cAMP, transduces a signal (receptor activation of G proteins) and amplifies the signal by generating large amounts of cAMP. Other types of signaling proteins include those that integrate multiple signals. Other proteins carry the signal from one region of the cell to another: for example, by translocating from the cytosol to the nucleus.

1	Cells can adjust rapidly to changing signals. Cells can respond quickly and in a graded manner to increasing concentrations of hormone, and the effect of a signaling molecule can be either long-or short-lived. Cells can also adjust their sensitivity to a signal by desensitization, whereby prolonged exposure to a hormone decreases the cell’s response over time. Desensitization is a reversible process that can involve a reduction in the number of receptors expressed in the plasma membrane, inactivation of receptors, or changes in signaling proteins that mediate the downstream effect of the receptors. Homologous desensitization involves a reduction in the response only to the signaling molecule that caused the response (e.g., opioid dependence and tolerance), whereas heterologous desensitization is when one ligand desensitizes the response to another ligand.

1	Table 3.1 summarizes the four general classes of receptors and provides a few examples of the signal transduction pathways associated with each class of receptors. This class of receptors transduces a chemical signal into an electrical signal, which elicits a response. For example, the ryanodine receptor, located in the membrane of the sarcoplasmic reticulum of skeletal muscle, is activated by Ca++ , caffeine, adenosine triphosphate (ATP), or metabolites of arachidonic acid to release Ca++ into the cytosol, which facilitates muscle contraction (see for details). In glutamergic synapses in which high levels of prior synaptic activity have led to partial membrane depolarization, activation of the N-methyl-D-aspartate receptor by glutamate stimulates Ca++ influx important for synaptic plasticity.

1	There are two classes of GTP-binding proteins (i.e., GTPases, which are named for their ability to hydrolyze GTP to guanosine diphosphate [GDP] and an inorganic phosphate): low-molecular-weight, monomeric G proteins and heterotrimeric G proteins composed of α, β, and γ subunits. GTP binding activates, whereas hydrolysis of GTP to GDP inactivates, GTP-binding proteins (Fig. 3.8A ). All GTPases are controlled by regulatory proteins, including GTPase-activating proteins, which induce the hydrolysis of GTP to GDP and thus inactivate the GTPase, and guanine nucleotide exchange factors (GEFs) that causes the GTPase to release GDP, which is rapidly replaced by GTP, thereby activating the GTPase (see Fig. 3.8B

1	Fig. 3.8B Monomeric G proteins are composed of a single 20to 40-kDa protein and can be membrane bound because of the addition of lipids posttranslationally. Monomeric G proteins have been classified into five families (Ras, Rho, Rab, Ran, and Arf), play a central role in many enzyme-linked receptor pathways, and regulate gene expression and cell proliferation, differentiation, and survival. Rho GTPases regulate actin cytoskeletal organization, cell cycle progression, and gene expression. The Rab GTPases regulate intravesicular transport and trafficking of proteins between organelles in the secretory and endocytic pathways. Ran GTPases regulate nucleocytoplasmic transport of RNA and

1	CHAPTER 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression 43 • Fig. 3.7 Illustration of How Intracellular Signals Are Amplified and Integrated. Signaling pathways often include dozens of proteins and small molecules that form complicated networks within the cell. Some signaling proteins relay the signal by passing the message to another protein. Many proteins amplify the signal either by producing large amounts of additional signaling molecules or by activating a large number of downstream signaling proteins. Other proteins carry the signal from one region of the cell to another. See text for more details. (Redrawn from Alberts B, et al: Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.).

1	• Fig. 3.8 GTP-Binding Proteins. GTP binding activates whereas hydrolysis of GTP to GDP inactivates GTP-binding proteins (A). All GTPase are controlled by regulatory proteins, including GTPase-activating proteins (GAP), which induce the hydrolysis of GTP to GDP, thus inactivating the GTPase, and guanine nucleotide exchange factors, (GEF) that which cause the GTPase to release GDP, which is rapidly replaced by GTP, thereby activating the GTPase (B). (Redrawn from Kantrowitx ER, Lipscomb WN. Escherichia coli aspartate transcarbamoylase: the molecular basis for a concerted allosteric transition. Trends Biochem Sci. 1990;15:53-59.) proteins. Ras GTPases are involved in many signaling pathways that control cell division, proliferation and death. Arf GTPases, like Rab GTPases, regulate vesicular transport.

1	Sci. 1990;15:53-59.) proteins. Ras GTPases are involved in many signaling pathways that control cell division, proliferation and death. Arf GTPases, like Rab GTPases, regulate vesicular transport. Heterotrimeric G proteins couple to more than 1000 different receptors and thereby mediate the cellular response to an incredibly diverse set of signaling molecules, including hormones, neurotransmitters, peptides, and odorants. Like monomeric G proteins, they can be membrane bound because of the addition of lipids posttranslationally. Heterotrimeric complexes are composed of three subunits: α, β, and γ. There exist 16 α subunits, 5 β subunits, and 11 γ subunits, which can assemble into hundreds of different combinations and thereby interact with a diverse number of receptors and effectors. The assembly of subunits and the association with receptors and effectors depend on the cell type. An overview of heterotrimeric G protein activation is illustrated in

1	An overview of heterotrimeric G protein activation is illustrated in Fig. 3.9 . In the absence of ligand, these G proteins are inactive and form a heterotrimeric complex in which GDP binds to the α subunit. Binding of a signal molecule to an inactive GPCR induces a conformational change in the G protein that promotes the release of GDP and the subsequent binding of GTP to the α subunit. Binding of GTP to the α subunit stimulates dissociation of the α subunit from the heterotrimeric complex and results in release of the α subunit from the βγ dimer, each of which can interact with and regulate downstream effectors such as adenylyl cyclase and phospholipases (see Fig. 3.9 ). Activation of downstream effectors by the α subunit and βγ dimer is terminated when the α subunit hydrolyzes the bound GTP to GDP and inorganic phosphate (Pi). The α subunit bound to GDP associates with the βγ dimer and terminates the activation of effectors.

1	Another way to attenuate or terminate signaling through a GPCR involves desensitization and endocytic removal of receptors from the plasma membrane. Binding of hormone to a GPCR increases the ability of GPCR kinases to phosphorylate the intracellular domain of GPCRs, which recruits proteins called β-arrestins to bind to the GPCR. β-Arrestins inactivate the receptor and promote endocytic

1	CHAPTER 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression 45 • Fig. 3.9 Activation of a G Protein–Coupled Receptor (GPCR) and Effector Activation. In the absence of ligand, heterotrimeric G proteins are in an inactive state because GDP binds to the α subunit. Binding of a signal molecule to an inactive G protein–coupled receptor (GPCR) induces a conformational change in the G protein that promotes the release of GDP and the subsequent binding of GTP to the α subunit. Binding of GTP to the α subunit stimulates dissociation of the α subunit from the heterotrimeric complex and results in release of the α subunit from the βγ dimer, each of which can interact with and regulate downstream effectors. (Redrawn from Alberts B, et al: Molecular Biology of the Cell. 6th ed. New York:

1	Garland Science; 2015.) removal of the GPCR from the plasma membrane. GPCR kinase/β-arrestin inactivation with endocytosis of GPCRs is an important mechanism whereby cells downregulate (desensitize) a response during prolonged exposure to elevated hormone levels. One of the major benefits of β blockers, given for congestive heart failure, is that they reverse chronic desensitization and the associated recovery of adrenergic responsiveness. Activated G protein α subunits couple to a variety of effector proteins, including adenylyl cyclase, phosphodiesterases, and phospholipases (A2, C, and D). A very common downstream effector of heterotrimeric G proteins is adenylyl cyclase, which facilitates the conversion of ATP to cAMP (

1	Fig. 3.10 ). When a signal molecule binds to a GPCR that interacts with a G protein composed of an α subunit of the αs class, adenylyl cyclase is activated, which causes an increase in cAMP levels and, as a result, activation of protein kinase A (PKA). By phosphorylating specific serine and threonine residues on downstream effector proteins, PKA regulates effector protein activity. In contrast, when a ligand binds to a receptor that interacts with a G protein composed of a α subunit of the αi class, adenylyl cyclase is inhibited, which causes reductions in cAMP levels and, consequently, in PKA activity.

1	Some effector proteins, such as ion-gated channels, are also regulated directly by cAMP. cAMP is degraded to AMP activated, phosphorylated CREB •Fig. 3.10 GPCR Stimulation of Adenylyl Cyclase, cAMP, and Protein Kinase A.Binding of a signal molecule to a GPCR mediates Gs stimulation of adenylyl cyclase, which increases cytosolic cAMP, which in turn activates protein kinase A (PKA). Activated PKA phosphorylates a number of target proteins to elicit many effects. PKA also enters the nucleus where it phosphorylates CREB (cyclic adenosine mono-phosphate [cyclic AMP] response element–binding protein). Phosphorylated CREB recruits coactivator CBP, which stimulates gene transcription. (Redrawn from Alberts B, etal: Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015.) by cAMP phosphodiesterases, which are inhibited by caf-and phosphorylate and activate the transcription factor feine and other methylxanthines. Thus by interfering with cAMP response element–binding (CREB)

1	phosphodiesterases, which are inhibited by caf-and phosphorylate and activate the transcription factor feine and other methylxanthines. Thus by interfering with cAMP response element–binding (CREB) protein (see a constitutive “off” signal, caffeine can prolong a cellular

1	Fig. 3.10 ). Phospho-CREB protein increases the transcripresponse mediated by cAMP and PKA. Because these effects tion of many genes, which can in turn produce a distinct set target existing proteins, they can be extremely rapid (e.g., of responses with much slower kinetics. Hence, cAMP has adrenaline response). In addition to cytoplasmic signaling, many cellular effects, including direct and indirect effects the catalytic subunit of PKA can enter the nucleus of cells mediated by PKA. CHAPTER 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression

1	CHAPTER 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression Cholera toxin, secreted by Vibrio cholera, catalyzes the ADP ribosylation of the G-protein αs subunit, which inhibits the GTPase activity of αs. Thus αs remains in its activated, GTP-bound state, which in turn causes activation of adenylyl cyclase and an increase in cAMP/PKA levels. In the intestines, elevated levels of PKA increase cystic fibrosis transmembrane conductance regulator (CFTR)–mediated chloride secretion, which leads to secretory diarrhea and extensive loss of fluids, characteristic of cholera. Bordetella pertussis, the bacterium that causes whooping cough, secretes pertussis toxin, which ADP ribosylates the αi subunit. In this case, the ribosylation inactivates αi, reducing the inhibition of adenylyl cyclase and thus also leading to increased levels of cAMP/PKA.

1	Heterotrimeric G proteins also regulate phototransduction. In rod cells in the eye, absorption of light by rhodopsin activates the G protein transducin, which via the αt subunit activates cGMP phosphodiesterase. Activation of this phosphodiesterase lowers the concentration of cGMP and thereby closes a cGMP-activated cation channel. The ensuing change in cation channel activity alters the membrane voltage. The exquisite sensitivity of rods to light—rods can detect a single photon of light—is due to the abundance of rhodopsin in rods and amplification of the signal (a photon of light) by the G protein–cGMP phosphodiesterase–cGMP channel signaling pathway (see for more details).

1	Heterotrimeric G proteins also regulate phospholipases, a family of enzymes that modulate a variety of signaling pathways. Ligands that activate receptors that are coupled to the αq subunit stimulate phospholipase C, an enzyme that converts phosphatidylinositol 4,5-biphosphate to inositol 1,4,5-triphosphate (InsP3) and diacylglycerol. InsP3 is a second messenger that diffuses to the endoplasmic reticulum, where it activates a ligand-activated Ca++ channel to release Ca++ into the cytosol, whereas diacylglycerol activates protein kinase C, which phosphorylates effector proteins. As noted earlier, both Ca++ and protein kinase C influence effector proteins, as well as other signaling pathways, to elicit responses.

1	Ligand binding to GPCRs can also activate phospholipase A2, an enzyme that releases arachidonic acid from membrane phospholipids Arachidonic acid, which can also be released from diacylglycerol via an indirect pathway, can be released from cells and thereby regulate neighboring cells or stimulate inflammation. It can also be retained within cells, where it is incorporated into the plasma membrane or is metabolized in the cytosol to form intracellular second messengers that affect the activity of enzymes and ion channels. In one pathway, cytosolic cyclooxygenases facilitate the metabolism of arachidonic acid to prostaglandins, thromboxanes, and prostacyclins. Prostaglandins mediate aggregation of platelets, cause constriction of the airways, and induce inflammation. Thromboxanes also induce platelet aggregation and constrict blood vessels, whereas prostacyclin inhibits platelet aggregation and causes dilation of blood vessels. In a second pathway of arachidonic acid metabolism, the

1	platelet aggregation and constrict blood vessels, whereas prostacyclin inhibits platelet aggregation and causes dilation of blood vessels. In a second pathway of arachidonic acid metabolism, the enzyme 5-lipoxygenase initiates the conversion of arachidonic acid to leukotrienes, which participate in allergic and inflammatory responses, including those causing asthma, rheumatoid arthritis, and inflammatory bowel disease. The third pathway of arachidonic acid metabolism is initiated by epoxygenase, an enzyme that facilitates the generation of hydroxyeicosatetraenoic acid (HETE) and cis-epoxyeicosatrienoic acid (cis-EET). HETE and cis-EET and their metabolites increase release of Ca++ from the endoplasmic reticulum, stimulate cell proliferation, and regulate inflammatory responses.

1	Ca++ is also an intracellular messenger that elicits cellular effects via Ca++-binding proteins, most notably calmodulin (CaM). When Ca++ binds to CaM, its conformation is altered, and the structural change in CaM allows it to bind to and regulate other signaling proteins, including cAMP phosphodiesterase, an enzyme that degrades cAMP to AMP, which is inactive and unable to activate PKA. By binding to CaM-dependent kinases, CaM also phosphorylates specific serine and threonine residues in many proteins, including myosin light-chain kinase, which facilitates smooth muscle contraction (see Protein Phosphatases and Phosphodiesterases Counteract the Activation of Cyclic Nucleotide Kinases

1	Protein Phosphatases and Phosphodiesterases Counteract the Activation of Cyclic Nucleotide Kinases There are two ways to terminate a signal initiated by cAMP and cGMP: enhancing degradation of these cyclic nucleotides by phosphodiesterases and dephosphorylation of effectors by protein phosphatases. Phosphodiesterases facilitate the breakdown of cAMP and cGMP to AMP and GMP, respectively, and are activated by ligand activation of GPCRs. Phosphatases dephosphorylate effector proteins that were phosphorylated by kinases such as PKA. The balance between kinase-mediated phosphorylation and phosphatase-mediated dephosphorylation allows rapid and exquisite regulation of the phosphorylated state and thus the activity of signaling proteins.

1	There are several classes of receptors that have enzymatic activity or are intimately associated with proteins that have enzymatic activity. Four of these classes are discussed next, including receptors that mediate the cellular responses to atrial natriuretic peptide (ANP) and nitric oxide (guanylyl cyclase receptors); transforming growth factor-β (TGF-β; threonine/serine kinase receptors); EGF, platelet-derived growth factor (PDGF), and insulin (tyrosine kinase receptors); and interleukins (tyrosine kinase–associated receptors).

1	There are two isoforms of cyclooxygenase: COX1 and COX2. When activated in endothelial cells, COX1 facilitates the production of prostacyclins, which inhibit blood clots (thrombin). In vascular smooth muscle cells and platelets, COX1 facilitates the production of thromboxane A2, which is prothrombotic. Thus cardiovascular health depends in part on the balance between prostacyclins and thromboxane A2, generated by distinct cell types. Low doses of aspirin, a nonsteroidal antiinflammatory drug (NSAID), reduce thromboxane A2 production by platelets with little effect on endothelial prostacyclin production. Thus low-dose aspirin is antithrombotic (i.e., reduces blood clots). COX2 is activated by inflammatory stimuli. Thus the ability of NSAIDs (e.g., aspirin, ibuprofen, naproxen, acetaminophen, indomethacin) to suppress the inflammatory response is due to inhibition of COX2. Both COX1 and COX2 facilitate the production of prostanoids that protect the stomach. Several lines of evidence

1	indomethacin) to suppress the inflammatory response is due to inhibition of COX2. Both COX1 and COX2 facilitate the production of prostanoids that protect the stomach. Several lines of evidence suggest that both COX1 and COX2 must be inhibited to elicit damage to the gastrointestinal tract. Consequently, the negative effects of NSAIDs on the gastric mucosa (e.g., increased incidence of gastrointestinal bleeding) are most likely due to inhibition of COX1 and COX2 by these nonselective COX inhibitors.

1	Selective COX2 inhibitors (e.g., celecoxib, rofecoxib) are very effective in selectively inhibiting COX2 and are used extensively to reduce the inflammatory response. Because COX2 inhibitors are thought to lack the negative effects elicited by NSAIDs on the gastrointestinal tract, their use has increased dramatically. However, in 2005, the U.S. Food and Drug Administration (FDA) announced that selective COX2 inhibitors, as well as nonselective NSAIDs, increase the risk for heart attacks and strokes and required that COX2selective and nonselective NSAIDs carry a warning label on product packaging that highlighted the potential for the increased risk for adverse cardiovascular events and stroke. In addition, although much evidence suggests that COX2selective inhibitors do not cause gastrointestinal bleeding, in 2005 the FDA also required the pharmaceutical industry to add to the warning label on COX2-selective drugs a caution about the potential for increased risk for gastrointestinal

1	bleeding, in 2005 the FDA also required the pharmaceutical industry to add to the warning label on COX2-selective drugs a caution about the potential for increased risk for gastrointestinal bleeding. In 2015, the FDA strengthened warnings that both COX2-selective and COX2-nonselective NSAIDs increase the risk of heart attacks and strokes.

1	bSee U.S. Food and Drug Administration. FDA Drug Safety Communication: FDA Strengthens Warning That Non-aspirin Nonsteroidal Anti-inflammatory Drugs (NSAIDs) Can Cause Heart Attacks or Strokes. < http://www.fda.gov/ Drugs/DrugSafety/ucm451800.htm >; 2015 Accessed 25.07.16. ANP binds to the extracellular domain of the plasma membrane receptor guanylyl cyclase and induces a conformational change in the receptor that causes receptor dimerization and activation of guanylyl cyclase, which metabolizes GTP to cGMP. cGMP activates cGMP-dependent protein kinase, which phosphorylates proteins on specific serine and threonine residues. In the kidney, ANP inhibits reabsorption of sodium and water by the collecting duct (see Nitric oxide activates a soluble receptor guanylyl cyclase that converts GTP to cGMP, which relaxes smooth muscle. Because nitroglycerin increases blood concentrations of

1	Nitric oxide activates a soluble receptor guanylyl cyclase that converts GTP to cGMP, which relaxes smooth muscle. Because nitroglycerin increases blood concentrations of Ras GTPases, monomeric G proteins, are involved in many signaling pathways that control cell division, proliferation, and death. Many mutations of proteins in the Ras signaling pathway are oncogenic (cancer causing) or inactivate tumor suppressors. Mutations in Ras genes that inhibit GTPase activity, as well as overexpression of Ras proteins as a result of transcriptional activation, lead to continuous cell proliferation, a major step in the development of cancer in many organs, including the pancreas, colon, and lungs. In addition, mutations in and overexpression of GEFs, which facilitate exchange of GTP for GDP, and GTPase-activating proteins, which accelerate GTP hydrolysis, may also be oncogenic (see

1	Fig. 3.8B nitric oxide, which increases cGMP and thereby relaxes smooth muscle in coronary arteries, it has long been used to treat angina pectoris (i.e., chest pain caused by inadequate blood flow to heart muscle; see The TGF-β receptor is a threonine/serine kinase that has two subunits. Binding of TGF-β to the type II subunit induces it to phosphorylate the type I subunit on specific serine and threonine residues, which in turn, phosphorylates other downstream effector proteins on serine and threonine residues and thereby elicits cellular responses, including cell growth, cell differentiation, and apoptosis.

1	There are two classes of tyrosine kinase receptors. Nerve growth factor (NGF) receptors are typical examples of one class. Ligand binding to two NGF receptors facilitates their dimerization and thus enables the cytoplasmic tyrosine kinase domain of each monomer to phosphorylate and activate the other monomer. Once the other monomer is phosphorylated, the cytoplasmic domains can recruit GEFs such as growth factor-receptor bound protein 2 to the plasma membrane, which in turn activates Ras and downstream kinases that regulate gene transcription programs important for cell survival and proliferation.

1	Activation of the insulin receptor (which is tetrameric and composed of two α and two β subunits) by insulin is an example of the other type of tyrosine kinase receptor. Binding of insulin to the α subunits produces a conformational change that facilitates interaction between the two α and β pairs. Binding of insulin to its receptor causes autophosphorylation of tyrosine residues in the catalytic domains of the β subunits, and the activated receptor then phosphorylates cytoplasmic proteins to initiate its cellular effects, including stimulating the absorption of glucose from the blood into skeletal muscle and fat tissue. The tyrosine kinase–associated receptors have no intrinsic kinase activity but associate with proteins that do have

1	The tyrosine kinase–associated receptors have no intrinsic kinase activity but associate with proteins that do have CHAPTER 3 Signal Transduction, Membrane Receptors, Second Messengers, and Regulation of Gene Expression tyrosine kinase activity, including tyrosine kinases of the Src family and Janus family. Receptors in this class bind several cytokines, including interleukin-6, a proinflammatory cytokine that is necessary for resistance to bacterial infections, and erythropoietin, which stimulates the production of red blood cells. Tyrosine kinase–associated receptor subunits assemble into homodimers (αα), heterodimers (αβ), or heterotrimers (αβγ) when ligands bind. Subunit assembly enhances the binding of tyrosine kinases, which induces kinase activity and thereby phosphorylates tyrosine residues on the kinases, as well as on the receptor. Most polypeptide growth factors bind to tyrosine-kinase-associated receptors. Regulation of Gene Expression by Signal Transduction Pathways

1	Regulation of Gene Expression by Signal Transduction Pathways Steroid and thyroid hormones, cAMP, and receptor tyrosine kinases are transcription factors that regulate gene expression and thereby participate in signal transduction pathways. This section discusses the regulation of gene expression by steroid and thyroid hormones, cAMP, and receptor tyrosine kinases. The family of nuclear receptors includes more than 30 genes and has been divided into two subfamilies on the basis of structure and mechanism of action: (1) steroid hormone receptors and (2) receptors that bind retinoic acid, thyroid hormones (iodothyronines), and vitamin D. When ligands bind to these receptors, the ligand-receptor complex activates transcription factors that bind to DNA and regulate the expression of genes (see Figs. 3.2B, 3.5, and 3.7

1	Figs. 3.2B, 3.5, and 3.7 The location of nuclear receptors varies. Glucocorticoid and mineralocorticoid receptors are located in the cytoplasm, where they interact with chaperones (i.e., heat shock proteins; see Fig. 3.2B ). Binding of hormone to these receptors results in a conformational change that causes chaperones to dissociate from the receptor, thereby revealing a nuclear localization motif that facilitates translocation of the hormone-bound receptor complex to the nucleus. Estrogen and progesterone receptors are located primarily in the nucleus, and thyroid hormone and retinoic acid receptors are located in the nucleus bound to DNA.

1	When activated by hormone binding, nuclear receptors bind to specific DNA sequences in the regulatory regions of responsive genes called hormone response elements. Ligand-receptor binding to DNA causes a conformational change in DNA that initiates transcription. Nuclear receptors also regulate gene expression by acting as transcriptional repressors. For example, glucocorticoids suppress the transcription activator protein-1 (AP-1) and nuclear factor κB, which stimulate the expression of genes that cause inflammation. By this mechanism glucocorticoids reduce inflammation. As noted previously, cAMP is an important second messenger. In addition to its importance in activating PKA, which phosphorylates specific serine and threonine residues on proteins, cAMP stimulates the transcription of many genes, including those that code for hormones, including somatostatin, glucagon, and vasoactive intestinal polypeptide (see

1	Fig. 3.10 ). Many genes activated by cAMP have a cAMP response element (CRE) in their DNA. Increases in cAMP stimulate PKA, which not only acts in the cytoplasm but also can translocate to the nucleus, where it phosphorylates CREB and thereby increases its affinity for CREB-binding protein (CBP). The CREB-CBP complex activates transcription. The response is terminated when PKA phosphorylates a phosphatase that dephosphorylates CREB (see Fig. 3.10). Many growth factors, including EGF, PDGF, NGF, and insulin, bind to and activate enzyme-linked receptors that have tyrosine kinase activity. Activation of tyrosine kinases initiates a cascade of events that enhance the activity of the small GTP-binding protein Ras, which in a series of steps and intermediary proteins phosphorylates the mitogenactivated protein kinase, which then translocates to the nucleus and stimulates transcription of genes that stimulate cell growth.

1	Tyrosine kinase–associated receptors, as noted earlier, are activated by a variety of hormones, including cytokines, growth hormone, and interferon. Although these receptors do not have tyrosine kinase activity, they are associated with Janus family proteins, which do have tyrosine kinase activity. Once activated, hormone tyrosine kinase– associated receptors activate Janus family protein, which phosphorylates latent transcription factors called signal transducers and activators of transcription (STATs). When phosphorylated on tyrosine residues, STATs dimerize and then enter the nucleus and regulate transcription.

1	1. The function of cells is tightly coordinated and integrated by external chemical signals, including hormones, neurotransmitters, growth factors, odorants, and products of cellular metabolism that serve as chemical messengers and provide cell-to-cell communication. Chemical and physical signals interact with receptors located in the plasma membrane, cytoplasm, and nucleus. Interaction of these signals with receptors initiates a cascade of events that mediate the response to each stimulus. These pathways ensure that the cellular response to external signals is specific, amplified, tightly regulated, and coordinated.

1	These pathways ensure that the cellular response to external signals is specific, amplified, tightly regulated, and coordinated. 2. There are two classes of GTP-binding proteins: monomeric G proteins and heterotrimeric G proteins composed of α, β, and γ subunits. Monomeric G proteins regulate actin cytoskeleton organization, cell cycle progression, intracellular vesicular transport, and gene expression. Heterotrimeric G proteins regulate ion channels, adenylyl cyclase and the cAMP-PKA signaling pathway, phosphodiesterases (which also regulate cAMP and cGMP signaling pathways), and Cheung E, Kraus WL. Genomic analyses of hormone signaling and gene regulation. Annu Rev Physiol. 2010;72:191-218. Huang P, Chandra V, Rastinejad F. Structural overview of the nuclear receptor superfamily: insights into physiology and therapeutics. Annu Rev Physiol. 2010;72:247-272. Levin ER. Extranuclear steroid receptors are essential for steroid hormone actions. Annu Rev Med. 2015;66:271-280.

1	Levin ER. Extranuclear steroid receptors are essential for steroid hormone actions. Annu Rev Med. 2015;66:271-280. Riccardi D, Kemp P. The calcium-sensing receptor beyond extracellular calcium homeostasis: conception, development, adult physiology, and disease. Annu Rev Physiol. 2012;74:271-297. Wu H. Higher-order assemblies in a new paradigm of signal transduction. Cell. 2013;153:287-292. phospholipases, which regulate the production of prostaglandins, prostacyclins, and thromboxanes. 3. There are four subtypes of enzyme-linked receptors that mediate the cellular response to a wide variety of signals, including ANP, nitric oxide, TGF-β, PDGF, insulin, and interleukins. 4. There are two types of nuclear receptors: (1) one type that in the absence of ligand is located in the cytoplasm and when bound to ligand translocates to the nucleus and (2) another type that permanently resides in the nucleus. Both classes of receptors regulate gene transcription.

1	Cantley L. Signal transduction. In: Boron WF, Boulpaep EL, eds. Medical Physiology. 3rd ed. Philadelphia: Elsevier; 2016 [Chapter 3]. Caplan MJ. Functional organization of the cell. In: Boron WF, Boulpaep EL, eds. Medical Physiology. 3rd ed. Philadelphia: Elsevier; 2016 [Chapter 2]. Heald R. Cell signaling. In: Alberts B, Johnson A, Lewis J, et al., eds. Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015 [Chapter 15]. Igarashi P. Regulation of gene expression. In: Boron WF, Boulpaep EL, eds. Medical Physiology. 3rd ed. Philadelphia: Elsevier; 2016 [Chapter 4]. SECTION 2The Nervous System ERIC J. LANG AND KALMAN RUBINSON Chapter 4 The Nervous System: Introduction to Cells and Systems Chapter 5 Generation and Conduction of Action Potentials The Somatosensory System The Special Senses Organization of Motor Function Chapter 10 Integrative Functions of the Nervous System Chapter 11 The Autonomic Nervous System and Its Central Control

1	The Somatosensory System The Special Senses Organization of Motor Function Chapter 10 Integrative Functions of the Nervous System Chapter 11 The Autonomic Nervous System and Its Central Control Upon completion of this chapter the student should be able to answer the following questions: 1. What are the major cell types of the central and peripheral nervous systems? 2. What are the major components of a neuron, and what are their functional roles? 3. What are the functional roles of the major glial cell types? 4. What are the main divisions of the central nervous system? 5. How and where is the cerebrospinal fluid formed, and how does it circulate and exit the ventricular system? 6. How is axon transport related to the response of the axon to transection?

1	5. How and where is the cerebrospinal fluid formed, and how does it circulate and exit the ventricular system? 6. How is axon transport related to the response of the axon to transection? he nervous system is a communications and control network that allows an organism to interact rapidly and adaptively with its environment, where environment includes both the external environment (the world outside the body) and the internal environment (the components and cavities of the body). To carry out its function the nervous system takes in sensory information from a variety of sources using specialized sensors (receptors), integrates this information with previously obtained information stored as memories and with the intrinsic goals and drives of the organism that have been embedded in its nervous system through evolution, decides on a course of action, and then issues commands to the effector organs (muscles and glands) to execute the chosen behavioral response.

1	Moreover, almost all behavioral responses require the coordination of many body parts. For example, even a simple reaching movement of the arm may require coactivation of axial muscles and possibly muscles in the lower extremity to maintain posture and balance, which themselves may be monitored by up to three different sensory systems (vision, vestibular, and proprioceptive) whose information has to be integrated. Furthermore, movements can alter the internal environment and thus can require compensatory changes in heart and breathing rates, blood vessel diameters, and other internal processes. All these variables are monitored and controlled by various specialized subsystems of the nervous system, all of which must work together for the organism to perform movements and more generally to survive. The succeeding chapters will describe these major subsystems individually; however, it should be remembered that in reality their activity is integrated to generate normal behavior.

1	To begin, it is useful to divide the nervous system into central and peripheral parts. The central nervous system (CNS) consists of the brain and spinal cord. The peripheral nervous system (PNS) consists of nerves and ganglia (small groups of neurons) that innervate all parts of the body and provide an interface between the environment and the CNS. The transition between the CNS and PNS occurs on the dorsal and ventral rootlets near to where they emerge from the spinal cord and on the cranial nerve fibers near to where they arise from the brain. Cellular Components of the Nervous System

1	Cellular Components of the Nervous System The nervous system is made up of cells, connective tissue, and blood vessels. The major cell types are neurons (nerve cells) and glia (neuroglia = “nerve glue”). In its most general form a neuron’s function can be defined as generation of signals (to be sent to other neurons or effector cells [e.g., muscle cells]) based on an integration of its own electrical properties with electrochemical signals from other neurons. The points where specific neuron-to-neuron communication occurs are known as synapses, and the process of synaptic transmission is critical to neuronal function (see ). Neuroglia, or just glia, traditionally have been characterized as supportive cells that sustain neurons both metabolically and physically, isolate individual neurons from each other, and help maintain the internal milieu of the nervous system; however, it is now known that they also have important roles in shaping the flow of activity through the nervous system.

1	The typical neuron consists of three main cellular compartments: a cell body (also referred to as a perikaryon or CHAPTER 4 The Nervous System: Introduction to Cells and Systems •Fig. 4.1 Schematicdiagramofanidealizedneuronanditsmajorcomponentsandconnections.A,Afferentinputfromaxonsofothercellsterminatesinsynapsesonthedendritesandcellbody.Theinitialsegmentoftheaxonattachesattheaxonhillock.Thisaxonismyelinated,asindicatedbythebluestructuresthatencapsulatesegmentsoftheaxon.Theaxonterminatesontwopostsynapticneuronsbyformingsynapticterminals.B,NodesofRanvierarethegapsbetweenthemyelinsegmentswheretheaxonmembraneisexposedtotheextracellularspace.C,Higher-magnificationviewofsynapse.(RedrawnfromBlumenfeldH.Neuroanatomy Through Clinical Cases. 2nded.Sunderland,MA:SinauerAssociates;2010.) soma), a variable number of processes that extend from the soma called dendrites, and an axon (

1	Fig. 4.1 ). A tremendous number of morphological variants of this basic template exist, including cases where dendrites or an axon may be absent ( Fig. 4.2 ). These variations do not occur randomly but rather relate to the distinct functional properties of each neuronal class. Indeed, neurons with similar morphologies often characterize specific regions of the CNS and reflect the distinct neuronal processing performed in each CNS region.

1	The cell body is the main genetic and metabolic center of the neuron. Correspondingly it contains the nucleus and nucleolus of the cell and also possesses a well-developed biosynthetic apparatus for manufacturing membrane constituents, synthetic enzymes, and other chemical substances needed for the specialized functions of nerve cells. The neuronal biosynthetic apparatus includes Nissl bodies, which are stacks of rough endoplasmic reticulum, and a prominent Golgi apparatus. The soma also contains numerous mitochondria and cytoskeletal elements, including neurofilaments and microtubules.

1	The cell body is also a region in which the neuron receives synaptic input (i.e., electrical and chemical signals from other neurons). Although quantitatively the synaptic input to the soma is usually much less than that to dendrites, it often differs qualitatively from dendritic inputs, and by virtue of the closeness of the soma to the axon, inputs to the soma can override those to the dendrites (see

1	Dendrites are tapering and branching extensions of the soma and are the main direct recipients of signals from other neurons. They can be thought of as a way to expand and specialize the surface area of a neuron, and indeed, they may account for more than 90% of the surface area available for synaptic contact (soma plus dendrites). Dendrites can be divided into primary dendrites (those that extend directly from the soma) and higher-order dendrites (daughter branches extending from a more proximal branch, in •Fig. 4.2 A,Purkinjecell.B,Pyramidalcell.C,Golgicell.D,Granulecell.E,Inferiorolivecells.F,Bipolarcells.(A,CourtesyofBorisBarbour.B,CourtesyofT.F.Fletcher,from http://vanat.cvm.umn.edu/ neurHistAtls/pages/neuron3.html .C,FigurewasprovidedbyCourtHullandWadeRegehr,DepartmentofNeurobiology,HarvardMedicalSchool.D,FromDelvendahlIetal.Front Cell Neurosci 2015;9:93,Fig.1A.E,FromMathyA,ClarkBA.In:MantoMetal[eds].Handbook of the Cerebellum and Cerebellar Disorders.

1	Cell Neurosci 2015;9:93,Fig.1A.E,FromMathyA,ClarkBA.In:MantoMetal[eds].Handbook of the Cerebellum and Cerebellar Disorders. Dordrecht,Netherlands:SpringerScience+BusinessMediaDordrecht;2013.F,FromLiW,DeVriesSH.Nat Neurosci 2006;9:669-675,Fig.2.) which proximal refers to closeness to the soma). The main cytoplasmic organelles in dendrites are microtubules and neurofilaments; however, the primary dendrites can also contain Nissl bodies and parts of the Golgi apparatus.

1	A neuron’s set of dendrites is termed its dendritic tree. Dendritic trees differ tremendously between different types of neurons in terms of the size, number, and spatial organization of the dendrites. A dendritic tree can consist of just a few unbranched dendrites or of many highly ramified dendrites. Individual dendrites can be longer than 1 mm or only 10 to 20 µm in length. Another major morphological variation is whether or not a dendrite has spines, which are small mushroomor lollipop-shaped protrusions from the main dendrite. Spines are sites specialized for synaptic contact (usually, but not always) from excitatory inputs. The shape and size of the dendritic tree, as well as the population and distribution of channels in the dendritic membrane, are all important determinants of how the synaptic input will affect the neuron (see Chapter 6).

1	Chapter 6). The axon is an extension of the cell that conveys the output of the cell to other neurons or, in the case of a motor neuron, to muscle cells as well. In general, each neuron has only one axon, and it is usually of uniform diameter. The length and diameter of axons vary with the neuronal type. Some axons do not extend much beyond the length of the dendrites, whereas others may be a meter or more long. Axons may have orthogonal branches en passant, but they often end in a spray of branches called a terminal arborization (represented by the four terminal branches and their synaptic terminals in

1	Fig. 4.1A ). The size, shape, and organization of the terminal arborization determine which other cells it will contact. The first part of the axon is known as the initial segment and arises from the soma (or sometimes from a proximal dendrite) in a specialized region called the axon hillock. The axon differs from the soma and proximal dendrites in that it lacks rough endoplasmic reticulum, free ribosomes, and a Golgi apparatus. The initial segment is usually the site where action potentials (spikes) that are propagated down the axon are initiated (see ). An axon may terminate in a synapse and/or it may make synapses along its length. Synapses will be described in detail in

1	CHAPTER 4 The Nervous System: Introduction to Cells and Systems •Fig. 4.3 Axonaltransport.Schematicofneuronandenlargementofaxonaltransportmechanism.Axonaltransportdependsonmovementofmaterialalongtransportfilamentssuchasmicrotubules.Transportedcomponentsattachtotransportfilamentsbymeansofcross-bridges.Differentobjectsaretransportedanterogradely(fromcellbodytoaxonterminal)andothersretrogradely(towardthecellbody).Thedirectionoftransport—retrogradeandanterograde—isdeterminedbyspecificproteinssuch asdyneinandkinesin,respectively.

1	Neurons are special because of their ability to control and respond to electricity. Moreover, the response and control mechanisms of each part of a neuron are distinct from those in other parts. This intraneuronal specialization is a consequence of the particular morphology and the ion channel composition of each part of the neuron. For example, dendrites have ligand gated ion channels that allow neurons to respond to chemicals released by other neurons, and their characteristic branching pattern allows for integration of multiple input signals. In contrast the axon typically has a long length and high concentration of voltage-gated channels that allows it to convey electrical signals (action potentials) rapidly over long distances without alteration.

1	Because the soma is the metabolic engine of the neuron, substances needed to support axonal and synaptic function are synthesized there. These substances must be distributed to replenish secreted or inactivated materials along the axon and especially to the presynaptic terminals. Most axons are too long to allow efficient movement of substances from the soma to the synaptic endings by simple diffusion. Thus special axonal transport mechanisms have evolved to accomplish this task ( Fig. 4.3). A consequence of this metabolic dependency is that axons degenerate when disconnected from the cell body, a fact that has been used by scientists tracing out neuronal pathways; they would cut an axonal pathway and then determine where the degenerating axons distal to the cut projected to.

1	Several types of axonal transport exist. Membrane-bound organelles and mitochondria are transported relatively rapidly by fast axonal transport. Substances that are dissolved in cytoplasm (e.g., proteins) are moved by slow axonal transport. In mammals, fast axonal transport proceeds as rapidly as 400 mm/day, whereas slow axonal transport occurs at about 1 mm/day. Synaptic vesicles, which travel by fast axonal transport, can travel from the soma of a motor neuron in the spinal cord to a neuromuscular junction in a person’s foot in about 2.5 days. In comparison the movement of some soluble proteins over the same distance can take nearly 3 years. Axonal transport requires metabolic energy and involves calcium ions. Microtubules provide a system of guidewires along which membrane-bound organelles move (see Fig.

1	Axonal transport requires metabolic energy and involves calcium ions. Microtubules provide a system of guidewires along which membrane-bound organelles move (see Fig. 4.3 ). Organelles attach to microtubules through a linkage similar to that between the thick and thin filaments of skeletal muscle fibers. Ca++ triggers movement of the organelles along the microtubules. Special microtubule-associated motor proteins called kinesin and dynein are required for axonal transport. Axonal transport occurs in both directions. Transport from the soma toward the axonal terminals is called anterograde axonal transport. This process involves kinesin, and it allows replenishment of synaptic vesicles and enzymes responsible for the synthesis of neurotransmitters in synaptic terminals. Transport in the opposite direction, which is driven by dynein, is called retrograde axonal transport. This process returns recycled synaptic vesicle membrane to the soma for lysosomal degradation.

1	Certainvirusesandtoxinscanbeconveyedbyaxonaltransportalongperipheralnerves.Forexample,herpeszoster,thevirusofchickenpox,invadesdorsalrootganglioncells.Thevirusmaybeharboredbytheseneuronsformanyyears.However,eventuallythevirusmaybecomeactivebecauseofachangeinimmunestatus.Thevirusmaythenbetransportedalongthesensoryaxonstotheskin,causingshingles,averypainfuldisease.Anotherexampleistheaxonaltransportoftetanustoxin.Clostridium tetani bacteriamaygrowinadirtywound,andifthepersonhadnotbeenvaccinatedagainsttetanustoxin,thetoxincanbetransportedretrogradelyintheaxonsofmotorneurons.Thetoxincanescapeintotheextracellularspaceofthespinalcordventralhornandblockthesynapticreceptorsforinhibitoryaminoacids.Thisprocesscanresultintetanicconvulsions. The major nonneuronal cellular elements of the nervous system are the glia (

1	The major nonneuronal cellular elements of the nervous system are the glia ( Fig. 4.4 ). Glial cells in the human CNS outnumber neurons by an order of magnitude: there are about 1013 glia and 1012 neurons. Glial cells in the CNS include astrocytes, oligodendrocytes, microglia, and ependymal cells (see Fig. 4.4 ); in the PNS the glial cells are Schwann cells and satellite cells. Traditionally, glial cells were thought of as supportive cells, and consistent with that conception, their functions include regulation of the microenvironment and myelination of axons. Glial cells are now also recognized to be important determinants of the flow of signals through neuronal circuits, and related to that they act to modulate synaptic and nonsynaptic transmission and have important roles in synaptogenesis and maintenance.

1	Astrocytes (named for their star shape) help regulate the microenvironment of the CNS, both under normal conditions and in response to damage to the nervous system. Astrocytes have a cell body from which several main branches arise. Through repeated branching these main processes give rise to hundreds to thousands of branchlets. Astrocyte processes contact neurons and surround synaptic endings, isolating them from adjacent synapses and the general extracellular space. Astrocytes also have foot •Fig. 4.4 SchematicrepresentationofcellularelementsintheCNS.Twoastrocytesareshownendingonasomaanddendritesofaneuron.Astrocytesalsocontactthepialsurfaceorcapillariesorboth.Anoligodendrocyteprovidesthemyelinsheathsforaxons.Alsoshownaremicrogliaandependymalcells. N,neuron.(RedrawnfromWilliamsPL,WarwickR.Functional Neuroanatomy of Man. Edinburgh:ChurchillLivingstone;1975.) processes that contact the capillaries and connective tissue at the surface of the CNS, the pia mater (see

1	Fig. 4.4 ). These foot processes may help mediate the entry of substances into the CNS. Astrocytes can actively take up K+ ions and neurotransmitter substances, which they metabolize, biodegrade, or recycle. Thus astrocytes serve to buffer the extracellular environment of neurons with respect to both ions and neurotransmitters. The cytoplasm of astrocytes contains glial filaments that provide mechanical support for CNS tissue. After injury the astrocytes undergo a variety of changes to become reactive astrocytes. One example is a class of reactive astrocytes that act to form a glial scar around an area of focal damage, which segregates the damaged tissue and thereby allows inflammatory processes to act selectively at the site of damage, minimizing the impact on surrounding normal tissue. Astrocytes can also affect the properties of synaptic transmission, which is discussed in

1	Oligodendrocytes and Schwann cells are critical for the function of axons. Many axons are surrounded by a myelin sheath, which is a spiral multilayered wrapping of glial cell membrane ( Fig. 4.5A–B ). In the CNS, myelin is formed by the oligodendrocytes, whereas in the PNS Schwann cells form myelin. Myelin increases the speed of action potential conduction, in part by restricting the

1	CHAPTER 4 The Nervous System: Introduction to Cells and Systems •Fig. 4.5 Axonal/glialassociations.A,MyelinatedaxonsintheCNS.Asingleoligodendrocyte(G)emitsseveralprocesses,eachofwhichwindsinaspiralfashionaroundanaxontoformamyelinsegment.Theaxonisshownincutaway.Themyelinfromasingleoligodendrocyteendsbeforethenextwrappingfromanotheroligodendrocyte.ThebareaxonbetweenthemyelinatedsegmentsisthenodeofRanvier(N).B,ElectronmicrographofmyelinatedaxoninthePNSshownincrosssection.Theaxon(Ax)isseenatthecenterwithinasheathconsistingofmultiplewrappingsoftheSchwanncell’scytoplasmicmembrane.TheSchwanncellsoma(SC)isattheupperright.mes,mesaxon,internalandexternal.C,Electronmicro-graphofunmyelinatedaxonsinPNS.Nineaxons(asterisks) cutincrosssectionareseenembeddedinaSchwanncellwhosenucleusisatthecenter(N).Atlowerrightaportionofamyelinatedaxonisvisible.(B,FromPetersAetal.The Fine Structure of the Nervous System. NewYork:OxfordUniversityPress;1991,Fig.6.5.C,FromPanneseE.Neurocytology.

1	Fine Structure of the Nervous System. NewYork:OxfordUniversityPress;1991,Fig.6.5.C,FromPanneseE.Neurocytology. 2nded.Basel,Switzerland:SpringerInternational;2015.)

1	Astrocytesarecoupledtoeachotherbygapjunctionssuchthattheyformasyncytiumthroughwhichsmallmoleculesandionscanredistributealongtheirconcentrationgradientsorbycurrentflow.Whennormalneuralactivitygivesrisetoalocalincreaseinextracellular[K+],thiscouplednetworkcanenablespatialredistributionofK+ overawideareaviacurrentflowinmanyastrocytes. Underconditionsofhypoxia,suchasmightbeassociatedwithischemiasecondarytoblockageofanartery(i.e.,astroke),[K+]intheextracellularspaceofabrainregioncanincreasebyafactorofasmuchas20.Thiswilldepolarizeneuronsandsynapticterminalsandresultinreleaseoftransmitterssuchasglutamate,whichwillcausefurtherreleaseofK+ fromneurons.Theadditionalreleaseonlyexacerbatestheproblemandcanleadtoneuronaldeath.Undersuchconditions,localastrogliawillprobablytakeuptheexcessK+ byK+-Cl− symportratherthanbyspatialbuffering,becausetheelevationinextracellular[K+]tendstobewidespreadratherthanlocal.

1	flow of ionic current to small unmyelinated portions of the axon between adjacent glial cells, the nodes of Ranvier (see ). Although both act to increase the speed of conduction, there are several important differences in the relationship between axons and either oligodendrocytes or Schwann cells. One major difference is that a single oligodendrocyte typically helps myelinate multiple axons in the CNS, whereas each Schwann cell helps myelinate only a single axon in the PNS. A second difference is that in the CNS, unmyelinated axons are bare, whereas in the PNS, unmyelinated axons are not. Rather, they are surrounded by Schwann cell processes; the Schwann cell, however, does not form a multilayered covering (i.e., myelin), but instead extends processes that surround parts of several axons (the Schwann cell with its set of unymyelinated axons is called a Remak bundle) (see Fig. 4.5C

1	Fig. 4.5C Satellite cells encapsulate dorsal root and cranial nerve ganglion cells and regulate their microenvironment in a fashion similar to that of astrocytes. Microglia are derived from erythromyeloid stem cells that migrate into the CNS early in development. They play an important role in immune responses within the CNS. When the CNS is damaged, microglia help remove the cellular products of the damage by phagocytosis. They are assisted by other glia and by other phagocytes that invade the CNS from the circulation. In addition to their role in immune responses, recent evidence suggests they are also active in healthy brain tissue and may have important roles in normal brain development and function, including pruning of excess synapses that are formed during development and synaptic plasticity.

1	Ependymal cells form the epithelium lining the ventricular spaces of the brain, which contain (cerebrospinal fluid) CSF. CSF is secreted in large part by specialized ependymal cells of the choroid plexuses located in the ventricular system. Many substances diffuse readily across the ependyma, which lies between the extracellular space of the brain and the CSF.

1	Mostneuronsintheadultnervoussystemarepostmitoticcells(althoughsomestemcellsmayalsoremainincertainsitesinthebrain).Manyglialprecursorcellsarepresentintheadultbrain,andtheycanstilldivideanddifferentiate.Thusthecellularelementsthatgiverisetomostintrinsicbraintumorsintheadultbrainaretheglialcells.Forexample,braintumorscanbederivedfromastrocytes(whichvaryinmalignancyfromtheslowlygrowingastrocytomatotherapidlyfatalglioblastomamultiforme),fromoligodendroglia(oligodendroglioma),orfromependymalcells(ependymoma).Meningealcellscanalsogiverisetoslowlygrowingtumors(meningiomas)thatcompressbraintissue,ascanSchwanncells(e.g.,acousticschwannomas,whicharetumorsformedbySchwanncellsoftheeighthcranialnerve).Inthebrainofinfants,neuronsthatarestilldividingcansometimesgiverisetoneuroblastomas(e.g.,oftheroofofthefourthventricle)orretinoblastomas(intheeye). The Peripheral Nervous System

1	The Peripheral Nervous System The PNS provides an interface between the environment and the CNS, both for sensory information flowing to the CNS and for motor commands issued from the CNS. It includes sensory (or primary afferent) neurons, somatic motor neurons, and autonomic motor neurons. Sensory pathways into the nervous system start with a receptor, which may simply be a specialized part of an axon in the PNS or may include additional cells. Each sensory receptor is organized so that it transduces a specific type of energy into an electrical signal. Thus they can be classified in terms of the type of energy they transduce (e.g., photoreceptors transduce light, mechanoreceptors transduce displacement and force). They may also be classified according to the source of the input (e.g., exteroceptors signal external events, proprioceptors signal the state of a body part such as the angle of elbow, and interoceptors signal the distension of the gut).

1	The transduction process leads to an electrical response in the primary afferent called a receptor potential, which triggers action potentials in the primary afferent fibers innervating the receptor. These action potentials contain information about the sensory stimulus that is conveyed to the CNS via the primary afferent. Somatic and autonomic motor neurons convey signals from the CNS to their respective effector targets. The somatic motor neurons innervate the skeletal muscles throughout the body. Their cell bodies lie in the ventral horn (or equivalent brainstem nuclei) and project out of the CNS via a ventral root or cranial nerve. The details of their relationship to muscles is covered in . The autonomic motor pathway is responsible for controlling the functioning of organs, smooth muscle, and glands. It is actually a two-neuron pathway, and its properties are covered in Chapter 11. The Central Nervous System

1	Chapter 11. The Central Nervous System The CNS is built from the cellular elements just described and includes the spinal cord and brain ( Fig. 4.6A ). These cellular elements are connected in a variety of complex ways to form the subsystems that underlie the multitude of functions performed by the CNS. The physiology of these systems is covered in ; however, a basic knowledge of CNS anatomy is needed to understand systems physiology and will be briefly discussed here.

1	Regions of the CNS containing high concentrations of axon pathways (and very few neurons) are called white matter because the axonal myelin sheaths of the axons are highly refractive to light. Regions containing high concentrations of neurons and dendrites are by contrast called gray matter. Note that axons are also present in gray matter. These axons may be related to local processing (i.e., either originating from local neurons or terminating on them) or may be fibers of passage. Thus effects of damage to an area may reflect either loss of local function or disconnection CHAPTER 4 The Nervous System: Introduction to Cells and Systems pallidus Fimbria of Temporal (fornix) Caudate Inferior horn nucleus of lateral (tail) Mammillary body ventricle

1	CHAPTER 4 The Nervous System: Introduction to Cells and Systems pallidus Fimbria of Temporal (fornix) Caudate Inferior horn nucleus of lateral (tail) Mammillary body ventricle OpticB Cerebral peduncle of hypothalamus tract Hippocampus •Fig. 4.6 A,SchematicofthemajorcomponentsoftheCNSasshowninalongitudinalmidlineview.B–F,Representativesectionsthroughthebrainandspinalcord,withthemajorlandmarkslabeled.B,Cerebrumandthalamus;C,midbrain;D,pons;E,medulla;F,cervicalspinalcord.Notethatmanypathways(e.g.,corticospinalfibers)crosssides(decussate)astheytravelthroughtheCNS,butthesedescussationsarenotindicatedinthefigure(seeChapters7and9fordetailsonthemotorandsensorypathwaycrossings).(A,FromHainesDE[ed].Fundamental Neuroscience for Basic and Clinical Applications. 3rded.Philadelphia:ChurchillLivingstone;2006.) of remote regions that had been linked by fibers of passage through the area that was damaged.

1	In the CNS, axons often travel in bundles or tracts. The names applied to tracts usually describe their origin and termination. For example, the spinocerebellar tracts convey information from the spinal cord to the cerebellum. The term pathway is similar to tract but is generally used to suggest a particular function (e.g., the auditory pathway: a series of neuron-to-neuron links across several synapses that convey and process auditory information). Gray matter exists in two main configurations in the CNS. A nucleus is a group of neurons in the CNS (in the PNS such a grouping is called a ganglion). Examples include the thalamic, cerebellar, and cranial nerve nuclei. A cortex is neurons that are organized into layers and usually found on the surface of the CNS. The most prominent are the cerebral and cerebellar cortices, which cover the surface of the cerebral hemispheres and the cerebellum, respectively ( Fig. 4.7

1	Fig. 4.7 In most nuclei and cortices, one can classify neurons into two broad categories: projection cells and local interneurons. Projection cells are neurons that send their axon to another region and thus are the origins of the various tracts of the nervous system. In contrast, local interneurons have axons that terminate in the same neural structure as their cell of origin and are involved with local computations rather than conveying signals from one region to another. These categories are not exclusive; many neurons have axons that both give off local branches and project to one or more distant regions. • Fig. 4.7 Lateral view of the human brain showing the left cerebral hemisphere, cerebellum, pons, and medulla. Note the division of the lobes of the cerebrum (frontal, parietal, occipital, and temporal) and the two major fissures (lateral and central). (From Nolte J, Angevine J. The Human Brain in Photographs and Diagrams. 2nd ed. St Louis: Mosby; 2000.)

1	Regional Anatomy of the CNS The spinal cord can be subdivided into a series of regions (see Fig. 4.6A ), each composed of a number of segments named for the vertebrae where their nerve roots enter or leave: 8 cervical, 12 thoracic, 5 lumbar, 5 sacral, and 1 coccygeal. Each portion maintains its tubular appearance, although its lumen, the spinal canal, may not remain patent. Within the gray matter the dorsal horn receives and processes sensory information from the dorsal roots, whereas the ventral horn is primarily a motor structure and contains the motor neurons whose axons project out via the ventral roots ( Fig. 4.8 The surrounding white matter consists of many tracts interconnecting spinal cord levels and for communication with the brain. Three major ones are the lateral corticospinal tract (motor), spinothalamic tract/anterolateral system (sensory), and dorsal column-medial lemniscus pathway (sensory) (see Fig. 4.6F

1	Fig. 4.6F The brainstem consists of the medulla, pons, and midbrain ( Fig. 4.9 Fig. 4.6 ). In addition to the longitudinal pathways interconnecting with the spinal cord, the brainstem contains nuclei and many additional pathways that vary by level. These structures have many functions, some of which are analogous to those of the spinal cord (e.g., conveying basic sensory information and motor commands) and others related to a variety of other brain functions, such as cardiac control and state of consciousness. The brainstem also receives input and sends motor output via cranial nerves ( Table 4.1 The cerebellum sits dorsal to the pons and medulla. It receives inputs from spinal cord, brainstem, and cerebral cortex and projects back to many of these same structures. The cerebellum is critical for motor coordination but is increasingly recognized as having key roles in other cognitive functions.

1	• Fig. 4.8 Diagram of the spinal cord, spinal roots, and spinal nerve. The spinal nerve begins where the dorsal and ventral roots fuse, and has multiple branches (rami), the first few of which are represented. A primary afferent neuron is shown with its cell body in the dorsal root ganglion and its central and peripheral processes distributed, respectively, to the spinal cord gray matter and to a sensory receptor in the skin. An α motor neuron is shown to have its cell body in the spinal cord gray matter and to project its axon out the ventral root to innervate a skeletal muscle fiber. CHAPTER 4 The Nervous System: Introduction to Cells and Systems Massa intermedia of Thalamus Interventricular foramen Fornix Anterior commissure Infundibular recess Lamina terminalis

1	CHAPTER 4 The Nervous System: Introduction to Cells and Systems Massa intermedia of Thalamus Interventricular foramen Fornix Anterior commissure Infundibular recess Lamina terminalis Choroid plexus of third ventricle •Fig. 4.9 Midsagittalviewofthebrainshowingthethirdandfourthventricles,thecerebralaqueductofthemidbrain,andthechoroidplexus.TheCSFformedbythechoroidplexusinthelateralventriclesentersthiscirculationviatheinterventricularforamen.Notealsothelocationofthecorpuscallosumandothercommissures.(FromHainesDE[ed]:Fundamental Neuroscience for Basic and Clinical Applications. 3rded.Philadelphia:ChurchillLivingstone;2006.) The thalamus sits at the upper end of the brainstem and is enclosed by the cerebrum with which it is highly interconnected (see

1	The thalamus sits at the upper end of the brainstem and is enclosed by the cerebrum with which it is highly interconnected (see Fig. 4.6B ). With a few exceptions, ascending information first reaches the thalamus, which conveys it to the cerebral cortex. These structures play a major role in many functions, including conscious awareness, volition, memory, and language. In addition to the cortex, the cerebrum contains a group of deep nuclei, the basal ganglia, that are interconnected with the cortex and thalamus and whose function will be described in The major functions of the different parts of the CNS are listed in Table 4.1

1	The major functions of the different parts of the CNS are listed in Table 4.1 CSF fills the ventricular system, a series of interconnected spaces within the brain, and the subarachnoid space directly surrounding the brain. The intraventricular CSF reflects the composition of the brain’s extracellular space via free exchange across the ependyma, and the brain “floats” in the subarachnoid CSF to minimize the effect of external mechanical forces. The volume of CSF within the cerebral ventricles is approximately 30 mL, and that in the sub-arachnoid space is about 125 mL. Because about 0.35 mL of CSF is produced each minute, CSF is turned over more than three times daily. CSF is a filtrate of capillary blood formed largely by the choroid plexuses, which comprise pia mater, invaginating capillaries, and ependymal cells specialized for transport. The choroid plexuses are located in the lateral, third, and fourth ventricles (see

1	Fig. 4.9 ). The lateral ventricles are situated within the two cerebral hemispheres. They each connect with the third ventricle through one of the inter-ventricular foramina (of Monro). The third ventricle lies in the midline between the diencephalon on the two sides. The cerebral aqueduct (of Sylvius) traverses the midbrain and connects the third ventricle with the fourth ventricle. The fourth ventricle is a space defined by the pons and medulla below and the cerebellum above. The central canal of the spinal cord continues caudally from the fourth ventricle, although in adult humans the canal is not fully patent and continues to close with age.

1	CSF escapes from the ventricular system through three apertures or foramina (a medial foramen of Magendie and two lateral foramina of Luschka) located in the roof of the fourth ventricle. After leaving the ventricular system, CSF circulates through the subarachnoid space that surrounds the brain and spinal cord. Regions where these spaces are expanded are called subarachnoid cisterns. An example is the lumbar cistern, which surrounds the lumbar and sacral spinal roots below the level of termination of the spinal cord. The lumbar cistern is the target for lumbar puncture, a clinical procedure to sample CSF. A large part of CSF is removed by bulk flow through the valvular arachnoid granulations into the dural venous sinuses in the cranium.

1	Because the extracellular fluid within the CNS communicates with the CSF, the composition of the CSF is a useful indicator of the composition of the extracellular environment of neurons in the brain and spinal cord. The main constituents of CSF in the lumbar cistern are listed in Table 4.2. For comparison the concentrations of the same constituents in blood are also given. CSF has a lower concentration of K+ , glucose, and protein but a greater concentration of Na+ and Cl− than blood does. Furthermore, CSF contains practically no blood cells. The increased concentration of Na+ and Cl− enables CSF to be isotonic to blood. The pressure in the CSF column is about 120 to 180 mm H2O when a person is recumbent. The rate at which CSF is formed is relatively independent of the pressure in the Na+ (mEq/L)148136–145K+ (mEq/L)2.93.5–5Cl− (mEq/L)120–130100–106Glucose(mg/dL)50–7570–100Protein(mg/dL)15–456.8× 103pH7.3 FromWillisWD,GrossmanRG.Medical Neurobiology. 3rded.StLouis:Mosby;1981.

1	Na+ (mEq/L)148136–145K+ (mEq/L)2.93.5–5Cl− (mEq/L)120–130100–106Glucose(mg/dL)50–7570–100Protein(mg/dL)15–456.8× 103pH7.3 FromWillisWD,GrossmanRG.Medical Neurobiology. 3rded.StLouis:Mosby;1981. ventricles and subarachnoid space, as well as systemic blood pressure. However, the absorption rate of CSF is a direct function of CSF pressure. ObstructionofthecirculationofCSFleadstoincreasedCSFpressureandhydrocephalus,anabnormalaccumulationoffluidinthecranium.Inhydrocephalustheventriclesbecomedistended,andiftheincreaseinpressureissustained,brainsubstanceislost.Whentheobstructioniswithintheventricularsystemorintheforaminaofthefourthventricle,theconditioniscalledanoncommunicating hydrocephalus. Iftheobstructionisinthesubarachnoidspaceorthearachnoidvilli,itisknownasacommunicating hydrocephalus. The Blood-Brain Barrier

1	The Blood-Brain Barrier The local environment of most CNS neurons is controlled such that neurons are normally protected from extreme variations in the composition of the extracellular fluid that bathes them. Part of this control is provided by the presence of a blood-brain barrier (other mechanisms are the buffering functions of glia, regulation of CNS circulation, and exchange of substances between the CSF and extracellular fluid of the CNS). Movement of large molecules and highly charged ions from blood into the brain and spinal cord is severely restricted. The restriction is at least partly due to the barrier action of the capillary endothelial cells of the CNS and the tight junctions between them. Astrocytes may also help limit the movement of certain substances. For example, astrocytes can take up potassium ions and thus regulate [K+] in the extracellular space. Some pharmaceutical agents, such as penicillin, are removed from the CNS by transport mechanisms.

1	Nervous Tissue Reactions to Injury Injury to nervous tissue elicits responses by neurons and glia. Severe injury causes cell death. Except in specific instances, once a neuron is lost it cannot be replaced because, in general, CHAPTER 4 The Nervous System: Introduction to Cells and Systems 63 The blood-brain barrier can be disrupted by pathology of the brain. For example, brain tumors may allow substances that are otherwise excluded to enter the brain from the circulation. Radiologists can exploit this by introducing a substance into the circulation that normally cannot penetrate the blood-brain barrier. If the substance can be imaged, its leakage into the region occupied by the brain tumor can be used to demonstrate the distribution of the tumor. neurons are postmitotic cells. In animals, two exceptions are olfactory bulb and hippocampal neurons; however, in humans, only for the hippocampus has evidence been found for significant levels of neurogenesis in the adult CNS.

1	When an axon is transected, the soma of the neuron may show chromatolysis, or “axonal reaction.” Normally, Nissl bodies stain well with basic aniline dyes, which attach to the RNA of ribosomes ( Fig. 4.10A ). After injury to the Fig. 4.10B), the neuron attempts to repair the axon by making new structural proteins, and the cisterns of the rough endoplasmic reticulum become distended with the products of protein synthesis. The ribosomes appear to be disorganized, and the Nissl bodies are stained weakly by basic aniline dyes. This process, called chromatolysis, alters the staining pattern (see Fig. 4.10C ). In addition, the soma may swell and become rounded, and the nucleus may assume an eccentric position. These morphological changes reflect the cytological processes that accompany increased protein synthesis. Because it cannot synthesize new protein, the axon distal to the transection dies (see

1	Because it cannot synthesize new protein, the axon distal to the transection dies (see Fig. 4.10C ). Within a few days the axon and all the associated synaptic endings disintegrate. If the axon had been a myelinated axon in the CNS, the myelin sheath would also fragment and eventually be removed by phagocytosis. However, in the PNS the Schwann cells that had formed the myelin sheath remain viable, and in fact they undergo cell division. This sequence of events was originally described by Waller and is called wallerian degeneration.

1	If the axons that provide the sole or predominant synaptic input to a neuron or to an effector cell are interrupted, the • Fig. 4.10 A, Normal motor neuron innervating a skeletal muscle fiber. B, A motor axon has been severed, and the motor neuron is undergoing chromatolysis. C, This is associated in time with sprouting and, in D, with regeneration of the axon. The excess sprouts degenerate. E, When the target cell is reinnervated, chromatolysis is no longer present. and even death. The best known example of this is atrophy of skeletal muscle fibers after their innervation by motor neurons has been interrupted. However, if only one or a few of the innervating axons are removed, the other surviving axons may sprout additional terminals, thereby taking up the synaptic space of the damaged axons and increasing their influence on the postsynaptic cell.

1	In the PNS, after an axon is lost through injury, many neurons can regenerate a new axon. The proximal stump of the damaged axon develops sprouts (see Fig. 4.10C ), these sprouts elongate, and they grow along the path of the original nerve if this route is available (see Fig. 4.10D ). The Schwann cells in the distal stump of the nerve not only survive the wallerian degeneration but also proliferate and form rows along the course previously taken by the axons. Growth cones of the sprouting axons find their way along these rows of Schwann cells, and they may eventually reinnervate the original peripheral target structures (see Fig. 4.10E ). The Schwann cells then remyelinate the axons. The rate of regeneration is limited by the rate of slow axonal transport to about 1 mm/day.

1	Fig. 4.10E ). The Schwann cells then remyelinate the axons. The rate of regeneration is limited by the rate of slow axonal transport to about 1 mm/day. In the CNS, transected axons also sprout. However, proper guidance for the sprouts is lacking, in part because the oligodendroglia do not form a path along which the sprouts can grow. This limitation may be a consequence of the fact that a single oligodendroglial cell myelinates many central axons, whereas a single Schwann cell provides myelin for only a single axon in the periphery. In addition, different chemical signals may affect peripheral and central attempts at regeneration differently. Other obstacles to successful CNS regeneration include formation of a glial scar by astrocytes and lack of trophic influences that guided axonal trajectories during development. 1. The functions of the nervous system include excitability, sensory detection, information processing, and behavior. 2.

1	1. The functions of the nervous system include excitability, sensory detection, information processing, and behavior. 2. The CNS includes the spinal cord and brain. The brain includes the medulla, pons, cerebellum, midbrain, thalamus, hypothalamus, basal ganglia, and cerebral cortex. 3. The neuron is the functional unit of the nervous system. Neurons have three major compartments: the dendrites, cell body, and axon. The first two receive and integrate signals, and the axon conveys the output signals of the neuron to other cells. 4. The PNS includes primary afferent neurons and the sensory receptors they innervate, the axons of somatic motor neurons, and autonomic neurons. 5. Information is conveyed through neural circuits by action potentials in the axons of neurons and by synaptic transmission between axons and the dendrites and somas of other neurons or between axons and effector cells. 6.

1	6. Different types of neurons are specialized as a consequence of their individual morphology and the ion channel distribution in the cell membrane of their soma, dendrites, and axons. 7. Sensory receptors include exteroceptors, interoceptors, and proprioceptors. Stimuli are environmental events that excite sensory receptors, responses are the effects of stimuli, and sensory transduction is the process by which stimuli are detected by transforming their energy into electrical signals. 8. Sensory receptors can be classified in terms of the type of energy they transduce or by the source of the input. Central pathways are usually named by their origin and termination or for the type of information conveyed. 9. Chemical substances are distributed along the axons by fast or slow axonal transport. The direction of axonal transport may be anterograde or retrograde. 10.

1	9. Chemical substances are distributed along the axons by fast or slow axonal transport. The direction of axonal transport may be anterograde or retrograde. 10. Glial cells include astrocytes (regulate the CNS microenvironment), oligodendroglia (form CNS myelin), Schwann cells (form PNS myelin), ependymal cells (line the ventricles), and microglia (CNS macrophages). Myelin sheaths increase the conduction velocity of axons. 11. The choroid plexus forms CSF. CSF differs from blood in having a lower concentration of K+ , glucose, and protein and a higher concentration of Na+ and Cl−; CSF normally lacks blood cells. 12. The extracellular fluid composition of the CNS is regulated by CSF, the blood-brain barrier, and astrocytes. 13. Damage to the axon of a neuron causes an axonal reaction (chromatolysis) in the cell body and wallerian degeneration of the axon distal to the injury. Regeneration of PNS axons is more likely than regeneration of CNS axons.

1	Squire L, Berg D. Fundamental Neuroscience. 4th ed. Waltham, MA: Kiernan JA, Rajakumar N. Barr’s The Human Nervous System: An Academic Press; 2012. Anatomical Viewpoint. 10th ed. Philadelphia: Lippincott Williams Vanderah T, Gould D. Nolte’s The Human Brain: An Introduction to its & Wilkins; 2013. Functional Anatomy. 7th ed. St. Louis: Mosby; 2015. Upon completion of this chapter, the student should be able to answer the following questions: 1. How is a nerve membrane’s response to small-amplitude stimuli like a passive electric circuit comprising batteries, resistors, and capacitors? 2. What factors determine the time and length constants of a nerve membrane? How do these constants shape the electric responses of the nerve membrane? 3. How does an action potential differ from the subthreshold responses of a membrane (i.e., the passive and local responses)? 4. What is the sequence of conductances that underlies the action potential? 5.

1	4. What is the sequence of conductances that underlies the action potential? 5. How are the responses of Na+ and K+ channels to membrane depolarization similar? How does the presence of the Na+ channel inactivation gate cause the responses to differ? 6. How do the gating properties of Na+ and K+ channels relate to the absolute and relative refractory periods of the action potential? 7. How is the action potential propagated without decrement? What are the factors that determine its propagation velocity? 8. What are the structural properties of myelin that underlie its ability to increase conduction velocity? 9. Given the all-or-none nature of action potentials, how are the characteristics of different stimuli distinguished by the central nervous system?

1	n action potential is a rapid, all-or-none change in the membrane potential, followed by a return to the resting membrane potential. This chapter describes how action potentials are generated by voltage-dependent ion channels in the plasma membrane and propagated with the same shape and size along the length of an axon. The influences of axon geometry, ion channel distribution, and myelin are discussed and explained. The ways in which information is encoded by the frequency and pattern of action potentials in individual cells and in groups of nerve cells are also described. Finally, because the nervous system provides important information about the external world through specific sensory receptors, general principles of sensory transduction and coding are introduced. More detailed information about these sensory mechanisms and systems is provided in other chapters.

1	When a microelectrode (tip diameter <0.5 µm) is inserted through the plasma membrane of a neuron, a difference in potential is observed between the tip of the microelectrode inside the cell and an electrode placed outside the cell. The internal electrode is approximately 70 mV negative with regard to the external electrode, and this difference is referred to as the resting membrane potential or, simply, the resting potential (see for details on the basis of the resting potential). (By convention, membrane potentials are expressed as the intracellular potential minus the extracellular potential.) Neurons have a resting potential that typically is around −70 mV.

1	One of the signature features of neurons is their ability to change their membrane potential rapidly from rest in response to an appropriate stimulus. Two such classes of responses are action potentials and synaptic potentials, which are described in this chapter and the next, respectively. Current knowledge about the ionic mechanisms of action potentials comes from experiments with many species. One of the most studied is the squid because the large diameter (up to 0.5 mm) of the squid giant axon makes it an excellent model for electrophysiological research with intracellular electrodes. The Passive Response

1	To understand how an action potential is generated and why it is needed, it is necessary to understand the passive electrical properties of the nerve cell membrane. The term passive properties refers to the fact that components of the cell membrane behave very similarly to some of the passive elements of electric circuits, including batteries, resistors, and capacitors. This is very useful because the properties of these elements are well understood. In particular, a piece of membrane containing ion channels responds to changes in voltage across it much as a circuit containing a resistor and capacitor in parallel (parallel RC circuit) would: The ion channels correspond to the resistor, and the lipid bilayer acts as a capacitor. When a battery is first connected across the two terminals of a parallel RC circuit, all of the current flows through the branch of the circuit with the capacitor, causing the voltage across it to begin changing (recall that for a capacitor, I ˜ dV/dt). Over

1	of a parallel RC circuit, all of the current flows through the branch of the circuit with the capacitor, causing the voltage across it to begin changing (recall that for a capacitor, I ˜ dV/dt). Over time, however, the current flow through the capacitor decreases, whereas that through the resistor increases. As this happens, the rate of voltage change across the capacitor (and resistor) slows, and the voltage approaches a steady-state value. This change in voltage has an exponential time course whose specific characteristics depend on the resistance (R) and capacitance (C) of the resistor and capacitor. Moreover, a time constant, τ, for this circuit can be defined by the equation τ= R * C, and it equals the time it takes for the voltage to rise (or fall) exponentially by approximately 63% of the difference between its initial and final values.

1	With regard to how an axon actually responds to electrical stimulation, Fig. 5.1 illustrates the results of an experiment in which the membrane potential of an axon is altered by passing rectangular pulses of depolarizing (upward-going 1.0 0.89 0.44 0.22 0.11 –0.11 –0.22 –0.44 –0.89 –1.0 0 5 10 15 20 25 Change in Voltage from baseline (mV)

1	Slope ˜ 4.6 MegaOhms Linear range Upward deviation due to activation of Na conductance 1.0 0.0 –1.0 –1.0 –0.5 0.0 0.5 1.0 •Fig. 5.1 (A) Voltageresponsesofanaxontorectangularpulsesofhyperpolarizingcurrent(negativenumbers)ordepolarizingcurrent(positivenumbers)asinjectedandrecordedfromanintracellularelectrode.Thechangesintransmembranepotentialaremirrorimagesofthesmallamplitudepulses.Atthethresholdlevel(current= 1.0),thereisa50:50chanceofreturningtorestingpotentialorofgeneratinganactionpotential.Forclarity,onlytherisingphaseoftheactionpotentialisshown.B, Current-voltage(I-V)plotderivedfromdatainA.Currentpulseamplitudeisplottedonthex-axis,andvoltageresponse(measuredatdottedline)isplottedonthey-axis.Notethedeviationfromlinearitywithlargedepolarizations,whichisduetoactivationofvoltage-gatedconductances.(RedrawnfromHodgkinAL,RushtonWAH:Theelectricalconstantsofacrustaceannervefibre.Proc R Soc Lond B Biol Sci. 1946;133:444-479.) pulses) or hyperpolarizing (downward-going pulses) current across

1	R Soc Lond B Biol Sci. 1946;133:444-479.) pulses) or hyperpolarizing (downward-going pulses) current across its cell membrane. The injection of positive charge is depolarizing because it makes the cell less negative (i.e., decreases the potential difference across the cell membrane). Conversely, the injection of negative charge makes the membrane potential more negative, and this change in potential is called hyperpolarization. The larger the current that is injected, the greater the change in the membrane potential will be. The responses to hyperpolarizing and small-amplitude depolarizing current pulses (see

1	Fig. 5.1A ) all have the same fundamental shape because of the passive properties of the membrane. In contrast, the shapes of the responses to the larger depolarizing stimulus pulses differ from those to hyperpolarizing and small-amplitude depolarizing current pulses because the larger stimuli activate nonpassive elements in the membrane. For the responses to hyperpolarizing current pulses, once a long enough time has elapsed from the start of the current pulse to allow the membrane voltage to plateau (essentially several times τ), virtually all of the injected current is flowing through the membrane resistance. If the difference between the initial and steady-state voltages is plotted against the amplitude of the current pulse (see Fig. 5.1B ), a linear relationship is observed for the hyperpolarizing pulses, which is exactly what is expected from Ohm’s law (V = I *

1	Fig. 5.1B ), a linear relationship is observed for the hyperpolarizing pulses, which is exactly what is expected from Ohm’s law (V = I * R) for current flowing through a resistor. The slope of this line (ΔV/ΔI) is referred to as the input resistance of the cell (Rin) and is determined experimentally, exactly as just described. Rin is related to the membrane resistance (rm) of the cell, but the exact relationship depends on the geometry of the cell and is complex in most cases. Next, note that although the current is injected as rectangular pulses, with vertical rising and falling edges, the shape of the membrane voltage responses just after the starts and ends of the pulses have slower rises and falls. Moreover, with regard to only the responses to hyperpolarizing and small-amplitude depolarizing current pulses (see

1	Fig. 5.1A ), the fall and rise in the membrane voltage have exponential shapes. This shows that the membrane is responding to these current pulses as a parallel RC circuit would; that is, the stimulus causes no change in membrane resistance or capacitance (cm), and thus the time course of the rise and fall in voltage is the same in all cases because it is governed by the same membrane time constant (τ).

1	The relationships between voltage and current just described show that within a certain range of stimulation, the cell membrane in one region of the axon can be modeled by a passive RC circuit. However, this model circuit, with only a single resistor and capacitor, takes no account of the fact that axons are spatially extended structures and that because of this, the resistance of the intracellular space is a significant factor in how electrical events in one region affect other regions. That is, if axons had no intracellular resistance, their intracellular space would be isoelectric, and voltage changes, like those just described, across one part of the axonal membrane would occur across all regions instantaneously. In this case, there would be no need for a

1	CHAPTER 5 Generation and Conduction of Action Potentials special mechanism (i.e., the action potential) to propagate signals actively down the axon. In actuality, axons (and neurons in general) are spatially extended structures with significant resistance to current flow between different regions (this is one reason the relationship of Rin and rm is complicated). Therefore, it is important to understand how current injected at one point along the axon affects the membrane potential at other points because this both helps explain why action potentials are needed and helps explain some of their characteristics. When current pulses that elicit only passive responses are passed across the plasma membrane, the size of the change in potential recorded depends on the distance of the recording electrode from the point of passage of the current ( Fig. 5.2).

1	Fig. 5.2). The closer the recording electrode is to the site of current passage, the larger and steeper the change in potential is. The magnitude of the change in potential decreases exponentially with distance from the 0.0 mm 0.5 mm Change of membrane potential 1.0 mm (mV) 1.5 2.0 2.5 mm 4.5mV •Fig. 5.2 Responsesofanaxonofashorecrabtoasubthresholdrectangularcurrentpulsebyanextracellularelectrodeappliedcloselytoitssurfaceandlocatedatdifferentdistancesfromthecurrent-passingelectrode.Astherecordingelectrodeismovedfartherfromthepointofstimulation,theresponseofthemembranepotentialisslowerandsmaller.(RedrawnfromHodgkinAL,RushtonWAH:Theelectricalconstantsofacrustaceannervefibre.Proc R Soc Lond B Biol Sci. 1946;133:444-479.) site of passage of the current, and the change in potential is said to reflect passive or electrotonic conduction. Such passively conducted changes in potential do not spread very far along the membrane before they become insignificant. As shown in

1	Fig. 5.2 , an electrotonically conducted signal dies away over a distance of a few millimeters. The distance over which the change in potential decreases to 1/e (37%) of its maximal value is called the length constant or space constant (where e is the base of natural logarithms and is equal to 2.7182). A length constant of 1 to 3 mm is typical for mammalian axons, which can be more than a meter long, which makes obvious the need for a mechanism to propagate information about electrical events generated at the soma to the far end of the axon.

1	The length constant can be related to the electrical properties of the axon according to cable theory because nerve fibers have many of the properties of an electrical cable. In a perfect cable, the insulation surrounding the core conductor prevents all loss of current to the surrounding medium, so that a signal is transmitted along the cable with undiminished strength. If an unmyelinated nerve fiber (discussed later) is compared to an electrical cable, the plasma membrane equates to the insulation and the cytoplasm as the core conductor, but the plasma membrane is not a perfect insulator. Thus the spread of signals depends on the ratio of the membrane resistance to the axial resistance of the axonal cytoplasm (ra). When the ratio of rm to ra is high, less current is lost across the plasma membrane per unit of axonal length, the axon can function better as a cable, and the distance that a signal can be conveyed electrotonically without significant decrement is longer. A useful analogy

1	membrane per unit of axonal length, the axon can function better as a cable, and the distance that a signal can be conveyed electrotonically without significant decrement is longer. A useful analogy is to think of the axon as a garden hose with holes poked in it. The more holes there are in the hose, the more water leaks out along its length (analogous to more loss of current when rm is low) and the less water is delivered to its nozzle. According to cable theory, the length constant can be related to axonal resistance and is equal to rm/ra . This relationship can be used to determine how changes in axonal diameter affect the length constant and, hence, how the decay of electrotonic potentials varies. An increase in the diameter of the axon reduces both ra and rm. However, rm is inversely proportional to diameter (because it is related to the circumference of the axon), whereas ra varies inversely to the diameter squared (because it is related to the cross-sectional area of the axon).

1	proportional to diameter (because it is related to the circumference of the axon), whereas ra varies inversely to the diameter squared (because it is related to the cross-sectional area of the axon). Thus ra decreases more rapidly than rm does as axonal diameter increases, and the length constant therefore increases (

1	Fig. 5.3 ˜ = Distance over which response decays to 1 or ~ 37% of original size (Vo) Vo/e Vo/e Vo/e Vo/e Large-diameter axon ra is lowra is high˜Vo ˜˜˜˜ = 3 mm ˜ = 1 mm 1. 2. •Fig. 5.3 Comparison of the Length Constant to Axon Diameter. Notethattheincreaseindiameterisassociatedwithadecreaseinaxialresistanceoftheaxonalcytoplasm(ra)andanincreaseinthelengthconstant(λ).(RedrawnfromBlankenshipJ.Neurophysiology. Philadelphia:Mosby;2002.) CHAPTER 5 Generation and Conduction of Action Potentials In sum, in the passive domain, the membrane response to electrical stimuli is essentially identical to that of a circuit composed of passive electrical elements, and it can thus be characterized by the length and time constants of the membrane, which will determine how far and how rapidly electrical signals at one point in the cell spread to other parts. The Local Response With regard to the experiment shown in

1	The Local Response With regard to the experiment shown in Fig. 5.1, if larger depolarizing current pulses are injected, the voltage response of the membrane no longer resembles that of a passive RC circuit. This is most easily observed with pulses that elicit depolarizations either just below or to the threshold membrane potential for an action potential but fail to evoke an action potential (tracings 0.89 and 1.0; the threshold membrane potential can be defined as the voltage at which the probability of evoking an action potential is 50%). In these cases, the voltage response shape is altered from that of the passive responses because the stimulus has changed the membrane potential sufficiently to cause the opening of significant numbers of voltage-sensitive Na+ channels (described later). Also, note the upward deviation from linearity for the corresponding points in the I-V curve (see

1	Also, note the upward deviation from linearity for the corresponding points in the I-V curve (see Fig. 5.1B ). Opening of these voltage-sensitive channels changes the membrane’s resistance and allows Na+ to enter more easily, driven by its electrochemical gradient. This entry of positive charge (Na+ current) enhances the depolarization by adding to the current pulse delivered by the electrode. The resulting depolarization is called a local response. The local response results from active changes in membrane properties (specifically, its Na+ conductance), whereas in a passive electrotonic response, the conductance to various ions remains constant. Nevertheless, the local response is not self-regenerating but, again, decreases in amplitude with distance. The change in membrane properties is insufficient for what is needed to generate an action potential. Suprathreshold Response: The Action Potential

1	Suprathreshold Response: The Action Potential Local responses will increase in size as the amplitude of the depolarizing current pulse is increased, until the threshold membrane potential is reached, at which point a different sort of response, the action potential (or spike), can occur. The threshold value is typically near −55 mV. Normally, when the membrane potential exceeds this value, an action potential is always triggered.

1	Fig. 5.4 shows the typical shape of an action potential. When the membrane is depolarized past threshold, the depolarization becomes explosive and overshoots in such a way that the membrane potential reverses from negative to positive and approaches, but does not reach, the Nernst equilibrium potential for Na+ (ENa; see ). The membrane potential then returns toward the resting membrane potential (repolarizes) almost as rapidly as it was depolarized, and in general, it hyperpolarizes beyond its resting potential (the afterhyperpolarization). The main phase of the action potential (from the onset to the return to the resting potential) typically has a duration of 1 to 2 msec, but the afterhyperpolarization, it can persist •Fig. 5.4 Components of the Action Potential With Regard to Time and Voltage.

1	return to the resting potential) typically has a duration of 1 to 2 msec, but the afterhyperpolarization, it can persist •Fig. 5.4 Components of the Action Potential With Regard to Time and Voltage. Markersindicatetheabsoluteandrelativerefractoryperiods.Notethatthetimescaleforthefirstfewmillisecondshasbeenexpandedforclarity.RMP,restingmembranepotential.(RedrawnfromBlankenshipJ.Neurophysiology. Philadelphia:Mosby;2002.) from a few to 100 msec, depending on the particular type of neuron.

1	The action potential differs from the subthreshold and passive responses in three important ways: (1) It is a response of much larger amplitude, in which the polarity of the membrane potential actually overshoots 0 mV (the cell interior becomes positive in relation to the exterior). (2) The action potential is generally propagated down the entire length of the axon without decrement (i.e., it maintains its size and shape because it is regenerated as it travels along the axon). (3) It is an all-or-none response, which means that a stimulus normally either produces a full-sized action potential or fails to produce one. This all-or-none nature is in contrast both to the graded nature of the passive and local responses described previously and to synaptic responses (see Ionic Basis of Action Potentials

1	Ionic Basis of Action Potentials Recall that the resting membrane potential is determined primarily by the weighted average of the Nernst potentials for Na+ (ENa) and K+ (EK), as defined by the chord conductance equation (see ). The weighting factors are the conductance (g = 1/resistance) to each ion. At rest the conductance to K+ (gK) is high in relation to that for Na+ (gNa), and so the resting membrane potential (Vr) is closer to EK (Vr ≅−70 mV). If, however, the relative conductances to these ions were to change, this would cause a corresponding change in the membrane potential. For example, an increase in gK would hyperpolarize the membrane, whereas a decrease in gK would depolarize the membrane because EK is approximately −100 mV. Conversely, an increase in gNa would depolarize the membrane and, if of sufficient magnitude, even a lead to reversal in membrane polarity because ENa is approximately +65 mV.

1	An axonic action potential is, in fact, the result of a rapid sequence of transient changes in gNa or gK, or both. In all axons there is a brief rise in gNa, followed by a decline back to baseline levels. In some axons, this change in gNa occurs against a fixed resting gK (because of leak channels, which are not voltage-gated; discussed later). In many other cases, however, both gNa and gK change. Thus as with the resting membrane potential, the action potential depends on the opposing tendencies of (1) the Na+ gradient to bring the resting membrane potential toward the Nernst potential for Na+ and (2) the K+ gradient to bring the resting membrane potential toward the Nernst potential for K+; but in contrast to when the neuron is at rest, the gK/gNa ratio is not constant but is changing continuously. One additional difference is that because the membrane potential is changing, a capacitative current also exists, and this must also be taken into account to describe the membrane

1	continuously. One additional difference is that because the membrane potential is changing, a capacitative current also exists, and this must also be taken into account to describe the membrane potential quantitatively during an action potential (as a corollary, note that the chord conductance equation is valid only when the membrane potential is constant because then there is no capacitative current).

1	The early phase of the action potential (the positive deflection of the membrane potential toward ENa) is a result of a rapid increase in gNa and thus of the Na+ current (INa). These changes cause the membrane potential to move toward the equilibrium potential for Na+ . The peak of the action potential does not reach ENa because the rise in gNa is not infinite (i.e., the gK/gNa ratio does not fall to zero).

1	Because of the nature of the underlying Na+ channels (described later), the rise in gNa with depolarization is transient. Moreover, in many cases, the depolarization leads to a rise in gK. These two factors cause the gK/gNa ratio to stop falling and start increasing; as a result, the membrane potential is driven back toward EK and thus repolarizes toward its resting value. In cases in which the repolarization involves a rise in gK, the membrane potential hyperpolarizes temporarily beyond its normal rest value (if gK does not change, the drop in gNa causes the membrane simply to return to its resting potential). This afterhyperpolarization occurs because gK remains elevated for a period of time after the action potential. As gK returns to its baseline level, the membrane potential returns to its rest value. These changes in conductance can be explained by the properties of Na+ and K+ ion channels, which are described next.

1	These changes in conductance can be explained by the properties of Na+ and K+ ion channels, which are described next. Early studies of the mechanism underlying action potentials indicated that ion currents pass through separate Na+ and K+ channels, each with distinct characteristics, in the cell membrane. Subsequent research has supported this interpretation. The amino acid sequences of the channel proteins and many of the functional and structural characteristics of the channels are now known in detail. The structure of a voltage-gated Na+ channel (

1	The structure of a voltage-gated Na+ channel ( Fig. 5.5 ) consists of four α subunits and two β subunits. The α subunit has four repeated motifs each of six transmembrane helices that surround a central ion pore. The pore walls are partly formed by the six helices in each motif. Most voltage-gated K+ channels are composed of four separate subunits, each consisting of a polypeptide with six membrane-spanning segments, similar to the motifs that make up the α subunit of the Na+ channel. An important characteristic of some channels, such as those that underlie the action potential, is that they are gated by the membrane voltage. These voltage-gated channels sense the potential across the membrane and then act to either open or close the pore according to the membrane potential. The gates are formed by groups of charged amino acid residues, and the voltage dependence of the Na+ and K+ channel gates can account for the complex changes in gNa and gK that occur during an action potential.

1	The Characteristics of the Na+ and K+ Channels Explain the Conductance Changes During the Action Potential Use of standard intracellular recording along with voltage-clamp techniques enabled investigators to characterize the underlying ionic currents and conductance changes

1	CHAPTER 5 Generation and Conduction of Action Potentials 71 •Fig. 5.5 A Model of the Voltage-Gated Na+ Channel. A, Thelargeredelementsrepresentthefourα subunitsandthetwoyellowelementsareβ subunitswiththereceptorsitesforα scorpiontoxin(ScTX)andtetrodotoxin(TTX)indicated.B, β1andβ2subunitsflankinganα subunitareshownwiththeirtransmembranehelices.(RedrawnfromCatterallWA:Structureandfunctionofvoltage-gatedsodiumchannelsatatomicresolution.Exp Physiol. 2014;99:35-51.) associated with the action potential. Detailed statistical analyses of these recordings also allowed remarkable inferences to be made about the nature of the channels that passed these Na+ and K+ currents. The development of the patch recording, however, enabled direct observation of the behavior of individual channels. In this technique, a specially shaped microelectrode is placed against the surface of a cell, and suction is applied to the microelectrode. As a result, a high-resistance seal is formed between the mem brane and

1	a specially shaped microelectrode is placed against the surface of a cell, and suction is applied to the microelectrode. As a result, a high-resistance seal is formed between the mem brane and the tip of the microelectrode (Fig. 5.6A ), which allows recording of the activity of whatever channels happen to be in the patch of membrane that is inside the seal. Under ideal conditions only one or a few ion channels of a single type are present in the membrane patch.

1	Patch recordings show that many ion channels flip spontaneously between open and closed conductance states as if they have gates that open and close the entrance to their pore. In the case of voltage-gated channels, the gate is sensitive to the voltage across the membrane, and thus the time a gate spends in each state is a probabilistic function of the membrane potential. A patch recording of a K+ channel demonstrates this probabilistic behavior (see Fig. 5.6B ). As the membrane potential is clamped to more depolarized levels, the channel spends more time in its open state, which reflects the voltage dependence of the probability that the channel will open (see Fig. 5.6C ). Also, the amplitude of the current in the open state increases with the level of depolarization; this is because the driving force for K+ is greater at more depolarized levels (i.e., the membrane potential is farther from the K+ Nernst potential).

1	The behavior of a Na+ channel is more complex than that of the K+ channel. Like the K+ channel, it has a 72 SECTION2Berne & Levy Physiology X˜X˜X˜X˜X˜Cytosol Pipette solution Patch pipette Ground Operational amplifier Feedback resistor Micropipette Cell membrane Ion channel A 1.0 0.8 0.6 0.4 0.2 C Po °80 °60 °40 °20 200 mV D Time (1ms/division)B ˜°°50 0 50 Membrane potential (mV) °1.0 0 Na+ current density (mA/cm2)

1	CHAPTER 5 Generation and Conduction of Action Potentials •Fig. 5.6 A, Amicropipetteisappliedtothecellmembraneandsufficientsuctionisappliedtoelectricallyisolateasinglechannelatthetip.Anamplifierrecordsthecurrentthatpassesthroughthechannel.B, EachlineshowsthecurrentpassedthroughaK+ channelasitopensspontaneously.Notethatasthetransmembranevoltageisprogressivelydepolarized(fromtoptobottom),boththeprobabilitythatthechannelwillopenandtheamplitudeofthecurrentareincreased.C, Agraphoftheprobabilitythatthechannelwillopenversusmembranedepolarization.D, Agraphoftransmembranevoltage(lowertracing,right-sidedscale)andthecurrentdensityfromapopulationofNa+ channels(uppertracing,left-sidedscale)isolatedinapatchsimilartothatinA (exceptthatitcontainsseveralNa+ channels).Initially,atrestingpotential,thereisnocurrentflow.Withdepolarizationto0mV,thereisaninwardNa+

1	(exceptthatitcontainsseveralNa+ channels).Initially,atrestingpotential,thereisnocurrentflow.Withdepolarizationto0mV,thereisaninwardNa+ currentthatiscurtailedevenwhilethedepolarizationcontinues.Thisisduetotheclosingofthechannels’inactivationgates.Afterabriefreturntorestingpotential,anotherdepolarizationto0mVevokesinwardcurrentflow,butitissmallerandbrieferbecausetherehasnotbeenenoughtimeformostoftheslowinactivationgatestoreopen.(B, C, andD areredrawnfrom http://www.physiologymodels.info/electrophysiology voltage-sensitive gate (activation gate) whose probability of being open increases with depolarization. However, unlike K+ channels, with maintained depolarization, Na+ channels open only at the onset of the depolarization and then remain closed. This suggests that Na+ channels have a second gate (inactivation gate) whose probability of being open goes down as the membrane is depolarized. Thus any Na+ current conducted by these channels will be transient (see

1	Fig. 5.6D ), because the same stimulus (depolarization) increases both the probabilities that the activation gate will open and that the inactivation gate will close. Note that the Na+ channel thus has two closed states, one in which the activation gate is closed, and the channel is said to be “closed,” and one in which the inactivation gate is closed, and the channel is referred to as “inactivated.” The reason the channel is called “inactivated” when the second gate closes is that once this gate closes, it will remain so until the membrane is repolarized. The activation gate, in contrast, can open and close at all membrane potentials, just with differing probabilities.

1	With the knowledge of the Na+ and K+ channel gating behavior just discussed, we can understand how the action potential is generated by the interaction of these channels (in the following, we assume that both gNa and gK change during the action potential). As stated previously, the action potential starts with a rapid increase in Na+ conductance (gNa; Fig. 5.7 ). This increase in Na+ conductance reflects the opening of many Na+ channels in response to the depolarization. The open channels allow the influx of Na+ ions, and the effect of this current is to depolarize the membrane further. Note that this is a positive feedback loop, which accounts for the explosive nature of the action potential: the Na+ current depolarizes the membrane, which causes more Na+ channels to open, which in turn increases the Na+ current. In sum, the voltage-dependent opening of Na+ channels and the depolarizing action of the Na+ current account for the rising phase of the action potential.

1	The end of the rising phase and the subsequent falling (repolarization) phase of the action potential is the result of two processes: a reduction in gNa and an increase in gK. The rise in gK is simply a consequence of membrane depolarization, which increases the probability that the K+ channel will be open. The decrease in gNa results from two •Fig. 5.7 The Action Potential and the Conductance and Currents That Underlie the Action Potential With Regard to Time. NotethattheincreasedconductanceforNa+ (gNa),aswellasitsinwardflow,isassociatedwiththerisingphaseoftheactionpotential,whereastheslowerincreaseinconductanceforK+ (gK),aswellasitsoutwardflow,isassociatedwithrepolarizationofthemembraneandwithafterhyperpolarization.ThereductionintheNa+ current(INa)beforethepeakoftheactionpotential(eventhoughgNaisstillhigh)isduetoinactivationoftheNa+ channels.(RedrawnfromSquiresLR,etal:Fundamental Neuroscience. 2nded.SanDiego,CA:AcademicPress;2002.) factors. First, Na+ channels are inactivated as a

1	channels.(RedrawnfromSquiresLR,etal:Fundamental Neuroscience. 2nded.SanDiego,CA:AcademicPress;2002.) factors. First, Na+ channels are inactivated as a result of the closing of the inactivation gate with depolarization. Unlike the activation gate, which can flip between states even when the membrane is depolarized, the inactivation gate, once closed, remains closed until significant repolarization occurs. Second, as the gK/gNa ratio increases (as a result of both inactivation of Na+ channels and opening of K+ channels), the membrane begins to repolarize, and this repolarization acts to shut the activation gate of the Na+ channel. The closure of both voltage-gated Na+ and K+ channels during the falling phase brings the membrane back to its resting state. If only Na+ channels had opened during the action potential (as is the case for some axons), the membrane would simply return to its rest potential. If voltage-gated K+ channels also had opened during the action potential, an

1	during the action potential (as is the case for some axons), the membrane would simply return to its rest potential. If voltage-gated K+ channels also had opened during the action potential, an afterhyperpolarization would be present because these K+ channels close slowly in response to hyperpolarization.

1	Knowledgeofthemolecularstructureofchannelshasincreasedtheunderstandingofthebasisoftheirproperties.Forexample,mostchannelsarehighlyselectiveforaparticularion.First,ifthechannelwallsarelinedwitheitherpositiveornegativecharges,theneithercationsoranionscanbeexcluded;however,mostchannelsarealsodifferentiallypermeablebydifferentionsofthesamecharge.Thisfurtherselectivityappearstobetheresultofrequiringionstobecomedehydratedastheypassthroughthenarrowestpartofachannel,knownastheselectivity filter. Ionsinsolutionarehydrated(aresurroundedbyashellofH2Omolecules),andtheradiusofthishydrationshellisdifferentforeachtypeofion.InNa+ andK+

1	filter. Ionsinsolutionarehydrated(aresurroundedbyashellofH2Omolecules),andtheradiusofthishydrationshellisdifferentforeachtypeofion.InNa+ andK+ channels,tomakedehydrationenergeticallypossible,theporeofthechannelislinedwithnegativelypolarizedaminoacidsubstituentsofaparticulargeometry,andthesesubstituentssubstituteforthewatermolecules.Suchsubstitution,however,requiresclosematchingofthefilter’ssizetotheion’shydrationshell.Becauseeachionhasadifferent-sizedshell,aparticularchannelwillbestallowpassageofoneparticularionicspecies.

1	Tetrodotoxin (TTX), oneofthemostpotentpoisonsknown,specificallyblockstheNa+ channel.TTXbindstotheextracellularsideofthesodiumchannel(see Fig.5.5A ).Tetraethylammonium (TEA+), anotherpoison,blocksK+ channels.TEA+ enterstheK+ channelfromthecytoplasmicsideandblocksthechannelbecauseTEA+ isunabletopassthroughit.Theovariesofcertainspeciesofpufferfish,alsoknownasblowfish,containTTX.RawpufferfishisahighlyprizeddelicacyinJapan.ConnoisseursofpufferfishenjoythetinglingnumbnessofthelipscausedbytheminusculequantitiesofTTXpresentintheflesh.Sushichefswhoaretrainedtoremovetheovariessafelyarelicensedbythegovernmenttopreparepufferfish.Despitetheseprecautions,severalpeopledieeachyearasaresultofeatingimproperlypreparedpufferfish.

1	SaxitoxinisanotherblockerofNa+ channelsthatisproducedbythereddishdinoflagellatesthatareresponsibleforso-calledredtides.Shellfisheatthedinoflagellatesandconcentratesaxitoxinintheirtissues.Apersonwhoeatstheseshellfishmayexperiencelife-threateningparalysiswithin30minutesafterthemeal.

1	When a nerve is depolarized very slowly, the normal threshold may be passed without the firing of an action potential; this phenomenon is called accommodation. Both Na+ and K+ channels are involved in accommodation. In response to membrane depolarization, gNa first increases and then, a short time later, decreases. This is due to the opening of the activation gates and closing of the inactivation gates of the Na+ channels. Normally, membrane depolarization to threshold or beyond triggers an action potential; however, the explosive depolarization of the action potential can occur only if a critical number of Na+ channels are recruited. Thus if a cell is slowly depolarized, Na+ channels can become inactivated even without the occurrence of an action potential, and the pool of available noninactivated Na+ channels (i.e., channels in the closed state) can be reduced to the point at which a stimulus to may not be able to recruit a sufficient number of Na+ channels to generate an action

1	Na+ channels (i.e., channels in the closed state) can be reduced to the point at which a stimulus to may not be able to recruit a sufficient number of Na+ channels to generate an action potential. An additional factor in causing accommodation is that K+ channels open slowly in response to the depolarization. The increased gK tends to oppose depolarization of the membrane, which makes it even less likely to fire an action potential.

1	When a cell is refractory, it is either completely unable to fire an action potential or it requires a much stronger stimulation than usual. During much of the action potential, the cell is completely refractory because it will not fire another action potential no matter how strongly it is stimulated. This absolute refractory period (see Fig. 5.4 ) occurs when a large fraction of the Na+ channels are inactivated and therefore cannot be reopened until the membrane is repolarized. During this period, the critical number of Na+ channels required to produce an action potential cannot be recruited.

1	Inaninheriteddisordercalledprimary hyperkalemic paralysis, patientshaveepisodesofpainfulspontaneousmusclecontractionsfollowedbyperiodsofparalysisoftheaffectedmuscles.Thesesymptomsareaccompaniedbyelevated[K+]inplasmaandextracellularfluid.Somepatientswiththisdisorderhavemutationsofvoltage-gatedNa+ channelsthatresultinadecreasedrateofvoltageinactivation.Thisresultsinlonger-lastingactionpotentialsinskeletalmusclecellsandincreasedeffluxofK+ duringeachactionpotential,whichcanraiseextracellular[K+]. Theelevationinextracellular[K+]causesdepolarizationofskeletalmusclecells.Initially,thedepolarizationbringsmusclecellsclosertothreshold,andsospontaneousactionpotentialsandcontractionsaremorelikelytooccur.Asdepolarizationofthecellsbecomesmoremarked,thecellsbecomerefractorybecauseincreasingnumbersofNa+ channelsbecomeinactivated.Consequently,thecellsbecomeunabletofireactionpotentialsandarenotabletocontractinresponsetoactionpotentialsintheirmotoraxons.

1	During the latter part of the action potential, and during the afterhyperpolarization period, the cell is able to fire a second action potential, but a stimulus stronger than normal is required. This period is called the relative refractory period. Early in the relative refractory period, before the

1	CHAPTER 5 Generation and Conduction of Action Potentials membrane potential has returned to the resting potential level, some Na+ channels are still voltage inactivated, but there are enough in the closed state (and therefore have the potential to open when the membrane is depolarized) to support the generation of an action potential if they are stimulated to open. However, a stimulus stronger than normal is necessary to recruit the critical number of Na+ channels needed to trigger an action potential (i.e., the reduction in the total number of available Na+ channels is countered by increasing the probability of opening). Throughout the relative refractory period, conductance to K+ is elevated, which opposes depolarization of the membrane. This increase in K+ conductance continues throughout the afterhyperpolarization and accounts for most of the duration of the relative refractory period. Conduction of Action Potentials

1	Conduction of Action Potentials Fundamental to nervous system function is the transmission of information along neuronal pathways. To accomplish this, neurons generate action potentials that propagate down the length of their axon without decrement in size in order to trigger neurotransmitter release from the presynaptic terminals. How action potentials propagate down an axon and how the characteristics of the axon affect this propagation are discussed in this section. How they trigger transmitter release is covered in Action Potential as a Self-Reinforcing Signal

1	Passive conduction will not transport a signal from one end of an axon to the other unless the axon is very short (i.e., on the order of its length constant) because passively conducted signals decrease in size rapidly with distance from their origin. Neurons with such short axons exist; for example, in the retina of the eye, the distance from one neuron to the next is so small that electrotonic (passive) conduction is sufficient. However, in most cases, axons are many times longer than their length constant. In fact, they can be up to 1 m or more in length (e.g., those of motor neurons) and thus hundreds of times their length constant. Nonetheless, if researchers were to record from points along a typical axon, they would find that as the action potential arrives at successive points traveling along the axon, its shape and size remain constant. This is because the action potential regenerates itself as it is conducted along the fiber and thus is said to be actively propagated.

1	Fig. 5.8 shows how in a local response the current that flows in through one part of the membrane acts to depolarize the neighboring membrane. The same thing happens when the Na+ channels are opened by an action potential at one site along the axon, except that in this case, the current will be large enough to depolarize the areas on either side past threshold and thus generate action potentials in these neighboring areas. The inward Na+ current in these areas can then provide the current to depolarize their neighbors past threshold so that they in turn generate action potentials, •Fig. 5.8 Mechanism of Electrotonic Spread of Depolarization. A, Thereversalofmembranepolaritythatoccurswithlocaldepolariza-tion.B, Thelocalcurrentsthatflowtodepolarizeadjacentareasofthemembraneandallowconductionofthedepolarization.B + – + – + – + – + – + – + – + – + – + – + – + – + – + – – + – + – + + – + – + – – + – + – + – + – + – + – + – + – + – + – + – + – + – + and so on. In short, action potential

1	+ – + – + – + – + – + – + – + – + – + – + – + – + – + – – + – + – + + – + – + – – + – + – + – + – + – + – + – + – + – + – + – + – + – + and so on. In short, action potential propagation along an axon involves recurring cycles of depolarization to provide sufficient local current flow for generation of an action potential in an adjacent region of the cell membrane. Thus the action potential is said to be propagated down the axon, with “new” action potentials being generated along its length. In this way, the action potential can propagate down the entire length of the axon while retaining the same size and shape.

1	Normally, action potentials are first generated at the axon’s initial segment (i.e., where the axon is attached to the neuron cell body or proximal dendrite) and then conducted to the terminal end. The reason for this is that the initial segment has a very high density of voltage-gated Na+ channels, and thus it has a lower threshold for spiking than does the soma or dendrites. However, axons are not inherently unidirectional conductors. For example, as implied by the local circuits shown in Fig. 5.8 , an action potential generated by a depolarization in the middle of an axon is conducted in both directions from its initiation site simultaneously.

1	Why does a spike that starts at the initial segment not propagate in both directions? In fact, it does. In addition to propagating down the axon, the current flowing from the initial segment back to the soma can cause a spike to be generated in the soma because the soma also has voltage-gated Na+ channels. Additional “backpropagating” spikes from the axon do not occur, however, nor does the somatic spike cause the initial segment of the axon to fire a second time (and thereby send another spike down the axon and start a repeating cycle). This does not happen because the refractory period of the membrane makes any area that has already spiked unable to fire a second spike for a short time. Thus for a spike that started at the initial segment and has begun traveling down the axon, current flowing in at the site of the spike depolarizes the membrane on both sides of that site. However, the side closer to the cell body, which has recently fired a spike, cannot respond to this

1	current flowing in at the site of the spike depolarizes the membrane on both sides of that site. However, the side closer to the cell body, which has recently fired a spike, cannot respond to this depolarization because its Na+ channels are still inactivated. By the time Na+ channels are de-inactivated (have returned to their closed state and would be able to open) the depolarization of membrane at that site has ended (because the action potential lasts only for ≈1 msec). Thus the inactivation gate of the Na+ channel not only helps determine the duration of the action potential but it also is responsible for its singular and unidirectional propagation from its origin at the initial segment.

1	The speed of conduction in a nerve fiber is determined by the electrical properties of the cytoplasm and the plasma membrane that surrounds the fiber, as well as by its geometry. In nonmyelinated fibers, conduction velocity is proportional to the square root of the cross-sectional diameter (Fig. 5.9 ). This effect is related to the changes in ra and rm with diameter. As the diameter of a fiber increases, ra decreases with the square of the diameter, and rm increases only linearly with diameter; as a result, resistance to current •Fig. 5.9 Conductionvelocitiesofunmyelinated(blue) andmyelinated(red) felineaxonsasfunctionsofaxondiameter.Solid lines representmeasureddata.Dotted lines representextrapolationsthatshowtheadvantageofmyelinationoversimplyincreasingaxondiameterasamechanismforincreasedconductionvelocity.(FromSchmidt-Nielsen K.

1	K. Animal Physiology: Adaptation and Environment. 5thed.Cambridge,UK:CambridgeUniversityPress;1997.) flow down the axon decreases more than it does to current flow across the membrane. This increases the length constant (see Fig. 5.3 ), which means that a greater amount of the current entering at one site is delivered to neighboring regions of the axon, which brings those regions to threshold more quickly, and thus the action potential is conducted faster along fibers with large diameters. However, increasing the diameter also increases the surface area of the plasma membrane over which inner negative and outer positive charges are held to each other. Discharging this increased capacitance tends to slow conduction and mitigate the increase in conduction velocity gained by increasing diameter.

1	In vertebrates, many nerve fibers are coated with myelin, and such fibers are said to be myelinated. Myelin consists of the plasma membranes of Schwann cells (in the peripheral nervous system) or oligodendroglia (in the central nervous system [CNS]), which wrap around and insulate the nerve fiber ( Fig. 5.10A and B ). The myelin sheath consists of several to more than 100 layers of glial cell plasma membrane. Gaps about 1-2 µm wide, known as nodes of Ranvier, separate the contribution of one Schwann cell (or oligodendrocyte) from that of another. For all but the axons of smallest diameter, a myelinated axon has much greater conduction velocity than does an unmyelinated fiber of the same caliber because the myelin sheath increases the effective membrane resistance of the axon, decreases the capacitance of the axon membrane, and limits the generation of action potentials to the nodes of Ranvier. In short, myelination greatly alters the electrical properties of the axon.

1	Because the many wrappings of membrane around the axon increase the effective membrane resistance rm/ra and the length constant are much greater. The increased membrane resistance means that less current is lost through the membrane per length of axon, and thus the amplitude of a conducted signal decreases less with distance along the axon and needs to be regenerated (by opening of Na+ channels) less often.

1	In addition, the thicker myelin-wrapped membrane results in a much larger separation of charges across it than exists across the bare membrane of an axon, so that the charges across it are much less tightly bound to each other. This is analogous to when the plates of a capacitor are moved apart and reduce its capacitance. Because the effect of membrane capacitance is to slow the rate at which the membrane potential can be changed, the reduced capacitance of myelinated axons means that the depolarization occurs more rapidly. For all these reasons, conduction velocity is greatly increased by myelination, and the current generated at one node of Ranvier is conducted at great speed to the next (see Fig. 5.10 In myelinated axons, the Na+ channels that bring about generation of an action potential are highly concentrated

1	Fig. 5.10 In myelinated axons, the Na+ channels that bring about generation of an action potential are highly concentrated CHAPTER 5 Generation and Conduction of Action Potentials •Fig. 5.10 A, Schematicillustrations,incrosssectionandlongitudinalsectionthroughanodeofRanvier,ofaSchwanncellwrappedaroundanaxontoformmyelin.NotethattheaxonisexposedtotheextracellularspaceonlyatthenodeofRanvier.B, Viewoftwonodesandtheinterveninginternodeofmyelin.C, Saltatoryconductioninamyelinatedaxonwithaplotoftheactionpotentiallocationalongtheaxon(x-axis)versustime(y-axis).Notetheshorttimetakenfortheactionpotentialtotraversethelargedistancebetweennodes(shallow sloped lines ontheplot)becauseofthehighresistanceandlowcapacitanceoftheinternodalregion.Incontrast,theactionpotentialslowsasitcrosseseachnode(steep sloped line segments). (B, RedrawnfromSquiresLRetal:Fundamental Neuroscience. 2nded.SanDiego,CA:AcademicPress;2002.C, RedrawnfromBlankenshipJ:Neurophysiology. Philadelphia:

1	Mosby;2002.) at the nodes of Ranvier and are not found between them. Thus the action potential is regenerated only at the nodes of Ranvier (0.3-2 mm apart) rather than being regenerated continuously along the fiber, as is the case in an unmyelinated fiber. Resistance to the flow of ions across the many layers that make up the myelin sheath is so high that transmembrane currents are largely restricted to the short stretches of naked plasma membrane that are present at the nodes of Ranvier (see Fig. 5.10C ). Therefore, the action potential is regenerated at each successive node. The local currents entering the node are almost entirely conducted from one node to the next node, bringing each node to threshold in about 20 µsec. Thus the action potential appears to “jump” from one node of Ranvier to the next, and the process is called saltatory (from the Latin word saltare, “to leap”) conduction ( Fig. 5.11 Functional Consequences of Myelination

1	Fig. 5.11 Functional Consequences of Myelination The functional consequences of myelination can be highlighted by a comparison of squid and mammalian axons. Although human nerve fibers are much smaller in diameter than squid giant axons, human axons conduct at comparable or even faster speeds because of myelination. The unmyelinated squid giant axon has a 500-µm diameter and a conduction velocity of about 20 m/sec. In mammals, axon diameters range from about 0.2 to 20 µm, and all fibers with diameters larger than 1-2 µm are myelinated. An unmyelinated mammalian nerve fiber, which has a diameter of less than 1 to 2 µm, has a conduction velocity of less than 2 m/sec (see

1	Fig. 5.9 ), as expected because of its smaller diameter in comparison to the squid giant axon. In contrast, a 10-µm myelinated mammalian fiber has a conduction velocity in the range of 50 m/sec, more than twice that of the 500-µm squid giant axon, despite being 1/50 of its diameter. Thus the high conduction velocity with far narrower axons achieved by myelination allows a tremendous increase in neuronal connectivity without enormously expanding the volume of the CNS. This is certainly one factor that enabled the evolution of mammalian nervous systems with their huge numbers of neurons that are able to generate everything from fast reflexes to efficient and complex mental processing.

1	To receive information about the world, the CNS contains a wide variety of sensory receptors, each of which is specialized •Fig. 5.11 Comparison of Action Potential Conduction in an Unmyelinated Axon and in a Myelinated Axon. Attheinitialtime(A andC),anactionpotentialisbeinggeneratedattheleftsideofeachaxon.Notethattheinwardcurrentintheunmyelinatedaxon(A) isdepolarizinganadjacentportion,whereastheinwardcurrentinthemyelinatedaxon(C) isdepolarizingallofthemembranetothenextnode.Atthesecondinstantintime(B andD),theactionpotentialintheunmyelinatedaxon(B) hasbeengeneratedintheadjacentportion,whereastheactionpotentialinthemyelinatedaxon(D) hasbeengeneratedatsubsequentnodesandisalreadydepolarizingthelastnodetotheright.(RedrawnfromCastroA,etal:Neuroscience: An Outline Approach. Philadelphia:Mosby;2002.)

1	Insomediseasesknownasdemyelinating disorders, themyelinsheathdeteriorates.Inmultiple sclerosis, scatteredprogressivedemyelinationofaxonsintheCNSresultsinlossofmotorcontrolandsensorydeficits.Theneuropathycommoninseverecasesofdiabetesmellitusiscausedbythedemyelinationofperipheralaxons.Whenmyelinislost,thelengthconstantbecomesmuchshorter.Hence,theactionpotentiallosesamplitudeasitiselectrotonicallyconductedfromonenodeofRanviertothenext.Ifdemyelinationissufficientlysevere,theactionpotentialmayarriveatthenextnodeofRanvierwithinsufficientstrengthtofireanactionpotentialatthatnode,leadingtopropagationfailure. to detect a particular type of energy (stimulus). When a stimulus activates a sensory receptor, it initiates a process called sensory transduction by which information about the stimulus (e.g., its intensity and duration) is converted into local electrical signals. These local signals are called

1	TheactionpotentialsofmyelinatedaxonsmaynothaveahyperpolarizingafterpotentialoranextendedrelativerefractoryperiodbecausetheirK+ channelsaredisplacedfromthenodesintothepartlyexposedflankingparanodes.Thatincreasestherateatwhichthesefast-conductingaxonscanfire.Myelinatedaxonsarealsomoremetabolicallyefficientthanunmyelinatedaxons.Na+,K+-ATPaseextrudestheNa+ thatentersthecellandcausestheK+ thatleavesthecelltoreaccumulateduringactionpotentials.Inmyelinatedaxons,ioniccurrentsarerestrictedtothesmallfractionofthemembranesurfaceatthenodesofRanvier.Forthisreason,farfewerionstraverseaunitlengthoffibermembrane,andmuchlessionpumping—andenergyexpenditure—isnecessarytomaintainthegradients.

1	Investigatorscanrecordanactionpotentialwithamicroelectrodewithoutpenetratingtheaxonbyplacingtwospacedelectrodesonitssurfaceandcomparingtheelectricalchargeateachpoint.Anelectrodelocatedwherethereisanactionpotentialwouldyieldasomewhatnegativesignalincomparisontoanelectrodewherethereisnoactionpotential.Astheactionpotentialisconductedtothesecondelectrode,thepolarityoftherecordingreverses.Thistechniqueisusedclinicallytoassessnervefunction.Peripheralnervesandmanycentralpathwaysconsistofapopulationofaxons ofvariousdiameters(Fig.5.12 );someoftheaxonsaremyelinated,andsomearenot.Consequently,actionpotentialstravelatdifferentvelocitiesintheindividualaxons.Asaresult,arecordingfromsuchanervewithexternalelectrodesdoesnotshowasinglesynchronouspeakbutaseriesofpeaksthatvaryintime(whichreflectstheconductionvelocityofgroupsofaxons)andinmagnitude(whichreflectsthenumberofaxonsineachvelocitygroup).Thisiscalledacompound action

1	action potential.Theclinicalvalueofsucharecordingisitsability,incertaindiseasestates,torevealthedysfunctionofaparticulargroupofaxonsassociatedwithspecificfunctions,aswellasthenoninvasivenatureofthetechniquebecauseitcanbeperformedwithskinsurfaceelectrodes( receptor or generator potentials. The receptor potentials can then be transformed into patterns of action potentials that are conducted over one or more axons into the CNS. In order for this to happen, the stimulus must produce receptor potentials that are large enough to change the spiking levels of one or more primary afferent fibers that are connected to the receptor. Weaker intensities of stimulation can produce subthreshold receptor potentials, but such stimuli do not change the activity of central sensory neurons and thus are not detected. Thus stimulus threshold is defined as the weakest stimulus that can be reliably detected.

1	Environmental events that evoke sensory transduction can be mechanical, thermal, chemical, or other forms of energy. However, the types of information used by a CHAPTER 5 Generation and Conduction of Action Potentials particular organism depend on its set of sensory receptors. For example, humans cannot sense electrical or magnetic fields, but other animals can sense such stimuli. In particular, many fish have electroreceptors, and various fish and birds use the earth’s magnetic field to orient themselves during migration. The transduction process varies with the type of environmental stimulus being detected. Fig. 5.13 shows three examples of how stimuli can alter the membrane properties of the specific sensory receptors that transduce such stimuli 0246 (further details for each of these examples are given in other 36 72 108 m/s chapters).

1	Fig. 5.13A illustrates how a chemoreceptor, such A as that used for taste and smell, might respond when a chemical stimulant reacts with receptor molecules on the plasma membrane of the sensory receptor. Binding of the •Fig. 5.12 Thedistributionofaxons,bysizeandconductionvelocity,inamixed(muscle)nerve(A) andacutaneousnerve(B). Notetheincreasednumberofsmall-diameterfibersandtheabsenceofAα fibersinthecutaneousnerve.(FromHainesDE[ed].Fundamental Neuroscience for Basic and Clinical Applications. 3rded.Philadelphia:ChurchillLivingstone;2006.) chemical stimulant to the receptor molecule opens an ion channel, which enables the influx of an ionic current that depolarizes the sensory receptor cell. (This is similar to what is described for ligand-gated channels in Chapter 6.)

1	Chapter 6.) Fig. 5.13B, the ion channel of a mechanoreceptor, such as those in the skin, opens in response to the application of a mechanical force along the membrane, and this allows an influx of current to depolarize the sensory receptor. In Fig. 5.13C, the ion channel of a retinal photoreceptor cell (so-called because it responds to light) is open in the dark and closed when a photon is absorbed by pigment on an internal disc membrane. In this case, an influx of current occurs in the dark; the current ceases when light is applied. When the current stops, the photoreceptor hyperpolarizes. (Because capture of the photon is distant from the ion channel that it influences, this process must involve an intracellular “second messenger” mechanism.) The nature of the receptor also can vary. In the simplest situation, a receptor is just a specialized portion of an axon,

1	The nature of the receptor also can vary. In the simplest situation, a receptor is just a specialized portion of an axon, Aα N/A8-1344-78ExtrafusalskeletalmusclefibersAγ N/A2-812-48IntrafusalmusclefibersBN/A1-36-18PreganglionicautonomicfibersCN/A0.2-20.5-2Postganglionicautonomicfibers FromHainesDE(ed).Fundamental Neuroscience for Basic and Clinical Applications. 3rded.Philadelphia:ChurchillLivingstone;2006.N/A,notapplicable. •Fig. 5.13 Models of Transducer Mechanisms in Three Types of Receptors. A, Chemoreceptor.B, Mechanoreceptor.C, Photoreceptor. in which case the transduction of a stimulus into receptor potential and the translation of this potential into a spike train all take place in the same cell. For example, a mechanical stimulus, such as pressure on the skin of a finger, can distort the membrane of an axon that forms part of a mechanoreceptor, as shown in

1	Fig. 5.14A . This distortion causes inward current flow at the end of the axon and longitudinal and outward current flow along the neighboring parts of the axon. The outward current produces a depolarization (the receptor potential) that might exceed the threshold for an action potential (see Fig. 5.14B ). If so, one or more action potentials are evoked and then travel along this primary afferent fiber to the CNS and thereby convey information about the mechanical stimulus. In many other cases, the receptor is composed of more than one cell. In this situation, transduction occurs in one cell, but spikes are generated in other cells that are •Fig. 5.14 A, Currentflow(thin arrows) producedbystimulation(thick arrow) ofamechanoreceptoratthetipofanaxon.AnintracellularrecordingelectrodeisplacedatthefirstnodeofRanvier.B, ThereceptorpotentialproducedbythecurrentandanactionpotentialthatwouldbesuperimposedonthereceptorpotentialifitweretoexceedthresholdatthefirstnodeofRanvier.

1	synaptically connected to it (see ). For example, in the cochlea, the primary afferent fibers get synaptic input from mechanoreceptive hair cells. Sensory transduction in such sense organs can be more complex in this arrangement. In photoreceptors, moreover, the receptor potential is hyperpolarizing, as mentioned earlier, and interruption of the dark current is the signal event. Information about each of these mechanisms is discussed in Chapter 8. Although the mechanisms of sensory transduction vary between stimulus types, the end result is typically a receptor potential in either the receptor cell or the primary afferent neuron (i.e., the first neuron in a sensory pathway) that has a synapse with the receptor cell.

1	The relationship between the location of a stimulus and activation of particular sensory neurons is a major theme in the field of sensory physiology. The receptive field of a sensory neuron is the region that, when stimulated, affects the activity of that neuron. For example, a sensory receptor might be activated by indentation of only a small area of skin. That area is the excitatory receptive field of the sensory receptor. Moreover, a neuron in the CNS might have a receptive field several times as large as that of a sensory receptor because it may receive information from many sensory receptors, each with a slightly different receptive field. The receptive field of that CNS neuron is thus the sum of the receptive fields of the sensory receptors that influence it. The location of the receptive field is determined by the location of the sensory transduction apparatus responsible for signaling information about the stimulus to the sensory neuron.

1	In general, sensory receptive fields are excitatory. However, a central sensory neuron can have either an excitatory or an inhibitory receptive field or, indeed, a complex receptive field that includes areas that excite it and areas that inhibit it. Examples of such complex receptive fields are discussed in Coding of Information by Action Potentials

1	Coding of Information by Action Potentials Central to CNS function is the transmission of information between neurons. This is accomplished primarily through action potentials, which propagate down the axon to the presynaptic terminals and cause neurotransmitter release, signaling the postsynaptic cells. As already explained, the regenerative nature of action potentials allows them to carry signals regardless of the length of the axon, whereas local signals, such as receptor or synaptic potentials (see ), decay with distance and are therefore not suitable for this purpose. The tradeoff, however, is that the all-or-none nature of action potentials means that their shape and size do not generally convey information in the way gradations of local potentials do. Instead, the variations in the rate or timing of action potentials appear to be used primarily as the “codes” for transmission of information between neurons.

1	Rate coding refers to information being coded in the firing rate of a neuron, where firing rate is defined as the number of spikes fired per unit time, usually expressed as spikes/second, also called hertz (Hz). For example, the force of a mechanical stimulus to the skin can be encoded in the firing rate of the primary afferent neuron that innervates the skin; the greater the force applied to the skin, the larger the resulting receptor potential in the primary afferent neuron will be and, as a consequence, the faster the rate of action potentials triggered by the receptor potential will be. Research has shown many neurons employ rate coding in the sense that the firing rate of a neuron shows a consistent relationship to particular parameters of sensory stimuli, upcoming movements, or other aspects of behavior.

1	The amount of information in such rate codes is constrained by several factors. One factor is a neuron’s range of firing rates. The upper limit of this range is set by the maximal frequency that a neuron can fire action potentials, which is determined by the duration of the absolute and relative refractory periods (see Fig. 5.4 ) and rarely exceeds 1000 Hz. The lower limit of the firing range is, of course, 0 Hz, as neurons cannot fire at negative rates. To avoid this problem, many neurons have spontaneous activity levels. These can be quite high (e.g., some Purkinje cells fire spontaneously at 100 Hz) and let a cell either increase or decrease its activity over a similar range in response to inputs. A second constraining factor is the variability of neuron’s firing rate, which determines the resolution of the neuron’s information coding. CHAPTER 5 Generation and Conduction of Action Potentials

1	CHAPTER 5 Generation and Conduction of Action Potentials Timing, or temporal coding, refers to spike codes in which the specific timing of spikes rather than the overall firing rate encodes information. One often-studied version of temporal coding is the synchronization of spikes across neurons. Synchronization of neuronal spiking has been shown to occur in a number of brain regions and has been related to function in a number of instances. An advantage of temporal coding is that it can convey information more quickly than can rate coding, inasmuch as it does not require averaging, which takes time. Moreover, rate coding and temporal coding are not mutually exclusive, inasmuch as overall firing rates can be varied while synchronous events are superimposed. Such multiplexing of codes may increase the information transmission capacity of neuronal pathways.

1	Sensory neurons encode information about stimuli. In the process of sensory transduction, one or more aspects of the stimulus must be encoded in a way that can be interpreted by the CNS. The encoded information is an abstraction based on (1) which sensory receptors are activated, (2) the responses of sensory receptors to the stimulus, and (3) information processing in the sensory pathway. Some stimulus parameters that can be encoded include sensory modality, location, intensity, frequency, and duration. Other aspects of stimuli that are encoded are described in relation to particular sensory systems in later chapters.

1	A sensory modality is a class of sensation. For example, sustained mechanical stimuli applied to the skin result in sensations of touch or pressure, and transient mechanical stimuli may evoke sensations of flutter or vibration. Other cutaneous modalities include cold, warmth, and pain. Vision, audition, taste, and smell are examples of noncutaneous sensory modalities. The specific sensory receptors define the normal energy associated with the modality of a sensory pathway. For example, the visual pathway includes photoreceptors, neurons in the retina, the lateral geniculate nucleus of the thalamus, and the visual areas of the cerebral cortex (see ). The normal means of activating the visual pathway is light striking the retina. However, mechanical stimulation (e.g., pressure on the eyeball) or electrical stimulation of neurons in the visual pathway also produce a visual sensation. Thus neurons of the visual system can be regarded as a labeled line, which, when activated by whatever

1	or electrical stimulation of neurons in the visual pathway also produce a visual sensation. Thus neurons of the visual system can be regarded as a labeled line, which, when activated by whatever means, results in a visual sensation.

1	The location of a stimulus is signaled by activation of the particular population of sensory neurons whose receptive fields are affected by the stimulus. The information may be encoded in the CNS as a neural map. For example, a somatotopic map is formed by arrays of neurons in the somatosensory cortex that receive information from corresponding locations on the body surface (see ). In the visual system, points on the retina are represented by neuronal arrays that form retinotopic maps (see

1	Intensity may be encoded in a number of ways. Because action potentials have a uniform magnitude, some sensory neurons encode intensity by their frequency of discharge (rate coding). The relationship between stimulus intensity and response can be plotted as a stimulus-response function. For many sensory neurons, the stimulus-response function approximates an exponential curve with an exponent that can be less than, equal to, or greater than 1. Stimulus-response functions with fractional exponents characterize many mechanoreceptors. Thermoreceptors, which detect changes in temperature, have linear stimulus-response curves (exponent of 1). Nociceptors, which detect painful stimuli, may have linear or positively accelerating stimulus-response functions (i.e., the exponent for these curves is 1 or greater). The positively accelerating stimulus-response functions of nociceptors help explain the urgency that is experienced as the pain sensation increases.

1	Another way in which stimulus intensity is encoded is according to the number of sensory receptors that are activated. A stimulus at the threshold for perception may activate only one or only a few primary afferent neurons of an appropriate class, whereas a strong stimulus of the same type may recruit many similar receptors. Central sensory neurons that receive input from sensory receptors of this particular class would be more powerfully affected as more primary afferent neurons discharge. Greater activity in central sensory neurons may be perceived as a stronger stimulus.

1	Stimuli of different intensities may also activate different sets of sensory receptors. The limit of a neuron’s firing rate of action potentials can also limit its range of response to a stimulus. However, mechanoreceptors with different thresholds can overcome this problem: Those with low thresholds can signal over a range of low input intensities, whereas others with higher thresholds can signal higher input intensities. Together they allow fine resolution over an extended range of intensities. In addition, still higher intensities might recruit nociceptors, and that will also change the perceived quality of the stimulus.

1	Stimulus frequency can sometimes be encoded by action potentials whose interspike intervals correspond exactly to the intervals between stimuli (e.g., at intervals corresponding •Fig. 5.15 Responses of Slowly and Rapidly Adapting Mechanoreceptors to Displacement of the Skin. A toC arethedischargesofprimaryafferentfibersduringaramp-and-holdstimulusshowninD. A, Theresponseofaslowlyadaptingreceptorthatsignalsthemagnitudeanddurationofdisplacement.B, Theresponseofarapidlyadaptingreceptorwhoseoutputsignalsthevelocityofdisplacement.C, Theresponseofadifferentrapidlyadaptingreceptorthatrespondstoacceleration.p,displacement;R,response;t,time.

1	to that of a low-frequency vibration). However, this mechanism is limited by the firing rate limits of neurons as discussed earlier. When higher frequencies need to be encoded (e.g., the auditory system, which in humans is capable of detecting frequencies up to 20,000 Hz; see ), other strategies are needed. Other candidate codes depend on the spatiotemporal patterns of firing across populations of neurons. The duration and the onset and offset of events are encoded by different populations of sensory neurons. For example, slowly adapting receptors in the skin produce a repetitive discharge throughout a prolonged stimulus. However, rapidly adapting receptors produce spikes at the onset (or offset) of the same stimulus.

1	Fig. 5.15 shows the responses of three types of receptors to the slow deflection of the skin, which is depicted in the graph at the bottom of the figure. The functional implication is that different temporal features of a stimulus can be signaled by receptors with different adaptation rates. 1. Ion channels are integral membrane proteins that have ion-selective pores. An ion channel typically has two states: high conductance (open) and zero conductance (closed). Different regions of an ion channel protein act as gates to open and close the channel. The channel flips spontaneously between the open and closed states. 2. For a voltage-dependent channel, the fraction of time that the channel spends in the open state is a function of the transmembrane potential difference. 3. The action potential is generated by the rapid opening and subsequent voltage inactivation of voltage-dependent Na+ channels and by the delayed opening and closing of voltage-dependent K+ channels. 4.

1	4. The absolute and relative refractory periods result from voltage inactivation of Na+ channels and the delayed closure of K+ channels in response to membrane repolarization. These refractory periods limit the firing rate of action potentials. 5. Subthreshold signals and action potentials are conducted along the length of a cell by local circuit currents. Subthreshold signals are conducted only electrotonically, and thus decrease with distance. 6. The action potential is propagated rather than merely conducted; it is regenerated as it moves along the axon. In this way, an action potential retains the same size and shape as it travels along the axon. 7. A large-diameter axon has greater propagation velocity because increased axon diameter lowers axial resistance and allows greater amounts of current to flow farther down the axon. 8.

1	7. A large-diameter axon has greater propagation velocity because increased axon diameter lowers axial resistance and allows greater amounts of current to flow farther down the axon. 8. Myelination dramatically increases the conduction velocity of a nerve axon because myelin increases membrane resistance and lowers membrane capacitance. Myelination allows an action potential to Fain GL. Molecular and Cellular Physiology of Neurons. 2nd ed. Cambridge, MA: Harvard University Press; 2014. Hille B. Ion Channels of Excitable Membranes. 3rd ed. Sunderland, MA: Sinauer Associates; 2001. Hodgkin AL, Huxley AF. A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol. 1952;117:500-544. CHAPTER 5 Generation and Conduction of Action Potentials be conducted very rapidly from one node of Ranvier to the next. This makes the action potential appear to jump from node to node in a form of conduction called saltatory conduction. 9.

1	9. A receptor responds preferentially to a particular form of stimulus energy. Its receptive field is that part of a sensory domain in which energy can affect the receptor. 10. Receptor potentials are the result of transduction of sensory stimuli. These potentials reflect the specific parameters of the stimulus and, if they exceed threshold, alter the action potential firing patterns of the afferent neurons. Johnston D, Wu SM-S. Foundations of Cellular Neurophysiology. Cambridge, MA: MIT Press; 1994. Sakmann B, Neher E. Single-Channel Recording. 2nd ed. Philadelphia: Springer; 1995. Upon completion of this chapter the student should be able to answer the following questions: 1. What are the characteristics of electrical synapses? 2. What are the specializations found in the presynaptic and postsynaptic elements of a chemical synapse? 3. What sequence of events connect the arrival of the action potential at the presynaptic terminal to the entry of calcium? 4.

1	3. What sequence of events connect the arrival of the action potential at the presynaptic terminal to the entry of calcium? 4. What sequence of events connect the entry of calcium at the presynaptic terminal to release of neurotransmitter? 5. What is the quantal hypothesis of synaptic transmission, and how does the presence of miniature end plate potentials support this hypothesis? 6. Why is the reversal potential of a typical EPSP near 0 mV? 7. What distinguishes EPSPs and IPSPs in terms of underlying ionic conductances, effect on membrane potential, and neuronal firing probability? 8. How does an IPSP still inhibit a neuron when its reversal potential is equal to or more positive than the neuron’s resting potential? 9. What are the mechanisms by which synaptic effects can change over time? 10. What are the criteria for determining a substance is a neurotransmitter, and what are the major excitatory and inhibitory neurotransmitters? 11.

1	10. What are the criteria for determining a substance is a neurotransmitter, and what are the major excitatory and inhibitory neurotransmitters? 11. What are the major classes of neurotransmitter receptors?

1	ynaptic transmission is the major process by which electrical signals are transferred between cells within the nervous system (or between neurons and muscle cells or sensory receptors). Within the nervous system, synaptic transmission is usually conceived of as an interaction between two neurons that occurs in a point-to-point manner at specialized junctions called synapses. Two main classes of synapses are distinguished: electrical and chemical. However, as the list of chemical neurotransmitters has grown and as understanding of their mechanisms of action has increased, the definition and conception of what constitutes synaptic transmission has had to be refined and expanded. We no longer think of synaptic transmission as a process that involves only neurons, but now realize that glia form an important element of the synapse and that signaling occurs between neurons and glia. Moreover, in some cases, neurotransmitter released at a synapse will act over a widespread territory rather

1	an important element of the synapse and that signaling occurs between neurons and glia. Moreover, in some cases, neurotransmitter released at a synapse will act over a widespread territory rather than just at the synapse from which it is released. Thus, we must either generalize the definition of synaptic transmission or consider classically defined synaptic transmission as but one of several mechanisms by which cells in the nervous system communicate with each other. In this chapter we first describe the classic conception of synaptic transmission (electrical and chemical) and then introduce some of the nontraditional neurotransmitters and discuss how they have forced modifications in our conception of chemical communication between cells in the nervous system.

1	Although their existence in the mammalian central nervous system (CNS) has been known for a long time, electrical synapses, or gap junctions, between neurons were thought to be of relatively little importance for the functioning of the adult mammalian CNS. Only recently has it become apparent that these synapses are quite common and that they may underlie important neuronal functions. An electrical synapse is effectively a low-resistance pathway between cells that allows current to flow directly from one cell to another and, more generally, allows the exchange of small molecules between cells. Electrical synapses are present in the CNS of animals from invertebrates to mammals. They are present between glial cells as well as between neurons. Electrical coupling of neurons has been demonstrated for most brain regions, including the inferior olive, cerebellum, spinal cord, neocortex, thalamus, hippocampus, olfactory bulb, retina, and striatum.

1	A gap junction is the morphological correlate of an electrical synapse (see also ). These junctions are plaque-like structures in which the plasma membranes of the coupled cells become closely apposed (the intercellular space narrows to ≈ 3 nm) and filled with electron-dense material (Fig. 6.1 ). Freeze-fracture electron micrographs of gap junctions display regular arrays of intramembrane particles that correspond to proteins that form the intercellular channels connecting the cells. The typical channel diameter is large (1 to 2 nm), thus making it permeable not only to ions but also other small molecules up to approximately 1 kDa in size.

1	.28 µm • Fig. 6.1 Gap junction structure. A, Schematic view of the gap junction showing narrowing of the intercellularspaceto3.5nmatthejunction.Thegapjunctionhasmultiplechannels,witheachchannel formed by two connexon hemichannels. Each connexon in turn comprises six connexin subunits. B,Electronmicrographofpartofacomplexsynapticarrangementcalledaglomerulusthatisfoundin theinferioroliveandsomeotherCNSregions.Twodendriticspinesarecoupledbyagapjunction(small black arrows).Anaxonterminalpackedwithsynapticvesiclesfillstheupperrightpartofthepanel.Large arrowheadspointtotheelectron-densematerialthatmarkstheactivezones.Blackdotsareimmunogold labelingforGABA,thusidentifyingthisterminalasGABAergic.Redarrowheadspointtosynapticvesicles. (FromDeZeeuwCIetal.J Neurosci1996;16:3420.Copyright1996bytheSocietyforNeuroscience.)

1	Eachgapjunctionchannelisformedbytwohemichannels(calledconnexons),onecontributedbyeachcell.Eachconnexon,inturn,isahexamerofconnexinproteinsubunits,whichareencodedforbyagenefamilyofatleast21differentmembersinmammals.(Asecondfamilyofproteinsthatformgapjunctions,thepannexins,hasalsobeenidentified.)Gapjunctionsformedbydifferentconnexinshavedistinctbiophysicalproperties(gatingandconductance)andcellulardistributions.Althoughatleast10connexintypesareexpressedintheCNS,connexin36(connexinsarenamedaccordingtotheirmolecularweight;thus,thenumberreferstotheapproximatemolecularweightoftheconnexininkilodaltons)isthemajorneuronalconnexinintheadultCNS.OtherconnexintypesfoundintheCNSformgapjunctionsbetweenglialcellsorareprimarilyexpressedtransientlyduringdevelopment.

1	Electrical synapses are fast (essentially no synaptic delay) and bidirectional (i.e., current generated in either cell can flow across the gap junction to influence the other cell). In addition they act as low-pass filters. That is, slow electrical events are much more readily transmitted than are fast signals such as action potentials. One important role for neuronal gap junctions appears to be synchronization of network activity. For example, the activity of inferior olivary neurons is normally synchronized but becomes uncorrelated when pharmacological blockers of gap junctions are injected into the inferior olive. It also appears that the patterns of electrical coupling by gap junctions may be highly specific. For example, neocortical interneurons almost exclusively couple to interneurons of the same type. This specific gap junction–coupling pattern suggests that multiple, independent, electrically coupled networks of interneurons may coexist across the neocortex.

1	Finally, although electrical synapses are generally regarded as relatively simple and static in comparison to chemical synapses, they may actually be fairly dynamic entities. For example, the properties of electrical synapses can be modulated by several factors, including voltage, intracellular pH, and [Ca++]. Moreover, they are subject to regulation by G protein–coupled receptors, and connexins (the protein subunits that form a gap junction, see At The Cellular ) contain sites for phosphorylation. These factors can change the coupling between cells by causing changes in single-channel conductance, formation of new gap junctions, or removal of existing ones.

1	Chemical synaptic transmission was first demonstrated between the vagus nerve and the heart by a simple experiment by Otto Loewi. The vagus nerve of a frog was stimulated to slow the heart rate down while the solution perfusing the heart was collected. This solution was then used to perfuse a second heart, whose beating then also slowed, demonstrating that the vagal nerve stimulation had caused a chemical to be released into the solution. The chemical responsible was found to be acetylcholine, which we now know is also a neurotransmitter at the neuromuscular junction and at other synapses in the peripheral and central nervous systems.

1	Unlike the situation at electrical synapses, at chemical synapses there is no direct communication between the cytoplasm of the two cells. Instead the cell membranes are separated by a synaptic cleft of some 20 nm, and interaction between the cells occurs via chemical intermediaries known as neurotransmitters. Chemical synapses are generally unidirectional, and thus one can refer to the presynaptic and postsynaptic elements that are diagramed in Fig. 6.2. The presynaptic element is often the terminal portion of an axon and is packed with small vesicles whose exact shape and size vary with the neurotransmitter they contain. In addition, the presynaptic membrane apposed to the postsynaptic element has regions, known as active zones, of electron-dense material that corresponds to the proteins involved in transmitter release (see

1	Fig. 6.1B). Moreover, mitochondria and rough endoplasmic reticulum are typically found in the presynaptic terminal. The postsynaptic membrane is also characterized by electron-dense material, which in this case corresponds to the receptors for the neurotransmitter.

1	Chemical synapses occur between different parts of neurons. Traditionally, focus has been placed on synapses formed by an axon onto the dendrites or soma of a second cell (axodendritic or axosomatic synapses), and our description will be based primarily on such synapses. However, there are many additional types of chemical synapses, such as axoaxonic (axon to axon), dendrodendritic (dendrite to dendrite), and dendrosomatic (dendrite to soma). Furthermore, complex synaptic arrangements are possible, such as mixed synapses, in which cells form both electrical and chemical synapses with each other; serial synapses, in which an axoaxonic synapse is made onto the axon terminal and influences the efficacy of that terminal’s synapse with yet a third element; and reciprocal synapses, in which both cells can release transmitter to influence the other.

1	Fig. 6.1B shows a complex synaptic arrangement called a glomerulus that involves both chemical and electrical synapses among the participating elements. Much of what we know about chemical synapses comes from the study of two classic preparations, the frog neuromuscular junction (the synapse from a motor neuron onto a muscle fiber) and the squid giant synapse (the synapse from a second-order neuron onto third-order neurons that innervate the muscle of the squid’s mantle; i.e., the motor neurons whose axons were used to characterize the conductances underlying the action potential [see ]). The principles governing transmission at these synapses mostly apply to synapses within the mammalian CNS as well, at least with regard to synapses using what are called the “classic” neurotransmitters (see the section mitters). Thus, much of the following discussion will be based on results from these two preparations; however, some differences in CNS synapses will also be pointed out.

1	3 Gaseous transmitters Arginine NO synthase Citrulline Mitochondria Peptides diffuse in extracellular space and bind to synaptic and extrasynaptic G protein–complex receptors Gaseous transmitters diffuse out of cell of origin and directly into other cells. They can act inside cell of origin or in cells distant from point of release G protein– coupled receptors NO Activates variety of enzymes Ionotropic and G protein– coupled receptors Acetyl CoA + Choline Reuptake by transporter 1 Small-molecule neurotransmitters AChAcetyl cholinesterase Other small-molecule neurotransmittersSmall-molecule neurotransmitters diffuse across synaptic cleft and bind to postsynaptic receptors Vesicle transporter concentrates neurotransmitter into vesicles Postsynaptic cell Presynaptic terminal Ca++ channels Large electron-dense vesicles NO O2 Nitrogen oxide (inactive) 2 Neuropeptides •Fig. 6.2

1	transporter concentrates neurotransmitter into vesicles Postsynaptic cell Presynaptic terminal Ca++ channels Large electron-dense vesicles NO O2 Nitrogen oxide (inactive) 2 Neuropeptides •Fig. 6.2 Schematicofachemicalsynapticterminalreleasingallthreemainclassesofneurotransmitter.Foreach,themechanismsofrelease,sitesofaction,andmechanismsforterminationofactivityareshown.Realsynapsesreleasetransmitterfromoneormoreclasses.

1	Synaptic transmission at a chemical synapse may be summarized as follows. Synaptic transmission is initiated by arrival of the action potential at the presynaptic terminal. The action potential depolarizes the terminal, which causes Ca++ channels to open. The subsequent rise in [Ca++] within the terminal triggers the fusion of vesicles containing neurotransmitter with the plasma membrane. The transmitter is then expelled into the synaptic cleft, diffuses across it, and binds to specific receptors on the postsynaptic membrane. Binding of transmitter to receptors then causes the opening (or less often, the closing) of ion channels in the postsynaptic membrane, which in turn results in changes in the potential and resistance of the postsynaptic membrane that alter the excitability of the cell. The changes in membrane potential of the postsynaptic cell are termed excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) (

1	Fig. 6.3 ), depending on whether they increase or decrease, respectively, the cell’s excitability, which can be defined as its probability of firing action potentials. The transmitter acts for only a very short time (milliseconds) because reuptake and degradation mechanisms rapidly clear the transmitter from the synaptic cleft.

1	The succeeding sections will amplify specific points of this summary. However, it is worth mentioning at this point that some of the nonclassic types of neurotransmitters (e.g., neuropeptides and gaseous neurotransmitters such as nitric oxide) and the discovery of metabotropic receptors have required modifications of several aspects of this basic conception. (Whereas an ionotropic receptor usually contains the ion channel as an integral part of itself, a metabotropic receptor does not contain an ion channel but instead is coupled to a G protein that initiates second messenger cascades that can ultimately affect ion channels.) Some of the differences between classic and peptide transmitters are listed in Table 6.1 . More details on the properties of peptide and gaseous transmitters are provided in the relevant parts of the section of this chapter, and metabotropic receptors are covered in the Receptors section. Calcium Entry Is the Signal for Transmitter Release

1	Depolarization of the presynaptic membrane by the action potential causes voltage-gated Ca++ channels to open, which makes it possible for Ca++ to flow into the terminal and trigger the release of transmitter. However, Ca++ will enter the terminal only if there is a favorable electrochemical gradient to do so. Recall that it is the combination of the concentration and voltage gradients that determines the direction of ion flow through open channels. Extracellular [Ca++] is high relative to intracellular [Ca++], which favors entry into the terminal; however, during the peak of the action potential, the membrane potential is positive, and the voltage gradient opposes the entry of Ca++ because of its positive charge. Thus, at the peak of the action potential, relatively little Ca++ enters the terminal because although the membrane is highly permeable to Ca++ , the overall driving force is small. In fact, by using a voltage clamp, one can experimentally make the membrane potential

1	the terminal because although the membrane is highly permeable to Ca++ , the overall driving force is small. In fact, by using a voltage clamp, one can experimentally make the membrane potential positive and equal to the Nernst equilibrium potential for Ca++ . If this is done, no Ca++ will enter the terminal despite Ca++ channels being open, and as a result no transmitter is released and no postsynaptic response is observed. This voltage is known as the suppression potential. If the membrane potential is rapidly made negative again (because of either the end of the action potential or by adjusting the voltage clamp), Ca++ rushes into the terminal as a result of the large driving force (which arises instantaneously on repolarization) and the high membrane permeability to Ca++ (which remains high because it takes the Ca++ channels several milliseconds to close in response to the new membrane potential), thereby resulting in release of transmitter and a postsynaptic response (

1	Fig. 6.4 •Fig. 6.3 IPSPsandEPSPsrecordedwithamicroelectrodeinacatspinalmotorneuroninresponsetostimulationofappropriateperipheralafferentfibers.Fortytracesaresuperimposed.NotethattheseIPSPsarehyperpolarizing,butinsomecasesIPSPscanbedepolarizing—seetextforanexplanation.(RedrawnfromCurtisDR,EcclesJC.J Physiol 1959;145:529.) Synaptic Vesicles and the Quantal Nature of Transmitter Release How neurotransmitter is stored and how it is released are questions fundamental to synaptic transmission. Answering these questions began with two observations. The first was the discovery of small round or irregularly shaped organelles known as synaptic vesicles in presynaptic terminals by electron microscopy (see

1	Figs. 6.1B 6.2 ). The second observation came from recordings of postsynaptic responses at the neuromuscular junction. Normally an action potential in a motor neuron causes a large depolarization in the postsynaptic muscle, termed an end plate potential (EPP), which is equivalent to an EPSP in a neuron. However, under conditions of low extracellular [Ca++], the EPP amplitude is reduced (because the presynaptic Ca++ current is reduced, leading to a smaller rise in intracellular [Ca++], and transmitter release is proportional to [Ca++]). In this condition, the EPP is seen to fluctuate among discrete values (

1	Fig. 6.5 ). Moreover, small spontaneous depolarizations of the postsynaptic membrane, termed miniature end plate potentials (mEPPs), are observable. The amplitude of the mEPP (≤1 mV) corresponds to that of the smallest EPP evoked under low [Ca++], and the amplitudes of other EPPs were shown to be integral multiples of the mEPP amplitude; thus, it was proposed that each mEPP corresponded

1	SynthesizedandpackagedinthenerveterminalSynthesizedandpackagedinthecellbody;transportedtothenerveterminalbyfastaxonaltransportSynthesizedinactiveformActivepeptideformedwhenitiscleavedfromamuchlargerpolypeptidethatcontainsseveralneuropeptidesUsuallypresentinsmallclearvesiclesUsuallypresentinlargeelectron-densevesiclesReleasedintoasynapticcleftMaybereleasedsomedistancefromthepostsynapticcellTheremaybenowell-definedsynapticstructureActionofmanyterminatedbecauseofuptakebypresynapticActionterminatedbyproteolysisorbythepeptidediffusingawayterminalsviaNa+-poweredactivetransportTypically,actionhasshortlatencyandshortduration(msec)Actionmayhavelonglatencyandmaypersistformanyseconds •Fig. 6.4 PresynapticCa++ currentanditsrelationshiptothepostsynapticresponse.A, Schematicofasquidgiantsynapsepreparation.Electrodes1and2areusedtovoltage-clampthepresynapticterminalandrecorditsvoltageandcurrent.(NotethattetrodotoxinandtetraethylammoniumwerepresenttoblockNa+ andK+ conductancetoisolatetheCa++

1	andK+ conductancetoisolatetheCa++ conductance.)Electrode3recordsthemembranepotentialofthepostsynapticaxon.Thepresynapticterminalwasvoltage-clampedtoincreasinglymoredepolarizedlevels(blue traces). Withasmalldepolarization(B), asmallCa++ currentstartsshortlyafterthevoltagestep,continuestogrowforthedurationofthestep(oncurrent),andthendecaysexponentiallyafteritstermination(offortailcurrent).Alargervoltagestep(C) increasesboththeonandtheoffcomponentsoftheCa++ current,andnowdistinctonandoffresponsesareobservedinthepostsynapticresponse.D, ThevoltagestepistotheNernstpotentialforCa++ ,sothereisnoCa++ currentduringthestep,butalargetailcurrentandoffresponseareobserved.(BasedondataofLlinasRetal.Biophys J 1981;33:323.) to the release of transmitter from a single vesicle and that EPPs represented the combined simultaneous release of transmitter from many vesicles.

1	This linking of mEPPs and vesicles implies that each mEPP is caused by the action of many molecules of neurotransmitter binding to postsynaptic receptors. The alternative that each mEPP could be caused by a single transmitter molecule binding to and opening a single postsynaptic receptor was rejected, in part because responses smaller in amplitude than mEPPs could be generated experimentally by directly applying dilute solutions of acetylcholine to the muscle. In fact, mEPPs were calculated to be caused by the action of approximately 10,000 molecules, which corresponds well to estimates of the number of neurotransmitter molecules contained within a single vesicle.

1	Many additional studies have confirmed the vesicle hypothesis of neurotransmitter release. For example, biochemical studies have shown that neurotransmitter is concentrated in vesicles, and fusion of vesicles to the plasma membrane and their depletion in the terminal cytoplasm after action potentials have been shown with electron microscopic techniques. 3.6 mV 47 msec A 1 mV 20 msec B •Fig. 6.5 A, SpontaneousmEPPsrecordedataneuromuscularjunctioninafiberoffrogextensordigitorumlongus.B, EPPsevokedbynervestimulationunderlow-[Ca++]conditions,whichreducetheprobabilityoftransmitterrelease.Thesmall-amplitudeEPPsevokedundertheseconditionsvaryinamplitudeinastep-likemanner,wherethesizeofthestepisequaltothesmallestEPP,whichinturnequalsthesizeofthemEPPs.(Notethatintheseconditionsthestimulusoftenfailstoevokeanyresponse,asindicatedbyaflatresponse.)(A, DatafromFattP,KatzB.Nature 1950;166:597;B, datafromFattP,KatzB.J Physiol 1952;117:109.)

1	The small vesicles that contain nonpeptide neurotransmitters can fuse with the presynaptic membrane only at specific sites called active zones. To become competent to fuse with the presynaptic membrane at an active zone, a small vesicle must first dock at the active zone and then undergo a priming process. Once primed the vesicle can fuse and release its transmitter into the synaptic cleft in response to an increase in local cytoplasmic [Ca++]. On the order of 25 proteins may play roles in docking, priming, and fusion. Some of these proteins are cytosolic, whereas others are proteins associated with the vesicle membrane or the presynaptic plasma membrane. The functions of most of these proteins are incompletely understood; however, knowledge of the molecular details of transmitter release has increased dramatically in recent years.

1	As with other exocytotic processes, neurotransmitter release involves SM (sec1/Munc18-like) and SNARE (soluble N-ethyl maleimide-sensitive factor attachment protein receptor) proteins: v-SNARES in the vesicle membrane and t-SNARES in the (target) presynaptic plasma membrane. Zipper-like interactions between synaptobrevin (a v-SNARE) and syntaxin and SNAP-25 (both are t-SNARES) with the assistance of SM proteins bring the vesicle membrane and the presynaptic plasma membrane close together before fusion. The SNARE proteins are targets for various botulinum toxins, which disrupt synaptic transmission, thus demonstrating their critical role in this process. Nevertheless, they do not bind Ca++ , so another protein must be the Ca++ sensor that triggers the actual fusion event. Evidence indicates that a synaptotagmin protein is almost certainly the Ca++ sensor and, even more specifically, that the second of its two cytoplasmic domains contains the Ca++ binding site. Interestingly,

1	indicates that a synaptotagmin protein is almost certainly the Ca++ sensor and, even more specifically, that the second of its two cytoplasmic domains contains the Ca++ binding site. Interestingly, synaptotagmins differ in their kinetics, and brain regions vary as to which synaptotagmin family member acts as the Ca++ sensor for vesicular fusion. Thus, differential expression of synaptotagmin genes in neurons may be a mechanism to adapt the kinetics of vesicle release and thereby tailor the specific characteristics of synaptic transmission to the functional needs of each CNS region.

1	Calcium channels are located in the active zone membrane at sites adjacent to the docked vesicles. When they open, a small area of high [Ca++], a microdomain is created at the active zone. This local high concentration (which lasts for less than a millisecond), allows the rapid binding of Ca++ to synaptotagmin, triggering the fusion of a docked vesicle and allowing release of its neurotransmitter. Despite the multiple steps involved, the process of vesicular release at a synapse is extremely rapid because of the close proximity of the molecular apparatuses involved to each other. Indeed, the time from Ca++ influx to vesicle fusion is about 0.2 msec.

1	“Reserve” pool Competence maturation Fusion and collapse Coated-vesicle retrieval “Recycling” pool “Docked” “Primed” (?) (2) (2') (3') ? ? (1) (3) (4) (5) “Cocked” (?) Armed Fire! ? ? “Readily releasable” pool (RRP) Kiss... Run! Kiss-and-run fusion Phosphorylations, Ca++ fusion machine assembly ? ?? Ca++ •Fig. 6.6 Vesiclerecyclingpathways.Synapticvesicleshavebeenthoughttofusewiththemembranewhileemptyingtheircontentsandthenberecycledbyformingclathrin-coatedpitsthatareendocytosedtoformcoatedvesicles(1→ [2or2′]→ 3′→ 1).Analternativepathwaythatmayallowmorerapidrecyclingofvesicleshasbeenproposed.Thispathway,called“kissandrun,”involvesonlytransientfusionofthevesicletothepresynapticmembranetoformaporethroughwhichthevesiclecontentsmaybeemptied,followedbydetachmentofthevesiclefromthemembrane(1→ 2→ 3→ 4→ 5→ 1).(RedrawnfromValtortaF,MeldolesiJ,FesceR.Trends Cell Biol 2001;11:324.)

1	During synaptic transmission, vesicles must fuse with the plasma membrane to release their contents into the synaptic cleft. However, there must be a reverse process; otherwise, not only would it be hard to sustain the vesicle population, but the presynaptic membrane’s surface area would also grow with each bout of synaptic transmission, and its molecular content and functionality would likewise change (because, as just discussed, the protein content of the vesicle membrane is distinct from that of the terminal membrane).

1	There appear to be two distinct mechanisms by which vesicles are retrieved after release of their neurotransmitter content (Fig. 6.6 ). One mechanism is the endocytotic pathway commonly found in most cell types. Coated pits are formed in the plasma membrane, which then pinch off to form coated vesicles within the cytoplasm of the presynaptic terminal. These vesicles then lose their coat and undergo further transformations (i.e., acquire the correct complement of membrane proteins and be refilled with neurotransmitter) to become once again synaptic vesicles ready for release. Evidence for a second, more rapid recycling mechanism has been obtained (see

1	Evidence for a second, more rapid recycling mechanism has been obtained (see Fig. 6.6 ). It involves transient fusion of the vesicle to the synaptic membrane and has been called “kiss and run.” In this case, fusion of the vesicle with the synaptic membrane leads to the formation of a pore through which the transmitter is expelled, but there is no wholesale collapse of the vesicle into the membrane. Instead, the duration of the fusion is very brief, after which the vesicle detaches from the plasma membrane and reseals itself. Thus, the vesicle membrane retains its molecular identity. Its contents can then simply be replenished, thereby making the vesicle ready for use again.

1	The relative importance of these two mechanisms is still being debated. However, at central synapses, which tend to be small and contain relatively few vesicles in comparison to the neuromuscular junction, the rapid time course of the kiss-and-run mechanism may help avoid the problem of vesicle depletion and the consequent failure of synaptic transmission during periods of high activity (many neurons in the CNS can show firing rates of several hundred hertz, and a few types of neuron can fire at rates of ≈ 1000 Hz).

1	Following vesicle fusion the neurotransmitter molecules are released and diffuse across the synaptic cleft (a very rapid process) and bind to receptors on the postsynaptic membrane. This binding leads to the opening (or less often the closing) of ion channels. These channels are termed ligand-gated because their opening and closing are primarily controlled by the binding of neurotransmitter. This mechanism can be contrasted with that of the voltage-gated channels underlying the action potential, whose opening and closing are determined by the membrane potential. However, there are some channels, most notably the NMDA (N-methyl-Daspartate) channel, that are both ligand and voltage gated.

1	It is also worth noting here that what follows in this section refers to what happens when neurotransmitter binds to receptors in which the ion channel is part of the receptor itself. These receptors are referred to as ionotropic receptors and underlie what is now called “fast” synaptic transmission. There is also “slow” synaptic transmission, mediated by what are called metabotropic receptors, in which the receptor and ion channel are not part of the same molecule, and binding of neurotransmitter to the receptor initiates biochemical cascades that lead to postsynaptic potentials with slow onsets (see the section Receptors for details). Despite the differing time courses, many of the same basic principles apply to both types of postsynaptic potential.

1	EPSPs. As stated earlier, the binding of neurotransmitter generally changes the membrane potential of the postsynaptic cell, and these changes are referred to as EPSPs when they increase the excitability of the neuron and IPSPs when they inhibit the neuron from firing action potentials. EPSPs are always depolarizing potentials, and IPSPs are usually hyperpolarizing.

1	Once a ligand-gated channel is open, the direction of current flow through it is determined by the electrochemical driving force for the permeant ion(s). It turns out that the pores of most channels that underlie EPSPs are relatively large and therefore allow passage of most cations with similar ease. As an example, consider the acetylcholinegated channel that is opened at the neuromuscular junction. Na+ and K+ are the major cations present (Na+ extracellularly and K+ intracellularly); therefore, the net current through the channel is approximately the sum of the Na+ and K+ currents (Inet = INa + IK). Recall that the current through a channel from a particular ion is dependent on two factors: the conductance of the channel to the ion and the driving force on the ion. This relationship is expressed by the equation

1	Equation 6.1 where gx is the conductance of the channel to ion x, Vm is the membrane potential, and Ex is the Nernst equilibrium potential for ion x. In this case gx is similar for Na+ and K+ , so the main determinant of net current is the relative driving forces (Vm − Ex). If the membrane is at its resting potential (typically around −70 mV), there is a strong driving force (Vm − ENa) for Na+ to enter the cell because this potential is far from the Na+ Nernst potential (about +55 mV), whereas there is only a small driving force for K+ to leave the cell because Vm is close to the K+ Nernst potential (about −90 mV). Thus, if acetylcholine-gated channels open when the membrane is at its resting potential, a large inward Na+ current and a small outward K+ current will flow through the acetylcholine channel, thereby resulting in a net inward current, which acts to depolarize the membrane.

1	The net inward current that results from opening such channels is called the excitatory postsynaptic current (EPSC).

1	Fig. 6.7A contrasts the time course of the EPSC and the resulting EPSP for fast synaptic transmission. The EPSC is much shorter (≈1 to 2 msec in duration) and corresponds to the time the channels are actually open. The short duration of the EPSC is due to the fact that the released neurotransmitter remains in the synaptic cleft for only a short while before being either enzymatically degraded or taken up by either glia or neurons. Binding and unbinding of a neurotransmitter to its receptor take place rapidly, so once its concentration falls in the cleft, the postsynaptic receptor channels rapidly close as well and terminate the EPSC. Note how the end of the EPSC corresponds to the peak of the EPSP, which is followed by a long tail. The duration of the tail and the rate of the decay in EPSP amplitude reflect the passive membrane properties of the cell (i.e., its RC properties) (see ). In slow synaptic transmission, the duration of the EPSP reflects the activation and deactivation of

1	amplitude reflect the passive membrane properties of the cell (i.e., its RC properties) (see ). In slow synaptic transmission, the duration of the EPSP reflects the activation and deactivation of biochemical processes more than the membrane properties. The long duration of even fast EPSPs (relative to EPSCs and action potentials) is functionally important because it allows EPSPs to overlap and thereby summate. Such summation is central to the integrative properties of neurons (see the next section,

1	Normally an EPSP depolarizes the membrane, and if this depolarization reaches threshold, an action potential is generated. However, consider what happens if the channels underlying the action potential are blocked and the membrane of the postsynaptic cell is experimentally depolarized by injecting current through an intracellular electrode. Because the membrane potential is now more positive, the driving force for Na+ is decreased and that for K+ increased. If the synapse is activated at this point, the net current through the receptor channel (the EPSC) will be smaller because of changes in the relative driving force. This implies that if the membrane potential is depolarized enough, there will be a point at which the Na+ and K+ currents through the channel are equal and opposite, and thus there is no net current and no EPSP. If the membrane is depolarized beyond this point, there is a net outward current through the receptor channels and the membrane will hyperpolarize (i.e., the

1	thus there is no net current and no EPSP. If the membrane is depolarized beyond this point, there is a net outward current through the receptor channels and the membrane will hyperpolarize (i.e., the EPSP will be negative). Thus, the potential at which there is no EPSP (or EPSC) is known as the reversal potential. For excitatory synapses, the reversal potential is usually around 0 mV (±10 mV), depending on the synapse (see

1	Fig. 6.7B–C It is worth noting that a reversal potential is a key criterion for demonstrating the chemical-gated as opposed to the voltage-gated nature of a synaptic response because currents through voltage-gated channels do not reverse, except at the Nernst potential of the ion for which they are selective (and then only if the channel is open at that potential). Consequently, beyond a certain membrane potential, no current will flow through voltage-gated channels because they will be closed. In contrast, ligand-gated channels can be opened at any membrane potential and thus can always have a net current flow through them, except at one specific voltage, the reversal potential.

1	0.1 msec/div Action potentials triggered by EPSP 5 mV ˜V •Fig. 6.7 PropertiesofEPSPs.A, TimecourseofafastEPSPcomparedwiththatoftheunderlyingEPSC.Inmanycases,suchasthisone,theEPSCismuchshorterthantheEPSP;however,sometimestheEPSCcanhaveafairlyextensivetail.B, IntracellularlyrecordedEPSPsatdifferentlevelsofdepolarization.EPSPswereevokedinmotorneuronsbystimulationofIaafferents.Thenumbertotheleftofeachtraceindicatesthemembranepotentialinducedbyinjectionofcurrentthroughtheelectrode.Atinitialmembranepotentialsof−42and−60mV,theEPSPtriggeredanactionpotential.Atmoredepolarizedlevels,Na+ channelsareinactivated,sonospikeoccurs.C, TodeterminetheEPSPreversalpotential,theinitialmembranepotentialisplottedagainstthesizeoftheEPSP(ΔV).ThisEPSPreversedat−7mV.(A, DatafromCurtisDR,EcclesJC.J Physiol 1959;145:529;B, datafromCoombsJSetal.J Physiol 1955;130:374.)

1	IPSPs. Like EPSPs, IPSPs are triggered by the binding an action potential (otherwise it would increase the prob-of neurotransmitter to receptors on the postsynaptic ability of the cell spiking and by definition be an EPSP). It membrane and typically involve an increase in membrane may seem counterintuitive that something that depolarizes permeability as a result of the opening of ligand-gated the membrane can still be considered inhibitory, but if channels. They differ in that IPSP channels are permeable it decreases the probability of spiking, then it is indeed to only a single ionic species, either Cl− or K+ . Thus, IPSPs inhibitory (a further explanation is given in the Synaptic will have a reversal potential equal to the Nernst potential Integration section). of the ion carrying the underlying current. Typically the In sum, starting from the resting membrane potential, Nernst potential for these ions is somewhat negative relative EPSPs are always depolarizing, IPSPs can be either

1	current. Typically the In sum, starting from the resting membrane potential, Nernst potential for these ions is somewhat negative relative EPSPs are always depolarizing, IPSPs can be either depolarto the resting potential, so when IPSP channels open, there izing or hyperpolarizing, and a hyperpolarizing potential is is an outward flow of current through them that results in always an IPSP. Thus, the key distinction between inhibitory hyperpolarization of the membrane (see

1	Fig. 6.3 ). and excitatory synapses (and IPSPs and EPSPs) is how they

1	However, in some cells, activation of an inhibitory affect the probability of the cell firing an action potential: synapse may produce no change in potential (if the mem-EPSPs increase the probability, whereas IPSPs decrease the brane potential equals the Nernst potential for Cl− or K+) or probability. may actually result in a small depolarization. Nevertheless, Safety factor. Synapses between cells vary in strength and in both these cases, the reversal potential for the IPSP thus in the size of the PSP generated in the postsynaptic cell. is still negative with regard to the threshold for eliciting Many factors determine synaptic strength, including the size and number of synaptic contacts between two cells, its activity level and past history, and the probability of vesicle fusion for the synapse. For excitatory synapses, the strength of the synapse may be quantified by what is known as its safety factor (the ratio of EPSP amplitude to the amplitude needed to reach the threshold to

1	synapse. For excitatory synapses, the strength of the synapse may be quantified by what is known as its safety factor (the ratio of EPSP amplitude to the amplitude needed to reach the threshold to trigger an action potential). Most synapses have low safety factors (<1), and thus it takes the summed EPSPs of multiple active synapses to trigger an action potential in the postsynaptic neuron. This summation process is at the core of synaptic integration, which is taken up in the next section. Synapses with high (>1) safety factors exist, however, and the neuromuscular junction is one prominent example. When a motor neuron action potential triggers release of neurotransmitter at the neuromuscular junction, an end plate potential (EPP; the equivalent of an EPSP in a neuron) is generated in the muscle fiber. The EPP is so large that under normal circumstances it depolarizes the sarcolemma well above the action potential threshold and thus always triggers a spike, leading to contraction of

1	muscle fiber. The EPP is so large that under normal circumstances it depolarizes the sarcolemma well above the action potential threshold and thus always triggers a spike, leading to contraction of the muscle cell. A high safety factor makes sense for the neuromuscular junction because each muscle cell is contacted by only a single motor neuron, and if that motor neuron is firing, the nervous system has basically made the decision to contract that muscle. In certain diseases of the neuromuscular junction, such as myasthenia gravis and Lambert-Eaton syndrome, the EPPs are reduced such that the safety factor can fall below 1, and thus the EPPs sometimes fail to trigger action potentials in the muscle fibers, leading to weakness.

1	The overall effect of a particular synapse is dependent on its location. To understand this concept fully, we must first recall that action potentials are typically generated at the initial segment of the cell because it has the highest density of voltage-gated Na+ channels and therefore the lowest threshold for initiation of a spike. Thus, it is the summed amplitudes of the synaptic potentials at this point, the initial segment, that is critical for the decision to spike. EPSPs generated by synapses close to the initial segment (i.e., synapses onto the soma or proximal dendrites) will result in a larger depolarization at the initial segment than will EPSPs generated by synapses on distal dendrites ( Fig.

1	6.8A single action potential in axon 2 versus 1). This is because the cell membrane is leaky and synaptic currents are generated locally at the synapse, so even if two synapses generate a local EPSC of the same size, less of the initial current will arrive at the initial segment from the more distal synapse than from the more proximal one, thereby resulting in the generation of a smaller EPSP at the initial segment by the distal synapse (see discussion of length constant in ). Thus, the synapse’s spatial location in the dendritic tree is an important determinant of its efficacy. However, as already mentioned, EPSPs generated by most CNS synapses, even those in favorable positions (i.e., close to the initial segment), are too small by themselves to reach •Fig. 6.8 SynapticintegrationofEPSPsrecordedattheaxonhillockadjacenttotheinitialsegment.A, ComparisonofEPSPsevokedbyproximalversusdistalsynapses(2versus1).B,

1	are too small by themselves to reach •Fig. 6.8 SynapticintegrationofEPSPsrecordedattheaxonhillockadjacenttotheinitialsegment.A, ComparisonofEPSPsevokedbyproximalversusdistalsynapses(2versus1).B, Temporalsummation.EPSPsinresponsetotwospikesinthesameaxonoccurringinrapidsuccession(axon2).C, Spatialsummation.Responsesevokedbysynapsesthatareelectricallydistantfromeachother(1and3).D, Sublinearsummationoftwosynapseslocatedneareachotherbecauseofshunting(2and4).

1	the spiking threshold in the postsynaptic cell, as illustrated in Fig. 6.8A , where an action potential in either axon 1 (distal) or 2 (proximal) both produce EPSPs that are too small to trigger a spike. Thus, generally the summed EPSPs from multiple synapses are required to reach threshold and trigger a spike. The requirement for multiple EPSPs to summate in order to trigger a spike is what makes the relatively long duration of EPSPs so important. Temporal summation refers to the fact that EPSPs that are separated by a latency less than their duration can sum. This is illustrated in Fig. 6.8B , where the same synapse is activated multiple times in rapid succession (axons can fire action potentials at rates well over 100 Hz); in this situation, successive EPSPs will be less than 10 msec apart and therefore overlap and sum. Note the higher amplitude of the second peak.

1	Spatial summation refers to the fact that synaptic potentials generated by different synapses can interact. For example, in Fig. 6.8 , suppose axon 1 and 3 each fire an action potential but at widely separated times. Each produces an EPSP that depolarizes the cell but is too small to reach threshold (see Fig. 6.8C , EPSP1, EPSP3). Instead, if both axons fire within a short enough time of each other, their effect can be additive, as shown in Fig. 6.8C (EPSP 1+3). The combined EPSP amplitude may then reach threshold and lead to spiking of the cell. If the EPSPs generated by axons 1 and 3 were simultaneous, then we would have an example of pure spatial summation. In the example shown, however, the times of the two EPSPs were slightly separated, thus we have both spatial and temporal summation present. The fact that EPSPs have a long time course (when compared with action potentials or the underlying EPSCs) facilitates both types of synaptic integration.

1	In the foregoing example, the combined EPSP was approximately the linear summation of the two individual EPSPs evoked by action potentials in axons 1 and 3. This is the case when two synapses are far apart. If the two synapses are close together, such as for axons 2 and 4 (see Fig. 6.8D ), the summation becomes less than linear because of what is known as a shunting effect. That is, when synapse 2 is active, channels are opened in the cell membrane, which means that it is more leaky. Therefore, when synapse 4 is also active, more of its EPSC will be lost (shunted) through the dendritic membrane, and less current will be left to travel down the dendrite to the initial segment. The result is that synapse 4 causes a smaller EPSP at the initial segment than it would have generated in isolation. Nevertheless, the combined EPSP is still larger than an EPSP caused by either synapse 2 or 4 alone.

1	Where do IPSPs fit into synaptic integration? In many cases one can think of them as negative EPSPs. Thus, whereas EPSPs add together to help bring the membrane potential up to and beyond the spiking threshold, IPSPs subtract from the membrane potential to make it more negative and therefore further from threshold. In deciding whether to spike, a cell adds up the ongoing EPSPs and subtracts the IPSPS to determine whether the sum reaches threshold. As with an EPSP, the efficacy of an IPSP varies with its location.

1	In addition to subtracting algebraically from the membrane potential, IPSPs exert an inhibitory action via the shunting mechanism, just as was described earlier for EPSPs. That is, while the IPSP channels are open, they make the membrane more leaky (i.e., lower its resistance) and thereby reduce the size of EPSPs, thus making them less effective. This shunting mechanism explains how IPSPs that do not change the membrane potential—or even those that slightly depolarize it—can still decrease the excitability of the cell. An alternative way to look at this effect is to view each synapse as a device that tries to bring the membrane potential to its own equilibrium potential. Because this potential is below the action potential threshold in the case of IPSPs, IPSPs make it harder for the cell to spike.

1	Thus far the interaction of synaptic potentials has been presented under the assumption that the postsynaptic cell membrane is passive (i.e., it acts as though it were simply resistors and capacitors in parallel with each other). However, it is clear that the dendrites and somas of most, if not all, neurons contain active elements (i.e., gated channels) that can amplify and alter EPSPs and IPSPs. For example, a distal EPSP can have a larger-than-expected effect if the EPSP activates dendritically-located voltage-gated Na+ or Ca++ channels that boost its amplitude or even generate propagated dendritic action potentials. Another example is Ca++-activated K+ channels that are present in the dendrites of some neurons. These channels are activated by the influx of Ca++ either through synaptic channels or via dendritic voltage-gated Ca++ channels opened by EPSPs and can cause long-lasting hyperpolarizations that effectively make the cell inexcitable for tens to hundreds of milliseconds. As

1	channels or via dendritic voltage-gated Ca++ channels opened by EPSPs and can cause long-lasting hyperpolarizations that effectively make the cell inexcitable for tens to hundreds of milliseconds. As a final example, there are some Ca++ channels that underlie a low-threshold Ca++ spike. These channels are normally inactive at resting membrane potentials, but the hyperpolarization that results from a large IPSP can de-inactivate them and allow them to open (and produce a spike) after termination of the IPSP. In this case “inhibition” actually increases the cell’s excitability. In sum, synaptic integration is a highly complex, nonlinear process. Nevertheless, the basic principles just described remain at its core.

1	Modulation of Synaptic Activity Integration of synaptic input by a postsynaptic neuron, as described in the previous section, represents one aspect of the dynamic nature of synaptic transmission. A second aspect is that the strength of individual synapses can vary as a function of their use or activity. That is, a synapse’s current functional state reflects, to some extent, its history.

1	Activation of a synapse typically produces a response in the postsynaptic cell (i.e., a postsynaptic potential) that will be roughly the same each time, assuming the postsynaptic cell is in a similar state. Certain patterns of synaptic activation, however, result in changes in the response to subsequent activation of the synapse. Such use-related changes may remain for short (milliseconds) or long (minutes to days) durations and may be either a potentiation or suppression of the synapse’s strength. These changes probably underlie cognitive abilities such as learning and memory. Thus, the processes by which activity results in changes in a synapse’s efficacy are a critical feature of synaptic transmission. When a presynaptic axon is stimulated twice in rapid succession, it is often found that the postsynaptic potential evoked by the second stimulus are larger in amplitude than the one evoked by the first (Fig. 6.9 ). This increase

1	A Facilitation B Facilitation C Posttetanic potentiation 3 msec 10 msec 1/sec 5/sec 10/sec 12.5/sec 31.2/sec 25/sec 1 mV 4 sec tetanic stimulation 8 sec 14 sec 27 sec Time after tetanic stimulation •Fig. 6.9 A, Facilitationataneuromuscularjunction.EPPsataneuromuscularjunctionintoadsartoriusmusclewereelicitedbysuccessiveactionpotentialsinthemotoraxon.Neuromusculartransmissionwasdepressedby5mMMg++ and2.1mMcuraresothatactionpotentialsdidnotoccur.B, EPPsatafrogneuromuscularjunctionelicitedbyrepetitivelystimulatingthemotoraxonatdifferentfrequencies.Notethatfacilitationfailedtooccuratthelowestfrequencyofstimulation(1/sec)andthatthedegreeoffacilitationincreasedwithincreasingfrequencyofstimulationintherangeoffrequencyused.Neuromusculartransmissionwasinhibitedbybathingthepreparationin12to20mMMg++ .C,

1	.C, Posttetanicpotentiationatafrogneuromuscularjunction.ThetoptwotracesindicatecontrolEPPsinresponsetosingleactionpotentialsinthemotoraxon.SubsequenttracesindicateEPPsinresponsetosingleactionpotentialsaftertetanicstimulation(50impulses/secfor20seconds)ofthemotorneuron.Thetimeintervalbetweentheendoftetanicstimulationandthesingleactionpotentialisshownoneachtrace.Themusclewastreatedwithtetrodotoxintopreventgenerationofactionpotentials.(A, RedrawnfromBelnaveRJ,GagePW.J Physiol 1977;266:435;B, redrawnfromMagelbyKL.J Physiol 1973;234:327;C, redrawnfromWeinrichD.J Physiol 1971;212:431.) is known as paired-pulse facilitation (PPF). Note PPF is distinct from temporal summation, in which two EPSPs overlap and sum to a larger response; with PPF the second EPSP itself is greater in size. If one plots the relative size of the two postsynaptic potentials (PSPs) as a function of the time between two stimuli, the amount of increase in the second PSP will be seen to depend on the time interval.

1	plots the relative size of the two postsynaptic potentials (PSPs) as a function of the time between two stimuli, the amount of increase in the second PSP will be seen to depend on the time interval. Maximal facilitation occurs at around 20 msec, followed by a gradual reduction in facilitation as the interstimulus interval continues to increase; with intervals of several hundred milliseconds, the two PSPs are equal in amplitude and no facilitation is observed. Thus, PPF is a relatively rapid and short-lasting change in synaptic efficacy.

1	Posttetanic potentiation (PTP) is similar to PPF; however, in this case the responses are compared before and after stimulation of the presynaptic neuron tetanically (tens to hundreds of stimuli at a high frequency). Such a tetanic stimulus train causes an increase in synaptic efficacy (see Fig. 6.9C ). PTP, like PPF, is an enhancement of the postsynaptic response, but it lasts longer: tens of seconds to several minutes after the cessation of tetanic stimulation.

1	Fig. 6.9C ). PTP, like PPF, is an enhancement of the postsynaptic response, but it lasts longer: tens of seconds to several minutes after the cessation of tetanic stimulation. Numerous experiments have shown that PPF and PTP are the result of changes in the presynaptic terminal and do not generally involve a change in the sensitivity of the postsynaptic cell to transmitter. Rather, the repeated stimulation leads to an increased number of quanta of transmitter being released. This increase is thought to be due to residual amounts of Ca++ that remain in the presynaptic terminal after each stimulus and help potentiate subsequent release of transmitter. However, the exact mechanism or mechanisms by which this residual Ca++ enhances release is not yet clear. The residual Ca++ does not, however, appear to act simply by binding to the same sites as the Ca++ that enters at the active zone and directly triggers vesicle fusion in response to the action potential.

1	Use of a synapse can also lead to a short-term depression in its efficacy. Most commonly, the postsynaptic cell at such a fatigued or depressed synapse responds normally to transmitter applied from a micropipette; hence, as was the case for PPF and PTP, the change is presynaptic. In general, the depression is thought to reflect depletion of the number of releasable presynaptic vesicles. Thus, short-term depression of synaptic transmission is most often and most easily seen at synapses in which the probability of release after a single stimulus is high and under conditions that favor release (i.e., high [Ca++]). A postsynaptically related cause of synaptic depression can be desensitization of the receptors in the postsynaptic membrane.

1	Both potentiation and depressive processes can occur at the same synapse. So in general, the type of modulation observed will depend on which process dominates. This in turn can reflect stimulus parameters, local ionic conditions, and the properties of the synapse. In particular, synapses have different baseline probabilities for releasing vesicles. Synapses with a high release probability will be more likely to show poststimulus depression, whereas those with low release probability are less likely to deplete their vesicle store and thus can be facilitated more easily. Sometimes mixed responses can occur. For example, during a tetanic stimulus train a synapse may show a depressed response, but after the train the synapse can show posttetanic facilitation once the vesicles are recycled.

1	Just as the postsynaptic membrane contains receptors for neurotransmitters, so does the presynaptic membrane. When these presynaptic receptors bind neurotransmitter, they cause events that can modulate subsequent release of transmitter by the terminal. There are several sources of transmitter that bind to presynaptic receptors: it can be the transmitter released by the terminal itself (i.e., self-modulation, in which case the receptors are referred to as autoreceptors), it can be released by another presynaptic terminal that synapses onto the terminal (a serial synapse), or it can be a nonsynaptically acting neurotransmitter (see the section

1	Presynaptic receptors can be either ionotropic or metabotropic. In the latter case, recall that their action will be relatively slow in onset and long in duration and the effect will depend on the specific second messenger cascades that are activated. Such cascades can ultimately regulate presynaptic voltage-gated Ca++ and K+ channels and other presynaptic proteins and thereby alter the probability of vesicle release. In contrast, activation of presynaptic ionotropic receptors will directly alter the electrical properties of the presynaptic terminal and cause rapid transient (millisecond time scale) changes in the probability of vesicle release (although they too can have much longer lasting effects). Binding of an ionotropic receptor will open channels in the presynaptic terminal and thereby alter the amount of transmitter released by an action potential.

1	Presynaptic inhibition refers to occasions when binding of presynaptic receptors leads to a decrease in release of transmitter, and it can be the result of one or more mechanisms (Fig. 6.10 ). First, opening of channels decreases membrane resistance and creates a current shunt. The shunt acts to divert the current associated with the action potential from the active zone membrane and thereby lessens the depolarization of the active zone, which results in less activation of Ca++ channels, less Ca++ entry, and less release of transmitter. A second mechanism is the change in membrane potential caused by the opening of presynaptic •Fig. 6.10 Presynapticinhibition.Activeregenerationofactionpotentialsinaxon2endsatthelastnode.Theactionpotentialisthenpassivelyconductedintotheterminal.Axon1makesanaxoaxonicsynapsewithaxon2.Activationofthissynapsereducesconductionoftheactionpotentialinaxon2totheactivezoneofitssynapticterminalbymechanismsdescribedinthetext.Thisreducestheopeningofvoltage-gatedCa++

1	channelsandthereforereleaseofneurotransmitter.

1	ionotropic channels. If a small depolarization is the result, there will be inactivation of voltage-gated Na+ channels and thereby lessening of the action potential–associated current and transmitter release. Presynaptic γ-aminobutyric acid A receptors (GABAA) occur in the spinal cord and mediate presynaptic inhibition by these mechanisms. They control Cl− channels. Generally, opening of Cl− channels generates a hyperpolarization. However, in the presynaptic terminal, the [Cl−] gradient is such that Cl− flows out of the cell and generates a small depolarization. This depolarization is small enough that it does not cause significant opening of voltage-gated Ca++ channels; otherwise, it would increase release of transmitter (presynaptic facilitation). In fact, there are other receptors that control cation channels and create large depolarizations, thereby increasing the release of transmitter. In addition, presynaptic nicotinic acetylcholine receptors control a cation channel that is

1	control cation channels and create large depolarizations, thereby increasing the release of transmitter. In addition, presynaptic nicotinic acetylcholine receptors control a cation channel that is permeable to Ca++ . By allowing additional entry of Ca++ , these receptors increase the release of transmitter from the terminal.

1	Repetitive stimulation of certain synapses in the brain can also produce more persistent changes in the efficacy of transmission at these synapses, a process called long-term potentiation or long-term depression. Such changes can persist for days to weeks and are believed to be involved in the storage of memories.

1	The increased synaptic efficacy that occurs in long-term potentiation probably involves both presynaptic (greater transmitter release) and postsynaptic (greater sensitivity to transmitter) changes, in contrast to the short-term changes that involve changes only in presynaptic function. Entry of calcium into the postsynaptic region is an early step required for initiating the changes that result in long-term enhancement of the response of the postsynaptic cell to neurotransmitter. Entry of calcium occurs through NMDA and some AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (classes of glutamate receptors; see the section Receptors). Entry of Ca++ is believed to activate Ca++-calmodulin kinase II, a multifunctional protein kinase that is present in very high concentrations in postsynaptic densities. In the presence of high [Ca++], this kinase can phosphorylate itself and thereby become active. Calciumcalmodulin kinase II is believed to phosphorylate proteins that

1	in postsynaptic densities. In the presence of high [Ca++], this kinase can phosphorylate itself and thereby become active. Calciumcalmodulin kinase II is believed to phosphorylate proteins that are essential for the induction of long-term potentiation. Long-term potentiation may also have an anatomic component. After appropriate stimulation of a presynaptic pathway, the number of dendritic spines and the number of synapses on the dendrites of postsynaptic neurons may increase rapidly. Changes in the presynaptic nerve terminal may also contribute to long-term potentiation. The postsynaptic neuron may release a signal (nitric oxide has been suggested) that enhances release of transmitter by the presynaptic nerve terminal.

1	Neurotransmitters are the substances that mediate chemical signaling between neurons. For a substance to be considered a neurotransmitter, it must meet several generally recognized criteria. First, the substance must be demonstrated to be present in the presynaptic terminal, and the cell must be able to synthesize the substance. It should be released on depolarization of the terminal. Finally, there should be specific receptors for it on the postsynaptic membrane. This last criterion is certainly true for substances that act as synaptic transmitters, but if we want to be inclusive and include substances that act over widespread territories rather than just at a single synapse, the last criterion needs to be relaxed to include situations in which receptors are located at sites outside the synapse. Neurotransmission has been suggested as a general term to describe both synaptic and nonsynaptic signaling between cells.

1	More than 100 substances have been identified as potential neurotransmitters because they have met some (hence the “potential” qualifier) or all of these criteria. These substances can be subdivided into three major categories: small-molecule transmitters, peptides, and gaseous transmitters. The small-molecule neurotransmitters may be further subdivided into acetylcholine, amino acids, biogenic amines, and purines. The first three groups of small-molecule transmitters contain what are considered the classic neurotransmitters. Remaining transmitters are substances that are more recent additions to the list of neurotransmitters, although many of them have been known as biologically important molecules in other contexts for a long time.

1	In the peripheral nervous system, acetylcholine is the transmitter at neuromuscular junctions, at sympathetic and parasympathetic ganglia, and of the postganglionic fibers from all parasympathetic ganglia and a few sympathetic ganglia. It is also a transmitter within the CNS, most prominently of neurons in some brainstem nuclei, in several parts of the basal forebrain (septal nuclei and nucleus basalis) and basal ganglia, and in the spinal cord (e.g., motor neuron axon collaterals). Cholinergic neurons from the basal forebrain areas project diffusely throughout the neocortex and to the hippocampus and amygdala, and they have been implicated in memory functions. Indeed, degeneration of these cells occurs in Alzheimer’s disease, a form of dementia in which memory function is gradually and progressively lost.

1	Anumberofdrugsknownasanticholinesterases interferewithacetylcholinesteraseandtherebyenhancetheactionofacetylcholinebyprolongingitspresenceatitssynapses.Suchdrugsincludeinsecticidesandchemicalwarfareagents,aswellassometherapeuticdrugs,suchasthoseusedtotreatmyasthenia gravis.

1	Myastheniagravisisanautoimmunediseaseinwhichantibodiesbindtoacetylcholinereceptorsattheneuromuscularjunction,therebydisruptingtheirfunctionalityandcausingthemtobemorerapidlydegraded.Thisreductioninreceptorsleadstosevereweaknessandultimatelyparalysis.Theweaknessischaracterizedbyrapidtiringofthemusclewithrepeateduse.Rapidtiringoccursbecausethenumberofpresynapticvesiclesavailableforreleasedropsduringthehigh-frequencytrainofmotorneuronactionpotentialsthatgeneratessuchcontractions.Normally,becauseofthehighsafetyfactoroftheneuromuscularjunction,smallerbutstillsuprathresholdEPPswouldstillbegeneratedandmaintainmusclecontractionduringrepeateduse.Inpeoplewithmyastheniagravis,thesafetyfactorissoreducedbythelossofacetylcholinereceptorsthatthedecreaseinreleaseofacetylcholinewithrepeatedactivityleadstoEPPsthatfailtotriggerspikes,andthusmuscularcontractionfails.Standardtreatmentsincludeanticholinesterases,whichallowagreaterconcentrationofacetylcholinetopartiallyovercomethedeficitcausedbythereducednu

1	EPPsthatfailtotriggerspikes,andthusmuscularcontractionfails.Standardtreatmentsincludeanticholinesterases,whichallowagreaterconcentrationofacetylcholinetopartiallyovercomethedeficitcausedbythereducednumberoffunctionalpostsynapticreceptors,andimmunosuppressivetherapiesandplasmaexchange,whichreducelevelsofautoantibodiesagainsttheacetylcholinereceptor.Thesetherapiesareallrelativelynonspecificandcanthereforehavemanysideeffects.PotentialfuturetherapiesarebeingdevelopedandincludeinducingtolerancetotheacetylcholinereceptorandselectivedestructionoftheBcellsthatmakeantibodiesagainstthereceptor.

1	Acetylcholine is synthesized from acetyl coenzyme A and choline by the enzyme choline acetyltransferase, which is located in the cytoplasm of cholinergic presynaptic terminals. After synthesis, acetylcholine is concentrated in vesicles. After release, the action of acetylcholine is terminated by the enzyme acetylcholinesterase, which is highly concentrated in the synaptic cleft. Acetylcholinesterase hydrolyzes acetylcholine into acetate and choline. The choline is then taken up by an Na+ symporter in the presynaptic membrane for the resynthesis of acetylcholine. The extracellular enzymatic degradation of acetylcholine is unusual for a neurotransmitter inasmuch as the synaptic action of other classic neurotransmitters is terminated via reuptake by a series of specialized transporter proteins.

1	•Fig. 6.11 Glutamatetransportcycle.A, Schematicshowsthefateofglutamatereleasedfromapresynapticterminal.Distinctglutamatetransportersexistonthepresynapticandpostsynapticcellmembranesforreuptake.Inaddition,glialcellstakeupglutamateandconvertittoglutamine.Theglutamineisthenreleasedandtakenintothepresynapticterminal,whereitisconvertedbacktoglutamatebeforebeingrepackagedintosynapticvesicles.B, Schematicoftransportershowingdirectionofionflowassociatedwiththemovementofglutamateacrossthemembrane. A variety of amino acids function as neurotransmitters. The three most important are glutamate, glycine, and GABA.

1	A variety of amino acids function as neurotransmitters. The three most important are glutamate, glycine, and GABA. Glutamate is the neurotransmitter at the overwhelming majority of excitatory synapses throughout the CNS. Despite its ubiquity, it was initially difficult to identify specific neurons as glutamatergic because glutamate is present in all cells; it has a key role in multiple metabolic pathways, and it is a precursor to GABA, the major inhibitory neurotransmitter. Nevertheless, experimental results have now clearly established glutamate as the major excitatory CNS neurotransmitter. When applied to cells, it causes depolarization and is released from neurons, and specific receptors and transporters for it have been identified.

1	In addition to being the main excitatory neurotransmitter, glutamate is a potent neurotoxin at high concentrations. Thus, strict limitation of glutamate’s activity after its release from the presynaptic terminal is necessary, not only to allow normal synaptic transmission but also to prevent cell death. This task is accomplished by specialized membrane transporter proteins ( Fig. 6.11 At the Cellular Level box). GABA and glycine act as inhibitory neurotransmitters. GABA is the major inhibitory transmitter throughout the nervous system. GABA is produced from glutamate by a specific enzyme (glutamic acid decarboxylase) that is present only in neurons that use GABA as a transmitter. Thus, experimentally, it is possible to identify cells as inhibitory GABAergic neurons by using antibodies to this enzyme to mark them (immunolabeling, see

1	Fig. 6.1B ). Many local interneurons are GABAergic. In addition, several brain regions contain large numbers of GABAergic projection neurons. The most notable are the spiny neurons of the striatum and the Purkinje cells of the cerebellar cortex. The inhibitory nature of Purkinje cells was especially surprising because they represent the entire output of the cerebellar cortex, and thus cerebellar cortical activity basically functions to suppress the activity of its downstream targets (cerebellar and vestibular nuclei).

1	Glycine functions as an inhibitory neurotransmitter in a much more restricted territory. Glycinergic synapses are predominantly found in the spinal cord, where they represent approximately half of the inhibitory synapses. They are likewise present in the lower brainstem, cerebellum, and retina in significant numbers. Interestingly, glycine also has another synaptic function. At excitatory NMDA-type glutamate receptors, glycine must also be bound for the ion channel to open. Thus, it acts as a cotransmitter at these synapses. It was generally thought that under physiological conditions the extracellular glycine concentration was high enough that the glycine binding sites of the NMDA channel were always saturated, but recent results suggest that this may not always be true, which implies that fluctuations in glycine levels may also be an important modulator of NMDA-mediated synaptic transmission.

1	After GABA and glycine are released from the presynaptic terminal, they are taken back up into the nerve terminal and neighboring glia by high-affinity Na+-Cl−–coupled membrane transporters. These Na+-Cl− transporters are part of what is called the solute carrier 6 (SLC6) family of transporters that also includes those for the biogenic amine neurotransmitters, but it is distinct from those for glutamate. Transport of the neurotransmitter into the cell is accomplished by symport with two Na+ and one Cl− ion. Four GABA transporter (GAT1, GAT2, GAT3, and BGT1) genes have been identified; however, GAT1 and GAT3 are the ones that are highly expressed in the CNS. Depending on region and species, they may be expressed in neurons and/or in glia. There are two main glycine transporters, GlyT1 and GlyT2. GlyT1 is found predominantly on astrocytes and is present throughout the CNS. In contrast, GlyT2 is located on glycinergic nerve terminals and is largely restricted to the spinal cord,

1	and GlyT2. GlyT1 is found predominantly on astrocytes and is present throughout the CNS. In contrast, GlyT2 is located on glycinergic nerve terminals and is largely restricted to the spinal cord, brainstem, and cerebellum.

1	Many of the neurotransmitters in this category may be familiar because they have roles outside the nervous system, often as hormones. Among the amines known to act as neurotransmitters are dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), serotonin (5-hydroxytryptamine [5-HT]), and histamine. Dopamine, norepinephrine, and epinephrine are catecholamines, and they share a common biosynthetic pathway that starts with the amino acid tyrosine. Tyrosine is converted to L-dopa by the enzyme tyrosine hydroxylase. L-Dopa is then converted to dopamine by dopa-decarboxylase. In dopaminergic neurons, the pathway stops here. In noradrenergic neurons, another enzyme, dopamine β-hydroxylase, converts dopamine to norepinephrine. Epinephrine is obtained by adding a methyl group to norepinephrine via phenylethanolamineN-methyl transferase. In serotoninergic neurons, serotonin is synthesized from the essential amino acid tryptophan. Tryptophan is first converted to 5-hydroxytryptophan

1	via phenylethanolamineN-methyl transferase. In serotoninergic neurons, serotonin is synthesized from the essential amino acid tryptophan. Tryptophan is first converted to 5-hydroxytryptophan by tryptophan 5-hydroxylase, which is then converted to serotonin by aromatic L-amino acid decarboxylase. Finally, in histaminergic neurons the conversion of histidine to histamine is catalyzed by histidine decarboxylase.

1	Removal of synaptically released biogenic amines is generally accomplished by reuptake into glia and neurons via transporters belonging to the Na+-Cl−–dependent transporter family. The catecholamines are then degraded by two enzymes, monoamine oxidase and catechol Omethyltransferase. Atleastfivetransporters(EAAT1toEAAT5,inwhichEAAT standsforexcitatory amino acid transporter)thatcarryglutamateacrosstheplasmamembranehavebeenidentified.TheyareallpartoftheNa+-K+–dependentfamilyoftransporters.InwardmovementofeachglutamatemoleculeisdrivenbythecotransportofthreeNa+ ionsandoneH+ ionandthecountertransportofoneK+ ionoutofthecell(see

1	Fig.6.11B ).Inaddition,thetransporterhasCl− conductance,althoughpassageofCl− ionsisnotstoichiometricallylinkedtoglutamatetransport.Glutamatetransportersarefoundonbothneuronsandglia.However,thetransportersdifferintheirregionalandcellulardistributionandintheirpharmacologicalandbiophysicalproperties.Forexample,EAAT2isfoundongliaandisgenerallyresponsibleformorethan90%ofglutamateuptakefromtheextracellularspace.TheglutamatetakenupintoglialcellsbyEAAT2iseventuallyreturnedtothepresynapticterminalbytheglutamateglutaminecycle(see

1	Fig.6.11 ).Insideglialcells,glutamateisconvertedtoglutamine.Glutamineisthentransportedoutoftheglialcellandbackintothepresynapticterminal,whereitissubsequentlyconvertedbacktoglutamate.GlutamateinsidethepresynapticterminalispackagedintosynapticvesiclesbyasecondsetofglutamatetransportersknownasvGLUTs (vesicular glutamate transporters),whicharepresentinthemembraneofglutamatergicvesicles.TransportofglutamateintosynapticvesiclesbyvGLUTisdrivenbythecountertransportofH+ ions,theelectrochemicalgradientforwhichhavingbeenestablishedbyanH+-ATPaseinthevesiclemembrane.

1	Within the CNS, nerve cells that use biogenic amines as neurotransmitters are primarily found within one of a few brainstem nuclei, most of which project rather diffusely throughout large areas of the brain. Noradrenergic neurons are primarily found in the locus coeruleus and nucleus subcoeruleus, which are located near each other in the dorsal part of the rostral pons. The neurons of the locus coeruleus project throughout the entire brain. Targets of the nucleus subcoeruleus are more limited but still widespread and include the pons, medulla, and spinal cord. (Norepinephrine is also important in the peripheral nervous system because it is used by postganglionic sympathetic cells.) Serotoninergic fibers arise from a series of nuclei located at the midline of the brainstem, known as the raphe nuclei. Similar to the noradrenergic fibers, serotoninergic fibers are distributed throughout most of the brain and spinal cord. Dopaminergic fibers arise from two main brainstem regions: the

1	raphe nuclei. Similar to the noradrenergic fibers, serotoninergic fibers are distributed throughout most of the brain and spinal cord. Dopaminergic fibers arise from two main brainstem regions: the substantia nigra pars compacta, which projects to the striatum, and the ventral tegmental area, which projects more widely to the neocortex and subcortical areas, including the nucleus accumbens. Histaminergic neurons are located within the tuberomammillary nucleus of the hypothalamus and project diffusely throughout the CNS. Finally, adrenergic neurons are relatively few in number when compared with the other biogenic amine transmitters. They have cell bodies localized to small cell groups in the rostral medulla. The largest group, termed C1, has projections to the locus coeruleus and down to the thoracic and lumbar levels of the spinal cord, where they terminate in the autonomic nuclei of the intermediolateral and intermediomedial cell columns. Thus, these neurons are important for

1	to the thoracic and lumbar levels of the spinal cord, where they terminate in the autonomic nuclei of the intermediolateral and intermediomedial cell columns. Thus, these neurons are important for autonomic functions, particularly vasomotor ones, such as control of arterial pressure.

1	The diffuse nature of the projection pattern of most of the amine systems is mirrored in their proposed functions. Activity in the different aminergic systems is believed to be important in setting global brain states. For example, these systems are involved in setting the level of arousal (sleep, waking), attention, and mood. Their involvement in pathways connected with the hypothalamus and other autonomic centers also indicates that they have important homeostatic functions. The role of dopamine in balancing the flow of activity through the basal ganglia pathways and how its loss leads to the motor symptoms observed in Parkinson’s disease are described in

1	ATP has the potential to act as a transmitter or cotransmitter at synapses in the peripheral and central nervous systems. ATP is found in all synaptic vesicles and thus is co-released during synaptic transmission. ATP has its own receptors, which like standard neurotransmitters, are coupled to ion channels, but it can also modify the action of other neurotransmitters with which it is co-released, including norepinephrine, serotonin, glutamate, dopamine, and GABA. Glial cells may also release ATP after certain types of stimulation. Once released, ATP is broken down by ATPases and 5-nucleotidase to adenosine, which can be taken up again by the presynaptic terminal. Peptide neurotransmitters consist of chains of between 3 and about 40 amino acids. Studies of neuropeptides

1	Peptide neurotransmitters consist of chains of between 3 and about 40 amino acids. Studies of neuropeptides Hyperactivityofdopaminergicsynapsesmaybeinvolvedinsomeformsofpsychosis.Chlorpromazine andrelatedantipsychoticdrugsinhibitdopaminereceptorsonpostsynapticmembranesandthusdiminishtheeffectsofdopaminereleasedfrompresynapticnerveterminals.Overdosesofsuchantipsychoticdrugscanproduceatemporaryparkinsonian-likestate. focused on the hypothalamus for many years. However, it is now clear that neuropeptides are released by neurons and act on receptors throughout the CNS and thus are a fundamental mechanism of neurotransmission throughout the CNS. To date, more than 100 neuropeptides have been identified. They can be classified into several functional groups, as shown in

1	Box 6.1 , which lists some of the known neuropeptides. It is now clear that many neurons that release classic neurotransmitters also release neuropeptides. As detailed later, understanding the interaction between coexisting classic and peptide transmitters has become an important area of research. In addition to being co-released with another transmitter, neuropeptides can also function as the sole or primary neurotransmitter at a synapse. In some ways neuropeptides are like the classic neurotransmitters: they are packaged into synaptic vesicles, their release is dependent on Ca++ , and they bind to specific receptors on target neurons. However, there are also significant differences, ones that have led to alternative names for the intercellular communication mediated by neuropeptides, such as nonsynaptic, parasynaptic, and volume transmission. Table 6.1 summarizes some of these differences between classic and peptide neurotransmitters.

1	Table 6.1 summarizes some of these differences between classic and peptide neurotransmitters. Unlike classic neurotransmitters, which are synthesized at the presynaptic terminal, neuropeptides are synthesized at the cell body and then transported to the terminal (see Fig. 6.2 ). Neuropeptides are packaged into large electron-dense vesicles that are scattered throughout the presynaptic terminal rather than in small electron-lucent vesicles docked at the active zone, where small-molecule transmitters are stored. (In neurons that make multiple neuropeptides, the various peptides are co-stored in the same vesicles.) Neuropeptide receptors are not confined to the synaptic region, and in general, peptide action is not limited by reuptake mechanisms.

1	Each of these differences has functional implications. For example, the separate storage of peptide and nonpeptide transmitters immediately raises the question of whether the two transmitters are co-released or differentially released in response to particular stimulation patterns.

1	In fact, differential release of peptide and classic transmitters from the same cell has been demonstrated for several types of neurons and is probably a result of the differences in vesicle storage described earlier. Because of their proximity to the active zones, nonpeptide vesicles can be released rapidly (<1 msec) in response to single action potentials • BOX 6.1 Some Neuroactive Peptides as a result of localized influx of Ca++ . Thus, low-frequency stimulation of the cell causes just the release of nonpeptide transmitter. In contrast, with higher-frequency stimulation of the presynaptic neuron, there is a more global increase in [Ca++] throughout the nerve terminal that leads to release of neuropeptide as well as neurotransmitter.

1	When neuropeptides are co-released with other transmitters, they may act synergistically or antagonistically. For example, in the spinal cord, tachykinins and calcitonin gene–related peptide (CGRP) act synergistically with glutamate and with substance P to enhance the action of serotonin. Conversely, tachykinins and CGRP antagonize norepinephrine’s action at other synapses. The interactions, however, are not simply a one-to-one synergism or antagonism at a particular synapse because of the differing temporal and spatial profiles of the action of peptides versus classic transmitters. In particular, the slower release and lack of rapid reuptake mean that neuropeptides can act for long durations, diffuse over a region of brain tissue, and affect all cells in that region (that have the appropriate receptors) rather than just acting at the specific synapse at which it was released. In fact, studies have shown that there is often a spatial mismatch between the presynaptic terminals that

1	receptors) rather than just acting at the specific synapse at which it was released. In fact, studies have shown that there is often a spatial mismatch between the presynaptic terminals that contain a particular neuropeptide and the sites of the receptors for that peptide. In sum, peptides released from a particular synapse probably affect the local neuronal population as a whole, whereas the co-released classic transmitters act in more of a point-to-point manner.

1	Opiates are drugs derived from the juice of the opium poppy. Compounds that are not derived from the opium poppy but that exert direct effects by binding to opiate receptors are called opioids and form a clinically and functionally important class of neuropeptides. Operationally, opioids are defined as compounds whose effects are stereospecifically antagonized by a morphine derivative called naloxone. The three major classes of endogenous opioid peptides in mammals are enkephalins, endorphins, and dynorphins. Enkephalins are the simplest opioids; they are pentapeptides. Dynorphins and endorphins are somewhat longer peptides that contain one or the other of the enkephalin sequences at their N-terminal ends.

1	Opioid peptides are widely distributed in neurons of the CNS and intrinsic neurons of the gastrointestinal tract. The endorphins are discretely localized in particular structures of the CNS, whereas the enkephalins and dynorphins are more widely distributed. Opioids inhibit neurons in the brain involved in the perception of pain. Indeed, opioid peptides are among the most potent analgesic (pain-relieving) compounds known, and opiates are used therapeutically as powerful analgesics. They exert their analgesic effect by binding to specific opiate receptors.

1	Substance P is a peptide consisting of 11 amino acids. It is present in specific neurons in the brain, in primary sensory neurons, and in plexus neurons in the wall of the gastrointestinal tract. The wall of the gastrointestinal tract is richly innervated with neurons that form networks or plexuses (see also ). The intrinsic plexuses of the gastrointestinal tract exert primary control over its motor and secretory activities. These enteric neurons contain many of the neuropeptides, including substance P, that are found in the brain and spinal column. Substance P is involved in pain transmission and has a powerful effect on smooth muscle. Substance P is probably the transmitter used at synapses made by primary sensory neurons (their cell bodies are in the dorsal root ganglia) with spinal interneurons in the dorsal horn of the spinal column, and thus it is an example of a peptide acting as a primary transmitter at a synapse.

1	Enkephalins act to decrease the release of substance P at these synapses and thereby inhibit the pathway for pain sensation at the first synapse in the pathway.

1	This is the newest category of neurotransmitter to be defined and stretches the usual definition of synaptic transmission even further than neuropeptides do. Gas neurotransmitters are neither packaged into synaptic vesicles nor released by exocytosis. Instead, gas neurotransmitters are highly permeant and simply diffuse from synaptic terminals to neighboring cells after synthesis, their synthesis being triggered by depolarization of the nerve terminal (the influx of Ca++ activates synthetic enzymes). Moreover, there are no specific reuptake mechanisms, nor do they undergo enzymatic destruction, so their action appears to be ended by diffusion or binding to superoxide anions or various scavenger proteins. Both nitric oxide (NO) and carbon monoxide (CO) are examples of gaseous neurotransmitters. NO is a transmitter at synapses between inhibitory motor neurons of the enteric nervous system and gastrointestinal smooth muscle cells (see ). NO also functions as a neurotransmitter in the

1	NO is a transmitter at synapses between inhibitory motor neurons of the enteric nervous system and gastrointestinal smooth muscle cells (see ). NO also functions as a neurotransmitter in the CNS. The enzyme NO synthase catalyzes the production of NO as a product of the oxidation of arginine to citrulline. This enzyme is stimulated by an increase in cytosolic [Ca++].

1	In addition to serving as a neurotransmitter, NO functions as a cellular signal transduction molecule both in neurons and in nonneuronal cells (e.g., vascular smooth muscle; see Chapter 14). One way that NO functions as a signal transduction molecule is by regulating guanylyl cyclase, the enzyme that produces cGMP from GTP. NO binds to a heme group in soluble guanylyl cyclase and potently stimulates the enzyme. Stimulation of this enzyme leads to an elevation in cGMP in the target cell. The cGMP can then influence multiple cellular processes.

1	The multitude of neurotransmitters used in the nervous system provides it with a specific and flexible interneuronal communications system. These characteristics are even further enhanced by the variety of receptors for each neurotransmitter. Receptors for a particular neurotransmitter were traditionally distinguished primarily by pharmacological differences in their sensitivity to particular agonists and antagonists. For example, acetylcholine receptors were split into muscarinic and nicotinic classes, depending on whether they bind muscarine or nicotine. Similarly, glutamate receptors were split into three main groups according to their sensitivity to the agonists NMDA, kainic acid, or AMPA. Though useful, this classification scheme has several limitations: some receptors fail to be activated by agonists, and it fails to disclose all the various receptor subtypes for a particular transmitter. Over the past 15 years or so, molecular biological approaches have been used to identify

1	by agonists, and it fails to disclose all the various receptor subtypes for a particular transmitter. Over the past 15 years or so, molecular biological approaches have been used to identify and sequence the receptor genes for many of the known neurotransmitters. It is thought that we now have a relatively complete catalog of the genes for these receptors. What this work has revealed is that there is a tremendous diversity of actual and potential receptor subtypes that are or could be used by the nervous system. Moreover, knowledge of the gene sequences has enabled an understanding of the relationship of different receptor proteins to each other and to other important proteins. This knowledge, combined with the results of biochemical, crystallographic, and other types of studies, has led to a much deeper understanding of the structural and functional workings of receptor proteins. In particular, various receptors can be grouped into families based on gene sequences, and members of

1	to a much deeper understanding of the structural and functional workings of receptor proteins. In particular, various receptors can be grouped into families based on gene sequences, and members of each family share various structural and functional features.

1	Neurotransmitter receptors are members of one of two large groups or families of proteins: ligand-gated ion channels, also known as ionotropic receptors, and G protein– coupled receptors, also referred to as metabotropic receptors (

1	Fig. 6.12A–B ). Almost all classic neurotransmitters and neuropeptides have at least one metabotropic-type receptor. Many of the classic neurotransmitters also have at least one ionotropic receptor. Ionotropic receptors are protein complexes that both have an extracellular binding site for the transmitter and form an ion channel (pore) through the cell membrane. The receptor is made up of several protein subunits, usually three to five, each of which typically has a series of membrane-spanning domains, some of which contribute to the wall of the ion channel. Binding of the neurotransmitter alters (usually increases) the probability of the ion channel being in the open state and thus typically results in postsynaptic events that are rapid in both onset and decay, with a duration of several milliseconds. Ionotropic receptors underlie fast synaptic EPSPs and IPSPs, as described earlier.

1	Theionotropicreceptorscanbedividedintoseveralsuperfamilies.Membersofthecys-loopsuperfamilyhavepeptidesubunitsthathaveanN-terminalextracellulardomainthatcontainsaloopdelimitedbycysteineresidues.Thisfamilyincludestheionotropicreceptorsforacetylcholine,serotonin,GABA,andglycine.Inadditiontotheirfamily-definingcysteineloop,thesereceptorssharethefollowingcommonfeatures:theyarepentamers,witheachpeptidesubunithavingfourtransmembranedomains;theneurotransmitterbindstotheN-terminaldomain;andthesecondtransmembranedomainsarethoughttoformthewalloftheionpore.

1	IonotropicglutamateandATPreceptorsformtwootherionotropicreceptorsuperfamilies;thedetailsforeacharegiveninthelatercorrespondingsections.Transientreceptorpotential(TRP)channels,whichareimportantfortransductionofpainandthermalsensations,formyetanotherfamily(see 1. Neurotransmitter Neurotransmitter Ions binds 1. Neurotransmitter binds 2. Channel opens 5. Ions flow across membrane 4. Ion 3. Ions flow Ions across membrane 2. G protein is 3. G protein subunits or • Fig.

1	binds 2. Channel opens 5. Ions flow across membrane 4. Ion 3. Ions flow Ions across membrane 2. G protein is 3. G protein subunits or • Fig. 6.12 Neurotransmitter receptors. The basic structure and mechanism of action are shown for ligand-gated ion channels (ionotropic receptors) (A) and G protein–coupled (metabotropic) receptors (B). Detailed structures of cys-loop and glutamate ionotropic receptors are shown in C and D, respectively. Cys-loop receptors include ionotropic receptors for GABA, glycine, serotonin, and acetylcholine. Note the differing membrane topologies of the individual subunits of these two classes of receptors: four transmembrane domains for cys-loop receptors and three plus a pore loop for glutamate receptors. Pore loops form the internal wall of the glutamate channel, whereas transmembrane domain 2 forms the internal wall of cys-loop receptors. (A and B, From Purves D, Augustine GJ, Fitzpatrick D. Neuroscience. 2nd ed. Sunderland, MA: Sinauer Associates; 2001.)

1	Metabotropic receptors are not ion channels. Instead, they are protein monomers that have an extracellular binding site for a particular transmitter and an intracellular site for binding a G protein. Binding of the receptor leads to activation of a G protein, which is the first step in a signal transduction cascade that alters the function of an ion channel in the postsynaptic membrane. In contrast to ionotropic receptors, metabotropic receptors mediate post-synaptic phenomena that have a slow onset and that may persist from hundreds of milliseconds to minutes. Because of the various biochemical cascades they initiate, they have great potential to cause changes in the neuron beyond just generating a postsynaptic potential.

1	Acetylcholine receptors were originally classified on a pharmacological basis (being sensitive to nicotine or muscarine) into two major groups. This grouping corresponds to groupings based on structural and molecular biological studies. Nicotinic receptors are members of the ionotropic cys-loop family, and muscarinic receptors are part of the metabotropic family of receptor proteins.

1	The nicotinic receptors mediate synaptic transmission at the neuromuscular junction as described earlier; however, nicotinic receptors are also present within the CNS. The nicotinic receptor contains a relatively nonselective cationic channel, so binding of acetylcholine produces an EPSP. Being members of the cys-loop family, acetylcholine receptors are pentamers constructed from a series of subunit types called α, β, γ, δ, ε, some of which contain multiple members. At the neuromuscular junction the channel is constructed from 2α, β, δ, ε, whereas in the CNS the composition is typically 3α, 2β. Furthermore, the junctional receptors all use the α1 subunit, whereas centrally located receptors use one of the α subunits between α2 and α10. As noted, the differing subunits result in receptors with differing pharmacological sensitivities and channel kinetics and selectivity.

1	There are five known muscarinic subtypes of acetylcholine receptors (M1 to M5). All are metabotropic receptors; however, they are coupled to different G proteins and can thus have distinct effects on the cell. M1, M3, and M5 are coupled to pertussis toxin–insensitive G proteins, whereas M2 and M4 are coupled to pertussis toxin–sensitive G proteins. Each set of G proteins is coupled to different enzymes and second messenger pathways (see for details of these pathways). Inhibitory Amino Acid Receptors: GABA and Glycine As noted, the most common inhibitory synapses in the CNS use either glycine or GABA as their transmitter. Glycine-mediated inhibitory synapses are common in the spinal cord, whereas GABAergic synapses make up the majority of inhibitory synapses in the brain.

1	Both glycine and GABA have ionotropic receptors that are members of the cys-loop family, thus sharing a number of characteristics as already described. In addition, each of these receptors has a Cl− channel, which opens while the receptor portion is bound. Therefore, the probability of these channels opening and the average time a channel stays open are controlled by the concentration of the neurotransmitter for which the receptor is specific.

1	Glycine receptors are pentamers and may be heteromers of α and β subunits (3:2 ratio) or homomers. Interestingly, the molecular composition appears to be related to its cellular location, with heteromers located postsynaptically and homomers located extrasynaptically. The β subunit seems to bind to an intracellular scaffold protein called gephyrin that appears to help localize receptors to the postsynaptic site. The α subunit contains the glycine binding site, and there are four genes coding for distinct α subunits (and splice variants of each). Each variant results in a receptor having distinct conductance, kinetics, agonist and antagonist affinity, and modulatory sites. Intriguingly, subunit variants are differentially expressed during development and in different brain regions.

1	GABA has two separate ionotropic receptors (GABAA and GABAC) coded for by distinct sets of genes. Like glycine receptors, both control a Cl− channel. GABAA receptors are heteromers generated from seven classes of subunits, three of which have multiple members. The most common configuration is α1, β2, γ2 in a 2:2:1 stoichiometry, which may account for 80% of the receptors; however, many other heteromers are found in the brain. As with glycine, different subunits confer distinct properties on the receptor. For example, GABAA receptors are the targets of two major classes of drugs: benzodiazepines and barbiturates. Benzodiazepines (e.g., diazepam) are widely used antianxiety and relaxant drugs. Barbiturates are used as sedatives and anticonvulsants. Both classes of drugs bind to distinct sites on the α subunits of GABAA receptors and enhance opening of the receptors’ Cl− channels in response to GABA. The sedative and anticonvulsant actions of benzodiazepines appear to be mediated by

1	sites on the α subunits of GABAA receptors and enhance opening of the receptors’ Cl− channels in response to GABA. The sedative and anticonvulsant actions of benzodiazepines appear to be mediated by receptors with the α1 subunit, whereas the anxiolytic effects reflect binding to receptors with the α2 subunit. GABAC receptors are structurally similar to GABAA receptors but have a distinct pharmacological profile (e.g., they are not affected by benzodiazepines) and are coded for by a separate set of genes (ρ1, ρ2, and ρ3).

1	The GABAB receptor is a metabotropic receptor. Binding of GABA to this receptor activates a heterotrimeric GTP-binding protein (G protein; see ), which leads to activation of K+ channels and hence hyperpolarization of the postsynaptic cell, as well as inhibition of Ca++ channels (when located presynaptically) and thus a reduction in release of transmitter. Excitatory Amino Acid Receptors: Glutamate

1	Glutamate has both ionotropic and metabotropic receptors. Based on pharmacological properties and subunit composition, several distinct ionotropic receptor subtypes are recognized: AMPA, kainate, and NMDA. Overall, there are 18 known genes that code for glutamate subunits for the ionotropic glutamate receptors. The genes are divided into several families (AMPA, kainate, NMDA, and δ) that essentially correspond to the pharmacological subtypes of receptors. Each glutamate receptor is a tetramer. Thus, there is a certain correspondence between the genes and the receptor types that are formed. For example, AMPA receptors are formed from GluR1 to GluR4 subunits, kainate receptors require either KA1 or KA2 and GluR5 to GluR7 subunits, and NMDA receptors all have NR1 subunits plus some combination of NR2 and NR3 subunits. As was mentioned for the other receptors, the receptor properties vary with subunit composition. Ionotropic glutamate receptors are excitatory and contain a

1	some combination of NR2 and NR3 subunits. As was mentioned for the other receptors, the receptor properties vary with subunit composition. Ionotropic glutamate receptors are excitatory and contain a cationic-selective channel. Thus, all the channels are permeable to Na+ and K+ , but only a subset allow Ca++ to pass.

1	AMPA and kainate receptors behave as classic ligand-gated channels as already discussed; on binding of glutamate to the receptor, the channel opens and allows current to flow, thereby generating an EPSP. NMDA channels are different. First, they require binding of both glutamate and glycine to open. Second, they display voltage sensitivity as a result of Mg++ blockade of the channel. That is, at resting (or more negative) membrane potentials, a Mg++ ion blocks the entrance to the channel so that even when glutamate and glycine are bound, no current flows through the channel. However, if the cell is depolarized (either experimentally by injection of current through an electrode or by other EPSPs), the Mg++ block is relieved and current can flow through the channel. A further interesting feature of NMDA channels is that they are generally permeable to Ca++, which can act as a second messenger. The combination of voltage sensitivity and Ca++ permeability of the NMDA channels has led to

1	of NMDA channels is that they are generally permeable to Ca++, which can act as a second messenger. The combination of voltage sensitivity and Ca++ permeability of the NMDA channels has led to hypotheses concerning their role in learning and memory-related functions (see

1	Eight genes coding for metabotropic glutamate receptors have been identified and classified into three groups. Group I receptors are found postsynaptically, whereas groups II and III are found presynaptically. These receptors generate slow EPSPs, but probably at least as importantly, they trigger second messenger cascades (see

1	Purines have two receptor families: an ionotropic (P2X) and a metabotropic (P2Y) family. There are seven identified P2X subunit types that form channels, and they represent their own superfamily of ligand-gated channels. Each subunit has only two transmembrane domains, with the loop between these two domains located extracellularly and containing the ATP binding site. The receptors are heterotrimers or homotrimers or hexamers. In general, these receptors form a cationic channel that is permeable to Na+ , K+ , and Ca++ . The distribution of subunits in the brain varies significantly, with some subunits having a widespread distribution (P2X2) and others being quite limited (P2X3 is present mostly on cells involved in pain-related pathways).

1	Metabotropic purine receptors are coded for by 10 genes, but only 6 are expressed in the human CNS. They have the typical features of G protein–coupled receptors and are known to activate K+ currents and modulate both NMDA and voltage-gated Ca++ currents. An interesting localization distinction between P2X and P2Y receptors is that although both are present on neurons, the latter dominates on astrocytes. Finally, in addition to the P2X and P2Y receptors, which respond to ATP, there are adenosine receptors that respond to the adenosine that is released after the enzymatic breakdown of ATP. These receptors are located presynaptically and act to inhibit synaptic transmission by inhibiting influx of Ca++ . Biogenic Amine Receptors: Serotonin, Dopamine, Noradrenaline, Adrenaline, Histamine

1	Biogenic Amine Receptors: Serotonin, Dopamine, Noradrenaline, Adrenaline, Histamine With the exception of one class of serotonin receptors (5-HT3) that are part of the cys-loop ionotropic family, receptors for the various biogenic amines are all metabotropic-type receptors. Thus, these neurotransmitters tend to act on relatively long time scales by generating slow synaptic potentials and by initiating second messenger cascades. Agonists and blockers of many of these receptors are important clinical tools for treating various neurological and psychiatric disorders. The role of different dopamine receptors in basal ganglia disorders will be covered in the motor systems (see

1	As is the case with the biogenic amines, receptors for the various peptides are essentially all of the metabotropic type and are coupled to G proteins that mediate effects via second messenger cascades. It is worth mentioning again that studies consistently show a mismatch between the locations of terminals containing a particular peptide and the receptors for it. Thus, these receptors are often activated by neurotransmitter diffusing through the extracellular space rather than at synapses. This implies that these receptors will experience much lower concentrations of agonist, and indeed, they are very sensitive to their agonists.

1	Unlike the other neurotransmitters that were covered, NO and CO do not bind to receptors. One way they do affect cell activity is to activate enzymes involved in second messenger cascades, such as guanylyl cyclase. In addition, NO has been shown to modify the activity of other proteins, such as NMDA receptors and the Na+,K+-ATPase pump, by nitrosylating them. 1. Both electrical and chemical synapses are important means of cellular communication in the mammalian nervous system. 2. Electrical synapses directly connect the cytosol of two neurons and allow rapid bidirectional current flow between neurons. They act as low-pass filters. 3. Gap junctions are the morphological correlate of electrical synapses. Gap junctions contain channels formed by hemichannels called connexons. Connexons are formed by proteins called connexins. 4.

1	Gap junctions are the morphological correlate of electrical synapses. Gap junctions contain channels formed by hemichannels called connexons. Connexons are formed by proteins called connexins. 4. Standard chemical synaptic transmission involves the release of transmitter from a presynaptic terminal, diffusion of transmitter across a synaptic cleft, and binding of the transmitter to receptors on the apposed postsynaptic membrane. 5. Entry of calcium into the presynaptic terminal triggers the release of neurotransmitter. Release of neurotransmitter is quantal, as first demonstrated by the recording of mEPPs at the frog neuromuscular junction. 6. Transmitter is packaged into synaptic vesicles in the presynaptic terminal. The vesicles are the quantal elements. That is, the release of transmitter from one vesicle causes a mEPP at the neuromuscular junction or, equivalently, one mPSP at a central synapse. 7.

1	7. Many proteins are involved in priming, docking, and fusion of synaptic vesicles. Synaptotagmin is the Ca++ sensor for triggering vesicle fusion. 8. Excitatory and inhibitory synapses increase or decrease, respectively, the probability that the postsynaptic neuron will spike. 9. The reversal potential is the membrane potential at which net current flow through a ligand-gated channel reverses. Excitatory synapses generate depolarizing potentials (EPSPs) that have reversal potentials positive to the spike threshold, most often as a result of the opening of nonselective cation channels. 10. Inhibitory synapses generate IPSPs that have reversal potentials more negative than the spike threshold but not necessarily negative to the resting potential. Inhibitory synapses can decrease spike probability by two mechanisms: hyperpolarization of the membrane and a decrease in the input resistance of the neuron, leading to a shunt of synaptic currents.

1	Alabi AA, Tsien RW. Perspectives on kiss-and-run: role in exocytosis, endocytosis, and neurotransmission. Annu Rev Physiol. 2013;75:393-422. 11. The process by which a neuron decides to fire an action potential as a result of its inputs is referred to as synaptic integration. The summation of EPSPs and IPSPs can be highly nonlinear and depends on many factors, including the geometry of the dendritic tree, location of the synaptic inputs relative to the initial segment, and the passive (RC) and active membrane properties of the cell. 12. The efficacy of synaptic transmission depends on the timing and frequency of action potentials in the presynaptic neuron. Facilitation, posttetanic potentiation, and long-term potentiation are examples of increased efficacy of synaptic transmission in response to previous multiple stimulations of a synapse. Long-term depression is an example of reduced efficacy resulting from previous activation of the synapse. 13.

1	13. The nervous system uses hundreds of neurotransmitters. Neurotransmitters can be subdivided into a few broad functional classes: small-molecule transmitters (acetylcholine, amino acids, biogenic amines, and purines), peptides, and gases (CO, NO). The action of a neurotransmitter depends on its postsynaptic receptors. Most nongaseous transmitters have both ionotropic and metabotropic receptors. 14. Small-molecule transmitters act locally, mainly across a single synapse, and their duration of action is limited by reuptake and enzymatic degradation. Peptides can diffuse from their presynaptic release site and thus have the potential to affect all cells within a local region. Gaseous transmitters are free to diffuse from their release site. 15. Ionotropic receptors contain an ion channel whose state (open versus closed) is gated by the binding of neurotransmitter to the receptor. Metabotropic receptors activate second messengers on binding neurotransmitter. 16.

1	16. Many synapses can release multiple types of transmitters, and which ones they release depends on the activity pattern of the terminal. Co-released transmitters may function independently or act synergistically or antagonistically. Fain GL. Molecular and Cellular Physiology of Neurons. 2nd ed. Cambridge, MA: Harvard University Press; 2014. Hille B. Ion Channels of Excitable Membranes. 3rd ed. Sunderland, MA: Sinauer Associates; 2001. Upon completion of this chapter the student should be able to answer the following questions: 1. What are the major modalities of somatosensory information, and what are the corresponding pathways that convey each from the periphery to the primary somatosensory cortex? 2. What body regions and categories of information are the exteroceptive, proprioceptive, and enteroceptive divisions of the somatosensory system associated with? 3. What are the main receptors for fine/discriminatory touch sensations? 4.

1	3. What are the main receptors for fine/discriminatory touch sensations? 4. What types of somatosensory information does the cerebellum receive? 5. What are the main receptors for pain and temperature touch sensations? 6. What is the phenomenon of referred pain? 7. What proteins are involved in transducing different categories of somatosensory information? 8. How do descending pathways act to regulate the flow of activity in ascending somatosensory pathways?

1	What proteins are involved in transducing different categories of somatosensory information? 8. How do descending pathways act to regulate the flow of activity in ascending somatosensory pathways? he somatosensory system provides information to the central nervous system (CNS) about the state of the body and its contact with the world. It does so by using a variety of sensory receptors that transduce mechanical (pressure, stretch, and vibrations) and thermal energies into electrical signals. These electrical signals are called generator or receptor potentials and occur in the distal ends of axons of first-order somatosensory neurons, where they trigger action potential trains that reflect information about the characteristics of the stimulus. The cell bodies of these neurons are located in dorsal root ( Fig. 7.1A Fig. 4.8 ) and cranial nerve ganglia.

1	Fig. 7.1A Fig. 4.8 ) and cranial nerve ganglia. Each ganglion cell gives off an axon that after a short distance divides into a peripheral process and a central process. The peripheral processes of the ganglion cells coalesce to form peripheral nerves. A purely sensory nerve will have only axons from such ganglion cells; however, mixed nerves, which innervate muscles, will contain both afferent (sensory) fibers and efferent (motor) fibers. At the target organ the peripheral process of an afferent axon divides repeatedly, with each terminal branch ending as a sensory receptor. In most cases the free nerve ending by itself forms a functional receptor, but in some the nerve ending is encapsulated by accessory cells and the entire structure (axon terminal plus accessory cells) forms the receptor.

1	The central axonal process of the ganglion cell either enters the spinal cord via a dorsal root or enters the brainstem via a cranial nerve. A central process typically gives rise to numerous branches that may synapse with a variety of cell types, including second-order neurons of the somatosensory pathways. The terminal location of these central branches varies depending on the type of information being transmitted. Some terminate at or near the segmental level of entry, whereas others project to brainstem nuclei.

1	Second-order neurons that are part of the pathway for the perception of somatosensory information project to specific thalamic nuclei where the third-order neurons reside. These neurons in turn project to the primary somatosensory cortex (S-I). Within the cortex, somatosensory information is processed in S-I and in numerous higher-order cortical areas. Somatosensory information is also transmitted by other second-order neurons to the cerebellum for use in its motor coordination function. The organization of the somatosensory system is quite distinct from that of the other senses, which has both experimental and clinical implications. In particular, other sensory systems have their receptors localized to a single organ, where they are present at high density (e.g., the eye for the visual system). In contrast, somatosensory receptors are distributed throughout the body (and head). Subdivisions of the Somatosensory System

1	Subdivisions of the Somatosensory System The somatosensory system receives three broad categories of information based on the distribution of its receptors. Its exteroceptive division is responsible for providing information about contact of the skin with objects in the external world, and a variety of cutaneous mechanoceptive, nociceptive (pain), and thermal receptors are used for this purpose. Understanding this division will be the main focus of this chapter. The proprioceptive component provides information about body and limb position and movement and relies primarily on receptors found in joints, muscles, and tendons. The ascending central pathways that originate CHAPTER 7 The Somatosensory System 109

1	Pain, temperature, crude touch 7 • Fig.

1	7.1 Ascending somatosensory pathways from the body. A, First-, second-, and third-order neurons are shown for the two main pathways conveying cutaneous information from the body to the cerebral cortex: the dorsal column/medial lemniscal and the spinothalamic pathways. Note that the axon of the second-order neuron crosses the midline in both cases, so sensory information from one side of the body is transmitted to the opposite side of the brain, but the levels in the neuraxis at which this takes place are distinct for each pathway. Homologous central pathways for the head originate in the trigeminal nucleus and are described in text, but they are not illustrated for clarity. B, Major spinocerebellar pathways carrying tactile and proprioceptive information to the cerebellum from the upper and lower parts of the body. Again, pathways from the head originate in the trigeminal nuclei but are not shown for clarity. A midsagittal view of the nervous system shows the levels of the spinal and

1	nd lower parts of the body. Again, pathways from the head originate in the trigeminal nuclei but are not shown for clarity. A midsagittal view of the nervous system shows the levels of the spinal and brainstem cross sections in panels A and B.

1	with them and that underlie conscious and unconscious proprioceptive functions will be covered in this chapter. However, because these receptors also initiate pathways that are intimately involved in the control of movement, they will be discussed again in . Finally, the enteroceptive division has receptors for monitoring the internal state of the body and includes mechanoreceptors that detect distention of the gut or fullness of the bladder. Aspects of enteroceptive division are also covered in as they are related to autonomic functions.

1	The somatosensory pathways can also be classified by the type of information they carry. Two broad functional categories are recognized, each of which subsumes several somatosensory submodalities. Fine discriminatory touch sensations include light touch, pressure, vibration, flutter (low-frequency vibration), and stretch or tension. The second major functional group of sensations is that of pain and temperature. Submodalities here include both noxious and innocuous cold and warm sensations and mechanical and chemical pain. Itch is also closely related to pain and appears to be carried by particular fibers associated with the pain system.

1	Thesensoryfunctionsofvariouscutaneoussensoryreceptorshavebeenstudiedinhumansubjectswithatechniqueknownasmicroneurography, inwhichafinemetalmicroelectrodeisinsertedintoanervetrunkinthearmorlegtorecordtheactionpotentialsfromsinglesensoryaxons.Whenarecordingcanbemadefromasinglesensoryaxon,thereceptivefieldofthefiberismapped.Mostofthevarioustypesofsensoryreceptorsthathavebeenstudiedinexperimentalanimalshavealsobeenfoundinhumanswiththistechnique. Afterthereceptivefieldofasensoryaxonhasbeencharacterized,theelectrodecanbeusedtostimulatethesamesensoryaxon.Intheseexperimentsthesubjectisaskedtolocatetheperceivedreceptivefieldofthesensoryaxon,whichturnsouttobeidenticaltothemappedreceptivefield.

1	Of great importance experimentally, the afferent fibers that convey these somatosensory submodalities to the CNS are different sizes. Recall that the compound action potential recorded from a peripheral nerve (see 5.1 ) consists of a series of peaks, thus implying that the diameters of axons in a nerve are grouped rather than being uniformly distributed. Information about tactile sensations is carried primarily by large-diameter myelinated fibers in the Aβ class, whereas pain and temperature information travels via small-diameter, lightly myelinated (Aδ) and unmyelinated (C) fibers. It is possible to block or stimulate selectively a class of axons of particular size, thereby allowing study of the different somatosensory submodalities in isolation. Innervation of the Skin

1	Innervation of the Skin The skin is an important sensory organ and not surprisingly is richly innervated with a variety of afferents. We first consider the afferent types related to fine or discriminatory touch sensations. These afferents are related to what are called low-threshold mechanoreceptors. Nociceptor and thermoceptor innervation will be considered separately in a later section of this chapter. To study the responsiveness of tactile receptors, a small-diameter rod or wire is used to press on a localized region of skin. With this technique, two basic types of responses may be seen when recording sensory afferent fibers: fast-adapting (FA) and slow-adapting (SA) responses (

1	Fig. 7.2). They are present in similar quantities. FA fibers will show a short burst of action potentials when the rod first pushes down on the skin, but then they will cease firing despite continued application of the rod. They may also burst at the cessation of the stimulus (i.e., when the rod is lifted off). In contrast, SA units will start firing action potentials (or increase their firing rate) at the onset of the stimulus and continue to fire until the stimulus ends.

1	Both the FA and SA afferent classes can be subdivided on the basis of other aspects of their receptive fields, where receptive field is defined as the region of skin from which stimuli can evoke a response (i.e., change the firing of the afferent axon). Type 1 units have small receptive fields with well-defined borders. Particularly for glabrous skin (i.e., hairless skin, such as on the palms of the hands and soles of the feet), the receptive field has a circular or ovoid shape within which there is relatively uniform and high sensitivity to stimuli that decreases sharply at the border ( Fig. 7.3). Type 1 units, particularly SA1 units, respond best to edges. That is, a larger response is elicited from them when the edge of a stimulus cuts through their receptive field than when the entire receptive field is indented by the stimulus.

1	Type 2 units have wider receptive fields with poorly defined borders and only a single point of maximal sensitivity, from which there is a gradual reduction in sensitivity with distance (see Fig. 7.3 ). For comparison a type 1 unit’s receptive field typically will cover approximately four papillary ridges in the fingertip, whereas a type 2 unit will have a receptive field that covers most or all of a finger. Thus, four main classes of low-threshold mechanosensitive afferents have been identified physiologically (FA1, FA2, SA1, and SA2). Peripherally these axons may terminate either as free nerve endings, associated with a hair follicle, or within a specialized receptor structure made up of supporting cells. For glabrous skin the four afferent classes have been associated with four specific types of histologically identified receptor structures whose locations and physical

1	CHAPTER 7 The Somatosensory System •Fig. 7.2 Cutaneousmechanoreceptorsandtheresponsepatternsofassociatedafferentfibers.A,Schematicviewsofglabrous(hairless)andhairyskinshowingthearrangementofthevariousmajormechanoreceptors.B,Firingpatternsofthedifferentcutaneouslow-thresholdmechanosensitiveafferentfibersthatinnervatethevariousencapsulatedreceptorsoftheskin.(TracesinBarebasedondatafromJohanssonRS,VallboÅB.Trends Neurosci 1983;6:27.) •Fig. 7.3 Receptivefieldcharacteristicsfortype1andtype2sensoryafferents.Plotsinthetoprowshowthethresholdlevelofforceneededtoevokearesponseasafunctionofthedistanceacrossthereceptivefield.Receptivefieldsizeisshownonthehandbeloweachplot.(DatafromJohanssonRS,VallboÅB.Trends Neurosci 1983;6:27.) characteristics help explain the firing properties of these sensory afferents. FA1 afferents terminate in Meissner’s corpuscles, whereas SA1 afferents terminate in Merkel’s disks. In both cases the receptor is located relatively superficially, either in the basal

1	afferents. FA1 afferents terminate in Meissner’s corpuscles, whereas SA1 afferents terminate in Merkel’s disks. In both cases the receptor is located relatively superficially, either in the basal epidermis (Merkel) or just below the epidermis (Meissner) (see

1	Fig. 7.2 ). These receptors are small and oriented to detect stimuli pressing down on the skin surface just above them, thus allowing SA1 and FA1 afferents to have small receptive fields. For glabrous skin, SA2 afferents terminate in Ruffini endings and FA2 afferents end in Pacinian corpuscles. Both of these receptors lie deeper in the dermis and connective tissue and therefore are sensitive to stimuli applied over much larger territory. The capsules of both Pacinian and Meissner receptors act to filter out slowly changing or steady stimuli, thus making these afferents selectively sensitive to changing stimuli.

1	For hairy skin, the relationship between receptors and afferent classes is similar to that of glabrous skin. SA1 and SA2 fibers connect to Merkel and Ruffini endings, the same as for glabrous skin. Pacinian corpuscles also underlie the properties of FA2 afferents; however, they are not found in hairy skin but instead are located in deep tissues surrounding muscles and blood vessels. There is not an exact analogue to the FA1 afferents. Rather there are hair units, which are afferents whose free endings wrap around hair follicles (see Fig. 7.2 ). Each such hair unit will connect with about 20 hairs to produce a large ovoid or irregularly shaped receptive field. These units are extremely sensitive to movement of even a single hair. There are also field units that respond to touch of the skin, but unlike FA1 units, they have large receptive fields.

1	Several psychophysical and neural coding questions can be related to the receptive field properties and sensitivities of the various categories of afferents. For example, is the threshold of perception of tactile stimuli due to the sensitivity of the peripheral receptors or to central processes? In fact, by using microneurography it is possible to show that a single spike in an FA1 afferent from the finger can be perceived, thus indicating that the receptors limit the sensitivity; however, for other skin regions, perception is more dependent on central factors such as attention.

1	An important behavioral and clinical measure of somatosensory function is spatial acuity or two-point discrimination. Clinically a doctor will apply two needle-like points simultaneously to the skin of a patient. The patient will generally perceive the points as two distinct stimuli as long as they are farther apart than some threshold distance, which varies across the body. The best discrimination (shortest threshold distance) is at the fingertips. Type 1 units underlie spatial acuity, which is not surprising given the smaller receptive fields of type 1 units than type 2 units. Moreover, the threshold distance for a region of skin is most closely related to its density of type 1 units because these units have similarly sized receptive fields throughout the glabrous skin, but their density falls off from fingertip to palm to forearm, and this fall-off correlates with the rise in threshold distance. Note that this variation in innervation density also matches the overall sensitivity of

1	falls off from fingertip to palm to forearm, and this fall-off correlates with the rise in threshold distance. Note that this variation in innervation density also matches the overall sensitivity of different skin regions to cutaneous stimuli.

1	The relationship of the firing rates in the various afferent classes to perceived stimulus quality is another important issue that has been addressed with microneurographic techniques. When a single SA fiber is stimulated with brief current pulses such that each pulse triggers a spike, a sensation of steady pressure is felt at the receptive field area of that fiber. As pulse frequency is increased a concurrent increase in pressure is perceived. Thus the firing rate in SA fibers codes for the force of the tactile stimulus. As another example, when an FA fiber is repetitively stimulated, a sensation of tapping results first, and as the frequency of the stimulus is increased, the sensation turns to one of vibration. Interestingly, in neither case does the stimulus change its qualitative character—for example, to a feeling of pain—so long as the stimulus activates only a particular fiber class. This is evidence that pain is a distinct submodality that uses a set of fibers distinct from

1	example, to a feeling of pain—so long as the stimulus activates only a particular fiber class. This is evidence that pain is a distinct submodality that uses a set of fibers distinct from those used by low-threshold mechanoreceptors.

1	These findings illustrate an important principle of sensory systems called labeled line. The idea is that the quality (i.e., modality) of a particular sensation results from the fact that it is conveyed to the CNS by a specific set of afferents that have a distinct set of targets in the nervous system. Alterations in activity in these afferents will therefore change only quantitative aspects of the sensation. As will be seen in more detail later, the various somatosensory submodalities (i.e., information arising from FA and SA mechanoreceptors, proprioceptors, and nociceptors) appear to use relatively separate dedicated cell populations even at relatively high levels of the CNS such as the thalamus and primary somatosensory cortex. Innervation of the Body

1	Innervation of the Body Axons of the peripheral nervous system (PNS) enter or leave the CNS through the spinal roots (or through cranial nerves). The dorsal root of a given spinal segment is composed entirely of the central processes of its associated dorsal root ganglion cells. The ventral root consists chiefly of motor axons, including α and γ motor neuron axons (see ), and at certain segmental levels, autonomic preganglionic axons (see CHAPTER 7 The Somatosensory System The pattern of innervation is determined during embryo-logical development. In adults a given dorsal root ganglion supplies a specific cutaneous region called a dermatome. Many dermatomes become distorted during development, chiefly because of rotation of the upper and lower extremities as they are formed, but also because humans maintain an upright posture. However, the sequence of dermatomes can readily be understood if depicted on the body of a person in a quadrupedal position ( Fig. 7.4).

1	Fig. 7.4). Although a dermatome receives its densest innervation from the corresponding spinal cord segment, collaterals of afferent fibers from the adjacent spinal segments also supply the dermatome. Thus transection of a single dorsal root causes little sensory loss in the corresponding dermatome. Anesthesia of any given dermatome requires interruption of several adjacent dorsal roots. Acommondiseasethatillustratesthedermatomalorganizationofthedorsalrootsisshingles. Shinglesistheresultofreactivationoftheherpeszostervirus,whichtypicallycauseschickenpoxduringtheinitialinfection.Duringtheinitialinfectionthevirusinfectsdorsalroot(andcranialnerve)ganglioncells,whereitcanremainlatentforyearstodecades.Whenthevirusreactivates,thecellsofthatparticulardorsalrootganglionbecomeinfected,andthevirustravelsalongtheperipheralaxonbranchesandgivesrisetoapainfuloritchyrashthatisconfinedtoonesideofthebody(endsatthemidline)inadermatomal,belt-likedistributionortothedistributionofacranialnerve.

1	Within the dorsal roots, fibers are not randomly distributed. Rather the large myelinated primary afferent fibers assume a medial position in the dorsal root, whereas the small myelinated and unmyelinated fibers are more lateral. The large medially placed afferent fibers enter the dorsal column, where they bifurcate into rostrally and caudally directed branches. These branches give off collaterals that terminate in several neighboring segments. The rostral branch also ascends to the medulla as part of the dorsal column–medial lemniscus pathway. The axonal branches that terminate locally in the spinal cord gray matter transmit sensory information to neurons in the dorsal horn and also provide the afferent limb of reflex pathways (see Chapter 9). Innervation of the Face

1	Chapter 9). Innervation of the Face The arrangement of primary afferent fibers that supply the face is comparable to that of fibers that supply the body and is provided for primarily by fibers of the trigeminal nerve. Peripheral processes of neurons in the trigeminal ganglion (also called the gasserian or semilunar ganglion) pass through the ophthalmic, maxillary, and mandibular divisions of the trigeminal nerve to innervate dermatome-like regions of the face. These fibers carry both tactile information and pain and temperature information. The trigeminal nerve •Fig. 7.4 A,Dermatomesrepresentedonadrawingofapersonassumingaquadrupedalposition.NotenerveC1generallyhaslittleornosensorycomponent,andtheunlabeledportionoftheheadandthefaceareinnervatedbysensoryfibersofthecranialnerves,primarilythetrigeminalnerve.B,SagittalviewofthespinalcordshowingtheoriginofnervescorrespondingtoeachofthedermatomesshowninA.

1	Thetrigeminalnuclearcomplexconsistsoffourmaindivisions,threeofwhicharesensory.Thethreesensorydivisions(fromrostraltocaudal)arethemesencephalic, chief (ormain)sensory, anddescending (orspinal)trigeminal nuclei. Thelattertwoaretypicalsensorynucleiinthatthecellbodiescontainedinthemaresecond-orderneurons.Themesencephalicnucleusactuallycontainsfirst-orderneuronsandthusisanalogoustoadorsalrootganglion.Thefourthdivisionofthetrigeminalcomplexisthemotornucleusofthetrigeminalnerve,whosemotorneuronsprojecttoskeletalmusclesoftheheadviathetrigeminalnerve(see Fig.4.6D–E also innervates the teeth, the oral and nasal cavities, and the cranial dura mater.

1	Fig.4.6D–E also innervates the teeth, the oral and nasal cavities, and the cranial dura mater. The central processes of trigeminal ganglion cells enter the brainstem at the midpontine level, which also corresponds to the level of the chief sensory trigeminal nucleus (nucleus of cranial nerve V). Some axons terminate in this nucleus (primarily large-caliber axons carrying the information needed for fine discriminative touch), whereas others (intermediateand small-caliber axons that carry information about touch as well as pain and temperature) form the descending trigeminal tract, which descends through the medulla just lateral to the descending trigeminal nucleus. As the tract descends, axons peel off and synapse in this nucleus.

1	Proprioceptive information is also conveyed via the trigeminal nerve; however, in this unique case the cell bodies of the first-order fibers are located within the CNS in the mesencephalic portion of the trigeminal nucleus. The central processes of these neurons terminate in the motor trigeminal nucleus (to subserve segmental reflexes equivalent to the segmental spinal cord reflexes [see ]), the reticular formation, and the chief sensory trigeminal nucleus.

1	As may already be clear, information related to the different somatosensory submodalities travels to a large extent into the CNS via distinct sets of axons and targets different structures in the spinal cord and brainstem. Within the CNS this segregation continues as the information travels via separate pathways up the spinal cord and brainstem. For example, from the body, fine discriminatory touch information is conveyed by the dorsal column–medial lemniscus pathway, whereas pain, temperature, and crude touch information is conveyed by the anterolateral system.

1	Proprioceptive information is transmitted by yet another route that partially overlaps with the dorsal column–medial lemniscal pathway. Note, however, that this functional segregation is not absolute, so, for example, there can be some recovery of discriminative touch ability after a lesion of the dorsal columns. The anterolateral system will be discussed in the section on pain because it is the critical pathway for that information. Here, the central pathways CHAPTER 7 The Somatosensory System for discriminatory touch and proprioception are considered in detail. This pathway is shown in its entirety in Fig. 7.1A.

1	Fig. 7.1A. The dorsal columns are formed by ascending branches of the large myelinated axons of dorsal root ganglion cells (the first-order neurons). These axons enter at each spinal segmental level and travel rostrally up to the caudal medulla to synapse in one of the dorsal column nuclei: the nucleus gracilis, which receives information from the lower part of the body and leg, and the nucleus cuneatus, which receives information from the upper part of the body and arm. Note that across the dorsal columns and across the dorsal column nuclei there is a somatotopic representation of the body, with the legs represented most medially, followed by the trunk and then the upper limb. This somatotopy is a consequence of newly entering afferents being added to the lateral border of the dorsal funiculus as the spinal cord is ascended. Such somatotopic maps are present at all levels in the somatosensory system, at least through the primary sensory cortices.

1	The dorsal column nuclei are located in the medulla and contain the second-order neurons of the pathway for discriminatory touch sensation. These cells respond similarly to the primary afferent fibers that synapse on them (see the earlier description of afferent types). The main differences between the responses of dorsal column neurons and primary afferent neurons are as follows: (1) dorsal column neurons have larger receptive fields because multiple primary afferent fibers synapse on a given dorsal column neuron, (2) dorsal column neurons sometimes respond to more than one class of sensory receptor because of the convergence of several different types of primary afferent fibers on the second-order neurons, and (3) dorsal column neurons often have inhibitory receptive fields that are mediated through local interneurons.

1	The axons of dorsal column nuclear projection neurons exit the nuclei and are referred to as the internal arcuate fibers as they sweep ventrally and then medially to cross the midline at the same medullary level as the nuclei. Immediately after crossing the midline, these fibers form the medial lemniscus (see Fig. 4.6C–E ), which projects rostrally to the thalamus. Knowledge of the level of this decussation is clinically important because damage to the dorsal column–medial lemniscal pathway below this level, which includes all of the spinal cord, will produce loss of fine somatosensory discriminatory abilities on the same, or ipsilateral, side of the lesion, whereas lesions above this level will produce contralateral deficits. Moreover, because there is a clear somatotopic arrangement of fibers in the medial lemniscus, localized lesions cause selective loss of fine-touch sensations limited to specific body regions.

1	The third-order neurons of the pathway are located in the ventral posterior lateral (VPL) nucleus of the thalamus and project to somatosensory areas of the cerebral cortex ( Fig. 7.5 •Fig. 7.5 Diagramofconnectionsfromthesomatosensoryreceivingnucleiofthethalamustothesomatosensorycortexoftheparietallobe.Notetheparallelflowofdifferenttypesofsomatosensoryinformationthroughthethalamusandontothecortex.CS,centralsulcus;S1andS2areprimaryandsecondarysomatosensoryareas,respectively.Note:collectively,areas3a,3b,1,and2arereferredtoasS1.

1	The dorsal column–medial lemniscus pathway conveys information about fine-touch and vibratory sensations. This information is critical for many of the discriminatory tactile abilities we have. For example, spatial acuity is lowered by damage to this pathway, and the ability to identify objects by their shape and texture can be lost by damage to this pathway. Clinically, one may test for impaired graphesthesia, or the ability to recognize letters or numbers traced on the skin, or for loss of the ability to tell the direction of a line drawn across the skin. Importantly, some tactile function remains even after complete loss of the dorsal columns, and awareness and localization of nonnoxious tactile stimuli can still occur. Thus at least some of the information carried by the dorsal column pathway is also conveyed by additional ascending pathways. In contrast to the severe deficits in discriminatory touch sensation, cutaneous pain and temperature sensations are unaffected by lesions of

1	pathway is also conveyed by additional ascending pathways. In contrast to the severe deficits in discriminatory touch sensation, cutaneous pain and temperature sensations are unaffected by lesions of the dorsal columns. However, visceral pain is substantially diminished by damage to the dorsal columns.

1	Trigeminal Pathway for Fine-Touch Sensation From the Face Primary afferent fibers that supply the face, teeth, oral and nasal cavities, and cranial meninges synapse in several brainstem nuclei, including the main sensory nucleus and the descending nucleus of the trigeminal nerve. The pathway through the main sensory nucleus resembles the dorsal column–medial lemniscus pathway. This sensory nucleus relays tactile information to the contralateral ventral posterior medial (VPM) thalamic nucleus by way of the trigeminothalamic tract. Third-order neurons in the VPM nucleus project to the facial area of the somatosensory cortex. Proprioceptors provide information about the positions and movement of parts of the body. In addition to being used for local reflexes (see

1	Proprioceptors provide information about the positions and movement of parts of the body. In addition to being used for local reflexes (see Chapter 9), this information has two main targets, the cerebellum and the cerebral cortex. The cerebellum uses this information for its motor coordination functions. The information sent to the cerebral cortex is the basis for conscious awareness of our body parts (e.g., position of our hand), which is referred to as kinesthesia. The major pathways by which somatosensory information is brought to the cerebellum are shown in

1	The major pathways by which somatosensory information is brought to the cerebellum are shown in Fig. 7.1B . These pathways carry both cutaneous and proprioceptive information to the cerebellum. For the trunk and lower limb, the pathway starts with dorsal root ganglion cells whose axons synapse in Clarke’s column (nucleus dorsalis). The cells of Clarke’s column send their axons into the ipsilateral lateral funiculus to form the dorsal spinocerebellar tract, which enters the cerebellum via the inferior cerebellar peduncle. The ventral spinocerebellar tract also provides somatosensory input from the lower limb to the cerebellum. Note the double decussation of the ventral spinocerebellar pathway (one decussation at the spinal cord levels and a second one in the cerebellar white matter). This double crossing highlights the general rule that each half of the cerebellum is functionally related to the ipsilateral side of the body.

1	To provide proprioceptive information from the lower limb to the cerebral cortex, the main axons of the dorsal spinocerebellar tract give off a branch in the medulla that terminates in nucleus z, which is just rostral to the nucleus gracilis. The axons of cells from nucleus z then form part of the internal arcuate fibers and medial lemniscus and ascend to the VPL nucleus of the thalamus. The ascending somatosensory pathways to the cerebellum for the upper limb are simpler than those from the lower limb (see Fig. 7.1B ). The route to the cerebellum starts with dorsal root ganglion fibers from the cervical spinal levels that ascend in the cuneate fasciculus to the external cuneate nucleus. The axons of the external cuneate nucleus then form the cuneocerebellar tract, which enters the cerebellum via its inferior peduncle.

1	The route to the cerebral cortex for proprioceptive information from the upper limb is identical to that for discriminative touch: the dorsal column–medial lemniscal pathway, with a synapse in the cuneate nucleus and then in the VPL nucleus of the thalamus. For the head, proprioceptive input is carried by cells of the mesencephalic nucleus of the trigeminal nerve. Recall that the neurons in this nucleus are actually the cell bodies of the primary afferents that innervate stretch receptors in the muscles of mastication (muscles that move the jaw) and in other muscles of the head. The central processes of these neurons project to the trigeminal motor nucleus for local reflexes or to the nearby reticular formation. Axons from these reticular formation neurons join the trigeminothalamic tract, which terminates in the VPM of the thalamus. There are also trigeminocerebellar pathways for conveying somatosensory (tactile and proprioceptive) information from the head to the cerebellum.

1	The ventroposterior nuclear complex of the thalamus represents the main termination site for ascending somatosensory information in the diencephalon. It consists of two major nuclei, the VPL and VPM, and a smaller nucleus called the ventral posterior inferior (VPI) (see Fig. 7.5 ). The medial lemniscus forms the main input to the VPL nucleus, and the equivalent trigeminothalamic tract from the main sensory nucleus of the trigeminal nerve forms the main input to the VPM nucleus. These nuclei also receive input conveying pain and temperature information from the spinothalamic or equivalent trigeminothalamic tracts, respectively. The VPI nucleus receives input from the spinothalamic tract. In addition, the spinothalamic tract terminates in parts of the posterior nuclear complex and several other thalamic nuclei.

1	Single-unit recordings from the ventroposterior complex of nuclei have shown that the responses of many of the neurons in these nuclei to stimuli resemble those of firstand second-order neurons in the ascending tracts. The receptive fields of thalamic cells are small but somewhat larger than those of primary afferent fibers. Moreover, the responses may be dominated by a particular type of sensory receptor. For example, VPL and VPM nuclei have cells whose receptive fields typically reflect input either from one type of cutaneous receptor (FA or SA) or from proprioceptive receptors, as expected from their medial lemniscal input. In contrast, cells of the VPI and posterior nuclei show responses to activation of nociceptors, the main input to the spinothalamic pathway.

1	Thalamic neurons often have inhibitory as well as excitatory receptive fields. The inhibition may actually take place in the dorsal column nuclei or in the dorsal horn of the spinal cord. However, inhibitory circuits are also situated within the thalamus. For example, the VPL and VPM nuclei contain GABAergic inhibitory interneurons (in primates but not in rodents), and GABAergic inhibitory interneurons in the reticular nucleus of the thalamus project into the VPL and VPM nuclei. (The reticular nucleus of the thalamus is a thin shell of neurons that surrounds much of the thalamus. Thalamocortical and corticothalamic neurons both send axon collaterals to the reticular nucleus, whose neurons then respectively complete feedback and/ or feed-forward inhibitory circuits with neurons in other thalamic nuclei.)

1	One difference between neurons in the VPL and VPM nuclei and sensory neurons at lower levels of the somatosensory system is that thalamic neuron excitability depends on the stage of the sleep/wake cycle and on the presence or absence of anesthesia. During a state of drowsiness or during barbiturate anesthesia, thalamic neurons tend to undergo an alternating sequence of excitatory and inhibitory postsynaptic potentials. The alternating bursts of discharges in turn intermittently excite neurons in the cerebral cortex. Such patterns of excitation and inhibition result in an α rhythm or in spindling on the electroencephalogram. This alternation of excitatory and inhibitory postsynaptic potentials during these two states may reflect the level of excitation of thalamic neurons by excitatory amino acids that act at NMDA and non-NMDA receptors. It may also reflect inhibition of the thalamic neurons by recurrent pathways through the reticular nucleus.

1	Thalamic neuron receptive fields are on the side of the body contralateral to the neuron, and the receptive field locations vary systematically across the ventroposterior nuclear complex. That is, the VPL and VPM nuclei are somatotopically organized such that the lower limb is represented most laterally and the upper limb most medially in the VPL nucleus, and the head is represented even more medially in the VPM nucleus. Moreover, the fact that thalamic neurons often receive input from only one class of receptor suggests that there are multiple somatotopic maps laid out across the ventroposterior nuclear complex. That is, there appear to be separate somatotopic maps for SA, FA, and proprioceptive and pain sensations laid out across the ventroposterior nuclear complex.

1	These maps are not randomly interspersed. As already mentioned, pain sensation is largely mapped across the VPI nucleus. In addition, the cutaneous receptors appear to drive cells located in a central “core” region of the VPL-VPM complex, whereas proprioceptive information is directed to cells that form a “shell” (VPS) around this core. This parallel flow of information into thalamus and then onto the cortex is diagramed in Fig. 7.5 The spinothalamic tract also projects to other thalamic regions, including the posterior nucleus and the central lateral nucleus of the intralaminar complex of the thalamus. The intralaminar nuclei of the thalamus are not somatotopically organized, and they project diffusely to the cerebral cortex as well as to the basal ganglia (see ). The projection of the central lateral nucleus to the S-I cortex may be involved in arousal of this part of the cortex and in selective attention.

1	Third-order sensory neurons in the thalamus project to the somatosensory cortex. The details of this projection pattern are shown in Fig. 7.5 . The main somatosensory receiving CHAPTER 7 The Somatosensory System areas of the cortex are called the S-I and S-II areas. The S-I cortex (or primary somatosensory cortex) is located on the postcentral gyrus, and the S-II cortex (secondary somatosensory cortex) is in the superior bank of the lateral fissure (see Fig. 7.5

1	Fig. 7.5 As previously discussed, the S-I cortex, like the somatosensory thalamus, has a somatotopic organization. The S-II cortex also contains a somatotopic map, as do several other less understood areas of the cortex. In the S-I cortex the face is represented in the lateral part of the postcentral gyrus, above the lateral fissure. The hand and the rest of the upper extremity are represented in the dorsolateral part of the postcentral gyrus, and the lower extremity on the medial surface of the hemisphere. A map of the surface of the body and face of a human on the postcentral gyrus is called a sensory homunculus. The map is distorted because the volume of neural tissue devoted to a body region is proportional to the density of its innervation. Thus in humans, the perioral area, the thumb, and other digits take up a disproportionately large expanse of cortex relative to their size.

1	The sensory homunculus is an expression of place coding of somatosensory information. A locus in the S-I cortex encodes the location of a somatosensory stimulus on the surface of the body or face. For example, the brain knows that a certain part of the body has been stimulated because certain neurons in the postcentral gyrus are activated. The S-I cortex has several morphological and functional subdivisions, and each subdivision has a somatotopic map. These subdivisions were originally described by Brodmann, and they were based on the arrangements of neurons in the various layers of the cortex, as seen in Nissl-stained preparations. The subdivisions are therefore known as Brodmann areas 3a, 3b, 1, and 2 (see ). Cutaneous input dominates in areas 3b and 1, whereas muscle and joint input (proprioceptive) dominates in areas 3a and 2. Thus separate cortical zones are specialized for processing tactile and proprioceptive information (see Fig. 7.5

1	Fig. 7.5 Within any particular area of the S-I cortex, all the neurons along a line perpendicular to the cortical surface have similar response properties and receptive fields. The S-I cortex is thus said to have a columnar organization. A comparable columnar organization has also been demonstrated for other primary sensory receiving areas, including the primary visual and auditory cortices (see ). Nearby cortical columns in the S-I cortex may process information for different sensory modalities. For example, the cutaneous information that reaches one cortical column in area 3b may come from FA mechanoreceptors, whereas the information that reaches a neighboring column might originate from SA mechanoreceptors.

1	Besides being responsible for the initial processing of somatosensory information, the S-I cortex also begins higher-order processing such as feature extraction. For example, certain neurons in area 1 respond preferentially to a stimulus that moves in one direction across the receptive field but not in the opposite direction (Fig. 7.6 ). Such neurons presumably contribute to the perceptual ability to recognize the direction of an applied stimulus and could help detect slippage of an object being grasped by the hand. 118 SECTION2Berne & Levy Physiology •Fig. 7.6 Featureextractionbycorticalneurons.Theresponseswererecordedfromaneuroninthesomatosensorycortexofamonkey.Thedirectionofastimuluswasvaried,asshownbythearrowsinthedrawing.NotethattheresponsesweregreatestwhenthestimulusmovedinthedirectionfromUWtoRFandleastfromRWtoUF.F,fingers;R,radialside;U,ulnarside;W,wrist.(FromCostanzoRM,GardnerEP.J Neurophysiol 1980;43:1319.)R W F UW RF RW UF U R U R F U W UF RW F W RF UW 1 second

1	Effects of Lesions of the Somatosensory Cortex A lesion of the S-I cortex in humans produces sensory changes similar to those produced by a lesion of the somatosensory thalamus. However, usually only a part of the cortex is involved, and thus the sensory loss may be confined, for example, to the face or to the leg, depending on the location of the lesion with respect to the sensory homunculus. The sensory modalities most affected are discriminative touch and position sense. Graphesthesia and stereognosis (i.e., the ability to recognize objects such as coins and keys as they are handled) are particularly disturbed. Pain and thermal sensation may be relatively unaffected, although loss of pain sensation may follow cortical lesions. Conversely, cortical lesions can result in a central pain state that resembles thalamic pain (see “ Effects of Interruption of the Spinotha lamic Tract and Lesions of the Thalamus on Somatosensory

1	Effects of Interruption of the Spinotha lamic Tract and Lesions of the Thalamus on Somatosensory The sensations of pain and temperature are related and often grouped together because they are mediated by overlapping sets of receptors and are conveyed by the same types of fibers in the PNS and the same pathways in the CNS. One consequence of these labeled lines is that pain sensations in particular are not due to stronger activation of touch pathways, as might naively be thought. This difference is borne out experimentally because if SA afferents, for example, are stimulated more and more frequently, the sensation of tactile pressure becomes stronger but not painful. The axons that carry painful and thermal sensations are members of the relatively slowly conducting Aδ and C classes. However, not all Aδ and C axons carry pain and temperature information; some respond to light touch in a manner similar to what was described for low-threshold mechanoreceptors.

1	Unlike the case for low-threshold mechanoreceptors in which morphologically distinct receptors correspond to response properties, the Aδ and C axons conveying pain and temperature information appear to originate mostly as “free nerve endings.” (This description is not entirely accurate; the endings are mostly but not entirely covered by Schwann cells.) Despite the lack of distinct morphological specialization associated with their endings, Aδ and C axons constitute a heterogeneous population that is differentially sensitive to a variety of tissue-damaging or thermal stimuli (or both). This ability to sense tissue-damaging stimuli (mechanical, thermal, or chemical) is mediated by what are called nociceptors. These receptors share some features with low-threshold mechanoreceptors but are distinct in many ways, such as the ability to become sensitized (see later). Indeed, there appear to be a significant number of C fibers that are silent or unresponsive to any stimuli until first

1	are distinct in many ways, such as the ability to become sensitized (see later). Indeed, there appear to be a significant number of C fibers that are silent or unresponsive to any stimuli until first sensitized.

1	The first functional distinction that may be made in the pain system is between Aδ and C axons. Aδ axons conduct signals faster than C fibers do and are thought to underlie what is called first pain, whereas C fibers are responsible for second pain. Thus after a damaging stimulus, one first feels an initial sharp, pricking, highly localized sensation (first pain) followed by a duller, more diffuse, burning sensation (second pain). Experiments in which Aδ or C fibers were selectively activated demonstrated that activity in Aδ fibers produces sensations similar to first pain and that activity in C fibers produces second pain–like sensations.

1	Each fiber class in turn forms a heterogeneous group with regard to sensitivity to stimuli. Thus afferents are classified according to both size and their sensitivity to mechanical, thermal, and chemical stimuli. Fibers may have a low or high threshold to mechanical stimulation or be completely insensitive to it. Thermal sensitivity has been classified as responsiveness to warmth, noxious heat, cool, and noxious cold. Note that 43°C and 15°C are the approximate limits above and below which, respectively, thermal stimuli are sensed as painful. Chemical sensitivity to a variety of irritating compounds has been tested, including capsaicin (found in chili peppers), mustard oil, and acids.

1	Afferent fibers may be sensitive to one or more types of stimuli and have been named accordingly. For example, C fibers sensitive only to high-intensity (damaging) mechanical stimuli are called C mechanosensitive fibers, whereas those sensitive to heat and mechanical stimuli are labeled C mechanoheat-sensitive fibers (also called polymodal fibers). Other identified fiber types include Aδ and C cold-sensitive, Aδ mechanosensitive, and mechanoheat-sensitive fibers. Thus there is quite a variety of afferent types; however, the most common afferent type is the C polymodal fiber, which accounts for nearly half of the cutaneous C fibers. Surprisingly the second most common type is the mechanoheatinsensitive afferent (i.e., an afferent that is not sensitive to noxious stimuli until sensitized—see later).

1	The central portion of the Aδ and C axons carrying pain and temperature information from the body terminates in the dorsal horn of the spinal cord. The Aδ fibers target lamina I (dorsalmost part of dorsal horn), V (base of dorsal horn), and X (area surrounding central canal) of the gray matter, whereas the C fibers terminate in lamina I and II. The distinct termination patterns of the Aδ and C fibers in the spinal cord suggest that the messages they are carrying to the CNS are kept separated, and this is consistent with our ability to feel two distinct types of pain. The primary afferent termination patterns in the spinal cord are also important because they may help determine the possible interactions pain fibers can have with other afferents and with descending control systems (see later). Indeed, the gate control theory of pain refers to the phenomenon that

1	CHAPTER 7 The Somatosensory System innocuous stimuli, such as rubbing a hurt area, can block or reduce painful sensations. Such stimulation activates the large-diameter (Aα and Aβ) fibers, and their activity leads to release of GABA and other neurotransmitters by inter-neurons within the dorsal horn. GABA then acts by both presynaptic and postsynaptic mechanisms to shut down the activity of spinothalamic tract cells. Presynaptically, GABA activates both GABAA and GABAB receptors, which leads to partial depolarization of the presynaptic terminal and blocking of Ca++ channels, respectively. Both actions will decrease release of transmitter by the afferent terminal and thereby lessen excitation of the tract cell (see section on presynaptic inhibition).

1	Nociceptive and thermoreceptive information that originates from regions of the head is processed in a fashion similar to that for the trunk and limbs. The primary afferent fibers of nociceptors and thermoreceptors in the head enter the brainstem through the trigeminal nerve (some also enter through the facial, glossopharyngeal, and vagus nerves). Of note, the trigeminal distribution includes both tooth and headache pain. These fibers then descend through the brainstem as far as the upper cervical spinal cord via the descending tract of the trigeminal nerve. Some mechanoreceptive afferent fibers also join the descending tract of the trigeminal nerve. Axons in the descending tract synapse on second-order neurons in the descending nucleus of the trigeminal nerve.

1	Elderlypeoplearesometimessusceptibletoaconditionofchronicpainknownastrigeminal neuralgia. Peoplewiththisconditionexperiencespontaneousepisodesofsevere,oftenlancinatingpaininthedistributionofoneormorebranchesofthetrigeminalnerve.Frequentlythepainistriggeredbyweakmechanicalstimulationinthesameregion.Amajorcontributingfactortothispainfulstateappearstobemechanicaldamagetothetrigeminalganglionbyanarterythatimpingesontheganglion.Surgicaldisplacementofthearterycanoftenresolvethecondition. The central pain pathways include the spinothalamic, spinoreticular, and spinomesencephalic tracts. The spinothalamic tract is the most important sensory pathway for somatic pain and thermal sensations from the body (see

1	Fig. 7.1A ). It also contributes to tactile sensation. The spinothalamic tract originates from second-order neurons located in the spinal cord (primarily laminae I and IV to VI). The axons of these cells cross to the opposite side of the cord at or near to their level of origin. They then ascend to the brain in the ventral part of the lateral funiculus and subsequently through the brainstem to the thalamus, where they terminate on third-order neurons (thalamocortical neurons), as described earlier. Spinothalamic cells conveying pain and temperature target the VPI portion of the ventroposterior complex (although some also end in the

1	Multireceptive spinothalamic VPL), the posterior nucleus, and the intralaminar nuclei tract cells of the thalamus. Nociceptive signals are then forwarded to 120 several cortical areas, including not only the somatosensory cortex but also cortical areas that are involved in affective responses, such as the cingulate gyrus and the insula, which have limbic system functions (see Fig. 7.5 Most spinothalamic tract cells receive excitatory input from nociceptors in the skin, but many can also be excited by noxious stimulation of muscle, joints, or viscera. Few receive input only from viscera. Effective cutaneous stimuli include noxious mechanical, thermal (hot or cold), and chemical stimuli. Thus different spinothalamic tract cells respond in a manner appropriate for signaling noxious, thermal, or mechanical events. A Some nociceptive spinothalamic tract cells receive convergent excitatory input from several different classes of cutaneous sensory receptors. For example, a given spino

1	Some nociceptive spinothalamic tract cells receive convergent excitatory input from several different classes of cutaneous sensory receptors. For example, a given spino Fig. 7.7A ). Such neurons are called wide–dynamic range cells or multirecep-120 tive cells because they are activated by stimuli with a wide range of intensities. Wide–dynamic range neurons signal mainly noxious events; weak responses to tactile stimuli appear to be ignored by the higher centers. However, in certain pathological conditions, these neurons may be suf ficiently activated by tactile stimuli to evoke a sensation of pain, possibly as a result of activity in sensitized afferents that were previously silent. This would explain some pain states in which activation of mechanoreceptors causes pain (mechanical allodynia). Other spinothalamic tract cells are activated only by noxious stimuli. Such neurons are often called high-threshold or nociceptive-specific cells (see Fig. 7.7B

1	Fig. 7.7B Because cells signaling visceral input also typically convey information from cutaneous receptors, the brain may misidentify the source of the pain. This phenomenon is called referred pain. A typical example is when the heart muscle becomes ischemic and pain is felt in the chest wall and left arm.

1	Neurotransmitters released by nociceptive afferents that activate spinothalamic tract cells include the excitatory amino acid glutamate and any of several peptides, such as SP, CGRP, and vasoactive intestinal polypeptide (VIP). Glutamate appears to act as a fast transmitter by its action on non-NMDA excitatory amino acid receptors. However, with repetitive stimulation, glutamate can also act through NMDA receptors. Peptides appear to act as neuromodulators. For example, through a combined action with an excitatory amino acid such as glutamate, SP can produce a long-lasting increase in the responses of spinothalamic tract cells; this enhanced responsiveness is called central sensitization. CGRP seems to increase release of SP and prolong the action of SP by inhibiting its enzymatic degradation.

1	Spinothalamic tract cells often have inhibitory receptive fields. Inhibition may result from weak mechanical stimuli, but usually the most effective inhibitory stimuli •Fig. 7.7 A,Responsesofawide–dynamicrangeormultireceptivespinothalamictractcell.B,Responsesofahigh-thresholdspinothalamictractcell.Graphsshowresponsestogradedintensitiesofmechanicalstimulation.Brushstimulusisacamel’shairbrushrepeatedlystrokedacrossthereceptivefield.Pressureisappliedbyattachmentofanarterialcliptotheskin.Thisisamarginallypainfulstimulustoahuman.Pinchisachievedbyattachmentofastiffarterialcliptotheskinandisdistinctlypainful.Squeezeisappliedbycompressingafoldofskinwithforcepsandisdamagingtotheskin.

1	are noxious ones. The nociceptive inhibitory receptive fields may be very large and may include most of the body and face. Such receptive fields may account for the ability of various physical manipulations, including transcutaneous electrical nerve stimulation and acupuncture, to suppress pain. Neurotransmitters that can inhibit spinothalamic tract cells include the inhibitory amino acids GABA and glycine, as well as monoamines and the endogenous opioid peptides. Spinoreticular tract neurons frequently have large, sometimes bilateral receptive fields, and effective stimuli include noxious ones. These dorsal horn neurons target multiple regions in the medullary and pontine reticular formation. The reticular formation, which projects to the intralaminar complex of the thalamus and thereby to wide areas of the cerebral cortex, is involved in attentional mechanisms and arousal (see Chapter 10).

1	Chapter 10). The reticular formation also gives rise to descending reticulospinal projections, which contribute to the descending systems that control transmission of pain. Many cells of the spinomesencephalic tract respond to noxious stimuli, and the receptive fields may be small or large. The terminations of this tract are in several midbrain nuclei, including the periaqueductal gray, which is an important component of the endogenous analgesia system. Motivational responses may also result from activation of the periaqueductal gray matter. For example, stimulation in the periaqueductal gray matter can cause vocalization and aversive behavior. Information from the midbrain is relayed not only to the thalamus but also to the amygdala. This provides one of several pathways by which noxious stimuli can trigger emotional responses.

1	Pain and temperature information originating from the face and head is conveyed along analogous ascending central pathways, as is such information from the body. Neurons in the descending trigeminal nucleus transmit pain and temperature information to specific nuclei (VPM, VPI) of the contralateral thalamus via the ventral trigeminothalamic tract, which runs in close association with the medial lemniscus. The descending nucleus also projects to the intralaminar complex and other thalamic nuclei in a fashion similar to that of the spinothalamic tract. The thalamic nuclei in turn project to the somatosensory cerebral cortex for sensory discrimination of pain and temperature and to other cortical regions responsible for motivational-affective responses. Effects of Interruption of the Spinothalamic Tract and Lesions of the Thalamus on Somatosensory Sensation

1	When the spinothalamic tract and accompanying ventral spinal cord pathways are interrupted, both the sensory-discriminative and the motivational-affective components of pain are lost on the contralateral side of the body. This result motivated development of the surgical procedure known as anterolateral cordotomy, which was used to treat pain in many individuals, especially those suffering from cancer. This operation is now used infrequently because of improvements in drug therapy and because pain often returns months to years after an initially successful cordotomy. Return of pain may reflect either extension of the disease or development of a central pain state. In addition to loss of pain sensation, anterolateral cordotomy produces loss of cold and warmth sensation on the contralateral side of the body. Careful testing may reveal a minimal tactile deficit as well, but the intact sensory pathways of the dorsal part of the spinal cord provide sufficient tactile information that any

1	side of the body. Careful testing may reveal a minimal tactile deficit as well, but the intact sensory pathways of the dorsal part of the spinal cord provide sufficient tactile information that any loss caused by interruption of the spinothalamic tract is insignificant.

1	Destruction of the VPL or VPM nuclei diminishes sensation on the contralateral side of the body or face. The sensory qualities that are lost reflect those that are

1	CHAPTER 7 The Somatosensory System transmitted mainly by the dorsal column–medial lemniscus pathway and its trigeminal equivalent. The sensory-discriminative component of pain sensation is also lost. However, the motivational-affective component of pain is still present if the medial thalamus is intact. Presumably, pain persists because of the spinothalamic and spinoreticulothalamic projections to this part of the thalamus. In some individuals a lesion of the somatosensory thalamus results in a central pain state known as thalamic pain. Patients with thalamic pain report that even the slightest touch feels painful, although the intensity of the touch is lower than the threshold of any pain receptor. It is thought that their pain sensitivity is due to a post-lesion sprouting of low-threshold dorsal column system fibers that synapse onto surviving thalamic neurons that normally mediate only pain. Pain that is indistinguishable from thalamic pain can also be produced by lesions in the

1	dorsal column system fibers that synapse onto surviving thalamic neurons that normally mediate only pain. Pain that is indistinguishable from thalamic pain can also be produced by lesions in the brainstem or cortex.

1	Pain sometimes occurs in the absence of nociceptor stimulation. This type of pain is most likely to occur after damage to peripheral nerves or to parts of the CNS that are involved in transmitting nociceptive information. Pain caused by damage to neural structures is called neuropathic pain. Neuropathic pain states include peripheral neuropathic pain, which may follow damage to a peripheral nerve, and central neuropathic pain, which sometimes occurs after damage to CNS structures.

1	Examples of pain secondary to damage to a peripheral nerve are causalgia and phantom limb pain. Causalgia may develop after traumatic damage to a peripheral nerve. Even though evoked pain is reduced, severe pain may develop in the area innervated by the damaged nerve. This pain may be very difficult to treat, even with strong analgesic drugs. The pain is caused in part by spontaneous activity that develops in dorsal root ganglion cells; such activity may be attributed to upregulation of Na+ channels. In some cases the pain seems to be maintained by sympathetic neural activity, because a sympathetic nerve block may alleviate the pain. Sympathetic involvement may relate to the sprouting of damaged sympathic postganglionic axons into the dorsal root ganglia, and it may be accompanied by upregulation of adrenoreceptors in primary afferent neurons. Phantom limb pain follows traumatic amputation in some individuals. Such phantom pain is clearly not caused by activation of nociceptors in the

1	of adrenoreceptors in primary afferent neurons. Phantom limb pain follows traumatic amputation in some individuals. Such phantom pain is clearly not caused by activation of nociceptors in the area in which pain is felt, because these receptors are no longer present.

1	Lesions of the thalamus or at other levels of the spinothalamocortical pathway may cause central pain, which is a severe spontaneous pain. However, interruption of the nociceptive pathway by the same lesion may simultaneously prevent or reduce the pain evoked by peripheral stimulation. The mechanism of such trauma-induced pain caused by neural damage is poorly understood. The pain appears to depend on changes in the activity and response properties of more distant neurons in the nociceptive system. Transduction in the Somatosensory System

1	Transduction in the Somatosensory System Unraveling the transduction processes for the somatosensory system has proven difficult because of a variety of factors. The heart of the transduction process occurs at the specialized endings of the peripheral branch of the axon of the sensory neuron (dorsal root ganglia and trigeminal ganglia cells). These endings have channels that are activated by mechanical, thermal, and/or chemical stimuli and that allow the current flow that underlies the local generator potential (see Fig. 5.13 ). The generator potential then triggers spikes in the afferent fiber (see Fig. 5.14 ). However, the fact is that somatosensory axons are found throughout the body at low densities, making purification of the proteins difficult (e.g., compare with the retina, in which photoreceptors are packed at high density onto a small surface).

1	Complicating matters, the generator potential can be modified by a host of voltage-gated channels, both excitatory (e.g., Na+ and Ca++ channels) and inhibitory (K+ channels). Moreover, as described earlier, in many cases the axon terminal is encapsulated (e.g., Pacinian corpuscles), and the properties of this accessory structure modify the transduction process. They may do so passively, as a result of the mechanical characteristics of the capsule, or actively by having the accessory cells release transmitter in response to a stimulus. An example of the latter is Merkel cells, where recent evidence suggests the Merkel cells and the axon innervating them both have mechanosensitive channels and the Merkel cells release neurotransmitter. Despite these complications, using a number of approaches, our knowledge of somatosensory transduction processes has begun to increase rapidly.

1	Despite these complications, using a number of approaches, our knowledge of somatosensory transduction processes has begun to increase rapidly. Until recently, ASIC (acid-sensing ion channel) proteins, which belong to the DEG/ENaC family, had been thought to be the channel proteins underlying the cutaneous mechanoactivated currents, because homologs of these proteins underlie the touch sensation in invertebrates such as Caenorhabditis elegans, and because some ASIC proteins are highly expressed in dorsal root ganglion cells. However, the mechanoactivated currents in dorsal root ganglion cells are not affected by knockdown of the ASIC proteins, indicating that they are not the channel underlying cutaneous mechanotransduction. Nevertheless, touch and pain sensitivity is altered in such knockdown mutants, so they may still play a modulatory role in the transduction process.

1	Currently, Piezo2 is thought to be channel protein underlying the transduction for cutaneous mechanical rapidly adapting responses, because it forms a nonselective cation pore that opens in response to mechanical stimuli. Moreover, the activation and inactivation kinetics of this channel are consistent with its causing the rapidly adapting mechanoactivated current, and it is blocked by agents (gadolinium, ruthenium red) that block these rapidly adapting currents. Furthermore, transfection of ganglion cells with small interfering (si)RNA for Piezo2 blocks the rapid mechanoactivated current in these cells. Lastly, messenger (m)RNA expression of Piezo2 has been shown in dorsal root ganglion cells.

1	Both low-threshold (light touch) and nociceptive-type ganglion cells have been found to express Piezo2, indicating that it plays a role in both innocuous and painful touch sensation. The Piezo proteins (Piezo1 and Piezo2) are both found in a variety of organs and thus may underlie visceral sensation as well. Piezo2 has been found in the main proprioceptors (muscle spindles and Golgi tendon organs [see ]) and appears to be the main mechanotransducer protein there as well. The receptor that binds capsaicin (the molecule in chili peppers responsible for their spiciness) has been identified, and either it or one of a family of related proteins has been found to be expressed in populations of dorsal root ganglion cells. These proteins belong to the TRP (transient receptor potential) protein family and are currently the most likely candidates for being the transducers of thermal sensations.

1	It is important to note that many ion channels (and other proteins, e.g., enzymes) are sensitive to temperature; however, in the case of TRP channels, temperature is acting directly as the gating mechanism. The temperatures at which specific TRP channels are active are indicated by arrows in Fig. 7.8 (bottom), in which the direction of each •Fig. 7.8 Temperaturedependenceoffiringratesindifferentthermo-sensitiveafferents.BelowthefiringratecurvesareshowntherangesoverwhichthedifferentTRPchannelsareactivated.Thedirectionofincreasingactivationisindicatedbyanarrowineachcase.Notehowinsomecasestherangeoverwhichanafferentisactive(topgraphs)correspondswelltotheactivationrangeofasingleTRPchannel,thussuggestingthattheafferentwouldneedtoexpressonlyasingletypeofchannel.InothercasestheactiverangeoftheafferentsuggeststhatmultipleTRPchannelswouldbeneededtounderliethecompleteresponsivenessoftheafferent. arrow indicates which temperatures cause greater activation. For comparison,

1	arrow indicates which temperatures cause greater activation. For comparison, Fig. 7.8 also plots the firing rates of several thermosensitive fibers as a function of temperature. Note how the response ranges of the afferents largely match up with those of individual heat-sensitive channels. The cold fibers, however, show firing over a wider range than any one TRP channel does. One possible explanation for this discrepancy is that dorsal root ganglion cells may express multiple classes of TRP, which would enable them to respond over a wider range of physiological temperatures.

1	MembersoftheTRPproteinfamilywerefirstidentifiedinDrosophila andwerefoundtobepartofthephototransductionprocessinDrosophila photoreceptors.Thusthename(TRP) referstothefactthatamutationinthegeneleadstoatransientdepolarizingresponsetoalightstimulusinsteadofthenormalsustainedresponse.Onthebasisofsequencehomology,anumberofgenesencodingTRPproteinshavebeenfoundinmammals(27inhumansalone),whicharecurrentlydividedintosevensubfamilies.TRPchannelsarecationpermeableandhaveastructuresimilartovoltage-gatedK+ channels.Theyarehomotetramersorheterotetramers.Eachsubunithassixtransmembranedomains.TRPproteinsappeartohaveavarietyoffunctions(e.g.,phototransduction,chemotransduction,andmechanotransduction)andareexpressedinanumberofcelltypes.Thoselistedin Table7.1 appeartoactastemperaturesensorswithdistinctthermalsensitivitiesthatspantherangeofphysiologicallyrelevanttemperatures. Modulation of the Transduction Process

1	Table7.1 appeartoactastemperaturesensorswithdistinctthermalsensitivitiesthatspantherangeofphysiologicallyrelevanttemperatures. Modulation of the Transduction Process As with the low-threshold mechanoreceptors for innocuous touch sensations, activation of the various nociceptor Thefourthletterinthenameidentifiesthesubfamilyandwaschosenbecauseofthefirstmemberofthesubfamilyidentified:V,vanilloid;M,melastatin;A,ankyrin-like.Eachoftheproteinslistedisexpressedinatleastsomedorsalrootganglioncells,buttheyarealsoexpressedinothercelltypes.

1	CHAPTER 7 The Somatosensory System transduction proteins leads to a generator potential that causes spiking of the afferent, which transmits information to the CNS. In addition, activation of nociceptors also leads to local release of various chemical compounds, including tachykinins (substance P [SP]) and calcitonin gene–related protein (CGRP). These substances and others released from the damaged cells cause neurogenic inflammation (edema and redness of the surrounding skin).

1	In addition to causing a local reaction, these substances may serve to activate the insensitive or silent nociceptors mentioned earlier, such that they can henceforth respond to any subsequent damaging stimuli. Sensitization of silent nociceptors has been suggested to underlie allodynia (elicitation of painful sensations by stimuli that were innocuous before an injury) and hyperalgesia (increase in the level of pain felt to already painful stimuli). Thermal hyperalgesia is due to sensitization of TRPV1 channels. The Piezo2 current is increased by substances known to cause mechanical hyperalgesia and allodynia, suggesting changes in this current underlie these phenomena. Centrifugal Control of Somatosensation

1	Centrifugal Control of Somatosensation Sensory experience is not just the passive detection of environmental events. Instead, it more often depends on exploration of the environment. Tactile cues are sought by moving the hand over a surface. Visual cues result from scanning targets with the eyes. Thus sensory information is often received as a result of activity in the motor system. Furthermore, transmission in pathways to the sensory centers of the brain is regulated by descending control systems. These systems allow the brain to control its input by filtering the incoming sensory messages. Important information can be attended to and unimportant information can be ignored.

1	The tactile and proprioceptive somatosensory pathways are regulated by descending pathways that originate in the S-I and motor regions of the cerebral cortex. For example, cortical projections to the dorsal column nuclei help control the sensory input that is transmitted by the dorsal column– medial lemniscus pathway.

1	Of particular interest is the descending control system that regulates transmission of nociceptive information. This system presumably suppresses excessive pain under certain circumstances. For example, it is well known that soldiers on the battlefield, accident victims, and athletes in competition often feel little or no pain at the time a wound occurs or a bone is broken. At a later time, pain may develop and become severe. Although the descending regulatory system that controls pain is part of a more general centrifugal control system that modulates all forms of sensation, the pain control system is so important medically that it is distinguished as a special system called the endogenous analgesia system.

1	Several centers in the brainstem and pathways descending from these centers contribute to the endogenous analgesia system. For example, stimulation in the midbrain •Fig. 7.9 A,

1	Modelsofdescendingcontrolofascendingpainpathways.A,Schematicshowingtheascendingnociceptivepathwaythatconveyspaininformationtothebrain,representedbythespinothalamictract(STT),andthedescendingpathwaythatmodulates(gates)theflowofinformationintoit.Thedescendingpathwaystartsintheperiaqueductalgray(PAG),whichprojectsviatherostroventromedial(RVM)medullatothedorsalhorn.ActivationofthePAGorRVMblockstransmissionofpaininformationatthespinalcordlevel.B,Thestandardmodelofdescendingcontrolofpainperception.TonicallyactiveGABAergicinterneuronsinthePAGandRVMinhibittheprojectioncells(notethatthePAGhaslocalGABAergicinterneuronsbutalsoreceivesGABAergicterminalsfromotherbrainregions,andbothmaybeinvolvedintonicallyinhibitingthePAGprojectioncells).OpioidsappeartoinhibittheGABAergicinterneurons,thusdisinhibitingtheprojectionneurons,whoseincreasedactivityleadstoblockingtheactivationofSTTcellsbynociceptivedorsalrootganglion(DRG)cells.C,Parallelinhibition-excitationmodel.Somestudiessuggestbothexcitatoryandinhib

1	theprojectionneurons,whoseincreasedactivityleadstoblockingtheactivationofSTTcellsbynociceptivedorsalrootganglion(DRG)cells.C,Parallelinhibition-excitationmodel.SomestudiessuggestbothexcitatoryandinhibitorycellsprojectfromthePAGandtheRVM.Inthismodel,gatingoftransmissionofpaininformationwouldbeduetoabalanceoftheexcitatoryandinhibitoryactivityinthedescendingpathways.Notethattheexcitatoryandinhibitorypathwaysareshownonoppositesidesjustforclarity.D,Possiblepresynapticandpostsynapticsitesofactionofenkephalin(Enk).ManyoftheterminalsfromthedescendingpathwaysforpainmodulationreleasebothGABAandenkephalin(EnK),whichappearstohavebothpre-andpostsynapticsitesofaction.ThepresynapticactionmightpreventreleaseofsubstanceP(SubP.)fromnociceptors.(A-C,RedrawnfromLauBK,VaughanCW.Curr

1	Opin Neurobiol 2014;29:159.D,RedrawnfromHenryJL.In:PorterR,O’ConnorM[eds]:CibaFoundationSymposium91.London:Pitman;1982.) periaqueductal gray, locus coeruleus, or medullary raphe nuclei inhibits nociceptive neurons at the spinal cord and brainstem level, including spinothalamic and trigeminothalamic tract cells (

1	Fig. 7.9A-C ). Other inhibitory pathways originate in the sensorimotor cortex, hypothalamus, and reticular formation. The endogenous analgesia system can be subdivided into two components: one component uses endogenous opioid peptides as neurotransmitters and the other does not. Endogenous opioids are neuropeptides that activate one of several types of opiate receptors. Some of the endogenous STT relay cell afferent Circulating opioid binding sites Opiate receptor Sub P. Enk Substance P receptor opioids include enkephalin, dynorphin, and β-endorphin. Opiate analgesia can generally be prevented or reversed by the narcotic antagonist naloxone. Therefore naloxone is frequently used to determine whether analgesia is mediated by an opioid mechanism.

1	The opioid-mediated endogenous analgesia system can be activated by exogenous administration of morphine or other opiate drugs. Thus one of the oldest medical treatments of pain depends on the triggering of a sensory control system. Opiates typically inhibit neural activity in nociceptive pathways. Two sites of action have been proposed for opiate inhibition, presynaptic and postsynaptic (see Fig. 7.9D ). The presynaptic action of opiates on nociceptive afferent terminals is thought to prevent release of excitatory transmitters such as SP. The postsynaptic action of opiates produces an inhibitory postsynaptic potential. How can an inhibitory neurotransmitter activate descending pathways? One hypothesis is that the descending analgesia system is under tonic inhibitory control by inhibitory interneurons in both the midbrain and medulla. The action of opiates would inhibit the inhibitory inter-neurons and thereby disinhibit the descending analgesia pathways.

1	Some endogenous analgesia pathways operate by neurotransmitters other than opioids and thus are unaffected by naloxone. One way of engaging a nonopioid analgesia CHAPTER 7 The Somatosensory System pathway is through certain forms of stress. The analgesia thus produced is a form of stress-induced analgesia.

1	CHAPTER 7 The Somatosensory System pathway is through certain forms of stress. The analgesia thus produced is a form of stress-induced analgesia. Many neurons in the raphe nuclei use serotonin as a neurotransmitter. Serotonin can inhibit nociceptive neurons and presumably plays an important role in the endogenous analgesia system. Other brainstem neurons release catecholamines, such as norepinephrine and epinephrine, in the spinal cord. These catecholamines also inhibit nociceptive neurons; therefore catecholaminergic neurons may contribute to the endogenous analgesia system. Furthermore, these monoamine neurotransmitters interact with endogenous opioids. Undoubtedly, many other substances are involved in the analgesia system. In addition, there is evidence for the existence of endogenous opiate antagonists that can prevent opiate analgesia. 1.

1	1. Sensory neurons have cell bodies in sensory nerve ganglia: (1) dorsal root ganglia for neurons innervating the body and (2) cranial nerve ganglia for neurons innervating the face, oral and nasal cavities, and dura, except for proprioceptive neurons, which are in the trigeminal mesencephalic nucleus. They connect peripherally to a sensory receptor and centrally to second-order neurons in the spinal cord or brainstem. 2. Skin contains low-threshold mechanoreceptors, thermoreceptors, and nociceptors. Muscle, joints, and viscera have mechanoreceptors and nociceptors. Low-threshold mechanoreceptors may be rapidly or slowly adapting. Thermoreceptors include cold and warm receptors. Aδ and C nociceptors detect noxious mechanical, thermal, and chemical stimuli and may be sensitized by release of chemical substances from damaged cells. Peripheral release of substances, such as peptides, from nociceptors themselves may contribute to inflammation. 3.

1	3. Large primary afferent fibers enter the dorsal funiculus through the medial part of the dorsal root; collaterals synapse in the deep dorsal horn, intermediate zone, and ventral horn. Small primary afferent fibers enter the spinal cord through the lateral part of the dorsal root; collaterals synapse in the dorsal horn. 4. Ascending branches of large primary afferent fibers synapse on second-order neurons in the dorsal column nuclei. These second-order neurons project via the medial lemniscus to the contralateral thalamus and synapse on third-order neurons of the VPL nucleus. The equivalent trigeminal pathway is relayed by the main sensory nucleus to the contralateral VPM nucleus. 5. The dorsal column spinal cord pathways signal the sensations of flutter-vibration, touch-pressure, and proprioception. They also contribute to visceral sensation, including visceral pain. 6.

1	5. The dorsal column spinal cord pathways signal the sensations of flutter-vibration, touch-pressure, and proprioception. They also contribute to visceral sensation, including visceral pain. 6. The spinothalamic tract includes nociceptive, thermoreceptive, and tactile neurons; its cells of origin are mostly in the dorsal horn, and the axons cross, ascend in the ventrolateral funiculus, and synapse in the VPL, VPI, and posterior and intralaminar nuclei of the thalamus. The equivalent trigeminal pathway is relayed by the descending trigeminal nucleus and projects to the contralateral VPM and intralaminar nuclei. 7. The spinothalamic relay in the VPL, VPM, and VPI nuclei helps account for the sensory-discriminative aspects of pain. Parallel nociceptive pathways in the ventrolateral funiculus are the spinoreticular and spinomesencephalic tracts; these tracts and the spinothalamic projection to the medial thalamus contribute to the motivational-affective aspects of pain. 8.

1	8. Referred pain is explained by convergent input to spinothalamic tract cells from the body wall and from viscera. 9. The VPL and VPM nuclei are somatotopically organized and contain inhibitory circuits. These nuclei contain multiple somatotopic maps, one for each somatosensory submodality. The somatosensory cortex includes the S-I and S-II regions; these regions are also somatotopically organized. 10. The S-I cortex contains columns of neurons with similar receptive fields and response properties. Some S-I neurons are involved in feature extraction. 11. Transmission in somatosensory pathways is regulated by descending control systems. The endogenous analgesia system regulates nociceptive transmission, and it uses transmitters such as endogenous opioid peptides, norepinephrine, and serotonin.

1	Additional Readings Lau BK, Vaughan CW. Descending modulation of pain: the GABA disinhibition hypothesis of analgesia. Curr Opin Neurobiol. Coste B, Xiao B, Santos JS, et al. Piezo proteins are pore-2014;29:159-164. forming subunits of mechanically activated channels. Nature. Squire L, Berg D, Bloom FE, et al., eds. Fundamental Neuroscience. 4th ed. Waltham, MA: Academic Press; 2013. 2012;483:176-181. Upon completion of this chapter, the student should be able to answer the following questions: 1. What is the dark current, and how does the absorption of a photon change it? 2. What are the synaptic pathways for the central and surround portions of the receptive field of an on-center bipolar cell? Of an off-center bipolar cell? 3. What are the receptive field properties of simple and complex cells in the visual cortex? 4. What is the frequency theory of sound encoding? Why is the place theory also required? 5.

1	3. What are the receptive field properties of simple and complex cells in the visual cortex? 4. What is the frequency theory of sound encoding? Why is the place theory also required? 5. What are the stimuli that are normally transduced by the hair cells in the semicircular canals and otolith organs? 6. What are the functional consequences of the differing numbers of different receptor molecules between olfactory and gustatory receptor cells?

1	6. What are the functional consequences of the differing numbers of different receptor molecules between olfactory and gustatory receptor cells? he evolution of vertebrates shows a trend called cephalization in which special sensory organs develop in the heads of animals, along with the corresponding development of the brain. These special sensory systems, which include the visual, auditory, vestibular, olfactory, and gustatory systems, detect and analyze light, sound, and chemical signals in the environment, as well as signal the position and movement of the head. The stimuli transduced by these systems are most familiar to humans when they provide conscious awareness of the environment, but they are equally important as the sensory basis for reflexive and subconscious behavior. The Visual System

1	The Visual System Vision is one of the most important special senses in humans and, along with audition, is the basis for most human communication. The visual system detects electromagnetic waves between 400 and 750 nm long as visible light, which enters the eye and impinges on photoreceptors in a specialized sensory epithelium, the retina. The photoreceptors, rods and cones, can distinguish two aspects of light: its brightness (or luminance) and its wavelength (or color). Rods have high sensitivity for detecting low light intensities but do not provide well-defined visual images, nor do they contribute to color vision. Rods operate best under conditions of reduced lighting (scotopic vision). Cones, in contrast, are not as sensitive to light as rods are and thus operate best under daylight conditions (photopic vision). Cones are responsible for high visual acuity and color vision.

1	The retina is an outgrowth of the thalamus. Thus information processing within the retina is performed by retinal neurons, and the output signals are carried to the brain by the axons of retinal ganglion cells in the optic nerves. There is a partial crossing of these axons in the optic chiasm that causes all input from one side of the visual space to pass to the opposite side of the brain. Posterior to the optic chiasm, the axons of retinal ganglion cells form the optic tracts and synapse in nuclei of the brain. The main visual pathway in humans targets the lateral geniculate nucleus (LGN) of the thalamus and this nucleus, through the visual radiations, projects the visual information to the visual cortex. Other visual pathways project to the superior colliculus, pretectum, and hypothalamus, structures that participate in orientation of the eyes, control of pupil size, and circadian rhythms, respectively. Structure of the Eye

1	Structure of the Eye The wall of the eye is composed of three concentric layers ( Fig. 8.1 ). The outer layer, or the fibrous coat, includes the transparent cornea, with its epithelium, and the opaque sclera. The middle layer, or vascular coat, includes the iris and the choroid. The iris contains both radially and circularly oriented smooth muscle fibers, which make up the pupillary dilator and constricter muscles, respectively. The choroid is rich in blood vessels that support the outer layers of the retina, and it also contains pigment. The innermost layer of the eye, the retina, is embryologically derived from the diencephalon and therefore is part of the central nervous system (CNS). The functional part of the retina covers the entire posterior aspect of the eye except for the optic nerve head, or optic disc, which is where the optic nerve axons leave the retina. Because there are no receptors at this location, it is often referred to as the anatomical “blind spot” (see

1	Fig. 8.1

1	A number of functions of the eyes are under muscular control. Externally attached extraocular muscles aim the eyes toward an appropriate visual target (see ). These muscles are innervated by the oculomotor nerve •Fig. 8.1 Illustrationofaviewofahorizontalsectionoftherighteye.(RedrawnfromWallGL.The Vertebrate Eye and Its Adaptive Radiation. BloomfieldHills,MI:CranbrookInstituteofScience;1942.)IrisPosterior chamber Anterior chamber Limbus Sclera Choroid Retina Optic disc Fovea Vitreous humor Optic nerve Temporal retina Nasal retina Ciliary body Zonule fibers Nodal point Lens Aqueous humor Cornea (cranial nerve [CN] III), the trochlear nerve (CN IV), and the abducens nerve (CN VI). Several muscles are also found within the eye (intraocular muscles). The muscles in the ciliary body control lens shape and thereby the focus of images on the retina. The pupillary dilator and sphincter muscles in the iris control the amount of light entering the eye, in a way similar to that of the diaphragm

1	shape and thereby the focus of images on the retina. The pupillary dilator and sphincter muscles in the iris control the amount of light entering the eye, in a way similar to that of the diaphragm of a camera. The dilator is activated by the sympathetic nervous system, whereas the sphincter and ciliary muscles are controlled by the parasympathetic nervous system (through the oculomotor nerve; see

1	Light enters the eye through the cornea and passes through a series of transparent fluids and structures that are collectively called the dioptric media. These fluids and structures consist of the cornea, aqueous humor, lens, and vitreous humor (see Fig. 8.1 ). The aqueous humor (located in the anterior and posterior chambers) and the vitreous humor (located in the space behind the lens) help maintain the shape of the eye. Although the geometrical optic axis of the human eye passes through the nodal point of the lens and reaches the retina at a point between the fovea and the optic disc (see Fig. 8.1 ), the eyes are oriented by the oculomotor system to a point, called the fixation point, on the visual target. Light from the fixation point passes through the nodal point of the lens and is focused on the fovea. Light from the remainder of the visual target falls on the retina surrounding the fovea.

1	Normally, light from a visual target is focused sharply on the retina by the cornea and lens, which bend or refract the light. The cornea is the major refractive element of the eye, with a refractive power of 43 diopters (D). However, unlike the cornea, the lens can change shape and vary its refractive power between 13 and 26 D. Thus the lens is responsible for adjusting the optical focus of the eye. Suspensory ligaments (or zonule fibers) attach to the wall of the eye at the ciliary body (see Fig. 8.1 ) and hold the lens in place. When the muscles in the ciliary body are relaxed, the tension exerted by the suspensory ligaments flattens the lens. When the ciliary muscles contract, the tension on the suspensory ligaments is reduced; this process allows the somewhat elastic lens to assume a more spherical shape. The ciliary muscles are activated by the parasympathetic nervous system via the oculomotor nerve.

1	In this way, the lens allows the eye to focus on, or accommodate to, either near or distant objects. For instance, when light from a distant visual target enters a normal eye (one with a relaxed ciliary muscle), the target image is in focus on the retina. However, if the eye is directed at a nearby visual target, the light is initially focused behind the retina (i.e., the image at the retina is blurred) until accommodation aA diopter is a unit of measurement of optical power that is equal to the reciprocal of the focal length measured in meters. Thus it is a unit of reciprocal length, and a 2-D lens would bring parallel rays of light into focus at a distance of 0.5 m. CHAPTER 8 The Special Senses occurs; that is, until the ciliary muscle contracts, causing the lens to become more spherical, the increased convexity causes the lens to refract the light waves more strongly, bringing the image into focus on the retina.

1	Proper imaging of light on the retina depends not only on the lens and cornea but also on the iris, which adjusts the amount of light that can enter the eye through the pupil. In this regard, the pupil is analogous to the aperture in a camera, which also controls the depth of field of the image and the amount of spherical aberration produced by the lens. When the pupil is constricted, the depth of field is increased, and the light is directed through the central part of the lens, where spherical aberration is minimal. Pupillary constriction occurs reflexively when the eye accommodates for near vision or adapts to bright light, or both. Thus when a person reads or does other fine visual work, the quality of the image is improved by adequate light. Layers of the Retina The 10 layers of the retina are shown in Fig. 8.2 . The outermost portion is the pigmented epithelium (layer 1), which is just inside the choroid. The pigment cells have

1	Layers of the Retina The 10 layers of the retina are shown in Fig. 8.2 . The outermost portion is the pigmented epithelium (layer 1), which is just inside the choroid. The pigment cells have Asanindividualages,theelasticityofthelensgraduallydeclines.Asaresult,accommodationofthelensfornearvisionbecomesprogressivelylesseffective,aconditioncalledpresbyopia. Ayoungpersoncanchangethepowerofthelensbyasmuchas14D.However,bythetimethatapersonreaches40yearsofage,theamountofaccommodationhalves,andafter50years,itcandecreaseto2Dorless.Presbyopiacanbecorrectedbyconvexlenses.

1	Defectsinfocuscanalsobecausedbyadiscrepancybetweenthesizeoftheeyeandtherefractivepowerofthedioptricmedia.Forexample,inmyopia (near-sightedness),theimagesofdistantobjectsarefocusedinfrontoftheretina.Concavelensescorrectthisproblem.Conversely,inhypermetropia (far-sightedness),theimagesofdistantobjectsarefocusedbehindtheretina;thisproblemcanbecorrectedwithconvexlenses.Inastigmatism, anasymmetryexistsintheradiiofcurvatureofdifferentmeridiansofthecorneaorlens(orsometimesoftheretina).Astigmatismcanoftenbecorrectedwithlensesthatpossesscomplementaryradiiofcurvature.

1	•Fig. 8.2 Layers of the Retina. Lightimpingingontheretinawouldbecomingfromthetopofthefigure,andwouldpassthroughallthesuperficiallayerstoreachthephotoreceptorrodsandcones.9 Nerve fiber layer Axons at surface of retina passing via optic nerve, chiasm and tract to lateral geniculate body Ganglion cell Bipolar cell Amacrine cell Horizontal cell Rod Cone Choroid Pigment cells Müller cell (supporting glial cell) 8 Ganglion cell layer 7 Inner plexiform layer 5 Outer plexiform layer 6 Inner nuclear layer 4 Outer nuclear layer 2 Photoreceptor layer 1 Pigment epithelium 3 Outer limiting membrane 10 Inner limiting membrane Inner segment Outer segment tentacle-like processes that extend into the photoreceptor layer (layer 2) and surround the outer segments of the rods and cones. These processes prevent transverse scatter of light between photoreceptors. In addition, they serve a mechanical function in maintaining contact between layers 1 and 2 so that the pigmented epithelium can (1) provide

1	transverse scatter of light between photoreceptors. In addition, they serve a mechanical function in maintaining contact between layers 1 and 2 so that the pigmented epithelium can (1) provide nutrients and remove waste from the photoreceptors; (2) phagocytose the ends of the outer segments of the rods, which are continuously shed; and (3) reconvert metabolized visual pigment into a form that can be reused after it is transported back to the photoreceptors.

1	Retinal glial cells, known as Müller cells, play an important role in maintaining the internal geometry of the retina. Müller cells are oriented radially, parallel to the light path through the retina. The outer ends of Müller cells form tight junctions with the inner segments of the photoreceptors, and these numerous connections have the appearance of a continuous layer, the outer limiting membrane (layer 3 of the retina).

1	Thejunctionbetweenlayers1and2oftheretinainadultsrepresentsthesurfaceofcontactbetweentheanteriorandposteriorwallsoftheembryonicopticcupduringdevelopmentandisstructurallyweak.Retinaldetachmentisseparationatthissurfaceandcancauselossofvisionbecauseofdisplacementoftheretinafromthefocalplaneoftheeye.Itcanalsoleadtothedeathofphotoreceptorcells,whicharemaintainedbythebloodsupplyofthechoroid(thephotoreceptorlayeritselfisavascular).Deteriorationofthepigmentedepitheliumcanalsoresultinmaculardegeneration,acriticallossofhigh-acuitycentralandcolorvisionthatdoesnotaffectperipheralvision.

1	Inside the external limiting membrane is the outer nuclear layer (layer 4) that contains the cell bodies and nuclei of the rods and cones. The outer plexiform layer (layer 5) contains synapses between the photoreceptors and retinal interneurons, including bipolar cells and horizontal cells, whose cell bodies are found in the inner nuclear layer (layer 6). This layer also contains the cell bodies of other retinal interneurons (the amacrine and interplexiform cells) and the Müller cells.

1	The inner plexiform layer (layer 7) contains synapses between the retinal neurons of the inner nuclear layer, including the bipolar and amacrine cells, and the ganglion cells, whose cell bodies lie in the ganglion cell layer (layer 8). As previously mentioned, the ganglion cells are the output cells of the retina; it is their axons that transmit visual information to the brain. These axons form the optic fiber layer (layer 9), pass along the inner surface of the retina while avoiding the fovea, and enter the optic disc, where they leave the eye as the optic nerve. The portions of the ganglion cell axons that are in the optic fiber layer remain unmyelinated, but they become myelinated after they reach the optic disc. The lack of myelin where the Folding of membrane •Fig. 8.3 Rods and Cones. Thedrawingsatthebottom showthegeneralfeaturesofarodandacone.Theinsets showtheoutersegments. axons cross the retina helps permit light to pass through the inner retina with minimal distortion.

1	axons cross the retina helps permit light to pass through the inner retina with minimal distortion. The innermost layer of the retina is the inner limiting membrane (layer 10) formed by the end-feet of Müller cells. Structure of Photoreceptors: Rods and Cones

1	Structure of Photoreceptors: Rods and Cones Each rod or cone photoreceptor cell is composed of a cell body (in layer 4), an inner and an outer segment that extend into layer 2, and a set of synaptic terminals that synapse in layer 5 onto other retinal cells (Fig. 8.3 ). The outer segments of cones are not as long as those of rods, and they contain stacks of disc membranes formed by infoldings of the plasma membrane. The outer segments of rods are longer, and they contain stacks of membrane discs that float freely in the outer segment, having completely disconnected from the plasma membrane when formed at the base. Both sets of discs are rich in visual pigment molecules, but rods have a greater visual pigment density, which partly accounts for their greater sensitivity to light. A single photon can elicit a rod response, whereas several hundred photons may be required for a cone response.

1	The outer segments of the photoreceptors are connected by a modified cilium to the inner segments, which contain a number of organelles, including numerous mitochondria. The inner segments are the sites where the visual pigment is synthesized before it is incorporated into the membranes of the outer segment. In rods, the pigment is inserted into new membranous discs, which are then displaced distally until they are eventually shed at the apex of the outer segment, where they undergo phagocytosis by cells of the pigmented

1	CHAPTER 8 The Special Senses •Fig. 8.4 Graph of a Plot of the Density of Cones and Rods as a Function of Retinal Eccentricity From the Fovea. Notethatconedensitypeaksatthefovea,roddensitypeaksatabout20degreeseccentricity,andnophotoreceptorsarefoundattheopticdisc,wheretheganglioncellaxonsleavetoformtheopticnerve.(DatafromCornsweetTN.Visual Perception. NewYork:AcademicPress;1970.) epithelium. This process determines the rod-like shape of the outer segments of rods. In cones, the visual pigment is inserted randomly into the membranous folds of the outer segment, and shedding, comparable to that seen in rods, does not take place. Regional Variations in the Retina The macula lutea is the area of central vision and is characterized by a slight thickening and a pale color. The thickness is due to the high concentration of photoreceptors and interneurons, which are needed for high-resolution vision. It is pale because both optic nerve fibers and blood vessels are routed around it.

1	The fovea, which is a depression in the macula lutea, is the region of the retina with the very highest visual resolution and, as noted previously, the light from the fixation point is focused on the fovea. (A major function of eye movements is to bring objects of interest into view on the fovea.) The retinal layers in the foveal region are unusual because several of them appear to be pushed aside into the surrounding macula. Consequently, light can reach the foveal photoreceptors without having to pass through the inner layers of the retina, and both image distortion and light loss are minimized. The fovea has cones with unusually long and thin outer segments, which allows for high packing density. In fact, cone density is maximal in the fovea, providing for high visual resolution, as well as high quality of the image ( Fig. 8.4

1	Fig. 8.4 The optic disc, where ganglion cell axons leave the retina, lacks photoreceptors and therefore lacks photosensitivity. Thus it is a so-called blind spot in the visual surface of the retina (see Figs. 8.4 8.9 ). A person is normally unaware of the blind spot because the corresponding part of the visual field can be seen by the contralateral eye and because of the psychological process in which incomplete visual images tend to be completed perceptually.

1	Asmentioned,theaxonsofretinalganglioncellscrosstheretinaintheopticfiberlayer(layer9)toentertheopticnerveattheopticdisc.Theseaxonsintheopticfiberlayerpassaroundthemaculaandfovea,asdothebloodvesselsthatsupplytheinnerlayersoftheretina.Theopticdisccanbevisualizedonphysicalexaminationwithanophthalmoscope. Thenormalopticdischasaslightdepressioninitscenter.Changesintheappearanceoftheopticdiscareimportantclinically.Forexample,thedepressionmaybeexaggeratedbylossofganglioncellaxons(optic atrophy), ortheopticdiscmayprotrudeintothevitreousspacebecauseofedema(papilledema) thatresultsfromincreasedintracranialpressure.

1	To be detected by the retina, light energy must be absorbed. This is the job primarily of the rods and cones (a small class of ganglion cells are also photosensitive) and is accomplished by visual pigment molecules located in their outer segments. For both rods and cones, the pigment molecule consists of a chromophore, 11-cis retinal, bound to an opsin protein. The visual pigment found in the outer segments of rods is rhodopsin, or visual purple (so named because it has a purple appearance when light has been absorbed). It absorbs light best at a wavelength of 500 nm. Three variants of visual pigment, resulting from the binding of different opsins to retinal, are found in cones (in most species, each cone expresses one of the three cone pigments). The cone pigments absorb best at 419 nm (blue), at 533 nm (green), and at 564 nm (red). However, the absorption spectrum of these visual pigments is broad so that they overlap considerably ( Fig. 8.5).

1	Fig. 8.5). •Fig. 8.5 The Spectral Sensitivity of the Three Types of Cone Pigments and of the Rod Pigment (Rhodopsin) in the Human Retina. Notethatthecurvesoverlapandthattheso-calledblueandredconesactuallyabsorbmaximallyinthevioletandyellowranges,respectively.(DatafromSquireLR,etal[eds].Fundamental Neuroscience. SanDiego,CA:AcademicPress;2002.) Despite the differences in spectral sensitivity, the transduction process is similar in rods and cones. The absorption of a photon by a visual pigment molecule leads to the isomerization of 11-cis retinal to all-trans retinal, release of the bond with the opsin, and conversion of retinal to retinol. These changes trigger a second messenger cascade that leads to a change in the electrical activity of the rod or cone (discussed later in this section).

1	The separation of all-trans retinal from opsin also causes both the loss of its ability to absorb light and bleaching (i.e., the visual pigment loses its color). In both rods and cones, regeneration of the visual pigment molecule is a multistep process: the all-trans retinal is transported to the retinal pigmented cell layer, where it is reduced to retinol, isomerized, and esterified back to 11-cis retinal. It is then transported back to the photoreceptor layer, taken up by outer segments, and recombined with opsin to regenerate the visual pigment molecule, which can again absorb light. There is evidence that cones also use a second pathway to regenerate visual pigment. This pathway is much more rapid and involves transport of the retinal molecule to and from the Müller cells (see Fig. 8.2 ) rather than the pigmented epithelial cells. The potential importance of this more rapid pathway is discussed in the section “

1	Fig. 8.2 ) rather than the pigmented epithelial cells. The potential importance of this more rapid pathway is discussed in the section “ Ultimately, the transduction process triggered by absorption of photons causes the photoreceptor to hyperpolarize. To understand this action and its consequences fully, it is necessary to know the baseline state of the photoreceptor in the dark (i.e., before it absorbs a photon). In darkness, photoreceptors are slightly depolarized (≈−40 mV) in relation to most neurons because cyclic guanosine monophosphate (cGMP)–gated cation channels in their outer segments are open (Fig. 8.6A ). These channels allow a steady influx of Na+ and Ca2+ . The resulting current is known as the dark current, and the depolarization it causes leads to the tonic release of the neurotransmitter glutamate at the photoreceptor’s synapses.

1	When light is absorbed in a rod (an equivalent sequence happens in cones), photoisomerization of rhodopsin activates a G protein called transducin (see Fig. 8.6B ). This G protein, in turn, activates cyclic guanosine monophosphate phosphodiesterase, which is associated with the rhodopsin-containing discs, hydrolyzes cGMP to 5′-GMP, and lowers the cGMP concentration in the rod cytoplasm. The reduction in cGMP leads to closing of the cGMP-gated cation channels, hyperpolarization of the rod cell membrane, and a reduction in the release of neurotransmitters. Thus cGMP acts as a “second messenger” to translate the absorption of a photon by rhodopsin into a change in membrane potential.

1	In sum, in all photoreceptors (cones undergo a process analogous to that described for rod transduction), capture of light energy leads to (1) hyperpolarization of the photoreceptor and (2) a reduction in the release of neurotransmitters. Because of the very short distance between the site of transduction and the synapse, the modulation of neurotransmitter release is accomplished without the generation of an action potential. Adaptation refers to the ability of the retina to adjust its sensitivity according to ambient light. This ability allows the retina to operate efficiently over a wide range of lighting conditions, and it reflects a switching between the use of the cone and rod systems for brightand low-light conditions, respectively.

1	CHAPTER 8 The Special Senses •Fig. 8.6 A, Drawingofarodwiththeflowofcurrentinthedark.WiththeassistanceoftheNa+,K+pump,therodiskeptdepolarized.B, SequenceofthesecondmessengereventsthatfollowtheabsorptionoflightthroughthereductionofcGMP.BecausecGMPmaintainsopenNa+channelsinthedark,theresultsoflightabsorptionaretheclosingoftheNa+channelsandhyperpolarizationoftherod.cGMP,cyclicguanosinemonophosphate;GC,guanylatecyclase;GTP,guanosinetriphosphate;PDE,phosphodiesterase;Rh,rhodopsin;T,transducin.Passive Na+ influx Na+ current Rh T + + + – PDE GC GMP Light Disc A B cGMP GTP Hyperpolarization Membrane Membrane Na+ Na+ Ca++ K+ Active Na+ efflux

1	Rhodopsincontainsachromophorecalledretinal, whichisthealdehydeofretinol, orvitaminA.Retinolisderivedfromcarotenoids,suchasβ-carotene,theorangepigmentfoundincarrots.Likeothervitamins,retinolcannotbesynthesizedbyhumans;instead,itisderivedfromfoodsources.IndividualswithaseverevitaminAdeficiencysufferfrom“nightblindness,”aconditioninwhichvisionisdefectiveinlow-lightsituations. Theextraordinarysensitivityofrods,whichcansignalthecaptureofasinglephoton,isenhancedbyanamplificationmechanisminwhichphotoactivationofonlyonerhodopsinmoleculecanactivatehundredsoftransducinmolecules.Inaddition,eachphosphodiesterasemoleculehydrolyzesthousandsofcGMPmoleculespersecond.Similareventsoccurincones,butthemembranehyperpolarizationoccursmuchmorequicklythaninrodsandrequiresthousandsofphotons.

1	As described previously, absorption of a photon causes 11-cis retinal to be converted to all-trans retinal, which then splits from the opsin (bleaching). The visual pigments in rods and cones are bleached at a similar rate; however, regeneration of the visual pigment occurs much more rapidly in cones than in rods. This difference is, at least in part, due to the cones’ ability to utilize a second pathway for regeneration (see previous section). This more rapid regeneration of visual pigment prevents cones from becoming unresponsive in bright light conditions. In contrast, the slowness of the regeneration of rhodopsin molecules means that at light levels not much above those found in evening hours, essentially all of the rhodopsin molecules are bleached. Thus in bright-light conditions, only the cone system is functioning, and the retina is said to be light-adapted.

1	When entering a darkened movie theater, a person can observe evidence of the existing light adaptation (decreased light sensitivity in association with the reduced amount of rhodopsin) in the inability to see the empty seats (or much else). The gradual return of the ability to see the seats while the person remains in the theater reflects the slow regeneration of rhodopsin and recovery of function of the rod system, a process known as dark adaptation.

1	This process refers to the gradual increase in light sensitivity of the retina when in low-light conditions. Rods adapt to darkness slowly as their rhodopsin levels are restored, and, indeed, it may take more than 30 minutes for the retina to become fully dark-adapted. In contrast, cones adapt rapidly to darkness, but their adapted threshold is relatively high, and so they do not function when the ambient light level is low. Thus within 10 minutes in a dark room, rod vision is more sensitive than cone vision and becomes the main system for seeing.

1	In sum, in the dark-adapted state, primarily rod vision is operative, and thus visual acuity is low and colors are not distinguished (this is called scotopic vision). However, when light levels are higher (e.g., when the movie is projected) and cone function resumes (this is called photopic vision), visual acuity and color vision are restored. There is an intermediate range of light levels at which rod and cones are both functional (mesopic vision). The visual pigments in the cone outer segments contain different opsins. As a result of these differences, the three types of cones absorb light best at different wavelengths. Although the cone pigments have maximum efficiency closer to violet, green, and yellow wavelengths, they are referred to as blue, green, and red pigments, respectively (see Fig. 8.5). The differences in the cone absorption spectra underlie humans’ ability to see colors, as opposed to only shades of gray.

1	Fig. 8.5). The differences in the cone absorption spectra underlie humans’ ability to see colors, as opposed to only shades of gray. According to the trichromacy theory, the differences in absorption efficiency of the cone visual pigments are presumed to account for color vision because a suitable mixture of three colors can produce any other color. However, a neural mechanism must also exist for the analysis of color brightness because the amount of light absorbed by a visual pigment, as well as the subsequent response of the cell, depends on both the wavelength and the intensity of the light (see

1	Fig. 8.5 ). Two or three of the cone pigments may absorb a particular wavelength of light, but the amount absorbed by each differs according to its efficiency at that wavelength. If the intensity of the light is increased (or decreased), all will absorb more (or less), but the ratio of absorption among them will remain constant. Consequently, there must be a neural mechanism to compare the absorption of light of different wavelengths by the different types of cones for the visual system to distinguish different colors. At least two different kinds of cones are required for color vision. The presence of three kinds decreases the ambiguity in distinguishing colors when all three absorb light, and it ensures that at least two types of cones will absorb most wavelengths of visible light.

1	The opponent process theory is based on observations that certain pairs of colors seem to activate opposing neural processes. Green and red are opposed, as are yellow and blue, as well as black and white. For example, if a gray area is surrounded by a green ring, the gray area appears to acquire a reddish color. Furthermore, a greenish red or a bluish yellow color does not exist. These observations are supported by findings that neurons activated by green wavelengths are inhibited by red wavelengths. Similarly, neurons excited by blue wavelengths may be inhibited by yellow wavelengths. Neurons with these characteristics are present both in the retina and at higher levels of the visual pathway and seem to serve to increase the ability to see the contrast between opposing colors. A diagram of the basic circuitry of the retina is shown in

1	A diagram of the basic circuitry of the retina is shown in Fig. 8.7 . Several features of this circuitry are noteworthy. Input to the retina is provided by light striking the photoreceptors. The output is carried by axons of the retinal ganglion cells to the brain. Information is processed within the retina by the interneurons. The most direct pathway through the retina is from a photoreceptor to a bipolar cell and then to a ganglion cell (see Fig. 8.7). More indirect pathways that provide for intraretinal signal processing involve photoreceptors, bipolar cells, amacrine cells, and ganglion cells, as well as horizontal cells to provide lateral interactions between adjacent pathways.

1	Observationsoncolorblindnessareconsistentwiththetrichromacytheory.Incolorblindness,ageneticdefect(sex-linkedrecessive),oneormoreconemechanismsarelost.Peoplewithnormalcolorvisionaretrichromatsbecausetheyhavethreeconemechanisms.Individualswholackoneoftheconemechanismsarecalleddichromats.Whenthelong-wavelengthconemechanismisabsent,theresultingconditioniscalledprotanopia;absenceofthemedium-wavelengthsystemcausesdeuteranopia;andabsenceoftheshort-wavelengthsystemcausestritanopia.Monochromatslacktwoormoreconemechanisms.

1	Rod and cone pathways have several important functional differences in their phototransduction mechanisms and their retinal circuitry. As described previously, rods have more visual pigment and a better signal amplification system than cones do, and there are many more rods than cones. Thus rods function better in dim light (scotopic vision), and loss of rod function results in night blindness. In addition, all rods contain the same visual pigment, so they cannot signal color differences. Furthermore, many rods converge onto individual bipolar cells and the results are very large receptive fields and low spatial resolution. Finally, in bright light, most rhodopsin is bleached, so that rods no longer function under photopic conditions.

1	Cones have a higher threshold to light and thus are not activated in dim light after dark adaptation. However, they operate very well in daylight. They provide high-resolution vision because only a few cones converge onto individual bipolar cells in cone pathways. Moreover, no convergence occurs in the fovea, where the cones make one-to-one connections to bipolar cells. As a result of the reduced convergence, cone pathways have very small receptive fields and can resolve stimuli that originate from sources very close to each other. Cones also respond to sequential stimuli with good temporal resolution. Finally, cones have three

1	CHAPTER 8 The Special Senses •Fig. 8.7 Basic Retinal Circuitry. Thearrow attheleftindicatesthedirectionoflightthroughtheretina.Photoreceptors(R)synapseonthedendritesofbipolarcells(B)andhorizontalcells(H)intheouterplexiformlayer.Thehorizontalcellsmakereciprocalsynapticconnectionswithphotoreceptorcellsandareelectricallycoupledtootherhorizontalcells.Bipolarcellsreachsynapseonthedendritesofganglioncells(G)andontheprocessesofamacrinecells(A)intheinnerplexiformlayer.Amacrinecellsconnectwithganglioncellsandotheramacrinecells. different visual pigments and therefore provide for color vision. Loss of cone function results in functional blindness; rod vision is not sufficient for normal visual requirements.

1	different visual pigments and therefore provide for color vision. Loss of cone function results in functional blindness; rod vision is not sufficient for normal visual requirements. The receptive field of an individual photoreceptor is circular. Light in the receptive field hyperpolarizes the photoreceptor cell and cause it to release less neurotransmitter. The receptive fields of photoreceptors and retinal interneurons determine the receptive fields of the retinal ganglion cells onto which their activity converges. The characteristics of the receptive fields of retinal ganglion cells constitute an important step in visual information processing because all the information about visual events that is conveyed to the brain is contained in ganglion cell activity. The bipolar cell, which receives input from a photoreceptor, can have either of two types of receptive fields, as shown in Fig. 8.8.

1	The bipolar cell, which receives input from a photoreceptor, can have either of two types of receptive fields, as shown in Fig. 8.8. Both are described as having a center-surround organization in which the light that strikes the central region of the receptive field either excites or inhibits the cell, whereas the light that strikes a region that surrounds the central portion has the converse effect. The receptive field with a centrally located excitatory region surrounded by an inhibitory annulus is called an on-center, off-surround receptive field (see Fig. 8.8A ). Bipolar cells with such a receptive field are described as “on” bipolar cells. The other type of receptive field has an off-center, on-surround arrangement, which characterizes “off” bipolar cells (see Fig. 8.8F

1	Fig. 8.8F The center response of a bipolar cell receptive field is due to only the photoreceptors that directly synapse with the bipolar cell. Photoreceptor cells respond to light with hyperpolarization and a decrease in glutamate release and respond to the removal of light with depolarization and increased glutamate release. This implies that the difference in the center responses of “on” and “off ” bipolar cells lies in their response to glutamate. In fact, off-center bipolar 136 SECTION2Berne & Levy Physiology A On-center field 0 Sec 0.5 1.0 1.5 Central spot of light Light Peripheral spot F Off-center field B On-center cell responses C Central illumination D Annular illumination E Diffuse illumination 0 Sec 0.5 1.0 1.5 H Central illumination I Annular illumination J Diffuse illumination G Off-center cell responses

1	Fig. 8.8 Thereceptivefieldsofon-center(A) andoff-center(F) bipolarcellsand,belowthem,thereceptivefieldsofganglioncellsB throughE andG throughJ towhichtheyareconnected.Ganglioncellresponsestocentralspots(upper recording)andperipheralspots(lower recording)areshowninB and G. Alsoshownareresponsestocentral(C andH),surround(D andG),anddiffusewhole-field(E and

1	J)illuminationintheirreceptivefields.Theganglioncellsandtheon-centerandoff-centerbipolarcellsprovidinginputtotheseganglioncellshavesimilarreceptivefields,butwhereasganglioncellsincreaseordecreasetheirspikefrequency,bipolarcellsdepolarizeorhyperpolarize,withoutgeneratingactionpotentials.(RedrawnfromSquireLRetal[eds].Fundamental Neuroscience. SanDiego,CA:AcademicPress;2002.) cells have ionotropic glutamate receptor channels that open in response to glutamate, and thus they are excited by the removal of light stimuli from the center of their receptive field. In contrast, on-center bipolar cells have metabotropic glutamate receptors that close their channels in response to glutamate. They are depolarized by light on the center of their receptive field, because the reduced release of glutamate by the photoreceptors results in more open metabotropic channels. Thus on-center bipolar cells are excited by light stimulation of the center of their receptive fields.

1	The antagonistic surround response of bipolar cells is due to photoreceptors that surround those that synapse directly on them. These photoreceptors (which also connect directly with their own bipolar cells) synapse with horizontal cells that participate in complex triadic synapses with many photoreceptors and bipolar cells. The pathway through •Fig. 8.9 Relationshipsamongavisualtarget(long arrow, top),imagesontheretinasofthetwoeyes(middle), andprojectionsoftheganglioncellscarryingvisualinformationabouttheseimages(bottom). Thetargetimageissolargethatitextendsintothemonocularsegmentsoftheeyes,whereonesideofitisseenbyonlytheipsilateraleye.Notehowtheaxonsaresortedinthechiasmsothatallinformationabouttheleftvisualfieldofbotheyesisconveyedtotherightsideofthebrainandallinformationabout therightvisualfieldisconveyedtotheleftside.

1	continued object movement within the field will not be signaled. P cells respond differently to different wavelengths of light. Because there are blue, green, and red cones, many combinations of color properties are possible, but in fact P cells have been shown to have opposing responses only to red and green or only to blue and yellow (a combination of red and green). These mechanisms can greatly reduce the ambiguity of color detection caused by the overlap in cone color sensitivity and may provide a substrate for the opponency process observations. M cells, on the other hand, respond with phasic bursts of action potentials to the redistribution of light, such as would be caused by the movement of an object within their large receptive fields. M cells are not sensitive to differences in wavelength but are more sensitive to luminance than P cells are.

1	Thus the output of the retina consists primarily of ganglion cell axons from (1) sustained, linear P cells with small receptive fields that convey information about color, form, and fine details and (2) phasic, nonlinear M cells with larger receptive fields that convey information about illumination and movement. Both exist in on-center and off-center varieties ( Fig. 8.8 The Visual Pathway Retinal ganglion cells transmit information to the brain by way of the optic nerve, optic chiasm, and optic tract. Fig. 8.9 shows the relationships among a visual target, the retinal images of the target in the two eyes, and the projections of retinal ganglion cells to the two hemispheres of the brain. The eyes and the optic nerves, chiasm, and tract are viewed from above. The visual target, an arrow, is in the visual fields of both eyes (see

1	The visual target, an arrow, is in the visual fields of both eyes (see Fig. 8.9 ) and, in this case, is so long that it extends into the monocular segments of each retina (i.e., one end of the target can be seen by only one eye and the other end by only the other eye). The shaded circle at the center of the target represents the fixation point. The image of the target on the retinas is reversed by the lens system. The left CHAPTER 8 The Special Senses half of the visual target is imaged on the nasal retina of the left eye and the temporal retina of the right eye. Thus the left visual field is seen by the left nasal retina and the right temporal retina. Similarly, the right half of the visual target is imaged on and seen by the left temporal retina and the right nasal retina. The lens system also causes an inversion in the vertical axis, with the upper visual field imaged on the lower retina and vice versa.

1	The axons of retinal ganglion cells may or may not cross in the optic chiasm, depending on the location of the ganglion cell in the retina (see Fig. 8.9 ). Axons from the temporal portion of each retina pass through the optic nerve, the lateral side of the optic chiasm, and the ipsilateral optic tract and terminate ipsilaterally in the brain. Axons from the nasal portion of each retina pass through the optic nerve, cross to the opposite side in the optic chiasm, and then pass through the contralateral optic tract to end in the contralateral side of the brain. As a result of this arrangement, objects in the left field of vision are represented in the right side of the brain, and those in the right field of vision are represented in the left side of the brain.

1	Retinal ganglion cell axons can synapse in several parts of the brain, but the main target for vision is the lateral geniculate nucleus (LGN) of the thalamus. There is a point-to-point projection from the retina to the LGN. The LGN thus has a retinotopic map. Cells that represent a particular retinal location are aligned along projection lines that can be drawn across the layers of the LGN. The projection from each eye is distributed to three of the layers of the LGN, one of the magnocellular layers (layers 1 and 2 receive M cell input) and two of the parvocellular layers (layers 3 to 6 receive P cell input). Color-coded ganglion cells project to groups of cells between the major layers, the intralaminar zones. Thus the properties of LGN neurons are very similar to those of retinal ganglion cells. For example, LGN neurons can be classified as P or M cells, and they have on-center or off-center receptive fields.

1	The LGN also receives input from the visual areas of the cerebral cortex, the thalamic reticular nucleus, and several nuclei of the brainstem reticular formation. The activity of LGN projection neurons is inhibited by interneurons both in the LGN and in the thalamic reticular nucleus. These cells use GABA as their inhibitory neurotransmitter. In addition, the activity of LGN neurons is influenced by corticofugal pathways and by brainstem neurons that transmit signals via monoamine neurotransmitters. These control systems filter visual information and may be important for selective attention. The LGN projects to the primary visual cortex or striate cortex by way of the visual radiations. The visual radiation fibers carrying information derived from the lower half of the appropriate hemiretinas (and therefore the contralateral upper visual field) project to the lingual gyrus, which lies Fig.

1	Fig. 8.9 ).Forexample,atinylesionintheretinawouldresultinablindspot(scotoma) inthateye,whereasasimilarlesioninthestriatecortexwouldproducecorrespondingscotomasinbotheyes.Interruptionoftheopticnerveononesideproducesblindnessinthateye.Damagetotheopticnervefibersastheycrossintheopticchiasmresultsinlossofvisioninbothtemporalfieldsofvision;thisconditionisknownasbitemporal hemianopsia andoccursbecausethecrossingfibersoriginatefromganglioncellsinthenasalhalvesofeachretina.Alesionoftheentireoptictract,LGN,visualradiation,orvisualcortexononesidecauseshomonymous hemianopsia, whichislossofvisionintheentirecontralateralvisualfield.Partiallesionsresultinpartialvisualfielddefects.Forexample,alesioninthelingualgyruscausesanupperhomonymous quadrantanopsia, whichinthiscaseislossofvisioninthecontralateral,uppervisualfield.

1	on the medial surface of the occipital lobe, just below the calcarine sulcus. Axons in the visual radiation that represent the contralateral lower visual field project to the adjacent cuneus gyrus, which lies just above the calcarine sulcus. Together, the portions of these two gyri that line and border the calcarine sulcus constitute the primary visual cortex (or Brodmann area 17; Fig. 8.10 Like the LGN, the striate cortex contains a retinotopic map. The representation of the macula occupies the most posterior and largest part of both gyri, and progressively more peripheral areas of the retina are projected to more anterior parts of these gyri. Overall, there is an orderly mapping of retinal loci across the surface of the striate cortex (see Fig. 8.10 The geniculostriate pathway ends chiefly in layer 4 of the striate cortex (

1	Fig. 8.10 The geniculostriate pathway ends chiefly in layer 4 of the striate cortex ( Fig. 8.11 ), whereas the projection from the intralaminar LGN terminates in so-called blobs in layers 2 and 3. Similarly, axons that represent one eye or the other terminate within layer 4C in alternate adjacent patches that define ocular dominance columns. Cortical neurons in such a column respond preferentially to input from one eye. Near the border between two ocular dominance columns, neurons respond about equally to input from the two eyes.

1	The receptive fields of neurons in the striate cortex, aside from the monocular cells in layer 4C, are more complex than those of LGN neurons. Neurons in other layers may be binocular and respond to stimulation of both eyes, although the input from one eye often dominates (see ). In addition, cortical neurons outside layer 4C often show orientation selectivity (i.e., they respond best when the stimulus, such as a bar or an edge, is oriented and positioned in a particular way;

1	Fig. 8.12 ). These “simple cells” appear to be responding as though they received input from cells whose concentric center-surround receptive fields were arranged in such a way that their “on” centers were aligned in a row flanked by antagonistic regions. “Complex” cortical •Fig. 8.10 Theleftvisualfieldisrelayed(viatheLGNandvisualradiation)totheprimaryvisualcortexoftherighthemisphere,asapoint-to-pointretinotopicmap.Therepresentationofeachpartofvisualspaceisproportionaltothenumberofafferentaxonswithreceptivefieldsinthatpartofspace.Asaresult,theareaofmacularrepresentation(neartheoccipitalpole)islargerthanthatfortherestofthebinocularandmonocularfields.Notethatthelowerhalfofthefieldisrepresentedinthecuneusgyrusabovethecalcarinesulcusandtheupperhalfofthefieldinthelingualgyrusbelowthesulcus.(RedrawnfromPurvesD,etal[eds].Neuroscience. 3rded.Sunderland,MA:Sinauer;2004.) •Fig. 8.11

1	3rded.Sunderland,MA:Sinauer;2004.) •Fig. 8.11 DiagramofvisualinformationflowintothevisualcortexfromtheLGNanditsprojectiontotheextrastriatecortex,tothesuperiorcolliculus(SC),andbacktotheLGN.M,magnocellularpath;P,parvocellularpath.(RedrawnfromSquireLR,etal[eds].Fundamental Neuroscience. SanDiego,CA:AcademicPress;2002.)LGN m cell LGN p cell LGN intralaminar p cell Cortex Cortex SC LGN 4B 2+3 5 6 4C˜4C°Blobs (2+3) neurons are similar to “simple” cells in that they are orientation specific, but instead of having flanking excitatory and inhibitory zones, they respond best to a particular stimulus orientation anywhere in their receptive field. They may also display direction selectivity; that is, they may respond when the stimulus is moved in one direction but not when it is moved in the opposite direction (see

1	Fig. 8.12 ). The receptive field of a “complex” cell may be thought of as a composite of adjacent “simple” cells with similar orientation selectivity. Because such neurons in a particular zone of the cortex all tend to have the same orientation selectivity, they are considered to form an orientation column (Fig. 8.13 As already discussed, color vision may depend on the presence in the retina of three different types of cones, as well as neurons in the visual pathway that show spectral opposition. Retinal ganglion cells, LGN neurons, and some P cells display spectral opponent properties. The spectral opponent neurons in the striate cortex are found in cortical blobs, and these show double-opponency, in which both the center and the surround portions respond antagonistically

1	CHAPTER 8 The Special Senses •Fig. 8.12 Simple and Complex Receptive Fields in the Visual Cortex Can Be Generated From Multiple Inputs With Concentric Fields. A andB representon-centerandoff-centerinput,respectively,fromtheretina.Ifthreeon-centercells(A) withadjacentreceptivefieldsconvergedontoonecorticalneuron(E), thatneuron,asimplecell,wouldrespondbesttoalongbarstimulusataspecificlocationandorientation(C). Forthreeoff-centerinputs(B), theresultingreceptivefieldisshowninD. Theconvergenceofmultiplesimplecellsontoanothercorticalneuron(F) wouldresultinacomplexcellthatrespondsbesttoabarstimuluswithaverticalorientationthatcanbeplacedanywherewithinitsreceptivefield.(RedrawnfromSquireLR,etal[eds].Fundamental Neuroscience. SanDiego,CA:AcademicPress;2002.) to two colors. Such a cell, whose center responds to red but not green (R+G−) and whose surround portion responds to green but not red (R−G+), is shown in

1	Fig. 8.13A . The relationships between the ocular dominance and orientation columns and the cortical color blobs are shown in Fig. 8.13B In animal studies, at least 25 different visual areas have been identified in the cerebral cortex, in addition to the striate cortex (Brodmann area 17, or V1). The extrastriate areas include several parallel pathways of visual processing. The P pathway originates with P cells and functions in the recognition of form and color. Structures in the P pathway include LGN layers 3 to 6, layer 4Cβ of the striate cortex, V4 (Brodmann area 19), and several areas in the inferotemporal region ( Fig. 8.14 ). Processing of form includes recognition of complex visual patterns, such as faces. Color information is processed separately from form. The M pathway originates with M cells and functions in motion detection and control of eye movement. Cortical structures in the M pathway include layers 4B and 4Cα of the striate cortex and areas MT (medial temporal) and

1	I Cortical blobs, concerned with color Stimulus duration Orientation columns I C C •Fig. 8.13 A, Thereceptivefieldandresponsesofadouble-opponent(R+G−/R−G+)neuroninablobofthestriatecortexasitrespondstovariouscombinationsofredandgreenbars.Thebest“on”responseistoaredbarflankedbytwogreenbars.B, Diagramofthecolumnararrangementofthevisualcortex.OculardominancecolumnsareindicatedbyI(foripsilateral)andC(forcontralateral).Orientationcolumnsareindicatedbythesmallercolumnsmarkedwithshortbarsatvaryingangles.ThecorticalblobscontainneuronslikethatinA andhavespectralopponent-receptivefields. •Fig. 8.14 DistributionofPandMcellinfluencesondifferentareasofthevisualcortex.IT,inferotemporalarea;MST,medialsuperiortemporalarea;MT,medialtemporalarea;V1,striatecortex;V2andV4,higherordervisualareas.MT7 V4 MST Visual cortex Laminae 3-6 Laminae 1,2 Dorsal LGNM-cell pathway P-cell pathway IT V2 V1

1	MST (medial superior temporal) on the lateral aspect of the temporal lobe, as well as Brodmann area 7a of the parietal lobe (see Fig. 8.14 Both P and M pathways contribute to depth perception or stereopsis, which is dependent on slight differences in the retinal images formed in the two eyes. Stereopsis is useful only for relatively nearby objects. However, in such cases, these disparities provide visual cues about depth. Interestingly, the anatomy of the visual pathways indicates that depth perception must be a cortical function because it depends on convergent input from the two eyes and the left/right eye inputs are parallel but segregated in the LGN and in Layer 4 of striate cortex.

1	The separation of M and P pathways from the retina through the thalamus and all the cortical regions raises the issue of how all the parts are combined to account for the clear, coherent images of events, objects, and persons that humans perceive. It seems unlikely that all the components that represent a percept, such as parts of a face and whether that face belongs to a familiar person, are somehow converged onto a single neuron that recognizes it. Rather, complex percepts probably arise from the coordinated activity of large sets of neurons across multiple regions of the CNS. The process by which a “binding” of such disparate neuronal elements into a percept is unclear, but one working hypothesis is that it may be accomplished by the temporal synchronization of many anatomically distributed neural events.

1	Lesionsoftheextrastriatevisualcortexcanproducevariousdeficits.Bilaterallesionsoftheinferotemporalcortexcanresultincorticalcolorblindness(achromatopsia) orinaninabilitytorecognizefaces,evenofclosemembersofthefamily(prosopagnosia). AlesioninareaMTorMSTcaninterferewithmotiondetectionandeyemovements. CHAPTER 8 The Special Senses •Fig. 8.15 Twopuretonesareshownbythesolid anddashed lines. Frequencyisdeterminedfromthewavelengthasindicated.Amplitudeisthepeak-to-peakchangeinsoundpressure.Thetwotoneshavethesamefrequencyandamplitudebutdifferinphase. The superior colliculus of the midbrain is a layered structure that is important for certain types of eye movements (see ). The three most superficial layers are involved exclusively in visual processing, whereas the deeper layers receive multimodal input from the somatosensory and auditory systems, as well as the visual system, particularly from cortical areas involved in eye movement.

1	Another retinal projection is to the pretectum, which bilaterally activates parasympathetic preganglionic neurons in the Edinger-Westphal nucleus that cause pupillary constriction in the pupillary light reflex. The pretectal areas are also interconnected through the posterior commissure, and thus the reflex causes both ipsilateral (direct) and contra-lateral (consensual) pupillary constriction when a light is shown in one eye. The visual pathways also include connections to nuclei that serve functions other than vision. For example, a retinal projection to the suprachiasmatic nucleus of the hypothalamus controls circadian rhythmicity (see The Auditory and Vestibular Systems

1	The Auditory and Vestibular Systems The peripheral parts of the auditory and vestibular systems share components of the bony and membranous labyrinths, use hair cells as mechanical transducers, and transmit information to the CNS through the vestibulocochlear nerve (CN VIII). However, the CNS processing and sensory functions of the auditory and vestibular systems are distinct. The function of the auditory system is to transduce sound. This allows us to recognize environmental cues and to communicate with other organisms. The most complex auditory functions are those involved in language. The function of the vestibular system is to provide the CNS with information related to the position and movements of the head in space. Control of eye movement by the vestibular system is discussed in

1	Sound is produced by compression and decompression waves in air or in other elastic media, such as water. Sound frequency is measured in cycles per second, or hertz (Hz). Each pure tone results from a sinusoidal wave at a particular frequency and is characterized not only by its frequency but also, instantaneously, by its amplitude and phase ( Fig. 8.15 ). Most naturally occurring sound, however, is a mixture of pure tones. Noise is unwanted sound and may have any composition of pure tones. Sound propagates at about 335 m/sec in air. The waves are associated with certain pressure changes, called sound pressure. The unit of sound pressure is Newtons per meter squared (N/m2), but sound pressure is more commonly expressed as the sound pressure level (SPL). The unit of SPL is the decibel (dB):

1	Equation 8.1 where P is sound pressure and PR is a reference pressure (0.0002 dyne/cm2, the absolute threshold for human hearing at 1000 Hz). A sound with intensity 10 times greater would be 20 dB; one 100 times greater would be 40 dB. The normal young human ear is sensitive to pure tones with frequencies that range between about 20 and 20,000 Hz. The threshold for detection of a pure tone varies with its frequency (

1	The normal young human ear is sensitive to pure tones with frequencies that range between about 20 and 20,000 Hz. The threshold for detection of a pure tone varies with its frequency ( Fig. 8.16 ). The lowest thresholds for human hearing are, for pure tones, approximately 3000 Hz. The threshold at these frequencies is approximately −3 to −5 dB, in comparison with the reference 0 dB at 1000 Hz. In reference to this scale, normal speech has an intensity of about 65 dB, and its main frequencies fall in the range of 300 to 3500 Hz. Sounds that exceed 100 dB can damage the peripheral auditory apparatus, and those higher than 120 dB can cause pain and permanent damage. As people age, their thresholds at high frequencies rise, thereby reducing their ability to hear such tones, a condition called presbycusis.

1	• Fig. 8.16 Sound Threshold Intensities at Different Frequencies. The bottom curve indicates the absolute intensity needed to detect a sound. The dashed curve represents the threshold for functional hearing.Thetop curveindicateslevelsatwhichsoundispainfuland damaging. The Ear The peripheral auditory apparatus is the ear, which can be subdivided into the external ear, the middle ear, and the inner ear (Fig. 8.17 The external ear includes the pinna and the external auditory meatus (auditory canal). The auditory canal contains glands that secrete cerumen, a waxy protective substance. The pinna helps direct sounds into the auditory canal and plays a role in sound localization. The auditory canal transmits the sound pressure waves to the tympanic membrane. In humans, the auditory canal has a resonant frequency of about 3500Hz, and this resonance contributes to the low perceptual threshold for sounds in that range.

1	The external ear is separated from the middle ear by the tympanic membrane (see Fig. 8.17A ). The middle ear contains air. Three ossicles are present and serve to link the tympanic membrane to the oval window of the inner ear. and organ of Corti Scala tympani Scala vestibuli • Fig. 8.17 Ear and Cochlear Structure. A, Location of the right human cochlea in relation to the vestibular apparatus, the middle ear, and the external ear. B,Relationshipsbetweentheouter, middle, and inner ear spaces; the cochlea is depicted unrolled for clarity. CHAPTER 8 The Special Senses 145 • Fig. 8.17, cont’d C, Drawing of a cross-section through the cochlea. The organ of Corti (see Fig. 8.18A and B ) is outlined. Adjacent to the oval window is the round window, another membrane-covered opening between the middle ear and inner ear (see Fig. 8.17A and B

1	Fig. 8.18A and B ) is outlined. Adjacent to the oval window is the round window, another membrane-covered opening between the middle ear and inner ear (see Fig. 8.17A and B The ossicles include the malleus, the incus, and the stapes. The stapes has a footplate that inserts into the oval window. Behind the oval window is a fluid-filled component of the inner ear, the vestibule. It is continuous with a tubular structure known as the scala vestibuli. Inward movement of the tympanic membrane by a sound pressure wave causes the chain of ossicles to push the footplate of the stapes into the oval window (see Fig. 8.17B ). This movement of the stapes footplate in turn displaces the fluid within the scala vestibuli. The pressure wave that ensues within the fluid is transmitted through the basilar membrane of the cochlea to the scala tympani (described later), and it causes the round window to bulge into the middle ear.

1	The tympanic membrane and the chain of ossicles serve as an impedance-matching device. The ear must detect sound waves traveling in air, but the neural transduction mechanism depends on movement in the fluid-filled cochlea, where acoustic impedance is much higher than that of air. Therefore, without a special device for impedance matching, most sound reaching the ear would simply be reflected, as are voices from shore when a person is swimming under water. Impedance matching in the ear depends on (1) the ratio of the surface area of the large tympanic membrane advantage of the lever system formed by the ossicles. This impedance matching is sufficient to increase the efficiency of energy transfer by nearly 30 dB in the range of hearing from 300 to 3500 Hz.

1	The middle ear also serves other functions. Two muscles are found in the middle ear: the tensor tympani attached to the malleus and the stapedius attached to the stapes. When these muscles contract, they damp movements of the ossicles and decrease the sensitivity of the acoustic apparatus. This action can protect the acoustic apparatus against damaging sounds that can be anticipated. However, a sudden explosion can still damage the acoustic apparatus because reflex contraction of the middle ear muscles does not occur quickly enough. The chamber of the middle ear connects to the pharynx through the eustachian tube. Pressure differences between the external ear and middle ear can be equalized through this passage. If fluid collects in the middle ear, as during an infection, the eustachian tube may become blocked. The resulting pressure difference between the external ear and middle ear can produce painful displacement of the tympanic membrane and, in extreme cases, cause rupture of the

1	ube may become blocked. The resulting pressure difference between the external ear and middle ear can produce painful displacement of the tympanic membrane and, in extreme cases, cause rupture of the tympanic membrane. Unequalized pressure changes as a result of flying or diving can also cause discomfort.

1	to that of the smaller oval window and (2) the mechanical The inner ear includes the bony and membranous labyrinths. The bony labyrinth is a complex but continuous series of spaces in the temporal bone of the skull, whereas the membranous labyrinth consists of a series of soft tissue spaces and channels lying inside the bony labyrinth. The cochlea and the vestibular apparatus are formed from these structures. The cochlea is a spiral-shaped organ (see Fig. 8.17A ). In humans, the spiral consists of 23 4 turns from a broad base to a narrow apex, although its internal lumen is small at the base and wide at the top. The apex of the cochlea faces laterally (see Fig. 8.17A ). The bony labyrinth component of the cochlea is subdivided into several chambers. The vestibule is the space facing the oval window (see

1	Fig. 8.17A ). The bony labyrinth component of the cochlea is subdivided into several chambers. The vestibule is the space facing the oval window (see Fig. 8.17A ). Continuous with the vestibule is the scala vestibuli, the spiral-shaped chamber that extends to the apex of the cochlea, where it meets and merges with the scala tympani at the helicotrema. The scala tympani is another spiral-shaped space that winds back down the cochlea and ends at the round window (see Fig. 8.17B ). Separating the two, except at the helicotrema, is the scala media enclosed in the membranous labyrinth. The scala media, or cochlear duct (see Fig. 8.17B and ), is a membrane-bound spiral tube that extends along the cochlea, between the scala vestibuli and scala tympani. One wall of the scala media is formed by the basilar membrane, another by Reissner’s membrane, and the third by the stria vascularis (see Fig. 8.17C

1	Fig. 8.17C The spaces within the cochlea are filled with fluid. The fluid in the bony labyrinth, including the scala vestibuli and scala tympani, is perilymph, which closely resembles cerebrospinal fluid. The fluid in the membranous labyrinth, including the scala media, is endolymph, which is very different from perilymph. Endolymph, generated by the stria vascularis, contains high [K+] (about 145 mM) and low [Na+] (about 2 mm) and has a high positive potential (about +80 mV) with regard to the perilymph. As a result, a very large potential gradient (about 140 mV) exists across the membranes of the hair cell cilia that extend into the endolymph. (These hair cells, which are the sensory receptors for sound, are discussed in more detail later.) The neural apparatus responsible for transduction of sound is the organ of Corti (see

1	The neural apparatus responsible for transduction of sound is the organ of Corti (see Fig. 8.17C ), which is located within the cochlear duct. It lies on the basilar membrane and consists of several components, including three rows of outer hair cells, a single row of inner hair cells, a gelatinous tectorial membrane, and a number of types of supporting cells. The organ of Corti in humans contains 15,000 outer and 3500 inner hair cells. The rods of Corti help provide a rigid scaffold. Located on the apical surface of the hair cells are stereocilia, which can be described as nonmotile cilia that contact the tectorial membrane. The organ of Corti is innervated by nerve fibers of the cochlear division of the vestibulocochlear nerve (CN VIII). The 32,000 auditory afferent fibers in humans originate in sensory ganglion cells in the spiral ganglion. These nerve fibers penetrate the organ of Corti and terminate at the bases of the hair cells ( Fig. 8.18

1	Fig. 8.18 Fig. 8.17C ). Approximately 90% of the fibers end on inner hair cells, and the remainder end on outer hair cells. Thus approximately 10 afferent fibers supply each inner hair cell, whereas other afferent fibers diverge to supply about five outer hair cells each. The inner hair cells clearly provide most of the neural information about acoustic signals that the CNS processes for hearing. The sensory function of the outer hair cells is less clear.

1	In addition to afferent fibers, the organ of Corti is supplied by efferent fibers, most of which terminate on the outer hair cells. These cochlear efferent fibers originate in the superior olivary nucleus of the brainstem and are often called olivocochlear fibers. The length of the outer hair cells varies; this characteristic suggests that changes in outer hair cell length may affect the sensitivity, or “tuning,” of the inner hair cells. The cochlear efferent fibers may control outer hair cell length. Such a mechanism could conceivably influence the sensitivity of the cochlea and the way that the brain recognizes sound. Other efferent fibers that end on cochlear afferent fibers may be inhibitory, and they may help improve frequency discrimination.

1	Sound is transduced by the organ of Corti. Sound waves that reach the ear cause the tympanic membrane to oscillate, and these oscillations are transmitted to the scala vestibuli by the ossicles. This creates a pressure difference between the scala vestibuli and the scala tympani (see Fig. 8.17B ) that serves to displace the basilar membrane and, with it, the organ of Corti (see Fig. 8.18A and B ). Because of the shear forces set up by the relative displacement of the basilar and tectorial membranes, the stereocilia of the hair cells bend. Upward displacement bends the stereocilia toward the tallest cilium, which depolarizes the hair cells; downward deflection bends the stereocilia in the opposite direction, which hyperpolarizes the hair cells.

1	Acommoncauseofdeafnessisthedestructionofhaircellsbyloudsounds.Haircellscanbedestroyed,forexample,byexposuretoindustrialnoiseorbylisteningtoloudmusic.Typically,haircellsincertainpartsofthecochleaareselectivelydamagedbyexposuretohighlevelsofsoundatparticularfrequencies(aspredictedbytheplacetheory),andthushearingmaybelostoveradiscretefrequencyrange.Presbycusis,orthelossofhigh-frequencyhearingwithage,isprobablyincreasedbythelossofhaircellsasaresultoflong-termnoiseexposureinurbanenvironments. In view of the wide range of frequencies and amplitudes of sound stimuli, it is no surprise that hair cell transduction must provide for a fast response. The fast response to deflection of the cilia is based on direct opening of ion channels by so-called tip links that connect the tip of each stereocilium with the shaft of the next taller one (see

1	CHAPTER 8 The Special Senses •Fig. 8.18 DetailoftheorganofCortiatrest(A) andwithupwardmovementofthebasilarmembrane(B). Theupwardmovementcausesthestereociliatobendbecauseofshearforcesproducedbyrelativedisplacementofthehaircellsandthetectorialmembrane.C, Diagramofahaircellwithtiplinkconnectionsbetweenthehaircellciliatoshowhowshearforcesopenmechanoreceptorchannelsand depolarizethehaircell.

1	Fig. 8.18C ). With deflection, the tip links are subjected to a lever action that transiently opens the channels, allows the entry of K+ (because of the high [K+] and high potential in endolymph), and depolarizes the hair cell. Several mechanisms have been proposed to account for the equally important rapid adaptation necessary for a high-frequency response. A “spring” response by the tip links would allow the attachment point of the tip link to be moved along the stereocilium’s shaft to reset the mechanical leverage of the tip link. In addition, it has been observed that Ca++ can enter and bind to the open channel, change it to require greater opening force, and thereby reduce the statistical probability of opening.

1	The potential gradient that induces movement of ions into hair cells includes both the resting potential of the hair cells and the positive potential of the endolymph. As noted previously, the total gradient across the apical membrane of hair cells is about 140 mV. Therefore, a change in K+ conductance in the apical membranes of hair cells results in a rapid current flow that produces the receptor potential in these cells. This current flow can be recorded extracellularly as a cochlear microphonic potential, an oscillatory event that has the same frequency as the acoustic stimulus. The cochlear microphonic potential represents the sum of the receptor potentials of a number of hair cells.

1	Hair cells, like retinal photoreceptors, release an excitatory neurotransmitter (probably glutamate) when depolarized. The neurotransmitter produces an excitatory postsynaptic potential (EPSP) in the cochlear afferent nerve fibers with which the hair cell synapses. In summary, sound is transduced when oscillatory movements of the basilar membrane cause transient changes in the transmembrane voltage of the hair cells and, finally, the generation of action potentials in cochlear afferent nerve fibers. The activity of a large number of cochlear afferent fibers in the auditory nerve can be recorded extracellularly as a compound action potential. On the basis of differences in width and tension, investigators originally concluded that different parts of the basilar membrane have different resonant frequencies.

1	On the basis of differences in width and tension, investigators originally concluded that different parts of the basilar membrane have different resonant frequencies. •Fig. 8.19 Different Frequencies of Sound Result in Different Amplitudes of Displacement at Different Sites Along the Organ of Corti. A, Travelingwaveproducedinthebasilarmembranebyasoundof200Hz.Thecurvesata,b,c,anddrepresentdisplacementofthebasilarmembraneatdifferenttimes,andthedashed line istheenvelopeformedbythepeaksofthewaveatdifferenttimes.Maximumdeflectionoccursatabout29mmfromtheovalwindow.B, Envelopesoftravelingwavesproducedbyseveralfrequenciesofsound.Notethatthemaximumdisplacementvarieswithfrequencyandisclosesttothestapeswhenthefrequencyishighest.(RedrawnfromvonBekesyG.Experiments in Hearing. NewYork:McGraw-Hill;1960.)

1	For example, the basilar membrane is about 100 µm wide at the base and 500 µm wide at the apex. It also has higher tension at the base. Thus, the investigators predicted that the base would vibrate at higher frequencies than would the apex, as do the shorter strings of musical instruments. Experiments have shown that the basilar membrane moves as a whole in traveling waves ( Fig. 8.19 ), but displacement of the basilar membrane is maximal nearer the base of the cochlea during high-frequency tones and maximal nearer the apex during low-frequency tones.

1	Fig. 8.19 ), but displacement of the basilar membrane is maximal nearer the base of the cochlea during high-frequency tones and maximal nearer the apex during low-frequency tones. In effect, the basilar membrane serves as a frequency analyzer; it distributes the stimulus along the organ of Corti, and different hair cells respond differentially to particular 50 Hz frequencies of sound. This is the basis of the place theory of hearing. In addition, hair cells located at different places along the organ of Corti may be tuned to different frequencies because of variations in their stereocilia and biophysical properties. As a result of these factors, the basilar mem brane and organ of Corti have a so-called tonotopic map ( Fig. 8.20).

1	Fig. 8.20). Neurotransmitter release by hair cells in the organ of Corti can evoke action potentials in the primary afferent fibers of the cochlear nerve. Afferent fibers in the vestibulocochlear nerve (CN VIII) are bipolar cells with a myelin sheath around the cell bodies, as well as around the axons. The cell bodies are in the spiral ganglion, their peripheral processes synapse at the base of hair cells, and their central processes 400 Hz synapse in the cochlear nuclei of the brainstem. acteristic frequency. The characteristic frequency can be determined from a tuning curve for the fiber (

1	acteristic frequency. The characteristic frequency can be determined from a tuning curve for the fiber ( Fig. 8.21 ). A tuning curve is a plot of the threshold for activation of the nerve fiber by different sound frequencies. The major factor that influences the activity of individual afferent fibers is the location along the basilar membrane of the hair cells that they innervate. The location of those hair cells is important because for any given sound frequency, there is Distance from stapes (mm) a site of maximum displacement of the basilar membrane B as the pressure wave travels along its length (see Fig. 8.19 ). Typically, tuning curves are sharp near the characteristic CHAPTER 8 The Special Senses

1	Fig. 8.19 ). Typically, tuning curves are sharp near the characteristic CHAPTER 8 The Special Senses Base20,0007,0005,0001,0004,0003,0006004002002,0001,500800Apex •Fig. 8.20 Thetonotopicmapofthecochlea.(RedrawnfromStuhlmanO.An Introduction to Biophysics. NewYork:JohnWiley&Sons;1943.) frequency, but they broaden at high sound pressure levels. Tuning curves can have excitatory and inhibitory areas (see Fig. 8.21A ). The sharpness of the excitatory regions may reflect inhibitory processes. The different features of an acoustic stimulus are encoded in the discharges of cochlear nerve fibers. Duration is signaled by the duration of activity; intensity is signaled both by the amount of neural activity and by the number of fibers that discharge. For low-frequency sounds, the frequency is signaled by the tendency of an afferent fiber to discharge in phase with the stimulus (phase locking; see

1	Fig. 8.22A ). If the tone is much more than 1 kHz, a single fiber cannot discharge with every cycle, but phase locking can also occur for sounds with periods shorter than the absolute refractory period of the afferent fiber. This allows the CNS to detect higher frequency information from the activity of a population of afferent fibers, each of which discharges in phase with the stimulus and which, as a group, signal the frequency of the stimulus (see Fig. 8.22B ). This observation is the basis of the frequency theory of hearing. For still higher frequencies (>5000 Hz), the place theory dominates: the CNS interprets sounds that activate afferent fibers supplying hair cells near the base of the cochlea as being of high frequency. Thus both the place and the frequency theories are necessary to explain the frequency coding of sound (duplex theory) across the entire range from 20 to 20,000 Hz.

1	2 A 7 10 154 –80 –60 –40 –20 0 Frequency (kHz) E I I Threshold intensity (dB) B 0.2 1 2052 100.5 Frequency (kHz) –100 –80 –60 –40 –20 0 Threshold intensity (dB) •Fig. 8.21 Tuning Curves of Neurons in the Auditory System. Tuningcurvescanbeconsideredasreceptivefieldplots.A, Tuningcurvewithcentralexcitatoryfrequencies(E)andflankinginhibitoryfrequencies(I).B, Tuningcurvesforcochlearnervefibers.(A, RedrawnfromArthurRM,etal.J Physiol [Lond] 1971;212:593.B, RedrawnfromKatsuiY.In:RosenblithWA[ed].Sensory Communication. Cambridge,MA:MITPress;1961.)

1	Animportant,althoughrelativelyuncommon,conditionthatcaninterruptthefunctionofcochlearnervefibersisanacoustic neuroma, atumorofSchwanncellsofthevestibulocochlearnerve(CNVIII).Asthetumorgrows,irritationofcochlearnervefibersmaycausearingingsoundintheaffectedear(tinnitus). Eventually,conductionincochlearnervefibersisblocked,andtheearbecomesdeaf.Thetumormaybeoperablewhilestillsmall;therefore,earlydiagnosisisimportant.Ifthetumorisallowedtoenlargesubstantially,itcouldinterrupttheentirevestibulocochlearnerveandcausevestibularaswellasauditorydifficulties.Itcouldalsoimpingeonordistortneighboringcranialnerves(e.g.,V,VII,IX,andX),anditcouldproducecerebellarsignsbycompressingthecerebellarpeduncles.

1	•Fig. 8.22 A, Atlowfrequencies,individualauditoryafferentfiberscanrespondateachcycletothesignalfrequency.B, Athigherfrequencies,eachafferentfibergeneratesanactionpotentialonlyatcertaincycles,limitedbyitsmaximumfiringfrequency.However,theoverallpopulationofafferentfiberscanstillsignalstimulusfrequencybytheiraggregatefiringfrequency.

1	Cochlear afferent fibers synapse on neurons of the dorsal and ventral cochlear nuclei. The neurons in these nuclei have axons that contribute to the central auditory pathways. Some of the axons from the cochlear nuclei cross to the contralateral side and ascend in the lateral lemniscus, the main ascending auditory tract. Others connect with various ipsilateral or contralateral nuclei, such as the superior olivary nuclei, which project through the ipsilateral and contralateral lateral lemnisci. Each lateral lemniscus ends in an inferior colliculus. Neurons of the inferior colliculus project to the medial geniculate nucleus of the thalamus, which gives rise to the auditory radiation. The auditory radiation ends in the primary auditory cortex (Brodmann areas 41 and 42), located on the superior surface of the temporal lobe.

1	The input from each ear is bilaterally represented in the ascending auditory system pathway at the level of the lateral lemniscus and above. Thus the representation of auditory space is complex, even at the brainstem level. Consequently, unilateral deafness may occur with isolated lesions of the cochlear nuclei or more peripheral structures. Central lesions do not cause unilateral deafness, although they may interfere with overall sensitivity to speech or with sound localization. Functional Organization of the Central Auditory System The responses of neurons in several structures that belong to the auditory system can be described by tuning curves (see

1	Functional Organization of the Central Auditory System The responses of neurons in several structures that belong to the auditory system can be described by tuning curves (see Fig. 8.21B ). By plotting the distribution of the characteristic frequencies of neurons within a nucleus or in the auditory cortex, a tonotopic map may be revealed in which neurons are ordered according to their “best” frequencies. Tonotopic maps have been found in the cochlear nuclei, superior olivary complex, inferior colliculus, medial geniculate nucleus, and auditory cortex. A given auditory structure may, in fact, contain several tonotopic maps.

1	Most auditory neurons at levels above the cochlear nuclei respond to stimulation of either ear (i.e., they have binaural receptive fields). Binaural receptive fields contribute to sound localization. A human can distinguish sounds originating from sources separated by as little as 1 degree. The auditory system uses several clues to judge the origin of sounds, including differences in the time (or phase) of arrival of the sound at the two ears and differences in sound intensity on the two sides of the head.

1	For example, neurons in the medial superior olivary nucleus have medial and lateral dendrites. The synapses on the medial dendrites are largely excitatory, and they originate from the contralateral ventral cochlear nucleus. Those on the lateral dendrites are mostly inhibitory and come from the ipsilateral ventral cochlear nucleus. Differences in the phase of the sound reaching the two ears affect the strength and timing of the excitation and inhibition reaching a particular medial superior olivary neuron. The lateral superior olivary nucleus processes differences in the sound intensity that reaches the two ears to provide information about the source of the sound. The activity of superior olivary neurons can provide information about sound localization.

1	Several features of the primary auditory cortex resemble those of other primary sensory areas. Not only are sensory maps—in this case, tonotopic maps—present in the auditory cortex but also this cortical region performs feature extraction. Neurons in the primary auditory cortex form isofrequency columns (in which the neurons in the column have the same characteristic frequency), and they also form alternating columns, known as summation and suppression columns. Neurons in summation columns are more responsive to binaural than to monaural input. Neurons in suppression columns are less responsive to binaural than to monaural stimulation, and, accordingly, the response to one ear is dominant. Some neurons are selective for the direction of frequency change.

1	Bilateral lesions of the auditory cortex have some effect on the ability to distinguish the frequency or intensity of different sounds, and they reduce the abilities to localize sound and to understand speech. Unilateral lesions, however, have little effect, especially if the nondominant (for language) hemisphere is involved (see ). Evidently, frequency discrimination depends on activity at lower levels of the auditory pathway, possibly the inferior colliculus. Twosimpletestsareoftenusedclinicallytodistinguishthemostimportanttypesofdeafness:conduction loss andsensorineural loss. Conductionhearinglossoccursindisordersoftheexternalear(e.g.,earcanalblockedbycerumen)ormiddleear(e.g.,ruptureoftheeardrum).Sensorineuralhearinglossreflectsdisordersoftheinnerear,thecochlearnerve,orcentralconnections.

1	TheWeber test isusedtoevaluatethemagnitudeofconductionhearingloss.Inthistest,thebaseofavibratingtuningforkisplacedagainstthemiddleoftheperson’sforehead,andthepersonisaskedtolocalizethesound.Normally,thesoundisnotlocalizedtoaparticularear.However,ifthepersonhasconductivehearingloss(e.g.,duetoapuncturedtympanicmembrane,fluidinthemiddleear,otosclerosis,orlossofcontinuityoftheossicularchain),thesoundislocalizedtothedeafearbecauseitisconductedtothecochleathroughbone.Thesoundisalsoconductedtothecochleaoftheundamagedear,butbone-conductedsounddoesnotactivatetheorganofCortiaswellasdoessoundconductednormallythroughthetympanicmembraneandossiclechain.OnereasonwhythesoundintheWebertestisnotlocalizedtothenormalearmaybethathearinginthenormalearisinhibitedbytheambientsoundlevel(auditory masking). Conversely,inpeoplewithsensorineuralhearingloss(e.g.,duetodamagetotheorganofCorti,thecochlearnerve,orthecochlearnuclei),thesoundislocalizedtothenormalside.

1	IntheRinne test, avibratingtuningforkisplacedagainstthebonebehindtheperson’sear,andthepersonisaskedtoindicatewhenthesounddiesout.Thetuningforkisthenheldneartheexternalauditorymeatusofthatear.Inpeoplewithnormalhearing,thesoundisagainheardbecausethesoundismoreeffectivelytransmittedtothecochleainair(i.e.,airconduction> boneconduction).Iftheconductionmechanismisdamaged,thesoundisnotheardwhenthetuningforkisheldneartheexternalauditorymeatus.Boneconductioninthiscaseisbetterthanairconduction.Ifthehearinglossissensorineural,thesoundisheardagainwhenthetuningforkisplacedbytheexternalauditorymeatusbecausewithsensorineuralhearingloss,theinnerearandcochlearnervearelessabletotransmitimpulsesregardlessofwhetherthesoundvibrationsreachthecochleaviaairorbone.Thusbecauseairconductionismoreeffectivethanboneconduction,theboneconductionpatternseenwithsensorineuralhearinglossisthesameasinanormalear. The Vestibular System

1	The Vestibular System The vestibular system detects angular and linear accelerations of the head. Signals from the vestibular system allow the body to make adjustments in posture that maintain balance and trigger head and eye movements to stabilize the visual image on the retina. The following description of the vestibular system emphasizes the sensory aspects of vestibular function, and it introduces the central vestibular pathways. The role of the vestibular apparatus in motor control is discussed in The Vestibular Apparatus Structure of the Vestibular Labyrinth The vestibular apparatus, like the cochlea, consists of a component of the membranous labyrinth located within CHAPTER 8 The Special Senses the bony labyrinth. The vestibular apparatus on each side is composed of three semicircular canals and two otolith organs ( Fig. 8.23 Fig. 8.17A).

1	CHAPTER 8 The Special Senses the bony labyrinth. The vestibular apparatus on each side is composed of three semicircular canals and two otolith organs ( Fig. 8.23 Fig. 8.17A). These structures contain endolymph and are surrounded by perilymph. The semicircular canals are named the horizontal, anterior, and posterior canals. The otolith organs are the utricle and the saccule (together indicated as “Vestibule” in Fig. 8.17A ). Each semicircular canal has a swelling called an ampulla at the point where it joins the utricle. The saccule connects with the cochlea, through which endolymph (produced by the stria vascularis of the cochlea) can reach the vestibular apparatus. The three semicircular canals on one side are matched with corresponding coplanar semicircular canals on the other side. The horizontal canals on each side of the head correspond, as do the anterior canal on one side and the posterior canal on the other side (see

1	Fig. 8.23B ). This arrangement allows the sensory epithelia, in corresponding pairs of canals on the two sides, to cooperate in sensing acceleration of the head about three nearly orthogonal axes in space. The horizontal canals are not truly horizontal; rather, they lie in the horizontal plane if the head is tilted down 30 degrees in relation to the horizon. The ampulla of each of the semicircular canals contains a sensory epithelium called a crista ampullaris, or ampullary crest ( Fig. 8.24 ). An ampullary crest consists of a ridge, transverse to the long axis of the canal, that is covered by epithelium containing vestibular hair cells. These hair cells are innervated by primary afferent fibers of the vestibular nerve, which is a subdivision of the vestibulocochlear nerve (CN VIII).

1	Like cochlear hair cells, each vestibular hair cell contains a set of stereocilia on its apical surface. However, unlike cochlear hair cells, vestibular hair cells also contain a large single kinocilium. The cilia on ampullary hair cells are embedded in a gelatinous structure called the cupula. The cupula and the crista occlude the lumen of the ampulla completely. Movement of endolymph, produced by angular acceleration of the head about an axis perpendicular to the plane of the canal, deflects the cupula and consequently bends the cilia on the hair cells. The cupula has the same specific gravity as endolymph, and thus it is unaffected by linear acceleratory forces, such as gravity. The sensory epithelia of the otolith organs are called the macula utriculi and the macula sacculi (

1	The sensory epithelia of the otolith organs are called the macula utriculi and the macula sacculi ( Fig. 8.25 ). The utricle is oriented nearly horizontally; the saccule is oriented vertically. Their hair cells are embedded in the epithelium that overlies each macula. As in the ampullary crests, the stereocilia and kinocilia of the macula project into a gelatinous mass. However, the gelatinous mass in the macula contains numerous otoliths (“ear stones”) composed of calcium carbonate crystals. Together, the gelatinous mass and its otoliths are known as an otolithic membrane. The otoliths increase the specific gravity of the otolithic membrane to about twice that of the endolymph. Hence, the otolithic membrane tends to move when subjected to acceleration, whether linear (such as that produced by gravity) or angular, particularly when the center of rotation is outside the head.

1	• Fig. 8.23 A, Lateral view of the right semicircular canals of a rhesus monkey that were dissected after being filled with plastic. Note the ampullae associated with each canal. Scale is in millimeters. B,Overheadviewofthebaseoftheskullshowingtheorientationofstructuresoftheinnerear.Coplanar pairsofsemicircularcanalsincludethehorizontalcanals,aswellastheanteriorandcontralateralposterior canals.(A,CourtesyofDr.JohnSimpson,New YorkUniversity SchoolofMedicine.B,Redrawnfrom Haines DE [ed]. Fundamental Neuroscience for Basic and Clinical Applications. 3rd ed. Philadelphia: ChurchillLivingstone;2006.) •Fig. 8.24 Drawing of an Ampullary Crest Inside an Ampulla.Thestereocilia andthekinocilium of eachhaircellextendintothecupula,whichextendsacrosstheentirecross-sectionoftheampulla.Head movement(acceleration)totherightwouldresultinendolymphpressuretotheleftanddeflectionofthe cupulatotheleft. Innervation of Sensory Epithelia of the Vestibular Apparatus

1	Innervation of Sensory Epithelia of the Vestibular Apparatus The cell bodies of the primary afferent fibers of the vestibular nerve are located in the Scarpa ganglion. The neurons are bipolar, and their cell bodies, as well as axons, are myelinated. Peripherally, the vestibular nerve gives off separate • Fig. 8.25 Structure of one of the otolith organs, the saccule. Note the orderly variation in kinocilium orientation, as well as their mirror symmetry with regard to the striola. (Redrawn from Lindeman HH. Adv Otorhinolaryngol 1973;20:405.) CHAPTER 8 The Special Senses 153 branches to each of the vestibular epithelia; centrally, it accompanies the cochlear and facial nerves as they enter the internal auditory meatus of the skull.

1	CHAPTER 8 The Special Senses 153 branches to each of the vestibular epithelia; centrally, it accompanies the cochlear and facial nerves as they enter the internal auditory meatus of the skull. Like cochlear hair cells, vestibular hair cells are functionally polarized, and the transduction mechanism is presumed to be similar. When the stereocilia are bent toward the longest cilium (in this case, the kinocilium), conductance of the apical membrane increases for cations and, because of the high K+ concentration of the endolymph, K+ enters, and the vestibular hair cell is depolarized (

1	Fig. 8.26 ). Conversely, when the cilia are bent away from the kinocilium, the hair cell is hyperpolarized. The hair cell releases an excitatory neurotransmitter (either glutamate or aspartate) tonically, so that the afferent fiber on which it synapses has a resting discharge. When the hair cell is depolarized, more neurotransmitter is released, and the discharge rate of the afferent fiber increases. Conversely, when the hair cell is hyperpolarized, less neurotransmitter is released, and the firing rate of the afferent fiber slows. Angular accelerations of the head produce minute movement of the endolymph in relation to the head (

1	Angular accelerations of the head produce minute movement of the endolymph in relation to the head ( Fig. 8.27 ). This happens because the inertia of the endolymph causes it to resist the initial acceleration of the membranous labyrinth. This lag pushes on the cupula, causes the cilia to bend, and consequently changes the discharge rates of the vestibular afferent fibers. All the cilia in a given ampullary crest are oriented in the same direction. In the horizontal canal, the cilia are oriented toward the utricle, and in the other ampullae, they are oriented away from the utricle. • Fig. 8.26 Functional Polarization of Vestibular Hair Cells. When the stereocilia are bent toward the kinocilium, the hair cell is depolarized, and the afferent fiber is excited. When the stereocilia are bent away from the kinocilium, the hair cell is hyperpolarized, and the afferent discharge slows or stops. (Redrawn from Kandel ER, Schwartz JH. Principles of Neural Science. New York: Elsevier; 1981.)

1	Turning motion of head AmpullaAxis of Axis of hair cells •Fig. 8.27 Effect of Leftward Head Movement on the Activity of Vestibular Afferent Fibers Supplying Hair Cells in the Horizontal Semicircular Canals. The perspective in the figure is from above the head looking down. In this case, head rotation (acceleration) to the left causes endolymph pressure to the right and the result is increased output from the left canal and decreased output from the right canal. Small arrows intheampullaindicatethefunctionalpolarityofthehaircells.Thelarge curved arrow atthetopindicatesmovementofthehead;smaller curved arrows indicaterelativemovementoftheendolymph. The way in which angular acceleration of the head affects the discharge of vestibular afferent fibers is exemplified by the activity that originates from the horizontal canals. Fig.

1	8.27 shows the horizontal canals and utricle, as seen from above as the head is rotated (accelerated) to the left. As acceleration to the left begins, the inertia of the endolymph in the horizontal canals increases pressure toward the right side. This causes the cilia to bend on hair cells of the ampulla of the left horizontal canal toward the utricle and bends the cilia of the right canal away from the utricle. These actions increase the firing rate in the afferent fibers on the left and decrease the firing rate of the afferent fibers on the right. Once the head is moving at a constant velocity of rotation (i.e., no acceleration), there would be no force on either cupula, and therefore the hair cells of both canals would be firing as they do at rest. However, when the indicated rotation is stopped, the inertia of the endolymph creates a force on both cupulas, but in the direction opposite to that caused by the original acceleration. This results in an increase in the discharge rate

1	is stopped, the inertia of the endolymph creates a force on both cupulas, but in the direction opposite to that caused by the original acceleration. This results in an increase in the discharge rate of afferent fibers on the right side and a decrease in the discharge rate on the left. This postrotatory effect is of functional and clinical significance.

1	Unlike the hair cells in the ampullary crests, not all the hair cells in the otolith organs are oriented in the same •Fig. 8.28 Functional Polarization of Hair Cells in the Otolith Organs. A, Thesaccule.B, Theutricle.Thestriolaineachcaseisindicatedbythedotted line. (RedrawnfromSpoendlinHH.In:WolfsonRJ[ed].The Vestibular System and Its Diseases. Philadelphia:UniversityofPennsylvaniaPress;1966.) Irritationofthevestibularlabyrinth,asinMeniere’s disease, canresultinrhythmicconjugatedeviationsoftheeyes,followedbyquickreturnsaccades.Thisconditionisknownasnystagmus (see Chapter9).Theseeye movementsareaccompaniedbyasenseofvertigo andoftennausea. Thebraininterpretsadifferenceintheinputfromthetwosidesofthevestibularsystemasheadmotion.Irritation(ordestruction)ofonelabyrinthproducesanasymmetryofinputthatresultsinabnormaleyemovementandassociatedpsychologicaleffects. direction. Instead, they are oriented in relation to a ridge, called the striola, along the otolith organ (see

1	direction. Instead, they are oriented in relation to a ridge, called the striola, along the otolith organ (see Fig. 8.25 ). In the utricle, the hair cells on either side of the striola are polarized toward the striola, whereas in the saccule they are polarized away from the striola. Because the striola in each otolith organ is curved, there are hair cells with all orientations in the plane of the organ (

1	Fig. 8.28 ). In any particular orientation of the head, the cilia of the hair cells are bent to varying extents according to their orientation in relation to the gravitational vector. This results in a particular pattern of input from the otolith organs to the CNS. When the head is tilted to a new position, the orientation of the otolithic membranes in relation to the gravitational vector changes, and so the cilia of the hair cells are bent in a new way. This change in the bending of the cilia of the hair cells changes the pattern of input from the otolith organs to the CNS and creates the sensation of movement, as well as possibly triggering various reflexes. Similarly, a linear acceleration caused by other forces, such as might occur in a fall or the angular acceleration when a car turns around a curve (angular accelerations have linear centripetal and instantaneous tangential components), also affects output from the otolith organs.

1	The vestibular afferent fibers project to the brainstem through the vestibular nerve. As previously mentioned, the cell bodies of these afferent fibers are located in the Scarpa ganglion. These primary afferent fibers terminate in the vestibular nuclei (see Fig. 4.6 D to E), which are located in the rostral medulla and caudal pons and in specific regions of the cerebellum, most prominently in the nodulus. The vestibular nuclei give rise to various projections, including projections through the medial longitudinal fasciculus (see

1	The vestibular nuclei give rise to various projections, including projections through the medial longitudinal fasciculus (see Fig. 4.6C to E ) to the oculomotor nuclei. Therefore, it is not surprising that the vestibular nuclei exert powerful control over eye movements (the vestibule-ocular reflex). Other projections give rise to the lateral and medial vestibulospinal tracts, which, respectively, provide for the activation of trunk and neck muscles and thereby contribute to equilibrium and to head movements (vestibulocolic reflex). There are vestibular pathways to the cerebellum, the reticular formation, and the contra-lateral vestibular complex, as well as to the thalamus. The pathway to the thalamus, via a projection to the cerebral cortex, mediates conscious sensation of vestibular activity. Vestibular reflexes and clinical tests of vestibular function are described in The Chemical Senses

1	The Chemical Senses The senses of gustation (taste) and olfaction (smell) help detect chemical stimuli that are present either in food and drink or in the air. In the evolution of humans, these chemical senses apparently did not have the survival value of some of the other senses, but they contribute considerably to quality of life and food selection, and they are important stimulants of digestion. In other animals, the chemical senses have greater survival value, and their activation evokes a number of social behaviors, including mating, territoriality, and feeding. The stimuli that we commonly know as tastes are actually mixtures of five elementary taste qualities: salty, sweet, sour, bitter, and umami. Taste stimuli that are particularly effective in eliciting these sensations are, respectively, sodium chloride, sucrose, hydrochloric acid, quinine, and mono-sodium glutamate. Umami has been described as having a proteinaceous, meaty character.

1	bThe existence of a sixth, taste, fat (free fatty acids), is currently being debated. CHAPTER 8 The Special Senses The sensation of taste depends on the activation of chemoreceptors located in taste buds. A taste bud consists of a group of 50 to 150 receptor cells, as well as supporting cells and basal cells (Fig. 8.29A ). The chemoreceptor cells synapse at their bases with primary afferent nerve fibers, and their apices have microvilli that extend toward a taste pore. Chemoreceptor cells live only about 10 days. They are continuously replaced by new chemoreceptor cells that differentiate from basal cells located near the base of the taste bud.

1	Chemoreceptor molecules, each specialized for one type of taste stimulus, sit on the microvilli of chemoreceptor cells and detect molecules that diffuse into the taste pore from the overlying mucus of the tongue, part of which originates from glands adjacent to the taste buds. Some stimuli can pass directly into the cell to depolarize it (Na+ for salty and H+ for sour) or open cation channels to generate a receptor potential (also salty and sour), whereas others (sucrose, quinine, and glutamate for sweet, bitter, and umami) activate a second messenger that can either open cation channels or directly activate intracellular Ca++ stores (see Fig. 8.29B ). In each case, depolarization of the receptor results in the release of an excitatory neurotransmitter and, consequently, action potentials in the primary afferent nerve fiber that are transmitted to the CNS.

1	Coding of taste, however, is not based entirely on the selectivity of the chemoreceptors for the different primary qualities because each cell responds to a range of stimuli, although most intensely to one. Because most natural tastes have chemicals that effect responses from a number of chemoreceptors, recognition of taste quality appears to depend on the patterned input from a population of chemoreceptors, each responding differentially to the components of the stimulus. The intensity of the stimulus is reflected in the total amount of activity evoked. Distribution and Innervation of Taste Buds Taste buds are located on different types of taste papillae found on the tongue, palate, pharynx, and larynx. Types of taste papillae include fungiform and foliate papillae on the anterior and lateral aspects, respectively, of the tongue and circumvallate papillae on the base of the tongue (see Fig. 8.29C).

1	Fig. 8.29C). The circumvallate papillae may contain several hundred taste buds. The tongue in humans may have several thousand taste buds. The sensitivity of different regions of the tongue for different taste qualities varies slightly because taste buds responding to each type of taste are widely distributed. The taste buds are innervated by three cranial nerves. The chorda tympani branch of the facial nerve (CN VII) innervates taste buds on the anterior two thirds of the tongue, and the glossopharyngeal nerve (CN IX) innervates taste buds on the posterior third of the tongue (see Fig. 8.29C ). The vagus nerve (CN X) innervates a few taste buds in the larynx and upper esophagus. The cell bodies of taste fibers in cranial nerves VII, IX, and X are located in the geniculate, petrosal, and nodose ganglia, respectively. The central processes of the afferent fibers enter the medulla, join the solitary tract, and synapse in the nucleus of the solitary tract (see Fig. 4.6

1	Fig. 4.6 D to E). In some animals, including several rodent species, the second-order taste neurons of the solitary nucleus project rostrally to the ipsilateral parabrachial nucleus. The parabrachial nucleus then projects to the small-celled (parvocellular) part of the ventroposterior medial (VPMpc) nucleus of the thalamus. In monkeys, the solitary nucleus projects directly to the VPMpc nucleus. The VPMpc nucleus is connected to two different gustatory areas of the cerebral cortex: one in the face area of the S1 cortex and the other in the insula. An unusual feature of the central gustatory pathway is that it is predominantly an uncrossed pathway (unlike the central somatosensory pathways, which are predominantly crossed).

1	The sense of smell is much better developed in some animals (macrosmatic animals) than in humans. The ability of dogs to track other animals on the basis of odor is legendary, as is the use of pheromones by insects to attract mates. However, olfaction contributes to humans’ emotional life, and odors can effectively conjure up memories. It also helps Release of • Fig. 8.29 A, A taste bud is shown with the taste pore at the top and its innervation below. B, basal cell; R, ciliated taste receptor cells; S, supporting cells. B, Taste receptor cell showing second messenger, ligand-gated, and direct depolarization resulting in depolarization of the cell.

1	• Fig. 8.29, cont’d C, Distribution of the taste buds on the tongue and their innervation. (Redrawn from Squire LR, et al [eds]. Fundamental Neuroscience. San Diego, CA: Academic Press; 2002.) people avoid consuming spoiled food and detect dangerous situations. For example, an unpleasant odorant is added to odorless, colorless natural gas so that people can easily detect a leak. Odor has more primary qualities than taste does. As many as 1000 different odor receptors are coded in the human genome, and although only approximately 350 types are functional, they represent the largest population of G protein–coupled receptors in the genome. The olfactory mucosa also contains somatosensory receptors of the trigeminal nerve. When performing clinical tests of olfaction, clinicians must avoid activating these somatosensory receptors with thermal or noxious stimuli, such as the ammonia used in “smelling salts.”

1	The olfactory chemoreceptor cells are located in the olfactory mucosa, a specialized part of the nasopharynx. Olfactory chemoreceptors are bipolar nerve cells ( Fig. 8.30 ). The cThe conscious perception of flavor, particularly of foods, is the result of both olfactory and gustatory input based on directly inhaled odor, taste from the food as it is macerated in the mouth, and retronasal odor from the volatile molecules that are released by maceration and pass up into the nasal cavity from the pharynx.

1	CHAPTER 8 The Special Senses 157 nonmotile cilia on the apical surface of these cells contain chemoreceptors that detect odorant chemicals dissolved in the overlying mucus layer. From its opposite side, the cell gives off an unmyelinated axon that joins other olfactory nerve filaments and penetrates the base of the skull through openings in the cribriform plate of the ethmoid bone. These olfactory nerves synapse in the olfactory bulb, a portion of the cerebral hemisphere of the brain located at the base of the cranial cavity, just below the frontal lobe ( Fig. 8.31 Humans have about 10 million olfactory chemoreceptors. Like taste cells, olfactory chemoreceptors have a short life span (about 60 days), and they are also continuously replaced. However, olfactory receptor cells are true neurons and, as such, are the only neurons that are continuously regenerated throughout life.

1	The olfactory mucosa is exposed to odorant molecules by ventilatory air currents or from the oral cavity during feeding. Sniffing increases the influx of odorants. The odorants are temporarily bound in mucus to an olfactory binding protein that is secreted by a gland in the nasal cavity. Olfactory coding resembles taste coding in that most natural odors are complex and consist of many molecules that excite a wide variety of olfactory chemoreceptors. Coding for a particular perceived odor depends on the responses of many olfactory chemoreceptors, and the strength of the odorant is represented by the overall amount of afferent neural activity. The initial synapse of the olfactory pathway is located in the olfactory bulb, which is a specialized portion of the cerebral cortex and located on the underside of the frontal lobe. It contains mitral cells, interneurons (granule cells; periglomerular cells), and distinct synaptic clusters (glomeruli; see

1	Fig. 8.31 ) in which theses interact with olfactory afferent fibers. As the olfactory afferent fibers reach the olfactory bulb from the olfactory mucosa, they branch as they approach an olfactory glomerulus to synapse on the dendrites of mitral cells. Each glomerulus is the target of thousands of olfactory afferent fibers, but all the afferent fibers to a single glomerulus convey input from the one type of olfactory receptor. This is all the more remarkable because olfactory receptor cells are being regenerated continuously and new axons must therefore navigate their way to a correct glomerulus.

1	The granule and periglomerular cells are inhibitory inter-neurons. They form dendrodendritic reciprocal synapses with the dendrites of mitral cells. Activity in a mitral cell depolarizes these inhibitory cells, and they in turn inhibit the original and adjacent glomeruli. Because each glomerulus is specialized by being the target of afferent fibers for a unique combination of odor qualities, this appears to be a way of enhancing stimulus contrast, much the way horizontal cells do in the retina. In addition, it provides a mechanism for adaptation to continuous stimulation.

1	• Fig. 8.30 Olfactory chemoreceptors and supporting cells. (Redrawn from de Lorenzo AJD. In: Zotterman Y [ed]. Olfaction and Taste. Elmsford, NY: Pergamon; 1963.) • Fig. 8.31 Drawing of a Sagittal Section Through an Olfactory Bulb, Showing Terminations of the Olfactory Chemoreceptor Cells in the Olfactory Glomeruli and the Intrinsic Neurons of the Olfactory Bulb. The axons of the mitral cells are shown exiting in the olfactory tract to the right. (Modified from House EL, Pansky B. A Functional Approach to Neuroanatomy. 2nd ed. New York: McGraw-Hill; 1967.) CHAPTER 8 The Special Senses

1	CHAPTER 8 The Special Senses The axons of mitral cells leave the olfactory bulb and enter the olfactory tracts. From here, the olfactory connections become highly complex. Within the olfactory tracts is a nucleus, called the anterior olfactory nucleus, that receives input from the olfactory bulb and projects to the contralateral olfactory bulb through the anterior commissure. As each olfactory tract approaches the base of the brain, it splits into the lateral and medial olfactory striae. Axons of the lateral olfactory stria synapse in the primary olfactory cortex, which includes the prepiriform cortex (and, in many animals, the piriform lobe). The medial olfactory stria includes projections to the amygdala, as well as to the basal forebrain. These structures are portions of, or directly connected to, the limbic system (see

1	Of note is that the olfactory pathway is the only sensory system that does not have an obligatory synaptic relay in the thalamus before signals reach the cortex. However, olfactory information does reach the mediodorsal nucleus of the thalamus, and it is then transmitted to the prefrontal and orbitofrontal cortex. The functional roles of olfaction, in addition to the conscious perception of odor, include providing much of the subtleties of taste by enhancing the 1. Light enters the eye through the cornea and lens and is focused on the retina, which lines the back of the eye. The cornea is the most powerful refractive surface, but the lens has a variable power that allows images of near objects to be focused on the retina. The iris regulates depth of field and the amount of illumination that enters the eye. 2.

1	2. The outer segments of the photoreceptor cells transduce light. Photoreceptors synapse on retinal bipolar cells, which in turn synapse on other interneurons and on ganglion cells. The ganglion cells project to the brain through the optic nerve. The optic disc, where the optic nerve leaves the retina, contains no photoreceptors and is therefore a blind spot. The portion of the retina with the highest degree of spatial resolution is the fovea and the surrounding macula. 3. Rod photoreceptors have high sensitivity, do not discriminate among colors, and function best under low light levels. Cone photoreceptors have lower sensitivity but higher spatial resolution. Color vision relies on the three types of cones that have different spectral sensitivities. 4.

1	4. Bipolar cells and many ganglion cells have concentric receptive fields with an on-center/off-surround or off-center/on-surround organization. Horizontal cells mediate this center-surround antagonism. Photoreceptor, bipolar, and horizontal cells respond to stimulation by modulating their membrane potential and their release of neurotransmitters,

1	Olfactionisnotgenerallyexaminedinaroutineneurologicalexamination.However,smellcanbetestedbyhavingthepatientinhaleandidentifyanodorant.Onenostrilshouldbeexaminedatatimewhiletheothernostrilisoccluded.Strongodorants,suchasammonia,shouldbeavoidedbecausetheyalsoactivatetrigeminalnervefibers.Smellsensationcanbelost(anosmia) afterabasalskullfractureorafterdamagetooneorbotholfactorybulbsortractsbyatumor(suchasanolfactory groove meningioma).Concussionscancauseanosmiabecausethesuddenmovementofthebraininsidetheskullcanshearthesmallunmyelinatedolfactorynervefibers.Anauraofadisagreeableodor,oftenthesmellofburningrubber,occursduringuncinate seizures, whichareepilepticseizuresthatoriginateinthemedialtemporallobe. narrow range of gustatory receptors with the wide repertoire of olfactory receptors. In addition, via its intimate connections with limbic and, by extension, hypothalamic structures, it provides input to subconscious mechanisms related to emotions, memory, and sexual behavior.

1	but ganglion cells respond by generating action potentials. 5. The axons of ganglion cells in the temporal retina project to the brain ipsilaterally; those in the nasal retina cross in the optic chiasm. Because the lens inverts the image that falls on the retina, each side of the visual field is projected to the contralateral side of the brain for both eyes. In the lateral geniculate nucleus (LGN) of the thalamus, the input from each eye terminates in separate layers, and the M ganglion cells (sensitive to movement) and P ganglion cells (sensitive to detail and color) project to separate layers as well. 6.

1	6. The LGN projects to primary visual (striate) cortex via the visual radiation and terminates largely in layer 4, where there is an orderly retinotopic map. Within the map, information from each eye maps to alternating adjacent points to create ocular dominance columns that extend vertically in the cortex. Striate cortical neurons outside of layer 4 respond best to bar or edge stimuli oriented in a particular way. Cells that “prefer” a particular stimulus orientation are grouped in orientation columns. 7. The extrastriate visual areas have different functions. Some in the inferotemporal cortex are influenced chiefly by P cells, and they function in form detection, color vision, and face discrimination. M cells influence regions of the middle temporal and parietal cortex, which function in motion detection and the control of eye movements. 8.

1	8. A pure tone is characterized in terms of its amplitude, frequency, and phase. Natural sounds are combinations of pure tones. Sound pressure is measured in decibels (dB), in relation to a reference level. 9. The pinna and auditory canal convey airborne sound waves to the tympanic membrane. The three small bones (ossicles) of the middle ear transmit the vibrations of the tympanic membrane to the oval window of the fluid-filled inner ear. Hearing is most sensitive at about 3000 Hz because of the dimensions of the auditory canal and the mechanics of the ossicles. 10. The cochlea of the inner ear has three main compartments: the scala vestibuli, the scala tympani, and the intervening scala media (cochlear duct). The cochlear duct is bounded on one side by the basilar membrane, on which lies the organ of Corti, the sound transduction mechanism. 11.

1	11. When the basilar membrane oscillates in response to pressure waves introduced into the scala vestibuli at the oval window, the stereocilia of the hair cells of the organ of Corti are subjected to shear forces, which open mechanoreceptor K+ channels. This results in a membrane conductance change that modulates the release of neurotransmitters on to cochlear nerve fibers. 12. High-frequency sounds best activate the hair cells near the base of the cochlea, and low-frequency sounds activate cells near the apex. Such a tonotopic organization is also present in central auditory structures, including the cochlear nuclei, superior olivary complex, inferior colliculus, medial geniculate nucleus, and primary auditory cortex. 13. Auditory processing at many sites in the central auditory pathway contributes to sound localization, frequency and intensity analysis, and speech recognition. 14.

1	13. Auditory processing at many sites in the central auditory pathway contributes to sound localization, frequency and intensity analysis, and speech recognition. 14. The vestibular apparatus is part of the inner ear. It includes three semicircular canals (horizontal, anterior, and posterior) and two otolith organs (utricle and saccule) on each side. These transduce, respectively, angular and linear accelerations of the head. The three semicircular canals are mutually orthogonal, so they can resolve angular acceleration of the head about any axis of rotation. 15. In each semicircular canal, there are sensory hair cells whose cilia extend into a cupula, which blocks the cross-section of the endolymph-filled canal. Angular head acceleration displaces the endolymph and the cupula, bending the cilia. If the stereocilia bend toward the kinocilium, the hair cell is depolarized, which causes an increase in the firing rate in the afferent fiber. 16.

1	16. In the otolith organs, the cilia project into an otolithic membrane. Acceleration of the head, as with linear movement, or change in position in relation to gravity displaces the otolithic membrane (because of the mass of the otoliths) and changes the firing patterns of the hair cells, depending on their orientation. 17. Central vestibular pathways include afferent connections to the vestibular nuclei and the cerebellum. Activation of the vestibular afferent fibers is detected by the brain as head acceleration or position change and is relayed via the vestibular nuclei to pathways that mediate compensatory eye movements, neck movements, and adjustments to posture. 18.

1	18. Taste buds contain chemoreceptor cells arranged around a taste pore. Taste buds are located on several kinds of papillae on the tongue and in the pharynx and larynx. Five types of taste-receptor cells detect the five elementary qualities of taste: salty, sweet, sour, bitter, and umami. Complex flavors are signaled by the patterned activity of multiple classes of taste receptor and by central correlation with accompanying olfactory input. 19. Afferent taste fibers synapse in the nucleus of the solitary tract. The thalamic relay is via a part of the ventroposterior medial nucleus to the taste-receiving areas located in the S1 cortex and the insula. 20. Odors are detected by olfactory chemoreceptor cells, which are continuously regenerated in the olfactory mucosa. These cells are true neurons that are endowed with a wide array of G protein–coupled receptors that enable the detection of hundreds of odor molecules. 21.

1	21. Individual olfactory axons project to olfactory glomeruli, specific for each stimulus type, in the olfactory bulb. They synapse on the dendrites of mitral cells, which have reciprocal synapse with inhibitory interneurons. This synaptic organization in the glomerulus underlies stimulus adaptation and contrast enhancement. Squire L, Berg D. Fundamental Neuroscience. 4th ed. New York: Academic Press; 2012. Upon completion of this chapter, the student should be able to answer the following questions: 1. What is a motor neuron, and how are α and γ motor neurons different? 2. What is a motor unit? How does the “size principle” apply to the orderly recruitment of motor units? 3. What is a reflex, and why are reflexes useful for clinical and scientific understanding? 4. What information about the state of the muscle is sensed by the muscle spindles, and what afferent fibers convey this information to the central nervous system (CNS)? 5.

1	4. What information about the state of the muscle is sensed by the muscle spindles, and what afferent fibers convey this information to the central nervous system (CNS)? 5. How do γ motor neurons modulate the responses of the muscle spindle? 6. What are the pathways and functions of the basic spinal reflexes? 7. What is a central pattern generator, and what types of movements can it be used for? 8. What distinguishes the pathways of the medial and lateral descending pathways in motor control? 9. What is decerebrate rigidity, and what are its implications for the control of muscle tone? 10. What distinguishes the cortical motor areas from each other? 11. What motor parameters are coded for in the activity of neurons in motor cortex? 12. How does the organization of the mossy and olivocerebellar (climbing) fiber afferent systems to the cerebellum differ in their origins, topography, and synaptic connections. 13.

1	12. How does the organization of the mossy and olivocerebellar (climbing) fiber afferent systems to the cerebellum differ in their origins, topography, and synaptic connections. 13. What is the geometric relationship between the major cellular elements of the cerebellar cortex? 14. What are simple and complex spikes in Purkinje cells? 15. What are the direct and indirect pathways in the basal ganglia, and how does their activity influence movement? 16. How is the balance of activity between the direct and indirect pathways altered in Parkinson’s disease and Huntington’s disease? 17. How do the vestibuloocular and optokinetic reflexes act to stabilize gaze? How do they complement each other? 18. What are the roles of saccades and smooth pursuit movements in visual tracking? 19. What is nystagmus, and what types of sensory stimulation can drive nystagmus in a normal individual? 20.

1	18. What are the roles of saccades and smooth pursuit movements in visual tracking? 19. What is nystagmus, and what types of sensory stimulation can drive nystagmus in a normal individual? 20. What is the somatotopic organization of the different CNS regions involved in motor control. ovements are the major way in which humans interact with the world. Most activities— including running, reaching, eating, talking, writing, and reading—ultimately involve motor acts. Thus motor control is a major task of the central nervous system (CNS), and from an evolutionary perspective, it is probably the reason that nervous systems first arose. Not surprisingly, a large amount of the CNS is devoted to motor control, which can be defined as the generation of signals to coordinate contraction of the musculature of the body and head, either to maintain a posture or to make a movement (transition between two postures).

1	Because large amounts of the nervous system are involved in motor control, damage or diseases of the nervous system often result in motor abnormalities. Conversely, particular motor symptoms help determine the location of the damaged or malfunctioning region; thus assessment of motor function is an important clinical tool.

1	In this chapter, each major CNS area involved in motor control is described, starting with the spinal cord and continuing with the brainstem, cerebral cortex, cerebellum, and basal ganglia. Eye movements are discussed at the end of the chapter because of their importance and the specialized circuits involved in their generation. Each CNS area is described separately; however, CNS regions do not function in isolation, and most movements result from the coordinated action of multiple brain regions. For example, even spinal reflexes, which are mediated by local circuits in the spinal cord, can be modified by descending motor commands, and virtually all voluntary movements, which arise from cerebral activity, are ultimately generated by activation of the spinal cord circuitry (or analogous brainstem nuclei for muscles in the head and face). Principles of Spinal Cord Organization

1	The spinal cord has a cylindrical shape in which the white matter is located superficially and the gray matter is found deep to the white matter shell. The gray matter forms a continuous column that runs the length of the cord. However, the nerve roots that enter and exit the spinal cord bundle into discrete nerves, which form the basis for naming the •Fig. 9.1 Musculotopic Organization of Motor Neurons in the Ventral Horn of the Spinal Cord. A, Schematicviewofthecervicothoracicspinalcordandassociatedcross-sections,showingtheloca-tionsofmotorneuronsthatinnervateaflexor(blue dots) andanextensor(red dots). B, Spinalcordcross-section,withlocationsofdifferentmusclesrepresentedbyadrawingofthearm.(RedrawnfromPurvesD,etal[eds].Neuroscience. 3rded.Sunderland,MA:Sinauer;2004.)B Distal muscles Proximal muscles specific levels (“segments”) of the spinal cord (8 cervical, 12 thoracic, 5 lumbar, 5 sacral, and 1 coccygeal). When viewed in cross-section, the gray matter column typically has an “H”

1	muscles specific levels (“segments”) of the spinal cord (8 cervical, 12 thoracic, 5 lumbar, 5 sacral, and 1 coccygeal). When viewed in cross-section, the gray matter column typically has an “H” or butterfly shape. The “butterfly wings” are divided into dorsal and ventral horns that are separated by an intermediate zone (

1	Fig. 9.1 ) (at some spinal cord levels, a small lateral horn is also present; see ). The ventral horn is where motor neurons reside, and thus it has primarily a motor function. Correspondingly, it is the main target of descending motor pathways from the brain. In contrast, the dorsal horn is the major recipient of incoming sensory information and the main source of ascending sensory pathways (e.g., the spinothalamic tract; see Chapter 7). The description of the motor function of the spinal cord therefore begins with the properties of motor neurons and their organization in the ventral horn. Similar organizational principles hold for cranial nerve nuclei that are involved in controlling the musculature of the head and face (e.g., the facial and motor trigeminal nuclei).

1	A motor neuron is a neuron that projects to muscle cells. Because motor neurons represent the only route for CNS activity to control muscle activity, motor neurons have been termed the final common pathway. There are both somatic motor neurons and autonomic motor neurons. However, in this chapter, the term motor neuron refers only to somatic motor neurons; autonomic motor neurons are discussed in . Somatic motor neurons innervate the skeletal (striated) muscles of the body. Two main classes are distinguished on the basis of their axonal diameters: α and γ motor neurons. The α motor neurons are large, multipolar neurons that range in size up to 70 µm in diameter (see

1	The α motor neurons are large, multipolar neurons that range in size up to 70 µm in diameter (see Fig. 4.10A ). Their axons leave the spinal cord through the ventral roots and the brainstem via several cranial nerves and are distributed to the appropriate skeletal muscles via peripheral nerves. The α motor neuron axon also projects to other neurons by giving off collateral axons before leaving the CNS. The main axon terminates by synapsing onto the extrafusal muscle fibers. These synapses are called neuromuscular junctions or end plates. Extrafusal fibers are large muscle fibers that make up the bulk of a skeletal muscle and essentially generate its contractile force (a muscle also contains intrafusal fibers whose functions are detailed later in this chapter; also see

1	A key functional aspect of the motor neuron projection pattern out of the CNS is that each neuron’s axon innervates only one muscle, but it branches to innervate multiple fibers within that muscle. Moreover, each extrafusal muscle fiber in mammals is supplied by only one α motor neuron. Thus a motor unit can be defined as an α motor neuron and all of the skeletal muscle fibers that its axon supplies. The motor unit can be regarded as the basic unit of movement because when an α motor neuron discharges under normal circumstances, all of the muscle fibers of the motor unit contract. That is, the safety factor (see ) of the neuromuscular junction is greater than 1, and so each action potential in the motor neuron axon triggers an action potential in every muscle fiber of the motor unit.

1	Of importance is that the average size of the motor unit (i.e., the number of muscle fibers innervated by an axon) varies between muscles, depending on how fine a control of the muscle is required. For finely controlled muscles, such as the eye muscles, an α motor neuron may supply only a few muscle fibers. However, in a proximal limb muscle, such as the quadriceps femoris, a single α motor neuron may innervate thousands of muscle fibers.

1	The muscle fibers that belong to a given motor unit are called a muscle unit. All the muscle fibers in a muscle unit are of the same histochemical type (i.e., they are all either slow-twitch [type I] or fast-twitch [type IIA or IIB] fibers). For an in-depth description of muscle fiber types, see . Of importance in this chapter is that a number of physiological properties are correlated with this histochemical classification scheme. In particular, slow-twitch fibers, which contract and relax slowly as implied by their name, also generate low-force levels but essentially never fatigue. In contrast, the fast-twitch fiber contract and relax rapidly, generate higher levels of force, and fatigue at varying rates. The first motor units to be activated in many cases, either by voluntary effort or during reflex action or just

1	The first motor units to be activated in many cases, either by voluntary effort or during reflex action or just CHAPTER 9 Organization of Motor Function to maintain posture, are those with the smallest motor axons. These motor units contain slow-twitch fibers and thus generate the smallest contractile force, allowing the initial contraction to be finely graded. These units tend to be active much of the time, if not continuously, and so their lack of fatigability makes good functional sense. As more motor units are recruited for a motor act, motor neurons with progressively larger axons become involved, and these axons synapse onto the fast-twitch fibers. Thus they generate progressively larger amounts of tension. The most powerful such motor units are typically recruited only for tasks requiring large amounts of force (e.g., sprinting, jumping, and lifting a heavy weight), tasks that people can perform for only for short periods of time.

1	The orderly recruitment of motor units helps the CNS generate a large range of forces and also maintain relatively precise control at the different force levels. This recruitment pattern is called the size principle because the motor units are recruited in order of motor neuron axon size. The size principle depends on the fact that small motor neurons are activated more easily than are large motor neurons. Recall that if an excitatory synapse is active, it opens channels in the postsynaptic membrane and causes an excitatory postsynaptic current. The same size excitatory postsynaptic current generates a larger potential change at the initial segment of an axon of a small motor neuron than it does at a larger motor neuron, simply as a consequence of Ohm’s law (V = IR) and the fact that smaller motor neurons have higher membrane resistance than larger motor neurons do. Thus because excitatory postsynaptic potentials in the CNS are small and need to summate to reach threshold for

1	smaller motor neurons have higher membrane resistance than larger motor neurons do. Thus because excitatory postsynaptic potentials in the CNS are small and need to summate to reach threshold for triggering spikes, as the level of synaptic bombardment rises from zero, the resulting depolarization will reach spiking threshold in smaller motor neurons first. As the size principle is usually obeyed, this assumption generally appears to hold; however, there can be exceptions, and in these cases, the descending motor pathways presumably must provide differing levels of synaptic drive to the different-sized motor neurons.

1	The γ motor neurons are smaller than α motor neurons; they have a soma diameter of about 35 µm. The γ motor neurons that project to a particular muscle are located in the same regions of the ventral horn as the α motor neurons that supply that muscle. γ Motor neurons do not supply extrafusal muscle fibers; instead, they synapse on specialized striated muscle fibers called intrafusal muscle fibers, which traverse receptors called muscle spindles that are embedded in skeletal muscles. The function of γ motor neurons is to regulate the sensitivity of these receptors (discussed later). Topographic Organization of Motor Neurons in the Ventral Horn The spatial distribution of motor neurons in the spinal cord is highly organized. (This is also true in the cranial nerve nuclei.) A given skeletal muscle is supplied by a group

1	Aclinicallyusefulwaytomonitortheactivityofmotorunitsiselectromyography. Anelectrodeisplacedwithinaskeletalmuscletorecordthesummedactionpotentialsoftheskeletalmusclefibersofamuscleunit.Ifnospontaneousactivityisnoted,thepatientisaskedtocontractthemusclevoluntarilytoincreasetheactivityofmotorunitsinthemuscle.Astheforceofvoluntarycontractionincreases,moremotorunitsarerecruited.Inadditiontotherecruitmentofmoremotorneurons,contractilestrengthincreaseswithincreasesintherateofdischargeoftheactiveα

1	motorneurons.Electromyographyisusedforvariouspurposes.Forexample,theconductionvelocityofmotoraxonscanbeestimatedasthedifferenceinlatencyofmotorunitpotentialswhenaperipheralnerveisstimulatedattwositesseparatedbyaknowndistance.Anotheruseistoobservefibrillationpotentialsthatoccurwhenmusclefibersaredenervated.Fibrillationpotentialsarespontaneouslyoccurringactionpotentialsinsinglemusclefibers.Thesespontaneouspotentialscontrastwithmotorunitpotentials,whicharelargerandhavealongerdurationbecausetheyrepresenttheactionpotentialsinasetofmusclefibersthatbelongtoamotorunit.

1	of α motor neurons, called a motor nucleus, located in the ventral horn. Each such motor nucleus takes the form of a rostrocaudally running column that can span several spinal cord levels (see Fig. 9.1A ). Motor neurons that supply the axial musculature collectively form a column of cells that extends the length of the spinal cord. In the cervical and lumbosacral enlargements, these cells are located in the most medial part of the ventral horn; at other levels, they essentially form the entire ventral horn. The motor neurons innervating the limb muscles are in the cervical and lumbosacral enlargements, where they form columns that are lateral to those for the axial muscles. Motor neurons to muscles of the distal part of the limb are located most laterally, whereas those that innervate more proximal muscles are located more medially (

1	Fig. 9.1B). Also, motor neurons to flexors are dorsal to those that innervate extensors. Note that the α and γ motor neurons to a given muscle are found intermixed within the same motor neuron column. The interneurons that connect with the motor neurons in the enlargements are also similarly topographically organized. In general, interneurons that supply the limb muscles are located mainly in the lateral parts of the deep dorsal horn and the intermediate region between the dorsal and ventral horns. Those that supply the axial muscles, however, are located in the medial part of the ventral horn. All of these interneurons receive synaptic connections from primary afferent fibers and from the axons of pathways that descend from the brain, and thus they are part of both spinal reflex arcs and descending motor control pathways.

1	An important aspect of interneuronal systems is that the laterally placed interneurons project ipsilaterally to motor neurons that supply the distal or the proximal limb muscles, whereas the medial interneurons project bilaterally. This arrangement of the lateral interneurons allows the limbs to be controlled independently. In contrast, the bilateral arrangement of the medial interneurons allows bilateral control of motor neurons to the axial muscles to provide postural support to the trunk and neck.

1	Although motor neurons are the final common pathway from the CNS to muscles and thus shape how neuronal activity is transformed into muscular contraction, each motor neuron directly acts on only a single muscle. Normal movements (or postures), however, are rarely, if ever, caused by the isolated contraction of an individual muscle. Rather, they reflect the coordinated activity of large groups of muscles. For example, elbow flexion involves an initial burst of activity in flexor muscles, such as the biceps, and in relaxation of extensor muscles, such as the triceps. This activity is then succeeded by a burst of activity of the triceps and then a second burst of activity in the biceps to stop the flexion movement at the desired position. Furthermore, other muscles are also activated during the elbow flexion to maintain overall balance and posture.

1	As the elbow flexion example shows, different roles are played by each muscle during a movement. The muscle that initiates, and is the prime cause of, the movement is called the agonist. Muscles that act similarly to the agonist are called synergists, whereas muscles whose activity opposes the action of the agonist are antagonists. In addition, muscles can act as fixators to immobilize a joint and in postural roles. Moreover, the relationship two muscles have to each other may depend on the specific movement being performed. For example, during elbow flexion, the triceps acts an antagonist to the biceps. In contrast, during supination of the forearm without rotation occurring about the elbow, the biceps (which also acts to supinate the forearm) is again an agonist, but the role of the triceps is that of an elbow fixator.

1	Thus motor control requires flexibly linking (and unlinking) the activity of groups of motor neurons that connect to different muscles. The circuits of the spinal cord are a major mechanism used by the CNS for this aspect of motor control. Indeed, descending pathways from the brain target primarily the interneurons of the spinal cord, although there are some descending axons that synapse directly onto motor neurons.

1	Spinal cord circuitry has several levels of organization. The most basic is the segmental level: that is, a circuit that is largely confined to a single or several neighboring segments and that is repeated again at many levels. The basic spinal reflexes covered below (i.e., the myotatic, inverse myotatic, and flexion reflexes) are mediated by such circuits. Superimposed on this segmental organization is the propriospinal system, which is a series of neurons whose axons run up and down the spinal cord to interconnect the different levels of the cord. This system allows the coordination of activity at different spinal levels, which is important for behavior involving both the forelimbs and the hind limbs, such as •Fig. 9.2 Muscle Proprioceptors. Skeletalmusclescontainsensoryreceptorsembeddedwithinthemuscle(spindles)andwithintheirtendons(Golgitendonorgans).A,

1	both the forelimbs and the hind limbs, such as •Fig. 9.2 Muscle Proprioceptors. Skeletalmusclescontainsensoryreceptorsembeddedwithinthemuscle(spindles)andwithintheirtendons(Golgitendonorgans).A, Schematicviewofamuscle,showingthearrangementofaspindleinparallelwithextrafusalmusclefibersandatendonorganinserieswithmusclefibers.B, Structureandinnervation(motorandsensory)ofamusclespindle.C, Structureandinnervationofatendonorgan.A B C Ib afferent neuron Muscle fibers Tendon 250 µm Bag fibers Chain fibersGolgi tendon organ

1	The neural innervation of an intrafusal fiber differs significantly from that of an extrafusal fiber, which is innervated by a single motor neuron. Intrafusal fibers are multiply innervated and receive both sensory and motor innervation. The sensory innervation typically includes a single group Ia afferent fiber and a variable number of group II afferent fibers (see

1	Fig. 9.2B). Group Ia fibers belong to the class of sensory nerve fibers with the largest diameters and conduct at 80 to 120 m/sec; group II fibers are intermediate in size and conduct at 35 to 75 m/sec. A group Ia afferent fiber forms a spiral-shaped termination, referred to as a primary ending, on each of the intrafusal muscle fibers in the spindle. Thus primary endings are found on both types of nuclear bag fibers and on nuclear chain fibers. The group II afferent fiber forms a secondary type ending on nuclear chain and bag2 fibers, but not on bag1 fibers. The primary and secondary endings have mechanosensitive channels that are sensitive to the level of tension on the intrafusal muscle fiber. The motor supply to a muscle spindle consists of two types of γ motor axons (see Fig. 9.2B ). Dynamic γ motor axons end on nuclear bag1 fibers, and static γ motor axons end on nuclear chain and bag2 fibers.

1	Fig. 9.2B ). Dynamic γ motor axons end on nuclear bag1 fibers, and static γ motor axons end on nuclear chain and bag2 fibers. Muscle spindles respond to changes in muscle length because they lie in parallel with the extrafusal fibers and therefore are also stretched or shortened along with the extrafusal fibers. Because intrafusal fibers, like all muscle fibers, display spring-like properties, a change in their length changes the tension that they are under, and this change is sensed by mechanoreceptors of the group Ia and group II spindle afferent fibers. The nonselective cation channel Piezo2 has been identified as the principal transduction channel that allows spindle sensory afferent fibers to sense changes in mechanical stress that occur when a muscle changes length.

1	Fig. 9.3 shows the changes in activity of the afferent fibers of a muscle spindle when the muscle is stretched. It is clear that group Ia and group II fibers respond differently to stretch. Group Ia fibers are sensitive both to the amount of muscle stretch and to its rate, whereas group II fibers respond chiefly to the amount of stretch. Thus when a muscle is stretched to a new longer length, group II firing increases in proportion to the amount of stretch (see Fig. 9.3 , left), and when the muscle is allowed to shorten, its firing rate decreases proportionately (see Fig. 9.3 , right). Group Ia fibers show this same static-type response, and thus under steady-state conditions (i.e., constant muscle length), their firing rate reflects the amount of muscle stretch, similar to that of group II fibers.

1	While muscle length is changing, however, group Ia firing also reflects the rate of stretch or shortening that the muscle is undergoing. Its activity overshoots during muscle stretch and undershoots (and possibly ceases) during muscle shortening. These are called dynamic responses. This dynamic sensitivity also means that the activity of group Ia fibers is much more sensitive to transient and oscillatory stretches, such as shown in the middle diagrams of

1	Fig. 9.3 . In particular, the tap profile is what occurs when a reflex hammer is used to hit the muscle tendon and thereby cause a brief stretching of the attached muscle. The change in muscle length is too brief for significant changes in group II firing to occur, but because the magnitude of the rate of change (slopes of the tap profile) is so high with this stimulus, large dynamic responses are elicited in the group Ia fibers. Thus the functionality of reflex arcs involving group Ia afferent fibers is what is being assessed when a reflex hammer is used to tap on tendons. γ Motor Neurons Adjust the Sensitivity of the Spindle Up to this point, we have described only how muscle spindles behave when there are no changes in γ motor neuron activity. The efferent innervation of muscle spindles is extremely important, however, because it determines the sensitivity of muscle spindles to stretch. For example, in Fig. 9.4A, the activity of a muscle spindle afferent fiber is shown during

1	Fig. 9.4A, the activity of a muscle spindle afferent fiber is shown during CHAPTER 9 Organization of Motor Function •Fig. 9.3 Responses of a Primary Ending (Group Ia) and a Secondary Ending (Group II) to Changes in Muscle Length. Notethedifferenceindynamicandstaticresponsivenessoftheseendings.Thewaveformsatthetoprepresentthechangesinmusclelength.ThemiddleandbottomrowsshowthedischargesofagroupIafiberandagroupIIfiber,respectively,duringthevariouschangesinmusclelength.

1	•Fig. 9.4 The Activity of γ Motor Neurons Can Counteract the Effects of Unloading on the Discharge of a Muscle Spindle Afferent Fiber. A, Theactivityofamusclespindleafferentfiberduringsteadystretch.B, Stimulationofα motorneuronat0mseccausescontractionoftheextrafusalfibers,whichleadstomuscleshorteningandincreasedmuscletensionbutunloadingofthetensionacrossthemusclespindle,whichinturninducestheafferentfibertostopfiring.Uponrelaxation,themusclereturnstoitsoriginallength,andtensionisrestoredontheintrafusalfibers,causingthereturnofactivityinthegroupIaafferentfiber.C, Coactivationofα andγ motorneuronscausesshorteningofbothextrafusalandintrafusalfibers.Thusthereisnounloadingofthespindle,andtheafferentfibermaintainsitsspontaneousactivity.(RedrawnfromKufflerSW,NichollsJG.From Neuron to Brain. Sunderland,MA:Sinauer;1976.) a steady stretch. If only the extrafusal muscle fibers were may stop discharging and become insensitive to further to contract (this can be done experimentally by selective

1	a steady stretch. If only the extrafusal muscle fibers were may stop discharging and become insensitive to further to contract (this can be done experimentally by selective decreases in muscle length. However, the unloading of the stimulation of α motor neurons; see

1	Fig. 9.4B ), the muscle spindle can be prevented if α and γ motor neurons are spindle would be unloaded by the resultant shortening of stimulated simultaneously. Such combined stimulation the muscle. If this happens, the muscle spindle afferent fiber causes the intrafusal muscle fibers of the spindle to shorten along with the extrafusal muscle fibers, maintaining the baseline tension on the equatorial portion of the intrafusal fibers (see Fig. 9.4C •Fig. 9.5 Effects of Static and Dynamic γMotor Neurons on the Responses of a Primary Ending to Muscle Stretch. A, Thetimecourseofthestretch.B, ThedischargeofgroupIafibersintheabsenceofγmotorneuronactivity.C, Stimulationofastaticγmotoraxon.D, Stimulationofadynamicγmotoraxon.(RedrawnfromCroweA,MatthewsPBC.J Physiol. 1964;174:109.)

1	Note that only the two polar regions of the intrafusal muscle contract; the equatorial region, where the nuclei are located, does not contract because it has little contractile protein. Nevertheless, when the polar regions contract, the equatorial region elongates and regains its sensitivity. Conversely, when a muscle relaxes (α motor neuron activity drops) and thus elongates (if its ends are being pulled), a concurrent decrease in γ motor neuron activity allows the intrafusal fibers to relax (and thus elongate) as well and thereby prevent the tension on the central portion of the intrafusal fiber from reaching a level at which firing of the afferent fibers is saturated. Thus the γ motor neuron system allows the muscle spindle to operate over a wide range of muscle lengths while retaining high sensitivity to small changes in length.

1	For voluntary movements, descending motor commands from the brain in fact typically activate α and γ motor neurons simultaneously, presumably to maintain spindle sensitivity as just described. This has two important functions. By maintaining the muscle spindle’s sensitivity as the muscle changes length, the spindle remains capable of sensing, and signaling to the CNS, any disturbances to the ongoing movement that cause an unexpected stretch of the muscle, and this in turn allows the CNS to initiate both reflex (see next section) and voluntary corrections. Second, if the spindle were to become unloaded during the movement, this would oppose the intended movement by decreasing the excitatory drive, via the group Ia reflex arc (see next section), to the α motor neurons driving the agonist muscles. As mentioned earlier, there are two types of γ motor neurons: dynamic and static (see Fig. 9.2).

1	As mentioned earlier, there are two types of γ motor neurons: dynamic and static (see Fig. 9.2). This allows the CNS to have very precise control over the sensitivity of the muscle spindle. Dynamic γ motor axons end on nuclear bag1 fibers, and static γ motor axons synapse on nuclear chain and bag2 fibers. Thus when a dynamic γ motor neuron is activated, the response of the group Ia afferent fiber is enhanced, but the activity of the group II afferent fibers is unchanged; when a static γ motor neuron discharges, the responsiveness of the group II afferent fibers and the static responsiveness of the group Ia afferent fibers are increased. The effects of stimulating the static and dynamic fibers on a group Ia afferent fiber’s response to stretch are illustrated in

1	Fig. 9.5 . Descending pathways can preferentially influence dynamic or static γ motor neurons and thereby alter the nature of reflex activity in the spinal cord and also, presumably, the functioning of the muscle spindle during voluntary movements. The Phasic (or Ia) Stretch Reflex The reflex arc responsible for the phasic stretch reflex is depicted in

1	Fig. 9.6 ; the rectus femoris muscle serves as an example. A rapid stretch of the rectus femoris muscle strongly activates the group Ia fibers of the muscle spindles, which then convey this signal into the spinal cord. In the spinal cord, each group Ia afferent fiber branches many times to form excitatory synapses directly (monosynaptically) on virtually all α motor neurons that supply the same (also known as the homonymous) muscle and with many α motor neurons that innervate synergists, such as the vastus intermedius muscle in this case, which also acts to extend the leg at the knee. If the excitation is powerful enough, the motor neurons discharge and cause a contraction of the muscle. Note that the group Ia fibers do not contact the γ motor neurons, possibly to avoid a positive-feedback loop situation. This selective targeting of α motor neurons is exceptional in that most other reflex and descending pathways target both α and γ motor neurons.

1	Other branches of group Ia fibers end on a variety of interneurons; however, one type, the reciprocal Ia inhibitory interneuron (black cell in Fig. 9.6 ), is particularly important with regard to the stretch reflex. These interneurons are identifiable because they are the only inhibitory interneurons •Fig. 9.6 Reflex Arc of the Stretch Reflex. Thepathwaybacktotherectusfemorisinthisarccontainsasinglesynapsewithinthecentralnervoussystem;hence,itisamonosynapticreflex.Theinterneuron,showninblack, isagroupIainhibitoryinterneuron.DRG,dorsalrootganglion. that receive input from both the group Ia afferent fibers and Renshaw cells (see

1	that receive input from both the group Ia afferent fibers and Renshaw cells (see Fig. 9.12 ). They end on α motor neurons that innervate the antagonist muscles—in this case, the hamstring muscles, including the semitendinosus muscle—which act to flex the knee. Other branches of the group Ia afferent fibers synapse with yet other neurons that originate ascending pathways that provide various parts of the brain (particularly the cerebellum and cerebral cortex) with information about the state of the muscle. The organization of the stretch reflex arc guarantees that one set of α motor neurons is activated and the opposing set is inhibited. This arrangement is known as reciprocal innervation. Although many reflexes involve such reciprocal innervation, this type of innervation is not the only possible organization of a motor control system; descending motor pathways can override such patterns.

1	The stretch reflex is quite powerful, in large part because of its monosynaptic nature. The power of this reflex also derives from the essentially maximal convergence and divergence that exist in this pathway, which is not apparent from the circuit diagrams, such as Fig. 9.6 , that are typically used to illustrate reflex pathways. That is, each group Ia fiber contacts virtually all homonymous α motor neurons, and each such α motor neuron receives input from every spindle in that muscle. Although its monosynaptic nature makes the group Ia reflex rapid and powerful, it also means that there is relatively little opportunity for direct control of CHAPTER 9 Organization of Motor Function activity flow through its reflex arc. The CNS overcomes this problem by controlling muscle spindle sensitivity via the γ motor neuron system as described previously. The Tonic Stretch Reflex

1	The Tonic Stretch Reflex The tonic stretch reflex can be elicited by passive bending of a joint. This reflex circuit includes both group Ia and group II afferent fibers from muscle spindles. Group II fibers make monosynaptic excitatory connections with α motor neurons, but they also excite them through disynaptic and polysynaptic pathways. Normally, there is ongoing activity in the group Ia and group II afferent fibers that helps maintain a baseline rate of firing of α motor neurons; therefore, the tonic stretch reflex contributes to muscle tone. Its activity also contributes to the ability to maintain a posture. For example, if the knee of a soldier standing at attention begins to flex because of fatigue, the quadriceps muscle is stretched, a tonic stretch reflex is elicited, and the quadriceps contracts more, thereby opposing the flexion and restoring the posture.

1	The foregoing discussion suggests that stretch reflexes can act like a negative-feedback system to control muscle length. By following the stretch reflex arc, it is possible to see that changes in its activity act to oppose changes in muscle length from a particular equilibrium point. For example, if the muscle’s length is increased, there will be an increase in firing by group Ia and group II fibers, which excites homonymous α motor neurons and leads to contraction of the muscle and reversal of the stretch. Similarly, passive shortening of the muscle unloads the spindles and leads to a decrease in the excitatory drive to the motor neurons and thus relaxation of the muscle. So how are humans able to rotate their joints? It is partly because the γ motor neurons are coactivated during a movement and thereby shift the equilibrium point of the spindle and partly because the gain or strength of the reflex is low enough that other input to the motor neuron can override the stretch reflex.

1	The inverse myotatic reflex acts to oppose changes in the level of force in the muscle. Just as the stretch reflex can be thought of as a feedback system to regulate muscle length, the inverse myotatic, or group Ib, reflex can be thought of as a feedback system to help maintain force levels in a muscle. With the upper part of the leg as an example, the group Ib reflex arc is depicted in Fig. 9.7. The arc starts with the Golgi tendon organ receptor, which senses the tension in the muscle. Golgi tendon organs are located at the junction of the tendon and the muscle fibers and thus lie in series with the muscle fibers, in contrast to the parallel arrangement of the muscle spindles (see Fig. 9.2 ). Golgi tendon organs have a diameter of about 100 µm and a length of about 1 mm. A Golgi tendon organ is innervated by the terminals of group Ib afferent fibers. These terminals wrap about bundles of collagen fibers in the tendon of a muscle (or in tendinous inscriptions within the muscle).

1	Hyperactivestretchreflexescanleadtotremorsandclonus,whicharetypesofinvoluntaryrhythmicmovements.Althoughthenegative-feedbackactionofthestretchreflexcanhelpstabilizethelimbataparticularposition,ifanexternalperturbationtothelimboccurs,theconductiondelaybetweentheinitiatingstimulus(musclestretch)andtheresponse(musclecontraction)cancausethestretchreflexcircuittobeasourceofinstabilitythatleadstorhythmicmovements.Specifically,clonusiselicitedbyasustainedstretchofamuscleinapersonwhohasspinalcorddamage.Normally,animposedsustainedstretchonamuscleelicitsanincreaseingroupIaandgroupIIfiberactivity,whichafteradelaycausesacontractioninthemusclethatopposesthestretchbutdoesnotcompletelyreturnthemuscletoitsinitiallengthbecausethegainofthestretchreflexismuchless than1.*

1	Thispartialcompensation,inturn,leadstoadecreaseingroupIaandgroupIIfiberactivity,whichcausesthelimbtolengthenagain,butnotfully.ThislengtheningonceagainincreasesgroupIaandgroupIIfiberactivity,andsoon.Thedelayisessentialinsettingupthisoscillationbecauseitcausesthefeedbacksignaltocontinueevenafterthemusclehascompensatedandthusresultsinanovercompensationthatleadstothenextovercorrection.However,becausethereflexgainisnormallymuchlessthan1,thisoscillationnormallydiesoutquickly(theovercompensationsdecreaseinamplituderapidly),andthemusclecomestorestatanintermediatelength.Incontrast,whendescendingmotorpathwaysaredamaged,theresultingchangesinspinalcordconnectivityandincreasesinneuronalexcitabilityresultinahyperactivereflex(whichisequivalenttoraisingthegainofthestretchreflexcloseto1).Inthiscase,thesuccessiveovercompensationsaremuchlarger,andanovertbuttransientoscillationcanbeobserved(clonus).Ifthegainequals1,theclonusdoesnotdieoutbutratherpersistsforaslongastheinitialstretchstimulusismaintained.

1	*Ingeneral,gain ofasystemisdefinedasitsoutputforagiveninput.Inthiscase,theinputtothesystemistheimposedstretch,andtheoutputismovementcausedbythestretchreflex–evokedcontraction. Because of their in-series relationship to the muscle, Golgi tendon organs can be activated either by muscle stretch or by muscle contraction. In both cases, however, the actual stimulus sensed by the Golgi tendon organ is the force that develops in the tendon to which it is linked. Thus the response to stretch is the result of the spring-like nature of the muscle (i.e., by Hooke’s law, the force on a spring is proportional to how much it is stretched). To distinguish between the responsiveness of the muscle spindles and Golgi tendon organs, the firing patterns of group Ia and group Ib fibers can be compared when a muscle is stretched and then held at a longer length (Fig.

1	9.8 ). The firing rate of the group Ia fibers maintains its increase until the stretch is reversed. In contrast, the group Ib fiber shows an initial large increase in firing, reflecting the increased tension on the muscle caused by the stretch, but then shows a gradual return toward its initial firing rate as the tension on the muscle is lowered because of cross-bridge recycling and the resultant lengthening of the •Fig. 9.7 Reflex Arc of the Inverse Myotatic Reflex. Theinterneuronsincludebothexcitatory(white) andinhibitory(black) interneurons.Thisisanexampleofadisynapticreflex.

1	•Fig. 9.8 Changes in Group Ia and Group Ib Firing Rates When Muscle Is Stretched to a New Length. Afteratransientburst,thefiringrateofthegroupIafiberremainsconstantatanewhigherlevelthatisproportionaltotheincreaseinlength(compareblue lines in upper and lower graphs).Incontrast,thegroupIbfibershowsaninitialrapidincreaseinfiringfollowedbyaslowdecreasebacktowarditsoriginallevel(lower graph, red line) andhasafiringprofilethatmatchesthetensionlevelinthemusclecausedbythestretch(upper graph, red line). sarcomeres. Therefore, Golgi tendon organs signal force, whereas spindles signal muscle length. Further evidence of this distinction is that group Ib firing is correlated with force level during isometric contraction even though muscle length and therefore group Ia activity are unchanged.

1	CHAPTER 9 Organization of Motor Function •Fig. 9.9 The Reflex Arc of the Flexion Reflex. Black interneuronsareinhibitory,andwhite onesareexcitatory.FRA,flexionreflexafferentfiber.++––FRASemitendinosusNociceptorFlexionExtensionRectusfemoris

1	The group Ib afferent fibers branch as they enter the spinal cord and end on interneurons. There are no mono-synaptic connections to α motor neurons. Rather, the group Ib afferent fibers synapse onto two classes of interneurons: interneurons that inhibit α motor neurons that supply the homonymous muscle (in this case the rectus femoris muscle) and excitatory interneurons that activate α motor neurons to the antagonist (the semitendinosus muscle). Because there are two synapses in series in the CNS, this is a disynaptic reflex arc. Because of these connections, group Ib fiber activity should have the opposite action of the group Ia stretch reflex during passive stretch of the muscle, which explains the group Ib reflex’s other name, the inverse myotatic reflex.

1	Functionally, however, the two reflex arcs can act synergistically, as the following example shows. Recall that the Golgi tendon organs monitor force levels across the tendon that they supply. If during maintained posture (such as standing at attention) knee extensors (such as the rectus femoris muscle) begin to fatigue, the force pulling on the patellar tendon declines. The decline in force reduces the activity of Golgi tendon organs in this tendon. Because the group Ib reflex normally inhibits the α motor neurons to the rectus femoris muscle, reduced activity of the Golgi tendon organs enhances the excitability of (i.e., disinhibits) the α motor neurons and thereby helps reverse the decrease in force caused by the fatigue. Simultaneously, bending of the knee stretches the knee extensors and activates the afferent fibers from the muscle spindles, which then excite the same α motor neurons. Thus coordinated action of afferent fibers from both the muscle spindle and Golgi tendon organ

1	and activates the afferent fibers from the muscle spindles, which then excite the same α motor neurons. Thus coordinated action of afferent fibers from both the muscle spindle and Golgi tendon organ help oppose the decrease in contraction of the rectus femoris muscle due to fatigue and thereby work together to maintain the standing posture.

1	The flexion reflex starts with activation of one or more of a variety of sensory receptors, including nociceptors, whose signals can be carried to the spinal cord via a variety of afferent fibers, including group II and group III fibers, collectively called the flexion reflex afferent (FRA) fibers. In flexion reflexes, afferent volleys (1) cause excitatory inter-neurons to activate the α motor neurons that supply the flexor muscles in the ipsilateral limb and (2) cause inhibitory interneurons to inhibit the α motor neurons that supply the antagonistic extensor muscles ( Fig. 9.9). This pattern of activity causes one or more joints in the stimulated limb to flex. In addition, commissural interneurons evoke the opposite pattern of activity in the contralateral side of the spinal cord (see

1	Fig. 9.9 ), which results in extension of the opposite limb, the crossed extension reflex. For lower limbs in humans (or for both forelimbs and hind limbs in quadrupeds), the crossed extension part of the reflex helps in maintaining balance by enabling the contralateral limb to be able to support the additional load that is transferred to it when the flexed limb is lifted.

1	Afterdamagetothedescendingmotorpathways,hyperactivestretchreflexesmayresultinspasticity,inwhichthereislargeresistancetopassiverotationofthelimbs.Inthiscondition,itmaybepossibletodemonstratewhatiscalledtheclasp-knife reflex.

1	reflex. Whenspasticityispresent,attemptstorotatealimbaboutajointinitiallymeethighresistance.However,iftheappliedforceisincreased,therecomesapointatwhichtheresistancesuddenlydissipatesandthelimbrotateseasily.Thischangeinresistanceiscausedbyreflexinhibition.ThegroupIbreflexarcsuggeststhatrisingactivityinthispathwaycouldunderliethesuddenreleaseofresistance,andindeed,theclasp-knifereflexwasonceattributedtotheactivationofGolgitendonorganswhenthesereceptorswerethoughttohaveahighthresholdtomusclestretch.However,thetendonorganshavesincebeenshowntobeactivatedatverylowlevelsofforceandarenolongerthoughttocausetheclasp-knifereflex.Itisnowthoughtthatthisreflexiscausedbytheactivationofotherhigh-thresholdmusclereceptorsthatsupplythefasciaaroundthemuscle.Signalsfromthesereceptorscausetheactivationofinterneuronsthatleadtoinhibitionofthehomonymousmotorneurons.

1	Because flexion typically brings the affected limb in closer to the body and away from a painful stimulus, flexion reflexes are a type of withdrawal reflex. In Fig. 9.9 , the neural circuit of the flexion reflex is shown for neurons that affect only the knee joint. Actually, however, considerable divergence of the primary afferent and interneuronal pathways occurs in the flexion reflex. In fact, all the major joints of a limb (e.g., hip, knee, and ankle) may be involved in a strong flexor withdrawal reflex. Details of the flexor withdrawal reflex vary, depending on the nature and location of the stimulus.

1	The interneurons subserving flexion reflexes also appear to be part of the central pattern generator (CPG) for generating locomotion and thus are an example of how reflex circuits are used for multiple purposes. A CPG is a set of neurons and circuits capable of generating the rhythmic activity that underlies motor acts, even in the absence of sensory input. For example, activation of the FRA inter-neurons leads to a pattern of flexor excitation and extensor inhibition on one side and the converse pattern on the opposite side, and if the FRA interneurons on each side of the spinal cord alternated in being active, a stepping pattern would emerge. That is, walking motion could result from alternately activating the FRA interneurons on each side. Note that such a rhythmic activity pattern in the FRA circuits need not be dependent on activity from the FRA fibers themselves (e.g., they could be activated by descending pathways from the brain).

1	To show that these circuits are actually involved in generating the locomotion rhythm, spinal cord preparations were made that showed spontaneous locomotion (i.e., if the brainstem is transected and weight is supported, the spinal cord circuits can generate activity that causes the limbs to perform a normal locomotion sequence). In one such preparation, the electromyographic signals from the flexors and extensors of a limb were recorded, and the FRA fibers then stimulated to demonstrate the effect on locomotion rhythm ( Fig. 9.10 ). Before any stimulus, a spontaneous alternating pattern of flexor and extensor electromyographic (EMG) activity exists. If the FRA fibers were not involved in the locomotion circuit, or at least were not a critical part of the circuits responsible for generating the rhythm (see Fig.

1	Fig. 9.10B ), the stimulus would be expected to produce only a transient response (i.e., a single burst in the flexor EMG record and brief inhibition of activity in the extensor EMG record) but to have no long-term effect on ongoing EMG pattern. Such a transient response is observed (see

1	Fig. 9.10A ; EMG records just after the stimulus). However, the stimulus also causes a permanent, approximately 180degree phase shift in locomotor rhythm, as can shown from a comparison of the times of contractions before and after the stimulus. The dashed vertical lines indicate the times at which a flexor EMG response would be expected if the stimulus had produced no phase shift from the EMG activity pattern. Before the stimulus, each vertical line is aligned with the onset of a flexor EMG burst, whereas after the stimulus, each vertical line occurs at the end of the flexor burst. Therefore, the stimulus affected the locomotor CPG itself and the FRA interneurons are a critical part of this CPG (see Fig. 9.10C

1	Fig. 9.10C A second important point illustrated by this experiment is that the locomotion CPG (and CPGs in general) can be influenced by strong afferent fiber activity. The afferent fiber’s influence ensures that the pattern generator adapts to changes in the terrain as locomotion proceeds. Such changes may occur rapidly during running, and locomotion must then be adjusted to ensure proper coordination. Determining Spinal Cord Organization Through the Use of Reflexes Convergence and divergence are important aspects of reflex pathways and of neuronal circuits in general. Several examples of these phenomena have been described in the previous discussion of the reflexes. Reflexes can be used to identify and characterize these phenomena in the spinal cord. For example, convergent input can be demonstrated through the phenomenon of spatial facilitation, which is illustrated in Fig. 9.11

1	Fig. 9.11 In this example, a monosynaptic reflex is elicited by electrical stimulation of the group Ia fibers in each of two nerves (see Fig. 9.11A ). The reflex response is characterized by a recording of the discharges of α motor axons from the appropriate ventral root (as a compound action potential). When nerve A is stimulated, a small compound action potential is recorded as reflex A. Similarly, when nerve B is stimulated, reflex B is recorded. Fig. 9.11B depicts the motor neurons contained within the motor nucleus.

1	Fig. 9.11B depicts the motor neurons contained within the motor nucleus. CHAPTER 9 Organization of Motor Function •Fig. 9.10 Phase Reset of Locomotion Rhythm by Flexion Reflex Afferent (FRA) Fiber Stimulation Helps Identify Neuronal Components of the Underlying Central Pattern Generator (CPG). A, EMGrecordsfromkneeflexorandextensormuscles.Notetherhythmicalternatingpatternbeforeapplicationofthestimulus.Thesolid vertical lines beloweachtraceindicatethetimesatwhichflexorcontractionisinitiated.Thedashed vertical lines indicatethetimesatwhichflexorcontractionwouldhavebeeninitiatedifthestimuluscausednolastingeffectontherhythmicpattern.B andC, TwopossiblemodelsfortheCPGunderlyingthelocomotorrhythmdepictedinA. B, TheFRAinterneurons(INs)intheCPGarenotshown.C, TheFRAinterneuronsareshown.ThedatashowninA supportthemodelshowninC. MN,motoneuron.(DatafromHultbornH,etal.Ann N Y Acad Sci. 1998;860:70.)

1	The α motor neurons in the discharge zones are activated above threshold when each nerve branch is stimulated separately. Thus a distinct pair of α motor neurons spike when each nerve is stimulated alone. In addition, each of these motor neuron pairs is surrounded by a subliminal fringe of eight additional motor neurons that are excited but not sufficiently to trigger spikes. When the two nerves are stimulated at the same time, a much larger reflex discharge is recorded (compare RA and RB with RA + RB recordings at the right of Fig. 9.11B ). As the figure demonstrates, this reflex represents the discharge of seven α motor neurons: the four that spiked after the singular stimulation of each nerve (two per nerve) and three additional α motor neurons (located in the facilitation zone) that are made to discharge only when the two nerves are stimulated simultaneously because they lie in the subliminal fringe for both nerves.

1	A similar effect could be elicited by repetitive stimulation of one of the nerves, provided that the stimuli occur close enough together that some of the excitatory effect of the first volley still persists after the second volley arrives. This effect is called temporal summation. Both spatial summation and temporal summation depend on the properties of the excitatory postsynaptic potentials evoked in α motor neurons by the group Ia afferent fibers (see Fig. 6.8). Convergence can also lead to inhibitory interactions between stimuli, a phenomenon called occlusion. If a volley in one of the two nerves in Fig. 9.11 reaches the motor nucleus at a time when the motor neurons are highly excitable, the reflex discharge is relatively large (see

1	Fig. 9.11 reaches the motor nucleus at a time when the motor neurons are highly excitable, the reflex discharge is relatively large (see Fig. 9.11C ). A similar volley in the other nerve might also produce a large reflex response. However, when the two nerves are excited simultaneously, the reflex can be less than the sum of the two independently evoked reflexes if the cells reaching threshold to activation of either of the two nerves alone overlap significantly. In this case, each afferent nerve activates 7 α motor neurons, but the volleys in the two nerves together cause only 12 α motor neurons to discharge because two motor neurons lie in the individual discharge zones of both afferent nerves.

1	The phenomena of spatial and temporal summation and occlusion can also be used to demonstrate interactions between spinal cord neurons and the various reflex circuits. To start, a monosynaptic reflex discharge can be evoked by stimulation of the group Ia afferent fibers in a muscle nerve. This is a test of the reflex excitability of a population of α motor neurons. The discharges of either extensor or flexor α motor neurons can be recorded if the proper muscle nerve to be stimulated is chosen. Other kinds of afferent fibers •Fig. 9.11 Spatial Facilitation. A, Arrangementforusingelectricallyevokedafferentvolleysandrecordingsfrommotoraxonsinaventralroottostudyreflexes.B, Experimentinwhichcombinedstimulationofafferentfibersintwomusclenervesresultedinspatialsummation(RAandRB).Thedischargezones(pink areas) encloseα motorneuronsthatareactivatedabovethresholdwheneachnervebranchisstimulatedseparately.InC, thecombinedvolleyscausedocclusion.(RedrawnfromEyzaguirreC,FidoneSJ.Physiology of the

1	areas) encloseα motorneuronsthatareactivatedabovethresholdwheneachnervebranchisstimulatedseparately.InC, thecombinedvolleyscausedocclusion.(RedrawnfromEyzaguirreC,FidoneSJ.Physiology of the Nervous System. 2nded.Chicago:Mosby–YearBook;1975.) are then stimulated along with the homonymous group Ia afferent fibers from the muscle to demonstrate whether the response to the group Ia stimulation changes. For example, stimulation of group Ia afferent fibers in the nerve to the antagonist muscles produces inhibition of the response to the homonymous group Ia stimulation (which is mediated by the reciprocal group Ia inhibitory interneuron described previously).

1	As another example, if the small afferent fibers of a cutaneous nerve are stimulated to evoke a flexion reflex, the responses to group Ia stimulation of the α motor neurons that innervate the extensor muscles are inhibited (and those of α motor neurons that innervate flexor muscles are potentiated). As a final example, stimulation of a ventral root causes inhibition of group Ia responses and inhibits the reciprocal group Ia inhibition. Because the ventral root contains only motor neuron axons, this result implies the presence of axon collaterals that excite inhibitory interneurons that feed back onto the same motor neuron population (

1	Fig. 9.12 ). These interneurons are named Renshaw cells. Because ventral root stimulation also inhibits the group Ia inhibition of antagonist motor neurons, but no other classes of interneurons, the reciprocal group Ia interneurons are uniquely inhibited by ventral root stimulation (and activated by group Ia stimulation). Experiments like these described have been used to provide a detailed knowledge of the circuitry of the spinal cord. •Fig. 9.12 Renshaw Cell (RC) Connections With Motor Neurons and Group Ia Inhibitory Interneurons. ThecircuitsshownmediategroupIareciprocalinhibitionofantagonistmuscles(inthiscase,anextensor)andinhibitionofthisreciprocalinhibitionbyRenshawcells.NotethatequivalentnumbersofRenshawcellsandgroupIainhibitoryinterneuronsareassociatedwithextensormotorneuronsandgroupIainputfromspindlesinextensormuscles,buttheyarenotshownforsimplicity.Orange cells areinhibitory,andblue andgreen cells areexcitatory. Classification of Descending Motor Pathways

1	Classification of Descending Motor Pathways Descending motor pathways were traditionally subdivided into pyramidal and extrapyramidal pathways. This terminology reflects a clinical dichotomy between pyramidal tract disease and extrapyramidal disease. In pyramidal tract disease, the corticospinal (pyramidal) tract is interrupted. The signs of this disease were originally attributed to the loss of function of the pyramidal tract (so named because the corticospinal tract passes through the medullary pyramid). However, in many cases of pyramidal tract disease, the functions of other pathways are also altered, and most signs of pyramidal tract disease (see the later section “ Caused by Lesions of Descending Motor Pathways ”) are apparently not caused solely by loss of the corticospinal tract but also reflect damage to additional motor pathways. The

1	Caused by Lesions of Descending Motor Pathways ”) are apparently not caused solely by loss of the corticospinal tract but also reflect damage to additional motor pathways. The CHAPTER 9 Organization of Motor Function term extrapyramidal is even more problematic. Thus this classification system is not used in this book. Another way of classifying the motor pathways is based on their sites of termination in the spinal cord and the consequent differences in their roles in the control of movement and posture. The lateral pathways terminate in the lateral portions of the spinal cord’s gray matter ( Fig. 9.13 ). The lateral pathways can excite motor neurons directly, although interneurons are their main target. They influence reflex arcs that control fine movement of the distal ends of limbs, as well as those that activate supporting musculature in the proximal ends of limbs. The medial pathways end in the medial ventral horn on the medial group of interneurons (see

1	Fig. 9.13 ). These interneurons connect bilaterally with motor neurons that control the axial musculature and thereby contribute to balance and posture. They also contribute to the control of proximal limb muscles. In this book, the terms lateral and medial are used to classify the descending motor pathways. However, even this terminology is not perfect, partly because although motor neuron cell bodies form localized columns, motor neuron dendritic trees are rather large and typically span most of the ventral horn. Thus any motor neuron can potentially receive input from so-called medial or lateral system pathways. The Lateral System

1	The Lateral System The corticospinal and corticobulbar tracts originate from a wide region of the cerebral cortex. This region includes the primary motor, premotor, supplementary, and cingulate motor areas of the frontal lobe and the somatosensory cortex of the parietal lobe. The cells of origin of these tracts include both large and small pyramidal cells of layer V of the cortex, including the giant pyramidal cells of Betz. Although Betz cells are a defining feature of the primary motor cortex, they represent a small minority (<5%) of the cells that contribute to these tracts, in part because they are found only in the primary motor cortex, and even there they represent a minority of the cells contributing to the tracts. These tracts leave the cortex and enter the internal capsule, then traverse the midbrain in the cerebral peduncle, pass through the basilar pons, and emerge to form the pyramids on the ventral surface of the medulla (see

1	Fig. 9.13A). The corticobulbar axons leave the tract as it descends in the brainstem and terminate in the motor nuclei of the various cranial nerves. The corticospinal fibers continue caudally, and in the most caudal region of the medulla, about 90% of them cross to the opposite side. They then descend in the contralateral lateral funiculus as the lateral corticospinal tract. The lateral corticospinal axons terminate at all spinal cord levels, primarily on interneurons, but also on motor neurons. The remaining uncrossed axons continue caudally in the ventral funiculus on the same side as the ventral corticospinal tract, which belongs to the medial system. Many of these fibers ultimately decussate (cross) at the spinal cord level at which they terminate.

1	•Fig. 9.13 Descending Motor Pathways. Majorpathwaysconnectingthecorticalandbrainstemmotorareastothespinalcordareshown.A, Lateralsystempathways,corticospinal(red) andrubrospinal(blue) pathways.NotethattheventralcorticospinalpathwayispartofthemedialsystembutisshowninA forsimplicity.B, Medialsystempathways,medullary(blue) andpontine(green) reticulospinalandlateralvestibulospinal(red) pathways.C-B,corticobulbar;C-P,corticopontine;C-S,corticospinal;LCST,lateralcorticospinaltract;VCST,ventralcorticospinaltract.

1	The lateral corticospinal tract is a relatively minor tract in lower mammals but is quantitatively and functionally very important in primates, particularly in humans, in which it contains more than 1 million axons. This number still represents a relatively small proportion of the outflow from the cortex because there are approximately 20 million axons in the cerebral peduncles. Nevertheless, the corticospinal pathway is critical for the fine independent control of finger movement, inasmuch as isolated lesions of the corticospinal tract typically lead to a permanent loss of this ability, even though other movement abilities are often recovered with such lesions. Indeed, in primates, corticospinal synapses directly onto motor neurons are particularly prevalent for the motor neurons controlling finger muscles and are probably the basis of the ability to make independent, finely controlled finger movements.

1	The corticobulbar tract, which projects to the cranial nerve motor nuclei, has subdivisions that are comparable with the lateral and ventral corticospinal tracts. For example, part of the corticobulbar tract ends contralaterally in the portion of the facial nucleus that supplies muscles of the lower part of the face and in the hypoglossal nucleus. This component of the corticobulbar tract is organized like the lateral corticospinal tract. The remainder of the corticobulbar tract ends bilaterally.

1	The rubrospinal tract originates in the magnocellular portion of the red nucleus, which is located in the midbrain tegmentum. These fibers decussate in the midbrain, descend through the pons and medulla, and then take up a position just ventral to the lateral corticospinal tract in the spinal cord. They preferentially affect motor neurons controlling distal musculature, as do the corticospinal fibers. Red nucleus neurons receive input from the cerebellum and from the motor cortex; thus making this an area of integration of activity from these two motor systems. The Medial System

1	The Medial System The ventral corticospinal tract and much of the corticobulbar tract can be regarded as medial system pathways. These tracts end on the medial group of interneurons in the spinal cord and on equivalent neurons in the brainstem. The axial muscles are controlled by these pathways. These muscles often contract bilaterally to provide postural support or some other bilateral function, such as swallowing or wrinkling of the brow. Other medial system pathways originate in the brainstem. These include the pontine and medullary reticulospinal tracts, the lateral and medial vestibulospinal tracts, and the tectospinal tract. The cells that give rise to the pontine reticulospinal tract are in the medial pontine reticular formation. The tract descends in the ventral funiculus, and it ends on the CHAPTER 9 Organization of Motor Function ipsilateral medial group of interneurons. Its function is to excite motor neurons to the proximal extensor muscles to support posture.

1	CHAPTER 9 Organization of Motor Function ipsilateral medial group of interneurons. Its function is to excite motor neurons to the proximal extensor muscles to support posture. The medullary reticulospinal tracts arise from neurons of the medial medulla, particularly those of the gigantocellularis reticular nucleus. The tracts descend bilaterally in the ventral lateral funiculus, and they end mainly on interneurons associated with cell groups of medial motor neurons. The function of the pathway is mainly inhibitory.

1	The lateral vestibulospinal tract originates in the lateral vestibular nucleus, also known as Deiter’s nucleus. This tract descends ipsilaterally through the ventral funiculus of the spinal cord and ends on interneurons associated with the medial motor neuron groups. The lateral vestibulospinal tract excites motor neurons that supply extensor muscles of the proximal part of the limb that are important for postural control. In addition, this pathway inhibits flexor motor neurons because it also excites the reciprocal group Ia interneurons that receive group Ia input from extensor muscles, which in turn inhibit flexor motor neurons. The excitatory input to the lateral vestibular nucleus is from both the semicircular canals and the otolith organs, whereas the inhibitory input is from the Purkinje cells of the anterior vermis region of the cerebellar cortex. An important function of the lateral vestibulospinal tract is to assist in postural adjustments after angular and linear

1	the Purkinje cells of the anterior vermis region of the cerebellar cortex. An important function of the lateral vestibulospinal tract is to assist in postural adjustments after angular and linear accelerations of the head.

1	The medial vestibulospinal tract originates from the medial vestibular nucleus. This tract descends in the ventral funiculus of the spinal cord to the cervical and midthoracic levels, and it ends on the medial group of interneurons. Sensory input to the medial vestibular nucleus from the labyrinth is chiefly from the semicircular canals. This pathway thus mediates adjustments in head position in response to angular acceleration of the head. The Tectospinal Tract The tectospinal tract originates in the deep layers of the superior colliculus. The axons cross to the contralateral side, just below the periaqueductal gray matter. They then descend in the ventral funiculus of the spinal cord to terminate on the medial group of interneurons in the upper cervical spinal cord. The tectospinal tract regulates head movement in response to visual, auditory, and somatic stimuli.

1	In addition to the lateral and medial systems, less specifically organized systems descend from the brainstem to the spinal cord. These include several pathways in which monoamines serve as synaptic transmitters. The locus coeruleus and the nucleus subcoeruleus are nuclei located in the rostral pons, and they are composed of norepinephrine-containing neurons. These nuclei project widely throughout the CNS, and their projection to the spinal cord travels in the lateral funiculus. Their terminals are on interneurons and motor neurons. The dominant effect of the pathway is inhibitory. The raphe nuclei of the medulla also project widely throughout the CNS and give rise to several raphe-spinal pathways. With regard to motor function, the ventral horn projection may enhance motor activity.

1	In general, the monoaminergic pathways act to alter the responsiveness of spinal cord circuits, including the reflex arcs. In this way, they induce widespread changes in excitability rather than discrete movements or specific changes in behavior. Motor Deficits Caused by Lesions of Descending Motor Pathways

1	A common cause of motor impairment in humans is interruption of the cerebral cortical efferent fibers in the internal capsule; such interruptions occur in capsular strokes. The resulting disorder is often termed a pyramidal tract syndrome, or upper motor neuron disease, although these names are misnomers. Motor changes characteristic of this disorder include (1) increased phasic and tonic stretch reflexes (spasticity); (2) weakness, usually of the distal muscles, especially the finger muscles; (3) pathological reflexes, including the sign of Babinski (dorsiflexion of the big toe and fanning of the other toes when the sole of the foot is stroked); and (4) a reduction in superficial reflexes, such as the abdominal and cremasteric reflexes. Of importance is that if only the corticospinal tract is interrupted, as can occur with a lesion of the medullary pyramid, most of these signs are much reduced or absent. In this situation, the most prominent deficits are weakness of the distal

1	tract is interrupted, as can occur with a lesion of the medullary pyramid, most of these signs are much reduced or absent. In this situation, the most prominent deficits are weakness of the distal muscles, especially those of the fingers, and a Babinski sign. Spasticity does not occur; instead, muscle tone may actually decrease. Evidently, the presence of spasticity requires the disordered function of other pathways, such as the reticulospinal tracts, as would occur after loss of the descending cortical influence to the brainstem nuclei of origin of these tracts.

1	The effects of interruption of the medial system pathways are quite different from those produced by corticospinal tract lesions. The main deficits associated with medial system interruption are an initial reduction in the tone of postural muscles and loss of righting reflexes. Long-term effects include locomotor impairment and frequent falling. However, manual manipulation of objects is perfectly normal. The Decerebrate Preparation The decerebrate preparation has been useful for experimentally investigating how various descending pathways interact with the spinal cord circuitry. Surgical decerebration is achieved either by transection of the midbrain, often at an intercollicular level, or by occlusion of the blood vessels feeding this area. In the latter case, a lesion also occurs in the anterior vermis of the cerebellum, an important distinction.

1	With the intercollicular transection, some descending pathways, such as those originating in the cerebral cortex, are interrupted, whereas others, such as those originating in the brainstem, remain intact.

1	However, remember that the corticospinal tract is only one component of the cortical descending fibers. Many other cortical fibers project to locations throughout the brainstem, including the nuclei of origin for the medial descending pathways. Loss of these cortical control systems results in altered activity in the intact descending pathways. As a result, affected animals show hypertonia and suppression of some spinal reflexes, such as the flexion reflex, and exaggeration of others, such as the stretch reflex; this condition is called decerebrate rigidity. Decerebrate animals maintain a posture that has been called exaggerated standing. Human patients with brainstem damage may also develop a decerebrate state that has many of the same reflex features as animal preparations. The prognosis in such patients is poor if signs of decerebration appear.

1	Loss of descending control on the reticular formation results in increased activity in the pontine reticulospinal pathway and decreased activity in the medullary reticulospinal pathway. Such increase and decrease in activity, respectively, produces increased excitation and decreased inhibition (disinhibition) of the motor neurons, which explains the observed rigidity. Interestingly, this hypertonia can be relieved by cutting the dorsal roots, which indicates that the reticulospinal tracts have a major effect on γ motor neurons. This is because γ motor neuron activity alters muscle stiffness by increasing muscle spindle sensitivity and thereby causes increased activity in the group Ia and group II afferent fibers, which travel through the dorsal roots into the spinal cord in order to innervate the α motor neurons.

1	When vessel occlusion is used to generate the decerebrate state, the lateral vestibulospinal tract becomes hyperactive because of damage to Purkinje cells in the anterior vermis of the cerebellum, which provide the major inhibitory projection to the lateral vestibular nucleus. This hypertonia is actually not lost after transection of the dorsal roots, which implies that the lateral vestibulospinal tract is acting to a significant extent directly on α motor neurons (either monosynaptically or via interneurons). Brainstem Control of Posture and Movement

1	Brainstem Control of Posture and Movement The importance of motor control pathways that originate in the brainstem is evident from observations of the extensor hypertonus and increased phasic stretch reflexes that occur in decerebrate animals. Particular brainstem systems have been identified as influencing posture and locomotion. Brainstem circuits are also critically involved in the control of eye movement; these circuits are discussed in a separate section at the end of the chapter. Several reflex mechanisms are evoked when the head is moved or the neck is bent. There are three types of postural reflexes: vestibular reflexes, tonic neck reflexes, and righting reflexes. The sensory receptors responsible for these reflexes include the vestibular apparatus (see ), which is stimulated by head movement, and stretch receptors in the neck.

1	The vestibular reflexes constitute one class of postural reflex. Rotation of the head activates sensory receptors of the semicircular canals (see ). In addition to generating eye movement, the sensory input to the vestibular nuclei results in postural adjustments. Such adjustments are mediated by commands transmitted to the spinal cord through the lateral and medial vestibulospinal tracts and the reticulospinal tracts. The lateral vestibulospinal tract activates extensor muscles that support posture. For instance, if the head is rotated to the left, postural support is increased on the left side. This increased support prevents the person from falling to the left as the head rotation continues. A person who has any disease that eliminates labyrinthine function in the left ear tends to fall to the left. Conversely, a person with a disease that irritates (stimulates) the left labyrinth tends to fall to the right. The medial vestibulospinal tract causes contractions of neck muscles that

1	to the left. Conversely, a person with a disease that irritates (stimulates) the left labyrinth tends to fall to the right. The medial vestibulospinal tract causes contractions of neck muscles that oppose the induced movement (vestibulocollic reflex).

1	Tilting the head also changes the linear acceleration on individual hair cells of the otolith organs of the vestibular apparatus. The resulting changes in hair cell activity can produce eye movement and postural adjustment. For example, when a quadruped, such as a cat, tilts the head and body forward (without bending the neck and consequently without evoking the tonic neck reflexes), the result is extension of the forelimbs and flexion of the hind limbs. This vestibular action tends to restore the body toward its original orientation. Conversely, if the quadruped tilts the head and body backward (without bending the neck), the forelimbs flex and the hind limbs extend. Otolithic organs also contribute to the vestibular placing reaction. If an animal, such as a cat, is dropped, stimulation of the utricles leads to extension of the forelimbs in preparation for landing.

1	The tonic neck reflexes are another type of positional reflex. These reflexes are activated by the muscle spindles found in neck muscles. These muscles contain the largest concentration of muscle spindles of any muscle in the body. If the neck is bent (without tilting of the head), the neck muscle spindles evoke tonic neck reflexes without interference from the vestibular system. When the neck is extended, the forelimbs extend and the hind limbs flex. The opposite effects occur when the neck is flexed. Note that these effects are opposite those evoked by the vestibular system. Furthermore, if the neck is bent to the left, the extensor muscles in the limbs on the left contract more, and the flexor muscles in the limbs on the right side relax. CHAPTER 9 Organization of Motor Function

1	CHAPTER 9 Organization of Motor Function The third class of postural reflex is the righting reflexes. These reflexes tend to restore an altered position of the head and body toward normal. The receptors responsible for righting reflexes include the vestibular apparatus, the neck stretch receptors, and mechanoreceptors of the body wall. Brainstem Control of Locomotion The spinal cord contains neural circuits that serve as central pattern generators for locomotion, as discussed earlier. These CPG circuits produce very regular rhythmic output that characterizes stereotyped behavior, such as walking. The irregularities of real-world environments, however, often require modification of this stereotyped output (e.g., if you are walking and see a hole in the floor where you are about to step, you can extend the forward swing of your leg past the hole onto solid ground beyond it). Such modifications can be the result of sensory input to the spinal cord, as shown in

1	Such modifications can be the result of sensory input to the spinal cord, as shown in Fig. 9.10 , in which stimulation of FRA fibers in a peripheral nerve caused a phase shift in the locomotor pattern. They can also be the result of descending commands along the motor pathways discussed earlier. In this case, sensory data (e.g., visual) can be used by the brain to make anticipatory modifications in CPG activity so that potential obstacles can be avoided. In addition, people can voluntarily control activation, or shutdown, of the CPG (i.e., deciding consciously when to start and stop walking). Such voluntary regulation of spinal CPGs originates in the cerebral cortex; however, much of the cortical influence on locomotion appears to be mediated via projections to brainstem regions known as locomotor regions. A locomotor region can be defined as a brain area that, when stimulated, leads to sustained locomotion.

1	There are several such locomotor regions in the brain-stem, and they are located at different levels ranging from the subthalamus to the medulla and are connected with each other. The best known is the midbrain locomotor region, which is thought to organize commands to initiate locomotion. It is located in the midbrain at the level of the inferior colliculus. Voluntary activity that originates in the motor cortex can trigger locomotion by the action of corticobulbar fibers projecting to the midbrain locomotor region. The commands are relayed through the reticular formation and then to the spinal cord via the reticulospinal tracts. Motor Control by the Cerebral Cortex

1	Motor Control by the Cerebral Cortex Thus far in this chapter, emphasis has been on reflexes and relatively automatic types of movement. We now discuss the neural basis for more complex, goal-directed voluntary movement. Such movement often varies when repeated and is frequently initiated as a result of cognitive processes rather than in direct response to an external stimulus. Thus it requires the participation of motor areas of the cerebral cortex. •Fig. 9.14 Motor Areas of the Frontal Cortex. A andB, Lateralandmedialviewsofahemisphere,showingthemajorcorticalmotorareas.FEF,frontaleyefield.Theinset inB showsthewallsofthecingulatesulcus,whichcontainthecingulatemotorareas.NumbersinparenthesesareBrodmannareanumbersforthecingulatemotorareas(CMAs).C, Diagramshowinginterconnectionsofthemotorareas.PMA,premotorarea;SMA,supplementarymotorarea.

1	First, consider what is necessary to generate a voluntary movement. For example, to make a reaching movement with your arm, you must first identify the target (or goal) and locate it in external space. Next, a limb trajectory must be determined on the basis of an internal representation of your arm and, in particular, your hand in relation to the target. Finally, a set of forces necessary to generate the desired trajectory must be computed. This process is often thought of as a series of transformations between coordinate systems. For example, the location of a visually identified target is measured in a retinotopic space, but its location is perceived in an external or world space (i.e., the position of a nonmoving target is perceived as stable, even when the eye, and thus the target’s image on the retina, changes). Next, calculation of a trajectory would involve a body-or hand-centered system, and finally, forces must ultimately be computed in a muscle-based reference frame.

1	These steps form a linear sequence, and traditionally it was thought that a hierarchy of motor areas carried out the successive steps. For example, the target of the movement was thought to be identified by pooling of sensory information in the posterior parietal cerebral cortex (Fig. 9.14A ). This information would then be transmitted to the supplementary motor and premotor areas, where a motor plan would be developed and then forwarded to the primary motor cortex, whose activity would be related to the final execution stage (e.g., generation of appropriate force levels). The motor cortex would then transmit commands, via the descending pathways discussed earlier, to the spinal cord and brainstem motor nuclei.

1	Although there is significant evidence in support of this hierarchical view of the generation of voluntary movement by the cortical motor system, more recent results have suggested a different conception: namely, that the various motor areas should be thought of as forming a parallel distributed network rather than a strict hierarchy (see Fig.

1	Fig. 9.14C ). For example, each cortical motor area makes its own significant contribution to the descending motor pathways; the primary motor cortex contributes only approximately half the fibers in the corticospinal tract that arise from the frontal lobe. Moreover, the various motor areas are all bidirectionally connected to each other, and results of the single-unit recording studies described later suggest that each of the areas plays a role in several of the stages of planning and executing a movement. This debate forms one of the themes of the following discussion because in its various guises, the distributed network versus hierarchical organization debate has been ongoing for decades and will probably continue for some time.

1	The motor areas in the cerebral cortex were originally defined on the basis of experiments in which electrical stimuli applied to the cortex evoked discrete, contralateral movement. Movement, however, can also be evoked when other cortical areas are stimulated more intensely. Thus motor areas are defined as those from which movement can be evoked by the lowest stimulus intensity. On the basis of these stimulation studies, the effects produced by lesions, results of anatomical experiments, electrophysiological recordings, and modern imaging studies in humans, many “motor” areas of the cerebral cortex have been recognized (see

1	Fig. 9.14 ), including the primary motor cortex in the precentral gyrus, the premotor area just rostral to the primary motor cortex, the supplementary motor cortex on the medial aspect of the hemisphere, and three cingulate motor areas located on the walls of the cingulate sulcus in the frontal lobe. There are also cortical regions in other lobes whose activity is related specifically to eye movement (see the section Somatotopic Organization of Cortical Motor Areas The primary motor cortex (or just motor cortex) can be defined as the region of cortex from which movements are elicited with the least amount of electrical stimulation. It is essentially congruent with the Brodmann cytoarchitectonic area 4 (see

1	Fig. 10.3). In humans it is located on the parts of the precentral gyrus that form the rostral wall of the central sulcus and the caudal half of the apex of the gyrus. On the basis of initial mapping studies, which were done with surface stimulation, the motor cortex was described as having a topographic organization that parallels that of the somatosensory cortex. The face, body, and upper limb were represented on the lateral surface with the face located inferiorly, near the lateral fissure, the torso most superiorly, and the lower extremity mostly on the medial aspect of the hemisphere. This somatotopic organization is often represented as a figurine or in a graphic form called a motor homunculus (

1	Fig. 9.15B ). The distortion of the various body parts in the homunculus indicates approximately how much of the cortex is devoted to their motor control. This simple homunculus was likened to a piano keyboard and fit well with traditional conceptions of the motor cortex being the final cortical stage and acting as a relay for sending motor commands to the spinal cord. Beginning in the 1960s and 1970s, investigators in mapping studies began using microelectrodes inserted to the deep, or output, layers of the cortex to apply stimuli. With this technique, called intracortical microstimulation, much lower stimulus intensities could be used to evoke movements and thus allowed higher resolution mapping of the motor cortex, which revealed a much more complex topography than was previously imagined (see CHAPTER 9 Organization of Motor Function

1	CHAPTER 9 Organization of Motor Function Fig. 9.15C ). Movement about each joint was found to be evoked by many noncontiguous columns throughout wide regions of the motor cortex. Thus cell columns related to movement about a particular joint are actually interspersed among columns that control movement about many other joints. In sum, although the motor cortex may have large subdivisions corresponding to a limb or the head, within each such area there is a complex intermingling of cell columns that control the muscles within that body part.

1	Such mixing of cell columns makes functional sense because most movement requires the coordinated action of muscles throughout a limb and most connectivity in the cortex is localized (i.e., axon collaterals that connect different cell columns are primarily confined to a 1to 3-mm region surrounding the column from which they originate). Thus when multiple cell columns that control movement about a joint are present and intermixed with columns controlling movement about other joints, multijoint movement can be generated as a whole.

1	Although the topographic map of the motor cortex is in part anatomically determined by the topography of the corticospinal pathway, it is also a dynamic map. Axon collaterals link the different cell columns, so that activity in one column could potentially lead to movement about multiple joints. In fact, this can happen, but these intercolumnar connections are modulated by inhibitory interneurons that transmit or secrete gamma-aminobutyric acid (GABA). This was shown by locally blocking GABA in one region of the motor cortex and then stimulating the neighboring region. Before the block, stimuli evoked contractions of one set of muscles, but once inhibition was blocked, contractions were also evoked in muscles controlled by the region that was no longer inhibited (

1	Fig. 9.16 ). Functional connections between cell columns can be controlled on a millisecond time scale, and depending on their state, the motor cortex map can be radically changed. Longer term plastic changes are also known to occur; for example, the use (or disuse) of a body part can affect the size of its somatotopic representation. The supplementary motor area (SMA) is located mainly on the medial surface of the hemisphere, just anterior to the primary motor cortex, and corresponds to the medial portion of Brodmann area 6 (see

1	The supplementary motor area (SMA) is located mainly on the medial surface of the hemisphere, just anterior to the primary motor cortex, and corresponds to the medial portion of Brodmann area 6 (see Fig. 9.14). It is subdivided into two regions: the more posterior part is referred to as the SMA proper (or just SMA), and the anterior portion is called the pre-SMA. The SMA proper is similar to the other motor areas already listed: it contains a complete somatotopic map, it contributes to the corticospinal tract, and it is interconnected with the other motor areas. In contrast, the pre-SMA is not strongly connected with the other motor areas and spinal cord but rather is connected to the prefrontal cortex. The results of stimulation studies show that as in the motor cortex, there is a complete somatotopic map in the SMA. Stimulation of the SMA can evoke isolated movement

1	The results of stimulation studies show that as in the motor cortex, there is a complete somatotopic map in the SMA. Stimulation of the SMA can evoke isolated movement Rostral wall of central sulcus •Fig. 9.15 Traditional and Modern Views of Motor Cortex Musculotopic Organization. A, Lateralviewofthecerebrum,showingaplaneofsectionthroughtheprecentralgyrus(primarymotorcortex)thatcorrespondstothesectionshowninB. B, Classicviewofmotorcortexmusculotopy(“muscularhomunculus”).C, Modernviewofmotorcortexorganizationinwhicheachbodypartisrepresentedmultipletimesacrossseveraldiscreteregions.

1	CHAPTER 9 Organization of Motor Function •Fig. 9.16 Dynamic Nature of a Motor Cortex Musculotopic Map. InhibitoryGABAergicinterneuronsplayanimportantroleinshapingmotorresponsestostimulationofeachregionofthemotorcortex.A, Schematicviewofexcitatoryconnectionsbetweentworegionsofprimarymotorcortexandlocalinhibitoryneuronswithinasingleregion.B, Schematicviewofaratbrain,indicatingmotorcortex(MC)regionswhereelectricalstimuliwereappliedtoevokemovements(Vibregion)andbicucullinewasappliedtoblockGABAergicsynapses(intheFLregion).FL,forelimb;Vib,vibrissa.C, ForelimbEMGrecordsshowingresponsetostimulationoftheVibregionbeforeandduringtheapplicationofbicucullineandafteritwaswashedout.NotethatVibstimulationevokedvibrissaemovementinallconditionsbutevokedforelimbmovementonlywheninhibitoryinterneuronsinFLwereblocked.(DatafromJacobsK,DonoghueJ.Science. 1991;251:944.) about single joints, similar to that after stimulation of the motor cortex, but stimulation must be of higher intensity and longer duration;

1	1991;251:944.) about single joints, similar to that after stimulation of the motor cortex, but stimulation must be of higher intensity and longer duration; moreover, the evoked movements are often more complex than those evoked by stimulation of the motor cortex. However, longer duration stimulation of the primary motor cortex can also evoke complex, apparently purposeful movement sequences, and so the distinction is not absolute. In addition, stimulation of the SMA can produce vocalization or complex postural movements, but it can also have the opposite result: namely, a temporary arrest of movement or speech. Removal of the supplementary motor cortex retards movement of the opposite extremities and may result in forced grasping movements with the contralateral hand.

1	This area lies rostral to the primary motor cortex and is contained in Brodmann area 6 on the lateral surface of the brain (see Fig. 9.14 ). It can be distinguished from the primary motor cortex by the higher stimulus intensities needed to evoke movement. The premotor area has been divided into two functionally distinct subdivisions: dorsal and ventral. Like the motor cortex, both subdivisions are somatotopically organized and both contribute to the corticospinal tract. The dorsal division (PMd) contains a relatively complete map representing the leg, trunk, arm, and face. In contrast, the somatotopic map of the ventral division (PMv) is mostly limited to the arm and face, with only a small leg representation. Thus the PMv appears to be specialized for control of upper limb and head movement. A second difference between the subdivisions is that the PMd contains a large representation of the proximal muscles, whereas the PMv has a large representation of the distal muscles.

1	These motor areas are located within the cingulate sulcus at approximately the same anterior-posterior level as the SMA. There are three cingulate motor areas (dorsal, ventral, and rostral; see Fig. 9.14B ). Each contains a somatotopic map and contributes to the corticospinal tract. Microstimulation in these areas evokes movement similar to that evoked by motor cortex stimulation, except that, again, higher stimulus intensities are needed. Single-cell recordings during movements have shown that the spontaneous activity of neurons in the cingulate motor areas is related to the preparation and execution of movements. Connections of the Cortical Motor Areas

1	Connections of the Cortical Motor Areas The motor areas of the cortex receive input from a number of sources, cortical and subcortical; however, the largest source of synapses in an area is the area itself: specifically, the local intrinsic connections. Moreover, all the motor areas described earlier are bidirectionally connected to each other with high topographic specificity (see

1	Fig. 9.14C ). For example, the arm regions of the primary motor cortex and the cingulate motor areas project to each other. Ascending pathways relay sensory information to the thalamus. This information can reach the motor cortex directly from the thalamus or indirectly by way of the somatosensory cortex. Both somatosensory information and visual information are conveyed to the motor areas from the posterior parietal cortex. The motor areas of the cortex also receive information through circuits that interconnect them with the other major brain regions involved in motor control: namely, the cerebellum and basal ganglia. These two structures project to distinct parts of the thalamus (the ventral lateral and ventral anterior nuclei), which then project to the cortical motor areas.

1	The output of the cortical motor areas to the spinal cord and brainstem is conducted through several descending pathways. These pathways include not only direct projections through the corticospinal and corticobulbar tracts (to the cranial nerve nuclei) but also indirect projections to the red nucleus and to various nuclei in the reticular formation. Descending projections from these brainstem sites were reviewed in the section .” Control of head and neck muscles is mediated by projections to the various cranial nerve nuclei. The motor regions also project to the cerebellum and basal ganglia, thus completing neuroanatomical loops with these structures. The major connection to the cerebellum is via the corticopontine projections to the basilar pontine nuclei, which in turn project to the cerebellum. In addition, the cortical motor areas project, mostly via disynaptic pathways that synapse in the midbrain, to the inferior olivary nucleus, another important precerebellar area. The

1	the cerebellum. In addition, the cortical motor areas project, mostly via disynaptic pathways that synapse in the midbrain, to the inferior olivary nucleus, another important precerebellar area. The cortical motor regions project directly to the striatum of the basal ganglia. Finally, there are major projections to the thalamus by which the cortex regulates the information that it receives.

1	Activity of Motor Cortex Cells The role of individual motor cortex neurons in the control of movement has been extensively investigated in trained monkeys. In these experiments, discharges from a neuron in the primary motor cortex are recorded during the execution of a previously learned simple movement, such as wrist flexion, made immediately in response to a sensory cue (Fig.

1	9.17 ). Motor cortex neurons were found to change their firing rates before initiation of the movement, and the onset of this change was correlated with the reaction time (i.e., the time from the cue to onset of the movement). Moreover, in this task, the change in firing of motor cortex neurons was often correlated with the contractile force of the muscle that generates the movement and with the rate of change in force rather than with the position of the joint. These findings suggest that these cells are involved in the final stages of planning and executing movements, which is consistent with the hierarchical view of the cortical motor areas.

1	However, even in these early experiments, the firing rates of some motor cortex cells appeared to relate to earlier planning stages. Moreover, even when a monkey was trained to withhold the movement for a certain period after the cue, the firing rates of motor cortex neurons still changed despite the absence of any movement. Such “set-related” activity has been amply confirmed in a variety of other tasks and suggests that motor cortex activity may be involved in the earlier planning stages along with activity in other motor areas of the cortex. It also suggests the possibility that other, perhaps subcortical, systems may be needed to generate a trigger signal for the initiation of movement. In subsequent studies, researchers have used tasks in which animals were trained to move a manipulandum (a device with a handle to hold and a small circle on the end) to capture lighted targets on a surface in front of them (Fig.

1	9.18A ). These experiments demonstrated that cells in the arm region of the motor cortex showed changes in their firing rates in response to movement in many different directions and thus were described as broadly tuned (see Fig. 9.18B ). That is, a cell that showed a maximal increase for movement in one particular direction, called its preferred direction, would also show somewhat smaller increases or even decreases for movement in other directions (see Fig. 9.18C ). Moreover, the preferred directions of the different cells were uniformly distributed across all 360 degrees of possible movement directions.

1	Fig. 9.18C ). Moreover, the preferred directions of the different cells were uniformly distributed across all 360 degrees of possible movement directions. These results implied that a particular cell is probably involved in most arm movements, but they also raised the issue of how precise movements could be made with such broadly tuned cells. It was suggested that although changes in the activity of individual cells could not precisely predict or specify the direction of the upcoming movement, the net activity of the population could. To test this idea, models were made in which the activity of each cell is represented as a vector (see Fig. 9.18D ). The direction of each cell vector is determined by the preferred direction of the cell, and the magnitude of the vector for a particular movement is proportional to the firing rate of the cell during the time preceding the movement. The individual cell vectors (see

1	Fig. 9.18D , black lines) from hundreds of cells can then be vectorially summed to get a resultant or population vector (see Fig. 9.18D , red lines) that accurately predicts the upcoming movement. One of the difficulties in assessing the relationship between firing of cortical cells and various movement parameters, such as force, velocity, displacement, and target location, is that these parameters are normally correlated with each other. Therefore, variations of the tasks described earlier have been used to decorrelate these various parameters (e.g., using weights to vary the force needed to make a movement without changing the displacement, as illustrated in

1	Fig. 9.17A , or rotating the starting position of the wrist so that different muscles are required to generate the same trajectory in external space). The results of these experiments showed that the activity of motor cortex cells may be related to each of the various motor planning stages. Furthermore, the activity of a single cell may be correlated with one parameter initially and then switch as the time for onset of movement approaches. Activity in the premotor and supplementary motor areas is in many ways similar to that in the primary motor cortex. Cells in these areas show activity related to upcoming

1	CHAPTER 9 Organization of Motor Function flexion movement results from relaxation of antagonist •Fig. 9.17 A, Experimentalarrangementforrecordingfromacorticospinalneuronwhileamonkeyperformstrainedwristmovements.Astimulationelectrodeisusedtoelicitantidromicspikesthatareusedtoidentifythemotorcortexneuronspecificallyasapyramidaltractneuron.Stimuliarenotappliedwhilethemonkeyisperformingmovements.B, Thepyramidaltractneuron(PTN)dischargesbeforetheonsetofmovementorEMGactivitywhenflexorsneedtogenerateforce(noloadandflexorloadconditions).Moreover,thefiringrateiscorrelatedwiththelevelofflexorforcethatisneeded.Intheextensorloadcondition,flexorsdonotneedtocontracttogeneratemovement,andthusthereisnoactivityinthisPTN.Thetoptraceshowswristmovement,whichisessentiallyidenticalforallthreeexperimentalconditions.Thusthiscell’sactivityinvolvesencodingforcemagnitudeanddirection,butnotdisplacement.(Figure basedonworkofEvartsandcolleagues.) movement, and the activity is correlated with movement parameters,

1	basedonworkofEvartsandcolleagues.) movement, and the activity is correlated with movement parameters, such as displacement, force, and target location, just as primary motor cortex activity can be, which is consistent with the distributed network view of the cortical motor areas. There do, however, appear to be some real differences between the areas as well, although these differences may be more quantitative than qualitative. For example, the percentage of cells in the premotor and supplementary motor areas that show activity related to earlier motor planning stages is higher than that of such cells in the primary motor cortex. In addition, the premotor and supplementary motor areas can be distinguished from each other by the apparently greater involvement of the premotor area in movements made to external cues (such as in the task shown in

1	Fig. 9.18 ) and the greater involvement of the supplementary motor area in movements made in response to internal cues (i.e., self-initiated). Research has also revealed that each of these areas is functionally heterogeneous and can therefore be further subdivided; however, such details are beyond the scope of this discussion. Motor Control by the Cerebellum Overview of the Role of the Cerebellum in Motor Control

1	In the early 1900s, scientists showed that damage to the cerebellum led to deficits in motor coordination. That is, damage or loss of the cerebellum does not lead to paralysis, loss of sensation, or an inability to understand the nature of a task; rather, it leads to an inability to perform movements well. Yet it has been hard to define the precise role or 45 135 225 315° Direction of movement • Fig.

1	movement • Fig. 9.18 A, Experimental setup in which a monkey holds onto the arm of the apparatus and captures light spots with the distal end of the arm. The monkey first captures the central light spot and then captures whichever of the surrounding targets that becomes illuminated. B, Raster plots showing the activity of one motor cortex cell during movement in eight different directions. T indicates the time at which the target light turns on, whereas M indicates the time at the onset of movement, which is at the center of each raster. Each mark on a raster represents a spike of a motor cortex cell, and each row of marks shows the cell’s activity during one trial. C, Cosine function was fit to the firing level as a function of the direction of movement. The horizontal bar indicates the average spontaneous firing rate in the absence of an upcoming movement. Note that for most directions, the activity in the periods just before and during movement changed significantly from baseline.

1	•Fig. 9.18, cont’d D, Vectormodelofpopulationactivityinthemotorcortex.Black lines representindividualcellvectors.Whenallofthemaresummedforaparticulardirectionofmovement,theresultingpopulationvector(red) pointsinessentiallythedirectionoftheupcomingmovement.(B andC, ModifiedfromGeorgopoulosAP,etal.J Neurosci. 1982;2:1527.D, ModifiedfromGeorgopoulosAP,etal.InMassionJ,etal[eds].Experimental Brain Research Series, vol.7:Neuronal Coding of Motor Performance. Berlin:Springer-Verlag;1983.) roles of the cerebellum in generating movement, although, paradoxically, scientists have more detailed knowledge of its deceptively simple anatomic and physiological organization than of any other CNS region. The cerebellum is proposed to play a critical role in the learning and execution of both voluntary and certain reflex movements. However, hypotheses about these roles face significant challenges that prevent their complete acceptance. In this section, the behavioral effects of damaging the cerebellum

1	certain reflex movements. However, hypotheses about these roles face significant challenges that prevent their complete acceptance. In this section, the behavioral effects of damaging the cerebellum are discussed, followed by a description of its connectivity, both intrinsic and with the rest of the CNS, and then finally a discussion of its activity.

1	Behavioral Consequences of Cerebellar Damage Damage to one side of the cerebellum impairs motor function on the ipsilateral side of the body. This reflects a double crossing of most cerebellum-related output as it travels to the motor neurons. The first crossing typically occurs in the cerebellar efferent pathway, whereas the second crossing takes place in the descending motor pathways. For example, the cerebellum projects to the contralateral motor cortex, via the thalamus, and the corticospinal pathway recrosses the midline at the lower medulla. The specific motor deficits that result from cerebellar lesions depend on which functional component of the

1	The specific motor deficits that result from cerebellar lesions depend on which functional component of the CHAPTER 9 Organization of Motor Function cerebellum is most affected. If the flocculonodular lobe is damaged, the motor disorders resemble those produced by a lesion of the vestibular apparatus; such disorders include difficulty in balance and in gait and often nystagmus. If the vermis is affected, the motor disturbance affects the trunk, and if the intermediate region or hemisphere is involved, motor disorders occur in the limbs. The part of the limbs affected depends on the site of damage; hemispheric lesions affect the distal muscles more than paravermal lesions do.

1	Types of motor dysfunction in cerebellar disease include disorders of coordination, equilibrium, and muscle tone. Incoordination is called ataxia and is often manifested as dysmetria, a condition in which errors in the direction and force of movement prevent a limb from being moved smoothly to a desired position. Ataxia may also be manifested as dysdiadochokinesia, in which rapid alternating of supination and pronation of the arm is difficult to execute. When more complicated movement is attempted, decomposition of movement occurs, in which the movement is accomplished in a series of discrete steps rather than as a smooth sequence. An intention tremor appears when the subject is asked to touch a target; the affected hand (or foot) develops a tremor that increases in magnitude as the target is approached. When equilibrium is disturbed, impaired balance may be seen, and the individual tends to fall toward the affected side and may walk with a wide-based stance (gait ataxia). Speech may

1	is approached. When equilibrium is disturbed, impaired balance may be seen, and the individual tends to fall toward the affected side and may walk with a wide-based stance (gait ataxia). Speech may be slow and slurred; such a defect is called scanning speech. Muscle tone may be diminished (hypotonia), except for lesions of the anterior vermis (see earlier section on decerebrate rigidity); the diminished tone may be associated with a pendular knee jerk. This can be demonstrated by eliciting a phasic stretch reflex of the quadriceps muscle by striking the patellar tendon. The leg continues to swing back and forth because of the hypotonia, in contrast to the highly damped oscillation in a normal person.

1	These disorders reflect, in part, abnormal timing of muscle contractions. Normally, limb movements involve precisely timed EMG bursts in both agonist and antagonist muscles. There is an initial agonist burst followed by a burst in the antagonist and, finally, a second agonist burst. With cerebellar damage, the relative timing of these bursts is abnormal ( Fig. 9.19

1	The cerebellum (“little brain”) is located in the posterior fossa of the cranium, just below the occipital lobe, and is connected to the brainstem via three cerebellar peduncles (superior, middle, and inferior). From the outer surface, only the cortex is visible. Deep to the cortex is the white matter of the cerebellum, and buried within the white matter are the four cerebellar nuclei: proceeding medially to laterally, the fastigial, globose, emboliform, and dentate nuclei. The middle two nuclei are often grouped together and referred to as the interpositus nucleus. For the most part, cerebellar afferent fibers to the cortex and nuclei enter the cerebellum •Fig. 9.19 Disruption of Cerebellar Activity Alters the Timing of EMG Responses During Movement.

1	nucleus. For the most part, cerebellar afferent fibers to the cortex and nuclei enter the cerebellum •Fig. 9.19 Disruption of Cerebellar Activity Alters the Timing of EMG Responses During Movement. Thecerebellarnucleiwerecooledtoblocktheirfunctioningtemporarilywhilemonkeysperformedmovementsabouttheirelbow.LossofcerebellaractivitydisruptstherelativetimingofagonistandantagonistEMGbursts.Thisleadstoabnormalaccelerationofthelimbandamovementtrajectorythatovershootsthetargetposition(hypermetria).(DatafromFlamentD,HoreJ.J Neurophysiol. 1986;55:1221.) via the inferior and middle peduncles, and efferent fibers from the cerebellar nuclei leave via the superior peduncle.

1	The cerebellar cortex is subdivided into three rostrocaudally arranged lobes: the anterior lobe, the posterior lobe, and the flocculonodular lobe ( Fig. 9.20A ). The cerebellar lobes are separated by two major fissures, the primary fissure and the posterolateral fissure, and each lobe is made up of one or more lobules. Each lobule of the cerebellar cortex is composed of a series of transverse folds called folia. The cerebellar cortex has also been divided into longitudinal compartments (see

1	The cerebellar cortex has also been divided into longitudinal compartments (see Fig. 9.20B and C). Initially, the cerebellar cortex was divided into three such compartments: the vermis, which lies on the midline; the paravermis, which lies adjacent to both sides of the vermis; and the lateral hemispheres. These regions have now been subdivided into many further compartments on the basis of myeloarchitectonics (patterns of axonal bundles in the white matter) and the expression patterns of specific molecules, such as aldolase C. Although the functional significance of these compartments is not fully known, the topography of cerebellar afferent fibers, particularly the olivocerebellar system, is precisely aligned with them, and the receptive field properties of cerebellar Purkinje cells also tend to follow this organizational scheme.

1	There are two major classes of cerebellar afferent systems: mossy fibers and olivocerebellar fibers. Mossy fibers are named for their distinctive appearance in the cerebellar cortex: as a mossy fiber courses through the granule layer, on occasion it swells and sends out a bunch of short twisted branchlets. These entities are called rosettes and are points of synaptic contact between these fibers and neurons in the granule cell layer. Mossy fibers arise from many sources, including the spinal cord (the spinocerebellar pathways), dorsal column nuclei, trigeminal nucleus, nuclei in the reticular formation, primary vestibular afferent fibers, vestibular nuclei, cerebellar nuclei, and the basilar pontine nuclei. The details of specific mossy fiber projection patterns are beyond the scope of this chapter; however, several general points are worth noting: 1. Mossy fibers are excitatory. 2.

1	Mossy fibers are excitatory. 2. They convey exteroceptive and proprioceptive information from the body and head and form at least two somatotopic maps of the body across the cerebellar cortex. However, like those of the motor cortex, these maps are fractured in the sense that contiguous body regions are not necessarily represented on contiguous areas of the cerebellar cortex; rather, the maps are complicated mosaics. 3. Mossy fibers conveying vestibular information are restricted to the flocculonodular lobe and regions of the vermis. As a result, the flocculonodular lobe and regions of the vermis are sometimes referred to as the vestibulocerebellum. However, these same regions also receive a variety of other information (e.g., visual, neck, oculomotor), and so their function is not exclusively vestibular. 4. The largest sources of mossy fibers are the basilar pontine nuclei, which serve to relay information from areas throughout much of the cerebral cortex. 5.

1	Mossy fibers enter the cerebellum via all three cerebellar peduncles and provide collateral fibers to the cerebellar nuclei before heading up to the cortex. In sum, via the mossy fiber system, the cerebellum receives a wide variety of sensory information, as well as descending motor-related and cognitive-related activity. In contrast to the diverse origins of mossy fibers, olivocerebellar fibers all originate from a single nucleus: the inferior olivary nucleus, which is located in the rostral medulla, just dorsal and lateral to the pyramids. Almost all the olivary neurons are projection cells whose axons leave the nucleus without giving off collaterals and then cross the brainstem to enter the cerebellum primarily via the inferior cerebellar peduncle. Like mossy fibers, olivocerebellar axons are excitatory and send collaterals to the cerebellar nuclei as they ascend through the cerebellar white matter to the cortex. In the cerebellar cortex, olivocerebellar axons may synapse with

1	axons are excitatory and send collaterals to the cerebellar nuclei as they ascend through the cerebellar white matter to the cortex. In the cerebellar cortex, olivocerebellar axons may synapse with basket, stellate, and Golgi cells, but they form a special synaptic arrangement with Purkinje cells. Each Purkinje cell receives input from only a single climbing fiber, which “climbs” up its proximal dendrites and makes hundreds of excitatory synapses. (The terminal portion of the olivocerebellar axon is referred to as a climbing fiber.) Conversely, each olivary axon branches to form about 10 to 15 climbing fibers.

1	CHAPTER 9 Organization of Motor Function •Fig. 9.20 Anatomic Divisions of the Cerebellum. A, Schematicmidsagittalviewofthefoldingofthecortexintolobe,lobules,andfolia.B, Schematicviewofanunfoldedferretcerebellarcortextoillustrateearliercompartmentationschemesforsubdividingthecerebellarcortexintothree(vermis,paravermis,andhemisphere)andthensevenlongitudinallyrunningzones(A,B,C1,C2,C3,D1,D2).Thelight yellow portionofeachhemisphereindicatesanareaforwhichnodatawereavailable.

1	C, Schematicviewofanunfoldedratcerebellum,showingitssubdivisionintomorethanI 1+2+3+3−3b−2b−*2a−5a−4−d−d−6−1−a−c−5−5−e2−e1−f−Par 4−5a−4a−4b−3−3−2−1−*3b+4+a+a+a+2+*2b+d+d+c+5+5+4+5a+6+4b+6+3+3+2+2+1+1+7+e2+*f+4+4+e1+b+2−1−b−III II IV V VII VIII IXa CP Cr IIb Cr IIa Cr Ic Cr Ib Cr Ia Sim b Sim a pf DPFL VPFL FL 5 mm 1 mm Rostral Left Right Caudal IXb IXc Xa Xb Vld Vla Vlb Vlc C •Fig. 9.20, cont’d 20compartments,accordingtostainingformolecularmarkers:inthiscase,zebrinII(aldolaseC).Letters and numbers on the right half ofthecerebellumindicatethezebrincompartmentnumber.Roman numerals down the center indicatecerebellarlobules.Names on left hemisphere indicatenamesofcerebellarlobules.CP,copulapyramis;Cr,crus;DPFL,dorsalparaflocculus;FL,flocculus;Par,paramedian;pf,primaryfissure;Sim,simplex;VPFL,ventralparaflocculus.(B, ModifiedfromVoogdJ.InLlinásRR(ed).Neurobiology of Cerebellar Evolution and Development. Chicago:AmericanMedicalAssociation;1969.C, CourtesyofDr.IzumiSugihara.)

1	The inferior olivary nucleus is a distinctive brain region for several reasons. As already noted, its neurons are virtually all projection cells, and so there is little local chemical synaptic interaction between the cells. Instead, olivary neurons are electrically coupled to each other by gap junctions. In fact, the inferior olivary nucleus has the highest density of neuronal gap junctions in the CNS. This allows olivary neurons to have synchronized activity that gets transmitted to the cerebellum. Afferent fibers to the inferior olivary nucleus may be divided into two main classes: those transmitting excitatory input, which arises from many regions throughout the CNS, and those transmitting inhibitory GABAergic input from the cerebellar nuclei and a few brainstem nuclei. Although these afferent fibers can modulate the firing rates of olivary neurons (as is typical in most brain regions), the membrane properties of olivary neurons limit this modulation to a range of a few hertz and

1	afferent fibers can modulate the firing rates of olivary neurons (as is typical in most brain regions), the membrane properties of olivary neurons limit this modulation to a range of a few hertz and endows these neurons with the potential to be intrinsic oscillators. Instead of just modulating firing rates, olivary afferent activity also acts to modify the effectiveness of the electrical coupling between olivary neurons and thus changes the patterns of synchronous activity delivered to the cerebellum. Afferent activity may also modulate expression of the oscillatory potential of olivary neurons. Thus the inferior olivary nucleus appears to be organized to generate patterns of synchronous activity across the cerebellar cortex. The functional significance of these patterns remains controversial. One hypothesis is that they provide a gating signal for synchronizing motor commands to various muscle combinations.

1	CHAPTER 9 Organization of Motor Function •Fig. 9.21 Three-Dimensional View of the Cerebellar Cortex, Showing Some of the Cerebellar Neurons. Thecutfaceattheleftisalongthelongaxisofthefolium;thecutfaceattherightisatrightanglestothelongaxis.BC,basketcell;BCA,basketcellaxon;CF,climbing(olivocerebellar)fiber;CN,cerebellarnuclearcell;GC,Golgicell;Glm,glomerulus;GrC,granulecell;MF,mossyfiber;PC,Purkinje cell;PF,parallelfiber;SC,stellatecell. Cellular Elements and Efferent Fibers of the Cortex

1	Cellular Elements and Efferent Fibers of the Cortex Despite its enormous expansion throughout vertebrate evolution, the basic anatomical organization of the cerebellar cortex has remained nearly invariant. The circuitry is also among the most regular and stereotyped of any brain region. The cerebellar cortex contains eight different neuronal types: Purkinje cells, Golgi cells, granule cells, Lugaro cells, basket cells, stellate cells, unipolar brush cells, and candelabrum cells. These cells are found in all regions of the cerebellar cortex, with the exception of unipolar brush cells, which are limited mainly to cerebellar areas receiving vestibular input. These eight cell types are distributed among the three layers that make up the cerebellar cortex of higher vertebrates (

1	Fig. 9.21 ). The outer or superficial layer is the molecular layer; stellate and basket cells are found there. The deepest layer is the granule cell layer; this layer has the highest cellular density in the CNS and contains granule, Golgi, and unipolar brush cells. Separating the molecular and granule cell layers is the Purkinje cell layer, formed by Purkinje cell somata, which are arranged as a one-cell-thick sheet of cells. Candelabrum cells are also located in this layer. Lugaro cells are situated slightly deeper at the upper border of the granule cell layer. The sole efferent fiber from the cortex is the Purkinje cell axon, which also has local collaterals and is GABAergic and inhibitory. Thus the remaining seven cell types are local interneurons. Of these, the stellate, basket, Golgi, Lugaro, and candelabrum cells are also inhibitory GABAergic neurons, whereas the granule and unipolar brush cells are excitatory. Microcircuitry of the Cortex

1	Microcircuitry of the Cortex The dendrites, axons, and patterns of synaptic connections of most neurons within the cerebellar cortex are organized with regard to the transverse (short) and longitudinal (long) axes of the folium (Fig. 9.21 ). In the vermis, where the folia run perpendicular to the sagittal plane, these axes lie in the sagittal and coronal planes, respectively. In the hemispheres, where the folia are oriented at various angles with regard to the sagittal plane, this correspondence is lost, and the local axes of the folia must then serve as the reference axes.

1	The Purkinje cell dendritic tree is the largest in the CNS. It extends from the Purkinje cell layer through the molecular layer to the surface of the cerebellar cortex and for several hundred microns along the transverse axis of the folium but for only 30 to 40 µm in the longitudinal direction. Thus it is like a flat pancake that lies in a plane parallel to the transverse axis of the folium. Accordingly, a set of Purkinje cell dendritic trees can be thought of as a stack of pancakes, with the stack running along the longitudinal axis of the folium.

1	•Fig. 9.22 Functional Connectivity of the Cerebellar Cortex. Becauseofthegeometricorganizationofthecerebellarcorticalcircuits,thefunctionalconnectivityofthecellularelementscanbedeterminedelectrophysiologically.Thefiguredepictsaclassicparadigminwhichstimulationofthecerebellarcortexactivatesabeamofparallelfibers(orange). Recordingsfromthestellateandbasketcells(green cells) andPurkinjecells(PCs;orange cells)inlinewiththisbeamshowthattheyareexcitedbytheparallelfibers.Incontrast,Purkinjecellsflankingthebeamreceiveonlyinhibition(purple areas) asaresultoftheperpendicularspatialrelationshipoftheparallelfibersandthestellateandbasketcellaxons.

1	The dendritic trees of the molecular layer interneurons (stellate and basket cells) are oriented in a manner similar to that of the Purkinje cell dendritic tree, although they are much less extensive. The axons of stellate and basket cells run transversely across the folium and form synapses with Purkinje cells. Stellate and basket cells synapse onto Purkinje cell dendrites. In addition, basket cells make synapses on the Purkinje cell soma and form a basket-like structure around the base of the soma, which gives the basket cell its name.

1	Granule cells are small neurons with four to five short unbranched dendrites, each ending in a claw-like expansion that synapses with a mossy fiber rosette and with terminals from Golgi cell axons in a complex arrangement known as a glomerulus. The axons of granule cells ascend through the Purkinje cell layer to the molecular layer, where they bifurcate and form parallel fibers. The parallel fibers run parallel to the cerebellar surface along the longitudinal axis of the folium (perpendicular to the planes of the Purkinje, stellate, and basket cell dendritic trees) and form excitatory synapses with the dendrites of the Purkinje, Golgi, stellate, and basket cells.

1	The orthogonal relationship between the parallel fibers and the dendritic trees of the Purkinje cells and molecular layer interneurons (basket and stellate cells) has significant functional consequences. This arrangement allows maximal convergence and divergence to occur. A single parallel fiber, which can be up to 6 mm long, passes through more than 100 Purkinje cell dendritic trees (and also interneuron dendrites); however, it has the chance to make only one or two synapses with any particular cell because it crosses through the short dimension of the dendritic tree. Conversely, a given Purkinje cell receives synapses from on the order of 100,000 parallel fibers. Thus a beam of parallel fibers can be excited experimentally, which excites a row of Purkinje cells and interneurons that are in line with this beam (

1	Fig. 9.22 ). In addition, because the axons of the interneurons run perpendicular to the parallel fibers, this beam of excitation is flanked by inhibition. Although this classic electrophysiological experiment clearly demonstrates the functional connectivity of the cerebellar cortex, whether such beams of excitation occur normally remains a controversial question.

1	The Golgi cells are inhibitory interneurons in the granule cell layer. The geometric organization of their axonal and dendritic arbors is an exception to the orthogonal and planar organization of the cortex in that their dendrites and axons carve out approximately conical territories: like two cones, tip to tip, in which the soma is at the point where the two cone tips meet. The dendritic tree forms the upper cone, which often extends into the molecular layer, and the axon forms the lower one. Golgi cells are excited by mossy and olivocerebellar fibers and by granule cell axons (parallel fibers) and inhibited by basket, stellate, and Purkinje cell axon collaterals. They in turn inhibit granule cells. Thus they participate both in feedback (when excited by parallel fibers) and in feedforward (when excited by mossy fibers) inhibitory loops that control activity in the mossy fiber–parallel fiber pathway to the Purkinje cell.

1	Lugaro cells have fusiform somata from which emerge two relatively unbranched dendrites, one from each side, that run along the transverse axis of the folium for several hundred microns, usually just under the Purkinje cell layer. Purkinje cell axon collaterals provide the main input to these neurons, and granule cell axons add minor input. The axon terminates mainly in the molecular layer on basket, stellate, and possibly Purkinje cells. Thus these cells appear to sample the activity of Purkinje cells and provide both positive-feedback signals (they inhibit the interneurons that inhibit Purkinje cells) and negative-feedback signals (they directly inhibit the Purkinje cell).

1	Unipolar brush cells have only a single dendrite that ends as a tight bunch of branchlets that resemble a brush. These cells receive excitatory input from mossy fibers and inhibitory input from Golgi cells. It is thought that they synapse with granule and Golgi cells, which would make these cells an excitatory feedforward link in the mossy fiber–parallel fiber pathway. Candelabrum cells are GABAergic cells located in the Purkinje layer. Their dendrites and axons terminate in the molecular layer, where the axonal arborization pattern resembles a candelabrum. The cerebellar nuclei are the main targets of the cerebellar cortex. This projection is topographically organized in such a way that each longitudinal strip of cortex targets a specific region of the cerebellar nuclei. The gross pattern is that the vermis projects to the fastigial and vestibular nuclei, the paravermal region projects to the interpositus, and the lateral hemisphere projects to the dentate nucleus.

1	The cerebellar nuclear neurons in turn provide the output from the cerebellum to the rest of the brain (with the primary exception of Purkinje cells that project to the

1	CHAPTER 9 Organization of Motor Function vestibular nuclei). In discussing the output of the cerebellar nuclei, it is useful to group the nuclear cells according to whether they are GABAergic because the GABAergic cells project back to the inferior olivary nucleus and form a negative-feedback loop to one of the cerebellum’s principal afferent sources. Of importance is that GABAergic cells project to the specific part of the inferior olivary nucleus from which they receive input and from which their overlying longitudinal strip of cortex receives climbing fibers. Thus the cerebellar cortex, cerebellar nuclei, and inferior olivary nucleus are functionally organized as a series of closed loops. The non-GABAergic, excitatory nuclear cells project to a variety of targets from the spinal cord to the thalamus. In general, each nucleus gives rise to crossed ascending and descending projections that leave the cerebellum via the superior cerebellar peduncle. The fastigial nucleus also gives

1	to the thalamus. In general, each nucleus gives rise to crossed ascending and descending projections that leave the cerebellum via the superior cerebellar peduncle. The fastigial nucleus also gives rise to significant uncrossed fibers, as well as a second crossed projection called the uncinate gyrus, or hook bundle, that leaves via the inferior cerebellar peduncle.

1	Although there are differences in the specific targets of each nucleus, in general, the ascending cerebellar projections target midbrain structures, such as the red nucleus and superior colliculus, and the ventral lateral nucleus of the thalamus, which connects to the primary motor cortex and thereby links the cerebellum to motor areas of the cerebrum. (The cerebral motor areas are likewise linked to the cerebellum by multiple pathways, including ones that relay in the basilar pons and inferior olivary nucleus.) It should also be mentioned that ascending cerebellar projections, particularly from the dentate nucleus, also target nonmotor regions of the cerebrum, particularly in the frontal lobe. The descending fibers target mainly the basilar pontine nuclei, inferior olivary nucleus, and several reticular nuclei. Lastly, a small cerebellospinal pathway arises principally from the fastigial nucleus. In addition, the fastigial nucleus has significant projections to the vestibular nuclei.

1	Activity of Purkinje Cells in the Cerebellar Cortex in the Context of Motor Coordination Mossy fiber input to the cerebellar cortex, via their excitation of granule cells, causes a Purkinje cell to discharge single action potentials, referred to as simple spikes (

1	Fig. 9.23 ). The spontaneous simple spike firing rate of a Purkinje cell typically is between 20 and 100 Hz but can be modulated over a much wider range (from 0 to >200 Hz), depending on the relative balance of excitation from parallel fiber input and inhibition from cerebellar cortex interneurons. Thus this activity reflects the state of the cerebellar cortex. Interestingly, evidence from studies performed in the early 2000s indicates that the spontaneous levels of simple spike activity vary systematically across the cerebellar cortex: Firing rates in zebrin-negative regions average twice those of the zebrin-positive regions. The full significance of this discovery is not known, but it suggests that despite the anatomical uniformity of the cerebellar cortical circuits, they might be functionally quite distinct.

1	•Fig. 9.23 Responses of a Purkinje Cell to Excitatory Input, Recorded Extracellularly. A, Granulecells,viatheirascendingaxonsandparallelfibers,excitePurkinjecellsandtriggersimplespikes.B, Climbingfiberactivityleadstohigh-frequency(≈500-Hz)burstsofspikesknownascomplex spikes inPurkinjecells.Notethatthespikesfollowingtheinitialonearesmallerandreferredtoasspikelets. In contrast, a climbing fiber discharge causes a high-frequency burst of action potentials, called a complex spike (see Fig. 9.23 ), in an all-or-none manner because of the massive excitation that is provided by the single climbing fiber that a Purkinje cell receives. This excitation is so powerful that there is essentially a one-to-one relationship between climbing fiber discharge and a complex spike. Thus complex spikes essentially override what is happening at the cortex level and reflect the state of the inferior olivary nucleus. The average firing rate of a spontaneous complex spike is only about 1 Hz.

1	Because the climbing fibers generate complex spikes at such a low frequency, they do not substantially change the average firing rates of Purkinje cells, and as a consequence, it is commonly argued that they have no direct role in shaping the output of the cerebellar cortex and are therefore not involved in ongoing motor control. Instead, it is commonly thought that their function is to alter the responsiveness of Purkinje cells to parallel fiber input. In particular, under certain circumstances, complex spike activity produces a prolonged depression in synaptic efficacy of parallel fibers, termed long-term depression (LTD). This phenomenon is the proposed mechanism by which climbing fibers act in motor-learning hypotheses. According to typical hypotheses about it, the parallel fiber system and hence simple spikes are involved in generating ongoing movement, and when there is a mismatch between the intended and actual movement, this error activates the inferior olivary nucleus and

1	and hence simple spikes are involved in generating ongoing movement, and when there is a mismatch between the intended and actual movement, this error activates the inferior olivary nucleus and complex spikes result, which then lead to LTD of the active parallel fiber synapses. This adjustment in synaptic weight changes the motor output in the future. If this change results in a properly executed movement, activation of the inferior olivary nucleus does not occur, and the motor program is unchanged, but if there is still an error, the olivocerebellar system triggers additional complex spikes that cause further changes in synaptic efficacy, and so on. Major challenges to this view are that motor learning can occur when LTD is chemically blocked and that learned behavior can remain after removal of portions of the cerebellum in which the memory is supposedly stored.

1	An alternative view is that the olivocerebellar system is directly involved in motor control (note that this does not preclude a role in motor learning as well) and, in particular, helps in the timing of motor commands. This view follows from the types of motor deficits observed in cerebellar damage and accounts for the special properties of the inferior olivary nucleus mentioned earlier: namely, that it can generate rhythmic, synchronous complex spike discharges across populations of Purkinje cells. These complex spikes would then produce synchronized inhibitory postsynaptic currents (IPSPs) on cerebellar nuclear neurons as a result of the convergence present in the Purkinje cell axon to the cerebellar nuclear projection. Because of the membrane properties of cerebellar nuclear neurons, these synchronized IPSPs could have a qualitatively different effect on nuclear cell firing than would the IPSPs caused by simple spikes, which are more numerous but largely asynchronous.

1	neurons, these synchronized IPSPs could have a qualitatively different effect on nuclear cell firing than would the IPSPs caused by simple spikes, which are more numerous but largely asynchronous. Specifically, they could trigger large precisely timed changes in the nuclear cell activity that would then be transmitted to other motor systems as a gating signal. In fact, voluntary movements appear to be composed of a series of periodic accelerations that reflect a central oscillatory process. However, determining whether the olivocerebellar system helps time motor commands requires further evidence.

1	Motor Control by the Basal Ganglia The basal ganglia are deep nuclei in the cerebrum. Like the cerebellum, a major function of the basal ganglia is to regulate motor activity and we will focus on that role in this section. However, it is worth noting that they, like the cerebellum, also contribute to affective and cognitive functions. To understand basal ganglia function in motor control, the following discussion is organized around two major themes: (1) the connections between basal ganglia and cortex form loops through which activity flows and (2) there are two functionally distinct pathways through the basal ganglia, the direct and indirect pathways. Organization of the Basal Ganglia and Related Nuclei The basal ganglia include the caudate nucleus, the putamen, and the globus pallidus ( Fig. 9.24 ). The term striatum, derived from the striated appearance of these

1	The basal ganglia include the caudate nucleus, the putamen, and the globus pallidus ( Fig. 9.24 ). The term striatum, derived from the striated appearance of these CHAPTER 9 Organization of Motor Function •Fig. 9.24 Components of Basal Ganglia and Other Closely Associated Brain Regions. Themaincomponentsofthebasalgangliaarethecaudatenucleus,putamen,globuspallidus,andsubstantianigraparsreticulata.Majorportionsofthebasalgangliaconnectwithmotorareasinthefrontalcortex,viatheventralanteriorandventrallateralthalamicnuclei,andwiththesuperiorcolliculus.Inputfromthesubstantianigraparscompactaiscriticalfornormalbasalgangliafunction.GPe,externalsegmentofglobuspallidus;GPi,internalsegmentofglobuspallidus.

1	nuclei, refers only to the caudate nucleus and putamen. The striations are produced by the fiber bundles formed by the anterior limb of the internal capsule as it separates the caudate nucleus and putamen. The globus pallidus typically has two parts: an external segment and an internal segment. The combination of putamen and globus pallidus is often referred to as the lentiform nucleus. Associated with the basal ganglia are several thalamic nuclei. These include the ventral anterior (VA) and ventral lateral (VL) nuclei and several components of the intralaminar complex. Other associated nuclei are the subthalamic nucleus of the diencephalon and the substantia nigra of the midbrain (see

1	Fig. 9.24 ). The substantia nigra (“black substance”) derives its name from its content of melanin pigment. Many of the neurons in the pars compacta of this nucleus contain melanin, a byproduct of dopamine synthesis. The other subdivision of the substantia nigra is the pars reticulata. This structure can be regarded as an extension of the internal segment of the globus pallidus because these nuclei have an identical origin and analogous connections. Connections and Operation of the Basal Ganglia With the exception of the primary visual and auditory cortices, most regions of the cerebral cortex project topographically to the striatum. The corticostriatal projection arises from neurons in layer V of the cortex. Glutamate appears to be the excitatory neurotransmitter of these neurons. The striatum then influences neurons in the nuclei of the thalamus by two pathways: direct and indirect ( Fig.

1	Fig. 9.25A ). The thalamic neurons in turn excite neurons of the cerebral cortex thereby forming closed loops with most of the cortex. Several distinct loops have been identified on the basis of cortical regions and function; however, here we will focus on the motor-related loops as a model for basal ganglia operation ( Fig. 9.25A

1	The overall action of the direct pathway through the basal ganglia to motor areas of the cortex is to enhance motor activity. In the direct pathway, the striatum projects to •Fig. 9.25 Functional Connectivity of the Basal Ganglia for Motor Control. A, Connectionsbetweenvariousbasalgangliacomponentsandotherassociatedmotorareas.TheexcitatorycorticalinputtothecaudateandputameninfluencesoutputfromtheGPiandthesubstantianigraparsreticulata(SNpr)viaadirectandanindirectpathway.Thetwoinhibitorystepsintheindirectpathwaymeanthatactivitythroughthispathwayhasaneffectonbasalgangliaoutputtothethalamusandsuperiorcolliculusoppositethatofthedirectpathway.Dopamine(DA)isaneuromodulatorthatactsonD1andD2receptorsonstriatalneuronsthatparticipateinthedirectandindirectpathways,respectively.B, ChangesinactivityflowthatoccurinParkinson’sdisease,inwhichthesubstantianigraparscompacta(SNpc)degenerates.C, ChangesinactivityflowinHuntington’sdisease,inwhichinhibitorycontroloftheGPeislost.Plus symbols (+)andminus

1	ChangesinactivityflowinHuntington’sdisease,inwhichinhibitorycontroloftheGPeislost.Plus symbols (+)andminus symbols (−),respectively,indicatetheexcitatoryorinhibitorynatureofasynapticconnection.glu,glutamate;GPe,externalglobuspallidus;GPi,internalglobuspallidus;VA/VL,ventralanterior/ventrallateralnucleiofthethalamus.

1	the internal segment of the globus pallidus (GPi). This projection is inhibitory, and the main transmitter is GABA. The GPi projects to the VA and VL nuclei of the thalamus. These connections also function with GABA and are inhibitory. The VA and VL nuclei send excitatory connections to the prefrontal, premotor, and supplementary motor cortex. This input to the cortex influences motor planning, and it also affects the discharge of corticospinal and corticobulbar neurons.

1	The direct pathway appears to function as follows: Neurons in the striatum have little background activity, but during movement they are activated by their input from the cortex. In contrast, neurons in the GPi have a high level of background activity. When the striatum is activated, its inhibitory projections to the globus pallidus slow the activity of pallidal neurons. However, the pallidal neurons themselves are inhibitory, and they normally provide tonic inhibition of neurons in the VA and VL nuclei of the thalamus. Therefore, activation of the striatum causes disinhibition of neurons of the VA and VL nuclei. When disinhibited, the VA/VL neurons increase their firing rates, exciting their target neurons in the motor areas of the cerebral cortex. Because the motor areas evoke movement by activating α and γ motor neurons in the spinal cord and brainstem, the basal ganglia can regulate movement by enhancing the activity of neurons in the motor cortex.

1	The overall effect of the indirect pathway is to reduce the activity of neurons in motor areas of the cerebral cortex. The indirect pathway involves inhibitory connections from the striatum to the external segment of the globus pallidus (GPe), which in turn sends an inhibitory projection to the subthalamic nucleus and to the GPi. The subthalamic nucleus then sends an excitatory projection back to the GPi (see Fig. 9.25A

1	Fig. 9.25A In this pathway, pallidal neurons in the external segment are inhibited by the GABA released from striatal terminals in the globus pallidus. The GPe normally releases GABA in the subthalamic nucleus and thereby inhibits the subthalamic neurons. Therefore, striatal inhibition of the GPe results in the disinhibition of neurons of the subthalamic nucleus. The subthalamic neurons are normally active, and they excite neurons in the GPi by releasing glutamate. When the neurons of the subthalamic nucleus become more active because of disinhibition, they release more glutamate in the GPi. This transmitter excites neurons in the GPi and consequently activates inhibitory projections that affect the VA and VL thalamic nuclei. The activity of the thalamic neurons consequently decreases, as does the activity of the cortical neurons that they influence.

1	The direct and indirect pathways thus have opposing actions; an increase in the activity of either one of these pathways might lead to an imbalance in motor control. Such imbalances, which are typical of basal ganglion diseases, may alter the motor output of the cortex. CHAPTER 9 Organization of Motor Function Actions of Neurons in the Pars Compacta of the Substantia Nigra on the Striatum

1	Dopamine is the neurotransmitter used by neurons of the substantia nigra pars compacta. In the nigrostriatal pathway, release of dopamine has an overall excitatory action on the direct pathway and an inhibitory action on the indirect pathway. This is, however, a modulatory type of effect; that is, dopamine is apparently causing its action not by triggering spikes directly but rather by altering the striatal cells’ response to other transmitters. The different actions on the direct and indirect pathways result from the expression of different types of dopamine receptors (D1 and D2) by the spiny projection cells of the striatum that contribute to the direct and indirect pathways. D1 receptors are found on striatal cells that form the direct pathway by projecting to the GPi, whereas D2 receptors are found on striatal cells that participate in the indirect pathway and project to the GPe. The overall consequence of dopamine release in both cases is facilitation of activity in the motor

1	are found on striatal cells that participate in the indirect pathway and project to the GPe. The overall consequence of dopamine release in both cases is facilitation of activity in the motor areas of the cerebral cortex.

1	Subdivision of the Striatum Into Striosomes and Matrix On the basis of the associated neurotransmitters, the stria-tum has been subdivided into zones called striosomes and matrix. The cortical projections related to motor control end in the matrix area. The limbic system projects to the striosomes. Striosomes are thought to synapse in the pars compacta of the substantia nigra and to influence the dopaminergic nigrostriatal pathway. Role of the Basal Ganglia in Motor Control The basal ganglia influence the cortical motor areas. Therefore, the basal ganglia have an important influence on the lateral system of motor pathways. Such an influence is consistent with some of the movement disorders observed in diseases of the basal ganglia. However, the basal ganglia must additionally regulate the medial motor pathways because diseases of the basal ganglia can also affect the posture and tone of proximal muscles.

1	The deficits seen in the various basal ganglia diseases include abnormal movement (dyskinesia), increased muscle tone (cogwheel rigidity), and slowness in initiating movement (bradykinesia). Abnormal movement includes tremor, athetosis, chorea, ballism, and dystonia. The tremor of basal ganglion disease is a 3-Hz “pill-rolling” tremor that occurs when the limb is at rest. Athetosis consists of slow, writhing movement of the distal parts of the limbs, whereas chorea is characterized by rapid, flicking movement of the extremities and facial muscles. Ballism is associated with violent, flailing movement of the limbs (ballistic movement). Finally, dystonic movements are slow involuntary movements that may cause distorted body postures.

1	Parkinson’s disease is a common disorder characterized by tremor, rigidity, and bradykinesia. This disease is caused by loss of neurons in the pars compacta of the substantia nigra. Consequently, the striatum suffers a severe loss of dopamine. Neurons of the locus coeruleus and the raphe nuclei, as well as other monoaminergic nuclei, are also lost. The loss of dopamine diminishes the activity of the direct pathway and increases the activity of the indirect pathway (see Fig. 9.25B ). The net effect is an increase in the activity of neurons in the internal segment of the globus pallidus. This results in greater inhibition of neurons in the VA and VL nuclei and less pronounced activation of the motor cortical areas. The consequence is slowed movement (bradykinesia).

1	Before the dopaminergic neurons are completely lost, administration of levodopa can relieve some of the motor deficits in Parkinson’s disease. Levodopa is a precursor of dopamine, and it can cross the blood-brain barrier. Currently, the possibility of transplanting dopamine-synthesizing neurons into the striatum is being explored. Future research will no doubt focus on the potential for human embryonic stem cells to play such a therapeutic role. Another basal ganglia disturbance is Huntington’s disease, which results from a genetic defect that involves an autosomal dominant gene. This defect leads to the preferential loss of striatal GABAergic and cholinergic neurons that project to the GPe as part of the indirect pathway (and also degeneration of the cerebral cortex, with resultant dementia). Loss of inhibition of the GPe presumably leads to diminished activity of neurons in the subthalamic nucleus (see

1	Fig. 9.25C ). Hence, the excitation of neurons of the GPi would be reduced. This would disinhibit neurons in the VA and VL nuclei. The resulting enhancement of activity in neurons in the motor areas of the cerebral cortex may help explain the choreiform movements of Huntington’s disease. The rigidity in Parkinson’s disease may in a sense be the opposite of chorea because overtreatment of patients with Parkinson’s disease with levodopa can result in chorea. Hemiballism is caused by a lesion of the subthalamic nucleus on one side of the brain. In this disorder, involuntary, violent flailing movements of the limbs may occur on the side of the body contralateral to the lesion. Because the subthalamic nucleus excites neurons of the GPi, a lesion of the subthalamic nucleus would reduce the activity of these pallidal neurons. Therefore, neurons in the VA and VL nuclei of the thalamus would be less inhibited, and the activity of neurons in the motor cortex would be increased.

1	In all these basal ganglia disorders, the motor dysfunction is contralateral to the diseased component. This is understandable because the main final output of the basal ganglia to the body is mediated by the corticospinal tract.

1	Eye movement has a number of features that distinguish it from other motor behavior. In comparison with the movement that limbs, with their multiple joints and muscles, can perform, eye movement is relatively simple. For example, each eye is controlled by only three agonistantagonist muscle pairs: the medial and lateral recti, the superior and inferior recti, and the superior and inferior oblique muscles. These muscles allow the eye to rotate about three axes. Assuming that the head is in an upright position, the axes are the vertical axis, a horizontal axis that runs left to right, and the torsional axis (which is directed along the axis of sight). The medial and lateral recti control movement about the vertical axis; the other four muscles generate movement about the horizontal and torsional axes. Another simplifying feature is that there are no external loads for which to be compensated. Furthermore, eye movement appears to be separable into a few distinct types, with each type

1	axes. Another simplifying feature is that there are no external loads for which to be compensated. Furthermore, eye movement appears to be separable into a few distinct types, with each type being controlled by its own specialized circuitry. Thus eye movement offers a number of advantages as a model system for studying motor control. Moreover, deficits in eye movement provide important clinical clues to the diagnosis of neurological problems. We first review the different eye movement types and then discuss the neural circuitry underlying their generation.

1	Types of Eye Movement Eye movement probably first evolved to hold the image of the external world still. (In contrast, limb movements evolved to generate changes in the position of the limb with regard to the external world.) The reason is that visual acuity degrades rapidly when there is eye movement in relation to the external world (i.e., the visual scene slips across the retina). A major cause of such slippage is movement of the head. The vestibuloocular reflex (VOR) is one of the main mechanisms by which head movement is compensated in order to maintain stability of the visual scene on the retina.

1	To maintain a stable visual scene on the retina, the VOR produces movements of the eyes that are equal and opposite the movement of the head. This reflex is initiated by stimulation of the receptors (hair cells) in the vestibular system (see ). Recall that the vestibular organs are sensitive to head acceleration, not visual cues, and thus the VOR occurs in both the light and dark. Functionally, it is what is called an open-loop system in that it generates an output (eye movement) in response to a stimulus (head acceleration), but its immediate behavior is not regulated by feedback about the success or failure of its output. It is worth noting, however, that in the light at least, any failure by the VOR to match eye and head rotation results in what is called retinal slip (i.e., slip of the visual image across the retina), and this error signal can be fed back to the VOR circuits by other neuronal pathways and over time can lead to adjustments in the strength of the VOR to eliminate

1	the visual image across the retina), and this error signal can be fed back to the VOR circuits by other neuronal pathways and over time can lead to adjustments in the strength of the VOR to eliminate the error. This adaptation of the VOR is a major model for studying plasticity in the brain.

1	As stated, acceleration signals initiate the VOR. The output of the VOR, however, must be a change in eye position in the orbit. Thus the problem to be solved by the nervous system is to translate the acceleration signals sensed by the vestibular organs into correct positional signals for the eyes. Mathematically, this can be thought of as a double integration. The first integration occurs in the vestibular receptor apparatus because although the hair cells respond to head acceleration, the signals in the vestibular afferent fibers are proportional to head velocity (at least for most stimuli that are encountered physiologically). The second integration, from velocity to position, occurs in the CNS in circuits described later.

1	The head can move in six different ways, often referred to as six degrees of freedom: three translational and three rotational. To compensate for these different types of movement, there are both translational and angular VORs, as well as separate subsystems for handling movement about different directions (e.g., rotation about a vertical or a horizontal axis).

1	The optokinetic reflex (OKR) is a second mechanism by which the CNS stabilizes the visual scene on the retina, and it often works in conjunction with the VOR. Whereas the VOR is activated only by head motion, the OKR is activated by movement of the visual scene, whether caused by motion of the scene itself or by head motion. Specifically, the sensory stimulus for this reflex is slip of the visual scene on the retina as detected by motion-sensitive retinal ganglion cells. An example of the former occurs when you are sitting in a train and a train on the adjacent track begins moving: Your eyes rotate to keep the image of the neighboring car stable. This often leads to a sensation that you are moving (this is not entirely surprising because OKR circuits feed into the same circuits as used by the vestibular system).

1	The OKR can work in conjunction with the VOR to stabilize the visual image and is particularly important for maintaining a stable image when head movements are slow because the VOR works poorly in these conditions. In animals whose eyes have a fovea, it becomes particularly advantageous to be able to move the eye in relation to the world (i.e., the main visual scene) so that objects of importance can be focused onto the fovea and scrutinized with this high-resolution part of the retina. Two classes of eye movement underlie this ability: saccadic and smooth pursuit. Very rapid discrete movements that bring a particular region of the visual world onto the fovea are called saccades. For example, to read this sentence, you are making a series of saccades to bring successive words onto your fovea to be read. However, even in animals that lack a fovea, the eyes make saccades, and thus saccades may also be used to rapidly scan the visual environment.

1	Saccades are extremely rapid eye movements. In humans, eye velocity during a saccade can reach 800 degrees/second, in comparison with movement velocity of less than 10 degrees/second generated in response to typical VOR and CHAPTER 9 Organization of Motor Function OKR stimuli (velocities of up to ≈120 degrees/second can be produced by OKR stimuli in humans; however, they are still much slower than the maximal saccade velocities). Saccades can be made voluntarily or reflexively. Moreover, although they are usually made in response to visual targets, they can also be made toward auditory or other sensory cues, in the dark, or toward memorized targets.

1	Interestingly, visual processing appears to be suppressed just before and during saccades, particularly in the magnocellular visual pathway that is concerned with visual motion. This phenomenon is known as saccadic suppression and may function to prevent sensations of sudden, rapid movement of the visual world that would result during a saccade in the absence of such suppression. The mechanisms underlying saccadic suppression are not fully known, but in areas of the cortex related to visual processing, the responsiveness of the cells to visual stimuli is reduced and altered during saccades.

1	Once a saccade has brought a moving object of interest onto the fovea, the smooth pursuit system allows the person to keep it stable on the fovea despite its continued motion. This ability appears to be limited to primates and allows prolonged continuous observation of a moving object. Note that in some respects, smooth pursuit might seem similar to the OKR; in fact, there may not be an absolute difference because as the target size grows, the distinction between target and background is lost; however, for small moving targets, smooth pursuit requires suppression of the OKR. You can see the effect of this suppression by moving your finger back and forth in front of this text while tracking it with your eyes. Your finger will be in focus, but the words on this page will be part of the background scene and will become illegible as they slip along your retina.

1	When there is a prolonged OKR or VOR stimulus (e.g., if you keep turning in one direction), these reflexes will initially counterrotate the eyes in an attempt to maintain a stable image on the retina, as described earlier. However, with a prolonged stimulus, the eyes will reach their mechanical limit, no further compensation will be possible, and the image will begin to slip on the retina. To avoid this situation, a fast saccade-like movement of the eyes occurs in the opposite direction, essentially resetting the eyes to begin viewing the visual scene again. Then the slow OKR-or VOR-induced counterrotation will start anew. This alternation of slow and fast movement in opposite directions is nystagmus and can be recorded on a nystagmogram ( Fig.

1	Fig. 9.26 ). Thus nystagmus can be defined as oscillatory or rhythmic movements of the eye in which there is a fast phase and a slow phase. The nystagmus is named according to the direction of the fast phase because the fast phase is more easily observed. In addition to being induced physiologically by VOR or OKR stimuli, nystagmus can result from damage to the vestibular circuits, either in the periphery (e.g., cranial nerve •Fig. 9.26 Nystagmogram Showing Eye Movements That Occur During Nystagmus. Theplotshowsaleftnystagmusbecausethefastphaseisdirectedtowardtheleft(downwardonthegraph). VIII) or centrally (e.g., vestibular nuclei), and can be an informative diagnostic symptom.

1	VIII) or centrally (e.g., vestibular nuclei), and can be an informative diagnostic symptom. Conjugate eye movement is movement of both eyes in the same direction and in an equal amount. Such coordination allows a target to be maintained on both foveae during eye movement and is necessary to maintain binocular vision without diplopia (double vision). However, when objects are close (<30 m), maintaining a target on both foveae requires non-identical movements of the two eyes. Such disjunctive, or vergence, movements are also necessary for fixation of both eyes on objects that are approaching or receding. Stimuli that trigger vergence movements are diplopia and blurry images. It should be noted that when tracking an approaching object in addition to convergence movements, the lens accommodates for near vision, and pupillary constriction occurs. Motor Neurons of the Extraocular Muscles

1	Motor Neurons of the Extraocular Muscles Three cranial nerve nuclei supply the extraocular muscles: oculomotor, trochlear, and abducens nuclei. These three nuclei are sometimes referred to collectively as the oculomotor nuclei; however, the context (the specific nucleus or all three) should be clear. Motor neurons for the ipsilateral medial and inferior recti, ipsilateral inferior oblique, and contralateral superior rectus muscles reside in the oculomotor nucleus; those for the contralateral superior oblique muscle reside in the trochlear nucleus; and those for the ipsilateral lateral rectus muscle are located in the abducens nucleus. These motor neurons form some of the smallest motor units (1 : 10 nerve-to-muscle ratio), which is consistent with the very fine control needed for precise eye movement.

1	An important point regarding motor neurons innervating the extraocular muscles is that most have spontaneous activity when the eye is in the primary position (looking straight ahead), and their firing rate is correlated with eye position and velocity. This spontaneous activity allows the antagonist muscle pairs to act in a push-pull manner, which increases the responsiveness of the system. That is, as motor neurons innervating one muscle are activated and cause increased contraction, those innervating its antagonist are inhibited, which leads to relaxation. In addition to motor neurons, the abducens nuclei have internuclear neurons. These neurons project, via the medial longitudinal fasciculus, to medial rectus motor neurons in the contralateral oculomotor nucleus. As described later, this projection facilitates the coordinated action of the medial and lateral recti muscles that is needed for conjugate movements, such as those that occur in the VOR.

1	Circuits Underlying the Vestibuloocular Reflex The VOR acts to counter head motion by causing rotation of the eyes in the opposite direction. There are separate circuits for rotational and translational movement of the head. The sensors for the former are the semicircular canals, and the sensors for the latter are the otoliths (the utricle and saccule). The circuits for the angular VOR are more straightforward (but still complex), and this section focuses on these pathways to illustrate how this reflex works; however, the basic scheme is the same: Vestibular afferent fibers go to vestibular nuclei, the vestibular nuclei in turn project to the various oculomotor nuclei, and motor neurons in the oculomotor nuclei give rise to axons that innervate the extraocular muscles. What varies are the specific vestibular and oculomotor nuclei that are involved.

1	With regard to the angular VOR pathways, the pathway for generating horizontal eye movement originates in the horizontal canals, and the analogous one for vertical movement originates in the anterior and posterior canals. Fig.

1	Fig. 9.27A shows the basic circuit for the horizontal VOR. Note that only the major central circuits originating in the left horizontal canal and vestibular nuclei are shown; however, mirror image pathways arise from the right canal and vestibular nuclei. Vestibular afferent fibers involved in the horizontal VOR pathway synapse primarily in the medial vestibular nucleus, which projects to the abducens nucleus bilaterally; inhibitory neurons project ipsilaterally, and excitatory ones project contralaterally. Control of the medial rectus muscle is achieved by abducens internuclear neurons that project from the abducens to the part of the oculomotor nucleus controlling the medial rectus muscle. Note the double decussation of this pathway results in aligning of the responses of functional synergists (e.g., the left medial rectus with the right lateral rectus).

1	The vertical VOR pathway involves primarily the superior vestibular nucleus, which has direct bilateral projections to the oculomotor nucleus. Consider what happens in the horizontal canal pathway when there is head rotation to the left, as shown in Fig. 9.27B. Leftward head rotation would cause the visual image to slip to the right. However, compensation by the VOR would be triggered by depolarization of the hair cells of the left canal in response to the angular acceleration

1	CHAPTER 9 Organization of Motor Function •Fig. 9.27 Circuits Underlying the Horizontal Vestibuloocular Reflex (VOR). A, Thevestibularnucleireceiveexcitatoryinputfromtheafferentfibersofthehorizontalcanalandprojecttotheabducens(cranialnerveVI)nucleus.Thisnucleusinnervatesthelateralrectusmuscleandprojectstothecontralateraloculomotor(cranialnerveIII)nucleus,whichcontrolsthemedialrectusmuscle.Excitatoryneuronsareshowninred; inhibitoryones,inblue. Notethatonlythemajorpathwaysoriginatingintheleftvestibularnucleiareshown.Forclarity,onlythebeginningsofmirrorimagepathwaysfromtherightvestibularnucleiareshown(dotted lines). B, FlowofactivityintheVORcircuitryinducedbyleftwardheadrotation.Increasedaxonalthicknessindicatesincreasedactivity;thinneraxonsindicatedecreasedactivityincomparisonwithlevelsatrest(A).

1	lines). B, FlowofactivityintheVORcircuitryinducedbyleftwardheadrotation.Increasedaxonalthicknessindicatesincreasedactivity;thinneraxonsindicatedecreasedactivityincomparisonwithlevelsatrest(A). Notethatleftwardrotationcausesbothanincreaseinactivityoftheleftvestibularafferentfibersandadecreaseinactivityoftherightones.MLF,mediallongitudinalfasciculus;vestibularnuclei:I,inferior;L,lateral;M,medial;S,superior.

1	Fig. 8.27 ). The depolarized hair cells cause increased activity in the left vestibular afferent fibers and thereby excite neurons of the left medial vestibular nucleus. These include excitatory neurons that project to the contralateral abducens nucleus and synapse with both motor neurons and internuclear neurons. Excitation of the motor neurons leads to contraction of the right lateral rectus muscle and rotation of the right eye to the right, whereas excitation of the internuclear neurons of the right abducens nucleus leads to excitation of the medial rectus motor neurons in the left oculomotor nucleus, thus causing the left eye to rotate to the right as well.

1	Along the pathway starting with the inhibitory vestibular neurons that project from the left medial vestibular nucleus to the ipsilateral abducens nucleus, the activity of these cells leads to inhibition of motor neurons to the left lateral rectus muscle and motor neurons to the right medial rectus muscle (the latter via internuclear neurons to the right oculomotor nucleus). Consequently, these muscles relax, thereby facilitating rotation of the eyes to the right. Thus the eye is being pulled by the increased tension of one set of muscles and “pushed” by the release of tension in the antagonist set of muscles. Note again that the mirror image pathways originating from the right canal have been left out of

1	Note again that the mirror image pathways originating from the right canal have been left out of Fig. 9.27 for clarity, but the changes in activity through them with leftward head rotation would be exactly the opposite, and thus they would function synergistically with those that are shown. As an exercise, work out the resulting changes in activity through these circuits. Remember that leftward head rotation hyper-polarizes the hair cells of the right canal, thereby leading to a decrease in right vestibular afferent activity and disfacilitation of the right vestibular nuclear neurons.

1	Now, consider the commissural fibers that connect the two medial vestibular nuclei are excitatory but end on local inhibitory interneurons of the contralateral vestibular nucleus and thus inhibit the projection neurons of that nucleus. This pathway reinforces the actions of the contra-lateral vestibular afferent fibers on their target vestibular nuclear neurons. In the aforementioned example, commissural cells in the left vestibular nucleus are activated and therefore cause active inhibition of the right medial vestibular nuclei projection neurons, which reinforces the disfacilitation caused by the decrease in right afferent activity. In fact, this commissural pathway is powerful enough to modulate the activity of the contralateral vestibular nuclei even after unilateral labyrinthectomy, which destroys the direct vestibular afferent input to these nuclei.

1	Of importance is that superimposed on the brainstem circuits is the cerebellum. Parts of the vermis and flocculonodular lobe receive primary vestibular afferent fibers or secondary vestibular afferent fibers (axons of the vestibular nuclear neurons), or both, and in turn project back to the vestibular nuclei directly and via a disynaptic pathway involving the fastigial nucleus. The exact role of these cerebellar circuits in generating the VOR is much debated, but they are critical inasmuch as damage to them leads to abnormal eye movement, such as spontaneous nystagmus, and other symptoms of vestibular dysfunction.

1	Whenalabyrinthisirritatedinoneear,asinMeniere’s disease, orwhenalabyrinthisrenderednonfunctional,asmayhappenasaresultofheadtraumaordiseaseofthelabyrinth,thesignalstransmittedthroughtheVORpathwaysfromthetwosidesbecomeunbalanced.Vestibularnystagmuscanthenresult.Forexample,irritationofthelabyrinthoftheleftearcanincreasethedischargeofafferentfibersthatsupplythelefthorizontalsemicircularduct.Thesignalproducedresemblesthatnormallygeneratedwhentheheadisrotatedtotheleft.Becausethestimulusisongoing,aleftnystagmusresults,withaslowphasetotheright(causedbytheVORpathway)andafastphasetotheleft.Destructionofthelabyrinthintherightearproduceseffectssimilartothoseinducedbyirritationoftheleftlabyrinth.Interestingly,thenystagmusistemporary,whichshowstheabilityofthesecircuitstoadaptovertime. Circuits Underlying the Optokinetic Reflex

1	The stimulus eliciting the OKR is visual (retinal slip), and so photoreceptors are the start of the reflex arc. Key brainstem centers for this reflex lie in the tegmentum and pretectal region of the rostral midbrain. They are the nucleus of the optic tract (NOT) and a group of nuclei collectively known as the accessory optic nuclei (AON). Direction-selective, motion-sensitive retinal ganglion cells are a major afferent source carrying visual information to these nuclei. In addition, input comes from primary and higher order visual cortical areas in the occipital and temporal lobes. These latter afferent sources are particularly important in primates and humans. Cells of the NOT and AON have large receptive fields, and their responses are selective for the direction and speed of movement of the visual scene. Of interest is that the preferred directions of movement of the NOT/AON cells correspond closely to motion caused by rotation about axes perpendicular to the semicircular canals,

1	of the visual scene. Of interest is that the preferred directions of movement of the NOT/AON cells correspond closely to motion caused by rotation about axes perpendicular to the semicircular canals, thereby facilitating coordination of the VOR and OKR to produce stable retinal images.

1	The efferent connections of these nuclei are numerous and complex and not fully understood. There are polysynaptic pathways to the oculomotor and abducens nuclei and monosynaptic input to the vestibular nuclei, which allow interaction with the VOR. There are projections to various precerebellar nuclei, including the inferior olivary nucleus and basilar pontine nuclei. These pathways then loop through the flocculus and back to the vestibular nuclei. In sum, via several pathways operating in parallel, activity ultimately arrives at the various oculomotor nuclei whose motor neurons are activated, and proper counterrotation of the eyes results. Saccades are generated in response to activity in the superior colliculus or the cerebral cortex (frontal eye fields and

1	Saccades are generated in response to activity in the superior colliculus or the cerebral cortex (frontal eye fields and ClinicaltestingoflabyrinthinefunctionisdoneeitherbyrotatingthepatientinaBáránychairtoactivatethelabyrinthsinbothearsorbyintroducingcoldorwarmwaterintotheexternalauditorycanalofoneear(caloric test). WhenapersonisrotatedinaBáránychair,nystagmusdevelopsduringtherotation.Thedirectionofthefastphaseofthenystagmusisinthesamedirectionastherotation.Whentherotationofthechairishalted,nystagmusintheoppositedirectiondevelops(postrotatorynystagmus)becausestoppingarotationhasthesameeffectasacceleratingintheoppositedirection.

1	Thecalorictestismoreusefulbecauseitcandistinguishbetweenmalfunctionofthelabyrinthsonthetwosides.Theneckisbentbackwardabout60degreessothatthetwohorizontalcanalsareessentiallyvertical.Ifwarmwaterisintroducedintotheleftear,theendolymphintheouterportionoftheloopoftheleftsemicircularcanaltendstoriseasthespecificgravityoftheendolymphdecreasesbecauseofheating.Thissetsupaconvectionflowofendolymph,andasaresult,thekinociliaoftheleftampullarycresthaircellsaredeflectedtowardtheutricle,asiftheheadhadrotatedtotheleft;thedischargeoftheafferentfibersthatsupplythiscanalincreases;andnystagmusoccurswiththefastphasetowardtheleft.Thenystagmusproducesasensationthattheenvironmentisspinningtotheright,andthepersontendstofalltotheright.Theoppositeeffectsareproducedifcoldwaterisplacedintheear.AmnemonicexpressionthatcanhelpinrememberingthedirectionofthenystagmusinthecalorictestisCOWS

1	(“coldopposite,warmsame”).Inotherwords,coldwaterinoneearresultsinafastphaseofnystagmustowardtheoppositeside,andwarmwatercausesafastphasetowardthesameside.

1	posterior parietal areas). Activity in the superior colliculus is related to computation of the direction and amplitude of the saccade. Indeed, the deep layers of the superior colliculus contain a topographic motor map of saccade locations. From the superior colliculus, information is forwarded to distinct sites for control of horizontal and vertical saccades, referred to as the horizontal and vertical gaze centers, respectively. The horizontal gaze center consists of neurons in the paramedian pontine reticular formation, in the vicinity of the abducens nucleus (

1	Fig. 9.28A ). The vertical gaze center is located in the reticular formation of the midbrain: specifically, the rostral interstitial nucleus of the medial longitudinal fasciculus and the interstitial nucleus of Cajal. Because the circuitry and operation of the horizontal gaze center are better understood than those of the vertical gaze center, it is discussed here in detail. However, cells showing analogous activity patterns have been described in the vertical gaze center. Fig. 9.28A is an overview of the neural circuitry by which saccades are generated, and Fig. 9.28B shows the activity of certain types of neurons found in the gaze center that are responsible for horizontal saccades. Each horizontal gaze center has excitatory burst neurons that project to motor neurons in the ipsilateral abducens nucleus and to the internuclear neurons (which excites medial rectus motor

1	CHAPTER 9 Organization of Motor Function neurons in the contralateral oculomotor nucleus). It also has inhibitory burst neurons that inhibit the contralateral abducens. These burst neurons are capable of extremely high bursts of spikes (up to 1000 Hz). Moreover, the gaze center has neurons showing tonic activity and burst-tonic activity. Normally, both inhibitory and excitatory burst neurons are inhibited by omnipause neurons located in the nucleus of the dorsal raphe. When a saccade is to be made, activity from the frontal eye fields or the superior colliculus, or both, leads to inhibition of the omnipause cells and excitation of the burst cells on the contralateral side. The resulting high-frequency bursts in the excitatory burst neurons provide a powerful drive to motor neurons of the ipsilateral lateral rectus and contralateral medial rectus muscles (see

1	Fig. 9.28A ); at the same time, inhibitory burst neurons enable relaxation of the antagonists. The initial bursts of these neurons allow strong contraction of the appropriate extraocular muscles, which overcomes the viscosity of the extraocular muscle and enables rapid movement to occur.

1	Smooth pursuit involves tracking a moving target with the eyes (Fig. 9.29 ). Visual information about target velocity is processed in a series of cortical areas, including the visual cortex in the occipital lobe, several temporal lobe areas, and the frontal eye fields. In the past, the frontal eye fields were thought to be related only to control of saccades, but more recent evidence has shown that there are distinct regions within the frontal eye fields dedicated to either saccade production or smooth pursuit. Indeed, there may be two distinct cortical networks, each specialized for one of these types of eye movement. Cortical activity from multiple cortical areas is fed to the cerebellum via parts of the pontine nuclei and nucleus reticularis tegmenti pontis. Specific areas in the cerebellum—namely, parts of the posterior lobe vermis, the flocculus, and the paraflocculus—receive this input, and they in turn project to the vestibular nuclei. From the vestibular nuclei, activity can

1	parts of the posterior lobe vermis, the flocculus, and the paraflocculus—receive this input, and they in turn project to the vestibular nuclei. From the vestibular nuclei, activity can then be forwarded to the oculomotor, abducens, and trochlear nuclei, as was described for the VOR earlier.

1	The neural circuits underlying vergence movements are not well known. There are premotor neurons (neurons that feed onto motor neurons) located in the brainstem areas surrounding the various oculomotor nuclei. In some cortical visual areas and the frontal eye fields, there are neurons whose activity is related to the disparity of the image on the two retinas or to the variation of the image during vergence movements. How vergence signals in these cortical areas feed into the brainstem premotor neurons is not clear. The cerebellum also appears to play a role in vergence movements because cerebellar lesions impair this type of eye movement. Note that lesions of the medial longitudinal fasciculus that result in a loss of the VOR do not compromise vergence.

1	•Fig. 9.28 Horizontal Saccade Pathways. A, Circuitdiagramofthemajorpathways.EBN,excitatoryburstneuron;FEF,frontaleyefield;IBN,inhibitoryburstneuron;LBN,longleadburstneuron;OPN,omnipauseneuron;PPRF,paramedianpontinereticularformation.B, Firingpatternsofsomeoftheneuronsinvolvedinmakingsaccades.Excitationofburstneuronsoftherighthorizontalgazecentercausesabducensmotorneuronsontherightandmedialrectusmotorneuronsonthelefttobeactivated.Theascendingpathwaytotheoculomotornucleusisthroughthemediallongitudinalfasciculus.Thelefthorizontalgazecenterissimultaneouslyinhibited.

1	CHAPTER 9 Organization of Motor Function •Fig. 9.29 Smooth Pursuit Pathways. Thestimulusforsmoothpursuiteyemovementisamovingvisualtarget.Thiscausesactivitytoflowthroughthecircuitrydiagramedinthefigureandleadstomaintenanceofthefoveaonthetarget.FEF,frontaleyefield;LGN,lateralgeniculatenucleus;MSTandMT,higherordervisualassociationareas;MVN,medialvestibularnucleus;NRTP,nucleusreticularistegmentipontis;SEF,supplementaryeyefield;V1,primaryvisualcortex.Retina LGN V1 MT MST Pontine nuclei FEF SEF NRTP Flocculus paraflocculus Vermis MVN y-group Fastigial nucleus Oculomotor nuclei Cortical areas involved in processing object motion Brainstem and cerebellar areas involved in transforming sensory-related signal into motor command Motor commands to eye muscles 1.

1	The extrafusal skeletal muscle fibers are innervated by α motor neurons. A motor unit consists of a single α motor neuron and all the muscle fibers with which it synapses. Motor unit size varies greatly among muscles; small motor units allow finer control of muscle force. 2. The size principle refers to the orderly recruitment of α motor neurons according to their size, from smallest to largest. Because smaller motor neurons connect to weaker motor units, the relative fineness of motor control is similar for weak and strong contractions. 3. A reflex is a simple, stereotyped motor response to a stimulus. A reflex arc includes the afferent fibers, interneurons, and motor neurons responsible for the reflex. 4.

1	3. A reflex is a simple, stereotyped motor response to a stimulus. A reflex arc includes the afferent fibers, interneurons, and motor neurons responsible for the reflex. 4. Muscle spindles are complex sensory receptors found in skeletal muscle. They lie parallel to extrafusal muscle fibers, and they contain nuclear bag and nuclear chain intrafusal muscle fibers. By being in parallel to the main muscle, the spindle can detect changes in muscle length. 5. Group Ia afferent fibers form primary endings on nuclear bag1, bag2, and chain fibers, and group II fibers form secondary endings on nuclear chain and bag2 fibers. 6. Primary endings demonstrate both static and dynamic responses that signal muscle length and rate of change in muscle length. Secondary endings demonstrate only static responses and signal only muscle length. 7.

1	7. The intrafusal muscle fibers associated with muscle spindles are innervated by γ motor neurons. Contraction of intrafusal fibers does not directly cause significant changes in muscle tension or length; however, when the level of tension in these fibers is adjusted, γ motor neurons influence the sensitivity of the muscle spindle to stretch. 8. Golgi tendon organs are located in the tendons of muscles and are thus arranged in series with the muscle. They are supplied by group Ib afferent fibers. Their in-series relationship means that tendon organs can detect the force level generated by the muscle, whether it is due to passive stretch or to active contraction of the muscle. 9. The phasic stretch (or myotatic) reflex includes (1) a monosynaptic excitatory pathway from group Ia afferent fibers in muscle spindles to α motor neurons that supply the same and synergistic muscles and (2) a disynaptic inhibitory pathway to antagonistic motor neurons. 10.

1	10. The inverse myotatic reflex is evoked by Golgi tendon organs. Afferent volleys in group Ib fibers from a given muscle cause disynaptic inhibition of α motor neurons to the same muscle, and they excite α motor neurons to antagonist muscles. 11. The flexion reflex is an important protective response because it acts to withdraw a limb from damaging stimuli. The reflex is evoked by volleys in afferent fibers that supply various receptors, particularly nociceptors. Via polysynaptic pathways, these volleys cause excitation of flexor motor neurons and inhibition of extensor motor neurons ipsilaterally. Concurrently, the opposite pattern of action (inhibition of flexor and excitation of extensor motor neurons) occurs contralaterally and is referred to as the crossed extension reflex. 12.

1	12. Descending pathways can be subdivided into (1) a lateral system, which ends on motor neurons to limb muscles and on the lateral group of interneurons, and (2) a medial system, which ends on the medial group of interneurons. 13. The lateral system includes the lateral corticospinal tract and part of the corticobulbar tract. These pathways influence the contralateral motor neurons that supply the musculature of the limbs, especially of the digits, and the muscles of the lower part of the face and the tongue. 14. The medial system includes the ventral corticospinal, lateral and medial vestibulospinal, reticulospinal, and tectospinal tracts. These pathways affect mainly posture and provide the motor background for movement of the limbs and digits. 15.

1	15. Locomotion is triggered by commands relayed through the midbrain locomotor center. However, central pattern generators formed by spinal cord circuits and influenced by afferent input provide for the detailed organization of locomotor activity. 16. Voluntary movements depend on interactions among motor areas of the cerebral cortex, the cerebellum, and the basal ganglia. 17. Motor areas of the cerebral cortex are arranged as a parallel distributed network, in which each contributes to the various descending motor pathways. The areas primarily involved in body and head movement include the primary motor cortex, the premotor area, the supplementary motor cortex, and the cingulate motor areas. The frontal eye fields are important for eye movement and help initiate voluntary saccades. 18.

1	18. Individual corticospinal neurons discharge before voluntary contractions of related muscles occur. The discharges are typically related to contractile force rather than to joint position. However, the activity of an individual neuron may encode different parameters of a movement at different times in relation to the execution of that movement. 19. The population activity of motor cortex neurons can be used to predict the direction of upcoming movements. 20. The cerebellum influences the rate, range, force, and direction of movements. It also influences muscle tone and posture, as well as eye movement and balance. 21. The intrinsic circuitry of the cerebellum is remarkably uniform. Differences in function of different parts of the cerebellum arise largely as a result of differing afferent sources and efferent targets. 22.

1	22. Anatomical and physiological techniques have shown that the cerebellar cortex may be divided into many functionally distinct, longitudinally running compartments. 23. Most of the input to the cerebellum is through pathways that end as mossy fibers. Mossy fibers excite granule cells, which in turn can evoke single action potentials, called simple spikes, in Purkinje cells, whose axons form the only output pathway from the cerebellar cortex. 24. The projections of the inferior olivary nucleus to the cerebellum end as climbing fibers and are the only source of them. Each Purkinje cell receives massive input from just one climbing fiber. As a result, each climbing fiber discharge produces a high-frequency burst of several action potentials, known as a complex spike, in the Purkinje cell. 25.

1	25. Although complex spike activity is relatively rare in comparison to simple spike activity, complex spikes are precisely synchronized across populations of Purkinje cells, and because of the convergence of these cells onto cerebellar nuclear neurons, this synchronization may allow complex spike activity to significantly affect cerebellar output. Synchronization of complex spikes is the result of electrical coupling of inferior olivary neurons by gap junctions. 26. The basal ganglia include several deep telencephalic nuclei (including the caudate nucleus, putamen, and globus pallidus). The basal ganglia interact with the cerebral cortex, subthalamic nucleus, substantia nigra, and thalamus. 27.

1	27. Activity transmitted from the cerebral cortex through the basal ganglia can either facilitate or inhibit the thalamic neurons that project to motor areas of the cortex, depending on the balance between direct and indirect basal ganglia pathways. When there is an imbalance of these two pathways, hyperkinetic or hypokinetic disorders occur. CHAPTER 9 Organization of Motor Function 28. Some types of eye movement help stabilize the view of the visual world. This is critical because visual acuity drops dramatically when the visual world moves, or slips, across the retina. Vestibuloocular and optokinetic movements help stabilize the visual world on the retina by compensating for movement of the head or external world (or both). Smooth pursuit movements allow tracking of a visual target so that it remains centered on the foveae. Eccles JC, Ito M, Szentagothai J. The Cerebellum as a Neuronal Machine. New York: Springer; 2013. (Original work published 1967.)

1	Eccles JC, Ito M, Szentagothai J. The Cerebellum as a Neuronal Machine. New York: Springer; 2013. (Original work published 1967.) Shadmehr R, Wise SP. The Computational Neurobiology of Reaching and Pointing: A Foundation for Motor Learning. Cambridge, MA: Bradford Books; 2004. Shepherd GM. Spinal cord. In: Synaptic Organization of the Brain. 5th ed. Oxford, UK: Oxford University Press; 2003: Chapter 3. 29. Saccades act to move a specific part of the visual scene to the fovea, the retinal area of highest acuity, for detailed inspection. 30. There are specialized circuits and areas in the brainstem for control of vertical and horizontal eye movements. These areas are used both by the cortex (when voluntary eye movements are made) and by the sensory input that initiates reflexive eye movement. Shepherd GM. Cerebellum. In: Synaptic Organization of the Brain. 5th ed. Oxford, UK: Oxford University Press; 2003: Chapter 7.

1	Shepherd GM. Cerebellum. In: Synaptic Organization of the Brain. 5th ed. Oxford, UK: Oxford University Press; 2003: Chapter 7. Shepherd GM. Basal ganglia. In: Synaptic Organization of the Brain. 5th ed. Oxford, UK: Oxford University Press; 2003: Chapter 9. Xiao J, Cerminara NL, Kotsurovskyy Y, et al. Systematic regional variations in Purkinje cell spiking patterns. PLoS ONE. 2014;9: e105633. Upon completion of this chapter, the student should be able to answer the following questions: 1. What is the basic layering pattern of the neocortex, and how do cortical inputs and outputs align with this layering pattern? What is the functional significance of the variation in the layering pattern between cortical areas? 2. What are the major functions of each of the lobes of the cerebrum? 3. How does the electroencephalogram (EEG) reflect cortical activity? What are evoked potentials? 4. How does cerebral dominance correlate with language and hand preference? 5.

1	3. How does the electroencephalogram (EEG) reflect cortical activity? What are evoked potentials? 4. How does cerebral dominance correlate with language and hand preference? 5. What is aphasia, and what is compromised in the different types of aphasia? 6. How do synaptic and cellular processes support learning and memory? How is memory distributed in the brain? 7. What role does plasticity play in neural development and in response to damage of the nervous system?

1	n earlier chapters, the interaction of the nervous system with the body and the outside world was discussed in terms of the transduction and analysis of sensory events, the organization of motor function, and relatively simple central processes that link them, such as reflexes (e.g., the stretch reflex and the vestibuloocular reflex). The nervous system has other capabilities, so-called integrative or higher functions, that are less directly tied to specific sensory modalities or motor behavior. These functions, in particular, require interactions between different parts of the cerebral cortex, and, as is being increasingly recognized, between the cerebral cortex and other parts of the brain. The neural basis for some of these higher functions is discussed in this chapter. Because these functions (as well as sensory perception and voluntary motor function) are so highly dependent on the cerebral cortex, its basic organization is described first. The Cerebral Cortex

1	The Cerebral Cortex The human cerebral cortex occupies a volume of about 600 cm3 and has a surface area of 2500 cm2. The surface of the cortex is highly convoluted and folded into ridges known as gyri. Gyri are separated by grooves called sulci (if shallow) or fissures (if deep; see Fig. 4.7 ). This folding greatly increases the surface area of cortex that can be fit into the limited and fixed volume within the skull. Indeed, most of the cortex cannot be seen from the brain surface because of this folding. The cerebral cortex can be divided into the left and right hemispheres and subdivided into a number of lobes (Fig. 10.1 Fig. 4.7 ), including the frontal, parietal, temporal, and occipital lobes. The frontal and parietal lobes are separated by the central sulcus; both are separated from the temporal lobe by the lateral fissure. The occipital and parietal lobes are separated (on the medial surface of the hemisphere) by the parieto-occipital fissure (see Fig.

1	Fig. 10.1 ). Buried within the lateral fissure is another lobe, the insula (see Fig. 4.6A). A group of structures that make up the limbic lobe is on the medial aspect of the hemisphere, and its largest part, the hippocampal formation, is folded into the parahippocampal gyrus of the temporal lobe and cannot be seen from the surface of the brain. Activity in the two hemispheres of the cerebral cortex is coordinated by interconnections through the cerebral commissures. The bulk of the cortex is connected through the massive corpus callosum (see Figs. 4.9 10.1), and parts of the temporal lobes connect through the anterior commissure. There are three types of cerebral cortex: neocortex, archicortex, and paleocortex. The neocortex has six cortical layers (Fig. 10.2 ). In contrast, the archicortex has only three layers, and the paleocortex has four to five layers. In humans, approximately 90% of the cerebral cortex is neocortex. The Neocortex Neuronal Cell Types in the Neocortex

1	The Neocortex Neuronal Cell Types in the Neocortex A number of different neuronal cell types in the neocortex have been described (see Fig. 10.2 ). Pyramidal cells are the most abundant cell type and account for approximately 75% of neocortical neurons. Stellate cells and various other types of nonpyramidal neurons make up the balance. Pyramidal cells have a large triangular cell body, a long •Fig. 10.1 Lateral(A) andmedial(B) illustrationsofthelefthemisphereofthehumancerebrumwiththemajorfeatureslabeledandthelobesindicatedbycolor.R,G,B,andSindicate,respectively,therostrum,genu,body,andspleniumofthecorpuscallosum.(FromHainesDE[ed].Fundamental Neuroscience for Basic and Clinical Applications. 3rded.Philadelphia:ChurchillLivingstone;2006.) I. Molecular layer II. External granular layer III.External pyramidal layer IV. V. VI.Polymorphic layer Outer band of Baillarger Inner band of Baillarger

1	I. Molecular layer II. External granular layer III.External pyramidal layer IV. V. VI.Polymorphic layer Outer band of Baillarger Inner band of Baillarger Fig. 10.2 An Area of Neocortex Stained by Three Different Methods. TheNisslstain(center) showsthecellbodiesofallneuronsandrevealshowdifferenttypesaredistributedamongthesixlayers.TheGolgistain(left) showsonlyasampleoftheneuronalpopulationbutrevealsdetailsoftheirdendrites.TheWeigertstainformyelin(right) demonstratesverticallyorientedbundlesofaxonsenteringandleavingthecortexandhorizontallycoursingfibersthatinterconnectneuronswithinalayer.(FromBrodmann K.

1	K. Zellenbaues. Leipzig:JABarth;1909.) apical dendrite directed toward the cortical surface, and several basal dendrites. The cell’s axon emerges from the body opposite the apical dendrite, and those from the larger pyramidal cells project into the subcortical white matter. The axon may give off collateral branches as it descends through the cortex. The neurotransmitter of pyramidal cells is an excitatory amino acid (glutamate or aspartate). Stellate cells, often called granule cells, are interneurons with local connections. They have a small soma and numerous branched dendrites, although many have an apical dendrite and thus look like small pyramidal cells. Some are excitatory interneurons; these cells are abundant in layer IV of the cortex (described in the next section). Their axons remain in the same cortical region, and many ascend into the upper cortical layers. Some stellate cells are inhibitory inter-neurons whose neurotransmitter is gamma-aminobutyric acid (GABA).

1	Cytoarchitecture of Cortical Layers Each of the six layers of the neocortex has a characteristic cellular content (see Fig. 10.2 ). Layer I (molecular layer) has few neuronal cell bodies and contains mostly axon terminals synapsing on apical dendrites. Layer II (external granular layer) contains mostly stellate cells. Layer III (external pyramidal layer) consists mostly of small pyramidal cells. Layer IV (internal granular layer) contains mostly stellate cells and a dense matrix of axons. Layer V (internal pyramidal layer) is dominated by large pyramidal cells, the main source of cortical efferents to most subcortical regions. Layer VI (multiform layer) contains pyramidal, fusiform, and other types of cells. Most input to the cortex from other regions of the central nervous system (CNS) is relayed by neurons in the thalamus, the paleocortex has four to five layers. The paleocortex is located at the border between the archicortex and neocortex.

1	In humans, the hippocampal formation is part of the archicortex. It is folded into the temporal lobe and can be viewed only when the brain is dissected. The hippocampal cortex has three layers: the molecular, pyramidal cell, and polymorphic layers. They resemble layers I, V, and VI of the neocortex. The white matter covering the hippocampus is called the alveus, which contains hippocampal afferent and efferent fibers. The efferent axons coalesce to form the fornix (Fig. 10.4 Functions of the Lobes of the Cerebral Cortex There is no exact correspondence between the folds (lobes and gyri) of the cerebral cortex and function; nevertheless, some with the individual lobes of the cerebral hemispheres have a general association with function that helps clarify cortical organization.

1	One of the main functions of the frontal lobe is motor behavior. As discussed in , the motor, premotor, cingulate motor, and supplementary motor areas are located •Fig. 10.3 Brodmann’sareasinthehumancerebralcortex.(RedrawnfromCrosbyEC,etal.Correlative Anatomy of the Nervous System. NewYork:Macmillan;1962.)6312578910113825273029262834363523312433321237201919181817B •Fig. 10.4 Thehippocampusandtheamygdalaarelocatedonthemedialaspectofthetemporallobe.Thefornix,themajoroutputpathwayfromthehippocampus,projectstothemammillarybody,whichinturnconnectstotheanteriornucleusofthethalamusviathemammillothalamictract.Alsoillustratedarethecingulategyrus,thebasalforebrainarea(septalnuclei,bednucleusofthestriaterminalis,nucleusaccumbens),andtheprefrontalcortex.(FromPurvesD.Sleepandwakefulness.In:PurvesD,etal[eds].Neuroscience. 3rded.Sunderland,MA:Sinauer;2004.) in the frontal lobe, as is the frontal eye field. These areas are crucial for planning and executing motor behavior. Broca’s area, essential for

1	3rded.Sunderland,MA:Sinauer;2004.) in the frontal lobe, as is the frontal eye field. These areas are crucial for planning and executing motor behavior. Broca’s area, essential for the generation of speech, is located in the inferior frontal gyrus of the dominant hemisphere for human language (almost always the left hemisphere, as explained later). In addition, the more anterior prefrontal cortex in plays a major role in personality and emotional behavior.

1	Bilateral lesions of the prefrontal cortex may be produced either by disease or by a surgical frontal lobotomy. Such lesions produce deficits in attention, difficulty in planning and problem solving, and inappropriate social behavior. Aggressive behavior is also lessened and the motivational-affective component of pain is reduced although pain sensation remains. Frontal lobotomies are rarely performed today because modern drug therapies provide more focal and effective management for mental illness and chronic pain.

1	The parietal lobe contains the somatosensory cortex (see ) and the adjacent parietal association cortex. The parietal association cortex gets information from somatosensory, visual, and auditory cortices and is involved in the processing, perception, and integration of sensory information. Connections with the frontal lobe allow somatosensory information to aid in voluntary motor activity. Somatosensory, visual, and auditory information can also be transferred to language centers, such as Wernicke’s area, as described later. Lesions in the left parietal lobe can result in Gerstmann’s syndrome, which includes a person’s inability to name his or her fingers (or those of another) and a loss of the ability to perform numerical calculations. The right parietal lobe is involved in determining spatial context. Localized lesions can result in the neglect syndrome, in which the patient seems unaware of the left side of his or her body and of persons, objects, and events on his or her left.

1	spatial context. Localized lesions can result in the neglect syndrome, in which the patient seems unaware of the left side of his or her body and of persons, objects, and events on his or her left. (See an “

1	In the Clinic ” box later in this chapter.) The major function of the occipital lobe is visual processing and perception (see ). The primary visual cortex (Brodmann area 17) lines the calcarine sulcus and is flanked by secondary (Brodmann area 18) and tertiary (Brodmann area 19) visual cortices. Lesions of these areas in the cuneus gyrus result in blindness in the lower contralateral visual field; those in the lingual gyrus result in blindness in the upper contralateral visual field. Connections to the frontal eye fields affect direction of gaze, and projections to the midbrain assists in the control of convergent eye

1	Twoareasimportantforplanningandexecutingmotortasksaretheparietal cortex andthefrontal cortex, theformerbecauseitintegratessensoryinformationneededtodefinethecontextofatask(see ).Mirrorneuronshavebeenfoundinboththeinferior parietal andtheinferior frontal cortices of macaques. Thesecellsrespondduringperformanceofaspecificmotortaskandalsoduringobservationofthesametaskperformedbyanotheranimal.Becausethesemirrorcellsseemtoencodeforandrespondtoveryspecificandparticulartasks,ithasbeenspeculatedthattheymayunderliesuchfunctionsasunderstandingtheintentionsofothersandempathy,aswellastheabilitytolearntasksfromobservation.Inhumans,EEGactivityconsistentwiththebehaviorofsuchmirrorneuronshasbeenlocalizedtotheinferior frontal andsuperior parietal lobes. Autism,whichinvolvesaninabilityto“read”theintentionsandemotionsofothers,hasbeenlinkedtoalackofmirrorneuronsbyEEGevidence. movements, pupillary constriction, and accommodation, all of which occur when the eyes adjust for near vision.

1	movements, pupillary constriction, and accommodation, all of which occur when the eyes adjust for near vision. The temporal lobe has many different functions, including the processing and perception of sounds and vestibular information and higher order visual processing (see ). For example, the infratemporal cortex, on its inferior surface, is involved in the recognition of faces. In addition, Meyer’s loop, which forms part of the optic pathway, passes through the temporal lobe. As a result, temporal lobe lesions can damage vision in upper part of the visual field. Similarly, a portion of Wernicke’s area, essential for the understanding of language, lies in the posterior region of the temporal lobe. The limbic system dominates the medial temporal lobe (see

1	The limbic system dominates the medial temporal lobe (see Fig. 10.4 ), and it participates in emotional behavior and in learning and memory (see the “Learning and Memory” section). The limbic system helps control emotional behavior, in part by an influence on the hypothalamus via the Papez circuit. This circuit projects from the cingulate gyrus to the entorhinal cortex and hippocampus, and from there, via the fornix, to the mammillary bodies in the hypothalamus. The mammillothalamic tract then connects the hypothalamus with the anterior thalamic nuclei, which project back to the cingulate gyrus (see Fig. 10.4 ). In addition, the hippocampus and amygdala are connected to the prefrontal cortex, the basal forebrain, and the anterior cingulate cortex.

1	Fig. 10.4 ). In addition, the hippocampus and amygdala are connected to the prefrontal cortex, the basal forebrain, and the anterior cingulate cortex. Bilateral temporal lobe lesions can produce Klüver-Bucy syndrome, which is characterized by loss of the ability to recognize the meaning of objects from visual cues (visual agnosia); a tendency to examine all objects, even dangerous ones, orally; attention to irrelevant stimuli; hypersexuality; a change in dietary habits; and decreased emotionality.

1	Although this syndrome was originally described as following large lesions of most or all of the temporal lobe, more recent studies have highlighted the role of the amygdala. The amygdala conditions the association of fear with painful stimuli and may trigger, via connections to the medial frontal cortex and anterior cingulate gyrus, emotional or avoidance responses when these stimuli recur. In addition, the amygdala projects to the nucleus accumbens, a region of the basal ganglia which has been called a “reward center.” The nucleus accumbens signals pleasurable events in response to dopaminergic input from the ventral tegmental area of the brainstem and is also the most common site of the plaques and tangles associated with Alzheimer’s disease.

1	Thefunctionsofthedifferentlobesofthecerebralcortexhavebeendefinedaccordingtoboththeeffectsoflesionsproducedbydiseaseorthoseofsurgicalinterventionstotreatdiseaseinhumansandfromexperimentsonanimals.Inanotherapproach,thephysicalmanifestationsofepileptic seizures havebeencorrelatedwiththebrainlocationsthatgiverisetoseizures(epileptic seizure foci). Forexample,epilepticfociinthemotorcortexcausemovementsonthecontralateralside;theexactmovementsrelatetothesomatotopiclocationoftheseizurefocus.Seizuresthatoriginateinthesomatosensorycortexcauseanepileptic aura inwhichatouchsensationisperceived.Similarly,seizuresthatstartinthevisualcortexcauseavisualaura(scintillations,colors),thoseintheauditorycortexcauseanauditoryaura(humming,buzzing,ringing),andthoseinthevestibularcortexcauseasensationofspinning.Complexbehaviorresultsfromseizuresthatoriginateintheassociationareasofthetemporallobe;inaddition,amalodorousauramaybeperceivediftheolfactorycortexisinvolved (uncinate fit).

1	The Electrical Activity of the Cortex

1	An electroencephalogram (EEG) is a recording of the neuronal electrical activity of the cerebral cortex made by electrodes placed on the skull. EEG waves normally reflect the summed extracellular currents that result from the generation of synaptic potentials in the pyramidal cells and are thus a type of field potential. Because the currents generated by a single cell are too small to be detected as discrete events by an electrode on the skull (to record the activity of a single neuron, a microelectrode must be placed within microns of the neuron), the EEG waves reflect the combined activity of many pyramidal cells. Moreover, for the activity of a group of neurons to generate an event detectable on EEG, they must be oriented so that their individual currents summate to produce a detectable field. The arrangement of pyramidal neurons, with their apical dendrites aligned in parallel to form a dipole sheet, is particularly favorable for generating large field potentials. One pole of this

1	field. The arrangement of pyramidal neurons, with their apical dendrites aligned in parallel to form a dipole sheet, is particularly favorable for generating large field potentials. One pole of this sheet is oriented toward the cortical surface and the other toward the subcortical white matter. Thus the currents generated •Fig. 10.5 Electroencephalographictracingsduringdrowsiness;stages1,2,and4ofslow-wave(non–rapideyemovement[non-REM])sleep;andREMsleep.(ModifiedfromShepherdGM.Neurobiology. London:OxfordUniversityPress;1983.) by cortical pyramidal cells have a similar orientation and can therefore summate to produce a field potential that can be detected. The need for summation also explains why EEG signals reflect primarily synaptic potentials rather than action potentials: electrical events must overlap in time in order to sum, and synaptic potentials have much longer durations than do action potentials.

1	The sign of an EEG wave can be positive or negative, but its direction alone does not indicate whether pyramidal cells are being excited or inhibited. For instance, a negative EEG potential may be generated at the surface of the skull (or cortex) by excitation of apical dendrites or by inhibition near the somas. Conversely, a positive EEG wave can be produced by inhibition of apical dendrites or by excitation near the somas.

1	A normal EEG tracing consists of waves of various frequencies. The dominant frequencies depend on several factors, including the state of wakefulness, the age of the subject, the location of the recording electrodes, and the absence or presence of drugs or disease. When a normal awake adult is relaxed with the eyes closed, the dominant frequencies of the EEG recorded over the parietal and occipital lobes are about 8 to 12 Hz, the alpha rhythm. If the subject is asked to open the eyes, the wave becomes less synchronized, and the dominant frequency increases to 13 to 30 Hz, which is called the beta rhythm. The delta (0.5 to 2 Hz) and theta (3 to 7 Hz) rhythms are observed during sleep (see the following discussion; Fig. 10.5 ). Also, brief EEG waves do exist and, because of their shape, are sometimes referred to as spikes, but this does not imply that they are associated with action potentials.

1	Fig. 10.5 ). Also, brief EEG waves do exist and, because of their shape, are sometimes referred to as spikes, but this does not imply that they are associated with action potentials. An EEG change that can be elicited by a stimulus is called a cortical evoked potential. A cortical evoked potential is best recorded from the part of the skull located over the cortical area being activated. For example, a visual stimulus results in an evoked potential that can be recorded best over the occipital bone, whereas a somatosensory evoked potential is recorded most effectively near the junction of the frontal and parietal bones. Evoked potentials reflect the activity in large numbers of cortical neurons. They may also reflect activity in subcortical structures.

1	Evoked potentials are small in comparison with the size of the EEG waves. However, their appearance can be enhanced by a process called signal averaging. In this process, the stimulation is repeated, and the EEGs recorded during each trial are electronically averaged. With each repetition of the stimulus, the evoked potential occurs at a fixed time after the stimulus. When the records are averaged, the components of the EEG that have a random temporal association with the stimulus cancel each other, whereas the evoked potentials sum. Evokedpotentialsareusedclinicallytoassesstheintegrityofasensorypathway,atleasttotheleveloftheprimarysensoryreceivingarea.Thesepotentialscanberecordedincomatoseindividuals,aswellasininfantstooyoungtoundergoasensoryexamination.Theinitialpartsoftheauditoryevokedpotentialactuallyreflectactivityinthebrainstem;therefore,thisevokedpotentialcanbeusedtoassessthefunctionofbrainstemstructures.

1	Sleep and wakefulness are among the many functions of the body that show circadian (about 1-day) periodicity. Characteristic changes in the EEG can be correlated with changes in the behavioral state during the sleep-wake cycle. Beta wave activity dominates in an awake, aroused individual. The EEG is said to be desynchronized; it displays low-voltage, high-frequency activity. In relaxed individuals with their eyes closed, the EEG is dominated by alpha waves (see Fig. 10.5 ). A person falling asleep passes sequentially through four stages of slow-wave sleep (called stages 1 through 4) over a period of 30 to 45 minutes (see

1	Fig. 10.5 ). A person falling asleep passes sequentially through four stages of slow-wave sleep (called stages 1 through 4) over a period of 30 to 45 minutes (see Fig. 10.5 ). In stage 1, alpha waves are interspersed with lower frequency waves called theta waves. In stage 2, the waves slow further, but the slow-wave activity is interrupted by sleep spindles, which are bursts of activity at 12 to 14 Hz, and by large K complexes (large, slow potentials). Stage 3 sleep is associated with delta waves and with occasional sleep spindles. Stage 4 is characterized by delta waves without spindles. During slow-wave sleep, the muscles of the body relax, but the posture is adjusted intermittently. The heart rate and blood pressure decrease, and gastrointestinal motility increases. The ease with which individuals can be awakened decreases progressively as they pass through these sleep stages. As individuals awaken, they pass through the sleep stages in reverse order.

1	About every 90 minutes, slow-wave sleep changes to a different form of sleep, called rapid eye movement (REM) sleep. In REM sleep, the EEG again becomes desynchronized. The low-voltage, fast activity of REM sleep resembles that seen in the EEG from an aroused subject (see Fig.

1	Fig. 10.5 , bottom trace). Because of the similarity of the EEG to that of an awake individual and the difficulty awaking the person, the term paradoxical sleep characterizes this type of sleep. Muscle tone is completely lost, but phasic contractions occur in a number of muscles, most notably the eye muscles. The resulting rapid eye movements are basis of the name for this type of sleep. Many autonomic changes also take place. Temperature regulation is lost, and meiosis occurs. Penile erection may occur during this type of sleep. Heart rate, blood pressure, and respiration change intermittently. Several episodes of REM sleep occur each night. Although it is difficult to arouse a person from REM sleep, internal arousal is common. Most dreaming occurs during REM sleep.

1	Thesleep-wakecyclehasanendogenousperiodicityofabout25hours,butitnormallybecomesentrainedtotheday-nightcycle.Thesourceofcircadianperiodicityappearstobethesuprachiasmaticnucleusofthehypothalamus.Thisnucleusreceivesprojectionsfromtheretina,anditsneuronsseemtoformabiologicalclockthatadaptstothelight-darkcycle.However,theentrainmentcanbedisruptedwhenthesubjectisisolatedfromtheenvironmentorchangestimezones(jetlag).Destructionofthesuprachiasmaticnucleusdisruptsanumberofbiologicalrhythms,includingthesleep-wakecycle. The proportion of slow-wave (non-REM) sleep to REM sleep varies with age. Newborns spend about half of their sleep time in REM sleep, whereas elderly people have little REM sleep. About 20% to 25% of the sleep of young adults is REM sleep.

1	The mechanism of sleep is incompletely understood. Stimulation in the brainstem in a large region known as the reticular activating system causes arousal and low-voltage, fast EEG activity. Sleep was once thought to be caused by a reduced level of activity in the reticular activating system. However, substantial data, including the observations that anesthesia of the lower brainstem results in arousal and that stimulation in the medulla near the nucleus of the solitary tract can induce sleep, suggest that sleep is an active process. Investigators have tried to find a relationship between sleep mechanisms and brainstem networks in which particular neurotransmitters, including serotonin, norepinephrine, and acetylcholine, are used; manipulations of the levels of these transmitters in the brain can affect the sleep-wake cycle. However, a detailed neurochemical explanation of the neural mechanisms of sleep is not yet available.

1	Similarly, the purpose of sleep is still unclear. However, it must have a high value because so much of life is spent in sleep and because lack of sleep can be debilitating. Medically important disorders of the sleep-wake cycle include insomnia, bed-wetting, sleepwalking, sleep apnea, and narcolepsy. •Fig. 10.6 Electroencephalographic (EEG) Abnormalities in Several Forms of Epilepsy. A, EEGtracingsduringthetonic(left) andclonic(right) phasesofatonic-clonic(grandmal)seizure.B, Spikeandwavecomponentsofanabsence(petitmal)seizure.C, EEGtracinginapersonwithtemporallobeepilepsy.D, EEGtracingofafocalseizure.(RedrawnfromEyzaguirreC,FidoneSJ.Physiology of the Nervous System. 2nded.St.Louis:Mosby;1975.) TheEEGbecomesabnormalinavarietyofpathologicalcircumstances.Forexample,duringcoma,theEEGisdominatedbydeltaactivity.Brain death isdefinedbyamaintainedflatEEGwave.

1	TheEEGbecomesabnormalinavarietyofpathologicalcircumstances.Forexample,duringcoma,theEEGisdominatedbydeltaactivity.Brain death isdefinedbyamaintainedflatEEGwave. Epilepsy commonlycauses,andcanbediagnosedby,specificEEGabnormalities.Therearemanyformsofepilepsy,andexamplesofEEGpatternsfromsomeofthesetypesofepilepsyareshownin Figure10.6 .Epilepticseizurescanbeeitherpartialorgeneralized. Oneformofpartialseizuresoriginatesinthemotorcortexandresultsinlocalizedcontractionsofcontralateralmuscles.Thecontractionsmaythenspreadtoothermuscles;suchspreadfollowsthesomatotopicsequenceofthemotorcortex(see ).ThisstereotypicalprogressioniscalledaJacksonian march. Complexpartialseizures(whichmayoccurinpsychomotor epilepsy)originateinthelimbicstructuresofthetemporallobeandresultinillusionsandsemipurposefulmotoractivity.Duringandbetweenfocalseizures,scalprecordingsmayrevealEEGspikes(see Fig. 10.6C andD Generalizedseizuresinvolvewideareasofthebrainandlossofconsciousness.Twomajortypesarepetit mal and

1	Fig. 10.6C andD Generalizedseizuresinvolvewideareasofthebrainandlossofconsciousness.Twomajortypesarepetit mal and Although right-handedness represents a sensorimotor dominance of the left hemisphere and left-handedness represents a sensorimotor dominance of the right hemisphere, cerebral dominance is assigned to the hemisphere in which language is to communicate; in humans, the left hemisphere is the dominant hemisphere in more than 90% of both rightand left-handed people. This dominance grand mal seizures. Inpetitmalepilepsy,consciousnessislosttransiently(typicallyforlessthan15seconds),andtheEEGdisplaysspike and wave activity (see Fig.10.6B ).Ingrandmalseizures,consciousnessislostforalongerperiod,andtheaffectedindividualmayfallifstandingwhentheseizurestarts.Theseizurebeginswithageneralizedincreaseinmuscletone(tonic phase), followedbyaseriesofjerkymovements(clonic phase). Thebowelandbladdermaybeevacuated.TheEEGshowswidelydistributedseizureactivity(see Fig.10.6A

1	Fig.10.6A EEGspikesthatoccurbetweenfull-blownseizuresarecalledinterictal spikes. Similareventscanbestudiedexperimentally.Thesespikesarisefromabrupt,long-lastingdepolarizations,calleddepolarization shifts, thattriggerrepetitiveactionpotentialsincorticalneurons.Thesedepolarizationshiftsmayreflectseveralchangesinepilepticfoci.SuchchangesincluderegenerativeCa++-mediateddendriticactionpotentialsincorticalneuronsandareductionininhibitoryinteractionsincorticalcircuits.ElectricalfieldpotentialsandthereleaseofK+ andexcitatoryaminoacidsfromhyperactiveneuronsmayalsocontributetotheincreasedcorticalexcitability.

1	has been demonstrated (1) by the effects of lesions of the left hemisphere that produce deficits in language function (aphasia) and (2) by the transient aphasia (inability to speak or write) that results when a short-acting anesthetic is introduced into the left carotid artery. Lesions of the nondominant hemisphere and injection of anesthetic into it do not usually affect language substantially.

1	Several areas in the left hemisphere are involved in language. Wernicke’s area is a large area in the posterior part of the superior temporal gyrus, extending from behind the auditory cortex into the parietal lobe. Another important language area, Broca’s area, is in the posterior part of the inferior frontal gyrus, close to the face representation of the motor cortex. Damage to Wernicke’s area results in receptive aphasia, in which the person has difficulty comprehending spoken and written language; however, speech production remains fluent, if meaningless. Conversely, a lesion in Broca’s area causes expressive aphasia, in which individuals have difficulty in generating speech and writing, although they can understand language relatively well.

1	The terms sensory aphasia and motor aphasia are often interchanged with receptive aphasia and expressive aphasia, respectively. The former terms, however, are misleading: A person with receptive aphasia may not have auditory or visual impairment, and one with expressive aphasia may have normal motor control of the muscles responsible for speech or writing. Aphasia does not depend on a deficit of sensation or of motor skill; rather, it is an inability to decode language-encoded sensory information into concepts or to encode concepts into language. However, lesions in the dominant hemisphere may be large enough to result in mixed forms of aphasia, as well as sensory changes or paralysis of some of the muscles used to express language. For example, the latter situation could occur with a lesion of the face representation portion of the motor cortex that results in an inability to manipulate the motor apparatus needed for speaking (vocal cords, jaws, tongue, lips) and would be manifest as

1	of the face representation portion of the motor cortex that results in an inability to manipulate the motor apparatus needed for speaking (vocal cords, jaws, tongue, lips) and would be manifest as unclear speech because of dysarthria, a mechanical deficit. An affected individual would, however, be able to write if the motor cortex serving the upper limb were unaffected.

1	Interhem﻿ispheric Com﻿m﻿unication and the Corpus Callosum﻿ The two cerebral hemispheres can function somewhat independently, as in the control of one hand. However, information must be transferred between the hemispheres to coordinate activity on the two sides of the body. Much of that information is transmitted through the corpus callosum, although some is transmitted through other commissures (e.g., the anterior commissure or the hippocampal commissure). The importance of the corpus callosum for interhemispheric transfer of information is illustrated in Figure 10.7A. An animal with an intact optic chiasm and corpus callosum and with the left eye closed learns a visual discrimination task (see Fig. 10.7A). The information is transmitted to both hemispheres through bilateral connections made by the optic chiasm or through the corpus callosum, or both. When the animal is tested with the left eye open and the right eye closed (see

1	Fig. 10.7A, center), the task can still be performed because both hemispheres have learned the task. If the optic chiasm is transected before the animal is trained, the result is the same (see Fig. 10.7B). Information is presumably transferred between the two hemispheres through the corpus callosum. This finding can be confirmed by cutting both the optic chiasm and the corpus callosum before training (see Fig. 10.7C ). Then the information is not transferred, and each hemisphere must learn the task independently. A similar experiment was conducted in human patients who had undergone surgical transection of the corpus callosum to prevent the interhemispheric spread of epilepsy (

1	Fig. 10.8 ). The optic chiasm remained intact, but visual information was directed to one or the other hemisphere by the patient’s fixing vision on the central point of the screen. A picture or name of an object was then flashed to one side of the fixation point, so that visual information about the picture reached only the contralateral hemisphere. An opening beneath the screen allowed the patient to manipulate objects that could not be seen. The objects included those shown in the projected pictures. Normal individuals would be able to locate the correct object with either hand. However, patients with a transected corpus callosum could locate the correct object only with the hand ipsilateral to the projected image (contralateral to the hemisphere that received the visual information). For the hand to explore and recognize the correct object, the visual information must have access to the somatosensory and motor areas of the cortex. With the corpus callosum cut, the visual and motor

1	the hand to explore and recognize the correct object, the visual information must have access to the somatosensory and motor areas of the cortex. With the corpus callosum cut, the visual and motor areas are interconnected only on the same side of the brain.

1	Another test was to ask the patient to verbally identify what object was seen in the picture. The patient would make a correct verbal response to a picture that was projected to the right of the fixation point because the visual information reached only the left (language-dominant) hemisphere. However, the patient could not verbally identify a picture that was presented to the left hemifield because visual information reached only the right hemisphere.

1	Similar observations can be made in patients with a transected corpus callosum when different forms of stimuli are used. For example, when such patients are given a verbal command to raise the right arm, they do so without difficulty. The language centers in the left hemisphere send signals to the ipsilateral motor areas, and these signals produce the movement of the right arm. However, these patients cannot respond to a command to raise the left arm. The language areas on the left side cannot influence the motor areas on the right unless the corpus callosum is intact. Somatosensory stimuli applied to the right side of the body can be described by patients with a transected corpus callosum, but these patients cannot describe the same stimuli applied to the left side of the body. Information that reaches the right somatosensory areas of the cortex cannot reach the language centers if the corpus callosum has been cut.

1	In addition to language, other differences in the functional capabilities of the two hemispheres can be compared by exploring the performance of individuals with a transected corpus callosum. Such patients solve CHAPTER 10 Integrative Functions of the Nervous System﻿ 219 • Fig. 10.7 Role of the Corpus Callosum in the Interhemispheric Transfer of Visual Information When Learning Involves One Eye.A, Discrimination depends on distinguishing between a cross and a circle. B, Discrimination is between triangles oriented with the apex up or down. C, Discrimination is between vertical and horizontal bars.

1	•Fig. 10.8 Illustration of Tests in a Patient With a Transected Corpus Callosum. A, Thepatientfixesonapointonarearprojectionscreen,andpicturesareprojectedtoeithersideofthefixationpoint.Thehandcanpalpateobjectsthatcorrespondtotheprojectedpictures,buttheseobjectscannotbeseen.B, Responsebythelefthandtoapictureofakeyintheleftfieldofview.However,theverbalresponseisthatthepatientseesapictureofaring.(RedrawnfromSperryRW.In:SchmittFO,WordenFG[eds].The Neurosciences: Third Study Program. Cambridge,MA:MITPress;1974.) three-dimensional puzzles better with the right than with the left hemisphere, which suggests that the right hemisphere has specialized functions for spatial tasks. Other functions that seem to be more associated with the right than the left hemisphere are facial expression, body language, and speech intonation (

1	Fig. 10.9 ). Patients with a transected corpus callosum lack normal interhemispheric coordination. When they are dressing, for example, one hand may button a shirt while the other tries to unbutton it. Observation of these patients indicates that the two hemispheres can operate quite independently when they are no longer interconnected. However, one hemisphere can express itself with language, whereas the other communicates only nonverbally.

1	Oneofthemorestrikingexamplesofinterhemisphericdifferencesisthephenomenonof“cortical neglect,” whichisaconsequenceofalesionintheparietalcortexofthenondominant(usuallyright)hemisphere.Insuchcases,thepatientignoresobjectsandindividualsintheleftvisualfield,drawsobjectsthatareincompleteontheleft,deniestheexistenceofhisorherleftarmandleg,andfailstodresstheleftsideofhisorherbody.Thepatientalsodenieshavinganysuchdifficulties(anosognosia). Althoughthepatientmayrespondtotouchandpinprickontheleftsideofthebody,heorshecannotidentifyobjectsplacedinthelefthand.Thelesionisadjacenttothefirstsomatosensory(SI)cortex,aswellasthevisualassociationcortex,anditsuggeststhatthisregionplaysaspecialroleintheperceptionofbodyimageandimmediateextrapersonalspace.Similarlesionsonthedominantsideresultonlyinlossofsomehigherordersomesthesias,suchasagraphesthesia (inabilitytoidentifycharactersdrawnonthepalm)andastereognosis (inabilitytoidentifyanobjectonlybytouch).

1	Major functions of the higher levels of the nervous system are learning and memory. Learning is a neural mechanism by which the organism’s behavior changes as a result of experience. Memory is the storage mechanism for what is learned.

1	The neural circuitry involved in memory and learning in mammals is complex and difficult to study. Alternative approaches are animal studies (especially in the simpler nervous systems of invertebrates), analysis of the functional consequences of lesions, and anatomical/physiological studies at the cellular and pathway level. For example, in the marine mollusk Aplysia, it has been possible to isolate a connection between a single sensory neuron and a motor neuron, which shows aspects of habituation (learning not to respond to repetitions of an insignificant stimulus), sensitization (increased responsiveness to innocuous stimuli that follow the presentation of a strong or noxious stimulus), and even associative conditioning (learning to respond to a previously insignificant event after it has been

1	Right-side olfaction Memory for shapes Stereognosis, left side Hearing (left-ear preference) Musical ability Recognition of forms, faces, and body image Left visual fieldRight visual field Mathematical ability Understanding language Hearing (right-ear preference) Motor speech Left-side olfaction Verbal memory Stereognosis, right side Right-hand skills (e.g., writing) L Left Right RLR 372 x 49 18228 •Fig. 10.9 Schematicillustrationofthefunctionalspecializationsoftheleftandrighthemispheres,asdeterminedinpatientsaftersectionofthecorpuscallosum.(ModifiedfromSiegelA,SapruHN.Essential Neuroscience. 5thed.Philadelphia:LippincottWilliams&Wilkins;2005.) paired with a significant one). In the case of habituation, the amount of transmitter released in successive responses gradually diminishes. The change involves an alteration in the Ca++ current that triggers release of neurotransmitter. The cause of this change is inactivation of presynaptic Ca++ channels by repeated action potentials.

1	The change involves an alteration in the Ca++ current that triggers release of neurotransmitter. The cause of this change is inactivation of presynaptic Ca++ channels by repeated action potentials. Long-term habituation can also be produced. In this case, the numbers of synaptic endings and active zones in the remaining terminals decreases.

1	Additional models of learning, provided by synaptic phenomena, are called long-term potentiation (LTP) and long-term depression (LTD). LTD has been studied most extensively in the cerebellum (see Chapter 9), but it also occurs in the hippocampus and in other regions of the CNS.

1	LTP has been studied most intensively in slices of the hippocampus in vitro, but it has also been studied in the neocortex, cerebellum, and other parts of the CNS. Repetitive activation of an afferent pathway to the hippocampus or repetitive activation of one of the intrinsic connections increases the responses of pyramidal cells. The increased responses (the LTP) last for hours in vitro (and even days to weeks in vivo). The forms of LTP differ, depending on the particular synaptic system. The mechanism of the enhanced synaptic efficacy seems to involve both presynaptic and postsynaptic events. The neurotransmitters involved in LTP include excitatory amino acids that act on N-methyl-D-aspartate (NMDA) receptors, the responses of which are associated with an influx of Ca++ into the post-synaptic neuron. Second messenger pathways (including G proteins, Ca++/calmodulin-dependent kinase II, protein kinase G, and protein kinase C) are also involved, and these kinases cause protein

1	post-synaptic neuron. Second messenger pathways (including G proteins, Ca++/calmodulin-dependent kinase II, protein kinase G, and protein kinase C) are also involved, and these kinases cause protein phosphorylation and changes in the responsiveness of neurotransmitter receptors. A retrograde messenger, perhaps nitric oxide (or carbon monoxide), may be released from postsynaptic neurons to act on presynaptic endings in such a way that transmitter release is enhanced. Immediate-early genes are also activated during LTP. Hence, changes in gene expression may also be involved.

1	With regard to the stages of memory storage, a distinction between short-term memory and long-term memory is useful. Recent events appear to be stored in short-term memory by ongoing neural activity because short-term memory persists for only minutes. Short-term memory is used, for instance, to remember page numbers in a book after looking them up in the index. Long-term memory can be subdivided into an intermediate form, which can be disrupted, and a long-lasting form, which is difficult to disrupt. Memory loss, or amnesia, can be caused by a loss of memory information per se, or it can result from interference with the mechanism for accessing the information. Long-term memory probably involves structural changes because it can remain intact even after events that disrupt short-term memory.

1	The temporal lobes appear to be particularly important for memory because bilateral removal of the hippocampal formation can severely and permanently disrupt recent memory. Short-term and long-term memories are unaffected, but new long-term memories can no longer be established. Thus patients with such amnesia remember events before their surgery but fail to recall new events, even with multiple exposures, and must be reintroduced repeatedly to people they meet after the surgery. This loss of declarative memory involves the conscious recall of personal events, places, and general history. Such patients, however, can still learn some tasks because they retain procedural memory, which involves associational and motor skills. If such patients are given a complex task to perform (e.g., mirror writing), they not only improve during the first training session but also perform better on subsequent days despite their denial of having any earlier experience with that task. The cerebral

1	mirror writing), they not only improve during the first training session but also perform better on subsequent days despite their denial of having any earlier experience with that task. The cerebral structures involved in procedural memory are not yet defined.

1	Plasticity most commonly refers to the ability of the CNS to change its connectivity. Such changes can occur in various contexts, including learning and memory (see previous discussion), damage, and development. Damage to the CNS can induce remodeling of neural pathways and thereby alter behavior. Plasticity is greatest in the developing brain, but

1	Cellularstudiesofthehippocampusandtheentorhinalcortex(whichisadjacentandparalleltothehippocampus)havedemonstratedtheexistenceof“placecells”thatfirewhenthesubjectentersaspecificplaceinatestenvironment.Althoughtherearemanyplacecells,theyarenotdistributedinanorderlymannerthatwouldresembleatopographicmap.Placecellsappearinveryyounganimalsassoonastheyareabletoexplore.Morerecentstudiesrevealthepresenceof“gridcells,”whichalsorespondtospecificsitesbutaredistributedinhexagonalarraysthatresemblesanorderlymapoftheenvironmentalspaceintheposteriorentorhinalcortex.Althoughthiscognitivemapisfixedforanycontext,changestotheenvironmentorremovalofthepersontoanewtestenvironmentcausesthegenerationofanewandappropriatemapofgridcells.

1	Becausetheentorhinalcortexisamajorsourceofinputtothehippocampus,itisinterestingthatstudiesofLondon’staxidrivers—whomustdemonstrateanextremelydetailedknowledgeofthecity’sstreetsandmostefficientroutesbeforebeinglicensed—indicatethattheposteriorhippocampusintrainedandexperienceddriversislargerthanthatinbeginnersorthegeneralpopulation.“Gettinglost,”acommoncomplaintassociatedwithamnesia,maybeduetothelossofspatialmemoryskills. some degree of plasticity remains in the adult brain, as evidenced by responses to certain manipulations, such as lesions of the brain, sensory deprivation, or even experience.

1	some degree of plasticity remains in the adult brain, as evidenced by responses to certain manipulations, such as lesions of the brain, sensory deprivation, or even experience. The capability for developmental plasticity may be maximal for some neural systems at a time referred to as the critical period. For example, it is possible to alter some connections formed in the visual pathways during their development by preventing one eye from providing input, but only during a specific critical period early in development. In such visually deprived animals, the visual connections become abnormal (

1	Fig. 10.10 ), and restoration of normal visual input after the critical period does not undo the abnormality, nor does it restore functional vision from the deprived eye. In contrast, similar visual deprivation later in life does not result in abnormal connections. The plastic changes seen in such experiments may reflect a competition for synaptic connections, whereby the less functional connections are pruned away. Research has shown a correlation between the form of a gene that modulates the efficacy of synaptic pruning and the probability of schizophrenia.

1	Plastic changes can also occur after injury to the brain in adults. Sprouting of new axons does occur in the damaged CNS; however, the sprouts do not necessarily restore normal function, and many neural pathways do not appear to produce sprouts. Additional knowledge concerning neural plasticity in the adult CNS is vital if medical therapy is to be improved for many diseases of the CNS and after neural trauma. Research is currently being conducted to explore •Fig. 10.10 Plasticity in the Visual Pathway as a Result of Sensory Deprivation During Development. Theoculardominancecolumnsaredemonstratedbyautoradiographyafterinjectionofaradioactivetracerintooneeye.Thetraceristransportedtothelateralgeniculatenucleusandthentransneurallytransportedtothestriatecortex.Thecortexislabeledinbandsthatalternatewithunlabeledbandswhoseinputisfromtheuninjectedeye.A, Normalpattern.B,

1	Normalpattern.B, Changedpatterninananimalraisedwithmonocularvisualdeprivation.Theinjectionwasmadeintothenondeprivedeye,andtheoculardominancecolumnsforthiseyewereclearlyexpanded.Otherexperimentsshowedthattheoculardominancecolumnsforthedeprivedeyecontracted.(A, FromHubelDH,WieselTN.Proc R Soc Lond B. 1977;198:1.B, FromLeVayS,etal.J Comp Neurol. 1980;191:1.) the potential of human embryonic stem cells for restoring CNS function.

1	Phantom limb sensation is an example of neural plasticity in adults. A patient whose limb has been amputated often perceives sensations on the missing limb when stimulated elsewhere on the body. Functional imaging studies suggest that this is a result of the spread of connections from the surrounding cortical territories into the cortical region that had served the amputated limb. Itwastraditionalpolicytodelaycorrectivesurgeryforachildbornwithacongenitalcataractuntilthechildwasolderandmoreabletocopewiththestressofsurgery.However,ifthecorrectionisdeferreduntilafterthe“criticalperiod,”fullrecoveryoffunctionisunlikely.Similarly,childrenbornwithamblyopia, aconditioncharacterizedbystrabismus(crosseye)becauseofrelativeweaknessofoneoftheextraocularmuscles,tendtousetheunaffectedeyeinpreference.Inbothcases,earlysurgeryisnowcommonpracticesothatthecorticalcircuitrycanbecorrectlysculptedbybalancedinputfromthetwoeyes.

1	Such remapping can also occur after surgical amputation of the second and third digits of the hand. Before surgery, each of the digits was represented in discrete and somatotopically organized areas of the postcentral gyrus (SI cortex). After surgery, the area that represented the amputated digits is now mapped with an enlarged representation of the adjacent digits (

1	Fig. 10.11 ). Conversely, individuals born with syndactyly (fusion of two or more digits of the hand) have a single or mostly overlapping representation of these digits in the SI cortex. After corrective surgery, the independent digits come to have distinctive representations. Even more remarkable is that monkeys that were trained on a sensory discrimination task requiring repeated daily use of their fingertips showed cortical differences after training. Not only were the SI cortical territories of their fingertips larger than before training but also the number of cortically recorded receptive fields on the fingertips was likewise increased.

1	•Fig. 10.11 Representationofthedigitregionoftheleftfirstsomatosensory(SI)cortex(A) andreorganizationofthisrepresentation (B) afteramputationofthesecondandthirddigits.(FromHainesDE[ed].Fundamental Neuroscience for Basic and Clinical Applications. 3rded.Philadelphia:ChurchillLivingstone;2006.) 1. The cerebral cortex can be divided into lobes on the basis of the pattern of gyri and sulci. Each lobe has distinctive functions, as shown by the effects of lesions. The left cerebral hemisphere is dominant for language in most individuals. Wernicke’s area (in the posterior temporal lobe) is responsible for the understanding of language, and Broca’s area (in the inferior frontal lobe) is responsible for its expression.

1	2. The neocortex contains pyramidal cells and several kinds of interneurons. Specific thalamocortical afferent fibers terminate mainly in layer IV of the neocortex; diffuse thalamocortical afferent fibers synapse in layers I and VI. Axons from pyramidal cells in layer V are the major source of output to subcortical targets, including the spinal cord, brainstem, striatum, and thalamus. 3. The cortical structure varies in different regions. Brodmann’s designations reflect these variations in cortical structure and correspond to functionally discrete areas. 4. The EEG reflects electrical fields generated by the activity of pyramidal and varies with the state of the sleep-wake cycle, disease, and other factors. Cortical evoked potentials are stimulus-triggered changes in the EEG and are useful clinical data about sensory transmission. 5.

1	5. EEG patterns during sleep are divided into slow-wave and REM forms. Slow-wave sleep progresses through stages 1 through 4, each with a characteristic EEG pattern. Most dreams occur in REM sleep. Sleep is produced actively by a brainstem mechanism, Squire L, Berg D. Fundamental Neuroscience. 4th ed. New York: Academic Press; 2012. and its circadian rhythmicity is controlled by the suprachiasmatic nucleus. 6. Information is transferred between the two hemispheres primarily through the corpus callosum. The right hemisphere is more capable than the left in spatial tasks, facial expression, body language, and speech intonation. The left hemisphere is specialized for the understanding and generation of language, for logic, and for mathematical computation. 7.

1	7. Learning and memory can be studied on the cellular level, in invertebrates, and in higher animals. Memory includes short-term (lasting minutes), recent, and long-term storage processes and a retrieval mechanism. The hippocampal formation is important for storing declarative and spatial memory. 8. Lesion studies and behavioral studies indicate that plasticity occurs in the brain throughout life. However, there appears to be more plasticity early in life, and synaptic competition in “critical periods” is important for the establishment of neural circuitry. Upon completion of this chapter, the student should be able to answer the following questions: 1. What are the similarities and differences in the general organizations of the parasympathetic and sympathetic systems? 2. What are the respective actions of the parasympathetic and sympathetic innervation of the eye, and what symptoms arise when the parasympathetic or sympathetic innervation is lost? 3.

1	2. What are the respective actions of the parasympathetic and sympathetic innervation of the eye, and what symptoms arise when the parasympathetic or sympathetic innervation is lost? 3. What are the changes in the balance of parasympathetic and sympathetic activity to the bladder that occur during micturation? 4. What is meant by a “servomechanism”? 5. What are the specific feedback loops that regulate body temperature, feeding and body weight, and water intake? 6. What is the role of the hypothalamus in each of these feedback loops?

1	5. What are the specific feedback loops that regulate body temperature, feeding and body weight, and water intake? 6. What is the role of the hypothalamus in each of these feedback loops? he main function of the autonomic nervous system is to assist the body in maintaining a constant internal environment (homeostasis). When internal stimuli signal that regulation of the body’s environment is required, the central nervous system (CNS) and its autonomic outflow issue commands that lead to compensatory actions. For example, a sudden increase in systemic blood pressure activates the baroreceptors, which in turn modify the activity of the autonomic nervous system so that the blood pressure is lowered toward its previous level (see

1	The autonomic nervous system has both sensory and motor divisions. The motor division is further divided into the sympathetic and parasympathetic divisions. Because much of the autonomic nervous system’s actions relate to control of the viscera, it is sometimes called the visceral nervous system. In service of its homeostatic function, the autonomic nervous system mediates visceral reflexes (e.g., the gastrocolic reflex, where stomach distention triggers peristalsis in the intestines) and provides sensory information to the CNS for the perception of the state of our viscera, a percept known to anyone who has eaten too much at a meal. More generally, activation of autonomic receptors can evoke a variety of sensory experiences such as pain, hunger, thirst, nausea, and a sense of visceral distention; these perceptions can then lead to compensatory voluntary behaviors that assist in maintaining homeostasis.

1	In addition to its central role in homeostasis, the autonomic nervous system also participates in appropriate and coordinated responses to external stimuli that are required for the optimal functioning of the somatic nervous system in performing voluntary behaviors. For example, the autonomic nervous system helps regulate pupil size in response to different intensities of ambient light, thus helping the visual system to operate over a large range of light intensity.

1	In this chapter, the enteric nervous system is also considered part of the autonomic nervous system, although it is sometimes considered a separate entity (see also ). In addition, because the autonomic nervous system is under CNS control, the central components of the autonomic nervous system are discussed in this chapter. These central components include the hypothalamus and higher levels of the limbic system, which are associated with emotions (see ) and with many visceral types of behavior (e.g., feeding, drinking, thermoregulation, reproduction, defense, and aggression) that have survival value. Organization of the Autonomic Nervous System

1	Organization of the Autonomic Nervous System The sensory autonomic neurons are located in the dorsal root ganglia and in the cranial nerve ganglia. Like the other neurons of the dorsal root ganglia, they are pseudounipolar cells with a peripheral axonal branch extended to one of the viscera and a central branch that enters the CNS. With regard to autonomic motor output, both the sympathetic and parasympathetic nervous systems use a two-neuron motor pathway, which consists of a preganglionic neuron, whose cell body is located in the CNS, and a postganglionic neuron, whose cell body is located in one of the autonomic ganglia ( Figs. 11.1 11.2 ). The targets of this motor CHAPTER 11 The Autonomic Nervous System and Its Central Control 227 Projections of Parasympathetic parasympathetic nervous system

1	Figs. 11.1 11.2 ). The targets of this motor CHAPTER 11 The Autonomic Nervous System and Its Central Control 227 Projections of Parasympathetic parasympathetic nervous system Facial (VII) nerve, Glossopharyngeal (IX) nerve • Fig. 11.1 Schematic Illustration of the Sympathetic and Parasympathetic Pathways. Sympathetic pathways are shown in red and parasympathetic pathways in blue. Preganglionic neurons are shown in darker shades, and postganglionic neurons, in lighter shades. pathway are smooth muscle, cardiac muscle, and glands. The enteric nervous system includes the neurons and nerve fibers in the myenteric and submucosal plexuses, which are located in the wall of the gastrointestinal tract.

1	Control of the sympathetic and parasympathetic nervous systems of many organs is often antagonistic. To highlight this contrast, the sympathetic and parasympathetic systems are sometimes referred to as the “fight or flight” and the “rest and digest” systems, respectively. Indeed, the fight-orflight response to a threat to the organism reflects an intense activation of the sympathetic nervous system, which leads to a variety of responses, including increased heart rate and blood pressure, redistribution of blood to the muscles, decreased peristalsis and gastrointestinal secretions, pupil dilation, and sweating.

1	However, under most conditions, the two parts of the autonomic control system work in a coordinated manner—sometimes acting reciprocally and sometimes synergistically—to regulate visceral function. Furthermore, not all visceral structures are innervated by both systems. For example, the smooth muscles and glands in the skin and most of the blood vessels in the body receive sympathetic innervation exclusively; only a small fraction of the blood vessels have parasympathetic innervation. Indeed, the parasympathetic nervous system innervates not the body wall but only structures in the head and in the thoracic, abdominal, and pelvic cavities. The Sympathetic Nervous System

1	The Sympathetic Nervous System The sympathetic preganglionic neurons are located in the thoracic and upper lumbar segments of the spinal cord. For this reason, the sympathetic nervous system is sometimes referred to as the thoracolumbar division of the autonomic nervous system. Specifically, sympathetic preganglionic neurons are concentrated in the intermediolateral cell column (lateral horn) in the thoracic and upper lumbar segments of the spinal cord (see

1	Fig. 11.2). Some neurons may also be found in the C8 segment. In addition to the intermediolateral cell column, groups of sympathetic preganglionic neurons are found in other locations, including the lateral funiculus, the intermediate gray matter, and the gray matter dorsal to the central canal. Sympathetic post-ganglionic neurons are generally found in the paravertebral •Fig. 11.2 Details of the Sympathetic Pathway at a Thoracic Spinal Segment. Autonomicsensoryfibersarerepresentedbyblue lines; sympatheticfibers,byred lines; preganglionicaxons,bysolid lines, andpostganglionicaxons,bydashed lines. (RedrawnfromParentA,CarpenterMB.Carpenter’s Human Neuroanatomy. 9thed.Philadelphia:Williams&Wilkins;1996:295.)White ramus Gray ramus Blood vessel Paravertebral ganglion Prevertebral ganglion Visceral sensory fiber Postganglionic fiber Postganglionic fibers Hair glands Skin Arrector pili muscle Preganglionic fibers Lateral horn Spinal ganglion Peripheral nerve Visceral afferent fiber

1	Visceral sensory fiber Postganglionic fiber Postganglionic fibers Hair glands Skin Arrector pili muscle Preganglionic fibers Lateral horn Spinal ganglion Peripheral nerve Visceral afferent fiber Digestive tube Sympathetic trunk or prevertebral ganglia. The paravertebral ganglia form two sets of ganglia, each lateral to one side of the vertebral column. The individual ganglia on each side are linked by longitudinally running axons that form a sympathetic trunk (see

1	Figs. 11.1 11.2 ). Prevertebral ganglia are located in the abdominal cavity and include the Celiac and Superior and Inferior Mesenteric Ganglia (see Fig. 11.1 ). Thus paravertebral and prevertebral ganglia are located at some distance from their target organs. The axons of preganglionic neurons are often small, myelinated nerve fibers known as B fibers (see Table 5.1 ). However, some are unmyelinated C fibers. They leave the spinal cord in the ventral root and enter the paravertebral ganglion at the same segmental level through a white communicating ramus. White rami are found only from the levels of T1 to L2. The preganglionic axon may synapse on postganglionic neurons in the ganglion at its level of entry; may travel rostrally or caudally within the sympathetic trunk and give off collaterals to the ganglia that it passes; or may pass through the ganglion, exit the sympathetic trunk, and enter a splanchnic nerve to travel to a prevertebral ganglion (see

1	Figs. 11.1 11.2 ). Splanchnic nerves innervate the viscera; they contain both visceral afferents and autonomic motor fibers (sympathetic or parasympathetic). Postganglionic neurons whose somata lie in paravertebral ganglia generally send their axons through a gray communicating ramus to enter a spinal nerve (see Fig. 11.2 ). Each of the 31 pairs of spinal nerves has a gray ramus. Postganglionic axons are distributed through the peripheral nerves to effectors, such as piloerector muscles, blood vessels, and sweat glands, located in the skin, muscle, and joints. Postganglionic axons are generally unmyelinated (C fibers), although some exceptions exist. The names white and gray rami reflect the relative contents of myelinated and unmyelinated axons in these rami.

1	Preganglionic axons in a splanchnic nerve often travel to a prevertebral ganglion and synapse, or they may pass through the ganglion and an autonomic plexus and end in a more distant ganglion. Some preganglionic axons pass through a splanchnic nerve and end directly on cells of the adrenal medulla, which are equivalent to postganglionic cells.

1	The sympathetic chain extends from the cervical to the coccygeal levels of the spinal cord. This arrangement serves as a distribution system that enables preganglionic neurons, which are limited to the thoracic and upper lumbar segments, to activate postganglionic neurons that innervate all body segments. However, there are fewer paravertebral ganglia than there are spinal segments because some of the segmental ganglia fuse during development. For example, the superior cervical sympathetic ganglion represents the fused ganglia of C1 through C4; the middle cervical sympathetic ganglion is the fused ganglia of C5 and C6; and the inferior cervical sympathetic ganglion is a combination of the ganglia at C7 and C8. The term stellate ganglion refers to fusion of the inferior cervical sympathetic ganglion with the ganglion of T1. The superior cervical sympathetic ganglion provides postganglionic innervation to the head and neck, and the middle cervical and stellate ganglia innervate the

1	ganglion with the ganglion of T1. The superior cervical sympathetic ganglion provides postganglionic innervation to the head and neck, and the middle cervical and stellate ganglia innervate the heart, lungs, and bronchi.

1	In general, the sympathetic preganglionic neurons are distributed to ipsilateral ganglia and thus control autonomic function on the same side of the body. Important exceptions are the sympathetic innervation of the intestines and the pelvic viscera, which are both bilateral. As with motor neurons to skeletal muscle, sympathetic preganglionic neurons that control a particular organ are spread over several segments. For example, the sympathetic preganglionic neurons that control sympathetic functions in the head and neck region are distributed at levels C8 to T5, whereas those that control the adrenal gland are distributed at levels T4 to T12. The Parasympathetic Nervous System The parasympathetic preganglionic neurons are found in several of the cranial nerve nuclei of brainstem and in the sacral spinal cord (S3-S4) gray matter (see

1	Fig. 11.1 ). Hence, this part of the autonomic nervous system is sometimes called the craniosacral division. The cranial nerve nuclei that contain parasympathetic preganglionic neurons are the Edinger-Westphal nucleus (cranial nerve III), the superior (cranial nerve VII) and inferior (cranial nerve IX) salivatory nuclei, and the dorsal motor nucleus of the vagus and nucleus ambiguus (cranial nerve X). Postganglionic parasympathetic cells are located in cranial ganglia, including the ciliary ganglion (preganglionic input is from the Edinger-Westphal nucleus), the pterygopalatine and submandibular ganglia (input is from the superior salivatory nucleus), and the otic ganglion (input is from the inferior salivatory nucleus). The ciliary ganglion innervates the pupillary sphincter and ciliary muscles in the eye. The pterygopalatine ganglion supplies the lacrimal gland, as well as glands in the nasal and oral pharynx. The submandibular ganglion projects to the submandibular and sublingual

1	muscles in the eye. The pterygopalatine ganglion supplies the lacrimal gland, as well as glands in the nasal and oral pharynx. The submandibular ganglion projects to the submandibular and sublingual salivary glands and to glands in the oral cavity. The otic ganglion innervates the parotid salivary gland and glands in the mouth.

1	Other parasympathetic postganglionic neurons are located near or in the walls of visceral organs in the thoracic, abdominal, and pelvic cavities. Neurons of the enteric plexus include cells that can also be considered parasympathetic postganglionic neurons. All of these cells receive input from the vagus or pelvic nerves. The vagus nerves innervate the heart, lungs, bronchi, liver, pancreas, and gastrointestinal tract from the esophagus to the splenic flexure of the colon. The remainder of the colon and rectum, as well as the urinary bladder and reproductive organs, is supplied by sacral parasympathetic preganglionic neurons that travel through the pelvic nerves to postganglionic neurons in the pelvic ganglia. The parasympathetic preganglionic neurons that project to the viscera of the thorax and part of the abdomen are located in the dorsal motor nucleus of the vagus (see Fig.

1	Fig. 4.6E ) and the nucleus ambiguus. The dorsal motor nucleus is largely secretomotor (it activates glands), whereas the nucleus ambiguus is visceromotor (it modifies the activity of cardiac muscle). The dorsal motor nucleus supplies visceral organs in the neck (pharynx, larynx), thoracic cavity (trachea, bronchi, lungs, heart, and esophagus), and abdominal cavity (including much of the gastrointestinal tract, liver, and pancreas). Electrical stimulation of the dorsal motor nucleus results in gastric acid secretion, as well as secretion of insulin and glucagon by the pancreas. Although projections to the heart have been described, their function is uncertain. The nucleus ambiguus contains two groups of neurons: (1) a dorsal group (branchiomotor) that activates striated muscle in the soft palate, pharynx, larynx, and esophagus and (2) a ventrolateral group that innervates and slows the heart (see also

1	The visceral motor fibers in the autonomic nerves are accompanied by visceral afferent fibers. Most of these afferent fibers supply information that originates from sensory receptors in the viscera. The activity of these sensory receptors only rarely reaches the level of consciousness; however, these receptors initiate the afferent limb of reflex arcs. Both viscerovisceral and viscerosomatic reflexes are elicited by these afferent fibers. Even though these visceral reflexes generally operate at a subconscious level, they are very important for homeostatic regulation and adjustment to external stimuli.

1	The fast-acting neurotransmitters released by visceral afferent fibers are not well documented, although many of these neurons release an excitatory amino acid transmitter such as glutamate. However, visceral afferent fibers also contain many neuropeptides or combinations of neuropeptides, including angiotensin II, arginine vasopressin, bombesin, calcitonin gene–related peptide, cholecystokinin, galanin, substance P, enkephalin, oxytocin, somatostatin, and vasoactive intestinal polypeptide.

1	Visceral afferent fibers that can mediate conscious sensation include nociceptors that travel in sympathetic nerves, such as the splanchnic nerves. Visceral pain is caused by excessive distention of hollow viscera, contraction against an obstruction, or ischemia. The origin of visceral pain is often difficult to identify because of the diffuse nature of the pain and its tendency to be referred to somatic structures (see ). Visceral nociceptors in sympathetic nerves reach the spinal cord via the sympathetic chain, white rami, and dorsal roots. The terminals of nociceptive afferent fibers project to the dorsal horn and to the region surrounding the central canal. They activate not only local interneurons, which participate in reflex arcs, but also projection cells, which include spinothalamic tract cells that signal pain to the brain.

1	A major visceral nociceptive pathway from the pelvis involves a relay in the gray matter of the lumbosacral spinal cord. These neurons send axons into the fasciculus gracilis that terminate in the nucleus gracilis. Thus the dorsal columns not only contain primary afferents for somatic sensation (their main component) but also second-order neurons of the visceral pain pathway (recall that second-order axons for somatic pain travel in the lateral funiculus as part of the spinothalamic tract). Visceral nociceptive signals are then transmitted to the ventral posterior lateral nucleus of the thalamus and presumably from there to the cerebral cortex. Interruption of this pathway accounts for the beneficial effects of surgically induced lesions of the dorsal column at lower thoracic levels to relieve pain produced by cancer of the pelvic organs.

1	Other visceral afferent fibers travel in parasympathetic nerves. These fibers are generally involved in reflexes rather than sensation (except for taste afferent fibers; see ). For example, the baroreceptor afferent fibers that innervate the carotid sinus are in the glossopharyngeal nerve. They enter the brainstem, pass through the solitary tract, and terminate in the nucleus of the solitary tract (see Fig. 4.6E ). These neurons connect with interneurons in the brainstem reticular formation. The interneurons, in turn, project to the autonomic preganglionic neurons that control heart rate and blood pressure (see The nucleus of the solitary tract receives information from all visceral organs, except those in the pelvis. This nucleus is subdivided into several areas that receive information from specific visceral organs. The Enteric Nervous System

1	The Enteric Nervous System The enteric nervous system, which is located in the wall of the gastrointestinal tract, contains about 100 million neurons. The enteric nervous system is subdivided into the myenteric plexus, which lies between the longitudinal and circular muscle layers of the gut, and the submucosal plexus, which lies in the submucosa of the gut. The neurons of the myenteric plexus primarily control gastrointestinal motility (see ), whereas those in the submucosal plexus primarily regulate body fluid homeostasis (see

1	The types of neurons found in the myenteric plexus include not only excitatory and inhibitory motor neurons (which can be considered parasympathetic postganglionic neurons) but also interneurons and primary afferent neurons. Afferent neurons supply mechanoreceptors within the wall of the gastrointestinal tract. These mechanoreceptors are the beginning of the afferent limb of reflex arcs within the enteric plexus. Local excitatory and inhibitory interneurons participate in these reflexes, and the output is sent through the motor neurons to smooth muscle cells. Excitatory motor neurons release acetylcholine and substance P; inhibitory motor neurons release dynorphin and vasoactive intestinal polypeptide. The circuitry of the enteric plexus is so extensive that it can coordinate the movements of an intestine that has been completely removed from the body. However, normal function requires innervation by the autonomic preganglionic neurons and regulation by the CNS.

1	Activity in the enteric nervous system is modulated by the sympathetic nervous system. Sympathetic post-ganglionic neurons that contain norepinephrine inhibit intestinal motility, those that contain norepinephrine and neuropeptide Y regulate blood flow, and those that contain norepinephrine and somatostatin control intestinal secretion. Feedback is provided by intestinofugal neurons that project back from the myenteric plexus to the sympathetic ganglia. The submucosal plexus regulates ion and water transport across the intestinal epithelium and glandular secretion. It also communicates with the myenteric plexus to ensure coordination of the functions of the two components of the enteric nervous system. The neurons and neural circuits of the submucosal plexus are not as well understood as those of the myenteric plexus, but many of the neurons contain neuropeptides, and the neural networks are well organized.

1	The main type of neuron in autonomic ganglia is the post-ganglionic neuron. These cells receive synaptic connections from preganglionic neurons, and they project to autonomic effector cells. However, many autonomic ganglia also contain interneurons. These interneurons process information within the autonomic ganglia; the enteric plexus can be regarded as an elaborate example of this kind of processing. One type of interneuron found in some autonomic ganglia contains a high concentration of catecholamines; hence, these interneurons have been called small, intensely fluorescent (SIF) cells. SIF cells are believed to be inhibitory.

1	The classic neurotransmitter of autonomic ganglia, whether sympathetic or parasympathetic, is acetylcholine. The two classes of acetylcholine receptors in autonomic ganglia are nicotinic and muscarinic receptors, so named because of their responses to the plant alkaloids nicotine and muscarine. Nicotinic acetylcholine receptors can be blocked by such agents as curare or hexamethonium, and muscarinic receptors can be blocked by atropine. Nicotinic receptors in autonomic ganglia differ somewhat from those on skeletal muscle cells.

1	Nicotinic and muscarinic receptors both mediate excitatory postsynaptic potentials (EPSPs), but these potentials have different time courses. Stimulation of preganglionic neurons elicits a fast EPSP, followed by a slow EPSP. The fast EPSP results from activation of nicotinic receptors, which cause ion channels to open. The slow EPSP is mediated by muscarinic receptors (primarily the M2 receptor; see ) that inhibit the M current, a current produced by potassium conductance. Neurons in autonomic ganglia also release neuropeptides that act as neuromodulators. Besides acetylcholine, sympathetic preganglionic neurons may release enkephalin, substance P, luteinizing hormone–releasing hormone, neurotensin, or somatostatin. Catecholamines such as norepinephrine and dopamine serve as the neurotransmitters of SIF cells in autonomic ganglia.

1	Catecholamines such as norepinephrine and dopamine serve as the neurotransmitters of SIF cells in autonomic ganglia. Sympathetic postganglionic neurons typically release nor-epinephrine, which excites some effector cells but inhibits others. The receptors on target cells may be either α-or β-adrenergic receptors. These receptors are further subdivided into α1, α2, β1, β2, and β3 receptor types on the basis of pharmacological and genetic features. The distribution of these types of receptors and the actions that they mediate when activated by sympathetic postganglionic neurons are listed for various target organs in

1	Table 11.1 α1 receptors are located postsynaptically, but α2 receptors may be either presynaptic or postsynaptic. Receptors located presynaptically are generally called autoreceptors; they usually inhibit release of transmitter. The effects of agents that excite α1 or α2 receptors can be distinguished through the use of antagonists to block these receptors specifically. For example, prazosin is a selective α1-adrenergic antagonist, and yohimbine is a selective α2-adrenergic antagonist. The effects of α1 receptors are mediated by activation of the inositol triphosphate/diacylglycerol second messenger system (see Chapter 3). In contrast, α2 receptors decrease the rate of synthesis of cyclic adenosine monophosphate (cAMP) through action on a G protein.

1	Chapter 3). In contrast, α2 receptors decrease the rate of synthesis of cyclic adenosine monophosphate (cAMP) through action on a G protein. β receptors were originally classified on the basis of the ability of antagonists to block them, but this has been supplemented by genetic studies. The β1 and β2 proteins have been much more extensively studied than has β3, but it is thought that the proteins that make up all three types of β receptors are similar, with seven membrane-spanning regions connected by intracellular and extracellular domains (see ). Agonist drugs that work on β receptors activate a G protein that stimulates adenylyl cyclase to increase the cAMP concentration. This action is terminated by the buildup of guanosine diphosphate. Radialmuscle,irisα Contraction(mydriasis)++ —Sphinctermuscle,irisα —Contraction(miosis)+++ Ciliarymuscleβ Relaxationforfarvision+ Contractionfornearvision+++

1	Radialmuscle,irisα Contraction(mydriasis)++ —Sphinctermuscle,irisα —Contraction(miosis)+++ Ciliarymuscleβ Relaxationforfarvision+ Contractionfornearvision+++ Sinoatrialnodeβ1Increaseinheartrate++ Decreaseinheartrate;vagalarrest+++ Atriaβ1IncreaseincontractilityandconductionDecreaseincontractilityand(usually)velocity++ increaseinconductionvelocity++ Atrioventricular(AV)nodeβ1IncreaseinautomaticityandDecreaseinconductionvelocity;AVblockconductionvelocity++ +++ His-Purkinjesystemβ1IncreaseinautomaticityandLittleeffectconductionvelocity+++ Ventriclesβ1Increaseincontractility,conductionSlightdecreaseincontractilityvelocity,automaticity,andrateofidioventricularpacemakers+++ Coronaryα,β2Constriction+;dilation Skeletalmuscleα,β2Constriction++;dilation c,e Pulmonaryα,β2Constriction+;dilation Abdominalviscera,renalα,β2Constriction+++;dilation + —Salivaryglandsα Constriction+++ Dilation++ Veins (Systemic) α,β2Constriction++;dilation++ —

1	Pulmonaryα,β2Constriction+;dilation Abdominalviscera,renalα,β2Constriction+++;dilation + —Salivaryglandsα Constriction+++ Dilation++ Veins (Systemic) α,β2Constriction++;dilation++ — Responses of Effector Organs to Autonomic Nerve Impulses—cont’d 11.1 Effector Organs Receptor Type Adrenergic Impulses,a Responsesb Cholinergic Impulses,a Responsesb Bronchialmuscleβ2Relaxation+ Contraction++ Bronchialglands?Inhibition(?)Stimulation+++ Motilityandtoneα2,β2Decrease(usually) + Increase+++ Sphinctersα Contraction(usually)+ Relaxation(usually)+ SecretionInhibition(?)Stimulation+++ Motilityandtoneα2,β2Decrease + Increase+++ Sphinctersα Contraction(usually)+ Relaxation(usually)+ SecretionInhibition(?)Stimulation+++ Gallbladder and Ducts Relaxation+ Contraction+

1	Motilityandtoneα2,β2Decrease + Increase+++ Sphinctersα Contraction(usually)+ Relaxation(usually)+ SecretionInhibition(?)Stimulation+++ Gallbladder and Ducts Relaxation+ Contraction+ Motilityandtoneα Increase(usually)Increase(?)Uterus α,β2Pregnant:contraction(α);Variable nonpregnant:relaxation(β)Sex Organs, Male α Ejaculation+++ Erection+++ + Generalizedsecretion+++ Spleen Capsule α,β2Contraction+++;relaxation+ — Adrenal Medulla —SecretionofepinephrineandnorepinephrineLiver α,β2Glycogenolysis,gluconeogenesis β2Increasedsecretion+ —Fatcellsα,β1Lipolysis

1	FromGoodmanLS,GilmanA.The Pharmacological Basis of Therapeutics. 6thed.NewYork:Macmillan;1980.aAlongdash(—)signifiesnoknownfunctionalinnervation.bResponsesaredesignated+ to+++ toprovideanapproximateindicationoftheimportanceofadrenergicandcholinergicnerveactivityincontrolofthevariousorgansandfunctionslisted.cDilationpredominatesinsitubecauseofmetabolicautoregulatoryphenomena.dCholinergicvasodilationatthesesitesisofquestionablephysiologicalsignificance.eOvertheusualconcentrationrangeofphysiologicallyreleasedcirculatingepinephrine,aβ receptorresponse(vasodilation)predominatesinbloodvesselsofskeletalmuscleandtheliver,andanα

1	receptorresponse(vasodilation)predominatesinbloodvesselsofskeletalmuscleandtheliver,andanα receptorresponse(vasoconstriction)predominatesinbloodvesselsofotherabdominalviscera.Therenalandmesentericvesselsalsocontainspecificdopaminergicreceptors,activationofwhichcausesdilation,buttheirphysiologicalsignificancehasnotbeenestablished.fThesympatheticcholinergicsystemcausesvasodilationinskeletalmuscle,butthisisnotinvolvedinmostphysiologicalresponses.gIthasbeenproposedthatadrenergicfibersterminateatinhibitoryβ receptorsonsmoothmusclefibersandatinhibitoryα receptorsonparasympatheticcholinergic(excitatory)ganglioncellsoftheAuerbachplexus.hDependsonthestageofthemenstrualcycle,theamountofcirculatingestrogenandprogesterone,andotherfactors.iPalmsofthehandsandsomeothersites(“adrenergicsweating”).jThereissignificantvariationamongspeciesinthetypeofreceptorthatmediatescertainmetabolicresponses.

1	β receptor activity is controlled in a number of ways. It can be antagonized by the action of α1 receptors. The β receptors can also be desensitized by phosphorylation with prolonged exposure to agonists. Regulation of β receptor numbers represents a third control mechanism. For example, β receptor numbers can be decreased by being internalized. Alternatively, β receptor numbers can be increased (upregulated) in certain circumstances: for example, after denervation. Note that the number of α receptors is likewise regulated. In addition to releasing norepinephrine, sympathetic postganglionic neurons release neuropeptides such as somatostatin and neuropeptide Y. For example, cells that release both norepinephrine and somatostatin supply the mucosa of the gastrointestinal tract, and cells that release both norepinephrine and neuropeptide Y innervate blood vessels in the gut and the limb. Another chemical mediator in sympathetic postganglionic neurons is adenosine triphosphate (ATP).

1	The endocrine cells of the adrenal medulla are similar in many ways to sympathetic postganglionic neurons (see also ). They receive input from sympathetic preganglionic neurons, are excited by acetylcholine, and release catecholamines. However, the cells of the adrenal medulla differ from sympathetic postganglionic neurons in that they release catecholamines into the circulation rather than into a synapse. Moreover, the main catecholamine released is epinephrine, not norepinephrine. In humans, 80% of the catecholamine released by the adrenal medulla is epinephrine, and 20% is norepinephrine.

1	Some sympathetic postganglionic neurons release acetylcholine rather than norepinephrine as their neurotransmitter. For example, sympathetic postganglionic neurons that innervate eccrine sweat glands are cholinergic. The acetylcholine receptors involved are muscarinic, and they are therefore blocked by atropine. Similarly, some blood vessels are innervated by cholinergic sympathetic postganglionic neurons. In addition to releasing acetylcholine, the postganglionic neurons that supply the sweat glands also release neuropeptides, including calcitonin gene–related peptide and vasoactive intestinal polypeptide. The neurotransmitter released by parasympathetic postganglionic neurons is acetylcholine. The effects of these neurons on various target organs are listed in

1	The neurotransmitter released by parasympathetic postganglionic neurons is acetylcholine. The effects of these neurons on various target organs are listed in Table 11.1 . Parasympathetic postganglionic actions are mediated by muscarinic receptors. On the basis of binding studies, the action of selective antagonists, and molecular cloning, five types of muscarinic receptors have been identified (see Chapter 6). Activation of M1 receptors enhances the secretion of gastric acid in the stomach. M2 receptors are the most abundant receptor type in smooth muscle, including smooth muscle in the intestines, uterus, trachea, and bladder. In addition, they are present in autonomic ganglia and in the heart, where they exert negative chronotropic and inotropic actions (see

1	Chapter 18). M 3 receptors are also present in the smooth muscle of a variety of organs, and although they are less abundant than M2 receptors, normal contractile patterns appear to require an interaction between the two types of receptors. M4 receptors, like M2 receptors, are present in autonomic ganglia and thus play a role in synaptic transmission at these sites. M5 receptors are present in the sphincter muscle of the pupil, in the esophagus, and in the parotid gland, as well as in cerebral blood vessels.

1	Muscarinic receptors, like adrenergic receptors, have diverse actions. Some of their effects are mediated by specific second messenger systems. For example, cardiac M2 muscarinic receptors may act by way of the inositol triphosphate system, and they may also inhibit adenylyl cyclase and thus cAMP synthesis. Muscarinic receptors also open or close ion channels, particularly K+ or Ca++ channels. This action on ion channels is likely to occur through activation of G proteins. A third action of muscarinic receptors is to relax vascular smooth muscle by an effect on endothelial cells, which produce endothelium-derived relaxing factor (EDRF). EDRF is actually nitric oxide, a gas released when arginine is converted to citrulline by nitric oxide synthase (see ). Nitric oxide relaxes vascular smooth muscle by stimulating guanylate cyclase and thereby increasing levels of cyclic guanosine monophosphate (cGMP), which in turn activates a cGMP-dependent protein kinase (see ). The number of

1	smooth muscle by stimulating guanylate cyclase and thereby increasing levels of cyclic guanosine monophosphate (cGMP), which in turn activates a cGMP-dependent protein kinase (see ). The number of muscarinic receptors is regulated, and exposure to muscarinic agonists decreases the number of receptors by internalization of the receptors.

1	Chagas disease istheresultofinfectionbytheparasiteTrypanosoma cruzi.

1	istheresultofinfectionbytheparasiteTrypanosoma cruzi. About18millionpeopleareinfectedworldwide,andapproximately50,000dieeachyearasaresultofcomplicationsfromthedisease.Themostseriousformsinvolveenlargementoftheesophagus,colon,andheart.Lossofparasympatheticcontrolisasignificantcomponentoftheinitialstagesofthedisease;shortlyaftertheinitialinfection,theparasympatheticneuronsinnervatingtheheart,esophagus,andcolonaredestroyed,whichleadstoarrhythmias(andpotentiallysuddendeath)andaperistalsis.Chroniccardiomyopathy(malfunctionoftheheartmuscle)thatcanleadtodeathoccursinapproximately30%ofthoseinfected.Althoughthepathogenesisofthecardiomyopathyisnotfullyunderstood,oneleadingideainvolvesautoimmunity.Antibodiesagainsttheparasiticantigenshavebeenfoundtobindtotheβ-adrenergicandM2acetylcholinereceptorsintheheart.Theseantibodiesnotonlytriggerautoimmuneresponsesthatdestroyheartmusclebutalsoactasagonistsatthesereceptorsandcauseinappropriateresponsesofthecardiovascularsystemtochangingexternaldemands.

1	Central Control of Autonomic Function The discharges of autonomic preganglionic neurons are controlled by pathways that synapse on autonomic preganglionic neurons. The pathways that influence autonomic activity include spinal cord and brainstem reflex pathways, as well as descending control systems originating at higher levels of the nervous system, such as the hypothalamus. Examples of Autonomic Control of Particular Organs The dilator and constrictor muscles of the iris, which are under the control of sympathetic and parasympathetic fibers, respectively, determine the size of the pupil. Activation of sympathetic innervation of the eye, via thoracic white rami and sympathetic trunk ganglia, dilates the pupil, which occurs during emotional excitement and also in response to painful stimulation. The neurotransmitter at the sympathetic postganglionic synapses is norepinephrine, and it acts at α receptors.

1	The parasympathetic nervous system exerts an action on pupillary size opposite that of the sympathetic nervous system. Whereas the sympathetic system elicits pupillary dilation, the parasympathetic system constricts the pupil. The preganglionic parasympathetic nerves that innervate the pupillary constrictor are in the Edinger-Westphal nucleus, which is in the midbrain, and travel in cranial nerve III, and so damage to this nerve can lead to a dilated pupil (mydriasis).

1	Sympatheticcontrolofthepupilissometimesaffectedbydisease.Forexample,interruptionofthesympatheticinnervationoftheheadandneckresultsinHorner’s syndrome. Thissyndromeischaracterizedbythetriadofmiosis(abnormalpupillaryconstriction),ptosis(causedbyparalysisofthesuperiortarsalmuscle),andanhydrosis(lossofsweating)ontheface.Enophthalmos (retractionoftheeyeintotheorbit)alsooccursinsomeanimals(rats,cats,anddogs,amongothers),butinhumansnotrueenophthalmosoccurs;however,thereisanapparentenophthalmos,anillusioncreatedbypartialclosureoftheeyelidfromtheptosis.Horner’ssyndromecanbeproducedbyalesionthat(1)destroysthesympatheticpreganglionicneuronsintheupperthoracicspinalcord,(2)interruptsthecervicalsympatheticchain,or(3)damagesthelowerbrainstemintheregionofthereticularformation,throughwhichpathwaysdescendtothespinalcordtoactivatesympatheticpreganglionicneurons.Inthelastcase,thereisalsoalossofsweatingonthesideofthebodyipsilateraltothelesion.

1	Pupil size is reduced by the pupillary light reflex and during accommodation for near vision. In the pupillary light reflex, light that strikes the retina is processed by retinal circuits that excite W-type retinal ganglion cells (see Chapter 8). These cells respond to diffuse illumination. The axons of some of the W-type cells project through the optic nerve and tract to the pretectal area, where they synapse in the olivary pretectal nucleus. This nucleus contains neurons that also respond to diffuse illumination. Activity of neurons of the olivary pretectal nucleus causes pupillary constriction by means of bilateral connections with parasympathetic preganglionic neurons in the Edinger-Westphal nuclei. The reflex results in contraction of the pupillary sphincter muscles in both eyes, even when light is shone into only one eye.

1	The accommodation response, which is important for focusing on near objects, involves pupillary constriction, increasing the curvature of the lens, and convergence of the eyes. This response is triggered by information from M cells of the retina that is transmitted to the striate cortex through the geniculostriate visual pathway (see ). The specific stimuli that trigger accommodation are thought to be a blurred retinal image and disparity of the image between the two eyes. After the information is processed in the visual cortex, signals are transmitted directly or indirectly to the middle temporal cortex, where they activate neurons in a visual area known as MT. Area MT neurons transmit signals to the midbrain that activate parasympathetic preganglionic neurons in the Edinger-Westphal nuclei, which results in pupillary constriction. At the same time, signals are transmitted to the ciliary muscle that cause it to contract. The ciliary muscle contraction allows the lens to round up and

1	which results in pupillary constriction. At the same time, signals are transmitted to the ciliary muscle that cause it to contract. The ciliary muscle contraction allows the lens to round up and increase its refractile power. (Convergence is a somatic response mediated by neurons in the oculomotor [cranial nerve III] nucleus of the midbrain.)

1	Thepupillarylightreflexissometimesabsentinpatientswithtertiary(advanced)syphilis,whichaffectstheCNS(i.e.,intheformoftabesdorsalis).Althoughthepupilfailstorespondtolight,ithasanormalaccommodationresponse.ThisconditionisknownastheArgyll Robertson pupil. Theexactmechanismiscontroversial.Oneexplanationrestsonthefactthatsomeoptictractfibersprojecttothepretectalareainthemidbrain.Thesefiberscanbedamagedinsyphiliticmeningitis,possiblybythepresenceofspirochetesinthesubarachnoidspace.NotethatthepretectalareaprojectstotheEdinger-Westphalnucleus,alsointhemidbrain,whosecellsoriginatetheparasympatheticinnervationoftheeye,whichcontrolsthepupillarysphinctermuscle.Althoughinputtotheolivarypretectalnucleusisinterrupted,theoptictractfibersprojectingtothelateralgeniculatenucleusarenotdestroyed,andthusvisionismaintained,asispupillaryconstrictionduringaccommodation. The urinary bladder is controlled by reflex pathways in the spinal cord and also by a supraspinal center (

1	The urinary bladder is controlled by reflex pathways in the spinal cord and also by a supraspinal center ( Fig. 11.3 ). The sympathetic innervation originates from preganglionic sympathetic neurons in the upper lumbar segments of the spinal cord. Postganglionic sympathetic axons act to inhibit the smooth muscle (detrusor muscle) throughout the body of the bladder, and they also act to excite the smooth muscle of the trigone region and the internal urethral sphincter. •Fig. 11.3 Illustration of Descending and Efferent Pathways for Reflexes That Control the Urinary Bladder. Forclarity,onlysomeofthemajorinvolvedpathwaysareshown.(RedrawnfromdeGroatWC,BoothAM.InDyckPJ,etal[eds].Peripheral Neuropathy. 2nded.Philadelphia:WBSaunders;1984.)

1	The detrusor muscle is tonically inhibited during filling of the bladder, and such inhibition prevents urine from being voided. Inhibition of the detrusor muscle is mediated by the action of norepinephrine on β receptors, whereas excitation of the trigone and internal urethral sphincter is elicited by the action of norepinephrine on α receptors. The external sphincter of the urethra also helps control voiding. This sphincter is a striated muscle, and it is innervated by motor axons in the pudendal nerves, which are somatic nerves. The motor neurons are located in the Onuf nucleus, in the ventral horn of the sacral spinal cord.

1	The parasympathetic preganglionic neurons that control the bladder are located in the sacral spinal cord (the S2 and S3 or S3 and S4 segments). These cholinergic neurons project through the pelvic nerves and are distributed to ganglia in the pelvic plexus and the bladder wall. Post-ganglionic parasympathetic neurons in the bladder wall innervate the detrusor muscle, as well as the trigone and sphincter. The parasympathetic activity contracts the detrusor muscle and relaxes the trigone and internal sphincter. These actions result in micturition, or urination. Some of the postganglionic neurons are cholinergic and others are purinergic (they release ATP). Micturition is normally controlled by the micturition reflex (see

1	Fig. 11.3 ). Mechanoreceptors in the bladder wall are excited by both stretch and contraction of the muscles in the bladder wall. Thus as urine accumulates and distends the bladder, the mechanoreceptor afferents begin to discharge. The pressure in the urinary bladder is low during filling (5 to 10 cm H2O), but it increases abruptly when micturition begins. Micturition can be triggered either reflexively or voluntarily. In reflex micturition, bladder afferent fibers excite neurons that project to the brainstem and activate the micturition center in the rostral pons (Barrington’s nucleus). The descending projections also inhibit sympathetic preganglionic neurons that prevent voiding. When a sufficient level of activity occurs in this ascending pathway, micturition is triggered by the micturition center. Commands reach the sacral spinal cord through a reticulospinal pathway. Activity in the sympathetic projection to the bladder is inhibited, and the parasympathetic projections to the

1	center. Commands reach the sacral spinal cord through a reticulospinal pathway. Activity in the sympathetic projection to the bladder is inhibited, and the parasympathetic projections to the bladder are activated. Contraction of muscle in the wall of the bladder causes a vigorous discharge of the mechanoreceptors that supply the bladder wall and thereby further activates the supraspinal loop. The result is complete emptying of the bladder.

1	A spinal reflex pathway also exists for micturition. This pathway is operational in newborn infants. However, with maturation, the supraspinal control pathways take on a dominant role in triggering micturition. After spinal cord injury, human adults lose bladder control during the period of spinal shock (urinary incontinence). As the spinal cord recovers from spinal shock, some degree of bladder function is recovered because of enhancement of the spinal cord micturition reflex. However, the bladder has increased muscle tone and fails to empty completely. These circumstances frequently lead to urinary infections. Autonomic Centers in the Brain

1	Autonomic Centers in the Brain Influence over autonomic output is maintained by autonomic centers, which consist of local networks of neurons, in a variety of brain regions. The micturition center in the pons, which was just discussed, is one example. Many other autonomic centers with diverse functions exist. Vasomotor and vasodilator centers are in the medulla, and respiratory centers are in the medulla and pons. Perhaps the greatest concentration of autonomic centers is found in the hypothalamus. The Hypothalamus and Preoptic Area The hypothalamus is part of the diencephalon. Some of the nuclei of the hypothalamus are shown in

1	The Hypothalamus and Preoptic Area The hypothalamus is part of the diencephalon. Some of the nuclei of the hypothalamus are shown in Fig. 11.4 . Located anteriorly from the hypothalamus are telencephalic structures: the preoptic region and septum, both of which help regulate autonomic function. Important fiber tracts that course through the hypothalamus are the fornix, the medial forebrain bundle, and the mammillothalamic tract. The fornix is used as a landmark to divide the hypothalamus into medial and lateral zones.

1	The hypothalamus has many functions; see for a discussion of hypothalamic control of endocrine function. Its control of autonomic function is emphasized here. In its control of autonomic function, the hypothalamus functions much like a control system that is termed, in •Fig. 11.4 Illustration of Main Nuclei of the Hypothalamus, Viewed From the Third Ventricle. Anterioristotheright.(RedrawnfromNautaWJH,HaymakerW.The Hypothalamus. Springfield,IL:CharlesCThomas;1969.) engineering, a servomechanism: that is, a system in which a particular physiological parameter is controlled through the use of negative feedback loops to maintain the parameter at a particular set point or value. The following examples illustrate this principle for body temperature, body weight and adiposity, and water intake.

1	Homeothermic animals maintain a relatively constant core body temperature in situations of fluctuating environmental temperatures and differing levels of bodily activity that cause endogenous heat production. This ability rests on information from three main groups of thermoreceptors located in the skin, CNS, and viscera. Information about the external temperature is provided by thermoreceptors in the skin. Core body temperature is monitored by central thermoreceptive neurons in the preoptic area (and possibly the spinal cord), which monitor the temperature of local blood. Thermoreceptors in the viscera monitor the temperature in these organs. All of these receptors provide temperature information to the preoptic area (pathways described later), along with parts of the hypothalamus, in which this information is used to keep core body temperature constant. Thus the preoptic area and hypothalamus act together as a servomechanism with a set point at the normal body temperature.

1	Although the signals from each of these sources are integrated, their relative importance may shift, depending on the situation. Changes in environmental temperature evoke more rapid and much larger changes in the temperature of the skin than in the body core, and so cutaneous receptors are probably the initial and most often used mechanism for compensating for external changes in temperature. Central thermoreceptors are more important for situations with internal causes of temperature change, such as during exercise, or in which external changes temperature are so severe or prolonged that core body temperature starts to change despite the signals from peripheral thermoreceptors. Last, alteration of body temperature by ingestion of hot or cold food or liquids is detected by the visceral thermoreceptors.

1	Error signals (i.e., cooling and warming of the body), which represent a deviation from the set point of the servomechanism, evoke responses that tend to restore body temperature toward the set point. These responses are mediated by the autonomic, somatic, and endocrine systems.

1	Situations involving cooling, for example, trigger a variety of responses that increase heat production (thermogenesis) and minimize heat loss. Heat production is increased by mechanisms that include shivering thermogenesis (asynchronous contractions of skeletal muscle that increases heat production) and brown adipose tissue (BAT) thermogenesis (in BAT thermogenesis, oxidative phosphorylation is uncoupled from ATP synthesis, which allows the energy released by the reaction to be dissipated as heat instead), and increased thyroid hormone levels lead to increased metabolism. Heat loss is reduced by cutaneous vasoconstriction and by piloerection. Piloerection is effective in animals with fur but not in humans; in the latter, the result is only goose bumps. In addition, tachycardia occurs, which may help provide metabolites to be used in thermogenesis to the thermogenic tissues (fat and muscle) and help distribute the heat generated throughout the body. Finally, the perception of being

1	which may help provide metabolites to be used in thermogenesis to the thermogenic tissues (fat and muscle) and help distribute the heat generated throughout the body. Finally, the perception of being cold influences the decision to initiate voluntary behaviors: in this case, possibly putting on a jacket.

1	Warming the body generally causes changes in the opposite direction. The activity of the thyroid gland diminishes, which leads to reduced metabolic activity and less heat production. Heat loss is increased by sweating, salivation (in some animals but not humans), and cutaneous vasodilation (because of decreased sympathetic activity). However, again tachycardia occurs, this time presumably to allow optimal perfusion of the cutaneous circulation for heat dissipation.

1	Early studies identified the preoptic region and anterior hypothalamus as a heat loss center and the posterior hypothalamus as a heat conservation center. For example, lesions in the preoptic region prevent sweating and cutaneous vasodilation, and if an individual with a lesion in this region is placed in a warm environment, hyperthermia occurs. Conversely, electrical stimulation of the heat loss center causes cutaneous vasodilation and inhibits shivering. In contrast, lesions in the area dorsolateral to the mammillary body interfere with heat production and conservation and can cause hypothermia when the person is in a cold environment. Electrical stimulation in this region of the brain evokes shivering.

1	Many details of the circuitry and physiologic processes underlying the temperature regulation responses are now known, and they indicate that the preoptic area and dorsal medial hypothalamic nucleus are key components in the regulation of body temperature. The preoptic area in particular appears to be the target of the various sources of sensory information. Cutaneous temperature information is conveyed by thermosensitive primary afferents that synapse in the dorsal horn of the spinal cord onto neurons that project up to and excite the parabrachial nucleus in the caudal midbrain. Information from visceral afferents is relayed by the solitary nucleus to the parabrachial nucleus as well. The parabrachial neurons in turn excite neurons within a specific part of the preoptic area, the median nucleus (of the preoptic area). Many median nucleus neurons are also sensitive to local changes in blood temperature and contain prostaglandin E2 (PGE2) receptor 3 (EP3), which mediates fever

1	median nucleus (of the preoptic area). Many median nucleus neurons are also sensitive to local changes in blood temperature and contain prostaglandin E2 (PGE2) receptor 3 (EP3), which mediates fever responses (see the following “In the Clinic” box).

1	Thus the median preoptic nucleus is a key component of the thermoregulatory control system in which information from the various types of thermoreceptors is integrated. Output from this nucleus is directed to the neighboring medial preoptic area, which projects to regions of the rostral medulla, both directly and via the dorsal medial hypothalamic nucleus. The rostral medulla has spinally projecting neurons that project to the lateral horn of the spinal cord, where the preganglionic sympathetic neurons are located, and the activity of these neurons regulates BAT thermogenesis and modulates cutaneous vasomotor tone. The rostral medulla also projects to the ventral horn of the spinal cord, which contains the somatic motor neurons that contract skeletal muscle and thus mediate the shivering response.

1	Fever,whichaccompaniessomeinfections,canbethoughtofasanelevationofthesetpointforbodytemperature.Thiselevationcanbecausedbythereleaseofpyrogens bymicroorganismsorbycellsmediatingtheinflammatoryresponse.Thepyrogen’seffecttoraisethesetpointismediatedprimarilybytheactionofprostaglandinPGE2’sbindingtoEP3receptorsonneuronsinthepreopticarea.PGE2isreleasedbyperipheraltissuesandbybloodvesselssupplyingthepreopticarea.ThebindingofPGE2toEP3receptorscausesareductionintheactivityofpreopticneurons.ThisreductioninneuronalactivityleadstoincreasedheatproductionthroughshiveringandBATthermogenesisandtoheatconservationbycutaneousvasoconstriction,thecombinedeffectofwhichistoraisebodytemperature.EvidenceofthismechanismoffeverproductionincludesstudiesinwhichinjectionofPGE2intothepreopticareainducedfeverandothersinwhichselectivedeletionoftheEP3receptorfrompreopticneuronsabolishedtheabilityofPGE2injectionstoinducefever. Regulation of Feeding and Body Weight

1	Regulation of Feeding and Body Weight Energy homeostasis is crucial for the survival of the animal. The challenge is that most cells need a continuous supply of nutrients to function, but most animals do not constantly eat; instead, they have periodic meals. Thus to achieve energy homeostasis, feeding behavior is controlled by many factors, which operate both on a short-term basis to control ingestion and on a long-term basis to control body weight in order to ensure sufficient energy stores. Both hedonic and homeostatic factors are involved; however, in this chapter, the focus is on the latter because of the central role that the hypothalamus plays in energy homeostasis.

1	In the short run, eating is controlled by a number of mechanisms. First, the stomach wall has stretch receptors that signal distention as food fills the stomach. These signals are conveyed by the afferents of the vagus nerve to the solitary nucleus in the medulla. From there the information is relayed to several brain areas, including the hypothalamus, either directly or via a relay in the parabrachial nucleus, to organize autonomic responses to the ingested material, and the thalamus and cortex, for conscious awareness of the fullness of the stomach. In the hypothalamus, the paraventricular, dorsomedial, and arcuate nuclei and the lateral hypothalamus are the major targets of these signals.

1	Sensory afferents also sense the concentrations of glucose and lipids in the intestines and the hepatic portal circulation and send this information to the solitary nucleus and, from there, to the hypothalamus, in a manner similar to that described for the stretch receptors. In addition, the stomach and gut release a number of hormones in response to feeding, including cholecystokinin, peptide YY, glucagonlike peptide-1 (GLP-1), and ghrelin. Hypothalamic cells have receptors for many of these hormones and can be influenced directly by them. In addition, cells in other brain areas have receptors for these hormones and thus may provide an indirect pathway to the hypothalamus. One such region is the area postrema, which is just dorsal to the solitary nucleus and projects to it. The area postrema is not protected by the blood-brain barrier (it is one of the circumventricular organs), and its neurons respond to cholecystokinin and GLP-1, which leads to decreased food intake.

1	Control of body weight over the long run is influenced by many factors and involves the interaction of the nervous and endocrine systems. In this section, the focus is on the role of hypothalamus and its control of the autonomic nervous system, which provides another example of how the hypothalamus is part of a servomechanism. In this case, adiposity is the controlled parameter. For further details on the endocrine system’s role, see

1	Early studies in which researchers used lesions and electrical stimulation provided evidence that the ventromedial and ventrolateral hypothalamus are involved in energy homeostasis. A lesion in the ventromedial region causes an increase in food intake (hyperphagia) that results in obesity, whereas electrical stimulation of the same region decreases feeding behavior. These lesions were also shown to alter autonomic activity, increasing parasympathetic and decreasing sympathetic tone, both of which lead to high blood insulin levels, which in turn promote energy conservation and storage (see ). These observations led to the idea that the ventromedial hypothalamus contains a satiety center. However, an alternative interpretation is that the primary controlled variable may not be simply eating behavior per se but rather body weight and, even more specifically, body fat levels (i.e., adiposity). Thus modulation of feeding behavior may be just one of several actions used to defend a body

1	behavior per se but rather body weight and, even more specifically, body fat levels (i.e., adiposity). Thus modulation of feeding behavior may be just one of several actions used to defend a body weight set point. Evidence for this is that although lesions cause an initial period of dynamic weight gain in which hyperphagia is present, this is followed by a static period in which the higher weight is maintained without hyperphagia. Moreover, animals with a lesion in the ventromedial hypothalamus that are fed a fixed (normal) amount of food to prevent hyperphagia nonetheless become obese, which implies changes in the regulation of other metabolic processes. Last, lesions of the ventromedial hypothalamus have been shown to alter levels of energy expenditure.

1	In contrast to lesions of the ventromedial hypothalamus, those of the lateral hypothalamus suppress food intake (hypophagia) and lead to a decrease in body weight; indeed, animals can starve to death after such lesions. Conversely, electrical stimulation of the medial forebrain bundle in the lateral hypothalamus evokes exploratory behavior and eating, if food is present. This stimulation also provides a dopamine-dependent reward that mediates the incentive effects of natural rewards (food, sex) as well as the rewarding effects of most drugs of abuse. These observations led to the view that the lateral hypothalamus contains a feeding center. This interpretation, however, is complicated by the fact that the dopaminergic axons of substantia nigra neurons pass just lateral to the lateral hypothalamus on their way to the striatum, and so loss or stimulation of these fibers could account for the effects produced in these experiments. However, lateral hypothalamic neurons have been found to

1	on their way to the striatum, and so loss or stimulation of these fibers could account for the effects produced in these experiments. However, lateral hypothalamic neurons have been found to synthesize peptides, such as orexin, that affect feeding behavior, and so the lateral hypothalamus probably does play a role in energy homeostasis.

1	In newer studies, investigators have identified a number of hormones and neuropeptides involved in feeding and control of body weight, and many of the interactions between the endocrine and nervous systems that underlie energy homeostasis have been clarified. In normal individuals, blood insulin levels are correlated with adiposity (in addition to varying acutely with blood levels of glucose and other substances). Similarly, the level of the protein leptin, a hormone released by adipocytes (primarily those forming white adipose tissue), is correlated with adiposity. High levels of leptin inhibit food intake and stimulate catabolic processes, including loss of fat tissue, whereas low leptin levels trigger the reverse actions. Similarly, high insulin levels promote energy storage processes.

1	The ability of leptin and insulin to regulate body weight has been linked to their actions on the hypothalamus, particularly the arcuate nucleus, whose neurons express receptors for both hormones (see also ). Two major classes of arcuate nucleus neurons that respond to leptin and insulin have been identified. Neurons that express proopiomelanocortin (POMC) and cocaine-and amphetamine-related transcript (CART) are stimulated by leptin and insulin, and their activity leads to increased catabolism. In contrast, the activity of a second group of neurons, those that express neuropeptide Y (NPY) and agouti-related peptide (AgRP), triggers anabolic processes but is inhibited by leptin and insulin. Thus increased body fat levels lead to high leptin and insulin levels, which in turn both (1) increase the activity of POMCand CART-expressing neurons, leading to increased catabolism, and (2) decrease the activity of NPYand AgRP-expressing neurons, leading to decreased anabolism; both of which act

1	the activity of POMCand CART-expressing neurons, leading to increased catabolism, and (2) decrease the activity of NPYand AgRP-expressing neurons, leading to decreased anabolism; both of which act to return body fat levels to their set point. Lowering body fat levels would result in a sequence of events opposite to that just described to increase body fat levels to their original level or set point.

1	The efferent limb that mediates the actions of these sets of arcuate neurons is not fully worked out. However, the arcuate nucleus projection to the paraventricular hypothalamic nucleus appears to be an important step in the pathway. Paraventricular neurons contain oxytocin. Many of them project to the posterior pituitary gland and are involved in lactation and uterine contractions during labor (see ). However, the paraventricular neurons involved in body weight regulation are a distinct subset of neurons that project down to the brainstem and spinal cord, where they probably synapse with autonomic and preautonomic nuclei that control parasympathetic vagal fibers to the pancreas, which act to stimulate insulin release, and sympathetic fibers, which act to inhibit its release. Regulation of Water Intake Water intake also depends on a servomechanism. Fluid intake is influenced by blood osmolality and volume ( Fig. 11.5

1	Regulation of Water Intake Water intake also depends on a servomechanism. Fluid intake is influenced by blood osmolality and volume ( Fig. 11.5 With water deprivation, the extracellular fluid becomes hyperosmotic, which in turn causes the intracellular fluid to become hyperosmotic. The brain contains neurons that serve as osmoreceptors for detection of increases in the osmotic pressure of extracellular fluid (see also ). The osmoreceptors appear to be located in the organum

1	Angiotensin II receptors Angiotensin II receptors Osmotic receptors Blood vessels Drinking circuit, including vasopressin-releasing neurons in the posterior pituitary B •Fig. 11.5 A, Structuresthoughttoplayaroleintheregulationofwaterintakeinrats.LHA,lateralhypothalamicarea.B, Neuralcircuitsthatsignalchangesinbloodosmolalityandvolume.(A, RedrawnfromShepherdGM.Neurobiology. NewYork:OxfordUniversityPress;1983.) vasculosum of the lamina terminalis, which is a circumventricular organ. Circumventricular organs surround the cerebral ventricles and lack a blood-brain barrier. The subfornical organ and the organum vasculosum are involved in thirst.

1	Water deprivation also causes a decrease in blood volume, which is sensed by receptors in the low-pressure side of the vasculature, including the right atrium of the heart (see also ). In addition, decreased blood volume triggers the release of renin by the kidneys. Renin breaks down angiotensinogen into angiotensin I, which is then hydrolyzed to angiotensin II (see ). This peptide stimulates drinking by an action on angiotensin II receptors in another of the circumventricular organs, the subfornical organ. Angiotensin II also causes vasoconstriction and release of aldosterone and antidiuretic hormone (ADH).

1	Insufficient water intake is usually a greater problem than excessive water intake. However, when more water is taken in than required, it is easily eliminated by inhibition of the release of ADH from neurons in the supraoptic nucleus at their terminals in the posterior pituitary gland (see ). As mentioned previously, signals that inhibit release of ADH include increased blood volume and decreased osmolality of extracellular fluid. Other areas of the hypothalamus, particularly the preoptic region and lateral hypothalamus, help regulate water intake, as do several structures outside the hypothalamus.

1	Several regions of the forebrain other than the hypothalamus also play a role in autonomic control. These regions include the central nucleus of the amygdala and the bed nucleus of the stria terminalis, as well as a number of areas of the cerebral cortex. Information reaches these higher autonomic centers from viscera through an ascending system that involves the nucleus of the solitary tract, the parabrachial nucleus, the periaqueductal gray matter, and the hypothalamus. Descending pathways that help control autonomic activity originate in such structures as the paraventricular nucleus of the hypothalamus, noradrenergic cell group A5, the rostral ventrolateral medulla, and the raphe nuclei and adjacent structures of the ventromedial medulla. Neural Influences on the Immune System

1	Neural Influences on the Immune System Environmentalstresscancauseimmunosuppression,inwhich the number of helper T cells and the activity of natural killer cells are reduced. Immunosuppression can even be the result of classical conditioning. One mechanism for such an effect involves the release of corticotropin-releasing factor from the hypothalamus. Corticotropin-releasing factor causes the release of adrenocorticotropic hormone (ACTH) from the pituitary gland; release of ACTH stimulates the secretion of adrenal corticosteroids, which cause immunosuppression (see ). Other mechanisms include direct neural actions on lymphoid tissue. The immune system may also influence neural activity. 1.

1	1. The autonomic nervous system controls smooth muscle, cardiac muscle, and glands. It helps maintain homeostasis and coordinates responses to external stimuli. It has sensory and motor components, and the motor component consists of sympathetic and parasympathetic divisions. The enteric nervous system is often considered as part of the autonomic nervous system but is concerned specifically with control of the gastrointestinal tract. 2.

1	2. Autonomic motor pathways have preganglionic and postganglionic neurons. Preganglionic neurons reside in the CNS, whereas postganglionic neurons lie in peripheral ganglia. Sympathetic preganglionic neurons are located in the thoracolumbar region of the spinal cord, and sympathetic postganglionic neurons are located in paravertebral and prevertebral ganglia. Parasympathetic preganglionic neurons are located in cranial nerve nuclei or in the sacral portion of the spinal cord. Parasympathetic postganglionic neurons reside in ganglia located in or near the target organs. 3. Autonomic afferent fibers innervate sensory receptors in the viscera. Most function to activate reflexes; for some, activation also leads to sensations that are experienced consciously. 4.

1	3. Autonomic afferent fibers innervate sensory receptors in the viscera. Most function to activate reflexes; for some, activation also leads to sensations that are experienced consciously. 4. The enteric nervous system includes the myenteric and submucosal plexuses in the wall of the gastrointestinal tract. The myenteric plexus regulates motility, and the submucosal plexus regulates ion and water transport and secretion. 5. Neurotransmitters at the synapses of preganglionic neurons in autonomic ganglia include acetylcholine (acting at both nicotinic and muscarinic receptors) and a number of neuropeptides. Interneurons in Nakamura K. Central circuitries for body temperature regulation and fever. Am J Physiol Regul Integr Comp Physiol. 2011;301: R1207-R1228.

1	Nakamura K. Central circuitries for body temperature regulation and fever. Am J Physiol Regul Integr Comp Physiol. 2011;301: R1207-R1228. the ganglia release catecholamines. Norepinephrine (acting on adrenergic receptors) is the neurotransmitter generally released by sympathetic postganglionic neurons; neuropeptides are also released. Sympathetic postganglionic neurons that supply sweat glands release acetylcholine. Parasympathetic postganglionic neurons release acetylcholine (acting on muscarinic receptors). 6. The pupil is controlled reciprocally by the sympathetic and parasympathetic nervous systems. Sympathetic activity causes pupillary dilation (mydriasis); parasympathetic activity causes pupillary constriction (meiosis). 7.

1	7. Emptying of the urinary bladder depends on parasympathetic outflow during the micturition reflex. Sympathetic constriction of the internal sphincter of the urethra prevents voiding. The micturition reflex is triggered by stretch receptors, and it is controlled in normal adults by a micturition center in the pons. 8. The hypothalamus contains many nuclei that have a variety of functions related to regulation of basic bodily functions, including body temperature, body weight, and fluid intake. 9. The goal of hypothalamic function is to maintain homeostasis of critical physiological parameters by acting as a servomechanism. The hypothalamus receives information about specific physiological parameters and uses this information to maintain each of these parameters at a specific set point. It does so via multiple mechanisms. This chapter illustrates how it maintains homeostasis via its control of the autonomic system.

1	Saper CB, Chou TC, Elmquist JK. The need to feed: homeostatic and hedonic control of eating. Neuron. 2002;36:199-211. Squire L, Berg D. Fundamental Neuroscience. 4th ed. New York: Academic Press; 2012. SECTION 3Muscle JAMES M. WATRAS Upon completion of this chapter, the student should be able to answer the following questions: 1. Describe the organization of skeletal muscle, including the structural features/proteins within the skeletal muscle fiber that link the contractile elements to the extracellular matrix and bone to effect movement. While describing the various linkages, identify congenital conditions that commonly affect particular structures and how they might contribute to a myopathy. 2. Describe the molecular mechanisms by which an action potential in the α motor neuron in the ventral horn of the spinal column can lead to contraction of a skeletal muscle. 3. Describe the mechanisms by which the force of skeletal muscle contraction increases. 4.

1	3. Describe the mechanisms by which the force of skeletal muscle contraction increases. 4. Compare skeletal muscle fiber types in terms of recruitment pattern, metabolic characteristics, contractile characteristics, and thus their suitability for various types of activity. 5. Discuss the signaling pathways that contribute to the expression of the slow-twitch muscle phenotype versus the fast-twitch muscle phenotype. 6. Describe general signaling pathways that contribute to hypertrophy or atrophy. 7. Discuss mechanisms underlying the development of muscular fatigue. 8. Describe mechanisms underlying the monosynaptic reflex. 9. Discuss the length-tension curves and force-velocity curves for skeletal muscle, including the molecular bases of both curves.

1	8. Describe mechanisms underlying the monosynaptic reflex. 9. Discuss the length-tension curves and force-velocity curves for skeletal muscle, including the molecular bases of both curves. Muscle cells are highly specialized for the conversion of chemical energy to mechanical energy. Specifically, muscle cells use the energy in adenosine triphosphate (ATP) to generate force or do work. Because work can take many forms (such as locomotion, pumping blood, or peristalsis), several types of muscle have evolved. The three basic types of muscle are skeletal muscle, cardiac muscle, and smooth muscle.

1	Skeletal muscle acts on the skeleton. In limbs, for example, skeletal muscle spans a joint, thereby allowing a lever action. Skeletal muscle is under voluntary control (i.e., controlled by the central nervous system) and plays a key role in numerous activities such as maintenance of posture, locomotion, speech, and respiration. When viewed under the microscope, skeletal muscle exhibits transverse striations (at intervals of 2 to 3 µm) that result from the highly organized arrangement of actin and myosin molecules within the skeletal muscle cells. Thus skeletal muscle is classified as a striated muscle. The heart is composed of cardiac muscle, and although it is also a striated muscle, it is an involuntary muscle (i.e., controlled by an intrinsic pacemaker and modulated by the autonomic nervous system). Smooth muscle (which lacks the striations evident in skeletal and cardiac muscle) is an involuntary muscle typically found lining hollow organs such as the intestine and blood vessels.

1	system). Smooth muscle (which lacks the striations evident in skeletal and cardiac muscle) is an involuntary muscle typically found lining hollow organs such as the intestine and blood vessels. In all three muscle types, force is generated by the interaction of actin and myosin molecules, a process that requires transient elevation of intracellular [Ca++].

1	In this chapter, attention is directed at the molecular mechanisms underlying contraction of skeletal muscle. Mechanisms for regulating the force of contraction are also addressed. To put this information into perspective, it is important to first examine the basic organization of skeletal muscle. Organization of Skeletal Muscle

1	Organization of Skeletal Muscle Fig. 12.1 illustrates skeletal muscles spanning the elbow joint. The muscles are attached to bone on either side of the joint. The point of attachment closest to the spine (proximal) is called the origin, whereas the point of attachment on the far side of the joint (distal) is called the insertion. These points of attachment occur through tendons (connective tissue) at the end of the muscle. Note that the point of insertion is close to the elbow joint, which enables a broad range of motion. Also note that the joint is spanned by a flexor muscle on one side and an extensor muscle on the opposite side of the joint. Thus contraction of the flexor muscle (see the biceps muscle in Fig. 12.1 ) results in a decrease in the angle of the elbow joint (bringing the forearm closer to the shoulder), whereas contraction of the extensor muscle (see the triceps muscle in Fig. 12.1 ) results in the reverse motion (extending the arm).

1	Fig. 12.1 ) results in the reverse motion (extending the arm). The basic structure of skeletal muscle is shown in Fig. 12.2 . Each muscle is composed of numerous cells called • Fig. 12.1. Skeletal Muscle Attaches to the Skeleton by Way of Tendons and Typically Spans a Joint. The proximal and distal points of attachment of the tendon are termed origin and insertion, respectively. Note that the insertion is close to the joint, which allows a broad range of motion. Also note that skeletal muscles span both sides of the joint, which allows both flexion and extension of the forearm.

1	• Fig. 12.2. Skeletal muscle is composed of bundles of muscle fibers; each such bundle is called a fasciculus. A muscle fiber represents an individual muscle cell and contains bundles of myofibrils. The striations are due to the arrangement of thick and thin filaments. See text for details. (Redrawn from Bloom W, Fawcett DW. A Textbook of Histology. 10th ed. Philadelphia: Saunders; 1975.) muscle fibers. A connective tissue layer called the endomysium surrounds each of these fibers. Individual muscle fibers are then grouped together into fascicles, which are surrounded by another connective tissue layer called the perimysium. Within the perimysium are the blood vessels and nerves that supply the individual muscle fibers. The fascicles are joined together to form the muscle. The connective tissue sheath that surrounds the muscle is called the epimysium. At the ends of the muscle, the connective tissue layers come together to form a tendon, which attaches the muscle to the skeleton. The

1	tissue sheath that surrounds the muscle is called the epimysium. At the ends of the muscle, the connective tissue layers come together to form a tendon, which attaches the muscle to the skeleton. The myotendinous junction is a specialized region of the tendon where the ends of the muscle fibers interdigitate with the tendon for the transmission of the force of contraction of the muscle to the tendon to effect movement of the skeleton (discussed later in this section). The tendon and the connective tissue layers are composed mainly of elastin and collagen fibers, and thus they also contribute to passive tension of muscle and prevent damage to the muscle fibers as a result of overstretching or contraction.

1	Individual skeletal muscle cells are narrow (≈10 to 80 µm in diameter), but they can be extremely long (up to 25 cm in length). Each skeletal muscle fiber contains bundles of filaments, called myofibrils, running along the axis of the cell. The gross striation pattern of the cell results from a repeating pattern in the myofibrils. Specifically, it is the regular arrangement of the thick and thin filaments within these myofibrils, coupled with the highly organized alignment of adjacent myofibrils, that gives rise to the striated appearance of skeletal muscle. Striations can be observed in intact muscle fibers and in the underlying myofibrils.

1	Fig. 12.3 ). The sarcomere is demarcated by two dark lines called Z lines and represents a repeating contractile unit in skeletal muscle. The average length of a sarcomere is 2 µm. On either side of the Z line is a light band (I band) that contains thin filaments composed primarily of the protein actin. The area between two I bands within a sarcomere is the A band, which contains thick filaments composed primarily of the protein myosin. The thin actin filaments extend from the Z line toward the center of the sarcomere and overlap a portion of the thick filaments. The dark area at the end of the A band represents this region of overlap between thick and thin filaments. A light area in the center of the sarcomere is called the H band. This area represents the portion of the A band that contains myosin thick filaments but no thin actin filaments. Thus thin actin filaments extend from the Z line to the edge of the H band and overlap a portion of the thick filament in the A band. A dark

1	myosin thick filaments but no thin actin filaments. Thus thin actin filaments extend from the Z line to the edge of the H band and overlap a portion of the thick filament in the A band. A dark line called the M line is evident in the center of the sarcomere and includes proteins that appear to be critical for organization and alignment of the thick filaments in the sarcomere.

1	Fig. 12.3, each myofibril in a muscle fiber is surrounded by sarcoplasmic reticulum (SR). The SR is an intracellular membrane network that plays a critical •Fig. 12.3. A, Myofibrilsarearrangedinparallelwithinamusclefiber.B, Eachfibrilissurroundedbysarcoplasmicreticulum(SR).TerminalcisternaeoftheSRarecloselyassociatedwithTtubulesandformatriadatthejunctionoftheIandAbands.TheZlinesdefinetheboundaryofthesarcomere.Thestriationsareformedbyoverlapofthecontractileproteins.Threebandscanbeobserved:theAband,Iband,andHband.AnMlineisvisibleinthemiddleoftheHband.C, Organizationoftheproteinswithinasinglesarcomere.Thecross-sectionalarrangementoftheproteinsisalsoillustrated.

1	role in the regulation of intracellular [Ca++]. Invaginations of the sarcolemma, called T tubules, pass into the muscle fiber near the ends of the A band (i.e., close to the SR). The SR and the T tubules, however, are distinct membrane systems: The SR is an intracellular network, whereas the T tubules are in contact with the extracellular space. A gap (≈15 nm in width) separates the T tubules from the SR. The portion of the SR nearest the T tubules is called the terminal cisternae, and it is the site of Ca++ release, which is critical for contraction of skeletal muscle (see the section “ ”). The longitudinal portions of the SR are continuous with the terminal cisternae and extend along the length of the sarcomere. This portion of the SR contains a high density of Ca++ pump protein (i.e., SERCA: Sarcoplasmic Endoplasmic Reticulum Ca++-ATPase), which is critical for reaccumulation of Ca++ in the SR and hence for relaxation of the muscle.

1	The thick and thin filaments are highly organized in the sarcomere of myofibrils (see Fig. 12.3 ). As mentioned, thin actin filaments extend from the Z line toward the center of the sarcomere, whereas thick myosin filaments are centrally located and overlap a portion of the opposing thin actin filaments. The thick and thin filaments are oriented in such a way that in the region of overlap within the sarcomere, each thick myosin filament is surrounded by a hexagonal array of thin actin filaments. The Ca++-dependent interaction of the thick myosin filaments and the thin actin filaments generate the force of contraction after stimulation of the muscle (see the section “Actin-Myosin Interaction:

1	The thin filament is formed by aggregation of actin molecules (termed globular actin or G-actin) into a two-stranded helical filament called filamentous actin or F-actin. The elongated cytoskeletal protein nebulin extends along the length of the thin filament and may participate in regulation of the length of the thin filament. Dimers of the protein tropomyosin extend over the entire actin filament and cover myosin binding sites on the actin molecules. Each tropomyosin dimer extends across seven actin molecules, with sequential tropomyosin dimers arranged in a head-to-tail configuration. A troponin complex consisting of three subunits (troponin T, troponin I, and troponin C) is present on each tropomyosin dimer and influences the position of the tropomyosin molecule on the actin filament and hence the ability of tropomyosin to inhibit binding of myosin to the actin filament at low cytosolic Ca concentrations (see the section “Actin-Myosin Interaction: Cross-Bridge Formation”).

1	Cross-Bridge Formation”). Binding of cytosolic Ca++ to troponin C promotes the movement of tropomyosin on the actin filament that exposes myosin binding sites on actin, thereby facilitating actin-myosin interaction, and hence contraction (see the section “Actin-Myosin Interaction: ”). Additional proteins associated with the thin filament include tropomodulin, α-actinin, and CapZ protein. Tropomodulin is located at the end of the thin filament, toward the center of the sarcomere, and may participate in setting the length of the thin filament.

1	• Fig.

1	12.4. The force of contraction of the muscle fiber is transmitted both longitudinally to the tendon (at the myotendinous junction) and laterally to adjacent extracellular connective tissue (at costameres). The force of contraction is transmitted from the end of the muscle fiber (M) to the tendon by connections with numerous collagen fibers (A, tip of arrow). Folds in the sarcolemma at the end of the muscle fiber (B) result in an interdigitation of the muscle fiber with the tendon, and represents the myotendinous junction. Costameres are located on the sides of the muscle fibers, and represent the bridges between the Z lines in the subsarcolemmal myofibrils and the extracellular connective tissue (C). Costameres facilitate the lateral transmission of force of contraction, which helps stabilize the sarcolemma. DGC, dystrophinassociated glycoprotein complex. (A and B, From Tidball JG. Myotendinous junction: morphological changes and mechanical failure associated with muscle cell atrophy.

1	lize the sarcolemma. DGC, dystrophinassociated glycoprotein complex. (A and B, From Tidball JG. Myotendinous junction: morphological changes and mechanical failure associated with muscle cell atrophy. Exp

1	Molec Pathol. 1984;40:1-12. C, From Hughes D, Wallace M, Baar K. Effects of aging, exercise, and disease on force transfer in skeletal muscle. Am J Physiol Endocrin Metab. 2015;309:E1-E10.)

1	CapZ protein and α-actinin serve to anchor the thin fila-of sarcomeres across muscles and the lateral transmission of ment to the Z line. force (described later in this section). The thick myosin filaments are tethered to the Z lines by The force of contraction is transmitted both longitua cytoskeletal protein called titin. Titin is a very large, elastic dinally to the tendon (via myotendinous junctions) and protein (molecular weight, >3000 kDa) that extends from laterally to connective tissue adjacent to the muscle fibers the Z line to the center of the sarcomere and appears to be (via costameres). The myotendinous junction represents a important for organization and alignment of the thick fila-specialized region where the muscle fiber connects to the ments in the sarcomere. Some forms of muscular dystrophy tendon ( Fig. 12.4A and B).

1	Fig. 12.4A and B). Folding of the sarcolemma have been attributed to defects in titin (i.e., titinopathies). at the myotendinous junction results in an interdigitation

1	The cytoskeleton (including the intermediate filament of the tendon with the end of the muscle fiber, which protein desmin) participates in the highly organized align-increases the contact area between the muscle fiber and ment of sarcomeres. Desmin extends from the Z lines of the connective tissue and hence reduces the force per adjacent sarcomeres to the integrin protein complexes on unit area at the end of the muscle fiber. Some forms of the sarcolemma and thus participates in both the alignment muscular dystrophy are associated with decreased folding at the myotendinous junction. Proteins involved in longitudinal transmission of force at the myotendinous junction include the talin-vinculin-integrin protein complexes and the dystrophin-glycoprotein complexes. The myotendinous junction also contains Z disk proteins that have been implicated in signaling.

1	Lateral transmission of the force of contraction involves costameres, which link the Z lines of subsarcolemmal sarcomeres to extracellular matrix through a series of proteins (see Fig. 12.4C ). This is particularly important for muscle fibers that do not extend the entire length of the muscle. The lateral transmission of force is also thought to stabilize the sarcolemma and to protect it from damage during contraction. The myotendinous junction and the costameres also contain signaling molecules.

1	Themusculardystrophiesconstituteagroupofgeneticallydetermineddegenerativedisorders.Duchenne’s muscular dystrophy (describedbyG.B.Duchennein1861)isthemostcommonofthemusculardystrophiesandaffects1per3500boys(3to5yearsofage).Severemusclewastingoccurs,andmostaffectedpatientsarewheelchairboundbytheageof12;manydieofrespiratoryfailureinadulthood(30to40yearsofage).Duchenne’smusculardystrophyisanX-linkedrecessivediseasethathasbeenlinkedtoadefectinthedystrophingenethatleadstoadeficiencyofthedystrophinproteininskeletalmuscle,brain,retina,andsmoothmuscle.Dystrophin isalarge(427-kDa)proteinthatispresentinlowabundance(0.025%)inskeletalmuscle.Itislocalizedontheintracellularsurfaceofthesarcolemmainassociationwithseveralintegralmembraneglycoproteins(formingadystrophin-glycoproteincomplex;

1	Fig.12.5 ).Thisdystrophin-glycoproteincomplexprovidesastructurallinkbetweenthesubsarcolemmalcytoskeletonofthemusclecellandtheextracellularmatrixandappearstostabilizethesarcolemmaandhencepreventcontraction-inducedinjury(rupture).Thedystrophin-glycoproteincomplexmayalsoserveasascaffoldforcellsignalingcascades.Theenzymenitricoxidesynthaseispresentinthedystrophin-glycoproteincomplex.

1	Althoughdefectsinthedystrophin-glycoproteincomplexareinvolvedinmanyformsofmusculardystrophy,someformsofmusculardystrophythatinvolveothermechanismshavebeenidentified.Specifically,adefectinsarcolemmarepair(attributedtoloss/mutationoftheproteindysferlin)appearstounderlieatleastoneformofmusculardystrophy(limb-girdle muscular dystrophy 2B, associatedwithmusclewastinginthepelvicregion).Defectsintheproteintitin(titinopathies)havebeenimplicatedinotherformsofmusculardystrophy(e.g.,limb-girdle muscular dystrophy 2J andtibial muscular dystrophy).Mutationsintheproteasecalpain 3 (resultinginlossofproteaseactivity)havealsobeenimplicatedinsometypesofmusculardystrophy(e.g.,limb-girdlemusculardystrophy2A),apparentlysecondarytoapoptosis. Organization of the thick filament is shown in

1	Fig. 12.6 . Myosin is a large protein (≈480 kDa) that consists of six different polypeptides with one pair of large heavy chains (≈200 kDa) and two pairs of light chains (≈20 kDa). The heavy chains are wound together in an α-helical configuration to form a long rod-like segment, and the N-terminal portions of each heavy chain form a large globular head. The head region extends away from the thick filament toward the actin thin filament and is the portion of the molecule that can bind to actin. Myosin is also able to hydrolyze ATP, and ATPase activity is located in the globular head as well. Two pairs of light chains are associated with the globular head. One of these pairs of light chains, termed essential light chains, is crucial for the ATPase activity of myosin. The other pair of light chains, called regulatory light chains, can be phosphorylated by Ca++/calmodulin-dependent myosin light chain protein kinase, which can influence the interaction of myosin with actin (see the section

1	chains, called regulatory light chains, can be phosphorylated by Ca++/calmodulin-dependent myosin light chain protein kinase, which can influence the interaction of myosin with actin (see the section “

1	Skeletal Muscle Types”). Thus myosin ATPase activity occurs in the globular head of myosin and requires the presence of light chains (namely, the “essential” light chains).

1	Myosin filaments form by a tail-to-tail association of myosin molecules, which results in a bipolar arrangement of the thick filament. The thick filament then extends on either side of the central bare zone by a head-to-tail association of myosin molecules, thus maintaining the filament’s bipolar organization centered on the M line. Such a bipolar arrangement is critical for drawing the Z lines together (i.e., shortening the length of the sarcomere) during contraction. The mechanisms controlling this highly organized structure of the myosin thick filament are not clear, although the cytoskeletal protein titin is thought to participate in the formation of a scaffold for organization and alignment of the thick filament in the sarcomere. Additional proteins found in the thick filaments (e.g., myomesin and C protein) may also participate in the bipolar organization or packing of the thick filament (or both). Control of Skeletal Muscle Activity

1	Control of Skeletal Muscle Activity Skeletal muscle is controlled by the central nervous system. Specifically, each skeletal muscle is innervated by an α motor neuron. The cell bodies of α motor neurons are located in the ventral horn of the spinal cord ( Fig. 12.7 ). The motor axons exit via the ventral roots and reach the muscle through mixed peripheral nerves. The motor nerves branch in the muscle, and each branch innervates a single muscle fiber. The specialized cholinergic synapse that forms the neuromuscular junction and the neuromuscular transmission process that generates an action potential in the muscle fiber are described in A motor unit consists of the motor nerve and all the muscle fibers innervated by the nerve. The motor unit is the functional contractile unit because all the muscle cells within a motor unit contract synchronously when the motor nerve fires. The size of motor units within a muscle varies, depending on the function of the muscle.

1	• Fig.

1	12.5. A, Organization of the dystrophin-glycoprotein complex in skeletal muscle. The dystrophinglycoprotein complex provides a structural link between the cytoskeleton of the muscle cell and the extracellular matrix, which appears to stabilize the sarcolemma and hence prevents contraction-induced injury (rupture). Duchenne’s muscular dystrophy is associated with loss of dystrophin. Numbers in dystrophin indicate hinge regions (e.g., H1, H2) and spectrin-like repeat domains (e.g., 4, 8, 12). ABD, actin-binding domain; C, carboxy terminus; CC, coiled-coil domain; DBD, dystroglycan-binding domain; N, amino terminus; nNOSµ, neuronal nitric oxide synthase µ; SBS, syntrophin-binding site; SSPN, sarcospan; Syn, syntrophin. Electron micrographs of a longitudinal view (B) and a cross-sectional view (C) show the distribution of dystrophin in skeletal muscle of a normal patient (CTRL). Another cross-sectional view (D) shows the loss of dystrophin from skeletal muscle in a patient with Duchenne’s

1	w (C) show the distribution of dystrophin in skeletal muscle of a normal patient (CTRL). Another cross-sectional view (D) shows the loss of dystrophin from skeletal muscle in a patient with Duchenne’s muscular dystrophy (DMD). (A, From Allen D, Whitehead N, Froehner S. Absence of dystrophin disrupts skeletal muscle signaling: roles of Ca2+, reactive oxygen species, and nitric oxide in the development of muscular dystrophy. Physiol

1	Rev. 2016;96:253-305. B, From Anastasi G, et al. Costameric proteins in human skeletal muscle during muscular inactivity. J Anat. 2008;213:284-295. C and D, From Beekman C, et al. A sensitive, reproducible and objective immunofluorescence analysis method of dystrophin in individual fibers in samples from patients with Duchenne muscular dystrophy. PLoS One. 2014;9[9]:e107494.)

1	Activation of motor units with a small number of fibers cardiac muscle, in contrast, is approximately 200 msec. facilitates fine motor control. Activation of varying numbers The short duration of the skeletal muscle action potential of motor units within a muscle is one way in which the allows very rapid contractions of the fiber and provides tension developed by a muscle can be controlled (see yet another mechanism by which the force of contrac“Recruitment” in the section “Modulation of the Force of tion can be increased. Increasing tension by repetitive ”). stimulation of the muscle is called tetany (see the section The neuromuscular junction formed by the α motor “

1	The neuromuscular junction formed by the α motor “ Modulation of the Force of Contraction ”). neuron is called an end plate (see for details). Acetylcholine released from the α motor neuron at the neuromuscular junction initiates an action potential in the muscle fiber that rapidly spreads along its length. When an action potential is transmitted along the sarco-The duration of the action potential in skeletal muscle is lemma of the muscle fiber and then down the T tubules, less than 5 msec. The duration of the action potential in Ca++ is released from the terminal cisternae SR into the

1	Globular heads (S-1) with light chains Proteolytic cleavage • Fig. 12.6. Organization of a Thick Filament. A thick filament is formed by the polymerization of myosin molecules in a tail-to-tail configuration extending from the center of the sarcomere (A). An individual myosin molecule has a tail region and a cross-bridge region. The cross-bridge region is composed of an arm and globular heads (B). The globular heads contain light chains that are important for the function of myosin ATPase activity. LMM, light meromyosin; S-1 and S-2, myosin subfragments 1 and 2. myoplasm. This release causes intracellular [Ca++] to rise, which in turn promotes actin-myosin interaction and contraction. The time course for the increase in intracellular [Ca++] in relation to the action potential and development of force is shown in

1	Fig. 12.8 . The action potential is extremely short-lived (≈5 msec). The elevation in intracellular [Ca++] begins slightly after the action potential and peaks at approximately 20 msec. This increase in intracellular [Ca++] initiates a contraction called a twitch. The mechanism underlying the elevation in intracellular [Ca++] involves an interaction between protein in the T tubule and the adjacent terminal cisternae of the SR. As previously described (see Fig. 12.3 ), the T tubule represents an invagination of the sarcolemma that extends into the muscle fiber and forms a close association with two terminal cisternae of the SR. The association of a T tubule with two opposing terminal cisternae is called a triad. Although there is a gap (≈15 nm in width) between the T tubule and the terminal cisternae, proteins bridge this gap. On the basis of their appearance on electron micrographs, these bridging proteins are called feet (

1	Fig. 12.9 ). These feet are the Ca++ release channels in the membrane of the terminal cisternae that are responsible for the elevation in intracellular [Ca++] in response to the action potential. Because this channel binds the drug ryanodine, it is commonly called the ryanodine receptor (RYR). RYR is a large protein (≈500 kDa) that exists as a homotetramer. Only a small portion of the RYR molecule is actually embedded in the SR membrane. Most of the RYR molecule appears to be in the myoplasm and spans the gap between the terminal cisternae and the T tubule ( Fig. 12.10

1	Fig. 12.10 At the T tubule membrane, the RYR is thought to interact with a protein called the dihydropyridine receptor (DHPR). DHPR is an L-type voltage-gated Ca++ channel with five subunits. One of these subunits binds the dihydropyridine class of channel-blocking drugs and appears to be crucial for the ability of the action potential in the T tubule to induce release of Ca++ from the SR. However, influx of Ca++ into the cell through the DHPR is not needed for the initiation of Ca++ release from the SR. In fact, skeletal muscle is able to contract in the absence of extracellular Ca++ or with a mutated DHPR that does not conduct Ca++ . Instead, release of Ca++ from the terminal cisternae of the SR is thought to result from a conformational change in the DHPR as the action potential passes down the T tubule, and this conformational change in the DHPR, by means of a protein-protein interaction, opens the RYR and releases Ca++ into the myoplasm.

1	Structural analysis, including the use of freeze-fracture techniques, provides evidence for a close physical association of DHPR and RYR (see Fig. 12.9 ). DHPR in the T tubule membrane appears to reside directly opposite the four corners of the underlying homotetrameric RYR channel in the SR membrane. Studies have shown that the SH3-cysteine rich domain 3 protein Stac3 is also critical for coupling of the DHPR to the RYR during excitation-contraction coupling in skeletal muscle SR. Stac3 is not present in cardiac muscle, which relies on Ca++ influx through the sarcolemma to initiate Ca++ release from the RYR instead of the direct coupling of the DHPR and the RYR in skeletal muscle. Other proteins that reside near the RYR include calsequestrin, triadin, and junctin (see

1	Fig. 12.10 ). Calsequestrin is a low-affinity Ca++-binding protein that is present in the lumen of the terminal cisternae. It allows Ca++ to be “stored” at high concentration and thereby establishes a favorable concentration gradient that facilitates the efflux of Ca++ from the SR into the myoplasm when the RYR opens. Triadin and junctin are in the terminal cisternae membrane and bind both RYR and calsequestrin; they could anchor calsequestrin near the RYR and thereby increase Ca++ buffering capacity at the site of Ca++ release. Histidine-rich calcium-binding protein is another low-affinity Ca++-binding protein in the SR lumen, although it is less abundant than calsequestrin. It appears to bind triadin in a Ca++-dependent manner, which raises the possibility that it has a role more important than serving simply as a Ca++ buffer. There is also evidence for the presence of store-operated Ca entry (SOCE) in skeletal muscle (e.g., via the Orai/Stim1 complex) during tetany. Inhibition of

1	than serving simply as a Ca++ buffer. There is also evidence for the presence of store-operated Ca entry (SOCE) in skeletal muscle (e.g., via the Orai/Stim1 complex) during tetany. Inhibition of Ca influx did not affect excitation-contraction coupling but did reduce maximal tetanic tension at high rates of electrical stimulation, which suggests that there may be some extrusion of intracellular Ca during tetany, which is compensated by Ca influx to maintain maximal tetanic tension.

1	•Fig. 12.7. Skeletal Muscle Is Voluntary Muscle Controlled by the Central Nervous System, With Efferent Signals (i.e., action potentials) Passing Through an α Motor Neuron to Muscle Fibers. Eachmotorneuronmayinnervatemanymusclefiberswithinamuscle,althougheachmusclefiberisinnervatedbyonlyonemotorneuron(A). Ascanningelectronmicrograph(B) showsinnervationofseveralmusclefibersbyasinglemotorneuron.(B, FromBloomW,FawcettDW:A Textbook of Physiology. 12thed.NewYork:Chapman&Hall;1994.) AvarietyofmutationalstudieshavebeenconductedtoascertaintheregionoftheDHPRthatiscriticalforopeningoftheRYR.Onepossiblesiteofinteraction(depictedin

1	AvarietyofmutationalstudieshavebeenconductedtoascertaintheregionoftheDHPRthatiscriticalforopeningoftheRYR.Onepossiblesiteofinteraction(depictedin Fig.12.10 )isthemyoplasmicloopbetweentransmembranedomainsIIandIIIintheα1subunitoftheDHPR.Thevoltage-sensingregionoftheDHPRinvolvedinintramembranouschargemovementisthoughttoresideintheS4transmembranesegmentsoftheα1subunit.GeneticmutationsintheRYRorDHPR,orinboth,havebeenassociatedwithpathologicaldisturbancesinmyoplasmic[Ca++].Suchdisturbancesincludemalignanthyperthermiaandcentralcoredisease,asdescribedlater.ThesemutationsaretypicallyobservedinthemyoplasmicportionoftheRYR,althoughmutationshavealsobeenobservedinamyoplasmicloopintheDHPR.ResultsofstudiesindicatethattheproteinStac3isalsoimportantforthecouplingoftheDHPRtotheRYRinskeletalmuscleandthatamutationinStac3thatispresentintherarecongenitaldisorderNativeAmericanmyopathyresultsinimpairedexcitation-contractioncouplinginskeletalmuscle.

1	a. Action potential potential Sarcolemma b. Myoplasmic [Ca++] c. Twitch force Calsequestrin Terminal cisternae of SRRYR Actin-myosin interaction DHPR T tubule Ca++ Ca++ SERCA ATP ADP + Pi 0 8040 120 160 200 •Fig. 12.8. A, StimulationofaskeletalmusclefiberinitiatesanactionpotentialinthemusclethattravelsdowntheTtubuleandinducesreleaseofCa++ fromtheterminalcisternaeofthesarcoplasmicreticulum(SR).Theriseinintracellular[Ca++]causesacontraction.AsCa++ ispumpedbackintotheSRbysarcoplasmicendoplasmicreticulumCa++-ATPase(SERCA),relaxationoccurs.DHPR,dihydropyridinereceptor;Pi,inorganicphosphate;RYR,ryanodinereceptor.B, Timecoursesoftheactionpotential,myoplasmicCa++ transient,andforceofthetwitchcontraction.

1	Terminal T tubule RYR DHPR cisternae of SR •Fig. 12.9. A, Electronmicrographofatriadillustratingthe“feet”(arrows) betweentheTtubuleandthesarcoplasmicreticulum(SR),whicharethoughttobetheryanodinereceptors(RYRs)intheSR.B, EachRYRintheSRisassociatedwithfourdihydropyridinereceptors(DHPRs)intheTtubule.(FromProtasiF,etal.RYR1andRYR3havedifferentrolesintheassemblyofcalciumreleaseunitsofskeletalmuscle.Biophys J. 2000;79:2494.)

1	Relaxation of skeletal muscle occurs as intracellular Ca++ is resequestered by the SR. Uptake of Ca++ into the SR is due to the action of a Ca++ pump (i.e., Ca++-ATPase). This pump is not unique to skeletal muscle; it is found in all cells in association with the endoplasmic reticulum. Accordingly, it is named SERCA, which stands for sarcoplasmic endoplasmic reticulum calcium ATPase. SERCA is the most abundant protein in the SR of skeletal muscle, and it is distributed throughout the longitudinal tubules and the terminal cisternae as well. It transports two molecules of Ca++ into its lumen for each molecule of ATP hydrolyzed. Thus the Ca++ transient seen during a twitch contraction (see

1	Thus the Ca++ transient seen during a twitch contraction (see Fig. 12.8 ) reflects release of Ca++ from the terminal cisternae via the RYR and reuptake primarily into the longitudinal portion of the SR by SERCA. The low-affinity Ca++-binding protein sarcalumenin is present throughout the longitudinal tubules of the SR and nonjunctional regions of the terminal cisternae and is thought to be involved in the transfer of Ca++ from sites of Ca++ uptake in the longitudinal tubules to sites of Ca++ release in the terminal cisternae. Results of studies suggest that sarcalumenin increases Ca++ uptake by SERCA, at least in part by buffering luminal Ca++ near the pump. The endogenous micropeptides phospholamban, sarcolipin, and myoregulin have been shown to regulate the activity of SERCA by decreasing the Ca sensitivity of Ca uptake (

1	Ca++ near the pump. The endogenous micropeptides phospholamban, sarcolipin, and myoregulin have been shown to regulate the activity of SERCA by decreasing the Ca sensitivity of Ca uptake ( Fig. 12.11 ). Protein kinase A–dependent phosphorylation of phospholamban in slow-twitch skeletal muscle has been reported to increase Ca transport in the SR, similar to the effect of phospholamban phosphorylation in the heart. Phospholamban and sarcolipin are present in slow-twitch muscle, whereas myoregulin is present in both fastand slow-twitch muscle. *During the transport of Ca++ , SERCA exchanges two Ca++ ions for two H+ ions (i.e., H+ is pumped out of the SR).

1	*During the transport of Ca++ , SERCA exchanges two Ca++ ions for two H+ ions (i.e., H+ is pumped out of the SR). •Fig. 12.10. Molecular Structure and Relationships Between the Dihydropyridine Receptor (DHPR) in the T Tubule Membrane and the Ryanodine Receptor (RYR) in the Sarcoplasmic Reticulum (SR) Membrane. TriadinisanassociatedSRproteinthatmayparticipateintheinteractionofRYRandDHPR.Calsequestrinisalow-affinityCa++-bindingproteinthathelpsaccumulateCa++ intheterminalcisternae.Seetextfordetails.COOH,carboxylicacid.(FromRossiAE,DirksenRT.Sarcoplasmicreticulum:thedynamiccalciumgovernorofmuscle.Muscle Nerve. 2006;33:715.) movement of the associated tropomyosin molecule toward

1	Actin-Myosin Interaction: the cleft of the actin filament. This movement of tropo myosin exposes myosin binding sites on the actin filament As noted, contraction of skeletal muscle requires an increase and allows a cross-bridge to form and thereby generate in intracellular [Ca++]. Moreover, the process of contraction tension (see section “ Cross-Bridge Cycling: Sarcomere is regulated by the thin filament. As shown in

1	Fig. 12.12 ”). Troponin C has four Ca++ binding sites. Two contractile force (i.e., tension) increases in a sigmoidal of these sites have high affinity for Ca++ but also bind manner as intracellular [Ca++] is elevated above 0.1 µM, Mg++ at rest. These sites seem to be involved in control-with half-maximal force occurring at less than 1 µM Ca++ . ling and enhancing the interaction between the troponin The mechanism by which Ca++ promotes this increase in I and troponin T subunits. The other two binding sites tension is as follows: Ca++ released from the SR binds to have lower affinity and bind Ca++ as its concentration rises troponin C. Once bound with Ca++ , troponin C facilitates after release from the SR. Binding of myosin to the actin • Fig.

1	the SR binds to have lower affinity and bind Ca++ as its concentration rises troponin C. Once bound with Ca++ , troponin C facilitates after release from the SR. Binding of myosin to the actin • Fig. 12.11. Identification of three endogenous micropeptides that inhibit sarcoplasmic endoplasmic reticulum Ca++-ATPase (SERCA; A), and their predicted organization in the SR membrane (B). MLN, myoregulin; PLN, phospholamban; SCL, sarcolamban; SLN, sarcolipin. (Modified from Anderson DM, et al. A micropeptide encoded by a putative long noncoding RNA regulates muscle performance. Cell. 2015;160:595-606.) 0 0.01 0.1 1 10 100 • Fig. 12.12. The contractile force of skeletal muscle increases in a Ca++-dependent manner as a result of binding of Ca++ to troponin C and the subsequent movement of tropomyosin away from myosin binding sites on the underlying actin molecules. See text for details. (Redrawn from Hartshorne DJ. In Lapedes DN. ed. Yearbook of Science and

1	of Science and Technology. New York: McGraw-Hill; 1976.) filaments appears to cause a further shift in tropomyosin. Although a given tropomyosin molecule extends over seven actin molecules, it is hypothesized that the strong binding of myosin to actin results in movement of an adjacent tropomyosin molecule, perhaps exposing myosin binding sites on as many as 14 actin molecules. This ability of one tropomyosin molecule to influence the movement of another may be a consequence of the close proximity of adjacent tropomyosin molecules.

1	Cross-Bridge Cycling: Sarcomere Shortening Once myosin and actin are bound, ATP-dependent conformational changes in the myosin molecule result in movement of the actin filaments toward the center of the sarcomere. Such movement shortens the length of the sarcomere and thereby contracts the muscle fiber. The mechanism by which myosin produces force and shortens the sarcomere is thought to involve four basic steps that are collectively termed the cross-bridge cycle (labeled a to d in Fig. 12.13 ). In the resting state, myosin is thought to have partially hydrolyzed ATP (state a). When Ca++ is released from the terminal cisternae of the SR, it binds to troponin C, which in turn promotes movement of tropomyosin on the actin filament in such a way that myosin-binding sites on actin are exposed. This then allows the “energized”

1	GeneticdiseasesthatcausedisturbancesinCa++ homeostasisinskeletalmuscleincludemalignant hyperthermia, central core disease, andBrody’s disease. Malignanthyperthermiaisanautosomaldominanttraitthathaslife-threateningconsequencesincertainsurgicalinstances.AnestheticssuchashalothaneoretherandthemusclerelaxantsuccinylcholinecanproduceuncontrolledreleaseofCa++ fromtheSR,therebyresultinginskeletalmusclerigidity,tachycardia,hyperventilation,andhyperthermia.Thisconditionislethalifnottreatedimmediately.Therearecurrentlyaseriesoftests(involvingcontractileresponsesofmusclebiopsyspecimens)toassesswhetherapatienthasmalignanthyperthermia.Theincidenceofthisconditionisapproximately1per15,000childrenand1per50,000adultstreatedwithanesthetics.MalignanthyperthermiaistheresultofadefectintheSRCa++ releasechannel(RYR),whichbecomesactivatedinthepresenceoftheaforementionedanesthetics,causesthereleaseofCa++

1	releasechannel(RYR),whichbecomesactivatedinthepresenceoftheaforementionedanesthetics,causesthereleaseofCa++ intothemyoplasm,andhenceprolongsmusclecontraction(rigidity).ThedefectintheRYRisnotrestrictedtoasinglelocus.Insomecases,malignanthyperthermiahasbeenlinkedtoadefectintheDHPRoftheTtubule.

1	Centralcorediseaseisarareautosomaldominanttraitthatresultsinmuscleweakness,lossofmitochondriainthecoreofskeletalmusclefibers,andsomedisintegrationofcontractilefilaments.Itisoftencloselyassociatedwithmalignanthyperthermia,andsopatientswithcentralcorediseasearetreatedasthoughtheyaresusceptibletomalignanthyperthermiainsurgicalsituations.ItishypothesizedthatcentralcoresdevoidofmitochondriarepresentareasofelevatedintracellularCa++ secondarytoamutationintheRYR.ThelossofmitochondriaisthoughttooccurwhentheytakeuptheelevatedCa++ ,whichleadstomitochondrialCa++ overload.

1	Brody’sdiseaseischaracterizedbypainlessmusclecrampingandimpairedmusclerelaxationduringexercise.Whileanaffectedpersonrunsupstairs,forexample,musclesmaystiffenandtemporarilycannotbeused.Thisrelaxation myosin head to bind to the underlying actin (state b). Myosin next undergoes a conformational change termed “ratchet action” that pulls the actin filament toward the center of the sarcomere (state c). Myosin releases adenosine diphosphate (ADP) and inorganic phosphate during the transition to state c. Binding of ATP to myosin decreases the affinity of myosin for actin, thereby resulting in the release of myosin from the actin filament (state d). Myosin then partially hydrolyzes the ATP, and part of the energy in the ATP is used to recock the head and return to the resting state.

1	If intracellular [Ca++] is still elevated, myosin undergoes another cross-bridge cycle and produces further contraction of the muscle. The ratchet action of the cross-bridge is capable of moving the thin filament approximately 10 nm. The cycle continues until the SERCA pumps Ca++ back into the SR. As [Ca++] falls, Ca++ dissociates from troponin C, and the troponin-tropomyosin complex moves and blocks the myosin binding sites on the actin filament. If the supply of ATP is exhausted, as occurs with death, the cycle stops abnormalityisseeninmusclesofthelegs,arms,andeyelid,andtheresponseisworsenedincoldweather.Brody’sdiseasecanbeeitherautosomalrecessiveorautosomaldominantandmayinvolvemutationsinuptothreegenes;however,itisrare(affecting1per10,000,000births).ItappearstoresultfromdecreasedactivityoftheSERCA1Ca++ pumpfoundinfast-twitchskeletalmuscle(seethesection

1	pumpfoundinfast-twitchskeletalmuscle(seethesection ”).ThedecreasedactivityofSERCA1hasbeenassociatedwithmutationsinthegenethatencodesSERCA1,althoughanotheraccessoryfactormaycontributetothedecreasedSRCa++ uptakeinthefast-twitchskeletalmuscleofindividualswithBrody’sdisease.

1	Myotoniacongenitaisalsoassociatedwithprolongedmusclecontractions(painlesscramping)aftervoluntarycontractions,asaresultofmutationsintheCLCN1 gene,whichencodesthechloridevoltage-gatedchannel1inskeletalmusclesarcolemmaandTtubules.Chlorideconductanceintheskeletalmuscleisimportantforrepolarizationandstabilizationofthemembranepotential,andsothereducedchlorideconductanceinskeletalmusclesofindividualswithmyotoniacongenitaresultsinhyperexcitabilityofthemusclefiber.Voluntarycontractionmaythereforebefollowedbyaseriesofactionpotentials(afterdepolarizations)inthemusclethatresultinprolongedcontractions(i.e.,cramping).Epinephrine(e.g.,duringstressfulsituations)oftenworsensthecondition,asshowninmyotonic(“fainting”)goats.Musclestiffnesscanberelievedbyrepeatedcontractions(i.e.,thewarm-upphenomenon),althoughthemechanismunderlyingthewarm-upphenomenonisnotknown.MutationsintheCLCN1

1	geneinmyotoniacongenitamaybetransmittedineitheranautosomalrecessivemanner(asinBecker’sdisease,onetypeofmyotoniacongenita)oranautosomaldominantmanner(asinThomsen’sdisease,theothertypeofmyotoniacongenita).Theprevalenceofmyotoniacongenitaisapproximately1per100,000worldwide;theincidenceishigher(≈1:10,000)innorthernScandinavia.

1	in state c with the formation of permanent actin-myosin complexes (i.e., the rigor state). In this state, the muscle is rigid, and the condition is termed rigor mortis. As already noted, formation of the thick filaments involves the association of myosin molecules in a tail-totail configuration to produce a bipolar orientation (see Fig. 12.6 ). Such a bipolar orientation allows myosin to pull the actin filaments toward the center of the sarcomere during the cross-bridge cycle. The myosin molecules are also oriented in a helical array in the thick filament in such a way that cross-bridges extend toward each of the six thin filaments surrounding the thick filament (see Fig.

1	Fig. 12.3 ). These myosin projections/cross-bridges can be seen on electron micrographs of skeletal muscle and appear to extend perpendicular from the thick filaments at rest. In the contracted state, the myosin cross-bridges slant toward the center of the sarcomere, which is consistent with the ratchet action of the myosin head. The cross-bridge cycling mechanism just described is called the sliding filament theory because the myosin Cycle stops here in relaxed living muscle (because of actions of regulatory mechanisms)

1	The cross-bridge cycling mechanism just described is called the sliding filament theory because the myosin Cycle stops here in relaxed living muscle (because of actions of regulatory mechanisms) Tension Time0 G. 40 S. 90 msec L.R. 7.5 0 0.5 1.0 •Fig. 12.13. Cross-Bridge Cycle. Intherelaxedstate(statea),ATPispartiallyhydrolyzed(M•ADP•Pi).InthepresenceofelevatedmyoplasmicCa++ (stateb),myosin(M)bindstoactin(A).HydrolysisofATPiscompleted(statec)andcausesaconformationalchangeinthemyosinmoleculethatpullstheactinfilamenttowardthecenterofthesarcomere.AnewATPmoleculebindstomyosinandcausesreleaseofthecross-bridge(stated).PartialhydrolysisofthenewlyboundATPrecocksthemyosinhead,whichisnowreadytobindagainandagain.Ifmyoplasmic[Ca++]isstillelevated,thecyclerepeats.Ifmyoplasmic[Ca++]islow,relaxationresults.

1	cross-bridge is pulling the actin thin filament toward the center of the sarcomere, which results in an apparent “sliding” of the thin filament past the thick filament. There is, however, uncertainty about how many myosin molecules contribute to the generation of force and whether both myosin heads in a given myosin molecule are involved. It has been calculated that there may be 600 myosin heads per thick filament, with a stoichiometry of 1 myosin head per 1.8 actin molecules. As a result of steric considerations, it is unlikely that all myosin heads can interact with actin, and calculations suggest that even during maximal force generation, only 20% to 40% of the myosin heads bind to actin.

1	The conversion of chemical energy (i.e., ATP) to mechanical energy by muscle is highly efficient. In isolated muscle preparations, maximum mechanical efficiency (≈65% efficiency) is obtained at a submaximal force of 30% maximal tension. In humans performing steady-state ergometer exercise, mechanical efficiencies range from 40% to 57%.

1	Skeletal muscle fibers can be classified into two main groups according to the speed of contraction: fast-twitch •Fig. 12.14. A, Musclesvaryintermsofthespeedofcontraction.G,gastrocnemiusmuscleoftheleg;LR,lateralrectusmuscleoftheeye;S,soleusmuscleoftheleg.B, ThespeedofshorteningiscorrelatedwithmyosinATPaseactivity.N-SOL,normalsoleusmuscle(slowtwitch);N-EDL,normalextensordigitorumlongusmuscle(fasttwitch);S-EDL,self-innervatedextensordigitorumlongusmuscle(EDLmotornervetransectedandresutured);S-SOL,self-innervatedsoleusmuscle(soleusmotornervetransectedandresutured);X-EDL,cross-innervatedextensordigitorumlongusmuscle(EDLinnervatedbysoleusmotornerve);X-SOL,cross-innervatedSOLmuscle(soleusinnervatedbyEDLmotornerve).(A, FromMontcastleV[ed].Medical Physiology. 12thed.St.Louis:Mosby;1974.B, FromBárányM,CloseRI.J Physiol. 1971;213:455.) and slow-twitch muscle fibers. As shown in

1	Fig. 12.14A , the lateral rectus of the eye contracts very quickly in response to an action potential, reaching peak tension within 8 msec, and then relaxes quickly, which results in a short duration of contraction. The soleus muscle of the leg, in contrast, requires 90 msec to reach peak tension in response to an action potential, and then it relaxes slowly. The gastrocnemius muscle requires an intermediate time to reach peak tension (40 msec) because of the presence of both fast-twitch and slow-twitch muscle fibers in this muscle. The difference in speed of contraction between fast-twitch and slow-twitch muscles is correlated with myosin ATPase activity (see Fig. 12.14B ), which in turn reflects the type of myosin present in the muscle fiber (Fig. 12.15 Table 12.1 ). Thus fast-twitch muscle fibers contain myosin isoforms that hydrolyze ATP quickly, whereas slow-twitch muscle fibers contain myosin isoforms that hydrolyze ATP

1	MHC expression: Type I MHC expression: MHC expression: Function: antigravity, IIa, IIx IIb, IIx weight bearing, and Function: sustained Function: burst •Fig. 12.15. Comparisonofthreebasicmotorunitphenotypesinskeletalmuscleofextremitiesandtrunk.MHC,myosinheavychain.(RedrawnfromBaldwinK,HaddadF,PandorfC,etal.Alterationsinmusclemassandcontractilephenotypeinresponsetounloadingmodels:roleoftranscriptional/pretranslationalmechanisms.Front Physiol. 2013;4:284.) *Human“fastglycolytic”skeletalmusclefiberstypicallyexpressthetypeIIxmyosinisoenzymeinsteadofthetypeIIbmyosinisoenzyme.Incontrast,rodentsexpressonetypeofslow-twitchmusclefiber(typeI)andthreetypesoffast-twitchmusclefibers(expressingtypeIIa,typeIIx,ortypeIIbmyosinisoenzymes);thetypeIIxmusclefibersexpressmetabolicpropertiesintermediatebetweenthoseoftypeIIaandtypeIIbmusclefibers.ThetypeIIxmyosinisoenzymemaybecoexpressedwithtypeIIamyosinisoenzymein“fastoxidative”musclefibers.Intext,thesimpledesignationtype II fiber

1	II fiber referstofastglycolyticmusclefibers.

1	slowly. These two types of myosin isoforms have the same basic structure described previously, with two heavy chains and two pairs of light chains, although they differ in amino acid composition. It is very difficult to convert a slow-twitch muscle fiber into a fast-twitch fiber, although it can be accomplished by cross-innervation, which involves surgically interconnecting two motor neurons. As shown in Fig. 12.14B , when the soleus muscle and extensor digitorum longus muscle underwent cross-innervation, so that contraction of the soleus muscle was controlled by the extensor digitorum motor neuron (and vice versa), the speed of the contraction and the myosin ATPase activity of the soleus muscle increased (labeled X-SOL in

1	Fig. 12.14 ), whereas the extensor digitorum longus exhibited a decrease in shortening velocity and myosin ATPase activity (labeled X-EDL). Thus the motor innervation of the muscle fiber plays an important role in determining which type of myosin isoform is expressed in the muscle fiber. Further study showed that the intracellular Ca concentration in the muscle (secondary to differences in the activity pattern of the motor neuron) was an important determinant of whether the muscle fiber expressed the slow myosin isoform or the fast myosin isoform (see the section The myosin isoforms expressed in skeletal muscle can be distinguished on the basis of myosin heavy chain composition (see Fig. 12.15 Table 12.1 ). Slow-twitch muscle fibers express type I myosin heavy chain, whereas fast-twitch skeletal muscle fibers could contain type IIa, type IIx, or type IIb myosin heavy chains. Some fast-twitch muscle fibers may contain a mixture of type II myosin isoforms.

1	Slow-twitch skeletal muscles are also characterized by a high oxidative capacity (see Table 12.1 ), which in combination with the low myosin ATPase activity contribute to the fatigue resistance of slow-twitch muscle fibers. The oxidative capacity of the fast-twitch muscle fiber ranges from relatively high (in muscle fibers expressing type IIa myosin heavy chain) to low (in muscle fibers expressing type IIb myosin heavy chains). The low oxidative capacity of fast type IIb muscle fibers, coupled with the high myosin ATPase activity, increases the susceptibility of these muscle fibers to fatigue. Humans, however, rarely express the type IIb myosin heavy chain. Type IIb myosins are expressed in small animals such as rabbit and rats. Motor units are generally composed of only one type of muscle fiber ( Table 12.2

1	Motor units are generally composed of only one type of muscle fiber ( Table 12.2 Fig. 12.15), unless the muscle fibers are undergoing a transition. Conditions that may trigger a change in the type of myosin expressed in a muscle fiber within a motor unit include chronic conditions such as microgravity (in space flight), denervation, and chronic unloading, which are associated with severe atrophy and promote the gradual transition from the expression of slow muscle myosin (type I) in the fiber to the expression of fast muscle myosin (types IIa and IIx). An important function of slow motor units is in the maintenance of posture (see

1	An important function of slow motor units is in the maintenance of posture (see Fig. 12.15 ). The low ATPase activity of myosin in slow motor units, coupled with their high oxidative capacity, facilitates the ability of these slow motor units to maintain posture at low energy cost and thus resist fatigue. The smaller diameter of slow muscle fibers, and the higher capillary density in slow muscle, also helps slow muscle resist fatigue. Fast muscle, in contrast, is recruited for activities that require faster movements, more force, or both (see Fig. 12.15 ). Weightlifting, for example, can require a lot of power for short duration. In order to meet the demands for more force, additional motor units are recruited. In comparison with slow motor units, the fast motor units typically contain more muscle fibers (see

1	Table 12.2 ). Fast muscle fibers also have a larger diameter than do slow muscle fibers. Thus recruitment of fast motor units can help meet the increased demands of burst activities such as weightlifting. The high myosin ATPase activity in fast muscle fibers and the increase in diffusion distance (resulting from the large diameter of the fast muscle fibers), however, increase the susceptibility of fast muscle fibers to fatigue. Additional differences between fast and slow muscles include the following: 1. The motor neuron in slow muscle is more easily excited than that in fast muscle, and so slow muscles are typically recruited first. As stated previously, the high oxidative capacity of slow muscle, coupled with the low myosin ATPase activity, helps reduce the susceptibility to fatigue in slow muscle. 2.

1	2. The neuromuscular junction of fast muscle differs from that in slow muscle in terms of acetylcholine vesicle content, the amount of acetylcholine released, the density of nicotinic acetylcholine receptors, the acetylcholine esterase activity, and Na channel density, all of which endow the fast muscle with a higher safety factor for initiation of an action potential. During repetitive stimulation, however, the safety factor in fast muscle drops quickly (faster than that seen in slow muscle). 3.

1	The SR is more highly developed in fast muscle than in slow muscle, with higher levels of RYR, SERCA, lumenal Ca, and a higher DHPR/RYR ratio, all of which promote the development of a larger, faster intracellular Ca transient in fast muscle, which is important for quick, forceful contraction. In addition to the differences between fast and slow fibers just noted, other muscle proteins are also expressed in a fiber type–specific manner. Such proteins include the three troponin subunits, tropomyosin, and C protein. The differential expression of troponin and tropomyosin isoforms influences the dependency of contraction on Ca++ . Slow fibers begin to develop tension at lower [Ca++] than fast fibers do. This difference in sensitivity to Ca++ is related in part to the fact that the troponin C isoform in slow fibers has only a single low-affinity Ca++-binding site, whereas the troponin C of fast fibers has two low-affinity binding sites. Changes in the dependence of contraction on Ca++ ,

1	C isoform in slow fibers has only a single low-affinity Ca++-binding site, whereas the troponin C of fast fibers has two low-affinity binding sites. Changes in the dependence of contraction on Ca++ , however, are not restricted to differences in the troponin C isoforms. Differences in troponin T and tropomyosin isoforms are also found. Thus regulation of the dependence of contraction on Ca++ is complex and involves contributions from multiple proteins on the thin filament. Phosphorylation of the regulatory light chain of myosin by Ca++/ calmodulin-dependent myosin light chain kinase, however, can increase Ca++ sensitivity of contraction, particularly in fast muscle fibers (partly because of the reported higher activity of myosin light chain kinase in fast muscle fibers).

1	•Fig. 12.16. Increasing the Frequency of Electrical Stimulation of Skeletal Muscle Results in an Increase in the Force of Contraction. ThisisattributabletoprolongationoftheintracellularCa++transientandistermedtetany. IncompletetetanyresultsfrominitiationofanotherintracellularCa++transientbeforethemusclehascompletelyrelaxed.Thusthereisasummationoftwitchforces.Seetextfordetails.Action potential Time 1 sec Myoplasmic Ca++ Incomplete tetanus Twitch force Tetanus force Modulation of the Force of Contraction

1	Modulation of the Force of Contraction A simple means of increasing the force of contraction of a muscle is to recruit more muscle fibers. Because all the muscle fibers within a motor unit are activated simultaneously, a muscle recruits more muscle fibers by recruiting more motor units. As already noted, muscle fibers can be classified as fast-twitch or slow-twitch. The type of fiber is determined by its innervation. Because all fibers in a motor unit are innervated by a single α motor neuron, all fibers within a motor unit are of the same type. Slow-twitch motor units tend to be small (100 to 500 muscle fibers) and are innervated by an α motor neuron that is easily excited (see

1	Table 12.2 ). Fast-twitch motor units, in contrast, tend to be large (containing 1000 to 2000 muscle fibers) and are innervated by α motor neurons that are more difficult to excite. Thus slow-twitch motor units tend to be recruited first. As more and more force is needed, fast-twitch motor units are recruited. The advantage of such a recruitment strategy is that the first muscle fibers recruited are those that have high resistance to fatigue. Moreover, the small size of slow-twitch motor units allows fine motor control at low levels of force. The process of increasing the force of contraction by recruiting additional motor units is termed spatial summation because forces from muscle fibers are being “summed” within a larger area of the muscle. This is in contrast to temporal summation, which is discussed later. Action potentials in skeletal muscles are quite uniform and lead to the release of a reproducible pulse of Ca++ from the SR (

1	Action potentials in skeletal muscles are quite uniform and lead to the release of a reproducible pulse of Ca++ from the SR ( Fig. 12.16 ). A single action potential releases sufficient Ca++ to cause a twitch contraction. However, the duration of this contraction is very short because Ca++ is very rapidly pumped back into the SR. If the muscle is stimulated a second time before it is fully relaxed, the force of contraction increases (see Fig. 12.16 , middle). Thus twitch forces are amplified as stimulus frequency increases. At a high level of stimulation, intracellular [Ca++] increases and is maintained throughout the period of stimulation (see Fig. 12.16 , right), and the amount of force developed greatly exceeds that observed during a twitch. The response is termed tetany. At intermediate stimulus frequency, intracellular [Ca++] returns to baseline just before the next stimulus. However, there is a gradual rise in force (see

1	Fig. 12.16 , middle). This phenomenon is termed incomplete tetany. In both cases, the increased frequency of stimulation is said to produce a fusion of twitches.

1	The low force generation during a twitch, in comparison with that during tetany, may be due to the presence of a series elastic component in the muscle. Specifically, when the muscle is stretched a small amount shortly after initiation of the action potential, the muscle generates a twitch force that approximates the maximal tetanic force. This result, coupled with the observation that the size of the intracellular Ca++ transient during a twitch contraction is comparable with that during tetany, suggests that enough Ca++ is released into the myoplasm during a twitch to allow the actin-myosin interactions to produce maximal tension. However, the duration of the intracellular Ca++ transient during a twitch is sufficiently short that the contractile elements may not have enough time to fully stretch the series elastic components in the fiber and muscle. As a result, the measured tension is submaximal.

1	An increase in the duration of the intracellular Ca++ transient, as occurs with tetany, provides the muscle with sufficient time to completely stretch the series elastic component and thereby results in expression of the full contractile force of the actin-myosin interactions (i.e., maximal tension). Partial stretching of the series elastic component (as might be expected during a single twitch), followed •Fig. 12.17. Slow-Twitch Muscles Exhibit Tetany at a Lower Stimulation Frequency Than Do Fast-Twitch Muscles. A, Fast-twitchmotorunitinthegastrocnemiusmuscle.B, Slow-twitchmotorunitinthegastrocnemiusmuscle.C, Slow-twitchmuscleunitinthesoleusmuscle.Themotorunitswerestimulatedatthefrequenciesindicatedontheleft.Thecalibrationbarfortension(ingrams)generatedduringconcentrationisindicatedbythevertical brackets underthecurves.Notethelargeforcegeneratedbythefast-twitchmotorunit(A).(FromMontcastleV[ed].Medical Physiology. 12thed.St.Louis:Mosby:1974.) by restimulation of the muscle before

1	brackets underthecurves.Notethelargeforcegeneratedbythefast-twitchmotorunit(A).(FromMontcastleV[ed].Medical Physiology. 12thed.St.Louis:Mosby:1974.) by restimulation of the muscle before complete relaxation, however, would be expected to yield an intermediate level of tension, similar to that seen with incomplete tetany. The location of the series elastic component in skeletal muscle is not known. One potential source is the myosin molecule itself. In addition, it is likely that there are other sources of the series elastic component, such as the connective tissue and titin.

1	The stimulus frequency needed to produce tetany depends on whether the motor unit consists of slow or fast fibers ( Fig. 12.17 ). Slow fibers can be tetanized at lower frequencies than can fast fibers. The ability of slow-twitch muscle to tetanize at lower stimulation frequencies reflects, at least in part, the longer duration of contraction seen in slow fibers. As also illustrated in Fig. 12.17 , fast fibers develop a larger maximal force than slow fibers do because fast fibers are larger in diameter than slow fibers and there are more fibers in fast motor units than in slow motor units. Modulation of Force by Reflex Arcs

1	Skeletal muscles contain sensory fibers (muscle spindles; also called intrafusal fibers) that run parallel to the skeletal muscle fibers. The muscle spindles assess the degree of stretch of the muscle, as well as the speed of contraction. In the stretch reflex, rapid stretching of the muscle (e.g., tapping the tendon) lengthens the spindles in the muscle and results in an increased frequency of action potentials in the afferent sensory neurons of the spindle. These afferent fibers in turn excite the α motor neurons in the spinal cord that innervate the stretched muscle. The result is that the reflex arc is a stretch-induced contraction of the muscle that does not require input from high centers in the brain. As the muscle shortens, efferent output is also sent to the spindle, which thereby takes the slack out of the spindle and ensures its ability to respond to stretch at all muscle lengths. By their action, muscle spindles provide feedback to the muscle in terms of its length and

1	takes the slack out of the spindle and ensures its ability to respond to stretch at all muscle lengths. By their action, muscle spindles provide feedback to the muscle in terms of its length and thus help maintain a joint at a given angle.

1	Golgi tendon organs are located in the tendons of muscles and provide feedback regarding contraction of the muscle. The main component of the tendon organ is an elongated fascicle of collagen bundles that is in series with the muscle fibers and can respond to contractions of individual muscle fibers. A given tendon organ may attach to several fast-twitch or slow-twitch muscle fibers (or both) and sends impulses through type Ib afferent nerve fibers in response to muscle contraction. The type Ib afferent impulses enter the spinal cord, which can promote inhibition of α motor neurons to the contracting (and synergistic) muscles while promoting excitation of α motor neurons to antagonistic muscles. The inhibitory actions are mediated through interneurons in the cord that release an inhibitory transmitter to the α motor neuron and create an inhibitory postsynaptic potential. The type Ib afferent impulses are also sent to higher centers of the brain (including the motor cortex and

1	transmitter to the α motor neuron and create an inhibitory postsynaptic potential. The type Ib afferent impulses are also sent to higher centers of the brain (including the motor cortex and cerebellum). It is hypothesized that feedback from the tendon organs in response to muscle contraction may smooth the progression of muscle contraction by limiting the recruitment of additional motor units. Of interest is that the response of the tendon organ is not linearly related to force; rather, it drops off at higher levels of force, which may facilitate the recruitment of motor units at higher levels of effort.

1	The skeletal system supports the body in an erect posture with the expenditure of relatively little energy. Nonetheless, even at rest, muscles normally exhibit some level of contractile activity. Isolated (i.e., denervated) unstimulated muscles are in a relaxed state and are said to be flaccid. However, relaxed muscles in the body are comparatively firm. This firmness, or tone, is caused by low levels of contractile activity in some of the motor units and is driven by reflex arcs from the muscle spindles. Interruption of the reflex arc by sectioning of the sensory afferent fibers abolishes this resting muscle tone. The tone in skeletal muscle is distinct from the “tone” in smooth muscle (see

1	Muscle cells convert chemical energy to mechanical energy. ATP is the energy source used for this conversion. The ATP pool in skeletal muscle is small and capable of supporting only a few contractions if not replenished. This pool, however, is continually replenished during contraction, as described later, so that even when the muscle fatigues, ATP stores are only modestly decreased. Muscle cells contain creatine phosphate, which is used to convert ADP to ATP and thus replenish the ATP store during muscle contraction. The creatine phosphate store represents the immediate high-energy source for replenishing the ATP supply in skeletal muscle, especially during intense exercise. The enzyme creatine phosphokinase catalyzes the reaction:

1	Although much of the creatine phosphokinase is present in the myoplasm, a small amount is located in the thick filament (near the M line). The creatine phosphokinase in the thick filament may participate in the rapid resynthesis of ATP near the myosin heads during muscle contraction. The phosphate store created, however, is only about five times the size of the ATP store and thus cannot support prolonged periods of contraction (less than a minute of maximal muscle activity). Skeletal muscle fatigue during intense exercise is associated with depletion of the creatine phosphate store, although as described subsequently, this does not necessarily imply that the fatigue is caused by depletion of the creatine phosphate store. Because the creatine phosphokinase–catalyzed reaction is reversible, the muscle cell replenishes the creatine phosphate pool during recovery from fatigue by using ATP synthesized through oxidative phosphorylation.

1	Muscle cells contain glycogen, which can be metabolized during muscle contraction to provide glucose for oxidative phosphorylation and glycolysis, both of which generate ATP to replenish the ATP store. Muscle cells can also take up glucose from blood, a process that is stimulated by insulin (see

1	Chapter 39). The cytosolic enzyme phosphory lase releases glucose 1-phosphate residues from glycogen, which are then metabolized by a combination of glycolysis (in the cytosol) and oxidative phosphorylation (in the mitochondria) to yield the equivalent of 37 mol of ATP per mole of glucose 1-phosphate. Blood glucose yields 36 mol of ATP per mole of glucose because 1 ATP is used to phosphorylate glucose at the start of glycolysis. These ATP yields, however, are dependent on an adequate oxygen supply. Under anaerobic conditions, in contrast, metabolism of glycogen and glucose yields only 3 and 2 mol of ATP per mole of glucose 1-phosphate and glucose, respectively (along with 2 mol of lactate). As discussed later, muscle fatigue during prolonged exercise is associated with depletion of glycogen stores in the muscle.

1	Fatty acids represent an important source of energy for muscle cells during prolonged exercise. Muscle cells contain fatty acids but can also take up fatty acids from blood. In addition, muscle cells can store triglycerides, which can be hydrolyzed when needed to produce fatty acids. The fatty acids are subjected to β oxidation within the mitochondria. For fatty acids to enter the mitochondria, however, they are converted to acylcarnitine in the cytosol and then transported into the mitochondria, where they are converted to acyl coenzyme A (CoA). Within the mitochondria, the acyl CoA is subjected to β oxidation and yields acetyl CoA, which then enters the citric acid cycle and ultimately produces ATP.

1	If the energy demands of exercise cannot be met by oxidative phosphorylation, an oxygen debt is incurred. After completion of exercise, respiration remains above the resting level in order to “repay” this oxygen debt. The extra oxygen consumption during this recovery phase is used to restore metabolite levels (such as creatine phosphate and ATP) and to metabolize the lactate generated by glycolysis. The increased cardiac and respiratory work during recovery also contributes to the increased oxygen consumption seen at this time and explains why more oxygen has to be “repaid” than was “borrowed.” Some oxygen debt occurs even with low levels of exercise because slow oxidative motor units consume considerable ATP, derived from creatine phosphate or glycolysis, before oxidative metabolism can increase ATP production to meet steady-state requirements. The oxygen debt is much greater with strenuous exercise, when fast glycolytic motor units are used (Fig. 12.18 ). The oxygen debt is

1	can increase ATP production to meet steady-state requirements. The oxygen debt is much greater with strenuous exercise, when fast glycolytic motor units are used (Fig. 12.18 ). The oxygen debt is approximately equal to the energy consumed during exercise minus that supplied by oxidative metabolism (i.e., the darkand light-colored areas in

1	Fig. 12.18 are approximately equal). As indicated earlier, the additional oxygen used during recovery from exercise represents the energy requirements for restoring normal cellular metabolite levels. The ability of muscle to meet energy needs is a major determinant of the duration of the exercise. However, fatigue is not the result of depletion of energy stores. Instead, metabolic byproducts seem to be important factors in the onset of fatigue. Fatigue may potentially occur at any of the points involved in muscle contraction, from the brain to the muscle cells, as well as in the cardiovascular and respiratory systems that maintain energy supplies (i.e., fatty acids and glucose) and oxygen delivery to the exercising muscle. Several factors have been implicated in muscle fatigue. During brief periods of tetany, the oxygen supply to the muscle is adequate as long as the circulation is intact. However, the force/stress generated during these brief

1	Rate of energy expenditure •Fig. 12.18. An Oxygen Debt Is Incurred by the Exercising of Muscle When the Rate of Energy Expenditure Exceeds the Rate of Energy Production by Oxidative Metabolism. Upper panel, Energyexpenditureduringstrenuousexercise.Lower panel, Energyexpenditureduringenduranceexercise.Seetextfordetails. tetanic periods decays rapidly to a level that can be maintained for long periods (

1	tetanic periods decays rapidly to a level that can be maintained for long periods ( Fig. 12.19 ). This decay represents the rapid and almost total failure of the fast motor units. The decline in force/stress is paralleled by depletion of glycogen and creatine phosphate stores and the accumulation of lactic acid. Of importance is that the decline in force/ stress occurs when the ATP pool is not greatly reduced, so that the muscle fibers do not go into rigor. In contrast, the slow motor units are able to meet the energy demands of fibers under this condition, and they do not exhibit significant fatigue, even after many hours. Evidently, some factor associated with energy metabolism can inhibit contraction (e.g., in the fast fibers), but this factor has not been clearly identified.

1	During intense exercise, accumulation of inorganic phosphate (Pi) and lactic acid in the myoplasm accounts for muscle fatigue. The accumulation of lactic acid, to levels as high as 15 to 26 mmol/L, decreases myoplasmic pH (from ≈7 to ≈6.2) and inhibits actin-myosin interactions. This decrease in pH reduces the sensitivity of the actin-myosin interaction to Ca++ by altering Ca++ binding to troponin C and by decreasing the maximum number of actin-myosin interactions. Pi has also been implicated as an important factor in the development of fatigue during intense exercise, inasmuch as phosphate concentrations can increase from approximately 2 mmol/L at rest to nearly 40 mmol/L in working muscle. Such an elevation in [Pi] can reduce tension by at least the following three different mechanisms: •Fig. 12.19.

1	can increase from approximately 2 mmol/L at rest to nearly 40 mmol/L in working muscle. Such an elevation in [Pi] can reduce tension by at least the following three different mechanisms: •Fig. 12.19. Aseriesofbrieftetanicstimulationsofskeletalmuscleresultinarapiddecreaseinforce(tetanicstress,exemplifiedbythe“Wholemuscle”lineinplot)thatisattributabletofatigueoffast-twitch(typeII)motorunitsinthemuscle.Undertheseconditions,however,slow-twitch(typeI)motorunitsareresistanttofatigue.3 2 1 0 10 2 3 4 5 10 20 30 Minutes Tetanus stress (kg/cm2 ) Type 1 motor units Whole muscle Type II motor units (1) inhibition of Ca++ release from the SR, (2) decrease in the sensitivity of contraction to Ca++ , and (3) alteration in actin-myosin binding. A number of other factors, including glycogen depletion from a specialized compartment, a localized increase in [ADP], extracellular elevation of [K+], and generation of oxygen free radicals, have also been implicated in various forms of exercise-induced

1	from a specialized compartment, a localized increase in [ADP], extracellular elevation of [K+], and generation of oxygen free radicals, have also been implicated in various forms of exercise-induced muscle fatigue. Finally, the central nervous system contributes to fatigue, especially in how fatigue is perceived by the individual.

1	Regardless of whether the muscle is fatigued as a consequence of high-intensity exercise or prolonged exercise, the myoplasmic ATP level does not decrease substantially. In view of the reliance of all cells on the availability of ATP to maintain viability, fatigue has been described as a protective mechanism to minimize the risk of muscle cell injury or death. Consequently, it is likely that skeletal muscle cells have developed redundant systems to ensure that ATP levels do not drop to dangerously low levels and hence risk the viability of the cell.

1	Most persons tire and cease exercise long before the motor unit fatigues. General physical fatigue may be defined as a homeostatic disturbance produced by work. The basis for the perceived discomfort (or even pain) probably involves many factors. These factors may include a decrease in plasma glucose levels and accumulation of metabolites. Motor system function in the central nervous system is not impaired. Highly motivated and trained athletes can withstand the discomfort of fatigue and may exercise to the point at which some motor unit fatigue occurs. Part of the enhanced performance observed after training involves motivational factors.

1	Skeletal muscle fibers differentiate before they are innervated, and some neuromuscular junctions are formed well after birth. Before innervation, the muscle fibers physiologically resemble slow (type I) cells. Acetylcholine receptors are distributed throughout the sarcolemma of these uninnervated cells and are supersensitive to that neurotransmitter. An end plate is formed when the first growing nerve terminal establishes contact with a muscle cell. The cell forms no further association with nerves, and receptors to acetylcholine become concentrated in the end plate membranes. Cells innervated by a small motor neuron form slow (type I) oxidative motor units. Fibers innervated by large motor nerves develop all the characteristics of fast (type II) motor units. Innervation produces major cellular changes, including synthesis of the fast and slow myosin isoforms, which replace embryonic or neonatal variants. Thus muscle fiber type is determined by the nerves that innervate the fiber.

1	An increase in muscle strength and size occurs during maturation. As the skeleton grows, the muscle cells lengthen. Lengthening is accomplished by the formation of additional sarcomeres at the ends of the muscle cells (

1	Fig. 12.20 ), a process that is reversible. For example, the length of a cell decreases when terminal sarcomeres are eliminated, which can occur when a limb is immobilized with the muscle in a shortened position or when improper setting of a fracture causes shortening of the limb segment. Changes in muscle length affect the velocity and extent of shortening but do not influence the amount of force that can be generated by the muscle. The gradual increase in strength and diameter of a muscle during growth is achieved mainly by hypertrophy. Doubling the myofibrillar diameter by adding more sarcomeres in parallel (hypertrophy, for example) may double the amount of force generated but has no effect on the maximal velocity of shortening. Resistance exercise can promote hypertrophy by activation of the Akt-mTOR signaling pathway and simultaneous inhibition of the forkhead box O protein (FoxO)–atrogene pathway, which results in an increase in net protein synthesis (

1	Fig. 12.21A ). The increase in intracellular Ca and decrease in ATP concentration can also stimulate mitochondrial biogenesis and expression of a slow muscle phenotype through activation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) signaling (see Fig. 12.21B Skeletal muscles have a limited ability to form new fibers (hyperplasia). These new fibers result from differentiation of satellite cells that are present in the tissues. However, major cellular destruction leads to replacement by scar tissue. Muscles not only must be used to maintain normal growth and development but must also experience loading.

1	Muscles not only must be used to maintain normal growth and development but must also experience loading. •Fig. 12.20. Effects of Growth on the Mechanical Output of a Muscle Cell. Typically,skeletalmusclecellgrowthinvolveseitherlengthening(addingmoresarcomerestotheendsofthemusclefibers)orincreasingmusclefiberdiameter(hypertrophyasaresultoftheadditionofmoremyofilaments/myofibrilsinparallelwithinthemusclefiber).Theformationofnewmusclefibersiscalledmuscle hyperplasia, anditisinfrequentinskeletalmuscle.

1	Muscles immobilized in a cast lose mass. In addition, space flight exposes astronauts to a microgravity environment that mechanically unloads their muscles. Such unloading leads to rapid loss of muscle mass (i.e., atrophy) and weakness. Disuse atrophy appears to involve both inhibition of protein synthesis and stimulation of protein degradation (with net activation of the FoxO-atrogene pathway). Other categories of skeletal muscle atrophy include sarcopenia (which is atrophy associated with the aging process) and cachexia (which is atrophy associated with an illness).

1	Muscles that frequently contract to support the body typically have a high number of slow (type I) oxidative motor units. These slow motor units atrophy more rapidly than the fast (type II) motor units during prolonged periods of unloading. This atrophy of slow motor units is associated with a decrease in maximal tetanic force but also an increase in maximal shortening velocity. The increase in velocity is correlated with expression of the fast myosin isoform in these fibers. An important aspect of space medicine is the design of exercise programs that minimize such phenotypic changes during prolonged space flight.

1	• Fig.

1	12.21. Basic molecular signaling pathways involved in contributing to net protein synthesis (A) and mitochondrial biogenesis (B) in skeletal muscle in response to exercise. AKT, a serine/threonine-specific protein kinase (protein kinase B); AMPK, 5′ adenosine monophosphate–activated protein kinase; ATP, adenosine triphosphate; CaM kinase, Ca++/calmodulin-dependent protein kinase; CREB, cAMP response element binding protein; ERRα, estrogen-related receptor-α; FoxO, forkhead box O class of transcription factors; FoxO-P, phosphorylated FoxO; Glut-4, glucose transporter type 4; HDAC5, histone deacetylase 5; IGF-1, insulin-like growth factor 1; MEF2, myocyte enhancer factor 2; mTOR, mammalian target of rapamycin; Nrf1, Nrf2, and Nrf2α, nuclear respiratory factors 1, 2, and 2α; PGC-1α, peroxisome proliferator-activated receptor γ coactivator 1α; PI3K, phosphatidylinositol-3-kinase; Tn I, troponin I. (A, From Sandri M, et al. PGC-1alpha protects skeletal muscle from atrophy by suppressing Fo

1	me proliferator-activated receptor γ coactivator 1α; PI3K, phosphatidylinositol-3-kinase; Tn I, troponin I. (A, From Sandri M, et al. PGC-1alpha protects skeletal muscle from atrophy by suppressing FoxO3 action and atrophy-specific gene transcription. PNAS. 2006;103:16260-16265. B, From Lin J, Handschin C, Spiegelman BM. Metabolic control through the PGC-1 family of transcription coactivators. Cell

1	Metab. 2005;1:361-370.)

1	Testosterone is a major factor responsible for the greater Denervation, Reinnervation, muscle mass in men because it has both myotrophic action ). A variety of synthetic molecules, called anabolic steroids, As already noted, innervation is crucial for the skeletal have been designed to enhance muscle growth while mini-muscle phenotype. If the motor nerve is cut, muscle fasmizing their androgenic action. These drugs are widely used ciculation occurs. Fasciculation is characterized by small, by bodybuilders and athletes in sports in which strength is irregular contractions caused by release of acetylcholine from important. The doses are typically 10-to 50-fold greater the terminals of the degenerating distal portion of the axon. than might be prescribed therapeutically for individuals Several days after denervation, muscle fibrillation begins. with impaired hormone production. Unfortunately, none Fibrillation is characterized by spontaneous, repetitive of these compounds lack

1	Several days after denervation, muscle fibrillation begins. with impaired hormone production. Unfortunately, none Fibrillation is characterized by spontaneous, repetitive of these compounds lack androgenic effects. Hence, at the contractions. At this time, the cholinergic receptors have doses used, they induce serious hormone disturbances, spread out over the entire cell membrane, in effect reverting including depression of testosterone production. A major to their preinnervation embryonic arrangement. The muscle issue is whether these drugs do in fact increase muscle and fibrillations reflect supersensitivity to acetylcholine. Affected athletic performance in individuals with normal circulating muscles also atrophy, with a decrease in the size of the levels of testosterone. Although they have been used since muscle and its cells. Atrophy is progressive in humans, with the 1950s, the scientific facts remain uncertain, and most degeneration of some cells 3 or 4 months after

1	they have been used since muscle and its cells. Atrophy is progressive in humans, with the 1950s, the scientific facts remain uncertain, and most degeneration of some cells 3 or 4 months after denervation. experimental studies in animals have not demonstrated Most of the muscle fibers are replaced by fat and connective any significant effects on muscle development. Reports of tissue after 1 to 2 years. These changes can be reversed if studies in humans remain controversial. Proponents claim reinnervation occurs within a few months. Reinnervation increases in strength that provide advantages in world-class is normally achieved by growth of the peripheral stump of performance. Critics argue that these increases are largely motor nerve axons along the old nerve sheath. placebo effects associated with expectations and motivational Reinnervation of formerly fast (type II) fibers by a small factors. The public debate on abuse of anabolic steroids has motor axon causes that cell to

1	associated with expectations and motivational Reinnervation of formerly fast (type II) fibers by a small factors. The public debate on abuse of anabolic steroids has motor axon causes that cell to redifferentiate into a slow led to their designation as controlled substances, along with (type I) fiber, and vice versa. This suggests that large and opiates, amphetamines, and barbiturates. small motor nerves differ qualitatively and that the nerves have a specific “trophic” effect on the muscle fibers. This “trophic” effect reflects the rate of fiber stimulation. For example, stimulation via electrodes implanted in the muscle can lessen denervation atrophy. More strikingly, chronic low-frequency stimulation of fast motor units causes these units to be converted to slow units. Some conversion toward a typical fast-fiber phenotype can occur when the frequency of contraction in slow units is greatly decreased by reducing the excitatory input. Excitatory input can be reduced by sectioning the

1	a typical fast-fiber phenotype can occur when the frequency of contraction in slow units is greatly decreased by reducing the excitatory input. Excitatory input can be reduced by sectioning the appropriate spinal or dorsal root or by severing the tendon, which functionally inactivates peripheral mechanoreceptors.

1	The frequency of contraction determines fiber development and phenotype through changes in gene expression and protein synthesis. Fibers that undergo frequent contractile activity form many mitochondria and synthesize the slow isoform of myosin. Fibers innervated by large, less excitable axons contract infrequently. Such relatively inactive fibers typically form few mitochondria, have large concentrations of glycolytic enzymes and synthesize the fast isoform of myosin.

1	Fig.12.22A ).Specifically,itappearsthatstimulationofadultfast-twitchmusclecellsatafrequencyconsistentwithslow-twitchmusclecellscanactivatetheCa++-dependentphosphatasecalcineurin,whichinturncandephosphorylateNFATandresultintranslocationofNFATfromthemyoplasmtothenucleus,followedbythetranscriptionofslow-twitchmusclegenes(andinhibitionoffast-twitchmusclegenes).Inaccordancewiththismechanism,expressionofconstitutivelyactiveNFATinfast-twitchmusclepromotestheexpressionofslow-twitchmyosinwhileinhibitingtheexpressionoffast-twitchmyosin.Thetranscriptionfactormyocyte enhancing factor 2 (MEF2) hasalsobeenimplicatedinthistransitionfromfast-twitchtoslow-twitchmuscle(see Fig.12.22B ).ActivationofMEF2isthoughttoresultfromCa++/calmodulin-dependentphosphorylationofaninhibitorofMEF2:namely,histonedeacetylase(HDAC).

1	Intracellular [Ca++] appears to play an important role in expression of the slow myosin isoform. Slow-twitch muscle fibers have a higher resting level of intracellular Ca++ than do fast-twitch muscle fibers. In addition, chronic electrical stimulation of fast-twitch muscle is accompanied by a 2.5fold increase in resting myoplasmic [Ca++] that precedes the increased expression of slow-twitch myosin and decreased expression of fast-twitch myosin. Similarly, chronic elevation of intracellular Ca++ (approximately fivefold) in muscle cells expressing fast-twitch myosin induces a change in gene expression from the fast muscle myosin isoform to the slow myosin isoform within 8 days. An increase in citrate synthetase activity (an indicator of oxidative capacity) and a decrease in lactate dehydrogenase activity (an indicator of glycolytic capacity) accompany this Ca++-dependent

1	Muscle: action potential Surface membrane •Fig. 12.22. Molecular Signaling Pathways Contributing to the Transition From Fast-Twitch Muscle to Slow-Twitch Muscle. Chronicelectricalstimulationofafast-twitchmuscleinapatternconsistentwithaslow-twitchmuscleresultsindevelopmentoftheslow-twitchmusclephenotypebecauseofdephosphorylationofthetranscriptionfactornuclearfactorfromactivatedTcells(NFAT)bytheCa++/calmodulin-dependentproteinphosphatasecalcineurin(CaN);thisinturnresultsinnucleartranslocationofNFATandexpressionofslow-twitchmusclefibergenes(A). Activationofthetranscriptionfactormyocyteenhancerfactor2(MEF2)alsoappearstocontributetothisfibertypetransition(B), inwhichactivationofMEF2involvesCa++/calmodulin-dependentphosphorylationofaninhibitor,histonedeacetylase(HDAC).CaMK,Ca++/calmodulin-dependentproteinkinase;ECC,excitation-contractioncoupling;P,phosphorylationofHDAC.(FromLiuY,etal:.Signalingpathwaysinactivity-dependentfibertypeplasticityinadultskeletalmuscle.J Muscle Res Cell Motil.

1	Muscle Res Cell Motil. 2005;26:13-21.) transition from fast-twitch to slow-twitch myosin. These Ca++-dependent changes are reversible by a reduction of intracellular [Ca++].

1	Response to Exercise Exercise physiologists identify three categories of training regimens and responses: learning, endurance, and strength training ( Table 12.3). Typically, most athletic endeavors involve elements of all three. The learning aspect of training involves motivational factors, as well as neuromuscular coordination. This aspect of training does not involve adaptive changes in the muscle fibers per se. However, motor skills can persist for years without regular training, unlike the responses of muscle cells to exercise.

1	All healthy persons can maintain some level of continuous muscular activity that is supported by oxidative metabolism. This level can be greatly increased by a regular exercise regimen that is sufficient to induce adaptive responses. The adaptive response of skeletal muscle fibers to endurance exercise is mainly the result of an increase in the oxidative metabolic capacity of the motor units involved. This demand places an increased load on the cardiovascular and respiratory systems and increases the capacity of the heart and respiratory muscles. The latter effects are responsible for the principal health benefits associated with endurance exercise.

1	Muscle strength can be increased by regular massive efforts that involve most motor units. Such efforts recruit fast glycolytic motor units, as well as slow oxidative motor units. During these efforts, blood supply to the working muscles may be interrupted as tissue pressures rise above intravascular pressure. The reduced blood flow limits the duration of the contraction. Regular maximal-strength exercise, such as weightlifting, induces the synthesis of more myofibrils and hence hypertrophy of the active muscle cells. The increased stress also induces the growth of tendons and bones. Endurance exercise does not cause fast motor units to become slow, nor does maximal muscular effort produce a shift from slow to fast motor units. Thus any practical exercise regimen, when superimposed on normal daily activities, probably does not alter muscle fiber phenotype.

1	Activities such as hiking or, in particular, downhill running, in which contracting muscles are stretched and lengthened too vigorously, are followed by more pain and stiffness than after comparable exercise that does not involve vigorous muscle stretching and lengthening (e.g., cycling). The resultant dull, aching pain develops slowly and reaches its peak within 24 to 48 hours. The pain is associated with reduced range of motion, stiffness, and weakness of the affected muscles. The prime factors that cause the pain are swelling and inflammation from injury to muscle cells, most commonly near the myotendinous junction. Type II motor units are affected more than type I motor units because the maximal force is highest in large cells, in which the loads imposed are approximately 60% greater than the maximal force that the cells can develop. Recovery is slow and depends on regeneration of the injured sarcomeres. Biophysical Properties of Skeletal Muscle

1	Biophysical Properties of Skeletal Muscle The molecular mechanisms of muscle contraction described earlier underlie and are responsible for the biophysical properties of muscle. Historically, these biophysical properties were well described before elucidation of the molecular mechanisms of contraction. They remain important ways of describing muscle function. When muscles contract, they generate force (often measured as tension or stress) and decrease in length. In examination of the biophysical properties of muscle, one of these parameters is usually held constant, and the other is measured after an experimental maneuver. Accordingly, an isometric contraction is one in which muscle length is held constant, and the force generated during the contraction is then measured. An isotonic contraction is one in which the force (or tone) is held constant, and the change in length of the muscle is then measured.

1	When a muscle at rest is stretched, it resists stretch by a force that increases slowly at first and then more rapidly as the extent of stretch increases (

1	Fig. 12.23 ). This purely passive property is due to the elasticity of the muscle tissue. If the muscle is stimulated to contract at these various lengths, a different relationship is obtained. Specifically, contractile force increases as muscle length is increased up to a point (designated LO to indicate optimal length). As the muscle is stretched beyond LO, contractile force decreases. This length-tension curve is consistent with the sliding filament theory, described previously. At a very 0 1 201 Length (fraction of optimum) Sarcomere length (µm) •Fig. 12.23. Length-Tension Relationship in Skeletal Muscle. A, Experimentalsetupinwhichmaximalisometrictetanictensionismeasuredatvariousmusclelengths.B, Howactivetensionwascalculatedatvariousmusclelengths(i.e.,bysubtractingpassivetensionfromtotaltensionateachmusclelength).C, Plotofactivetensionasafunctionofmusclelength,withthepredictedoverlapofthickandthinfilamentsatselectedpoints.

1	•Fig. 12.24. Force-Velocity Relationship of Skeletal Muscle. Theexperimentalsetupisshownontheright.Theinitialmusclelengthwaskeptconstant,buttheamountofweightthatthemusclehadtoliftduringtetanicstimulationvaried.Whilethesevariousamountsofweightwerelifted,muscle-shorteningvelocitywasmeasured.Seetextfordetails.F,force;V,velocity. long sarcomere length (3.7 µm), actin filaments no longer overlap with myosin filaments, and so there is no contraction. As muscle length is decreased toward LO, the amount of overlap increases, and contractile force progressively increases. As sarcomere length decreases below 2 µm, the thin filaments collide in the middle of the sarcomere, the actin-myosin interaction is disturbed, and hence contractile force decreases. For construction of the length-tension curves, muscles were maintained at a given length, and then contractile force was measured (i.e., isometric contraction).

1	Thus the length-tension relationship supports the sliding filament theory of muscle contraction. The velocity at which a muscle shortens is strongly dependent on the amount of force that the muscle must develop (

1	Fig. 12.24 ). In the absence of any load, the shortening velocity of the muscle is maximal (denoted as V0). V0 corresponds to the maximal cycling rate of the cross-bridges (i.e., it is proportional to the maximal rate of energy turnover [ATPase activity] by myosin). Thus V0 for fast-twitch muscle is higher than that for slow-twitch muscle. Increasing the load decreases the velocity of muscle shortening until, at maximal load, the muscle cannot lift the load and hence cannot shorten (zero velocity). Further increases in load result in stretching the muscle (negative velocity). The maximal isometric tension (i.e., force at which shortening velocity is zero) is proportional to the number of active cross-bridges between actin and myosin, and it is usually greater for fast-twitch motor units (because of the larger diameter of fast-twitch muscle fibers and greater number of muscle fibers in a typical fast-twitch motor unit). In

1	Fig. 12.24 , the power-stress curve reflects the rate of work done at each load and shows that the maximal rate of work was done at a submaximal load (namely, when the force of contraction was approximately 30% of the maximal tetanic tension). To calculate the latter curve, the x-and y-coordinates were simply multiplied, and then the product was plotted as a function of the x-coordinate. 1.

1	1. Skeletal muscle is composed of numerous muscle cells (muscle fibers) that are typically 10 to 80 µm in diameter and up to 25 cm in length. The appearance of striations in skeletal muscle is due to the highly organized arrangement of thick and thin filaments in the myofibrils of skeletal muscle fibers. The sarcomere is a contractile unit in skeletal muscle. Each sarcomere is approximately 2 µm in length at rest and is bounded by two Z lines. Sarcomeres are arranged in series along the length of the myofibril. Thin filaments, containing actin, extend from the Z line toward the center of the sarcomere. Thick filaments, containing myosin, are positioned in the center of the sarcomere and overlap the actin thin filaments. Muscle contraction results from the Ca++-dependent interaction of myosin and actin, in which myosin pulls the thin filaments toward the center of the sarcomere. 2.

1	2. Contraction of skeletal muscle is under control of the central nervous system (i.e., voluntary). Motor centers in the brain control the activity of α motor neurons in the ventral horns of the spinal cord. These α motor neurons, in turn, synapse on skeletal muscle fibers. Whereas each skeletal muscle fiber is innervated by only one motor neuron, a motor neuron innervates several muscle fibers within the muscle. A motor unit refers to all the muscle fibers innervated by a single motor neuron. 3.

1	3. The motor neuron initiates contraction of skeletal muscle by producing an action potential in the muscle fiber. As the action potential passes down the T tubules of the muscle fiber, dihydropyridine receptors (DHPRs) in the T tubules undergo conformational changes that result in the opening of neighboring SR Ca++ channels called ryanodine receptors (RYRs), which then release Ca++ to the myoplasm from the SR. The increase in myoplasmic Ca++ promotes muscle contraction by exposing myosin-binding sites on the actin thin filaments (a process that involves binding of Ca++ to troponin C, followed by movement of tropomyosin toward the groove in the thin filament). Myosin cross-bridges then appear to undergo a ratchet action, with the thin filaments pulled toward the center of the sarcomere and contracting the skeletal muscle fiber. Relaxation of the muscle follows as myoplasmic Ca++ is resequestered by Ca++-ATPase (SERCA) in the SR. 4.

1	4. The force of contraction can be increased by the activation of more motor neurons (i.e., recruiting of more muscle fibers) or by an increase in the frequency of action potentials in the muscle fiber, which produces tetany. The increase in force during tetanic contractions is due to prolonged elevation of intracellular [Ca++]. 5. The two basic types of skeletal muscle fibers are distinguished on the basis of their speed of contraction (i.e., fast-twitch versus slow-twitch). The difference in speed of contraction is attributed to the expression of different myosin isoforms that differ in myosin ATPase activity. In addition to the difference in myosin ATPase activity, fast-and slow-twitch muscles also differ in metabolic activity, fiber diameter, motor unit size, sensitivity to tetany, and recruitment pattern. 6.

1	6. Typically, slow-twitch muscles are recruited before fast-twitch muscle fibers because of the greater excitability of motor neurons innervating slow-twitch muscles. The high oxidative capacity of slow-twitch muscle fibers supports sustained contractile activity. Fast-twitch muscle fibers, in contrast, tend to be large and typically have low oxidative capacity and high glycolytic capacity. The fast-twitch motor units are thus best suited for short periods of activity when high levels of force are required. 7.

1	7. Fast-twitch muscle fibers can be converted to slow-twitch muscle fibers (and vice versa), depending on the stimulation pattern. Chronic electrical stimulation of a fast-twitch muscle results in the expression of slow-twitch myosin and decreased expression of fast-twitch myosin, along with an increase in oxidative capacity. The mechanism or mechanisms underlying this change in gene expression are unknown, but the change appears to be secondary to an elevation in resting intracellular [Ca++]. The Ca++-dependent phosphatase calcineurin and the transcription factor NFAT have been implicated in this transition from the fast-twitch to the slow-twitch phenotype. Ca++/calmodulin-dependent kinase and the transcription factor MEF2 may also participate in the phenotype transition. 8.

1	8. Skeletal muscle fibers atrophy after denervation. Muscle fibers depend on the activity of their motor nerves for maintenance of the differentiated phenotype. Reinnervation by axon growth along the original nerve sheath can reverse these changes. Skeletal muscle has a limited capacity to replace cells lost as a result of trauma or disease. Inhibition of the PI3K/Akt signaling pathways and activation of the FoxO pathway appears to contribute to the decreased rate of protein synthesis and the increased rate of protein degradation (respectively) observed during disuse atrophy. The increased protein degradation during atrophy is attributed to increases in both protease activity (e.g., activation of caspase 3) and ubiquitination (through elevated levels of ubiquitin ligases). 9.

1	9. Skeletal muscle exhibits considerable phenotypic plasticity. Normal growth is associated with cellular hypertrophy, caused by the addition of more myofibrils and more sarcomeres at the ends of the cell to match skeletal growth. Strength training induces cellular hypertrophy, whereas endurance training increases the Allen DG, Whitehead NP, Froehner SC. Absence of dystrophin disrupts skeletal muscle signaling: roles of Ca2+ , reactive oxygen species, and nitric oxide in the development of muscular dystrophy. Physiol Rev. 2016;96(1):253-305. Anderson DM, Anderson KM, Chang CL, et al. A micropeptide encoded by a putative long noncoding RNA regulates muscle performance. Cell. 2015;160(4):595-606. Baldwin KM, Haddad F, Pandorf CE, Roy RR, Edgerton VR. Alterations in muscle mass and contractile phenotype in response to unloading models: role of transcriptional/pretranslational mechanisms. Front Physiol. 2013;4:284.

1	Burr AR, Molkentin JD. Genetic evidence in the mouse solidifies the calcium hypothesis of myofiber death in muscular dystrophy. Cell Death Differ. 2015;22:1402-1412. oxidative capacity of all involved motor units. Training regimens cannot alter fiber type or the expression of myosin isoforms. 10. Muscle fatigue during exercise is not due to depletion of ATP. The mechanism or mechanisms underlying exercise-induced fatigue are not known, although the accumulation of various metabolic products (lactate, Pi, ADP) has been implicated. In view of the importance of preventing depletion of myoplasmic ATP, which would affect the viability of the cell, it is likely that multiple mechanisms may have been developed to induce fatigue and hence lower the rate of ATP hydrolysis before the individual risks injury or death of the skeletal muscle cell. 11.

1	11. When the energy demands of an exercising muscle cannot be met by oxidative metabolism, an oxygen debt is incurred. Increased breathing during the recovery period after exercise reflects this oxygen debt. The greater the reliance on anaerobic metabolism to meet the energy requirements of muscle contraction, the greater the oxygen debt. Gordon AM, Homsher E, Regnier M. Regulation of contraction in striated muscle. Physiol Rev. 2000;80(2):853-924. Imbrici P, Altamura C, Pessia M, Mantegazza R, Desaphy JF, Camerino DC. ClC-1 chloride channels: state-of-the-art research and future challenges. Front Cell Neurosci. 2015;9:156. Joyner MJ, Casey DP. Regulation of increased blood flow (hyperemia) to muscles during exercise: a hierarchy of competing physiological needs. Physiol Rev. 2015;95(2):549-601.

1	Joyner MJ, Casey DP. Regulation of increased blood flow (hyperemia) to muscles during exercise: a hierarchy of competing physiological needs. Physiol Rev. 2015;95(2):549-601. Nelson CR, Fitts RH. Effects of low cell pH and elevated inorganic phosphate on the pCa-force relationship in single muscle fibers at near-physiological temperatures. Am J Physiol Cell Physiol. 2014;306(7):C670-C678. Sandri M. Protein breakdown in cancer cachexia. Semin Cell Dev Biol. 2016;54:11-19. Schiaffino S, Reggiani C. Fiber types in mammalian skeletal muscles. Physiol Rev. 2011;91(4):1447-1531. Upon completion of this chapter, the student should be able to answer the following questions: 1. Describe the organization of cardiac muscle and how it meets the demands of the organ. 2. Describe the molecular mechanisms involved in excitation-contraction coupling in cardiac muscle and its suitability for this organ. 3.

1	2. Describe the molecular mechanisms involved in excitation-contraction coupling in cardiac muscle and its suitability for this organ. 3. Describe the molecular mechanisms that lead to an increase in the force of contraction of the heart. 4. Discuss the length-tension relationship and the force-velocity curve for cardiac muscle, including the molecular basis for both curves. If the student has already completed on skeletal muscle, the student will be able to compare cardiac and skeletal muscle for each of the learning objectives just listed.

1	If the student has already completed on skeletal muscle, the student will be able to compare cardiac and skeletal muscle for each of the learning objectives just listed. he function of the heart is to pump blood through the circulatory system, and this is accomplished by the highly organized contraction of cardiac muscle cells. Specifically, the cardiac muscle cells are connected together to form an electrical syncytium, with tight electrical and mechanical connections between adjacent cardiac muscle cells. An action potential initiated in a specialized region of the heart (e.g., the sinoatrial node) is therefore able to pass quickly throughout the heart to facilitate synchronized contraction of the cardiac muscle cells, which is important for the pumping action of the heart. Likewise, refilling of the heart requires synchronized relaxation of the heart; abnormal relaxation often results in pathological conditions.

1	This chapter begins with a description of the organization of cardiac muscle cells within the heart, including discussion of the tight electrical and mechanical connections. The mechanisms that underlie contraction, relaxation, and regulation of the force of contraction of cardiac muscle cells are also addressed. Although cardiac muscle and skeletal muscle are both striated muscles, they are significantly different in terms of organization, electrical and mechanical coupling, excitation-contraction coupling, and mechanisms to regulate the force of contraction. These differences are also highlighted. Basic Organization of Cardiac Muscle Cells Cardiac muscle cells are much smaller than skeletal muscle cells. Typically, cardiac muscle cells measure 10 µm in diameter and approximately 100 µm in length. As shown in

1	Cardiac muscle cells are much smaller than skeletal muscle cells. Typically, cardiac muscle cells measure 10 µm in diameter and approximately 100 µm in length. As shown in Fig. 13.1A , cardiac cells are connected to each other through intercalated disks, which include a combination of mechanical junctions and electrical connections. The mechanical connections, which keep the cells from pulling apart when contracting, include the fascia adherens and desmosomes. Gap junctions between cardiac muscle cells, on the other hand, provide electrical connections between cells to allow propagation of the action potential throughout the heart. Thus the arrangement of cardiac muscle cells within the heart is said to form an electrical and mechanical syncytium that allows a single action potential (generated within the sinoatrial node) to pass throughout the heart so that the heart can contract in a synchronous, wave-like manner. Blood vessels course through the myocardium.

1	The basic organization of thick and thin filaments in cardiac muscle cells is comparable with that in skeletal muscle (see ). Electron microscopy reveals repeating light and dark bands that represent I bands and A bands, respectively (see Fig. 13.1B Fig. 12.3 ). Thus cardiac muscle is classified as a striated muscle. The Z line transects the I band and represents the point of attachment of the thin filaments. The region between two adjacent Z lines represents the sarcomere, which is the contractile unit of the muscle cell. The thin filaments are composed of actin, tropomyosin, and troponin and extend into the A band. The A band is composed of thick filaments, along with some overlap of thin filaments. The thick filaments are composed of myosin and extend from the center of the sarcomere toward the Z lines.

1	Myosin filaments are formed by a tail-to-tail association of myosin molecules in the center of the sarcomere, followed by a head-to-tail association as the thick filament extends toward the Z lines. Thus the myosin filament is polarized and poised for pulling the actin filaments toward the center of the sarcomere. A cross-section view of the sarcomere near the end of the A band shows that each thick filament is surrounded by six thin filaments, and each thin filament receives cross-bridge attachments from three thick filaments. This complex array of thick and thin filaments is characteristic of both cardiac and skeletal muscle and helps • Fig.

1	each thin filament receives cross-bridge attachments from three thick filaments. This complex array of thick and thin filaments is characteristic of both cardiac and skeletal muscle and helps • Fig. 13.1 A, Photomicrograph of cardiac muscle cells (magnification, ×210). Intercalated disks at either end of a muscle cell are identified in the lower left portion of the micrograph. The intercalated disk physically connects adjacent myocytes and, because of the presence of gap junctions, electrically couples the cells as well so that the muscle functions as an electrical and mechanical syncytium. B, Schematic representation of the organization of a sarcomere within a cardiac muscle cell. (A, From Telser A. Elsevier’s Integrated Histology. St. Louis: Mosby; 2007. B, Redrawn from Fawcett D, McNutt NS. The ultrastructure of the cat myocardium. I. Ventricular papillary muscle. J Cell Biol. 1969;42:1-45.) stabilize the filaments during muscle contraction (see

1	Fig. 12.3B , for the hexagonal array of thick and thin filaments in the sarcomere of striated muscle). Several proteins may contribute to the organization of the thick and thin filaments, including meromyosin and C protein (in the center of the sarcomere), which appear to serve as a scaffold for organization of the thick filaments. Similarly, nebulin extends along the length of the actin filament and may serve as a scaffold for the thin filament. The actin filament is anchored to the Z line by α-actinin, whereas the protein tropomodulin resides at the end of the actin filament and regulates the length of the thin filament. These proteins are present in both cardiac and skeletal muscle cells.

1	The thick filaments are tethered to the Z lines by a large elastic protein called titin. Although titin was postulated to tether myosin to the Z lines and thus prevent overstretching of the sarcomere, there is evidence indicating that titin may participate in cell signaling (perhaps by acting as a stretch sensor and thus modulating protein synthesis in response to stress). Such signaling by titin has been observed in both cardiac and skeletal muscle cells. Moreover, genetic defects in titin result in atrophy of both cardiac and skeletal muscle cells and may contribute to both cardiac dysfunction and skeletal muscle dystrophies (termed titinopathies). Titin is also thought to contribute to the ability of cardiac muscle to increase force upon stretch (discussed in the later section “

1	Although both cardiac muscle and skeletal muscle contain an abundance of connective tissue, there is more connective tissue in the heart. The abundance of connective tissue in the heart helps prevent muscle rupture (as in skeletal muscle), but it also prevents overstretching of the heart. Length-tension analysis of cardiac muscle, for example, shows a dramatic increase in passive tension as cardiac muscle is stretched beyond its resting length. Skeletal muscle, in contrast, tolerates a much greater degree of stretch before passive tension increases to a comparable level. The reason for this difference between cardiac and skeletal muscle is not known, although one possibility is that stretch of skeletal muscle is typically limited by the range of motion of the joint, which in turn is limited by the ligaments/connective tissue surrounding the joint.

1	The heart, on the other hand, appears to rely on the abundance of connective tissue around cardiac muscle cells to prevent overstretching during periods of increased venous return. During intense exercise, for example, venous return may increase fivefold. However, the heart is capable of pumping this extra volume of blood into the arterial system with only minor changes in the ventricular volume of the heart (i.e., end-diastolic volume increases less than 20%). Although the abundance of connective tissue in the heart limits stretch of the heart during these periods of increased venous return, additional regulatory mechanisms help the heart pump the extra blood that it receives (as discussed in the section “ ”). Conversely, if the heart were to be overstretched, the contractile ability of cardiac muscle cells would be expected to decrease (because of decreased overlap of the thick and thin filaments), which would result in insufficient pumping, increased venous pressure, and perhaps

1	cardiac muscle cells would be expected to decrease (because of decreased overlap of the thick and thin filaments), which would result in insufficient pumping, increased venous pressure, and perhaps pulmonary edema.

1	Within cardiac muscle cells, myofibrils are surrounded by the sarcoplasmic reticulum (SR), an internal network of membranes (see Fig. 13.1B ). This is similar to the SR in skeletal muscle except that the SR in the heart is less dense and not as well developed. Terminal regions of the SR abut the T tubule or lie just below the sarcolemma (or both) and play a key role in the elevation of intracellular [Ca++] during an action potential. The mechanism by which an action potential initiates release of Ca++ in the heart, however, differs significantly from that in skeletal muscle (as discussed in the section “ ”). The heart contains an abundance of mitochondria; up to 30% of the volume of the heart is occupied by these organelles. The high density of mitochondria provides the heart with great oxidative capacity, more so than is typical in skeletal muscle.

1	The sarcolemma of cardiac muscle also contains invaginations (T tubules) comparable to those seen in skeletal muscle. In cardiac muscle, however, T tubules are positioned at the Z lines, whereas in mammalian skeletal muscle, T tubules are positioned at the ends of the I bands. In cardiac muscle, the connections between the T tubules and the SR are fewer than, and not as well developed as, those in skeletal muscle.

1	Familial cardiomyopathic hypertrophy (FCH) occursinapproximately0.2%ofthegeneralpopulationbutisaleadingcauseofsuddendeathinotherwisehealthyadults.Ithasbeenlinkedtogeneticdefectsinavarietyofproteinsincardiacsarcomeres,includingmyosin,troponin,tropomyosin,andmyosin-bindingproteinC,astructuralproteinlocatedinthemiddleoftheAbandofthesarcomere.FCHisanautosomaldominantdisease,andtransgenicstudiesindicatethatexpressionofonlyasmallamountofthemutatedproteincanresultindevelopmentofthecardiomyopathicphenotype.Moreover,mutationofasingleaminoacidinthemyosinmoleculeissufficienttoproducecardiomyopathichypertrophy.ThepathogenesisofFCH,however,isvariable,evenwithinafamilywithasinglegenedefect,intermsofbothonsetandseverity;thisvariabilitysuggeststhepresenceofmodifyingloci. Control of Cardiac Muscle Activity

1	Control of Cardiac Muscle Activity Cardiac muscle is an involuntary muscle with an intrinsic pacemaker. The pacemaker represents a specialized cell (located in the sinoatrial node of the right atrium) that is able to undergo spontaneous depolarization and generate action potentials. Of importance is that although several cells in the heart are able to depolarize spontaneously, the fastest spontaneous depolarizations occur in cells in the sinoatrial node. Moreover, once a given cell spontaneously depolarizes and fires an action potential, this action potential is then propagated throughout the heart (by specialized conduction pathways and cell-to-cell contact). Thus depolarization from only one cell is needed to initiate a wave of contraction in the heart (i.e., a heartbeat). The mechanisms underlying this spontaneous depolarization are discussed in depth in

1	Fig. 16.17 , once an action potential is initiated in the sinoatrial node, it is propagated between atrial cells via gap junctions, as well as through specialized conduction fibers in the atria. The action potential can pass throughout the atria within approximately 70 msec. For the action potential to reach the ventricles, it must pass through the atrioventricular node, after which the action potential passes throughout the ventricle via specialized conduction pathways (the bundle of His and the Purkinje system) and gap junctions in the intercalated disks of adjacent cardiac myocytes. The action potential can pass through the entire heart within 220 msec after initiation in the sinoatrial node. Because contraction of a cardiac muscle cell typically lasts 300 msec, this rapid conduction promotes nearly synchronous contraction of heart muscle cells. This is a very different scenario from that of skeletal muscle, in which cells are grouped into motor units that are recruited

1	promotes nearly synchronous contraction of heart muscle cells. This is a very different scenario from that of skeletal muscle, in which cells are grouped into motor units that are recruited independently as the force of contraction is increased.

1	Blood and extracellular fluids typically contain 1 to 2 mmol/L of free Ca++ , and it has been known since the days of the physiologist Sidney Ringer (ca. 1882) that the heart requires extracellular Ca++ to contract. Thus an isolated heart typically continues to beat when perfused with a warm (37°C), oxygenated, physiological salt solution that contains approximately 2 mmol/L Ca++ (e.g., Tyrode’s solution), but it stops beating in the absence of extracellular . This cessation of contractions in Ca++-deficient media is also observed in hearts that are electrically stimulated, which further demonstrates the importance of extracellular Ca++ for contraction of cardiac muscle. This situation is quite different from that of skeletal muscle, which can contract in the total absence of extracellular Ca++ . Action potentials in cardiac muscle are prolonged, lasting 150 to 300 msec (

1	Fig. 13.2 inset), which is substantially longer than the action potentials in skeletal muscle (≈5 msec). The long duration of the action potential in cardiac muscle is due to a slow inward Ca++ current through a voltage-gated L-type calcium channel in the sarcolemma. The amount of Ca++ coming into the cardiac muscle cell is relatively small and serves as a trigger for release of Ca++ from the SR. In the absence of extracellular Ca++ , an action potential can still be initiated in cardiac muscle, although it • Fig.

1	is relatively small and serves as a trigger for release of Ca++ from the SR. In the absence of extracellular Ca++ , an action potential can still be initiated in cardiac muscle, although it • Fig. 13.2 Excitation-contraction coupling in the heart requires Ca++ influx through L-type calcium channels in the sarcolemma and T tubules. See text for details. Inset shows time course of action potential (AP), intracellular Ca transient (Ca), and contraction. ATP, adenosine triphosphate; NCX, sarcolemmal 3Na+-Ca++ antiporter; PLN, phospholamban; RYR, ryanodine receptor. (Modified from Bers DM. Cardiac excitation-contraction coupling. Nature. 2002;415:198-205. Inset modified from Mountcastle VB: Medical Physiology, 13 ed. St Louis, Mosby, 1974; Brooks CM, Hoffman BF, Suckling EE, Orias O: Excitability of the Heart. New York, Grune & Stratton, 1955.) is considerably shorter in duration and unable to initiate a contraction. Thus influx of Ca++ during the action potential is crucial for triggering

1	is considerably shorter in duration and unable to initiate a contraction. Thus influx of Ca++ during the action potential is crucial for triggering release of Ca++ from the SR and thus initiating contraction.

1	The L-type calcium channel is composed of five subunits (α1, α2, β, γ, and δ). The α1 subunit is also called the dihydropyridine receptor (DHPR) because it binds the dihydropyridine class of calcium channel–blocking drugs (e.g., nitrendipine and nimodipine). Although this channel complex is present in both skeletal and cardiac muscle, it serves very different functions in the two muscle types (discussed in the next paragraph). In each cardiac muscle sarcomere, terminal regions of the SR abut T tubules and the sarcolemma (see

1	Figs. 13.1B 13.2 ). These junctional regions of the SR are enriched in ryanodine receptors (RYRs; a Ca++ release channel in the SR). The RYR is a Ca++-gated calcium channel, and so influx of Ca++ during an action potential is able to initiate release of Ca++ from the SR in cardiac muscle. The amount of Ca++ released into the cytosol from the SR is much greater than that entering the cytosol from the sarcolemma, although release of Ca++ from the SR does not occur without this entry of “trigger” Ca++ . In contrast, in skeletal muscle, release of Ca++ from the SR does not involve entry of Ca++ across the sarcolemma but instead results from a voltage-induced conformational change in the DHPR. Thus excitation-contraction coupling in cardiac muscle is termed electrochemical coupling (involving Ca++-induced release of Ca++), whereas excitation-contraction coupling in skeletal muscle is termed electromechanical coupling (involving direct interactions between the DHPR in the T tubule and the

1	Ca++-induced release of Ca++), whereas excitation-contraction coupling in skeletal muscle is termed electromechanical coupling (involving direct interactions between the DHPR in the T tubule and the RYR in the SR). The basis for this difference in Ca++ release mechanisms appears to depend on the DHPR isoform because expression of cardiac DHPR in skeletal muscle cells results in a requirement for extracellular Ca++ for contraction of these modified skeletal muscle cells.

1	As in skeletal muscle, contraction of cardiac muscle is regulated by thin filaments, and an elevation in intracellular [Ca++] is necessary to promote actin-myosin interaction. At low (<50 nmol/L) intracellular [Ca++], binding of myosin to actin is blocked by tropomyosin. As cytosolic [Ca++] increases during an action potential, however, binding of Ca++ to troponin C results in a conformational change in the troponin/tropomyosin complex in which tropomyosin slips into the groove of the actin filament and exposes myosin binding sites on the actin filament. As long as cytosolic [Ca++] remains elevated, and hence myosin binding sites are exposed, myosin will bind to actin, undergo a ratchet action, and contract the cardiac muscle cell. Note that because myosin binding sites on actin are blocked at low [Ca++] and exposed during a rise in intracellular [Ca++], contraction of cardiac muscle is termed thin filament regulated. This is identical to the situation in skeletal muscle; in smooth

1	at low [Ca++] and exposed during a rise in intracellular [Ca++], contraction of cardiac muscle is termed thin filament regulated. This is identical to the situation in skeletal muscle; in smooth muscle, in contrast, contraction is thick filament regulated (see

1	During a rise in intracellular [Ca++] and exposure of myosin-binding sites on actin, the myosin cross-bridges undergo a series of steps that result in contraction of the cardiac muscle cell. At rest, the myosin molecules are energized in that they have partially hydrolyzed adenosine triphosphate (ATP) to “cock the head” and are thus ready to interact with actin. An elevation in intracellular [Ca++] then exposes myosin-binding sites on actin and thus allows myosin to bind actin (step 1). The bound myosin subsequently undergoes a powerstroke in which the actin filament is pulled toward the center of the sarcomere (step 2). Adenosine diphosphate (ADP) and inorganic phosphate (Pi) are released from the myosin head during this step as the energy from ATP is used to contract the muscle. The myosin head moves approximately 70 nm during each ratchet action (cross-bridge cycle). Binding of ATP to myosin decreases the affinity of myosin for actin and thus allows myosin to release from actin

1	myosin head moves approximately 70 nm during each ratchet action (cross-bridge cycle). Binding of ATP to myosin decreases the affinity of myosin for actin and thus allows myosin to release from actin (step 3). Myosin then partially hydrolyzes the bound ATP to reenergize (“cock”) the head (step 4) and ready the cross-bridge for another cycle. This four-step cycle is identical to that described for skeletal muscle (see

1	Fig. 12.13). Cardiac muscle and skeletal muscle differ, however, in the level of intracellular [Ca++] attained after an action potential and hence in the number of actin-myosin interactions. In skeletal muscle, intracellular [Ca++] rises and the number of actin-myosin interactions is high after an action potential. In cardiac muscle, the rise in intracellular [Ca++] can be regulated, which affords the heart an important means of modulating the force of contraction without recruiting more muscle cells or undergoing tetany. Recall that in the heart, all the muscle cells are activated during a contraction, and so recruiting more muscle cells is not an option. Moreover, tetany of cardiac muscle cells would prevent any pumping action and thus be fatal. Consequently, the heart relies on different means of increasing the force of contraction, including varying the amplitude of the intracellular Ca++ transient. Relaxation of Cardiac Muscle

1	Relaxation of skeletal muscle simply requires reaccumulation of Ca++ by the SR through the action of the sarcoplasmic endoplasmic reticulum calcium-ATPase (SERCA), also known as the SR Ca++ pump. Although SERCA plays a key role in the decrease in cytosolic [Ca++] in cardiac muscle, the process is more complex than that in skeletal muscle because some trigger Ca++ enters the cardiac muscle cell through the sarcolemmal calcium channels during each action potential. A mechanism must therefore exist to extrude this trigger Ca++; otherwise, the amount of Ca++ in the SR would continuously increase, and Ca++ overload would result. In particular, some Ca++ is extruded from the cardiac muscle cell though the sarcolemmal 3Na+-Ca++ antiporter and a sarcolemmal Ca++ pump (Fig. 13.2 ). The extracellular [Ca++] is in the millimolar range, whereas the amount of intracellular [Ca++] is submicromolar, and so extrusion of Ca++ is accomplished against a large chemical gradient. Similarly, [Na+] is

1	[Ca++] is in the millimolar range, whereas the amount of intracellular [Ca++] is submicromolar, and so extrusion of Ca++ is accomplished against a large chemical gradient. Similarly, [Na+] is considerably higher in the extracellular media than within the cell. The antiporter uses the Na+ gradient across the cell to power the uphill movement of Ca++ out of the cell. Because three Na+ ions enter the cell in exchange for one Ca++ ion, the 3Na+-Ca++ antiporter is electrogenic and creates a depolarizing current. The sarcolemmal Ca++ pump, on the other hand, uses the energy in ATP to extrude Ca++ from the cell. Both extrusion mechanisms and SERCA thus contribute to the relaxation of cardiac muscle by decreasing cytosolic [Ca++].

1	Although the interaction of actin and myosin requires a relatively small increase in free intracellular [Ca++], the abundance of Ca++-binding proteins in the myoplasm necessitates a much larger increase in total intracellular [Ca++]. The resting intracellular [Ca++] is approximately 50 to 100 nmol/L; half-maximal force of contraction requires approximately 600 nmol/L of free Ca++ . However, because of Ca++-binding proteins such as parvalbumin and troponin C, the total myoplasmic concentration must increase by 70 µmol/L. As already noted, much of this increase in total myoplasmic [Ca++] occurs through release of Ca++ from the SR. In a number of species, including rabbits, dogs, cats, guinea pigs, and humans, uptake and release of Ca++ by the SR account for approximately 70% of the intracellular Ca++ transient. Thus up to 30% of the rise in intracellular [Ca++] may be attributable to influx of Ca++ through voltage-gated calcium channels in the sarcolemma, and the 3Na+-Ca++ antiporter

1	Ca++ transient. Thus up to 30% of the rise in intracellular [Ca++] may be attributable to influx of Ca++ through voltage-gated calcium channels in the sarcolemma, and the 3Na+-Ca++ antiporter contributes significantly to Ca++ extrusion during relaxation.

1	The sarcolemmal Ca++ pump is in lower abundance than the 3Na+-Ca++ antiporter but has a higher affinity for Ca++ and thus may contribute more to the regulation of resting intracellular [Ca++] (see Fig. 13.2 ). The relative contribution of the Ca++ extrusion mechanisms, however, varies between species. For example, rat and mouse myocytes rely primarily on Ca++ reuptake by the SR (i.e., the SR accounts for 92% of Ca++ transport). Regulation of the Force of Contraction

1	Regulation of the Force of Contraction Because the heart represents an electrical syncytium, in which all the cardiac muscle cells contract during a single beat, it is not possible to increase the force of contraction by recruiting more muscle cells. Moreover, tetany of the heart would be lethal because it would defeat the critical pumping action of the heart. The heart has therefore developed alternative strategies to increase the force of contraction. The long duration of the action potential found in cardiac muscle, which is due to activation of the voltage-gated L-type calcium channel, results in a long refractory period, which in turn prevents tetany. Modulation of Ca++ influx through L-type calcium channels during an action potential, however, provides the heart with a mechanism to alter cytosolic [Ca++] and hence the force of contraction.

1	A simple means of modulating the force of contraction of cardiac muscle cells in vitro is to vary extracellular [Ca++]. As noted previously, contraction of the heart requires extracellular Ca++ . Decreasing extracellular [Ca++] from a normal range of 1 to 2 mmol/L to 0.5 mmol/L, for example, reduces the force of the contraction. This reduction in force of contraction is not associated with a change in the duration of the contraction because the kinetic characteristics of Ca++ sequestration by the SR and Ca++ extrusion have not been modified. Although this approach of varying extracellular [Ca++] to alter the force of contraction is demonstrable in vitro, it is not a common means of modulating the force of cardiac contraction in vivo. In vivo, an increase in the size of the intracellular Ca++ transient and hence the force of contraction occurs in response to sympathetic stimulation (see the section

1	In vivo, an increase in the size of the intracellular Ca++ transient and hence the force of contraction occurs in response to sympathetic stimulation (see the section Chapter 18). Sympathetic stimulation often occurs during periods of excitement or fright and involves activation of β-adrenergic receptors on the heart by norepinephrine (released from nerve terminals in the heart) or epinephrine (released from the adrenal medulla into the bloodstream). As shown in

1	Fig. 13.3 , the β-adrenergic agonist isoproterenol results in a dramatic increase in the size of the intracellular Ca++ transient and, consequently, a more forceful contraction. An increase in the force of contraction is termed positive inotropy. Typically, the rate of relaxation accompanying this β-adrenergic stimulation also increases, which results in a shorter contraction. The increase in the rate of muscle relaxation is termed positive lusitropy. The frequency of contractions of the heart also increases with β-adrenergic stimulation and is termed positive chronotropy. Thus β-adrenergic stimulation of the heart produces stronger, briefer, and more frequent contractions.

1	The sympathetic nervous system is stimulated when a human or an animal becomes excited, and it is said to prepare the individual for “fight or flight.” In the case of the heart, increased levels of the adrenal medullary hormone epinephrine or the sympathetic neurotransmitter norepinephrine activate β-adrenergic receptors on the cardiac muscle cells, which in turn activates adenylate cyclase, increases cyclic adenosine monophosphate (cAMP), and thus promotes cAMP-dependent phosphorylation of numerous proteins in cardiac muscle cells ( Fig. 13.4

1	Fig. 13.4 Both voltage-gated L-type calcium channels (responsible for the trigger Ca++) and a protein associated with SERCA, called phospholamban, are phosphorylated by cAMPdependent protein kinase. The combined action of these phosphorylations increases the amount of Ca++ in the SR. Specifically, phosphorylation of the sarcolemmal calcium channel causes more trigger Ca++ to enter the cell, and phosphorylation of phospholamban increases the activity of SERCA, thereby allowing the SR to accumulate more Ca++ before it is extruded by the 3Na+-Ca++ antiporter and the sarcolemmal Ca++ pump. The net result is that the SR releases more Ca++ into the cytosol during the next action potential, which promotes more actin-myosin interactions and hence greater force of contraction (see

1	Fig. 13.3 ). The increased activity of SERCA after sympathetic stimulation also results in a shortened contraction because of the rapid reaccumulation of Ca++ by the SR. This in turn allows the heart to increase its rate of relaxation. An additional consequence of sympathetic stimulation is an increase in heart rate through a direct effect on the pacemaker cells (see •Fig. 13.3 Stimulationofβ-adrenergicreceptorsintheheartincreasestheforceofcontraction.Electricalstimulationofmyocardiumresultsinatransientriseinintracellular[Ca++]andproductionofforce(A). Isoproterenol(aβ-adrenergicreceptoragonist)increasestheamplitudeoftheintracellularCa++transientandhencetheamountofforcegenerated(B). A Control B +Isoproterenol Ca Ca Force Force 0.5 sec Additional proteins and some micropeptides also appear to be associated with SERCA and influence SR calcium transport. This includes the 34–amino acid peptide dwarf open reading frame (DWORF), which increases SERCA calcium affinity (

1	Fig. 13.5 ), apparently by displacing phospholamban. DWORF was identified in putative non-coding RNA. Themechanismsunderlyingtheresponseofthehearttoβ-adrenergicstimulationarecomplexandinvolvecAMPdependentphosphorylationofseveralproteins.AnA kinase anchoring protein (AKAP) hasbeenshowntobecloselyassociatedwiththeL-typecalciumchannelintheheart,therebypositioningcAMP-dependent protein kinase closetothechannelandfacilitatingcAMP-dependentphosphorylationofthischannelduringsympatheticstimulation.HowthesecAMP-dependentphosphorylationsincreasetheamplitudeoftheintracellularCa++ transientand,insodoing,resultinamoreforceful,briefercardiaccontractionisdiscussedingeneraltermslater(seealso

1	Mutationsinthecardiacryanodinereceptor(RYR2)havebeenassociatedwithcardiacarrhythmias.Specifically,catecholaminergicpolymorphicventriculartachycardia(CPVT)isaninheritedautosomaldominantdiseasethatistypicallymanifestedduringchildhoodasanexercise-inducedtachycardiathatcanprogresstoarrhythmiasduringexercise(orstress)andresultinsuddendeath.Approximately40%ofpatientswithCPVTexhibitadefectinRYR2thathasbeenassociatedwithincreasedreleaseofCa++ fromtheSR.ThemutationinRYR2mayinvolvesubstitutionofahighlyconservedaminoacid,whichdiffersfrommalignanthyperthermia,inwhichsplicingerrorsordeletionswithinRYRhavebeenreported.Itishypothesizedthatduringperiodsofexerciseorstress,increasedlevelsofintracellularCa++ (becauseofthecombinedeffectsofβ-adrenergicstimulationandincreasedactivityofthemutatedRYR2)promotethedevelopmentofdelayedafterdepolarizationsandhencearrhythmias.Elevationofintracellular[Ca++]duringdiastoleisthoughttopromotethedevelopmentofdelayedafterdepolarizationsthroughactivationofthe3Na+-Ca++

1	antiporter,whereinCa++ extrusionduringdiastoleresultsinanetinwardcurrentsufficienttodepolarizethecelltothethresholdforanactionpotential.TreatmentofCPVTinvolvesantiadrenergictherapy(withβ-adrenergicantagonists)or(forunresponsivepatients)animplanteddefibrillator.

1	Stretching of the heart increases the force of contraction both in vivo and in vitro and is an intrinsic mechanism for regulating contractile force. In contrast, skeletal muscle typically exhibits maximal tension at resting length. Stretching of the heart in vivo occurs during times of increased venous return of blood to the heart (e.g., during exercise or when the heart rate is slowed, or both). The Frank-Starling law of the heart refers to this ability of the heart to increase its force of contraction when stretched, which occurs at •Fig. 13.4

1	or when the heart rate is slowed, or both). The Frank-Starling law of the heart refers to this ability of the heart to increase its force of contraction when stretched, which occurs at •Fig. 13.4 Sympatheticstimulationoftheheartresultsinanincreaseincytosoliccyclicadenosinemonophosphate(cAMP)andhencephosphorylationofseveralproteinsbyproteinkinaseA(PKA).AnAkinaseanchorprotein(AKAP)adjacenttotheL-typecalciumchannelfacilitatesphosphorylationofthischannelandpossiblynearbysarcoplasmicreticulumcalciumchannels.OtherproteinsphosphorylatedbyPKAincludephospholamban(PLN)andtroponinI.Muscarinicagonists(e.g.,acetylcholine[ACh]),ontheotherhand,inhibitthissympatheticcascadebyinhibitingtheproductionofcAMPbyadenylatecyclase(AC).β-AR,β-adrenergicreceptor;ATP,adenosinetriphosphate;Gi,inhibitoryGprotein;M2Rec,muscarinicacetylcholineM2receptor;Reg,regeneratinggenereceptor.(RedrawnfromBersDM.Cardiacexcitation-contractioncoupling.Nature 2002;415:198.) times of increased venous return (Fig. 13.6A

1	The importance of this mechanism is that it helps the heart pump whatever volume of blood it receives. Thus when the heart receives a lot of blood, the ventricles are stretched, and the force of contraction is increased, which ensures ejection of this extra volume of blood. Stretching of cardiac muscle also increases passive tension, which helps prevent overstretching of the heart. This passive resistance in the heart is greater than that in skeletal muscle and is attributed to both extracellular matrix (connective tissue) and intracellular elastic proteins (titin).

1	This stretch-induced increase in force of contraction of cardiac muscle occurs over a narrow range of sarcomere lengths (ca. 1.6-2.3 um), resulting in a steep length-dependent activation of contraction. This ascending limb of the length-tension relationship in cardiac muscle is much steeper than that seen in skeletal muscle. It is important to note that this stretch-induced increase in force can occur within a singe heart beat. The mechanism(s) underlying this stretch-induced increase in force of contraction of cardiac muscle is controversial, but appears to involve changes in the overlap of the thick and thin filaments as well as stretch-induced increase in the Ca++ sensitivity of contraction (

1	Fig. 13.6B of the stretch-induced increase in force of contraction has been attributed to an increase in Ca++ sensitivity, whereas the remaining 40% of the stretch-induced increase in force of contraction has been attributed to changes in the overlap of the thick and thin filaments. The changes in myofilament overlap, however, are less likely to contribute to the continued increase in force of cardiac contraction as sarcomere length increases from 2.0um to 2.3 um, as this region is thought to represent a region of optimal overlap of myofilaments (and represents a plateau in the length tension relationship in skeletal muscle). The mechanism(s) contributing to the stretch-induced increase in Ca++ sensitivity of cardiac contraction are not clear, but appears to involve the intracellular elastic protein titin, as well as regulatory proteins (e.g., troponinC).

1	As in skeletal muscle, myosin uses the energy in ATP to generate force, so the ATP pool, which is small, must be continually replenished. Typically, this replenishment of ATP pools is accomplished by aerobic metabolism, including the oxidation of fats and carbohydrates. During times of ischemia, the creatine phosphate pool, which converts ADP to ATP, may decrease. As in skeletal muscle, the In rat ventricular trabecular muscle, approximately 60% creatine phosphate pool is small.

1	276 SECTION3Berne & Levy Physiology ContractionSarcomereCa2˜Ca2˜Ca2˜Sarcomere RelaxationSarcoplasm MyocyteSarcoplasmic reticulum RyRSERCASERCAPLNDWORF A B C 125 100 75 50 25 0 250 200 150 100 50 0 WT Tg KO % Vmax 8.0 7.5 7.0 6.5 6.0 5.5 pCa * * WT Tg KO KCa[Ca2˜]free (nM) •Fig. 13.5 Asmall(34–aminoacid)peptideencodedbyalongnoncodingRNAappearstoimprovesarcoplasmicreticulumfunctionandmyocyteperformancebycounteringtheinhibitoryeffectofphospholamban(PLN)(asdepictedintheworkingmodelshowninpanelA).TheabilityofDWORFtoincreasecardiacSRCauptakeisshowninpanelsBandC,whereoverexpressionofDWORFincardiomyocytes(labeledTg)increasedtheCasensitivityofSRCatransport,whereasknockoutofDWORF(labeledKO)hadthereverseeffect.DWORF,dwarfopenreadingframe;KO,knockout;SERCA,sarcoplasmicendoplasmicreticulumcalcium-ATPase;Tg,transgenic;WT,wild-type.(FromNelsonBR,MakarewichCA,AndersonDM,etal.ApeptideencodedbyatranscriptannotatedaslongnoncodingRNAenhancesSERCAactivityinmuscle.Science. 2016;351:271-275.)

1	When cardiac muscle is completely deprived of O2 because of occlusion of a coronary vessel (i.e., stopped-flow ischemia), contractions quickly cease (within 30 seconds). This is not due to depletion of either ATP or creatine phosphate because these levels decline more slowly. Even after 10 minutes of stopped-flow ischemia, when creatine phosphate levels are near zero and only 20% of the ATP remains, reperfusion can restore these energy stores, as well as contractile ability. However, prolonging the stopped-flow ischemia for 20 minutes results in further drops in ATP, so that reperfusion has considerably less effect, with only limited restoration of ATP and creatine phosphate levels or contractile activity.

1	Exercise such as endurance running can increase the size of the heart as a result of hypertrophy of individual cardiac muscle cells. Concomitant with this enlarged so-called athlete’s heart is improved cardiac performance, as assessed by an increase in stroke volume, increased oxygen consumption, and preserved relaxation. Thus the athlete’s heart represents an example of “physiological hypertrophy,” with beneficial contractile effects. In contrast, if exposed to chronic pressure overload, the heart may undergo either concentric left ventricular hypertrophy or dilated left ventricular hypertrophy, which causes impairment of function. Details regarding the morphological, functional, and mechanistic differences between these various types of hypertrophy can be found elsewhere in this textbook (see Chapter 18).

1	Concentric hypertrophy is characterized by thickening of the left ventricular wall and represents a compensatory hypertrophy to the increased load. Dilated hypertrophy is characterized by increased ventricular volume (end-diastolic volume). Both concentric/compensatory left ventricular hypertrophy and dilated left ventricular hypertrophy have been shown to exhibit decreased contractile response to β-adrenergic stimulation, which limits the contractile reserve. In dilated left ventricular hypertrophy, normal •Fig. 13.6 Stretchingoftheheartincreasestheforceofcontraction(A). ThisisattributabletobothanincreaseinthemaximalforceofcontractionandanincreaseinthesensitivityofcontractiontoCa++ (B). ItreflectsanintrinsicregulatoryprocessreferredtoastheFrank-Starling law of the heart. (B,RedrawnfromDobeshD,KonhilasJ,deTombeP.Cooperativeactivationincardiacmuscle:impactofsarcomerelength.Am J Physiol Heart Circ Physiol. 2002;282:H1055-H1062.) contractile function, along with the Frank-Starling

1	J Physiol Heart Circ Physiol. 2002;282:H1055-H1062.) contractile function, along with the Frank-Starling response, may also be impaired.

1	The cellular and molecular mechanisms underlying the development of cardiac hypertrophy are not clear, although an elevation in intracellular [Ca++] has been implicated. The link or links between cardiac hypertrophy, decreased cardiac performance, and impaired β-adrenergic response during chronic pressure overload are unclear. Decreased cardiac performance has been attributed to dysregulation of intracellular [Ca++]. Alterations in the level, activity, and phosphorylation status of a variety of proteins, including L-type calcium channels, phospholamban, SERCA, and RYR, have all been implicated in the Ca++ dysregulation associated with a failing heart (pathological hypertrophy). A microRNA (miR-222) has been shown to be important for cardiac growth in response to exercise. It also appeared to inhibit maladaptive remodeling of the heart after ischemia/ reperfusion injury.

1	A microRNA (miR-222) has been shown to be important for cardiac growth in response to exercise. It also appeared to inhibit maladaptive remodeling of the heart after ischemia/ reperfusion injury. Amodestelevationinintracellular[Ca++](asaresultofincreasedcontractileactivity,forexample),hasbeenproposedtoactivateaCa++/calmodulin-dependentproteinphosphatase(calcineurin) thatcandephosphorylatethetranscriptionfactornuclear factor of activated T cells (NFAT), therebyfacilitatingtranslocationofNFATtothenucleusandultimatelypromotingproteinsynthesisandthushypertrophy( Fig.13.7 ).ActivationofCa++/calmodulin-dependentproteinkinasehasalsobeenimplicatedinactivationofthetranscriptionfactormyocyte enhancer factor 2 (MEF2) bypromotingthedissociation(nuclearexport)ofaninhibitorofMEF2(namely,histone deacetylase [HDAC]).

1	Theimpairedβ-adrenergicresponseofcardiacmuscleafterchronicpressureoverloadinvolves,atleastinpart,adecreaseinβ-adrenergicreceptorsbecauseofinternalization.Bothphosphatidylinositol-3-kinase (PI3K) andβ-adrenergic receptor kinase 1 havebeenimplicatedintheinternalizationofβ-adrenergicreceptors. Highbloodpressure,defectsinheartvalves,andventricularwallsweakenedasaresultofmyocardialinfarctioncanallleadtoheartfailure,aleadingcauseofdeath.Heartfailuremaybeseenwiththickeningofthewallsoftheventricleorwithdilation(i.e.,increasedvolume)oftheventricles.

1	Resultsofstudiessuggestthatdilatedcardiomyopathycanbepreventedinananimalmodelbydownregulatingphospholamban.ThemechanismunderlyingthispreventiveeffectofphospholambandownregulationisthoughttoinvolveanincreaseinCa++ uptakeactivityintheSRbecausephospholambantypicallyinhibitsSERCA.IncreasedactivityofSERCAwouldfacilitaterelaxationoftheheartasaresultofrapidCa++ uptakebytheSR.Inaddition,theforceofcontractionisincreasedbecausemoreCa++ isavailableforrelease.IncreasedCa++ uptakebytheSRmayalsodecreaseactivationoftheCa++-dependentphosphatasesthathavebeenimplicatedinthedevelopmentofcardiachypertrophy. There is evidence that cardiac hypertrophy may not be associated with some functional impairments. Intermittent aortic constrictions, for example, result in decreased β-adrenergic signaling, decreased capillary density, and decreased SERCA2 levels, without evidence of hypertrophy. Activation of PI3K appears to be involved in this response.

1	278 SECTION3Berne & Levy Physiology Activity Ca++ Other transducers? Other effectors? Reprogramming Gene Expression Myocyte Hypertrophy/Tissue Remodeling CalcineurinCaMK Other targets? NFAT NFAT GSK3 HDAC CsA Cabin MCIP Agonists MEF2 GATA P •Fig. 13.7 Calcium-dependentactivationofcalcineurinandcalmodulin-dependentproteinkinasehavebeenimplicatedinthedevelopmentofcardiachypertrophyandinvolveactivationofthefollowingtranscriptionfactors:nuclearfactorofactivatedTcells(NFAT),transcriptionfactorbindingtoDNAsequenceGATA(GATA),andmyocyteenhancerfactor2(MEF2).Cabin,calcineurin-bindingprotein/inhibitor;CaMK,Ca++/calmodulin-dependentproteinkinase;CsA,cyclosporine;GSK3,glycogensynthasekinase3;HDAC,histonedeacetylase;MCIP,modulatorycalcineurin-interactingprotein.(RedrawnfromOlsonEN,WilliamsRS.Calcineurinsignalingandmuscleremodeling.Cell. 2000;101:689-692.) 1.

1	Cardiac muscle is an involuntary, striated muscle. Cardiac muscle cells are relatively small (10 µm × 100 µm) and form an electrical syncytium with tight electrical and mechanical connections between adjacent cardiac muscle cells. Action potentials are initiated in the sinoatrial node and spread quickly throughout the heart to allow synchronous contraction, a feature important for the pumping action of the heart. 2. Contraction of cardiac muscle involves the Ca++dependent interaction of actin and myosin filaments, as in skeletal muscle. However, unlike skeletal muscle, cardiac muscle requires an influx of extracellular Ca++ . Specifically, the influx of Ca++ during an action potential triggers release of Ca++ from the SR, which then promotes actin-myosin interaction and contraction. 3.

1	3. Relaxation of cardiac muscle involves reaccumulation of Ca++ by the SR and extrusion of Ca++ from the cell via the 3Na+-Ca++ antiporter and the sarcolemmal Ca++ pump. The Ca++ pump of the SR is associated with numerous proteins (forming a regulosome), including some endogenous micropeptide inhibitors and activators. 4. The force of contraction of cardiac muscle is increased by stretch (Frank-Starling Law of the Heart) and by sympathetic stimulation. Skeletal muscle, in contrast, increases force by recruiting more muscle fibers or by tetany. 5.

1	5. Hypertrophy of the heart can occur in response to exercise, chronic pressure overload, or genetic mutations. The cardiac hypertrophy resulting from exercise is typically beneficial, with improved cardiac performance, increased oxygen consumption, and normal relaxation. Chronic pressure overload, on the other hand, can result in cardiac hypertrophy that is initially associated with a decreased β-adrenergic response but may progress to dilated cardiac hypertrophy, characterized by decreased contractile ability. Genetic mutations resulting in cardiac hypertrophy include familial hypertrophic Anderson DM, Anderson KM, Chang CL, et al. A micropeptide encoded by a putative long noncoding RNA regulates muscle performance. Cell. 2015;160:595-606. Endoh M. Cardiac Ca2+ signaling and Ca2+ sensitizers. Circ J. 2008;72:1915-1925.

1	Endoh M. Cardiac Ca2+ signaling and Ca2+ sensitizers. Circ J. 2008;72:1915-1925. Haghighi K, Bidwell P, Kranias EG. Phospholamban interactome in cardiac contractility and survival: a new vision of an old friend. J Mol Cell Cardiol. 2014;77:160-167. Hidalgo C, Granzier H. Tuning the molecular giant titin through phosphorylation: role in health and disease. Trends Cardiovasc Med. 2013;23:165-171. cardiomyopathy, in which a mutation in a single intracellular protein may alter contractile function and promote a hypertrophic response. Researchers have identified a microRNA that appears to contribute to the exercise-induced hypertrophy of the heart and to inhibit maladaptide remodeling after ischemia/reperfusion injury. Kobirumaki-Shimozawa F, Inoue T, Shintani SA, et al. Cardiac thin filament regulation and the Frank–Starling mechanism. J Physiol Sci. 2014;64:221-232. Marks AR. Calcium cycling proteins and heart failure: mechanisms and therapeutics. J Clin Invest. 2013;123(1):46-52.

1	Marks AR. Calcium cycling proteins and heart failure: mechanisms and therapeutics. J Clin Invest. 2013;123(1):46-52. Tao L, Bei Y, Zhang H, Xiao J, Li X. Exercise for the heart: signaling pathways. Oncotarget. 2015;6:20773-20784. Williams GS, Boyman L, Lederer WJ. Mitochondrial calcium and the regulation of metabolism in the heart. J Mol Cell Cardiol. 2015;78:35-45. Upon completion of this chapter the student should be able to answer the following questions: 1. Describe the organization of smooth muscle in various tissues, and how it meets the demands of each tissue/ organ. 2. Discuss the mechanisms that promote contraction and relaxation of smooth muscle in the vasculature and various organs. 3. Describe the autoregulatory mechanism by which an artery can maintain relatively constant blood flow to a tissue over a broad range of perfusion pressures. 4. Describe the basis and utility of a transition from phasic contraction to tonic contraction. 5.

1	4. Describe the basis and utility of a transition from phasic contraction to tonic contraction. 5. Discuss the length-tension curves and force-velocity curves for smooth muscle, and the molecular basis for each of these curves. If the student has already completed on skeletal muscle and cardiac muscle, comparison of all three tissues for each of the learning objectives listed should be possible.

1	onstriated, or smooth, muscle cells are a major component of hollow organs such as the alimentary canal, airways, vasculature, and urogenital tract. Contraction of smooth muscle serves to alter the dimensions of the organ, which may result in either propelling the contents of the organ (as in peristalsis of the intestine) or increasing the resistance to flow (as in vasoconstriction). The basic mechanism underlying contraction of smooth muscle involves an interaction of myosin with actin (as in striated muscle), although there are some important differences. Specifically, contraction of smooth muscle is thick-filament regulated and requires an alteration in myosin before it can interact with actin, whereas contraction of striated muscle is thin-filament regulated and requires movement of the troponin-tropomyosin complex on the actin filament before myosin can bind to actin. Smooth muscle can contract in response to either electrical or hormonal signals and exhibits the ability to

1	of the troponin-tropomyosin complex on the actin filament before myosin can bind to actin. Smooth muscle can contract in response to either electrical or hormonal signals and exhibits the ability to remain contracted for extended periods at low levels of energy consumption, which is important for functions such as maintaining vascular tone and hence blood pressure. Thus regulation of contraction of smooth muscle is complex, sometimes involving multiple intracellular signaling cascades. In the present chapter, effort is made to identify mechanisms underlying this diverse regulation of smooth muscle contraction and, when appropriate, compare these regulatory mechanisms with those observed in striated muscle. Alterations in smooth muscle function/ regulation that have been implicated in various pathological conditions are also discussed.

1	Overview of Smooth Muscle Types of Smooth Muscle

1	Smooth muscle has been subdivided into two groups: single unit and multiunit. In single-unit smooth muscle the smooth muscle cells are electrically coupled such that electrical stimulation of one cell is followed by stimulation of adjacent smooth muscle cells. This results in a wave of contraction, as in peristalsis. Moreover, this wave of electrical activity, and hence contraction, in single-unit smooth muscle may be initiated by a pacemaker cell (i.e., a smooth muscle cell that exhibits spontaneous depolarization). In contrast, multiunit smooth muscle cells are not electrically coupled, so stimulation of one cell does not necessarily result in activation of adjacent smooth muscle cells. Examples of multiunit smooth muscle include the vas deferens of the male genital tract and the iris of the eye. Smooth muscle, however, is even more diverse, with the single-unit and multiunit classifications representing ends of a spectrum. The terms single-unit and multiunit represent an

1	the iris of the eye. Smooth muscle, however, is even more diverse, with the single-unit and multiunit classifications representing ends of a spectrum. The terms single-unit and multiunit represent an oversimplification, however; many smooth muscles are modulated by a combination of neural elements with at least some degree of cell-to-cell coupling and locally produced activators or inhibitors that also promote a somewhat coordinated response of smooth muscles.

1	A second consideration when discussing types of smooth muscle is the activity pattern ( Fig. 14.1 ). In some organs the smooth muscle cells contract rhythmically or intermittently, whereas in other organs the smooth muscle cells are continuously active and maintain a level of “tone.” Smooth muscle exhibiting rhythmic or intermittent activity is termed phasic smooth muscle and includes smooth muscles in the walls of the gastrointestinal (GI) and urogenital tracts. Such phasic smooth muscle corresponds to the single-unit category described earlier because the smooth muscle cells contract in response to action potentials that propagate from cell to cell. Smooth muscle that is continuously active, on the other hand, is termed tonic smooth muscle.

1	Vascular smooth muscle, respiratory smooth muscle, and some sphincters are continuously active. The continuous partial activation of tonic smooth muscle is not associated with action potentials, although it is proportional to membrane potential. Tonic smooth muscle would thus correspond to the multiunit smooth muscle described earlier. Phasic and tonic contractions of smooth muscle result from interactions of actin and myosin filaments, although as discussed later in this chapter, there is a change in cross-bridge cycling kinetics during tonic contraction, such that the smooth muscle can maintain force at low energy cost.

1	•Fig. 14.1 Somecontractileactivitypatternsexhibitedbysmoothmuscles.Tonicsmoothmusclesarenormallycontractedandgenerateavariablesteady-stateforce.Examplesaresphincters,bloodvessels,andairways.Phasicsmoothmusclescommonlyexhibitrhythmiccontractions(e.g.,peristalsisintheGItract)butmaycontractintermittentlyduringphysiologicalactivitiesundervoluntarycontrol(e.g.,voidingofurinefromurinarybladder,swallowing). Structure of Smooth Muscle Cells

1	Fig. 14.2 ). Blood vessels and airways exhibit a simple tubular structure in which the smooth muscle cells are arranged circumferentially, so contraction reduces the diameter of the tube. This contraction increases resistance to the flow of blood or air but has little effect on the length of the organ. Smooth muscle cell organization is more complex in the GI tract. Layers of smooth muscle in both circumferential and longitudinal orientations provide the mechanical action for mixing food and also propelling the luminal contents from the mouth to the anus. Coordination between these layers depends on a complex system of autonomic nerves linked by plexuses. These plexuses cells in the walls of these organs contributes to their ability to reduce internal volume to almost zero during urination are located between the two muscle layers. The smooth muscle in the walls of saccular structures such as the urinary bladder or rectum allows the organ to increase in size with accumulation of urine

1	are located between the two muscle layers. The smooth muscle in the walls of saccular structures such as the urinary bladder or rectum allows the organ to increase in size with accumulation of urine or feces. The varied arrangement of

1	Normally partially contracted (tone) Blood vessels, airways Stomach, intestines Esophagus, urinary bladder Phasically active or defecation. Smooth muscle cells in hollow organs occur in a spectrum of forms, depending on their function and mechanical loads. In all hollow organs the smooth muscle is separated from the contents of the organ by other cellular elements, which may be as simple as vascular endothelium or as complex as the mucosa of the digestive tract. The walls of hollow organs also contain large amounts of connective tissue that bear an increasing share of the wall stress as organ volume increases.

1	The following sections describe the structural components that enable smooth muscle to set or alter hollow organ volume. These components include contractile and regulatory proteins, force-transmitting systems such as the •Fig. 14.2 Scanningelectronmicrographsofsmoothmuscle.A,Musculararteriolewithfusiformsmoothmusclecellsinacircularorientation(bar, 20µm).B,Superimposedimagesofcircular(below) andlongitudinal(above) layersofintestinalsmoothmusclesandwichingneuralcomponentsofthemyentericplexus(asterisk) (bar, 50mm).C,Rectangularsmoothmusclecellswiththinprojectionstoadjacentcellsinasmalltesticularduct(bar, 5µm).(FromMottaPM[ed].Ultrastructure of Smooth Muscle. Norwell,MA:KluwerAcademic;1990.) cytoskeleton, linkages between cells and the extracellular matrix, and membrane systems that transduce extracellular signals into changes in myoplasmic [Ca++].

1	A variety of specialized contacts exists between smooth muscle cells. Such contacts allow mechanical linkage and communication between cells. In contrast to skeletal muscle cells, which are normally attached at either end to a tendon, smooth (and cardiac) muscle cells are connected to each other. Because smooth muscle cells are anatomically arranged in series, they not only must be mechanically linked but must also be activated simultaneously and to the same degree. This mechanical and functional linkage is crucial to smooth muscle function. If such linkage did Dense bodies c Caveolae Thick filaments Mitochondria Sarcoplasmic reticulum Thick Thin Filaments 0.1 µm0.1 µm C 0.5 mµ not exist, contraction in one region would simply stretch another region without a substantial decrease in radius or increase in pressure. The mechanical connections are provided by attachments to sheaths of connective tissue and by specific junctions between muscle cells.

1	Several types of junctions are found in smooth muscle. Functional linkage of the cells is provided by gap junctions ( Fig. 14.3A ). Gap junctions form low-resistance pathways between cells (see ). They also allow chemical communication by diffusion of low-molecular-weight compounds. In certain tissues, such as the outer longitudinal layer of smooth muscle in the intestine, large numbers of such junctions exist. Action potentials are readily propagated from cell to cell through such tissues.

1	B Surface couplings Sarcoplasmic reticulum 0.5 mµ0.2 mµ 0.5 mµ •Fig. 14.3 Junctions,membranes,andmyofilaments,insmoothmuscle.A,TransmissionelectronmicrographofGapjunctionbetweenintestinalsmoothmusclecells.B,Longitudinalviewofapulmonaryarterysmoothmusclecell.Thesarcoplasmicreticulumisstainedwithosmiumferricyanideandappearstoformacontinuousnetworkthroughoutthecellconsistingoftubules,fenestratedsheets(long arrows), andsurfacecouplingsatthecellmembrane(short arrows). C,Transversesectionofabundleofvenoussmoothmusclecellsillustratingtheregularspacingofthickfilaments(long line) andtherelativelylargenumberofsurroundingthin(actin)filaments(inset). Densebodies(arrowheads) aresitesofattachmentforthethinactinfilamentsandequivalenttotheZlinesofstriatedmuscles.Elementsofsarcoplasmicreticulum(short line) occurattheperipheryofthesecells.D,Scanningelectronmicrographoftheinnersurfaceofthesarcolemmaofanintestinalsmoothmusclecell.Longitudinalrowsofcaveolaeprojectintothemyoplasm(small, light-colored

1	line) occurattheperipheryofthesecells.D,Scanningelectronmicrographoftheinnersurfaceofthesarcolemmaofanintestinalsmoothmusclecell.Longitudinalrowsofcaveolaeprojectintothemyoplasm(small, light-colored spheres), surroundedbydarkerelementsofthetubularsarcoplasmicreticulum.Theattachmentsofthinfilamentstothesarcolemmabetweentherowsofmembraneelementswereremovedduringpreparationofthespecimen.(AandD,FromMottaPM[ed].Ultrastructure of Smooth Muscle. Norwell,MA:KluwerAcademic;1990.BandC,FromSomlyoAP,SomlyoAV.Smoothmusclestructureandfunction.In:FozzardHAetal.[eds].The Heart and Cardiovascular System.2nded.NewYork:RavenPress;1992.) muscle cells. The adherens junction appears as thickened regions of opposing cell membranes that are separated by a small gap (≈60 nm) containing dense granular material. Thin filaments extend into the adherens junction to allow the contractile force generated in one smooth muscle cell to be transmitted to adjacent smooth muscle cells.

1	Embryonic smooth muscle cells do not fuse, and each differentiated cell has a single centrally located nucleus. Though dwarfed by skeletal muscle cells, smooth muscle cells are nevertheless quite large (typically 40–600 µm long). These cells are 2 to 10 µm in diameter in the region of the nucleus, and most taper toward their ends. Contracting cells become quite distorted as a result of the force exerted on the cell by attachments to other cells or to the extracellular matrix, and cross sections of these cells are often very irregular. Smooth muscle cells lack T tubules, the invaginations of the skeletal muscle sarcolemma that provide electrical links to the sarcoplasmic reticulum (SR). However, the sarcolemma of smooth muscle has longitudinal rows of tiny sac-like inpocketings called caveolae (see

1	Fig. 14.3B–D ). Caveolae increase the surface-to-volume ratio of the cells and are often closely apposed to the underlying SR. A gap of approximately 15 nm has been observed between the caveolae and the underlying SR, comparable to the gap between the T tubules and terminal SR in skeletal muscle. Moreover, “Ca++ sparks” and a variety of Ca++-handling proteins have been observed in the vicinity of caveolae, thus raising the possibility that the caveolae and the underlying SR may contribute to regulation of intracellular [Ca++] in smooth muscle. The voltage-gated L-type Ca++ channel and the 3Na+-1Ca++ antiporter, for example, are associated with caveolae. The proteins caveolin and cholesterol are both critical for the formation of caveolae, and it is hypothesized that the caveolae reflect a specialized region of the sarcolemma that may also contain various signaling molecules in addition to the Ca++ signaling mentioned earlier.

1	Smooth muscle also has an intracellular membrane network of SR that serves as an intracellular reservoir for Ca++ (see

1	Fig. 14.3B–D ). Calcium can be released from the SR into the myoplasm when stimulatory neurotransmitters, hormones, or drugs bind to receptors on the sarcolemma. Importantly, intracellular Ca++ channels in the SR of smooth muscle include the ryanodine receptor (RYR), which is similar to that found in skeletal muscle SR, and the inositol 1,4,5-trisphosphate (InsP3)-gated Ca++ channel. The RYR is typically activated by a rise in intracellular [Ca++] (i.e., Ca++-induced release of Ca++ in response to an influx of Ca++ through the sarcolemma). The InsP3-gated Ca++ channel is activated by InsP3, which is produced when a hormone or hormones bind to various Ca++-mobilizing receptors on the sarcolemma. Intracellular [Ca++] is lowered through the action of an SR Ca++-ATPase (SERCA) and extrusion of Ca++ from the cell via a 3Na+-1Ca++ antiporter and a sarcolemmal Ca++-ATPase. The amount of SR in smooth muscle cells varies from 2% to 6% of cell volume and approximates that of skeletal muscle.

1	of Ca++ from the cell via a 3Na+-1Ca++ antiporter and a sarcolemmal Ca++-ATPase. The amount of SR in smooth muscle cells varies from 2% to 6% of cell volume and approximates that of skeletal muscle. Chemical signals such as InsP3 or a localized increase in intracellular [Ca++] (e.g., within the gap between the caveolae and SR) functionally link the sarcolemma and the SR.

1	Smooth muscle cells contain a prominent rough endoplasmic reticulum and Golgi apparatus, which are located centrally at each end of the nucleus. These structures reflect significant protein synthetic and secretory functions. The scattered mitochondria are sufficient for oxidative phosphorylation to generate the increased adenosine triphosphate (ATP) consumed during contraction.

1	The thick and thin filaments of smooth muscle cells are about 10,000 times longer than their diameter and are tightly packed. Therefore the probability of observing an intact filament by electron microscopy is extremely low. In contrast to skeletal muscle, which contains a transverse alignment of thick and thin filaments that results in striations, the contractile filaments in smooth muscle are not in uniform transverse alignment, and thus smooth muscle has no striations. The lack of striations in smooth muscle does not imply a lack of order. The thick and thin filaments are organized in contractile units that are analogous to sarcomeres.

1	The thin filaments of smooth muscle have an actin and tropomyosin composition and structure similar to that in skeletal muscle. However, the cellular content of actin and tropomyosin in smooth muscle is about twice that of striated muscle. Smooth muscle lacks troponin and nebulin but contains two proteins not found in striated muscle: caldesmon and calponin. The precise roles of these proteins are unknown, but they do not appear to be fundamental to cross-bridge cycling. It has been suggested that both calponin and caldesmon may regulate the contractility of smooth muscle. (in part by inhibiting actomyosin ATPase activity). Most of the myoplasm is filled with thin filaments that are roughly aligned along the long axis of the cell. The myosin content of smooth muscle is only a fourth that of striated muscle. Small groups of three to five thick filaments are aligned and surrounded by many thin filaments. These groups of thick filaments with interdigitating thin filaments are connected

1	of striated muscle. Small groups of three to five thick filaments are aligned and surrounded by many thin filaments. These groups of thick filaments with interdigitating thin filaments are connected to dense bodies or areas (

1	Fig. 14.4 Fig. 14.3A–B ) and represent the equivalent of the sarcomere. The contractile apparatus of adjacent cells is mechanically coupled by the links between membrane-dense areas.

1	The cytoskeleton in smooth muscle cells serves as an attachment point for the thin filaments and permits transmission of force to the ends of the cell. In contrast to skeletal muscle, the contractile apparatus in smooth muscle is not organized into myofibrils, and Z lines are lacking. The functional equivalents of the Z lines in smooth muscle cells •Fig. 14.4 Apparentorganizationofcell-to-cellcontacts,cytoskeleton,andmyofilamentsinsmoothmusclecells.Smallcontractileelementsfunctionallyequivalenttoasarcomereunderliethesimilaritiesinmechanicsbetweensmoothandskeletalmuscle.Linkagesconsistingofspecializedjunctionsorinterstitialfibrillarmaterialfunctionallycouplethecontractileapparatusofadjacentcells.Densebodies,thefunctionalequivalentofZlinesinstriatedmuscle,areinterconnectedbyintermediatefilaments.Themyofilamentsareorientedlargelyinparallelwiththelongitudinalaxisofthecell,thoughanobliqueorientationhasbeenobservedinsomearteries.

1	are ellipsoidal dense bodies in the myoplasm and dense areas that form bands along the sarcolemma (see Figs. 14.3A–B 14.4 ). These structures serve as attachment points for the thin filaments and contain α-actinin, a protein also found in the Z lines of striated muscle. Intermediate filaments with diameters between those of thin filaments (7 nm) and thick filaments (15 nm) are prominent in smooth muscle. These filaments link the dense bodies and areas into a cytoskeletal network. The intermediate filaments consist of protein polymers of desmin or vimentin. Control of Smooth Muscle Activity

1	Control of Smooth Muscle Activity The contractile activity of smooth muscle can be controlled by numerous factors, including hormones, autonomic nerves, pacemaker activity, and a variety of drugs. Like skeletal or cardiac muscle, contraction of smooth muscle is dependent on Ca++ , and the agents just listed induce smooth muscle contraction by increasing intracellular [Ca++]. However, in contrast to skeletal or cardiac muscle, action potentials in smooth muscle are highly variable and not always needed to initiate contraction. Moreover, several agents can increase intracellular [Ca++] and hence contract smooth muscle without changing the membrane potential.

1	Fig. 14.5 shows various types of action potentials in smooth muscle and the corresponding changes in force. An action potential in smooth muscle can be associated with a slow twitch-like response, and the twitch forces can summate during periods of repetitive action potentials (i.e., similar to tetany in skeletal muscle). Such a pattern of activity is characteristic of single-unit smooth muscle in many viscera.

1	Periodic oscillations in membrane potential can occur as a result of changes in the activity of Na+,K+-ATPase in the sarcolemma. These oscillations in membrane potential can trigger multiple action potentials in the cell. Alternatively the contractile activity of smooth muscle may not be associated with generation of action potentials or even a change in membrane potential. In many smooth muscles the resting membrane potential is sufficiently depolarized (−60 to −40 mV) that a small decrease in membrane potential can significantly inhibit influx of Ca++ through voltage-gated Ca++ channels in the sarcolemma. By decreasing Ca++ influx, the force developed by smooth muscle decreases. Such a graded response to slight changes in the resting membrane potential is common in multiunit smooth muscles that maintain constant tension (e.g., vascular smooth muscle).

1	Contraction of smooth muscle in response to an agent that does not produce a change in membrane potential is termed pharmacomechanical coupling and typically reflects the ability of the agent to increase the level of the intracellular second messenger InsP3. Other agents result in a decrease in tension, also without a change in membrane potential. These agents typically increase levels of the intracellular second messengers cyclic guanosine monophosphate (cGMP) or cyclic adenosine monophosphate (cAMP). The molecular mechanisms by which InsP3, cGMP, cAMP, and Ca++ alter the contractile force of smooth muscle are presented later.

1	Phosphorylation of a myosin light chain is required for the interaction of myosin with actin, and although Ca++dependent phosphorylation plays a key role in this process, the level of myosin phosphorylation (and hence the degree of contraction) is dependent on the relative activities of both myosin light-chain kinase (MLCK, which promotes phosphorylation) and myosin phosphatase (MP, which promotes dephosphorylation). Several agonists/hormones increase the level of myosin light-chain phosphorylation by simultaneously activating MLCK through an increase in intracellular [Ca++] and inhibiting MP through a signaling cascade involving the monomeric G protein RhoA and its effector Rho kinase (ROK). Moreover, hyperactivity of this RhoA/ROK signaling cascade has been implicated in various pathological conditions such as hypertension and vasospasm (discussed later). Innervation of Smooth Muscle

1	Neural regulation of smooth muscle contraction depends on the type of innervation and neurotransmitters released, the proximity of the nerves to the muscle cells, and the •Fig. 14.5

1	depends on the type of innervation and neurotransmitters released, the proximity of the nerves to the muscle cells, and the •Fig. 14.5 Relationshipsbetweenmembranepotential(Em)andgenerationofforce(F)indifferenttypesofsmoothmuscle.A,Actionpotentialsmaybegeneratedandleadtoatwitchorlargersummedmechanicalresponses.Actionpotentialsarecharacteristicofsingle-unitsmoothmuscles(manyviscera).Gapjunctionspermitthespreadofactionpotentialsthroughoutthetissue.B,Rhythmicactivityproducedbyslowwavesthattriggeractionpotentials.Thecontractionsaregenerallyassociatedwithaburstofactionpotentials.Slowoscillationsinmembranepotentialusuallyreflecttheactivityofelectrogenicpumpsinthecellmembrane.C,Toniccontractileactivitymayberelatedtothevalueofthemembranepotentialintheabsenceofactionpotentials.GradedchangesinEmarecommoninmultiunitsmoothmuscles(e.g.,vascular),whereactionpotentialsarenotgeneratedandpropagatedfromcelltocell.D,Pharmacomechanicalcoupling;changesinforceproducedbytheadditionorremoval(arrows)

1	ofdrugsorhormonesthathavenosignificanteffectonmembranepotential.

1	type and distribution of the neurotransmitter receptors on the muscle cell membranes ( Fig. 14.6 ). In general, smooth muscle is innervated by the autonomic nervous system. The smooth muscle in arteries is innervated primarily by sympathetic fibers, whereas the smooth muscle in other tissues can have both sympathetic and parasympathetic innervation. In the GI tract, smooth muscle is innervated by nerve plexuses that make up the enteric nervous system. The smooth muscle cells of some tissues (e.g., uterus) have no innervation. The neuromuscular junctions and neuromuscular transmission in smooth muscle are functionally comparable to that of skeletal muscle but structurally less complex. The autonomic nerves that supply smooth muscle have a series of swollen areas, or varicosities, that are spaced at intervals along the axon. These varicosities contain vesicles for the neurotransmitter (see Fig. 14.6).

1	Fig. 14.6). The postsynaptic membrane of smooth muscle exhibits little specialization when compared with that of skeletal muscle (see ). The synaptic cleft is typically about 80 to 120 nm wide but can be as narrow as 6 to 20 nm or even greater than 120 nm. In synapses in which a wide synaptic cleft is found, release of neurotransmitter can affect multiple smooth muscle cells. There are a large number of neurotransmitters that affect smooth muscle activity. A partial listing is provided in Table 14.1 Regulation of Contraction Contraction of smooth muscle requires phosphorylation of a myosin light chain. Typically this phosphorylation occurs in response to a rise in intracellular [Ca++], either after an action potential or in the presence of a hormone/agonist. As depicted in

1	Fig. 14.7 , a rise in intracellular [Ca++] in smooth muscle results in the binding of 4 Ca++ ions to the protein calmodulin, and then the Ca++-calmodulin complex activates MLCK, which phosphorylates the regulatory light chain of myosin. This phosphorylation step is critical for the interaction of smooth muscle myosin with actin. In addition to this phosphorylation step in smooth muscle,

1	Varicosity with neurotransmitter vesicles •Fig. 14.6 Controlsystemsofsmoothmuscle.Inbothmultiunitsmoothmuscle(A)andsingleunitsmoothmuscle(B),neurallyreleasedtransmittersorcirculatingorlocallygeneratedhormonesorsignalingmoleculescaninducecontractionorrelaxationofthesmoothmuscle.Thecombinationofaneurotransmitter,hormone,ordrugwithspecificreceptorsactivatescontractionbyincreasingcellCa++

1	.Theresponseofthecellsdependsontheconcentrationofthetransmittersorhormonesatthecellmembraneandthenatureofthereceptorspresent.Hormoneconcentrationsdependondiffusiondistance,release,reuptake,andcatabolism.Consequently,cellslackingcloseneuromuscularcontactswillhavealimitedresponsetoneuralactivityunlesstheyareelectricallycoupledsothatdepolarizationistransmittedfromcelltocell.A,Multiunitsmoothmusclesresemblestriatedmusclesinthatthereisnoelectricalcouplingandneuralregulationisimportant.B,Single-unitsmoothmusclesarelikecardiacmuscle,andelectricalactivityispropagatedthroughoutthetissue.Mostsmoothmusclesprobablyliebetweenthetwoendsofthesingleunit–multiunitspectrum.C,Scanningmicrographofvaricosenervelyingoverratsmallintestinescalebar= 3µm.D,Fluorescentimageofcatecholaminesinsingleadrenergicaxoninguineapigmesentery.Scalebar= 10µm.E,Depictionofdistributionofsympatheticnerves(in black)

1	3µm.D,Fluorescentimageofcatecholaminesinsingleadrenergicaxoninguineapigmesentery.Scalebar= 10µm.E,Depictionofdistributionofsympatheticnerves(in black) invasculature.Sympatheticnervesareassociatedwithsmallarteries(SA),largearterioles(la),smallveins(SV),largevenules(lv),smallarterioles(sa),andterminalarterioles(TA).Capillaries(c),post-capillaryvenules(pcv),andsmallvenules(svl)appeartolacksympatheticinnervation.(C,FromBurnstockG:Autonomicneuralcontrolmechanisms.Withspecialreferencetotheairways.InKalinerMA,BarnesPJ(Eds.),TheAirways.NeuralControlinHealthandDisease,MarcelDekker,NewYork(1988),pp.1–22.D,FromChamleyJH,MarkGE,CampbellGR,BurnstockG.(1972)Sympatheticgangliainculture.I.Neurons.Z.Zellforsch.Mikrosk.Anat.135,287-314.)

1	aThepredominanteffectofsympatheticstimulationissmoothmusclecontractioncausedbytheabundanceofα1-ARrelativetoβ2-ARinsmoothmuscle.bActivationofβ2-ARonsmoothmusclemodulatesthedegreeofsmoothmusclecontractionduringsympatheticstimulation.Therapeuticβ2-ARagonistsareimportantfortherelaxationofbronchialsmoothmuscleduringasthmaticattacks.cVascularsmoothmusclesarepoorlyinnervatedbytheparasympatheticsystem.Duringvagalstimulation,however,acetylcholine(ACh)canbecomeelevatedinthecoronarycirculationandresultincoronaryrelaxation(mediatedbybindingofAChtoendothelialcells).NotethatthiseffectofAChisindirectbecausebindingofAChtoendothelialcellsresultsinreleaseofthesmoothmusclerelaxantnitricoxidefromtheendothelialcells.Inregionsofthecoronarycirculationwithdamagedendothelium,bindingofAChtocoronarysmoothmusclecouldpromotecontraction(vasospasm;directeffect).dAvarietyofhormonescanelevateInsP3insmoothmuscleandtherebyresultinsmoothmusclecontraction.SuchhormonesincludeangiotensinII,vasopressin,andendothelin,alongwi

1	dpromotecontraction(vasospasm;directeffect).dAvarietyofhormonescanelevateInsP3insmoothmuscleandtherebyresultinsmoothmusclecontraction.SuchhormonesincludeangiotensinII,vasopressin,andendothelin,alongwiththeneurotransmittersnorepinephrineandacetylcholine.Asnotedabove,however,eachhormone/transmitterbindstoaspecificreceptortype.eDuringperiodsofintensemuscularactivity,adenosinecanbereleasedfromtheworkingmuscle,diffusetotheneighboringvasculature,andpromotevasodilation.Thus,adenosineisactingasalocalfactortoincreasebloodflowtoaspecificregion(i.e.,workingmuscle).AR,adrenergicreceptor;EC,endothelialcell;InsP3,inositol1,4,5-trisphosphate;SMC,smoothmusclecell.

1	an ATP molecule is also needed to energize the myosin cross-bridge for the development of force. TheentericnervoussystemcontrolsmanyaspectsofGIfunction,includingmotility.Somechildrenarebornwithoutentericnervesinthedistalportionofthecolon.Theabsenceofnervesiscausedbymutantgenesthatdisruptthesignalsnecessaryfortheembryonicnervestomigratetothecolon.Inthesechildren,normalmotilityofthecolondoesnotoccurandsevereconstipationresults.ThisconditioniscalledHirschsprung’s disease. Itcanbecorrectedbysurgicallyremovingtheportionofthecolonthatdoesnotcontainentericnerves. Contraction of smooth muscle is thus said to be thick-filament regulated, which contrasts with the thin-filament regulation of contraction of striated muscle, where binding of Ca++ to troponin exposes myosin binding sites on the actin thin filament. The thick-filament regulation is attributable to expression of a distinct myosin isoform in smooth muscle.

1	The myosin cross-bridge cycle in smooth muscle is similar to that in striated muscle in that after attachment to the actin filament, the cross-bridge undergoes a ratchet action in which the thin filament is pulled toward the center of the thick filament and force is generated. ADP and Pi are released from the myosin head at this time, thereby allowing ATP to bind. ATP decreases the affinity of myosin for actin, which allows release of myosin from actin. Energy from the newly bound ATP is then used to produce a conformational change in the myosin head (i.e., recocking the head) so that the cross-bridge is ready for another contraction cycle. The cross-bridge cycle continues as long as the myosin cross-bridge remains phosphorylated. Note that although the four basic steps of the cross-bridge cycle appear to be the same for striated and smooth muscle, the kinetics of cross-bridge cycling is much slower for smooth muscle.

1	Cross-bridge cycling continues with the hydrolysis of 1 ATP molecule per cycle until myoplasmic [Ca++] falls. With the decrease in [Ca++], MLCK becomes inactive, and the cross-bridges are dephosphorylated by MP (see Fig. 14.7 Fig. 14.4 , the thin filaments in smooth muscle are attached to dense bodies, and the myosin thick filaments appear to reside between two dense bodies and overlap a portion of the thin filaments, much like the overlap of thick and thin filaments in the sarcomere of striated muscle. A bipolar arrangement of myosin molecules within the thick filament is thought to allow the myosin cross-bridges to pull the actin filaments toward the center of the thick filament, thus contracting the smooth muscle and hence developing force.

1	From a structural standpoint, smooth muscle myosin is similar to striated muscle myosin in that they both contain a pair of heavy chains and two pairs of light chains. Despite this similarity, they represent different gene products and thus have different amino acid sequences. As noted, smooth muscle myosin, unlike skeletal muscle myosin, is unable to interact with the actin thin filament unless the regulatory light chain of myosin is phosphorylated. Moreover, the thin filament in smooth muscle lacks troponin, which plays a •Fig. 14.7

1	is unable to interact with the actin thin filament unless the regulatory light chain of myosin is phosphorylated. Moreover, the thin filament in smooth muscle lacks troponin, which plays a •Fig. 14.7 RegulationofsmoothmusclemyosininteractionswithactinbyCa++-stimulatedphosphorylation.Intherelaxedstate,cross-bridgesarepresentasahigh-energymyosin-ADP-PicomplexinthepresenceofATP.Attachmenttoactindependsonphosphorylationofthecross-bridgebyaCa++-calmodulin–dependentmyosinlight-chainkinase(MLCK).Phosphorylatedcross-bridgescycleuntiltheyaredephosphorylatedbymyosinphosphatase.Notethatcross-bridgephosphorylationataspecificsiteonamyosinregulatorylightchainrequiresATPinadditiontothatusedineachcyclicinteractionwithactin.

1	critical role in the thin-filament regulation of contraction in striated muscle (see Although intracellular Ca++ is required for smooth muscle contraction, the sensitivity of contraction to Ca++ is variable. Several hormones/agonists, for example, increase the force of contraction at a given submaximal intracellular [Ca++], thereby resulting in Ca++ sensitization ( Fig. 14.8 ). Ca++ sensitization is depicted as a leftward shift in the Ca++ dependence of smooth muscle contraction (see Fig. 14.8B ) and can occur in response to a decrease in the activity of MP activity at a given intracellular [Ca++]. Likewise an increase in MP activity at a given intracellular [Ca++] promotes a rightward shift in the Ca++ dependence of smooth muscle contraction, resulting in Ca++ desensitization (see Fig. 14.8B ). Reciprocal changes in the activities of MP and MLCK can occur, as shown in

1	Fig. 14.8B ). Reciprocal changes in the activities of MP and MLCK can occur, as shown in Fig. 14.8A , where stimulation of the ROK signaling cascade by an agonist can simultaneously inhibit MP and stimulate MLCK at a given intracellular [Ca++], thereby increasing the Ca++ sensitivity of contraction (i.e., Ca++ sensitization). Fig. 14.8A ).RhoAactivatesRhokinase(ROK),whichinturninhibitsMPbybothdirectandindirectmechanisms.DirectinhibitionofMPbyactivatedROKinvolvesROKphosphorylationofthemyosin-bindingsubunit(MBS)ofMP.IndirectinhibitionofMPbyactivatedROKinvolvesphosphorylationofCPI-17, anendogenous17-kDaprotein,whichtheninhibitsMP.Hormones/agonistssuchascatecholamines(actingonα1-adrenergicreceptors),vasopressin,endothelin,angiotensin,andmuscarinicagonistsincreasethesensitivityofsmoothmusclecontractiontoCa++ throughactivationofRhoA/ROKsignaling.ROKcanalsobeactivatedbyarachidonicacidandinhibitedbyY-27632,ahighlyspecificinhibitor(see Fig.14.8A ).Thoughnotshownin

1	Fig.14.8A ).Thoughnotshownin Fig.14.8 ,inactiveRhoAistypicallylocatedinthecytosol,boundtoGDPandaninhibitoryprotein(Rho-GDP dissociation inhibitor [GDI]). BindingofagonisttovariousG-coupledreceptorscanactivateRhoAbystimulatingguanine nucleotide exchange factor (GEF) toyieldRhoA-GTP,whichlocalizestothesarcolemmaandactivatesROK.Conversely,stimulationofMPactivityreducestheCa++ sensitivityofcontractionofsmoothmuscle,therebypromotingrelaxation(andhencevasodilation).

1	HyperactivityoftheRhoA/ROKsignalingcascadehasbeenimplicatedinvariouspathologicalconditionssuchashypertensionandvasospasm.HyperactivityofRhoA/ROKinthevascularsmoothmuscleofhypertensiveanimals,forexample,wasmanifestedbyincreasedlevelsofactivatedRhoA,upregulationofROK,enhancementofagonist-inducedCa++

1	sensitizationofcontraction,andagreaterreductioninbloodpressurebyROKinhibitorsascomparedwithnormotensivecontrols.Asimilartrendwasobservedinhumans,inthatROKinhibitorsdecreasedforearmvascularresistanceinhypertensivepatientstoagreaterextentthaninnormotensivecontrols.ROKinhibitorshavealsobeenshowntoreverseorpreventexperimentallyinducedcerebralvasospasmandcoronaryvasospasm,aswellastheassociatedupregulationofRhoA/ROKandincreasedmyosinlight-chainphosphorylation.HyperactivityofRhoA/ROKhasadditionallybeenimplicatedinbronchialasthma,erectiledysfunction,andpretermlabor,asevidencedbytheeffectsofROKinhibitors.Inaddition,ROKinhibitorshavedecreasedvascularsmoothmuscleproliferationandreducedrestenosisafterballoonangioplastyinratcarotidartery.

1	During a phasic contraction, myoplasmic [Ca++], cross-bridge phosphorylation, and force reach a peak and then return to baseline ( Fig. 14.9 ). In contrast, during a tonic contraction, myoplasmic [Ca++] and cross-bridge phosphorylation decline after an initial spike but do not return to baseline levels. During this later phase, force slowly increases and is sustained at a high level (see Fig. 14.9). This sustained force is maintained with only 20% to 30% of the cross-bridges phosphorylated, and thus ATP utilization is reduced. The term latch state refers to this condition of tonic contraction during which force is maintained at low energy expenditure.

1	P P Ins(1,4,5)P3 Ca++ SR Ca++ CaM MLCK MLC MLC P GEFs Rho Rho-kinase CPI-17 CPI-17 MBS M20 cat MBS Inactive Active Contraction M20 cat MP Y27632 GTP ? AA PLC GqG12/13 •Fig. 14.8 Rho-kinase(ROK)signalinginsmoothmuscle.A,AvarietyofagonistsofG-coupledreceptorssimultaneouslystimulateInsP3productionandactivateRhoA-ROKsignaling.InsP3(Ins[1,4,5]P3)isproducedbyphospholipaseC(PLC)-mediatedhydrolysisofPIP2.InsP3increasesintracellular[Ca++]byopeningInsP3-gatedCa++

1	channelsinthesarcoplasmicreticulum(SR),therebyresultinginCa++-calmodulin–dependentactivationofmyosinlight-chainkinase(MLCK)andsubsequentphosphorylationofthemyosinregulatorylightchain(MLC)andpromotionofactin-myosininteraction(contraction).ActivatedRhoA(depictedasRho-GTP)stimulatesROK,whichinhibitsmyosinphosphatase(MP)byphosphorylatingthemyosin-bindingsubunit(MBS)ofMP.ROKalsoinhibitsMPindirectlybyphosphorylating/activatingCPI-17,a17-kDainhibitorofMP.TheneteffectofROKphosphorylationsisadecreaseinMPactivity,whichresultsinanincreasedlevelofmyosinlight-chainphosphorylation(MLCP)andhencegreaterforceofcontractionatagivenintracellular[Ca++](i.e.,increasedsensitivityofcontractiontoCa++).B,Ca++ sensitization referstoanincreaseintheforceofcontractionatagivenintracellular[Ca++]andisdepictedasaleftwardshiftintheCa++ dependenceofcontractionofsmoothmuscle.Ca++

1	sensitization referstoanincreaseintheforceofcontractionatagivenintracellular[Ca++]andisdepictedasaleftwardshiftintheCa++ dependenceofcontractionofsmoothmuscle.Ca++ sensitizationcanresultfromadecreaseinMPactivityand/oranincreaseinMLCKactivityatagivenintracellular[Ca++](asshownintheROKsignalingcascadeinpanelA).Conversely,Ca++ desensitization referstoadecreaseintheforceofcontractionatagivenintracellular[Ca++]andisdepictedasarightwardshiftintheCa++ dependenceofcontraction.AA,arachidonicacid;CaM,Ca++-calmodulincomplex;cat,catalyticsubunitofmyosinphosphatase;G12/13andGq,heterotrimericG-proteins;GEF,guanine-nucleotideexchangefactor;M20,subunitofmyosinphosphatase;MP,myosinphosphatasecomplex;Y27632,commercialinhibitorofROK.(FromFukataYetal.Trends Pharmacol Sci 2001;22:32-39.) •Fig. 14.9 Timecourseofeventsincross-bridgeactivationandcontractioninsmoothmuscle.A,AbriefperiodofstimulationisassociatedwithCa++

1	Pharmacol Sci 2001;22:32-39.) •Fig. 14.9 Timecourseofeventsincross-bridgeactivationandcontractioninsmoothmuscle.A,AbriefperiodofstimulationisassociatedwithCa++ mobilization,followedbycross-bridgephosphorylationandcyclingtoproduceabriefphasic,twitch-likecontraction.B,Inasustainedtoniccontractionproducedbyprolongedstimulation,theCa++ andphosphorylationlevelstypicallyfallfromaninitialpeak.Forceismaintainedduringtoniccontractionsatareduced[Ca++](andhencealowlevelofmyosinlight-chainphosphorylation),withlowercross-bridgecyclingratesmanifestedbylowershorteningvelocitiesandATPconsumption.

1	The latch state is thought to reflect a slowing of the cross-bridge cycle, so that the myosin heads remain in contact with the actin filament for a longer time, thereby maintaining tension at low energy cost. Note that the intracellular [Ca++] falls to a low level during the tonic phase of contraction, though it is still above the resting/ basal [Ca++]. The mechanism contributing to the ability of smooth muscle to maintain force at a low intracellular [Ca++] during tonic contraction is thought to involve dephosphorylation of the myosin regulatory light chain while the myosin cross-bridge is attached to the actin filament, resulting in slowing of the rate of dissociation of the myosin from the actin, allowing the myosin to spend more time in an attached, force-generating conformation. Relaxation of smooth muscle following tonic contraction occurs when intracellular [Ca++] decreases to a level that prevents a net phosphorylation of the regulatory light chain by MLCK. It has also been

1	of smooth muscle following tonic contraction occurs when intracellular [Ca++] decreases to a level that prevents a net phosphorylation of the regulatory light chain by MLCK. It has also been proposed that caldesmon may participate in the transition to the latch state.

1	As already noted, ATP consumption is reduced during the latch state. Under this condition, smooth muscle uses 300fold less ATP than would be required by skeletal muscle to generate the same force. Smooth muscle, like skeletal muscle, requires ATP for ion transport to maintain the resting membrane potential, sequester Ca++ in the SR, and extrude Ca++ from the cell. All these metabolic needs are readily met by oxidative phosphorylation. Fatigue of smooth muscle does not occur unless the cell is deprived of oxygen. However, aerobic glycolysis with lactic acid production normally supports membrane ion pumps even when oxygen is plentiful. Regulation of Myoplasmic Calcium Concentration

1	Regulation of Myoplasmic Calcium Concentration The mechanisms that couple activation to contraction in smooth muscle involve two Ca++ sources: one involving the sarcolemma and the other involving the SR. The sarcolemma regulates Ca++ influx and efflux from the extracellular Ca++ pool. The SR membranes determine Ca++ movement between the myoplasm and the SR pool. Skeletal muscle contraction does not require extracellular Ca++ (see ). In contrast, extracellular Ca++ is important for smooth muscle contraction. Thus regulation of myoplasmic [Ca++] involves not only the SR but also the sarcolemma ( Fig. 14.10). A number of factors can alter the myoplasmic Na+-K+ pump 2K+ 3 Na+ Ca++ MLCK off CaCM CM ATP Receptor-activated CaCM*MLCK ATP G G Sarcoplasmic reticulum Time IP3 PIP2 3Na Cell Ca++ Neurotransmitter or hormone Neurotransmitter or hormone Ca++ channel Refilling ATP ?

1	•Fig. 14.10 Principalmechanismsdeterminingmyoplasmic[Ca++]insmoothmuscle.Releaseofcalciumfromthesarcoplasmicreticulum(SR)isarapidinitialeventinactivation,whereasboththeSRandthesarcolemmaparticipateinthesubsequentstimulus-dependentregulationofmyoplasmic[Ca++].Thesarcolemmaintegratesmanysimultaneousexcitatoryandinhibitoryinputstogovernthecellularresponse.Higher-orderregulatorymechanismscanaltertheactivityofvariouspumps,exchangers,orenzymes(theasterisk designateswell-establishedinstances).ThelabelATP indicatesthattheprocessrequiresATPhydrolysis;thequestion mark onthesarcolemmareferstotheCa++ pathwayimportantforrefillingtheSR(i.e.,store-operatedCa++ entry[SOCE]),whichappearstoinvolvetheinteractionofaCa++-sensitivestromalinteractionmolecule(STIM)ontheSRandthesarcolemmalCa++ channelOrai.CaCM,Ca++-calmodulincomplex;G,guaninenucleotide–bindingproteins;IP3,inositol1,4,5-trisphosphate;MLCK,myosinlight-chainkinase;PIP2,phosphatidylinositolbisphosphate;PLC,phospholipaseC.

1	Inappropriatecontractionofsmoothmuscleisassociatedwithmanypathologicalsituations.Oneexampleissustainedvasospasmofacerebralarterythatdevelopsseveralhoursafterasubarachnoidhemorrhage.Itisthoughtthatfreeradicalsgeneratedasaresultofthehemorrhageraisemyoplasmic[Ca++]insurroundingarterialsmoothmusclecells.Theriseinmyoplasmic[Ca++]activatesMLCK,whichleadstocross-bridgephosphorylationandcontraction.Thevasoconstrictiondeprivesotherareasofthebrainofoxygenandmayleadtopermanentinjuryordeathofsurroundingneurons.Forafewdaysthecerebralarteryremainssensitivetovasoactiveagents,andthereforetreatmentwithvasodilatorsmayrestoreflow.AnincreaseinROKactivityandMPphosphorylationhasbeenobservedduringcerebralvasospasm.AdministrationofROKinhibitorspromotesrelaxationofthevasospasmanddecreasesthelevelofmyosinlight-chainphosphorylation.Thesmoothmusclecellsceasetorespondtothevasodilatorsafterseveraldays,andtheylosecontractileproteinsandsecreteextracellularcollagen.Thelumenofthearteryremainsconstrictedasaresultofstru

1	inphosphorylation.Thesmoothmusclecellsceasetorespondtothevasodilatorsafterseveraldays,andtheylosecontractileproteinsandsecreteextracellularcollagen.Thelumenofthearteryremainsconstrictedasaresultofstructuralandmechanicalchangesthatdonotinvolveactivecontraction.

1	[Ca++] of smooth muscle. This differs from skeletal muscle, in which action potential–induced release of Ca++ from the SR fully activates the contractile apparatus.

1	The role of smooth muscle SR in regulating myoplasmic [Ca++] is comparable to that of skeletal muscle. Stimulation of the cell opens SR Ca++ channels, and myoplasmic [Ca++] increases rapidly. This release is not linked to voltage sensors, as is the case in skeletal muscle, but to binding of the second messenger InsP3 to receptors in the SR. InsP3 is generated by a stimulus that acts on sarcolemmal receptors that are coupled via a guanine nucleotide–binding protein (G protein) to activate phospholipase C (PLC) (see ). PLC hydrolyzes the membrane phospholipid phosphatidylinositol bisphosphate (PIP2) into InsP3 and diacylglycerol. InsP3 then diffuses to the SR and opens the InsP3-gated Ca++ channel, thereby resulting in release of Ca++ from the SR into the myoplasm. This complex process may permit graded release of Ca++ from the SR and also enable many different neurotransmitters and hormones to effect smooth muscle contraction. Calcium is reaccumulated by the SR through the activity of

1	permit graded release of Ca++ from the SR and also enable many different neurotransmitters and hormones to effect smooth muscle contraction. Calcium is reaccumulated by the SR through the activity of the SERCA, although as indicated later, extrusion of Ca++ from the smooth muscle cell also contributes to the reduction in myoplasmic [Ca++]. Refilling of the SR with Ca++ not only involves reaccumulation of cytosolic Ca++ but also depends on the extracellular [Ca++]. The dependence on extracellular [Ca++] is thought to reflect the operation of a “store-operated” Ca++ channel present in the sarcolemma at points near underlying SR called junctional SR.

1	A variety of hormones and neurotransmitters elevate myoplasmic [Ca++] by stimulating InsP3 production. Vascular smooth muscle, for example, is innervated by sympathetic fibers of the autonomic nervous system. These fibers use norepinephrine as a neurotransmitter, which when released binds to α1-adrenergic receptors on vascular smooth muscle cells and results in G protein–dependent activation of PLC. Activation of PLC results in production of InsP3, which activates the InsP3-gated Ca++ channel in the SR, thereby elevating myoplasmic [Ca++] and causing vasoconstriction. Other agents that promote vasoconstriction by activating the InsP3 cascade include angiotensin II and vasopressin. Development of drugs that block production of angiotensin II (e.g., angiotensin-converting enzyme [ACE] inhibitors) provides a means of promoting vasodilation that is important for individuals with hypertension or congestive heart failure. As mentioned previously, a variety of agents can produce contraction

1	provides a means of promoting vasodilation that is important for individuals with hypertension or congestive heart failure. As mentioned previously, a variety of agents can produce contraction of smooth muscle without altering membrane potential (i.e., pharmacomechanical coupling). Agonist-induced activation of the InsP3 cascade represents an example of pharmacomechanical coupling. Many of the hormones/agonists that activate PLC through G protein– coupled receptors also promote sarcolemmal Ca++ influx and activation of RhoA/ROK. The net effect is a rise in intracellular [Ca++], which activates MLCK, concomitant with a rise in ROK activity, which inhibits MP, both of which act complementarily to increase the level myosin light-chain phosphorylation.

1	Fig.14.11 ).Specifically,EDHFappearstobeanarachidonicacidmetabolite(e.g.,epoxyeicosatrienoic acid [EET])thatisproducedbyendothelialcellsinresponsetovariousstimuliandthenreleasedtotheunderlyingvascularsmoothmuscle.EEThasbeenshowntoactivateatransient receptor channel (e.g.,TRPV4)inthesarcolemmaofsmoothmusclethatleadstotheinfluxofCa++ ,whichthenopensRYRchannelsintheSRandresultsinCa++ sparks.TheCa++ sparksinturnactivatealarge-conductanceK+ channelinthesarcolemma(BKCa),andthesmoothmusclecellbecomeshyperpolarized.HyperpolarizationinturndecreasesbasalCa++ influxthroughvoltage-gatedCa++ channelsinthesmoothmuscle,therebydecreasingintracellular[Ca++]andhencerelaxingthesmoothmuscle,asdescribedearlier.

1	In addition to the InsP3 receptor, the SR also contains the Ca++-gated Ca++ channel, also called the RYR, which may be activated during periods of Ca++ influx through the sarcolemma. Short-lived spontaneous opening of the RYR 11,12 EET COOH •Fig. 14.11 Anarachidonicacidmetabolite(11,12-epoxyeicosatrienoicacid[11,12EET])releasedfromendothelialcellscanopenthetransientreceptorchannelTRPV4intheunderlyingsmoothmuscletopermittheinfluxofCa++ ,whichinturninitiatesbriefopeningsoftheSRryanodinereceptor(Ca++ sparks)localizednearthesarcolemma.OpeningofCa++-activatedK+ channelsinthesarcolemmabycalciumsparksresultsinhyperpolarizationofthesmoothmuscleandhencevasodilation.BKCa,largeconductanceCa++-activatedpotassiumchannel.(FromEarleySetal.Circ Res 2005;97:1270-1279.) resulting in localized elevations in myoplasmic [Ca++] occurs in many cells, including smooth muscle. When observed with Ca++-sensitive fluorescent dyes, these spontaneous localized elevations in myoplasmic [Ca++] produce brief light

1	in myoplasmic [Ca++] occurs in many cells, including smooth muscle. When observed with Ca++-sensitive fluorescent dyes, these spontaneous localized elevations in myoplasmic [Ca++] produce brief light flashes and as a result are named Ca++ sparks. In smooth muscle an increase in cAMP has been associated with an increase in the frequency of Ca++ sparks, particularly in situations in which the SR is in close proximity to the sarcolemma (i.e., junctional SR, perhaps near caveolae). An increase in the frequency of these sparks hyperpolarizes vascular smooth muscle by activation of a large-conductance Ca++-gated K+ channel in the sarcolemma. This hyperpolarization then decreases overall myoplasmic [Ca++], and relaxation occurs.

1	Calcium is extruded from the smooth muscle cell by the activity of sarcolemmal Ca++-ATPase and by a 3Na+-1Ca++ antiporter (i.e., 3 Na+ ions enter the cell for each Ca++ ion extruded). Extrusion of Ca++ from the cell competes with sequestration of Ca++ in the SR by SERCA and thus reduces the accumulation of Ca++ in the SR. When the [Ca++] in the SR decreases, the SR is thought to initiate Ca++ influx into the cell through a process called store-operated Ca++ entry (SOCE) to facilitate refilling of the SR. Specifically, stromal interaction molecule 1 (STIM1) in the SR is hypothesized to monitor SR [Ca++] and then initiate Ca++ influx through the sarcolemmal channel protein Orai through a protein-protein interaction. Thus the influx of Ca++ during SOCE is thought to occur in the confined space between the caveolae and peripheral SR of smooth muscle. Recent studies raise the possibility that STIM1-Orai–mediated SOCE may also contribute to the rise in intracellular Ca++ transient in smooth

1	between the caveolae and peripheral SR of smooth muscle. Recent studies raise the possibility that STIM1-Orai–mediated SOCE may also contribute to the rise in intracellular Ca++ transient in smooth muscle following α1-adrenergic receptor stimulation. The Stim1-Orai complex has also been implicated in remodeling of smooth muscle in pathological conditions such as restenosis following balloon angioplasty.

1	In addition to the stimulatory effects of various agents on sarcolemma Ca++ channels and InsP3 cascades, there are several inhibitory factors that lower myoplasmic [Ca++] and thereby relax smooth muscle. For example, the dihydropyridine class of Ca++ channel blocking drugs decreases the influx of Ca++ through sarcolemmal L-type voltage-gated Ca++ channels and reduces vasomotor tone. Similarly, drugs that open K+ channels in the sarcolemma (e.g., hydralazine) promote relaxation (e.g., vasodilation) by hyperpolarizing the membrane potential, which reduces the influx of Ca++ through voltage-gated Ca++ channels. Conversely, agents that decrease K+ permeability of the sarcolemma may promote vasoconstriction by inducing membrane depolarization, which then increases influx of Ca++ through these same voltage-gated Ca++ channels. Smooth muscle also contains receptor-activated Ca++ channels. Conductance of these receptor-activated Ca++ channels is linked to receptor occupancy.

1	A variety of drugs and hormones relax smooth muscle by increasing the cellular concentrations of cAMP or cGMP. Nitric oxide (NO) is produced by nerves and vascular endothelial cells and relaxes smooth muscle by increasing cGMP ( Fig. 14.12 ). Acetylcholine released from parasympathetic fibers causes vasodilation in some vascular beds as a result of stimulating the production of NO by vascular endothelial cells. Shear stress and adenosine (e.g., released from exercising muscle) may also promote NO release from vascular endothelial cells. The molecular mechanisms underlying the cGMP-dependent relaxation of vascular smooth muscle are complex and have been reported to involve (1) inhibition of InsP3 production, (2) inhibition of the InsP3 receptor, (3) activation of myosin light-chain phosphatase, and (4) activation of the Ca++-activated K+ channel (BKCa), which promotes hyperpolarization of the cells and thus inhibits Ca++ influx through voltage-gated Ca++ channels.

1	Similarly, elevation of cAMP in vascular smooth muscle by activation of β-adrenergic receptors or adenosine receptors promotes vasodilation through multiple mechanisms, including (1) decreased affinity of MLCK for Ca++/ calmodulin, (2) decreased cytosolic [Ca++] concentration, and/or (3) increased MP activity. The decreased affinity of MLCK for Ca++/calmodulin following elevation of cAMP involves protein kinase A (PKA)-dependent phosphorylation of MLCK. The ability of PKA to reduce cytosolic [Ca++] is complex and can involve activation of potassium channels (e.g., ATP-dependent potassium channels), resulting in hyperpolarization of the smooth muscle and hence decreased Ca++ influx through voltage-gated Ca++ channels. cAMP has also been shown to increase the frequency of Ca++ sparks in vascular smooth muscle, which as described earlier, hyperpolarizes the membrane potential by activation of Ca++-gated K+ channels, thereby reducing the influx of Ca++ through voltage-gated Ca++ channels.

1	smooth muscle, which as described earlier, hyperpolarizes the membrane potential by activation of Ca++-gated K+ channels, thereby reducing the influx of Ca++ through voltage-gated Ca++ channels. cAMP can also promote relaxation of smooth muscle by increasing the activity of MP (through either PKA-dependent phosphorylation of a subunit of MP or through inhibition of ROK). Inhibition of ROK can occur through PKA-dependent phosphorylation or through a signaling pathway involving the cAMP-modulated guanine nucleotide exchange factor Epac (exchange protein directly activated by cAMP)

1	Relaxation of smooth muscle by elevation of cAMP has afforded asthmatics a means of reversing bronchiolar constriction with use of β2-adrenergic agonists. The local vasodilatory effect of adenosine produced in working muscle during periods of intense exercise has also been attributed at least in part to elevated cAMP levels in vascular smooth muscle secondary to adenosine-induced stimulation of purinergic receptors on the sarcolemma of vascular smooth muscle. Adenosine may also activate a sarcolemmal K+ channel to induce membrane hyperpolarization, which as already noted will decrease the influx of Ca++ through voltage-gated Ca++ channels and cause vasodilation. Thus regulation of smooth muscle tone may be under the influence of not only the autonomic nervous system and circulating hormones but also neighboring endothelial cells and skeletal muscle cells via diffusible substances such as NO and adenosine.

1	Cat.Cat.Ca2˜Ca2˜IP3RICa2˜Ca2˜K˜CaV1.2BKCaCaMMLCK PDE5 RGS2 MYPT1 MLCP GMP pGC cGMP Hyper-polarization NucleuscGMP-A/B cGMP-A/B LZ LZ ˜˜˜°PKG1 NO sGC GTPGPCR IRAG ER Actin/myosin °˛G˜q IP3 PLC°12354 •Fig. 14.12 Signalingmechanismsbywhichnitricoxide(NO)andnitroprusside(NP)promoterelaxationofvascularsmoothmuscle.Nitricoxidestimulatessolubleguanylcyclase(sGC)inthevascularsmoothmusclecell,resultinginproductionofcGMP,activationofproteinkinaseG(PKG1),whichinturnphosphorylatesvarioussubstratesthat(1)inhibitInP3production,(2)inhibitInsP3-inducedCa++ release, (3)activatemyosinlight-chainphosphatase,(4)activatethelargeconductanceCa++-activatedK+ channel(BKCa),whichhyperpolarizesthecellandthusinhibitsthevoltage-gatedCa++

1	release, (3)activatemyosinlight-chainphosphatase,(4)activatethelargeconductanceCa++-activatedK+ channel(BKCa),whichhyperpolarizesthecellandthusinhibitsthevoltage-gatedCa++ channel(Cav1.2)(5).BKCa,calcium-activatedmaxi-Kchannel;CaM,calmodulin;Cat,catalyticdomain;Cav1.2,L-typecalciumchannel;ER,endoplasmicreticulum;GPCR,Gprotein–coupledreceptor;IP3RI,inositol1,4,5-trisphosphate(IP3)receptorI;IRAG,inositoltrisphosphatereceptor-associatedcGMP-kinasesubstrate;LZ,leucine/isoleucinezipper;MLCK,myosinlight-chainkinase;MLCP,myosinlight-chainphosphatase;MYPT,regulatorymyosinphosphatasetargetingsubunit1;PDE5,phosphodiesterase5;pGC,particulateguanylylcyclase;PLCβ,phospholipaseCbeta;RGS2,regulatorofG-proteinsignaling2;sGC,solubleguanylylcyclase.(ModifiedfromSchlossmannJ,DeschM.Am J Physiol Heart Circ Physiol 2011;301:H672–H682.)

1	Blood flow to tissues such as the brain is maintained at a relatively constant flow over a wide range of blood pressures through a process called autoregulation. The mechanism underlying autoregulation of blood flow involves the myogenic response, wherein an increase in distending pressure in an artery results in vasoconstriction, whereas a decrease in transmural pressure results in a vasodilation (over a given range of pressures). As shown in Fig. 14.13 , distention of the resistance artery resulted in an immediate elevation of intracellular [Ca++], followed by vasoconstriction in an effort to maintain relatively constant flow. The mechanism underlying this elevation of intracellular [Ca++] and subsequent vasoconstriction is complex. The rise in intracellular [Ca++] in response to stretch been reported to involve stretch-activated channels, InsP3-signaling, and voltage-gated Ca++ channels. During development and growth, the number of smooth muscle cells increases (

1	During development and growth, the number of smooth muscle cells increases ( Fig. 14.14 ). Smooth muscle tissue mass also increases if an organ is subjected to a sustained increase in mechanical work. This increase in mass is called compensatory hypertrophy. A striking example occurs with arterial smooth muscle cells (i.e., in the tunica media of the artery) in hypertensive patients. The increased mechanical load on the muscle cells appears to be the common factor that induces this hypertrophy. Chromosomal replication can result in significant numbers of polyploid muscle cells. The polyploid cells contain multiple sets of the normal number of chromosomes. They synthesize more contractile proteins and thus increase the size of the cell (see Fig. 14.14

1	Fig. 14.14 The myometrium, which is the smooth muscle component of the uterus, undergoes hypertrophy as parturition (birth) approaches. Hormones play an important role in this response. The smooth muscle is quiescent during pregnancy when the hormone progesterone predominates, and few gap junctions that electrically couple the smooth muscle cells are present. At term, under the dominant influence of estrogen, the myometrium undergoes marked hypertrophy. Large numbers of gap junctions form just before birth and convert the myometrium to a single-unit tissue to coordinate contraction during parturition.

1	The growth and development of tissues that contain smooth muscle are associated with increases in the connective tissue 1.4 1.2 1.0 0.8 0.6 0.0 •Fig. 14.14 Smoothmusclecellscarryoutmanyactivities.A,Theyretainthecapacitytodivideduringnormalgrowthorincertainpatho logicalresponsessuchasformationofatheroscleroticplaque.B,Cellsmayalsohypertrophyinresponsetoincreasedloads.Chromosomalreplicationnotfollowedbycelldivisionyieldscellswithagreatercontentofcontractileproteins.C,Smoothmusclecellsalsosynthesizeandsecretetheconstituentsoftheextracellularmatrix.

1	Althoughsmoothmuscleisinvolvedinphysiologicaladjustmentstoexercise,sustainedchangesinthe mechanicalloadingthatinducecellularadaptationsareusuallytheresultofapathologicalcondition(e.g.,hypertension).Afairlycommonexampleinmenisurinary bladder hypertrophy causedbybenignorcancerous enlargementoftheprostategland,whichobstructsthebladderoutlet.Theclinicalresultisdifficultyurinating, •Fig. 14.13 Myogenicresponseinvascularsmoothmuscleisanautoregulatoryresponsetomaintainconstantflowtoatissue.Anincreaseintransmuralpressure(TMP; top panel) elevatesintracellular[Ca++](middle panel), resultinginavasoconstriction(bottom panel). Notethattheincreaseintransmuralpressureinitiallystretchestheskeletalmuscleresistanceartery(bottom panel), butthisisquicklyfollowedbyvasoconstriction.(FromSchubertRetal.Cardiovasc Res 2008;77:8-18.) matrix. Smooth muscle cells can synthesize and secrete the materials that make up this matrix, including collagen, elastin, and proteoglycans (see

1	Fig. 14.14 ). The synthetic and secretory capacities are evident when smooth muscle cells are isolated and placed in tissue culture. The cells rapidly distentionofthebladder,andimpairedemptying.Inthissituationtheabilityofthebladdersmoothmuscletocontractanddevelopstressisdiminished.Thereasonsforthisremainunexplained,butphenotypicmodulationofthesmoothmusclecellswithalteredcontractileproteinisoformexpressionandgrossanatomicdistortionofthebladderwalloccurs.NeuromuscularchangesalsoaffectmyoplasmicCa++ mobilizationandcross-bridgephosphorylation.Fortunately,normalstructureandfunctionareusuallyrestoredaftertheobstructionisalleviated.

1	lose thick myosin filaments and much of the thin-filament lattice, and there is expansion of the rough endoplasmic reticulum and Golgi apparatus. The phenotypically altered cells multiply and lay down connective tissue. This process is reversible, and some degree of redifferentiation with the formation of thick filaments occurs after cell replication ceases. Determinants of the smooth muscle cell phenotype are largely unknown, but hormones and growth factors in blood, as well as mechanical loads on cells, have been implicated in the control of phenotypic modulation.

1	Atherosclerosis

1	isadiseasecharacterizedbylesionslocatedinthewallofbloodvessels.Thelesionsareinducedbydisordersthatinjuretheendothelium,suchashypertension,diabetes,andsmoking.Threeformedelements(monocytes,Tlymphocytes,andplatelets)thatcirculateinthebloodstreamactonthedamagedvascularendothelium.There,theygeneratechemotacticfactorsandmitogensthatmodifythestructureofthesurroundingsmoothmusclecells.ThelatterlosemostoftheirthickandthinfilamentsanddevelopanextensiveroughendoplasmicreticulumandGolgicomplex.Thesecellsmigratetothesubendothelialspace(i.e.,thetunicamediaoftheartery),proliferate,andparticipateinformationofthefattylesionsorthefibrousplaquesthatcharacterizeatherosclerosis.InhibitionordownregulationofRhokinase(ROK)hasbeenshowntopromoteregressionofatherosclerotic-likelesionsinananimalmodel.ThemechanismormechanismsunderlyingthisbeneficialeffectofROKinhibitionareunclearbutmayberelatedtoregulationofbothendothelialpermeabilityandmonocytemigrationbyROK.Thatis,hyperactivityofROKhasbeenimplicatedinvariouspa

1	mechanismsunderlyingthisbeneficialeffectofROKinhibitionareunclearbutmayberelatedtoregulationofbothendothelialpermeabilityandmonocytemigrationbyROK.Thatis,hyperactivityofROKhasbeenimplicatedinvariouspathologicalconditions,includingincreasedtransendothelialpermeability(perhapssecondarytoincreasedactomyosinactivity),whereasinhibitionofROKhasbeenshowntodecreasetransendothelialmigrationofmonocytesandneutrophils.

1	Biophysical Properties of Smooth Muscle Smooth muscle contains large amounts of connective tissue composed of extensible elastin fibrils and inextensible collagen fibrils. Because this extracellular matrix can withstand high distending forces or loads, it is responsible for the passive length-tension curve measured in relaxed tissues. This ability of the matrix also limits organ volume. When lengths are normalized to the optimal length for development of force (i.e., L0), the length-tension curves for smooth and skeletal muscle are very similar (

1	Fig. 14.15 ). However, the length-tension curves of striated and smooth muscle differ quantitatively. For example, smooth muscle cells shorten more than skeletal muscle cells do. In addition, smooth muscle is characteristically only partially activated, and the peak isometric force attained varies with the stimulus. In skeletal muscle the stimulus (i.e., action potential) always produces a full twitch contraction. Smooth muscle can generate active force comparable to that of skeletal muscle, even though smooth muscle contains only about a fourth as much myosin. This does not imply that the cross-bridges in smooth muscle have greater force-generating capacity. Instead, active cross-bridges in smooth muscle are much more likely to be in 2.4 2.0 1.6 1.2 0.8 0.4 0.6 0.8 1.0 1.2 Po = 2.22 ± 0.17 × 106 dynes/cm2 N = 11 •Fig. 14.15

1	force-generating capacity. Instead, active cross-bridges in smooth muscle are much more likely to be in 2.4 2.0 1.6 1.2 0.8 0.4 0.6 0.8 1.0 1.2 Po = 2.22 ± 0.17 × 106 dynes/cm2 N = 11 •Fig. 14.15 Lengthdependenceofcontractionofsmoothmuscleshowsabell-shapedresponsesimilartothatseeninskeletalmuscle.Thesmoothmusclestripswereobtainedfromhogcarotidartery.Contractionwasinducedbypotassiumdepolarization.Optimalmusclelength(Lo)wasgraphicallydeterminedasthelengthatwhichmaximaltension(Po)wasdeveloped.Redcircles,passivetension;bluecircles,activetension,triangles,totaltension;(FromHerlihyJT,MurphyRA.Circ. Res. 1973;33:275-283.) the attached force-generating configuration because of their slow cycling kinetics.

1	Smooth and striated muscles both exhibit a hyperbolic dependence of shortening velocity on load. However, contraction velocities are far slower in smooth muscle than in striated muscle. One factor that underlies these slow velocities is that the myosin isoform in smooth muscle cells has low ATPase activity. Skeletal muscle cells have a force-velocity curve in which shortening velocities are determined only by load and the myosin isoform (see ). In contrast, both force and shortening velocity, which reflect the number of cycling cross-bridges and their cycling rates, vary in smooth muscle. When activation of smooth muscle is altered, for example, by different frequencies of nerve stimulation or changing hormone concentrations, a “family” of velocity-stress curves can be derived ( Fig. 14.16 ). This implies that both cross-bridge cycling rates and the number of active cross-bridges in smooth muscle are regulated in some way, Vo(% of max) Velocity (% of max)

1	Fig. 14.16 ). This implies that both cross-bridge cycling rates and the number of active cross-bridges in smooth muscle are regulated in some way, Vo(% of max) Velocity (% of max) Stress (% of max) •Fig. 14.16 A,Force-velocitycurvesforfastandslowhumanskeletalmusclecellsandsmoothmuscle.B,Smoothmuscleshavevariableforce-velocityrelationshipsthataredeterminedbythelevelofCa++-stimulatedcross-bridgephosphorylation.C,Maximalshorteningvelocitieswithnoload(Vo,whichrepresentstheinterceptsontheordinateinB)aredirectlydependentoncross-bridgephosphorylationbyMLCK.D,Activeforce/stress(abscissainterceptsinB)risesrapidlywithphosphorylation;nearmaximalstress,itmaybegeneratedwithonly20%to30%ofthecross-bridgesinthephosphorylatedstate.

1	which is in marked contrast to striated muscle. This difference is conferred by a regulatory system that depends on phosphorylation of cross-bridges, which in turn depends on myoplasmic [Ca++]. Because myosin light-chain phosphorylation is required for actin-myosin interaction in smooth muscle, a dependence of maximal force on the degree of myosin phosphorylation is expected (i.e., phosphorylation of more myosin molecules results in more actin-myosin interactions and hence more force generated). The variation in maximal shortening velocity as a function of the degree of myosin phosphorylation may reflect dephosphorylation of the myosin light chain while the myosin is still attached to the actin, thus slowing the rate of detachment (i.e., latch state) at low levels of phosphorylation. At higher levels of phosphorylation, the likelihood of latch states would be reduced and the myosin cross-bridges would be released more quickly from actin, thereby yielding a higher shortening velocity

1	higher levels of phosphorylation, the likelihood of latch states would be reduced and the myosin cross-bridges would be released more quickly from actin, thereby yielding a higher shortening velocity at all loads (see

1	Fig. 14.16B 1. Smooth muscle cells are linked by a variety of junctions that serve both mechanical and communication roles. These linkages are essential in cells that must contract uniformly. 2.

1	The sarcolemma plays an important role in Ca++ exchange between extracellular fluid and myoplasm. The sarcolemma of smooth muscle contains numerous caveolae that contribute to regulation of intracellular [Ca++] and also appear to serve as a scaffold for signaling molecules. The sarcoplasmic reticulum (SR) contains an intracellular Ca++ pool that can be mobilized to transiently increase myoplasmic [Ca++]. Myoplasmic [Ca++] is dependent on extracellular Ca++ . Transporters in the sarcolemma that regulate myoplasmic [Ca++] include receptor-mediated Ca++ channels, voltage-gated Ca++ channels, Ca++-ATPase, and the 3Na+1Ca++ antiporter. The SR also regulates myoplasmic [Ca++]. The Ca++ channels in the SR open in response to a chemical. Neurotransmitters or hormones that act via receptors in the sarcolemma can activate phospholipase C (PLC), followed by generation of the second messenger inositol 1,4-triphosphate (InsP3). InsP3 then activates InsP3-gated Ca++ channels on the SR. Many

1	in the sarcolemma can activate phospholipase C (PLC), followed by generation of the second messenger inositol 1,4-triphosphate (InsP3). InsP3 then activates InsP3-gated Ca++ channels on the SR. Many agonists that activate PLC through G protein–coupled receptors also activate the RhoA/Rho kinase (ROK) signaling cascade, thereby increasing the sensitivity of smooth muscle contraction to Ca++ . Smooth muscle SR also contains Ca++-gated Ca++ channels (ryanodine receptor [RYR]). Ca++ reaccumulates in the SR via SERCA (sarcoplasmic endoplasmic reticulum Ca++ ATPase).

1	3. Smooth muscles contain contractile units that consist of small groups of thick myosin filaments that interdigitate with large numbers of thin filaments attached to Z-line equivalents termed dense bodies or membrane-dense areas. No striations are evident. Contraction is caused by a sliding filament–crossbridge mechanism. 4. Contraction of smooth muscle is dependent on both release of Ca++ from the SR and entry of Ca++ across the sarcolemma. Smooth muscle lacks troponin. Phosphorylation of cross-bridges by a Ca++-dependent myosin light chain kinase (MLCK) is necessary for attachment to the thin filament. Dephosphorylation of an attached cross-bridge by myosin phosphatase (MP) slows its cycling rates. Higher myoplasmic [Ca++] increases the ratio of MLCK to MP activity, with the result that more of the cross-bridges remain phosphorylated throughout a cycle. This increases shortening velocities. 5.

1	5. Smooth muscle activity is controlled by nerves (principally autonomic), circulating hormones, locally generated signaling substances, junctions with other smooth muscle cells, and even junctions with other non–smooth muscle cells. A variety of hormones/ agonists increase the sensitivity of smooth muscle contraction to Ca++ by reducing the activity of MP and reciprocally increasing the activity of MLCK at a given intracellular [Ca++]. Activation of the RhoA/ROK signaling cascade contributes to this inhibition of MP and stimulation of MLCK, and hence to the increase in sensitivity of smooth muscle contraction to Ca++ . Smooth muscle also has an intrinsic ability to respond to stretch, which is important for autoregulation of blood flow to various tissues. 6.

1	6. The response to sustained or tonic stimulation is a rapid contraction followed by sustained maintenance of force with reduced cross-bridge cycling rates and ATP consumption. This behavior, called the latch state, is advantageous for muscles that may need to withstand continuous external force, such as blood vessels, which must be able to withstand blood pressure. During the latch state, ATP is consumed at less than 1/300 the rate needed to maintain the same force in skeletal muscle. 7.

1	7. The length-tension relationships, hyperbolic velocity-load relationships, power output curves, and ability to resist imposed loads are comparable to those of skeletal muscle. Shortening velocities and ATP consumption rates are very low in smooth muscle, in keeping with expression of a myosin isoform with low activity. Smooth muscles also have the unusual ability to alter velocity-stress relationships, which reflects regulation of both the number of active cross-bridges (determining force) and their average cycling rates for a given load (determining velocity). Burnstock G, Ralevic V. Purinergic signaling and blood vessels in health and disease. Pharmacol Rev. 2014;66:102-192. Joyner MJ, Casey DP. Regulation of increased blood flow (hyperemia) to muscles during exercise: a hierarchy of competing physiological needs. Physiol Rev. 2015;95:549-601. Longden TA, et al. Ion channel networks in the control of cerebral blood flow. J Cereb Blood Flow Metab. 2016;36:492-512.

1	Longden TA, et al. Ion channel networks in the control of cerebral blood flow. J Cereb Blood Flow Metab. 2016;36:492-512. Momotani K, Somlyo AV. p63RhoGEF: a new switch for G q-mediated activation of smooth muscle. Trends Cardiovasc Med. 2012;22:122-127. Roberts OL, Dart C. cAMP signalling in the vasculature: the role of Epac (exchange protein directly activated by cAMP). Biochem Soc Trans. 2014;42:89-97. 8. Smooth muscle is also a synthetic and secretory cell with a major role in formation of the extensive extracellular matrix that surrounds and links the cells. Cellular hypertrophy occurs in response to physiological needs, and smooth muscle cells retain the potential to divide. Schlossmann J, Desch M. IRAG and novel PKG targeting in the cardiovascular system. Am J Physiol Heart Circ Physiol. 2011;301: H672-H682. Shimokawa H, et al. RhoA/Rho-Kinase in the Cardiovascular System. Circ Res. 2016;118:352-366.

1	Shimokawa H, et al. RhoA/Rho-Kinase in the Cardiovascular System. Circ Res. 2016;118:352-366. Somlyo AP, Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev. 2003;83:1325-1358. Spinelli AM, Trebak M. ORAI channel-mediated Ca2+ signals in vascular and airway smooth muscle. Am J Physiol Cell Physiol. 2016;310:C402-C413. Tinker A, et al. The role of ATP-sensitive potassium channels in cellular function and protection in the cardiovascular system. Br J Pharmacol. 2014;171:12-23. SECTION 4The Cardiovascular System ACHILLES J. PAPPANO AND WITHROW GIL WIER Overview of Circulation Regulation of the Heart and Vasculature Chapter 16 Elements of Cardiac Function Chapter 19 Integrated Control of the Chapter 17 Cardiovascular System Properties of the Vasculature Upon completion of this chapter, the student should be able to answer the following questions: 1.

1	Upon completion of this chapter, the student should be able to answer the following questions: 1. How does the arrangement of heart and vessels enable unidirectional flow of well-oxygenated blood to the body? 2. What is the advantage of the reciprocal relation between blood flow velocity and vascular cross-sectional area? 3. How do the differing compositions (smooth muscle, fibrous and elastic tissue) of blood vessels contribute to their respective functions?

1	he circulatory system transports and distributes essential substances to tissues and removes metabolic byproducts. This system also participates in homeostatic mechanisms such as regulation of body temperature, maintenance of fluid balance, and adjustment of O2 and nutrient supply in various physiological states. The cardiovascular system, which accomplishes these tasks, is composed of a pump (the heart), a series of distributing and collecting tubes (blood vessels), and an extensive system of thin vessels (capillaries) that enable rapid exchange between the tissues and vascular channels. Blood vessels throughout the body are filled with a heterogeneous fluid (blood) that is essential for the transport processes performed by the heart and blood vessels. This chapter is a general, functional overview of the heart and blood vessels, whose functions are analyzed in much greater detail in subsequent chapters. The Heart

1	The Heart The heart consists of two pumps in series: one pump propels blood through the lungs for exchange of O2 and CO2 (the pulmonary circulation) and the other pump propels blood to all other tissues of the body (the systemic circulation). Flow of blood through the heart is in one direction (unidirectional). Unidirectional flow through the heart is achieved by the appropriate arrangement of flap valves. Although cardiac output is intermittent, continuous flow to body tissues (periphery) occurs by distention of the aorta and its branches during ventricular contraction (systole) and by elastic recoil of the walls of the large arteries with forward propulsion of the blood during ventricular relaxation (diastole). The Cardiovascular Circuit

1	The Cardiovascular Circuit In the normal intact circulation, the total volume of blood is constant, and an increase in the volume of blood in one area must be accompanied by a decrease in another. However, the distribution of blood circulating to the different regions of the body is determined by the output of the left ventricle and by the contractile state of the resistance vessels (arterioles) of these regions. The circulatory system is composed of conduits arranged in series and in parallel ( Fig. 15.1 ). This arrangement, which is discussed in subsequent chapters, has important implications in terms of resistance, flow, and pressure in blood vessels.

1	Fig. 15.1 ). This arrangement, which is discussed in subsequent chapters, has important implications in terms of resistance, flow, and pressure in blood vessels. Blood entering the right ventricle via the right atrium is pumped through the pulmonary arterial system at a mean pressure about one seventh that in the systemic arteries. The blood then passes through the lung capillaries, in which CO2 in the blood is released and O2 is taken up. The O2-rich blood returns via the pulmonary veins to the left atrium, where it is pumped from the ventricle to the periphery, which thus completes the cycle. Blood moves rapidly through the aorta and its arterial branches. As these branches approach the periphery, the branches narrow, and their walls become thinner. They also change histologically. The aorta is a predominantly elastic structure, but the peripheral arteries become more muscular until, at the arterioles, the muscular layer predominates ( Fig. 15.2

1	Fig. 15.2 In the large arteries, frictional resistance is relatively small, and pressures are only slightly less than those in the aorta. The small arteries, on the other hand, offer moderate resistance to blood flow. This resistance reaches a maximal level in the arterioles, which are sometimes referred to as the “stopcocks” of the vascular system. Hence, the pressure drop is greatest across the terminal segment of the small arteries and the arterioles ( Fig. 15.3 ). Adjustment in the degree of contraction of the circular muscle of these small vessels allows regulation of tissue blood flow and aids in the control of arterial blood pressure. In addition to the reduction in pressure along the arterioles, there is a change from pulsatile to steady blood flow (see

1	Fig. 15.3 ). Pulsatile arterial blood flow, caused by the intermittent ejection of blood from the heart, is damped • Fig. 15.3 Phasic Pressure, Velocity of Flow, and Cross-Sectional • Fig. 15.1 Schematic Diagram of the Parallel and Series Arrange-Area of the Systemic Circulation. The important features are the major ment of the Vessels That Constitute the Circulatory System. The pressure drop across the small arteries and arterioles, the inverse capillary beds are represented by thin lines connecting the arteries relationship between blood flow velocity and cross-sectional area, and (on the right) with the veins (on the left). The crescent-shaped thicken-the maximal cross-sectional area and minimal flow rate in the capilings proximal to the capillary beds represent the arterioles (resistance laries. (From Levick JR. An Introduction to Cardiovascular Physiology. vessels). (Redrawn from Green HD. In: Glasser O [ed]. Medical

1	Introduction to Cardiovascular Physiology. vessels). (Redrawn from Green HD. In: Glasser O [ed]. Medical Physics. 5th ed. London: Hodder Arnold; 2010.)Vol 1. Chicago: Year Book; 1944.) • Fig. 15.2 Internal Diameter, Wall Thickness, and Relative Amounts of the Principal Components of the Vessel Walls of the Various Blood Vessels That Constitute the Circulatory System. Cross-sections of the vessels are not drawn to scale because of the huge range in size from aorta and venae cavae to capillary. (Redrawn from Burton AC. Relation of structure to function of the tissues of the wall of blood vessels. Physiol Rev. 1954;34:619.) at the capillary level by a combination of two factors: distensibility of the large arteries and frictional resistance in the small arteries and arterioles.

1	Many capillaries arise from each arteriole. The total cross-sectional area of the capillary bed is very large despite the fact that the cross-sectional area of each capillary is less than that of each arteriole. As a result, blood flow velocity becomes quite slow in the capillaries (see Fig. 15.3 ), which is analogous to the decrease in velocity of flow in the wide regions of a river. Conditions in the capillaries are ideal for the exchange of diffusible substances between blood and tissue because capillaries consist of short tubes with walls that are only one cell thick and in which flow velocity is low. On its return to the heart from the capillaries, blood passes through venules and then through veins of increasing size. Pressure within these vessels progressively decreases until the blood reaches the right atrium (see Fig. 15.3 ). Near the heart, the number of veins decreases, the thickness and composition of the vein walls change (see

1	Fig. 15.3 ). Near the heart, the number of veins decreases, the thickness and composition of the vein walls change (see Fig. 15.2), the total cross-sectional area of the venous channels diminishes, and the velocity of blood flow increases (see Fig. 15.3 ). The velocity of blood flow and the cross-sectional area at each level of the vasculature are essentially mirror images (see Fig. 15.3

1	Fig. 15.3 ). The velocity of blood flow and the cross-sectional area at each level of the vasculature are essentially mirror images (see Fig. 15.3 Table 15.1 ) indicate that between the aorta and the capillaries, the total cross-sectional area increases about 500-fold. The volume of blood in the systemic vascular system is greatest in the veins and venules (64%). Only 6% of total blood volume exists in the capillaries, and 14% of total blood volume is found in the aorta, arteries, and arterioles. In contrast, blood volume in the pulmonary vascular bed is about equally divided among the arterial, capillary, and venous vessels. The cross-sectional area of the venae cavae is larger than that of the aorta. Therefore, the velocity of flow is slower in the venae cavae than in the aorta (see Fig. 15.3 Aortaandlargearteries3006.0Smallarteries4008.0Capillaries3006.0Smallveins230046.0Largeveins90018.0Total420084.0

1	Fig. 15.3 Aortaandlargearteries3006.0Smallarteries4008.0Capillaries3006.0Smallveins230046.0Largeveins90018.0Total420084.0 Arteries1302.6Capillaries1102.2Veins2004.0Total4408.8Heart (End-Diastole) 3607.2 DatafromBoronWF,BoulpaepEL.Medical Physiology. 2nded.Philadelphia:ElsevierSaunders;2009.*Valuesrefertoa70-kgwoman. Inapatientwithhyperthyroidism(Graves’ disease), basalmetabolismiselevatedandoftenassociatedwitharteriolarvasodilation.Thisreductioninarteriolarresistancediminishesthedampingeffectonpulsatilearterialpressureandismanifestedaspulsatileflowinthecapillaries,asobservedinthefingernailbedsofpatientswiththisailment. Upon completion of this chapter, the student should be able to answer the following questions: 1. How does the action potential contribute to excitability and contraction in heart muscle? 2. What is automaticity, and how does it differ from excitability? How do derangements of these properties contribute to arrhythmias? 3.

1	2. What is automaticity, and how does it differ from excitability? How do derangements of these properties contribute to arrhythmias? 3. What is the structural basis of the electrocardiogram? 4. How are the concepts of preload and afterload, developed for skeletal muscle, applied to the contraction of the heart? 5. What is the role of Ca++ in excitation-contraction coupling? 6. What changes occur in atrial and ventricular pressures and volumes during a cardiac cycle, and what is their temporal relation to the electrocardiogram? 7. How does the relation between end-diastolic volume and left ventricular developed pressure define the Frank-Starling law of the heart and regulate the force of cardiac contraction? 8. What is the pressure-volume loop of the left ventricle, and how does it define changes in left ventricular function? 9. How is cardiac metabolism linked to O2 consumption, and how are these processes affected by changes in cardiac work?

1	9. How is cardiac metabolism linked to O2 consumption, and how are these processes affected by changes in cardiac work? Electrical Properties of the Heart The cells of the heart, like neurons, are excitable and generate action potentials. These action potentials initiate contraction and thus determine the heart rate. Disorders in electrical activity can induce serious and sometimes lethal disturbances in cardiac rhythm. In this section, the electrical properties of cardiac cells are described. In addition, how these electrical properties account for the electrocardiogram (ECG) is considered. The initiation of contraction as a result of the electrical properties of cardiac cells is considered in a later section. The Cardiac Action Potential

1	Fig. 16.1 illustrates action potentials found in different cardiac cells. Two main types of action potentials occur in the heart and are depicted. One type, the fast response, occurs in normal atrial and ventricular myocytes and in the specialized conducting fibers (Purkinje fibers of the heart) and is divided into five phases. The rapid upstroke of the action potential is designated phase 0. The upstroke is followed by a brief period of partial, early repolarization (phase 1) and then by a plateau (phase 2) that persists for approximately 0.1 to 0.2 second. The membrane then repolarizes (phase 3) until the resting state of polarization (phase 4) is again attained. Final repolarization (phase 3) develops more slowly than depolarization (phase 0). The other type of action potential, the slow response, occurs in the sinoatrial (SA) node, which is the natural pacemaker region of the heart, and in the atrioventricular (AV) node, which is the specialized tissue that conducts the cardiac

1	slow response, occurs in the sinoatrial (SA) node, which is the natural pacemaker region of the heart, and in the atrioventricular (AV) node, which is the specialized tissue that conducts the cardiac impulse from the atria to the ventricles. The slow-response cells lack the early repolarization phase (phase 1).

1	Other differences between the electrical properties of the fast-response and slow-response cells include the following: The resting membrane potential (phase 4) of the fast-response cells is considerably more negative than that of the slow-response cells. Moreover, the slope of the upstroke (phase 0), the amplitude of the action potential, and the overshoot (membrane voltage positive to 0 mV) are greater in the fast-response cells than in the slow-response cells. The action potential amplitude and the steepness of the upstroke are important determinants of propagation velocity along the myocardial fibers. In slow-response cardiac tissue, the action potential is propagated more slowly and conduction is more likely to be blocked than in fast-response cardiac tissue. Slow conduction and a tendency toward conduction block increase the likelihood of some rhythm disturbances (see the section “

1	The action potential initiates contraction of the myocyte. The relationship between the action potential and contraction of a cardiac myocyte is shown in Fig. 16.2 . Rapid depolarization (phase 0) precedes cell shortening, and completion of repolarization occurs just before peak shortening. Relaxation of the muscle takes place mainly during phase 4 of the action potential. The duration of contraction usually parallels the duration of the action potential. The various phases of the cardiac action potential are associated with changes in cell membrane permeability, mainly by Na+, K+, and Ca++ ions. Changes in cell membrane permeability alter the rate of movement of these ions across the membrane and thereby change the membrane voltage (Vm). These changes in permeability are accomplished by the opening and closing of ion channels that are specific for individual ions (see

1	As with all other cells in the body, the concentration of K+ inside a cardiac muscle cell (intracellular [K+]) exceeds the concentration outside the cell (extracellular [K+]). The concentration gradient is reversed for Na+ and Ca++ . Estimates of the extracellular and intracellular concentrations of Na+, K+, and Ca++ and the Nernst equilibrium potentials (see Eq. 1.5b ) for these ions are compiled in Table 16.1 The resting cell membrane has a relatively high permeability for K+; permeability for Na+ and Ca++ is much less. Because of the existing chemical gradient for K+ and Vm, K+ tends to diffuse from the inside to the outside of the cell. 3 Any diffusion of K+ that occurs at the resting membrane potential (i.e., during phase 4) takes place mainly through 4 specific potassium channels. Several types of potassium channels exist in cardiac cell membranes. Opening and –120

1	ERP RRP closing of some of these channels are regulated by Vm, whereas others are controlled by a chemical signal (e.g., the extracellular acetylcholine concentration). The specific 4 is a voltage-regulated channel that conducts the inward rectifying K+ current (iK1), which is discussed in more detail later. For now, it is necessary only to know how this current is established.

1	The dependence of Vm on conductance and the intracellular and extracellular concentrations of K+, Na+, and other ions are described by the chord conductance equation •Fig. 16.2 Temporalrelationshipbetweenthechangesintrans-membranepotential(top trace) andcellshortening(bottom trace) inasingleventricularmyocyte.Thevalueof0mVmembranepotentialisindicatedby“0.”(FromPappanoA.unpublishedrecord,1995.)0 50 mV 400 msec 7˜m •Fig. 16.1 Action Potentials of Cardiac Fibers. A, Fast-responsecardiacfibers.B, Slow-responsecardiacfibers.Thephasesoftheactionpotentialsarelabeled(seetextfordetails),asaretheeffectiverefractoryperiod(ERP)andtherelativerefractoryperiod(RRP).Notethatincomparisonwithfast-responsefibers,therestingpotential ofslowfibersislessnegative,theupstroke(phase0)oftheactionpotentialislesssteep,theamplitudeoftheactionpotentialissmaller,phase1isabsent,andtheRRPextendswellintophase4afterthefibershavefullyrepolarized.

1	Eq. 2.7 ). In a resting cardiac cell, K+ conductance (gK) is approximately 100 times greater than Na+ conductance (gNa). Therefore, Vm is similar to the Nernst equilibrium potential for K+ . As a result, alterations in extracellular [K+] can significantly change Vm: Hypokalemia causes hyperpolarization, and hyperkalemia causes depolarization. In contrast, because gNa is so small in the resting cell, changes in extracellular [Na+] do not significantly affect Vm. Fastresponsesofcardiacmusclemaychangetoslowresponsesincertainpathologicalconditions.Forexample,incoronaryarterydisease,aregionofcardiacmusclemaybedeprivedofitsnormalbloodsupply.Asaresult,[K+]intheinterstitialfluidthatsurroundstheaffectedmusclecellsrisesbecauseK+ islostfromtheinadequatelyperfused(orischemic)cells.Theactionpotentialsinsomeofthesecellsmaythenbeconvertedfromfasttoslowresponses.Conversionfromafasttoaslowresponseasaresultofincreasinginterstitial[K+]isillustratedlaterin Fig.16.13 Genesis of the Upstroke (Phase 0)

1	Fig.16.13 Genesis of the Upstroke (Phase 0) Any stimulus that abruptly depolarizes Vm to a critical value (the threshold) elicits an action potential. The characteristics of fast-response action potentials are shown in Fig. 16.1A. The rapid depolarization (phase 0) is related almost exclusively to the influx of Na+ into the myocyte as a result of a sudden increase in gNa. The action potential amplitude (the membrane potential change during phase 0) is dependent on extracellular [Na+]. When extracellular [Na+] is decreased, the amplitude of the action potential decreases, and when it is reduced from its normal value of approximately 140 mEq/L to approximately 20 mEq/L, the cell is no longer excitable.

1	When the resting membrane potential, Vm, is suddenly depolarized from −90 mV to the threshold level of approximately −65 mV, the cell membrane properties change dramatically. Na+ enters the myocyte through specific fast voltage-activated sodium channels that exist in the membrane (see Fig. 5.7). These channels can be blocked by the puffer fish toxin tetrodotoxin. In addition, many drugs used to treat certain cardiac rhythm disturbances (cardiac arrhythmias) act by blocking these fast sodium channels.

1	The sodium channels open very rapidly, or activate (in ≈0.1 msec), thereby resulting in an abrupt increase in gNa. However, once open, the sodium channels inactivate (time course ≅ 1 to 2 msec), and gNa rapidly decreases (Fig. 16.3 ). The sodium channels remain in the inactivated state until the membrane begins to repolarize. With repolarization, the channel transitions to the closed state, from which it can then be reopened by another depolarization of Vm to the threshold. These properties of the sodium channel are the basis of the refractory period of the action potential. When the sodium channels are in the inactivated state, they cannot be reopened, and another action potential cannot be generated. During this period, the cell is said to be in the effective refractory period. This prevents a sustained, tetanic contraction of cardiac muscle, which would retard ventricular relaxation and therefore interfere with the normal intermittent pumping action of the heart. As the cell

1	prevents a sustained, tetanic contraction of cardiac muscle, which would retard ventricular relaxation and therefore interfere with the normal intermittent pumping action of the heart. As the cell repolarizes (phase 3), the inactivated channels begin to transition to the closed state. During this period, called the relative refractory period, another action potential can be generated, but it requires a larger-than-normal depolarization of Vm. Only when Vm has returned to the resting level (phase 4) are all the sodium channels closed and thus able to be reactivated by the normal depolarization of Vm.

1	Anioniccurrentthroughonemembranechannelcanbemeasuredwiththepatchclamptechnique.Theindividualchannelsopenandcloserepeatedlyinarandommanner.Thisprocessisillustratedin Fig.16.4 ,whichshowsthecurrentflowthroughsodiumchannelsinamyocardialcell.Beforethetimeindicatedbythearrow,themembranepotentialwasclampedat−85mV.Atthetimeindicatedbythearrow,thepotentialwassuddenlychangedto−45mV,atwhichvalueitwasheldfortheremainderoftherecording. Fig.16.4 indicatesthatimmediatelyafterthemembranepotentialwasmadelessnegative,onesodiumchannelopened(1.5pAinamplitude),andthenasecondsodiumchannelopened(3pAtotalcurrentfrombothchannels),whereuponbothchannelsclosed(i.e.,thecurrentreturnedto0).Bothchannelsclosedforapproximately4or5msec,andthenbothchannelsopenedatthesametime.Thereafter,onechannelclosedandthenthesecondrapidlyclosed.Finally,afterseveralseconds,onechannelopenedandthenclosed.Thereafter,bothchannelsremainedclosedfortherestoftherecording,eventhoughthemembranevoltagewasheldconstantat−45mV.

1	Genesis of Early Repolarization (Phase 1) In many cardiac cells that have a prominent plateau, phase 1 is an early, brief period of limited repolarization. This brief repolarization results in the notch between the end of the upstroke and the beginning of the plateau (see Figs. 16.1 16.3). Repolarization is brief because of activation of a transient outward current (ito) carried mainly by K+ . Activation of potassium channels during phase 1 causes a brief efflux of K+ from the cell because the cell interior is positively charged and intracellular [K+] greatly exceeds extracellular [K+] (see Fig. 16.3 ). The cell is briefly and partially repolarized as a result of this transient efflux of K+ . The size of the phase 1 notch varies among cardiac cells. It is prominent in myocytes in the epicardial and midmyocardial regions of the left ventricular wall (

1	The size of the phase 1 notch varies among cardiac cells. It is prominent in myocytes in the epicardial and midmyocardial regions of the left ventricular wall ( Fig. 16.5 ) and in ventricular Purkinje fibers. However, the notch is negligible in myocytes from the endocardial region of the left ventricle (see Fig. 16.5) because the density of i to channels is less in these cells. The notch is also less prominent in the presence 4 Chemical Electrostatic 3 2 1 0 Fast Na+ Channel Na+ Na+ – – – – – – – – – – – – – – – – – – – – K+ Channel (ito) K+ K+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Ca++ Ca++ Ca++ K+ K+ K+ Channels (iK, iK1) K+ K+ – – – – – – – – – – – – – – – – – – – – K+ Channels (iK, iK1) K+ K+ – – – – – – – – – – – – – – – – – – – – 0 1 3 4 2

1	K+ Channels (iK, iK1, ito) •Fig. 16.3 Principal Ionic Currents and Channels That Generate the Various Phases of the Action Potential in a Cardiac Cell. Phase 0, ThechemicalandelectrostaticforcesbothfavortheentryofNa+ intothecellthroughfastsodiumchannelstogeneratetheupstroke.Phase 1, BoththechemicalandelectrostaticforcesfavortheeffluxofK+ throughtransientoutwardcurrent(ito)channelstogenerateearly,partialrepolarization.Phase 2, Duringtheplateau,thenetinfluxofCa++ throughcalciumchannelsisbalancedbytheeffluxofK+ throughoutwardrectifyingcurrent(iK),inwardrectifyingcurrent(iK1),anditochannels.Phase 3, ThechemicalforcesthatfavortheeffluxofK+ throughiKandiK1channelspredominateovertheelectrostaticforcesthatfavortheinfluxofK+ throughthesesamechannels.Phase 4, ThechemicalforcesthatfavortheeffluxofK+ throughiKandiK1channelsveryslightlyexceedtheelectrostaticforcesthatfavortheinfluxofK+ throughthesesamechannels.

1	0 1.5 3 4.5 10 msec pA Channel #1 current Channel #2 current •Fig. 16.4 Current (in Picoamperes [pA]) Through Two Individual Sodium Channels in a Cultured Heart Cell, Recorded With the Patch Clamp Technique. Membranevoltagewasheldat−85mV,thenabruptlychangedto−45mVatthetimeindicatedbythearrow, andheldatthispotentialfortheremainderoftherecord.(RedrawnfromCachelinAB,etal.J Physiol. 1983;340:389.) of 4-aminopyridine, which blocks the potassium channels that carry ito. Genesis of the Plateau (Phase 2) During the action potential plateau, Ca++ enters myocardial cells through calcium channels (described in next paragraph) that activate and inactivate much more slowly than the fast sodium channels do. During the flat portion of phase 2 (see

1	Figs. 16.1 16.3), this influx of Ca ++ is counterbalanced by the efflux of K+ . K+ exits through channels that conduct mainly the ito, iK, and iK1 currents. The ito current is responsible for phase 1, as described previously, but it is not completely inactivated until after phase 2 has expired. The iK and iK1 are described later in this section of the chapter. Ca++ enters the cell via voltage-regulated calcium channels, which are activated as Vm becomes progressively less negative during the action potential upstroke. Two types of calcium channels (L-type and T-type) have been identified in cardiac tissue. Some of their important characteristics are illustrated in Fig. 16.6 . L-type channels are so designated because once open, they inactivate slowly (see

1	Fig. 16.6 . L-type channels are so designated because once open, they inactivate slowly (see Fig. 16.6, lower panel) and enable a long-lasting Ca++ current. They are the predominant type of calcium channel in the heart, and they are activated during the action potential upstroke when Vm reaches approximately −20 mV. L-type channels are blocked by calcium channel antagonists such as verapamil, amlodipine, and diltiazem ( Fig. 16.7 T-type (or “transient”) calcium channels are much less abundant in the heart. They are activated at membrane potentials that are more negative (approximately −70 mV) than the potential that activates L-type channels (−20 mV). They also inactivate more quickly than do L-type channels (see Fig. 16.6 ; compare the upper and lower panels).

1	Fig. 16.6 ; compare the upper and lower panels). Opening of calcium channels results in an increase in Ca++ conductance (gCa) and calcium current (iCa) soon after •Fig. 16.5 Actionpotentialsrecordedfromtheepicardial(Epi)(A), midmyocardial(Mid)(B), andendocardial(Endo)(C) regionsofthefreewalloftheleftventricle.Thepreparationsweredrivenatabasiccyclelength(BCL):thatis,atinterstimulusintervalsof300and8000msec.Thevalueof0mVmembranepotentialisindicatedby“0.”(FromLiuD-W,etal.Circ Res. 1993;72:671.) the action potential upstroke (see Fig. 16.3 ). Because the intracellular concentration of Ca++ is much lower than the extracellular concentration of Ca++ (see Table 16.1 ), the increase in gCa promotes the influx of Ca++ into the cell throughout the plateau. This Ca++ influx during the plateau is involved in excitation-contraction coupling, as described in the section “

1	Various neurotransmitters and drugs may substantially influence gCa. The adrenergic neurotransmitter norepinephrine, the β-adrenergic receptor agonist isoproterenol, and various other catecholamines enhance gCa, whereas the parasympathetic neurotransmitter acetylcholine decreases gCa. Enhancement of gCa by catecholamines is the principal mechanism by which they enhance cardiac muscle contractility. During the plateau (phase 2) of the action potential, the concentration gradient for K+ across the cell membrane is virtually the same as it is during phase 4; at that time, however, Vm is positive. Therefore, there is a large gradient that favors efflux of K+ from the cell (see Fig. 16.3). If g K were the same during the plateau as it is during phase 4, efflux of K+ during phase 2 would greatly exceed the influx of Ca++ , and a sustained plateau could not be achieved.

1	Fig. 16.3). If g K were the same during the plateau as it is during phase 4, efflux of K+ during phase 2 would greatly exceed the influx of Ca++ , and a sustained plateau could not be achieved. •Fig. 16.6 Effects of Isoproterenol on the Ca++ Currents. ThecurrentswereconductedbyT-type(upper panel) andL-type(lower panel) calciumchannelsinatrialmyocytes.Upper panel, Themembranepotentialwaschangedfrom−80to−20mV.Lower panel, Themembranepotentialwaschangedfrom−30to+30mV.(RedrawnfromBeanBP.J Gen Physiol. 1985;86:1.) •Fig. 16.7 Effectsofdiltiazem,acalciumchannelantagonist,ontheactionpotentials(inmillivolts[mV])andisometriccontractileforces(inmillinewtons[mN])recordedfromapapillarymuscleinvitro.Thetrac-ingswererecordedundercontrolconditions(C)andinthepresenceofdiltiazeminconcentrationsof3,10,and30µmol/L.(RedrawnfromHirthC,etal.J Mol Cell Cardiol. 1983;15:799.)20 50 msec Action potential Force 0 mV 10 30 C and 3 C 3 10 30 mN 0.5 50 msec

1	However, as Vm approaches and then attains positive values near the peak of the action potential upstroke, gK suddenly decreases ( Fig. 16.8 ). The diminished K+ current associated with the reduction in gK prevents excessive loss of K+ from the cell during the plateau. ToenhancegCa,catecholaminesfirstbindtoβ-adrenergicreceptorsinthecardiaccellmembrane.Thisinteractionstimulatesthemembrane-boundenzymeadenylatecyclase,whichraisestheintracellularconcentrationofcyclicadenosinemonophosphate(cAMP;seealso ).TheriseincAMPactivatescAMP-dependentproteinkinase,whichinturnpromotesphosphorylationofL-typecalciumchannelsinthecellmembraneandthusaugmentstheinfluxofCa++ intothecells(see Fig.16.6 ).Conversely,acetylcholineinteractswithmuscarinicreceptorsinthecellmembranetoinhibitadenylatecyclase.Inthisway,acetylcholineantagonizestheactivationofcalciumchannelsandtherebydiminishesgCa.

1	Fig.16.6 ).Conversely,acetylcholineinteractswithmuscarinicreceptorsinthecellmembranetoinhibitadenylatecyclase.Inthisway,acetylcholineantagonizestheactivationofcalciumchannelsandtherebydiminishesgCa. Calciumchannelantagonistsaresubstancesthatblockcalciumchannels.Examplesincludethedrugsverapamil,amlodipine,anddiltiazem.ThesedrugsdecreasegCaandtherebyimpedetheinfluxofCa++ intomyocardialcells.Calciumchannelantagonistsdecreasethedurationoftheactionpotentialplateauanddiminishthestrengthofthecardiaccontraction(see Fig.16.7 ).Calciumchannelantagonistsalsodepressthecontractionofvascularsmoothmuscleandtherebyinducegeneralizedvasodilation.Thisdiminishedvascularresistancereducesthecounterforce(afterload)thatopposesthepropulsionofbloodfromtheventriclesintothearterialsystem,asexplainedin .Hence,vasodilatordrugssuchasthecalciumchannelantagonistsareoftenreferredtoasafterload-reducing drugs.

1	The reduction in gK at both positive and low negative values of Vm is called inward rectification. Inward rectification is a characteristic of several K+ currents, including iK1 ( Fig. 16.9 ). For these channels, large K+ currents flow at negative values of Vm (i.e., gK is large). However, when Vm is near 0 mV or positive, as occurs during the plateau (phase 2), little or no K+ current flows (i.e., gK is small). Thus the substantial gK that prevails during phase 4 of the cardiac action potential (see Fig. 16.8 ) is large because of the iK1 channels, but current through these channels is greatly diminished during the plateau (see Fig. 16.9

1	Fig. 16.8 ) is large because of the iK1 channels, but current through these channels is greatly diminished during the plateau (see Fig. 16.9 Other potassium channels play a role in phase 2 of the action potential. These potassium channels are characterized as delayed iK channels. They are closed during phase 4 and are activated very slowly by the potentials that prevail toward the end of phase 0. Hence, activation of these channels tends to increase gK very gradually during phase 2. These channels play only a minor role during phase 2, but they contribute to the process of final repolarization (phase 3), as described in the section “ Genesis of Repolarization (Phase 3) .” There exist two types of iK channels, classified according to their rates of activation: The more slowly activating channel is designated the iKs channel, and the more rapidly activating channel is designated the iKr channel (see Fig. 16.8). The Nav1.5 SCN5A Cav1.2 CACNA1C ICa,L IK1 Ito,1 Ito,2 IKr IKs

1	Fig. 16.8). The Nav1.5 SCN5A Cav1.2 CACNA1C ICa,L IK1 Ito,1 Ito,2 IKr IKs Kir2.1/2.2 Kv4.2/4.3 Kv1.4/1.7 •Fig. 16.8 Changes in Ion Currents During the Various Phases of the Action Potential in a Fast-Response Cardiac Ventricular Cell. Upper panels, Depolarizingioncurrents.Lower panels, Repolarizingioncurrents.TheinwardcurrentsincludethefastNa+ (iNa)andL-typeCa++ (iCa,L)currents.OutwardcurrentsareiK1,ito,andtherapid(iKr)andslow(iKs)delayedrectifierK+ currents.Theclonesandgenesfortheprincipalioniccurrentsarealsoincluded.(RedrawnfromTomaselliG,MarbánE.Cardiovasc Res. 1999;42:270.) duration of the action potential in myocytes in various regions of the ventricular myocardium is determined in part by the relative distributions of these iKr and iKs channels.

1	The action potential plateau persists as long as the efflux of charge carried mainly by K+ is balanced by the influx of charge carried mainly by Ca++ . The effects of altering this balance are exemplified by the action of the calcium channel antagonist diltiazem in a papillary muscle preparation (see Fig. 16.7 ). With increasing concentrations of diltiazem, the plateau voltage becomes progressively less positive and the plateau duration diminishes. Conversely, administration of certain potassium channel antagonists prolongs the plateau substantially. Genesis of Final Repolarization (Phase 3) The process of final repolarization (phase 3) starts at the end •Fig. 16.9 Inward Rectified K+ of phase 2, when efflux of K+ from the cardiac cell begins to tricular Myocyte When the Potential Was Changed From a Holding exceed influx of Ca++ . As noted, at least three outward K+

1	Potential of −80 mV to Various Test Potentials. Positivevaluesalong currents (ito, iK, and iK1) contribute to the final repolariza tion (phase 3) of the cardiac cell (see Figs. 16.3 16.8 inwardcurrents.Thepointatwhichthecurrenttraceintersectsthe x-axisisthereversalpotential(open circle); itdenotestheNernstThe ito, iKr, and iKs currents help initiate repolarization. equilibriumpotential(EK),atwhichpointthechemicalandelectrostaticThese currents are therefore important determinants of forcesareequal.Vm,membranepotential.(RedrawnfromGilesWR, the duration of the plateau. For example, the duration of ImaizumiY.J Physiol [Lond]. 1988;405:123.) the plateau is substantially less in atrial myocites than in ventricular myocytes (

1	ImaizumiY.J Physiol [Lond]. 1988;405:123.) the plateau is substantially less in atrial myocites than in ventricular myocytes ( Fig. 16.10 ) because the magnitude •Fig. 16.10 Typical Action Potentials (in Millivolts, y-Axis). Thesepotentialswererecordedfromcellsintheventricle(A), sinoatrial(SA)node(B), andatrium(C). NotethatthetimecalibrationinB differsfromthatinA andC. Numbersonthecurvesdenotethephasesoftheactionpotential.(FromHoffmanBF,CranefieldPF.Electrophysiology of the Heart. NewYork:McGraw-Hill;1960.) of ito during the plateau is greater in atrial myocites than in ventricular myocytes. As already noted, the duration of the action potential in ventricular myocytes varies considerably with the location of these myocytes in the ventricular walls (see

1	Fig. 16.5). The i to and iK currents mainly account for these differences. In endocardial myocytes, in which the duration of the action potential is least, the magnitude of iK is greatest. The converse applies to the midmyocardial myocytes. The magnitude of iK and the duration of the action potential are intermediate for epicardial myocytes. The iK1 current does not participate in the initiation of repolarization because the conductance of these channels is very small over the range of Vm values that prevail during the plateau. However, the iK1 channels contribute substantially to the rate of repolarization once phase 3 has been initiated. As Vm becomes increasingly negative during phase 3, the conductance of the channels that carry the iK1 current progressively increases and thereby accelerates repolarization (see Fig. 16.3 Restoration of Ionic Concentrations (Phase 4)

1	Fig. 16.3 Restoration of Ionic Concentrations (Phase 4) The steady inward leak of Na+ that enters the cell rapidly during phase 0 and more slowly throughout the cardiac cycle would gradually depolarize the resting membrane voltage were it not for Na+,K+-ATPase, which is located in the cell membrane (see ). Similarly, most of the excess Ca++ ions that had entered the cell mainly during phase 2 are eliminated principally by a 3Na+-Ca++ antiporter, which exchanges three Na+ ions for one Ca++ ion. However, some of the Ca++ ions are eliminated by an ATP-driven Ca++ pump.

1	Cardiacionchannelsareconnectedtocellularproteinstoformmacromolecularcomplexes.Thesecomplexesareinvolvedinmodulatingthetransport,membranelocalization,operation,posttranslationalmodificationandturnoverofparticularionchannels.ThecarboxyterminalsofionchannelslinkthemwithseveralintracellularproteinssuchasPDZ(postsynapticdensity,disclarge,andzonulaoccludens-1)domainproteinswhosebindingsitesinteractwithsynapse-associatedprotein(SAP97),syntrophin,andA-kinaseanchoringprotein(AKAP5)amongothers.Differentmacromolecularcomplexesarefoundindistinctcellularlocations,andthisisthoughttounderlieionchanneldistribution.Thevoltage-sensitivesodiumchannel(Nav1.5)islinkedwithsyntrophin/dystrophinatlateralcellmembranesandwithankyrinB,plakophilin-2,andcalmodulin-dependentproteinkinaseIIattheintercalateddisk.Also,differentionchannelscanfoundinthesamecomplex.ThusNav1.5channelsandinward-rectifyingK(Kir2.1)channelscanbeconnectedinacomplex,orchannelosome,

1	withSAP97.ThislinknotonlyaffectsthelocalizationbutalsoallowschangesintheabundanceofKir2.1channelstoproducereciprocalchangesinNav1.5abundance;theconverseisalsoobserved.ThusthecomplexcontainsKir2.1,whichsetstherestingmembranepotential,andNav1.5,whichaccountsforrapidexcitation.Colocalizationofthesetwochannelsthereforeexertsapowerfuleffectonexcitabilityanditsregulationundernormalandpathologicalconditions(arrhythmias).

1	Fig. 16.1A ) consist of four principal components: an upstroke (phase 0), an early partial repolarization (phase 1), a plateau (phase 2), and a final repolarization (phase 3). However, in the slow-response action potential (see Fig. 16.1B ), the upstroke is much less steep, early repolarization (phase 1) is absent, the plateau is less prolonged and not as flat, and the transition from the plateau to the final repolarization is less distinct. Blocking fast sodium channels with tetrodotoxin in a fast-response fiber can generate slow responses under appropriate conditions. The Purkinje fiber action potentials shown in

1	Blocking fast sodium channels with tetrodotoxin in a fast-response fiber can generate slow responses under appropriate conditions. The Purkinje fiber action potentials shown in Fig. 16.11 clearly exhibit the two response types. In the control tracing (A), the typical fast-response action potential displays a prominent notch, as a result of ito, that separates the upstroke from the plateau. Progressively higher concentrations of tetrodotoxin produce a graded blockade of the fast sodium channels, as demonstrated in tracings B to E. The upstroke and notch are progressively less prominent in tracings B to D. In tracing E, the notch has disappeared, and the upstroke is very gradual; this action potential resembles a typical slow response.

1	•Fig. 16.11 Effect of Tetrodotoxin, Which Blocks Fast Sodium Channels, on the Action Potentials (Tracings A to E) Recorded in a Purkinje Fiber. Theconcentrationsoftetrodotoxinwere0mol/LintracingA,3×10−8mol/LintracingB,3×10−7mol/LintracingC,and3×10−6mol/LintracingsDandE;EwasrecordedlaterthanD.(RedrawnfromCarmelietE,VereeckeJ.Pflügers Arch. 1969;313:300.)100 mV 1 sec A B C D E Certain cells in the heart, notably those in the SA and AV nodes, exhibit slow-response action potentials. In these cells, depolarization is achieved mainly by influx of Ca++ through L-type calcium channels instead of influx of Na+ through fast sodium channels. Repolarization is accomplished in these fibers by inactivation of the calcium channels and by the increased K+ conductance through the iK1 and iK channels (see Fig. 16.3).

1	Fig. 16.3). An action potential traveling along a cardiac muscle fiber is propagated by local circuit currents, much as it is in nerve and skeletal muscle fibers (see ). When the wave of depolarization reaches the end of the cell, the impulse is conducted to adjacent cells through gap junctions (see Chapter 2). Impulses pass more readily along the length of the cell (isotropic) than laterally from cell to cell (anisotropic) because gap junctions are preferentially located at the ends of the cell. Gap junctions are rather nonselective in their permeability by ions and have a low electrical resistance that allows ionic current to pass from one cell to another. The electrical resistance of gap junctions is similar to that of cytoplasm. The flow of charge from cell to cell follows the principles of local circuit currents and therefore allows intercellular propagation of the impulse. Conduction of the Fast Response

1	Conduction of the Fast Response In fast-and slow-response fibers, the characteristics of conduction differ. In fast-response fibers, fast sodium channels are activated when the transmembrane potential •Fig. 16.12 Theroleoflocalcurrentsinthepropagationofawaveofexcitationdownacardiacfiber. of one region of the fiber suddenly changes from a resting value of approximately −90 mV to the threshold value of approximately −65 mV. The inward Na+ current then rapidly depolarizes the cell at that site. This portion of the fiber subsequently becomes part of the depolarized zone, and the border is displaced accordingly. The same process then begins at the new border. This process is repeated again and again, and the border moves continuously down the fiber as a wave of depolarization ( Fig. 16.12

1	Fig. 16.12 The conduction velocity along the fiber varies directly with the action potential amplitude and the rate of change of the potential (dVm/dt) during phase 0. The action potential amplitude is the potential difference between the fully depolarized and the fully polarized regions of the cell interior. The magnitude of the local current is proportional to this potential difference (see ). Because these local currents shift the potential of the resting zone toward the threshold value, they are local stimuli that depolarize the adjacent resting portion of the fiber to its threshold potential. The greater the potential difference between the depolarized and polarized regions (i.e., the greater the action potential amplitude), the more effectively local stimuli can depolarize adjacent parts of the membrane, and the more rapidly the wave of depolarization is propagated down the fiber.

1	The dVm/dt during phase 0 is also an important determinant of conduction velocity. If the active portion of the fiber depolarizes gradually, the local currents between the resting region and the neighboring depolarizing region are small. The resting region adjacent to the active zone is depolarized gradually, and more time is therefore required for each new section of the fiber to reach threshold. This allows some sodium channels to inactivate.

1	The resting membrane potential is another important determinant of conduction velocity. Changes in the resting membrane potential influence both the amplitude of the action potential and dVm/dt, which in turn alter the conduction velocity (Fig. 16.13 ). Depolarization of Vm inactivates the fast sodium channels, which in turn decreases the amplitude of the action potential and the dVm/dt, and as a consequence conduction velocity is slowed. In addition to changes in extracellular [K+], premature excitation of a cell that has not completely repolarized also results in a •Fig. 16.13 Effect of Changes in Extracellular Potassium Concentration (Extracellular [K+]) on the Action Potentials Recorded From a Purkinje Fiber. Thestimulusartifact(StintracingD)appearsasabiphasicspiketotheleftoftheupstrokeoftheactionpotential.Thehorizontal dashed lines

1	From a Purkinje Fiber. Thestimulusartifact(StintracingD)appearsasabiphasicspiketotheleftoftheupstrokeoftheactionpotential.Thehorizontal dashed lines nearthepeaksoftheactionpotentialsdenote0mV.Whenextracellular[K+]is3mmol/L(“mM”;tracingsAandF),therestingmembranevoltage(Vm)is−82mV,andtheslopeofphase0issteep.Attheendofphase0,theovershootattainsavalueof+30mV.Hence,theactionpotentialamplitudeis112mV.Thedistancefromthestimulusartifacttothebeginningofphase0isinverselyproportionaltotheconductionvelocity.Whenextracellular[K+]isincreasedgraduallyto16mmol(tracingsBtoE),therestingVmbecomesprogressivelylessnegative.Atthesametime,theamplitudesanddurationsoftheactionpotentialsandthesteepnessoftheupstrokesalldiminish.Asaconsequence,conductionvelocitydecreasesprogressively.Atextracellular[K+]levelsof14and16mmol(tracingsDandE),therestingVmattainslevelssufficienttoinactivateallthefastsodiumchannelsandleadtothecharacteristicslow-responseactionpotentials.(FromMyerburgRJ,LazzaraR.In:FischE[ed].Complex

1	Electrocardiography. Philadelphia:FADavis;1973.) decrease in conduction velocity. This too reflects the fact that when Vm is depolarized, more fast sodium channels are inactivated, and thus only a fraction of the sodium channels are available to conduct the inward Na+ current during phase 0.

1	Conduction of the Slow Response Fig. 16.12 ) also propagate the slow response, whose conduction characteristics differ quantitatively from those of the fast response. The threshold potential is approximately −40 mV for the slow response, and conduction is much slower than for the fast response. The conduction velocities of the slow response in the SA and AV nodes are approximately 0.02 to 0.10 m/sec. The fast-response conduction velocities are approximately 0.30 to 1.00 m/sec for myocardial cells and 1.00 to 4.00 m/sec for the specialized conducting (Purkinje) fibers in the ventricles. Slow responses are more readily blocked than are fast responses; that is, conduction ceases before the impulse reaches the end of the myocardial fiber. Also, fast-response fibers can respond at repetition rates that are much faster than those of slow-response fibers.

1	Fig.16.13 alsotakeplaceinthecardiactissueofpatientswithcoronaryarterydisease.Whenregionalmyocardialbloodflowisdiminished,thesupplyofO2andmetabolicsubstratesdeliveredtotheischemictissuesisinsufficient.TheNa+,K+-ATPaseinthemembraneofcardiacmyocytesrequiresconsiderablemetabolicenergytomaintainthenormaltransmembranegradientsofNa+ andK+ .Whenbloodflowisinadequate,theactivityofNa+,K+-ATPaseisimpaired,andtheischemicmyocytesgainexcessNa+ andloseK+ tothesurroundinginterstitialspace.Asaconsequence,[K+]intheextracellularfluidsurroundingtheischemicmyocytesiselevated.Suchchangesinextracellular[K+]may,ifsufficientlylarge,disturbcardiacrhythmandconductioncritically(see Fig.16.13

1	Fig.16.13 Because of the rapid development of artificial pacemakers and other electrical devices for correcting cardiac rhythm disturbances, detailed knowledge of cardiac excitability is essential. The excitability characteristics of various types of cardiac cells differ considerably, depending on whether the action potentials are fast or slow responses. Once the fast response has been initiated, the depolarized cell is no longer excitable until it has partially repolarized (see

1	Once the fast response has been initiated, the depolarized cell is no longer excitable until it has partially repolarized (see Fig. 16.1A ). In the fast response, the interval from the beginning of the action potential until the fiber is able to conduct another action potential is called the effective refractory period which extends from the beginning of phase 0 to a point in phase 3 at which repolarization has reached approximately −50 mV. At approximately this value of Vm, many of the fast sodium channels have transitioned from the inactivated state to the closed state. However, the cardiac fiber is not fully excitable until it has been completely repolarized. Before complete repolarization (i.e., during the relative refractory period), an action potential may be evoked only when the stimulus is stronger than a stimulus that could elicit a response during phase 4.

1	When a fast response is evoked during the relative refractory period of a previous excitation, its characteristics vary with the membrane potential that exists at the time of stimulation ( Fig. 16.14). The later in the relative refractory period that the fiber is stimulated, the greater are the increases in the amplitude of the response and the slope of the upstroke because the number of fast sodium channels that have recovered from inactivation increases as repolarization proceeds. As a consequence, propagation velocity also increases the later in the relative refractory period that the fiber is stimulated. Once the fiber is fully repolarized, the response is constant no matter what time in phase 4 the stimulus is applied.

1	•Fig. 16.15 Effects of Excitation at Various Times After the Initiation of an Action Potential in a Slow-Response Fiber. Inthis fiber,excitationverylateinphase3(orearlyinphase4)inducesa small,nonpropagated(local)response(wavea).Laterinphase4,a •Fig. 16.14 Changesinactionpotentialamplitudeandupstrokeslopeasactionpotentialsareinitiatedatdifferentstagesoftherelativerefractoryperiodoftheprecedingexcitation.(RedrawnfromRosenMR,etal.Am Heart J. 1974;88:380.) In slow-response fibers, the relative refractory period frequently extends well beyond phase 3 (see Fig. 16.1B ). Even after the cell has completely repolarized, it may be difficult to evoke a propagated response for some time. This characteristic of slow-response fibers is called postrepolarization refractoriness.

1	propagatedresponse(waveb)canbeelicited,butitsamplitudeissmallandtheupstrokeisnotverysteep;thisresponseisconductedveryslowly.Stilllaterinphase4,fullexcitabilityisregained,andtheresponse(wavec)displaysnormalcharacteristics.(ModifiedfromSingerDH,etal.Prog Cardiovasc Dis. 1981;24:97.) Fig.16.32 ),thetimingoftheseearlybeatsmaydeterminetheirclinicalconsequence.Iftheyoccurlateintherelativerefractoryperiodoftheprecedingdepolarizationorafterfullrepolarization,theprematuredepolarizationisinconsequential.However,iftheprematuredepolarizationsoriginateearlyintherelativerefractoryperiodoftheventricles,conductionoftheprematureimpulsefromthesiteoforiginisslow,andhenceacardiacimpulseismorelikelytoreexcitesomemyocardialregionthroughwhichithadpassedpreviously(aphenomenonknownasreentry).Ifthatreentryisirregular(i.e.,ifventricularfibrillationensues),theheartcannotpumpeffectively,anddeathmayresult.

1	Action potentials evoked early in the relative refractory period are small, and the upstrokes are not very steep (Fig. 16.15 ). The amplitudes and upstroke slopes progressively improve as action potentials are elicited later in the relative refractory period. Recovery of full excitability is much slower than recovery of the fast response. Impulses that arrive early in the relative refractory period are conducted much more slowly than those that arrive late in that period. The long refractory periods also lead to conduction blocks. Even when slow responses recur at low frequency, the fiber may be able to conduct only a fraction of these impulses; for example, in certain conditions, only alternate impulses may be propagated. •Fig. 16.16 Effectofchangesincyclelength(CL)ontheactionpotentialduration(APD)ofPurkinjefibers.(ModifiedfromSingerD,TenEickRE.Am J Cardiol. 1971;28:381.) Effects of Cycle Length

1	•Fig. 16.16 Effectofchangesincyclelength(CL)ontheactionpotentialduration(APD)ofPurkinjefibers.(ModifiedfromSingerD,TenEickRE.Am J Cardiol. 1971;28:381.) Effects of Cycle Length Cycle length is the time between successive action potentials. Changes in cycle length alter the duration of the action potential in cardiac cells ( Fig. 16.16 Fig. 16.5 ) and thus change their refractory periods. As a consequence, changes in cycle length are often important factors in the initiation or termination of certain arrhythmias (irregular heart rhythms). The changes in action potential duration produced by stepwise reductions in cycle length from 2000 to 200 msec in a Purkinje fiber are shown in

1	The changes in action potential duration produced by stepwise reductions in cycle length from 2000 to 200 msec in a Purkinje fiber are shown in Fig. 16.16 . Note that as cycle length diminishes, the duration of the action potential decreases. This direct correlation between action potential duration and cycle length is mediated by changes in gK that involve at least two types of potassium channels: namely, those that conduct the delayed rectifier K+ currents (iKr and iKs) and those that conduct the transient outward K+ current (ito).

1	The iK current is activated at values of Vm near zero, but the current activates slowly, remains activated for hundreds of milliseconds, and also inactivates very slowly. As a consequence, as the basic cycle length diminishes, each action potential tends to occur earlier in the inactivation period of the iK current initiated by the preceding action potential. Therefore, the shorter the basic cycle length, the greater the outward K+ current during phase 2 and hence the shorter the action potential duration. The ito current also influences the relationship between cycle length and action potential duration. The ito current is also activated at near zero potential, and its magnitude varies inversely with cardiac frequency. Therefore, as cycle length decreases, the consequent increase in the outward K+ current shortens the plateau. Natural Excitation of the Heart and the Electrocardiogram

1	Natural Excitation of the Heart and the Electrocardiogram Excitation of the heart normally occurs in an ordered manner, which allows effective pumping of blood. This ordered excitation occurs via the heart’s conduction system ( Fig. 16.17 ). The SA node is the pacemaker of the heart and initiates the spread of action potentials throughout the atria. This spread of excitation reaches the AV node, where conduction is slowed so that atrial contraction can occur and the ventricles can be adequately filled. Excitation then spreads rapidly throughout the ventricles via the Purkinje fibers so that the ventricular myocytes contract in a coordinated manner. The properties of each component of the heart’s conduction system are described in the next sections. •Fig. 16.17 Thecardiacconductionsystem. CHAPTER 16 Elements of Cardiac Function

1	•Fig. 16.17 Thecardiacconductionsystem. CHAPTER 16 Elements of Cardiac Function The autonomic nervous system controls various aspects of cardiac function, such as the heart rate and contraction strength. However, cardiac function does not require intact innervation. Indeed, a patient with a cardiac transplant, whose heart is completely denervated, may still adapt well to stressful situations. The ability of a denervated, transplanted heart to adapt to changing conditions lies in certain intrinsic properties of cardiac tissue, especially its automaticity.

1	The properties of automaticity (the ability to initiate its own beat) and rhythmicity (the regularity of pacemaking activity) allow a perfused heart to beat even when it is completely removed from the body. The vertebrate heartbeat is myogenic in origin. If the coronary vasculature of an excised heart is artificially perfused with blood or an oxygenated electrolyte solution, rhythmic cardiac contractions may persist for many hours. Some cells in the atria and ventricles can initiate beats; such cells reside mainly in nodal tissues or specialized conducting fibers of the heart.

1	As noted, the region of the mammalian heart that ordinarily generates impulses at the greatest frequency is the SA node; it is the main cardiac pacemaker. Detailed mapping of the electrical potentials on the surface of the right atrium has revealed that two or three sites of automaticity, located 1 or 2 cm from the SA node itself, serve along with the SA node as an atrial pacemaker complex. At times, all these loci initiate impulses simultaneously. At other times, the site of earliest excitation shifts from locus to locus, depending on certain conditions, such as the level of autonomic neural activity.

1	In humans, the SA node is approximately 8 mm long and 2 mm thick, and it lies posteriorly in the groove at the junction between the superior vena cava and the right atrium. The sinus node artery runs lengthwise through the center of the node. The SA node contains two principal cell types: (1) small, round cells that have few organelles and myofibrils and (2) slender, elongated cells that are intermediate in appearance between the round and “ordinary” atrial myocardial cells. The round cells are probably the pacemaker cells; the slender, elongated cells probably conduct the impulses within the node and to the nodal margins. A typical transmembrane action potential recorded from an SA node cell is depicted in Fig. 16.10B. In comparison with the transmembrane potential recorded from a ventricular myocardial cell (see

1	A typical transmembrane action potential recorded from an SA node cell is depicted in Fig. 16.10B. In comparison with the transmembrane potential recorded from a ventricular myocardial cell (see Fig. 16.10A ), the resting potential of the SA node cell is usually less negative, the upstroke of the action potential (phase 0) is less steep, the plateau is not sustained, and repolarization (phase 3) is more gradual. These are characteristic attributes of the slow response. Tetrodotoxin (which blocks the fast Na+ current) has no influence on the SA nodal action potential because the action potential upstroke is not produced by an inward Na+ current through fast channels. Thus tetrodotoxin has no effect on cells that exhibit the slow response.

1	Ordinarily,thefrequencyofpacemakerfiringiscontrolled system.Increasedsympatheticnervousactivity,throughthe releaseofnorepinephrine,raisestheheartrateprincipallyby increasingtheslopeoftheslowdiastolicdepolarization.Thismechanismofincreasingheartratefunctionsduringphysical exertion,anxiety,orcertainillnessessuchasfebrileinfectiousA diseases.Increasedvagalactivity,throughthereleaseof acetylcholine,diminishestheheartratebyhyperpolarizingthepacemakercellmembraneandreducingtheslope oftheslowdiastolicdepolarization.Thesemechanisms predominantoversympatheticactivity.Anextremeexample isvasovagalsyncope,abriefperiodoflightheadednessorlossofconsciousnesscausedbyanintenseburstofvagal activity.Thistypeofsyncopeisareflexresponsetopainor tocertainpsychologicalstimuli.

1	•Fig. 16.18 Mechanisms Involved in the Changes in Frequency of Pacemaker Firing. A, Areductionintheslope(fromwaveatowave b)ofslowdiastolicdepolarizationdiminishesthefiringfrequency.TP,thresholdpotential.B, Anincreaseinthethresholdpotential(fromTP-1toTP-2)oranincreaseinthemagnitudeofthemaximumdiastolicpotential(fromwavesegmentsatod)alsodiminishesthefiringfrequency.(FromHoffmanBF,CranefieldPF.Electrophysiology of the Heart. NewYork:McGraw-Hill;1960.) The transmembrane potential during phase 4 is much less negative in SA (and AV) nodal automatic cells than in atrial or ventricular myocytes because nodal cells lack the iK1 (inward rectifying) type of potassium channel. Thus the ratio of gK to gNa during phase 4 is much less in nodal cells than in myocytes. Hence, during phase 4, Vm deviates much more from the K+ Nernst equilibrium potential (EK) in nodal cells than it does in myocytes.

1	The principal feature of a pacemaker cell that distinguishes it from the other cells manifests in phase 4. In nonautomatic cells, the potential remains constant during this phase, whereas a pacemaker fiber is characterized by slow diastolic depolarization throughout phase 4. Depolarization proceeds at a steady rate until a threshold is attained, and an action potential is then triggered. Pacemaker cell frequency may be varied by a change in (1) the rate of depolarization during phase 4, (2) the maximal negativity during phase 4, or (3) the threshold potential (

1	Pacemaker cell frequency may be varied by a change in (1) the rate of depolarization during phase 4, (2) the maximal negativity during phase 4, or (3) the threshold potential ( Fig. 16.18). When the rate of slow diastolic depolarization is increased, the threshold potential is attained earlier, and the heart rate increases. A rise in the threshold potential delays the onset of phase 0, and the heart rate is reduced. Similarly, when the maximal negative potential is increased, more time is required to reach the threshold potential, when the slope of phase 4 remains unchanged, and the heart rate therefore diminishes. Ionic Basis of Automaticity Several ionic currents contribute to the slow diastolic depolarization that characteristically occurs in the automatic

1	Ionic Basis of Automaticity Several ionic currents contribute to the slow diastolic depolarization that characteristically occurs in the automatic ChangesinautonomicneuralactivitydonotusuallychangetheheartratebyalteringthethresholdlevelofVminthenodalpacemakercells.However,certainantiarrhythmicdrugs,suchasquinidineandprocainamide,doshiftthethresholdpotentialoftheautomaticcellstolessnegativevalues. cells in the heart. In the pacemaker cells of the SA node, at least three ionic currents mediate the slow diastolic depolarization: (1) an outward K+ current, iK; (2) a hyperpolarization-induced inward current, if; and (3) an inward Ca++ current, ICa ( Fig. 16.19 The repetitive firing of the pacemaker cell begins with the iK current. Efflux of K+ tends to repolarize the cell after the upstroke of the action potential. K+ continues to move out well beyond the time of maximal repolarization, but its efflux diminishes throughout phase 4 (see

1	Fig. 16.19 ). As the current diminishes, its opposition to the depolarizing effects of the two inward currents (if and iCa) also gradually decreases. The progressive diastolic depolarization is mediated by the if and iCa currents, which oppose the repolarizing effect of the iK current. The inward current if is activated near the end of repolarization and is carried mainly by Na+ through specific channels that differ from the fast sodium channels. The current was dubbed “funny” because its discoverers had not expected to detect an inward Na+ current in pacemaker cells at the end of repolarization. This current is activated as the membrane potential becomes hyperpolarized beyond −50 mV. The more negative the membrane potential at this time, the greater the activation of if.

1	The second current responsible for diastolic depolarization is the inward rectifying Ca++ current, iCa. This current is activated toward the end of phase 4 as the membrane potential reaches a value of approximately −35 mV (see

1	Fig. 16.19 ). Once the calcium channels are activated, influx of Ca++ into the cell increases. This influx accelerates the rate of diastolic depolarization, which then leads to the action potential upstroke. A decrease in extracellular •Fig. 16.19 Themembranepotentialchanges(blue trace, top half) thatoccurinsinoatrialnodecellsareproducedbythreeprincipalcurrents(green trace, bottom half): theinwardCa++ current(iCa);ahyperpolarization-inducedinwardcurrent(if),andanoutwardK+ current(iK).Thethinnoisygreen trace showsnetmembranecurrentandtheapproximatetimecourseoftherepolarizingoutwardK+ current(iK),thehyperpolarization-inducedinwardcurrent(if),andtheL-typeCa++ current(iCa).Thethick bold red line inthecurrenttraceindicatesthemagnitudeanddirectionofestimatedIf.(RedrawnfromvanGinnekenACG,GilesW.J Physiol. 1991;434:57.)

1	Theso-calledfunnycurrent(if)incardiacSAnodecellsisactivatedbyhyperpolarizationandgatedbycyclicnucleotides,anditschannelisdesignatedHCN.TherearefourmembersoftheHCN genefamily,andsuchchannelsarefoundincentralnervoussystemneuronsthatgenerateactionpotentialsrepetitively.Transmembranesegment4(S4)ofHCNhasmanypositivelychargedaminoacidsthatactasvoltagesensors,whicharealsopresentinvoltage-gatedsodium,potassium,andcalciumchannels.ThedominantchannelexpressedintheheartisderivedfromtheHCN4 gene.MutationsinaminoacidsinS4andintheS4-to-S5linkercausemarkedchangesinthevoltagedependenceofactivation,insuchawaythatgreaterhyperpolarizationisneededtoopenthechannel.Thiseffectislikethatofacetylcholine,andithasbeenpredictedthattheoccurrenceofsuchmutationsinthehumanheartcouldunderliesinusbradycardiaandsicksinussyndrome.

1	•Fig. 16.20 Membrane Action Potentials Recorded From an Sino-atrial Node Pacemaker Cell. TheconcentrationofCa++inthebathwasreducedfrom2to0.2mmol/L(“mM”).(ModifiedfromKohlhardtM,etal.Basic Res Cardiol. 1976;71:17.)–60 0 Ca++ 2 mM 500 msec Ca++ 0.2 mM mV Fig. 16.20 ) or the addition of calcium channel antagonists diminishes the amplitude of the action potential and the slope of the slow diastolic depolarization in SA node cells. Research evidence indicates that additional ion currents—including a sustained (background) inward Na+ current (iNa), the T-type Ca++ current, and the Na+/Ca++ exchange current triggered by spontaneous release of Ca++ from the sarcoplasmic reticulum—may also be involved in pacemaking. These observations illustrate the many ways to sustain this vital function.

1	RegionsoftheheartotherthantheSAnodemayinitiatebeatsinspecialcircumstances.Suchsitesarecalledectopic foci orectopic pacemakers. Ectopicfocimaybecomepacemakerswhen(1)theirownrhythmicitybecomesenhanced,(2)therhythmicityofthehigherorderpacemakersbecomesdepressed,or(3)allconductionpathwaysbetweentheectopicfocusandregionswithgreaterrhythmicitybecomeblocked.Ectopicpacemakersmayactasasafetymechanismwhennormalpacemakingcentersfail.However,ifanectopiccenterfireswhilethenormalpacemakingcenterstillfunctions,theectopicactivitymayinduceeithersporadicrhythmdisturbances,suchasprematuredepolarizations,orcontinuousrhythmdisturbances,suchasparoxysmaltachycardias(seethesection“

1	The autonomic neurotransmitters affect automaticity by altering membrane ionic currents. The adrenergic transmitters increase all three currents involved in SA nodal automaticity. To increase the slope of diastolic depolarization, the augmentation of if and iCa by adrenergic transmitters must exceed the enhancement of iK by these same transmitters. The hyperpolarization induced by acetylcholine released from vagus nerve endings in the heart is achieved by the activation of specific potassium channels: the aThe ionic basis for automaticity in AV node pacemaker cells resembles that in SA node cells. Similar mechanisms also account for automaticity in ventricular Purkinje fibers, except that the fast Na+ current rather than iCa is involved. Also, a voltageand time-dependent K+ current rather than the hyperpolarization-induced inward current if has been suggested to mediate the slow diastolic depolarization; however, this remains to be clarified.

1	acetylcholine-regulated potassium channels (KACh). Acetylcholine also depresses the if and iCa currents. The autonomic neural effects on cardiac cells are described in greater detail in When the SA node or other components of the atrial pacemaker complex are excised or destroyed, pacemaker cells in the AV junction generally take over the pacemaker function for the entire heart. After some time, which may vary from minutes to days, automatic cells in the atria usually become dominant again and resume their pacemaker function. Purkinje fibers in the specialized conduction system of the ventricles also display automaticity. These fibers characteristically fire at a very slow rate. When the AV junction cannot conduct cardiac impulses from the atria to the ventricles, these idioventricular pacemakers in the Purkinje fiber network initiate the ventricular contractions, but at a frequency of only 30 to 40 beats per minute.

1	Ifanectopicfocusinoneoftheatriasuddenlybegantofireatahighrate(e.g.,150impulsesperminute)inanindividualwithanormalheartrateof70beatsperminute,theectopicsitewouldbecomethepacemakerfortheentireheart.Ifthatrapidectopicfocussuddenlystoppedfiring,theSAnodewouldremainbrieflyquiescentbecauseofoverdrivesuppression.TheintervalfromtheendoftheperiodofoverdriveuntiltheSAnoderesumesfiringiscalledthesinus node recovery time. Inpatientswithsicksinussyndrome,thesinusnoderecoverytimeisprolonged.Theconsequentperiodofasystole(absenceofaheartbeat)maycauselossofconsciousness. The automaticity of pacemaker cells diminishes after these cells have been excited at a high frequency. This phenomenon is known as overdrive suppression. Because the intrinsic rhythmicity of the SA node is greater than that of the other latent pacemaking sites in the heart, firing of the SA node tends to suppress the automaticity in other loci.

1	Overdrive suppression results from the activity of membrane Na+,K+-ATPase. A certain amount of Na+ enters the cardiac cell during each depolarization. The more frequently the cell is depolarized, the more Na+ enters the cell per minute. At high excitation frequencies, the activity of Na+,K+-ATPase increases to extrude this larger amount of Na+ from the cell. The activity of Na+,K+-ATPase hyperpolarizes the cell because three Na+ ions are extruded by the pump in exchange for two K+ ions that enter the cell (see ). Therefore, slow diastolic depolarization requires more time to reach the firing threshold. In addition, when the overdrive suddenly ceases, the activity of Na+,K+-ATPase does not slow instantaneously but temporarily remains overactive. This continued extrusion of Na+ opposes the gradual depolarization of the pacemaker cell during phase 4, and it temporarily suppresses the cell’s intrinsic automaticity.

1	From the SA node, the cardiac impulse spreads radially throughout the right atrium (see Fig. 16.17 ) along ordinary atrial myocardial fibers at a conduction velocity of approximately 1 m/second. A special pathway, the anterior interatrial myocardial band (or Bachmann’s bundle), conducts the SA node impulse directly to the left atrium. The wave of excitation proceeds inferiorly through the right atrium and ultimately reaches the AV node (see Fig. 16.17 ), which is normally the sole entry route of the cardiac impulse to the ventricles.

1	SomepeoplehaveaccessoryAVpathways.Becausethesepathwaysoftenserveasapartofareentryloop(seethesection“

1	”),theycanbeassociatedwithseriouscardiacrhythmdisturbances.Wolff-Parkinson-Whitesyndrome,acongenitaldisturbance,isthemostcommonclinicaldisorderinwhichabypasstractofmyocardialfibersbecomesanaccessorypathwaybetweentheatriaandventricles.Ordinarily,thesyndromecausesnofunctionalabnormality.ThedisturbanceiseasilydetectedonanECGbecauseaportionoftheventricleisexcitedviathebypasstractbeforetheremainderoftheventricleisexcitedviatheAVnodeandtheHis-Purkinjesystem.Thispreexcitationappearsasabizarreconfigurationintheventricular(QRS)complexoftheECG.Onoccasion,however,areentryloopdevelopsinwhichtheatrialimpulsetravelstotheventriclesviaoneofthetwoAVpathways(AVnodeorbypasstract)andthenbacktotheatriathroughtheotherofthesetwopathways.Continuouscirclingaroundtheloopleadstoaveryrapidrhythm(supraventriculartachycardia).Thisrapidrhythmmaybeincapacitatingbecauseitmightnotallowsufficienttimeforventricularfilling.TransientblockoftheAVnodebyaninjectionofadenosineintravenouslyorbyareflexiveincreaseinvagalactivity

1	ycardia).Thisrapidrhythmmaybeincapacitatingbecauseitmightnotallowsufficienttimeforventricularfilling.TransientblockoftheAVnodebyaninjectionofadenosineintravenouslyorbyareflexiveincreaseinvagalactivity(bypressingontheneckoverthecarotidsinusregion)usuallyabolishesthetachycardiaandrestoresanormalsinusrhythm.

1	The atrial plateau (phase 2) is briefer and less developed, and repolarization (phase 3) is slower (see Fig. 16.10 ) than in a typical ventricular fiber. The action potential duration in atrial myocytes is briefer than that in ventricular myocytes because efflux of K+ is greater during the plateau in atrial myocytes than in ventricular myocytes. The presence of an ultrarapid K+ current (IKur) in atrial myocytes, but not in ventricular myocytes, contributes to greater K+ efflux and shorter action potentials in atrial myocytes.

1	The atrial excitation wave reaches the ventricles via the AV node. In adult humans, this node is approximately 15 mm long, 10 mm wide, and 3 mm thick. The node is situated posteriorly on the right side of the interatrial septum near the ostium of the coronary sinus. The AV node contains the same two cell types as the SA node, but the round cells •Fig. 16.21 Membranepotentialsrecordedfromanatrioventricularnodecellundercontrolconditions(C)andinthepresenceofthecalciumchannelantagonistdiltiazematconcentrationsof0.1,0.3,and1µmol/L.(RedrawnfromHirthC,etal.J Mol Cell Cardiol. 1983;15:799.)–25 0 25 msec C 0.1 0.3 1 mV in the AV node are less abundant and the elongated cells predominate. The AV node is made up of three functional regions: the atrionodal (AN) region, or the transitional zone between the atrium and the remainder of the node; (2) the nodal (N) region, or the midportion of the AV node; and the nodal-His (NH) region, or the zone in which nodal fibers gradually merge with the bundle

1	the atrium and the remainder of the node; (2) the nodal (N) region, or the midportion of the AV node; and the nodal-His (NH) region, or the zone in which nodal fibers gradually merge with the bundle of His, which is the upper portion of the specialized conducting system for the ventricles (see

1	Fig. 16.17). Normally, the AV node and the bundle of His are the only pathways along which the cardiac impulse travels from atria to ventricles. Several features of AV conduction are of physiological and clinical significance. The principal delay in conduction of impulses from the atria to the ventricles occurs in the AN and N regions of the AV node. Conduction velocity is actually less in the N region than in the AN region. However, the path length is substantially greater in the AN region than in the N region. Conduction times through the AN and N regions account for the delay between the start of the P wave (the electrical manifestation of atrial excitation) and the QRS complex (the electrical manifestation of ventricular excitation) on an ECG (see the section “ Electrocardiography”). In terms of function, the delay between atrial and ventricular excitation enables optimal ventricular filling during atrial contraction.

1	Electrocardiography”). In terms of function, the delay between atrial and ventricular excitation enables optimal ventricular filling during atrial contraction. In the N region, slow-response action potentials prevail. The resting potential is approximately −60 mV, the upstroke velocity is low (≈5 V/sec), and the conduction velocity is approximately 0.05m/sec. Tetrodotoxin, which blocks the fast sodium channels, has virtually no effect on action potentials in this region (or on any other slow-response fibers). Conversely, calcium channel antagonists decrease the amplitude and duration of the action potentials ( Fig. 16.21 ) and depress AV conduction. bThe shapes of the action potentials in the AN region are intermediate between those in the N region and those in the atria. Similarly, action potentials in the NH region are transitional between those in the N region and those in the bundle of His. CHAPTER 16 Elements of Cardiac Function

1	CHAPTER 16 Elements of Cardiac Function Like other slow-response action potentials, the relative refractory period of cells in the N region extends well beyond the period of complete repolarization; that is, these cells display postrepolarization refractoriness (see Fig. 16.15 ). As the heart rate increases, the time between successive atrial depolarizations is decreased, and conduction through the AV junction slows. Abnormal prolongation of the AV conduction time is called a first-degree AV block (see the section ”). Most of the prolongation of AV conduction induced by an increase of atrial frequency takes place in the N region of the AV node.

1	Impulses tend to be blocked in the AV node at stimulation frequencies that are easily conducted in other regions of the heart. If the atria are depolarized at a high repetition rate, only a fraction (e.g., half) of the atrial impulses might be conducted through the AV junction to the ventricles. The conduction pattern in which only a fraction of the atrial impulses are conducted to the ventricles is called a second-degree AV block (see the section ”). This type of block may protect the ventricles from excessive contraction frequencies, wherein the filling time between contractions might be inadequate. Retrograde conduction can occur through the AV node. However, the conduction time is significantly longer, and the impulse is blocked at lower repetition rates when the impulse is conducted in the retrograde instead of the ante-grade direction. In addition, the AV node is a common site for reentry (see the section “

1	As in the SA node, the autonomic nervous system regulates AV conduction. Weak vagal activity may simply prolong the AV conduction time. Thus for any given atrial cycle length, the atrium-to-His or atrium-to-ventricle conduction time is prolonged by vagal stimulation. Stronger vagal activity may cause some or all of the impulses arriving from the atria to be blocked in the node. The conduction pattern in which none of the atrial impulses reaches the ventricles is called a third degree, or complete, AV block (see the section ”). The vagally induced delay or absence of conduction through the AV node occurs mainly in the N region. This effect of vagal stimulation reflects the action of acetylcholine to hyperpolarize the membrane of the conducting fibers in the N region (Fig. 16.22 ). The greater the hyperpolarization at the time of arrival of the atrial impulse, the more impaired the AV conduction.

1	Cardiac sympathetic nerves, in contrast, facilitate AV conduction. They decrease the AV conduction time and enhance the rhythmicity of latent pacemakers in the AV junction. The norepinephrine released at the postganglionic sympathetic nerve terminals increases the amplitude and slope of the upstroke of the AV nodal action potentials, principally in the AN and N regions of the node.

1	The bundle of His passes subendocardially down the right side of the interventricular septum for approximately 1 cm •Fig. 16.22 Effects of a Brief Vagal Stimulus (St) on the Transmembrane Potential. Thisresponsewasrecordedfromanatrioventricular(AV)nodalfiber(red trace). Compoundactionpotentialswererecordedfromtheatrium(A1,A2,A3;upper blue trace)andfromtheHis-Purkinjesystem(H;lower blue trace).Notethatshortlyaftervagalstimulation,themembraneofthefiberwashyperpolarized.Theatrialexcitation(A2)thatarrivedattheAVnodewhenthecellwashyperpolarizedfailedtobeconducted,asdenotedbytheabsenceofadepolarizationintheHiselectrogram(H).Theatrialexcitationsthatpreceded(A1)andfollowed(A3)excitationA2wereconductedtotheHisbundleregion.(RedrawnfromMazgalevT.etal.Am J Physiol. 1986;251:H631.) and then divides into the right and left bundle branches (see

1	Fig. 16.17 ). The right bundle branch, a direct continuation of the bundle of His, proceeds down the right side of the interventricular septum. The left bundle branch, which is considerably thicker than the right, arises almost perpendicular from the bundle of His and perforates the interventricular septum. On the subendocardial surface of the left side of the interventricular septum, the left bundle branch splits into a thin anterior division and a thick posterior division. Conductionofimpulsesintherightorleftbundlebranchorineitherdivisionoftheleftbundlebranchmaybeimpaired.Conductionblocksmaydevelopinoneormoreoftheseconductionpathwaysasaconsequenceofcoronaryarterydiseaseordegenerativeprocessesassociatedwithaging,andtheygiverisetocharacteristicECGpatterns.Blockofeitherofthemainbundlebranchesisknownasright orleft bundle branch block. Blockofeitherdivisionoftheleftbundlebranchiscalledleft anterior orleft posterior hemiblock.

1	The right bundle branch and the two divisions of the left bundle branch ultimately subdivide into a complex network of conducting fibers, called Purkinje fibers, that spread out over the subendocardial surfaces of both ventricles. Purkinje fibers have abundant, linearly arranged sarcomeres, as do myocytes. However, the transverse (T) tubular system, which is well developed in myocytes, is absent in the Purkinje fibers of many species. Purkinje fibers are the broadest cells in the heart: 70 to 80 µm in diameter, in comparison with diameters of 10 to 15 µm for ventricular myocytes. Partly because of the large diameter of the Purkinje fibers, conduction velocity (1 to 4 m/second) in these fibers exceeds that in any other fiber type within the heart. The increased conduction velocity enables rapid activation of the entire endocardial surface of the ventricles.

1	The action potentials recorded from Purkinje fibers resemble those of ordinary ventricular myocardial fibers. However, because of the long refractory period of Purkinje fiber action potentials, many premature excitations of the atria are conducted through the AV junction but are then blocked by the Purkinje fibers. Blockade of these atrial excitations prevents premature contraction of the ventricles. This function of protecting the ventricles against the effects of premature atrial depolarization is especially pronounced at slow heart rates because the action potential duration, and hence the effective refractory period, of the Purkinje fibers vary inversely with the heart rate (see Fig. 16.16 ). At slow heart rates, the effective refractory period of the Purkinje fibers is especially prolonged.

1	Fig. 16.16 ). At slow heart rates, the effective refractory period of the Purkinje fibers is especially prolonged. In contrast to Purkinje fibers, the effective refractory period of AV node cells does not change appreciably over the normal range of heart rates and actually increases at very rapid heart rates. Therefore, when the atrium is excited at high repetition rates, it is the AV node that normally protects the ventricles from these excessively high frequencies.

1	The first portions of the ventricles to be excited by impulses arriving from the AV node are the interventricular septum (except the basal portion) and the papillary muscles. The activation wave spreads into the substance of the septum from both its left and right endocardial surfaces. Early contraction of the septum makes it more rigid and allows it to serve as an anchor point for contraction of the remaining ventricular myocardium. Furthermore, early contraction of the papillary muscles prevents eversion of the AV valves into the atria during ventricular systole.

1	The endocardial surfaces of both ventricles are activated rapidly, but the wave of excitation spreads from endocardium to epicardium at a slower velocity (≈0.3 to 0.4 m/sec). The epicardial surface of the right ventricle is activated earlier than that of the left ventricle because the right ventricular wall is appreciably thinner than the left. In addition, the apical and central epicardial regions of both ventricles are activated somewhat earlier than their respective basal regions. The last portions of the ventricles to be excited are the posterior basal epicardial regions and a small zone in the basal portion of the interventricular septum. The conditions necessary for reentry (defined in the following box) are illustrated in

1	The conditions necessary for reentry (defined in the following box) are illustrated in Fig. 16.23 . In each of the four panels, a single bundle of cardiac fibers splits into a left branch and a right branch. A connecting bundle runs cSimilar directional changes in the refractory period also occur in ventricular myocytes in response to changes in heart rate. •Fig. 16.23 The Role of Unidirectional Block in Reentry. A, Anexcitationwavetravelingdownasinglebundle(S)offiberscontinuesdowntheleft(L)andright(R)branches.Thedepolarizationwaveenterstheconnectingbranch(C)frombothendsandisextinguishedatthezoneofcollision.B, ThewaveisblockedintheLandRbranches.C, AbidirectionalblockexistsintheRbranch.D, AunidirectionalblockexistsintheRbranch.Theantegradeimpulseisblocked,buttheretrogradeimpulseisconductedthroughandreenterstheSbundle. between the two branches. Normally, the impulse moving down the single bundle is conducted along the left and right branches (see

1	between the two branches. Normally, the impulse moving down the single bundle is conducted along the left and right branches (see Fig. 16.23A ). As the impulse reaches the link to the connecting bundle ( Fig. 16.23C), it enters from both sides and becomes extinguished at the point of collision. The impulse from the left side cannot proceed because the tissue beyond is absolutely refractory; it has just been depolarized from the other direction. The impulse also cannot pass through the connecting bundle from the right for the same reason. Fig. 16.23B shows that the impulse cannot complete the circuit if an antegrade block exists in the left and right branches of the fiber bundle. Also, if a bidirectional block exists at any point in the loop (e.g., branch R in Fig. 16.23C ), the impulse also cannot reenter.

1	Fig. 16.23C ), the impulse also cannot reenter. Incertainconditions,acardiacimpulsemayreexcitesomemyocardialregionthroughwhichithadpassedpreviously.Thisphenomenon,knownasreentry, isresponsibleformanyclinicalarrhythmias(disturbancesincardiacrhythm).Thereentrymaybeorderedorrandom.Inorderedreentry,theimpulsetraversesafixedanatomicalpath,whereasinrandomreentry,thepathcontinuestochange. A necessary condition for reentry is that at some point in the loop the impulse can pass in one direction but not in the other. This phenomenon is called unidirectional block. As shown in Fig. 16.23D , the impulse may travel down

1	CHAPTER 16 Elements of Cardiac Function the left branch normally but is blocked in the antegrade direction in the right branch because of some pathological change in the myocardial cells in that branch. The impulse that was conducted down the left branch and through the connecting branch of the connecting bundle may then be able to penetrate the depressed region in the right branch from the retrograde direction, even though the antegrade impulse had been blocked previously at this same site. Why is the antegrade impulse blocked but not the retrograde impulse? The reason is that the antegrade impulse arrives at the depressed region in the right branch earlier than the retrograde impulse because the path length of the ante-grade impulse is very short, whereas the retrograde impulse traverses a much longer path. Therefore, the antegrade impulse may be blocked simply because it arrives at the depressed region during its effective refractory period. If the retrograde impulse is delayed

1	a much longer path. Therefore, the antegrade impulse may be blocked simply because it arrives at the depressed region during its effective refractory period. If the retrograde impulse is delayed sufficiently, the refractory period may have ended in the affected region, and the impulse can then be conducted back through this region and return to the single bundle.

1	Although unidirectional block is a necessary condition for reentry, it alone cannot cause reentry. For reentry to occur, the effective refractory period of the reentered region must be shorter than the conduction time around the loop. In Fig. 16.23D , if the tissue just beyond the depressed zone in the right branch is still refractory from the ante-grade depolarization, the retrograde impulse will not be conducted into the single bundle branch. Therefore, the conditions that promote reentry are those that prolong the conduction time or shorten the effective refractory period.

1	The functional characteristics of the various components of the reentry loops responsible for specific cardiac arrhythmias are diverse. Some loops are large and involve entire specialized conduction bundles, whereas others are microscopic. The loop may include myocardial fibers, specialized conducting fibers, nodal cells, and junctional tissues in almost any conceivable arrangement. In addition, the various cardiac cells in the loop may be normal or abnormal.

1	The propagation velocity along a multicellular cardiac conduction fiber is normally facilitated by gap junctions that lie between consecutive conducting fibers. Variations in the protein structure of the connexins in the gap junctions can affect the propagation velocity along these fibers. The chemical structure of the specific connexins can vary locally in cardiac tissues and, as a result, can establish local variations in propagation velocity. Such topical variations in velocity might include regions of unidirectional block that induce reentrant rhythm disturbances.

1	Triggered activity is so named because it is always coupled to a preceding action potential. Because reentrant activity is also coupled to a preceding action potential, the arrhythmias induced by triggered activity are usually difficult to distinguish from those induced by reentry. Triggered activity is caused by afterdepolarizations, of which two types •Fig. 16.24 Effect of Pacing at Different Cycle Lengths (CL) on Cesium-Induced Early Afterdepolarizations (EADs) in a Purkinje Fiber. A, EADsarenotevident.B, EADsfirstappear(arrows). ThethirdEADreachesthresholdandtriggersanactionpotential(third arrow). C, EADsthatappearaftereachdrivendepolarizationtriggeranactionpotential.D, Triggeredactionpotentialsoccurinsalvos.(ModifiedfromDamianoBP,RosenM.Circulation. 1984;69:1013.) are recognized: early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs). EADs may appear either at the end of the action potential plateau (phase 2) or approximately midway through repolarization

1	early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs). EADs may appear either at the end of the action potential plateau (phase 2) or approximately midway through repolarization (phase 3), whereas DADs occur near the very end of repolarization or just after full repolarization (phase 4).

1	EADs are more likely to occur when the prevailing heart rate is slow; a rapid heart rate suppresses EADs ( Fig. 16.24 ). EADs are also more likely to occur in cardiac cells with prolonged action potentials than in cells with shorter action potentials. For example, EADs can be induced more readily in myocytes from the midmyocardial region of the ventricular walls than in myocytes from the endocardial or epicardial regions because of the longer action potential of midmyocardial myocytes (see Fig. 16.5 ). Certain antiarrhythmic drugs, such as quinidine, prolong the action potential. As a consequence, such drugs increase the likelihood that EADs may occur. Hence, some antiarrhythmic drugs are also proarrhythmic. Insomecases,mutationsintheconnexin40gene(GJA5) underliethedevelopmentofatrialfibrillation.Thismutationimpairstheassemblyofgapjunctionsinmyocytesandthereforereduceselectricalcouplingofthecells,asdescribedbyGollobMHandcolleagues(2006).

1	The direct correlation between a cell’s action potential duration and its susceptibility to EADs is related to the time required for calcium channels in the cell membranes to recover from inactivation. When action potentials are sufficiently prolonged, the calcium channels that were activated at the beginning of the plateau have sufficient time to recover from inactivation and thus may be reactivated before the cell fully repolarizes. This secondary activation could then trigger an EAD. ThepathologicalsignificanceofEADswasrecognizedinconnectionwiththecongenitalanddrug-inducedlongQTsyndrome.Astheactionpotentiallengthens,EADsoccurandcausetriggeredautomaticity.Intheelectrocardiogram,thisproblemismanifestedaspolymorphicventriculartachycardia,alsocalledtorsades de pointes. Episodesoftorsadesdepointescanbeself-limitedbutmayprogresstoventricularfibrillationandsuddendeath.Hypokalemiaandbradycardia(see

1	Fig.16.24) areclinicalhallmarksofthiscondition.RestoringextracellularK+ tonormallevelsandincreasingheartratearetwoapproachesusedtoovercomethepropensityfordevelopmentoftorsadesdepointes.

1	In contrast to EADs, DADs are more likely to occur when the heart rate is high (Fig. 16.25 ). DADs are associated with elevated intracellular [Ca++]. The amplitudes of DADs are increased by interventions that raise intracellular [Ca++], such as increasing extracellular [Ca++] and administering toxic amounts of digitalis glycosides. The elevated levels of intracellular Ca++ provoke the oscillatory release of Ca++ from the sarcoplasmic reticulum. Hence, in myocardial cells, DADs are accompanied by small rhythmic changes in the force developed. The high intracellular [Ca++] also activates certain membrane channels that allow the passage of Na+ and K+ . The net flux of these cations constitutes a transient inward current (iti), that contributes to the appearance of DADs. The elevated intracellular [Ca++] may also activate the 3Na+-Ca++ antiporter. This electrogenic antiporter, which moves three Na+ ions into the cell for each Ca++ ion that it releases, also creates a net inward cation

1	[Ca++] may also activate the 3Na+-Ca++ antiporter. This electrogenic antiporter, which moves three Na+ ions into the cell for each Ca++ ion that it releases, also creates a net inward cation current that contributes to the appearance of DADs.

1	The ECG enables physicians to infer the course of cardiac electrical impulses by recording the variations in electrical potential at various loci on the surface of the body. By analyzing the details of these fluctuations in electrical potential, the physician gains valuable insight into (1) the anatomical orientation of the heart; (2) the relative sizes of its chambers; (3) various disturbances in rhythm and conduction; (4) the extent, location, and progress of ischemic damage to the myocardium; (5) the effects of •Fig. 16.25 Transmembrane Action Potentials Recorded From Purkinje Fibers. Acetylstrophanthidin,adigitalisglycoside,wasaddedtothebath,andsequencesofsixdrivenbeats(denotedbythedots)wereeachproducedatabasiccyclelength(BCL)of800(A), 700(B), 600(C), and500(D) msec.NotethatdelayedafterpotentialsoccurredafterthedrivenbeatsandthattheseafterpotentialsreachedthresholdafterthelastdrivenbeatinB toD. (FromFerrierGR,etal.Circ Res. 1973;32:600.) altered electrolyte concentrations; and (6)

1	toD. (FromFerrierGR,etal.Circ Res. 1973;32:600.) altered electrolyte concentrations; and (6) the influence of certain drugs (notably digitalis, antiarrhythmic agents, and calcium channel antagonists). Because electrocardiography is an extensive and complex discipline, only the elementary principles are considered in this section.

1	P-R QRS Q P R S T QT ST 0.1 sec •Fig. 16.26 Importantdeflectionsandintervalsofatypicalscalarelectrocardiogram.

1	In electrocardiography, a lead is the electrical connection from the patient’s skin to a recording device (electrocardiograph) that measures the electrical activity of the heart. The system of leads used to record routine ECGs is oriented in certain planes of the body. The diverse electrical events that exist in the heart at any moment can be represented by a three-dimensional vector (a quantity with magnitude and direction). A system of recording leads oriented in a given plane detects only the projection of the three-dimensional vector on that plane. The potential difference between two recording electrodes represents the projection of the vector on the line between the two leads. Components of vectors projected on such lines are not vectors but scalar quantities (having magnitude but not direction). Hence, a recording of changes in the difference in potential between two points on the skin surface over time is called a scalar ECG.

1	A scalar ECG detects temporal changes in the electrical potential between some point on the surface of the skin and an indifferent electrode or between pairs of points on the skin surface. The cardiac impulse progresses through the heart in a complex three-dimensional pattern. Hence, the precise configuration of the ECG varies from individual to individual, and in any given individual, the pattern varies with the anatomical location of the leads. The graphic display of the electrical impulse recorded in an ECG is called a tracing. In general, a tracing consists of P, QRS, and T waves (

1	In general, a tracing consists of P, QRS, and T waves ( Fig. 16.26). The P wave reflects the spread of depolarization through the atria, the QRS wave (or complex) reflects depolarization of the ventricles, and the T wave represents repolarization of the ventricles (repolarization of the atria occurs, and is therefore masked, during ventricular depolarization). The PR interval (or more precisely, the PQ interval) is a measure of the time from the onset of atrial activation to the onset of ventricular activation; it normally ranges from 0.12 to 0.20 second. A large fraction of this time involves passage of the impulse through the AV conduction system. Pathological prolongations of the PR interval are associated with disturbances in AV conduction. Such disturbances may be produced by inflammatory, circulatory, pharmacological, or neuronal mechanisms.

1	The configuration and amplitude of the QRS complex vary considerably among individuals. The duration is usually between 0.06 and 0.10 second. An abnormally prolonged QRS complex may indicate a block in the normal conduction pathways through the ventricles (such as a block of the left or right bundle branch). During the ST interval, the entire ventricular myocardium is depolarized. Therefore, the ST segment normally lies on the isoelectric line. Any appreciable deviation of the ST segment from the isoelectric line may indicate ischemic damage to the myocardium. The QT interval, sometimes referred to as the period of “electrical systole” of the ventricles, is closely correlated with the mean action potential duration of the ventricular myocytes. The duration of the QT interval is approximately 0.4 second, but it varies inversely with the heart rate, mainly because the duration of the myocardial cell’s action potential varies inversely with the heart rate (see Fig. 16.16

1	Fig. 16.16 In most leads, the T wave is deflected in the same direction from the isoelectric line as the major component of the QRS complex, although biphasic (i.e., oppositely directed) T waves are perfectly normal in certain leads. Deviation of the T wave and QRS complex in the same direction from the isoelectric line indicates that the repolarization process is proceeding in a direction counter to that of the depolarization process. T waves that are abnormal either in direction or in amplitude may indicate myocardial damage, electrolyte disturbances, or cardiac hypertrophy. The original ECG lead system was devised by Willem Einthoven at the beginning of the 20th century. In this system, the vector sum of all cardiac electrical activity at any moment is called the resultant cardiac vector. This directional electrical force is considered to lie in the center of an equilateral triangle whose apices are located in the left and right shoulders and the pubic region (

1	Fig. 16.27 ). This triangle, called Einthoven’s triangle, is oriented in the frontal plane of the body. Hence, only the projection of the resultant cardiac vector on the frontal plane is detected by this system of leads. For convenience, the electrodes are connected to the right and left forearms rather than to the corresponding shoulders because the arms represent simple electrical extensions of leads from the shoulders. Similarly, the leg represents an extension of the lead system from the pubis, and thus the third electrode is generally connected to an ankle (usually the left one). Certain conventions dictate the manner in which these standard limb leads are connected to the electrocardiograph. Lead I records the potential difference between the left arm and the right arm. The connections are such that when the •Fig. 16.27 Einthoven’striangle,illustratingtheelectrocardiographicconnectionsforstandardlimbleadsI,II,andIII.

1	potential at the left arm (VLA) exceeds the potential at the right arm (VRA), the tracing is deflected upward from the isoelectric line. In Fig. 16.27 Fig. 16.28 , this arrangement of connections for lead I is designated by a plus sign at the left arm and by a minus sign at the right arm. Lead II records the potential difference between the right arm and the left leg, and the tracing is deflected upward when the potential at the left leg (VLL) exceeds VRA. Finally, lead III registers the potential difference between the left arm and the left leg, and the tracing is deflected upward when VLL exceeds VLA. These connections were chosen arbitrarily so that the QRS complexes are upright in all three standard limb leads in most normal individuals. If the frontal projection of a resultant cardiac vector at some moment is represented by an arrow (tail negative, head positive), as in

1	If the frontal projection of a resultant cardiac vector at some moment is represented by an arrow (tail negative, head positive), as in Fig. 16.27 , the potential difference, VLA − VRA, recorded in lead I is represented by the component of the vector projected along the horizontal line between the left arm and the right arm, also shown in Fig. 16.27 . If the vector makes an angle (θ) of 60 degrees with the horizontal line (as in Fig. 16.28A), the deflection recorded in lead I is upward because the positive arrowhead lies closer to the left arm than to the right arm. The deflection in lead II is also upright because the arrowhead lies closer to the left leg than •Fig. 16.28 MagnitudeanddirectionoftheQRScomplexesinlimbleadsI,II,andIIIwhenthemeanelectricalaxis(θ)is60degrees(A), 120degrees(B), and0degrees(C).

1	to the right arm. The magnitude of the lead II deflection is greater than that in lead I because in this example, the direction of the vector parallels that of lead II; therefore, the magnitude of the projection on lead II exceeds that on lead I. Similarly, in lead III, the deflection is upright and its magnitude equals that in lead I. If the vector in Fig. 16.27A is the result of electrical events that occur during the peak of the QRS complex, the orientation of this vector is said to represent the mean electrical axis of the heart in the frontal plane. The positive rotatory direction of this axis is assumed to be in the clockwise direction from the horizontal plane (contrary to the usual mathematical convention). In normal individuals, the average mean electrical axis is approximately +60 degrees (as in Fig. 16.28A ). Therefore, QRS complexes are usually upright in all three leads and largest in lead II. If the mean electrical axis shifts substantially to the right (as in

1	Fig. 16.28A ). Therefore, QRS complexes are usually upright in all three leads and largest in lead II. If the mean electrical axis shifts substantially to the right (as in Fig. 16.28B , in which θ= 120 degrees), projections of the QRS complexes on the standard leads change considerably. In this case, the largest upright deflection is in lead III, and the deflection in lead I is inverted because the arrowhead is closer to the right arm than to the left arm. Such a shift is termed right axis deviation and occurs with hypertrophy (i.e., increased thickness) of the right ventricle. When the axis shifts to the left, as occurs with hypertrophy of the left ventricle (see Fig. 16.28C , where θ= 0 degrees), the largest upright deflection is in lead I, and the QRS complex in lead III is inverted.

1	Fig. 16.28C , where θ= 0 degrees), the largest upright deflection is in lead I, and the QRS complex in lead III is inverted. In addition to limb leads I, II, and III, other limb leads that are also oriented in the frontal plane are routinely recorded in patients. These leads are (1) aVR, for which the right arm is defined as the positive lead and the middle of the heart is defined as the negative lead (i.e., the left arm and ankle leads are connected together); (2) aVL, for which the left arm is the positive lead and the middle of the heart is defined as the negative lead (i.e., the right arm and ankle leads are connected together); and (3) aVF, for which the ankle (foot) lead is defined as positive and the middle of the heart is defined as the negative lead (i.e., the two arm leads are connected together). The axes of these leads form angles of +90 degrees for aVF, −30 degrees for aVL, and −150 degrees for aVR (all with respect to the horizontal axis).

1	Leads can also be applied to the surface of the chest, so-called precordial leads, to determine the projections of the cardiac vector on the sagittal and transverse planes of the body. These precordial leads are recorded from six selected points on the anterior and lateral surfaces of the chest in the vicinity of the heart. The leads extend from the right border of the sternum in the fourth intercostal space (lead V1) to under the left arm (midaxillary line) in the fifth intercostal space (lead V6). Each precordial lead (V1 to V6) is defined as a positive lead, whereas the middle of the heart is defined as the negative lead. Detailed analysis of the ECG, as detected by the various lead systems just described, is beyond the scope of this book. Interested students are referred to textbooks on electrocardiography for more information.

1	Changesinthemeanelectricalaxismayoccuriftheanatomicalpositionoftheheartisalteredoriftherelativemassoftherightandleftventriclesisabnormal,asitisincertaincardiovasculardisturbances.Forexample,theaxistendstoshifttowardtheleft(morehorizontal)inshort,stockyindividualsandtowardtheright(morevertical)intall,thinpersons.Inaddition,inleftorrightventricularhypertrophy(increasedmyocardialmassofeitherventricle),theaxisshiftstowardthehypertrophiedside. Cardiac arrhythmias are disturbances in either impulse initiation or impulse propagation. Disturbances in impulse initiation include those that arise from the SA node and •Fig. 16.29 Electrocardiographic Tracings of Heart Rhythms. A, Normalsinusrhythm.B, Sinus tachycardia.C, Sinusbradycardia. those that originate from various ectopic foci. The principal disturbances in impulse propagation are reentrant rhythms and conduction blocks.

1	those that originate from various ectopic foci. The principal disturbances in impulse propagation are reentrant rhythms and conduction blocks. Mechanisms that vary the firing frequency of cardiac pacemaker cells were described previously. Changes in the firing rate of the SA node are usually produced by cardiac autonomic nerves. When the firing rate of the SA node is decreased, the heart rate also decreases (bradycardia). Conversely, increased SA node firing results in elevation of the heart rate (tachycardia). Examples of ECGs of sinus tachycardia and sinus bradycardia are shown in Fig. 16.29 . The P, QRS, and T deflections are all normal, but duration of the cardiac cycle (the PP interval) is altered. The changes in cardiac frequency are characteristically gradual. A rhythmic variation of the PP interval at the respiratory frequency (i.e., a respiratory sinus arrhythmia) is a normal, common occurrence (see Fig. 18.7

1	Fig. 18.7 Various physiological, pharmacological, and pathological processes can impede transmission of an impulse through the AV node. The site of block can be localized more precisely by an electrocardiogram of the His bundle (Fig. 16.30 ). To obtain such tracings, an electrode catheter is inserted into a peripheral vein and advanced centrally into the right side of the heart until the electrode lies in the AV junctional region. When the electrode is properly positioned, a distinct deflection (H in Fig. 16.30 ) is registered as the cardiac impulse passes through the bundle of His. The time intervals required for propagation from the atrium to the bundle of His and from the bundle of His to the ventricles (His-to-ventricle interval) may be measured accurately. Abnormal prolongation of the atrium-to-His or His-to-ventricle interval indicates block above or below the bundle of His, respectively.

1	Premature depolarizations occur occasionally in most normal individuals, but they arise more commonly in certain abnormal conditions. Such depolarizations may originate in the atria, AV junction, or ventricles. One type of premature depolarization follows a normally conducted depolarization at a constant time interval (the coupling interval). If the normal depolarization is suppressed in some way (e.g., by vagal stimulation), the premature depolarization is also abolished. Such premature depolarizations are called coupled extrasystoles, or simply extrasystoles, and they

1	P A H V T QRS 0.2 sec •Fig. 16.30 ElectrocardiogramoftheHisbundle(lower tracing, retouched)andleadIIrecordingofthescalarelectrocardiogram(upper tracing). ThedeflectionH,whichrepresentsconductionoftheimpulseoverthebundleofHis,isclearlyvisiblebetweentheatrial andtheventricular(V)deflections.TheconductiontimefromtheatriatothebundleofHisisdenotedbytheA-Hinterval;thatfromthebundleofHistotheventricles,bytheH-Vinterval.(CourtesyofDr. J. Edelstein.) ThreedegreesofAVblockcanbedistinguished,asshownin

1	Fig.16.31 .First-degreeAVblockischaracterizedbyaprolongedPRinterval.Inmostcasesoffirst-degreeblock,theatrium-to-HisintervalisprolongedandtheHis-toventricleintervalisnormal.Hence,thedelayinafirst-degreeAVblockislocatedabovetheHisbundle(i.e.,intheAVnode).Insecond-degreeAVblock,allQRScomplexesareprecededbyPwaves,butnotallPwavesarefollowedbyQRScomplexes.TheratioofPwavestoQRScomplexesisusuallytheratiooftwosmallintegers(suchas2:1,3:1,or 3:2).ThesiteofblockmaybelocatedaboveorbelowtheHisbundle.Ablockbelowthebundleisusuallymoreseriousthanoneabovethebundlebecausetheformerismorelikelytoevolveintoathird-degreeblock.Anartificialpacemakerisfrequentlyimplantedwhentheblockisbelowthebundle.Third-degreeAVblockisoftenreferredtoascomplete heart block

1	heart block becausetheimpulseiscompletelyunabletotraversetheAVconductionpathwayfromatriatoventricles.ThemostcommonsitesofcompleteblockaredistaltothebundleofHis.Incompleteheartblock,theatrialandventricularrhythmsareentirelyindependent.Becauseoftheslowventricularrhythmthatresults,thevolumeofbloodpumpedbytheheartisofteninadequate,especiallyduringmuscularexercise.Third-degreeblockisfrequentlyassociatedwithsyncope(pronouncedlightheadedness),whichiscausedprincipallybyinsufficientcerebralbloodflow.Third-degreeblockisoneofthemostcommonconditionsthatnecessitatetheimplantationofartificialpacemakers.

1	•Fig. 16.31 Atrioventricular (AV) Blocks. A, First-degreeblock;thePRintervalis0.28second(normal, <0.20sec).B, Second-degreeblock(ratioofPwavestoQRScomplexes,2:1).C, Third-degreeblock;notethedissociationbetweenthePwavesandtheQRScomplexes. •Fig. 16.32 Premature Atrial Depolarization and Premature Ventricular Depolarization. Theprematureatrialdepolarization(A; secondbeat)ischaracterizedbyaninvertedPwave(justbelowthesecond“P”)andnormalQRScomplexesandTwaves.Theintervalaftertheprematureatrialdepolarizationisnotmuchlongerthantheusualintervalbetweenbeats.Thebriefrectangulardeflectionjustbeforethelastatrialdepolarizationisastandardizationsignal.Theprematureventriculardepolarization(B) ischaracterizedbybizarre,invertedQRScomplexesandelevatedTwavesandisfollowedbyacompensatorypause.

1	generally reflect a reentry phenomenon. A second type of premature depolarization occurs as the result of enhanced automaticity in some ectopic focus. This ectopic center may fire regularly, and a zone of tissue that conducts unidirectionally may protect this center from being depolarized by the normal cardiac impulse. If this premature depolarization occurs at a regular interval or at an integral multiple of that interval, the disturbance is called parasystole. A tracing of a premature atrial depolarization is shown in

1	A tracing of a premature atrial depolarization is shown in Fig. 16.32A . With a premature atrial depolarization, the normal interval between beats is shortened. In addition, the configuration of the premature P wave differs from that of the other normal P waves because the course of atrial excitation, which originates at some ectopic focus in the atrium, differs from the normal spread of excitation, which originates at the SA node. The QRS complex of the premature depolarization is generally normal because the ventricular excitation spreads over the usual pathways. A tracing of a premature ventricular depolarization is shown in

1	A tracing of a premature ventricular depolarization is shown in Fig. 16.32B . Propagation of the impulse is abnormal, and the configuration of the QRS complex and T wave is entirely different from the normal ventricular deflections because the premature excitation originates at some ectopic focus in the ventricles. The time interval between the premature QRS complex and the preceding normal QRS complex is shortened, whereas the interval after the premature QRS complex and the next normal QRS complex is prolonged by a compensatory pause. The interval from the QRS complex just before the premature excitation to the QRS complex just after it is virtually equal to the duration of two normal cardiac cycles. •Fig. 16.33 Paroxysmaltachycardias.A, supraventriculartachycardia;PwavesprecedeeachQRScomplex.B, ventriculartachycardia;Pwavesnotreadilyobserved.

1	•Fig. 16.33 Paroxysmaltachycardias.A, supraventriculartachycardia;PwavesprecedeeachQRScomplex.B, ventriculartachycardia;Pwavesnotreadilyobserved. As noted, a compensatory pause usually follows a premature ventricular depolarization. This pause occurs because the ectopic ventricular impulse does not disturb the natural rhythm of the SA node. There are two possible reasons for this: The ectopic ventricular impulse is not conducted in a retrograde direction through the AV conduction system, or the SA node had already fired at its natural interval before the ectopic impulse could have reached and depolarized it prematurely. Likewise, the SA nodal impulse generated just before or after the ventricular extrasystole generally does not affect the ventricle because the AV junction and perhaps also the ventricles are still refractory from the premature ventricular excitation.

1	Fig.16.33A )areusuallyindistinguishable,andthereforethetermparoxysmal supraventricular tachycardia referstobothtypes.Inthistachycardia,theimpulseoftencirclesareentryloopthatincludesatrialandAVjunctionaltissue.TheQRScomplexesarefrequentlynormalbecauseventricularactivationproceedsovertheusualpathways.Asitsnameimplies,paroxysmalventriculartachycardiaoriginatesfromanectopicfocusintheventricles.TheECGischaracterizedbyrepeatedbizarreQRScomplexesthatreflecttheabnormalintraventricularimpulseconduction(see Fig.16.33B ).Paroxysmalventriculartachycardiaismuchmoreominousthansupraventriculartachycardiabecausetheformerisfrequentlyaprecursorofventricularfibrillation,alethalarrhythmiadescribedinthenextsection.

1	In contrast to the gradual rate changes that characterize sinus tachycardia, tachycardias that originate from an ectopic focus typically begin and end abruptly. Such ectopic tachycardias are generally called paroxysmal tachycardias. Episodes of paroxysmal tachycardia may persist for only a few beats or for many hours or days, and episodes often recur. Paroxysmal tachycardias may result from (1) rapid firing of an ectopic pacemaker, (2) triggered activity secondary to afterpotentials that reach threshold, or (3) an impulse that circles a reentry loop repetitively. Under certain conditions, cardiac muscle undergoes an irregular type of contraction that is ineffectual in propelling blood. Such an arrhythmia is termed fibrillation, and the disturbance may involve either the atria or the ventricles. Fibrillation probably represents a reentry phenomenon in which the reentry loop fragments into multiple, irregular circuits.

1	The electrocardiographic changes in atrial fibrillation are shown in

1	Fig. 16.34A . This arrhythmia occurs in various types of chronic heart disease. The atria do not contract and relax sequentially during each cardiac cycle, and thus they do not contribute to ventricular filling. Instead, the atria undergo a continuous, uncoordinated rippling motion. P waves do not appear on the ECG; instead, the tracing shows continuous irregular fluctuations in potential called f waves. The AV node is activated at intervals that may vary considerably from cycle to cycle. Hence, no constant interval occurs between successive QRS complexes or between successive ventricular contractions. Because the strength of ventricular contraction depends on the interval between beats (see ), the volume and rhythm of the pulse are irregular. In many affected patients, the atrial reentry loop and the pattern of AV conduction are more regular than they are in atrial fibrillation. The rhythm is then referred to as atrial flutter. •Fig. 16.34 Atrial(A)andventricular(B)fibrillation.

1	•Fig. 16.34 Atrial(A)andventricular(B)fibrillation. Atrialfibrillationandflutterarenotusuallylife-threatening;somepeoplewiththesedisturbancescanfunctionnormally.However,becausetheatriadonotcontractandrelaxrhythmically,bloodclotstendtoformintheatria.Suchclots,ifdislodged,maythentraveltothepulmonaryorsystemicvascularbeds.Patientswithatrialfibrillationorflutteraregenerallytreatedwithanticoagulantdrugssuchasdicumaroltopreventtheformationofsuchclots.Ventricularfibrillation,incontrast,leadstolossofconsciousnesswithinafewseconds.Theirregular,continuous,uncoordinatedtwitchingoftheventricularmusclefiberspumpsnoblood.Deathensuesunlessimmediateresuscitationisachievedortherhythmspontaneouslyrevertstonormal,whichrarelyoccurs.Ventricularfibrillationmaysupervenewhentheentireventricle,orsomeportionofit,isdeprivedofitsnormalbloodsupply.Itmayalsooccurasaresultofelectrocutionorinresponsetocertaindrugsandanesthetics.OntheECG(see Fig.16.34B ),thefluctuationsinpotentialarehighlyirregular.

1	Fig.16.34B ),thefluctuationsinpotentialarehighlyirregular. Ventricular fibrillation is often initiated when a premature impulse arrives during the vulnerable period of the cardiac cycle. This period coincides with the downslope of the T wave on the ECG. During this period, the excitability of cardiac cells varies spatially. Some fibers are still in their effective refractory periods, others have almost fully recovered their excitability, and still others are able to conduct impulses, but only at very slow conduction velocities. As a consequence, the action potentials are propagated over the chambers in many irregular wavelets that travel along circuitous paths and at various conduction velocities. As a region of cardiac cells becomes excitable again, it is ultimately activated by one of the wave fronts traveling around the chamber. Hence, the process is self-sustaining.

1	•Fig. 16.35 Electrocardiogramsrecordedfromanormalsubject(A) andfromapatientwithlongQTsyndrome(B). Q SQ S P PT T R R 1 sec. Normal Q SQ S P P T R R Long QT A B Insomeindividuals,theintervalbetweentheQRScomplexandtheTwaveisabnormallyprolonged,aconditiontermedlong QT syndrome (Fig.16.35 ).SeveralcongenitalformsoflongQTsyndromehavebeenidentifiedinhumansubjects.TwoofthemanygenesthathavebeenidentifiedasabasisforthissyndromearetheHERG gene(aK+ channelgene),locatedonchromosome7,andtheSCN5A gene(aNa+ gene),locatedonchromosome3.PatientswithcongenitalformsoflongQTsyndromemayhaveperiodicepisodesofsyncope(fainting),andapproximately10%ofpediatricsubjectswiththisdisordermaydiesuddenly,withoutanyprecedingsymptoms.Insomeindividualswithsilentionchannelmutations,longQTsyndromemaynotbeevidentuntiladrugistakenthataffectstheionchannelinvolved.Manydrugs,includingseveralantiarrhythmicagents,havebeenidentifiedascausingacquiredlongQTsyndrome.

1	Atrial fibrillation may be changed to a normal sinus rhythm by drugs that prolong the refractory period. As the cardiac impulse completes the reentry loop, it may then encounter refractory myocardial fibers. When atrial fibrillation does not respond adequately to drugs, electrical defibrillation may be used to correct this condition. Dramatic therapy is required for ventricular fibrillation. Conversion to a normal sinus rhythm is accomplished by means of a strong electrical current that places the entire myocardium briefly in a refractory state. Techniques have been developed to administer the current safely through the intact chest wall. In successful cases, the SA node again takes over the normal pacemaker function for the entire heart. The Cardiac Pump The great amount of work performed by the heart over an individual’s lifetime is impressive. A useful way to

1	The Cardiac Pump The great amount of work performed by the heart over an individual’s lifetime is impressive. A useful way to Implantablecardioverter-defibrillator(ICD)deviceshavebeendevelopedtopreventdeathinpatientsinwhomeitherventricularfibrillationorparoxysmalventriculartachycardiahassuddenlydeveloped.Theformerislethalunlessitistreatedimmediately,andthelatteroftenleadstoventricularfibrillationandsuddendeath.TheICDdeviceisimplantedsubcutaneouslyintheleftsubclavicularregionofthechestwall.Atrialandventricularleadsenablerecordingoftherightatrialandrightventricularelectrocardiogramsandprovidetheabilityforrightatrialorrightventricularpacing,orboth.Thedefibrillationcoilintherightventricleenablestheapplicationofastrongelectricalcurrenttotheventricleandtherebyusuallyterminatesthelethalarrhythmia. understand how the heart accomplishes its important task is to consider the relationships between the structure and function of its components. Relationship of Heart Structure to Function

1	understand how the heart accomplishes its important task is to consider the relationships between the structure and function of its components. Relationship of Heart Structure to Function The Myocardial Cell ). Of importance is that both are striated as a result of regular arrangement of the contractile proteins actin and myosin, and generation of force and contraction of muscle fiber occur as a result of their interactions (i.e., sliding filament mechanism). Skeletal muscle and cardiac muscle show similar length-force relationships. As shown in , this relationship for the heart may be expressed graphically, as in Fig. 16.36, by substituting ventricular systolic pressure for force and end-diastolic ventricular volume for resting myocardial fiber (and hence sarcomere) length. The lower curve in Fig.

1	Fig. 16.36, by substituting ventricular systolic pressure for force and end-diastolic ventricular volume for resting myocardial fiber (and hence sarcomere) length. The lower curve in Fig. 16.36 represents the increment in pressure produced by each increment in volume when the heart is in diastole. The upper curve represents the peak force or pressure developed by the ventricle during systole as a function of initial fiber length (or diastolic filling pressure). This curve illustrates the Frank-Starling law of the heart. The pressure-volume curve during diastole is initially quite flat (compliant), which indicates that large increases in volume can be accommodated with only small increases in pressure. In contrast, the development of systolic pressure is considerable at the lower filling pressures. However, the ventricle becomes much less distensible with greater filling, as evidenced by the sharp rise in the diastolic pressure curve at large intraventricular volumes.

1	In a normal intact heart, peak force may be attained at a filling pressure of approximately 12 mm Hg. At this intraventricular diastolic pressure, which is near the upper limit observed in a normal heart, sarcomere length is near its resting length of 2.2 µm. However, the force causes filling pressures to peak as high as 30 mm Hg. At even higher diastolic pressures (>50 mm Hg), sarcomere length is no greater than 2.6 µm. This ability of the myocardium to resist stretch at high filling pressures probably resides in the noncontractile constituents of the heart tissue (connective tissue), and it may serve as a safety factor against overloading of the heart in diastole. Usually, ventricular diastolic pressure is approximately 0 to 7 mm Hg, and the average diastolic sarcomere length is approximately 2.2 µm. Thus a normal heart operates on the ascending portion of the Frank-Starling curve, as depicted in

1	Fig. 16.36 •Fig. 16.36 Relationshipofmyocardialrestingfiberlength(sarco-merelength)orend-diastolicvolumetotheforcedevelopedorpeaksystolicventricularpressureduringventricularcontractioninanintactheart.(RedrawnfromPattersonSW,etal.J Physiol. 1914;48:465.) Cardiac muscle functions as a syncytium; that is, a stimulus applied to any part of the cardiac muscle results in contraction of the entire muscle. Gap junctions with high conductance are present in the intercalated disks between adjacent cells and facilitate conduction of the cardiac impulse from one cell to the next. Cardiac muscle must contract repetitively for a lifetime, and hence it requires a continuous supply of O2. Cardiac muscle is therefore very rich in mitochondria. The large number of mitochondria (see Fig. 13.1 B), which have the enzymes necessary for oxidative phosphorylation, allows rapid oxidation of substrates and synthesis of ATP and thus sustains the myocardial energy requirements.

1	Fig. 13.1 B), which have the enzymes necessary for oxidative phosphorylation, allows rapid oxidation of substrates and synthesis of ATP and thus sustains the myocardial energy requirements. CHAPTER 16 Elements of Cardiac Function To provide adequate O2 and substrate for its metabolic machinery, the myocardium is also endowed with a rich capillary supply, approximately one capillary per fiber. Thus diffusion distances are short, and O2, CO2, substrates, and waste material can move rapidly between the myocardial cell and capillary. The transverse (T) tubular system within myocardial cells participates in this exchange of substances between capillary blood and myocardial cells (as described later, the T tubule system also plays a key role in excitation-contraction coupling). The T tubular system is absent or poorly developed in the atrial cells of many mammals.

1	Results of the earliest studies on isolated hearts indicated that optimal concentrations of Na+ , K+ , and Ca++ in extracellular fluid are necessary for contraction of cardiac muscle. Without Na+ , the heart is not excitable and does not beat. As already described, the resting membrane potential is independent of the extracellular [Na+] gradient across the membrane but very much dependent on extracellular [K+]. Decreases or increases in extracellular [K+], especially if they are large or occur quickly, can lead to arrhythmias, loss of excitability of the myocardial cells, and even cardiac arrest. Ca++ is also essential for cardiac contraction. Removal of Ca++ from the extracellular fluid results in decreased contractile force and eventual arrest in diastole. Conversely, an increase in extracellular [Ca++] enhances contractile force, and very high extracellular [Ca++] induces cardiac arrest in systole (rigor). The free intracellular [Ca++] is the factor principally responsible for the

1	[Ca++] enhances contractile force, and very high extracellular [Ca++] induces cardiac arrest in systole (rigor). The free intracellular [Ca++] is the factor principally responsible for the contractile state of the myocardium.

1	The process by which the action potential of the cardiac myocyte leads to contraction is termed excitation-contraction coupling (see also Chapter 13). Cardiac muscle is excited when a wave of excitation spreads rapidly along the myocardial sarcolemma from cell to cell via gap junctions. Excitation also spreads into the interior of the cells via the T tubules, which invaginate the cardiac fibers at the Z lines. Electrical stimulation at the Z line or the application of Ca++ to the Z lines in a skinned cardiac fiber (whose sarcolemma is removed) elicits localized contraction of the adjacent myofibrils. During the plateau (phase 2) of the action potential, permeability of the sarcolemma by Ca++ increases. Ca++ flows down its electrochemical gradient and enters the cell through calcium channels in the sarcolemma and in the T tubules.

1	During the action potential, Ca++ enters the cell via calcium channels (L-type). However, the amount of Ca++ that enters the cell interior from the extracellular/ interstitial fluid is not sufficient to induce contraction of the myofibrils. Instead, it acts as a trigger (trigger Ca++) to release Ca++ from the sarcoplasmic reticulum, where the intracellular Ca++ is stored (see Fig. 13.2 ). Ca++ leaves the sarcoplasmic reticulum through Ca++ release channels, which are called ryanodine receptors because the channel protein binds ryanodine avidly. Cytoplasmic [Ca++] increases from a resting level of approximately 10−7 mol to levels of approximately 10−5 mol during excitation. This Ca++ then binds to the protein troponin C. The Ca++–troponin C complex interacts with tropomyosin to unblock active sites between the actin and myosin filaments. This unblocking initiates cross-bridge cycling and hence contraction of the myofibrils.

1	Mechanisms that raise cytosolic [Ca++] increase the force developed, and those that lower cytosolic [Ca++] decrease the force developed. For example, catecholamines increase the movement of Ca++ into the cell by phosphorylation of the sarcolemmal calcium channels via a cAMP-dependent protein kinase (see Fig. 13.4). This in turn causes the release of more Ca++ from the sarcoplasmic reticulum, and as a result, contractile force increases. Increasing extracellular [Ca++] increases the amount of Ca++ that enters the cell via the calcium channels and thereby increases contractile force as just described. Reducing the Na+ gradient across the sarcolemma also increases contractile force, an effect mediated by the 3Na+-Ca++ antiporter that normally extrudes Ca++ from the cell (see

1	Fig. 13.2 ). For example, reducing extracellular [Na+] causes less Na+ to enter the cell in exchange for Ca++ , which results in an increase in intracellular [Ca++] and thus contractile force. Raising intracellular [Na+] has a similar effect. In fact, this is the mechanism by which cardiac glycosides increase contractile force. Cardiac glycosides inhibit Na+,K+-ATPase and thereby raise intracellular [Na+] in the cells. The elevated cytosolic [Na+] reverses the direction of the 3Na+-Ca++ antiporter, and therefore less Ca++ is removed from the cell. The increase in intracellular [Ca++] results in an increase in contractile force. Finally, contractile force is diminished when intracellular [Ca++] is decreased by a reduction in extracellular [Ca++], by an increase in the Na+ gradient across the sarcolemma, or by the administration of a calcium channel antagonist that prevents Ca++ from entering the myocardial cell.

1	At the end of systole, the influx of Ca++ stops, and the sarcoplasmic reticulum is no longer stimulated to release Ca++ . In fact, the sarcoplasmic reticulum avidly takes up Ca++ by means of Ca++-ATPase. The Ca++-ATPase in the sarcoplasmic reticulum is similar to but distinct from the Ca++-ATPase found in the sarcolemma. Cytosolic [Ca++] is also reduced during diastole through the action of the 3Na+-Ca++ antiporter in the sarcolemma, as well as by a sarcolemmal Ca++-ATPase (see Fig. 13.2 Both cardiac contraction and relaxation are accelerated by catecholamines (see

1	Fig. 13.3 ). When catecholamines bind to their receptor (β1-adrenoceptor), adenylate cyclase is activated, thereby increasing intracellular cAMP levels, which then leads to activation of cAMP-dependent protein kinase A. Protein kinase A has multiple effects in the cell. As already described, it phosphorylates the calcium channel in the sarcolemma and causes increased entry of Ca++ into the cell, thus increasing the force of contraction. In addition, protein kinase A phosphorylates other proteins that facilitate relaxation. One such protein is phospholamban. Phospholamban normally inhibits the Ca++-ATPase of the sarcoplasmic reticulum. However, when phospholamban is phosphorylated, its inhibitory action is reduced, and uptake of Ca++ into the sarcoplasmic reticulum is enhanced. The increased activity of the Ca++-ATPase in the sarcoplasmic reticulum decreases intracellular [Ca++], thereby causing relaxation. Protein kinase A also phosphorylates troponin I, which in turn inhibits binding

1	activity of the Ca++-ATPase in the sarcoplasmic reticulum decreases intracellular [Ca++], thereby causing relaxation. Protein kinase A also phosphorylates troponin I, which in turn inhibits binding of Ca++ by troponin C. As a result, tropomyosin returns to its position of blocking the myosin-binding sites on the actin filaments, and relaxation results.

1	Contraction of cardiac muscle is influenced by both preload and afterload ( Fig. 16.37 ). Preload is the force that stretches the relaxed muscle fibers, thereby increasing their resting length (see Fig. 16.37B ). In the left ventricle, for example, the blood filling and thus the stretching of the wall during diastole represents the preload. Afterload is the force added to the muscle (see Fig. 16.37C ) against which the contracting muscle must act (see Fig. 16.37D ). Again from the perspective of the left ventricle, afterload is the pressure in the aorta that must be overcome by the contracting left ventricular muscle to open the aortic valve and eject the blood. Preload can be increased by greater filling of the left ventricle during diastole (i.e., increasing end-diastolic volume). From lower initial end-diastolic volumes, increments in filling pressure during diastole increase resting fiber length and elicit a greater systolic pressure during the subsequent contraction (see

1	Fig. 16.36 ). Systolic pressure increases until a maximal systolic pressure is reached at the optimal preload (see Fig. 16.36 ). If diastolic filling continues beyond this point, the pressure developed will no longer increase. At very high filling pressures, peak pressure development in systole is actually reduced.

1	At a constant preload, higher systolic pressure can be reached during ventricular contractions by an increase in the afterload (e.g., increasing aortic pressure by restricting the runoff of arterial blood to the periphery). Incremental increases in afterload produce progressively higher peak systolic pressures. However, if the afterload continues to increase, it becomes so great that the ventricle can no longer generate enough force to open the aortic valve. At this point, ventricular systole is totally isometric (i.e., there is no ejection of blood), and thus no change occurs in ventricular volume during systole. The maximal pressure developed by the left ventricle under these conditions is the maximal isometric force that the ventricle is capable of generating at a given preload. At preloads below the optimal filling volume, an increase in preload can yield greater maximal isometric force (see Fig. 16.36

1	Fig. 16.36 Preload and afterload depend on certain characteristics of the vascular system and the behavior of the heart. With regard to the vasculature, the degree of venomotor tone and peripheral resistance influences preload and afterload. With regard to the heart, a change in rate or stroke volume can also alter preload and afterload. Hence, cardiac and •Fig. 16.37 Preload and Afterload in a Papillary Muscle. A, Restingstage(noload)intheintactheartjustbeforeopeningoftheAVvalves.B, Preload(PL)intheintactheartattheendofventricularfilling.C, Supportedpreloadplusafterload(AL)intheintactheartjustbeforeopeningoftheaorticvalve.D, Liftingpreloadplusafterloadintheintactheartduringventricularejection,withadecreaseinventricularvolume.PL+ AL,totalload. vascular factors interact with each other to affect preload and afterload (see

1	vascular factors interact with each other to affect preload and afterload (see Contractility defines cardiac performance at a given preload and afterload. Contractility determines the change in peak isometric force (isovolumic pressure) at a given initial fiber length (end-diastolic volume). Contractility can be augmented by drugs, such as norepinephrine or digitalis, or by an increase in contraction frequency (tachycardia). The increase in contractility (positive inotropic effect) produced by these interventions is reflected by incremental increases in the force developed and in the velocity of contraction. Indices of Contractility A reasonable index of myocardial contractility can be derived from the contour of ventricular pressure curves ( Fig. 16.38 ). A hypodynamic heart is characterized by elevated end-diastolic pressure, slowly rising ventricular pressure, and a somewhat reduced ejection phase (curve C in Fig. 16.38 ). A hyperdynamic heart (curve B in Fig.

1	Fig. 16.38 ). A hyperdynamic heart (curve B in Fig. 16.38 ) is characterized by reduced end-diastolic pressure, fast-rising ventricular pressure, and a brief ejection phase. The slope of the ascending limb of the ventricular pressure curve indicates the maximal rate of force development by the ventricle. The maximal rate of change in pressure with time—that is, the maximum dP/dt—is illustrated by the tangents to the steepest portion of the ascending limbs of the ventricular pressure curves in Fig. 16.38 . The slope of the ascending limb is maximal during the isovolumic phase of systole (see Fig. 16.38 ). At any given degree of ventricular filling, the slope provides an index of the initial contraction velocity and hence an index of contractility. Similarly, the contractile state of the myocardium can be calculated from the velocity of blood flow that occurs initially in the ascending aorta during the cardiac cycle ( Fig.

1	Similarly, the contractile state of the myocardium can be calculated from the velocity of blood flow that occurs initially in the ascending aorta during the cardiac cycle ( Fig. 16.39 ). In addition, the ejection fraction, which is the ratio of the volume of blood ejected from the left ventricle per beat (stroke volume) to the volume of blood in the left ventricle at the end of diastole (end-diastolic volume), is widely used clinically as an index of contractility. •Fig. 16.38 Leftventricularpressurecurveswithtangentsdrawntothesteepestportionsoftheascendinglimbstoindicatemaximalratesofchangeinpressurewithtime(dP/dtvalues).A(blue curve), control;B(dashed red curve), hyperdynamicheart,aswithadministrationofnorepinephrine;C(green dashed curve), hypodynamicheart,asincardiacfailure.160 120 80 40 0 0 0.2 0.4 0.6 Time (sec) Left ventricular pressure (mm Hg) Max dP/dt B A C

1	The atria are thin-walled, low-pressure chambers that function more as large-reservoir conduits of blood for their respective ventricles than as important pumps for the forward propulsion of blood. The ventricles comprise a continuum of muscle fibers originating from the fibrous skeleton at the base of the heart (chiefly around the aortic orifice). These fibers sweep toward the cardiac apex at the epicardial surface. They pass toward the endocardium and gradually undergo a 180-degree change in direction to lie parallel to the epicardial fibers and to form the endocardium and papillary muscles.

1	At the apex of the heart, the fibers twist and turn inward to form papillary muscles. At the base of the heart and around the valve orifices, these myocardial fibers form a thick, powerful muscle mass that not only decreases the ventricular circumference to implement the ejection of blood but also narrows the AV valve orifices, which aids in closure of the valve. Ventricular ejection is also accomplished by a decrease in the longitudinal axis as the heart begins to narrow toward the base. The early contraction of the apical part of the ventricles, coupled with the approximation of the ventricular walls, propels the blood toward the ventricular outflow tracts. The right ventricle, which develops a mean pressure that is approximately one seventh that developed by the left ventricle, is considerably thinner than the left ventricle.

1	The cardiac valve leaflets consist of thin flaps of flexible, tough, endothelium-covered fibrous tissue that are firmly attached at the base to the fibrous valve rings. Movement of the valve leaflets is essentially passive, and the orientation of the cardiac valves is responsible for the unidirectional flow of blood through the heart. There are two types of valves in the heart: atrioventricular and semilunar ( Figs. 16.40 16.41

1	Figs. 16.40 16.41 The tricuspid valve, located between the right atrium and the right ventricle, is made up of three cusps, whereas the mitral valve, which lies between the left atrium and the left ventricle, has two cusps. The total area of the cusps of each AV valve is approximately twice that of the respective AV orifice, and so considerable overlap of the leaflets occurs when the valves are in the closed position. Attached to the free edges of these valves are fine, strong ligaments (chordae tendineae cordis) that arise from the powerful papillary muscles of the respective ventricles. These ligaments prevent the valves from becoming everted during ventricular

1	Atrial systoleIsovol. contractionRapid ejectionReduced ejectionIsovol. relaxationRapid ventricular filling 0 0.1 0.2 0.3 0.4 0.60.5 0.7 0.8 Time (sec) Ventricular systole S P P R c a 4 1 2 3 v T Q Left atrial pressure Mitral valve opens Mitral valve closes Left ventricular pressure Aortic valve opens Aortic valve closes Aortic pressure Heartsounds VenouspulseElectrocardiogram •Fig. 16.39 Leftatrial,aortic,andleftventricularpressurepulsescorrelatedintimewithaorticflow,ventricularvolume,heartsounds,venouspulse,andtheelectrocardiogramforacompletecardiaccycleinahumansubject.Isovol,isovolumic. systole. In a normal heart, the valve leaflets remain relatively close together during ventricular filling. The partial approximation of the valve surfaces during diastole is caused by eddy currents that prevail behind the leaflets and by tension that is exerted by the chordae tendineae cordis and papillary muscles.

1	CHAPTER 16 Elements of Cardiac Function •Fig. 16.40 Illustrationofaheartsplitperpendiculartotheinterventricularseptumtodepicttheanatomicalrelationshipsoftheleafletsoftheatrioventricularandaorticvalves.Pulmonary artery Left atrium Left auricular appendage Mitral valve—anterior cusp Pulmonary veins Superior vena cava Aorta Aorta Mitral valve— posterior cusp Right ventricle Left ventricle Left ventricle Papillary muscle Papillary muscles appendage Orifices of coronary arteries Posterior cusp Anterior cusp •Fig. 16.41 Illustrationoffourcardiacvalvesasviewedfromthebaseoftheheart.Notehowtheleafletsoverlapintheclosedvalves.Anterior cusp Right cusp Left cusp Posterior cusp Anulus fibrosus Medial cusp valve The pulmonic and aortic valves are located between the right ventricle and the pulmonary artery and between the left ventricle and the aorta, respectively. These valves consist of three cup-like cusps that are attached to the valve rings (see

1	Figs. 16.40 16.41 ). At the end of the reduced ejection phase of ventricular systole, blood flow briefly reverses toward the ventricles. This reversal of blood flow snaps the cusps together and prevents regurgitation of blood into the ventricles. During ventricular systole, the cusps do not lie back against the walls of the pulmonary artery and aorta; instead, they float in the bloodstream at a point approximately midway between the vessel walls and their closed position. Behind the semilunar valves are small outpocketings (sinuses of Valsalva) of the pulmonary artery and aorta. In these sinuses, eddy currents develop, which tend to keep the valve cusps away from the vessel walls. Furthermore, the orifices of the right and left coronary arteries are behind the right and the left cusps, respectively, of the aortic valve. Were it not for the presence of the sinuses of Valsalva and the eddy currents developed therein, the coronary ostia could be blocked by the valve cusps, and coronary

1	of the aortic valve. Were it not for the presence of the sinuses of Valsalva and the eddy currents developed therein, the coronary ostia could be blocked by the valve cusps, and coronary blood flow would cease.

1	The Pericardium The pericardium invests the entire heart and the cardiac portion of the great vessels, and it is reflected onto the cardiac surface as the epicardium. The sac normally contains a small amount of fluid, which provides lubrication for the continuous movement of the enclosed heart. The pericardium is not very distensible; it strongly resists a large, rapid increase in cardiac size and hence prevents sudden overdistention of the chambers of the heart. However, in congenital absence of the pericardium or after its surgical removal, cardiac function is not seriously affected. Nevertheless, with the pericardium intact, an increase in diastolic pressure in one ventricle increases the pressure and decreases the compliance of the other ventricle.

1	Four sounds are usually generated by the heart, but only two are ordinarily audible through a stethoscope. With electronic amplification, the less intense sounds can be detected and recorded graphically as a phonocardiogram. This means of registering faint heart sounds helps delineate the precise timing of the heart sounds in relation to other events in the cardiac cycle. The first heart sound is initiated at the onset of ventricular systole ( Fig. 16.42 ) and reflects closure of the AV valves. It is the loudest and longest of the heart sounds, has a crescendo-decrescendo quality, and is heard best over the apical region of the heart. The tricuspid valve sounds are heard best in the fifth intercostal space just to the left of the sternum; the mitral sounds are heard best in the fifth intercostal space at the cardiac apex.

1	•Fig. 16.42 Thefirstandsecondheartsoundsofthephonocardio-gram(bottom tracing) areshowninregardtotheirrelationshiptotheP,R,andTwavesoftheelectrocardiogram(top tracing). 0 0.2 1.2 1.40.8 1.00.4 0.6 1.6 Time (sec) Phonocardiogram ElectrocardiogramP R T 2 1 The second heart sound, which occurs with abrupt closure of the semilunar valves (see

1	The second heart sound, which occurs with abrupt closure of the semilunar valves (see Fig. 16.42 ), is composed of higher frequency vibrations (higher pitch) and is of shorter duration and lower intensity than is the first heart sound. The portion of the second sound caused by closure of the pulmonic valve is heard best in the second thoracic interspace just to the left of the sternum, whereas that caused by closure of the aortic valve is heard best in the same intercostal space but to the right of the sternum. The aortic valve sound is generally louder than the pulmonic valve, but in cases of pulmonary hypertension, the reverse is true. The nature of the second heart sound changes with respiration. During expiration, a single heart sound is heard that reflects simultaneous closing of the pulmonic and aortic valves. However, during inspiration, closure of the pulmonic valve is delayed, mainly as a result of increased blood flow from an inspiration-induced increase in venous return.

1	With this delayed closure of the pulmonic valve, the second heart sound can be heard as two components; this is termed physiological splitting of the second heart sound. A third heart sound is sometimes heard in children with thin chest walls or in patients with left ventricular failure. It consists of a few low-intensity, low-frequency vibrations heard best in the region of the cardiac apex. The vibrations occur in early diastole and are caused by the abrupt cessation of ventricular distention and by the deceleration of blood entering the ventricles. A fourth, or atrial, sound consists of a few low-frequency oscillations. This sound is occasionally heard in individuals with normal hearts. It is caused by the oscillation of blood and cardiac chambers as a result of atrial contraction. The Cardiac Cycle

1	The Cardiac Cycle The phase between the start of ventricular systole and opening of the semilunar valves (when ventricular pressure rises abruptly) is called the isovolumic (literally, “same volume”) contraction period. This term is appropriate because ventricular volume remains constant during this brief period (see Fig. 16.39 ). The onset of isovolumic contraction also coincides with the peak of the R wave on an ECG, initiation of the first heart sound, and the earliest rise in ventricular pressure on the ventricular pressure curve after atrial contraction. Opening of the semilunar valves marks the onset of the ventricular ejection phase, which may be subdivided dWith inspiration, intrathoracic pressure is reduced (see Chapter 21), which then increases venous blood flow to the right atrium.

1	Chapter 21), which then increases venous blood flow to the right atrium. Inoverloadedhearts,asincongestiveheartfailure,whenventricularvolumeisverylargeandtheventricularwallsarestretchedmaximally,athirdheartsoundisoftenheard.Athirdheartsoundinpatientswithheartdiseaseisusuallyagravesign.Whenthethirdandfourth(atrial)soundsareaccentuated,asoccursincertainabnormalconditions,tripletsofsoundsresemblingthesoundofagallopinghorse(calledgallop rhythms)mayoccur.Mitralinsufficiencyandmitralstenosisproduce,respectively,systolicanddiastolicmurmursthatareheardbestatthecardiacapex.Aorticinsufficiencyandaorticstenosis,incontrast,produce,respectively,diastolicandsystolicmurmursthatareheardbestinthesecondintercostalspacejusttotherightofthesternum.Thecharacteristicsofthemurmursserveasanimportantguideinthediagnosisofvalvulardisease.

1	into an earlier, shorter phase (rapid ejection) and a later, longer phase (reduced ejection). The rapid ejection phase is distinguished from the reduced ejection phase by three characteristics: (1) a sharp rise in ventricular and aortic pressure that terminates at peak ventricular and aortic pressure, (2) an abrupt decrease in ventricular volume, and (3) a pronounced increase in aortic blood flow (see Fig. 16.39 ). The sharp decrease in left atrial pressure at the onset of ventricular ejection results from descent of the base of the heart and consequent stretching of the atria. During the reduced ejection period, runoff of blood from the aorta to the peripheral blood vessels exceeds the rate of ventricular output, and aortic pressure therefore declines. Throughout ventricular systole, the blood returning from the peripheral veins to the atria produces a progressive increase in atrial pressure.

1	During the rapid ejection period, left ventricular pressure slightly exceeds aortic pressure and aortic blood flow accelerates (continues to increase), whereas during the reduced ventricular ejection phase, aortic pressure is higher and aortic blood flow decelerates. This reversal of the ventricular-aortic pressure gradient in the presence of continuous flow of blood from the left ventricle to the aorta is the result of storage of potential energy in the stretched arterial walls. This stored potential energy causes blood flow from the left ventricle into the aorta to decelerate. The peak of the flow curve coincides with the point at which the left ventricular pressure curve intersects the aortic pressure curve during ejection. Thereafter, flow decelerates (continues to decrease) because the pressure gradient has been reversed.

1	Fig. 16.39 shows a tracing of a venous pulse curve recorded from a jugular vein. Three waves are apparent. The a wave occurs with the rise in pressure caused by atrial contraction. The c wave is caused by impact of the common carotid artery with the adjacent jugular vein and, to some extent, by the abrupt closure of the tricuspid valve in early ventricular systole. The v wave reflects the rise in pressure associated with atrial filling. Except for the c wave, the CHAPTER 16 Elements of Cardiac Function venous pulse curve closely resembles the left atrial pressure curve. At the end of ventricular ejection, a volume of blood approximately equal to that ejected during systole remains in the ventricular cavities. This residual volume is fairly constant in normal hearts. However, residual volume decreases somewhat when the heart rate increases or when peripheral vascular resistance has diminished.

1	Closure of the aortic valve produces the characteristic incisura (notch) on the descending limb of the aortic pressure curve, and it also produces the second heart sound (with some vibrations evident on the atrial pressure curve). The incisura marks the end of ventricular systole. The period between closure of the semilunar valves and opening of the AV valves is termed isovolumic relaxation. It is characterized by a precipitous fall in ventricular pressure without a change in ventricular volume. The major portion of ventricular filling occurs immediately after the AV valves open. At this point, the blood that returned to the atria during the previous ventricular systole is abruptly released into the relaxing ventricles. This period of ventricular filling is called the rapid filling phase. In Fig.

1	Fig. 16.39 , the onset of the rapid filling phase is indicated by the decrease in left ventricular pressure below left atrial pressure. This pressure reversal opens the mitral valve. The rapid flow of blood from atria to relaxing ventricles produces transient decreases in atrial and ventricular pressures and a sharp increase in ventricular volume. The rapid ventricular filling phase is followed by a phase of slow ventricular filling called diastasis. During diastasis, blood returning from the peripheral veins flows into the right ventricle and blood from the lungs flows into the left ventricle. This small, slow addition to ventricular filling is indicated by gradual rises in atrial, ventricular, and venous pressures and in ventricular volume (see Fig. 16.39

1	Fig. 16.39 Anincreaseinmyocardialcontractility,asproducedbycatecholaminesorbydigitalisinapatientwithafailingheart,maydecreaseresidualventricularvolumeandincreasethestrokevolumeandejectionfraction.Inseverelyhypodynamicanddilatedhearts,residualvolumecanbecomemuchgreaterthanstrokevolume. The onset of atrial systole occurs soon after the beginning of the P wave (atrial depolarization) of the ECG. The transfer of blood from atrium to ventricle achieved by atrial contraction completes the period of ventricular filling. Atrial systole is responsible for the small increases in atrial, ventricular, and venous pressure, as well as in ventricular volume (see Fig. 16.39 ). Throughout ventricular diastole, atrial pressure barely exceeds ventricular pressure. This small pressure difference indicates that the pathway through the open AV valves during ventricular filling has low resistance.

1	Because there are no valves at the junction of the venae cavae and right atrium or at the junction of the pulmonary veins and left atrium, atrial contraction may force blood in both directions. However, little blood is actually pumped back into the venous tributaries during the brief atrial contraction, mainly because of the inertia of the inflowing blood.

1	The contribution of atrial contraction to ventricular filling is governed to a great extent by the heart rate and the position of the AV valves. At slow heart rates, filling practically ceases toward the end of diastasis, and atrial contraction contributes little additional filling. During tachycardia, however, diastasis is abbreviated and the atrial contribution can become substantial. Should tachycardia become so severe that the rapid filling phase is attenuated, atrial contraction assumes great importance in rapidly propelling blood into the ventricle during this brief period of the cardiac cycle. If the period of ventricular relaxation is so brief that filling is seriously impaired, even atrial contraction cannot provide adequate ventricular filling. The consequent reduction in cardiac output may result in syncope (fainting).

1	Atrialcontractionisnotessentialforventricularfilling,ascanbeobservedinpatientswithatrialfibrillationorcompleteheartblock.Inatrialfibrillation,theatrialmyofiberscontractinacontinuous,uncoordinatedmannerandthereforecannotpumpbloodintotheventricles.Incompleteheartblock,theatriaandventriclesbeatindependentlyofeachother.However,ventricularfillingmaybenormalinpatientswiththesetwoarrhythmias.Incertaindiseasestates,theAVvalvesmaybemarkedlynarrowed(stenotic).Insuchconditions,atrialcontractionplaysamuchmoreimportantroleinventricularfillingthanitdoesinanormalheart. The changes in left ventricular pressure and volume throughout the cardiac cycle are summarized in Fig. 16.43 . Diastolic filling starts when the mitral valve opens (point A in

1	The changes in left ventricular pressure and volume throughout the cardiac cycle are summarized in Fig. 16.43 . Diastolic filling starts when the mitral valve opens (point A in Fig. 16.43 ), and it terminates when the mitral valve closes (point C). The initial decrease in left ventricular pressure (from points A to B), despite the rapid inflow of blood from the left atrium, is attributed to progressive ventricular relaxation and distensibility. During the remainder of diastole (from points B to C), the increase in ventricular pressure reflects ventricular filling and changes in the passive elastic characteristics of the ventricle. Note that only a small increase in pressure accompanies the substantial increase in ventricular volume during diastole (from points B to C). •Fig. 16.43 Schematizedpressure-volumeloopoftheleftventricleforasinglecardiaccycle.

1	The small pressure increase reflects the compliance of the left ventricle during diastole. The small increase in pressure just before the mitral valve closes (to the left of point C) is caused by the contribution of atrial contraction to ventricular filling. With isovolumic contraction (from points C to D), pressure rises steeply, but ventricular volume does not change because the mitral and aortic valves are both closed. When the aortic valve opens (point D), and during the first (rapid) phase of ejection (from points D to E), the large reduction in volume is associated with a steady increase in ventricular pressure. This reduction in volume is followed by reduced ejection (from points E to F) and a small decrease in ventricular pressure. Closure of the aortic valve (point F) is followed by isovolumic relaxation (from points F to A), which is characterized by a sharp drop in pressure. Ventricular volume does not change during the interval between closing of the aortic valve and

1	by isovolumic relaxation (from points F to A), which is characterized by a sharp drop in pressure. Ventricular volume does not change during the interval between closing of the aortic valve and opening of the mitral valve (from points F to A) because both the mitral and aortic valves are closed. The mitral valve opens (point A) to complete one cardiac cycle.

1	Several key cardiovascular system parameters are evident on a left ventricular pressure-volume loop (P-V loop), or may be calculated from it. End-diastolic volume is obtained at mitral valve closure (point C in Fig. 16.43 ) and the end-systolic volume at mitral valve opening (point A). The stroke volume is then apparent as the “width” of the P-V loop and is calculated as follows: The preload of the left ventricle, considered here as left ventricular end-diastolic pressure, is the pressure coordinate when the mitral valve closes (point C in

1	Fig. 16.43 ). The •Fig. 16.44 A, Leftventricularpressure-volumeloopsrecordedinahuman.Theleftventriclewassubjectedtodifferentpreloadsbytransientocclusionofbloodflowintheinferiorvenacava.Aspreload(leftventricularend-diastolicpressure[LVEDP])wasdecreased,bothend-diastolicvolumeandend-systolicvolumedecreased,butend-diastolicvolumedecreasedmore,whichresultedindecreasedstrokevolume.B, CardiacfunctioncurveswhenstrokevolumeisplottedasafunctionofLVEDPforanormalheartinthebasalstate(blue line), aheartwithincreasedcontractility(red line), andaheartwithreducedcontractility(green line). Atanygivenpreload,strokevolumeishigherinheartswithincreasedcontractilityandlowerinheartswithdecreasedcontractilitythanstrokevolumeinthenormalbasalstate.(A, FromSenzakiH,etal.Single-beatestimationofend-systolicpressure-volumerelationinhumans.Anewmethodwiththepotentialfornoninvasiveapplication.Circulation. 1996;94[10]:2497-2506.) approximate “diastolic” arterial blood pressure may be read when the aortic valve opens

1	1996;94[10]:2497-2506.) approximate “diastolic” arterial blood pressure may be read when the aortic valve opens (point D), and the approximate “systolic” arterial blood pressure is read during systole (point E). Left ventricular P-V loops recorded from a human (

1	Fig. 16.44A ) are similar to the schematized version (see Fig. 16.43 ). The slope of the end-systolic P-V relation (line extending from the dot in Fig. 16.44A ) defines contractility; a steeper slope indicates increased contractility. In this subject, partial occlusion of the inferior vena cava reduced the preload of the left ventricle on successive beats (as the inflow of blood to the left ventricle was reduced, as a consequence of the reduced return of blood to the right ventricle) and the effect was successively smaller stroke volume of the left ventricle. This illustrates, in humans, the operation of the Frank-Starling law of the heart ( Chapter 13), whereby changes in preload (left ventricular volume) change myocardial fiber length, and thus the strength of the subsequent contraction, as well as stroke volume produced. This important phenomenon is characterized by the cardiac function curve (see Fig. 16.44B).

1	As preload increases, stroke volume increases (solid blue line). If contractility of the heart is increased, as in the action of norepinephrine, the slope of the end-systolic P-V relation becomes steeper, and the entire cardiac function curve shifts upward (solid red line), which reflects the fact that the ventricle is now able to produce a larger stroke volume at a given preload. The increased stroke volume at any given preload is largely produced by a decreased end-systolic volume: Hearts with increased contractility are able to “squeeze down” to a greater extent. Conversely, if the ventricle is damaged, as after cardiac ischemia, or if its contractility is otherwise reduced from normal (as after calcium channel blockade), the cardiac function curve is shifted downward (green line), which reflects a reduced stroke volume at any given preload. The cardiac function curve is also known as ventricular function curves or Starling curves. To assess the integrated functioning of the

1	which reflects a reduced stroke volume at any given preload. The cardiac function curve is also known as ventricular function curves or Starling curves. To assess the integrated functioning of the cardiovascular system ( ), cardiac output, rather than stroke volume, is usually measured; preload of the heart is considered to be the filling pressure of the right ventricle, typically measured as mean right atrial pressure (Pra), or central venous pressure.

1	Measurement of Cardiac Output The Fick Principle In 1870 the German physiologist Adolph Fick contrived the first method for measuring cardiac output in intact animals and people. The basis for this method, called the Fick principle, is simply an application of the law of conservation of mass. The principle is derived from the fact that the quantity of O2 delivered to the pulmonary capillaries via Terminal bronchiole To pulmonary veins From pulmonary artery Pulmonary artery q1 q2 O2 consumption 250 mL O2 /min [O2 ] pv 0.20 mL O2 /mL blood [O2 ] pa 0.15 mL O2 /mL blood q1 + q2 = q3 Alveolus q3 Pulmonary vein Alveoli •Fig. 16.45 Schema Illustrating the Fick Principle for Measuring Cardiac Output. Thechangeincolorfrompulmonaryarterytopulmonaryveinrepresentsthechangeincolorofthebloodasvenousbloodbecomesfullyoxygenated.

1	the pulmonary artery, plus the quantity of O2 that enters the pulmonary capillaries from the alveoli, must equal the quantity of O2 that is carried away by the pulmonary veins. The Fick principle is depicted schematically in Fig. 16.45. The rate of O2 delivery to the lungs (q1) equals the O2 concentration in pulmonary arterial blood ([O2]pa), multiplied by pulmonary arterial blood flow (Q), which equals cardiac output; that is, Equation 16.1 If q2 is the net rate of O2 uptake by the pulmonary capillaries from the alveoli, then at steady state, q2 equals the O2 consumption of the body. The rate at which O2 is carried away by the pulmonary veins (q3) equals the O2 concentration in pulmonary venous blood ([O2]pv), multiplied by total pulmonary venous flow, which is virtually equal to pulmonary arterial blood flow (Q); that is, Equation 16.2 From the law of conservation of mass, Equation 16.3 Therefore, Equation 16.4 When this is solved for cardiac output, Equation 16.5

1	Equation 16.2 From the law of conservation of mass, Equation 16.3 Therefore, Equation 16.4 When this is solved for cardiac output, Equation 16.5 Eq. 16.5 is the statement of the Fick principle.

1	To determine cardiac output by this method, three values must be known: (1) O2 consumption of the body, (2) the O2 concentration in pulmonary venous blood ([O2]pv), and (3) the O2 concentration in pulmonary arterial blood ([O2]pa). O2 consumption is computed from measurements of the volume and O2 content of expired air over a given interval. Because the O2 concentration of peripheral arterial blood is essentially identical to that in the pulmonary veins, [O2]pv is determined with a sample of peripheral arterial blood withdrawn by needle puncture. The compositions of pulmonary arterial blood and mixed systemic venous blood are virtually identical to one another. Samples for O2 analysis are obtained from the pulmonary artery or right ventricle through a catheter. A very flexible catheter with a small balloon near the tip can be inserted into a peripheral vein. As the flexible tube is advanced, the flowing blood carries it toward the heart. By monitoring the pressure changes, the

1	with a small balloon near the tip can be inserted into a peripheral vein. As the flexible tube is advanced, the flowing blood carries it toward the heart. By monitoring the pressure changes, the physician can advance the catheter tip into the pulmonary artery.

1	With the values depicted in Fig. 16.45 , cardiac output can be calculated as follows: If the O2 consumption is 250 mL/minute, the arterial (pulmonary venous) O2 content is 0.20 mL of O2 per milliliter of blood, and the mixed venous (pulmonary arterial) O2 content is 0.15 mL of O2 per milliliter of blood, cardiac output equals 250/ (0.20 − 0.15) = 5000 mL/minute. The Fick principle is also used to estimate the O2 consumption of organs when blood flow and the O2 content of arterial and venous blood can be determined. Algebraic rearrangement reveals that O2 consumption equals blood flow multiplied by the difference in the arteriovenous O2 concentration. For example, if blood flow through one kidney is 700 mL/minute, the arterial O2 content is 0.20 mL of O2 per milliliter of blood, and the renal venous O2 content is 0.18 mL of O2 per milliliter of blood, the rate of O2 consumption by that kidney must be 700 (0.20 − 0.18) = 14 mL of O2 per minute.

1	In the clinic, cardiac output is most commonly measured noninvasively with Doppler echocardiography. By this method, the velocity of blood in the ascending aorta is measured. Obtaining the cross-sectional area of the aorta (also measured by echocardiography) allows the volume of blood ejected in a single beat (i.e., stroke volume) to be determined (see Eq. 17.1 ). Multiplying stroke volume by the heart rate then yields a value for cardiac output in liters per minute.

1	Eq. 17.1 ). Multiplying stroke volume by the heart rate then yields a value for cardiac output in liters per minute. Consumption of O2 by the heart depends on the amount and type of activity that the heart performs. Under basal conditions, myocardial O2 consumption (V˜O2) is approximately 8 to 10 mL/minute/100 g of heart. It can increase severalfold during exercise and decrease moderately in such conditions as hypotension and hypothermia. The O2 content of cardiac venous blood is normally low (≈5 mL/ dL), and the myocardium can receive little additional O2 by further extraction of O2 from coronary blood. Therefore, increased O2 demands of the heart must be met mainly by an increase in coronary blood flow (see ). In experiments in which the heartbeat is arrested but coronary perfusion is maintained, O2 consumption falls to 2 mL/ minute/100 g or less, which is still six to seven times greater than the O2 consumption of resting skeletal muscle.

1	Cardiac work has external and internal components. Left ventricular work per beat (stroke work) is approximately equal to the product of stroke volume and the mean aortic pressure against which the blood is ejected by the left ventricle. External cardiac work (We) may be defined as follows: Equation 16.6 That is, each small increment in volume that is pumped, (dV) is multiplied by the associated pressure (P), and the products (PdV) are integrated over the time interval of interest (t2 − t1) to calculate total work. Added to this is kinetic work due to the velocity (v) of blood flow and the density (ρ) of blood. The mean pressure during expulsion is used to simplify this to Equation 16.7 At resting levels of cardiac output, the kinetic energy component is negligible. However, with high cardiac output, as in strenuous exercise, the kinetic energy component can account for up to 50% of total cardiac work. The internal work (Wi) of the heart can be written as

1	The internal work (Wi) of the heart can be written as Equation 16.8 where α is a proportionality constant that converts T dt into units of work, T is wall tension, and dt is time. Clinically, V˜O2 and left ventricular power are difficult to measure; however, both are closely related to the systolic pressure–time index, the integral of left ventricular pressure, and time during systole. Such measurements are important because internal work is a large determinant of myocardial O2 need. An alternative approach to evaluate cardiac work and its relation to O2 consumption has been developed in which P-V loops (see Fig. 16.44 ) are examined in conditions with varied preload and afterload; contractility is maintained constant.

1	Fig. 16.44 ) are examined in conditions with varied preload and afterload; contractility is maintained constant. Simultaneously halving aortic pressure and doubling cardiac output, or vice versa, result in the same value for cardiac work. However, the O2 requirements are greater for any given amount of cardiac work when a major proportion of the work is pressure work, as opposed to volume work. An increase in cardiac output at a constant aortic pressure (volume work) is accomplished with only a small increase in left ventricular O2 consumption, whereas increased

1	CHAPTER 16 Elements of Cardiac Function arterial pressure at constant cardiac output (pressure work) is accompanied by a large increase in myocardial O2 consumption. Thus myocardial O2 consumption may not be well correlated with overall cardiac work. The magnitude and duration of left ventricular pressure are correlated with left ventricular O2 consumption. The work of the right ventricle is one-seventh that of the left ventricle because pulmonary vascular resistance is much less than systemic vascular resistance.

1	The efficiency of the heart may be calculated as the ratio of the work accomplished to the total energy used. If the average O2 consumption is assumed to be 9 mL/minute/100 g for the two ventricles, a 300-g heart will consume 27 mL of O2 per minute. This value is equivalent to 130 small calories when the respiratory quotient is 0.82. Together, the two ventricles do approximately 8 kg-m of work per minute, which is equivalent to 18.7 small calories. Therefore, the gross efficiency of the heart is approximately 14%: Equation 16.9 Thegreaterenergydemandofpressureworkthanofvolumeworkisclinicallyimportant,especiallyinaorticstenosis.Inthiscondition,leftventricularO2consumptionisincreased,mainlybecauseofthehighintraventricularpressuredevelopedduringsystole.However,coronaryperfusionpressure(andhenceO2supply)iseithernormalorreducedbecauseofthepressuredropacrossthenarroworificeofthediseasedaorticvalve.

1	The actual gross mechanical efficiency of the heart is slightly higher (18%) than the value calculated and is determined through subtracting the O2 consumption of the nonbeating (asystolic) heart (≈2 mL/minute/100 g) from the total cardiac O2 consumption in the calculation of efficiency. The efficiency of the heart as a pump is relatively low. During physical exercise, efficiency improves because mean blood pressure does not change appreciably, whereas cardiac output and work increase considerably, without a proportional increase in myocardial O2 consumption. Of interest is that the chemical efficiency of the heart is rather high, as indicated by the estimate of 60% for the efficiency of generating ATP from oxidative phosphorylation. The energy expended in cardiac metabolism that does not contribute to the propulsion of blood through the body appears in the form of heat. The energy of flowing blood is also dissipated as heat.

1	Myocardial Adenosine Triphosphate and Its Relation to Mechanical Function The chemical energy that fuels cardiac contractile work and relaxation is derived from ATP hydrolysis (

1	The chemical energy that fuels cardiac contractile work and relaxation is derived from ATP hydrolysis ( Fig. 16.46 •Fig. 16.46 Overall Scheme for Production and Utilization of Adenosine Triphosphate (ATP) Within a Cardiac Myocyte. Pathwaysforutilizationofglucose,fattyacids(FA),andlactateareindicated,asaretherequirementsforO2andH+bytheelectrontransportchaininmitochondria.ADP,adenosinediphosphate;CoA,coenzymeA;CPT-I,carnitinepalmitoyltransferase;G-6-P,glucose-6-phosphate;GLUT,glucosetransporter;NADH,nicotinamideadenosinedehydrogenase;PDH,pyruvatedehydrogenase;Pi,inorganicphosphate;SR,sarcoplasmicreticulum;TG,triglyceride.ATPase FACPT-I Cardiomyocyte Citric Acid CycleCO2 Acetyl-CoA Acetyl-CoA NADH NADH Lactate GLUT Glycogen Cytosol Contractile work, SR Ca++ uptake, lon homeostasis G-6-P PDH Pyruvate Electron Transport Chain TGATP ATP ATP ADP + Pi ADP + Pi ADP + Pi H+ O2 Mitochondrion — GLYCOLYSIS Fatty Acid ˛-Oxidation

1	The healthy heart has a relatively constant level of ATP (≈5µmol/g wet weight) despite an extremely high rate of ATP hydrolysis (≈0.3µmol/g−1/second−1). The tissue content of ATP is low in relation to the rate of breakdown and production; complete turnover of the myocardial ATP content occurs approximately every 12 seconds in the heart at rest. ATP hydrolysis (see

1	Fig. 16.46) provides energy for contractile work (actin-myosin interaction and cell shortening), pumping Ca++ back into the sarcoplasmic reticulum at the end of systole and maintaining normal ion gradients (low Na+ and high K+ in the cell). Approximately two thirds of the ATP hydrolyzed by the heart is used to fuel contractile work, and the remaining third is used for ion pumps and “housekeeping” functions such as synthesis of proteins and nucleic acids. The Ca++-ATPase of the sarcoplasmic reticulum is the primary ion pump consuming ATP. This process occurs during the end of systole, when cytosolic Ca++ is rapidly sequestered into the sarcoplasmic reticulum to initiate diastolic relaxation. In the healthy heart, the rate of ATP hydrolysis is exquisitely matched to the rate of ATP resynthesis. There is no significant change in the concentration of ATP or ADP even with the onset of heavy exercise.

1	ATP resynthesis occurs primarily through oxidative phosphorylation in mitochondria (>98%) and, to a small degree, through glycolysis (<2%). Oxidative phosphorylation requires O2 and H2. The O2 is delivered to the myocardium and consumed in the mitochondria to make H2O, and the H2 is from the metabolism of carbon fuels (mainly fatty acids, glucose, and lactate) and the generation of the reduced form of nicotinamide adenine dinucleotide (NADH). Pyruvate dehydrogenase (PDH) regulates the oxidation of glucose and lactate. The activity of PDH is inhibited by product inhibition from acetyl–coenzyme A and NADH. Also, PDH activity is inhibited when phosphorylated by PDH kinase and activated when dephosphorylated by PDH phosphatase. ATP is formed from ADP and inorganic phosphate with the use of H+ . Of importance is that the rates of ATP formation and breakdown depend on an adequate delivery of O2 to the myocardium, which is a function of myocardial blood flow and oxygenation of arterial

1	of H+ . Of importance is that the rates of ATP formation and breakdown depend on an adequate delivery of O2 to the myocardium, which is a function of myocardial blood flow and oxygenation of arterial blood. An increase in ATP breakdown in the myocardium, such as occurs when heart rate, systolic blood pressure, and contractility are increased (as during exercise), necessitates an increase in O2 delivery to the myocardium so that the mitochondria can generate sufficient ATP by oxidative phosphorylation to meet the demand for ATP. Thus the rate of myocardial O2 consumption is tightly linked to the work rate (or power) of the myocardium.

1	The heart is versatile in its use of substrates, and within certain limits, uptake of a particular substrate is directly proportional to its arterial concentration. The use of one substrate by the heart is also influenced by the presence or absence of other substrates. For example, the addition of lactate to the blood that perfuses a heart metabolizing glucose leads to a reduction in glucose uptake and vice versa. At normal blood concentrations, glucose and lactate are consumed at approximately equal rates.

1	In contrast, uptake of pyruvate is very low, as is its arterial concentration. For glucose, the threshold concentration is approximately 4 mmol/L. Below this blood level, no glucose is taken up by the myocardium. Insulin reduces the glucose threshold and increases the rate of glucose uptake by the heart. Cardiac utilization of lactate occurs in most circumstances; insulin does not affect its uptake by the myocardium. Under hypoxic conditions, glucose utilization is facilitated by an increase in the rate of transport across the myocardial cell wall. However, lactate cannot be metabolized by the hypoxic heart and is produced by the heart under anaerobic conditions. Associated with lactate production by the hypoxic heart is the breakdown of cardiac glycogen.

1	Of the total cardiac O2 consumption, only 35% to 40% can be accounted for by the oxidation of carbohydrate. Thus the heart derives the major part of its energy from the oxidation of noncarbohydrate sources: namely, esterified and nonesterified fatty acids, which account for approximately 60% of the myocardial O2 consumption in people in the postabsorptive state. Various fatty acids have different thresholds for myocardial uptake, but these

1	CHAPTER 16 Elements of Cardiac Function acids are generally used in direct proportion to their arterial concentration. Ketone bodies, especially acetoacetate, are readily oxidized by the heart, and they are a major source of energy in diabetic acidosis. As is true of carbohydrate substrates, use of a specific noncarbohydrate is influenced by the presence of other substrates, whether noncarbohydrate or carbohydrate. Therefore, within certain limits, the heart preferentially uses the substrate that is available in the largest concentration. The oxidation of amino acids makes a small contribution to myocardial energy expenditure. Normally, the heart derives its energy by oxidative phosphorylation, in which each mole of glucose yields 36 mol of ATP. However, during hypoxia, glycolysis takes over, and 2 mol of ATP is provided by each mole of glucose; β-oxidation of fatty acids is also curtailed. If hypoxia is prolonged, cellular creatine phosphate and eventually ATP are depleted.

1	In ischemia, lactic acid accumulates and decreases intracellular pH. This condition inhibits glycolysis, fatty acid use, and protein synthesis, and therefore it results in cellular damage and eventually necrosis of myocardial cells.

1	1. The transmembrane action potentials recorded from cardiac myocytes may contain the following five phases: Phase 0: The action potential upstroke is initiated when a suprathreshold stimulus rapidly depolarizes the membrane by activating the fast sodium channels. Phase 1: The notch is an early partial repolarization that is achieved by the efflux of K+ through channels that conduct the transient outward current (ito). Phase 2: The plateau represents a balance between the influx of Ca++ through calcium channels and the efflux of K+ through several types of potassium channels. Phase 3: Final repolarization is initiated when the efflux of K+ exceeds the influx of Ca++ . The resultant partial repolarization rapidly increases K+ conductance and restores full repolarization. Phase 4: The resting potential of the fully repolarized cell is determined by conductance of the cell membrane to K+ , mainly through iK1 channels.

1	Phase 4: The resting potential of the fully repolarized cell is determined by conductance of the cell membrane to K+ , mainly through iK1 channels. 2. Fast-response action potentials are recorded from atrial and ventricular myocardial fibers and from ventricular specialized conducting (Purkinje) fibers. Such an action potential is characterized by a large amplitude, a steep upstroke, and a relatively long plateau. The effective refractory period of fast-response fibers begins at the upstroke of the action potential and persists until midway through phase 3. The fiber is relatively refractory during the remainder of phase 3, and it regains full excitability soon after it is fully repolarized (phase 4). 3.

1	3. Slow-response action potentials are recorded from normal SA and AV nodal cells and from abnormal myocardial cells that have been partially depolarized. The action potential is characterized by a less negative resting potential, a smaller amplitude, a less steep upstroke, and a shorter plateau than is typical of the fast-response action potential. The upstroke in slow-response fibers is produced by the activation of calcium channels. Slow-response fibers become absolutely refractory at the beginning of the upstroke, and partial excitability may not be regained until very late in phase 3 or until after the fiber is fully repolarized. 4.

1	4. Normally, the SA node serves as the cardiac pacemaker to initiate the cardiac impulse. This impulse is propagated from the SA node to the atria and ultimately reaches the AV node. After a delay in the AV node, the cardiac impulse is propagated throughout the ventricles. Ectopic foci in the atrium, the AV node, or the His-Purkinje system may initiate propagated cardiac impulses if the normal pacemaker cells in the SA node are suppressed or if the rhythmicity of the ectopic automatic cells is abnormally enhanced. 5.

1	5. Under certain abnormal conditions, afterdepolarizations may be triggered by an otherwise normal action potential. EADs arise early in phase 3 of a normal action potential. They are more likely to occur when the basic cycle length of the initiating beats is very long and when the cardiac action potentials are abnormally prolonged. DADs appear late in phase 3 or in phase 4. They are more likely to occur when the basic cycle length of the initiating beats is short and when the cardiac cells are overloaded with Ca++ . 6. Reentrant arrhythmias occur when a cardiac impulse traverses a loop of cardiac fibers and reenters previously excited tissue, when the impulse is conducted slowly around the loop, and when the impulse is blocked unidirectionally in some section of the loop. 7.

1	7. The ECG, which is recorded from the surface of the body, traces the conduction of the cardiac impulse throughout the heart. The ECG may be used to detect and analyze certain cardiac arrhythmias, such as altered SA rhythms, AV conduction blocks, premature depolarizations, ectopic tachycardias, and atrial and ventricular fibrillation. 8. On excitation, voltage-gated calcium channels open to admit extracellular Ca++ into the cardiac myocytes. The influx of Ca++ triggers the release of Ca++ from the sarcoplasmic reticulum. The elevated intracellular [Ca++] elicits contraction of the myofilaments. Relaxation is accomplished through the restoration of resting cytosolic [Ca++] by the pumping of Ca++ back into the sarcoplasmic reticulum and the exchange of Ca++ for extracellular Na+ across the sarcolemma. Velocity and force of contraction are functions of intracellular [Ca++]. Force and velocity are inversely related, and so with no load, velocity is maximal. In

1	Abriel H, Rougier J-S, Jalife J. Ion channel macromolecular complexes in cardiomyocites: Roles in sudden cardiac death. Circ Res. 2015;116:1971. Bers DM, Guo T. Calcium signaling in cardiac ventricular myocytes. Ann N Y Acad Sci. 2005;1047:86. Cannell MB, Kong CH. Local control in cardiac E-C coupling. J Mol Cell Cardiol. 2012;52:298. an isovolumic contraction, no external shortening occurs. 9. In ventricular contraction, preload is stretch of the fibers by blood during ventricular filling. Afterload is the arterial pressure against which the ventricle ejects the blood. An increase in myocardial fiber length, as occurs with augmented ventricular filling (preload) during diastole, produces a more forceful ventricular contraction. This relationship between fiber length and strength of contraction is known as the Frank-Starling law of the heart. 10.

1	10. Contractility is an expression of cardiac performance at a given preload and afterload. Contractility can be modulated by the autonomic nervous system. 11. To determine cardiac output, according to the Fick principle, the O2 consumption of the body (q2) and the oxygen content of arterial blood ([O2]a) and mixed venous blood ([O2]v) are measured. Cardiac output = q2/([O2]a − [O2]v). It can also be measured noninvasively by Doppler echocardiography. 12. In the normal heart, the rate of ATP hydrolysis is matched by the rate of ATP synthesis. ATP is generated by oxidative phosphorylation of fatty acids and glucose, a process that requires O2. When the rate of ATP hydrolysis increases, as during exercise, the consumption of O2 for oxidative phosphorylation also increases. The rate of O2 utilization is thereby coupled to the rate of cardiac work. The myocardium functions only aerobically, and in general it uses substrates in proportion to their arterial concentration.

1	Gima K, Rudy Y. Ionic current basis of electrocardiographic waveforms. Circ Res. 2003;90:889. Priori SG. The fifteen years of discoveries that shaped molecular electrophysiology: time for appraisal. Circ Res. 2010;107: 451. Upon completion of this chapter, the student should be able to answer the following questions: 1. What physical properties of blood vessels and blood determine hemodynamics, and how are they defined by Poiseuille’s law? 2. How is arterial compliance related to stroke volume and pulse pressure? How does arterial compliance affect the arterial pulse wave and cardiac work? 3. What are mean, systolic, diastolic, and pulse pressures, and how are they measured? 4. What vessels constitute the microcirculation? How is pulsatile blood flow in large arteries converted into steady flow in the microcirculation? 5. What are the hydrostatic and osmotic factors that underlie Starling’s hypothesis for capillary function? 6.

1	5. What are the hydrostatic and osmotic factors that underlie Starling’s hypothesis for capillary function? 6. How do intrinsic and extrinsic factors modulate peripheral circulation, and how do these factors affect blood flow in particular organs? 7. How does the myogenic hypothesis account for autoregulation of blood flow? What is the effect of tissue metabolism on autoregulation?

1	7. How does the myogenic hypothesis account for autoregulation of blood flow? What is the effect of tissue metabolism on autoregulation? he vasculature consists of a closed system of tubes or vessels that distributes blood from the heart to the tissues and returns blood from the tissues to the heart. It can be divided into three components: the arterial system, which takes blood from the heart and distributes it to the tissues; the venous system, which returns blood from the tissues to the heart; and the microcirculation, which separates the arterial and venous systems and is the site where nutrients and cellular waste products are exchanged between blood and tissues. These components of the vasculature are described in this chapter. In addition, the properties of blood flow to specific vascular beds and tissues are considered. As an introduction to this material, the physics of blood/fluid flow through the vasculature (i.e., hemodynamics) is reviewed.

1	The physics of fluid flow through rigid tubes provides a basis for understanding the flow of blood through blood vessels, even though the blood vessels are not rigid tubules (i.e., they are distensible) and blood is not a simple homogeneous fluid. Knowledge of these physical principles underlies understanding of the interrelationships among velocity of blood flow, blood pressure, and the dimensions of the various components of the systemic circulation. Velocity of the Bloodstream Velocity, as relates to fluid movement, is the distance that a particle of fluid travels with regard to time, and it is expressed in units of distance per unit time (e.g., centimeters per second). Flow, in contrast, is the rate of displacement of a volume of fluid, and it is expressed in units of volume per unit time (e.g., cubic centimeters per second). In a rigid tube, velocity (v) and flow (Q) are related to one another by the cross-sectional area (A) of the tube: Equation 17.1

1	Equation 17.1 The interrelationships among velocity, flow, and area are shown in Fig. 17.1 . Because conservation of mass requires that the fluid flowing through a rigid tube be constant, the velocity of the fluid varies inversely with the cross-sectional area. Thus fluid flow velocity is greatest in the section of the tube with the smallest cross-sectional area and slowest in the section of the tube with the greatest cross-sectional area. Fig. 15.3 , velocity decreases progressively as blood traverses the arterial system. In the capillaries, velocity decreases to a minimal value. As the blood then passes centrally through the venous system toward the heart, velocity progressively increases again. The relative velocities in the various components of the circulatory system are related only to the respective cross-sectional areas.

1	The total energy in a hydraulic system consists of three components: pressure, gravity, and velocity. The velocity of blood flow can have an important effect on the pressure within the tube. Consider the effect of velocity on pressure in a tube with different cross-sectional areas ( Fig. 17.2 ). In this system, the total energy remains constant. The total pressure within the tube equals the lateral (static) pressure plus the dynamic pressure. The gravitational component can be neglected because the tube is horizontal. The total pressures in segments A, B, and C are equal, provided that •Fig. 17.1 Asfluidflowsthroughatubeofvariablecross-sectionalarea(A),thelinearvelocity(v)variesinverselywiththecross-sectionalarea.Q,flow.

1	˜v2/2 = 3.8 mm Hg 15 mm Hg 3.8 mm Hg •Fig. 17.2 Inanarrowsection(B)ofatube,thelinearvelocity(v)andhencethedynamiccomponentofpressure(ρv2/2)aregreaterthaninthewidesections(AandC),ofthesametube.Ifthetotalenergyisvirtuallyconstantthroughoutthetube(i.e.,iftheenergylossbecauseofviscosityisnegligible),thelateralpressureinthenarrowsectionislowerthanthelateralpressureinthewidesectionsofthetube(seetheheightsofthecolumnoffluidabovecompartmentsA,B,andCeach,whichreflectpressure). the energy loss from viscosity is negligible (i.e., this fluid is an “ideal fluid”). The effect of velocity on the dynamic component (Pdyn) can be estimated as follows: Equation 17.2 where ρ is the density of the fluid (grams per cubic centimeters) and v is velocity (centimeters per second). Assume that the fluid has a density of 1 g/cm3. In section A in Fig. 17.2 , the lateral pressure is 100 mm Hg; note that 1 mm Hg equals 1330 dynes/cm2. According to

1	Eq. 17.2, P dyn = 5000 dynes/cm2, or 3.8 mm Hg. In the narrow section B of the tube, where the velocity is twice as high, Pdyn = 20,000 dynes/ cm2, or 15 mm Hg. Thus the lateral pressure in section B is 15 mm Hg lower than the total pressure, whereas the lateral pressures in sections A and C are only 3.8 mm Hg lower. In most arterial locations, the dynamic component is a negligible fraction of the total pressure. However, at sites of an arterial constriction or obstruction, the high flow velocity is associated with large kinetic energy, and the dynamic pressure component may therefore increase significantly. Hence, the pressure would be reduced, and perfusion of distal segments would be correspondingly decreased. This example helps explain how pressure changes in a vessel that is narrowed by atherosclerosis or spasm of the blood vessel •Fig. 17.3 Laminar and Turbulent Flow. A,

1	be correspondingly decreased. This example helps explain how pressure changes in a vessel that is narrowed by atherosclerosis or spasm of the blood vessel •Fig. 17.3 Laminar and Turbulent Flow. A, Whenflowislaminar,allelementsofthefluidmoveinstreamlinesthatareparalleltotheaxisofthetube;thefluiddoesnotmoveinaradialorcircumferentialdirection.Thelayeroffluidincontactwiththewallismotionless;thefluidthatmovesalongthecentralaxisofthetubehasthemaximalvelocity.B, Inturbulentflow,theelementsofthefluidmoveirregularlyinaxial,radial,andcircumferentialdirections.Vorticesfrequentlydevelop.

1	wall: that is, in narrowed sections of a tube, the dynamic component increases significantly because the flow velocity is associated with large kinetic energy. The most fundamental law that governs the flow of fluids through cylindrical tubes was derived empirically by the French physiologist Jean Léonard Marie Poiseuille in the 1840s. He was interested primarily in the physical determinants of blood flow, but he replaced blood with simpler liquids in his measurements of flow through glass capillary tubes. His work was so precise and important that his observations have been designated Poiseuille’s law. Poiseuille’s law applies to the steady (i.e., nonpulsatile) laminar flow of newtonian fluids through rigid cylindrical tubes. A newtonian fluid is one whose viscosity remains constant, and laminar flow is the type of motion in which the fluid moves as a series of individual layers, with each layer moving at a velocity different from that of its neighboring layers (

1	Fig. 17.3A ). In the case of laminar flow through a tube, the fluid consists of a series of infinitesimally thin concentric tubes sliding past one another, of which the central tube has the highest velocity. The velocities of the concentric laminae decrease parabolically towards the vessel wall. Despite the differences between the vascular system (i.e., flow is pulsatile, the vessels are not rigid cylinders, and blood is not a newtonian fluid), Poiseuille’s law does provide valuable insight into the determinants of blood flow through the vascular system. In certain unusual situations, however, flow can become turbulent (see Fig. 17.3B ), rather than laminar. Under these conditions, vortices (swirls) are present, and the distribution of flow velocities is chaotic. This condition is described in more detail later in this chapter.

1	Poiseuille’s law describes the laminar flow of fluids through cylindrical tubes in terms of pressure, the dimensions of the tube, and the viscosity of liquid: Equation 17.3 Pi − Po = pressure gradient from the inlet (i) of the tube to the outlet (o) r = radius of the tube η= viscosity of the fluid l = length of the tube As is clear from the equation, flow through the tube increases as the pressure gradient is increased, and it decreases as either the viscosity of the fluid or the length of the tube increases. The radius of the tube is a critical factor in determining flow because it is raised to the fourth power. Resistance to Flow In electrical theory, Ohm’s law is that the resistance (R) equals the ratio of voltage drop (E) to current flow (I). Equation 17.4 Similarly, in fluid mechanics, hydraulic resistance (R) may be defined as the ratio of the pressure drop (Pi − Po,) to flow (Q): Equation 17.5

1	Equation 17.4 Similarly, in fluid mechanics, hydraulic resistance (R) may be defined as the ratio of the pressure drop (Pi − Po,) to flow (Q): Equation 17.5 For the steady, laminar flow of a newtonian fluid through a cylindrical tube, the physical components of hydraulic resistance may be appreciated by the rearranging of Poiseuille’s law to yield the hydraulic resistance equation: Equation 17.6 Thus when Poiseuille’s law applies, the resistance to flow depends on only the dimensions of the tube and the characteristics of the fluid. The principal determinant of resistance to blood flow through any vessel is the caliber of the vessel because resistance varies inversely as the fourth power of the radius of the tube. In

1	Fig. 17.4 , the resistance to flow through small blood vessels is measured, and the resistance per unit length of vessel (R/l) is plotted against the vessel diameter. As shown, resistance is highest in the capillaries (diameter of 7 µm), and it diminishes as the vessels increase in diameter 1000 100 10 1 0.1 0.01 58 5850 5042 4234 3426 2618 187 Vessel diameter (µm) R/l(mm Hg/[mm3/sec])/µm Capillaries Arterial side Venous side •Fig. 17.4 Resistance Per Unit Length (R/l) of Individual Small Blood Vessels. Thecapillaries,withadiameterof7µm,aredenotedbythevertical dashed line. Resistancesofarteriolesareplottedtotheleftoftheverticaldashedline,andresistancesofvenules,totherightoftheverticaldashedline.Forbothtypesofvessels,theresistanceperunitlengthisinverselyproportionaltothefourthpowerofthevesseldiameter.(RedrawnfromLipowskyHH,etal.Circ Res. 1978;43:738.) on the arterial and venous sides of the capillaries. Values of R/l are virtually inversely proportional to the fourth power of the

1	Res. 1978;43:738.) on the arterial and venous sides of the capillaries. Values of R/l are virtually inversely proportional to the fourth power of the diameter (or radius) of the larger vessels on both sides of the capillaries.

1	Changes in vascular resistance occur when the caliber of vessels changes. The most important factor that leads to a change in vessel caliber is contraction of the circular smooth muscle cells in the vessel wall. Changes in internal pressure also alter the caliber of blood vessels and therefore alter the resistance to blood flow through these vessels. Blood vessels are elastic tubes. Hence, the greater the transmural pressure (i.e., the difference between internal and external pressure) across the wall of a vessel, the greater the caliber of the vessel and the less its hydraulic resistance. It is apparent from Fig. 15.3 that the greatest drop in pressure occurs in the very small arteries and arterioles. However, capillaries, which have a mean diameter of approximately 7 µm, have the greatest resistance to blood flow. Nevertheless, of all the different varieties of blood vessels that lie in series with one another (as in

1	Fig. 15.3 ), the arterioles, not the capillaries, have the greatest resistance. This seeming paradox is related to the relative numbers of parallel capillaries and parallel arterioles: There are far more capillaries than arterioles in the systemic circulation, and total resistance across the many capillaries arranged in parallel is much less than total resistance across the fewer arterioles arranged in parallel. In addition, arterioles have a thick coat of circularly arranged smooth muscle fibers that can vary the lumen radius. Even small changes in radius alter resistance greatly, as can be seen from the hydraulic •Fig. 17.5 Forresistances(R1,R2,andR3)arrangedinseries,totalresistance(Rt)equalsthesumoftheindividualresistances.P,pressure;Q,flow. Eq. 17.6 ), wherein R varies inversely with r4. In the cardiovascular system, the various types of vessels listed along the horizontal axis in

1	Eq. 17.6 ), wherein R varies inversely with r4. In the cardiovascular system, the various types of vessels listed along the horizontal axis in Fig. 15.3 lie in series with one another. The individual members of each category of vessels are ordinarily arranged in parallel with one another (see Fig. 15.1 ). Thus capillaries are in most instances parallel elements throughout the body, except in the renal vasculature (in which the peritubular capillaries are in series with the glomerular capillaries) and the splanchnic vasculature (in which the intestinal and hepatic capillaries are aligned in series with each other). The total hydraulic resistance of components arranged in series or in parallel can be derived in the same manner as those for analogous combinations of electrical resistance. Resistance of Vessels in Series In the system depicted in

1	Resistance of Vessels in Series In the system depicted in Fig. 17.5 , three hydraulic resistances, R1, R2, and R3, are arranged in series. The pressure drop across the entire system (i.e., the difference between inflow pressure [Pi] and outflow pressure [Po]) consists of the sum of the pressure drops across each of the individual resistances (equation a in Fig. 17.5 ). In the steady state, the flow (Q) through any given cross-section must equal the flow through any other cross-section. When each component in equation (a) is divided by Q (equation [b] in Fig. 17.5 ), it is evident from the definition of resistance ( Eq. 17.5 ) that for resistances in series, the total resistance (Rt) of the entire system equals the sum of the individual resistances; that is, Equation 17.7 Resistance of Vessels in Parallel For resistances in parallel, as illustrated in

1	Equation 17.7 Resistance of Vessels in Parallel For resistances in parallel, as illustrated in Fig. 17.6 , inflow and outflow pressure is the same for all tubes. In steady state, the total flow (Qt) through the system equals the sum of the flows through the individual parallel elements (equation [a] in Fig. 17.5). Because the pressure gradient (Pi − Po) is identical for all parallel elements, each term •Fig. 17.6 Forresistances(R1,R2,andR3)arrangedinparallel,thereciprocalofthetotalresistance(Rt)equalsthesumofthereciprocalsoftheindividualresistances.P,pressure;Q,flow. in equation (a) may be divided by that pressure gradient to yield equation (b). From the definition of resistance, equation (c) in Fig. 17.5 may be derived. According to this equation, for resistances in parallel, the reciprocal of the total resistance (Rt) equals the sum of the reciprocals of the individual resistances; that is, Equation 17.8

1	Equation 17.8 In a few simple illustrations, some of the fundamental properties of parallel hydraulic systems become apparent. For example, if the resistances of the three parallel elements in Fig. 17.6 were all equal, then Equation 17.9 Therefore, from Eq. 17.8, Equation 17.10 When the reciprocals of these terms, are equated, Equation 17.11 Thus the total resistance is less than the individual resistances. For any parallel arrangement, the total resistance must be less than that of any individual component. For example, consider a system in which a tube with very high resistance is added in parallel to a low-resistance tube. The total resistance of the system must be less than that of the low-resistance component by itself because the high-resistance component affords an additional pathway, or conductance, for flow of fluid.

1	Consider the physiological relationship between the total peripheral resistance (TPR) of the entire systemic vascular bed and the resistance of one of its components, such as the renal vasculature. TPR is the ratio of the arteriovenous (AV) pressure difference (arterial pressure [Pa] − venous pressure [Pv]) to the flow through the entire systemic vascular bed (i.e., the cardiac output [Qt]). For example, the renal vascular resistance (Rr) would be the ratio of the same AV pressure difference (Pa − Pv) to renal blood flow (Qr).

1	In an individual with an Pa of 100 mm Hg, a peripheral Pv of 0 mm Hg, and a cardiac output of 5000 mL/min, TPR is 0.02 mm Hg/mL/minute, or 0.02 peripheral resistance units (PRUs). Normally, the rate of blood flow through one kidney would be approximately 600 mL/ minute. Renal resistance would therefore be 100 mm Hg ÷ 600 mL/minute, or 0.17 PRUs, which is 8.5 times greater than the TPR. In an organ such as the kidney, which weighs only approximately 1% as much as the whole body, the vascular resistance is much greater than that of the entire systemic circulation. Hence, it is not surprising that the resistance to flow would be greater for a component organ, such as the kidney, than for the entire systemic circulation because the systemic circulation has not only one kidney but also many more alternative pathways for blood to flow.

1	Fig. 17.3A ), a thin layer of fluid in contact with the tube wall adheres to the wall and hence is motionless. The layer of fluid just central to the external lamina must shear against this motionless layer, and therefore that layer moves slowly but with a finite velocity. Similarly, the next more central layer moves still more rapidly; the longitudinal velocity profile is that of a paraboloid (see Fig. 17.3A ). The fluid elements in any given lamina remain in that lamina as the fluid moves longitudinally along the tube. The velocity at the center of the stream is maximal and equal to twice the mean velocity of flow across the entire cross-section of the tube. Irregular motions of the fluid elements may develop in the flow of fluid through a tube; such flow is called turbulent. In this condition, fluid elements do not remain confined to a specific laminae; instead, rapid, radial mixing occurs (see

1	Fig. 17.3B ). Greater pressure is necessary to force a given flow of fluid through the same tube when the flow is turbulent than when it is laminar. In turbulent flow, the pressure drop is approximately proportional to the square of the flow rate, whereas in laminar flow, the pressure drop is proportional to the first power of the flow rate. Hence, to produce a given flow, a pump such as the heart must do considerably more work if turbulent flow develops. Whether turbulent or laminar flow exists in a tube under given conditions may be predicted on the basis of a dimensionless number called Reynold’s number (NR). This number represents the ratio of inertial to viscous forces. For a fluid flowing through a cylindrical tube,

1	Equation 17.12 where ρ= fluid density, D = tube diameter, v = mean velocity, and η= viscosity. When NR is 2000 or less, the flow is usually laminar; when NR is 3000 or greater, the flow is turbulent; and when NR is between 2000 and 3000, the flow is transitional between laminar and turbulent. Eq. 17.12 indicates that high fluid densities, small tube diameters, high flow velocities, and low fluid viscosities predispose to turbulence. In addition to these factors, abrupt variations in tube dimensions or irregularities in the tube walls may produce turbulence. Shear Stress on the Vessel Wall As blood flows through a vessel, it exerts a force on the vessel wall parallel to the wall. This force is called a shear stress (τ). Shear stress is directly proportional to the flow rate and viscosity of the fluid: Equation 17.13

1	Equation 17.13 Turbulenceisusuallyaccompaniedbyaudiblevibrations.Turbulentflowwithinthecardiovascularsystemmaybedetectedthroughastethoscopeduringphysicalexamination.Whentheturbulenceoccursintheheart,theresultantsoundistermedamurmur; whenitoccursinavessel,thesoundistermedabruit. Insevereanemia,functionalcardiacmurmurs(murmursnotcausedbystructuralabnormalities)arefrequentlydetectable.Thephysicalbasesforsuchmurmursresidesare(1)thereducedviscosityofbloodinanemiaand (2)thehighflowvelocitiesassociatedwiththehighcardiacoutputthatusuallyprevailsinanemicpatients.Bloodclots,orthrombi,aremorelikelytodevelopinturbulentflowthaninlaminarflow.Aproblemwiththeuseofartificialvalvesinthesurgicaltreatmentofvalvularheartdiseaseisthatthrombimayoccurinassociationwiththeprostheticvalve.Thethrombimaybedislodgedandoccludeacrucialbloodvessel.Itisimportanttodesignsuchvalvestoavertturbulenceandtoincludeanticoagulantsasapartoftherapy.

1	Incertaintypesofarterialdisease,particularlyhypertension,thesubendotheliallayersofvesselstendtodegeneratelocally,andsmallregionsoftheendotheliummaylosetheirnormalsupport.Theviscousdragonthearterialwallmaycauseatearbetweenanormallysupportedregionandanunsupportedregionoftheendotheliallining.Bloodmaythenflowfromthevessellumenthroughtheriftintheliningandbecomedissectedbetweenthevariouslayersoftheartery.Suchalesioniscalledadissecting aneurysm.

1	aneurysm. Itoccursmostoftenintheproximalportionsoftheaortaandisextremelyserious.Onereasonforitspredilectionforthissiteisthehighvelocityofbloodflow,withassociatedlargeshearratevaluesattheendothelialwall.Shearstressatthevesselwallalsoinfluencesmanyothervascularfunctions,suchasthepermeabilityofthevesselwallsbylargemolecules,thebiochemicalactivityofendothelialcells,theintegrityoftheformedelementsinblood,andbloodcoagulation.Anincreaseinshearstressontheendothelialwallisalsoaneffectivestimulusforthereleaseofnitricoxide(NO)fromvascularendothelialcells;NOisapotentvasodilator(seethesection“ •Fig. 17.7 Therelativeviscosityofwholebloodincreasesataprogressivelygreaterrateasthehematocritratioincreases.Foranygivenhematocritratio,theapparentviscosityofbloodislowerwhenmeasuredinabiologicalviscometer(suchasahindlegbloodvessel)thaninaconventionalcapillarytubeviscometer.(RedrawnfromLevyMN,ShareL.Circ Res. 1953;1:247.)9010 1 2 3 4 5 6 7 8 7030 50 Hematocrit ratio Relative viscosity Hind leg Capillary tube

1	Res. 1953;1:247.)9010 1 2 3 4 5 6 7 8 7030 50 Hematocrit ratio Relative viscosity Hind leg Capillary tube viscometer

1	Rheologic Properties of Blood The viscosity of a given newtonian fluid at a specified temperature stays constant over a wide range of tube dimensions and flows. However, for a nonnewtonian fluid such as blood, viscosity may vary considerably as a function of tube dimensions and flows. Therefore, the term viscosity does not have a unique meaning for blood. The term apparent viscosity is frequently used for the derived value of blood viscosity obtained under the particular conditions of measurement. Rheologically, blood is a suspension of formed elements, principally erythrocytes, in a relatively homogeneous liquid, the blood plasma. Because blood is a suspension, the apparent viscosity of blood varies as a function of the hematocrit (ratio of the volume of red blood cells to the volume of whole blood). The viscosity of plasma is 1.2 to 1.3 times that of water. The upper curve in

1	Fig. 17.7 shows that the apparent viscosity of blood with a normal hematocrit ratio of 45% is 2.4 times that of plasma. In severe anemia, blood viscosity is low. As the hematocrit increases, the slope of the curve increases progressively; it is especially steep at the upper range of erythrocyte concentrations (see Fig. 17.7 For any given hematocrit, the apparent viscosity of blood depends on the dimensions of the tube used in estimating the viscosity. Fig. 17.8 demonstrates that the apparent viscosity of blood diminishes progressively as tube diameter decreases to less than approximately 0.3 mm. The diameters of the blood vessels with the highest resistance, the arterioles, are considerably less than this critical value. This phenomenon Fig. 17.7 also illustrates that the apparent viscosity of blood, when measured in living tissues, is considerably less than the apparent viscosity of the same blood measured in a conventional capillary tube viscometer.

1	0.60 6 5 4 3 2 1 0.50.20.1 0.40.3 Tube diameter (mm) Relative viscosity •Fig. 17.8 Therelativeviscosityofbloodrelativetothatofwaterincreasesasafunctionoftubediameteruptoadiameterofapproximately0.3mm.(RedrawnfromFåhraeusR,LindqvistT.Am J Physiol. 1931;96:562.) therefore reduces the resistance to flow in blood vessels that possess the greatest resistance. The influence of tube diameter on apparent viscosity is explained in part by the actual change in blood composition as it flows through small tubes. The composition of blood changes because the red blood cells tend to accumulate in the faster axial stream, whereas plasma tends to flow in the slower marginal layers. Because the axial portions of the bloodstream contain a greater proportion of red cells and this axial portion moves at greater velocity, the red blood cells tend to traverse the tube in less time than plasma does. Furthermore, the hematocrit of the blood contained in small blood vessels is lower than that in blood in large

1	velocity, the red blood cells tend to traverse the tube in less time than plasma does. Furthermore, the hematocrit of the blood contained in small blood vessels is lower than that in blood in large arteries or veins.

1	The physical forces responsible for the drift of erythrocytes toward the axial stream and away from the vessel walls when blood is flowing at normal rates are not fully understood. One factor is the great flexibility of red blood cells. At low flow rates, like those in the microcirculation, rigid particles do not migrate toward the central axis of a tube, whereas flexible particles do. The concentration of flexible particles near the tube’s central axis is enhanced by an increase in the shear rate. The apparent viscosity of blood diminishes as the shear rate is increased (

1	The apparent viscosity of blood diminishes as the shear rate is increased ( Fig. 17.9 ), a phenomenon called shear thinning. The greater the amount of flow, the greater the rate that one lamina of fluid shears against an adjacent lamina. The greater tendency for erythrocytes to accumulate in the axial laminae at higher flow rates is partly responsible for this nonnewtonian behavior. However, a more important factor is that at very slow flow rates, the suspended cells tend to form aggregates; such aggregation increases blood viscosity. As flow is increased, this aggregation decreases, and so does the apparent viscosity of blood (see Fig. 17.9

1	Fig. 17.9 The tendency for erythrocytes to aggregate at low flow rates depends on the concentration of the larger protein molecules in plasma, especially fibrinogen. For this reason, changes in blood viscosity with flow rate are much more pronounced when the concentration of fibrinogen is high. In addition, at low flow rates, leukocytes tend to adhere to the endothelial cells of the microvessels and thereby increase the apparent viscosity of the blood.

1	•Fig. 17.9 Decreaseintheviscosityofblood(cp,centipoise)atincreasingratesofshear(sec−1).Theshearrateisthevelocityofonelayeroffluidinrelationtothatoftheadjacentlayersandisdirectionallyrelatedtotherateofflow.(RedrawnfromAminTM,SirsJA.Q J Exp Physiol. 1985;70:37.)2400 7 6 5 4 3 18060 120 Shear rate/sec–1 Viscosity (cp) •Fig. 17.10 Effectoftheplasmafibrinogenconcentrationontheflexibilityofhumanerythrocytes.(RedrawnfromAminTM,SirsJA.Q J Exp Physiol. 1985;70:37.)40 8 6 4 2 0 31 2 Fibrinogen concentration (mg/mL) Erythrocyte flexibility (% min–1)

1	The deformability of erythrocytes is also a factor in shear thinning, especially when the hematocrit is high. The mean diameter of human red blood cells is approximately 7 µm, but they are able to pass through openings with a diameter of only 3 µm. As blood with densely packed erythrocytes flows at progressively greater rates, the erythrocytes become more and more deformed. Such deformation diminishes the apparent viscosity of blood. The flexibility of human erythrocytes is enhanced as the concentration of fibrinogen in plasma increases ( Fig. 17.10 ). If the red blood cells become hardened, as they are in certain spherocytic anemias, shear thinning may diminish. The Arterial System

1	Fig. 17.10 ). If the red blood cells become hardened, as they are in certain spherocytic anemias, shear thinning may diminish. The Arterial System The systemic and pulmonary arterial systems distribute blood to the capillary beds throughout the body. The arterioles are high-resistance vessels of this system that regulate the distribution of flow to the various capillary beds. The aorta, the pulmonary artery, and their major branches have a large amount of elastin in their walls, which makes these vessels highly distensible (i.e., compliant). This distensibility serves to dampen the pulsatile nature of blood flow that results as the heart pumps blood intermittently. When blood is ejected from the ventricles during systole, these vessels distend, and during diastole, they recoil and propel the blood forward (Fig. 17.11 ). Thus the intermittent output of the heart is converted to a steady flow through the capillaries.

1	The elastic nature of the large arteries also reduces the work of the heart. If these arteries were rigid rather than compliant, the pressure would rise dramatically during systole. This increased pressure would require the ventricles to pump against a large load (i.e., afterload) and thus increase the work of the heart. Instead, as blood is ejected into these vessels, they distend, and the resultant increase in systolic pressure, and thus the work of the heart, are reduced. Aspeopleage,theelastincontentofthelargearteriesisreducedandreplacedbycollagen.Thisreducesarterialcompliance( Fig.17.12 ).Thuswithage,systolicpressureincreases,asdoesthedifferencebetweensystolicanddiastolicbloodpressure,calledthepulse pressure (describedinthenextsection). Determinants of Arterial Blood Pressure

1	Determinants of Arterial Blood Pressure Arterial blood pressure is routinely measured in patients, and it provides a useful estimate of their cardiovascular status. Arterial pressure can be defined as mean arterial pressure (Pa), which is the pressure averaged over time, and as systolic (maximal) and diastolic (minimal) arterial pressure within the cardiac cycle ( Fig. 17.13 ). The difference between systolic and diastolic pressure is termed pulse pressure. The determinants of arterial blood pressure are arbitrarily divided into “physical” and “physiological” factors. The two Systole Arterial blood flows through the Diastole Arterial blood continues to flow through capillaries throughout systole. the capillaries throughout diastole.

1	Systole Arterial blood flows through the Diastole Arterial blood continues to flow through capillaries throughout systole. the capillaries throughout diastole. A When the arteries are normally compliant, a B During ventricular diastole the previously substantial fraction of the stroke volume is stretched arteries recoil. The volume of blood stored in the arteries during ventricular systole. that is displaced by the recoil furnishes The arterial walls are stretched. continuous capillary flow throughout diastole. Left atrium Left atrium Left ventricle Left ventricle Aorta Aorta Capillaries Capillaries Systole A volume of blood equal to the entire stroke volume must flow through the capillaries during systole. Diastole Flow through the capillaries ceases during diastole. C When the arteries are rigid, virtually none D Rigid arteries cannot recoil appreciably during of the stroke volume can be stored in the diastole. arteries.

1	C When the arteries are rigid, virtually none D Rigid arteries cannot recoil appreciably during of the stroke volume can be stored in the diastole. arteries. •Fig. 17.11 Whenarteriesarenormallycompliant(A andB),bloodflowsthroughthecapillariesthroughoutthecardiaccycle.Whenthearteriesarerigid,bloodflowsthroughthecapillariesduringsystole (C),butflowceasesduringdiastole(D). physical factors, or fluid mechanical characteristics, are fluid volume (i.e., blood volume) within the arterial system and the static elastic characteristics (compliance) of the system. The physiological factors are cardiac output (which equals heart rate × stroke volume) and peripheral resistance. To estimate Pa from an arterial blood pressure tracing, the area under the pressure curve is divided by the time interval involved (see Fig. 17.13 ). Alternatively, Pa can be approximated from the measured values of systolic pressure (Ps) and diastolic pressure (Pd) by means of the following formula: Equation 17.14

1	Fig. 17.13 ). Alternatively, Pa can be approximated from the measured values of systolic pressure (Ps) and diastolic pressure (Pd) by means of the following formula: Equation 17.14 Consider that Pa depends on only two physical factors: mean blood volume in the arterial system and arterial compliance ( Fig. 17.14 ). Arterial volume (Va), in turn, depends on the rate of inflow, (Qh) into the arteries from the heart (cardiac output) and on the rate of outflow (Qr) from the arteries through the resistance vessels (peripheral runoff). These relationships are expressed mathematically as

1	Equation 17.15 where dVa/dt is the change in arterial blood volume per unit of time. If Qh exceeds Qr, arterial volume increases, the arterial walls are stretched further, and pressure rises. The converse happens when Qr exceeds Qh. When Qh equals Qr, Pa remains constant. Thus increases in cardiac output raise Pa, as do increases in peripheral resistance. Conversely, decreases in cardiac output or peripheral resistance decrease Pa. Arterial pulse pressure is systolic pressure minus diastolic pressure. It is principally a function of just one physiological factor, stroke volume, which determines the change in arterial blood volume (a physical factor) during ventricular systole. This physical factor, in addition to a second physical factor (arterial compliance), determines the arterial pulse pressure (see Fig. 17.14

1	Fig. 17.14 As described previously, Pa depends on cardiac output and peripheral resistance. During the rapid ejection phase of systole, the volume of blood introduced into the arterial system exceeds the volume that exits the system through the arterioles. Arterial pressure and volume therefore peak; the peak arterial pressure is systolic pressure. During the remainder of the cardiac cycle (i.e., ventricular diastole), exceeds cardiac ejection. The resultant decrement in arterial blood volume thus causes pressure to fall to a minimum, which is diastolic pressure. Fig. 17.15 illustrates the effect cardiac ejection is zero, and peripheral runoff now greatly of stroke volume on pulse pressure when arterial compliance is constant.

1	Fig. 17.15 illustrates the effect cardiac ejection is zero, and peripheral runoff now greatly of stroke volume on pulse pressure when arterial compliance is constant. •Fig. 17.12 Pressure-volumerelationshipsofaortasobtainedat autopsyfromhumansindifferentagegroups(denotedbythenumbers•Fig. 17.13 Arterial Systolic, Diastolic, Pulse, and Mean Pres-attherightendofeachofthecurves).Notethatcompliance(ΔV/ΔP)sure. Meanarterialpressure(P)representstheareaunderthearterialdecreaseswithage.(RedrawnfromHallockP,BensonIC.J Clin Invest. pressurecurve(dark red) dividedbythedurationofthecardiaccycle1937;16:595.)(t2− t1). •Fig. 17.14 Thetwophysicaldeterminantsofpulsepressurearearterialcompliance(Ca)andthechangeinarterialvolume.Thetwophysiologicaldeterminantsofmeanarterialpressure(P)arecardiacoutputandtotalperipheralresistance. Arterial compliance (Ca), the ratio of blood volume to mean blood pressure (see Eq. 19.1 ), also affects pulse pressure. This relationship is illustrated in

1	Arterial compliance (Ca), the ratio of blood volume to mean blood pressure (see Eq. 19.1 ), also affects pulse pressure. This relationship is illustrated in Fig. 17.16 . When cardiac output and TPR are constant, a decrease in arterial compliance results in an increase in pulse pressure. Diminished arterial compliance also imposes a greater workload on the left ventricle (i.e., increased afterload), even if stroke volume, TPR, and Pa are equal in the two individuals. As previously discussed, if the heart rate and stroke volume remain constant, an increase in TPR causes Pa to increase. •Fig. 17.15 Effect of a Change in Stroke Volume on Pulse Pressure in a System in Which Arterial Compliance Remains Constant Over the Prevailing Range of Pressures and Volumes. Alargerincrementinbloodvolume,whereby(V4− V3)> (V2− V1),resultsingreatermeanbloodpressure(PB > PA)andagreaterpulsepressure,sothat(P4− P3)> (P2− P1).

1	When arterial compliance is constant, an increase in TPR leads to proportional increases in systolic and diastolic pressure so that the pulse pressure is unchanged (Fig. 17.17A ). However, arterial compliance is not linear. As Pa increases and the artery is stressed, compliance decreases (see Fig. 17.17B ). Because of the decrease in arterial compliance with increased Pa, pulse pressure increases when Pa is elevated. Effect of Arterial Compliance on Myocardial Energy Consumption The increased cardiac energy requirement imposed by a rigid arterial system is illustrated in Fig. 17.18 . In the data depicted in

1	Effect of Arterial Compliance on Myocardial Energy Consumption The increased cardiac energy requirement imposed by a rigid arterial system is illustrated in Fig. 17.18 . In the data depicted in Fig. 17.18 , the cardiac output from the left ventricle either was allowed to flow through the natural route (the aorta) or was directed through a stiff plastic tube to the peripheral arteries. In this experiment, the TPR values were virtually identical, regardless of which pathway •Fig. 17.16 Foragivenvolumeincrement(V2− V1),reducedarterialcompliance(complianceB[LowCa]< complianceA[HighCa])resultsinincreasedpulsepressure,whereby(P4− P1)> (P3− P2).Pa,meanarterialpressure.

1	•Fig. 17.17 Comparisonoftheeffectsofagivenchangeinperipheralresistanceonpulsepressure(P)whenthepressure-volumecurveforthearterialsystemiseitherrectilinear(A) orcurvilinear(B). Theincrementinarterialvolumeisthesameforbothconditions;thatis,(V4−V3)=(V2−V1).PressureABVolumeP1V1V2V3V4P3P6P4P2–P5–PressureP1V1V2V3V4P3P6P4P2–P5– •Fig. 17.18 Therelationshipbetweenmyocardialoxygencon-sumption(1mL/100g/beat)andstrokevolume(inmilliliters)inananesthetizeddogwhosecardiacoutputcouldbepumpedbytheleftventricleeitherthroughtheaortaorthroughastiffplastictubetotheperipheralarteries.(ModifiedfromKellyRP,TuninR,KassDA.Circ Res. 1992;71:490.)Plastic tubing Native aorta Stroke volume (mL) 5 0 0.05 O 2 consumption (mL) O2/100 g/beat)0.1 10 15 was selected. The results showed that for any given stroke volume, myocardial oxygen consumption was substantially greater when the blood was diverted through the plastic tubing than when it flowed through the aorta. The increased oxygen consumption indicates that the

1	oxygen consumption was substantially greater when the blood was diverted through the plastic tubing than when it flowed through the aorta. The increased oxygen consumption indicates that the left ventricle has to expend significantly more energy to pump blood through a less compliant conduit than through a more compliant conduit.

1	Arterialpulsepressureprovidesvaluableinformationaboutaperson’sstrokevolume,providedthatarterialcomplianceisessentiallynormal.Patientswhohaveseverecongestiveheartfailureorwhohavesufferedaseverehemorrhagearelikelytohaveaverylowarterialpulsepressurebecausetheirstrokevolumesareabnormallysmall.Conversely,individualswithlargestrokevolumes,asinaorticvalveregurgitation,arelikelytohaveanincreasedarterialpulsepressure.Similarly,well-trainedathletesatresttendtohavelargestrokevolumesbecausetheirheartratesareusuallylow.Theprolongedventricularfillingtimesintheseindividualsinducetheventriclestopumpalargestrokevolume,andhencetheirpulsepressureislarge. The radial stretch of the ascending aorta brought about by left ventricular ejection initiates a pressure wave that is propagated down the aorta and its branches. The pressure wave travels much faster (≈4 to 12 m/second) than the blood itself does. This pressure wave is the “pulse” that can be detected through palpation of a peripheral artery.

1	Inchronichypertension,aconditioncharacterizedbyapersistentelevationinTPR,thearterialpressure-volumecurveresemblesthatshownin Fig.17.17B . BecausearteriesbecomesubstantiallylesscompliantwhenParises,anincreaseinTPRcausessystolicpressuretobemoreelevatedthandiastolicpressure.Diastolicpressureiselevatedinsuchindividualsbutordinarilynotmorethan10to40mmHgabovetheaveragenormallevelof80mmHg.Notuncommonly,however,systolicpressureiselevatedby50to100mmHgabovetheaveragenormallevelof120mmHg. Thevelocityofthepressurewavevariesinverselywitharterialcompliance.Ingeneral,transmissionvelocityincreaseswithage,whichconfirmstheobservationthatthearteriesbecomelesscompliantwithadvancingage.Velocityalsoincreasesprogressivelyasthepulsewavetravelsfromtheascendingaortatowardtheperiphery.Thisincreaseinvelocityreflectsthedecreaseinvascularcomplianceinthemoredistalportionsthaninthemoreproximalportionsofthearterialsystem.

1	ThePacontourbecomesdistortedasthewaveistransmitteddownthearterialsystem.Thisdistortioninthepressurewavecontourofthehumanarterialtreeisdemonstratedasafunctionofageandofrecordingsitein Fig.17.19 .Dampingofthehigh-frequencycomponentsofthearterialpulseiscausedlargelybytheviscoelasticpropertiesofthearterialwalls.Thepulsepressurewavetravelsmorerapidlyinolderpeoplethanintheyoungerpeople,asaconsequenceofreducedcompliance.Severalfactors—includingwavereflectionandresonance,vasculartapering,andpressure-inducedchangesintransmissionvelocity—contributetopeakingofthePawave.

1	Most commonly, blood pressure is estimated indirectly by means of a sphygmomanometer. In hospital intensive care units, needles or catheters may be introduced into the peripheral arteries of patients to measure arterial blood pressure directly by means of strain gauges. When blood pressure readings are taken from the arm, systolic pressure may be estimated by palpation of the radial artery at the wrist (palpatory method). While pressure in the cuff exceeds the systolic level, no pulse is perceived. As pressure falls just below the systolic level ( Fig. 17.20A ), a spurt of blood passes through the brachial artery under the cuff during the peak of systole, and a slight pulse is felt at the wrist.

1	Fig. 17.20A ), a spurt of blood passes through the brachial artery under the cuff during the peak of systole, and a slight pulse is felt at the wrist. The auscultatory method is a more sensitive and therefore more precise technique for measuring systolic pressure, and it also enables diastolic pressure to be estimated. The practitioner listens with a stethoscope applied to the skin of the antecubital space over the brachial artery. While the pressure in the cuff exceeds systolic pressure, the brachial artery is occluded, and no sounds are heard (see Fig. 17.20B ). When the inflation pressure falls just below the systolic level (120 mm Hg in

1	Fig. 17.20A ), a small spurt of blood escapes the occluding pressure of the cuff, and slight tapping sounds (called Korotkoff sounds) are heard with each heartbeat. The pressure at which the first sound is detected •Fig. 17.19 Pulse Pressure Curves Recorded From Various Sites in the Arterial Trees of Humans at Different Ages. Inthe24-yearold,thearterialpulsedisplaysstrikingchangesinthepulsepressureamplitudeandcontourasitpassesdownthearterialtree.Thepulsepressurewaveinthe68-year-oldshowslittleamplificationandisrelativelyunchangedasthepulsetravelsbecausethereislesswavereflection.(ReproducedbypermissionofHodderEducationfromNicholsWW,O’RourkeM,eds.McDonald’s Blood Flow in Arteries: Theoretical, Experimental and Clinical Principles. 5thed.London:Arnold;2005.) represents systolic pressure. It usually corresponds closely to the directly measured systolic pressure. As the inflation pressure of the cuff continues to fall, more blood escapes under the cuff per beat and the sounds become louder.

1	corresponds closely to the directly measured systolic pressure. As the inflation pressure of the cuff continues to fall, more blood escapes under the cuff per beat and the sounds become louder. When the inflation pressure approaches the diastolic level, the Korotkoff sounds become muffled. When the inflation pressure falls just below the diastolic level (80 mm Hg in

1	Fig. 17.20A ), the sounds disappear; the pressure reading at this point indicates diastolic pressure. The origin of the Korotkoff sounds is related to the discontinuous spurts of blood that pass under the cuff and meet a static column of blood beyond the cuff; the impact and turbulence generate audible vibrations. Once the inflation pressure is less than diastolic pressure, flow is continuous in the brachial artery, and sounds are no longer heard (see Fig. 17.20C The Venous System Veins are elements of the circulatory system that return blood to the heart from tissues. Moreover, veins constitute a very large reservoir that contains up to 70% of the blood in the circulation. The reservoir function of veins makes them able to adjust the volume of blood returning to the heart,

1	Theankle-brachialindex(ABI)istheratioofsystolicbloodpressuresattheankle(dorsalispedisartery)tothatinthebrachialartery.TheABI,whichisobtainedbysimplemeasurements,isanindicatorofpossibleperipheralarterydisease.TheABIhasalsobeenproposedasapredictorofriskforcardiovascularandcerebrovasculardisease.PeoplewithanormalABIratioof1.1to1.4havealowerincidenceofeithercoronaryorcerebrovasculareventsthandothosewitharatioof0.9orlower.Inaddition,astherateofABIincreaseswithtime,theincidencesofcardiovascularmorbidityandmortalityalsoincrease. or preload, so that the needs of the body can be matched when cardiac output is altered (see Chapter 19). This high capacitance is an important property of veins.

1	or preload, so that the needs of the body can be matched when cardiac output is altered (see Chapter 19). This high capacitance is an important property of veins. The hydrostatic pressure in postcapillary venules is approximately 20 mm Hg, and it decreases to approximately 0 mm Hg in the thoracic venae cavae and right atrium. Hydrostatic pressure in the thoracic venae cavae and right atrium is also termed central venous pressure. Veins are very distensible and have very low resistance to blood flow. Such low resistance allows movement of blood from Consider that the arterial blood pressure is being measured in a patient whose blood pressure is 120/80 mm Hg. The pressure (represented by the oblique line) in a cuff around the patient’s arm is allowed to fall from greater than 120 mm Hg (point B) to below 80 mm Hg (point C) in about 6 seconds.

1	When the cuff pressure exceeds the systolic arterial pressure (120 mm Hg), no blood progresses through the arterial segment under the cuff, and no sounds can be detected by a stethoscope bell placed on the arm distal to the cuff. When the cuff pressure falls below the diastolic arterial pressure, arterial flow past the region of the cuff is continuous, and no sounds are audible. When the cuff pressure is between 120 and 80 mm Hg, spurts of blood traverse the artery segment under the cuff with each heartbeat, and the Korotkoff sounds are heard through the stethoscope. •Fig. 17.20 A toC, Measurementofarterialbloodpressurewithasphygmomanometer. peripheral veins to the heart with only small reductions in central venous pressure. Moreover, veins control filtration and absorption by adjusting postcapillary resistance (see the section “ ”) and assist in the cardiovascular adjustments that accompany changes in body position.

1	The ability of veins to participate in these various functions depends on their distensibility, or compliance. Venous compliance varies with the position in the body in such a way that veins in the lower limb are less compliant than those at or above the level of the heart. Veins in the lower limbs are also thicker than those in the brain or upper limbs. The compliance of veins, like that of arteries, decreases with age, and the vascular thickening that occurs is accompanied by a reduction in elastin and an increase in collagen content. Variations in venous return are achieved by adjustments in venomotor tone, respiratory activity (see Chapter 19), and orthostatic stress or gravity.

1	Variations in venous return are achieved by adjustments in venomotor tone, respiratory activity (see Chapter 19), and orthostatic stress or gravity. Gravitational forces influence the amount of blood in the venous system and therefore may profoundly affect cardiac output. For example, soldiers standing at attention for a long time may faint because gravity causes blood to pool in the dependent blood vessels, which reduces cardiac output. Warm ambient temperatures interfere with the compensatory vasomotor reactions, and the absence of muscular activity exaggerates these effects. Gravitational effects are amplified in airplane pilots during pullout from dives. The centrifugal force in the footward direction may be several times greater than the force of gravity. Pilots characteristically black out momentarily during the pullout maneuver as blood is drained from the cephalic regions and pooled in the lower parts of the body.

1	Some explanations have been advanced to explain the gravitationally induced reduction in cardiac output, but they are inaccurate. For example, it has been argued that when an individual is standing, the force of gravity impedes venous return to the heart from the dependent regions of the body. This explanation is incomplete because it does not account for the gravitational counterforce on the arterial side of the same vascular circuit, and this counterforce facilitates venous return. Moreover, it does not account for the effect of gravity in causing venous pooling. When a person is standing upright, gravity causes blood to accumulate in the lower extremities and distend both the arteries and veins. Because venous compliance is so much greater than arterial compliance, this distention occurs more on the venous side than on the arterial side of the circuit.

1	The hemodynamic effects of such venous distention (venous pooling) resemble those caused by the hemorrhage of an equivalent volume of blood from the body. When an adult shifts from a supine position to a relaxed standing position, 300 to 800 mL of blood pools in the legs. This pooling may reduce cardiac output by approximately 2 L/min. The compensatory adjustments made to assume a standing position are similar to the adjustments to blood loss (see also ): There are reflex increases in heart rate and cardiac contractility. In addition, both arterioles and veins constrict; the arterioles are affected to a greater extent than are the veins. When a recumbent person stands but remains at rest, the pressure in the veins rises in the dependent regions of the body (

1	When a recumbent person stands but remains at rest, the pressure in the veins rises in the dependent regions of the body ( Fig. 17.21 ). The Pv in the legs increases gradually and does not reach an equilibrium value until almost 1 minute after the person begins standing. The slowness of this rise in Pv is attributable to the venous valves, which allow flow only toward the heart. When a person stands, the valves prevent blood in the veins from falling toward the feet. Hence, the column of venous blood is supported at numerous levels by these valves. Because of these valves, the venous column can be thought of as consisting of many discontinuous segments. However, blood continues to enter the column from many venules and small tributary veins, and the pressure continues to rise. As soon as the pressure in one segment exceeds that in the segment just above it, the intervening valve is forced open. Ultimately, all the valves are open, and the column is continuous.

1	•Fig. 17.21 Meanpressures(±95%confidenceintervals)inthefootveinsofhumansubjectsduringquietstanding,duringwalking,andduringrunning.(FromStickC,etal.J Appl Physiol. 1992;72:2063.)100 80 60 40 20 0 Venous pressure (mm Hg) Walking (3 km/hr) Walking (6 km/hr) Running (10 km/hr) Standing Someofthedrugsusedtotreatchronichypertensioninterferewiththereflexadaptationtostanding.Similarly,astronautsexposedtoweightlessnesslosetheiradaptationstogravityafterafewdaysinspace,andtheyexperiencepronounceddifficultieswhentheyfirstreturntoearth.Whensuchastronautsandotherindividualswithimpairedreflexadaptationsstand,theirbloodpressuremaydropsubstantially.Thisresponseiscalledorthostatic hypotension, whichmaycauselightheadednessorfainting.

1	Thesuperficialveinsintheneckordinarilyarepartiallycollapsedwhenanormalindividualissittingorstanding.Venousreturnfromtheheadisconductedlargelythroughthedeepercervicalveins,whichareprotectedfromcollapsebecausetheyaretetheredtosurroundingstructures.Whencentralvenouspressureisabnormallyelevated,thesuperficialneckveinsaredistended,andtheydonotcollapseevenwhenthepersonsitsorstands.Suchcervicalvenousdistentionisanimportantclinicalsignofcongestiveheartfailure. Theauxiliarypumpingmechanismgeneratedbyskeletalmusclecontractionsismuchlesseffectiveinpeoplewithvaricoseveinsintheirlegs.Thevalvesinthesedefectiveveinsdonotfunctionproperly,andthereforewhenthelegmusclescontract,thebloodinthelegveinsisforcedinboththeretrogradeandantegradedirections.Thuswhenanindividualwithvaricoseveinsstandsorwalks,Pvintheanklesandfeetisexcessivelyhigh.Theconsequenthighcapillarypressureleadstotheaccumulationofedematousfluidintheanklesandfeet.

1	Precise measurement reveals that the final level of Pv in the feet during quiet standing is only slightly greater than that in a static column of blood extending from the right atrium to the feet. This finding indicates that the pressure drop caused by blood flow from the foot veins to the right atrium is very small. Because of this very low resistance, all the veins can be viewed as having a common venous compliance in the model of the circulatory system illustrated in . When an individual who has been standing quietly begins to walk, Pv in the legs decreases appreciably (see

1	Fig. 17.21 ). Because of the intermittent venous compression exerted by the contracting leg muscles, and because of the operation of the venous valves, blood is forced from the veins toward the heart. Hence, muscular contraction lowers the mean Pv in the legs and serves as an auxiliary pump. Furthermore, muscular contraction prevents venous pooling and lowers capillary hydrostatic pressure. In this way, muscular contraction reduces the tendency for edematous fluid to collect in the feet during standing.

1	The circulatory system supplies the tissues with blood in amounts that meet the body’s requirements for O2 and nutrients. The capillaries, whose walls consist of a single layer of endothelial cells, allow rapid exchange of gases, water, and solutes with interstitial fluid. The muscular arterioles, which are the major resistance vessels, regulate regional blood flow to the capillary beds. Venules and veins serve primarily as collecting channels and storage vessels. The lymphatic system is composed of lymphatic vessels, nodes, and lymphoid tissue. This system collects the fluid and proteins that have escaped from blood and transports them back into the veins for recirculation in blood. In this section, the network of the smallest blood vessels of the body, as well as the lymphatic vessels, are examined in detail.

1	The microcirculation is defined as the circulation of blood through the smallest vessels of the body: arterioles, capillaries, and venules. Arterioles (5 to 100 µm in diameter) have a thick smooth muscle layer, a thin adventitial layer, and an endothelial lining (see Fig. 15.2 ). Arterioles give rise directly to capillaries (5 to 10 µm in diameter) or, in some tissues, to metarterioles (10 to 20 µm in diameter), which then give rise to capillaries ( Fig. 17.22 ). Metarterioles can bypass the capillary bed and connect to venules, or they can connect directly to the capillary bed. Arterioles that give rise directly to capillaries regulate flow through these capillaries by constriction or dilation. The capillaries form an interconnecting network of tubes with an average length of 0.5 to 1 mm. Functional Properties of Capillaries

1	Functional Properties of Capillaries In metabolically active organs, such as the heart, skeletal muscle, and glands, capillary density is high. In less active tissues, such as subcutaneous tissue or cartilage, capillary density is low. Capillary diameter also varies. Some capillaries have diameters smaller than those of erythrocytes. Passage through these tiny vessels requires the erythrocytes to become temporarily deformed. Fortunately, normal erythrocytes are quite flexible. Blood flow in capillaries depends chiefly on the contractile state of arterioles. The average velocity of blood flow •Fig. 17.22 Composite Schematic Illustration of the Microcirculation. Thecircular structures onthearterioleandvenulerepresentsmoothmusclefibers,andthebranching solid lines representsympatheticnervefibers.Thearrows indicatethedirectionofbloodflow.AV,arteriovenous.

1	in capillaries is approximately 1 mm/second; however, it can vary from zero to several millimeters per second in the same vessel within a brief period. These changes in capillary blood flow may be random or rhythmic. The rhythmic oscillatory behavior of capillaries is caused by contraction and relaxation (vasomotion) of the precapillary vessels (i.e., the arterioles and small arteries).

1	Vasomotion is an intrinsic contractile behavior of vascular smooth muscle and is independent of external input. Changes in transmural pressure (intravascular pressure minus extravascular pressure) also influence the contractile state of precapillary vessels. An increase in transmural pressure, caused either by an increase in Pv or by dilation of arterioles, results in contraction of the terminal arterioles. A decrease in transmural pressure causes precapillary vessel relaxation. Humoral and possibly neural factors also affect vasomotion. For example, when increased transmural pressure causes the precapillary vessels to contract, the contractile response can be overridden and vasomotion abolished. This effect is accomplished by metabolic (humoral) factors when the O2 supply becomes too low for the requirements of parenchymal tissue, as occurs in skeletal muscle during exercise.

1	Although a reduction in transmural pressure relaxes the terminal arterioles, blood flow through the capillaries cannot increase if the reduction in intravascular pressure is caused by severe constriction of the upstream microvessels. Large arterioles and metarterioles also exhibit vasomotion. However, their contraction usually does not completely occlude the lumen of the vessel and arrest blood flow, whereas contraction of the terminal arterioles may arrest blood flow. Thus the flow rate in capillaries may be altered by contraction and relaxation of small arteries, arterioles, and metarterioles. Blood flow through the capillaries has been called nutritional flow because it provides for exchange of gases and solutes between blood and tissue. Conversely, blood flow that bypasses the capillaries as it passes from the arterial to the venous side of the circulation via metarterioles has been termed nonnutritional, or shunt, flow (see

1	Fig. 17.22 ). In some areas of the body (e.g., fingertips, ears), true AV shunts exist (see Fig. 17.37 ). However, in many tissues, such as muscle, anatomical shunts are lacking. Even in the absence of these shunts, nonnutritional flow can occur. In tissues with metarterioles, nonnutritional flow may be continuous from arteriole to venule during low metabolic activity, when many precapillary vessels are closed. When metabolic activity increases in these tissues, more precapillary vessels open to allow capillary perfusion. True capillaries lack smooth muscle and are therefore incapable of active constriction. Nevertheless, the endothelial cells that form the capillary wall contain actin and myosin, and they can alter their shape in response to certain chemical stimuli. Because of its narrow lumen (i.e., small radius), a thin-walled capillary can withstand high internal pressures without bursting. This property can be explained in terms of the law of Pierre-Simon Laplace:

1	Equation 17.16 T = tension in the vessel wall r = radius of the vessel Laplace’s equation applies to very thin-walled vessels, such as capillaries. Wall tension opposes the distending force (ΔPr) that tends to pull apart a theoretical longitudinal slit in the vessel (

1	Fig. 17.23 ). Transmural pressure in a blood vessel in vivo is essentially equal to intraluminal pressure because extravascular pressure is generally negligible. To calculate wall tension, pressure in mm Hg is converted to dynes per square centimeter according to the equation P = hρg, where h is the height of an Hg column in centimeters, ρ is the density of Hg in g/cm3, and g is gravitational acceleration in cm/s2. For a capillary with a pressure of 25mmHg and a radius of 5 × 10−4 cm, the pressure (2.5 cm Hg × 13.6 g/cm3 × 980 cm/sec2) is 3.33 × 104 •Fig. 17.23 Diagram of a Small Blood Vessel to Illustrate the Law of Laplace. T=Pr,whereP=intraluminalpressure,r=radiusofthevessel,andT=walltensionastheforceperunitlengthtangentialtothevesselwall.Walltensionpreventsrupturealongatheoreticallongitudinalslitinthevessel.

1	dyne/cm2. Wall tension is then 16.7 dyne/cm. For an aorta with a pressure of 100 mm Hg and a radius of 1.5 cm, wall tension is 2 × 105 dyne/cm. Thus at the pressures normally found in the aorta and capillaries, the wall tension of the aorta is approximately 12,000 times greater than that of the capillaries. In a person standing quietly, capillary pressure in the feet may reach 100 mm Hg. Even under such conditions, capillary wall tension increases to a value that is still only one three-thousandth of the wall tension in the aorta at the same internal pressure.

1	The diameter of the resistance vessels (arterioles) is determined from the balance between the contractile force of the vascular smooth muscle and the distending force produced by intraluminal pressure. The greater the contractile activity of the vascular smooth muscle of an arteriole, the smaller its diameter. In small arterioles, contraction can continue to the point at which the vessel is completely occluded. Occlusion is caused by infolding of the endothelium and by trapping of blood cells in the vessel.

1	With a progressive reduction in intravascular pressure, vessel diameter decreases (as does vessel wall tension, according to the law of Laplace) and blood flow eventually ceases, although pressure within the arteriole is still greater than tissue pressure. The pressure that causes flow to cease has been called the critical closing pressure, and its mechanism is still unclear. The critical closing pressure is low when vasomotor activity is reduced by inhibition of sympathetic nerve activity in the vessel and is increased when vasomotor tone is enhanced by activation of the vascular sympathetic nerve fibers.

1	Iftheheartbecomesgreatlydistendedwithbloodduringdiastole,asmayoccurwithcardiacfailure,itfunctionslessefficiently.Toejectagivenvolumeofbloodperbeat,moreenergyisrequired(walltensionmustbegreater)forthedistendedheartthanforanormalundilatedheart.ThelessefficientpumpingofadistendedheartisanexampleofLaplace’slaw,accordingtowhichthetensioninthewallofavesselorchamber(inthiscase,theventricles)equalstransmuralpressure(pressureacrossthewall,ordistendingpressure)multipliedbytheradiusofthevesselorchamber.Laplace’srelationshipordinarilyappliestoinfinitelythin-walledvessels,butitcanbeappliedtothespherical,dilatedheartifcorrectionismadeforwallthickness.Undertheseconditions,theequationisσ=ΔPr/2w,whereσ= wallstress,ΔP= transmuralpressuredifference,r= radius,andw= wallthickness. Vasoactive Role of the Capillary Endothelium

1	Vasoactive Role of the Capillary Endothelium The endothelium is an important source of substances that cause contraction or relaxation of vascular smooth muscle. One of these substances is prostacyclin, or prostaglandin I2 (PGI2). PGI2 can relax vascular smooth muscle via an increase in cyclic adenosine monophosphate (cAMP; Fig. 17.24 ). PGI2 is formed in the endothelium from muscle; its action is not endothelium mediated. Vasodilator agents such as adenosine, H+, CO2, and K+ may be released from parenchymal tissue and act locally on resistance vessels (see Fig. 17.24 Lumen Endothelium Interstitial space Relaxation Basement membrane ATP ADP AMP Adenosine Parenchymal tissue cAMP RelaxationcGMP NO NP ACh GCyc L-arg. Vascular smooth muscle Cyc OxPGI2Syn PGI2 AA H+, CO2, K+

1	Injurytotheendotheliumofbloodvesselsprecedesatherosclerosis.Theprotective(antiatherogenic)effectoftheendotheliumresidesinseveralproperties.Forexample,theendotheliumregulatesadhesionofleukocytestothevesselwall,suppressestheproliferationofvascularsmoothmusclecells,maintainsavesselliningthatresiststheformationofthrombi,andregulatesvascularsmoothmuscletone.AllthesefunctionsinvolvetheactionofNO.Asindicatedpreviously,productionofNOisregulatedbymanysubstancesandbyshearstressactingonthevesselwall.

1	•Fig. 17.24 Endothelium-Mediated and Non–Endothelium-Mediated Vasodilation. Prostacyclin(PGI2)isformedfromarachidonicacid(AA)bytheactionofcyclooxygenase(CycOx)andprostacyclinsynthase(PGI2Syn)intheendotheliumandelicitsrelaxationoftheadjacentvascularsmoothmuscleviaincreasesincAMP.Stimulationoftheendothelialcellswithacetylcholine(ACh)orotheragents(seetext)resultsintheformationandreleaseofanendothelium-derivedrelaxingfactoridentifiedasnitricoxide(NO).NOstimulatesguanylylcyclase(GCyc)toincreasecGMPinthevascularsmoothmuscletoproducerelaxation.Thevasodilatornitroprusside(NP)actsdirectlyonvascularsmoothmuscle.Substancessuchasadenosine,H+ ,CO2,andK+ canariseintheparenchymaltissueandelicitvasodilationbydirectactiononvascularsmoothmuscle.ADP,adenosinediphosphate;AMP,adenosinemonophosphate;ATP,adenosinetriphosphate;cAMP,cyclicadenosinemonophosphate;cGMP,cyclicguanosinemonophosphate;L-arg,L-arginine.

1	arachidonic acid, and the process is catalyzed by PGI2 synthase. The mechanism that triggers synthesis of PGI2 is not known. However, PGI2 may be released by an increase in shear stress caused by accelerated blood flow. The primary function of PGI2 is to inhibit platelet adherence to the endothelium and platelet aggregation and thus prevent intravascular clot formation. PGI2 also causes relaxation of vascular smooth muscle. Of far greater importance in endothelium-mediated vascular dilation is the formation and release of nitric oxide (NO), a component of endothelium-derived relaxing factor (see

1	Of far greater importance in endothelium-mediated vascular dilation is the formation and release of nitric oxide (NO), a component of endothelium-derived relaxing factor (see Fig. 17.24 ). When endothelial cells are stimulated by acetylcholine or other vasodilator agents (e.g., adenosine triphosphate [ATP], bradykinin, serotonin, substance P, histamine), NO is released. These agents do not cause vasodilation in blood vessels lacking the endothelium. NO (synthesized from L-arginine) activates guanylyl cyclase in vascular smooth muscle to increase the concentration of cyclic guanosine monophosphate (cGMP), which produces relaxation by decreasing myofilament sensitivity to [Ca++]. Release of NO can be stimulated by the shear stress of blood flow on the endothelium. The drug nitroprusside also increases cGMP by acting directly on vascular smooth

1	Acetylcholine also stimulates the release of an endothelium-dependent hyperpolarizing factor that underlies the relaxation of adjacent smooth muscle. Although arachidonic acid metabolites have been suggested, the factor remains unknown. Moreover, how the factor reaches vascular smooth muscle (diffusion through the extracellular space or passage via myoepithelial junctions) is unclear. Nevertheless, there are diverse ways by which endothelial cells communicate with vascular smooth muscle. The endothelium can also synthesize endothelin, a potent vasoconstrictor peptide. Endothelin affects vascular tone and blood pressure and may be involved in pathological states, including atherosclerosis, pulmonary hypertension, congestive heart failure, and renal failure. Passive Role of the Capillary Endothelium

1	Passive Role of the Capillary Endothelium Solvent and solute move across the capillary endothelial wall by three processes: diffusion, filtration, and pinocytosis. Diffusion is the most important process for transcapillary exchange, and pinocytosis is the least important. Diffusion. Under normal conditions, only approximately 0.06 mL of water per minute moves across the capillary wall per 100 g of tissue as a result of filtration. In contrast, 300 mL of water per minute per 100 g of tissue moves across the capillary wall by diffusion. Thus diffusion is the key factor in providing exchange of gases, substrates, and waste products between capillaries and tissue cells. The process of diffusion is described by Fick’s law (see also Equation 17.17 J = quantity of a substance moved per unit time A = cross-sectional area of the diffusion pathway

1	ΔC = concentration gradient of the solute •Fig. 17.25 Flowand Diffusion-Limited Transport From Capillaries (Cap) to Tissue. A, Flow-limitedtransport.Thesmallestwater-solubleinerttracerparticles(blue dots) reachnegligibleconcentrationsafterpassingonlyashortdistancedownthecapillary.Largerparticles(brown dots) withsimilarpropertiestravelfartheralongthecapillarybeforereachinganinsignificantintracapillaryconcentration.Bothsubstancescrosstheinterstitialfluid(ISF)andreachtheparenchymaltissue(Cell).Becauseoftheirsize,moreofthesmallerparticlesaretakenupbythetissuecells.Thelargestparticles(red circles) cannotpenetratethecapillaryporesandhencedonotescapefromthecapillarylumenexceptbypinocytoticvesicletransport.Anincreaseinthevolumeofbloodfloworanincreaseincapillarydensityincreasestissuesupplyofthediffusiblesolutes.Notethatcapillarypermeabilityisgreateratthevenousendofthecapillary(alsointhevenule,notshown)becauseofthelargernumberofporesinthisregion.B,

1	Diffusion-limitedtransport.Whenthedistancebetweencapillariesandparenchymaltissueislargeasaresultofedemaorlowcapillarydensity,diffusionbecomesalimitingfactorinsolutetransportfromcapillarytotissue,evenathighratesofcapillaryflow.

1	For diffusion across a capillary wall, Fick’s law can also be expressed as Equation 17.18 P = capillary permeability by the substance Co = concentration of the substance outside the Ci = concentration of the substance inside the capillary The PS product provides a convenient expression of available capillary surface area because the intrinsic permeability of the capillary is rarely altered much under physiological conditions. However, in pathological conditions, as with a bee sting, capillary permeability may be altered.

1	In capillaries, diffusion of lipid-insoluble molecules is restricted to water-filled channels or pores. Movement of solute across the capillary endothelium is complex and involves corrections for attractions between solute and solvent molecules, interactions between solute molecules, pore configuration, and charge on the molecules in relation to charge on the endothelial cells. Such solute motion is not simply a matter of random thermal movement of molecules that seemingly run down a concentration gradient. For small molecules, such as water, NaCl, urea, and glucose, the capillary pores offer little restriction to diffusion (i.e., they have a low reflection coefficient; see the section “ ”). Diffusion of these substances is so rapid that the mean concentration gradient across the capillary endothelium is extremely small. The larger the lipid-insoluble molecules are, the more restricted is their diffusion through capillaries. Diffusion eventually becomes minimal when the molecular

1	endothelium is extremely small. The larger the lipid-insoluble molecules are, the more restricted is their diffusion through capillaries. Diffusion eventually becomes minimal when the molecular weight of the molecules exceeds approximately 60,000. With small molecules, the only limitation to net movement across the capillary wall is the rate at which blood flow transports the molecules to the capillary. Transport of these molecules is said to be flow limited.

1	With flow-limited small molecules, the concentration of the molecule in blood reaches equilibrium with its concentration in interstitial fluid at a location near the origin of the capillary from its parent arteriole. Its concentration falls to negligible levels near the arterial end of the capillary ( Fig. 17.25A ). If the flow is large, the small molecule can still be present at a distant locus downstream in the capillary. A somewhat larger molecule moves farther along the capillary before it reaches an insignificant concentration in blood. Furthermore, the number of still larger molecules that enter the arterial end of the capillary but cannot pass through the capillary pores equals the number that leaves the venous end of the capillary (see Fig. 17.25A

1	Fig. 17.25A With large molecules, diffusion across the capillaries becomes the limiting factor (diffusion limited); that is, the permeability of a capillary to a large solute molecule limits its transport across the capillary wall. Diffusion of small lipid-insoluble molecules is so rapid that diffusion limits blood-tissue exchange only when distances between capillaries and parenchymal cells are great (e.g., as in tissue edema or very low capillary density; see Fig. 17.25B Movement of lipid-soluble molecules across the capillary wall is not limited to capillary pores (only ≈0.02% of the capillary surface); it also occurs directly through the lipid membranes of the entire capillary endothelium. Consequently, lipid-soluble molecules move rapidly between blood and tissue. The degree of lipid solubility (oil-to-water partition coefficient) provides a good index of the ease of transfer of lipid molecules through the capillary endothelium.

1	Both O2 and CO2 are lipid soluble, and they readily pass through endothelial cells. Calculations based on (1) the diffusion coefficient for O2, (2) capillary density and diffusion distances, (3) blood flow, and (4) tissue O2 consumption indicate that the O2 supply of normal tissue at rest and during activity is not limited by diffusion or by the number of open capillaries.

1	Measurements of the partial pressure of O2 (PO2) and O2 saturation of blood in microvessels indicate that in many tissues, O2 saturation at the entrance of capillaries has decreased to approximately 80% as a result of diffusion of O2 from arterioles and small arteries. Moreover, CO2 loading and the resulting intravascular shifts in the oxyhemoglobin dissociation curve occur in the precapillary vessels. Hence, in addition to gas exchange at the capillaries, O2 and CO2 pass directly between adjacent arterioles and venules and possibly between arteries and veins (countercurrent exchange). The countercurrent exchange represents a diffusional shunting of gas away from the capillaries; this shunting may limit the supply of O2 to the tissue at low blood flow rates.

1	Capillary Filtration. The permeability of the capillary endothelial membrane is not uniform. For example, liver capillaries are quite permeable, and albumin escapes from them at a rate several times greater than that from the less permeable muscle capillaries. Furthermore, permeability is not uniform along the length of the capillary. The venous ends are more permeable than the arterial ends, and permeability is greatest in the venules, a property attributed to the greater number of pores in these regions. Where does filtration occur? Some water passes through the capillary endothelial cell membranes, but most flows through apertures (pores) in the endothelial walls of the capillaries (

1	Where does filtration occur? Some water passes through the capillary endothelial cell membranes, but most flows through apertures (pores) in the endothelial walls of the capillaries ( Figs. 17.26 17.27 ). The pores in skeletal and cardiac muscle capillaries have diameters of approximately 4 nm. There are clefts between adjacent endothelial cells in cardiac muscle, and the gap at the narrowest point is approximately 4 nm. The clefts (pores) are sparse and represent only approximately 0.02% of the capillary surface area. Pores are absent in cerebral capillaries, where the blood-brain barrier blocks the entry of many small molecules. In addition to clefts, some of the more porous capillaries (e.g., those in the kidneys and intestines) contain fenestrations 20 to 100 nm wide, whereas other capillaries (e.g., those in the liver) have a discontinuous endothelium (see

1	Fig. 17.27 ). Fenestrations and discontinuous endothelia allow the passage of molecules that are too large to pass through the intercellular clefts of the endothelium. The direction and magnitude of water movement across the capillary wall can be estimated as the algebraic sum of the hydrostatic and osmotic pressure that exists across the wall. An increase in intracapillary hydrostatic pressure favors movement of fluid from the vessel interior to the interstitial space, whereas an increase in the concentration CHAPTER 17 Properties of the Vasculature of osmotically active particles within vessels favors movement of fluid into the vessels from the interstitial space ( Fig. 17.28

1	CHAPTER 17 Properties of the Vasculature of osmotically active particles within vessels favors movement of fluid into the vessels from the interstitial space ( Fig. 17.28 Hydrostatic Forces. Hydrostatic pressure (blood pressure) within capillaries is not constant. Instead, it depends on arterial and venous pressure and on precapillary resistance (in the arterioles) and postcapillary resistance (in the venules and small veins). An increase in arterial or venous pressure elevates capillary hydrostatic pressure, whereas a reduction in arterial or venous pressure has the opposite effect. An increase in arteriolar resistance or closure of arteries reduces capillary pressure, whereas a greater resistance to flow in venules and veins increases capillary pressure.

1	Hydrostatic pressure is the principal force in capillary filtration. A given change in Pv produces a greater effect on capillary hydrostatic pressure than does the same change in Pa. Approximately 80% of an increase in Pv is transmitted back to the capillaries. Capillary hydrostatic pressure (Pc) varies from tissue to tissue. Average values, obtained from direct measurements in human skin, are approximately 32 mm Hg at the arterial end of capillaries and approximately 15 mm Hg at the venous end of capillaries at the level of the heart (see Fig. 17.28 ). As discussed previously, when a person stands, hydrostatic pressure increases in the legs and decreases in the head. Tissue pressure, or, more specifically, interstitial fluid pressure (Pi) outside the capillaries, opposes capillary filtration. The difference between Pc and Pi constitutes the driving force for filtration. Normally, Pi is close to zero, and so Pc essentially represents the hydrostatic driving force.

1	Osmotic Forces. The key factor that restrains fluid loss from capillaries is the osmotic pressure of plasma proteins (such as albumin). This osmotic pressure is called colloid osmotic pressure or oncotic pressure (πp). The total osmotic pressure of plasma is approximately 6000 mm Hg (reflecting the presence of electrolytes and other small molecules, as well as plasma proteins), whereas oncotic pressure is only approximately 25 mm Hg. This low level of oncotic pressure is an important factor in fluid exchange across the capillary because plasma proteins are essentially confined to the intravascular space, whereas electrolytes are virtually equal in concentration on both sides of the capillary endothelium. The relative permeability of solute by water influences the actual magnitude of osmotic pressure. The reflection coefficient (σ) is the relative impediment to the passage of a substance through the capillary membrane. The reflection coefficient of water is 0, and that of albumin (to

1	pressure. The reflection coefficient (σ) is the relative impediment to the passage of a substance through the capillary membrane. The reflection coefficient of water is 0, and that of albumin (to which the endothelium is essentially impermeable) is 1. Filterable solutes have reflection coefficients between 0 and 1. In addition, different tissues have different reflection coefficients for the same molecule. Hence, movement of a given solute across the endothelial wall varies with the tissue. The actual oncotic pressure of the plasma (πp) is defined by the following equation (see also

1	Equation 17.19 1 µm 0.5 µm 0.1 µm0.1 µm GJTJ CT Nu PC V BM * V A B C D •Fig. 17.26 A, Electronmicrographofacross-sectionofacapillaryinamouseventricle.Theluminaldiameterisapproximately4µm.Inthissection,thecapillarywallisformedbyasingleendothelialcell(Nu,endothelialnucleus).Thethinpericapillaryspaceisoccupiedbyapericyte(PC)andaconnectivetissue(CT)cell(“fibroblast”),whichformsafunctionalcomplex(arrow)withitself.V,plasmalemmalvesicles.B, DetailoftheendothelialcellinA showingplasmalemmalvesicles(V)attachedtotheendothelialcellsurface.Thesevesiclesareespeciallyprominentinvascularendotheliumandareinvolvedintransportofsubstancesacrossthebloodvesselwall.Notethecomplexalveolarvesicle(asterisk). BM,basementmembrane.C, Junctionalcomplexinacapillaryofamouseheart.“Tight”junctions(TJ)typicallyforminthesesmallbloodvesselsandappeartoconsistoffusionsbetweenapposedendothelialcellsurfacemembranes.D,

1	BM,basementmembrane.C, Junctionalcomplexinacapillaryofamouseheart.“Tight”junctions(TJ)typicallyforminthesesmallbloodvesselsandappeartoconsistoffusionsbetweenapposedendothelialcellsurfacemembranes.D, Interendothelialjunctioninamusculararteryofapapillarymuscle.Althoughtightjunctionssimilartothoseofcapillariesarefoundintheselargebloodvessels,extensivejunctionsthatresemblegapjunctionsintheintercalateddisksbetweenmyocardialcellsoftenappearinarterialendothelium(exampleshownatGJ).

1	CHAPTER 17 Properties of the Vasculature 365 Junction of two Discontinuousendothelial cells endothelium • Fig. 17.27 Illustration of an electron micrograph of a capillary in cross-section. With prolonged standing, particularly when associated with elevation of Pv in the legs (such as that caused by pregnancy and congestive heart failure), filtration across capillaries is greatly enhanced, exceeding the capacity of the lymphatic system to remove the filtrate from the interstitial space and thus leading to edema.

1	The concentration of plasma proteins may change in several pathological states and thus alter the osmotic force and movement of fluid across the capillary membrane. The plasma protein concentration is increased in conditions of dehydration (e.g., water deprivation, prolonged sweating, severe vomiting, diarrhea). In this condition, less water moves by osmotic force from the tissues to the vascular compartment, thereby decreasing the volume of the interstitial fluid. In contrast, the plasma protein concentration is reduced in some renal diseases because of its loss in urine, and edema may occur. When capillary injury is extensive, as in severe burns, intravascular fluid and plasma protein leak into the interstitial space in the damaged tissues. The protein that escapes from the vessel lumen increases the oncotic pressure of the interstitial fluid. This greater osmotic force outside the capillaries leads to additional fluid loss and possibly to severe dehydration.

1	Balance of Hydrostatic and Osmotic Forces. The pressure sure and the role of these forces in regulating fluid passage across the capillary endothelium were expounded by Frank Starling in 1896. This relationship constitutes Starling’s • Fig. 17.28 Schematic representation of the factors responsible for filtration and absorption across the capillary wall and the formation of lymph.

1	Albumin is the most important plasma protein that determines oncotic pressure. Its molecular weight is 69,000 D. Albumin exerts an osmotic force greater than can be accounted for solely on the basis of its concentration in plasma. Therefore, it cannot be replaced on a mole-bymole basis by inert substances of appropriate molecular size, such as dextran. This additional osmotic force becomes disproportionately great at high concentrations of albumin (as in plasma), and this force is weak to absent in dilute solutions of albumin (as in interstitial fluid). The reason for this activity of albumin is its negative charge at normal blood pH and the attraction and retention of cations (principally Na+) in the vascular compartment (Gibbs-Donnan effect). hypothesis. It can be expressed as follows:

1	hypothesis. It can be expressed as follows: Equation 17.20 k = filtration constant for the capillary membrane πi= interstitial fluid oncotic pressure Filtration occurs when the algebraic sum is positive; absorption occurs when it is negative. Traditionally, filtration was thought to occur at the arterial end of the capillary, and absorption was thought to occur at its venous end because of the gradient of hydrostatic pressure along the capillary. This scheme is true for an idealized capillary (see

1	Fig. 17.28 ). However, in well-perfused capillaries, arteriolar vasoconstriction can reduce Pc in such a way that absorption at the arteriolar end can occur transiently. With continued vasoconstriction, absorption diminishes with time because Pi increases. In some vascular beds (e.g., the renal glomerulus), hydrostatic pressure in the capillary is high enough to cause filtration along the entire length of the capillary. In other vascular beds (e.g., the intestinal mucosa), the hydrostatic and oncotic forces are such that absorption occurs along the whole capillary.

1	In the steady-state Pa, Pv, postcapillary resistance, hydrostatic and oncotic pressure of interstitial fluid, and oncotic pressure of plasma are relatively constant. Hence, in the normal state, filtration and absorption across the capillary wall are well balanced. However, a change in precapillary resistance influences fluid movement across the capillary wall. Vasoconstriction reduces net filtration, and vasodilation increases filtration.

1	Inthelungs,meancapillaryhydrostaticpressureisonlyapproximately8mmHg(see ).Becauseplasmaoncoticpressureis25mmHgandpressureoftheinterstitialfluidinthelungsisapproximately15mmHg,thenetforceslightlyfavorsnetabsorption(i.e.,fluidleavestheinterstitialspace).Despitenetabsorption,pulmonarylymphisformed.Thislymphconsistsoffluidthatisosmoticallywithdrawnfromthecapillariesbythesmallamountofplasmaproteinthatescapesthroughthecapillaryendothelium.Inpathologicalconditions,suchasleftventricularfailureormitralvalvestenosis,pulmonarycapillaryhydrostaticpressuremayexceedplasmaoncoticpressure.Whenthisoccurs,itmaycausepulmonaryedema,aconditioninwhichexcessivefluidaccumulatesinthepulmonaryinterstitium.Thisfluidaccumulationseriouslyinterfereswithgasexchangeinthelungs.

1	Capillary Filtration Coefficient. The rate of fluid movement (Qf) across the capillary membrane depends not only on the algebraic sum of the hydrostatic and osmotic forces across the endothelium (ΔP) but also on the area (Am) of the capillary wall available for filtration, the distance (Δx) across the capillary wall, the viscosity (η) of the filtrate, and the filtration constant (k) of the membrane. These factors may be expressed as follows: Equation 17.21 This expression, which describes the flow of fluid through the membrane pores, is essentially Poiseuille’s law for flow through tubes. Because the thickness of the capillary wall and the viscosity of the filtrate are relatively constant, they can be included in the filtration constant k. If the area of the capillary membrane is not known, the rate of filtration can be expressed per unit weight of tissue. Hence, the equation can be simplified as

1	Equation 17.22 where kt is the capillary filtration coefficient for a given tissue and the units for Qf are milliliters per minute per 100 g of tissue. In any given tissue, the filtration coefficient per unit area of capillary surface, and hence capillary permeability, is not changed by various physiological conditions, such as arteriolar dilation and capillary distention, or by such adverse conditions as hypoxia, hypercapnia, or reduced pH. When capillaries are injured (as by toxins or severe burns), significant amounts of fluid and protein leak out of the capillaries into the interstitial space. This increase in capillary permeability is reflected by an increase in the filtration coefficient.

1	Because capillary permeability is constant under normal conditions, the filtration coefficient can be used to determine the relative number of open capillaries (i.e., the capillary surface area available for filtration in tissue). For example, the increased metabolic activity of contracting skeletal muscle relaxes the precapillary resistance vessels and hence opens more capillaries. This process, called capillary recruitment, increases the filtering surface area.

1	Disturbances in Hydrostatic-Osmotic Balance. Relatively small changes in Pa may have little effect on filtration. The change in pressure may be countered by adjustments in precapillary resistance vessels (autoregulation; see ) so that hydrostatic pressure remains constant in the open capillaries. However, a severe reduction in Pa usually evokes arteriolar constriction mediated by the sympathetic nervous system. This response may occur in hemorrhage, and it is often accompanied by a fall in Pv. These changes reduce capillary hydrostatic pressure. However, the lowering of blood pressure in hemorrhage causes a decrease in blood flow (and hence in O2 supply) to the tissue, with the result that vasodilator metabolites accumulate and relax the arterioles. Precapillary vessel relaxation also occurs because of the reduced transmural pressure (autoregulation; see ). Consequently, absorption predominates over filtration, and fluid moves from the interstitium into the capillary. These responses

1	because of the reduced transmural pressure (autoregulation; see ). Consequently, absorption predominates over filtration, and fluid moves from the interstitium into the capillary. These responses to hemorrhage constitute one of the compensatory mechanisms used by the body to restore blood volume (see

1	An increase in Pv alone, as occurs in the feet when a person stands up, would elevate capillary pressure and enhance filtration. However, the increase in transmural pressure closes precapillary vessels (myogenic mechanism; see ), and hence the capillary filtration coefficient actually decreases. This reduction in capillary surface available for filtration prevents large amounts of fluid from leaving the capillaries and entering the interstitial space.

1	In a healthy individual, the filtration coefficient (kt) for the whole body is approximately 0.006 mL/minute/100 g of tissue/mm Hg. For a 70-kg man, an elevation in Pv of 10 mm Hg for 10 minutes would increase filtration from capillaries by 420 mL. Edema does not usually occur because the fluid is returned to the vascular compartment by the lymphatic vessels. When edema develops, it usually appears in the dependent parts of the body, where the hydrostatic pressure is greatest, but its location and magnitude are also determined by the type of tissue. Loose tissues, such as the subcutaneous tissue around the eyes or in the scrotum, are more prone than firm tissues, as in a muscle, or encapsulated structures, as in a kidney, to collect larger quantities of interstitial fluid. Pinocytosis. Some transfer of substances across the capillary wall can occur in tiny pinocytotic vesicles. These vesicles (see

1	Pinocytosis. Some transfer of substances across the capillary wall can occur in tiny pinocytotic vesicles. These vesicles (see Figs. 17.26 17.27 ), formed by the pinching off of the endothelial cell membrane, can take up substances on one side of the capillary wall, move them across the cell by kinetic energy, and deposit their contents on the other side. This process is termed transcytosis. The amount of material transported in this way is very small in relation to that moved by diffusion. However, pinocytosis may be responsible for the movement of large (30-nm) lipid-insoluble molecules between blood and interstitial fluid. The number of pinocytotic vesicles in endothelium varies among tissues (amount in muscle > amount in lung > amount in brain), and the number increases from the arterial end to the venous end of the capillary.

1	The terminal vessels of the lymphatic system consist of a widely distributed, closed-end network of highly permeable lymphatic capillaries. These lymphatic capillaries resemble blood capillaries, with two important differences: tight junctions are not present between endothelial cells, and fine filaments anchor lymphatic vessels to the surrounding connective tissue. With muscular contraction, these fine strands pull on the lymphatic vessels to open spaces between the endothelial cells and enable the entrance of protein and large particles into the lymphatic vessels. The lymphatic capillaries drain into larger vessels that finally enter the right and left subclavian veins, where they connect with the respective internal jugular veins.

1	Only cartilage, bone, epithelia, and tissues of the central nervous system lack lymphatic vessels. These vessels return the plasma capillary filtrate to the circulation. This task is accomplished by means of tissue pressure, and it is facilitated by intermittent skeletal muscle activity, lymphatic vessel contractions, and an extensive system of one-way valves. In this regard, lymphatic vessels resemble veins, although the larger lymphatic vessels do have thinner walls than do the corresponding veins, and they contain only a small amount of elastic tissue and smooth muscle.

1	The volume of fluid transported through the lymphatic vessels in 24 hours is approximately equal to the body’s total plasma volume. The lymphatic vessels return all of the proteins filtered back to the blood; these proteins account for approximately one fourth to half of the circulating plasma proteins in the blood. The lymphatic vessels are the only means by which the protein that leaves the vascular compartment can be returned to blood. Net backward diffusion of protein into the capillaries cannot occur against the large protein concentration gradient. If the protein were not removed by the lymph vessels, it would accumulate in interstitial fluid and act as an oncotic force that draws fluid from the blood capillaries and produces edema. In addition to returning fluid and protein to the vascular bed, the lymphatic system filters the lymph at the lymph nodes and removes foreign particles such as bacteria. The

1	In addition to returning fluid and protein to the vascular bed, the lymphatic system filters the lymph at the lymph nodes and removes foreign particles such as bacteria. The CHAPTER 17 Properties of the Vasculature largest lymphatic vessel, the thoracic duct, not only drains the lower extremities but also returns the protein lost through the permeable liver capillaries. Moreover, the thoracic duct carries substances absorbed from the gastrointestinal tract. The principal substance is fat, in the form of chylomicrons.

1	Lymph flow varies considerably. The flow from resting skeletal muscle is almost nil, and it increases during exercise in proportion to the degree of muscular activity. It is increased by any mechanism that enhances the rate of blood capillary filtration; such mechanisms include increased capillary pressure or permeability and decreased plasma oncotic pressure. When the volume of interstitial fluid exceeds the drainage capacity of the lymphatic vessels, or when the lymphatic vessels become blocked, interstitial fluid accumulates and gives rise to clinical edema. Functional Anatomy of Coronary Vessels

1	Functional Anatomy of Coronary Vessels The right and left coronary arteries arise at the root of the aorta behind the right and left cusps of the aortic valve, respectively. These arteries provide the entire blood supply to the myocardium. The right coronary artery supplies mainly the right ventricle and atrium. The left coronary artery, which divides near its origin into the anterior descending and the circumflex branches, supplies mainly the left ventricle and atrium. There is some overlap between the regions supplied by the left and right arteries. In humans, the right coronary artery is dominant (supplying most of the myocardium) in approximately 50% of individuals. The left coronary artery is dominant in another 20%, and the flow delivered by each main artery is approximately equal in the remaining 30%. The epicardial distribution of the coronary arteries and veins is illustrated in Fig. 17.29

1	Coronary arterial blood passes through the capillary beds; most of it returns to the right atrium through the coronary sinus. Of the coronary arteries, epicardial arteries are largest (2 to 5 mm in diameter), large arterioles are medium in size (1.0 to 0.5 mm in diameter), and small arterioles are smallest (<0.1 mm in diameter). Some of the coronary venous blood reaches the right atrium via the anterior coronary veins. In addition, vascular communications directly link the myocardial vessels with the cardiac chambers; these communications are the arteriosinusoidal, arterioluminal, and thebesian vessels. The arteriosinusoidal channels consist of small arteries or arterioles that lose their arterial structure as they penetrate the chamber walls, where they divide into irregular, endothelium-lined sinuses. These sinuses anastomose with other sinuses and with capillaries, and they communicate with the cardiac chambers. The arterioluminal vessels are small arteries or arterioles that open

1	sinuses. These sinuses anastomose with other sinuses and with capillaries, and they communicate with the cardiac chambers. The arterioluminal vessels are small arteries or arterioles that open directly into the atria and ventricles. The thebesian vessels are small veins that connect capillary beds directly with the cardiac chambers and also communicate with the cardiac veins. All the minute vessels of the myocardium communicate in the form of an extensive

1	Posterior descending branch of right coronary artery • Fig. 17.29 Illustrations of the anterior and posterior surfaces of the heart, depicting the location and distribution of the principal coronary vessels. plexus of subendocardial vessels. However, the myocardium does not receive significant nutritional blood flow directly from the cardiac chambers. The primary factor responsible for perfusion of the myocardium is aortic pressure. Changes in aortic pressure generally evoke parallel directional changes in coronary blood flow. This is caused in part by changes in coronary perfusion pressure. However, the major factor in the regulation of coronary blood flow is a change in arteriolar resistance engendered by changes in the metabolic activity of the heart. When the metabolic activity of the heart increases, coronary resistance decreases; when cardiac metabolism decreases, coronary resistance increases (see

1	Blood flow in the heart is autoregulated. If a cannulated coronary artery is perfused by blood from a pressure-controlled reservoir, perfusion pressure can be altered without a change in aortic pressure and cardiac work. The relationship between initial and steady-state blood flow is shown in the experiment depicted in Fig. 17.30 . This is an example of autoregulation of blood flow, which is mediated by a myogenic mechanism in large and small arterioles ( ). The metabolic activity of cardiac muscle in small arterioles and the endothelium modulate autoregulation. The coronary circulation adjusts serial resistances within the microvasculature thereby adapting blood flow to O2 requirements. Blood pressure is kept within narrow 0.2 0.4 0.6 0.8 1 Time (sec) •Fig. 17.30 Pressure-Flow Relationships in the Coronary Vascular

1	Bed. Asaorticpressurewasheldconstant,thecardiacoutput,heart rate,andcoronaryarteryperfusionpressurewereabruptlyincreased ordecreasedfromthecontrollevel,whichisindicatedbythepointatwhichthetwolinescross.Thesolid circles representtheflowsthat resultedimmediatelyafterthechangeinperfusionpressure;theopen circles representthesteady-stateflowsatthenewpressures.Thereisatendencyforflowtoreturntowardthecontrollevel(autoregulationofbloodflow),andthisismostprominentovertheintermediatepressurerange(≈60to180mmHg).(FromBerneRM,RubioR.Coronarycirculation.InPageE,ed.Handbook of Physiology: Section 2: The Cardiovascular System: The Heart. Vol1.Bethesda,MD:AmericanPhysiologicalSociety;1979.) limits by baroreceptor reflex mechanisms. Hence, changes in coronary blood flow are caused mainly by changes in the diameter of coronary resistance vessels in response to the metabolic demands of the heart.

1	•Fig. 17.31 Comparison of Phasic Coronary Blood Flow in the Left and Right Coronary Arteries. Extravascularcompressionissogreatduringearlyventricularsystolethatthedirectionofbloodflowinthelargecoronaryarteriessupplyingtheleftventricleisbrieflyreversed.Maximalinflowintheleftcoronaryarteryoccursinearlydiastole,whentheventricleshaverelaxedandextravascularcompressionofthecoronaryvesselsisvirtuallyabsent.Afteraninitialreversalinearlysystole,bloodflowintheleftcoronaryarteryfollowstheaorticpressureuntilearlydiastole,whenitrisesabruptlyandthendeclinesslowlyasaorticpressurefallsduringtheremainderofdiastole. In addition to providing the pressure to move blood through the coronary vessels, the heart also affects its blood supply by the squeezing effect (extravascular compression) of the contracting myocardium on its own blood vessels. The patterns of flow in the left and right coronary arteries are shown in

1	Fig. 17.31 . In the left ventricle, coronary perfusion pressure is the difference between aortic diastolic pressure and left ventricular end-diastolic pressure. Left ventricular myocardial pressure (pressure within the wall of the left ventricle) is highest near the endocardium and lowest near the epicardium. This pressure gradient does not normally impair endocardial blood flow because the greater blood flow to the endocardium during diastole compensates for the greater blood flow to the epicardium during systole. Measurements of coronary blood flow indicate that the epicardial and endocardial halves of the left ventricle receive approximately equal blood flow under normal conditions. Because extravascular compression is greatest at the endocardial surface of the ventricle, the equality of epicardial and endocardial blood flow indicates that the tone of the endocardial resistance vessels is less than that of the epicardial vessels.

1	Theminimalextravascularresistanceandabsenceofleftventricularworkduringdiastolecanbeusedtoimprovemyocardialperfusioninpatientswithadamagedmyocardiumandlowbloodpressure.Inamethodcalledcounterpulsation, aninflatableballoonisinsertedintothethoracicaortathroughafemoralartery.Theballoonisinflatedduringeachventriculardiastoleanddeflatedduringeachsystole.Thisprocedureenhancescoronarybloodflowduringdiastolebyraisingdiastolicpressureatatimewhencoronaryextravascularresistanceislowest.Furthermore,itreducescardiacenergyrequirementsbyloweringaorticpressure(afterload)duringventricularejection. The flow pattern in the right coronary artery is similar to that in the left coronary artery (see

1	The flow pattern in the right coronary artery is similar to that in the left coronary artery (see Fig. 17.31 ). In contrast to the left ventricle, reversal of blood flow does not occur in the right ventricle in early systole because pressure in the thin right ventricle is lower during systole. Hence, systolic blood flow constitutes a much greater proportion of total coronary inflow than it does in the left coronary artery.

1	•Fig. 17.32 A, Unmaskingoftherestrictingeffectofventricularsystoleonmeancoronarybloodflowbyinductionofventricularfibrillationduringperfusionoftheleftcoronaryarteryatconstantpressure.Withtheonsetofventricularfibrillation,coronarybloodflowincreasesabruptlybecauseextravascularcompressionisremoved.Flowthengraduallyreturnstowardandoftenfallsbelowtheprefibrillationlevel.Thisincreaseincoronaryresistancethatoccursdespitetheremovalofextravascularcompressiondemonstratestheheart’sabilitytoadjustitsbloodflowtomeetitsenergyrequirements.B, EffectofcardiacsympatheticnervestimulationoncoronarybloodflowandonbloodO2tension(PO2)inthecoronarysinusinafibrillatingheartduringperfusionoftheleftcoronaryarteryatconstantpressure.(BerneRM.Unpublishedobservations.) The extent to which extravascular compression restricts coronary inflow can be readily observed when the heart is suddenly arrested in diastole or with the induction of ventricular fibrillation.

1	The extent to which extravascular compression restricts coronary inflow can be readily observed when the heart is suddenly arrested in diastole or with the induction of ventricular fibrillation. Fig. 17.32A depicts mean left coronary flow when the vessel was perfused with blood at a constant pressure from a reservoir. When ventricular fibrillation was electrically induced, blood flow increased immediately and substantially. A subsequent increase in coronary resistance over a period of many minutes reduced myocardial blood flow to below the level that existed before induction of ventricular fibrillation (see Fig. 17.32B , just before stellate ganglion stimulation).

1	Fig. 17.32B , just before stellate ganglion stimulation). When diastolic pressure in the coronary arteries is abnormally low (as in severe hypotension, partial coronary artery occlusion, or severe aortic stenosis), the ratio of endocardial to epicardial blood flow falls below a value of 1. This ratio indicates that blood flow to the endocardial regions is more severely impaired than that to the epicardial regions of the ventricle. There is also an increase in the gradient of myocardial lactic acid and myocardial adenosine concentrations from epicardium to endocardium. For this reason, the myocardial damage observed in atherosclerotic heart disease (e.g., after coronary occlusion) is greatest in the inner wall of the left ventricle.

1	Tachycardia and bradycardia have dual effects on coronary blood flow. A change in heart rate mainly alters diastole. In tachycardia, the proportion of time spent in systole, and consequently the period of restricted inflow, increases. However, this mechanical effect is overridden by the dilation of coronary resistance vessels associated with the increased metabolic activity of the more rapidly beating heart. With bradycardia, the opposite occurs: Coronary inflow is less restricted (more time spent in diastole), but so are the metabolic (O2) requirements of the myocardium.

1	Stimulation of cardiac sympathetic nerves markedly increases coronary blood flow. However, the increase in flow is associated with an increased heart rate and more forceful systole. The stronger contraction and the tachycardia tend to restrict coronary flow. The increase in myocardial metabolic activity, however, tends to dilate coronary resistance vessels. The increase in coronary blood flow evoked by cardiac sympathetic nerve stimulation reflects the sum of these factors. In perfused hearts in which the mechanical effect of extravascular compression is eliminated by cardiac arrest or by ventricular fibrillation, an initial coronary vasoconstriction of the coronary vessels is often observed. After this initial vasoconstriction, the metabolic effect evokes vasodilation (see Fig. 17.32B

1	Fig. 17.32B Furthermore, when β-adrenergic receptor blockade eliminates the positive chronotropic and inotropic effects, activation of the cardiac sympathetic nerves increases coronary resistance. These observations indicate that the direct action of the sympathetic nerve fibers on the coronary resistance vessels is vasoconstriction. Both α-adrenergic receptors (constrictors) and β2adrenergic receptors (dilators) are present on the coronary vessels. Coronary resistance vessels also participate in the baroreceptor and chemoreceptor reflexes, and the sympathetic constrictor tone of the coronary arterioles can be modulated by such reflexes. Nevertheless, coronary resistance is predominantly under local nonneural control.

1	Vagus nerve stimulation causes slight dilation of the coronary resistance vessels, and activation of the carotid and aortic chemoreceptors can cause a slight decrease in coronary resistance via the vagus nerves to the heart. Failure of strong vagal stimulation to increase coronary blood flow is not due to lack of muscarinic receptors on the coronary resistance vessels because intracoronary administration of acetylcholine elicits marked vasodilation. In the human heart, acetylcholine caused vasodilation when administered directly into the left anterior descending coronary artery of subjects with no evidence of coronary artery disease. However, acetylcholine caused vasoconstriction in the coronary artery of subjects whose endothelium had been damaged and rendered dysfunctional by atherosclerosis. A striking characteristic of the coronary circulation is the close relationship between the level of myocardial metabolic activity and the magnitude of coronary blood flow (Fig.

1	A striking characteristic of the coronary circulation is the close relationship between the level of myocardial metabolic activity and the magnitude of coronary blood flow (Fig. 17.33 ). This relationship is also found in a denervated heart and in a completely isolated heart, either in the beating state or in the fibrillating state. Ventricles can fibrillate for many hours when the coronary arteries are perfused with arterial blood from some external source. As already noted, a fibrillating heart uses less O2 than a pumping heart does, and blood flow to the myocardium is reduced accordingly.

1	The mechanisms that link the cardiac metabolic rate and coronary blood flow remain unsettled. However, it appears that a decrease in the ratio of O2 supply to O2 demand releases vasodilator substances from the myocardial cells into the interstitial fluid, where they relax the coronary resistance vessels. Decreases in arterial blood O2 content or in coronary blood flow and increases in metabolic rate all decrease the O2 supply/demand ratio ( Fig. 17.34 ). As a consequence, substances are released that dilate the arterioles and thereby adjust the O2 supply to the O2 demand. A decrease in O2 demand diminishes the release of vasodilators and enables greater expression of basal tone.

1	Numerous metabolites participate in the vasodilation that accompanies increased cardiac work. Accumulation of vasoactive metabolites can also account for the increase in blood flow that results from a brief period of ischemia (i.e., reactive hyperemia; see ). The duration of the enhanced coronary flow after release of the briefly occluded vessel is, within certain limits, proportional to the duration of the period of occlusion. Among the factors implicated in reactive hyperemia are ATP-sensitive potassium (KATP) channels, NO, CO2, H+, K+, hypoxia, H2O2, and adenosine.

1	Of these agents, the key factors appear to be adenosine, NO, opening of the KATP channels, and H2O2. The contributions of each of these agents and their interaction under basal conditions and during increased myocardial •Fig. 17.33 RelationshipbetweenmyocardialO2consumptionandcoronarybloodflowduringavarietyofinterventionsthatincreaseordecreasethemyocardialmetabolicrate.(FromBerneRM,RubioR:Coronarycirculation.InPageE,ed.Handbook of Physiology: Section 2: The Cardiovascular System: The Heart. Vol1.Bethesda,MD:AmericanPhysiologicalSociety;1979.)18 16 14 12 10 8 6 4 2 20 30 40 50 60 70 80 90 100 110 120 Coronary blood flow (mL/min/100 g) Myocardial oxygen consumption(mL/min/100 g) •Fig. 17.34 ImbalanceintheO2supply–O2demandratioalterscoronarybloodflowbytherateofreleaseofavasodilatormetabolitefromcardiomyocytes.Adecreaseintheratioelicitsanincreaseinvasodilatorrelease,whereasanincreaseintheratiohastheoppositeeffect.

1	372 SECTION4Berne & Levy Physiology •Fig. 17.35 Schematic Representation of Factors That Increase (+) or Decrease (−) Coronary Vascular Resistance. Intravascularpressure(arterialbloodpressure)stretchesthevesselwall.KATPchannels,adenosinetriphosphate–sensitivepotassiumchannels;NO,nitricoxide;PCO2,partialpressureofcarbondioxide;PO2,partialpressureofoxygen.˜ Myocardial contractile activity Autonomic control Sympathetic nerves: °-Receptors ˛-Receptors Vagus nerves Oxygen supply Myogenic mechanism ˜ Metabolism ˝ Myocardial PO2 KATP channels H2O2 NO Adenosine – + +± + – – – Other metabolic factors PO2, PCO2, H+, K+ Systolic compression activity are complex. A reduction in oxidative metabolism in vascular smooth muscle reduces ATP synthesis, which in turn opens KATP channels and causes hyperpolarization. This change in potential reduces entry of Ca++ and relaxes coronary vascular smooth muscle to increase flow. A reduction in ATP also opens KATP channels in cardiac muscle and generates an

1	This change in potential reduces entry of Ca++ and relaxes coronary vascular smooth muscle to increase flow. A reduction in ATP also opens KATP channels in cardiac muscle and generates an outward current that reduces action potential duration and limits Ca++ entry during phase 2 of the action potential. This action may be protective during periods of imbalance between O2 supply and demand. In addition, as cardiac work increases, H2O2 production rises, which activates Kv1.5 channels and thereby causes hyperpolarization of muscle membrane and relaxation of vascular smooth muscle. Moreover, the release of NO and adenosine dilates the arterioles and thereby adjusts the O2 supply to the O2 demand. At low concentrations, adenosine appears to activate endothelial KATP channels and to enhance release of NO. Conversely, at higher concentrations, adenosine acts directly on vascular smooth muscle by activating KATP channels. Decreased O2 demand would sustain the ATP level, as well as reduce the

1	of NO. Conversely, at higher concentrations, adenosine acts directly on vascular smooth muscle by activating KATP channels. Decreased O2 demand would sustain the ATP level, as well as reduce the amount of vasodilator substances released, and allows greater expression of basal tone. If production of all these agents is inhibited, coronary blood flow is reduced, both at rest and during exercise. Furthermore, contractile dysfunction and signs of myocardial ischemia become evident.

1	According to the adenosine hypothesis, a reduction in myocardial O2 tension produced by inadequate coronary blood flow, hypoxemia, or increased metabolic activity of the heart leads to release of adenosine from the myocardium. Adenosine enters the interstitial fluid space to reach the coronary resistance vessels and induces vasodilation by activating adenosine receptors. However, it cannot be responsible for the increased coronary flow observed during prolonged enhancement of cardiac metabolic activity because release of adenosine from cardiac muscle is transitory. Factors that alter coronary vascular resistance are illustrated in Fig. 17.35 Effects of Diminished Coronary Blood Flow

1	Fig. 17.35 Effects of Diminished Coronary Blood Flow Most of the O2 in coronary arterial blood is extracted during one passage through the myocardial capillaries. Thus the supply of O2 to myocardial cells is flow limited; any substantial reduction in coronary blood flow curtails O2 delivery to the myocardium because O2 extraction is nearly maximal even when blood flow is normal.

1	A reduction in coronary flow that is neither too prolonged nor too severe to induce myocardial necrosis can nonetheless cause substantial (but temporary) dysfunction of the heart. A relatively brief period of severe ischemia followed by reperfusion can result in pronounced mechanical dysfunction (myocardial stunning). However, the heart eventually recovers fully from the dysfunction. The pathophysiological basis for myocardial stunning appears to be intracellular Ca++ overload, initiated during the period of ischemia, combined with the generation of OH− and superoxide free radicals early in the period of reperfusion. These changes impair the responsiveness of myofilaments to Ca++ .

1	In the normal human heart, there are virtually no functional intercoronary channels. Abrupt occlusion of a coronary artery or one of its branches leads to ischemic necrosis and eventual fibrosis of the areas of myocardium supplied by the occluded vessel. However, if a coronary artery narrows slowly and progressively over a period of days or weeks, collateral vessels develop and may furnish sufficient blood to the ischemic myocardium to prevent or reduce the extent of necrosis. Collateral vessels may develop between branches of occluded and nonoccluded arteries. They originate from preexisting small vessels that undergo proliferative changes of the endothelium and smooth muscle. These changes may occur in response to wall stress and to chemical agents, including vascular endothelial growth factors (VEGFs) released by the ischemic tissue. The VEGFs, of which there are at least five in mammals, are glycoproteins. The VEGFs induce angiogenesis, elicit vasodilation and increase endothelial

1	factors (VEGFs) released by the ischemic tissue. The VEGFs, of which there are at least five in mammals, are glycoproteins. The VEGFs induce angiogenesis, elicit vasodilation and increase endothelial permeability. By causing vasodilation, VEGFs enable perfusion of more capillaries and increase capillary permeability by opening tight junctions between endothelial cells and by adding fenestrations.

1	Myocardial stunning, prolongedventriculardysfunctionwithoutmyocardialnecrosis,maybeevidentinpatientswhohavesufferedanacutecoronaryarteryocclusion.Ifthepatientistreatedsufficientlyearlybycoronarybypasssurgeryorballoonangioplasty,andifadequatebloodflowisrestoredtotheischemicregion,themyocardialcellsinthisregionmayrecoverfully.However,formanydaysorevenweeks,thecontractilityofthemyocardiumintheaffectedregionmaybegrosslysubnormal.

1	Prolongedreductionsincoronarybloodflow(myocardialischemia)maycriticallyandpermanentlyimpairthemechanicalandelectricalbehavioroftheheart.Diminishedcoronarybloodflowasaconsequenceofcoronaryarterydisease(usuallycoronaryatherosclerosis)isoneofthemostcommoncausesofseriouscardiacdisease.Theischemiamaybeglobal(affectsanentireventricle)orregional(affectssomefractionoftheventricle).TheimpairmentinmechanicalcontractionoftheaffectedmyocardiumisproducednotonlybythediminisheddeliveryofO2andmetabolicsubstratesbutalsobytheaccumulationofpotentiallyharmfulsubstances(e.g.,K+ ,lacticacid,H+)inthecardiactissues.Ifthereductionincoronaryflowtoanyregionoftheheartissufficientlysevereandprolonged,necrosisoftheaffectedcardiaccellsresults.

1	Myocardial hibernation describesthephenomenoninwhichcellularmetabolismisdownregulatedincellswhosefunctionisimpairedbyinadequatedeliveryofO2andnutrients.Myocardialhibernationoccursmainlyinpatientswithcoronaryarterydisease.Thecoronarybloodflowinsuchpatientsisdiminishedpersistentlyandsignificantly,andthemechanicalfunctionoftheheartisimpaired.Ifcoronarybloodflowisrestoredtonormalbybypasssurgeryorangioplasty,mechanicalfunctionreturnstonormal. The O2 and nutrient requirements of the skin are relatively small. Unlike other body tissues, the supply of O2 and nutrients is not the chief factor in the regulation of cutaneous blood flow. The primary function of the cutaneous circulation is to maintain a constant body temperature. Thus the skin undergoes wide fluctuations in blood flow, depending on whether the body needs to lose or conserve heat. Changes in ambient and internal body temperature activate mechanisms responsible for alterations in skin blood flow.

1	Numeroussurgicalattemptshavebeenmadetoenhancethedevelopmentofcoronarycollateralvessels.However,thetechniquesuseddonotincreasethecollateralcirculationoverandabovethatproducedbycoronaryarterynarrowingalone.Whendiscreteocclusionsorseverenarrowingoccursincoronaryarteries,asincoronaryatherosclerosis,thelesionscanbebypassedwithanarteryoraveingraft.Frequently,thenarrowsegmentcanbedilatedbyinsertionofaballoon-tippedcatheterintothediseasedvesselviaaperipheralarteryandtheninflationoftheballoon.Distentionofthevesselbyballooninflation(angioplasty)canproducelastingdilationofanarrowed coronaryartery(Fig.17.36 ),particularlywhenadrug-elutingstent(thedrugshelppreventrestenosis)isinsertedduringangioplasty.

1	Manydrugsareavailableforuseinpatientswithcoronaryarterydiseasetorelieveanginapectoris,thechestpainassociatedwithmyocardialischemia.Thesecompoundsincludeorganicnitrates/nitrites,calciumchannelantagonists,andβ-adrenoceptorantagonists.Organicnitrates/nitritesaremetabolizedtoNO.NOdilatesthegreatveinstoreducevenousreturn(preload),therebyreducingcardiacwork(see )andmyocardialO2requirements.Inaddition,NOdilatesthecoronaryarteriestoincreasecollateralflow.Ofimportanceisthatorganicnitrates/nitritesdonotinterferewithcoronaryautoregulation.Calciumchannelantagonistsalsocausevasodilation;noneselectivelydilatesthecoronaryvessels.Theβ-adrenoceptorantagonistsreducetheheartratetoindirectlyincreasecoronaryflowandopposethereflextachycardiathathasbeenobservedwithorganicnitrates/nitrites.

1	Inpatientswithmarkednarrowingofacoronaryartery,administrationofdipyridamole,avasodilator,canfullydilatenormalvesselbranchesthatareparalleltothenarrowedsegmentandtherebyreducethepressureonthepartiallyoccludedvessel.Thereducedpressureonthenarrowedvesselfurthercompromisesbloodflowtotheischemicmyocardium.Thisphenomenon,knownascoronary steal, occursbecausedipyridamoleactsbyblockingthecellularuptakeandmetabolismofendogenousadenosine.Ofnoteisthatdipyridamoleinterfereswithcoronaryautoregulation. Regulation of Skin Blood Flow

1	Regulation of Skin Blood Flow The skin contains essentially two types of resistance vessels: arterioles and arteriovenous anastomoses. AV anastomoses shunt blood from the arterioles to the venules and venous plexuses; hence, they bypass the capillary bed. Such anastomoses are found in the fingertips, palms of the hands, toes, soles of the feet, ears, nose, and lips. AV anastomoses differ morphologically from arterioles; the anastomoses are either short and straight or long coiled vessels, approximately 20 to 40 µm in luminal diameter, and they have thick muscular walls richly supplied with nerve fibers ( Fig. 17.37 ). These vessels are almost exclusively under sympathetic neural control, and they dilate maximally when their nerve

1	Fig. 17.37 ). These vessels are almost exclusively under sympathetic neural control, and they dilate maximally when their nerve Fig. 17.36 A, Angiogram(withintracoronaryradiopaquedye)ofmarkednarrowingoftheleftanteriordescendingbranchoftheleftcoronaryartery(white arrow). B, Thesamesegmentofthecoronaryartery(black arrow) afterangioplastyandinsertionofadrug-elutingstent.(CourtesyofDr.MichaelAzrin.)

1	Fig. 17.37 Arteriovenous (AV) Anastomosis in the Ear Injected With Berlin Blue Dye. A,artery;V,vein;arrowpointstoanAVanasto-mosis.ThewallsoftheAVanastomosisinthefingertipsarethickerandmorecellular.(FromPritchardMML,DanielPM.J Anat. 1956;90:309.) supply is interrupted. Conversely, reflex stimulation of the sympathetic fibers to these vessels may constrict them and obliterate the vascular lumen. Although AV anastomoses do not exhibit basal tone, they are highly sensitive to vasoconstrictor agents such as epinephrine and norepinephrine. Furthermore, AV anastomoses are not under metabolic control, and they do not show reactive hyperemia or auto-regulation of blood flow. Thus regulation of blood flow through these anastomotic channels is governed principally by the nervous system in response to reflex activation by temperature receptors or from higher centers of the central nervous system.

1	Most of the resistance vessels in the skin exhibit some basal tone and are under dual control of the sympathetic nervous system and local regulatory factors. However, neural control predominates. Stimulation of sympathetic nerve fibers Thefingersandtoesofsomeindividualsareverysensitivetocold.Onexposuretocold,thearteriolestothefingersandtoesconstrict.Theconsequentischemiaresultsinlocalizedblanchingoftheskinassociatedwithtingling,numbness,andpain.Theblanchingisfollowedbycyanosis(adarkbluecoloroftheskin)andlaterbyrednessasthearterialspasmsubsides.Thecauseofthiscondition,calledRaynaud’sdisease,isunknown.

1	induces vasoconstriction, and cutting of the sympathetic nerves induces vasodilation. After chronic denervation of the cutaneous blood vessels, the degree of tone that existed before denervation is gradually regained over a period of several weeks. This restoration of tone is accomplished by an enhancement of basal tone. Denervation of the skin vessels results in enhanced sensitivity to catecholamines in circulation (denervation hypersensitivity). Parasympathetic vasodilator nerve fibers do not innervate cutaneous blood vessels. However, stimulation of the sweat glands, which are innervated by sympathetic cholinergic fibers, dilates the skin resistance vessels. Sweat contains an enzyme that lyses a protein (kallidin) in the tissue fluid to produce bradykinin, a polypeptide with potent vasodilator properties. Bradykinin, formed locally, dilates the arterioles and increases blood flow to the skin.

1	Certain skin vessels, particularly those in the head, neck, shoulders, and upper part of the chest, are regulated by higher centers in the brain. Blushing, in response to embarrassment or anger, and blanching, in response to fear or anxiety, are examples of cerebral inhibition and stimulation, respectively, of the sympathetic nerve fibers to the affected cutaneous regions. In contrast to AV anastomoses in the skin, the resistance vessels display autoregulation of blood flow and reactive hyperemia. If the arterial inflow to a limb is stopped briefly by inflation of a blood pressure cuff, the skin becomes bright red below the point of vascular occlusion when the cuff is subsequently deflated. The increased cutaneous blood flow (reactive hyperemia) is also manifested by distention of the superficial veins in the affected extremity. The Role of Temperature in the Regulation of Skin Blood Flow

1	The primary function of the skin is to maintain a constant internal environment and protect the body from adverse changes. Ambient temperature is one of the most important external variables with which the body must contend. Exposure to cold elicits a generalized cutaneous vasoconstriction that is especially pronounced in the hands and feet. This response is chiefly mediated by the nervous system. Arrest of the circulation to a hand by a pressure cuff plus immersion of that hand in cold water induces vasoconstriction in the skin of the other extremities that are exposed to room temperature. When the circulation to the chilled hand is not occluded, the reflex-generalized vasoconstriction is caused in part by the cooled blood that returns to the general circulation. This returned blood then stimulates the temperature-regulating center in the anterior hypothalamus, which then activates heat preservation centers in the posterior hypothalamus to evoke cutaneous vasoconstriction.

1	The skin vessels of the cooled hand also respond directly to cold. Moderate cooling or a brief exposure to severe cold (0°C to 15°C) constricts the resistance and capacitance vessels, including the AV anastomoses. Prolonged exposure to severe cold evokes a secondary vasodilator response. Prompt vasoconstriction and severe pain are elicited by immersion of the hand in ice water. However, this response is soon followed by dilation of the skin vessels, with reddening of the immersed part and alleviation of the pain. With continued immersion of the hand, alternating periods of constriction and dilation occur, but the skin temperature rarely drops as much as it did in response to the initial vasoconstriction. Prolonged severe cold, of course, damages tissue. The rosy faces of people exposed to a cold environment are examples of cold-induced vasodilation. However, blood flow through the skin of the face may be greatly reduced despite the flushed appearance. The red color of the slowly

1	to a cold environment are examples of cold-induced vasodilation. However, blood flow through the skin of the face may be greatly reduced despite the flushed appearance. The red color of the slowly flowing blood is mainly caused by reduced O2 uptake by the cold skin and the cold-induced shift of the oxyhemoglobin dissociation curve to the left (see

1	Direct application of heat to the skin not only dilates the local resistance and capacitance vessels and the AV anastomoses but also reflexively dilates blood vessels in other parts of the body. The local effect is independent of the vascular nerve supply, whereas the reflex vasodilation is a combined response to stimulation of the anterior hypothalamus by the returning warmed blood and stimulation of cutaneous heat receptors in the heated regions of the skin.

1	The close proximity of the major arteries and veins allows countercurrent heat exchange between them. Cold blood that flows in veins from a cooled hand toward the heart takes up heat from adjacent arteries; this warms the venous blood and cools the arterial blood. Heat exchange takes place in the opposite direction when the extremity is exposed to heat. Thus heat conservation is enhanced during exposure of extremities to cold environments, and heat conservation is minimized during exposure of the extremities to warm environments. Skin Color: Relationship to Skin Blood Volume, Oxyhemoglobin, and Blood Flow

1	Skin Color: Relationship to Skin Blood Volume, Oxyhemoglobin, and Blood Flow Skin color is determined mainly by the pigment content. However, the degree of pallor or ruddiness is mainly a function of the amount of blood in the skin, except when the skin is very dark. With little blood in the venous plexus, the skin appears pale, whereas with moderate to large quantities of blood in the venous plexus, the skin displays a color. This color may be red, blue, or some shade between, depending CHAPTER 17 Properties of the Vasculature on the degree of oxygenation of the blood. A combination of vasoconstriction and reduced hemoglobin can impart an ashen gray color to the skin. A combination of venous engorgement and reduced hemoglobin content can impart a dark purple hue.

1	Skin color provides little information about the rate of cutaneous blood flow. Rapid blood flow may be accompanied by skin pallor when the AV anastomoses are open, and slow blood flow may be associated with skin ruddiness when the skin is exposed to cold. The rate of blood flow in skeletal muscle varies directly with the contractile activity of the tissue and the type of muscle. Blood flow and capillary density are greater in red muscle (slow-twitch muscle with high oxidative capacity) than in white muscle (fast-twitch muscle with low oxidative capacity). In resting muscle, the precapillary arterioles contract and relax intermittently. Thus at any given moment, most of the capillary bed is not perfused, and total blood flow through quiescent skeletal muscle is low (1.4 to 4.5 mL/ minute/100 g). During exercise, the resistance vessels relax, and muscle blood flow may increase to 15 to 20 times the resting level, depending on the intensity of the exercise.

1	Regulation of Skeletal Muscle Blood Flow Neural and local factors regulate muscle circulation. Physical factors such as Pa, tissue pressure, and blood viscosity influence muscle blood flow. However, another physical factor, the squeezing effect of the active skeletal muscle, affects blood flow in the vessels. With intermittent contractions, inflow is restricted, and as previously described, venous outflow is enhanced. The venous valves prevent backward flow of blood between contractions and thereby aid in the forward propulsion of blood. With strong sustained contractions, as occur during exercise, the vascular bed can be compressed to the point at which blood flow actually ceases temporarily.

1	The resistance vessels of muscle possess a high degree of basal tone; they also display tone in response to continuous low-frequency activity in the sympathetic vasoconstrictor nerve fibers. The basal firing frequency of sympathetic vasoconstrictor fibers is only approximately 1 to 2 per second, and maximal vasoconstriction occurs at frequencies of approximately 10 per second. Vasoconstriction evoked by sympathetic nerve activity is caused by the local release of norepinephrine. Intra-arterially injected norepinephrine elicits only vasoconstriction (α1adrenergic receptor). In contrast, low doses of epinephrine produce vasodilation (β2-adrenergic receptor), whereas large doses cause vasoconstriction. Baroreceptor reflexes greatly influence the tonic activity of the sympathetic nerves. An increase in carotid sinus pressure causes the muscle vascular bed to dilate, whereas a decrease in carotid sinus pressure elicits vasoconstriction (

1	Fig. 17.38 ). When sympathetic constrictor tone is high, the decrease in blood flow evoked by common carotid artery occlusion is small, but the increase in flow after the release of occlusion is large. The vasodilation produced by baroreceptor stimulation is caused by inhibition of sympathetic vasoconstrictor activity. The resistance vessels in skeletal muscle contribute significantly to maintenance of arterial blood pressure because skeletal muscle constitutes a large fraction of the body’s mass, and the muscle vasculature thus constitutes the largest vascular bed. Participation of the skeletal muscle vessels in vascular reflexes is important in maintaining normal arterial blood pressure. A comparison of the sympathetic neural effects on the blood vessels of muscle and skin is summarized in Fig. 17.39. Note that the lower the basal tone of the skin vessels, the greater their constrictor response; also note the absence of active cutaneous vasodilation.

1	Fig. 17.39. Note that the lower the basal tone of the skin vessels, the greater their constrictor response; also note the absence of active cutaneous vasodilation. In active skeletal muscle, blood flow is regulated by meta- bolic factors. In resting muscle, neural factors predominate, Fig. 17.39 ). Cutting of the sympathetic nerves to muscle abolishes the neural component of vascular tone, and it unmasks the intrinsic basal tone of the blood vessels. The neural and local mechanisms that regulate blood flow oppose each other, and during muscle contraction, the local vasodilator mechanism supervenes. However, during exercise, strong sympathetic nerve stimulation slightly attenuates the vasodilation induced by locally released metabolites.

1	•Fig. 17.38 Evidence of Participation of the Muscle Vascular Bed in Vasoconstriction and Vasodilation Mediated by the Carotid Sinus Baroreceptors After Occlusion and Release of the Common Carotid Artery. Inthispreparation,thesciaticandfemoralnervesconstitutedtheonlydirectinnervationofthehindlegmusclemass.Themusclewasperfusedwithbloodataconstantpressure.(RedrawnfromJonesRD,BerneRM.Am J Physiol. 1963;204:461.)

1	Blood reaches the brain through the internal carotid and vertebral arteries. The vertebral arteries join to form the basilar artery, which, in conjunction with branches of the •Fig. 17.39 Basaltoneandtherangeofresponseofresistancevesselsinmuscle(dashed lines) andskin(shaded areas) tostimulationandsectionofsympatheticnerves.Peripheralresistanceisplottedonalogarithmicscale.(RedrawnfromCelanderO,FolkowB.Acta Physiol Scand. 1953;29:241.) internal carotid arteries, forms the circle of Willis. Arteries on the brain surface differ from those that penetrate the brain parenchyma. Pial arteries and arterioles have an extrinsic innervation (e.g., via superior cervical ganglion, sphenopalatine nerves, trigeminal nerve); parenchymal arterioles have an intrinsic innervation (via cerebral neurons). Pial arteries have more smooth muscle cells than do parenchymal arterioles. Also, pial arteries and arterioles have collateral branches, whereas parenchymal arterioles do not. Therefore, parenchymal

1	Pial arteries have more smooth muscle cells than do parenchymal arterioles. Also, pial arteries and arterioles have collateral branches, whereas parenchymal arterioles do not. Therefore, parenchymal arterioles regulate blood flow to discrete cortical regions, and their occlusion can reduce blood flow significantly.

1	The cerebral circulation is unique because it lies within a rigid structure, the cranium. Any increase in arterial inflow must be associated with a comparable increase in venous outflow because the intracranial contents cannot be compressed. The volume of blood and extravascular fluid can vary considerably in most body tissues. In the brain, however, the volume of blood and extravascular fluid is relatively constant; a change in one of these fluid volumes must be accompanied by a reciprocal change in the other. The rate of cerebral blood flow is maintained within a narrow range; in humans, it averages 55 mL/minute/100 g of brain tissue. Regulation of Cerebral Blood Flow

1	Regulation of Cerebral Blood Flow At rest, the brain consumes 20% of total body oxygen and 25% of total body glucose. Of all body tissues, the brain is the least tolerant of ischemia. Interruption of cerebral blood flow for as little as 5 seconds results in loss of consciousness. Ischemia that lasts just a few minutes may cause irreversible tissue damage. Fortunately, regulation of the cerebral circulation is primarily under the direction of the brain itself. Local regulatory mechanisms and reflexes that originate in the brain tend to maintain a relatively constant cerebral circulation in the presence of such adverse effects as sympathetic vasomotor nerve activity, circulating humoral vasoactive agents, and changes in arterial blood pressure. Under certain conditions, the brain also regulates its blood flow by initiating changes in systemic blood pressure.

1	Changes in cerebral blood flow are associated with “functional recruitment” of capillaries. Thus the rate of flow through each capillary is adjusted to meet the needs of the organ. In “capillary recruitment,” in contrast, more capillaries are open to accommodate greater blood flow. The brain has several protective mechanisms that regulate blood flow. These mechanisms include the blood-brain barrier, extrinsic regulation of central cardiovascular centers, intrinsic control (autoregulation) of circulation, and functional hyperemia, in which blood flow increases to a brain region that is active. The blood-brain barrier regulates ion and nutrient transport between the blood and the brain and also limits the entry of harmful substances from the blood into the brain. The •Fig. 17.40 Diagram of a Neurovascular Unit With an Astrocyte Linking a Neuron to an Arteriole of the Brain Microcirculation.

1	Arteriolartoneismodulatedbyvascularsmoothmuscleandbytheactionofpericytes.Theendothelialcellrestrictsdiffusionofsubstancesbyvirtueoftightjunctions.Theneurovascularunitisacomponentoftheblood-brainbarrierandalsoservesasaregulatorofbloodflowduringneuronalactivity. blood-brain barrier includes tight junction proteins (junctional adhesion molecule-1, occludins, claudins), which are connected to the endothelial cell cytoskeleton to form a barrier that opposes paracellular movement of substances from blood to brain. In addition, the blood-brain barrier includes the neurovascular unit (microcirculation, pericytes, the extracellular matrix, astrocytes, and neurons; Fig.

1	Fig. 17.40 ). Pericytes regulate blood flow by adjusting vascular diameter, and they secrete angiopoetin, a growth factor that stimulates the expression of occludins in endothelial cells. Occludins are prominently expressed in brain endothelial cells, in contrast to their sparse distribution in nonneural endothelium. The neurovascular unit regulates blood flow and capillary permeability. Thus the neurovascular unit is involved in pathological states, including hypoxia, neurodegenerative diseases, and inflammation, that are characterized by dysfunction of the blood-brain barrier.

1	The extrinsic innervation of cerebral (pial) vessels consists of components of the autonomic nervous system. Cervical sympathetic nerve fibers that accompany the internal carotid and vertebral arteries into the cranial cavity innervate the cerebral vessels. In comparison to other vascular beds, sympathetic control of the cerebral vessels is weak, and the contractile state of the cerebrovascular smooth muscle depends primarily on local metabolic factors. The density of α1-adrenergic receptors is less than in other vascular beds. Cerebral vessels receive parasympathetic fibers from the facial nerve that produce a slight vasodilation

1	Controls: rest Sens 1 Sens 2 •Fig. 17.41 Effects of Different Stimuli on Regional Blood Flow in the Contralateral Human Cerebral Cortex. Sens1,low-intensityelectricalstimulationofthehand;Sens2,high-intensity(painful)electricalstimulationofthehand.Otherstimuliareasnoted.(RedrawnfromIngvarDH.Brain Res. 1976;107:181.) on stimulation. The sympathetic nervous system exerts the most prominent effect on cerebral blood flow during pathophysiological conditions.

1	In general, total cerebral blood flow is relatively constant and is autoregulated. Autoregulation of cerebral blood flow involves interplay among myogenic, metabolic, and neural mechanisms much as described for peripheral vessels (see ). However, regional blood flow in the brain is associated with regional neural activity. For example, movement of one hand results in increased blood flow only in the hand area of the contralateral sensorimotor and premotor cortex. Talking, reading, and other stimuli to the cerebral cortex are also associated with increased blood flow in the appropriate regions of the contralateral cortex ( Fig. 17.41 ). Glucose uptake also corresponds to regional cortical neuronal activity. Thus when the retina is stimulated by light, uptake of glucose is enhanced in the visual cortex.

1	Fig. 17.41 ). Glucose uptake also corresponds to regional cortical neuronal activity. Thus when the retina is stimulated by light, uptake of glucose is enhanced in the visual cortex. The neurovascular unit plays an integral role in the discrete regulation of blood flow. Production of vasoactive compounds couples increased neuronal activity to greater uptake of oxygen and glucose. Within the neurovascular unit, astrocytes link neurons with the microcirculation (see

1	Fig. 17.40 ). At one pole, astrocytes surround presynaptic and postsynaptic neurons at synapses. At the other pole, astrocytes converge on vascular smooth muscle and endothelial cells of cerebral vessels. When activated by the neurotransmitter glutamate or acetylcholine, astrocytes produce inositol trisphosphate (IP3), which causes the release of Ca++, which, in turn, activates large conductance potassium (BKCa) channels. The released K+ raises extracellular [K+] to 8 to 15 mEq/L in the space between the astrocyte and arteriolar smooth muscle. The elevated extracellular [K+] causes hyperpolarization of smooth muscle by activating Na+/K+-ATPase and also by increasing the conductance of inward-rectifying K+ channels. The hyperpolarization reduces Ca++ entry into vascular smooth muscle because the membrane potential is shifted away from the threshold. Hence, the parenchymal arteriole dilates, and blood flow increases.

1	With regard to K+ , stimuli such as hypoxia, electrical stimulation of the brain, and seizures elicit rapid increases in cerebral blood flow, and they are associated with increases in perivascular K+ . The increments in K+ are similar to those that produce pial arteriolar dilation when K+ is applied topically to these vessels. When extracellular K+ exceeds 15 mEq/L, smooth muscle cells depolarize, and Ca++ entry is increased to cause contraction and vasoconstriction. Thus the extracellular [K+] has a dual effect on smooth muscle function that is derived from its actions on Na+/K+-ATPase, K+ conductance, and the K+ concentration gradient.

1	The cerebral vessels are also regulated by CO2. Increases in arterial blood CO2 tension (PaCO2) elicit marked cerebral vasodilation; for example, inhalation of 7% CO2 increases cerebral blood flow twofold. Conversely, decreases in PaCO2, caused by hyperventilation, diminish cerebral blood flow. CO2 evokes changes in arteriolar resistance by altering perivascular pH. When PaCO2 and the HCO3 − concentration are independently changed, pial vessel diameter and blood flow are inversely related to pH, regardless of the level of PaCO2 or [HCO3 −]. Acidosis initiates a marked vasodilation of brain arterioles. The vasodilation is mediated by a very localized release of Ca++ from the endoplasmic reticulum (Ca++ “sparks”). This local Ca++ signal activates large conductance BKCa channels; the ensuing hyperpolarization stabilizes the vascular smooth muscle cell and opposes vasoconstriction.

1	Elevationinintracranialpressure,causedbyabraintumor,resultsinanincreaseinsystemicbloodpressure.Thisresponse,calledCushing’s phenomenon, isevokedbyischemicstimulationofvasomotorregionsinthemedulla.Cushing’sphenomenonhelpsmaintaincerebralbloodflowinconditionssuchasexpandingintracranialtumors. Carbon dioxide diffuses into vascular smooth muscle from brain tissue or from the lumen of blood vessels, whereas H+ in blood is prevented from reaching arteriolar smooth muscle by the blood-brain barrier. Hence, cerebral vessels dilate when the [H+] of cerebrospinal fluid is increased, but these vessels dilate only minimally in response to an increase in the [H+] of arterial blood. Chemical regulation of cerebral blood flow by PaCO2 is impaired in humans with endothelial dysfunction (e.g., diabetes, hypertension); the relative roles of H+ and NO in response to changes of PaCO2 are not clear.

1	Potassium concentration also affects cerebral blood flow. Hypoxia, electrical stimulation of the brain, and seizures elicit rapid increases in cerebral blood flow and in perivascular [K+]. The increases in [K+] are similar in magnitude to those that produce pial arteriolar dilation when K+ is applied topically to these vessels. However, the increase in [K+] is not sustained throughout the period of cerebral stimulation. Thus only the initial increase in cerebral blood flow can be attributed to the release of K+ .

1	Adenosine also has a major effect on cerebral blood flow. Adenosine levels in the brain increase in response to ischemia, hypoxemia, hypotension, hypocapnia, electrical stimulation of the brain, and induced seizures. Topically applied adenosine is a potent dilator of the pial arterioles. Any intervention that either reduces the O2 supply to the brain or increases the O2 requirements of the brain results in the rapid (within 5 seconds) formation of adenosine in cerebral tissue. Unlike the changes in pH or [K+], the adenosine concentration in the brain increases with initiation of the change in O2 supply, and it remains elevated throughout the period of O2 imbalance. The adenosine that is released into cerebrospinal fluid during cerebral ischemia becomes incorporated into adenine nucleotides in cerebral tissue. These local factors, including pH, K+, and adenosine, act in concert to adjust cerebral blood flow to the metabolic activity of the brain. The cerebral circulation displays

1	in cerebral tissue. These local factors, including pH, K+, and adenosine, act in concert to adjust cerebral blood flow to the metabolic activity of the brain. The cerebral circulation displays reactive hyperemia

1	CHAPTER 17 Properties of the Vasculature and excellent autoregulation when arterial blood pressure is between 60 and 160 mm Hg. Mean arterial pressures below 60 mm Hg result in reduced cerebral blood flow and then syncope, whereas mean pressures above 160 mm Hg may lead to increased permeability of the blood-brain barrier and consequently to cerebral edema. Hypercapnia or any other potent vasodilator abolishes autoregulation of cerebral blood flow. Autoregulation of cerebral blood flow is probably mediated by a myogenic mechanism that is modulated by a metabolic component.

1	The gastrointestinal tract is supplied by the celiac, superior mesenteric, and inferior mesenteric arteries. The superior mesenteric artery carries more than 10% of the cardiac output. Small mesenteric arteries form an extensive vascular network in the submucosa of the gastrointestinal tract. The arterial branches penetrate the longitudinal and circular muscle layers of the tract, and they give rise to thirdand fourth-order arterioles. Some third-order arterioles in the submucosa supply the tips of the villi ( Fig. 17.42 The direction of blood flow in the capillaries and venules in a villus is opposite that in the main arteriole (see Fig.

1	Fig. 17.42 The direction of blood flow in the capillaries and venules in a villus is opposite that in the main arteriole (see Fig. 17.42 ). This arrangement is a countercurrent exchange system. Effective countercurrent exchange enables diffusion of O2 from arterioles to venules. At low blood flow rates, a substantial portion of the O2 may be shunted from arterioles to venules near the base of the villus. This reduces the O2 supply to the mucosal cells at the tip of the villus. When intestinal blood flow is very low, shunting of O2 is so great that extensive necrosis of the intestinal villi takes place. Neural control of the mesenteric circulation is almost exclusively sympathetic. Increased sympathetic activity, through α1-adrenergic receptors, constricts the mesenteric arterioles and capacitance vessels. These receptors are preeminent in the mesenteric circulation. However, β2-adrenergic receptors are also present, and so the agonist isoproterenol causes vasodilation.

1	In response to aggressive behavior or to artificial stimulation of the hypothalamic “defense” area, pronounced vasoconstriction occurs in the mesenteric vascular bed. This vasoconstriction shifts blood flow from the less important intestinal circulation to the more crucial skeletal muscles, heart, and brain. Autoregulation of blood flow is not as well developed in the intestinal circulation as in other vascular beds. The principal mechanism responsible for autoregulation is metabolic, although a myogenic mechanism probably also

1	Simple columnar epithelium with goblet cells •Fig. 17.42 Microcirculation Pattern of the Small Intestine. A, Capillaryplexusesarisefromarteriolesinthevillusandalsointheintestinalcrypt.Bloodleavestheintestinalcryptviavenulesthatentertheportalcirculation.B, Lymphaticvessels(lacteals)originatewithinthevillusandeventuallyformaplexusatthebaseofthevillus.(RedrawnfromKierszenbaumA.Histology and Cell Biology: An Introduction to Pathology. Philadelphia:Mosby;2002.) ). The adenosine concentration in mesenteric venous blood rises fourfold after brief arterial occlusion. It also rises during enhanced metabolic activity of the intestinal mucosa, such as during absorption of food. Adenosine, a potent vasodilator in the mesenteric vascular bed, may be the principal metabolic mediator of autoregulation. However, [K+] and altered plasma osmolality may also contribute to autoregulation.

1	Oxygen consumption by the small intestine is more rigorously controlled than is blood flow. Experiments have shown that O2 uptake of the small intestine remains constant when arterial perfusion pressure is varied between 30 and 125 mm Hg. Food ingestion increases intestinal blood flow. Secretion of certain gastrointestinal hormones contributes to this hyperemia. Gastrin and cholecystokinin augment intestinal blood flow, and they are secreted when food is ingested. Absorption of food also affects intestinal blood flow. Undigested food has no vasoactive influence, whereas several products of digestion are potent vasodilators. Among the various constituents of chyme, the principal mediators of mesenteric hyperemia are glucose and fatty acids.

1	Normally, blood flow to the liver is approximately 25% of cardiac output. Hepatic blood flow is supplied by two sources: the portal vein (≈75%) and the hepatic artery. Because portal venous blood has already passed through the gastrointestinal capillary bed, much of the O2 of the portal vein blood flow has already been extracted. The hepatic artery delivers the remaining 25% of the blood, which is fully saturated with O2. Hence, approximately three fourths of the O2 used by the liver is derived from hepatic arterial blood. The small branches of the portal vein and hepatic artery give rise to terminal portal venules and hepatic arterioles (

1	The small branches of the portal vein and hepatic artery give rise to terminal portal venules and hepatic arterioles ( Fig. 17.43 ). These terminal vessels enter the hepatic acinus (the functional unit of the liver) at its center. Blood flows from these terminal vessels into the sinusoid capillaries, which constitute the capillary network of the liver. The sinusoid capillaries radiate toward the periphery of the acinus, where they connect with the terminal hepatic venules. Blood from these terminal venules drains into progressively larger branches of the hepatic veins, which are tributaries of the inferior vena cava.

1	•Fig. 17.43 Microcirculation of the Hepatic Acinus. Arrows indicatethedirectionofbloodflowfromtheterminalportionsofthehepaticarteryandportalveintothesinusoidcapillaries.Themixtureofarterialandvenousbloodflowsintothecentralveinandthenpassesintothesublobularvein.(RedrawnfromRossMH,PawlingW.Histology:A Text and Atlas: With Correlated Cell and Molecular Biology. Philadelphia:LippincottWilliams&Wilkins;2006.) Periportal space (space of Mall) Capillary Terminal branch of hepatic artery Terminal branch of portal vein Portal canal Arteriosinusoidal branch Lymphatic vessel Bile ductule Inlet venule Hepatic sinusoid Terminal hepatic venule (central vein) Sublobular vein

1	Mean blood pressure in the portal vein is approximately 10 mm Hg, and mean blood pressure in the hepatic artery is approximately 90 mm Hg. The resistance of the vessels upstream to the hepatic sinusoid capillaries is considerably greater than that of the downstream vessels. Consequently, the pressure in the sinusoid capillaries is only 2 or 3 mm Hg greater than that in the hepatic veins and inferior vena cava. The ratio of presinusoidal to postsinusoidal resistance is much greater in the liver than in almost any other vascular bed. Hence, drugs and other interventions that alter presinusoidal resistance usually affect pressure in the sinusoid capillaries and fluid exchange across the sinusoidal wall only slightly. However, changes in hepatic and central venous pressure are transmitted almost quantitatively to the hepatic sinusoid capillaries, and they profoundly affect the transsinusoidal exchange of fluids. Regulation of Flow

1	Regulation of Flow Blood flow in the portal venous and hepatic arterial systems varies reciprocally. When blood flow is curtailed in one system, flow increases in the other but does not fully compensate for the decreased flow in the first system. The portal venous system is not autoregulated. As portal Pv and flow are raised, resistance either remains constant or decreases. The hepatic arterial system is autoregulated, however, and adenosine may be involved in this adjustment of blood flow. The liver tends to maintain constant O2 consumption because O2 extraction from hepatic blood is very efficient. As the rate of O2 delivery to the liver varies, the liver compensates by an appropriate change in the fraction of O2 extracted from blood. Such extraction is facilitated by the distance between the presinusoidal vessels at the acinar center and the postsinusoidal vessels at the periphery of the acinus (see

1	Fig. 17.43 ). The substantial distance between these types of vessels prevents countercurrent exchange of O2, in contrast to the countercurrent exchange that occurs in an intestinal villus. The sympathetic nerves constrict the presinusoidal resistance vessels in the portal venous and hepatic arterial systems. Neural effects on the capacitance vessels are more important, however. The liver contains approximately 15% of the total blood volume of the body. In appropriate conditions, as in response to hemorrhage, approximately half of the hepatic blood volume can be rapidly expelled by constriction of the capacitance vessels (see also ). Hence, the liver is an important blood reservoir in humans.

1	Whencentralvenouspressureiselevated,asincongestiveheartfailure,largevolumesofplasmawaterdiffusefromtheliverintotheperitonealcavity;thisaccumulationoffluidintheabdomenisknownasascites. Extensivefibrosisoftheliver,asinhepaticcirrhosis,markedlyincreaseshepaticvascularresistanceandtherebyraisespressuresubstantiallyintheportalvenoussystem.Theconsequentincreaseincapillaryhydrostaticpressurethroughthesplanchniccirculationalsoleadstoextensivefluidtransudationintotheabdominalcavity.Thepressuremaylikewiserisesubstantiallyinotherveinsthatanastomosewiththeportalvein.Forexample,theesophagealveinsmayenlargeconsiderablytoformesophagealvarices.Thesevaricesmayruptureandleadtosevere,frequentlyfatalinternalbleeding.TopreventthesegraveproblemsassociatedwithelevatedportalPvincirrhosisoftheliver,ananastomosis(portacavalshunt)isofteninsertedsurgicallybetweentheportalveinandtheinferiorvenacavatolowerportalPv.

1	Fetal circulation differs from the circulation in postnatal infants. Of most importance is that the fetal lungs are functionally inactive, and the fetus depends completely on the placenta for O2 and nutrients. Oxygenated fetal blood from the placenta passes through the umbilical vein to the fetal liver. Approximately half the flow from the placenta passes through the liver, and the remainder bypasses the fetal liver and reaches the inferior vena cava through the ductus venosus (Fig. 17.44 ). Blood from the ductus venosus joins the blood returning from the lower part of the fetal trunk and the extremities in the inferior vena cava. This blood merges with blood from the fetal liver through the hepatic veins.

1	The streams of blood tend to maintain their characteristics in the inferior vena cava and are divided into two streams of unequal size by the edge of the interatrial septum (crista dividens). The larger stream, which contains mainly blood from the umbilical vein, is shunted from the inferior vena cava to the left atrium through the foramen ovale (see Fig. 17.44 ). The other stream passes into the right atrium, where it merges with blood returning from the upper parts of the fetal body through the superior vena cava and with blood from the myocardium.

1	Unlike the ventricles in adults, those in a fetus operate essentially in parallel. Only a tenth of right ventricular output passes through the lungs because the pulmonary vascular resistance in the fetus is high. The remainder passes from the fetal pulmonary artery through the ductus arteriosus to the aorta at a point distal to the origins of the arteries to the fetal head and upper extremities. Blood flows from the pulmonary artery to the aorta because pulmonary vascular resistance is high, and the diameter of the ductus arteriosus is as large as that of the descending aorta.

1	The large volume of blood that passes through the foramen ovale into the fetal left atrium is joined by blood returning from the lungs, and it is pumped out by the left ventricle into the aorta. Most of the blood in the ascending aorta goes to the fetal head, upper thorax, and arms; the remainder joins blood from the ductus arteriosus and supplies the rest of the body. The amount of blood pumped by the left ventricle is approximately half that pumped by the right ventricle. The major fraction of the blood that passes down the descending aorta comes from the ductus arteriosus and right ventricle and flows by way of the two umbilical arteries to the placenta. Oxygen saturation of fetal blood occurs at various loci (see Fig. 17.44 ). Thus the fetal tissues that receive the most highly saturated blood are the liver, heart, and upper parts of the body, including the head.

1	Fig. 17.44 ). Thus the fetal tissues that receive the most highly saturated blood are the liver, heart, and upper parts of the body, including the head. At the placenta, the chorionic villi dip into the maternal sinuses, and O2, CO2, nutrients, and metabolic waste products are exchanged across the membranes. The barrier to exchange prevents equilibration of O2 between the two circulations at normal rates of blood flow. Therefore, the PO2 of the fetal blood that leaves the placenta is very low. Were it not for the fact that fetal hemoglobin has a greater affinity for O2 than adult hemoglobin does, the fetus would not receive an adequate O2 supply. The fetal oxyhemoglobin dissociation curve is shifted to the left. Therefore, at equal pressures of O2, fetal blood carries significantly more O2 than maternal blood does.

1	In early gestation, the high glycogen levels that prevail in cardiac myocytes may protect the heart from acute periods of hypoxia. Glycogen levels decrease in late gestation, and they reach adult levels by term. The umbilical vessels have thick muscular walls that react to trauma, tension, sympathomimetic amines, bradykinin, angiotensin, and changes in PO2. In animals in which the umbilical cord is not tied, hemorrhage of the newborn is minimized by constriction of these large umbilical vessels in response to stretching of the umbilical arteries and by an associated increase in PO2 in systemic arteries. Closure of the umbilical vessels increases total peripheral resistance and the arterial blood pressure of the infant. When blood flow through the umbilical vein ceases, the ductus venosus, a thick-walled vessel with a muscular sphincter, closes. The factor that initiates closure of the ductus venosus is unknown.

1	Ifapregnantwomanissubjectedtohypoxia,thereducedbloodPO2inthefetusevokestachycardiaandanincreaseinbloodflowthroughtheumbilicalvessels.Ifthehypoxiapersistsorifflowthroughtheumbilicalvesselsisimpaired,fetaldistressoccursandismanifestedinitiallyasbradycardia.

1	•Fig. 17.44 Schematic Diagram of the Fetal Circulation. Thenumbers representthepercentageofO2saturationofthebloodflowingintheindicatedbloodvessel.Fetalbloodthatleavestheplacentais80%saturated,butthesaturationofthebloodthatpassesthroughtheforamenovaleisreducedto67%.ThisreductioninO2saturationiscausedbythemixingofsaturatedbloodwithdesaturatedbloodreturningfromthelowerpartofthefetalbodyandtheliver.AdditionofthedesaturatedbloodfromthefetallungsreducestheO2saturationofleftventricularbloodto62%,whichisthelevelofsaturationofthebloodreachingthefetalheadandupperextremities.Thebloodintherightventricle—whichisamixtureofdesaturatedsuperiorvenacavalblood,coronaryvenousblood,andinferiorvenacavalblood—isonly52%saturatedwithO2.Whenthemajorportionofthisbloodtraversestheductusarteriosusandjoinsthatpumpedbytheleftventricle,theresultingO2saturationofthebloodtravelingtothelowerpartofthefetalbodyandbacktotheplacentais58%.Theinset

1	atupperleftillustratesthedirectionofflowofamajorportionoftheinferiorvenacavalbloodthroughtheforamenovaletotheleftatrium.Arrows indicatethedirectionsofflow.(DatafromDawesGS,etal.J Physiol. 1954;126:563.)

1	Immediately after birth, the asphyxia caused by constriction or clamping of the umbilical vessels, together with cooling of the body, activates the respiratory center of the newborn infant. As the lungs fill with air, pulmonary vascular resistance decreases to approximately 10% of the value that existed before lung expansion. This change in vascular resistance is not caused by the presence of O2 in the lungs because the change is just as great if the lungs are filled with N2. However, filling the lungs with liquid does not reduce pulmonary vascular resistance.

1	After birth, left atrial pressure is raised above that in the inferior vena cava and right atrium by (1) the decrease in pulmonary resistance, with the consequent large flow of blood through the lungs to the left atrium; (2) the reduction of flow to the right atrium caused by occlusion of the umbilical vein; and (3) the increased resistance to left ventricular output produced by occlusion of the umbilical arteries. Reversal of the pressure gradient across the atria abruptly closes the valve over the foramen ovale, and the septal leaflets fuse over a period of several days.

1	The decrease in pulmonary vascular resistance causes the pressure in the pulmonary artery to fall to approximately half its previous level (to ≈35 mm Hg). This change in pressure, coupled with a slight increase in aortic pressure, reverses the flow of blood through the ductus arteriosus. However, within several minutes, the large ductus arteriosus begins to constrict. This constriction produces turbulent flow, which is manifested as a murmur in newborn infants. Constriction of the ductus arteriosus is progressive and usually complete within 1 to 2 days after birth. Closure of the ductus arteriosus appears to be initiated by the high PO2 of the arterial blood passing through it; pulmonary ventilation with O2 closes the ductus, whereas ventilation with air low in O2 opens this shunt vessel. Whether O2 acts directly on the ductus or through the release of a vasoconstrictor substance is not known.

1	At birth, the walls of the two ventricles have approximately equal thickness. In addition, the muscle layer of the pulmonary arterioles is thick; this thickness is partly responsible for the high pulmonary vascular resistance of the fetus. After birth, the thickness of the walls of the right ventricle diminishes, as does the muscle layer of the pulmonary arterioles. In contrast, the left ventricular walls become thicker. These changes progress over a period of weeks after birth and reflect the effects of different hemodynamic forces (e.g., vascular resistance) on the two ventricles. Cardiac hypertrophy underlies the increase of heart weight during the normal growth period after birth. The physical demands imposed by the developing cardiovascular system, together with increased levels of soluble factors (e.g., growth hormone, insulin-like growth factor-1), account for physiological hypertrophy by which left ventricular mass more than doubles during the period from birth to early

1	of soluble factors (e.g., growth hormone, insulin-like growth factor-1), account for physiological hypertrophy by which left ventricular mass more than doubles during the period from birth to early adulthood.

1	Theductusarteriosusoccasionallyfailstocloseafterbirth.Inthenewborn,thiscongenitalcardiovascularabnormality,calledpatent ductus arteriosus, cansometimesbecorrectedbytheadministrationofnonsteroidalanti-inflammatoryagentssuchasibuprofen.Ifthisdoesnotresultinclosureoftheductusorifthechildisolder,closuremustbeachievedsurgically. 1. The vascular system is composed of two major subdivisions: the systemic circulation and the pulmonary circulation. These subdivisions are in series with each other and are composed of a number of vessel types (e.g., arteries, arterioles, capillaries) that are aligned in series with one another. In general, the vessels of a given type are arranged in parallel with each other. 2. The mean velocity (v) of blood flow in a given type of vessel is directly proportional to the total blood flow being pumped by the heart, and it is inversely proportional to the cross-sectional area of all the parallel vessels of that type. 3.

1	3. Poiseuille’s law characterizes blood flow that is steady and laminar in vessels larger than arterioles. However, blood flow is nonnewtonian in very small blood vessels (i.e., Poiseuille’s law is not applicable). 4. Flow tends to become turbulent when (1) flow velocity is high, (2) fluid viscosity is low, (3) fluid density is great, (4) vessel diameter is large, or (5) the wall of the vessel is irregular. 5. Arteries not only conduct blood from the heart to the capillaries but also store some of the ejected blood during each cardiac systole. Hence, blood flow continues through the capillaries during cardiac diastole. Veins return blood to the heart from the capillaries and have a relatively low resistance and high capacitance that enables them to serve as reservoirs for blood. 6.

1	6. The aging process diminishes compliance of the arteries, as well as of the veins. The less compliant the arteries are, the more work the heart must do to achieve a given cardiac output. The less compliant the veins are, the poorer is their ability to store blood. 7. Mean arterial pressure varies directly with cardiac output and total peripheral resistance. Arterial pulse pressure varies directly with stroke volume but inversely with arterial compliance. 8. Blood flow through capillaries is regulated chiefly by contraction of arterioles (resistance vessels). The capillary endothelium is the source of NO and PGI2, which relax vascular smooth muscles. 9.

1	9. Water and small solutes move between the vascular and interstitial fluid compartments through capillary pores mainly by diffusion but also by filtration and absorption. Molecules larger than approximately 60 kD are essentially confined to the vascular compartment. Lipid-soluble substances, such as CO2 and O2, pass directly through the lipid membranes of the capillary; the rate of transfer is directly proportional to their lipid solubility. Large molecules can move across the capillary wall in vesicles by pinocytosis. The vesicles are formed from the lipid membrane of the capillaries. 10. Capillary filtration and absorption are described by Starling’s equation: Filtration occurs when the algebraic sum of these terms is positive; absorption occurs when it is negative. 11. Fluid and protein that have escaped from blood capillaries enter lymphatic capillaries and are transported via the lymphatic system back to the blood vascular compartment. 12.

1	Physical factors that influence coronary arterial blood flow are the viscosity of the blood, frictional resistance of the vessel walls, aortic pressure, and extravascular compression of the vessels within the walls of the left ventricle. Left coronary arterial blood flow is restricted during ventricular systole by extravascular compression, and the flow is greatest during diastole, when the intramyocardial vessels are not compressed. Neural regulation of coronary arterial blood flow is much less important than metabolic regulation. Activation of the cardiac sympathetic nerves constricts the coronary resistance vessels. However, the enhanced myocardial metabolism caused by the associated increase in heart rate and contractile force produces vasodilation, which overrides the direct constrictor effect of sympathetic nerve stimulation. Stimulation of the cardiac branches of the vagus nerves causes slight dilation of the coronary arterioles. A striking parallelism exists between metabolic

1	effect of sympathetic nerve stimulation. Stimulation of the cardiac branches of the vagus nerves causes slight dilation of the coronary arterioles. A striking parallelism exists between metabolic activity of the heart and coronary arterial blood flow. A decrease in O2 supply or an increase in O2 demand apparently releases vasodilators that decrease coronary arterial resistance. Of the known factors (CO2, O2, H+ , K+ , H2O2, adenosine) that can mediate this response, KATP channels, NO, H2O2 and adenosine are the most likely candidates, although CO2, O2, and H+ cannot be ruled out.

1	13. Most of the resistance vessels in the skin are under dual control of the sympathetic nervous system and local vasodilator metabolites. The AV anastomoses found in the hands, feet, and face, however, are solely under neural control. The main function of skin blood vessels is to aid in the regulation of body temperature by constricting to conserve heat and by dilating to lose heat. Skin blood vessels dilate directly and reflexively in response to heat, and they constrict directly and reflexively in response to cold. 14. Blood flow in skeletal muscle is regulated centrally by sympathetic nerves and locally by the release of vasodilator metabolites. In persons at rest, neural Camici PG, d’Amati G, Rimoldi O. Coronary microvascular dysfunction: mechanisms and functional assessment. Nat Rev Cardiol. 2015;12:48. Chiu J-J, Chien S. Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives. Physiol Rev. 2011;91:327.

1	Chiu J-J, Chien S. Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives. Physiol Rev. 2011;91:327. Edwards G, Félétou M, Weston AH. Endothelium-derived hyper-polarization factors and associated pathways: a synopsis. Pflügers Arch. 2010;459:863. Kara T, Narkiewicz K, Somers VK. Chemoreflexes—physiology and clinical implications. Acta Physiol Scand. 2003;177:377. regulation of blood flow is paramount, but it yields to metabolic regulation during muscle contractions (as during exercise). 15.

1	regulation of blood flow is paramount, but it yields to metabolic regulation during muscle contractions (as during exercise). 15. Cerebral blood flow is regulated predominantly by metabolic factors, especially CO2, K+ , and adenosine. The increased regional cerebral activity produced by stimuli such as touch, pain, hand motion, talking, reading, reasoning, and problem solving are associated with enhanced blood flow in the activated area of the contralateral cerebral cortex. The neurovascular unit (microcirculation, pericytes, the extracellular matrix, astrocytes and neurons), a component of the blood-brain barrier, is thought to link brain activity with increased blood flow and oxygenation. 16.

1	16. The microcirculation in intestinal villi constitutes a countercurrent exchange system for O2. Because of the presence of this countercurrent exchange system, the villi are in jeopardy in states of low blood flow. The splanchnic resistance and capacitance vessels are very responsive to changes in sympathetic neural activity. 17. The liver receives approximately 25% of cardiac output; approximately three fourths of this output is from the portal vein and approximately a fourth from the hepatic artery. When flow is diminished in either the portal or hepatic system, flow in the other system usually increases, but not proportionately. The liver tends to maintain constant O2 consumption, in part because its mechanism for extracting O2 from blood is so efficient. The liver normally contains approximately 15% of the total blood volume. It serves as an important blood reservoir for the body. 18.

1	18. In the fetus, a large percentage of right atrial blood passes through the foramen ovale to the left atrium, and a large percentage of pulmonary arterial blood passes through the ductus arteriosus to the aorta. At birth, the umbilical vessels, ductus venosus, and ductus arteriosus close by contraction of their muscle layers. The reduction in pulmonary vascular resistance caused by lung inflation is the main factor that reverses the pressure gradient between the atria and thereby causes the foramen ovale to close. Ludmer PL, Selwyn AP, Shook TL, et al. Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic coronary arteries. N Engl J Med. 1986;315:1046. Ohanyan V, Yin L, Bardakjian R, et al. Requisite role of Kv1.5 channels in coronary metabolic dilation. Circ Res. 2015;117:612. Potente M, Gerhardt H, Carmeliet P. Basic and therapeutic aspects of angiogenesis. Cell. 2011;146:873.

1	Potente M, Gerhardt H, Carmeliet P. Basic and therapeutic aspects of angiogenesis. Cell. 2011;146:873. Resnick N, Yahav H, Shay-Salit A, et al. Fluid shear stress and the vascular endothelium: for better and for worse. Prog Biophys Mol Biol. 2003;81:177. Upon completion of this chapter, the student should be able to answer the following questions: 1. How do the parasympathetic and sympathetic nervous systems regulate the functions of heart and vasculature? 2. What factors affect the differential sympathetic regulation of resistance and capacitance vessels? 3. How does the baroreceptor-mediated reflex mimic the operation of skeletal muscle proprioceptor reflex? 4. What are the two major mechanisms, intrinsic to heart muscle, that regulate myocardial performance? 5. What are the major hormones that regulate myocardial performance? 6. How is myocardial performance affected by changes in the arterial blood concentrations of O2, CO2, and H+? 7.

1	5. What are the major hormones that regulate myocardial performance? 6. How is myocardial performance affected by changes in the arterial blood concentrations of O2, CO2, and H+? 7. What is the myogenic mechanism of vascular smooth muscle, and how does it participate in regulation of tissue blood flow? 8. What are the humoral factors that participate in regulation of blood flow, and what are their actions? Regulation of Heart Rate and Myocardial Performance Cardiac output is defined as the quantity of blood pumped by the heart each minute. Cardiac output may be varied by a change in the heart rate or the volume of blood ejected from either ventricle with each heartbeat; this volume is called the stroke volume. Mathematically, cardiac output (CO) can be expressed as the product of heart rate (HR) and stroke volume (SV): Equation 18.1

1	Equation 18.1 Thus to understand how cardiac activity is controlled, consider how the heart rate and stroke volume are regulated. Heart rate is regulated by the activity of the autonomic nervous system to modulate the intrinsic cardiac pacemaker. Stroke volume is determined by myocardial performance (which is determined by cardiac cell contractility) and by the hemodynamic loads on the heart. All of these determinants are interdependent, inasmuch as a change in one determinant of cardiac output almost invariably alters another. Nervous Control of the Heart Rate Although certain local factors, such as temperature changes and stretching of tissue, can affect the heart rate, the autonomic nervous system is the principal means by which the heart rate is controlled.

1	The average resting heart rate is approximately 70 beats per minute in normal adults, and it is significantly faster in children. During sleep, the heart rate decreases by 10 to 20 beats per minute. It may increase during emotional excitement, and during muscular exercise, it may increase to rates well above 150 beats per minute. In well-trained athletes, the usual resting rate is only approximately 50 beats per minute. Both divisions of the autonomic nervous system tonically influence the cardiac pacemaker, which is normally the sinoatrial (SA) node. The sympathetic nervous system enhances automaticity, whereas the parasympathetic nervous system inhibits it. Changes in heart rate usually involve a reciprocal action of these two divisions of the autonomic nervous system. Thus the heart rate ordinarily increases with a combined decrease in parasympathetic activity and increase in sympathetic activity; the heart rate decreases with the opposite changes in autonomic neural activity.

1	Parasympathetic tone usually predominates in healthy, resting individuals. When a resting individual is given atropine, a muscarinic receptor antagonist that blocks parasympathetic effects, the heart rate generally increases substantially. If a resting individual is given propranolol, a β-adrenergic receptor antagonist that blocks sympathetic effects, the heart rate usually decreases only slightly ( Fig. 18.1 ). When both divisions of the autonomic nervous system are blocked, the heart rate of young adults averages approximately 100 beats per minute. The rate that prevails after complete autonomic blockade is called the intrinsic heart rate.

1	•Fig. 18.1 Effectsoffourequaldosesofatropine(muscarinicrecep (β-adrenergicreceptorantagonistthatblockssympatheticeffects)ontheheartratesof10healthyyoungmen.Inhalfthetrials,atropine wasgivenfirst(top curve); intheotherhalf,propranololwasgivenfirst(bottom curve). (RedrawnfromKatonaPG,etal.J Appl Physiol. •Fig. 18.2 Changesinheartrateevokedbystimulation(horizontal 1982;52:1652.) bars) ofthevagus(A) andsympatheticnerves(B). (ModifiedfromWarnerHR,CoxA.J Appl Physiol. 1962;17:349.)

1	The cardiac parasympathetic fibers originate in the medulla oblongata, in cells that lie in the dorsal motor nucleus of the vagus nerve or in the nucleus ambiguus (see ). In humans, centrifugal vagal fibers pass inferiorly through the neck near the common carotid arteries and then through the mediastinum to synapse with postganglionic vagal cells. These cells are located either on the epicardial surface or within the walls of the heart. Most of the vagal ganglion cells are located in epicardial fat pads near the SA and atrioventricular (AV) nodes.

1	The right and left vagus nerves are distributed to different cardiac structures. The right vagus nerve affects the SA node predominantly; stimulation of this nerve slows SA nodal firing and can even stop the firing for several seconds. The left vagus nerve mainly inhibits AV conduction tissue to produce various degrees of AV block (see ). However, the distribution of the efferent vagal fibers is overlapping in such a way that left vagal stimulation also •Fig. 18.3 Changes in Heart Rate When the Vagus and Cardiac Sympathetic Nerves Are Stimulated Simultaneously. Thesympa depresses the SA node and right vagal stimulation impedes AV conduction. The SA and AV nodes are rich in acetylcholinesterase, an enzyme that rapidly hydrolyzes the neurotransmitter acetylcholine (ACh). The effects of a given vagal stimulus decay very quickly (

1	The SA and AV nodes are rich in acetylcholinesterase, an enzyme that rapidly hydrolyzes the neurotransmitter acetylcholine (ACh). The effects of a given vagal stimulus decay very quickly ( Fig. 18.2A ) when vagal stimulation is discontinued because ACh is rapidly destroyed. In addition, vagal effects on SA and AV nodal function have a very short latency (≈50 to 100 msec) because the ACh released quickly activates special ACh-regulated potassium (KACh) channels in the cardiac cells. These channels open quickly because the muscarinic receptor is coupled directly to the KACh channel by a guanine nucleotide–binding protein. These two features of the vagus nerves—brief latency and rapid theticnerveswerestimulatedat0,2,and4Hzinthepresenceofvagalnervestimulation(Vag)at0,4,and8Hz.(ModifiedfromLevyMN,ZieskeH.J Appl Physiol. 1969;27:465.) decay of the response—enable them to exert beat-by-beat control of SA and AV nodal function.

1	Parasympathetic influences usually predominate over sympathetic effects at the SA node, as shown in Fig. 18.3 . When the frequency of sympathetic stimulation increases from 0 to 4 Hz, the heart rate increases by approximately 80 beats per minute in the absence of vagal nerve stimulation (0 Hz). However, when the vagus nerves are stimulated at 8 Hz, increasing the sympathetic stimulation frequency from 0 to 4 Hz has only a negligible influence on heart rate.

1	The cardiac sympathetic fibers originate in the intermediolateral columns of the upper five or six thoracic segments and the lower one or two cervical segments of the spinal cord (see ). These fibers emerge from the spinal column through the white communicating branches and enter the paravertebral chains of ganglia. The preganglionic and postganglionic neurons synapse mainly in the stellate or middle cervical ganglia, depending on the species. In the mediastinum, postganglionic sympathetic and preganglionic parasympathetic fibers join to form a complicated plexus of mixed efferent nerves to the heart. The postganglionic cardiac sympathetic fibers in this plexus approach the base of the heart along the adventitial surface of the great vessels. From the base of the heart, these fibers are distributed to the various chambers as an extensive epicardial plexus. They then penetrate the myocardium, usually accompanying the coronary vessels.

1	In contrast to abrupt termination of the response after vagal activity, the effects of sympathetic stimulation decay gradually after stimulation is stopped (see

1	Fig. 18.2B ). Nerve terminals take up to 70% of the norepinephrine released during sympathetic stimulation; much of the remainder is carried away by the bloodstream. These processes are slow. Furthermore, the facilitatory effects of sympathetic stimulation on the heart attain steady-state values much more slowly than do the inhibitory effects of vagal stimulation. The onset of the cardiac response to sympathetic stimulation begins slowly for two main reasons. First, norepinephrine appears to be released slowly from the sympathetic nerve terminals. Second, the cardiac effects of the neurally released norepinephrine are mediated mainly by a relatively slow second messenger system involving cyclic adenosine monophosphate (cAMP; see ). Hence, sympathetic activity alters the heart rate and AV conduction much more slowly than vagal activity does. Whereas vagal activity can exert beat-by-beat control of cardiac function, sympathetic activity cannot.

1	Stimulation of various brain regions can have significant effects on cardiac rate, rhythm, and contractility (see ). In the cerebral cortex, centers that regulate cardiac function are located in the anterior half of the brain, principally in the frontal lobe, the orbital cortex, the motor and premotor cortex, the anterior portion of the temporal lobe, the insula, and the cingulate gyrus. Stimulation of the midline, ventral, and medial nuclei of the thalamus elicits tachycardia. Stimulation of the posterior and posterolateral regions of the hypothalamus can also change the heart rate. Stimuli applied to the H2 field of Forel in the posterior hypothalamus evoke various cardiovascular responses, including tachycardia and associated limb movements; these changes resemble those observed during muscular exercise. Undoubtedly, the cortical and hypothalamic centers initiate the cardiac reactions that occur during excitement, anxiety, and other emotional states. The hypothalamic centers also

1	muscular exercise. Undoubtedly, the cortical and hypothalamic centers initiate the cardiac reactions that occur during excitement, anxiety, and other emotional states. The hypothalamic centers also initiate the cardiac response to alterations in environmental temperature. Experimentally induced temperature changes in the preoptic anterior hypothalamus alter the heart rate and peripheral resistance.

1	Stimulation of the parahypoglossal area of the medulla reciprocally activates cardiac sympathetic pathways and inhibits cardiac parasympathetic pathways. In certain dorsal regions of the medulla, distinct cardiac accelerator sites (increase the heart rate) and augmentor sites (increase cardiac contractility) have been detected in animals with transected vagus nerves. The accelerator regions are more abundant on the right side, whereas the augmentor sites are more prevalent on the left. A similar distribution also exists in the hypothalamus. Therefore, the sympathetic fibers mainly descend ipsilaterally through the brainstem.

1	Corticalcentershaveimportanteffectsonautonomicfunction.Theinsulaexertsdistinctregulationofthebalancebetweensympatheticandparasympatheticactionsonthecardiovascularsystem.Inpatientssubjectedtoelectricalstimulation,stimuliappliedtotheleftinsularcortexelicitpredominantlyparasympatheticresponses(bradycardiaandvasodepression),whereasstimuliappliedtotherightinsularcortexevokesympatheticactions(tachycardiaandvasopression).Aspredicted,patientswithacute,stroke-induceddamageoftheleftinsularcortexdisplayincreasedsympathetictoneandanincreasedriskofarrhythmiasandcardiovascularmortality.Whentherightinsularcortexisacutelyinvolvedinthestroke,theincidenceofcardiovascularmortality/morbidityisunchanged.

1	Fig. 18.4 ). Baroreceptors located in the aortic arch and carotid sinuses are responsible for this reflex (see the section ”). The inverse relationship between heart rate and arterial blood pressure is generally most pronounced over an intermediate range of arterial blood pressures. Below this intermediate range, the heart rate maintains a constant, high value; above this pressure range, the heart rate maintains a constant, low value. The effects of changes in carotid sinus pressure on the activity in cardiac autonomic nerves are described in Fig. 18.5 , which shows that over an intermediate range of carotid sinus pressures (100 to 180 mm Hg), reciprocal changes are evoked in efferent vagal and sympathetic neural activity. Below this range of carotid sinus pressure, sympathetic activity is intense, and vagal activity is virtually absent. Conversely, above the intermediate range of carotid sinus pressure, vagal activity is intense and sympathetic activity is minimal.

1	Bainbridge Reflex, Atrial Receptors, and Atrial Natriuretic Peptide In 1915, Francis A. Bainbridge reported that infusing blood or saline into dogs accelerated their heart rate. This increase did not seem to be tied to arterial blood pressure because the heart rate rose regardless of whether arterial blood pressure did or did not change. However, Bainbridge also noted that the heart rate increased whenever central venous pressure rose sufficiently to distend the right side of the heart. This response is termed the Bainbridge reflex. Bilateral transection of the vagus nerves abolished this response.

1	Many investigators have confirmed Bainbridge’s observations and have noted that the magnitude and direction of the response depend on the prevailing heart rate. When the heart rate is slow, intravenous infusions of blood or electrolyte solutions usually accelerate the heart. At more rapid heart rates, however, such infusions ordinarily slow the heart. What accounts for these different responses? Increases in blood volume not only evoke Bainbridge reflex but also activate other reflexes (of note, the baroreceptor reflex). These other reflexes tend to elicit opposite changes in heart rate. Therefore, changes in heart rate evoked by an alteration in blood volume are the result of these antagonistic reflex effects ( Fig. 18.6 ). Evidently, the Bainbridge reflex predominates over the baroreceptor reflex when blood volume rises, but the baroreceptor reflex prevails over the Bainbridge reflex when blood volume diminishes.

1	•Fig. 18.4 Experimentaldatashowingthatheartratedecreasesasarterialpressureisincreased.(AdaptedfromCornishKG,etal.Am J Physiol. 1989;257:r595.)250 200 150 100 50 50 75 100 125 150 Arterial pressure (mm Hg) Heart rate (beats/min) Both atria have receptors that are affected by changes in blood volume and that influence the heart rate. These receptors are located principally in the venoatrial junctions: in the right atrium at its junctions with the venae cavae and in the left atrium at its junctions with the pulmonary veins. Distention of these atrial receptors sends afferent impulses to the brainstem in the vagus nerves. The efferent impulses are carried from the brainstem to the SA node by fibers from both autonomic divisions.

1	The cardiac response to these changes in autonomic neural activity is highly selective. Even when the reflex increase in heart rate is large, changes in ventricular contractility are generally negligible. Furthermore, the neurally induced increase in heart rate is not usually accompanied by an increase in sympathetic activity in the peripheral arterioles.

1	•Fig. 18.5 Experimentaldatashowingthatincreasesincarotidsinuspressureresultindecreasedsympatheticefferentnerve(Symp)activityandincreasedcardiacvagalnerveactivity.(AdaptedfromKollaiM,KoizumiK.Pflügers Arch. 1989;413:365.)100 50 0 60 100 140 180 220 Sinus pressure (mm Hg) Vagal activity (% of max) 100 50 0 Symp. activity (% of max) •Fig. 18.6 Intravenous Infusions of Blood or Electrolyte Solutions Tend to Increase the Heart Rate Through the Bainbridge Reflex and to Decrease the Heart Rate Through the Baroreceptor Reflex. Theactualchangeinheartrateinducedbysuchinfusionsistheresultofthesetwoopposingeffects.Intravenous infusion Increases right atrial pressure Atrial receptors stimulated Bainbridge reflex – + Increases cardiac output Increases arterial pressure Baroreceptor reflex Heart rate

1	Stimulation of the atrial receptors increases not only the heart rate but also urine volume. Reduced activity in the renal sympathetic nerve fibers may partially account for this diuresis. However, the principal mechanism appears to be a neurally mediated reduction in vasopressin (antidiuretic hormone) secretion by the posterior pituitary gland (see ). Stretch of the atrial walls also releases atrial natriuretic peptide (ANP) from the atria. ANP, a 28–amino acid peptide, exerts potent diuretic and natriuretic effects on the kidneys (see also ) and vasodilator effects on the resistance and capacitance vessels. Thus ANP is an important regulator of blood volume and blood pressure.

1	Incongestiveheartfailure,NaClandwaterareretained,mainlybecausestimulationbytherenin-angiotensinsystemincreasesthereleaseofaldosteronefromtheadrenalcortex.TheplasmalevelofANPisalsoincreasedincongestiveheartfailure.ByenhancingtherenalexcretionofNaClandwater,ANPgraduallyreducesfluidretentionandtheconsequentelevationsincentralvenouspressureandcardiacpreload.

1	Rhythmic variations in heart rate, occurring at the frequency of respiration, are detectable in most individuals and tend to be more pronounced in children. The heart rate typically accelerates during inspiration and decelerates during expiration (Fig. 18.7 *The myocytes of the ventricles secrete a related peptide in response to stretch. This peptide, termed brain natriuretic peptide (BNP) because of its initial discovery in the central nervous system, has actions similar to those of ANP (see •Fig. 18.7 Respiratory Sinus Arrhythmia. Notethattheheartrateincreasesduringinspirationanddecreasesduringexpiration.(ModifiedfromWarnerMR,etal.Am J Physiol. 1986;251:H1134.)50 80 110 140 Expiration Time (sec) Inspiration 3 6 9 12 150 Heart rate(beats/min)

1	Recordings from cardiac autonomic nerves reveal that neural activity increases in the sympathetic fibers during inspiration and increases in the vagal fibers during expiration. The heart rate response to cessation of vagal stimulation is very quick because, as already noted, ACh released from the vagus nerves is rapidly hydrolyzed by acetylcholinesterase. This short latency enables the heart rate to vary rhythmically at the respiratory frequency. Conversely, the norepinephrine released periodically at the sympathetic endings is removed very slowly. Therefore, the rhythmic variations in sympathetic activity that accompany inspiration do not induce any appreciable oscillatory changes in heart rate. Thus respiratory sinus arrhythmia is brought about almost entirely by changes in vagal activity. In fact, respiratory sinus arrhythmia is exaggerated when vagal tone is enhanced.

1	Fig. 18.8 ). Stretch receptors in the lungs are stimulated during inspiration, and this action leads to a reflex increase in heart rate. The afferent and efferent limbs of this reflex are located in the vagus nerves. Intrathoracic pressure also decreases during inspiration and thereby increases venous return to the right side of the heart (see ). The consequent stretch of the right atrium elicits the Bainbridge reflex. After the time delay required for the increased venous return to reach the left side of the heart, left ventricular output increases and raises arterial blood pressure. This rise in blood pressure in turn reduces the heart rate through the baroreceptor reflex. Central factors are also responsible for respiratory cardiac arrhythmia. The respiratory center in the medulla directly influences the cardiac autonomic centers (see

1	Central factors are also responsible for respiratory cardiac arrhythmia. The respiratory center in the medulla directly influences the cardiac autonomic centers (see Fig. 18.8). In heart-lung bypass studies, the chest is opened, the lungs are collapsed, venous return is diverted to a pump-oxygenator, and arterial blood pressure is maintained at a constant level. In such studies, rhythmic movement of the rib cage attests to the activity of the medullary respiratory centers, and is often accompanied by rhythmic changes in heart rate at the respiratory frequency. This respiratory cardiac arrhythmia is almost certainly induced by a direct interaction between the respiratory and cardiac centers in the medulla.

1	The cardiac response to peripheral chemoreceptor stimulation illustrates the complex interactions that may ensue when one stimulus excites two organ systems simultaneously. Stimulation of carotid chemoreceptors consistently increases ventilatory rate and depth (see ), but ordinarily it changes the heart rate only slightly. The magnitude of the ventilatory response determines whether the heart rate increases or decreases as a result of carotid chemoreceptor stimulation. Mild chemoreceptor-induced stimulation of respiration decreases the heart rate moderately; more pronounced stimulation increases the heart rate only slightly. If the pulmonary response to chemoreceptor stimulation is blocked, the heart rate response may be greatly exaggerated, as described later.

1	•Fig. 18.9 The Primary Effect of Stimulation of Peripheral Chemoreceptors on the Heart Rate Is to Excite the Cardiac Vagal Center in the Medulla and Thus to Decrease the Heart Rate. Peripheralchemoreceptorstimulationalsoexcitestherespiratorycenterinthemedulla.Thiseffectproduceshypocapniaandincreaseslunginflation,bothofwhichsecondarilyinhibitthemedullaryvagalcenter.Thusthesesecondaryinfluencesattenuatetheprimaryreflexeffectofperipheralchemoreceptorstimulationonheartrate.Medullary vagal center Peripheral chemoreceptors Respiratory activity Primary effect (+) Secondary effects (–) (+) (–) (–) Hypocapnia Increased lung stretch Heart rate •Fig. 18.8 Respiratorysinusarrhythmiaisgeneratedbyadirectinteractionbetweentherespiratoryandcardiaccentersinthemedulla,aswellasbyreflexesthatoriginatefromstretchreceptorsinthelungs,fromstretchreceptorsintherightatrium(theBainbridgereflex),andfrombaroreceptorsinthecarotidsinusesandaorticarch.Cardiac vagal center (medulla) Respiratory center (medulla)

1	vagal center (medulla) Respiratory center (medulla) Change in intrathoracic pressure Change in venous return Change in arterial pressure Baroreceptor reflex Heart rate Change in lung volume (stretch receptors) Bainbridge reflex

1	The cardiac response to peripheral chemoreceptor stimulation is the result of primary and secondary reflex mechanisms ( Fig. 18.9). The principal effect of the primary reflex stimulation is to excite the medullary vagal center and thereby decrease the heart rate. The respiratory system mediates secondary reflex effects. The respiratory stimulation by arterial chemoreceptors tends to inhibit the medullary vagal center. This inhibition varies with the level of concomitant stimulation of respiration; small increases in respiration inhibit the vagal center slightly, whereas large increases in ventilation inhibit the vagal center more profoundly. An example of the primary inhibitory influence is shown in

1	An example of the primary inhibitory influence is shown in Fig. 18.10 . In this example, the lungs are completely collapsed, and blood oxygenation is accomplished with an artificial oxygenator. When the carotid chemoreceptors are stimulated, intense bradycardia and some degree of AV block ensue. Such effects are mediated primarily by efferent vagal fibers. The pulmonary hyperventilation that is ordinarily evoked by carotid chemoreceptor stimulation influences the heart rate secondarily, both by initiating more pronounced pulmonary inflation reflexes and by producing hypocapnia (see Fig. 18.9). Both influences tend to depress the primary cardiac response to chemoreceptor stimulation and thereby accelerate the heart rate. Hence, when pulmonary hyper- ventilation is not prevented, the primary and secondary effects neutralize each other, and carotid chemoreceptor stimulation affects the heart rate only moderately.

1	Sensory receptors located near the endocardial surfaces of the ventricles initiate reflex effects similar to those elicited •Fig. 18.10 Changes in Heart Rate With Carotid Chemoreceptor Stimulation During Total Heart Bypass. Thelungsremaindeflated,andrespiratorygasexchangeisaccomplishedbyanartificialoxygenator.Thelowertracingrepresentstheoxygensaturationofthebloodperfusingthecarotidchemoreceptors.Thebloodperfusingtheremainderofthebody,includingthemyocardium,isfullysaturatedwithoxygen.(ModifiedfromLevyMN,etal.Circ Res. 1966;18:67.)

1	Fig.18.11 wasrecordedfromaquadriplegicpatientwhocouldnotbreathespontaneouslyandrequiredtrachealintubationandartificialrespiration.Whenthetrachealcatheterwasbrieflydisconnected(nearthebeginningofthetopstripinthefigure,indicatedbythearrow)toallownursingcare,profoundbradycardiadevelopedafter9heartbeats.Thepatient’sheartratewas65beatsperminutejustbeforethetrachealcatheterwasdisconnected.Inlessthan10secondsaftercessationofartificialrespiration,hisheartratedroppedtoapproximately20beatsperminute.Thisbradycardiacouldbepreventedbyblockingtheeffectsofefferentvagalactivitywithatropine,anditsonsetcouldbedelayedconsiderablybyhyperventilationofthepatientbeforethetrachealcatheterisdisconnected.

1	by the arterial baroreceptors. Excitation of these endocardial receptors causes the heart rate and peripheral resistance to diminish. Other sensory receptors have been identified in the epicardial regions of the ventricles. Although all these ventricular receptors are excited by various mechanical and chemical stimuli, their exact physiological functions remain unclear. Regulation of Myocardial Performance Intrinsic Regulation of Myocardial Performance

1	As noted previously, the heart can initiate its own beat in the absence of any nervous or hormonal control. The myocardium can also adapt to changing hemodynamic conditions by means of mechanisms that are intrinsic to cardiac muscle itself. For example, racing greyhounds with •Fig. 18.11 Electrocardiogram of a 30-Year-Old Quadriplegic Man Who Could Not Breathe Spontaneously and Required Tracheal Intubation and Artificial Respiration. Thetwostripsarecontinuous.(ModifiedfromBerkJL,LevyMN.Eur Surg Res. 1977;9:75.) denervated hearts perform almost as well as those with intact innervation. Their maximal running speed decreases by only 5% after complete cardiac denervation. In these dogs, the threefold to fourfold increase in cardiac output during a race is achieved principally by an increase in stroke volume. Normally, the increase in cardiac output with exercise is accompanied by a proportionate increase in heart rate; stroke volume does not change much (see ). This adaptation in the

1	in stroke volume. Normally, the increase in cardiac output with exercise is accompanied by a proportionate increase in heart rate; stroke volume does not change much (see ). This adaptation in the denervated heart is not achieved entirely by intrinsic mechanisms; circulating catecholamines undoubtedly contribute. For example, if β-adrenergic receptor antagonists are given to greyhounds with denervated hearts, their racing performance is severely impaired.

1	Ventricularreceptorshavebeenimplicatedintheinitiationofvasovagal syncope, afeelingoflightheadednessorbrieflossofconsciousnessthatmaybetriggeredbypsychologicalororthostaticstress.Theventricularreceptorsarebelievedtobestimulatedbyreducedventricularfillingvolumeincombinationwithvigorousventricularcontraction.Inapersonstandingquietly,ventricularfillingisdiminishedbecausebloodtendstopoolintheveinsintheabdomenandlegs,asexplainedin Chapter17. Consequently,thereductionincardiacoutputandarterialbloodpressureleadstoageneralizedincreaseinsympatheticneuralactivitythroughthebaroreceptorreflex(see Fig.18.5 ).Theenhancedsympatheticactivitytotheheartevokesavigorousventricularcontractionthatstimulatestheventricularreceptors.Excitationoftheventricularreceptorsinitiatestheautonomicneuralchangesthatevokevasovagalsyncope:namely,acombinationofprofound,vagallymediatedbradycardiaandgeneralizedarteriolarvasodilationmediatedbyareductioninsympatheticneuralactivity.

1	Theheartispartiallyorcompletelydenervatedinvariousclinicalsituations:(1)asurgicallytransplantedheartistotallydenervated,althoughtheintrinsic,postganglionicparasympatheticfiberspersist;(2)atropineblocksvagaleffectsontheheart,andpropranololblockssympatheticβ-adrenergicinfluences;(3)certaindrugs,suchasreserpine,depletecardiacnorepinephrinestoresandtherebyrestrictorabolishsympatheticcontrol;and(4)inchroniccongestiveheartfailure,cardiacnorepinephrinestoresareoftenseverelydiminished,andanysympatheticinfluencesareattenuated. Two principal intrinsic mechanisms, the Frank-Starling mechanism and rate-induced regulation, enable the myocardium to adapt to changes in hemodynamic conditions. The Frank-Starling mechanism (Frank-Starling law of the heart) is invoked in response to changes in the resting length of myocardial fibers. Rate-induced regulation is evoked by changes in the frequency of the heartbeat. CHAPTER 18 Regulation of the Heart and Vasculature

1	CHAPTER 18 Regulation of the Heart and Vasculature In the 1910s, the German physiologist Otto Frank and the English physiologist Ernest Starling independently studied the response of isolated hearts to changes in preload and afterload (see Chapter 16). When ventricular filling pressure (preload) is increased, ventricular volume increases progressively, and after several beats, becomes constant and larger. At equilibrium, the volume of blood ejected by the ventricles (stroke volume) with each heartbeat increases to equal the greater quantity of venous return to the right atrium. The increased ventricular volume facilitates ventricular contraction and enables the ventricles to pump a greater stroke volume. This increase in ventricular volume is associated with an increase in length of the individual ventricular cardiac fibers. The increase in fiber length alters cardiac performance mainly by altering the number of myofilament cross-bridges that interact (see Chapter 16).

1	Chapter 16). More recent evidence indicates that the principal mechanism involves a stretch-induced change in the sensitivity of cardiac myofilaments to Ca++ (see ). There exists an optimal fiber length, however. Excessively high filling pressures that overstretch the myocardial fibers may depress rather than enhance the pumping capacity of the ventricles (see Fig. 16.36 Starling also showed that isolated heart preparations could adapt to changes in the counterforce to the ventricular ejection of blood during systole (i.e., afterload). As the left ventricle contracts, it does not eject blood into the aorta until the ventricle has developed a pressure that just exceeds the prevailing aortic pressure (see

1	Fig. 16.39 ). The aortic pressure during ventricular ejection essentially constitutes the left ventricular afterload. In Starling’s experiments, arterial pressure was controlled by a hydraulic device in the tubing that led from the ascending aorta to the right atrial blood reservoir. To hold venous return to the right atrium constant, the hydrostatic level of the blood reservoir was maintained. As Starling raised arterial pressure to a new constant level, the left ventricle responded at first to the increased afterload by pumping a diminished stroke volume. Because venous return was held constant, the diminution in stroke volume was accompanied by a rise in ventricular diastolic volume, as well as by an increase in the length of the myocardial fibers. This change in end-diastolic fiber length finally enabled the ventricle to pump a normal stroke volume against the greater peripheral resistance. As mentioned, a change in the number of cross-bridges between the thick and thin filaments

1	finally enabled the ventricle to pump a normal stroke volume against the greater peripheral resistance. As mentioned, a change in the number of cross-bridges between the thick and thin filaments probably contributes to this adaptation, but the major factor appears to be a stretch-induced change in the sensitivity of the contractile proteins to Ca++ .

1	Cardiac adaptation to alterations in heart rate also involves changes in ventricular volume. During bradycardia, for example, the increased duration of diastole allows greater ventricular filling. The consequent increase in myocardial fiber length increases stroke volume. Therefore, the reduction in heart rate may be fully compensated by the increase in stroke volume, and cardiac output may therefore remain constant. When cardiac compensation involves ventricular dilation, the effect of the increased size of the ventricle on the generation of intraventricular pressure must be considered. According to Laplace’s relationship (see

1	Chapter 17), if the ventricle enlarges, the force required by each myocardial fiber to generate a given intraventricular systolic pressure must be appreciably greater than that developed by the fibers in a ventricle of normal size. Thus more energy is required for a dilated heart to perform a given amount of external work than for a normal-sized heart to do so. Hence, computation of afterload on contracting myocardial fibers in the walls of the ventricles must account for ventricular dimensions along with intraventricular (and aortic) pressure.

1	The relatively rigid pericardium that encloses the heart determines the pressure-volume relationship at high levels of pressure and volume. The pericardium limits heart volume even under normal conditions, when an individual is at rest and the heart rate is slow. In patients with chronic congestive heart failure, the sustained cardiac dilation and hypertrophy may stretch the pericardium considerably. In such patients, the pericardial limitation of cardiac filling is exerted at pressures and volumes entirely different from those in normal individuals.

1	To assess changes in ventricular performance, the Frank-Starling mechanism is often represented by a family of ventricular function curves. To construct a control ventricular function curve, for example, blood volume is altered over a range of values, and stroke work (i.e., stroke volume × mean arterial pressure) and end-diastolic ventricular pressure are measured at each step. Similar observations are then made during the desired experimental intervention. For example, the ventricular function curve obtained during infusion of norepinephrine lies above and to the left of the control ventricular function curve (

1	Fig. 18.12). Clearly, for a given level of left ventricular end-diastolic pressure (an index of preload), the left ventricle performs more work during the norepinephrine infusion than during control conditions. Hence, the upward and leftward shift of the ventricular function curve signifies improved ventricular contractility. Conversely, a shift downward and to the right indicates impaired contractility and a tendency toward cardiac failure.

1	The Frank-Starling mechanism is well suited to match cardiac output to venous return. Any sudden, excessive output by one ventricle soon causes an increase in venous return to the second ventricle. The consequent increase in diastolic fiber length in the second ventricle augments the output of that ventricle to correspond to the output of its mate. In this way, the Frank-Starling mechanism maintains a precise balance between the output of the right and left ventricles. If the two ventricles were not arranged in series in a closed circuit, any small but maintained imbalance in output of the two ventricles would be catastrophic. •Fig. 18.12 Aconstantinfusionofnorepinephrineshiftstheventricu-larfunctioncurveupandtotheleft.Thisshiftsignifiesanenhancementinventricularcontractility.(RedrawnfromSarnoffSJ,etal.Circ Res. 1960;8:1108.)0 5 10 15 20 25 30 35 40 Norepinephrine Control 0 10 20 30 40 50 60 Left ventricular end-diastolic pressure (cm H2O) Left ventricular stroke work (g•m)

1	The curves that relate cardiac output to mean atrial pressure for the two ventricles do not coincide; the curve for the left ventricle usually lies below that for the right ventricle ( Fig. 18.13). At equal right and left atrial pressures Fig. 18.13 ), right ventricular output exceeds left ventricular output. Hence, venous return to the left ventricle (a function of right ventricular output) exceeds left ventricular output, and left ventricular diastolic volume and pressure rise. According to the Frank-Starling mechanism, left ventricular output therefore increases (from point B toward point C in Fig. 18.13 ). Only when the output of both ventricles is identical (points A and C) is equilibrium reached. Under such conditions, however, left atrial pressure (point C) exceeds right atrial pressure (point A). This is precisely the relationship that ordinarily prevails.

1	Thefactthatleftatrialpressureexceedsrightatrialpressureaccountsfortheobservationthatinindividualswithcongenitalatrialseptaldefectsinwhichthetwoatriacommunicatewitheachotherviaapatentforamenovale,thedirectionofshuntflowisusuallyfromlefttotherightsideoftheheart. •Fig. 18.13 Relationships Between the Output of the Right and Left Ventricles and Mean Pressure in the Right and Left Atria, Respectively. Atanygivenlevelofcardiacoutput,meanleftatrialpressure(e.g.,pointC)exceedsmeanrightatrialpressure(pointA).Atrial pressure Right Left A C B Cardiac output Force0.632020 •Fig. 18.14 Changesindevelopmentofforce(y-axis)inanisolatedpapillarymuscleastheintervalbetweencontractionsisvariedfrom20secondsto0.63secondandthenbackto20seconds.(RedrawnfromKoch-WeserJ,BlinksJR.Pharmacol Rev. 1963;15:601.)

1	Myocardial performance is also regulated by changes in the frequency at which the myocardial fibers contract. The effects of changes in contraction frequency on the force developed in an isometrically contracting papillary muscle are shown in Fig. 18.14 . Initially, the cardiac muscle is stimulated to contract once every 20 seconds. When the muscle is suddenly made to contract once every 0.63 seconds, the force developed increases progressively over the next several beats. At the new steady state, the force developed is more than five times greater than the force at the larger contraction interval. A return to the larger interval (20 seconds) has the opposite influence on the development of force.

1	The rise in the force developed when the contraction interval is decreased is caused by a gradual increase in intracellular [Ca++]. Two mechanisms contribute to the rise in intracellular [Ca++]: an increase in the number of depolarizations per minute and an increase in the inward Ca++ current per depolarization.

1	In the first mechanism, Ca++ enters the myocardial cell during each action potential plateau (see ). As the interval between beats is diminished, the number of plateaus per minute increases. Although the duration of •Fig. 18.15 Calcium Currents Induced in a Myocyte During the First (Labeled 1) and Seventh (Labeled 7) Depolarizations in a Consecutive Sequence of Depolarizations. Thearrows indicatethehalf-timesofinactivationofthecalciumcurrent,asobtainedfromkineticanalysis.Bytheseventhdepolarization,themaximalinwardrectifyingcalciumcurrenthadincreasedbymorethan50%,andthehalf-timeofinactivationhadincreasedby20msec.(ModifiedfromLeeKS.Proc Natl Acad Sci U S A. 1987;84:3941.) each action potential (and of each plateau) decreases as the interval between beats is reduced, the overriding effect of the increased number of plateaus per minute on the influx of Ca++ prevails, and intracellular [Ca++] increases.

1	In the second mechanism, as the interval between beats is suddenly diminished, the inward rectifying calcium current (iCa) progressively increases with each successive beat until a new steady state is attained at the new basic cycle length. In an isolated ventricular myocyte, influx of Ca++ into the myocyte increases on successive depolarizations ( Fig. 18.15 ). Both the increased magnitude and the slowed inactivation of iCa result in greater Ca++ influx into the myocyte during the later depolarizations than during the first depolarization. This greater Ca++ influx strengthens contraction. Transient changes in the intervals between beats also profoundly affect the strength of contraction. When the left ventricle contracts prematurely (

1	Transient changes in the intervals between beats also profoundly affect the strength of contraction. When the left ventricle contracts prematurely ( Fig. 18.16 , beat A), the premature contraction (extrasystole) itself is weak, whereas contraction B (postextrasystolic contraction) after the compensatory pause is very strong. In the intact circulatory system, this response depends partly on the Frank-Starling mechanism. Inadequate time for ventricular filling just before the premature beat results in the weak premature contraction. Subsequently, the exaggerated degree of filling associated with the long compensatory pause (see Fig. 18.16 , beat B) contributes to the vigorous postextrasystolic contraction.

1	Fig. 18.16 , beat B) contributes to the vigorous postextrasystolic contraction. The weakness of the premature beat is directly related to its degree of prematurity: The earlier the premature beat, the weaker its force of contraction. The curve that represents strength of contraction of a premature beat in relation to the coupling interval is called a mechanical restitution curve. Fig. 18.17 shows the restitution curve obtained when the coupling intervals of test beats were isolated in an isolated ventricular muscle preparation. 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 Time (sec) •Fig. 18.16 Inanisovolumicleftventriclepreparation,aprematureventricularsystoliccontraction(beat

1	0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 Time (sec) •Fig. 18.16 Inanisovolumicleftventriclepreparation,aprematureventricularsystoliccontraction(beat A)istypicallyweak,whereasthepostextrasystoliccontraction(beatB)ischaracteristicallystrong,andtheenhancedcontractilitymaydiminishoverafewcontractions(e.g.,beatC).(FromLevyMN.Unpublishedtracing.) 100 80 60 40 20 0 0 0.2 0.4 0.6 0.8 1.0 Test beat interval (sec) Test beat force (% of max) relatively large amount of Ca++ taken up by it during the time that had elapsed from the end of the last regular beat until the beginning of the postextrasystolic beat. Extrinsic Regulation of Myocardial Performance Although a completely isolated heart can adapt well to changes in preload and afterload, various extrinsic factors also influence the heart in an individual. Often, these extrinsic regulatory mechanisms may overwhelm the intrinsic mechanisms. The extrinsic regulatory factors may be subdivided into nervous and chemical components.

1	•Fig. 18.17 Force Generated During Premature Contractions in an Isolated Ventricular Muscle Preparation. Themusclewasstimulatedtocontractoncepersecond.Periodically,themusclewasstimulatedprematurely.Thescalealongthex-axisdenotesthetimebetweenthedrivenandtheprematurebeat.Thescalealongthey-axisdenotestheratioofthecontractileforceoftheprematurebeattothatofthedrivenbeat.(ModifiedfromSeedWA,WalkerJM.Cardiovasc Res. 1988;22:303.)

1	Restitution of the force of contraction depends on the time course of the intracellular circulation of Ca++ in cardiac myocytes during contraction and relaxation. During relaxation, the Ca++ that dissociates from the contractile proteins is taken up by the sarcoplasmic reticulum for subsequent release. However, there is a lag of approximately 500 to 800 msec before this Ca++ is available for release from the sarcoplasmic reticulum in response to the next depolarization. Thus the strength of the premature beat is reduced because the time during the preceding relaxation is insufficient to allow much of the Ca++ taken up by the sarcoplasmic reticulum to become available for release during the premature beat. Conversely, the postextrasystolic beat is considerably stronger than normal because more Ca++ is released from the sarcoplasmic reticulum as a result of the

1	Sympathetic nervous activity enhances atrial and ventricular contractility. The alterations in ventricular contraction evoked by electrical stimulation of the left stellate ganglion in an isovolumic left ventricle preparation are shown in Fig. 18.18 . Note that the duration of systole is reduced and the rate of ventricular relaxation is increased during the early phases of diastole; both these effects assist ventricular filling. For any given cardiac cycle length, the abbreviated systole allows more time for diastole and hence for ventricular filling. ). Neurally released norepinephrine or circulating catecholamines interact with β-adrenergic receptors on the cardiac cell membranes (

1	). Neurally released norepinephrine or circulating catecholamines interact with β-adrenergic receptors on the cardiac cell membranes ( Fig. 18.19 ). This interaction activates adenylate cyclase, which raises intracellular levels of cAMP (see ). Consequently, protein kinases that promote the phosphorylation of various proteins are activated within the myocardial cells. Phosphorylation of phospholamban facilitates reuptake of Ca++ by the sarcoplasmic reticulum, and phosphorylation of troponin I reduces the sensitivity of contractile proteins to Ca++ . These effects facilitate relaxation and reduce end-diastolic pressure (see ). Phosphorylation of specific sarcolemmal proteins also activates calcium channels in the membranes of myocardial cells.

1	Activation of calcium channels increases the influx of Ca++ during the action potential plateau, and more Ca++ is released from the sarcoplasmic reticulum in response to each cardiac excitation. The contractile strength of the heart is thereby increased. Fig. 18.20 shows the correlation between the contractile force in a thin strip of ventricular muscle and the free [Ca++] (indicated by the aequorin light signal) in the myoplasm as the concentration of isoproterenol (a β-adrenergic agonist) is increased (see also Fig. 13.3

1	Fig. 13.3 The overall effect of increased cardiac sympathetic activity in intact animals can best be appreciated in terms of 0.1 0.2 0.3 0.4 0.5 0.6 0 0.1 0.2 0.3 0.4 0.5 0.6 Time (sec) •Fig. 18.18 Inanisovolumicleftventriclepreparation,stimulationofcardiacsympatheticnervesevokesasubstantialriseinpeakleftventricularpressureandinthemaximalratesofriseandfallinintraventricularpressure(dP/dt).(FromLevyMN.Unpublishedtracing.) families of ventricular function curves. When the frequency of electrical stimulation applied to the left stellate ganglion increases, the ventricular function curves shift progressively to the left. The changes parallel those produced by infusions of norepinephrine (see

1	Fig. 18.12 ). Hence, for any given left •Fig. 18.19 Interneuronal and Intracellular Mechanisms Responsible for Interactions Between the Sympathetic and Parasympathetic Systems in the Neural Control of Cardiac Function. AC,adenylatecyclase;ACh,acetycholine;ATP,adenosinetriphosphate;β,β-adrenergicreceptor;βγ,beta/gamma;cAMP,cyclicadenosinemonophosphate;GsandGi,stimulatoryandinhibitoryGproteins;M,muscarinicreceptor;NE,norepinephrine;NPY,neuropeptideY.(ModifiedfromLevyMN.In:KulbertusHE,FranckG,eds.Neurocardiology. Mt.Kisco,NY:Futura;1988.) •Fig. 18.20 Effects of Various Concentrations of Isoproterenol (Iso) on the Aequorin Light Signal (in Nanoamperes) and Contractile Force (in Millinewtons Per Square Millimeter) in a Rat Ventricular Muscle Injected With Aequorin. Theaequorinlightsignalreflectstheinstantaneouschangesinintracel-lular[Ca++].µM,micromolar.(ModifiedfromKuriharaS,KonishiM.Pflügers Arch. 1987;409:427.)0.1 sec Force Aequorin light signal 20 nA 0.2 µM Iso.0.1 µM Iso.0.05 µM

1	Arch. 1987;409:427.)0.1 sec Force Aequorin light signal 20 nA 0.2 µM Iso.0.1 µM Iso.0.05 µM Iso.Control 5 mN mm2 A B C D ventricular end-diastolic pressure, the ventricle can perform more work as sympathetic nervous activity is increased.

1	The vagus nerves inhibit the cardiac pacemaker, atrial myocardium, and AV conduction tissue. The vagus nerves also depress the ventricular myocardium, but the effects are less pronounced than in the atria. In pumping heart preparations, the ventricular function curve shifts to the right during vagal stimulation, an indication of reduced contractility. Vagus nerve stimulation suppresses the contractile force in the left ventricle. This is shown in pressure-volume curves obtained at constant ventricular rate ( Fig. 18.21 ). The negative inotropic effect of vagus nerve stimulation, depicted as the reduced slope of the end-systolic pressure-volume relation, is opposed by a muscarinic receptor antagonist and diminished by a β-adrenoceptor antagonist. The results indicate that vagus nerve stimulation reduces contractility in the heart and does so by at least two pathways. Mechanisms for the vagal effects on the ventricular myocardium are shown in

1	Mechanisms for the vagal effects on the ventricular myocardium are shown in Fig. 18.19 . ACh released by the vagal nerves interact with muscarinic receptors on the cardiac ventricular cell membrane to inhibit adenylate cyclase and the cAMP/protein kinase A cascade. This direct inhibition diminishes the Ca++ conductance of the cell membrane, reduces phosphorylation of the calcium channel, and hence decreases myocardial contractility. The ACh released from vagal nerves can also inhibit norepinephrine release by activating muscarinic receptors on neighboring sympathetic nerves, a mechanism of indirect inhibition. Thus vagal activity can decrease ventricular contractility partly by antagonizing any stimulatory effects that concomitant sympathetic activity may be exerting on ventricular contractility. Similarly, sympathetic nerves release norepinephrine and certain neuropeptides, including neuropeptide Y, which inhibits the release of ACh from neighboring vagal fibers (see Fig. 18.19

1	Fig. 18.19 The adrenal medulla is essentially a component of the auto ). The principal hormone secreted by the adrenal medulla is epinephrine; some norepinephrine is also released. The rate of secretion of these catecholamines by the adrenal medulla is regulated by mechanisms that control the activity of the sympathetic nervous system. Concentrations of catecholamines in blood thus rise under the same conditions that activate the sympathetic nervous system. However, the cardiovascular effects of circulating catecholamines are probably minimal under normal conditions. Moreover, the pronounced changes in myocardial contractility with exercise, for example, are mediated mainly by the norepinephrine released from cardiac sympathetic nerve fibers rather than by the catecholamines released from the adrenal medulla.

1	How adrenocortical steroids influence myocardial contractility is controversial. Cardiac muscle taken from adrenalectomized animals and placed in a tissue bath is more likely to fatigue in response to stimulation than is cardiac muscle obtained from normal animals. In some species, however, adrenocortical hormones enhance contractility. In addition, the glucocorticoid hydrocortisone potentiates the cardiotonic effects of catecholamines. This potentiation is mediated in part by the ability of adrenocortical steroids to inhibit the extraneuronal catecholamine uptake mechanisms.

1	•Fig. 18.21 Vagus Nerve Stimulation Reduces Ventricular Con tractility. Pressure-volumecurvesobtainedatconstantventricular ratebeforeopenheartsurgeryinhuman.A, Controlpressure-volumecurveswerecalculatedduringocclusionoftheinferiorvenacava.Theend-systolicpressure-volumerelation,definedbytheslopeofthestraight line, measuredapproximately4mmHg/mL.B, Duringstimulationoftheleftvagusnerve,theslopeoftheend-systolicpressure-volumerelationdecreasedtoapproximately3mmHg/mL,anindicationthatcontractilityhaddecreased.LV,leftventricular.(RedrawnfromLewisME,Al-KhalidiAH,BonserRS,etal.Vagusnervestimulationdecreasesleftventricularcontractilityinvivointhehumanandpigheart.J Physiol. 2001;534:547.)

1	Thyroid hormones enhance myocardial contractility. Rates of ATP hydrolysis and Ca++ uptake by the sarcoplasmic reticulum are increased in hyperthyroidism; the opposite effects occur in hypothyroidism. Thyroid hormones increase cardiac protein synthesis, and this response leads to cardiac hypertrophy. These hormones also affect the composition of myosin isoenzymes in cardiac muscle. By increasing isoenzymes with the greatest ATPase activity, thyroid hormones enhance myocardial contractility. Cardiovascularproblemsarecommoninpatientswithadrenocorticalinsufficiency(Addison’sdisease).Bloodvolume The cardiovascular changes in thyroid dysfunction also depend on indirect mechanisms. Thyroid hyperactivity tendstofall,whichmayleadtoseverehypotensionandcardiovascularcollapse,theso-calledaddisoniancrisis(see

1	Thyroidhormoneexertsitscardiacactionsbytwopaths:genomicandnongenomic.Thegenomicrouteinvolvesinteractionofthyroxine(T3)withnuclearreceptorsthatregulatethetranscriptionofT3-responsivegenes.Inhyperthyroidism,messengermRNAforcardiacmyocyteproteinsinvolvedinregulatingintracellular[Ca++](e.g.,sarcoplasmicendoplasmicreticulumcalcium-ATPase[SERCA],ryanodinechannel)isincreased,asareamountsofcontractileproteins(e.g.,myosinheavychain,actin,troponinI).Consequently,theratesofcontractionandrelaxationincreaseasATPhydrolysisandO2consumptionincrease.Inthehyperthyroidstate,theuseofATPislessefficient,andthefractionallossofheatisgreater.Ifuntreated,severehyperthyroidismcanresultinheartfailure.

1	Cardiacactivityisdepressedinpatientswithinadequatethyroidfunction(hypothyroidism).Theconverseistrueinpatientswithoveractivethyroidglands(hyperthyroidism).Characteristically,patientswithhyperthyroidismexhibittachycardia,highcardiacoutput,andarrhythmiassuchasatrialfibrillation.Insuchpatients,sympatheticneuralactivitymaybeincreased,orthesensitivityofthehearttosuchactivitymaybeenhanced.Studieshaveshownthatthyroidhormoneincreasesthedensityofβ-adrenergicreceptorsincardiactissue(seealso ).Inexperimentalanimals,thecardiovascularmanifestationsofhyperthyroidismmaybesimulatedbytheadministrationofexcessthyroxine. increases the body’s metabolic rate, which in turn results in arteriolar vasodilation. The consequent reduction in total peripheral resistance increases cardiac output, as explained in Insulin has a positive inotropic effect on the heart. The effect of insulin is evident even when hypoglycemia is prevented by glucose infusions and when β-adrenergic receptors are

1	Insulin has a positive inotropic effect on the heart. The effect of insulin is evident even when hypoglycemia is prevented by glucose infusions and when β-adrenergic receptors are CHAPTER 18 Regulation of the Heart and Vasculature blocked. Indeed, the positive inotropic effect of insulin is potentiated by β-adrenergic receptor antagonists. The enhanced contractility cannot be explained satisfactorily by the concomitant augmentation of glucose transport into myocardial cells.

1	Glucagon has potent positive inotropic and chronotropic effects on the heart. This endogenous hormone is probably not important in normal regulation of the cardiovascular system, but it has been used clinically to enhance cardiac performance. The effects of glucagon on the heart and certain metabolic effects are similar to those of catecholamines. Both glucagon and catecholamines activate adenylate cyclase to increase myocardial levels of cAMP. The catecholamines activate adenylate cyclase by interacting with β-adrenergic receptors, but glucagon activates this enzyme by a different mechanism. Nevertheless, the rise in cAMP increases influx of Ca++ through calcium channels in the sarcolemma and facilitates release and reuptake of Ca++ by the sarcoplasmic reticulum, just as catecholamines do.

1	The cardiovascular derangements in hypopituitarism are related principally to the associated deficiencies in adrenocortical and thyroid function. Growth hormone affects the myocardium, at least in combination with thyroxine. In hypophysectomized animals, growth hormone alone has little effect on the depressed heart, whereas thyroxine by itself restores adequate cardiac performance under basal conditions. However, when blood volume or peripheral resistance is increased, thyroxine alone does not restore adequate cardiac function, but the combination of growth hormone and thyroxine reestablishes normal cardiac performance. In certain animal models of heart failure, administration of growth hormone alone increases cardiac output and myocardial contractility.

1	Changes in cardiac performance as a result of stimulation of central and peripheral chemoreceptors have been described previously in this chapter. These effects usually predominate. However, O2 and CO2 do have direct effects on the myocardium. Hypoxia has a biphasic effect on myocardial performance. Mild hypoxia stimulates performance, but more severe hypoxia depresses performance because oxidative metabolism is limited. An increase in partial pressure of carbon dioxide (PCO2)— which results in a decrease in pH—has a direct depressant effect on the heart. This effect is mediated by changes in intracellular pH. A reduction in intracellular pH, induced by an increase in PCO2, diminishes the amount of Ca++ released from the sarcoplasmic reticulum in response to excitation. The diminished pH also decreases the sensitivity of the myofilaments to Ca++ . Increases in intracellular pH have the opposite effect; that is, they enhance sensitivity to Ca++ .

1	Regulation of the Peripheral Circulation The peripheral circulation is essentially under dual control: centrally through the nervous system and locally by conditions in tissues surrounding the blood vessels. Nervous and humoral regulation of vascular smooth muscle is described in Fig. 14.7 Table 14.1 ), in which transmitters, hormones and their receptors are discussed. Aspects of local control are discussed in , in which the relative importance of these two control mechanisms is shown to vary in different tissues. The arterioles are involved in regulating the rate of blood flow throughout the body. These vessels offer the greatest resistance to the flow of blood pumped to the tissues by the heart, and thus these vessels are important in the maintenance of arterial blood pressure. The arteriole walls are composed in large part of smooth muscle fibers that allow the diameter of the vessel lumen to vary (see Fig.

1	Fig. 15.2 ). When this smooth muscle contracts strongly, the endothelial lining folds inward and completely obliterates the vessel lumen. When the smooth muscle is completely relaxed, the vessel lumen is maximally dilated. Some resistance vessels are closed at any given time. In addition, the smooth muscle in these vessels is partially contracted (which accounts for the tone of these vessels). If all the resistance vessels in the body dilated simultaneously, arterial blood pressure would fall precipitously. Vascular smooth muscle controls total peripheral resistance, arterial and venous tone, and the distribution of blood flow throughout the body. The properties of vascular smooth muscle are discussed in . In the following sections, intrinsic and extrinsic control of vascular smooth muscle tone, and thus perfusion of peripheral tissues, is reviewed. Intrinsic or Local Control of Peripheral Blood Flow

1	Intrinsic or Local Control of Peripheral Blood Flow In certain tissues, blood flow is adjusted to the existing metabolic activity of the tissue. Furthermore, when tissue metabolism is steady, changes in perfusion pressure (arterial blood pressure) evoke changes in vascular resistance that tend to maintain a constant blood flow. This myogenic mechanism, which is illustrated graphically in Fig. 18.22 , is commonly referred to as autoregulation of blood flow. When pressure is abruptly increased or decreased from a control pressure of 100 mm Hg, flow increases or decreases, respectively. However, even with pressure maintained at its new level, blood flow returns toward the control level within 30 to 60 seconds.

1	•Fig. 18.22 Pressure-flowrelationshipinthevascularbedoftheskeletalmuscle.Filled (red) circles representtheflowratesobtainedimmediatelyafterabruptchangesinperfusionpressurefromthecontrollevel(thepointwherelinescross).Open (blue) circles representthesteady-stateflowratesobtainedatthenewperfusionpressure.(RedrawnfromJonesRD,BerneRM.Circ Res. 1964;14:126.)0 20 40 60 10080 120 140 160 180 0 2 4 6 8 10 12 Perfusion pressure (mm Hg) Muscle blood flow (mL/min/100 g)

1	Over the pressure range of 20 to 120 mm Hg, the steady-state flow is relatively constant. Calculation of hydraulic resistance (pressure/flow) across the vascular bed during steady-state conditions shows that the resistance vessels constrict with an elevation in perfusion pressure but dilate with a reduction in perfusion pressure. This response to perfusion pressure is independent of the endothelium because it is identical in intact vessels and in vessels that have been stripped of their endothelium. According to the myogenic mechanism, vascular smooth muscle contracts in response to an increase in the pressure difference across the wall of a blood vessel (transmural pressure), and it relaxes in response to a decrease in transmural pressure. The signaling mechanisms that allow distention of a vessel to elicit contraction are unknown. However, because stretch of vascular smooth muscle has been shown to raise intracellular [Ca++], an increase in transmural pressure is believed to

1	of a vessel to elicit contraction are unknown. However, because stretch of vascular smooth muscle has been shown to raise intracellular [Ca++], an increase in transmural pressure is believed to activate membrane calcium channels.

1	Transientreceptorpotential(TRP)channelshavebeenimplicatedinthemyogenicmechanism.ThesechannelsaremammalianhomologuesofaDrosophila melanogaster genethat,whenmutated,allowsonlyatransientresponsetoasustainedlightstimulus.Thepressure-inducedvasoconstrictiveresponseofanartery(myogenicresponse)appearstohavethefollowingsignalpath:pressure → increasedphospholipaseCactivity→ synthesisofdiacylglycerol→ activationofTRPchannel→ smoothmuscledepolarizationandopeningofL-typecalciumchannelsthatincreaseintracellular[Ca++]andmuscletone.Thisisameansofregulatingvascularresistance.OtherTRPchanneltypeshavebeenproposedtoparticipateinchronichypoxicpulmonaryhypertensionandinthevasoconstrictioncausedbytheα-adrenergicagonistnorepinephrine.

1	In normal subjects, blood pressure is maintained at a fairly constant level via the baroreceptor reflex. Hence, the myogenic mechanism may play little role in regulating blood flow to tissues under normal conditions. However, when a person changes from a lying to a standing position, transmural pressure rises in the lower extremities, and the precapillary vessels constrict in response to this imposed stretch. , the endothelium lining the vasculature produces a number of substances that can relax (e.g., nitric oxide) or contract (e.g., angiotensin II and endothelin) vascular smooth muscle. Thus the endothelium plays an important role in regulating blood flow to specific vascular beds.

1	The metabolic activity of a tissue governs blood flow in that tissue. Any intervention that results in inadequate O2 supply prompts the formation of vasodilator metabolites that are released from the tissue and act locally to dilate the resistance vessels. When the metabolic rate of the tissue increases, or when O2 delivery to the tissue decreases, more vasodilator substances are released (see Chapter 17).

1	Chapter 17). Potassium, inorganic phosphate ions, and interstitial fluid osmolarity induce vasodilation. During skeletal muscle contraction, both (1) K+ and phosphate are released and (2) osmolarity is increased. Therefore, these factors may contribute to active hyperemia (increase in blood flow caused by enhanced tissue activity). However, significant increases in the phosphate concentration and in osmolarity are not always observed during muscle contraction, and they may increase blood flow only transiently. Therefore, they probably do not mediate the vasodilation observed during muscular activity.

1	Potassium is released at the onset of skeletal muscle contraction or with an increase in cardiac muscle activity. Hence, release of K+ could underlie the initial decrease in vascular resistance observed in response to physical exercise or to increased cardiac work. However, release of K+ is not sustained, but arteriolar dilation persists throughout the period of enhanced muscle activity. Furthermore, reoxygenated venous blood obtained from active cardiac and skeletal muscles does not elicit vasodilation when the blood is infused into a test vascular bed. It is unlikely that oxygenation of venous blood alters its K+ or phosphate content or its osmolarity and thereby neutralizes its vasodilator effect. Therefore, some agent other than K+ must mediate the vasodilation associated with metabolic activity of the tissue.

1	Adenosine, which contributes to the regulation of coronary blood flow, may also participate in control of the resistance vessels in skeletal muscle. In addition, some prostaglandins may be important vasodilator mediators in certain vascular beds. Many prostaglandins have thus been CHAPTER 18 Regulation of the Heart and Vasculature proposed as mediators of metabolic vasodilation, and the relative contribution of each remains to be determined.

1	CHAPTER 18 Regulation of the Heart and Vasculature proposed as mediators of metabolic vasodilation, and the relative contribution of each remains to be determined. Metabolic control of vascular resistance by the release of a vasodilator substance requires the existence of a basal vessel tone. Tonic activity in vascular smooth muscle is readily demonstrable, but in contrast to tone in skeletal muscle, the tone in vascular smooth muscle is independent of the nervous system. Thus some metabolic factor must be responsible for maintaining this tone. The following factors may be involved: (1) the myogenic response to the stretch imposed by blood pressure, (2) the high partial pressure of oxygen in arterial blood (PaO2), or (3) the presence of Ca++ .

1	If arterial inflow to a vascular bed is stopped temporarily, blood flow on release of the occlusion immediately exceeds the flow that prevailed before occlusion, and the flow gradually returns to the control level. This increase in blood flow is called reactive hyperemia. This type of event provides evidence for the existence of a local metabolic factor that regulates tissue blood flow. In the experiment shown in Fig. 18.23 , blood flow to the leg was stopped by clamping of the femoral artery for 15, 30, and 60 seconds. Release of the 60-second occlusion resulted in a peak blood flow that was 70% greater than the control flow, and the flow returned to the control level within 110 seconds. Within limits, peak flow and particularly the duration of reactive hyperemia are proportional to the duration of the occlusion (see

1	Within limits, peak flow and particularly the duration of reactive hyperemia are proportional to the duration of the occlusion (see Fig. 18.23 ). If the extremity is exercised during the occlusion period, reactive hyperemia is increased. These observations and the close relationship between metabolic activity and blood flow in an unoccluded limb are consistent with the notion of a metabolic mechanism in the local regulation of tissue blood flow. •Fig. 18.23 Graphsofreactivehyperemiainthehindlimbofthelegafter15-,30-,and60-secondocclusionofthefemoralartery.(FromBerneRM.Unpublishedobservations.)0 2 4 6 8 10 0 20 40 60 80 100 Time (min) Femoral blood flow(mL/min) 0 20 40 60 80 100 120 Femoral arterial pressure(mm Hg) Coordination of Arterial and Arteriolar Dilation

1	When the vascular smooth muscle of arterioles relaxes in response to vasodilator metabolites whose release is caused by a decrease in the ratio of O2 supply to O2 demand of the tissue, resistance may diminish concomitantly in the small upstream arteries that feed these arterioles. The result is blood flow greater than that produced by arteriolar dilation alone. There are two possible mechanisms for this coordination of arterial and arteriolar dilation. First, the vasodilation in the microvessels may be propagated, and when dilation is initiated in the arterioles, it can propagate along the vessels from the arterioles back to the small arteries. Second, the metabolite-mediated dilation of the arterioles accelerates blood flow in the feeder arteries. This greater blood flow velocity increases the shear stress on the arterial endothelium, which in turn can induce flow-mediated vasodilation by release of one or more vasodilators (e.g., nitric oxide, prostacyclin, H2O2, epoxyeicosatrienoic

1	the shear stress on the arterial endothelium, which in turn can induce flow-mediated vasodilation by release of one or more vasodilators (e.g., nitric oxide, prostacyclin, H2O2, epoxyeicosatrienoic acid).

1	Inthelegs,diseaseofthearterialwallscanleadtoobstructionofthearteriesandsymptoms,aconditioncalledintermittent claudication. Thesymptomsconsistoflegpainwhenthepersonwalksorclimbsstairs,andthepainisrelievedbyrest.Thediseaseiscalledthromboangiitis obliterans, anditappearsmostfrequentlyinmenwhoaresmokers.Withminimalwalking,theresistancevesselsbecomemaximallydilatedbylocalreleaseofmetabolites;whentheO2demandofthemusclesincreaseswithmorerapidwalking,bloodflowcannotincreasesufficientlytomeetthemuscleneedsforO2,andpaincausedbymuscleischemiaresults. Extrinsic Control of Peripheral Blood Flow

1	Extrinsic Control of Peripheral Blood Flow Several regions in the cerebral medulla influence cardiovascular activity. Stimulation of the dorsal lateral medulla (pressor region) evokes vasoconstriction, cardiac acceleration, and enhanced myocardial contractility. Stimulation of cerebral centers caudal and ventromedial to the pressor region decreases arterial blood pressure. This depressor area exerts its effect by direct inhibition of spinal regions and by inhibition of the medullary pressor region. These areas are not true anatomical centers in which a discrete group of cells is discernible, but they constitute “physiological” centers.

1	The cerebrospinal vasoconstrictor regions are tonically active. Reflexes or humoral stimuli that enhance this activity increase the frequency of impulses that reach the terminal neural branches to the vessels. A constrictor neurohumor (norepinephrine) is released at the terminals to elicit a constrictive α-adrenergic effect on the resistance vessels. Inhibition of the vasoconstrictor areas diminishes the impulse frequency in the efferent nerve fibers, and vasodilation results. Thus neural regulation of the peripheral circulation is achieved mainly by alteration in the impulse frequency in the sympathetic nerves to the blood vessels. Surgical section of the sympathetic nerves to an extremity abolishes sympathetic vascular tone and thereby increases blood flow to that limb. With time, vascular tone is regained by an increase in basal (intrinsic) tone.

1	Both the pressor and depressor regions may undergo rhythmic changes in tonic activity that are manifested as oscillations in arterial pressure. Some rhythmic changes (Traube-Hering waves) occur at the frequency of respiration and are caused by a cyclic fluctuation in sympathetic impulses to the resistance vessels. Other fluctuations in sympathetic activity (Mayer waves) occur at a frequency lower than that of respiration.

1	Vasoconstrictor fibers of the sympathetic nervous system supply the arteries, arterioles, and veins; the neural influence is much less on larger vessels than on arterioles and small arteries. Capacitance vessels (veins) respond more to sympathetic nerve stimulation than do resistance vessels; the capacitance vessels are maximally constricted at a lower stimulation frequency than the are resistance vessels. However, capacitance vessels lack β-adrenergic receptors, and they respond less to vasodilator metabolites. Norepinephrine is the neurotransmitter released at the sympathetic nerve terminals in blood vessel. Factors such as circulating hormones and particularly locally released substances mediate the release of norepinephrine from the nerve terminals. The response of the resistance and capacitance vessels to stimulation of sympathetic fibers is illustrated in Fig.

1	The response of the resistance and capacitance vessels to stimulation of sympathetic fibers is illustrated in Fig. 18.24 . When arterial pressure is held constant, stimulation of sympathetic fibers reduces blood flow (constriction of resistance vessels) and decreases the blood volume of the tissue (constriction of capacitance vessels). Constriction of the resistance vessels establishes a new equilibrium of the forces responsible for filtration and absorption across the capillary wall (see Eq. 17.20 In addition to active changes (contraction and relaxation of vascular smooth muscle) in vessel caliber, passive changes are also caused by alterations in intraluminal pressure. An increase in intraluminal pressure distends the vessels, and a decrease reduces the caliber of the vessels as a consequence of elastic recoil of the vessel walls.

1	At basal vascular tone, approximately a third of the blood volume of a tissue can be mobilized when the sympathetic nerves are stimulated at physiological frequencies. Basal tone is very low in capacitance vessels; if these vessels are denervated experimentally, the increases in volume evoked

1	Change of tissue Arterial pressureBlood flow volume (mL) (mm Hg) •Fig. 18.24 Effect of Sympathetic Nerve Stimulation (2 Hz) on Blood Flow and Tissue Volume of the Lower Limb. Theupward arrow denotesthechangeinslopeofthetissuevolumecurveatthepointatwhichthedecreaseinvolumecausedbyemptyingofcapacitancevesselsceasesandlossofextravascularfluidbecomesevident.Theabruptdecreaseintissuevolumeiscausedbymovementofbloodoutofthecapacitancevesselsandoutofthelowerlimb.Thelate,slow,progressivedeclineinvolume(totherightofthearrow)iscausedbythemovementofextravascularfluidintothecapillariesandhenceawayfromthetissue.Thelossoftissuefluidresultsfromtheloweringofthecapillaryhydrostaticpressuresecondarytoconstrictionoftheresistancevessels.(FromMellanderS.Acta Physiol Scand Suppl. 1960;50[176]:1.) by maximal doses of ACh are small. Therefore, at basal vascular tone, blood volume is close to the maximal blood volume of the tissue. More blood can be mobilized from the capacitance vessels in the skin than from

1	of ACh are small. Therefore, at basal vascular tone, blood volume is close to the maximal blood volume of the tissue. More blood can be mobilized from the capacitance vessels in the skin than from those in the muscle. This disparity depends in part on the greater sensitivity of the skin vessels to sympathetic stimulation, but it also occurs because basal tone is lower in skin vessels than in muscle vessels. Therefore, in the absence of a neural influence, skin capacitance vessels contain more blood than do muscle capacitance vessels.

1	Physiological stimuli mobilize blood from capacitance vessels. For example, during physical exercise, activation of sympathetic nerve fibers constricts the peripheral veins and hence augments cardiac filling pressure. In arterial hypo-tension (as occurs in hemorrhage), the capacitance vessels constrict and thereby correct the decreased central venous pressure associated with blood loss. The efferent fibers of the cranial division of the parasympathetic nervous system innervate the blood vessels of the head and some of the viscera, whereas fibers of the sacral division innervate blood vessels of the genitalia, bladder, CHAPTER 18 Regulation of the Heart and Vasculature and large bowel. Skeletal muscle and skin do not receive parasympathetic innervation. The effect of cholinergic fibers on total vascular resistance is small because only a small proportion of the resistance vessels of the body receive parasympathetic fibers.

1	Inhemorrhagicshock,theresistancevesselsconstrictandtherebyassistinthemaintenanceofnormalarterialbloodpressure.Witharterialhypotension,theenhancedarteriolarconstrictionalsoleadstoasmallmobilizationofbloodfromthetissuebyvirtueofrecoilofthepostarteriolarvesselswhenintraluminalpressureisreduced.Furthermore,extravascularfluidismobilizedbecauseofgreaterfluidabsorptionintothecapillariesinresponsetotheloweredcapillaryhydrostaticpressure. Stimulation of the parasympathetic fibers to the salivary glands induces marked vasodilation. A vasodilator polypeptide, bradykinin, formed locally from the action of an enzyme on a plasma protein substrate in the glandular lymphatic vessels, mediates this vasodilation. Bradykinin is formed in other exocrine glands, such as the lacrimal and sweat glands. Its presence in sweat may be partly responsible for the dilation of cutaneous blood vessels.

1	Epinephrine and norepinephrine exert a powerful effect on peripheral blood vessels. In skeletal muscle, low concentrations of epinephrine dilate resistance vessels (β2-adrenergic effect), but high concentrations produce constriction (α1adrenergic effect), as noted in Table 14.1. In all vascular beds, the primary effect of norepinephrine is vasoconstriction. When stimulated, the adrenal gland can release epinephrine and norepinephrine into the systemic circulation. However, under physiological conditions, the effect of catecholamine release from the adrenal medulla is less important than norepinephrine release from sympathetic nerve endings. Table 14.1 ). Some are released from the endothelium (e.g., nitric oxide, endothelin, thromboxane A2), whereas others are derived from perivascular tissues (e.g., histamine, adenosine, angiotensin II).

1	Areas of the cerebral medulla that mediate sympathetic and vagal effects are under the influence of neural impulses that originate in the baroreceptors, chemoreceptors, hypothalamus, cerebral cortex, and skin. These areas of the medulla are also affected by changes in the blood concentrations of CO2 and O2. The baroreceptors (or pressoreceptors) are stretch receptors located in the carotid sinuses and in the aortic arch ( Figs. 18.25 18.26 ). The carotid sinuses are the slightly Fig. 18.25 Diagrammaticrepresentationofthecarotidsinusandcarotidbodyandtheirinnervation.(RedrawnfromAdamsWE.The Comparative Morphology of the Carotid Body and Carotid Sinus. Fig. 18.26 Anteriorviewoftheaorticarchshowingtheinnervationoftheaorticbodiesandbaroreceptors.(ModifiedfromNonidezJF.Anat Rec. 1937;69:299.)

1	Springfield,IL:CharlesCThomas;1958.)Left vagus nerveRight vagus nerve Right inferior cervical ganglion Right stellate ganglion Recurrent branch of right vagus Vagosympathetic fibers Brachiocephalic artery Baroreceptor fibers Cardiac nerves Aorta Right coronary artery Left inferior cervical ganglion Left subclavian ansa Left subclavian artery Recurrent branch of left vagus Aortic bodies Ductus arteriosus Pulmonary artery Left stellate ganglion Vagosympathetic fibers Vagosympathetic fibers widened areas at the origins of the internal carotid arteries. Impulses that arise in the carotid sinus travel up the carotid sinus nerve (nerve of Hering) to the glossopharyngeal nerve (cranial nerve IX) and, via the latter, to the nucleus of the tractus solitarius (NTS) in the medulla. The NTS is the site of the central projections of the chemoreceptors and baroreceptors. Stimulation of the NTS inhibits sympathetic nerve outflow to the peripheral blood vessels (depressor effect), whereas lesions of

1	of the central projections of the chemoreceptors and baroreceptors. Stimulation of the NTS inhibits sympathetic nerve outflow to the peripheral blood vessels (depressor effect), whereas lesions of the NTS produce vasoconstriction (pressor effect). Impulses that arise in the aortic arch baroreceptors reach the NTS via afferent fibers in the vagus nerves.

1	Baroreceptor nerve terminals in the walls of the carotid sinus and aortic arch respond to the vascular stretch and deformation induced by changes in arterial blood pressure. The frequency of firing of these nerves is enhanced by an increase in arterial blood pressure and diminished by a reduction in arterial blood pressure. An increase in impulse frequency, as occurs with a rise in arterial pressure, inhibits the cerebral vasoconstrictor regions and results in peripheral vasodilation and lowering of arterial blood pressure. Bradycardia brought about by activation of the cardiac branches of the vagus nerves contributes to this lowering of blood pressure. The carotid sinus baroreceptors are more sensitive than those in the aortic arch. Changes in carotid sinus pressure evoke greater changes in systemic arterial pressure and peripheral resistance than do equivalent changes in aortic arch pressure.

1	The receptors in the carotid sinus walls respond more to pulsatile pressure than to constant pressure. This is illustrated in Fig. 18.27 , which shows that at normal levels 0 0.5 1.0 1.5 2.0 •Fig. 18.27 Relationshipofphasicaorticbloodpressureinthefiring CHAPTER 18 Regulation of the Heart and Vasculature of mean arterial blood pressure (≈100 mm Hg), a barrage of impulses from a single fiber of the sinus nerve is initiated in early systole by the pressure rise; only a few spikes occur during late systole and early diastole. At lower arterial pressure, these phasic changes are even more evident, but the overall discharge frequency is reduced. The blood pressure threshold for evoking sinus nerve impulses is approxi mately 50 mm Hg; maximal sustained firing is reached at approximately 200 mm Hg. Because the baroreceptors adapt, their response at any mean arterial pressure level is greater to a high pulse pressure than to a low pulse pressure.

1	The increases in resistance that occur in response to reduced pressure in the carotid sinus vary from one peripheral vascular bed to another. These variations allow blood flow to be redistributed. The resistance changes elicited by altering carotid sinus pressure are greatest in the femoral vessels, less in the renal vessels, and least in the mesenteric and celiac vessels.

1	In addition, the sensitivity of the carotid sinus reflex can be altered. Local application of norepinephrine or stimulation of sympathetic nerve fibers to the carotid sinuses enhances the sensitivity of its receptors in such a way that a given increase in intrasinus pressure produces a greater depressor response. Baroreceptor sensitivity decreases in hypertension because the carotid sinuses become stiffer as a result of the high intra-arterial pressure. Consequently, a given increase in carotid sinus pressure elicits a smaller decrease in systemic arterial pressure than it does at a normal level of blood pressure. Thus the set point of the baroreceptors is raised in hypertension in such a way that the threshold is increased and the pressure receptors are less sensitive to changes in transmural pressure. As would be expected, denervation of the carotid sinus can produce temporary and, in some instances, prolonged hypertension.

1	The arterial baroreceptors play a key role in short-term adjustments in blood pressure in response to relatively abrupt changes in blood volume, cardiac output, or peripheral resistance (as in exercise). However, long-term control of blood pressure—over a period of days or weeks—is determined by the fluid balance of the individual: namely, the balance between fluid intake and fluid output. By far, the most important organ in the control of body fluid volume, and hence blood pressure, is the kidney (see also Cardiopulmonary receptors are located in the atria, ventricles, and pulmonary vessels. These baroreceptors are innervated by vagal and sympathetic afferent nerves. Cardiopulmonary reflexes are tonically active and can alter peripheral resistance in response to changes in intracardiac, venous, or pulmonary vascular pressure.

1	The atria contain two types of cardiopulmonary baroreceptors: those activated by the tension developed during atrial systole (type A receptors) and those activated by ofmeanarterialpressure.stretch of the atria during atrial diastole (type B receptors). Stimulation of these atrial receptors sends impulses up vagal fibers to the vagal center in the medulla. Consequently, sympathetic activity is decreased to the kidney and increased to the sinus node. These changes in sympathetic activity increase renal blood flow, urine flow, and heart rate. Activation of the cardiopulmonary receptors can also initiate a reflex that lowers arterial blood pressure by inhibiting the vasoconstrictor center in the cerebral medulla. Stimulation of the cardiopulmonary receptors inhibits release of angiotensin, aldosterone, and vasopressin (antidiuretic hormone); interruption of the reflex pathway has the opposite effects.

1	The role that activation of these baroreceptors plays in the regulation of blood volume is apparent in the body’s responses to hemorrhage. The reduction in blood volume (hypovolemia) enhances sympathetic vasoconstriction in the kidney and increases the secretion of renin, angiotensin, aldosterone, and vasopressin (see also ). The renal vasoconstriction (primarily afferent arterioles) reduces glomerular filtration and increases release of renin from the kidney. Renin acts on a plasma substrate to yield angiotensin II, which stimulates aldosterone secretion by the adrenal cortex. The enhanced release of vasopressin decreases renal water excretion, and the release of aldosterone decreases renal NaCl excretion. The kidneys retain salt and water, and hence blood volume increases. Angiotensin II (formed from angiotensin I by angiotensin-converting enzyme) also raises systemic arteriolar tone (see Table 14.1

1	Table 14.1 Insomeindividuals,thecarotidsinusisabnormallysensitivetoexternalpressure.Hence,tightcollarsorotherformsofexternalpressureovertheregionofthecarotidsinusmayelicitmarkedhypotensionandfainting.Suchhypersensitivityisknownasthecarotid sinus syndrome. Peripheral chemoreceptors consist of small, highly vascular bodies in the region of the aortic arch (aortic bodies; see Fig. 18.26 ) and just medial to the carotid sinuses (carotid bodies; see Fig. 18.25 ). These vascular bodies are sensitive to changes in the PO2, PCO2, and pH of arterial blood. Although they primarily regulate respiration, they also influence the vasomotor regions. A reduction in PaO2 stimulates the chemoreceptors. The increased activity in afferent nerve fibers from the carotid and aortic bodies stimulates the vasoconstrictor regions and thereby increases the tone of resistance and capacitance vessels.

1	The chemoreceptors are also stimulated by increased arterial blood PCO2 (PaCO2) and by reduced pH. However, the reflex effect is small in comparison to the direct effects of hypercapnia (high PaCO2) and acidosis on the vasomotor regions in the medulla. When hypoxia and hypercapnia occur simultaneously, the effects of the chemoreceptors are greater than the sum of the effects of each of the two stimuli when they act alone. Chemoreceptors are also located in the heart. These cardiac chemoreceptors are activated by ischemia of cardiac muscle, and they transmit the precordial pain (angina pectoris) associated with an inadequate blood supply to the myocardium.

1	Optimal function of the cardiovascular reflexes requires integrity of the pontine and hypothalamic structures. Furthermore, these structures are responsible for behavioral and emotional control of the cardiovascular system (see also ). Stimulation of the anterior hypothalamus produces both a fall in blood pressure and bradycardia, whereas stimulation of the posterolateral region of the hypothalamus increases both blood pressure and the heart rate. The hypothalamus also contains a temperature-regulating center that affects blood vessels in the skin. Stimulation by the application of cold to the skin or by cooling of the blood perfusing the hypothalamus results in constriction of the skin vessels and heat conservation, whereas warm stimuli to the skin result in cutaneous vasodilation and enhanced heat loss.

1	The cerebral cortex also affects blood flow distribution in the body. Stimulation of the motor and premotor areas affects blood pressure; usually, a pressor response occurs. However, vasodilation and depressor responses may be evoked, as in blushing or fainting, in response to an emotional stimulus. Painful stimuli can elicit either pressor or depressor responses, depending on the magnitude and location of the stimulus. Distention of the viscera often evokes a depressor response, whereas painful stimuli to the body surface generally evoke a pressor response.

1	Inflation of the lungs initiates a reflex that induces systemic vasodilation and a decrease in arterial blood pressure. Conversely, collapse of the lungs evokes systemic vasoconstriction. Afferent fibers that mediate this reflex are in the vagus nerves and possibly also in the sympathetic nerves. Stimulation of these fibers by stretch of the lungs inhibits the vasomotor areas. The magnitude of the depressor response to lung inflation is directly related to the degree of inflation and to the existing level of vasoconstrictor tone (see also

1	Increases in PCO2 stimulate chemosensitive regions of the medulla (the central chemoreceptors), and they elicit vasoconstriction and increased peripheral resistance. A reduction in PCO2 to subnormal levels (in response to hyperventilation) decreases tonic activity in these areas in the medulla and thereby decreases peripheral resistance. The chemosensitive regions are also affected by changes in pH. Lowering of blood pH stimulates these cerebral areas, and a rise in blood pH inhibits them. These effects of changes in PCO2 and blood pH may operate through changes in cerebrospinal fluid pH, as may also the respiratory center.

1	PaO2 has little direct effect on the medullary vasomotor region. The primary effect of hypoxia is mediated by reflexes via the carotid and aortic chemoreceptors. A moderate reduction in PaO2 stimulates the vasomotor region, but a severe reduction depresses vasomotor activity in the same manner by which other areas of the brain are depressed by very low O2 tension. Balance Between Extrinsic and Intrinsic Factors in Regulation of Peripheral Blood Flow Dual control of peripheral vessels by intrinsic and extrinsic mechanisms evokes a number of important vascular adjustments. Such regulatory mechanisms enable the body to direct blood flow to areas where it is most needed and away from areas that have fewer requirements. In some tissues, the effects of the extrinsic and intrinsic mechanisms are fixed; in other tissues, the ratio is changeable and depends on the state of activity of that tissue.

1	Cerebralischemia,whichmayoccurbecauseofexcessivepressureexertedbyanexpandingintracranialtumor,resultsinamarkedincreaseinperipheralvasoconstriction.ThestimulationisprobablycausedbylocalaccumulationofCO2andareductioninO2andpossiblybyexcitationofintracranialbaroreceptors.Withprolonged,severeischemia,centraldepressioneventuallysupervenes,andbloodpressurefalls. CHAPTER 18 Regulation of the Heart and Vasculature In the brain and heart, which are vital structures with limited tolerance for a reduced blood supply, intrinsic flow-regulating mechanisms are dominant. For instance, massive discharge of the vasoconstrictor region via the sympathetic nerves, which might occur in severe, acute hemorrhage, has negligible effects on the cerebral and cardiac resistance vessels, whereas the cutaneous, renal, and splanchnic blood vessels become greatly constricted.

1	In the skin, extrinsic vascular control is dominant. The cutaneous vessels not only participate strongly in a general vasoconstrictor discharge but also respond selectively via hypothalamic pathways to subserve the functions of heat loss and heat conservation required for regulation of body temperature. However, intrinsic control can be elicited by local temperature changes that modify or override the central influence on resistance and capacitance vessels (see also

1	In skeletal muscle, the extrinsic and intrinsic mechanisms interact. In resting skeletal muscle, neural control (vasoconstrictor tone) is dominant, as can be demonstrated by the large increase in blood flow that occurs immediately after section of the sympathetic nerves to the tissue. After the onset of exercise, the intrinsic flow-regulating mechanism assumes control, and vasodilation occurs in the active muscles because of the local increase in metabolites. Vasoconstriction occurs in the inactive tissues as a manifestation of the general sympathetic discharge. However, constrictor impulses that reach the resistance vessels of the active muscles are overridden by the local metabolic effect. Operation of this dual control mechanism thus increases blood flow where it is required and shunts it away from relatively inactive areas (see also Chapter 17).

1	Chapter 17). Similar effects may be achieved in response to an increase in PCO2. Normally, the hyperventilation associated with exercise keeps PCO2 at normal levels. However, if PCO2 is increased, generalized vasoconstriction would occur because CO2 stimulates the medullary vasoconstrictor region. In active muscles, where [CO2] would be highest, the smooth muscle of the arterioles would relax in response to the local PCO2. Factors that affect and are affected by the vasomotor region are summarized in Fig. 18.28 408 SECTION4Berne & Levy Physiology •Fig. 18.28 Schematic Diagram Illustrating Neural Input and Output of the Vasomotor Region (VR). Arrows indicatedirectionofneuralinputandoutput.IX,glossopharyngealnerve;↑PCO2,increasedpartialpressureofcarbondioxide;↓PCO2,increasedPCO2;↑PO2,increasedpartialpressureofoxygen;↓PO2,increasedPO2;SA,sinoatrial;X,vagusnerve.Aortic bodies SA node Sympathetic chain V R Carotid body X X IX ˜ PCO2 ° PO2 ˜ PCO2 ° PO2 ˜ PCO2 ° PO2 1.

1	Cardiac function is regulated by a number of intrinsic and extrinsic mechanisms. The principal intrinsic mechanisms that regulate myocardial contraction are the Frank-Starling mechanism and rate-induced regulation. 2. The heart rate is regulated mainly by the autonomic nervous system. Sympathetic nervous activity increases the heart rate, whereas parasympathetic (vagal) activity decreases the heart rate. When both systems are active, the vagal effects usually dominate. The autonomic nervous system regulates myocardial performance mainly by varying the Ca++ conductance of the cell membrane via the adenylate cyclase system. 3. The reflexes that regulate the heart rate are the baroreceptor, chemoreceptor, pulmonary inflation, atrial receptor (Bainbridge), and ventricular receptor reflexes. 4.

1	3. The reflexes that regulate the heart rate are the baroreceptor, chemoreceptor, pulmonary inflation, atrial receptor (Bainbridge), and ventricular receptor reflexes. 4. Certain hormones—such as epinephrine, adrenocortical steroids, thyroid hormones, insulin, glucagon, and anterior pituitary hormones—regulate myocardial performance. Changes in the arterial blood concentrations of O2, CO2, and H+ alter cardiac function directly and, through the chemoreceptors, indirectly.

1	5. The arterioles (resistance vessels) regulate blood flow mainly through their downstream capillaries. Smooth muscle, which makes up most of the walls of arterioles, contracts and relaxes in response to neural and humoral stimuli. Neural regulation of blood flow is almost completely accomplished by the sympathetic nervous system. Sympathetic nerves to blood vessels are tonically active; inhibition of the vasoconstrictor center in the medulla reduces peripheral vascular resistance. Stimulation of the sympathetic nerves constricts the resistance and capacitance (veins) vessels. Parasympathetic fibers innervate the head, viscera, and genitalia; they do not innervate the skin and muscle. 6.

1	6. Autoregulation of blood flow occurs in most tissues. This process is characterized by a constant blood flow in the presence of a change in perfusion pressure. Autoregulation is mediated by a myogenic mechanism whereby an increase in transmural pressure elicits a contraction of vascular smooth muscle and a decrease in transmural pressure elicits a relaxation. 7. The striking parallelism between tissue blood flow and tissue O2 consumption indicates that blood flow is regulated largely by a metabolic mechanism. A decrease in the ratio of O2 supply to O2 demand of a tissue releases vasodilator metabolites that dilate arterioles and thereby enhance the O2 supply. 8.

1	8. The baroreceptors in the internal carotid arteries and aorta are tonically active and regulate blood pressure on a moment-to-moment basis. An increase in arterial pressure stretches these receptors to initiate a reflex that inhibits the medullary vasoconstrictor center and induces vasodilation. Conversely, a decrease in arterial pressure disinhibits the vasoconstrictor center and induces vasoconstriction. The baroreceptors in the internal carotid arteries Fukuda K, Kanazawa H, Aizawa Y, et al. Cardiac innervation and sudden cardiac death. Circ Res. 2015;116:2005. Iancu RV, Jones SW, Harvey RD. Compartmentation of cAMP signaling in cardiac myocytes. Biophys J. 2007;92:3317. Liu Y, Bubolz AH, Mendoza S, et al. H2O2 is the transferrable factor mediating flow-induced dilation in human coronary arterioles. Circ Res. 2011;108:566. predominate over those in the aorta, and they respond more vigorously to changes in pressure (stretch) than to elevated or reduced nonpulsatile pressure.

1	predominate over those in the aorta, and they respond more vigorously to changes in pressure (stretch) than to elevated or reduced nonpulsatile pressure. 9. Peripheral chemoreceptors (carotid and aortic bodies) and central chemoreceptors in the medulla oblongata are stimulated by a decrease in blood PaO2 and by an increase in blood PCO2. Stimulation of these chemoreceptors increases the rate and depth of respiration, but it also produces peripheral vasoconstriction. Cardiopulmonary baroreceptors are also present in the cardiac chambers and large pulmonary vessels. They have less influence on blood pressure but do participate in regulation of blood volume. 10.

1	10. Peripheral resistance and hence blood pressure are affected by stimuli that arise in the skin, viscera, lungs, and brain. The combined effect of neural and local metabolic factors distributes blood to active tissues and diverts it from inactive tissues. In vital structures, such as in the heart and brain and in contracting skeletal muscle, the metabolic factors predominate. Llewellyn-Smith IJ, Verberne AJM, eds. Central Regulation of Autonomic Functions. New York: Oxford University Press; 2011. Timmers HJ, Wieling W, Karemaker JM, et al. Cardiovascular responses to stress after carotid baroreceptor denervation in humans. Ann N Y Acad Sci. 2004;1018:515. Zhang P, Mende U. Regulators of G-protein signaling in the heart and their potential as therapeutic targets. Circ Res. 2011;109: 320. Upon completion of this chapter, the student should be able to answer the following questions: 1.

1	Upon completion of this chapter, the student should be able to answer the following questions: 1. What are the four major factors that determine cardiac output? Which two of these factors are said to be “coupling factors,” and what is the reason for that description? 2. What is a cardiac function curve, and how is it related to the Frank-Starling mechanism? 3. What is a vascular function curve, and how is it affected by changes in total peripheral resistance, blood volume, and venous tone? 4. Why does the operating point of the cardiovascular system occur at the intersection of the vascular and cardiac function curves? 5. How does evaluation of the cardiac function curve and the vascular function curve enable clinicians to determine the effect of changes in blood volume, vascular tone, and contractility on cardiac output? 6.

1	6. What mechanisms in the central nervous system, heart, and systemic vasculature allow cardiac output to increase to the necessary levels during vigorous exercise? 7. What are the cardiovascular consequences of hemorrhage, and what are the compensatory mechanisms that tend to restore arterial pressure and cardiac output? Regulation of Cardiac Output and Blood Pressure Four factors control cardiac output: heart rate, myocardial contractility, preload, and afterload (

1	Regulation of Cardiac Output and Blood Pressure Four factors control cardiac output: heart rate, myocardial contractility, preload, and afterload ( Fig. 19.1). Heart rate and myocardial contractility are strictly cardiac factors, although they are controlled by various neural and humoral mechanisms. Preload and afterload are factors that are mutually dependent on function of the heart and the vasculature and are important determinants of cardiac output. Preload and afterload are themselves determined by cardiac output and by certain vascular characteristics. Preload and afterload are called coupling factors because they constitute a functional coupling between the heart and blood vessels. To understand regulation of cardiac output, the nature of the coupling between the heart and the vascular system must be appreciated.

1	In this chapter, two kinds of graphed curves are used to analyze interactions between the cardiac and vascular components of the circulatory system. The first curve, the cardiac function curve, is an expression of the well-known Frank-Starling relationship, and it illustrates the dependence of cardiac output on preload (i.e., central venous or right atrial pressure). The cardiac function curve is a characteristic of the heart itself and is usually studied in hearts completely isolated from the rest of the circulation. This curve has already been discussed in detail in . Later in this chapter, this curve is discussed in association with the other characteristic curve, the vascular function curve, to analyze interactions between the heart and the vasculature. The vascular function curve defines the dependence of central venous pressure on cardiac output. This relationship depends only on several vascular system characteristics, including peripheral vascular resistance, arterial and

1	defines the dependence of central venous pressure on cardiac output. This relationship depends only on several vascular system characteristics, including peripheral vascular resistance, arterial and venous compliance, and blood volume. The vascular function curve is entirely independent of the characteristics of the heart. Because of this independence, it can be derived experimentally even if a mechanical pump replaces the heart.

1	The vascular function curve defines the changes in central venous pressure (Pv) that are caused by changes in cardiac output. In this curve, Pv is the dependent variable (or response), and cardiac output is the independent variable (or stimulus). These variables are opposite those of the cardiac function curve, in which Pv (or preload) is the independent variable and cardiac output is the dependent variable. The simplified model of the circulation shown in Fig. 19.2 helps explain how cardiac output determines the level of Pv. In this model, all essential components of the cardiovascular system have been lumped into four basic elements. The right and left sides of the heart, as well as the pulmonary vascular bed, constitute a pump-oxygenator, much like an artificial heart-lung machine used to perfuse •Fig. 19.1 Thefourfactors(inblue squares)thatdeterminecardiacoutput.

1	the body during open heart surgery. The high-resistance microcirculation is designated the peripheral resistance. Finally, the compliance of the system is subdivided into arterial compliance (Ca) and venous compliance (Cv). As defined in , the compliance (C) of a blood vessel is the change in volume (ΔV) that is accommodated in that vessel per unit change in transmural pressure (ΔP); that is, Equation 19.1 Venous compliance is approximately 20 times greater than arterial compliance. In the example in Fig. 19.2, the ratio of Cv to Ca is set at 19:1 to simplify calculations. To show how a change in cardiac output causes an inverse change in Pv, the hypothetical model has certain characteristics that mimic those of an average adult (see

1	To show how a change in cardiac output causes an inverse change in Pv, the hypothetical model has certain characteristics that mimic those of an average adult (see Fig. 19.2A ). The flow generated by the heart (i.e., cardiac output; Qh) is 5 L/minute; mean arterial pressure (Pa) is 102 mm Hg; and Pv is 2 mm Hg. Peripheral resistance (R) is the ratio of the arteriovenous pressure difference (Pa − Pv) to flow (Qr) through the resistance vessels; this ratio is equal to 20 mm Hg/L/minute. An arteriovenous pressure difference of 100 mm Hg is sufficient to force a flow rate (Qr) of 5 L/minute through a peripheral resistance of 20 mm Hg/L/minute (see Fig.

1	An arteriovenous pressure difference of 100 mm Hg is sufficient to force a flow rate (Qr) of 5 L/minute through a peripheral resistance of 20 mm Hg/L/minute (see Fig. 19.2A ). Under equilibrium conditions, this flow rate (Qr) is precisely equal to the flow rate (Qh) pumped by the heart. From heartbeat to heartbeat, the volume of blood in the arteries (Va) and the volume of blood in the veins (Vv) remain constant because the volume of blood transferred from the veins to the arteries by the heart is equal to the volume of blood that flows from the arteries through the resistance vessels and into the veins. *Thus if it were necessary to add x mL of blood to the arterial system to produce a 1–mm Hg increase in arterial pressure, 19x mL of blood would need to be added to the venous system to raise venous pressure by the same amount. Effects of Cardiac Arrest on Arterial and Venous Pressure

1	Effects of Cardiac Arrest on Arterial and Venous Pressure Fig. 19.2B depicts the circulation at the very beginning of an episode of cardiac arrest; that is, Qh = 0. In the instant immediately after arrest of the heart, the volume of blood in the arteries (Va) and veins (Vv) has not had time to change appreciably. Because arterial pressure and venous pressure depend on Va and Vv, respectively, these pressures are identical to the respective pressures in

1	Fig. 19.2A (i.e., Pa = 102 and Pv = 2). This arteriovenous pressure gradient of 100 mm Hg forces a flow rate (Qr) of 5 L/minute through the peripheral resistance of 20 mm Hg/L/minute. Thus although cardiac output (Qh) at that point is 0 L/ minute, the rate of flow through the microcirculation (Qt) is 5 L/minute because the potential energy stored in the arteries by the preceding pumping action of the heart causes blood to be transferred from arteries to veins. This transfer occurs initially at the control (steady-state) rate, even though the heart can no longer transfer blood from the veins to the arteries.

1	As cardiac arrest continues, blood flow through the resistance vessels causes the blood volume in the arteries to decrease progressively and the blood volume in the veins to increase progressively at the same absolute rate. Because the arteries and veins are elastic structures, arterial pressure falls gradually, and the venous pressure rises gradually. This process continues until arterial and venous pressures become equal (see Fig. 19.2C ). Once this condition is reached, the rate of flow (Qr) from the arteries to the veins through the resistance vessels is 0 L/minute, as is Qh. When the effects of cardiac arrest reach this equilibrium state (see

1	When the effects of cardiac arrest reach this equilibrium state (see Fig. 19.2C ), the pressure attained in the arteries and veins depends on the relative compliance of these vessels. If arterial compliance (Ca) and venous compliance (Cv) are equal, the decline in Pa is equal to the rise in Pv because the decrease in arterial volume would be equal to the increase in venous volume (according to the principle of conservation of mass). Both Pa and Pv would attain the average of their combined values in Fig. 19.2A ; that is, Pa = Pv = (102 + 2)/2 = 52 mm Hg. However, Ca and Cv in a living person are not equal. Veins are much more compliant than arteries; the compliance ratio (Cv/Ca) is approximately 19, the ratio assumed for the model in

1	Fig. 19.2 . When the effects of cardiac arrest reach equilibrium in an intact subject, the pressure in the arteries and veins is much lower than the average value of 52 mm Hg that occurs when Ca and Cv are equal. Hence, transfer of blood from arteries to veins at equilibrium induces a fall in arterial pressure 19 times greater than the concomitant rise in venous pressure. As Fig. 19.2C shows, Pv would increase by 5 mm Hg (to 7 mm Hg), whereas Pa would fall by 95 (i.e., 19 × 5) mm Hg (to 7 mm Hg). This equilibrium pressure, which prevails in the absence of flow, is referred to as either mean circulatory pressure or static pressure. The pressure in the static system reflects the total blood volume in the system and the overall compliance of the system. •Fig. 19.2 A toD, Simplifiedmodelofthecardiovascularsystem,consistingofapump,arterialcompliance(Ca),peripheralresistance,andvenouscompliance(Cv).

1	•Fig. 19.2 A toD, Simplifiedmodelofthecardiovascularsystem,consistingofapump,arterialcompliance(Ca),peripheralresistance,andvenouscompliance(Cv). The example of cardiac arrest aids in the understanding of the vascular function curve. The clinician can now begin to assemble a vascular function curve ( Fig. 19.3 ). The independent variable (plotted along the x-axis) is cardiac output, and the dependent variable (plotted along the y-axis) is Pv. Two important points on this curve can be derived from the example in Fig. 19.2 . One point (A in Fig. 19.3 ) represents the control state; that is, when cardiac output is 5 L/minute, Pv is 2 mm Hg. When the heart is arrested (cardiac output = 0), Pv becomes 7 mm Hg at equilibrium (see Fig. 19.2C ); this pressure is the mean circulatory pressure.

1	Fig. 19.2C ); this pressure is the mean circulatory pressure. The inverse relationship between Pv and cardiac output simply means that when cardiac output is suddenly decreased, the rate at which blood flows from arteries to veins through the capillaries is temporarily greater than the rate at which the heart pumps blood from the veins back •Fig. 19.3 Changes in Central Venous Pressure Produced by Changes in Cardiac Output. Themeancirculatory(orstatic)pressure(Pmc)istheequilibriumpressurethroughoutthecardiovascularsystemwhencardiacoutputis0.PointsBandArepresentthevaluesofvenouspressureatcardiacoutputsof1and5L/minute,respectively. into the arteries. During that transient period, a net volume of blood is transferred from arteries to veins; hence, Pa falls and Pv rises. Now suppose that cardiac output suddenly increases. This example illustrates how a third point (B in

1	Now suppose that cardiac output suddenly increases. This example illustrates how a third point (B in Fig. 19.3 ) on the vascular function curve is derived. Consider that the arrested heart is suddenly restarted and immediately begins pumping blood from the veins into the arteries at a rate of 1 L/minute (see

1	Fig. 19.2D ). When the heart first begins to beat, the arteriovenous pressure gradient is 0, and no blood is transferred from the arteries through the capillaries and into the veins. Thus when beating resumes, blood is depleted from the veins at the rate of 1 L/minute, and arterial blood volume is replenished from venous blood volume at that same absolute rate. Hence, Pv begins to fall and Pa begins to rise. Because of the difference in arterial and venous compliance, Pa rises at a rate 19 times faster than the rate at which Pv falls. The resultant arteriovenous pressure gradient causes blood to flow through the peripheral resistance vessels. If the heart maintains a constant output of 1 L/minute, Pa continues to rise and Pv continues to fall until the pressure gradient becomes 20 mm Hg. This gradient forces a rate of flow of 1 L/minute through a peripheral resistance of 20 mm Hg/L/minute. This gradient is achieved by a 19–mm Hg rise (to 26 mm Hg) in Pa and a 1–mm Hg fall (to 6 mm Hg)

1	gradient forces a rate of flow of 1 L/minute through a peripheral resistance of 20 mm Hg/L/minute. This gradient is achieved by a 19–mm Hg rise (to 26 mm Hg) in Pa and a 1–mm Hg fall (to 6 mm Hg) in Pv. This equilibrium value of Pv (6 mm Hg) for a cardiac output of 1 L/minute also appears on the vascular function curve of

1	Fig. 19.3 (point B). The 1–mm Hg reduction in Pv reflects a net transfer of blood from the veins to the arteries of the circuit. The reduction in Pv that can be evoked by a sudden increase in cardiac output is limited. At some critical maximal value of cardiac output, sufficient fluid is transferred from the veins to the arteries of the circuit for Pv to fall below ambient pressure. In a system of very distensible vessels, such as the venous system, the greater external pressure causes the vessels to collapse (see ). This •Fig. 19.4 Effectsofincreasedbloodvolume(transfusion curve) anddecreasedbloodvolume(hemorrhage curve) onthevascularfunctioncurve.Similarshiftsinthevascularfunctioncurvecanbeproducedbyincreasesanddecreases,respectively,invenomotortone. venous collapse impedes venous return to the heart. Hence, it limits the maximal value of cardiac output to 7 L/minute in this example (see Fig. 19.3 ), regardless of the capabilities of the pump.

1	venous collapse impedes venous return to the heart. Hence, it limits the maximal value of cardiac output to 7 L/minute in this example (see Fig. 19.3 ), regardless of the capabilities of the pump. Factors That Influence the Vascular Function Curve Dependence of Venous Pressure on Cardiac Output According to experimental and clinical observations, changes in cardiac output do indeed evoke the alterations in Pa and Pv that have been predicted by the simplified model in Fig. 19.2 The vascular function curve is affected by variations in total blood volume. During circulatory standstill (zero cardiac output), mean circulatory pressure depends only on total vascular compliance and blood volume. For a given vascular compliance, mean circulatory pressure is increased when blood volume is expanded (hypervolemia) and is decreased when blood volume is diminished (hypovolemia). This relationship is illustrated by the y-axis intercepts in Fig.

1	Fig. 19.4 , in which mean circulatory pressure is 5 mm Hg after hemorrhage and 9 mm Hg after transfusion, in comparison with a value of 7 mm Hg at normal blood volume (normovolemia or euvolemia). The effects of changes in venomotor tone on the vascular function curve closely resemble those of changes in blood volume. In Fig. 19.4 , for example, the transfusion curve could also represent increased venomotor tone, whereas the hemorrhage curve could represent decreased tone. During Cardiacoutputmaydecreaseabruptlywhenamajorcoronaryarterysuddenlybecomesoccluded.Theacute heart failure thatoccursasaresultofmyocardial infarction (deathofmyocardialtissue)isusuallyaccompaniedbyafallinarterialbloodpressureandariseinPv.In

1	Fig.19.4 itisalsoapparentthatthecardiacoutputatwhichPv= 0variesdirectlywithbloodvolume.Therefore,themaximalvalueofcardiacoutputbecomesprogressivelymorelimitedasthetotalbloodvolumeisreduced.However,thePvatwhichtheveinscollapse(illustratedbythesharpchangeinslopeofthevascularfunctioncurve)isnotsignificantlyalteredbychangesinbloodvolume.Thispressuredependsonlyontheambientpressuresurroundingthecentralveins.Ambientpressureisthepleuralpressureinthethorax(see circulatory standstill, for a given blood volume, the pressure within the vascular system rises as smooth muscle tension exerted within the vascular walls increases (these contractile changes in arteriolar and venous smooth muscle are under nervous and humoral control). The fraction of the blood volume located within the arterioles is very small, whereas the blood volume in the veins is large percentage of total blood volume (see

1	Table 15.1 ). Thus changes in peripheral resistance (arteriolar tone) have no significant effect on mean circulatory pressure, but changes in venous tone can alter mean circulatory pressure appreciably. Hence, mean circulatory pressure rises with increased venomotor tone and falls with diminished venomotor tone. In experiments, the mean circulatory pressure attained approximately 1 minute after abrupt circulatory standstill is usually substantially above 7 mm Hg, even when blood volume is normal. The elevation to this pressure level is attributable to the generalized venoconstriction that is caused by cerebral ischemia, activation of chemoreceptors, and reduced excitation of baroreceptors. If resuscitation fails, this reflex response subsides as central nervous activity ceases, and mean circulatory pressure then usually falls to a value close to 7 mm Hg.

1	Venoconstriction is considerably greater in certain regions of the body than in others. In effect, vascular beds that undergo significant venoconstriction constitute blood reservoirs. The skin’s vascular bed is one of the major blood reservoirs in humans. Blood loss evokes profound subcutaneous venoconstriction, which gives rise to the characteristically pale appearance of the skin in response to hemorrhage. Diversion of blood away from the skin frees several hundred milliliters of blood that can be perfused through more vital regions of the body. The vascular beds of the liver, lungs, and spleen are also important blood reservoirs. In humans, however, the volume changes in the spleen are considerably less extensive (see also the sections “ •Fig. 19.5 Effectsofarteriolardilationandconstrictiononthevascularfunctioncurve. The changes in the vascular function curve induced by alterations in arteriolar tone are depicted in

1	The changes in the vascular function curve induced by alterations in arteriolar tone are depicted in Fig. 19.5 . The amount of blood in the arterioles is small; these vessels contain only approximately 3% of total blood volume (see ). Changes in the contractile state of arterioles do not significantly alter mean circulatory pressure. Thus vascular function curves that represent different peripheral resistances converge at a common point on the y-axis (see Fig. 19.5

1	Fig. 19.5 Pv varies inversely with total peripheral resistance (TPR) when all other factors remain constant. Physiologically, the relationship between Pv and TPR can be explained as follows: if cardiac output is held constant, a sudden increase in TPR causes a progressively greater volume of blood to be retained in the arterial system. Blood volume in the arterial system continues to increase until Pa rises sufficiently to force a flow of blood equal to cardiac output through the resistance vessels. If total blood volume does not change, this increase in arterial blood volume is accompanied by an equivalent decrease in venous blood volume. Hence, an increase in TPR diminishes Pv proportionately. This relationship between TPR and Pv, together with the inability of peripheral resistance to affect mean circulatory pressure, accounts for the clockwise rotation of the vascular function curves in response to increased arteriolar constriction (see

1	Fig. 19.5 ). Similarly, arteriolar dilation produces a counterclockwise rotation from the same vertical axis intercept. A higher maximal level of cardiac output is attainable when the arterioles are dilated than when they are constricted (see Fig. 19.5). Cardiac output and venous return are tightly linked. Except for small, transient disparities, the heart cannot pump any more blood than is delivered to it through the venous system. Similarly, because the circulatory system is a closed circuit, venous return to the heart must equal cardiac output over any appreciable time interval. The flow around the entire closed circuit depends on the capability of the pump, the characteristics of the circuit, and the total fluid volume of the system.

1	Thus cardiac output and venous return are simply two terms for the flow around this closed circuit. Cardiac output is the volume of blood being pumped by the heart per unit time. Venous return is the volume of blood returning to the heart per unit time. At equilibrium, these two volumes are equal. In the following section, certain techniques of circuit analysis are discussed to provide some insight into the control of flow around the circuit. Relating the Cardiac Function Curve to the Vascular Function Curve Coupling Between the Heart and the Vasculature

1	Relating the Cardiac Function Curve to the Vascular Function Curve Coupling Between the Heart and the Vasculature In accordance with the Frank-Starling law of the heart, cardiac output depends closely on right atrial (or central venous) pressure. Furthermore, right atrial pressure is approximately equal to right ventricular end-diastolic pressure because the normal tricuspid valve acts as a low-resistance junction between the right atrium and ventricle. Graphs of cardiac output as a function of Pv are called cardiac function curves; extrinsic regulatory influences may be expressed as shifts in such curves. A typical cardiac function curve is plotted on the same coordinates as those for a normal vascular function curve in

1	A typical cardiac function curve is plotted on the same coordinates as those for a normal vascular function curve in Fig. 19.6 . The cardiac function curve is plotted according to the usual convention; that is, the independent variable (Pv) is plotted along the x-axis, and the dependent variable (cardiac output) is plotted along the y-axis. In accordance with the Frank-Starling mechanism, the cardiac function curve reveals that a rise in Pv increases cardiac output. Conversely, the vascular function curve characterizes an inverse relationship between cardiac output and Pv; that is, a rise in cardiac output diminishes Pv. Pv is the dependent variable (or response) and cardiac output is the independent variable (or stimulus) for the vascular function curve. Therefore, to plot a vascular function curve in the conventional manner, Pv should be scaled along the y-axis and cardiac output along the x-axis.

1	To plot the cardiac and vascular function curves on the same set of axes requires a modification of the plotting convention for one of these curves. The convention for the vascular function curve is violated arbitrarily in this chapter. Note that the vascular function curve in Fig. 19.6 is intended to reflect how Pv (scaled along the x-axis) varies in response to a change in cardiac output (scaled along the y-axis). When the cardiovascular system is represented by a given pair of cardiac and vascular function curves, the intersection of these two curves defines the equilibrium point of that system. The coordinates of this equilibrium point represent •Fig. 19.6 Typical Vascular and Cardiac Function Curves Plotted on the Same Coordinate Axes. Toplotbothcurvesonthesamegraph,thex-axisandy-axisforthevascularfunctioncurveshadtobeswitched;comparetheassignmentofaxeswiththosein Figs. 19.3,19.4,and19.5.

1	Figs. 19.3,19.4,and19.5. Thecoordinatesoftheequilibriumpoint,attheintersectionofthecardiacandvascularfunctioncurves,representthestablevaluesofcardiacoutputandcentralvenouspressureatwhichthesystemtendstooperate.Anyperturbation(e.g.,asuddenincreaseinvenouspressuretopointA)institutesasequenceofchangesincardiacoutputandvenouspressurethatrestorethesevariablestotheirequilibriumvalues. the values of cardiac output and Pv at which the system tends to operate. Only transient deviations from such values of cardiac output and Pv are possible, as long as the given cardiac and vascular function curves characterize the system accurately. The tendency to operate about this equilibrium point may best be illustrated by the response to a sudden change. Consider the changes caused by a sudden rise in Pv from the equilibrium point to point A in

1	Fig. 19.6 . This change in Pv might be caused by the rapid injection, during ventricular diastole, of a given volume of blood on the venous vessels of the circuit and simultaneous withdrawal of an equal volume from the arterial vessels of the circuit. Thus although Pv rises, total blood volume remains constant. As defined by the cardiac function curve, this elevated Pv would increase cardiac output (from point A to point B in

1	Fig. 19.6 ) during the next ventricular systole. The increased cardiac output would then cause the transfer of a net quantity of blood from the veins to the arteries of the circuit, with a consequent reduction in Pv. In one heartbeat, the reduction in Pv would be small (from point B to point C) because the heart would transfer only a fraction of the total venous blood volume to the arteries. As a result of this reduction in Pv, cardiac output during the very next beat diminishes (from point C to point D) by an amount dictated by the cardiac function curve. Because point C is still above the intersection point, the heart pumps blood from the veins to the arteries at a rate greater than that at which blood flows across the peripheral resistance from arteries to veins. Hence, Pv continues to fall. This process continues in diminishing steps until the point of •Fig. 19.7

1	greater than that at which blood flows across the peripheral resistance from arteries to veins. Hence, Pv continues to fall. This process continues in diminishing steps until the point of •Fig. 19.7 Enhancementofmyocardialcontractility,asbystimulationofcardiacsympatheticnerves,causestheequilibriumvaluesofcardiacoutputandcentralvenouspressure(Pv)toshiftfromtheintersection(pointA)ofthecontrolvascularandcardiacfunctioncurves(continuous curve) totheintersection(pointD)ofthesamevascularfunctioncurvewiththecardiacfunctioncurve(dashed curve) thatrepresentstheresponsetosympatheticstimulation.

1	intersection is reached. Only one specific combination of cardiac output and venous pressure—the equilibrium point, denoted by the coordinates of the point at which the curves intersect—satisfies the requirements of the cardiac and vascular function curves simultaneously. At the equilibrium point, cardiac output equals venous return, and the system is stable. Combinations of cardiac and vascular function curves also help explain the effects of alterations in ventricular contractility on cardiac output and Pv. In Fig. 19.7, the lower cardiac function curve represents the control state, whereas the upper curve reflects the influence of increased myocardial contractility. This pair of curves is analogous to the ventricular function curves shown in Fig. 18.12. The enhanced ventricular contractility represented by the upper curve in

1	Fig. 18.12. The enhanced ventricular contractility represented by the upper curve in Fig. 19.7 can be produced by electrical stimulation of the cardiac sympathetic nerves. When the effects of such neural stimulation are restricted to the heart, the vascular function curve is unaffected. Therefore, only one vascular function curve is needed for this hypothetical intervention (see Fig. 19.7 During the control state of the model, the equilibrium values for cardiac output and Pv are designated by point A in

1	Fig. 19.7 During the control state of the model, the equilibrium values for cardiac output and Pv are designated by point A in Fig. 19.7 . Cardiac sympathetic nerve stimulation abruptly raises cardiac output to point B because of the enhanced myocardial contractility. However, this high cardiac output causes an increase in the net transfer of blood from the veins to the arteries of the circuit, and as a consequence, Pv subsequently begins to fall (to point C). The reduction in Pv then leads to a small decrease in cardiac output. However, cardiac output is still sufficiently high to effect •Fig. 19.8 Duringelectricalstimulationofthecardiacsympatheticnervefibers,aorticbloodflow(cardiacoutput)increased,whereaspressuresintheleftatrium(PLA)andrightatrium(PRA)diminished.Thesedataconformtotheconclusionsderivedfrom

1	Fig.19.7 ,inwhichtheequilibriumvaluesofcardiacoutputandvenouspressureareobservedtoshiftfrompointAtopointD(i.e.,cardiacoutputincreased,butcentralvenouspressuredecreased)duringcardiacsympatheticnervestimulation.(RedrawnfromSarnoffSJ,etal.Circ Res. 1960;8:1108.) the net transfer of blood from the veins to the arteries of the circuit. Thus both Pv and cardiac output continue to fall gradually until a new equilibrium point (point D) is reached. This equilibrium point is located at the intersection of the vascular function curve and the new cardiac function curve. In Fig. 19.7 , point D lies above and to the left of the control equilibrium point (point A) and indicates that sympathetic stimulation can evoke greater cardiac output despite the lower level of Pv. The biological response to enhancement of myocardial contractility is mimicked by the hypothetical change predicted by the model in this chapter. As depicted in

1	The biological response to enhancement of myocardial contractility is mimicked by the hypothetical change predicted by the model in this chapter. As depicted in Fig. 19.8 , sympathetic nerves innervating the heart are stimulated during the time denoted by the double-headed arrow. During neural stimulation, cardiac output (aortic flow) rises quickly to a peak value and then falls gradually to a steady-state value significantly higher than the control level. The increase in aortic flow is accompanied by reductions in right and left atrial pressures. Changes in blood volume do not directly affect myocardial contractility, but they do influence the vascular function curve in the manner shown in Fig. 19.4 . Thus to understand how changes in blood volume affect cardiac output and Pv, the appropriate cardiac function curve is plotted along with the vascular function curves that represent the control and experimental states (

1	Fig. 19.9 ). When blood volume is increased by a blood transfusion, the equilibrium point (point B in Fig. 19.9 ), which denotes the values of cardiac output and Pv after transfusion, lies above and to the right of the control equilibrium point (point A). Thus transfusion increases both cardiac output and Pv. Hemorrhage causes the opposite effect. Mechanistically, the change in ventricular filling pressure (Pv) evoked by a given change in blood volume alters cardiac output by changing the sensitivity of the contractile proteins to the prevailing concentration of intracellular Ca++ (see ). For reasons explained earlier, pure increases or decreases in venomotor tone elicit responses that are like those evoked by increases or decreases, respectively, in total blood volume.

1	•Fig. 19.9 Afterabloodtransfusion,thevascularfunctioncurveisshiftedtotheright.Therefore,bothcardiacoutputandvenouspressureareincreased,asdenotedbytranslocationoftheequilibriumpointfrompointAtopointB.–2 A B 1086420 0 2 4 6 8 10 Central venous pressure (mm Hg) Transfusion ControlCardiac output (L/min) Analysis of the effects of changes in peripheral resistance on cardiac output and Pv is complex because both the cardiac and vascular function curves shift. When peripheral resistance increases ( Fig. 19.11 ), the vascular function curve is rotated counterclockwise, but it converges on the same Pv axis intercept as the control curve does. Note that vasoconstriction causes a counterclockwise rotation of the vascular function curve in Fig. 19.11 Fig. 19.5 . The direction of rotation differs because the axes for the vascular function curves were switched in these two figures, as explained earlier. The cardiac function curve in

1	Fig. 19.11 Fig. 19.5 . The direction of rotation differs because the axes for the vascular function curves were switched in these two figures, as explained earlier. The cardiac function curve in Fig. 19.11 is also shifted downward because at any given Pv, the heart is able to pump less blood against the greater cardiac afterload imposed by the increased peripheral resistance. Because both curves in Fig. 19.11 are displaced downward, the new equilibrium point (point B) is below the control point (point A); that is, an increase in peripheral resistance diminishes cardiac output.

1	Fig. 19.11 are displaced downward, the new equilibrium point (point B) is below the control point (point A); that is, an increase in peripheral resistance diminishes cardiac output. Whether point B falls directly below point A or lies slightly to the right or left of it depends on the magnitude of the shift in each curve. For example, if a given increase •Fig. 19.10 Moderateorsevereheartfailureshiftsthecardiacfunctioncurvesdownwardandtotheright.Beforechangesinbloodvolume,cardiacoutputdecreasesandcentralvenouspressurerises(fromcontrolequilibriumpointAtopointBorpointC).Aftertheincreaseinbloodvolumethatusuallyoccursinheartfailure,thevascularfunctioncurveisshiftedtotheright.Hence,centralvenouspressuremaybeelevatedwithnoreductionincardiacoutput(pointD)or,insevereheartfailure,withsomereductionincardiacoutput(pointE).

1	Heart failure isageneraltermthatappliestoconditionsinwhichthepumpingcapabilityoftheheartisimpairedtotheextentthatthetissuesofthebodyarenotadequatelyperfused.Inheartfailure,myocardialcontractilityisimpaired.Heartfailuremaybeacuteorchronic.Consequently,inagraphofcardiacandvascularfunctioncurves,thecardiacfunctioncurveisshifteddownwardandtotheright,asdepictedin Fig.19.10 Acuteheartfailuremaybecausedbytoxicconcentrationsofdrugsorbycertainpathologicalconditionssuchascoronaryarteryocclusion.Inacuteheartfailure,bloodvolumedoesnotchangeimmediately.In Fig.19.10 ,therefore,theequilibriumpointshiftsfromtheintersection(pointA)ofthenormalcurvestotheintersection(pointBorpointC)ofthenormalvascularfunctioncurve.

1	Fig.19.10 ,therefore,theequilibriumpointshiftsfromtheintersection(pointA)ofthenormalcurvestotheintersection(pointBorpointC)ofthenormalvascularfunctioncurve. Chronicheartfailuremayoccurinconditionssuchasessentialhypertensionorischemicheartdisease.Inchronicheartfailure,boththecardiacfunctionandvascularfunctioncurvesshift.Thevascularfunctioncurveshiftsbecauseofanincreaseinbloodvolumecausedinpartbyfluidretentionbythekidneys.ThefluidretentionisrelatedtotheconcomitantreductioninglomerularfiltrationrateandthedecreasedrenalexcretionofNaClandwater(seealso ).Theresultanthypervolemiaisreflectedbyarightwardshiftofthevascularfunctioncurve,asshownin Fig.19.10. Hence,withmoderatedegreesofheartfailure,Pviselevated,butcardiacoutputmaybenormal(pointD).Withmoreseveredegreesofheartfailure,Pvisstillelevated,butcardiacoutputissubnormal(pointE).

1	Fig.19.10. Hence,withmoderatedegreesofheartfailure,Pviselevated,butcardiacoutputmaybenormal(pointD).Withmoreseveredegreesofheartfailure,Pvisstillelevated,butcardiacoutputissubnormal(pointE). in peripheral resistance shifts the vascular function curve more than it does the cardiac function curve, equilibrium point B is below and to the left of point A; that is, both cardiac output and Pv diminish. Conversely, if the cardiac function curve is displaced more than the vascular function •Fig. 19.11 Anincreaseinperipheralresistanceshiftsthecardiacandvascularfunctioncurvesdownward.Atequilibrium,cardiacoutputisless(pointB)whenperipheralresistanceishighthanwhenperipheralresistanceisnormal(pointA).

1	•Fig. 19.12 Simplifiedmodelofthecardiovascularsystemthatconsistsoftheleftventricle(LV)andrightventricle(RV),systemicvascularresistance(Rs)andpulmonaryvascularresistance(Rp),systemicarterialandvenouscompliance,andpulmonaryarterialandvenouscompliance.PsaandPsvarethepressuresinthesystemicarteriesandveins,respectively;PpaandPpvarethepressuresinthepulmonaryarteriesandveins,respectively. curve, point B falls below and to the right of point A; that is, cardiac output decreases, but Pv rises. A More Com﻿plete Theoretical Model: The Two-Pum﻿p System﻿

1	curve, point B falls below and to the right of point A; that is, cardiac output decreases, but Pv rises. A More Com﻿plete Theoretical Model: The Two-Pum﻿p System﻿ The preceding discussion shows that the interrelationships between cardiac output and Pv are complex, even in an oversimplified circulation model that includes only one pump and just the systemic circulation. In reality, the cardiovascular system includes the systemic and pulmonary circulations and two pumps: the left and right ventricles. Thus the interrelationships among ventricular output, arterial pressure, and atrial pressure are much more complex.

1	Fig. 19.12 depicts a more complete (but still oversimplified) cardiovascular system model that has two pumps in series (the left and right ventricles) and two vascular beds in series (the systemic and pulmonary vasculature). The series arrangement requires that the flow pumped by the two ventricles be virtually equal to each other over any substantial period; otherwise, all the blood would ultimately accumulate in one or the other of the vascular systems. Because the cardiac function curves for the two ventricles differ substantially, the filling (atrial) pressures for the two ventricles must differ appropriately to ensure equal stroke volumes (see Fig. 18.13

1	Anychangeincontractilitythataffectsthetwoventriclesdifferentlyaltersthedistributionofbloodvolumeinthetwovascularsystems.Ifacoronaryarterytotheleftventriclebecomesoccluded,leftventricularcontractilityisimpaired,andacute left ventricular failure

1	left ventricular failure ensues.Intheinstantafterocclusion,leftatrialpressuredoesnotchange,andtheleftventriclebeginstopumpatadiminishedrateofflow.Iftherightventricleisnotaffectedbytheacutecoronaryarteryocclusion,therightventricleinitiallycontinuestopumpthenormalflow.Thedisparaterightandleftventricularoutputsresultinaprogressiveincreaseinleftatrialpressureandaprogressivedecreaseinrightatrialpressure.Therefore,leftventricularoutputincreasestowardthenormalvalue,andrightventricularoutputfallsbelowthenormalvalue.Thisprocesscontinuesuntiltheoutputsofthetwoventriclesagainbecomeequal.Atthisnewequilibrium,theoutputofthetwoventriclesissubnormal.Theelevationinleftatrialpressureisaccompaniedbyanequalelevationinpulmonaryvenouspressure,whichcanhaveseriousclinicalconsequences.Thehighpulmonaryvenouspressurecanincreaselungstiffnessandleadtorespiratorydistressbyincreasingthemechanicalworkofpulmonaryventilation(see

1	).Furthermore,thehighpulmonaryvenouspressurecausesanelevatethehydrostaticpressureinthepulmonarycapillariesandmayleadtothetransudationoffluidfromthepulmonarycapillariestothepulmonaryinterstitiumorintothealveoli(pulmonary edema), whichmaybelethal.

1	Two basic principles to remember about ventricular function are that (1) the left ventricle pumps blood through the systemic vasculature, and (2) the right ventricle pumps blood through the pulmonary vasculature. However, these principles do not necessarily imply that both ventricles are essential to perfuse the systemic and pulmonary vascular beds adequately. To better understand the relationships between the two ventricles and the two vascular beds, the right ventricular function is examined in more detail as follows. In the circulatory system model shown in

1	In the circulatory system model shown in Fig. 19.12, consider the hemodynamic consequences that would occur if the right ventricle suddenly ceased its pump function but instead served merely as a passive, low-resistance conduit between the systemic veins and the pulmonary arteries. Under these conditions, the only functional pump would be the left ventricle, which would then be required to pump blood through both the systemic and pulmonary resistances (for the purposes of this discussion, consider the resistance to the flow of blood through the inactive right ventricle to be negligible).

1	Normally, pulmonary vascular resistance is approximately 10% as great as systemic vascular resistance. Because the two resistances are in series with one another, total resistance would be 10% greater than systemic resistance alone (see ). In a normal cardiovascular system, a 10% (and hence left ventricular afterload) by approximately 10%. This increase would not drastically affect left ventricular function. Under certain conditions, however, this increase in Pa could significantly alter the function of the cardiovascular system. If the 10% increase in total resistance is achieved by adding a small degree of resistance (i.e., pulmonary vascular resistance) to that of the much larger systemic resistance, and if the pulmonary vascular resistance is separated from the systemic resistance by a large degree of compliance (the combined systemic venous and pulmonary arterial compliance), the 10% increase in total resistance could drastically impair operation of the cardiovascular system.

1	The simulated effects of inactivating the pumping action of the right ventricle in a hydraulic analogue of the circula tory system are shown in Fig. 19.13 . In the model, the right and left ventricles generate cardiac outputs that vary directly with their respective filling pressures. Under control conditions (when the right ventricle is functioning normally), the outputs of the left and right ventricles are equal (5 L/ minute). The right ventricular pumping action causes the pressure in the pulmonary artery (not shown) to exceed the pressure in the pulmonary veins (Ppv) by an amount that forces fluid through the pulmonary vascular resistance at a rate of 5 L/minute. When the right ventricle ceases pumping (arrow 1 in

1	Fig. 19.13 ), the systemic venous and pulmonary arterial systems, along with the right ventricle itself, become a common passive conduit with a large compliance. When the right ventricle ceases to transfer blood actively from the systemic veins to the pulmonary arteries, pulmonary arterial pressure (Ppa) decreases rapidly (not shown) and systemic venous pressure (Psv) rises rapidly to a common value (≈5 mm Hg). At this low pressure, however, fluid flows from the pulmonary arteries to the pulmonary veins at a greatly reduced rate.

1	At the start of right ventricular arrest, the left ventricle is pumping fluid from the pulmonary veins to the systemic arteries at the control rate of 5 L/minute, which greatly exceeds the rate at which blood returns to the pulmonary veins once the right ventricle ceases to operate. Hence, pulmonary venous pressure (Ppv) drops sharply. Because pulmonary venous pressure is the preload for the left ventricle, left ventricular (cardiac) output drops abruptly as well and attains a steady-state value of approximately 2.5 L/minute. This effect in turn leads to a rapid reduction in systemic arterial pressure (Psa). In short, stoppage of right ventricular pumping markedly curtails cardiac output, systemic arterial pressure, and pulmonary venous •Fig. 19.13 Changesincardiacoutput,systemicarterialpressure(Psa),systemicvenouspressure(Psv),andpulmonaryvenouspressure(Ppv)evokedbysimulatedrightventricular(RV)failureandbysimulatedinfusionoffluidinthecirculatorymodelshownin

1	Fig.19.12 .Atarrow 1, thepumpingactionoftherightventriclewasdiscontinued(simulatedRVfailure),andtherightventricleservedonlyasalow-resistanceconduit.Atarrow 2, thefluidvolumeinthesystemwasexpanded,andtherightventriclecontinuedtoserveonlyasaconduit.(ModifiedfromFureySA,etal.Am Heart J. 1984;107:404.) Fig. 19.13). Most of the hemodynamic problems induced by inactivation of the right ventricle can be reversed by an increase in the fluid (blood) volume of the system (arrow 2 in Fig.

1	Fig. 19.13). Most of the hemodynamic problems induced by inactivation of the right ventricle can be reversed by an increase in the fluid (blood) volume of the system (arrow 2 in Fig. 19.13). If fluid is added until pulmonary venous pressure (left ventricular preload) is raised to its control value, cardiac output and systemic arterial pressure are restored almost to normal, but systemic venous pressure is abnormally elevated. If left ventricular function is normal, adding a normal left ventricular preload evokes normal left ventricular output. The 10% increase in peripheral resistance caused by adding the pulmonary vascular resistance to that of the systemic vascular resistance does not impose a serious burden on left ventricular pumping capacity.

1	When the right ventricle is inoperative, however, pulmonary blood flow is not normal unless the usual pulmonary arteriovenous pressure gradient (≈10 to 15 mm Hg) prevails. Hence, systemic venous pressure (Psv) must exceed pulmonary venous pressure (Ppv) by this amount.

1	Maintenance of high systemic venous pressure may lead to the accumulation of tissue fluid (edema) in dependent regions of the body, a characteristic finding in patients with right ventricular heart failure. With this information, the principal function of the right ventricle may be characterized as as follows. From the viewpoint of providing sufficient flow of blood to all tissues in the body, the left ventricle alone can carry out this function. Operation of the two ventricles in series is not essential to provide adequate blood flow to the tissues. The crucial function of the right ventricle is to prevent the rise in systemic venous (and pulmonary arterial) pressure that would be required to force the normal cardiac output through the pulmonary vascular resistance. A normal right ventricle, by preventing an abnormal rise in systemic venous pressure, prevents the development of extensive edema in dependent regions of the body.

1	Clinically,right ventricular heart failure maybecausedbyocclusivediseasepredominantlyofthecoronaryvesselstotherightventricle.Thesevesselsareaffectedmuchlesscommonlythanthevesselstotheleftventricle.Themajorhemodynamiceffectsofacuteright-sidedheartfailurearepronouncedreductionsincardiacoutputandarterialbloodpressure,andtheprincipaltreatmentisinfusionofbloodorplasma.Bypassoftherightventricle(byanastomosisoftherightatriumtothepulmonaryartery)maybeperformedsurgicallyinpatientswithcertaincongenital cardiac defects, suchasseverenarrowingofthetricuspidvalveormaldevelopmentoftherightventricle.Theeffectsofacuteright-sidedheartfailureorrightventricularbypassaredirectionallysimilartothosepredictedpreviouslyfromanalysisofthemodelshownin Fig.19.13 Role of the Heart Rate in Control of Cardiac Output

1	Fig.19.13 Role of the Heart Rate in Control of Cardiac Output Cardiac output is the product of stroke volume and heart rate. Analysis of the control of cardiac output has thus far been restricted to the control of stroke volume, and the role of heart rate has not been considered. Analysis of the effect of a change in heart rate on cardiac output is complex because a change in heart rate alters the other three factors (preload, afterload, and myocardial contractility) that determine stroke volume (see Fig. 19.1 ). An increase in heart rate, for example, shortens the duration of diastole. Hence, ventricular filling is diminished; that is, preload is reduced. If an increase in heart rate altered cardiac output, arterial pressure would change; that is, afterload would be altered. A rise in heart rate would increase the net influx of Ca++ per minute into myocardial cells (see also ), and this influx would enhance myocardial contractility.

1	The effects of changes in heart rate on cardiac output have been studied extensively, and the results are similar to those shown in Fig. 19.14 . As atrial pacing frequency is •Fig. 19.14 Changesinstrokevolume(A) andcardiacoutput (B) inducedbychangesintherateofatrialpacing.(RedrawnfromKumadaM,etal.Jpn J Physiol. 1967;17:538.) gradually increased, stroke volume progressively diminishes (see Fig. 19.14A ). The decrease in stroke volume is caused by the reduced time for ventricular filling. The change in stroke volume is not inversely proportional to the change in heart rate because the direction of the change in cardiac output (Qh) is markedly influenced by the actual heart rate (see Fig. 19.14B ). For example, as pacing frequency is increased from 50 to 100 beats/minute, the increase in heart rate augments Qh. Because Qh = SV × HR, the decrease in stroke volume (SV) over this frequency range must be proportionately less than the increase in heart rate (HR).

1	Over the frequency range from approximately 100 to 200 beats/minute, however, cardiac output is not affected significantly by changes in pacing frequency (see Fig. 19.14B ). Hence, as pacing frequency is increased, the decrease in stroke volume must be approximately equal to the increase in heart rate. In addition, generalized vascular autoregulation tends to keep tissue blood flow constant (see also ). This adaptation leads to changes in preload and afterload that also keep cardiac output nearly constant. Moreover, at excessively high pacing frequencies (above 200 beats/minute; see

1	Moreover, at excessively high pacing frequencies (above 200 beats/minute; see Fig. 19.14 ), further increases in heart rate decrease cardiac output. Therefore, the induced decrease in stroke volume must have exceeded the increase in heart rate at this high range of pacing frequencies. At such high pacing frequencies, the ventricular filling time is so severely restricted that compensation is inadequate, and cardiac output decreases sharply. Although the relationship of cardiac output to heart rate is characteristically that of an inverted U in the general population, the relationship varies quantitatively among subjects and among physiological states.

1	Strong correlations between heart rate and cardiac output must be interpreted cautiously. In people who are exercising, for example, cardiac output and heart rate usually increase proportionately, and stroke volume may remain constant or increase only slightly (see the later section ”). It is tempting to conclude that the increase in cardiac output during exercise must be caused solely by the observed increase in heart rate. However,

1	Fig. 19.14 shows that over a wide range of heart rates, a change in heart rate may have little influence on cardiac output. The principal increase in cardiac output during exercise must therefore be attributed to other factors. Such ancillary factors include the pronounced reduction in peripheral vascular resistance because of the vasodilation in the active skeletal muscles and the increased contractility of cardiac muscle associated with the generalized increase in sympathetic neural activity. Nevertheless, the increase in heart rate is still an important factor. Abundant data show that if the heart rate cannot increase normally during exercise, the augmentation in cardiac output and the capacity for exercise are severely limited. Because stroke volume changes only slightly during exercise, the increase in heart rate may play an important permissive role in augmenting cardiac output during physical exercise.

1	Thecharacteristicrelationshipbetweencardiacoutputandheartrateexplainstheurgentneedoftreatmentbypatientswhohaveexcessivelysloworexcessivelyfastheartrates.Profoundbradycardia (slowrate)mayoccurasaresultofaveryslowsinusrhythminpatientswithsick sinus syndrome orasaresultofaslowidioventricularrhythminpatientswithcomplete atrioventricular block. Ineitherrhythmdisturbance,thecapacityoftheventriclestofillduringprolongeddiastoleislimited(oftenbythenoncompliantpericardium).Hence,cardiacoutputusuallydecreasessubstantiallybecausetheveryslowheartratecannotbecounterbalancedbyasufficientlylargestrokevolume.Asaconsequence,suchbradycardiaoftennecessitatestheimplantationofanartificialpacemaker.Inpatientswithsupraventricular orventricular tachycardia,

1	orventricular tachycardia, excessivelyhighheartratesfrequentlynecessitateemergencytreatmentbecauseinsuchpatients,cardiacoutputmaybecriticallylow,andthefillingtimeissorestrictedatveryhighheartratesthatevensmalladditionalreductionsinfillingtimecausedisproportionatelyseverereductionsinfillingvolume.Slowingtheheartratetoamorenormalrhythmcangenerallybeaccomplishedpharmacologically,butelectricalcardioversionmayberequiredinemergencies(see

1	Ancillary Factors That Affect the Venous System﻿ and Cardiac Output In earlier sections of this chapter, the descriptions of the interrelationships between Pv and cardiac output were simplified by restricting the discussion to the effects evoked by individual variables. However, because the cardiovascular system is regulated by so many feedback control loops, its responses are rarely simple. A change in blood volume, for example, not only affects cardiac output directly through the Frank-Starling mechanism but also triggers reflexes that alter other aspects of cardiac function (such as the heart rate, atrioventricular conduction, and myocardial contractility) and other characteristics of the vascular system (such as peripheral resistance and venomotor tone). Several other factors, especially gravity (see ) and respiration, also regulate cardiac output. Circulatory Effects of Respiratory Activity

1	Circulatory Effects of Respiratory Activity The normal, periodic activity of the respiratory muscles causes rhythmic variations in vena caval flow (Fig. 19.15 ). During respiration, the reduction in intrathoracic pressure is transmitted to the lumens of the thoracic blood vessels. The reduction in Pv during inspiration increases the pressure gradient between extrathoracic and intrathoracic veins. The consequent acceleration in venous return to the right atrium is shown in Fig. 19.15 as an increase in superior vena caval blood flow from 5.2 mL/sec during expiration to 11 mL/ sec during inspiration.

1	Fig. 19.15 as an increase in superior vena caval blood flow from 5.2 mL/sec during expiration to 11 mL/ sec during inspiration. The exaggerated reduction in intrathoracic pressure achieved by a strong inspiratory effort against a closed glottis (called Müller’s maneuver) does not increase venous return proportionately. The extrathoracic veins collapse near their entry into the chest when their internal pressures fall below the ambient level. As the veins collapse, flow into the chest momentarily stops. The cessation of flow causes pressure upstream to rise, which forces the collapsed segment to reopen.

1	During normal expiration, flow into the central veins decelerates. However, the mean rate of venous return during normal respiration exceeds the flow during a brief period of apnea (cessation of respiration). Hence, normal inspiration apparently facilitates venous return more than normal expiration impedes it. In part, venous return is facilitated by the valves in the veins of the extremities. These valves prevent any reversal of flow during expiration. Thus the respiratory muscles and venous valves constitute an auxiliary pump for venous return. •Fig. 19.15 Duringanormalinspiration,intrathoracicpressure,rightatrialpressure(RAP),andjugularvenous(JVP)pressuredecrease,andflowinthesuperiorvenacava(SVCF)increases(from5.2to11mL/sec).AllpressuresaregiveninmmH2O.Femoralarterialpressure(notshown)didnotchangesubstantiallyduringthenormalinspiration.Jugular vein 50 5.2 mL/sec 11 mL/sec SVCF JVP RAP 0 Atrial 0 –100

1	Thedramaticincreaseinintrathoracicpressureinducedbycoughingconstitutesanauxiliarypumpingmechanismfortheblooddespiteitsconcurrenttendencytoimpedevenousreturn.Certaindiagnosticprocedures,suchascoronaryangiographyorelectrophysiologicaltestingofcardiacfunction,increasetheriskforventricularfibrillation;therefore,patientsundergoingsuchproceduresaretrainedtocoughrhythmicallyoncommandduringthestudy.Ifventricularfibrillationdoesoccur,eachcoughcangeneratesubstantialincreasesinarterialbloodpressure,andenoughcerebralbloodflowmaybepromotedtosustainconsciousness.Thecoughraisesintravascularpressureequallyintheintrathoracicarteriesandveins.Bloodispropelledthroughtheextrathoracictissuesbecausetheincreasedpressureistransmittedtotheextrathoracicarteriesbutnottotheextrathoracicveinsbecausethevenousvalvespreventbackwardflowfromtheintrathoracictotheextrathoracicveins.

1	Inmostformsofartificialrespiration(mouth-to-mouthresuscitation,mechanicalrespiration),endotrachealpressureaboveatmosphericpressureisusedtoinflatethelungs,andexpirationoccursbypassiverecoilofthethoraciccage(see ).Thuslunginflationisaccompaniedbyanappreciableriseinintrathoracicpressure.Venacavalflowdecreasessharplyduringthephaseofpositive-pressurelunginflationwhentheendotrachealpressureprogressivelyrises.Whennegativeendotrachealpressureisusedtofacilitatedeflation,venacavalflowacceleratesmorethanwhenthelungsareallowedtodeflatepassively.

1	Sustained expiratory efforts increase intrathoracic pressure and thus impede venous return. Straining against a closed glottis (Valsalva’s maneuver) regularly occurs during coughing, defecation, and heavy lifting. Intrathoracic pressures in excess of 100 mm Hg have been recorded in trumpet players, and pressures higher than 400 mm Hg have been observed during paroxysms of coughing. Such increases in pressure are transmitted directly to the lumens of the intrathoracic arteries. After coughing stops, arterial blood pressure may fall precipitously because of the preceding impediment to venous return. Interplay of Central and Peripheral Factors in Control of the Circulation

1	Interplay of Central and Peripheral Factors in Control of the Circulation The primary function of the circulatory system is to deliver the nutrients needed for tissue metabolism and growth and to remove the products of metabolism. The contributions of the components of the cardiovascular system to maintain adequate tissue perfusion under different physiological conditions were discussed previously. In this section, the interrelationships among the various components of the circulatory system are explored. The autonomic nervous system and the baroreceptors and chemoreceptors play key roles in regulating the cardiovascular system. Control of fluid balance by the kidneys, with maintenance of a constant blood volume, is also very important.

1	In any well-regulated system, one way to evaluate the extent and sensitivity of its regulatory mechanisms is to disturb the system and to observe how it restores the preexisting steady state. Two such disturbances, physical exercise and hemorrhage, are discussed in the following sections to illustrate operation of the various regulatory factors.

1	The cardiovascular adjustments that occur during exercise consist of a combination of neural and local (chemical) factors. Neural factors include (1) central command, (2) reflexes that originate in the contracting muscle, and (3) the baroreceptor reflex. Central command is the cerebrocortical activation of the sympathetic nervous system that produces cardiac acceleration, increased myocardial contractile force, and peripheral vasoconstriction. Reflexes are activated intramuscularly by stimulation of mechanoreceptors (by stretch, tension) and chemoreceptors (by metabolic products) in response to muscle contraction. Impulses from these receptors travel centrally via small myelinated (group III) and unmyelinated (group IV) afferent nerve fibers. Group IV unmyelinated fibers may represent the muscle chemoreceptors, inasmuch as no morphological chemoreceptor has been identified. The central connections of this reflex are unknown, but the efferent limb consists of sympathetic nerve fibers

1	muscle chemoreceptors, inasmuch as no morphological chemoreceptor has been identified. The central connections of this reflex are unknown, but the efferent limb consists of sympathetic nerve fibers to the heart and peripheral blood vessels. The baroreceptor reflex is described in , and local factors that influence skeletal muscle blood flow (metabolic vasodilators) are described in . Vascular chemoreceptors are important in regulation of the cardiovascular system during exercise. Evidence for this assertion comes from the observations that the PaCO2, the PaO2, and the pH of arterial blood remain normal during exercise.

1	Mild to Moderate Exercise In humans or trained animals, anticipation of physical activity inhibits vagal nerve impulses to the heart and increases sympathetic discharge. The result is an increase in heart rate and myocardial contractility. The tachycardia and enhanced contractility increase cardiac output. When cardiac stimulation occurs, the sympathetic nervous system also changes vascular resistance in the periphery. Sympathetic nervous system–mediated vasoconstriction increases vascular resistance and thereby diverts blood away from the skin, kidneys, splanchnic regions, and inactive Cardiac output (L/min) 22 18 14 10 6 2 0.25 1 2 3 4 O2 uptake (L/min) Skin Heart and brain Viscera Muscle Maximum Vo2 .

1	Cardiac output (L/min) 22 18 14 10 6 2 0.25 1 2 3 4 O2 uptake (L/min) Skin Heart and brain Viscera Muscle Maximum Vo2 . •Fig. 19.16 ApproximatedistributionofcardiacoutputatrestandatdifferentlevelsofexerciseuptothemaximalO2consumption(V˜O2)in 0 0 5 10 15 800 1600 0.006 0.010 0.014 60 100 140 180 5 10 15 90 100 110 60 100 140 180 Mean Systolic pressure Diastolic pressure anormalyoungman.(RedrawnfromRuchHP,PattonTC.Physiology and Biophysics. 12thed.Philadelphia:Saunders;1974.) Fig. 19.16 ). This increase in vascular resistance persists throughout the period of exercise.

1	Fig. 19.16 ). This increase in vascular resistance persists throughout the period of exercise. Cardiac output and blood flow to active muscles increase as the intensity of exercise increases. Blood flow to the myocardium increases, whereas flow to the brain is unchanged. Blood flow in the skin initially decreases during exercise, then increases as body temperature rises with increments in the duration and intensity of exercise, and finally decreases when the skin vessels constrict as total body O2 consumption nears its maximal value (see Fig. 19.16

1	The major circulatory adjustment to prolonged exercise occurs in the vasculature of the active muscles. Local formation of vasoactive metabolites causes marked dilation of the resistance vessels. This dilation progresses with increases in the intensity of exercise. Potassium is one of the vasodilator substances released by the contracting muscle, and this ion may be partly responsible for the initial decrease in vascular resistance in the active muscles. Other contributing factors may be the release of adenosine and a decrease in tissue pH during sustained exercise. The local accumulation of metabolites causes the terminal arterioles to relax, and blood flow through the muscle may increase 15to 20-fold above the resting level. This metabolic vasodilation of the precapillary vessels in active muscles occurs very soon after the onset of exercise. The decrease in TPR enables the heart to pump more blood at a lesser load, and it pumps more efficiently than if TPR were unchanged (see •Fig.

1	muscles occurs very soon after the onset of exercise. The decrease in TPR enables the heart to pump more blood at a lesser load, and it pumps more efficiently than if TPR were unchanged (see •Fig. 19.17 Effectsofdifferentlevelsofexercise(i.e.,work)onseveralcardiovascularvariables.(DatafromCarlstenA,GrimbyG.The Circulatory Response to Muscular Exercise in Man. Springfield,

1	IL:CharlesCThomas;1966.) Marked changes in the capillary circulation also occur during exercise. At rest, only a small percentage of the capillaries are perfused, whereas in actively contracting muscle, all or nearly all of the capillaries contain flowing blood (capillary recruitment). The surface area available for exchange of gases, water, and solutes is increased many times. Furthermore, hydrostatic pressure in the capillaries is increased because of relaxation of the resistance vessels. Hence, water and solutes move into the muscle tissue. Tissue pressure rises and remains elevated during exercise as fluid continues to move out of the capillaries; this tissue fluid is carried away by the lymphatic vessels. Lymph flow is increased as a result of the rise in capillary hydrostatic pressure and the massaging effect of the contracting muscles on the valve-containing lymphatic vessels (see

1	Contracting muscle avidly extracts O2 from the perfusing blood and thereby increases the arteriovenous O2 difference (

1	Fig. 19.17 ). This release of O2 from blood is facilitated by the shift in the oxyhemoglobin dissociation curve during exercise. During exercise, the high concentration of CO2 and the formation of lactic acid cause a reduction in tissue pH. This decrease in pH, in addition to the increase in temperature in the contracting muscle, shifts the oxyhemoglobin dissociation curve to the right (see ). Therefore, at any given PO2, less O2 is held by the hemoglobin in the red blood cells, and consequently more O2 is available for the tissues. Oxygen consumption may increase as much as 60-fold, with only a 15-fold increase in muscle blood flow. Muscle myoglobin may serve as a limited O2 store during exercise, and it can release the attached O2 at very low partial pressures. However, myoglobin can also facilitate O2 transport from capillaries to mitochondria by serving as an O2 carrier.

1	Because the enhanced sympathetic drive and the reduced parasympathetic inhibition of the sinoatrial node continue during exercise, tachycardia persists. If the workload is moderate and constant, the heart rate reaches a certain level and remains there throughout the period of exercise. However, if the workload increases, the heart rate increases concomitantly until a plateau of approximately 180 beats per minute is reached during strenuous exercise. In contrast to the large increase in heart rate, the increase in stroke volume is only approximately 10% to 35%, the larger values occurring in trained individuals (see Fig. 19.17 ). In well-trained distance runners, whose cardiac output can reach six to seven times the resting level, stroke volume attains approximately twice the resting value.

1	Cardiacmusclesize(growth)isdirectlyrelatedtotheamountofworkthatisimposeduponit.Duringdevelopmentandinenduranceexercise,cardiacgrowthisachievedataconstantrelationbetweensystolicbloodpressureandtheratioofwallthicknesstoventricularchamberradius.Anechocardiographicmeasurementusedtodistinguishphysiologicalfrompathologicalhypertrophyisrelativewallthickness(ratioofleftventricularwallthicknesstochamberradius).Inphysiologicalhypertrophy,leftventricularmassandradiusincreaseproportionatelysothatrelativewallthicknessdoesnotchangesignificantly.Examplesofphysiologicalhypertrophyoccurinenduranceathletesandinpregnantwomen,inwhomleftventricularenlargementoccurswithvolumeoverloadatconstantrelativewallthickness.Physiologicalhypertrophyisassociatedwithanincreasedarteriolardiameterinexperimentalanimals.Also,capillarydensityincreasesinproportiontothedegreeofhypertrophy.Thisisincontrasttothesituationinpathologicalhypertrophy,inwhichareductionofcapillarydensity(rarefaction)canoccur.Neithermyocardialfibrosisn

1	apillarydensityincreasesinproportiontothedegreeofhypertrophy.Thisisincontrasttothesituationinpathologicalhypertrophy,inwhichareductionofcapillarydensity(rarefaction)canoccur.Neithermyocardialfibrosisnorderangementofmusclefiberorientationisdetectedinphysiologicalhypertrophy,incontrasttothefindingsinpathologicalhypertrophy.

1	Thus the increase in cardiac output observed during exercise is correlated principally with an increase in heart rate. If the baroreceptors are denervated, the cardiac output and heart rate responses to exercise are small in comparison with those in individuals with normally innervated baroreceptors. However, with total cardiac denervation, exercise still increases cardiac output as much as it does in normal individuals. This increase in cardiac output is achieved chiefly by means of an elevated stroke volume. However, if a β-adrenergic receptor antagonist is given to dogs with denervated hearts, exercise performance is impaired. The β-adrenergic receptor antagonist prevents the cardiac acceleration and enhanced contractility caused by increased amounts of circulating catecholamines. Therefore, the increase in cardiac output necessary for maximal exercise performance is limited.

1	In addition to the contribution made by sympathetically mediated constriction of the capacitance vessels in both exercising and nonexercising parts of the body, venous return is aided by the auxiliary pumping action of the working skeletal muscles and the muscles of respiration (see also ). The intermittently contracting muscles compress the veins that course through them. Because the venous valves are oriented toward the heart, the contracting muscle pumps blood back toward the right atrium (see ). In exercise, the flow of venous blood to the heart is also aided by the deeper and more frequent respirations that increase the pressure gradient between the abdominal and thoracic veins (intrathoracic pressure becomes more negative during exercise).

1	In humans, blood reservoirs do not contribute much to the circulating blood volume. In fact, blood volume is usually reduced slightly during exercise, as evidenced by a rise in the hematocrit ratio. This decrease in blood volume is caused by water loss externally through sweating and enhanced ventilation and by fluid movement into the contracting muscle. However, fluid loss is counteracted in several ways. Fluid loss from the vascular compartment into the contracting muscles eventually reaches a plateau as interstitial fluid pressure rises and opposes the increased hydrostatic pressure in capillaries of the active muscle. Fluid loss is partially offset by movement of fluid from the splanchnic regions and inactive muscle into the bloodstream. This influx of fluid results from (1) a decrease in hydrostatic pressure in the capillaries of these tissues and (2) an increase in plasma osmolarity because of movement of osmotically active molecules into blood from the contracting muscle.

1	in hydrostatic pressure in the capillaries of these tissues and (2) an increase in plasma osmolarity because of movement of osmotically active molecules into blood from the contracting muscle. Reduction in urine formation by the kidneys also helps conserve body water.

1	The large volume of venous blood returning to the heart is so effectively pumped through the lungs and out into the aorta that Pv remains essentially constant. Thus the Frank-Starling mechanism of a greater initial fiber length does not account for the greater stroke volume in moderate exercise. Radiographs of individuals at rest and during exercise reveal a decrease in heart size during exercise. However, during maximal or near-maximal exercise, right atrial pressure and end-diastolic ventricular volume do increase, and the Frank-Starling mechanism contributes to the enhanced stroke volume in very vigorous exercise.

1	•Fig. 19.18 Cardiacandvascularfunctioncurvesaregreatlyalteredduringstrenuousexercise,whichallowscardiacoutputtoincreasefourfoldtofivefold.TheoperatingpointofthecardiovascularsystemmovesfrompointAtopointB.Thecardiacfunctioncurveduringstrenuousexerciseistheresultofincreasedheartrate,strokevolume,andcontractility.Thevascularfunctioncurvereflectsgreatlydecreasedtotalperipheralresistanceandincreasedmeancirculatorypressure.Atthenewoperatingpoint(pointB),cardiacoutputisincreasedmorethanfourfold,butfillingpressureisincreasedonlyslightly. In an active, healthy (untrained) individual, the mechanisms previously described typically lead to a fourfold to fivefold increase in cardiac output during vigorous exercise ( Fig. 19.18 ). Increased cardiac output is the fundamental means by which more O2 is delivered to exercising muscles (see

1	Fig. 19.18 ). Increased cardiac output is the fundamental means by which more O2 is delivered to exercising muscles (see Fig. 19.17 ). The cardiac function curve during exercise reflects increased stroke volume (up to ≈1.5-fold) and heart rate (up to ≈3-fold). The vascular function curve during dynamic exercise reflects a marked decrease in peripheral resistance (changed slope) and an increase in mean circulatory filling pressure (changed intercept), which result from increased venous constriction (tone) in the skeletal muscle “pump,” and the respiratory “pump.” In these conditions, the cardiovascular system is able to operate at a new point (point B in Fig. 19.18 ), in which cardiac output is increased while filling pressure is little changed. The graphical analysis (see Fig. 19.18 ) shows that without the systemic changes in vascular function, even a heart beating strongly and fast would be able to produce only a small increase in cardiac output.

1	Fig. 19.18 ) shows that without the systemic changes in vascular function, even a heart beating strongly and fast would be able to produce only a small increase in cardiac output. If exercise involves a large proportion of the body musculature, as in running or swimming, the reduction in total vascular resistance can be considerable. Nevertheless, arterial pressure starts to rise with the onset of exercise, and the increase in blood pressure approximately parallels the severity of the exercise performed (see

1	Fig. 19.17 ). Therefore, the increase in cardiac output is proportionally greater than the decrease in TPR. The vasoconstriction produced in the inactive tissues by the sympathetic nervous system (and to some extent by the release of catecholamines from the adrenal medulla) is important for maintenance of normal or increased blood pressure. Sympathectomy or drug-induced blockade of the adrenergic sympathetic nerve fibers decreases arterial pressure (hypotension) during exercise. Sympathetic neural activity also elicits vasoconstriction in active skeletal muscle when additional muscles are recruited. In experiments in which one leg is working at maximal levels and then the other leg starts to work, blood flow decreases in the first working leg. Furthermore, blood levels of norepinephrine rise significantly during exercise, and most of the norepinephrine is released from sympathetic nerves to the active muscles.

1	As body temperature rises during exercise, the skin vessels dilate in response to thermal stimulation of the heat-regulating center in the hypothalamus, and TPR decreases further. This reduction in TPR would reduce blood pressure were it not for the increased cardiac output and the constriction of arterioles in the renal, splanchnic, and other tissues. In general, Pa rises during exercise as a result of the increase in cardiac output. However, the effect of enhanced cardiac output is offset by an overall decrease in TPR, and therefore mean blood pressure increases only slightly. Vasoconstriction in the inactive vascular beds helps maintain normal arterial blood pressure for adequate perfusion of the active tissues. The actual Pa attained during exercise thus represents a balance between cardiac output and TPR (see ). Systolic pressure usually increases more than diastolic pressure, which results in an increase in pulse pressure (see

1	Fig. 19.17 ). The larger pulse pressure is primarily attributable to a greater stroke volume, but also to more rapid ejection of blood by the left ventricle and diminished peripheral runoff during the brief ventricular ejection period (see also

1	During exhaustive exercise, the compensatory mechanisms begin to fail. The heart rate attains a maximal level of approximately 180 beats per minute, and stroke volume reaches a plateau. The heart rate may then decrease, which results in a fall in blood pressure. The exercising individual also frequently becomes dehydrated. Sympathetic vasoconstrictor activity supersedes the vasodilator influence on vessels of the skin so that the rate of heat loss is decreased. Body temperature is normally elevated during exercise. A reduction in heat loss through cutaneous vasoconstriction can lead to very high body temperatures and to acute distress during severe exercise. Tissue pH and blood pH decrease as a result of increased production of lactic acid and CO2. The reduced pH may be a key factor that determines •Fig. 19.19 Cardiovascularadjustmentsinexercise.Plus signs indicateincreasedactivity,andminus signs

1	of increased production of lactic acid and CO2. The reduced pH may be a key factor that determines •Fig. 19.19 Cardiovascularadjustmentsinexercise.Plus signs indicateincreasedactivity,andminus signs indicatedecreasedactivity.C,vasoconstrictoractivity;D,vasodilatoractivity;IX,glossopharyngeal nerve;VR,vasomotorregion;X,vagusnerve.

1	the maximal amount of exercise that a given individual can tolerate. Muscle pain, a subjective feeling of exhaustion, and loss of the will to continue determine exercise tolerance. A summary of the neural and local effects of exercise on the cardiovascular system is diagrammed in Fig. 19.19 When exercise stops, the heart rate and cardiac output quickly decrease: The sympathetic drive to the heart is essentially removed. In contrast, TPR remains low for some time after the exercise is stopped, presumably because vasodilator metabolites have accumulated in the muscles during the exercise period. As a result of the reduced cardiac output and persistence of vasodilation in the muscles, arterial pressure falls, often below preexercise levels, for brief periods. Blood pressure is then stabilized at normal levels by the baroreceptor reflexes. Limits of Exercise Performance

1	The two main factors that limit skeletal muscle performance in humans are the rate of O2 use by the muscles and the O2 supply to the muscles. However, O2 use by muscle is probably not a critical factor. During exercise, maximal O2 consumption (maximal V˜O2) by a large percentage of the body’s muscle mass is unchanged or increases only slightly when additional muscles are activated. In fact, during exercise of a large muscle mass, as in vigorous bicycling, the addition of bilateral arm exercise without change in the cycling effort produces only a small increase in cardiac output and maximal VO2. However, the additional arm exercise decreases blood flow to the legs. This centrally mediated (baroreceptor reflex) vasoconstriction during maximal cardiac output prevents the fall in blood pressure that would otherwise be caused by metabolically induced vasodilation in the active muscle. If use of O2 by muscles were a significant limiting factor, recruitment of more contracting muscles would

1	that would otherwise be caused by metabolically induced vasodilation in the active muscle. If use of O2 by muscles were a significant limiting factor, recruitment of more contracting muscles would entail the use of much more O2 to meet the enhanced O2 requirements.

1	Limitation of the O2 supply could be caused by inadequate oxygenation of blood in the lungs or limitation of the supply of O2-laden blood to the muscles. Failure by the lungs to oxygenate blood fully can be ruled out because even with the most strenuous exercise at sea level, arterial blood is fully saturated with O2. Therefore, O2 delivery to the active muscles (or blood flow because the arterial blood O2 content is normal) appears to be the limiting factor in muscle performance. This limitation could be caused by the inability to increase cardiac output beyond a critical level. In turn, this inability is caused by a limitation in stroke volume because the heart rate reaches maximal levels before maximal V˜O2 is reached. Hence, the major factor that limits muscle performance is the pumping capacity of the heart.

1	The response of the cardiovascular system to regular exercise is to increase its capacity to deliver O2 to the active muscles and improve the ability of the muscle to use O2. Maximal

1	VO2 varies with the level of physical conditioning. Train ing progressively increases maximal VO2, which reaches a plateau at the highest level of conditioning. Highly trained athletes have a lower resting heart rate, a greater stroke volume, and lower peripheral resistance than they had before training or after deconditioning. The low resting heart rate is caused by a higher vagal tone and a lower sympathetic tone. During exercise, the maximal heart rate of a trained individual is the same as that in an untrained person, but it is attained at a higher level of exercise. A trained person also exhibits low vascular resistance in the muscles. If an individual exercises one leg regularly over an extended period and does not exercise the other leg, vascular resistance is lower and maximal V˜O2 is higher in the “trained” leg than in the “untrained” leg.

1	Physical conditioning is also associated with greater extraction of O2 from the blood (greater arteriovenous O2 difference) by the muscles. With long-term training, capillary density in skeletal muscle increases. Also, an increase in the number of arterioles may account for the decrease in muscle vascular resistance. The number of mitochondria increases, as does the number of oxidative enzymes in mitochondria. In addition, levels of adenosine triphosphatase (ATPase) activity, myoglobin, and enzymes involved in lipid metabolism increase in response to physical conditioning. Endurancetraining,suchasrunningorswimming,increasesleftventricularvolumewithoutincreasingleftventricularwallthickness.Incontrast,strengthexercises,suchasweightlifting,increaseleftventricularwallthickness(hypertrophy)withlittleeffectonventricularvolume.However,thisincreaseinwallthicknessissmallinrelationtothatobservedinchronichypertension,inwhichafterloadispersistentlyelevatedbecauseofhighperipheralresistance.

1	The cardiovascular system is the system mainly affected in an individual who has lost a large quantity of blood. Arterial systolic, diastolic, and pulse pressures decrease, and the arterial pulse is rapid and feeble. The cutaneous veins collapse, and they fill slowly when compressed centrally. The skin is pale, moist, and slightly cyanotic. Respiration is rapid but may be shallow or deep. Course of Arterial Blood Pressure Changes Cardiac output decreases as a result of blood loss. The amount of blood removed when it is donated is approximately 10% of total blood volume; its removal is well tolerated, and mean arterial blood pressure changes little. This is not the case when greater amounts are lost from the circulation. The changes in Pa evoked by acute hemorrhage are illustrated in

1	Fig. 19.19 . If sufficient blood is rapidly withdrawn to decrease Pa to 50 mm Hg, the pressure then tends to rise spontaneously toward the control level over the next 20 or 30 minutes. In some individuals (curve A in Fig. 19.20 ), this trend continues, and normal pressure is regained within a few hours. In others (curve B in Fig. 19.20 ), the pressure rises initially after the cessation of hemorrhage. The pressure then begins to decline, and it continues to fall at an accelerating rate until death ensues. This progressive deterioration in cardiovascular function is termed hemorrhagic shock. At some time after the hemorrhage, the deterioration in the cardiovascular system becomes irreversible. A lethal outcome in patients with hemorrhagic shock can be prevented only temporarily by any known therapy, including massive transfusions of donor blood.

1	•Fig. 19.20 Changes in Mean Arterial Pressure After Rapid Hem-orrhage. Attime0,rapidlossofbloodcausesareductioninthemeanarterialpressureto50mmHg.Afteraperiodinwhichthepressurereturnstowardthecontrollevel,someindividualscontinuetoimprove(curveA)untilthecontrolpressureisattained.However,inotherindividuals,thepressurebeginstodecline(curveB)untildeathensues.–2 –1 0 0 25 50 75 100 1 2 3 4 5 A B 6 7 Time (hr) Mean arterial pressure (mm Hg) The changes in arterial pressure immediately after acute blood loss (see

1	The changes in arterial pressure immediately after acute blood loss (see Fig. 19.20 ) indicate that certain compensatory mechanisms must be operative. Any mechanism that raises arterial blood pressure toward normal in response to a reduction in pressure is designated a negative feedback mechanism. This mechanism is termed negative because the direction of the secondary change in pressure is opposite the direction of the initiating change after the acute blood loss. The following negative feedback responses are evoked: baroreceptor reflexes, (2) chemoreceptor reflexes, (3) cerebral ischemia responses, (4) reabsorption of tissue fluids, release of endogenous vasoconstrictor substances, and renal conservation of salt and water.

1	The reductions in Pa and pulse pressure during hemorrhage decrease stimulation of the baroreceptors in the carotid •Fig. 19.21 Changes in Mean Aortic Pressure in Response to an 8% Blood Loss in Three Conditions. A, Thecarotidsinusbaroreceptorswereintactandtheaorticreflexeswereinterrupted.B, Theaorticreflexeswereintactandthecarotidsinusreflexeswereinterrupted.C, Allsinoaorticreflexeswereabrogated.(DatafromShepherdJT.Circulation. 1974;50:418.DerivedfromthedataofEdisAJ.Am J Physiol. 1971;221:1352.) ). Several cardiovascular responses are thus evoked, all of which tend to restore arterial pressure to the normal level. Such responses include reduction of vagal tone and enhancement of sympathetic tone, increased heart rate, and enhanced myocardial contractility. The increased sympathetic tone also produces generalized venoconstriction, which has the same hemodynamic consequences as transfusion of blood (see

1	The increased sympathetic tone also produces generalized venoconstriction, which has the same hemodynamic consequences as transfusion of blood (see Fig. 19.9). Sym pathetic activation constricts certain blood reservoirs. This vasoconstriction acts as an autotransfusion of blood into the circulation. In humans, the cutaneous, pulmonary, and hepatic branches of the vasculature constitute the principal blood reservoirs. Generalized arteriolar constriction is a prominent response to the reduced baroreceptor stimulation during hemorrhage. The reflex increase in peripheral resistance minimizes the fall in arterial pressure caused by the reduction in cardiac output. Fig. 19.21 shows the effect of an 8% blood loss on mean aortic pressure. When both vagus nerves were cut to eliminate the influence of the aortic arch baroreceptors and only the carotid sinus baroreceptors were operative (see

1	Fig. 19.21A), this hemorrhage decreased mean aortic pressure by 14%. This pressure change did not differ significantly from the decline in pressure (12%) evoked by the same hemorrhage before vagotomy (not shown). When the carotid sinuses were denervated and the aortic baroreceptor reflexes were intact, the 8% blood loss decreased mean aortic pressure by 38% (see Fig. 19.21B). Hence, the carotid sinus baroreceptors were more effective than the aortic baroreceptors in attenuating the fall in pressure. However, when both sets of afferent baroreceptor pathways were interrupted (see Fig. 19.21C), an 8% blood loss reduced arterial pressure by 48%.

1	Fig. 19.21C), an 8% blood loss reduced arterial pressure by 48%. Arteriolar constriction is widespread during hemorrhage but it is by no means uniform. Vasoconstriction is most pronounced in the cutaneous, skeletal muscle, and splanchnic vascular beds, and it is slight or absent in the cerebral and coronary circulations in response to hemorrhage. In many instances, cerebral and coronary vascular resistance is diminished. The reduced cardiac output is redistributed to favor flow through the brain and the heart. In the early stages of mild to moderate hemorrhage, renal resistance changes only slightly. The tendency for increased sympathetic activity to constrict the renal vessels is counteracted by autoregulatory mechanisms (see ). With more prolonged and severe hemorrhage, however, renal vasoconstriction becomes intense.

1	The renal and splanchnic vasoconstriction during hemorrhage is least severe in the heart and brain. However, if such constriction persists too long, it may be detrimental. Frequently, patients survive the acute hypotensive period of a prolonged, severe hemorrhage, only to die several days later from the kidney failure that results from renal ischemia. Intestinal ischemia may also have dire effects. For example, intestinal bleeding and extensive sloughing of the mucosa can occur after only a few hours of hemorrhagic hypotension. Furthermore, the diminished splanchnic flow swells the centrilobular cells in the liver. The resulting obstruction of the hepatic sinusoids causes portal venous pressure to rise, and this response intensifies intestinal blood loss.

1	Reductions in arterial pressure below approximately 60 mm Hg do not evoke any additional responses through the baroreceptor reflexes because this pressure level constitutes the threshold for stimulation (see ). However, low arterial pressure may stimulate peripheral chemoreceptors because inadequacy of local blood flow leads to hypoxia in the chemoreceptor tissue. Chemoreceptor excitation may then enhance the already existent peripheral vasoconstriction evoked by the baroreceptor reflexes. In addition, respiratory stimulation assists venous return by the auxiliary pumping mechanism described earlier (see also

1	When arterial pressure falls below approximately 40 mm Hg as a consequence of blood loss, the resulting cerebral ischemia activates the sympathoadrenal system. The discharge by sympathetic nerves is several times greater than the maximal neural activity that occurs when the baroreceptors cease to be stimulated. The vasoconstriction and increase in myocardial contractility may be pronounced. With more severe degrees of cerebral ischemia, however, the vagal centers also become activated. The resulting bradycardia aggravates the hypotension that initiated the cerebral ischemia Reabsorption of Tissue Fluids The arterial hypotension, arteriolar constriction, and reduced venous pressure during hemorrhagic hypotension cause the hydrostatic pressure in the capillaries to drop. The balance of these forces promotes the net reabsorption of interstitial fluid into the vascular compartment (see ). The rapidity of this response is displayed in

1	Fig. 19.22 . When 45% of the estimated blood volume is removed over a 30-minute period, mean arterial blood pressure declines rapidly and then is largely restored to nearly the control level. Plasma colloid osmotic pressure declines markedly during the bleeding and continues to decrease more gradually for several hours. The reduction in colloid osmotic pressure reflects dilution of the blood by tissue fluids that contain little protein.

1	Considerable quantities of fluid may thus be drawn into the circulation during hemorrhage. Approximately 0.25 mL of fluid per minute per kilogram of body weight may be reabsorbed by the capillaries. Thus approximately 1 L of fluid per hour might be autoinfused from the interstitial spaces into the circulatory system of an average individual after acute blood loss. Substantial quantities of fluid may shift slowly from the intracellular space to the extracellular space. This fluid exchange is probably mediated by secretion of cortisol from the adrenal cortex in response to hemorrhage. Cortisol is essential for the full restoration of plasma volume after hemorrhage. The catecholamines epinephrine and norepinephrine are released from the adrenal medulla in response to the same stimuli that evoke widespread sympathetic nervous discharge (see

1	The catecholamines epinephrine and norepinephrine are released from the adrenal medulla in response to the same stimuli that evoke widespread sympathetic nervous discharge (see Chapter 43). Blood levels of catecholamines are high during and after hemorrhage. When blood loss is such that arterial pressure is reduced to 40 mm Hg, the level of catecholamines increases as much as 50-fold. Epinephrine comes almost exclusively from the adrenal medulla, whereas norepinephrine is derived from both the adrenal medulla and peripheral sympathetic nerve endings. These humoral substances reinforce the effects of the sympathetic nervous activity listed previously. Vasopressin (antidiuretic hormone), a potent vasoconstrictor, is secreted by the posterior pituitary gland in response to hemorrhage (see 41). The plasma concentration of vasopressin rises progressively as arterial blood pressure diminishes (

1	The plasma concentration of vasopressin rises progressively as arterial blood pressure diminishes ( Fig. 19.23 ). The receptors responsible for the augmented release of vasopressin are the aortic arch and carotid sinus baroreceptors (high pressure) and stretch receptors in the left atrium (low pressure). The diminished renal perfusion during hemorrhagic hypotension leads to the secretion of renin from the juxtaglomerular apparatus (see Chapter 35). This enzyme acts on a plasma protein, angiotensinogen, to form the decapeptide angiotensin I, which in turn is cleaved to the active octapeptide angiotensin II by angiotensin-converting enzyme; angiotensin II is a very powerful vasoconstrictor. Renal Conservation of Salt and Water Fluid and electrolytes are conserved by the kidneys during hemorrhage in response to various stimuli, including increased secretion of vasopressin, as noted previously (see

1	Fig. 19.23 ), and increased renal sympathetic nerve activity, which enhances NaCl reabsorption by the nephron (decreased excretion). The lower arterial pressure decreases •Fig. 19.22 Changesinarterialbloodpressureandplasmacolloid osmoticpressureinresponsetowithdrawalof45%oftheestimated•Fig. 19.23 Meanpercentagechangesinarterialbloodpressureandbloodvolumeovera30-minuteperiod,beginningattime0.(Redrawnplasmavasopressinconcentrationinresponsetobloodloss.(RedrawnfromZweifachBW.Anesthesiology. 1974;41:157.)fromShenYT,etal.Circ Res. 1991;68:1422.) the glomerular filtration rate, which also curtails the excretion of water and electrolytes. In addition, the elevated levels of angiotensin II, as described earlier, stimulate the release of aldosterone from the adrenal cortex. Aldosterone, in turn, stimulates reabsorption of NaCl by the nephrons. Thus NaCl and water excretion is decreased (see also Chapter 35).

1	Chapter 35). In contrast to negative feedback mechanisms, hemorrhage also evokes latent positive feedback mechanisms. These mechanisms exaggerate any primary change initiated by the blood loss. Specifically, positive feedback mechanisms aggravate the hypotension induced by blood loss and tend to initiate vicious cycles, which may lead to death.

1	Whether a positive feedback mechanism will lead to a vicious cycle depends on the gain of that mechanism. Gain is the ratio of the secondary change evoked by a given mechanism to the initiating change itself. A gain greater than 1 induces a vicious cycle; a gain of less than 1 does not. Consider a positive feedback mechanism with a gain of 2. If Pa were to decrease by 10 mm Hg, a positive feedback mechanism with a gain of 2 would then evoke a secondary reduction in pressure of 20 mm Hg, which in turn would cause a further decrease of 40 mm Hg. Thus each change would induce a subsequent one that is twice as great. Hence, Pa would decline at an ever-increasing rate until death occurred. This process is depicted in curve B in Fig. 19.20

1	Fig. 19.20 Conversely, a positive feedback mechanism with a gain of 0.5 also exaggerates any change in Pa, but the change would not necessarily lead to death. If arterial pressure suddenly decreased by 10 mm Hg, a positive feedback mechanism would initiate a secondary, additional fall of 5 mm Hg. This decrease, in turn, would provoke a further decrease of 2.5 mm Hg. The process would continue in ever-diminishing steps until arterial pressure approached an equilibrium value. Some of the more important positive feedback mechanisms that are evident during hemorrhage include (1) cardiac failure, (2) acidosis, (3) central nervous system depression, (4) aberrations in blood clotting, and (5) depression of the mononuclear phagocytic system (MPS). These mechanisms are discussed next. Shifts to the right in ventricular function curves, particularly in the later stages of hemorrhagic shock (

1	These mechanisms are discussed next. Shifts to the right in ventricular function curves, particularly in the later stages of hemorrhagic shock ( Fig. 19.24 ), provide evidence of a progressive depression in myocardial contractility during hemorrhage. *The MPS (previously called the reticuloendothelial system) consists of macrophages that are distributed throughout the body. They are derived from bone marrow and exist for a short period in circulating blood as monocytes. They then migrate into tissues, where they phagocytose foreign material and present antigens to lymphocytes to initiate the adaptive immune response. Cells of the MPS include the Kupffer cells of the liver, alveolar macrophages, microglia, and Langerhans cells.

1	•Fig. 19.24 Ventricular Function Curves for the Left Ventricle During the Course of Hemorrhagic Shock. Curve A representsthecontrolfunctioncurve;curves B toF representtimeafterthehemor-rhage:117minutes(curve B), 247minutes(curve C), 280minutes(curve D), 295minutes(curve E), and310minutes(curve F). (RedrawnfromCrowellJW,GuytonAC.Am J Physiol. 1962;203:248.)–2 0 2 4 6 8 10 12 0 200 400 600 800 1000 1200 1400 1600 A E F C D B Left atrial pressure (mm Hg) Cardiac output (mL/min) The hypotension induced by hemorrhage reduces coronary blood flow and therefore depresses ventricular function. The consequent reduction in cardiac output further reduces arterial pressure, a classic example of a positive feedback mechanism. Furthermore, reduced blood flow to peripheral tissues leads to an accumulation of vasodilator metabolites that decrease peripheral resistance and therefore aggravate the fall in arterial pressure.

1	The inadequate blood flow during hemorrhage affects the metabolism of all cells. The decreased O2 delivery to cells accelerates tissue production of lactic acid and other acid metabolites. Moreover, impaired kidney function prevents adequate excretion of the excess H+ , and generalized metabolic acidosis ensues. The resulting depressant effect of acidosis on the heart is a further reduction in tissue perfusion, which aggravates the metabolic acidosis. Acidosis also reduces the reactivity of the heart and resistance vessels to neurally released and circulating catecholamines and thereby intensifies the hypotension.

1	The hypotension in shock reduces cerebral blood flow. Moderate degrees of cerebral ischemia induce pronounced sympathetic nervous stimulation of the heart, arterioles, and veins, as noted earlier. In severe hypotension, however, the cardiovascular centers in the brainstem eventually become depressed because of inadequate cerebral blood flow. The resulting loss of sympathetic tone then reduces cardiac output and peripheral resistance. The consequent reduction in Pa intensifies the inadequate cerebral perfusion.

1	Endogenous opioids, such as enkephalins and βendorphin, may be released into the brain substance and into the circulation in response to the same stresses that provoke circulatory shock. Opioids are stored, along with catecholamines, in secretory granules in the adrenal medulla and in sympathetic nerve terminals, and they are released together in response to stress. Similar stimuli cause the release of β-endorphin and adrenocorticotropic hormone from the anterior pituitary gland. Opioids depress the brainstem centers that mediate some of the compensatory autonomic adaptations to blood loss, endotoxemia, and other shock-provoking stress. Conversely, the opoid antagonist naloxone improves cardiovascular function and rates of survival in various forms of shock.

1	The alterations in blood clotting after hemorrhage are typically biphasic. An initial phase of hypercoagulability is followed by a secondary phase of hypocoagulability and fibrinolysis. In the initial phase, platelets and leukocytes adhere to the vascular endothelium, and intravascular clots, or thrombi, develop within a few minutes of the onset of severe hemorrhage. This phenomenon, called disseminated intravascular coagulation (DIC), occurs when thrombin is activated and causes widespread deposition of fibrin within narrow and medium-diameter vessels.

1	The initial phase is further enhanced by the release of thromboxane A2 from various ischemic tissues. Thromboxane A2 aggregates platelets. As more platelets aggregate, more thromboxane A2 is released and more platelets are trapped. This form of positive feedback intensifies and prolongs the clotting tendency. Inflammatory cytokines (interleukin-6, tumor necrosis factor) also contribute to DIC. The rate of mortality from certain standard shock-provoking procedures has been reduced considerably by the administration of anticoagulants such as heparin. In the later stages of hemorrhagic hypotension, the clotting time is prolonged, and fibrinolysis is prominent. Fibrinolysis occurs when clotting factors and platelets are depleted. Depression of the Mononuclear Phagocytic System﻿

1	During the course of hemorrhagic hypotension, MPS function becomes depressed. The phagocytic activity of the MPS is modulated by an opsonic protein. The opsonic activity in plasma diminishes during shock, and this change may account in part for the depression in MPS function. As a result, antibacterial and antitoxin defense mechanisms are impaired. Hypoperfusion also suppresses the barrier function of the adherens junctions and tight junctions in the 1. Two important relationships between cardiac output (Qh) and central venous pressure (Pv) prevail in the cardiovascular system. With regard to the heart, Qh varies directly with Pv (or preload) over a very wide range of Pv. This relationship is represented by the cardiac function curve, and it expresses the Frank-Starling mechanism. In the vascular system, Pv varies intestinal epithelium. Endotoxins from the normal bacterial flora of the intestine constantly enter the circulation. Ordinarily, they are inactivated by the MPS, principally

1	system, Pv varies intestinal epithelium. Endotoxins from the normal bacterial flora of the intestine constantly enter the circulation. Ordinarily, they are inactivated by the MPS, principally in the liver. Disruption of the intestinal epithelial barrier, together with depression of the MPS, allows these endotoxins to invade the general circulation. Endotoxins produce profound, generalized vasodilation, mainly by inducing the synthesis of an isoform of nitric oxide synthase in the smooth muscle of blood vessels throughout the body. The profound vasodilation aggravates the hemodynamic changes caused by blood loss.

1	In addition to their role in inactivating endotoxin, macrophages release many of the mediators associated with shock. These mediators include acid hydrolases, neutral proteases, oxygen free radicals, certain coagulation factors, and the following arachidonic acid derivatives: prostaglandins, thromboxanes, and leukotrienes. Macrophages also release certain monokines that modulate temperature regulation, intermediary metabolism, hormone secretion, and the immune system. Interactions of Positive and Negative Feedback Mechanisms

1	Interactions of Positive and Negative Feedback Mechanisms Hemorrhage provokes a multitude of circulatory and metabolic derangements. Some of these changes are compensatory, and others are decompensatory. Some of these feedback mechanisms possess high gain and others possess low gain. Furthermore, the gain of any specific mechanism varies with the severity of the hemorrhage. For example, with only a slight loss of blood, Pa is maintained within the normal range and the gain of the baroreceptor reflexes is high. With greater losses of blood, when Pa is below 60 mm Hg (i.e., below the threshold for the baroreceptors), further reductions in pressure have no additional influence through the baroreceptor reflexes. Hence, below this critical pressure, the baroreceptor reflex gain is zero or near zero.

1	In general, with minor degrees of blood loss, the gains of negative feedback mechanisms are high, whereas those of positive feedback mechanisms are low. The opposite is true with more severe hemorrhage. The gains of the various mechanisms add algebraically. Therefore, whether a vicious cycle develops depends on whether the sum of the positive and negative gains exceeds 1. Total gains in excess of 1 are, of course, more likely to occur with severe losses of blood. Therefore, to avert a vicious cycle, serious hemorrhages must be treated quickly and intensively, preferably by whole blood transfusion, before the process becomes irreversible. inversely with Qh. This relationship is represented by the vascular function curve, and it reflects the fact that as Qh increases, a greater fraction of the total blood volume resides in the arteries and a smaller volume resides in the veins.

1	2. The principal cardiac mechanisms that govern cardiac output are the changes in numbers of myocardial cross-bridges that interact and in the affinity of the contractile proteins for Ca++ . The principal factors that govern the vascular function curve are arterial and venous compliance, peripheral vascular resistance, and total blood volume. 3. The equilibrium values of Qh and Pv that prevail under a given set of conditions are determined by the intersection of the cardiac and vascular function curves. At very low and very high heart rates, the heart is unable to produce adequate Qh. At very low heart rates, the increase in filling during diastole cannot compensate for the small number of cardiac contractions per minute. At very high heart rates, the large number of contractions per minute cannot compensate for the inadequate filling time. 4.

1	4. Gravity influences Qh because the veins are so compliant, and substantial quantities of blood tend to pool in the veins of dependent portions of the body. Respiration changes the pressure gradient between the intrathoracic and extrathoracic veins. Hence, respiration serves as an auxiliary pump, which may affect the mean level of Qh and induce rhythmic changes in stroke volume during the various phases of the respiratory cycle. 5. In anticipation of exercise, vagus nerve impulses to the heart are inhibited and the sympathetic nervous system is activated by central command. The result is an increase in heart rate, myocardial contractile force, and regional vascular resistance. In addition, vascular resistance increases in the skin, kidneys, splanchnic regions, and inactive muscles and decreases markedly in the active muscles. The overall effect is a pronounced reduction in total peripheral resistance,

1	Eijsvogels TMH, Fernandez AB, Thompson PD. Are there deleterious cardiac effects of acute and chronic endurance exercise? Physiol Rev. 2016;96:99. Fernandes T, Baraúna VG, Negrão CE, et al. Aerobic exercise training promotes physiological cardiac remodeling involving a set of microRNAs. Am J Physiol. 2015;309:H543. Gruen RL, Brohi K, Schreiber M, et al. Haemorrhage control in severely injured patients. Lancet. 2012;380:1099.

1	Gruen RL, Brohi K, Schreiber M, et al. Haemorrhage control in severely injured patients. Lancet. 2012;380:1099. which, along with the auxiliary pumping action of the contracting skeletal muscles, greatly increases venous return. The increases in heart rate and myocardial contractility, both induced by the activation of cardiac sympathetic nerves, enables the heart to transfer blood to the pulmonary and systemic circulations, thereby increasing cardiac output. Stroke volume increases only slightly. O2 consumption and blood O2 extraction increase, and systolic pressure and mean blood pressure increase slightly. As body temperature rises during exercise, the skin blood vessels dilate. However, when the heart rate becomes maximal during severe exercise, the skin vessels constrict. This increases the effective blood volume but causes greater increases in body temperature and a feeling of exhaustion. The limiting factor in exercise performance is delivery of blood to the active muscles.

1	6. Acute blood loss induces tachycardia, hypotension, generalized arteriolar constriction, and generalized venoconstriction. Acute blood loss invokes a number of negative feedback (compensatory) mechanisms, such as baroreceptor and chemoreceptor reflexes, responses to moderate cerebral ischemia, reabsorption of tissue fluids, release of endogenous vasoconstrictors, and renal conservation of water and electrolytes. Acute blood loss also invokes a number of positive feedback (decompensatory) mechanisms, such as cardiac failure, acidosis, central nervous system depression, aberrations in blood coagulation, and depression of the mononuclear phagocytic system. The outcome of acute blood loss depends on the sum of gains of the positive and negative feedback mechanisms and on the interactions between these mechanisms.

1	Laughlin MH, Korthuis RJ, Duncker DJ, et al. Control of blood flow to cardiac and skeletal muscles during exercise. In: Rowell LB, Shepherd JT, eds. Handbook of Physiology, Section 12: Exercise: Regulation and Integration of Multiple Systems. Bethesda, MD: Oxford University Press; 1996. Lavie CJ, Arena R, Swift DL, et al. Exercise and the cardiovascular system: clinical science and cardiovascular outcomes. Circ Res. 2015;117:207. Valparaiso AP, Vicente DA, Bograd BA, et al. Modeling acute traumatic injury. J Surg Res. 2015;194:220. SECTION 5The Respiratory System MICHELLE M. CLOUTIER AND ROGER S. THRALL Upon completion of this chapter, the student should be able to answer the following questions: 1. Explain the anatomical structure/function relationships of the upper and lower components of the respiratory system. 2. Compare and contrast the pulmonary and bronchial circulatory systems. 3.

1	Explain the anatomical structure/function relationships of the upper and lower components of the respiratory system. 2. Compare and contrast the pulmonary and bronchial circulatory systems. 3. Explain the relationships between innervation and muscles in the control of respiration. 4. Compare and contrast the roles of the conducting airways and components of the respiratory unit. 5. Compare and contrast the effects of stimulation of the parasympathetic and sympathetic nervous systems on respiratory responses. 6 Describe lung development and alveolar repair after injury to regeneration of normal architecture.

1	6 Describe lung development and alveolar repair after injury to regeneration of normal architecture. he primary function of the lungs is gas exchange, which consists of movement of oxygen (O2) into the body and removal of carbon dioxide (CO2). This chapter provides an overview of lung anatomical structure/ function relationships (i.e., upper and lower airways, the two circulatory systems, innervation, and muscles), lung development (at the embryo stage and throughout life), and lung repair. This chapter is designed to provide a broad conceptual understanding of structure/function interactions and is not intended to afford a comprehensive understanding of individual lung structures and anatomy.

1	The lungs are contained in a space with a volume of approximately 4 L, but they have a surface area for gas exchange that is the size of a tennis court (≈85 m2). This large surface area is composed of myriads of independently functioning respiratory units. Unlike the heart, but like the kidneys, the lungs demonstrate functional unity; that is, each unit is structurally identical and functions just like every other unit. Because the divisions of the lung and the sites of disease are designated by their anatomical locations (e.g., right upper lobe, left lower lobe), it is essential to understand pulmonary anatomy in order to comprehend respiratory physiology and pathophysiological alterations in respiratory diseases. Upper Airways: Nose, Sinuses, and Pharynx

1	Upper Airways: Nose, Sinuses, and Pharynx The respiratory system begins at the nose and ends in the most distal alveolus. Thus the nasal cavity, the posterior pharynx, the glottis and vocal cords, the trachea, and all divisions of the tracheobronchial tree are included in the respiratory system. The upper airway consists of all structures from the nose to the vocal cords, including sinuses and the larynx, whereas the lower airway consists of the trachea, airways, and alveoli. The upper airways “condition” inspired air so that by the time air reaches the trachea, inspired air is at body temperature and fully humidified. The nose also functions to filter, entrap, and clear particles larger than 10 µm in size. The interior of the nose is lined by respiratory epithelial cells interspersed with surface secretory cells. These secretory cells produce important immunoglobulins, inflammatory mediators, and interferons, which are the first line of host defense.

1	The paranasal sinuses (frontal, maxillary, sphenoid, and ethmoid) are lined by ciliated epithelial cells and surround the nasal passages ( Fig. 20.1A).

1	Fig. 20.1A). The cilia facilitate the movement of mucus from the upper airways and clear the main nasal passages approximately every 15 minutes. The functions of the sinuses are (1) to lessen the weight of the skull, which makes upright posture easier; (2) to offer resonance to the voice; and (3) to protect the brain from frontal trauma. The fluid covering their surfaces is continually being propelled into the nose. In some sinuses (e.g., the maxillary sinus), the opening (ostium) is at the upper edge, which makes them particularly susceptible to retention of mucus. The ostia are readily obstructed by nasal edema (swelling), and retention of secretions and secondary infection (sinusitis) can result. The volume of the nose in an adult is approximately 20 mL, but its surface area is greatly increased by the nasal turbinates, which are a series of three continuous ribbons of tissue that protrude into the nasal cavity (see

1	Fig. 20.1B ). The nose enables the sense of smell. Neuronal endings in the roof of the nose above the superior turbinate carry impulses through the cribriform plate to the olfactory bulb. The pharynx is divided into three sections: the nasopharynx, oropharynx, and laryngopharynx. Important • Fig. 20.1 Illustrations of Upper Airway Anatomy.A, Anterior view of the paranasal sinuses. B, Lateral view of the nasal passage structures demonstrating the superior, middle, and inferior turbinates and the sinus ostia. C, Lateral midsagittal section view of the head and neck, showing the three divisions of the pharynx (nasopharynx, oropharynx, and laryngopharynx) and surrounding upper airway structures. CHAPTER 20 Introduction to the Respiratory System 435 structures within these regions include the epiglottis, vocal cords, and arytenoid cartilage which is attached to the vocal cords (see

1	CHAPTER 20 Introduction to the Respiratory System 435 structures within these regions include the epiglottis, vocal cords, and arytenoid cartilage which is attached to the vocal cords (see Fig. 20.1C ). The nasopharynx (2 to 3 cm wide and 3 to 4 cm long) is the most anterior and lies behind the nose. In this region, the nose and mouth are connected via an isthmus (canals) that enables both oral and nasal breathing. Also, the nasopharynx contains small masses of lymphoid tissue (adenoids), also known as pharyngeal tonsils, which fight infections. The nasopharynx is connected to the middle ear cavity via the eustachian tubes, which aid in equalizing pressure in the ear to atmospheric pressure; thus they represent a drainage pathway of lymphatic fluid between the throat, nose, and ears. This network of structures provides a means of fighting infections but also is a common location for infections in the head.

1	The soft palate separates the nasopharynx and the oropharynx, which ends at the epiglottis. The laryngopharynx begins at the epiglottis and ends at the esophagus. Its major role is to help regulate the passage of food into the esophagus and air into the lungs. With some infections, these structures can become edematous and contribute significantly to airflow resistance. The epiglottis and arytenoid cartilage (attached to the vocal cords) cover or act as a hood over the vocal cords during swallowing. Thus under normal circumstances, the epiglottis and arytenoid cartilage function to prevent aspiration of food and liquid into the lower respiratory tract. The act of swallowing food after mastication (chewing) usually occurs within 2 seconds, and it is closely synchronized with muscle reflexes that coordinate opening and closing of the airway. Hence, air is allowed to enter the lower airways, and food and liquids are kept out. Patients with some neuromuscular diseases have altered muscle

1	that coordinate opening and closing of the airway. Hence, air is allowed to enter the lower airways, and food and liquids are kept out. Patients with some neuromuscular diseases have altered muscle reflexes and can lose this coordinated swallowing mechanism. Such patients may become susceptible to aspiration of food and liquid, which poses a risk for pneumonia.

1	Lower Airways: Trachea, Bronchi, Bronchioles, and Respiratory Unit The right lung, located in the right hemithorax, is divided into three lobes (upper, middle, and lower) by two interlobular fissures (oblique, horizontal), whereas the left lung, located in the left hemithorax, is divided into two lobes (upper, including the lingula, a tongue-like projection of the anterior aspect of the upper lobe, and lower) by an oblique fissure (

1	Fig. 20.2 ). Both the right and left lungs are covered by a thin membrane called the visceral pleura and are encased by another membrane called the parietal pleura. The interface of these two pleurae allows for smooth gliding of the lung as it expands in the chest and produces a potential space. Air can enter between the visceral and parietal pleurae by trauma, surgery, or rupture of a group of alveoli; the resulting condition is a pneumothorax. Fluid can also enter this space and create a pleural effusion or, in the case of severe infection, an empyema. •Fig. 20.2 Illustrations of the Topography of the Lung With Anterior and Posterior Views, Demon-strating the Lobes, Segments, and Fissures. Thefissures(orchasms)demarcatethelobesineachlung.Numbersrefertospecificbronchopulmonarysegments,asdepictedin

1	Fig.20.3 .SVC,superiorvenacava.Lateral 4 Anterior 3 Anterior 3 1 1 Apical 1 Apical 1 Medial 5 Inferior 5 Superior 4 Superior 6 Posterior basal 10 POSTERIOR VIEW Left Right ANTERIOR VIEW LeftRight Posterior 2 2 Lateral basal 9 Lateral basal 9 Posterior basal 10 Superior 6 8 8 8 Heart Anterior basal Lower lobe Anterior basal Oblique fissure SVC Trachea Upper lobe Middle lobe Lower lobe Anterior basal Oblique fissure Aortic arch Apical Upper lobe Lower lobe Upper lobe Lower lobe Upper lobe Posterior Apical Horizontalfissure The trachea branches into two main stem bronchi ( Fig. 20.3 ). These main stem bronchi then divide (like the branches of a tree) into lobar bronchi (one for each lobe), which in turn divide into segmental bronchi (Fig. 20.4 Fig. 20.3 ) and then into smaller and smaller branches (bronchioles) until ending in the alveolus ( Fig.

1	20.4 Fig. 20.3 ) and then into smaller and smaller branches (bronchioles) until ending in the alveolus ( Fig. 20.5 ). Bronchi and bronchioles differ not only in size but also by the presence of cartilage, the type of epithelium, and their blood supply ( Table 20.1). Beyond the segmental bronchi, the airways divide in a dichotomous or asymmetrical branching pattern. Bronchi, distinguished by their size and the presence of cartilage, eventually become terminal bronchioles, which are the smallest airways without alveoli. Each branching of an airway results in an increase in the number of airways with smaller diameters; as a result, the total surface area for the next generation of branches increases. Terminal bronchioles terminate in an opening (duct) to a group of alveoli and are called respiratory bronchioles.

1	The region of the lung supplied by a segmental bronchus is called a bronchopulmonary segment and is the functional anatomical unit of the lung. Because of their structure, bronchopulmonary segments that have become irreversibly diseased can easily be removed surgically. The basic physiological unit of the lung is the gas-exchanging unit (respiratory unit), which consists of the respiratory bronchioles, the alveolar ducts, and the alveoli (see Figs. 20.4 20.5). The bronchi, which contain cartilage, and the terminal bronchioles (i.e., lacking alveoli), in which cartilage is absent, serve to move gas from the airways to the alveoli and are referred to as the conducting airways. This area of the lung (≈150 mL in volume) does not participate in gas exchange and forms the anatomical dead space. The respiratory bronchioles with alveoli and the area beginning •Fig. 20.3 Illustration of Bronchopulmonary Segments, Anterior View. Thenumberscorrespondtothosein

1	Fig.20.2 :1,apical;2,posterior;3,anterior;4,lateral(superior);5,medial(inferior);6,superior;7,medialbasal;8,anteriorbasal;9,lateralbasal;10,posteriorbasal.Themedialbasalregions(7)arelocatedintheupperregionoftheposteriorbasalregions(10)in

1	Fig.20.2 .Upper lobe Middle lobe Lower lobe Upper lobe Lingula Thyroid cartilage Cricoid cartilage Trachea Left main stem bronchus Right main stem bronchus 12 3 4 8 9 10 5 1 2 3 4 8 77 6 6 9 10 5 •Fig. 20.4 Illustration of Conducting Airways and Alveolar Units of the Lung. Therelativesizeofthealveolarunitisgreatlyenlarged.Numbersatthebottomindicatetheapproximatenumberofgenerationsfromtracheatoalveoli,whichmayvaryfromasfewas10toasmanyas23.(FromWeibelER.Morphometry of the Human Lung. Heidelberg:Springer-Verlag;1963.) with the respiratory bronchioles to the alveoli are where all gas exchange occurs. This region is only approximately 5 mm long, but it is the largest volume of the lung, at a volume of approximately 2500 mL and with a surface area of 70 m2 when the lung and chest wall are at the resting volume (see Table 20.1

1	Table 20.1 The alveoli are polygonal in shape and approximately 250 µm in diameter. Alveolar spaces are responsible for most of the lung’s volume; these spaces are divided by tissue known collectively as the interstitium. The interstitium is composed primarily of lung collagen fibers and is a space in which fluid and cells can potentially accumulate. An adult has approximately 5 × 108 alveoli ( Fig. 20.6 ), which are composed of type I and type II epithelial cells. Under normal conditions, type I and type II cells exist in a 1 : 1 ratio. The type I cell occupies 96% to 98% of the surface area of the alveolus, and it is the primary site for gas exchange. The thin cytoplasm of type I cells is ideal for optimal gas diffusion. In addition, the basement membrane of type I cells and the capillary endothelium are fused, which minimizes the distance for gas diffusion and thereby facilitates gas exchange.

1	Type II cells are cuboidal and usually found in the “corners” of the alveolus, where they occupy 2% to 4% of the surface area. During embryonic development, the alveolar epithelium is composed entirely of type II cells, and only very late in gestation do they differentiate into type I cells and form the “normal” alveolar epithelium for optimal gas exchange. Also, type II cells synthesize pulmonary surfactant (see ), which reduces surface tension (nonsticking properties) in the alveolus and thereby promotes less resistance during inhalation and exhalation. Gas exchange occurs in the alveoli through a dense mesh-like network of capillaries and alveoli called the alveolar-capillary network. The barrier between gas in the alveoli and the red blood cell is only 1 to 2 µm thick and consists of type I alveolar epithelial cells, capillary endothelial cells, and their respective basement membranes. O2 and CO2 passively diffuse across this barrier into plasma and red blood cells (see

1	Chapter 24). Red blood cells pass through the network in less than 1 second, which is sufficient time for CO2 and O2 gas exchange. Circulatory Systems in the Lung The circulation to the lung is unique in its duality and ability to accommodate large volumes of blood at low pressure. The lung has two separate blood supplies, one •Fig. 20.5 The Airway From the Terminal Bronchiole to the Alveo-lus. Notetheabsenceofalveoliintheterminalbronchiole.A,alveolus;RB,respiratorybronchiole;TB,terminalbronchiole.

1	Theconductingairwaysareinvolvedinseveralmajorpulmonarydiseasescollectivelyreferredtoasobstructive pulmonary disease, includingasthma,bronchiolitis,chronicbronchitis,andcysticfibrosis.Obstructionofairflowthroughtheairwaysiscommonlycausedbyincreasedamountsofmucus,airwayinflammation,andsmoothmuscleconstriction.Asthma isachronicinflammatorydiseaseofthelargeandsmallairways,mediatedpredominantlybylymphocytesandeosinophils,andisassociatedwithincreasedamountsofmucusintheairwaysandwithreversibleconstrictionoftheairwaysmoothmuscle(bronchospasm).Bronchiolitis isadiseaseofthebronchiolesthatusuallyoccurinyounginfantsandiscausedbyviruses,primarilyrespiratory syncytial virus. Chronic bronchitis, adiseasetypicallyofpeoplewhosmoke,isassociatedwithamarkedincreaseinmucus-secretingcellsintheairwaysandanincreaseinmucusproduction.Cystic fibrosis isanautosomalrecessivegeneticdiseasecausedbymutationsintheCl− ionchannelofthecysticfibrosistransmembraneconductanceregulator(CFTR).MutationsintheCFTR

1	fibrosis isanautosomalrecessivegeneticdiseasecausedbymutationsintheCl− ionchannelofthecysticfibrosistransmembraneconductanceregulator(CFTR).MutationsintheCFTR genecauseareductioninchlorideandwatersecretionintothemucusoverlyingtheepitheliacells,whichincreasestheviscosityofmucus.Thissituationresultsinmucusaccumulationandchronicpulmonaryinfections,primarilybyPseudomonas aeruginosa.

1	Notallimportantobstructivelungdiseasesinvolvetheairwaysdirectly.Emphysema isanirreversible,obstructivelungdiseasecommoninpeoplewhosmoke.Thepathogenesisinvolvesaprogressivedestructionoftheelastictissuesinthelungwithalossofalveolar/capillarystructure.Themechanismsoftissuedestructionareunclearbutmayinvolveproteolyticenzymesandothertoxiccompoundsincigarettesmoke.Emphysemaalsooccursinnonsmokingindividualswiththegeneticdisorderα1-antitrypsin deficiency, causedbytheinabilitytoregulateproteolyticenzymes,particularlyelastase. for the uptake of O2 and removal of CO2 from the body (pulmonary circulation) and the other to supply O2 to lung tissue (bronchial circulation; Fig. 20.7

1	The pulmonary circulation begins in the right atrium of the heart. Deoxygenated blood from the right atrium enters the right ventricle via the tricuspid valve, and it is then pumped under low pressure (9 to 24 mm Hg) into the pulmonary artery (pulmonary trunk), which is approximately 3 cm 200 µmPVCTB PARB AD A PV PV A I A A C C C II M10 µm 200 µm PAPA A A A I I I I EL C R COL E * E Nu 1.0 µmB •Fig. 20.6 Alveoli. A. Theterminalrespiratoryunitconsistsofthealveoli(A)andthealveolarducts(AD)arisingfromarespiratorybronchiole(RB).Eachunitisapproximatelyspherical,assuggestedbythedashed outline. Pulmonaryvenousvessels(PV)haveaperipherallocation.PA,pulmonaryartery;TB,terminalbronchiole.Inset, typeIandtypeIIalveolarepithelialcells.Alargefractionofthealveolarwallconsistsofcapillaries(C)andtheircontents.B.Illustrationofacross-sectionofanalveolarwall,showingthepathofdiffusionofO2andCO2.Thethinsideofthealveolarwallbarrier(short double arrow) consistsoftypeIepithelium(I),interstitium(asterisk)

1	double arrow) consistsoftypeIepithelium(I),interstitium(asterisk) formedbythefusedbasallaminaeoftheepithelialandendothelialcells,capillaryendothelium(E),plasmainthealveolarcapillary(C),andthecytoplasmoftheredbloodcell(R).Thethicksideofthegas-exchangebarrier(long double arrow) hasanaccumulationofelastin(EL),collagen(COL),andmatrixthatjointlyseparatethealveolarepitheliumfromthealveolarcapillaryendothelium.Nu,nucleusofthecapillaryendothelialcell.

1	in diameter, and branches quickly (5 cm from the right progressively smaller series of branching vessels—arteries ventricle) into the right and left main pulmonary arteries, (diameter, >500 µm); arterioles (diameter, 10 to 200 µm); which supply blood to the right and left lungs, respectively. and capillaries (diameter, <10 µm)—that end in a complex The arteries of the pulmonary circulation are the only arter-mesh-like network of capillaries. The sequential branching ies in the body that carry deoxygenated blood. The deoxy-pattern of the pulmonary arteries follows the pattern of genated blood in the pulmonary arteries passes through a airway branching.

1	•Fig. 20.7 Illustrationoftheanatomicalrelationshipofthepulmonaryartery,thebronchialartery,theairways,andthelymphaticvessels.A,alveoli;AD,alveolarducts;RB,respiratorybronchioles;TB,terminalbronchioles.AD AD AD AD RB TB Lymphatics Lymphatics Vasomotor nerves Pulmonary vein Bronchomotor nerve Pulmonary artery Bronchial artery Bronchus A A A A A The functions of the pulmonary circulatory system are (1) to reoxygenate the blood and release CO2, (2) to aid in fluid balance in the lung, and (3) to distribute metabolic products to and from the lung. Red blood cells are oxygenated in the capillaries that surround the alveoli, where the pulmonary capillary bed and the alveoli come together in the alveolar wall in a unique configuration for optimal gas exchange. Gas exchange occurs through this alveolar-capillary network (see

1	The total blood volume of the pulmonary circulation is approximately 500 mL, which is approximately 10% of the circulating blood volume. Approximately 75 mL of blood is present in the alveolar-capillary network of healthy adults at any one time. The pulmonary capillary bed is the largest vascular bed in the body. It covers a surface area of 70 to 80 m2, which is nearly as large as the alveolar surface area. During exercise, the pulmonary capillary blood volume increases from 75 mL to as high as 200 mL because of the recruitment of new capillaries as the result of an increase in pressure and flow. This recruitment of new capillaries is a unique feature of the lungs, and it allows for compensation in periods of stress, as in the case of exercise.

1	The oxygenated blood leaves the alveolus through a network of small pulmonary venules (15 to 500 µm in diameter) and veins. These small vessels quickly coalesce to form larger pulmonary veins (>500 µm in diameter), through which the oxygenated blood returns to the left atrium of the heart. In contrast to arteries, arterioles, and capillaries, which closely follow the branching patterns of the airways, venules and veins run quite distant from the airways. Structure of the Pulmonary Circulation

1	The arteries of the pulmonary circulation have thin walls, with minimal smooth muscle. They are seven times more compliant than systemic vessels, and they are easily distensible. This highly compliant state of the pulmonary arterial vessels requires lower pressure for blood flow through the pulmonary circulation than do the more muscular, noncompliant arterial walls of the systemic circulation. The vessels in the pulmonary circulation, under normal circumstances, are in a dilated state and have larger diameters than do similar arteries in the systemic system. All of these factors contribute to a very compliant, low-resistance circulatory system, which aids in the flow of blood through the pulmonary circulation via the relatively weak pumping action of the right ventricle. This low-resistance, low-work system also explains why the right ventricle is less muscular than the left ventricle. The pressure gradient differential for the pulmonary circulation from the pulmonary artery to the

1	low-work system also explains why the right ventricle is less muscular than the left ventricle. The pressure gradient differential for the pulmonary circulation from the pulmonary artery to the left atrium is only 6 mm Hg (14 mm Hg in the pulmonary artery minus 8 mm Hg in the left atrium). This pressure gradient differential is less than 7% of the pressure gradient differential of 87 mm Hg present in the systemic circulation (90 mm Hg in the aorta minus 3 mm Hg in the right atrium).

1	Structures of the Extra-Alveolar and Alveolar Vessels and the Pulmonary Microcirculation

1	Although not well defined anatomically, vessels in the pulmonary circulation can be divided into three categories (extra-alveolar, alveolar, and microcirculation) on the basis of differences in their physiological properties. The extra-alveolar vessels (arteries, arterioles, veins, and venules) are larger than their systemic counterparts. They are not influenced by alveolar pressure changes, but they are affected by intrapleural and interstitial pressure changes. Thus the caliber of extra-alveolar vessels is affected by lung volume and by lung elastin. At high lung volumes, the decrease in pleural pressure increases the caliber of extra-alveolar vessels, whereas at low lung volumes, an increase in pleural pressure decreases vessel caliber. In contrast, alveolar capillaries reside within the interalveolar septa, and they are very sensitive to changes in alveolar pressure but not to changes in pleural or interstitial pressure. Positive-pressure ventilation increases alveolar pressure

1	the interalveolar septa, and they are very sensitive to changes in alveolar pressure but not to changes in pleural or interstitial pressure. Positive-pressure ventilation increases alveolar pressure and compresses these capillaries and thus blocks blood flow. The pulmonary microcirculation comprises the small vessels that participate in liquid and solute exchange in maintenance of fluid balance in the lung.

1	Structure of the Alveolar-Capillary Network The sequential branching of the pulmonary arteries culminates in a dense mesh-like network of capillaries that surround alveoli. This alveolar-capillary network is composed of thin epithelial cells of the alveolus and endothelial cells of the vessels and their supportive matrix, and it has an alveolar surface area of approximately 85 m2 (the approximate size of a tennis court). The structural matrix and the tissue components of this alveolar-capillary network provide the only barrier between gas in the airway and blood in the capillary. The cells of this barrier, which is 1 to 2 µm thick, include type I alveolar epithelial cells, capillary endothelial cells, and their respective basement membranes, which are back to back (see

1	Fig. 20.6B ). Surrounded mostly by air, this alveolar-capillary network is an ideal environment for gas exchange. Red blood cells pass through the capillary component of this network in single file in less than 1 second, which is sufficient time for CO2 and O2 gas exchange. In addition to gas exchange, the alveolar-capillary network regulates the amount of fluid within the lung. At the pulmonary capillary level, the balance between hydrostatic and oncotic pressure across the wall of the capillary results in a small net movement of fluid out of the vessels into the interstitial space. The fluid is then removed from the lung interstitium by the lymphatic system and enters the circulation via the vena cava in the area of the lung hilus. In normal adults, an average of 30 mL of fluid per hour is returned to the circulation via this route.

1	The bronchial circulation is a distinct system, separate from the pulmonary circulation in the lung, that provides systemic arterial blood to the trachea, upper airways, surface secretory cells, glands, nerves, visceral pleural surfaces, lymph nodes, pulmonary arteries, and pulmonary veins. The bronchial circulation is similar in structure to the systemic circulatory system and perfuses the upper respiratory tract; it does not reach the terminal or respiratory bronchioles or the

1	CHAPTER 20 Introduction to the Respiratory System alveoli. Venous blood from the capillaries of the bronchial circulation flows to the heart through either true bronchial veins or bronchopulmonary veins. True bronchial veins are present in the region of the lung hilus, and blood flows into the azygos, hemiazygos, or intercostal veins before entering the right atrium. The bronchopulmonary veins are formed through a network of tributaries from the bronchial and pulmonary circulatory vessels that anastomose and form vessels with an admixture of blood from both circulatory systems. Blood from these anastomosed vessels returns to the left atrium through pulmonary veins. Approximately two thirds of the total bronchial circulation is returned to the heart via the pulmonary veins and this anastomosis route.

1	The bronchial circulation receives only approximately 1% of total cardiac output; in comparison, the pulmonary circulation receives almost 100%. In the presence of diseases such as cystic fibrosis, the bronchial arteries, which normally receive only 1% to 2% of cardiac output, increase in size (hypertrophy) and receive as much as 10% to 20% of the cardiac output. The erosion of inflamed tissue into these vessels as a result of bacterial infection is responsible for the hemoptysis (coughing up blood) that can occur in this disease. Breathing is automatic and under control of the central nervous system (CNS). The lungs are innervated by the autonomic nervous system of the peripheral nervous system (PNS), which is under CNS control ( Fig. 20.8). The autonomic nervous system has four distinct components: parasympathetic, sympathetic, nonadrenergic noncholinergic inhibitory, and nonadrenergic noncholinergic stimulatory.

1	Fig. 20.8). The autonomic nervous system has four distinct components: parasympathetic, sympathetic, nonadrenergic noncholinergic inhibitory, and nonadrenergic noncholinergic stimulatory. Stimulation of the parasympathetic system leads to airway smooth muscle constriction, blood vessel dilation, and increased glandular cell secretion, whereas stimulation of the sympathetic system causes relaxation of the airway smooth muscle, constriction of blood vessels, and inhibition of glandular secretion (see Fig. 26.1 ). The functional unit of the autonomic nervous system is composed of preganglionic and postganglionic neurons in the CNS and postganglionic neurons in the ganglia of the specific organ. As with most organ systems, the CNS and PNS work in concert to maintain homeostasis. There is no voluntary motor innervation in the lung, nor are there pain fibers. Pain fibers are found only in the pleura.

1	The parasympathetic innervation of the lung originates from the medulla in the brainstem (cranial nerve X, the vagus nerve). Preganglionic fibers from the vagal nuclei descend in the vagus nerve to ganglia adjacent to airways and blood vessels in the lung. Postganglionic fibers from the ganglia then complete the network by innervating smooth muscle cells, blood vessels, and bronchial epithelial cells (including goblet cells and submucosal glands). In the lungs, both preganglionic and postganglionic fibers •Fig. 20.8 Innervation of the Lung. Theautonomicinnervation(motorandsensory)ofthelungandthesomatic(motor)nervesupplytotheintercostalmusclesanddiaphragmaredepicted.

1	contain excitatory (cholinergic) and inhibitory (nonadrenergic) motor neurons. Acetylcholine and substance P are neurotransmitters of excitatory motor neurons; dynorphin and vasoactive intestinal peptide are neurotransmitters of inhibitory motor neurons. Parasympathetic stimulation through the vagus nerve is responsible for the slightly constricted smooth muscle tone in a normal resting lung. Parasympathetic fibers also innervate the bronchial glands, and these fibers, when stimulated, increase the synthesis of mucus glycoprotein, which increases the viscosity of mucus. Parasympathetic innervation is greatest in the larger airways and most limited in the smaller conducting airways in the periphery.

1	Whereas the response of the parasympathetic nervous system is very specific and local, the response of the sympathetic nervous system tends to be more general. Mucous glands and blood vessels are heavily innervated by the sympathetic nervous system; however, airway smooth muscle is not. Neurotransmitters of the adrenergic nerves include norepinephrine and dopamine, although dopamine has no influence on the lung. Stimulation of the sympathetic nerves in mucous glands increases water secretion. This disrupts the balanced response of increased water and increased viscosity between the sympathetic and parasympathetic pathways. Adrenergic fibers are absent in humans. In addition to those in the sympathetic and parasympathetic systems, afferent nerve endings are present in the epithelium and in smooth muscle cells in the lung. Central Control of Respiration

1	Central Control of Respiration Breathing is an automatic, rhythmic, and centrally regulated process with voluntary control. The CNS, particularly the brainstem, functions as the main control center for respiration ( Fig. 20.9 ). Regulation of respiration requires (1) generation and maintenance of a respiratory rhythm; (2) modulation of this rhythm by sensory feedback loops and reflexes that allow adaptation to various conditions while minimizing energy costs; and (3) recruitment of respiratory muscles that can contract appropriately for gas exchange. Control of respiration is described in greater detail in

1	Cerebral cortex Respiratory center, medulla Spinal cord Respiratory muscles Lung and chest wall Alveolar-capillary barrier BloodPerfusion Mechano-receptors Chemo-receptors PCO2, PO2, pH Diffusion Ventilation Nerve impulses Nerve impulses Force displacement •Fig. 20.9 Block Diagram of the Respiratory Control System, Demonstrating Relationships Between the Respiratory Control Center and Muscles of Respiration. Therespiratorycenterneurons,dispersedintoseveralgroupsinthemedulla,demonstratespontaneouscyclicactivitybutarestronglyinfluencedbystimulidescendingfromthecerebralcortex(volitionalcontrol)andfromtwosensoryloops:mechanoreceptorandchemoreceptorpathways.Ventilationandperfusionoccurtogetherneartheendofthecycle,andtheiroutputdeterminespartialpressuresofarterialandalveolarcarbondioxide(PCO2)andoxygen(PO2)and,inpart,arterialhydrogenionconcentration(pH).Theseoutputsfeedbacktotherespiratorycenterviachemoreceptorandmechanoreceptorsensorypathways. Muscles of Respiration

1	Muscles of Respiration The major muscles of respiration include the diaphragm, the external intercostal muscles, and the scalene muscles, all of which are skeletal muscles. Skeletal muscles provide the driving force for ventilation; the force of contraction increases when they are stretched and decreases when they are shortened. The force of contraction of respiratory muscles increases at larger lung volumes. The diaphragm is the major muscle of respiration, and it divides the thoracic cavity from the abdominal cavity ( Fig.

1	The diaphragm is the major muscle of respiration, and it divides the thoracic cavity from the abdominal cavity ( Fig. 20.10). Contraction of the diaphragm forces the abdominal contents downward and forward. This increases the vertical dimension of the chest cavity and creates a pressure difference between the thorax and abdomen. In adults, the diaphragm can generate airway pressures of up to 150 to 200 cm H2O during maximal inspiratory effort. During quiet breathing (tidal breathing), the diaphragm moves approximately 1 cm; however, during deep-breathing maneuvers (vital capacity), the diaphragm can move as much as 10 cm. The diaphragm CHAPTER 20 Introduction to the Respiratory System is innervated by the right and left phrenic nerves, whose origins are at the third to fifth cervical segments of the spinal cord (C3 to C5). The other important muscles of inspiration are the external intercostal muscles, which pull the ribs upward and forward during inspiration (see

1	The other important muscles of inspiration are the external intercostal muscles, which pull the ribs upward and forward during inspiration (see Fig. 20.10 ). This causes an increase in both the lateral and anteroposterior diameters of the thorax. Innervation of the external intercostal muscles originates from intercostal nerves that arise from the same level of the spinal cord (T1 and T2). Paralysis of these muscles has no significant effect on respiration because respiration is dependent primarily on the diaphragm. This is why individuals with injuries to the lower spinal cord can breathe on their own; it is only when the injury is above C3 that an individual is completely dependent on a ventilator.

1	Accessory muscles of inspiration (the scalene muscles, which elevate the sternocleidomastoid muscles; the alae nasi, which cause nasal flaring; and small muscles in the neck and head) do not contract during normal breathing. However, they do contract vigorously during exercise, and when airway obstruction is significant, they actively pull up the rib cage. During normal breathing, they anchor the sternum and upper ribs. Because the upper airway must remain patent during inspiration, the pharyngeal wall muscles (genioglossus and arytenoid) are also considered muscles of inspiration. All the rib cage muscles are voluntary muscles that are supplied by intercostal arteries and veins and innervated by motor and sensory intercostal nerves.

1	Exhalation during normal breathing is passive, but it becomes active during exercise and hyperventilation. The most important muscles of exhalation are those of the abdominal wall (rectus abdominis, internal and external oblique, and transversus abdominis) and the internal intercostal muscles, which oppose the external intercostal muscles (i.e., they pull the ribs downward and inward). The inspiratory muscles do the work of breathing. During normal breathing, this workload is low, and the inspiratory muscles have significant reserve. Respiratory muscles can be trained to do more work, but there is a limit to the work that they can perform. Respiratory muscle weakness can impair movement of the chest wall, and respiratory muscle fatigue is a major factor in the development of respiratory failure. Lung Embryology, Development, Aging, and Repair

1	Lung Embryology, Development, Aging, and Repair The epithelium of the lung arises as a pouch from the primitive foregut at approximately 22 to 26 days after fertilization of the ovum. This single lung bud branches into primitive right and left lungs. Over the next 2 to 3 weeks, further branching occurs to create the irregular dichotomous branching pattern. The pathologist Lynne Reid described “three laws of lung development”: (1) The bronchial tree has developed by week 16 of intrauterine Diaphragm (domes descend, increasing longitudinal dimension of chest and elevating lower ribs) A Muscles of inspiration Internal intercostals, except parasternal intercartilaginous muscles (depress ribs) Abdominal muscles (depress lower ribs, compress abdominal contents) Muscles of expiration

1	Abdominal muscles (depress lower ribs, compress abdominal contents) Muscles of expiration Because respiratory muscles provide the driving force for ventilation, diseases that affect the mechanical properties of the lung affect the muscles of respiration. For example, in chronic obstructive pulmonary disease (COPD), the work of breathing is increased secondary to airflow obstruction. Exhalation no longer is passive but instead requires active, expiratory muscle contraction. In addition, total lung capacity is increased (see ). The larger total lung capacity forces the diaphragm downward, shortens the muscle fibers, life; (2) alveoli develop after birth, the number of alveoli increases until the age of 8 years, and the size of alveoli increases until growth of the chest wall is completed at adulthood; and (3) the development of preacinar vessels (arteries and veins) parallels that of the airways, whereas that of intra-acinar vessels parallels that of the alveoli.

1	and decreases the radius of curvature. As a result, the function and efficiency of the diaphragm are decreased. Respiratory muscles can fatigue just as other skeletal muscles do when the workload increases. Respiratory muscles can also weaken in patients with neuromuscular diseases (e.g., Guillain-Barré syndrome, myasthenia gravis). In these diseases, sufficient weakness of the respiratory muscles can impair movement of the chest wall and result in respiratory failure, even though the mechanical properties of the lung and chest wall are normal.

1	Thus intrauterine events that occur before 16 weeks of gestation will affect the number of airways. A condition known as congenital diaphragmatic hernia is an example of a congenital lung disease. It occurs at 6 to 8 weeks of gestation and is due to failure of the pleuroperitoneal canal to close and thereby separate the chest and abdominal cavities; the presence of the abdominal contents in the lung hemithorax results in abnormal lung growth with a decrease in the number of airways and alveoli. Before the birth of an affected infant, the alveolar epithelium is composed solely of type II epithelial cells, and it is not until birth that these cells differentiate into type I epithelial cells.

1	Growth of the lungs is similar and relatively proportional to growth in body length and stature. The rate of development is fastest in the neonatal and preadolescent periods (≈11 years of age), and girls’ lungs mature earlier than boys’. Although the growth rate of the lung slows after adolescence, the body and lung increase in size steadily until adulthood. Improvement in lung function occurs at all stages of growth development; however, once optimal size has been attained in early adulthood (20 to 25 years of age), lung function starts to decline with age. The decrease in lung function with age, estimated at less than 1% per year, appears to begin earlier and proceed faster in individuals who smoke or are exposed to toxic environmental factors. The major physiological insufficiencies caused by aging involve ventilatory capacity and responses, especially during exercise, and they result in abnormal ventilation with normal perfusion. In addition, gas diffusion decreases with age,

1	caused by aging involve ventilatory capacity and responses, especially during exercise, and they result in abnormal ventilation with normal perfusion. In addition, gas diffusion decreases with age, probably as a result of a decrease in alveolar surface area. Age-related decreases in lung function and altered structure parallel biochemical observations of increased levels of elastin within the lung, which could explain some of the functional abnormalities.

1	TypeIcellslackfreeradicalscavengers(i.e.,superoxidedismutase)andaresusceptibletoinjuryanddeathinducedbytoxicO2compoundsandfreeradical(i.e.,H2O2,OH− ,andO2−).Invariousinflammatorylungdiseases,typeIcellsdie,andthealveolarepitheliumtherebybecomesdenuded,withincreasedvascularpermeabilityandensuingfluidaccumulation(impairedgasexchange).TypeIIcellshavesuperoxidedismutaseandarethusmoreresistanttothetoxicoxygenradicals.TheycansurvivetoproliferateanddifferentiateintotypeIcellstorestorethenormalalveolararchitecture.ThistypeofresponseisdependentonanintactbasementmembranetosupporttheproliferationoftypeIIcellsandisanexampleof“phylogenyrecapitulatingontogeny.”Ifthebasementmembranecannotberepopulated,thenthebody’srecourseforrepairiscollagendepositionandscarformation,whichisnotconducivetogas 1. The lungs demonstrate anatomical and physiological unity; that is, each unit (bronchopulmonary segment) is structurally identical and functions just like every other unit. 2.

1	The lungs demonstrate anatomical and physiological unity; that is, each unit (bronchopulmonary segment) is structurally identical and functions just like every other unit. 2. The upper airways (nose, sinuses, pharynx) condition inspired air for temperature, humidity, and atmospheric pressure, and they control, via the epiglottis, the flow of air into the lungs and food/fluids into the esophagus. 3. Components of the lower airways (trachea, bronchi, bronchioles) are considered conducting airways in which air is transported to the gas-exchanging respiratory units composed of respiratory bronchioles, alveolar ducts, and alveoli. 4.

1	The lungs have unique, dual circulatory systems. The pulmonary circulatory system has the ability to accommodate large volumes of blood at low pressure and brings deoxygenated blood from the right ventricle to the gas-exchanging units in the lung. The

1	pulmonary circulatory system has the ability to accommodate large volumes of blood at low pressure and brings deoxygenated blood from the right ventricle to the gas-exchanging units in the lung. The exchange.Inlungdiseasethatinvolvesscarformation(i.e.,pulmonaryfibrosis),thetotallungvolumedecreasesasaresultofthelossofalveoliandimpairmentofO2diffusionintothecapillariesbyathickened,nonpermeablematrix.Historically,idiopathicpulmonaryfibrosishasbeenverydifficulttotreatbecauseofthelackofspecifictherapeutics,whichcaninhibitcollagendeposition.Twotherapeuticcompounds(pirfenidoneandnintedanib)havebeenshowninclinicaltrialstoslowtheprogressionofthediseaseandimproveoutcomesinpatientswithidiopathicpulmonaryfibrosis.Pirfenidoneisasmalllow-molecular-weightcompoundwithanti-inflammatoryproperties(itdecreasesprocollagentypesIandIIsynthesis),andnintedanibisatyrosinekinaseinhibitor(inhibitsvascularendothelialgrowthfactorandfibroblast-derivedgrowthfactor).

1	bronchial circulation arises from the aorta and provides nourishment (O2) to the lung parenchyma. 5. Breathing is automatic; the lungs are innervated by the autonomic nervous system of the PNS while under the control of the CNS. Parasympathetic stimulation results in constriction of airway smooth muscles (airway narrowing) whereas sympathetic stimulation results in relaxation of airway smooth muscles (airway opening). 6. Inspiration is the active phase of breathing. The diaphragm is the major muscle of respiration, and its contraction creates a pressure difference (mechanoreceptor response) between the thorax and diaphragm (negative pressure in the chest), which induces inspiration. 7. The respiratory center is located in the medulla and regulates respiration with input from sensory (mechanoreceptor and chemoreceptor) feedback loops. Burri PH. Structural aspects of postnatal lung development—alveolar formation and growth. Biol Neonate. 2006;89:313-322.

1	Burri PH. Structural aspects of postnatal lung development—alveolar formation and growth. Biol Neonate. 2006;89:313-322. Hameed A, Sherkheli MA, Hussain A, Ul-haq R. Molecular and physiological determinants of pulmonary developmental biology: a review. Am J Biomed Res. 2013;1:13-24. Harding R, Pinkertton KE, eds. The Lung: Development, Aging and the Environment. 2nd ed. London: Academic Press; 2014. Hattrup CL, Gendler SJ. Structure and function of the cell surface (tethered) mucins. Annu Rev Physiol. 2007;70:431-457. Reynolds HY. Lung inflammation and fibrosis: an alveolar macrophage-centered perspective from the 1970s to 1980s. Am J Respir Crit Care Med. 2005;171:98-102. Satir P, Christensen ST. Overview of structure and function of mammalian cilia. Annu Rev Physiol. 2007;69:377-400. Shannon JM, Hyatt BA. Epithelial cell-mesenchymal interactions in the developing lung. Annu Rev Physiol. 2004;66:625-645.

1	Shannon JM, Hyatt BA. Epithelial cell-mesenchymal interactions in the developing lung. Annu Rev Physiol. 2004;66:625-645. Warburton D, El-Hashash A, Carraro G, et al. Lung organogenesis. Curr Top Devel Bio. 2010;90:73-158. Upon completion of this chapter, the student should be able to answer the following questions: 1. Define the different pressures in the respiratory system. 2. Explain how a pressure gradient is created. 3. Define the different volumes in the lung, and describe how they are measured. 4. Explain how static lung mechanics determines lung volumes. 5. Define lung compliance. 6. Explain how surfactant affects lung compliance, and describe its importance in maintaining unequal alveolar volumes.

1	o achieve its primary function of gas exchange, air must be moved in and out of the lung. The mechanical properties of the lung and chest wall determine the ease or difficulty of this air movement. Lung mechanics is the study of the mechanical properties of the lung and chest wall (including the diaphragm, abdominal cavity, and anterior abdominal muscles). Lung mechanics is important for how the lungs work both normally and in the presence of disease, inasmuch as most lung diseases affect the mechanical properties of the lungs, chest wall, or both. In addition, death from lung disease is almost always due to respiratory muscle fatigue, which results from an inability of the respiratory muscles to overcome the altered mechanical properties of the lungs, chest wall, or both. Lung mechanics includes static mechanics (the mechanical properties of a lung whose volume is not changing with time) and dynamic mechanics (properties of a lung whose volume is changing with time). Dynamic

1	includes static mechanics (the mechanical properties of a lung whose volume is not changing with time) and dynamic mechanics (properties of a lung whose volume is changing with time). Dynamic mechanics of the lung and chest wall are described in

1	Chapter 22. Pressures in the Respiratory System

1	In healthy people, the lungs and chest wall move together as a unit. Between these structures is the pleural space, which under normal conditions is best thought of as a potential (or virtual) space. Because the lungs and chest wall move together, changes in their respective volumes are equal during inspiration and exhalation. Volume changes in the lungs and chest wall are driven by changes in the surrounding pressure. In accordance with convention, pressures inside the lungs and chest wall are referenced in relation to atmospheric pressure, which is considered 0. Thus a negative pressure in the pleural space is a pressure that is lower than atmospheric pressure. Also in accordance with convention, pressures across surfaces such as the lungs or chest wall have been defined as the difference between the pressure inside and the pressure outside the surface. The pressure differences across the lung and across the chest wall are defined as the transmural (across a wall or surface)

1	between the pressure inside and the pressure outside the surface. The pressure differences across the lung and across the chest wall are defined as the transmural (across a wall or surface) pressures. For the lung, this transmural pressure is called the transpulmonary (or translung) pressure (PL), and it is defined as the pressure difference between the air spaces (alveolar pressure [PA]) and the pressure surrounding the lung (pleural pressure [Ppl]):

1	Equation 21.1 The transmural pressure across the chest wall (Pw) is the difference between pleural (inside) pressure (Ppl) and the pressure surrounding the chest wall (Pb), which is the atmospheric pressure or body surface pressure: Equation 21.2 The pressure across the respiratory system (Prs) is the sum of the pressure across the lung and the pressure across the chest wall: Equation 21.3

1	Equation 21.3 Air flows into and out of the lungs from areas of higher pressure to areas of lower pressure. In the absence of a pressure gradient, there is no airflow. Thus at end inspiration and at the end of exhalation, which are periods of time when there is no airflow, alveolar pressure (PA) is the same as atmospheric pressure (Pb), and there is no pressure gradient (Pb − PA = 0). Pleural pressure at these same times, however, is not 0. Before inspiration begins, the pleural pressure in normal individuals is approximately −3 to −5 cm H2O. Therefore, the pressure in the pleural space is negative in relation to atmospheric pressure. This negative pressure is created by the inward elastic recoil pressure of the lung, and it acts to “pull the lung” away from the chest wall. The lung is not able, however, to pull away from the chest wall, inasmuch as the two function as a unit. Thus the inward elastic recoil pressure of the lung is balanced by the outward recoil of the chest wall.

1	With the onset of inspiration, the muscles of the diaphragm and chest wall contract, which causes a downward movement of the diaphragm and an outward and upward movement of the rib cage. As a result, pleural pressure decreases during inspiration. This negative pleural pressure is transmitted across the lung tissue and results in a decrease in alveolar pressure. As alveolar pressure decreases below 0 (i.e., from atmospheric pressure to a lower pressure), gas moves into the airways when the glottis is open. As gas flows into the airways to the alveoli, the pressure gradient along the airways decreases, and flow stops when there is no longer a pressure gradient from atmospheric to alveolar pressure. The decrease in pleural pressure at the start of inspiration secondary to inspiratory muscle contraction is and gas again flows from a higher (alveolar) pressure to a lower (atmospheric) pressure. In the alveoli, the driving force for exhalation is the sum of the elastic recoil of the lungs

1	contraction is and gas again flows from a higher (alveolar) pressure to a lower (atmospheric) pressure. In the alveoli, the driving force for exhalation is the sum of the elastic recoil of the lungs and pleural pressure (see ). This relationship between changes in pressure, changes in airflow, and changes in volume during inspiration and exhalation is displayed in

1	Fig. 21.2. During tidal volume breathing in normal individuals, the decrease in alveolar pressure at the start of inspiration is small (1 to 3 cm H2O). It is much larger in individuals with airway obstruction because of the larger pressure drop that occurs across obstructed airways. Airflow stops in the absence of a pressure gradient, which occurs whenever alveolar pressure and atmospheric pressure are equal. (Fig. 21.3 ) and the factors that determine these volumes are important components of lung mechanics greater than the transmitted fall in alveolar pressure, and, as a result, transpulmonary pressure at the start of inspiration 0.5 is positive (see Eq. 21.1). Positive transpulmonary pressure 0.4 is necessary to increase lung volume, and lung volume increases with increasing transpulmonary pressure ( Fig.

1	Eq. 21.1). Positive transpulmonary pressure 0.4 is necessary to increase lung volume, and lung volume increases with increasing transpulmonary pressure ( Fig. 21.1 ). Similarly, during inspiration, the chest wall expands to a larger volume. Because pleural pressure is negative in 0.1 relation to atmospheric pressure during quiet breathing, 0 the transmural pressure across the chest wall is negative Eq. 21.2

1	Eq. 21.2 On exhalation, the diaphragm moves higher into the chest, pleural pressure increases (i.e., becomes less nega tive), alveolar pressure becomes positive, the glottis opens, +0.5 –0.5 •Fig. 21.1 Volume of the Lung as a Function of the Transpulmonary Pressure in Health and Disease. Asthetranspulmonarypressureincreases,lungvolumeincreases.Alsoshownarethechangesinlungvolumeinthepresenceofemphysemaandpulmonaryfibrosis.Notethatforthesamechangeintranspulmonarypressure,inthepresenceofeitherofthesetypesofdiseases,thechangesinlungvolumearedifferent.TLC,totallungcapacity(thetotalvolumeofgasinthelung).

1	0.3 0.2 •Fig. 21.2 Changes in Alveolar and Pleural Pressure During Quiet Breathing (Tidal Volume). Inspirationisrepresentedtotheleftofthevertical dotted line, andexhalationisrepresentedtotherightofit.Positive(inrelationtoatmospheric)pressuresarerepresentedabovethehorizontal dotted line, andnegativepressuresarerepresentedbelowit.Seetextfordetails.Atpointsofnoairflow(pointsAandC),alveolarpressureis0.FRC,functionalresidualcapacity. 2 1.2 •Fig. 21.3 The Various Lung Volumes and Capacities. ERV,expiratoryreservevolume;FRC,functionalresidualcapacity;FVC,forcedvitalcapacity;IC,inspiratorycapacity;IRV,inspiratoryreservevolume;RV,residualvolume;TLC,totallungcapacity;VC,vitalcapacity;VT,tidalvolume.

1	and play a major role in the work of breathing (see ). All lung volumes are subdivisions of total lung capacity (TLC), the total volume of air that is contained in the lung at the point of maximal inspiration. Lung volumes are reported in liters either as volumes or as capacities. A capacity is composed of two or more volumes. Many lung volumes are measured with a spirometer. The patient is asked to first breathe normally into the spirometer, and the volume of air (the tidal volume [VT]) that is moved into and out of the lungs with each quiet breath is measured. The patient then inhales maximally and exhales forcefully and completely, and the volume of exhaled air is measured. The total volume of exhaled air, from a maximal inspiration to a maximal exhalation, is the vital capacity (VC). Residual volume (RV) is the air remaining in the lungs after a complete exhalation. TLC is the sum of VC and RV; it is the total volume of air contained in the lungs at the end of maximal inspiration,

1	volume (RV) is the air remaining in the lungs after a complete exhalation. TLC is the sum of VC and RV; it is the total volume of air contained in the lungs at the end of maximal inspiration, and it includes the volume of air that can be moved (VC) and the volume of air that is always present (trapped) in the lungs (RV). Functional residual capacity (FRC) is the volume of air in the lungs at the end of exhalation during quiet breathing and is also called the resting volume of the lungs. FRC is composed of RV and the expiratory reserve volume (the volume of air that can be exhaled from FRC to RV).

1	Measurement of Lung Volumes RV and TLC can be measured in two ways: by helium dilution and by body plethysmography. Both methods are used clinically and provide valuable information about lung function and lung disease. The helium dilution technique is the older and simpler method, but it is often less accurate than body plethysmography, which requires sophisticated and expensive equipment. 21.1 Pulmonaryfunctiontestsareoftenusedtodiagnoseabnormalitiesinlungfunctionandtoassesstheprogressionoflungdisease.Theycandistinguishthetwomajortypesofpulmonarypathophysiologicprocesses:obstructivelungdiseasesandrestrictivelungdiseases.Forexample,innormalindividuals,theratioofRVtoTLCislessthan 0.25.Thusinahealthyindividual,approximately25%ofthetotalvolumeofairinthelungistrapped.Inobstructive pulmonary diseases, anelevationinRV/TLCratioissecondarytoanincreaseinRVoutofproportiontoanyincreaseinTLC.Incontrast,inrestrictive lung diseases, theelevationintheRV/TLCratioiscausedbyadecreaseinTLC.

1	In normal individuals, the FRC measured by helium dilution and the FRC measured by plethysmography are the same ( Table 21.1 ). This is not true in individuals with lung disease. The FRC measured by helium dilution is the volume of gas in the lung that communicates with the •Fig. 21.4 Measurement of Lung Volume by Helium Dilution. C1,knownconcentrationofaninertgas;C2,new(previouslyunknown)concentrationofthegas;V1,knownvolumeofabox;V2,lungvolume(initiallyunknown). airways, whereas the FRC measured by plethysmography is the total volume of gas in the lungs at the end of a normal exhalation. If a significant amount of gas is trapped in the lungs (because of premature airway closure; see ), the FRC determined by plethysmography is considerably higher than that determined by helium dilution.

1	Intheheliumdilutiontechnique,aknownconcentration(C1)ofaninertgas(suchashelium)isaddedtoaboxofknownvolume(V1).Theboxisthenconnectedtoavolume(V2)thatisunknown(thelungvolumetobemeasured).Afteradequatetimefordistributionoftheinertgas,thenewconcentration(C2)oftheinertgasismeasured.Thechangeinconcentrationoftheinertgasisthenusedtodeterminethenewvolumeinwhichtheinertgashasbeendistributed( Fig.21.4 ).Specifically, Inbodyplethysmograph(bodybox),RobertBoyle’sgaslaw—thatpressuremultipliedbyvolumeisconstant(ataconstanttemperature)—isusedtomeasurelungvolumes.Thepatientsitsinanairtightbox(

1	Fig.21.4 ).Specifically, Inbodyplethysmograph(bodybox),RobertBoyle’sgaslaw—thatpressuremultipliedbyvolumeisconstant(ataconstanttemperature)—isusedtomeasurelungvolumes.Thepatientsitsinanairtightbox( Fig.21.5 )andbreathesthroughamouthpiecethatisconnectedtoaflowsensor(pneumotach).Thepatientthenmakesapantingrespiratoryeffortagainstaclosedmouthpiece.Duringtheexpiratoryphaseofthemaneuver,thegasinthelungbecomescompressed,lungvolumedecreases,andthepressureinsidetheboxfallsbecausethegasvolumeintheboxincreases.Oncethevolumeoftheboxandthechangeinpressureoftheboxatthemouthareknown,thechangeinvolume(ΔV)ofthelungcanbecalculated: whereP1andP2aremouthpressuresandVisFRC.FromthemeasurementofFRC,inspiratorycapacitycanberecordedasthevolumeofairinspiredaboveFRC,andexpiratoryreservevolumecanbedeterminedasthevolumeofgasexhaledfromFRC.Thesemeasurementscanthenbeusedtodeterminetheotherlungvolumes. •Fig. 21.5 The Body Plethysmograph. Notethattheboxinwhichthepatientsitsisnotdepicted.

1	•Fig. 21.5 The Body Plethysmograph. Notethattheboxinwhichthepatientsitsisnotdepicted. Determinants of Lung Volume

1	Determinants of Lung Volume What determines the volume of air in the lung at TLC or at RV? The answer lies in the properties of the lung parenchyma and in the interaction between the lungs and the chest wall. The lungs and chest wall always move together as a unit in healthy individuals. The lung contains elastic fibers that (1) stretch when stress is applied, which results in an increase in lung volume, and (2) recoil passively when this stress is released, which results in a decrease in lung volume. The elastic recoil of the lung parenchyma is very high. In the absence of external forces (such as the force generated by the chest wall), the lungs become almost airless (10% of TLC). Similarly, chest wall volume can increase when the respiratory muscles are stretched and decrease when respiratory muscle length is shortened. In the theoretical absence of the lung parenchyma, the resting volume of the chest wall increases and is approximately 60% of TLC.

1	Lung volumes are determined by the balance between the lung’s elastic properties and the properties of the muscles of the chest wall. The maximum volume of air contained within the lung and the chest wall (i.e., TLC) is controlled by the muscles of inspiration (see ). With increasing lung volume, the chest wall muscles lengthen progressively. As these muscles lengthen, their ability to generate force decreases. TLC occurs when the inspiratory chest wall muscles are unable to generate the additional force needed to further distend the lung and chest wall. Similarly, the minimal volume of air in the –40–20 0 20 40 Pressure (cm H2O) (PL, P , or P ) •Fig. 21.6 Relaxation Pressure-Volume Curve of the Lung, Chest Wall, and Respiratory System. Thecurvefortherespiratorysystemisthesumoftheindividualcurves.Thecurveforthelungisthesameastheoneforthenormallungin

1	Fig.21.1 .FVC,forcedvitalcapacity;PL,transpulmonary(ortranslung)pressure(PL);Prs,thepressureacrosstherespiratorysystem;Pw,thetransmuralpressureacrossthechestwall;RV,residualvolume;TLC,totallungcapacity. lung (i.e., RV) is controlled by the expiratory muscle force. Decreasing lung volume results in shortening of the expiratory muscles, which, in turn, results in a decrease in muscle force. The decrease in lung volume is also associated with an increase in the outward recoil pressure of the chest wall. RV occurs when expiratory muscle force is insufficient to further reduce chest wall volume.

1	FRC, or the volume of the lung at the end of a normal exhalation, is determined by the balance between the elastic recoil pressure generated by the lung parenchyma to become smaller (inward recoil) and the pressure generated by the chest wall to become larger (outward recoil). When the chest wall muscles are weak, FRC decreases (lung elastic recoil > chest wall muscle force). In the presence of airway obstruction, FRC increases because of premature airway closure, which traps air in the lung (see

1	A number of important observations can be made from an examination of the pressurevolume curves of the lung, chest wall, and respiratory system (Fig. 21.6 ). At the resting volume of the lung (FRC), the elastic recoil of the lung acts to decrease lung volume, but this inward recoil is offset by the outward recoil of the chest wall, which acts to increase lung volume. At FRC, these forces are equal and opposite, and the muscles are relaxed. As a result, the transmural pressure across the respiratory system (Prs) at FRC is 0. At TLC, both lung pressure and chest wall pressure are positive, and both require positive transmural distending pressure. The resting volume of the chest wall, in the absence of the lungs, is the volume at which the transmural pressure for the chest wall is 0, and it is approximately 60% of TLC. At volumes greater than 60% of TLC, the chest wall recoils inward and positive transmural pressure is needed, whereas at volumes below 60% of TLC, the chest wall tends to

1	is approximately 60% of TLC. At volumes greater than 60% of TLC, the chest wall recoils inward and positive transmural pressure is needed, whereas at volumes below 60% of TLC, the chest wall tends to recoil outward.

1	The lungs alone are smallest when transpulmonary pressure is 0. The lungs, however, are not totally devoid of air when transpulmonary pressure is 0 because of the surface tension–lowering properties of surfactant (see the section “ ”). The transmural pressure for a healthy lung alone flattens at pressures higher than 20 cm H2O because the elastic limits of the lung have been reached. Thus further increases in transmural pressure produce little change in volume, and compliance (see the section ”) is low. Further distention is limited by the connective tissue (collagen, elastin) of the lung. If further pressure is applied, the alveoli near the lung surface can rupture, and air can escape into the pleural space. This is called a pneumothorax. In a pneumothorax or when the chest is opened, as during thoracic surgery, the lungs and chest wall no longer function as a single unit. The lungs recoil until transpulmonary pressure is 0; the chest wall then increases in size until trans chest

1	as during thoracic surgery, the lungs and chest wall no longer function as a single unit. The lungs recoil until transpulmonary pressure is 0; the chest wall then increases in size until trans chest wall pressure is 0.

1	The relationship between transpulmonary pressure and pleural, alveolar, and elastic recoil pressures is depicted in Fig. 21.7 . Alveolar pressure is the sum of the pleural pressure and elastic recoil pressure (Pel) of the lung: Equation 21.4 Because transpulmonary pressure (PL) = PA + Ppl, Equation 21.5 •Fig. 21.7 Relationshipbetweentranspulmonarypressure(PL)andthepleural(Ppl),alveolar(PA),andelasticrecoil(Pel)pressuresofthelung.Alveolarpressureisthesumofpleuralpressureandelasticrecoilpressure.Transpulmonarypressureisthedifferencebetweenalveolarpressureandpleuralpressure. In general, PL is the pressure distending the lung, whereas Pel is the pressure that tends to collapse the lung. Lung elastic recoil increases as the lung inflates.

1	In general, PL is the pressure distending the lung, whereas Pel is the pressure that tends to collapse the lung. Lung elastic recoil increases as the lung inflates. Lung compliance (CL) is a measure of the elastic properties of the lung. It reflects how easily the lung is distended. Lung compliance is defined as the change in lung volume resulting from a 1–cm H2O change in the distending pressure of the lung. The units of compliance are in milliliters (or liters) per centimeter of water. When lung compliance is high, the lung is readily distended. When lung compliance is low (“stiff” lung), the lung is not easily distended. The compliance of the lung (CL) is expressed as Equation 21.6 where ΔV is the change in volume and ΔP is the change in pressure. Graphically, lung compliance is the slope of the line between any two points on the deflation limb of the pressurevolume loop (

1	Fig. 21.8 ). The compliance of a normal human lung is approximately 0.2 L/cm H2O, but it varies with lung volume. Note that the lung is less distensible at high lung volumes. For this reason, compliance is corrected for the lung volume at which it is measured (specific compliance; Fig. 21.9 ). Compliance is not often measured for clinical purposes because it requires placement of an esophageal balloon. The esophageal balloon, which is connected to a pressure transducer, is an excellent surrogate marker for pleural pressure, which is very difficult to measure directly. The change in pleural pressure (Ppl) is measured as a function of the change in lung volume; that is, CL =ΔV/ΔPplor .ΔPpl =ΔCL.

1	In addition to the elastic properties of the lungs, another major determinant of lung compliance is surfactant and its •Fig. 21.8 Deflation Pressure-Volume Curve. Thepatientinhalestototallungcapacity,andtranspulmonarypressureismeasuredwiththeuseofanesophagealballoon(whichmeasurespleuralpressure).Thepatientthenexhalesslowly,andpressureismeasuredatpointsofnoairflow,whentherespiratorymusclesarerelaxed.Thepressure-volumecurveofthelungisnotthesameininspiration(notshown)andexhalation.Thisdifferenceiscalledhysteresis, anditiscausedbytheactionofsurfactant.Inaccordancewithconvention,thedeflationpressure-volumecurveisusedformeasurements.Complianceatanypointalongthiscurveisthechangeinvolumeperchangeinpressure.Thecurvedemonstratesthatlungcompliancevarieswithlungvolume.Alinecanbedrawnbetweentwodifferentvolumesonthecurve,andtheslopeofthislinerepresentsthechangeinvolume(ΔV)forachangeinpressure(ΔP).Comparethecomplianceatline1versusline

1	2.Theslopeofline2islesssteepthantheslopeofline1,andsothecomplianceislessatthishigherlungvolumethanitisatthelowerlungvolume.Inaccordancewithconvention,lungcomplianceisthechangeinpressurefromfunctionalresidualcapacity(FRC)toFRC+1L.MV,minimalvolume;RV,residualvolume;TLC,totallungcapacity.

1	Thecomplianceofthelungsisaffectedbyseveralrespiratorydisorders.Inemphysema,anobstructivelungdiseasethatusuallyoccursinpeoplewhosmokeandisassociatedwithdestructionofthealveolarseptaandpulmonarycapillarybed,thelungismorecompliant;thatis,forevery1–cmH2Oincreaseinpressure,theincreaseinvolumeisgreaterthanthatinanormallung(see Fig.21.1 ).Incontrast,inpulmonaryfibrosis,arestrictivelungdiseaseassociatedwithincreasedcollagenfiberdepositionintheinterstitialspace,thelungisnoncompliant;thatis,forevery1–cmH2Ochangeinpressure,thechangeinvolumeisless.Thesechangesincomplianceareofclinicalsignificancebecausealungwithlowcompliancerequireslargerchangesinpleuralpressuretoeffectchangesinlungvolumes;asresult,theworkofbreathingisincreasedforeverybreaththattheindividualtakes.

1	1 liter = 0.2 0.5 liter = 0.1 0.1 liter = 0.02 = 0.2 5 cm H2O 0.1 liter 0.2 = 0.2 0.1 = 0.2 0.5 liter 0.02 •Fig. 21.9 Relationship Between Compliance and Lung Volume. Imaginelungsinwhicha5–cmH2Ochangeinpressureresultsina1-Lchangeinvolume(situation1).Ifonelungisremoved(situation2),thecompliancedecreases,butwhencorrectedforvolumeofthelung,thereisnochange(specificcompliance).Evenwhentheremaininglungisreducedby90%(situation3),thespecificcomplianceisunchanged.

1	effect on surface tension. Surface tension is a force caused by water molecules at the airliquid interface that tends to minimize surface area, which makes inflating the lungs more difficult. The effect of surface tension on lung inflation is illustrated by a comparison of the volumepressure curves of a salinefilled lung and of an airfilled lung. Higher pressure is necessary to fully inflate the lung with air than with saline because of the higher surface tension forces in airfilled lungs than in salinefilled lungs. Surface tension is a measure of the attractive force of the surface molecules per unit length of material to which they are attached. The units of surface tension are those of a force applied per unit length. For a sphere (such as an alveolus), the relationship between the pressure within the sphere (Ps) and the tension in the wall is described by the law of Laplace: Equation 21.7 where T is the wall tension (in dynes per centimeter) and r is the radius of the sphere.

1	Equation 21.7 where T is the wall tension (in dynes per centimeter) and r is the radius of the sphere. The alveoli are lined with a predominantly lipidbased substance called surfactant. Pulmonary surfactant serves several physiological roles, including (1) reducing the work of breathing by decreasing surface tension forces; (2) preventing collapse and sticking of alveoli on exhalation; and (3) stabilizing alveoli, especially those that tend to deflate at low surface tension. In the absence of surfactant, the surface tension at the airliquid interface would remain constant, and the transalveolar pressure needed to keep it at that volume would be higher when alveolar volumes are lower (

1	Fig. 21.10A ). Therefore, higher transalveolar pressure would be necessary to produce a given increase in alveolar volume at lower lung volumes than at higher lung volumes. Surfactant stabilizes the inflation of alveoli because it allows the surface tension to increase as the alveoli become larger (see Fig. 21.10B ). As a result, the transalveolar pressure necessary to keep an alveolus inflated increases as lung volume and transpulmonary pressure increases, and it decreases as lung volume decreases. In the presence of surfactant, surface tension is increased at high lung volume and decreased at low lung volume. The result is that the lungs can maintain alveoli at many different volumes. Otherwise, the gas in small alveoli would empty into large alveoli.

1	Pulmonary surfactant is synthesized by alveolar type II cells, stored in the cell in lamellar bodies, and secreted into the alveolar space in a precursor form (tubular myelin), from where it spreads throughout the entire alveolar surface and attains its ability to decrease surface tension. Surfactant is 85% to 90% lipids, predominantly phospholipids, and 10% to 15% proteins (

1	Table 21.2). The major phospholipid is phosphatidylcholine, approximately 75% of which is present as dipalmitoyl phosphatidylcholine (DPPC). DPPC decreases surface tension and is the major surfaceactive component in surfactant. The second most abundant phospholipid is phosphatidylglycerol, which accounts for •Fig. 21.10 Surface Forces in a Sphere Attempt to Reduce the Area of the Surface and Generate Pressure Within the Sphere. ByLaplace’slaw,thepressuregeneratedisinverselyproportionaltotheradiusofthesphere.A, Intheabsenceofsurfactant,surfaceforcesinthesmallerspheregeneratehigherpressure(heavier purple arrows) thandothoseinthelargersphere(lighter purple arrows). Asaresult,airmovesfromthesmallsphere(higherpressure)tothelargersphere(lowerpressure;black arrow).Thiscausesthesmallspheretocollapseandthelargespheretobecomeoverdistended.B, Surfactant(shaded layer)

1	Asaresult,airmovesfromthesmallsphere(higherpressure)tothelargersphere(lowerpressure;black arrow).Thiscausesthesmallspheretocollapseandthelargespheretobecomeoverdistended.B, Surfactant(shaded layer) lowerssurfacetensionandlowersitmoreinthesmallerspherethaninthelargersphere.Thenetresultsarethatthepressuresinthesmallandlargerspheresaresimilar,andthevolumesofthespheresarestabilized.

1	1% to 10% of total surfactant. These lipids are important in formation of the monolayer on the alveolarair interface, and phosphatidylglycerol is important in the spreading of surfactant over a large surface area. Surfactant protein A, which is the protein most studied, is expressed in alveolar type II cells and in Clara cells in the lungs. Surfactant protein A is involved in the regulation of surfactant turnover, in immunoregulation within the lungs, and in the formation of tubular myelin.

1	Surfactant is secreted into the airway through exocytosis of the lamellar body by constitutive and regulated mechanisms. Numerous agents, including βadrenergic agonists, activators of protein kinase C, leukotrienes, and purinergic agonists, stimulate the exocytosis of surfactant. The major routes of clearance of pulmonary surfactant within the lung are reuptake by type II cells, absorption into the lymphatic vessels, and clearance by alveolar macrophages. Surfactant is readily inactivated by hypoxia, infection, and edema fluid, which results in a decrease in lung compliance.

1	In addition to surfactant, another mechanism, interdependence, contributes to stability of the alveoli. Alveoli, except for those on the pleural surface, are surrounded by other alveoli. The tendency of one alveolus to collapse is opposed by the traction exerted by the surrounding alveoli. Thus collapse of a single alveolus causes stretching and distortion of the surrounding alveoli, which in turn are connected to other alveoli. Small openings (pores of Kohn) in the alveolar walls connect adjacent alveoli, whereas the canals of Lambert connect the terminal airways to adjacent alveoli. The pores of Kohn and the canals of Lambert provide collateral ventilation and prevent alveolar collapse (atelectasis).

1	In1959,AveryandMeaddiscoveredthatinprematureinfantswhodiedofhyalinemembranedisease(HMD),thelungsweredeficientinsurfactant.HMD,alsoknownasinfantrespiratory distress syndrome, ischaracterizedbyprogressiveatelectasisandrespiratoryfailureinprematureinfants.Itisamajorcauseofmorbidityandmortalityintheneonatalperiod.Themajorsurfactantdeficiencyinpremature 1. Gas flows from areas of higher pressure to areas of lower pressure. Positive transpulmonary pressure is needed to increase lung volume. The pressure across the respiratory system is 0 at points of no airflow (end inspiration and end exhalation). At functional residual capacity (FRC), the pressure difference across the respiratory system is 0, and lung elastic recoil pressure, which operates to decrease lung volume, and the pressure generated by the chest wall to become larger are equal and opposite. 2.

1	2. Pressure gradients in the respiratory system are created by the active contraction and subsequent relaxation of the muscles of respiration. 3. Lung volumes are determined by the balance between the lung’s elastic recoil properties and the properties of the muscles of the chest wall. Gibson GJ, Pride NB. Lung distensibility: the static pressurevolume curve of the lungs and its use in clinical assessment. Br J Dis Chest. 1976;70:143184. Jobe AH. The alveolar lining layer: a review of studies on its role in pulmonary mechanics and in the pathogenesis of atelectasis, by Mary Ellen Avery, MD, Pediatrics, 1962:30:324330. Pediatrics. 1998;102(S1):234235. infantsislackofphosphatidylglycerol.Ingeneral,asthelevelofphosphatidylglycerolincreasesinamnioticfluid,theinfantmortalityratedecreases.ResearchinthisfieldhasculminatedinsuccessfulattemptstotreatHMDinprematureinfantswithsurfactantreplacementtherapy.Today,surfactantreplacementtherapyisstandardcareforprematureinfants. 4.

1	4. Total lung capacity (TLC) is equal to the total volume of air that can be exhaled after a maximal inspiration (vital capacity [VC]) and the air remaining in the lung after a maximal exhalation (residual volume [RV]). 5. Lung compliance is a measure of the elastic properties of the lung. Elastic recoil is lost in patients with emphysema, and this loss is associated with an increase in lung compliance, whereas in diseases associated with pulmonary fibrosis, lung compliance is decreased. 6. The surface tensionreducing and antisticking properties of surfactant increase lung compliance, decrease the work of breathing, and help stabilize alveoli of different size. Lumb AB. Nunn’s Applied Respiratory Physiology. 8th ed. St. Louis: Elsevier; 2016. Mead J, Macklem PT, vol eds. American Physiological Society Handbook of Physiology: The Respiratory System, vol. 3: Mechanics. Bethesda, MD: American Physiological Society; 1986.

1	Mead J, Macklem PT, vol eds. American Physiological Society Handbook of Physiology: The Respiratory System, vol. 3: Mechanics. Bethesda, MD: American Physiological Society; 1986. Otis AB. A perspective of respiratory mechanics. J Appl Physiol. 1983;54:11831187. Otis AB, Fenn WO, Rahn H. Mechanics of breathing in man. J Appl Physiol. 1950;2:592607. Upon completion of this chapter the student should be able to answer the following questions: 1. Describe air flow in the airways. 2. Define resistance and its affect upon airflow in the airways. 3. List and describe two categories of factors that contribute to airway resistance. 4. List the features of a spirogram and flow volume curve. 5. Describe how flow limitation occurs at the equal pressure point and the role of dynamic airway compression in flow limitation. 6. Define the components of work of breathing. 7. Understand how dynamic compliance is different from static compliance and its contribution to work of breathing.

1	6. Define the components of work of breathing. 7. Understand how dynamic compliance is different from static compliance and its contribution to work of breathing. In this chapter, the principles that control air movement into and out of the lungs are examined. Dynamic mechanics is the study of physical systems in motion, and for the respiratory system it is the study of the properties of a lung whose volume is changing with time. Air flows into and out of an airway when there is a pressure difference at the two ends of the airway. By way of review, during inspiration the diaphragm contracts, pleural pressure becomes more negative, and gas flows into the lung (see

1	Fig. 21.2 ). To meet the changing metabolic needs of the body, gas exchange depends on the speed at which fresh gas is brought to the alveoli and the rapidity with which the metabolic products of respiration (i.e., CO2) are removed. Two major factors determine the speed at which gas flows into the airways for a given pressure change: the pattern of gas flow and the resistance to airflow by the airways. Patterns of Airflow There are two major patterns of gas flow in the airways— laminar and turbulent. Laminar flow is parallel to the airway walls and is present at low flow rates. As the flow rate increases and particularly as the airways divide, the flow stream becomes unsteady and small eddies develop. At higher flow rates the flow stream is disorganized and turbulence occurs.

1	The pressure-flow characteristics of laminar flow were first described by the French physician Poiseuille and apply to both liquids and air. In straight circular tubes the flow rate (V̇ ) is defined by the following equation: Equation 22.1 where P is the driving pressure, r is the radius of the tube, η is the viscosity of the fluid, and l is the length of the tube. It can be seen that driving pressure (P) is proportional to the flow rate (V̇ ); thus the greater the pressure, the greater the flow. The flow resistance (R) across a set of tubes is defined as the change in driving pressure (ΔP) divided by the flow rate, or: Equation 22.2

1	The flow resistance (R) across a set of tubes is defined as the change in driving pressure (ΔP) divided by the flow rate, or: Equation 22.2 The units of resistance are cm H2O/L•sec. This equation is for laminar flow and demonstrates that the radius of the tube is the most important determinant of resistance. If the radius of the tube is reduced by half, the resistance will increase 16-fold. If, however, tube length is increased twofold, the resistance will increase only twofold. Thus the radius of the tube is the principal determinant of resistance. Stated another way, resistance is inversely proportional to the fourth power of the radius, and it is directly proportional to the length of the tube and to the viscosity of the gas.

1	In turbulent flow, gas movement occurs both parallel and perpendicular to the axis of the tube. Pressure is proportional to the flow rate squared. The viscosity of the gas increases with increasing gas density, and therefore the pressure drop increases for a given flow. Overall, gas velocity is blunted because energy is consumed in the process of generating eddies and chaotic movement. As a consequence, higher driving pressure is needed to support a given turbulent flow than to support a similar laminar flow. Whether flow through a tube is laminar or turbulent depends on the Reynolds number. The Reynolds number (Re) is a dimensionless value that expresses the ratio of two dimensionally equivalent terms (kinematic/viscosity), as seen in the equation:

1	Equation 22.3 where d is the fluid density, v is the average velocity, r is the radius, and η is the viscosity. In straight tubes, turbulence occurs when the Reynolds number is greater than 2000. From this relationship it can be seen that turbulence is most likely to occur when the average velocity of the gas flow is high and the radius is large. In contrast, a low-density gas such as helium is less likely to cause turbulent flow. This is clinically relevant in states of increased airway resistance where a decrease in gas density (e.g., substituting helium for nitrogen in inspired air) can improve airflow.

1	Although these relationships apply well to smooth cylindrical tubes, application of these principles to a complicated system of tubes such as the airways is difficult. As a result, much of the flow in the airways demonstrates characteristics of both laminar and turbulent flow. In the trachea, for example, even during quiet breathing the Reynolds number is greater than 2000. Hence turbulent flow occurs in the trachea even during quiet breathing. Turbulence is also promoted by the glottis and vocal cords, which produce some irregularity and obstruction in the airways. As gas flows distally the total cross-sectional area increases dramatically, and gas velocities decrease significantly. As a result, gas flow becomes more laminar in the smaller airways even during maximal ventilation. Overall the gas flow in the larger airways (nose, mouth, glottis, and bronchi) is turbulent, whereas the gas flow in the smaller airways is laminar. Breath sounds heard with a stethoscope reflect turbulent

1	the gas flow in the larger airways (nose, mouth, glottis, and bronchi) is turbulent, whereas the gas flow in the smaller airways is laminar. Breath sounds heard with a stethoscope reflect turbulent airflow. Laminar flow is silent, which is why it is difficult to “hear” small airway disease with a stethoscope.

1	Airflow resistance is the second major factor that determines rates of airflow in the airways. Airflow resistance in the airways (Raw) differs in airways of different size. In moving from the trachea toward the alveolus, individual airways become smaller while the number of airway branches increases dramatically. Raw is equal to the sum of the resistance of each of these airways (i.e., Raw = Rlarge + Rmedium + Rsmall). From Poiseuille’s equation, one might conclude that the major site of airway resistance is in the smallest airways. In fact, however, the major site of resistance along the bronchial tree is in the first eight generations of airways. .08 .06 Respiratory zoneConducting zone Airway generation .02 .04 0 0 5 10 15 20 Resistance (cm H2 O/L/sec) •Fig. 22.1 Airwayresistanceasafunctionoftheairwaygeneration.Inanormallung,mostoftheresistancetoairflowoccursinthefirsteightairwaygenerations.

1	The smallest airways contribute very little to the overall total resistance of the bronchial tree (Fig. 22.1 ). The reason for this is twofold: (1) airflow velocity decreases substantially as the effective cross-sectional area increases (i.e., flow becomes laminar), and (2) most importantly, the airway branches in each generation exist in parallel rather than in series. The resistance of airways in parallel is the inverse of the sum of the individual resistances; therefore the overall contribution to resistance of the small airways is very small. As an example, assume that each of three tubes has a resistance of 3 cm H2O. If the tubes are in series, the total resistance (Rtot) is the sum of the individual resistances: Equation 22.4 If the tubes are in parallel (as they are in small airways), the total resistance is the sum of the inverse of the individual resistances: Equation 22.5

1	Equation 22.4 If the tubes are in parallel (as they are in small airways), the total resistance is the sum of the inverse of the individual resistances: Equation 22.5 This relationship is in marked contrast to the pulmonary blood vessels, in which most of the resistance is located in the small vessels (see ). Thus as airway diameter decreases, the resistance offered by each individual airway increases, but the large increase in the number of parallel pathways and cross-sectional area reduces the resistance at each generation of branching.

1	During normal breathing, approximately 80% of the resistance to airflow at functional residual capacity (FRC) of efferent vagal fibers, either directly or reflexively, increases ) secondary to airway constriction (recall that the vagus nerve innervates airway smooth muscle). In con trast, stimulation of sympathetic nerves and release of the postganglionic neurotransmitter norepinephrine inhibits airway constriction. Reflex stimulation of the vagus nerve by inhalation of smoke, dust, cold air, or other irritants can also result in airway constriction and coughing. Agents such as histamine, acetylcholine, thromboxane A2, prostaglandin F2, and leukotrienes (LTB4, LTC4, and LTD4) are released by resident cells (e.g., mast cells, airway epithelial cells) and recruited cells (e.g., neutrophils, eosinophils) in response to various triggers such as allergens and viral infections. These agents act directly on airway smooth muscle to cause constriction and an increase in airway resistance.

1	eosinophils) in response to various triggers such as allergens and viral infections. These agents act directly on airway smooth muscle to cause constriction and an increase in airway resistance. Inhalation •Fig. 22.2 Airwayresistance(AWR)andconductance(Cond.)asafunctionoflungvolume.

1	occurs in airways with diameters greater than 2 mm. Because the small airways contribute so little to total lung resistance, measurement of airway resistance is a poor test for detecting small airway obstruction. Factors That Contribute to Airway Resistance In healthy individuals, airway resistance is approximately 1 cm H2O/L • sec. One of the most important factors affecting resistance is lung volume. Increasing lung volume increases the caliber of the airways because it creates a positive transairway pressure. As a result, resistance to airflow decreases with increasing lung volume and increases with decreasing lung volume. If the reciprocal of resistance (i.e., conductance) is plotted against lung volume, the relationship between lung volume and conductance is linear (Fig. 22.2 ). Other factors that increase airway resistance include airway mucus, edema, and contraction of bronchial smooth muscle, all of which decrease the caliber of the airways.

1	22.2 ). Other factors that increase airway resistance include airway mucus, edema, and contraction of bronchial smooth muscle, all of which decrease the caliber of the airways. The density and viscosity of the inspired gas also affect airway resistance. When scuba diving, gas density rises and results in an increase in airway resistance; this increase can cause problems for individuals with asthma and obstructive pulmonary disease. Breathing a low-density gas such as an oxygen-helium mixture results in a decrease in airway resistance and has been exploited in the treatment of status asthmaticus, a condition associated with increased airway resistance due to a combination of bronchospasm, airway inflammation, and hypersecretion of mucus. Neurohumoral Regulation of Airway Resistance

1	Neurohumoral Regulation of Airway Resistance In addition to the effects of disease, airway resistance is regulated by various neural and humoral agents. Stimulation of methacholine, a derivative of acetylcholine, is used to diagnose airway hyperresponsiveness, which is one of the cardinal features of certain asthma phenotypes. Although everyone is capable of responding to methacholine, airway obstruction develops in individuals with asthma at much lower concentrations of inhaled methacholine. Measurement of Expiratory Flow Measurement of expiratory flow rates and expiratory volumes is an important clinical tool for evaluating and monitoring respiratory diseases. Commonly used clinical tests have the patient inhale maximally to total lung capacity (TLC) and then exhale as rapidly and completely as possible to residual volume (RV). The test results are displayed either as a spirogram ( Fig. 22.3A ) or as a flow-volume curve/ loop (

1	Fig. 22.3A ) or as a flow-volume curve/ loop ( Fig. 22.3B ). Results from individuals with suspected lung disease are compared with results predicted from normal healthy volunteers. Predicted or normal values vary with age, sex, ethnicity, height, and to a lesser extent, weight ( Table 22.1 ). Abnormalities in values indicate abnormal pulmonary function and can be used to predict abnormalities in gas exchange. These values can detect the presence of abnormal lung function long before respiratory symptoms develop, and they can be used to determine disease severity and the response to therapy. The Spirogram A spirogram displays the volume of gas exhaled as a function of time (see Fig. 22.3A ) and measures: (1) forced vital capacity (FVC), (2) forced expiratory volume in 1 second (FEV1), (3) the ratio of FEV1 to FVC (FEV1/ FVC), and (4) the average midmaximal expiratory flow (FEF25-75).

1	The total volume of air that is exhaled during a maximal forced exhalation from TLC to RV is called the FVC. The volume of air that is exhaled in the first second during the maneuver is called the FEV1. In normal individuals, 70% to 85% (depending on age) of the FVC can be exhaled in the first second. Thus the normal FEV1/FVC ratio is greater than 70% in healthy adults. A ratio less than 70% suggests •Fig. 22.3 Theclinicalspirogram(A)andflow-volumeloop(B).Theindividualtakesamaximalinspirationandthenexhalesasrapidly,asforcibly,andasmaximallyaspossible.Thevolumeexhaledisplottedasafunctionoftime.Inthespirogramthatisreportedinclinicalsettings,exhaledvolumeincreasesfromthebottomofthetracetothetop(A).Thisisincontrasttothephysiologist’sviewofthesamemaneuver(see

1	Fig.21.3 ),inwhichtheexhaledvolumeincreasesfromthetoptothebottomofthetrace.Intheflow-volumeloop(B),exhaledvolumeisplottedasafunctionoftheinstantaneousflowrate,whichismeasuredusingapneumotachometer.Themaximalexpiratoryflowrateachievedduringthemaneuveriscalledthepeak expiratory flow rate. NotethelocationsofTLCandRVonbothtracings. difficulty exhaling because of obstruction and is a hallmark of obstructive pulmonary disease. One expiratory flow rate—the average flow rate over the middle section of the VC—can be calculated from the spirogram. This expiratory flow rate has several names, including MMEF (midmaximal expiratory flow) and FEF25-75 (forced expiratory flow from 25%–75% of VC). Although it can be calculated from the spirogram, today’s spirometers automatically calculate FEF25-75.

1	Another way of measuring lung function clinically is the flow-volume curve or loop. A flow-volume curve or loop is created by displaying the instantaneous flow rate during a forced maneuver as a function of the volume of gas. This instantaneous flow rate can be displayed both during exhalation (expiratory flow-volume curve) and during inspiration (inspiratory flow-volume curve) (see

1	Fig. 22.3B ). Expiratory flow rates are displayed above the horizontal line, and inspiratory flow rates are displayed below the horizontal line. The flow-volume loop measures: (1) the FVC, (2) the greatest flow rate achieved during the expiratory maneuver, called the peak expiratory flow rate (PEFR), and (3) multiple expiratory flow rates at various lung volumes. When the expiratory flow-volume curve is divided into quarters, the instantaneous flow rate at which 50% of the VC remains to be exhaled is called the FEF50 (also known as the V̇ max50), the instantaneous flow rate at which 75% of the VC has been exhaled is called the FEF75 (V̇ max75), and the instantaneous flow rate at which 25% of the VC has been exhaled is called the FEF25 (V̇ max25). •Fig. 22.4 Isovolumecurves.Threesuperimposedexpiratoryflowmaneuversaremadewithincreasingeffort.Notethatpeakinspiratoryandexpiratoryflowratesaredependentoneffort,whereasexpiratoryflowrateslaterinexpirationareindependentofeffort.

1	Inamethacholinechallengetest,spirometrymeasurementsaremadeafterthepatientinhalesincreasingconcentrationsofthemuscarinicagonistmethacholine.ThetestisstoppedwhenFEV1fallsby20%ormoreorwhenamaximumconcentration(25mg/mL)ofmethacholinehasbeeninhaled.Theconcentrationofmethacholinethatproducesa20%decreaseinFEV1iscalledtheprovocation concentration (PC)20. ThelowerthePC20,themoresensitiveanindividualistomethacholine.MostindividualswithasthmahaveaPC20lessthan8mg/mLofmethacholine. Determinants of Maximal Flow

1	Determinants of Maximal Flow The shape of the flow-volume loop reveals important information about normal lung physiology that can be altered by disease. Inspection of the flow-volume loop reveals that the maximum inspiratory flow is the same or slightly greater than the maximum expiratory flow. Three factors are responsible for the maximum inspiratory flow. First, the force generated by the inspiratory muscles decreases as lung volume increases above RV. Second, the recoil pressure of the lung increases as the lung volume increases above RV. This opposes the force generated by the inspiratory muscles and reduces maximum inspiratory flow. However, airway resistance decreases with increasing lung volume as the airway caliber increases. The combination of inspiratory muscle force, recoil of the lung, and changes in airway resistance causes maximal inspiratory flow to occur about halfway between TLC and RV.

1	During exhalation, maximal flow occurs early (in the first 20%) in the maneuver, and flow rates decrease progressively toward RV. Even with increasing effort, maximal flow decreases as RV is approached. This is known as expiratory flow limitation and can be demonstrated by asking an individual to perform three forced expiratory maneuvers with increasing effort.

1	Fig. 22.4 shows the results of these three maneuvers. As effort increases, peak expiratory flow increases. However, the flow rates at lower lung volumes converge; this indicates that with modest effort, maximal expiratory flow is achieved. No amount of effort will increase the flow rates as lung volume decreases. For this reason, expiratory flow rates at lower lung volumes are said to be effort independent and flow limited because maximal flow is achieved with modest effort, and no amount of additional effort can increase the flow rate beyond this limit. In contrast, events early in the expiratory maneuver are said to be effort dependent; that is, increasing effort generates increasing flow rates. In general the first 20% of the flow in the expiratory flow-volume loop is effort dependent. Flow Limitation and the Equal Pressure Point

1	Flow Limitation and the Equal Pressure Point Why is expiratory flow limited and reasonably effort independent? Factors that limit expiratory flow are important because many lung diseases affect these factors and thus affect the volume and speed with which air is moved into and out of the lung. Flow limitation occurs when the airways, which are intrinsically floppy distensible tubes, become compressed. The airways become compressed when the pressure outside the airway exceeds the pressure inside the airway. How and when this occurs is important to understanding lung disease. Fig. 22.5 shows the events that TLC, no flow •Fig. 22.5 Flowlimitation.A,Endinspiration,beforethestartofexhalation.B,Atthestartofaforcedexhalation.C,Expiratoryflowlimitationlaterinaforcedexhalation.Expiratoryflowlimitationoccursatlocationswhereairwaydiameterisnarrowedasaresultofnegativetransmuralpressure.Seetextfordetails.

1	occur during expiratory flow limitation at two different lung volumes. The airways and alveoli are surrounded by the pleural space and the chest wall. The airways are shown as tapered tubes because the total or collective airway cross-sectional area decreases from the alveoli to the trachea. At the start of exhalation but before any gas flow occurs, the pressure inside the alveolus (PA) is zero (no airflow) and pleural pressure (in this example) is −30 cm H2O. Transpulmonary pressure is thus +30 cm H2O (PL = PA − Ppl). Because there is no flow, the pressure inside the airways is zero and the pressure across the airways (Pta, transairway pressure) is +30 cm H2O [Pta = Pairway − Ppl = 0 − (−30 cm H2O)]. This positive transpulmonary and transairway pressure holds the alveoli and airways open.

1	When an active exhalation begins and the expiratory muscles contract, pleural pressure rises to +60 cm H2O (in this example). Alveolar pressure also rises, in part because of the increase in pleural pressure (+60 cm H2O) and in part because of the elastic recoil pressure of the lung at that lung volume (which in this case is 30 cm H2O). Alveolar pressure is the sum of pleural pressure and elastic recoil pressure (i.e., PA = Pel + Ppl = 30 cm H2O + 60 cm H2O = 90 cm H2O in this example). This is the driving pressure for expiratory gas flow. Because alveolar pressure exceeds atmospheric pressure, gas begins to flow from the alveolus to the mouth when the glottis opens. As gas flows out of the alveoli, the transmural pressure across the airways decreases (i.e., the pressure head for expiratory gas flow dissipates). This occurs for three reasons: (1) there is a resistive pressure drop caused by the frictional pressure loss associated with flow (expiratory airflow resistance); (2) as the

1	gas flow dissipates). This occurs for three reasons: (1) there is a resistive pressure drop caused by the frictional pressure loss associated with flow (expiratory airflow resistance); (2) as the cross-sectional area of the airways decreases toward the trachea, gas velocity increases and this acceleration of gas flow further decreases the pressure; and (3) as lung volume decreases, the elastic recoil pressure decreases.

1	Thus as air moves out of the lung, the driving pressure for expiratory gas flow decreases. In addition, the mechanical tethering that holds the airways open at high lung volumes diminishes as lung volume decreases. There is a point between the alveoli and the mouth at which the pressure inside the airways equals the pressure that surrounds the airways. This point is called the equal pressure point. Airways toward the mouth but still inside the chest wall become compressed because the pressure outside is greater than the pressure inside (dynamic airway compression). As a consequence the transairway pressure now becomes negative [Pta = Paw − Ppl = 58 − (+60) =−2 cm H2O] just beyond the equal pressure point. No amount of effort will increase the flow further because the higher pleural pressure tends to collapse the airway at the equal pressure point, just as it also tends to increase the gradient for expiratory gas flow. Under these conditions, airflow is independent of the total driving

1	tends to collapse the airway at the equal pressure point, just as it also tends to increase the gradient for expiratory gas flow. Under these conditions, airflow is independent of the total driving pressure. Hence the expiratory flow is effort independent and flow limited. It is also why airway resistance is greater during exhalation than during inspiration. In the absence of lung disease, the equal pressure point occurs in airways that contain cartilage, and thus they resist collapse. The equal pressure point, however, is not static. As lung volume decreases and as elastic recoil pressure decreases, the equal pressure point moves closer to the alveoli.

1	One additional measurement of dynamic lung mechanics should be mentioned, and this is the measurement of dynamic compliance. A dynamic pressure-volume curve can be created by having an individual breathe over a normal lung volume range (usually from FRC to FRC +1 L). The Whathappensinindividualswithlungdisease?Imagineanindividualwithairwayobstructionsecondarytoacombinationofmucus accumulationandairwayinflammation(Fig.

1	22.6A ).Atthestartofexhalationthedrivingpressureforexpiratorygasflowisthesameasinanormalindividual;thatis,thedrivingpressureisthesumoftheelasticrecoilpressureandpleuralpressure.Asexhalationproceeds,however,theresistivedropinpressureisgreaterthaninthenormalindividualbecauseofthegreaterdecreaseinairwayradiussecondarytotheaccumulationofmucusandtheinflammation.Asaresulttheequalpressurepointnowoccursinsmallairwaysthataredevoidofcartilage.Theseairwayscollapse.Thiscollapseisknownaspremature airway closure, whichresultsinaless-than-maximalexpiratoryvolumeandproducesanincreaseinlungvolumeknownasair trapping.Theincreaseinlungvolumeinitiallyhelpsoffsettheincreaseinairwayresistancecausedbytheaccumulationofmucusandinflammationbecauseitresultsinanincreaseinairwaycaliberandelasticrecoil.Asthediseaseprogresses,however,inflammationandaccumulationofmucusincreasefurther,thereisagreaterincreaseinexpiratoryresistance,andmaximalexpiratoryflowratesdecrease.

1	Fig.22.6B ).Atthestartofexhalationthedrivingpressureforexpiratorygasflowisreducedsecondarytoalossofelasticrecoil.Whiletheresistivedropinpressureisnormal,thesmallerinitialdrivingpressureresultsinanequalpressurepointthatoccursclosertothealveolusinairwaysthatdonotcontaincartilage.Prematureairwayclosureagainoccursbutforaverydifferentreasonthantheprematureairwayclosureobservedinindividualswithanincreaseinairwayresistance.

1	Individualswithprematureairwayclosurefrequentlyhavecrackles, alsosometimescalledrales, apoppingsoundusuallyheardduringinspirationonauscultation.Thesecracklesareduetotheopeningofairwaysduringinspirationthatclosed(i.e.,werecompressed)duringthepreviousexhalation.Cracklescanbeduetomucusaccumulation,airwayinflammation,fluidintheairways,oranymechanismresponsibleforairwaynarrowingorcompression.Theyarealsoheardinindividualswithemphysema,inwhichthereisadecreaseinlungelasticrecoil.Infact,acuteandchroniclungdiseasescanchangetheexpiratoryflow-volumerelationshipbychangesin(1)staticlungrecoilpressure,(2)airwayresistanceandthedistributionofresistancealongtheairways,(3)lossofmechanicaltetheringofintraparenchymalairways,(4)changesinthestiffnessormechanicalpropertiesoftheairways,and(5)differencesinseverityoftheaforementionedchangesinvariouslungregions.

1	mean dynamic compliance of the lung (dyn CL) is calculated as the slope of the line that joins the end-inspiratory and end-expiratory points of no flow ( Fig. 22.7 Dynamic compliance is always less than static compliance, and it increases during exercise. This is because during tidal volume breathing, a small change in alveolar surface area is insufficient to bring additional surfactant molecules to the surface, and thus the lung is less compliant. During exercise the opposite occurs; there are large changes in Mucus, Edema, Bronchoconstriction PL = 90˜60 = 30 cm H2O Fig. 22.6 A,Flowlimitationinthepresenceofincreasedairwayresistance.B,Flowlimitationinthepresenceofalossofelasticrecoil.

1	Mucus, Edema, Bronchoconstriction PL = 90˜60 = 30 cm H2O Fig. 22.6 A,Flowlimitationinthepresenceofincreasedairwayresistance.B,Flowlimitationinthepresenceofalossofelasticrecoil. Fig. 22.7 Inflation-deflationpressure-volumecurve.Thedirectionofinspirationandexhalationisshownbythearrows.Thedifferencebetweentheinflationanddeflationpressure-volumecurvesisduetothevariationinsurfacetensionwithchangesinlungvolume.Notetheslopeofthelinejoiningpointsofnoairflow.Thisslopeislesssteepthantheslopefromthedeflationpressure-volumecurveatthesamelungvolume. tidal volume, and more surfactant material is incorporated into the air-liquid interface. Therefore the lung is more compliant.

1	tidal volume, and more surfactant material is incorporated into the air-liquid interface. Therefore the lung is more compliant. Sighing and yawning increase dynamic compliance by increasing tidal volume and restoring the normal surfactant layer. Both of these respiratory activities are important for D maintaining normal lung compliance. In contrast to the 1000 lung, the dynamic compliance of the chest wall is not significantly different from its static compliance. EF Insp. Exp. B Work of Breathing

1	EF Insp. Exp. B Work of Breathing Breathing requires the use of respiratory muscles (diaphragm, intercostals, etc.), which expends energy. Work is required to overcome the inherent mechanical properties of the lung (i.e., elastic and flow-resistive forces) and to move both the lungs and the chest wall. This work is known as the work of breathing. Changes in the mechanical properties of the lung or chest wall (or both) in the presence of disease result in an increase in the work of breathing. Respiratory muscles can perform increased work over long periods. However, the body. In the respiratory system, the work of breathing is calculated by multiplying the change in volume by the pressure exerted across the respiratory system:

1	Work of breathing (W) = Pressure (P) DC like other skeletal muscles they can fatigue, and respiratory failure may ensue. Respiratory muscle fatigue is the most common cause of respiratory failure, a process in which gas exchange is inadequate to meet the metabolic needs of Although methods are not available to measure the total amount of work involved in breathing, one can estimate the mechanical work by measuring the volume and pressure changes during a respiratory cycle. Analysis of pressure-volume curves can be used to illustrate these points. Fig. 22.8A represents a respiratory cycle of a normal lung. The static inflation-deflation curve is represented by line ABC. The total mechanical workload is represented by the trapezoidal area OAECD. In restrictive lung diseases, such as pulmonary fibrosis, lung compliance is decreased and the pressure-volume curve is shifted to the right. This results in a significant increase in the work of breathing (see

1	Fig. 22.8B ), as indicated by the increase in the trapezoidal area of OAECD. In obstructive lung diseases, such as asthma during an exacerbation or chronic bronchitis, airway resistance is elevated (see Fig. 22.8C ) and greater negative pleural pressure is needed to maintain normal inspiratory flow rates. In addition to the increase in total inspiratory work (OAECD), individuals with obstructive lung disease have an increase in positive pleural pressure during exhalation because of the increase in resistance and the increased expiratory workload, which is visualized as area DFO. The stored elastic energy, represented by area ABCF of

1	Fig. 22.8A , is not sufficient, and additional energy is needed for exhalation. With time or disease progression, these respiratory muscles can fatigue and result in respiratory failure. The work of breathing is also increased when deeper breaths are taken (an increase in tidal volume requires more elastic work to overcome) and when the respiratory rate increases (an increase in minute •Fig. 22.8 Mechanicalworkdoneduringarespiratorycycleinanormallung(A),alungwithreducedcompliance(B),andalungwithincreasedairwayresistance(C).Breakdownofthetrapezoidalareasenablesonetoappreciatetheindividualaspectsofthemechanicalworkload,whichincludethefollowing:OABCD,worknecessarytoovercomeelasticresistance;AECF,worknecessarytoovercomenonelasticresistance;AECB,worknecessarytoovercomenonelasticresistanceduringinspiration;ABCF,worknecessarytoovercomenonelasticresistanceduringexhalation(representsstoredelasticenergyfrominspiration). ventilation requires more flow resistance force to overcome) (

1	ventilation requires more flow resistance force to overcome) ( Fig. 22.9 ). Normal individuals and individuals with lung disease adopt respiratory patterns that minimize the work of breathing. For this reason, individuals with pulmonary fibrosis (increased elastic work) breathe more shallowly and rapidly, and those with obstructive lung disease (normal elastic work but increased resistive work) breathe more slowly and deeply. 60 50 40 Total work Elastic work Nonelastic work 20 10 30 10 20 30 40 Mechanical work (kg-m/min) Respiratory rate (no./min)

1	60 50 40 Total work Elastic work Nonelastic work 20 10 30 10 20 30 40 Mechanical work (kg-m/min) Respiratory rate (no./min) Chronic obstructive pulmonary disease (COPD)isageneraltermthatincludesdiseasessuchasemphysemaandchronicbronchitis.COPDmostcommonlyoccursinindividualswhosmoke,inwhompathologicchangesinthelungconsistentwithbothemphysemaandchronicbronchitiscancoexist.ForindividualswithCOPDinwhomemphysemaisamajorcomponent,theelastictissueinthealveolarandcapillarywallsisprogressivelydestroyed,whichresultsinincreasedlungcomplianceanddecreasedelasticrecoil.Thedecreaseinelasticrecoilresultsinmovementoftheequalpressurepointtowardthealveolusandprematureairwayclosure.ThisproducesairtrappingandincreasesinRV,FRC,andTLC.Airwayresistanceisalsoincreased.Theseincreasesinlungvolumesincreasetheworkofbreathingbystretchingtherespiratorymusclesanddecreasingtheirefficiency.

1	Inchronicbronchitis,accumulationofmucusandairwayinflammationcausetheequalpressurepointtomovetowardthealveolus,whichleadstoprematureairwayclosureandincreasesinRV,FRC,andTLC.Airwayresistanceandtheworkofbreathingareincreased,butlungcomplianceisnormal. Inrestrictivelungdiseasessuchaspulmonaryfibrosis,lungcomplianceisdecreased.Lungvolumesaredecreased,butflowratesarereasonablynormal.Someofthechangesinpulmonaryfunctionvaluesinobstructiveandrestrictivepulmonarydiseasesareshownin Table22.1 Inthethirdtrimesterofpregnancy,theenlargeduterusincreasesintraabdominalpressureandrestrictsmovementofthediaphragm.TheFRC,asaresult,decreases.Thischangeinlungvolumeresultsindecreasedlungcomplianceandincreasedairwayresistanceinotherwisehealthywomen. •Fig. 22.9 Effectoftherespiratoryrateontheelastic,nonelastic,andtotalmechanicalworkofbreathingatagivenlevelofalveolarventilation.Individualstendtoadopttherespiratoryrateatwhichthetotalworkofbreathingisminimal(arrow) forthosewithoutlungdisease. 1.

1	1. There are two major patterns of gas flow in the airways: turbulent and laminar. 2. Resistance to airflow is the change in pressure per unit of flow. Airway resistance varies with the inverse of the fourth power of the radius and is higher in turbulent than in laminar flow. The major site of airway resistance is the first eight airway generations. Airway resistance decreases with increases in lung volume and with decreases in gas density. Airways resistance is also regulated by neural and humoral agents. 3.

1	3. Pulmonary function tests (spirometry, flow-volume loop, body plethysmography) can detect abnormalities in lung function before individuals become symptomatic. Test results are compared with results obtained in normal individuals and vary with sex, ethnicity, age, and height. COPD is characterized by increases in lung volumes and airway resistance and by decreases in expiratory flow rates. Emphysema, a specific type of COPD, is further characterized by increased lung compliance. Restrictive lung diseases are characterized by decreases in lung volume, normal expiratory flow rates and resistance, and a marked decrease in lung compliance. 4.

1	4. The equal pressure point is the point at which the pressure inside and surrounding the airway is the same. The location of the equal pressure point is dynamic. Specifically, as lung volume and elastic recoil decrease, the equal pressure point moves toward the alveolus in normal individuals. In individuals with chronic obstructive pulmonary disease (COPD), the equal pressure point at any lung volume is closer to the alveolus. Expiratory flow limitation occurs at the equal pressure point. 5. Energy is expended during breathing to overcome the inherent mechanical properties of the lung. Respiratory muscle fatigue is the most common cause of respiratory failure. Individuals breathe at a respiratory rate to minimize work. For individuals with increased airway resistance, work is minimized by breathing at lower frequencies. For individuals with restrictive lung diseases, work is minimized by shallow breathing at high frequencies. 6.

1	6. The dynamic compliance of the lung is always less than the static compliance and increases during exercise, sighing, and yawning. Calverley PMA, Koulouris NG. Flow limitation and dynamic hyperinflation: key concepts in modern respiratory physiology. Eur Respir J. 2005;25:186-199. Crapo RO, et al. Reference spirometric values using techniques and equipment that meet ATS recommendations. Am Rev Respir Dis. 1981;123:659-664. Otis AB. A perspective of respiratory mechanics. J Appl Physiol. 1983;54:1183-1187. Otis AB, et al. Mechanics of breathing in man. J Appl Physiol. 1950;2:592-607. Leff AR, Schumacker PT. Respiratory Physiology: Basics and Applications. Philadelphia: Saunders; 1993. Lumb AB. Nunn’s Applied Respiratory Physiology. 8th ed. St. Louis: Elsevier; 2016. Mead J, MacKlem PT. Mechanics of breathing. In: Handbook of Physiology. Section 3, The Respiratory System. Bethesda, MD: American Physiological Society; 1986. Wiley Online Library doi:10.1002/cphys.cp0303fmo1.

1	Ventilation, Perfusion, and Ventilation/ Perfusion Relationships Upon completion of this chapter, the student should be able to answer the following questions: 1. Define two types of dead space ventilation, and describe how dead space ventilation changes with tidal volume. 2. Describe the composition of gas in ambient air, the trachea, and the alveolus, and understand how this composition changes with changes in oxygen fraction and barometric pressure. 3. Use the alveolar air equation to calculate the alveolar-arterial difference for oxygen (AaDO2). 4. Understand the alveolar carbon dioxide equation and identify how it changes with alterations in alveolar ventilation. 5. Compare the distribution of pulmonary blood flow to the distribution of ventilation. 6. List and define the four categories of hypoxia and the six causes of hypoxic hypoxia. 7. Distinguish the causes of hypoxic hypoxia on the basis of the response to 100% O2. 8. Describe the two causes of hypercapnia.

1	7. Distinguish the causes of hypoxic hypoxia on the basis of the response to 100% O2. 8. Describe the two causes of hypercapnia. he major determinant of normal gas exchange and thus the level of PO2 and PCO2 in blood is the relationship between ventilation (V̇ ) and perfusion (Q̇ ). This relationship is called the ventilation/perfusion (V̇ /Q̇ ) ratio. Ventilation is the process by which air moves in and out of the lungs. The incoming air is composed of a volume that fills the conducting airways (dead space ventilation) and a portion that fills the alveoli (alveolar ventilation). Minute (or total) ventilation (V̇ E) is the volume of air that enters or leaves the lung per minute:

1	Equation 23.1 where f is the frequency or number of breaths per minute and VT (also known as TV) is the tidal volume, or volume of air inspired (or exhaled) per breath. Tidal volume varies with age, sex, body position, and metabolic activity. In an average-sized adult at rest, tidal volume is 500 mL. In children, it is 3 to 5 mL/kg. Dead Space Ventilation: Anatomical and Physiological Dead space ventilation is ventilation to airways that do not participate in gas exchange. There are two types of dead space: anatomical dead space and physiological dead space. Anatomical dead space (VD) is composed of the volume of gas that fills the conducting airways: Equation 23.2 where V refers to volume and the subscripts T, D, and A refer to tidal, dead space, and alveolar. A “dot” above V denotes a volume per unit of time (n): Equation 23.3

1	Equation 23.2 where V refers to volume and the subscripts T, D, and A refer to tidal, dead space, and alveolar. A “dot” above V denotes a volume per unit of time (n): Equation 23.3 Equation 23.4 where V̇ E is the total volume of gas in liters expelled from the lungs per minute (also called exhaled minute volume), V̇ D is the dead space ventilation per minute, and V̇ A is alveolar ventilation per minute. In a healthy adult, the volume of gas contained in the conducting airways at functional residual capacity (FRC) is approximately 100 to 200 mL, in comparison with the 3 L of gas in an entire lung. The ratio of the volume of the conducting airways (dead space) to tidal volume represents the fraction of each breath that is “wasted” in filling the conducting airways. This volume is related to tidal volume (VT) and to exhaled minute ventilation (V̇ E) in the following way: Equation 23.5 CHAPTER 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships

1	Equation 23.5 CHAPTER 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships If the dead space volume is 150 mL and tidal volume increases from 500 to 600 mL for the same exhaled minute ventilation, what is the effect on dead space ventilation? = 0 3. × VE and, similarly, Increasing tidal volume is an effective way to increase alveolar ventilation (and thus normal blood gas values), as might occur during exercise or periods of stress. As tidal volume increases, the fraction of the dead space ventilation decreases for the same exhaled minute ventilation.

1	Dead space ventilation (VD) varies inversely with tidal volume (VT). The larger the tidal volume, the smaller the proportion of dead space ventilation. Normally, VD/VT is 20% to 30% of exhaled minute ventilation. Changes in dead space are important contributors to work of breathing. If the dead space increases, the individual must inspire a larger tidal volume to maintain normal levels of blood gases. This adds to the work of breathing and can contribute to respiratory muscle fatigue and respiratory failure. If metabolic demands increase (e.g., during exercise or with fever), individuals with lung disease may not be able to increase tidal volume sufficiently.

1	The second type of dead space is physiological dead space. Often in diseased lungs, some alveoli are perfused but not ventilated. The total volume of gas in each breath that does not participate in gas exchange is called the physiological dead space. This volume includes the anatomical dead space and the dead space secondary to perfused but unventilated alveoli. The physiological dead space is always at least as large as the anatomical dead space, and in the presence of disease, it may be considerably larger. Both anatomical and physiological dead space can be measured, but they are not measured routinely in the course of patient care. Composition of Air Inspiration brings ambient or atmospheric air to the alveoli, where O2 is taken up and CO2 is excreted. Ambient air is a

1	Composition of Air Inspiration brings ambient or atmospheric air to the alveoli, where O2 is taken up and CO2 is excreted. Ambient air is a In individuals with certain types of chronic obstructive pulmonary disease (COPD), such as emphysema, physiological dead space is increased. If dead space doubles, tidal volume must increase in order to maintain the same level of alveolar ventilation. If tidal volume is 500 mL and VD/VT is 0.25, then If VD increases to 250 mL in this example, tidal volume (VT) must increase to 625 mL to maintain a normal alveolar ventilation (i.e., VA = 375 mL): gas mixture composed of N2 and O2, with minute quantities of CO2, argon, and inert gases. The composition of this gas mixture can be described in terms of either gas fractions or the corresponding partial pressure.

1	Because ambient air is a gas, the gas laws can be applied, from which two important principles arise. The first is that when the components are viewed in terms of gas fractions (F), the sum of the individual gas fractions must equal one: Equation 23.6 1 0. = FN2 + FO2 + Fargon and other gases It follows, then, that the sum of the partial pressures (in millimeters of mercury) of a gas, also known as the gas tension (in torr), must be equal to the total pressure. Thus at sea level, where atmospheric pressure (also known as barometric pressure [Pb]) is 760 mm Hg, the partial pressures of the gases in air are as follows: Equation 23.7 Three important gas laws govern ambient air and alveolar ventilation. According to Boyle’s law, when temperature is constant, pressure (P) and volume (V) are inversely related; that is, Boyle’s law is used in the measurement of lung volumes (see

1	Boyle’s law is used in the measurement of lung volumes (see Fig. 21.4 ). Dalton’s law is that the partial pressure of a gas in a gas mixture is the pressure that the gas would exert if it occupied the total volume of the mixture in the absence of the other components. Eq. 23.7 is an example of how Dalton’s law is used in the lung. According to Henry’s law, the concentration of a gas dissolved in a liquid is proportional to its partial pressure. The second important principle is that the partial pressure of a gas (Pgas) is equal to the fraction of that gas in the gas mixture (Fgas) multiplied by the atmospheric (barometric) pressure: Equation 23.8 Ambient air is composed of approximately 21% O2 and 79% N2. Therefore, the partial pressure of O2 in inspired ambient air (PO2) is calculated as follows:

1	Equation 23.8 Ambient air is composed of approximately 21% O2 and 79% N2. Therefore, the partial pressure of O2 in inspired ambient air (PO2) is calculated as follows: Equation 23.9 where (FiO2) is the fraction of oxygen in inspired air. The partial pressure of O2, or oxygen tension, in ambient air at the mouth at the start of inspiration is therefore 159 mm Hg, or 159 torr. The O2 tension at the mouth can be altered in one of two ways: by changing the fraction of O2 in inspired air (FiO2) or by changing barometric pressure. Thus ambient O2 tension can be increased through the administration of supplemental O2 and is decreased at high altitude.

1	The partial pressure of O2 in ambient air varies with altitude. The highest and lowest points in the contiguous United States are Mount Whitney in Sequoia National Park/ Inyo National Forest (14,505 feet; barometric pressure, 437 mm Hg) and Badwater Basin in Death Valley National Park (282 feet; barometric pressure, 768 mm Hg). On Mount Whitney, the partial pressure of O2 in ambient air is calculated as follows: . = 92 mm Hg whereas in Death Valley Badwater Basin, the partial pressure of oxygen is calculated as follows: . = 161mm Hg Note that the FiO2 does not vary at different altitudes; only the barometric pressure varies. These differences in oxygen tension have profound effects on arterial blood gas values.

1	Note that the FiO2 does not vary at different altitudes; only the barometric pressure varies. These differences in oxygen tension have profound effects on arterial blood gas values. As inspiration begins, ambient air is brought into the nasopharynx and laryngopharynx, where it becomes warmed to body temperature and humidified. Inspired air becomes saturated with water vapor by the time it reaches the glottis. Water vapor exerts a partial pressure and dilutes the total pressure in which the other gases are distributed. Water vapor pressure at body temperature is 47 mm Hg. To calculate the partial pressures of O2 and N2 in a humidified mixture, the water vapor partial pressure must be subtracted from the total barometric pressure. Thus in the conducting airways, which begin in the trachea, the partial pressure of O2 is calculated as follows: Equation 23.10 = . = 150 mm Hg and the partial pressure of N2 is calculated similarly:

1	Equation 23.10 = . = 150 mm Hg and the partial pressure of N2 is calculated similarly: Equation 23.11 = 563 mm Hg Note that the total pressure remains constant at 760 mm Hg (150 + 563 + 47 mm Hg) and that the fractions of O2 and N2 are unchanged. Water vapor pressure, however, reduces the partial pressures of O2 and N2. Note also that in the calculation of the partial pressure of ambient air ( Eq. 23.9), water vapor is ignored, and ambient air is considered “dry.” The conducting airways do not participate in gas exchange. Therefore, the partial pressures of O2, N2, and water vapor remain unchanged in the airways until the air reaches the alveolus.

1	When the inspired air reaches the alveolus, O2 is transported across the alveolar membrane into the capillary bed, and CO2 moves from the capillary bed into the alveolus. The process by which this occurs is described in . At the end of inspiration and with the glottis open, the total pressure in the alveolus is atmospheric; thus, the partial pressures of the gases in the alveolus must equal the total pressure, which in this case is atmospheric. The composition of the gas mixture, however, is changed and can be described as follows:

1	Equation 23.12 1 0. = FO2 + FN2 + FH2O + FCO2 + Fargon and other gases where N2 and argon are inert gases, and therefore the fraction of these gases in the alveolus does not change from ambient fractions. The fraction of water vapor also does not change because the inspired gas is already fully saturated with water vapor and is at body temperature. As a consequence of gas exchange, however, the fraction of O2 in the alveolus decreases, and the fraction of CO2 in the alveolus increases. Because of changes in the fractions of O2 and CO2, the partial pressures exerted by these gases also change. The partial pressure of O2 in the alveolus (PAO2) is given by the alveolar gas equation, which is also called the ideal alveolar oxygen equation: Equation 23.13 CHAPTER 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships PO2 159 150 102 90 40 PCO2 0 040 40 46 PH2O, 37°C 0 47 47 47 47 PN2 601 563 571

1	Equation 23.13 CHAPTER 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships PO2 159 150 102 90 40 PCO2 0 040 40 46 PH2O, 37°C 0 47 47 47 47 PN2 601 563 571 PCO2, partial pressure of carbon dioxide; PH2O, partial pressure of water; PN2, partial pressure of nitrogen; PO2, partial pressure of oxygen; PTOTAL, partial pressure of all parameters; R, respiratory quotient. *PN2 is increased in alveolar gas by 1% because R is normally less than 1. †Ptotal is less in venous than in arterial blood because PO2 has decreased more than PCO2 has increased.

1	where PiO2 is the partial pressure of inspired O2, which is equal to the fraction of O2 (FiO2) multiplied by the barometric pressure (Pb) minus water vapor pressure (PH2O); PACO2 is the partial pressure of alveolar CO2; and R is the respiratory exchange ratio, or respiratory quotient. The respiratory quotient is the ratio of the amount of CO2 excreted (V̇ CO2) to the amount of O2 taken up (V̇ O2) by the lungs. This quotient is the amount of CO2 produced in relation to the amount of O2 consumed by metabolism and is dependent on caloric intake. The respiratory quotient varies between 0.7 and 1.0; it is 0.7 in states of exclusive fatty acid metabolism and 1.0 in states of exclusive carbohydrate metabolism. Under normal dietary conditions, the respiratory quotient is assumed to be 0.8. Thus the quantity of O2 taken up exceeds the quantity of CO2 that is released in the alveoli. The partial pressures of O2, CO2, and N2 from ambient air to the alveolus at sea level are shown in Table 23.1

1	Table 23.1 A similar approach can be used to calculate the estimated PACO2. The fraction of CO2 in the alveolus is a function of the rate of CO2 production by the cells during metabolism and the rate at which the CO2 is eliminated from the alveolus. This process of elimination of CO2 is known as alveolar ventilation. The relationship between CO2 production and alveolar ventilation is defined by the alveolar carbon dioxide equation: Equation 23.14 where V̇ CO2 is the rate of CO2 production by the body, V̇ A is alveolar ventilation per minute, and FACO2 is the fraction of CO2 in dry alveolar gas. This relationship demonstrates that the rate of elimination of CO2 from the alveolus is related to alveolar ventilation and to the fraction of CO2 in the alveolus. Like the partial pressure of any other gas (see Eq. 23.8 ), PACO2 is defined by the following: Equation 23.15 Substituting for FACO2 in the previous equation yields the following relationship: Equation 23.16

1	Eq. 23.8 ), PACO2 is defined by the following: Equation 23.15 Substituting for FACO2 in the previous equation yields the following relationship: Equation 23.16 This equation demonstrates several important relationships. First, there is an inverse relationship between the partial pressure of CO2 in the alveolus (PACO2) and alveolar ventilation per minute (V̇ A), regardless of the exhaled CO2. Specifically, if ventilation is doubled, PACO2 decreases by 50%. Conversely, if ventilation is decreased by half, the PACO2 doubles. Second, at a constant alveolar ventilation per minute (V̇ A), doubling of the metabolic production of CO2 (V̇ CO2) causes the PACO2 to double. The relationship between V̇ A and PACO2 is depicted in Fig. 23.1

1	Fig. 23.1 In normal lungs, PaCO2 is tightly regulated and maintained at 40 ± 2 mm Hg. Increases or decreases in PaCO2, particularly when associated with changes in arterial pH, have profound effects on cell function, including enzyme and protein activity. Specialized chemoreceptors monitor PaCO2 in the brainstem ( ), and exhaled minute ventilation ( Eq. 23.1 ) varies in accordance with the level of PaCO2. An acute increase in PaCO2 results in respiratory acidosis (pH < 7.35), whereas an acute decrease in PaCO2 results in respiratory alkalosis (pH > 7.45). Hypercapnia is defined as an elevation in PaCO2, and it occurs when CO2 production exceeds alveolar ventilation (hypoventilation). Conversely, hyperventilation occurs when alveolar ventilation exceeds CO2 production, and it decreases PaCO2 (hypocapnia).

1	• Fig. 23.1 The Alveolar Partial Pressure of Carbon Dioxide (PCO2; y-axis) as a Function of Alveolar Ventilation per Minute (VA; x-axis) • Fig. 23.2 Regional Distribution of Lung Volume, Including in the Lung. Each line corresponds to a given metabolic rate asso- Alveolar Size (Circles) and Location on the Pressure-Volume ciated with a constant production of CO2 CO2 isometabolic line).

1	Alveolar Size (Circles) and Location on the Pressure-Volume ciated with a constant production of CO2 CO2 isometabolic line). Curve of the Lung at Different Lung Volumes. Because the lungs are suspended in the upright position, the pleural pressure (Ppl) and translung pressure (PL) of lung units at the apex are greater than those at the base. These lung units are larger at any lung volume than are those at the base. The effect is greatest at residual volume (RV), less so at functional residual capacity (FRC), and absent at total lung capacity (TLC). Note also that because of their “location” on the pressure-volume curve, inspired air is differentially distributed to these lung units; those at the apex are less compliant and receive a smaller Normally, alveolar ventilation is controlled to maintain an alveolar PCO2 of approximately 40 mm Hg. Thus at rest, when VCO2 is approximately 250 mL/minute, alveolar ventilation of 5 L/minute results in an alveolar

1	PCO2 of 40 mm Hg. A 50% decrease in ventilation at rest (i.e., from 5 to 2.5 L/minute) results in doubling of alveolar PCO2. During exercise, CO2 production is increased (V̇ CO2 = 750 mL/min), and to maintain normal alveolar PCO2, ventilation must increase (in this case, to 15 L/ minute). Again, however, a 50% reduction in ventilation (from 15 to 7.5 L/minute) results in doubling of the alveolar PCO2. proportion of the inspired air than do the lung units at the base, which are more compliant (i.e., are represented at a steeper part of the Distribution of Ventilation

1	proportion of the inspired air than do the lung units at the base, which are more compliant (i.e., are represented at a steeper part of the Distribution of Ventilation Ventilation is not uniformly distributed in the lung, largely because of the effects of gravity. In the upright position, at most lung volumes, alveoli near the apex of the lung are more expanded than are alveoli at the base. Gravity pulls the lung downward and away from the chest wall. As a result, pleural pressure is lower (i.e., more negative) at the apex than at the base of the lung, and static translung pressure (PL = PA − Ppl) is increased; this results in an increase in alveolar volume at the apex. Because of the difference in alveolar volume at the apex and at the base of the lung (Fig.

1	23.2 ), alveoli at the lung base are represented along the steep portion of the pressure-volume curve, and they receive more of the ventilation (i.e., they have greater compliance). In contrast, the alveoli at the apex are represented closer to the top or flat portion of the pressure-volume curve. They have lower compliance and thus receive proportionately less of the tidal volume. The effect of gravity is less pronounced when a person is supine rather than upright, and it is less pressure-volume curve). Alveolar units with long time constants fill and empty slowly. Thus an alveolar unit with increased airway resistance or increased compliance takes longer to fill and longer to empty. In adults, the normal respiratory rate is approximately 12 breaths per minute, the inspiratory time is approximately 2 seconds, and the expiratory time is approximately 3 seconds. In normal lungs, this time is sufficient to approach volume equilibrium (

1	Fig. 23.3 ). In the presence of increased resistance or increased compliance, however, volume equilibrium is not reached. Adults with COPD have a very long time constant as a result when a person is supine rather than prone. This is because the diaphragm is pushed in a cephalad direction when a person is supine, and it affects the size of all of the alveoli. In addition to gravitational effects on the distribution of ventilation, ventilation in alveoli is not uniform. The reason for this is variable airway resistance (R) or compliance (C), and it is described quantitatively by the time constant (τ):

1	Equation 23.17 of an increase in resistance and, in the case of individuals with emphysema, an increase in compliance. As a result, such affected adults tend to breathe at a low respiratory rate. Imagine now what happens when individuals with COPD climb a flight of stairs. The increase in respiratory rate does not allow sufficient time for a full exhalation, and a process called dynamic hyperinflation occurs ( Fig. 23.4 ); lung volumes, which are already increased, increase further, the lung becomes less compliant, and the work of breathing is very high.

1	Fig. 23.4 ); lung volumes, which are already increased, increase further, the lung becomes less compliant, and the work of breathing is very high. CHAPTER 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships ˛=0.28 ˛=0.56 C=0.4 Decreased compliance C=0.8 Increased resistance R=0.7 R=1.4 R=0.7 ˛=1.12 • Fig. 23.4 Dynamic Hyperinflation. The total time for respiration (Ttot) is composed of the time for inspiration (Ti) and the time for exhalation (Te). When the respiratory rate increases (e.g., during exercise), Ttot decreases. In individuals with chronic obstructive pulmonary disease

1	C=0.8 Normal (COPD), the effect of the increase in Ttot on Te may not allow for complete emptying of the alveoli with a long time constant, and with each succeeding breath, there is an increase in the lung volume (air trapping). This increase in lung volume eventually results in such a degree of hyperinflation that the affected person is no longer able to do the work needed to overcome the decreased compliance of the lung at this high lung volume. In such individuals, it is a major cause of shortness of breath with activity. FRC, functional residual capacity.

1	• Fig. 23.3 Examples of Local Regulation of Ventilation as a Result of Variation in the Resistance (R) or Compliance (C) of Individual Lung Units. Top, The individual resistance and compliance values of three different lung units are illustrated. Bottom, The graph illustrates the volume of these three lung units as a function of time. In the upper schema, the normal lung has a time constant (τ) of 0.56 second. This lung unit reaches 97% of final volume equilibrium in 2 seconds, which is the normal inspiratory time. The lung unit at the right has a twofold increase in resistance; hence its time constant is doubled. That lung unit fills more slowly and reaches only 80% volume equilibrium during a normal inspiratory time (see graph); thus this lung unit is underventilated. The lung unit on the left has decreased compliance (is “stiff”), which acts to reduce its time constant. This lung unit fills quickly, reaching its maximum volume within 1 second, but receives only half the ventilation

1	left has decreased compliance (is “stiff”), which acts to reduce its time constant. This lung unit fills quickly, reaching its maximum volume within 1 second, but receives only half the ventilation of a normal lung unit.

1	Blood flow in the pulmonary circulation is pulsatile and influenced by pulmonary vascular resistance (PVR), gravity, alveolar pressure, and the arterial-to-venous pressure gradient. PVR is calculated as the change in pressure from the pulmonary artery (PPA) to the left atrium (PLA), divided by the flow (QT), which is cardiac output: Equation 23.18 Under normal circumstances, Equation 23.19 This resistance is about 10 times less than that in the systemic circulation. The pulmonary circulation has two unique features that allow increased blood flow on demand without an increase in pressure: (1) With increased demand, as during exertion or exercise, pulmonary vessels that are normally closed are recruited; and (2) the blood vessels in the pulmonary circulation are highly distensible, and their diameter increases with only a minimal increase in pulmonary arterial pressure.

1	Fig. 23.5). At end inspiration, the air-filled alveoli compress the alveolar capillaries and increase PVR. In contrast to the capillary beds in the systemic circulation, the capillary beds in the lungs account for approximately 40% of PVR. The diameters of the larger extra-alveolar vessels increase at end inspiration because of radial traction and elastic recoil, and their PVR is lower at higher lung volume. During exhalation, the deflated alveoli apply the least resistance to the alveolar capillaries and their PVR is diminished, whereas the higher pleural pressure during exhalation increases the PVR of extra-alveolar vessels. As a result of these opposite effects of lung volume on PVR, total PVR in the lung is lowest at FRC.

1	• Fig. 23.5 Schematic Representation of the Effects of Changes in Vital Capacity on Total Pulmonary Vascular Resistance and the Contributions to the Total Afforded by Alveolar and Extra-Alveolar Vessels. During inflation from residual volume (RV) to total lung capacity (TLC), resistance to blood flow through alveolar vessels increases, whereas resistance through extra-alveolar vessels decreases. Thus changes in total pulmonary vascular resistance are plotted as a U-shaped curve during lung inflation, with the nadir at functional residual capacity (FRC). Distribution of Pulmonary Blood Flow

1	Distribution of Pulmonary Blood Flow Because the pulmonary circulation is a low-pressure/lowresistance system, it is influenced by gravity much more dramatically than is the systemic circulation. This gravitational effect contributes to an uneven distribution of blood flow in the lungs. In normal upright persons at rest, the volume of blood flow increases from the apex of the lung to the base of the lung, where it is greatest. Similarly, in a supine individual, blood flow is least in the uppermost (anterior) regions and greatest in the lower (posterior) regions. Under conditions of stress, such as exercise, the difference in blood flow in the apex and base of the lung in upright persons becomes less, mainly because of the increase in arterial pressure.

1	On leaving the pulmonary artery, blood must travel against gravity to the apex of the lung in upright people. For every 1-cm increase in location of a pulmonary artery segment above the heart, there is a corresponding decrease in hydrostatic pressure equal to 0.74 mm Hg. Thus the pressure in a pulmonary artery segment that is 10 cm above the heart is 7.4 mm Hg less than the pressure in a segment at the level of the heart. Conversely, a pulmonary artery segment 5 cm below the heart has a 3.7–mm Hg increase in pulmonary arterial pressure. This effect of gravity on blood flow affects arteries and veins equally and results in wide variations in arterial and venous pressure from the apex to • Fig. 23.6 Model to Explain the Uneven Distribution of Blood Flow in the Lung According to the Pressures Affecting the Capillaries.

1	PA, pulmonary alveolar pressure; Pa, pulmonary arterial pressure; Pv, pulmonary venous pressure. (From West JB, et al. J Appl Physiol. 1964;19:713.) the base of the lung. These variations influence both flow and ventilation/perfusion relationships. In addition to the pulmonary arterial pressure (Pa) to pulmonary venous pressure (Pv) gradients, differences in pulmonary alveolar pressure (PA) also influence blood flow in the lung. Classically, the lung has been thought to be divided into three functional zones (

1	Fig. 23.6 ). Zone 1 represents the lung apex, where Pa is so low that it can be exceeded by PA. The capillaries collapse because of the greater external PA, and blood flow ceases. Under normal conditions, this zone does not exist; however, this state could be reached during positive-pressure mechanical ventilation or if Pa decreases sufficiently (such as might occur with a marked decrease in blood volume). In zone 2, or the upper third of the lung, Pa is greater than PA, which is in turn is greater than Pv. Because PA is greater than Pv, the greater external PA partially collapses the capillaries and causes a “damming” effect. This phenomenon is often referred to as the waterfall effect. In zone 3, Pa is greater than Pv, which is greater than PA, and blood flows in this area in accordance with the pressure gradients. Thus, pulmonary blood flow is greater in the base of the lung because the increased trans-mural pressure distends the vessels and lowers the resistance.

1	Active Regulation of Blood Flow Blood flow in the lung is regulated primarily by the passive mechanisms described previously. There are, however, several active mechanisms that regulate blood flow. Although the smooth muscle around pulmonary vessels is much thinner than that around systemic vessels, it is sufficient to affect vessel caliber and thus PVR. Oxygen levels have a major effect on blood flow. Hypoxic vasoconstriction occurs in arterioles in response to decreased PAO2. The response is local, and the result is the shifting of blood flow from CHAPTER 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships

1	CHAPTER 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships AT THE CELLULAR LEVEL • BOX 23.1 Factors and Hormones That Regulate Pulmonary Blood Flow hypoxic areas to well-perfused areas in an effort to enhance gas exchange. Isolated, local hypoxia does not alter PVR; approximately 20% of the vessels must be hypoxic before a change in PVR can be measured. Low inspired O2 levels as a result of high altitude have a greater effect on PVR because all vessels are affected. High levels of inspired O2 can dilate pulmonary vessels and decrease PVR. Other factors and some hormones (

1	Box 23.1 ) can also influence vessel caliber, but their effects are usually local, brief, and important only in pathological conditions. Pulmonary capillaries lack smooth muscle and are thus not affected by these mechanisms. In some individuals, as a consequence of chronic hypoxia or collagen vascular disease, or for no apparent reason, pulmonary artery vascular resistance and subsequently pulmonary artery pressures rise (pulmonary artery hypertension).

1	Both ventilation (V̇ ) and lung perfusion (Q̇ ) are essential components of normal gas exchange, but a normal relationship between the two components is insufficient to ensure normal gas exchange. The ventilation/perfusion ratio (also referred to as the V̇ /Q̇ ratio) is defined as the ratio of ventilation to blood flow. This ratio can be defined for a single alveolus, for a group of alveoli, or for the entire lung. At the level of a single alveolus, the ratio is defined as alveolar ventilation per minute (V̇ A) divided by capillary flow (Q̇ c). At the level of the lung, the ratio is defined as total alveolar ventilation divided by cardiac output. In normal lungs, alveolar ventilation is approximately 4.0 L/min, whereas pulmonary blood flow is approximately 5.0 L/min. Thus

1	Endothelin-1 is an amino acid peptide that is produced by the vascular endothelium. Endothelin regulates the tone of pulmonary arteries, and increased expression of endothelin-1 has been found in individuals with pulmonary artery hypertension. Endothelin-1 also decreases endothelial expression of nitric oxide synthase, which reduces levels of nitric oxide, an endothelial vasodilator. Endothelin-1 antagonists (e.g., bosentan, sitaxentan) have been produced and are important drugs in the treatment of pulmonary arterial hypertension.

1	in a normal lung, the overall ventilation/perfusion ratio is approximately 0.8, but the range of V̇ /Q̇ ratios varies widely in different lung units. When ventilation exceeds perfusion, the ventilation/perfusion ratio is greater than 1 (V̇ /Q̇ > 1), and when perfusion exceeds ventilation, the ventilation/ perfusion ratio is less than 1 (V̇ /Q̇ < 1). Mismatching of pulmonary blood flow and ventilation results in impaired O2 and CO2 transfer. In individuals with cardiopulmonary disease, mismatching of pulmonary blood flow and alveolar ventilation is the most frequent cause of systemic arterial hypoxemia (reduced PaO2). In general, V̇ /Q̇ ratios greater than 1 are not associated with hypoxemia.

1	A normal ventilation/perfusion ratio does not mean that ventilation and perfusion of that lung unit are normal; it simply means that the relationship between ventilation and perfusion is normal. For example, in lobar pneumonia, ventilation to the affected lobe is decreased. If perfusion to this area remains unchanged, perfusion would exceed ventilation; that is, the ventilation/perfusion ratio would be less than 1 (V̇ /Q̇ < 1). However, the decrease in ventilation to this area produces hypoxic vasoconstriction in the pulmonary arterial bed supplying this lobe. This results in a decrease in perfusion to the affected area and a more “normal” ventilation/perfusion ratio. Nonetheless, neither the ventilation nor the perfusion to this area is normal (both are decreased), but the relationship between the two could approach the normal range.

1	The ventilation/perfusion ratio varies in different areas of the lung. In an upright individual, although both ventilation and perfusion increase from the apex to the base of the lung, the increase in ventilation is less than the increase in blood flow. As a result, the normal V̇ /Q̇ ratio at the apex of the lung is much greater than 1 (ventilation exceeds perfusion), whereas the V̇ /Q̇ ratio at the base of the lung is much less than 1 (perfusion exceeds ventilation). The relationship between ventilation and perfusion from the apex to the base of the lung is depicted in

1	Fig. 23.7 • Fig. 23.7 Ventilation/Perfusion Relationships in a Normal Lung in the Upright Position. Only the apical and basal values are shown for clarity. In each column, the number on top represents values at the apex of the lung, and the number on the bottom represents values at the base. PCO2, partial pressure of carbon dioxide; PN2, partial pressure of nitrogen; PO2, partial pressure of oxygen; Q̇ , perfusion per minute; VA, alveolar ventilation per minute.

1	PACO2 and PaCO2 are equal because of the solubility properties of CO2 (see ). The same is not true for alveolar and arterial O2. Even in individuals with normal lungs, PAO2 is slightly greater than PaO2. The difference between PAO2 and PaO2 is called the alveolar-arterial difference for oxygen (AaDO2). An increase in the AaDO2 is a hallmark of abnormal O2 exchange. This small difference in healthy individuals is not caused by “imperfect” gas exchange, but by the small number of veins that bypass the lung and empty directly into the arterial circulation. The thebesian vessels of the left ventricular myocardium drain directly into the left ventricle (rather than into the coronary sinus in the right atrium), and some bronchial and mediastinal veins drain into the pulmonary veins. This results in venous admixture and a decrease in PaO2. (This is an example of an anatomical shunt; see the section .”) Approximately 2% to 3% of the cardiac output is shunted in this way.

1	To measure the clinical effectiveness of gas exchange in the lung, PaO2 and PaCO2 are measured. PAO2 is calculated from the alveolar air equation ( Eq. 23.13 ). The difference between the calculated PAO2 and the measured PaO2 is the AaDO2. In individuals with normal lungs who are breathing room air, the AaDO2 is less than 15 mm Hg. The mean value rises approximately 3 mm Hg per decade of life after 30 years of age. Hence, an AaDO2 lower than 25 mm Hg is considered the upper limit of normal. Assume that an individual with pneumonia is receiving 30% supplemental O2 by face mask. Arterial blood gas pH is 7.40, PaCO2 is 44 mm Hg, and PaO2 is 70 mm Hg. What is the patient’s AaDO2? (Assume that the patient is at sea level and the patient’s respiratory quotient is 0.8.) According to the alveolar air equation ( Eq. 23.13 R 44PAO2 = [0 3. × (760 − 47)] − 0 8. = 159 mm Hg Therefore, This high AaDO2 suggests that the patient has lung disease (in this case, pneumonia).

1	Eq. 23.13 R 44PAO2 = [0 3. × (760 − 47)] − 0 8. = 159 mm Hg Therefore, This high AaDO2 suggests that the patient has lung disease (in this case, pneumonia). Abnormalities in PaO2 can occur with or without an elevation in AaDO2. Hence, the relationship between PaO2 and AaDO2 is useful in determining the cause of an abnormal PaO2 and in predicting the response to therapy (particularly to supplemental O2 administration). Causes of a reduction in PaO2 (arterial hypoxemia) and their effect on AaDO2 are listed in Table 23.2 . Each of these causes is discussed in greater detail in the following sections. Arterial Blood Hypoxemia, Hypoxia, and Hypercarbia

1	Table 23.2 . Each of these causes is discussed in greater detail in the following sections. Arterial Blood Hypoxemia, Hypoxia, and Hypercarbia Arterial hypoxemia is defined as a PaO2 lower than 80 mm Hg in an adult who is breathing room air at sea level. Hypoxia is defined as insufficient O2 to carry out normal metabolic functions; hypoxia often occurs when the PaO2 is less than 60 mm Hg. There are four major categories of hypoxia. The first, hypoxic hypoxia, is the most common. The six main pulmonary conditions associated with hypoxic hypoxia—anatomical shunt, physiological shunt, decreased FiO2, V̇ /Q̇ mismatching, diffusion abnormalities, and hypoventilation—are described in the following sections and in

1	Table 23.2 . A second category is anemic hypoxia, which is caused by a decrease in the amount of functioning hemoglobin as a result of too little hemoglobin, abnormal hemoglobin, or interference with the chemical combination of oxygen and hemoglobin (e.g., carbon monoxide poisoning; see the following “ In the Clinic” box). The third category is hypoperfusion hypoxia, which results from low CHAPTER 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships AaDO2, alveolar-arterial difference for oxygen; FiO2, fraction of inspired oxygen; PaO2, partial pressure of arterial oxygen. blood flow (e.g., decreased cardiac output) and reduced oxygen delivery to the tissues. Histotoxic hypoxia, the fourth category of hypoxia, occurs when the cellular machinery that uses oxygen to produce energy is poisoned, as in cyanide poisoning. In this situation, arterial and venous PO2 are normal or increased because oxygen is not being utilized.

1	Carbon monoxide can be generated from a malfunctioning space heater, from car exhaust, or from a burning building. Individuals exposed to carbon monoxide experience headache, nausea, and dizziness, and if it is not recognized, such individuals may die. They often have a cherry-red appearance, and oxygen saturation as measured with an oximeter is high (approaching 100%). Even on an arterial blood gas, the PAO2 may be normal. Nevertheless, the tissues are depleted of O2. Thus it is imperative that the clinician recognize a potential case of carbon monoxide poisoning and order an oxygen saturation measurement with the use of a carbon monoxide oximeter. If a patient has carbon monoxide poisoning, there will be a marked difference between the measurement of oxygen saturation by oximetry and that measured with a carbon monoxide oximeter. A useful way to examine the relationship between ventila tion and perfusion is with the two–lung unit model (Fig.

1	23.8 ). Two alveoli are ventilated, each of which is supplied by blood from the heart. When ventilation is uniform, half the inspired gas goes to each alveolus, and when perfusion is uniform, half the cardiac output goes to each alveolus. In this normal unit, the ventilation/perfusion ratio in each of the alveoli is the same and is equal to 1. The alveoli are perfused by mixed venous blood that is deoxygenated and contains increased PaCO2. PAO2 is higher than mixed venous O2, and this provides a gradient for movement of O2 into blood. In contrast, mixed venous CO2 is greater than PACO2, and this provides a gradient for movement • Fig. 23.8 Simplified Lung Model of Two Normal Parallel Lung Units. Both units receive equal volumes of air and blood flow for their size. The blood and alveolar gas partial pressures are normal values in a resting person at sea level. PACO2, partial pressure of alveolar carbon dioxide; PAO2, partial pressure of alveolar oxygen; PiCO2, partial pressure of

1	gas partial pressures are normal values in a resting person at sea level. PACO2, partial pressure of alveolar carbon dioxide; PAO2, partial pressure of alveolar oxygen; PiCO2, partial pressure of inspired carbon dioxide; PiO2, partial pressure of inspired oxygen; PpvCO2, partial pressure of carbon dioxide in portal venous blood; PpvO2, partial pressure of oxygen in portal venous blood; PvCO2, partial pressure of carbon dioxide in mixed venous blood; PvO2, partial pressure of oxygen in mixed venous blood.

1	of CO2 into the alveolus. Note that in this ideal model, alveolar-arterial O2 values do not differ. An anatomical shunt occurs when mixed venous blood bypasses the gas-exchange unit and goes directly into the arterial circulation (

1	Fig. 23.9 ). Alveolar ventilation, the distribution of alveolar gas, and the composition of alveolar gas are normal, but the distribution of cardiac output is changed. Some of the cardiac output goes through the pulmonary capillary bed that supplies the gas-exchange units, but the rest of it bypasses the gas-exchange units and goes directly into the arterial circulation. The blood that bypasses the gas-exchange unit is thus shunted, and because the blood is deoxygenated, this type of bypass is called a right-to-left shunt. Most anatomical shunts develop within the heart, and they develop when deoxygenated blood from the right atrium or ventricle crosses the septum and mixes with blood from the left atrium or ventricle. The effect of this right-to-left shunt is to mix deoxygenated blood with oxygenated blood, and it results in varying degrees of arterial hypoxemia.

1	• Fig. 23.9 Right-to-Left Shunt. Alveolar ventilation is normal, but a portion of the cardiac output bypasses the lung and mixes with oxygenated blood. PaO2 varies according to the size of the shunt. PACO2, partial pressure of alveolar carbon dioxide; PAO2, partial pressure of alveolar oxygen; PiCO2, partial pressure of inspired carbon dioxide; PiO2, partial pressure of inspired oxygen; PpvCO2, partial pressure of carbon dioxide in portal venous blood; PpvO2, partial pressure of oxygen in portal venous blood; PvCO2, partial pressure of carbon dioxide in mixed venous blood; PvO2, partial pressure of oxygen in mixed venous blood.

1	An important feature of an anatomical shunt is that if an affected individual is given 100% O2 to breathe, the response is blunted severely. The blood that bypasses the gas-exchanging units is never exposed to the enriched O2, and thus it continues to be deoxygenated. The PO2 in the blood that is not being shunted increases and it mixes with the deoxygenated blood. Thus the degree of persistent hypoxemia in response to 100% O2 varies with the volume of the shunted blood. Normally, the hemoglobin in the blood that perfuses the ventilated alveoli is almost fully saturated. Therefore, most of the added O2 is in the form of dissolved O2 (see

1	The PaCO2 in an anatomical shunt is not usually increased even though the shunted blood has an elevated level of CO2. The reason for this is that the central chemoreceptors (see ) respond to any elevation in CO2 with an increase in ventilation and reduce PaCO2 to the normal range. If the hypoxemia is severe, the increased respiratory drive secondary to the hypoxemia increases the ventilation and can decrease PaCO2 to below the normal range. A physiological shunt (also known as venous admixture) can develop when ventilation to lung units is absent in the presence of continuing perfusion (

1	Fig. 23.10 ). In this situation, in the two–lung unit model, all the ventilation goes to the other lung unit, whereas perfusion is equally distributed between both lung units. The lung unit without ventilation but with perfusion has a V̇ /Q̇ ratio of 0. The blood perfusing this unit is mixed venous blood; because • Fig. 23.10 Schema of a Physiological Shunt (Venous Admixture). Notice the marked decrease in PaO2 in comparison to PCO2. The alveolar-arterial difference for oxygen (AaDO2) in this example is 85 mm Hg. PACO2, partial pressure of alveolar carbon dioxide; PAO2, partial pressure of alveolar oxygen; PiCO2, partial pressure of inspired carbon dioxide; PiO2, partial pressure of inspired oxygen; PpvCO2, partial pressure of carbon dioxide in portal venous blood; PpvO2, partial pressure of oxygen in portal venous blood; PvCO2, partial pressure of carbon dioxide in mixed venous blood; PvO2, partial pressure of oxygen in mixed venous blood.

1	there is no ventilation, no gas is exchanged in the unit, and the blood leaving this unit continues to resemble mixed venous blood. The effect of a physiological shunt on oxygenation is similar to the effect of an anatomical shunt; that is, deoxygenated blood bypasses a gas-exchanging unit and admixes with arterial blood. Clinically, atelectasis (which is obstruction to ventilation of a gas-exchanging unit with subsequent loss of volume) is an example of a situation in which the lung region has a V̇ /Q̇ of 0. Causes of atelectasis include mucous plugs, airway edema, foreign bodies, and tumors in the airway. Mismatching between ventilation and perfusion is the most frequent cause of arterial hypoxemia in individuals with respiratory disorders. In the most common example, the composition of mixed venous blood, total blood flow (cardiac output), and the distribution of blood flow are normal. However, when alveolar ventilation is distributed unevenly between the two gas-exchange units (

1	Fig. 23.11 ) and blood flow is equally distributed, the unit with decreased ventilation has a V̇ /Q̇ ratio of less than 1, whereas the unit with the increased ventilation has a V̇ /Q̇ of greater than 1. This causes the alveolar and end-capillary gas compositions to vary. Both the arterial O2 content and CO2 content are abnormal in the blood that has come from the unit with the decreased ventilation (V̇ /Q̇ , <1). The unit with the increased ventilation (V̇ /Q̇ , >1) has a lower CO2 content and a higher O2 content because it is being overventilated. The actual PaO2 and PaCO2 vary, depending on the relative contribution of each of these units to arterial blood. The alveolar-arterial O2 gradient (AaDO2) is increased because the relative overventilation of one unit does not fully

1	PlO2 = 150 PlCO2 = 0 PAO2 = 77 PACO2 = 45 PCO2= 45PO2= 77 PAO2 = 105 PACO2 = 36 PpvO2 = 89 PpvCO2 = 40.5 Pulmonary vein Pulmonary artery PvO2 = 40 PvCO2 = 46 – – Anatomic dead space • Fig. 23.11 Effects of Ventilation/Perfusion Mismatching on Gas Exchange. The decrease in ventilation to the one lung unit could be due to mucus obstruction, airway edema, bronchospasm, a foreign body, or a tumor. PACO2, partial pressure of alveolar carbon dioxide; PAO2, partial pressure of alveolar oxygen; PiCO2, partial pressure of inspired carbon dioxide; PiO2, partial pressure of inspired oxygen; PpvCO2, partial pressure of carbon dioxide in portal venous blood; PpvO2, partial pressure of oxygen in portal venous blood; PvCO2, partial pressure of carbon dioxide in mixed venous blood; PvO2, partial pressure of oxygen in mixed venous blood.

1	compensate (either by the addition of extra O2 or by the removal of extra CO2) for underventilation of the other unit. The failure to compensate is greater for O2 than for CO2, as indicated by the flatness of the upper part of the oxyhemoglobin dissociation curve, in contrast to the slope of the CO2 dissociation curve (see Chapter 24). In other words, increased ventilation increases PAO2, but it adds little extra O2 content to the blood because hemoglobin is close to being 100% saturated in the overventilated areas. This is not the case for CO2, for which the steeper slope of the CO2 curve indicates removal of more CO2 when ventilation increases. Thus inasmuch as CO2 moves by diffusion, then as long as a CO2 gradient is maintained, CO2 diffusion will occur.

1	The PAO2 is determined by a balance between the rate of O2 uptake and the rate of O2 replenishment by ventilation. Oxygen uptake depends on blood flow through the lung and the metabolic demands of the tissues. If ventilation decreases, PAO2 decreases, and PaO2 subsequently decreases. In addition, VA and PACO2 are directly but inversely related. When ventilation is halved, the PACO2 doubles and thus so does the PaCO2 (see Eq. 23.16 ). Ventilation insufficient to maintain normal levels of CO2 is called hypoventilation. Hypoventilation always decreases PaO2 and increases PaCO2. One of the hallmarks of hypoventilation is a normal AaDO2. Hypoventilation reduces PAO2, which in turn results in a decrease in PaO2. Because gas exchange is normal, the AaDO2 remains normal. Hypoventilation accompanies diseases associated with muscle weakness and is associated with drugs that reduce the respiratory drive. In the presence of hypoventilation, however, areas of atelectasis develop

1	Ventilation, Perfusion, and Ventilation/Perfusion Relationships rapidly; atelectasis creates regions with V̇ /Q̇ ratios of 0, and when this occurs, the AaDO2 rises.

1	Abnormalities in diffusion of O2 across the alveolar-capillary barrier could potentially result in arterial hypoxia. Equilibration between alveolar and capillary O2 and CO2 content occurs rapidly: in a fraction of the time that it takes for red blood cells to transit the pulmonary capillary network. Hence, diffusion equilibrium almost always occurs in normal people, even during exercise, when the transit time of red blood cells through the lung increases significantly. An increased AaDO2 attributable to incomplete diffusion (diffusion disequilibrium) has been observed in normal persons only during exercise at high altitude (≥10,000 feet). Even in individuals with an abnormal diffusion capacity, diffusion disequilibrium at rest is unusual but can occur during exercise and at altitude. Alveolar capillary block, or thickening of the air-blood barrier, is an uncommon cause of hypoxemia. Even when the alveolar wall is thickened, there is usually sufficient time for gas diffusion unless the

1	capillary block, or thickening of the air-blood barrier, is an uncommon cause of hypoxemia. Even when the alveolar wall is thickened, there is usually sufficient time for gas diffusion unless the red blood cell transit time is increased.

1	Mechanisms of Hypercapnia

1	Two major mechanisms account for the development of hypercapnia (elevated PCO2): hypoventilation and wasted, or increased, dead space ventilation. As noted previously, alveolar ventilation and alveolar CO2 are inversely related. When ventilation is halved, PACO2 and PaCO2 double. Hypoventilation always decreases PaO2 and increases PaCO2 and thereby results in a hypoxemia that responds to an enriched source of O2. Dead space ventilation is wasted, or increased, when pulmonary blood flow is interrupted in the presence of normal ventilation. This is most often caused by a pulmonary embolus that obstructs blood flow. The embolus halts blood flow to pulmonary areas with normal ventilation (V̇ /Q̇ =∞). In this situation, the ventilation is wasted because it fails to oxygenate any of the mixed venous blood. The ventilation to the perfused regions of the lung is less than ideal (i.e., there is relative “hypoventilation” to this area because in this situation, it receives all the pulmonary

1	venous blood. The ventilation to the perfused regions of the lung is less than ideal (i.e., there is relative “hypoventilation” to this area because in this situation, it receives all the pulmonary blood flow with “normal” ventilation). If compensation does not occur, PaCO2 increases and PaO2 decreases. Compensation after a pulmonary embolus, however, begins almost immediately; local bronchoconstriction occurs, and the distribution of ventilation shifts to the areas being perfused. As a result, changes in arterial CO2 and O2 content are minimized.

1	Effect of 100% Oxygen on Arterial Blood Gas Abnormalities One of the ways that a right-to-left shunt can be distinguished from other causes of hypoxemia is for the individual to breathe 100% O2 through a non-rebreathing face mask for approximately 15 minutes. When the individual breathes 100% O2, all of the N2 in the alveolus is replaced by O2. Thus the PAO2, according to the alveolar air equation ( Eq. 23.13), is calculated as follows: Equation 23.20 PAO2 =[1 0. × (Pb − PH2O)]− PaCO2 0 8. =[1 0 × (760 4 . − 47)]− 40 08

1	In a normal lung, the PAO2 rapidly increases, and it provides the gradient for transfer of O2 into capillary blood. This is associated with a marked increase in PaO2 (see 23.2 ). Similarly, over the 15-minute period of breathing enriched with O2, even areas with very low V̇ /Q̇ ratios develop high alveolar O2 pressure as the N2 is replaced by O2. In the presence of normal perfusion to these areas, there is a gradient for gas exchange, and the end-capillary blood is highly enriched with O2. In contrast, in the presence of a right-to-left shunt, oxygenation is not corrected because mixed venous blood continues to flow through the shunt and mix with blood that has perfused normal units. The poorly oxygenated blood from the shunt lowers the arterial O2 content and maintains the AaDO2. An elevated AaDO2 during a properly conducted study with 100% O2 signifies the presence of a shunt (anatomical or physiological); the magnitude of the AaDO2 can be used to quantify the proportion of the

1	elevated AaDO2 during a properly conducted study with 100% O2 signifies the presence of a shunt (anatomical or physiological); the magnitude of the AaDO2 can be used to quantify the proportion of the cardiac output that is being shunted.

1	The regional differences in ventilation and perfusion and the relationship between ventilation and perfusion were discussed earlier in this chapter. The effects of various physiological abnormalities (e.g., shunt, V̇ /Q̇ mismatch, and hypoventilation) on arterial O2 and CO2 levels were also described. In addition, however, it should be noted that because the V̇ /Q̇ ratio varies in different regions of the lung, the end-capillary blood coming from these regions has different O2 and CO2 levels. These differences are shown in

1	Fig. 23.7 , and they demonstrate the complexity of the lung. First, recall that the volume of the lung at the apex is less than the volume at the base. As previously described, ventilation and perfusion are less at the apex than at the base, but the differences in perfusion are greater than the differences in ventilation. Thus the V̇ /Q̇ ratio is high at the apex and low at the base. This difference in ventilation/perfusion ratios is associated with a difference in alveolar O2 and CO2 content between the apex and the base. The PAO2 is higher and the PACO2 is lower in the apex than in the base. This results in differences in end-capillary contents for these gases. End-capillary PO2 is lower, and, as a consequence, the O2 content is lower in end-capillary blood at the lung base than at the apex. In addition, there is significant variation in blood pH in the end capillaries in these regions because of the variation in CO2 content. During exercise, blood flow to the apex increases and

1	apex. In addition, there is significant variation in blood pH in the end capillaries in these regions because of the variation in CO2 content. During exercise, blood flow to the apex increases and becomes more uniform in the lung; as a result, the difference between the content of gases in the apex and in the base of the lung diminishes with exercise.

1	1. The volume of air in the conducting airways is called the anatomical dead space. Dead space ventilation varies inversely with tidal volume. The total volume of gas in each breath that does not participate in gas exchange is called the physiological dead space. It includes the anatomical dead space and the dead space secondary to ventilated but unperfused alveoli. 2. The sum of the partial pressures of a gas is equal to the total pressure. The partial pressure of a gas (Pgas) is equal to the fraction of the gas in the gas mixture (Fgas) multiplied by the total pressure (Ptotal). The conducting airways do not participate in gas exchange. Therefore, the partial pressures of O2, N2, and water vapor in humidified air remain unchanged in the airways until the gas reaches the alveolus. 3. The partial pressure of O2 in the alveolus is given by the alveolar air equation ( Eq. 23.13 ). This equation is used to calculate the AaDO2, a useful measurement of abnormal arterial O2. 4.

1	3. The partial pressure of O2 in the alveolus is given by the alveolar air equation ( Eq. 23.13 ). This equation is used to calculate the AaDO2, a useful measurement of abnormal arterial O2. 4. The relationship between CO2 production and alveolar ventilation is defined by the alveolar carbon Eq. 23.14 ). There is an inverse relationship between the PACO2 and VA, regardless of the exhaled quantity of CO2. In normal lungs, PaCO2 is tightly regulated to remain constant at around 40 mm Hg.

1	Eq. 23.14 ). There is an inverse relationship between the PACO2 and VA, regardless of the exhaled quantity of CO2. In normal lungs, PaCO2 is tightly regulated to remain constant at around 40 mm Hg. 5. Because of the effects of gravity, there are regional differences in ventilation and perfusion. The ventilation/perfusion (V̇ /Q̇ ) ratio is defined as the ratio of ventilation to blood flow. In a normal lung, the overall ventilation/perfusion ratio is approximately 0.8. When ventilation exceeds perfusion, the ventilation/perfusion ratio is greater than 1 (V̇ /Q̇ > 1), and when perfusion exceeds ventilation, the ventilation/perfusion ratio is less than 1 (V̇ /Q̇ < 1). The V̇ /Q̇ ratio at the apex of the lung is high (ventilation is increased in relation to very little blood flow), whereas the V̇ /Q̇ ratio at the base of the lung is low. In individuals with normal lungs who are breathing room air, the AaDO2 is less than 15 mm Hg; the upper limit of normal is 25 mm Hg.

1	CHAPTER 23 Ventilation, Perfusion, and Ventilation/Perfusion Relationships 6. The pulmonary circulation is a low-pressure, low-resistance system. Recruitment of new capillaries and dilation of arterioles without an increase in pressure are unique features of the lung and allow for adjustments during stress, as in the case of exercise. Pulmonary vascular resistance is the change in pressure from the pulmonary artery (PPA) to the left atrium (PLA), divided by cardiac output (QT). This resistance is about 10 times less than in the systemic circulation. Leff AR, Schumacker PT. Respiratory Physiology: Basics and Applications. Philadelphia: WB Saunders; 1993. Lumb AB. Nunn’s Applied Respiratory Physiology. 8th ed. St. Louis: Elsevier; 2016. Mead J, Macklem PT, vol eds. American Physiological Society Handbook of Physiology: The Respiratory System. Vol. 3. Mechanics. Bethesda, MD: American Physiological Society; 1986. 7.

1	Mead J, Macklem PT, vol eds. American Physiological Society Handbook of Physiology: The Respiratory System. Vol. 3. Mechanics. Bethesda, MD: American Physiological Society; 1986. 7. There are four categories of hypoxia (hypoxic hypoxia, anemic hypoxia, diffusion hypoxia, and histotoxic hypoxia) and six mechanisms of hypoxic hypoxia and hypoxemia: anatomical shunt, physiological shunt, decreased FiO2, V̇ /Q̇ mismatching, diffusion abnormalities, and hypoventilation. 8. There are two mechanisms of the development of hypercapnia: increase in dead space ventilation and hypoventilation. Wasserman K, Beaver WL, Whipp BI. Gas exchange theory and the lactic acidosis (anaerobic) threshold. Circulation. 1990;81(1 suppl):1114-1130. West JB. Ventilation/Blood Flow and Gas Exchange. 5th ed. New York: Blackwell Scientific; 1991. Upon completion of this chapter, the student should be able to answer the following questions: 1.

1	West JB. Ventilation/Blood Flow and Gas Exchange. 5th ed. New York: Blackwell Scientific; 1991. Upon completion of this chapter, the student should be able to answer the following questions: 1. Describe the basic gas diffusion principles and how they affect O2 and CO2 absorption and expiration. 2. Compare and contrast the chemical transport mechanisms of O2 and CO2 in blood. 3. Describe the basic principles and clinical significances of the O2 and CO2 dissociation curves. 4. Describe the chemical synthesis of H+ ions and their role in the regulation of acid-base balance. 5. Explain why the diffusion of O2 and that of CO2 are considered perfusion limited and that of CO is considered diffusion limited. 6. Compare and contrast factors that shift the oxyhemoglobin dissociation curve. 7. Explain the clinical significance of the differences in the oxyhemoglobin and carboxyhemoglobin dissociation curves. 8.

1	7. Explain the clinical significance of the differences in the oxyhemoglobin and carboxyhemoglobin dissociation curves. 8. Compare and contrast the chloride shift and the Haldane effect on CO2 transport.

1	8. Compare and contrast the chloride shift and the Haldane effect on CO2 transport. he respiratory and circulatory systems function together to transport oxygen (O2) from the lungs to the tissues to sustain normal cellular activity and to transport carbon dioxide (CO2) from the tissues to the lungs for expiration. CO2, a product of active cellular metabolism, is transported from the tissues via systemic veins to the lungs, where it is expired (Fig. 24.1 ). To enhance uptake and transport of these gases between the lungs and tissues, specialized mechanisms (e.g., binding of O2 and hemoglobin and HCO3 − transport of CO2) have evolved that enable O2 uptake and CO2 expiration to occur simultaneously. Moreover, these specialized mechanisms facilitate uptake of O2 and expiration of CO2. To understand the mechanisms involved in the transport of these gases, gas diffusion properties, as well as transport and delivery mechanisms, must be considered.

1	Gas movement throughout the respiratory system occurs predominantly via diffusion. The respiratory and circulatory systems contain several unique anatomical and physiological features to facilitate gas diffusion: (1) large surface areas for gas exchange (alveolar to capillary and capillary to tissue membrane) with short distances to travel, (2) substantial partial pressure gradient differences, and (3) gases with advantageous diffusion properties. Transport and delivery of O2 from the lungs to the tissue and vice versa for CO2 are dependent on basic gas diffusion laws. Diffusion of Gases From Regions of Higher to Lower Partial Pressure in the Lungs

1	Diffusion of Gases From Regions of Higher to Lower Partial Pressure in the Lungs The process of gas diffusion is passive and similar whether diffusion occurs in a gaseous or liquid state. The rate of diffusion of a gas through a liquid is described by Graham’s law, which states that the rate is directly proportional to the solubility coefficient of the gas and inversely proportional to the square root of its molecular weight. Calculation of the diffusion properties for O2 and CO2 reveals that CO2 diffuses approximately 20 times faster than O2. Rates of O2 diffusion from the lungs into blood and from blood into tissue, and vice versa for CO2, are predicted by Fick’s law of gas diffusion ( Fig. 24.2). The ratio AD:T represents the con ductance of a gas from the alveolus to blood. The diffusing capacity of the lung (DL) is its conductance (A•D/T) when considered for the entire lung; thus, with Fick’s equation, DL can be calculated as follows:

1	Equation 24.1 where V̇ gas = gas diffusion. Fick’s law of diffusion could be used to assess the diffusion properties of O2 in the lungs, except that the capillary partial pressure of oxygen cannot be measured. This limitation can be overcome with the use of carbon monoxide (CO) rather than O2. Because CO has low solubility in the capillary membrane, the rate of CO equilibrium across the capillary is slow, and the partial pressure of CO in capillary blood remains close to 0. In contrast, the solubility of CO in blood is high. Thus the only limitation for diffusion of CO is the alveolar-capillary membrane, and thus CO is a •Fig. 24.1 Oxygen (O2) and Carbon Dioxide (CO2) Transport in Arterial and Venous Blood. Oxygeninarterialbloodistransferredfromarterialcapillariestotissues.TheflowratesforO2andCO2areshownfor1Lofblood.

1	•Fig. 24.2 AccordingtoFick’slaw,diffusionofagasacrossasheetoftissue(Vgas)isdirectlyrelatedtothesurfacearea(A)ofthetissue, thediffusionconstant(D)ofthespecificgas,andthepartialpressuredifference(P1− P2)ofthegasoneachsideofthetissue,anditisinverselyrelatedtotissuethickness(T). useful gas for calculating DL. The capillary partial pressure (P2 in Eq. 24.1 ) is essentially 0 for CO, and therefore DL can be measured from the diffusion of carbon monoxide (V̇ CO) and the average partial pressure of CO in the alveolus; that is, Equation 24.2 where DLCO = diffusion capacity of the lung for carbon monoxide. Assessment of DLCO has become a classic measurement of the diffusion barrier of the alveolar-capillary membrane. It is useful in the differential diagnosis of certain obstructive lung diseases, such as emphysema.

1	Apatientwithinterstitialpulmonaryfibrosis(arestrictivelungdisease)inhalesasinglebreathof0.3%COfromresidualvolumetototallungcapacity.Heholdshisbreathfor10secondsandthenexhales.Afterdiscardingtheexhaledgasfromthedeadspace,arepresentativesampleofalveolargasfromlateinexhalationiscollected.TheaveragealveolarCOpressureis0.1mmHg,and0.25mLofCOhasbeentakenup.ThediffusioncapacityforCOinthispatientis . mL/10 seconds × 0 1. mm Hg = 15 mL/minute/mm Hg ThenormalrangeforDLCOis20to30mL/minute/mmHg.Patientswithinterstitialpulmonaryfibrosishaveaninitialalveolarinflammatoryresponsewithsubsequentscarformationwithintheinterstitialspace.Theinflammationandscarreplacethealveolianddecreasethesurfaceareaforgasdiffusiontooccur,whichresultsindecreasedDLCO.Thisisaclassiccharacteristicofcertaintypesofrestrictivelungdisease. Oxygen and Carbon Dioxide Exchange in the Lung Is Perfusion Limited

1	Different gases have different solubility factors. Gases that are insoluble in blood (i.e., anesthetic gases such as nitrous oxide and ether) do not chemically combine with proteins in blood and equilibrate rapidly between alveolar gas and blood. The equilibration occurs in less time than the 0.75 seconds that the red blood cell spends in the capillary bed (capillary transit time). The diffusion of insoluble gases between alveolar gas and blood is considered perfusion limited because the partial pressure of gas in the blood leaving the capillary has reached equilibrium with alveolar gas and is limited only by the amount of blood perfusing the alveolus. In contrast, a gas that is diffusion limited, such as CO, has low solubility in the alveolar-capillary membrane but high solubility in blood because of its high affinity for hemoglobin (Hgb). These features prevent the equilibration of CO between alveolar gas and blood during the red blood cell transit time.

1	The high affinity of CO for Hgb enables large amounts of CO to be taken up in blood with little or no appreciable increase in its partial pressure. Gases that are chemically bound to Hgb do not exert a partial pressure in blood. Like CO, both CO2 and O2 have relatively low solubility in the alveolar-capillary membrane but high solubility in blood because of their ability to bind to Hgb. However, their rate of equilibration is sufficiently rapid for complete equilibration to occur during the transit time of the red blood cell within the capillary. Equilibration for O2 and CO2 usually occurs within 0.25 seconds. Thus O2 and CO2 transfer is normally perfusion limited. The partial pressure of a gas that is diffusion limited (i.e., CO) does not reach equilibrium with the alveolar pressure over the time that it spends in the capillary (

1	Fig. 24.3 ). Although CO2 has a greater rate of diffusion in blood than O2 does, it has a lower membrane-blood solubility ratio and consequently takes approximately the same amount of time to reach equilibration in blood. Start of End of 0 .25 .50 .75 Time in capillary (sec) •Fig. 24.3 Uptake of Nitrous Oxide (N2O), Carbon Monoxide (CO), and O2in Blood in Relation to Their Partial Pressures and the Transit Time of the Red Blood Cell in the Capillary. Forgasesthatareperfusionlimited(N2OandO2),theirpartialpressureshaveequilibratedwithalveolarpressurebeforeexitingthecapillary.Incontrast,thepartialpressureofCO,agasthatisdiffusionlimited,doesnotreachequilibriumwithalveolarpressure.Inrareconditions,O2uptakecanbecomediffusionlimited. Diffusion limitation for O2 and CO2 would occur if red blood cells spent less than 0.25 seconds in the capillary bed. This is occasionally the case in very fit athletes during vigorous exercise and in healthy subjects who exercise at high altitude.

1	Oxygen is carried in blood in two forms: dissolved O2 and O2 bound to Hgb. The dissolved form is measured clinically in an arterial blood gas sample as the partial pressure of arterial oxygen (PaO2). Only a small percentage of O2 in blood is in the dissolved form, and its contribution to O2 transport under normal conditions is almost negligible. However, dissolved O2 can become a significant factor in conditions of severe hypoxemia. Binding of O2 to Hgb to form oxyhemoglobin within red blood cells is the primary transport mechanism of O2. Hgb not bound to O2 is referred to as deoxyhemoglobin or reduced Hgb. The O2-carrying capacity of blood is enhanced about 65 times by its ability to bind to Hgb.

1	Hgb is the major transport molecule for O2. The Hgb molecule is a protein with two major components: four nonprotein heme groups, each containing iron in the reduced ferric (Fe+++) form, which is the site of O2 binding, and a globin portion consisting of four polypeptide chains. Normal adults have two α-globin chains and two β-globin chains (HgbA), whereas children younger than 6 months of age have predominantly fetal Hgb (HgbF), which consists of two α chains and two γ chains. This difference in the structure of HgbF increases its affinity for O2 and aids in the transport of O2 across the placenta. In addition, HgbF is not inhibited by 2,3-diphosphoglycerate (2,3DPG), a product of glycolysis; thus O2 uptake is further enhanced.

1	Binding of O2 to Hgb alters the ability of Hgb to absorb light. This effect of O2 on Hgb is responsible for the change in color between oxygenated arterial blood (bright red) and deoxygenated venous blood (dark red-bluish). Binding and dissociation of O2 with Hgb occur in milliseconds, thus facilitating O2 transport because red blood cells spend only 0.75 seconds in the capillaries. There are approximately 280 million Hgb molecules per red blood cell, which provides an efficient mechanism to transport O2. Myoglobin, a protein similar in structure and function to Hgb, has only one subunit of the Hgb molecule. It aids in the transfer of O2 from blood to muscle cells and in the storage of O2, which is especially critical in O2-deprived conditions.

1	Abnormalities of the Hgb molecule occur with mutations in the amino acid sequence (i.e., sickle cell disease) or in the spatial arrangement of the globin polypeptide chains and result in abnormal function. Compounds such as CO, nitrites (nitric oxide), and cyanides can oxidize the •Fig. 24.4 Oxyhemoglobin Dissociation Curve Showing the Relationship Between the Partial Pressure of Oxygen (Po2) in Blood and the Percentage of Hemoglobin (Hgb) Binding Sites That Are Occupied by Oxygen Molecules (Percentage Saturation). Adulthemoglobin(HgbA)isabout50%saturatedwithoxygenataPO2of27mmHg,90%saturatedat60mmHg,andabout98%saturatedat100mmHg.TheP50isthepartialpressureatwhichHgbis50%saturatedwithO2.WhentheO2dissociationcurveshiftstotheright,P50increases.Whenthecurveshiftstotheleft,P50decreases. iron molecule in the heme group and change it from the reduced ferrous state (Fe++) to the ferric state (Fe+++), which reduces the ability of O2 to bind to Hgb.

1	iron molecule in the heme group and change it from the reduced ferrous state (Fe++) to the ferric state (Fe+++), which reduces the ability of O2 to bind to Hgb. In the alveoli, the majority of O2 in plasma quickly diffuses into red blood cells and chemically binds to Hgb. This process is reversible, so that Hgb quickly gives up its O2 to tissue through passive diffusion (the concentration of O2 in Hgb decreases). The oxyhemoglobin dissociation curve illustrates the relationship between PO2 in blood and the number of O2 molecules bound to

1	Hgb (Fig. 24.4 ). The S shape of the curve demonstrates the dependence of Hgb saturation on PO2, especially at partial pressures lower than 60 mm Hg. The clinical significance of the flat portion of the oxyhemoglobin dissociation curve (>60 mm Hg) is that a drop in PO2 over a wide range of partial pressures (100 to 60 mm Hg) has a minimal effect on Hgb saturation, which remains at 90% to 100%, a level sufficient for normal O2 transport and delivery. The clinical significance of the steep portion (<60 mm Hg) of the curve is that a large amount of O2 is released from Hgb with only a small change in PO2, which facilitates the release and diffusion of O2 into tissue. The point on the curve at which Hgb is 50% saturated with O2 is called the P50, and it is 27 mm Hg in normal adults.

1	˜˜˜˜°°°°Temperature PCO2 2,3-DPG pH Decreased P50 (increased affinity) Temperature PCO2 2,3-DPG pH Increased P50 (decreased affinity) •Fig. 24.5 Factorsthatshifttheoxyhemoglobindissociationcurvetotheright(decreasedaffinityofHgbforO2)ortotheleft(increasedaffinity).2,3-DPG,2,3-diphosphoglycerate;PCO2,partialpressureofcarbondioxide. Physiological Factors That Shift the Oxyhemoglobin Dissociation Curve The oxyhemoglobin dissociation curve can shift in numerous clinical conditions, either to the right or to the left ( Fig. 24.5 ). The curve is shifted to the right when the affinity of

1	Intheinheritedhomozygousconditionknownassickle cell disease, affectedindividualshaveanaminoacidsubstitution(valineforglutamicacid)ontheβ chainoftheHgbmolecule.ThiscreatesasicklecellHgb(HgbS),which,whennotboundtooxygen(deoxyhemoglobinordesaturatedHgb),cantransformintoastiffgelatinousmaterialthatdistortsthenormalbiconcaveshapeoftheredbloodcelltoacrescent,orsickle-shaped,form.Thischangeinappearancefromsphericaltoasickleshapeincreasesthetendencyoftheredbloodcelltoformthrombiorclotsthatobstructsmallvesselsandcreatesaclinicalconditionknownasacute sickle cell episode.

1	sickle cell episode. Thesymptomsofsuchanepisodevary,dependingonthesiteoftheobstruction(e.g.,inthebrain,stroke;inthelungs,pulmonaryinfarction)andarecommonlyassociatedwithintensepain.Thespleenisacommonsiteofobstruction/infarction,andtheensuingtissuedamagecompromisestheimmunecapabilitiesofaffectedindividualsandrendersthemsusceptibletorecurrentinfections.Inthehomozygousform,thisconditionislife-shortening;however,individualswiththeheterozygousformareresistanttomalaria.Thusanindividualwithheterozygousalleleshasasurvivaladvantageinregionsoftheworldwheremalariaisprevalent,whichmayexplainwhythesicklecellmutationhasbeenpreservedthroughevolution.TheincreasedaffinityofHgbFforO2confersadvantagestoindividualswithsicklecelldiseaseinthatthecellsdonotdesaturateasmuchwhenO2isreleasedfromHgbtothetissueandthusarelesslikelytobecomedeformedinthesickleshape.SicklecelldiseaseismostprevalentamongindividualsofAfricanAmericandescent,butitisalsoobservedinHispanic,Turkish,Asian,andotherethnicgroups.

1	Hgb for O2 decreases, which enhances O2 dissociation. This results in decreased Hgb binding to O2 at a given PO2, which causes the P50 to increase. When the affinity of Hgb for O2 increases, the curve is shifted to the left, which causes the P50 to decrease. In this state, O2 dissociation and delivery to tissue are inhibited. Shifts to the right or left of the dissociation curve have little effect when they occur at O2 partial pressures within the normal range (80 to 100 mm Hg). However, at O2 partial pressures below 60 mm Hg (steep part of the curve), shifts in the oxyhemoglobin dissociation curve can dramatically influence O2 transport.

1	Changes in blood hydrogen ion concentration (pH) shift the oxyhemoglobin dissociation curve. An increase in CO2 production by tissue and its release into blood results in the generation of hydrogen ions (H+) and a decrease in pH. This shifts the dissociation curve to the right, which has a beneficial effect by aiding in the release of O2 from Hgb for diffusion into tissues. The shift to the right in the dissociation curve is due to the decrease in pH and to a direct effect of CO2 on Hgb. Conversely, as blood passes through the lungs, CO2 is exhaled, which results in an increase in pH, which in turn causes the oxyhemoglobin dissociation curve to shift to the left. This effect of CO2 on the affinity of Hgb for O2 is known as the Bohr effect, and it serves to enhance O2 uptake in the lungs and delivery of O2 to tissues. An increase in body temperature, as occurs during exercise, shifts the oxyhemoglobin dissociation curve to the right and enables more O2 to be released to tissues, where

1	delivery of O2 to tissues. An increase in body temperature, as occurs during exercise, shifts the oxyhemoglobin dissociation curve to the right and enables more O2 to be released to tissues, where it is needed because the demand increases. During cold weather, a decrease in body temperature, especially in the extremities (lips, fingers, toes, and ears), shifts the O2 dissociation curve to the left (higher Hgb affinity). In this instance, PaO2 may be normal, but release of O2 in these extremities is not facilitated.

1	2,3-Diphosphoglycerate Mature red blood cells do not have mitochondria, and therefore they depend on anaerobic glycolysis. Large quantities of a metabolic intermediary, 2,3-DPG, are formed in red blood cells during glycolysis, and the affinity of Hgb for O2 decreases as 2,3-DPG levels increase. Thus the oxyhemoglobin dissociation curve shifts to the right. Although the binding sites of 2,3-DPG and O2 differ on the Hgb molecule, binding of 2,3-DPG creates an allosteric effect that inhibits the binding of O2. Conditions that increase 2,3-DPG include hypoxia, decreased Hgb, and increased pH. Decreased levels of 2,3-DPG are observed in stored blood samples and thus may present a problem to transfusion recipients because of the greater affinity of Hgb for O2, which inhibits the unloading of O2 to tissues. As discussed previously, HgbF has a greater affinity for O2 than does adult Hgb, and the oxyhemoglobin dissociation curve thus shifts to the left.

1	As discussed previously, HgbF has a greater affinity for O2 than does adult Hgb, and the oxyhemoglobin dissociation curve thus shifts to the left. •Fig. 24.6 Theoxyhemoglobinandcarboxyhemoglobindissociationcurvesclearlyillustratetheincreasedaffinitythatcarbonmonoxide(CO)hasforHgb,incomparisonwithO2.100 80 60 40 20 0 0 20 40 60 PO2 (mm Hg) Hemoglobin saturation (%) 80 100 O2 + hemoglobin CO + hemoglobin CO binds to the heme group of the Hgb molecule at the same site as O2 and forms carboxyhemoglobin (HgbCO). A major difference between the ability of CO and that of O2 to bind to Hgb is illustrated by a comparison of the oxyhemoglobin and carboxyhemoglobin dissociation curves. The affinity of CO for Hgb is about 200 times greater than it is for O2 (

1	Fig. 24.6 ). Thus small amounts of CO can greatly influence the binding of O2 to Hgb. In the presence of CO, the affinity of Hgb for O2 is enhanced. This causes the dissociation curve to shift to the left, which further prevents the unloading and delivery of O2 to tissues. As the PCO of blood approaches 1.0 mm Hg, all of the Hgb binding sites are occupied by CO, and Hgb is unable to bind to O2. This situation is not compatible with life and is the cause of death in cases of CO poisoning. In healthy individuals, HgbCO occupies about 1% to 2% of the Hgb binding sites; however, in cigarette smokers and in individuals who reside in high-density urban traffic areas, occupation of Hgb binding sites can be increased to 10%. Levels above 5% to 7% are considered hazardous. Treatment of individuals with high levels of CO, such as those who have inhaled car exhaust or smoke from a burning building, consists of administering high concentrations of O2 to displace CO from Hgb. Increasing the

1	with high levels of CO, such as those who have inhaled car exhaust or smoke from a burning building, consists of administering high concentrations of O2 to displace CO from Hgb. Increasing the ambient pressure above atmospheric pressure, through the use of a barometric chamber, substantially increases the O2 tension, which promotes the dissociation of CO from Hgb. Another gas, nitric oxide, has great affinity (200,000 times greater than O2) for Hgb, and it binds irreversibly to Hgb at the same site that O2 does. Endothelial cells synthesize nitric oxide, which has vasodilation properties. Thus nitric oxide is used therapeutically as an inhalant in patients with pulmonary hypertension to reduce pressure. Although nitric oxide poisoning is not common, the clinician should be cautious when administering nitric oxide therapy for long periods. Hgb-bound CO and nitric oxide is referred to as methemoglobin. Under normal conditions, about 1% to 2% of Hgb is bound to CO and nitric oxide.

1	Oxygen Saturation, Content, and Delivery Each Hgb molecule can bind up to four O2 atoms, and each gram of Hgb can bind up to 1.34 mL of O2. The term O2 saturation (SO2) refers to the amount of O2 bound to Hgb in relation to the maximal amount of O2 (100% O2 capacity) that can bind Hgb. At 100% O2 capacity, the heme groups of the Hgb molecules are fully saturated with O2, and at 75% O2 capacity, three of the four heme groups are occupied. Binding of O2 to each heme group increases the affinity of the Hgb molecule to bind additional O2. The O2 content in blood is the sum of the O2 bound to Hgb and the dissolved O2. Oxygen content is decreased in the presence of increased CO2 and CO and in individuals with anemia ( Fig. 24.7

1	Fig. 24.7 Oxygen delivery from the lungs to tissues is dependent on several factors, including cardiac output, the Hgb content of blood, and the ability of the lung to oxygenate the blood. Not all of the O2 carried in blood is unloaded at the tissue level. The actual O2 extracted from blood by the tissue is the difference between the arterial O2 content and the venous O2 content, multiplied by cardiac output. Under normal conditions, Hgb leaves the tissue 75% saturated with O2, and only about 25% is actually used by tissues. Hypothermia, relaxation of skeletal muscles, and an increase in cardiac output reduce O2 extraction. Conversely, a decrease in cardiac output, anemia, hyperthermia, and exercise increase O2 extraction.

1	•Fig. 24.7 AcomparisonofO2contentcurvesunderthreeconditionsshowswhycarboxyhemoglobin(HgbCO)dramaticallyreducestheO2transportsystem.FiftypercentHgbCOrepresentsthebindingofhalfthecirculatingHgbwithcarbonmonoxide(CO).The50%hemoglobinand50%HgbCOcurvesshowthesamedecreasedO2contentinarterialblood.However,COhasaprofoundeffectinloweringvenouspartialpressureofoxygen.Thearterial(a)andmixedvenous(v)pointsofconstantcardiacoutputareindicated.

1	Tissue hypoxia is a condition in which O2 available to cells is insufficient for maintaining adequate aerobic metabolism. Thus anaerobic metabolism is stimulated and results in the increases in levels of lactate and H+ and the subsequent formation of lactic acid. The net result can lead to a significant decrease in blood pH. In cases of severe hypoxia, the extremities, toes, and fingertips may appear blue-gray (cyanotic) because of lack of O2 and increased deoxyhemoglobin levels. There are four major types of tissue hypoxia (hypoxic hypoxia, circulatory hypoxia, anemic hypoxia, histotoxic hypoxia), discussed in detail in Chapter 23.

1	Chapter 23. Tissue oxygenation depends on the concentration of Hgb and thus on the number of red blood cells available in the circulation. Red blood cell production (erythropoiesis) in the bone marrow is controlled by the hormone erythropoietin, which is synthesized in the kidneys by cortical interstitial cells. Although Hgb levels are normally very stable, decreased O2 delivery, low Hgb concentration, and low PaO2 stimulate the secretion of erythropoietin. This increases the production of red blood cells. Chronic renal disease damages the cortical interstitial cells and thereby suppresses their ability to synthesize erythropoietin. This causes anemia, along with decreased Hgb because of the lack of erythropoietin. Erythropoietin replacement therapy using epoetin alfa (Epogen, Procrit) or darbepoetin alfa (Aranesp) effectively increases red blood cell production.

1	CO2 is produced at a rate of approximately 200 mL/minute under healthy conditions, and typically, 80 molecules of CO2 are expired by the lung for every 100 molecules of O2 that enter the capillary bed. The ratio of expired CO2 to O2 uptake is referred to as the respiratory exchange ratio and, under normal conditions, is 0.8 (80 molecules of CO2 to 100 molecules of O2). This ratio is similar at the tissue to blood compartment, where it is referred to as the respiratory quotient. The body has enhanced storage capabilities for CO2, in comparison with O2, and hence PaO2 is much more sensitive to changes in ventilation than is PaCO2. Whereas PaO2 is dependent on several factors, in addition to alveolar ventilation, arterial PaCO2 is solely dependent on alveolar ventilation and CO2 production. There is an inverse relationship between alveolar ventilation and PaCO2. In blood, CO2 is transported in red blood cells primarily as bicarbonate (HCO3 −) but also as dissolved CO2 and as

1	In blood, CO2 is transported in red blood cells primarily as bicarbonate (HCO3 −) but also as dissolved CO2 and as H2CO3 = PCO2 ˜ 0.0301 •Fig. 24.8 Mechanisms of CO2Transport in Blood. v. ThepredominantmechanismbywhichCO2istransportedfromtissuecellstothelungisintheformofbicarbonateanion(HCO3−).H2CO3,carbonicacid;HgbO,oxyhemoglobin;NaHCO3,sodiumbicarbonate;RBC,redbloodcell. carbamino protein complexes (i.e., CO2 binds to plasma proteins and to Hgb; Fig. 24.8 ). Once CO2 diffuses through the tissue and enters plasma, it quickly dissolves. The reaction of CO2 with H2O to form carbonic acid (H2CO3) is the major mechanism for the generation of HCO3 − in red blood cells: Equation 24.3

1	Equation 24.3 The reaction normally proceeds quite slowly; however, it is catalyzed within red blood cells by the enzyme carbonic anhydrase. The HCO3 − diffuses out of the red blood cell in exchange for Cl−, in a process known as the chloride shift, which helps the cell maintain its osmotic equilibrium. This chemical reaction (see Fig. 24.8 ) is reversible and can be shifted to the right to generate more HCO3 − in response to more CO2 entering the blood from tissues, or it can be shifted to the left as CO2 is exhaled in the lungs, which reduces HCO3 − levels. The free H+ is quickly buffered within the red blood cell by binding to Hgb. Buffering of H+ is critical for keeping the reaction moving toward the synthesis of HCO3; high levels of free H+ (low pH) cause the reaction to shift to the left. Regulation of Hydrogen Ion Concentration and Acid-Base Balance

1	Regulation of Hydrogen Ion Concentration and Acid-Base Balance The H+ concentration (pH) has a dramatic effect on many metabolic processes within cells, and regulation of pH is essential for normal homeostasis. In the clinical setting, blood pH is measured to assess the concentration of H+ . The normal pH range for adults is 7.35 to 7.45 and is maintained by the lungs, kidneys, and chemical buffer systems. In the respiratory system, conversion of CO2 to HCO3 − , illustrated as follows, is a major mechanism of buffering and regulating the H+ concentration (pH): Equation 24.4 As PACO2 changes, so does the concentration of HCO3 − and H2CO3, as well as PaCO2. The Henderson-Hasselbalch equation is used to calculate how changes in CO2 and HCO3 − affect pH: Equation 24.5 where α= solubility. Thus Equation 24.6 log[HCO3 − ]pH = 6 1. + 0 03 × PCO2

1	Equation 24.5 where α= solubility. Thus Equation 24.6 log[HCO3 − ]pH = 6 1. + 0 03 × PCO2 In these equations, the amount of CO2 is determined from the PCO2 and its solubility (α) in solution. For plasma at 37°C, α has a value of 0.03. Also, pK′ is the negative logarithm of the overall dissociation constant for the reaction and has a logarithmic value of 6.1 for plasma at 37°C. Acute hyperventilation secondary to exercise or anxiety reduces PCO2 and thereby causes an increase in pH (respiratory alkalosis). Conversely, if PCO2 increases because of hypoventilation secondary to an overdose of a respiratory depressant, the pH decreases (respiratory acidosis). Acid-base imbalances are also caused by metabolic disorders such as metabolic acidosis (e.g., lactic acidosis, ketoacidosis, and renal failure) and metabolic alkalosis (e.g., hypokalemia, hypochloremia, vomiting, high doses of steroids).

1	In contrast to O2, the dissociation curve for CO2 in blood is linear and directly related to PCO2 ( Fig. 24.9). The degree of Hgb saturation with O2 has a major effect on the CO2 dissociation curve. Although O2 and CO2 bind to Hgb 1. Gases (nitrous oxide, ether, helium) that have a rapid rate of air-to-blood equilibration are perfusion limited. Gases (CO) that have a slow air-to-blood equilibration rate are diffusion limited. Under normal conditions, O2 and CO2 exchange are perfusion limited but can be diffusion limited in some situations. 2. The major transport mechanism of O2 in blood is within the red blood cell bound to Hgb, and for CO2, it is within red blood cells in the form of HCO3 − . 3. The reversible reaction of CO2 with H2O to form H2CO3, with its subsequent dissociation to HCO3 − and H+ , is catalyzed by the enzyme carbonic anhydrase within red blood cells and is the major mechanism for generation of HCO3 − . 4.

1	4. The O2 dissociation curve is S shaped. In the plateau area (>60 mm Hg), increasing or decreasing PO2 has only a minimal effect on Hgb saturation from 100% •Fig. 24.9 Equilibrium Curves for CO2in Arterial and Venous Blood. VenousbloodcantransportmoreCO2thanarterialbloodcanatanygivenPCO2.IncomparisonwiththeHgbO2equilibriumcurve,theCO2curvesareessentiallystraightlinesbetweenaPCO2of20andaPCO2of80mmHg.Long dashes representthearterialequilibriumcurve;short dashes representthevenousequilibriumcurve.

1	at different sites, deoxygenated Hgb has greater affinity for CO2 than oxygenated Hgb. Thus deoxygenated blood (venous blood) freely takes up and transports more CO2 than oxygenated arterial blood does. The deoxygenated Hgb more readily forms carbamino compounds and also more readily binds free H+ released during the formation of HCO3 − . The effect of changes in oxyhemoglobin saturation on the relationship of CO2 content to PCO2 is referred to as the Haldane effect and is reversed in the lungs when O2 is transported from the alveoli to red blood cells. This effect is illustrated by a shift to the left in the CO2 dissociation curve in venous blood in comparison with arterial blood. to 90%. This ensures adequate Hgb saturation over a large range of PO2 values. 5. The CO2 dissociation curve is linear and directly related to PCO2. PCO2 is solely dependent on alveolar ventilation and CO2 production. 6.

1	5. The CO2 dissociation curve is linear and directly related to PCO2. PCO2 is solely dependent on alveolar ventilation and CO2 production. 6. The CO2 to HCO3 − pathway plays a critical role in the regulation of H+ ions and in maintaining acid-base balance in the body. 7. Tissue oxygenation is dependent on Hgb within red blood cells and subsequently the number (and production) of red blood cells, which is controlled by the hormone erythropoietin. Low O2 delivery, low Hgb concentration, and low PaO2 stimulate the secretion of erythropoietin in the kidneys. 8. Tissue hypoxia occurs when insufficient amounts of O2 are supplied to the tissue to conduct normal levels of aerobic metabolism. Butler JP, Tsuda A. Transport of gases between the environment and alveoli—theoretical foundations. Compr Physiol. 2011;1: 1301-1316. Calverley PMA, Koulouris NG. Flow limitation and dynamic hyperinflation: key concepts in modern respiratory physiology. Eur Respir J. 2005;25:186-199.

1	Calverley PMA, Koulouris NG. Flow limitation and dynamic hyperinflation: key concepts in modern respiratory physiology. Eur Respir J. 2005;25:186-199. Hillman SS, Hancock TV, Hedrick MS. A comparative meta-analysis of maximal aerobic metabolism of vertebrates: implications for respiratory and cardiovascular limits to gas exchange. J Comp Physiol [B]. 2013;183:167-179. Hughes JM. Assessing gas exchange. Chron Respir Dis. 2007;4:205-214. Petersson J, Glenny RW. Gas exchange and ventilation-perfusion relationships in the lung. Eur Respir J. 2014;44:1023-1041. Sheel AW, Romer LM. Ventilation and respiratory mechanics. Compr Physiol. 2012;2:1093-1142. Stickland MK, Lindinger MI, Olfert IM, et al. Pulmonary gas exchange and acid-base balance during exercise. Compr Physiol. 2013;3:693-739. Whipp BJ. Physiological mechanisms dissociating pulmonary CO2 and O2 exchange dynamics during exercise in humans. Exp Physiol. 2007;92:347-355.

1	Whipp BJ. Physiological mechanisms dissociating pulmonary CO2 and O2 exchange dynamics during exercise in humans. Exp Physiol. 2007;92:347-355. Upon completion of this chapter, the student should be able to answer the following questions: 1. Describe the central organization of breathing. 2. Explain the role of central and peripheral chemoreceptors in regulating respiration. 3. Compare and contrast the roles of chemoreceptors and pulmonary mechanoreceptors in regulating respiration. 4. Describe ventilatory control during special circumstances (e.g., exercise and high altitude). 5. Describe the effects of abnormalities in ventilatory control.

1	eople breathe without thinking, and they can willingly modify their breathing pattern and even hold their breath. Control of ventilation includes the generation and regulation of rhythmic breathing by the respiratory center in the brainstem and its modification by the input of information from higher brain centers and from systemic receptors. The goals of breathing are, from a mechanical perspective, to minimize work and, from a physiological perspective, to maintain and regulate positive pressures of arterial blood O2 (PaO2) and CO2 (PaCO2). Another goal of breathing is to maintain acid-base balance in the brain by regulating PaCO2. Automatic respiration begins at birth. In utero, the placenta, not the lung, is the organ of gas exchange in the fetus. Its microvilli interdigitate with the maternal uterine circulation, and PaO2 transport and PaCO2 removal from the fetus occur by passive diffusion across the maternal circulation. Ventilatory Control: An Overview

1	Ventilatory Control: An Overview There are four major sites of ventilatory control: (1) the respiratory control center, (2) central chemoreceptors, (3) peripheral chemoreceptors, and (4) pulmonary mechanoreceptors/sensory nerves. The respiratory control center is located in the medulla oblongata of the brainstem and is composed of multiple nuclei that generate and modify the basic ventilatory rhythm. This center consists of two main parts: (1) a ventilatory pattern generator, which sets the rhythmic pattern, and (2) an integrator, which controls generation of the pattern, processes input from higher brain centers and chemoreceptors, and controls the rate and amplitude of the ventilatory pattern. Input to the integrator arises from higher brain centers, including the cerebral cortex, hypothalamus, limbic system including the amygdalae, and cerebellum.

1	Central chemoreceptors are located in the central nervous system just below the ventrolateral surface of the medulla. These central chemoreceptors detect changes in the PaCO2 and pH of interstitial fluid in the brainstem, and they modulate ventilation. Peripheral chemoreceptors are located on specialized cells in the aortic arch (aortic bodies) and at the bifurcation of the internal and external carotid arteries (carotid bodies) in the neck. These peripheral chemoreceptors sense the PaO2, PaCO2, and pH of arterial blood, and they feed information back to the integrator nuclei in the medulla through the vagus nerves and carotid sinus nerves, which are branches of the glossopharyngeal nerves. Pulmonary mechanoreceptors and sensory nerve stimulation, in response to lung inflation or to stimulation by irritants or release of local mediators in the airways, modify the ventilatory pattern.

1	The collective output of the respiratory control center to motor neurons located in the anterior horn of the spinal column controls the muscles of respiration, and this output determines the automatic rhythmic pattern of respiration. Motor neurons located in the cervical region of the spinal column control the activity of the diaphragm through the phrenic nerves, whereas other motor neurons located in the thoracic region of the spine control the intercostal muscles and the accessory muscles of respiration.

1	In contrast to automatic respiration, voluntary respiration bypasses the respiratory control center in the medulla. The neural activity controlling voluntary respiration originates in the motor cortex, and signaling passes directly to motor neurons in the spine through the corticospinal tracts. The motor neurons to the respiratory muscles act as the final site of integration of the voluntary (corticospinal tract) and automatic (ventrolateral tracts) control of ventilation. Voluntary control of these muscles competes with automatic influences at the level of the spinal motor neurons, and this competition can be demonstrated by breath holding. At the start of the breath hold, voluntary control dominates the spinal motor neurons. However, as the breath hold continues, the automatic ventilatory control eventually overpowers the voluntary effort and limits the duration of the breath hold. Motor neurons also innervate muscles 490 SECTION5Berne & Levy Physiology 15 10 5 0 25 35 45 55 65

1	control eventually overpowers the voluntary effort and limits the duration of the breath hold. Motor neurons also innervate muscles 490 SECTION5Berne & Levy Physiology 15 10 5 0 25 35 45 55 65 PaCO2 (mm Hg) Alveolar ventilation (L/min) Metabolic acidosis Awake normal Sleep Morphine, barbiturates COPD Anesthetic agents •Fig. 25.1 Relationship between Partial Pressure of Arterial Carbon Dioxide (PaCO2) and Alveolar Ventilation in Awake Normal States, during Sleep, after Narcotic Ingestion and Deep Anesthesia, and in the Presence of Metabolic Acidosis. Boththeslopesoftheresponse(sensitivity)andthepositionoftheresponsecurves(threshold,thepointatwhichthecurvecrossesthex-axis[notshown])arechanged,whichindicatesdifferencesinventilatoryresponsesandresponsethresholds.COPD,chronicobstructivepulmonarydisease.

1	of the upper airway. These neurons are located within the medulla near the respiratory control center. They innervate muscles in the upper airways through the cranial nerves. When activated, they dilate the pharynx and large airways at the initiation of inspiration. Response to Carbon Dioxide Ventilation is regulated by PaCO2, PaO2, and pH in arterial blood. PaCO2 is the most important of these regulators. Both the rate and depth of breathing are controlled to maintain PaCO2 close to 40 mm Hg. In a normal awake individual, there is a linear rise in ventilation as PaCO2 reaches and exceeds 40 mm

1	Hg (Fig. 25.1 ). The ventilatory drive or response to changes in PaCO2 can be reduced by hyperventilation and by drugs that depress the respiratory center and decrease the ventilatory response to both CO2 and O2, such as morphine, barbiturates, and anesthetic agents. In these instances, the stimulus is inadequate to stimulate the motor neurons that innervate the muscles of respiration. It is also depressed during sleep. In addition, the ventilatory response to changes in PaCO2 is reduced if the work of breathing is increased, which can occur in individuals with chronic obstructive pulmonary disease (COPD). This effect occurs primarily because the neural output of the respiratory center is less effective in promoting ventilation as a result of the mechanical limitation to ventilation.

1	Changes in PaCO2 are sensed by central and peripheral chemoreceptors, and they transmit this information to the medullary respiratory centers. The respiratory control center then regulates minute ventilation and thereby maintains PaCO2 within the normal range. In the presence of a normal PaO2, ventilation increases by approximately 3 L/minute for each 1 mm Hg rise in PaCO2. The response to an increase in PaCO2 is further increased when the PaO2 is low ( Fig. 25.2, ). With a low PaO2, ventilation is greater for any given PaCO2, and the increase in ventilation for a given increment in PaCO2 is enhanced. The slope of the minute ventilation response as a function of the inspired CO2 is termed the ventilatory response to CO2 and is a test of CO2 sensitivity. It is important to recognize that this relationship is amplified by low O2 (see

1	Fig. 25.2, B ). The responsiveness to low O2 is enhanced because different mechanisms are responsible for sensing PaO2 and PaCO2 in the peripheral chemoreceptors. Thus the presence of both hypercapnia-elevated CO2 and hypoxemia-low O2 (often called asphyxia when both changes are present) has an additive effect on chemoreceptor output and on the resulting ventilatory stimulation. Control of Ventilation: The Details The Respiratory Control Center When the brain is transected experimentally between the medulla and the pons, periodic breathing is maintained, thus demonstrating that the inherent rhythmicity of breathing originates in the medulla. Although no single group of neurons in the medulla has been found to be the breathing “pacemaker,” two distinct nuclei within the medulla are involved in generation of the respiratory pattern (

1	Fig. 25.3 ). One nucleus is the dorsal respiratory group (DRG), which is composed of cells in the nucleus tractus solitarius and is located in the dorsomedial region of the medulla. Cells in the DRG receive afferent input from cranial nerves IX •Fig. 25.2 The Effects of Hypoxia and Hypercapnia on Ventilation as the Other Respiratory Gas Partial Pressures Are Varied. A, Atagivenpartialpressureofarterialcarbondioxide(PaCO2),ventilationincreasesmoreandmoreaspartialpressureofarterialoxygen(PaO2)decreases.WhenPaCO2isallowedtodecrease(thenormalcondition)duringhypoxia,thereislittlestimulationofbreathinguntilPaO2fallsbelow60mmHg.Thehypoxicresponseismediatedthroughthecarotidbodychemoreceptors.B, ThesensitivityoftheventilatoryresponsetoCO2isenhancedbyhypoxia.

1	and X, which originate from airways and the lungs and constitute the initial intracranial processing station for this afferent input. The second group of medullary cells is the ventral respiratory group (VRG), located in the ventrolateral region of the medulla. The VRG is composed of three cell groups: the rostral nucleus retrofacialis, the caudal nucleus retroambiguus, and the nucleus para-ambiguus. The VRG contains both inspiratory and expiratory neurons. The nucleus retrofacialis and the caudally located cells of the nucleus retroambiguus are active during exhalation, whereas the rostrally located cells of the nucleus retroambiguus are active during inspiration. The nucleus para-ambiguus has inspiratory and expiratory neurons that travel in the vagus nerve to the laryngeal and pharyngeal muscles. Discharges •Fig. 25.3 The Respiratory Control Center is Located in the Medulla, the Most Primitive Portion of the Brain.

1	that travel in the vagus nerve to the laryngeal and pharyngeal muscles. Discharges •Fig. 25.3 The Respiratory Control Center is Located in the Medulla, the Most Primitive Portion of the Brain. Theneuronsaremainlyintwoareas:thedorsalrespiratorygroup(DRG),whichconsistsofthenucleustractussolitarius,andtheventralrespiratorygroup(VRG),whichconsistsoftherostralnucleusretrofacialis,nucleusparaambiguus,andthecaudalnucleusretroambiguus.C1referstothefirstcervicalsignalsegmenttothecaudalborderofthepons.Thefourthventricleofthebrainislocatedbelowthecerebellumandabove,andbetween,theponsandthemedulla.

1	•Fig. 25.4 Diagram of the Basic Wiring of the Brainstem Ventilatory Controller. Thesignsofthemainoutput(arrows) oftheneuronpoolsindicatewhethertheoutputisexcitatory(+)orinhibitory(−).PoolAprovidestonicinspiratorystimulitothemusclesofbreathing.PoolBisstimulatedbypoolAandprovidesadditionalstimulationtothemusclesofbreathing,andpoolBstimulatespoolC.OtherbraincentersfeedintopoolC(inspiratorycutoffswitch),whichsendsinhibitoryimpulsestopoolA.Afferentinformation(feedback)fromvarioussensorsactsatdifferentlocations:ChemoreceptorsactonpoolA,andintrapulmonarysensoryfibersactviathevagusnervesonpoolB.Apneumotaxiccenterintheanteriorponsreceivesinputfromthecerebralcortex,anditactsonpoolC. from cells in these areas excite some cells and inhibit other cells. At the level of the respiratory control center, inspiration and exhalation involve three phases: one inspiratory and two expiratory (

1	Fig. 25.4 ). Inspiration begins with an abrupt increase in discharge from cells in the nucleus tractus solitarius, the nucleus retroambiguus, and the nucleus para-ambiguus, followed by a steady ramp-like increase in firing rate throughout inspiration. This leads to progressive contraction of the respiratory muscles during automatic breathing. At the end of inspiration, an “off-switch” event causes neuron firing to decrease markedly, at which point exhalation begins. At the start of exhalation (phase I of expiration), a paradoxical increase in inspiratory neuron firing slows the expiratory phase down by increasing inspiratory muscle tone and expiratory neuron firing. This inspiratory neuron firing decreases and stops during phase II of exhalation. Although many different neurons in the DRG and VRG are involved in ventilation, each cell type appears to have a specific function. For example, the Hering-Breuer reflex is an inspiratory-inhibitory reflex that arises from afferent stretch

1	and VRG are involved in ventilation, each cell type appears to have a specific function. For example, the Hering-Breuer reflex is an inspiratory-inhibitory reflex that arises from afferent stretch receptors located in the smooth muscles of the airways. Increasing lung inflation stimulates these stretch receptors and results in early exhalation by stimulating the neurons associated with the off-switch phase of inspiratory muscle control. Thus rhythmic breathing depends on a continuous (tonic) inspiratory drive from the DRG and an intermittent (phasic) expiratory drive from the cerebrum, thalamus, cranial nerves, and ascending sensory tracts in the spinal cord.

1	A chemoreceptor is a receptor that responds to a change in the chemical composition of blood or other fluid around it. Central chemoreceptors are specialized cells on the ventrolateral surface of the medulla. Chemoreceptors are sensitive to the pH of the surrounding extracellular fluid. Because this extracellular fluid is in contact with cerebrospinal fluid (CSF), changes in the pH of CSF affect ventilation by acting on these chemoreceptors CSF is an ultrafiltrate of plasma that is secreted continuously by the choroid plexus and is reabsorbed by the arachnoid villi. Because it is in contact with the extracellular fluid in the brain, the composition of CSF is influenced by the metabolic activity of the cells in the surrounding area and the composition of the blood. Although the origin of CSF is plasma, the composition of CSF is not the same as that of plasma because the blood-brain barrier exists between the two sites (

1	Fig. 25.5 ). The blood-brain barrier is composed of endothelial cells, smooth muscle, and the pial and arachnoid membranes, and it regulates the movement of ions between blood and CSF. In addition, the choroid plexus also determines the ionic composition of CSF by transporting ions into and out of CSF. The blood-brain barrier is relatively impermeable by H+ and HCO3 − ions, but it is very permeable by CO2. Thus the PCO2 in CSF parallels the arterial PCO2 tension. CO2 is also produced by cells of the brain as a product of metabolism. As a consequence, the PCO2 in CSF is usually a few millimeters of mercury higher than that in arterial blood, and so the pH is slightly more acidic (7.33) in CSF than in plasma (

1	Table 25.1 •Fig. 25.5 Carbon Dioxide and the Blood-Brain Barrier. PaCO2crossestheblood-brainbarrierandrapidlyequilibrateswithCO2incerebrospinalfluid(CSF).H+ andHCO3− ionscrossthebarrierslowly.Thepartialpressureofarterialcarbondioxide(PaCO2)combineswithCO2generatedbymetabolismtodilatethesmoothmuscle.Incomparisonwitharterialblood,thepHofCSFislowerandthePCO2ishigher,withlittleproteinbuffering. PCO2,partialpressureofcarbondioxide.

1	PCO2,partialpressureofcarbondioxide. The carotid and aortic bodies are peripheral chemoreceptors that respond to changes in PaO2 (not the O2 content), PaCO2, and pH, and they transmit afferent information to the central respiratory control center. The peripheral chemoreceptors are the only chemoreceptors that respond to changes in PaO2. The peripheral chemoreceptors are also responsible for approximately 40% of the ventilatory response to PaCO2. These chemoreceptors are small, highly vascularized structures. They consist of type I (glomus) cells that are rich in mitochondria and endoplasmic reticulum. They also have several types of cytoplasmic granules (synaptic vesicles) that contain various neurotransmitters, including dopamine, acetylcholine, norepinephrine, and neuropeptides. Afferent nerve fibers synapse with type I cells, and they transmit information to the brainstem through the carotid sinus nerve (carotid body) and vagus

1	TheHenderson-Hasselbalch equation relatesthepHofCSFtotheconcentrationofbicarbonate([HCO3−]): whereα isthesolubilitycoefficient(0.03mmol/L/mmHg)andpKisthenegativelogarithmofthedissociationconstantforcarbonicacid(6.1).TheHenderson-HasselbalchequationdemonstratesthatanincreaseinCSFPCO2causesthepHofCSFtodecreaseatanygiven[HCO3−].ThedecreaseinpHstimulatesthecentralchemoreceptorsandtherebyincreasesventilation.ThusCO2inbloodregulatesventilationbyitseffectonthepHofCSF.TheresultinghyperventilationreducesthePaCO2,andthereforethePCO2ofCSF,andreturnsthepHofCSFtowardanormalvalue.Furthermore,cerebralvasodilationaccompaniesanincreaseinPaCO2,andthisenhancesthediffusionofCO2intoCSF.Incontrast,anincreaseinCSF[HCO3−]causesanincreaseinthepHofCSFatanygivenPaCO2.

1	ChangesinPaCO2thatresultfromalterationsinpHactivatehomeostaticmechanismsthatreturnthepHbacktowardanormalvalue.Theblood-brainbarrierregulatesthepHofCSFbyadjustingtheioniccompositionand[HCO3 −]ofCSF.ThesechangesinCSF[HCO3−],however,occurslowly,overaperiodofseveralhours,whereaschangesinCSFPCO2canoccurwithinminutes.ThuscompensationforchangesinthepHofCSFrequireshourstodevelopfully. Several reflexes that arise from the chest wall and lungs affect ventilation and ventilatory patterns (

1	Several reflexes that arise from the chest wall and lungs affect ventilation and ventilatory patterns ( Table 25.2 ). The Hering-Breuer inspiratory-inhibitory reflex is stimulated by increases in lung volume, especially those associated with an increase in both ventilatory rate and tidal volume. This stretch reflex is mediated by vagal fibers, and when nerve (aortic body). Type I cells are the cells primarily responsible for sensing PaO2, PaCO2, and pH. In response to even small decreases in PaO2, there is an increase in chemoreceptor discharge, which enhances respiration. The response is robust when PaO2 decreases below 75 mm Hg. Thus ventilation is regulated by changes in arterial and CSF pH through effects on peripheral and central chemoreceptors (

1	Fig. 25.6 •Fig. 25.6 The Ventilatory Response to Partial Pressure of Arterial Carbon Dioxide (PaCO2) is Affected by the Concentration of Hydrogen ([H+]) in Cerebrospinal Fluid (CSF) and Brainstem Interstitial Fluid. Duringchronicmetabolicacidosis(e.g.,diabeticketoacidosis),the[H+]inCSFisincreased,andtheventilatoryresponsetoinspiredPaCO2isincreased(steeperslope).Conversely,duringchronicmetabolicalkalosis(arelativelyuncommoncondition),the[H+]inCSFisdecreasedandtheventilatoryresponsetoinspiredPaCO2isdecreased(reducedslope).Thepositionsoftheresponselinesarealsoshifted,whichindicatesalteredthresholds.

1	ImagineflyingfromNewYorkCitytoDenver.ThebarometricpressureinNewYorkisapproximately760mmHg,whereasinthemountainssurroundingDenver,Colorado,itis600mmHg.Atsealevel,thePaO2isapproximately95mmHgandPAO2= [(760− 47)× 0.21]− [40/0.8]= 100mmHg(accordingtothealveolarairequation;see ).Ifthealveolar-arterialPO2difference[AaDO2]is5mmHg,PaO2= 100mmHg− 5mmHg= 95mmHg.IntheCSF,pHwouldbeapproximately7.33,PaCO2wouldbe44mmHg(PaCO2+ CO2producedbymetabolismofbraincells),andHCO3− wouldbeapproximately22mEq/L.

1	WhenyouarriveinDenver,thereisanabruptdecreaseinthepartialpressureofinspiredO2(PiO2):PiO2= (600− 47)× 0.21= 116mmHg;therearealsodecreasesinthepartialpressuresofalveolarandarterialO2:PAO2= 116− (40/0.8)= 66mmHg,andPaO2= 61mmHgifthereisnochangeinAaDO2).ThisdecreaseinarterialO2stimulatestheperipheralchemoreceptorsandtherebyincreasesventilation.TheincreaseinventilationdecreasesPaCO2andelevatesarterialpH.TheresultofthisincreaseinventilationistominimizethehypoxemiabyincreasingPAO2.Forexample,assumethatPACO2decreasesto30mmHg.ThenPAO2= [(600− 47)× 0.21]− [30/0.8]= 78mmHg,a12–mmHgincreaseinPAO2.

1	ThedecreaseinPaCO2alsocausesadecreaseinthePCO2ofCSF.Because[HCO3−]isunchanged,thepHofCSFincreases.ThisincreaseinthepHofCSFattenuatestherateofdischargeofthecentralchemoreceptorsanddecreasestheircontributiontotheventilatorydrive.Overthenext12to36hours,[HCO3−]inCSFdecreasesasacid-basetransporterproteinsintheblood-brainbarrierreduce[HCO3−].Asaconsequence,thepHofCSFreturnstowardnormal.Centralchemoreceptordischargeincreases,andminuteventilationisfurtherincreased.Atthesametimethat[HCO3−]inCSFdecreases,HCO3−

1	isgraduallyexcretedfromplasmabythekidneys.ThisresultsinagradualreturnofarterialpHtowardnormalvalues.PeripheralchemoreceptorstimulationincreasesfurtherasarterialpHbecomesnormal(peripheralchemoreceptorsareinhibitedbytheelevatedarterialpH).Finally,within36hoursofarrivingathighaltitude,minuteventilationincreasessignificantly.Thisdelayedresponseisgreaterthantheimmediateeffectofthehypoxemiaonventilation.Thisfurtherincreaseinventilationisduetobothcentralandperipheralchemoreceptorstimulation.Thusafter36hours,botharterialpHandCSFpHareapproachingnormalvalues;minuteventilationisincreased,PaO2isdecreased,andPaCO2isdecreased.

1	Younowreturnhome.WhenyoulandinNewYork,thePiO2returnstoanormalvalue,andthehypoxicstimulustoventilationisremoved.PaO2returnstoanormalvalue,andtheperipheralchemoreceptorstimulationtoventilationdecreases.Thiscausesanincreaseinarterial[CO2]towardnormalvalues,whichinturncausesanincreaseinCSF[CO2].ThisincreaseisassociatedwithadecreaseinthepHofCSFas[HCO3−]inCSFisreducedandventilationisaugmented.Overthenext12to36hours,theacid-basetransportersintheblood-brainbarriertransportHCO3− backintoCSF,andthepHofCSFgraduallyreturnstowardnormalvalues.Similarly,thepHofblooddecreasesasPaCO2risesbecausearterial[HCO3−]decreases.Thisstimulatestheperipheralchemoreceptors,andminuteventilationremainsaugmented.Overthenext12to36hours,blood[HCO3−]increasesinthekidneys(see ),arterialpHreturnstoanormalvalue,andminuteventilationreturnstoanormallevel.

1	elicited, it results in cessation of inspiration by stimulating the off-switch neurons in the medulla. This reflex is inactive during quiet breathing and appears to be most important in newborns. Stimulation of nasal or facial receptors with cold water initiates the diving reflex. When this reflex is elicited, apnea, or cessation of breathing, and bradycardia occur. This reflex protects individuals from aspirating water in the initial stages of drowning. Activation of receptors in the nose is responsible for the sneeze reflex.

1	The aspiration or sniff reflex can be elicited by stimulation of mechanical receptors in the nasopharynx and pharynx. This is a strong, short-duration inspiratory effort that brings material from the nasopharynx to the pharynx, where it can be swallowed or expectorated. The mechanical receptors responsible for the sniff reflex are also important in swallowing by inhibiting respiration and causing laryngeal closure. For anatomical reasons, only newborns can breathe and swallow simultaneously, which allows more rapid ingestion of nutrients. The larynx contains both superficial and deep receptors. Activation of the superficial receptors results in apnea, cough, and expiratory movements that protect the lower respiratory tract from aspirating foreign material. The deep receptors are located in the skeletal muscles of the larynx, and they control muscle fiber activation, as in other skeletal muscles.

1	Three major types of sensory receptors located in the tracheobronchial tree respond to a variety of different stimuli, and those responses result in changes in the lung’s mechanical properties, alterations in the respiratory pattern, and the development of respiratory symptoms. Inhaled dust, noxious gases, and cigarette smoke stimulate irritant receptors in the trachea and large airways that transmit information through myelinated vagal afferent fibers. Stimulation of these receptors results in an increase in airway resistance, reflex apnea, and coughing. These receptors are also known as rapidly adapting pulmonary stretch receptors. Slowly adapting pulmonary stretch receptors respond to mechanical stimulation, and they are activated by lung inflation. They also transmit information through myelinated, vagal afferent fibers. The increase in lung volume in people with COPD stimulates these pulmonary stretch receptors and delays the onset of the next inspiratory effort. This explains

1	myelinated, vagal afferent fibers. The increase in lung volume in people with COPD stimulates these pulmonary stretch receptors and delays the onset of the next inspiratory effort. This explains the long, slow expiratory effort in affected individuals, and it is essential to minimize dynamic, expiratory airway compression.

1	In addition, specialized sensory receptors located in the lung parenchyma respond to chemical or mechanical stimulation in the lung interstitium. These receptors are called juxta-alveolar, (or J) receptors. They transmit their afferent input through unmyelinated, vagal C fibers. They may be responsible for the sensation of dyspnea (abnormal shortness of breath) and the rapid, shallow ventilatory patterns that occur in interstitial lung edema and some inflammatory lung states. VO2 (L/min) . 1 1.5 2 VCO2 . PaCO2 pH Ventilation PaO2 Lactate •Fig. 25.8 The Two Main Categories of Sleep Apnea. A, Obstruc-tivesleepapnea,thepleuralpressureoscillationsincreaseasCO2levelrises.Thisindicatesthatresistancetoairflowisveryhighasaresultofupperairwayobstruction.B, Centralsleepapneaischaracterizedbynoattempttobreathe,asdemonstratedbynooscillationsinpleuralpressure.

1	•Fig. 25.7 Oxygen Consumption (V̇ O2) as a Function of the Metabolic Changes That Occur During Exercise. Theanaerobicthreshold(arrow) isthepointatwhichtheillustratedvariableschangeandisduetolacticacidosis.PaCO2,partialpressureofarterialcarbondioxide;PaO2,partialpressureofarterialoxygen;VCO2,carbondioxide consumption. Somatic receptors are also located in the intercostal muscles, rib joints, accessory muscles of respiration, and tendons, and they respond to changes in the length and tension of the respiratory muscles. Although they do not directly control respiration, they do provide information about lung volume and play a role in terminating inspiration. They are especially important in individuals with increased airway resistance and decreased pulmonary compliance because they can augment muscle force within the same breath. Somatic receptors also help minimize the chest wall distortion during inspiration in newborns, who have very compliant rib cages.

1	The ability to exercise depends on the capacity of the cardiac and respiratory systems to increase delivery of O2 to tissues and remove CO2 from the body. Ventilation increases immediately when exercise begins, and this increase in minute ventilation closely matches the increases in O2 consumption and CO2 production that accompany exercise ( Fig. 25.7 ). Ventilation is linearly related to both CO2 production and O2 consumption at low to moderate levels (see Fig. 25.7 ). During maximal exercise, a physically fit individual can achieve an O2 consumption of 4 L/minute with a minute ventilation volume of 120 L/minute, which is almost 15 times the resting level.

1	Exercise is remarkable because of the lack of significant changes in blood gases. Except at maximal exertion, changes in PaCO2 and PaO2 are minimal during exercise. Arterial pH remains within normal values during moderate exercise. During strenuous exercise, arterial pH begins to fall as lactic acid is liberated from muscles during anaerobic metabolism. This decrease in arterial pH stimulates ventilation that is out of proportion to the level of exercise. The level of exercise at which sustained metabolic (lactic) acidosis begins is called the anaerobic threshold (see Fig. 25.7 Abnormalities in the Control of Breathing

1	Fig. 25.7 Abnormalities in the Control of Breathing Changes in the ventilatory pattern can occur for both primary and secondary reasons. During sleep, approximately one third of normal individuals have brief episodes of apnea or hypoventilation that have no significant effects on PaO2 or PaCO2. The apnea usually lasts less than 10 seconds, and it occurs in the lighter stages of slow-wave and rapid eye movement (REM) sleep. In sleep apnea syndromes, the duration of apnea is abnormally prolonged, and it changes PaO2 and PaCO2. There are two major categories of sleep apnea ( Fig. 25.8 ). The first, obstructive sleep apnea (OSA), is the most common of the sleep apnea syndromes, and it occurs when the upper airway (generally the hypopharynx) closes during inspiration. Although the process is similar to what happens during snoring, it is more severe, inasmuch as it obstructs the airway and causes cessation of airflow.

1	The second sleep apnea syndrome is central sleep apnea. This variant of apnea occurs when the ventilatory drive to TheclinicalhistoriesofindividualswithOSAareverysimilar.Aspouseusuallyreportsthattheaffectedindividualsnores.Thesnoringbecomeslouderandlouderandthenstopswhiletheindividualcontinuestomakevigorousrespiratoryefforts(see Fig.25.8 ).Theindividualthenawakens,fallsbacktosleep,andcontinuesthesameprocessrepetitivelythroughoutthenight.IndividualswithOSAawakenwhenthearterialhypoxemiaandhypercapniastimulatebothperipheralandcentralchemoreceptors.Respirationisrestoredbrieflybeforethenextapneiceventoccurs.IndividualswithOSAcanhavehundredsoftheseeventseachnightthatinterruptsleep.ComplicationsofOSAincludesleepdeprivation,polycythemia,right-sidedcardiacfailure(corpulmonale),andpulmonaryhypertensionsecondarytotherecurrent,hypoxicevents.OSAiscommoninindividualswithobesityandinthosewithexcessivecomplianceofthehypopharynx,upperairwayedema,andstructuralabnormalitiesoftheupperairway.

1	the respiratory motor neurons decreases. Individuals with central sleep apnea have repeated episodes of apnea, during which time they make no respiratory effort, every night (see Fig. 25.8 ). The degree of hypercapnia and hypoxemia in individuals with central sleep apnea is less than that in individuals with OSA, but the same complications (e.g., polycythemia) can occur when central sleep apnea is recurrent and severe. Cheyne-Stokes ventilation is another abnormality of breathing that is characterized by varying tidal volume and ventilatory frequency (

1	Cheyne-Stokes ventilation is another abnormality of breathing that is characterized by varying tidal volume and ventilatory frequency ( Fig. 25.9 ). After a period of apnea, tidal volume and respiratory frequency increase progressively over several breaths, and then they progressively decrease until apnea recurs. This irregular breathing pattern is seen in some individuals with central nervous system diseases, head trauma, and increased intracranial pressure. It is also present on occasion in normal individuals during sleep at high altitude. The mechanism underlying Cheyne-Stokes respiration is not known. In some individuals, it appears to be due to slow blood flow in the brain in association with periods of overshooting and undershooting ventilatory effort in response to changes in PCO2.

1	Central alveolar hypoventilation, alsoknownasOndine’s curse, isararediseaseinwhichvoluntarybreathingisintactbutabnormalitiesinautomaticityexist.Itisthemostsevereofthecentralsleepapneasyndromes.Asaresult,peoplewithcentralalveolarhypoventilationcanbreatheaslongastheydonotfallasleep.Fortheseindividuals,mechanicalventilationor,morerecently,bilateraldiaphragmaticpacing(similartoacardiacpacemaker)canbelifesaving. Apneustic breathing is another abnormal breathing pattern that is characterized by sustained periods of inspiration separated by brief periods of exhalation ( Fig. 25.10, C •Fig. 25.9 In Cheyne-Stokes Breathing, Tidal Volume and, as a Consequence, Arterial Blood Gas Levels Wax and Wane. Ingeneral,Cheyne-Stokesbreathingisasignofvasomotorinstability,particularlylowcardiacoutput.PaCO2,partialpressureofarterialcarbondioxide;PaO2,partialpressureofarterialoxygen.

1	Lung volumeRemove input from cortex, thalamus, and vagus nerves A B Remove sensory input from lungs •Fig. 25.10 Some Patterns of Breathing. A, Normalrateofbreathingisintherangeof12to20breathsperminute.B, Whensensoryinputisremovedfromvariouslungreceptors(mainlystretch),eachbreathingcycleislengthenedandtidalvolumeisincreased,sothatalveolarventilationisnotsignificantlyaffected.C, Wheninputfromthecerebralcortexandthalamusisalsoeliminated,togetherwithvagalblockade,theresultisprolongedinspiratoryactivitybrokenafterseveralsecondsbybriefexpirations(apneusis). The mechanism underlying this ventilatory pattern appears to be a loss of inspiratory-inhibitory activities that results in augmentation of the inspiratory drive. The pattern sometimes occurs in individuals with central nervous system injury.

1	Sudden infant death syndrome (SIDS)isthemostcommoncauseofdeathininfantsinthefirstyearoflifeaftertheperinatalperiod.AlthoughthecauseofSIDSisnotknown,abnormalitiesinventilatorycontrol,particularlyin 1. Ventilatory control is composed of the respiratory control center, central chemoreceptors, peripheral chemoreceptors, and pulmonary mechanoreceptors/ sensory nerves. PaCO2 is the major factor that influences ventilation. 2. The respiratory control center is composed of the dorsal respiratory group and the ventral respiratory group. Rhythmic breathing depends on a continuous (tonic) inspiratory drive from the dorsal respiratory group and on intermittent (phasic) expiratory input from the cerebrum, thalamus, cranial nerves, and ascending spinal cord sensory tracts. The peripheral and central chemoreceptors respond to changes in PaCO2 and pH. The peripheral chemoreceptors (carotid and aortic bodies) are the only chemoreceptors that respond to changes in PaO2.

1	Calverley PMA, Koulouris NG. Flow limitation and dynamic hyperinflation: key concepts in modern respiratory physiology. Eur Respir J. 2005;25:186-199. Canning BJ. Afferent nerves regulating the cough reflex: mechanisms and mediators of cough in disease. Otolaryngol Clin N Am. 2010;43:15-25. Leff AR, Schumacker PT. Respiratory Physiology: Basics and Applications. Philadelphia: WB Saunders; 1993. Nishino T. Dyspnoea underlying mechanisms and treatment. Br J Anaesth. 2011;106:463-474. CO2responsiveness,havebeenimplicated.Placinginfantsontheirbackstosleep(whichreducesthepotentialforCO2rebreathing)hasdramaticallydecreased(butnoteliminated)therateofdeathfromthissyndrome. 3. Acute hypoxia and chronic hypoxia affect breathing differently because the slow adjustments in CSF [H+] in chronic hypoxia alter sensitivity to CO2. 4.

1	3. Acute hypoxia and chronic hypoxia affect breathing differently because the slow adjustments in CSF [H+] in chronic hypoxia alter sensitivity to CO2. 4. Irritant receptors protect the lower respiratory tract from particles, chemical vapors, and physical factors, primarily by inducing cough. C fiber J receptors in the terminal respiratory units are stimulated by distortion of the alveolar walls (by lung congestion or edema). 5. The two most important clinical abnormalities of breathing are obstructive and central sleep apnea. 6. PaO2, PaCO2, and pH remain within normal limits during moderate exercise; however, during strenuous exercise, pH falls, which stimulates ventilation, whereas PaO2 and PaCO2 remain relatively normal. Otis AB. A perspective of respiratory mechanics. J Appl Physiol. 1983;54:1183-1187. Sheel AW, Romer LM. Ventilation and respiratory mechanics. Compr Physiol. 2012;2:1093-1142.

1	Otis AB. A perspective of respiratory mechanics. J Appl Physiol. 1983;54:1183-1187. Sheel AW, Romer LM. Ventilation and respiratory mechanics. Compr Physiol. 2012;2:1093-1142. Wasserman K, Beaver WL, Whipp BI. Gas exchange theory and the lactic acidosis (anaerobic) threshold. Circulation. 1990;81(1 suppl):1114-1130. Wine JJ. Parasympathetic control of airway submucosal glands: central reflexes and the airway intrinsic nervous system. Auton Neurosci. 2007;133:35-54. Upon completion of this chapter, the student should be able to answer the following questions: 1. Describe the components of the mucociliary clearance system and their role in the removal of xenobiotic substances and particulates. 2. Explain how particle characteristics and properties influence their deposition and clearance. 3. Compare and contrast the major mechanisms of particle deposition. 4. Compare and contrast the mucosal and systemic immune responses. 5.

1	3. Compare and contrast the major mechanisms of particle deposition. 4. Compare and contrast the mucosal and systemic immune responses. 5. Describe unique features of immunoglobulin A and why it is well suited for the mucosal environment and protection. 6. Compare and contrast adaptive and innate immune cells in the respiratory system. 7. Describe the various metabolic processes in the lung. n addition to their primary function of gas exchange, the lungs act as a primary barrier between the outside world and the inside of the body, with host defense functions. They are also active organs in the metabolism of xenobiotic and endogenous compounds. To cope with the inhalation of foreign substances, the respiratory system and, in particular, the conducting airways have developed unique structural features: the mucociliary clearance system and specialized adaptive and innate immune response mechanisms.

1	The mucociliary clearance system protects the conducting airways by trapping and removing inhaled pathogenic viruses and bacteria, in addition to nontoxic and toxic particulates (e.g., pollen, ash, mineral dust, mold spores, and organic particles), from the lungs. These particulates are inhaled with each breath and must be removed. The three major components of the mucociliary clearance system are two fluid layers, referred to as the sol (periciliary fluid) and gel (mucus) layers, and cilia, which are positioned on the surface of bronchial epithelial cells ( Fig. 26.1 ). Inhaled material is trapped on the viscoelastic (sticky) mucus layer, whereas the watery periciliary fluid allows the cilia to move freely and establish an upward flow to clear particulates from the lung. Effective clearance requires both ciliary activity and the appropriate balance of periciliary fluid and mucus.

1	The periciliary fluid layer is composed of nonviscous serous fluid, which is produced by the pseudostratified ciliated columnar epithelial cells that line the airways. These cells have the ability to either secrete fluid, a process that is mediated by activation of cystic fibrosis transmembrane regulator (CFTR) chloride (Cl−) ion channels (Na+ secretion follows passively between cells across the tight junctions) or reabsorb fluid, a process that is mediated by activation of epithelial sodium channels (ENaC; Cl− absorption follows passively between cells across the tight junctions). NaCl secretion or reabsorption temporarily establishes an osmotic gradient across the pseudostratified epithelium, which provides the driving force for passive water movement. The balance between CFTR-mediated Cl− secretion and ENaC-mediated Na+ absorption is regulated by a variety of hormones and determines the volume of the periciliary fluid, which in the healthy lung is 5 to 6 µm deep, a level that is

1	Cl− secretion and ENaC-mediated Na+ absorption is regulated by a variety of hormones and determines the volume of the periciliary fluid, which in the healthy lung is 5 to 6 µm deep, a level that is optimal for rhythmic beating of the cilia and mucociliary clearance.

1	The mucus layer lies on top of the periciliary fluid layer and is composed of a complex mixture of macromolecules and electrolytes. Because the mucus layer is in direct contact with air, it entraps inhaled substances, including pathogens. The mucus layer is predominantly water (95% to 97%), 5 to 10 µm thick and exists as a discontinuous blanket (i.e., islands of mucus). Mucus has low viscosity and high elasticity and is composed of glycoproteins with groups of oligosaccharides attached to a protein backbone. Healthy •Fig. 26.1 Overview of the Epithelial Lining and Innervation of the Tracheobronchial Tree. Theciliaoftheepithelialcellresideinthepericiliaryfluidlayer,andthemucuslayerisontop.Interspersedbetweentheciliatedepithelialcellsaresurfacesecretory(goblet)cellsandsubmucosalglands.Sympatheticandparasympatheticnervefibersdescendintothesubmucosalglandsandsmoothmuscles.

1	Cystic fibrosis (CF)isthemostcommonlethalinheriteddiseaseamongwhitepeople.ItisanautosomalrecessivediseasecausedbymutationsintheCFTR gene.Itischaracterizedbychronicbacteriallunginfection,progressivedeclineinlungfunction,andprematuredeathatanaverageageof38years.Morethan1000mutationsoftheCFTR genehavebeendescribed,but70%ofaffectedindividualshaveadeletionofphenylalanineatcodon508(ΔF508-CFTR)inatleastoneallele.ThismutationresultsinalackofCl− secretionandanincreaseinENaC-mediatedNa+ reabsorption,whichinturnresultsinareductioninthevolumeofthepericiliaryfluid.

1	DetailedstudyofthedifferentCFTR mutationshasresultedinanunderstandingofvariousdisease-relatedphenotypes,someofwhichareassociatedwithmilderdiseaseandsomewithmoreseveredisease.Sincethe1980s,researchfindingshaveelucidatedhowmanyofthemostcommonmutationsintheCFTR genecauseCF,andthishasledtothedevelopmentofdrugsthattargetspecificmutationsandreversethe individuals produce approximately 100 mL of mucus each day. Four cell types contribute to the quantity and composition of the mucus layer: goblet cells and Clara cells within the tracheobronchial epithelium, and mucous cells and serous cells within the tracheobronchial submucosal glands. Goblet cells, also referred to as surface secretory cells, represent approximately 15% to 20% of the tracheobronchial epithelium, and are found in the tracheobronchial tree up to the 12th division. In many respiratory diseases, goblet cells appear further down the tracheobronchial tree; thus the smaller airways are more susceptible to obstruction

1	tracheobronchial tree up to the 12th division. In many respiratory diseases, goblet cells appear further down the tracheobronchial tree; thus the smaller airways are more susceptible to obstruction progressivereductioninlungfunction.Forexample,inonemutation(G551D-CFTR,whichaffects≈5%ofpatientswithCF),theCFTRCl− channelreachestheplasmamembraneofairwayepithelialcellsbutdoesnotsecreteCl− .Throughprecisionmedicine,adrug,ivacaftor(Kalydeco),hasbeenfoundtostimulateCl− secretionviatheG551D-CFTR,therebyimprovinglungfunctionanddecreasingtherateofdiseaseprogression.WhentheallelesarehomozygousfortheΔF508CFTRmutation(whichaffects≈50%ofpatientswithCF),theCFTRCl− channeldoesnotreachtheplasmamembraneofairwayepithelialcells.

1	In2015,theU.S.FoodandDrugAdministrationapprovedacombinationdrugtherapy,lumacaftor/ivacaftor(Orkambi),thathasbeenshowntocorrectthegenedefect,increasetheamountofΔF508-CFTRintheplasmamembrane,andimproveCl− transport.Clinically,bothivacaftorandlumacaftor/ivacaftorhavebeenshowntoimprovelungfunctionsignificantlyandtodecreasetherateofdeclineinlungfunction. by mucus plugging. Goblet cells secrete neutral and acidic glycoproteins rich in sialic acid in response to chemical stimuli. In the presence of infection or cigarette smoke or in patients with chronic bronchitis, goblet cells can increase in size and number, extend above the 12th division of the tracheobronchial tree, and secrete copious amounts of mucus. Injury and infection increase the viscosity of the mucus secreted by goblet cells, which reduces mucociliary clearance of inhaled particles and pathogens.

1	Submucosal tracheobronchial glands are present wherever there is cartilage in the upper regions of the conducting airways, and they secrete water, ions, and mucus into the airway lumen through a ciliated duct (see Fig. 26.1 ). Although both mucous and serous cells secret mucus, their cellular structure and mucus composition are distinctly different (

1	Fig. 26.1 ). Although both mucous and serous cells secret mucus, their cellular structure and mucus composition are distinctly different ( Table 26.1 ). In several lung diseases, including chronic bronchitis, the number and size of submucosal glands are increased, which leads to increases in mucus production, alterations in chemical composition of mucus (i.e., increased viscosity and decreased elasticity), and the formation of mucus plugs that cause airway obstruction. Mucus secretion from submucosal tracheobronchial glands is stimulated by parasympathetic (cholinergic) compounds such as acetylcholine and substance P and inhibited by sympathetic (adrenergic) compounds such as norepinephrine and vasoactive intestinal polypeptide. Local inflammatory mediators such as histamine and arachidonic acid metabolites also stimulate mucus production.

1	Clara cells, located in the epithelium of bronchioles, also contribute to the composition of mucus through secretion of a nonmucinous material containing carbohydrates and proteins. These cells play a role in bronchial regeneration after injury. As noted previously, the respiratory tract to the level of the bronchioles is lined by a pseudostratified, ciliated columnar epithelium (see

1	As noted previously, the respiratory tract to the level of the bronchioles is lined by a pseudostratified, ciliated columnar epithelium (see Fig. 26.1 ). These cells maintain the level of the periciliary fluid in which cilia and the mucociliary transport system function. Mucus and inhaled particles are removed from the airways by the rhythmic beating of the cilia. There are approximately 250 cilia per airway epithelial cell, and each is 2 to 5 µm in length. Cilia are composed of nine microtubular doublets that surround two central microtubules held together by dynein arms, nexin links, and spokes. The central microtubule doublet contains an adenosine triposphatase (ATPase) that is responsible for the contractile beat of the cilium. Cilia beat with a coordinated oscillation in a characteristic, biphasic, and wave-like rhythm called metachronism. They beat at approximately

1	Sputumisexpectoratedmucus.However,inadditiontomucus,sputumcontainsserumproteins,lipids,electrolytes,Ca++ ,DNAfromdegeneratedwhitebloodcells(collectivelyknownasbronchial secretions),andextrabronchialsecretions;includingnasal,oral,lingual,pharyngeal,andsalivarysecretions.Thecolorofsputumismorecloselycorrelatedwiththeamountoftimethatithasbeenpresentinthelowerrespiratorytractthanwiththepresenceofinfection.Althoughnotpreciselyidentifiablewithdiseasediagnosis,thecolorofsputumcanbeinformativeinhelpingleadtoadiagnosisandstageofdisease.Mucushasmanycolors:white,yellow,green,red,pink,brown,gray,andblack.Thecolorationiscommonlyduetothetypeofcellpresentintheairways(inflammatorycells,suchasneutrophilsoreosinophils,orredbloodcells)andhowlongtheyhavebeenthere.Clearorcloudywhite

1	thinmucusisconsiderednormal;however,ifamountsandthicknessareincreased,itmayrepresentanearlysignofinfection.Thickwhitemucuscanbetheonlyidentifiablefeatureofgastroesophagealrefluxdiseasecausedbygastricacidrefluxintotheairways.Yellow andgreen colorationofmucusisduetothepresenceandbreakdownofneutrophilsandeosinophilsininfectiousandallergicdiseases.Yellowistypicallyassociatedwithmoreacutedisease(infection,allergy)andgreenusuallyindicatesamorechronicstagewiththepresenceofbacteria(chronicbronchitis,bronchiectasis,cysticfibrosis,andlungabscess).Red mucusindicatesthepresenceofredbloodcellsintheairwaysandisassociatedwithpneumococcalpneumonia,lungcancer,tuberculosis,andpulmonaryemboli.Pink mucusistypicallyassociatedwiththebreakdownofeosinophilsinindividualswithallergies.Gray,brown,andblackmucusisoftenassociatedwithcigaretteormarijuanasmoking,cocaineuse,airpollution(workplaceenvironment,suchascoalmines),andoldblood.

1	1000 strokes per minute, with a power forward stroke and a slow return or recovery stroke. During their power forward stroke, the tips of the cilia extend upward into the viscous mucus layer and thereby move it and the entrapped particles. On the reverse beat, the cilia release the mucus and withdraw completely into the sol layer. Cilia in the nasopharynx beat in the direction that propels the mucus into the pharynx, whereas cilia in the trachea propel mucus upward toward the pharynx, where it is swallowed.

1	In general, deposition of particles in the lung depends on the particle’s size, density, and shape; the distance over which it has to travel; airflow speed; and the relative humidity of the air. The four major mechanisms for deposition are impaction, sedimentation, interception, and brownian movement. Particle characteristics and properties, which influence the mechanism of deposition, are listed in 26.2 . In general, particles larger than 10 µm are deposited by impaction in the nasal passages and do not penetrate into the lower respiratory tract. Particles 2 to 10 µm in size Sedimentation0.2to2.0InterceptionNABrownianmovement<0.2 NA,notapplicable.

1	are deposited in the lower respiratory tract predominantly by inertial impaction at points of turbulent airflow (i.e., nasopharynx, trachea, and bronchi) and at airway bifurcations because their tendency to move in a straight direction prevents them from changing directions rapidly. In more distal areas, where airflow is slower, smaller particles (0.2 to 2 µm) are deposited on the surface by sedimentation as a result of gravity. For substances with elongated shapes (i.e., asbestos, silica), the mechanism of deposition is interception. The elongated particle’s center of gravity is compatible with the flow of air; however, when the distal tip of the particulate comes in contact with a cell or mucus layer, deposition is facilitated. Particles smaller than 0.2 µm are deposited in the smaller airways and alveoli and are influenced mainly by their diffusion coefficient and brownian motion. Unlike the deposition of larger particles in the upper airways, particle density does not influence

1	airways and alveoli and are influenced mainly by their diffusion coefficient and brownian motion. Unlike the deposition of larger particles in the upper airways, particle density does not influence diffusion of these smaller particles, and deposition is enhanced with decreased size. These smaller particles come in contact with the alveolar epithelium, where cilia and the mucociliary transport system do not exist; thus they are removed by the phagocytic activity of alveolar macrophages or absorption into the interstitium with subsequent clearance by lymphatic drainage. Although most alveolar macrophages are adjacent to the epithelium of the alveolus, some are located in the terminal airways and interstitial space.

1	In the conducting airways, the mucociliary clearance system transports deposited particles from the terminal bronchioles to the major airways, where they are coughed up and either expectorated or swallowed. Deposited particles can be removed in a matter of minutes to hours. In the trachea and main bronchi, the rate of particle clearance is 5 to 20 µm/minute, but it is slower in the bronchioles (0.5 to 1 µm/minute). In general, the longer an inhaled material remains in the airways, the greater is the probability that the material will cause lung damage. The region from the terminal bronchioles to the alveoli is devoid of ciliated cells and is considered the “Achilles heel” in what is otherwise a highly effective system. The relatively slow rate of particle clearance in this area, which is mediated by macrophages, renders it the most common location for many occupational lung diseases.

1	Insomelungdiseases—forexample,thosecausedbyinhalationofsilicaparticles(silicosis) orcoaldustparticles(pneumoconiosis, the“blacklung”diseaseofcoalminers)—alveolarmacrophagesphagocytizetheparticlesbutareunabletodestroythem,andthemacrophageseventuallydie.Alveolarmacrophageshavelocalizedandconcentratedtheparticlesinthe“Achillesheel”regionofthelung.Theseparticlesarenotremovedviamucociliaryclearanceandeventuallyenterthelunginterstitium.Theensuinginflammatoryresponseleadstoagranulomatouslikelesionwithfibrosis,arestrictivelungdisease.Silicosisandpneumoconiosisareclassicalexamplesofdiseasesoriginatingthroughenvironmentalworkplaceexposure.Increasedawarenessofthecauseofthesediseasesandimprovedworkplaceenvironmentshaveledtoreductionintheincidenceofthesetypesoflungdiseases. Mucosal Immune System: Adaptive and Innate Immunity

1	In nonmucosal tissues (e.g., spleen, liver, kidney), the body’s primary defense is the classical proinflammatory adaptive, antigen-specific immune response orchestrated in the local draining lymph nodes with afferent and efferent lymph flow. The major adaptive immune cells in the systemic immune system are T lymphocytes with αβ T cell receptors (TCRαβ T cells) for specific antigen recognition and plasma B cells that synthesize immunoglobulin M (IgM) and immunoglobulin G (IgG) complement–binding antibodies, which can induce inflammation. However, mucosal tissues (i.e., those of the respiratory, gastrointestinal, and urinary systems, as well as the eyes, nose, throat, and mouth) must constantly discriminate between what is harmful and what is not, and although inflammation is protective, it usually disrupts the normal physiologic processes and is not desirable unless absolutely necessary. Accordingly, mucosal tissues have developed specialized “noninflammatory” defense mechanisms, which

1	usually disrupts the normal physiologic processes and is not desirable unless absolutely necessary. Accordingly, mucosal tissues have developed specialized “noninflammatory” defense mechanisms, which form the basis of the mucosal immune system and can function independently of the systemic immune system. The mucosal immune system contains both specialized innate lymphoid cells (macrophages, natural killer cells, dendritic cells [DCs]) and adaptive T lymphocytes with γδ T cell receptors (TCRγδ T cells) and plasma B cells that synthesize immunoglobulin A (IgA), a nonclassical complement-binding antibody. These innate and unique adaptive responses can prevent or limit responses to foreign nonpathological agents while eliminating pathological agents/substances with little or no inflammation. In addition, if this front-line defense system fails or is bypassed, the lungs do have a classical adaptive immune response system in which lymphatic drainage is via the mediastinal lymph node located

1	addition, if this front-line defense system fails or is bypassed, the lungs do have a classical adaptive immune response system in which lymphatic drainage is via the mediastinal lymph node located in the upper region of the thoracic cavity adjacent to the main left-right lung bifurcation.

1	A distinctive feature of the mucosal immune system is that antigens are processed through lymphoid aggregates rather than through a true lymph node. Unlike a true lymph node, which has afferent and efferent lymph flow, lymphoid aggregates have only afferent drainage of material into the aggregate without efferent flow. This lymphoid network is commonly referred to as mucosa-associated lymphoid tissue (MALT); in the gastrointestinal tract, it is referred to as gut-associated lymphoid tissue (GALT); and in the lungs, it is known as bronchus-associated lymphoid tissue (BALT;

1	Fig. 26.2 ). BALT is present in the conducting airways, where the epithelium is composed mainly of ciliated cells with consistent mucus flow. However, an interesting feature of BALT is that its airway epithelium is not ciliated; it is referred to as a lymphoepithelium, which creates a break in the mucus flow (like a drain) and allows the substances/ particulates to be processed in the lymphoid aggregate (or follicle).

1	It appears that there is communication between mucosal tissues and that sensitization via one organ is transposed to all MALT/BALT tissues via a lymphatic-like drainage network. The systemic immune system and MALT/BALT may work independently of each other, and the fact that one is sensitized may not be true of the other. This may serve as a defense mechanism in limiting sensitization only to mucosal tissue. Lymphocytes also cluster in smaller numbers and density in what is referred to as tertiary ectopic lymphoid tissue (TELT), in which they can also process antigens (see Fig. 26.2, A ). Another prominent feature is a diffuse submucosal and intraepithelial network of solitary lymphocytes and innate lymphoid cells scattered throughout the respiratory tract. Because inhaled particles are broadly dispersed throughout the respiratory tract, each type of lymphoid cell and tissue (BALT, TELT, and solitary lymphocytes) play important and unique roles in the overall defense of the lungs.

1	One of the specialized features of MALT, GALT, and BALT is a unique antibody system in which specialized features • Fig. 26.2 Representation of Bronchus-Associated Lymphoid Tissue (BALT)/Tertiary Ectopic Lymphoid Tissue (TELT), M Cells, and Immunoglobulin a (IgA) Synthesis.A, M cells located in mucosal epithelium endocytose antigen in the lumen and transport it for processing to loosely organized submucosal pockets of lymphoid cells, predominantly lymphocytes and macrophages (TELT). B, Diagram of a mucous membrane, showing secretion of IgA antibodies in response to antigen endocytosed by M cells. Activated B cells differentiate into IgA-producing plasma cells (lymphocytes) and migrate from the densely organized lymphoid follicle (in bronchus-associated lymphoid tissue [BALT]) to the nearby submucosa, where they secret IgA.

1	of the IgA antibody (non–complement-fixing, J chain for transport, and dimeric structure for stability in the airway lumen) are used. In submucosal areas, plasma cells synthesize and secrete IgA, which migrates to the submucosal surface of epithelial cells, where it binds to a surface protein •Fig. 26.3 Structure and Formation of Secretory Immunoglobulin a (IgA). A, SecretoryIgAconsistsofatleasttwoIgAmoleculesthatarecovalentlylinkedviaJchainandcovalentlyassociatedwiththesecretorycomponent.Thesecretorycomponentcontainsfiveimmunoglobulin-likedomainsandislinkedtodimericIgAthroughbindingtoanIgAheavychain.B, SecretoryIgAisformedduringtransportthroughepithelialcells.Poly-Ig,polymericimmunoglobulin. receptor, polymeric immunoglobulin (poly-Ig;

1	receptor, polymeric immunoglobulin (poly-Ig; Fig. 26.3 ). The poly-Ig receptor aids in the pinocytosis of IgA into the epithelial cell and its eventual secretion into the airway lumen. During exocytosis of the IgA complex, the poly-Ig is enzymatically cleaved, and a portion of it, the secretory piece, remains associated with the complex. The secretory piece stays attached to the IgA complex in the airway and helps protect it from proteolytic cleavage. The IgA-antigen immune complex does not bind complement in the same classical manner as do other immune complexes, and thus its proinflammatory properties are limited. The IgA-antibody system is very effective in binding particulates and viruses to form a large complex, which promotes its removal via the mucociliary clearance system, before they invade epithelial cells.

1	Most classical adaptive immune T lymphocytes are CD3+ cells with TCRs that are composed of α and β chains (TCRαβ T cells). These cells mature in the thymus and egress mostly to lymph nodes and the spleen. The classical activation of TCRαβ cells requires antigen processing/ presentation, typically via the major histocompatibility complex, in a DC, usually to induce an inflammatory response. CD3+ T lymphocytes with TCRs expressing γ and δ chains (TCRγδ T cells) also mature in the thymus, but the majority of these cells egress to mucosal tissues (i.e., lung, intestines, and skin) and represent only a minority of T cells in the peripheral blood and systemic lymphoid tissues. TCRγδ T cells, often referred to as intraepithelial lymphocytes, preferentially localize to submucosal sites and

1	BALT,bronchus-associatedlymphoidtissue;IFN-γ,interferonγ;IgA,IgE,IgG,andIgM,immunoglobulinsA,E,G,andM;IL-10,interleukin10;iNKT,invariantnaturalkillerT;NK,naturalkiller;TCR,Tcellreceptor;TELT,tertiaryectopiclymphoidtissue;Th1andTh2,Thelper1andThelper2. epithelium and are considered a first line of defense of epithelial surfaces.

1	In contrast to TCRαβ T cells, the classical antigen activation of TCRγδ T cells does not require antigen processing or presentation by DCs. Of interest is that TCRγδ T cells have been shown to be capable of responding to antigen either in the manner of an innate cellular response, via pathogen-associated molecular patterns (PAMPs, described later in this chapter), or, in some circumstances, in the manner of a classical TCRαβ T cell response. Whether this variation in responsiveness is due to the presence of subpopulations of TCRγδ T cells or to plasticity (alterations) of the same cell is not clear. Because these cells express this unique duality, they are considered to be a link between adaptive and innate immune responses. Furthermore, TCRγδ cells can respond immediately to antigen and generally do not demonstrate memory. In contrast to the typical TCRαβ T cell proinflammatory response, TCRγδ T cells can provide protection without inducing inflammation. TCRγδ T cells have been shown

1	do not demonstrate memory. In contrast to the typical TCRαβ T cell proinflammatory response, TCRγδ T cells can provide protection without inducing inflammation. TCRγδ T cells have been shown to expand in response to viral and bacterial infections and synthesize either proinflammatory cytokines (interleukin [IL]–17, interferon [IFN]–γ) or regulatory/ suppressive cytokines (transforming growth factor β [TGFβ], lymphocyte-activation gene 3). TCRγδ T cells also suppress the immunoglobulin E (IgE) response to inhaled antigen, further preventing allergen-induced inflammation.

1	Innate lymphoid cells (ILCs) generally fall into four categories, TCRγδ T cells, dendritic cells (DCs), macrophages, natural killer (NK) cells, and subpopulations of each ( 26.3 ). ILCs are distinctively different from adaptive immune cells by their lack of “antigen” specificity for the offending agents and their ability to distinguish self from non-self through recognition of PAMPs present on the pathogens. Since most inhaled substances are nonpathogenic, the body has developed a specialized recognition system to identify harmful pathogenic substances and organisms. Rather than specific antigen recognition, this system enables discrimination of self from non-self material through the recognition of PAMPs on the pathogenic organisms/substances, which are then recognized by a family of receptors on host defense cells (i.e., ILCs) called pattern recognition receptors (PRRs). PAMPs are common distinguishing features on many pathogens and are composed of peptidoglycans,

1	by a family of receptors on host defense cells (i.e., ILCs) called pattern recognition receptors (PRRs). PAMPs are common distinguishing features on many pathogens and are composed of peptidoglycans, lipopolysaccharides, and unmethylated 5′-C-phosphate-G-3′ (CpG) DNA, which bind to their corresponding PPRs on ILCs. There are two major classifications for PRRs: toll-like receptors (TLRs), which are expressed mainly on cell surfaces, and nucleotide-oligomerization domain (NOD) receptors, which are expressed intracellularly in the cytoplasma of ILCs. Both of these pathways activate nuclear factor Kβ (NFKβ), a transcription factor, which stimulates release of proinflammatory cytokines, as well the antiviral type I interferons (IFN-α and IFN-β). The TLRs are a family of transmembrane proteins with different specificities for various pathogens. TLR-2 is specific for lipoteichoic acids associated with gram-positive bacteria, whereas TLR-4 is specific for lipopolysaccharide (endotoxin), a

1	with different specificities for various pathogens. TLR-2 is specific for lipoteichoic acids associated with gram-positive bacteria, whereas TLR-4 is specific for lipopolysaccharide (endotoxin), a product of gram-negative bacteria. In the lung, bronchial epithelial cells, macrophages, DCs, mast cells, eosinophils, and alveolar type II epithelial cells express both TLR-2 and TLR-4. Other TLRs are specific for viruses: TLR-3 binds to double-stranded RNA viruses, and TLR-7 and TLR-8 bind to single-stranded RNA viruses. NK cells express TLR-3, TLR-7, and TLR-8 and plasmacytoid DCs, eosinophils, and B cells express TLR-7. A variety of phagocytic cells and DCs in the lung and other mucosal tissues also express TLRs. Thus in addition to classical phagocytic cells, bronchial and alveolar epithelial cells play active roles in host defense by means of the PAMP-PRR nexus.

1	Perhaps the most unique and intriguing aspect of this specialized mucosal defense system is the ability of ILCs to respond to pathogens immediately, as opposed to the days or weeks it takes to mount a classical TCRαβ T cell adaptive immune response with clonal expansion and memory. The fast response, and the lack thereof for innocuous substances, are highly advantageous in the mucosal tissues, which are common sites for parasitic invasion and toxic chemical exposure. In addition, many of the ILCs demonstrate plasticity, meaning that they can be induced by the environment (i.e., cytokines) to alter their functionality to either a proinflammatory or regulatory (suppressive) phentoype.

1	Both macrophages and DCs originate in the bone marrow but vary somewhat in their differentiation lineage: Macrophages develop through the common myeloid-granulocyte/ macrophage progenitor linage with other granulocytic cells (basophils, mast cells, eosinophils, neutrophils), and DCs develop through either the common lymphoid progenitor or the common myeloid progenitor lineages. Macrophages and DCs are the first nonepithelial cells to contact and respond to a foreign substance. In the lungs, there are three major types of macrophages (alveolar, M-1 [proinflammatory], and M-2 [regulatory]) and two types of DCs (conventional and plasmacytoid).

1	If the inhaled foreign material stays within the airspace in the lower respiratory system (alveolar ducts and alveoli), it will probably be phagocytized by alveolar macrophages. However, if the foreign material/organism penetrates and reaches submucosal areas, it will come into contact with M-1 or M-2 macrophages and conventional or plasmacytoid DCs. Alveolar macrophages are found mostly in the alveolus adjacent to the epithelium and less frequently in the terminal airways and interstitial space. They migrate freely throughout the alveolar spaces and serve as a first line of defense in the terminal bronchioles and alveoli. They phagocytize foreign particles and substances, as well as surfactant and cellular debris from dead cells. For a particle/organism that is phagocytized by a macrophage, the major mechanisms of destruction include formation of O2 radicals, enzyme activity, and halogen derivatives within lysosomes.

1	The phagocytic activity of the macrophage inhibits the binding of particulates to the alveolar epithelium and their subsequent penetration into the interstitium. The alveolar macrophage also transports engulfed particles to ciliated regions of the mucociliary transport system for elimination and thus provides an important link between the alveolar spaces, the postterminal bronchiole “Achilles heel” region, and the mucociliary clearance system.

1	In addition, alveolar macrophages and M-2 macrophages present in the submucosa can also suppress T cell activity by direct contact or by the secretion of soluble factors such as nitric acid, prostaglandin E2, and the immunosuppressive cytokines (IL-10 and TGF-β). M-1 macrophages are also located in submucosal sites and represent the classical “proinflammatory” phagocytic cell, with similar killing capabilities as those of alveolar macrophages. In addition, the T helper 2 (Th2) proinflammatory (and proasthma) cytokines IL-4 and IL-13 have been shown to promote the differentiation of M-1 macrophages into regulatory/suppressive M-2 macrophages, which demonstrates the plasticity of these cells. The ability of these macrophage populations to demonstrate plasticity and dispose of foreign material rapidly with either a proinflammatory or regulatory response considerably enhances the lung defense system and is a unique contributor to the overall mucosal defense system.

1	Conventional DCs reside in the submucosa of lung and other mucosa tissues and are considered the major antigen-presenting cells. They are usually in a resting immature state in which they function as sentinels to capture and process antigen, after which they mature and migrate to the local draining lymph node (mediastinal node for the lungs). At this node, they present antigen to T cells, which initiates an adaptive immune response, either proinflammatory or suppressive, depending on the antigen and DC. CD103+ DCs have been shown to induce regulatory/suppressor T cells by means of their synthesis and secretion of TGF-β and indoleamine dioxygenase. Plasmacytoid DCs are located in similar submucosal areas but function more in an antiviral role, as opposed to antigen presentation; upon viral activation, they rapidly secrete the antiviral cytokine IFN-γ. Macrophages and DCs commonly synthesize and secrete many similar cytokines, depending on the stimulus; such cytokines include IL-1β

1	activation, they rapidly secrete the antiviral cytokine IFN-γ. Macrophages and DCs commonly synthesize and secrete many similar cytokines, depending on the stimulus; such cytokines include IL-1β (which activates vascular endothelium and lymphocytes), IL-6 (which activates lymphocytes and enhances antibody production), IL-12 (which activates NK cells, CD4 T cells to Th1 T cells), tumor necrosis factor α (which activates vascular endothelium and increases permeability), IL-8 (which recruits neutrophis, basophils, and T cells), and IFN-γ (which has antiviral effects).

1	NK cells originate in the bone marrow and differentiate through the common lymphoid progenitor lineage . They are neither T or B cells, and resident populations of functionally active NK and invariant natural killer T (iNKT) cells are present in mucosal sites and the lung interstitium. NK cells are a major component of the body’s innate immune system of defense against invading pathogens such as herpesviruses, various bacterial infections, and tumor cells. NK cells can be activated by interferons (IFN-α and IFN-β) and other macrophage-derived cytokines (IL-12 and IL-18), which enhances their killing capabilities. The mechanism of killing is through the release of granular enzymes (granzymes, perforins, and serine esterases), which create holes or pores within the target cell membranes and thereby cause cell death. In addition, NK cells can synthesize IFN-γ, which is capable of inhibiting viral infections and stimulating CD4 and CD8 T cell responses.

1	NK cells have a complicated set of receptors to recognize a wide array of infectious bacteria and viruses. Individuals who lack NK cells are very prone to herpesvirus infections and often have recurrent infection. The iNKT cells belong to a category of lymphoid cells termed innate-like lymphocytes, reside together with TCRγδ cells in mucosal tissues, and have antigen receptors with limited diversity, unlike the adaptive immune cells. These types of cells are thought to be links between innate and adaptive immunity. A critical aspect of the link is that these cells can respond must faster than cells of the classical adaptive immune response, limiting early damage until the adaptive response can mobilize a stronger defense. The iNKT cells have an invariant T cell receptor α chain and, on activation, secrete the regulatory cytokines IL-10 and IFN-γ. Epithelial Cells and Commensal Microbiota Protect the Lumen of the Airways

1	Epithelial Cells and Commensal Microbiota Protect the Lumen of the Airways Airway epithelial cells can produce and secret both antibacterial enzymes and amphipathic (hydrophilic–positively charged) antimicrobial peptides, which generally target and disrupt cell wall components of the bacteria, which leads to cell death. Lysozyme, elastase, hydrolases, and secretory phospholipase A2 are examples of antibacterial enzymes targeted at various pathogens. Antimicrobial peptides are typically cationic peptides synthesized as propeptides and activated through cleavage of an anionic propiece. Peptides secreted from epithelial cells and alveolar macrophages usually enter the mucosal fluid, whereas peptides secreted from submucosal macrophages typically stay within the local tissue.

1	There are three classes of antimicrobial peptides: defensins, cathelicidins, and histatins. Defensins, which are categorized as α and β can disrupt cell membranes within minutes and are very effective on bacteria, fungi, and viruses. β–Defensins are synthesized in alveolar type II cells, are stored in lamellar bodies, and are a component of surfactant. α–Defensins are synthesized and stored within epithelial cells, macrophages, and neutrophils. Cathelicidins are stored in secondary granules and activated intracellularly by fusion of the phagosomes with the granule or also directly by elastase. In the lungs, cathelicidins are secreted from lung type II epithelial cells, as well as from neutrophils and macrophages. Histatins are present mainly in the oral cavity and are more specific for pathogenic fungi. Although it is not clear whether they exist in the respiratory tract, the carbodydrate-binding proteins (lectins) in the gut can directly kill Gram-positive bacteria by binding to

1	pathogenic fungi. Although it is not clear whether they exist in the respiratory tract, the carbodydrate-binding proteins (lectins) in the gut can directly kill Gram-positive bacteria by binding to their cell walls. Furthermore, lactoferrins and lysozyme present in epithelial cells have been shown to have bacteriostatic and bactericidal effects; the precise mechanism by which this occurs is not clearly understood, although it is postulated that they may work synergistically with iron and calcium.

1	The human microbiome, especially the gut, has received a lot of attention, and exploration into the role of these commensal microorganisms in health and disease is rapidly developing. Although much is known about the gut microbiome, the lung microbiome has been difficult to explore. Once thought to be a sterile environment, the lung remains a difficult site to sample for a variety of reasons, including subject safety during sampling (invasive procedures), complexity and variety of epithelial surfaces throughout the respiratory tract, and the simple fact that far fewer bacteria are available to sample. Furthermore, the lumen ecosystem and epithelium of the respiratory tract differ considerably along the respiratory tract, from the upper airways to the alveoli; it is likely that the density and types of bacteria residing in these various regions also differ. It is clear, however, that the lung does have a microbiome, and three primary phyla appear to be represented: Bacteroidetes

1	and types of bacteria residing in these various regions also differ. It is clear, however, that the lung does have a microbiome, and three primary phyla appear to be represented: Bacteroidetes (Prevotella, Bacteroides), Firmicutes (Veillonella, Streptococcus, Staphylococcus), and Proteobacteria (Pseudomonas, Haemophilus, Moraxella, Neisseria, Acinetobacter).

1	Alterations in the lung microbiome have been shown to be associated with several pulmonary conditions (including asthma, cystic fibrosis, and chronic obstructive pulmonary disease) and with lung transplantation. The increased rate of asthma in children has been linked to the increased rate of antibiotic use, which has a vast effect on the microbiome. Organisms in the Proteobacteria (Haemophilus, Moraxella) and Firmicutes (Streptococcus) phyla have been shown to correlate with asthma. An experimental study in mice showed an improvement in asthma when Proteobacteria organisms were replaced with Bacteriodetes organisms, which coincided with an increase in regulatory T cells and suppression of the Th2 proinflammatory cytokines prominent in the pathogenesis of asthma.

1	From a therapeutic standpoint, the use of probiotics to maintain a “normal” microbiome has proved useful in treating infectious diseases in the gut, and the use of probiotic therapy in patients with chronic lung infections (e.g., cystic fibrosis, upper respiratory tract infections) have been promising. The precise mechanism or mechanisms by which the bacteria may “protect” the lungs is still poorly understood but probably varies considerably according to the microorganism.

1	It has been shown that Bifidobacterium can activate DCs and TLRs to secrete IL-10 and retinoic acid, mediators capable of inducing regulatory T cells, which downregulate the Th2 inflammatory response. From a causative perspective, histamine, a major component of mast cell granules and secreted from Lactobacillus organisms, has been shown to have an association with priming DCs to stimulate Th2 inflammatory responses, as seen in individuals with asthma. In addition, it has been shown that ILCs can play a role in influencing the microbiome through their ability to bind to receptors on epithelial cells, thus altering the interaction of the epithelium with the microorganisms and possibly promoting colonization of either pathogenic or nonpathogenic microorganisms. Much is still to be learned and absorbed from future studies of the lung microbiome.

1	By far the most common pathological conditions associated with mucosal tissue are allergic responses (e.g., allergic asthma, allergic rhinitis, and food and skin allergies). Some common human allergens include components of inhaled house dust mites, cockroaches, and cat dander, as well as bee stings and ingestion of peanuts. One of the most common drug allergens is penicillin, which can bind to many endogenous proteins and also alter their antigenicity.

1	As previously described, the predominant antibody response in MALT is IgA; however, in an allergic response, IgE is the predominant antibody. It is generated by a switchover mechanism induced by the synthesis of IL-4 from Th2-primed CD4 T cells. The IL-4 induces the antibody-producing B cells to switch over from synthesizing IgG antibodies to IgE. IgE binds to the surface of mast cells in the submucosa through its crystallizable fragment (constant) region of the antibody molecule. Upon reexposure to the inhaled antigen and its subsequent migration from the airway lumen to the submucosa, the allergen then binds to the antigen-binding fragment region of the IgE molecule, which forms an immune complex (IgE-Ag) on the surface of the mast cell.

1	The final step is that IgE-Ag complexes must crosslink with other IgE-Ag complexes on the surface of the mast cell, which induce intracellular signaling pathways to initiate degranulation and immediate release of preformed Th2 mediators (histamine, heparin, prostaglandins, leukotrienes, IL-4, IL-5, IL-13, proteases). These mediators induce the classical signs of asthma: smooth muscle constriction

1	The inflammatory response can resolve spontaneously or as a result of therapy (bronchodilator or anti-inflammatory drugs, such as corticosteroids). Low-grade inflammation may persist and result in a process called airway remodeling, manifested by permanent, irreversible structural changes such as submucosal fibrosis and airway smooth muscle hypertrophy. The mechanisms responsible for airway remodeling in allergic diseases are not clearly understood, but chemokines and cytokines such as TGF-β, a potent profibrotic cytokine, play important roles. Thus a very elegant, highly effective defensive system against infectious and parasitic organisms is “tricked” into responding to an innocuous substance, an allergen, as if it were harmful and initiates its defenses; the result is allergic airway disease. Metabolic Functions of the Lung

1	Metabolic Functions of the Lung The lungs are exposed to and metabolize a wide variety of xenobiotic substances. The endothelial cells within the lung capillary bed have a large surface area that receives very high blood flow, and lung endothelial cells have developed various mechanisms and cell surface receptors to metabolize xenobiotics. Most of the metabolic processing of inhaled or ingested xenobiotic compounds occurs enzymatically within the liver and intestinal tract with members of the cytochrome P-450 (CYP) enzyme families (e.g., CYP1, CYP2, CYP3). The lungs and other organs also selectively participate in the processing of xenobiotics and typically have lower levels of cytochrome P-450 enzymes. Prominent cytochrome P-450 enzymes in the lung include CYP1B1, CYP2B6, CYP2E1, CYP2J2, CYP3A5, and CYP1A1, the last of which is present in high levels in people who smoke cigarettes.

1	Drugs for the treatment of asthma and chronic obstructive pulmonary disease—such as corticosteroids, long-acting β2 receptor agonists, leukotriene receptor antagonists, and methylxanthines—are degraded enzymatically in the lungs. In addition, a wide array of endogenous substances are metabolized by endothelial cells within the pulmonary capillary bed, including vasoactive amines, cytokines, lipid mediators, and proteins. Box 26.1 provides a list of compounds metabolized in the lung. Metabolism can occur • BOX 26.1 Compounds Metabolized in the Lungs (bronchoconstriction), eosinophil recruitment and activa-Vasoactiveaminestion (inflammation), and connective tissue remodeling. Cytokines Symptoms of wheezing, coughing, and shortness of breath occur within minutes, followed by a late response of eosinophilia and airway inflammation.

1	Symptoms of wheezing, coughing, and shortness of breath occur within minutes, followed by a late response of eosinophilia and airway inflammation. through either intracellular or extracellular processing of endogenous substances that pass though the capillaries or by direct synthesis and secretion by endothelial cells. For example, angiotensin I is activated by angiotensinconverting enzyme, which is on the surface of endothelial cells. Serotonin, a vasoconstrictor, binds to a specific receptor on the surface of endothelial cells and is internalized and metabolized inside cells. Approximately 80% of the serotonin entering the lungs is metabolized in a single pass through the pulmonary capillary bed.

1	Endothelial cells also have surface receptors for bradykinin, tumor necrosis factor, components of the complement system, immunoglobulin crystallizable fragments, and adhesion molecules. In addition, vascular endothelial cells synthesize and secrete prostacyclin, endothelin, clotting factors, nitric oxide, prostaglandins, and cytokines. Vascular endothelial cells, however, lack 5-lipoxygenase and are not able to synthesize leukotrienes (smooth muscle constrictors). Compounds not metabolized by the pulmonary capillary bed include epinephrine, dopamine, histamine, isoproterenol, angiotensin II, and substance P. Angiotensin-convertingenzyme(ACE)ispresentinsmallindentations(caveolae)onthesurfaceofpulmonaryendothelialcellsandcatalyzestheconversionofthephysiologicalinactiveangiotensinItotheactiveangiotensinII,apotentvasoconstrictor.Thisisamajormechanisminthebody’sabilitytosupplysystemiclevelsofangiotensinIIandthusinfluencebloodpressure(see Chapter36).

1	Chapter36). ThetherapeuticuseofACEinhibitorsisimportantinthemanagementofpatientswithhighbloodpressure. Thelungsplayakeyroleinthemetabolismofmanyprodrugs,whichareinactive,intoactivedrugs.Administrationofprodrugsimprovestheamountofactivedrugdeliveredtotheirtargetsinthebody.Inmanycases,inactiveprecursorprodrugsaredeliveredsystemicallyorlocally(throughinhalation)tothelungs,wheretheyareactivatedinsitu.Anexampleofthisisbeclomethasonedipropionate(Qvar,BeconaseAQ),amedicationinhaledbypatientswithasthma,whichisactivatedbyesteraseswithinthelungtotheactiveform17-beclamethasonemonopropionate. 1. The respiratory system has developed unique structural (mucociliary transport system) and immunological (mucosal immune system) features to cope with the constant environmental exposure to foreign substances; these features limit or inhibit inflammation. 2. The three components of the mucociliary transport system are the sol phase (periciliary fluid), the gel phase (mucus), and cilia. 3.

1	2. The three components of the mucociliary transport system are the sol phase (periciliary fluid), the gel phase (mucus), and cilia. 3. The depth of the periciliary fluid layer is maintained by the balance between Cl− secretion and Na+ absorption and is essential to normal ciliary beating. 4. Mucus is a complex macromolecule composed of glycoproteins, proteins, electrolytes, and water. It has low viscosity and high elastic mechanical properties. 5. Goblet cells, Clara cells, and the mucous and serous cells residing in the tracheobronchial glands produce mucus. 6.

1	5. Goblet cells, Clara cells, and the mucous and serous cells residing in the tracheobronchial glands produce mucus. 6. Particle deposition in the lung is dependent on their size, density, and shape; the distance traveled; airflow speed; and relative humidity. The major mechanisms for particle deposition are impaction (particles larger than 10 µm, in nasal passages, and particles 2 to 10 µm, in the nasopharynx, trachea, and bronchi), sedimentation (particles 0.2 to 2 µm in size, in distal airways), interception (particles with elongated shape, in the lower airways), and brownian movement (particles smaller than 0.2 µm, in the alveoli). 7. The respiratory system is part of the mucosal immune system, which is composed of the intestinal (GALT), the respiratory (BALT), and urinary tract systems. These systems do not contain true lymph nodes with afferent and efferent lymph flow; they are composed mainly of nonencapsulated lymph nodules without true lymphatic drainage. 8.

1	8. The nonciliated lymphoepithelium of BALT establishes a break in the mucociliary blanket that acts as a drain to facilitate the collection and immune processing of foreign particulates throughout the conducting airways. 9. TCRγδ T cells, IgA synthesizing plasma cells, NK cells, and alveolar macrophages are highly specialized innate and adaptive immune cells unique to the anti-inflammatory defense system in the lung and other mucosal tissues. Cua DJ, Tato CM. Innate IL-17–producing cells: the sentinels of the immune system. Nat Rev Immunol. 2010;10:479-489. Dickson RP, Erb-Downward JR, Martinez FJ, Huffnagle GB. The microbiome and the respiratory tract. Annu Rev Physiol. 2016;78:481-504. Martin TR, Frevert CW. Innate immunity in the lungs. Proc Am Thorac Soc. 2005;2:403-411. McKenize AN, Spits H, Eberl G. Innate lymphoid cells in inflammation and immunity. Immunity. 2014;41:366-374.

1	McKenize AN, Spits H, Eberl G. Innate lymphoid cells in inflammation and immunity. Immunity. 2014;41:366-374. Monticelli LA, Sonnenberg GF, Abt MC, et al. Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus. Nat Immunol. 2011;12:1045-1054. Olsson B, Bondesson E, Borgström L, et al. Pulmonary drug metabolism, clearance, and adsorption. In: Smyth HDC, Hickey AJ, eds. Controlled Pulmonary Drug Delivery. New York: Springer; 2011:21-51. Spits H, Artis D, Colonna M, et al. Innate lymphoid cells—a proposal for uniform nomenclature. Nat Rev Immunol. 2013;13: 145-149. Vantourout P, Hayday A. The inbetweeners: innate-like lymphocytes. Six of the best: unique contributions of γδ T cells to immunology. Nat Rev Immunol. 2014;13:88-100. Wine JJ. Parasympathetic control of airway submucosal glands: central reflexes and the airway intrinsic nervous system. Auton Neurosci. 2007;133:35-54.

1	Wine JJ. Parasympathetic control of airway submucosal glands: central reflexes and the airway intrinsic nervous system. Auton Neurosci. 2007;133:35-54. SECTION 6Gastrointestinal Physiology KIM E. BARRETT AND HELEN E. RAYBOULD Chapter 27 Functional Anatomy and General Principles of Regulation in the Gastrointestinal Tract Chapter 28 The Cephalic, Oral, and Esophageal Phases of the Integrated Response to a Meal Chapter 29 The Gastric Phase of the Integrated Response to a Meal Chapter 30 The Small Intestinal Phase of the Integrated Response to a Meal Chapter 31 The Colonic Phase of the Integrated Response to a Meal Chapter 32 Transport and Metabolic Functions of the Liver Upon completion of this chapter the student should be able to answer the following questions: 1. What is the neural innervation of the GI tract, and how is GI function regulated? 2. What are some examples of neural, paracrine, and humoral regulation of GI function?

1	he gastrointestinal (GI) tract consists of the alimentary tract from the mouth to the anus and includes the associated glandular organs that empty their contents into the tract. The overall function of the GI tract is to absorb nutrients and water into the circulation and eliminate waste products. The major physiological processes that occur in the GI tract are motility, secretion, digestion, and absorption. Most of the nutrients in the diet of mammals are taken in as solids and as macromolecules that are not readily transported across cell membranes to enter the circulation. Thus digestion consists of physical and chemical modification of food such that absorption can occur across intestinal epithelial cells. Digestion and absorption require motility of the muscular wall of the GI tract to move the contents along the tract and to mix the food with secretions. Secretions from the GI tract and associated organs consist of enzymes, biological detergents, and ions that provide an

1	tract to move the contents along the tract and to mix the food with secretions. Secretions from the GI tract and associated organs consist of enzymes, biological detergents, and ions that provide an intraluminal environment optimized for digestion and absorption. These physiological processes are highly regulated to maximize digestion and absorption, and the GI tract is endowed with complex regulatory systems to ensure this occurs. In addition the GI tract absorbs drugs administered by the oral or rectal routes.

1	The GI tract also serves as an important organ for excretion of substances. It stores and excretes waste substances from ingested food materials and excretes products from the liver such as cholesterol, steroids, and drug metabolites (all sharing the common property of being lipid-soluble molecules). When considering the physiology of the GI tract, it is important to remember that it is a long tube that is in contact with the body’s external environment. As such, it is vulnerable to infectious microorganisms that can enter along with food and water. To protect itself the GI tract possesses a complex system of defenses consisting of immune cells and other nonspecific defense mechanisms. In fact the GI tract represents the largest immune organ of the body. This chapter provides an overview of the functional anatomy and general principles of regulation in the GI system.

1	The structure of the GI tract varies greatly from region to region, but there are common features in the overall organization of the tissue. Essentially the GI tract is a hollow tube divided into major functional segments; the major structures along the tube are the mouth, pharynx, esophagus, stomach, duodenum, jejunum, ileum, colon, rectum, and anus ( Fig. 27.1 ). Together the duodenum, jejunum, and ileum make up the small intestine, and the colon is sometimes referred to as the large intestine. Associated with the tube are blind-ending glandular structures that are invaginations of the lining of the tube; these glands empty their secretions into the gut lumen (e.g., Brunner’s glands in the duodenum, which secrete copious amounts of HCO3 −). Additionally there are glandular organs attached to the tube via ducts through which secretions empty into the gut lumen—for example, the salivary glands and pancreas.

1	The major structures along the GI tract have many functions. One important function is storage; the stomach and colon are important storage organs for processed food (also referred to as chyme) and exhibit specialization in terms of both their functional anatomy (e.g., shape and size) and control mechanisms (characteristics of smooth muscle to produce tonic contractions) that enable them to perform this function efficiently. The predominant function of the small intestine is digestion and absorption; the major specialization of this region of the GI tract is a large surface area over which absorption can occur. The colon reabsorbs water and ions to ensure they do not get eliminated from the body. Ingested food is moved along the GI tract by the action of muscle in its walls. Separating the regions of the GI tract are also specialized muscle structures called sphincters. These function to isolate one region from the next and provide selective retention of contents or prevent backflow,

1	regions of the GI tract are also specialized muscle structures called sphincters. These function to isolate one region from the next and provide selective retention of contents or prevent backflow, or both.

1	The blood supply to the intestine is important for carrying absorbed nutrients to the rest of the body. Unlike other organ systems of the body, venous drainage from the GI tract does not return directly to the heart but first enters • Fig. 27.1 General anatomy of the GI system and its division into functional segments. the portal circulation leading to the liver. Thus the liver is unusual in receiving a considerable part of its blood supply from other than the arterial circulation. GI blood flow is also notable for its dynamic regulation. Splanchnic blood flow receives about 25% of cardiac output, an amount disproportionate to the mass of the GI tract it supplies. After a meal, blood can also be diverted from muscle to the GI tract to subserve the metabolic needs of the gut wall and also to remove absorbed nutrients.

1	The lymphatic drainage of the GI tract is important for the transport of lipid-soluble substances that are absorbed across the GI tract wall. As we will see later, lipids and other lipid-soluble molecules (including some vitamins and drugs) are packaged into particles that are too large to pass into the capillaries and instead pass into lymph vessels in the intestinal wall. These lymph vessels drain into larger lymph ducts, which finally drain into the thoracic duct and thus into the systemic circulation on the arterial side. This has major physiological implications in lipid metabolism and also in the ability of drugs to be delivered straight into the systemic circulation. The wall of the tubular gut is made up of layers consisting of specialized cells ( Fig. 27.2

1	The wall of the tubular gut is made up of layers consisting of specialized cells ( Fig. 27.2 The mucosa is the innermost layer of the GI tract. It consists of the epithelium, the lamina propria, and the muscularis mucosae. The epithelium is a single layer of specialized cells that line the lumen of the GI tract. It forms a continuous layer along the tube and with the glands and organs that drain into the lumen of the tube. Within this cell layer are • Fig. 27.2 General organization of the layers composing the wall of the GI tract.

1	a number of specialized epithelial cells; the most abundant are cells termed absorptive enterocytes, which express many proteins important for digestion and absorption of macronutrients. Enteroendocrine cells contain secretory granules that release regulatory peptides and amines to help regulate GI function. In addition, cells in the gastric mucosa are specialized for production of protons, and mucin-producing cells throughout the GI tract produce a glycoprotein (mucin) that helps protect the GI tract and lubricate the luminal contents.

1	The columnar epithelial cells are linked together by intercellular connections called tight junctions. These junctions are complexes of intracellular and transmembrane proteins, and the tightness of these junctions is regulated throughout the postprandial period. The nature of the epithelium varies greatly from one part of the digestive tract to another, depending on the predominant function of that region. For example, the intestinal epithelium is designed for absorption; these cells mediate selective uptake of nutrients, ions, and water. In contrast, the esophagus has a squamous epithelium that has no absorptive role. It is a conduit for transportation of swallowed food and thus needs some protection, provided by the squamous epithelium, from rough food such as fiber.

1	The surface area of the epithelium is arranged into villi and crypts (Fig. 27.3 ). Villi are finger-like projections that serve to increase the surface area of the mucosa. Crypts are invaginations or folds in the epithelium. The epithelium lining the GI tract is continuously renewed and replaced by dividing cells; in humans this process takes about 3 days. These proliferating cells are localized to the crypts, where there is a proliferative zone of intestinal stem cells. •Fig. 27.3 Comparisonofthemorphologyoftheepitheliumofthesmallintestineandcolon. The lamina propria immediately below the epithelium consists largely of loose connective tissue that contains collagen and elastin fibrils (see

1	The lamina propria immediately below the epithelium consists largely of loose connective tissue that contains collagen and elastin fibrils (see Fig. 27.2 ). The lamina propria is rich in several types of glands and contains lymph vessels and nodules, capillaries, and nerve fibers. The muscularis mucosae is the thin innermost layer of intestinal smooth muscle. When seen through an endoscope, the mucosa has folds and ridges that are caused by contractions of the muscularis mucosae. The next layer is the submucosa (see Fig. 27.2 ), which consists largely of loose connective tissue with collagen and elastin fibrils. In some regions of the GI tract, glands (invaginations or folds of the mucosa) are present in the submucosa. The larger nerve trunks, blood vessels, and lymph vessels of the intestinal wall lie in the submucosa, together with one of the plexuses of the enteric nervous system (ENS), the submucosal plexus.

1	The muscularis externa, or muscularis propria, typically consists of two substantial layers of smooth muscle cells: an inner circular layer and an outer longitudinal layer (see Fig. 27.2 ). Muscle fibers in the circular muscle layer are oriented circumferentially, whereas muscle fibers in the longitudinal muscle layer are oriented along the longitudinal axis of the tube. In humans and most mammals, the circular muscle layer of the small intestine is subdivided into an inner dense circular layer that consists of smaller, more closely packed cells, and an outer circular layer. Between the circular and longitudinal layers of muscle lies the other plexus of the ENS, the myenteric plexus. Contractions of the muscularis externa mix and circulate the contents of the lumen and propel them along the GI tract.

1	The wall of the GI tract contains many interconnected neurons. The submucosa contains a dense network of nerve cells called the submucosal plexus (also referred to as Meissner’s plexus). The prominent myenteric plexus (Auerbach’s plexus) is located between the circular and longitudinal smooth muscle layers. These intramural plexuses constitute the ENS. The ENS helps integrate the motor and secretory activities of the GI system. If the sympathetic and parasympathetic nerves to the gut are cut, many motor and secretory activities continue because the ENS directly controls these processes. The serosa, or adventitia, is the outermost layer of the GI tract and consists of a layer of squamous mesothelial cells (see

1	The serosa, or adventitia, is the outermost layer of the GI tract and consists of a layer of squamous mesothelial cells (see Fig. 27.2 ). It is part of the mesentery that lines the surface of the abdominal wall and suspends the organs within the abdominal cavity. The mesenteric membranes secrete a thin viscous fluid that helps lubricate the abdominal organs so movement of the organs can occur as the muscle layers contract and relax. Regulatory Mechanisms in the Gastrointestinal Tract

1	Regulatory Mechanisms in the Gastrointestinal Tract Unlike the cardiovascular or respiratory systems, the GI tract undergoes periods of relative quiescence (intermeal period) and periods of intense activity after the intake of food (postprandial period). Consequently the GI tract has to detect and respond appropriately to food intake. In addition the macronutrient content of a meal can vary considerably, and there have to be mechanisms that can detect this and mount appropriate physiological responses. Thus the GI tract has to communicate with associated organs such as the pancreas. Finally, because the GI tract is essentially a long tube, there have to be mechanisms by which events occurring in the proximal portion of the GI tract are signaled to the more distal parts, and vice versa. There are three principal control mechanisms involved in the regulation of GI function: endocrine, paracrine, and neurocrine (Fig. 27.4

1	There are three principal control mechanisms involved in the regulation of GI function: endocrine, paracrine, and neurocrine (Fig. 27.4 Endocrine regulation describes the process whereby the sensing cell in the GI tract, an enteroendocrine cell (EEC), responds to a stimulus by secreting a regulatory peptide or hormone that travels via the bloodstream to target cells removed from the point of secretion. Cells responding to a GI hormone express specific receptors for the hormone. Hormones released from the GI tract have effects on cells located in other regions of the GI tract and also on glandular structures associated with the GI tract, such as the pancreas. In addition, GI hormones have effects on other tissues that have no direct role in digestion and absorption, including endocrine cells in liver and brain. EECs are packed with secretory granules, the products of which are released from the cell in response to chemical and mechanical stimuli to the wall of the GI tract (Fig.

1	EECs are packed with secretory granules, the products of which are released from the cell in response to chemical and mechanical stimuli to the wall of the GI tract (Fig. 27.5 ). In addition, EECs can be stimulated by neural input or other factors not associated with a meal. The most common EECs in the gut wall are referred to as the “open” type; these cells have an apical membrane that is in contact with the lumen of the GI tract (generally regarded as the location where sensing occurs) and a basolateral membrane through which secretion occurs. There are also “closed”-type EECs that do not have part of their membrane in contact with the luminal surface of the gut; an example is the enterochromaffin-like (ECL) cell in the gastric epithelium, which secretes histamine. There are many examples of hormones secreted by the GI tract (see

1	There are many examples of hormones secreted by the GI tract (see Table 27.1 ); it is worth remembering that the first hormone ever identified was the GI hormone secretin. One of the most well-characterized GI hormones is gastrin, which is released from endocrine cells located in the wall of the distal part of the stomach. Release of gastrin is stimulated by activation of parasympathetic outflow to the GI tract, and gastrin potently stimulates gastric acid secretion in the postprandial period. •Fig. 27.4 ThethreemechanismsbywhichfunctionintheGItractisregulatedintheintegratedresponsetoameal.

1	•Fig. 27.4 ThethreemechanismsbywhichfunctionintheGItractisregulatedintheintegratedresponsetoameal. Paracrine regulation describes the process whereby a chemical messenger or regulatory peptide is released from a sensing cell (often an EEC) in the intestinal wall that acts on a nearby target cell by diffusion through the interstitial space. Paracrine agents exert their actions on several different cell types in the wall of the GI tract, including smooth muscle cells, absorptive enterocytes, secretory cells in glands, and even on other EECs. There are several important paracrine agents, and they are listed in

1	Table 27.1 along with their site of production, site of action, and function. An important paracrine mediator in the gut wall is histamine. In the stomach, histamine is stored and released by ECL cells located in the gastric glands. Histamine diffuses through the interstitial space in the lamina propria to neighboring parietal cells and stimulates production of acid. Serotonin (5-hydroxytryptamine [5-HT]), released from enteric neurons, mucosal mast cells, and specialized EECs called enterochromaffin cells, regulates smooth muscle function and water absorption across the intestinal wall. There are other paracrine mediators in the gut wall, including prostaglandins, adenosine, and nitric oxide (NO); the functions of these mediators are not well described, but they are capable of producing changes in GI function. Fig. 27.5 Electronmicrographofanopen-typeendocrinecellintheGItract.Notethemicrovilliattheapicalprojectionandthesecretorygranulesinthebasolateralportionofthecell.(FromBarrett

1	Fig. 27.5 Electronmicrographofanopen-typeendocrinecellintheGItract.Notethemicrovilliattheapicalprojectionandthesecretorygranulesinthebasolateralportionofthecell.(FromBarrett K. Gastrointestinal Physiology [Lange Physiology Series]. NewYork:McGraw-Hill;2005.)(CourtesyofLeonardR.Johnson,Ph.D.) Many substances can be both paracrine and endocrine regulators of GI function. For example, cholecystokinin, which is released from the duodenum in response to dietary protein and lipid, acts locally on nerve terminals in a paracrine fashion and also affects the pancreas. This will be discussed in more detail in Neural Regulation of Gastrointestinal Function

1	Neural Regulation of Gastrointestinal Function Nerves and neurotransmitters play an important role in regulating the function of the GI tract. In its simplest form, neural regulation occurs when a neurotransmitter is released from a nerve terminal located in the GI tract and the neurotransmitter has an effect on the cell that is innervated. However, in some cases there are no synapses between motor nerves and effector cells in the GI tract. Neural regulation of GI function is very important within an organ, as well as between distant parts of the GI tract.

1	Therearemultiplereceptorsubtypesfortheregulatorypeptidehormonesreleasedfromendocrinecellsinthewallofthegut.Theselectivityofreceptorstopeptidehormonesisdeterminedbyposttranslationalmodifications,whichthenconfersreceptorselectivity.AnexampleofthisispeptideYY(PYY).TherearemultiplereceptorsubtypesforPYY,classifiedasY1toY7.PYYisreleasedfromendocrinecellsinthewallofthegut,mainlyinresponsetofattyacids.Itisreleasedasa36–aminoacidpeptideandbindstotheY1,Y2,andY5receptors;however,itcanbecleavedtoPYY3-36bytheenzymedipeptidylpeptidaseIV,amembranepeptidase.ThisformofthepeptideismoreselectivefortheY2receptor.ThusthepresenceoftheenzymethatcleavesthepeptidecanalterthebiologicalresponsetoPYYsecretion.

1	Glucagon-likepeptide1(GLP-1)isaregulatorypeptidereleasedfromEECscellsinthegutwallinresponsetothepresenceofluminalcarbohydrateandlipids.GLP-1arisesfromdifferentialprocessingoftheglucagongene,thesamegenethatisexpressedinthepancreasandthatgivesrisetoglucagon.GLP-1isinvolvedinregulationofthebloodglucoselevelviastimulationofinsulinsecretionandalsoinsulinbiosynthesis.AgonistsoftheGLP-1receptorimproveinsulinsensitivityindiabeticanimalmodelsandhumansubjects.AdministrationofGLP-1alsoreducesappetiteandfoodintakeanddelaysgastricemptying,responsesthatmaycontributetoimprovingglucosetolerance.Long-actingagonistsfortheGLP-1receptor(e.g.,exenatide)havebeenapprovedforthetreatmentoftype2diabetes.

1	Neural regulation of the GI tract is surprisingly complex. The gut is innervated by two sets of nerves, the extrinsic and intrinsic nervous systems. The extrinsic nervous system is defined as nerves that innervate the gut, with cell bodies located outside the gut wall; these extrinsic nerves are part of the autonomic nervous system (ANS). The intrinsic nervous system, also referred to as the enteric nervous system, has cell bodies that are contained within the wall of the gut (submucosal and myenteric plexuses). Some GI functions are highly dependent on the extrinsic nervous system, yet others can take place independently of the extrinsic nervous system and are mediated entirely by the ENS. However, extrinsic nerves can often modulate intrinsic nervous system function (

1	Fig. 27.6 •Fig. 27.6 HierarchicalneuralcontrolofGIfunction.StimulitotheGItractfromameal(e.g.,chemical,mechanical,osmotic)willactivateboththeintrinsicandextrinsicsensory(afferent)pathways,whichinturnwillactivatetheextrinsicandintrinsicneuralreflexpathways. Extrinsic neural innervation to the gut is via the two major subdivisions of the ANS, namely, parasympathetic and sympathetic innervation ( Fig. 27.7 ). Parasympathetic innervation to the gut is via the vagus and pelvic nerves. The vagus nerve, the 10th cranial nerve, innervates the esophagus, stomach, gallbladder, pancreas, first part of the intestine, cecum, and the proximal part of the colon. The pelvic nerves innervate the distal part of the colon and the anorectal region, in addition to the other pelvic organs that are not part of the GI tract.

1	Consistent with the typical organization of the parasympathetic nervous system, the preganglionic nerve cell bodies lie in the brainstem (vagus) or the sacral spinal cord (pelvic). Axons from these neurons run in the nerves to the gut (vagus and pelvic nerves, respectively), where they synapse with postganglionic neurons in the wall of the organ, which in this case are enteric neurons in the gut wall. There is no direct innervation of these efferent nerves to effector cells within the wall of the gut; the transmission pathway is always via a neuron in the ENS. Consistent with transmission in the ANS, the synapse between preganglionic and postganglionic neurons is an obligatory nicotinic synapse. That is, the synapse between preganglionic and postganglionic neurons is mediated via acetylcholine released from the nerve terminal and acting at nicotinic receptors localized on the postganglionic neuron, which in this case is an intrinsic neuron.

1	Sympathetic innervation is supplied by cell bodies in the spinal cord and fibers that terminate in the prevertebral ganglia (celiac, superior, and inferior mesenteric ganglia); these are the preganglionic neurons. These nerve fibers synapse with postganglionic neurons in the ganglia, and the fibers leave the ganglia and reach the end organ along the major blood vessels and their branches. Rarely there is a synapse in the paravertebral (chain) ganglia, as seen with sympathetic innervation of other organ systems. Some vasoconstrictor sympathetic fibers directly innervate blood vessels of the GI tract, and other sympathetic fibers innervate glandular structures in the wall of the gut. The ANS, both parasympathetic and sympathetic, also carries the fibers of afferent (toward the central nervous system [CNS]) neurons; these are sensory in nature. The cell bodies for the vagal afferents are in the nodose

1	SuperiorcervicalganglionPrevertebral ganglia1. Celiac2. Superior mesenteric3. Inferior mesenteric 123 •Fig. 27.7 ExtrinsicinnervationoftheGItract,consistingoftheparasympathetic(A)andsympathetic(B)subdivisionsoftheautonomicnervoussystem.

1	ganglion. These neurons have a central projection terminating in the nucleus of the tractus solitarius in the brainstem and the other terminal in the gut wall. The cell bodies of the spinal afferent neurons that run with the sympathetic pathway are segmentally organized and are found in the dorsal root ganglia. Peripheral terminals of the spinal and vagal afferents are located in all layers of the gut wall, where they detect information about the state of the gut. Afferent neurons send this information to the CNS. Information sent to the CNS relays the nature of the luminal contents (e.g., acidity, nutrient content, osmolality of the luminal contents), as well as the degree of stretch or contraction in smooth muscle. Afferent innervation is also responsible for transmitting painful stimuli to the CNS.

1	The components of a reflex pathway—afferents, inter-neurons, and efferent neurons—exist within the extrinsic innervation to the GI tract. These reflexes can be mediated entirely via the vagus nerve (termed a vagovagal reflex), which has both afferent and efferent fibers. The vagal afferents send sensory information to the CNS, where they synapse with an interneuron, which then drives activity in the efferent motor neuron. These extrinsic reflexes are very important in regulating GI function after ingestion of a meal. An example of an important vagovagal reflex is the gastric receptive relaxation reflex, in which distention of the stomach results in relaxation of the smooth muscle in the stomach; this allows filling of the stomach to occur without an increase in intraluminal pressure.

1	In general, as with other visceral organ systems, the parasympathetic and sympathetic nervous systems tend to work in opposition. However, this is not as simple as in the cardiovascular system, for example. Activation of the parasympathetic nervous system is important in the integrative response to a meal and is discussed in the following chapters. The parasympathetic nervous system generally results in activation of physiological processes in the gut wall, although there are notable exceptions. In contrast, the sympathetic nervous system tends to be inhibitory to GI function and is more frequently activated in pathophysiological circumstances. Overall, sympathetic activation inhibits smooth muscle function. The exception to this is the sympathetic innervation of GI sphincters, in which sympathetic activation tends to induce contraction of smooth muscle. Moreover, the sympathetic nervous system is notably important in regulation of blood flow in the GI tract.

1	The ENS is made up of two major plexuses, which are collections of nerve cell bodies (ganglia) and their fibers, all originating in the wall of the gut ( Fig. 27.8). The myenteric plexus lies between the longitudinal and circular muscle layers, and the submucosal plexus lies in the submucosa. Interganglionic strands link neurons in the two plexuses.

1	Neurons in the ENS are characterized functionally as afferent neurons, interneurons, or efferent neurons, similar to neurons in the extrinsic part of the ANS. Thus all components of a reflex pathway can be contained within the ENS. Stimuli in the wall of the gut are detected by afferent neurons, which activate interneurons and then efferent neurons to alter function. In this way the ENS can act autonomously from extrinsic innervation. However, neurons in the ENS, as we have already seen, are innervated by extrinsic neurons, and thus the function of these reflex pathways can be modulated by the extrinsic nervous system. Because the ENS is capable of performing its own integrative functions and complex reflex pathways, it is sometimes referred to as the “little brain in the gut” as a result of its importance and complexity. It is estimated that there are as many neurons in the ENS as in the spinal cord. In addition, many GI hormones also act as neurotransmitters in the ENS and in the

1	of its importance and complexity. It is estimated that there are as many neurons in the ENS as in the spinal cord. In addition, many GI hormones also act as neurotransmitters in the ENS and in the brain in regions involved in autonomic outflow. These mediators and regulatory peptides are thus • Fig. 27.8 The enteric nervous system in the wall of the GI tract.

1	referred to as brain-gut peptides, and the extrinsic and intrinsic components innervating the gut are sometimes referred to as the brain-gut axis. Response of the GI Tract to a Meal

1	Response of the GI Tract to a Meal This introductory chapter provides a broad overview of the anatomy and regulatory mechanisms in the GI tract. In the following chapters there will be discussion of the integrated response to a meal to provide the details of GI physiology. The response to a meal is classically divided into phases: cephalic, oral, esophageal, gastric, duodenal, and intestinal. In each phase the meal presents certain stimuli (e.g., chemical, mechanical, and osmotic) that activate different pathways (e.g., neural, paracrine, and humoral reflexes) that result in changes in effector function (e.g., secretion and motility). There is considerable crosstalk between the regulatory mechanisms that have been outlined, and this will be discussed in the next chapters. As with maintenance of homeostasis in other systems of the body, control of GI function requires complex regulatory mechanisms to sense and act in a dynamic fashion. 1.

1	1. The GI tract is a tube subdivided into regions that subserve different functions associated with digestion and absorption. 2. The lining of the GI tract is subdivided into layers— the mucosal, submucosal, and muscle layers. 3. There are three major control mechanisms: hormonal, paracrine, and neurocrine. 4. The innervation of the GI tract is particularly interesting because it consists of two interacting components, extrinsic and intrinsic. 5. Extrinsic innervation (cell bodies outside the wall of the GI tract) consists of the two subdivisions of the Baldwin GS. Posttranslational modification of gastrointestinal peptides. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012. Brierley SM, et al. Innervation of the gastrointestinal tract by spinal and vagal afferent nerves. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012.

1	Brierley SM, et al. Innervation of the gastrointestinal tract by spinal and vagal afferent nerves. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012. Brookes SJ, et al. Extrinsic primary afferent signalling in the gut. Nat Rev Gastroenterol Hepatol. 2013;10:286-296. Chao C, Hellmich MR. Gastrointestinal peptides: gastrin, cholecystokinin, somatostatin and ghrelin. In: Johnson LR, ed. ANS: parasympathetic and sympathetic. Both have an important sensory (afferent) component. 6. The intrinsic or enteric nervous system (cell bodies in the wall of the GI tract) can act independently of extrinsic neural innervation. 7. When a meal is in different regions of the tract, sensory mechanisms detect the presence of the nutrients and mount appropriate physiological responses in that region of the tract, as well as in more distal regions. These responses are mediated by endocrine, paracrine, and neurocrine pathways.

1	Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012. Furness JB. The enteric nervous system and neurogastroenterology. Nat Rev Gastroenterol Hepatol. 2012;9:286-294. Gomez GA, et al. Postpyloric gastrointestinal peptides. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012. Parker HE, et al. The role of gut endocrine cells in control of metabolism and appetite. Exp Physiol. 2014;99:1116-1120. Sandoval DA, D’Alessio DA. Physiology of proglucagon peptides: role of glucagon and GLP-1 in health and disease. Physiol Rev. 2015;95:513-548. 28 The Cephalic, Oral, and Esophageal Phases of the Integrated Response to a Meal Upon completion of this chapter the student should be able to answer the following questions: 1. What are the structures of the functional anatomy of salivary glands, including their secretory elements? 2. What are the cephalic and oral phases (what, why, how it happens) of the response to a meal? 3.

1	2. What are the cephalic and oral phases (what, why, how it happens) of the response to a meal? 3. What are the general principles of secretion along the gastrointestinal (GI) tract (where do secretions come from, what are the components)? 4. How do the components of secretion vary with the gland or region of the GI tract? 5. What is the correlation between the composition and functions of salivary secretion? 6. How are primary and secondary secretion within salivary glands generated and regulated? 7. What is the sequence of events in swallowing? 8. What are the stimulus and neural pathways generating primary and secondary esophageal peristalsis? 9. What changes in gastric motility take place during swallowing, and what is the significance? 10. What are the major functions of the esophagus and associated structures in terms of protection and propulsion?

1	10. What are the major functions of the esophagus and associated structures in terms of protection and propulsion? his chapter will describe the processes that occur in the gastrointestinal (GI) tract in the early stages of the integrated response to a meal. There are changes in GI tract physiology (1) before food is ingested (the cephalic phase), (2) when ingested food is in the mouth (the oral phase), and (3) when food is transferred from the mouth to the esophagus (the esophageal phase). The responses of the GI tract to the presence of food are mainly associated with preparing the GI tract for digestion and absorption.

1	The main feature of the cephalic phase is activation of the GI tract in readiness for the meal. The stimuli involved are cognitive and include anticipation or thinking about the consumption of food, olfactory input, visual input (seeing or smelling appetizing food when hungry), and auditory input. The latter may be an unexpected link but was clearly demonstrated in the classic conditioning experiments of Pavlov, in which he paired an auditory stimulus to the presentation of food to dogs; eventually the auditory stimulus alone could stimulate secretion. A real-life analogy is presumably being told that dinner is ready. All these stimuli result in an increase in excitatory parasympathetic neural outflow to the gut. Sensory input (e.g., smell) stimulates sensory nerves that activate parasympathetic outflow from the brainstem. Higher brain sites (e.g., limbic system, hypothalamus, cortex) are also involved in the cognitive components of this response. The response can be both positive and

1	outflow from the brainstem. Higher brain sites (e.g., limbic system, hypothalamus, cortex) are also involved in the cognitive components of this response. The response can be both positive and negative; thus anticipation of food and a person’s psychological status, such as anxiety, can alter the cognitive response to a meal. However, the final common pathway is activation of the dorsal motor nucleus in the brainstem, the region where the cell bodies of the vagal preganglionic neurons arise. Activation of the nucleus leads to increased activity in efferent fibers passing to the GI tract in the vagus nerve. In turn the efferent fibers activate the postganglionic motor neurons (referred to as motor because their activation results in change of function of an effector cell). Increased parasympathetic outflow enhances salivary secretion, gastric acid secretion, pancreatic enzyme secretion, gallbladder contraction, and relaxation of the sphincter of Oddi (the sphincter between the common

1	outflow enhances salivary secretion, gastric acid secretion, pancreatic enzyme secretion, gallbladder contraction, and relaxation of the sphincter of Oddi (the sphincter between the common bile duct and duodenum). All these responses enhance the ability of the GI tract to receive and digest the incoming food. The salivary response is mediated via the ninth cranial nerve; the remaining responses are mediated via the vagus nerve.

1	Many of the features of the oral phase are indistinguishable from the cephalic phase. The only difference is that food is in contact with the surface of the GI tract. Thus there are additional stimuli generated from the mouth, both mechanical and chemical (taste). However, many of CHAPTER 28 The Cephalic, Oral, and Esophageal Phases of the Integrated Response to a Meal the responses initiated by the presence of food in the oral cavity are identical to those initiated in the cephalic phase, because the efferent pathway is the same. The responses specifically initiated in the mouth, which consist mainly of the stimulation of salivary secretion, will be discussed next.

1	The mouth is important for the mechanical disruption of food and for initiation of digestion. Chewing subdivides and mixes the food with the enzymes salivary amylase and lingual lipase and with the glycoprotein mucin, which lubricates food for chewing and swallowing. Minimal absorption occurs in the mouth, although alcohol and some drugs are absorbed from the oral cavity, and this can be clinically important. However, as with the cephalic phase, it is important to realize that stimulation of the oral cavity initiates responses in the more distal GI tract, including increased gastric acid secretion, increased pancreatic enzyme secretion, gallbladder contraction, and relaxation of the sphincter of Oddi, mediated via the efferent vagal pathway. Properties of Secretion

1	Secretions in the GI tract come from glands associated with the tract (salivary glands, pancreas, and liver), from glands formed by the gut wall itself (e.g., submucosal glands in esophagus and duodenum), and from the intestinal mucosa itself. The exact nature of the secretory products can vary tremendously, depending on the function of that region of the GI tract. However, these secretions have several characteristics in common. Secretions from the GI tract and associated glands include water, electrolytes, protein, and humoral agents. Water is essential for generating an aqueous environment for efficient enzyme action. Secretion of electrolytes is important for generation of osmotic gradients to drive the movement of water. Digestive enzymes in secreted fluid catalyze the breakdown of macronutrients in ingested food. Moreover, many additional proteins secreted along the GI tract have specialized functions, some of which are fairly well understood, such as those of mucin and

1	of macronutrients in ingested food. Moreover, many additional proteins secreted along the GI tract have specialized functions, some of which are fairly well understood, such as those of mucin and immunoglobulins, and others that are only just beginning to be understood, such as those of trefoil peptides.

1	Secretion is initiated by multiple signals associated with the meal, including chemical, osmotic, and mechanical components. Secretion is elicited by the action of specific effector substances called secretagogues acting on secretory cells. Secretagogues work in one of the three ways that have already been described in Chapter 27—endocrine, paracrine, and neurocrine. Constituents of Secretions Inorganic secretory components are region or gland specific, depending on the particular conditions required in that part of the GI tract. The inorganic components are electrolytes, including H+ and HCO3 − . Two examples of different secretions include acid (HCl) in the stomach, which is important to activate pepsin and start protein digestion, and HCO3 − in the duodenum, which neutralizes gastric acid and provides optimal conditions for the action of digestive enzymes in the small intestine.

1	Organic secretory components are also gland or organ specific and depend on the function of that region of the gut. The organic constituents are enzymes (for digestion), mucin (for lubrication and mucosal protection), and other factors such as growth factors, immunoglobulins, bile acids, and absorptive factors. During the cephalic and oral phases of the meal, considerable stimulation of salivary secretion takes place. Saliva has a variety of functions, including those important for the integrative responses to a meal and for other physiological processes ( Box 28.1 ). The main functions of saliva in digestion include lubrication and moistening of food for swallowing, solubilization of material for taste, initiation of carbohydrate digestion, and clearance and neutralization of refluxed gastric secretions in the esophagus. Saliva also has antibacterial actions that are important for overall health of the oral cavity and teeth. Functional Anatomy of the Salivary Glands

1	Functional Anatomy of the Salivary Glands There are three pairs of major salivary glands: parotid, submandibular, and sublingual. In addition, many smaller glands are found on the tongue, lips, and palate. These glands are the typical tubuloalveolar structures of glands located in the GI tract ( Fig. 28.1 ). The acinar portion of the gland is classified according to its major secretion: serous (“watery”), mucous, or mixed. The parotid gland produces mainly serous secretion, the sublingual gland secretes mainly mucus, and the submandibular gland produces a mixed secretion.

1	Cells in the secretory end pieces, or acini, are called acinar cells and are characterized by basally located nuclei, abundant rough endoplasmic reticulum, and apically located secretory granules that contain the enzyme amylase and other secreted proteins. There are also mucous cells in the acinus; the granules in these cells are larger and contain the specialized glycoprotein mucin. There are three kinds of ducts in the gland that transport secretions from the acinus to the opening in the mouth and also modify the secretion: intercalated ducts drain acinar fluid into larger •BOX 28.1FunctionsofSalivaandChewing

1	Disruption of food to produce smaller particles Formation of a bolus for swallowing Initiation of starch and lipid digestion Facilitation of taste Production of intraluminal stimuli in the stomach Regulation of food intake and ingestive behavior Cleansing of the mouth and selective antibacterial action Neutralization of refluxed gastric contents Mucosal growth and protection in the rest of the GI tract Aid in speech 522 SECTION6Berne & Levy Physiology • Fig.28.1General structure of tubuloalveolar secretory glands (e.g., salivary glands, pancreas) associ-ated with the digestive tract. Demilune of serous cells Serous cell Basement membrane Intercalated duct Mucous cell Salivary duct (secretory) ducts, the striated ducts, which then empty into even larger excretory ducts. In addition, a single large duct from each gland drains saliva to the mouth. The ductal cells lining the striated ducts, in particular, modify the ionic composition and osmolarity of saliva. Composition of Saliva

1	Composition of Saliva The important properties of saliva are a large flow rate relative to the mass of gland, low osmolarity, high K+ concentration, and organic constituents, including enzymes (amylase, lipase), mucin, and growth factors. The latter are not important in the integrated response to a meal but are essential for long-term maintenance of the lining of the GI tract. The inorganic composition is entirely dependent on the stimulus and the rate of salivary flow. In humans, salivary secretion is always hypotonic. The major components are − , Ca++ , Mg++ Na+ , K+ , HCO3 , and Cl− . Fluoride can be secreted in saliva, and fluoride secretion forms the basis of oral fluoride treatment for prevention of dental caries. The concentration of ions varies with the rate of secretion; the flow rate of salivary secretion is stimulated during the postprandial period.

1	The primary secretion is produced by acinar cells in the secretory end pieces (or acini) and is modified by duct cells as saliva passes through the ducts. The primary secretion is isotonic, and the concentration of the major ions is similar to that in plasma. Secretion is driven predominantly by Ca++-dependent signaling, which opens apical Cl− channels in the acinar cells. Cl− therefore flows out into the duct lumen and establishes an osmotic and electrical gradient. Because the epithelium of the acinus is relatively leaky, Na+ and water then follow across the epithelium via the tight junctions (i.e., via paracellular transport). Transcellular water movement may also occur, mediated by aquaporin 5 water channels. The amylase content and rate of fluid secretion vary with the type and level of stimulus. As the fluid passes along the ducts, the excretory and striated duct cells modify the ionic composition of the primary secretion to produce the secondary secretion. The duct cells

1	level of stimulus. As the fluid passes along the ducts, the excretory and striated duct cells modify the ionic composition of the primary secretion to produce the secondary secretion. The duct cells reabsorb Na+ and Cl− , and secrete K+ and HCO3 − into the lumen. Na+ is exchanged for protons, but some of the secreted protons are then reabsorbed in exchange for K+ . HCO3 − on the other hand is secreted only in exchange for Cl−, thereby alkalinizing salivary secretion.

1	At rest, final salivary secretion is hypotonic and slightly alkaline. The alkalinity of saliva is important in restricting microbial growth in the mouth, as well as in neutralizing refluxed gastric acid once the saliva is swallowed. When salivary secretion is stimulated, there is a small decrease in the K+ concentration (but it always remains above plasma concentrations), the Na+ concentration increases toward plasma levels, and Cl− and HCO3 − concentrations increase, thus the secreted fluid becomes even more alkaline ( Fig. 28.2 ). Note that HCO3 − secretion can be directly stimulated by the action of secretagogues on duct cells. The duct epithelium is relatively tight and lacks expression of aquaporin, and therefore water cannot follow the ions rapidly enough to maintain isotonicity at moderate or high flow rates during stimulated salivary secretion. Thus with

1	CHAPTER 28 The Cephalic, Oral, and Esophageal Phases of the Integrated Response to a Meal 1.0 2.0 3.0 4.0 Flow of saliva (mL/min) Na+ Saliva Plasma Cl– K+ Cl– Na+ K+ HCO– 3 HCO– 3 Amylase-containing PRIMARY SECRETION (nearly isotonic; levels of Na+, K+, Cl–, and probably HCO3 similar to plasma) Modification of ionic content • Fig. 28.2 A, The composition of salivary secretion as a function of the salivary flow rate compared with the concentration of ions in plasma. Saliva is hypotonic to plasma at all flow rates. [HCO3 −] in saliva exceeds that in plasma except at very low flow rates. B, Schematic representation of the two-stage model of salivary secretion. The primary secretion containing amylase and electrolytes is produced in the acinar cell. The concentration of electrolytes in plasma is similar to that in the primary secretion, but it is modified as it passes through ducts that absorb Na+ and Cl− and secrete K+ and HCO3 − .

1	an increase in secretion rate, there is less time for ionic modification by the duct cells, and the resulting saliva more closely resembles the primary secretion and therefore plasma. However, [HCO3 −] remains high because secretion from duct cells and possibly acinar cells is stimulated (see Fig. 28.2). The organic constituents of saliva—proteins and glycoproteins—are synthesized, stored, and secreted by the acinar cells. The major products are amylase (an enzyme that initiates starch digestion), lipase (important for lipid digestion), glycoprotein (mucin, which forms mucus when hydrated), and lysozyme (attacks bacterial cell walls to limit colonization of bacteria in the mouth). Although salivary amylase begins the process of digestion of carbohydrates, it is not required in healthy adults because of the excess of pancreatic amylase. Similarly the importance of lingual lipase is unclear. Metabolism and Blood Flow of Salivary Glands

1	Metabolism and Blood Flow of Salivary Glands The salivary glands produce a prodigious flow of saliva. The maximal rate of saliva production in humans is about 1 mL/ min/g of gland; thus at this rate, the glands are producing their own weight in saliva each minute. Salivary glands have a high rate of metabolism and high blood flow; both are proportional to the rate of saliva formation. Blood flow to maximally secreting salivary glands is approximately 10 times that of an equal mass of actively contracting skeletal muscle. Stimulation of the parasympathetic nerves to salivary glands increases blood flow by dilating the vasculature of the glands. Vasoactive intestinal polypeptide (VIP) and acetylcholine are released from parasympathetic nerve terminals in the salivary glands and are vasodilatory during secretion. Regulation of Salivary Secretion

1	Regulation of Salivary Secretion Control of salivary secretion is exclusively neural. In contrast, control of most other GI secretions is primarily hormonal. Salivary secretion is stimulated by both the sympathetic and parasympathetic subdivisions of the autonomic nervous system. Excitation of either sympathetic or parasympathetic nerves to the salivary glands stimulates salivary secretion. Primary physiological control of the salivary glands during the response to a meal is by the parasympathetic nervous system. If the parasympathetic supply is interrupted, salivation is severely impaired and the salivary glands atrophy.

1	Sympathetic fibers to the salivary glands stem from the superior cervical ganglion. Preganglionic parasympathetic fibers travel via branches of the facial and glossopharyngeal nerves (cranial nerves VII and IX, respectively). These fibers form synapses with postganglionic neurons in ganglia in or near the salivary glands. The acinar cells and ducts are supplied with parasympathetic nerve endings. Parasympathetic stimulation increases synthesis and secretion of salivary amylase and mucins, enhances the transport activities of the ductular epithelium, greatly increases blood flow to the glands, and stimulates glandular metabolism and growth. Ionic Mechanisms of Salivary Secretion

1	Ionic Mechanisms of Salivary Secretion Fig. 28.3 shows a simplified view of the mechanisms of ion secretion by serous acinar cells. The basolateral membrane of the cell contains Na+,K+-ATPase and an Na+-K+-2Cl− symporter. The concentration gradient for Na+ across the basolateral membrane, which is dependent on Na+,K+-ATPase, provides the driving force for entry of Na+ , K+ , and Cl− into the cell. Cl− and HCO3 − leave the acinar cell and enter the lumen via an anion channel located in the apical membrane of the acinar cell. This secretion of anions drives the entry of Na+ and thus water into the acinar lumen across the relatively leaky tight junctions. Acinar cell fluid secretion is strongly enhanced in response to elevations in intracellular [Ca++] as a result of activation of the muscarinic receptor for acetylcholine.

1	Acinar cell fluid secretion is strongly enhanced in response to elevations in intracellular [Ca++] as a result of activation of the muscarinic receptor for acetylcholine. Fig. 28.4 shows a simplified model of ion transport processes in epithelial cells of the excretory and striated ducts. Na+,K+-ATPase located in the basolateral membrane maintains the electrochemical gradients for Na+ and K+ that drive most of the other ionic transport processes of the cell. In the apical membrane the parallel operation of the Na+/ H+ antiporter, the Cl−/HCO3 − antiporter, and the H+/K+ antiporter results in absorption of Na+ and Cl− from the lumen and secretion of K+ and HCO3 − into the lumen. The relative impermeability of the ductular epithelium to water prevents the ducts from absorbing too much water by osmosis. • Fig.28.3Ionic transport mechanism involved in the secretion of amylase and electrolytes in salivary acinar cells. ATP2Cl–HCO3LumenofacinusNa˜Na˜Na˜Cl°Na˜K˜K˜K˜K˜°H˜

1	• Fig.28.3Ionic transport mechanism involved in the secretion of amylase and electrolytes in salivary acinar cells. ATP2Cl–HCO3LumenofacinusNa˜Na˜Na˜Cl°Na˜K˜K˜K˜K˜°H˜ Swallowing can be initiated voluntarily, but thereafter it is almost entirely under reflex control. The swallowing reflex is a rigidly ordered sequence of events that propel food from the mouth to the pharynx and from there to the stomach. This reflex also inhibits respiration and prevents entrance of food into the trachea during swallowing. The afferent limb of the swallowing reflex begins when touch receptors, most notably those near the opening of the pharynx, are stimulated. Sensory impulses from these receptors are transmitted to an area in the medulla and lower pons called the swallowing center. Motor impulses travel from the swallowing center to the musculature of the pharynx and upper esophagus via various cranial nerves and to the remainder of the esophagus by vagal motor neurons.

1	The timing of events in swallowing is shown in Fig. 28.5 . The voluntary phase of swallowing is initiated when the tip of the tongue separates a bolus of food from the mass of food in the mouth. First the tip of the tongue and later the more posterior portions of the tongue press against the hard palate. The action of the tongue moves the bolus upward and then backward into the mouth. The bolus is forced into the pharynx, where it stimulates the touch receptors CHAPTER 28 The Cephalic, Oral, and Esophageal Phases of the Integrated Response to a Meal • Fig.28.4Ionic transport mechanism involved in secretion and absorption in epithelial cells of the striated and excretory duct of the salivary gland. ATPHCO3LumenofductNa˜Na˜Na˜Na˜Cl°Cl°K˜K˜K˜°H˜H˜H˜

1	The acinar cells and duct cells of the salivary glands respond to both cholinergic and adrenergic agonists. Nerves stimulate the release of acetylcholine, norepinephrine, substance P, and VIP by salivary glands, and these hormones increase the secretion of amylase and the flow of saliva. These neurotransmitters act mainly by elevating the intracellular concentration of cyclic adenosine monophosphate (cAMP) and by increasing the concentration of Ca++ in the cytosol. Acetylcholine and substance P, acting on muscarinic and tachykinin receptors, respectively, increase the cytosolic concentration of Ca++ in serous acinar cells. In contrast, norepinephrine, acting on β receptors, and VIP, binding to its receptor, elevate the cAMP concentration in acinar cells. Agonists that elevate the cAMP concentration in serous acinar cells elicit a secretion that is rich in amylase; agonists that mobilize Ca++ elicit a secretion that is more voluminous but has a lower concentration of amylase.

1	cAMP concentration in serous acinar cells elicit a secretion that is rich in amylase; agonists that mobilize Ca++ elicit a secretion that is more voluminous but has a lower concentration of amylase. Ca++-mobilizing agonists may also elevate the concentration of cyclic guanosine monophosphate (cGMP), which may mediate the trophic effects evoked by these agonists.

1	that initiate the swallowing reflex. The pharyngeal phase of swallowing involves the following sequence of events, which occur in less than 1 second: 1. The soft palate is pulled upward and the palatopharyngeal folds move inward toward one another; these movements 0.2 0.4 0.6 0.8 1.0 1.2 Time (sec) Bolus in mouth Bolus moves through pharynx and UES Bolus enters esophagus Tongue thrust up and back Nasopharynx closed Larynx elevated Airway closed UES open Pharynx contracts • Fig. 28.5 Timing of motor events in the pharynx and UES during a swallow. Xerostomia, or dry mouth, is caused by impaired salivary secretion. It can be congenital or develop as part of an autoimmune process. The decrease in secretion reduces the pH in the oral cavity, which causes tooth decay and is associated with esophageal erosions. Reduced secretion also causes difficulty swallowing.

1	The ability to measure and monitor a wide range of molecular components that are indicative of overall health is useful in diagnosis and monitoring. Saliva is easy to access, and collection of it is noninvasive. It is used to identify individuals with disease (presence of biomarkers) and to monitor the progress of affected individuals under treatment. In endocrinology, levels of steroids can be measured in the free form rather than as the free and bound form, as in plasma (e.g., the stress hormone cortisol and the sex hormones estradiol, progesterone, and testosterone). Viral infections such as human immunodeficiency virus (HIV), herpes, hepatitis C, and Epstein-Barr virus infection can be detected by polymerase chain reaction (PCR) techniques. Bacterial infections, such as Helicobacter pylori, can likewise be detected in saliva, and saliva is also used for monitoring drug levels.

1	prevent reflux of food into the nasopharynx and open a narrow passage through which food moves into the pharynx. 2. The vocal cords are pulled together and the larynx is moved forward and upward against the epiglottis; these actions prevent food from entering the trachea and help open the upper esophageal sphincter (UES). 3. The UES relaxes to receive the bolus of food. 4. The superior constrictor muscles of the pharynx then contract strongly to force the bolus deeply into the pharynx. A peristaltic wave is initiated with contraction of the pharyngeal superior constrictor muscles, and the wave moves toward the esophagus. This wave forces the bolus of food through the relaxed UES. During the pharyngeal stage of swallowing, respiration is also reflexively inhibited. After the bolus of food passes the UES, a reflex action causes the sphincter to constrict.

1	Gastroesophageal reflux disease (GERD) is commonly referred to as heartburn or indigestion. It occurs when the lower esophageal sphincter allows the acidic contents of the stomach to reflux back into the distal part of the esophagus. This region of the esophagus, unlike the stomach, does not have a robust system to protect the mucosal lining. Thus the acid will activate pain fibers and thereby result in discomfort and pain. This is not an uncommon phenomenon, even in healthy individuals. In the long term, continual reflux can result in damage to the esophageal mucosa. In this case, this condition is classed as GERD and can be treated by H2 receptor antagonists that reduce gastric acid secretion (e.g., ranitidine [Zantac]) or by proton pump inhibitors (e.g., omeprazole [Prilosec]). The esophagus, the UES, and the lower esophageal sphincter (LES) serve two main functions (

1	The esophagus, the UES, and the lower esophageal sphincter (LES) serve two main functions ( Fig. 28.6 ). First, they propel food from the mouth to the stomach. Second, the sphincters protect the airway during swallowing and protect the esophagus from acidic gastric secretions.

1	The stimuli that initiate the changes in smooth muscle activity that result in these propulsive and protective functions are mechanical and consist of pharyngeal stimulation during swallowing and distention of the esophageal wall itself. The pathways are exclusively neural and involve both extrinsic and intrinsic reflexes. Mechanosensitive afferents in both the extrinsic (vagus) nerves and intrinsic neural pathways respond to esophageal distention. These pathways include activated reflex pathways via the brain-stem (extrinsic, vagus) or solely intrinsic pathways. The striated muscle is regulated from the nucleus ambiguus in the brainstem, and the smooth muscle is regulated by parasympathetic outflow via the vagus nerve. The changes in function resulting from mechanosensitive stimuli and activation of reflex pathways are peristalsis of striated and smooth muscle, relaxation of the LES, and relaxation of the proximal portion of the stomach.

1	Functional Anatomy of the Esophagus and Associated Structures The esophagus, like the rest of the GI tract, has two muscle layers—circular and longitudinal—but the esophagus is one of two places in the gut where striated muscle occurs, the other being the external anal sphincter. The type of muscle (striated or smooth) in the esophagus varies along its length. The UES and LES are formed by thickening of striated or circular smooth muscle, respectively. Motor Activity During the Esophageal Phase The UES, esophagus, and LES act in a coordinated manner to propel material from the pharynx to the stomach. At the end of a swallow, a bolus passes through the UES, and the presence of the bolus, via stimulation of mechanoreceptors and reflex pathways, initiates a peristaltic wave (alternating contraction and relaxation of the muscle) along the esophagus that is called primary peristalsis (

1	Fig. 28.7 ). This wave moves down the esophagus slowly (3–5 cm/s). Distention of the esophagus by the moving bolus initiates another • Fig.28.6The esophagus and associated sphincters have multiple functions involved in movement of food from the mouth to the stomach and also in protection of the airway and esophagus. PropulsivefunctionsProtectiveeffectsFood transfer to esophagus Allows entry of food into esophagus Transports bolus from pharynx to stomach Allows entry of food into stomach Protects airway from swallowed material Protects airway from gastric reflux Clears material refluxed from stomach Protects esophagus from gastric reflux LES Esophagus UES Pharynx

1	CHAPTER 28 The Cephalic, Oral, and Esophageal Phases of the Integrated Response to a Meal • Fig. 28.7 Changes in pressure in the different regions of the pharynx, esophagus, and associated sphincters initiated during a swallow. The pressure trace is a diagrammatic representation from that obtained during manometry in an awake human. Stimulation of the pharynx by the presence of a bolus initiates a decrease in pressure (= opening) of the UES and a peristaltic wave of contraction along the esophagus. Stimulation of the pharynx also relaxes the smooth muscle of the LES to prepare for entry of food. • Fig. 28.8 Swallowing in the form of pharyngeal stimulation induces neural reflex relaxation of the LES and the proximal part of the stomach to allow entry of food.

1	• Fig. 28.8 Swallowing in the form of pharyngeal stimulation induces neural reflex relaxation of the LES and the proximal part of the stomach to allow entry of food. wave called secondary peristalsis. Frequently, repetitive secondary peristalsis is required to clear the esophagus of the bolus. Stimulation of the pharynx by the swallowed bolus also produces reflex relaxation of the LES and the most proximal region of the stomach. Thus when the bolus reaches the LES, it is already relaxed to allow passage of the bolus into the stomach. Similarly the portion of the stomach that receives the bolus is relaxed. In addition, esophageal distention produces further receptive relaxation of the stomach. The proximal part of the stomach relaxes at the same time as the LES; this occurs with each swallow, and its function is to allow the stomach to accommodate large volumes with a minimal rise in intragastric pressure. This process is called receptive relaxation ( Fig. 28.8

1	Fig. 28.8 The LES also has important protective functions. It is involved in preventing acid reflux from the stomach back into the esophagus. An insufficient tonic contraction of the LES is associated with reflux disease, a gradual erosion of the esophageal mucosa, which is not as well protected as the gastric and duodenal mucosa. There is also some evidence that peristalsis in the absence of swallowing (secondary peristalsis) is important for clearing refluxed gastric contents. 1. The cephalic and oral phases of the meal share many characteristics and prepare the remainder of the GI tract for the meal; these responses are neurally mediated, predominantly by the efferent vagus nerve. 2. Salivary secretion has important functions and, together with chewing of the food, allows the formation of a bolus that can be swallowed and passed along the esophagus to the stomach. 3.

1	2. Salivary secretion has important functions and, together with chewing of the food, allows the formation of a bolus that can be swallowed and passed along the esophagus to the stomach. 3. The ionic composition of salivary secretion varies with the flow rate, which is stimulated during a meal. The primary secretion comes from cells in the acini and is modified by epithelial cells as it passes through the ducts. 4. Regulation of salivary secretion is exclusively neural; parasympathetic innervation is most important in the response to food. Catalan MA, et al. Salivary gland secretion. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012. 5. The swallowing reflex is a rigidly ordered sequence of events that propel food from the mouth to the pharynx and from there to the stomach. 6.

1	5. The swallowing reflex is a rigidly ordered sequence of events that propel food from the mouth to the pharynx and from there to the stomach. 6. The major function of the esophagus is to propel food from the mouth to the stomach. The esophagus has sphincters at either end that are involved in protective functions important in swallowing and preserving the integrity of the esophageal mucosa. 7. Esophageal peristalsis (primary) is stimulated by mechanical stimulation of the pharynx, and secondary peristalsis is stimulated by distention of the esophageal wall. 8. Esophageal function and the associated sphincters are regulated by extrinsic and intrinsic neural pathways. Lee MG, et al. Molecular mechanism of pancreatic and salivary gland fluid and HCO3 secretion. Physiol Rev. 2012;92:39-74. Mittal RK. Motor Function of the Pharynx, Esophagus, and its Sphincters. San Rafael, CA: Morgan & Claypool Life Sciences; 2011.

1	Mittal RK. Motor Function of the Pharynx, Esophagus, and its Sphincters. San Rafael, CA: Morgan & Claypool Life Sciences; 2011. Upon completion of this chapter the student should be able to answer the following questions: 1. What are the major functions of the monogastric stomach? 2. What are the gross functional regions of the stomach? 3. What is the role of the gastric epithelium in digestion and absorption? 4. What is the role of the proton pump in parietal cell function? 5. What are some examples of how gastric acid secretion is regulated during the postprandial period? 6. What are the differences between gastric mucosal protection and defense? 7. What are the components of the functional anatomy of GI smooth muscle? 8. What is the significance of gap junctions, interstitial cells of Cajal, and pacemaker cells in the functioning of GI smooth muscle? 9.

1	8. What is the significance of gap junctions, interstitial cells of Cajal, and pacemaker cells in the functioning of GI smooth muscle? 9. How is the basic electrical rhythm (slow wave) generated, how is it regulated by chemical messengers (hormones, paracrine, neurotransmitters), and what causes contractions associated with the slow wave to occur? 10. What physiological events in gastric motility occur in the gastric phase? n this chapter, gastrointestinal (GI) tract physiology when food is in the stomach (i.e., the gastric phase of digestion) will be discussed. This includes gastric function and its regulation, in addition to changes in function that occur in more distal regions of the GI tract. The main functions of the stomach are to act as a temporary reservoir for the meal and to initiate protein digestion through secretion of acid and the enzyme precursor pepsinogen. Other functions are listed in Box 29.1

1	Box 29.1 Food entering the stomach from the esophagus causes mechanical stimulation of the gastric wall via distention and stretching of smooth muscle. Food—predominantly oligopeptides and amino acids—also provides chemical stimulation when present in the gastric lumen. Regulation of gastric function during the gastric phase is dependent on endocrine, paracrine, and neural pathways. These pathways are activated by mechanical and chemical stimuli, which result in intrinsic and extrinsic neural reflex pathways that are important for regulation of gastric function. Afferent neurons that pass from the GI tract to the central nervous system (and to a lesser extent to the spinal cord) via the vagus nerve respond to these mechanical and chemical stimuli and activate parasympathetic outflow.

1	The endocrine pathways include the release of gastrin, which stimulates gastric acid secretion, and the release of somatostatin, which inhibits gastric secretion. Important paracrine pathways include histamine release, which stimulates gastric acid secretion. The responses elicited by activation of these pathways include both secretory and motor responses; secretory responses include secretion of acid, pepsinogen, mucus, intrinsic factor, gastrin, lipase, and HCO3 − . Overall these secretions initiate protein digestion and protect the gastric mucosa. Motor responses (changes in activity of smooth muscle) include inhibition of motility of the proximal part of the stomach (receptive relaxation) and stimulation of motility of the distal part of the stomach, which causes antral peristalsis. These changes in motility play important roles in storage and mixing of the meal with secretions and are also involved in regulating the flow of contents out of the stomach.

1	Functional Anatomy of the Stomach The stomach is divided into three regions: the cardia, the corpus (also referred to as the fundus or body), and the antrum ( Fig. 29.1 ). However, when discussing the physiology of the stomach, it is helpful to think of it as subdivided into two functional regions: the proximal and distal parts of the stomach. The proximal portion of the stomach (called proximal because it is the most cranial) and the distal portion of the stomach (furthest away from the mouth) have quite different functions in the postprandial response to a meal, which will be discussed later. The lining of the stomach is covered with a columnar epithelium folded into gastric pits; each pit is the opening of a duct into which one or more gastric glands empty (Fig.

1	The lining of the stomach is covered with a columnar epithelium folded into gastric pits; each pit is the opening of a duct into which one or more gastric glands empty (Fig. 29.2 ). The gastric pits account for a significant fraction of the total surface area of the gastric mucosa. The gastric mucosa is divided into three distinct regions based on the structure of the glands. The small cardiac glandular region, located just below the lower esophageal sphincter (LES), primarily contains mucus-secreting gland cells. The remainder of the gastric mucosa is divided into the oxyntic or parietal (acid-secreting) gland region, located above the gastric notch (equivalent to the proximal part of the stomach), and the pyloric gland region, located below the notch (equivalent to the distal part of the stomach). The structure of a gastric gland from the oxyntic glandular region is illustrated in

1	The structure of a gastric gland from the oxyntic glandular region is illustrated in Fig. 29.2 . Surface epithelial cells extend slightly into the duct opening. The opening of the gland is called the isthmus and is lined with surface mucous cells and a few parietal cells. Mucous neck cells are located in the narrow neck of the gland. Parietal or oxyntic cells, which secrete HCl and intrinsic factor (involved in absorption of vitamin B12), and chief or peptic cells, which secrete pepsinogens, are located deeper in the gland. Oxyntic glands also contain enterochromaffin-like (ECL) cells that secrete histamine, and D cells that secrete somatostatin. Parietal cells are particularly numerous in glands in the fundus, whereas mucus-secreting cells are more numerous in glands •BOX 29.1FunctionsoftheStomach Storage—acts as temporary reservoir for the meal Secretion of H+ to kill microorganisms and convert pepsinogen to its active form

1	Storage—acts as temporary reservoir for the meal Secretion of H+ to kill microorganisms and convert pepsinogen to its active form Secretion of intrinsic factor to absorb vitamin B12 (cobalamin) Secretion of mucus and HCO3 − to protect the gastric mucosa Secretion of water for lubrication and to provide aqueous suspension of nutrients Motor activity for mixing secretions (H+ and pepsin) with ingested food Coordinated motor activity to regulate the emptying of contents into the duodenum of the pyloric (antral) glandular region. In addition the pyloric glands contain G cells that secrete the hormone gastrin. The parietal glands are also divided into regions: the neck (mucous neck cells and parietal cells) and the base (peptic/chief and parietal cells). Endocrine cells are scattered throughout the glands.

1	The fluid secreted into the stomach is called gastric juice. Gastric juice is a mixture of the secretions of the surface epithelial cells and the secretions of gastric glands. One of the most important components of gastric juice is H+, which is secreted against a very large concentration gradient. Thus H+ secretion by the parietal mucosa is an energy-intensive process. The cytoplasm of the parietal cell is densely packed with mitochondria, which have been estimated to fill 30% to 40% of the cell’s volume. One major function of H+ is conversion of inactive pepsinogen (the major enzyme product of the stomach) to pepsins, which initiate protein digestion in the stomach. Additionally, H+ ions are important for preventing invasion and colonization of the gut by bacteria and other pathogens that may be ingested with food. The stomach also secretes significant amounts of HCO3 − and mucus, which are important for protection of the gastric mucosa against the acidic and peptic luminal

1	that may be ingested with food. The stomach also secretes significant amounts of HCO3 − and mucus, which are important for protection of the gastric mucosa against the acidic and peptic luminal environment. The gastric epithelium also secretes intrinsic factor, which is necessary for absorption of vitamin B12 (cobalamin). The functions of other components of gastric juice are redundant with secretions provided more distally in the GI tract.

1	Composition of Gastric Secretions Like other GI secretions, gastric juice consists of inorganic and organic constituents together with water. Among the important components of gastric juice are HCl, salts, • Fig.29.1The three functional regions of the stomach. The regions have different luminal secretions and patterns of smooth muscle activity indicative of their unique functions in response to food. CHAPTER 29 The Gastric Phase of the Integrated Response to a Meal 531 Opening of gastric pit Opening of gastric pit • Fig. 29.2 Representation of the structure of the gastric mucosa showing a section through the wall of the stomach (A) and detail of the structure of gastric glands and cell types in the mucosa (B). pepsins, intrinsic factor, mucus, and HCO3 − . Secretion of all these components increases after a meal. Inorganic Constituents of Gastric Juice

1	pepsins, intrinsic factor, mucus, and HCO3 − . Secretion of all these components increases after a meal. Inorganic Constituents of Gastric Juice The ionic composition of gastric juice depends on the rate of secretion. The higher the secretory rate, the higher the concentration of H+ ions. At lower secretory rates, [H+] decreases and [Na+] increases. [K+] is always higher in gastric juice than in plasma. Consequently, prolonged vomiting may lead to hypokalemia. At all rates of secretion, Cl− is the major anion of gastric juice. Gastric HCl converts pepsinogens to active pepsins and provides the acid pH at which pepsins are active.

1	The rate of gastric H+ secretion varies considerably among individuals. In humans, basal (unstimulated) rates of gastric H+ production typically range from about 1 to 5 mEq/hr. During maximal stimulation, HCl production rises to 6 to 40 mEq/hr. The basal rate is greater at night and lowest in the early morning. The total number of parietal cells in the stomach of normal individuals varies greatly, and this variation is partly responsible for the wide range in basal and stimulated rates of HCl secretion. Organic Constituents of Gastric Juice The predominant organic constituent of gastric juice is pepsinogen, the inactive proenzyme of pepsin. Pepsins, often collectively called “pepsin,” are a group of proteases secreted by the chief cells of the gastric glands. Pepsinogens are contained in membrane-bound zymogen granules in the chief cells. Zymogen granules release their contents by exocytosis when chief cells are stimulated to secrete 29.1 Response to a Meal

1	Table 29.1 ). Pepsinogens are converted to active pepsins by the cleavage of acid-labile linkages. The lower the pH, the more rapid the conversion. Pepsins also act proteolytically on pepsinogens to form more pepsin. Pepsins are most proteolytically active at pH 3 and below. Pepsins may digest as much as 20% of the protein in a typical meal but are not required for digestion, because their function can be replaced by that of pancreatic proteases. When the pH of the duodenal lumen is neutralized, pepsins are inactivated by the neutral pH. Intrinsic factor, a glycoprotein secreted by parietal cells of the stomach, is required for normal absorption of vitamin B12. Intrinsic factor is released in response to the same stimuli that elicit secretion of HCl by parietal cells. Cellular Mechanisms of Gastric Acid Secretion Fig. 29.3 ). Branching secretory canaliculi course through the cytoplasm • Fig. 29.3

1	Cellular Mechanisms of Gastric Acid Secretion Fig. 29.3 ). Branching secretory canaliculi course through the cytoplasm • Fig. 29.3 Parietal cell ultrastructure. A, A resting parietal cell showing the tubulovesicular apparatus in the cytoplasm and the intracellular canaliculus. B, An activated parietal cell that is secreting acid. The tubulovesicles have fused with the membranes of the intracellular canaliculus, which is now open to the lumen of the gland and lined with abundant long microvilli. • Fig. 29.4 Mechanism of H+ and Cl− secretion by an activated parietal cell in the gastric mucosa. ATP, adenosine triphosphate.

1	• Fig. 29.4 Mechanism of H+ and Cl− secretion by an activated parietal cell in the gastric mucosa. ATP, adenosine triphosphate. and are connected by a common outlet to the cell’s luminal surface. Microvilli line the surfaces of the secretory canaliculi. The cytoplasm of unstimulated parietal cells contains numerous tubules and vesicles called the tubulovesicular system. The membranes of tubulovesicles contain the transport proteins responsible for secretion of H+ and Cl− into the lumen of the gland. When parietal cells are stimulated to secrete HCl (see

1	Fig. 29.3 ), tubulovesicular membranes fuse with the plasma membrane of the secretory canaliculi. This extensive membrane fusion greatly increases the number of H+/K+ antiporters in the plasma membrane of the secretory canaliculi. When parietal cells secrete gastric acid at the maximal rate, H+ is pumped against a concentration gradient that is about 1 million–fold. Thus the pH is 7 in the parietal cell cytosol and 1 in the lumen of the gastric gland. The cellular mechanism of H+ secretion by the parietal cell is depicted in

1	The cellular mechanism of H+ secretion by the parietal cell is depicted in Fig. 29.4 . Cl− enters the cell across the basolateral membrane in exchange for HCO3 − generated in the cell by the action of carbonic anhydrase, which produces HCO3 − and H+ . H+ is secreted across the luminal membrane by H+,K+-ATPase in exchange for K+ . K+ recycles across the luminal membrane via a K+ channel. Cl− enters the lumen via an ion channel (a CLC family Cl− channel) located in the luminal membrane. Increased intracellular Ca++ and cyclic adenosine monophosphate (cAMP) stimulate luminal membrane conduction of Cl− and K+ . Increased K+ conductance hyperpolarizes the luminal membrane

1	CHAPTER 29 The Gastric Phase of the Integrated Response to a Meal 533 potential, which increases the driving force for efflux of Cl− across the luminal membrane. The K+ channel in the basolateral membrane also mediates the efflux of K+ that accumulates in the parietal cell via the activity of H+,K+-ATPase. In addition, cAMP and Ca++ promote trafficking of Cl− channels into the luminal membrane, as well as fusion of cytosolic tubulovesicles containing H+,K+-ATPase with the membrane of the secretory canaliculi (see Figs. 29.3 29.4 ). Parietal cell secretion of H+ is also accompanied by transport of HCO3 − into the bloodstream to maintain intracellular pH. Secretion of HCO3 −

1	Figs. 29.3 29.4 ). Parietal cell secretion of H+ is also accompanied by transport of HCO3 − into the bloodstream to maintain intracellular pH. Secretion of HCO3 − The surface epithelial cells also secrete a watery fluid that contains Na+ and Cl− in concentrations similar to those in plasma but with higher K+ and HCO3 − concentrations. HCO3 − is entrapped by the viscous mucus that coats the surface of the stomach; thus the mucus secreted by the resting mucosa lines the stomach with a sticky alkaline coat. When food is eaten, rates of secretion of both mucus and HCO3 − increase. Secretion of Mucus Secretions that contain mucins are viscous and sticky and are collectively termed mucus. Mucins are secreted by mucous neck cells located in the necks of gastric glands and by the surface epithelial cells of the stomach. Mucus is stored in large granules in the apical cytoplasm of mucous neck cells and surface epithelial cells and is released by exocytosis.

1	Gastric mucins are about 80% carbohydrate by weight and consist of four similar monomers of about 500,000 Da each that are linked together by disulfide bonds (Fig. 29.5 These tetrameric mucins form a sticky gel that adheres to the surface of the stomach. This gel is subject to proteolysis by pepsins that cleave disulfide bonds near the center of the tetramers. Proteolysis releases fragments that do not form gels and thus dissolves the protective mucous layer. Maintenance of the protective mucous layer requires continuous synthesis of new tetrameric mucins to replace the mucins cleaved by pepsins. Mucus is secreted at a significant rate in the resting stomach. Secretion of mucus is stimulated by some of the same stimuli that enhance acid and pepsinogen secretion, especially acetylcholine released from parasympathetic nerve endings near the gastric glands. If the gastric mucosa is mechanically deformed, neural reflexes are evoked to enhance mucus secretion.

1	Regulation of Gastric Secretion Parasympathetic innervation via the vagus nerve is the strongest stimulant of gastric H+ secretion. Extrinsic efferent fibers terminate on intrinsic neurons that innervate parietal cells, ECL cells that secrete the paracrine mediator histamine, and endocrine cells that secrete the hormone gastrin. In addition, vagal stimulation results in secretion of pepsinogen, mucus, HCO3 − , and intrinsic factor. Stimulation of the parasympathetic nervous system also occurs during the cephalic and oral phase of the meal. However, the gastric phase produces the largest stimulation of gastric secretion of the postprandial period ( Fig. 29.6 Stimulation of gastric acid secretion is an excellent example of a “feed-forward” (or cascade) response that uses endocrine, paracrine, and neural pathways. Activation of intrinsic neurons by vagal efferent activity results in release

1	Protein core: protected from further proteolysisGlycosylated part of by carbohydrate chains peptide cores (resistant to Sheath of branched proteolysis) carbohydrate chains with average of 15 Nonglycosylated part of sugars per chain peptide cores with disulfide bridges joining subunits (site of proteolysis) • Fig.29.5Schematic representation of the structure of gastric mucins before and after hydrolysis by pepsin. Intact mucins are tetramers of four similar monomers of about 500,000 Da. Each monomer is largely covered by carbohydrate side chains that protect it from proteolytic degradation. The central portion of the mucin tetramer, near the disulfide cross-links, is more susceptible to proteolytic digestion. Pepsins cleave bonds near the center of the tetramers to release fragments about the size of monomers.

1	• Fig.29.6Neural regulation of gastric acid secretion in the gastric phase of the meal is mediated by the vagus nerve. The stimulation that occurs in the cephalic and oral phases (before food reaches the stomach) results in stimulation of parietal cells to secrete acid and chief cells to secrete pepsinogen. Thus when food reaches the stomach, protein digestion is initiated by generating protein hydrolysate, which further stimulates secretion of gastrin from the mucosa of the gastric antrum. In addition, gastric distention activates a vagovagal reflex that further stimulates gastric acid and pepsinogen secretion. ˜ H+ ˜ Pepsinogen Endocrine (gastrin) Oligopeptides Protein Pepsin Pepsin pH 2.0 Pepsinogen ˜ H+ ˜ Pepsinogen ˜ Gastrin • Intrinsic neural pathways are also activated by distention Distention Dorsal vagal complex Neural (vagovagal) of acetylcholine from nerve terminals, which activates cells in the gastric epithelium. Parietal cells express muscarinic receptors and are

1	Distention Dorsal vagal complex Neural (vagovagal) of acetylcholine from nerve terminals, which activates cells in the gastric epithelium. Parietal cells express muscarinic receptors and are activated to secrete H+ in response to vagal efferent nerve activity. In addition, parasympathetic activation, via gastrin-releasing peptide from intrinsic neurons, releases gastrin from G cells located in the gastric glands in the gastric antrum (see

1	Fig. 29.6 ). Gastrin enters the bloodstream and, via an endocrine mechanism, further stimulates the parietal cell to secrete H+ . Parietal cells express cholecystokinin type 2 (CCK2) receptors for gastrin. Histamine is also secreted in response to vagal nerve stimulation, and ECL cells express muscarinic and gastrin receptors. Thus gastrin and vagal efferent activity induce release of histamine, which potentiates the effects of both gastrin and acetylcholine on the parietal cell. Hence activation of parasympathetic (vagal) outflow to the stomach is very efficient at stimulating the parietal cell to secrete acid ( Fig. 29.7

1	Fig. 29.7 In the gastric phase the presence of food in the stomach is detected and activates vagovagal reflexes to stimulate secretion. Food in the stomach results in distention and stretch, which are detected by afferent (or sensory) nerve endings in the gastric wall. These are the peripheral terminals of vagal afferent nerves that transmit information to the brainstem and thereby drive activity in vagal efferent fibers, a vagovagal reflex (see Fig. 29.6 ). In addition, digestion of proteins increases the concentration of oligopeptides and free amino acids in the lumen, which are detected by chemosensors in the gastric mucosa. Oligopeptides and amino acids also stimulate vagal afferent activity. The exact nature of the

1	ACETYLCHOLINE, GASTRIN, AND HISTAMINE STIMULATE THE PARIETAL CELL • Fig.29.7The parietal cell is regulated by neural, hormonal, and paracrine pathways. Activation of vagal parasympathetic preganglionic outflow to the stomach acts in three ways to stimulate gastric acid secretion. There is direct neural innervation and activation of the parietal cell via release of acetylcholine (A) from enteric neurons, which acts on the parietal cell via muscarinic receptors. In addition, neural activation of the ECL cell stimulates release of histamine (H), which acts via a paracrine pathway to stimulate the parietal cell. Finally, G cells located in gastric glands in the gastric antrum are activated by release of gastrin-releasing peptide (GRP) from enteric neurons, which acts on the G cell to stimulate release of gastrin (G). Gastrin thereafter acts via a humoral pathway to stimulate the parietal cell.

1	chemosensors is not clear but may involve endocrine cells that release their contents to activate nerve endings. This topic will be discussed in more detail in Chapter 30. There is also an important negative feedback mechanism whereby the presence of acid in the distal part of the stomach (antrum) induces a feedback loop to inhibit the parietal cell such that meal-stimulated H+ secretion does not go unchecked. When the concentration of H+ in the lumen reaches a certain threshold (<pH 3), somatostatin is released from endocrine cells in the antral mucosa. Somatostatin has a paracrine action on neighboring G cells to decrease the release of gastrin and thereby decrease gastric acid secretion (Fig. 29.8 The receptors on the parietal cell membrane for acetylcholine, gastrin, and histamine, as well as the intracellular second messengers by which these secretagogues act, are shown in

1	Fig. 29.9 . Histamine is the strongest agonist of H+ secretion, whereas gastrin and acetylcholine are much • Fig.29.8Feedback regulation of gastric acid secretion by release of somatostatin and its action on G cells in the gastric antrum. Endocrine cells in the mucosa of the gastric antrum sense the presence of H+and secrete somatostatin. This in turn acts on specific receptors on G cells to inhibit release of gastrin and thus bring about inhibition of gastric acid secretion. Circulation G cell – + D cell Antral lumen GastrinSomatostatin weaker agonists. However, histamine, acetylcholine, and gastrin potentiate one another’s actions on the parietal cell. Antagonists of H2 histamine receptors (e.g., cimetidine [Tagamet]) block secretagogue-stimulated acid secretion. Thus much of the response to gastrin results from gastrinstimulated release of histamine. Gastrin also has important trophic effects; elevation of gastrin levels causes ECL cells to increase in size and number. Binding of

1	to gastrin results from gastrinstimulated release of histamine. Gastrin also has important trophic effects; elevation of gastrin levels causes ECL cells to increase in size and number. Binding of histamine to H2 receptors on parietal cell plasma membranes activates adenylyl cyclase and elevates the cytosolic concentration of cAMP. These events stimulate H+ secretion by activating basolateral K+ channels and apical Cl− channels and by causing more H+,K+-ATPase molecules and Cl− channels to be inserted into the apical plasma membrane (see

1	Fig. 29.4 ). Acetylcholine binds to M3 muscarinic receptors and opens Ca++ channels in the apical plasma membrane. Acetylcholine also elevates intracellular [Ca++] by promoting release of Ca++ from intracellular stores, which enhances H+ secretion by activating basolateral K+ channels and causing more H+,K+-ATPase molecules and Cl− channels to be inserted into the apical plasma membrane. Gastrin enhances acid secretion by binding to CCK2 receptors (Fig. 29.10 Digestion in the Stomach Some digestion of nutrients occurs in the stomach. However, this is not required for full digestion of a meal; intestinal digestion is sufficient. Some amylase-mediated digestion of carbohydrates occurs in the stomach. Amylase is sensitive to pH and inactivated at low pH; however, some amylase is active even in the acidic gastric environment of the stomach because of substrate protection. Thus when carbohydrate occupies the active site of amylase, it protects the enzyme from degradation.

1	• Fig.29.9Vagal parasympathetic stimulation of gastric secretions via enteric neurons. Vagal preganglionic neurons innervate the myenteric and submucosal plexus. The terminals of the vagal preganglionic neurons innervate many enteric neurons and thus bring about changes in function as described in Fig. 29.7 . ACh, acetylcholine; GRP, gastrin-releasing peptide.

1	Fig. 29.7 . ACh, acetylcholine; GRP, gastrin-releasing peptide. • Fig.29.10Signal transduction mechanisms showing the mechanism of action of agonists (secretagogues) and antagonists that regulate secretion in parietal cells. Acetylcholine (ACh) binds to muscarinic M3 receptors. Histamine acts via the H2 receptor. Gastrin binds to the cholecystokinin type 2 (CCK2) receptor. Activation of M3 and CCK2 receptors results in opening of Ca++ channels and release of Ca++ from intracellular stores and thus an increase in cytosolic [Ca++]. Activation of H2 receptors activates adenylyl cyclase to increase intracellular levels of cAMP. Ac, adenylyl cyclase; ACh, acetylcholine; CCK, cholecystokinin; DAG, diacylglycerol; EGF, epidermal growth factor; IP3, inositol triphosphate; PGE2, prostaglandin E2; PIP2, phosphatidylinositol 4,5-diphosphate; PKC, protein kinase C; PLC, protein lipase C; TGF-α, transforming growth factor α.

1	Digestion of lipids also starts in the stomach. The mixing patterns of gastric motility result in formation of an emulsion of lipids and gastric lipase, which attaches to the surface of lipid droplets in the emulsion and generates free fatty acids and monoglyceride from dietary triglyceride. However, the extent of hydrolysis of triglyceride is approximately 10%, and hydrolysis is not essential for normal digestion and absorption of dietary lipids. Moreover, as discussed in the next chapter, the products of lipolysis are not available for absorption in the stomach because of its low luminal pH. Mucus and HCO3 − protect the surface of the stomach from the effects of H+ and pepsins. The protective mucus gel that forms on the luminal surface of the stomach, as well as alkaline secretions entrapped within it, constitute a gastric mucosal barrier that prevents damage to the mucosa by gastric contents (

1	Fig. 29.11 ). The mucus gel layer, which is about 0.2 mm thick, effectively separates the HCO3 −-rich secretions of the surface epithelial cells from the acidic contents of the gastric lumen. The mucus allows the pH of epithelial cells to be maintained at nearly neutral despite a luminal pH of about 2. Mucus also slows the diffusion of acid and pepsins to the epithelial cell surface. Protection of the gastric epithelium depends on both mucus and HCO3 − secretion. To understand GI motility it is necessary to review some properties of smooth muscle function. The motion of the gut wall governs the flow of the luminal contents along its length; the main patterns of motility are mixing (segmentation) and propulsion (peristalsis). In addition, smooth muscle activity in the stomach and colon subserves a storage function.

1	• Fig.29.11The surface of the stomach is protected by the gastric mucosal barrier. Buffering by the HCO3 −-rich secretions and the high viscosity of the layer of mucus allow the pH at the cell surface to remain near 7, whereas the pH in the gastric juice in the lumen is 2. Functional Anatomy of Gastrointestinal Smooth Muscle The smooth muscle in the GI tract is similar in structure to other smooth muscle found in the body. Fusiform cells are packed together in bundles surrounded by a connective tissue sheath. Gap junctions functionally couple the smooth muscle cells so that contraction of bundles occurs synchronously. The interstitial cells of Cajal (ICCs) are a specialized group of cells in the intestinal wall that are involved in transmission of information from enteric neurons to smooth muscle cells (

1	Fig. 29.12 ). It is also thought that ICCs are “pacemaker” cells that have the capacity to generate the basic electrical rhythm, or slow-wave activity, that is a consistent feature of GI smooth muscle ( Fig. 29.13). Electrophysiology of Gastrointestinal Smooth Muscle The cyclic variation in resting membrane potential of GI smooth muscle is called the basic electrical rhythm or slow wave. The frequency of slow waves is 3 to 5 per minute in the stomach and about 12 to 20 per minute in the small intestine; it decreases to 6 to 8 per minute in the colon. The frequency of the slow wave is set by a pacemaker region in the different regions of the GI tract (see Fig. 29.13 ). Slow waves are thought to be generated by ICCs. These cells are located in a thin layer between the longitudinal and Slow waves are generated in interstitial cells of Cajal

1	Fig. 29.13 ). Slow waves are thought to be generated by ICCs. These cells are located in a thin layer between the longitudinal and Slow waves are generated in interstitial cells of Cajal Slow wave conducted to smooth muscle cells • Fig.29.12Diagrammatic representation of the interstitial cells of Cajal network in the smooth muscle wall of the GI tract. circular layers of the muscularis externa and in other places in the wall of the GI tract. Interstitial cells have properties of both fibroblasts and smooth muscle cells. Their long processes form gap junctions with the longitudinal and circular smooth muscle cells; the gap junctions enable the slow waves to be conducted rapidly to both muscle layers. Because gap junctions electrically and chemically couple the smooth muscle cells of both longitudinal and circular layers, the slow wave spreads throughout the smooth muscle of each segment of the GI tract.

1	538 SECTION6Berne & Levy Physiology • Fig.29.13Amplitude of slow wave determines the strength of muscle contraction. The slow wave will initiate a contraction in smooth muscle when it reaches a threshold amplitude. The amplitude of the slow wave is altered by release of neurotransmitters from enteric neurons. At rest Stimulated Inhibited Membrane potential Muscle tone Membrane potential Muscle tone Membrane potential Muscle tone

1	There are times when the gastric mucosal barrier fails. Superficial breakdowns of the GI lining not involving the submucosa are called erosions. They generally heal without intervention. In contrast, breakdowns of the GI lining involving the muscularis and deeper layers are called ulcers. Gastric and duodenal erosions and ulcers occur as a result of an imbalance between the mechanisms that protect the mucosa and aggressive factors that can break it down. A healthy stomach/duodenum has ample natural protection against the destructive effects of H+ . Factors that magnify the harmful effect of H+ on the stomach/duodenum or act separately from H+ include pepsin, bile, the bacterium Helicobacter pylori, and the class of drugs known as nonsteroidal antiinflammatory drugs (NSAIDs). Indeed, ulcer disease is becoming more common as the population ages and has more need of NSAIDs for non-GI complaints such as arthritis. Alcohol, tobacco, and caffeine are also risk factors for ulcers. Infectious

1	disease is becoming more common as the population ages and has more need of NSAIDs for non-GI complaints such as arthritis. Alcohol, tobacco, and caffeine are also risk factors for ulcers. Infectious agents can also cause gastritis (inflammation of the gastric epithelium). H. pylori is a spiral bacterium that has now become widely recognized as one factor that can lead to gastritis, ulcer formation, and in humans, gastric carcinoma. H. pylori exists in the stomach because it secretes an enzyme, urease, that converts urea to NH3, which is used to buffer H+ by forming NH4 + . An aggressive regimen of antibiotic treatment, sometimes in combination with an H+,K+-ATPase inhibitor, can often eliminate the infection, after which the gastritis and ulcer symptoms improve.

1	The amplitude and, to a lesser extent, the frequency of the slow wave can be modulated by the activity of intrinsic and extrinsic nerves and by hormones and paracrine substances. If the depolarization of the slow wave exceeds the threshold, a train of action potentials may be triggered during the peak of the slow wave.

1	Action potentials in GI smooth muscle are more prolonged (10–20 msec) than those in skeletal muscle and have little or no overshoot. The rising phase of the action potential is caused by flow of ions through channels that conduct both Ca++ and Na+ and are relatively slow to open. The Ca++ that enters the cell during the action potential helps initiate contraction. The extent of depolarization of the cells and the frequency of action potentials are enhanced by some hormones and paracrine agonists and by neurotransmitters from excitatory enteric nerve endings (e.g., acetylcholine and substance P). Inhibitory hormones and neuroeffector substances (e.g., vasoactive intestinal polypeptide and nitric oxide) hyperpolarize the smooth muscle cells and may diminish or abolish action potential spikes.

1	Slow waves that are not accompanied by action potentials elicit little or no contraction of the smooth muscle cells. Much stronger contractions are evoked by the presence of action potentials. The greater the number of action potentials that occur at the peak of a slow wave, the more intense the contraction of the smooth muscle. Because smooth muscle cells contract rather slowly (about a 10th as fast as skeletal muscle cells), the individual contractions caused by each action potential in a train do not cause distinct twitches; rather they sum temporally to produce a smoothly increasing level of tension.

1	Between the trains of action potentials, the tension developed by GI smooth muscle falls, but not to zero. This nonzero resting, or baseline, tension of smooth muscle is called tone. The tone of GI smooth muscle is altered by neuroeffectors, hormones, paracrine substances, and drugs and is important in the sphincters and also in regions where storage of contents is important, such as the stomach and colon. Specialized Patterns of Motility Peristalsis is a moving ring of contraction that propels material along the GI tract. It involves neurally mediated contraction and relaxation of both muscle layers. Peristalsis occurs in the pharynx, esophagus, gastric antrum, and the small and large intestine.

1	Segmental contractions produce narrow areas of contracted segments between relaxed segments. These movements allow mixing of the luminal contents with GI tract secretions and increase exposure to the mucosal surfaces where absorption occurs. Segmentation occurs predominantly in the small and large intestine. There are also characteristic pathological patterns of motility. During spasm, maximal contractile activity occurs continuously in a dysregulated manner. In ileus, contractile activity is markedly decreased or absent; it often results from irritation of the peritoneum, such as occurs in surgery, peritonitis, and pancreatitis. Functional Anatomy of the Stomach

1	Functional Anatomy of the Stomach As discussed, the stomach is divided into two functional regions—proximal and distal, with sphincters at either end. The LES and cardia (defined as the region of the stomach immediately surrounding the LES) have important functions. Relaxation of the LES and cardia allows entry of food from the esophagus into the stomach and the release of gas, called belching. By maintaining tone, reflux of contents from the stomach back into the esophagus is prevented. The proximal part of the stomach (the fundus together with the corpus or body) produces slow changes in tone compatible with its reservoir function. It is important for receiving and storing food and for mixing the contents with gastric juice (

1	Table 29.2 ). Generation of tone in the proximal portion of the stomach is also an important driving force in the regulation of gastric emptying. Low tone and consequently low intragastric pressure are associated with delayed or slow gastric emptying, and an increase in tone in this region is required for gastric emptying to occur.

1	The distal part of the stomach is important in the mixing of gastric contents and for propulsion through the pylorus and into the duodenum. The muscle layers in the region of the gastric antrum are much thicker than in the more proximal regions of the stomach, and thus the antrum is capable of producing strong phasic contractions. Contractions initiated by the slow wave begin in the midportion of the stomach and move toward the pylorus. The strength of these contractions varies during the postprandial period. In the gastric phase of the meal the pylorus is usually closed, and these antral contractions serve to mix the gastric contents and reduce the size of solid particles (grinding). However, eventually these antral contractions are also important in emptying the stomach of its contents.

1	The pyloric sphincter is the gastroduodenal junction and is defined as an area of thickened circular muscle. This is a region of high pressure generated by tonic smooth muscle contraction. It is important in regulating gastric emptying. Control of Gastric Motility in the Gastric Phase Gastric motility is highly regulated and coordinated to perform the functions of storage and mixing. Regulation of emptying of contents into the small intestine, an important part of gastric motor function, will be considered in detail in the discussion of the duodenal phase of the meal, because the controls are generated in the duodenum.

1	The stimuli regulating gastric motor function that result from the presence of the meal in the stomach are both mechanical and chemical and include distention and the presence of products of protein digestion (amino acids and small peptides). The pathways regulating these processes are predominantly neural and consist of vagovagal reflexes initiated by extrinsic vagal afferent fibers that terminate in the muscle and mucosa. Mucosal afferents respond to chemical stimuli, and mechanosensitive afferents respond to distention and contraction of smooth muscle. This afferent stimulation results in reflex activation of vagal efferent (parasympathetic) outflow and activation of enteric neurons that innervate the smooth muscle. Activation of enteric neurons produces both inhibitory and excitatory effects on gastric smooth muscle; these effects vary depending on the region of the stomach. Thus distention of the gastric wall results in inhibition of smooth muscle in the proximal portion of the

1	effects on gastric smooth muscle; these effects vary depending on the region of the stomach. Thus distention of the gastric wall results in inhibition of smooth muscle in the proximal portion of the stomach and subsequent reflex accommodation, which allows entry and storage of the meal to occur with minimal increase in intragastric pressure.

1	In contrast, the predominant motor pattern of the distal part of the stomach in the gastric phase of the meal is activation of smooth muscle to produce and strengthen the antral contractions. The rate of antral contractions is set by the gastric pacemaker; however, the magnitude of the contractions is regulated by release of neurotransmitters from enteric neurons, including substance P and acetylcholine, which increase the level of depolarization of the smooth muscle and therefore produce stronger contractions. In this phase of the meal the pylorus is mostly closed. Thus antral contractions will tend to move the contents toward the pylorus; however, because the pylorus is closed, the contents will be returned to the more proximal part of the stomach. In this way the gastric contents will be mixed. In addition, antral contractions can occlude the lumen, and thus larger particles will be dispersed, a process referred to as grinding ( Fig. 29.14

1	Fig. 29.14 Onset of terminal Complete terminal antral contraction antral contraction • Force for retropulsion is increased pressure in terminal antrum as the antral contraction approaches the closed pylorus. •Fig.29.14Coordinated activity in the smooth muscle of the proximal and distal portions of the stomach and the pyloric sphincter results in mixing and grinding in the gastric antrum. The peristaltic wave moves down the gastric body and antrum toward the pylorus. If the pylorus is closed, the contents of the gastric antrum are retropulsed back into the more proximal part of the stomach. This pattern of motility results in grinding and mixing of the food with secretions from the gastric wall and eventually leads to a reduction in particle size and the presence of digestive products that will empty into the duodenum. 1. The main functions of the stomach are storage and initiation of protein digestion. 2.

1	1. The main functions of the stomach are storage and initiation of protein digestion. 2. Regulation of gastric function is driven by extrinsic and intrinsic neural pathways together with key humoral (gastrin) and paracrine (histamine) mediators. 3. The key secretions from the stomach are acid and pepsinogen, which together begin protein digestion. 4. H+ is secreted across the apical plasma membrane of parietal cells via H+,K+-ATPase. 5. The stomach also secretes intrinsic factor, which is involved in absorption of vitamin B12. Huizinga JD, Chen JH. Interstitial cells of Cajal: update on basic and clinical science. Curr Gastroenterol Rep. 2014;16:363. Hunt RH, et al. The stomach in health and disease. Gut. 2015;64:1650-1668. Said H, et al. Gastroduodenal mucosal defense mechanisms. Curr Opin Gastroenterol. 2015;31:486-491. 6.

1	Hunt RH, et al. The stomach in health and disease. Gut. 2015;64:1650-1668. Said H, et al. Gastroduodenal mucosal defense mechanisms. Curr Opin Gastroenterol. 2015;31:486-491. 6. The gastric epithelium secretes HCO3 − and mucus to form a gel-like mucosal barrier that protects it against the acidic and peptic luminal contents. 7. The smooth muscle of the gut wall undergoes cyclic changes in membrane potential, termed the basic electrical rhythm or the slow wave. 8. The interstitial cells of Cajal are pacemakers in the gut wall, and they set the frequency of the slow wave. 9. The proximal part of the stomach undergoes a slow change in tone compatible with its storage function. 10. The distal part of the stomach undergoes phasic contractions that can vary considerably in strength. 11. Gastric emptying is regulated by vagovagal reflexes. Sanders KM, et al. Regulation of gastrointestinal smooth muscle function by interstitial cells. Physiology (Bethesda). 2016;31:316-326.

1	11. Gastric emptying is regulated by vagovagal reflexes. Sanders KM, et al. Regulation of gastrointestinal smooth muscle function by interstitial cells. Physiology (Bethesda). 2016;31:316-326. Schubert ML. Functional anatomy and physiology of gastric secretion. Curr Opin Gastroenterol. 2015;31:479-485. Schubert ML. Regulation of gastric acid secretion. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012. Upon completion of this chapter the student should be able to answer the following questions: 1. How are the various components of a mixed meal digested and absorbed in the small intestine? 2. What are the constituents and functions of pancreatic juice, and how is their secretion controlled? 3. How do bile acids assist with digestion and assimilation of lipids? 4. What are the mechanisms that provide for appropriate levels of fluidity of the intestinal contents? 5.

1	3. How do bile acids assist with digestion and assimilation of lipids? 4. What are the mechanisms that provide for appropriate levels of fluidity of the intestinal contents? 5. What are the motor patterns of the small intestine in the postprandial period as well as during fasting, and what functions do these patterns subserve?

1	he small intestine is the critical portion of the intestinal tract for assimilation of nutrients. In this site the meal is mixed with a variety of secretions that permit its digestion and absorption, and motility functions ensure adequate mixing and exposure of the intestinal contents (chyme) to the absorptive surface. The small intestine has many specializations that enable it to perform its functions efficiently. One of the most obvious specializations is the substantial surface area of the mucosa; this is achieved in a number of different ways. The small intestine is essentially a long tube that is coiled inside the abdominal cavity, there are folds of the full thickness of the mucosa and submucosa, the mucosa has finger-like projections called villi, and finally, each epithelial cell has microvilli on its apical surface. Thus a large surface area exists over which digestion and absorption occur.

1	The main characteristic of the small intestinal phase of the response to a meal is controlled delivery of chyme from the stomach to match the digestive and absorptive capacity of the intestine. In addition there is further stimulation of pancreatic and biliary secretion and emptying of these secretions into the small intestine. Therefore the function of this region is highly regulated by feedback mechanisms that involve hormonal, paracrine, and neural pathways.

1	The stimuli that regulate these processes are both mechanical and chemical and include distention of the intestinal wall and the presence of increased [H+], high osmolarity, and nutrients in the intestinal lumen. These stimuli result in a set of changes that represent the intestinal phase of the response to the meal: (1) increased pancreatic secretion, (2) increased gallbladder contraction, (3) relaxation of the sphincter of Oddi, (4) regulation of gastric emptying, (5) inhibition of gastric acid secretion, and (6) interruption of the migrating motor complex (MMC). The goal of this chapter is to discuss how such changes are brought about and how they result ultimately in assimilation of nutrients. Changes in small intestinal function that occur after the meal has passed through will also be addressed. Gastric Emptying in the Small Intestinal Phase

1	Gastric Emptying in the Small Intestinal Phase Immediately after a meal the stomach may contain up to a liter of material that will empty slowly into the small intestine. The rate of gastric emptying is dependent on the macronutrient content of the meal and the amount of solids it contains. Thus solids and liquids of similar nutritional composition will empty at different rates. Liquids empty rapidly, but solids do so only after a lag phase, which means that after a solid meal, there is a period of time during which little or no emptying occurs (Fig. 30.1

1	Regulation of gastric emptying is achieved by alterations in motility of the proximal part of the stomach (fundus and corpus) and distal part of the stomach (antrum and pylorus) as well as in the duodenum. Motor function in these regions is highly coordinated. Recall that during the esophageal and gastric phase of the meal, the predominant reflex response is receptive relaxation. At the same time, peristaltic movements in the more distal part of the stomach (antrum) mix the gastric contents with gastric secretions. The pyloric sphincter is largely closed. Even if it opens periodically, little emptying will occur, because the proximal portion of the stomach is relaxed and the antral pump (antral contractions) is not very strong. Subsequently, gastric emptying is brought about by an increase in tone (intraluminal pressure) in the proximal portion of the stomach, increased strength of antral contractions (increased strength of the •Fig. 30.1

1	emptying is brought about by an increase in tone (intraluminal pressure) in the proximal portion of the stomach, increased strength of antral contractions (increased strength of the •Fig. 30.1 Ratesofemptyingofdifferentmealsfromadog’sstomach.Asolution(1%glucose)isemptiedfasterthanadigestiblesolid(cubedliver).Notethelagphaseforemptyingofthesolids,whichisrelatedtothetimeneededtoreduceparticlesbelow2mminsize.(AdaptedfromHinderRA,KellyKA.Am J Physiol 1977;233:E335.)100 80 60 40 20 21 3 4 % emptied Hours400 mL 1% glucose 50 g cubed liver Protein Glucose

1	Thegastrointestinal(GI)tractplaysamajorroleinthesensingandsignalingofingestednutrientsbyactivatingneuralandendocrinepathwaysthatconnectwithothersignals,suchasfatenergystorageandutilization,whichtogetherregulateenergyhomeostasis.SatietysignalsfromtheGItractaregenerallyinvolvedinshort-termregulationoffoodintake,suchasindividualmealsizeandmealduration.Forexample,theluminalcontentsactivatevagalafferentpathways,leadingtosuppressionofmealsize.Inaddition,severalGIhormonesreleasedbynutrientsalsoinfluencefoodintake.Cholecystokinin(CCK)isawell-describedsatietyhormone;itisreleasedbynutrientsanddecreasesfoodintakeafterexogenousadministration.OtherGIhormonesinthisclassincludeglucagon-likepeptide1(GLP-1)andpeptideYY(PYY).Inbothleanandobesehumans,injectionofexogenousPYYinhibitsfoodintake.Along-actinganalogueofGLP-1,exendin-4,iscurrentlybeingusedasanagentforweightcontrolinhumans.

1	antral pump), opening of the pylorus to allow the contents to pass, and simultaneous inhibition of duodenal segmental contractions. Liquids and the semiliquid chyme flow down the pressure gradient from the stomach to the duodenum.

1	As the meal enters the small intestine, it feeds back via both neural and hormonal pathways to regulate the rate of gastric emptying based on the chemical and physical composition of the chyme. Afferent neurons, predominantly of vagal origin, respond to nutrients, [H+], and the hyperosmotic content of chyme as it enters the duodenum. Reflex activation of vagal efferent outflow decreases the strength of antral contractions, contracts the pylorus, and decreases proximal gastric motility (with a decrease in intragastric pressure), thereby resulting in inhibition (slowing) of gastric emptying. This same pathway is responsible for the inhibition of gastric acid secretion that occurs when nutrients are in the duodenal lumen. Cholecystokinin (CCK) is released from endocrine cells in the duodenal mucosa in response to such nutrients. This hormone is physiologically important, in addition to its role in neural pathways, in the regulation of gastric emptying, gallbladder contraction, relaxation

1	mucosa in response to such nutrients. This hormone is physiologically important, in addition to its role in neural pathways, in the regulation of gastric emptying, gallbladder contraction, relaxation of the sphincter of Oddi, and pancreatic secretion. Recent evidence suggests that CCK acts both directly (inhibits gastric emptying) and indirectly (stimulates vagal afferent fiber discharge to produce an indirect vagovagal reflex–mediated decrease in gastric emptying).

1	How then can gastric emptying proceed in the face of these inhibitory pathways? The amount of chyme in the duodenum decreases as it passes further down the small intestine into the jejunum; thus the strength of intestinal feedback inhibition fades as there is less activation of duodenal sensory mechanisms by nutrients. At this time, intragastric pressure in the proximal portion of the stomach increases, thereby moving material into the antrum and toward the antral pump. Antral peristaltic contractions again deepen and culminate in opening of the pylorus and release of gastric contents into the duodenum.

1	Surgicaltreatmentofobesity,so-calledbariatricsurgery,canachievesubstantialandlastingweightlossandalsohelpameliorateassociatedhealthproblemssuchasinsulinresistance,hyperlipidemia,andelevatedbloodpressure.Initially,surgeryinvolvedjejunoileal bypass, theremovalofasubstantialpartoftheabsorptivesmallintestine,butthisprocedureisassociatedwithmalabsorptionandsubsequentundesirablesequelaesuchasdiarrhea.Avarietyofrevisedsurgicalapproachestoobesityhavebeendevised,includingRoux-en-Ygastricbypassandverticalsleevegastrectomy.Themechanismsbywhichtheseproceduresarethoughttobesuccessfullieinthesmallsizeoftheresidualgastricpouch,wherebymealsizeisdecreasedbecauseofearlysatiety,andabeneficialeffectofthebypassontheprofilesofGIhormones.Recentdataimplythateffectsofsurgeryonbileacidsandthemicrobiomemayalsocontributetobothweightlossandmetabolicbenefits.

1	Most of the nutrients ingested by humans are in the chemical form of macromolecules. However, such molecules are too large to be assimilated across the epithelial cells that line the intestinal tract and must therefore be broken down into their smaller constituents by processes of chemical and enzymatic digestion. Secretions arising from the pancreas are quantitatively the largest contributors to enzymatic digestion of the meal. The pancreas also provides additional important secretory products that are vital for normal digestive function. Such products include substances that regulate the function or secretion (or both) of other pancreatic products, as well as water and bicarbonate ions. The latter are involved in neutralizing gastric acid so that the small intestinal lumen has a pH approaching 7.0. This is important because pancreatic enzymes are inactivated by high levels of acidity and also because neutralization of gastric acid reduces the likelihood that the small intestinal

1	7.0. This is important because pancreatic enzymes are inactivated by high levels of acidity and also because neutralization of gastric acid reduces the likelihood that the small intestinal mucosa will be injured by such acid acting in combination with pepsin. Quantitatively the pancreas is the largest contributor to the supply of bicarbonate ions needed to neutralize the gastric acid load, although the biliary ductules and duodenal epithelial cells also contribute.

1	As in the salivary glands, the pancreas has a structure that consists of ducts and acini. The pancreatic acinar cells line the blind ends of a branching ductular system that eventually empties into the main pancreatic duct and from there into the small intestine under control of the sphincter of Oddi. Also in common with salivary glands, a primary secretion arises in the acini, which is subsequently modified as it passes through the pancreatic ducts. In general the acinar cells supply the organic constituents of the pancreatic juice in a primary secretion whose ionic composition is comparable to that of plasma, whereas the ducts dilute and alkalinize the pancreatic juice while reabsorbing chloride ions (Fig. 30.2 ). The major constituents of pancreatic juice, which amounts to approximately 1.5 L/day in adult humans, are listed in

1	Box 30.1 . This list also outlines the functions of pancreatic secretory products. Many of the digestive enzymes produced by the pancreas, particularly the proteolytic enzymes, are produced as inactive precursor forms. Storage in these inactive forms is critically important in preventing the pancreas from digesting itself.

1	Pancreatitis canresultwhenenzymessecretedbypancreaticacinarcellsbecomeproteolyticallyactivatedbeforetheyhavereachedtheirappropriatesiteofactioninthesmallintestinallumen.Indeed,pancreaticjuicecontainsavarietyoftrypsininhibitorstoreducetheriskofprematureactivation,becausetrypsinistheactivatorofotherpro-formsofenzymessecretedinpancreaticjuice.Asecondlevelofprotectionliesinthefactthattrypsincanbedegradedbyothertrypsinmolecules.Despitethesedefenses,someindividualsaresusceptibletohereditarypancreatitisthatoccursspontaneouslyintheabsenceofknownriskfactors.Insomeofthesepatientsthereisamutationintrypsinthatrendersitresistanttodegradationbyothertrypsinmolecules.Othersharbormutationsintrypsininhibitors,renderingtheinhibitorsinactive.Inanyevent,ifotherdefenseshavebeenbreachedandtrypsinbecomesactiveprematurely,aviciouscycleofenzymeactivationensuesandboutsofpancreatitisfollow. Characteristics and Control of Ductular Secretion

1	Characteristics and Control of Ductular Secretion In this section we consider how the pancreatic ductular cells contribute to the flow and composition of pancreatic juice in the postprandial period. The ducts of the pancreas can be considered the effector arm of a pH regulatory Lobules of acinar and ductal parenchyma Secretion of H2O and – Extralobular HCO3 in response to ductal system absorption of Cl˜ Cl˜

1	Lobules of acinar and ductal parenchyma Secretion of H2O and – Extralobular HCO3 in response to ductal system absorption of Cl˜ Cl˜ Direction of fluid movement •Fig. 30.2 Locationsofimportanttransportprocessesinvolvedintheelaborationofpancreaticjuice.AcinarfluidisisotonicandresemblesplasmainitsconcentrationsofNa+ ,K+ ,Cl− ,andHCO3− .SecretionofacinarfluidandtheproteinsitcontainsisstimulatedprimarilybyCCK.Thehormonesecretinstimulatessecretionofwaterandelectrolytesfromthecellsthatlinetheextralobularducts.Thesecretin-stimulatedsecretionisricherinHCO3− thantheacinarsecretionbecauseofCl−/HCO3− exchange.(AdaptedfromSwansonCH,SolomonAK.J Gen Physiol 1973;62:407.) system designed to respond to luminal acid in the small intestine and secrete just enough bicarbonate to restore pH to neutrality (

1	Fig. 30.3 ). This regulatory function also requires mechanisms to sense luminal pH and convey this information to the pancreas as well as other epithelia (e.g., biliary ductules and the duodenal epithelium itself) capable of secreting bicarbonate. The pH-sensing mechanism is embodied in specialized endocrine cells known as S cells, localized within the small intestinal epithelium. When luminal pH falls below approximately 4.5, S cells are triggered to release secretin in response to the increase in [H+]. The components of this regulatory loop constitute a self-limited system. Thus as secretin evokes secretion of bicarbonate, pH in the small intestinal lumen will rise and the signal for release of secretin from S cells will be terminated.

1	At the cellular level, secretin stimulates epithelial cells to secrete bicarbonate into the ductular lumen, with water following via the paracellular route to maintain osmotic equilibrium. Secretin increases cyclic adenosine monophosphate (cAMP) in the ductular cells and thereby opens cystic fibrosis transmembrane conductance regulator (CFTR) • BOX 30.1 Products of Pancreatic Acinar Cells

1	Precursors of Proteases channels (Fig. 30.4 ) and causes an outflow of Cl− into the duct lumen. This secondarily drives the activity of an adjacent antiporter that exchanges the chloride ions for bicarbonate. CFTR is also permeable to bicarbonate. Thus the bicarbonate secretory process is dependent on CFTR, which provides an explanation for the defects in pancreatic function seen in the disease cystic fibrosis, in which CFTR is mutated. The bicarbonate needed for this secretory process is derived from two sources. Some is taken up across the basolateral membrane of the ductular epithelial cells via the sodium-bicarbonate cotransporter type 1 (NBC-1) symporter. Recall that the process of gastric acid secretion results in an increase in circulating bicarbonate ions, which can serve as a source of bicarbonate to be secreted by the pancreas. However, bicarbonate can also be generated intracellularly via the activity of the enzyme carbonic anhydrase. The net effect is to move HCO3 − into

1	of bicarbonate to be secreted by the pancreas. However, bicarbonate can also be generated intracellularly via the activity of the enzyme carbonic anhydrase. The net effect is to move HCO3 − into the lumen and thereby increase the pH and volume of pancreatic juice.

1	•Fig. 30.3 ParticipationofsecretinandHCO3−secretioninaclassicnegative-feedbackloopthatrespondstoafallinluminalpHintheduodenum.˜ pH in duodenum S cells Ductular bicarbonate secretion Secretin release ˜°°° •Fig. 30.4 Iontransportpathwaysinpancreaticductcells.CA,carbonicanhydrase;CFTR,cysticfibrosistransmembraneconductanceregulator;NBC-1,sodium/bicarbonatecotransporter(symporter)type1;NHE-1,sodium-hydrogenexchanger(antiporter)type1.NBC-1 NHE-1 Duct cell 2K˜Cl °HCO3 Duct lumen CFTR Na˜Na˜3Na˜2HCO3 H2CO3 CO2 ˜ H2O H2O ˜CO2 ˜H˜H2CO3 CA Cl °Na˜K˜H2O °2HCO3 °H˜˜ HCO3 °°HCO3° Characteristics and Control of Acinar Secretion In contrast to the pancreatic ductules, where secretin is the most important physiological agonist, CCK plays the predominant role at the level of the acinar cells. Thus it is important to understand how release of CCK is controlled during the small intestinal phase of the response to a meal.

1	Cysticfibrosis(CF)isageneticdiseasethataffectsthefunctionofavarietyofepithelialorgans,includingthelung,intestine,biliarysystem,andpancreas.Previouslythediseasewasalmostuniformlyfatalduringadolescenceasaresultofsevererespiratoryinfections.However,improvedantibiotics,drugsthatimproveclearanceofmucusfromthelungs,correctionofpancreaticinsufficiencyandundernutrition,andrecentUSFoodandDrugAdministration(FDA)approvalofdrugssuchasivacaftor(Kalydeco)andlumacaftor/ivacaftor(Orkambi)nowextendlifeintothefifthdecadeorlaterinsomepatients.CFiscausedbyamutationinCFTR,

1	whichinthegutimpairstheabilitytohydrateandalkalinizetheluminalcontents.IntheGIsystemspecifically,thiscanresultinintestinalobstruction,duodenalmucosalinjury,anddamagetotheliverandbiliarysystemaswellasthepancreas.InmanyCFpatientstheendocrinepancreasisdysfunctional,andtheymustbegivendigestiveenzymesupplementstomaintainadequatenutrientdigestion.Inotherpatientswithmildermutations,pancreatitismaydeveloplaterinlifeintheabsenceofotherclassicCFsymptoms,presumablybecauseofretentionofdigestiveenzymesinthepancreas.Ineithercase,improvedrecognitionandtreatmentofthepulmonarycomplicationsofCFmeanthatGIsymptomssuchasliverfailure,reducedbileflow,pancreatitis,obstruction,andmaldigestion/malabsorptionofnutrientsareacquiringincreasedimportanceasfacetsofthediseasethatmustbemanagedinadults,oftenbymultidisciplinaryteamsofphysiciansandotherhealthcareprofessionals.

1	CCK is the product of I cells, which are also localized in the small intestinal epithelium. These classic enteroendocrine cells release CCK into the interstitial space when specific food components are present in the lumen, particularly free fatty acids and certain amino acids. Release of CCK may occur following direct interaction of fatty acids or amino acids, or both, with the I cells. Release of CCK is also regulated by two luminally acting releasing factors that can stimulate the I cell. The first of these, CCK-releasing peptide, is secreted by paracrine cells within the epithelium into the small intestinal lumen in response to products of fat •Fig. 30.5 MechanismsresponsibleforcontrollingreleaseofCCKfromduodenalIcells.ACh,acetylcholine;CCK-RP,CCK-releasingpeptide;GRP,gastrin-releasingpeptide.Solidarrowsrepresentstimulatoryeffects,whereasdashedarrowsindicateinhibition.(RedrawnfromBarrettKE.Gastrointestinal Physiology. NewYork:McGraw-Hill;2006.) and protein digestion. The second

1	Physiology. NewYork:McGraw-Hill;2006.) and protein digestion. The second releasing factor, monitor peptide, is released by pancreatic acinar cells into pancreatic juice. Both CCK-releasing peptide and monitor peptide can also be released in response to neural input, which is particularly important in initiating pancreatic secretion during the cephalic and gastric phases, thereby preparing the system to digest the meal as soon as it enters the small intestine.

1	What is the significance of these CCK-releasing factors? Their primary role is to match CCK release, as well as the resulting availability of pancreatic enzymes, to the need for these enzymes to digest the meal in the small intestinal lumen ( Fig. 30.5 ). Because the releasing factors are peptides, they will be subject to proteolytic degradation by enzymes such as pancreatic trypsin in exactly the same way as dietary protein. However, when dietary protein is ingested, it is present in much greater amounts in the lumen than the releasing factors and thus “competes” with the releasing factors for proteolytic degradation. The net effect is that the releasing factors will be protected from breakdown while the meal is in the small intestine and are therefore available to continue stimulation of CCK release from I cells. However, once the meal has been digested and absorbed, the releasing factors are degraded and the signal for release of CCK is shut off.

1	CCK evokes secretion by pancreatic acinar cells in two ways. First, it is a classic hormone that travels through the bloodstream to encounter acinar cell CCK1 receptors. However, CCK also stimulates neural reflex pathways that impinge on the pancreas. Vagal afferent nerve endings in •Fig. 30.6 Receptorsofthepancreaticacinarcellandregulationofsecretion.ThethickblackarrowindicatesthatCa++-dependentsignalingpathwaysplaythemostprominentrole.ACh,acetylcholine;CCK,cholecystokinin;GRP,gastrin-releasingpeptide;VIP,vasoactiveintestinalpolypeptide;M3,M3muscarinicreceptor;CCK-1;CCKreceptortype1.(RedrawnfromBarrettKE.Gastrointestinal Physiology. NewYork:McGraw-Hill;2006.) the wall of the small intestine are responsive to CCK by virtue of their expression of CCK1 receptors. As described earlier for the effect of CCK on gastric emptying, binding of CCK activates a vagovagal reflex that can further enhance acinar cell secretion via activation of pancreatic enteric neurons and release of a series of

1	effect of CCK on gastric emptying, binding of CCK activates a vagovagal reflex that can further enhance acinar cell secretion via activation of pancreatic enteric neurons and release of a series of neurotransmitters such as acetylcholine, gastrin-releasing peptide, and vasoactive intestinal polypeptide (VIP).

1	The secretory products of pancreatic acinar cells are largely presynthesized and stored in granules that cluster toward the apical pole of acinar cells (

1	Fig. 30.6 ). The most potent stimuli of acinar cell secretion, including CCK, acetylcholine, and gastrin-releasing peptide (GRP), act by mobilizing intracellular Ca++ . Stimulation of acinar cells results in phosphorylation of a series of regulatory and structural proteins within the cell cytosol that move the granules closer to the apical membrane, where the granules fuse with the plasma membrane. The contents of the granule are then discharged into the acinar lumen and washed out by an exudate of plasma crossing the tight junctions, linking the acinar cells together, and subsequently by ductular secretions. In the period between meals the granule constituents are resynthesized by the acinar cells and then stored until needed to digest the next meal. Resynthesis may be stimulated by the same agonists that evoke the initial secretory response.

1	Another important digestive juice that is mixed with the meal in the small intestinal lumen is bile. Bile is produced by the liver, and the mechanisms that are involved, as well as the specific constituents, will be discussed in greater detail in when we address the transport and metabolic functions of the liver. However, for purposes of the current discussion, bile is a secretion that serves to aid in digestion and absorption of lipids. Bile flowing out of the liver is stored and concentrated in the gallbladder until it is released in response to ingestion of a meal. Contraction of the gallbladder, as well as relaxation of the sphincter of Oddi, are evoked predominantly by CCK.

1	When considering the small intestinal phase of meal assimilation, the bile constituents we are most concerned with are the bile acids. These form structures known as micelles that serve to shield the hydrophobic products of lipid digestion from the aqueous environment of the lumen. Bile acids are in essence biological detergents, and large quantities are needed on a daily basis for optimal lipid absorption—as much as 1 to 2 g/day. The majority of the bile acid pool is recycled from the intestine back to the liver after each meal via the enterohepatic circulation (Fig. 30.7 ). Thus bile acids are synthesized in a conjugated form that limits their ability to passively cross the epithelium •Fig. 30.7 Enterohepaticcirculationofbileacids.Activeuptakeofconjugatedbileacidsoccursviatheapicalsodium-dependentbileacidtransporter(asbt).

1	lining the intestine so that they are retained in the lumen to participate in lipid assimilation (discussed in detail under that heading). However, when the meal contents reach the terminal ileum, after lipid absorption has been completed, the conjugated bile acids are reabsorbed by a symporter, the apical Na+-dependent bile acid transporter (asbt), that specifically takes up conjugated bile acids in association with sodium ions. Only a minor portion of the bile acid pool is left to spill over into the colon in health, and here bile acids become deconjugated and subject to passive reabsorption (see Fig. 30.7).

1	Fig. 30.7). The net effect is to cycle the majority of the bile acid pool between the liver and intestine on a daily basis, coincident with signals arising in the postprandial period. Bile acids also exert biological actions beyond their role as detergents by binding to both cell surface and nuclear receptors in a variety of cell types throughout the body. In this way they regulate their own synthesis as well as other metabolic processes. The most important physiological function of the small intestine is to take up the products of digestion of ingested nutrients. Quantitatively the most significant nutrients (macronutrients) fall into three classes: carbohydrates, proteins, and lipids. The small intestine is critical not only

1	The Small Intestinal Phase of the Integrated Response to a Meal for absorption of nutrients into the body but also for the final stages of their digestion into molecules that are simple enough to be transported across the intestinal epithelium. We will consider the processes involved in assimilation of each of these nutrients in turn, beginning with carbohydrates. Carbohydrate digestion occurs in two phases: in the lumen of the intestine and then on the surface of enterocytes in a process known as brush border digestion. The latter is important in generating simple absorbable sugars only at the point where they can be absorbed. This may therefore limit their exposure to the small number of bacteria present in the small intestinal lumen that might otherwise use these sugars as nutrients. Digestion of Carbohydrates

1	Digestion of Carbohydrates Dietary carbohydrates are composed of several different molecular classes. Starch, the first of these, is a mixture of both straightand branched-chain polymers of glucose. The straight-chain polymers are called amylose and the branched-chain molecules are called amylopectin (Fig.

1	30.8 ). Starch is a particularly important source of calories, especially in developing countries, and is found predominantly in cereal products. Disaccharides are a second class of carbohydrate nutrients that includes sucrose (consisting of glucose and fructose) and lactose (consisting of glucose and galactose), the latter being an important caloric source in infants. It is, however, a key principle that the intestine can absorb only monosaccharides and not larger carbohydrates. Finally, many food items of vegetable origin contain dietary fiber, which consists of carbohydrate polymers that cannot be digested by human enzymes. These polymers are instead digested by bacteria present largely in the colonic lumen (see ), thereby allowing salvage of their caloric value.

1	Dietary disaccharides are hydrolyzed to their component monomers directly on the surface of small intestinal epithelial cells by brush border digestion, mediated by a family of membrane-bound, heavily glycosylated hydrolytic enzymes synthesized by small intestinal epithelial cells. Brush border hydrolases critical to the digestion of dietary carbohydrates include sucrase, isomaltase, glucoamylase, and lactase ( Table 30.1). Glycosylation of these hydrolases is believed to protect them to some extent from degradation by luminal pancreatic proteases. However, between meals the hydrolases are degraded and must therefore be resynthesized by the enterocyte to participate in digesting the next carbohydrate meal. Sucrase/isomaltase and glucoamylase are synthesized in quantities that are in excess of requirements, and assimilation of their products into the body is limited by the availability of specific membrane transporters for these monosaccharides (see

1	Uptake of Carbohydrates). Lactase, in contrast, shows a developmental decline in expression after weaning. The relative paucity of lactase means that digestion of lactose, rather than uptake of the resulting products, is rate limiting for assimilation. If lactase levels fall below a certain threshold, the disease of lactose intolerance results. •Fig. 30.8 Structureofamylopectinandtheactionofamylase.Thebluecirclesrepresentglucosemonomerslinkedbyα-1,4bonds.Theblackcirclesrepresentglucoseunitslinkedbyα-1,6bondsatthebranchpoints.

1	•Fig. 30.8 Structureofamylopectinandtheactionofamylase.Thebluecirclesrepresentglucosemonomerslinkedbyα-1,4bonds.Theblackcirclesrepresentglucoseunitslinkedbyα-1,6bondsatthebranchpoints. Lactose intolerance isrelativelycommoninadultsfromspecificethnicgroups,suchasAsians,AfricanAmericans,andHispanics.Thedisorderreflectsanormaldevelopmentaldeclineintheexpressionoflactasebyenterocytes,particularlywhenlactoseisnotaconsistentcomponentofthediet.Insuchindividuals,consumptionoffoodscontaininglargequantitiesoflactose(e.g.,milk,icecream)canresultinabdominalcramping,gas,anddiarrhea.Thesesymptomsreflectarelativeinabilitytodigestlactose;thusitremainsinthelumen,andwaterisretained.Somelactose-intolerantpatientsbenefitfromoraladministrationofabacteriallyderivedlactaseenzymebeforeingestingdairyproducts.

1	Digestion of starch occurs in two phases. The first takes place in the lumen and is actually initiated in the oral cavity via the activity of salivary amylase, as discussed in . Salivary amylase, however, is not essential for starch digestion, although it may assume greater importance in neonates or patients in whom the output of pancreatic enzymes is impaired by disease. Quantitatively the most significant contributor to the luminal digestion of starch is pancreatic amylase. Both enzymes hydrolyze internal α-1,4 bonds in both amylose and amylopectin, but not external bonds nor the α-1,6 bonds that form the branch points in the amylopectin molecule (see

1	Fig. 30.8 ). Thus digestion of starch by amylase is of necessity incomplete and results in short oligomers of glucose, including dimers (maltose) and trimers (maltotriose), as well as the simplest branching structures, which are called α-limit dextrins. Thus to allow absorption of its constituent monosaccharides, starch must also undergo brush border digestion. At the brush border, straight-chain glucose oligomers can be digested by the hydrolases glucoamylase, sucrase, or isomaltase (see Table 30.1 ). All yield free glucose monomers, which can then be absorbed by the mechanisms discussed later. For α-limit dextrins, on the other hand, isomaltase activity is critical because it is the only enzyme that can cleave α-1,6 bonds that make up the branch points as well as α-1,4 bonds. Uptake of Carbohydrates

1	Uptake of Carbohydrates Water-soluble monosaccharides resulting from digestion must next be transported across the hydrophobic plasma membrane of the enterocyte. The sodium/glucose transporter 1 (SGLT1) is a symporter that takes up glucose (and galactose) against its concentration gradient by coupling its transport to that of Na+ (

1	Fig. 30.9 ). Once inside the cytosol, glucose and galactose can be retained for the epithelium’s metabolic needs or can exit the cell across its basolateral pole via a transporter known as GLUT2. Fructose, in contrast, is taken up across the apical membrane by GLUT5. However, •Fig. 30.9 Absorptionofglucose,galactose,andfructoseinthesmallintestine.GLUT,glucosetransporter;SGLT1,sodium/glucosetransporter1.GlucoseGalactoseFructoseFructoseGLUT2GLUT5 GLTI?K+ATP because fructose transport is not coupled to that of Na+, its uptake is relatively inefficient and can easily be overwhelmed if large quantities of food containing this sugar are ingested. The symptoms that occur from this malabsorption are similar to those experienced by a lactose-intolerant patient who consumes lactose.

1	Proteins are also water-soluble polymers that must be digested into their smaller constituents before absorption is possible. Their absorption is more complicated than that of carbohydrates because they contain 20 different amino acids and short oligomers of these amino acids (dipeptides, tripeptides, and perhaps even tetrapeptides) can also be transported by enterocytes. The body, particularly the liver (see ), has substantial ability to interconvert various amino acids subject to the body’s needs. However, some amino acids, termed the essential amino acids, cannot be synthesized by the body either de novo or from other amino acids and thus must be obtained from the diet. The amino acids that must be obtained in this way in humans are shown in Fig. 30.10

1	Fig. 30.10 Araregeneticdisorderresultsinaninabilityoftheintestinetoabsorbglucoseorgalactose.ThisdiseasehasbeenmappedtoavarietyofmutationsintheSGLT1 genethatresultinafaultyorunexpressedproteinor,morecommonly,failureoftheproteintotrafficappropriatelytotheapicalmembraneofenterocytes.Inpatientscarryingsuchmutations,malabsorbedglucosecontributestodiarrhealandothersymptoms,asdiscussedearlierforlactoseintolerance.Despitetherarityofthedisease,itisimportantintermsoftheinsightitprovidedintoacriticalprocessofintestinalepithelialtransport. •Fig. 30.10 Naturallyoccurringdietaryaminoacids.Thoseinboxesareessentialaminoacidsthatcannotbesynthesizedbyhumansandthusmustbeobtainedfromthediet.(RedrawnfromBarrettKE.Gastrointestinal Physiology. NewYork:McGraw-Hill;2006.)Amino acids NeutralBasicArg HisLys AcidicGlu, Gln Asp, Asn AllphaticGly, Ala AromaticTyr Phe Try HydroxylSer Thr SulfurCys Met IminoPro HydroxyproLeu lleVal , , , , Digestion of Proteins

1	Digestion of Proteins Proteins can be hydrolyzed to long peptides simply by virtue of the acidic pH that exists in the gastric lumen. However, for assimilation of proteins into the body, three phases of enzymatically mediated digestion are required (

1	Fig. 30.11 ). Like acid hydrolysis, the first of these phases takes place in the gastric lumen and is mediated by pepsin, the product of chief cells localized to the gastric glands. When gastric secretion is activated by signals coincident with ingestion of a meal, pepsin is released from the chief cells as the inactive precursor pepsinogen. At acidic pH, this precursor is autocatalytically cleaved to yield the active enzyme. Pepsin is highly specialized to act in the stomach, since it is activated by low pH. The enzyme cleaves proteins at sites of neutral amino acids, with a preference for aromatic or large aliphatic side chains. Because such amino acids occur only relatively infrequently in a given protein, pepsin is not capable of digesting protein fully into a form that can be absorbed by the intestine. Instead it yields a mixture of intact protein, large peptides (the majority), and a limited number of free amino acids.

1	On moving into the small intestine, the partially digested protein encounters the proteases provided in pancreatic juice. Recall that these enzymes are secreted in inactive forms. How then are they activated to begin the process of protein digestion? In fact, activation of proteases is delayed until these enzymes are in the lumen by virtue of the localized presence of an activating enzyme, enterokinase, only on the brush border of small intestinal epithelial cells (

1	Fig. 30.12 ). Enterokinase cleaves trypsinogen to yield active trypsin. Trypsin in turn cleaves all the other protease precursors secreted by the pancreas, thereby resulting in a mixture of enzymes that can almost completely digest the vast majority of dietary proteins. Trypsin is an endopeptidase that cleaves proteins only at internal bonds within the peptide chain rather than releasing individual amino acids from the end of the chain. Trypsin is specific for cleavage at basic amino acids, and such cleavage results in a set of shorter peptides with a basic amino acid at their C-terminus. The two other pancreatic endopeptidases, chymotrypsin and elastase, have a similar mechanism of action but cleave at sites of neutral •Fig. 30.11 Hierarchyofproteasesandpeptidasesthatfunctioninthestomachandsmallintestinetodigestdietaryprotein.Proteinsareabsorbedaseithersingleaminoacids(70%)orshortpeptides(30%).(AdaptedfromVanDykeRW.In:SleisengerMH,FordtranJS[eds].Gastrointestinal Disease. 4thed.

1	Philadelphia:Saunders;1989.) •Fig. 30.12 Conversionoftheinactiveproenzymesofpancreaticjuicetoactiveenzymesbytheactionoftrypsin.Trypsinogeninpancre-aticjuiceisproteolyticallyconvertedtoactivetrypsinbyenterokinaseexpressedonthesurfaceofepithelialcellsoftheduodenumandjejunum.Trypsinthenactivatestheotherproenzymesasshown. amino acids. The peptides that result from endopeptidase activity are then acted on by pancreatic ectopeptidases. These enzymes cleave single amino acids from the end of a peptide chain, and those present in pancreatic juice are specific for either neutral (carboxypeptidase A) or basic (carboxypeptidase B) amino acids situated at the C-terminus. Thus the products that result after digestion of a protein meal by gastric and pancreatic secretions include neutral and basic amino acids, as well as short peptides that have acidic amino acids at their C-termini and thus are resistant to carboxypeptidase A or B (Fig. 30.13

1	B (Fig. 30.13 The final phase of protein digestion takes place at the brush border. Mature enterocytes express a variety of peptidases on their brush borders, including both amino-peptidases and carboxypeptidases that generate products suitable for uptake across the apical membrane (see Fig.

1	Fig. 30.11 ). However, it should be noted that even with the substantial complement of active proteolytic enzymes, some dietary peptides are either relatively or totally resistant to hydrolysis. In particular, peptides containing either proline or glycine are digested very slowly. Fortunately the intestine can take up short peptides in addition to single amino acids. The majority of peptides taken up into the enterocyte in their intact form are then subjected to a final stage of digestion in the cytosol of the enterocyte to liberate their constituent amino acids for use in the cell or elsewhere in the body (Fig. 30.14 ). However, some di-and tripeptides may also be transported into the blood in their intact form.

1	•Fig. 30.13 Luminaldigestionofpeptidesresultingfrompartialproteolysisinthestomach.AA,aminoacid.(RedrawnfromBarrettKE.Gastrointestinal Physiology. NewYork:McGraw-Hill;2006.)Peptide with C-terminal neutral AA Carboxypeptidase AChymotrypsin Elastase Large peptides Trypsin Peptide with C-terminal basic AA Short peptides free neutral and basic AAs SerArg Carboxypeptidase B Ser Arg •Fig. 30.14 Awidevarietyofdipeptidesandtripeptidesaretakenupacrossthebrushbordermembranebytheproton-coupledsymporterknownaspeptide transporter 1 (PepT1).Theprotongradientiscreatedbytheactionofsodium/hydrogenexchangers(NHEs)intheapicalmembrane.Peptidesarelargelydigestedinthecytosoltotheirconstituentaminoacidsforexporttothebody,butasmallproportionmaybeexportedintact. Uptake of Peptides and Amino Acids

1	Uptake of Peptides and Amino Acids The body is also endowed with a series of plasma membrane transporters capable of promoting uptake of the water-soluble products of protein digestion. Given the large number of amino acids, there is a relatively large number of specific transporters (see Figs. 30.11 30.14 ). Amino acid transporters are of clinical interest because their absence in a variety of genetic disorders results in diminished ability to transport the relevant amino acid or acids. However, such mutations are often clinically silent, at least from a nutritional standpoint, because the amino acid in question can be assimilated by other transporters with overlapping specificity or in the form of peptides. This does not rule out the possibility of pathology in other organ systems in

1	The Small Intestinal Phase of the Integrated Response to a Meal which the transporter of interest may normally be expressed (e.g., cystinuria). In general, amino acid transporters have reasonably broad specificity and usually transport a subset of amino acids (e.g., neutral, anionic, or cationic) but with some overlap in their affinity for particular amino acids. Furthermore, some (but not all) of the amino acid transporters are symporters that carry their substrate amino acids in conjunction with obligatory uptake of Na+ . The small intestine is also notable for its ability to take up short peptides (see

1	The small intestine is also notable for its ability to take up short peptides (see Fig. 30.14 ). The primary transporter responsible for such uptake is called peptide transporter 1 (PepT1) and is a symporter that transports peptides in conjunction with protons. Amino acids liberated from these peptides that are not required by the enterocyte are in turn exported across the basolateral membrane and enter blood capillaries to be transported to the liver via the portal vein. PepT1 is also of clinical interest because it can mediate the uptake of so-called peptidomimetic drugs, which include a variety of antibiotics as well as cancer chemotherapeutic agents. The mechanisms by which amino acids and peptidomimetic drugs exit the enterocyte are not fully understood but are presumed to involve additional transport proteins.

1	Theredundancyinuptakemechanismsfortheproductsofproteindigestionunderscorestheimportanceofthisprocessandalsomeansthatdeficienciesinspecificaminoacidassimilationacrosstheintestinearerelativelyrare.However,undercertaincircumstances,mutationsinproteinsresponsibleforspecificaminoacidtransportcanleadtopathologyinotherorgans.Oneexampleisthediseaseofcysteinuria,whichisamolecularlyheterogeneousdiseaseinvolvingmutationsinavarietyofaminoacidtransporterscapableoftransportingcysteine.Becausecysteinecanalsobeassimilatedacrossthegutintheformofpeptides,nutritionaldeficienciesdonotoccurdespitealackofintestinaluptakemechanismsforthisparticularaminoacid.Incontrast,cysteinecanonlybepoorlyreabsorbedfromtheurineofpatientssufferingfromcystinuria,andkidneystonescanformbecausethisaminoacidisrelativelyinsoluble.PathophysiologycanalsoarisesecondarytomutationsinSLC6A19, aNa+-independenttransporterofneutralaminoacids,andresultinaconditionknownasHartnup disease,

1	aNa+-independenttransporterofneutralaminoacids,andresultinaconditionknownasHartnup disease, arelativelyrareinbornerrorofmetabolismthatcauseslossofneutralaminoacidsinurine,resultinginpsychiatricissues,intellectualdisability,shortstature,headaches,andunsteadygait,althoughnotnutritionaldeficienciesperse.

1	Lipids, defined as substances that are more soluble in organic solvents than in water, are the third major class of macronutrients making up the human diet. Lipids supply more significant calories on a per-gram basis than proteins or carbohydrates do and are thus of major nutritional significance; they also have a propensity to contribute to obesity if consumed in excessive amounts. Lipids also dissolve volatile compounds that contribute to food’s taste and aroma.

1	The predominant form of lipid in the human diet is triglyceride, found in oils and other fats. The majority of these triglycerides have long-chain fatty acids (carbon chains > 12 carbons) esterified to the glycerol backbone. Additional lipid is supplied in the form of phospholipids and cholesterol, mostly arising from cell membranes. It is also important to consider that the intestine is presented daily not only with dietary lipid but also with lipid originating from the liver in biliary secretions, as described in more detail in . Indeed, the cholesterol supplied in bile exceeds that provided in the diet on a daily basis in all but the most egg-loving individuals. Finally, though present in only trace amounts, the fat-soluble vitamins (A, D, E, K) are essential nutrients that should be supplied in the diet to avoid disease. These substances are almost entirely insoluble in water and thus require special handling to promote their uptake into the body.

1	Emulsification and Solubilization of Lipids When a fatty meal is ingested, the lipid becomes liquefied at body temperature and floats on the surface of the gastric contents. This would limit the area of the interface between the aqueous and lipid phases of the gastric contents and thus restrict access of enzymes capable of breaking down the lipid to forms that can be absorbed. This is because the lipolytic enzymes, as proteins, reside in the aqueous phase. Therefore an early stage in the assimilation of lipid is its emulsification. The mixing action of the stomach churns the dietary lipid into a suspension of fine droplets, which vastly increases the surface area of the lipid phase. Lipid absorption is also facilitated by formation of a micellar solution with the aid of bile acids supplied in biliary secretions. Details of this process will be discussed subsequently. Digestion of Lipids

1	Digestion of Lipids Lipid digestion begins in the stomach. Gastric lipase is released in large quantities from gastric chief cells; it adsorbs to the surface of fat droplets dispersed in the gastric contents and hydrolyzes component triglycerides to diglycerides and free fatty acids. However, little lipid assimilation can take place in the stomach because of the acidic pH of the lumen, which results in protonation of the free fatty acids released by gastric lipase. Lipolysis is also incomplete in the stomach because gastric lipase, despite its optimum catalytic activity at acidic pH, is not capable of hydrolyzing the second position of the triglyceride ester, which means that the molecule cannot be fully broken down into components that can be absorbed into the body. There is also little if any breakdown of cholesterol esters or the esters of fat-soluble vitamins. Indeed, gastric lipolysis is dispensable in healthy individuals because of the marked excess of pancreatic enzymes.

1	The majority of lipolysis takes place in the small intestine in health. Pancreatic juice contains three important lipolytic enzymes that are optimized for activity at neutral pH. The first of these is pancreatic lipase. This enzyme differs from the stomach enzyme in that it is capable of hydrolyzing both the 1 and 2 positions of triglyceride to yield a large quantity of free fatty acids and monoglycerides. At neutral pH, the head groups of the free fatty acids are charged, and thus these molecules migrate to the surface of the oil droplets. Lipase also displays an apparent paradox in that it is inhibited by bile acids, which also form part of the small intestinal contents. Bile acids adsorb to the surface of the oil droplets and cause lipase to dissociate. However, lipase activity is sustained by an important cofactor, colipase, which is also supplied in pancreatic juice. Colipase is a bridging molecule that binds to bile acids and to lipase; it anchors lipase to the oil droplet even

1	by an important cofactor, colipase, which is also supplied in pancreatic juice. Colipase is a bridging molecule that binds to bile acids and to lipase; it anchors lipase to the oil droplet even in the presence of bile acids.

1	Pancreatic juice also contains two additional enzymes that are important in fat digestion. The first of these is phospholipase A2, which hydrolyzes phospholipids such as those present in cell membranes. Predictably this enzyme would be quite toxic in the absence of dietary substrates, and thus it is secreted as an inactive pro-form that is activated only when it reaches the small intestine. Furthermore, pancreatic juice contains a relatively nonspecific cholesterol esterase that can break down esters of cholesterol, as its name implies, as well as esters of fat-soluble vitamins and even triglycerides. Interestingly this enzyme requires bile acids for activity (contrast with lipase, discussed earlier), and it is related to an enzyme produced in breast milk that plays an important role in lipolysis in neonates.

1	As lipolysis proceeds, the products are abstracted from the lipid droplet, first into a lamellar (membrane) phase and subsequently into mixed micelles composed of lipolytic products as well as bile acids. The amphipathic (meaning they have both a hydrophobic and hydrophilic face) bile acids serve to shield the hydrophobic regions of lipolytic products from water while presenting their own hydrophilic faces to the aqueous environment (

1	Fig. 30.15 ). Micelles are truly in solution and thus markedly increase lipid solubility in the intestinal contents. This increases the rate at which molecules such as fatty acids diffuse to the absorptive epithelial surface. Nevertheless, given the very large surface area of the small intestine and the appreciable molecular solubility of the products of triglyceride hydrolysis, micelles are not essential for the absorption of triglyceride. Thus patients who have insufficient output of bile acids (caused, for example, by a gallstone that obstructs bile output) do not normally show fat malabsorption. On the other hand, cholesterol and the fat-soluble vitamins are almost totally insoluble in water and accordingly require micelles to be absorbed even after they have been digested. Thus if luminal bile acid concentrations fall below the critical micellar concentration, patients can become deficient in fat-soluble vitamins.

1	•Fig. 30.15 Schematicdepictionofbileacids(A)andmixedmicelles(B).Bileacidsinsolutionareamphipathic.Mixedmicellesarecylindricalassemblagesofbileacidswithotherdietarylipids. Uptake of Lipids and Subsequent Handling

1	The products of fat digestion are believed to be capable of crossing cell membranes readily because of their lipophilicity. However, recent evidence suggests that their uptake may alternatively or additionally be regulated via the activity of specific membrane transporters. A microvillus membrane fatty acid–binding protein (MVM-FABP) provides for the uptake of long-chain fatty acids across the brush border. Likewise, Niemann-Pick C1–like 1 (NPC1L1) has recently been identified as an uptake pathway for cholesterol in enterocytes and may be a therapeutic target in patients who suffer from pathological increases in circulating cholesterol (hypercholesterolemia). However, uptake of cholesterol overall is relatively inefficient because this molecule, along with plant sterols, can also be actively effluxed from enterocytes back into the cytosol by a heterodimeric complex of two ATP-binding cassette (ABC) transporters termed ABC G5 and G8. Finally, the glycerol backbone of triglycerides may

1	effluxed from enterocytes back into the cytosol by a heterodimeric complex of two ATP-binding cassette (ABC) transporters termed ABC G5 and G8. Finally, the glycerol backbone of triglycerides may be transported into intestinal epithelial cells by a number of different aquaglyceroporins.

1	Arelativelynewtreatmentforhypercholesterolemiatargetsabsorptionofcholesterol,eitherderivedfromthedietorinbile,acrossthesmallintestinalepithelium.EzetimibeisadrugthatspecificallyblockscellularuptakeofcholesterolbyinhibitingtheactivityoftheNPC1L1proteinexpressedintheapicalmembraneofenterocytes.Inconjunctionwithotherdrugsdesignedtocounteratherosclerosis,thismaybeausefuladjunctinthatitcaninterrupttheenterohepaticcirculationaswellaspreventabsorptionofdietarycholesterol.Clinicalstudiessuggestthatezetimibemaysynergisticallyimprovetheefficacyofotherstrategiesdesignedtoreducecirculatinglevelsoflow-densitylipoproteincholesterolinthoseatriskforcardiovasculardisease,suchastheuseofstatindrugs.

1	Lipids also differ from carbohydrates and proteins in terms of their fate after absorption into the enterocyte. Unlike monosaccharides and amino acids, which leave the enterocyte in molecular form and enter the portal circulation, the products of lipolysis are reesterified in the enterocyte to form triglycerides, phospholipids, and cholesterol esters. These metabolic events take place in the smooth endoplasmic reticulum. Concurrently the enterocyte synthesizes a series of proteins known as apolipoproteins in the rough endoplasmic reticulum. These proteins are then combined with the resynthesized lipids to form a structure known as a chylomicron, which consists of a lipid core (predominantly triglyceride with much less cholesterol, phospholipid, and fat-soluble vitamin esters) coated by the apolipoproteins. The chylomicrons are then exported from the enterocyte by a process of exocytosis. However, on entering the lamina propria, they are too large (≈750–5000 Å in diameter) to permeate

1	apolipoproteins. The chylomicrons are then exported from the enterocyte by a process of exocytosis. However, on entering the lamina propria, they are too large (≈750–5000 Å in diameter) to permeate through the intercellular spaces of the mucosal capillaries. Instead they are taken up into lymphatics in the lamina propria and therefore bypass the portal circulation and, at least for their first pass, the liver. Eventually, chylomicrons in the lymph enter the bloodstream via the thoracic duct and then serve as the vehicle to transport lipids around the body for use by cells in other organs. The only exception to this chylomicron-mediated transport is for medium-chain fatty acids. These acids are relatively water soluble and can also permeate enterocyte tight junctions appreciably, which means that they bypass the intracellular processing steps described earlier and are not packaged into chylomicrons. They therefore enter the portal circulation and are more readily available to other

1	means that they bypass the intracellular processing steps described earlier and are not packaged into chylomicrons. They therefore enter the portal circulation and are more readily available to other tissues. A diet rich in medium-chain triglycerides may be of particular benefit in patients with inadequate bile acid pools.

1	The foregoing description of digestion has stressed that these processes take place in the small intestine in an aqueous •Fig. 30.16 OverallfluidbalanceinthehumanGItract.About1to2Lofwaterisingested,and8LofvarioussecretionsenterstheGItract.Ofthistotal,mostisabsorbedinthesmallintestine.About2Lispassedontothecolon,thevastmajorityofwhichisabsorbedinhealth.(FromVanderAJetal.Human Physiology. 6thed.NewYork:McGraw-Hill;1994.) milieu. The fluidity of intestinal contents, especially in the small intestine, is important in allowing the meal to be propelled along the length of the intestine and to permit digested nutrients to diffuse to their site of absorption. Part of this fluid is derived from oral intake, but in most adults this consists of only about 1 to 2 L/day derived from both food and drink (Fig. 30.16 ). Additional fluid is supplied by the stomach and the small intestine themselves, as well as the organs that drain into the GI tract. In total, these secretions add another 8 L, which

1	(Fig. 30.16 ). Additional fluid is supplied by the stomach and the small intestine themselves, as well as the organs that drain into the GI tract. In total, these secretions add another 8 L, which means that the intestine is presented with approximately 9 to 10 L of fluid on a daily basis. However, in health only about 2 L of this load is passed to the colon for reabsorption, and eventually only 100 to 200 mL exits in stool. Thus the intestine absorbs water overall. During the postprandial period, such absorption is promoted in the small intestine predominantly via the osmotic effects of nutrient absorption. An osmotic gradient is established across the intestinal epithelium that simultaneously drives movement of water across the tight junctions. The generic mechanism for nutrient-driven Na+ and water absorption in the small intestine is diagrammed in

1	Fig. 30.17 . Moreover, in the period between meals, when nutrients are absent, fluid absorption can still occur via the coupled uptake of Na+ and Cl− mediated by the cooperative interaction of a Na+/H+ antiporter (NHE-3) and a Cl−/ HCO3 − antiporter (see Fig. 30.17

1	Even though net water and electrolyte transport in the small intestine is predominantly absorptive, this does not imply that the tissue fails to participate in electrolyte secretion. Secretion is regulated in response to signals derived from the luminal contents and in response to deformation of the mucosa and intestinal distention. Critical secretagogues include acetylcholine, VIP, prostaglandins, and serotonin. Secretion ensures that the intestinal contents are appropriately fluid while digestion and absorption are ongoing and is important to lubricate the passage of food particles along the length of the intestine. For example, some clinical evidence suggests that constipation and intestinal obstruction, the latter observed in cystic fibrosis, can result when secretion is abnormally low. The majority of the intestinal •Fig. 30.17

1	evidence suggests that constipation and intestinal obstruction, the latter observed in cystic fibrosis, can result when secretion is abnormally low. The majority of the intestinal •Fig. 30.17 MechanismsofNaClabsorptioninthesmallintestine.DRA,downregulatedinadenoma;GLUT2,glucosetransporter2;KCC1,potassium/chloridecotransporter1;NHE-3,sodium/hydrogenexchanger3;PAT1,putativeaniontransporter1;SGLT1,sodium/glucosetransporter1.SGLT1H˜2K˜KCC1NHE 3DRA?PAT1?ATPHCO3°GLUT2Glucose secretory flow of fluid into the lumen is driven by active secretion of chloride ions via the mechanism diagrammed in

1	Fig. 30.18 . In cystic fibrosis the loss of CFTR Cl channels can partially be compensated for by accessory chloride channels, such as the TMEM16A channel that is activated by elevations in cytoplasmic calcium. Some segments of the intestine may engage in additional secretory mechanisms, such as secretion of bicarbonate ions via the mechanisms shown in Fig. 30.19. Bicarbonate secretion protects the epithelium, particularly in the most proximal portions of the duodenum immediately downstream from the pylorus, from damage caused by acid and pepsin. Absorption of Minerals and Water-Soluble Vitamins

1	Absorption of Minerals and Water-Soluble Vitamins The small intestine is also an important site for absorption of water-soluble vitamins (e.g., vitamin C, cobalamin) and minerals such as calcium, magnesium, and iron. In general these substances are taken up from the luminal contents via the activity of specific transporters. Their uptake is •Fig. 30.18 MechanismofCl−secretioninthesmallandlargeintestines.CFTR,cysticfibrosistransmembraneconductanceregula-tor;NKCC1,sodium/potassium/2chloridecotransporter1.Notethatadditionalchloridechannels(notshown),suchasthoseregulatedbyintracellularcalciumconcentration,mayalsoexistintheapicalmembraneandcontributetooveralltransport.˜Ca˜˜Cl°2K˜˜˜cAMPNKCC1ATP˜ also controlled by feedback mechanisms that sense the concentration of the relevant substrate in the circulation and adjust the expression of transporters and accessory molecules accordingly. Motor Patterns of the Small Intestine

1	Motor Patterns of the Small Intestine The smooth muscle layers in the small intestine produce motility patterns that mix chyme with the various digestive secretions and propel fluid along the length of the intestine so that nutrients (along with water and electrolytes) can be absorbed. Motor patterns of the small intestine during the postprandial period are directed predominantly toward mixing and consist largely of segmenting and retropulsive contractions that retard the meal while digestion is still ongoing. Segmentation is a stereotypical pattern of rhythmic contractions that is displayed in

1	Fig. 30.20 and presumably reflects programmed activity of the enteric nervous system superimposed on the basic electrical rhythm. Hormonal mediators of this fed pattern of motility are poorly defined, although CCK contributes. CCK also plays important roles in slowing gastric emptying when the meal is in the small intestine, as described at the beginning of this chapter. This makes sense as a mechanism to match nutrient delivery to the available capacity to digest and absorb the components of the meal. •Fig. 30.19 Mechanismsofbicarbonatesecretionintheduodenum.CA,carbonicanhydrase;CFTR,cysticfibrosistransmembranecon-ductanceregulator;DRA,downregulatedinadenoma;NBC-1,sodium/bicarbonatecotransporter1;NHE-1,sodium/hydrogenexchanger-1;PAT1,putativeaniontransporter1.3Na˜Na˜Na˜H˜Cl°Cl°K˜NBC-1 NHE-1 DRA? PAT1? CFTR ATP CO2 + H2O CA HCO3 °HCO3 °

1	After the meal has been digested and absorbed, it is desirable to clear any undigested residues from the lumen to prepare the intestine for the next meal. Such clearance is effected by peristalsis ( Fig. 30.21 ), a coordinated sequence of contraction occurring above the intestinal contents and relaxation below that permits the contents to be conveyed over considerable distances. Peristalsis reflects the action of acetylcholine and substance P released orad to a site of intestinal distention, which serves to contract the circular muscle, as well as the inhibitory effects of VIP and nitric oxide on the caudad side. Like segmentation, peristalsis originates when action potentials generated by intrinsic innervation are superimposed on sites of cellular depolarization dictated by the basic electrical rhythm. The peristaltic motor patterns that occur during fasting, moreover, are organized into a sequence of phases known as the migrating motor complex (

1	Fig. 30.22 ). Phase I of the MMC is characterized by relative quiescence, whereas small disorganized contractions begin to occur during phase II. During phase III, which lasts about 10 minutes, large contractions that •Fig. 30.20 A,Radiographicviewshowingthestomachandsmallintestinefilledwithbariumcontrastmediuminanormalindividual.Notethesegmentationoftheintestine.B,Sequenceofsegmentalcontractionsinthesmallintestine.Lines1to4representsequentialtimepoints.Thedottedlinesindicatewherecontractionswilloccurnext;thearrowsdepictthedirectionofmovementoftheintestinalcontents.(A,FromGardenerEMetal.Anatomy: A Regional Study of Human Structure. 4thed.Philadelphia:Saunders;1975;B,redrawnfromCannonWB. Am J Physiol 1902;6:251.) •Fig. 30.21 Peristalticmotilityintheintestinepropelsintestinalcontentsalongthelengthofthesmallintestine.

1	propagate along the length of the intestine are stimulated by the hormone motilin and sweep any remaining gastric and intestinal contents out into the colon. The pylorus and ileocecal valve open fully during this phase, so even large undigested items can eventually pass from the body. Motility of the intestine then reverts to phase I of the MMC, with the entire cycle taking about 90 minutes in adults unless a meal is ingested, in which case the MMC is suspended. After the meal, motilin levels fall, and the MMC cannot be resumed until they rise again. 1. On leaving the stomach, the meal enters the small intestine, which consists (sequentially) of the duodenum, jejunum, and ileum. The principal function of the small intestine is to digest and absorb the nutrients contained in the meal. 2. The presence of chyme in the duodenum retards additional gastric emptying, thus helping match nutrient delivery to the ability of the small intestine to digest and absorb such substances. 3.

1	2. The presence of chyme in the duodenum retards additional gastric emptying, thus helping match nutrient delivery to the ability of the small intestine to digest and absorb such substances. 3. Digestion and absorption in the small intestine are aided by two digestive juices derived from the pancreas (pancreatic juice) and liver (bile). These secretions are triggered by hormonal and neural signals activated by the presence of the meal in the small intestine. 4. Pancreatic secretions arise from the acini and contain various proteins capable of digesting the meal or acting as important cofactors. The secretion is diluted and alkalinized as it passes through the pancreatic ducts. 5. Bile is produced by the liver and stored in the gallbladder until needed in the postprandial period. Bile acids, important components of bile, are biological detergents that solubilize the products of lipid digestion.

1	•Fig. 30.22 Migratingmotorcomplexesintheduodenumandjejunumasrecordedfromafastinghumansubjectbymanometry.D1,D2,J1,J2,andJ3indicatesequentialrecordingpointsalongthelengthoftheduodenumandjejunum.Theintensecontractions(phaseIII)propagateaborally.(RedrawnfromSofferEEetal. Am J Gastroenterol 1998;93:1318.) 6. Carbohydrates and proteins, water-soluble macromolecules, are digested and absorbed by broadly analogous mechanisms. Lipids, the third macronutrient, require special mechanisms to transfer the products of lipolysis to the epithelial surface where they can be absorbed. The small intestine also absorbs fatand water-soluble vitamins, as well as minerals such as calcium, magnesium, and iron. 7. The small intestine transfers large volumes of fluid into and out of the lumen on a daily basis to facilitate digestion and absorption of nutrients, driven by active transport of ions and other electrolytes. 8.

1	8. The motor patterns of the small intestine vary depending on whether a meal has been ingested. Immediately after a meal, motility is directed to retaining the meal in the small intestine, mixing it with digestive juices, and providing sufficient time for absorption of nutrients. During fasting, a “housekeeper” complex of intense contractions (the migrating motor complex) sweeps periodically along the length of the stomach and small intestine to clear them of undigested residues. Abumrad NA, Davidson NO. Role of the gut in lipid homeostasis. Physiol Rev. 2012;92:1061-1085. Argent BE, et al. Cell physiology of the pancreatic ducts. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012. Barrett KE, Keely SJ. Electrolyte secretion and absorption in the small intestine and colon. In: Podolsky DK, et al., eds. Yamada’s Textbook of Gastroenterology. 6th ed. Hoboken, NJ: Wiley Blackwell; 2015.

1	Bharucha AE, Hasler WL. Motility of the small intestine and colon. In: Podolsky DK, et al., eds. Yamada’s Textbook of Gastroenterology. 6th ed. Hoboken, NJ: Wiley Blackwell; 2015. Ganapathy V. Protein digestion and absorption. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012. Liddle RA. Regulation of pancreatic secretion. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012. Wright EM, Sala-Rabanal M, Loo DDF, Hirayama BA. Sugar absorption. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012. Upon completion of this chapter the student should be able to answer the following questions: 1. What are the structures of the anatomy of the colon and rectum, and what is the role of the large intestine in storing and desiccating the residues of a meal? 2.

1	What are the structures of the anatomy of the colon and rectum, and what is the role of the large intestine in storing and desiccating the residues of a meal? 2. What are the motility patterns of the colon that provide for its storage function, and what reflexes signal to the colon from more proximal portions of the GI tract? 3. What is the role of intestinal microorganisms in metabolism and host defense? 4. What are the mechanisms that provide for defecation, and how it can be delayed until convenient? Overview of the Large Intestine The most distal segment of the gastrointestinal (GI) tract, the large intestine, comprises the cecum; ascending, transverse, and descending portions of the colon; rectum; and anus (

1	Fig. 31.1 ). The primary functions of the large intestine are to digest and absorb components of the meal that cannot be digested or absorbed more proximally, reabsorb the remaining fluid that was used during movement of the meal along the GI tract, and store the waste products of the meal until they can conveniently be eliminated from the body. In fulfilling these functions, the large intestine uses characteristic motility patterns and expresses transport mechanisms that drive the absorption of fluid, electrolytes, and other solutes from the stool. The large intestine also contains a unique biological ecosystem known as the microbiota, consisting of many trillions of commensal bacteria and other microorganisms that engage in a lifelong symbiotic relationship with their human host. These microorganisms can metabolize components of the meal that are not digested by host enzymes and make their products available to the body via a process known as fermentation. Colonic bacteria also

1	microorganisms can metabolize components of the meal that are not digested by host enzymes and make their products available to the body via a process known as fermentation. Colonic bacteria also metabolize other endogenous substances such as bile acids and bilirubin, thereby influencing their disposition. There is emerging evidence that the colonic microbiota is critically involved in promoting development of the normal colonic epithelium and in stimulating its differentiated functions.

1	In addition the microbiota can detoxify xenobiotics (substances originating outside the body, such as drugs) and protect the colonic epithelium from infection by invasive pathogens. Finally, the colon is both the recipient and the source of signals that allow it to communicate with other GI segments to optimally integrate function. For example, when the stomach is filled with freshly masticated food, the presence of the meal triggers a long reflex arc that results in increased colonic motility (the gastrocolic reflex) and eventually evacuation of the colonic contents to make way for the residues of the next meal. Similarly the presence of luminal contents in the colon causes release of both endocrine and neurocrine mediators that slow propulsive motility and decrease electrolyte secretion in the small intestine. This negative feedback mechanism matches the rate of delivery of colonic contents to the segment’s capacity to process and absorb the useful components. Details of the signals

1	in the small intestine. This negative feedback mechanism matches the rate of delivery of colonic contents to the segment’s capacity to process and absorb the useful components. Details of the signals that mediate this crosstalk between the colon and other components of the GI system are reviewed in the next section.

1	The colon is regulated primarily, though not exclusively, by neural pathways. Colonic motility is influenced by local reflexes that are generated by filling of the lumen, thereby initiating distention and the activation of stretch receptors. These regulatory pathways exclusively involve the enteric nervous system. Local reflexes triggered by distortion of the colonic epithelium and produced, for example, by passage of a bolus of fecal material stimulate short bursts of Cl− and fluid secretion mediated by 5-hydroxytryptamine (5-HT) from enteroendocrine cells and acetylcholine from enteric secretomotor nerves. On the other hand, colonic function and motility responses in particular are also regulated by long reflex arcs originating more proximally in the GI tract or in other body systems. One example is the gastrocolic reflex. Distention of the stomach activates a generalized increase in colonic motility and mass movement of fecal material, as described in more detail later. This reflex

1	example is the gastrocolic reflex. Distention of the stomach activates a generalized increase in colonic motility and mass movement of fecal material, as described in more detail later. This reflex has both chemosensitive and mechanosensitive components at its site of origin and involves release of 5-HT and acetylcholine. Similarly the orthocolic reflex is activated on rising from bed and promotes a morning urge to defecate in many individuals.

1	The colon is relatively poorly supplied with cells that release bioactive peptides and other regulatory factors. Exceptions are enterochromaffin cells, which release 5-HT, and cells that synthesize peptide YY, so named because its sequence contains two adjacent tyrosine residues. Peptide YY is synthesized by enteroendocrine cells localized in the terminal ileum and colon and is released in response to lipid in the lumen. It decreases gastric emptying and intestinal propulsive motility. Peptide YY also reduces Cl− and thus fluid secretion by intestinal epithelial cells. Thus peptide YY has been characterized as an “ileal brake” in that it is released if nutrients, especially fat, are not absorbed by the time the meal reaches the terminal ileum and proximal part of the colon. By reducing propulsion of the intestinal contents, in part by limiting their fluidity and distention-induced motility, peptide YY provides more time for the meal to be retained in the small intestine where its

1	propulsion of the intestinal contents, in part by limiting their fluidity and distention-induced motility, peptide YY provides more time for the meal to be retained in the small intestine where its constituent nutrients can be digested and absorbed.

1	Patterns of Colonic Motility To appreciate colonic motility the functional anatomy of the colonic musculature will be reviewed first, followed by a discussion of the regulation of colonic motility. Functional Anatomy of the Colonic Musculature

1	Functional Anatomy of the Colonic Musculature As in other segments of the intestine, the colon consists of functional layers, with a columnar epithelium most closely apposed to the lumen, which is then underlaid by the lamina propria, serosa, and muscle layers. Similarly the colonic mucosa is surrounded by continuous layers of circular muscle that can occlude the lumen. Indeed, at intervals •Fig. 31.1 Majoranatomicsubdivisionsofthecolon.Sigmoid colon Cecum Rectum Ileocecal valve Terminal ileum •Fig. 31.2 Radiographshowingaprominenthaustralpatterninthecolonofanormalindividual.(FromKeatsTE.An Atlas of Normal Roentgen Variants. 2nded.StLouis:Mosby–YearBook;1979.) the circular muscle contracts to divide the colon into segments called haustra. These haustra are readily appreciated if the colon is viewed at laparotomy or by x-ray imaging as shown in

1	Fig. 31.2 . The arrangement of the majority of the longitudinal muscle fibers, however, is distinct from •Fig. 31.3 Anatomyoftherectumandanalcanal. that in the small intestine. Three nonoverlapping bands of longitudinal muscle known as the taeniae coli extend along the length of the colon. Although the circular and longitudinal muscle layers of the colon are electrically coupled, this process is less efficient than in the small intestine. Thus propulsive motility in the colon is less effective than in the small intestine. Activity of the enteric nervous system also provides for the segmenting contractions that form the haustra. Contents can be moved back and forward between haustra, which is a means of retarding passage of the colonic contents and maximizing their contact time with the epithelium. In contrast, when rapid propulsion is called for, the contractions forming the haustra relax and the contour of the colon is smoothened.

1	The colon terminates in the rectum, which is joined to the colon at an acute angle (the rectosigmoid junction ) (Fig.

1	31.3 ). The rectum lacks circular muscle and is surrounded only by longitudinal muscle fibers. It is a reservoir wherein feces can be stored before defecation. Muscular contractions also form functional “valves” in the rectum that retard movement of feces and are important in delaying the loss of feces until it is convenient, at least in adults. The rectum in turn joins the anal canal, distinguished by the fact that it is surrounded not only by smooth muscle but also by striated (skeletal) muscle. The combination of these muscle layers functionally accounts for two key sphincters that control evacuation of solid waste and flatus from the body. The internal anal sphincter is composed of a thickened band of circular muscle, whereas the external anal sphincter is made up of three different striated muscle structures in the pelvic cavity that wrap around the anal canal. These latter muscles are distinctive because they maintain a significant level of basal tone and can be contracted

1	striated muscle structures in the pelvic cavity that wrap around the anal canal. These latter muscles are distinctive because they maintain a significant level of basal tone and can be contracted further either voluntarily or reflexively when abdominal pressure increases abruptly (e.g., when lifting a heavy object).

1	Hirschsprung’s disease isaconditioninwhichasegmentofthecolonremainspermanentlycontractedandresultsinobstruction.Itistypicallydiagnosedininfancyandaffectsupto1in5000livebirthsintheUnitedStates.Thebasisofthediseaseisfailureoftheentericnervoussystemtodevelopnormallyduringfetallife.Duringorganogenesis,cellsdestinedtobecomeentericneuronsmigrateoutfromtheneuralcrestandpopulatethegutsequentiallyfrommouthtoanus.Insomeindividualsthismigrationterminatesprematurelybecauseofabnormalitiesinthemechanismsthatwouldotherwisedrivethisprocess.Mutationsinglial-derivedneurotrophicfactorandendothelinIII,aswellasintheirreceptors,havebeendescribedinindividualssufferingfromthisdisease,andtheaffectedsegmentcompletelylackstheplexusesoftheentericnervoussystemandassociatedganglia.ArelativedeficiencyofinterstitialcellsofCajalisalsoseenintheaffectedsegment,andoverallcontrolofmotilityismarkedlyimpaired.Inmostindividualsthesymptomscanbecompletelyalleviatedbysurgicalexcisionoftheaffectedsegment.

1	Contraction of the smooth muscle layers in the proximal part of the colon is stimulated by vagal input as well as by the enteric nervous system. On the other hand, the remainder of the colon is innervated by the pelvic nerves, which also control the caliber of the internal anal sphincter. Voluntary input from the spinal cord via branches of the pudendal nerves regulates contraction of the external anal sphincter and muscles of the pelvic floor. The ability to control these structures is learned during toilet training. This voluntary control distinguishes the anal canal from most of the GI system, with the exception of the striated muscle in the esophagus that regulates swallowing.

1	Consistent with its primary function, the two predominant motility patterns of the large intestine are directed not to propulsion of the colonic contents but rather to mixing of the contents and retarding their movement, thereby providing them with ample time in contact with the epithelium. Two distinctive forms of colonic motility have been identified. The first is referred to as short-duration contractions, which are designed to provide for mixing. These contractions originate in the circular muscle and are stationary pressure waves that persist for 8 seconds on average. Long-duration contractions, in contrast, are produced by the taeniae coli, last for 20 to 60 seconds, and may propagate over short distances. Notably, however, propagation may move orally as well as aborally, particularly in the more proximal segments of the colon. Both motility patterns are thought to originate largely in response to local conditions such as distention. Note that the basal electrical rhythm that

1	in the more proximal segments of the colon. Both motility patterns are thought to originate largely in response to local conditions such as distention. Note that the basal electrical rhythm that governs the rate and origination sites of smooth muscle contraction in the small intestine does not traverse the ileocecal valve to continue into the colon.

1	On the other hand, probably as a result of both local influences and long reflex arcs, approximately 10 times per day in healthy individuals the colon engages in a motility pattern that is of high intensity and sweeps along the length of the large intestine from the cecum to the rectum. Such contractions, called high-amplitude propagating contractions, move exclusively in an aboral direction and are designed to clear the colon of its contents. However, although such a motility pattern can clearly be associated with defecation, it does not necessarily result in defecation for reasons discussed later.

1	It is also important to note that there is considerable variability among individuals with respect to the rate at which colonic contents are transported from the cecum to the rectum. Although small intestinal transit times are relatively constant in healthy adults, the contents may be retained in the large intestine anywhere from hours to days without signifying dysfunction. This also accounts for significant variation among individuals in their normal patterns of defecation and mandates careful elicitation of a patient’s history before diagnosing bowel dysfunction. Transport Mechanisms in the Colon

1	Transport Mechanisms in the Colon The major role of the colonic epithelium is to either absorb or secrete electrolytes and water rather than nutrients. Secretion, which is confined to the crypts, maintains the sterility of the crypts, which might otherwise become stagnant. This is important because the stem cells that renew the epithelium are located at the base of the crypt. The stem cells give rise to daughter cells that migrate out of the crypts and acquire the differentiated properties of surface cells that are responsible for water and electrolyte absorption. The colonic epithelium also absorbs short-chain fatty acids salvaged from nonabsorbed carbohydrates by colonic bacteria. Indeed, one such short-chain fatty acid, butyrate, is a critical energy source for colonocytes. A reduction in butyrate levels in the lumen (as a result of changes in colonic microbiota caused by administration of broad-spectrum antibiotics) may induce epithelial dysfunction.

1	The colon receives 2 L of fluid each day and absorbs 1.8 L, thus leaving 200 mL of fluid to be lost in stool. The colon has a considerable reserve capacity for fluid absorption and can absorb up to three times its normal fluid load without loss of excessive fluid in the stool. Therefore any illness that results in stimulation of active fluid secretion in the small intestine will cause diarrhea only when the reserve capacity of 4 to 6L is exceeded. Absorption and secretion of water by the colon are passive processes driven by absorption or secretion of electrolytes and other solutes. Quantitatively, fluid absorption by the

1	Irritable bowel syndrome isthenamegiventoaheterogeneouscollectionoffunctionaldisorderswhosesuffererscomplainofdiarrhea,constipation,oralternatingpatternsofboth,oftenwithaccompanyingpainanddistention.Theetiologyofthesedisordersisstillnotfullyunderstoodbutmayinvolveinpartaconditionofvisceral hypersensitivity inwhichtheindividualperceivesnormalsignalsoriginatingfromthebowel(e.g.,inresponsetodistention)aspainful.Thishypersensitivitymaybeattheleveloftheentericorcentralnervoussystem(orboth)andcanbetriggeredbyavarietyoffactorssuchaspreviousinfections,childhoodabuse,orpsychiatricdisorders.Mosttreatmentsfocusonsymptomaticrelief,butthereisthepromiseofmoreeffectivetherapiesaswelearnmoreabouttheunderlyingcausesofthecondition.Treatmentofpatientswithirritableboweldisorders,whichareoftenrefractorytotherapy,formsamajorpartofthepracticeofmanygastroenterologists.

1	Thecolonicepitheliumturnsoverrapidlyinhealth,thuslimitingtheaccumulationofgeneticdamage.However,thisrapidturnover,aswellasfrequent/prolongedexposuretobacteriallysynthesizedorenvironmentaltoxins,orboth,makesthelargeintestineespeciallyvulnerabletomalignancy.ColoncancerissecondinprevalenceonlytolungcancerinmenintheUnitedStatesandthirdbehindlungandbreastcancerinwomen.Withthedecreasedincidenceofcigarettesmokingandthereforelungcancer,coloncancermayassumeevengreatersignificance.Coloncancerariseswhennormalgeneticcontrolsontherateofepithelialproliferationaresubverted;initiallythisleadstogrowthofapolypandeventually,ifnotremoved,toaninvasivetumorthatmaymetastasizetootherpartsofthebody.Coloncancercanbesubdividedaccordingtothebasicnatureoftheunderlyingmoleculardefect,whichcanincludeoverexpressionofgrowthstimulatoryfactorsoramutationthatpreventsthecellsfromrespondingtofactorsthatwouldnormallybegrowthsuppressive.However,coloncancermortalitycanbereducedverysubstantiallybyearlydetectionandremovalofpol

1	wthstimulatoryfactorsoramutationthatpreventsthecellsfromrespondingtofactorsthatwouldnormallybegrowthsuppressive.However,coloncancermortalitycanbereducedverysubstantiallybyearlydetectionandremovalofpolypswithmalignantpotential.Thishasdrivencurrentguidelinesforincreasedscreeningofevenasymptomaticmiddle-agedindividualsforcolonicabnormalitiesviacolonoscopy(inwhichaflexiblefiberoptictubeisinsertedintothecolontoinspectitsinterior),screeningforthepresenceofso-calledoccult(orhidden)bloodinthestoolderivedfromableedingpolyportumor,ornoninvasiveimagingtechniquessuchascomputedtomographyscans.

1	colon is driven by three transport processes. The first is electroneutral NaCl absorption, which is mediated by the same mechanism that drives NaCl absorption in the intestine (see Fig. 30.17 ). NaCl absorption is stimulated by various growth factors, such as epidermal growth factor, and is inhibited by hormones and neurotransmitters that increase levels of cyclic adenosine monophosphate (cAMP) in colonic surface epithelial cells. •Fig. 31.4 Mechanismofshort-chainfattyacid(SCFA)uptakebycolonocytes.ATP,adenosinetriphosphate;SMCT1,sodium/monocarboxylatecotransporter1.3Na˜SMCT1 SCFA used by colonocyte for intracellular metabolism ATP

1	The second transport process that drives fluid absorption in the colon is absorption of short-chain fatty acids, including acetate, propionate, and butyrate. These molecules are absorbed from the lumen by surface (and perhaps crypt) epithelial cells in an Na+-dependent fashion by a family of symporters related to the Na+-glucose symporter in the small intestine, and known as sodium-monocarboxylate transporters (SMCTs). Uptake of short-chain fatty acids by SMCTs located in the apical plasma membrane is driven by the low intracellular [Na+] established by the basolateral Na+,K+-ATPase ( Fig. 31.4 ). These short-chain fatty acids are used for energy by colonocytes. In addition, butyrate regulates expression of specific genes in colonic epithelial cells and may suppress development of a malignant phenotype. Expression of SMCT1 (also identified as SLC5A8) is reduced in some colon cancers, thereby leading to a reduction in butyrate uptake, which may contribute to malignant transformation.

1	The third absorptive process of major significance in the colon is absorption of Na+ ( Fig. 31.5 ). This transport process is predominantly localized to the distal part of the colon and is driven by the epithelial Na+ channel (ENaC), which is also involved in reabsorption of Na+ in the kidney. When the channel is opened in response to activation by neurotransmitters or hormones, or both, Na+ flows into the colonocyte cytosol and is then transported across the basolateral membrane by Na+,K+-ATPase. Cl− ions follow passively via the intercellular tight junctions to maintain electrical neutrality. Water is absorbed across the tight junctions as a result of the transepithelial osmotic gradient due to solute absorption. This mode of Na+ absorption is the •Fig. 31.5 ElectrogenicNa+ absorptioninthecolon.ENaC,epithelialsodiumchannel.

1	last line of defense to prevent excessive loss of water in stool, given its strategic location in the distal part of the colon. Indeed, patients suffering from bowel inflammation often show markedly diminished expression of ENaC, perhaps accounting for their diarrheal symptoms. We also know that expression of ENaC can be acutely regulated in response to whole-body Na+ balance. Thus in situations of reduced Na+ intake, the hormone aldosterone increases ENaC expression in both the colon and kidney, thereby promoting retention of Na+ . Adequate hydration of the colonic contents is determined by the balance between water absorption and secretion. Fluid secretion in the colon is driven by Cl− ion secretion, by the same mechanism driving fluid secretion in the small intestine, and is subject to the same regulation (see Fig. 30.18).

1	Fig. 30.18). Indeed, some cases of constipation may reflect abnormalities in epithelial transport, and constipation that results from abnormally slow motility can be treated by agents that stimulate Cl− secretion. Conversely, excessive Cl− secretion can be one mechanism underlying diarrhea.

1	The remnants of the meal entering the colon interact with a vast assortment of bacteria and other microorganisms. This enteric microbial ecosystem is established shortly after birth, matures as the child grows, and fluctuates in predictable ways in healthy individuals depending on factors such as diet or circadian rhythms. More drastic perturbations in the microbiota can be provoked by antibiotics or introduction of an aggressive pathogen and may then take considerable time to resolve. The enteric bacterial ecosystem contributes to GI physiology in a surprising number of ways. Indeed, the large intestine (and to a lesser extent the distal portion of the small intestine) is an unusual organ in that it maintains a symbiotic relationship with such an extensive microbiota, whereas other body compartments are largely sterile.

1	Diarrhealdiseasesareamajorcauseofinfantmortalityworldwideandareusuallytheresultofinadequateaccesstocleanfoodandwater.Evenindevelopedcountries,diarrhealdiseasescausesubstantialsufferingandoccasionalwell-publicizeddeathsandcarryasubstantialeconomicburdenbecauseoftheirprevalence.Infectiousdiarrheaiscausedbyanumberoforganisms,withseveral(e.g.,choleraorpathogenicstrainsofEscherichia coli)capableofelaboratingtoxinsthattriggerexcessiveincreasesinactiveCl− secretionbysmallandlargeintestinalepithelialcells.Diarrheacanalsoresultwhennutrientsarenotappropriatelydigestedandabsorbedinthesmallintestine(e.g.,lactoseintolerance)orasaresultofcolonicinflammation.Inmostdiarrhealdiseases,colonicNaClandNa+ absorptionaredownregulatedatthesametimeCl− secretionmaybestimulated,thusfurtherworseningfluidloss.Ontheotherhand,nutrient-linkedNa+ absorptiveprocessestypicallyremainintact.Thisprovidestherationalefortheeffectivenessoforalrehydrationsolutions,whichareprepackagedmixturesofsaltandglucose.UptakeofNa+

1	absorptiveprocessestypicallyremainintact.Thisprovidestherationalefortheeffectivenessoforalrehydrationsolutions,whichareprepackagedmixturesofsaltandglucose.UptakeofNa+ andglucosefromthesesolutions,mediatedbySGLT1(see ),restoresfluidabsorption.Thesesolutionssavelivesinareaswherediarrheaisprevalentandtheabilitytorehydratepatientswithsterileintravenoussolutionsislimitedorabsent.

1	The colonic microbiota is not essential to life because animals raised in germ-free conditions apparently develop normally and are able to reproduce. However, in such animals the mucosal immune system is immature, and intestinal epithelial cells differentiate more slowly. Importantly the colonic microbiota provides benefits to the host in that the constituent microbes are capable of performing metabolic reactions that do not take place in mammalian cells. Bacterial enzymes act on both endogenous and exogenous substrates. They form secondary bile acids and deconjugate bile acids that have escaped uptake in the terminal ileum, so they can be reabsorbed. They convert bilirubin into urobilinogen (see ) and salvage nutrients that are resistant to pancreatic and brush border hydrolases, such as dietary fiber. A summary of the metabolic contributions of the colonic microbiota is provided in

1	Table 31.1 . Microbial metabolism can also be exploited for pharmacological purposes. A drug targeted to the colon, for example, can be conjugated in such a way that it will become bioavailable only after it is acted on by bacterial enzymes. Bacterial enzymes may also detoxify some dietary carcinogens, but equally they may generate toxic or carcinogenic compounds from dietary substrates.

1	Commensal microorganisms also play a critical role in limiting the growth or invasion (or both) of pathogenic microorganisms. They fulfill this antimicrobial role via a number of different mechanisms—by synthesizing and secreting compounds that inhibit the growth of competitor organisms or that are microbicidal, by functioning as a physical barrier to prevent attachment of pathogens and their subsequent entry into colonic epithelial cells, and by triggering patterns of gene expression in the epithelium that counteract the adverse effects of pathogens on epithelial function. These mechanisms provide a basis for understanding why patients who have received broad-spectrum antibiotics, which temporarily disrupt the colonic microbiota, are susceptible to overgrowth of pathogenic organisms and associated intestinal and systemic infections. They may also shed light on the efficacy of probiotics, commensal bacteria selected for their resistance to gastric acid and proteolysis that are

1	and associated intestinal and systemic infections. They may also shed light on the efficacy of probiotics, commensal bacteria selected for their resistance to gastric acid and proteolysis that are intentionally ingested to prevent or treat a variety of digestive disorders.

1	Atoxinknownasheat-stable toxin of E. coli, orSTa,isamajorcausativeagentoftraveler’sdiarrhea,whichcanbecontractedbyconsumptionofinfectedfoodorwater.Thistoxinbindstoareceptorontheapicalsurfaceofintestinalepithelialcellsknownasguanylyl cyclase C (GC-C).Inturnthisenzymegenerateslargequantitiesofintracellularcyclicguanosinemonophosphate(cGMP)thattriggerincreasedCl− secretionviaactivationofthecysticfibrosistransmembraneconductanceregulator(CFTR)Cl−

1	secretionviaactivationofthecysticfibrosistransmembraneconductanceregulator(CFTR)Cl− channel.However,onecouldofcoursequestionwhyhumansexpressareceptorforthistoxininasitethatwouldbeaccessibletoluminalbacteriaandtheirproducts.Indeed,thisledtothehypothesisthatthereisanativeligandforGC-Cthatcouldplayaphysiologicalrole.Researcherssubsequentlypurifiedandidentifiedguanylin,ahormonesynthesizedintheintestine.Togetherwitharelatedmolecule,uroguanylin,secretedbythekidney,guanylinisanimportantregulatorofsaltandwaterhomeostasisinthebody.STahasstructuralsimilaritiestoguanylin,butithasmodificationsthatpermitittopersistintheintestinallumenforprolongedperiods.Thisisanexampleofmolecularmimicry,inwhichabacterialproducthijacksareceptorandassociatedsignalingforitsownpurposes(presumablytopropagatethetoxin-producingbacteriatoadditionalhosts).

1	The colonic microbiota is also notable for its contribution to formation of intestinal gas. Although large volumes of air may be swallowed in conjunction with meals, the majority of this gas returns up the esophagus via belching. However, during fermentation of unabsorbed dietary components, the microbiota generates large volumes of nitrogen, hydrogen, and carbon dioxide. Approximately 1 L of these nonodorous gases is excreted on a daily basis via the anus in all individuals, even those who do not complain of flatulence. Some individuals may generate appreciable concentrations of methane. Trace amounts of odorous compounds are also present, such as hydrogen sulfide, indole, and skatole.

1	The final stage in the journey taken by a meal after its ingestion is expulsion of its indigestible residues from the body in the process known as defecation. The feces also contain the remnants of dead bacteria, dead and dying epithelial cells that have been desquamated from the lining of the intestine, biliary metabolites specifically targeted for excretion (e.g., conjugates of xenobiotics [see ]), and a small amount of water. In health, the stool contains few if any useful nutrients. The presence of nutrients in stool, particularly lipid (known as steatorrhea), signifies maldigestion, malabsorption, or both. Fat in the stool is a sensitive marker of small intestinal dysfunction because it is poorly used by the colonic microbiota, but loss of carbohydrate and protein in stool can also be seen if the underlying condition worsens.

1	The process of defecation requires coordinated action of the smooth and striated muscle layers in the rectum and anus, as well as surrounding structures such as the pelvic floor muscles. During the mass movement of feces produced by high-amplitude propagating contractions, the rectum fills with fecal material. Expulsion of this material from the body is controlled by the internal and external anal sphincters, which contribute approximately 70% to 80% and 20% to 30% of anal tone at rest, respectively. Filling of the rectum causes relaxation of the internal anal sphincter via release of vasoactive intestinal polypeptide and generation of nitric oxide. Relaxation of the inner sphincter permits the anal sampling mechanism, which can distinguish whether the rectal contents are solid, liquid, or gaseous in nature. After toilet training, sensory nerve endings in the anal mucosa then generate reflexes that initiate appropriate activity of the external sphincter to either retain the rectal

1	or gaseous in nature. After toilet training, sensory nerve endings in the anal mucosa then generate reflexes that initiate appropriate activity of the external sphincter to either retain the rectal contents or permit voluntary expulsion (e.g., of flatus). If defecation is not convenient, the external sphincter contracts to prevent the loss of stool. Then with time the rectum accommodates to its new volume, the internal anal sphincter contracts again, and the external anal sphincter relaxes (

1	Fig. 31.6 When defecation is desired, on the other hand, adoption of a sitting or squatting position alters the relative orientation of the intestine and surrounding muscular structures by straightening the path for exit of either solid or liquid feces. Relaxation of the puborectalis muscle likewise increases the rectoanal angle. After voluntary relaxation of the external anal sphincter, rectal contractions move the fecal material out of the body, sometimes followed by additional mass movements of feces from more proximal segments of the colon ( Fig. 31.7 ). Evacuation is assisted by simultaneous contraction of muscles that increase abdominal pressure, such as the diaphragm. Voluntary expulsion of flatus, on the other hand, involves a similar sequence of events, except that there is no relaxation of the puborectalis muscle. This permits flatus to be squeezed past the acute rectoanal angle while retaining fecal material.

1	Cooperative activity of the external anal sphincter, puborectalis muscle, and sensory nerve endings in the anal canal is required to delay defecation until it is appropriate, even if the rectum is acutely distended with stool or intraabdominal pressure rises sharply. This explains why incontinence can develop in individuals in whom the integrity of such structures has been compromised, such as after trauma, surgical or obstetrical injuries, prolapse of the rectum, or neuropathic diseases such as long-standing diabetes. Surgical intervention may be necessary to correct muscle abnormalities in patients with the distressing condition of fecal incontinence, although many can be helped to increase external anal sphincter tone with the use of biofeedback exercises.

1	•Fig. 31.6 Responsesoftheinternalandexternalanalsphincterstoprolongeddistentionoftherectum.Notethattheresponsesofthesphinctersaretransientbecauseofaccommodation.(RedrawnfromShusterMMetal.Bull Johns Hopkins Hosp 1965;116:79.) •Fig. 31.7 Motilityoftherectumandanalsphinctersinresponsetorectalfillingandduringdefecation.Notethatfillingoftherectumcausesaninitialdecreaseininternalsphinctertonethatiscounterbalancedbycontractionoftheexternalsphincter.Theinternalsphincterthenaccommodatestothenewrectalvolume,therebyallowingtheexternalsphinctertorelax.Finally,defecationoccurswhentheexternalanalsphincterisrelaxedvoluntarily.(DatafromChangEBetal.Gastrointestinal, Hepatobiliary and Nutritional Physiology. Philadelphia:Lippincott-Raven;1996.) 1.

1	The final segment of the intestine through which the meal traverses is the large intestine, which is composed of the cecum, colon, rectum, and anus. The primary role of the large intestine is to reclaim water used during the process of digestion and absorption and to store the residues of the meal until defecation is socially convenient. 2. Colonic motility primarily serves to mix and delay passage of the luminal contents, other than during periodic large-amplitude contractions that convey fecal material to the rectum. Bharucha AE, Brookes SJR. Neurophysiologic mechanisms of human large intestinal motility. In: Johnson LR, ed. Physiology of the GI Tract. 5th ed. Waltham, MA: Academic Press; 2012. Bharucha AE, Hasler WL. Motility of the small intestine and colon. In: Podolsky DK, et al., eds. Yamada’s Textbook of Gastroenterology. 6th ed. Hoboken, NJ: Wiley Blackwell; 2015. 3.

1	Bharucha AE, Hasler WL. Motility of the small intestine and colon. In: Podolsky DK, et al., eds. Yamada’s Textbook of Gastroenterology. 6th ed. Hoboken, NJ: Wiley Blackwell; 2015. 3. The colon is highly active in transporting water and electrolytes as well as products salvaged from undigested components of the meal by colonic bacteria. 4. The colon maintains a lifelong mutually beneficial relationship with a vast microbial ecosystem that metabolizes endogenous substances, nutrients, and drugs and protects the host from infection with pathogens. 5. Defecation involves both involuntary and voluntary relaxation of muscle structures surrounding the anus and reflex pathways that control these structures. Kau AL, et al. Human nutrition, the gut microbiome and the immune system. Nature. 2011;474:327-336. Kendig DM, Grider JR. Serotonin and colonic motility. Neurogastroenterol Motil. 2015;27:899-905.

1	Kau AL, et al. Human nutrition, the gut microbiome and the immune system. Nature. 2011;474:327-336. Kendig DM, Grider JR. Serotonin and colonic motility. Neurogastroenterol Motil. 2015;27:899-905. Koren A, Ley RE. The human intestinal microbiota and microbiome. In: Podolsky DK, et al., eds. Yamada’s Textbook of Gastroenterology. 6th ed. Hoboken, NJ: Wiley Blackwell; 2015. Upon completion of this chapter the student should be able to answer the following questions: 1. How does the liver contribute metabolic, detoxification, and excretory functions that are vital to life? 2. What cell types make up the liver, what is their unusual arrangement, and what is the liver’s unique portal blood supply? 3. What is the function of bile as an excretory fluid, what are the mechanisms that underlie its formation, and what is the role of the enterohepatic circulation? 4.

1	3. What is the function of bile as an excretory fluid, what are the mechanisms that underlie its formation, and what is the role of the enterohepatic circulation? 4. How is bile is stored and concentrated in the gallbladder, and what are the mechanisms that coordinate gallbladder emptying with ingestion of a meal? 5. How are ammonia and bilirubin eliminated from the body? Overview of the Liver and Its Functions

1	The liver is a large multilobed organ located in the abdominal cavity whose function is intimately associated with that of the gastrointestinal system. The liver serves as the first site of processing for most absorbed nutrients and also secretes bile acids, which as we learned in , play a critical role in absorption of lipids from the diet. In addition the liver is a metabolic powerhouse, critical for disposing of a variety of metabolic waste products and xenobiotics from the body by converting them to forms that can be excreted. The liver stores or produces numerous substances needed by the body, such as glucose, amino acids, and plasma proteins. In general, key functions of the liver can be divided into three areas: (1) contributions to whole-body metabolism, (2) detoxification, and (3) excretion of protein-bound/lipid-soluble waste products. In this chapter we discuss the structural and molecular features of the liver and biliary system that subserve these functions, as well as

1	(3) excretion of protein-bound/lipid-soluble waste products. In this chapter we discuss the structural and molecular features of the liver and biliary system that subserve these functions, as well as their regulation. Although the liver contributes in a pivotal way to the maintenance of whole-body biochemical status, a complete discussion of all the underpinning reactions is beyond the scope of this chapter. We will confine our discussion primarily to hepatic functions that relate to gastrointestinal physiology.

1	Metabolic Functions of the Liver Hepatocytes contribute to metabolism of the major nutrients: carbohydrates, lipids, and proteins. Thus the liver plays an important role in glucose metabolism by engaging in gluconeogenesis, conversion of other sugars to glucose. The liver also stores glucose as glycogen at times of glucose excess (e.g., postprandial period) and then releases stored glucose into the bloodstream as it is needed. This process is referred to as the glucose buffer function of the liver. When hepatic function is impaired, glucose concentrations in blood may rise excessively after ingestion of carbohydrate; conversely, between meals, hypoglycemia may be seen because of an inability of the liver to contribute to carbohydrate metabolism and interconversion of one sugar to another.

1	Hepatocytes also participate in lipid metabolism. They are a particularly rich source of the metabolic enzymes engaged in fatty acid oxidation to supply energy for other body functions. Hepatocytes also convert products of carbohydrate metabolism to lipids that can be stored in adipose tissue and synthesize large quantities of lipoproteins, cholesterol, and phospholipids, the latter two being important in the biogenesis of cell membranes. In addition, hepatocytes convert a considerable portion of synthesized cholesterol to bile acids, which will be discussed in more detail later in this chapter.

1	The liver also plays a vital role in protein metabolism. The liver synthesizes all the nonessential amino acids (see ) that do not need to be supplied in the diet, in addition to participating in interconverting and deaminating amino acids so the products can enter biosynthetic pathways for carbohydrate synthesis. With the exception of immunoglobulins, the liver synthesizes almost all the proteins present in plasma (especially albumin, which determines plasma oncotic pressure) as well as most of the important clotting factors. Patients suffering from liver disease may develop peripheral edema secondary to hypoalbuminemia and are also susceptible to bleeding disorders. Finally, the liver is the critical site for disposal of the ammonia generated from protein catabolism. This is accomplished by converting ammonia to urea, which is •Fig. 32.1 Typicalbloodflowthroughthesplanchniccirculationinafastingadulthuman.

1	excreted by the kidneys. The details of this process will be discussed later in this chapter. The Liver and Detoxification The liver serves both as a gatekeeper, by limiting entry of toxic substances into the bloodstream, and as a garbage disposal, by extracting potentially toxic metabolic products produced elsewhere in the body and converting them to chemical forms that can be excreted. The liver fulfills these functions in part because of its unusual blood supply. Unlike all other organs, the majority of blood arriving at the liver is venous in nature and is supplied via the portal vein from the intestine (

1	Fig. 32.1 ). As such, the liver is strategically located to receive both absorbed nutrients and potentially harmful absorbed molecules such as drugs and bacterial toxins. Depending on the efficiency with which these molecules are extracted by hepatocytes and subjected to first-pass metabolism, little or none of the absorbed substance may make it into the systemic circulation. This is a major reason why not all pharmaceutical agents can achieve therapeutic concentrations in the bloodstream if administered orally. CHAPTER 32 Transport and Metabolic Functions of the Liver

1	The liver has two levels at which it removes and metabolizes/detoxifies substances originating from the portal circulation. The first of these is physical. Blood arriving in the liver percolates among cells of the macrophage lineage, known as Kupffer cells. These cells are phagocytic and particularly important in removing particulate material from portal blood, including bacteria that may enter blood from the colon even under normal conditions. The second level of defense is biochemical. Hepatocytes are endowed with a broad array of enzymes that modify both endogenous and exogenous toxins so the products are in general more water soluble and less susceptible to reuptake by the intestine. The metabolic reactions involved are broadly divided into two classes. Phase I reactions (oxidation, hydroxylation, and other reactions catalyzed by cytochrome P450 enzymes) are followed by phase II reactions that conjugate the resulting products with another molecule (e.g., glucuronic acid, sulfate,

1	and other reactions catalyzed by cytochrome P450 enzymes) are followed by phase II reactions that conjugate the resulting products with another molecule (e.g., glucuronic acid, sulfate, amino acids, glutathione) to promote their excretion. The products of these reactions are then excreted into bile or returned to the bloodstream to ultimately be excreted by the kidneys. We will return to the precise mechanisms involved in detoxification of some key metabolic waste products later in this chapter.

1	Role of the Liver in Excretion The kidneys play an important role in excretion of water-soluble catabolites, as discussed in the renal section. Only relatively small water-soluble catabolites can be excreted by the process of glomerular filtration. However, larger water-soluble catabolites and molecules bound to plasma proteins, including lipophilic metabolites and xenobiotics, steroid hormones, and heavy metals, cannot be filtered by the glomerulus. All these substances are potentially harmful if allowed to accumulate, so a mechanism must exist for their excretion. The mechanism involves the liver, which excretes these substances in bile. Hepatocytes take up these substances with high affinity by virtue of an array of basolateral membrane transporters, and the substances are subsequently metabolized at the level of microsomes and in the cytosol (

1	Table 32.1 ). Ultimately, substances destined for excretion in bile are exported across the canalicular membrane of hepatocytes via a different array of transporters. The features of bile allow solubilization of even lipophilic substances, which can then be excreted into the intestine and ultimately leave the body in feces. Structural Features of the Liver and Biliary System Hepatocytes, the major cell type in the liver, are arranged in anastomosing cords that form plates around which large volumes of blood circulate (

1	Hepatocytes, the major cell type in the liver, are arranged in anastomosing cords that form plates around which large volumes of blood circulate ( Fig. 32.2 ). The liver receives a high blood flow that is disproportionate to its mass, which ensures that hepatocytes receive high quantities of both O2 and nutrients. Hepatocytes receive more than 70% of their •Fig. 32.2 Diagrammaticrepresentationofahepaticlobule.Platesofhepatocytesarearrayedradiallyaroundacentralvein.Branchesoftheportalveinandhepaticarteryarelocatedontheperipheryofthelobuleandformthe“portaltriad”togetherwiththebileduct.Bloodfromtheportalveinandhepaticarterypercolatesaroundthehepatocytesviathesinusoidsbeforedrainingintothecentralvein.(ModifiedfromBloomW,FawcettDW.A Textbook of Histology. 10thed.Philadelphia:Saunders;1975.) blood supply at rest via the portal vein (rising to more than 90% in the postprandial period).

1	The plates of hepatocytes that constitute the liver parenchyma are supplied by a series of sinusoids, which are low-resistance cavities supplied by branches of both the portal vein and hepatic artery. The sinusoids are unlike the capillaries that perfuse other organs. During fasting, many sinusoids are collapsed, but more can gradually be recruited as portal blood flow increases during the period after a meal when absorbed nutrients are transported to the liver. The low resistance of the sinusoidal cavities means that blood flow through the liver can increase considerably without •Fig. 32.3 Interrelationshipsofthemajorcelltypesintheliver. a concomitant increase in pressure. Eventually the blood drains into central branches of the hepatic vein. The sinusoids are also unusual in terms of the endothelial cells that line their walls (

1	The sinusoids are also unusual in terms of the endothelial cells that line their walls ( Fig. 32.3 ). Hepatic endothelial cells contain specialized openings known as fenestrations that are large enough to permit passage of molecules as big as albumin. Sinusoidal endothelial cells also lack a basement membrane, which might otherwise pose a diffusion barrier. These features allow access of albumin-bound substances to the hepatocytes that will eventually take them up. The sinusoids also contain Kupffer cells. Beneath the sinusoidal endothelium and separating the endothelium from the hepatocytes is a thin layer of loose connective tissue called the space of Disse, which in health likewise poses little resistance to the movement of molecules even as large as albumin. The space of Disse is also the location of another important hepatic cell type, the stellate cell. Stellate cells serve as storage sites for retinoids and in addition are the source of key growth factors for hepatocytes.

1	Under abnormal conditions, stellate cells are activated to synthesize large quantities of collagen, which contributes to the hepatic dysfunction.

1	Ifthecirculationoftheliver(particularlyitssinusoids)iscompressedbyfibrosis,theliverlosesitsabilitytoaccommodatetheincreasesinbloodflowthatoccurafteramealwithoutaconcomitantincreaseinpressure.Becauseofthefenestrations,albuminescapesfromthecirculationandalbumin-richfluidweepsfromthesurfaceoftheliverintotheabdominalcavity,whereitoverwhelmsthelymphaticdrainage.Thisconditionisknownasascites

1	andisreflectedinaconsiderableincreaseinthegirthofmanypatientswithliverdisease.Aspressureintheliverbuilds,newcollateralbloodvesselsforminanattempttocircumventtheobstructionandreducetheportalhypertension.Someofthesevesselsaredirectedtoabdominalstructuresand,becauseoftheirthinweakwalls,arepronetorupture.Aparticularexampleisformationofhigh-pressurecollateralstotheesophagus,whichcanthenbecomevaricesthatbleedintothelumen.Bleedingintotheesophageallumenisveryhardtocontrolandisthusamedicalemergency.Evenintheabsenceofbleeding,moreover,theformationofcollateralbloodvesselsbypassestheremainingmetaboliccapacityoftheliver,andlevelsoftoxinssuchasammoniaareincreasedandcanexertadverseeffectselsewhereinthebody.

1	Infectionoftheliverwithcertainvirusesoroverexposuretotoxicsubstancessuchasalcoholkillshepatocytesandactivateshepaticstellatecells,whichsynthesizeexcessiveamountsofcollagenthatresultinthehistologicappearanceoffibrosis.Iftheinsultischronic,thefibrosiseventuallybecomesirreversible,aconditionknownascirrhosis. Thefibroticscarredareascrowdoutthehepatocytemass,therebyreducingthesynthetic,metabolic,andexcretorycapacityoftheliver.Fibroticmassespressonthesinusoidsandpreventthemfromexpandingasportalbloodflowtotheliverincreasesduringthepostprandialperiod.Edemamaydevelopinpatientswithchronicliverinjuryasaresultofreducedlevelsofalbumininblood,andascitesmaythendevelop,inwhichfluidaccumulatesintheperitonealcavitysecondarytoincreasedportalpressure.Eventually,accumulationoftoxicsubstancesinthebloodstreamcanleadtojaundice,itching,andneurologicalcomplications.Ifhepaticfunctionbecomescompromisedbeyondacertainlevel,theonlyeffectivetreatmentislivertransplantation.

1	Hepatocytes are also the origination point for the biliary system. Although hepatocytes are considered to be epithelial cells with basolateral and apical membranes, the spatial arrangement of these two cell domains differs from that seen in simple columnar epithelium, such as that lining the gastrointestinal tract. Rather, in the liver the apical surface of the hepatocyte occupies only a small fraction of the cell membrane, and the apical membranes of adjacent cells oppose each other to form a channel between the cells known as the canaliculus (see

1	Fig. 32.3 ). The role of canaliculi is to drain bile from the liver, and these canaliculi drain into biliary ductules that are lined by classic columnar epithelial cells known as cholangiocytes. Ultimately the biliary ductules drain into large bile ducts that coalesce into the right and left hepatic ducts to permit exit of bile from the liver. These in turn form the common hepatic duct, from which bile can flow into either the gallbladder (via the cystic duct) or the intestine (via the common bile duct; Fig. 32.4 ) on the basis of prevailing pressure relationships.

1	One other feature of the structural organization of the liver bears emphasis because of its clinical significance. Branches of the hepatic vein, hepatic artery, and bile ducts run in parallel in the so-called hepatic triad. Hepatocytes lying closest to this triad are referred to as periportal, or zone 1, and have the greatest supply of oxygen and nutrients. In contrast, hepatocytes lying closest to branches of the hepatic vein are referred to as pericentral, or zone 3. The latter cells are more sensitive to ischemia, whereas the former are more sensitive to oxidative injury. Thus the location of damaged cells on biopsy may provide clues to the cause of a given case of liver injury. Zone 1 cells are most active in detoxification functions in normal circumstances, but •Fig. 32.4 Functionalanatomyofthebiliarysystem.Right and left hepatic ducts from liver Common hepatic duct Common bile duct Cystic duct Sphincter of Oddi Duodenum Gallbladder zone 2 (intermediate between zones 1 and 3) and

1	and left hepatic ducts from liver Common hepatic duct Common bile duct Cystic duct Sphincter of Oddi Duodenum Gallbladder zone 2 (intermediate between zones 1 and 3) and zone 3 cells can progressively be recruited in cases of liver disease. Conversely, zone 3 cells are thought to be most active in bile acid synthesis.

1	Bile is the excretory fluid of the liver that plays an important role in lipid digestion. Bile formation begins in hepatocytes, which actively transport solutes into bile canaliculi across their apical membranes. Bile is a micellar solution in which the major solutes are bile acids, phosphatidylcholine, and cholesterol in an approximate ratio of 10:3:1, respectively. Secretion of these solutes drives concomitant movement of water and electrolytes across the tight junctions that link adjacent hepatocytes to form canalicular bile. The majority of bile flow is driven by the secretion of bile acids across the apical membrane of hepatocytes via an adenosine triphosphatase (ATPase) transporter known as the bile salt export pump (BSEP; see

1	Table 32.1 ). The composition of the resulting fluid can be modified further as it flows through the biliary ductules (resulting in hepatic bile) and still further on storage in the gallbladder (gallbladder bile). Ultimately bile becomes a concentrated solution of biological detergents that aids in solubilization of the products of lipid digestion in the aqueous environment of the intestinal lumen, thereby enhancing the rate at which lipids are transferred to the absorptive epithelial surface. It also serves as a medium in which metabolic waste products are exported from the body.

1	Thoughrare,avarietyoffamilialsyndromesthataremanifestedasprogressivecholestasishavetaughtusagreatdealaboutthemolecularnatureofthetransportersthatdeliverbileconstituentsintothecanaliculus.Forexample, (PFICII)hasbeenmappedtoamutationinBSEP thatresultsinanalmosttotalabsenceofbileacidsinbile.Cholestasisdevelopsinpatientswiththisdisorder,buttheyhaverelativelylittleifanyevidenceofbileductinjury.TypeIIIPFIContheotherhandisamuchmoreaggressivediseaseinwhichcholestasisisaccompaniedbyearlyincreasesincirculatingγ-glutamyltranspeptidase.Themolecularculpritisamutationthatabolishesexpressionofmultidrug resistance protein 3 (MDR3).Intheabsenceofthistransporter,phosphatidylcholineisnolongerabletoenterbile,thusillustratingtheimportanceofthislipidinprotectingcholangiocytesfromtheinjuriouseffectsofbileacids,becausemixedmicellescannotforminitsabsence.

1	Bile acids are produced by hepatocytes as end products of cholesterol metabolism. Cholesterol is selectively metabolized by a series of enzymes that result in formation of bile acid ( Fig. 32.5 ). The initial and rate-limiting step is addition of a hydroxyl group to the 7 position of the steroid nucleus by the enzyme cholesterol 7α-hydroxylase. The side chain of the product of this reaction is then shortened and a carboxylic acid function added by C27 dehydroxylase to yield chenodeoxycholic acid, a dihydroxy bile acid. Alternatively the product is further hydroxylated at the 12 position and then acted on by C27 dehydroxylase to yield cholic acid, a trihydroxy bile acid. Bile acid synthesis can be up-or downregulated depending on the body’s requirements (Fig.

1	32.6 ). For example, if bile acid levels are reduced in the blood flowing to the liver, synthesis can be increased up to 10-fold. Conversely, feeding of bile acids profoundly suppresses new synthesis of bile acids by hepatocytes. The mechanisms underlying these changes in bile acid synthesis relate to changes in expression of the enzymes involved. Bile acids activate a variety of cell surface and nuclear receptors in hepatocytes, leading ultimately to activation of specific transcription factors that regulate enzyme abundance. Chenodeoxycholic acid and cholic acid are defined as primary bile acids because they are synthesized by the hepatocyte (see

1	Chenodeoxycholic acid and cholic acid are defined as primary bile acids because they are synthesized by the hepatocyte (see Fig. 32.5 ). However, each can be metabolized in the colonic lumen by bacterial enzymes to yield ursodeoxycholic and deoxycholic acid, respectively. Chenodeoxycholic acid is also converted by bacterial enzymes to form lithocholic acid, which is relatively cytotoxic. Collectively these three products of bacterial metabolism are referred to as secondary bile acids. One additional important biochemical modification occurs for both primary and secondary bile acids in the hepatocyte (see Fig.

1	Fig. 32.5 ). These molecules are conjugated with either glycine or taurine, which significantly depresses their pKa. The result is that conjugated bile acids are almost totally ionized at the pH prevailing in the small intestinal lumen and thus cannot passively traverse cell membranes. Consequently the conjugated bile acids are retained in the intestinal lumen until they are actively absorbed in the terminal ileum via the apical sodium-dependent bile salt transporter (asbt). Conjugated bile acids that escape this uptake step are deconjugated by bacterial enzymes in the colon, and the resulting unconjugated forms are passively reabsorbed across the colonic epithelium because they are no longer charged. Hepatic Aspects of Enterohepatic Circulation of Bile Acids

1	Hepatic Aspects of Enterohepatic Circulation of Bile Acids Bile acids assist in digestion and absorption of lipids by acting as detergents rather than enzymes, and thus a significant mass of these molecules is required to solubilize all dietary lipids. Via the enterohepatic circulation, actively reabsorbed conjugated bile acids travel through the portal blood back to the hepatocyte, where they are efficiently taken up by basolateral transporters that may be Na+ dependent or independent (see Table 32.1 ). Similarly, bile acids that are deconjugated in the colon also return to the hepatocyte, where they are reconjugated to be secreted into bile. In this way a pool of circulating primary and secondary

1	Cholesterol Major bile acids Cholic acid HO Primary Secondary OH OH COOH C27 dehydroxylase 7˜-Hydroxylase (12˜-hydroxylase) HO HO OH COOH Chenodeoxycholic acid Deoxycholic acid Conjugation of bile acids HO OH COOH HO OH 3 7 12 OH C O O OH C O–H CH2N H Glycine, pKa˜3.7 Taurine, pKa˜1.5 or SO2O–HN CH2CH2 H HO OH COOH Ursodeoxycholic acid HO COOH Lithocholic acid Liver Colon (bacteria) •Fig. 32.5 Structuresofthemajorprimaryandsecondarybileacidsofbile.Primarybileacidsaresynthesizedintheliver.Secondarybileacidsareproducedwhenintestinalbacteriaactonprimarybileacids.Atthebottomofthefiguretheconjugationofcholicacidwithglycineortaurineisshown. bile acids is produced, and daily synthesis is then equal only to the minor fraction (≈10%/day, or 200–400 mg) that escapes uptake and is lost in stool (

1	bile acids is produced, and daily synthesis is then equal only to the minor fraction (≈10%/day, or 200–400 mg) that escapes uptake and is lost in stool ( Fig. 32.7 ). The only exception to this rule is lithocholic acid, which is preferentially sulfated in the hepatocyte rather than being conjugated with glycine or taurine. The majority of the sulfate conjugates are lost from the body after each meal because they are not substrates for asbt, thereby avoiding accumulation of a potentially toxic molecule. Some comment should also be made with respect to the role of bile acids in whole-body cholesterol homeostasis. The pool of cholesterol in the body reflects its daily synthesis as well as the relatively minor component derived from inefficient dietary uptake, balanced against loss from the body, which can only occur in health via bile (

1	Fig. 32.8 ). Cholesterol can be excreted in two forms, either as the native molecule or after its conversion to bile acids. The latter account for up to a third of the cholesterol excreted per day despite enterohepatic recycling. Thus one strategy for treating hypercholesterolemia is to interrupt the enterohepatic circulation of bile acids, which drives increased conversion of cholesterol to bile acids; the bile acids are then lost from the body in feces.

1	As noted earlier, bile also contains cholesterol and phosphatidylcholine. Cholesterol transport across the canalicular membrane is mediated at least in part by a heterodimer of the active transporters we discussed in as participating in the efflux of cholesterol from the small intestinal epithelial cells, namely, ABC G5 and ABC G8 (see 32.1 ). Phosphatidylcholine derives from the inner leaflet of the canalicular membrane and is specifically “flipped” across the membrane by another ABC family transporter called multidrug resistance protein 3 (MDR3). Furthermore, 5.0

1	Limit of intestinal absorption 0.5 30 0.0 0.25 0.5 0.75 1.0 1.25 Diet Hepatic and extra-hepatic synthesis To bile As bile acids As cholesterol In health, input = output Cholesterol (g/day) 0.5 •Fig. 32.6 Relationshipbetweenratesofbileacidsynthesisandsecretion.Thebileacidsecretionratenormallyaverages30g/24h,whereasthesynthesisrateaverages0.5g/24h.Thepairsofverticalandhorizontaldottedlinesdepictthenormalrangeforbileacidsecretionandsynthesis,respectively.Increasedsecretion(simulatedbybileacidfeeding)increasestherateofreturnofbileacidstotheliverviaportalblood,whichexertsanegativefeedbackonsynthesis.Conversely,interruptionoftheenterohepaticcirculation,suchasafterilealresection,canincreasesynthesistovaluesmorethan10-foldhigherthannormal.(FromCareyMC,CahalaneMJ.In:AriasIMetal.[eds].The Liver: Biology and Pathobiology. 2nded.NewYork:RavenPress;1988.) •Fig. 32.8 Dailycholesterolbalanceinhealthyadulthumans.

1	•Fig. 32.9 Pathwaysforentryofsolutesintobile.(ModifiedfromBarrettKE.Gastrointestinal Physiology. NewYork:McGraw-Hill;2006.) because mixed micelles composed of bile acids, phosphatidylcholine, and cholesterol are osmotically active and the tight junctions that link adjacent hepatocytes are relatively leaky, water is drawn into the canalicular lumen, as well as other plasma solutes (e.g., Ca++, glucose, glutathione, amino acids, urea) at concentrations essentially approximating •Fig. 32.7 Relativeamountsofbileacidsindifferentbodypoolsand theenterohepaticcirculation.Therelativeamountsareindicatedbythe widthofthegreenlines.Thusthefigureillustratesthatthemajorityof thebileacidpoolcirculatesbetweentheliver,gallbladder,andsmall Fig. 32.9 ). Finally, conjugated bilirubin, which is water soluble, and a variety of additional organic and xenobiotics are secreted into bile across the apical intestine,ratherthanbeinginthesystemiccirculation.membrane of the hepatocyte.

1	•Fig. 32.10 Themajortransportprocessesofcholangiocytesthatsecreteanalkaline-richfluidandreclaimusefulsubstances.AQP,aquaporin;CFTR,cysticfibrosistransmembraneconductanceregulator;GGT,γ-glutamyltranspeptidase;NKCC1,sodium/potassium/2chloridecotransporter1;NBC,sodium/bicarbonatecotransporter;NHE1,sodium/hydrogenexchanger1,SGLT-1,sodium-glucosecotransporter1;GLUT1,glucosetransporter1. The cholangiocytes lining the biliary ductules are specifically designed to modify the composition of bile (

1	The cholangiocytes lining the biliary ductules are specifically designed to modify the composition of bile ( Fig. 32.10 ). Useful solutes (e.g., glucose, amino acids) are reclaimed by the activity of specific transporters. Chloride ions in bile are also exchanged for HCO3 − , rendering the bile slightly alkaline and reducing the risk of precipitation of Ca++ . Glutathione is broken down on the surface of cholangiocytes into its constituent amino acids by the enzyme γ-glutamyl transpeptidase (GGT), and the products are reabsorbed. Bile is also diluted at this site, in concert with ingestion of a meal, in response to hormones such as secretin that increase HCO3 − secretion and stimulate insertion of aquaporin water channels into the cholangiocyte’s apical membrane. Flow of bile is thereby increased during the postprandial period when bile acids are needed to aid in assimilation of lipid. Role of the Gallbladder

1	Role of the Gallbladder Finally, bile enters the ducts and is conveyed toward the intestine. However, in the period between meals, outflow is blocked by constriction of the sphincter of Oddi, and thus bile is redirected to the gallbladder. The gallbladder is a muscular sac lined with high-resistance epithelial cells. During gallbladder storage, bile becomes concentrated because sodium ions are actively absorbed in exchange for protons, and bile acids, as the major anions, are too large to exit across the gallbladder epithelial tight junctions (Fig. 32.11 ). However, although the concentration of bile acids can rise more than 10-fold, bile remains isotonic because a •Fig. 32.11 Mechanismsaccountingforconcentrationofbileduringstorageinthegallbladder.NHE,sodium/hydrogenexchanger;BA−,bileacidanion;AQP1,aquaporin1.

1	single micelle acts as only one osmotically active particle. Any additional bile acid monomers that become available as a result of concentration are thus immediately incorporated into existing mixed micelles. This also reduces to some extent the risk that cholesterol will precipitate from bile. However, cholesterol is supersaturated in the bile of many adults, with precipitation normally being inhibited by the presence of antinucleating proteins. Prolonged storage of bile increases the chance that nucleation can occur, thus making a good case for never skipping breakfast and perhaps explaining why gallstone disease is relatively prevalent in humans.

1	Humansareunusuallysusceptibletogallstones, whichrepresentprecipitatedbileconstituentsthataccumulateinthegallbladderorelsewhereinthebiliarytree.GallstonesarecomposedpredominantlyofcholesterolorCa++

1	bilirubinate(cholesterolvs.pigmentstones,respectively).Theirsignificanceliesintheirpropensitytoobstructbiliaryflowandtherebyresultinpain,poortoleranceoflargefattymeals,retentionofbiliaryconstituents,and(ifleftuntreated)liverinjury.Insusceptibleindividuals,mechanismsthatnormallypreventnucleationofsaturatedbileareeitherdefectiveorovercome,andsmallcrystalsformandcangrowintogallstones.Humanbileisoftensupersaturatedintermsofitscholesterolcontent,thusincreasingtheriskforstoneformation,particularlyduringprolongedfasting.Cholesterolgallstonesareespeciallycommoninmiddle-agedwomenwhoareobese,particularlythosewhohavebornechildren.Thisincreasedprevalenceisapparentlydueatleastinparttotheabilityofestrogentoincreasehepaticcholesterolsecretion.Inseverecasesofgallstonediseasethegallbladdermayberemovedsurgically,whichisusuallyaccomplishedlaparoscopically.SmallgallstonesthathavelodgedinthebiliarytreecansometimesberetrievedendoscopicallybyinsertingasmallsnarethroughthesphincterofOddifromanendoscope.

1	Bile is secreted from the gallbladder in response to signals that simultaneously relax the sphincter of Oddi and contract the smooth muscle that encircles the gallbladder epithelium ( Fig. 32.12 ). A critical mediator of this response is cholecystokinin. In addition, intrinsic neural reflexes and vagal pathways, some of which themselves are stimulated by the ability of cholecystokinin to bind to vagal afferents, also contribute to gallbladder contractility. The net result is ejection of a concentrated bolus of bile into the duodenal lumen, where the constituent mixed micelles can aid in lipid uptake. Then, when no longer needed, the bile acids are reclaimed and reenter the enterohepatic circulation to begin the cycle again. However, the other components of bile are largely lost in stool, thus providing for their excretion from the body. Bilirubin Formation and Excretion by the Liver

1	Bilirubin Formation and Excretion by the Liver The liver is also important for excretion of bilirubin, which is a metabolite of heme that is potentially toxic to the body. Bilirubin is an antioxidant and also serves as a way to eliminate the excess heme released from the hemoglobin of senescent red blood cells. Indeed, red blood cells account for 80% of bilirubin production, with the remainder coming from additional heme-containing proteins in other tissues such as skeletal muscle and the liver. Bilirubin can cross the blood-brain barrier and, if present in excessive levels, results •Fig. 32.12 Neurohumoralcontrolofgallbladdercontractionandbiliarysecretion.ThepathwayalsoinvolvesrelaxationofthesphincterofOdditopermitoutflowofbileintotheduodenum.ACh,acetylcholine;CCK,cholecystokinin;NO,nitricoxide;VIP,vasoactiveintestinalpolypeptide.

1	in brain dysfunction secondary to neuronal cell death and the activation of astrocytes and microglia; it can be fatal if left untreated. Bilirubin and its metabolites are also notable for the fact that they provide color to bile, feces, and to a lesser extent urine. By the same token, when bilirubin accumulates in the circulation as a result of liver disease, it is responsible for the common symptom of jaundice, or yellowing of the skin and conjunctiva. Bilirubin is synthesized from heme by a two-stage reaction that takes place in phagocytic cells of the reticuloendothelial system, including Kupffer cells and cells in the spleen (

1	Bilirubin is synthesized from heme by a two-stage reaction that takes place in phagocytic cells of the reticuloendothelial system, including Kupffer cells and cells in the spleen ( Fig. 32.13 ). The enzyme heme oxygenase that is present in these cells liberates iron from the heme molecule and produces the green pigment biliverdin. This in turn can be reduced to form yellow bilirubin. Because bilirubin is essentially insoluble in aqueous solutions at neutral pH, it is transported through the bloodstream bound to albumin. When this complex reaches the liver, it enters the space of Disse, where bilirubin is selectively taken up across the basolateral membrane of hepatocytes via an OATP transporter (see

1	Table 32.1 ). In the microsomal compartment, bilirubin is then conjugated with one or two molecules of glucuronic acid to enhance its aqueous solubility. The reaction is catalyzed by UDP glucuronyl transferase (UGT). This enzyme is synthesized only slowly after birth, which explains why mild jaundice is relatively common in •Fig. 32.13 Conversionofhemetobilirubin.Thereactionsinsidethedashedboxoccurincellsofthereticuloendothelialsystem.NADP+,oxidizedformofnicotinamideadeninedinucleotidephosphate;NADPH,reducedformofnicotinamideadeninedinucleotidephosphate.

1	newborn infants. The bilirubin conjugates are then secreted into bile by a multidrug resistance–associated protein (MRP2) located in the canalicular membrane. Notably the conjugated forms of bilirubin cannot be reabsorbed from the intestine, thereby ensuring they can be excreted. However, transport of bilirubin across the hepatocyte (and indeed its initial uptake from the bloodstream) is relatively inefficient, so some conjugated and unconjugated bilirubin is present in plasma even under normal conditions. Both circulate bound to albumin, but the conjugated form is bound more loosely and thus can enter the urine. In the colon, bilirubin conjugates are deconjugated by bacterial enzymes, whereupon the bilirubin liberated is metabolized by bacteria to yield urobilinogen, which is reabsorbed, and urobilins and stercobilins, which are excreted. Absorbed urobilinogen in turn can be taken up by hepatocytes and reconjugated, thus giving the molecule yet another chance to be excreted.

1	Measurement of bilirubin in plasma, as well as assessment of whether it is unconjugated or conjugated, is an important tool in the evaluation of liver disease. The presence of unconjugated bilirubin, which is essentially fully albumin bound and cannot be excreted in urine, reflects either loss of UGT (or a normal temporary delay in its maturation in infants) or a sudden oversupply of heme that overwhelms the conjugation mechanism (such as occurs in transfusion reactions or in Rhesus-incompatible newborns). Conjugated bilirubinemia on the other hand is characterized by the presence of bilirubin in urine, to which it imparts a dark coloration. This is indicative of genetic defects in the transporter that mediates bilirubin glucuronide/diglucuronide secretion into the canaliculus, or it may be due to blockage to the flow of bile, perhaps

1	CHAPTER 32 Transport and Metabolic Functions of the Liver caused by an obstructing gallstone. In both cases, bilirubin conjugates are formed in the liver, but with no means of exit they regurgitate back into plasma for urinary excretion.

1	Crigler-Najjar syndrome isaconditionassociatedwithmutationsinthehepatocyteenzymeUGT.IntypeICrigler-Najjarsyndrome,acongenitalmissensemutationresultsincompletelackofthisenzyme,whereaspatientswithtypeIICrigler-NajjarsyndromehaveamildermutationthatreducesUGTlevelstoabout10%ofthoseseeninnormalindividuals.Thuswithvaryingdegreesofseverity,Crigler-Najjarsyndromeimpairstheabilityofhepatocytestoconjugatebilirubin.Theunconjugatedbilirubinregurgitatesbackintothecirculationandbindstoalbumin,withanassociatedriskofneurologicalinjuryiflevelsriseprecipitously.TheonlyeffectivetreatmentoftypeICrigler-Najjarsyndromeatpresentislivertransplantation,althoughgenetherapymaybeapromisingoptioninthenearfuture.ThosewithtypeIIdiseasecansometimesbemanagedeffectivelywithbluelight.Thisconvertscirculatingunconjugatedbilirubintoformsthataremorewatersolubleandthuslessfirmlyboundtoalbumin,whichcanbeexcretedinurine. Ammonia Handling by the Liver

1	Ammonia Handling by the Liver Ammonia (NH3) is a small neutral metabolite that arises from protein catabolism and bacterial activity and is highly membrane permeant. The liver is a critical contributor to prevention of ammonia accumulation in the circulation, which is important because like bilirubin, ammonia is toxic to the central nervous system. The liver eliminates ammonia from the body by converting it to urea via a series of enzymatic reactions known as the urea, or Krebs-Henseleit, cycle (Fig. 32.14 ). The liver is the only tissue in the body that can convert ammonia to urea.

1	Ammonia is derived from two major sources. Approximately 50% is produced in the colon by bacterial ureases. Because the colonic lumen is normally slightly acidic, some of this ammonia is converted to the ammonium ion (NH4 +), which renders it impermeant to the colonic epithelium and therefore allows it to be excreted in stool. However, the remainder of the ammonia generated crosses the colonic epithelium passively and is transported to the liver via the portal circulation. The other major source of ammonia (≈40%) is the kidney (see ). A small amount of ammonia (≈10%) is derived from deamination of amino acids in the liver, by metabolic processes in muscle cells, and via release of glutamine from senescent red blood cells. The “mass balance” for ammonia handling in a healthy adult is presented in

1	The “mass balance” for ammonia handling in a healthy adult is presented in Fig. 32.15 . As just noted, ammonia is a small neutral molecule that readily crosses cell membranes without the benefit of a specific transporter, although some membrane proteins transport ammonia, including certain aquaporins. Whatever the mechanism for transport, the physicochemical properties of ammonia ensure that it is efficiently extracted from portal and systemic circulation •Fig. 32.14 Theureacycle.ADP,adenosinediphosphate;ATP,adenosinetriphosphate.

1	If the metabolic capacity of the liver is compromised acutely, coma and death can rapidly ensue. In chronic liver disease, patients may experience a gradual decline in mental function that reflects the action of both ammonia and other toxins that cannot be cleared by the liver, in a condition known as hepatic encephalopathy. Development of confusion, dementia, and eventually coma in a patient with liver disease is evidence of significant progression, and these symptoms can prove fatal if left untreated. Clinical Assessment of Liver Function

1	Clinical Assessment of Liver Function Given the importance of the liver for homeostasis, tests of liver function are a mainstay of clinical diagnosis. Such tests have several goals: (1) to assess whether hepatocytes have been injured or are dysfunctional, (2) to determine whether bile excretion has been interrupted, and (3) to evaluate whether cholangiocytes have been injured or are dysfunctional. Liver function tests are also used to monitor responses to therapy or rejection reactions after liver trans •Fig. 32.15 Ammoniahomeostasisinhealth.(RedrawnfromBarrettKE.Gastrointestinal Physiology. NewYork:McGraw-Hill;2006.) by hepatocytes, where it then enters the urea cycle to be converted to urea (see

1	Fig. 32.14 ) and is subsequently transported back into the systemic circulation. Urea is a small neutral molecule that is readily filtered at the glomerulus, and it is reabsorbed by the kidney tubules such that approximately 50% of the filtered urea is excreted in urine (see ). Urea that enters the colon is either excreted or metabolized to ammonia via colonic bacteria, with the resulting ammonia being reabsorbed or excreted. plantation. However, not all such tests measure function directly. Nevertheless, liver function tests are discussed briefly because of their link to hepatic physiology.

1	plantation. However, not all such tests measure function directly. Nevertheless, liver function tests are discussed briefly because of their link to hepatic physiology. Tests for hepatocyte injury rely on markers that are specific for this cell type. When hepatocytes are killed by necrotic responses to inflammation or infection, for example, they release enzymes that include alanine aminotransferase (ALT) and aspartate aminotransferase (AST). These enzymes, which are essential to interconvert amino acids, are easily measured in serum and indicate hepatocyte injury, although AST may also be released after injury to other tissues, including the heart. Two other tests are markers of injury to the biliary system. Alkaline phosphatase is expressed in the canalicular membrane, and elevations of this enzyme in plasma suggest localized obstruction to bile flow. Similarly, increased levels of GGT are seen when there is damage to cholangiocytes.

1	Measurement of bilirubin in the circulation or in urine also provides insight into liver function. In addition, measurement of any of the other characteristic secreted products of the liver can be used to diagnose liver disease. Clinically the most common tests are measurements of serum albumin and a blood clotting parameter, the prothrombin time. If results of these tests are abnormal, when considered together with other aspects of the clinical picture, a diagnosis of liver CHAPTER 32 Transport and Metabolic Functions of the Liver disease may be established. Blood glucose and ammonia levels are frequently monitored in patients with chronic liver disease. Finally, imaging tests and histological examination of biopsy specimens of liver parenchyma, usually obtained percutaneously, are also important in evaluating and monitoring patients with suspected or proven liver disease. 1.

1	1. Vital functions of the liver include carbohydrate, lipid, and protein metabolism and synthesis; detoxification of unwanted substances; and excretion of circulating substances that are lipid soluble and carried in the bloodstream bound to albumin. The liver also synthesizes the majority of plasma proteins, including albumin. 2. Liver function depends on its unique anatomy, its constituent cell types (especially hepatocytes), and the unusual arrangement of its blood supply. 3. Substances are excreted from the liver in bile. Bile flow is driven by the presence of bile acids, which are Feranchak AP. Bile secretion and cholestasis. In: Podolsky DK, et al., eds. Yamada’s Textbook of Gastroenterology. 6th ed. Hoboken, NJ: Wiley Blackwell; 2015. Kanel GC. Liver: anatomy, microscopic structure, and cell types. In: Podolsky DK, et al., eds. Yamada’s Textbook of Gastroenterology. 6th ed. Hoboken, NJ: Wiley Blackwell; 2015.

1	Kanel GC. Liver: anatomy, microscopic structure, and cell types. In: Podolsky DK, et al., eds. Yamada’s Textbook of Gastroenterology. 6th ed. Hoboken, NJ: Wiley Blackwell; 2015. amphipathic end products of cholesterol metabolism that are produced by hepatocytes. Bile acids circulate between the liver and intestine to conserve their mass, and water-insoluble metabolites (e.g., cholesterol) are carried in bile in the form of mixed micelles. 4. Bile is stored in the gallbladder between meals, where it is concentrated and released when hormonal and neural signals simultaneously contract the gallbladder and relax the sphincter of Oddi. 5. The liver is critical for disposing of certain substances that would be toxic if allowed to accumulate in the bloodstream, including bilirubin and ammonia. Parekh PJ, Balart LA. Ammonia and its role in the pathogenesis of hepatic encephalopathy. Clin Liver Dis. 2015;19:529-537.

1	Parekh PJ, Balart LA. Ammonia and its role in the pathogenesis of hepatic encephalopathy. Clin Liver Dis. 2015;19:529-537. Wolkoff AW. Organic anion uptake by hepatocytes. Compr Physiol. 2014;4:1715-1735. Wu T, et al. Gut motility and enteroendocrine secretion. Curr Opin Pharmacol. 2013;13:928-934. SECTION 7The Renal System BRUCE A. STANTON AND BRUCE M. KOEPPEN Chapter 36 Potassium, Calcium, and Phosphate Homeostasis Elements of Renal Function Chapter 34 Solute and Water Transport Along the Nephron: Tubular Function Chapter 35 Control of Body Fluid Osmolality and Volume Chapter 37 Role of the Kidneys in the Regulation of Acid-Base Balance Upon completion of this chapter the student should be able to answer the following questions: 1. Which structures in the glomerulus are filtration barriers to plasma proteins? 2. What is the physiological significance of the juxtaglomerular apparatus? 3. What blood vessels supply the kidneys? 4. What nerves innervate the kidneys? 5.

1	2. What is the physiological significance of the juxtaglomerular apparatus? 3. What blood vessels supply the kidneys? 4. What nerves innervate the kidneys? 5. What is the location of the kidneys, and what are their gross anatomical features? 6. What are the different parts of the nephron, and what is their locations within the cortex and medulla? 7. What are the major components of the glomerulus, and what are the cell types located in each component? 8. How can the concepts of mass balance be used to measure the glomerular filtration rate (GFR)? 9. Why can inulin clearance and creatinine clearance be used to measure GFR? 10. Why is plasma creatinine concentration used clinically to monitor GFR? 11. What are elements of the glomerular filtration barrier, and how do they determine how much protein enters Bowman’s space? 12. What Starling forces are involved in formation of the glomerular ultrafiltrate, and how do changes in each force affect GFR? 13.

1	12. What Starling forces are involved in formation of the glomerular ultrafiltrate, and how do changes in each force affect GFR? 13. What is autoregulation of renal blood flow and GFR, and which factors and hormones are responsible for autoregulation? 14. Which hormones regulate renal blood flow? 15. Why do hormones influence renal blood flow despite autoregulation? Overview of Renal Function The kidney presents in the highest degree the phenomenon of sensibility, the power of reacting to various stimuli in a direction, which is appropriate for the survival of the organism; a power of adaptation which almost gives one the idea that its component parts must be endowed with intelligence. E. STARLING—1909 Certainly, mental integrity is a sine qua non of the free and independent life. But let the composition of our internal environment suffer change, let our kidneys fail for even a short time to fulfill their tasks, and our mental integrity, or personality is destroyed.

1	HOMER W. SMITH—1939 As both Starling and Smith recognized, the kidneys are more appropriately considered regulatory rather than excretory organs. The kidneys regulate (1) body fluid osmolality and volumes, (2) electrolyte balance, and (3) acid-base balance. In addition the kidneys excrete metabolic products and foreign substances and produce and secrete hormones.

1	Control of body fluid osmolality is important for maintenance of normal cell volume in all tissues of the body. Control of body fluid volume is necessary for normal function of the cardiovascular system. The kidneys are also essential in regulating the amount of several important inorganic ions in the body, including Na+, K+, Cl−, bicarbonate (HCO3 −), hydrogen (H+), Ca++ , and inorganic phosphate (Pi). Excretion of these electrolytes must be equal to daily intake to maintain appropriate total body balance. If intake of an electrolyte exceeds its excretion, the amount of this electrolyte in the body increases and the individual is in positive balance for that electrolyte. Conversely if excretion of an electrolyte exceeds its intake, its amount in the body decreases and the individual is in negative balance for that electrolyte. For many electrolytes the kidneys are the sole or principal route for excretion from the body.

1	Another important function of the kidneys is regulation of acid-base balance. Many metabolic functions of the body are exquisitely sensitive to pH. Thus the pH of body fluids must be maintained within narrow limits. Normal pH is maintained by buffers within body fluids and by the coordinated action of the lungs, liver, and kidneys. The kidneys excrete a number of the end products of metabolism. These waste products include urea (from amino acids), uric acid (from nucleic acids), creatinine (from muscle creatine), end products of hemoglobin metabolism, and metabolites of hormones. The kidneys eliminate these substances from the body at a rate that matches their production. Thus the kidneys regulate hormone concentrations within body fluids. The kidneys also represent an important route for elimination of foreign substances such as drugs, toxins (e.g., pesticides), and other chemicals from the body.

1	Finally, the kidneys are important endocrine organs that produce and secrete renin, calcitriol, and erythropoietin. Renin is not a hormone but an enzyme that activates the renin-angiotensin-aldosterone system, which helps regulate blood pressure and Na+ and K+ balance. Calcitriol, a metabolite of vitamin D3, is necessary for normal absorption of Ca++ by the gastrointestinal tract and for its deposition in bone (see ). In patients with renal disease the kidneys’ ability to produce calcitriol is impaired and levels of this hormone are reduced. As a result, Ca++ absorption by the intestine is decreased, which over time contributes to the bone formation abnormalities seen in patients with chronic renal disease. Another consequence of many kidney diseases is a reduction in erythropoietin production and secretion. Erythropoietin stimulates red blood cell formation by bone marrow. Decreased erythrocyte production contributes to the anemia that occurs in chronic kidney disease (CKD), a

1	production and secretion. Erythropoietin stimulates red blood cell formation by bone marrow. Decreased erythrocyte production contributes to the anemia that occurs in chronic kidney disease (CKD), a progressive loss in kidney function over a period of months or years.

1	A large variety of diseases impair kidney function and result in renal failure. In some instances the impairment in renal function is transient, but in many cases renal function declines progressively. Patients in whom the glomerular filtration rate (GFR) is less than 10% of normal are said to have end-stage renal disease (ESRD) and must receive renal replacement therapy in the form of either dialysis or kidney transplantation to survive. To understand the mechanisms that contribute to renal disease, it is first necessary to understand the normal physiology of renal function. Thus in the following chapters in this section of the book, various aspects of renal function are considered. Functional Anatomy of the Kidneys Structure and function are closely linked in the kidneys. Consequently an appreciation of the gross anatomical and histological features of the kidneys is a prerequisite for understanding their functions.

1	The kidneys are paired organs that lie on the posterior wall of the abdomen behind the peritoneum on either side of the vertebral column. In an adult human, each kidney weighs between 115 and 170 g and is approximately 11 cm long, 6 cm wide, and 3 cm thick. The gross anatomical features of the human kidney are illustrated in Fig. 33.1 . The medial side of each kidney contains an indentation through which pass the renal artery and vein, nerves, and pelvis. If a kidney were cut in half, two regions would be evident: an outer region called the cortex and an inner region called the medulla. The cortex and medulla are composed of nephrons (the functional units of the kidney), blood vessels, lymphatics, and nerves. The Kidney disease is a major health problem worldwide. In the United States alone: CKD affects over 23 million patients and accounts for more than 90,000 deaths annually. Kidney diseases represent the ninth leading cause of death.

1	CKD affects over 23 million patients and accounts for more than 90,000 deaths annually. Kidney diseases represent the ninth leading cause of death. The health care cost for CKD in Medicare patients alone exceeds $44 billion per year. Each year, kidney disease is diagnosed in over 3 million new patients. Over 500,000 people are treated for ESRD every year. Approximately 275,000 patients with ESRD are maintained on either hemodialysis or peritoneal dialysis. Diabetes and hypertension are the leading causes of ESRD. ESRD secondary to diabetes is increasing at an annual rate of more than 11% per year. More than 17,000 kidney transplants are performed each year. Unfortunately, in excess of 100,000 patients are awaiting kidney transplants.

1	Urinary tract infections (8.3 million visits annually), urolithiasis (i.e., kidney and urinary tract stones; 1.3 million visits annually), interstitial cystitis (i.e., inflammation of the urinary bladder; 700,000 patients), and urinary incontinence (13 million adults affected, mostly older than 65) are also major health care problems. Individuals with ESRD must undergo renal replacement therapy, which includes peritoneal dialysis, hemodialysis, and kidney transplantation. Both peritoneal dialysis and hemodialysis, as their names suggest, rely on the ability to remove small dialyzable molecules from the blood—including metabolic waste products normally removed by intact kidneys—via diffusion across a selectively permeable membrane into a solution lacking these substances, thereby mitigating both their accumulation and associated adverse health effects. In addition, dialysis helps reestablish both fluid and electrolyte balance via removal of excess fluid, correction of acid-base

1	both their accumulation and associated adverse health effects. In addition, dialysis helps reestablish both fluid and electrolyte balance via removal of excess fluid, correction of acid-base changes, and normalization of plasma electrolyte concentrations). In peritoneal dialysis, the peritoneal membrane lining the abdominal cavity acts as a dialyzing membrane. Several liters of a defined dialysis solution are typically introduced into the abdominal cavity, and small molecules in blood diffuse across the peritoneal membrane into the solution, which can then be iteratively removed, discarded, and replaced. In hemodialysis, a patient’s blood is pumped through an extracorporeal artificial kidney in which blood is separated from a defined dialysis solution by an artificial semipermeable membrane that allows small molecules to diffuse from the blood down their concentration gradient into the dialysis solution, thereby removing small molecules associated with adverse health effects if

1	membrane that allows small molecules to diffuse from the blood down their concentration gradient into the dialysis solution, thereby removing small molecules associated with adverse health effects if allowed to accumulate in patients without functioning kidneys. Patients who are candidates for renal transplantation are often treated with dialysis until an appropriate donor kidney can be procured. Although anemia has historically been a significant problem in ESRD patients owing to severely reduced endogenous erythropoietin production, this problem can now be easily corrected in patients undergoing chronic dialysis via administration of erythropoiesis-stimulating agents (e.g., recombinant human erythropoietin).

1	CHAPTER 33 Elements of Renal Function 583 • Fig. 33.1 Structure of a human kidney, cut open to show the internal structures. (Modified From Boron WF, Boulpaep EL. Medical Physiology. 2nd ed. Philadelphia: Saunders Elsevier; 2009.) medulla in the human kidney is divided into conical masses called renal pyramids. The base of each pyramid originates at the corticomedullary border, and the apex terminates in a papilla, which lies within a minor calyx. Minor calyces collect urine from each papilla. The numerous minor calyces expand into two or three open-ended pouches, the major calyces. The major calyces in turn feed into the pelvis. The pelvis represents the upper expanded region of the ureter, which carries urine from the pelvis to the urinary bladder. The walls of the calyces, pelvis, and ureters contain smooth muscle that contracts to propel the urine toward the urinary bladder.

1	Blood flow to the two kidneys is equivalent to about 25% (1.25 L/min) of the cardiac output in resting individuals. However, the kidneys constitute less than 0.5% of total body weight. As illustrated in Fig. 33.2 (left), the renal artery branches progressively to form the interlobar artery, the arcuate artery, the interlobular artery, and the afferent arteriole, which leads into the glomerular capillaries. The glomerular capillaries come together to form the efferent arteriole, which leads into a second capillary network, the peritubular capillaries, which supply blood to the nephron. The vessels of the venous system run parallel to the arterial vessels and progressively form the interlobular vein, arcuate vein, interlobar vein, and renal vein, which courses beside the ureter. Ultrastructure of the Nephron

1	Ultrastructure of the Nephron The functional unit of the kidneys is the nephron. Each human kidney contains approximately 1.2 million nephrons, which are essentially hollow tubes composed of a single epithelial cell layer. The nephron consists of a renal corpuscle, proximal tubule, loop of Henle, distal tubule, and collecting duct system (Fig. 33.3

1	Fig. 33.2 ). The renal corpuscle consists of glomerular capillaries enclosed within Bowman’s capsule. The proximal tubule exits this structure and initially forms several coils, followed by a straight piece that descends toward the medulla. The next segment is the loop of Henle, which is composed of aThe organization of the nephron is actually more complicated than presented here. However, for simplicity and clarity of presentation in subsequent chapters, the nephron is divided into five segments. The collecting duct system is not actually part of the nephron. However, again for simplicity, we consider the collecting duct system part of the nephron. bAlthough the renal corpuscle is composed of glomerular capillaries and Bowman’s capsule, the term glomerulus is commonly used to described the renal corpuscle.

1	• Fig. 33.2 Left, Organization of the vascular system of the human kidney. 1, interlobar arteries; 1a, interlobar vein; 2, arcuate arteries; 2a, arcuate veins; 3, interlobular arteries; 3a, interlobular veins; 4, stellate vein; 5, afferent arterioles; 6, efferent arterioles; 7a, 7b, glomerular capillary networks; 8, descending vasa recta; 9, ascending vasa recta. Right, Organization of the human nephron. A superficial nephron is illustrated on the left and a juxtamedullary (JM) nephron is illustrated on the right. The loop of Henle includes the straight portion of the proximal tubule (PT), descending thin limb (DTL), ascending thin limb (ATL), and thick ascending limb (TAL). B, Bowman’s capsule; CCD, cortical collecting duct; DT, distal tubule; IMCD, inner medullary collecting duct; MD, macula densa; OMCD, outer medullary collecting duct; P, pelvis. (Modified from Kriz W, Bankir LA. Am J Physiol 1988;254:F1; and Koushanpour E, Kriz W. Renal Physiology: Principles, Structure, and

1	MD, macula densa; OMCD, outer medullary collecting duct; P, pelvis. (Modified from Kriz W, Bankir LA. Am J Physiol 1988;254:F1; and Koushanpour E, Kriz W. Renal Physiology: Principles, Structure, and Function. 2nd ed. New York: Springer-Verlag; 1986.) the straight part of the proximal tubule, the descending thin limb (which ends in a hairpin turn), the ascending thin limb (only in nephrons with long loops of Henle), and the thick ascending limb. Near the end of the thick ascending limb, the nephron passes between the afferent and efferent arterioles of the same nephron. This short segment of the thick ascending limb abutting the glomerulus is called the macula densa (see

1	Figs. 33.2 33.3 ). The distal tubule begins a short distance beyond the macula densa and extends to the point in the cortex where two or more nephrons join to form a cortical collecting duct. The cortical collecting duct enters the medulla and becomes the outer medullary collecting duct and then the inner medullary collecting duct. Each nephron segment is made up of cells that are uniquely suited to perform specific transport functions (see

1	Each nephron segment is made up of cells that are uniquely suited to perform specific transport functions (see Fig. 33.3 ). Proximal tubule cells have an extensively amplified apical membrane (the ultrafiltrate or urine side of the cell) called the brush border, which is present only in the proximal tubule. The basolateral membrane (the interstitial or blood side of the cell) is highly invaginated. These invaginations contain many mitochondria. In contrast, the descending and ascending thin limbs of the loop of Henle have poorly developed apical and basolateral surfaces and few mitochondria. The cells of the thick ascending limb and the distal tubule have abundant mitochondria and extensive infoldings of the basolateral membrane. The collecting duct is composed of two cell types: principal cells and intercalated cells. Principal cells have a moderately invaginated basolateral membrane and contain few mitochondria (see

1	The collecting duct is composed of two cell types: principal cells and intercalated cells. Principal cells have a moderately invaginated basolateral membrane and contain few mitochondria (see Fig. 33.3 ). Principal cells play an important role in reabsorption of NaCl (see ) and secretion of K+ (see ). Intercalated cells, which play an important role in regulating acid-base balance, have a high density of mitochondria (see Fig. 33.3 ). One population of intercalated cells secretes H+ (i.e., reabsorbs HCO3 −), and a second population secretes HCO3 − (see ). The final segment of the nephron, the inner medullary collecting duct, is composed of inner medullary collecting duct cells, which have poorly developed apical and basolateral surfaces and few mitochondria. All cells in the nephron except intercalated cells have in their apical plasma membrane a single nonmotile primary cilium that protrudes into the tubule fluid (

1	All cells in the nephron except intercalated cells have in their apical plasma membrane a single nonmotile primary cilium that protrudes into the tubule fluid ( Fig. 33.4 ). Primary cilia are mechanosensors (i.e., they sense changes in the rate of flow of tubule fluid) and chemosensors (i.e., they sense or respond to compounds in the surrounding fluid), and they initiate Ca++-dependent signaling pathways, including those that control kidney cell function, proliferation, differentiation, and apoptosis (i.e., programmed cell death).

1	Fig. 33.2 ), with approximately 10 superficial nephrons for each juxtamedullary nephron. The 585 • Fig. 33.3 Diagram of a nephron including cellular ultrastructure. Inner medulla collecting duct Ascending thin limbThick ascending limb Descending thin limb Proximal tubule Macula densa Cortical collecting duct Distal tubule Principal cell Intercalated cell OutermedullaInner medulla Cortex • Fig. 33.4 Scanning electron micrograph illustrating primary cilia (C, ≈2 to 30 µm long and 0.5 µm in diameter) in the apical plasma membrane of principal cells in the cortical collecting duct. Note that intercalated cells (IC1 and IC2) do not have cilia but have numerous microvilli. CD, collecting duct principal cells with short microvilli (arrowhead); straight ridges (open arrow) represent the cell borders between principal cells; IC1 and IC2, intercalated cells with numerous long microvilli in the apical membrane. (From Kriz W, Kaissling B. Structural organization of the mammalian kidney. In:

1	borders between principal cells; IC1 and IC2, intercalated cells with numerous long microvilli in the apical membrane. (From Kriz W, Kaissling B. Structural organization of the mammalian kidney. In: Seldin DW, Giebisch G [eds]. The Kidney: Physiology and Pathophysiology. 3rd ed. Philadelphia: Lippincott Williams & Wilkins; 2000.) glomerulus of each superficial nephron is located in the outer region of the cortex. The corresponding loops of Henle are short, and associated efferent arterioles branch into peritubular capillaries that surround its associated nephron segments as well as adjacent nephrons. This capillary network conveys oxygen and important nutrients to the nephron segments in the cortex, delivers substances to individual nephron segments for secretion (i.e., movement of a substance from blood into tubular fluid), and serves as a pathway for return of reabsorbed water and solutes to the circulatory system. A few species, including humans,

1	Polycystin 1 (encoded by the PKD1 gene) and polycystin 2 (encoded by the PKD2 gene) are expressed in the membrane of primary cilia and mediate entry of Ca++ into cells. PKD1 and PKD2 are thought to play an important role in flow-dependent K+ secretion by principal cells of the collecting duct. As described in more detail in Chapter 36, increased flow of tubule fluid in the collecting duct is a strong stimulus for secretion of K+ . Increased flow bends the primary cilium in principal cells, which activates the PKD1/PKD2 Ca++-conducting channel complex and allows Ca++ to enter the cell and increase intracellular [Ca++]. The increase in [Ca++] activates K+ channels in the apical plasma membrane, which enhances secretion of K+ from the cell into the tubule fluid.

1	Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, occurring in about 1 in 1000 people. Approximately 12.5 million people worldwide have ADPKD, which is caused primarily by mutations in PKD1 (85%–90% of cases) or PKD2 (≈15% of cases). The major phenotype of ADPKD is enlargement of the kidneys due to the presence of hundreds or thousands of space-occupying renal cysts that can be as large as 20 cm in diameter. Cysts are also seen in the liver and other organs in this condition. About 50% of patients with ADPKD progress to renal failure by the age of 60. Although it is not clear how mutations in PKD1 and PKD2 cause ADPKD, renal cyst formation may result from defects in Ca++ uptake that alter Ca++-dependent signaling pathways, including those controlling kidney cell proliferation, differentiation, and apoptosis. also possess very short superficial nephrons whose loops of Henle never enter the medulla.

1	also possess very short superficial nephrons whose loops of Henle never enter the medulla. The glomerulus of each juxtamedullary nephron is located in the region of the cortex adjacent to the medulla (see Fig. 33.2, right). When compared with superficial nephrons, juxtamedullary nephrons differ anatomically in two important ways: the loop of Henle is longer and extends deeper into the medulla, and the efferent arteriole forms not only a network of peritubular capillaries but also a series of accompanying vascular loops called the vasa recta.

1	Fig. 33.2 (left), the vasa recta descend into the medulla, where they form capillary networks that surround the collecting ducts and ascending limbs of the loop of Henle. The blood returns to the cortex via the ascending vasa recta. Although less than 0.7% of the renal blood flow (RBF) enters the vasa recta, these vessels serve important functions in the renal medulla that include (1) conveying oxygen and important metabolic substrates to support nephron function, (2) delivering substances to the nephron for secretion, (3) serving as a pathway for return of reabsorbed water and solutes to the circulatory system, and (4) concentrating and diluting urine (urine concentration and dilution are discussed in more detail in Ultrastructure of the Glomerulus

1	Ultrastructure of the Glomerulus The first step in urine formation begins with passive movement of a plasma ultrafiltrate from the glomerular capillaries (i.e., glomerulus) into Bowman’s space. The term ultrafiltration refers to this passive movement of fluid—similar in composition to plasma, except for the fact that the ultrafiltrate protein concentration is much lower than that in the plasma—from the glomerular capillaries into Bowman’s space. To appreciate this process, one must understand the anatomy of the glomerulus, which consists of a network of capillaries supplied by the afferent arteriole and drained by the efferent arteriole ( Figs. 33.5 33.6 ). During embryological development, the glomerular capillaries press into the closed end of the proximal tubule, forming Bowman’s capsule. As the epithelial cells thin on the outside circumference of Bowman’s capsule, they form the parietal epithelium (see

1	Fig. 33.5 ). The epithelial cells in contact with the capillaries thicken and develop into podocytes, which form the visceral layer of Bowman’s capsule ( Figs. 33.7–33.9 ). The visceral cells face outward at the vascular pole (i.e., where afferent and efferent arterioles enter and exit Bowman’s capsule) to form the parietal layer of Bowman’s capsule. The space between the visceral layer and the parietal layer is Bowman’s space, which at the urinary pole (i.e., where the proximal tubule joins Bowman’s capsule) of the glomerulus becomes the lumen of the proximal tubule. The endothelial cells of glomerular capillaries are covered by a basement membrane surrounded by podocytes. The capillary endothelium, basement membrane, and foot processes of podocytes form the so-called filtration barrier (see Figs. 33.5 and 33.7–33.9 ). The endothelium is fenestrated

1	Fig. 33.5 Anatomy of the glomerulus and juxtaglomerular apparatus. The juxtaglomerular apparatus is composed of the macula densa (MD) of the thick ascending limb, extraglomerular mesangial cells (EGM), and renin-and angiotensin II–producing granular cells (G) of the afferent arterioles (AA). BM, basement membrane; BS, Bowman’s space; EA, efferent arteriole; EN, endothelial cell; FP, foot processes of the podocyte; M, mesangial cells between capillaries; P, podocyte cell body (visceral cell layer); PE, parietal epithelium; PT, proximal tubule cell. (Modified from Kriz W, Kaissling B. Structural organization of the mammalian kidney. In Alpern RJ, Moe OW, Caplan M [eds]: Seldin and Giebisch’s The Kidney, 5th ed. London, Elsevier, 2013. Figure in that source based on Kriz W, Sakai T, et al. [1988]. Morphological aspects of glomerular function. In “Nephrology,” A. M. Davison, Vol. 1, Proceedings of the 10th International Congress of Nephrology, 323. Bailliere Tindall, London.)

1	Fig. 33.6 Scanning electron micrograph of the interlobular artery, afferent arteriole (af), efferent arteriole (ef), and glomerulus. The white bars on the afferent and efferent arterioles indicate that they are about 15 to 20 µm in diameter. (From Kimura K et al. Am J Physiol 1990;259:F936.) • Fig. 33.7 A, Electron micrograph of a podocyte surrounding a glomerular capillary. The cell body of the podocyte contains a large nucleus with three indentations. Cell processes of the podocyte form the interdigitating foot processes (FP). The arrows in the cytoplasm of the podocyte indicate the well-developed Golgi apparatus, and the asterisks indicate Bowman’s space. C, capillary lumen; GBM, glomerular basement membrane. B, Electron micrograph of the filtration barrier of a glomerular capillary. The filtration barrier is composed of three layers: the endothelium, basement membrane, and foot processes of the podocytes. Note the filtration slit diaphragm bridging the floor of the filtration

1	The filtration barrier is composed of three layers: the endothelium, basement membrane, and foot processes of the podocytes. Note the filtration slit diaphragm bridging the floor of the filtration slits (arrows). CL, capillary lumen. (From Kriz W, Kaissling B: Structural organization of the mammalian kidney. In: Alpern RJ, Moe OW, Caplan M [eds]: Seldin and Giebisch’s The Kidney, 5th ed. London, Elsevier, 2013.) (i.e., contains 700-Å holes, where 1 Å = 10−10 m) and freely permeable to water, small solutes (e.g., Na+ , urea, glucose), and most proteins but is not permeable to red blood cells, white blood cells, or platelets. Because endothelial cells express negatively charged glycoproteins on their surface, they minimize the filtration into Bowman’s space of albumin, the most abundant plasma protein, and most • Fig. 33.8 A, Scanning electron micrograph showing the outer surface of glomerular capillaries. This is the view that would be seen from Bowman’s space. Processes (P) of

1	plasma protein, and most • Fig. 33.8 A, Scanning electron micrograph showing the outer surface of glomerular capillaries. This is the view that would be seen from Bowman’s space. Processes (P) of podocytes run from the cell body (CB) toward the capillaries, where they ultimately split into foot processes. Interdigitation of the foot processes creates the filtration slits. B, Scanning electron micrograph of the inner surface (blood side) of a glomerular capillary. This view would be seen from the lumen of the capillary. The fenestrations of the endothelial cells are seen as small 700-Å holes. (From Kriz W, Kaissling B: Structural organization of the mammalian kidney. In: Alpern RJ, Moe OW, Caplan M [eds]: Seldin and Giebisch’s The Kidney, 5th ed. London, Elsevier, 2013.) other plasma proteins. In addition to their role as a barrier to filtration, the endothelial cells synthesize a number of vasoactive substances (e.g., nitric oxide [NO], a vasodilator, and endothelin 1 [ET-1], a

1	proteins. In addition to their role as a barrier to filtration, the endothelial cells synthesize a number of vasoactive substances (e.g., nitric oxide [NO], a vasodilator, and endothelin 1 [ET-1], a vasoconstrictor) that are important in controlling renal plasma flow (RPF). The basement • Fig. 33.9 Electron micrograph of the mesangium, the area between the glomerular capillaries containing mesangial cells. C, glomerular capillaries; cGBM, capillary glomerular basement membrane surrounded by foot processes of podocytes (PO) and endothelial cells; M, mesangial cell that gives rise to several processes, some marked by stars; mGBM, mesangial glomerular basement membrane surrounded by foot processes of podocytes and mesangial cells; US, urinary space. Note the extensive extracellular matrix surrounded by mesangial cells (triangles) (×4100). (From Kriz W, Kaissling B: Structural organization of the mammalian kidney. In: Alpern RJ, Moe OW, Caplan M [eds]: Seldin and Giebisch’s The Kidney,

1	surrounded by mesangial cells (triangles) (×4100). (From Kriz W, Kaissling B: Structural organization of the mammalian kidney. In: Alpern RJ, Moe OW, Caplan M [eds]: Seldin and Giebisch’s The Kidney, 5th ed. London, Elsevier, 2013.) membrane, which is a porous matrix of negatively charged proteins (type IV collagen, laminin, the proteoglycans agrin and perlecan, and fibronectin), is an important filtration barrier to plasma proteins. The basement membrane is thought to function primarily as a charge-selective filter in which the ability of proteins to cross the filter is based on charge.

1	The podocytes, which are endocytic, have long finger-like processes that completely encircle the outer surface of the capillaries (see Figs. 33.7 33.8A ). The processes of the podocytes interdigitate to cover the basement membrane and are separated by apparent gaps called filtration slits (see Figs. 33.7 33.8A ). Each filtration slit is bridged by a thin diaphragm that contains pores with a dimension of 40 × 140 Å. The filtration slit diaphragm, which appears as a continuous structure when viewed by electron microscopy (see Fig. 33.7B ), is composed of several proteins, including nephrin (NPHS1), NEPH-1, and podocin (NPHS2), and intracellular proteins that associate with the slit diaphragm, including α-actinin-4 (ACTN4) and CD2-AP (

1	Figs. 33.10 33.11 ). Filtration slits, which function primarily as a size-selective filter, minimize the filtration of proteins and macromolecules that cross the basement membrane from entering Bowman’s space. Because both the basement membrane and filtration slits contain negatively charged glycoproteins, some proteins are held back (i.e., not filtered into Bowman’s space) on the basis of size and charge. For molecules with an effective molecular radius between 18 and 42 Å, cationic molecules are filtered more readily than anionic molecules. Another important component of the renal corpuscle is the mesangium, which consists of mesangial cells and the mesangial matrix (see

1	Another important component of the renal corpuscle is the mesangium, which consists of mesangial cells and the mesangial matrix (see Fig. 33.9 ). Mesangial cells, which possess many properties of smooth muscle cells, provide structural support for the glomerular capillaries, secrete extracellular matrix, exhibit phagocytic activity by removing macromolecules from the mesangium, and secrete prostaglandins and proinflammatory cytokines. Because they also contract and are adjacent to glomerular capillaries, mesangial cells may influence GFR by regulating blood flow through the glomerular capillaries or by altering the capillary surface area. Mesangial cells located outside the glomerulus (between the afferent and efferent arterioles) are called extraglomerular mesangial cells.

1	The nephrotic syndrome is produced by a variety of disorders and is characterized by increased permeability of the glomerular capillaries to proteins and by loss of normal podocyte structure, including effacement (i.e., thinning) of foot processes. The augmented permeability to proteins results in increased urinary protein excretion (proteinuria). Thus the appearance of proteins in urine can indicate kidney disease. Individuals with this syndrome often develop hypoalbuminemia as a result of the proteinuria. In addition, generalized edema is commonly seen in nephrotic individuals. Mutations in several genes encoding either slit diaphragm proteins (see

1	Figs. 33.10 33.11), including nephrin, NEPH-1, and podocin, or intracellular proteins that functionally interact with slit diaphragm proteins, such as CD2-AP and α-actinin 4 (ACTN4), result in proteinuria and kidney disease. For example, mutations in the nephrin gene (NPHS1) in congenital nephrotic syndrome lead to abnormal or absent slit diaphragms, causing massive proteinuria and renal failure. In addition, mutations in the podocin gene (NPHS2) cause autosomal recessive, steroid-resistant nephrotic syndrome. These naturally occurring mutations and knockout studies in mice demonstrate that nephrin, NEPH-1, podocin, CD2-AP, and α-actinin-4 play key roles in normal glomerular podocyte structure and function.

1	• Fig. 33.10 Anatomy of podocyte foot processes. This figure illustrates the proteins that make up the slit diaphragm between two adjacent foot processes. Nephrin and NEPH1 are membrane-spanning proteins that have large extracellular domains that interact. Podocin, also a membrane-spanning protein, organizes nephrin and NEPH1 in specific microdomains in the plasma membrane, which is important for signaling events that determine the structural integrity of podocyte foot processes. Many of the proteins that compose the slit diaphragm interact with adapter proteins inside the cell, including CD2-AP. The adapter proteins bind to the filamentous actin (F-actin) cytoskeleton, which in turn binds either directly or indirectly to proteins such as α3β1 and MAGI-1 that interact with proteins expressed by the glomerular basement membrane (GBM). α-act-4, α-actinin 4; α3β1, α3β1 integrin; α-DG, α-dystroglycan; CD2-AP, an adapter protein that links nephrin and podocin to intracellular proteins;

1	by the glomerular basement membrane (GBM). α-act-4, α-actinin 4; α3β1, α3β1 integrin; α-DG, α-dystroglycan; CD2-AP, an adapter protein that links nephrin and podocin to intracellular proteins; FAT, a protocadherin that organizes actin polymerization; MAGI-1, a membrane-associated guanylate kinase protein; NHERF-2, Na+-H+ exchanger regulatory factor 2; P, paxillin; P-Cad, P-cadherin; Synpo, synaptopodin; T, talin; V, vinculin; Z, zona occludens. (Adapted from Mundel P, Shankland SJ. J Am Soc Nephrol 2002;13:3005.)

1	Mesangial cells are involved in development of immune complex–mediated glomerular disease. Because the glomerular basement membrane does not completely surround all glomerular capillaries (see Fig. 33.9 ), some immune complexes can escape the blood and enter the mesangium without crossing the glomerular basement membrane. Accumulation of immune complexes induces mesangial infiltration of inflammatory cells and promotes local proinflammatory cytokine and autocoid production. These cytokines and autocoids enhance the immune complex– initiated inflammatory response, which can ultimately lead to mesangial expansion, scarring, and obliteration of the glomerulus. Ultrastructure of the Juxtaglomerular Apparatus The juxtaglomerular apparatus is one component of an important feedback mechanism, the tubuloglomerular feedback mechanism, described later in this chapter. Structures that make up the juxtaglomerular apparatus (see Fig. 33.5 ) include: 1.

1	Fig. 33.5 ) include: 1. the macula densa of the thick ascending limb 2. 3. reninand angiotensin II–producing granular cells of the afferent arteriole

1	3. reninand angiotensin II–producing granular cells of the afferent arteriole Alport syndrome is characterized by hematuria (i.e., blood in the urine) and progressive glomerulonephritis (i.e., inflammation of the glomerular capillaries), and it accounts for 1% to 2% of all cases of ESRD. Alport syndrome is caused by mutations in type IV collagen, a major component of the glomerular basement membrane. In about 80% of patients with Alport syndrome the disease is X-linked with mutations in the COL4A5 gene. Some 15% of patients also have mutations in type IV collagen genes (COL4A3 and COL4A4); six have been identified, but the mode of inheritance is autosomal recessive. The remaining 5% of patients with Alport syndrome have autosomal dominant disease that arises from heterozygous mutations in the COL4A3 or COL4A4 genes. In Alport syndrome, the glomerular basement membrane becomes irregular in thickness and fails to serve as an effective filtration barrier to blood cells and protein.

1	The cells of the macula densa represent a morphologically distinct region of the thick ascending limb. This region passes through the angle formed by the afferent and efferent arterioles of the same nephron. The cells of the macula densa contact the extraglomerular mesangial cells and the granular cells of the afferent arterioles. The granular cells of the afferent arterioles contain smooth muscle myofilaments and—importantly—manufacture, store, and release renin in response to signals associated with decreased effective • Fig. 33.11 Overview of the major proteins that form the slit diaphragm. Nephrins (red) from opposite foot processes interdigitate in the center of the slit. In the slit, nephrin interacts with NEPH1 and NEPH2 (blue), FAT1 and FAT2 (green), and P-cadherin. The intracellular domains of nephrin, NEPH1, and NEPH2 interact with podocin and CD2-AP, which connect these slit diaphragm proteins with ZO-1, α-actinin 4, and actin. (Modified from Tryggvason K et al. N Engl J

1	domains of nephrin, NEPH1, and NEPH2 interact with podocin and CD2-AP, which connect these slit diaphragm proteins with ZO-1, α-actinin 4, and actin. (Modified from Tryggvason K et al. N Engl J Med 2006;354:1387.) circulating volume and reduced renal perfusion. Renin is involved in proteolytic generation of angiotensin II and ultimately in secretion of aldosterone (see ). The juxtaglomerular apparatus is one component of the tubuloglomerular feedback mechanism involved in autoregulation of RBF and GFR.

1	Innervation of the Kidneys Renal nerves regulate RBF, GFR, and salt and water reabsorption by the nephron. The nerve supply to the kidneys consists of sympathetic nerve fibers that originate in the celiac plexus. There is no corresponding parasympathetic innervation. Adrenergic fibers innervating the kidneys release norepinephrine and lie adjacent to the smooth muscle cells of the major branches of the renal artery (interlobar, arcuate, and interlobular arteries) as well as the afferent and efferent arterioles. In addition, sympathetic nerves innervate the renin-producing granular cells of the afferent arterioles. Renin secretion is stimulated by increased sympathetic activity. Nerve fibers also innervate the proximal tubule, loop of Henle, distal tubule, and collecting duct; activation of these nerves enhances Na+ reabsorption by these nephron segments. Assessment of Renal Function

1	Assessment of Renal Function The coordinated actions of the nephron’s various segments determine the final amount of a substance that appears in urine. This represents three general processes (1) glomerular CHAPTER 33 Elements of Renal Function This relationship permits quantification of the amount of substance x excreted in urine versus the amount returned to the systemic circulation in renal venous blood. Thus for any substance that is neither synthesized nor metabolized, the amount that enters the kidneys is equal to the amount that leaves the kidneys in urine plus the amount that leaves the kidneys in renal venous blood. The principle of renal clearance emphasizes the excretory function of the kidneys; it considers only the rate at which a substance is excreted into urine and not its rate of return to the systemic circulation in the renal vein. Therefore in terms of mass balance (

1	Eq. 33.1 ) the urinary excretion rate of substance x (Ux × V̇ ) is proportional to the plasma concentration of substance x ( Equation 33.2 To equate the urinary excretion rate of substance x to • Fig. 33.12 Mass balance relationships for the kidney. See text for definition of symbols. its renal arterial plasma concentration, it is necessary to determine the rate at which it is removed from plasma by filtration, (2) reabsorption of the substance from tubular the kidneys. This removal rate is the clearance (Cx): fluid back into blood, and (3) (in some cases) secretion of Equation 33.3 the substance from blood into tubule fluid. The first step in P × C = U × V˜ urine formation by the kidneys is production of an ultrafil-xa x x trate of plasma across the glomerulus. The process of glo-If

1	Eq. 33.3 is rearranged and the concentration of merular filtration and regulation of GFR and RBF substance x in renal artery plasma ( ) is assumed to beare discussed later in this chapter. The concept of renal clearance, which is the theoretical basis for measurement of GFR and RBF, is presented in the following section. Reabsorption and secretion are discussed in subsequent chapters. The concept of renal clearance is based on the Fick principle (i.e., mass balance or conservation of mass). Fig.

1	The concept of renal clearance is based on the Fick principle (i.e., mass balance or conservation of mass). Fig. 33.12 illustrates the various factors required to describe the mass balance relationships of a kidney. The renal artery is the single input source to the kidney for substances not synthesized by this organ, whereas the renal vein and ureter constitute the two principal output routes. In other words a nonmetabolized substance entering the renal circulation via the renal artery may only exit this circulation via the renal vein (i.e., the unfiltered fraction plus any filtered amount subsequently reabsorbed back into blood) or the ureter (the combined filtered and secreted fractions less any tubular reabsorption). The following equation defines the mass balance relationship: Equation 33.1 where identical to its concentration in a plasma sample from any peripheral blood vessel (Px), the following relationship is obtained: Equation 33.4

1	Equation 33.1 where identical to its concentration in a plasma sample from any peripheral blood vessel (Px), the following relationship is obtained: Equation 33.4 Clearance has the dimensions of volume/time, and it represents a volume of plasma from which all the substance has been removed and excreted into urine per unit time. This last point is best illustrated by considering the following example. If a substance is present in urine at a concentration of 100 mg/mL and the urine flow rate is 1 mL/min, the excretion rate for this substance is calculated as follows: Equation 33.5 If this substance is present in plasma at a concentration of 1 mg/mL, its clearance according to Eq. 33.4 is as follows: Equation 33.6 are the concentrations of substance x in the renal artery and renal vein plasma, respectively

1	Eq. 33.4 is as follows: Equation 33.6 are the concentrations of substance x in the renal artery and renal vein plasma, respectively RPFa and RPFv are renal plasma flow rates in the artery and vein, respectively In other words, 100 mL of plasma will be completely Ux is the concentration of substance x in urine cleared of substance x each minute. The definition of clearV̇ is the urine flow rate ance as a volume of plasma from which all the substance . UCr x V Amount filtered PCr x GFR = Amount excreted UCr x V .

1	Amount filtered PCr x GFR = Amount excreted UCr x V . • Fig. 33.13 Renal handling of creatinine. Creatinine is freely filtered across the glomerulus and is, to a first approximation, not reabsorbed, secreted, or metabolized by the nephron. Note that all the creatinine coming to the kidney in the renal artery does not get filtered at the glomerulus (normally, 15%–20% of plasma creatinine is filtered). The portion that is not filtered is returned to the systemic circulation in the renal vein. PCr, plasma creatinine concentration; RPF, renal plasma flow; Ucr, urinary concentration of creatinine; V., urine flow rate. has been removed and excreted into urine per unit time is somewhat misleading because it is not a real volume of plasma; rather it is a virtual volume. The concept of clearance is important because it can be used to measure GFR and RPF and determine whether a substance is reabsorbed or secreted along the nephron.

1	The concept of clearance is important because it can be used to measure GFR and RPF and determine whether a substance is reabsorbed or secreted along the nephron. The GFR is equal to the sum of the filtration rates of all functioning nephrons. Thus it is an aggregate index of kidney function. A fall in GFR generally means the kidney disease is progressing, whereas recovery generally suggests recuperation. Thus serial assessment of a patient’s GFR is essential to evaluate the severity and course of kidney disease.

1	Creatinine is a byproduct of normal skeletal muscle creatine metabolism and is freely filtered across the glomerulus into Bowman’s space. It is normally generated by the body at a fairly constant rate, and—to a first approximation—it is not appreciably reabsorbed, secreted, or metabolized by the cells of the nephron after its filtration. Accordingly the amount of creatinine excreted in urine per minute is fairly constant at steady state (i.e., when [creatinine] is constant) and equals the amount of creatinine filtered at the glomerulus each minute ( Fig. 33.13): cFor most substances cleared from plasma by the kidneys, only a portion is actually removed and excreted in a single pass through the kidneys. Equation 33.7 where PCr = plasma concentration of creatinine UCr = urine concentration of creatinine V̇ = urine flow Eq. 33.7 is solved for GFR: Equation 33.8 This equation is the same form as that for clearance (see Eq.

1	Eq. 33.7 is solved for GFR: Equation 33.8 This equation is the same form as that for clearance (see Eq. 33.4 ). Thus measured creatinine clearance (CrCl) can be used clinically to determine GFR at steady state. Clearance has the dimensions of volume/time, and it represents an equivalent volume of plasma from which all of the substance has been removed and excreted into urine per unit time. Creatinine is not the only substance that can be used to measure GFR; any substance that meets the following criteria can serve as an appropriate marker. The substance must: 1. 2. be freely filtered across the glomerulus into Bowman’s space 3. not be reabsorbed or secreted by the nephron 4. not be metabolized or produced by the kidney 5.

1	2. be freely filtered across the glomerulus into Bowman’s space 3. not be reabsorbed or secreted by the nephron 4. not be metabolized or produced by the kidney 5. Not all creatinine (or other substances used to measure GFR) that enters the kidney in renal arterial plasma is filtered at the glomerulus. Likewise not all plasma coming into the kidneys is filtered. Although nearly all plasma that enters the kidneys in the renal artery passes through the glomerulus, approximately 10% does not. The portion of filtered plasma is termed the filtration fraction and is determined as: Equation 33.9 Under normal conditions the filtration fraction averages 0.15 to 0.20, which means that only 15% to 20% of the plasma that enters the glomerulus is actually filtered. The remaining 80% to 85% continues on through the glomerular capillaries and into the efferent arterioles and peritubular capillaries before finally returning to the systemic circulation via the renal vein.

1	The first step in the formation of urine is ultrafiltration of plasma by the glomerulus. In normal adults, GFR ranges from 90 to 140 mL/min in males and from 80 to 125 mL/ min in females. Thus in 24 hours as much as 180 L of plasma is filtered by the glomeruli. The plasma ultrafiltrate is devoid of cellular elements (i.e., red and white blood cells and

1	Creatinine clearance (CrCl) is used to estimate GFR in clinical practice. It is synthesized at a relatively constant rate, and the amount produced is proportional to the total muscle mass. However, creatinine is not a perfect substance for measuring GFR because it is secreted to a small extent by the organic cation secretory system in the proximal tubule (see ). The error introduced by this secretory component is approximately 10%. Thus the amount of creatinine excreted in urine exceeds the amount expected from filtration alone by 10%. However, the method used to measure the plasma creatinine concentration (PCr) overestimates the true value by 10%. Consequently the two errors cancel each other, and in most clinical situations, CrCl provides a reasonably accurate measure of GFR.

1	A fall in GFR may be the first and only clinical sign of kidney disease. Thus measuring GFR is important when kidney disease is suspected. A 50% loss of functioning nephrons reduces GFR only by about 25%. The decline in GFR is not 50% because the remaining nephrons compensate. Because measurements of GFR are cumbersome, kidney function is usually assessed in the clinical setting by measuring the plasma concentration of creatinine (PCr),which is inversely related to GFR ( Fig. 33.14 ). However, as

1	Fig. 33.14 shows, GFR must decline substantially before an increase in PCr can be detected in a clinical setting. For example, a fall in GFR from 120 to 100 mL/min is accompanied by an increase in PCr from 1.0 to 1.2 mg/dL. This does not appear to be a significant change in PCr, but GFR has actually fallen by almost 20%. In the clinical setting, estimated GFR (eGFR) also includes consideration of several other factors in addition to the plasma concentration of creatinine, including age, sex, body size, and race. A free app to calculate eGFR can be downloaded at: https://www.kidney.org/apps/professionals/ platelets) and is essentially protein free. The concentration • Fig. 33.14 Relationship between GFR and plasma [creatinine] (Pcr). The amount of creatinine filtered is equal to the amount excreted; thus GFR × PCr = UCr × V. Because the production of creatinine is constant, excretion must be constant to maintain creatinine balance. Therefore if GFR falls from 120 to 60 mL/min, PCr must

1	excreted; thus GFR × PCr = UCr × V. Because the production of creatinine is constant, excretion must be constant to maintain creatinine balance. Therefore if GFR falls from 120 to 60 mL/min, PCr must increase from 1 to 2 mg/dL to keep filtration of creatinine and its excretion equal to the production rate.

1	1.0 0.8 0.6 0.4 of salts and organic molecules (e.g., glucose, amino acids) is similar in plasma and the ultrafiltrate. Starling forces drive ultrafiltration across the glomerular capillaries, and changes in these forces alter GFR. GFR and RPF are normally held within very narrow ranges by a phenomenon called autoregulation. The next sections of this chapter review the composition of the glomerular filtrate, the dynamics of its formation, and the relationship between RPF and GFR. In addition, factors that contribute to autoregulation and regulation of GFR and RBF are discussed. Determinants of Ultrafiltrate Composition The glomerular filtration barrier determines the composition of the plasma ultrafiltrate. It restricts filtration of molecules on the basis of both size and electric charge (

1	Fig. 33.15 ). In general, neutral molecules with a radius smaller than about 0.2 • Fig. 33.15 Influence of the size and electric charge of dextran on its filterability. A value of 1 indicates that it is filtered freely, whereas a value of zero indicates that it is not filtered. The filterability of dextrans between approximately 18 and 42 Å depends on charge. Dextrans larger than 42 Å are not filtered regardless of charge, and polycationic dextrans and neutral dextrans smaller than 18 Å are freely filtered. The major proteins in plasma are albumin and immunoglobulins. Because the effective molecular radii of immunoglobulin (Ig)G (53 Å) and IgM (>100 Å) are greater than 42 Å, they are not filtered. Although the effective molecular radius of albumin is 35 Å, it is a polyanionic protein, so it does not cross the filtration barrier to a significant degree.

1	18 Å are freely filtered, molecules larger than about 42 Å are not filtered, and molecules between about 18 and 42 Å are filtered to varying degrees.

1	Fig. 33.15 shows how electric charge affects filtration of macromolecules (e.g., dextrans) by the glomerulus. Dextrans are a family of exogenous polysaccharides manufactured in various molecular weights. They can be electrically neutral or have either negative (polyanionic) or positive (polycationic) charges. As the size (i.e., effective molecular radius) of a dextran molecule increases, the rate at which it is filtered decreases. For any given molecular radius, cationic molecules are more readily filtered than anionic molecules. The reduced filtration rate for anionic molecules is explained by the presence of negatively charged glycoproteins on the surface of all components of the glomerular filtration barrier. These charged glycoproteins repel similarly charged molecules. Because most plasma proteins are negatively charged, the negative charge on the filtration barrier restricts filtration of anionic proteins more than the filtration of neutral and polyanionic proteins with a

1	most plasma proteins are negatively charged, the negative charge on the filtration barrier restricts filtration of anionic proteins more than the filtration of neutral and polyanionic proteins with a molecular radius between approximately 18 to 42 Å. For example, serum albumin, an anionic protein that has an effective molecular radius of 35.5 Å, is poorly filtered. Because the small amount of filtered albumin is normally reabsorbed avidly by the proximal tubule, almost no albumin appears in urine.

1	The importance of the negative charges on the filtration barrier in restricting filtration of plasma proteins is shown in Figs. 33.15 33.16 . Removal of the negative charges from the filtration barrier causes proteins to be filtered solely on the basis of their effective molecular radius ( Fig. 33.16 ). Hence at any molecular radius between approximately 18 and 42 Å, filtration of polyanionic proteins will exceed the filtration that prevails in the normal state (in which the filtration barrier has anionic charges). In a number of glomerular diseases the negative charges on the filtration barrier are reduced because of immunological damage and inflammation. As a result, filtration of anionic proteins between approximately 18 and 42 Å in radius is increased. When the filtered proteins exceed the ability of the proximal tubule to reabsorb and catabolize them, anionic proteins begin to appear in urine (proteinuria), which is a marker of kidney disease. Dynamics of Ultrafiltration

1	Dynamics of Ultrafiltration The forces responsible for glomerular filtration of plasma are the same as those in other capillary beds. Ultrafiltration occurs because the Starling forces (i.e., hydraulic and oncotic pressures) combine to drive fluid from the lumen of glomerular capillaries across the filtration barrier and into Bowman’s space (

1	Fig. 33.17 ). The hydraulic pressure inside the glomerular capillary (PGC) is oriented to promote movement of fluid from the glomerular capillary into Bowman’s space. Because the glomerular ultrafiltrate is essentially protein free under normal conditions, owing in large part to the paucity of proteins in serum smaller than 18 Å in radius 1.0 0.8 0.6 0.4 0.2 0 • Fig. 33.16 Reduction of the negative charges on the glomerular wall results in filtration of proteins on the basis of size only. In this situation the relative filterability of proteins depends only on the molecular radius. Accordingly, excretion of polyanionic proteins (18–42 Å) in urine increases because more proteins of this size are filtered.

1	that can be effectively filtered, the reflection coefficient (σ) for proteins across the glomerular capillary is essentially 1. Thus the oncotic pressure in Bowman’s space (πBS) is near zero. Therefore PGC is the principal force favoring filtration. In contrast, the hydraulic pressure in Bowman’s space (PBS) and the oncotic pressure in the glomerular capillary (πGC) both oppose filtration.

1	Fig. 33.17 , a net ultrafiltration pressure (PUF) of 17 mm Hg exists at the afferent end of the glomerulus, whereas at the efferent end it is 8 mm Hg (where PUF = PGC − PBS −πGC). Two additional points concerning Starling forces and this pressure change are important. First, PGC decreases slightly along the length of the capillary because of the resistance to flow along the length of the capillary. Second, πGC increases as plasma is filtered while protein is retained within the glomerular capillary, thereby progressively increasing the protein concentration along the length of the capillary. GFR is proportional to the sum of the Starling forces that exist across the capillaries [(PGC − PBS) −σ(πGC −πBS)] multiplied by the ultrafiltration coefficient (Kf). That is: Equation 33.10

1	Equation 33.10 Kf is the product of the intrinsic permeability of the glomerular capillary and the glomerular surface area available for filtration. The rate of glomerular filtration is considerably greater in glomerular capillaries than in systemic capillaries, mainly because Kf is approximately 100 times greater in glomerular capillaries. Furthermore PGC is approximately twice as great as the hydraulic pressure in systemic capillaries.

1	GFR can be altered by changing Kf or by changing any of the Starling forces. In normal individuals, GFR is regulated by alterations in PGC that are mediated mainly by changes • Fig. 33.17 Idealized glomerular capillary and the Starling forces across it. The reflection coefficient (σ) for protein across the glomerular capillary is approximately 1. PBS, hydraulic pressure in Bowman’s space; PGC, hydraulic pressure in the glomerular capillary; PUF, net ultrafiltration pressure; πBS, oncotic pressure in Bowman’s space; πGC, oncotic pressure in the glomerular capillary. The negative signs for PBS and πGC indicate that these forces oppose formation of the glomerular filtrate. in afferent or efferent arteriolar resistance. PGC is affected in three ways: 1. Changes in afferent arteriolar resistance: A decrease in resistance increases PGC and GFR, whereas an increase in resistance decreases PGC and GFR. 2.

1	Changes in afferent arteriolar resistance: A decrease in resistance increases PGC and GFR, whereas an increase in resistance decreases PGC and GFR. 2. Changes in efferent arteriolar resistance: A decrease in resistance reduces PGC and GFR, whereas an increase in resistance elevates PGC and GFR. 3. Changes in renal arteriolar pressure: An increase in blood pressure transiently increases PGC (which enhances GFR), whereas a decrease in blood pressure transiently decreases PGC (which reduces GFR). Blood flow through the kidneys serves several important functions: 1. 2. modifies the rate of solute and water reabsorption by the proximal tubule 3. participates in concentration and dilution of urine 4. delivers O2, nutrients, and hormones to cells along the nephron and returns CO2, reabsorbed fluid, and solutes to the general circulation 5. Blood flow through any organ may be represented by the following equation:

1	Blood flow through any organ may be represented by the following equation: A reduction in GFR in disease states is most often due to decreases in Kf because of the loss of filtration surface area. GFR also changes in pathophysiological conditions because of changes in PGC, πGC, and PBS. 1. Changes in Kf: Increased Kf enhances GFR, whereas decreased Kf reduces GFR. Some kidney diseases reduce Kf by decreasing the number of filtering glomeruli (i.e., diminished surface area). Some drugs and hormones that dilate the glomerular arterioles also increase Kf. Similarly, drugs and hormones that constrict the glomerular arterioles also decrease Kf. 2. Changes in PGC: With decreased renal perfusion, GFR declines because PGC falls. As previously discussed, a reduction in PGC is caused by a decline in renal arterial pressure, an increase in afferent arteriolar resistance, or a decrease in efferent arteriolar resistance. 3.

1	3. Changes in πGC: An inverse relationship exists between πGC and GFR. Alterations in πGC result from changes in protein synthesis outside the kidneys. In addition the protein loss in urine caused by some renal diseases can lead to a decrease in the plasma protein concentration and thus in πGC. 4. Changes in PBS: Increased PBS reduces GFR, whereas decreased PBS enhances GFR. Acute obstruction of the urinary tract (e.g., a kidney stone occluding the ureter) increases PBS. Equation 33.11 organ R = resistance to flow through that organ Accordingly, RBF is equal to the pressure difference between the renal artery and the renal vein divided by renal vascular resistance: Equation 33.12

1	Accordingly, RBF is equal to the pressure difference between the renal artery and the renal vein divided by renal vascular resistance: Equation 33.12 The afferent arteriole, efferent arteriole, and interlobular artery are the major resistance vessels in the kidneys and thereby determine renal vascular resistance. Like most other organs, the kidneys regulate their blood flow by adjusting vascular resistance in response to changes in arterial pressure. As shown in

1	Fig. 33.18 these adjustments are so precise that blood flow remains relatively constant as arterial blood pressure changes between 90 and 180 mm Hg. GFR is also regulated over the same range of arterial pressures. The phenomenon whereby RBF and GFR are maintained relatively constant between blood pressures of 90 and 180 mm Hg, namely autoregulation, is achieved by changes in vascular resistance, mainly through the afferent arterioles of the kidneys. Because both RBF and GFR are regulated over the • Fig. 33.18 Relationship between arterial blood pressure and RBF and between arterial blood pressure and GFR. Autoregulation maintains GFR and RBF relatively constant as blood pressure changes from 90 to 180 mm Hg. same range of pressures and because RBF is an important determinant of GFR, it is not surprising that the same mechanisms regulate both flows.

1	same range of pressures and because RBF is an important determinant of GFR, it is not surprising that the same mechanisms regulate both flows. Two mechanisms are responsible for autoregulation of RBF and GFR: one mechanism that responds to changes in arterial pressure and another that responds to changes in [NaCl] in tubular fluid. Both regulate the tone of the afferent arteriole. The pressure-sensitive mechanism, the so-called myogenic mechanism, is related to an intrinsic property of vascular smooth muscle: the tendency to contract when stretched. Accordingly, when arterial pressure rises and the renal afferent arteriole is stretched, the smooth muscle contracts in response. Because the increase in resistance of the arteriole offsets the increase in pressure, RBF, and therefore GFR, remains constant. (That is, RBF is constant if ΔP/R is kept constant [see Eq. 33.11

1	The second mechanism responsible for autoregulation of GFR and RBF is the [NaCl]-dependent mechanism known as tubuloglomerular feedback. This mechanism involves a feedback loop in which a change in GFR leads to alteration in the concentration of NaCl in tubular fluid, which is sensed by the macula densa of the juxtaglomerular apparatus and converted into signals that affect afferent arteriolar resistance and thus the GFR (Fig. 33.19 ). For example, when the GFR increases and causes [NaCl] in tubular fluid in the loop of Henle to rise, more NaCl enters the macula densa cells in this segment (Fig. 33.20 ). This leads to an increase in formation and release of adenosine triphosphate (ATP) and adenosine (a metabolite of ATP) by macula densa cells, which causes vasoconstriction of the afferent arteriole and normalization of GFR. In contrast, when GFR and [NaCl] in tubule fluid decrease, less NaCl enters the macula densa cells, and both ATP and adenosine • Fig. 33.19 Tubuloglomerular

1	arteriole and normalization of GFR. In contrast, when GFR and [NaCl] in tubule fluid decrease, less NaCl enters the macula densa cells, and both ATP and adenosine • Fig. 33.19 Tubuloglomerular feedback. An increase in GFR (1) increases [NaCl] in tubule fluid in the loop of Henle (2). The increase in [NaCl] is sensed by the macula densa and converted to a signal (3) that increases the resistance of the afferent arteriole (RA) (4), which decreases GFR. A decrease in GFR has the opposite effects. (Modified from Cogan MG. Fluid and Electrolytes: Physiology and Pathophysiology. Norwalk, CT: Appleton & Lange; 1991.) production and release decline. The fall in [ATP] and [adenosine] results in afferent arteriolar vasodilation, which returns GFR to normal. NO, a vasodilator produced by the macula densa, attenuates tubuloglomerular feedback, whereas angiotensin II enhances tubuloglomerular feedback. Thus the macula densa may release both vasoconstrictors (e.g., ATP and adenosine) and a

1	macula densa, attenuates tubuloglomerular feedback, whereas angiotensin II enhances tubuloglomerular feedback. Thus the macula densa may release both vasoconstrictors (e.g., ATP and adenosine) and a vasodilator (e.g., NO) that oppose each other’s action at the level of the afferent arteriole. Production plus release of either vasoconstrictors or vasodilators ensures exquisite control over tubuloglomerular feedback.

1	Fig. 33.20 also illustrates the role of the macula densa in controlling renin secretion by granular cells of the afferent arteriole. This aspect of function of the juxtaglomerular apparatus is considered in detail in Because animals engage in many activities that can change arterial blood pressure, mechanisms that maintain RBF and GFR relatively constant despite changes in arterial pressure are highly desirable. If GFR and RBF were to rise or fall suddenly in proportion to changes in blood pressure, urinary excretion of fluid and solute would also change suddenly. Such changes in excretion of water and solutes without comparable changes in intake would alter fluid and electrolyte balance (the reason for which is discussed in ). Accordingly, autoregulation of GFR and RBF provides an effective means for uncoupling renal function from arterial pressure, and it ensures that fluid and solute excretion remain relatively constant.

1	Three points concerning autoregulation should be noted: 1. Autoregulation is absent when arterial pressure is less than 90 mm Hg.

1	• Fig. 33.20 Cellular mechanism whereby an increase in delivery of NaCl to the macula densa causes vasoconstriction of the afferent arteriole of the same nephron (i.e., tubuloglomerular feedback). An increase in GFR elevates [NaCl] in tubule fluid at the macula densa. This in turn enhances uptake of NaCl across the apical cell membrane of macula densa cells via the 1Na+-1K+-2Cl− (NKCC2) symporter, which leads to an increase in [ATP] and [adenosine] (ADO) release. ATP binds to P2X receptors and adenosine binds to adenosine A1 receptors in the plasma membrane of smooth muscle cells surrounding the afferent arteriole, both of which increase intracellular [Ca++]. The rise in [Ca++] induces vasoconstriction of the afferent arteriole, thereby returning GFR to normal levels. Note that ATP and adenosine also inhibit renin release by granular cells in the afferent arteriole. This too results from an increase in intracellular [Ca++] as a reflection of electrical coupling of the granular and

1	adenosine also inhibit renin release by granular cells in the afferent arteriole. This too results from an increase in intracellular [Ca++] as a reflection of electrical coupling of the granular and vascular smooth muscle (VSM) cells. When GFR is reduced, [NaCl] in tubule fluid falls, as does uptake of NaCl into macula densa cells. This in turn decreases release of ATP and adenosine by the macula densa, which decreases intracellular [Ca++] in smooth muscle cells and thereby increases GFR and stimulates renin release by granular cells. In addition a decrease in entry of NaCl into macula densa cells enhances production of PGE2, which also stimulates renin secretion by granular cells. As discussed in detail in , renin increases plasma [angiotensin II], a hormone that enhances NaCl and water retention by the kidneys. (Modified from Persson AEG et al. Acta Physiol Scand 2004;181:471.) 2.

1	Autoregulation is not perfect; RBF and GFR do change slightly as arterial blood pressure varies. 3. Despite autoregulation, RBF and GFR can be changed by several hormones and by alterations in sympathetic nerve activity that change in response to alterations in the extracellular fluid volume (ECFV) ( Table 33.1 Regulation of Renal Blood Flow and Glomerular Filtration Rate Table 33.1 ). As already discussed, the myogenic mechanism and tubuloglomerular feedback play key roles in maintaining RBF and GFR constant when blood pressure is greater that 90 mm Hg and ECFV is in the normal range. However, when the ECFV changes sympathetic nerves, angiotensin II, prostaglandins, NO, endothelin, bradykinin, ATP, and adenosine exert major control over RBF and GFR. Fig. 33.21 shows how changes in efferent and afferent arteriolar resistance, mediated by changes in the hormones listed in Table 33.1 , modulate both RBF and GFR.

1	Fig. 33.21 shows how changes in efferent and afferent arteriolar resistance, mediated by changes in the hormones listed in Table 33.1 , modulate both RBF and GFR. The afferent and efferent arterioles are innervated by sympathetic neurons; however, sympathetic tone is minimal when ECFV is normal (see ). When ECFV is reduced, sympathetic nerves release norepinephrine and dopamine, and circulating epinephrine (a catecholamine-like norepinephrine and dopamine) is secreted by the adrenal medulla. Norepinephrine and epinephrine cause vasoconstriction by Sympathetic nerves ↓ ECFV ↓↓ Angiotensin II ↓ ECFV ↓↓ Endothelin ↑ Stretch, A-II, bradykinin, epinephrine; ↓ ECFV ↓↓ Prostaglandins (PGE1, PGE2, PGI2) ↓ ECFV; ↑ shear stress, A-II No change/↑↑ Nitric oxide (NO) ↑ Shear stress, acetylcholine, histamine, bradykinin, ↑↑ ATP Bradykinin ↑ Prostaglandins, ↓ ACE ↑↑ Natriuretic peptides (ANP, BNP) ↑ ECFV ↑ No change

1	ATP Bradykinin ↑ Prostaglandins, ↓ ACE ↑↑ Natriuretic peptides (ANP, BNP) ↑ ECFV ↑ No change A-II, angiotensin II; ACE, angiotensin-converting enzyme; ECFV, extracellular fluid volume. Tubuloglomerular feedback (TGF) is absent in mice that do not express the adenosine receptor (A1). This underscores the importance of adenosine signaling in TGF. Studies have shown that when GFR increases and causes the concentration of NaCl in tubular fluid at the macula densa to rise, more NaCl enters cells via the 1Na+-1K+-2Cl− symporter (NKCC2) located in the apical plasma membrane (see Fig.

1	Fig. 33.20 ). Increased intracellular [NaCl] in turn stimulates release of ATP via ATP-conducting ion channels located in the basolateral membrane of macula densa cells. In addition, adenosine production is also enhanced. Adenosine binds to A1 receptors and ATP binds to P2X receptors located on the plasma membrane of smooth muscle cells in the afferent arteriole. Both hormones increase intracellular [Ca++], which causes vasoconstriction of the afferent artery and therefore a fall in GFR. Although adenosine is a vasodilator in most other vascular beds, it constricts the afferent arteriole in the kidney. binding to α1-adrenoceptors, which are located mainly in afferent arterioles. Activation of α1-adrenoceptors decreases RBF and GFR. Dehydration or strong emotional stimuli (e.g., fear, pain) also activate sympathetic nerves and reduce RBF and GFR.

1	Angiotensin II is produced systemically as well as locally within the kidneys. It constricts the afferent and efferent arterioles and decreases both RBF and GFR. Fig. 33.22 shows how norepinephrine, epinephrine, and angiotensin II act together to decrease RBF and GFR and thereby dThe efferent arteriole is more sensitive than the afferent arteriole to angiotensin II. Therefore with low concentrations of angiotensin II, constriction of the efferent arteriole predominates, and GFR increases and RBF decreases. However, with high concentrations of angiotensin II, constriction of both afferent and efferent arterioles occurs, and GFR and RBF both decrease (see Fig. 33.21

1	Fig. 33.21 Individuals with renal artery stenosis (narrowing of the lumen of the artery) caused by atherosclerosis, for example, often have elevated systemic arterial blood pressure mediated by the renin-angiotensin system. Pressure in the renal artery proximal to the stenosis is increased, but pressure distal to the stenosis is normal or reduced. Autoregulation is important in maintaining RBF, PGC, and GFR in the presence of this stenosis. Administration of drugs to lower systemic blood pressure also lowers the pressure distal to the stenosis; accordingly, RBF, PGC, and GFR fall. Significant hemorrhage decreases ECFV and arterial blood pressure and therefore activates sympathetic innervation of the kidneys via the baroreceptor reflex (

1	Significant hemorrhage decreases ECFV and arterial blood pressure and therefore activates sympathetic innervation of the kidneys via the baroreceptor reflex ( Fig. 33.22 ). Norepinephrine causes intense vasoconstriction of the afferent and efferent glomerular arterioles and thereby decreases both RBF and GFR. The rise in sympathetic activity also increases release of epinephrine and angiotensin II, which cause further vasoconstriction and a fall in RBF. The rise in vascular resistance of the kidneys and other vascular beds increases total peripheral resistance. The resulting tendency for blood pressure to increase (blood pressure = cardiac output × total peripheral resistance) offsets the tendency of blood pressure to decrease in response to hemorrhage. Hence this system works to preserve arterial pressure at the expense of maintaining normal RBF and GFR. increase blood pressure and ECFV (e.g., as would occur with hemorrhage).

1	increase blood pressure and ECFV (e.g., as would occur with hemorrhage). Prostaglandins do not play a major role in regulating RBF in healthy resting individuals. However, during • Fig. 33.21 Relationship between selective changes in resistance of either the afferent arteriole or the efferent arteriole on RBF and GFR. Constriction of either the afferent or efferent arteriole increases resistance, and according to

1	Eq. 33.11 (Q =ΔP/R), an increase in resistance (R) decreases flow (Q) (i.e., RBF). Dilation of either the afferent or afferent arteriole increases flow (i.e., RBF). Constriction of the afferent arteriole (A) decreases PGC because less of the arterial pressure is transmitted to the glomerulus, thereby reducing GFR. In contrast, constriction of the efferent arteriole (B) elevates PGC and thus increases GFR. Dilation of the efferent arteriole (C) decreases PGC and thus decreases GFR. Dilation of the afferent arteriole (D) increases PGC because more of the arterial pressure is transmitted to the glomerulus, thereby increasing GFR. (Modified from Rose BD, Rennke KG. Renal Pathophysiology: The Essentials. Baltimore: Williams & Wilkins; 1994.) pathophysiological conditions such as hemorrhage and reduced ECFV, prostaglandins (PGI2, PGE1, and PGE2) are produced locally within the kidneys and serve to increase RBF without changing GFR. Prostaglandins increase RBF by dampening the

1	hemorrhage and reduced ECFV, prostaglandins (PGI2, PGE1, and PGE2) are produced locally within the kidneys and serve to increase RBF without changing GFR. Prostaglandins increase RBF by dampening the vasoconstrictor effects of both sympathetic activation and angiotensin II. These effects are important because they prevent severe and potentially harmful vasoconstriction and renal ischemia. Synthesis of prostaglandins is stimulated by ECFV depletion and stress (e.g., surgery, anesthesia), angiotensin II, and sympathetic nerves. Nonsteroidal antiinflammatory drugs (NSAIDs), such as ibuprofen and naproxen, potently inhibit prostaglandin synthesis. Thus administration of these drugs during renal ischemia and hemorrhagic shock is contraindicated because, by blocking the production of prostaglandins, they decrease RBF and increase renal ischemia. Prostaglandins also play an increasingly important role in maintaining RBF and GFR as individuals age. Accordingly, NSAIDs can significantly reduce

1	they decrease RBF and increase renal ischemia. Prostaglandins also play an increasingly important role in maintaining RBF and GFR as individuals age. Accordingly, NSAIDs can significantly reduce RBF and GFR in the elderly.

1	NO, an endothelium-derived relaxing factor, is an important vasodilator under basal conditions, and it counteracts the vasoconstriction produced by angiotensin II and catecholamines. When blood flow increases, greater shear force acts on endothelial cells in the arterioles and increases production of NO. In addition a number of vasoactive hormones, including acetylcholine, histamine, bradykinin, and ATP, facilitate release of NO from endothelial cells. Increased production of NO causes dilation of the afferent and efferent arterioles in the kidneys. Whereas increased levels of NO decrease total peripheral resistance, inhibition of NO production increases total peripheral resistance.

1	Abnormal production of NO is observed in individuals with diabetes mellitus and hypertension. Excess renal NO production in diabetes may be responsible for glomerular hyperfiltration (i.e., increased GFR) and damage to the glomerulus, problems characteristic of this disease. Elevated NO levels increase glomerular capillary pressure secondary to a fall in resistance of the afferent arteriole. The ensuing hyperfiltration is thought to cause glomerular damage. The normal response to an increase in dietary salt intake includes stimulation of renal NO production, which prevents an increase in blood pressure. In some individuals, however, NO production may not increase appropriately in response to an elevation in salt intake, so blood pressure rises.

1	Endothelin is a potent vasoconstrictor secreted by endothelial cells of the renal vessels, mesangial cells, and distal tubular cells in response to angiotensin II, bradykinin, epinephrine, and endothelial shear stress. Endothelin causes profound vasoconstriction of the afferent and efferent arterioles and decreases GFR and RBF. Although this potent vasoconstrictor may not influence GFR and RBF in resting subjects, production of endothelin is elevated in a number of glomerular disease states (e.g., renal disease associated with diabetes mellitus). Kallikrein is a proteolytic enzyme produced in the kidneys. Kallikrein cleaves circulating kininogen to bradykinin, which is a vasodilator that acts by stimulating the release of NO and prostaglandins. Bradykinin increases RBF and GFR.

1	600 SECTION7Berne & Levy Physiology • Fig. 33.22 Pathway by which hemorrhage activates renal sympathetic nerve activity and stimulates production of angiotensin II. (Modified from Vander AJ. Renal Physiology. 2nd ed. New York: McGraw-Hill; 1980.) Hemorrhage ˜ Arterial blood pressure ˜ RBF and GFR (Intrarenal receptors) ° Renin secretion ° Plasma and renal renin ° Activity of renal sympathetic nerves ° Plasma and renal angiotensin II ° Constriction of renal arterioles Carotid sinus and aortic arch reflexes ° Tubular sodium and water reabsorption ˜ Renal excretion of sodium and water Adenosine is produced within the kidneys and causes vasoconstriction of the afferent arteriole, thereby reducing RBF and GFR. As previously mentioned, adenosine plays an important role in tubuloglomerular feedback.

1	Secretion of atrial natriuretic peptide (ANP) by the cardiac atria and brain natriuretic peptide (BNP) by the cardiac ventricle increases when ECFV is expanded and myocardial wall tension is increased. Both ANP and BNP dilate the afferent arteriole and constrict the efferent arteriole. Therefore ANP and BNP produce a modest increase in GFR with little change in RBF. Cells release ATP into the renal interstitial fluid. ATP can have bidirectional effects on both RBF and GFR. Under some conditions, ATP constricts the afferent arteriole, reduces RBF and GFR, and may play a role in tubuloglomerular feedback. Under other conditions, ATP may stimulate NO production and have directionally opposite effects, increasing both RBF and GFR. Administration of therapeutic doses of glucocorticoids increases GFR and RBF.

1	Administration of therapeutic doses of glucocorticoids increases GFR and RBF. Local release of histamine modulates RBF during the resting state and during inflammation and injury. Histamine decreases the resistance of the afferent and efferent arterioles and thereby increases RBF without elevating GFR. The proximal tubule produces the vasodilator substance dopamine. Dopamine has several actions within the kidney, such as increasing RBF and inhibiting renin secretion. • Fig. 33.23 Examples of the interactions of endothelial cells with smooth muscle and mesangial cells. ACE, angiotensin-converting enzyme; AI, angiotensin I; AII, angiotensin II. (Modified from Navar LG et al. Physiol Rev 1996;76:425.) Smooth muscle cell or mesangial cellVasodilation PGI2 PGE2 Nitric oxide Stretch Histamine Acetylcholine Bradykinin ATP A I A II Endothelin A C E Endothelial cell Vasoconstriction Finally, as illustrated in

1	Fig. 33.23 , endothelial cells play an important role in regulating the resistance of the renal afferent and efferent arterioles by producing a number of paracrine hormones, including NO, prostacyclin (PGI2), endothelin, and angiotensin II. These hormones regulate contraction or relaxation of smooth muscle cells in afferent and efferent arterioles and mesangial cells. Shear stress, acetylcholine, histamine, bradykinin, and ATP stimulate production of NO, which increases GFR and RBF. Angiotensin-converting enzyme (ACE), located on the surface of endothelial cells lining the afferent arteriole and glomerular capillaries, converts angiotensin I to angiotensin II, which decreases GFR and RBF. Angiotensin II is also produced locally by granular cells in the afferent arteriole and by proximal tubular cells. PGI2 and PGE2 release by endothelial cells is stimulated by both sympathetic nerve activity and angiotensin II, resulting in increased GFR and RBF. Finally, endothelin release from

1	tubular cells. PGI2 and PGE2 release by endothelial cells is stimulated by both sympathetic nerve activity and angiotensin II, resulting in increased GFR and RBF. Finally, endothelin release from endothelial cells decreases both RBF and GFR.

1	ACE proteolytically inactivates the vasodilatory hormone bradykinin and converts angiotensin I, an inactive hormone, to angiotensin II, an active vasoconstrictive hormone. Thus ACE increases angiotensin II levels and decreases bradykinin levels. ACE inhibitors (e.g., lisinopril, enalapril, and captopril) are used clinically to reduce systemic blood pressure in patients with hypertension by decreasing angiotensin II levels and elevating bradykinin levels. Both effects lower systemic vascular resistance, reduce blood pressure, and decrease renal vascular resistance, thereby increasing GFR and RBF. Angiotensin II receptor antagonists (e.g., losartan) are also used to treat hypertension. As their name suggests, they block the binding of angiotensin II to the angiotensin II receptor (AT1). These antagonists block the vasoconstrictor effects of angiotensin II on the afferent arteriole; thus they increase RBF and GFR. In contrast to ACE inhibitors, angiotensin II receptor antagonists do not

1	antagonists block the vasoconstrictor effects of angiotensin II on the afferent arteriole; thus they increase RBF and GFR. In contrast to ACE inhibitors, angiotensin II receptor antagonists do not inhibit kinin metabolism (e.g., bradykinin).

1	1. The first step in urine formation is passive movement of a plasma ultrafiltrate from the glomerular capillaries into Bowman’s space. The term ultrafiltration refers to passive movement of a plasma-like fluid that has a very low concentration of proteins from the glomerular capillaries into Bowman’s space. The endothelial cells of glomerular capillaries are covered by a basement membrane that is surrounded by podocytes. The capillary endothelium, basement membrane, and foot processes of podocytes form the so-called filtration barrier. 2. The juxtaglomerular apparatus is one component of an important feedback mechanism (i.e., tubuloglomerular feedback) that regulates RBF and GFR. The structures that make up the juxtaglomerular apparatus include the macula densa, extraglomerular mesangial cells, and reninand angiotensin II–producing granular cells. 3. Clinically, GFR is frequently estimated using measures of plasma [creatinine] or CrCl. 4.

1	3. Clinically, GFR is frequently estimated using measures of plasma [creatinine] or CrCl. 4. Autoregulation allows GFR and RBF to remain constant despite changes in arterial blood pressure between 90 and 180 mm Hg. When ECFV is altered, sympathetic nerves, catecholamines, angiotensin II, prostaglandins, NO, endothelin, natriuretic peptides, bradykinin, and adenosine exert substantial control over GFR and RBF. Patel A. The primary cilium calcium channels and their role in flow sensing. Pflugers Arch. 2015;467:157-165. Pollak MR, et al. The glomerulus: the sphere of influence. Clin J Am Soc Nephrol. 2014;9:1461-1469. Schlondorff J. How many Achilles’ heels does a podocyte have? An update on podocyte biology. Nephrol Dial Transplant. 2015;30: 1091-1097. Schnermann J. Concurrent activation of multiple vasoactive signaling pathways in vasoconstriction caused by tubuloglomerular feedback: a quantitative assessment. Annu Rev Physiol. 2015;77:301-322.

1	Schnermann J. Concurrent activation of multiple vasoactive signaling pathways in vasoconstriction caused by tubuloglomerular feedback: a quantitative assessment. Annu Rev Physiol. 2015;77:301-322. Taylor AE, Moore TM. Capillary fluid exchange. Adv Physiol Educ. 1999;22:s203-s210. Arendshorst WJ, Navar LG. Renal circulation and glomerular hemodynamics. In: Schrier RW, et al., eds. Diseases of the Kidney and Urinary Tract. 9th ed. Philadelphia: Lippincott Williams & Wilkins; 2012. Dworkin LD, et al. The renal circulations. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012. Kriz W, Kaissling B. Structural organization of the mammalian kidney. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2012.

1	Lafayette RA, et al. Laboratory evaluation of renal function. In: Schrier RW, et al., eds. Diseases of the Kidney and Urinary Tract. 9th ed. Philadelphia: Lippincott Williams & Wilkins; 2012. Madsen K, et al. Anatomy of the kidney. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012. Upon completion of this chapter the student should be able to answer the following questions: 1. What three processes are involved in the production of urine? 2. What is the composition of “normal” urine? 3. What transport mechanisms are responsible for NaCl reabsorption by the nephron? Where are they located along the nephron? 4. How is water reabsorption “coupled” to NaCl reabsorption in the proximal tubule? 5. Why are solutes but not water reabsorbed by the thick ascending limb of Henle’s loop? 6. What transport mechanisms are involved in secretion of organic anions and cations? What is the physiological relevance of these transport processes?

1	6. What transport mechanisms are involved in secretion of organic anions and cations? What is the physiological relevance of these transport processes? 7. What is glomerulotubular balance, and what is its physiological importance? 8. What are the major hormones that regulate NaCl and water reabsorption by the kidneys? What is the nephron site of action of each hormone? 9. What is the aldosterone paradox? he formation of urine involves three basic processes: (1) ultrafiltration of plasma by the glomerulus, (2) reabsorption of water and solutes from the ultra-filtrate, and (3) secretion of selected solutes into tubular fluid. Although an average of 115 to 180 L/day in women and 130 to 200 L/day in men of essentially protein-free fluid is filtered by the human glomeruli each day, less than 1% of the filtered water and sodium chloride (NaCl) and variable amounts of other solutes are typically excreted in urine (

1	Table 34.1 ). By the processes of reabsorption and secretion, the renal tubules determine the volume and composition of urine ( Table 34.2 ), which in turn allows the 1Normal glomerular filtration rate (GFR) averages 115–180 L/day in women and 130–200 L/day in men. Thus the volume of the ultrafiltrate represents a volume that is approximately 10 times that of the extracellular fluid volume (ECFV). For simplicity, we assume throughout the remainder of this section that GFR is 180 L/day. kidneys to precisely control the volume, osmolality, composition, and pH of the extracellular and intracellular fluid compartments. Transport proteins in cell membranes of the nephron mediate reabsorption and secretion of solutes and water in the kidneys. Approximately 5% to 10% of all human genes code for transport proteins, and genetic and acquired defects in transport proteins are the cause of many kidney diseases (

1	Table 34.3 ). In addition, numerous transport proteins are important drug targets. This chapter discusses NaCl and water reabsorption, transport of organic anions and cations, the transport proteins involved in solute and water transport, and some of the factors and hormones that regulate NaCl transport. Details on acid-base transport and on K+ , Ca++ , and inorganic phosphate (Pi) transport and their regulation are provided in through 37. Solute and Water Reabsorption Along the Nephron The general principles of solute and water transport across epithelial cells were discussed in Quantitatively, reabsorption of NaCl and water represent the major function of nephrons. Approximately 25,000 mEq/day of Na+ and 179 L/day of water are reabsorbed by the renal tubules (see Table 34.1).

1	Table 34.1). In addition, renal transport of many other important solutes is linked either directly or indirectly to reabsorption of Na+ . In the following sections, the NaCl and water transport processes of each nephron segment and their regulation by hormones and other factors are presented. The proximal tubule reabsorbs approximately 67% of filtered water, Na+ , Cl− , K+ , and most other solutes. In addition the proximal tubule reabsorbs virtually all the glucose and amino acids filtered by the glomerulus, as well as most of the HCO3 − . The key element in proximal tubule reabsorption is Na+,K+-ATPase in the basolateral membrane. Reabsorption of every substance, including water, is linked in some manner to the operation of Na+,K+-ATPase.

1	WaterL/day1801.5178.599.2Na+ mEq/day25,20015025,05099.4K+ mEq/day72010062086.1Ca++ mEq/day5401053098.2HCO3− mEq/day43202431899.9+ Cl− mEq/day18,00015017,85099.2Glucosemmol/day8000800100.0Ureag/day56282850.0 aThe filtered amount of any substance is calculated by multiplying the concentration of that substance in the ultrafiltrate by the glomerular filtration rate (GFR); for example, the filtered load of Na+ is calculated as [Na+]ultrafiltrate (140mEq/L) × GFR (180L/day) = 25,200mEq/day. The composition and volume of urine can vary widely in the healthy state. These values represent average ranges. Normal water excretion typically ranges between 0.5 and 1.5L/day.Data from Valtin HV. Renal Physiology. 2nd ed. Boston: Little, Brown; 1983.

1	Na+ is reabsorbed by different mechanisms in the first and the second halves of the proximal tubule. In the first half of the proximal tubule, Na+ is reabsorbed primarily with bicarbonate (HCO3 −) and a number of other solutes (e.g., glucose, amino acids, Pi, lactate). In contrast, in the second half, Na+ is reabsorbed mainly with Cl− . This disparity is mediated by differences in the Na+ transport systems in the first and second halves of the proximal tubule and by differences in the composition of tubular fluid at these Bilirubin50–130mEq/L20–70mEq/L30–50mEq/L5–12mEq/L2–18mEq/L50–130mEq/L20–40mEq/L200–400mmol/L6–20mmol/L5.0–7.0500–800mOsm/kgH2O0000000 sites. In absolute terms the first half of the proximal tubule reabsorbs significantly more Na+ than the second half.

1	In the first half of the proximal tubule, Na+ uptake into the cell is coupled with either H+ or organic solutes, includ ing glucose (Fig. 34.1 ). Specific transport proteins mediate entry of Na+ into the cell across the apical membrane. For example, the Na+/H+ antiporter, NHE3, (see Fig. 34.1A ) couples entry of Na+ with extrusion of H+ from the cell. H+ secretion results in reabsorption of sodium bicarbonate (NaHCO3) (see ). Na+ also enters proximal tubule cells via several symporter mechanisms, including Na+/glucose (SGLT2), Na+/amino acid, Na+/Pi, and Na+/ lactate (see

1	Fig. 34.1B ). The glucose and other organic solutes that enter the cell with Na+ leave the cell across the basolateral membrane via passive transport mechanisms. Any Na+ that enters the cell across the apical membrane leaves the cell and enters the blood via Na+,K+-ATPase. Thus reabsorption of Na+ in the first half of the proximal tubule is coupled to that of HCO3 − and a number of organic molecules, and this generates a negative transepithelial voltage across the proximal tubule that provides the driving force for the paracellular reabsorption of Cl− . Reabsorption of many organic molecules, including glucose and lactate, is so avid they are almost completely removed from the tubular fluid in the first half of the proximal tubule (

1	Fig. 34.2 ). Reabsorption of NaHCO3 and Na+–organic solutes across the proximal tubule establishes a transtubular osmotic gradient (i.e., the osmolality of the interstitial fluid bathing the basolateral side of the cells is a few mOsm/L higher than the osmolality of tubule fluid) that provides the driving force for the passive reabsorption of water by osmosis. Because more water than Cl− is reabsorbed in the first half of the proximal tubule, the [Cl−] in tubular fluid rises along the length of the proximal tubule (see Fig. 34.2 In the second half of the proximal tubule, Na+ reabsorption is largely accompanied by Cl− reabsorption via both transcellular and paracellular pathways ( Fig. 34.3 ). Na+ is primarily reabsorbed with Cl− rather than organic solutes or HCO3 − as the accompanying anion because the Na+ transport mechanisms in the second half of the proximal tubule

1	Fig. 34.3 ). Na+ is primarily reabsorbed with Cl− rather than organic solutes or HCO3 − as the accompanying anion because the Na+ transport mechanisms in the second half of the proximal tubule There are over 300 different solute transporter genes that form the so-called SLC (solute carrier) family of genes.AD, autosomal dominant; AR, autosomal recessive; TAL, thick ascending limb of Henle’s loop; XLR, X-linked recessive.Modified from Nachman RH, Glassock RJ. NephSAP 2010;9(3). differ from those in the first half, and because the tubular fluid that enters the second half contains very little glucose or amino acids. In addition the high [Cl−] (140 mEq/L)

1	differ from those in the first half, and because the tubular fluid that enters the second half contains very little glucose or amino acids. In addition the high [Cl−] (140 mEq/L) Fanconi syndrome, a renal disease that is either hereditary in tubule fluid, which is due to preferential reabsorption − or acquired, results from an impaired ability of the proximal of Na+ with HCO3 and organic solutes in the first half − tubule to reabsorb HCO3, Pi, amino acids, glucose, and of the proximal tubule, facilitates reabsorption of Cl− low-molecular-weight proteins. Because other downstream with Na+ . nephron segments cannot reabsorb these solutes and The mechanism of transcellular Na+ reabsorption in the protein, Fanconi syndrome results in increased urinary excretion of HCO3− , amino acids, glucose, Pi, and low- second half of the proximal tubule is shown in Fig. 34.3 molecular-weight proteins.

1	Na+ enters the cell across the luminal membrane primarily via the parallel operation of a Na+/H+ antiporter (NHE3) •Fig. 34.1 Na+ transport processes in the first half of the proximal tubule. These transport mechanisms are present in all cells in the first half of the proximal tubule but are separated into different cells to simplify the discussion. A, Operation of the Na+/H+ antiporter (NHE3) in the apical membrane and the Na+,K+-ATPase and HCO3− transporters, including the Na+/HCO3− symporter (NBC1; see also ) in the basolateral membrane mediates reabsorption of NaHCO3. Carbon dioxide and water combine inside the cells to form H+ and HCO3− in a reaction facilitated by the enzyme carbonic anhydrase (CA). B, Operation of the Na+/glucose symporter (SGLT2) in the apical membrane, in conjunction with Na+,K+-ATPase and the glucose transporter (GLUT2) in the basolateral membrane, mediates Na+-glucose reabsorption. Inactivating mutations in the GLUT2 gene lead to decreased glucose

1	conjunction with Na+,K+-ATPase and the glucose transporter (GLUT2) in the basolateral membrane, mediates Na+-glucose reabsorption. Inactivating mutations in the GLUT2 gene lead to decreased glucose reabsorption in the proximal tubule and glucosuria (i.e., glucose in the urine). Though not shown, Na+ reabsorption is also coupled with other solutes, including amino acids, Pi, and lactate. Reabsorption of these solutes is mediated by the Na+/amino acid, Na+/Pi, and Na+/lactate symporters, respectively, located in the apical membrane and the Na+,K+-ATPase, amino acid, Pi, and lactate transporters, respectively, located in the basolateral membrane. Three classes of amino acid transporters have been identified in the proximal tubule: two that transport Na+ in conjunction with either acidic or basic amino acids and one that does not require Na+ and transports basic amino acids.

1	•Fig. 34.2 Concentration of solutes in tubule fluid as a function of length along the proximal tubule. [TF] is the concentration of the substance in tubular fluid; [P] is the concentration of the substance in plasma. Values above 100 indicate that relatively less of the solute than water is reabsorbed, and values below 100 indicate that relatively more of the substance than water is reabsorbed.

1	•Fig. 34.3 Na+ transport processes in the second half of the proximal tubule. Na+ and Cl− enter the cell across the apical membrane through the operation of parallel Na+/H+ (NHE3) and Cl−-base (e.g., formate, oxalate, and bicarbonate) antiporters (CFEX). More than one Cl−-base antiporter is involved in this process, but only one is depicted. The secreted H+ and base combine in the tubular fluid to form an H-base complex that can recycle across the plasma membrane. Accumulation of the H-base complex in tubular fluid establishes a H-base concentration gradient that favors H-base recycling across the apical plasma membrane into the cell. Inside the cell, H+ and the base dissociate and recycle back across the apical plasma membrane. The net result is uptake of NaCl across the apical membrane. The base may be hydroxide ions (OH−), formate (HCO2−), oxalate, HCO3− , or sulfate. The positive transepithelial voltage in the lumen, indicated by the plus sign inside the circle in the tubular

1	The base may be hydroxide ions (OH−), formate (HCO2−), oxalate, HCO3− , or sulfate. The positive transepithelial voltage in the lumen, indicated by the plus sign inside the circle in the tubular lumen, is generated by diffusion of Cl− (lumen to blood) across the tight junction. The high [Cl−] of tubular fluid provides the driving force for diffusion of Cl− . Some glucose is also reabsorbed in the second half of the proximal tubule by a mechanism similar to that described in the first half of the proximal tubule, except that the Na+/glucose symporter (SGLT1 gene) transports 2Na+ with one glucose and has higher affinity and lower capacity than the Na+/glucose symporter in the first part of the proximal tubule, depicted in

1	Fig. 34.1 . In addition, glucose exits the cell across the basolateral membrane via GLUT1 rather than via GLUT2 as in the first part of the proximal tubule (not shown). KCC, KCl symporter. aAtrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) inhibit vasopressin (AVP)-stimulated water permeability. and one or more Cl−-base antiporters (e.g., CFEX). Because the secreted H+ and base combine in the tubular fluid and reenter the cell, operation of the Na+/H+ and Cl−-base antiporters is equivalent to uptake of NaCl from tubular fluid into the cell. Na+ leaves the cell via Na+,K+-ATPase, and Cl− leaves the cell and enters the blood via a K+/ Cl− symporter (KCC) and a Cl− channel in the basolateral membrane.

1	Some NaCl is also reabsorbed across the second half of the proximal tubule via a paracellular route. Paracellular NaCl reabsorption occurs because the rise in [Cl−] in tubule fluid in the first half of the proximal tubule creates a [Cl−] gradient (140 mEq/L in the tubule lumen and 105 mEq/L in the interstitium). This concentration gradient favors diffusion of Cl− from the tubular lumen across the tight junctions into the lateral intercellular space. Movement of the negatively charged Cl− results in the tubular fluid becoming positively charged relative to blood. This positive transepithelial voltage causes diffusion of positively charged Na+ out of the tubular fluid across the tight junction into blood. Thus in the second half of the proximal tubule, some Na+ and Cl− are reabsorbed across the tight junctions via passive diffusion.

1	In summary, reabsorption of Na+ and Cl− in the proximal tubule occurs via both paracellular and transcellular pathways. Approximately 67% of the NaCl filtered each day is reabsorbed in the proximal tubule. Of this amount, two-thirds moves across the transcellular pathway, whereas the remaining one-third moves across the paracellular pathway ( Table 34.4 The proximal tubule reabsorbs 67% of the filtered water ( Table 34.5 ). The driving force for water reabsorption is a transtubular osmotic gradient established by reabsorption of solute (e.g., NaCl, Na+-glucose). Reabsorption of Na+ along with organic solutes, HCO3 − , and Cl− from tubular fluid into the lateral intercellular spaces reduces the osmolality of the tubular fluid and increases the osmolality of the lateral intercellular space. The osmotic gradient across the proximal tubule established by these transport processes is only a few mOsm/L (

1	Fig. 34.4 ). Because the proximal tubule is highly permeable to water, primarily owing to expression of aquaporin water channels (AQP1) in the apical and basolateral membranes, water is reabsorbed across cells by osmosis. In addition the tight junctions in the proximal tubule are also water permeable, so some water is also reabsorbed across the paracellular pathway between proximal tubular cells. Accumulation of fluid and solutes within the lateral intercellular space increases hydrostatic pressure in this compartment. The increased hydrostatic pressure forces fluid and solutes into the capillaries. 2In addition, protein oncotic pressure in the peritubular capillaries (πpc) is elevated because of the process of glomerular filtration (see ). The elevated πpc facilitates uptake of fluid and solute into the capillary.

1	•Fig. 34.4 Routes of reabsorption of water and solute across the proximal tubule. Transport of solutes, including Na+ , Cl− , and organic solutes, into the lateral intercellular space increases the osmolality of this compartment, which establishes the driving force for osmotic reabsorption of water across the proximal tubule. This occurs because some Na+,K+-ATPase and some transporters of organic solutes, HCO3− , and Cl− are located on the lateral cell membranes and deposit these solutes between cells. Furthermore, some NaCl also enters the lateral intercellular space via diffusion across the tight junction (i.e., paracellular pathway). An important consequence of osmotic water flow across the transcellular and paracellular pathways in the proximal tubule is that some solutes, especially K+ and Ca++ , are entrained in the reabsorbed fluid and thereby reabsorbed by the process of solvent drag.

1	reabsorption in the proximal tubule. The reabsorbed fluid is slightly hyperosmotic relative to plasma. However, this difference in osmolality is so small it is commonly said that proximal tubule reabsorption is isosmotic (i.e., ≈67% of both the filtered load of solute and water are reabsorbed). Indeed, there is little difference in the osmolality of tubular fluid at the start and end of the proximal tubule. An important consequence of osmotic water flow across the proximal tubule is that some solutes, especially K+ and Ca++ , are entrained in the reabsorbed fluid and thereby reabsorbed by the process of solvent drag (see

1	Fig. 34.4 ). Reabsorption of virtually all organic solutes, Cl− and other ions, and water is coupled to Na+ reabsorption. Therefore changes in Na+ reabsorption influence reabsorption of water and other solutes by the proximal tubule. This point will be discussed later, notably in , and is especially relevant during volume depletion when increased Na+ reabsorption by the proximal tubule is accompanied by a parallel increase in HCO3 − reabsorption, which can contribute to metabolic alkalosis (i.e., volume contraction alkalosis).

1	Proteins filtered by the glomerulus are reabsorbed in the proximal tubule. As mentioned previously, peptide hormones, small proteins, and small amounts of larger proteins such as albumin are filtered by the glomerulus. Overall, only a small percentage of proteins cross the glomerulus and enter Bowman’s space (i.e., the concentration of proteins in the glomerular ultrafiltrate is only ≈ 40 mg/L). However, the total amount of protein filtered per day is significant because the glomerular filtration rate (GFR) is so high: Equation 34.1 Filtered protein = GFR ×[Protein] in the ultrafiltrate = 7200 mg/day, or 7 2. g/day

1	Filtered proteins are reabsorbed in the proximal tubule by endocytosis either as intact proteins or after being partially degraded by enzymes on the surface of proximal tubule cells. Once the proteins and peptides are inside the cell, enzymes digest them into their constituent amino acids, which then leave the cell across the basolateral membrane by transport proteins and are returned to the blood. Normally this mechanism reabsorbs virtually all the proteins filtered, and hence the urine is essentially protein free. However, because the mechanism is easily saturated, an increase in filtered proteins can result in proteinuria (appearance of protein in urine). Disruption of the glomerular filtration barrier to proteins increases the filtration of proteins and 610 SECTION7Berne & Levy Physiology • BOX 34.2 Some Organic Cations Secreted by Proximal Tubule • BOX 34.1 Some Organic Anions Secreted by Proximal Tubule cAMP, cGMPBile saltsHippuratesOxalateProstaglandins: PGE2, PGF2α

1	Levy Physiology • BOX 34.2 Some Organic Cations Secreted by Proximal Tubule • BOX 34.1 Some Organic Anions Secreted by Proximal Tubule cAMP, cGMPBile saltsHippuratesOxalateProstaglandins: PGE2, PGF2α UrateVitamins: ascorbate, folate

1	AcetazolamideAcyclovirAmoxicillinCaptoprilChlorothiazideFurosemideLosartanPenicillinProbenecidSalicylate (aspirin)HydrochlorothiazideSimvastatinBumetanideNonsteroidal antiinflammatory drugs (NSAIDs): indomethacin cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate. results in proteinuria, which is frequently seen with kidney disease. Secretion of Organic Anions and Organic Cations Cells of the proximal tubule also secrete organic anions and organic cations into the tubule fluid. Secretion of organic anions and cations by the proximal tubule plays a key role in regulating the plasma levels of xenobiotics (e.g., a variety of antibiotics, diuretics, statins, antivirals, antineoplastics, immunosuppressants, neurotransmitters, and nonsteroidal antiinflammatory drugs [NSAIDs]) and toxic compounds derived from endogenous and exogenous sources. Many of the organic anions and cations (

1	Boxes 34.1 34.2 ) secreted by the proximal tubule are end products of metabolism that circulate in plasma. Many of these organic compounds are bound to plasma proteins and thus are not readily filtered. Therefore only a small fraction of these potentially toxic substances are eliminated from the body by excretion resulting from filtration alone. Thus secretion of organic anions and cations, including many toxins from the peritubular capillary into the tubular fluid, promote elimination of these compounds from plasma entering the kidneys. Hence these substances are removed from plasma by both filtration and secretion. It is important to note that when kidney function is reduced by disease, urinary excretion of organic anions and cations is severely reduced, which can lead to increased plasma levels of xenobiotics and potentially toxic accumulation of organic anions and cations.

1	Water channels called aquaporins (AQPs) mediate transcellular reabsorption of water across many nephron segments. To date, 13 aquaporins have been identified. The AQP family is divided into two groups based on their permeability characteristics. One group (aquaporins) is permeable to water (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, AQP8, AQP11, and AQP12). The other group (aquaglyceroporins) is permeable to water and small solutes, especially glycerol (AQP3, AQP7, AQP9, AQP10). Aquaporins form tetramers in the plasma membrane of cells, with each subunit forming a water channel. In the kidneys, AQP1 is expressed in the apical and basolateral membranes of the proximal tubule and in portions of the descending thin limb of Henle’s loop. The importance of AQP1 in renal water reabsorption is underscored by studies in which the AQP1 gene was “knocked out” in mice. These mice exhibit increased urine output (polyuria) and reduced ability to concentrate urine. In addition the osmotic water

1	underscored by studies in which the AQP1 gene was “knocked out” in mice. These mice exhibit increased urine output (polyuria) and reduced ability to concentrate urine. In addition the osmotic water permeability of the proximal tubule is fivefold less in mice lacking APQ1 than in normal mice. AQP7 and AQP8 are also expressed in the proximal tubule. AQP2 is expressed in the apical plasma membrane of principal cells in the collecting duct, and its abundance in the membrane is regulated by arginine vasopressin (AVP) (see ). AQP3 and AQP4 are expressed in the basolateral membrane of principal cells in the collecting duct, and mice deficient in these AQPs (i.e., AQP3 and AQP4 knockout mice) have defects in the ability to concentrate urine (see ). AQPs are also expressed in many other organs in the body, including the lung, eye, skin, secretory glands, and brain, where they play key physiological roles. For example, AQP4 is expressed in cells that form the blood-brain barrier. Knockout of

1	the body, including the lung, eye, skin, secretory glands, and brain, where they play key physiological roles. For example, AQP4 is expressed in cells that form the blood-brain barrier. Knockout of AQP4 affects the water permeability of the blood-brain barrier such that brain edema is reduced in AQP4-deficient mice after acute water loading and subsequent development of hyponatremia.

1	ATP ATP ADP ˜-KG NaDC3 OAT 1,2,3 OAT4 OA– ˜-KG MRP2/4 BCRP K+ •Fig. 34.5 Secretion of organic anion (OA−) across the proximal tubule. OA−s enter the cell across the basolateral membrane by one of three OA−/α-ketoglutarate (α-KG) antiporter mechanisms (organic anion transporters, OAT1, OAT2, OAT3). Uptake of α-KG into the cell against its chemical concentration gradient is driven by movement of Na+ into the cell via the Na+-dicarboxylate transporter (NaDC3). The [Na+] inside the cell is low because of the Na+,K+-ATPase in the basolateral membrane, which transports Na+ out of the cell in exchange for K+ . The α-KG recycles across the basolateral membrane on the OATs in exchange for OA− . OA−s leave the cell across the apical membrane by multidrug drug resistance proteins (MRP2 and 4), and by breast cancer resistance protein (BCRP), which require ATP. OAT4 in the apical membrane reabsorbs urate, an organic anion.

1	The endocytosis of proteins by the proximal tubule is mediated by apical membrane proteins that specifically bind proteins and peptides in tubule fluid. These receptors, called multiligand endocytic receptors, can bind a wide range of peptides and proteins and thereby mediate their endocytosis. Megalin and cubilin mediate protein and peptide endocytosis in the proximal tubule. Both are glycoproteins, with megalin being a member of the low-density lipoprotein receptor gene family. Fig. 34.5 illustrates the mechanisms of organic anion (OA−) transport across the proximal tubule. These secretory pathways have maximum transport rates, low specificity (i.e., they transport many OA−s), and are responsible for secretion of the OA−s listed in Box 34.1.

1	Box 34.1. OA−s are taken up into the cell across the basolateral membrane against their chemical gradient in exchange for α-ketoglutarate (α-KG) via several OA−/α-KG antiporters, including OAT1, OAT2, and OAT3. α-KG accumulates inside the cells via metabolism of glutamate and by a Na+/α-KG symporter (i.e., the Na+/dicarboxylate transporter [NaDC3]) also present in the basolateral membrane. Thus uptake of OA− into the cell against an electrochemical gradient is coupled to the

1	Urinalysis is an important and routine tool for detection of kidney disease. A thorough analysis of urine includes macroscopic, microscopic, and biochemical assessments. This is performed by visual assessment of the urine, microscopic examination of urinary sediment, and biochemical evaluation of urinary composition using dipstick reagent strips. The dipstick test is both inexpensive and fast (i.e., <5 minutes) and tests urine for both pH and the presence of many substances (e.g., bilirubin, blood, glucose, ketones, protein). It is normal to find trace amounts of protein in urine, particularly concentrated urine. Urinary proteins are derived from two principal sources: (1) filtration exceeding the reabsorptive capacity of the proximal tubule and (2) synthesis and secretion of Tamm-Horsfall glycoprotein by the thick ascending limb of Henle’s loop. Because the mechanism for protein reabsorption is “upstream” of the thick ascending limb (i.e., in the proximal tubule), the secreted

1	glycoprotein by the thick ascending limb of Henle’s loop. Because the mechanism for protein reabsorption is “upstream” of the thick ascending limb (i.e., in the proximal tubule), the secreted Tamm-Horsfall glycoprotein appears in urine. However, proteinuria in greater than trace amounts is often indicative of renal disease.

1	exit of α-KG out of the cell, down its chemical gradient generated by the Na+/α-KG symporter mechanism. The exit of OA−s across the luminal membrane into the tubular fluid are mediated by multidrug resistance proteins 2 and 4 (MRP2/4) and breast cancer resistance protein 1 (BCRP), which require adenosine triphosphate (ATP) for their operation. Recent studies reveal that OAT4 mediates reabsorption of the organic anion urate, the end product of purine catabolism, by the proximal tubule (see

1	Fig. 34.5 •Fig. 34.6 Organic cation (OC+) secretion across the proximal tubule. OC+s enter the cell across the basolateral membrane primarily by OCT2. Uptake of OC+s into the cell against their chemical concentration gradient is driven by the cell-negative potential difference. OC+s leave the cell across the apical membrane in exchange with H+by electrically neutral multidrug and toxin transporters (MATE1 and MATE2-K) and by multidrug resistance protein (MDR1), which requires ATP. ATP OCT2MATE MDR1 Na+ OC+ OC+ H+ K+ Blood Tubular fluid OC+ ATP ADP

1	Fig. 34.6 illustrates the mechanism of organic cation (OC+) transport across the proximal tubule. Organic cations, including xenobiotics such as the antidiabetic agent metformin, the antiviral agent lamivudine, and the anticancer drug oxaliplatin, and many important monoamine neurotransmitters including dopamine, epinephrine, histamine, and norepinephrine are secreted by the proximal tubule. Organic cations are taken up into the cell across the basolateral membrane, primarily by the organic cation transporter 2 (OCT2). Uptake of organic cations is driven by the magnitude of the cell-negative potential difference across the basolateral membrane. Organic cation transport across the luminal membrane into the tubular fluid, which is the rate-limiting step in secretion, is mediated primarily by electroneutral multidrug and toxin extrusion transporters (MATEs) and MDR1 (also known as P-glycoprotein), which requires ATP for its operation. These transport mechanisms are nonspecific, and

1	by electroneutral multidrug and toxin extrusion transporters (MATEs) and MDR1 (also known as P-glycoprotein), which requires ATP for its operation. These transport mechanisms are nonspecific, and several organic cations usually compete for secretion via a given transport pathway.

1	Henle’s loop reabsorbs approximately 25% of the filtered NaCl and 15% of the filtered water. Reabsorption of NaCl

1	Because many organic anions compete for the same secretory pathways, elevated plasma levels of one transported anion often inhibit secretion of the others. For example, infusing p-aminohippuric acid (PAH) can reduce secretion of penicillin by the proximal tubule. Because the kidneys are responsible for eliminating penicillin, infusion of PAH into individuals receiving penicillin reduces penicillin excretion and thereby extends its biological half-life. In World War II, when penicillin was in short supply, hippurates were given with penicillin to extend its therapeutic effect. Similar competition is observed for organic cation secretion by the proximal tubule, and elevated plasma levels of one transported cation species can inhibit secretion of the other competing cations. For example, the histamine H2antagonist cimetidine used to treat gastric ulcers is secreted via organic cation transport mechanisms in the proximal tubule. If cimetidine is given to patients receiving procainamide (a

1	histamine H2antagonist cimetidine used to treat gastric ulcers is secreted via organic cation transport mechanisms in the proximal tubule. If cimetidine is given to patients receiving procainamide (a drug used to treat cardiac arrhythmias), cimetidine reduces urinary excretion of procainamide (also an organic cation) by direct competition for a common secretory pathway. As a consequence, coadministration of cationic drugs competing for the same pathway can increase the plasma concentration of both drugs to levels much higher than those observed when the drugs are given alone. This effect can lead to drug toxicity.

1	•Fig. 34.7 Transport mechanisms for NaCl reabsorption in the thick ascending limb of the loop of Henle. The positive voltage in the lumen plays a major role in driving the passive paracellular reabsorption of cations. Because the apical membrane is conductive primarily to K+ , the apical membrane voltage is more negative than the basolateral membrane voltage, which is conductive to K+ and Cl− , thereby resulting in a lumen positive transepithelial potential. Mutations in the apical membrane K+ channel (ROMK), the apical membrane 1Na+/1K+/2Cl− symporter (NKCC2), or the basolateral Cl− channel (ClCNKB) cause Bartter syndrome (see the clinical box on Bartter syndrome). CA, carbonic anhydrase.

1	in the loop of Henle occurs in both the thin and thick ascending limbs, whereas the descending thin limb does not reabsorb NaCl. In contrast, water reabsorption mediated by AQP1 water channels is exclusively restricted to the descending thin limb, whereas the ascending limb is impermeable to water. In addition, divalent cations (e.g., Ca++ and Mg++) and HCO3 − are also reabsorbed in the loop of Henle (see for more details). The thin ascending limb reabsorbs NaCl by a passive mechanism. Reabsorption of water, but not NaCl, in the descending thin limb increases [NaCl] in the tubule fluid entering the ascending thin limb. As the NaCl-rich fluid moves toward the cortex, NaCl diffuses out of the tubule lumen across the ascending thin limb and into the medullary interstitial fluid, down a concentration gradient directed from the tubule fluid to the interstitium (see for details).

1	The key element in reabsorption of solute by the thick ascending limb is Na+,K+-ATPase in the basolateral membrane (Fig. 34.7 ). As with reabsorption in the proximal tubule, reabsorption of every solute by the thick ascending limb is linked to Na+,K+-ATPase activity. This transporter maintains a low intracellular [Na+], which provides a favorable chemical gradient for the movement of Na+ from tubular fluid into the cell. This movement of Na+ across the apical membrane into the cell is mediated by the 1Na+/1K+/2Cl− symporter (NKCC2), which couples the movement of 1Na+ with 1K+ and 2Cl− . Using the potential energy released by the downhill movement of Na+ and Cl− , this symporter drives the uphill movement of K+ into the cell. K+ channels (ROMK and Maxi-K) in the apical plasma membrane play an important role in reabsorption of NaCl by the thick ascending limb. These K+ channels allow the K+ transported into the cell via the 1Na+/1K+/2Cl− symporter to recycle back into tubule fluid.

1	play an important role in reabsorption of NaCl by the thick ascending limb. These K+ channels allow the K+ transported into the cell via the 1Na+/1K+/2Cl− symporter to recycle back into tubule fluid. Because the [K+] in tubule fluid is relatively low, K+ recycling is required for continued operation of the 1Na+/1K+/2Cl− symporter. A Na+/H+ anti-porter (NHE3) in the apical cell membrane also mediates Na+ reabsorption as well as H+ secretion (HCO3 − reabsorption) in the thick ascending limb (see also ). The operation of the Na+/H+ antiporter in the apical membrane results in cellular uptake of Na+ in exchange for H+ . The production of H+ inside cells generates HCO3 −, which exits the cell across the basolateral membrane via a Cl−/HCO3 − antiporter (AE2). Na+ leaves the cell across the basolateral membrane via the Na+,K+-ATPase, whereas K+ and Cl− leave the cell via separate pathways in the basolateral membrane (i.e., K+ and Cl− channels and the K+/Cl− symporter).

1	614 SECTION7Berne & Levy Physiology •Fig. 34.8 Transport mechanism for NaCl reabsorption in the early segment of the distal tubule. This segment is impermeable to water. Mutations in the apical membrane NaCl symporter (NCC) cause Gitelman syndrome. ATP H2O NCC Na+ K+ K+ K+ Na+ Cl– Blood Tubular fluid Cl– The voltage across the thick ascending limb is important for reabsorption of several cations. The tubular fluid is positively charged relative to blood because of the unique location of transport proteins in the apical and basolateral membranes. Two points are important: (1) increased NaCl transport by the thick ascending limb increases the magnitude of the positive voltage in the lumen, and (2) this voltage is an important driving force for reabsorption of several cations, including Na+ , K+ , Mg++ , and Ca++ , across the paracellular pathway (see

1	Fig. 34.7 ). The importance of the paracellular pathway to solute reabsorption is underscored by the observation that inactivating mutations of the tight junction protein claudin-16 reduce reabsorption of Mg++ and Ca++ by the ascending thick limb, even in the presence of a lumen positive transepithelial voltage. In summary, NaCl reabsorption across the thick ascending limb occurs via transcellular and paracellular pathways. Fifty percent of NaCl reabsorption is transcellular, and 50% is paracellular. Because the thick ascending limb does not reabsorb water, owing to a lack of water channels (i.e., AQPs), reabsorption of NaCl and other solutes reduces the osmolality of tubular fluid to less than 150 mOsm/ kg H2O. Thus because the thick ascending limb of Henle’s loop produces a fluid that is dilute relative to plasma, this segment and the adjacent distal tubule (as discussed next) are often collectively referred to as the “diluting segments.”

1	The distal tubule and collecting duct reabsorb approximately 8% of the filtered NaCl, secrete variable amounts of K+ and H+ , and reabsorb a variable amount of water (≈8%–17%). The initial segment of the distal tubule (early distal tubule) reabsorbs Na+, Cl−, and Ca++ and is impermeable to water ( Fig. 34.8 ). Entry of NaCl into the cell across the apical membrane is mediated by a Na+/Cl− symporter,

1	Chapter 2, epithelial cells are joined at their apical surfaces by tight junctions (zonula occludens). A number of proteins have now been identified as components of the tight junction, including proteins that span the membrane of one cell and link to the extracellular portion of the same molecule in the adjacent cell (e.g., occludin and claudins), as well as cytoplasmic linker proteins (e.g., ZO-1, ZO-2, and ZO-3) that link the membrane-spanning proteins to the cytoskeleton of the cell. Of these junctional proteins, claudins appear to be major determinants of the permeability characteristics of tight junctions. For example, claudin-16 and claudin-19 are critical determinants of divalent cation permeability of the tight junctions in the thick ascending limb of Henle’s loop. Mutations in human claudin-16 and claudin19 cause familial hypomagnesemia (i.e., low plasma [Mg++]) with hypercalciuria (i.e., increased Ca++ in the urine) and nephrocalcinosis (i.e., calcification of the kidney).

1	claudin-16 and claudin19 cause familial hypomagnesemia (i.e., low plasma [Mg++]) with hypercalciuria (i.e., increased Ca++ in the urine) and nephrocalcinosis (i.e., calcification of the kidney). Claudin-2 is permeable to water and may be responsible for paracellular water reabsorption across the proximal tubule. Claudin-4 has been shown in cultured kidney cells to control the permeability of the tight junction to Na+ , whereas claudin-15 determines whether a tight junction is permeable to cations or anions. Thus the permeability characteristics of the tight junctions in different nephron segments are determined at least in part by the specific claudins expressed by the cells in that segment.

1	AVP, water is reabsorbed. By contrast, in the absence of AVP the late distal tubule and collecting duct reabsorb little Bartter syndrome is a set of autosomal recessive genetic diseases characterized by hypokalemia, metabolic alkalosis, and hyperaldosteronism (see Table 34.3 ). Inactivating mutations in the gene coding for the 1Na+/1K+/2Cl− symporter (NKCC2), the apical K+ channel (ROMK), or the basolateral Cl− channel (ClCNKB) decrease both NaCl reabsorption and K+ reabsorption by the ascending thick limb, which in turn causes hypokalemia (i.e., low plasma [K+]) and a decrease in ECFV. The fall in ECFV stimulates aldosterone secretion, which in turn stimulates NaCl reabsorption and H+ secretion by the distal tubule and collecting duct.

1	Fig. 34.8 ). Na+ leaves the cell via the action of Na+,K+-ATPase, and Cl− leaves the cell via diffusion through Cl− channels and a K+/Cl− symporter (KCC4). Thus dilution of tubular fluid begins in the thick ascending limb and continues in the early segment of the distal tubule. The last segment of the distal tubule (late distal tubule) and the collecting duct are composed of three cell types: principal cells and two types of intercalated cells. As illustrated in

1	Fig. 34.9, principal cells reabsorb NaCl and water and secrete K+ . Both Na+ reabsorption and K+ secretion by these cells depend on the activity of Na+,K+-ATPase in the basolateral membrane. By maintaining a low intracellular [Na+], the Na+,K+-ATPase provides a favorable chemical gradient for movement of Na+ from tubular fluid into the cell. Because Na+ enters the cell across the apical membrane via diffusion through epithelial Na+-selective channels (ENaCs), the negative voltage inside the cell facilitates entry of Na+, which then exits the cell and enters the blood via the basolateral membrane Na+,K+-ATPase. Reabsorption of Na+ generates a negative luminal voltage across the late distal tubule and collecting duct, which provides the driving force for paracellular reabsorption of Cl− . Intercalated cells secrete either H+ or HCO3 − and play important roles in acid-base homeostasis (see ). The α-intercalated cell (see

1	Fig. 34.9 , center) secretes H+ and reabsorbs both HCO3 − and K+ and is thus important in regulating acid-base balance (see ). α-Intercalated cells reabsorb K+ by the operation of an H+,K+-ATPase (HKA) located in the apical plasma membrane. In contrast, β-intercalated cells (see Fig. 34.9 , bottom) secrete HCO3 − and reabsorb both H+ and Cl− . Chloride enters the β-intercalated cell across the apical membrane via a Cl−/HCO3 − antiporter (pendrin) and leaves the cell across the basolateral membrane via a Cl− channel. A variable amount of water is reabsorbed across principal cells in the late distal tubule and collecting duct. Water reabsorption in these segments is mediated by the AVP-regulated AQP2 water channel located in the apical plasma membrane and by AQP3 and AQP4 located in the basolateral membrane of principal cells. In the presence of water (see K+ is secreted from blood into tubular fluid by principal cells in two steps (see Fig. 34.9, top).

1	K+ is secreted from blood into tubular fluid by principal cells in two steps (see Fig. 34.9, top). First, uptake of K+ across the basolateral membrane is mediated by the action of Na+,K+-ATPase. Second, K+ leaves the cell via passive diffusion. Because [K+] inside principal cells is high (≈150 mEq/L) and [K+] in tubular fluid is low (≈10 mEq/L), K+ diffuses down its concentration gradient through apical cell membrane K+ channels (ROMK and BK) into tubular fluid. Although the negative potential inside these cells favors intracellular K+ retention, the electrochemical gradient across the apical membrane promotes secretion of K+ from the cell into tubular fluid (see ). In contrast, K+ reabsorption by α cells is mediated by an H+,K+-ATPase (HKA) located in the apical cell membrane (see

1	Fig. 34.9 , center). As a consequence these distal nephron segments possess the ability to both secrete and reabsorb K+ via independently regulated mechanisms, which contrasts with the general tendency to reabsorb Na+ along most nephron segments. Regulation of NaCl and Water Reabsorption Quantitatively, angiotensin II, aldosterone, catecholamines, natriuretic peptides, and uroguanylin are the most important hormones regulating NaCl reabsorption and thereby urinary NaCl excretion ( Table 34.6). However, other hormones (including dopamine and adrenomedullin), Starling forces, and the phenomenon of glomerulotubular balance also influence NaCl reabsorption. AVP is the only major hormone that directly regulates the amount of water excreted by the kidneys.

1	Angiotensin II has a potent stimulatory effect on the isosmotic reabsorption of NaCl and water in the proximal tubule. It also stimulates reabsorption of Na+ in the thick ascending limb of Henle’s loop, as well as the late distal tubule and collecting duct. A decrease in ECFV activates the renin-angiotensin-aldosterone system (see for more details), thereby increasing the plasma concentration of angiotensin II.

1	Aldosterone is synthesized by the glomerulosa cells of the adrenal cortex and stimulates reabsorption of NaCl by the thick ascending limb of Henle’s loop, the late distal tubule, and the collecting duct. Most of aldosterone’s effect on NaCl reabsorption reflects its action on the late distal tubule and collecting duct. Aldosterone enhances reabsorption of NaCl across principal cells in these segments by four mechanisms: (1) increasing the amount of Na+,K+-ATPase in the basolateral membrane; (2) increasing expression of the sodium channel (ENaC) in the apical cell membrane; (3) elevating Sgk1 (serum glucocorticoid-stimulated kinase; see the molecular box) levels, which also increases the expression of ENaC in the apical cell membrane; and (4) stimulating •Fig. 34.9 Transport pathways in principal cells, α-intercalated cells, and β-intercalated cells of the late segment of the distal tubule and collecting duct. CA, carbonic anhydrase. Principal cells reabsorb Na+and secrete K+. K+is

1	principal cells, α-intercalated cells, and β-intercalated cells of the late segment of the distal tubule and collecting duct. CA, carbonic anhydrase. Principal cells reabsorb Na+and secrete K+. K+is secreted by two types of K+channels (ROMK and BK) and by a K+/Cl−symporter (KCC1). α-Intercalated cells secrete H+and reabsorb HCO3−and K+, and β-intercalated cells secrete HCO3−and reabsorb H+and Cl−. CO2 + H2O HCO− 3 CA Cl− Cl− H+ ˙-Intercalated cell Pendrin V-ATPase Blood

1	CAP1 (channel-activating protease, also called prostatin), a serine protease that directly activates ENaCs by proteolysis. Taken together, these actions increase Na+ uptake across the apical cell membrane and facilitate Na+ exit from the cell interior into blood. The increase in reabsorption of Na+ generates a negative transepithelial luminal voltage across the late distal tubule and the collecting duct. This negative voltage in the lumen provides the electrochemical driving force for reabsorption of Cl− across the tight junctions. Aldosterone also stimulates secretion of K+ by the late distal tubule and collecting duct (collectively referred to as the aldosterone-sensitive distal nephron (ASDN) Angiotensin II↑ReninPT, TAL, DT/CD↑NaCl and H2O reabsorptionAldosterone↑Angiotensin II, ↑[K+]pTAL, DT/CD↑NaCl and H2O reabsorption

1	Angiotensin II↑ReninPT, TAL, DT/CD↑NaCl and H2O reabsorptionAldosterone↑Angiotensin II, ↑[K+]pTAL, DT/CD↑NaCl and H2O reabsorption ANP, BNP, urodilatin↑ECFVCD↓H2O and NaCl reabsorptionUroguanylin, guanylinOral ingestion of NaClPT, CD↓H2O and NaCl reabsorptionSympathetic nerves↓ECFVPT, TAL, DT/CD↑NaCl and H2O reabsorption Dopamine↑ECFVPT↓H2O and NaCl reabsorptionAVP↑Posm, ↓ECFVDT/CD↑H2O reabsorption aAll these hormones act within minutes, except aldosterone, which exerts its action on reabsorption of NaCl with a delay of 1 hour. Aldosterone achieves its maximal effect after a few days.bThe effect on reabsorption of H2O does not include the thick ascending limb.ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide, BP, blood pressure; CD, collecting duct; DT, distal tubule; ECFV, extracellular fluid volume; [K+]p, plasma K+ concentration; Posm, plasma osmolality; PT, proximal tubule; TAL, thick ascending limb. Chapter 36]).

1	Chapter 36]). Aldosterone secretion is increased by hyperkalemia and by hypovolemia (i.e., reduced ECFV via increased angiotensin II following activation of the renin-angiotensin system [RAS]; see for more details). Aldosterone secretion is decreased by hypokalemia and natriuretic peptides (discussed in more detail next). Through its stimulation of NaCl reabsorption in the collecting duct, aldosterone also indirectly increases water reabsorption by this nephron segment.

1	The aldosterone paradox. As noted earlier, aldosterone stimulates both NaCl reabsorption and K+ secretion by the collecting duct. Although both a reduction in the ECFV (i.e., hypovolemia, see ) increase aldosterone levels, the physiological response of the kidneys differs in these two conditions. In the setting of ECFV depletion, NaCl excretion by the kidneys is reduced to restore ECFV, without an accompanying change in K+ excretion. By contrast, during hyperkalemia, K+ excretion by the kidneys is increased to normalize plasma [K+], albeit without an accompanying change in NaCl excretion. This phenomenon—the apparent independent effects of aldosterone on urinary Na+ and K+ excretion—is called the aldosterone paradox. The paradox can be explained by the observation that ECFV depletion increases aldosterone release via activation of the RAS, whereas hyperkalemia directly stimulates adrenal release of aldosterone without a requirement for RAS activation. As such, aldosterone increases in

1	aldosterone release via activation of the RAS, whereas hyperkalemia directly stimulates adrenal release of aldosterone without a requirement for RAS activation. As such, aldosterone increases in both conditions, whereas angiotensin II levels increase only during ECFV depletion and not during hyperkalemia. It is the differential regulation of transport processes in the distal tubule and collecting duct by aldosterone and angiotensin II that accounts for this paradox. The integrated physiological response to a reduction in ECFV is depicted in

1	Fig. 34.10A . During hypovolemia, angiotensin II stimulates NaCl reabsorption by the proximal tubule (not shown in Figure 34.10A ), and by the early distal tubule by activating WNK (with no lysine [K] kinase), which enhances NaCl reabsorption by activating the Na+/Cl− symporter (NCC). Aldosterone stimulates ENaC-mediated Na+ reabsorption in principal cells of the collecting duct by activating SGK1, which increases ENaC abundance in the apical plasma membrane. In parallel, angiotensin II activates WNK in principal cells, which inhibits K+ secretion via ROMK, thereby preventing increased K+ excretion despite elevated aldosterone levels, which would be expected to promote K+ secretion. Angiotensin II stimulation of proximal tubule NaCl and water reabsorption also reduces delivery of NaCl and fluid to the collecting duct, which also suppresses K+ secretion in this segment (see for more details). The corresponding integrated physiological response to hyperkalemia is depicted in

1	Fig. 34.10B . During hyperkalemia, aldosterone stimulates ROMK mediated K+ secretion by principal cells in the collecting duct by activating WNK. Because the early distal tubule is not directly responsive to aldosterone, this hormone does not stimulate NaCl reabsorption in this segment. In fact, because angiotensin II levels are not elevated by hyperkalemia, the basal activity of WNK in the early distal tubule is low, resulting in decreased NaCl reabsorption via NCC (see Fig. 34.10B ). This effect of WNK on early distal tubule NaCl reabsorption offsets the stimulatory effect of aldosterone, via SGK1, on ENaC-mediated Na+ reabsorption in principal cells of the collecting duct.

1	Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) inhibit NaCl and water reabsorption. Secretion of ANP by the cardiac atria and BNP by the cardiac ventricles are both stimulated by increased ECFV and increased myocardial wall pressure. ANP and BNP reduce blood pressure by decreasing total peripheral resistance and enhancing urinary excretion of both NaCl and water, primarily by increasing RBF and GFR. These natriuretic peptides vasodilate the afferent arterioles and vasoconstrict the efferent arterioles, which increases GFR and thus filtration of NaCl, thereby increasing NaCl excretion (see later discussion of glomerulotubular balance for the mechanism). In addition the increase in RBF decreases the concentration of NaCl in the medullary interstitium, which in turn reduces passive NaCl reabsorption by the thin ascending •Fig. 34.10 Aldosterone paradox (see text for details). +, stimulate; −, inhibit; AII, angiotensin II; NCC, NaCl symporter; ENaC, epithelial sodium

1	passive NaCl reabsorption by the thin ascending •Fig. 34.10 Aldosterone paradox (see text for details). +, stimulate; −, inhibit; AII, angiotensin II; NCC, NaCl symporter; ENaC, epithelial sodium channel; ROMK, K+ channel; WNK, with no lysine[K] kinases. There are four WNKs in the kidney; details are not given in the figure for clarity and because some of the details of WNK regulation of NCC, ENaC, and ROMK have not been elucidated.

1	limb of Henle’s loop (see earlier discussion for details on NaCl reabsorption by this segment). ANP and BNP also inhibit NaCl reabsorption by the medullary portion of the collecting duct and inhibit AVP-stimulated water reabsorption across the collecting duct. Moreover, ANP and BNP also reduce secretion of AVP from the posterior pituitary. These actions of ANP and BNP are mediated by the activation of membrane-bound guanylyl cyclase receptors, which increases intracellular levels of the second messenger cyclic guanine monophosphate (cGMP). ANP is a more profound natriuretic and diuretic agent than BNP.

1	Urodilatin and ANP are encoded by the same gene and have similar amino acid sequences. Urodilatin is a 32–amino acid hormone that differs from ANP by the addition of four amino acids to the amino terminus. Urodilatin is secreted by the distal tubule and collecting duct and is not present in the systemic circulation; thus urodilatin influences only the function of the kidneys. Secretion of urodilatin is stimulated by a rise in blood pressure and an increase in ECFV. It inhibits NaCl and water reabsorption across the medullary portion of the collecting duct. Urodilatin is a more potent natriuretic and diuretic hormone than ANP because some of the ANP that enters the kidneys in blood is degraded by a neutral endopeptidase that has no corresponding effect on urodilatin.

1	Uroguanylin and guanylin are produced by neuroendocrine cells in the intestine in response to oral ingestion of NaCl. These hormones enter the circulation and inhibit NaCl and water reabsorption by the kidneys via activation of membrane-bound guanylyl cyclase receptors, which

1	Sgk1 (serum glucocorticoid-stimulated kinase), a serine/threonine kinase, plays an important role in maintaining NaCl and K+ homeostasis by regulating excretion of both NaCl and K+ by the kidneys. Studies in Sgk1 knockout mice reveal that this kinase is required for animals to survive severe NaCl restriction and K+ loading. NaCl restriction and K+ loading enhance plasma [aldosterone], which rapidly (in minutes) increases Sgk1 protein expression and phosphorylation. Phosphorylated Sgk1 enhances ENaC-mediated Na+ reabsorption in the collecting duct, primarily by increasing the number of ENaCs in the apical plasma membrane of principal cells and also by increasing the number of Na+,K+-ATPase pumps in the basolateral membrane. Phosphorylated Sgk1 inhibits Nedd4-2, a ubiquitin ligase that monoubiquitinylates ENaC subunits, thereby targeting them for endocytic removal from the plasma membrane and subsequent destruction in lysosomes. Inhibition of Nedd4-2 by Sgk1 reduces the

1	ligase that monoubiquitinylates ENaC subunits, thereby targeting them for endocytic removal from the plasma membrane and subsequent destruction in lysosomes. Inhibition of Nedd4-2 by Sgk1 reduces the monoubiquitinylation of ENaC, thereby reducing endocytosis and increasing the number of channels in the membrane. Sgk1 induces the translocation of K+ channels (ROMK) from an intracellular pool to the plasma membrane, and thereby enhances ROMK-mediated K+ secretion by principal cells. These effects of Sgk1 precede the aldosterone-stimulated increase in ENaC, ROMK, and Na+,K+-ATPase abundance, which leads to a delayed (>4 hours) secondary increase in NaCl and K+ transport by the collecting duct. Activating polymorphisms in Sgk1 cause an increase in blood pressure, presumably by enhancing NaCl reabsorption by the collecting duct, which increases the ECFV and thereby blood pressure.

1	increase intracellular [cGMP]. The involvement of these gut-derived hormones helps explain why the natriuretic response of the kidneys to an oral NaCl load is more pronounced than when delivered intravenously. Catecholamines stimulate reabsorption of NaCl. Catecholamines released from the sympathetic nerves (norepinephrine) and the adrenal medulla (epinephrine) stimulate reabsorption of NaCl and water by the proximal tubule, thick ascending limb of the loop of Henle, distal tubule, and collecting duct. Although sympathetic nerves are not active when ECFV is normal, when ECFV declines (e.g., after hemorrhage), sympathetic nerve activity rises and dramatically stimulates reabsorption of NaCl and water by these four nephron segments.

1	Dopamine, a catecholamine, is released from dopaminergic nerves in the kidneys and is also synthesized by cells of the proximal tubule. The action of dopamine is opposite that of norepinephrine and epinephrine. Secretion of dopamine is stimulated by an increase in ECFV, and its secretion directly inhibits reabsorption of NaCl and water in the proximal tubule. Adrenomedullin is a 52–amino acid peptide hormone that is produced by a variety of organs, including the kidneys. Adrenomedullin induces a marked diuresis and natriuresis, and its secretion is stimulated by congestive heart failure and hypertension. The major effect of adrenomedullin on the kidneys is to increase GFR and renal blood flow and

1	Liddle syndrome is a rare genetic disorder characterized by an increase in blood pressure (i.e., hypertension) secondary to an increase in ECFV. Liddle syndrome is caused by activating mutations in either the β or γ subunit of the epithelial Na+ channel (ENaC). These mutations increase the number of Na+ channels in the apical cell membrane of principal cells and thereby the amount of Na+ reabsorbed. In Liddle syndrome, the rate of renal Na+ reabsorption is inappropriately high, which leads to an increase in ECFV and hypertension. There are two different forms of pseudohypoaldosteronism (PHA) (i.e., the kidneys reabsorb NaCl as they do when aldosterone levels are low; however, in PHA, aldosterone levels are elevated). The autosomal recessive form is caused by inactivating mutations in the α, β, or γ subunit of ENaC. The cause of the autosomal dominant form is an inactivating mutation in the mineralocorticoid receptor. Pseudohypoaldosteronism is characterized by an increase in Na+

1	the α, β, or γ subunit of ENaC. The cause of the autosomal dominant form is an inactivating mutation in the mineralocorticoid receptor. Pseudohypoaldosteronism is characterized by an increase in Na+ excretion, a reduction in ECFV, hyperkalemia, and hypotension. Some individuals with expanded ECFV and elevated blood pressure are treated with drugs that inhibit angiotensin-converting enzyme (ACE) (e.g., captopril, enalapril, lisinopril) and thereby lower fluid volume and blood pressure. Inhibition of ACE blocks degradation of angiotensin I to angiotensin II and thereby lowers plasma angiotensin II levels. The decline in plasma angiotensin II concentration has three effects. First, NaCl and water reabsorption by the nephron (especially the proximal tubule) falls. Second, aldosterone secretion decreases, thus reducing NaCl reabsorption in the thick ascending limb, distal tubule, and collecting duct. Third, because angiotensin is a potent vasoconstrictor, a reduction in its concentration

1	decreases, thus reducing NaCl reabsorption in the thick ascending limb, distal tubule, and collecting duct. Third, because angiotensin is a potent vasoconstrictor, a reduction in its concentration permits the systemic arterioles to dilate and thereby lower arterial blood pressure. ACE also degrades the vasodilator hormone bradykinin; thus ACE inhibitors increase the concentration of bradykinin, a vasodilatory hormone. ACE inhibitors decrease ECFV and the arterial blood pressure by promoting renal NaCl and water excretion and by reducing total peripheral resistance.

1	thereby indirectly stimulate excretion of NaCl and water (see earlier discussion about ANP and BNP). Arginine vasopressin (AVP) regulates water reabsorption. It is the most important hormone that regulates reabsorption of water in the kidneys (see ). This hormone is secreted by the posterior pituitary gland in response to an increase in plasma osmolality (1% or more) or a decrease in ECFV (>5%–10% of normal). AVP increases the permeability of the collecting duct to water. It increases reabsorption of water by the collecting duct because of the osmotic gradient that exists across the wall of the collecting duct (see Chapter 35). AVP has little effect on urinary NaCl excretion.

1	Starling forces regulate reabsorption of NaCl and water across the proximal tubule. As previously described, Na+ , Cl−, HCO3 −, amino acids, glucose, and water are transported into the intercellular space of the proximal tubule. Starling forces between this space and the peritubular capillaries facilitate movement of the reabsorbed fluid into the capillaries. Starling forces across the wall of peritubular capillaries •Fig. 34.11 Starling forces modify proximal tubule solute and water reabsorption. (1) Solute and water are reabsorbed across the apical membrane. This solute and water then cross the lateral cell membrane. Some solute and water reenters the tubule fluid (3), and the remainder enters the interstitial space and then flows into the capillary (2). The width of the arrows is directly proportional to the amount of solute and water moving by pathways 1 to 3. Starling forces across the capillary wall determine the amount of fluid flowing through pathway 2 versus pathway 3.

1	directly proportional to the amount of solute and water moving by pathways 1 to 3. Starling forces across the capillary wall determine the amount of fluid flowing through pathway 2 versus pathway 3. Transport mechanisms in the apical cell membranes determine the amount of solute and water entering the cell (pathway 1). Pi, interstitial hydrostatic pressure; Ppc, peritubular capillary hydrostatic pressure; πi, interstitial fluid oncotic pressure; πpc, peritubular capillary oncotic pressure. Thin arrows across the capillary wall indicate the direction of water movement in response to each force.

1	consist of hydrostatic pressure in the peritubular capillary (Ppc) and lateral intercellular space (Pi) and oncotic pressure in the peritubular capillary (πpc) and lateral intercellular space (πi). Thus reabsorption of water as a result of transport of Na+ from tubular fluid into the lateral intercellular space is modified by the Starling forces. Accordingly:

1	Equation 34.2 where J is flow (positive numbers indicate flow from the intercellular space into blood). Starling forces that favor movement from the interstitium into the peritubular capillaries are πpc and Pi (Fig. 34.11 ). The opposing Starling forces are πi and Ppc. Normally the sum of the Starling forces favors movement of solute and water from the interstitial space into the capillary. However, some of the solutes and fluid that enter the lateral intercellular space leak back into the proximal tubular fluid. Starling forces do not affect transport by the loop of Henle, distal tubule, and collecting duct because these segments are less permeable to water than the proximal tubule is. A number of factors can alter the Starling forces across the peritubular capillaries surrounding the proximal tubule. For example, dilation of the efferent arteriole increases Ppc, whereas constriction of the efferent arteriole decreases it.

1	An increase in Ppc inhibits solute and water reabsorption by increasing back-leak of NaCl and water across the tight junction, whereas a decrease stimulates reabsorption by decreasing back-leak across the tight junction.

1	Peritubular capillary oncotic pressure (πpc) is partially determined by the rate of formation of the glomerular ultrafiltrate. For example, if one assumes a constant plasma flow in the afferent arteriole, the plasma proteins become less concentrated in the plasma that enters the efferent arteriole and peritubular capillary as less ultrafiltrate is formed (i.e., as GFR decreases). Hence, πpc decreases. Thus πpc is directly related to the filtration fraction (FF = GFR/renal plasma flow [RPF]). A fall in the FF resulting from a decrease in GFR, at constant RPF, decreases πpc. This in turn increases the backflow of NaCl and water from the lateral intercellular space into tubular fluid and thereby decreases net reabsorption of solute and water across the proximal tubule. An increase in FF has the opposite effect.

1	The importance of Starling forces in regulating solute and water reabsorption by the proximal tubule is underscored by the phenomenon of glomerulotubular (G-T) balance. Spontaneous changes in GFR markedly alter the filtered amount of Na+ (filtered Na+= GFR × [Na+] in the filtered fluid). Without rapid adjustments in Na+ reabsorption to counter the changes in filtration of Na+ , urinary excretion of Na+ would fluctuate widely and disturb the Na+ balance of the body and thus alter ECFV and blood pressure (see for more details). However, spontaneous changes in GFR do not alter Na+ excretion in urine or Na+ balance when ECFV is normal because of the phenomenon of G-T balance. When body Na+ balance is normal (i.e., ECFV is normal), G-T balance refers to the fact that reabsorption of Na+ and water increases in proportion to the increase in GFR and filtered amount of Na+ . Thus a constant fraction of the filtered Na+ and water is reabsorbed from the proximal tubule despite variations in GFR.

1	increases in proportion to the increase in GFR and filtered amount of Na+ . Thus a constant fraction of the filtered Na+ and water is reabsorbed from the proximal tubule despite variations in GFR. The net result of G-T balance is to reduce the impact of changes in GFR on the amount of Na+ and water excreted in urine when ECFV is normal.

1	Two mechanisms are responsible for G-T balance. One is related to the oncotic and hydrostatic pressure differences between the peritubular capillaries and the lateral intercellular space (i.e., Starling forces). For example, an increase in the GFR (at constant RPF) raises the protein concentration in glomerular capillary plasma above normal. This protein-rich plasma leaves the glomerular capillaries, flows through the efferent arterioles, and enters the peritubular capillaries. The increased πpc augments the movement of 1. The four major segments of the nephron (proximal tubule, Henle’s loop, distal tubule, and collecting duct) determine the composition and volume of urine by the processes of selective reabsorption of solutes and water and secretion of some solutes. 2.

1	2. Tubular reabsorption of substances filtered by the glomerulus allows the kidneys to retain substances that are essential and regulate their levels in plasma by altering the degree to which they are reabsorbed. Reabsorption of Na+ , Cl− , other anions, and organic anions and cations together with water constitutes the major function of the nephron. Approximately 25,200 mEq of Na+ and 179 L of water are reabsorbed each day. Proximal tubule cells reabsorb 67% of the glomerular ultrafiltrate, and cells of Henle’s loop reabsorb about 25% of the NaCl that was filtered and about 15% of the water that was filtered. The distal segments of the nephron (distal tubule and collecting duct system) have a more limited reabsorptive capacity. However, although the proximal tubule reabsorbs Brown D, et al. New insights into the dynamic regulation of water and acid-base balance by renal epithelial cells. Am J Physiol Cell Physiol. 2012;302:C1421-C1433.

1	Brown D, et al. New insights into the dynamic regulation of water and acid-base balance by renal epithelial cells. Am J Physiol Cell Physiol. 2012;302:C1421-C1433. Dantzler WH, et al. Urine-concentrating mechanism in the inner medulla: function of the thin limbs of the loops of Henle. Clin J Am Soc Nephrol. 2014;9:1781-1789. solute and fluid from the lateral intercellular space into the peritubular capillaries. This action increases net solute and water reabsorption by the proximal tubule. The second mechanism responsible for G-T balance is initiated by an increase in the filtered amount of glucose and amino acids. As discussed earlier, reabsorption of Na+ in the first half of the proximal tubule is coupled to that of glucose and amino acids. The rate of Na+ reabsorption therefore partially depends on the filtered amount of glucose and amino acids. As the GFR and filtered amount of glucose and amino acids increase, reabsorption of Na+ and water also rises.

1	In addition to G-T balance, another mechanism minimizes changes in the filtered amount of Na+ . As discussed in , an increase in GFR (and thus in the amount of Na+ filtered by the glomerulus) activates the tubuloglomerular feedback mechanism. This action returns the GFR and filtration of Na+ to normal values. Thus spontaneous changes in GFR (e.g., caused by changes in posture and blood pressure) increase the amount of Na+ filtered for only a few minutes. The mechanisms that underlie G-T balance maintain urinary Na+ excretion constant and thereby maintain Na+ homeostasis (and ECFV and blood pressure) until the GFR returns to normal. the largest fraction of the filtered solutes and water (i.e., 67%), final adjustments in the composition and volume of urine and most of the regulation by hormones and other factors occur primarily in the distal tubule and collecting duct. 3.

1	3. Secretion of substances from the blood into tubular fluid is a means for excreting various byproducts of metabolism, and it also serves to eliminate exogenous organic anions and cations (e.g., drugs) and toxins from the body. Many organic anions and cations are bound to plasma proteins and are therefore unavailable for ultrafiltration. Thus secretion is their major route of excretion in urine. 4. Various hormones (including angiotensin II, aldosterone, AVP, natriuretic peptides [ANP, BNP, and urodilatin], uroguanylin, and guanylin), sympathetic nerves, dopamine, and Starling forces regulate reabsorption of NaCl by the kidneys. AVP is the major hormone that regulates water reabsorption. Divers J, Freedman BI. Genetics in kidney disease in 2013: susceptibility genes for renal and urological disorders. Nat Rev Nephrol. 2014;10:69-70. Hoenig MP, Zeidel ML. Homeostasis, the milieu interieur, and the wisdom of the nephron. Clin J Am Soc Nephrol. 2014;9: 1272-1281.

1	Hoenig MP, Zeidel ML. Homeostasis, the milieu interieur, and the wisdom of the nephron. Clin J Am Soc Nephrol. 2014;9: 1272-1281. Knepper MA, et al. Molecular physiology of water balance. N Engl J Med. 2015;372:1349-1358. Kortenoeven ML, et al. Vasopressin regulation of sodium transport in the distal nephron and collecting duct. Am J Physiol Renal Physiol. 2015;309:F280-F299. McCormick JA, Ellison DH. Distal convoluted tubule. Compr Physiol. 2015;5:45-98. Mount DB. Thick ascending limb of the loop of Henle. Clin J Am Soc Nephrol. 2014;9:1974-1986. Palmer BF. Regulation of potassium homeostasis. Clin J Am Soc Nephrol. 2015;10:1050-1060. Palmer LG, Schnermann J. Integrated control of Na transport along the nephron. Clin J Am Soc Nephrol. 2015;10:676-687. Pearce D, et al. Collecting duct principal cell transport processes and their regulation. Clin J Am Soc Nephrol. 2015;10:135-146.

1	Pearce D, et al. Collecting duct principal cell transport processes and their regulation. Clin J Am Soc Nephrol. 2015;10:135-146. Pelis RM, Wright SH. Renal transport of organic anions and cations. Compr Physiol. 2011;1:1795-1835. Pluznick JL, Caplan MJ. Chemical and physical sensors in the regulation of renal function. Clin J Am Soc Nephrol. 2015;10:1626-1635. Roy A, et al. Collecting duct intercalated cell function and regulation. Clin J Am Soc Nephrol. 2015;10:305-324. Theilig F, Wu Q. ANP-induced signaling cascade and its implications in renal pathophysiology. Am J Physiol Renal Physiol. 2015;308:F1047-F1055. Weiner ID, et al. Urea and ammonia metabolism and the control of renal nitrogen excretion. Clin J Am Soc Nephrol. 2015;10: 1444-1458. Brown D, Nielsen S. The cell biology of vasopressin action. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012.

1	Brown D, Nielsen S. The cell biology of vasopressin action. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012. Burckhardt G, Koepsell H. Organic anion and cation transporters in renal elimination of drugs. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2012. Christensen EI, et al. Renal filtration, transport, and metabolism of albumin and albuminuria. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2012. Gamba G, Schild L. Sodium chloride transport in the loop of Henle, distal convoluted tubule, and collecting duct. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2012.

1	Giebisch G, Satlin L. Regulation of potassium excretion. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2012. Moe OW, et al. Renal transport of glucose, amino acids, sodium, chloride, and water. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012. Preisig P, et al. Cellular mechanisms of renal tubular acidification. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2012. Sands JM, Layton H. The urine concentrating mechanism and urea transporters. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2012. Upon completion of this chapter the student should be able to answer the following questions: 1.

1	Upon completion of this chapter the student should be able to answer the following questions: 1. Why do changes in water balance result in alterations in the [Na+] of the extracellular fluid (ECF)? 2. How is the secretion of arginine vasopressin (AVP) controlled by changes in the osmolality of body fluids and in blood volume and pressure? 3. What are the cellular events associated with the action of AVP on the collecting duct, and how do they lead to an increase in the water permeability of this segment of the nephron? 4. What is the role of Henle’s loop in the production of both dilute and concentrated urine? 5. What is the composition of the medullary interstitial fluid, and how does it participate in the process of producing concentrated urine? 6. What are the roles of the vasa recta in the process of diluting and concentrating urine? 7. How is the diluting and concentrating ability of the kidneys quantitated? 8.

1	6. What are the roles of the vasa recta in the process of diluting and concentrating urine? 7. How is the diluting and concentrating ability of the kidneys quantitated? 8. Why do changes in Na+ balance alter the volume of extracellular fluid? 9. What is the effective circulating volume, how is it influenced by changes in Na+ balance, and how does it influence renal Na+ excretion? 10. What are the mechanisms by which the body monitors the effective circulating volume? 11. What are the major signals acting on the kidneys to alter their excretion of Na+? 12. How do changes in extracellular fluid volume alter Na+ transport in the different segments of the nephron, and how do these changes in transport regulate renal Na+ excretion? 13. What are the mechanisms involved in formation of edema, and what role do the kidneys play in this process?

1	13. What are the mechanisms involved in formation of edema, and what role do the kidneys play in this process? he kidneys maintain the osmolality and volume of the body fluids within a narrow range by regulating excretion of water and NaCl, respectively. This chapter discusses the regulation of renal water excretion (urine concentration and dilution) and NaCl excretion. The composition and volumes of the various body fluid compartments are reviewed in

1	Control of Body Fluid Osmolality: Urine Concentration and Dilution , water constitutes approximately 60% of the healthy adult human body. Body water is divided into two major compartments—intracellular fluid (ICF) and extracellular fluid (ECF)—that are in osmotic equilibrium because of the high permeability of most cell membranes to water via aquaporins (e.g., AQP1). Water intake into the body generally occurs orally. This may be water contained in beverages as well as water generated during metabolism of ingested foods (e.g., carbohydrates). In many clinical situations, intravenous infusion is an important route of water entry. The kidneys are responsible for regulating water balance and under most conditions are the major route for elimination of water from the body (

1	The kidneys are responsible for regulating water balance and under most conditions are the major route for elimination of water from the body ( Table 35.1 ). Other routes of water loss from the body include evaporation from cells of the skin and respiratory passages. Collectively, water loss by these routes is termed insensible water loss because the individual is unaware of its occurrence. Production of sweat accounts for the loss of additional water. Water loss by this mechanism can increase dramatically in a hot environment, with exercise, or in the presence of fever ( 35.2 ). Finally, water can be lost from the gastrointestinal tract. Fecal water loss is normally small (≈100 mL/day) but can increase dramatically with diarrhea (e.g., 20 L/ day with cholera). Vomiting can also cause gastrointestinal water losses.

1	Although water loss from sweating, defecation, and evaporation from the lungs and skin can vary depending on the environmental conditions or during pathological conditions, loss of water by these routes cannot be regulated. In contrast, renal excretion of water is tightly regulated to maintain whole-body water balance. Maintenance of water balance requires that water intake and loss from the body be precisely matched. If intake exceeds losses, positive water balance exists. Conversely, when intake is less than losses, for review of (with its anions Cl− and HCO3 −), these disorders also result steady-state balance). in alterations in plasma or serum [Na+] ( Fig. 35.2 ). One

1	Fig. 35.2 ). One When water intake is low or water losses increase, the kidneys conserve water by producing a small volume of urine that is hyperosmotic with respect to plasma. When water intake is high, a large volume of hypoosmotic urine is produced. In a normal individual, urine osmolality (Uosm) can vary from approximately 50 to 1200 mOsm/kg H2O, and the corresponding urine volume can vary from approximately 18 L/day to 0.5 L/day. Importantly the kidneys can regulate excretion of water separately from excretion of total solute ( Fig. 35.1 ). The ability to regulate water excretion separate from excretion of solutes (e.g., Na+ , K+ , urea, etc.) is necessary for survival because it allows water balance to be achieved without upsetting the other homeostatic functions of the kidneys.

1	It is important to recognize that disorders of water balance are manifested by alterations in body fluid osmolality, which are usually measured by changes in plasma osmolality (POSM). Because the major determinant of plasma osmolality is Na+ aFluid intake varies widely for both social and cultural reasons. of the most common fluid and electrolyte disorders seen in clinical practice is an alteration in serum [Na+]. When an abnormal serum [Na+] is found in an individual, it is tempting to suspect a problem in Na+ balance. However, the problem most often relates to water balance, not Na+ balance. As described later, changes in Na+ balance result in alterations in the volume of ECF, not its osmolality.

1	The following sections discuss the mechanisms by which the kidneys excrete either hypoosmotic (dilute) or hyperosmotic (concentrated) urine. Control of arginine vasopressin secretion and its important role in regulating excretion of water by the kidneys are also explained (see also • Fig. 35.1 Relationships between plasma AVP levels, and urine osmolality, urine flow rate, and total solute excretion. Max, maximum; Min, minimum. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) aIn hot weather and during prolonged heavy exercise, water balance is maintained by increased water ingestion. Decreased excretion of water by the kidneys alone is insufficient to maintain water balance. Kidneys excrete 1 L of water Kidneys excrete hyperosmotic in hypoosmotic urine, urine as the individual drinks returning volume to 14 L and water, returning volume to 14 L restoring [Na˜] and osmolality and restoring [Na˜] and osmolality to normal. to normal.

1	• Fig. 35.2 Response to changes in water balance. Illustrated are the effects of adding or removing 1 L of water from the ECF of a 70-kg individual. Positive Water Balance: (1) Addition of 1 L of water increases the ECFV and reduces its osmolality. The [Na+] is also decreased (hyponatremia). (2) The normal renal response is to excrete 1 L of water as hypoosmotic urine. (3) As a result of the renal excretion of water, the ECFV, osmolality, and [Na+] are returned to normal. Negative Water Balance: (4) The loss of 1 L of water from the ECF decreases its volume and increases its osmolality. The [Na+] is also increased (hypernatremia). (5) The renal response is to conserve water by excreting a small volume of hyperosmotic urine. (6) With ingestion of water, stimulated by thirst, and conservation of water by the kidneys, the ECFV, osmolality, and [Na+] are returned to normal. Size of the boxes indicates relative ECFV. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia:

1	of water by the kidneys, the ECFV, osmolality, and [Na+] are returned to normal. Size of the boxes indicates relative ECFV. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.)

1	In the clinical setting, hypoosmolality (a reduction in plasma osmolality) shifts water into cells, and this process results in cell swelling (see ). Symptoms associated with hypoosmolality are related primarily to swelling of brain cells. For example, a rapid fall in Posm can alter neurological function and thereby cause nausea, malaise, headache, confusion, lethargy, seizures, and coma. When Posm is increased (i.e., hyperosmolality), water is lost from cells. Symptoms of an increase in Posm are also primarily neurological and include lethargy, weakness, seizures, coma, and even death.

1	Symptoms associated with changes in body fluid osmolality vary depending on how quickly osmolality is changed. Rapid changes in osmolality (i.e., over hours) are less well tolerated than changes that occur more gradually (i.e., over days to weeks). Indeed, individuals who have developed alterations in their body fluid osmolality over an extended period of time may be entirely asymptomatic. This reflects the ability of cells over time to either eliminate intracellular osmoles, as occurs with hypoosmolality, or to generate new intracellular osmoles in response to hyperosmolality and thus minimize changes in cell volume of the neurons (see several nephron segments express a different AVP receptor (V2) that mediates the kidneys’ ability to regulate the volume and osmolality of urine. When plasma AVP levels are low, a large volume of urine is excreted (diuresis), and the urine osmolality is less than that of plasma (i.e., dilute).

1	When plasma levels of AVP are high, a small volume of urine is excreted (antidiuresis), and the urine osmolality is greater than that of plasma (i.e., concentrated). AVP is a small peptide that is 9 amino acids in length (arginine is found at position 8). It is synthesized in neuroendocrine cells located within the supraoptic and paraventricular nuclei of the hypothalamus. The synthesized hormone is packaged in granules that are transported down the axon of the cell and stored in nerve terminals located in the neurohypophysis (posterior pituitary). The anatomy of the hypothalamus and pituitary gland are shown in Fig. 35.3 Secretion of AVP by the posterior pituitary can be influenced by several factors. The primary physiological regulators of AVP secretion are (1) the osmolality of body fluids (osmotic) and (2) the volume and pressure of the vascular system (hemodynamic, or nonosmotic). Other factors that can alter AVP secretion include nausea (stimulates), atrial

1	The human form of vasopressin is arginine vasopressin (AVP), which is also known as antidiuretic hormone (ADH). AVP, acting though V1 receptors, causes vascular smooth muscle contraction. As described subsequently, aDiuresis is the term used for excretion of a large volume of urine. This may reflect either excretion of a large volume of water (water diuresis), or excretion of a large amount of solute (solute diuresis). bNeurons within the supraoptic and paraventricular nuclei synthesize either AVP or the related peptide oxytocin. AVP-secreting cells predominate in the supraoptic nucleus, whereas oxytocin-secreting neurons are primarily found in the paraventricular nucleus.

1	626 SECTION7Berne & Levy Physiology • Fig. 35.3 Anatomy of the hypothalamus and pituitary gland (midsagittal section) depicting the pathways for AVP section. Also shown are pathways involved in regulating AVP secretion. Afferent fibers from the baroreceptors are carried in the vagus and glossopharyngeal nerves. The inset box illustrates an expanded view of the hypothalamus and pituitary gland. Vagus and glossopharyngeal nerves Vasomotor center (medulla oblongata) Cerebellum Baroreceptor input Pituitary Posterior lobe AVP Paraventricular neuronsOsmoreceptors Supraoptic neurons Optic chiasm Anterior lobe natriuretic peptide (inhibits), and angiotensin II (stimulates). A number of drugs, prescription and nonprescription, also affect AVP secretion. For example, nicotine stimulates secretion, whereas ethanol inhibits secretion. Osmotic Control of AVP Secretion

1	Osmotic Control of AVP Secretion Changes in the osmolality of body fluids (changes as minor as 1% are sufficient) play the most important role in regulating AVP secretion. The receptors that monitor changes in osmolality of body fluids (termed osmoreceptors) are distinct from the cells that synthesize and secrete AVP, and are located in the organum vasculosum of the lamina terminalis (OVLT) of the hypothalamus. The osmoreceptors sense changes in body fluid osmolality by either shrinking or swelling. Recent studies have provided evidence that transient receptor potential vanilloid (TRVP) cation channels are involved in the response of the cells to changes in body fluid osmolality. The osmoreceptors respond only to solutes in plasma that are effective osmoles (see ). For example, urea is an ineffective osmole when the function of osmoreceptors is considered. Thus elevation of the plasma urea concentration alone has little effect on AVP secretion.

1	When the effective osmolality of the plasma increases, the osmoreceptors send signals to the AVP synthesizing/ secreting cells located in the supraoptic and paraventricular nuclei of the hypothalamus, and AVP synthesis and secretion are stimulated. Conversely, when the effective cAngiotensin II also stimulates AVP secretion; the cells that mediate this response are located in the subfornical organ (SFO).

1	The gene for AVP is found on chromosome 20. It contains approximately 2000 base pairs with three exons and two introns. The gene codes for a preprohormone that consists of a signal polypeptide, the AVP molecule, neurophysin, and a glycopeptide (copeptin). As the cell processes the preprohormone the signal peptide is cleaved off in the rough endoplasmic reticulum. Once packaged in neurosecretory granules, the preprohormone is further cleaved into AVP, neurophysin, and copeptin molecules. The neurosecretory granules are then transported down the axon to the posterior pituitary and stored in the nerve endings until released. When the neurons are stimulated to secrete AVP, the action potential opens Ca++ channels in the nerve terminal, which raises the intracellular [Ca++] and causes exocytosis of the neurosecretory granules. All three peptides are secreted in this process. Neurophysin and copeptin do not have an identified physiological function.

1	osmolality of the plasma is reduced, secretion is inhibited. Because AVP is rapidly degraded in the plasma, circulating levels can be reduced to zero within minutes after secretion is inhibited. As a result the AVP system can respond rapidly to fluctuations in body fluid osmolality. Fig. 35.4A illustrates the effect of changes in plasma osmolality on circulating AVP levels. The slope of the relationship is quite steep and accounts for the sensitivity of this system. The set point of the system is the plasma osmolality value at which AVP secretion begins to increase. Below this set point, virtually no AVP is released. The set point varies

1	CHAPTER 35 Control of Body Fluid Osmolality and Volume among individuals and is genetically determined. In healthy adults it varies from 275 to 290 mOsm/kg H2O (average ≈ 280–285 mOsm/kg H2O). Several physiological factors can also change the set point in a given individual. As discussed later, alterations in blood volume and pressure can shift it. In addition, pregnancy is associated with a decrease in the set point. The mechanism responsible for the set-point shift with pregnancy is not completely understood but is likely due to hormone levels (e.g., relaxin and chorionic gonadotropin) that are elevated during pregnancy. Hemodynamic (Nonosmotic) Control of AVP Secretion

1	A decrease in blood volume or pressure also stimulates AVP secretion. The receptors responsible for this response are located in both the low-pressure (left atrium and large pulmonary vessels) and the high-pressure (aortic arch and carotid sinus) sides of the circulatory system. Because the low-pressure receptors are located in the high-compliance side of the circulatory system (i.e., venous), and because the majority of blood is in the venous side of the circulatory system, these low-pressure receptors can be viewed as responding to overall vascular volume. The high-pressure receptors respond to arterial pressure. Both groups of receptors are sensitive to stretch of the wall of the structure in which they are located (e.g., cardiac atrial and aortic arch) and are termed baroreceptors. Signals from these receptors are carried in afferent fibers of the vagus and glossopharyngeal nerves to the brainstem (solitary tract nucleus of the medulla oblongata), which is part of the center that

1	from these receptors are carried in afferent fibers of the vagus and glossopharyngeal nerves to the brainstem (solitary tract nucleus of the medulla oblongata), which is part of the center that ). Signals are then relayed from the brainstem to the

1	AVP secretory cells of the supraoptic and paraventricular hypothalamic nuclei. The sensitivity of the baroreceptor system is less than that of the osmoreceptors, and a 5% to 10% decrease in blood volume or pressure is required before AVP secretion is stimulated. This is illustrated in Fig. 35.4B . A number of substances have been shown to alter the secretion of AVP through their effects on blood pressure. These include bradykinin and histamine, which lower pressure and thus stimulate AVP secretion, and norepinephrine, which increases blood pressure and inhibits AVP secretion. Alterations in blood volume and pressure also affect the response to changes in body fluid osmolality (see Fig.

1	Alterations in blood volume and pressure also affect the response to changes in body fluid osmolality (see Fig. 35.4C ). With a decrease in blood volume or pressure, the set point is shifted to lower osmolality values and the slope of the relationship is steeper. In terms of survival of the individual this means that faced with circulatory collapse, the kidneys will continue to conserve water, even though by doing so they reduce the osmolality of the body fluids. With an increase in blood volume or pressure, the opposite occurs. The set point is shifted to higher osmolality values and the slope is decreased. AVP Actions on the Kidneys

1	AVP Actions on the Kidneys The primary action of AVP on the kidneys is to enhance absorption of water from the tubular fluid by increasing • Fig. 35.4 Osmotic and hemodynamic (nonosmotic) control of AVP secretion. A, Effect of changes in plasma osmolality (constant blood volume and pressure) on plasma AVP levels. B, Effect of changes in blood volume or pressure (constant plasma osmolality) on plasma AVP levels. C, Interactions between osmolar and blood volume and pressure stimuli on plasma AVP levels.

1	Inadequate release of AVP from the posterior pituitary results in excretion of a large volume of dilute urine (polyuria). To compensate for this loss of water the individual must ingest a large volume of water (polydipsia) to maintain constant body fluid osmolality. If the individual is deprived of water, body fluids will become hyperosmotic. This condition is called central diabetes insipidus or pituitary diabetes insipidus. Central diabetes insipidus can be inherited, although this is rare. It occurs more commonly after head trauma and with brain neoplasms or infections. Individuals with central diabetes insipidus have a urine-concentrating defect that can be corrected by administration of exogenous AVP. The inherited (autosomal dominant) form of central diabetes insipidus is due to numerous mutations in all regions of the AVP gene (i.e., AVP, copeptin and neurophysin). The human placenta produces a cysteine aminopeptidase that degrades AVP. In some women the levels of this

1	to numerous mutations in all regions of the AVP gene (i.e., AVP, copeptin and neurophysin). The human placenta produces a cysteine aminopeptidase that degrades AVP. In some women the levels of this vasopressinase result in diabetes insipidus. The associated polyuria can be treated by administration of the synthetic AVP analogue desmopressin (DDAVP).

1	The syndrome of inappropriate AVP (ADH) secretion (SIADH) is a common clinical problem characterized by plasma AVP levels that are elevated above what would be expected on the basis of body fluid osmolality and blood volume and pressure—hence the term inappropriate AVP (ADH) secretion (this is alternatively named syndrome of inappropriate antidiuresis [SIAD]). In addition the collecting duct overexpresses water channels (see below), thus augmenting the effect of AVP to stimulate water retention by the kidney. Individuals with SIADH retain water, and their body fluids become progressively hypoosmotic. In addition, their urine is more hyperosmotic than expected based on the low body-fluid osmolality. SIADH can be caused by infections and neoplasms of the brain, drugs (e.g., antitumor drugs), pulmonary diseases, and carcinoma of the lung. Many of these conditions stimulate AVP secretion by altering neural input to the AVP secretory cells. By contrast, small cell carcinoma of the lung

1	pulmonary diseases, and carcinoma of the lung. Many of these conditions stimulate AVP secretion by altering neural input to the AVP secretory cells. By contrast, small cell carcinoma of the lung produces and secretes a number of peptides including AVP. Recently, AVP receptor antagonists (e.g., the nonpeptide antagonists conivaptan [Vaprisol] and tolvaptan [Samsca and Jinarc]) have been developed that can be used to treat SIADH and other conditions in which AVP-dependent water retention by the kidneys occurs (e.g., congestive heart failure and hepatic cirrhosis).

1	the water permeability of the latter portion of the distal tubule and collecting duct. In addition, and importantly, AVP increases the permeability of the medullary portion of the collecting duct to urea. Finally, AVP stimulates NaCl reabsorption by the thick ascending limb of Henle’s loop, distal tubule, and collecting duct.

1	In the absence of AVP, the apical membrane of principal cells (see ), located in the latter portion of the distal tubule and along the collecting duct, is relatively impermeable to water. This reflects the fact that in the absence of AVP the apical membrane of these cells contains few water channels (aquaporins). Thus in the absence of AVP, little water is reabsorbed by these nephron segments. Binding of AVP to the V2 receptor located in the basolateral membrane of principal cells results in the insertion of aquaporin (AQP2) water channels into the apical membrane, allowing water to enter the cell from the tubule lumen. This water then exits the cell across the basolateral membrane, which is always freely permeable to water owing to the presence of AQP3 and AQP4 water channels. Thus in the presence of AVP, water is reabsorbed from the tubule lumen.

1	The gene for the V2 receptor is located on the X chromosome. It codes for a 371–amino acid protein that is in the family of receptors that have seven membrane spanning domains and are coupled to heterotrimeric G proteins. As shown in

1	Fig. 35.5 , binding of AVP to its receptor on the basolateral membrane activates adenylcyclase. The increase in intracellular cyclic adenosine monophosphate (cAMP) then activates protein kinase A (PKA), which results in phosphorylation of AQP2 water channels and also results in increased transcription of the AQP2 gene via activation of a cAMP-response element (CRE). Vesicles containing phosphorylated AQP2 move toward the apical membrane along microtubules driven by the molecular motor dynein. Once near the apical membrane, proteins called SNAREs interact with vesicles containing AQP2 and facilitate fusion of these vesicles with the membrane. Addition of AQP2 to the membrane allows water to enter the cell driven by the osmotic gradient (lumen osmolality < cell osmolality). The water then exits the cell across the basolateral membrane through AQP3 and AQP4 water channels, which are constitutively present in the basolateral membrane. When the V2 receptor is not occupied by AVP, the AQP2

1	the cell across the basolateral membrane through AQP3 and AQP4 water channels, which are constitutively present in the basolateral membrane. When the V2 receptor is not occupied by AVP, the AQP2 water channels are removed from the apical membrane by clathrinmediated endocytosis, thus rendering the apical membrane impermeable to water. The endocytosed AQP2 molecules may be either stored in cytoplasmic vesicles, ready for reinsertion into the apical membrane when AVP levels in the plasma increase, or degraded.

1	AVP also regulates long-term expression of AQP2 (and AQP3). When large volumes of water are ingested over an extended period of time (e.g., psychogenic polydipsia), the abundance of AQP2 and AQP3 in principal cells is reduced. As a consequence, when water ingestion is restricted, these individuals cannot maximally concentrate their urine. Conversely, in states of restricted water ingestion, AQP2 and AQP3 protein expression in principal cells increases, thereby facilitating excretion of maximally concentrated urine. It is also clear that expression of AQP2 (and in some instances also AQP3) varies in pathological conditions associated with disturbances in urine concentration and dilution. AQP2 expression is reduced in a number of conditions associated with impaired urine concentrating ability (e.g., hypercalcemia, hypokalemia). By contrast, in conditions associated with water retention (e.g., congestive heart failure, hepatic cirrhosis, pregnancy) AQP2 expression is increased.

1	CHAPTER 35 Control of Body Fluid Osmolality and Volume 629 • Fig. 35.5 Action of AVP via the V2 receptor on the principal cell of the late distal tubule and collecting duct. See text for details. AC, adenylcyclase; cAMP, cyclic adenosine monophosphate; CREB-P, phosphorylated cAMP response element binding protein; CRE, cAMP response element; P, phosphorylated proteins; AQP2, aquaporin 2; AP2, aquaporin 2 gene. (Adapted from Brown D, Nielsen S. The cell biology of vasopressin action. In: Brenner BM [ed]. The Kidney. 7th ed. Philadelphia: Saunders; 2004.)

1	AVP also increases the permeability of the terminal portion of the inner medullary collecting duct to urea. This results in an increase in urea reabsorption and an increase in the osmolality of the medullary interstitial fluid, which as described below is needed for maximal urine concentration. The cells of the collecting duct express two types of urea transporters, UT-A1 and UT-A3. UT-A1 is localized to the apical membrane, and UT-A3 is localized primarily to the basolateral membrane. AVP, acting through the cAMP/PKA cascade, increases expression of both UT-A1 and UT-A3. Increasing the osmolality of the interstitial fluid of the renal medulla also increases the permeability of the inner medullary collecting duct to urea. This effect is mediated by the phospholipase C/protein kinase C (PKC) pathway, which increases UT-A1 and UT-A3 expression.

1	AVP also stimulates reabsorption of NaCl by the thick ascending limb of Henle’s loop and by the distal tubule and cortical segment of the collecting duct. This increase in Na+ reabsorption is associated with increased abundance of three Na+ transporters: the 1Na+/1K+/2Cl− symporter (thick ascending limb of Henle’s loop), the Na+/Cl− symporter (distal tubule), and the Na+ channel (ENaC, in the latter portion of the distal tubule and collecting duct). Stimulation of thick ascending limb NaCl transport may help maintain the hyperosmotic medullary interstitium that is necessary for absorption of water from the medullary portion of the collecting duct (see below).

1	In addition to affecting the secretion of AVP, changes in plasma osmolality and blood volume or pressure lead to alterations in the perception of thirst. When body fluid osmolality is increased or the blood volume or pressure is reduced, the individual perceives thirst. Of these stimuli, hypertonicity is the more potent. An increase in plasma osmolality of only 2% to 3% produces a strong desire to drink, whereas decreases in blood volume and pressure in the range of 10% to 15% are required to produce the same response.

1	As already discussed, there is a genetically determined threshold for AVP secretion (i.e., a body fluid osmolality above which AVP secretion increases). Similarly there is a genetically determined threshold for triggering the sensation of thirst. However, the thirst threshold is higher than the threshold for AVP secretion. On average the threshold for AVP secretion is approximately 285 mOsm/kg H2O, whereas the thirst threshold is approximately 295 mOsm/ kg H2O. Because of this difference, thirst is stimulated at a body fluid osmolality where AVP secretion is already maximal. The neural centers involved in regulating water intake (the thirst center) are located in the same region of the hypothalamus involved with regulating AVP secretion. However, it is not certain whether the same cells serve both functions. Indeed the thirst response, like the regulation of

1	The collecting ducts of some individuals do not respond normally to AVP. These individuals cannot maximally concentrate their urine and consequently have polyuria and polydipsia. This clinical entity is termed nephrogenic diabetes insipidus to distinguish it from central diabetes insipidus. Nephrogenic diabetes insipidus can result from a number of systemic disorders and more rarely occurs as a result of inherited disorders. Many of the acquired forms of nephrogenic diabetes insipidus are the result of decreased expression of AQP2 in the collecting duct. Decreased expression of AQP2 has been documented in the urine concentrating defects associated with hypokalemia, lithium ingestion (35% of individuals who take lithium for bipolar disorder develop some degree of nephrogenic diabetes insipidus), ureteral obstruction, low-protein diet, and hypercalcemia. The inherited forms of nephrogenic diabetes insipidus reflect mutations in the AVP receptor (V2 receptor) gene or the AQP2 gene.

1	insipidus), ureteral obstruction, low-protein diet, and hypercalcemia. The inherited forms of nephrogenic diabetes insipidus reflect mutations in the AVP receptor (V2 receptor) gene or the AQP2 gene. Approximately 90% of hereditary forms of nephrogenic diabetes insipidus are the result of mutations in the V2 receptor gene, with the other 10% the result of mutations in the AQP2 gene. Since the gene for the V2 receptor is located on the X chromosome, these inherited forms of nephrogenic diabetes insipidus are X-linked. The gene coding for AQP2 is located on chromosome 12 and is inherited as both an autosomal recessive as well as an autosomal dominant defect. As noted in , aquaporins exist as homotetramers. This homotetramer formation explains the difference between the two forms of AQP2-related nephrogenic diabetes insipidus. In the recessive form, heterozygotes produce both normal AQP2 and defective AQP2 molecules. The defective AQP2 monomer is retained in the endoplasmic reticulum of

1	nephrogenic diabetes insipidus. In the recessive form, heterozygotes produce both normal AQP2 and defective AQP2 molecules. The defective AQP2 monomer is retained in the endoplasmic reticulum of the cell, and thus the homotetramers that do form only contain normal molecules. Thus mutations in both alleles are required to produce nephrogenic diabetes insipidus. In the autosomal dominant form, the defective monomers can form tetramers with normal monomers as well as defective monomers. However, tetramers containing defective monomers are unable to traffic to the apical membrane.

1	Recently, individuals have been found that have activating (gain-of-function) mutations in the V2 receptor gene. Thus the receptor is constitutively activated even in the absence of AVP. These individuals have laboratory findings similar to those seen in SIADH, including reduced plasma osmolality, hyponatremia (reduced plasma [Na+]), and urine more concentrated than would be expected from the reduced body fluid osmolality. However, unlike SIADH where circulating levels of AVP are elevated and thus responsible for water retention by the kidneys, these individuals have undetectable levels of AVP in their plasma. This new clinical entity has been termed nephrogenic syndrome of inappropriate antidiuresis.

1	AVP secretion, only occurs in response to effective osmoles (e.g., NaCl). Even less is known about the pathways involved in the thirst response to decreased blood volume or pressure, but it is believed that the pathways are the same as those involved in the volume and pressure-related regulation of AVP secretion. Angiotensin II acting on cells of the thirst center also evokes the sensation of thirst. Because angiotensin II levels are increased when blood volume and pressure are reduced, this effect of angiotensin II contributes to the homeostatic response that restores and maintains body fluids at their normal volume.

1	The sensation of thirst is satisfied by the act of drinking, even before sufficient water is absorbed from the gastrointestinal tract to correct the plasma osmolality. It is interesting to note that cold water is more effective in reducing the thirst sensation. Oropharyngeal and upper gastrointestinal receptors appear to be involved in this response. However, relief of the thirst sensation via these receptors is short lived, and thirst is only completely satisfied when the plasma osmolality or blood volume or pressure is corrected.

1	It should be apparent that the AVP and thirst systems work in concert to maintain water balance. An increase in plasma osmolality evokes drinking and, via AVP action on the kidneys, conservation of water. Conversely, when plasma osmolality is decreased, thirst is suppressed and, in the absence of AVP, renal water excretion is enhanced. However, most of the time fluid intake is dictated by cultural factors and social situations. This is especially the case when thirst is not stimulated. In this situation, maintaining normal body fluid osmolality relies solely on the ability of the kidneys to excrete water. How the kidney accomplishes this is discussed in detail in the following sections of this chapter. Renal Mechanisms for Dilution and Concentration of Urine

1	As already noted, water excretion is regulated separately from solute excretion. For this to occur the kidneys must be able to excrete urine that is either hypoosmotic or hyper-osmotic with respect to body fluids. This ability to excrete urine of varying osmolality in turn requires that solute be separated from water at some point along the nephron. As discussed in , reabsorption of solute in the proximal tubule results in reabsorption of a proportional amount of water. Hence solute and water are not separated in this portion of the nephron. Moreover, this proportionality between proximal tubule water and solute reabsorption occurs regardless of whether the kidneys excrete dilute or concentrated urine. Thus the proximal tubule reabsorbs a large portion of the filtered amount of solute and water, but it does not produce dilute or concentrated tubular fluid. The loop of Henle, in particular the thick ascending limb, is the major site where solute and water are separated. Thus excretion

1	water, but it does not produce dilute or concentrated tubular fluid. The loop of Henle, in particular the thick ascending limb, is the major site where solute and water are separated. Thus excretion of both dilute and concentrated urine requires normal function of the loop of Henle.

1	Excretion of hypoosmotic urine is relatively easy to understand. The nephron must simply reabsorb solute from the tubular fluid and not allow water reabsorption to also occur. As just noted, and as described in greater detail later, reabsorption of solute without concomitant water reabsorption occurs in the ascending limb of Henle’s loop. Under appropriate conditions (i.e., in the absence of AVP) the distal tubule and collecting duct also dilute the tubular fluid by reabsorbing solute but not water. Excretion of hyperosmotic urine is more complex and thus more difficult to understand. This process in essence

1	Excretion of hyperosmotic urine is more complex and thus more difficult to understand. This process in essence With adequate access to water, the thirst mechanism can prevent development of hyperosmolality. This mechanism is responsible for the polydipsia seen in response to the polyuria of both central and nephrogenic diabetes insipidus. Most individuals ingest water/beverages even in the absence of the thirst sensation. Normally the kidneys are able to excrete this excess water because they can excrete up to 18 L/day of urine. However, in some instances, the volume of water ingested exceeds the kidneys’ capacity to excrete water, especially over short periods of time. When this occurs, body fluids become hypoosmotic. An example of how water intake can exceed the capacity of the kidneys to excrete water is long-distance running. A study of participants in the Boston Marathon found that 13% of the runners developed hyponatremia during the course of the race.

1	This reflected the practice of some runners of ingesting water or other hypotonic drinks during the race to remain “well hydrated.” In addition, water is produced from the metabolism of glycogen and triglycerides used as fuels by the exercising muscle. Because over the course of the race they ingested (and generated through metabolism) more water than their kidneys were able to excrete or was lost by sweating, hyponatremia developed. In some racers the hyponatremia was severe enough to elicit the neurological symptoms described previously. The maximum amount of water that can be excreted by the kidneys (e.g., 18 L/day) depends on the amount of solute excreted, which in turn depends on food intake. For example, with maximally dilute urine (Uosm = 50 mOsm/kg H2O), the maximum urine output of 18 L/day will be achieved only if the solute excretion rate is 900 mmol/day:

1	If solute excretion is reduced, as commonly occurs in the elderly with reduced food intake, the maximum urine output will decrease. For example, if solute excretion is only 400 mmol/day, a maximum urine output (at Uosm = 50 mOsm/kg H2O) of only 8 L/day can be achieved. Thus individuals with reduced food intake have a reduced capacity to excrete water. dAlmond CS et al. Hyponatremia among runners in the Boston Marathon. N Engl J Med 2005;352:1150-1556.

1	dAlmond CS et al. Hyponatremia among runners in the Boston Marathon. N Engl J Med 2005;352:1150-1556. involves removing water from the tubular fluid without solute. Because water movement is passive, driven by an osmotic gradient, the kidney must generate a hyperosmotic compartment into which water is reabsorbed, without solute, osmotically from the tubular fluid. The hyperosmotic compartment in the kidney that serves this function is the interstitium of the renal medulla. Henle’s loop is critical for generating the hyperosmotic medullary interstitium. Once established, this hyperosmotic compartment drives water reabsorption from the collecting duct and thereby concentrates urine. Fig. 35.6 summarizes tubular fluid osmolality at several points along the nephron, both in the absence and presence

1	Fig. 35.6 summarizes tubular fluid osmolality at several points along the nephron, both in the absence and presence CHAPTER 35 Control of Body Fluid Osmolality and Volume of AVP. Note that tubular fluid entering the loop of Henle from the proximal tubule is isoosmotic with respect to plasma and is so regardless of the absence or presence of AVP. Also, tubular fluid leaving the thick ascending limb is hypoosmotic with respect to plasma, both in the absence and presence of AVP. The osmolality of tubular fluid along the collecting duct is hypoosmotic with respect to plasma in the absence of AVP and becomes progressively hyperosmotic (i.e., from cortex to inner medulla) in the presence of AVP.

1	Establishment and maintenance of the hyperosmotic medullary interstitium has been a subject of study for more than 50 years. Despite this intense study, the most accepted model for how the medullary osmotic gradient is established, especially within the inner medulla, is incomplete and not consistent with more recent experimental findings regarding the transport properties of the nephron segments in this region of the kidney. With the caveat that the current model needs refinement, it is presented here because it embodies some fundamental concepts that underlie the process.

1	In the current model the medullary interstitial osmotic gradient is established by a process termed countercurrent multiplication. By this process, solute (principally NaCl) is reabsorbed without water from the ascending limb of Henle’s loop into the surrounding medullary interstitium. This decreases the osmolality in the tubular fluid and raises the osmolality of the interstitium at this point. The increased osmolality of the interstitium then causes water to be reabsorbed from the descending limb of Henle’s loop, thus increasing the tubular fluid osmolality in this segment. Thus at any point along the loop of Henle the fluid in the ascending limb has an osmolality less than fluid in the adjacent descending limb. This osmotic difference was termed the single effect. Because of the countercurrent flow of tubular fluid in the descending (fluid flowing into the medulla) and ascending (fluid flow out of the medulla) limbs, this single effect could be multiplied, resulting in an osmotic

1	flow of tubular fluid in the descending (fluid flowing into the medulla) and ascending (fluid flow out of the medulla) limbs, this single effect could be multiplied, resulting in an osmotic gradient within the medullary interstitium, where the tip of the papilla has an osmolality of 1200 mOsm/kg H2O compared to 300 mOsm/kg H2O at the corticomedullary junction.

1	Fig. 35.7 schematically depicts the processes for excreting a dilute urine (water diuresis) as well as a concentrated urine (antidiuresis). Three key concepts underlie these processes: 1. Urine is concentrated by AVP-dependent reabsorption of water from the collecting duct. 2. Reabsorption of NaCl from the ascending limb of Henle’s loop dilutes the tubular fluid and at the same time generates a high [NaCl] in the medullary interstitium (up to 600 mmol/L at the tip of the papilla), which then drives water reabsorption from the collecting duct. 3. Urea accumulates in the medullary interstitium (up to 600 mmol/L), which allows the kidneys to excrete urine with the same high urea concentration. This allows large amounts of urea to be excreted with relatively little water. First, how the kidneys excrete dilute urine (water diure sis) when AVP levels are low or zero is considered. The following numbers refer to those encircled in

1	Fig. 35.7A + AVP: 140 − AVP: 140 + AVP: 290 Cortex + AVP: 1200 + AVP: 1200 − AVP: 600 − AVP: 50 • Fig. 35.6 Tubular fluid osmolality along the nephron in the presence (+AVP) and in the absence (−AVP) of arginine vasopressin. See text for details. (Adapted from Sands JM et al. Urine concentration and dilution. In: Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012.) 1. Fluid entering the descending thin limb of the loop of Henle from the proximal tubule is isosmotic with respect to plasma. This reflects the essentially isosmotic nature of solute and water reabsorption in the proximal tubule (see ). (NOTE: Water is reabsorbed from the segments of the proximal tubule via AQP1.) 2.

1	Water is reabsorbed from the thin descending limb of Henle’s loop. Most of this water is reabsorbed in the outer medulla, thereby limiting the amount of water added to the deepest part of the inner medullary interstitial space and thus preserving the hyperosmolality of this region of the medulla. (NOTE: Water is reabsorbed via AQP1.) 3. In the inner medulla the terminal portion of the descending thin limb and all of the thin ascending limb is impermeable to water. (NOTE: AQP1 is not expressed.) These same nephron segments express the Cl− channel CLC-K1, which mediates Cl− reabsorption, with Na+ following via the paracellular pathway. This passive reabsorption of NaCl without concomitant water reabsorption begins the process of diluting the tubular fluid. 4.

1	4. The thick ascending limb of the loop of Henle is also impermeable to water and actively reabsorbs NaCl from the tubular fluid and thereby dilutes it further (see ). Dilution occurs to such a degree that this segment is often referred to as the diluting segment of the kidney. Fluid leaving the thick ascending limb is hypoosmotic with respect to plasma (see Fig. 35.6 5. The distal tubule and cortical portion of the collecting duct actively reabsorb NaCl. In the absence of AVP these segments are not permeable to water (i.e., AQP2 is not present in the apical membrane of the cells). Thus when AVP is absent or present at low levels (i.e., decreased plasma osmolality), the osmolality of tubule fluid in these segments is reduced further because NaCl is reabsorbed without water. Under this condition, fluid leaving the cortical portion of the collecting duct is hypoosmotic with respect to plasma (see Fig. 35.6 6.

1	Fig. 35.6 6. The medullary collecting duct actively reabsorbs NaCl. Even in the absence of AVP, this segment is slightly permeable to water and some water is reabsorbed. 7. The urine has an osmolality as low as approximately 50 mOsm/kg H2O and contains low concentrations of NaCl. The volume of urine excreted can be as much as 18 L/day, or approximately 10% of the glomerular filtration rate (GFR). Next, how the kidneys excrete concentrated urine (antidiuresis) when plasma osmolality and plasma AVP levels are high is considered. The following numbers refer to those encircled in Fig. 35.7B : 1–4. These steps are similar to those for production of dilute urine. An important point in understanding how a concentrated urine is produced is to recognize that while reabsorption of NaCl by the ascending thin and thick limbs of the loop of Henle dilutes the tubular fluid, the reabsorbed NaCl accumulates in the medullary interstitium and raises the osmolality of this compartment.

1	• Fig. 35.7 Schematic of nephron segments involved in urine dilution and concentration. Henle’s loops of juxtamedullary nephrons are shown. A, Mechanism for excretion of dilute urine (water diuresis). AVP) is absent and the collecting duct is essentially impermeable to water. Note also that during a water diuresis the osmolality of the medullary interstitium is reduced as a result of increased vasa recta blood flow and entry of some urea into the medullary collecting duct. B, Mechanism for excretion of a concentrated urine (antidiuresis). Plasma AVP levels are maximal and the collecting duct is highly permeable to water. Under this condition the medullary interstitial gradient is maximal. See text for details.

1	Accumulation of NaCl in the medullary interstitium is crucial for production of urine hyperosmotic to plasma, because it provides the osmotic driving force for water reabsorption by the medullary collecting duct. As already noted, AVP stimulates NaCl reabsorption by the thick ascending limb of Henle’s loop. This is thought to maintain the medullary interstitial gradient at a time when water is being added to this compartment from the medullary collecting duct, which would tend to dissipate the gradient. 5.

1	5. Because of NaCl reabsorption by the ascending limb of the loop of Henle, the fluid reaching the collecting duct is hypoosmotic with respect to the surrounding interstitial fluid. Thus an osmotic gradient is established across the collecting duct. In the presence of AVP, which increases the water permeability of the latter portion of the distal tubule and the collecting duct by causing insertion of AQP2 into the luminal membrane of the cells, water diffuses out of the tubule lumen and the tubule fluid osmolality increases. This diffusion of water out of the lumen of the collecting duct begins the process of urine concentration. The maximum osmolality the fluid in the distal tubule and cortical portion of the collecting duct can attain is approximately 290 mOsm/kg H2O (i.e., the same as plasma), which is the osmolality of the interstitial fluid and plasma within the cortex of the kidney. 6.

1	6. As the tubular fluid descends deeper into the medulla, water continues to be reabsorbed from the collecting duct, increasing the tubular fluid osmolality to 1200 mOsm/ kg H2O at the tip of the papilla. 7. The urine produced when AVP levels are elevated has an osmolality of 1200 mOsm/kg H2O and contains high concentrations of urea and other nonreabsorbed solutes. Urine volume under this condition can be as low as 0.5 L/day.

1	In comparing the two conditions just described, it should be apparent that a relatively constant volume of dilute tubular fluid is delivered to the AVP-sensitive portions of the nephron (latter portion of the distal tubule and collecting duct). Plasma AVP levels then determine the amount of water reabsorbed by these segments. When AVP levels are low, a relatively small volume of water is reabsorbed by these segments and a large volume of hypoosmotic urine is excreted (up to 10% of the filtered water). When AVP levels are high, a large volume of water is reabsorbed by these same segments and a small volume of hyperosmotic urine is excreted (<1% of filtered water). During antidiuresis, most of the water is reabsorbed in the distal tubule and cortical and outer medullary portions of the collecting duct. Thus a relatively small volume of fluid reaches the inner medullary collecting duct where it is then reabsorbed. This distribution of water reabsorption along the length of the collecting

1	duct. Thus a relatively small volume of fluid reaches the inner medullary collecting duct where it is then reabsorbed. This distribution of water reabsorption along the length of the collecting duct (i.e., cortex > outer medulla > inner medulla) allows for maintenance of a hyperosmotic interstitial environment in the inner medulla by minimizing the amount of water entering this compartment.

1	• Fig. 35.8 The medullary interstitial gradient comprises primarily NaCl and urea. The concentrations for NaCl and urea depicted reflect those found in the antidiuretic state (i.e., excretion of hyperosmotic urine). See text for details. (Adapted from Sands JM et al. Urine concentration and dilution. In: Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Elsevier; 2012.) 100200300400500600Concentration(mmol/L)CortexO termed llaInnerMed llaUreaNacl

1	As noted earlier, the interstitial fluid of the renal medulla is critically important in concentrating urine. The osmotic pressure of the interstitial fluid provides the driving force for reabsorbing water from both the descending thin limb of the loop of Henle and the collecting duct. The principal solutes of the medullary interstitial fluid are NaCl and urea, but the concentration of these solutes is not uniform throughout the medulla (i.e., a gradient exists from cortex to papilla). Other solutes also accumulate in the medullary interstitium (e.g., NH4 + and K+), but the most abundant solutes are NaCl and urea. For simplicity, this discussion assumes that NaCl and urea are the only solutes.

1	Fig. 35.8 , NaCl and urea accumulate in the renal medulla, and the interstitial fluid at the tip of the papilla of the inner medulla reaches a maximum osmolality of 1200 mOsm/kg H2O, with approximately 600 mOsm/kg H2O attributable to NaCl (300 mmol/L) and 600 mOsm/ kg H2O attributable to urea (600 mmol/L). Establishment of the NaCl gradient is essentially complete at the transition between the outer and inner medulla. The medullary gradient for NaCl results from accumulation of NaCl reabsorbed by the nephron segments in the medulla during countercurrent multiplication. The most important segment in this regard is the ascending limb of the loop of Henle. Urea accumulation within the medullary interstitium is more complex and occurs most effectively when hyperosmotic urine is excreted (i.e., antidiuresis). When dilute urine is produced, especially over extended periods, the osmolality of the medullary interstitium declines (see

1	Fig. 37.7A ). This reduced osmolality is almost entirely caused by a decrease in the concentration of urea. This decrease reflects washout by the vasa recta (discussed later) and diffusion of urea from the interstitium into the tubular fluid within the medullary portion of the collecting duct, which is permeable to urea even in the absence of AVP. (NOTE: The cortical and outer medullary portions of the collecting duct have a low permeability to urea, whereas the inner medullary portion has a relatively high permeability because of the presence of the urea transporters UT-A1 and UT-A3, the expression of which is increased by AVP.) Some of this reabsorbed urea is secreted into the thin descending limbs of Henle’s loops via the urea transporter UT-A2, and some enters the vasa recta via the UT-B transporter. The urea that is secreted into the descending thin limbs of Henle’s loops is then trapped in the nephron until it again reaches the medullary collecting duct, where it can reenter the

1	transporter. The urea that is secreted into the descending thin limbs of Henle’s loops is then trapped in the nephron until it again reaches the medullary collecting duct, where it can reenter the medullary interstitium. Thus urea recycles from the interstitium to the nephron and back into the interstitium. This process of urea recycling facilitates accumulation of urea in the medullary interstitium, where it can attain a concentration at the tip of the papilla of 600 mmol/L.

1	As described, the hyperosmotic medulla is essential for concentrating the tubular fluid within the collecting duct. Because water reabsorption from the collecting duct is driven by the osmotic gradient established in the medullary interstitium, urine can never be more concentrated than that of the interstitial fluid in the papilla. Thus any condition that reduces the medullary interstitial osmolality impairs the ability of the kidneys to maximally concentrate urine. Urea within the medullary interstitium contributes to the total osmolality of the urine. However, because the inner medullary collecting duct is highly permeable to urea, especially in the presence of AVP, urea cannot drive water reabsorption across this nephron segment. Instead, urea in the tubular fluid and medullary interstitium equilibrate, and a small volume of urine with a high concentration of urea is excreted.

1	It is the medullary interstitial NaCl concentration that is responsible for reabsorbing water from the medullary collecting duct and thereby concentrating the nonurea solutes (e.g., NH4 + salts, K+ salts, creatinine) in the urine.

1	The vasa recta, the capillary networks that supply blood to the medulla, are highly permeable to solute and water. As with the loop of Henle, the vasa recta form a parallel set of hairpin loops within the medulla (see ). Not only do the vasa recta bring nutrients and oxygen to the medullary nephron segments, but more importantly they also remove the excess water and solute that is continuously added to the medullary interstitium by these nephron seg eOn a typical diet the kidneys must excrete 450 mmol/day of urea. At a maximal urine [urea] of 600 mmol/L this amount of urea can be excreted in less than 1 L of urine. However, if the maximal urine [urea] is reduced because of a decrease in the medullary interstitial fluid [urea], a larger urine volume would be needed to excrete the 450 mmol/day of urea (e.g., 2.25 L of urine would be required if the maximal urine [urea] was only 200 mM).

1	CHAPTER 35 Control of Body Fluid Osmolality and Volume ments. The ability of the vasa recta to maintain the medullary interstitial gradient is flow dependent. A substantial increase in vasa recta blood flow dissipates the medullary gradient (i.e., washout of osmoles from the medullary interstitium). Alternatively, reduced blood flow reduces oxygen delivery to the nephron segments within the medulla. Because transport of salt and other solutes requires oxygen and ATP, reduced medullary blood flow decreases salt and solute transport by nephron segments in the medulla. As a result the medullary interstitial osmotic gradient cannot be maintained. Assessment of Renal Diluting and Concentrating Ability

1	Assessment of Renal Diluting and Concentrating Ability Assessment of renal water handling includes measurements of urine osmolality and the volume of urine excreted. The range of urine osmolality is from 50 to 1200 mOsm/kg H2O. The corresponding range in urine volume is 18 L to as little as 0.5 L/day. These ranges are not fixed and vary from individual to individual. They can also be affected by disease processes, and as noted previously they are dependent on the amount of solute the kidneys must also excrete. The ability of the kidneys to dilute or concentrate urine requires the separation of solute and water (i.e., the single effect of the countercurrent multiplication process). This separation of solute and water in essence generates a volume of water that is “free of solute.” When urine is dilute, solute-free water is excreted from the body. When urine is concentrated, solute-free water is returned to the body (i.e., conserved).

1	For the kidneys to maximally excrete solute-free water (i.e., 18 L/day) the following conditions must be met: 1. AVP must be absent; without it the collecting duct does not reabsorb a significant amount of water. 2. The tubular structures that separate solute from water (i.e., dilute the tubule fluid) must function normally. In the absence of AVP the following nephron segments can dilute the luminal fluid: ascending thin limb of Henle’s loop thick ascending limb of Henle’s loop Because of its high transport rate, the thick ascending limb is quantitatively the most important nephron segment involved in the separation of solute and water.

1	Because of its high transport rate, the thick ascending limb is quantitatively the most important nephron segment involved in the separation of solute and water. 3. An adequate amount of tubular fluid must be delivered to the aforementioned nephron sites for maximal separation of solute and water. Factors that reduce delivery (e.g., decreased GFR or enhanced proximal tubule reabsorption) impair the kidneys’ ability to maximally excrete solute-free water. Similar requirements also apply to conservation of water by the kidneys. For the kidneys to conserve water maximally (6–8 L/day) the following conditions must be met: 1. An adequate amount of tubular fluid must be delivered to those nephron segments that separate solute from water; the most important segment in this regard is the

1	In response to elevated AVP levels In response to decreased AVP levelsthe kidneys retain 1 L of water the kidneys excrete 1 L of waterreturning osmolality and [Na˜] returning osmolality and [Na˜]to normal, but increasing ECF volume. to normal, but reducing ECF volume.

1	• Fig. 35.9 Impact of changes in Na+ balance on ECFV. (1) Addition of NaCl (without water) to the ECF increases the [Na+] and osmolality. (2) The increase in ECF osmolality stimulates secretion of AVP from the posterior pituitary, which than acts on the kidneys to conserve water. (3) Decreased renal excretion of water together with water ingestion restore plasma osmolality and plasma [Na+] to normal. However, the ECFV is now increased by 1 L. (4) Removal of NaCl (without water) from the ECF decreases plasma [Na+] and plasma osmolality. (5) The decrease in ECF osmolality inhibits AVP secretion. In response to the decrease in plasma AVP the kidneys excrete water. (6) Increased renal excretion of water returns the plasma [Na+] and plasma osmolality to normal. However, ECFV is now decreased by 1 L. As illustrated, changes in Na+ balance alter ECFV because of the efficiency of the AVP system to maintain normal body fluid osmolality. (Adapted from Koeppen BM, Stanton BA. Renal Physiology.

1	by 1 L. As illustrated, changes in Na+ balance alter ECFV because of the efficiency of the AVP system to maintain normal body fluid osmolality. (Adapted from Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) thick ascending limb of Henle’s loop. Delivery of tubular fluid to Henle’s loop depends on GFR and proximal tubule reabsorption.

1	2. Reabsorption of NaCl by the nephron segments must be normal; again, the most important segment is the thick ascending limb of Henle’s loop. 3. A hyperosmotic medullary interstitium must be present. The interstitial fluid osmolality is maintained by NaCl reabsorption by Henle’s loop (conditions 1 and 2) and by effective accumulation of urea. Urea accumulation in turn depends on adequate dietary protein intake. 4. Maximum levels of AVP must be present and the collecting duct must respond normally to AVP. Control of Extracellular Fluid Volume and Regulation of Renal NaCl Excretion The major solutes of ECF are the salts of Na+ (see also the major determinant of ECF osmolality, alterations in Na+ balance are commonly assumed to disturb ECF osmolality. However, under normal circumstances this is not the case because the AVP and thirst systems maintain body fluid osmolality within a very narrow range (discussed earlier). As illustrated in

1	Fig. 35.9 , adding or removing NaCl from ECF changes this body fluid compartment’s volume and not the [Na+] (compare initial condition and final conditions). For example, addition of NaCl to the ECF (without water) increases the [Na+] and osmolality of this compartment. (ICF osmolality also increases because of osmotic equilibration with the ECF.) In response, AVP secretion and thirst are stimulated, and as a result water is ingested and renal water loss is reduced. This restores plasma osmolality (and serum [Na+]) to their initial values, but the volume of the ECF is now increased. The opposite occurs when NaCl is lost from the ECF. Changes in ECF volume (ECFV) can be monitored by measuring body weight, because 1 L of ECF equals 1 kg of ). Of these, NaCl is the most abundant. Because NaCl is body weight.

1	The kidneys are the major route for excretion of NaCl from the body. Only about 10% of the Na+ lost from the body each day does so by nonrenal routes (e.g., in perspiration and feces). As such, the kidneys are critically important in regulating ECFV. Under normal conditions the kidneys keep ECFV constant (a state termed euvolemia) by adjusting the excretion of NaCl to match the amount ingested in the diet. If ingestion exceeds excretion, ECFV increases above normal (volume expansion), whereas the opposite occurs if excretion exceeds ingestion (volume contraction).

1	The typical diet contains approximately 140 mEq/day of Na+ (8 g of NaCl), and thus daily Na+ excretion in urine is also about 140 mEq/day. However, the kidneys can vary excretion of Na+ over a wide range. Excretion rates as low as 10 mEq/day can be attained when individuals are placed on a low-salt diet. Conversely, the kidneys can increase their excretion rate to more than 1000 mEq/day when challenged by ingestion of a high-salt diet. These changes in Na+ excretion can occur with only modest changes in the ECFV and Na+ content of the body.

1	The response of the kidneys to abrupt changes in NaCl intake typically takes several hours to several days, depending on the magnitude of the change. During this transition period the intake and excretion of Na+ are not matched as they are in the steady state. Thus the individual experiences either positive Na+ balance (intake > excretion) or negative Na+ balance (intake < excretion). However, by the end of the transition period a new steady state is established and intake once again equals excretion. This section reviews the physiology of the receptors that monitor ECFV and explains the various signals that act on the kidneys to regulate NaCl excretion and thereby ECFV. In addition, responses of the various portions of the nephron to these signals is considered.

1	Concept of Effective Circulating Volume , the ECF is subdivided into two compartments: blood plasma and interstitial fluid. Plasma volume is a determinant of vascular volume and thus blood pressure and cardiac output. Maintenance of Na+ balance, and thus ECFV, involves a complex system of sensors and effector signals that act primarily on the kidneys to regulate NaCl excretion. As can be appreciated from the dependency of vascular volume, blood pressure, and cardiac output on ECFV, this complex system is designed to ensure adequate tissue perfusion. Because the primary sensors of this system are located in the large vessels of the vascular system, changes in vascular volume, blood pressure, and cardiac output are the principal factors regulating renal NaCl excretion (discussed later). In a healthy individual, changes in ECFV result in parallel changes in vascular volume, blood pressure, and cardiac output. Thus a decrease in ECFV results in reduced vascular volume, blood pressure, and

1	healthy individual, changes in ECFV result in parallel changes in vascular volume, blood pressure, and cardiac output. Thus a decrease in ECFV results in reduced vascular volume, blood pressure, and cardiac output. Conversely, an increase in ECFV results in increased vascular volume, blood pressure, and cardiac output. The degree to which these cardiovascular parameters

1	CHAPTER 35 Control of Body Fluid Osmolality and Volume change is dependent upon the degree of ECFV contraction or expansion and the effectiveness of cardiovascular reflex mechanisms (see ). When a person is in negative Na+ balance, ECFV is decreased and renal NaCl excretion is reduced. Conversely, with positive Na+ balance there is an increase in ECFV, which results in enhanced renal NaCl excretion (i.e., natriuresis).

1	However, in some pathological conditions (e.g., congestive heart failure, hepatic cirrhosis), renal excretion of NaCl does not reflect the ECFV. In both of these situations the volume of the ECF is increased. However, instead of increased renal NaCl excretion, as would be expected, renal excretion of NaCl is reduced. To explain renal Na+ handling in these situations, it is necessary to understand the concept of effective circulating volume (ECV). Unlike ECF, ECV is not a measurable and distinct body fluid compartment. Effective circulating volume refers to that portion of the ECF that is contained within the vascular system and is “effectively” perfusing the tissues (effective blood volume and effective arterial blood volume are other commonly used terms). More specifically the ECV reflects the activity of volume sensors located in the vascular system (discussed later).

1	Patients with congestive heart failure frequently have an increase in ECFV that manifests as increased plasma volume and accumulation of interstitial fluid in the lungs (pulmonary edema) and peripheral tissues (generalized edema). This excess fluid is the result of NaCl and water retention by the kidneys. The kidneys’ response (i.e., retention of NaCl and water) is paradoxical because the ECFV is increased. However, this fluid is not in the vascular system but rather in the interstitial fluid compartment. In addition, blood pressure and cardiac output may be reduced because of poor cardiac performance. Therefore the sensors located in the vascular system respond as they do in ECFV contraction and cause NaCl and water retention by the kidneys. In this situation the ECV, as monitored by the volume sensors, is decreased.

1	In healthy individuals, ECV varies directly with ECFV and in particular the volume of the vascular system (arterial and venous), the arterial blood pressure, and cardiac output. However, as noted this is not the case in certain pathological conditions. In the remaining sections of this chapter the relationship between ECFV and renal NaCl excretion in healthy adults—where changes in ECV and ECFV occur in parallel—are examined. ECFV (or ECV) is monitored by multiple sensors. A number of these sensors are located in the vascular system, and they monitor its fullness and pressure. These receptors are typically called vascular volume receptors, or because they respond to pressure-induced stretch of the walls of the structure in which they are located (e.g., blood vessels or the cardiac atria and ventricles), they are also referred to as baroreceptors. Volume Sensors in the Low-Pressure Cardiopulmonary Circuit

1	Volume Sensors in the Low-Pressure Cardiopulmonary Circuit Volume sensors (i.e., baroreceptors) are located within the walls of the left and right atria, right ventricle, and large pulmonary vessels, and they respond to distention of these structures (see 19). Because the low-pressure side of the circulatory system has a high compliance, these sensors respond mainly to the “fullness” of the vascular system. These baroreceptors send signals to the brainstem via afferent fibers in the glossopharyngeal and vagus nerves (cranial nerves IX and X). The activity of these sensors modulates both sympathetic nerve outflow and AVP secretion. For example, a decrease in filling of the pulmonary vessels and cardiac atria increases sympathetic nerve activity and stimulates AVP secretion. Conversely, distention of these structures decreases sympathetic nerve activity. In general, 5% to 10% changes in blood volume and pressure are necessary to evoke a response.

1	The cardiac atria possess an additional mechanism related to control of renal NaCl excretion. The myocytes of the atria synthesize and store a peptide hormone, atrial natriuretic peptide (ANP). It is released when the atria are distended and, via mechanisms outlined later in this chapter, reduces blood pressure and increases excretion of NaCl and water by the kidneys. The ventricles of the heart also produce a natriuretic peptide, brain natriuretic peptide (BNP), so named because it was first isolated from the brain. Like ANP, BNP is released from myocytes by distention of the ventricles. Its actions are similar to those of ANP. Volume Sensors in the High-Pressure Arterial Circuit

1	Volume Sensors in the High-Pressure Arterial Circuit Baroreceptors are also present in the arterial side of the circulatory system, located in the wall of the aortic arch, carotid sinus, and afferent arterioles of the kidneys. The aortic arch and carotid baroreceptors send input to the brainstem via afferent fibers in the glossopharyngeal and vagus nerves (cranial nerves IX and X). The response to this input alters sympathetic outflow and AVP secretion. Thus a decrease in blood pressure increases sympathetic nerve activity and AVP secretion. An increase in pressure tends to reduce sympathetic nerve activity (and activate parasympathetic nerve activity). The sensitivity of the high-pressure baroreceptors is similar to that in the low-pressure side of the vascular system; 5% to 10% changes in pressure are needed to evoke a response.

1	The juxtaglomerular apparatus (JGA) of the kidneys (see ), particularly the afferent arteriole, responds fThe liver and central nervous system also have sensors that respond to changes in blood pressure and [Na+] and then signal the kidneys to alter NaCl excretion. These systems do not appear to be as important as vascular receptors in monitoring changes in ECFV and effecting changes in renal NaCl excretion and are not considered here. directly to changes in pressure. If perfusion pressure in the afferent arteriole is reduced, renin is released from the granular cells. By contrast, renin secretion is suppressed when perfusion pressure is increased. As described later in this chapter, renin determines blood levels of angiotensin II and aldosterone, both of which reduce renal NaCl excretion.

1	Constriction of a renal artery (e.g., by an atherosclerotic plaque) reduces perfusion pressure to that kidney. This reduced perfusion pressure is sensed by the afferent arteriole of the JGA and results in renin secretion. The elevated renin levels increase the production of angiotensin II, which in turn increases systemic blood pressure via its vasoconstrictor affect on arterioles throughout the vascular system. The increased systemic blood pressure is sensed by the JGA of the contralateral kidney (i.e., the kidney without stenosis of its renal artery), and renin secretion from that kidney is suppressed. In addition the high levels of angiotensin II also inhibit renin secretion by the contralateral kidney (negative feedback). Treatment of patients with constricted renal arteries includes surgical repair of the stenotic artery, administration of angiotensin II receptor blockers, or administration of an inhibitor of angiotensin-converting enzyme (ACE). The ACE inhibitor blocks

1	includes surgical repair of the stenotic artery, administration of angiotensin II receptor blockers, or administration of an inhibitor of angiotensin-converting enzyme (ACE). The ACE inhibitor blocks conversion of angiotensin I to angiotensin II.

1	When the vascular volume sensors have detected a change in ECFV, they send signals to the kidneys, which results in an appropriate adjustment in NaCl and water excretion. Accordingly, when the ECFV is expanded, renal NaCl and water excretion are increased. Conversely, when the ECFV is contracted, renal NaCl and water excretion are reduced. The signals involved in coupling the volume sensors to the kidneys are both neural and hormonal. These are summarized in Box 35.1 , as are their effects on renal NaCl and water excretion. , sympathetic nerve fibers innervate the afferent and efferent arterioles of the glomerulus as well as the cells of the nephron. With ECFV contraction, activation of the lowand high-pressure vascular baroreceptors results in stimulation of sympathetic nerve activity, including those fibers innervating the kidneys. This stimulation has the following effects: 1.

1	The afferent and efferent arterioles are constricted (mediated by α-adrenergic receptors). This vasoconstriction (the effect is greater on the afferent arteriole) decreases the hydrostatic pressure within the glomerular capillary lumen, which results in a decrease in GFR. With this decrease in GFR, the filtered amount of Na+ is reduced. 2. Renin secretion is stimulated by the cells of the afferent arterioles (mediated by β-adrenergic receptors). As • BOX 35.1 Signals Involved in Control of Renal NaCl and Water Excretion Renal Sympathetic Nerves (↑Activity: reabsorption along the nephron Renin-Angiotensin-Aldosterone (↑Secretion: II stimulates Na+ reabsorption along the nephron stimulates Na+ reabsorption in the distal tubule and collecting duct and to a lesser degree in the thick ascending limb of Henle’s loop

1	II stimulates Na+ reabsorption along the nephron stimulates Na+ reabsorption in the distal tubule and collecting duct and to a lesser degree in the thick ascending limb of Henle’s loop Natriuretic Peptides: ANP, BNP & Urodilatin (↑Secretion: ↑NaCl Excretion) and water reabsorption by the collecting duct secretion and inhibition of AVP action on the distal tubule and collecting duct AVP (↑Secretion: ↓H2O Excretion) ↑H2O reabsorption by the distal tubule and collecting duct described later, renin ultimately increases the circulating levels of angiotensin II and aldosterone, both of which stimulate Na+ reabsorption by the nephron.

1	3. NaCl reabsorption along the nephron is directly stimulated (mediated by α-adrenergic receptors on the cells of the nephron). Because of the large amount of Na+ reabsorbed by the proximal tubule, the effect of increased sympathetic nerve activity is quantitatively most important for this segment. As a result of these actions, increased renal sympathetic nerve activity decreases NaCl excretion, an adaptive response that works to restore ECFV to normal. With ECFV expansion, renal sympathetic nerve activity is reduced. This generally reverses the effects just described. Cells in the afferent arterioles (juxtaglomerular cells, also known as granular cells) are the site of synthesis, storage, and release of the proteolytic enzyme renin. Three factors are important in stimulating renin secretion: 1.

1	Perfusion pressure. The afferent arteriole behaves as a high-pressure baroreceptor. When perfusion pressure to the kidneys is reduced, renin secretion is stimulated. Conversely, an increase in perfusion pressure inhibits renin release. 2. Sympathetic nerve activity. Activation of the sympathetic nerve fibers that innervate the afferent arterioles increases renin secretion (mediated by β-adrenergic receptors). CHAPTER 35 Control of Body Fluid Osmolality and Volume Renin secretion is decreased as renal sympathetic nerve activity is decreased.

1	Renin secretion is decreased as renal sympathetic nerve activity is decreased. 3. Delivery of NaCl to the macula densa. Delivery of NaCl to the macula densa regulates GFR by a process termed tubuloglomerular feedback (see ). In addition the macula densa plays a role in renin secretion. When NaCl delivery to the macula densa is decreased, renin secretion is enhanced. Conversely, an increase in NaCl delivery inhibits renin secretion. It is likely that macula densa–mediated renin secretion helps maintain systemic arterial pressure under conditions of a reduced vascular volume. For example, when vascular volume is reduced, perfusion of body tissues (including the kidneys) decreases. This in turn decreases GFR and the filtered amount of NaCl. The reduced delivery of NaCl to the macula densa stimulates renin secretion, which acts through angiotensin II (a potent vasoconstrictor) to increase blood pressure and thereby maintain tissue perfusion.

1	Although many tissues express renin (e.g., brain, heart, adrenal gland), juxtaglomerular (JG) cells are the primary source of circulating renin; they are located in the afferent arterioles of the kidneys. Renin secretion is stimulated by a decrease in intracellular [Ca++], a response opposite that of most secretory cells, where secretion is stimulated by an increase in intracellular [Ca++]. Renin secretion is also stimulated by an increase in intracellular cAMP levels. Conversely, anything that increases intracellular [Ca++] will inhibit renin secretion. Renin secretion is also inhibited by increases in intracellular cyclic guanosine monophosphate (cGMP).

1	Stretch of the afferent arteriole, angiotensin-II, and endothelin increase intracellular [Ca++] and thus inhibit renin secretion by JG cells. The stimulatory effect of sympathetic nerve activity on renin secretion is mediated by norepinephrine, which increases intracellular cAMP (via β-adrenergic receptors). Prostaglandin E2 also increases JG cell cAMP levels and therefore stimulates renin secretion. Natriuretic peptides and nitric oxide (NO) inhibit renin secretion by increasing intracellular cGMP. Control of renin secretion by the macula densa is complex and appears to involve several paracrine factors, including ATP, adenosine, and prostaglandin E2 (see

1	Fig. 35.10 summarizes the essential components of the renin-angiotensin-aldosterone system (RAAS). Renin alone does not have a physiological function; it functions solely as a proteolytic enzyme. Its substrate is a circulating protein, angiotensinogen, which is produced by the liver. Angiotensinogen is cleaved by renin to yield a 10–amino acid peptide, angiotensin I. Angiotensin I also has no known physiological function, and it is cleaved to an 8–amino acid peptide, angiotensin II, by angiotensin-converting enzyme (ACE) found on the surface of vascular endothelial cells. Lung and renal endothelial cells are important sites for the conversion of angiotensin I to angiotensin II. ACE also • Fig. 35.10 Schematic representation of the essential components of the renin-angiotensin-aldosterone system (RAAS). Activation of this system results in a decrease in excretion of Na+ and water by the kidneys. NOTE: Angiotensin I is converted to angiotensin II by an angiotensin-converting enzyme that

1	(RAAS). Activation of this system results in a decrease in excretion of Na+ and water by the kidneys. NOTE: Angiotensin I is converted to angiotensin II by an angiotensin-converting enzyme that is present on all vascular endothelial cells. As shown, the endothelial cells within the lungs play a significant role in this conversion process. See text for details.

1	degrades bradykinin, a potent vasodilator. Angiotensin II has several important physiological functions: 1. stimulation of aldosterone secretion by the adrenal cortex 2. arteriolar vasoconstriction, which increases blood pressure 3. stimulation of AVP secretion and thirst 4. enhancement of NaCl reabsorption by the proximal tubule, thick ascending limb of Henle’s loop, the distal tubule, and the collecting duct (Of these segments the effect on the proximal tubule is quantitatively the largest.) Angiotensin II is an important secretagogue for aldoste rone. An increase in plasma K+ concentration is the other important stimulus for aldosterone secretion (see ). Aldosterone is a steroid hormone produced by the glomerulosa cells of the adrenal cortex and acts in a number of ways on the kidneys (see also

1	Chapters 34, 36, and ). With regard to regulation of the ECFV, aldosterone reduces NaCl excretion by stimulating its reabsorption by several nephron segments. Most importantly it stimulates NaCl reabsorption in the aldosterone-sensitive distal nephron (ASDN), which consists of the latter portion of the distal tubule and the collecting duct. To a lesser extent it also stimulates NaCl reabsorption by the thick ascending limb of Henle’s loop and the early portion of the distal tubule. Aldosterone sensitivity is conferred by the presence of mineralocorticoid receptors as well as the enzyme 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2). Because the mineralocorticoid receptor also binds glucocorticoids, 11β-HSD2 is required for aldosterone specificity because it metabolizes glucocorticoids and thus prevents them from binding to the mineralocorticoid receptor. The aldosterone-sensitive distal nephron expresses both the mineralocorticoid receptor and 11β-HSD2.

1	). In the ASDN it increases the abundance and activity of ENaC in the apical membrane of principal cells. This increases Na+ entry into cells across the apical membrane. Extrusion of Na+ from cells across the basolateral membrane occurs by Na+,K+-ATPase, the abundance of which is also increased by aldosterone. Thus aldosterone increases reabsorption of NaCl from the tubular fluid by distal nephron segments, whereas reduced levels of aldosterone decreases the amount of NaCl reabsorbed by these segments. Although the principal site of action of aldosterone is the ASDN, aldosterone also increases the abundance of the Na+/Cl− symporter in the distal tubule and the Na+/K+/2Cl− symporter in the thick ascending limb of Henle’s loop. Box 35.1 , activation of the RAAS, as occurs with ECFV depletion, decreases excretion of NaCl

1	Diseases of the adrenal cortex can alter aldosterone levels and thereby impair the ability of the kidneys to maintain Na+ balance and euvolemia. With decreased secretion of aldosterone (hypoaldosteronism), the reabsorption of NaCl, mainly by the aldosterone-sensitive distal nephron, is reduced and NaCl is lost in the urine. Because urinary NaCl loss can exceed the amount of NaCl ingested in the diet, negative Na+ balance ensues and ECFV decreases. In response to ECFV contraction, sympathetic tone is increased and levels of renin, angiotensin II, and AVP are elevated. With increased aldosterone secretion (hyperaldosteronism) the effects are the opposite; NaCl reabsorption by the aldosterone-sensitive distal nephron is enhanced and excretion of NaCl is reduced. Consequently, ECFV is increased, sympathetic tone is decreased, and levels of renin, angiotensin II, and AVP are decreased. As described later, ANP and BNP levels are also elevated in this setting.

1	by the kidneys. The RAAS is suppressed with ECFV expansion, and renal NaCl excretion is therefore enhanced. The body produces a number of substances that act on the kidneys to increase NaCl excretion (see ). Of these, natriuretic peptides produced by the heart and kidneys are best understood and will be the focus of the following discussion. The heart produces two natriuretic peptides. Atrial myocytes produce and store the peptide hormone ANP, and ventricular myocytes produce and store BNP. Both peptides are secreted when the heart dilates (i.e., during volume expansion, with heart failure), and they relax vascular smooth muscle and promote NaCl and water excretion by the kidneys. The kidneys also produce a related natriuretic peptide termed urodilatin. Its actions are limited to promoting NaCl excretion by the kidneys. In general the actions of these natriuretic peptides as they relate to renal NaCl and water excretion antagonize those of the RAAS. These actions include: 1.

1	Vasodilation of the afferent and vasoconstriction of the efferent arterioles of the glomerulus. This increases the GFR and the filtered amount of NaCl. 2. Inhibition of renin secretion by the afferent arterioles. 3. Inhibition of aldosterone secretion by the glomerulosa cells of the adrenal cortex. This occurs via two mechanisms: (a) inhibition of renin secretion by the juxtaglomerular cells, thereby reducing angiotensin II– induced aldosterone secretion, and (b) direct inhibition of aldosterone secretion by the glomerulosa cells of the adrenal cortex. 4. Inhibition of NaCl reabsorption by the collecting duct, which is also caused in part by reduced levels of aldosterone. However, natriuretic peptides increase cGMP, which inhibits cation channels in the apical membrane of medullary collecting duct cells and thereby decreases NaCl reabsorption.

1	CHAPTER 35 Control of Body Fluid Osmolality and Volume 5. Inhibition of AVP secretion by the posterior pituitary and AVP action on the collecting duct. These effects decrease water reabsorption by the collecting duct and thus increase excretion of water in the urine. The net effect of natriuretic peptides is to increase excre tion of NaCl and water by the kidneys. Hypothetically a reduction in circulating levels of these peptides would be expected to decrease NaCl and water excretion, but convincing evidence for this has not been reported. As discussed previously, a decreased ECFV stimulates AVP secretion by the posterior pituitary. Elevated levels of AVP decrease water excretion by the kidneys, which serves to reestablish euvolemia. Control of NaCl Excretion During Euvolemia

1	Control of NaCl Excretion During Euvolemia Maintenance of Na+ balance and therefore euvolemia requires precise matching of the amount of NaCl ingested with the amount excreted from the body. As already noted the kidneys are the major route for NaCl excretion. Accordingly, in a euvolemic individual we can equate daily urine NaCl excretion with daily NaCl intake. As noted, the amount of NaCl excreted by the kidneys can vary widely. Under conditions of salt restriction (i.e., low-NaCl diet), virtually no NaCl appears in the urine. Conversely, in individuals who ingest large quantities of NaCl, renal NaCl excretion can exceed 1000 mEq/day. The time course for adjustment of renal NaCl excretion varies (hours to days) and depends on the magnitude of the change in NaCl intake. Acclimation to large changes in NaCl intake requires a longer time than acclimation to small changes in intake. The general features of NaCl transport along the nephron are illustrated in

1	The general features of NaCl transport along the nephron are illustrated in Fig. 35.11 . Most (67%) of the Na+ filtered by the glomerulus is reabsorbed by the proximal tubule. An additional 25% is reabsorbed by the thick ascending limb of the loop of Henle, and the remainder by the distal tubule and collecting duct. In a normal adult the filtered amount (load) of Na+ is approximately 25,000 mEq/day: Equation 35.1 Filtered load of Na+= (GFR) × (Plasma [Na+ ]) = (180 L/day) × (140 mEq/L) , mEq/day With a typical diet, less than 1% of this filtered load is excreted in urine (≈140 mEq/day). Because of the large filtered load of Na+ , small changes in its reabsorption by the nephron can profoundly affect Na+ balance and thus ECFV. For example, an increase in Na+ excretion from 1% gThe percentage of the filtered load excreted in urine is termed fractional excretion. In this example the fractional excretion of Na+ is 140 mEq/day ÷ 25,200 mEq/day = 0.005, or 0.5%.

1	• Fig. 35.11 Segmental Na+ reabsorption. The percentage of the filtered load of Na+ reabsorbed by each nephron segment is indicated. PT, proximal tubule; TAL, thick ascending limb; DT, distal tubule; CD, cortical collecting duct. to 3% of the filtered load represents an additional loss of approximately 500 mEq/day of Na+ . Because the ECF Na+ concentration is 140 mEq/L, such a Na+ loss would decrease the ECFV by more than 3 L (i.e., water excretion would parallel the loss of Na+ to maintain body fluid osmolality constant: 500 mEq/day ÷ 140 mEq/L = 3.6 L/day of fluid loss). Such fluid loss in a 70-kg individual would represent a 26% decrease in ECFV.

1	In euvolemic individuals the nephron segments distal to the loop of Henle (distal tubule and collecting duct) are the main nephron segments where Na+ reabsorption is adjusted to maintain excretion at a level appropriate for dietary intake. However, this does not mean the other portions of the nephron are not involved in this process. Because the reabsorptive capacity of the distal tubule and collecting duct is limited, these other portions of the nephron (i.e., proximal tubule and loop of Henle) must reabsorb the bulk of the filtered load of Na+ . Thus during euvolemia, Na+ handling by the nephron can be explained by two general processes: 1.

1	Na+ reabsorption by the proximal tubule and loop of Henle is regulated so that a relatively constant portion of the filtered load of Na+ is delivered to the distal tubule. The combined action of the proximal tubule and loop of Henle reabsorbs approximately 92% of the filtered load of Na+ , and thus 8% of the filtered load is delivered to the distal tubule. 2. Reabsorption of this remaining portion of the filtered load of Na+ by the distal tubule and collecting duct is regulated so that the amount of Na+ excreted in the urine matches the amount ingested in the diet. Thus these later nephron segments make final adjustments in Na+ excretion to maintain the euvolemic state. Mechanisms for Maintaining Constant Delivery of NaCl to the Distal Tubule

1	Mechanisms for Maintaining Constant Delivery of NaCl to the Distal Tubule A number of mechanisms maintain a constant delivery of Na+ to the beginning of the distal tubule. These processes are autoregulation of the GFR (and thus the filtered load of Na+), glomerulotubular balance, and load dependency of Na+ reabsorption by the loop of Henle. Autoregulation of the GFR (see ) allows maintenance of a relatively constant filtration rate over a wide range of perfusion pressures. Because the filtration rate is constant, the filtered load of Na+ is also constant.

1	Despite the autoregulatory control of GFR, small variations occur. If these changes are not compensated for by an appropriate adjustment in Na+ reabsorption by the nephron, Na+ excretion would change markedly. Fortunately, Na+ reabsorption in the euvolemic state, especially by the proximal tubule, changes in parallel with changes in GFR. This phenomenon is termed glomerulotubular balance. Thus if GFR increases, the amount of Na+ reabsorbed by the proximal tubule also increases. The opposite occurs if GFR decreases (see for a more detailed description of glomerulotubular balance). The final mechanism that helps maintain constant delivery of Na+ to the distal tubule and collecting duct involves the ability of the loop of Henle to increase its reabsorptive rate in response to increased delivery of Na+ . Regulation of Distal Tubule and Collecting Duct NaCl Reabsorption

1	Regulation of Distal Tubule and Collecting Duct NaCl Reabsorption When delivery of Na+ is constant, small adjustments in Na+ reabsorption, primarily by the ASDN, are sufficient to balance excretion with intake. Aldosterone is the primary regulator of Na+ reabsorption by the ASDN and thus of NaCl excretion. When aldosterone levels are elevated, Na+ reabsorption by these segments is increased (Na+ excretion is decreased). When aldosterone levels are decreased, Na+ reabsorption is decreased (NaCl excretion is increased). Other factors have been shown to alter Na+ reabsorption by the ASDN (e.g., angiotensin II and natriuretic peptides), but their role during euvolemia is unclear.

1	As long as variations in dietary intake of NaCl are minor, the mechanisms previously described can regulate renal Na+ excretion appropriately and thereby maintain euvolemia. However, these mechanisms cannot effectively handle significant changes in NaCl intake. When NaCl intake changes significantly, ECFV expansion or contraction occurs. In such cases, additional factors act on the kidneys to adjust Na+ excretion and thereby reestablish the euvolemic state. Control of NaCl Excretion With Volume Expansion

1	Control of NaCl Excretion With Volume Expansion During ECFV expansion, the high-pressure and low-pressure vascular volume sensors send signals to the kidneys that result in increased excretion of NaCl and water. The signals acting on the kidneys include: ˜ AVP ° ANP and BNP Heart Brain ˜ Aldosterone Lung Adrenal gland ° Na+, H2O excretion ˜ Renin ° Urodilatin ˜ Sympathetic activity 2 1 3 ˜ Angiotensin I ˜ Angiotensin II Volume expansion UNa+V = °GFR ˜PNa+ – ˜R • Fig. 35.12 Integrated response to ECFV expansion. Numbers refer to the description of the response in the text. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; GFR, glomerular filtration rate; PNa+, plasma [Na+]; R, tubular reabsorption of Na+; UNa+V, Na+ excretion rate. 1. decreased activity of renal sympathetic nerves 2. release of ANP and BNP from the heart and urodilatin in the kidneys 3. inhibition of AVP secretion from the posterior pituitary and decreased AVP action on the collecting duct 4.

1	release of ANP and BNP from the heart and urodilatin in the kidneys 3. inhibition of AVP secretion from the posterior pituitary and decreased AVP action on the collecting duct 4. decreased renin secretion and thus decreased production of angiotensin II 5. decreased aldosterone secretion, which is caused by reduced angiotensin II levels, and elevated natriuretic peptide levels The integrated response of the nephron to these signals is Fig. 35.12 . Three general responses to ECFV expansion occur (the numbers match those encircled in

1	Fig. 35.12 . Three general responses to ECFV expansion occur (the numbers match those encircled in Fig. 35.12 1. GFR increases. GFR increases mainly as a result of the decrease in sympathetic nerve activity. Sympathetic fibers innervate the afferent and efferent arterioles of the glomerulus and control their diameter. Decreased sympathetic nerve activity leads to arteriolar dilation and an increase in renal plasma flow (RPF). Because the effect appears to be greater on the afferent arterioles, the hydrostatic pressure within the glomerular capillary is increased, thereby increasing the GFR. Because RPF increases to a greater degree than GFR, the filtration fraction (GFR/RPF) decreases. Natriuretic peptides also increase GFR by dilating the afferent arterioles and constricting the efferent arterioles. Thus the increased natriuretic peptide levels that occur during ECFV expansion contribute to this response. With the increase in GFR, the filtered load of Na+ increases.

1	2. The reabsorption of Na+ decreases in the proximal tubule and loop of Henle. Several mechanisms reduce Na+ reabsorption by the proximal tubule. Because activation of the sympathetic nerve fibers that innervate this nephron segment stimulates proximal tubule Na+ reabsorption, the decreased sympathetic nerve activity that results from ECFV expansion decreases Na+ reabsorption. In addition, angiotensin II directly stimulates Na+ reabsorption by the proximal tubule. Because angiotensin II levels are also reduced under this condition, proximal tubule Na+ reabsorption decreases as a result. Starling forces across the proximal tubule also change. The elevated hydrostatic pressure within the glomerular capillaries in ECFV expansion also tends to increase the hydrostatic pressure within the peritubular capillaries. In addition the decrease in filtration fraction reduces the peritubular oncotic pressure. These alterations in the capillary Starling forces (i.e., hydrostatic and oncotic) reduce

1	capillaries. In addition the decrease in filtration fraction reduces the peritubular oncotic pressure. These alterations in the capillary Starling forces (i.e., hydrostatic and oncotic) reduce the absorption of solute (e.g., NaCl) and water from the lateral intercellular space and thus reduce tubular reabsorption of NaCl (see for a complete description of this mechanism). Both the increase in the filtered load and the decrease in NaCl reabsorption by the proximal tubule result in delivery of more NaCl to the loop of Henle. Because activation of sympathetic nerves and angiotensin II and aldosterone stimulate NaCl reabsorption by the thick ascending limb of the loop of Henle, the reduced nerve activity and low angiotensin II and aldosterone levels that occur with ECFV expansion reduce NaCl reabsorption by the thick ascending limb. Thus the fraction of the filtered load delivered to the distal tubule is increased.

1	3. Na+ reabsorption decreases in the distal tubule and collecting duct. As noted, the amount of Na+ delivered to the distal tubule exceeds that observed in the euvolemic state (i.e., the amount of Na+ delivered to the distal tubule varies in proportion to the degree of ECFV expansion). This increased load of Na+ overwhelms the reabsorptive capacity of the distal tubule and the collecting duct, and this capacity is also impaired by reduced levels of angiotensin II and aldosterone, as well as increased levels of natriuretic peptides. The final component in the response to ECFV expansion is increased excretion of water. As Na+ excretion increases, plasma osmolality begins to fall. This decreases secretion of AVP. AVP secretion is also decreased in response to the elevated levels of natriuretic peptides. In addition, natriuretic peptides inhibit the action of AVP on the collecting duct. Together, these effects decrease water reabsorption by the collecting duct and thereby increase water

1	peptides. In addition, natriuretic peptides inhibit the action of AVP on the collecting duct. Together, these effects decrease water reabsorption by the collecting duct and thereby increase water excretion by the kidneys. Thus excretion of NaCl and water occurs in concert; euvolemia is restored and body fluid osmolality remains constant. The time course of this response (hours to days) depends on the magnitude of the ECFV expansion. Thus if the degree of ECFV expansion is small, the mechanisms just described generally restore euvolemia within 24 hours. However, with large degrees of ECFV expansion, the response can take several days.

1	In brief, the renal response to ECFV expansion involves the integrated action of all parts of the nephron; (1) the amount of Na+ filtered is increased, (2) Na+ reabsorption by the proximal tubule and loop of Henle is reduced (glomerulotubular balance does not occur under this condition), and (3) reabsorption of Na+ by the distal tubule and collecting duct is decreased (while the delivery of Na+ is increased), primarily secondary to a reduction in aldosterone. In summary, ECFV expansion leads to a reduction in Na+ reabsorption by both the proximal tubule and the ASDN, which results in excretion of a larger fraction of the filtered Na+ , thereby restoring euvolemia. Control of NaCl Excretion With Volume Contraction During ECFV contraction, the high-pressure and low-pressure vascular volume sensors send signals to the kidneys to reduce NaCl and water excretion and thereby restore ECFV. The signals that act on the kidneys include: 1. 2.

1	2. increased secretion of renin, which results in elevated angiotensin II levels and thus increased secretion of aldosterone by the adrenal cortex 3. inhibition of ANP and BNP secretion by the heart and urodilatin production by the kidneys 4. stimulation of AVP secretion by the posterior pituitary The integrated response of the nephron to these signals is illustrated in Fig. 35.13 . The general response is as follows (the numbers correlate with those encircled in Fig. 35.13): 1. GFR decreases. Afferent and efferent arteriolar constriction occurs as a result of increased renal sympathetic nerve activity. The effect appears to be greater on the afferent than on the efferent arteriole, because the hydrostatic pressure in the glomerular capillaries fall, which thereby decreases GFR. Because RPF decreases more than GFR, filtration fraction increases. The decrease in GFR reduces the filtered amount of Na+ . 2.

1	2. Na+ reabsorption by the proximal tubule and loop of Henle is increased. Several mechanisms augment Na+ reabsorption in the proximal tubule. For example, increased sympathetic nerve activity and angiotensin II levels directly stimulate Na+ reabsorption. The decreased hydrostatic pressure within the glomerular capillaries also leads to a decrease in the hydrostatic pressure within the peritubular capillaries. In addition, as just noted, the increased filtration fraction results in an increase in the peritubular oncotic pressure. These alterations in the capillary Starling forces facilitate movement of fluid from the lateral intercellular space into the capillary and thereby stimulate reabsorption of NaCl and water by the proximal tubule (see for a complete description of this mechanism). Increased sympathetic nerve activity as well as elevated levels of angiotensin II and aldosterone stimulate Na+ reabsorption by the thick ascending limb. 3.

1	Na+ reabsorption by the distal tubule and collecting duct is enhanced. The small amount of Na+ that is delivered to the ASDN, owing to decreased filtration and increased reabsorption by the proximal tubule and loop of Henle, is almost completely reabsorbed because transport in the segments of the ASDN is enhanced. Stimulation of Na+ reabsorption by the ASDN is primarily the result of increased aldosterone levels, although increased sympathetic nerve activity and increased angiotensin II levels may also contribute to this response. Lastly, levels of natriuretic peptides, which inhibit collecting duct reabsorption, are reduced. Finally, water reabsorption by the latter portion of the distal tubule and the collecting duct is enhanced by AVP, the levels of which are elevated through activation of the lowand high-pressure vascular volume sensors as well as by the elevated levels of angiotensin II. As a result, water excretion is reduced. Because both water and Na+ are retained by the

1	of the lowand high-pressure vascular volume sensors as well as by the elevated levels of angiotensin II. As a result, water excretion is reduced. Because both water and Na+ are retained by the kidneys in equal proportions, euvolemia is reestablished and ˜ AVP ° ANP and BNP Heart Brain ˜ Aldosterone Lung Adrenal gland ° Na+, H2O excretion ˜ Renin ˜ Sympathetic activity 2 1 3 ˜ Angiotensin I ˜ Angiotensin II Volume contraction • Fig. 35.13 Integrated response to ECFV contraction. Numbers refer to the description of the response in the text. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; GFR, glomerular filtration rate; PNa+, plasma [Na+]; R, tubular reabsorption of Na+; UNa+V, Na+ excretion rate.

1	body fluid osmolality remains constant. The time course of this restoration of ECFV (hours to days) and the degree to which euvolemia is attained depend on the magnitude of ECFV contraction as well as the dietary intake of Na+ . Euvolemia can be restored more rapidly if additional NaCl is ingested in the diet. In brief, the nephron’s response to ECFV contraction involves the integrated action of all its segments: (1) the 1.

1	In brief, the nephron’s response to ECFV contraction involves the integrated action of all its segments: (1) the 1. Regulation of body fluid osmolality (i.e., steady-state balance) requires that the amount of water added to the body exactly matches the amount lost from the body. Water is lost from the body by several routes (e.g., during respiration, with sweating, and in feces). The kidneys are the only regulated route of water excretion. Excretion of water by the kidneys is regulated by AVP secreted from the posterior pituitary. When AVP levels are high, the kidneys excrete a small volume of hyperosmotic urine. When AVP levels are low, a large volume of hypoosmotic urine is excreted. 2.

1	2. Disorders of water balance alter body fluid osmolality. Because Na+ with its anions are the major determinant filtered amount of Na+ is decreased, (2) reabsorption in the proximal tubule and loop of Henle is enhanced (GFR is decreased, whereas proximal reabsorption is increased, and thus glomerulotubular balance does not occur under this condition), and (3) delivery of Na+ to the ASDN is reduced. This decreased delivery, together with enhanced Na+ and water reabsorption by the ASDN, virtually eliminates Na+ from the urine and reduces urine volume. of ECF osmolality, disorders of water balance manifest as changes in ECF [Na+]. Positive water balance (intake > excretion) results in a decrease in body fluid osmolality and hyponatremia. Negative water balance (intake < excretion) results in an increase in body fluid osmolality and hypernatremia.

1	3. The volume of the ECF is determined by the amount of Na+ in this compartment. To maintain a constant ECFV (i.e., euvolemia) NaCl excretion must match NaCl intake. The kidneys are the major route for regulating excretion of NaCl from the body. Volume sensors located primarily in the vascular system monitor volume and pressure. When ECFV expansion occurs, neural and hormonal signals are sent to the kidneys to increase excretion of NaCl and water and thereby restore euvolemia. When ECFV contraction occurs, neural and hormonal signals are sent to the kidneys to decrease NaCl and water excretion and Berl T. Vasopressin antagonists. N Engl J Med. 2015;372:2207-2216. Bichet DG. Polyuria and diabetes insipidus. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013.

1	Bichet DG. Polyuria and diabetes insipidus. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013. Brown D, Fenton RA. The cell biology of vasopressin action. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012. Dantzler WH, et al. Urine-concentrating mechanism in the inner medulla: function of the thin limbs of the loops of Henle. Clin J Am Soc Nephrol. 2014;9:1781-1789. Danziger J, Zeidel M. Osmotic homeostasis. J Am Soc Nephrol. 2015;10:852-862. Knepper MA, et al. Molecular physiology of water balance. N Engl J Med. 2015;372:1349-1358. Nielsen S, et al. Aquaporin water channels in mammalian kidney. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013.

1	Robertson GL. Thirst and vasopressin. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013. Sands JM, et al. Urine concentration and dilution. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012. Sands JM, Layton HE. The urine concentrating mechanism and urea transporters. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013. Sands JM, Layton HE. Advances in understanding the urine-concentrating mechanism. Annu Rev Physiol. 2014;76:387-409. Schrier RW. Systemic arterial vasodilation, vasopressin, and vasopres sinase in pregnancy. J Am Soc Nephrol. 2010;21:570-572. Sterns RH. Disorders of plasma sodium–causes, consequences, and correction. N Engl J Med. 2015;372:55-65.

1	thereby restore euvolemia. The sympathetic nervous system, the RAAS, and natriuretic peptides are important components of the system that maintain steady-state Na+ balance. Verbalis JG. Disorders of water balance. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012. Eladari D, et al. A new look at electrolyte transport in the distal tubule. Annu Rev Physiol. 2012;74:325-349. Gamba G, et al. Sodium chloride transport in the loop of Henle, distal convoluted tubule, and collecting duct. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013. Mount DB. Transport of sodium, chloride, and potassium. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012. Mount DB. Thick ascending limb of the loop of Henle. Clin J Am Soc Nephrol. 2014;9:1974-1986.

1	Mount DB. Thick ascending limb of the loop of Henle. Clin J Am Soc Nephrol. 2014;9:1974-1986. Palmer LG, Schnermann J. Integrated control of Na transport along the nephron. Clin J Am Soc Nephrol. 2015;10:676-687. Pearce D, et al. Aldosterone regulation of transport. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012. Pearce D, et al. Collecting duct principal cell transport processes and their regulation. Clin J Am Soc Nephrol. 2015;10:135-146. Slotki IN, Skorecki KL. Disorders of sodium balance. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012. Staub O, Loffing J. Mineralocorticoid action in aldosterone sensitive distal nephron. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013. Subramanya AR, Ellison DH. Distal convoluted tubule. Clin J Am Soc Nephrol. 2014;9:2147-2163.

1	Subramanya AR, Ellison DH. Distal convoluted tubule. Clin J Am Soc Nephrol. 2014;9:2147-2163. Vesley DL. Natriuretic hormones. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013. Weinstein AM. Sodium and chloride transport: proximal nephron. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Philadelphia: Academic Press; 2013. Potassium, Calcium, and Phosphate Homeostasis Upon completion of this chapter the student should be able to answer the following questions: 1. How does the body maintain K+ homeostasis? 2. What is the distribution of K+ within the body compartments? Why is this distribution important? 3. What are the hormones and factors that regulate plasma K+ levels? Why is this regulation important? 4.

1	3. What are the hormones and factors that regulate plasma K+ levels? Why is this regulation important? 4. How do the various segments of the nephron transport K+ , and how does the mechanism of K+ transport by these segments determine how much K+ is excreted in the urine? 5. Why are the distal tubule and collecting duct so important in regulating K+ excretion? 6. How do plasma K+ levels, aldosterone, vasopressin, tubular fluid flow rate, and acid-base balance influence K+ excretion? 7. What is the physiological importance of calcium (Ca++) and inorganic phosphate (Pi)? 8. How does the body maintain Ca++ and Pi homeostasis? 9. What roles do the kidneys, intestinal tract, and bone play in maintaining plasma Ca++ and Pi levels? 10. What hormones and factors regulate plasma Ca++ and Pi levels? 11. What are the cellular mechanisms responsible for Ca++ and Pi reabsorption along the nephron? 12. What hormones regulate renal Ca++ and Pi excretion by the kidneys? 13.

1	11. What are the cellular mechanisms responsible for Ca++ and Pi reabsorption along the nephron? 12. What hormones regulate renal Ca++ and Pi excretion by the kidneys? 13. What is the role of the calcium-sensing receptor (CaSR)? 14. What are some of the more common clinical disorders of Ca++ and Pi homeostasis? 15. What is the role of the kidneys in the production of calcitriol (active form of vitamin D)? 16. What effects do loop and thiazide diuretics have on Ca++ excretion?

1	15. What is the role of the kidneys in the production of calcitriol (active form of vitamin D)? 16. What effects do loop and thiazide diuretics have on Ca++ excretion? Potassium (K+) is one of the most abundant cations in the body, and it is critical for many cell functions, including regulation of cell volume, regulation of intracellular pH, synthesis of DNA and protein, growth, enzyme function, resting membrane potential, and cardiac and neuromuscular activity. Despite wide fluctuations in dietary K+ intake, [K+] in cells and extracellular fluid (ECF) remains remarkably constant. Two sets of regulatory mechanisms safeguard K+ homeostasis. First, several mechanisms regulate [K+] in the ECF. Second, other mechanisms maintain the amount of K+ in the body constant by adjusting renal K+ excretion to match dietary K+ intake. It is the kidneys that regulate excretion of K+ .

1	Total body [K+] is 50 mEq/kg of body weight, or 3600 mEq for a 70-kg individual. Ninety-eight percent of the K+ in the body is located within cells, where the average [K+] is 150 mEq/L. High intracellular [K+] is required for many cell functions, including cell growth and division and volume regulation. Only 2% of total body [K+] is located in the ECF, where its normal concentration is approximately 4 mEq/L. A [K+] in ECF that exceeds 5.0 mEq/L constitutes hyperkalemia. Conversely, a [K+] in ECF of less than 3.5 mEq/L constitutes hypokalemia.

1	Hypokalemia is one of the most common electrolyte disorders in clinical practice and can be observed in as many as 20% of hospitalized patients. The most frequent causes of hypokalemia include administration of diuretic drugs, surreptitious vomiting (e.g., bulimia), and severe diarrhea. Gitelman syndrome (a genetic defect in the Na+/ Cl− symporter in the apical membrane of distal tubule cells) also causes hypokalemia (see Chapter 36). Hyperkalemia is also a common electrolyte disorder and is seen in 1% to 10% of hospitalized patients. Hyperkalemia often occurs in patients with renal failure, in patients taking drugs such as angiotensin-converting enzyme (ACE) inhibitors and K+-sparing diuretics, in patients with hyperglycemia (i.e., high blood sugar), and in the elderly. Pseudohyperkalemia, a falsely high plasma [K+], is caused by traumatic lysis of red blood cells during blood drawing. Red blood cells, like all cells, contain K+ , and lysis of red blood cells releases K+ into plasma,

1	a falsely high plasma [K+], is caused by traumatic lysis of red blood cells during blood drawing. Red blood cells, like all cells, contain K+ , and lysis of red blood cells releases K+ into plasma, thereby artificially elevating plasma [K+].

1	The large concentration difference of K+ across cell membranes (≈146 mEq/L) is maintained by the operation of Na+,K+-ATPase. This [K+] gradient is important in • Fig. 36.2 Electrocardiographs from individuals with varying plasma • Fig. 36.1 Effects of variations in plasma [K+] on resting membrane potential of skeletal muscle. Hyperkalemia causes membrane potential to become less negative, which decreases excitability by inactivating the fast Na+ channels responsible for the depolarizing phase of the action potential. Hypokalemia hyperpolarizes the membrane potential and thereby reduces excitability because a larger stimulus is required to depolarize the membrane potential to the threshold potential. Resting indicates “normal” resting membrane potential. Normal threshold indicates the membrane threshold potential.

1	maintaining the potential difference across cell membranes. Thus K+ is critical for the excitability of nerve and muscle cells, as well as for the contractility of cardiac, skeletal, and smooth muscle cells ( Fig. 36.1 [K+]. See text for details. (Modified from Barker L et al. Principles of Ambulatory Medicine. 5th ed. Baltimore: Williams & Wilkins; 1999.) This rise in plasma [K+], which could have deleterious effects on the electrical activity of the heart and other excitable tissues, is prevented by the rapid (minutes) uptake of K+ into cells. Because excretion of K+ by the kidneys Cardiac arrhythmias are produced by both hypokalemia and hyperkalemia. The electrocardiogram (ECG;

1	Cardiac arrhythmias are produced by both hypokalemia and hyperkalemia. The electrocardiogram (ECG; Fig. 36.2 ) monitors the electrical activity of the heart and is a fast and easy way to determine whether changes in plasma [K+] influence the heart and other excitable cells. In contrast, measurement of plasma [K+] by the clinical laboratory requires a blood sample, and values are often not immediately available. The first sign of hyperkalemia is the appearance of tall, thin T waves on the ECG. Further increases in plasma [K+] prolong the PR interval, depress the ST segment, and lengthen the QRS interval of the ECG. Finally, as plasma [K+] approaches 10 mEq/L, the P wave disappears, the QRS interval broadens, the ECG appears as a sine wave, and the ventricles fibrillate (i.e., manifest rapid, uncoordinated contractions of muscle fibers). Hypokalemia prolongs the QT interval, inverts the T wave, and lowers the ST segment of the ECG.

1	After a meal the K+ absorbed by the gastrointestinal (GI) tract enters the ECF within minutes (Fig. 36.3 ). If the K+ ingested during a normal meal (≈33 mEq) were to remain in the ECF compartment (14 L), plasma [K+] would increase by 2.4 mEq/L (33 mEq added to 14 L of ECF): Equation 36.1 14L = 2 4. mEq after a meal is relatively slow (hours), uptake of K+ by cells is essential to prevent life-threatening hyperkalemia. Maintaining total body [K+] constant requires that all the K+ absorbed by the GI tract eventually be excreted by the kidneys. This process requires about 6 hours. Regulation of Plasma [K+] Several hormones, including epinephrine, insulin, and aldosterone, increase uptake of K+ into skeletal muscle, liver, bone, and red blood cells (Box 36.1

1	Regulation of Plasma [K+] Several hormones, including epinephrine, insulin, and aldosterone, increase uptake of K+ into skeletal muscle, liver, bone, and red blood cells (Box 36.1 Fig. 36.3 ) by stimulating Na+,K+-ATPase, the 1Na+/1K+/2Cl− symporter, and the Na+/Cl− symporter in these cells. Acute stimulation of K+ uptake (i.e., within minutes) is mediated by increased activity of existing Na+,K+-ATPase, 1Na+/1K+/2Cl− , and Na+/Cl− transporters, whereas a chronic increase in K+ uptake (i.e., within hours to days) is mediated by an increase in the quantity of Na+,K+-ATPase. The rise in plasma [K+] that follows K+ absorption by the GI tract stimulates secretion of insulin from the pancreas, release of aldosterone from the adrenal cortex, and secretion of epinephrine from the adrenal medulla (see

1	Fig. 36.3 ). In contrast, a decrease in plasma [K+] inhibits release of these hormones. Whereas insulin and epinephrine act within a few minutes, aldosterone requires about an hour to stimulate uptake of K+ into cells. • BOX 36.1 Major Factors, Hormones, and Drugs Influencing Distribution of K+Between Intracellular and Extracellular Fluid Compartments Physiological: Keep Plasma [K+] Constant Pathophysiological: Displace Plasma [K+] From Normal CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis Catecholamines affect the distribution of K+ across cell membranes by activating α-and β2-adrenergic receptors. Stimulation of α-adrenoceptors releases K+ from cells, especially in the liver, whereas stimulation of β2-adrenoceptors promotes K+ uptake by cells.

1	For example, activation of β2-adrenoceptors after exercise is important in preventing hyperkalemia. The rise in plasma [K+] after a K+-rich meal is greater if the patient has been pretreated with propranolol, a β-adrenoceptor antagonist. Furthermore, release of epinephrine during stress (e.g., myocardial ischemia) can rapidly lower plasma [K+]. Insulin also stimulates uptake of K+ into cells. The importance of insulin is illustrated by two observations. First, the rise in plasma [K+] after a K+-rich meal is greater in patients with diabetes mellitus (i.e., insulin deficiency) than in healthy people. Second, insulin (and glucose to prevent insulin-induced hypoglycemia) can be infused to correct hyperkalemia. Insulin is the most important hormone that shifts K+ into cells after ingestion of K+ in a meal. In patients with chronic kidney disease, although insulin-stimulated glucose uptake into cells is impaired, insulin stimulation of K+ uptake into cells is normal.

1	• Fig. 36.3 Overview of K+homeostasis. An increase in plasma insulin, epinephrine, or aldosterone stimulates movement of K+into cells and decreases plasma [K+], whereas a fall in plasma concentration of these hormones has the opposite effect and increases plasma [K+]. The amount of K+in the body is determined by the kidneys. An individual is in K+balance when dietary intake and urinary output (plus output by the GI tract) are equal. Excretion of K+by the kidneys is regulated by plasma [K+], aldosterone, and AVP. Diet 100 mEq of K+/day Feces 5–10 mEq of K+/day Urine 90–95 mEq of K+/day Intestinal absorption 90 mEq of K+/day Extracellular fluid 65 mEq of K+ Tissue stores 3435 mEq of K+ Insulin Epinephrine Aldosterone Plasma [K+] AVP Aldosterone

1	Aldosterone, like catecholamines and insulin, also promotes uptake of K+ into cells. A rise in aldosterone levels (e.g., primary aldosteronism) causes hypokalemia, whereas a fall in aldosterone levels (e.g., Addison’s disease) causes hyperkalemia. As discussed later and as illustrated in Fig. 36.3 , aldosterone also stimulates urinary K+ excretion. Thus aldosterone alters plasma [K+] by acting on uptake of K+ into cells and altering urinary K+ excretion. Box 36.1 ). These factors are not involved in regulation of plasma [K+] but rather alter the movement of K+ between the intracellular fluid (ICF) and ECF and thus cause development of hypokalemia or hyperkalemia.

1	Metabolic acidosis increases plasma [K+], whereas metabolic alkalosis decreases it. Respiratory alkalosis causes hypokalemia. In contrast, respiratory acidosis has little or no effect on plasma [K+]. Metabolic acidosis produced by addition of inorganic acids (e.g., HCl, H2SO4) increases plasma [K+] much more than an equivalent acidosis produced by accumulation of organic acids (e.g., lactic acid, acetic acid, ketoacids). The reduced pH (i.e., increased [H+]) promotes movement of H+ into cells and the reciprocal movement of K+ out of cells to maintain electroneutrality. This effect of acidosis occurs in part because acidosis inhibits the transporters that accumulate K+ inside cells, including Na+,K+-ATPase and the 1Na+/1K+/2Cl− symporter. In addition, movement of H+ into cells occurs as the cells buffer changes in [H+] of the ECF (see Chapter 37).

1	Chapter 37). As H+ moves across cell membranes, K+ moves in the opposite direction, and thus cations are neither gained nor lost across cell membranes. Metabolic alkalosis has the opposite effect; plasma [K+] decreases as K+ moves into cells and H+ exits. Although organic acids produce a metabolic acidosis, they do not cause significant hyperkalemia. Two explanations have been suggested for the reduced ability of organic acids to cause hyperkalemia. First, the organic anion may enter the cell with H+ and thereby eliminate the need for K+-H+ exchange across the membrane. Second, organic anions may stimulate insulin secretion, which moves K+ into cells. This movement may counteract the direct effect of the acidosis, which moves K+ out of cells.

1	The osmolality of plasma also influences the distribution of K+ across cell membranes. An increase in the osmolality of ECF enhances the release of K+ by cells and thus increases extracellular [K+]. Plasma [K+] may increase by 0.4 to 0.8 mEq/L with a 10 mOsm/kg H2O elevation in plasma osmolality. In patients with diabetes mellitus who do not take insulin, plasma [K+] is often elevated, in part because of the lack of insulin and in part because of the increase in plasma [glucose] (i.e., from a normal value of ≈ 100 mg/ dL to as high as ≈ 1200 mg/dL in some cases), which increases plasma osmolality. Hypoosmolality has the opposite action. The alterations in plasma [K+] associated with changes in osmolality are related to changes in cell volume. For example, as plasma osmolality increases, water leaves cells because of the osmotic gradient across the plasma membrane (see Chapter 1).

1	Chapter 1). Water leaves cells until the intracellular osmolality equals that of ECF. This loss of water shrinks cells and causes [K+] in cells to rise. The rise in intracellular [K+] provides a driving force for the exit of K+ from cells. This sequence increases plasma [K+]. A fall in plasma osmolality has the opposite effect. Cell lysis causes hyperkalemia as a result of addition of intracellular K+ to ECF. Severe trauma (e.g., burns) and some conditions such as tumor lysis syndrome (i.e., chemotherapy-induced destruction of tumor cells) and rhabdomyolysis (i.e., destruction of skeletal muscle) destroy cells and release K+ and other cell solutes into ECF. In addition, gastric ulcers may cause seepage of red blood cells into the GI tract. The blood cells are digested, and the K+ released from the cells is absorbed and can cause hyperkalemia.

1	More K+ is released from skeletal muscle cells during exercise than during rest. The ensuing hyperkalemia depends on the degree of exercise. In people walking slowly, plasma [K+] increases by 0.3 mEq/L. With vigorous exercise, plasma [K+] may increase by 2.0 or more mEq/L. K+ Excretion by the Kidneys The kidneys play a major role in maintaining K+ balance. As illustrated in

1	K+ Excretion by the Kidneys The kidneys play a major role in maintaining K+ balance. As illustrated in Fig. 36.3 the kidneys excrete 90% to 95% of the K+ ingested in the diet. Excretion equals intake even when intake increases by as much as 10-fold. This balance in urinary excretion and dietary intake underscores the importance of the kidneys in maintaining K+ homeostasis. Although small amounts of K+ are lost each day in feces and sweat (≈5%–10% of the K+ ingested in the diet), except during severe diarrhea this amount is essentially constant, is not regulated, and therefore is relatively less important than the K+ excreted by the kidneys. K+ secretion from blood into tubular fluid by cells of the distal tubule and collecting duct system is the key factor in determining urinary K+ excretion ( Fig. 36.4

1	Fig. 36.4 CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis 3% 10% to 50% 9% 5% to 30% • Fig. 36.4 K+ transport along the nephron. Excretion of K+ depends on the rate and direction of K+ transport by the late segment of the distal tubule and collecting duct. Percentages refer to the amount of filtered K+ reabsorbed or secreted by each nephron segment. Arrows indicate direction of transport. Left, Dietary K+ depletion. An amount of K+ equal to 1% of the filtered load of K+ is excreted. Right, Normal and increased dietary K+ intake. An amount of K+ equal to 15% to 80% of the filtered load is excreted. CCD, cortical collecting duct; DT, distal tubule; IMCD, inner medullary collecting duct; PT, proximal tubule; TAL, thick ascending limb.

1	TAL, thick ascending limb. Exercise-induced changes in plasma [K+] do not usually produce symptoms and are reversed after several minutes of rest. However, vigorous exercise can lead to life-threatening hyperkalemia in individuals (1) who have endocrine disorders that affect release of insulin, epinephrine (a β-adrenergic agonist), or aldosterone; (2) whose ability to excrete K+ is impaired (e.g., renal failure); or (3) who take certain medications, such as β1-adrenergic blockers. For example, during vigorous exercise, plasma [K+] may increase by at least 2 to 4 mEq/L in individuals who take β1-adrenergic receptor antagonists for hypertension. Because acid-base balance, plasma osmolality, cell lysis, and exercise do not maintain plasma [K+] at a normal value, they do not contribute to K+ homeostasis (see

1	Box 36.1 ). The extent to which these pathophysiological states alter plasma [K+] depends on the integrity of the homeostatic mechanisms that regulate plasma [K+] (e.g., secretion of epinephrine, insulin, and aldosterone). Because K+ is not bound to plasma proteins, it is freely filtered by the glomerulus. When individuals ingest 100 mEq of K+ per day, urinary K+ excretion is about 15% of the amount filtered. Accordingly K+ must be reabsorbed along the nephron. When dietary K+ intake increases, however, K+ excretion can exceed the amount filtered. Thus K+ can also be secreted.

1	The proximal tubule reabsorbs about 67% of the filtered K+ under most conditions. Approximately 20% of the filtered K+ is reabsorbed by the loop of Henle, and as with the proximal tubule, the amount reabsorbed is a constant fraction of the amount filtered. In contrast to these segments, which can only reabsorb K+ , the distal tubule and collecting duct are able to reabsorb or secrete K+ . The rate of K+ reabsorption or secretion by the distal tubule and collecting duct depends on a variety of hormones and factors. When 100 mEq/day of K+ is ingested, it is secreted by these nephron segments. A rise in dietary K+ intake increases K+ secretion. K+ secretion can increase the amount of K+ that appears in urine so that it approaches 80% of the amount filtered (see Fig. 36.4 , right panel). In contrast, a low-K+ diet activates K+ reabsorption along the distal tubule and collecting duct so that urinary excretion falls to about 1% of the K+ filtered by the glomerulus (see

1	Fig. 36.4 , left panel). The kidneys cannot reduce K+ excretion to the same low levels as they can for Na+ (i.e., 0.2%). Therefore hypokalemia can develop in individuals placed on a K+-deficient diet. Because the magnitude and direction of K+ transport by the distal tubule and collecting duct are variable, the overall rate of urinary K+ excretion is determined by these tubular segments. Cellular Mechanism of K+ Secretion by Principal Cells and Intercalated Cells Fig. 36.5A illustrates the cellular mechanisms of K+ secretion by principal cells in the late segment of the distal • Fig. 36.5 Cellular mechanism of K+ secretion by principal cells (A) and α-intercalated cells (B) in the late segment of the distal tubule and collecting duct. α-Intercalated cells contain very low levels of Na+,K+-ATPase in the basolateral membrane (not shown). K+ depletion increases K+ reabsorption by α-intercalated cells by stimulating the H+,K+-ATPase (HKA).

1	In individuals with advanced renal disease, the kidneys are unable to eliminate ingested K+ from the body. Therefore plasma [K+] rises. The resulting hyperkalemia reduces the resting membrane potential (i.e., the voltage becomes less negative), and this reduced potential decreases the excitability of neurons, cardiac cells, and muscle cells by inactivating fast Na+ channels, which are critical for the depolarization phase of the action potential (see

1	Fig. 36.1 ). Severe rapid increases in plasma [K+] can lead to cardiac arrest and death. In contrast, in patients taking diuretic drugs for hypertension, urinary K+ excretion often exceeds dietary K+ intake. Accordingly K+ balance is negative and hypokalemia develops. This decline in extracellular [K+] hyperpolarizes the resting cell membrane (i.e., the voltage becomes more negative) and reduces the excitability of neurons, cardiac cells, and muscle cells. Severe hypokalemia can lead to paralysis, cardiac arrhythmias, and death. Hypokalemia can also impair the ability of the kidneys to concentrate urine and can stimulate renal production of NH4 + , which affects acid-base balance (see ). Therefore maintenance of high intracellular [K+], low extracellular [K+], and a high [K+] gradient across cell membranes is essential for a number of cellular functions.

1	CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis tubule and the collecting duct. Secretion from blood into the tubule lumen is a two-step process: (1) uptake of K+ from blood across the basolateral membrane by Na+,K+-ATPase and (2) diffusion of K+ from the cell into tubular fluid via K+ channels (ROMK and BK). A K+/ Cl− symporter (KCC1) in the apical plasma membrane also secretes K+ . Na+,K+-ATPase creates a high intracellular [K+] that provides the chemical driving force for exit of K+ across the apical membrane through K+ channels. Although K+ channels are also present in the basolateral membrane, K+ preferentially leaves the cell across the apical membrane and enters the tubular fluid. K+ transport follows this route for two reasons. First, the electrochemical gradient of K+ across the apical membrane favors its downhill movement into tubular fluid. Second, the permeability of the apical membrane to K+ is greater than that of the basolateral membrane. Therefore K+

1	of K+ across the apical membrane favors its downhill movement into tubular fluid. Second, the permeability of the apical membrane to K+ is greater than that of the basolateral membrane. Therefore K+ preferentially diffuses across the apical membrane into tubular fluid. K+ secretion across the apical membrane via the K+/Cl− symporter is driven by the favorable concentration gradient of K+ between the cell and tubular fluid. The three major factors that control the rate of K+ secretion by the late segment of the distal tubule and the collecting duct are: 1.

1	the activity of Na+,K+-ATPase 2. the driving force (electrochemical gradient for K+ channels and the chemical concentration gradient for the K+/Cl− symporter) for movement of K+ across the apical membrane 3. the permeability of apical membrane K+ channels to K+ Every change in K+ secretion by principal cells results from an alteration in one or more of these factors (Fig. 36.6 α-Intercalated cells reabsorb K+ by a H+ , K+-ATPase transport mechanism (HKA) located in the apical membrane (see Fig. 36.5B ). This transporter mediates K+ uptake across the apical plasma membrane in exchange for H+ . K+ exit from intercalated cells into the blood is mediated by a K+ channel. Reabsorption of K+ is activated by a low-K+ diet. Regulation of K+ Secretion by the Distal Tubule and Collecting Duct

1	Regulation of K+ Secretion by the Distal Tubule and Collecting Duct Regulation of K+ excretion is achieved mainly by alterations in K+ secretion by principal cells of the late segment of the distal tubule and collecting duct. Plasma [K+] and aldosterone are the major physiological regulators of K+ secretion. Ingestion of a K+-rich meal also stimulates renal K+ excretion by a mechanism involving an unknown gut-dependent mechanism. Arginine vasopressin (AVP), also stimulates K+ secretion; however, it is less important than plasma [K+] and aldosterone. Other factors, including the flow rate of tubular fluid and acid-base balance, influence secretion of K+ by the distal tubule and collecting duct. However, they are not homeostatic mechanisms, because they disturb K+ balance ( Box 36.2 Plasma [K+] is an important determinant of K+ secretion by the distal tubule and collecting duct. Hyperkalemia (e.g., resulting from a high-K+ diet or from rhabdomyolysis)

1	Box 36.2 Plasma [K+] is an important determinant of K+ secretion by the distal tubule and collecting duct. Hyperkalemia (e.g., resulting from a high-K+ diet or from rhabdomyolysis) Fig. 36.6 Cellular mechanism of K+ secretion by principal cells. The numbers indicate where K+ secretion is regulated. 1, Na+,K+-ATPase; 2, electrochemical gradient of K+ across the apical membrane; 3, permeability of the apical membrane to K+ . Fig. 36.7 Effects of aldosterone on secretion of K+ by principal cells in the late segment of the distal tubule and collecting duct. Numbers refer to the five effects of aldosterone discussed in text. K+ K+ ˜ K+ permeability + ˜ CAP ˜ SGK˜ ENaC ˜Na+,K+-ATPase + + Na+ ATP 2 1 3 4 5 IN THE CLINIC • BOX 36.2 Major Factors and Hormones Influencing K+Excretion Physiological: Keep K+ Balance Constant Pathophysiological: Displace K+ Balance

1	IN THE CLINIC • BOX 36.2 Major Factors and Hormones Influencing K+Excretion Physiological: Keep K+ Balance Constant Pathophysiological: Displace K+ Balance Flow rate of tubule fluid Acid-base disorders Glucocorticoids stimulates secretion of K+ within minutes. Several mechanisms are involved. First, hyperkalemia stimulates Na+ , K+-ATPase and thereby increases K+ uptake across the basolateral membrane. This uptake raises intracellular [K+] and increases the electrochemical driving force for exit of K+ across the apical membrane. Second, hyperkalemia also increases the permeability of the apical membrane to K+ . Third, hyperkalemia stimulates secretion of aldosterone by the adrenal cortex, which as discussed later, acts synergistically with plasma [K+] to stimulate secretion of K+ . Fourth, hyperkalemia also increases the flow rate of tubular fluid, which as discussed later, stimulates secretion of K+ by the distal tubule and collecting duct.

1	Hypokalemia (e.g., caused by a low-K+ diet or loss of K+ in diarrhea fluid) decreases K+ secretion via actions opposite those described for hyperkalemia. Hence hypokalemia inhibits Na+,K+-ATPase, decreases the electrochemical driving force for efflux of K+ across the apical membrane, reduces permeability of the apical membrane to K+ , and decreases plasma aldosterone levels. Chronic hypokalemia (plasma [K+] < 3.5 mEq/L) occurs most often in patients who receive diuretics for hypertension. Hypokalemia also occurs in patients who vomit, undergo nasogastric suction, have diarrhea, abuse laxatives, or have hyperaldosteronism. Hypokalemia occurs because excretion of K+ by the kidneys exceeds dietary intake of K+ . Vomiting, nasogastric suction, diuretics, and diarrhea can all decrease ECF volume (ECFV), which in turn stimulates secretion of aldosterone (see ). Because aldosterone stimulates excretion of K+ by the kidneys, its action contributes to development of hypokalemia.

1	Chronic hyperkalemia (plasma [K+] > 5.0 mEq/L) occurs most frequently in individuals with reduced urine flow, low plasma aldosterone levels, and renal disease in which the glomerular filtration rate (GFR) falls below 20% of normal. In these individuals, hyperkalemia occurs because excretion of K+ by the kidneys is less than dietary intake of K+ . Less common causes of hyperkalemia occur in people with deficiencies in insulin, epinephrine, and aldosterone secretion or in people with metabolic acidosis caused by inorganic acids.

1	Chronically elevated (i.e., ≥24 hours) plasma aldosterone levels enhance secretion of K+ across principal cells in the late segment of the distal tubule and collecting duct (i.e., aldosterone-sensitive distal nephron [ASDN]) via five mechanisms (Fig. 36.7 ): (1) by increasing the amount of Na+,K+-ATPase in the basolateral membrane; (2) by increasing expression of the epithelial sodium channel (ENaC) in the apical cell membrane; (3) by elevating SGK1 (serum glucocorticoid-stimulated kinase) levels, which also increases expression of ENaC in the apical membrane and activates K+ channels; (4) by stimulating CAP1 (channelactivating protease, also called prostatin), which directly activates ENaC; and (5) by stimulating the permeability of the apical membrane to K+ . Aldosterone increases the permeability of the apical membrane to K+ by increasing the number of K+ channels in the membrane. However, the cellular mechanisms involved in this response are not completely known. Increased

1	the permeability of the apical membrane to K+ by increasing the number of K+ channels in the membrane. However, the cellular mechanisms involved in this response are not completely known. Increased expression of Na+,K+-ATPase facilitates uptake of K+ across the basolateral membrane into cells and thereby elevates intracellular [K+]. The increase in the number and activity of Na+ channels enhances entry of Na+ into the cell from tubular fluid, an effect that depolarizes the apical membrane voltage. Depolarization of the apical membrane and increased intracellular [K+] enhance the electrochemical driving force for secretion of K+ from the cell into the tubule fluid. Taken together, these actions increase uptake of K+ into the cell across the basolateral membrane and enhance exit of K+ from the cell across the apical membrane. Secretion of aldosterone is increased by hyperkalemia and by angiotensin II (after activation of the renin-angiotensin system). Secretion of aldosterone is

1	from the cell across the apical membrane. Secretion of aldosterone is increased by hyperkalemia and by angiotensin II (after activation of the renin-angiotensin system). Secretion of aldosterone is decreased by hypokalemia and natriuretic peptides released from the heart.

1	CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis Although an acute (e.g., within hours) increase in aldosterone levels enhances the activity of Na+,K+-ATPase, K+ excretion does not increase immediately. The reason for this relates to the effect of aldosterone on Na+ reabsorption and tubular flow. Aldosterone stimulates reabsorption of Na+ and water and thus decreases tubular flow. Reduction in flow in turn decreases K+ secretion (as discussed in more detail later). However, chronic stimulation of Na+ reabsorption increases the ECFV and thereby returns tubular flow to normal. These actions allow a direct stimulatory effect of aldosterone on the ASDN to enhance K+ excretion. Although AVP does not affect urinary K+ excretion, this hormone does promote secretion of K+ by the ASDN (Fig.

1	Although AVP does not affect urinary K+ excretion, this hormone does promote secretion of K+ by the ASDN (Fig. 36.8 ). AVP increases the electrochemical driving force for exit of K+ across the apical membrane of principal cells by stimulating uptake of Na+ across the apical membrane of these cells. The increased Na+ uptake reduces the electrical potential difference across the apical membrane (i.e., the interior of the cell becomes less negatively charged). Despite this effect, AVP does not change K+ secretion by these nephron segments. The reason for this relates to the effect of AVP on tubular fluid flow. AVP decreases flow of tubular fluid by stimulating water reabsorption. The decrease in tubular flow in turn reduces secretion of K+ (explained later). The inhibitory effect of decreased flow of tubular fluid offsets the stimulatory effect of AVP on the electrochemical driving force for exit of K+ across the apical membrane (see

1	Fig. 36.8 ). If AVP did not increase the electrochemical gradient favoring K+ secretion, urinary K+ excretion would fall as AVP levels increased and urinary flow rates decreased. Hence K+ balance would change in • Fig. 36.8 Opposing effects of AVP and urine flow rate on secretion of K+by the ASDN. K+secretion is stimulated by an increase in urinary flow rate and reduced by a fall in AVP levels. In contrast, K+secretion is reduced by a decrease in urinary flow rate and increased by a rise in AVP levels. Because the effects of flow and AVP oppose each other, net K+secretion is not affected by water diuresis or antidiuretics. ˜ Urinary flow rate Water diuresis DT/CCD K+ secretion ˛ AVP levels Constant K+ balance ˛ Urinary flow rate DT/CCD K+ secretion ˜ AVP levels Antidiuresis + _ _ + response to alterations in water balance. Thus the effects of AVP on the electrochemical driving force for exit of K+ across the apical membrane and on tubule flow enable urinary K+ excretion to be

1	+ response to alterations in water balance. Thus the effects of AVP on the electrochemical driving force for exit of K+ across the apical membrane and on tubule flow enable urinary K+ excretion to be maintained constant despite wide fluctuations in water excretion.

1	Plasma [K+], aldosterone, and AVP play important roles in regulating K+ balance; however, the factors and hormones discussed next perturb K+ balance (see Box 36.2 Flow of Tubular Fluid A rise in the flow of tubular fluid (e.g., with diuretic treatment, ECFV expansion) stimulates secretion of K+ within minutes, whereas a fall (e.g., ECFV contraction caused by hemorrhage, severe vomiting, or diarrhea) reduces secretion of K+ by the late segment of the ASDN. Increments in tubular fluid flow are more effective in stimulating secretion of K+ as dietary K+ intake is increased. Recent studies on the primary cilium in principal cells have elucidated some of the mechanisms whereby increased flow stimulates secretion of K+ (

1	Fig. 36.9 ). Increased flow bends the primary cilium in principal cells, which activates the PKD1/PKD2 Ca++-conducting channel complex. This allows more Ca++ to enter principal cells and increases intracellular [Ca++]. The increase in [Ca++] activates BK K+ channels in the apical plasma membrane, which enhances K+ secretion from the cell into the tubule fluid. Increased flow may also stimulate secretion of K+ by other mechanisms. As flow increases, such as after administration of diuretics or as the result of an increase in ECFV, so does the [Na+] of tubule fluid. This increase in [Na+] facilitates entry of Na+ across the apical membrane of ASDN cells, thereby decreasing the cells’

1	K+ K+ ° Flow stimulates Na+ entry, which reduces Vm ° Flow Cilia bend activates PKD1/PKD2 and Ca++ entry ° Flow bends cilia ° Ca++ activates Na+ Ca++ ATP 1 25 4 3 • Fig. 36.9 Cellular mechanism whereby an increased flow rate of tubule fluid stimulates secretion of K+ by principal cells. See text for details. interior negative membrane potential. This depolarization of the cell membrane potential increases the electrochemical driving force that promotes secretion of K+ across the apical cell membrane into tubule fluid. In addition, increased uptake of Na+ into cells activates the Na+,K+-ATPase in the basolateral membrane, thereby increasing uptake of K+ across the basolateral membrane and consequently elevating [K+]. However, it is important to note that an increase in flow rate during a water diuresis does not have a significant effect on excretion of K+ , most likely because during a water diuresis the [Na+] of tubule fluid does not increase as flow rises.

1	Another factor that modulates secretion of K+ is the [H+] of ECF. Acute alterations (within minutes to hours) in the pH of plasma influences secretion of K+ by the ASDN. Alkalosis (i.e., plasma pH above normal) increases secretion of K+ , whereas acidosis (i.e., plasma pH below normal) decreases it. An acute acidosis reduces K+ secretion via two mechanisms: (1) it inhibits Na+,K+-ATPase and thereby reduces cell [K+] and the electrochemical driving force for exit of K+ across the apical membrane, and (2) it reduces the permeability of the apical membrane to K+ . Alkalosis has the opposite effects. The effect of metabolic acidosis on excretion of K+ is time dependent. As already noted, and illustrated in Fig. 36.10 , acute metabolic acidosis reduces K+ excretion. However, when metabolic acidosis lasts for several days, urinary K+ excretion is stimulated (see

1	Fig. 36.10 , acute metabolic acidosis reduces K+ excretion. However, when metabolic acidosis lasts for several days, urinary K+ excretion is stimulated (see Fig. 36.10 ). This occurs because chronic metabolic acidosis decreases reabsorption of water and solutes (e.g., NaCl) by the proximal tubule by

1	Fig. 36.10 ). This occurs because chronic metabolic acidosis decreases reabsorption of water and solutes (e.g., NaCl) by the proximal tubule by ROMK is the primary channel in the apical membrane of principal cells that mediates K+ secretion. Four ROMK subunits make up a single channel. In addition the BK K+ channel, which is activated by elevations in intracellular [Ca++], is also expressed in the apical membrane. The BK channel mediates the flow-dependent increase in K+ secretion discussed earlier. Interestingly, knockout of the KCNJ1 gene (ROMK) causes increased excretion of NaCl and K+ by the kidneys, thereby leading to reduced ECFV and hypokalemia. Although this effect is somewhat perplexing, it should be noted that ROMK is also expressed in the apical membrane of the thick ascending limb (TAL) of Henle’s loop, where it plays a very important role in recycling of K+ across the apical membrane, an effect that is critical for operation of the Na+/ K+/2Cl− symporter (see

1	Fig. 36.7 ). In the absence of ROMK, reabsorption of NaCl by the TAL is reduced, which leads to loss of NaCl in urine. Reduction of NaCl reabsorption by the TAL also reduces the positive transepithelial luminal voltage, which is the driving force for reabsorption of K+ by this nephron segment. Thus the reduction in paracellular K+ reabsorption by the TAL increases urinary K+ excretion even when the cortical collecting duct is unable to secrete the normal amount of K+ because of a lack of ROMK channels. The cortical collecting duct, however, does secrete K+ even in ROMK knockout mice via the flow and Ca++-dependent BK K+ channel expressed in the apical membrane of principal cells.

1	inhibiting Na+,K+-ATPase. Hence the flow of tubular fluid is augmented along the ASDN. Inhibition of water and NaCl reabsorption by the proximal tubule also decreases ECFV and thereby stimulates secretion of aldosterone. In addition, chronic acidosis caused by inorganic acids increases plasma [K+], which stimulates secretion of aldosterone. The rise in tubular fluid flow, plasma [K+], and aldosterone levels offsets the effects of acidosis on cell [K+] and apical membrane permeability, and K+ secretion rises. Thus metabolic acidosis may either inhibit or stimulate excretion of K+ , depending on the duration of the disturbance. As noted, acute metabolic alkalosis stimulates excretion of K+ . Chronic metabolic alkalosis, especially in association with ECFV contraction, significantly increases renal K+ excretion because of the associated increased levels of aldosterone.

1	CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis • Fig. 36.10 Acute versus chronic effect of metabolic acidosis on excretion of K+. See text for details. ECV, effective circulating volume. ˛ Na+,K+-ATPase activity ˛ K+ permeability of apical membrane ˛ Apical membrane K+ gradient Metabolic acidosis Acute Chronic Distal tubule and collecting duct principal cells ˜ Na+,K+-ATPase activity ˜ K+ permeability of apical membrane ˜ K+ secretion ˜ K+ excretion Skeletal muscle cell Proximal tubule cell ˛ H+/K+ exchange ˜ NaCl and H2O reabsorption ˜ ECV ˛ Plasma [K+] ˛ Tubule fluid flow rate Distal tubule and collecting duct principal cells ˛ Aldosterone ˛ Aldosterone ˛ K+ secretion ˛ K+ excretion Glucocorticoids increase urinary K+ excretion. This effect is mediated in part by an increase in GFR, which enhances the urinary flow rate, a potent stimulus of K+ excretion, and by stimulation of SGK1 activity (see earlier).

1	As discussed, the rate of urinary K+ excretion is frequently determined by simultaneous changes in hormone levels, acid-base balance, or the flow rate of tubule fluid ( 36.1 ). The powerful effect of flow often enhances or opposes the response of the ASDN to hormones and changes in acid-base balance. This interaction can be beneficial in the case of hyperkalemia, in which the increase in flow enhances excretion of K+ and thereby restores K+ homeostasis. However, this interaction can also be detrimental, as in the case of alkalosis, in which changes in flow and acid-base status alter K+ homeostasis.

1	Overview of Calcium and Inorganic Phosphate Homeostasis are multivalent ions that subserve many complex and vital functions. Ca++ is an important cofactor in many enzymatic reactions; it is a key second messenger in numerous signaling pathways; it plays an important role in neural transduction, blood clotting, and muscle contraction; and it is a critical component of the extracellular matrix, cartilage, teeth, and bone. Pi, like Ca++ , is a key component of bone. Pi is essential for metabolic aAt physiological pH, inorganic phosphate exists as HPO4 − and H2PO4 − (pK = 6.8). For simplicity, we collectively refer to these ion species as Pi. Modified from Field MJ et al. In Narins R (ed). Textbook of Nephrology: Clinical Disorders of Fluid and Electrolyte Metabolism. 5th ed. New York: McGraw-Hill; 1994.

1	The cellular mechanisms whereby changes in the K+ content of the diet and acid-base balance regulate secretion of K+ by the early segment of the ASDN have recently been elucidated. Elevated K+ intake increases secretion of K+ by several mechanisms, all related to increased serum [K+]. Hyperkalemia increases the activity of the ROMK channel in the apical plasma membrane of principal cells. Moreover, hyperkalemia inhibits reabsorption of NaCl and water by the proximal tubule, thereby increasing the ASDN flow rate, a potent stimulus to secretion of K+ . Hyperkalemia also enhances [aldosterone], which increases K+ secretion by three mechanisms. First, aldosterone increases the number of K+ channels in the apical plasma membrane. Second, aldosterone stimulates uptake of K+ across the basolateral membrane by increasing the number of Na+,K+-ATPase pumps, thereby enhancing the electrochemical gradient driving secretion of K+ across the apical membrane. Third, aldosterone increases movement of

1	membrane by increasing the number of Na+,K+-ATPase pumps, thereby enhancing the electrochemical gradient driving secretion of K+ across the apical membrane. Third, aldosterone increases movement of Na+ across the apical membrane, which depolarizes the apical plasma membrane voltage and thus increases the electrochemical gradient, promoting secretion of K+ . A low-K+ diet dramatically reduces secretion of K+ by the ASDN by increasing the activity of protein tyrosine kinase, which causes ROMK channels to be endocytosed from the apical plasma membrane, thereby reducing K+ secretion. Acidosis decreases secretion of K+ by inhibiting the activity of ROMK channels, whereas alkalosis stimulates secretion of K+ by enhancing ROMK channel activity.

1	processes, including formation of adenosine triphosphate (ATP), and it is an important component of nucleotides, nucleosides, and phospholipids. Phosphorylation of proteins is an important mechanism of cellular signaling, and Pi is an important buffer in cells, plasma, and urine.

1	In adults the kidneys play important roles in regulating total body Ca++ and Pi by excreting the amount of Ca++ and Pi that is absorbed by the intestinal tract (normal bone remodeling results in no net addition of Ca++ and Pi to, or Ca++ and Pi release from, bone). If plasma concentrations of Ca++ and Pi decline substantially, intestinal absorption, bone resorption (i.e., loss of Ca++ and Pi from bone), and renal tubular reabsorption increase and return plasma concentrations of Ca++ and Pi to normal levels. During growth and pregnancy, intestinal absorption exceeds urinary excretion, and these ions accumulate in newly formed fetal tissue and bone. In contrast, bone disease (e.g., osteoporosis) or a decline in lean body mass increases urinary Ca++ and Pi loss without a change in intestinal absorption. These conditions produce a net loss of Ca++ and Pi from the body. Finally, during chronic renal failure, Pi accumulates in the body because absorption by the intestinal tract exceeds

1	absorption. These conditions produce a net loss of Ca++ and Pi from the body. Finally, during chronic renal failure, Pi accumulates in the body because absorption by the intestinal tract exceeds excretion in the urine. This can lead to accumulation of Pi in the body and changes in bone (see the In The Clinic box discussion of chronic renal failure).

1	This brief introduction reveals that the kidneys, in conjunction with the GI tract and bone, play a major role in maintaining plasma Ca++ and Pi levels as well as Ca++ and Pi balance (see ). Accordingly this section of the chapter discusses Ca++ and Pi handling by the kidneys, with an emphasis on the hormones and factors that regulate urinary excretion.

1	Cellular processes in which Ca++ plays an important role include bone formation, cell division and growth, blood coagulation, hormone-response coupling, and electrical stimulus-response coupling (e.g., muscle contraction, neurotransmitter release). Nearly 99% of Ca++ is stored in bone and teeth, approximately 1% is found in ICF, and 0.1% in ECF. The total Ca++ concentration ([Ca++]) in plasma is 10 mg/dL (2.5 mM or 5 mEq/L), and its concentration is normally maintained within very narrow limits. Approximately 50% of the Ca++ in plasma is ionized, 40% is bound to plasma proteins (mainly albumin), and 10% is complexed to several anions, including Pi, HCO3 − , citrate, and SO42− (Fig. 36.11 ). The pH of plasma influences this distribution ( Fig. 36.12). Acidemia increases the

1	Fig. 36.12). Acidemia increases the CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis 1.25 mmol/L E A S 0.25 mmol/L R 1.0 mmol/L D • Fig. 36.12 Effect of pH on plasma [Ca++]. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) • Fig. 36.11 Distribution of Ca++ in plasma. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) • Fig. 36.13 Effect of Ca++ on nerve and muscle excitability. (From Koeppen BM, Stanton BA. Renal

1	Physiology. 5th ed. Philadelphia: Elsevier; 2013.) percentage of ionized Ca++ at the expense of Ca++ bound to proteins, whereas alkalemia decreases the percentage of ionized Ca++, again by altering the Ca++ bound to proteins. Individuals with alkalemia are susceptible to tetany (tonic muscular spasms), whereas individuals with acidemia are less susceptible to tetany, even when total plasma Ca++ levels are reduced. The increase in [H+] in patients with metabolic acidosis causes more H+ to bind to plasma proteins, Pi, HCO3 − , citrate, and SO42− , thereby displacing Ca++ . This displacement increases the plasma concentration of ionized Ca++ . In alkalemia the [H+] of plasma decreases. Some H+ ions dissociate from plasma proteins, Pi, HCO3 − , citrate, and SO42− in exchange for Ca++ , thereby decreasing the plasma concentration of ionized Ca++ . In addition, the plasma albumin concentration also affects ionized plasma [Ca++].

1	Hypoalbuminemia increases the ionized [Ca++], whereas hyperalbuminemia decreases ionized plasma [Ca++]. The total measured plasma [Ca++] does not reflect the total ionized [Ca++], which is the physiologically relevant measure of plasma [Ca++]. A low ionized plasma [Ca++] (hypocalcemia) increases the excitability of nerve and muscle cells and can lead to hypocalcemic tetany. Tetany associated with hypocalcemia occurs because hypocalcemia causes the threshold potential to shift to more negative values (i.e., closer to the resting membrane voltage) ( Fig. 36.13). An elevated ionized plasma [Ca • Fig. 36.14 Overview of Ca++ homeostasis. PTH, parathyroid hormone. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) arrhythmias, lethargy, disorientation, and even death.

1	This effect of hypercalcemia occurs because an elevated plasma [Ca++] causes the threshold potential to shift to less negative values (i.e., farther from the resting membrane voltage). Plasma [Ca++] is regulated within a very narrow range, primarily by parathyroid hormone (PTH), calcitriol (1,25-dihydroxyvitamin D), the active metabolite of vitamin D3, and plasma Ca++ , and this regulation will be discussed next. Within cells, Ca++ is sequestered in the endoplasmic reticulum and mitochondria, or it is bound to proteins. Thus the free intracellular [Ca++] is very low (≈100 nM). The large concentration gradient for [Ca++] across cell membranes is maintained by a Ca++-ATPase pump (PMCa1b) in all cells and by a 3Na+/Ca++ exchanger (NCX1) in some cells. Overview of Calcium Homeostasis

1	Overview of Calcium Homeostasis Ca++ homeostasis depends on two factors: (1) the total amount of Ca++ in the body and (2) the distribution of Ca++ between bone and ECF. The total body Ca++ level is determined by the relative amounts of Ca++ absorbed by the intestinal tract and excreted by the kidneys (Fig.

1	36.14 ). The intestinal tract absorbs Ca++ through an active carrier-mediated transport mechanism that is stimulated by calcitriol, the active metabolite of vitamin D3 that is produced in the proximal tubule of the kidneys. Net Ca++ absorption by the intestine is normally 200 mg/day, but it can increase to 600 mg/day when calcitriol levels rise. In adults, Ca++ excretion by the kidneys equals the amount absorbed by the GI tract (200 mg/day), and it changes in bIn clinical practice the terms hypercalcemia and hypocalcemia are often used to describe a high or low total plasma [Ca++], respectively, even though this usage is not the physiologically correct usage of hypercalcemia and hypocalcemia.

1	parallel with intestinal absorption. Thus in adults, Ca++ balance is maintained because the amount of Ca++ ingested in an average diet (1000 mg/day) equals the amount lost in feces (800 mg/day, the amount that escapes absorption by the intestinal tract) plus the amount excreted in urine (200 mg/day). The second factor that controls Ca++ homeostasis is the distribution of Ca++ between bone and ECF (see

1	Fig. 36.14 ). Two hormones (PTH and calcitriol ) regulate the distribution of Ca++ between bone and ECF and thereby, in concert with the kidneys, regulate plasma [Ca++]. PTH is secreted by the parathyroid glands, and its secretion is stimulated by a decline in plasma [Ca++] (i.e., hypocalcemia). Plasma Ca++ is an agonist of the calcium sensing receptor (CaSR), which is located in the plasma membrane of chief cells in parathyroid glands (discussed later). Hypercalcemia activates the CaSR, which decreases PTH release, whereas hypocalcemia reduces CaSR activity that in turn increases PTH release. PTH increases plasma [Ca++] by: (1) stimulating bone resorption, (2) increasing Ca++ reabsorption by the distal tubule of the kidney, and (3) stimulating the production of calcitriol, which in turn increases Ca++ absorption by the intestinal tract and facilitates PTH-mediated bone resorption. Production of calcitriol in the kidney is stimulated by hypocalcemia and hypophosphatemia. Calcitriol

1	increases Ca++ absorption by the intestinal tract and facilitates PTH-mediated bone resorption. Production of calcitriol in the kidney is stimulated by hypocalcemia and hypophosphatemia. Calcitriol increases plasma [Ca++], primarily by stimulating Ca++ absorption from the intestinal tract. It also facilitates the action of PTH on bone and enhances Ca++ reabsorption in the kidneys by increasing expression of key Ca++ transport and binding proteins in the kidneys (details discussed later). In addition, cCalcitonin is secreted by thyroid C cells (parafollicular cells), and its secretion is stimulated by hypercalcemia. Calcitonin decreases plasma [Ca++], mainly by stimulating bone formation (i.e., deposition of Ca++ in bone). Although it plays an important role in Ca++ homeostasis in lower vertebrates, calcitonin plays only a minor role in Ca++ homeostasis in humans, so it will not be discussed further.

1	hypercalcemia activates the CaSR in the TAL of Henle’s loop, inhibiting Ca++ reabsorption in this segment, resulting in an increase in urinary Ca++ excretion and thereby a reduction in plasma [Ca++]. Hypocalcemia has the opposite effect. Importantly, regulation of Ca++ excretion by the kidneys is one of the major ways the body regulates plasma [Ca++].

1	Conditions that lower PTH levels (i.e., hypoparathyroidism after parathyroidectomy for an adenoma) reduce plasma [Ca++], which can cause hypocalcemic tetany (intermittent muscular contractions). In severe cases, hypocalcemic tetany can cause death by asphyxiation. Hypercalcemia can also cause lethal cardiac arrhythmias and decreased neuromuscular excitability. Clinically the most common causes of hypercalcemia are primary hyperparathyroidism and malignancy-associated hypercalcemia. Primary hyperparathyroidism results most often from overproduction of PTH caused by a benign tumor of the parathyroid glands. In contrast, malignancy-associated hypercalcemia, which occurs in 10% to 20% of all patients with cancer, is caused by secretion of parathyroid hormone–related peptide (PTHrP), a PTH-like hormone secreted by carcinomas in various organs. Increased levels of PTH and PTHrP cause hypercalcemia and hypercalciuria. Calcium Transport Along the Nephron

1	Calcium Transport Along the Nephron The Ca++ available for glomerular filtration consists of the ionized fraction and the amount complexed with anions. Thus about 60% of the Ca++ in plasma is available for glomerular filtration. Normally 99% of filtered Ca++ is reabsorbed by the nephron (Fig. 36.15 ). The proximal tubule reabsorbs about 50% to 60% of the filtered Ca++ . Another 15% is reabsorbed in the loop of Henle (mainly the cortical • Fig. 36.15 Overview of Ca++ transport along the nephron. Percentages refer to amount of filtered Ca++ reabsorbed by each segment. CCD, cortical collecting duct; DT, distal tubule; IMCD, inner medullary collecting duct; PT, proximal tubule; TAL, thick ascending limb.

1	CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis portion of the TAL), about 10% to 15% is reabsorbed by the distal tubule, and less than 1% is reabsorbed by the collecting duct. About 1% (200 mg/day) is excreted in urine. This fraction is equal to the net amount absorbed daily by the intestinal tract. Ca++ reabsorption by the proximal tubule occurs primarily via the paracellular pathway. This passive paracellular reabsorption of Ca++ is driven by the lumen-positive trans-epithelial voltage (Vte) across the second half of the proximal tubule and by a favorable concentration gradient of Ca++ , both of which are established by transcellular sodium and water reabsorption in the first half of the proximal tubule (see

1	Ca++ reabsorption by the loop of Henle also occurs primarily via the paracellular pathway. Like the proximal tubule, Ca++ and Na+ reabsorption in the TAL parallel each other. These processes are parallel because of the significant component of Ca++ reabsorption that occurs via passive paracellular reabsorption secondary to Na+ reabsorption that generates a lumen-positive Vte. Loop diuretics inhibit Na+ reabsorption by the TAL of the loop of Henle, and in so doing reduce the magnitude of the lumen-positive Vte (see ). This action in turn inhibits reabsorption of Ca++ via the paracellular pathway. Thus loop diuretics are used to increase renal Ca++ excretion in patients with hypercalcemia.

1	Mutations in the tight junction protein claudin-16 (CLDN16) reduce the permeability of the paracellular pathway to Ca++ and Mg++ and thereby reduce the diffusive reabsorptive movement of Ca++ and Mg++ across tight junctions in the TAL of Henle’s loop. Familial hypomagnesemic hypercalciuria is caused by mutations in claudin-16, which is a component of the tight junctions in TAL cells. This disorder is characterized by enhanced excretion of Ca++ and Mg++ due to a fall in passive reabsorption of these ions across the paracellular pathway in the TAL. Affected individuals have high levels of Ca++ in their urine, which leads to stone formation (nephrolithiasis).

1	In the distal tubule where the voltage in the tubule lumen is electrically negative with respect to blood, reabsorption of Ca++ is entirely active because Ca++ is reabsorbed against its electrochemical gradient (Fig. 36.16 ). Thus Ca++ reabsorption by the distal tubule is exclusively transcellular. Calcium enters the cell across the apical membrane through Ca++-permeable epithelial ion channels (TRPV5). Inside the cell, Ca++ binds to calbindin-D28K. The calbindin-Ca++ complex carries Ca++ across the cell and delivers Ca++ to the basolateral membrane, where it is extruded from the cell primarily by the 3Na+/1Ca++ antiporter (NCX1); however, plasma membrane Ca++-ATPase isoform 1b (PMCA1b) may also contribute. Urinary Na+ and Ca++ excretion usually change in parallel. However, excretion of these ions does not always change in parallel, because reabsorption of Ca++ and Na+ by the distal tubule is independent and differentially • Fig. 36.16 Cellular mechanism of Ca++ reabsorption by the

1	these ions does not always change in parallel, because reabsorption of Ca++ and Na+ by the distal tubule is independent and differentially • Fig. 36.16 Cellular mechanism of Ca++ reabsorption by the distal tubule. Ca++ is reabsorbed exclusively by a cellular pathway. Ca++ enters the cell across the apical membrane via a Ca++-permeable ion channel (TRPV5). Inside cells, Ca++ binds to calbindin (calbindin-D28K), and the Ca++-calbindin complex diffuses across the cell to deliver Ca++ to the basolateral membrane. Ca++ is transported across the basolateral membrane primarily by a 3 (or 4) Na+/Ca++ antiporter (NCX1) and also by a Ca++-H+-ATPase (PMCa1b). Claudin 8 (CLDN8) is a tight junction protein that is impermeable to Ca++ and thereby prevents the back diffusion of Ca++ across the tight junction into the tubule lumen, which is electrically negative compared to the blood side of the cell.

1	regulated. For example, thiazide diuretics inhibit Na+ reabsorption by the distal tubule and stimulate Ca++ reabsorption by this segment. Accordingly the net effects of thiazide diuretics are to increase urinary Na+ excretion and reduce urinary Ca++ excretion. Because thiazide diuretics reduce urinary Ca++ excretion, they are often given to reduce urinary [Ca++] in individuals who produce Ca++-containing kidney stones. Regulation of Urinary Calcium Excretion

1	Table 36.2 ). Of these, PTH exerts the most powerful control on renal Ca++ excretion; it is the primary hormone/ factor responsible for maintaining Ca++ homeostasis. Overall this hormone stimulates Ca++ reabsorption by the kidneys (i.e., reduces Ca++ excretion). Although PTH inhibits reabsorption of NaCl and fluid, and therefore Ca++ reabsorption by the proximal tubule, PTH stimulates Ca++ reabsorption by the TAL of the loop of Henle and the distal tubule. Thus the net effect of PTH is to enhance renal Ca++ reabsorption. Changes in plasma [Ca++] also regulate urinary Ca++ excretion, with hypercalcemia increasing excretion and hypocalcemia decreasing excretion. Hypercalcemia increases urinary Ca++ excretion by: (1) reducing proximal tubule Ca++ reabsorption (reduced paracellular reabsorption due to increased interstitial fluid [Ca++]); (2) inhibiting Ca++ reabsorption by the TAL of the loop of Henle via activation of the CaSR located in the basolateral membrane of these cells (NaCl

1	due to increased interstitial fluid [Ca++]); (2) inhibiting Ca++ reabsorption by the TAL of the loop of Henle via activation of the CaSR located in the basolateral membrane of these cells (NaCl reabsorption is decreased, thereby reducing the magnitude of the lumen-positive Vte); and (3) suppressing Ca++ reabsorption by the distal tubule by reducing PTH levels. As a result, urinary Ca++ excretion increases. Hypocalcemia has the opposite effect on urinary Ca++ excretion, primarily by increasing Ca++ reabsorption by the proximal tubule and TAL. Calcitriol enhances Ca++ reabsorption by the distal tubule, but it is less effective than PTH.

1	Several factors disturb Ca++ excretion. An increase in plasma [Pi] concentration (e.g., caused by a dramatic increase in dietary intake of Pi or by reduced kidney function) elevates PTH levels both directly and by decreasing the ionized plasma [Ca++] and thereby decreases Ca++ excretion. A decline in plasma [Pi] (e.g., caused by dietary Pi depletion) has the opposite effect (NOTE: with normal kidney function, changes in dietary Pi intake over a sevenfold range has no effect on plasma [Pi]). Changes in ECFV alter Ca++ excretion mainly by affecting NaCl and fluid reabsorption in the proximal tubule. Volume contraction increases NaCl and water reabsorption by the proximal tubule and thereby enhances Ca++ reabsorption. Accordingly, urinary Ca++ excretion declines. Volume expansion has the opposite effect. Acidemia increases Ca++ excretion, whereas alkalemia decreases excretion. Regulation of Ca++ reabsorption by pH

1	CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis aPTH inhibits Ca++ reabsorption by the proximal tubule but stimulates reabsorption by the TAL and distal tubule. Overall the net effect is to increase Ca++ reabsorption and thereby reduce urinary Ca++ excretion. CaSR, calcium-sensing receptor. Modified from Mount DB, Yu A. Transport of inorganic solutes: sodium, chloride, potassium, magnesium, calcium and phosphate. In: Brenner BM (ed). Brenner and Rector’s The Kidney. 8th ed. Philadelphia: Saunders; 2008. occurs primarily in the distal tubule. Alkalosis stimulates the apical membrane Ca++ channel (TRPV5), thereby increasing Ca++ reabsorption. By contrast, acidosis inhibits the same channel, thereby reducing Ca++ reabsorption. Finally, as noted earlier, loop diuretics inhibit Ca++ reabsorption by the TAL, and thiazide diuretics stimulate Ca++ reabsorption by the distal tubule.

1	The calcium-sensing receptor (CaSR) is a receptor expressed in the plasma membrane of cells involved in regulating Ca++ homeostasis; it senses small changes in extracellular [Ca++]. Ca++ binds to CaSR receptors in PTH-secreting cells of the parathyroid gland and calcitriol-producing cells of the proximal tubule. Activation of the receptor by an increase in plasma [Ca++] results in inhibition of PTH secretion and production of calcitriol by the proximal tubule. Moreover, reduction in PTH secretion also contributes to decreased production of calcitriol because PTH is a potent stimulus of calcitriol synthesis. By contrast, a fall in plasma [Ca++] has the opposite effect on PTH and calcitriol secretion.

1	The CaSR also maintains Ca++ homeostasis by directly regulating Ca++ excretion by the kidneys. CaSRs in the TAL respond directly to changes in plasma [Ca++] and regulate Ca++ absorption. An increase in plasma [Ca++] activates CaSR in the TAL and inhibits Ca++ absorption, thereby stimulating urinary Ca++ excretion. By contrast, a fall in plasma [Ca++] leads to an increase in Ca++ absorption by the TAL and a corresponding decrease in urinary Ca++ excretion. Thus the direct effect of plasma [Ca++] on CaSRs in the TAL acts in concert with changes in PTH, which regulates Ca++ reabsorption by the distal tubule to regulate urinary Ca++ excretion and thereby maintain Ca++ homeostasis.

1	Mutations in the gene coding for CaSR cause disorders in Ca++ homeostasis. Familial hypocalciuric hypercalcemia (FHH) is a haploinsufficient state caused by an inactivating mutation of CaSR. The hypercalcemia is caused by deranged Ca++-regulated PTH secretion (i.e., the set point for Ca++-regulated PTH secretion is shifted such that PTH levels are elevated at any level of plasma [Ca++], and are not suppressed in the setting of hypercalcemia). The hypocalciuria is caused by enhanced Ca++ reabsorption in the TAL and distal tubule owing to elevated PTH levels and defective CaSR regulation of Ca++ transport in the kidneys. Autosomal-dominant hypoparathyroidism is caused by an activating mutation in CaSR. Activation of CaSRs causes deranged Ca++-regulated PTH secretion (i.e., the set point for Ca++-regulated PTH secretion is shifted such that PTH levels are decreased at any level of plasma [Ca++]). Hypercalciuria results and is caused by decreased PTH levels and defective CaSR-regulated

1	Ca++-regulated PTH secretion is shifted such that PTH levels are decreased at any level of plasma [Ca++]). Hypercalciuria results and is caused by decreased PTH levels and defective CaSR-regulated Ca++ transport in the kidneys.

1	Pi is an important component of many organic molecules, including DNA, RNA, ATP, nucleotides, nucleosides, and phospholipids and intermediates of metabolic pathways. Like Ca++ it is a major constituent of bone. Its concentration in plasma is an important determinant of bone formation and resorption. In addition, urinary Pi is an important buffer (i.e., it is one of many titratable acids) involved in the maintenance of acid-base balance (see ). Some 85% of Pi is located in bone and teeth, 14% is located in the ICF, and 1% is located in the ECF. Normal plasma • Fig. 36.17 Overview of Pi homeostasis. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) [Pi] is 3 to 4 mg/dL (1–1.5 mM). Pi in plasma is ionized (45%), complexed (30%), and bound to protein (25%). Phosphate deficiency causes muscle weakness, rhabdomyolysis, and reduced bone mineralization resulting in rickets (in children) and osteomalacia (in adults). Overview of Phosphate Homeostasis

1	Overview of Phosphate Homeostasis A general scheme of Pi homeostasis is shown in

1	Fig. 36.17 . Maintenance of Pi homeostasis depends on two factors: (1) the amount of Pi in the body and (2) the distribution of Pi between the ICF and ECF compartments. Total body Pi levels are determined by the relative amount of Pi absorbed by the intestinal tract versus the amount excreted by the kidneys. Pi absorption by the intestinal tract occurs via active and passive mechanisms; Pi absorption increases as dietary Pi rises, and it is stimulated by calcitriol. Despite variations in Pi intake between 800 and 1500 mg/day, in adults (i.e., the steady state) the kidneys maintain total body Pi balance constant by excreting an amount of Pi in the urine equal to the amount absorbed by the intestinal tract (normal bone remodeling results in no net addition of Pi to, or Pi release from, bone). By contrast, during growth, Pi is accumulated in the body. Renal Pi excretion is the primary mechanism by which the body regulates Pi balance and thereby Pi homeostasis.

1	The second factor that maintains Pi homeostasis is distribution of Pi among bone and the ICF and ECF compartments. Regulation of plasma [Pi] is controlled by two hormones, PTH and calcitriol, in concert with the kidneys (see Fig. 36.17 ). Release of Pi from bone is stimulated by the same hormones (i.e., PTH and calcitriol) that release Ca++ from this pool. Thus release of Pi from bone is always accompanied by release of Ca++ . The kidneys also make an important contribution to maintaining plasma [Pi] within a narrow range (1–1.5 mM). Pi excretion by the kidneys is regulated by PTH and calcitriol (see Fig. 36.17 ). PTH increases Pi excretion, whereas calcitriol inhibits Pi excretion. Plasma [Pi] is determined by: (1) intestinal absorption, (2) storage in bone, and (3) Pi excretion by the kidneys. Maintenance of plasma [Pi] is essential for optimal Ca++-Pi complex formation required for bone mineralization without deposition of Ca++-Pi in vascular and other soft tissues.

1	A rise in plasma [Pi] directly stimulates PTH synthesis and release and also decreases the ionized [Ca++], which stimulates PTH release by its interaction with the CaSR. PTH enhances urinary Pi excretion by inhibiting proximal tubule Pi reabsorption. Hyperphosphatemia also decreases calcitriol production by the proximal tubule, which leads to a reduction in Pi absorption by the intestine. Both the increase in PTH and the decrease in calcitriol reduce plasma [Pi]. Phosphate Transport Along the Nephron Fig. 36.18 summarizes Pi transport by the various portions of the nephron. The proximal tubule reabsorbs 80% of the Pi filtered by the glomerulus; the loop of Henle, distal tubule, and collecting duct reabsorb negligible amounts of Pi. Therefore approximately 20% of the Pi filtered across the glomerular capillaries is excreted in urine. Pi reabsorption by the proximal tubule occurs by a transcellular route (

1	Pi reabsorption by the proximal tubule occurs by a transcellular route ( Fig. 36.19 ). Pi uptake across the apical membrane of the proximal tubule occurs via two Na+/Pi symporters (IIa and IIc). Type IIa transports 3Na+ with one divalent Pi (HPO42−), and carries positive charge into the cell. Type IIc transports 2Na+ with one monovalent Pi (H2PO4 −) and is electrically neutral. Pi exits across the basolateral membrane by a Pi-inorganic anion antiporter that has not been characterized. CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis • Fig. 36.18 Pi transport along the nephron. Pi is reabsorbed primarily by the proximal tubule. Percentages refer to the amount of the filtered Pi reabsorbed by each nephron segment. Approximately 20% of the filtered Pi is excreted. CCD, cortical collecting duct; DT, distal tubule; IMCD, inner medullary collecting duct; PT, proximal tubule; TAL, thick ascending limb.

1	In patients with chronic renal failure, the kidneys cannot excrete Pi. Because of continued Pi absorption by the intestinal tract, Pi accumulates in the body and plasma [Pi] rises. The excess Pi complexes with Ca++ and reduces the ionized plasma [Ca++]. Pi accumulation also decreases production of calcitriol. This response reduces Ca++ absorption by the intestine, an effect that further reduces plasma [Ca++]. This reduction in plasma [Ca++] increases PTH secretion and Ca++ release from bone. These actions result in renal osteodystrophy (i.e., increased bone resorption with replacement by fibrous tissue, which renders bone more susceptible to fracture). Chronic hyperparathyroidism (i.e., elevated PTH levels due the fall in plasma [Ca++]) during chronic renal failure can lead to metastatic calcifications in which Ca++ and Pi precipitate in arteries, soft tissues, and viscera. Deposition of Ca++ and Pi in the heart may cause myocardial failure. Prevention and treatment of

1	to metastatic calcifications in which Ca++ and Pi precipitate in arteries, soft tissues, and viscera. Deposition of Ca++ and Pi in the heart may cause myocardial failure. Prevention and treatment of hyperparathyroidism and Pi retention include a low-Pi diet or administration of a “phosphate binder” (i.e., an agent that forms insoluble Pi salts and thereby renders Pi unavailable for absorption by the intestinal tract) in the diet. Supplemental Ca++ and calcitriol are also prescribed to increase plasma [Ca++].

1	• Fig. 36.19 Cellular mechanisms of Pi reabsorption by the proximal tubule. The apical transport pathway contains two Na+/Pi symporters, one that transports three Na+for each Pi (IIa) and one that transports two Na+for each Pi (IIc). Pi leaves the cell across the basolateral membrane by an unknown mechanism. ATP, adenosine triphosphate. TubularfluidBloodPiPiK˜Na˜2Na˜Pi3Na˜ATPIIaIIc? Fibroblast growth factor 23 (FGF-23) increases renal Pi excretion and thereby contributes to regulation of plasma [Pi] (see Fig. 36.17 ). FGF-23 is secreted by osteocytes and osteoblasts and inhibits Pi reabsorption and calcitriol production by the proximal tubule. Secretion of FGF-23 is stimulated by sustained hyperphosphatemia, PTH, and calcitriol. Activating mutations in the FGF23 gene cause hypophosphatemia, low plasma calcitriol, and rickets/osteomalacia, whereas inactivating mutations cause hyperphosphatemia, high serum calcitriol, and calcification of soft tissue.

1	Regulation of Urinary Phosphate Excretion Table 36.3 Fig. 36.20 ). Increased plasma Pi reduces TABLE Summary of Hormones and Factors 36.3 Affecting Pi Reabsorption by Proximal Tubule

1	PTH Decrease FGF-23 Decrease Phosphate loading Decrease Phosphate depletion Increase Metabolic acidosis: chronic Decrease Metabolic alkalosis: chronic Increase ECFV expansion Decrease Growth hormone Increase Glucocorticoids Decrease • Fig. 36.20 Overview of the major hormones regulating plasma [Pi]. FGF-23, fibroblast growth factor 23; PTH, parathyroid hormone. Dashed lines indicate negative feedback. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) plasma [Ca++] and therefore increases plasma PTH, which increases Pi excretion by the kidneys. PTH, the most important hormone that controls Pi excretion, inhibits Pi reabsorption by the proximal tubule and thereby increases Pi excretion. PTH reduces Pi reabsorption by stimulating endocytic removal of Na+/Pi transporters from the brush border membrane of the proximal tubule. Increased plasma Pi also increases FGF-23, which inhibits Pi reabsorption and calcitriol production by the proximal tubule.

1	Na+/Pi transporters from the brush border membrane of the proximal tubule. Increased plasma Pi also increases FGF-23, which inhibits Pi reabsorption and calcitriol production by the proximal tubule. Elevated plasma Pi also suppresses calcitriol production, which results in a decrease in intestinal Pi reabsorption. Dietary Pi intake also regulates Pi excretion by mechanisms unrelated to changes in PTH levels. Pi loading increases excretion, whereas Pi depletion decreases it. Changes in dietary Pi intake modulate Pi transport by altering the transport rate of each Na+/Pi symporter and the number of symporters in the apical membrane of the proximal tubule.

1	ECFV also affects Pi excretion. Expansion of the ECF enhances Pi excretion by: (1) increasing GFR and thus the filtered load of Pi; (2) decreasing Na/Pi coupled reabsorption, which reduces ECFV, and (3) reducing plasma [Ca++], thereby increasing PTH, which inhibits Pi reabsorption in the proximal tubule. Acid-base balance also influences Pi excretion; chronic acidosis increases Pi excretion, and chronic alkalosis decreases it. These effects of acid-base balance, like the effect of PTH, are mediated by changes of expression of the Na /Pi symporters in the apical membrane. Systemic acidosis increases glucocorticoid secretion, and glucocorticoids increase excretion of Pi by inhibiting Pi reabsorption by the proximal tubule. This inhibition, together with the direct effect of acidosis on Pi reabsorption by the proximal tubule, enables the distal tubule and collecting duct to secrete more H+ as titratable acid and to generate more HCO3 − because Pi is an important urinary buffer. Growth

1	reabsorption by the proximal tubule, enables the distal tubule and collecting duct to secrete more H+ as titratable acid and to generate more HCO3 − because Pi is an important urinary buffer. Growth hormone decreases Pi excretion.

1	Klotho, which was identified in 1997, is highly expressed in the early distal tubule of the kidney. Klotho knockout mice have a phenotype that resembles chronic kidney disease (CKD), including soft tissue calcification, hyperphosphatemia, and elevated plasma FGF-32. Klotho exists as both a membrane-bound and a soluble protein. The membrane-bound form is a coreceptor for FGF-23, thus Klotho promotes Pi excretion by the kidneys and reduces serum levels of 1,25-dihydroxyvitamin D3. Soluble circulating Klotho has a number of additional functions, including modulating ion transport and Wnt signal transduction, inhibiting reninangiotensin signaling, and modulating FGF-23 regulation of PTH production. A considerable body of experimental data suggests that Klotho may be a biomarker for CKD, and that Klotho deficiency may contribute to development of CKD. Moreover, experimental data also suggest that Klotho therapy may slow the progression of CKD.

1	CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis 667 • Fig. 36.21 Overview of the major hormones regulating plasma [Ca++]. Dashed lines indicate negative feedback. FGF-23, fibroblast growth factor 23; PTH, parathyroid hormone. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) Integrative Review of Parathyroid Hormone and Calcitriol on Ca++ and Pi Homeostasis

1	Integrative Review of Parathyroid Hormone and Calcitriol on Ca++ and Pi Homeostasis Fig. 36.21 , PTH has numerous effects on Ca++ and Pi homeostasis. Hypocalcemia is the major stimulus of PTH secretion. PTH stimulates bone resorption, increases urinary Pi excretion, decreases urinary Ca++ excretion, and stimulates production of calcitriol, which stimulates Ca++ and Pi absorption by the intestine. Because changes in Pi handling in bone, intestines, and kidneys tend to balance out, PTH increases plasma [Ca++] while having little effect on plasma [Pi]. Overall a rise in plasma PTH levels in response to hypocalcemia returns plasma [Ca++] to the normal range. A decline in plasma [Ca++] has the opposite effect. Calcitriol (the active form of vitamin D) also plays an important role in Ca++ and Pi homeostasis ( Fig. 36.22

1	Calcitriol (the active form of vitamin D) also plays an important role in Ca++ and Pi homeostasis ( Fig. 36.22 The primary action of calcitriol is to stimulate Ca++ and Pi absorption by the intestine. To a lesser degree it acts with PTH to release Ca++ and Pi from bone and to decrease Ca++ excretion by the kidneys. The net effect of calcitriol is to increase plasma [Ca++] and [Pi]. Thus the major stimuli of calcitriol production are hypocalcemia via PTH and hypophosphatemia (i.e., a low plasma [Pi]). In the absence of glucocorticoids (e.g., in Addison’s disease), excretion of Pi is depressed, as is the ability of the kidneys to excrete titratable acid and to generate new HCO3 − . Growth hormone increases reabsorption of Pi by the proximal tubule. As a result, growing children are in positive Pi balance and have a higher plasma [Pi] than adults, and this elevated [Pi] is important for bone formation.

1	• Fig. 36.22 Activation of calcitriol (vitamin D3) and its effect on Ca++ and Pi homeostasis. Hypocalcemia (via PTH) and hypophosphatemia are the major stimuli of the metabolism of calcifediol to calcitriol in the kidneys. The net effect of calcitriol is to increase plasma [Ca++] and [Pi]. (From Koeppen BM, Stanton BA. Renal Physiology. 5th ed. Philadelphia: Elsevier; 2013.) 1. K+ homeostasis is maintained by the kidneys, which adjust K+ excretion to match dietary K+ intake, and by the hormones insulin, epinephrine, and aldosterone, which regulate the distribution of K+ between the ICF and ECF compartments. Other events, such as cell lysis, exercise, and changes in acid-base balance and plasma osmolality, disturb K+ homeostasis and plasma [K+]. 2.

1	2. Excretion of K+ by the kidneys is determined by the rate and direction of K+ transport by the distal tubule and collecting duct. Secretion of K+ by these tubular segments is regulated by plasma [K+], aldosterone, and AVP. In contrast, changes in tubular fluid flow and acid-base disturbances perturb K+ excretion by the kidneys. In K+-depleted states, K+ secretion is inhibited and the distal tubule and collecting duct reabsorb K+ . 3. The kidneys, in conjunction with the intestinal tract and bone, play a vital role in regulating plasma [Ca++] and [Pi]. 4. Plasma [Ca++] is regulated by PTH and calcitriol. Calcitonin is not a major regulatory hormone in humans. Ca++ excretion by the kidneys is regulated by PTH, plasma [Ca++], and calcitriol and is altered by changes in acid-base status, ECFV, and plasma Pi. 5. Ca++ reabsorption by the TAL and distal tubule are regulated by PTH and calcitriol, both of which stimulate Ca++ reabsorption, and by plasma [Ca++]. 6.

1	5. Ca++ reabsorption by the TAL and distal tubule are regulated by PTH and calcitriol, both of which stimulate Ca++ reabsorption, and by plasma [Ca++]. 6. Plasma [Pi] is regulated by PTH, FGF-23, and calcitriol. Pi excretion is regulated by PTH, FGF-23, dietary phosphate, and growth hormone and is altered by acid base balance, ECFV expansion, and glucocorticoids. Bone tumors secrete FGF-23, which enhances renal Pi excretion and thereby causes hypophosphatemia, hyperphosphatemia, and a defect in bone mineralization (i.e., osteomalacia). Biber J, et al. Phosphate transporters and their function. Annu Rev Physiol. 2013;75:535-550. Christov M, Juppner H. Insights from genetic disorders of phosphate homeostasis. Semin Nephrol. 2013;33:143-157. Miller T. Control of renal calcium, phosphate, electrolyte, and water excretion by the calcium-sensing receptor. Best Pract Res Clin Endocrinol Metab. 2013;27:345-358.

1	Miller T. Control of renal calcium, phosphate, electrolyte, and water excretion by the calcium-sensing receptor. Best Pract Res Clin Endocrinol Metab. 2013;27:345-358. Palmer BF. Regulation of potassium homeostasis. Clin J Am Soc Nephrol. 2015;10:1050-1060. Patel A. The primary cilium calcium channels and their role in flow sensing. Pflugers Arch. 2015;467:157-165. Pearce D, et al. Collecting duct principal cell transport processes and their regulation. Clin J Am Soc Nephrol. 2015;10:135-146. Bernardo JF, Friedman PA. Renal calcium metabolism. In. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: CHAPTER 36 Potassium, Calcium, and Phosphate Homeostasis Physiology and Pathophysiology. 5th ed. Waltham, MA: Elsevier; 2013:2225. Berndt TJ, Kumar R. Clinical disturbances of phosphate homeostasis. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Waltham, MA: Elsevier; 2013:2369.

1	Malnic G, et al. Regulation of K+ excretion. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Waltham, MA: Elsevier; 2013:1659. Murer H, et al. Proximal tubular handling of phosphate. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Waltham, MA: Elsevier; 2013:2351. Smogorzewski MJ, et al. Disorders of calcium, magnesium, and phosphate balance. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012:689. Wang W, Huang CL. The molecular biology of renal K+ channels. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Waltham, MA: Elsevier; 2013: 1601. Upon completion of this chapter the student should be able to answer the following questions: 1. How does HCO3 − operate as a buffer, and why is it an important buffer of the extracellular fluid? 2.

1	How does HCO3 − operate as a buffer, and why is it an important buffer of the extracellular fluid? 2. How does metabolism of food produce acid and alkali, and what effect does the composition of the diet have on systemic acid-base balance? 3. What is the difference between volatile and nonvolatile acids, and what is net endogenous acid production (NEAP)? 4. How do the kidneys and lungs contribute to systemic acid-base balance, and what is renal net acid excretion (RNAE)? 5. Why are urinary buffers necessary for excretion of acid by the kidneys? 6. What are the mechanisms for H+ transport in the various segments of the nephron, and how are these mechanisms regulated? 7. How do the various segments of the nephron contribute to the process of reabsorbing the filtered HCO3 −? 8. How do the kidneys produce new HCO3 −? 9. How is ammonium produced by the kidneys, and how does its excretion contribute to renal acid excretion? 10.

1	8. How do the kidneys produce new HCO3 −? 9. How is ammonium produced by the kidneys, and how does its excretion contribute to renal acid excretion? 10. What are the major mechanisms by which the body defends itself against changes in acid-base balance? 11. What are the differences between simple metabolic and respiratory acid-base disorders, and how are they differentiated by arterial blood gas measurements? he concentration of H+ in body fluids is low compared with that of other ions. For example, Na+ is present at a concentration some three million times greater than that of H+ ([Na+] = 140 mEq/L; [H+] = 40 nEq/L). Because of the low [H+] of the body fluids, it is commonly expressed as the negative logarithm, or pH.

1	Virtually all cellular, tissue, and organ processes are sensitive to pH. Indeed, life cannot exist outside of a range of extracellular fluid (ECF) pH from 6.8 to 7.8 (160–16 nEq/L of H+). Normally the pH of ECF is maintained between 7.35 and 7.45. As described in the pH of intracellular fluid (ICF) is slightly lower (7.1–7.2) but also tightly regulated.

1	Each day, acid and alkali are ingested in the diet. Also, cellular metabolism produces a number of substances that have an impact on the pH of body fluids. Without appropriate mechanisms to deal with this daily acid and alkali load and thereby maintain acid-base balance, many processes necessary for life could not occur. This chapter reviews the maintenance of whole-body acid-base balance. Although the emphasis is on the role of the kidneys in this process, the roles of the lungs and liver are also considered. In addition the impact of diet and cellular metabolism on acid-base balance is presented. Finally, disorders of acid-base balance are considered, primarily to illustrate the physiological processes involved. Throughout this chapter, acid is defined as any substance that adds H+ to body fluids, whereas alkali is defined as a substance that removes H+ from body fluids. The HCO3 − Buffer System

1	The HCO3 − Buffer System Bicarbonate (HCO3 −) is an important buffer of the ECF. With a normal plasma [HCO3 −] of 23 to 25 mEq/L and a volume of 14 L (for a 70-kg individual) the ECF can potentially buffer 350 mEq of H+ . The HCO3 − buffer system differs from the other buffer systems of the body (e.g., phosphate) because it is regulated by both the lungs and kidneys. This is best appreciated by considering the following reaction: Equation 37.1 As indicated the first reaction (hydration/dehydration of CO2) is the rate-limiting step. This normally slow reaction is greatly accelerated in the presence of carbonic anhydrase. The second reaction, ionization of H2CO3 to H+ and HCO3 − , is virtually instantaneous.

1	The second reaction, ionization of H2CO3 to H+ and HCO3 − , is virtually instantaneous. aCarbonic anhydrase (CA) actually catalyzes the following reaction: (e.g., citrate) •Fig. 37.1 Overviewofacid-basebalance.Thelungsandkidneysworktogethertomaintainacid-basebalance.ThelungsexcreteCO2(volatileacid),andthekidneysexcreteacid(renalnetacidexcretion[RNAE])equaltonetendogenousacidproduction(NEAP),whichreflectsdietaryintake,cellularmetabolism,andlossofacidandalkali(e.g.,HCO3− lossinfeces)fromthebody.Seetextfordetails.(FromKoeppenBM,StantonBA.Renal Physiology. 5thed.Philadelphia:Elsevier;2013.) The Henderson-Hasselbalch equation is used to quantitate how changes in CO2 and HCO3 − effect pH: Equation 37.2 Equation 37.3 [HCO3 − ]pH = 6 1. + log 0 03. PCO2

1	The Henderson-Hasselbalch equation is used to quantitate how changes in CO2 and HCO3 − effect pH: Equation 37.2 Equation 37.3 [HCO3 − ]pH = 6 1. + log 0 03. PCO2 In these equations the amount of CO2 is determined from the partial pressure of CO2 (PCO2) and its solubility (α) in solution. For plasma at 37°C, α has a value of 0.03. Also, pK′ is the negative logarithm of the overall dissociation constant for the reaction in Eq. 37.1 and has a value for plasma at 37°C of 6.1. Alternatively the relationship between HCO3 − and CO2 on the [H+] can be determined as follows: Equation 37.4

1	Eq. 37.1 and has a value for plasma at 37°C of 6.1. Alternatively the relationship between HCO3 − and CO2 on the [H+] can be determined as follows: Equation 37.4 Inspection of Eqs. 36.3 and 36.4 show that pH and [H+] vary when either [HCO3 −] or PCO2 are altered. Disturbances of acid-base balance that result from a change in [HCO3 −] are termed metabolic acid-base disorders, whereas those resulting from a change in PCO2 are termed respiratory acid-base disorders. These disorders are considered in more detail in a subsequent section. The kidneys are primarily responsible for regulating the [HCO3 −] of ECF, whereas the lungs control the PCO2. Overview of Acid-Base Balance

1	Overview of Acid-Base Balance The diet of humans contains many constituents that are either acid or alkali. In addition, cellular metabolism produces acid and alkali. Finally, alkali is normally lost each day in feces. As described later, although diet dependent, the net effect of these processes is the addition of acid to body fluids. For acid-base balance to be maintained, acid must be excreted from the body at a rate equivalent to its addition. If acid addition exceeds excretion, acidosis results. Conversely, if acid excretion exceeds addition, alkalosis results.

1	Fig. 37.1 , the major constituents of the diet are carbohydrates and fats. When tissue perfusion is adequate, O2 is available to tissues, and insulin is present at normal levels, carbohydrates and fats are metabolized to CO2 and H2O. On a daily basis, 15 to 20 moles of CO2 are generated through this process. Normally this large quantity of CO2 is effectively eliminated from the body by the lungs. Therefore this metabolically derived CO2 has no impact on acid-base balance. CO2 is usually termed volatile acid because it has the potential to generate H+ after hydration with H2O (see Eq. 36.1 ). Acid not derived directly from hydration of CO2 is termed nonvolatile acid (e.g., lactic acid).

1	The cellular metabolism of other dietary constituents also has an impact on acid-base balance. For example, cysteine and methionine, sulfur-containing amino acids, yield sulfuric acid when metabolized, whereas hydrochloric acid results from metabolism of lysine, arginine, and histidine. A portion of this nonvolatile acid load is offset by production of HCO3 − through metabolism of the amino acids aspartate and glutamate. On average the metabolism of dietary amino acids yields net nonvolatile acid production. Metabolism of certain organic anions (e.g., citrate) results in production of HCO3 − , which offsets nonvolatile acid production to some degree. Overall in individuals ingesting a meat-containing diet, acid production exceeds HCO3 − production. In contrast, a vegetarian diet produces less nonvolatile acid. In addition to the metabolically derived acids and alkalis, the foods ingested contain acid and alkali. For example, the presence of phosphate (H2PO4 −) in ingested food

1	less nonvolatile acid. In addition to the metabolically derived acids and alkalis, the foods ingested contain acid and alkali. For example, the presence of phosphate (H2PO4 −) in ingested food increases the dietary acid load. Finally, during digestion, some HCO3 − is normally lost in feces. This loss is equivalent to the addition of nonvolatile acid to the body. In an individual ingesting a meat-containing diet, dietary intake, cellular metabolism, and fecal HCO3 − loss result in addition of approximately 0.7 to 1.0 mEq/kg body weight of nonvolatile acid to the body each day (50–100 mEq/day for most adults). This acid, referred to as net endogenous acid production (NEAP), results in an equivalent loss of HCO3 − from the body that must be replaced.

1	Wheninsulinlevelsarenormal,carbohydratesandfatsarecompletelymetabolizedtoCO2+ H2O.However,ifinsulinlevelsareabnormallylow(e.g.,diabetesmellitus),cellularmetabolismleadstoproductionofseveralorganicketoacids(e.g.,β-hydroxybutyricacidandacetoaceticacidfromfattyacids). IntheabsenceofadequatelevelsofO2(hypoxia),anaerobicmetabolismbycellscanalsoleadtoproductionoforganicacids(e.g.,lacticacid)ratherthanCO2+ H2O.Thisfrequentlyoccursinnormalindividualsduringvigorousexercise.Poortissueperfusion,suchasoccurswithreducedcardiacoutput,canalsoleadtoanaerobicmetabolismbycellsandthustoacidosis.IntheseconditionstheorganicacidsaccumulateandthepHofbodyfluidsdecreases(acidosis).Treatment(e.g.,administrationofinsulininthecaseofdiabetes)orimproveddeliveryofadequatelevelsofO2totissues(e.g.,inthecaseofpoortissueperfusion)resultsinthemetabolismoftheseorganicacidstoCO2+ H2O,whichconsumesH+ andtherebyhelpscorrecttheacid-basedisorder.

1	Nonvolatile acids do not circulate throughout the body but are immediately neutralized by the HCO3 − in ECF. Equation 37.5 Equation 37.6 This neutralization process yields the Na+ salts of the strong acids and removes HCO3 − from the ECF. Thus HCO3 − minimizes the effect of these strong acids on the pH of ECF. As noted previously, ECF contains approximately 350 mEq of HCO3 − . If this HCO3 − were not replenished, the daily production of nonvolatile acids (≈70 mEq/day) would deplete the ECF of HCO3 − within 5 days. To maintain acid-base balance the kidneys must replenish the HCO3 − lost by neutralization of the nonvolatile acids, a process termed renal net acid excretion (RNAE). Net Acid Excretion by the Kidneys

1	Net Acid Excretion by the Kidneys Under steady-state conditions, NEAP must equal RNAE to maintain acid-base balance. Although NEAP varies from individual to individual and from day to day in anyone individual, it is not regulated. Instead the kidneys regulate RNAE to match NEAP and in so doing replenish the HCO3 − (new HCO3 −) lost by neutralization of nonvolatile acids. In addition the kidneys must prevent the loss of HCO3 − in urine. This latter task is quantitatively more important because the filtered load of HCO3 − is approximately 4320 mEq/day (24 mEq/L × 180 L/day = 4320 mEq/day), compared with only 50 to 100 mEq/day needed to balance NEAP.

1	Both reabsorption of filtered HCO3 − and excretion of acid are accomplished via H+ secretion by the nephrons. Thus in a single day the nephrons must secrete approximately 4390 mEq of H+ into the tubular fluid. Most of the secreted serves to reabsorb the filtered load of HCO3 . Only 50 to 100mEq of H+ , an amount equivalent to NEAP, is excreted in urine. As a result of this acid excretion, urine is normally acidic. The kidneys cannot excrete urine more acidic than pH 4.0 to 4.5. Even at a pH of 4.0, only 0.1 mEq/L of H+ can be excreted. Therefore to excrete sufficient acid, the kidneys excrete H+ with urinary buffers such as phosphate (Pi).

1	Other constituents of urine can also serve as buffers (e.g., creatinine), although their role is less important than Pi. Collectively the various urinary buffers are termed titratable acid. This term is derived from the method by which these buffers are quantitated in the laboratory. Typically, alkali (OH−) is added to a urine sample to titrate its pH to that of plasma (i.e., 7.4). The amount of alkali added is equal to the H+ titrated by these urine buffers and is termed titratable acid.

1	Excretion of H+ as a titratable acid is insufficient to balance NEAP. An additional and important mechanism by which the kidneys contribute to maintenance of acid-base balance is through synthesis and excretion of ammonium (NH4 +). The mechanisms involved in this process are discussed in more detail later in this chapter. With regard to renal regulation of acid-base balance, each NH4 + excreted in urine results in the return of one HCO3 − to the systemic circulation, which replenishes the HCO3 − lost during neutralization of the nonvolatile acids. Thus production and excretion of NH4 + , like excretion of titratable acid, is equivalent to excretion of acid by the kidneys. In brief the kidneys contribute to acid-base homeostasis by reabsorbing the filtered load of HCO3 − and excreting an bThe titration reaction is: HPO42−+ H+↔ H2PO4 − . This reaction has a pK of approximately 6.8. amount of acid equivalent to NEAP. This process can be quantitated as follows:

1	amount of acid equivalent to NEAP. This process can be quantitated as follows: Equation 37.7 where (UNH4+× V˜ ) and (UTA × V˜ ) are the rates of excretion (mEq/day) of NH4 + and titratable acid (TA), and (UHCO3−× V˜ ) is the amount of HCO3 − lost in urine (equivalent to adding H+ to the body). Again, maintenance of acid-base balance means that net acid excretion must equal nonvolatile acid production. Under most conditions, very little HCO3 − is excreted in urine. Thus net acid excretion essentially reflects titratable acid and NH4 + excretion. Quantitatively, titratable acid accounts for approximately one-third and NH4 + for two-thirds of RNAE. HCO3 − Reabsorption Along the Nephron Eq. 37.7 , net acid excretion is maximized when little or no HCO3 − is excreted in urine. Indeed, under most circumstances, very little HCO3 − appears in urine. Because HCO3 − is freely filtered at the glomerulus, approximately 4320 mEq/day is delivered to the nephrons and is then reabsorbed.

1	Fig. 37.2 summarizes the contribution of each nephron segment to reabsorption of filtered HCO3 − . The proximal tubule reabsorbs the largest portion of the filtered load of HCO3 − . Fig. 37.3 summarizes the primary cThis equation ignores the small amount of free H+ excreted in urine. As already noted, urine with a pH = 4.0 contains only 0.1 mEq/L of H+ .

1	Fig. 37.3 summarizes the primary cThis equation ignores the small amount of free H+ excreted in urine. As already noted, urine with a pH = 4.0 contains only 0.1 mEq/L of H+ . transport processes involved. H+ secretion across the apical membrane of the cell occurs by both a Na+/H+ antiporter and H+-ATPase (V-type). The Na+/H+ antiporter (NHE3) is the predominant pathway for H+ secretion (accounts for ≈ two-thirds of HCO3 − reabsorption) and uses the lumen-to-cell [Na+] gradient to drive this process (i.e., •Fig. 37.2 SegmentalreabsorptionofHCO3− .ThefractionofthefilteredloadofHCO3− reabsorbedbythevarioussegmentsofthenephronisshown.NormallytheentirefilteredloadofHCO3− isreabsorbedandlittleornoHCO3− appearsintheurine.CCD,corticalcollectingduct;DT,distaltubule;IMCD,innermedullarycollectingduct;PT,proximaltubule;TAL,thickascendinglimb.

1	•Fig. 37.3 CellularmechanismforreabsorptionoffilteredHCO3− bycellsoftheproximaltubule.OnlytheprimaryH+ andHCO3− transportersareshown.ATP,adenosinetriphosphate;CA,carbonicanhydrase. secondary active secretion of H+). Within the cell, H+ and HCO3 − are produced in a reaction catalyzed by carbonic anhydrase (CA-II). The H+ is secreted into the tubular fluid, whereas the HCO3 − exits the cell across the basolateral membrane and returns to the peritubular blood. HCO3 − movement out of the cell across the basolateral membrane is coupled to other ions. The majority of HCO3 − exits via a symporter that couples the efflux of Na+ with HCO3 − (sodium bicarbonate symporter, NBC1). Some HCO3 − exits the cell by other transporters, but they are not as important as the Na+/HCO3 − symporter. As noted in

1	Fig. 37.3 , carbonic anhydrase (CA-IV) is also present in the brush border and basolateral membrane of the cell. The brush border enzyme catalyzes dehydration of H2CO3 in the luminal fluid, whereas the enzyme localized to basolateral membrane facilitates HCO3 − exit from the cell. The movement of CO2 into and out of the cell occurs via AQP1, which is localized to both the luminal and basolateral membranes. The cellular mechanism for HCO3 − reabsorption by the thick ascending limb (TAL) of the loop of Henle is very similar to that in the proximal tubule. H+ is secreted by a Na+/H+ antiporter and H+-ATPase. Like in the proximal tubule, the Na+/H+ antiporter (NHE3) is the predominant pathway for H+ secretion. HCO3 − exit from the cell involves both a Na+/HCO3 − symporter (NBC1) and a Cl−/HCO3 − antiporter (anion exchanger, AE-2). Some HCO3 − may also exit the cell through Cl− channels present in the basolateral membrane.

1	The distal tubule and collecting duct reabsorb the small amount of HCO3 − that escapes reabsorption by the proximal tubule and loop of Henle. Fig. 37.4 shows the cellular mechanism of H+/HCO3 − transport by the intercalated cells located within these segments (see also One type of intercalated cell secretes H+ (reabsorbs HCO3 −) and is called the A-or α-intercalated cell. Within this cell, H+ and HCO3 − are produced by hydration of CO2; this reaction is catalyzed by carbonic anhydrase (CA-II). H+ is secreted into the tubular fluid via two mechanisms. The first involves an apical membrane H+-ATPase (V-type). The second couples secretion of H+ with reabsorption of via an H+-K+-ATPase similar to those found in the stomach and colon (HKα1 and HKα2). HCO3 − exits the cell across the basolateral membrane in exchange for Cl− (via a Cl−/HCO3 − antiporter, AE-1) and enters the peritubular capillary blood.

1	A second population of intercalated cells secretes HCO3 − rather than H+ into the tubular fluid (also called B-or dHere and in the remainder of the chapter we focus on the function of intercalated cells. The early portion of the distal tubule, which does not contain intercalated cells, also reabsorbs HCO3 − . The cellular mechanism appears to involve an apical membrane Na+/H+ antiporter (NHE2) and a basolateral Cl−/HCO3 − antiporter (AE2). β-intercalated cells). In these cells the H+-ATPase (V-type) is located in the basolateral membrane and the Cl−/HCO3 − antiporter is located in the apical membrane (see Fig. 37.4).

1	β-intercalated cells). In these cells the H+-ATPase (V-type) is located in the basolateral membrane and the Cl−/HCO3 − antiporter is located in the apical membrane (see Fig. 37.4). However, the apical membrane Cl−/HCO3 − antiporter is different from the one found in the basolateral membrane of the H+-secreting intercalated cells and has been identified as pendrin. The activity of the HCO3 −-secreting intercalated cell is increased during metabolic alkalosis, when the kidneys must excrete excess HCO3 − . However, under most conditions (e.g., ingestion of a meat-containing diet) H+ secretion predominates in these segments.

1	The apical membrane of collecting duct cells is not very permeable to H+ , and thus the pH of tubular fluid can become quite acidic. Indeed, the most acidic tubular fluid along the nephron (pH = 4.0–4.5) is produced there. In comparison the permeability of the proximal tubule to H+ and HCO3 − is much higher, and the tubular fluid pH falls to only 6.5 in this segment. As explained later the ability of the collecting duct to lower the pH of the tubular fluid is critically important for excretion of urinary titratable acids and NH4 + . Regulation of H+ Secretion A number of factors influence secretion of H+ and thus reabsorption of filtered HCO3 − by the cells of the nephron. From a physiological perspective the primary factor that regulates H+ secretion by the nephron is a change in systemic acid-base balance. Thus acidosis stimulates RNAE, whereas RNAE is reduced during alkalosis.

1	The response of the kidneys to changes in acid-base balance includes both immediate changes in the activity and/or number of transporters in the membrane and longer-term changes in the synthesis of transporters. For example, with metabolic acidosis, H+ secretion is stimulated by multiple mechanisms, depending on the particular nephron segment. First, the decrease in intracellular pH that occurs with acidosis will create a more favorable cell-to–tubular fluid H+ gradient and thereby make secretion of H+ across the apical membrane more energetically favorable. Second, the decrease in pH may lead to allosteric changes in transport proteins, thereby altering their kinetics. Lastly, transporters may be shuttled to the membrane from intracellular vesicles. With long-term acidosis the abundance of transporters increases, either by increased transcription of appropriate transporter genes or by increased translation of transporter mRNA.

1	eA third group of intercalated cells shares features of both H+-secreting and HCO3 −-secreting intercalated cells. The precise function of this cell type in acid-base transport is not fully understood. fTraditionally it was believed that intercalated cells were only involved in acid-base transport. There is now good evidence that NaCl reabsorption is also carried out by intercalated cells (B type). Reabsorption of NaCl occurs by the tandem operation of an apical membrane Cl−/HCO3 − anti-porter (pendrin) and an apical membrane Na+/HCO3 −/2Cl− antiporter (NDCBE). This mechanism of NaCl reabsorption is inhibited by thiazide diuretics. •Fig. 37.4 CellularmechanismsforreabsorptionandsecretionofHCO3− byintercalatedcellsofthedistaltubuleandcollectingduct.OnlytheprimaryH+ andHCO3− transportersareshown.ATP,adenosinetriphosphate;CA,carbonicanhydrase.

1	•Fig. 37.5 ExamplesofcellularH+ andHCO3− sensors.ATP,adenosinetriphosphate;cAMP,cyclicadenosinemonophosphate;GPCR,Gprotein–coupledreceptor;IRR,insulinreceptor–relatedreceptor;Pyk2,nonreceptortyrosinekinase;sAC,solubleadenylylcyclase.(Adaptedfrom:LevinLR,BuckJ.Annu Rev Physiol 2015;77:347.)

1	Fig.37.5 ).Forexample,aGprotein–coupledH+ receptor(GPCR–GPR4)hasbeenlocalizedtothecollectingduct.ActivationofthisreceptorbyanincreaseinECF[H+]stimulatesH+ secretion.Alsointhecollectingduct,HCO3−-secretingintercalatedcells(B-orβ-ICs)expressabasolateralinsulin-relatedreceptor(IRR)thatisatyrosinekinase.ItisactivatedbyanincreaseinECF[HCO3−]andstimulatesHCO3− secretionbythecell.Asolubleadenylylcyclase(sAC)regulatedbyintracellularHCO3− appearstoalsoplayarollinregulatingcollectingductH+ secretion.Intheproximaltubule,basolateralmembranereceptortyrosinekinases(ErbB1andErbB2)sensechangesinECFPCO2.ActivationofthesereceptorsbyanincreaseinPCO2resultsingenerationofangiotensinII,whichactingfromthelumenviaAT-1Areceptors,stimulatesH+ secretion/HCO3− reabsorption.Alsointheproximaltubule,thenonreceptortyrosinekinase(Pyk2)sensesintracellular[H+].Whenitisactivatedbyanincreaseinintracellular[H+],H+ secretion/HCO3− reabsorptionisstimulated.Finally,thegatingofseveralionchannels(e.g.,therenaloutermedullaryK+

1	secretion/HCO3− reabsorptionisstimulated.Finally,thegatingofseveralionchannels(e.g.,therenaloutermedullaryK+ channel[ROMK])iseffectedbychangesineitherECForICFpH.Thesetoohavethepotentialtoserveascellularacid-basesensors.

1	Although some of the effects just described may be attributable directly to acidosis, many of these changes in cellular H+ transport are mediated by hormones or other factors. Three known mediators of the renal response to acidosis are endothelin, cortisol, and angiotensin II. Endothelin (ET-1) Intheproximaltubule,metabolicacidosisincreasesthetransportkineticsoftheNa+/H+ antiporter(NHE3)andincreasesapicalmembraneexpressionoftheNa+/H+ antiporter,H+-ATPase,andthebasolateralNa+/3HCO3− symporter(NBCe1).Inthecollectingduct,acidosisleadstoexocyticinsertionofH+-ATPaseintotheapicalmembraneofintercalatedcells.Withlong-termacidosistheabundanceofkeyacid-basetransportersisincreasedintheproximaltubule(NHE3andNBCe1)andincollectingductintercalatedcells(H+-ATPaseandAE1).Lastly,acidosisdecreasesexpressionoftheCl−/HCO3− antiporterpendrininHCO3−-secretingintercalatedcells.

1	is produced by endothelial and proximal tubule cells. With acidosis, ET-1 secretion is enhanced. In the proximal tubule, ET-1 stimulates phosphorylation and subsequent insertion of the Na+/H+ antiporter into the apical membrane, and insertion of the Na+/3HCO3 − symporter into the basolateral membrane. ET-1 may mediate the response to acidosis in other nephron segments as well. Acidosis also stimulates secretion of the glucocorticoid hormone cortisol by the adrenal cortex. Cortisol increases the abundance of the Na+/ H+ antiporter and Na+/3HCO3 − symporter in the proximal tubule. Angiotensin II is produced in proximal tubule cells in response to acidosis. It is secreted into the tubular fluid, where it binds to the angiotensin I receptor and thereby stimulates H+ secretion/HCO3 − reabsorption by the proximal tubule. Both cortisol and angiotensin II also stimulate production and secretion of NH4 + by the proximal tubule, which as described later is an important component of the kidney’s

1	by the proximal tubule. Both cortisol and angiotensin II also stimulate production and secretion of NH4 + by the proximal tubule, which as described later is an important component of the kidney’s response to acidosis.

1	Acidosis also stimulates secretion of parathyroid hormone (PTH). PTH inhibits phosphate (Pi) reabsorption by the proximal tubule (see ). In so doing, more Pi is delivered to the distal nephron, where it serves as a urinary buffer and thus increases the capacity of the kidneys to excrete titratable acid. The response of the kidneys to alkalosis is less well characterized. RNAE is decreased because of increased urinary HCO3 − excretion and because excretion of titratable acid and NH4 + are reduced. The factors that regulate this response are not well characterized.

1	Other factors not necessarily related to maintaining acid-base balance can influence secretion of H+ by the cells of the nephron. Because a significant H+ transporter in the nephron is the Na+/H+ antiporter, factors that alter Na+ reabsorption can secondarily affect H+ secretion. For example, with volume contraction (negative Na+ balance), Na+ reabsorption by the nephron is increased (see ), including reabsorption of Na+ via the Na+/H+ antiporter. As a result, H+ secretion is enhanced. This occurs by several mechanisms. One mechanism involves the renin-angiotensinaldosterone system, which is activated by volume contraction. As noted earlier, angiotensin II acts on the proximal tubule to stimulate the apical membrane Na+/H+ antiporter as well as the basolateral Na+/3HCO3 − symporter. To a lesser degree, angiotensin II stimulates H+ secretion in the TAL of Henle’s loop and the early portion of the distal tubule, a process also mediated by the Na+/H+ antiporter. Aldosterone’s primary

1	a lesser degree, angiotensin II stimulates H+ secretion in the TAL of Henle’s loop and the early portion of the distal tubule, a process also mediated by the Na+/H+ antiporter. Aldosterone’s primary action on the distal tubule and collecting duct is to stimulate Na+ reabsorption by principal cells (see ). However, it also stimulates intercalated cells in these segments to secrete H+ . This effect is both indirect and direct. By stimulating Na+ reabsorption by principal cells, aldosterone hyperpolarizes the transepithelial voltage (i.e., the lumen becomes more electrically negative). This change in transepithelial voltage then facilitates secretion of H+ by intercalated cells. In addition to this indirect effect, aldosterone (and angiotensin II) act directly on intercalated cells to stimulate H+ secretion via H+-ATPase and H+,K+-ATPase.

1	Another mechanism by which ECF volume (ECFV) contraction enhances H+ secretion (HCO3 − reabsorption) is through changes in peritubular capillary Starling forces. As described in , ECFV contraction alters the peritubular capillary Starling forces such that overall proximal tubule reabsorption is enhanced. With this enhanced reabsorption, more of the filtered load of HCO3 − is reabsorbed. Potassium balance influences secretion of H+ by the proximal tubule. Hypokalemia stimulates and hyperkalemia inhibits H+ secretion. It is thought that K+-induced changes in intracellular pH are responsible at least in part for this effect, with hypokalemia acidifying and hyperkalemia alkalinizing the cells. Hypokalemia also stimulates H+ secretion by the collecting duct. This occurs as a result of increased expression of the H+,K+-ATPase in intercalated cells. Formation of New HCO3 −

1	Formation of New HCO3 − As discussed previously, reabsorption of the filtered load of HCO3 − is important for maximizing RNAE. However, HCO3 − reabsorption alone does not replenish the HCO3 − lost during neutralization of the nonvolatile acids produced during metabolism. To maintain acid-base balance, the kidneys must replace this lost HCO3 − with new HCO3 − . Generation of new HCO3 − occurs by excretion of titratable acid and by synthesis and excretion of NH4 + . Production of new HCO3 − as a result of titratable acid excretion is depicted in

1	Production of new HCO3 − as a result of titratable acid excretion is depicted in Fig. 37.6 . Because of HCO3 − reabsorption by the proximal tubule and loop of Henle, fluid reaching the distal tubule and collecting duct normally contains little HCO3 − . Thus when H+ is secreted it will combine with non-HCO3 − buffers (primarily Pi) and be excreted as titratable acid. Because the H+ was produced inside the cell from hydration of CO2, a HCO3 − is also produced. This HCO3 − is returned to the ECF as new HCO3 − . As noted, Pi excretion increases with acidosis. However, even with increased Pi available for titratable acid formation, this response is insufficient to generate the required amount of new HCO3 − . The remainder of new HCO3 − generation occurs as a result of NH4 + generation and excretion. NH4 + is produced by the kidneys, and its synthesis and subsequent excretion adds HCO3 − to ECF. Importantly, this process is regulated in response to the acid-base requirements of the body.

1	NH4 + is produced by the kidneys, and its synthesis and subsequent excretion adds HCO3 − to ECF. Importantly, this process is regulated in response to the acid-base requirements of the body. NH4 + is produced in the kidneys via metabolism of glutamine. Essentially the kidneys metabolize glutamine, excrete NH4 + , and add HCO3 − to the body. However, formation of new HCO3 − via this process depends on the kidneys’ ability to excrete NH4 + in urine. If NH4 + is not excreted in urine but instead enters the systemic circulation, it is converted into urea by the liver. This conversion process generates H+ , which is then buffered by HCO3 − . Thus production of urea from renal-generated NH4 + consumes HCO3 − and negates formation of HCO3 − through synthesis and excretion of NH4 + by the kidneys. However, normally the kidneys excrete NH4 + in urine and thereby produce new HCO3 − . The process by which the kidneys excrete NH4 + is complex.

1	The process by which the kidneys excrete NH4 + is complex. Fig. 37.7 illustrates the essential features of this process. NH4 + is produced from glutamine in the cells of the proximal tubule, a process termed ammoniagenesis. Each glutamine molecule produces two molecules of NH4 + and the divalent anion 2-oxoglutarate2− . Metabolism of this anion ultimately provides two molecules of HCO3 − . HCO3 − exits the cell across the basolateral membrane and enters the peritubular blood as new HCO3 − . NH4 + exits the cell across the apical membrane and enters the tubular fluid. The primary mechanism for NH4 + secretion into the tubular fluid involves the Na+/H+ antiporter, with NH4 + substituting for H+ . In addition, some NH3 can diffuse out of the cell into the tubular fluid, where it is protonated to NH4 + .

1	A significant portion of the NH4 + secreted by the proximal tubule is reabsorbed by the loop of Henle. The TAL is the primary site of this NH4 + reabsorption, with NH4 + substituting for K+ on the 1Na+/1K+/2Cl− symporter. In addition the lumen-positive transepithelial voltage in this segment drives paracellular reabsorption of NH4 + . 678 SECTION7Berne & Levy Physiology •Fig. 37.6 GeneralschemeforexcretionofH+withnon-HCO3−urinarybuffers(titratableacid).Theprimaryurinarybufferisphosphate(HPO42−).AH+-secretingintercalatedcellisshown.Forsimplicity,onlytheH+-ATPaseisdepicted.H+secretionbytheH+,K+-ATPasealsotitratesluminalbuffers.ATP,adenosinetriphosphate;CA,carbonicanhydrase.Tubular fluid Blood Cl– CO2 + H2O Buffer + H+ H-Buffer H˜ATP CA HCO3 – HCO3 – The NH4 + reabsorbed by the TAL of the loop of Henle accumulates in the medullary interstitium. From there it is then secreted into the tubular fluid by the collecting duct.

1	The NH4 + reabsorbed by the TAL of the loop of Henle accumulates in the medullary interstitium. From there it is then secreted into the tubular fluid by the collecting duct. The cells of the collecting duct express two NH3 membrane transporters known as Rhesus (Rh) glycoproteins (RhBG and RhCG). RhBG is present in the basolateral membrane of H+-secreting intercalated cells and principal cells, and RhCG is present in both the apical and basolateral membranes of these cells. As depicted in Fig. 37.7 , NH3 is transported across the collecting duct, a process traditionally termed nonionic diffusion. The secreted NH3 is protonated in the tubule lumen as a result of intercalated H+ secretion. Because the apical membrane has a low permeability to NH4 + it is effectively trapped in the tubular lumen, a process traditionally termed diffusion trapping.

1	H+ secretion by the collecting duct is critical for excretion of NH4 + . If collecting duct H+ secretion is inhibited, the NH4 + reabsorbed by the TAL of Henle’s loop will not be excreted in the urine. Instead it will be returned to the systemic circulation, where as described previously, it will be converted to urea by the liver and consume HCO3 − in the process. Thus new HCO3 − is produced during the metabolism of glutamine by cells of the proximal tubule. However, the overall process is not complete until the NH4 + is excreted (i.e., production of urea from NH4 + by the liver is prevented). Thus NH4 + excretion in urine can be used as a marker of glutamine metabolism in the proximal tubule.

1	AssessingNH4+ excretionbythekidneysisdoneindirectlybecauseassaysofurineNH4+ arenotroutinelyavailable.Considerforexamplethesituationofmetabolicacidosis,whereintheappropriaterenalresponseistoincreasenetacidexcretion.Accordingly,littleornoHCO3− willappearinurine,urinewillbeacidic,andNH4+ excretionwillbeincreased.Toassessthis,andespeciallytheamountofNH4+ excreted,the“urinarynetcharge”or“urineaniongap”canbecalculatedbymeasuringurinaryconcentrationsofNa+ ,K+ ,andCl−: TheconceptofurineaniongapduringametabolicacidosisassumesthatthemajorcationsinurineareNa+ ,K+ ,andNH4+ andthatthemajoranionisCl− (withurinepH< 6.5,virtuallynoHCO3− ispresent).AsaresulttheurineaniongapwillyieldanegativevaluewhenNH4+ isbeingexcreted.Indeed,theabsenceofaurineaniongaportheexistenceofapositivevalueindicatesarenaldefectinNH4+ productionandexcretion. gBoth RhBG and RhCG transport NH3. There is some evidence that RhBG may also transport some NH4 + .

1	gBoth RhBG and RhCG transport NH3. There is some evidence that RhBG may also transport some NH4 + . •Fig. 37.7 Production,transport,andexcretionofNH4+bythenephron.GlutamineismetabolizedtoNH4+andHCO3−intheproximaltubule.TheNH4+issecretedintothelumen,andtheHCO3−enterstheblood.ThesecretedNH4+isreabsorbedinHenle’sloop,primarilybythethickascendinglimb,andaccumulatesinthemedullaryinterstitium.NH3issecretedbythecollectingductviarhesusglycoproteins,andH+secretiontrapsNH4+inthelumen.ForeachmoleculeofNH4+excretedintheurine,amoleculeof“new”HCO3−isaddedbacktotheECF.CA,carbonicanhydrase.Tubular fluid Tubular fluid CO2 + H2O NH4 + NH4 + 2NH4 + NH4 + NH3 NH3 NH4 + NH4 + NH3 + H+ NH4 + NH4 + NH4 + NH4 + H˜NH3HCO3 – Blood Blood Na+ Na+ H+H+ Glutamine A= 2HCO3 – CA In the net, one new HCO3 − is returned to the systemic circulation for each NH4 + excreted in the urine.

1	In the net, one new HCO3 − is returned to the systemic circulation for each NH4 + excreted in the urine. An important feature of the renal NH4 + system is that it is regulated by systemic acid-base balance. As already described, cortisol levels increase with acidosis, as does angiotensin II secretion into the lumen of the proximal tubule. Both cortisol and angiotensin II stimulate ammoniagenesis (i.e., NH4 + production from glutamine). During systemic acidosis, the enzymes in the proximal tubule cell responsible for metabolism of glutamine are stimulated. This involves synthesis of new enzyme and requires several days for complete adaptation. With increased levels of these enzymes, NH4 + production is increased, allowing enhanced production of new HCO3 − . Conversely, glutamine metabolism is reduced with alkalosis. Acidosis also increases the abundance of RhBG and RhCG in the collecting duct. Thus the ability to secrete NH4 + is also enhanced. Response to Acid-Base Disorders

1	Response to Acid-Base Disorders The pH of the ECF is maintained within a very narrow range (7.35–7.45). Inspection of Eq. 37.3 shows that the pH of hFor simplicity of presentation in this chapter, the value of 7.40 for body fluid pH is used as normal, even though the normal range is from 7.35 to 7.45. Similarly the normal range for PCO2 is 35 to 45 mm Hg. However, a PCO2 of 40 mm Hg is used as the normal value. Finally, a value of 24 mEq/L is considered a normal ECF [HCO3 −], even though the normal range is 22 to 28 mEq/L. ECF varies when either [HCO3 −] or PCO2 are altered. As already noted, disturbances of acid-base balance that result from a change in [HCO3 −] of ECF are termed metabolic acid-base disorders, whereas those resulting from a change in PCO2 are termed respiratory acid-base disorders. The kidneys are primarily responsible for regulating [HCO3 −], whereas the lungs regulate PCO2.

1	When an acid-base disturbance develops, the body uses several mechanisms to defend against the change in ECF pH. These defense mechanisms do not correct the acid-base disturbance but merely minimize the change in pH imposed by the disturbance. Restoration of the blood pH to its normal value requires correction of the underlying process or processes that produced the acid-base disorder. The body has three general mechanisms to defend against changes in body fluid pH produced by acid-base disturbances: (1) extracellular and intracellular buffering, (2) adjustments in blood PCO2 via alterations in the ventilatory rate of the lungs, and (3) adjustments in RNAE. The first line of defense against acid-base disorders is extracellular and intracellular buffering. The response of the extracellular buffers is virtually instantaneous, whereas the response to intracellular buffering is slower and can take several minutes.

1	Metabolic disorders that result from addition of nonvolatile acid or alkali to body fluids are buffered in both the extracellular and intracellular compartments. The HCO3 − buffer system is the principal ECF buffer. When nonvolatile acid is added to body fluids (or alkali is lost from the body), HCO3 − is consumed during the process of neutralizing the acid load, and the [HCO3 −] of ECF is reduced. Conversely, when nonvolatile alkali is added to body fluids (or acid is lost from the body), H+ is consumed, causing more HCO3 − to be produced from the dissociation of H2CO3. Consequently, [HCO3 −] increases.

1	Although the HCO3 − buffer system is the principal ECF buffer, Pi and plasma proteins provide additional extracellular buffering. The combined action of the buffering processes for HCO3 − , Pi, and plasma protein accounts for approximately 50% of the buffering of a nonvolatile acid load and 70% of a nonvolatile alkali load. The remainder of the buffering under these two conditions occurs intracellularly. Intracellular buffering involves movement of H+ into cells (during buffering of nonvolatile acid) or movement of H+ out of cells (during buffering of nonvolatile alkali). H+ is titrated inside the cell by HCO3 − , Pi, and the histidine groups on proteins. Bone represents an additional source of extracellular buffering. However, with acidosis, buffering by bone results in its demineralization.

1	Bone represents an additional source of extracellular buffering. However, with acidosis, buffering by bone results in its demineralization. When respiratory acid-base disorders occur, the pH of body fluid changes as a result of alterations in PCO2. Virtually all buffering in respiratory acid-base disorders occurs intracellularly. When PCO2 rises (respiratory acidosis), CO2 moves into the cell, where it combines with H2O to form H2CO3. H2CO3 then dissociates to H+ and HCO3 − . Some of the H+ is buffered by cellular proteins, and HCO3 − exits the cell and raises the ECF [HCO3 −]. This process is reversed when PCO2 is reduced (respiratory alkalosis). Under this condition the hydration reaction (H2O + CO2 ↔ H2CO3) is shifted to the left by the decrease in PCO2. As a result the dissociation reaction (H2CO3 ↔ H++ HCO3 −) also shifts to the left, thereby reducing the ECF [HCO3 −].

1	The lungs are the second line of defense against acid-base disorders. As indicated by the Henderson-Hasselbalch equation (see Eq. 37.2 ), changes in PCO2 alter the blood pH: a rise decreases the pH, and a reduction increases the pH. The ventilatory rate determines the PCO2. Increased ventilation decreases PCO2, whereas decreased ventilation increases it (Fig. 37.8 ). The blood PCO2 and pH are important regulators of the ventilatory rate. Chemoreceptors located in the brainstem (ventral surface of the medulla) and periphery (carotid and aortic bodies) sense changes in PCO2 and [H+] and alter the ventilatory rate appropriately. Thus when metabolic acidosis occurs, a rise in the [H+] (decrease in pH) stimulates the ventilatory rate. Conversely, during metabolic alkalosis, a decreased [H+] (increase in pH) reduces the ventilatory rate. With maximal hyperventilation, the PCO2 can be reduced to approximately •Fig. 37.8 EffectofventilatoryrateonalveolarPCO2andthusarterialbloodPCO2.

1	10 mm Hg. Because hypoxia, a potent stimulator of ventilation, also develops with hypoventilation, the degree to which the PCO2 can be increased is limited. In an otherwise normal individual, hypoventilation cannot raise the PCO2 above 60 mm Hg. The respiratory response to metabolic acid-base disturbances may be initiated within minutes but may require several hours to complete.

1	The third and final line of defense against acid-base disorders involves the kidneys. In response to an alteration in plasma pH and PCO2, the kidneys make appropriate adjustments in the excretion of RNAE. The renal response may require several days to reach completion because it takes hours to days to increase the synthesis and activity of the proximal tubule enzymes involved in NH4 + production. In the case of acidosis (increased [H+] or PCO2), the secretion of H+ by the nephron is stimulated and the entire filtered load of HCO3 − is reabsorbed. Titratable acid excretion is increased and production and excretion of NH4 + is also stimulated, and thus RNAE is increased ( Fig. 37.9). The new HCO3 − generated during the process of net acid excretion is added to the body, and the plasma [HCO3 −] increases.

1	Fig. 37.9). The new HCO3 − generated during the process of net acid excretion is added to the body, and the plasma [HCO3 −] increases. When metabolic alkalosis exists (decreased [H+]), the filtered load of HCO3 − is increased (plasma [HCO3 −] is elevated). With respiratory alkalosis (decreased PCO2), plasma [HCO3 −] is decreased and thus the filtered load is decreased. In both conditions, secretion of H+ by the nephron is inhibited. As a result, HCO3 − excretion is increased. At the same time, excretion of both titratable acid and NH4 + is decreased. Thus RNAE is decreased and HCO3 − appears in urine. Also, some HCO3 − is secreted into urine by the HCO3 −-secreting intercalated cells of the distal tubule and collecting duct. With enhanced excretion of HCO3 − , plasma [HCO3 −] decreases. •Fig. 37.9 Responseofthenephrontoacidosis.Pi,phosphate;PTH,parathyroidhormone;Rhbg&Rhcg,rhesusglycoproteins;RNAE,renalnetacidexcretion;TA,titratableacid;V,urineflowrate.

1	Lossofgastriccontentsfromthebody(e.g.,vomiting,nasogastricsuctioning)producesmetabolicalkalosissecondarytothelossofHCl.Ifthelossofgastricfluidissignificant,ECFVcontractionoccurs.UnderthisconditionthekidneyscannotexcretesufficientquantitiesofHCO3− tocompensateforthemetabolicalkalosis.TheinabilityofthekidneystoexcreteHCO3− isaresultoftheneedtoreduceNa+ excretiontocorrecttheECFVcontraction.Asdescribedpreviously(see fordetails)theresponseofthekidneystovolumecontractionistoreducetheglomerularfiltrationrate,whichreducesthefilteredloadofHCO3− ,andtoincreaseNa+ reabsorptionalongthenephron.BecausealargeamountofNa+ reabsorptionoccursviatheNa+/H+ antiporter,thisresultsinanincreaseinH+ secretion(HCO3− reabsorption)bytheproximaltubule.InthissettingtheentirefilteredloadofHCO3− isreabsorbedandnewHCO3− generationmayevenbeenhanced.Thelatterresponseoccursbecausealdosterone,thelevelsofwhichareelevatedinvolumecontraction,notonlystimulatesdistalNa+ reabsorptionbutalsoH+

1	isreabsorbedandnewHCO3− generationmayevenbeenhanced.Thelatterresponseoccursbecausealdosterone,thelevelsofwhichareelevatedinvolumecontraction,notonlystimulatesdistalNa+ reabsorptionbutalsoH+ secretionbyintercalatedcells.ThisstimulationofH+ secretiongeneratesnewHCO3− byenhancingtitratableacidandNH4+ excretion.Thusinindividualswholosegastriccontents,metabolicalkalosisandparadoxicallyacidicurinecharacteristicallyoccur.Correctionofthealkalosisoccursonlywheneuvolemiaisreestablished.

1	Table 37.1 summarizes the primary alterations and the subsequent compensatory or defense mechanisms of the various simple acid-base disorders. In all acid-base disorders the compensatory response does not correct the underlying disorder but simply reduces the magnitude of the change in pH. Correction of the acid-base disorder requires treatment of its cause. Types of Acid-Base Disorders Metabolic acidosis is characterized by a decreased ECF [HCO3 −] and pH. It can develop via the addition of nonvolatile acid to the body (e.g., diabetic ketoacidosis), the loss of nonvolatile base (e.g., HCO3 − loss caused by diarrhea), or failure of the kidneys to excrete titratable acid and NH4 + (e.g., renal failure). As previously described, the buffering of H+ occurs in both the ECF and ICF compartments. When the pH falls, the respiratory centers are stimulated and the ventilatory rate is increased (respiratory compensation).

1	Finally, in metabolic acidosis, RNAE is increased. This occurs via elimination of all HCO3 − from urine (enhanced reabsorption of filtered HCO3 −) and via increased titratable acid and NH4 + excretion (enhanced production of new HCO3 −). If the process that initiated the acid-base disturbance is corrected, the enhanced RNAE will ultimately return the pH and [HCO3 −] to normal. After correction of the pH, the ventilatory rate also returns to normal. Metabolic alkalosis is characterized by an increased ECF [HCO3 −] and pH. It can occur via the addition of nonvolatile base to the body (e.g., ingestion of antacids), as a result of volume contraction (e.g., hemorrhage), or more commonly from loss of nonvolatile acid (e.g., loss of gastric HCl because of prolonged vomiting). Buffering occurs predominantly in the ECF and to a lesser degree in the Respiratoryacidosis↓ Respiratoryalkalosis↑ ECF,extracellularfluid;ICF,intracellularfluid;RNAE,renalnetacidexcretion

1	Respiratoryacidosis↓ Respiratoryalkalosis↑ ECF,extracellularfluid;ICF,intracellularfluid;RNAE,renalnetacidexcretion Whennonvolatileacidisaddedtobodyfluids,asindiabetic ketoacidosis,the[H+]increases(pHdecreases),andthe[HCO3−]decreases.Inadditiontheconcentrationoftheanionassociatedwiththenonvolatileacidincreases.Thischangeinanionconcentrationprovidesaconvenientwayofanalyzingthecauseofametabolicacidosisbycalculatingwhatistermedtheanion gap. TheaniongaprepresentsthedifferencebetweentheconcentrationofthemajorECFcation(Na+)andthemajorECFanions(Cl− andHCO3−): Undernormalconditionstheaniongaprangesfrom8to16mEq/L.Itisimportanttorecognizethatananiongapdoesnotactuallyexist.Allcationsarebalancedbyanions.Thegapsimplyreflectstheparametersthataremeasured.Inreality:

1	IftheanionofthenonvolatileacidisCl− ,theaniongapwillbenormal(i.e.,thedecreasein[HCO3−]ismatchedbyanincreasein[Cl−]).Metabolicacidosisassociatedwithdiarrheaorrenaltubularacidosis(i.e.,defectinrenalH+ secretion)hasanormalaniongap.Incontrast,iftheanionofthenonvolatileacidisnotCl− (e.g.,lactate,β-hydroxybutyrate),theaniongapwillincrease(i.e.,thedecreasein[HCO3−]isnotmatchedbyanincreasein[Cl−]butratherbyanincreaseintheconcentrationoftheunmeasuredanion).Theaniongapisincreasedinmetabolicacidosis–associatedketoacidosis(e.g.,diabetesmellitus)withrenalfailure,lacticacidosis,oringestionoftoxinsorcertaindrugs(e.g.,largequantitiesofaspirin).Thuscalculationoftheaniongapisausefulwayofidentifyingtheetiologyofmetabolicacidosisintheclinicalsetting.

1	Albuminisanegativelychargedmacromolecule,anditmakesaconsiderablecontributionto“unmeasuredanions.”Asaresulttheaniongapmustbeadjustedinpatientswhohaveanabnormalserum[albumin].Foreach1g/dLchangeinserum[albumin],theaniongapneedstobeadjustedinthesamedirectionby2.5mEq/L. ICF. The increase in the pH inhibits the respiratory centers, which reduces the ventilatory rate, thus the PCO2 is elevated (respiratory compensation). The renal compensatory response to metabolic alkalosis is to increase excretion of HCO3 − by reducing its reabsorption along the nephron. Normally this occurs quite rapidly (minutes to hours) and effectively. Enhanced renal excretion of HCO3 − eventually returns the pH and [HCO3 −] to normal, provided the underlying cause of the initial acid-base disturbance is corrected. When the pH is corrected, the ventilatory rate also returns to normal.

1	Respiratory acidosis is characterized by an elevated PCO2 and reduced ECF pH. It results from decreased gas exchange across the alveoli as a result of either inadequate ventilation (e.g., drug-induced depression of the respiratory centers) or impaired gas diffusion (e.g., pulmonary edema, such as occurs in cardiovascular and lung disease). In contrast to the metabolic disorders, buffering during respiratory acidosis occurs almost entirely in the ICF compartment. The increase in PCO2 and the decrease in pH stimulate both HCO3 − reabsorption by the nephron and titratable acid and NH4 + excretion (renal compensation). Together these responses increase RNAE and generate new HCO3 − . The renal compensatory response takes several days to occur. Consequently, respiratory acid-base disorders are commonly divided into acute and chronic phases. In the acute phase the time for the renal compensatory response is not sufficient, and the body relies on ICF buffering to minimize the change in pH. In

1	divided into acute and chronic phases. In the acute phase the time for the renal compensatory response is not sufficient, and the body relies on ICF buffering to minimize the change in pH. In the chronic phase, renal compensation occurs. Correction of the underlying disorder returns the PCO2 to normal and RNAE decreases to its initial level.

1	Respiratory alkalosis is characterized by a reduced PCO2 and an increased ECF pH. It results from increased gas exchange in the lungs, usually caused by increased ventilation from stimulation of the respiratory centers (e.g., via drugs or disorders of the central nervous system). Hyperventilation also occurs at high altitude and as a result of anxiety, pain, or fear. Buffering is primarily in the ICF compartment. As with respiratory acidosis, respiratory alkalosis has both acute and chronic phases reflecting the time required for renal compensation to occur. The acute phase of respiratory alkalosis reflects intracellular buffering, whereas the chronic phase reflects renal compensation. With renal compensation, the elevated pH and reduced PCO2 inhibit HCO3 − reabsorption by the nephron and reduce titratable acid and NH4 + excretion. As a result of these effects, RNAE is reduced. Correction of the underlying disorder returns the PCO2 to normal, and renal excretion of acid then increases

1	reduce titratable acid and NH4 + excretion. As a result of these effects, RNAE is reduced. Correction of the underlying disorder returns the PCO2 to normal, and renal excretion of acid then increases to its initial level.

1	Analysis of Acid-Base Disorders Analysis of an acid-base disorder is directed at identifying the underlying cause so appropriate therapy can be initiated. The patient’s medical history and associated physical findings often provide valuable clues about the nature and origin of an acid-base disorder. In addition, analysis of an arterial blood sample is frequently required. Such an analysis is straightforward if approached systematically. For example, consider the following data: pH = . The acid-base disorder represented by these values or any other set of values can be determined using the following three-step approach: 1.

1	The acid-base disorder represented by these values or any other set of values can be determined using the following three-step approach: 1. Examination of the pH. When pH is considered first, the underlying disorder can be classified as either an acidosis or an alkalosis. The defense mechanisms of the body cannot correct an acid-base disorder by themselves. Thus even if the defense mechanisms are completely operative, the change in pH indicates the acid-base disorder. In the example provided, the pH of 7.35 indicates acidosis. 2. Determination of metabolic versus respiratory disorder. Simple acid-base disorders are either metabolic or

1	2. Determination of metabolic versus respiratory disorder. Simple acid-base disorders are either metabolic or Role of the Kidneys in the Regulation of Acid-Base Balance respiratory. To determine which disorder is present the clinician must next examine the ECF [HCO3 −] and PCO2. As previously discussed, acidosis could be the result of a decrease in [HCO3 −] (metabolic) or an increase in PCO2 (respiratory). Alternatively, alkalosis could be the result of an increase in ECF [HCO3 −] (metabolic) or a decrease in PCO2 (respiratory). For the example provided, the ECF [HCO3 −] is reduced from normal (normal = 24 mEq/L), as is the PCO2 (normal = 40 mm Hg). The disorder must therefore be metabolic acidosis; it cannot be a respiratory acidosis because the PCO2 is reduced. 3. Analysis of a compensatory response. Metabolic disorders result in compensatory changes in ventilation and thus in PCO2, whereas respiratory disorders result in compensatory changes in RNAE and thus in ECF [HCO3 −] (

1	Table 37.2 ). In an appropriately compensated metabolic acidosis, the PCO2 is decreased, whereas it is elevated in compensated metabolic alkalosis. With respiratory acidosis, compensation results in an elevation of the [HCO3 −]. Conversely, ECF [HCO3 −] is reduced in response to respiratory alkalosis. In this example, the PCO2 is reduced from normal, and the magnitude of this reduction (10 mm Hg decrease in PCO2 for an 8 mEq/L decrease in ECF [HCO3 −]) is as expected (see Table 37.2 ). Therefore the acid-base disorder is a simple metabolic acidosis with appropriate respiratory compensation. A mixed acid-base disorder reflects the presence of two or more underlying causes for the acid-base disturbance. For example, consider the following data: pH = .6 96

1	When the three-step approach is followed, it is evident that the disturbance is an acidosis that has both a metabolic component (ECF [HCO3 −] < 24 mEq/L) and a respiratory component (PCO2 > 40 mm Hg). Thus this disorder is mixed. Mixed acid-base disorders can occur, for example, in an individual who has a history of a chronic pulmonary disease such as emphysema (i.e., chronic respiratory acidosis) and who develops an acute gastrointestinal illness with diarrhea. Because diarrhea fluid contains HCO3 − , its loss from the body results in the development of metabolic acidosis.

1	A mixed acid-base disorder is also indicated when a patient has abnormal PCO2 and ECF [HCO3 −] values but the pH is normal. Such a condition can develop in a patient who has ingested a large quantity of aspirin. Salicylic acid (active ingredient in aspirin) produces metabolic acidosis and at the same time stimulates the respiratory centers, causing hyperventilation and respiratory alkalosis. Thus the patient has a reduced ECF [HCO3 −] and a reduced PCO2. (NOTE: The PCO2 is lower than would occur with normal respiratory compensation of a metabolic acidosis.) 1.

1	The kidneys maintain acid-base balance through excretion of an amount of acid equal to the amount of nonvolatile acid produced by metabolism and the quantity ingested in the diet (termed renal net acid excretion [RNAE]). The kidneys also prevent loss of HCO3 − in urine by reabsorbing virtually all the HCO3 − filtered at the glomeruli. Both reabsorption of filtered HCO3 − and excretion of acid are accomplished by secretion of H+ by the nephrons. Acid is excreted by the kidneys in the form of titratable acid (primarily as Pi) and NH4 + . Both titratable acid and NH4 + excretion result in generation of new HCO3 − , which replenishes the ECF HCO3 − lost during neutralization of nonvolatile acids. 2. The body uses three lines of defense to minimize the impact of acid-base disorders on body fluid pH: (1) ECF and ICF buffering, (2) respiratory compensation, and (3) renal compensation.

1	2. The body uses three lines of defense to minimize the impact of acid-base disorders on body fluid pH: (1) ECF and ICF buffering, (2) respiratory compensation, and (3) renal compensation. Curthoys NP. Renal ammonium ion production and excretion. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Waltham, MA: Elsevier; 2013. Dubose TD Jr. Disorders of acid-base balance. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012. Hamm LL, et al. Cellular mechanisms of renal tubule acidification. In: Alpern RJ, et al., eds. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 5th ed. Waltham, MA: Elsevier; 2013. Weiner ID, Verlander JW. Renal acidification mechanisms. In: Taal MW, et al., eds. Brenner and Rector’s The Kidney. 9th ed. Philadelphia: Saunders; 2012. Berend K, et al. Physiological approach to assessment of acid-base disturbances. N Engl J Med. 2014;371:1434-1445. 3.

1	Berend K, et al. Physiological approach to assessment of acid-base disturbances. N Engl J Med. 2014;371:1434-1445. 3. Metabolic acid-base disorders result from primary alterations in ECF [HCO3 −], which in turn results from addition of acid to or loss of alkali from the body. In response to metabolic acidosis, pulmonary ventilation is increased, which decreases PCO2. The pulmonary response to metabolic acid-base disorders occurs in a matter of minutes. RNAE is also increased, but this takes several days. An increase in ECF [HCO3 −] causes alkalosis. This rapidly (minutes to hours) decreases pulmonary ventilation, which elevates PCO2 as a compensatory response. RNAE is also decreased, but this takes several days. 4.

1	4. Respiratory acid-base disorders result from primary alterations in PCO2. Elevation of PCO2 produces acidosis, and the kidneys respond with an increase in RNAE. Conversely, a reduction of PCO2 produces alkalosis, and RNAE is reduced. The kidneys respond to respiratory acid-base disorders over several hours to days. Breton S, Brown D. Regulation of liminal acidification by the V-ATPase. Physiology (Bethesda). 2013;28:318-329. Brown D, Wagner CA. Molecular mechanisms of acid-base sensing in the kidney. J Am Soc Nephrol. 2012;23:774-780. Curthoys NP, Moe OW. Proximal tubule function and response to acidosis. Clin J Am Soc Nephrol. 2014;9:1627-1638. Hamm LL, et al. Acid-base homeostasis. Clin J Am Soc Nephrol. 2015;10:2232-2242. Levin LR, Buck J. Physiological roles of acid-base sensors. Annu Rev Physiol. 2015;77:347-362. Roy A, et al. Collecting duct intercalated cell function and regulation. Clin J Am Soc Nephrol. 2015;10:305-324.

1	Roy A, et al. Collecting duct intercalated cell function and regulation. Clin J Am Soc Nephrol. 2015;10:305-324. Seifter JL. Integration of acid-base and electrolyte disorders. N Engl J Med. 2014;371:1821-1831. Weiner ID, Verlander JW. Ammonia transport in the kidney by rhesus glycoproteins. Am J Physiol Renal Physiol. 2014;306:F1107-F1120. SECTION 8The Endocrine and Reproductive Systems BRUCE A. WHITE AND JOHN R. HARRISON Chapter 39 Hormonal Regulation of Energy Metabolism Introduction to the Endocrine System Chapter 40 Hormonal Regulation of Calcium and Phosphate Metabolism Chapter 41 The Hypothalamus and Pituitary Gland The Thyroid Gland The Adrenal Gland Chapter 44 The Male and Female Reproductive Systems Upon completion of this chapter, the student should be able to answer the following questions: 1. Name the major endocrine glands and their hormonal product or products. 2.

1	Upon completion of this chapter, the student should be able to answer the following questions: 1. Name the major endocrine glands and their hormonal product or products. 2. Map out and differentiate a simple endocrine negative feedback loop and one involving the hypothalamus, anterior pituitary and peripheral endocrine gland, and list the major endocrine glands under each type of feedback loop. 3. Define a releasing hormone and a tropic hormone. 4. Explain the chemical nature and the characteristics of protein/peptide hormones, catecholamine hormones, steroid hormones, and iodothyronines (thyroid hormones). Include such characteristics as site of regulation (synthesis or secretion), circulating form of hormone, subcellular localization of hormone receptor, and metabolic clearance. 5. Integrate the concept of peripheral conversion with the function/action of a secreted hormone. 6. Integrate the intracellular steps associated with a hormone response in a target cell.

1	5. Integrate the concept of peripheral conversion with the function/action of a secreted hormone. 6. Integrate the intracellular steps associated with a hormone response in a target cell. he ability of cells to communicate with each other is an underpinning of human biology. As discussed in , cell-to-cell communication exists at various levels of complexity and distance. Endocrine signaling involves (1) the regulated secretion of an extracellular signaling molecule, called a hormone, into the extracellular fluid; (2) diffusion of the hormone into the vasculature and its circulation throughout the body; and (3) diffusion of the hormone out of the vascular compartment into the extracellular space and binding to a specific receptor within cells of a target organ. Because of the spread of hormones throughout the body, one hormone often regulates the activity of several target organs. Conversely, cells frequently express receptors for multiple hormones.

1	The endocrine system is a collection of glands whose function is to regulate multiple organs within the body to (1) meet the growth and reproductive needs of the organism and (2) respond to fluctuations within the internal environment, including various types of stress. The endocrine system comprises the following major glands ( Fig. 38.1 Endocrine tissues of the pancreas Parathyroid glands Pituitary gland (in association with hypothalamic nuclei) Gonads (testes or ovaries) These endocrine glands synthesize and secrete bioactive hormones and, with the exception of gonads, which perform both endocrine and gametogenic functions, are dedicated to hormone production ( Table 38.1 ). A transitory organ, the placenta, also performs a major endocrine function. In addition to dedicated endocrine glands, there are endocrine cells within organs whose primary function is not endocrine (see

1	In addition to dedicated endocrine glands, there are endocrine cells within organs whose primary function is not endocrine (see Table 38.1 ). These include cells within the heart that produce atrial natriuretic peptide, liver cells that produce insulin-like growth factor type 1 (IGF-1), cells within the kidney that produce erythropoietin, and numerous cell types within the gastrointestinal tract that produce gastrointestinal hormones. There also exist collections of cell bodies (called nuclei) within the hypothalamus that secrete peptides, called neurohormones, into capillaries associated with the pituitary gland. A third subset of the endocrine system is represented by numerous cell types that express intracellular enzymes, ectoenzymes, or secreted enzymes that modify inactive precursors or less active hormones into highly active hormones (see

1	Table 38.1). An example is the generation of angiotensin II from the inactive polypeptide angiotensinogen by two subsequent proteolytic cleavages (see ). Another example is activation of vitamin D by two subsequent hydroxylation reactions in the liver and kidneys to produce the highly bioactive hormone 1,25-dihydroxyvitamin D (vitamin D). Configuration of Feedback Loops Within the Endocrine System The predominant mode of a closed feedback loop among endocrine glands is negative feedback. In a negative feedback loop, a hormone acts on one or more target organs to induce a change (either a decrease or increase) in circulating levels of a specific component, and the change in this component then inhibits secretion of the hormone. Negative feedback loops confer stability by keeping a physiological parameter (e.g., blood glucose level) within a normal range. There are also a few examples of positive feedback

1	CHAPTER 38 Introduction to the Endocrine System 687 • Fig. 38.1 Glands of the endocrine system. in endocrine regulation. A closed positive feedback loop, in which a hormone increases levels of a specific component and this component stimulates secretion of the hormone, confers instability. Under the control of positive feedback loops, something has got to give; for example, positive feedback loops control processes that lead to rupture of a follicle through the ovarian wall or expulsion of a fetus from the uterus. There are two basic configurations of negative feedback loops within the endocrine system: a physiological response–driven feedback loop (referred to simply as a response-driven feedback loop) and an endocrine axis–driven feedback loop ( Fig. 38.2).

1	The response-driven feedback loop is observed in endocrine glands that control blood glucose levels (pancreatic islet cells), blood Ca++ and Pi levels (parathyroid glands, kidneys), blood osmolarity and volume (hypothalamus/posterior pituitary gland), and blood Na+ , K+ , and H+ levels (zona glomerulosa of the adrenal cortex and atrial cells). In the response-driven configuration, secretion of a hormone is stimulated or inhibited by a change in the level of a specific extracellular parameter (e.g., an increase in blood glucose level stimulates insulin secretion). Alterations in hormone levels lead to changes in the physiological characteristics of target organs (e.g., decreased hepatic gluconeogenesis, increased uptake of glucose by muscle) that directly regulate the parameter (in this case, blood glucose level) in question. The change in the parameter (decreased blood glucose level) then inhibits further secretion of the hormone (i.e., insulin secretion drops as blood glucose level

1	blood glucose level) in question. The change in the parameter (decreased blood glucose level) then inhibits further secretion of the hormone (i.e., insulin secretion drops as blood glucose level falls).

1	Much of the endocrine system is organized into endocrine axes; each axis consists of the hypothalamus, the pituitary gland, and the peripheral endocrine glands (see

1	Fig. 38.2 ). Thus the endocrine axis–driven feedback loop involves a three-tiered configuration. The first tier is represented by hypothalamic neuroendocrine neurons that secrete releasing hormones. Releasing hormones stimulate (or, in a few cases, inhibit) the production and secretion of tropic hormones from the pituitary gland (second tier). Tropic hormones stimulate the production and secretion of hormones from peripheral endocrine glands (third tier). The peripherally produced hormones—namely, thyroid hormone, cortisol, sex steroids, and IGF-1—typically have pleiotropic actions (e.g., multiple phenotypic effects) on numerous cell types. However, in endocrine axis–driven feedback, the primary feedback loop involves feedback inhibition of pituitary tropic hormones and hypothalamic releasing hormones by the peripherally produced hormone. In contrast to response-driven feedback, the physiological responses to the peripherally produced hormone play only a minor role in regulation of

1	hormones by the peripherally produced hormone. In contrast to response-driven feedback, the physiological responses to the peripherally produced hormone play only a minor role in regulation of feedback within endocrine axis–driven feedback loops. From a clinical perspective, endocrine diseases are described as primary, secondary, or tertiary diseases (e.g., secondary hyperthyroidism, tertiary hypogonadism). Primary disease is a lesion in the peripheral endocrine gland; secondary disease is a lesion in the anterior pituitary gland; and tertiary disease is a lesion in the hypothalamus.

1	An important aspect of the endocrine axes is the ability of descending and ascending neuronal signals to modulate release of the hypothalamic releasing hormones and thereby control the activity of the axis. A major neuronal input to releasing hormone–secreting neurons comes from another region of the hypothalamus called the suprachiasmatic nucleus (SCN). SCN neurons impose a daily rhythm, called a circadian rhythm, on the secretion of hypothalamic releasing hormones and the endocrine axes that they control ( Fig. 38.3). SCN neurons represent an intrinsic circadian clock, as evidenced by the fact that they demonstrate a spontaneous peak of electrical activity at the same time every 24 to 25 hours. The 24-to 25-hour cycle can be “entrained” by the normal environmental light-dark cycle created by the earth’s rotation, so that the periodicity of the clock appears to be environmentally controlled ( Fig. 38.4 Hormones Produced to a Significant Degree by Peripheral Conversion

1	Fig. 38.4 Hormones Produced to a Significant Degree by Peripheral Conversion LungsAngiotensinIIKidney1,25-DihydroxyvitaminD(vitaminD)Adipose,mammaryglands,otherorgansEstradiol-17β Liver,sebaceousgland,otherorgansTestosteroneGenitalskin,prostate,otherorgans5-Dihydrotestosterone(DHT)ManyorgansTriiodothyronine •Fig. 38.2 Physiologicalresponse–drivenandendocrineaxis–drivennegativefeedbackloops.Endocrine gland Pituitary gland Releasing hormone Peripheral endocrine gland Hormone Hormone Negative feedback Negative feedback Circulating component (e.g., blood glucose) Tropic hormone Physiological effects Physiological effects Hypothalamic neuroendocrine neurons Target organ(s) Target organs ENDOCRINE AXIS–DRIVEN NEGATIVE FEEDBACK PHYSIOLOGICAL RESPONSE–DRIVEN NEGATIVE FEEDBACK

1	Neural input is generated from specialized light-sensitive retinal cells that are distinct from rods and cones and from signals to the SCN via the retinohypothalamic tract. Under constant conditions of light or dark, however, the SCN clock becomes “free running” and slightly drifts away from a 24-hour cycle each day. The pineal gland forms a neuroendocrine link between the SCN and various physiological processes that require circadian control. This tiny gland, close to the hypothalamus, synthesizes the hormone melatonin from the neurotransmitter serotonin, of which tryptophan is the precursor. The rate-limiting enzyme for melatonin synthesis is N-acetyltransferase. The amount and activity of this enzyme in the pineal gland vary markedly in a cyclic manner, which accounts for the cycling of melatonin secretion and its plasma levels. Synthesis of melatonin is inhibited by light and markedly stimulated by darkness (see

1	Fig. 38.4 ). Thus melatonin may transmit the information that nighttime has arrived, and body functions are regulated accordingly. Melatonin feedback to the SCN at dawn or dusk may also help evoke day-night entrainment of the SCN 24to 25-hour clock. Melatonin has numerous other actions, including induction of sleep.

1	Another important input to hypothalamic neurons and the pituitary gland is stress, either as systemic stress (e.g., hemorrhage, inflammation) or as processive stress (e.g., fear, anxiety). Major medical or surgical stress overrides the circadian clock and causes a pattern of persistent and exaggerated hormone release and metabolism that mobilizes endogenous fuels, such as glucose and free fatty acids, and augments their delivery to critical organs. Growth and reproductive processes, in contrast, are suppressed. In addition, cytokines released during inflammatory or immune responses, or both, directly regulate the release of hypothalamic releasing hormones and pituitary hormones. Chemical Nature of Hormones

1	Chemical Nature of Hormones Hormones are classified biochemically as proteins/peptides, catecholamines, steroid hormones, or iodothyronines. The chemical nature of a hormone determines (1) how it is synthesized, stored, and released; (2) how it is transported in blood; (3) its biological half-life and mode of clearance; and (4) its cellular mechanism of action. Protein and peptide hormones can be grouped into structurally related molecules that are encoded by gene families. Protein/peptide hormones obtain their specificity from their primary amino acid sequence and from posttranslational modifications, especially glycosylation. Because protein/peptide hormones are destined for secretion outside the cell, their synthesis and processing are differently from those of proteins destined to remain within the cell or to be continuously added to the membrane (

1	Fig. 38.5 ). These hormones are synthesized on the polyribosome as larger preprohormones or prehormones. The nascent peptides have at their N-terminus a group of 15 to 30 amino acids called the signal peptide. The signal peptide interacts with a ribonucleoprotein particle, which ultimately directs the growing peptide chain through a pore •Fig. 38.4 Originofcircadianrhythmsinendocrineglandsecretion,metabolicprocesses,andbehavioralactivity.(ModifiedfromTurekFW.Recent Prog Horm Res. 1994;49:43.) a membrane-bound secretory vesicle that is subsequently released into the cytoplasm. The carbohydrate moiety of glycoproteins is added in the Golgi apparatus. Most hormones are produced as prohormones. Prohormones harbor the peptide sequence of the active hormone within their primary sequence. However, prohormones are inactive or less active and require the action of endopeptidases to trim away the neighboring inactive sequences.

1	D Plasma ACTH Protein/peptide hormones are stored in the gland as membrane-bound secretory vesicles and are released by exocytosis through the regulated secretory pathway. Thus these hormones are not continually secreted. Rather, they are secreted in response to a stimulus through a mechanism of stimulus-secretion coupling. Regulated exocytosis requires energy, Ca++ , an intact cytoskeleton (microtubules, microfilaments), and the presence of coat proteins that specifically deliver secretory vesicles to the cell membrane.

1	The ultrastructure of protein hormone–producing cells is characterized by abundant rough endoplasmic reticulum and Golgi membranes and the presence of secretory vesicles •Fig. 38.3 Acircadianpacemakerdirectsnumerousendocrineand bodyfunctions,eachwithitsowndailyschedule.Thenighttimerise inplasmamelatoninmaymediatecertainothercircadianpatterns.ACTH,adrenocorticotropichormone.(DatafromSchwartzWJ.Adv Intern Med. 1994;38:81.) in the membrane of the endoplasmic reticulum located on the cisternal (i.e., inner) surface of the endoplasmic reticular membrane. Removal of the signal peptide by a signal peptidase generates a hormone or prohormone, which is then transported from the cisternae of the endoplasmic reticulum to the Golgi apparatus, where it is packaged into ( Fig. 38.6

1	Fig. 38.6 Protein/peptide hormones are soluble in body fluids and, with the notable exceptions of IGFs and growth hormone, circulate in blood predominantly in an unbound form and therefore have short biological half-lives. Protein hormones are removed from blood primarily by endocytosis and lysosomal degradation of hormone-receptor complexes (see the section “ Cellular Responses to Hormones”). Many protein hormones are small enough to appear in urine in a physiologically active form. For example, follicle-stimulating hormone and luteinizing hormone are present in urine.

1	Fig. 38.5 Schematic Representation of Peptide Hormone Synthesis. Inthenucleus,theprimarygenetranscript,amessengerRNAprecursormolecule,undergoesexcisionofintrons,splicingofexons,cappingofthe5′ end,andadditionofpolyadenylation(poly-A)atthe3′ end.TheresultantmaturemessengerRNAentersthecytoplasm,whereitdirectsthesynthesisofapreprohormonepeptidesequenceonribosomes.Inthisprocess,theN-terminussignalisremoved,andtheresultantprohormoneistransferredvectoriallyintotheendoplasmicreticulum.TheprohormoneundergoesfurtherprocessingandpackagingintheGolgiapparatus.Afterfinalcleavageoftheprohormonewithinthegranules,theycontainthehormoneandcopeptidesreadyforsecretionbyexocytosis.NH2,amidogen. Fig. 38.6 Ultrastructure of a Protein Hormone–Producing Cell. Notethepresenceofsecretoryvesiclesandroughendoplasmicreticulumintheproteinhormone–secretingcell.(FromKierszenbaumAL.Histology and Cell Biology: An Introduction to Pathology. 2nded.Philadelphia:Mosby;2007.) •Fig. 38.7 Chemicalstructuresofcatecholamines.

1	Proteins/peptides are readily digested in the gastrointestinal tract if administered orally. Hence they must be administered by injection or, in the case of small peptides, through a mucous membrane (sublingually or intranasally). Because proteins/peptides do not cross cell membranes readily, they signal through membrane receptors (see Catecholamines are synthesized by the adrenal medulla and neurons and include norepinephrine, epinephrine, and dopamine (

1	Fig. 38.7 ). The primary hormonal products of the adrenal medulla are epinephrine and, to a lesser extent, norepinephrine. Catecholamines obtain their specificity through enzymatic modifications of the amino acid tyrosine. Catecholamines are stored in secretory vesicles that are part of the regulated secretory pathway. They are copackaged with adenosine triphosphate, Ca++, and proteins called chromogranins. Chromogranins play a role in the biogenesis of secretory vesicles and in the organization of components within the vesicles. Catecholamines are soluble in blood and circulate either unbound or loosely bound to albumin. They are similar to protein/peptide hormones in that they do not cross cell membranes readily and hence produce their actions through cell membrane receptors. Catecholamines have short biological half-lives (1 to 2 minutes) and are removed from blood primarily by cell uptake and enzymatic modification.

1	Steroid hormones are made by the adrenal cortex, ovaries, testes, and placenta. Steroid hormones from these glands belong to five categories: progestins, mineralocorticoids, glucocorticoids, androgens, and estrogens. Progestins,

1	Bioactivehormonesaregeneratedfromprohormonesthroughproteolyticcleavageoftheprohormonebyprohormone (alsocalledproprotein)convertases. Theseenzymesareexpressedinacell-specificmanner.Forexample,insulin-producingcells(betacells)ofthepancreaticisletsexpressbothPC1andPC2.Insulinisproducedaspreproinsulin,cleavedtoproinsulinintheendoplasmicreticulum,andpackagedinsecretoryvesiclesasproinsulin.Whileinthesecretoryvesicle,aportionofthecenterofthesinglechain(connectingpeptide)iscleavedsequentiallybyPC1andPC2.Thematuresecretoryvesiclecontainsandsecretesequimolaramountsofinsulinandconnectingpeptide.Sometimesprohormonescontainthesequenceofmultiplehormones.Forexample,theproteinproopiomelanocortin(POMC)containstheaminoacidsequencesofadrenocorticotropichormone(ACTH)andmelanocyte-stimulatinghormones(MSHs).PituitarycellsexpressonlyPC1andreleaseonlyACTHasabioactivepeptide.Incontrast,certainneuronalcelltypesandkeratinocytesexpressbothPC1andPC2andcanproduceMSHs.Therearealsoprohormones,calledpolyproteins,

1	thatcontainmultiplecopiesofthesamebioactivepeptide.Forexample,thesequenceforthyrotropin-releasinghormoneisreiteratedsixtimeswithintheprepro–thyrotropin-releasinghormonesequence.RaremutationsinPC1havebeenidentifiedinhumansandareassociatedwithextremeobesityinchildhood,defectsinglucosehomeostasis,lowglucocorticoidlevels,lossofmenstrualcyclesandhypogonadism,andproblemsingastrointestinalfunction.

1	mineralocoricoids, and glucocorticoids are 21-carbon steroids, whereas androgens are 19-carbon steroids and estrogens are 18-carbon steroids ( Table 38.2 ). Steroid hormones also include the active metabolite of vitamin D (see ), which is a secosteroid (i.e., one of the rings has an open conformation).

1	Table 38.2 ). Steroid hormones also include the active metabolite of vitamin D (see ), which is a secosteroid (i.e., one of the rings has an open conformation). Gonadotropinsarepituitaryhormones,whichareluteinizinghormoneandfollicle-stimulatinghormone.Thesehormonesareheterodimersthatconsistofacommonα subunitandauniqueβ subunit(see ).Theurineofpostmenopausalwomenisanexcellentsourceofgonadotropinsbecausepostmenopausalserumgonadotropinlevelsarehighasaresultofthelossofnegativefeedbackbyovariansteroids(see ),andthehormonesarefilteredandexcretedasintactmoleculesinurine.Athirdgonadotropinistheplacentalhormonehumanchorionicgonadotropin(hCG;see ),whichhasthesamecommonα subunitandanhCG-specificβ subunit.Itisanextremelystablehormone,andbloodhCGlevelsdoubleevery2daysduringthefirsttrimester.Accordingly,urinarylevelsofhCGalsoincreaserapidly.Pregnancy tests arebasedonimmunologicaldetectioninurineofthehCGspecificβ subunitaspartoftheintacthCGheterodimer.

1	Steroid hormones are synthesized by a series of enzymatic modifications of cholesterol, and a cyclopentanoperhydrophenanthrene ring (or a derivative thereof) serves as their core (

1	Fig. 38.8 ). The enzymatic modifications of cholesterol are of three general types: hydroxylation, dehydrogenation/ reduction, and lyase reactions. The purpose of these modifications is to produce a cholesterol derivative that is sufficiently unique to be recognized by a specific receptor. Thus progestins bind to the progesterone receptor, mineralocorticoids bind to the mineralocorticoid receptor, glucocorticoids bind to the glucocorticoid receptor, androgens bind to the androgen receptor, estrogens bind to the estrogen receptor, and the active vitamin D metabolite binds to the vitamin D receptor. The complexity of steroid hormone action is increased by the expression of multiple forms of each receptor. In addition, there is some degree of nonspecificity between steroid hormones and the receptors to which they bind. For example, glucocorticoids bind to the mineralocorticoid receptor with high affinity, and progestins, glucocorticoids, and androgens can all interact with the

1	and the receptors to which they bind. For example, glucocorticoids bind to the mineralocorticoid receptor with high affinity, and progestins, glucocorticoids, and androgens can all interact with the progesterone, glucocorticoid, and androgen receptors to some degree. As discussed later, steroid hormones are hydrophobic and pass through cell membranes easily. Accordingly, classic steroid hormone receptors are localized intracellularly and act by regulating gene expression. There is mounting evidence of the presence of plasma membrane and juxtamembrane steroid hormone receptors that mediate rapid, nongenomic actions of steroid hormones.

1	Steroidogenic cell types are defined as cells that can convert cholesterol to pregnenolone, which is the first reaction common to all steroidogenic pathways. Steroidogenic cells have some capacity for cholesterol synthesis but often obtain cholesterol from cholesterol-rich lipoproteins (low-density lipoproteins and high-density lipoproteins). Pregnenolone is then further modified by several enzymatic reactions. Because of their hydrophobic nature, steroid hormones and precursors can leave the steroidogenic cell easily and thus are not stored. Therefore, steroidogenesis is regulated at the level of uptake, storage, and mobilization of cholesterol and at the level of steroidogenic enzyme gene expression and activity. Steroids are not regulated at the •Fig. 38.8 A, Structureofcholesterol,theprecursorofsteroidhormones.B, Structureofsteroidhormones.

1	Mitochondria with tubular cristae Nucleus •Fig. 38.9 Ultrastructure of a Steroidogenic Cell. Notetheabundanceoflipiddroplets,smoothendoplasmicreticulum,andmitochondriawithtubularcristae.(FromKierszenbaumAL.Histology and Cell Biology: An Introduction to Pathology. 2nded.Philadelphia:Mosby;2007.) level of secretion of the preformed hormone. A clinical implication of this mode of secretion is that high levels of steroid hormone precursors are easily released into blood when a steroidogenic enzyme within a given pathway is inactive or absent. The ultrastructure of steroidogenic cells is distinct from proteinand catecholamine-secreting cells. Steroidogenic enzymes reside within the inner mitochondrial membrane or the membrane of the smooth endoplasmic reticulum. Thus steroidogenic cells typically contain extensive mitochondria and smooth endoplasmic reticulum ( Fig. 38.9 ). These cells also contain lipid droplets, which represent a store of cholesterol esters.

1	An important feature of steroidogenesis is that steroid hormones often undergo further modifications (apart from those involved in deactivation and excretion) after their release from the original steroidogenic cell. For example, estrogen synthesis by the ovary and placenta requires at least two cell types to complete the conversion of cholesterol to estrogen. This means that one cell secretes a precursor and a second cell converts the precursor to estrogen. There is also considerable peripheral conversion of active steroid hormones. For example, the testes secrete little estrogen. However, adipose, muscle, and other tissues express the enzyme for converting testosterone (a potent androgen) to estradiol-17β (a potent estrogen). Thus the overall production of a particular steroid hormone is equivalent to the sum of the secretion of this steroid hormone from a steroidogenic cell type and peripheral conversion of other steroids to the particular steroid hormone (

1	Fig. 38.10 ). Peripheral conversion can produce (1) a more active but similar class of hormone (e.g., conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D); (2) a less active hormone that can be reversibly activated by another tissue (e.g., conversion of cortisol to cortisone in the kidneys, followed by conversion of cortisone to cortisol in abdominal adipose tissue); or (3) a different class of hormone (e.g., conversion of testosterone to estrogen). Peripheral conversion of steroids plays an important role in several endocrine disorders (see Because of their nonpolar nature, steroid hormones are not readily soluble in blood. Therefore, steroid hormones circulate bound to transport proteins, including albumin, but also the specific transport proteins sex hormone– binding globulin and corticosteroid-binding globulin (see the section “

1	Transport of Hormones in the Circulation ”). Excretion of hormones from the body typically involves inactivating modifications, followed by glucuronide or sulfate conjugation in the liver, which is often coupled to biliary excretion. These modifications also increase the water solubility of the steroid and decrease its affinity for transport proteins, thereby allowing the inactivated steroid hormone to be excreted by the kidneys. Steroid compounds are absorbed fairly readily in the gastrointestinal tract and may therefore be administered orally. Thyroid hormones are iodothyronines (Fig. 38.11 ) that are made by the coupling of iodinated tyrosine residues through an ether linkage. Their specificity is determined by the thyronine structure, as well as by where the thyronine is iodinated. Thyroid hormones cross cell membranes by •Fig. 38.10 Peripheralconversionofsteroidhormones.

1	3,5,3˜5˜-Tetraiodothyronine (thyroxine, or T4) 3,5,3˜-Triiodothyronine (T3) •Fig. 38.11 Structureofthyroidhormones,whichareiodothyronines. transport systems. They are stored extracellularly in the thyroid as an integral part of the glycoprotein molecule thyroglobulin. Thyroid hormones are sparingly soluble in blood and aqueous fluids and are transported in blood that is bound (>99%) to serum-binding proteins. A major transport protein is thyroid hormone–binding globulin. Thyroid hormones have long half-lives (7 days for thyroxine; 18 hours for triiodothyronine). Thyroid hormones are similar to steroid hormones in that the thyroid hormone receptor is intracellular and acts as a transcription factor. In fact, the thyroid hormone receptor belongs to the same gene family that includes steroid hormone receptors and vitamin D receptor. Thyroid hormones can be administered orally; the amount absorbed intact is sufficient for this to be an effective mode of therapy.

1	Transport of Hormones in the Circulation A significant fraction of steroid and thyroid hormones is transported in blood that is bound to plasma proteins that are produced in a regulated manner by the liver. Protein and polypeptide hormones are generally transported free in blood. The concentrations of bound hormone, free hormone, and plasma transport protein are in equilibrium. If free hormone levels drop, hormone will be released from the transport proteins. This relationship may be expressed as follows: Equation 38.1 where [H] = concentration of free hormone, [P] = concentration of plasma transport protein, [HP] = concentration of bound hormone, and K = the dissociation constant.

1	Equation 38.1 where [H] = concentration of free hormone, [P] = concentration of plasma transport protein, [HP] = concentration of bound hormone, and K = the dissociation constant. Free hormone is the biologically active form for action on the target organ, feedback control, and clearance by cellular uptake and metabolism. As a consequence, when hormonal status is evaluated, sometimes free hormone levels must be determined in addition to total hormone levels. This is particularly important because hormone transport proteins themselves are regulated by altered endocrine and disease states.

1	Protein binding serves several purposes. It prolongs the circulating half-life of the hormone. Many hormones cross cell membranes readily and would either enter cells or be excreted by the kidneys if they were not protein bound. The bound hormone represents a reservoir of hormone and, as such, can serve to buffer acute changes in hormone secretion. Some hormones, such as steroids, are sparingly soluble in blood, and protein binding facilitates their transport. Cellular Responses to Hormones

1	Cellular Responses to Hormones Hormones are also referred to as ligands, in the context of ligand-receptor binding, and as agonists, in that their binding to the receptor is transduced into a cellular response. Receptor antagonists typically bind to a receptor and lock it in an inactive state, in which the receptor is unable to induce a cellular response. Loss or inactivation of a receptor results in hormonal resistance. Constitutive activation of a receptor leads to unregulated, hormone-independent activation of cellular processes.

1	Hormones regulate essentially every major aspect of cellular function in every organ system. Hormones control the growth of cells, ultimately determining their size and competency for cell division. Hormones regulate the differentiation of cells and their ability to survive or to undergo programmed cell death. They influence cellular metabolism, the ionic composition of body fluids, and cell membrane potential. Hormones orchestrate several complex cytoskeleton-associated events, including cell shape, migration, division, exocytosis, recycling/endocytosis, and cell-cell and cell-matrix adhesion. Hormones regulate the expression and function of cytosolic and membrane proteins, and a specific hormone may determine the level of its own receptor or the receptors for other hormones.

1	Although hormones can exert coordinated, pleiotropic control on multiple aspects of cell function, any given hormone does not regulate every function in every cell type. Rather, a single hormone controls a subset of cellular functions in only the cell types that express receptors for that hormone. Thus selective receptor expression determines which cells respond to a given hormone. Moreover, the differentiated state of a cell determines how it responds to a hormone. Thus the specificity of hormonal responses resides in the structure of the hormone itself, the receptor for the hormone, and the cell type in which the receptor is expressed. Serum hormone concentrations are typically extremely low (10−11 to 10−9 mol/L). Therefore, a receptor must have high affinity, as well as specificity, for its cognate hormone.

1	How does hormone-receptor binding get transduced into a cellular response? Hormone binding to a receptor induces conformational changes in the receptor. These changes are collectively referred to as a signal. The signal is transduced into the activation of one or more intracellular messengers. Messenger molecules then bind to effector proteins, which in turn modify specific cellular functions. The combination of hormone-receptor binding (signal), activation of messengers (transduction), and regulation of one or more effector proteins is referred to as a signal transduction pathway (also called simply a signaling pathway), and the final outcome is referred to as the cellular response. Signaling pathways are usually characterized by the following properties: 1.

1	Signaling pathways are usually characterized by the following properties: 1. Multiple, hierarchical steps in which “downstream” effector proteins are dependent on and driven by “upstream” receptors, transducers, and effector proteins. This means that loss or inactivation of one or more components within the pathway leads to general resistance to the hormone, whereas constitutive activation or overexpression of components can drive a pathway in an unregulated manner. 2. Amplification of the initial hormone-receptor binding. Amplification can be so great that maximal response to a hormone is achieved when the hormone binds to a small percentage of receptors. 3.

1	2. Amplification of the initial hormone-receptor binding. Amplification can be so great that maximal response to a hormone is achieved when the hormone binds to a small percentage of receptors. 3. Activation of multiple pathways, or at least regulation of multiple cell functions, from one hormone-receptor binding event. For example, binding of insulin to its receptor activates three separate signaling pathways. Even in fairly simple pathways (e.g., glucagon activation of adenylate cyclase), divergent downstream events allow the regulation of multiple functions (e.g., post-translational activation of glycogen phosphorylase and increased phosphoenolpyruvate carboxykinase gene transcription). 4.

1	Antagonism by constitutive and regulated negative feedback reactions. This means that a signal is dampened or terminated (or both) by opposing reactions and that loss or gain of function of opposing components can cause hormone-independent activation of a specific pathway or hormone resistance. As discussed in , hormones signal to cells through membrane or intracellular receptors. Membrane receptors have rapid effects on cellular processes (e.g., enzyme activity, cytoskeletal arrangement) that are independent of the synthesis of new protein. Membrane receptors can also rapidly regulate gene expression through either mobile kinases (e.g., cyclic adenosine monophosphate–dependent protein kinase [PKA], mitogen-activated protein kinases [MAPKs]) or mobile transcription factors (e.g., signal transducer and activator of transcription proteins [STATs], Mothers against decapentaplegic homologs [Smads]). Steroid hormones have slower, longer term effects that involve chromatin remodeling and

1	transducer and activator of transcription proteins [STATs], Mothers against decapentaplegic homologs [Smads]). Steroid hormones have slower, longer term effects that involve chromatin remodeling and changes in gene expression. Increasing evidence indicates that steroid hormones have rapid, nongenomic effects as well, but these pathways are still being elucidated.

1	The presence of a functional receptor is an absolute requirement for hormone action, and loss of a receptor produces essentially the same symptoms as loss of hormone. In addition to the receptor, there are fairly complex pathways involving numerous intracellular messengers and effector proteins. Accordingly, endocrine diseases can arise from abnormal expression or abnormal activity, or both, of any of these signal transduction pathway components. Finally, hormonal signals can be terminated in several ways, including hormone/receptor internalization, phosphorylation/ dephosphorylation, proteosomal destruction of receptor, and generation of feedback inhibitors.

1	Endocrinediseasescanbebroadlycategorizedashyperfunctionorhypofunctionofaspecifichormonalpathway.Hypofunctioncanbecausedbylackofactivehormoneorbyhormone resistance asaresultofinactivationofhormonereceptorsorpostreceptordefects.Testicular feminization syndrome isadramaticformofhormoneresistanceinwhichtheandrogenreceptorismutatedandcannotbeactivatedbyandrogens.Inpatientsinwhomthediagnosisisnotmadebeforepuberty,thetestisbecomeshyperstimulatedbecauseofabrogationofthenegativefeedbackbetweenthetestisandthepituitarygland.Theincreasedandrogenlevels 1. Endocrine signaling involves (1) regulated secretion of an extracellular signaling molecule, called a hormone, into the extracellular fluid; (2) diffusion of the hormone into the vasculature and circulation throughout the body; and (3) diffusion of the hormone out of the vascular compartment into the extracellular space and binding to a specific receptor within cells of a target organ. 2.

1	2. The endocrine system is composed of the endocrine tissue of the pancreas, the parathyroid glands, the pituitary gland, the thyroid gland, the adrenal glands, and the gonads (testes or ovaries). 3. Negative feedback represents an important control mechanism that confers stability on endocrine systems. Hormonal rhythms are imposed on negative feedback loops. 4. Protein/peptide hormones are produced on ribosomes and stored in endocrine cells in membrane-bound secretory granules. They typically do not cross cell Bae YJ, Kratzsch J. Corticosteroid-binding globulin: modulating mechanisms of bioavailability of cortisol and its clinical implications. Best Pract Res Clin Endocrinol Metab. 2015;29:761-772.

1	Bae YJ, Kratzsch J. Corticosteroid-binding globulin: modulating mechanisms of bioavailability of cortisol and its clinical implications. Best Pract Res Clin Endocrinol Metab. 2015;29:761-772. havenodirectbiologicaleffectasaresultofthereceptordefect.However,theandrogensareperipherallyconvertedtoestrogens.Thusaffectedindividualsaregeneticallymale(i.e.,46,XY)buthaveastronglyfeminizedexternalphenotype,afemalesexualidentity,andusuallyasexualpreferenceformen(i.e.,heterosexualinrelationtosexualidentity).Treatmentinvolvesremovalofthehyperstimulatedtestes(whichresideintheabdomenandposeariskforcancer),estrogenreplacementtherapy,andcounselingforthepatientand,ifoneexists,thepartner/spousetoaddressinfertilityandsocial/psychologicaldistress. membranes readily and act through cell membrane– associated receptors. 5. Catecholamines are synthesized in the cytosol and secretory granules and do not readily cross cell membranes. They act through cell membrane– associated receptors. 6.

1	5. Catecholamines are synthesized in the cytosol and secretory granules and do not readily cross cell membranes. They act through cell membrane– associated receptors. 6. Steroid hormones are not stored in tissues and generally cross cell membranes relatively readily. They act through intracellular receptors. 7. Thyroid hormones are synthesized in follicular cells and stored in follicular colloid as thyroglobulin. They cross cell membranes and associate with nuclear receptors. 8. Some hormones act through membrane receptors, and their responses are mediated by rapid intracellular signaling pathways. 9. Other hormones bind to nuclear receptors and act by directly regulating gene transcription.

1	9. Other hormones bind to nuclear receptors and act by directly regulating gene transcription. Vija L, Ferlicot S, Paun D. Testicular histological and immunohistochemical aspects in a post-pubertal patient with 5 alpha-reductase type 2 deficiency: case report and review of the literature in a perspective of evaluation of potential fertility of these patients. BMC Endocr Disord. 2014;14:43. Upon completion of this chapter the student should be able to answer the following questions: 1. Explain the different requirements for and utilization by different cells of fuels during the digestive phase as opposed to the interdigestive and fasting phases. 2. Integrate the structure, synthesis, and secretion of insulin with circulating fuel levels, especially glucose. 3. Utilize the different signaling pathways regulated by insulin to link insulin to itscellular effects at the molecular level. 4.

1	3. Utilize the different signaling pathways regulated by insulin to link insulin to itscellular effects at the molecular level. 4. Integrate the structure, synthesis, and secretion of glucagon with the levels of circulating fuels, insulin, and catecholamines. 5. Map out and integrate the actions of insulin on the utilization and storage of glucose, free fatty acids (FFAs), and amino acids (AAs) by hepatocytes, skeletal muscle, and adipocytes during the digestive phase. 6. Map out and integrate the actions of counterregulatory hormones (glucagon, catecholamines) on the utilization of glucose, the sparing of glucose, and the utilization of FFAs and AAs by hepatocytes, skeletal muscle, and adipocytes during the interdigestive and fasting phases. 7. Integrate the changes in fuel utilization and hormonal signaling in hepatocytes during the interdigestive and fasting phases that allow for and promote hepatic glucose production and ketogenesis. 8.

1	8. Compare signaling pathways that have orexigenic and anorexigenic actions via the hypothalamus. 9. Link several pathologies related to metabolism, especially those caused by the absolute or relative absence of insulin and by obesity. Continual Energy Supply and Demand: The Challenge

1	There are an estimated 40 trillion cells in the human body, not including the approximately 40 trillion nonhuman cells that comprise the human microbiome. All these cells must continually perform work to stay alive. This work includes maintenance of cellular composition and structural integrity, along with the integrated synthesis and breakdown (i.e., turnover) of macromolecules and organelles. This work also involves the functions of cells that contribute to the human body as a whole (e.g., contraction of the muscle fibers of the diaphragm). Additional work is required of cells when the human body is engaged in a variety of activities, including (but not limited to) manual labor, exercise, and outdoor play; body growth spurt and maturation of the reproductive systems at puberty; pregnancy and breastfeeding; combating a serious infection or cancer; and the healing of damaged tissues/organs (e.g., a broken bone or healing from surgery). On average the resting metabolic rate of a

1	and breastfeeding; combating a serious infection or cancer; and the healing of damaged tissues/organs (e.g., a broken bone or healing from surgery). On average the resting metabolic rate of a relaxed, awake, stationary, healthy adult human accounts for about 70% of their total energy expenditure each day (

1	Fig. 39.1 To perform this work, cells need fuels, along with the capability to convert fuels into potential chemical energy in the form of adenosine triphosphate (ATP). Cells then convert the energy within ATP into chemical and mechanical work (see

1	Fig. 39.1 ). This means that the need for ATP is immediate and unending, and consequently all living cells must continually synthesize ATP. In fact, humans produce about the equivalent of their body weight in ATP daily. This places a demand on the body to continually supply fuel in some form to all cells. All fuel originates from the diet, but humans do not eat in a nonstop manner all day long. Thus the constant cellular demand for fuels to make ATP and perform work is paired with an intermittent intake of fuels. Diet-derived fuels are oxidized for ATP, but some are also stored for future use, and some are converted to other fuels that can be used by other cell types, depending on the metabolic phase, type of fuel, and cell type in question.

1	In trying to make sense of energy metabolism, it is important to organize one’s thinking around the following: 1. Metabolic phases. Metabolic phases refer to the hourly and daily differences in fuel usage and energy metabolism, which are dictated largely by the abundance or scarcity of certain fuels and orchestrated by phase-specific hormones. In general there are three metabolic phases ( Fig. 39.2 ): (1) the digestive or absorptive phase, which occurs during the 2 to 3 hours it takes to digest a meal; (2) the interdigestive or postabsorptive phase, which normally occurs between meals; and (3) the Sources of fuels 1. 2. Diet—Release of stored fuel during postabsorptive stage (especially during sleep on a daily basis, or during a fast or prolonged exercise)

1	Sources of fuels 1. 2. Diet—Release of stored fuel during postabsorptive stage (especially during sleep on a daily basis, or during a fast or prolonged exercise) ATP constant amount universal not stored Structural integrity Differentiated function Growth and division Response to stimuli and stress Output 60%–70% = Resting metabolic rate 25%–30% = Movement Glucose, FFAs, AAs, ketone bodies EnergyOxidation of fuels 60% Heat 40% •Fig. 39.1 Overviewofenergymetabolism.

1	•Fig. 39.2 Bloodglucoselevelsduringthethreemetabolicphases:D,digestivephase;F,fastingphase;I,interdigestivephase.B,break-fast;D,dinner;L,lunch.Red arrow indicatestheupperlimitfornormalfastingglucose;green arrow indicatesthelowerlimitfornormalfastingglucose.FFB150100BloodGlucose(mg/dL)50LDDDDII fasting phase, which most commonly occurs between the last snack before bedtime and breakfast. (In fact, physicians refer to a blood value as “fasting,” e.g., “fasting blood glucose,” if the patient abstains from eating after midnight and has blood drawn about 8 AM; prolonged fasting and starvation are more extreme forms of fasting.) Physical exertion, which imposes a heightened energy demand, is another type of metabolic phase that occurs with some frequency and regularity for some individuals. This chapter primarily compares how metabolism differs between the digestive phase and the fasting phase, and how different hormones orchestrate these metabolic differences.

1	2, Metabolic actions of hepatocytes, adipocytes, and skeletal myocytes. All cells are involved in energy metabolism, but these three cell types have a profound impact on whole-body metabolism. Key features with respect to metabolism of these three cell types are listed in Table 39.1 3, Insulin and the counterregulatory hormones. Metabolism during the digestive phase is orchestrated almost entirely by insulin. During the fasting phase, insulin drops to low levels, and this alone allows for some of the metabolic adaptions associated with fasting. In addition, glucagon and catecholamines (epinephrine, norepinephrine) stimulate metabolic pathways that integrate the body’s response to an absence of ingested and absorbed fuels. These hormones are referred to as counterregulatory hormones based on their opposition to insulin. Growth hormone (see ) also contribute somewhat to fasting-phase metabolism. Integrated Overview of Energy Metabolism

1	Integrated Overview of Energy Metabolism Fuels enter the body from the diet during the digestive phase. Our diet includes both monomeric and polymeric forms (the latter are converted into monomeric forms during digestion and absorption) of the following fuels: (1) monosaccharides, including glucose, fructose, and galactose; (2) long-chain free fatty acids (referred to in this chapter as simply FFAs); and (3) amino acids (AAs). The diet may also include other fuels such as ethanol. Glucose,fed•UtilizationforATPphase•Storageasglycogen Glucose,fasting•Breakdownofglycogenandphasereleaseofglucoseintoblood FFA/TG,fedphase•MakeFFAsfromglucosebyDNL FFA/TG,fasting•UtilizationforATPphase•UtilizationtoproduceKBs SecretionofVLDLAAs,fedphase•Utilizationformultipleanabolic pathwaysAAs,fastingphase•Utilizationforgluconeogenesis UtilizationforketogenesisKBs,fedphase•ShouldbeabsentKBs,fastingphase•SynthesisfromFFAsandsome

1	UtilizationforketogenesisKBs,fedphase•ShouldbeabsentKBs,fastingphase•SynthesisfromFFAsandsome AA,aminoacid;ATP,adenosinetriphosphate;DNL,denovolipogenesis;FFA,freefattyacid;G3P,glycerol-3-phosphate;KB,ketonebody;TG,triglyceride;VLDL,verylow-densitylipoprotein. During the digestive phase, absorbed fuels are partitioned and used for different purposes. Insulin (discussed in detail later) regulates essentially every aspect of metabolism during the digestive phase. Glucose is the primary fuel used for energy (i.e., ATP production) during the digestive phase ( Fig. 39.3A ). Glucose is considered a universal fuel in that most cells can perform the following: 1. 2. “trap” and “activate” imported glucose by converting glucose into glucose-6-phosphate (G6P) through the activity of one or more hexokinases. G6P cannot pass through GLUT transporters (“trapping”) and is now a substrate for several enzymatic pathways (“activation”). 3.

1	metabolize G6P to pyruvate via the glycolytic pathway, which yields a small amount of ATP without requiring mitochondria or O2. Cells without mitochondria ferment pyruvate to lactate and export lactate as waste. Most cells import pyruvate into mitochondria, convert it to acetyl CoA by pyruvate dehydrogenase, and then condense acetyl CoA with oxaloacetate to form citrate. Citrate is cycled through the tricarboxylic acid (TCA) cycle back to oxaloacetate. This metabolism of pyruvate through the TCA cycle releases CO2 as waste and generates guanosine triphosphate (GTP) along with flavin adenine dinucleotide hydride (FADH2) and nicotine adenine dinucleotide hydride (NADH). FADH2 and NADH are used by the electron transport system and oxidative phosphorylation to ultimately generate relatively large amounts of ATP through a process that is absolutely dependent on O2. Cells with no or very few mitochondria (e.g., erythrocytes, lens cells of the eye) are absolutely dependent on glucose for

1	large amounts of ATP through a process that is absolutely dependent on O2. Cells with no or very few mitochondria (e.g., erythrocytes, lens cells of the eye) are absolutely dependent on glucose for energy. Additionally the brain can only use glucose under normal conditions, in that it has essentially no capacity for oxidation of FFAs and only marginal capacity for the use of lactate, ketone bodies (KBs), and AAs as fuels. Thus in all metabolic phases, maintenance of blood glucose above a certain minimal threshold is absolutely necessary to avoid central nervous system (CNS)-related symptoms, beginning with those caused by a hypoglycemia-activated autonomic response (e.g., nausea, sweating, cardiac arrhythmias). If blood glucose continues to fall, progression to symptoms caused by neuroglycopenia (e.g., cognitive dysfunction, loss of coordinated motor function, and ultimately even coma

1	RBCCNSDietG6PG6P GLY HEPATOADIPOSKM2,4,5 11 2,3 2,4,5 8 7,82,4,52,3,4,5 6 G6P G6P G3G1 1 1 1 G2 G4-mG4-mG4-m ATP A. Digestive Phase ATP ATP TG G’OL3P G6P TG GlucoseATP 6 ATP GLY ATP G6P AAs G’OL ATP ATP13 TG 11 11 12 12 FFA A,4,5 GLY ATP RBCSKMG6P KBs CNS2,4,5112 3 HEPATOFFA 9 11 10 KBs A,4,5 A,4,5 B,4,5 G4-iG4-i B,4,5 ADIPOB,4,5 GLY G6P LACT ATP G6P Glucose PROT G4-iG4-i G1G1 G3G3 G2G2 B. Fasting Phase •Fig. 39.3 A, Overview of glucose utilization during the digestive phase. GLUTtransporters:G1,GLUT1;G2,GLUT2;G3,GLUT3;G4-m,functionalGLUT4localizedtocellmembrane.Celltypes:ADIPO,adipocyte;CNS,centralnervoussystemneuronsandglia;HEPATO,hepatocyte;RBC,redbloodcell;SKM,skeletalmyocyte.Metabolites:G6P,glucose-6-phosphate;GLY,glycogen;G’OL3P,glycerol-3-phosphate;TG,triglyceride.Metabolicreactions/pathways:1,hexokinase/glucokinase;2,glycolysis;3,lactatedehydrogenase;4,TCAcycle±

1	pyruvatedehydrogenase;5,oxidativephosphorylation;6,glycogensynthesis;7,denovolipogenesis;8,esterificationofFFAstoG’OL3PtoformTG.B, Overview of energy metabolism during the fasting phase. GLUTtransporters:seelegendforA,plus:G4-I,inactiveGLUT4withintracellularlocalization.Celltypes:seelegendforAMetabolites:seelegendforA,plus:AAs,aminoacids;FFA,freefattyacid;G’OL,glycerol;KBs,ketonebodies;LACT,lactate;PROT,protein.Metabolicreactions/pathways:seelegendforA,plus:9,glycogenolysis;10,gluconeogenesis;11,G6Pase;12,ketogenesis;13,proteolysis;A,β-oxidation;B,ketolysis.

1	and death) can occur. This means that whole-body metabolism during the interdigestive and fasting phases must meet the challenge of maintaining blood glucose above 60 mg/ dL (see Fig. 39.2 , green arrow). On the other hand, glucose levels must be maintained below a certain maximal threshold (i.e., fasting blood glucose < 100 mg/dL) to avoid glucose-induced side reactions (both enzymatic and nonenzymatic) as well as osmotic imbalances that lead to multiple forms of damage to cell function (see Fig. 39.2 , red arrow).

1	Theendotheliumofthemicrovasculatureofthekidneyandretina,aswellastheendotheliumofthevasanervosumoftheautonomicnervoussystem,areparticularlysensitivetohyperglycemia.Chronicallyhighbloodglucoseresultsinpathologicallyhighintracellularlevelsofglucoseintheseendothelialcells,resultinginalteredproteinandlipidstructure,oxidativestress,andalteredsignalingpathways.Theseinsults,collectivelyreferredtoasglucotoxicity, causepathologicalchangesinintracellularandmembranecomponentsaswellasinsecretedmoleculesthateithersignaland/ormakeuptheextracellularmatrix.Indeed,glucotoxicityistherootcauseofthenephropathy, retinopathy, and peripheral neuropathy thatoccurinpoorlycontrolleddiabetesmellitus.Thereforewhole-bodymetabolismduringallmetabolicphasesmustmeetthechallengeofminimizingthemagnitudeanddurationoftheriseinbloodglucoseassociatedwithingestionofamealandmustmaintainbloodglucosebelowasafemaximalthresholdof100mg/dLduringallothertimes.Fastingbloodglucosebetween100and124mg/dLisindicativeofimpaired glucose

1	glucose tolerance, andvaluesat125mg/dLandaboveareevidenceofdiabetes mellitus.

1	Glucose is consumed by erythrocytes and the brain continually throughout all metabolic phases. In contrast, hepatocytes, skeletal myocytes, and adipocytes primarily use glucose during the digestive phase only. Insulin stimulates glycolysis and entry of pyruvate (end product of glycolysis) into the TCA cycle and oxidative phosphorylation for ATP production in hepatocytes, skeletal myocytes, and adipocytes (see Table 39.1). Hepatocytes express the GLUT2 isoform of the glucose transporter, which is not regulated by insulin for its insertion into the cell membrane. In contrast, skeletal myocytes and adipocytes express the GLUT4 isoform. Newly synthesized GLUT4 exists in an intracellular inactive state with GLUT4 storage vesicles (G4-i in Fig. 39.3B ). Insulin induces translocation of GLUT4 to the cell membrane, where it can function as an active glucose transporter (G4-m in Fig. 39.3A).

1	Fig. 39.3B ). Insulin induces translocation of GLUT4 to the cell membrane, where it can function as an active glucose transporter (G4-m in Fig. 39.3A). After its phosphorylation to G6P by glucokinase, hepatocytes convert some of the imported glucose into the storage form, glycogen, during the digestive phase (see Fig. 39.3A ). Similarly, skeletal muscle converts some of the G6P from imported glucose into glycogen. Hepatocytes can only store a finite amount of glucose as glycogen. Hepatocytes also convert excess glucose into FFAs through the process of de novo lipogenesis (DNL). These FFAs are typically esterified to glycerol-3-phosphate (G3P) to form triglyceride (TG), which accumulates as intrahepatic TG during the digestive phase (see Fig. 39.3A ). As discussed later for insulin signaling, an excessive accumulation of intrahepatic TG (i.e., fatty liver, hepatic steatosis) can result in insulin resistance.

1	Fig. 39.3A ). As discussed later for insulin signaling, an excessive accumulation of intrahepatic TG (i.e., fatty liver, hepatic steatosis) can result in insulin resistance. During the digestive phase, AAs are used in multiple anabolic pathways to regenerate degraded molecules, including other AAs, proteins, nucleotides and nucleic acids, glutathione, and complex lipids.

1	FFAs represent the most efficient fuel type in terms of ATP molecules made per carbon of fuel. However, utilization of FFAs competes effectively with glucose utilization in the mitochondria. High FFA levels during the digestive phase would promote a greater magnitude and duration of the glucose surge, thereby contributing to hyperglycemia. Thus most of the FFAs in an average meal are prevented from entering the circulation by their reesterification into TG and packaging into chylomicrons within the intestinal enterocyte. Chylomicrons are secreted, enter lymphatic vessels, and ultimately enter the blood. Once in the general circulation, chylomicrons convey the FFAs (as TG) to the adipose tissue where they are released from chylomicronassociated TG through lipolysis, imported by adipocytes, and reesterified as intracellular TG for storage (discussed in detail later).

1	During the digestive phase, hepatocytes, skeletal myocytes, and adipocytes function largely independently of each other. In contrast the actions of these three cell types become highly integrated during the fasting phase to maintain adequate blood glucose levels while providing alternative energy substrates for each cell type (see Fig. 39.3B Table 39.1

1	Fig. 39.3B Table 39.1 As stated earlier, cells without mitochondria (e.g., erythrocytes) and cells of the CNS require glucose for ATP production during all metabolic phases, and thus the body must maintain blood glucose above 60 mg/dL even days or weeks after the last ingestion of food (an exception to this rule is that the brain can use KBs [discussed later] during a prolonged fasting phase). Two general processes contribute to maintenance of blood glucose during the fasting phase: hepatic glucose production and glucose sparing. Hepatic glucose production is in turn based on two metabolic pathways. The first is the rapid catabolic process of glycogenolysis (pathway 9 in Fig. 39.3B ). Hepatocytes express the enzyme glucose-6-phosphatase (G6Pase) (pathway 11 in Fig. 39.3B ), allowing them to convert G6P back to glucose, which can then exit the cell through a bidirectional GLUT transporter. Release of glucose derived from glycogenolysis is relatively short Source of blood

1	Source of blood DietaryGluconeogenesis0 Breakfast 12.00 24.00Glycogenolysis •Fig. 39.4 Relativecontributionsofthethreesourcesofbloodglucoserelativetomealsandtimeofday.Theinsetboxstressesreplacementofglycogenolysiswithgluconeogenesisduringthefastingphase(i.e.,sleep).(AdaptedfromBaynesJW,DominiczakJH[eds].Medical Biochemistry. 3rded.Philadelphia:Mosby/Elsevier;2009.) lived because the liver glycogen supply becomes exhausted by about 8 hours. The second metabolic contribution to hepatic glucose production during the fasting phase is the gradual pathway of gluconeogenesis (pathway 10 in Fig. 39.3B ). The onset of gluconeogenesis during fasting is slower than glycogenolysis, but gluconeogenesis continues essentially nonstop throughout a fasting phase (

1	Fig. 39.3B ). The onset of gluconeogenesis during fasting is slower than glycogenolysis, but gluconeogenesis continues essentially nonstop throughout a fasting phase ( Fig. 39.4 ). Gluconeogenesis requires precursors, especially lactate, “gluconeogenic” AAs, and glycerol. How are these precursors supplied during the fasting phase? Lactate is continually produced by erythrocytes. Lactate is also produced by glycolytic skeletal muscle fibers during exercise (exercise tends to occur more frequently during the interdigestive and fasting phases as opposed to “on a full stomach”), although much of this lactate is utilized by aerobic skeletal muscle and cardiac muscle during exercise. But additionally the overall anabolism of the digestive phase switches over to a general catabolism during the fasting phase (see Fig.

1	Fig. 39.3B ). TGs within adipocytes undergo lipolysis to FFAs and glycerol, and there is a general net proteolysis with the release of AAs during the fasting state. The glycerol and gluconeogenic AAs are released from cells and circulate to the liver, where they are subsequently used for gluconeogenesis. Thus gluconeogenesis requires an integration of catabolic pathways in adipocytes and skeletal myocytes with anabolic gluconeogenesis in hepatocytes. Gluconeogenesis eventually supplants glycogenolysis and can continue as long as precursors flow into the liver.

1	Glucose sparing represents the other general process that contributes to maintenance of adequate circulating glucose levels during the fasting phase. Glucose sparing means the switching of fuel utilization from glucose to a nongluconeogenic fuel in most cell types, but especially in skeletal muscle, which represents the potentially largest single consumer of glucose. First, the uptake of glucose by skeletal muscle and adipocytes is greatly reduced because the GLUT4 transporter isoform exists in an intracellular CHAPTER 39 Hormonal Regulation of Energy Metabolism Fig. 39.3B) during the fasting phase. Thus alternative fuels need to be delivered to skeletal muscle and adipocytes. The nongluconeogenic fuels (i.e., cannot be used for gluconeogenesis by the liver) are FFAs and KBs. FFAs are Fig. 39.3B are also released after packaging of intrahepatic TGs into cussed later). KBs are produced by hepatocytes from acetyl CoA, which in turn originates from FFAs and ketogenic

1	Fig. 39.3B are also released after packaging of intrahepatic TGs into cussed later). KBs are produced by hepatocytes from acetyl CoA, which in turn originates from FFAs and ketogenic AAs, both of which become abundant during the fasting phase. Thus glucose sparing depends on catabolic adipocyte metabolism, which results in lipolysis of stored TGs and release of FFAs. FFAs are imported by hepatocytes, which use FFAs to produce acetyl CoA. Protein degradation in available for ketogenesis. High levels of intramitochondrial acetyl CoA in the hepatocyte not only provides ample carbons for ATP synthesis but serves to: (1) inhibit conver sion of pyruvate to acetyl CoA, (2) promote conversion of pyruvate to oxaloacetate for gluconeogenesis, and (3) promote synthesis of KBs (see

1	Fig. 39.3B ). After several days of fasting, the CNS can start using KBs for energy, thereby further sparing glucose for erythrocytes. Many other cell types with mitochondria use KBs along with FFAs for ATP production, especially skeletal muscle. The hormones that drive glycogenolysis, gluconeogen esis, lipogenesis, and hepatic ketogenesis as well as VLDL production by the liver during the fasting phase are glucagon and catecholamines. In the presence of low glucose, insulin levels fall, and that removes the inhibition by insulin of the secretion of another pancreatic hormone, glucagon. Thus diminished blood glucose causes a rise in the circulating glucagon-to-insulin ratio. Hepatocytes are the primary target organ of glucagon, which directly drives glyco genolysis, gluconeogenesis, FFA oxidation, and ketogenesis by the hepatocyte ( Fig. 39.5B).

1	Fig. 39.5B). from sympathetic innervation and epinephrine from the ) can reinforce the actions of glucagon. Adipocytes also express the glucagon receptor, as well as the β2-and β3-adrenergic receptors that respond to catecholamines in response to hypoglycemia, exertion, or certain stresses. Skeletal muscle is not a target of glucagon but does respond to catecholamines stimulation through β2-adrenergic receptors. Skeletal muscle is very responsive to intracellular Ca++ , which increases during physical exertion/movement, and to an increase in the intracellular adenosine monophosphate (AMP):ATP ratio, which activates AMP kinase.

1	Finally, it is important to understand that the pathways upregulated during the fasting phase are opposed by insulin-dependent pathways that are most active during the digestive phase (discussed later). Thus attenuation of insulin signaling also contributes to the ability of hepatocytes, skeletal myocytes, and adipocytes to display an integrated response to the metabolic challenges of the fasting phase. •Fig. 39.5 TheisletsofLangerhans(endocrinepancreas)fromrat.A, Pancreashistologyshowingexocrineaciniwheredigestiveenzymesareproducedtobedeliveredtotheduodenumviathepancreaticduct,andanendocrineisletwhereinsulinandglucagonareproducedanddeliveredtothecirculationuponuptakebyarichcapillarybed.B, Stainingofendocrineisletforinsulinwithinbetacells;thesearethemostnumerouscelltypeandareprimarilylocatedcentrallywithintheislet.C, Stainingofendocrineisletforglucagonwithalphacells;thesearemuchlessnumerousthanbetacellsandareprimarilylocatedalongtheperipheryoftheislet.

1	•Fig. 39.6 ProinsulinisprocessedbyprohormoneconvertasesintoamatureinsulinmoleculewithtwopeptidestrandslinkedbyH-bondsandaCpeptide.Botharesecretedinequimolarratios.(FromWhiteBA,PorterfieldSP[eds].Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby/Elsevier;2013.)C peptide C peptide Insulin B B Converting enzymes Proinsulin “folded” S-S bonds formed Endoplasmic reticulum Golgi Secretory granule A A S S S S S S S S S S S S The islets of Langerhans constitute the endocrine pancreas ( Fig. 39.6 ). Approximately 1 million islets making up about 1% to 2% of the pancreatic mass are spread throughout the exocrine pancreas (see ). The islets are composed of several cell types, each producing a different hormone. Beta cells make up about three-fourths of the cells of the islets and produce the hormone insulin (see Fig. 39.5B ). Alpha cells account for about 10% of islet cells and secrete glucagon (see

1	Fig. 39.5B ). Alpha cells account for about 10% of islet cells and secrete glucagon (see Fig. 39.5C ). Other endocrine cell types reside within islets, but their respective hormone products are of marginal or unclear importance and thus will not be discussed further. Blood flow to the islets is somewhat autonomous from blood flow to the surrounding exocrine pancreatic tissue. Blood flow through the islets passes from beta cells, which predominate in the center of the islet, to alpha and delta cells, which predominate in the periphery (see Fig. 38.5B-C ). Consequently the first cells affected by circulating insulin are the alpha cells, in which insulin inhibits glucagon secretion.

1	Fig. 38.5B-C ). Consequently the first cells affected by circulating insulin are the alpha cells, in which insulin inhibits glucagon secretion. Insulin is the primary anabolic hormone that dominates regulation of metabolism during the digestive phase. Insulin is a protein hormone that belongs to the gene family that includes insulinlike growth factors I and II (IGF-I, IGF-II) and relaxin. The insulin gene encodes preproinsulin. Insulin is synthesized as preproinsulin, which is converted to proinsulin as the hormone enters the endoplasmic reticulum. Proinsulin is packaged in the CHAPTER 39 Hormonal Regulation of Energy Metabolism 705 4 (-) ATP of cell membrane • Fig. 39.7 Glucose is the primary stimulus of insulin secretion and is enhanced by sulfonylurea drugs as well as GLP-1 analogues/DPP-4 inhibitors. See text for explanation of numbered steps in glucose-stimulated insulin secretion (GSIS).

1	Golgi apparatus into membrane-bound secretory granules. Proinsulin contains the AA sequence of insulin plus the C (connecting) peptide. The proteases that cleave proinsulin (proprotein convertases) are packaged with proinsulin within secretory vesicles. Proteolytic processing clips out the C peptide and generates the mature hormone, which consists of two chains, an α chain and a β chain, connected by two disulfide bridges (

1	Fig. 39.7). A third disulfide bridge is contained within the α chain. Insulin is stored in secretory granules in zinc-bound crystals. On stimulation, the granule’s contents are released to the outside of the cell by exocytosis. Equimolar amounts of mature insulin and C peptide are released, along with small amounts of proinsulin. C peptide has no known biological activity but is useful in assessing endogenous insulin production. C peptide is more stable in blood than insulin (making it easier to assay) and helps distinguish endogenous insulin production from injected insulin, insofar as the latter has been purified from C peptide.

1	Insulin has a short half-life of about 5 minutes and is cleared rapidly from the circulation. It is degraded by insulin-degrading enzyme (IDE; also called insulinase) in the liver, kidney, and other tissues. Because insulin is secreted into the hepatic portal vein, it is exposed to liver IDE before it enters the peripheral circulation. About half the insulin is degraded before leaving the liver. Thus peripheral tissues are exposed to significantly less serum insulin concentrations than the liver. Recombinant human insulin and insulin analogues with different characteristics of speed of onset and duration of action and peak activity are now available. Serum insulin levels normally begin to rise within 10 minutes after ingestion of food and reach a peak in 30 to 45 minutes. The higher serum insulin level rapidly lowers blood glucose to baseline values.

1	Glucose is the primary stimulus of insulin secretion (“steps” in glucose-stimulated insulin secretion described in the discussion that follows refer to

1	Fig. 39.8 ). Entry of glucose into beta cells is facilitated by the GLUT2 transporter (step 1). Once glucose enters the beta cell, it is phosphorylated to G6P by the low-affinity hexokinase glucokinase (step 2). Glucokinase is referred to as the “glucose sensor” of the beta cell because the rate of glucose entry is correlated to the rate of glucose phosphorylation, which in turn is directly related to insulin secretion. Metabolism of G6P through glycolysis, the TCA cycle, and oxidative phosphorylation by beta cells increases the intracellular ATP : ADP ratio (step 3) and closes an ATP-sensitive K+ channel (step 4). This results in depolarization of the beta cell membrane (step 5), which opens voltage-gated Ca++ channels (step 6). Increased intracellular [Ca++] activates microtubule-mediated exocytosis of insulin/proinsulincontaining secretory granules (step 7).

1	Ingested glucose has a greater effect on insulin secretion than injected glucose. This phenomenon, called the incretin effect, is due to stimulation by glucose of incretin hormones from the gastrointestinal tract. One clinically relevant incretin hormone is glucagon-like peptide 1 (GLP-1), which is released by L cells of the ileum in response to glucose in the ileal lumen ( Fig. 39.9 ). As a hormone, GLP-1 enters the circulation and ultimately binds to the Gs-coupled GLP1 receptor (GLP1R) on beta cells. This GLP1R/Gs/adenylyl cyclase/protein kinase A (PKA) signaling pathway amplifies the intracellular effects of Ca++ on insulin secretion. GLP-1 is rapidly degraded in the circulation by dipeptidyl peptidase 4 (DPP-4).

1	706 SECTION8Berne & Levy Physiology •Fig. 39.8 Secondaryregulatorsofinsulinsecretion.Seetextforexplanationofabbreviations.Specific amino acids (Eating a meal) AA transporter ATP ˜CA2+ °cAMP ˛ CELL ˜cAMP M3R (Gq)ACh Parasympathetic innervation (Anticipating & ingesting a meal) Norepinephrine, epinephrine Sympathetic innervation adrenal medulla (Exercise, Hypoglycemia) ? ˛2AR (Gs) ˛2AR Agonists ˙2AR (Gi) ˙2AR Agonists Insulin Fig. 39.10 ). Insulin secretion is primarily dampened by sympathetic autonomic regulation through α2-adrenergic receptors. Binding of norepinephrine or epinephrine to α2-adrenergic receptors decreases cyclic adenosine mono-phosphate (cAMP), possibly by closing Ca++ channels (see

1	Fig. 39.6 ). Adrenergic inhibition of insulin serves to protect against hypoglycemia, especially during exercise. Beta cells also express Gs-coupled β2-adrenergic receptors that normally play a minor role in promoting insulin secretion. Note, however, that β-adrenergic receptor agonists oppose the actions of insulin on overall metabolism and may antagonize the actions of administered insulin in diabetics. Conversely, β-blockers (i.e., β-adrenergic receptor antagonists) may increase the severity of hypoglycemic episodes in patients receiving exogenous insulin.

1	TheATP-sensitiveK+ channelisanoctamericproteincomplexthatcontainsfourATP-bindingsubunitscalledSUR subunits. Thesesubunitsareboundbysulfonylurea drugs, whichalsoclosetheK+ channelandarewidelyusedasoralhypoglycemicstotreathyperglycemiainpatientswithpartiallyimpairedbetacellfunction.Hypoglycemiaisasignificantsideeffectofsulfonylureadrugsifusedinexcessorincorrectlyincombinationwithotherdrugs,owingtoinappropriatelyhighreleaseofinsulin. BothDPP-4–resistant analogues of GLP-1 andinhibitors of DPP-4 arecurrentlyapprovedfortreatmentofpatientswithtype2DMwithsomebetacellfunction.Importantlythesedrugsarepermissive totheactionsofglucoseonthebetacellandthusonlyweaklyincreaseinsulinsecretionintheabsenceofglucose.ThusGLP-1analoguesinducehypoglycemiamuchlessfrequentlythansulfonylureadrugs.

1	The insulin receptor (InsR) is a member of the receptor tyrosine kinase (RTK) gene family (see ). Most of the actions of insulin on metabolism involve activation of the protein kinase Akt, which in turn has pleiotropic actions on cell metabolism. The InsR is expressed on the cell membrane as a homodimer, with each monomer containing a tyrosine kinase B InsRec IRS protein Metabolic actions Anabolic actions of insulin • Utilization of glucose for ATP & anabolic pathways • Storage of FFAs as TG • Protein synthesis from AAs • Macromolecular synthesis (e.g., RNA, DNA, lipids) • Organelle formation • Cell proliferation/renewal Mitogenic actions Merabolic actions PIP2PIP3 RAS/ RAF MAPK/ ERK PI3K Akt

1	GLUT 4 Translocation Glucose import Glycolysis, Glycogenesis, PDH, DNL Glucogenolysis Gluconeogensis FFA ˜ oxidation Ketogenesis Lipolysis Proteolysis autophagy Protein synthesis Gycolysis, PPP, DNL Gluconeo-genesis, VLDL export Protein phoshatases mTORC1 SREBP-1C FOX01 Akt •Fig. 39.9 A,Structureofdimerizedinsulinreceptorincellmembrane.B,SimplifieddiagramoftheAktkinaseandMAPKpathwaysdownstreamoftheInsR.C,Summarizedactionsofinsulin/InsR-activatedAktkinase.

1	Insulingeneexpressionandisletcellbiogenesisaredependentonseveraltranscriptionfactorsspecifictothepancreas,liver,andkidney.Thesetranscriptionfactorsincludehepatocyte nuclear factor 4α (HNF-4α), HNF-1α, insulin promoter factor 1 (IPF-1), HNF-1β, and neurogenic differentiation 1/beta cell E-box trans-activator 2 (NeuroD1/β2). Aheterozygousnullmutationofoneofthesefactorsresultsinprogressivelyinadequateproductionofinsulinandmaturity-onset diabetes of the young (MODY) beforetheageof 25.MODYischaracterizedbynonketotichyperglycemia,oftenasymptomatic,thatbeginsinchildhoodoradolescence.Inadditiontothefivetranscriptionfactors,mutationsinglucokinase alsogiverisetoMODY. •Fig. 39.10 Divergentproteolyticcleavagepatternsoftheproglucagonmolecule.GLUC,glucagon;GLP,glucagon-likepeptide;GRPP,glucagon-relatedpolypeptide. domain on the cytosolic side (see

1	•Fig. 39.10 Divergentproteolyticcleavagepatternsoftheproglucagonmolecule.GLUC,glucagon;GLP,glucagon-likepeptide;GRPP,glucagon-relatedpolypeptide. domain on the cytosolic side (see Fig. 39.10A ). Binding of insulin to the receptor induces cross-phosphorylation of the subunits. These phosphotyrosine residues are then bound by the insulin receptor substrate (IRS) proteins (i.e., IRS proteins are “recruited” to the InsR). The IRS proteins themselves are phosphorylated by the InsR on specific tyrosines, which then recruits phosphoinositide-3-kinase (PI3K) to the IRS protein bound to the InsR (see

1	Fig. 39.10B ). PI3K converts phosphoinositol-4,5-bisphosphate (PIP2) to phosphoinositol-3,4,5-trisphosphate (PIP3). PIP3 is an informational lipid that recruits proteins to the membrane. In this pathway, PIP3 recruits Akt protein kinase to the cell membrane where it becomes activated. This pleiotropic Akt protein kinase signaling pathway orchestrates the numerous metabolic actions of insulin in hepatocytes, skeletal muscle, and adipocytes, including (see Fig. 39.10C 1. translocation of the GLUT4 glucose transporter to the cell membrane, thereby allowing import of glucose into skeletal myocytes and adipocytes; 2. activation of protein phosphatases, which in turn regulate the activity of multiple metabolic enzymes in all insulin target cells; 3. activation of the protein complex mechanistic target of rapamycin complex 1 (mTORC1), which promotes protein synthesis and may inhibit proteosomal-mediated protein degradation in insulin target cells; 4.

1	activation of the transcription factor sterol response element binding protein 1 (SREBP1). SREBP1 is especially important for insulin effects on the liver, where it orchestrates glycolysis and de novo lipogenesis (DNL) for production of phospholipids, FAs, and TGs from excessive ingested glucose and fructose. InsR/Akt signaling stimulates SREBP1 directly as well as indirectly through activation of mTORC1, which also activates SREBP1. SREBP1 also induces the enzyme that catalyzes the first reaction in the oxidative arm of the pentose phosphate pathway (PPP). This reaction generates the coenzyme NADPH, which is required for DNL. 5. inactivation of the transcription factor FOXO1. Aktmediated phosphorylation of FOXO1 promotes nuclear exclusion of FOXO1. In the absence of insulin/Akt signaling, FOXO1 induces expression of genes encoding

1	VLDL assembly and export. All these actions of Akt will be discussed in more detail later. The InsR also promotes proliferation/renewal of some target cells through the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway (see Fig. 39.10B ). The MAPK pathway also participates in some metabolic regulation. Glucagon is the primary counterregulatory hormone that increases blood glucose levels, primarily through its effects on liver glucose output. Glucagon also enhances intramitochondrial fatty acid oxidation and ketogenesis in hepatocytes. Glucagon is a member of the secretin gene family. The precursor preproglucagon harbors the AA sequences for glucagon, GLP-1, and GLP-2 (

1	Glucagon is a member of the secretin gene family. The precursor preproglucagon harbors the AA sequences for glucagon, GLP-1, and GLP-2 ( Fig. 39.11 ). Preproglucagon is proteolytically cleaved in the alpha cell in a cell-specific manner to produce the peptide glucagon. Glucagon circulates in an unbound form and has a short half-life of about 6 minutes. The predominant site of glucagon degradation is the liver, which degrades as much as 80% of circulating glucagon in one pass. Because glucagon enters the hepatic portal vein and is carried to the liver before reaching the systemic circulation, a large portion of the hormone never reaches the systemic circulation. The liver is the primary target organ of glucagon, with lesser effects on adipocytes. Skeletal muscle does not express the glucagon receptor.

1	The glucagon receptor is is a Gs-linked G protein– coupled receptor that increases adenylyl cyclase activity and thus cAMP levels. Glucagon exerts many rapid actions through PKA signaling. Glucagon also exerts some transcription effects through phosphorylation and activation of transcription factors such as CREB (cAMP response element binding) protein. The insulin-glucagon ratio determines the net effect of metabolic pathways on blood glucose. A major stimulus for secretion of glucagon is a decline in blood glucose. Insulin inhibits glucagon secretion, so low blood glucose has an CHAPTER 39 Hormonal Regulation of Energy Metabolism 709 indirect effect on glucagon secretion through removal of ˜ Cell Insulin inhibition by insulin ( Fig. 39.12 ). Some recent evidence also indicates that low glucose has a direct effect on alpha (+) cells to increase glucagon secretion.

1	Fig. 39.12 ). Some recent evidence also indicates that low glucose has a direct effect on alpha (+) cells to increase glucagon secretion. Circulating catecholamines, which inhibit secretion of glucose insulin via α2-adrenergic receptors, stimulate secretion of glucagon via β2-adrenergic receptors (see Fig. 39.12 (If low) Serum AAs promote secretion of glucagon. This means a glucose protein meal will increase postprandial levels of both insulin and glucagon (which protects against hypoglycemia), whereas a carbohydrate meal stimulates only insulin. Catecholamines: Epinephrine (–) and Norepinephrine

1	Catecholamines: Epinephrine (–) and Norepinephrine The other major counterregulatory factors are the catechol- amines epinephrine and norepinephrine. Epinephrine is the primary product of the adrenal medulla (see ), whereas norepinephrine is released from postganglionic sympathetic nerve endings (see ). Catecholamines are released in response to decreased glucose concentrations, various forms of stress, and exercise. Decreased glucose levels (i.e., hypoglycemia) are primarily • Fig. 39.11 Integrated regulation of blood glucose by insulin and sensed by neurons in the CNS, which initiate an integrated the counterregulatory factors glucagon and catecholamines (norepinephrine, epinephrine).

1	Fructose-1,6-bisP OA Acetyl CoA • Fig. 39.12 Metabolic pathways in hepatocytes during digestive (“D” numbers) and fasting (“F” numbers) phases. Reactions/pathways: 1D, glucokinase; 1F; G6Pase; 2D, glycogen synthesis; 2F, glycogenolysis; 3D, phosphofructokinase 1; 3F, fructose-1,6-bisphosphatase; 4D, glucose-6-phosphate dehydrogenase; 5D, pyruvate kinase; 6D, pyruvate dehydrogenase; 6F, pyruvate carboxylase; 7F, phosphoenolpyruvate carboxykinase; 8D, ATP-citrate lyase; 9D, acetyl CoA carboxylase; 9F, malonyl CoA decarboxylase; 10D fatty acid synthase; 11D, glycerol phosphate/fatty acyl CoA transferase and other enzymes involved in esterification and formation of TG; 12D, inhibition by malonyl CoA (Mal CoA) of fatty acyl CoA transporter, carnitine/palmitoyl transporter 1 (CPT1) on outer mitochondrial membrane; 13F, movement of fatty acyl CoA into mitochondrion through CPT1 and beta-oxidation to acetyl CoA.

1	sympathetic response through the hypothalamus. The direct metabolic actions of catecholamines are mediated primarily by α1-, β2-, and β3-adrenergic receptors located on muscle, adipose, and liver tissue (see later). Like the glucagon receptor, β-adrenergic receptors (β2 and β3) increase intracellular cAMP. The α1-adrenergic receptor is also expressed by some organs, especially the liver. Hormonal Regulation of Specific Metabolic Reactions and Pathways This section discusses the main pathways in hepatocytes, skeletal myocytes, and adipocytes that contribute to integrated metabolism. For even more detailed description, the student is referred to biochemistry textbooks. Hepatocyte Metabolism: Digestive vs. Fasting Phases Some of the key metabolic steps regulated by insulin and glucagon (and catecholamines) in the liver are as follows (refer to

1	Fig. 39.13 for numbered reactions; D denotes digestive phase, F denotes fasting phase): 1. Trapping vs. releasing intracellular glucose. Although glucose enters hepatocytes through insulin-independent GLUT2 transporters, insulin increases hepatic retention and utilization of glucose by increasing expression of glucokinase (reaction 1D). Insulin increases glucokinase gene expression through increased expression and activation of the transcription factor sterol regulatory element–binding protein 1C (SREBP1C), which acts as a “master switch” in the fed state to coordinately increase levels of several enzymes involved in glucose utilization and TG synthesis. Hepatocytes also express the enzyme G6Pase (reaction 1F), which converts G6P back to glucose, which can then exit the hepatocyte via the GLUT2 transporter. Insulin prevents the futile cycle of glucose phosphorylation-dephosphorylation by repressing gene expression of the enzyme G6Pase. The transcription factor FOXO1 stimulates gene

1	GLUT2 transporter. Insulin prevents the futile cycle of glucose phosphorylation-dephosphorylation by repressing gene expression of the enzyme G6Pase. The transcription factor FOXO1 stimulates gene expression of G6Pase. Insulin-activated Akt kinase phosphorylates and inactivates FOXO1. During the fasting phase, FOXO1 is active and promotes G6Pase expression, whereas SREBP1C is inactive and does not stimulate glucokinase expression. The reciprocal regulation of SREBP1C and FOXO1 are thus regulated primarily by the presence or absence of insulin.

1	2. Glycogen synthesis vs. breakdown. Insulin indirectly increases glycogen synthesis through increased expression of glucokinase because high levels of G6P allosterically increase glycogen synthase activity. Through stimulation of specific protein phosphatases, insulin promotes dephosphorylation and thereby activation of glycogen synthase (reaction 2D). Insulin also prevents the futile cycle of glycogen synthesis to glycogenolysis through inhibition of glycogen phosphorylase (reaction 2F). Glucagon-activated PKA phosphorylates phosphorylase kinase, which in turn phosphorylates and activates glycogen phosphorylase. 3. Increasing glycolysis. A. Activating phosphofructokinase 1 (PFK1) and inhibiting fructose-2,6-bisphosphatase. Insulin increases the activity of PFK1, which phosphorylates •Fig. 39.13 Insulinandcounterregulatoryhormoneregulationofphosphofructokinase1(PFK1;reaction3Din Fig.39.12) andfructose-1,6-bisphosphatase(F1,6BPase;reaction3Fin

1	Fig.39.12) throughchangingtheactivityofthebifunctionalenzymephosphofructokinase2/fructose-2,6-bisphosphatase(PKFBP)andthusthelevelsoftheallostericregulatorymetabolitefructose-2,6-bisphosphate(F-2,6-bisP).Insulin/Akt-activated protein phosphatase F-2,6BPase F-1,6BPase F-1,6-bisPATP, citrate AMP PKFBP-P PFK1 (–) (–) (+) Glucagon & NE/E-activated protein kinase A (PKA) HepatocytecytoplasmPKFBP PFK2 F-2,6-bisP F6P •Fig. 39.14 Metabolisminskeletalmuscleduringdigestive(“D”reactions)vs.fasting(“F”reactions)phases.Reactions/pathways:1D,translocationofGLUT4transportertocellmembrane;1F,lossoftranslocationofGLUT4transportertocellmembrane;2D,glycogensynthesis;2F,glycogenolysis;3D,glycolysisandlactatedehydrogenase,orpyruvatedehydrogenase/TCAcycle/oxidativephosphorylation(OxPhos),dependingonmusclefibertype;3F,β-oxidationofFFAsorketolysisfollowedbytheTCAcycleandOxPhos.GLUT4 GLUT4 Transporters FFAs, KBsAcetyl CoAATP GLUT4 storage vesicle Cell membrane GlucoseGlucoseGlycogen 1D 1F 2F 2D 3D 3F GLUT4

1	GLUT4 Transporters FFAs, KBsAcetyl CoAATP GLUT4 storage vesicle Cell membrane GlucoseGlucoseGlycogen 1D 1F 2F 2D 3D 3F GLUT4 Skeletal myocyte fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (reaction 3D). This reaction is referred to as the “commitment” reaction for glycolysis. Insulin also inhibits the reverse reaction, as catalyzed by the gluconeogenic enzyme fructose-1,6-bisphosphatase. Insulin regulates these two enzymes through an indirect two-step mechanism that is diagramed in

1	Fig. 39.14 . This mechanism involves the bifunctional enzyme phosphofructokinase-2/ fructose bisphosphatase (PKFBP; see Fig. 39.14 ). Insulin/Akt-activated protein phosphatases promote dephosphorylation of PKFBP, thereby activating the kinase function and lessening the phosphatase function. This phosphorylates F6P to fructose-2,6bisphosphate (F-2,6-bisP). F-2,6-bisP in turn allosterically activates PFK1, thereby driving glycolysis. F-2,6-bisP also competitively inhibits fructose-1,6bisphosphatase, thereby blocking the futile cycle of F6P to fructose-1,6-bisphosphate to F6P. B. Activatingpyruvatekinase(PK).PKcatalyzestheirreversibleconversionofphosphoenolpyruvate(PEP)topyruvate(seereaction5Din Fig. 39.12 ).Again,insulin/AktkinaseactivationofaproteinphosphatasedephosphorylatesPK,whichactivatestheenzyme.InsulinalsoincreasesPK geneexpressionthroughSREBP1C.Finally,fructose-1,6-bisphosphate(productofreaction3D)allostericallyactivatesPK. 4.

1	4. Activating pyruvate dehydrogenase (PDH) complex. PDH converts pyruvate to acetyl CoA, which can then enter the TCA cycle upon condensation with oxaloacetate (OA) to form citrate. Insulin increases PDH activity through Akt kinase activation of PDH phosphatase, which in turn dephosphorylates and activates PDH (reaction 6D). 5.

1	Increasing synthesis of intrahepatic TG. During the digestive phase, some acetyl CoA is transferred from the mitochondria to the cytosol in the form of citrate, which is then converted back to acetyl CoA and oxaloacetate by the cytosolic enzyme ATP-citrate lyase (reaction 8D). Insulin increases ATP-citrate lyase gene expression through transcription factor SREBP1C. Once in the cytoplasm, acetyl CoA can enter fatty acid synthesis. The first step involves conversion of acetyl CoA to malonyl CoA by the enzyme acetyl-CoA carboxylase (reaction 9D). Insulin stimulates acetyl-CoA carboxylase gene expression through the transcription factor SREBP1C. Insulin also promotes dephosphorylation of acetyl-CoA carboxylase, which activates the enzyme. Malonyl CoA is converted to the 16-carbon fatty acid palmitoyl CoA by repetitive additions of acetyl groups (contributed by malonyl CoA) by the fatty acid synthase (FASN) complex (reaction 10D). FASN gene expression is enhanced by insulin through the

1	palmitoyl CoA by repetitive additions of acetyl groups (contributed by malonyl CoA) by the fatty acid synthase (FASN) complex (reaction 10D). FASN gene expression is enhanced by insulin through the transcription factor SREBP1C. Insulin also stimulates palmitoyl-CoA desaturase, which produces unsaturated fatty acids, and glycerol phosphate–fatty acyl transferases that esterify FFAs to G3P to form intrahepatic TG.

1	Palmitate synthesis requires the coenzyme NADPH. A major source of NADPH is the pentose phosphate pathway (PPP; see Fig. 39.12 ). The first reaction converts G6P to 6-phosphogluconolactone by the enzyme glucose-6-phosphate dehydrogenase (G6PD; reaction 4D). Insulin increases G6PD gene expression through the transcription factor SREBP1C. By activating steps that lead to generation of malonyl CoA, insulin indirectly inhibits oxidation of FFAs. Malonyl CoA inhibits the activity of CPT-I, which transports FFAs from the cytosol into the mitochondria (reaction 12D). As a result, FFAs that are synthesized by DNL cannot be transported into mitochondria, where they undergo β-oxidation (reaction 13F). Thus increased malonyl CoA prevents the futile cycle of FFA synthesis to FFA oxidation.

1	FFAs are converted to TGs by the liver (reaction 11D) and are either stored in the liver or transported to adipose tissue and muscle in the form of VLDL (see later). Insulin acutely promotes degradation of the VLDL apoprotein apoB-100. This keeps the liver from secreting VLDL during a meal when the blood is rich with chylomicrons. Thus the lipid made in response to insulin during a meal is released as VLDL during the interdigestive and fasting phases and provides an important source of energy to skeletal and cardiac muscle.

1	6. Activation vs. inhibition of the gluconeogenic enzymes pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK). Pyruvate can also be converted to OA by PC (reaction 6F). However, this reaction is indirectly inhibited by insulin in several ways. First, insulin activates PDH as just discussed, thereby diverting pyruvate away from the PC reaction. Additionally, PC is allosterically activated by high levels of intramitochondrial acetyl CoA. Insulin keeps intramitochondrial levels of acetyl CoA low by activation of cytosolic DNL, which promotes removal of acetyl CoA via citrate from the mitochondria. Another key mechanism is to prevent β-oxidation of FFAs within the mitochondria, which generates high levels of acetyl CoA. By stimulating DNL, insulin also increases levels of cytosolic malonyl CoA, which inhibits transport of FFAs into the mitochondria (reaction 12D). Also, inhibitory actions of insulin on glucagon secretion and on lipolysis of TG within adipocytes

1	of cytosolic malonyl CoA, which inhibits transport of FFAs into the mitochondria (reaction 12D). Also, inhibitory actions of insulin on glucagon secretion and on lipolysis of TG within adipocytes prevents release of FFAs by adipose tissue and their import into hepatocytes.

1	In contrast, during the fasting phase, low insulin coupled with high glucagon and/or catecholamines stimulate release of FFAs from adipocytes (see later), which increases the flow of FFAs into hepatocytes. Glucagon also phosphorylates and activates the enzyme malonyl decarboxylase, which converts malonyl CoA back to acetyl CoA (reaction 9F). Enhanced malonyl CoA decarboxylase, along with generally low DNL due to low insulin, reduces malonyl CoA levels and thus removes the inhibition on the CPT1 transporter. This allows FFAs to enter the mitochondria and undergo β-oxidation (reaction 13F), generating high levels of intramitochondrial acetyl CoA, activating PC (reaction 6F) and also allosterically inhibiting PDH (reaction 6D).). The enzymes involved in β-oxidation are activated by PKA signaling. Glucagon also activates the transcription factor PPARα, which further induces expression of enzymes involved in β-oxidation. Fibrate drugs activate PPARα, promoting oxidation of intrahepatic TG

1	Glucagon also activates the transcription factor PPARα, which further induces expression of enzymes involved in β-oxidation. Fibrate drugs activate PPARα, promoting oxidation of intrahepatic TG and ameliorating insulin resistance.

1	Insulin also represses gene expression of the gluconeogenic enzyme PEPCK (reaction 7F), which converts pyruvate (by way of malate transferase out of the mitochondria) to phosphoenolpyruvate. PEPCK is primarily regulated at the level of transcription. Similar to its actions on G6Pase, FOXO1 stimulates transcription of PEPCK during the fasting phase, and insulin/Akt kinase signaling inactivates FOXO1 during the digestive phase. Glucagon and catecholamines also increase PEPCK gene expression through PKA-CREB signaling during the fasting phase. Skeletal Muscle and Adipose Tissue Metabolism: Digestive vs. Fasting Phases 1.

1	Fig. 39.15 ). Glucose tolerance refers to the ability of an individual to minimize the increase in blood glucose concentration after a meal. A primary way by which insulin promotes glucose tolerance is activation of glucose transporters in skeletal muscle. Insulin stimulates translocation of preexisting GLUT4 transporters to the cell membrane (reaction 1D). Insulin also promotes storage of glucose in muscle as glycogen (reaction 2D) and promotes oxidation of glucose through glycolysis (reaction 3D). During the fasting phase, low insulin results in a low number of GLUT4 transporters at the membrane (reaction 1F), so these cells consume less glucose (glucose sparing). Skeletal muscle fibers with mitochondria switch to the use of FFAs from adipocytes and KBs from hepatocytes (reaction 3F). Skeletal myocytes do not express glucagon receptors. Uptake of FFAs and KBs and their oxidation for ATP is largely upregulated by intracellular Ca++ levels and a high AMP:ATP ratio. Exercise also

1	Skeletal myocytes do not express glucagon receptors. Uptake of FFAs and KBs and their oxidation for ATP is largely upregulated by intracellular Ca++ levels and a high AMP:ATP ratio. Exercise also activates these pathways, as does glycogenolysis (reaction 2F), through adrenergic receptor stimulation.

1	2. (Fig. 39.16A ). Insulin also stimulates GLUT4-dependent uptake of glucose and subsequent glycolysis in adipose tissue (reactions 1D and 2D). Adipose tissue uses glycolysis for energy needs but also for generating G3P (reaction 3D), which is required for esterification of FFAs into TGs (reaction 4D). During the fasting phase, insulin is low, so GLUT4 movement to the cell membrane is blocked (reaction 1F). 2. Adipocytes → FFAs and TG (see Fig. 39.16B ). Insulin stimulates expression of lipoprotein lipase (LPL) •Fig. 39.15 A, Glucose metabolism in an adipocyte during digestive (“D” reactions) and fasting (“F” reactions) phases. Reactions/pathways:1Dand1F(alsosee

1	Fig.39.14 legend);2D,glycolysis,pyruvatedehydrogenase/TCAcycle/OxPhos;3D,glycerol-3-phosphatedehydrogenase;4D,esterificationofFFAstoG3Ptoformtriglyceride.B, Lipid metabolism in adipocyte during digestive (“D” reactions) and fasting (“F” reactions) phases. Reactions/pathways:1D,synthesisoflipoproteinlipase(LPL)andLPLsecretionintosubcapillaryspace,bindingtoGPI-anchoredprotein(red box),

1	during digestive (“D” reactions) and fasting (“F” reactions) phases. Reactions/pathways:1D,synthesisoflipoproteinlipase(LPL)andLPLsecretionintosubcapillaryspace,bindingtoGPI-anchoredprotein(red box), andmigrationtoluminalsideofcapillaryendothelialcell;2D,lipolysisofchylomicronTGandreleasingfreeFFA(afterdigestion,chylomicronremnantisclearedfromcirculationbyliver);3D,activationofimportedFFAsbytransfertoacetylCoAtoformfattyacylCoA;4D,esterificationoffattyacylCoAstoG3PtoformTG(TGdropletsarecoveredandstabilizedbyperilipins[PL]);5D,dephosphorylationandinactivationofhormone-sensitivelipase(HSL),whichpromotesstorageofTG;1F,phosphorylationandactivationofHSL,whichcontributestocompletelipolysisofTG(ATGL,adipocyteTGlipase;HSL,diacylglyceridelipase;MGL,monoacylglyceridelipase);2F,finalstepinTGlipasebyMGLreleasesFFAandglycerol.

1	714 SECTION8Berne & Levy Physiology •Fig. 39.16 Roleoflipoproteinsinenergymetabolism.A,Digestivephase.B,Fastingphase.Dietary fats Adipose capillaries Dietary sugar & carbs Glucose fructose CM Adipose capillaries LPL CR Cardiac muscle capillaries LPL FFAs FFAs A B FFAs FFAs Cardiac & skel muscle capillaries LPL Cardiac & skeletal muscle Steroidogenic cells Growing cells FFAs TG TG Adipocyte Adipocyte TG Liver VLDL Glucose fructose Cardiac muscle ATP Skeletal muscle ATP KBs TG Liver ATP CHOL ATP IDL LDL IDL within adipocytes and its migration to the apical side of endothelia in adipose capillaries (reaction 1D). This action of insulin allows LPL to extract FFAs from chylomicrons within adipose tissue capillary beds (reaction 2D). The chylomicron remnants (discussed later) are removed by the liver. Insulin also stimulates activation of imported FFAs by their conversion to fatty acyl CoAs (reaction 3D). Insulin stimulates glycolysis in adipocytes, which generates the G3P required for

1	the liver. Insulin also stimulates activation of imported FFAs by their conversion to fatty acyl CoAs (reaction 3D). Insulin stimulates glycolysis in adipocytes, which generates the G3P required for reesterification of FFAs into TGs (reaction 4D). Insulin directly inhibits hormone-sensitive lipase (HSL; reaction 5D), thereby promoting storage of FFAs as opposed to their release. During the fasting phase, glucagon and catecholamines phosphorylate and activate HSL (reaction 1F), thereby promoting release of FFAs and glycerol from stored TG (reaction 2F). In the absence of insulin these two products of lipolysis are exported into the blood.

1	Protein Metabolism in All Hormone Target Cells: Digestive vs. Fasting Phases Insulin promotes protein synthesis in muscle and adipose tissue by stimulating AA uptake and mRNA translation. Insulin also inhibits proteolysis. Although the liver uses AAs for ATP synthesis, insulin also promotes synthesis of proteins during the digestive phase and attenuates the activity of urea cycle enzymes in the liver. Glucagon and catecholamines activate proteosomal degradation of proteins and release of AAs during the fasting phase. Metabolic Roles of Lipoproteins: Digestive vs. Fasting Phases This section deals with lipoproteins in some detail. For more details, consult a biochemistry textbook.

1	FFAs circulate in the blood primarily bound to albumin. However, TG, free cholesterol, cholesterol esters, phospholipids and some lipid-soluble vitamins, all of which are hydrophobic and would partition into the membranes of endothelial cells instead of circulating, are transported through blood within lipid aggregations (i.e., a mix of the above) bound by specific apoproteins. These lipid-protein complexes are referred to as lipoproteins. The TG-rich lipoproteins are chylomicrons and VLDL and primarily function to deliver FFAs (as TG) to skeletal and cardiac muscle for energy and to adipocytes for storage. The cholesterol-rich lipoproteins include low-density lipoprotein (LDL) and high-density lipoprotein (HDL), which deliver cholesterol to proliferating cells, steroidogenic cells, and bile-producing hepatocytes. HDL also removes excess cholesterol (i.e., from macrophage-engulfed dead cells) from the periphery. There are also “remnants” of lipoproteins that have their lipid cargo

1	bile-producing hepatocytes. HDL also removes excess cholesterol (i.e., from macrophage-engulfed dead cells) from the periphery. There are also “remnants” of lipoproteins that have their lipid cargo partially digested and then cleared from the circulation by the liver.

1	Digestive Phase: Chylomicrons and Chylomicron Remnants TGs in a meal are enzymatically digested to FFAs and 2-monoglycerides within the lumen of the intestine. •Fig. 39.17 Leptinandhypothalamiccentersinvolvedinregulationofappetite.Seetextforexplanationsofabbreviations. Intestinal enterocytes import both of these lipids and reesterify them to form TGs. TGs, along with fat-soluble vitamins, cholesterol, cholesterol esters, and phospholipids, are complexed with the protein ApoB48 to form chylomicrons. Chylomicrons are secreted, move into lymphatics, and then ultimately enter the circulation. While in the blood, other apoproteins such as ApoE and ApoC2 are transferred to the chylomicrons from HDL particles (one function of HDL is to provide a circulating reservoir of various apoproteins). This converts nascent chylomicrons into mature chylomicrons.

1	When chylomicrons enter the capillaries of adipose tissue during the digestive phase, they are partially digested by lipoprotein lipase (LPL). LPL is synthesized by adipocytes and secreted into the subendothelial space. LPL then binds to the endothelial membrane GPI-anchored protein GPIHBP1, which transports LPL to the luminal (apical) surface of the capillary endothelial cell. Once in this position, LPL molecules come into contact with chylomicrons. ApoC2 within chylomicrons is an activator of LPL dimerization and activity. FFAs are released from chylomicrons by LPL-mediated lipolysis of TG. (See earlier discussion and Fig. 39.16B for an explanation of the processing of FFAs to stored TG within adipocytes. LPL is also expressed in cardiac and skeletal muscle. Cardiac muscle preferentially uses FFA for energy and obtains most FFAs from lipoprotein particles (

1	LPL is also expressed in cardiac and skeletal muscle. Cardiac muscle preferentially uses FFA for energy and obtains most FFAs from lipoprotein particles ( Fig. 39.17 ). Thus cardiac muscle also extracts FFA from chylomicrons during the digestive phase. The activity of LPL in cardiomyocytes is highly regulated by local factors such as the local concentration of FFAs within the coronary capillary beds. LPL activity in skeletal muscle is relatively low during the digestive phase.

1	After lipolytic digestion within adipose tissue capillary beds, chylomicrons are converted to smaller, denser chylomicron remnants (CR) that now have reduced TG content. CR particles are able to penetrate the tunica intima of blood vessels at sites with endothelial dysfunction and thus are atherogenic. Because they still have ApoE protein associated with them, they can bind to one of several membrane receptors that recognize ApoE. CRs can also bind to specific glycolipids. Bound CRs are then endocytosed by hepatocytes (see Fig. 39.17 ). Remaining FFAs that are released after endocytosis of CRs are reesterified into intrahepatic TG. Fasting Phase: VLDL, IDL, and LDL The source of circulating TG during the fasting phase is primarily the liver (see

1	Fasting Phase: VLDL, IDL, and LDL The source of circulating TG during the fasting phase is primarily the liver (see Fig. 39.17 ). During the digestive phase, intrahepatic TGs accumulate from de novo lipogenesis using the carbons in cytosolic citrate and from endocytosed CRs. Intrahepatic TG, along with other lipids including cholesterol and cholesterol esters, is exported by hepatocytes as VLDL. VLDL particles are assembled as lipids complexed to the ApoB100 protein. Expression of ApoB100, along with other components involved in VLDL assembly, is stimulated by transcription factor FOXO1. FOXO1 in turn is inhibited by the insulin signaling pathway. This means that hepatic VLDL production is minimal during the time when blood is rich in chylomicrons. During the fasting phase, insulin levels are low, so FOXO1 activity is high, and VLDL assembly and secretion resumes. Once VLDL particles enter the circulation, they accept other apoproteins (e.g., ApoE, ApoC2) and become mature VLDL.

1	Adipocytes display low LPL activity during the fasting phase, in part owing to low insulin levels. However, cardiomyocytes and skeletal myocytes express LPL, which digests VLDL and provides FFA to these muscle cell types during the fasting phase. Lipolytic extraction of some FFAs from VLDL generates a remnant particle called intermediate-density lipoprotein (IDL). IDL circulates to the liver, where it s processed in one of two ways (see

1	Fig. 39.17 ). About half of the IDL binds to one of several ApoErecognizing receptors, undergoes receptor-mediated endocytosis, and is digested in endolysosomes. Released lipids can be reassembled into VLDL particles and returned to the circulation to provide fuel for cardiac and skeletal muscle as the fasting phase progresses. The other half of the IDL undergoes further digestion from the hepatocyte-specific LPL-related enzyme hepatic lipase. Hepatic lipase extracts most of the remaining TG in the IDL, forming the final remnant of VLDL, LDL. LDL is TG poor but cholesterol rich. It should be noted that both mature chylomicrons and VLDL can receive additional cholesterol from HDL while in the circulation through the action of cholesterol ester transport protein (CETP), so cholesterol content of the remnant particles (ChyR, IDL, and LDL) can vary. In any case the LDL particle is a small, dense, cholesterol-rich particle that is potentially very atherogenic in the face of endothelial

1	of the remnant particles (ChyR, IDL, and LDL) can vary. In any case the LDL particle is a small, dense, cholesterol-rich particle that is potentially very atherogenic in the face of endothelial damage. LDL particles are safely imported into cells through the LDL receptor. It should be noted that in the conversion of IDL to LDL, the ApoE protein disassociates from the particle. This means that only receptors for which ApoB100 is a ligand can remove LDL from the blood. In contrast to the multiple ApoE receptors, only one receptor, the LDL receptor, can recognize and bind ApoB100. Thus loss or decrease of a functional LDL receptor has significant clinical consequences (see

1	At the Cellular box). LDL receptor is expressed on proliferating cells, including some cancer cells, which need to synthesize new cell membranes. LDL receptor is also expressed on steroidogenic cells, which use cholesterol to make steroid hormones. The major site of LDL uptake is the liver, which secretes cholesterol as well as cholesterol-based bile acids, as bile into the biliary tree. Some cholesterol is excreted by the intestines. Other cholesterol byproducts (e.g., steroid hormones) are excreted primarily at the kidney.

1	White adipose tissue (WAT) is composed of several cell types. The TG-storing cell is called the adipocyte. These cells develop from preadipocytes during gestation in humans. This process of adipocyte differentiation, which may continue throughout life, is promoted by several transcription factors. One of these factors is SREBP1C, which is activated by lipids as well as insulin and several growth factors and cytokines. Another important transcription factor in WAT is PPARγ. Activated PPARγ promotes expression of genes involved in TG storage. Thus an increase in food consumption leads to activation of SREBP1C and PPARγ, which increase the differentiation of preadipocytes into small adipocytes and upregulation of enzymes within these cells to allow storage of excess fat. Adipose tissue produces paracrine and endocrine factors, including adiponectin, TNF-α, resistin, interleukin-6, angiotensinogen, and acylation-stimulating protein.

1	Adipose tissue produces paracrine and endocrine factors, including adiponectin, TNF-α, resistin, interleukin-6, angiotensinogen, and acylation-stimulating protein. Leptin is an adipocyte-derived protein that signals information to the hypothalamus about the degree of adiposity and nutrition, which in turn controls eating behavior and energy expenditure. Leptin-deficient mice and humans become morbidly obese. These findings originally raised hope that leptin therapy could be used to combat morbid obesity. However, administration of leptin to individuals who suffer from diet-induced obesity does not have a significant anorectic or energy-consuming effect. In fact, obese individuals already have elevated endogenous circulating levels of leptin and appear to have developed leptin resistance.

1	Leptin has an important role in liporegulation in peripheral tissues. Leptin protects peripheral tissues (e.g., liver, skeletal muscle, cardiac muscle, beta cells) from accumulation of too much lipid by directing storage of excess caloric intake into adipose tissue. This action of leptin, though opposing the lipogenic actions of insulin, contributes significantly to maintenance of insulin sensitivity (as defined by insulin-dependent glucose uptake) in peripheral tissues. Leptin also acts as a signal that the body has sufficient energy stores to allow reproduction and to enhance erythropoiesis, lymphopoiesis, and myelopoiesis. For example, in women suffering from anorexia nervosa, leptin levels are extremely low and result in low ovarian steroids, amenorrhea (lack of menstrual bleeding), anemia from low red blood cell production, and immune dysfunction. Structure, Synthesis, and Secretion

1	Structure, Synthesis, and Secretion Leptin, a 16-kDa protein secreted by mature adipocytes, is structurally related to cytokines. Thus it is sometimes SREBP2 wasdiscoveredasatranscriptionfactorthatresidesinthemembraneoftheendoplasmicreticulum(ER).Inthepresenceofhighintracellularcholesterol,SREBP2isheldintheERbyalipid-sensingproteincalledSCAP (SREBPcleavage–activatingprotein).Inresponsetodepletedsterols,SCAPescortsSREBP2totheGolgi,whereSREBPiscleavedsequentiallybyproteasesandreleasedintothecytoplasm.SREBP2thentranslocatestothenucleusandincreasesthetranscriptionofgenesinvolvedinsynthesisanduptakeofcholesterol.AmorerecentlydiscoveredmemberofthistranscriptionfactorfamilyisSREBP1C, whichishighlyexpressedinadiposeandliver.IncontrasttoSREBP2,SREBP1CstimulatesgenesinvolvedinsynthesisofFAandTG.RegulationofSREBP1CoccursatthetranscriptionalleveloftheSREBP1C gene,withcleavageinducedbypolyunsaturatedfattyacidsandactivationbytheMAPKpathway.

1	Peroxisome proliferation activator receptors (PPARs) belongtothenuclearhormonereceptorsuperfamilythatalsoincludessteroidhormonereceptorsandthyroidhormonereceptors.PPARsheterodimerizewiththeretinoid X receptors (RXRs). Unlikesteroidandthyroidhormonereceptors,PPARsbindtoligandsinthemicromolarrange(i.e.,withloweraffinity).PPARsbindsaturatedandunsaturatedfattyacidsaswellasnaturalandsyntheticprostanoids.PPARγ ishighlyexpressedinadiposetissueandatalowerlevelinskeletalmuscleandliver.Itsnaturalligandsincludeseveralpolyunsaturatedfattyacids.PPARγ regulatesgenesthatpromotefatstorage.ItalsosynergizeswithSREBP1Ctopromotedifferentiationofadipocytesfrompreadipocytes.Tissue-specificknockoutofPPARγ inmiceandPPARγ-dominantnegativemutationsinhumansgiverisetolipodystrophy (i.e.,lackofwhiteadiposetissue),whichleadstodepositsofTGinmuscleandliver(calledsteatosis),insulinresistance,diabetes,andhypertension.

1	referred to as an adipocytokine. Circulating levels of leptin have a direct relationship with adiposity and nutritional status. Leptin output is increased by insulin, which prepares the body for correct partitioning of incoming nutrients. Leptin is inhibited by fasting and weight loss and by lipolytic signals (e.g., increased cAMP and β3-agonists). Diet-induced obesity, advanced age, and T2DM are associated with leptin resistance. Thus mechanisms that turn off leptin signaling are potential therapeutic targets. The amount of energy stored by an individual is determined by caloric intake and calories expended as energy per day. In many individuals, input and output are in balance, so weight remains relatively constant. However, the abundance of inexpensive high-fat, high-carbohydrate food, along with more sedentary lifestyles, is currently contributing to a pandemic of obesity and the pathological sequelae of obesity, including T2DM and cardiovascular disease.

1	The preponderance of stored energy consists of fat, and individuals vary greatly in the amount and percentage Thethiazolidinediones areexogenousligandsforPPARγ.Althoughtheypromoteweightgain,moderatelevelsofthiazolidinedionessignificantlyimproveinsulinsensitivity.PPARγ alsostimulatessecretionofadiponectin, whichpromotesoxidationoflipidsinmuscleandfatandtherebyimprovesinsulinsensitivity.PPARα isabundantlyexpressedinliverandtoalesserextentinskeletalandcardiacmuscleandkidney.PPARα promotesuptakeandoxidationofFFAs.ThusPPARα isanantisteatoticmolecule.Thefibrates areexogenousligandsofPPARα andareusedtoreduceTGdepositsinmuscleandliver,therebyimprovinginsulinsensitivity.Athirdmember,PPARδ, similarlypromotesfattyacidoxidationinadiposeandmuscletissue.PPARδ promotesdevelopmentofslow-twitchoxidativemusclefibersandincreasesmusclestamina.PPARδ hasapositiveeffectonlipoproteinmetabolismbyincreasingproductionofApoAapoproteinsandthenumberofHDLparticles.

1	Anotherfamilyoflipid-sensingtranscriptionfactorsistheliver X receptor (LXR) family,whichiscomposedofLXRα andLXRβ.LXRα isexpressedprimarilyinadiposetissue,liver,intestine,andkidney,whereasLXRβ isubiquitouslyexpressed.LXRsarerelatedtoPPARsinthattheyaremembersofthenuclearhormonereceptorfamilyandheterodimerizewithRXR.LXRsarecholesterolsensors.Inhigh-cholesterolconditions,LXRsupregulateexpressionofATP-bindingcassette(ABC)proteins.Inthefaceofexcesscholesterol,LXRsalsoincreaseABCproteinexpressioninthegastrointestinaltract,whichpromoteseffluxofcholesterolfromenterocytestothelumenforexcretion.Mutationsinthesetransporters(ABCG5andABCG8)causesitosterolemia, characterizedbyexcessiveabsorptionofcholesterolandplantsterols.Intheliver,LXRspromoteconversionofcholesteroltobileacidsforexcretionortocholesterolestersforstorage.Inthelatteraction,LXRsincreaseSREBP1Cexpression,therebyincreasingthefattyacylCoAsneededforesterification.

1	of body weight that is accounted for by adipose tissue. About 25% of the variance in total body fat appears to be due to genetic factors. A genetic influence on fat mass is supported by (1) the tendency for the body mass of adopted children to correlate better with that of their biological parents than with that of their adoptive parents; (2) the greater similarity of adipose stores in identical (monozygotic) twins, whether reared together or apart, than in fraternal (dizygotic) twins; (3) the greater correlation between gains in body weight and abdominal fat in identical twins than in fraternal twins when they are fed a caloric excess; and (4) the discovery of several genes that cause obesity.

1	In addition, the gestational environment has a profound effect on body mass of the adult. The effect of maternal diet on the weight and body composition of offspring is called fetal programming. Low birth weights correlate with increased risk for obesity, cardiovascular disease, and diabetes. These findings suggest that the efficiency of fetal metabolism has plasticity and can be altered by the in utero environment. The development of a “thrifty” metabolism would be advantageous to an individual born to a mother

1	Diabetes mellitus

1	isadiseaseinwhichinsulinlevelsorresponsivenessoftissuestoinsulin(orboth)isinsufficienttomaintainnormallevelsofplasmaglucose.Althoughthediagnosisofdiabetesisbasedprimarilyonplasmaglucose,diabetesalsopromotesimbalancesincirculatinglevelsoflipidsandlipoproteins(i.e.,dyslipidemia).Majorsymptomsofdiabetesmellitusincludehyperglycemia,polyuria,polydipsia,polyphagia,musclewasting,electrolytedepletion,andketoacidosis(inT1DM).Withnormalfasting(i.e.,nocaloricintakeforatleast8hours),plasmaglucoselevelsshouldbebelow110mg/dL.Apatientisconsideredtohaveimpairedglucosecontroliffastingplasmaglucoselevelsarebetween110and126mg/dL,andthediagnosisofdiabetesismadeiffastingplasmaglucoseexceeds126mg/dLon2successivedays.Anotherapproachtothediagnosisofdiabetesistheoralglucosetolerancetest.Afterovernightfastingthepatientisgivenabolusofglucose(usually75g)orally,andbloodglucoselevelsaremeasuredat2hours.A2-hourplasmaglucoseconcentrationgreaterthan200mg/dLon2consecutivedaysissufficienttomakethediagnosisofdiabetes.Th

1	tientisgivenabolusofglucose(usually75g)orally,andbloodglucoselevelsaremeasuredat2hours.A2-hourplasmaglucoseconcentrationgreaterthan200mg/dLon2consecutivedaysissufficienttomakethediagnosisofdiabetes.Thediagnosisofdiabetesisalsoindicatedifthepatienthassymptomsassociatedwithdiabetesandhasanonfastingplasmaglucoselevelgreaterthan200mg/dL.

1	Diabetesmellitusiscurrentlyclassifiedastype 1 (T1DM) ortype 2 (T2DM). T2DMisbyfarthemorecommonformandaccountsfor90%ofdiagnosedcases.However,T2DMisusuallyaprogressivediseasethatremainsundiagnosedinasignificantpercentageofpatientsforseveralyears.T2DMisoftenassociatedwithvisceralobesityandlackofexercise—indeed,obesity-relatedT2DMisreachingepidemicproportionsworldwide.UsuallytherearemultiplecausesforthedevelopmentofT2DMinagivenindividualthatareassociatedwithdefectsintheabilityoftargetorganstorespondtoinsulin(i.e.,insulin resistance),alongwithsomedegreeofbeta cell deficiency. InsulinsensitivitycanbecompromisedattheleveloftheInsRoratthelevelofpostreceptorsignaling.T2DMappearstobetheconsequenceofinsulinresistance,followedbyreactivehyperinsulinemia,butultimatelybyrelative hypoinsulinemia (i.e.,inadequatereleaseofinsulintocompensatefortheend-organresistance)andbeta cell failure.

1	Theunderlyingcausesofinsulinresistancedifferamongpatients.Threemajorunderlyingcausesofobesity-inducedinsulinresistanceare: 1.Decreased ability of insulin to increase GLUT4-mediated uptake of glucose, especially by skeletal muscle. Thisfunction,whichisspecificallyapartofglucometabolic regulation by insulin, maybeduetoexcessiveaccumulationofTGinmuscleinobeseindividuals.Excessivecaloricintakeinduceshyperinsulinemia.Initiallythisleadstoexcessiveglucoseuptakeintoskeletalmuscle.Justasintheliver,excessivecaloriesintheformofglucosepromotelipogenesisand,throughgenerationofmalonylCoA,repressionoffattyacylCoAoxidation.ByproductsoffattyacidandTGsynthesis(e.g.,diacylglycerol,ceramide)mayaccumulateandstimulatesignalingpathways(e.g.,proteinkinaseC–dependentpathways)thatantagonizesignalingfromtheInsRorIRSproteins,orboth.Thusinsulinresistanceintheskeletalmuscleofobeseindividualsmaybeduetolipotoxicity. 2.

1	2. Decreased ability of insulin to repress hepatic glucose production. Thelivermakesglucosebyglycogenolysisintheshorttermandbygluconeogenesisinthelongterm.Theabilityofinsulintorepresskeyhepaticenzymesinboththesepathwaysisattenuatedininsulin-resistantindividuals.Insulinresistanceinthelivermayalsobeduetolipotoxicityinobeseindividuals(e.g.,fatty liver orhepatic steatosis).Visceraladiposetissueislikelytoaffectinsulinsignalingattheliverinseveralways,inadditiontotheeffectsoflipotoxicity.Forexample,visceraladiposetissuereleasesthecytokinetumor necrosis factor (TNF)-α, whichhasbeenshowntoantagonizeinsulinsignalingpathways.Also,TGinvisceraladiposetissuehasahighrateofturnover(possiblybecauseofrichsympatheticinnervation),sotheliverisexposedtohighlevelsofFFAs,whichfurtherexacerbateshepaticlipotoxicity. 3.

1	3. Inability of insulin to repress hormone-sensitive lipase or increase LPL in adipose tissue (or both). HighHSLandlowLPLaremajorfactorsinthedyslipidemiaassociatedwithinsulinresistanceanddiabetes.AlthoughthefactorsthatresisttheactionsofinsulinonHSLandLPLarenotcompletelyunderstood,thereisevidenceforincreasedproductionofparacrinediabetogenicfactorsinadiposetissue,suchasTNF-α.ThedyslipidemiaischaracterizedashypertriglyceridemiawithlargeTG-richVLDLparticlesproducedbytheliver.BecauseoftheirhighTGcontent,largeVLDLsandIDLsaredigestedveryefficiently,therebygivingrisetosmall,denseLDLparticlesthatareveryatherogenic.Inaddition,HDLtakesonexcessTGinexchangeforcholesterolesters,whichappearstoshortenthecirculatinghalf-lifeofHDLandApoAproteins.ThustherearelowerlevelsofHDLparticles,whichnormallyplayaprotectiveroleagainstvasculardisease.T1DM ischaracterizedbydestructionofbetacells,almost alwaysbyanautoimmunemechanism.T1DMisalsotermedinsulin-dependent diabetes mellitus. CharacteristicsofT1DMare: 1.

1	PeoplewithT1DMneedexogenousinsulintomaintainlifeandpreventketosis;virtuallynopancreaticinsulinisproduced. 2. Thereispathologicaldamagetothepancreaticbetacells.Insulinitiswithpancreaticmononuclearcellinfiltrationisacharacteristicfeatureattheonsetofthedisorder.Cytokinesmaybeinvolvedintheearlydestructionofthepancreas. 3. PeoplewithT1DMarepronetoketoacidosis. 4. Ninetypercentofcasesbegininchildhood,mostlybetween10and14yearsofage.Thiscommonobservationledtoapplicationofthetermjuvenile diabetes tothedisorder.ThistermisnolongerusedbecauseT1DMcanariseatanytimeoflife,althoughjuvenileonsetisthetypicalpattern. 5.

1	5. Isletcellautoantibodiesarefrequentlypresentaroundthetimeofonset.IfT1DMisinducedbyavirus,theautoantibodiesaretransient.Occasionallyantibodieswillpersistlongterm,particularlyiftheyareassociatedwithotherautoimmunedisorders.About50%ofT1DMisrelatedtoproblemswiththemajorhistocompatibilitycomplexonchromosome6.Itiscorrelatedwithanincreasedfrequencyofcertainhumanleukocyteantigen(HLA)alleles.TheHLAtypesDR3andDR4aremostcommonlyassociatedwithdiabetes. who received poor nutrition and into a life that meant chronic undernourishment. A measure of adiposity is the body mass index (BMI). The BMI of an individual is calculated as: Equation 38.1 The BMI of healthy lean individuals ranges from 20 to 25. A BMI greater than 25 indicates that the individual is overweight, whereas a BMI higher than 30 indicates obesity. The condition of being overweight or obese is a risk factor for multiple pathologies, including insulin resistance, dyslipidemia, diabetes, cardiovascular disease, and hypertension.

1	WAT tissue is divided into subcutaneous and intraabdominal (visceral) depots. Intraabdominal WAT refers primarily to omental and mesenteric fat and is the smaller of the two depots. These depots receive different blood supplies that are drained in a fundamentally different way in that venous return from intraabdominal fat leads into the hepatic portal system. Thus intraabdominally derived FFAs are mostly cleared by the liver, whereas subcutaneous fat is the primary site for providing FFAs to muscle during exercise or fasting. Regulation of intraabdominal and subcutaneous adipose tissue also differs. Abdominal fat is highly innervated by autonomic neurons and has a greater turnover rate. Furthermore, these two depots display differences in hormone production and enzyme activity.

1	Men tend to gain fat in the intraabdominal depot (android [apple-shaped] adiposity), whereas women tend to gain fat in the subcutaneous depot, particularly in the thighs and buttocks (gynecoid [pear-shaped] adiposity). Clearly an excess of abdominal fat poses a greater risk factor for the pathologies mentioned earlier. Thus another indicator of body composition is circumference of the waist (measured in inches around the narrowest point between the ribs and hips when viewed from the front after exhaling) divided by the circumference of the hips (measured at the point where the buttocks are largest when viewed from the side). This waist-hip ratio may be a better indicator of body fat than BMI, especially as it relates to risk for development of diseases. A waist-hip ratio of greater than 0.95 in men or 0.85 in women is linked to a significantly higher risk for development of diabetes and cardiovascular disease.

1	In recent years, numerous hormones and neuropeptides have been implicated in both chronic and acute regulation of appetite, satiety, and energy expenditure in humans. One CHAPTER 39 Hormonal Regulation of Energy Metabolism simplified model involves two peptide hormones, leptin Fig. 39.17 ), already discussed. Leptin acts on at least two neuron types in the arcuate nucleus of the hypothalamus. In the first, leptin represses production of neuropeptide Y (NPY), a very potent stimulator of food- seeking behavior (energy intake) and an inhibitor of energy expenditure. Norepinephrine, another appetite stimulator, co-localizes with NPY in some of these neurons. At the same time, leptin represses production of agouti-related peptide (AGRP), an endogenous antagonist that acts on

1	MC4R, a hypothalamic receptor for the anorexigenic peptide α-melanocyte–stimulating hormone (α-MSH), which inhibits food intake. In another type of arcuate neuron, leptin stimulates production of proopiomelano cortin (POMC) products, one of which is α-MSH, and production of cocaine-amphetamine–regulated transcript (CART), both of which inhibit food intake. Thus leptin diture by simultaneously inhibiting NPY and the α-MSH Fig. 39.17 ). These second-order neuropeptides are transmitted to and interact with receptors in neurons of the and lateral hypothalamic nucleus (“hunger” neurons). In activity (especially sympathetic outflow) with diverse endo crine actions on thyroid gland function, reproduction, and growth.

1	Another regulator of food intake and body energy stores is melanin-concentrating hormone (MCH). This antagonizing the satiety effect of α-MSH downstream from the interaction of α-MSH with its MC4R receptor. The probable importance of this molecule is demonstrated by the fact that it is the only regulator whose ablation by gene knockout actually results in leanness. To maintain overall energy homeostasis, the system must example, carbohydrate intake with carbohydrate oxidation.

1	To maintain overall energy homeostasis, the system must example, carbohydrate intake with carbohydrate oxidation. neurotransmitter responses to meals. Serotonin produces satiety after ingestion of glucose. Gastrointestinal hormones such as cholecystokinin and GLP-1 produce satiety by humoral effects, but their local production in the brain may participate in nutrient and caloric regulation. The recently discovered hormone ghrelin is an acylated peptide with potent orexigenic activity that arises in cells of the oxyntic glands in the stomach. Plasma levels of ghrelin rise in humans in the 1 to 2 hours that precede their normal meals. Plasma levels of ghrelin fall drastically to minimum values about 1 hour after eating. Ghrelin appears to stimulate food intake by reacting with its receptor in hypothalamic neurons that express NPY. 1. Cells make ATP to meet their energy needs. ATP is made by glycolysis and by the TCA cycle coupled to oxidative phosphorylation. 2.

1	1. Cells make ATP to meet their energy needs. ATP is made by glycolysis and by the TCA cycle coupled to oxidative phosphorylation. 2. Cells can oxidize carbohydrate (primarily in the form of glucose), AAs, and FFAs to make ATP. Additionally the liver makes KBs for other tissues to oxidize for energy in times of fasting. 3. Some cell types are limited in the energy substrates they can oxidize for energy. The brain is normally exclusively dependent on glucose for energy. Thus blood glucose must be maintained above 60 mg/ dL for normal autonomic and CNS function. Conversely, inappropriately high levels of glucose (i.e., fasting glucose > 100 mg/dL) promote glucotoxicity and thereby lead to the long-term complications of diabetes. 4. The endocrine pancreas produces the hormones insulin, glucagon, somatostatin, gastrin, and pancreatic polypeptide. 5.

1	4. The endocrine pancreas produces the hormones insulin, glucagon, somatostatin, gastrin, and pancreatic polypeptide. 5. Insulin is an anabolic hormone that is secreted in times of excess nutrient availability. It allows the body to use carbohydrates as energy sources and store nutrients. 6. Major stimuli for insulin secretion include increased serum glucose and some AAs. Activation of cholinergic (muscarinic) receptors also increases insulin secretion, whereas activation of α2-adrenergic receptors inhibits insulin secretion. The gastrointestinal tract releases incretin hormones that stimulate pancreatic insulin secretion. GLP-1 is particularly potent in augmenting glucose-dependent stimulation of insulin secretion (GSIS). GLP-1 is degraded by dipeptidyl peptidase (DPP)-4. DPP-4– resistant GLP-1 analogues and inhibitors of DPP-4 are currently used to increase GSIS in patients with type 2 diabetes. 7.

1	7. Insulin binds to the insulin receptor (InsR), which is linked to multiple pathways that mediate the metabolic (Akt kinase) and growth effects (MAPK) of insulin. 8. During the digestive phase, insulin acts on the liver to promote trapping of glucose as G6P. Insulin also increases glycogenesis, glycolysis, and de novo lipogenesis (DNL) in the liver. Insulin inhibits gluconeogenesis, glycogenolysis, and assembly of lipids into VLDL. 9. Insulin increases GLUT4-mediated glucose uptake in muscle and adipose tissue. 10. Insulin increases glycogenesis, glycolysis, and in the presence of caloric excess, lipogenesis in muscle. 11. Insulin increases glycolysis and generation of G3P in adipocytes. Insulin induces expression of LPL and its transport to the luminal side of capillary endothelial cells. Insulin promotes uptake and activation of FFAs and esterification of fatty acyl CoAs to G3P to form TG, and it decreases hormone-sensitive lipase activity in adipocytes. 12.

1	TG, and it decreases hormone-sensitive lipase activity in adipocytes. 12. Insulin increases AA uptake and protein synthesis in skeletal muscle but also essentially all insulin target cells. Insulin/Akt kinase signaling activates mTORC1 and S6K to promote synthesis of ribosomal proteins and proteins involved in mRNA translation, as well as other types of proteins. Insulin inhibits proteosomal degradation of protein. 13. Glucagon is a catabolic counterregulatory hormone. Its secretion increases during periods of food deprivation, and it acts to mobilize nutrient reserves. It also mobilizes glycogen, fat, and even protein. 14. Glucagon is released in response to decreased serum glucose (and therefore decreased insulin) and increased serum AA levels and β-adrenergic signaling. 15.

1	14. Glucagon is released in response to decreased serum glucose (and therefore decreased insulin) and increased serum AA levels and β-adrenergic signaling. 15. Glucagon binds to the glucagon receptor, which is linked to PKA-dependent pathways. The primary target organ for glucagon is the liver. Glucagon increases liver glucose output by increasing glycogenolysis and gluconeogenesis. It increases β-oxidation of fatty acids and ketogenesis. 16. Glucagon regulates hepatic metabolism both by regulation of gene expression and through posttranslational PKA-dependent pathways. 17. The major counterregulatory factors in muscle and adipose tissue are the adrenal hormone epinephrine and the sympathetic neurotransmitter norepinephrine. These two factors act through β2-and β3-adrenergic receptors to increase cAMP levels. Epinephrine and norepinephrine enhance glycogenolysis and fatty acyl oxidation in muscle and increase hormone-sensitive lipase in adipose tissue. 18.

1	18. Diabetes mellitus is classified as type 1 (T1DM) and type 2 (T2DM). T1DM is characterized by destruction of pancreatic beta cells, and exogenous insulin is required for treatment. T2DM can be due to numerous factors but is usually characterized as insulin resistance coupled to some degree of beta cell deficiency. Patients with T2DM may require exogenous insulin at some point to maintain blood glucose levels. 19. Obesity-associated T2DM is currently at epidemic proportions worldwide and is characterized by insulin resistance due to lipotoxicity, hyperinsulinemia, and inflammatory cytokines produced by adipose tissue. T2DM is often associated with obesity, insulin resistance, hypertension, and coronary artery disease. This constellation of risk factors is referred to as the metabolic syndrome. 20. Major symptoms of diabetes mellitus include hyperglycemia, polyuria, polydipsia, polyphagia, muscle wasting, electrolyte depletion, and ketoacidosis (in T1DM).

1	20. Major symptoms of diabetes mellitus include hyperglycemia, polyuria, polydipsia, polyphagia, muscle wasting, electrolyte depletion, and ketoacidosis (in T1DM). 21. The long-term complications of poorly controlled diabetes are due to excess intracellular glucose (glucotoxicity), especially in the retina, kidney, and peripheral nerves. This leads to retinopathy, nephropathy, and neuropathy. Bailey CJ, et al. Future glucose-lowering drugs for type 2 diabetes. Lancet Diabetes Endocrinol. 2016;4:350-359. Davidson JA, et al. Glucagon therapeutics: dawn of a new era for diabetes care. Diabetes Metab Res Rev. 2016;doi:10.1002/dmrr.2773; [Epub ahead of print]. Font-Burgada J, et al. Obesity and cancer: the oil that feeds the flame. Cell Metab. 2016;23:48-62. 22. Adipose tissue has an endocrine function, especially in terms of energy homeostasis. Hormones produced by adipose tissue include leptin and adiponectin. Leptin acts on the hypothalamus to promote satiety.

1	Klein MS, Shearer J. Metabolomics and type 2 diabetes: translating basic research into clinical application. J Diabetes Res. 2016;2016:3898502. doi:10.1155/2016/3898502; [Epub 2015 Nov 9]. Marion-Letellier R, et al. Fatty acids, eicosanoids and PPAR gamma. Eur J Pharmacol. 2016;785:44-49. Pawlak M, et al. Molecular mechanism of PPARα action and its impact on lipid metabolism, inflammation and fibrosis in nonalcoholic fatty liver disease. J Hepatol. 2015;62:720-733. Upon completion of this chapter the student should be able to answer the following questions: 1. Describe the pool of serum calcium and phosphate, including ionized, complexed, and protein bound. Describe the normal concentration ranges of these ions and the major routes of influx and efflux. 2. Discuss the role of the parathyroid gland in the regulation of serum calcium and explain the role of the calcium-sensing receptor in the regulation of parathyroid hormone (PTH) secretion. 3.

1	2. Discuss the role of the parathyroid gland in the regulation of serum calcium and explain the role of the calcium-sensing receptor in the regulation of parathyroid hormone (PTH) secretion. 3. Describe the production of 1,25-dihydroxyvitamin D, including sources of vitamin D precursor, sites and key regulators of vitamin D hydroxylation, and transport of vitamin D metabolites in the blood. 4. List the target organs of PTH and describe its effects on calcium and phosphate mobilization or handling at each of these sites. 5. List the target organs and key actions of 1,25-dihydroxyvitamin D. 6. Discuss the regulation of phosphate metabolism by FGF23. 7. Predict the hormone responses that would be triggered by perturbations of serum calcium and phosphate or by vitamin D deficiency, and discuss the consequences of these compensatory hormone actions.

1	Predict the hormone responses that would be triggered by perturbations of serum calcium and phosphate or by vitamin D deficiency, and discuss the consequences of these compensatory hormone actions. alcium (Ca) and phosphate are essential to human life because they play important structural roles in hard tissues (i.e., bones and teeth) and important regulatory roles in metabolic and signaling pathways. In biological systems, inorganic phosphate (Pi) consists of a mixture of dihydrogen phosphate (H2PO4 −) and hydrogen phosphate (HPO4 −). The two primary sources of circulating Ca and Pi are the diet and the skeleton ( Fig. 40.1 ). Two hormones, 1,25-dihydroxyvitamin D (also called calcitriol) and parathyroid hormone (PTH), regulate intestinal absorption of Ca and Pi and release of Ca and Pi into the circulation after bone resorption. The primary processes for removal of Ca and Pi from blood are renal excretion and bone mineralization (see

1	Fig. 40.1 ). 1,25-Dihydroxyvitamin D and PTH regulate both processes. Other hormones and paracrine growth factors also regulate Ca and Pi homeostasis. Crucial Roles of Calcium and Phosphate in Cellular Physiology Ca is an essential dietary element. In addition to obtaining Ca from the diet, humans contain a vast store (i.e., >1 kg) of Ca in bone mineral, which can be called upon to maintain normal circulating levels of Ca in times of dietary restriction and during the increased demands of pregnancy and nursing. Circulating Ca exists in three forms (

1	Table 40.1): free ionized Ca++ , protein-bound Ca, and Ca complexed with anions (e.g., phosphates, HCO3 − , citrate). The ionized form represents about 50% of circulating Ca. Since it is critical to so many cellular functions, [Ca++] in both the extracellular and intracellular compartments is tightly controlled. Circulating Ca++ is under direct hormonal control and normally maintained within a relatively narrow range. Either too little calcium (hypocalcemia; total serum calcium < 8.7 mg/dL [2.2 mM]) or too much Ca (hypercalcemia; total serum Ca > 10.4 mg/dL [2.6 mM]) in blood can lead to a broad range of pathophysiological changes, including neuromuscular dysfunction, central nervous system dysfunction, renal insufficiency, calcification of soft tissue, and skeletal pathology.

1	Pi is also an essential dietary element, and it is stored in large quantities in mineral. Most circulating Pi is in the free ionized form, but some Pi (<20%) circulates as a protein-bound form or complexed with cations (see Table 40.1 ). Because soft tissues contain 10-fold more Pi than Ca, tissue damage (e.g., crush injury with massive muscle cell death) can result in hyperphosphatemia, whereupon the increased Pi complexes with Ca++ to cause acute hypocalcemia. Pi is a key intracellular component. Indeed, it forms the high-energy phosphate bonds of adenosine triphosphate (ATP) that maintain life. Phosphorylation and dephosphorylation of proteins, lipids, second messengers, and cofactors represent key regulatory steps in numerous metabolic and signaling pathways, and phosphate also serves as the backbone for nucleic acids. •Fig. 40.1 DailyCa++ andPiflux. Ca8.5–10.250%45%5%Pi3–4.584%10%6%

1	•Fig. 40.1 DailyCa++ andPiflux. Ca8.5–10.250%45%5%Pi3–4.584%10%6% Ca++ isbound(i.e.,complexed)tovariousanionsinplasma,includingHCO3− ,citrate,andSO42− .Piiscomplexedtovariouscations,includingNa+ andK+ .FromKoeppenBM,StantonBA.Renal Physiology. 4thed.Philadelphia:Mosby;2007. Physiological Regulation of Calcium and Phosphate: Parathyroid Hormone and 1,25-Dihydroxyvitamin D C PTH and 1,25-dihydroxyvitamin D are the two physiologically most important hormones dedicated to mainte- nance of normal blood Ca and Pi in humans. As such they are referred to as calciotropic hormones. The structure, synthesis, and secretion of these two hormones and their receptors will be discussed first. In the following section, the detailed actions of PTH and 1,25-dihydroxyvitamin D on the three key sites of Ca/Pi homeostasis (i.e., gut, bone, and kidney) are discussed. The predominant parenchymal cell type in the parathyroid B gland is the principal (also called chief) cell ( Fig. 40.2

1	The predominant parenchymal cell type in the parathyroid B gland is the principal (also called chief) cell ( Fig. 40.2 PTH is the primary hormone that protects against hypocalcemia. The primary targets of PTH are bone and the kidneys. PTH also functions in a positive feed-forward loop by stimulating production of 1,25-dihydroxyvitamin D. •Fig. 40.2 AandB,Histologyofparathyroidglands.A,adiposetissuewithinparathyroidglands;C,capillaries;O,oxyphilcells;P,principalorchiefcells.(FromYoungBetal.Wheater’s Functional Histology. 5thed.Philadelphia:ChurchillLivingstone;2006.) Structure, Synthesis, and Secretion PTH is secreted as an 84–amino acid polypeptide and is synthesized as a prepro-PTH, which is proteolytically processed to pro-PTH in the endoplasmic reticulum and then to PTH in the Golgi and secretory vesicles. PTH has a short half-life in the circulation (2 minutes), consistent with its role in minute-to-minute regulation of plasma calcium.

1	Because the PTH receptor also binds PTH-related peptide (PTHrP), it is usually referred to as the PTH/PTHrP receptor. The PTH/PTHrP receptor is expressed on osteoblasts in bone and in the proximal and distal tubules of the kidney, and it is the receptor that mediates the systemic actions of PTH. However, the PTH/PTHrP receptor is also expressed in many developing organs in which PTHrP has important paracrine functions. One such example is regulation of chondrocyte proliferation in the growth plate during endochondral bone growth. Vitamin D is a prohormone that must undergo two successive hydroxylation reactions to become the active form known as 1,25-dihydroxyvitamin D or calcitriol ( Fig. 40.5 ). This hormone plays a critical role in Ca absorption Extracellular[Ca++]issensedbytheparathyroidchiefcellthroughaplasmamembranecalcium-sensing receptor (CaSR). TheprimarysignalthatstimulatesPTHsecretionisadecreaseincirculating[Ca++](

1	Extracellular[Ca++]issensedbytheparathyroidchiefcellthroughaplasmamembranecalcium-sensing receptor (CaSR). TheprimarysignalthatstimulatesPTHsecretionisadecreaseincirculating[Ca++]( Fig.40.3 ).Conversely,increasingamountsofextracellularCa++ bindtotheCaSRandstimulatesignalingpathwaysthatrepressPTHsecretion.AlthoughtheCaSRbindstoextracellularCa++ withrelativelylowaffinity,theCaSRisextremelysensitivetominutechangesinextracellular[Ca++].Therelationshipbetween[Ca++]andtherateofPTHsecretionisdescribedbyasteepinversesigmoidalcurve.A0.2-mMdifferenceinblood[Ca++]spansthefullrangeofthecurve,alteringPTHsecretionfrombasal(5%ofmaximum)tomaximumlevels( Fig.40.4).Thesteady-state“setpoint” willvarybetweenindividualsbuttypicallyresidesbelowthemidpointofthecurve(i.e.,half-maximalPTHsecretion).ThustheCaSRisarapid,robust,andcontinuousregulatorofPTHoutputinresponsetosubtle[Ca++]fluctuations.

1	InadditiontoinhibitingPTHsecretion,activationoftheCaSRalsopromotesdegradationofstoredPTHintheparathyroidchiefcell.Asaresult,biologicallyinactivecarboxyterminalPTHfragmentsaresecretedfromtheparathyroidglandandarealsoproducedbyperipheralmetabolismofPTHbytheliverandkidney.ThereforecurrentPTHassaysusetwoantibodiesthatrecognizeepitopesfrombothendsofthemoleculetoaccuratelymeasureintactPTH[1-84]. Overalongertimeframe,PTHproductionisalsoregulatedatthelevelofmRNAstabilityandgenetranscription(see Fig. 40.3 ).Decreased[Ca++]leadstoproductionofproteinsthatbindthe3-untranslatedregionofPTHmRNAandstabilizeit,leadingtoincreasedPTHtranslation.PTH genetranscriptionisrepressedby1,25-dihydroxyvitaminDinanegativefeedbackloop(actingthroughvitaminDresponseelements—seelater).Theabilityof1,25-dihydroxyvitaminDtoholdPTH geneexpressionincheckisreinforcedbythecoordinatedupregulationofCASR geneexpressionbypositivevitaminDresponseelementsinthepromoterregionoftheCASR gene(see

1	Fig.40.3 ).Itshouldbenoted,however,thatduringahypocalcemicchallenge,thedecreasein[Ca++]overridestheinhibitoryeffectof1,25-dihydroxyvitaminDonPTHtranscription,allowingbothofthesehormonestobeelevatedsimultaneously. •Fig. 40.3 RegulationofPTH geneexpressionandsecretion.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.)Gq + Gi PTH PTH CaSR Downstream signaling pathway 1,25 Vitamin D CaSR Ca++ proPTH preproPTH preCaSR CaSR mRNA (+) (–) (–) (–) PTH mRNA Parathyroid cell membrane Nucleus CaSR gene PTH gene •Fig. 40.4 Sigmoidalrelationshipbetweenserum[Ca++]andserumPTH,whichreflectstherateofPTHsecretion.(ModifiedfromPor-terfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.)100 50 5 1.00 1.301.201.10 1.40 Serum PTH (% of maximum) Free Ca++ in blood (mM)

1	Patientswithbenign familial hypocalciuric hypercalcemia (FHH) areheterozygousforinactivatingmutationsoftheCaSR.Inthesepatients,becauseofcompleteorpartiallossofoneCaSRallele,higherlevelsof[Ca++]arerequiredtosuppressPTHsecretion.Thisresultsinanelevated[Ca++]setpointforPTHsecretion,accountingforthehypercalcemia.TheCaSRisalsoexpressedinthethickascendinglimboftherenaltubule,whereitnormallyinhibitsCa++ reabsorptionwhenbloodCa++ rises.ThehypocalciuriainthefaceofhypercalcemiainFHHisduetothereducedabilityoftheCaSRinthekidneytosenseandrespondtoelevatedblood[Ca++]byincreasingCaexcretion.

1	Parathyroid hormone–related peptide (PTHrP) isapeptideparacrinehormoneproducedbyseveraladulttissues(skin,hair,breast),whereitmayregulateproliferationanddifferentiation.Italsoplaysaroleinrelaxationofsmoothmuscleinresponsetostretchinbloodvessels,uterus,andbladder.Duringlactation,PTHrPpromotesmaternalboneresorptionandthetransportofcalciumintomilk.Duringdevelopment,PTHrPregulatescalciumtransportacrosstheplacentaandisakeyregulatorofchondrocyteproliferationanddifferentiationinthegrowthplateoflongbones.The30aminoacidsattheN-terminusofPTHrPhavesignificantstructuralhomologywithPTH.PTHrPisnotregulatedbycirculatingCa++ andnormallydoesnotplayaroleinCa/Pihomeostasisinadults.However,certaintumorssecretehighlevelsofPTHrP,whichcauseshypercalcemia of malignancy andsymptomsthatresemblehyperparathyroidism.

1	Hormonal Regulation of Calcium and Phosphate Metabolism 1,25-(OH)2D3 24,25-(OH)2D3 •Fig. 40.5 Biosynthesisof1,25-dihydroxyvitaminD.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.) and to a lesser extent Pi absorption by the small intestine. It also regulates bone remodeling and renal reabsorption of Ca and Pi. Structure, Synthesis, and Transport of Active Vitamin D Metabolites

1	Structure, Synthesis, and Transport of Active Vitamin D Metabolites Vitamin D3 (also called cholecalciferol) is synthesized via conversion of 7-dehydrocholesterol by ultraviolet B •Fig. 40.6 VitaminDmetabolism.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.)7-Dehydrocholesterol Vit D-DBP and Vit D-chylomicron 25(OH) Vit D-DBP Vit D-chylomicron Lymphatic Vit D-DBP 1,25-Dihydroxyvitamin D Portal blood Caval blood Peripheral blood Vitamin D3 Enterocyte Dietary vitamin D3 and vitamin D2 Vit D UV-B Hepatocyte Vit D 25-Hydroxyvitamin D 25-Hydroxylase 24-Hydroxylase 24-Hydroxylase25-Hydroxyvitamin D 24,25-Dihydroxyvitamin D 1, 25-Dihydroxyvitamin D 1,24,25-Trihydroxyvitamin D Biological effects at gut, bone, kidney, etc. 1˜-Hydroxylase (UVB) light in the more basal layers of the skin (Fig. 40.6 ). Chemically, vitamin D3 is a secosteroid in which one of the cholesterol rings is opened (see

1	Fig. 40.5 ). Vitamin D2 (ergocalciferol) is produced in plants. Vitamin D3 and to a lesser extent vitamin D2 are absorbed from the diet and are equally effective after conversion to active hydroxylated forms. The balance between UVB-dependent endogenously synthesized vitamin D3 and absorption of the dietary forms of vitamin D becomes important in certain situations. Individuals with higher melanin content in skin who live at higher latitudes convert less 7-dehydrocholesterol to vitamin D3 and thus are more dependent on vitamin supplements or dietary sources of vitamin D (natural or fortified, e.g., milk). Institutionalized elderly patients who stay indoors and avoid dairy products are particularly at risk for development of vitamin D deficiency. Vitamin D is transported in blood from the skin to the liver. Dietary vitamin D reaches the liver directly via transport in the portal circulation and indirectly via chylomicrons (see

1	Vitamin D is transported in blood from the skin to the liver. Dietary vitamin D reaches the liver directly via transport in the portal circulation and indirectly via chylomicrons (see Fig. 40.6 ). In the liver, vitamin D is hydroxylated at the 25-carbon position to yield 25-hydroxyvitamin D. The hepatic 25-hydroxylase is constitutively expressed and unregulated, so circulating levels of 25-hydroxyvitamin D reflect the amount of precursor available for 25-hydroxylation. For this reason, and because of its relatively long half-life in the circulation (2–3 weeks), measurement of 25-hydroxyvitamin D levels is used to assess vitamin D status. 25-Hydroxyvitamin D undergoes further hydroxylation in the proximal tubule of the kidney (see Figs. 40.5 40.6).

1	25-Hydroxyvitamin D undergoes further hydroxylation in the proximal tubule of the kidney (see Figs. 40.5 40.6). Hydroxylation at the 1α position generates 1,25-dihydroxyvitamin D, the most active form of vitamin D. Hydroxylation at the 24 position generates 24,25-dihydroxyvitamin D, which does not play a major biological role and serves as an inactivation pathway. Renal 1α-hydroxylase is tightly regulated by a number of factors ( Fig. 40.7). PTH and hypophosphatemia are the primary inducers of 1α-hydroxylase activity, resulting in 25(OH)D3 1˜-hydroxylase 1,25(OH)2D3 25(OH)D3 CYP27B1 FGFRKIotho •Fig. 40.7 Regulationof1α-hydroxylasegene(CYP27B1) expressionintheproximaltubule,showingstimulationbyPTHandinhibitionbyFGF23and1,25-dihydroxyvitaminD.Hypophosphatemiaprobablystimulates1α-hydroxylasebyreducingFGF23levelsatleastinpart.

1	increased levels of 1,25-dihydroxyvitamin D. Conversely, [Ca++] and 1,25-dihydroxyvitamin D, the enzyme product, inhibit it. Fibroblast growth factor (FGF)23, a major regulator of Pi metabolism (see later), also represses 1α-hydroxylase activity; a reduction of FGF23 levels likely mediates the effect of hypophosphatemia on 1,25-dihydroxyvitamin D production at least in part. Vitamin D and its metabolites circulate in blood primarily bound to vitamin D–binding protein (DBP). DBP is a serum glycoprotein that is synthesized by the liver. DBP binds more than 85% of 1,25-hydroxyvitamin D and 24,25-dihydroxyvitamin D. Because of binding to other proteins, only 0.4% of 1,25-dihydroxyvitamin D circulates as free hormone. DBP transports the highly lipophilic vitamin D in blood and provides a reservoir of vitamin D that protects against vitamin D deficiency.

1	1,25-Dihydroxyvitamin D Receptor 1,25-Dihydroxyvitamin D exerts its actions primarily through binding to the nuclear vitamin D receptor (VDR), which is a member of the nuclear hormone receptor family. The VDR is a ligand-dependent transcription factor that binds to cognate DNA sequences (vitamin D response elements) as a heterodimer with the retinoid X receptor (RXR). Thus the primary action of 1,25-dihydroxyvitamin D is to regulate gene expression in its target tissues, including the small intestine, bone, kidneys, and parathyroid gland.

1	The genomic actions of 1,25-dihydroxyvitamin D mediated by the VDR occur over a period of hours to days. 1,25-Dihydroxyvitamin D also has rapid effects (seconds to minutes). For example, 1,25-dihydroxyvitamin D rapidly induces absorption of Ca++ by the duodenum. The VDR is also expressed in the plasma membrane of cells and is linked to rapid signaling pathways (e.g., G proteins, phosphatidylinositol-3′-kinase). Current molecular modeling has led to development of ligands that specifically bind Hormonal Regulation of Calcium and Phosphate Metabolism to the nuclearversus the membrane-localized VDR, paving the way for selective treatment of disorders related to the rapid versus slow actions of 1,25-dihydroxyvitamin D with synthetic vitamin D analogues. Regulation of [Ca++] and [Pi] by Small Intestine and Bone An overview of the regulation of [Ca++] and Pi] by the action of PTH and 1,25-dihydroxyvitamin D on the small intestine, bone, and parathyroid glands is summarized in

1	An overview of the regulation of [Ca++] and Pi] by the action of PTH and 1,25-dihydroxyvitamin D on the small intestine, bone, and parathyroid glands is summarized in Table 40.2 and in the following paragraphs. For details on renal handling of Ca++ , consult Chapter 36.

1	Table 40.2 and in the following paragraphs. For details on renal handling of Ca++ , consult Chapter 36. Calcitonin isapeptidehormoneproducedbythemedullarycells,orC-cells,ofthethyroidgland.Calcitoninsecretionispositivelyregulatedbyserum[Ca++]viatheCaSR.Thecalcitoninreceptorisexpressedinosteoclasts,wherecalcitoninactsrapidlyanddirectlytoinhibitboneresorption.However,inhumans,calcitonindoesnotappeartoplayamajorroleinregulatingserumCa.Insupportofthisview,productionofexcesscalcitoninorcompleteabsenceofcalcitonin(e.g.,followingthyroidectomy)doesnotperturbserumCalevels.Morepotentformsofthehormone(e.g.,salmoncalcitonin)havebeenusedtherapeuticallyasanantiresorptiveinthetreatmentofPaget’s disease (characterizedbyexcessiveosteoclasticboneresorption)andinosteoporosis.Calcitoninisalsoausefulhistochemicalmarkerofmedullarythyroidcancer.

1	Dietary intake of Ca can vary widely among individuals and from day to day. Assuming an intake of 1000 mg (the RDA for ages 19–50), 350 mg would typically be absorbed, counterbalanced by 150 mg secreted by the intestine, for a net intake of 200 mg. Most Ca++ absorption takes place in the proximal small intestine. Importantly, absorption of Ca++ is stimulated by 1,25-dihydroxyvitamin D, so absorption is more efficient in the face of declining dietary Ca++ . Ca++ is absorbed from the duodenum and jejunum by both a Ca++-regulated and a hormonally regulated transcellular route and by a passive paracellular route. The transcellular route of Ca++ absorption is summarized in Fig.

1	Fig. 40.8 . Movement of Ca++ from the gastrointestinal lumen into the enterocyte, which is driven by both chemical and electrical gradients, occurs via apical calcium channels called TRPV5 and TRPV6. Once inside the cell, Ca++ ions bind to calbindin-D9K, which maintains a low cytoplasmic [Ca++], preserving the favorable transluminal membrane Ca++ gradient. Calbindin-D9K also plays a role in apical-to-basolateral shuttling of Ca++, which is transported across the basolateral membrane against an electrochemical gradient by plasma membrane calcium ATPase (PMCA). The Na+/Ca++ exchanger (NCX) also contributes to the CASR,calcium-sensingreceptor;M-CSF,monocytecolony-stimulatingfactor;NPT2,Na+/Picotransporter;OPG,osteoprotegerin;PTH,parathyroidhormone; RANKL,receptoractivatorofnuclearfactorκ-B.

1	CASR,calcium-sensingreceptor;M-CSF,monocytecolony-stimulatingfactor;NPT2,Na+/Picotransporter;OPG,osteoprotegerin;PTH,parathyroidhormone; RANKL,receptoractivatorofnuclearfactorκ-B. •Fig. 40.8 IntestinalabsorptionofCa++viathetranscellularroute.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.)LuminalsideTRPV5/6SerosalsideCa++Ca++Ca++Ca++-cal indin9KCal indin9KPMCA transport of Ca++ out of enterocyte. 1,25-Dihydroxyvitamin D stimulates expression of all the components involved in absorption of Ca++ by the small intestine. The fraction of dietary Pi absorbed by the jejunum remains relatively constant at about 70% and is under minor hormonal control by 1,25-dihydroxyvitamin D. The limiting process in transcellular Pi absorption is transport across the apical brush border, which is mediated by the Na+/Pi cotransporter (NPT2).

1	Bone stores vast amounts of Ca and Pi. Once peak bone mass has been achieved in an adult, the skeleton is constantly remodeled through the concerted activities of bone cells. The processes of bone formation and bone resorption are in balance in a healthy, physically active, and well-nourished individual. Of the 1 kg of Ca immobilized in bone, about 500 mg (i.e., 0.5%) is mobilized from and deposited into bone each day. However, the process of bone remodeling can be modulated to provide a net gain or loss of Ca++ and Pi into blood and is responsive to physical activity (loading), diet, age, and hormonal regulation. Because the integrity of bone is absolutely dependent on Ca and Pi, chronic dysregulation of these ions or the hormones that regulate them lead to pathological changes in bone. Physiology of Bone

1	Physiology of Bone The processes of pattern formation, growth, and remodeling of the skeleton is complex and beyond the scope of this chapter. The key elements required for understanding the role of adult bone in the hormonal regulation of Ca and Pi metabolism are discussed. In adults, bone remodeling involves (1) destruction of fatigued or microdamaged bone with the release of Ca++, Pi, and hydrolyzed fragments of bone matrix into blood and (2) synthesis of osteoid (yet to be mineralized bone matrix) at the site of resorption, followed by controlled mineralization of the osteoid by Ca++ and Pi to form new bone. Bone remodeling occurs continually at about 2 million discrete sites throughout the skeleton by packets of bone cells referred to as the basic multicellular units (BMU).

1	The cells involved in bone remodeling fall into two major classes: cells that form bone (osteoblasts) and cells that destroy or resorb bone (osteoclasts). The process of bone remodeling is a highly integrated process (

1	Fig. 40.9 ). Osteoblast-lineage cells express factors that induce differentiation of osteoclasts from progenitors of the monocyte/ macrophage lineage and also promote mature osteoclast function. Osteoblasts release monocyte colony-stimulating factor (M-CSF), which expands and differentiates early hematopoietic progenitors (CFU-GM) into preosteoclasts that express a cell surface receptor called RANK (receptor •Fig. 40.9 Osteoblastregulationofosteoclastdifferentiationandfunction.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.) activator of nuclear factor [NF]-κB). Osteoblast-lineage cells display RANK ligand (RANKL) on their cell surface. RANKL then binds to RANK on preosteoclasts and induces osteoclastogenesis. This process involves fusion of several osteoclast precursors, giving rise to a large multinucleated osteoclast. The perimeter of the osteoclast membrane facing mineralized bone adheres tightly to the bone and seals off the area of

1	several osteoclast precursors, giving rise to a large multinucleated osteoclast. The perimeter of the osteoclast membrane facing mineralized bone adheres tightly to the bone and seals off the area of osteoclast-bone contact (see

1	Fig. 40.9 ). The region within the sealed zone forms a highly invaginated membrane called the ruffled border, from which HCl and hydrolytic lysosomal enzymes are secreted. The acidic enzyme-rich microenvironment beneath the osteoclast dissolves the bone mineral, thereby releasing Ca++ and Pi into blood, and also degrades the bone matrix. There is an additional inhibitory component of the RANK/RANKL system. Osteoblast-lineage cells can also produce a soluble factor called osteoprotegerin (OPG), which acts as a decoy receptor for RANKL and inhibits osteoclast differentiation and function (see Fig. 40.9 ). Therefore the balance between RANKL and OPG expression by osteoblasts determines how much osteoclast differentiation and bone resorption will occur.

1	Following bone resorption in the BMU, there is a brief reversal phase, then adjacent osteoblasts migrate into the resorbed area and begin to lay down osteoid. Several components within osteoid (pyrophosphate, alkaline phosphatase, specific glycoproteins) promote slow, controlled mineralization, a process that removes Ca++ and Pi from blood. As the osteoblasts become surrounded by and entrapped within bone, they become osteocytes that sit within small spaces called lacunae. Osteocytes remain interconnected through cell processes that run within canaliculi and form communicating junctions with adjacent cell processes. The new concentric layers of bone, along with the interconnected osteocytes and the central canal, are referred to collectively as a Haversian system or osteon. Emerging evidence indicates that osteocytes are able to sense mechanical stress in bone and signal that additional local bone formation is needed. They can also detect microdamage in bone that serves to initiate

1	indicates that osteocytes are able to sense mechanical stress in bone and signal that additional local bone formation is needed. They can also detect microdamage in bone that serves to initiate remodeling at that location.

1	Asacalciotropichormone,PTHisapotentregulatorofboneresorptioninadults.ThePTH/PTHrPreceptorisexpressedonosteoblastsbutnotonosteoclasts.ThereforePTHactsonosteoblaststoincreaseexpressionofosteoblastparacrinefactors(i.e.,M-CSF,RANKL)thatupregulateosteoclastdifferentiationandboneresorption.1,25-DihydroxyvitaminDalsostimulatesboneresorptionbyupregulatingRANKLexpressioninosteoblasts. ItisimportanttorecognizethatPTH(alongwith1,25-dihydroxyvitaminD)willpromoteboneresorptionwhenPTHlevelsarehigh(i.e.,duringahypocalcemicchallenge).WhenPTHlevelsarenormal,however,boneremodelingisalocallycontrolledprocessbywhicholddamagedboneisreplaced.Interestinglyithasbeenshownthatintermittentadministrationoflow-dosePTHpromotesosteoblastsurvivalandboneanabolicfunctions,increasesbonedensity,andreducestheriskoffractureinhumans.

1	DiscoveryoftheRANK/RANKL/OPGsystemhaspresentednewtherapeuticopportunitiesfortreatingosteoporosis.AbiologicalantiresorptivedrugbasedonahumanizedantibodydirectedagainstRANKLisnowavailablefortreatmentofpostmenopausalosteoporosis.Thishasproventobeaneffectivetreatmentthatimprovesbonedensityandreducestheriskoffracture.

1	Fig.40.10C )producesahypocalcemicchallengebydecreasinggastrointestinalabsorptionofCa++ andPi.Adropinserum[Ca++]increasescompensatoryPTH geneexpression,PTHsecretion,parathyroidcellproliferation,andPTH-mediatedupregulationofrenal1-hydroxylase.Intheabsenceofsufficient25-hydroxyvitaminDprecursor,however,1,25-dihydroxyvitaminDlevelsfall.ThesecondaryelevationofPTHmobilizesCa++ fromboneandkidneybutpromotesrenalexcretionofPi,causinghypophosphatemia.BecausetheCa++ × Piproductinserumislow,bonemineralizationisimpaired.Inchildrenthisleadstorickets, inwhichthegrowthoflongbonesisabnormalandimpaired.Theribcage,wrists,andanklesshowcharacteristicbonedeformities,andtheimpairedmineralizationcausesbowingofthelegs.Inadults,vitaminDdeficiencyleadstoosteomalacia, whichischaracterizedbypoormineralizationofnewlyformedosteoid,visibleonradiographsaspseudofractures.Inseverecases,osteomalaciaresultsinweakness,bonepain,andincreasedriskoffracture. °PTH °1,25(OH)2D3 ˜1,25(OH)2D3

1	°PTH °1,25(OH)2D3 ˜1,25(OH)2D3 Regulation of Serum Phosphate by FGF23

1	Study of hypophosphatemic disorders has led to the discovery that FGF23, a peptide hormone produced by osteocytes, is a regulator of Pi metabolism. FGF23 binds to a receptor complex in proximal tubule cells and, like PTH, inhibits NPT2 to promote Pi excretion. Several diseases are associated with excess production of FGF23, including rickets in children and osteomalacia secondary to hypophosphatemia in adults. In autosomal dominant hypophosphatemic rickets (ADHR), a mutation in FGF23 prevents its cleavage and inactivation. X-linked hypophosphatemic rickets (XLHR) is caused by a mutation of the PHEX gene (protein with homology to endopeptidases on the X chromosome), which also causes overproduction of FGF23. Finally, FGF23 is sometimes ectopically produced by slow-growing occult mesenchymal tumors, causing a hypophosphatemic paraneoplastic syndrome. In addition to inhibition of Pi reabsorption, FGF23 also inhibits expression of 1α-hydroxylase in the proximal tubule, thereby inhibiting

1	tumors, causing a hypophosphatemic paraneoplastic syndrome. In addition to inhibition of Pi reabsorption, FGF23 also inhibits expression of 1α-hydroxylase in the proximal tubule, thereby inhibiting production of 1,25-dihydroxyvitamin D and exacerbating hypophosphatemia. The physiological role of this pathway is not completely understood, and many questions remain, °GI °1,25(OH)2D3 absorption •Fig. 40.10 IntegratedhormoneresponsestoperturbationsofCa++ (A),Pi(B),andvitaminD(C).

1	including how and where Pi levels are sensed. Pi is not as tightly regulated as calcium, either temporally or with respect to concentration range, but recent evidence suggest that long-term elevation of Pi is associated with increased production of FGF23. In what appears to be an emerging negative feedback loop, 1,25-dihydroxyvitamin D decreases production of FGF23 by osteocytes.

1	Gonadal and adrenal steroid hormones have profound effects on bone. 17β-Estradiol (E2; see has important anabolic effects on bone and is a potent regulator of osteoblast and osteoclast function. Estrogen promotes survival of osteoblasts and apoptosis of osteoclasts, thereby favoring bone formation over resorption. Androgens also have bone anabolic effects, although some of these effects are due to local conversion of testosterone to E2 in men (see ). The combined effects of testosterone and E2 account for the higher peak bone mass observed in men. In postmenopausal women, estrogen deficiency results in an initial phase of rapid bone loss that lasts about 5 years, followed by a second phase of slower age-related bone loss that is similar in both sexes. For this reason, women are susceptible to postmenopausal osteoporosis.

1	Glucocorticoids at high therapeutic doses promote bone resorption and inhibit intestinal Ca absorption. However, the most critical adverse effect is inhibition of osteoblast differentiation, which impairs bone formation. Therefore patients treated with high levels of a glucocorticoid as an antiinflammatory or immunosuppressive drug are at risk for glucocorticoid-induced osteoporosis and should be monitored carefully. Integrated Physiological Regulation of Ca++/Pi Metabolism The integrated response of PTH and 1,25-dihydroxyvitamin D to a hypocalcemic challenge is shown in

1	Fig. 40.10A . A decrease in serum [Ca++] detected by the CaSR on parathyroid chief cells stimulates secretion of PTH. In the kidney, PTH increases Ca++ reabsorption in the distal tubule and to a lesser extent in the distal thick ascending limb of the loop of Henle. In bone, elevated PTH stimulates osteoblast lineage cells to express RANKL, which increases osteoclast activity and leads to increased bone resorption and release of Ca++ and Pi into blood. PTH stimulates 1α-hydroxylase expression in the proximal renal tubule, thereby increasing 1,25-dihydroxyvitamin D levels. 1,25-Dihydroxyvitamin D stimulates absorption of Ca and Pi in the small intestine and upregulates osteoblast expression of RANKL, thereby amplifying the effect of PTH on bone resorption. In the kidney, PTH inhibits NPT2 in the proximal tubule to lower Pi reabsorption and increase Pi clearance, thereby counterbalancing Pi mobilized from the bone and gut.

1	Although not as tightly regulated as Ca++ , perturbations in serum Pi will also elicit hormonal responses (see Fig. 40.10B ). Low serum Pi stimulates production of 1,25-dihydroxyvitamin D in the kidney, which in turn will mobilize Ca and Pi from the intestine. The rise in Ca++ will suppress PTH secretion to prevent hypercalcemia. This drop in PTH will enhance Pi reabsorption in the proximal tubule to help restore serum Pi. Over a longer time course, a decrease in serum Pi will inhibit FGF23 production, which will favor Pi reabsorption in the proximal tubule. These integrated responses will allow correction of hypophosphatemia while maintaining normocalcemia. For hormonal responses to vitamin D deficiency, see the In the Clinic box and Fig. 40.10C

1	Primary hyperparathyroidism iscausedbyexcessiveproductionofPTHbytheparathyroidglands.Itismostfrequentlycausedbyasingleadenoma confinedtooneoftheparathyroids.OwingtoelevatedPTH,patientswithprimaryhyperparathyroidismhavehighserum[Ca++]and,inmostcases,lowserum[Pi].Hypercalcemia isaresultofboneresorption,increasedgastrointestinalCaabsorption(mediatedby1,25-dihydroxyvitaminD),andincreasedrenalCa++ reabsorption.Themajorsymptomsofthedisorderarerelatedtoincreasedboneresorption,hypercalcemia,andhypercalciuria. Theseincluderadiographicmanifestationsofexcessiveboneresorptionandpsychologicaldisorders,particularlydepression.Progressiveneurologicalsymptomsincludefatigue,mentalconfusion,andatveryhighlevels(>15mg/dL),coma.Kidneystones(nephrolithiasis) composedofcalciumphosphatearecommonbecausehypercalcemialeadstohypercalciuria,andincreasedPiclearancecausesphosphaturia.

1	composedofcalciumphosphatearecommonbecausehypercalcemialeadstohypercalciuria,andincreasedPiclearancecausesphosphaturia. Fortunately,routinebloodchemistryscreeningoverthepastseveraldecadeshasresultedinearlierdetectionofprimaryhyperparathyroidism,precludingdevelopmentofseveresymptomsinmostcases.

1	1. Serum [Ca++] is determined by the rate of Ca 2. Serum [Pi] is determined by the rate of Pi absorption absorption by the gastrointestinal tract, bone by the gastrointestinal tract, soft tissue influx and formation and resorption, and renal excretion. Serum efflux, bone formation and resorption, and renal [Ca++] is normally maintained within a very narrow excretion. Serum [Pi] normally fluctuates over a range. relatively wider range. 3. The major physiological hormones regulating serum [Ca++] and [Pi] are PTH, 1,25-dihydroxyvitamin D (calcitriol), and FGF23. 4. Vitamin D is synthesized from 7-dehydrocholesterol in skin in the presence of UVB light or acquired in the diet. It is hydroxylated to 25-hydroxycholecalciferol in the liver and activated by renal 1α-hydroxylase to 1,25-dihydroxyvitamin D. 5. 1,25-Dihydroxyvitamin D promotes intestinal Ca++ absorption and modestly increases Pi absorption. 6.

1	5. 1,25-Dihydroxyvitamin D promotes intestinal Ca++ absorption and modestly increases Pi absorption. 6. The flux of Ca++ and Pi into and out of bone is determined by the relative rates of osteoblastic Bhattacharyya N, et al. Fibroblast growth factor 23: state of the field and future directions. Trends Endocrinol Metab. 2012;23:610-618. Boyce BF. Advances in the regulation of osteoclasts and osteoclast functions. J Dent Res. 2013;92:860-867. Christakos S, et al. Vitamin D: metabolism, molecular mechanism of action, and pleiotropic effects. Physiol Rev. 2016;96:365-408. bone formation and osteoclastic bone resorption. 7. The PTH/PTHrP receptor is expressed on osteoblasts, not on osteoclasts. PTH has both anabolic and catabolic actions in bone depending on the dose and timing of administration. PTH promotes bone resorption by upregulation of M-CSF and RANKL in osteoblasts. 8.

1	8. 1,25-Dihydroxyvitamin D binds to the VDR in osteoblasts to support osteoclast differentiation via RANKL and promotes bone mineralization by maintaining appropriate serum [Ca++] and [Pi]. Upon completion of this chapter the student should be able to answer the following questions: 1. Describe the structure and composition of the pituitary gland and its structural and functional relationship to magnocellular and parvicellular hypothalamic neurons. 2. Discuss the mechanisms by which the neurohormones antidiuretic hormone (ADH) and oxytocin are synthesized, transported, and released by magnocellular neurons. 3. Diagram a basic scheme illustrating the components and feedback loops of a typical endocrine axis, including central input, hypothalamic releasing factors, pituitary hormones, and a peripheral endocrine gland. Explain the concept of a set point. 4.

1	4. List the endocrine cell types of the adenohypophysis and the tropic hormones they produce, noting hormones that share a common subunit. 5. Contrast the axes of somatotropes and lactotropes with the classic endocrine axes and explain how they differ. 6. Discuss the actions of growth hormone (GH) and insulinlike growth factor I (IGF-I) inthe regulation of growth, and the role of growth hormone in the fasted state. 7. Describe the role of prolactin in the initiation and maintenance of lactation. he pituitary gland (also called the hypophysis) is a small (≈0.5 g in weight) yet complex endocrine structure at the base of the forebrain (

1	he pituitary gland (also called the hypophysis) is a small (≈0.5 g in weight) yet complex endocrine structure at the base of the forebrain ( Fig. 41.1 ). It is composed of an epithelial component called the adenohypophysis and a neural structure called the neurohypophysis. The adenohypophysis is composed of five cell types that secrete six hormones. The neurohypophysis releases several neurohormones. All endocrine functions of the pituitary gland are regulated by the hypothalamus and by negativeand positive-feedback loops. Microscopic examination of the pituitary reveals two distinct types of tissue: epithelial and neural (

1	Microscopic examination of the pituitary reveals two distinct types of tissue: epithelial and neural ( Fig. 41.2 ). The epithelial portion of the human pituitary gland is called the adenohypophysis. The adenohypophysis makes up the anterior portion of the pituitary and is often referred to as the anterior lobe of the pituitary, and its hormones are referred to as anterior pituitary hormones. The adenohypophysis is composed of three parts: (1) the pars distalis, which makes up about 90% of the adenohypophysis, (2) the pars tuberalis, which wraps around the stalk, and (3) the pars intermedia, which regresses and is absent in adult humans.

1	The neural portion of the pituitary is called the neurohypophysis and represents a downgrowth of the hypothalamus. The most inferior portion of the neurohypophysis is called the pars nervosa, also called the posterior lobe of the pituitary (or simply posterior pituitary). At the superior end of the neurohypophysis, a funnel-shaped swelling called the median eminence develops. The portion of the neurohypophysis that extends from the median eminence down to the pars nervosa is called the infundibulum. The infundibulum and the pars tuberalis make up the pituitary stalk—a physical connection between the hypothalamus and pituitary gland (see Fig. 41.2).

1	Fig. 41.2). The pituitary gland (anterior and posterior lobes) is situated within a depression of the sphenoid bone called the sella turcica. Generally, cancers of the pituitary have only one way to expand, which is up into the brain and against the optic chiasma. Thus any increase in size of the pituitary is commonly associated with visual field or visual acuity abnormalities and headaches. The sella turcica is sealed off from the brain by a membrane called the diaphragma sellae. The Neurohypophysis

1	The Neurohypophysis The pars nervosa is a neurovascular structure that is the site of neurohormone release adjacent to a rich capillary bed. The peptide hormones that are released are antidiuretic hormone (ADH, or arginine vasopressin) and oxytocin. The cell bodies of the neurons that project to the pars nervosa are located in the supraoptic nuclei (SON) and paraventricular nuclei (PVN) of the hypothalamus (a nucleus refers to a collection of neuronal cell bodies residing within the central nervous system (CNS); a ganglion is a collection of neuronal cell bodies residing outside the CNS). The large cell bodies of these neurons are described as magnocellular, and they project axons down the infundibular stalk as the hypothalamohypophyseal tracts. Individual magnocellular neurons are hormone specific, producing •Fig. 41.1 Cross-sectionalimageoftheheaddemonstratingtheproximityofthehypothalamusandpituitaryglandandtheirconnectionbyaneurohypophyseal(pituitary)stalk.

1	either ADH or oxytocin. These axons terminate in the pars nervosa ( Fig. 41.3 ). In addition to axonal processes and termini from the SON and PVN, there are glial-like support cells called pituicytes. The posterior pituitary is extensively vascularized and the capillaries are fenestrated, thereby facilitating diffusion of hormones into the systemic circulation. Synthesis of ADH and Oxytocin ADH and oxytocin are small peptides (nine amino acids) that differ in only two amino acids, yet they have limited overlapping activity. ADH and oxytocin are synthesized as preprohormones (

1	ADH and oxytocin are small peptides (nine amino acids) that differ in only two amino acids, yet they have limited overlapping activity. ADH and oxytocin are synthesized as preprohormones ( Fig. 41.4 ). Each prohormone harbors the structure of oxytocin or ADH and a co-secreted peptide, either neurophysin I (associated with ADH) or neurophysin II (associated with oxytocin). These preprohormones are called preprovasophysin and preprooxyphysin. The N-terminal signal peptide is cleaved as the peptide is transported into the endoplasmic reticulum. In cell bodies within the SON and PVN, the prohormones are packaged in the endoplasmic reticulum and Golgi apparatus in membrane-bound secretory granules (

1	Fig. 41.5 ). The secretory granules are conveyed through a “fast” (i.e., millimeters per hour) adenosine triphosphate (ATP)-dependent axonal transport mechanism down the infundibular stalk to axonal termini in the pars nervosa. During transit of the secretory granule, the prohormones are proteolytically cleaved to produce equimolar amounts of hormone and neurophysin. Secretory granules containing fully processed peptides are stored in the axonal termini. Expansions of the termini due to the presence of stored secretory granules can be observed by light microscopy and are termed Herring bodies.

1	ADH and oxytocin are released from the pars nervosa in response to stimuli that are primarily detected at the cell body and its dendrites in the SON and PVN. These stimuli are mainly in the form of neurotransmitters released from hypothalamic interneurons. With sufficient stimulus the neurons will depolarize and propagate an action potential down the axon. At the axonal termini the action potential increases intracellular [Ca++] and results in a stimulus-secretion response, with exocytosis of ADH or oxytocin along with neurophysins into the extracellular fluid of the pars nervosa (see Fig. 41.5). Hormones and neurophysins enter the peripheral circulation, and both can be measured in blood. Actions and Regulation of ADH and Oxytocin

1	Fig. 41.5). Hormones and neurophysins enter the peripheral circulation, and both can be measured in blood. Actions and Regulation of ADH and Oxytocin ADH acts primarily at the kidney to retain water (antidiuresis). The actions of ADH and regulation of ADH secretion were described in . Oxytocin primarily acts on the pregnant uterus to induce labor and on myoepithelial cells of the breast to promote milk letdown during nursing. The actions and regulation of oxytocin are discussed in The Adenohypophysis The pars distalis is composed of five endocrine cell types that produce six hormones ( Table 41.1 ). Because of the histological staining properties of the cell types, the corticotropes, thyrotropes, and gonadotropes are referred to as pituitary basophils, whereas the somatotropes and lactotropes are referred to as pituitary acidophils (see Fig. 41.2B

1	Before discussing the individual hormones of the adenohypophysis, it is important to understand the structural •Fig. 41.2 A,Grossstructureofthepituitarygland.Thepituitaryglandisbelowthehypothalamusandisconnectedtoitbythepituitarystalk.Theglandsitswithinthesellaturcica,afossawithinthesphenoidbone,andiscoveredbyaduralreflection,thediaphragmasellae.Theparsdistalismakesupmostoftheanteriorpituitary.B,Theparsdistalisisderivedfromepithelialtissuethatiscomposedofacidophils (A)(somatotropesandlactotropes)andbasophils(B)(thyrotropes,gonadotropes,andcorticotropes).C,Theposteriorpituitaryisderivedfromneuraltissueandhasahistologicalappearanceofnonmyelinatednerves.Cp,chromophobes;H,Herringbodies.(A,ModifiedfromStevensA.In:LoweJS[ed].Human Histology. 3rded.Philadelphia:Elsevier;2005.BandC,FromYoungBetal[eds].Wheater’s Functional Histology. 5thed.Philadelphia:ChurchillLivingstone;2006.) •Fig. 41.3

1	Histology. 3rded.Philadelphia:Elsevier;2005.BandC,FromYoungBetal[eds].Wheater’s Functional Histology. 5thed.Philadelphia:ChurchillLivingstone;2006.) •Fig. 41.3 Magnocellularneuronsofthehypothalamus(paraven-tricularandsupraopticnuclei)projecttheiraxonsdowntheinfundibularprocessandterminateintheparsnervosa(posteriorlobe),wheretheyreleasetheirhormones(eitherADHoroxytocin)intoacapillarybed.(ModifiedfromLarsenPRetal[eds].Williams Textbook of Endocrinol-ogy. 10thed.Philadelphia:Saunders;2003.)Capilary bed Anterior lobe of pituitary Optic chiasm Hypothalamus Posterior lobe of pituitary Magnocellular neurosecretory cells

1	Becauseposteriorpituitaryhormonesaresynthesizedinthehypothalamusratherthanthepituitary,hypophysectomy (pituitaryremoval)doesnotnecessarilypermanentlydisruptsynthesisandsecretionofthesehormones.Immediatelyafterhypophysectomy,secretionofthehormonesdecreases.However,overaperiodofweekstheseveredproximalendofthetractwillshowhistologicalmodificationandpituicyteswillformaroundtheneuronterminals.Secretoryvacuolesareseen,andsecretionofhormoneresumesfromthisproximalend.Secretionofhormonecanevenpotentiallyreturntonormallevels.Incontrast,alesionhigheruponthepituitarystalkcanleadtolossofneuronalcellbodiesinthePVNandSON. and functional organization of the adenohypophysis in the context of the endocrine axes (

1	and functional organization of the adenohypophysis in the context of the endocrine axes ( Fig. 41.6 41.1 ). Each endocrine axis is composed of three levels of endocrine cells: (1) hypothalamic neurons, (2) anterior pituitary cells, and (3) peripheral endocrine glands. Hypothalamic neurons release specific hypothalamic releasing hormones (designated XRH in this generic scheme) that stimulate secretion of specific pituitary tropic hormones (XTH). In some cases, production of a pituitary tropic hormone is secondarily regulated by a release-inhibiting hormone (XIH). Pituitary tropic hormones then act on specific peripheral target endocrine glands and stimulate them to release peripheral hormones (X). The peripheral hormone X has two general functions: it regulates several aspects of human physiology, and it negatively feeds back on the pituitary gland and hypothalamus to inhibit production and secretion of tropic hormones and releasing hormones, respectively (see Fig. 41.6

1	Significantprogresshasbeenmadeinunderstandingthedifferentiationofthefiveendocrinecellsoftheparsdistalisfromoneprecursorcell.ThehomeodomaintranscriptionfactorPROP-1 isexpressedsoonafterRathke’spouch(embryologicalprecursoroftheadenohypophysis)formsandpromotesthecelllineagesofsomatotropes,lactotropes,thyrotropes,andgonadotropes.Inhumans,raremutationsinthePROP1 generesultinatypeofcombined pituitary hormone deficiency. TheseindividualsdisplaydwarfismduetolackofGH,cognitivedeficitssecondarytohypothyroidism,andinfertilityduetolackofgonadotropins.Asubsequentlyexpressedpituitary-specifichomeodomaintranscriptionfactorcalledPOU1F1 (formerlyknownasPit-1)isrequiredfordifferentiationofthyrotropes,somatotropes,andlactotropes,anditdirectlystimulatestranscriptionandexpressionofTSH,GH,andprolactin.AffectedindividualswithPOU1F1 mutationshavedwarfismandintellectualdisability.Thenuclearhormonereceptor–relatedtranscriptionfactorsteroidogenic factor-1 (SF-1)

1	mutationshavedwarfismandintellectualdisability.Thenuclearhormonereceptor–relatedtranscriptionfactorsteroidogenic factor-1 (SF-1) wasoriginallyidentifiedintheadrenalcortexandgonadsasaregulatorofsteroidogenicenzymegeneexpression.SF-1isalsoexpressedinGnRHneuronsinthehypothalamusandinpituitarygonadotropes,whereitregulatestranscriptionofLHandFSH.MutationsintheSF1 genedisruptadrenalandgonadalfunction,includinglossofgonadotropesinthepituitarygland.TPIT isatranscriptionfactorinvolvedinthedifferentiationofcorticotropes.TPIT,actingwithothertranscriptionfactors,promotesdifferentiationofcorticotropesandexpressionofthePOMC gene(seeCorticotropessection).MutationsinthehumanTPIT generesultinisolated ACTH deficiency. Thisresultsinaformofsecondary adrenal insufficiency thatrequireslifelongreplacementwithglucocorticoids(see

1	The hypothalamic regulation of anterior pituitary function is neurohormonal. An area of the hypothalamus collectively referred to as the hypophysiotropic (i.e., stimulatory to the hypophysis) region contains nuclei composed of small, or parvocellular, cell bodies that project axons to the Transcription, excision, splicing •Fig. 41.4 Synthesisandprocessingofpreprovasopressinorpreprooxytocin. median eminence. They are distinct from the magnocellular neurons of the PVN and SON that project to the pars nervosa. Parvocellular neurons secrete releasing hormones from their axonal termini at the median eminence (Fig.

1	41.7 ). The releasing hormones enter a primary plexus of fenestrated capillaries and are then conveyed to a second capillary plexus located in the pars distalis by the hypothalamohypophyseal portal vessels (a portal vessel is defined as a vessel that begins and ends in capillaries without going through the heart). At the secondary capillary plexus, the releasing hormones diffuse out of the vasculature and bind to their cognate receptors on specific cell types within the pars distalis. The neurovascular link (i.e., pituitary stalk) between the hypothalamus and pituitary is somewhat fragile and can be disrupted by physical trauma, surgery, or hypothalamic disease. Damage to the stalk and subsequent functional isolation of the anterior pituitary result in a decline in all anterior pituitary tropic hormones except prolactin (discussed later).

1	The cells of the adenohypophysis make up the intermediate level of the endocrine axes. The adenohypophysis secretes protein hormones that are referred to as tropic hormones—adrenocorticotropic hormone (ACTH, also called corticotropin), thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), growth hormone (GH), and prolactin (PRL) (see Table 41.1). With a few exceptions, tropic hormones bind to their cognate receptors on peripheral endocrine glands. Because of this arrangement, pituitary tropic hormones generally do not directly regulate physiological responses (see Chapter 38). The endocrine axes have the following important features: 1.

1	Chapter 38). The endocrine axes have the following important features: 1. The activity of a specific axis is normally maintained at a set point, which varies from individual to individual, usually within a normal range. The set point is determined by the integration of hypothalamic stimulation and peripheral hormone negative feedback. Importantly, negative feedback generally is not exerted by the physiological responses regulated by a specific endocrine axis but by the peripheral hormone itself acting on the pituitary and hypothalamus (see

1	Fig. 41.6 ). Thus if the level of a peripheral hormone drops, secretion of hypothalamic releasing hormones and pituitary tropic hormones will increase. As the level of peripheral hormone rises, the hypothalamus and pituitary will decrease secretion because of negative feedback. Although certain nonendocrine physiological parameters (e.g., acute hypoglycemia) can regulate some endocrine axes, the axes function semiautonomously with respect to the physiological changes they produce. This configuration means a peripheral hormone (e.g., thyroid hormone) can regulate multiple organ systems without these organ systems exerting competing negative-feedback regulation of the hormone. Clinically this partial autonomy means that multiple aspects of a patient’s physiology are at the mercy of whatever derangements might exist within a specific axis. 2.

1	Hypothalamic hypophysiotropic neurons are often secreted in a pulsatile manner and are entrained to daily and seasonal rhythms through CNS input. Additionally, hypothalamic nuclei receive a variety of neuronal inputs from higher and lower levels of the brain. These can be • Fig. 41.5 Synthesis, processing, and transport of preprovasopressin. Human ADH (also called arginine vasopressin [AVP]) is synthesized in the hypothalamic magnocellular cell bodies and packaged into neurosecretory granules. During intraaxonal transport of the granules down the infundibular process to the pars nervosa, provasopressin is proteolytically cleaved into the active hormone (AVP = ADH), neurophysin (NP), and a C-terminal glycoprotein (GP). NP arranges into tetramers that bind five AVP molecules. All three fragments are secreted from axonal termini in the pars nervosa (posterior pituitary) and enter the systemic blood. Only AVP (ADH) is biologically active. (Modified from Larsen PR et al [eds]. Williams

1	Textbook of Endocrinology. 10th ed. Philadelphia: Saunders; 2003.)

1	X Primary Endocrine Disease (Thyroid, adrenal cortex, gonads, liver) •Fig. 41.6 Negative-feedbackloopsregulatinghormonesecretioninatypicalhypothalamus-pituitaryperipheralglandaxis.X,peripheralglandhormone;XIH,hypothalamic-inhibitinghormone;XRH,hypothalamic-releasinghormone;XTH,pituitarytropichormone. short-term (e.g., various stress/infections) or long-term (e.g., onset of reproductive function at puberty) inputs. Thus inclusion of the hypothalamus in an endocrine axis allows integration of a considerable amount of information for setting or changing the set point of that axis. Clinically this means a broad range of complex neurogenic states can alter pituitary function. Psychosocial dwarfism is a striking example in which children subject to abuse or intense emotional stress have lower growth rates as a result of decreased growth hormone secretion by the pituitary gland.

1	3. Abnormally low or high levels of a peripheral hormone (e.g., thyroid hormone) may be due to a defect at the level of the peripheral endocrine gland (e.g., thyroid), the pituitary gland, or the hypothalamus. Such lesions are referred to as primary, secondary, and tertiary endocrine disorders, respectively (see Fig. 41.6 ). A thorough understanding of the feedback relationships within an axis allows the physician to determine where the defect lies. Primary endocrine deficiencies tend to be the most severe because they often involve complete absence of the peripheral hormone. Endocrine Function of the Adenohypophysis The adenohypophysis consists of the following endocrine cell types: corticotropes, thyrotropes, gonadotropes, somatotropes, and lactotropes (see Table 41.1

1	The adenohypophysis consists of the following endocrine cell types: corticotropes, thyrotropes, gonadotropes, somatotropes, and lactotropes (see Table 41.1 Corticotropes stimulate the adrenal cortex as part of the hypothalamic-pituitary-adrenal (HPA) axis. Corticotropes produce the hormone ACTH (corticotropin), which stimulates two zones of the adrenal cortex (see ). ACTH is a 39–amino acid peptide that is synthesized as part of a larger prohormone called proopiomelanocortin (POMC). Thus corticotropes are also referred to as POMC cells. POMC harbors the peptide sequence for ACTH, two isoforms of melanocyte-stimulating hormone (MSH), endorphins (endogenous opioids), and enkephalins ( Fig.

1	Fig. 41.8 ). However, the human corticotrope expresses only pro-hormone convertase-1, which produces ACTH as the sole active hormone secreted by these cells. The other fragments that are cleaved from POMC are the N-terminal fragment and β-lipotropic hormone (β-LPH), neither of which plays a physiological role in humans.

1	740 SECTION8Berne & Levy Physiology •Fig. 41.7 Neurovascularlinkbetweenthehypothalamusandtheanteriorlobe(parsdistalis)ofthepitu-itary.Parvicellular“hypophysiotropic”neurosecretoryneuronswithinvarioushypothalamicnucleiprojectaxonstothemedianeminence,wheretheysecretereleasinghormones(RHs).RHsflowdownthepituitarystalkinthehypothalamohypophysealportalvesselstotheanteriorpituitary.RHs(andrelease-inhibitinghormones[seetext])regulatesecretionoftropichormonesfromthefivecelltypesoftheanteriorpituitary.(FromLarsenPRetal[eds].Williams Textbook of Endocrinology. 10thed.Philadelphia:Saunders;2003.)Hormone-secreting cells Anterior lobe of pituitary Capillary beds Hypothalamohypophyseal portal vessels Hypothalamus Median eminence Parvocellular neurosecretory cells Hypophysiotropic hormones released Hormone transport in axons Hormone transport in blood Hormone transport in blood Stimulation or inhibition of anterior pituitary hormone release Action on endocrine glands

1	ACTH circulates as an unbound hormone and has a short half-life of about 10 minutes. It binds to the melanocortin-2 receptor (MC2R) on cells in the adrenal cortex ( Fig. 41.9 ). ACTH acutely increases cortisol and adrenal androgen production by increasing expression of steroidogenic enzyme genes. In the long term, ACTH promotes growth and survival of two zones within the adrenal cortex (see ACTH is under stimulatory control by the hypothalamus. A subset of parvocellular hypothalamic neurons expresses the peptide procorticotropin-releasing hormone (pro-CRH) (see Table 41.1). Pro-CRH is processed to CRH, an amidated 41–amino acid peptide. CRH acutely stimulates ACTH secretion and increases transcription of the POMC gene. The parvocellular neurons that express CRH also express ADH, which potentiates the action of CRH on corticotropes. ACTH secretion has a pronounced diurnal pattern, with a peak in early morning and a nadir

1	Atsupraphysiologicallevels,ACTH causesdarkeningoftheskin(e.g.,inCushing’sdisease).KeratinocytesinthebasallayeroftheepidermisalsoexpressthePOMCgenebutprocessittoα-MSH insteadofACTH.Keratinocytessecreteα-MSHinresponsetoultravioletlight,andα-MSHactsasaparacrinefactoronneighboringmelanocytestodarkentheskin.α-MSHbindstotheMC1R onmelanocytes.Atveryhighlevels,ACTHcancross-reactwiththeMC1Rreceptoronskinmelanocytes(see Fig.41.9 ).ThusincreasedskinpigmentationisoneindicatorofexcesscirculatingACTH. Fig. 41.10). In addition, secretion of CRH—and hence secretion of ACTH—is pulsatile. There are multiple regulators of the HPA axis, and many of them are mediated through the CNS ( Fig. 41.11). Many types of stress, both neurogenic (e.g., fear) and systemic TABLE 41.1

1	There are multiple regulators of the HPA axis, and many of them are mediated through the CNS ( Fig. 41.11). Many types of stress, both neurogenic (e.g., fear) and systemic TABLE 41.1 Adenohypophysis Cell Types: Hormonal Production and Action, Hypothalamic Regulation, and Feedback Regulation •Fig. 41.8 Theoriginalgenetranscriptofproopiomelanocortincontainsstructuresofmultiplebioactivecompounds.ACTH,adrenocorticotropichormone;CLIP,corticotropin-likeintermediatepeptide;MSH,melanocyte-stimulatinghormone.NotethatACTHistheonlybioactivepeptidereleasedbythehumancorticotrope.

1	•Fig. 41.9 NormallevelsofACTHactontheMC2Rtoincreasecortisol.SupraphysiologicallevelsofACTHduetodecreasedcortisolproductionactonboththeMC2RandtheMC1Ronmelanocytesandcauseskindarkening.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.)UV light Keratinocytes in skin Melanocytes in skin PKA Pituitary corticotrope POMC expression ACTH secretion ACTH (physiological levels) Cortisol Diverse physiological effects ACTH (supraphysiological levels) In particular ° Blood glucose ˛ Inflammatory response High affinity binding to MC2R Low affinity binding to MC1R • Loss of feedback in Addison’s disease Physiological, high affinity PKA Steroidogenesis Cell growth ˝-MSH ° POMC expression Skin darkening Release of CRH at median eminence CRH neurons Cortisol (−) Cortisol (−) CRH CRH-R1 Adrenal cortex PKA ° Melanin synthesis and dispersal (e.g., infection), stimulate ACTH. The stress effects are mediated through CRH and ADH via the CNS. The response to many forms of

1	(−) CRH CRH-R1 Adrenal cortex PKA ° Melanin synthesis and dispersal (e.g., infection), stimulate ACTH. The stress effects are mediated through CRH and ADH via the CNS. The response to many forms of severe stress can persist despite negative feedback from high cortisol levels. This means the hypothalamus has the ability to alter the set point of the HPA axis in response to stress. Severe chronic depression can reset the HPA axis as a result of hypersecretion of CRH and is a factor in the development of tertiary hypercortisolism.

1	Cortisol exerts negative feedback on the pituitary, where it suppresses POMC gene expression and ACTH secretion, and on the hypothalamus, where it decreases pro-CRH gene expression and release of CRH. Because cortisol has profound effects on the immune system (see ), the HPA axis and the immune system are closely coupled. Moreover, cytokines—particularly interleukin (IL)-1, IL-2, and IL-6—stimulate the HPA axis. Thyrotropes regulate thyroid function by secreting the hormone TSH (thyrotropin) as part of the hypothalamic-pituitary-thyroid axis. TSH is one of three pituitary glycoprotein hormones (see Table 41.1) that also include FSH and LH (discussed later). TSH is a heterodimer composed of an α subunit, called the α-glycoprotein subunit (α-GSU), and a β subunit (β-TSH) (

1	Table 41.1) that also include FSH and LH (discussed later). TSH is a heterodimer composed of an α subunit, called the α-glycoprotein subunit (α-GSU), and a β subunit (β-TSH) ( Fig. 41.12 ). The α-GSU is common to TSH, FSH, and LH, whereas the β subunit is hormone specific (i.e., β-TSH, β-FSH, and β-LH are all unique). Glycosylation of the subunits increases their stability in circulation and enhances the affinity and specificity of the hormones for their receptors. The half-lives of TSH, FSH, and LH (and an LH-like placental glycoprotein hormone, human chorionic gonadotropin [hCG]) are relatively long, ranging from tens of minutes to several hours. TSH binds to the TSH receptor on thyroid follicle cells (see Chapter 42). As discussed in

1	TSH binds to the TSH receptor on thyroid follicle cells (see Chapter 42). As discussed in Chapter 42, production of thyroid hormones is a complex multistep process, and TSH stimulates essentially every aspect of thyroid function. TSH also has a strong tropic effect and stimulates hypertrophy, hyperplasia, and survival of thyroid epithelial cells. In geographical regions where the availability of iodide is limited (iodide is required for the synthesis of thyroid hormone), TSH levels are elevated because of reduced negative feedback. Elevated TSH levels can produce striking growth of the thyroid, producing a bulge in the neck called a goiter. •Fig. 41.10 DiurnalpatternofserumACTH.(ModifiedfromPor-terfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.)Awake Sleep Serum ACTH level The pituitary thyrotrope is stimulated by the releasing hormone thyrotropin-releasing hormone (TRH) (see

1	The pituitary thyrotrope is stimulated by the releasing hormone thyrotropin-releasing hormone (TRH) (see Table 41.1). TRH, produced by a subset of parvocellular •Fig. 41.11 Hypothalamic-pituitary-adrenalaxisillustratingfactorsregulatingsecretionofcorticotropin-releasinghormone(CRH).ACTH,adrenocorticotropichormone.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.)CRH Physical Diurnal rhythms Emotional Others Set point ACTH Corticotroph Long feedback (cortisol) Short feedback (ACTH) Chemical (hypoglycemia) •Fig. 41.12 Pituitaryglycoproteinhormones.hCGismadebytheplacenta(see )andbindstotheLHreceptor.FSH,follicle-stimulatinghormone;hCG,humanchorionicgonadotropin;LH,luteinizinghormone;TSH,thyroid-stimulatinghormone.˜-TSH subunit ˜-FSH subunit ˜-LH subunit ˜-hCG subunit TSH FSH LH hCG TSH receptor FSH receptor LH receptor LH receptor Plus °-glycoprotein subunit (°-GSU)

1	Ca++ and PKC TSH synthesis TSH secretion TSH PKA • All aspects of thyroid hormone synthesis and secretion • Cell growth CNS inputs Diurnal rhythm T3 (−) T4, T 3 T3 (−) TRH TRH receptor TSH receptor Pituitary thyrotrope Thyroid epithelial cell •Fig. 41.13 Hypothalamic-pituitary-thyroidaxis.PKA,proteinkinaseA;PKC,proteinkinaseC;T3,triiodothyronine(activeformofthyroidhormone);T4,thyroxine;TRH,thyrotropin-releasinghormone;TSH,thyroid-stimulatinghormone.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.) hypothalamic neurons, is a tripeptide with cyclization of a glutamine at its N-terminus (pyro-Glu) and an amidated C-terminus. TRH is synthesized as a larger prohormone that contains six copies of TRH within its sequence. It binds to the TRH receptor on thyrotropes (Fig. 41.13 ). TRH neurons are regulated by numerous CNS-mediated stimuli, and TRH is released according to a diurnal rhythm (highest during overnight hours, lowest around dinner time). TRH

1	(Fig. 41.13 ). TRH neurons are regulated by numerous CNS-mediated stimuli, and TRH is released according to a diurnal rhythm (highest during overnight hours, lowest around dinner time). TRH secretion is also regulated by stress, but in contrast to CRH, stress inhibits secretion of TRH. This includes physical stress, starvation, and infection. Triiodothyronine (T3) and thyroxine (T4) (the latter via type 2 deiodinase-mediated conversion to T3; see ) negatively feed back on both pituitary thyrotropes and TRH-producing neurons. Thyroid hormone represses both β-TSH expression and the sensitivity of pituitary thyrotropes to TRH while also inhibiting TRH production and secretion by parvocellular neurons.

1	Duringembryonicdevelopment,GnRHneuronsmigratetothemediobasalhypothalamusfromthenasalplacode.PatientswithKallmann syndrome havetertiary hypogonadotropic hypogonadism, oftenassociatedwithlossofthesenseofsmell(anosmia).ThisisduetoamutationintheKAL gene, whichresultsinfailureoftheGnRHneuronalprecursorstoproperlymigratetothehypothalamusandestablishaneurovascularlinktotheparsdistalis. The Gonadotrope The gonadotrope secretes FSH and LH (collectively called gonadotropins) and regulates the function of gonads in both sexes. As such the gonadotrope plays an integral role in the hypothalamic-pituitary-testis axis and the hypothalamic-pituitary-ovarian axis ( Fig. 41.14

1	Fig. 41.14 FSH and LH are segregated into different secretory granules and are not co-secreted in equimolar amounts (in contrast to ADH and neurophysin, for example). This allows independent regulation and secretion of FSH/LH by gonadotropes. The actions of FSH and LH on gonadal function are complex, especially in women, and will be discussed in detail in . In general, gonadotropins promote testosterone secretion in men and estrogen and progesterone secretion in women. FSH also increases secretion of a transforming growth factor (TGF)-β–related protein hormone called inhibin in both sexes. FSH and LH secretion are regulated by one hypothalamic releasing hormone, gonadotropin-releasing hormone (GnRH; formerly called LHRH). GnRH is a 10–amino acid peptide produced by a subset of parvocellular hypothalamic GnRH neurons (see

1	Fig. 41.14 ). GnRH is produced as a larger prohormone and, as part of its processing to a decapeptide, is modified with a cyclized glutamine (pyro-Glu) at its N-terminus and an amidated C-terminus. GnRH is released in a pulsatile manner ( Fig. 41.15 ), and both the pulsatile secretion and the frequency of the pulses have important effects on the gonadotrope. Continuous infusion of GnRH downregulates the GnRH receptor, thereby resulting in a decrease in FSH and LH secretion. In contrast, pulsatile secretion does not desensitize the gonadotrope to GnRH, and FSH and LH secretion is normal. At a frequency of one pulse per hour, GnRH preferentially increases LH secretion ( Fig. 41.16 ). At a slower frequency of one pulse per 3 hours, GnRH preferentially increases FSH secretion. Gonadotropins increase sex steroid synthesis (see Fig. 41.14 ). In men, testosterone and estrogen negatively feed back at the level of the pituitary and the hypothalamus.

1	Fig. 41.14 ). In men, testosterone and estrogen negatively feed back at the level of the pituitary and the hypothalamus. Ca++, PKC, other pathways? SlowFast GnRH (+) pulses GnRH pulses LH synthesis and secretion FSH synthesis and secretion FSHLH PKA Inhibin Pulsatile release of GnRH at median eminence PubertyCNS Opioids Stress Prolactin (−)Sex steroids GnRH neurons inputs (+) (−) (−) GnRH Sex steroids GnRH receptor FSH receptorLH receptor Pituitary gonadotrope Gonadal cell types Steroidogenesis Gametogenesis •Fig. 41.14 Hypothalamic-pituitary-gonadalaxis.FSH,follicle-stimulatinghormone;GnRH,gonadotropin-releasinghormone;LH,luteinizinghormone.(ModifiedfromPorterfieldSP,WhiteBA.Endocrine Physiology. 3rded.Philadelphia:Mosby;2007.)

1	Physiology. 3rded.Philadelphia:Mosby;2007.) Exogenous progesterone also inhibits gonadotropin function in men and has been considered as a possible component of a male contraceptive pill. Additionally, inhibin negatively feeds back selectively on FSH secretion in men and women. In women, progesterone and testosterone negatively feed back on gonadotropic function at the level of the hypothalamus and pituitary. At low doses, estrogen also exerts negative feedback on FSH and LH secretion. However, high estrogen levels maintained for 3 days cause a surge in LH and to a lesser extent FSH secretion. This positive feedback, which is critical in promoting ovulation, is observed at the hypothalamus and pituitary. At the hypothalamus, GnRH pulse amplitude and frequency increase. At the pituitary, high estrogen levels greatly increase the sensitivity of the gonadotrope to GnRH, both by increasing GnRH receptor levels and by enhancing postreceptor signaling (see The Somatotrope

1	The Somatotrope The somatotrope produces GH (somatotropin) and is part of the hypothalamic-pituitary-liver axis ( Fig. 41.17 ). A major target of GH is the liver, where it stimulates production of insulinlike growth factor (IGF)-I. GH is a 191–amino acid protein that is similar to PRL and human placental lactogen (hPL); accordingly there is some overlap in activity among these hormones. Multiple forms of GH 746 SECTION8Berne & Levy Physiology 1.8 1.2 0.6 0 12 10 8 6 1200 1300 1400 1500 1600 Hours GnRH(pg/10 min) LH(ng/mL) Fig. 41.15 FluctuationofperipheralveinplasmaLHlevelsandportalveinplasmaGnRHlevelsinunanesthetized,ovariectomizedfemalesheep.EachpulseofLHiscoordinatedwithapulseofGnRH.ThissupportstheviewthatpulsatilityofLHreleaseisdependentonpulsatilestimulationofthepituitarybyGnRH.(FromLevineJetal.Endocrinology 1982;111:1449.)

1	Fig. 41.16 Frequency-encodedregulationofFSHandLHsecretionfromgonadotropes.AhighfrequencyofGnRH(1pulse/hr)preferentiallystimulatesLHsecretion,whereasaslowerfrequencyofGnRHpromotesFSHsecretion.(FromLarsenPRetal[eds].Williams Textbook of Endocrinology. 10th ed.Philadelphia:Saunders;2003.) are present in serum, with the 191–amino acid (22-kDa) form representing approximately 75% of circulating GH. The GH receptor is a member of the cytokine/GH/PRL/ erythropoietin receptor family and as such is linked to the JAK/STAT signaling pathway (see ). Human GH can also act as an agonist of the PRL receptor. About 50% of the 22-kDa form of GH in serum is bound to GH-binding protein (GHBP), which is derived from the N-terminal portion (the extracellular domain) of the GH receptor. Individuals with Laron syndrome, who lack normal GH receptors but have normal GH secretion, do not have detectable GHBP in their serum. GHBP reduces renal clearance and thus increases the biological half-life of GH, which

1	syndrome, who lack normal GH receptors but have normal GH secretion, do not have detectable GHBP in their serum. GHBP reduces renal clearance and thus increases the biological half-life of GH, which is about 20 minutes. The liver and kidney are major sites of GH degradation.

1	GH secretion is under dual positive/negative control by the hypothalamus (see Fig. 41.17 ). The hypothalamus predominantly stimulates GH secretion via the peptide growth •Fig. 41.17 Hypothalamic-pituitary-liveraxis.ALS,acidlabilesubunit;GHBP,growthhormone–bindingprotein;GHRH,growthhormone–releasinghormone;IGFBP,insulinlikegrowthfactor–bindingprotein;IGF-I,insulinlikegrowthfactorI;SS,somatostatin.(FromPorterfieldSP,WhiteBA.Endocrine Physiology.

1	3rded.Philadelphia:Mosby;2007.) hormone–releasing hormone (GHRH). This hormone is a member of the vasoactive intestinal polypeptide (VIP)/ secretin/glucagon family and is processed from a larger prohormone into a 44–amino acid peptide with an amidated C-terminus. GHRH enhances GH secretion and GH gene expression. The hypothalamus inhibits pituitary GH synthesis and release via the peptide somatostatin. In the anterior pituitary, somatostatin inhibits release of GH and TSH. GH secretion is also stimulated by ghrelin, which acts through the GH secretagogue receptor on somatotropes. Ghrelin is primarily produced by the stomach but is also expressed in the hypothalamus. Ghrelin increases appetite and may serve as a signal to coordinate nutrient acquisition with growth. The primary negative feedback on the somatotrope is exerted by IGF-I (see

1	The primary negative feedback on the somatotrope is exerted by IGF-I (see Fig. 41.17). GH stimulates IGF-I pro duction by the liver, and IGF-I then inhibits GH synthesis and secretion by the pituitary and hypothalamus in a classic “long feedback” loop. In addition, GH itself exerts negative feedback on release of GHRH through a “short feedback” loop. GH also increases somatostatin release.

1	GH secretion, like ACTH, shows prominent diurnal rhythms, with peak secretion occurring in the early morning just before awakening. Its secretion is stimulated during deep slow-wave sleep (stages III and IV). GH secretion is lowest during the day. This rhythm is entrained to sleep-wakefulness patterns rather than light-dark patterns, so a phase shift occurs in people who work night shifts. As is typical of anterior pituitary hormones, GH secretion is pulsatile. Levels of GH in serum vary widely (0–30 ng/ mL, with most values usually falling between 0 and 3). Because of this marked variation and the heterogeneity of circulating GH, measurement of serum GH levels is of limited clinical utility. Since IGF-I secretion is regulated by GH and possesses a longer half-life that buffers pulsatile and diurnal changes in GH secretion, it may be used to assess the status of the GH axis, especially in young patients.

1	GH secretion is differentially regulated depending on the physiological state. GH is classified as one of the “stress hormones” and is increased by neurogenic and physical stress. It promotes lipolysis, increases protein synthesis, and antagonizes the ability of insulin to reduce blood glucose levels. It is not surprising, therefore, that acute hypoglycemia is a stimulus for GH secretion and GH is classified as a hyperglycemic hormone. A rise in the serum concentration of some amino acids also stimulates GH secretion; administration of arginine is used for provocative testing of GH secretion. In contrast, an increase in blood glucose or free fatty acids inhibits secretion of GH. Obesity also inhibits GH secretion, in part because of insulin resistance (relative hyperglycemia) and increased circulating free fatty acids. Conversely, exercise and starvation stimulate GH secretion. The lifetime pattern of GH secretion is shown in Fig.

1	The lifetime pattern of GH secretion is shown in Fig. 41.18 . GH secretion increases in the neonatal period as growth becomes GH and IGF-I dependent. It remains high throughout childhood and peaks during puberty, when estrogen (in females and also males via aromatization) promotes even higher rates of GH secretion. Thyroid hormone also enhances GH and IGF-I secretion to support bone growth and maturation. Adults continue to produce GH, consistent with its role in metabolism, before levels fall during senescence.

1	IGFs are multifunctional hormones that regulate cellular proliferation, differentiation, and metabolism. These protein hormones resemble insulin in structure and function. The two hormones in this family, IGF-I and IGF-II, are produced in many tissues and have autocrine, paracrine, and endocrine actions. IGF-I is the major form produced in most adult tissues. IGF-II is the major form produced in the fetus, where it regulates growth of both the fetus and the placenta in a GH-independent manner. Both hormones are structurally similar to proinsulin, with IGF-I exhibiting 42% structural homology with proinsulin. IGFs and insulin show receptor cross-reactivity; IGFs in high concentration mimic the metabolic actions of insulin. Both IGF-I and IGF-II act through type I IGF receptors, which are similar to insulin and epidermal growth factor (EGF) receptors and contain intrinsic tyrosine kinase activity. However, IGF-II also binds to the type II IGF/mannose-6-phosphate receptor. This receptor

1	to insulin and epidermal growth factor (EGF) receptors and contain intrinsic tyrosine kinase activity. However, IGF-II also binds to the type II IGF/mannose-6-phosphate receptor. This receptor does not resemble the insulin receptor, does not have intrinsic tyrosine kinase activity, and probably functions to limit IGF-II signaling through the type I receptor. IGFs stimulate glucose and amino acid uptake and protein and DNA synthesis. They were initially called somatomedins because of their growth-mediating actions on cartilage, bone, and other organs. It was originally proposed that IGF-I is produced exclusively in the liver upon GH stimulation. During puberty, when GH levels increase (

1	Fig. 41.19 ), IGF-I levels increase in parallel. However, it is now known that IGFs are produced in many extrahepatic tissues, exhibiting both autocrine and paracrine actions. Some of these are under the control of GH, whereas others are not. In bone, for example, IGF-I has both endocrine and paracrine effects on linear growth, some of which are GH independent. Hormones such as parathyroid hormone (PTH) and estradiol are also effective stimuli for IGF-I production by osteoblasts. At the same time, GH exerts stimulatory effects on the growth plate that are independent of IGF-I. The liver appears to be the predominant source of the circulating pool of IGF-I (see Fig. 41.19 •Fig. 41.18 LifetimepatternofGHsecretion.GHlevelsarehigherinchildrenthaninadults,withapeakperiodduringpuberty.GHsecretiondeclineswithaging.

1	Fig. 41.19 •Fig. 41.18 LifetimepatternofGHsecretion.GHlevelsarehigherinchildrenthaninadults,withapeakperiodduringpuberty.GHsecretiondeclineswithaging. Essentially all circulating IGFs are transported in serum bound to IGF binding proteins (IGFBP). IGFBP-3 binds to IGF then associates with another protein called the acid labile subunit (ALS) (see Fig. 41.19 ). GH stimulates hepatic production of IGF-I, IGFBP-3, and ALS. The IGFBP-3/ALS/IGF-I complex mediates transport and bioavailability of IGF-I. Although IGFBPs generally inhibit IGF action, they greatly increase the biological half-life of IGFs (up to 12 hours). IGFBP proteases degrade IGFBP and play a role in locally generating free (i.e., active) IGFs. This is of interest in the context of IGF-responsive cancers (e.g., prostate cancer), which may overexpress one or more IGFBP proteases.

1	GH plays a dual role in metabolism that is highly dependent on physiological context. At the risk of oversimplification, its dual roles are to: (1) promote growth and protein anabolism when nutritional status is favorable, and (2) switch fuel consumption to lipids, sparing glucose in the fasted state. •Fig. 41.19 RelationshipofGHandIGF-I.GHhasdirectendocrineactionsongrowthandstimulatestheproductionofIGF-I,IGFBP-3andALSintheliver.CirculatingIGF-Iexertsendocrineactionsontargetorgans.IGF-1isalsoproducedlocallyinbone,whereitexertsparacrineeffects.Part,butnotallofthislocalIGF-IproductionisGH-dependent. GH acts through a specific GH receptor (GHR) that is a member of the cytokine receptor family. A GHR dimer binds to GH, which triggers activation of the JAK/STAT signaling pathway (see

1	GH acts through a specific GH receptor (GHR) that is a member of the cytokine receptor family. A GHR dimer binds to GH, which triggers activation of the JAK/STAT signaling pathway (see Fig. 41.17 ). This results in phosphorylation of STAT5b, which translocates to the nucleus to stimulate transcription of GH-responsive genes. Additional signaling pathways activated by GH include MAPK and PI3K among others.

1	In the fed state, GH is a protein anabolic hormone that increases cellular amino acid uptake and incorporation into protein. Consequently it produces nitrogen retention (positive nitrogen balance) and decreases urea production. The muscle wasting that occurs concomitant with aging has been proposed to be caused at least in part by the decrease in GH secretion that occurs during senescence. In children, GH increases skeletal, muscular, and visceral growth; children without GH show growth stunting or dwarfism. GH promotes cartilage growth and both linear and appositional growth of long bones ( Fig. 41.20 , green arrowheads). Although GH is an effective stimulator of IGF production, this response requires insulin, which supports GH receptor expression and signaling in hepatocytes. When a balanced supply of nutrients is available, high serum glucose levels stimulate insulin secretion and high serum amino acid levels promote GH secretion (

1	Fig. 41.21, top ). These conditions are appropriate for growth, and GH in turn stimulates IGF-I production by the liver. IGFs are mitogenic and have profound anabolic effects on many organs and tissues, including muscle, cartilage, and bone. Together, GH and IGF-I promote chondrocyte proliferation, differentiation, and hypertrophy during the process of endochondral ossification (see Fig. 41.20, green arrowheads). After closure of the epiphyses, longitudinal growth ceases but appositional growth of long bones continues. IGF-I stimulates osteoblast replication and synthesis of collagen and bone matrix. Not surprisingly, serum IGF levels correlate well with growth in children. The role of GH changes with alteration of nutritional status. If the diet is high in calories but low in amino acids, for example, high carbohydrate availability promotes insulin secretion but low serum amino acid levels inhibit GH and IGF production (see

1	Fig. 41.21, middle). These responses allow dietary carbohydrates and fats to be stored, but conditions are unfavorable for growth. In the fasted state, on the other hand, when nutrient availability wanes, serum GH levels rise and serum insulin levels fall in response to hypoglycemia (see

1	Fig. 41.21, bottom). In the absence of insulin, peripheral glucose utilization decreases, thereby conserving glucose for essential tissues such as the brain. In these circumstances the rise in 750 SECTION8Berne & Levy Physiology •Fig. 41.20 BiologicaleffectsofGHandIGF-I.Anabolicgrowth-promotingeffectsthatoccurwhennutritionalstatusisfavorableareindicatedbygreenarrowheads.MetaboliceffectsofGHthatmobilizefatwhilesparingglucoseandproteinduringfastingaredenotedbyredarrowheads.IGFBP,insulinlikegrowthfactor–bindingprotein.Adipose tissue GH Liver Muscle ˜ Lean body mass ˛ Adiposity ˛ Glucose uptake ˜ Lipolysis ˜ RNA synthesis ˜ Protein synthesis ˜ Gluconeogenesis ˜ IGFBP ˜ IGFs ˛ Glucose uptake ˜ Amino acid uptake ˜ Protein synthesis IGF-I fed state fasted state Kidney Pancreas Intestine Islets Parathyroids Skin Connective tissue Bone, heart, lung ˜ Protein synthesis ˜ RNA synthesis ˜ DNA synthesis ˜ Cell size and number ˜ Amino acid uptake ˜ Protein synthesis ˜ RNA synthesis ˜ DNA synthesis

1	Skin Connective tissue Bone, heart, lung ˜ Protein synthesis ˜ RNA synthesis ˜ DNA synthesis ˜ Cell size and number ˜ Amino acid uptake ˜ Protein synthesis ˜ RNA synthesis ˜ DNA synthesis ˜ Collagen ˜ Chondroitin sulfate ˜ Cell size and number ˜ Linear growth Chondrocytes ˜ Organ size ˜ Organ function ˜ Caloric mobilization (e.g., lipids) •Fig. 41.21 DifferentialregulationofGH,insulin,andIGF-Isecretioncoordinatesavailabilityofnutrientswithgrowthandproteinanabolism,caloricstorage,orcaloricmobilization(primarilylipids).

1	GH secretion is beneficial because it shifts metabolism to lipid as an energy source, thereby conserving carbohydrate and protein. This involves coordinated direct actions of GH on the liver, muscle, and adipose tissue (see Fig. 41.20 , red arrowheads).

1	GH is a lipolytic hormone. In adipocytes it mobilizes fatty acids and glycerol from triacylglycerol by combined direct and indirect activation of adipocyte lipases. An important indirect action of GH is sensitization of adipocytes to the lipolytic actions of catecholamines, which are also elevated during fasting. Serum fatty acid levels rise as a result of GH action, and more fats are used for energy production. Fatty acid uptake and β-oxidation increase in skeletal muscle and liver. GH can be ketogenic as a result of the increase in fatty acid oxidation when insulin is absent. GH also alters carbohydrate metabolism, causing blood glucose levels to rise. Many of its actions may be secondary to increased fat mobilization and oxidation. For example, an increase in serum free fatty acids inhibits uptake of glucose in skeletal muscle and adipose tissue. The hyperglycemic effects of GH are mild and slower than those of glucagon and epinephrine. Liver glucose output increases, but this is

1	uptake of glucose in skeletal muscle and adipose tissue. The hyperglycemic effects of GH are mild and slower than those of glucagon and epinephrine. Liver glucose output increases, but this is not an effect of GH on glycogenolysis. The increase in fatty acid oxidation and hence the rise in liver acetyl CoA stimulate gluconeogenesis. GH also directly stimulates expression of the gluconeogenic enzyme PEPCK through activation of STAT5b. These actions increase glucose production by the liver from substrates such as lactate and glycerol. The latter is released into the circulation as a result of GH-induced lipolysis in adipocytes.

1	GH antagonizes the action of insulin at the postreceptor level in skeletal muscle and adipose tissue (but not the liver). Hypophysectomy (removal of the pituitary gland) can improve diabetic management because GH, like cortisol, decreases insulin sensitivity. Because GH produces insulin insensitivity, it is considered a diabetogenic hormone. Therefore when secreted in excess (e.g., in acromegaly), GH can cause diabetes mellitus, and the insulin levels necessary to maintain normal metabolism increase. Excessive insulin secretion resulting from an excess of GH can cause damage to pancreatic beta cells. In the absence of GH, insulin secretion declines. Thus normal levels of GH are required for normal pancreatic function and insulin secretion.

1	GH deficiency in adults is becoming recognized as a pathological syndrome. If GH deficiency occurs after the epiphyses close, growth is not impaired. GH deficiency is one of many possible causes of hypoglycemia. Recent studies have shown that extended deficiencies of GH lead to changes in body composition. Fat as a percentage of body weight increases, whereas lean body mass declines. In addition, muscle weakness and early exhaustion are symptoms of GH deficiency. There has been interest in using GH in elderly populations to reverse age-related physical decline and body composition, but studies to date have shown small changes in body composition, no functional benefits, and increased risk of adverse events.

1	GHisnecessaryforgrowthbeforeadulthood.GHdeficienciesresultinseveregrowthdeficits,andexcessesresultingigantism.ExcessGHinadulthoodafterepiphysealclosurecausesacromegaly, characterizedbyinsidiousenlargementofthehandsandfeet,coarseningoffacialfeatures,insulinresistance,anddiabetes.GeneticdisordersoftheGH–IGF-Iaxiscauseseveregrowthimpairment.IdentifiedmutationscausingisolatedGHdeficiencymostcommonlyoccurintheGHandGHRHreceptorgenes.ThesepatientscanbetreatedwithrecombinanthGHtorestorefunctionofthedownstreamaxis.InLaronsyndrome,amutationoftheGHreceptorcausesGHresistance.InthisinstancetheliverdoesnotproduceIGF-I;thisisduetoalackofresponsivenesstoGH.ThesepatientscanbetreatedwithIGF-I,butwithoutthedirectactionsofGH,theeffectivenessofthetreatmentislimited.OtherdownstreamgeneticmutationsthathavebeenreportedincludethoseinSTAT5B,IGF-I,andALS. The Lactotrope

1	The Lactotrope The lactotrope produces the hormone prolactin, which is a 199–amino acid single-chain protein. PRL is structurally related to GH and hPL (see ). Like GH, the PRL receptor is a member of the cytokine family coupled to the JAK/STAT signaling pathways. Because the primary action of PRL in humans is related to breast development and function during pregnancy and lactation, the regulation and actions of prolactin will be discussed in detail in In the context of the pituitary gland, it should be appreciated that the lactotrope differs from the other endocrine cell types of the adenohypophysis in two major ways: 1. The lactotrope is not part of an endocrine axis. This means PRL acts directly on nonendocrine cells (primarily of the breast) to induce physiological changes. 2.

1	2. Production and secretion of PRL are predominantly under inhibitory control by the hypothalamus. Thus disruption of the pituitary stalk and the hypothalamohypophyseal portal vessels (e.g., secondary to surgery or physical trauma) results in an increase in PRL levels but a decrease in ACTH, TSH, FSH, LH, and GH. PRL circulates unbound to serum proteins and thus has a relatively short half-life of about 20 minutes. Normal basal serum concentrations are similar in men and women. Release of PRL is normally under tonic inhibition by the hypothalamus. This is exerted by dopaminergic tracts that secrete dopamine in the median eminence. There is also evidence for existence of a prolactin-releasing factor (PRF). The exact nature of this compound is not known, although many factors, including TRH and hormones in the glucagon family (secretin, glucagon, VIP, and gastric inhibitory polypeptide [GIP]) can stimulate release of PRL.

1	PRL is one of the many hormones released in response to stress. Surgery, fear, stimuli causing arousal, and exercise are Upon completion of this chapter the student should be able to answer the following questions: 1. Describe the anatomy and histology of the thyroid gland, including the structure of the thyroid follicle. 2. Explain how thyroid hormones are synthesized within the thyroid gland, including the processes of iodine uptake, iodination of tyrosine residues in thyroglobulin by thyroid peroxidase/dual oxidase, and coupling to form T4 and T3. 3. Describe the process of endocytosis by which thyroglobulin is retrieved from the follicle lumen and processed to yield T3 and T4, which are secreted into the circulation. 4. Diagram the hypothalamic-pituitary-thyroid axis to show how TSH regulates thyroid function and how thyroid hormones feed back to regulate the axis. List examples of how central input can alter the set point of the axis. 5.

1	5. Discuss the role of thyroid-binding proteins in the transport and stability of thyroid hormones, and the role of peripheral deiodinases in the activation of T4 to T3 or inactivation to reverse T3. Contrast the cellular location and function of the D1 and D2 deiodinases. 6. Describe the mechanisms of thyroid hormone action, including the nature and location of the thyroid hormone receptor and its ability to either repress or activate target gene transcription. 7. Discuss the actions of thyroid hormone during development, especially on the central nervous system (CNS) and skeleton, including the consequences of severe hypothyroidism. 8. Describe the effects of thyroid hormone on basal metabolic rate and thermogenesis, on the cardiovascular system (heart rate, cardiac output, systemic vascular resistance), and on other organ systems (skin, skeletal muscle, digestive tract).

1	he thyroid gland produces the prohormone tetraiodothyronine (T4, also called thyroxine) and the active hormone triiodothyronine (T3). Synthesis of T4 and T3 requires iodine, which can be a limiting factor in some parts of the world. Much of T3 is also made by peripheral conversion of T4 to T3. Thyroid hormone acts primarily through a nuclear receptor that regulates gene transcription. T3 is critical for normal brain and bone development and has broad effects on metabolism and cardiovascular function in adults. Anatomy and Histology of the Thyroid Gland The thyroid gland is composed of right and left lobes that sit anterolateral to the trachea ( Fig. 42.1). Typically the two lobes are connected by a midventral isthmus. The thyroid gland receives a rich blood supply. It is drained by three sets of veins on each side: the superior, middle, and inferior thyroid veins. The thyroid gland receives sympathetic innervation that is vasomotor but not secretomotor.

1	The functional unit of the thyroid gland is the thyroid follicle, a spherical structure about 200 to 300 µm in diameter that is surrounded by a single layer of thyroid epithelial cells ( Fig. 42.2 ). The epithelium sits on a basal lamina, the outermost structure of the follicle, and is surrounded by a rich capillary supply. The apical side of the follicular epithelium faces the lumen of the follicle. The follicular lumen itself is filled with colloid, which is composed of thyroglobulin. This large (660 kDa) protein is secreted into the lumen and iodinated by the thyroid epithelial cells, serving as a scaffold for production of thyroid hormones. The size of the epithelial cells and the amount of colloid are dynamic features that change with activity of the gland. The thyroid gland contains another type of cell in addition to follicular cells. Scattered within the gland are parafollicular cells, or C cells, which are the source of the polypeptide hormone calcitonin (see

1	Production of Thyroid Hormones The secretory products of the thyroid gland are iodothyronines ( Fig. 42.3 ), a class of hormones formed by the coupling of two iodinated tyrosine molecules. Approximately 90% of the thyroid output is 3,5,3′,5′-tetraiodothyronine (thyroxine, or T4). T4 is primarily a prohormone. About 10% is 3,5,3′-triiodothyronine (T3), which is the active form of thyroid hormone. Less than 1% of thyroid output is 3,3′,5′-triiodothyronine (reverse T3, or rT3), which is inactive. Normally these three products are secreted in the same proportions at which they are stored in the gland. Because the primary product of the thyroid gland is T4, yet the active form of thyroid hormone is T3, the thyroid axis relies heavily on peripheral conversion through the action of thyronine-specific deiodinases (see

1	Fig. 42.3 ). Most • Fig. 42.1 A and B, Anatomy of the thyroid gland. C, Image of pertechnetate uptake by a normal thyroid gland. (Modified from Drake RL et al. Gray’s Anatomy for Students. Philadelphia: Churchill Livingstone; 2005.) conversion of T4 to T3 by type 1 deiodinase (D1) occurs in tissues with high blood flow and rapid exchange with plasma, such as the liver and kidneys. This process supplies basal circulating T3 for uptake by other tissues in which local T3 generation is low or absent. D1 is also expressed in the thyroid (again, where T4 is abundant) and has relatively low affinity (i.e., a Km of 1 µM) for T4. Levels of D1 are paradoxically increased in hyperthyroidism and contribute to the elevated circulating T3 levels in this disease.

1	The brain maintains constant intracellular levels of T3 by a high-affinity deiodinase called type 2 deiodinase (D2) that is expressed in glial cells of the CNS. D2 has a Km of 1 nM and maintains intracellular concentrations of T3 even when circulating T4 falls to low levels. D2 is also present in pituitary thyrotropes. There, D2 acts as a “thyroid axis sensor” that mediates the ability of circulating T4 to feed back on secretion of thyroid stimulating hormone (TSH). Expression of D2 is increased during hypothyroidism, which helps maintain constant T3 levels in the brain. There is also an “inactivating” deiodinase called type 3 deiodinase (D3). D3 is a high-affinity inner ring deiodinase that converts T4 to the inactive rT3. Type 3 deiodinase is increased during hyperthyroidism, which helps blunt overproduction of T4. All forms of iodothyronines are eventually further deiodinated to noniodinated thyronine.

1	Because iodide plays a unique role in thyroid physiology, a description of thyroid hormone synthesis requires some understanding of iodide turnover (

1	Fig. 42.4 ). An average of 400 µg of iodide per person is ingested daily in the United States, versus a minimum daily requirement of 150 µg for adults, 90 to 120 µg for children, and 200 µg for pregnant women. In the steady state, the same amount, 400 µg, is excreted in urine. Iodide is actively concentrated in the thyroid gland, salivary glands, gastric glands, lacrimal glands, mammary glands, and choroid plexus. About 70 to 80 µg of iodide is taken up daily by the thyroid gland from a circulating pool that contains approximately 250 to 750 µg of iodide. The total iodide content of the thyroid gland averages 7500 µg, virtually all of which is in the form of stored iodothyronine in colloid thyroglobulin. In the steady state, 70 to 80 µg of iodide, or about 1% of the total, is released from the gland daily. Of this amount, 75% is secreted as thyroid hormone and the remainder as free iodide. The large ratio (100:1) of iodide stored in the form of hormone to the amount turned over daily

1	the gland daily. Of this amount, 75% is secreted as thyroid hormone and the remainder as free iodide. The large ratio (100:1) of iodide stored in the form of hormone to the amount turned over daily protects against iodide deficiency for about 2 months. Iodide is also conserved by a marked reduction in renal excretion of iodide as its concentration in serum falls.

1	•Fig. 42.2 Histologyofthethyroidglandatlow(upper panel) andhigh(lower panel) magnification.C,colloid;F,thyroidfollicles;S,connectivetissuesepta.(FromYoungBetal.Wheater’s Functional Histology. 5thed.Philadelphia:ChurchillLivingstone;2006.) CHAPTER 42 The Thyroid Gland Overview of Thyroid Hormone Synthesis To understand thyroid hormone synthesis and secretion, one must appreciate the directionality of each process as it relates to the polarized thyroid epithelial cell ( Fig. 42.5 ). Synthesis of thyroid hormone requires two precursors: iodide and thyroglobulin. Iodide is transported across cells from the basal (vascular) side to the apical (follicular luminal) side of the thyroid epithelium. Thyroglobulin is synthesized and secreted across the apical membrane into the follicular lumen. Thus synthesis involves a basal-to-apical movement of these precursors into the follicular lumen (see

1	Fig. 42.5 ). Actual synthesis of iodothyronines occurs enzymatically within the follicular lumen close to the apical membrane of the epithelial cells (see Synthesis of Iodothyronines on ). Secretion of thyroid hormone involves endocytosis of iodinated thyroglobulin and apicalto-basal movement of the endocytotic vesicles, which fuse with lysosomes. Thyroglobulin is enzymatically degraded by lysosomal enzymes, resulting in release of thyroid hormones from the thyroglobulin backbone. Finally, thyroid hormones move across the basolateral membrane, probably through a specific transporter, and ultimately into the blood. Thus secretion involves apical-to-basal movement (see Fig. 42.5 Synthesis of Iodothyronines on a Thyroglobulin Backbone Iodide is actively transported into the gland against chemical and electrical gradients by a sodium-iodide symporter (NIS) located in the basolateral membrane of thyroid epithelial cells (see

1	Iodide is actively transported into the gland against chemical and electrical gradients by a sodium-iodide symporter (NIS) located in the basolateral membrane of thyroid epithelial cells (see Fig. 42.5 ). NIS is highly expressed in the thyroid gland, but it is also expressed at lower levels in the placenta, salivary glands, and actively lactating breast. One iodide ion is transported uphill against an iodide gradient while two sodium ions move down their electrochemical gradient from extracellular fluid into the thyroid cell. The driving force for this secondary active transporter is provided by plasma membrane Na+,K+-ATPase. Expression of the NIS gene is inhibited by iodide and stimulated by TSH. A reduction in dietary iodide intake depletes the circulating iodide pool and greatly enhances the activity of the iodide trap. When dietary iodide intake is low, the percentage of thyroid uptake of iodide can reach 80% to 90%. The steps in thyroid hormone synthesis are shown in

1	The steps in thyroid hormone synthesis are shown in Fig. 42.6 . After entering the gland, iodide rapidly moves to the apical plasma membrane of epithelial cells. From there, iodide is transported into the lumen of the follicles by a sodium-independent iodide/chloride transporter called pendrin. Iodide is immediately oxidized and incorporated into tyrosine residues within thyroglobulin (see Fig. 42.5 ). A single iodination forms a monoiodotyrosine (MIT); a second iodination of the same residue produces diiodotyrosine (DIT) (see Fig. 42.6 ). After iodination, two DIT molecules are coupled to form T4; one MIT and one DIT are coupled to form T3. Coupling occurs between iodinated tyrosines that remain part of the primary structure of

1	I I 3,5,3¢ 5¢ -Tetraiodothyronine (thyroxine, or T4) NH2 HO O CH2CHCOOH Outer ring deiodination (activation) Inner ring deiodination Deiodinases type 1 and 2 I I Deiodinases type 3 I 3,5,3¢ -Triiodothyronine (T3) I NH2 CH2CHCOOHO (inactivation) I I 3,3¢ 5¢ -Triiodothyronine (reverse T3) NH2 CH2CHCOOHHO O •Fig. 42.3 StructureoftheiodothyroninesT4,T3,andreverseT3. •Fig. 42.4 Iodinedistributionandturnoverinhumans.HI,hormone-associatediodine.Stool 10 µg HIUrine 390 µg ITissues Thyroid Extracellular fluid 10 µg HI60 µg HI80 µg I400 µg IDiet 320 µg I20 µg I50 µg I thyroglobulin. This entire sequence of reactions is catalyzed by thyroid peroxidase (TPO), an enzyme complex that spans the apical membrane. The immediate oxidant (electron acceptor) for the reaction is hydrogen peroxide (H2O2). Generation of H2O2 in the follicular lumen is catalyzed by dual oxidases (DUOX1, DUOX2) that are also localized in the apical plasma membrane.

1	When iodide availability is restricted, formation of T3 is favored. Because T3 is three times as potent as T4, this response provides more active hormone per molecule of organified iodide. The proportion of T3 also increases when the thyroid gland is hyperstimulated by TSH or other activators. Secretion of Thyroid Hormones Once thyroglobulin has been iodinated, it is stored in the lumen of the follicle as colloid (see Fig. 42.2 ). Release of T4 and T3 into the bloodstream is initiated by endocytosis of colloid from the follicular lumen by the processes of macroand micropinocytosis. Endocytotic vesicles then fuse with lysosomes and thyroglobulin is degraded ( Fig. 42.7 Fig. 42.5 ). MIT and DIT molecules, which also are released during proteolysis of thyroglobulin, are rapidly deiodinated within the follicular cell by the enzyme iodotyrosine deiodinase (see

1	42.7 Fig. 42.5 ). MIT and DIT molecules, which also are released during proteolysis of thyroglobulin, are rapidly deiodinated within the follicular cell by the enzyme iodotyrosine deiodinase (see Fig. 42.5 ). This deiodinase is specific for MIT and DIT and cannot use T4 and T3 as substrates. The iodide is then recycled into synthesis of T4 and T3. Amino acids from the digestion of thyroglobulin reenter the intrathyroidal amino acid pool and can be reused for protein synthesis. Only minor amounts of intact thyroglobulin leave the follicular cell under normal circumstances. Enzymatically released T4 and T3 are transported across the basal side of the cell and enter the blood. •Fig. 42.5 Synthesisandsecretionofthyroidhormonesbythethyroidepithelialcell.

1	•Fig. 42.5 Synthesisandsecretionofthyroidhormonesbythethyroidepithelialcell. I I I I 3,5,3¢ 5¢ -Tetraiodothyronine (thyroxine, or T4) NH2 CH2CHCOOHHO O I I NH2 CH2CHCOOHHO O I NH2 CH2CHCOOHHO I NH2 CH2CHCOOH + I I DIT DIT NH2 CH2CHCOOH + I I NH2 CH2CHCOOHHO I Diiodotyrosine (DIT) or NH2 CH2CHCOOHHO Monoiodotyrosine (MIT) NH2 CH2CHCOOHHO Tyrosine NH2 CH2CHCOOHI2 + HO DIT MIT 3,5,3¢ -Triiodothyronine (T3) •Fig. 42.6 Reactionsinvolvedinthegenerationofiodide,MIT,DIT,T3,andT4.

1	DIT MIT 3,5,3¢ -Triiodothyronine (T3) •Fig. 42.6 Reactionsinvolvedinthegenerationofiodide,MIT,DIT,T3,andT4. Transport and Metabolism tissues in addition to exerting negative feedback on the pituitary and hypothalamus. The major binding protein is thyroxine-binding globulin (TBG), which is synthesized Secreted T4 and T3 circulate in the bloodstream almost in the liver and binds one molecule of T4 or T3. About 70% entirely bound to proteins. Normally only about 0.03% of circulating T4 and T3 is bound to TBG; 10% to 15% of total plasma T4 and 0.3% of total plasma T3 exist in is bound to another specific thyroid-binding protein called the free state (

1	Fig. 42.8 ). Free T3 is biologically active and transthyretin (TTR). Albumin binds 15% to 20%, and mediates the effects of thyroid hormone on peripheral 3% is bound to lipoproteins. Ordinarily only alterations in ).CirculatingthyroidhormonesfeedbackonthepituitaryglandtodecreaseTSHsecretion,primarilybyrepressingTSHβ subunitgeneexpression.Thepituitaryglandexpressesthehigh-affinityD2,whichconvertsT4enteringthesecellstoT3.ThusfeedbackinthyrotropesmediatedbyintracellularT3representsanintegratedmeasureofcirculatingfreeT4andT3(see ).BecausethediurnalvariationinTSHsecretionissmall,thyroidhormonesecretionandplasmaconcentrationsarerelativelyconstant.OnlysmallnocturnalincreasesinsecretionofTSHandreleaseofT4occur.Thyroidhormonesalsofeedbackonhypothalamicthyroid-releasinghormone(TRH)secretingneurons.Intheseneurons,T3inhibitsexpressionoftheprepro-TRHgene.

1	Autoregulationofthyroidglandfunctioniscausedbyiodideitself,whichhasabiphasicaction.Atrelativelylowlevelsofiodideintake,therateofthyroidhormonesynthesisisdirectlyrelatedtotheavailabilityofiodide.However,iftheintakeofiodideexceeds2mg/day,theintraglandularconcentrationofiodidereachesalevelthatparadoxicallysuppressesTPOactivity,blockinghormonebiosynthesis.ThisphenomenonisknownastheWolff-Chaikoff effect. AdaptationtohighiodideintakenormallyoccursbyreducingexpressionofNIS,whichcausestheintrathyroidaliodideleveltofall.TPOactivitythenreturnstonormal,andthyroidhormonesynthesisresumeswithindaystoweeks.Inunusualinstances,failureofNIStodownregulateleadstoprolongedinhibitionofhormonesynthesisbyiodideandresultanthypothyroidism.Thetemporaryreductioninhormonesynthesisbyexcessiodidehasalsobeenusedtherapeuticallyinhyperthyroidism.

1	Becauseofitsabilitytotrap andincorporateiodineintothyroglobulin(calledorganification),theactivityofthethyroidcanbeassessedbyradioactive iodine uptake (RAIU). Inthistestatracerdoseof123IisadministeredandRAIUismeasuredbyplacingagammadetectorontheneckat4to6hoursandat24hours.IntheUnitedStates,wherethedietisrelativelyrichiniodine,RAIUistypicallyaround15%after6hoursand25%after24hours( Fig.42.9 ).AbnormallyhighRAIU(>60%)after24hoursindicateshyperthyroidism.AbnormallylowRAIU(<5%)after24hoursindicateshypothyroidism.Inindividualswithextremechronicstimulationofthethyroid(Gravesdisease–associatedthyrotoxicosis),iodideistrapped,organified,andreleasedashormoneveryrapidly.Inthesecasesofelevatedturnover,6-hourRAIUwillbeveryhighbut24-hourRAIUwillbelower(see

1	Fig.42.8 ).Anumberofanions,suchasthiocyanate(CNS−),perchlorate(HClO4−),andpertechnetate(TcO4−),arecompetitiveornoncompetitiveinhibitorsofiodidetransportviaNIS.Ifiodidecannotberapidlyincorporatedintotyrosine(organification defect) afteritsuptakebythecell,administrationofoneoftheseanionswill,byblockingfurtheriodideuptake,causerapidreleaseofiodidefromthegland(see Fig.42.9 Thisreleaseoccursasaresultofthehighthyroid-plasmaconcentrationgradient. Thethyroidcanbeimagedwitharectilinearscannerorgammacameraafteradministrationofatracer,123I,131I,ortheiodine-mimicpertechnetate(99mTc).Imagingcandisplaythesizeandshapeofthethyroid(see

1	Thethyroidcanbeimagedwitharectilinearscannerorgammacameraafteradministrationofatracer,123I,131I,ortheiodine-mimicpertechnetate(99mTc).Imagingcandisplaythesizeandshapeofthethyroid(see Fig.42.1C )aswellasheterogeneitiesofactiveversusinactivetissuewithinthethyroidgland.Suchheterogeneitiesareoftenduetothedevelopmentofthyroid nodules, whichareregionsofenlargedfollicleswithevidenceofregressivechangesduetocyclesofstimulationandinvolution.“Hot” nodules (i.e.,nodulesthatdisplayhighRAIUonimaging)arenotusuallycancerousbutmayleadtothyrotoxicosis(hyperthyroidism).“Cold” nodules are10timesmorelikelytobecancerous.Suchnodulescanbesampledforpathologicalanalysisbyfine-needle aspiration biopsy. Thethyroidcanalsobeimagedbyultrasonography, whichissuperiorinresolutiontoRAIUimaging.Ultrasonographyisusedtoguidethephysicianduringfine-needleaspirationbiopsyofanodule.Thehighestresolutionofthethyroidisachievedwithmagnetic resonance imaging (MRI).

1	A B Colloid in endocytic vesicles Colloid in lumen of thyroid follicle •Fig. 42.7 Before(A)andminutesafter(B)rapidinductionofthy-roglobulinendocytosisbyTSH.(FromWollmanSHetal.J Cell Biol 1964;21:191.) TBG concentration significantly affect total plasma T4 and T3 levels. Two important biological functions have been ascribed to TBG. First, it maintains a large circulating reservoir of T4 that buffers any acute changes in thyroid gland function. Second, binding of plasma T4 and T3 to proteins prevents loss of these relatively small hormone molecules in urine and thereby helps conserve iodide. TTR transports T4 in cerebrospinal fluid and provides thyroid hormones to the CNS. Regulation of Thyroid Function

1	Regulation of Thyroid Function The most important regulator of thyroid gland function and growth is the hypothalamic-pituitary thyroid releasing •Fig. 42.8 TransportofT4andT3inserumbytransportproteinsandpercentagesofboundandfreehormone.Thyroid secretion Protein-Bound T4 (99.98%) & T3 (99.7%) Thyroid-binding globulin (TBG) Transthyretin (TTR) Albumin • Feedback • Activation to T3 • Inactivation • Excretion • Tissue actions • Feedback • Inactivation • Excretion Free T4 (0.02%) & T3 (0.3%) Total T4 8 µg/dL Total T3 0.14 µg/dL Free T4 1.6 ng/dl Free T3 – 0.42 ng/dl T3 T4 123I Uptake (% of dose) •Fig. 42.9 Thyroidglandiodothyronineuptakedefectiveorganificationstates. ). TSH stimulates every aspect of thyroid function. TSH has immediate, intermediate, and long-term actions on the thyroid epithelium. Rapid actions of TSH include pinocytosis of colloid droplets in the cytoplasm, which represent thyroglobulin within endocytic vesicles (see

1	Fig. 42.7 ). TSH stimulates the proteolysis of thyroglobulin and release of T4 and T3 from the gland. Iodide uptake and TPO activity increase. TSH also stimulates entry of glucose into the hexose monophosphate shunt pathway, which generates the reduced nicotinamide adenine dinucleotide phosphate (NADPH) needed for the peroxidase reaction. Intermediate effects of TSH on the thyroid gland occur after hours to days and involve protein synthesis and expression of numerous genes, including those encoding NIS, thyroglobulin, and TPO. Sustained TSH stimulation leads to the long-term effects of hypertrophy and hyperplasia of follicular cells. Capillaries proliferate and thyroid blood flow increases. These actions, which underlie the growth-promoting effects of TSH on the gland, are supported by local production of growth factors. A noticeably enlarged thyroid gland is called a goiter (

1	Fig. 42.10 ). Endemic goiter is due to lack of adequate iodine in the diet, which results in low thyroid hormone and elevated TSH levels. Physiological Effects of Thyroid Hormone Thyroid hormone acts on essentially all cells and tissues, and imbalances in thyroid function constitute some of the most common endocrine diseases. Thyroid hormone has many direct actions, but it also acts in more subtle ways to optimize the actions of several other hormones and neurotransmitters. curvesfornormal,hypothyroid,hyperthyroid,and •Fig. 42.10 Thethyroidglandislocatedintheanterioraspectoftheneck,whereitiseasilyvisualizedwhenenlarged(goiter). ˜ Cardiac muscle Myosin heavy chain ˝/˙ ratio Na+, K+-ATPase Sarcoplasma Ca-ATPase ˙-Adrenergic signaling G-protein stimulatory/ •Fig. 42.11 Mechanismsbywhichthyroidhormoneincreasescardiacoutput.Theindirectmechanismsareprobablyquantitativelymoreimportant.

1	Graves disease isthemostcommonformofhyperthyroidism. Itoccursmostfrequentlybetweentheagesof20and50andis10timesmorecommoninwomenthaninmen.GravesdiseaseisanautoimmunedisorderinwhichactivatingautoantibodiesareproducedagainsttheTSHreceptor.Hyperthyroidismdrivenbytheantibodyisoftenaccompaniedbyadiffusegoiterasaresultofhyperplasiaandhypertrophyofthegland.Thefollicularepithelialcellsbecometallcolumnarcells,andthecolloidshowsascallopedperipheryindicativeofrapidturnover.

1	TheprimaryclinicalstatefoundinGravesdiseaseisthyrotoxicosis—thestateofexcessivethyroidhormoneinbloodandtissues.Apatientwiththyrotoxicosispresentsoneofthemoststrikingpicturesinclinicalmedicine.Thelargeincreaseinmetabolicrateismanifestedasweightlossdespiteincreasedfoodintake.Excessheatproductioncausesdiscomfortinwarmenvironments,sweating,andgreaterintakeofwater.Theincreaseinadrenergicactivityproducesarapidheartrate,hyperkinesis,tremor,nervousness,andawide-eyedstare.Weaknessiscausedbyalossofmusclemassaswellasimpairedmusclefunction.Othersymptomsincludealabileemotionalstate,breathlessnessduringexercise,anddifficultyswallowingorbreathingbecauseofcompressionoftheesophagusortracheabytheenlargedthyroidgland.Themostcommoncardiovascularsignissinustachycardia.There Perhaps the most clinically important actions of thyroid hormone are those on cardiovascular physiology. T3 increases cardiac output, thereby ensuring sufficient O2 delivery to tissues (

1	Perhaps the most clinically important actions of thyroid hormone are those on cardiovascular physiology. T3 increases cardiac output, thereby ensuring sufficient O2 delivery to tissues ( Fig. 42.11). The resting heart rate and stroke volume are increased. The speed and force of myocardial contractions are enhanced (positive chronotropic and inotropic effects, respectively), and the diastolic relaxation isincreasedcardiacoutputassociatedwithawidenedpulsepressuresecondarytoapositiveinotropiceffectcoupledwithdecreasedsystemicvascularresistance.AmajorclinicalsigninGravesdiseaseisexophthalmos (abnormalprotrusionoftheeyeball)andperiorbital edema. ThisiscausedbyautoantibodybindingtotheTSHreceptorexpressedonorbitalfibrocytes,leadingtoproductionofinflammatorycytokines.

1	GravesdiseaseisusuallydiagnosedbyelevatedserumfreeandtotalT4andT3andtheclinicalsignsofdiffusegoiterandophthalmopathy.SerumTSHlevelsarelowbecausethehypothalamusandpituitaryareinhibitedbythehighlevelsofT4andT3.Inmostcases,radioiodineuptakebythethyroidisexcessiveanddiffuse.AssayofTSHlevelsandthepresenceofcirculatingthyroidstimulatingimmunoglobulinwilldistinguishGravesdisease(aprimarydisorder)fromarareadenomaofpituitarythyrotrophs(asecondarydisorder)thatproduceshighlevelsofTSH. TreatmentofGravesdiseaseisusuallyremovalofthethyroidtissue,followedbylifelongreplacementtherapywithT4.Thyroidtissuecanberemovedeitherbyradioablationwith131Iorbysurgery.Withsurgicalremovalofthegland,precautionsmustbetakentoavoidamassive,potentiallylife-threateningreleaseofthyroidhormonesknownasthyroid storm. Analternativetoremovalofthyroidtissueisadministrationofantithyroid drugs thatinhibitTPOactivity.

1	time is shortened (positive lusitropic effect). Systolic blood pressure is modestly augmented and diastolic blood pressure is decreased. The resultant widened pulse pressure reflects the combined effects of the increased stroke volume and the reduction in systemic vascular resistance secondary to blood vessel dilation in skin, muscle, and heart. These effects in turn are partly due to the increase in tissue production of heat and CO2 that thyroid hormone induces (see on Basal Metabolic Rate and Thermogenesis). In addition, however, thyroid hormone decreases systemic resistance by dilating arterioles in the peripheral circulation. Total blood volume is increased by activation of the renin-angiotensinaldosterone axis, thereby increasing renal tubular sodium reabsorption (see The cardiac inotropic effects of T3 are both direct and indirect. The latter are due primarily to enhanced responsiveness to catecholamines (see ). Direct inotropic effects (see

1	The cardiac inotropic effects of T3 are both direct and indirect. The latter are due primarily to enhanced responsiveness to catecholamines (see ). Direct inotropic effects (see Fig. 42.11 ) involve regulation of multiple proteins that enhance contractility, including increased α-myosin heavy chain expression and inhibition of the plasma membrane Na+/Ca++ exchanger. Sarcoplasmic reticulum Ca++-ATPase (SERCA) is increased by T3, whereas phospholamban is decreased. As a result, sequestration of calcium during diastole is enhanced and the relaxation time is shortened. Increased ryanodine Ca++ channels in the sarcoplasmic reticulum promote release of Ca++ from the sarcoplasmic reticulum during systole.

1	Thyroidhormonelevelsinthenormalrangearenecessaryforoptimumcardiacperformance.Adeficiencyofthyroidhormoneinhumansreducesstrokevolume,leftventricularejectionfraction,cardiacoutput,andtheefficiencyofcardiacfunction.Thelatterdefectisshownbythefactthatthestrokeworkindex[(strokevolume/leftventricularmass)× peaksystolicbloodpressure]isdecreasedtoagreaterextentthanmyocardialoxidativemetabolism.Theriseinsystemicvascularresistancemaycontributetothiscardiacdebility.Incontrast,excessthyroidhormoneenhancescardiacoutputbyincreasingbothheartrateandstrokevolume.Pulsepressureiswidenedbyincreasedsystolicpressureanddecreaseddiastolicpressureduetodecreasedsystemicvascularresistance.Thyrotoxicosisisassociatedwithpalpitations,atrialfibrillation,andmitralvalveprolapse(see

1	Increased O2 use ultimately depends on an increased supply of substrates for oxidation. T3 augments glucose absorption from the gastrointestinal tract and increases glucose turnover (glucose uptake, oxidation, and synthesis). In adipose tissue, thyroid hormone induces enzymes for the synthesis of fatty acids, including acetyl-CoA carboxylase and fatty acid synthase, and enhances lipolysis by increasing the number of β-adrenergic receptors (see the Autonomic Nervous System ). Thyroid hormone also enhances the clearance of chylomicrons. Thus lipid turnover (free fatty acid release from adipose tissue and oxidation) is augmented. Protein turnover (release of muscle amino acids, protein degradation, and to a lesser extent protein synthesis and urea formation) is also increased. T3 potentiates the respective stimulatory effects of epinephrine, norepinephrine, glucagon, cortisol, and growth hormone on gluconeogenesis,

1	CHAPTER 42 The Thyroid Gland lipolysis, ketogenesis, and proteolysis of the labile protein pool. The overall metabolic effect of thyroid hormone has been aptly described as accelerating the physiological response to starvation. In addition, thyroid hormone stimulates synthesis of bile acids from cholesterol and promotes biliary secretion. The net effect is a decrease in the body pool and plasma levels of total and low-density lipoprotein cholesterol. Metabolic clearance of adrenal and gonadal steroid hormones, some B vitamins, and certain administered drugs is also increased by thyroid hormone.

1	Thyroid hormones stimulate thermogenesis by affecting both adenosine triphosphate (ATP) utilization and the efficiency of ATP synthesis. ATP utilization is enhanced by upregulation of several energy-dependent processes, including Na+,K+-ATPase and SERCA, particularly in skeletal muscle, where calcium cycling between the cytoplasm and sarcoplasmic reticulum uses ATP and generates heat. Recently it has been demonstrated that brown fat in humans, once thought to be important only in neonates, appears to play a role in facultative thermogenesis in adults. Imaging studies have demonstrated the presence of brown fat in the mediastinum, particularly in lean individuals, and metabolic activity in brown fat is enhanced by exposure to cold. Brown fat expresses uncoupling protein-1 (UCP1), also called thermogenin, which causes the proton gradient across the inner mitochondrial membrane to be dissipated as heat, which is then disseminated to the rest of the body by the circulation. UCP1 is

1	called thermogenin, which causes the proton gradient across the inner mitochondrial membrane to be dissipated as heat, which is then disseminated to the rest of the body by the circulation. UCP1 is regulated by thyroid hormone, and brown fat expresses D2, providing intracellular conversion of T4 to T3. Brown fat thermogenesis involves a synergistic interaction between thyroid hormones and the sympathetic nervous system. Catecholamines promote lipolysis and upregulate expression of D2. T3 in turn upregulates adrenergic receptors and enhances catecholamine responsiveness. Hyperthyroidism is accompanied by heat intolerance, whereas hypothyroidism is accompanied by cold intolerance.

1	Thyroid hormone stimulates O2 utilization and enhances O2 delivery. Appropriately, T3 increases the resting respiratory rate, minute ventilation, and the ventilatory response to hypercapnia and hypoxia. These actions maintain a normal arterial PO2 when O2 utilization is increased and a normal PCO2 when CO2 production is increased. Additionally the hematocrit increases slightly to enhance O2-carrying capacity. This increase results from stimulation of erythropoietin production by the kidney. Normal function of skeletal muscles also requires optimal amounts of thyroid hormone. This requirement may be related to regulation of energy production and storage. Glycolysis and glycogenolysis are increased, whereas glycogen and creatine phosphate are reduced by thyroid hormone excess. The inability of muscle to take up and phosphorylate creatine leads to its increased urinary excretion. Effects on the Autonomic Nervous System and Catecholamine Action

1	Effects on the Autonomic Nervous System and Catecholamine Action As already mentioned, there is important synergism between catecholamines and thyroid hormones. Thyroid hormones are synergistic with catecholamines in increasing the metabolic rate, heat production, heart rate, motor activity, and excitation of the CNS. T3 may enhance sympathetic nervous system activity by increasing the number of β-adrenergic receptors in heart muscle and the generation of intracellular second messengers such as cyclic adenosine monophosphate (cAMP).

1	A major effect of thyroid hormone is to promote growth and maturation. A small but crucial amount of thyroid hormone crosses the placenta, and the fetal thyroid axis becomes functional at midgestation. Thyroid hormone is extremely important for normal neurological development and proper bone formation in the fetus. In infants, insufficient fetal thyroid hormone causes congenital hypothyroidism, characterized by irreversible intellectual disability and short stature (see In the Clinic box). Effects on Bone, Hard Tissue, and Dermis Thyroid hormone promotes endochondral ossification, linear bone growth, and maturation of the epiphyseal bone centers. T3 enhances maturation and activity of chondrocytes in the cartilage growth plate, in part by increasing local growth factor production and action. During linear postnatal growth, T3 supports the actions of growth hormone, insulinlike growth factor (IGF)-I, and other growth factors. T3 also supports normal adult bone remodeling.

1	The progression of tooth development and eruption depends on thyroid hormone, as does the normal cycle of growth and maturation of the epidermis, its hair follicles, and nails. The normal degradative processes in these structural and integumentary tissues are stimulated by thyroid hormone. Thus either too much or too little thyroid hormone can lead to hair loss and abnormal nail formation. Thyroid hormone regulates the structure of subcutaneous tissue by inhibiting synthesis and increasing degradation of mucopolysaccharides (glycosaminoglycans) and fibronectin in the extracellular connective tissue (see later description of myxedema). Effects on the Nervous System

1	Effects on the Nervous System Thyroid hormone regulates the timing and pace of development of the CNS. Thyroid hormone deficiency in utero and in early infancy inhibits growth of the cerebral and cerebellar cortex, proliferation of axons and branching of dendrites, synaptogenesis, myelination, and cell migration. Irreversible CNS impairment results when neonatal thyroid hormone deficiency is not recognized and treated promptly. These morphological defects are paralleled by biochemical abnormalities. Decreased thyroid hormone levels reduce cell size, RNA and protein content, tubulinand microtubuleassociated protein, protein and lipid content of myelin, local production of critical growth factors, and rates of protein synthesis.

1	Thyroid hormone also enhances wakefulness, alertness, responsiveness to various stimuli, auditory sense, awareness of hunger, memory, and learning capacity. In addition, normal emotional tone depends on proper thyroid hormone availability. Furthermore the speed and amplitude of peripheral nerve reflexes are increased by thyroid hormone, as is motility of the gastrointestinal tract. In both women and men, thyroid hormone plays an important permissive role in regulation of reproductive function. The normal ovarian cycle of follicular development, maturation, and ovulation, the homologous testicular process of spermatogenesis, and maintenance of the healthy pregnant state are all disrupted by significant deviations in thyroid hormone levels from the normal range. In part these deleterious effects may be caused by alterations in the metabolism or availability of steroid hormones. For example, thyroid hormone stimulates hepatic synthesis and release of sex steroid–binding globulin.

1	Hypothyroidism referstoinsufficientproductionofthyroidhormonesandcanoccurasprimary,secondary,ortertiaryendocrinedisease(see ).Inprimaryhypothyroidism,T4andT3levelsareabnormallylowandTSHishigh.Insecondaryandtertiaryhypothyroidism,boththyroidhormonesandTSHarelow.TheresponseofTSHlevelstosyntheticTRHcanbeusedtodistinguishbetweenpituitaryandhypothalamicdisease.

1	Fig.42.12 ]).Affectedindividualshavesevereintellectualdisability,shortstaturewithincompleteskeletaldevelopment,coarsefacialfeatures,andaprotrudingtongue.Themostcommoncauseofhypothyroidisminchildrenworldwideisiodidedeficiency.Historically,iodidedeficiencywasviewedasamajorcauseofhypothyroidismincertainmountainousregionsofSouthAmerica,Africa,andAsia,butrecentevidencesuggeststhattheproblemisevenmorewidespread.Thistragicformofendemic hypothyroidism canbepreventedbypublichealthprogramsthataddiodidetotablesaltorprovideyearlyinjectionsofaslowlyabsorbediodidepreparation.Congenital defects arealesscommoncauseofneonatal/childhypothyroidism.Inmostcasesthethyroidglandsimplydoesnotdevelop(thyroid gland dysgenesis). Lessfrequentcausesofchildhood •Fig. 42.12 A,Normal6-year-oldchild(left) andacongenitallyhypothyroid17-year-old(right)

1	gland dysgenesis). Lessfrequentcausesofchildhood •Fig. 42.12 A,Normal6-year-oldchild(left) andacongenitallyhypothyroid17-year-old(right) fromthesamevillageinanareaofendemichypothyroidism.Notetheshortstature,obesity,malformedlegs,anddullexpressionoftheintellectuallydisabledhypothyroidchild.Otherfeaturesareaprominentabdomen,aflatbroadnose,ahypoplasticmandible,dryscalyskin,delayedpuberty,andmuscleweakness.Radiographsofthehandcomparinganormal13-year-old(B)tothatofa13-year-oldsufferingfromhypothyroidism(C).Notethatthepatientwithhypothyroidismhasamarkeddelayindevelopmentofthesmallbonesofthehands,ingrowthplatesateitherendofthefingers,andinthegrowthplateofthedistalradius.(A,FromDelangeFM.In:BravermanLE,UtigerRD[eds].Werner and Ingbar’s The Thyroid. 7thed.Philadelphia:Lippincott-Raven;1996.B,FromTannerJMetal.

1	Assessment of Skeletal Maturity and Prediction of Adult Height (TW2 Method). NewYork:AcademicPress;1975.C,FromAndersenHJ.In:GardnerLI[ed].Endocrine and Genetic Diseases of Childhood and Adolescence. Philadelphia:Saunders;1975.) hypothyroidismaremutationsingenesinvolvedinthyroidhormoneproduction(e.g.NIS,TPO,thyroglobulin,pendrin)orblockingantibodiestotheTSHreceptor.Theseverityoftheneurologicalandskeletaldefectsiscloselylinkedtothetimingofdiagnosisandthyroidhormone(T4)replacement,withearlytreatmentresultinginanormalcognitiveabilityandsubtleneurologicaldeficits.Ontheotherhand,ifhypothyroidismatbirthremainsuntreatedforonly2to4weeks,theCNSwillnotmaturenormallyinthefirstyearoflife.Developmentalmilestonessuchassitting,standing,andwalkingwillbelate,andsevereirreversiblecognitivedeficitscanresult.Hypothyroidbabiesusuallyappearnormalatbirthbecauseofprotectionbymaternalthyroidhormones.Neonatal screening

1	screening (T4andTSHlevels)hasthereforeplayedacriticalroleindiagnosisandpreventionofcongenitalhypothyroidism.

1	HypothyroidisminadultswhoarenotiodidedeficientmostoftenresultsfromanotherautoimmunedisorderknownasHashimoto’s disease (formerlycalledlymphocytic thyroiditis).IncontrasttothestimulatoryeffectofautoantibodiesseeninGravesdisease,thyroidautoantibodiesinHashimoto’sdisease(againstTPO,thyroglobulin,orTSHreceptor)causeapoptosisofthyroidcellsanddestructionofthyroidfollicles.Theseantibodiesfixcomplementandpromotelysisofthyroidcells,causingreleaseofthyroglobulinintothecirculation.ThethyroidglandbecomesinfiltratedbybothBandTlymphocytes,whichmaycauseenlargementofthegland. Othercausesofhypothyroidismincludeiatrogeniccauses(e.g.,radiochemicaldamageorsurgicalremovalfortreatmentofhyperthyroidism),nodulargoiters,andpituitaryorhypothalamicdisease.Treatmentofpatientswiththeantiarrhythmicdrugamiodarone,whichcontainsalargeamountofiodine,maycauseeitherhypo-orhyperthyroidism.Thyroidfunctionmustbecarefullymonitoredinpatientstakingthismedication.

1	Theclinicalpictureofhypothyroidisminadultsisinmanyrespectstheexactoppositeofthatseeninhyperthyroidism.Thelower-than-normalmetabolicrateleadstoweightgainwithoutanappreciableincreaseincaloricintake.Thedecreasedthermogenesislowersbodytemperatureandcausesintolerancetocold,decreasedsweating,anddryskin.Adrenergicactivityisdecreased,andthereforebradycardiamayoccur.Movement,speech,andthoughtareallslowed,andlethargy,sleepiness,andloweringoftheuppereyelids(ptosis)occur.Accumulationofnegativelychargedmucopolysaccharidesinconnectivetissuesattractssodiumandfluid.Theresultingnonpittingmyxedema

1	producespuffyfeatures,anenlargedtongue,hoarseness,jointstiffness,effusionsinthepleural,pericardial,andperitonealspaces,andpressureonperipheralandcranialnervesentrappedbyexcessgroundsubstance.Constipation,lossofhair,menstrualdysfunction,andanemiaareothersigns.Inadultslackingthyroidhormone,positronemissiontomographydemonstratesageneralizedreductionincerebralbloodflowandglucosemetabolism.Thisabnormalitymayexplainthepsychomotorimpairmentanddepressedaffectofhypothyroidindividuals.

1	ReplacementtherapywithadailydoseofT4thatnormalizesTSHlevelsisusuallycurativeinadults.Inmostpatients,T3isnotneededbecauseitisgeneratedasneededbyperipheralD1andD2.Furthermore,administrationofT3iscomplicatedbyitshighpotencyandshorthalf-life,requiringfrequentdosingandcausingdifficultyinmaintainingconsistentphysiologicallevelsofT3. Thyroidhormonealsohassignificanteffectsonotherpartsoftheendocrinesystem.Pituitaryproductionofgrowthhormoneisincreasedbythyroidhormone,whereasthatofprolactinisdecreased.Adrenocorticalsecretionofcortisol(see )aswellasmetabolicclearanceofthishormoneisstimulated,butplasmafreecortisollevelsremainnormal.Theratioofestrogenstoandrogens(see )isincreasedinmen(inwhombreastenlargementmayoccurwithhyperthyroidism).Decreasesinbothparathyroidhormoneand1,25-(OH)2-vitaminDproductionarecompensatoryconsequencesoftheeffectsofthyroidhormoneonboneresorption(see

1	Chapter40).Kidneysize, renalplasmaflow,glomerularfiltrationrate,andtransportratesforanumberofsubstancesarealsoincreasedbythyroidhormone. Non-thyroidal illness syndrome (NTIS), also known as euthyroid sick syndrome, occurs frequently in severely ill patients who require hospitalization. NTIS is characterized by decreased levels of both circulating thyroid hormone and TSH caused by CNS-mediated suppression of the hypothalamic-pituitary-thyroid axis. In addition, peripheral metabolism of T4 to inactive rT3 is also increased. A similar pattern is seen upon prolonged fasting. Although it remains incompletely understood, NTIS has been proposed to represent a physiological energy-sparing adaptation to chronic illness or starvation. Mechanism of Thyroid Hormone Action

1	Mechanism of Thyroid Hormone Action Formanyyearsitwasthoughtthatthyroidhormonesdiffusepassivelyacrosscellmembranes,butthisisnowknowntorequiretransportproteins.TheseincludethemonocarboxylatetransportersMCT8andMCT10,whicharecapableoftransportingbothT4andT3acrosstheplasmamembrane( Fig. 42.13 Fig.42.5 ).Recently,mutationsinMCT8havebeenshowntocauseanX-linkeddevelopmentalsyndromeinhumanscharacterizedbyelevatedT3levels,musclehypoplasia,andsevereneurologicalimpairment.Anothertransporter,OATP1C1,appearstoplayaroleintransportofT4acrosstheblood-brainbarrier.

1	ManybutnotallT3actionsaremediatedthroughitsbindingtooneofthemembersofthethyroid hormone receptor (TR) family. TheTRfamilybelongstothenuclearhormonereceptorsuperfamilyoftranscriptionfactors(seealso ).Inhumanstherearetwothyroidhormonereceptorgenes,THRA andTHRB, locatedonchromosomes17and3,respectively,thatencodethenuclearthyroidhormonereceptors.THRAencodesTRα, whichisalternativelysplicedtoformtwomainisoforms.TRα1isabonafideTR,whereastheotherisoformdoesnotbindT3.THRBencodesTRβ1 andTRβ2, whicharehigh-affinityreceptorsforT3.ThetissuedistributionofTRα1andTRβ1iswidespread.TRα1isstronglyexpressedincardiacandskeletalmuscle.TRα1istheprimarymediatorofthyroidhormoneactionontheheart.Incontrast,TRβ1isexpressedmainlyinbrain,liver,andkidney.TRβ2expressionisrestrictedtothepituitaryandcriticalareasofthehypothalamus,aswellasthecochleaandretina.T3actingviaTRβ2isresponsibleforinhibitingexpressionoftheprepro-TRHgeneintheparaventricularneuronsofthehypothalamusandtheβ

1	subunitTSHgeneinpituitarythyrotropes.Thusthenegative-feedbackeffectsofthyroidhormoneonbothTRHandTSHsecretionarelargelymediatedbyTRβ2.

1	•Fig. 42.13 Mechanismsofthyroidhormoneaction,includingtheroleofMCTtransporters,D2deiodinase,andTR-RXRheterodimers.CoA,coactivator;CoR,co-repressor;RXR,retinoidXreceptor. Fig.42.13 ).UnligandedTR-RXRbindstothyroidresponseelementsintargetgenesandrecruitsco-repressorsthatinhibitgenetranscription.UponT3binding,theco-repressorsarereleased,andcoactivatorsarerecruitedtothehormone-receptorcomplex,inducinggenetranscription.

1	AnunderstandingofTRsubtypesisimportantbecauseinactivatingTRmutationshavebeenfoundtocausethyroid hormone resistance syndromes.ThemostcommonmutationsoccurintheTRβ2subtype,resultinginincompletenegativefeedbackatthehypothalamic-pituitarylevel.ThusT4levelsareelevated,butTSHisnotsuppressed.Whenresistanceispredominantlyatthehypothalamic-pituitarylevel,thepatientmayexhibitsignsofhyperthyroidismduetoeffectsofelevatedthyroidhormonelevelsonperipheraltissues,particularlyontheheart,mediatedbyTRα1.TRisoformsmayalsoofferpotentialtherapeutictargets.Forexample,researchisunderwaytodevelopTRβ-specificagoniststhathavebeneficialeffectsonlipidandcholesterolmetabolismwithouttheriskofadversecardiovascularsideeffects.

1	ThereisemergingevidencefornongenomicactionsofT3andT4thataremediatedbyreceptorsactingintheplasmamembrane,mitochondria,orcytoplasm.Insomecasesthesearemodifiedversionsofthenuclearthyroidreceptors.Forexample,truncatedisoformsofTRα1havebeenreportedthatbindT3intheplasmamembranetomediatenongenomiceffectsinboneorthatbindT4inthecytoplasmtoregulatemicrofilamentorganization.Ithasalsobeenreportedthatanintegrin,αvβ3,canactasaT4receptoratthecellsurfacetoregulatecellularproliferationandangiogenesisbyanongenomicmechanism.Theinterplaybetweentheclassicalgenomicandnongenomicactionsofthyroidhormonesislikelytobeanotheractiveareaoffutureresearch. 1. The thyroid gland is situated in the ventral aspect of the neck and is composed of right and left lobes anterolateral to the trachea and connected by an isthmus. 2. The thyroid gland is the source of tetraiodothyronine (thyroxine, T4) and triiodothyronine (T3). 3.

1	2. The thyroid gland is the source of tetraiodothyronine (thyroxine, T4) and triiodothyronine (T3). 3. The basic endocrine unit in the gland is a follicle that consists of a single spherical layer of epithelial cells surrounding a central lumen that contains colloid or stored hormone. 4. Iodide is taken up into thyroid cells by a sodium-iodide symporter in the basolateral plasma membrane. 5. T4 and T3 are synthesized from tyrosine and iodide by the enzyme complex of dual oxidase and thyroid peroxidase. Tyrosine residues in thyroglobulin undergo iodination, after which two iodotyrosine molecules are coupled to yield the iodothyronines. 6. Secretion of stored T4 and T3 requires retrieval of thyroglobulin from the follicle lumen by endocytosis. Thyroglobulin is then degraded in endolysosomes to liberate T4 and T3. Iodide is conserved by recycling any iodotyrosine molecules that did not undergo coupling within thyroglobulin. 7.

1	7. TSH acts on the thyroid gland via its plasma membrane receptor to stimulate all steps in the production of T4 and T3. These steps include iodide uptake, iodination and coupling, and retrieval from thyroglobulin. TSH also stimulates glucose oxidation, protein synthesis, and growth of epithelial cells. 8. More than 99.5% of T4 and T3 circulates bound to the following proteins: thyroid-binding globulin, Bernal J, et al. Thyroid hormone transporters–functions and clinical implications. Nat Rev Endocrinol. 2015;11:406-417. Bianco AC. Minireview: cracking the metabolic code for thyroid hormone signaling. Endocrinology. 2011;152:3306-3311. transthyretin, and albumin. Only the free fractions of T4 and T3 are biologically active. 9.

1	transthyretin, and albumin. Only the free fractions of T4 and T3 are biologically active. 9. T4 functions largely as a prohormone whose disposition is regulated by three types of deiodinases. Monodeiodination of the outer ring yields 75% of the daily production of T3, which is the principal active hormone. Alternatively, monodeiodination of the inner ring yields reverse T3, which is biologically inactive. Proportioning of T4 between T3 and reverse T3 regulates the availability of active thyroid hormone. 10. Thyroid hormone is a major positive regulator of the basal metabolic rate and thermogenesis. Other important actions of thyroid hormone are increased heart rate, cardiac output, and ventilation and decreased systemic vascular resistance. Substrate mobilization and disposal of metabolic products are enhanced. 11.

1	11. Thyroid hormone action on the CNS and skeleton are crucial for normal growth and development. Absence of the hormone causes congenital hypothyroidism, characterized by poor brain development, short stature, and immature skeletal development. In adults, thyroid hormone supports bone remodeling and degradation of skin and hair. 12. T3 binds to thyroid hormone receptor subtypes responsible for the various actions of thyroid hormone. The thyroid hormone receptor heterodimerizes with RXR to regulate thyroid response elements on target genes, resulting in induction or repression in the presence or absence of T3, respectively. Brent GA. Mechanisms of thyroid hormone action. J Clin Invest. 2012;122:3035-3043. de Vries EM, et al. The molecular basis of the non-thyroidal illness syndrome. J Endocrinol. 2015;225:R67-R81. Krude H, et al. Treatment of congenital thyroid dysfunction: achievements and challenges. Best Pract Res Clin Endocrinol Metab. 2015;29:399-413.

1	Krude H, et al. Treatment of congenital thyroid dysfunction: achievements and challenges. Best Pract Res Clin Endocrinol Metab. 2015;29:399-413. Upon completion of this chapter the student should be able to answer the following questions: 1. Describe the anatomy and microscopic anatomy of the adrenal gland, including the chromaffin cells of the adrenal medulla and the three zones of the adrenal cortex. 2. Explain the enzymatic reactions involved in generating norepinephrine and epinephrine and integrate those reactions with the regulation of epinephrine synthesis and secretion by the adrenal medulla. 3. Utilize the specific actions of catecholamines to explain an overall sympathetic response to a stress imposed on the body. 4. Describe the first two common reactions of the steroidogenic pathway, and their subcellular locations, and the function of StAR protein in the first reaction. 5.

1	4. Describe the first two common reactions of the steroidogenic pathway, and their subcellular locations, and the function of StAR protein in the first reaction. 5. Compare the steroidogenic pathways within the zona glomerulosa, zona fasciculata, and zona reticularis with respect to common and zona-specific reactions. 6. Describe the mechanism of action of glucocorticoids and mineralocorticoids, including the cross-reactivity of cortisol with the mineralocorticoid receptor, and the mechanism to prevent this. 7. Integrate the multiple actions of cortisol throughout the body to explain the hormone’s role during normal development and physiology, and to describe the multiple aspects of the pathophysiology of Addison’s disease and Cushing’s syndrome. 8.

1	8. Map out the hypothalamic-pituitary-adrenal axis, including the “loophole” in the feedback mechanisms that leads to excessive androgen production (e.g., in congenital adrenal hyperplasia) in the face of an enzyme deficiency specific to the zona fasciculata and cortisol synthesis. 9. Review the regulation and actions of aldosterone.

1	9. Review the regulation and actions of aldosterone. n adults the adrenal glands emerge as fairly complex endocrine structures that produce two structurally distinct classes of hormones: steroids and catecholamines. The catecholamine hormone epinephrine acts as a rapid responder to stresses such as hypoglycemia and exercise to regulate multiple parameters of physiology, including energy metabolism and cardiac output. Stress is also a major secretagogue of the longer-acting steroid hormone cortisol, which regulates glucose utilization, immune and inflammatory homeostasis, and numerous other processes. In addition the adrenal glands regulate salt and volume homeostasis through the steroid hormone aldosterone. Finally, the adrenal gland secretes large amounts of the androgen precursor dehydroepiandrosterone sulfate (DHEAS), which plays a major role in fetoplacental estrogen synthesis and as a substrate for peripheral androgen synthesis in women.

1	The adrenal glands are bilateral structures located immediately above the kidneys (ad, near; renal, kidney) ( Fig. 43.1 ). In humans they are also referred to as the suprarenal glands because they sit on the superior pole of each kidney. The adrenal glands are similar to the pituitary in that they are derived from both neuronal tissue and epithelial (or epithelial-like) tissue. The outer portion of the adrenal gland, called the adrenal cortex ( Fig. 43.2 ), develops from mesodermal cells in the vicinity of the superior pole of the developing kidney. These cells form cords of epithelial endocrine cells. The cells of the cortex develop into steroidogenic cells (see ). In adults the adrenal cortex is composed of three zones—the zona glomerulosa, the zona fasciculata, and the zona reticularis—that produce mineralocorticoids, glucocorticoids, and adrenal androgens, respectively (see Fig. 43.2B

1	Fig. 43.2B Soon after the cortex forms, neural crest–derived cells associated with the sympathetic ganglia, called chromaffin cells, migrate into the cortex and become encapsulated by cortical cells. Thus the chromaffin cells establish the inner portion of the adrenal gland, which is called the adrenal medulla (see Fig. 43.2). The chromaffin cells of the adrenal medulla have the potential to develop into postganglionic sympathetic neurons. They are innervated by cholinergic preganglionic sympathetic neurons and can synthesize the catecholamine neurotransmitter norepinephrine from tyrosine. However, high levels of cortisol that drain into the medulla from the adrenal cortex induce expression of the enzyme phenylethanolamine N-methyl transferase (PNMT), which transfers a methyl group onto norepinephrine to produce the catecholamine hormone epinephrine, the primary hormonal product of the adrenal medulla (see Fig. 43.2B).

1	Fig. 43.2B). CHAPTER 43 The Adrenal Gland 767 • Fig. 43.1 The adrenal glands sit on the superior poles of the kidneys and receive a rich arterial supply from the inferior, middle, and superior suprarenal arteries. The adrenals are drained by a single suprarenal vein. (Modified from Drake RL et al. Gray’s Anatomy for Students. Philadelphia: Churchill Livingstone; 2005.)

1	Instead of being secreted near a target organ and acting as neurotransmitters, adrenomedullary catecholamines are secreted into blood and act as hormones. About 80% of the cells of the adrenal medulla secrete epinephrine, and the remaining 20% secrete norepinephrine. Although circulating epinephrine is derived entirely from the adrenal medulla, only about 30% of the circulating norepinephrine comes from the medulla. The remaining 70% is released from postganglionic sympathetic nerve terminals and diffuses into the vascular system. Because the adrenal medulla is not the sole source of catecholamine production, this tissue is not essential for life. Synthesis of Epinephrine The enzymatic steps in epinephrine synthesis are shown in

1	Synthesis of Epinephrine The enzymatic steps in epinephrine synthesis are shown in Fig. 43.4 . Synthesis begins with transport of the amino acid tyrosine into the chromaffin cell cytoplasm and subsequent hydroxylation of tyrosine by the rate-limiting enzyme tyrosine hydroxylase to produce dihydroxyphenylalanine (DOPA). DOPA is converted to dopamine by a cytoplasmic enzyme, aromatic amino acid decarboxylase, and is then transported into the secretory vesicle (also called the chromaffin granule). Within the granule, all dopamine is completely converted to norepinephrine by the enzyme dopamine β-hydroxylase. In most adrenomedullary cells, essentially all of the norepinephrine diffuses out of the chromaffin granule by facilitated transport and is methylated by the cytoplasmic enzyme PNMT to form epinephrine.

1	The high local concentration of cortisol in the medulla is maintained by the vascular configuration within the adrenal gland. The outer connective tissue capsule of the adrenal gland is penetrated by a rich arterial supply coming from three main arterial branches: the inferior, middle, and superior suprarenal arteries (see

1	Fig. 43.1 ). These give rise to two types of blood vessels that carry blood from the cortex to the medulla (Fig. 43.3 ): (1) relatively few medullary arterioles, which provide high oxygen- and nutrient-laden blood directly to the medullary chromaffin cells, and (2) relatively numerous cortical sinusoids into which cortical cells secrete steroid hormones (including cortisol). Both vessel types fuse to give rise to the medullary plexus of vessels that ultimately drains into a single suprarenal vein. Thus secretions of the adrenal cortex percolate through the chromaffin cells and bathe them in high concentrations of cortisol before leaving the gland and entering the inferior vena cava. Cortisol inhibits neuronal differentiation of the medullary cells, so they fail to form dendrites and axons. Additionally, cortisol induces expression of the enzyme PNMT, which converts norepinephrine to epinephrine (

1	Fig. 43.4 ). Glucocorticoid receptor–knockout mice have an enlarged cortex, but the size of the medulla is decreased and PNMT activity is undetectable. Epinephrine is then transported back into the granule for storage and to undergo regulated exocytosis. Secretion of epinephrine and norepinephrine from the adrenal medulla is regulated primarily by descending sympathetic signals in response to various forms of stress, including exercise, hypoglycemia, and hemorrhagic hypovolemia •Fig. 43.2 Histologyoftheadrenalgland.A,Lowmagnificationillustratingtheoutercortex(C)andinnermedulla(M;notethecentralvein[V]).B,Highermagnificationclearlyillustratingthezonationofthecortex.Thecorrespondingendocrinefunctionandthedifferentzonesofthecortexandthemedullaarenoted.(FromYoungBetal.Wheater’s Functional Histology. 5thed.Philadelphia:ChurchillLivingstone;2006.)

1	Fig. 43.5 ). The primary autonomic centers that initiate sympathetic responses reside in the hypothalamus and brainstem, and they receive input from the cerebral cortex, the limbic system, and other regions of the hypothalamus and brainstem. The chemical signal for secretion of catecholamine from the adrenal medulla is acetylcholine (ACh), which is secreted from preganglionic sympathetic neurons and binds to nicotinic receptors on chromaffin cells (see Fig. 43.5 ). ACh increases the activity of the rate-limiting enzyme tyrosine hydroxylase in chromaffin cells (see Fig. 43.4 ). It also increases the activity of dopamine β-hydroxylase and stimulates exocytosis of the chromaffin granules. Synthesis of epinephrine and norepinephrine is closely coupled to secretion so that levels of intracellular catecholamines do not change significantly even in the face of changing sympathetic activity. Mechanism of Action of Catecholamines

1	Mechanism of Action of Catecholamines Adrenergic receptors are generally classified as α-and β-adrenergic receptors, with the α-adrenergic receptors • Fig. 43.3 Blood flow through the adrenal gland. Capsular arteries give rise to sinusoidal vessels that carry blood centripetally through the cortex to the medulla. (Modified from Young B et al. Wheater’s Functional Histology. 5th ed. Philadelphia: Churchill Livingstone; 2006.) further divided into α1 and α2 receptors and the β-adrenergic receptors divided into β1, β2, and β3 receptors ( Table 43.1 ). These receptors can be characterized according to: 1. Relative potency of endogenous and pharmacological agonists and antagonists. Epinephrine and norepinephrine are potent agonists for α receptors and for β1 and β3 receptors, whereas epinephrine is more potent than norepinephrine for β2 receptors. A large number of synthetic selective and nonselective adrenergic agonists and antagonists now exist. 2. Downstream signaling pathways.

1	2. Downstream signaling pathways. Table 43.1 shows the primary pathways that are coupled to the different adrenergic receptors. This is an oversimplification, because differences in signaling pathways for a given receptor have been linked to the duration of agonist exposure and cell type. 3. Location and relative density of receptors. Importantly, different receptor types predominate in different tissues. For example, although both α and β receptors are expressed by pancreatic islet beta cells, the predominant response to a sympathetic discharge is mediated by α2 receptors. Physiological Actions of Adrenomedullary Catecholamines

1	Physiological Actions of Adrenomedullary Catecholamines Because the adrenal medulla is directly innervated by the autonomic nervous system, adrenomedullary responses are very rapid. Furthermore, because of the involvement of several centers in the central nervous system (CNS), most notably the cerebral cortex, adrenomedullary responses can precede onset of the actual stress (i.e., they can be anticipated) (see Fig. 43.5 ). In many cases the adrenomedullary output, which is primarily epinephrine, is coordinated with sympathetic nervous activity as determined by the release of norepinephrine from postganglionic sympathetic neurons. However, some stimuli (e.g., hypoglycemia) evoke a stronger adrenomedullary than sympathetic nervous response and vice versa. Many organs and tissues are affected by a sympathoadrenal response (

1	Many organs and tissues are affected by a sympathoadrenal response ( Table 43.2). An informative example of the major physiological roles of catecholamines is the sympathoadrenal response to exercise. Exercise is similar to the “fight-or-flight” response but without the subjective element of fear, and it involves a greater adrenomedullary response (i.e., endocrine role of epinephrine) than a sympathetic nervous response (i.e., neurotransmitter role of norepinephrine). The overall goal of the sympathoadrenal system during exercise is to meet the increased energy demands of skeletal and cardiac muscle while maintaining sufficient oxygen and glucose supply to the brain. The response to exercise includes the following major physiological actions of epinephrine ( Fig. 43.6): 1. Increased blood flow to muscles is achieved by the integrated action of norepinephrine and epinephrine on the heart, veins and lymphatics, and nonmuscular (e.g., splanchnic) and muscular arteriolar beds.

1	•Fig. 43.4 Stepsinsynthesisandsecretionofcatecholaminesfromadrenalmedullarychromaffincells. 2. Epinephrine promotes glycogenolysis in muscle. Exercising muscle can also utilize free fatty acids (FFAs), and epinephrine and norepinephrine promote lipolysis in adipose tissue. Epinephrine increases blood glucose by increasing hepatic glycogenolysis and gluconeogenesis. The promotion of lipolysis in adipose tissue is also coordinated with an epinephrine-induced increase in hepatic ketogenesis. Finally, the effects of catecholamines on metabolism are reinforced by the fact that they stimulate glucagon secretion (β2 receptors) and inhibit insulin secretion (α2 receptors). Efficient production of adenosine triphosphate (ATP) during normal exercise •Fig. 43.5 Stimulithatenhancecatecholaminesecretion. cAMP,cyclicadenosinemonophosphate;DAG,diacylglycerol. Arteriolardilation:↓ BP↑ Arteriolarvasoconstriction; (β2)(muscle)↑ BP(α1)(splanchnic,renal,cutaneous,genital) BP,bloodpressure.

1	cAMP,cyclicadenosinemonophosphate;DAG,diacylglycerol. Arteriolardilation:↓ BP↑ Arteriolarvasoconstriction; (β2)(muscle)↑ BP(α1)(splanchnic,renal,cutaneous,genital) BP,bloodpressure. (i.e., a 1-hour workout) also requires efficient exchange of gases with an adequate supply of oxygen to exercising muscle. Catecholamines promote this by relaxation of bronchiolar smooth muscle. 3. Catecholamines decrease energy demand by visceral smooth muscle. In general a sympathoadrenal response decreases overall motility of the smooth muscle in the gastrointestinal (GI) and urinary tracts, thereby conserving energy where it is not immediately needed. Metabolism of Catecholamines

1	Metabolism of Catecholamines Two primary enzymes are involved in the degradation of catecholamines: monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). The neurotransmitter norepinephrine is degraded by MAO and COMT after uptake into the presynaptic terminal. This mechanism is also involved in the catabolism of circulating adrenal catecholamines. However, the predominant fate of adrenal catecholamines is methylation by COMT in nonneuronal tissues such as the liver and kidney. Urinary vanillylmandelic acid (VMA) and metanephrine are sometimes used clinically to assess the level of catecholamine production in a patient. Much of the urinary VMA and metanephrine is derived from neuronal rather than adrenal catecholamines.

1	Pheochromocytoma isatumorofchromaffintissuethatproducesexcessivequantitiesofcatecholamines.Thesearecommonlyadrenalmedullarytumors,buttheycanoccurinotherchromaffincellsoftheautonomicnervoussystem.Althoughpheochromocytomasarenotcommontumors,theyarethemostcommoncauseofhyperfunctioningoftheadrenalmedullary.Thecatecholaminemostfrequentlyelevatedinpheochromocytomaisnorepinephrine.Forunknownreasonsthesymptomsofexcessivecatecholaminesecretionareoftensporadicratherthancontinuous.Symptomsincludehypertension,headaches(fromhypertension),sweating,anxiety,palpitations,andchestpain.Inaddition,patientswiththisdisordermayshoworthostatichypotension(despitethetendencyforhypertension).Thisoccursbecausehypersecretionofcatecholaminescandecreasethepostsynapticresponsetonorepinephrineasaresultofdownregulationofthereceptors(see ).Consequentlythebaroreceptorresponsetobloodshiftsthatoccursonstandingisblunted.

1	The zona fasciculata produces the glucocorticoid hormone cortisol. This zone is an actively steroidogenic tissue composed of straight cords of large cells. These cells have a “foamy” cytoplasm because they are filled with lipid droplets that represent stored cholesterol esters (CEs). These cells make some cholesterol de novo but import a significant amount of cholesterol from the blood in the form of low-density lipoprotein (LDL). LDL particles bind to their receptor (LDLR) and are endocytosed. Within endolysosomes, free cholesterol (FC) is released from CEs by a lysosomal lipase, and the FC is transported out of the endolysosome by Niemann-Pick C (NPC) proteins. Free cholesterol is stored in lipid droplets in the cytoplasm after esterification by acyl CoA-cholesterol acyltransferase (ACAT) (

1	Fig. 43.7 ). The stored cholesterol is continually turned back into free cholesterol by hormone-sensitive lipase (HSL), a process that is increased in response to adrenocorticotropic hormone (ACTH; see Regulation of All steroid hormone synthesis begins in the mitochondria, where the first enzyme, CYP11A1, is attached to the inner mitochondrial membrane. Although several proteins appear to be involved in the transfer of FC into the inner mitochondrial matrix, one protein called steroidogenic acute regulatory protein (StAR protein) is indispensable in this process (see

1	Fig. 43.7). StAR protein is short-lived and rapidly activated posttranslationally (phosphorylation) and transcriptionally by pituitary tropic hormones. In patients with inactivating mutations in StAR protein, cells of the zona fasciculata become excessively laden with lipid (“lipoid”) because cholesterol cannot be accessed by CYP11A1 within the mitochondria and used for cortisol •Fig. 43.6 Someoftheindividualactionsofcatecholaminesthatcontributetotheintegratedsympa-thoadrenalresponsetoexercise.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.)Bronchiole (˜2) Dilation ° Exchange of O2 and CO2 ° Cardiac output ° Venous return GI and urinary tracts (˜2) ˝ Motility ˝ Energy usage ° Blood glucose ° Blood ketones ° Blood FFAs ° Blood lactate ° Blood glycerol Adipose (˜2, ˜3) ° Lipolysis ˝ Glucose uptake Liver (˜2) ° Glycogenolysis ° Gluconeogenesis ° Ketogenesis Hormonal reinforcement Skeletal muscle (˜2) ° Glycogenolysis ˝ Glucose

1	° Blood glycerol Adipose (˜2, ˜3) ° Lipolysis ˝ Glucose uptake Liver (˜2) ° Glycogenolysis ° Gluconeogenesis ° Ketogenesis Hormonal reinforcement Skeletal muscle (˜2) ° Glycogenolysis ˝ Glucose uptake ˜ Cells (˙2) ˝ Insulin secretion ˙ Cells (˜2) ° Glucagon secretion ° Blood glucagon/insulin ratio ° Nutrient supply to muscle and adequate supply of oxygen and glucose to brain ˝ Blood flow to GI tract ° Blood flow to skeletal muscle Splanchnic arterioles (˙) Vasoconstriction Skeletal muscle arterioles (˜2) Vasodilation Veins and lymphatics (˙) Vasoconstriction Heart (˜1) (+)ˆve Inotropic (+)ˆve Chronotropic (+)ˆve Lusitropic •Fig. 43.7 Eventsinvolvedinthefirsttworeactionsinthesteroidogenicpathway:conversionofcholesteroltopregnenolone;conversionofpregnenolone(P5)toprogesterone(P4)inzonafasciculatacells.ACAT,acylCoA:cholesterolacyltransferase;3β HSD,3β hydroxysteroiddehydrogenase;CE,cholesterolesters;CYP11A1alsocalledP450 side-chain cleavage enzyme;

1	HSD,3β hydroxysteroiddehydrogenase;CE,cholesterolesters;CYP11A1alsocalledP450 side-chain cleavage enzyme; FC,freecholesterol;HSL,hormone-sensitivelipase;LAL,lysosomalacidhydrolase;LDL,low-densitylipoprotein;LDLR,low-densitylipoproteinreceptor;P5,pregnenolone;StAR,steroidogenicacuteregulatoryprotein.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.) •Fig. 43.8 Summaryofthesteroidogenicpathwaysforeachofthethreezonesoftheadrenalcortex.Theenzymaticreactionsarecolorcodedacrosszones.sER,smoothendoplasmicreticulum.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.) synthesis. Moreover, these individuals cannot synthesize gonadal steroid hormones. The placenta does not express StAR, so these individuals have normal placental steroid production in utero.

1	Inthezonafasciculata,cholesterolisconvertedsequentially to pregnenolone, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, and cortisol (Figs. 43.8 43.9 ). A parallel pathway in the zona fasciculata involves a pathway that bypasses 17-hydroxylation, in which progesterone is converted to 11-deoxycorticosterone (DOC) and then to corticosterone (see Fig. 43.9C ). This pathway is minor in humans, but in the absence of active CYP11B1 (11-hydroxylase activity), the production of DOC is significant. Because DOC acts as a weak mineralocorticoid ( 43.3 ), elevated levels of DOC cause hypertension. Transport and Metabolism of Cortisol

1	Transport and Metabolism of Cortisol Cortisol is transported in blood predominantly bound to corticosteroid-binding globulin [CBG] (also called transcortin), which binds about 90% of cortisol, and albumin, which binds 5% to 7% of cortisol. The liver is the predominant site of steroid inactivation. It both inactivates cortisol and conjugates active and inactive steroids with glucuronide or sulfate so that they can be excreted more readily by the kidney. The circulating half-life of cortisol is about 70 minutes. Cortisol is reversibly inactivated by conversion to cortisone. This action is catalyzed by the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). Inactivation of cortisol by 11β-HSD2 occurs in cells that also express the mineralocorticoid receptor (MR)and are

1	Steroidogenicenzymesfallintotwosuperfamilies.Mostbelongtothecytochrome P-450 monooxidase gene family andarethusreferredtoasCYPs. Theseenzymesarelocatedeitherintheinnermitochondrialmatrix,wheretheyusemolecularoxygenandaflavoproteinelectrondonor,orinthesmoothendoplasmicreticulum,wheretheyuseadifferentflavoproteinforelectrontransfer.DifferentCYPenzymesactashydroxylases,lyases(desmolases),oxidases,oraromatases.Twooftheseenzymeshavemultiplefunctions.CYP17hasbotha17-hydroxylasefunctionanda17,20-lyase(desmolase)function.CYP11B2,alsocalledaldosterone synthase, hasthreefunctions:11-hydroxylase,18-hydroxylase,and18-oxidase.

1	Theotherenzymesinvolvedinsteroidogenesisbelongtothreehydroxysteroid dehydrogenase (HSD)families.3β-HSDs havetwoisoformsthatconvertthehydroxylgrouponcarbon3ofthecholesterolringtoaketoneandshiftthedoublebondfromthe5-6(Δ5) positiontothe4-5(Δ4) position.AllactivesteroidhormonesmustbeconvertedtoΔ4structuresby3β-HSD.The17β-HSDs haveatleastfivemembersandcanactaseitheroxidasesorreductases.17β-HSDsprimarilyactonsexsteroidsandcanbeactivatingordeactivating.Finally,the11β-HSDs havetwoisoformsthatcatalyzetheinterchangebetweencortisol(active)andcortisone(inactive). •Fig. 43.9 A,Reaction1,catalyzedbyCYP11A1,inmakingcortisol.B,Reactions2a/bandreactions3a/b,involvingCYP17(17-hydroxylasefunction)and3β-hydroxysteroiddehydrogenase(3β-HSD),inmakingcortisol.ThisfigureshowstheΔ5versusΔ4pathway.

1	•Fig. 43.9, cont’d C,Reactions4and5,involvingCYP21BandCYP11B1,inwhichthelasttwostepsincortisolsynthesisarecarriedout.Alsoshownistheminorpathwayleadingtocorticosteronesynthesisinthezonafasciculata.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.) target cells of aldosterone (see below). The conversion of cortisol to cortisone prevents the binding of cortisol to the MR and having inappropriate mineralocorticoid actions on these cells. Inactivation of cortisol by 11β-HSD2 is reversible in that another enzyme, 11β-HSD1, converts cortisone back to cortisol. This conversion occurs in tissues expressing the glucocorticoid receptor (GR), including liver, adipose tissue, and the CNS, as well as in skin. Mechanism of Action of Cortisol

1	Mechanism of Action of Cortisol Cortisol acts primarily through the glucocorticoid receptor, which regulates gene transcription (see ). In the absence of hormone, the GR resides in the cytoplasm in a stable complex with several molecular chaperones, including heat shock proteins and cyclophilins. Cortisol-GR binding promotes dissociation of the chaperone proteins, followed by: aAllvaluesarerelativetotheglucocorticoidandmineralocorticoidpotenciesofcortisol,whichhaveeachbeenarbitrarilysetat1.0.Cortisolactuallyhasonly1/500thepotencyofthenaturalmineralocorticoidaldosterone. 1. rapid translocation of the cortisol-GR complex into the nucleus, 2. dimerization and binding to glucocorticoid response elements (GREs, both “positive” GREs and “negative” GREs) near the basal promoters of cortisol-regulated genes, and 3. recruitment of coactivator proteins and assembly of general transcription factors leading to increased or decreased transcription of the targeted genes.

1	recruitment of coactivator proteins and assembly of general transcription factors leading to increased or decreased transcription of the targeted genes. In some cases the GR interacts with other transcription factors, such as the proinflammatory nuclear factor (NF)κB transcription factor, and interferes with their ability to activate gene expression. Physiological Actions of Cortisol

1	Physiological Actions of Cortisol Cortisol has a broad range of actions and is often characterized as a “stress hormone.” In general, cortisol maintains blood glucose levels, CNS function, and cardiovascular function during fasting and increases blood glucose during stress at the expense of muscle protein. Cortisol protects the body against the self-injurious effects of unbridled inflammatory and immune responses. Cortisol also partitions energy to cope with stress by inhibiting reproductive function. As stated later, cortisol has several other effects on bone, skin, connective tissue, the GI tract, and the developing fetus that are independent of its stress-related functions. As the term glucocorticoid implies, cortisol is a steroid hormone from the adrenal cortex that regulates blood glucose. It increases blood glucose by stimulating gluconeogenesis (

1	As the term glucocorticoid implies, cortisol is a steroid hormone from the adrenal cortex that regulates blood glucose. It increases blood glucose by stimulating gluconeogenesis ( Fig. 43.10 ). Cortisol enhances gene expression of the key hepatic gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Cortisol also decreases GLUT4-mediated glucose uptake in skeletal muscle and adipose tissue. During the interdigestive period (low insulin-glucagon ratio), cortisol promotes glucose sparing by potentiating the effects of catecholamines on lipolysis, thereby making FFAs available as energy sources. Cortisol inhibits protein synthesis and increases proteolysis, especially in skeletal muscle, thereby providing a rich source of carbon for hepatic gluconeogenesis.

1	Fig. 43.10 also contrasts the normal role of cortisol in response to stress and the effects of chronically elevated cortisol as a result of pathological conditions. As discussed later, there are important differences in the overall metabolic effects of cortisol between these two states, particularly with respect to lipid metabolism. During stress, cortisol synergizes with catecholamines and glucagon to promote a lipolytic, gluconeogenic, ketogenic, and glycogenolytic metabolic response while synergizing with catecholamines to promote an appropriate cardiovascular response. During chronically elevated cortisol secondary to pathological overproduction, cortisol synergizes with insulin in the context of elevated levels of glucose (from increased appetite) and hyperinsulinemia (from elevated glucose and glucose intolerance) to promote lipogenesis and truncal (abdominal/ visceral) adiposity.

1	Cortisol reinforces its effects on blood glucose by its positive effects on the cardiovascular system. Cortisol has permissive actions on catecholamines by increasing adrenergic receptor expression and thereby contributes to cardiac output and blood pressure. Cortisol stimulates erythropoietin synthesis and hence increases red blood cell production. Anemia occurs when cortisol is deficient, and polycythemia occurs when cortisol levels are excessive. Inflammation and immune responses are often part of the response to stress. However, inflammation and immune responses have the potential for significant harm and may cause death if they are not held in homeostatic balance. As a stress hormone, cortisol plays an important role in maintaining immune homeostasis. Cortisol, along with epinephrine and norepinephrine, represses production of proinflammatory cytokines and stimulates production of antiinflammatory cytokines.

1	Stress during interdigestive period (e.g., elderly person slips on ice and dislocates hip) Liver ˜ Glycogenolysis ˜ Gluconeogenesis Metabolic responses to stress ensure sufficient energy to meet increased demand on body, and will maintain adequate blood glucose levels necessary for conscious, deliberate action by the individual. The metabolic responses are supported by cardiovascular ones, first by those induced by the sympathoadrenal output (see Fig. 43.6) but also by cortisol, which optimizes adrenergic receptor function. Cortisol will also contribute to providing energy for the incipient inflammatory and immune response to this stress, but will also protect the individual from the potential damage of unregulated inflammation.

1	Metabolic responses to chronically elevated cortisol in patients with Cushing’s disease who are otherwise healthy tend to promote localized obesity (abdominal, neck, and face), with muscle wasting and weakness, especially at the extremities. Note that the elevated cortisol in this case usually occurs in the context of an elevated insulin/glucagon ratio. Elevated cortisol stimulates appetite, which increases the insulin/glucagon ratio. High levels of cortisol antagonize insulin’s effect on GLUT-4mediated glucose uptake into skeletal muscle and adipose tissue, so that glucose intolerance is frequently a symptom in patients with Cushing’s disease. This further contributes to hyperglycemia and hyperinsulinemia. Both cortisol and insulin promote the differentiation of preadipocytes into adipocytes, and lipogenesis.

1	Chronically elevated levels of cortisol in otherwise well-fed person (e.g., Cushing’s disease) •Fig. 43.10 Metabolicactionsofcortisol(integratedwithcatecholaminesandglucagon)inresponsetostress(upper panel) andcontrastedtotheactionsofchronicallyelevatedcortisol(integratedwithinsulin)inanotherwisehealthyindividual(lower panel). (ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.)

1	The inflammatory response to injury consists of local dilation of capillaries and increased capillary permeability, with resultant local edema and accumulation of white blood cells. These steps are mediated by prostaglandins, thromboxanes, and leukotrienes. Cortisol inhibits phospholipase A2, a key enzyme in prostaglandin, leukotriene, and thromboxane synthesis. Cortisol also stabilizes lysosomal membranes, thereby decreasing release of the proteolytic enzymes that augment local swelling. In response to injury, leukocytes normally leave the vascular system and migrate to the site of injury. This complex process is generally inhibited by cortisol, as is the phagocytic activity of neutrophils, although release of neutrophils from bone marrow is stimulated. Analogues of glucocorticoid are frequently used pharmacologically because of their antiinflammatory properties.

1	Cortisol inhibits the immune response, and for this reason glucocorticoid analogues have been used as immunosuppressants in organ transplants. High cortisol levels decrease the number of circulating T lymphocytes (particularly helper T lymphocytes) and reduce their ability to migrate to the site of antigenic stimulation. Glucocorticoids promote atrophy of the thymus and other lymphoid tissue. Although corticosteroids inhibit cellular-mediated immunity, antibody production by B lymphocytes is not impaired. Effects of Cortisol on the Reproductive Systems Reproduction exacts a considerable anabolic cost on the organism. In humans, reproductive behavior and function are dampened in response to stress. Cortisol decreases the function of the reproductive axis at the hypothalamic, pituitary, and gonadal levels. Effects of Cortisol on Bone

1	Effects of Cortisol on Bone Glucocorticoids increase bone resorption. They have multiple actions that alter bone metabolism. Glucocorticoids decrease intestinal Ca++ absorption and renal Ca++ reabsorption. Both mechanisms serve to lower serum [Ca++]. As serum [Ca++] drops, secretion of parathyroid hormone (PTH) increases, and PTH mobilizes Ca++ from bone by stimulating resorption of bone. In addition to this action, glucocorticoids directly inhibit osteoblast bone-forming functions (see ). Although glucocorticoids are useful for treating the inflammation associated with arthritis, excessive use will result in bone loss (osteoporosis). Actions of Cortisol on Connective Tissue Cortisol inhibits fibroblast proliferation and collagen formation. In the presence of excessive amounts of cortisol, the skin thins and is more readily damaged. The connective tissue support of capillaries is impaired, and capillary injury, or bruising, is increased. Actions of Cortisol on the Kidney

1	Actions of Cortisol on the Kidney Cortisol inhibits the secretion and action of antidiuretic hormone (ADH), and thus it is an ADH antagonist. In the absence of cortisol, the action of ADH is potentiated, which makes it difficult to increase free water clearance in response to a water load and increases the likelihood of water intoxication. Although cortisol binds to the mineralocorticoid receptor with high affinity, this action is normally blocked by inactivation of cortisol to cortisone by the enzyme 11βHSD2. However, the mineralocorticoid activity (i.e., renal Na+ and H2O retention, K+ and H+ excretion) of cortisol depends on the relative amount of cortisol (or synthetic glucocorticoids) and the activity of 11β-HSD2. Certain agents (e.g., compounds in black licorice) inhibit 11βHSD2 and thereby increase the mineralocorticoid activity of cortisol. Cortisol increases the glomerular filtration rate by both increasing cardiac output and acting directly on the kidney.

1	Actions of Cortisol on Muscle When cortisol levels are excessive, muscle weakness and pain are common symptoms. The weakness has multiple origins. In part it is a result of the excessive proteolysis cortisol produces. High cortisol levels can result in hypokalemia (via mineralocorticoid actions), which can produce muscle weakness because it hyperpolarizes and stabilizes the muscle cell membrane and thus makes stimulation more difficult. Actions of Cortisol on the Gastrointestinal Tract Cortisol exerts a trophic effect on the GI mucosa. In the absence of cortisol, GI motility decreases, GI mucosa degenerates, and GI acid and enzyme production decreases. Because cortisol stimulates appetite, hypercortisolism is frequently associated with weight gain. The cortisol-mediated stimulation of gastric acid and pepsin secretion increases the risk for development of ulcers. Psychological Effects of Cortisol

1	Psychological Effects of Cortisol Psychiatric disturbances are associated with either excessive or deficient levels of corticosteroids. Excessive corticosteroids can initially produce a feeling of well-being, but continued excessive exposure eventually leads to emotional lability and depression. Frank psychosis can occur with either excessive or deficient hormone. Cortisol increases the tendency for insomnia and decreases rapid eye movement (REM) sleep. People who are deficient in corticosteroids tend to be depressed, apathetic, and irritable. Effects of Cortisol During Fetal Development Cortisol is required for normal development of the CNS, retina, skin, GI tract, and lungs. The best studied system is the lungs, in which cortisol induces differentiation and maturation of type II alveolar cells. During late gestation these cells produce surfactant, which reduces surface tension in the lungs and thus allows the onset of breathing at birth. Regulation of Cortisol Production

1	Regulation of Cortisol Production Cortisol production by the zona fasciculata is regulated by a standard hypothalamic-pituitary-adrenal axis involving corticotropin-releasing hormone (CRH), ACTH, and cortisol (see ). The hypothalamus and pituitary stimulate cortisol production, and cortisol negatively feeds back on the hypothalamus and pituitary to maintain its set point. Both neurogenic (e.g., fear) and systemic (e.g., hypoglycemia, hemorrhage, cytokines) forms of stress stimulate release of CRH. CRH is also under strong diurnal rhythmic regulation emerging from the suprachiasmatic nucleus, such that cortisol levels surge during the early predawn and morning hours and then continually decline throughout the day and evening. CRH acutely stimulates release of ACTH and chronically increases proopiomelanocortin (POMC) gene expression and corticotrope hypertrophy and proliferation. Some parvicellular neurons coexpress CRH and ADH, which potentiates the actions of CRH.

1	ACTH binds to the melanocortin 2 receptor (MC2R) located on cells in the zona fasciculata ( Fig. 43.11 ). The effects of ACTH can be subdivided into three phases: 1. The acute effects of ACTH occur within minutes. Cholesterol is rapidly mobilized from lipid droplets by posttranslational activation of cholesterol ester hydrolase and transported to the outer mitochondrial membrane. ACTH both rapidly increases StAR protein gene expression and activates StAR protein through protein kinase A (PKA)-dependent phosphorylation. Collectively, these acute actions of ACTH increase pregnenolone levels. 2. The chronic effects of ACTH occur over a period of several hours. These effects involve increasing transcription of the genes encoding the steroidogenic enzymes and their coenzymes. ACTH also increases expression of the LDL receptor and scavenger receptor BI (SR-BI; the HDL receptor). 3.

1	3. The trophic actions of ACTH on the zona fasciculata and zona reticularis occur over a period of weeks and •Fig. 43.11 OverviewoftheactionsofACTHontargetadrenocorticalcells.Notethatthemajorsecondmessenger,cAMP,activatesimmediateproteinmediatorsandalsoinducesproductionoflaterproteinmediators.HDL,high-densitylipoprotein;LDL,low-densitylipoprotein. months. This effect is exemplified by atrophy of the zona fasciculata in patients receiving therapeutic (i.e., supraphysiological) levels of glucocorticoid analogues for at least 3 weeks. Under these conditions the exogenous corticosteroids completely repress CRH and ACTH production, thereby resulting in atrophy of the zona fasciculata and a decline in endogenous cortisol production (

1	Fig. 43.12 ). At the end of therapy, these patients need to be slowly weaned off exogenous glucocorticoids to allow the hypothalamic-pituitary-adrenal axis to reestablish itself and the zona fasciculata to enlarge and produce adequate amounts of cortisol. Cortisol inhibits both POMC gene expression at the corticotropes and pro-CRH gene expression at the hypothalamus. However, intense stress can override the negative-feedback effects of cortisol at the hypothalamus and reset the “set point” at a higher level. The innermost zone, the zona reticularis, begins to appear after birth at about 5 years of age. Adrenal androgens, especially DHEAS, the main product of the zona reticularis, become detectable in the circulation at about 6 years of age. This onset of adrenal androgen production is called adrenarche, and it contributes to the appearance of axillary and pubic hair at about age 8. DHEAS levels continue to increase, peak during the mid-20s, and then progressively decline with age.

1	Androgen Synthesis by the Zona Reticularis The zona reticularis differs from the zona fasciculata in several important ways with respect to steroidogenic enzyme activity (see Fig. 43.8 ). First, 3β-HSD is expressed at very low levels in the zona reticularis; thus the Δ5 pathway predominates in the zona reticularis. Second, the zona reticularis expresses cofactors or conditions that enhance the 17,20-lyase function of CYP17, thereby generating the 19-carbon androgen precursor molecule dehydroepiandrosterone (DHEA) from 17-hydroxypregnenolone. Additionally the zona reticularis expresses DHEA sulfotransferase (SULT2A1 gene), which converts DHEA into DHEAS (

1	Fig. 43.13 ). A limited amount of the Δ4 androgen androstenedione is also made in the zona reticularis. Although small amounts of potent androgens (e.g., testosterone) or 18-carbon estrogens are normally produced by the human adrenal cortex, most active sex steroids are produced primarily from peripheral conversion of DHEAS and androstenedione. Metabolism and Fate of DHEAS and DHEA DHEAS can be converted back to DHEA by peripheral sulfatases, and DHEA and androstenedione can be converted to active androgens (testosterone, dihydrotestosterone) peripherally in both sexes. DHEA binds to albumin and other globulins in blood with low affinity, so it is excreted efficiently by the kidney. The half-life of DHEA is 15 to 30 minutes. In contrast, DHEAS binds to albumin with very high affinity and has a half-life of 7 to 10 hours. Quiescent HPA axis in patient undergoing long-term (>3 wk) Normal HPA axis treatment with glucocorticoid

1	Quiescent HPA axis in patient undergoing long-term (>3 wk) Normal HPA axis treatment with glucocorticoid Hypothalamus CRH ACTH Pituitary Zona fasciculata Atrophied zona fasciculata Cortisol Exogenous corticosteroid (supraphysiological levels) Biological actions Biological actions and therapeutic actions (e.g., antiinflammatory or immunosuppressive) Pituitary Hypothalamus (–) (–) (–) (–) •Fig. 43.12 Comparisonofanormalhypothalamic-pituitary-adrenal(HPA)axistoaquiescentHPAaxisinindividualreceivingexogenousglucocorticoidtherapy.Thelattercausesthezonafasciculatatoatrophyafter3weeks,thusrequiringacarefulwithdrawalregimentoallowrebuildingoftheadrenaltissuebeforetotalcessationofexogenouscorticosteroidadministration.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.) Physiological Actions of Adrenal Androgens

1	Physiological Actions of Adrenal Androgens In men the contribution of adrenal androgens to active androgens is negligible. However, in women the adrenal contributes to about 50% of circulating active androgens, which are required for the growth of axillary and pubic hair and for libido.

1	Apart from providing androgen precursors, it is not clear what other role or roles if any the zona reticularis plays in adult humans. DHEAS is the most abundant circulating hormone in young adults. It increases steadily until it peaks in the mid-20s and then steadily declines thereafter. Thus there has been considerable interest in the possible role of DHEAS in the aging process. However, the function of this abundant steroid in young adults and the potential impact of its gradual disappearance on aging are still poorly understood. It should be noted that the age-related decline in DHEA and DHEAS has led to the popular use of these steroids as dietary supplements, even though recent studies indicate no beneficial effects.

1	Duringadrenalandrogenexcess(e.g.,adrenaltumor,Cushing’ssyndrome,congenitaladrenalhyperplasia),masculinization of women canoccur.Thisinvolvesmasculinizationoftheexternalgenitalia(e.g.,enlargedclitoris)inuteroandexcessivefacialandbodyhair(calledhirsutism)andacneinadultwomen.Excessiveadrenalandrogensalsoappeartoplayaroleinovariandysovulation(i.e.,polycysticovariansyndrome). Acrucialclinicalaspectofregulationofthezonareticularisisthatneitheradrenalandrogensnortheirmorepotentmetabolites(e.g.,testosterone,dihydrotestosterone,estradiol-17β)negativelyfeedbackonACTHorCRH( Fig. 43.14 ).Thismeansthatanenzymaticdefectassociatedwiththesynthesisofcortisol(e.g.,CYP21Bdeficiency)isassociatedwithadramaticincreaseinbothACTH(nonegativefeedbackfromcortisol)andadrenalandrogens(becauseoftheelevatedACTH).Itisthis“loophole”inthehypothalamic-pituitary-adrenalaxisthatgivesriseto congenital adrenal hyperplasia (CAH). Regulation of Zona Reticularis Function

1	Regulation of Zona Reticularis Function ACTH is the primary regulator of the zona reticularis. Both DHEA and androstenedione display the same diurnal rhythm as cortisol (DHEAS does not because of its long circulating half-life). Moreover, the zona reticularis shows the same atrophic changes as the zona fasciculata in conditions typified by little or no ACTH. However, other factors must regulate adrenal androgen function. Adrenarche occurs in the face of constant ACTH and cortisol levels, and the rise and decline of DHEAS is not associated with a similar pattern of ACTH or cortisol production. However, the other factors, whether extraadrenal or intraadrenal, remain unknown.

1	The thin outermost zone of the adrenal, the zona glomerulosa, produces the mineralocorticoid aldosterone, which regulates salt and volume homeostasis (see ). The zona glomerulosa is minimally influenced by ACTH. Rather it is regulated primarily by the reninangiotensin system, plasma [K+], and atrial natriuretic peptide (ANP).

1	An important feature in the steroidogenic capacity of the zona glomerulosa is that it does not express CYP17. Therefore zona glomerulosa cells never make cortisol, nor do they make adrenal androgens in any form. Pregnenolone is converted to progesterone and DOC by 3β-HSD and •Fig. 43.13 Steroidogenicpathwaysinthezonareticularis.Thefirstcommonreactioninthepathway,conversionofcholesteroltopregnenolonebyCYP11A1,isnotshown.Expressionof3β-hydroxysteroiddehydrogenase(3β-HSD)isrelativelylowinthezonareticularis,soandrostenedioneisaminorproductincomparisontoDHEAandDHEAS.Thezonareticularisalsomakesasmallamountoftestosteroneandestrogens(notshown).(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.) CYP21, respectively (

1	CYP21, respectively ( Fig. 43.15 ). A completely unique last three reactions from DOC to aldosterone within the feature of the zona glomerulosa among the steroidogenic zona glomerulosa. These reactions are 11-hydroxylation of glands is its expression of CYP11B2, which is regulated DOC to form corticosterone, 18-hydroxylation to form by different signaling pathways. Furthermore the enzyme 18-hydroxycorticosterone, and 18-oxidation to form aldocoded by CYP11B2, aldosterone synthase, catalyzes the sterone (see Figs. 43.8 43.15). •Fig. 43.14 The“loophole”inthehypothalamic-pituitary-adrenalaxis.ACTHstimulatesproductionofbothcortisolandadrenalandrogens,butonlycortisolnegativelyfeedsbackonACTHandCRH.Thusifcortisolproductionisblocked(i.e.,CYP11B1deficiency),ACTHlevelsincreasealongwithadrenalandrogens.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.)

1	CYP11B1(expressedonlyinthezonafasciculata)andCYP11B2(expressedonlyinthezonaglomerulosa)arelocatedonchromosome8inhumans,display95%similarity,andareseparatedfromeachotherbyonlyabout50kilobases.Thisincreasesthepossibilityofunevencrossingoverduringgametogenesis,withtheformationofhybridgenes.Inonecasethepromoterregionand5′ endoftheCYP11B1 geneisfusedtothe3′ endoftheCYP11B2 gene.ThisarrangementleadstoaldosteronesynthasebeingexpressedinthezonaefasciculataandreticularisunderthecontrolofACTH.Becausealdosteroneisnolongerunderfeedbackcontrolbytherenin-angiotensinsystem(see ),aldosteronelevelsarehighandhypertensionensues.Thisformofprimaryaldosteronismiscalledglucocorticoid-remediable aldosteronism, anditisinheritedinanautosomaldominantmanner.Thisdiseasecanbeconfirmedbythepolymerasechainreactiontechniqueandbymeasurementof18-hydroxycortisoland18-oxicortisolina24-hoururinesample.Thediseaseistreatedbytheadministration of glucocorticoid, whichsuppressesACTHandthusexpressionofthehybridgene.

1	Transport and Metabolism of Aldosterone Aldosterone binds to albumin and corticosteroid-binding protein in blood with low affinity and therefore has a biological half-life of about 20 minutes. Almost all aldosterone is inactivated by the liver in one pass, conjugated to a glucuronide group, and excreted by the kidney. Mechanism of Aldosterone Action Aldosterone acts much like cortisol (and other steroid hormones) in that its primary mechanism of action is mediated by binding to a specific intracellular receptor (i.e., mineralocorticoid receptor [MR]). After dissociation of chaperone proteins, nuclear translocation, dimerization, and binding to the mineralocorticoid response element (MRE), the aldosterone-MR complex regulates expression of specific genes (see ). As discussed earlier, cortisol binds to the MR with significant affinity. However, as also discussed, cells that express MR also express 11β-HSD2, which converts cortisol to the inactive steroid cortisone (

1	Fig. 43.16 ). Cortisone can be converted back to cortisol by 11β-HSD1, which is expressed in several glucocorticoidresponsive tissues, including the liver and skin. Clinicalstudiesinhumanshaverevealedadeleteriouseffectofaldosteroneoncardiovascularfunctionindependentofitseffectsonrenalsodiumandwaterreabsorption.Aldosteronehasaproinflammatory, profibrotic effect onthecardiovascularsystemandcausesleftventricularhypertrophyandremodeling.Thiseffectofaldosteroneisassociatedwithincreasedmorbidityandmortalityinpatientswithessentialhypertension. Physiological Actions of Aldosterone The actions and regulation of aldosterone are discussed in

1	Physiological Actions of Aldosterone The actions and regulation of aldosterone are discussed in CHAPTER 43 The Adrenal Gland •Fig. 43.15 Steroidogenicpathwaysinthezonaglomerulosa.Thefirstcommonreactioninthepathway,conversionofcholesteroltopregnenolonebyCYP11A1,isnotshown.NotethatthelastthreereactionsarecatalyzedbyCYP11B2.(ModifiedfromWhiteBA,PorterfieldSP.EndocrineandReproductivePhysiol-ogy.4thed.Philadelphia:Mosby;2013.)CH3 CH3 CH3 Pregnenolone C O 3˜-HSD CH3 CH3 CH3 Progesterone C O CYP21B HO CH3 CH3 CH2OH 11-Deoxycorticosterone (DOC) C O CYP11B2 CH3 CH3 CH2OH Corticosterone C O CYP11B2 O HO HO CH3 CH3 CH2OH 18(OH) Corticosterone C O CYP11B2 O HO CH3 CH2OH Aldosterone C O O O CH

1	Addison’s disease isdefinedbyprimary adrenal insufficiency, withbothmineralocorticoidsandglucocorticoidsusuallybeingdeficient.InNorthAmericaandEurope,themostprevalentcauseofAddison’sdiseaseisautoimmune destruction of the adrenal cortex. Becauseofthecortisoldeficiency,ACTHsecretionincreases.ElevatedlevelsofACTHcancompeteforMC1Rinmelanocytesandcauseanincreaseinskinpigmentation,particularlyinskincreases,scars,andgums(see

1	Fig.43.14 ).Thelossofmineralocorticoidsresultsincontractionofextracellularvolume,whichproducescirculatoryhypovolemiaandthereforeadropinbloodpressure.Becauselossofcortisoldecreasesthevasopressiveresponsetocatecholamines,peripheralvascularresistancedrops,therebyfacilitatingthedevelopmentofhypotension.IndividualswithAddison’sdiseasearealsopronetohypoglycemiawhenstressedorfasting,andwaterintoxicationcandevelopifexcesswaterisingested.Becausecortisolisimportantformusclefunction,muscleweaknessalsooccursincortisoldeficiency.Lossofcortisolresultsinanemia,decreasedGImotilityandsecretion,andreducedironandvitaminB12absorption.Appetitedecreaseswithcortisoldeficiency,andthisdecreasedappetitecoupledwiththeGIdysfunctionpredisposestheseindividualstoweightloss.Thesepatientsoftenhavedisturbancesinmoodandbehaviorandaremoresusceptibletodepression.

1	•Fig. 43.16 Themineralocorticoidreceptor(MR)isprotectedfromactivationbycortisolbytheenzyme11β-hydroxysteroiddehydrogenasetype2(11β-HSD2),whichconvertscortisoltoinactivecortisone.Cortisonecanbeconvertedbacktocortisolinglucocorticoidtargetcellsbytheenzyme11β-HSDtype1.GRE,glucocorticoidresponseelement;GTF,generaltranscriptionfactors;MRE,mineralocorticoidresponseelement.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine and Reproductive Physiology. 4thed.Philadelphia:Mosby;2013.)Aldosterone Aldosterone 11˜-HSD2 MR-chaperone complex Dissociated chaperones Dissociated chaperonesMR-aldosterone complex Mineralocorticoid-specific biological response Glucocorticoid-specific biological response Mineralocorticoid target cell 11˜-HSD1 Glucocorticoid target cell • Dimerization • Nuclear transport • Binding to MREs • Recruitment of coactivator proteins and GTFs • Altered transcription of aldosterone-regulated genes • Dimerization • Nuclear transport • Binding to GREs • Recruitment of coactivator proteins

1	MREs • Recruitment of coactivator proteins and GTFs • Altered transcription of aldosterone-regulated genes • Dimerization • Nuclear transport • Binding to GREs • Recruitment of coactivator proteins and GTFs • Altered transcription of cortisol-regulated genes Cortisol Cortisol Cortisol GR-chaperone complex GR-cortisol complex Cortisone Cortisone Cortisone Blood

1	Adrenocortical hormone excess istermedCushing’s syndrome. Pharmacological useofexogenouscorticosteroidsisnowthemostcommoncauseofCushing’ssyndrome.ThenextmostprevalentcauseisACTH-secreting tumors. TheformofCushing’ssyndromecausedbyafunctionalpituitaryadenomaiscalledCushing’s disease. ThefourthmostcommoncauseofCushing’ssyndromeisprimary hypercortisolism resultingfromafunctionaladrenaltumor.Ifthedisorderisprimaryorifitisaresultofcorticosteroidtreatment,secretionofACTHwillbesuppressedandincreasedskinpigmentationwillnotoccur.However,ifhypersecretionoftheadrenalistheresultofanACTH-secretingnonpituitarytumor,ACTHlevelssometimesbecomehighenoughtoincreaseskinpigmentation. Increasedcortisolsecretioncausesweightgainwithacharacteristiccentripetalfatdistributionanda“buffalohump.”

1	Increasedcortisolsecretioncausesweightgainwithacharacteristiccentripetalfatdistributionanda“buffalohump.” Thefaceappearsrounded(fatdeposition),andthecheeksmaybereddened,inpartbecauseofthepolycythemia.Thelimbsarethinowingtoskeletalmusclewasting(fromincreasedproteolysis),andmuscleweaknessisevident(frommuscleproteolysisandhypokalemia).Proximalmuscleweaknessisapparent,sothepatientmayhavedifficultyclimbingstairsorrisingfromasittingposition.Theabdominalfataccumulationcoupledwithatrophyoftheabdominalmusclesandthinningoftheskinproducesalargeprotrudingabdomen.Purpleabdominalstriaeareseenasaresultofdamagetotheskinbytheprolongedproteolysis,increasedintraabdominalfat,andlossofabdominalmuscletone.Capillaryfragilityoccursbecauseofdamagetotheconnectivetissuesupportingthecapillaries.Patientsarelikelytoshowsignsofosteoporosisandpoorwoundhealing.Theyhavemetabolicdisturbancesthatincludeglucoseintolerance,hyperglycemia,andinsulinresistance(see

1	Fig.43.10 ).Prolongedhypercortisolismcanleadtomanifestationsofdiabetesmellitus.Becauseofsuppressionoftheimmunesystemcausedbyglucocorticoids,patientsaremoresusceptibletoinfection.Themineralocorticoidactivitiesofglucocorticoidsandthepossibleincreaseinaldosteronesecretionproducesaltretentionandsubsequentwaterretentionthatresultinhypertension.Excessiveandrogensecretioninwomencanproducehirsutism,malepatternbaldness,andclitoralenlargement(adrenogenitalsyndrome). AnyenzymeblockagethatdecreasescortisolsynthesiswillincreaseACTHsecretionandproduceadrenalhyperplasia.Themostcommonformofcongenitaladrenalhyperplasiaisduetodeficiencyoftheenzyme21-hydroxylase (CYP21). Theseindividualscannotproducenormalquantitiesofcortisol,deoxycortisol, DOC,corticosterone,oraldosterone(see Figs.

1	Figs. 43.8 43.10C ).BecauseofimpairedcortisolproductionandresultantelevatedACTHlevels,steroidogenesisisstimulated,therebyincreasingthesynthesisproducts“upstream”ofthemissingenzyme,aswellasproductsofthezonareticularis.Becausethelatterincludetheadrenalandrogens,afemale 1. The adrenal gland is composed of a cortex that is of mesodermal origin and a medulla that is of neuroectodermal origin. The cortex produces steroid hormones, and the medulla produces catecholamines. 2. The rate-limiting enzymes in medullary catecholamine synthesis are tyrosine hydroxylase and dopamine β-hydroxylase, which are induced by sympathetic stimulation, and phenylethanolamine-Nmethyltransferase, which is induced by cortisol. 3. Catecholamines increase serum glucose and fatty acid levels. They stimulate gluconeogenesis, glycogenolysis, and lipolysis. Catecholamines increase cardiac output but have selective effects on blood flow to different organs. 4.

1	4. Pheochromocytoma is a tumor of chromaffin tissue that produces excessive quantities of catecholamines. Symptoms of pheochromocytoma are often sporadic and include hypertension, headaches, sweating, anxiety, palpitations, chest pain, and orthostatic hypotension. 5. The adrenal cortex displays clear structural and functional zonation: the zona glomerulosa produces the mineralocorticoid aldosterone, the zona fasciculata produces the glucocorticoid cortisol, and the zona reticularis produces the weak androgens DHEA and DHEAS. 6.

1	6. Cortisol binds to the glucocorticoid receptor. During stress, cortisol increases blood glucose by increasing gluconeogenesis in the liver and breaking muscle protein down to supply gluconeogenic precursors. Cortisol also decreases glucose uptake by muscle and adipose tissue and has permissive actions on glucagon and catecholamines. Cortisol has multiple effects on other tissue. From a pharmacological point of view, the most important is the immunosuppressive/ antiinflammatory effect. 7.

1	7. Cortisol is regulated by the CRH-ACTH-cortisol axis. Cortisol negatively feeds back at the hypothalamus on both CRH-producing neurons and pituitary corticotropes. CRH is regulated by several fetuswillbemasculinized.Becausetheyareunabletoproducethemineralocorticoids,aldosterone,DOC,andcorticosterone,patientswiththisdisorderhavedifficultyretainingsaltandmaintainingextracellularvolume.Consequentlytheyarelikelytobehypotensive.Iftheblockageisatthenextstep,11β-hydroxylase (CYP11B1),DOC willbeformedandlevelsofDOCwillaccumulate(see Figs.43.8 43.9C ).BecauseDOChassignificantmineralocorticoid activity anditslevelsbecomehigh,theseindividualstendtoretainsaltandwaterandbecomehypertensive. forms of stress, including proinflammatory cytokines, hypoglycemia, neurogenic stress, and hemorrhage, and by diurnal input. 8.

1	forms of stress, including proinflammatory cytokines, hypoglycemia, neurogenic stress, and hemorrhage, and by diurnal input. 8. The adrenal androgens DHEA, DHEAS, and androstenedione are androgen precursors. They can be converted to active androgens peripherally and provide about 50% of circulating androgens in women. In men the role of adrenal androgens if any remains obscure. In women, adrenal androgens promote pubic and axillary hair growth and libido. Excessive adrenal androgens in women can lead to various degrees of virilization and ovarian dysfunction. 9.

1	9. The zona glomerulosa of the adrenal cortex is the site of aldosterone production. Aldosterone is the strongest naturally occurring mineralocorticoid in humans. It promotes Na+ and water reabsorption by the distal tubule and collecting duct while promoting renal K+ and H+ secretion. Aldosterone promotes Na+ and water absorption in the colon and salivary glands. It also has a proinflammatory, profibrotic effect on the cardiovascular system and causes left ventricular hypertrophy and remodeling. 10. Major actions of angiotensin II on the adrenal cortex are increased growth and vascularity of the zona glomerulosa, increased StAR and CYP11B2 enzyme activity, and increased aldosterone synthesis. 11. Major stimuli for aldosterone production are a rise in angiotensin II and a rise in serum [K+]. The major inhibitory signal is ANP. 12.

1	11. Major stimuli for aldosterone production are a rise in angiotensin II and a rise in serum [K+]. The major inhibitory signal is ANP. 12. Addison’s disease is adrenocortical insufficiency. Common symptoms include hypotension, hyperpigmentation, muscle weakness, anorexia, hypoglycemia, and hyperkalemic acidosis. 13. Cushing’s syndrome results from hypercortisolemia. If the basis of the disorder is increased pituitary adrenocorticotropin secretion, the disorder is called Cushing’s disease. Common symptoms of Cushing’s syndrome include centripetal fat distribution, muscle wasting, proximal muscle weakness, thin skin with abdominal striae, capillary fragility, insulin resistance, and polycythemia. 14. Congenital adrenal hyperplasia is caused by a congenital enzyme deficiency that blocks production of cortisol. The enzyme blockage results in elevated

1	14. Congenital adrenal hyperplasia is caused by a congenital enzyme deficiency that blocks production of cortisol. The enzyme blockage results in elevated De Bosscher K, et al. Activation of the glucocorticoid receptor in acute inflammation: the SEDIGRAM concept. Trends Pharmacol Sci. 2016;37:4-16. Guerineau NC, et al. Functional chromaffin cell plasticity in response to stress: focus on nicotinic, gap junction, and voltage-gated Ca2+ channels. J Mol Neurosci. 2012;48:368-386. Namsolleck P, Unger T. Aldosterone synthase inhibitors in cardiovascular and renal diseases. Nephrol Dial Transplant. 2014;29(suppl 1): i62-i68. ACTH secretion, which stimulates adrenal cortical growth and secretion of precursors produced before the block. 21-Hydroxylase (CYP21B) deficiency is the most common form. Rafacho A, et al. Glucocorticoid treatment and endocrine pancreas function: implications for glucose homeostasis, insulin resistance and diabetes. J Endocrinol. 2014;223:R49-R62.

1	Rafacho A, et al. Glucocorticoid treatment and endocrine pancreas function: implications for glucose homeostasis, insulin resistance and diabetes. J Endocrinol. 2014;223:R49-R62. Sundahl N, et al. Selective glucocorticoid receptor modulation: new directions with non-steroidal scaffolds. Pharmacol Ther. 2015;152:28-41. Turcu AF, Auchus RJ. Adrenal steroidogenesis and congenital adrenal hyperplasia. Endocrinol Metab Clin North Am. 2015;44: 275-296. Whittier X, Saag KG. Glucocorticoid-induced osteoporosis. Rheum Dis Clin North Am. 2016;42:177-189. Upon completion of this chapter the student should be able to answer the following questions: 1. Describe the general anatomical components of the male and female reproductive system. 2. Map out the organization of the testis, with the Sertoli cells and developing sperm cells within the intralobular compartment, and the Leydig cells and capillary plexus within the interlobular/interstitial compartment. 3.

1	3. Describe the processes of spermatogenesis and spermiogenesis. 4. List the functions of the Sertoli cell. 5. Diagram the process of testosterone synthesis within the Leydig cells, and the peripheral conversion of testosterone to estradiol or dihydrotestosterone. 6. Diagram the male hypothalamus/pituitary/testis axis, including all cell types and hormones involved. 7. Map out the organization of the ovary, and describe the various stages of follicular development, ovulation and corpus luteum formation. 8. List the stages and control of female germ cell progression from oogonia to egg. 9. Map out the steroidogenic pathways in the corresponding cell types that lead to androgen, estrogen and progesterone synthesis. 10. Diagram the female hypothalamus/pituitary/ovarian axis during the menstrual cycle, including all cell types and hormones involved. 11. Explain changes in the female tract, with emphasis on the uterine endometrium, during the menstrual cycle. 12.

1	11. Explain changes in the female tract, with emphasis on the uterine endometrium, during the menstrual cycle. 12. List the events involved in fertilization. 13. Describe the development and function of the placenta. 14.

1	14. Describe the development and function of the mammary glands he two most basic components of the reproductive system are the gonads and the reproductive tract. The gonads (testes and ovaries) perform an endocrine function that is regulated within a hypothalamic-pituitary-gonadal axis. The gonads are distinct from other endocrine glands in that they also perform gametogenesis. The reproductive tract is involved in several aspects of gamete development, function, and transport and in women allows fertilization, implantation, and gestation. Normal gametogenesis in the gonads and development and physiology of the reproductive tract are absolutely dependent on the endocrine function of the gonads. The clinical ramifications of this hormonal dependence include infertility in the face of low sex hormone production, ambiguous genitalia in dysregulated hormone or receptor expression, and hormone-responsive cancers, especially uterine and breast cancer in women and prostate cancer in men.

1	The male reproductive system has evolved for continuous lifelong gametogenesis coupled with occasional internal insemination with a high density of sperm (>60 × 106/ mL in 3–5 mL of semen). In adult men the basic roles of gonadal hormones are (1) support of gametogenesis (spermatogenesis), (2) maintenance of the male reproductive tract and production of semen, and (3) maintenance of secondary sex characteristics and libido. There is no overall cyclicity of this activity in men. The Testis Unlike the ovaries, the testes reside outside the abdominal cavity in the scrotum ( Fig. 44.1). This location maintains testicular temperature at about 2°C lower than body temperature, which is crucial for optimal sperm development. The human testis is covered by a connective tissue capsule and divided into about 300 lobules by fibrous septa ( Fig.

1	Fig. 44.2 ). Within each lobule are two to four loops of seminiferous tubules. Each loop empties into an anastomosing network of tubules called the rete testis. The rete testis is continuous with small ducts, the efferent ductules, that lead the sperm out of the testis into the head of the epididymis on the superior pole of the testis (see Fig. 44.2 ). Once in the epididymis, the sperm pass from the head, to the body, to the tail of the epididymis and then to the vas (ductus) deferens. Viable sperm can be stored in the tail of the epididymis and vas deferens for several months. • Fig. 44.1 Anatomy of the male reproductive system. (Modified from Drake RL et al. Gray’s Anatomy for Students. Philadelphia: Churchill Livingstone; 2005.)

1	• Fig. 44.1 Anatomy of the male reproductive system. (Modified from Drake RL et al. Gray’s Anatomy for Students. Philadelphia: Churchill Livingstone; 2005.) The presence of the seminiferous tubules creates two compartments within each lobule: an intratubular compartment, which is composed of the avascular seminiferous epithelium of the seminiferous tubule, and a peritubular compartment, which is composed of neurovascular elements, connective tissue cells, immune cells, and the interstitial cells of Leydig, whose main function is to produce testosterone ( Fig. 44.3). The seminiferous tubule is lined by a complex seminiferous epithelium composed of two cell types: sperm cells in various stages of spermatogenesis and the Sertoli cell, which is a “nurse cell” in intimate contact with all sperm cells (Fig. 44.4 Spermatogenesis involves the processes of mitosis and meiosis. Stem cells called spermatogonia reside at the basal level of the seminiferous epithelium (see Fig. 44.4,

1	Spermatogenesis involves the processes of mitosis and meiosis. Stem cells called spermatogonia reside at the basal level of the seminiferous epithelium (see Fig. 44.4, SA and SB). Spermatogonia divide mitotically to generate daughter spermatogonia (spermatocytogenesis). One or more spermatogonia remain within the stem cell population, firmly adherent to the basal lamina. However, the majority of these daughter spermatogonia enter meiotic division, which results in haploid spermatozoa on completion of meiosis. These divisions are accompanied by incomplete cytokinesis such that all daughter cells remain interconnected by a cytoplasmic bridge. This configuration contributes to the synchrony of development of a clonal population of sperm cells. Spermatogonia migrate apically away from the basal lamina as they enter the first meiotic prophase. At this time they are called primary spermatocytes (see

1	Fig. 44.4 , S1). During the first meiotic prophase the hallmark processes of sexual reproduction involving chromosomal reduplication, synapsis, crossing over, and homologous recombination take place. Completion of the first meiotic division gives rise to secondary spermatocytes, which quickly (i.e., within 20 minutes) complete the second meiotic division. The initial products of meiosis are haploid spermatids (see Fig. 44.4, S3). Spermatids are small round cells that undergo a remarkable metamorphosis called spermiogenesis ( Fig. 44.5). The products of spermiogenesis are the streamlined spermatozoa see

1	Fig. 44.4, S3). Spermatids are small round cells that undergo a remarkable metamorphosis called spermiogenesis ( Fig. 44.5). The products of spermiogenesis are the streamlined spermatozoa see Fig. 44.4 , S4). As the spermatid matures into a spermatozoon the size of the nucleus decreases and a prominent tail is formed. The tail contains microtubular structures that propel sperm, similar to a flagellum. The chromatin material in the sperm nucleus condenses, and most of the cytoplasm is lost. The acrosome is a membrane-enclosed structure on the head of the sperm that acts as a lysosome CHAPTER 44 The Male and Female Reproductive Systems 789 Ligamentous remnant Vas deferens of processus vaginalis Fibrous septum Head of epididymis Body of epididymis

1	CHAPTER 44 The Male and Female Reproductive Systems 789 Ligamentous remnant Vas deferens of processus vaginalis Fibrous septum Head of epididymis Body of epididymis Tail of epididymis • Fig. 44.3 Histology of a testicular lobule. (From Young B etal. Wheater’s Functional Histology. A Text and Colour Atlas. 5th ed. London:ChurchillLivingstone;2006.) and contains hydrolytic enzymes that are important for fertilization. These enzymes remain inactive until the acrosomal reaction occurs (see

1	Fig. 44.4 , S4) are found at the luminal surface of the seminiferous tubule. Release of sperm, or spermiation, is controlled by Sertoli cells. The process of • Fig. 44.4 Histology of a seminiferous tubule. M, myoid cell just outside the basal lamina; S1, primary spermatocyte; S3, spermatid; S4, mature spermatid or spermatozoon; SB and SA, spermatogonia; St,Sertolicell.(FromYoungBetal.Wheater’s Functional Histology. A Text and Colour Atlas.5thed.London:ChurchillLivingstone;2006.) spermatogenesis takes about 72 days. A cohort of adjacent spermatogonia enter the process every 16 days so that the process is staggered at one point along a seminiferous tubule. In addition the process is staggered along the length of a seminiferous tubule (i.e., not all spermatogonia enter the process of spermatogenesis at the same time along the •Fig. 44.5 Structureofspermcellsduringtheprocessofspermatogenesisandspermiogenesis.

1	entire length of the tubule or in synchrony with every other tubule; there are about 500 seminiferous tubules per testis; see later). Because the seminiferous tubules within one testis are about 400 m in length, spermatozoa are continually being generated at many sites within the testis at any given time. The Sertoli Cell Sertoli cells are the true epithelial cells of the seminiferous epithelium and extend from the basal lamina to the lumen (see Fig. 44.4 , St). Sertoli cells surround sperm cells and provide structural support within the epithelium, and they form adhering and gap junctions with all stages of sperm cells. Through formation and breakdown of these junctions, Sertoli cells guide sperm cells toward the lumen as they advance to later stages of spermatogenesis. Spermiation requires the final breakdown of Sertoli–sperm cell junctions. Another important structural feature of Sertoli cells is the formation of tight junctions between adjacent Sertoli cells (

1	Another important structural feature of Sertoli cells is the formation of tight junctions between adjacent Sertoli cells ( Fig. 44.6 ). These Sertoli-Sertoli cell occluding junctions divide the seminiferous epithelium into a basal compartment containing the spermatogonia and early-stage primary spermatocytes and an adluminal compartment containing later-stage primary spermatocytes and all subsequent stages of sperm cells. As early primary spermatocytes move apically from the basal to the adluminal compartment, the tight junctions need to be disassembled and reassembled. These tight junctions form the physical basis for the blood-testis barrier (see

1	Fig. 44.6 ), which creates a specialized immunologically safe microenvironment for developing sperm. By blocking paracellular diffusion the tight junctions restrict movement of substances between blood and the developing germ cells through a trans–Sertoli cell transport pathway and in this manner allow the Sertoli cell to control the availability of nutrients to germ cells. Healthy Sertoli cell function is essential for sperm cell viability and development. In addition, spermatogenesis is absolutely dependent on testosterone produced by peritubular Leydig cells (see

1	Healthy Sertoli cell function is essential for sperm cell viability and development. In addition, spermatogenesis is absolutely dependent on testosterone produced by peritubular Leydig cells (see The Leydig Cell ), yet it is the Sertoli cells that express the androgen receptor and respond to testosterone, not the developing sperm cells. Similarly the pituitary hormone follicle-stimulating hormone (FSH) is also required for maximal sperm production, and again it is the Sertoli cell that expresses the FSH receptor, not the developing sperm. Thus these hormones support spermatogenesis indirectly through stimulation of Sertoli cell function.

1	Sertoli cells have multiple additional functions. They express the enzyme CYP19 (also called aromatase), which converts Leydig cell–derived testosterone to the potent estrogen estradiol-17β (see ). This local production of estrogen may enhance spermatogenesis in humans. Sertoli cells also produce androgen-binding protein (ABP), which maintains a high androgen level within the adluminal compartment, the lumens of the seminiferous tubules, and the proximal part of the male reproductive tract. Sertoli cells also produce a large amount of fluid. This fluid provides an appropriate bathing medium for the sperm and assists in moving the immotile spermatozoa from the seminiferous tubule into the epididymis. Sertoli cells perform an important phagocytic function by engulfing residual bodies, which represent cytoplasm shed by spermatozoa during spermiogenesis.

1	Finally, the Sertoli cell has an important endocrine role. During development, Sertoli cells produce antimüllerian hormone (AMH; also called müllerian inhibitory substance), which induces regression of the embryonic müllerian duct that is programmed to give rise to the female reproductive tract (discussed later). The Sertoli cells also produce the hormone inhibin. Inhibin is a heterodimer protein hormone related to the transforming growth factor-β family. FSH stimulates inhibin production, which then negatively feeds back on gonadotropes to inhibit FSH production. Thus inhibin keeps FSH levels within a set point. The peritubular compartment contains the primary endocrine cell of the testis, the Leydig cell (

1	The peritubular compartment contains the primary endocrine cell of the testis, the Leydig cell ( Fig. 44.7 ). This compartment also contains the common cell types of loose connective tissue and an extremely rich peritubular capillary network that provides nutrients to the seminiferous tubules (by way of Sertoli cells) while conveying testosterone away from the testes to the peripheral circulation. The Leydig Cell Leydig cells are steroidogenic stromal cells. These cells synthesize cholesterol de novo, as well as acquire it through low-density lipoprotein (LDL) receptors and high-density lipoprotein (HDL) receptors (also called scavenger receptor BI [SR-BI]), and store cholesterol as cholesterol esters as

1	CHAPTER 44 The Male and Female Reproductive Systems 791 • Fig. 44.6 Placement of germ cells within seminiferous tubule as they progress through spermatogenesis (From Carlson BM. Human Embryology and Developmental Biology. Philadelphia: Mosby; 2004.) ). Free to progesterone, 17-hydroxyprogesterone, and androstenecholesterol is generated by a cholesterol ester hydrolase and dione by 3β-hydroxysteroid dehydrogenase (3β-HSD) and transferred to the outer mitochondrial membrane and then CYP17 ( Fig. 44.8).

1	CYP17 to the inner mitochondrial membrane in a steroidogenic is a bifunctional enzyme with 17-hydroxylase activity acute regulatory (StAR) protein–dependent manner. and 17,20-lyase activity. CYP17 displays a robust level As in all steroidogenic cells, cholesterol is converted to of both activities in the Leydig cell. In this respect the pregnenolone by CYP11A1. Pregnenolone is then processed Leydig cell is similar to the zona reticularis cell, except that it expresses a higher level of 3β-HSD, so the Δ4 pathway is ultimately favored. Another major difference is that the Leydig cell expresses a Leydig cell–specific isoform of •Fig. 44.7 Histologyoftheperitubularspace(betweenthreesemi-niferoustubules)containingLeydigcells(L)andrichlyvascularizedbyperitubularcapillaries(cap).(ModifiedfromYoungBetal.Wheater’s Functional Histology. A Text and Colour Atlas.

1	Functional Histology. A Text and Colour Atlas. 5thed.London:ChurchillLivingstone;2006.)capSeminiferoustubuleSeminiferoustubuleSeminiferoustubuleLcapSeminiferoustubuleSeminiferoustubuleSeminiferoustubuleL 17β-hydroxysteroid dehydrogenase (17β-HSD type 3), which efficiently converts androstenedione to testosterone (see

1	Fig. 44.8 Fates and Actions of Androgens The testosterone produced by Leydig cells has several fates and multiple actions. Because of the proximity of Leydig cells to the seminiferous tubules, significant amounts of testosterone diffuse into the seminiferous tubules and become concentrated within the adluminal compartment by ABP (see Fig. 44.8 ). Testosterone levels within the seminiferous tubules that are greater than 100 times more concentrated than circulating testosterone levels are absolutely required for normal spermatogenesis. As mentioned earlier, Sertoli cells express the enzyme CYP19 (aromatase), which converts a small amount of testosterone into the highly potent estrogen estradiol-17β. Human sperm cells express at least one isoform of the estrogen receptor, and there is some evidence from aromatase-deficient men that this locally produced estrogen optimizes spermatogenesis in humans. Peripheral Conversion to Estrogen

1	Peripheral Conversion to Estrogen In several tissues (especially adipose tissue), testosterone is converted to estrogen (see Fig. 44.8 ). Studies involving men with aromatase deficiency have shown that an inability to produce estrogen results in tall stature because of the lack of epiphyseal closure in long bones, as well as osteoporosis. Thus peripheral estrogen plays an important role in bone maturation and biology in men. These studies also implicated estrogen in promoting insulin sensitivity, improving lipoprotein profiles (i.e., increasing HDL, decreasing triglycerides and LDL), and exerting negative feedback on gonadotropins at the pituitary and hypothalamus. Peripheral Conversion to Dihydrotestosterone Testosterone can also be converted into a potent nonaromatizable androgen, 5αα-dihydrotestosterone (DHT), by the enzyme 5α-reductase (see

1	Peripheral Conversion to Dihydrotestosterone Testosterone can also be converted into a potent nonaromatizable androgen, 5αα-dihydrotestosterone (DHT), by the enzyme 5α-reductase (see Fig. 44.8 ). There are two isoforms of 5α-reductase, type 1 and type 2. Major sites of 5α-reductase 2 expression are the male urogenital tract, genital skin, hair follicles, and liver. 5α-Reductase 2 generates DHT, which is required for masculinization of the external genitalia in utero and for many of the changes associated with puberty, including growth and activity of the prostate gland (see ), growth of the penis, darkening and folding of the scrotum, growth of pubic and axillary hair, growth of facial and body hair, and increased muscle mass (

1	Fig. 44.9 ). The onset of 5α-reductase 1 expression occurs at puberty. This isozyme is expressed primarily in the skin and contributes to sebaceous gland activity and the acne associated with puberty. Because DHT has strong growth-promoting (i.e., trophic) effects on its target organs, development of selective 5α-reductase 2 inhibitors has benefited the treatment of prostatic hypertrophy and prostatic cancer. Testosterone has a direct action (i.e., without conversion to DHT) in several cell types (see Fig. 44.9).

1	Testosterone has a direct action (i.e., without conversion to DHT) in several cell types (see Fig. 44.9). As mentioned earlier, testosterone regulates Sertoli cell function. It induces development of the male tract from the mesonephric duct in the absence of 5α-reductase. Testosterone has several metabolic effects, including increasing very low-density lipoprotein (VLDL) and LDL while decreasing HDL, promoting deposition of abdominal adipose tissue, increasing red blood cell production, promoting bone growth and health, and exerting a protein anabolic effect on muscle. Testosterone is sufficient to maintain erectile function and libido. Mechanism of Androgen Action Testosterone and DHT act through the same androgen receptor (AR). The AR resides in the cytoplasm bound to chaperone proteins in the absence of ligand.Testosterone-AR

1	HO 3˜-HSD CYP17 (17-hydroxylase) CH3 CH3 CH3 C O CH3 OH Pregnenolone OH CH3 CH3 CH3 O C O CH3 CH3 CH3 O H C O Progesterone 17(OH) Progesterone Type III 17˜-HSD Into seminiferous tubule T + ABP T • ABP (predominantly as T) SHBG + T CH3 CH3 O O Androstenedione CH3 CH3 O OH Testosterone (T) CH3 CH3 O OH Dihydrotestosterone (DHT) Into peripheral circulation CYP19 (adipose tissue) 5°-Reductase (genital skin, prostate) Estradiol-17˜Leydig cell HO SHBG • T •Fig. 44.8 SteroidogenicpathwayinLeydigcells(thefirststepofconvertingcholesteroltopregnenoloneisomitted).Testosteroneissequesteredbybindingtoandrogen-bindingprotein(ABP)withintheseminiferoustubulesorcirculateswithintheperipheralcirculationboundtosexhormone–bindingglobulin(SHBG)andcanbeperipherallyconvertedtodihydrotestosterone(DHT)orestradiol-17β (E2).(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.) binding or DHT-AR binding causes dissociation of the vicinity of a specific gene’s promoter. It

1	(E2).(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.) binding or DHT-AR binding causes dissociation of the vicinity of a specific gene’s promoter. It remains unclear how chaperone proteins, followed by nuclear translocation of testosterone and DHT differ in their ability to activate the the androgen-AR complex, dimerization, binding to an AR in the context of different cell types, although the pres-androgen response element (ARE), and recruitment of ence of different coactivator proteins in different cell types coactivator proteins and general transcription factors to the is probably involved. Such coactivator proteins would have

1	Feedback suppression of gonadotropin secretion Imprint male pattern of gonadotropins, sex drive, behavior • Fig. 44.9 Spectrum of effects of testosterone (T). Note that some effects result from the action of testosterone itself, whereas others are mediated by dihydrotestosterone (DHT) and estradiol (E2) after they are produced from testosterone. VLDL, LDL, HDL, very-low-density, low-density, and high-density lipoproteins, respectively. different affinities for the testosterone-induced configuration of the AR versus the DHT-induced configuration of the AR. Transport and Metabolism of Androgens

1	different affinities for the testosterone-induced configuration of the AR versus the DHT-induced configuration of the AR. Transport and Metabolism of Androgens As testosterone enters the peripheral circulation, it binds to and quickly reaches equilibrium with serum proteins. About 60% of circulating testosterone is bound to sex hormone–binding globulin (SHBG), 38% is bound to albumin, and about 2% remains as “free” hormone. Testosterone and its metabolites are primarily excreted in urine. Approximately 50% of excreted androgens are found as urinary 17-ketosteroids, with most of the remainder being conjugated androgens or diol or triol derivatives. Only about 30% of the 17-ketosteroids in urine are from the testis; the rest are produced from adrenal androgens. Androgens are conjugated with glucuronate or sulfate in the liver, and these conjugated steroids are excreted in urine.

1	The testis is regulated by an endocrine axis (Fig. 44.10 ) involving parvicellular hypothalamic gonadotropin-releasing hormone (GnRH) neurons and pituitary gonadotropes that produce both luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Regulation of Leydig Cell Function The Leydig cell expresses the LH receptor, which acts on Leydig cells much like adrenocorticotropic hormone (ACTH, or corticotropin) does on zona fasciculata cells in the adrenal cortex (see ). Rapid effects include hydrolysis of cholesterol esters and new expression of StAR protein. Less acute effects include an increase in steroidogenic enzyme gene expression and expression of the LDL. Over the long term, LH promotes Leydig cell growth and proliferation.

1	•Fig. 44.10 Thehypothalamic-pituitary-testicularaxis.Abbreviationsasinotherfigures.Kisspeptin hypothalamic neurons GnRH hypothalamic neurons GnRH Pituitary gonadotropes LH FSH Sertoli cell Sperm cells E2 T DHT Leydig cell T (–) (–) (–)(–) (–) Inhibin B Testosterone, and estradiol produced from peripheral conversion of testosterone, negatively feed back on GnRH hypothalamic neurons indirectly through inhibition of kisspeptin-producing neurons (see Fig. 44.10 ). Testosterone and estradiol also negatively feed back on pituitary gonadotropes. DHT, the other major product of peripheral conversion of testosterone, has little effect on LH or FSH levels. Regulation of Sertoli Cell Function

1	Regulation of Sertoli Cell Function The Sertoli cell is stimulated by both testosterone and FSH. In addition to stimulating synthesis of proteins involved in the “nurse cell” aspect of Sertoli cell function (e.g., ABP), FSH stimulates synthesis of the dimeric protein inhibin. Inhibin is induced by FSH and negatively feeds back on the gonadotrope to selectively inhibit FSH production (see Fig. 44.10 Once spermatozoa emerge from the efferent ductules, they leave the gonad and enter the male reproductive tract (see

1	Fig. 44.10 Once spermatozoa emerge from the efferent ductules, they leave the gonad and enter the male reproductive tract (see Fig. 44.1 ). The segments of the tract are the: epididymis (head, body, and tail), vas deferens, ejaculatory duct, prostatic urethra, membranous urethra, and penile urethra. Unlike the female tract, there is a contiguous lumen from the seminiferous tubule to the end of the male tract (i.e., the tip of the penile urethra), and the male reproductive tract connects to the distal urinary tract (i.e., male urethra). In addition to conveying sperm, the primary functions of the male reproductive tract are: 1. Sperm maturation. Sperm spend about a month in the epididymis, where they undergo further maturation.

1	Thereisanimportant“loophole”inthemalereproductiveaxisthatisbasedonthefactthatintratesticular levels of testosterone needtobegreaterthan100-foldhigherthancirculatinglevelsofthehormonetomaintainnormalratesofspermatogenesis,yetitisthecirculating levels of testosterone (and estradiol) thatprovidethenegativefeedbacktothepituitaryandhypothalamus.ThismeansthatexogenousadministrationoftestosteronecanraisecirculatinglevelsoftestosteroneandestradiolsufficienttoinhibitLHbutnotsufficienttoaccumulateinthetestisattherequiredconcentrationfornormalspermatogenesis.However,thedecreasedLHlevelswilldiminishintratesticularproductionoftestosteronebyLeydigcells,whichresultsinreducedlevels ofspermatogenesis(Fig.44.11 ).This“loophole”iscurrentlybeinginvestigatedasapossiblestrategyfordevelopingamale oral contraceptive. Itisalsothebasisforsterility insomecasesofsteroid abuse inmen.

1	The epithelium of the epididymis is secretory and adds numerous components to the seminal fluid. Spermatozoa that enter the head of the epididymis are weakly motile but are strongly unidirectionally motile by the time they exit the tail. Spermatozoa also undergo the process of decapacitation, which involves changes in the cell membrane to prevent spermatozoa from undergoing the acrosome reaction before contact with an egg (see later). Sperm become capacitated by the female reproductive tract within the oviduct. The function of the epididymis is dependent on luminal testosterone-ABP complexes that come from the seminiferous tubules and on testosterone from blood.

1	Normal set point determined by relatively low levels of circulating T, not intratesticular T •Fig. 44.11 Thedifferenceinintratesticulartestosteroneversuscirculatingtestosteroneconcentrationsanditsimportanceinthehypothalamic-pituitary-testisaxis.Upper panel, Feedbackloopinanormaladultman.Lower panel, Administrationoftestosterone(oranandrogenicanalogue)increasescirculatingtestosterone(androgen)levels,whichinturnincreasenegativefeedbackonreleaseofLH.DecreasedLHlevelsdiminishLeydigcellactivityandintratesticularproductionofandrogen.Loweredintratesticulartestosteronelevelsresultinreducedspermproductionandcancauseinfertility.(Theinhibinfeedbackloophasbeenomittedfromthisdiagram.)(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.) 2.

1	Sperm storage and emission. Sperm are stored in the tail The vas deferens has a very thick muscularis that is richly of the epididymis and vas deferens for several months innervated by sympathetic nerves. Normally in response without loss of viability. The primary function of the vas to repeated tactile stimulation of the penis during coitus, deferens, besides providing a storage site, is to propel the muscularis of the vas deferens receives bursts of symsperm during sexual intercourse into the male urethra. pathetic stimulation that cause peristaltic contractions. Emptying of the contents of the vas deferens into the prostatic urethra is called emission. Emission immediately precedes ejaculation, which is the propulsion of semen out of the male urethra. 3.

1	Production and mixing of sperm with seminal contents. During emission, contraction of the vas deferens coincides with contraction of the muscular coats of the two accessory sex glands, the seminal vesicles (right and left) and the prostate gland (which surrounds the prostatic urethra). At this point, sperm become mixed with all the components of semen. The seminal vesicles secrete approximately 60% of the volume. These glands are the primary source of fructose, a critical nutrient for sperm. The seminal vesicles also secrete semenogelins, which induce coagulation of semen immediately after ejaculation. The alkaline secretions of the prostate, which make up about 30% of the volume, are high in citrate, zinc, spermine, and acid phosphatase. Prostate-specific antigen (PSA) is a serine protease that liquefies coagulated semen after a few minutes. PSA can be detected in blood under conditions of prostatic infection, benign prostatic hypertrophy, and prostatic carcinoma and is currently

1	that liquefies coagulated semen after a few minutes. PSA can be detected in blood under conditions of prostatic infection, benign prostatic hypertrophy, and prostatic carcinoma and is currently used as one indicator of prostatic health. The predominant buffers in semen are phosphate and bicarbonate. A third accessory gland, the bulbourethral glands (also called Cowper’s glands), empty into the penile urethra in response to sexual excitement before emission and ejaculation. This secretion is high in mucus, which lubricates, cleanses, and buffers the urethra. Average sperm counts are between 60 and 100 million/mL semen. Men with sperm counts below 20 million/mL, less than 50% motile sperm, or less than 60% normally conformed sperm are usually infertile.

1	4.

1	Erection, penetration, and ejaculation. Emission and ejaculation occur during coitus in response to a reflex arc that involves sensory stimulation from the penis (via the pudendal nerve) followed by sympathetic motor stimulation to the smooth muscle of the male tract and somatic motor stimulation to the musculature associated with the base of the penis. However, for sexual intercourse to occur in the first place, the man has to achieve and maintain an erection of the penis. The penis has evolved as an intromittent organ designed to separate the walls of the vagina, pass through the potential space of the vaginal lumen, and deposit semen at the distal end of the vaginal lumen near the cervix. This process of internal insemination can be performed only if the penis is stiffened from the process of erection. Erection is a neurovascular event. The penis is composed of three erectile bodies: two corpora cavernosa and one corpus spongiosum (Fig. 44.12A ). The penile urethra runs through the

1	of erection. Erection is a neurovascular event. The penis is composed of three erectile bodies: two corpora cavernosa and one corpus spongiosum (Fig. 44.12A ). The penile urethra runs through the corpus spongiosum. These three bodies are composed of erectile tissue—an anastomosing network of potential cavernous vascular spaces lined with continuous endothelia within a loose connective tissue support. During the flaccid state, blood flow to the cavernous spaces is minimal (see

1	Fig. 44.12A). This is due CHAPTER 44 The Male and Female Reproductive Systems of the vasculature (called the helicine arteries) and shunting of blood flow away from the cavernous spaces. In response to sexual arousal the parasympathetic cavernous nerves innervating the vascular smooth muscle of the helicine arteries release nitric oxide (NO). NO activates guanylyl cyclase, thereby increasing cyclic guanosine monophosphate (cGMP), which decreases intracellular [Ca++] and causes muscular relaxation (see Fig. 44.12B ). Vasodilation allows blood to flow into the cavernous spaces to induce engorgement and erection. It also presses on veins in the penis and reduces venous drainage (see Fig. 44.12B

1	Fig. 44.12B ). Vasodilation allows blood to flow into the cavernous spaces to induce engorgement and erection. It also presses on veins in the penis and reduces venous drainage (see Fig. 44.12B Inabilitytoachieveormaintainanerectionistermederectile dysfunction (ED) andisonecauseofinfertilityinmen.MultiplefactorscanleadtoED,includinginsufficientandrogenproduction;neurovasculardamage(e.g.,fromdiabetesmellitus,spinalcordinjury);structuraldamagetothepenis,perineum,orpelvis;psychogenicfactors(e.g.,depression,performanceanxiety);andprescribedmedicationsandrecreationaldrugs,includingalcoholandtobacco.AmajordevelopmentinthetreatmentofsomeformsoferectiledysfunctionisuseofselectivecGMPphosphodiesteraseinhibitorsthatassistinmaintaininganerection(see Fig.44.12B

1	Fig.44.12B There is no distinct andropause in men. However, as men age, gonadal sensitivity to LH decreases and androgen production drops. As this occurs, serum LH and FSH levels rise. Although sperm production typically begins to decline after age 50, many men can maintain reproductive function and spermatogenesis throughout life. The female reproductive system is composed of the gonads, called ovaries, and the female reproductive tract, which includes the oviducts, uterus, cervix, vagina, and external genitalia. The Ovary The ovary is located within a fold of peritoneum called the broad ligament, usually close to the lateral wall of the pelvic cavity ( Fig. 44.13 ). Because the ovary extends into the peritoneal cavity, ovulated eggs briefly reside within the peritoneal cavity before they are captured by the oviducts. The ovary is divided into an outer cortex and inner medulla (

1	The ovary is divided into an outer cortex and inner medulla ( Fig. 44.14 ). Neurovascular elements innervate the medulla of the ovary. The cortex of the ovary is composed of a densely cellular stroma. Within this stroma reside the ovarian follicles (see Fig. 44.14, F ), which contain a primary oocyte surrounded by follicle cells. The cortex is covered by a connective tissue capsule, the tunica albuginea, (tactile, visual, auditory, psychic) Collapse of venous return from cavernous spaces Contraction of muscles around base of penis • Fig. 44.12 A, Arrangement of the vasculature and cavernous tissue within the penis. During the flaccid state, blood flow into the cavernous spaces is limited by contraction of the helicine arteries. B, Outline of neurovascular events leading to penile erection. (A, From Bhasun S et al. In: Larsen P et al [eds]. Williams Textbook of Endocrinology. 10th ed. Philadelphia: Saunders; 2003.) CHAPTER 44 The Male and Female Reproductive Systems 799

1	CHAPTER 44 The Male and Female Reproductive Systems 799 Labia minora Recto-uterine pouch of douglas • Fig. 44.13 Anatomy of the female reproductive system. (Modified from Drake RL et al. Gray’s Anatomy for Students. Philadelphia: Churchill Livingstone; 2005.) • Fig. 44.14 Histology of the ovary. CL, corpus luteum; F, follicle. (Modified from Young B et al. Wheater’s Functional Histology. A Text and Colour Atlas. 5th ed. London: Churchill Livingstone; 2006.) and a layer of simple epithelium consisting of ovarian surface epithelial cells. There are no ducts emerging from the ovary to convey its gametes to the reproductive tract. Thus the process of ovulation involves an inflammatory event that erodes the wall of the ovary. After ovulation the ovarian surface epithelial cells rapidly divide to repair the wall. The majority of ovarian cancer originates from this highly proliferative epithelium. Growth, Development, and Function of the Ovarian Follicle

1	Growth, Development, and Function of the Ovarian Follicle The ovarian follicle is the functional unit of the ovary, and it performs both gametogenic and endocrine functions. A histological section of the ovary from a premenopausal cycling woman contains follicular structures at many different stages of development. The life history of a follicle can be divided into the following stages: 1. 2. 3. 4. dominant (preovulatory, graafian) follicle 5. dominant follicle within the periovulatory period 6. corpus luteum (of menstruation or of pregnancy) 7.

1	2. 3. 4. dominant (preovulatory, graafian) follicle 5. dominant follicle within the periovulatory period 6. corpus luteum (of menstruation or of pregnancy) 7. Fig. 44.15 ) represent the earliest and simplest follicular structure in the ovary. Primordial follicles appear during midgestation through the interaction of gametes and somatic cells. Primordial germ cells that have migrated to the gonad continue to divide mitotically as oogonia until the fifth month of gestation in humans. At this point the approximately 7 million oogonia enter the process of meiosis and become primary oocytes. During this time the primary oocytes become surrounded by a simple epithelium of somatic follicle cells, thereby creating the primordial follicles (see

1	Fig. 44.15 ). The follicle cells establish gap junctions with each other and the oocyte. The follicle cells themselves represent a true avascular epithelium surrounded by a basal lamina. Similar to Sertoli cell–sperm interactions, a subpopulation of granulosa cells remains intimately attached to the oocytes throughout their development. Granulosa cells provide nutrients such as amino acids, nucleic acids, and pyruvate to support oocyte maturation. •Fig. 44.15 Developmentofaprimordialfollicleuptoasecondarypreantralfollicle.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)Primordial follicle Primary oocyte Basal lamina Pregranulosa cells Germinal vesicle with nucleolus Zona pellucida Cuboidal granulosa cells Primary follicle Secondary preantral follicle Theca Stratified granulosa cells The primordial follicles represent the ovarian reserve of follicles (

1	Fig. 44.16). This reserve is reduced from a starting number of about 7 million to less than 300,000 follicles at reproductive maturity. Of these, a woman will ovulate about 450 between menarche (first menstrual cycle) and menopause (cessation of menstrual cycles). At menopause, less than 1000 primordial follicles are left in the ovary. Primordial follicles are lost primarily from death as a result of follicular atresia. However, a small subset of primordial follicles will enter follicular growth in waves. Because the ovarian follicular reserve represents a fixed finite number, the rate at which resting primordial follicles die or begin to develop (or both) will determine the reproductive life span of a woman. Age at the onset of menopause has a strong genetic component but is also influenced by environmental factors. For example, cigarette smoking significantly depletes the ovarian reserve. An overly rapid rate of atresia or development will deplete the reserve and give rise to

1	by environmental factors. For example, cigarette smoking significantly depletes the ovarian reserve. An overly rapid rate of atresia or development will deplete the reserve and give rise to premature ovarian insufficiency.

1	Pituitary gonadotropins maintain a normal ovarian reserve by promoting the general health of the ovary. However, the rate at which resting primordial follicles enter the growth process appears to be independent of pituitary gonadotropins. The decision of a resting follicle to enter the early growth phase is primarily dependent on intraovarian paracrine factors produced by both the follicle cells and oocytes. The Gamete In primordial follicles the gamete is derived from oogonia that have entered the first meiotic division; such oogonia are referred to as primary oocytes. Primary oocytes progress through most of prophase of the first meiotic division

1	Ovarian reserve ~300,000 primordial follicles at menarche Atresia ~270,000 primordial follicles Growth ~30,000 primordial follicles Atresia About 30,000 primary, secondary, or tertiary follicles Ovulation ~450 dominant follicles •Fig. 44.16 Fateofovarianfollicles.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.) (termed prophase I) over a 2-week period and then arrest in the diplotene stage. This stage is characterized by the decondensation of chromatin, which supports the transcription needed for oocyte maturation. Meiotic arrest at this stage, which may last for up to 50 years, appears to be due to “maturational incompetence,” or lack of the cell cycle proteins needed to support the completion of meiosis. The nucleus of the oocyte, called the germinal vesicle, remains intact at this stage.

1	The first stage of follicular growth is preantral, which refers to the development that occurs before the formation of a fluid-filled antral cavity. One of the first visible signs of follicle growth is the appearance of cuboidal granulosa cells. At this point the follicle is referred to as a primary follicle (see Fig. 44.15 ). As granulosa cells proliferate, they form a multilayered (i.e., stratified) epithelium around the oocyte. At this stage the follicle is referred to as a secondary follicle (see Fig. 44.15 Once a secondary follicle acquires three to six layers of granulosa cells, it secretes paracrine factors that induce nearby stromal cells to differentiate into epithelioid thecal cells. Thecal cells form a flattened layer of cells around the follicle. Once a thecal layer forms, the follicle is referred to as a mature preantral follicle (see Fig. 44.15 ). In humans it takes several months for a primary follicle to reach the mature preantral stage.

1	Fig. 44.15 ). In humans it takes several months for a primary follicle to reach the mature preantral stage. Follicular development is associated with an inward movement of the follicle from the outer cortex to the inner cortex, closer to the vasculature of the ovarian medulla. Follicles release angiogenic factors that induce development of one to two arterioles that form a vascular wreath around the follicle. The Gamete During the preantral stage, the oocyte begins to grow and produce cellular and secreted proteins. The oocyte initiates secretion of extracellular matrix glycoproteins called ZP1, ZP2, and ZP3 that form the zona pellucida (see

1	Fig. 44.15 ). The zona pellucida increases in thickness and provides a species-specific binding site for sperm during fertilization (see ). Importantly, granulosa cells and the oocyte maintain gap junctional contact via cellular projections through the zona pellucida. The oocyte also continues to secrete paracrine factors that regulate follicle cell growth and differentiation.

1	Granulosa cells express the FSH receptor during this period, but they are primarily dependent on factors from the oocyte to grow. They do not produce ovarian hormones at this early stage of follicular development. The newly acquired thecal cells are analogous to testicular Leydig cells in that they reside outside the epithelial “nurse” cells, express the LH receptor, and produce androgens. The main difference •Fig. 44.17 Developmentofanearlyantralfollicletoamaturepreovulatoryfollicle.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.) between Leydig cells and thecal cells is that thecal cells do not express high levels of 17β-HSD. Thus the major product of theca cells is androstenedione as opposed to testosterone. Androstenedione production at this stage is minimal.

1	Fig. 44.17 ) over a period of about 25 days, during which they grow from a diameter of about 0.1 mm to a diameter of 0.2 mm. Once the granulosa epithelium increases to six to seven layers, fluid-filled spaces appear between cells and coalesce into the antrum. Over a period of about 45 days, this wave of small antral follicles will continue to grow to large recruitable antral follicles that are 2 to 5mm in diameter. This period of growth is characterized by about a 100-fold increase in granulosa cells (from about 10,000 to 1,000,000 cells). It is also characterized by swelling of the antral cavity, which increasingly divides the granulosa cells into two discrete populations: mural granulosa cells and cumulus cells (see Fig. 44.17

1	Fig. 44.17 Mural granulosa cells (also called the stratum granulosum) form the outer wall of the follicle. The basal layer is adherent to the basal lamina and in close proximity to 802 SECTION8Berne & Levy Physiology Oogonium Primary oocyte arrested at prophase I Meiosis begins, but levels of proteins required for completion of meiosis are too low – oocyte arrests at prophase I. As oocyte grows, it synthesizes enough proteins (e.g., CDK1, cyclin B) to complete meiosis (i.e., meiotic competence), but high cAMP levels generated by GPR3 actively maintain arrest. A few hours before ovulation, the oocyte completes meiosis I and extrudes the first polar body. It has synthesized enough MAPK pathway proteins to arrest at metaphase II.

1	A few hours before ovulation, the oocyte completes meiosis I and extrudes the first polar body. It has synthesized enough MAPK pathway proteins to arrest at metaphase II. The secondary oocyte completes meiosis at fertilization and extrudes second polar body. Haploid ovum •Fig. 44.18 Eventsinvolvedinmeioticarrestandmaturationoftheoocyte.MAPK,mitogen-activatedproteinkinase.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.) the outer-lying thecal layers. Mural granulosa cells become highly steroidogenic and remain in the ovary after ovulation to differentiate into the corpus luteum.

1	Cumulus cells are the inner cells that surround the oocyte (they are also referred to as the cumulus oophorus and corona radiata). The innermost layer of cumulus cells maintains gap and adhesion junctions with the oocyte. Cumulus cells are released with the oocyte (collectively referred to as the cumulus-oocyte complex) during the process of ovulation. Cumulus cells are crucial for the ability of the fimbriated end of the oviduct to “capture” and move the oocyte by a ciliary transport mechanism along the length of the oviduct to the site of fertilization (see Early antral follicles are dependent on pituitary FSH for normal growth. Large antral follicles become highly dependent on pituitary FSH for their growth and sustained viability. As discussed later, 2to 5-mm follicles are recruited to enter a rapid growth phase via the transient increase in FSH that occurs toward the end of the previous menstrual cycle. The Gamete

1	The Gamete The oocyte grows rapidly in the early stages of antral follicles; growth then slows in larger follicles. During the antral stage the oocyte synthesizes sufficient amounts of cell cycle components so it becomes competent to complete meiosis I at ovulation. (Note that the human egg arrests after ovulation at a second point, metaphase II, until it is fertilized by sperm.) Thus in early primary and secondary follicles, the oocyte fails to complete meiosis I because of a dearth of specific meiosis-associated proteins. However, larger antral follicles gain meiotic competence but still maintain meiotic arrest until the midcycle LH surge. Meiotic arrest is achieved by maintenance of elevated cyclic adenosine monophosphate (cAMP) levels in the mature oocyte ( Figs.

1	Figs. •Fig. 44.19 ModelofhowLHsurgeleadstoresumptionofmeiosis.PDE,phosphodiesterase.Granulosa & cumulus cells ZP GPR3 AMP cAMP PKA PDE 3A MEIOTIC ARREST (–) (–) LH SURGE LHSURGEcGMPcAMPMEIOTICRESUMPTIONGsACPDE3AcGMP cGMP cGMP 44.18 44.19 ). The constitutively active (i.e., not requiring a ligand) Gs protein-coupled receptor GPR3 maintains high cAMP. The oocyte-specific phosphodiesterase PDE3A degrades cAMP to inactive AMP. Before the LH surge, PDE3A is inhibited by cGMP, which is produced within cumulus and granulosa cells and enters the oocyte via gap junctions.

1	•Fig. 44.20 Two-cellmodelforsteroidogenesisinthedominantfollicle.Top panel: MG,muralgranulosa;T,theca.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)LHreceptor LH LDLR and HDLR-mediated uptake FSH receptor Expression of LH receptor Expression of CYP19 (aromatase) LH receptor FSH Expression and activation of StAR protein Expression of steroidogenic enzymes Acetate Cholesterol Pregnenolone Progesterone 17 (OH) Progesterone Androstenedione Testosterone Thecal cell Granulosa cell StAR protein CYP11A1 CYP17 CYP17 CYP19 CYP19 EstroneAndrostenedione Testosterone Estradiol-17˜3˜-HSD 17˜-HSD 17˜-HSD 17˜-HSD Blood vessel Basal lamina MG A a C C T The thecal cells of large antral follicles produce significant amounts of androstenedione and less testosterone. Androgens are converted to estradiol-17β by the granulosa cells (

1	The thecal cells of large antral follicles produce significant amounts of androstenedione and less testosterone. Androgens are converted to estradiol-17β by the granulosa cells ( Fig. 44.20 ). At this stage, FSH stimulates proliferation of granulosa cells and induces the expression of CYP19 (aromatase) required for estrogen synthesis. Additionally the mural granulosa cells of the large antral follicles produce increasing amounts of inhibin during the early follicular phase. Low levels of estrogen and inhibin negatively feed back on FSH secretion, thereby contributing to selection of the follicle with the most FSH-responsive cells. As already discussed, at the end of a previous menstrual cycle, a crop of large (2–5 mm) antral follicles (see Fig.

1	As already discussed, at the end of a previous menstrual cycle, a crop of large (2–5 mm) antral follicles (see Fig. 44.17 ) are recruited by a rise in FSH to begin rapid gonadotropin-dependent development. The total number of recruited follicles in both ovaries can be as high as 20 in a younger woman (<33 years old) but rapidly declines at older ages. The number of recruited follicles is reduced to the ovulation quota (one in humans) by the process of selection. As FSH levels decline the rapidly growing follicles progressively undergo atresia until one follicle is left. Generally the largest follicle with the most FSH receptors of the recruited crop becomes the dominant follicle. Selection occurs during the early follicular phase. By midcycle the dominant follicle becomes a large preovulatory follicle that is 20 mm in diameter and contains about 50 million granulosa cells by the midcycle gonadotropin surge. The Gamete

1	The Gamete The oocyte is competent to complete meiosis I but remains arrested in the dominant follicle until the LH surge. Growth of the oocyte continues but at a slower rate until the oocyte reaches a diameter of about 140 µm by ovulation (i.e., ≈20 times the diameter of an erythrocyte). The newly selected follicle emerges as a significant steroidogenic “gland.” Ovarian steroidogenesis requires both theca and granulosa cells. As discussed earlier, thecal cells (see

1	The newly selected follicle emerges as a significant steroidogenic “gland.” Ovarian steroidogenesis requires both theca and granulosa cells. As discussed earlier, thecal cells (see Fig. 44.20, T ) express LH receptors and produce primarily androstenedione. Basal levels of LH stimulate expression of steroidogenic enzymes in thecal cells. The thecal cells are richly vascularized and thus have access to cholesterol within the lipoprotein articles LDL and HDL. LH promotes expression of the LDL receptor and HDL receptor (SR-B1), which import cholesterol. LH also promotes robust expression of CYP11A1 (side chain cleavage enzyme), 3β-HSD, and CYP17 with both 17-hydroxylase activity and 17,20-lyase activity. Androgens (primarily androstenedione but also some testosterone) released from the theca diffuse into the mural granulosa cells or enter the vasculature surrounding the follicle. The mural granulosa cells (see

1	The mural granulosa cells (see Fig. 44.20, MG) of the selected follicle have a high number of FSH receptors and are very sensitive to FSH, which upregulates CYP19 (aromatase) gene expression and activity. CYP19 converts androstenedione to the weak estrogen estrone and converts testosterone to the potent estrogen estradiol-17β. Granulosa cells express activating isoforms of 17β-HSD, which converts the less active estrone to highly active estradiol17β. In addition, FSH induces expression of inhibin B during the follicular phase. Importantly, FSH also induces expression of LH receptors in mural granulosa cells during the second half of the follicular phase (see Fig. 44.20). Thus mural granulosa cells acquire the ability to respond to LH, which allows these cells to maintain high levels of CYP19 in the face of declining FSH levels. Acquisition of LH receptors also ensures that mural granulosa cells respond to the LH surge. The Dominant Follicle During the Periovulatory Period

1	The Dominant Follicle During the Periovulatory Period The periovulatory period is defined as the time from the onset of the LH surge to expulsion of the cumulus-oocyte complex out of the ovary (i.e., ovulation). This process lasts for 32 to 36 hours in women. Starting at the same time and superimposed on the process of ovulation is a change in the steroidogenic function of theca and mural granulosa cells. This process is called luteinization and culminates in formation of a corpus luteum that is capable of producing high amounts of progesterone, along with estrogen, within a few days after ovulation. Thus the LH surge induces the onset of complex processes during the periovulatory period that complete the gametogenic function of the ovary for a given month and switches the endocrine function to prepare the female reproductive tract for implantation and gestation.

1	The LH surge induces dramatic structural changes in the dominant follicle that involves its rupture, ovulation of the cumulus-oocyte complex, and biogenesis of a new structure called the corpus luteum from the remaining thecal and mural granulosa cells. Major structural changes occur during this transition: 1. Before ovulation the large preovulatory follicle presses against the ovarian surface and generates a poorly vascularized bulge of the ovarian wall called the stigma. The LH surge induces release of inflammatory cytokines and hydrolytic enzymes from the theca and granulosa cells. These secreted components lead to breakdown of the follicle wall, tunica albuginea, and surface epithelium in the vicinity of the stigma ( Fig. 44.21 ). At the end of this process the antral cavity becomes continuous with the peritoneal cavity.

1	Fig. 44.21 ). At the end of this process the antral cavity becomes continuous with the peritoneal cavity. •Fig. 44.21 Ovulation.GVBD,germinalvesiclebreakdown.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadel-phia:Mosby;2013.)Surface epithelium Tunica albugineaLuteinizing theca Blood vessels invading granulosa Luteinizing mural granulosa without basal lamina Peritoneal cavity Ruptured antrum Expanded cumulus Zona pellucida First polar body 2° oocyte after GVDB 2. The attachment of the cumulus cells to the mural granulosa cells degenerates, and the cumulus-oocyte complex becomes free-floating within the antral cavity (see Fig. 44.21 ). Cumulus cells also respond to the LH surge by secreting hyaluronic acid and other extracellular matrix components. These substances enlarge the entire cumulus-oocyte complex, a process called cumulus expansion (see

1	Fig. 44.21 ). This enlarged cumulusoocyte complex is more easily captured and transported by the oviduct. The expanded cumulus also makes the cumulus-oocyte complex easier for spermatozoa to find. Sperm express a membrane hyaluronidase that allows them to penetrate the expanded cumulus. The cumulusoocyte complex is released through the ruptured stigma through a relatively slow process. 3. The basal lamina of mural granulosa cells is broken down so that blood vessels and outer-lying theca cells can push into the granulosa cells. Granulosa cells secrete angiogenic factors such as vascular endothelial growth factor (VEGF), angiopoietin 2, and basic fibroblast growth factor (bFGF), which significantly increase the blood supply to the new corpus luteum. The Gamete Before ovulation the primary oocyte is competent to complete meiosis, but it is arrested in prophase I (see Fig.

1	The Gamete Before ovulation the primary oocyte is competent to complete meiosis, but it is arrested in prophase I (see Fig. 44.18 ). The LH surge inhibits production of cGMP by granulosa and cumulus cells, thereby removing inhibition of the oocyte-specific PDE3A. PDE3A proceeds to degrade cAMP to inactive AMP, thereby removing the brake on meiotic progression. The oocyte then progresses to meta-phase II and subsequently arrests at metaphase II until fertilization. Both theca and mural granulosa cells express LH receptors at the time of the LH surge. The LH surge induces differentiation of the granulosa cells—a process that continues for several days after ovulation. During the periovulatory period, the LH surge induces the following shifts in steroidogenic activity of the mural granulosa cells (now turning into granulosa lutein cells). 1.

1	1. Transient inhibition of CYP19 expression and consequently estrogen production. The rapid decline in estrogen helps turn off the positive feedback on LH secretion. 2. Breakdown of the basal lamina and vascularization of the granulosa cells. This makes LDL and HDL cholesterol accessible to these cells for steroidogenesis. The LH surge also increases expression of the LDL receptor and HDL receptor (SR-BI) in granulosa cells. 3. Onset of expression of StAR protein, CYP11A1 (side chain cleavage enzyme), and 3β-HSD ( Fig. 44.22 ). Expression of these enzymes are key to the onset of production of high levels of progesterone by these cells. As discussed later, high progesterone synthesis is absolutely necessary for maintenance of pregnancy. Because CYP17 activity, especially its 17,20-lyase function, is largely absent in granulosa lutein cells, progesterone is not further metabolized to another steroid but instead exits the cells and enters the circulation.

1	•Fig. 44.22 Steroidogenicpathwaysinthecorpusluteum(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)LH LH receptor LDLR and HDLR-mediated uptake Acetate Cholesterol Stimulation of steroidogenesis LH LH receptor LDLR and HDLR-mediated uptake Cholesterol Pregnenolone Stimulation of steroidogenesis StAR protein CYP11A1 Androstenedione Androstenedione Progesterone Estradiol-17˜3˜-HSD The Corpus Luteum

1	The Corpus Luteum After ovulation the remnant of the antral cavity fills with blood from damaged blood vessels in the vicinity of the stigma. This gives rise to a corpus hemorrhagicum. Within a few days, red blood cells and debris are removed by macrophages, and fibroblasts fill in the antral cavity with a hyaline-like extracellular matrix. In the mature corpus luteum the granulosa cells, now called granulosa lutein cells, enlarge and become filled with lipid (cholesterol esters). The enlarged granulosa lutein cells collapse into and partially fill in the old antral cavity. The theca along with blood vessels, mast cells, macrophages, leukocytes, and other resident connective tissue cells infiltrate the granulosa layer at multiple sites.

1	The human corpus luteum is programmed to live for 14 days, plus or minus 2 days (corpus luteum of menstruation), unless “rescued” by the LH-like hormone human chorionic gonadotropin (hCG), which originates from an implanting embryo. If rescued, the corpus luteum of pregnancy will remain viable during the pregnancy (usually ≈ 9 months). The mechanism by which the corpus luteum of menstruation regresses in 14 days is not fully understood. The corpus luteum appears to become progressively less sensitive to basal levels of LH, so that hCG binding to the LH receptor is needed for continued health and function of the corpus luteum. Regression appears to involve release of the prostaglandin PGF2α from both granulosa lutein cells and the uterus in response to declining levels of progesterone during the second week of the luteal phase. Several paracrine factors (endothelin, monocyte chemotactic protein-1) from immune and vascular cells are likely to play a role in the demise and removal of

1	the second week of the luteal phase. Several paracrine factors (endothelin, monocyte chemotactic protein-1) from immune and vascular cells are likely to play a role in the demise and removal of granulosa lutein cells. The corpus luteum is ultimately turned into a scarlike body called the corpus albicans, which sinks into the medulla of the ovary and is slowly absorbed.

1	The Gamete The LH surge induces two parallel events, ovulation and luteinization. If ovulation occurs normally, the corpus luteum is devoid of a gamete. Before the LH surge, the granulosa cells have very low capacity to convert cholesterol into a steroid hormone. The LH surge induces the onset of expression of CYP11A1, 3β-HSD and StAR protein, allowing granulosa lutein cells to convert cholesterol into progesterone. Because CYP17 expression is extremely low, progesterone accumulates and moves out of the granulosa lutein cells and enters the vasculature. Progesterone production by the corpus luteum (see

1	Fig. 44.22 ) increases steadily from the onset of the LH surge and peaks during the midluteal phase. The main purpose of this timing is to transform the uterine lining into an adhesive and supportive structure for implantation and early pregnancy. As discussed later the midluteal phase is synchronized with early embryogenesis so the uterus is optimally primed when a blastocyst tumbles into the uterus around day 22 of the menstrual cycle. Estradiol continues to be produced by theca lutein cells and granulosa lutein cells. Estrogen production transiently decreases in response to the LH surge but then rebounds and peaks at the midluteal phase. Estradiol induces the progesterone receptor in progesterone target cells, such as the uterine endometrium, and ensures a full response to progesterone. Luteal hormonal output is absolutely dependent on basal LH levels (see

1	Luteal hormonal output is absolutely dependent on basal LH levels (see Fig. 44.22 ). In fact, progesterone output is closely correlated with the pulsatile pattern of LH release in women. Both FSH and LH are reduced to basal levels during the luteal phase by negative feedback from progesterone and estrogen. In addition, granulosa lutein cells secrete inhibin, which selectively represses FSH secretion.

1	The corpus luteum must generate large amounts of progesterone to support implantation and early pregnancy. Accordingly the life of the corpus luteum is very regular, and a shortened luteal phase typically leads to infertility. The quality of the corpus luteum is largely dependent on the size and health of the dominant follicle from which it developed, which in turn is dependent on normal hypothalamic and pituitary stimulation during the follicular phase. Numerous factors that perturb hypothalamic and pituitary output during the follicular phase, including heavy exercise, starvation, high prolactin levels, and abnormal thyroid function, can lead to luteal phase deficiency and infertility.

1	Follicular atresia refers to the demise of an ovarian follicle. During atresia the granulosa cells and oocytes undergo apoptosis. The thecal cells typically persist and repopulate the cellular stroma of the ovary. The thecal cells retain LH receptors and the ability to produce androgens and are collectively referred to as the “interstitial gland” of the ovary. Follicles can undergo atresia at any time during development. Follicular Development With Respect to the Monthly Menstrual Cycle

1	The human menstrual cycle strictly refers to the monthly discharge of discarded uterine lining as menstrual blood or menstrual flow (a period referred to as menses) through the process of menstruation (see later). In fact it is the onset or lack thereof of menses, as detected by the woman herself, that is the primary evidence for the cessation of menstruation (e.g., due to pregnancy or menopause) or a change in the duration and/or frequency of the menstrual cycle. However, it is useful from an endocrinological perspective to consider the human menstrual cycle as having an ovarian cycle and a uterine cycle, with the latter being driven by the former. As discussed later, there are also hypothalamic, pituitary, oviductal, and vaginal components of the human menstrual cycle. The reproductive function of the menstrual cycle is the collective orchestration by ovarian hormones of the functions of the hypothalamus, pituitary, uterus, oviduct, cervix, and vagina—and even the ovary itself—to:

1	function of the menstrual cycle is the collective orchestration by ovarian hormones of the functions of the hypothalamus, pituitary, uterus, oviduct, cervix, and vagina—and even the ovary itself—to: (1) produce a fertilizable gamete (egg), (2) provide a supportive environment for intercourse, reception of sperm, fertilization of egg and early embryogenesis, (3) prepare the uterine lining for implantation, placentation and pregnancy, and (4) minimize the possibility of a superimplantation (i.e., a second implantation) from occurring, and/or prevent an ascending infection from moving up to the uterus from the vagina.

1	The first half of the monthly menstrual cycle is referred to as the follicular phase of the ovary and is characterized by recruitment and growth of a large antral follicle, selection of the dominant follicle, and growth of the dominant follicle until ovulation. The dominant follicle must contain a fully developed oocyte and somatic follicle cells that secrete high levels of estrogen. The second half of the monthly menstrual cycle is referred to as the luteal phase of the ovary and is dominated by hormonal secretions of the corpus luteum. The corpus luteum must secrete both progesterone and estradiol for progression of the normal cycle. Regulation of Late Stages of Follicular Development, Ovulation, and Luteinization: The Human Menstrual Cycle

1	Regulation of Late Stages of Follicular Development, Ovulation, and Luteinization: The Human Menstrual Cycle As stated earlier, late stages of follicular development and luteal function are absolutely dependent on normal hypothalamic and pituitary function. As in the male, hypothalamic neurons secrete GnRH in a pulsatile manner. GnRH in turn stimulates LH and FSH production by pituitary gonadotropes. A high frequency of GnRH pulses (1 pulse per 60–90 minutes) selectively promotes LH production, whereas a slow frequency promotes FSH production. A major difference between the male and female reproductive axes is the midcycle gonadotropin surge, which is dependent on a constant high level of estrogen coming from the dominant follicle. A highly dynamic “conversation” occurs among the ovary, pituitary, and hypothalamus in which the events of the menstrual cycle are orchestrated, beginning with the ovary at the end of the luteal phase of a previous nonfertile cycle (

1	Fig. 44.23 ). Events that follow are numbered according to Fig. 44.24 : Event 1: In the absence of fertilization and implantation, the corpus luteum regresses and dies (called luteolysis). This leads to a dramatic decline in levels of progesterone, estrogen, and inhibin by day 24 of the menstrual cycle. Event 2: The pituitary gonadotrope perceives the end of luteal function as a release from negative feedback (see Fig. 44.23B , Late Luteal Phase). This leads to an increase in FSH about 2 days before the onset of menstruation. The basis for the selective increase in FSH is incompletely understood, but it may be due to the slow frequency of GnRH pulses during the luteal phase, which in turn is due to high progesterone levels. CHAPTER 44 The Male and Female Reproductive Systems

1	CHAPTER 44 The Male and Female Reproductive Systems Event 3: The rise in FSH levels recruits a crop of large (2–5 mm in diameter) antral follicles to begin rapid, highly gonadotropin-dependent growth. These follicles produce low levels of estrogen and inhibin B. Event 4: The gonadotrope responds to the slowly rising levels of estrogen and inhibin B by decreasing FSH secretion (see Fig. 44.23A , Early Follicular Phase). The absence of progesterone promotes an increase in the frequency of GnRH pulses, thereby selectively increasing LH synthesis and secretion by the gonadotrope. Thus the LH:FSH ratio slowly increases throughout the follicular phase. Event 5: The ovary’s response to declining FSH levels is follicular atresia of all of the recruited follicles except for one dominant follicle (see

1	Event 5: The ovary’s response to declining FSH levels is follicular atresia of all of the recruited follicles except for one dominant follicle (see Fig. 44.23A , Early Follicular Phase). Thus the process of selection is driven by an extreme dependency of follicles on FSH in the face of declining FSH secretion. Usually only the largest follicle with the most FSH receptors and best blood supply can survive. This follicle produces increasing amounts of estradiol-17β and inhibin B. A critical action of FSH at this time is induction of the expression of LH receptors in the mural granulosa cells of the dominant follicle (see Fig. 44.23A , Late Follicular Phase).

1	Fig. 44.23A , Late Follicular Phase). Event 6: Once the dominant follicle causes circulating estrogen levels to exceed 200 pg/mL for about 50 hours in women, estrogen exerts a positive feedback on the gonadotrope to produce the midcycle LH surge. This is enhanced by the small amount of progesterone secreted at midcycle. The exact mechanism of the positive feedback is unknown, but it occurs largely at the level of the pituitary. GnRH receptors and the sensitivity to GnRH signaling increase dramatically in the gonadotropes. The hypothalamus contributes to the gonadotropin surge by increasing the frequency of GnRH pulses. Event 7: The LH surge drives meiotic maturation, ovulation, and differentiation of granulosa cells into progesterone-producing cells (see Fig. 44.23A , Late Follicular Phase).

1	Event 7: The LH surge drives meiotic maturation, ovulation, and differentiation of granulosa cells into progesterone-producing cells (see Fig. 44.23A , Late Follicular Phase). Event 8: Rising levels of progesterone, estrogen, and inhibin A by the mature corpus luteum negatively feed back on pituitary gonadotropes. Even though estradiol levels exceed the 200-pg/mL threshold for positive feedback, the high progesterone levels now produced by the corpus luteum block any positive feedback of estradiol. Consequently both FSH and LH levels decline to basal levels (see Fig. 44.23B , Mid-Luteal Phase).

1	Fig. 44.23B , Mid-Luteal Phase). Event 9: Basal levels of LH (but not FSH) are absolutely required for normal corpus luteum function. However, the corpus luteum becomes progressively insensitive to LH signaling and will die unless LH-like activity (i.e., hCG from an implanted embryo) increases. In a nonfertile cycle the corpus luteum of menstruation will regress in 14 days, and progesterone and estrogen levels will start to decline by about 10 days, thereby cycling back to event 1 (see Fig. 44.23B , Late Luteal Phase).

1	808 SECTION8Berne & Levy Physiology •Fig. 44.23 A,Endocrinesignalingleadingtotheovulationofadominantfollicleattheendofthefollicularphaseofthemenstrualcycle.B,Endocrinesignalingduringthelutealphaseofanon-pregnantmenstrualcycleleadingtothedeathofthecorpusluteumandrecruitmentoffolliclestobeginnextcycle.LATE FOLLICULAR d8-14 EARLY FOLLICULAR d3-7 Dominant follicle COC Ruptured follicle Growing large antral follicles recruited by FSH Decreasing FSH basal LH (–)(–)(+)(+)High E2 no P4 Gonado-tropes GnRH neurons GnRH neurons Kisspeptin neurons Kisspeptin neurons Moderate E2 & inhibin no P4 LH surge @ midcycle GnRH rapid pulses Gonado-tropes GnRH slow pulses • Increasing (–) feedback causing lower FSH • Follicular atresia • Selection of dominant follicle Switch to (+) feedback-especially at level of pituitary sensitivity to rapid GnRH pulses • Meiotic maturation • Luteinization • Ovulation of cumulus-oocyte complex (COC) A. Follicular Phase LATE LUTEAL (d25-28) MID-LUTEAL (d17-24) Corpus

1	of pituitary sensitivity to rapid GnRH pulses • Meiotic maturation • Luteinization • Ovulation of cumulus-oocyte complex (COC) A. Follicular Phase LATE LUTEAL (d25-28) MID-LUTEAL (d17-24) Corpus luteum from ovulated dominant follicle Large antral recruitable follicles Dying corpus luteum (corpus albicans) If no implantation no rescue by hCG Maintains active corpus luteum FSH recruits large antral follicles new cycle starts Loss of negative feedack from ovary after corpus luteum dies Gonado-tropes GnRH neurons Kisspeptin neurons GnRH rapid pulses Gonado-tropes GnRH neurons Kisspeptin neurons GnRH slow pulses (–)(–)High P4 moderate & inhibition FSH Peak Basal LH & FSH No E2 inhibin or P4 B. Luteal Phase

1	CHAPTER 44 The Male and Female Reproductive Systems 809 1. Corpus luteum dies, E and P levels fall. 5. Declining FSH levels progressively cause atresia of all but 1 follicle – leading to selection of dominant follicle, which produces high levels of E. 9. The corpus luteum progressively becomes less sensitive to basal LH – dies if levels of LH-like activity (i.e., hCG) do not increase. 3. FSH recruits a cohort of large antral follicles to enter rapid growth phase. Follicles secrete low amounts of E and inhibin. 7. LH surge induces meiotic maturation, ovulation and luteinization. The corpus luteum produces high P, along with E and inhibin. 4. E and inhibin negatively feed back on FSH. FSH LH Pituitary Progesterone OvaryEstradiol-17˜2. Pituitary responds to falling E and P by increasing FSH secretion. 6. High E has positive feedback on gonadotropes— LH (and some FSH) surges. 8. High P, E and inhibin negatively feed back on LH and FSH, returning them to basal levels.

1	•Fig. 44.24 Thehumanmenstrualcycle,withemphasisonthe“dialogue”betweenovaryandpituitarygonadotropes.NotethattherelativechangesinthelevelsofE2andinhibinareshownbythesameline. From this sequence of events it is evident that the ovary is the primary clock for the menstrual cycle. The timing of the two main pituitary-based events—the transient rise in FSH that recruits large antral follicles and the LH surge that induces ovulation—is determined by two ovarian events. These are respectively the highly regular life span of the corpus luteum and its demise after 14 days, and growth of the dominant follicle to the point at which it can maintain a sustained high production of estrogen that induces a switch to positive feedback at the pituitary. In essence the dominant follicle tells the pituitary it is ready to undergo ovulation and luteinization. The Oviduct

1	The Oviduct The oviducts (also called the uterine tubes and the fallopian tubes) are muscular tubes with the distal ends close to the surface of each ovary and the proximal ends traversing the wall of the uterus. The oviducts are divided into four sections (going from distal to proximal): the infundibulum, or open end of the oviduct, which has fingerlike projections called fimbriae that sweep over the surface of the ovary; the ampulla, which has a relatively wide lumen and extensive folding of the mucosa; the isthmus, which has a relatively narrow lumen and less mucosal folding; and the intramural or uterine segment, which extends through the uterine wall at the superior corners of the uterus ( Fig. 44.25 The main functions of the oviducts are to: 1.

1	Fig. 44.25 The main functions of the oviducts are to: 1. Capture the cumulus-oocyte complex at ovulation and transfer the complex to a midway point (the ampullary-isthmus junction), where fertilization takes place. Oviductal secretions coat and infuse the cumulusoocyte complex and are likely required for viability and fertilizability. 2. Provide a site for sperm storage. Women who ovulate up to 5 days after sexual intercourse can get pregnant. Sperm remain viable by adhering to the epithelial cells lining the isthmus. The secretions of the oviduct also induce capacitation and hyperactivity of sperm. 3. Secrete fluids that provide nutritional support to the preimplantation embryo. The timing of movement of the embryo into the uterus is critical because the uterus has an implantation window of about 3 days. The oviduct needs to hold the early embryo until it reaches the blastocyst stage (5 days after fertilization) and then allow it to pass into the uterine cavity.

1	810 SECTION8Berne & Levy Physiology •Fig. 44.25 Schematicofthefemalereproductivesystem.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)Fundus of uterusIsthmusAmpulla Infundibulum with fimbriae Uterine (fallopian) tube Ovary Body (corpus) of uterus Internal genitalia External genitalia Cervix of uterus Vagina Clitoris Vaginal introitus Opening of female urethra Mons pubis Labia majora Labia minora

1	The wall of the oviduct is composed of a mucosa (called the endosalpinx), a two-layered muscularis (called the myosalpinx), and an outer-lying connective tissue (the perisalpinx). The endosalpinx is thrown into many folds, almost to the extent that the lumen is obliterated, and is lined by a simple epithelium made up of two cell types: ciliated cells and secretory cells. The cilia are most numerous at the infundibular end and propel the cumulus-oocyte complex toward the uterus. The cilia on the fimbriae are the sole mechanism for transport of the ovulated cumulus-oocyte complex. Once the complex passes through the ostium of the oviduct and enters the ampulla, it is moved by both cilia and peristaltic contractions of the muscularis.

1	The secretory cells produce a protein-rich mucus that is conveyed along the oviduct to the uterus by the cilia. This ciliary-mucus escalator maintains a healthy epithelium, moves the cumulus-oocyte complex toward the uterus, and may provide directional cues for swimming sperm. Movement of the cumulus-oocyte complex slows at the ampullary-isthmus junction, where fertilization normally takes place. This appears to be due in part to thick mucus produced by the isthmus and increased tone of the muscularis of the isthmus. The composition of oviductal secretions is complex and includes growth factors, enzymes, and oviduct-specific glycoproteins. Note that the clinical process of in vitro fertilization has shown that secretions of the oviduct are not absolutely necessary for fertility. However, normal oviductal function is absolutely required for both fertilization and implantation after in vivo insemination (i.e., natural sexual intercourse). Normal oviductal function also minimizes the

1	normal oviductal function is absolutely required for both fertilization and implantation after in vivo insemination (i.e., natural sexual intercourse). Normal oviductal function also minimizes the risk of ectopic implantation and ectopic pregnancy, which occurs most often within the oviduct.

1	Hormonal Regulation During the Menstrual Cycle

1	In general, estrogen secreted during the follicular phase increases epithelial cell size and height in the endosalpinx. Estrogen increases blood flow to the lamina propria of the oviducts, promotes production of oviduct-specific glycoproteins (whose functions are poorly understood), and increases ciliogenesis throughout the oviduct. Estrogen promotes secretion of thick mucus in the isthmus and increases the tone of the muscularis of the isthmus, thereby keeping the cumulus-oocyte complex at the ampullary-isthmus junction for fertilization. High progesterone, along with estrogen, during the early luteal to midluteal phase decreases epithelial cell size and function. Progesterone promotes deciliation. It also decreases the secretion of thick mucus and relaxes the tone in the isthmus. In addition it should be noted that oviductal epithelial cells express the LH receptor, which may synergize with estrogen to optimize oviductal function during the periovulatory period. The Uterus

1	The Uterus The uterus is a single organ that sits in the midline of the pelvic cavity between the bladder and the rectum. The mucosa of the uterus is called the endometrium, the three-layered thick muscularis is called the myometrium, and the outer connective tissue and serosa are called the perimetrium. The parts of the uterus are (1) the fundus, which is the portion that rises superiorly from the entrance of the oviducts, (2) the body of the uterus, which makes up most of the uterus, (3) the isthmus, a short narrowed part of the body at its inferior end, and (4) the cervix, which extends into the vagina (see Figs. 44.13 and 43.25). Because the cervical mucosa is distinct from the rest of the uterus and does not undergo the process of menstruation, it will be discussed separately later. The established functions of the uterus are all related to fertilization and pregnancy (discussed later). The main functions of the uterus are to: 1.

1	The established functions of the uterus are all related to fertilization and pregnancy (discussed later). The main functions of the uterus are to: 1. assist movement of sperm from the vagina to the oviducts 2. provide a suitable site for attachment and implantation of the blastocyst, including a thick nutrient-rich stroma 3. limit the invasiveness of the implanting embryo so it stays in the endometrium and does not reach the myometrium 811 •Fig. 44.26 Diagramoftheorganizationofglandsandbloodflowwithintheuterineendometrium.(FromStrausIII.In:YenSSCetal[eds].Reproductive Endocrinology. 4thed.Philadelphia:Saunders;1999.)Radial branch Spiral artery Uterine gland ENDOMETRIUM MYOMETRIUM Uterine artery Functional Zone Basal Zone Venous lakes Arcuate CHAPTER 44 The Male and Female Reproductive Systems 4.

1	CHAPTER 44 The Male and Female Reproductive Systems 4. provide a maternal side of the mature placental architecture, including the basal plate to which the fetal side attaches, and large intervillous spaces that become filled with maternal blood after the first trimester 5. grow and expand with the growing fetus so it develops within an aqueous nonadhesive environment 6.

1	provide strong muscular contractions to expel the fetus and placenta at term To understand the function of the uterus and uterine changes during nonfertile menstrual cycles, the fine structure of the endometrium and the relationship of uterine blood supply to the endometrium will be reviewed (Fig. 44.26). The luminal surface of the endometrium is covered with a simple cuboidal/columnar epithelium. The epithelium is •Fig. 44.27 Themenstrualcycleoftheuterineendometrium.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.) continuous with mucosal glands (called uterine glands) that extend deep into the endometrium. The mucosa is vascularized by spiral arteries, which are branches of the uterine artery that run through the myometrium. The terminal arterioles of the spiral arteries project just beneath the surface epithelium. These arterioles give rise to a subepithelial plexus of capillaries and venules that have ballooned thin-walled segments called

1	of the spiral arteries project just beneath the surface epithelium. These arterioles give rise to a subepithelial plexus of capillaries and venules that have ballooned thin-walled segments called venous lakes or lacunae. The lamina propria itself is densely cellular. The stromal cells of the lamina propria play important roles during both pregnancy and menstruation.

1	About two-thirds of the luminal side of the endometrium is lost during menstruation and is called the functional zone (also called the stratum functionalis) (see Fig. 44.26 ). The basal third of the endometrium that remains after menstruation is called the basal zone (also called the stratum basale). The basal zone is fed by straight arteries that are separate from the spiral arteries, and it contains all the cell types of the endometrium (i.e., epithelial cells from the remaining tips of glands, stromal cells, and endothelial cells). Hormonal Regulation of the Uterine Endometrium During the Menstrual Cycle Monthly oscillations in ovarian steroids induce the uterine endometrium to enter different stages. At the time of selection of the dominant follicle and its elevating production of estradiol, the uterine endometrium is just ending menstruation. The stratum functionalis has been shed and only the stratum basale remains ( Fig. 44.27).

1	Fig. 44.27). The rising levels of estrogen during the mid to late follicular phase of the ovary induce the proliferative phase of the uterine endometrium. Estrogen induces all cell types in the stratum basale to grow and divide. In fact the definition of an “estrogenic” compound has historically been one that is “uterotropic.” Estrogen increases cell proliferation directly through its cognate receptors (ER-α and ER-β), which regulate gene expression ( Fig. 44.28 ). Estrogen also controls uterine growth indirectly through local production of growth factors. In addition, estrogen induces expression of progesterone receptors, thereby “priming” the uterine endometrium so it can respond to progesterone during the luteal phase of the ovary. By ovulation, the thickness of the stratum functionalis has been reestablished under the proliferative actions of estradiol-17β (see

1	By ovulation, the thickness of the stratum functionalis has been reestablished under the proliferative actions of estradiol-17β (see Fig. 44.27 ). After ovulation the corpus luteum produces high levels of progesterone along with estradiol-17β. The luteal phase of the ovary switches the proliferative phase of the uterine endometrium to the secretory phase. In general, progesterone inhibits further endometrial growth and induces differentiation of epithelial and stromal cells. Progesterone induces the uterine glands to secrete a nutrient-rich product that supports blastocyst viability. As the secretory phase proceeds the mucosal uterine glands become corkscrewed and sacculated (see Fig.

1	Fig. 44.27 ). Progesterone also induces changes in adhesivity of the surface epithelium, thereby generating the “window of receptivity” for implantation of an embryo (see nancy). Additionally, progesterone promotes differentiation of stromal cells into “predecidual cells,” which must be prepared to form the decidua of pregnancy or to orchestrate menstruation in the absence of pregnancy.

1	•Fig. 44.28 Molecularmechanismbywhichtheestrogenreceptor(ER)regulatesgeneexpression.Left, Estradiol-17β bindstotheERandchangesitsconformationsothatitbindsasadimertoestrogen-responseelement(ERE)andrecruitscoactivatorproteins(Co-Act),whichleadstostimulationofgeneexpression.Right, Selectiveestrogenreceptormodulators(SERMs),suchastamoxifeninthebreast,alterERconformationsothatitrecruitsco-repressorproteins(Co-Rep),therebyinhibitinggeneexpression.InthiscasetheSERMactsasanERantagonist,butinsometissuesthesameSERMcanactasanERagonist.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)

1	Progesteroneopposestheproliferativeactionsofestradiol-17β anddownregulatestheestrogenreceptor(ER).Progesteronealsoinducesinactivating isoforms of 17β-HSD, therebyconvertingtheactiveestradiol-17β intotheinactiveestrone.Thisoppositionofthemitogenicactionsofestradiol-17β byprogesteroneisimportanttoprotecttheuterineendometriumfromestrogen-induceduterinecancer.Incontrast,administrationof“unopposed estrogen” towomensignificantlyincreasestheriskforuterinecancer. Fig.44.28 ).Forexample,theSERMtamoxifen isusedasanERantagonistforthetreatmentofbreastcancer(whoseearlyprogressionispromotedbyestrogen).BindingofSERMtotheERinducesconformationalchangesthatallowco-repressorstobindtotheERorpromotedegradationoftheER(orboth;see Fig.

1	Fig. 44.28 ).Becausetamoxifenhassomeuterotropicactivity(i.e.,makesuterineendometrialtissuegrow),newerSERMssuchasraloxifene havebeendevelopedtohaveERantagonistactivityonthebreast,beneficialERagonistactivityonbone(seelater),andnoactivityorERantagonisticactivityontheuterineendometrium. In a nonfertile cycle, death of the corpus luteum results in sudden withdrawal of progesterone, which leads to changes in the uterine endometrium that result in loss of the lamina functionalis (see Fig. 43.27). Menstruation normally lasts for 4 to 5 days (called a period), and the volume of blood loss ranges from 25 to 35 mL. Menstruation coincides with the early follicular phase of the ovary. Hormonal Regulation of the Myometrium

1	Hormonal Regulation of the Myometrium The smooth muscle cells of the myometrium are also responsive to changes in steroid hormones. Peristaltic contractions of the myometrium favor movement of the luminal contents from the cervix to the fundus at ovulation, and these contractions probably play a role in rapid bulk transport of ejaculated sperm from the cervix to the oviducts. During menstruation, contractions propagate from the fundus to the cervix, thereby promoting expulsion of sloughed stratum functionalis. The size and number of smooth muscle cells are determined by estrogen and progesterone. Healthy cycling women maintain a robust myometrium, whereas the myometrium progressively thins in postmenopausal women. The most drastic changes are seen during pregnancy, when the smooth muscle cells increase from 50 to 500 µm in length. The pregnant myometrium also has a greater number of smooth muscle cells and more extracellular matrix.

1	Disordersofmenstruationarerelativelycommonandincludemenorrhagia (heavymenstrualflowleadingtolossofmorethan80mLofblood),metrorrhagia irregularandsometimesprolongedmenstrualflowbetweennormalperiods),anddysmenorrhea (painfulperiods).Theexistenceofafewirregularperiods,calledoligomenorrhea, andtheabsenceofperiods,calledamenorrhea, areoftenduetodysfunctionofthehypothalamic-pituitary-ovarianaxisasopposedtolocalpelvicpathophysiology. Because endometrial tissue is naturally sloughed in fragments that contain viable cells, endometrial tissue occasionally gains access to other parts of the female tract (e.g., oviducts, ovary), as well as the lower part of the abdomen and associated structures (e.g., rectouterine pouch of Douglas, as shown in

1	Fig. 44.13 ). These implants give rise to endometriosis—a foci of hormonally responsive endometrial tissue outside the uterus. The spread of endometriosis may be due to reflux of menstrual tissue into the oviducts or movement of tissue through lymphatics, or both. Endometriosis frequently exhibits cyclic bleeding and is associated with infertility, pain on defecation, pain on urination, pain with sexual intercourse, or generalized pelvic pain. The Cervix The cervix is the inferior extension of the uterus that projects into the vagina (see

1	Figs. 44.13 44.25 ). It has a mucosa that lines the endocervical canal, which has a highly elastic lamina propria and a muscularis that is continuous with the myometrium. The part of the cervix that extends into the vaginal vault is called the ectocervix, whereas the part surrounding the endocervical canal is called the endocervix. The openings of the endocervical canal at the uterus and vagina are called the internal cervical os and the external cervical os, respectively. The cervix acts as a gateway to the upper female tract—at midcycle the endocervical canal facilitates sperm viability and entry. During the luteal phase the endocervical canal impedes passage of sperm and microbes, thereby inhibiting superimplantation of a second embryo or ascending infection into the placenta, fetal membranes, and fetus. The cervix physically supports the weight of the growing fetus. At term, cervical softening and dilation allow passage of the newborn and placenta from the uterus into the vagina.

1	Hormonal Regulation of Cervical Mucus During the Menstrual Cycle The endocervical canal is lined by simple columnar epithelium that secretes cervical mucus in a hormonally responsive manner. Estrogen stimulates production of a copious quantity of thin, watery, slightly alkaline mucus that is an ideal environment for sperm. Progesterone stimulates production of a scant, viscous, slightly acidic mucus that is hostile to sperm. During the normal menstrual cycle the conditions of the cervical mucus are ideal for sperm penetration and viability at the time of ovulation. The Vagina The vagina is one of the copulatory structures in women and acts as the birth canal (see

1	The Vagina The vagina is one of the copulatory structures in women and acts as the birth canal (see Figs. 44.13 44.25 ). Its mucosa is lined by a nonkeratinized stratified squamous epithelium. The mucosa has a thick lamina propria enriched with elastic fibers and is well vascularized. There are no glands in the vagina, so lubrication during intercourse comes from (1) cervical mucus (especially with intercourse that occurs midcycle), (2) a transudate (i.e., ultrafiltrate) from the blood vessels of the lamina propria, and (3) the vestibular glands. The mucosa is surrounded by a relatively thin (i.e., relative to the uterus and cervix) two-layered muscularis and an outer connective tissue. The vaginal wall is innervated by branches of the pudendal nerve, which contribute to sexual pleasure and orgasm during intercourse. Hormonal Regulation During the Menstrual Cycle

1	Hormonal Regulation During the Menstrual Cycle The superficial cells of the vaginal epithelium are continually desquamating, and the nature of these cells is influenced by the hormonal environment. Estrogen stimulates proliferation of the vaginal epithelium and increases its glycogen content (referred to as “cornification”—but in humans, true cornification or keratinization does not occur). The glycogen is metabolized to lactic acid by commensal lactobacilli, thereby maintaining an acidic environment. This inhibits infection by noncommensal bacteria and fungi. Progesterone increases the desquamation of epithelial cells. The External Genitalia The female external genitalia are surrounded by the labia majora (homologues of the scrotum) laterally and the mons pubis anteriorly (see

1	The External Genitalia The female external genitalia are surrounded by the labia majora (homologues of the scrotum) laterally and the mons pubis anteriorly (see Fig. 44.25 ). The vulva collectively refers to an area that includes the labia majora and mons pubis plus the labia minora, the clitoris, the vestibule of the vagina, the vestibular bulbs (glands), and the external urethral orifice. The vulva is also referred to as the pudendum by clinicians. The structures of the vulva serve the functions of sexual arousal and climax, directing the flow of urine, and partially covering the opening of the vagina, thereby inhibiting entry of pathogens.

1	The clitoris is the embryological homologue of the penis and is composed of two corpora cavernosa, which attach the clitoris to the ischiopubic rami, and a glans. These structures are composed of erectile tissue and undergo the process of erection in essentially the same manner as the penis. Unlike the penis, clitoral tissue is completely separate from the urethra. Thus the clitoris is involved in sexual arousal and climax at orgasm. The vagina is likewise involved in sexual satisfaction but also serves as the copulatory organ and birth canal. Hormonal Regulation During the Menstrual Cycle The structures of the vulva do not show marked changes during the menstrual cycle. However, the health and function of these structures are dependent on hormonal support. The external genitalia and vagina are responsive to androgens (testosterone and dihydrotestosterone) and estrogen. Androgens also act on the central nervous system (CNS) to increase libido in women.

1	Biology of Estradiol-17β and Progesterone Biological Effects of Estrogen and Progesterone

1	Biological Effects of Estrogen and Progesterone Estradiol-17β and progesterone fluctuate during the menstrual cycle, and they have multiple effects that can be categorized according to whether they are directly related to the reproductive system or not. Both hormones have profound effects on the ovary, oviduct, uterus, cervix, vagina, and external genitalia and on the hypothalamus and pituitary. Estrogen and progesterone also have important effects on nonreproductive tissues: Bone: Estrogen is required for closure of the epiphyseal plates of long bones in both sexes. Estradiol-17β has a bone anabolic and calciotropic effect (see ). It stimulates intestinal Ca++ absorption. Estradiol-17β is also one of the most potent regulators of osteoblast and osteoclast function. Estrogen promotes survival of osteoblasts and apoptosis of osteoclasts, thereby favoring bone formation over resorption. Low estrogen levels associated with menopause leads to bone loss and osteoporosis.

1	Liver: The overall effect of estradiol-17β on the liver is to improve circulating lipoprotein profiles. Estrogen increases expression of the LDL receptor, thereby increasing clearance of cholesterol-rich LDL particles by the liver. Estrogen also increases circulating levels of HDL. Estrogen regulates hepatic production of several transport proteins, including cortisol-binding protein, thyroid hormone–binding protein, and SHBG. CHAPTER 44 The Male and Female Reproductive Systems Cardiovascular organs: Premenopausal women have significantly less cardiovascular disease than men or post-menopausal women do. Estrogen promotes vasodilation through increased production of nitric oxide, which relaxes vascular smooth muscle and inhibits platelet activation. Single-nucleotide polymorphisms in the estrogen receptor have been associated with increased cardiovascular disease.

1	Integument: Estrogen and progesterone maintain healthy smooth skin with normal epidermal and dermal thickness. Estrogen stimulates proliferation and inhibits apoptosis of keratinocytes. In the dermis, estrogen and progesterone increase collagen synthesis and inhibit breakdown of collagen by suppressing matrix metalloproteinases. Estrogen also increases glycosaminoglycan production and deposition in the dermis and promotes wound healing.

1	CNS: Estrogen is neuroprotective—that is, it inhibits neuronal cell death in response to hypoxia or other insults. Estrogen’s positive effects on angiogenesis may account for some of the beneficial and stimulant-like actions of estrogen on the CNS. Progesterone acts on the hypothalamus to increase the set point for thermoregulation, thereby elevating body temperature approximately 0.5°F. This is the basis for using body temperature measurements to determine whether ovulation has occurred. Progesterone is a CNS depressant. Loss of progesterone on demise of the corpus luteum of menstruation is the basis for premenstrual dysphoria (premenstrual syndrome [PMS]). Progesterone also acts on the brainstem to sensitize the ventilatory response to PCO2 so that ventilation increases and PCO2 decreases.

1	Adipose tissue: Estrogen decreases adipose tissue by decreasing lipoprotein lipase activity and increasing hormone-sensitive lipase (i.e., it has a lipolytic effect). Loss of estrogen results in accumulation of adipose tissue, especially in the abdomen. Transport and Metabolism of Ovarian Steroids Steroid hormones are slightly soluble in blood and are bound to plasma proteins. Approximately 60% of the estrogen is transported bound to SHBG, 20% is bound to albumin, and 20% is in the free form. Progesterone binds primarily to cortisol-binding globulin (transcortin) and albumin. Because it has relatively low binding affinity for these proteins, its circulating half-life is about 5 minutes.

1	Although the ovary is the primary site of estrogen production, peripheral aromatization of androgens to estrogens can generate locally high levels of estradiol-17β in some tissues. Peripheral conversion of adrenal and ovarian androgens serves as an important source of estrogen after menopause (discussed later). The fact that CYP19 (aromatase) is expressed in the breast is the basis for the use of aromatase inhibitors in the treatment of estrogen-dependent breast cancer in postmenopausal women. •Fig. 44.29 Patternofgonadotropinsecretionthroughoutlife.Notethetransientpeaksduringgestationandearlyinfancyandthelowlevelsthereafterinchildhood.Womensubsequentlyhavemonthlycyclicbursts,withluteinizinghormone(LH)exceedingfollicle-stimulatinghormone(FSH);mendonot.Bothgendersshowincreasedgonadotropinproductionafterage50,withFSHexceedingLH.

1	Estrogens and progestins are degraded in the liver to inactive metabolites, conjugated with sulfate or glucuronide, and excreted in urine. Major metabolites of estradiol include estrone, estriol, and catecholestrogens (2-hydroxyestrone and 2-methoxyestrone). The major metabolite of progesterone is pregnanediol, which is conjugated with glucuronide and excreted in urine. Ontogeny of the Reproductive Systems Unlike most other organ systems, the reproductive systems undergo significant changes in their activity during the life span of a man or woman (

1	Unlike most other organ systems, the reproductive systems undergo significant changes in their activity during the life span of a man or woman ( Fig. 44.29 ). Development of the reproductive systems occurs in utero and results in female or male fetuses. After birth and during infancy, the reproductive systems are largely quiescent. At puberty the hypothalamic-pituitary-gonadal axes “wake up” and the gonads begin producing sex steroids, which in turn induce the sexually dimorphic changes in appearance and behavior associated with men and women. The reproductive life span of women is set by their ovarian reserve and degree of follicular development (see earlier) and ends at menopause, usually in the fifth decade of life. Loss of estrogen production by the ovaries has a clear clinical impact on many postmenopausal women. Men continue to produce sperm throughout life but can experience a decline in androgen production (andropause), which is associated with its own clinical sequelae.

1	The reproductive system of women undergoes dramatic changes during pregnancy. Production of gonadotropin and gonadal steroids is switched from the maternal hypothalamic-pituitary-ovarian axis, which is strongly repressed during pregnancy, to the fetal placenta. Indeed, it is the endocrine function of fetal placental tissue that maintains a quiescent gravid uterus, (2) alters maternal physiology to ensure fetal nutrition in utero, (3) alters maternal pituitary function and mammary gland development to ensure ongoing fetal nutrition after birth, and determines the time of labor and delivery (also called parturition). The placenta also plays an important role in fetal testosterone production and male differentiation of the reproductive system before the fetal hypothalamus and pituitary develop into a functional axis. Fertilization, Early Embryogenesis, Implantation, and Placentation

1	Fertilization, Early Embryogenesis, Implantation, and Placentation Fertilization, early embryogenesis, implantation, and early gestation are all synchronized with the human menstrual cycle ( Fig. 44.30 ). Just before ovulation the ovary is in the late follicular stage and produces high levels of estrogen. Estrogen promotes growth of the uterine endometrium and induces expression of the progesterone receptor. Estrogen ultimately induces the LH surge, which in turn induces meiotic maturation of the oocyte and ovulation of the cumulus-oocyte complex. The events between fertilization and implantation take about 6 days to complete, so implantation occurs at about day 22 of the menstrual cycle. At this time the ovary is in the midluteal phase and secreting large amounts of progesterone. Progesterone stimulates secretion from the uterine glands, which provide nutrients to the embryo. This is referred to as histiotropic nutrition and is an important

1	CHAPTER 44 The Male and Female Reproductive Systems 817 •Fig. 44.30 Synchronizationofeventsofthemenstrualcycle(ovaryandendometrium)withfertiliza-tion,earlyembryonicdevelopmentwithintheoviduct,andimplantationofembryo(blastocyst)intotheuterineendometrium.E2,estradiol;P4,progesterone.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)d13 Peak E2 levels Onset of LH surge Dominant follicle-high E2 output d7 Ovary and pituitary Late follicular phase d13 Resumption of meiosis d20 Blastocyst in uterus d14 d15 Early luteal phase d15 First polar body and arrest at metaphase II 35 hours after onset of LH surge d21 Late luteal phase Growth of functional zone Induction of P4 receptors P4 induces secretion from E2-primed glands, and opposition of E2 mitogenic effects Receptive period Decreased antiadhesion protein expression Increased adhesion protein expression Uterine endometrium Arrested primary oocyte Egg and embryo Late proliferative phase Early secretory

1	Receptive period Decreased antiadhesion protein expression Increased adhesion protein expression Uterine endometrium Arrested primary oocyte Egg and embryo Late proliferative phase Early secretory phase Late secretory phase d28 Rescue of CL by hCG d20 d24 d16 Fertilization pronuclear stage d23 Blastocyst hatches from zona pellucida and implantation begins on day 22–23 (6–7 days after fertilization) d22d17 2-cell embryo d26 Completion of interstitial implantation mode of maternal-to-fetal transfer of nutrients for about the first trimester of pregnancy, after which it is replaced by hemotropic nutrition (see later). Progesterone inhibits myometrial contraction and prevents release of paracrine factors (e.g., cytokines, prostaglandins, chemokines, and vasoconstrictors) that lead to menstruation. Progesterone induces the “window of receptivity” in the uterine endometrium, which exists from about day 20 to day 24 of the menstrual cycle. This receptive phase is associated with increased

1	Progesterone induces the “window of receptivity” in the uterine endometrium, which exists from about day 20 to day 24 of the menstrual cycle. This receptive phase is associated with increased adhesivity of the endometrial epithelium and involves formation of cellular extensions called pinopodes on the apical surface of endometrial epithelia, along with increased expression of adhesive proteins (e.g., integrins, cadherins) and decreased expression of antiadhesive proteins (e.g., mucins) in the apical cell membrane.

1	When a fertilized egg implants in the uterus, the uterine endometrium is at its full thickness, is actively secreting, and is capable of tightly adhering to the implanting embryo. Fertilization accomplishes both recombination of genetic material to form a new genetically distinct organism and initiation of events that begin embryonic development. Several steps must occur to achieve successful (unassisted) fertilization (Fig. 44.31 3,4 ˜Ca++ Zona Egg plasma pellucida ZP2 membrane 3 • Fig. 44.31 Events involved in fertilization (see text for details). (Modified from White BA, Porterfield SP. Endocrine Physiology. 4th ed. Philadelphia: Mosby; 2013.) Step 1: Penetration of the expanded cumulus by the sperm. This involves digestion of the extracellular matrix of the cumulus by a membrane hyaluronidase, PH-20.

1	Step 1: Penetration of the expanded cumulus by the sperm. This involves digestion of the extracellular matrix of the cumulus by a membrane hyaluronidase, PH-20. Step 2: Penetration of the zona pellucida by the sperm. This involves binding of the sperm to the zona protein ZP3 (step 2a), which induces release of acrosomal enzymes (called the acrosomal reaction (step 2b). The sperm secondarily bind to another zona protein, ZP2 (step 2c), as the zona pellucida is digested and the sperm swims through to the egg (step 2d). Step 3: Fusion of the sperm and egg membrane takes place. Step 4: A Ca++ signaling cascade (see occurs.

1	Step 3: Fusion of the sperm and egg membrane takes place. Step 4: A Ca++ signaling cascade (see occurs. Step 5: The signaling cascade activates the exocytosis of enzyme-filled vesicles called cortical granules that reside in the outermost, or cortical, region of the unfertilized egg. The enzymes contained in the cortical granules are released to the outside of the egg upon exocytosis. These enzymes modify both ZP2 and ZP3 of the zona pellucida such that ZP2 can no longer bind acrosomereacted sperm, and ZP3 can no longer bind capacitated acrosome-intact sperm. Thus only one sperm usually enters the egg. Occasionally, more than one sperm does enter the egg. This results in a triploid cell that is unable to develop further. Therefore prevention of polyspermy is critical for normal development of the fertilized egg.

1	Step 6: The entire sperm enters the egg during fusion. The flagellum and mitochondria disintegrate, so most of the mitochondrial DNA in cells is maternally derived. Once inside the egg, decondensation of the sperm DNA occurs. A membrane called the pronucleus forms around the sperm DNA as the newly activated egg completes the second meiotic division. In mammalian eggs a large initial release of Ca++ is fol lowed by a series of subsequent smaller Ca++ oscillations that can last for hours. A major consequence of this signaling pathway is that it “wakes up” the metabolically quiescent •Fig. 44.32 Overviewofgeneticeventsafterfertilizationuptothefirstembryoniccleavage.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.) egg so it can resume meiosis and begin embryonic development. This process is called egg activation. The activated egg completes the second meiotic division as the sperm DNA decondenses and a pronucleus forms around it (

1	The activated egg completes the second meiotic division as the sperm DNA decondenses and a pronucleus forms around it ( Fig. 44.32). Once the egg has completed meiosis, CHAPTER 44 The Male and Female Reproductive Systems a pronucleus forms around the female chromosomes as well. A centrosome contributed by the sperm becomes a microtubule organizing center from which microtubules extend until they contact the female pronucleus. The male and female DNA replicate as the two pronuclei are pulled together. Once the pronuclei contact each other, the nuclear membranes break down, the chromosomes align on a common metaphase plate, and the first cleavage occurs. Fertilization typically occurs on day 16 to 17 of the men strual cycle, and implantation occurs about 6 days later. Thus the first week of embryogenesis takes place within the lumens of the oviduct and uterus. For most of this time the embryo remains encapsulated by the zona pellucida.

1	Thus the first week of embryogenesis takes place within the lumens of the oviduct and uterus. For most of this time the embryo remains encapsulated by the zona pellucida. The first two cleavages take about 2 days, and the embryo reaches a 16-cell morula by 3 days. The outer cells of the morula become tightly adhesive with each other and begin transporting fluid into the embryonic mass. During days 4 and 5 the transport of fluid generates a cavity called the blastocyst cavity, and the embryo is now called a blas Fig. 44.33 ). The blastocyst is composed of two subpopulations of cells: an eccentric inner cell mass and an outer epithelial-like layer of trophoblasts. The region of the trophoblast layer immediately adjacent to the inner cell mass is referred to as the embryonic pole, and it is this region that attaches to the uterine endometrium at Fig. 44.33

1	Fig. 44.33 The embryo resides within the oviduct during the first 3 days and then enters the uterus. By 5 to 6 days of develop ment the trophoblasts of the blastocyst secrete proteases that digest the outer-lying zona pellucida. At this point, corresponding to about day 22 of the menstrual cycle, the “hatched” blastocyst is able to adhere to and implant into the receptive uterine endometrium (see Fig. 44.33 At the time of attachment and implantation the tro phoblasts differentiate into two cell types: an inner layer of cytotrophoblasts and an outer layer of multinuclear/ Fig. 44.33).

1	Fig. 44.33 At the time of attachment and implantation the tro phoblasts differentiate into two cell types: an inner layer of cytotrophoblasts and an outer layer of multinuclear/ Fig. 44.33). The cytotrophoblasts initially provide a feeder layer of continu ously dividing cells. Syncytiotrophoblasts initially perform three general types of function: adhesive, invasive, and endocrine. Syncytiotrophoblasts express adhesive surface proteins (i.e., cadherins and integrins) that bind to uterine surface epithelia and, as the embryo implants, to compo nents of the uterine extracellular matrix. In humans the embryo completely burrows into the superficial layer of the endometrium (see

1	Fig. 44.33). This mode of implanta tion, called interstitial implantation, is the most invasive among placental mammals. Invasive implantation involves adhesion-supported migration of syncytiotrophoblasts into the endometrium, along with the breakdown of extracel lular matrix by secretion of matrix metalloproteinases and other hydrolytic enzymes. The endocrine function begins with the onset of implantation, when syncytiotrophoblasts start secreting the LH-like protein hCG, which maintains the viability of

1	Extraembryonic Primary Secondary Extraembryonic Remains of primary mesoderm yolk sac yolk sac forming coelom yolk sac •Fig. 44.33 A,Beginningofimplantation.Thetrophoblasthasdifferentiatedintocytotrophoblastand syncytiotrophoblast layers. B, As the syncytiotrophoblast layer increases in size and invades deeper, this layer begins to surround and erode maternal vessels, forming lacunae filled with maternal blood. C, Interstitial implantation is almost complete. Extensions of cytotrophoblasts have formed that will becomecoveredbyalayerofsyncytiotrophoblast.Atthispoint,theyarecalled“primaryvilli”.D,Interstitial implantationiscomplete.Extraembryonicmesodermalhasdevelopedfromtheepitheliallayers(amnion, primaryyolksac),andwillformaninnerlayerofthevilli,forming“secondaryvilli”.Ultimately,themesoderm willgiverise toumbilicalbloodvesselswithinthecoreofthe villus,therebyformingtertiaryvilli.(From CarlsonBM.Human Embryology and Developmental Biology.Philadelphia:Mosby;2004.) the corpus luteum and

1	Embryology and Developmental Biology.Philadelphia:Mosby;2004.) the corpus luteum and thus progesterone secretion. Syn-As implantation and placentation progress, syncytiotrocytiotrophoblasts also become highly steroidogenic. By 10 phoblasts take on the important functions of phagocytosis weeks the syncytiotrophoblasts acquire the ability to make (during histiotropic nutrition) and bidirectional placental progesterone at sufficient levels to maintain pregnancy transfer of gases, nutrients, and wastes. Exchange across independently of a corpus luteum. Syncytiotrophoblasts the syncytiotrophoblasts involves diffusion (e.g., gases), produce several other hormones as well as enzymes that facilitated transport (e.g., GLUT1-mediated transfer of modify hormones. glucose), active transport (e.g., amino acids by specific transporters), and pinocytosis/transcytosis (e.g., of irontransferrin complexes).

1	There is also a maternal response to implantation that involves transformation of the endometrial stroma. This response, called decidualization, involves an enlargement of stromal cells as they become lipid-and glycogen-filled decidual cells (at this time the endometrium is referred to as the decidua). The decidua forms an epithelial-like sheet with adhesive junctions that inhibit migration of the implanting embryo. The decidua also secretes factors such as tissue inhibitors of metalloproteinases (TIMPs) that moderate the activity of syncytiotrophoblast-derived hydrolytic enzymes in the endometrial matrix. Consequently, decidualization allows regulated invasion during implantation. Normally the implanting embryo and placenta do not extend to and involve the myometrium.

1	Placenta accreta istheburrowingoftheembryocompletelythroughtheendometriumandadhesionoftheplacentatothemyometrium,aconditionassociatedwithpotentiallylife-threateningpostpartum hemorrhage. Thedecidualresponseoccursonlyintheuterus.Thusthehighlyinvasivenatureofthehumanembryoposesconsiderablerisktothemotherinthecaseofectopic implantation. Ectopic implantation referstoimplantationofanembryoatasiteotherthantheuterus,andectopic pregnancy referstoadevelopingembryoatasiteofectopicimplantation.Mostectopicpregnancies(>90%)occurwithintheoviducts(calledtubal pregnancies),buttheycanalsooccurintheovaryandabdominalcavity.Implantationintheoviductsisoftenassociatedwithlong-terminfectionandinflammation(calledpelvic inflammatory

1	pregnancies),buttheycanalsooccurintheovaryandabdominalcavity.Implantationintheoviductsisoftenassociatedwithlong-terminfectionandinflammation(calledpelvic inflammatory disease)andobstructionofthetube.Inatubalpregnancythehighlyinvasivenatureofthehumansyncytiotrophoblast,whichisnormallymoderatedbytheuterinedecidualresponse,usuallyleadstoburrowingoftheimplantedembryothroughthewalloftheoviduct.Althoughabdominalpregnanciescanproceedtoterm,undetectedoviductalpregnanciesusuallyleadtoruptureoftheoviductalwall.Theresultinginternalhemorrhagecanbecatastrophictothemotherandrequiresimmediatesurgicalintervention.

1	The first hormone produced by syncytiotrophoblasts is hCG, which is structurally related to the pituitary glycoprotein hormones (see ). As such, hCG is composed of a common α-glycoprotein subunit (α-GSU) and a hormone-specific β subunit (β-hCG). Antibodies used to detect hCG (i.e., in laboratory assays and over-the-counter pregnancy tests) are designed to specifically detect the β subunit. hCG is most similar to LH and binds with high affinity to the LH receptor. The β subunit of hCG is longer than that of LH and contains more sites for glycosylation, which greatly increases the half-life of hCG to 24 to 30 hours. The stability of hCG allows it to rapidly accumulate in the maternal circulation such that hCG is detectable within maternal serum within 24 hours of implantation. Serum hCG levels double every 2 days for the first 6 weeks and peak at about 10 weeks. Serum hCG then declines to a constant level at about 50% of the peak value (Fig. 44.34A •Fig. 44.34

1	Serum hCG levels double every 2 days for the first 6 weeks and peak at about 10 weeks. Serum hCG then declines to a constant level at about 50% of the peak value (Fig. 44.34A •Fig. 44.34 CirculatinglevelsofhCGandhPLinmaternalbloodduringpregnancy.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)100 80 60Human chorionic gonadotropin(mIU/mL)40 20 10 0 10 20 30 40 Weeks of gestationA B 10 8Human placental lactogen(µg/mL)6 4 2 0 10 20 30 40 Weeks of gestation

1	The primary action of hCG is to stimulate LH receptors on the corpus luteum. This prevents luteolysis and maintains a high level of luteal-derived progesterone production during the first 10 weeks. The rapid increase in hCG is responsible for the nausea of “morning sickness” associated with early pregnancy. A small amount (i.e., 1%–10%) of hCG enters the fetal circulation. hCG stimulates fetal Leydig cells to produce testosterone before the fetal gonadotropic axis is fully mature. hCG also stimulates the fetal adrenal cortex (see later) during the first trimester. The placenta produces a high amount of progesterone, which is absolutely required to maintain a quiescent myometrium and a pregnant uterus. Progesterone production by the placenta is largely unregulated—the placenta produces as much progesterone as the supply of cholesterol and the levels of CYP11A1 and 3β-HSD allow (

1	Fig. 44.35 ). Notably, placental steroidogenesis differs from that in the adrenal cortex, ovaries, and testis in that cholesterol is transported into the placental mitochondria by a mechanism that is independent of StAR protein. Thus this first step in steroidogenesis is not a regulated rate-limiting step in the placenta as it is in other steroidogenic glands. This means that fetuses with an inactivating mutation in StAR protein will develop lipoid congenital adrenal hyperplasia (see ) and hypogonadism but will have normal progesterone levels produced by their placenta. Progesterone production by the placenta does not require fetal tissue. Consequently, progesterone levels are largely independent of fetal health and cannot be used as a measure of fetal health. Maternal progesterone levels continue to increase throughout pregnancy.

1	•Fig. 44.35 Productionofprogesteronebysyncytiotrophoblastandestrogensbyfetoplacentalunit.Intervillous space (Maternal) Sycytiotrophoblast Cholesterol pregnenolone Fetal zone adrenal cortex Pregnenolone Pregnenolone-s 17OH-Pregnenolone-s Sulfo-Transferase CYP11A1 Star-related protein CYP11A1 Star protein CYP17 (Hydroxylase) CYP17 (17/20 lyase) ACTH Cholesterol Fetal pituitary CYP19 Aromatase E3 E2 E1 CYP19 Aromatase Sulfatase 16OH DHEA 16OH A'DIONE 16OH T Progesterone 16OH DHEAS DHEAS DHEAS DHEAS DHEAS16OH DHEAS Fetal liver DHEA A'DIONE T 3˜HSD 3˜HSD 17˜HSD Actions in maternal compartment Actions in maternal compartment Peripheral conversion to E2 MC2R

1	Progesterone is released primarily into the maternal circulation and is required for implantation and maintenance of pregnancy. Progesterone also has several effects on maternal physiology and induces breast growth and differentiation. The switch from corpus luteum–derived progesterone to placental-derived progesterone (referred to as the luteal-placental shift) is complete at about the eighth week of pregnancy. Progesterone (and pregnenolone) are used by the transitional zone of the fetal cortex to make cortisol late in pregnancy. Estrogens are also produced by syncytiotrophoblasts. Syncytiotrophoblasts are similar to ovarian granulosa cells in that they lack CYP17 and are dependent on another cell type to provide 19-carbon androgens for aromatization (see Fig. 44.35 ). The ancillary androgen-producing cells reside in the fetal adrenal cortex.

1	Fig. 44.35 ). The ancillary androgen-producing cells reside in the fetal adrenal cortex. The fetal adrenal cortex contains an outer definitive zone, a middle transitional zone, and an inner fetal zone. The definitive and transitional zones give rise to the zona glomerulosa and zona fasciculata, respectively. Aldosterone synthesis is initiated close to parturition. Synthesis of cortisol begins at about 6 months and increases during late gestation. The fetal zone is the predominant portion of the adrenal cortex in the fetus; it constitutes as much as 80% of the bulk of the large fetal adrenal and is the site of most fetal adrenal steroidogenesis. The fetal zone strongly resembles the zona reticularis in that it expresses little or no 3β-HSD (see

1	Fig. 44.35 ). The fetal zone primarily releases the sulfated form of the inactive androgen dehydroepiandrosterone sulfate (DHEAS) throughout most of gestation. Production of DHEAS from the fetal adrenal is absolutely dependent on fetal ACTH from the fetal pituitary by the end of the first trimester. The DHEAS released from the fetal zone has two fates. First, DHEAS can go directly to the syncytiotrophoblast, where it is desulfated by a placental steroid sulfatase and used as a 19-carbon substrate for the synthesis of estradiol-17β and estrone (see Fig. 44.35 ). The second fate of DHEAS is 16-hydroxylation in the fetal liver by the enzyme CYP3A7. 16-Hydroxyl-DHEAS is then converted by syncytiotrophoblasts to the major estrogen of pregnancy, estriol (see

1	Fig. 44.35 ). In X-linked ichthyosis, the steroid sulfatase is low or missing, resulting in loss of active (i.e., desulfated) estrogen production by the fetoplacental unit. Pregnancy is normal, but because estrogens promote parturition, the pregnancy is prolonged and usually ends with physician-induced labor. The baby boy has a skin disorder of varying degrees of severity that is called ichthyosis (scaly skin), owing to buildup of layers of shed cells within the stratum corneum. This form of ichthyosis is readily treatable by topical creams.

1	Maternal estrogen levels increase throughout pregnancy. Because estrogen production is dependent on a healthy fetus, estriol levels can be used as one measure of fetal health. The collective term used for the placental syncytiotrophoblasts and fetal organs in the context of estrogen production is the fetoplacental unit. Estrogens increase uteroplacental blood flow, enhance LDL receptor expression in syncytiotrophoblasts, and induce several components (e.g., prostaglandins, oxytocin receptors) involved in parturition. Estrogens increase breast growth directly and indirectly through stimulation of maternal pituitary prolactin production. Estrogens also increase lactotrope size and number, thereby increasing overall pituitary mass by more than twofold by term. Estrogens also affect several other aspects of maternal physiology.

1	Human placental lactogen (hPL), also called human chorionic somatomammotropin (hCS), is a 191–amino acid protein hormone produced in the syncytiotrophoblast that is structurally similar to growth hormone (GH) and prolactin (PRL). Its function overlaps those of both GH and PRL. It can be detected within the syncytiotrophoblast by 10 days after conception and in maternal serum by 3 weeks’ gestation (see Fig. 44.34 ). Maternal serum levels rise progressively throughout the remainder of the pregnancy. The quantity of hormone produced is directly related to the size of the placenta, such that as the placenta grows during gestation, hPL secretion increases. As much as 1 g/day of hPL can be secreted late in gestation.

1	Like GH, hPL is protein anabolic and lipolytic. Its antagonistic action to insulin is the major basis for the diabetogenicity of pregnancy. Like PRL, it stimulates mammary gland growth and development. Mammary gland development in pregnancy results from the actions of hPL, PRL, estrogens, and progestins. hPL inhibits maternal glucose uptake and use, thereby increasing serum glucose levels. Glucose is a major energy substrate for the fetus, and hPL increases fetal glucose availability. CHAPTER 44 The Male and Female Reproductive Systems As with hCG, far less hPL is found in the fetal circulation than in the maternal circulation. This suggests that the hormones may play a more important role in the mother than in the fetus. hPL is not essential for the pregnancy. Both hPL and PRL act as fetal growth hormones and stimulate production of the fetal growth-promoting hormones insulinlike growth factor (IGF)-I and IGF-II.

1	Both hPL and PRL act as fetal growth hormones and stimulate production of the fetal growth-promoting hormones insulinlike growth factor (IGF)-I and IGF-II. Ironically, fetal GH does not appear to regulate growth, and have normal birth weight. Diabetogenicity of Pregnancy Fig.

1	44.36 ). During the last half of pregnancy when hPL levels are highest, maternal energy metabolism shifts from an anabolic state in which nutrients are stored to a catabolic state sometimes described as accelerated starvation, in which maternal energy metabolism shifts toward fat utilization with sparing of glucose. As maternal use of glucose for energy decreases, lipolysis increases and fatty acids become major energy sources. Peripheral responsiveness to insulin decreases and pancreatic insulin secretion increases. Beta cell hyperplasia occurs in pregnancy. Although this does not usually lead to a clinical condition, pregnancy aggravates existing diabetes mellitus, and diabetes can develop for the first time in pregnancy. If the diabetes resolves spontaneously with delivery, the condition is referred to as gestational diabetes. Other hormones contributing to the diabetogenicity of pregnancy are estrogens and progestins, because both these hormones decrease insulin sensitivity.

1	Human pregnancy lasts an average of 40 weeks from the beginning of the last menstrual period (gestational age). This corresponds to an average fetal age of 38 weeks. Par turition is the process whereby uterine contractions lead to childbirth. Labor consists of three stages: strong uterine contractions that force the fetus against the cervix, with dilation and thinning of the cervix (several hours); delivery of the fetus (<1 hour); and delivery of the placenta, along with contractions of the myometrium to halt bleeding (<10 minutes). Control of parturition in humans is complex, and the exact mechanisms underlying its control are not well understood. Placental CRH and the Fetal Adrenal Axis

1	The placenta produces corticotropin-releasing hormone (CRH), which is identical to the 41–amino acid peptide produced by the hypothalamus. Placental CRH production and maternal serum CRH levels increase rapidly during late pregnancy and labor. Moreover, circulating CRH is either in the form of free CRH, which is bioactive, or complexed to a CRH-binding protein. Maternal levels of CRH-binding protein plummet during late pregnancy and labor, so free CRH levels increase. Placental CRH also accumulates in the fetal circulation and stimulates fetal ACTH secretion. ACTH stimulates both fetal adrenal cortisol production and fetoplacental estrogen production. In contrast to the inhibitory effect of cortisol on hypothalamic CRH production, cortisol stimulates placental CRH production. This establishes a self-amplifying positive feedback. CRH itself promotes myometrial contractions by sensitizing the uterus to oxytocin and prostaglandins (see ). Estrogens also directly and indirectly stimulate

1	a self-amplifying positive feedback. CRH itself promotes myometrial contractions by sensitizing the uterus to oxytocin and prostaglandins (see ). Estrogens also directly and indirectly stimulate myometrial contractility. Moreover, this model correlates the onset of parturition with cortisol-induced maturation of fetal systems, including the lungs and gastrointestinal tract.

1	824 SECTION8Berne & Levy Physiology •Fig. 44.36 Overviewofenergyusebythematernalandfetalcompartments.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)Decreased maternal glucose use Antiinsulin action hPL Placenta Glucose Amino acids Fetus Lipolysis Proteolysis Amino acidsFatty acids for maternal use Although a rise in maternal serum estrogen and a drop in progesterone levels occur late in gestation in some species, no change in the ratio of the two hormones is seen in human serum. However, “functional” progesterone withdrawal involving changes in uterine progesterone receptor and progesterone metabolism has been proposed.

1	Oxytocin is secreted from the pars nervosa (posterior pituitary) (see ). Oxytocin, which stimulates powerful uterine contractions, plays a major role in progression and completion of parturition. Oxytocin is released in response to stretch of the cervix through a neuroendocrine reflex; it stimulates uterine contractions and thereby facilitates delivery. Oxytocin can be used to induce parturition, and uterine sensitivity to oxytocin increases before parturition. Because maternal serum oxytocin levels do not increase until after parturition has begun, oxytocin is not thought to initiate parturition. However, progesterone inhibits and estrogen stimulates synthesis of oxytocin receptors, and although maternal serum progesterone levels do not decrease immediately before human parturition, estrogen levels rise and oxytocin receptor synthesis increases.

1	Prostaglandins and other cytokines increase uterine motility, and levels of these compounds increase during parturition, thereby facilitating delivery. Their exact role in initiating parturition is not known. Prostaglandin levels in amniotic fluid, fetal membranes, and uterine decidua increase before the onset of labor. Prostaglandin F2α and prostaglandin E2 increase uterine motility. Large doses of these compounds have been used to induce labor. Because estrogens stimulate prostaglandin synthesis in the uterus, amnion, and chorion, the rising estrogen levels late in gestation can increase uterine prostaglandin formation before parturition.

1	Uterine size is thought to be a factor regulating parturition because stretch of smooth muscle, including the uterus, increases muscle contraction. In addition, uterine stretch stimulates uterine prostaglandin production. Multiple births generally occur prematurely. The tendency for early delivery can be a result of increased uterine size, increased fetal production of chemicals stimulating delivery, or both. CHAPTER 44 The Male and Female Reproductive Systems 825 • Fig. 44.37 Diagram of the structure of the breast, along with some pathological conditions of the breast and where they occur. (From Crum CP et al. In: Kumar V et al [eds]. Robbins Basic Pathology. 7th ed. Philadelphia: Saunders; 2003.) Structure of the Mammary Gland The mammary gland is composed of 15 to 20 lobes, each with an excretory lactiferous duct that opens at the nipple (

1	Fig. 44.37 ). The lobes in turn are composed of several lobules that contain secretory structures called alveoli and the terminal portions of the ducts. The epithelium of the alveoli and ducts is composed of two cell layers: apical luminal epithelial cells and basal myoepithelial cells. There is strong evidence for the presence of adult mammary stem cells within this epithelium. The luminal epithelial cells of the alveoli are the producers of milk, and the luminal cells of the ducts convey and modify the secreted milk. Myoepithelial cells are stellate smooth muscle–like cells, and contraction of these cells in response to a stimulus (i.e., milk let-down) expels milk from the lumens of the alveoli and ducts. Lobes and lobules are supported within a connective tissue matrix. The other major tissue component of the breast is adipose tissue. The lactiferous ducts empty at the nipple, a highly innervated hairless protrusion of the breast designed for suckling by an infant. The nipple is

1	tissue component of the breast is adipose tissue. The lactiferous ducts empty at the nipple, a highly innervated hairless protrusion of the breast designed for suckling by an infant. The nipple is surrounded by a pigmented hairless areola that is lubricated by sebaceous glands. Protrusion of the nipple, called erection, is mediated by sympathetic stimulation of smooth muscle fibers in response to suckling and other mechanical stimulation, erotic stimulation, and cold.

1	Hormonal Regulation of Mammary Gland Development At puberty, estrogen increases ductal growth and branching. With onset of the luteal phases of the ovary, progesterone and estrogen induce ductal growth and formation of rudimentary alveoli. During nonpregnant cycles the breasts develop somewhat and then regress. Estrogen also increases deposition of adipose tissue, which makes a major contribution to breast size and overall form. Adipose tissue expresses CYP19/aromatase, so accumulation of this tissue in the breast increases local production of estrogens from circulating androgens.

1	Breast development is facilitated by pregnancy, during which extensive ductal growth and branching and lobuloalveolar development occur. The parenchymal growth of the breast during development occurs at the expense of stroma, which is degraded to make room for enlarging lobuloalveolar structures. Several placental hormones stimulate breast development, including estrogen, progesterone, placental lactogen, and a growth hormone variant (GH-V). Estrogen acts on the breast both directly and indirectly through increasing maternal pituitary PRL. Estrogen increases PRL secretion from pituitary lactotropes. Estrogen also stimulates lactotrope hypertrophy and proliferation, which accounts for the twofold increase in pituitary volume during pregnancy in humans. Although epithelial cells express genes encoding milk protein and enzymes involved in milk production, progesterone inhibits the onset of milk production and secretion 826 SECTION8Berne & Levy Physiology •Fig. 44.38

1	cells express genes encoding milk protein and enzymes involved in milk production, progesterone inhibits the onset of milk production and secretion 826 SECTION8Berne & Levy Physiology •Fig. 44.38 Neuroendocrinereflexcausedbysucklingatthenippleandleadingtosecretionofoxytocinandprolactin.Inturnthesehormonesinducecontinuedmilkproduction(galactopoiesis)andmilklet-down.Prolactinalsoinduceslactationalamenorrhea.(ModifiedfromWhiteBA,PorterfieldSP.Endocrine Physiology. 4thed.Philadelphia:Mosby;2013.)Magnocellular neurons Parvicellular neurons Lactotropes Gonadotropes Lactational amenorrhea Parvicellular neurons Hypothalamus Breast Ovary Pars distalis Pars nervosa ˜ GnRH˜ Dopamine ˜ LH and FSH ° PRF?? ° Prolactin GalactopoiesisConcentration of myoepithelial cells Milk let-down Sucking at nipple ° Oxytocin (lactogenesis).

1	After parturition, the human breast produces colostrum, which is enriched with antimicrobial and antiinflammatory proteins. In the absence of placental progesterone, normal breast milk production occurs within a few days. The lobuloalveolar structures produce milk, which is subsequently modified by the ductal epithelium. Lactogenesis and maintenance of milk production (galactopoiesis) require stimulation by pituitary PRL in the presence of normal levels of other hormones, including insulin, cortisol, and thyroid hormone. Although placental estrogen stimulates PRL secretion during pregnancy, the stimulus for PRL secretion during the nursing period is suckling by the infant (

1	Fig. 44.38 ). Levels of PRL are directly correlated with frequency and duration of suckling at the nipple. The link between suckling at the nipple and PRL secretion involves a neuroendocrine reflex in which dopamine secretion at the median eminence is inhibited (the PRL release inhibitory factor; see ). It is also possible suckling increases secretion of unidentified PRL-releasing hormones. PRL also inhibits release of GnRH, and consequently, nursing can be associated with lactational amenorrhea (see

1	PRL also inhibits release of GnRH, and consequently, nursing can be associated with lactational amenorrhea (see Fig. 44.38 ). This effect of prolactin has been called “nature’s contraceptive,” and it may play a role in spacing out pregnancies. However, only regular nursing over a 24-hour period is sufficient to induce a PRL-induced anovulatory state in the mother. Thus lactational amenorrhea is not an effective or reliable form of birth control for most women. Inhibition of GnRH by high levels of PRL is important clinically. A prolactinoma is the most common form of hormone-secreting pituitary tumor, and hyperprolactinemia is a significant cause of infertility in both sexes. Hyperprolactinemia can likewise be associated with galactorrhea (inappropriate flow of breast milk) in men and women. Suckling at the nipple also stimulates release of oxytocin from the pars nervosa (see Fig. 44.38). Contraction of myoepithelial cells induces milk let-down, or expulsion of milk from the

1	Suckling at the nipple also stimulates release of oxytocin from the pars nervosa (see Fig. 44.38). Contraction of myoepithelial cells induces milk let-down, or expulsion of milk from the Invasive breast cancer (IBC) isamajorcancerinwomenandcanbeclassifiedintoseveralcategories.MostnewlydiagnosedIBCisclassifiedasluminal A, whichisusuallyderivedfromluminalcellsofterminalductsoralveoli.LuminalAIBCdisplayssomeepithelialorganization,includingEcadherin–mediatedcell-cellcontacts,andispoorlymotileandpoorlyaggressive.Thisformalsoexpressestheestrogen receptor α (ERα) andisdependentonestrogenicstimulationforgrowth.EarlydiagnosisofluminalAIBCconfersagoodprognosis.Treatmentforearlysmalltumorsthatare“node-negative”(i.e.,havenotspreadtonearbylymphnodes)typicallyinvolvessurgicalremoval(“lumpectomy”),followedbyradiationtreatment,followedby5yearsofdailytamoxifentreatment.TamoxifenisaSERMthatopposesestrogeninthebreast.

1	Therearemultiplebehavioralmethodsofcontraception. Totalabstinenceisthebestwaytoavoidgettingpregnant.Othermethodsincludetherhythmmethod,whichreliesonabstinencefromsexualintercourseduringfertileperiodsaroundthetimeofovulation.Thefertileperiodextendsfrom3to4daysbeforethetimeofovulationuntil3to4daysafterward.Asecondmethodiswithdrawalbeforeejaculation,coitus interruptus. Boththesemethodshavehigherfailurerates(20%–30%)thanbarrier methods (2%–12%),intrauterine devices (IUDs) (<2%),andoral contraceptives (<1%)do.Barrierssuchascondoms ordiaphragms aremoreeffectivewhenusedwithspermicidal jellies. Ofallmethods,onlycondomsprovideeffectiveprotectionfromsexuallytransmitteddiseasesinsexuallyactiveindividuals.IUDsarerelativelyeffective.Theypreventimplantationbylocallyproducinganinflammatoryresponseintheendometrium.SomeformsofIUDscontaincopper,zinc,orprogestins,whichinhibitspermtransportorviabilityinthefemalereproductivetract.

1	OralcontraceptiveshavebeenmarketedintheUnitedStatessincetheearly1960s.Thedosesofsteroidsusedtodayaresignificantlylowerthanthoseused35yearsago.Properlyused,oralcontraceptiveshavealowfailurerate.Manyformsoforalcontraceptivesaremarketedtoday.Thetrendovertheyearshasbeentodecreasethedosageofsteroidsusedbecausethesideeffectsaredosedependent.Alloralsteroidalcontraceptivescontaineitheracombinationofanestrogenandaprogestinoraprogestinalone.Oralcontraceptivesworkthroughmultiplemechanisms.MostblocktheLHsurgethattriggersovulation.However,somepills(e.g.,progestin-onlyminipill)donotpreventLHsurges.Fertilityisalsoblockedbychangingthenatureofcervicalmucus,alteringendometrialdevelopment,orregulatingfallopiantubemotility.BecausethesecontraceptivessuppressFSH,theyimpairearlyfolliculardevelopment.

1	Emergency contraception involveshormonaltreatmentdesignedtoinhibitordelayovulation,inhibitcorpusluteumfunction,disruptthefunctionoftheoviductsanduterus,oranycombinationofthesemechanisms.Forexample,candidatesforemergencycontraceptionincludewomenwhoaresexuallyassaultedorwhoexperiencedfailureofabarriermethod(e.g.,rupturedcondom).Therearemorethan20typesofcommerciallyavailable“morning-after”pills.Thecurrentlypreferredmedicationislevonorgestrel (Plan B), whichisasyntheticprogestin-onlypill.Theefficacyofthepillisinverselycorrelatedwiththetimeitistakenafterintercourse.Theexactmechanismofactionisnotknown.Treatmenthasnoeffectifimplantationhasoccurred. Medical(hormonal) termination ofpregnancy(abortion)canbeachievedupto49days’gestationbyadministrationofmifepristone (RU-486), aprogesteronereceptorantagonistthatinducescollapseofthepregnantendometrium.Mifepristoneisfollowed48hourslaterbyingestionorvaginalinsertionofasynthetic prostaglandin E (e.g.,misoprostol),whichinducesmyometrialcontractions.

1	alveolar and ductal lumens. Thus the nursing infant does not gain milk by applying negative pressure to the breast from suckling. Rather, milk is actively ejected through a neuroendocrine reflex. Oxytocin release and milk let-down can be induced by psychogenic stimuli such as the mother hearing a baby crying on television or thinking about her baby. Such psychogenic stimuli do not affect PRL release. Though related to depletion of ovarian follicles, the causes and process of menopause are poorly understood. Age-related changes in the CNS, including critical patterns of GnRH secretion, precede follicular depletion and may play an important role in menopause. Because follicles do not develop in response to LH and FSH secretion, estrogen and progesterone levels drop. Loss of the negative-feedback inhibition of estrogen on GnRH and LH/FSH results in a marked rise in serum LH and FSH. FSH levels rise more than LH levels. This could result from loss of ovarian inhibin.

1	Menopause typically occurs between 45 and 55 years of age. It extends over a period of several years. Initially the cycles become irregular and are periodically anovulatory. The cycles tend to shorten, primarily in the follicular phase. Eventually the woman ceases to cycle altogether. Serum estradiol levels drop to about a sixth the mean levels for younger cycling women, and progesterone levels drop to about a third those in the follicular phase of younger women. Production of these hormones does not cease entirely, but the primary source of these hormones in postmenopausal women becomes the adrenal, although interstitial cells of the ovarian stroma continue to produce some steroids. Most circulating estrogens are now produced peripherally from androgens. Because estrone is the primary estrogen produced in adipose tissue, it becomes the predominant estrogen in postmenopausal women.

1	Most symptoms associated with menopause result from estrogen deficiency. The vaginal epithelium atrophies and becomes dry, and bone loss is accelerated and may lead to osteoporosis. The incidence of coronary artery disease increases markedly after menopause. Hot flashes result from periodic increases in core temperature, which produces peripheral vasodilation and sweating. Hot flashes are thought to be linked to increases in LH release and are probably associated not with the pulsatile rise in LH secretion but rather with central mechanisms controlling GnRH release. Hot flashes typically subside within 1 to 5 years of the onset of menopausal symptoms. 1. The reproductive systems are composed of gonads, an internal reproductive tract with associated glands, and external genitalia. Mammary glands are accessory reproductive glands in women. 2.

1	1. The reproductive systems are composed of gonads, an internal reproductive tract with associated glands, and external genitalia. Mammary glands are accessory reproductive glands in women. 2. Gonads have two main functions: production of gametes and production of hormones. Hormones (primarily sex steroids) are absolutely necessary for normal function of the reproductive system, and their production is regulated by a hypothalamic-pituitarygonadal axis. 3. Seminiferous tubules in the testis contain Sertoli cells and developing sperm cells. 4. Spermatogenesis refers to the progression of sperm cells from spermatogonia through the processes of meiosis and spermiogenesis to form mature spermatozoa. 5.

1	4. Spermatogenesis refers to the progression of sperm cells from spermatogonia through the processes of meiosis and spermiogenesis to form mature spermatozoa. 5. Testosterone and pituitary FSH are required for normal sperm production. Only Sertoli cells express the androgen receptor and the FSH receptor, so these hormones regulate spermatogenesis indirectly through their actions on Sertoli cells. Sertoli cells produce the hormone inhibin, which negatively feeds back on pituitary FSH production. 6. Sertoli cells have many functions, including production of androgen-binding protein (ABP) and fluid and creation of the blood-testis barrier. 7. Leydig cells are stromal cells that reside outside the seminiferous tubules. They respond to LH by producing testosterone. 8. Testosterone is an active androgen. It can be converted peripherally to DHT, which is more active in certain tissues (e.g., prostate), or to estradiol. 9.

1	8. Testosterone is an active androgen. It can be converted peripherally to DHT, which is more active in certain tissues (e.g., prostate), or to estradiol. 9. Leydig cells are regulated within a hypothalamicpituitary-testicular axis. The hypothalamus produces GnRH, which stimulates pituitary gonadotropes to secrete LH and FSH. Testosterone, DHT, and estradiol negatively feed back at the pituitary and hypothalamus and inhibit LH more than FSH secretion. Inhibin from the Sertoli cells selectively inhibits FSH. 10. Testosterone, DHT, and estradiol have numerous actions on the male reproductive tract, external genitalia, and male secondary sex characteristics, as well as on other organ systems (e.g., blood cell production, lipoprotein production, bone maturation). 11.

1	11. The male tract includes tubal structures (epididymis, ductus deferens, and male urethra), accessory sex glands (seminal vesicles, prostate), and the penis. The seminal vesicles and the prostate produce most of the ejaculate, which nourishes, buffers, and protects sperm. 12. Penile erection involves a complex neurovascular response leading to engorgement of the erectile tissue within the penis base and shaft with blood. 13. The follicle is the functional unit of the ovary. Follicles contain epithelial cells (granulosa and cumulus) and outer stromal cells (thecal). All these cells surround a primary oocyte that remains arrested in the first meiotic prophase until just before ovulation. 14. Follicles develop from the smallest (primordial) to a large antral follicle over a period of months. The latter part of follicular development requires gonadotropins. 15.

1	14. Follicles develop from the smallest (primordial) to a large antral follicle over a period of months. The latter part of follicular development requires gonadotropins. 15. The menstrual cycle refers to an approximately 28-day cycle that is driven by the following ovarian events: development of one large antral follicle to a preovulatory follicle (follicular phase), ovulation, and formation and death of a corpus luteum of menstruation (luteal phase). 16. The follicular phase of the ovary corresponds to the menstrual and proliferative phases of the uterine endometrium. The luteal phase of the ovary corresponds to the secretory phase of the uterine endometrium. 17. One dominant follicle is selected per menstrual cycle—usually the largest follicle with the most FSH receptors. 18.

1	17. One dominant follicle is selected per menstrual cycle—usually the largest follicle with the most FSH receptors. 18. High levels of estradiol occur around midcycle and exert positive feedback on gonadotropin secretion. This induces the LH (and a smaller FSH) surge. The midcycle gonadotropin surge induces (a) meiotic maturation of the primary oocyte so that it progresses to a secondary oocyte (with one polar body) arrested at metaphase of the second meiotic division, (b) breakdown of the ovarian and follicular wall so that the oocyte-cumulus complex is extruded (called ovulation), and (c) differentiation of the remaining follicular cells into a corpus luteum. The corpus luteum produces high levels of progesterone, estradiol, and inhibin. 19. If pregnancy does not occur, the corpus luteum will die in 14 days. This constitutes the luteal phase of the menstrual cycle. 20.

1	19. If pregnancy does not occur, the corpus luteum will die in 14 days. This constitutes the luteal phase of the menstrual cycle. 20. The oviducts capture the ovulated cumulus-oocyte complex and transport it medially into the oviduct and toward the uterus. Estrogen promotes ciliation and transport; progesterone inhibits transport. 21. The uterine mucosa, called the endometrium, is the normal site of embryonic implantation. The mucosa is increased in thickness in preparation for implantation and is sloughed away if no pregnancy occurs. 22. During the mid to late follicular phase (days 6–14 of the menstrual cycle), the ovary produces estradiol, which induces all cells of the endometrium to proliferate (called the proliferative phase of the uterus). 23. After ovulation the ovary enters the luteal phase (days 16–28) and produces progesterone. Progesterone stimulates secretion from the uterine glands (called the secretory phase of the uterus). 24.

1	After ovulation the ovary enters the luteal phase (days 16–28) and produces progesterone. Progesterone stimulates secretion from the uterine glands (called the secretory phase of the uterus). 24. In the absence of an implanting embryo the corpus luteum dies, progesterone production ceases, and the uterine endometrium is sloughed (called the menstrual phase, or period, of the uterus—this corresponds to days 1 to 5 of the follicular phase of the ovary). 25. The cervix is the lower portion of the uterus. Cervical mucus is hormonally regulated so that at midcycle in response to estrogen, cervical mucus promotes entry of sperm into the uterus from the vagina. During the luteal phase in response to progesterone, cervical mucus becomes thick and poses a barrier to entry of sperm and microbes into the uterus. We wish to thank Dr. Lisa Mehlmann for her advice on this chapter, and especially for help in drawing Figures 44.31 44.32

1	We wish to thank Dr. Lisa Mehlmann for her advice on this chapter, and especially for help in drawing Figures 44.31 44.32 Achar S, et al. Cardiac and metabolic effects of anabolic-androgenic steroid abuse on lipids, blood pressure, left ventricular dimensions, and rhythm. Am J Cardiol. 2010;106:893-901. Coticchio G, et al. Oocyte maturation: gamete-somatic cells interactions, meiotic resumption, cytoskeletal dynamics and cytoplasmic reorganization. Hum Reprod Update. 2015;21:427-454. Defeudis G, et al. Erectile dysfunction and its management in patients with diabetes mellitus. Rev Endocr Metab Disord. 2015; 16:213-231. Franca LR, et al. The Sertoli cell: one hundred fifty years of beauty and plasticity. Andrology. 2016;4:189-212. Kosaka T, et al. Is DHT production by 5alpha-reductase friend or foe in prostate cancer? Front Oncol. 2014;4:article 247. 26. Fertilization is a complex series of events that occur in the oviduct and lead to penetration of the oocyte by sperm. 27.

1	26. Fertilization is a complex series of events that occur in the oviduct and lead to penetration of the oocyte by sperm. 27. Early embryogenesis (up to day 6 after fertilization) occurs in the oviduct and gives rise to a blastocyst that hatches from the zona pellucida. 28. The placenta develops from the outer extraembryonic trophoblast. The endocrine function of the placenta includes production of hCG, progesterone, estrogens, and placental lactogen. Estrogen production requires placental cells (syncytiotrophoblasts) as well as the fetal adrenal and liver—collectively called the fetoplacental unit. 29.

1	29. Pregnancy and the hormones of pregnancy induce major changes in maternal physiology, including an increase in insulin resistance, an increase in the use of free fatty acids by the mother, and development of the mammary glands. Mammary gland development (but not lactation) is promoted by estrogen, progesterone, and placental lactogen but also by maternal pituitary prolactin, whose secretion is stimulated by placental estrogens. 30. Oxytocin is a pituitary hormone that promotes contraction of certain smooth muscles, including myometrial contractions during labor and myoepithelial contractions in the breasts that lead to let-down of milk in response to suckling. 31. Menopause results from exhaustion of the ovarian reserve and is characterized by low ovarian hormone and elevated gonadotropin levels. Larney C, et al. Switching on sex: transcriptional regulation of the testis-determining gene Sry. Development. 2014;141:21952205.

1	Larney C, et al. Switching on sex: transcriptional regulation of the testis-determining gene Sry. Development. 2014;141:21952205. Mehlmann LM. Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation. Reproduction. 2005;130:791-799. Pasqualini JR. Enzymes involved in the formation and transformation of steroid hormones in the fetal and placental compartments. J Steroid Biochem Mol Biol. 2005;97:401-415. Sreekumar A, et al. The mammary stem cell hierarchy: a looking glass into heterogeneous breast cancer landscapes. Endocr Relat Cancer. 2015;22:T161-T176. Umetani M, Shaul PW. 27-Hydroxycholesterol: the first identified endogenous SERM. Trends Endocrinol Metab. 2011;22: 130-135.

1	For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Proteins are the most abundant and functionally diverse molecules in living systems. Virtually every life process depends on this class of macromolecules. For example, enzymes and polypeptide hormones direct and regulate metabolism in the body, whereas contractile proteins in muscle permit movement. In bone, the protein collagen forms a framework for the deposition of calcium phosphate crystals, acting like the steel cables in reinforced concrete. In the bloodstream, proteins, such as hemoglobin and albumin, transport molecules essential to life, whereas immunoglobulins fight infectious bacteria and viruses. In short, proteins display an incredible diversity of functions, yet all share the common structural feature of being linear polymers of amino acids. This chapter describes the properties of amino acids. Chapter 2 explores how these simple building blocks are joined to form proteins that have unique three-dimensional structures, making them capable of performing specific biologic

1	of amino acids. Chapter 2 explores how these simple building blocks are joined to form proteins that have unique three-dimensional structures, making them capable of performing specific biologic functions.

1	II. STRUCTURE

1	Although >300 different amino acids have been described in nature, only 20 are commonly found as constituents of mammalian proteins. [Note: These standard amino acids are the only amino acids that are encoded by DNA, the genetic material in the cell (see p. 411). Nonstandard amino acids are produced by chemical modification of standard amino acids (see p. 45).] Each amino acid has a carboxyl group, a primary amino group (except for proline, which has a secondary amino group), and a distinctive side chain (R group) bonded to the α carbon atom. At physiologic pH (~7.4), the carboxyl group is dissociated, forming the negatively charged carboxylate ion (−COO−), and the amino group is protonated (−NH3+) (Fig. 1.1A). In proteins, almost all of these carboxyl and amino groups are combined through peptide linkage and, in general, are not available for chemical reaction except for hydrogen bond formation (Fig. 1.1B). Thus, it is the nature of the side chains that ultimately dictates the role

1	peptide linkage and, in general, are not available for chemical reaction except for hydrogen bond formation (Fig. 1.1B). Thus, it is the nature of the side chains that ultimately dictates the role an amino acid plays in a protein. Therefore, it is useful to classify the amino acids according to the properties of their side chains, that is, whether they are nonpolar (have an even distribution of electrons) or polar (have an uneven distribution of electrons, such as acids and bases) as shown in Figures 1.2 and 1.3.

1	and polarity of their side chains at acidic pH (continued from Fig. 1.2). [Note: At physiologic pH (7.35 to 7.45), the α-carboxyl groups, the acidic side chains, and the side chain of free histidine are deprotonated.] A. Amino acids with nonpolar side chains Each of these amino acids has a nonpolar side chain that does not gain or lose protons or participate in hydrogen or ionic bonds (see Fig. 1.2). The side chains of these amino acids can be thought of as “oily” or lipid-like, a property that promotes hydrophobic interactions (see Fig. 2.10, p. 19).

1	1. Location in proteins: In proteins found in aqueous solutions (a polar environment), the side chains of the nonpolar amino acids tend to cluster together in the interior of the protein (Fig. 1.4). This phenomenon, known as the hydrophobic effect, is the result of the hydrophobicity of the nonpolar R groups, which act much like droplets of oil that coalesce in an aqueous environment. By filling up the interior of the folded protein, these nonpolar R groups help give the protein its three-dimensional shape. However, for proteins that are located in a hydrophobic environment, such as a membrane, the nonpolar R groups are found on the outside surface of the protein, interacting with the lipid environment (see Fig. 1.4). The importance of these hydrophobic interactions in stabilizing protein structure is discussed on p. 19.

1	Sickle cell anemia, a disease of red blood cells that causes them to become sickle shaped rather than disc shaped, results from the replacement of polar glutamate with nonpolar valine at the sixth position in the β subunit of hemoglobin A (see p. 36). 2. Proline: Proline differs from other amino acids in that its side chain and α-amino nitrogen form a rigid, five-membered ring structure (Fig. 1.5). Proline, then, has a secondary (rather than a primary) amino group. It is frequently referred to as an “imino acid.” The unique geometry of proline contributes to the formation of the fibrous structure of collagen (see p. 45), but it interrupts the α-helices found in globular proteins (see p. 16). B. Amino acids with uncharged polar side chains

1	B. Amino acids with uncharged polar side chains These amino acids have zero net charge at physiologic pH, although the side chains of cysteine and tyrosine can lose a proton at an alkaline pH (see Fig. 1.3). Serine, threonine, and tyrosine each contain a polar hydroxyl group that can participate in hydrogen bond formation (Fig. 1.6). The side chains of asparagine and glutamine each contain a carbonyl group and an amide group, both of which can also participate in hydrogen bonds. 1. Disulfide bond: The side chain of cysteine contains a sulfhydryl (thiol) group (−SH), which is an important component of the active site of many enzymes. In proteins, the –SH groups of two cysteines can be oxidized to form a covalent cross-link called a disulfide bond (−S–S–). Two disulfide-linked cysteines are referred to as cystine. (See p. 19 for a further discussion of disulfide bond formation.)

1	Many extracellular proteins are stabilized by disulfide bonds. Albumin, a blood protein that functions as a transporter for a variety of molecules, is an example. 2. Side chains as attachment sites for other compounds: The polar hydroxyl group of serine, threonine, and (rarely) tyrosine can serve as a site of attachment for structures such as a phosphate group. In addition, the amide group of asparagine, as well as the hydroxyl group of serine or threonine, can serve as a site of attachment for oligosaccharide chains in glycoproteins (see p. 165). C. Amino acids with acidic side chains The amino acids aspartic acid and glutamic acid are proton donors. At physiologic pH, the side chains of these amino acids are fully ionized, containing a negatively charged carboxylate group (−COO−). The fully ionized forms are called aspartate and glutamate. D. Amino acids with basic side chains

1	D. Amino acids with basic side chains The side chains of the basic amino acids accept protons (see Fig. 1.3). At physiologic pH, the R groups of lysine and arginine are fully ionized and positively charged. In contrast, the free amino acid histidine is weakly basic and largely uncharged at physiologic pH. However, when histidine is incorporated into a protein, its R group can be either positively charged (protonated) or neutral, depending on the ionic environment provided by the protein. This important property of histidine contributes to the buffering role it plays in the functioning of such proteins as hemoglobin (see p. 30). [Note: Histidine is the only amino acid with a side chain that can ionize within the physiologic pH range.] E. Abbreviations and symbols for commonly occurring amino acids letter symbol (Fig. 1.7). The one-letter codes are determined by the following rules. 1.

1	E. Abbreviations and symbols for commonly occurring amino acids letter symbol (Fig. 1.7). The one-letter codes are determined by the following rules. 1. Unique first letter: If only one amino acid begins with a given letter, then that letter is used as its symbol. For example, V = valine. 2. Most commonly occurring amino acids have priority: If more than one amino acid begins with a particular letter, the most common of these amino acids receives this letter as its symbol. For example, glycine is more common than glutamate, so G = glycine. 3. Similar sounding names: Some one-letter symbols sound like the amino acid they represent. For example, F = phenylalanine, or W = tryptophan (“twyptophan” as Elmer Fudd would say). 4.

1	3. Similar sounding names: Some one-letter symbols sound like the amino acid they represent. For example, F = phenylalanine, or W = tryptophan (“twyptophan” as Elmer Fudd would say). 4. Letter close to initial letter: For the remaining amino acids, a one-letter symbol is assigned that is as close in the alphabet as possible to the initial letter of the amino acid, for example, K = lysine. Furthermore, B is assigned to Asx, signifying either aspartic acid or asparagine; Z is assigned to Glx, signifying either glutamic acid or glutamine; and X is assigned to an unidentified amino acid. F. Amino acid isomers

1	F. Amino acid isomers Because the α-carbon of an amino acid is attached to four different chemical groups, it is an asymmetric (chiral) atom. Glycine is the exception because its α-carbon has two hydrogen substituents. Amino acids with a chiral αcarbon exist in two different isomeric forms, designated D and L, which are enantiomers, or mirror images (Fig. 1.8). [Note: Enantiomers are optically active. If an isomer, either D or L, causes the plane of polarized light to rotate clockwise, it is designated the (+) form.] All amino acids found in mammalian proteins are of the L configuration. However, D-amino acids are found in some antibiotics and in bacterial cell walls (see p. 252). [Note: Racemases enzymatically interconvert the D-and L-isomers of free amino acids.] III. ACIDIC AND BASIC PROPERTIES

1	III. ACIDIC AND BASIC PROPERTIES Amino acids in aqueous solution contain weakly acidic α-carboxyl groups and weakly basic α-amino groups. In addition, each of the acidic and basic amino acids contains an ionizable group in its side chain. Thus, both free amino acids and some amino acids combined in peptide linkages can act as buffers. Acids may be defined as proton donors and bases as proton acceptors. Acids (or bases) described as weak ionize to only a limited extent. The concentration of protons ([H+]) in aqueous solution is expressed as pH, where pH = log 1/[H+] or –log [H+]. The quantitative relationship between the pH of the solution and concentration of a weak acid (HA) and its conjugate base (A−) is described by the Henderson-Hasselbalch equation. A. Equation derivation

1	A. Equation derivation Consider the release of a proton by a weak acid represented by HA: The salt or conjugate base, A−, is the ionized form of a weak acid. By definition, the dissociation constant of the acid, Ka, is: [Note: The larger the Ka, the stronger the acid, because most of the HA has dissociated into H+ and A−. Conversely, the smaller the Ka, the less acid has dissociated and, therefore, the weaker the acid.] By solving for the [H+] in the above equation, taking the logarithm of both sides of the equation, multiplying both sides of the equation by −1, and substituting pH = −log [H+] and pKa = −log Ka, we obtain the Henderson-Hasselbalch equation: B. Buffers

1	B. Buffers A buffer is a solution that resists change in pH following the addition of an acid or base. A buffer can be created by mixing a weak acid (HA) with its conjugate base (A−). If an acid such as HCl is added to a buffer, A− can neutralize it, being converted to HA in the process. If a base is added, HA can likewise neutralize it, being converted to A− in the process. Maximum buffering capacity occurs at a pH equal to the pKa, but a conjugate acid-base pair can still serve as an effective buffer when the pH of a solution is within approximately ±1 pH unit of the pKa. If the amounts of HA and A− are equal, the pH is equal to the pKa. As shown in Figure 1.9, a solution containing acetic acid (HA = CH3 – COOH) and acetate (A− = CH3 – COO−) with a pKa of 4.8 resists a change in pH from pH 3.8 to 5.8, with maximum buffering at pH 4.8. At pH values less than the pKa, the protonated acid form (CH3 – COOH) is the predominant species in solution.

1	At pH values greater than the pKa, the deprotonated base form (CH3 – COO−) is the predominant species. C. Amino acid titration The titration curve of an amino acid can be analyzed in the same way as described for acetic acid. 1. Carboxyl group dissociation: Consider alanine, for example, which contains an ionizable α-carboxyl and α-amino group. [Note: Its –CH3 R group is nonionizable.] At a low (acidic) pH, both of these groups are protonated (Fig. 1.10). As the pH of the solution is raised, the −COOH group of form I can dissociate by donating a H+ to the medium. The release of a H+ results in the formation of the carboxylate group, −COO−. This structure is shown as form II, which is the dipolar form of the molecule (see Fig. 1.10). This form, also called a zwitterion (from the German word for “hybrid”), is the isoelectric form of alanine, that is, it has an overall (net) charge of zero. 2.

1	2. Application of the Henderson-Hasselbalch equation: The dissociation constant of the carboxyl group of an amino acid is called K1, rather than Ka, because the molecule contains a second titratable group. The Henderson-Hasselbalch equation can be used to analyze the dissociation of the carboxyl group of alanine in the same way as described for acetic acid: where I is the fully protonated form of alanine and II is the isoelectric form of alanine (see Fig. 1.10). This equation can be rearranged and converted to its logarithmic form to yield: 3. Amino group dissociation: The second titratable group of alanine is the amino (−NH3+) group shown in Figure 1.10. Because this is a much weaker acid than the –COOH group, it has a much smaller dissociation constant, K2. [Note: Its pKa is, therefore, larger.] Release of a H+ from the protonated amino group of form II results in the fully deprotonated form of alanine, form III (see Fig. 1.10). 4.

1	4. Alanine pKs: The sequential dissociation of H+ from the carboxyl and amino groups of alanine is summarized in Figure 1.10. Each titratable group has a pKa that is numerically equal to the pH at which exactly one half of the H+ have been removed from that group. The pKa for the most acidic group (−COOH) is pK1, whereas the pKa for the next most acidic group (−NH3+) is pK2. [Note: The pKa of the α-carboxyl group of amino acids is ~2, whereas that of the α-amino group is ~9.] 5. Alanine titration curve: By applying the Henderson-Hasselbalch equation to each dissociable acidic group, it is possible to calculate the complete titration curve of a weak acid. Figure 1.11 shows the change in pH that occurs during the addition of base to the fully protonated form of alanine (I) to produce the completely deprotonated form (III). Note the following: a.

1	Buffer pairs: The –COOH/–COO− pair can serve as a buffer in the pH region around pK1, and the –NH3+/–NH2 pair can buffer in the region around pK2. b. When pH = pK: When the pH is equal to pK1 (2.3), equal amounts of forms I and II of alanine exist in solution. When the pH is equal to pK2 (9.1), equal amounts of forms II and III are present in solution. c.

1	c. Isoelectric point: At neutral pH, alanine exists predominantly as the dipolar form II in which the amino and carboxyl groups are ionized, but the net charge is zero. The isoelectric point (pI) is the pH at which an amino acid is electrically neutral, that is, in which the sum of the positive charges equals the sum of the negative charges. For an amino acid, such as alanine, that has only two dissociable hydrogens (one from the α-carboxyl and one from the α-amino group), the pI is the average of pK1 and pK2 (pI = [2.3 + 9.1]/2 = 5.7) as shown in Figure 1.11. The pI is, thus, midway between pK1 (2.3) and pK2 (9.1). pI corresponds to the pH at which the form II (with a net charge of zero) predominates and at which there are also equal amounts of forms I (net charge of +1) and III (net charge of −1).

1	Separation of plasma proteins by charge typically is done at a pH above the pI of the major proteins. Therefore, the charge on the proteins is negative. In an electric field, the proteins will move toward the positive electrode at a rate determined by their net negative charge. Variations in the mobility pattern are suggestive of certain diseases. 6. Net charge at neutral pH: At physiologic pH, amino acids have a negatively charged group (−COO−) and a positively charged group (−NH3+), both attached to the α-carbon. [Note: Glutamate, aspartate, histidine, arginine, and lysine have additional potentially charged groups in their side chains.] Substances such as amino acids that can act either as an acid or a base are defined as amphoteric and are referred to as ampholytes (amphoteric electrolytes). D. Other applications of the Henderson-Hasselbalch equation

1	D. Other applications of the Henderson-Hasselbalch equation The Henderson-Hasselbalch equation can be used to calculate how the pH of a physiologic solution responds to changes in the concentration of a weak acid and/or its corresponding salt form. For example, in the bicarbonate buffer system, the Henderson-Hasselbalch equation predicts how shifts in the bicarbonate ion concentration, [HCO3−], and the carbon dioxide concentration [CO2] influence pH (Fig. 1.12A). The equation is also useful for calculating the abundance of ionic forms of acidic and basic drugs. For example, most drugs are either weak acids or weak bases (Fig. 1.12B). Acidic drugs (HA) release a H+, causing a charged anion (A−) to form. Weak bases (BH+) can also release a H+. However, the protonated form of basic drugs is usually charged, and the loss of a proton produces the uncharged base (B).

1	A drug passes through membranes more readily if it is uncharged. Thus, for a weak acid, such as aspirin, the uncharged HA can permeate through membranes, but A− cannot. Likewise, for a weak base, such as morphine, the uncharged B form permeates through the cell membrane, but BH+ does not. Therefore, the effective concentration of the permeable form of each drug at its absorption site is determined by the relative concentrations of the charged (impermeant) and uncharged (permeant) forms. The ratio between the two forms is determined by the pH at the site of absorption and by the strength of the weak acid or base, which is represented by the pKa of the ionizable group. The Henderson-Hasselbalch equation is useful in determining how much drug is found on either side of a membrane that separates two compartments that differ in pH, for example, the stomach (pH 1.0–1.5) and blood plasma (pH 7.4). IV. CONCEPT MAPS

1	Students sometimes view biochemistry as a list of facts or equations to be memorized, rather than a body of concepts to be understood. Details provided to enrich understanding of these concepts inadvertently turn into distractions. What seems to be missing is a road map—a guide that provides the student with an understanding of how various topics fit together to “tell a story.” Therefore, in this text, a series of biochemical concept maps have been created to graphically illustrate relationships between ideas presented in a chapter and to show how the information can be grouped or organized. A concept map is, thus, a tool for visualizing the connections between concepts. Material is represented in a hierarchic fashion, with the most inclusive, most general concepts at the top of the map, and the more specific, less general concepts arranged beneath. The concept maps ideally function as templates or guides for organizing information, so the student can readily find the best ways to

1	and the more specific, less general concepts arranged beneath. The concept maps ideally function as templates or guides for organizing information, so the student can readily find the best ways to integrate new information into knowledge they already possess. Concept map construction is described below.

1	A. Concept boxes and links Educators define concepts as “perceived regularities in events or objects.” In the biochemical maps, concepts include abstractions (for example, free energy), processes (for example, oxidative phosphorylation), and compounds (for example, glucose 6-phosphate). These broadly defined concepts are prioritized with the central idea positioned at the top of the page. The concepts that follow from this central idea are then drawn in boxes (Fig. 1.13A). The size of the type indicates the relative importance of each idea. Lines are drawn between concept boxes to show which are related. The label on the line defines the relationship between two concepts, so that it reads as a valid statement (that is, the connection creates meaning). The lines with arrowheads indicate in which direction the connection should be read (Fig. 1.14). B. Cross-links

1	B. Cross-links Unlike linear flow charts or outlines, concept maps may contain cross-links that allow the reader to visualize complex relationships between ideas represented in different parts of the map (Fig. 1.13B) or between the map and other chapters in this book (Fig. 1.13C). Cross-links can, thus, identify concepts that are central to more than one topic in biochemistry, empowering students to be effective in clinical situations and on the United States Medical Licensure Examination (USMLE) or other examinations that require integration of material. Students learn to visually perceive nonlinear relationships between facts, in contrast to cross-referencing within linear text. V. CHAPTER SUMMARY Each amino acid has an α-carboxyl group and a primary α-amino group (except for proline, which has a secondary amino group). At physiologic pH, the α-carboxyl group is dissociated, forming the negatively charged carboxylate ion (−COO−), and the α-amino group is protonated (−NH3+).

1	Each amino acid also contains one of 20 distinctive side chains attached to the α-carbon atom. The chemical nature of this R group determines the function of an amino acid in a protein and provides the basis for classification of the amino acids as nonpolar, uncharged polar, acidic (polar negative), or basic (polar positive). All free amino acids, plus charged amino acids in peptide chains, can serve as buffers. The quantitative relationship between the pH of a solution and the concentration of a weak acid (HA) and its conjugate base (A−) is described by the Henderson-Hasselbalch equation. Buffering occurs within ±1 pH unit of the pKa and is maximal when pH = pKa, at which [A−] = [HA]. Because the α-carbon of each amino acid (except glycine) is attached to four different chemical groups, it is asymmetric (chiral), and amino acids exist in D-and L-isomeric forms that are optically active mirror images (enantiomers). The L-form of amino acids is found in proteins synthesized by the

1	it is asymmetric (chiral), and amino acids exist in D-and L-isomeric forms that are optically active mirror images (enantiomers). The L-form of amino acids is found in proteins synthesized by the human body.

1	Choose the ONE best answer. .1. Which one of the following statements concerning the titration curve for a nonpolar amino acid is correct? The letters A through D designate certain regions on the curve below. A. Point A represents the region where the amino acid is deprotonated. B. Point B represents a region of minimal buffering. C. Point C represents the region where the net charge on the amino acid is zero. D. Point D represents the pK of the amino acid’s carboxyl group. E. The amino acid could be lysine. Correct answer = C. Point C represents the isoelectric point, or pI, and as such is midway between pK1 and pK2 for a nonpolar amino acid. The amino acid is fully protonated at Point A. Point B represents a region of maximum buffering, as does Point D. Lysine is a basic amino acid, and free lysine has an ionizable side chain in addition to the ionizable α-amino and α-carboxyl groups.

1	.2. Which one of the following statements concerning the peptide shown below is correct?Val-Cys-Glu-Ser-Asp-Arg-Cys A. The peptide contains asparagine. B. The peptide contains a side chain with a secondary amino group. C. The peptide contains a side chain that can be phosphorylated. D. The peptide cannot form an internal disulfide bond. E. The peptide would move to the cathode (negative electrode) during electrophoresis at pH 5. Correct answer = C. The hydroxyl group of serine can accept a phosphate group. Asp is aspartate. Proline contains a secondary amino group. The two cysteine residues can, under oxidizing conditions, form a disulfide (covalent) bond. The net charge on the peptide at pH 5 is negative, and it would move to the anode.

1	.3. A 2-year-old child presents with metabolic acidosis after ingesting an unknown number of flavored aspirin tablets. At presentation, her blood pH was 7.0. Given that the pKa of aspirin (salicylic acid) is 3, calculate the ratio of its ionized to unionized forms at pH 7.0. Correct answer = 10,000 to 1. pH = pKa + log [A−]/[HA]. Therefore, 7 = 3 + × and × = 4. The ratio of A− (ionized) to HA (unionized), then, is 10,000 to 1 because the log of 10,000 is 4. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	I. OVERVIEW The 20 amino acids commonly found in proteins are joined together by peptide bonds. The linear sequence of the linked amino acids contains the information necessary to generate a protein molecule with a unique three-dimensional shape that determines function. The complexity of protein structure is best analyzed by considering the molecule in terms of four organizational levels: primary, secondary, tertiary, and quaternary (Fig. 2.1). An examination of these hierarchies of increasing complexity has revealed that certain structural elements are repeated in a wide variety of proteins, suggesting that there are general rules regarding the ways in which proteins achieve their native, functional form. These repeated structural elements range from simple combinations of α-helices and β-sheets forming small motifs to the complex folding of polypeptide domains of multifunctional proteins (see p. 19). II. PRIMARY STRUCTURE

1	II. PRIMARY STRUCTURE The sequence of amino acids in a protein is called the primary structure of the protein. Understanding the primary structure of proteins is important because many genetic diseases result in proteins with abnormal amino acid sequences, which cause improper folding and loss or impairment of normal function. If the primary structures of the normal and the mutated proteins are known, this information may be used to diagnose or study the disease. A. Peptide bond

1	A. Peptide bond In proteins, amino acids are joined covalently by peptide bonds, which are amide linkages between the α-carboxyl group of one amino acid and the αamino group of another. For example, valine and alanine can form the dipeptide valylalanine through the formation of a peptide bond (Fig. 2.2). Peptide bonds are resistant to conditions that denature proteins, such as heating and high concentrations of urea (see p. 20). Prolonged exposure to a strong acid or base at elevated temperatures is required to break these bonds nonenzymically (see p. 14). 1.

1	1. Naming the peptide: By convention, the free amino end (N-terminal) of the peptide chain is written to the left and the free carboxyl end (C-terminal) to the right. Therefore, all amino acid sequences are read from the N-to the C-terminal end. For example, in Figure 2.2A, the order of the amino acids in the dipeptide is valine, alanine. Linkage of ≥50 amino acids through peptide bonds results in an unbranched chain called a polypeptide, or protein. Each component amino acid is called a residue because it is the portion of the amino acid remaining after the atoms of water are lost in the formation of the peptide bond. When a peptide is named, all amino acid residues have their suffixes (-ine, -an, -ic, or -ate) changed to -yl, with the exception of the C-terminal amino acid. For example, a tripeptide composed of an N-terminal valine, a glycine, and a C-terminal leucine is called valylglycylleucine. 2.

1	2. Peptide bond characteristics: The peptide bond has a partial double-bond character, that is, it is shorter than a single bond and is rigid and planar (Fig. 2.2B). This prevents free rotation around the bond between the carbonyl carbon and the nitrogen of the peptide bond. However, the bonds between the α-carbons and the α-amino or α-carboxyl groups can be freely rotated (although they are limited by the size and character of the R groups). This allows the polypeptide chain to assume a variety of possible conformations. The peptide bond is almost always in the trans configuration (instead of the cis; see Fig. 2.2B), in large part because of steric interference of the R groups (side chains) when in the cis position. 3.

1	3. Peptide bond polarity: Like all amide linkages, the −C = O and −NH groups of the peptide bond are uncharged, and neither accept nor release protons over the pH range of 2–12. Thus, the charged groups present in polypeptides consist solely of the N-terminal (α-amino) group, the C-terminal (α-carboxyl) group, and any ionized groups present in the side chains of the constituent amino acids. The −C = O and −NH groups of the peptide bond are polar, however, and are involved in hydrogen bonds (for example, in α-helices and β-sheets), as described on pp. 16–17. B. Determining the amino acid composition of a polypeptide

1	The first step in determining the primary structure of a polypeptide is to identify and quantitate its constituent amino acids. A purified sample of the polypeptide to be analyzed is first hydrolyzed by strong acid at 110°C for 24 hours. This treatment cleaves the peptide bonds and releases the individual amino acids, which can be separated by cation-exchange chromatography. In this technique, a mixture of amino acids is applied to a column that contains a resin to which a negatively charged group is tightly attached. [Note: If the attached group is positively charged, the column becomes an anion-exchange column.] The amino acids bind to the column with different affinities, depending on their charges, hydrophobicity, and other characteristics. Each amino acid is sequentially released from the chromatography column by eluting with solutions of increasing ionic strength and pH (Fig. 2.3). The separated amino acids contained in the eluate from the column are quantitated by heating them

1	the chromatography column by eluting with solutions of increasing ionic strength and pH (Fig. 2.3). The separated amino acids contained in the eluate from the column are quantitated by heating them with ninhydrin (a reagent that forms a purple compound with most amino acids, ammonia, and amines). The amount of each amino acid is determined spectrophotometrically by measuring the amount of light absorbed by the ninhydrin derivative. The analysis described above is performed using an amino acid analyzer, an automated machine whose components are depicted in Figure 2.3.

1	C. Sequencing the peptide from its N-terminal end Sequencing is a stepwise process of identifying the specific amino acid at each position in the peptide chain, beginning at the N-terminal end. Phenylisothiocyanate, known as Edman reagent, is used to label the amino-terminal residue under mildly alkaline conditions (Fig. 2.4). The resulting phenylthiohydantoin (PTH) derivative introduces an instability in the N-terminal peptide bond such that it can be hydrolyzed without cleaving the other peptide bonds. The identity of the amino acid derivative can then be determined. Edman reagent can be applied repeatedly to the shortened peptide obtained in each previous cycle. Automated sequencers are now used. D. Cleaving the polypeptide into smaller fragments

1	D. Cleaving the polypeptide into smaller fragments Many polypeptides have a primary structure composed of >100 amino acids. Such molecules cannot be sequenced directly from end to end. However, these large molecules can be cleaved at specific sites and the resulting fragments sequenced. By using more than one cleaving agent (enzymes and/or chemicals) on separate samples of the purified polypeptide, overlapping fragments can be generated that permit the proper ordering of the sequenced fragments, thereby providing a complete amino acid sequence of the large polypeptide (Fig. 2.5). Enzymes that hydrolyze peptide bonds are termed peptidases (proteases). [Note: Exopeptidases cut at the ends of proteins and are divided into aminopeptidases and carboxypeptidases. Carboxypeptidases are used in determining the C-terminal amino acid. Endopeptidases cleave within a protein.] E. Determining a protein’s primary structure by DNA sequencing

1	E. Determining a protein’s primary structure by DNA sequencing The sequence of nucleotides in a protein-coding region of the DNA specifies the amino acid sequence of a polypeptide. Therefore, if the nucleotide sequence can be determined, knowledge of the genetic code (see p. 447) allows the sequence of nucleotides to be translated into the corresponding amino acid sequence of that polypeptide. This indirect process, although routinely used to obtain the amino acid sequences of proteins, has the limitations of not being able to predict the positions of disulfide bonds in the folded chain and of not identifying any amino acids that are modified after their incorporation into the polypeptide (posttranslational modification; see p. 459). Therefore, direct protein sequencing is an extremely important tool for determining the true character of the primary sequence of many polypeptides. III. SECONDARY STRUCTURE

1	III. SECONDARY STRUCTURE The polypeptide backbone does not assume a random three-dimensional structure but, instead, generally forms regular arrangements of amino acids that are located near each other in the linear sequence. These arrangements are termed the secondary structure of the polypeptide. The α-helix, β-sheet, and βbend (or, β-turn) are examples of secondary structures commonly encountered in proteins. Each is stabilized by hydrogen bonds between atoms of the peptide backbone. [Note: The collagen α-chain helix, another example of secondary structure, is discussed on p. 45.] A. α-Helix

1	A. α-Helix Several different polypeptide helices are found in nature, but the α-helix is the most common. It is a rigid, right-handed spiral structure, consisting of a tightly packed, coiled polypeptide backbone core, with the side chains of the component L-amino acids extending outward from the central axis to avoid interfering sterically with each other (Fig. 2.6). A very diverse group of proteins contains α-helices. For example, the keratins are a family of closely related, rigid, fibrous proteins whose structure is nearly entirely αhelical. They are a major component of tissues such as hair and skin. In contrast to keratin, myoglobin, whose structure is also highly α-helical, is a globular, flexible molecule (see p. 26) found in muscles. 1.

1	1. Hydrogen bonds: An α-helix is stabilized by extensive hydrogen bonding between the peptide bond carbonyl oxygens and amide hydrogens that are part of the polypeptide backbone (see Fig. 2.6). The hydrogen bonds extend up and are parallel to the spiral from the carbonyl oxygen of one peptide bond to the –NH group of a peptide linkage four residues ahead in the polypeptide. This insures that all but the first and last peptide bond components are linked to each other through intrachain hydrogen bonds. Hydrogen bonds are individually weak, but they collectively serve to stabilize the helix. 2. Amino acids per turn: Each turn of an α-helix contains 3.6 amino acids. Thus, amino acids spaced three or four residues apart in the primary sequence are spatially close together when folded in the α-helix. 3.

1	3. Amino acids that disrupt an α-helix: The R group of an amino acid determines its propensity to be in an α-helix. Proline disrupts an α-helix because its rigid secondary amino group is not geometrically compatible with the right-handed spiral of the α-helix. Instead, it inserts a kink in the chain, which interferes with the smooth, helical structure. Glycine is also a “helix breaker” because its R group (a hydrogen) confers high flexibility. Additionally, amino acids with charged or bulky R groups (such as glutamate and tryptophan, respectively) and those with a branch at the β-carbon, the first carbon in the R group (for example, valine), have low α-helix propensity. B. β-Sheet

1	B. β-Sheet The β-sheet is another form of secondary structure in which all of the peptide bond components are involved in hydrogen bonding (Fig. 2.7A). Because the surfaces of β-sheets appear “pleated,” they are often called βpleated sheets. [Note: Pleating results from successive α-carbons being slightly above or below the plane of the sheet.] Illustrations of protein structure often show β-strands as broad arrows (Fig. 2.7B). 1.

1	1. Formation: A β-sheet is formed by two or more peptide chains (βstrands) aligned laterally and stabilized by hydrogen bonds between the carboxyl and amino groups of amino acids that either are far apart in a single polypeptide (intrachain bonds) or are in different polypeptide chains (interchain bonds). The adjacent β-strands are arranged either antiparallel to each other (with the N-termini alternating as shown in Fig. 2.7B) or parallel to each other (with the N-termini together as shown in Fig. 2.7C). On each β-strand, the R groups of adjacent amino acids extend in opposite directions, above and below the plane of the β-sheet. [Note: β-sheets are not flat and have a right-handed curl (twist) when viewed along the polypeptide backbone.] 2.

1	Comparing α-helices and β-sheets: In β-sheets, the β-strands are almost fully extended and the hydrogen bonds between the strands are perpendicular to the polypeptide backbone (see Fig. 2.7A). In contrast, in α-helices, the polypeptide is coiled and the hydrogen bonds are parallel to the backbone (see Fig. 2.6). The orientation of the R groups of the amino acid residues in both the αhelix and the β-sheet can result in formation of polar and nonpolar sides in these secondary structures, thereby making them amphipathic.

1	C. β-Bends (reverse turns, β-turns) β-Bends reverse the direction of a polypeptide chain, helping it form a compact, globular shape. They are usually found on the surface of protein molecules and often include charged residues. [Note: β-Bends were given this name because they often connect successive strands of antiparallel βsheets.] β-Bends are generally composed of four amino acids, one of which may be proline, the amino acid that causes a kink in the polypeptide chain. Glycine, the amino acid with the smallest R group, is also frequently found in β-bends. β-Bends are stabilized by the formation of hydrogen bonds between the first and last residues in the bend. D. Nonrepetitive secondary structure

1	D. Nonrepetitive secondary structure Approximately one half of an average globular protein is organized into repetitive structures, such as the α-helix and β-sheet. The remainder of the polypeptide chain is described as having a loop or coil conformation. These nonrepetitive secondary structures are not random but rather simply have a less regular structure than those described above. [Note: The term “random coil” refers to the disordered structure obtained when proteins are denatured (see p. 20).] E. Supersecondary structures (motifs)

1	E. Supersecondary structures (motifs) Globular proteins are constructed by combining secondary structural elements (that is, α-helices, β-sheets, and coils), producing specific geometric patterns, or motifs. These form primarily the core (interior) region of the molecule. They are connected by loop regions (for example, β-bends) at the surface of the protein. Supersecondary structures are usually produced by the close packing of side chains from adjacent secondary structural elements. For example, α-helices and β-sheets that are adjacent in the amino acid sequence are also usually (but not always) adjacent in the final, folded protein. Some of the more common motifs are illustrated in Figure 2.8. Motifs may be associated with particular functions. Proteins that bind to DNA contain a limited number of motifs. The helix–loop–helix motif is an example found in a number of proteins that function as transcription factors (see p. 438). IV. TERTIARY STRUCTURE

1	IV. TERTIARY STRUCTURE The primary structure of a polypeptide chain determines its tertiary structure. “Tertiary” refers both to the folding of domains (the basic units of structure and function; see A. below) and to the final arrangement of domains in the polypeptide. The tertiary structure of globular proteins in aqueous solution is compact, with a high density (close packing) of the atoms in the core of the molecule. Hydrophobic side chains are buried in the interior, whereas hydrophilic groups are generally found on the surface of the molecule. A. Domains

1	A. Domains Domains are the fundamental functional and three-dimensional structural units of polypeptides. Polypeptide chains that are >200 amino acids in length generally consist of two or more domains. The core of a domain is built from combinations of supersecondary structural elements (motifs). Folding of the peptide chain within a domain usually occurs independently of folding in other domains. Therefore, each domain has the characteristics of a small, compact globular protein that is structurally independent of the other domains in the polypeptide chain. B. Stabilizing interactions The unique three-dimensional structure of each polypeptide is determined by its amino acid sequence. Interactions between the amino acid side chains guide the folding of the polypeptide to form a compact structure. The following four types of interactions cooperate in stabilizing the tertiary structures of globular proteins.

1	1. Disulfide bonds: A disulfide bond (–S–S–) is a covalent linkage formed from the sulfhydryl group (−SH) of each of two cysteine residues to produce a cystine residue (Fig. 2.9). The two cysteines may be separated from each other by many amino acids in the primary sequence of a polypeptide or may even be located on two different polypeptides. The folding of the polypeptide(s) brings the cysteine residues into proximity and permits covalent bonding of their side chains. A disulfide bond contributes to the stability of the three-dimensional shape of the protein molecule and prevents it from becoming denatured in the extracellular environment. For example, many disulfide bonds are found in proteins such as immunoglobulins that are secreted by cells. [Note: Protein disulfide isomerase breaks and reforms disulfide bonds during folding.] 2.

1	Hydrophobic interactions: Amino acids with nonpolar side chains tend to be located in the interior of the polypeptide molecule, where they associate with other hydrophobic amino acids (Fig. 2.10). In contrast, amino acids with polar or charged side chains tend to be located on the surface of the molecule in contact with the polar solvent. [Note: Recall that proteins located in nonpolar (lipid) environments, such as a membrane, exhibit the reverse arrangement (see Fig. 1.4, p. 4).] In each case, a segregation of R groups occurs that is energetically most favorable. 3.

1	3. Hydrogen bonds: Amino acid side chains containing oxygen-or nitrogen-bound hydrogen, such as in the alcohol groups of serine and threonine, can form hydrogen bonds with electron-rich atoms, such as the oxygen of a carboxyl group or carbonyl group of a peptide bond (Fig. 2.11; see also Fig. 1.6, p. 4). Formation of hydrogen bonds between polar groups on the surface of proteins and the aqueous solvent enhances the solubility of the protein. 4. Ionic interactions: Negatively charged groups, such as the carboxylate group (−COO−) in the side chain of aspartate or glutamate, can interact with positively charged groups such as the amino group (−NH3+) in the side chain of lysine (see Fig. 2.11). C. Protein folding

1	C. Protein folding Interactions between the side chains of amino acids determine how a linear polypeptide chain folds into the intricate three-dimensional shape of the functional protein. Protein folding, which occurs within the cell in seconds to minutes, involves nonrandom, ordered pathways. As a peptide folds, secondary structures form, driven by the hydrophobic effect (that is, hydrophobic groups come together as water is released). These small structures combine to form larger structures. Additional events stabilize secondary structure and initiate formation of tertiary structure. In the last stage, the peptide achieves its fully folded, native (functional) form characterized by a low-energy state (Fig. 2.12). [Note: Some biologically active proteins or segments thereof lack a stable tertiary structure. They are referred to as intrinsically disordered proteins.] D. Protein denaturation

1	D. Protein denaturation Denaturation results in the unfolding and disorganization of a protein’s secondary and tertiary structures without the hydrolysis of peptide bonds. Denaturing agents include heat, urea, organic solvents, strong acids or bases, detergents, and ions of heavy metals such as lead. Denaturation may, under ideal conditions, be reversible, such that the protein refolds into its original native structure when the denaturing agent is removed. However, most proteins remain permanently disordered once denatured. Denatured proteins are often insoluble and precipitate from solution. E. Chaperones in protein folding

1	The information needed for correct protein folding is contained in the primary structure of the polypeptide. However, most denatured proteins do not resume their native conformations even under favorable environmental conditions. This is because, for many proteins, folding is a facilitated process that requires a specialized group of proteins, referred to as molecular chaperones, and ATP hydrolysis. The chaperones, also known as heat shock proteins (HSP), interact with a polypeptide at various stages during the folding process. Some chaperones bind hydrophobic regions of an extended polypeptide and are important in keeping the protein unfolded until its synthesis is completed (for example, Hsp70). Others form cage-like macromolecular structures composed of two stacked rings. The partially folded protein enters the cage, binds the central cavity through hydrophobic interactions, folds, and is released (for example, mitochondrial Hsp60). [Note: Cage-like chaperones are sometimes

1	folded protein enters the cage, binds the central cavity through hydrophobic interactions, folds, and is released (for example, mitochondrial Hsp60). [Note: Cage-like chaperones are sometimes referred to as chaperonins.] Chaperones, then, facilitate correct protein folding by binding to and stabilizing exposed, aggregation-prone hydrophobic regions in nascent (and denatured) polypeptides, preventing premature folding.

1	V. QUATERNARY STRUCTURE Many proteins consist of a single polypeptide chain and are defined as monomeric proteins. However, others may consist of two or more polypeptide chains that may be structurally identical or totally unrelated. The arrangement of these polypeptide subunits is called the quaternary structure of the protein. Subunits are held together primarily by noncovalent interactions (for example, hydrogen bonds, ionic bonds, and hydrophobic interactions). Subunits either may function independently of each other or may work cooperatively, as in hemoglobin, in which the binding of oxygen to one subunit of the tetramer increases the affinity of the other subunits for oxygen (see p. 29). Isoforms are proteins that perform the same function but have different primary structures. They can arise from different genes or from tissue-specific processing of the product of a single gene. If the proteins function as enzymes, they are referred to as isozymes (see p. 65).

1	VI. PROTEIN MISFOLDING Protein folding is a complex process that can sometimes result in improperly folded molecules. These misfolded proteins are usually tagged and degraded within the cell (see p. 247). However, this quality control system is not perfect, and intracellular or extracellular aggregates of misfolded proteins can accumulate, particularly as individuals age. Deposits of misfolded proteins are associated with a number of diseases. A. Amyloid diseases

1	Misfolding of proteins may occur spontaneously or be caused by a mutation in a particular gene, which then produces an altered protein. In addition, some apparently normal proteins can, after abnormal proteolytic cleavage, take on a unique conformation that leads to the spontaneous formation of long, fibrillar protein assemblies consisting of β-pleated sheets. Accumulation of these insoluble fibrous protein aggregates, called amyloids, has been implicated in neurodegenerative disorders such as Parkinson disease and Alzheimer disease (AD). The dominant component of the amyloid plaque that accumulates in AD is amyloid β (Aβ), an extracellular peptide containing 40–42 amino acid residues. X-ray crystallography and infrared spectroscopy demonstrate a characteristic βpleated sheet secondary structure in nonbranching fibrils. This peptide, when aggregated in a β-pleated sheet conformation, is neurotoxic and is the central pathogenic event leading to the cognitive impairment characteristic

1	in nonbranching fibrils. This peptide, when aggregated in a β-pleated sheet conformation, is neurotoxic and is the central pathogenic event leading to the cognitive impairment characteristic of the disease. The Aβ that is deposited in the brain in AD is derived by enzymic cleavages (by secretases) from the larger amyloid precursor protein, a single transmembrane protein expressed on the cell surface in the brain and other tissues (Fig. 2.13). The Aβ peptides aggregate, generating the amyloid that is found in the brain parenchyma and around blood vessels. Most cases of AD are not genetically based, although at least 5% of cases are familial. A second biologic factor involved in the development of AD is the accumulation of neurofibrillary tangles inside neurons. A key component of these tangled fibers is an abnormal form (hyperphosphorylated and insoluble) of the tau (τ) protein, which, in its healthy version, helps in the assembly of the microtubular structure. The defective τ appears

1	fibers is an abnormal form (hyperphosphorylated and insoluble) of the tau (τ) protein, which, in its healthy version, helps in the assembly of the microtubular structure. The defective τ appears to block the actions of its normal counterpart. [Note: In Parkinson disease, amyloid is formed from α-synuclein protein.]

1	B. Prion (proteinaceous infectious particle) diseases

1	The prion protein (PrP) is the causative agent of transmissible spongiform encephalopathies (TSE), including Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle (popularly called “mad cow” disease). After an extensive series of purification procedures, scientists were surprised to find that the infectivity of the agent causing scrapie in sheep was associated with a single protein species that was not complexed with detectable nucleic acid. This infectious protein is designated PrPSc (Sc = scrapie). It is highly resistant to proteolytic degradation and tends to form insoluble aggregates of fibrils, similar to the amyloid found in some other diseases of the brain. A noninfectious form of PrPC (C = cellular), encoded by the same gene as the infectious agent, is present in normal mammalian brains on the surface of neurons and glial cells. Thus, PrPC is a host protein. No primary structure differences or alternate posttranslational

1	as the infectious agent, is present in normal mammalian brains on the surface of neurons and glial cells. Thus, PrPC is a host protein. No primary structure differences or alternate posttranslational modifications have been found between the normal and the infectious forms of the protein. The key to becoming infectious apparently lies in changes in the three-dimensional conformation of PrPC. Research has demonstrated that a number of αhelices present in noninfectious PrPC are replaced by β-sheets in the infectious form (Fig. 2.14). This conformational difference is presumably what confers relative resistance to proteolytic degradation of infectious prions and permits them to be distinguished from the normal PrPC in infected tissue. The infective agent is, thus, an altered version of a normal protein, which acts as a template for converting the normal protein to the pathogenic conformation. The TSE are invariably fatal, and no treatment is currently available that can alter this

1	a normal protein, which acts as a template for converting the normal protein to the pathogenic conformation. The TSE are invariably fatal, and no treatment is currently available that can alter this outcome.

1	VII. CHAPTER SUMMARY

1	Central to understanding protein structure is the concept of the native conformation (Fig. 2.15), which is the functional, fully folded protein structure (for example, an active enzyme or structural protein). The unique three-dimensional structure of the native conformation is determined by its primary structure, that is, its amino acid sequence. Interactions between the amino acid side chains guide the folding of the polypeptide chain to form secondary, tertiary, and (sometimes) quaternary structures, which cooperate in stabilizing the native conformation of the protein. In addition, a specialized group of proteins named chaperones is required for the proper folding of many species of proteins. Protein denaturation results in the unfolding and disorganization of the protein’s structure, which are not accompanied by hydrolysis of peptide bonds. Denaturation may be reversible or, more commonly, irreversible. Disease can occur when an apparently normal protein assumes a conformation

1	which are not accompanied by hydrolysis of peptide bonds. Denaturation may be reversible or, more commonly, irreversible. Disease can occur when an apparently normal protein assumes a conformation that is cytotoxic, as in the case of Alzheimer disease (AD) and the transmissible spongiform encephalopathies (TSE), including Creutzfeldt-Jakob disease. In AD, normal proteins, after abnormal chemical processing, take on a unique conformational state that leads to the formation of neurotoxic amyloid β peptide (Aβ) assemblies consisting of β-pleated sheets. In TSE, the infective agent is an altered version of a normal prion protein that acts as a template for converting normal protein to the pathogenic conformation.

1	Choose the ONE best answer. .1. Which one of the following statements concerning protein structure is correct? A. Proteins consisting of one polypeptide have quaternary structure that is stabilized by covalent bonds. B. The peptide bonds that link amino acids in a protein most commonly occur in the cis configuration. C. The formation of a disulfide bond in a protein requires the participating cysteine residues to be adjacent in the primary structure. D. The denaturation of proteins leads to irreversible loss of secondary structural elements such as the α-helix. E. The primary driving force for protein folding is the hydrophobic effect.

1	D. The denaturation of proteins leads to irreversible loss of secondary structural elements such as the α-helix. E. The primary driving force for protein folding is the hydrophobic effect. Correct answer = E. The hydrophobic effect, or the tendency of nonpolar entities to associate in a polar environment, is the primary driving force of protein folding. Quaternary structure requires more than one polypeptide, and, when present, it is stabilized primarily by noncovalent bonds. The peptide bond is almost always trans. The two cysteine residues participating in disulfide bond formation may be a great distance apart in the amino acid sequence of a polypeptide (or on two separate polypeptides) but are brought into close proximity by the three-dimensional folding of the polypeptide. Denaturation may be reversible or irreversible.

1	.2. A particular point mutation results in disruption of the α-helical structure in a segment of the mutant protein. The most likely change in the primary structure of the mutant protein is: A. glutamate to aspartate. B. lysine to arginine. C. methionine to proline. D. valine to alanine. Correct answer = C. Proline, because of its secondary amino group, is incompatible with an α-helix. Glutamate, aspartate, lysine, and arginine are charged amino acids, and valine is a branched amino acid. Charged and branched (bulky) amino acids may disrupt an α-helix. [Note: The flexibility of glycine’s R group can also disrupt an α-helix.] .3. In comparing the α-helix to the β-sheet, which statement is correct only for the β-sheet? A. Extensive hydrogen bonds between the carbonyl oxygen (C=O) and the amide hydrogen (N−H) of the peptide bond are formed. B. It may be found in typical globular proteins. C. It is stabilized by interchain hydrogen bonds.

1	B. It may be found in typical globular proteins. C. It is stabilized by interchain hydrogen bonds. D. It is an example of secondary structure. E. It may be found in supersecondary structures. Correct answer = C. The β-sheet is stabilized by interchain hydrogen bonds formed between separate polypeptide chains and by intrachain hydrogen bonds formed between regions of a single polypeptide. The α-helix, however, is stabilized only by intrachain hydrogen bonds. Statements A, B, D, and E are true for both of these secondary structural elements. .4. An 80-year-old man presented with impairment of intellectual function and alterations in behavior. His family reported progressive disorientation and memory loss over the last 6 months. There is no family history of dementia. The patient was tentatively diagnosed with Alzheimer disease (AD). Which one of the following best describes AD? A. It is associated with β-amyloid, an abnormal protein with an altered amino acid sequence.

1	A. It is associated with β-amyloid, an abnormal protein with an altered amino acid sequence. B. It results from accumulation of denatured proteins that have random conformations. C. It is associated with the accumulation of amyloid precursor protein. D. It is associated with the deposition of neurotoxic amyloid β peptide aggregates. E. It is an environmentally produced disease not influenced by the genetics of the individual. F. It is caused by the infectious β-sheet form of a host-cell protein.

1	E. It is an environmentally produced disease not influenced by the genetics of the individual. F. It is caused by the infectious β-sheet form of a host-cell protein. Correct answer = D. Alzheimer disease (AD) is associated with long, fibrillar protein assemblies consisting of β-pleated sheets found in the brain and elsewhere. The disease is associated with abnormal processing of a normal protein. The accumulated altered protein occurs in a β-pleated sheet conformation that is neurotoxic. The amyloid β that is deposited in the brain in AD is derived by proteolytic cleavages from the larger amyloid precursor protein, a single transmembrane protein expressed on the cell surface in the brain and other tissues. Most cases of AD are sporadic, although at least 5% of cases are familial. Prion diseases, such as Creutzfeldt-Jakob, are caused by the infectious β-sheet form (PrPSc) of a host-cell protein (PrPC).

1	For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW The previous chapter described the types of secondary and tertiary structures that are the bricks and mortar of protein architecture. By arranging these fundamental structural elements in different combinations, widely diverse proteins can be constructed that are capable of various specialized functions. This chapter examines the relationship between structure and function for the clinically important globular hemeproteins. Fibrous structural proteins are discussed in Chapter 4. II. GLOBULAR HEMEPROTEINS

1	II. GLOBULAR HEMEPROTEINS Hemeproteins are a group of specialized proteins that contain heme as a tightly bound prosthetic group. (See p. 54 for a discussion of prosthetic groups.) The role of the heme group is dictated by the environment created by the three-dimensional structure of the protein. For example, the heme group of a cytochrome functions as an electron carrier that is alternately oxidized and reduced (see p. 75). In contrast, the heme group of the enzyme catalase is part of the active site of the enzyme that catalyzes the breakdown of hydrogen peroxide (see p. 148). In hemoglobin and myoglobin, the two most abundant hemeproteins in humans, the heme group serves to reversibly bind oxygen (O2). A. Heme structure

1	A. Heme structure Heme is a complex of protoporphyrin IX and ferrous iron (Fe2+), as shown in Figure 3.1. The iron is held in the center of the heme molecule by bonds to the four nitrogens of the porphyrin ring. The heme Fe2+ can form two additional bonds, one on each side of the planar porphyrin ring. In myoglobin and hemoglobin, one of these positions is coordinated to the side chain of a histidine residue of the globin molecule, whereas the other position is available to bind O2 (Fig. 3.2). (See pp. 278 and 282, respectively, for a discussion of heme synthesis and degradation.) B. Myoglobin structure and function

1	B. Myoglobin structure and function Myoglobin, a hemeprotein present in heart and skeletal muscle, functions both as an oxygen reservoir and as an oxygen carrier that increases the rate of oxygen transport within the muscle cell. [Note: Surprisingly, mouse myoglobin double knockouts (see p. 502) have an apparently normal phenotype.] Myoglobin consists of a single polypeptide chain that is structurally similar to the individual polypeptide chains of the tetrameric hemoglobin molecule. This homology makes myoglobin a useful model for interpreting some of the more complex properties of hemoglobin. 1.

1	1. α-Helical content: Myoglobin is a compact molecule, with ~80% of its polypeptide chain folded into eight stretches of α-helix. These α-helical regions, labeled A to H in Figure 3.2A, are terminated either by the presence of proline, whose five-membered ring cannot be accommodated in an α-helix (see p. 16) or by β-bends and loops stabilized by hydrogen bonds and ionic bonds (see p. 19). [Note: Ionic bonds are also termed electrostatic interactions or salt bridges.] 2. Location of polar and nonpolar amino acid residues: The interior of the globular myoglobin molecule is composed almost entirely of nonpolar amino acids. They are packed closely together, forming a structure stabilized by hydrophobic interactions between these clustered residues (see p. 19). In contrast, polar amino acids are located almost exclusively on the surface, where they can form hydrogen bonds, both with each other and with water. 3.

1	3. Binding of the heme group: The heme group of the myoglobin molecule sits in a crevice, which is lined with nonpolar amino acids. Notable exceptions are two histidine residues (see Fig. 3.2B). One, the proximal histidine (F8), binds directly to the Fe2+ of heme. The second, or distal histidine (E7), does not directly interact with the heme group but helps stabilize the binding of O2 to Fe2+ . Thus, the protein, or globin, portion of myoglobin creates a special microenvironment for the heme that permits the reversible binding of one oxygen molecule (oxygenation). The simultaneous loss of electrons by Fe2+ (oxidation to the ferric [Fe3+] form) occurs only rarely. C. Hemoglobin structure and function

1	C. Hemoglobin structure and function Hemoglobin is found exclusively in red blood cells (RBC), where its main function is to transport O2 from the lungs to the capillaries of the tissues. Hemoglobin A, the major hemoglobin in adults, is composed of four polypeptide chains (two α chains and two β chains) held together by noncovalent interactions (Fig. 3.3). Each chain (subunit) has stretches of αhelical structure and a hydrophobic heme-binding pocket similar to that described for myoglobin. However, the tetrameric hemoglobin molecule is structurally and functionally more complex than myoglobin. For example, hemoglobin can transport protons (H+) and carbon dioxide (CO2) from the tissues to the lungs and can carry four molecules of O2 from the lungs to the cells of the body. Furthermore, the oxygen-binding properties of hemoglobin are regulated by interaction with allosteric effectors (see p. 29).

1	Obtaining O2 from the atmosphere solely by diffusion greatly limits the size of organisms. Circulatory systems overcome this, but transport molecules such as hemoglobin are also required because O2 is only slightly soluble in aqueous solutions such as blood.

1	1. Quaternary structure: The hemoglobin tetramer can be envisioned as composed of two identical dimers, (αβ)1 and (αβ)2. The two polypeptide chains within each dimer are held tightly together primarily by hydrophobic interactions (Fig. 3.4). [Note: In this instance, hydrophobic amino acid residues are localized not only in the interior of the molecule but also in a region on the surface of each subunit. Multiple interchain hydrophobic interactions form strong associations between α-subunits and β-subunits in the dimers.] In contrast, the two dimers are held together primarily by polar bonds. The weaker interactions between the dimers allow them to move with respect to one other. This movement results in the two dimers occupying different relative positions in deoxyhemoglobin as compared with oxyhemoglobin (see Fig. 3.4). a.

1	a. T form: The deoxy form of hemoglobin is called the “T,” or taut (tense) form. In the T form, the two αβ dimers interact through a network of ionic bonds and hydrogen bonds that constrain the movement of the polypeptide chains. The T conformation is the low-oxygen-affinity form of hemoglobin. b. R form: The binding of O2 to hemoglobin causes the rupture of some of the polar bonds between the two αβ dimers, allowing movement. Specifically, the binding of O2 to the heme Fe2+ pulls the iron into the plane of the heme (Fig. 3.5). Because the iron is also linked to the proximal histidine (F8), the resulting movement of the globin chains alters the interface between the αβ dimers. This leads to a structure called the “R,” or relaxed form (see Fig. 3.4). The R conformation is the high-oxygen-affinity form of hemoglobin. oxygen (O2) is not bound. B. Into the plane of the heme upon O2 binding. D. Oxygen binding to myoglobin and hemoglobin

1	Myoglobin can bind only one molecule of O2, because it contains only one heme group. In contrast, hemoglobin can bind four molecules of O2, one at each of its four heme groups. The degree of saturation (Y) of these oxygen-binding sites on all myoglobin or hemoglobin molecules can vary between zero (all sites are empty) and 100% (all sites are full), as shown in Figure 3.6. [Note: Pulse oximetry is a noninvasive, indirect method of measuring the oxygen saturation of arterial blood based on differences in light absorption by oxyhemoglobin and deoxyhemoglobin.] 1. Oxygen-dissociation curve: A plot of Y measured at different partial pressures of oxygen (pO2) is called the oxygen-dissociation curve. [Note: pO2 may also be represented as PO2.] The curves for myoglobin and hemoglobin show important differences (see Fig. 3.6). This graph illustrates that myoglobin has a higher oxygen affinity at all pO2 values than does hemoglobin. The partial pressure of oxygen needed to achieve half

1	important differences (see Fig. 3.6). This graph illustrates that myoglobin has a higher oxygen affinity at all pO2 values than does hemoglobin. The partial pressure of oxygen needed to achieve half saturation of the binding sites (P50) is ~1 mm Hg for myoglobin and 26 mm Hg for hemoglobin. The higher the oxygen affinity (that is, the more tightly O2 binds), the lower the P50.

1	a. Myoglobin: The oxygen-dissociation curve for myoglobin has a hyperbolic shape (see Fig. 3.6). This reflects the fact that myoglobin reversibly binds a single molecule of O2. Thus, oxygenated (MbO2) and deoxygenated (Mb) myoglobin exist in a simple equilibrium:

1	The equilibrium is shifted to the right or to the left as O2 is added to or removed from the system. [Note: Myoglobin is designed to bind O2 released by hemoglobin at the low pO2 found in muscle. Myoglobin, in turn, releases O2 within the muscle cell in response to oxygen demand.] b. Hemoglobin: The oxygen-dissociation curve for hemoglobin is sigmoidal in shape (see Fig. 3.6), indicating that the subunits cooperate in binding O2. Cooperative binding of O2 by the four subunits of hemoglobin means that the binding of an oxygen molecule at one subunit increases the oxygen affinity of the remaining subunits in the same hemoglobin tetramer (Fig. 3.7). Although it is more difficult for the first oxygen molecule to bind to hemoglobin, the subsequent binding of oxygen molecules occurs with high affinity, as shown by the steep upward curve in the region near 20–30 mm Hg (see Fig. 3.6). E. Allosteric effectors

1	The ability of hemoglobin to reversibly bind O2 is affected by the pO2, the pH of the environment, the partial pressure of carbon dioxide (pCO2), and the availability of 2,3-bisphosphoglycerate (2,3-BPG). These are collectively called allosteric (“other site”) effectors, because their interaction at one site on the tetrameric hemoglobin molecule causes structural changes that affect the binding of O2 to the heme iron at other sites on the molecule. [Note: The binding of O2 to monomeric myoglobin is not influenced by allosteric effectors.] 1. Oxygen: The sigmoidal oxygen-dissociation curve reflects specific structural changes that are initiated at one subunit and transmitted to other subunits in the hemoglobin tetramer. The net effect of this cooperativity is that the affinity of hemoglobin for the last oxygen molecule bound is ~300 times greater than its affinity for the first oxygen molecule bound. Oxygen, then, is an allosteric effector of hemoglobin. It stabilizes the R form.

1	a. Loading and unloading oxygen: The cooperative binding of O2 allows hemoglobin to deliver more O2 to the tissues in response to relatively small changes in the pO2. This can be seen in Figure 3.6, which indicates pO2 in the alveoli of the lung and the capillaries of the tissues. For example, in the lung, oxygen concentration is high, and hemoglobin becomes virtually saturated (or “loaded”) with O2. In contrast, in the peripheral tissues, oxyhemoglobin releases (or “unloads”) much of its O2 for use in the oxidative metabolism of the tissues (Fig. 3.8).

1	b. Significance of the sigmoidal oxygen-dissociation curve: The steep slope of the oxygen-dissociation curve over the range of oxygen concentrations that occur between the lungs and the tissues permits hemoglobin to carry and deliver O2 efficiently from sites of high to sites of low pO2. A molecule with a hyperbolic oxygen-dissociation curve, such as myoglobin, could not achieve the same degree of O2 release within this range of pO2. Instead, it would have maximum affinity for O2 throughout this oxygen pressure range and, therefore, would deliver no O2 to the tissues.

1	2. Bohr effect: The release of O2 from hemoglobin is enhanced when the pH is lowered (proton concentration [H+] is increased) or when the hemoglobin is in the presence of an increased pCO2. Both result in decreased oxygen affinity of hemoglobin and, therefore, a shift to the right in the oxygen-dissociation curve (Fig. 3.9). Both, then, stabilize the T (deoxy) form. This change in oxygen binding is called the Bohr effect. Conversely, raising the pH or lowering the concentration of CO2 results in a greater oxygen affinity, a shift to the left in the oxygen-dissociation curve, and stabilization of the R (oxy) form. a. Source of the protons that lower pH: The concentration of both H+ and

1	a. Source of the protons that lower pH: The concentration of both H+ and CO2 in the capillaries of metabolically active tissues is higher than that observed in alveolar capillaries of the lungs, where CO2 is released into the expired air. In the tissues, CO2 is converted by zinc-containing carbonic anhydrase to carbonic acid: which spontaneously loses a H+, becoming bicarbonate (the major blood buffer): The H+ produced by this pair of reactions contributes to the lowering of pH. This differential pH gradient (that is, lungs having a higher pH and tissues a lower pH) favors the unloading of O2 in the peripheral tissues and the loading of O2 in the lung. Thus, the oxygen affinity of the hemoglobin molecule responds to small shifts in pH between the lungs and oxygen-consuming tissues, making hemoglobin a more efficient transporter of O2.

1	b. Mechanism of the Bohr effect: The Bohr effect reflects the fact that the deoxy form of hemoglobin has a greater affinity for H+ than does oxyhemoglobin. This is caused by ionizable groups such as specific histidine side chains that have a higher pKa (see p. 6) in deoxyhemoglobin than in oxyhemoglobin. Therefore, an increase in the concentration of H+ (resulting in a decrease in pH) causes these groups to become protonated (charged) and able to form ionic bonds (salt bridges). These bonds preferentially stabilize the deoxy form of hemoglobin, producing a decrease in oxygen affinity. [Note: Hemoglobin, then, is an important blood buffer.] The Bohr effect can be represented schematically as: where an increase in H+ (or a lower pO2) shifts the equilibrium to the right (favoring deoxyhemoglobin), whereas an increase in pO2 (or a decrease in H+) shifts the equilibrium to the left.

1	3. 2,3-BPG effect on oxygen affinity: 2,3-BPG is an important regulator of the binding of O2 to hemoglobin. It is the most abundant organic phosphate in the RBC, where its concentration is approximately that of hemoglobin. 2,3-BPG is synthesized from an intermediate of the glycolytic pathway (Fig. 3.10; see p. 101 for a discussion of 2,3-BPG synthesis in glycolysis). a. 2,3-BPG binding to deoxyhemoglobin: 2,3-BPG decreases the oxygen affinity of hemoglobin by binding to deoxyhemoglobin but not to oxyhemoglobin. This preferential binding stabilizes the T conformation of deoxyhemoglobin. The effect of binding 2,3-BPG can be represented schematically as: b.

1	2,3-BPG binding site: One molecule of 2,3-BPG binds to a pocket, formed by the two β-globin chains, in the center of the deoxyhemoglobin tetramer (Fig. 3.11). This pocket contains several positively charged amino acids that form ionic bonds with the negatively charged phosphate groups of 2,3-BPG. [Note: Replacement of one of these amino acids can result in hemoglobin variants with abnormally high oxygen affinity that may be compensated for by increased RBC production (erythrocytosis).] Oxygenation of hemoglobin narrows the pocket and causes 2,3-BPG to be released. c. Oxygen-dissociation curve shift: Hemoglobin from which 2,3-BPG has been removed has high oxygen affinity. However, as seen in the RBC, the presence of 2,3-BPG significantly reduces the oxygen affinity of hemoglobin, shifting the oxygen-dissociation curve to the right (Fig. 3.12). This reduced affinity enables hemoglobin to release O2 efficiently at the partial pressures found in the tissues.

1	d. 2,3-BPG levels in chronic hypoxia or anemia: The concentration of 2,3-BPG in the RBC increases in response to chronic hypoxia, such as that observed in chronic obstructive pulmonary disease (COPD) like emphysema, or at high altitudes, where circulating hemoglobin may have difficulty receiving sufficient O2. Intracellular levels of 2,3-BPG are also elevated in chronic anemia, in which fewer than normal RBC are available to supply the body’s oxygen needs. Elevated 2,3-BPG levels lower the oxygen affinity of hemoglobin, permitting greater unloading of O2 in the capillaries of tissues (see Fig. 3.12).

1	e. 2,3-BPG in transfused blood: 2,3-BPG is essential for the normal oxygen transport function of hemoglobin. However, storing blood in the currently available media results in the gradual depletion of 2,3BPG. Consequently, stored blood displays an abnormally high oxygen affinity and fails to unload its bound O2 properly in the tissues. Thus, hemoglobin deficient in 2,3-BPG acts as an oxygen “trap” rather than as an oxygen delivery system. Transfused RBC are able to restore their depleted supplies of 2,3-BPG in 6–24 hours. However, severely ill patients may be compromised if transfused with large quantities of such 2,3-BPG–depleted blood. Stored blood, therefore, is treated with a “rejuvenation” solution that rapidly restores 2,3-BPG. [Note: Rejuvenation also restores ATP lost during storage.] 4. CO2 binding: Most of the CO2 produced in metabolism is hydrated and transported as bicarbonate ion (see Fig. 1.12 on p. 9). However, some CO2 is carried as carbamate bound to the terminal

1	4. CO2 binding: Most of the CO2 produced in metabolism is hydrated and transported as bicarbonate ion (see Fig. 1.12 on p. 9). However, some CO2 is carried as carbamate bound to the terminal amino groups of hemoglobin (forming carbaminohemoglobin as shown in Fig. 3.8), which can be represented schematically as follows:

1	The binding of CO2 stabilizes the T, or deoxy, form of hemoglobin, resulting in a decrease in its oxygen affinity (see p. 28) and a right shift in the oxygen-dissociation curve. In the lungs, CO2 dissociates from the hemoglobin and is released in the breath.

1	5. CO binding: Carbon monoxide (CO) binds tightly (but reversibly) to the hemoglobin iron, forming carboxyhemoglobin. When CO binds to one or more of the four heme sites, hemoglobin shifts to the R conformation, causing the remaining heme sites to bind O2 with high affinity. This shifts the oxygen-dissociation curve to the left and changes the normal sigmoidal shape toward a hyperbola. As a result, the affected hemoglobin is unable to release O2 to the tissues (Fig. 3.13). [Note: The affinity of hemoglobin for CO is 220 times greater than for O2. Consequently, even minute concentrations of CO in the environment can produce toxic concentrations of carboxyhemoglobin in the blood. For example, increased levels of CO are found in the blood of tobacco smokers. CO toxicity appears to result from a combination of tissue hypoxia and direct CO-mediated damage at the cellular level.] CO poisoning is treated with 100% O2 at high pressure (hyperbaric oxygen therapy), which facilitates the

1	from a combination of tissue hypoxia and direct CO-mediated damage at the cellular level.] CO poisoning is treated with 100% O2 at high pressure (hyperbaric oxygen therapy), which facilitates the dissociation of CO from the hemoglobin. [Note: CO inhibits Complex IV of the electron transport chain (see p. 76).] In addition to O2, CO2, and CO, nitric oxide gas (NO) also is carried by hemoglobin. NO is a potent vasodilator (see p. 151). It can be taken up (salvaged) or released from RBC, thereby modulating NO availability and influencing vessel diameter.

1	F. Minor hemoglobins It is important to remember that human hemoglobin A (HbA) is just one member of a functionally and structurally related family of proteins, the hemoglobins (Fig. 3.14). Each of these oxygen-carrying proteins is a tetramer, composed of two α-globin (or α-like) polypeptides and two βglobin (or β-like) polypeptides. Certain hemoglobins, such as HbF, are normally synthesized only during fetal development, whereas others, such as HbA2, are synthesized in the adult, although at low levels compared with HbA. HbA can also become modified by the covalent addition of a hexose (see 3. below). 1. Fetal hemoglobin: HbF is a tetramer consisting of two α chains identical to those found in HbA, plus two γ chains (α2γ2; see Fig. 3.14). The γ chains are members of the β-globin gene family (see p. 34).

1	a. HbF synthesis during development: In the first month after conception, embryonic hemoglobins such as Hb Gower 1, composed of two α-like zeta (ζ) chains and two β-like epsilon (ε) chains (ζ2ε2), are synthesized by the embryonic yolk sac. In the fifth week of gestation, the site of globin synthesis shifts, first to the liver and then to the marrow, and the primary product is HbF. HbF is the major hemoglobin found in the fetus and newborn, accounting for ~60% of the total hemoglobin in the RBC during the last months of fetal life (Fig. 3.15). HbA synthesis starts in the bone marrow at about the eighth month of pregnancy and gradually replaces HbF. Figure 3.15 shows the relative production of each type of hemoglobin chain during fetal and postnatal life. [Note: HbF represents <2% of the hemoglobin in most adults and is concentrated in RBC known as F cells.] b. 2,3-BPG binding to HbF: Under physiologic conditions, HbF has a higher oxygen affinity than does HbA as a result of HbF only

1	hemoglobin in most adults and is concentrated in RBC known as F cells.] b. 2,3-BPG binding to HbF: Under physiologic conditions, HbF has a higher oxygen affinity than does HbA as a result of HbF only weakly binding 2,3-BPG. [Note: The γ-globin chains of HbF lack some of the positively charged amino acids that are responsible for binding 2,3BPG in the β-globin chains.] Because 2,3-BPG serves to reduce the oxygen affinity of hemoglobin, the weaker interaction between 2,3BPG and HbF results in a higher oxygen affinity for HbF relative to HbA. In contrast, if both HbA and HbF are stripped of their 2,3-BPG, they then have a similar oxygen affinity. The higher oxygen affinity of HbF facilitates the transfer of O2 from the maternal circulation across the placenta to the RBC of the fetus.

1	2. Hemoglobin A2: HbA2 is a minor component of normal adult hemoglobin, first appearing shortly before birth and, ultimately, constituting ~2% of the total hemoglobin. It is composed of two α-globin chains and two δ-globin chains (α2δ2; see Fig. 3.14). 3. Hemoglobin A1c: Under physiologic conditions, HbA is slowly glycated (nonenzymically condensed with a hexose), the extent of glycation being dependent on the plasma concentration of the hexose. The most abundant form of glycated hemoglobin is HbA1c. It has glucose residues attached predominantly to the amino groups of the N-terminal valines of the βglobin chains (Fig. 3.16). Increased amounts of HbA1c are found in RBC of patients with diabetes mellitus, because their HbA has contact with higher glucose concentrations during the 120-day lifetime of these cells. (See p. 340 for a discussion of the use of HbA1c levels in assessing average blood glucose levels in patients with diabetes.) III. GLOBIN GENE ORGANIZATION

1	III. GLOBIN GENE ORGANIZATION To understand diseases resulting from genetic alterations in the structure or synthesis of hemoglobin, it is necessary to grasp how the hemoglobin genes, which direct the synthesis of the different globin chains, are structurally organized into gene families and also how they are expressed. A. α-Gene family The genes coding for the α-globin and β-globin subunits of the hemoglobin chains occur in two separate gene clusters (or families) located on two different chromosomes (Fig. 3.17). The α-gene cluster on chromosome 16 contains two genes for the α-globin chains. It also contains the ζ gene that is expressed early in development as an α-globin-like component of embryonic hemoglobin. [Note: Globin gene families also contain globinlike genes that are not expressed, that is, their genetic information is not used to produce globin chains. These are called pseudogenes.] B. β-Gene family

1	B. β-Gene family A single gene for the β-globin chain is located on chromosome 11 (see Fig. 3.17). There are an additional four β-globin-like genes: the ε gene (which, like the ζ gene, is expressed early in embryonic development), two γ genes (Gγ and Aγ that are expressed in HbF), and the δ gene that codes for the globin chain found in the minor adult hemoglobin HbA2. C. Steps in globin chain synthesis

1	C. Steps in globin chain synthesis Expression of a globin gene begins in the nucleus of RBC precursors, where the DNA sequence encoding the gene is transcribed. The ribonucleic acid (RNA) produced by transcription is actually a precursor of the messenger RNA (mRNA) that is used as a template for the synthesis of a globin chain. Before it can serve this function, two noncoding stretches of RNA (introns) must be removed from the mRNA precursor sequence and the remaining three fragments (exons) joined in a linear manner. The resulting mature mRNA enters the cytosol, where its genetic information is translated, producing a globin chain. (A summary of this process is shown in Figure 3.18. A more detailed description of gene expression is presented in Unit VII, Chapters 30–32.) IV. HEMOGLOBINOPATHIES

1	IV. HEMOGLOBINOPATHIES Hemoglobinopathies are defined as a group of genetic disorders caused by production of a structurally abnormal hemoglobin molecule, synthesis of insufficient quantities of normal hemoglobin, or, rarely, both. Sickle cell anemia (HbS), hemoglobin C disease (HbC), hemoglobin SC disease (HbS + HbC = HbSC), and the thalassemias are representative hemoglobinopathies that can have severe clinical consequences. The first three conditions result from production of hemoglobin with an altered amino acid sequence (qualitative hemoglobinopathy), whereas the thalassemias are caused by decreased production of normal hemoglobin (quantitative hemoglobinopathy). A. Sickle cell anemia (hemoglobin S disease)

1	Sickle cell anemia, the most common of the RBC sickling diseases, is a genetic disorder caused by a single nucleotide substitution (a point mutation, see p. 449) in the gene for β-globin. It is the most common inherited blood disorder in the United States, affecting 50,000 Americans. It occurs primarily in the African American population, affecting 1 in 500 newborn African American infants. Sickle cell anemia is an autosomalrecessive disorder. It occurs in individuals who have inherited two mutant genes (one from each parent) that code for synthesis of the β chains of the globin molecules. [Note: The mutant β-globin chain is designated βS, and the resulting hemoglobin, α2βS2, is referred to as HbS.] An infant does not begin showing symptoms of the disease until sufficient HbF has been replaced by HbS so that sickling can occur (see p. 36). Sickle cell anemia is characterized by lifelong episodes of pain (“crises”), chronic hemolytic anemia with associated hyperbilirubinemia (see p.

1	by HbS so that sickling can occur (see p. 36). Sickle cell anemia is characterized by lifelong episodes of pain (“crises”), chronic hemolytic anemia with associated hyperbilirubinemia (see p. 284), and increased susceptibility to infections, usually beginning in infancy. [Note: The lifetime of a RBC in sickle cell anemia is <20 days, compared with 120 days for normal RBC, hence, the anemia.] Other symptoms include acute chest syndrome, stroke, splenic and renal dysfunction, and bone changes due to marrow hyperplasia. Life expectancy is reduced. Heterozygotes, representing 1 in 12 African Americans, have one normal and one sickle cell gene. The blood cells of such heterozygotes contain both HbS and HbA, and these individuals have sickle cell trait. They usually do not show clinical symptoms (but may under conditions of extreme physical exertion with dehydration) and can have a normal life span.

1	1. Amino acid substitution in HbS β chains:: A molecule of HbS contains two normal α-globin chains and two mutant β-globin chains (βS), in which glutamate at position six has been replaced with valine (Fig. 3.19). Therefore, during electrophoresis at alkaline pH, HbS migrates more slowly toward the anode (positive electrode) than does HbA (Fig. 3.20). This altered mobility of HbS is a result of the absence of the negatively charged glutamate residues in the two β chains, thereby rendering HbS less negative than HbA. [Note: Electrophoresis of hemoglobin obtained from lysed RBC is routinely used in the diagnosis of sickle cell trait and sickle cell anemia (or, sickle cell disease). DNA analysis also is used (see p. 493).] 2.

1	Sickling and tissue anoxia: The replacement of the charged glutamate with the nonpolar valine forms a protrusion on the β chain that fits into a complementary site on the β chain of another hemoglobin molecule in the cell (Fig. 3.21). At low oxygen tension, deoxyhemoglobin S polymerizes inside the RBC, forming a network of insoluble fibrous polymers that stiffen and distort the cell, producing rigid, misshapen RBC. Such sickled cells frequently block the flow of blood in the narrow capillaries. This interruption in the supply of O2 leads to localized anoxia (oxygen deprivation) in the tissue, causing pain and eventually ischemic death (infarction) of cells in the vicinity of the blockage. The anoxia also leads to an increase in deoxygenated HbS. [Note: The mean diameter of RBC is 7.5 µm, whereas that of the microvasculature is 3–4 µm. Compared to normal RBC, sickled cells have a decreased ability to deform and an increased tendency to adhere to vessel walls. This makes moving through

1	whereas that of the microvasculature is 3–4 µm. Compared to normal RBC, sickled cells have a decreased ability to deform and an increased tendency to adhere to vessel walls. This makes moving through small vessels difficult, thereby causing microvascular occlusion.] 3.

1	Variables that increase sickling: The extent of sickling and, therefore, the severity of disease are enhanced by any variable that increases the proportion of HbS in the deoxy state (that is, reduces the oxygen affinity of HbS). These variables include decreased pO2, increased pCO2, decreased pH, dehydration, and an increased concentration of 2,3-BPG in RBC. 4.

1	4. Treatment: Therapy involves adequate hydration, analgesics, aggressive antibiotic therapy if infection is present, and transfusions in patients at high risk for fatal occlusion of blood vessels. Intermittent transfusions with packed RBC reduce the risk of stroke, but the benefits must be weighed against the complications of transfusion, which include iron overload that can result in hemosiderosis (see p. 404), bloodborne infections, and immunologic complications. Hydroxyurea (hydroxycarbamide), an antitumor drug, is therapeutically useful because it increases circulating levels of HbF, which decreases RBC sickling. This leads to decreased frequency of painful crises and reduces mortality. Stem cell transplantation is possible. [Note: The morbidity and mortality associated with sickle cell anemia has led to its inclusion in newborn screening panels to allow prophylactic antibiotic therapy to begin soon after the birth of an affected child.] 5.

1	Possible selective advantage of the heterozygous state: The high frequency of the βS mutation among black Africans, despite its damaging effects in the homozygous state, suggests that a selective advantage exists for heterozygous individuals. For example, heterozygotes for the sickle cell gene are less susceptible to the severe malaria caused by the parasite Plasmodium falciparum. This organism spends an obligatory part of its life cycle in the RBC. One theory is that because these cells in individuals heterozygous for HbS, like those in homozygotes, have a shorter life span than normal, the parasite cannot complete the intracellular stage of its development. This may provide a selective advantage to heterozygotes living in regions where malaria is a major cause of death. For example, in Africa, the geographic distribution of sickle cell anemia is similar to that of malaria. B. Hemoglobin C disease

1	B. Hemoglobin C disease Like HbS, HbC is a hemoglobin variant that has a single amino acid substitution in the sixth position of the β-globin chain (see Fig. 3.19). In HbC, however, a lysine is substituted for the glutamate (as compared with a valine substitution in HbS). [Note: This substitution causes HbC to move more slowly toward the anode than HbA or HbS does (see Fig. 3.20).] Rare patients homozygous for HbC generally have a relatively mild, chronic hemolytic anemia. They do not suffer from infarctive crises, and no specific therapy is required. C. Hemoglobin SC disease

1	C. Hemoglobin SC disease HbSC disease is another of the RBC sickling diseases. In this disease, some β-globin chains have the sickle cell mutation, whereas other β-globin chains carry the mutation found in HbC disease. [Note: Patients with HbSC disease are doubly heterozygous. They are called compound heterozygotes because both of their β-globin genes are abnormal, although different from each other.] Hemoglobin levels tend to be higher in HbSC disease than in sickle cell anemia and may even be at the low end of the normal range. The clinical course of adults with HbSC anemia differs from that of sickle cell anemia in that symptoms such as painful crises are less frequent and less severe. However, there is significant clinical variability. D. Methemoglobinemias

1	Oxidation of the heme iron in hemoglobin from Fe2+ to Fe3+ produces methemoglobin, which cannot bind O2. This oxidation may be acquired and caused by the action of certain drugs, such as nitrates, or endogenous products such as reactive oxygen species (see p. 148). The oxidation may also result from congenital defects, for example, a deficiency of NADH-cytochrome b5 reductase (also called NADH-methemoglobin reductase), the enzyme responsible for the conversion of methemoglobin (Fe3+) to hemoglobin (Fe2+), leads to the accumulation of methemoglobin (Fig. 3.22). [Note: The RBC of newborns have approximately half the capacity of those of adults to reduce methemoglobin.] Additionally, rare mutations in the α-or β-globin chain can cause the production of HbM, an abnormal hemoglobin that is resistant to the reductase. The methemoglobinemias are characterized by “chocolate cyanosis” (a blue coloration of the skin and mucous membranes and brown-colored blood) as a result of the dark-colored

1	to the reductase. The methemoglobinemias are characterized by “chocolate cyanosis” (a blue coloration of the skin and mucous membranes and brown-colored blood) as a result of the dark-colored methemoglobin. Symptoms are related to the degree of tissue hypoxia and include anxiety, headache, and dyspnea. In rare cases, coma and death can occur. Treatment is with methylene blue, which is oxidized as Fe3+ is reduced.

1	E. Thalassemias The thalassemias are hereditary hemolytic diseases in which an imbalance occurs in the synthesis of globin chains. As a group, they are the most common single-gene disorders in humans. Normally, synthesis of the αand β-globin chains is coordinated, so that each α-globin chain has a βglobin chain partner. This leads to the formation of α2β2 (HbA). In the thalassemias, the synthesis of either the α-or the β-globin chain is defective, and hemoglobin concentration is reduced. A thalassemia can be caused by a variety of mutations, including entire gene deletions, or substitutions or deletions of one of many nucleotides in the DNA. [Note: Each thalassemia can be classified as either a disorder in which no globin chains are produced (α0-or β0-thalassemia), or one in which some chains are synthesized but at a reduced level (α+-or β+-thalassemia).] 1.

1	β-Thalassemias: In these disorders, synthesis of β-globin chains is decreased or absent, typically as a result of point mutations that affect the production of functional mRNA. However, α-globin chain synthesis is normal. Excess α-globin chains cannot form stable tetramers and so precipitate, causing the premature death of cells initially destined to become mature RBC. Increase in α2δ2 (HbA2) and α2γ2 (HbF) also occurs. There are only two copies of the β-globin gene in each cell (one on each chromosome 11). Therefore, individuals with β-globin gene defects have either β-thalassemia trait (β-thalassemia minor) if they have only one defective β-globin gene or β-thalassemia major (Cooley anemia) if both genes are defective (Fig. 3.23). Because the β-globin gene is not expressed until late in prenatal development, the physical manifestations of β-thalassemias appear only several months after birth. Those individuals with β-thalassemia minor make some β chains and usually do not require

1	in prenatal development, the physical manifestations of β-thalassemias appear only several months after birth. Those individuals with β-thalassemia minor make some β chains and usually do not require specific treatment. However, those infants born with β-thalassemia major are seemingly healthy at birth but become severely anemic, usually during the first or second year of life, due to ineffective erythropoiesis. Skeletal changes as a result of extramedullary hematopoiesis also are seen. These patients require regular transfusions of blood. [Note: Although this treatment is lifesaving, the cumulative effect of the transfusions is iron overload. Use of iron chelation therapy has improved morbidity and mortality.] The only curative option available is hematopoietic stem cell transplantation.

1	2. α-Thalassemias: In these disorders, synthesis of α-globin chains is decreased or absent, typically as a result of deletional mutations. Because each individual’s genome contains four copies of the α-globin gene (two on each chromosome 16), there are several levels of α-globin chain deficiencies (Fig. 3.24). If one of the four genes is defective, the individual is termed a “silent” carrier of α-thalassemia, because no physical manifestations of the disease occur. If two α-globin genes are defective, the individual is designated as having α-thalassemia trait. If three α-globin genes are defective, the individual has hemoglobin H (β4) disease, a hemolytic anemia of variable severity. If all four α-globin genes are defective, hemoglobin Bart (γ4) disease with hydrops fetalis and fetal death results, because α-globin chains are required for the synthesis of HbF. [Note: Heterozygote advantage against malaria is seen in both α-and β-thalassemias.] V. CHAPTER SUMMARY

1	Hemoglobin A (HbA), the major hemoglobin in adults, is composed of four polypeptide chains (two α chains and two β chains, α2β2) held together by noncovalent interactions (Fig. 3.25). The subunits occupy different relative positions in deoxyhemoglobin compared with oxyhemoglobin. The deoxy form of Hb is called the “T,” or taut (tense), conformation. It has a constrained structure that limits the movement of the polypeptide chains. The T form is the low-oxygen-affinity form of Hb. The binding of oxygen (O2) to the heme iron causes rupture of some of the ionic and hydrogen bonds and movement of the dimers. This leads to a structure called the “R,” or relaxed, conformation. The R form is the high-oxygen-affinity form of Hb. The oxygen-dissociation curve for Hb is sigmoidal in shape (in contrast to that of myoglobin, which is hyperbolic), indicating that the subunits cooperate in binding O2. The binding of an oxygen molecule at one heme group increases the oxygen affinity of the remaining

1	to that of myoglobin, which is hyperbolic), indicating that the subunits cooperate in binding O2. The binding of an oxygen molecule at one heme group increases the oxygen affinity of the remaining heme groups in the same Hb molecule (cooperativity). Hb’s ability to bind O2 reversibly is affected by the partial pressure of oxygen (pO2), the pH of the environment, the partial pressure of carbon dioxide (pCO2), and the availability of 2,3-bisphosphoglycerate (2,3-BPG). For example, the release of O2 from Hb is enhanced when the pH is lowered or the pCO2 is increased (the Bohr effect), such as in exercising muscle, and the oxygen-dissociation curve of Hb is shifted to the right. To cope long-term with the effects of chronic hypoxia or anemia, the concentration of 2,3-BPG in red blood cells increases. 2,3-BPG binds to the Hb and decreases its oxygen affinity. It therefore also shifts the oxygen-dissociation curve to the right. Fetal hemoglobin (HbF) binds 2,3-BPG less tightly than does HbA

1	2,3-BPG binds to the Hb and decreases its oxygen affinity. It therefore also shifts the oxygen-dissociation curve to the right. Fetal hemoglobin (HbF) binds 2,3-BPG less tightly than does HbA and has a higher oxygen affinity. Carbon monoxide (CO) binds tightly (but reversibly) to the Hb iron, forming carboxyhemoglobin. Hemoglobinopathies are disorders primarily caused either by production of a structurally abnormal Hb molecule as in sickle cell anemia or synthesis of insufficient quantities of normal Hb subunits as in the thalassemias (Fig. 3.26).

1	Choose the ONE best answer. .1. Which one of the following statements concerning the hemoglobins is correct? A. HbA is the most abundant hemoglobin in normal adults. B. Fetal blood has a lower affinity for oxygen than does adult blood because HbF has an increased affinity for 2,3-bisphosphoglycerate. C. The globin chain composition of HbF is α2δ2. D. HbA1c differs from HbA by a single, genetically determined amino acid substitution. E. HbA2 appears early in fetal life.

1	C. The globin chain composition of HbF is α2δ2. D. HbA1c differs from HbA by a single, genetically determined amino acid substitution. E. HbA2 appears early in fetal life. Correct answer = A. HbA accounts for over 90% of the hemoglobin in a normal adult. If HbA1c is included, the percentage rises to ~97%. Because 2,3bisphosphoglycerate (2,3-BPG) reduces the affinity of hemoglobin for oxygen, the weaker interaction between 2,3-BPG and HbF results in a higher oxygen affinity for HbF relative to HbA. HbF consists of α2γ2. HbA1c is a glycated form of HbA, formed nonenzymically in red blood cells. HbA2 is a minor component of normal adult hemoglobin, first appearing shortly before birth and rising to adult levels (~2% of the total hemoglobin) by age 6 months. .2. Which one of the following statements concerning the ability of acidosis to precipitate a crisis in sickle cell anemia is correct? A. Acidosis decreases the solubility of HbS.

1	.2. Which one of the following statements concerning the ability of acidosis to precipitate a crisis in sickle cell anemia is correct? A. Acidosis decreases the solubility of HbS. B. Acidosis increases the oxygen affinity of hemoglobin. C. Acidosis favors the conversion of hemoglobin from the taut to the relaxed conformation. D. Acidosis shifts the oxygen-dissociation curve to the left. E. Acidosis decreases the ability of 2,3-bisphosphoglycerate to bind to hemoglobin. Correct answer = A. HbS is significantly less soluble in the deoxygenated form, compared with oxyhemoglobin S. Decreased pH (acidosis) causes the oxygen-dissociation curve to shift to the right, indicating decreased oxygen affinity (increased delivery). This favors the formation of the deoxy, or taut, form of hemoglobin and can precipitate a sickle cell crisis. The binding of 2,3bisphosphoglycerate is increased, because it binds only to the deoxy form of hemoglobin.

1	.3. Which one of the following statements concerning the binding of oxygen by hemoglobin is correct? A. The Bohr effect results in a lower oxygen affinity at higher pH values. B. Carbon dioxide increases the oxygen affinity of hemoglobin by binding to the C-terminal groups of the polypeptide chains. C. The oxygen affinity of hemoglobin increases as the percentage saturation increases. D. The hemoglobin tetramer binds four molecules of 2,3bisphosphoglycerate. E. Oxyhemoglobin and deoxyhemoglobin have the same affinity for protons.

1	D. The hemoglobin tetramer binds four molecules of 2,3bisphosphoglycerate. E. Oxyhemoglobin and deoxyhemoglobin have the same affinity for protons. Correct answer = C. The binding of oxygen at one heme group increases the oxygen affinity of the remaining heme groups in the same molecule. A rise in pH results in increased oxygen affinity. Carbon dioxide decreases oxygen affinity because it lowers the pH. Moreover, binding of carbon dioxide to the N-termini stabilizes the taut, deoxy form. Hemoglobin binds one molecule of 2,3-bisphosphoglycerate. Deoxyhemoglobin has a greater affinity for protons than does oxyhemoglobin. .4. β-Lysine 82 in HbA is important for the binding of 2,3-bisphosphoglycerate. In Hb Helsinki, this amino acid has been replaced by methionine. Which of the following should be true concerning Hb Helsinki? A. It should be stabilized in the taut, rather than the relaxed, form.

1	A. It should be stabilized in the taut, rather than the relaxed, form. B. It should have increased oxygen affinity and, consequently, decreased oxygen delivery to tissues. C. Its oxygen-dissociation curve should be shifted to the right relative to HbA. D. It results in anemia. Correct answer = B. Substitution of lysine by methionine decreases the ability of negatively charged phosphate groups in 2,3-bisphosphoglycerate (2,3-BPG) to bind the β subunits of hemoglobin. Because 2,3-BPG decreases the oxygen affinity of hemoglobin, a reduction in 2,3-BPG should result in increased oxygen affinity and decreased oxygen (O2) delivery to tissues. The relaxed form is the high-oxygen-affinity form of hemoglobin. Increased oxygen affinity (decreased delivery) results in a left shift in the oxygen-dissociation curve. Decreased delivery of O2 is compensated for by increased RBC production.

1	.5. A 67-year-old man presented to the emergency department with a 1-week history of angina and shortness of breath. He complained that his face and extremities had taken on a blue color. His medical history included chronic stable angina treated with isosorbide dinitrate and nitroglycerin. Blood obtained for analysis was brown. Which one of the following is the most likely diagnosis? A. Carboxyhemoglobinemia B. Hemoglobin SC disease C. Methemoglobinemia D. Sickle cell anemia E. β-Thalassemia

1	A. Carboxyhemoglobinemia B. Hemoglobin SC disease C. Methemoglobinemia D. Sickle cell anemia E. β-Thalassemia Correct answer = C. Oxidation of the ferrous (Fe2+) iron to the ferric (Fe3+) state in the heme prosthetic group of hemoglobin forms methemoglobin. This may be caused by the action of certain drugs such as nitrates. The methemoglobinemias are characterized by chocolate cyanosis (a blue coloration of the skin and mucous membranes and chocolate-colored blood) as a result of the dark-colored methemoglobin. Symptoms are related to tissue hypoxia and include anxiety, headache, and dyspnea. In rare cases, coma and death can occur. [Note: Benzocaine, an aromatic amine used as a topical anesthetic, is a cause of acquired methemoglobinemia.] .6. Why is hemoglobin C disease a nonsickling disease? In HbC, the polar glutamate is replaced by polar lysine rather than by nonpolar valine as in HbS.

1	In HbC, the polar glutamate is replaced by polar lysine rather than by nonpolar valine as in HbS. .7. What would be true about the extent of red blood cell sickling in individuals with HbS and hereditary persistence of HbF? It would be decreased because HbF reduces HbS concentration. It also inhibits polymerization of deoxy HbS. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW Collagen and elastin are examples of common, well-characterized fibrous proteins of the extracellular matrix (ECM) that serve structural functions in the body. For example, collagen and elastin are found as components of skin, connective tissue, blood vessel walls, and the sclera and cornea of the eye. Each fibrous protein exhibits special mechanical properties, resulting from its unique structure, which is obtained by combining specific amino acids into regular, secondary structural elements. This is in contrast to globular proteins (discussed in Chapter 3), whose shapes are the result of complex interactions between secondary, tertiary, and, sometimes, quaternary structural elements. II. COLLAGEN

1	II. COLLAGEN Collagen is the most abundant protein in the human body. A typical collagen molecule is a long, rigid structure in which three polypeptides (referred to as α chains) are wound around one another in a rope-like triple helix (Fig. 4.1). Although these molecules are found throughout the body, their types and organization are dictated by the structural role collagen plays in a particular organ. In some tissues, collagen may be dispersed as a gel that gives support to the structure, as in the ECM or the vitreous humor of the eye. In other tissues, collagen may be bundled in tight, parallel fibers that provide great strength, as in tendons. In the cornea of the eye, collagen is stacked so as to transmit light with a minimum of scattering. Collagen of bone occurs as fibers arranged at an angle to each other so as to resist mechanical shear from any direction. A. Types

1	A. Types The collagen superfamily of proteins includes >25 collagen types as well as additional proteins that have collagen-like domains. The three polypeptide α chains are held together by interchain hydrogen bonds. Variations in the amino acid sequence of the α chains result in structural components that are about the same size (~1,000 amino acids long) but with slightly different properties. These α chains are combined to form the various types of collagen found in the tissues. For example, the most common collagen, type I, contains two chains called α1 and one chain called α2 (α12α2), whereas type II collagen contains three α1 chains (α13). The collagens can be organized into three groups, based on their location and functions in the body (Fig. 4.2). 1.

1	1. Fibril-forming collagens: Types I, II, and III are the fibrillar collagens and have the rope-like structure described above for a typical collagen molecule. In the electron microscope, these linear polymers of fibrils have characteristic banding patterns, reflecting the regular staggered packing of the individual collagen molecules in the fibril (Fig. 4.3). Type I collagen fibers (composed of collagen fibrils) are found in supporting elements of high tensile strength (for example, tendons and corneas), whereas fibers formed from type II collagen molecules are restricted to cartilaginous structures. The fibers derived from type III collagen are prevalent in more distensible tissues such as blood vessels. 2. Network-forming collagens: Types IV and VIII form a three-dimensional mesh, rather than distinct fibrils (Fig. 4.4). For example, type IV molecules assemble into a sheet or meshwork that constitutes a major part of basement membranes.

1	Basement membranes are thin, sheet-like structures that provide mechanical support for adjacent cells and function as a semipermeable filtration barrier to macromolecules in organs such as the kidney and the lung. 3. Fibril-associated collagens: Types IX and XII bind to the surface of collagen fibrils, linking these fibrils to one another and to other components in the ECM (see Fig. 4.2). B. Structure Unlike most globular proteins that are folded into compact structures, collagen, a fibrous protein, has an elongated, triple-helical structure that is stabilized by interchain hydrogen bonds. 1.

1	1. Amino acid sequence: Collagen is rich in proline and glycine, both of which are important in the formation of the triple-stranded helix. Proline facilitates the formation of the helical conformation of each α chain because its ring structure causes “kinks” in the peptide chain. [Note: The presence of proline dictates that the helical conformation of the α chain cannot be an α helix (see p. 16).] Glycine, the smallest amino acid, is found in every third position of the polypeptide chain. It fits into the restricted spaces where the three chains of the helix come together. The glycine residues are part of a repeating sequence, −Gly–X–Y–, where X is frequently proline, and Y is often hydroxyproline (but can be hydroxylysine, Fig. 4.5). Thus, most of the α chain can be regarded as a polytripeptide whose sequence can be represented as (−Gly–Pro– Hyp–)333. 2.

1	2. Hydroxyproline and hydroxylysine: Collagen contains hydroxyproline and hydroxylysine, which are nonstandard amino acids (see p. 1) not present in most other proteins. They result from the hydroxylation of some of the proline and lysine residues after their incorporation into polypeptide chains (Fig. 4.6). Therefore, the hydroxylation is a posttranslational modification (see p. 460). [Note: Generation of hydroxyproline maximizes formation of interchain hydrogen bonds that stabilize the triple-helical structure.] 3. Glycosylation: The hydroxyl group of the hydroxylysine residues of collagen may be enzymatically glycosylated. Most commonly, glucose and galactose are sequentially attached to the polypeptide chain prior to triple-helix formation (Fig. 4.7). C. Biosynthesis

1	C. Biosynthesis The polypeptide precursors of the collagen molecule are synthesized in fibroblasts (or in the related osteoblasts of bone and chondroblasts of cartilage). They are enzymically modified and form the triple helix, which gets secreted into the ECM. After additional enzymic modification, the mature extracellular collagen fibrils aggregate and become cross-linked to form collagen fibers. 1.

1	1. Pro-α chain formation: Collagen is one of many proteins that normally function outside of cells. Like most proteins produced for export, the newly synthesized polypeptide precursors of α chains (prepro-α chains) contain a special amino acid sequence at their amino (N)-terminal ends. This sequence acts as a signal that, in the absence of additional signals, targets the polypeptide being synthesized for secretion from the cell. The signal sequence facilitates the binding of ribosomes to the rough endoplasmic reticulum (RER) and directs the passage of the prepro-α chain into the lumen of the RER. The signal sequence is rapidly cleaved in the lumen to yield a precursor of collagen called a pro-α chain (see Fig. 4.7). 2.

1	Hydroxylation: The pro-α chains are processed by a number of enzymic steps within the lumen of the RER while the polypeptides are still being synthesized (see Fig. 4.7). Proline and lysine residues found in the Y-position of the –Gly–X–Y– sequence can be hydroxylated to form hydroxyproline and hydroxylysine residues. These hydroxylation reactions require molecular oxygen, ferrous iron (Fe2+), and the reducing agent vitamin C (ascorbic acid, see p. 381), without which the hydroxylating enzymes, prolyl hydroxylase and lysyl hydroxylase, are unable to function (see Fig. 4.6). In the case of ascorbic acid deficiency (and, therefore, a lack of proline and lysine hydroxylation), interchain H-bond formation is impaired, as is formation of a stable triple helix. Additionally, collagen fibrils cannot be cross-linked (see 7. below), greatly decreasing the tensile strength of the assembled fiber. The resulting deficiency disease is known as scurvy. Patients with scurvy often show ecchymoses

1	cannot be cross-linked (see 7. below), greatly decreasing the tensile strength of the assembled fiber. The resulting deficiency disease is known as scurvy. Patients with scurvy often show ecchymoses (bruise-like discolorations) on the limbs as a result of subcutaneous extravasation (leakage) of blood due to capillary fragility (Fig. 4.8).

1	3. Glycosylation: Some hydroxylysine residues are modified by glycosylation with glucose or glucosyl-galactose (see Fig. 4.7). 4. Assembly and secretion: After hydroxylation and glycosylation, three pro-α chains form procollagen, a precursor of collagen that has a central region of triple helix flanked by the nonhelical N-and carboxyl (C)terminal extensions called propeptides (see Fig. 4.7). The formation of procollagen begins with formation of interchain disulfide bonds between the C-terminal extensions of the pro-α chains. This brings the three α chains into an alignment favorable for triple helix formation. The procollagen molecules move through the Golgi apparatus, where they are packaged in secretory vesicles. The vesicles fuse with the cell membrane, causing the release of procollagen molecules into the extracellular space. 5.

1	5. Extracellular cleavage of procollagen molecules: After their release, the triple-helical procollagen molecules are cleaved by N-and Cprocollagen peptidases, which remove the terminal propeptides, producing tropocollagen molecules. 6. Collagen fibril formation: Tropocollagen molecules spontaneously associate to form collagen fibrils. They form an ordered, parallel array, with adjacent collagen molecules arranged in a staggered pattern, each overlapping its neighbor by a length approximately three quarters of a molecule (see Fig. 4.7). 7.

1	7. Cross-link formation: The fibrillar array of collagen molecules serves as a substrate for lysyl oxidase. This copper-containing extracellular enzyme oxidatively deaminates some of the lysine and hydroxylysine residues in collagen. The reactive aldehydes that result (allysine and hydroxyallysine) can spontaneously condense with lysine or hydroxylysine residues in neighboring collagen molecules to form covalent cross-links and, thus, mature collagen fibers (Fig. 4.9). [Note: Cross-links can form between two allysine residues.] peroxide. Lysyl oxidase is one of several copper-containing enzymes. Others include ceruloplasmin (see p. 404), cytochrome c oxidase (see p. 76), dopamine hydroxylase (see p. 286), superoxide dismutase (see p. 148), and tyrosinase (see p. 273). Disruption in copper homeostasis causes copper deficiency (X-linked Menkes syndrome) or overload (Wilson disease) (see p. 402). D. Degradation

1	D. Degradation Normal collagens are highly stable molecules, having half-lives as long as several years. However, connective tissue is dynamic and is constantly being remodeled, often in response to growth or injury of the tissue. Breakdown of collagen fibers is dependent on the proteolytic action of collagenases, which are part of a large family of matrix metalloproteinases. For type I collagen, the cleavage site is specific, generating three-quarter and one-quarter length fragments. These fragments are further degraded by other matrix proteinases. E. Collagenopathies

1	E. Collagenopathies Defects in any one of the many steps in collagen fiber synthesis can result in a genetic disease involving an inability of collagen to form fibers properly and, therefore, an inability to provide tissues with the needed tensile strength normally provided by collagen. More than 1,000 mutations have been identified in 23 genes coding for 13 of the collagen types. The following are examples of diseases (collagenopathies) that are the result of defective collagen synthesis.

1	1. Ehlers-Danlos syndrome: Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective tissue disorders that result from heritable defects in the metabolism of fibrillar collagen molecules. EDS can be caused by a deficiency of collagen-processing enzymes (for example, lysyl hydroxylase or N-procollagen peptidase) or from mutations in the amino acid sequences of collagen types I, III, and V. The classic form of EDS, caused by defects in type V collagen, is characterized by skin extensibility and fragility and joint hypermobility (Fig. 4.10). The vascular form, due to defects in type III collagen, is the most serious form of EDS because it is associated with potentially lethal arterial rupture. [Note: The classic and vascular forms show autosomaldominant inheritance.] Collagen that contains mutant chains may have altered structure, secretion, or distribution, and it frequently is degraded. [Note: Incorporation of just one mutant chain may result in degradation of the triple

1	contains mutant chains may have altered structure, secretion, or distribution, and it frequently is degraded. [Note: Incorporation of just one mutant chain may result in degradation of the triple helix. This is known as a dominant-negative effect.] 2. Osteogenesis imperfecta: This syndrome, known as “brittle bone disease,” is a genetic disorder of bone fragility characterized by bones that fracture easily, with minor or no trauma (Fig. 4.11). Over 80% of cases of osteogenesis imperfecta (OI) are caused by dominant mutations to the genes that encode the α1 or α2 chains in type I collagen. The most common mutations cause the replacement of glycine (in –Gly–X–Y–) by amino acids with bulky side chains. The resultant structurally abnormal α chains prevent the formation of the required triple-helical conformation. Phenotypic severity ranges from mild to lethal. Type I OI, the most common form, is characterized by mild bone fragility, hearing loss, and blue sclerae. Type II, the most severe

1	conformation. Phenotypic severity ranges from mild to lethal. Type I OI, the most common form, is characterized by mild bone fragility, hearing loss, and blue sclerae. Type II, the most severe form, is typically lethal in the perinatal period as a result of pulmonary complications. In utero fractures are seen (see Fig. 4.11). Type III is also a severe form and is characterized by multiple fractures at birth, short stature, spinal curvature leading to a humped-back (kyphotic) appearance, and blue sclerae. Dentinogenesis imperfecta, a disorder of tooth development, may be seen in OI.

1	III. ELASTIN In contrast to collagen, which forms fibers that are tough and have high tensile strength, elastin is a connective tissue fibrous protein with rubber-like properties. Elastic fibers composed of elastin and glycoprotein microfibrils are found in the lungs, the walls of large arteries, and elastic ligaments. They can be stretched to several times their normal length but recoil to their original shape when the stretching force is relaxed. A. Structure

1	Elastin is an insoluble protein polymer generated from a precursor, tropoelastin, which is a soluble polypeptide composed of ~700 amino acids that are primarily small and nonpolar (for example, glycine, alanine, and valine). Elastin is also rich in proline and lysine but contains scant hydroxyproline and hydroxylysine. Tropoelastin is secreted by the cell into the ECM. There, it interacts with specific glycoprotein microfibrils, such as fibrillin, which function as a scaffold onto which tropoelastin is deposited. Some of the lysyl side chains of the tropoelastin polypeptides are oxidatively deaminated by lysyl oxidase, forming allysine residues. Three of the allysyl side chains plus one unaltered lysyl side chain from the same or neighboring polypeptides form a desmosine cross-link (Fig. 4.12). This produces elastin, an extensively interconnected, rubbery network that can stretch and bend in any direction when stressed, giving connective tissue elasticity (Fig. 4.13). Mutations in the

1	This produces elastin, an extensively interconnected, rubbery network that can stretch and bend in any direction when stressed, giving connective tissue elasticity (Fig. 4.13). Mutations in the fibrillin-1 protein are responsible for Marfan syndrome, a connective tissue disorder characterized by impaired structural integrity in the skeleton, the eye, and the cardiovascular system. With this disease, abnormal fibrillin protein is incorporated into microfibrils along with normal fibrillin, inhibiting the formation of functional microfibrils. [Note: Patients with Marfan syndrome, OI, or EDS may have blue sclerae due to tissue thinning that allows underlying pigment to show through.]

1	B. α1-Antitrypsin in elastin degradation Blood and other body fluids contain a protein, α1-antitrypsin (AAT), which inhibits a number of proteolytic enzymes (called peptidases, proteases, or proteinases) that hydrolyze and destroy proteins. [Note: The inhibitor was originally named AAT because it inhibits the activity of trypsin, a proteolytic enzyme synthesized as trypsinogen by the pancreas (see p. 248).] AAT has the important physiologic role of inhibiting neutrophil elastase, a powerful protease that is released into the extracellular space and degrades elastin of alveolar walls as well as other structural proteins in a variety of tissues (Fig. 4.14). Most of the AAT found in plasma is synthesized and secreted by the liver. Extrahepatic synthesis also occurs. 1.

1	1. α1-Antitrypsin in the lungs: In the normal lung, the alveoli are chronically exposed to low levels of neutrophil elastase released from activated and degenerating neutrophils. The proteolytic activity of elastase can destroy the elastin in alveolar walls if unopposed by the action of AAT, the most important inhibitor of neutrophil elastase (see Fig. 4.14). Because lung tissue cannot regenerate, the destruction of the connective tissue of alveolar walls caused by an imbalance between the protease and its inhibitor results in pulmonary disease. 2.

1	α1-Antitrypsin deficiency and emphysema: In the United States, ~2%– 5% of patients with emphysema are predisposed to the disease by inherited defects in AAT. A number of different mutations in the gene for AAT are known to cause a deficiency of the protein, but one single purine base mutation (GAG to AAG, resulting in the substitution of lysine for glutamic acid at position 342 of the protein) is clinically the most widespread and severe. [Note: The mutated protein is termed the Z variant.] The mutation causes the normally monomeric AAT to misfold, polymerize, and aggregate within the RER of hepatocytes, resulting in decreased secretion of AAT by the liver. AAT deficiency is, therefore, a misfolded protein disease. Consequently, blood levels of AAT are reduced, decreasing the amount that gets to the lung. The polymer that accumulates in the liver may result in cirrhosis (scarring of the liver). In the United States, the AAT mutation is most common in Caucasians of Northern European

1	gets to the lung. The polymer that accumulates in the liver may result in cirrhosis (scarring of the liver). In the United States, the AAT mutation is most common in Caucasians of Northern European ancestry. An individual must inherit two abnormal AAT alleles to be at risk for the development of emphysema. In a heterozygote, with one normal and one defective gene, the levels of AAT are sufficient to protect the alveoli from damage. [Note: Methionine 358 in AAT is required for the binding of the inhibitor to its target proteases. Smoking causes the oxidation and subsequent inactivation of the methionine, thereby rendering the inhibitor powerless to neutralize elastase. Smokers with AAT deficiency, therefore, have a considerably elevated rate of lung destruction and a poorer survival rate than nonsmokers with the deficiency.] The deficiency of elastase inhibitor can be treated by weekly augmentation therapy, that is, intravenous administration of AAT. The AAT diffuses from the blood

1	than nonsmokers with the deficiency.] The deficiency of elastase inhibitor can be treated by weekly augmentation therapy, that is, intravenous administration of AAT. The AAT diffuses from the blood into the lung, where it reaches therapeutic levels in the fluid surrounding the lung epithelial cells.

1	IV. CHAPTER SUMMARY Collagen and elastin are structural fibrous proteins of the extracellular matrix (Fig. 4.15). Collagen contains an abundance of proline, lysine, and glycine, the latter occurring at every third position in the primary structure. It also contains hydroxyproline, hydroxylysine, and glycosylated hydroxylysine, each formed by posttranslational modification. Fibrillar collagen has a long, rigid structure, in which three collagen polypeptide α chains are wound around one another in a rope-like triple helix stabilized by interchain hydrogen bonds. Diseases of fibrillar collagen synthesis affect bones, joints, skin, and blood vessels. Elastin is a connective tissue protein with rubber-like properties in tissues such as the lung. α1-Antitrypsin (AAT), produced primarily by the liver, inhibits elastase-catalyzed degradation of elastin in the alveolar walls. A deficiency of AAT increases elastin degradation and can cause emphysema and, in some cases, cirrhosis of the liver.

1	Choose the ONE best answer. .1. A 30-year-old woman of Northern European ancestry presents with progressive dyspnea (shortness of breath). She denies the use of cigarettes. Family history reveals that her sister also has problems with her lungs. Which one of the following etiologies most likely explains this patient’s pulmonary symptoms? A. Deficiency in dietary vitamin C B. Deficiency of α1-antitrypsin C. Deficiency of prolyl hydroxylase D. Decreased elastase activity E. Increased collagenase activity

1	A. Deficiency in dietary vitamin C B. Deficiency of α1-antitrypsin C. Deficiency of prolyl hydroxylase D. Decreased elastase activity E. Increased collagenase activity Correct answer = B. α1-Antitrypsin (AAT) deficiency is a genetic disorder that can cause pulmonary damage and emphysema even in the absence of cigarette use. A deficiency of AAT permits increased elastase activity to destroy elastin in the alveolar walls. AAT deficiency should be suspected when chronic obstructive pulmonary disease develops in a patient younger than age 45 years who does not have a history of chronic bronchitis or tobacco use or when multiple family members develop obstructive lung disease at an early age. Choices A, C, and E refer to collagen, not elastin.

1	.3. A 7-month-old child “fell over” while crawling and now presents with a swollen leg. Imaging reveals a fracture of a bowed femur, secondary to minor trauma, and thin bones (see x-ray at right). Blue sclerae are also noted. At age 1 month, the infant had multiple fractures in various states of healing (right clavicle, right humerus, and right radius). A careful family history has ruled out nonaccidental trauma (child abuse) as a cause of the bone fractures. Which pairing of a defective (or deficient) molecule and the resulting pathology best fits this clinical description? A. Elastin and emphysema B. Fibrillin and Marfan disease C. Type I collagen and osteogenesis imperfecta D. Type V collagen and Ehlers-Danlos syndrome E. Vitamin C and scurvy

1	A. Elastin and emphysema B. Fibrillin and Marfan disease C. Type I collagen and osteogenesis imperfecta D. Type V collagen and Ehlers-Danlos syndrome E. Vitamin C and scurvy Correct answer = C. The child most likely has osteogenesis imperfecta. Most cases arise from a defect in the genes encoding type I collagen. Bones in affected patients are thin, osteoporotic, often bowed, and extremely prone to fracture. Pulmonary problems are not seen in this child. Individuals with Marfan syndrome have impaired structural integrity of the skeleton, eyes, and cardiovascular system. Defects in type V collagen cause the classic form of Ehlers-Danlos syndrome characterized by skin extensibility and fragility and joint hypermobility. Scurvy caused by vitamin C deficiency is characterized by capillary fragility. .2. What is the differential basis of the liver and lung pathology seen in α1antitrypsin deficiency?

1	.2. What is the differential basis of the liver and lung pathology seen in α1antitrypsin deficiency? With α1-antitrypsin (AAT) deficiency, the cirrhosis that can result is due to polymerization and retention of AAT in the liver, its site of synthesis. The alveolar damage is due to the retention-based deficiency of AAT (a serine protease inhibitor) in the lung such that elastase (a serine protease) is unopposed. .4. How and why is proline hydroxylated in collagen? Proline is hydroxlyated by prolyl hydroxylase, an enzyme of the endoplasmic reticulum that requires oxygen, ferrous iron, and vitamin C. Hydroxylation increases interchain hydrogen bond formation, strengthening the triple helix of collagen. Vitamin C deficiency impairs hydroxylation. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW Virtually all reactions in the body are mediated by enzymes, which are protein catalysts that increase the rate of reactions without being changed in the overall process. Among the many biologic reactions that are energetically possible, enzymes selectively channel reactants (called substrates) into useful pathways. Thus, enzymes direct all metabolic events. This chapter examines the nature of these catalytic molecules and their mechanisms of action. II. NOMENCLATURE Each enzyme is assigned two names. The first is its short, recommended name, convenient for everyday use. The second is the more complete systematic name, which is used when an enzyme must be identified without ambiguity. A. Recommended name

1	A. Recommended name Most commonly used enzyme names have the suffix “-ase” attached to the substrate of the reaction (for example, glucosidase and urease) or to a description of the action performed (for example, lactate dehydrogenase and adenylyl cyclase). [Note: Some enzymes retain their original trivial names, which give no hint of the associated enzymic reaction, for example, trypsin and pepsin.] B. Systematic name In the systematic naming system, enzymes are divided into six major classes (Fig. 5.1), each with numerous subgroups. For a given enzyme, the suffix ase is attached to a fairly complete description of the chemical reaction catalyzed, including the names of all the substrates, for example, lactate:nicotinamide adenine dinucleotide (NAD+) oxidoreductase. [Note: Each enzyme is also assigned a classification number. Lactate:NAD+ oxidoreductase is 1.1.1.27.] The systematic names are unambiguous and informative but are frequently too cumbersome to be of general use.

1	inorganic phosphate. Potentially confusing enzyme nomenclature includes synthetase (requires ATP), synthase (no ATP required), phosphatase (uses water to remove a phosphate group), phosphorylase (uses inorganic phosphate to break a bond and generate a phosphorylated product), dehydrogenase (NAD+ or flavin adenine dinucleotide [FAD] is an electron acceptor in a redox reaction), oxidase (oxygen is the acceptor, and oxygen atoms are not incorporated into substrate), and oxygenase (one or both oxygen atoms are incorporated). III. PROPERTIES Enzymes are protein catalysts that increase the velocity of a chemical reaction and are not consumed during the reaction. [Note: Some ribonucleic acids (RNA) can catalyze reactions that affect phosphodiester and peptide bonds. RNAs with catalytic activity are called ribozymes (see p. 434) and are much less common than protein catalysts.] A. Active site

1	A. Active site Enzyme molecules contain a special pocket or cleft called the active site. The active site, formed by folding of the protein, contains amino acid side chains that participate in substrate binding and catalysis (Fig. 5.2). The substrate binds the enzyme noncovalently, forming an enzyme–substrate (ES) complex. Binding is thought to cause a conformational change in the enzyme (induced fit model) that allows catalysis. ES is converted to an enzyme–product (EP) complex that subsequently dissociates to enzyme and product. B. Efficiency Enzyme-catalyzed reactions are highly efficient, proceeding from 103 to 108 times faster than uncatalyzed reactions. The number of substrate molecules converted to product per enzyme molecule per second is called the turnover number, or kcat, and typically is 102–104 s−1 . [Note: kcat is the rate constant for the conversion of ES to E + P (see p. 58).] C. Specificity

1	C. Specificity Enzymes are highly specific, interacting with one or a few substrates and catalyzing only one type of chemical reaction. The set of enzymes made in a cell determines which reactions occur in that cell. D. Holoenzymes, apoenzymes, cofactors, and coenzymes

1	D. Holoenzymes, apoenzymes, cofactors, and coenzymes Some enzymes require nonproteins for enzymic activity. The term holoenzyme refers to the active enzyme with its nonprotein component, whereas the enzyme without its nonprotein moiety is termed an apoenzyme and is inactive. If the nonprotein moiety is a metal ion, such as zinc (Zn2+) or iron (Fe2+), it is called a cofactor (see Chapter 29). If it is a small organic molecule, it is termed a coenzyme. Coenzymes that only transiently associate with the enzyme are called cosubstrates. Cosubstrates dissociate from the enzyme in an altered state (NAD+ is an example; see p. 101). If the coenzyme is permanently associated with the enzyme and returned to its original form, it is called a prosthetic group (FAD is an example; see p. 110). Coenzymes commonly are derived from vitamins. For example, NAD+ contains niacin, and FAD contains riboflavin (see Chapter 28). E. Regulation

1	E. Regulation Enzyme activity can be regulated, that is, increased or decreased, so that the rate of product formation responds to cellular need. F. Location within the cell Many enzymes are localized in specific organelles within the cell (Fig. 5.3). Such compartmentalization serves to isolate the reaction substrate or product from other competing reactions. This provides a favorable environment for the reaction and organizes the thousands of enzymes present in the cell into purposeful pathways. IV. MECHANISM OF ENZYME ACTION The mechanism of enzyme action can be viewed from two different perspectives. The first treats catalysis in terms of energy changes that occur during the reaction. That is, enzymes provide an alternate, energetically favorable reaction pathway different from the uncatalyzed reaction. The second perspective describes how the active site chemically facilitates catalysis. A. Energy changes occurring during the reaction

1	A. Energy changes occurring during the reaction Virtually all chemical reactions have an energy barrier separating the reactants and the products. This barrier, called the activation energy (Ea), is the energy difference between that of the reactants and a high-energy intermediate, the transition state (T*), which is formed during the conversion of reactant to product. Figure 5.4 shows the changes in energy during the conversion of a molecule of reactant A to product B as it proceeds through the transition state. 1. Activation energy: The peak of energy in Figure 5.4 is the difference in free energy between the reactant and T*, in which the high-energy, short-lived intermediate is formed during the conversion of reactant to product. Because of the high Ea, the rates of uncatalyzed chemical reactions are often slow. 2.

1	2. Rate of reaction: For molecules to react, they must contain sufficient energy to overcome the energy barrier of the transition state. In the absence of an enzyme, only a small proportion of a population of molecules may possess enough energy to achieve the transition state between reactant and product. The rate of reaction is determined by the number of such energized molecules. In general, the lower the Ea, the more molecules have sufficient energy to pass through the transition state and, therefore, the faster the rate of the reaction. 3. Alternate reaction pathway: An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower Ea (see Fig. 5.4). The enzyme does not change the free energies of the reactants (substrates) or products and, therefore, does not change the equilibrium of the reaction (see p. 70). It does, however, accelerate the rate by which equilibrium is reached.

1	B. Active site chemistry The active site is not a passive receptacle for binding the substrate but, rather, is a complex molecular machine employing a diversity of chemical mechanisms to facilitate the conversion of substrate to product. A number of factors are responsible for the catalytic efficiency of enzymes, including the following examples. 1. Transition-state stabilization: The active site often acts as a flexible molecular template that binds the substrate and initiates its conversion to the transition state, a structure in which the bonds are not like those in the substrate or the product (see T* at the top of the curve in Fig. 5.4). By stabilizing the transition state, the enzyme greatly increases the concentration of the reactive intermediate that can be converted to product and, thus, accelerates the reaction. [Note: The transition state cannot be isolated.] 2.

1	Catalysis: The active site can provide catalytic groups that enhance the probability that the transition state is formed. In some enzymes, these groups can participate in general acid–base catalysis in which amino acid residues provide or accept protons. In other enzymes, catalysis may involve the transient formation of a covalent ES complex. [Note: The mechanism of action of chymotrypsin, an enzyme of protein digestion in the intestine, includes general base, general acid, and covalent catalysis. A histidine at the active site of the enzyme gains (general base) and loses (general acid) protons, mediated by the pK of histidine in proteins being close to physiologic pH. Serine at the active site forms a transient covalent bond with the substrate.] 3.

1	Transition-state visualization: The enzyme-catalyzed conversion of substrate to product can be visualized as being similar to removing a sweater from an uncooperative infant (Fig. 5.5). The process has a high Ea because the only reasonable strategy for removing the garment (short of ripping it off) requires that the random flailing of the baby results in both arms being fully extended over the head, an unlikely posture. However, we can envision a parent acting as an enzyme, first coming in contact with the baby (forming ES) and then guiding the baby’s arms into an extended, vertical position, analogous to the transition state. This posture (conformation) of the baby facilitates the removal of the sweater, forming the disrobed baby, which here represents product. [Note: The substrate bound to the enzyme (ES) is at a slightly lower energy than unbound substrate (S) and explains the small dip in the curve at ES.] V. FACTORS AFFECTING REACTION VELOCITY

1	V. FACTORS AFFECTING REACTION VELOCITY Enzymes can be isolated from cells and their properties studied in a test tube (that is, in vitro). Different enzymes show different responses to changes in substrate concentration, temperature, and pH. This section describes factors that influence the reaction velocity of enzymes. Enzymic responses to these factors give us valuable clues as to how enzymes function in living cells (that is, in vivo). A. Substrate concentration 1. Maximal velocity: The rate or velocity of a reaction (v) is the number of substrate molecules converted to product per unit time. Velocity is usually expressed as µmol of product formed per minute. The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax) is reached (Fig. 5.6). The leveling off of the reaction rate at high substrate concentrations reflects the saturation with substrate of all available binding sites on the enzyme molecules present. 2.

1	2. Shape of the enzyme kinetics curve: Most enzymes show Michaelis-Menten kinetics (see p. 58), in which the plot of initial reaction velocity (vo) against substrate concentration is hyperbolic (similar in shape to that of the oxygen-dissociation curve of myoglobin; see p. 29). In contrast, allosteric enzymes do not follow Michaelis-Menten kinetics and show a sigmoidal curve (see Fig. 5.6) that is similar in shape to the oxygen-dissociation curve of hemoglobin (see p. 29). B. Temperature 1. Velocity increase with temperature: The reaction velocity increases with temperature until a peak velocity is reached (Fig. 5.7). This increase is the result of the increased number of substrate molecules having sufficient energy to pass over the energy barrier and form the products of the reaction. 2.

1	2. Velocity decrease with higher temperature: Further elevation of the temperature causes a decrease in reaction velocity as a result of temperature-induced denaturation of the enzyme (see Fig. 5.7). The optimum temperature for most human enzymes is between 35°C and 40°C. Human enzymes start to denature (see p. 20) at temperatures above 40°C, but thermophilic bacteria found in hot springs have optimum temperatures of 70°C. C. pH 1. pH effect on active site ionization: The concentration of protons ([H+]) affects reaction velocity in several ways. First, the catalytic process usually requires that the enzyme and substrate have specific chemical groups in either an ionized or unionized state in order to interact. For example, catalytic activity may require that an amino group of the enzyme be in the protonated form (−NH3+). Because this group is deprotonated at alkaline pH, the rate of the reaction declines. 2.

1	2. pH effect on enzyme denaturation: Extremes of pH can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic character of the amino acid side chains. 3. Variable pH optimum: The pH at which maximal enzyme activity is achieved is different for different enzymes and often reflects the [H+] at which the enzyme functions in the body. For example, pepsin, a digestive enzyme in the stomach, is maximally active at pH 2, whereas other enzymes, designed to work at neutral pH, are denatured by such an acidic environment (Fig. 5.8). VI. MICHAELIS-MENTEN KINETICS

1	VI. MICHAELIS-MENTEN KINETICS Leonor Michaelis and Maude Menten proposed a simple model that accounts for most of the features of many enzyme-catalyzed reactions. In this model, the enzyme reversibly combines with its substrate to form an ES complex that subsequently yields product, regenerating the free enzyme. The reaction model, involving one substrate molecule, is represented below: where S is the substrate. E is the enzyme. ES is the enzyme–substrate complex. P is the product. k1, k−1, and k2 (or, kcat) are rate constants. A. Michaelis-Menten equation The Michaelis-Menten equation describes how reaction velocity varies with substrate concentration: The following assumptions are made in deriving the Michaelis-Menten rate equation. 1. Enzyme and substrate relative concentrations: The substrate concentration ([S]) is much greater than the concentration of enzyme so that the percentage of total substrate bound by the enzyme at any one time is small. 2.

1	2. Steady-state assumption: The concentration of the ES complex does not change with time (the steady-state assumption), that is, the rate of formation of ES is equal to that of the breakdown of ES (to E + S and to E + P). In general, an intermediate in a series of reactions is said to be in steady state when its rate of synthesis is equal to its rate of degradation. 3. Initial velocity: Initial reaction velocities (vo) are used in the analysis of enzyme reactions. This means that the rate of the reaction is measured as soon as enzyme and substrate are mixed. At that time, the concentration of product is very small, and therefore, the rate of the back reaction from product to substrate can be ignored.

1	B. Important conclusions 1. Km characteristics: Km, the Michaelis constant, is characteristic of an enzyme and its particular substrate and reflects the affinity of the enzyme for that substrate. Km is numerically equal to the substrate concentration at which the reaction velocity is equal to one half Vmax. Km does not vary with enzyme concentration. a. Small Km: A numerically small (low) Km reflects a high affinity of the enzyme for substrate, because a low concentration of substrate is needed to half-saturate the enzyme—that is, to reach a velocity that is one half Vmax (Fig. 5.9). b. Large Km: A numerically large (high) Km reflects a low affinity of enzyme for substrate because a high concentration of substrate is needed to half-saturate the enzyme. 2.

1	b. Large Km: A numerically large (high) Km reflects a low affinity of enzyme for substrate because a high concentration of substrate is needed to half-saturate the enzyme. 2. Velocity relationship to enzyme concentration: The rate of the reaction is directly proportional to the enzyme concentration because [S] is not limiting. For example, if the enzyme concentration is halved, the initial rates of the reaction (vo) and that of Vmax are reduced to half that of the original. 3. Reaction order: When [S] is much less (<<) than Km, the velocity of the reaction is approximately proportional to the substrate concentration (Fig. 5.10). The rate of reaction is then said to be first order with respect to substrate. When [S] is much greater (>>) than Km, the velocity is constant and equal to Vmax. The rate of reaction is then independent of substrate concentration (the enzyme is saturated with substrate) and is said to be zero order with respect to substrate concentration (see Fig. 5.10).

1	D. Lineweaver-Burk plot When vo is plotted against [S], it is not always possible to determine when Vmax has been achieved because of the gradual upward slope of the hyperbolic curve at high substrate concentrations. However, if 1/vo is plotted versus 1/[S], a straight line is obtained (Fig. 5.11). This plot, the Lineweaver-Burk plot (also called a double-reciprocal plot) can be used to calculate Km and Vmax as well as to determine the mechanism of action of enzyme inhibitors. The equation describing the Lineweaver-Burk plot is: where the intercept on the x axis is equal to − 1/Km, and the intercept on the y axis is equal to 1/Vmax. [Note: The slope = Km/Vmax.] VII. ENZYME INHIBITION

1	VII. ENZYME INHIBITION Any substance that can decrease the velocity of an enzyme-catalyzed reaction is called an inhibitor. Inhibitors can be reversible or irreversible. Irreversible inhibitors bind to enzymes through covalent bonds. Lead, for example, forms covalent bonds with the sulfhydryl side chain of cysteine in proteins. Ferrochelatase, an enzyme involved in heme synthesis (see p. 279), is irreversibly inhibited by lead. [Note: An important group of irreversible inhibitors are the mechanism-based inhibitors that are converted by the enzyme itself to a form that covalently links to the enzyme, thereby inhibiting it. They also are referred to as “suicide” inhibitors.] Reversible inhibitors bind to enzymes through noncovalent bonds and, thus, dilution of the enzyme–inhibitor complex results in dissociation of the reversibly bound inhibitor and recovery of enzyme activity. The two most commonly encountered types of reversible inhibition are competitive and noncompetitive.

1	A. Competitive inhibition This type of inhibition occurs when the inhibitor binds reversibly to the same site that the substrate would normally occupy and, therefore, competes with the substrate for that site. 1. Effect on Vmax: The effect of a competitive inhibitor is reversed by increasing the concentration of substrate. At a sufficiently high [S], the 2. Effect on Km: A competitive inhibitor increases the apparent Km for a given substrate. This means that, in the presence of a competitive inhibitor, more substrate is needed to achieve one half Vmax. 3. Effect on the Lineweaver-Burk plot: Competitive inhibition shows a characteristic Lineweaver-Burk plot in which the plots of the inhibited and uninhibited reactions intersect on the y axis at 1/Vmax (Vmax is reaction velocity reaches the Vmax observed in the absence of inhibitor, that is, Vmax is unchanged (Fig. 5.12).

1	unchanged). The inhibited and uninhibited reactions show different x-axis intercepts, indicating that the apparent Km is increased in the presence of the competitive inhibitor because − 1/Km moves closer to zero from a negative value (see Fig. 5.12). [Note: An important group of competitive inhibitors are the transition state analogs, stable molecules that approximate the structure of the transition state, and, therefore, bind the enzyme more tightly than does the substrate.] 4. Statin drugs as examples of competitive inhibitors: This group of antihyperlipidemic agents competitively inhibits the rate-limiting (slowest) step in cholesterol biosynthesis. This reaction is catalyzed by hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase; see p. 221). Statins, such as atorvastatin (Lipitor) and pravastatin (Pravachol), are structural analogs of the natural substrate for this enzyme and compete effectively to inhibit HMG CoA reductase. By doing so, they inhibit de novo cholesterol

1	and pravastatin (Pravachol), are structural analogs of the natural substrate for this enzyme and compete effectively to inhibit HMG CoA reductase. By doing so, they inhibit de novo cholesterol synthesis, thereby lowering plasma cholesterol levels (Fig. 5.13).

1	B. Noncompetitive inhibition This type of inhibition is recognized by its characteristic effect on Vmax (Fig. 5.14). Noncompetitive inhibition occurs when the inhibitor and substrate bind at different sites on the enzyme. The noncompetitive inhibitor can bind either free enzyme or the enzyme–substrate complex, thereby preventing the reaction from occurring (Fig. 5.15). 1. Effect on Vmax: Noncompetitive inhibition cannot be overcome by increasing the concentration of substrate. Therefore, noncompetitive inhibitors decrease the apparent Vmax of the reaction. 2. Effect on Km: Noncompetitive inhibitors do not interfere with the binding of substrate to enzyme. Therefore, the enzyme shows the same Km in the presence or absence of the noncompetitive inhibitor, that is, Km is unchanged. 3.

1	3. Effect on Lineweaver-Burk plot: Noncompetitive inhibition is readily differentiated from competitive inhibition by plotting 1/vo versus 1/[S] and noting that the apparent Vmax decreases in the presence of a noncompetitive inhibitor, whereas Km is unchanged (see Fig. 5.14). [Note: Oxypurinol, a metabolite of the prodrug allopurinol, is a noncompetitive inhibitor of xanthine oxidase, an enzyme of purine degradation (see p. 301).] C. Enzyme inhibitors as drugs

1	C. Enzyme inhibitors as drugs At least half of the ten most commonly prescribed drugs in the United States act as enzyme inhibitors. For example, the widely prescribed βlactam antibiotics, such as penicillin and amoxicillin, act by inhibiting enzymes involved in bacterial cell wall synthesis. Drugs may also act by inhibiting extracellular reactions. This is illustrated by angiotensinconverting enzyme (ACE) inhibitors. They lower blood pressure by blocking plasma ACE that cleaves angiotensin I to form the potent vasoconstrictor, angiotensin II. These drugs, which include captopril, enalapril, and lisinopril, cause vasodilation and, therefore, a reduction in blood pressure. Aspirin, a nonprescription drug, irreversibly inhibits prostaglandin and thromboxane synthesis by inhibiting cyclooxygenase (see p. 214). VIII. ENZYME REGULATION

1	VIII. ENZYME REGULATION The regulation of the reaction velocity of enzymes is essential if an organism is to coordinate its numerous metabolic processes. The rates of most enzymes are responsive to changes in substrate concentration, because the intracellular level of many substrates is in the range of the Km. Thus, an increase in substrate concentration prompts an increase in reaction rate, which tends to return the concentration of substrate toward normal. In addition, some enzymes with specialized regulatory functions respond to allosteric effectors and/or covalent modification or they show altered rates of enzyme synthesis (or degradation) when physiologic conditions are changed. A. Allosteric enzymes

1	A. Allosteric enzymes Allosteric enzymes are regulated by molecules called effectors that bind noncovalently at a site other than the active site. These enzymes are almost always composed of multiple subunits, and the regulatory (allosteric) site that binds the effector is distinct from the substrate-binding site and may be located on a subunit that is not itself catalytic. Effectors that inhibit enzyme activity are termed negative effectors, whereas those that increase enzyme activity are called positive effectors. Positive and negative effectors can affect the affinity of the enzyme for its substrate (K0.5), modify the maximal catalytic activity of the enzyme (Vmax), or both (Fig. 5.16). [Note: Allosteric enzymes frequently catalyze the committed step, often the rate-limiting step, early in a pathway.] 1.

1	Homotropic effectors: When the substrate itself serves as an effector, the effect is said to be homotropic. Most often, an allosteric substrate functions as a positive effector. In such a case, the presence of a substrate molecule at one site on the enzyme enhances the catalytic properties of the other substrate-binding sites. That is, their binding sites exhibit cooperativity. These enzymes show a sigmoidal curve when vo is plotted against substrate concentration, as shown in Figure 5.16. This contrasts with the hyperbolic curve characteristic of enzymes following Michaelis-Menten kinetics, as previously discussed. [Note: The concept of cooperativity of substrate binding is analogous to the binding of oxygen to hemoglobin (see p. 29).] 2.

1	Heterotropic effectors: The effector may be different from the substrate, in which case the effect is said to be heterotropic. For example, consider the feedback inhibition shown in Figure 5.17. The enzyme that converts D to E has an allosteric site that binds the end product, G. If the concentration of G increases (for example, because it is not used as rapidly as it is synthesized), the first irreversible step unique to the pathway is typically inhibited. Feedback inhibition provides the cell with appropriate amounts of a product it needs by regulating the flow of substrate molecules through the pathway that synthesizes that product. Heterotropic effectors are commonly encountered. For example, the glycolytic enzyme phosphofructokinase-1 is allosterically inhibited by citrate, which is not a substrate for the enzyme (see p. 99). Figure5.17Feedbackinhibitionofametabolicpathway. B. Covalent modification

1	Figure5.17Feedbackinhibitionofametabolicpathway. B. Covalent modification Many enzymes are regulated by covalent modification, most often by the addition or removal of phosphate groups from specific serine, threonine, or tyrosine residues of the enzyme. Protein phosphorylation is recognized as one of the primary ways in which cellular processes are regulated. 1. Phosphorylation and dephosphorylation: Phosphorylation reactions are catalyzed by a family of enzymes called protein kinases that use ATP as the phosphate donor. Phosphate groups are cleaved from phosphorylated enzymes by the action of phosphoprotein phosphatases (Fig. 5.18). 2.

1	2. Enzyme response to phosphorylation: Depending on the specific enzyme, the phosphorylated form may be more or less active than the unphosphorylated enzyme. For example, hormone-mediated phosphorylation of glycogen phosphorylase (an enzyme that degrades glycogen) increases activity, whereas phosphorylation of glycogen synthase (an enzyme that synthesizes glycogen) decreases activity (see p. 132). C. Enzyme synthesis

1	The regulatory mechanisms described above modify the activity of existing enzyme molecules. However, cells can also regulate the amount of enzyme present by altering the rate of enzyme degradation or, more typically, the rate of enzyme synthesis. The increase (induction) or decrease (repression) of enzyme synthesis leads to an alteration in the total population of active sites. Enzymes subject to regulation of synthesis are often those that are needed at only one stage of development or under selected physiologic conditions. For example, elevated levels of insulin as a result of high blood glucose levels cause an increase in the synthesis of key enzymes involved in glucose metabolism (see p. 105). In contrast, enzymes that are in constant use are usually not regulated by altering the rate of enzyme synthesis. Alterations in enzyme levels as a result of induction or repression of protein synthesis are slow (hours to days), compared with allosterically or covalently regulated changes in

1	of enzyme synthesis. Alterations in enzyme levels as a result of induction or repression of protein synthesis are slow (hours to days), compared with allosterically or covalently regulated changes in enzyme activity, which occur in seconds to minutes. Figure 5.19 summarizes the common ways that enzyme activity is regulated.

1	IX. Enzymes in Clinical Diagnosis Plasma enzymes can be classified into two major groups. First, a relatively small group of enzymes are actively secreted into the blood by certain cell types. For example, the liver secretes zymogens (inactive precursors) of the enzymes involved in blood coagulation. Second, a large number of enzyme species are released from cells during normal cell turnover. These enzymes almost always function intracellularly and have no physiologic use in the plasma. In healthy individuals, the levels of these enzymes are fairly constant and represent a steady state in which the rate of release from damaged cells into the plasma is balanced by an equal rate of removal from the plasma. Increased plasma levels of these enzymes may indicate tissue damage (Fig. 5.20). (B) cells.

1	(B) cells. Plasma is the fluid, noncellular part of blood. Laboratory assays of enzyme activity most often use serum, which is obtained by centrifugation of whole blood after it has been allowed to coagulate. Plasma is a physiologic fluid, whereas serum is prepared in the laboratory. A. Plasma enzyme levels in disease states Many diseases that cause tissue damage result in an increased release of intracellular enzymes into the plasma. The activities of many of these enzymes are routinely determined for diagnostic purposes in diseases of the heart, liver, skeletal muscle, and other tissues. The level of specific enzyme activity in the plasma frequently correlates with the extent of tissue damage. Therefore, determining the degree of elevation of a particular enzyme activity in the plasma is often useful in evaluating the prognosis for the patient. B. Plasma enzymes as diagnostic tools

1	B. Plasma enzymes as diagnostic tools Some enzymes show relatively high activity in only one or a few tissues. Therefore, the presence of increased levels of these enzymes in plasma reflects damage to the corresponding tissue. For example, the enzyme alanine aminotransferase (ALT; see p. 251) is abundant in the liver. The appearance of elevated levels of ALT in plasma signals possible damage to hepatic tissue. [Note: Measurement of ALT is part of the liver function test panel.] Increases in plasma levels of enzymes with a wide tissue distribution provide a less specific indication of the site of cellular injury and limits their diagnostic value. C. Isoenzymes and heart disease

1	C. Isoenzymes and heart disease Isoenzymes (also called isozymes) are enzymes that catalyze the same reaction. However, they do not necessarily have the same physical properties because of genetically determined differences in amino acid sequence. For this reason, isoenzymes may contain different numbers of charged amino acids, which allows electrophoresis (the movement of charged particles in an electric field) to separate them (Fig. 5.21). Different organs commonly contain characteristic proportions of different isoenzymes. The pattern of isoenzymes found in the plasma may, therefore, serve as a means of identifying the site of tissue damage. For example, the plasma levels of creatine kinase (CK) are commonly determined in the diagnosis of myocardial infarction (MI). They are particularly useful when the electrocardiogram (ECG) is difficult to interpret such as when there have been previous episodes of heart disease. 1.

1	1. Isoenzyme quaternary structure: Many isoenzymes contain different subunits in various combinations. For example, CK occurs as three isoenzymes. Each isoenzyme is a dimer composed of two polypeptides (called B and M subunits) associated in one of three combinations: CK1 = BB, CK2 = MB, and CK3 = MM. Each CK isoenzyme shows a characteristic electrophoretic mobility (see Fig. 5.21). [Note: Virtually all CK in the brain is the BB isoform, whereas it is MM in skeletal muscle. In cardiac muscle, about one third is MB with the rest as MM.] 2.

1	Diagnosis of myocardial infarction: Measurement of blood levels of proteins with cardiac specificity (biomarkers) is used in the diagnosis of MI. Myocardial muscle is the only tissue that contains >5% of the total CK activity as the CK2 (MB) isoenzyme. Appearance of this hybrid isoenzyme in plasma is virtually specific for infarction of the myocardium. Following an acute MI, CK2 appears in plasma within 4–8 hours following onset of chest pain, reaches a peak of activity at ~24 hours, and returns to baseline after 48–72 hours (Fig. 5.22). Troponins T (TnT) and I (TnI) are regulatory proteins involved in muscle contractility. Cardiac-specific isoforms (cTn) are released into the plasma in response to cardiac damage. They are highly sensitive and specific for damage to cardiac tissue. cTn appear in plasma within 4–6 hours after an MI, peak in 24–36 hours, and remain elevated for 3–10 days. Elevated cTn, in combination with the clinical presentation and characteristic changes in the ECG,

1	in plasma within 4–6 hours after an MI, peak in 24–36 hours, and remain elevated for 3–10 days. Elevated cTn, in combination with the clinical presentation and characteristic changes in the ECG, are currently considered the “gold standard” in the diagnosis of an MI.

1	X. CHAPTER SUMMARY

1	Enzymes are protein catalysts that increase the velocity of a chemical reaction by lowering the energy of the transition state (Fig. 5.23). They are not consumed during the reaction. Enzyme molecules contain a special cleft called the active site, which contains amino acid side chains that participate in substrate binding and catalysis. The active site binds the substrate, forming an enzyme–substrate (ES) complex. Binding is thought to cause a conformational change in the enzyme (induced fit) that allows catalysis. ES is converted to enzyme and product. An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower activation energy (Ea). Because the enzyme does not change the free energies of the reactants or products, it does not change the equilibrium of the reaction. Most enzymes show Michaelis-Menten kinetics, and a plot of the initial reaction velocity (vo) against substrate concentration ([S]) has a

1	products, it does not change the equilibrium of the reaction. Most enzymes show Michaelis-Menten kinetics, and a plot of the initial reaction velocity (vo) against substrate concentration ([S]) has a hyperbolic shape similar to the oxygen-dissociation curve of myoglobin. A Lineweaver-Burk plot of 1/v and 1/[S] allows determination of Vmax (maximal velocity) and Km (Michaelis constant, which reflects affinity for substrate). Any substance that can decrease the velocity of an enzyme-catalyzed reaction is called an inhibitor. The two most common types of reversible inhibition are competitive (which increases the apparent Km) and noncompetitive (which decreases the apparent Vmax). In contrast, the multisubunit allosteric enzymes show a sigmoidal curve similar in shape to the oxygen-dissociation curve of hemoglobin. They typically catalyze the committed step of a pathway. Allosteric enzymes are regulated by molecules called effectors that bind noncovalently at a site other than the active

1	curve of hemoglobin. They typically catalyze the committed step of a pathway. Allosteric enzymes are regulated by molecules called effectors that bind noncovalently at a site other than the active site. Effectors can be either positive (increase enzyme activity) or negative (decrease enzyme activity). An allosteric effector can alter the affinity of the enzyme for its substrate (K0.5), the maximal catalytic activity of the enzyme (Vmax), or both. Enzymes can also be regulated by covalent modification and by changes in the rate of synthesis or degradation.

1	Enzymes have diagnostic and therapeutic value in medicine. Choose the ONE best answer. .1. In cases of ethylene glycol poisoning and its characteristic metabolic acidosis, treatment involves correction of the acidosis, removal of any remaining ethylene glycol, and administration of an inhibitor of alcohol dehydrogenase (ADH), the enzyme that oxidizes ethylene glycol to the organic acids that cause the acidosis. Ethanol (grain alcohol) frequently is the inhibitor given to treat ethylene glycol poisoning. Results of experiments using ADH with and without ethanol are shown to the right. Based on these data, what type of inhibition is caused by the ethanol? A. Competitive B. Feedback C. Irreversible D. Noncompetitive

1	A. Competitive B. Feedback C. Irreversible D. Noncompetitive Correct answer = A. A competitive inhibitor increases the apparent Km for a given substrate. This means that, in the presence of a competitive inhibitor, more substrate is needed to achieve one half Vmax. The effect of a competitive inhibitor is reversed by increasing substrate concentration ([S]). At a sufficiently high [S], the reaction velocity reaches the Vmax observed in the absence of inhibitor. .2. Alcohol dehydrogenase (ADH) requires oxidized nicotinamide adenine dinucleotide (NAD+) for catalytic activity. In the reaction catalyzed by ADH, an alcohol is oxidized to an aldehyde as NAD+ is reduced to NADH and dissociates from the enzyme. The NAD+ is functioning as a/an: A. apoenzyme. B. coenzyme–cosubstrate. C. coenzyme–prosthetic group. D. cofactor. E. heterotropic effector.

1	A. apoenzyme. B. coenzyme–cosubstrate. C. coenzyme–prosthetic group. D. cofactor. E. heterotropic effector. Correct answer = B. A Coenzymes–cosubstrates are small organic molecules that associate transiently with an enzyme and leave the enzyme in a changed form. Coenzyme–prosthetic groups are small organic molecules that associate permanently with an enzyme and are returned to their original form on the enzyme. Cofactors are metal ions. Heterotropic effectors are not substrates. For Questions 5.3 and 5.4, use the graph below that shows the changes in free energy when a reactant is converted to a product in the presence and absence of an enzyme. Select the letter that best represents: .3. the activation energy of the catalyzed forward reaction. .4. the free energy of the reaction.

1	Correct answers = B; D. Enzymes (protein catalysts) provide an alternate reaction pathway with a lower activation energy. However, they do not change the free energy of the reactant or product. A is the activation energy of the uncatalyzed reaction. C is the activation energy of the catalyzed reverse reaction. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW Bioenergetics describes the transfer and utilization of energy in biologic systems. It concerns the initial and final energy states of the reaction components, not the reaction mechanism or how much time it takes for the chemical change to occur. Bioenergetics makes use of a few basic ideas from the field of thermodynamics, particularly the concept of free energy. Because changes in free energy provide a measure of the energetic feasibility of a chemical reaction, they allow prediction of whether a reaction or process can take place. In short, bioenergetics predicts if a process is possible, whereas kinetics measures the reaction rate (see p. 54). II. FREE ENERGY

1	II. FREE ENERGY The direction and extent to which a chemical reaction proceeds are determined by the degree to which two factors change during the reaction. These are enthalpy (∆H, a measure of the change [∆] in heat content of the reactants and products) and entropy (∆S, a measure of the change in randomness or disorder of the reactants and products), as shown in Figure 6.1. Neither of these thermodynamic quantities by itself is sufficient to determine whether a chemical reaction will proceed spontaneously in the direction it is written. However, when combined mathematically (see Fig. 6.1), enthalpy and entropy can be used to define a third quantity, free energy (G), which predicts the direction in which a reaction will spontaneously proceed. III. FREE ENERGY CHANGE

1	III. FREE ENERGY CHANGE The change in free energy is represented in two ways, ∆G and ∆G0. The first, ∆G (without the superscript “0”), represents the change in free energy and, thus, the direction of a reaction at any specified concentration of products and reactants. ∆G, then, is a variable. This contrasts with the standard free energy change, ∆G0 (with the superscript “0”), which is the energy change when reactants and products are at a concentration of 1 mol/l. [Note: The concentration of protons (H+) is assumed to be 10−7 mol/l (that is, pH = 7). This may be shown by a prime sign (ʹ ), for example, ∆G0ʹ.] Although ∆G0, a constant, represents energy changes at these nonphysiologic concentrations of reactants and products, it is nonetheless useful in comparing the energy changes of different reactions. Furthermore, ∆G0 can readily be determined from measurement of the equilibrium constant (see p. 71). [Note: This section outlines the uses of ∆G, and ∆G0 is described in D. below.]

1	A. ∆G and reaction direction The sign of ∆G can be used to predict the direction of a reaction at constant temperature and pressure. Consider the reaction: 1. Negative ∆G: If ∆G is negative, then there is a net loss of energy, and the reaction goes spontaneously as written (that is, A is converted into B) as shown in Figure 6.2A. The reaction is said to be exergonic. 2. Positive ∆G: If ∆G is positive, then there is a net gain of energy, and the reaction does not go spontaneously from B to A (Fig. 6.2B). Energy must be added to the system to make the reaction go from B to A. The reaction is said to be endergonic. 3. Zero ∆G: If ∆G = 0, then the reaction is in equilibrium. [Note: When a reaction is proceeding spontaneously (that is, ∆G is negative), the reaction continues until ∆G reaches zero and equilibrium is established.] B. ∆G of the forward and back reactions

1	B. ∆G of the forward and back reactions The free energy of the forward reaction (A → B) is equal in magnitude but opposite in sign to that of the back reaction (B → A). For example, if ∆G of the forward reaction is −5 kcal/mol, then that of the back reaction is +5 kcal/mol. [Note: ∆G can also be expressed in kilojoules per mole or kJ/mol (1 kcal = 4.2 kJ).] C. ∆G and reactant and product concentrations The ∆G of the reaction A → B depends on the concentration of the reactant and product. At constant temperature and pressure, the following relationship can be derived: where ∆G0 is the standard free energy change (see D. below) R is the gas constant (1.987 cal/mol K) T is the absolute temperature (K) [A] and [B] are the actual concentrations of the reactant and product ln represents the natural logarithm.

1	A reaction with a positive ∆G0 can proceed in the forward direction if the ratio of products to reactants ([B]/[A]) is sufficiently small (that is, the ratio of reactants to products is large) to make ∆G negative. For example, consider the reaction: D. Standard free energy change The standard free energy change, ∆G0, is so called because it is equal to the free energy change, ∆G, under standard conditions (that is, when reactants and products are at 1 mol/l concentrations; Fig. 6.3B). Under these conditions, the natural logarithm of the ratio of products to reactants is zero (ln1 = 0), and, therefore, the equation shown at the bottom of the previous page becomes: 1.

1	∆G0 and reaction direction: Under standard conditions, ∆G0 can be used to predict the direction a reaction proceeds because, under these conditions, ∆G0 is equal to ∆G. However, ∆G0 cannot predict the direction of a reaction under physiologic conditions because it is composed solely of constants (R, T, and Keq [see 2. below]) and is not, therefore, altered by changes in product or substrate concentrations. 2.

1	2. Relationship between ∆G0 and Keq: In a reaction A ⇄ B, a point of equilibrium is reached at which no further net chemical change takes place (that is, when A is being converted to B as fast as B is being converted to A). In this state, the ratio of [B] to [A] is constant, regardless of the actual concentrations of the two compounds: where Keq is the equilibrium constant, and [A]eq and [B]eq are the concentrations of A and B at equilibrium. If the reaction A ⇄ B is allowed to go to equilibrium at constant temperature and pressure, then, at equilibrium, the overall ∆G is zero (Fig. 6.3C). Therefore, where the actual concentrations of A and B are equal to the equilibrium concentrations of reactant and product ([A]eq and [B]eq), and their ratio is equal to the Keq. Thus, This equation allows some simple predictions: 3. ∆G0s of two consecutive reactions: The ∆G0s are additive in any sequence of consecutive reactions, as are the ∆Gs. For example: 4.

1	This equation allows some simple predictions: 3. ∆G0s of two consecutive reactions: The ∆G0s are additive in any sequence of consecutive reactions, as are the ∆Gs. For example: 4. ∆Gs of a pathway: The additive property of ∆G is very important in biochemical pathways through which substrates (reactants) must pass in a particular direction (for example, A → B → C → D → …). As long as the sum of the ∆Gs of the individual reactions is negative, the pathway can proceed as written, even if some of the individual reactions of the pathway have a positive ∆G. However, the actual rates of the reactions depend on the lowering of activation energies (Ea) by the enzymes that catalyze the reactions (see p. 55). IV. ATP: AN ENERGY CARRIER

1	IV. ATP: AN ENERGY CARRIER Reactions or processes that have a large positive ∆G, such as moving ions against a concentration gradient across a cell membrane, are made possible by coupling the endergonic movement of ions with a second, spontaneous process with a large negative ∆G such as the exergonic hydrolysis of ATP (see p. 87). [Note: In the absence of enzymes, ATP is a stable molecule because its hydrolysis has a high Ea.] Figure 6.4 shows a mechanical model of energy coupling. The simplest example of energy coupling in biologic reactions occurs when the energy-requiring and the energy-yielding reactions share a common intermediate. A. Common intermediates Two chemical reactions have a common intermediate when they occur sequentially in that the product of the first reaction is a substrate for the second. For example, given the reactions

1	Two chemical reactions have a common intermediate when they occur sequentially in that the product of the first reaction is a substrate for the second. For example, given the reactions D is the common intermediate and can serve as a carrier of chemical energy between the two reactions. [Note: The intermediate may be linked to an enzyme.] Many coupled reactions use ATP to generate a common intermediate. These reactions may involve the transfer of a phosphate group from ATP to another molecule. Other reactions involve the transfer of phosphate from an energy-rich intermediate to adenosine diphosphate (ADP), forming ATP. B. Energy carried by ATP

1	B. Energy carried by ATP ATP consists of a molecule of adenosine (adenine + ribose) to which three phosphate groups are attached (Fig. 6.5). Removal of one phosphate produces ADP, and removal of two phosphates produces adenosine monophosphate (AMP). For ATP, the ∆G0 of hydrolysis is approximately – 7.3 kcal/mol for each of the two terminal phosphate groups. Because of this large negative ∆G0 of hydrolysis, ATP is called a high-energy phosphate compound. [Note: Adenine nucleotides are interconverted (2 ADP ⇄ ATP + AMP) by adenylate kinase.] V. ELECTRON TRANSPORT CHAIN

1	Energy-rich molecules, such as glucose, are metabolized by a series of oxidation reactions ultimately yielding carbon dioxide and water (H2O), as shown in Figure 6.6. The metabolic intermediates of these reactions donate electrons to specific coenzymes, nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD), to form the energy-rich reduced forms, NADH and FADH2. These reduced coenzymes can, in turn, each donate a pair of electrons to a specialized set of electron carriers, collectively called the electron transport chain (ETC), described in this section. As electrons are passed down the ETC, they lose much of their free energy. This energy is used to move H+ across the inner mitochondrial membrane, creating a H+ gradient that drives the production of ATP from ADP and inorganic phosphate (Pi), described on p. 77. The coupling of electron transport with ATP synthesis is called oxidative phosphorylation, sometimes denoted as OXPHOS. It proceeds continuously in all

1	inorganic phosphate (Pi), described on p. 77. The coupling of electron transport with ATP synthesis is called oxidative phosphorylation, sometimes denoted as OXPHOS. It proceeds continuously in all tissues that contain mitochondria. [Note: The free energy not trapped as ATP is used to drive ancillary reactions such as transport of calcium ions into mitochondria and to generate heat.]

1	A. Mitochondrial electron transport chain The ETC (except for cytochrome c, see p. 75) is located in the inner mitochondrial membrane and is the final common pathway by which electrons derived from different fuels of the body flow to oxygen (O2), reducing it to H2O (see Fig. 6.6).

1	1. Mitochondrial membranes: The mitochondrion contains an outer and an inner membrane separated by the intermembrane space. Although the outer membrane contains special channels (formed by the protein porin), making it freely permeable to most ions and small molecules, the inner membrane is a specialized structure that is impermeable to most small ions, including H+, and small molecules such as ATP, ADP, pyruvate, and other metabolites important to mitochondrial function (Fig. 6.7). Specialized carriers or transport systems are required to move ions or molecules across this membrane. The inner mitochondrial membrane is unusually rich in proteins, over half of which are directly involved in oxidative phosphorylation. It also contains convolutions, called cristae, which greatly increase its surface area.

1	2. Mitochondrial matrix: The gel-like solution of the matrix (interior) of mitochondria is also rich in proteins. These include the enzymes responsible for the oxidation of pyruvate, amino acids, and fatty acids (by β-oxidation) as well as those of the tricarboxylic acid (TCA) cycle. The synthesis of glucose, urea, and heme occurs partially in the matrix of mitochondria. In addition, the matrix contains NAD+ and FAD (the oxidized forms of the two coenzymes that are required as electron acceptors), and ADP and Pi, which are used to produce ATP. [Note: The matrix also contains mitochondrial deoxyribonucleic acid (mtDNA), ribonucleic acid (mtRNA), and ribosomes.] B. Organization

1	B. Organization The inner mitochondrial membrane contains four separate protein complexes, called Complexes I, II, III, and IV that each contain part of the ETC (Fig. 6.8). These complexes accept or donate electrons to the relatively mobile electron carrier coenzyme Q (CoQ) and cytochrome c. Each carrier in the ETC can receive electrons from an electron donor and can subsequently donate electrons to the next acceptor in the chain. The electrons ultimately combine with O2 and H+ to form H2O. This requirement for O2 makes the electron transport process the respiratory chain, which accounts for the greatest portion of the body’s use of O2. C. Reactions

1	C. Reactions With the exception of CoQ, which is a lipid-soluble quinone, all members of the ETC are proteins. These may function as enzymes as is the case with the flavin-containing dehydrogenases, may contain iron as part of an iron-sulfur (Fe-S) center, may contain iron as part of the porphyrin prosthetic group of heme as in the cytochromes, or may contain copper (Cu) as does the cytochrome a + a3 complex. 1. NADH formation: NAD+ is reduced to NADH by dehydrogenases that remove two hydrogen atoms from their substrate. [Note: For examples of these reactions, see the discussion of the dehydrogenases of the TCA cycle, p. 112.] Both electrons but only one H+ (that is, a hydride ion [:H−]) are transferred to the NAD+, forming NADH plus a free H+. 2.

1	2. NADH dehydrogenase: The free H+ plus the hydride ion carried by NADH are transferred to NADH dehydrogenase, a protein complex (Complex I) embedded in the inner mitochondrial membrane. Complex I has a tightly bound molecule of flavin mononucleotide (FMN), a coenzyme structurally related to FAD (see Fig. 28.15, p. 384) that accepts the two hydrogen atoms (2 electrons + 2 H+), becoming FMNH2. NADH dehydrogenase also contains peptide subunits with Fe-S centers (Fig. 6.9). At Complex I, electrons move from NADH to FMN to the iron of the Fe-S centers and then to CoQ. As electrons flow, they lose energy. This energy is used to pump four H+ across the inner mitochondrial membrane, from the matrix to the intermembrane space. 3. Succinate dehydrogenase: At Complex II, electrons from the succinate dehydrogenase–catalyzed oxidation of succinate to fumarate move from the coenzyme, FADH2, to an Fe-S protein, and then to CoQ. [Note:

1	Because no energy is lost in this process, no H+ are pumped at Complex II.] 4. Coenzyme Q: CoQ is a quinone derivative with a long, hydrophobic isoprenoid tail. It is made from an intermediate of cholesterol synthesis (see p. 221). [Note: It is also called ubiquinone because it is ubiquitous in biologic systems.] CoQ is a mobile electron carrier and can accept electrons from NADH dehydrogenase (Complex I), from succinate dehydrogenase (Complex II) and from other mitochondrial dehydrogenases, such as glycerol 3-phosphate dehydrogenase (see p. 80) and acyl CoA dehydrogenases (see p. 192). CoQ transfers electrons to Complex III (cytochrome bc1). Thus, a function of CoQ is to link the flavoprotein dehydrogenases to the cytochromes. 5.

1	80) and acyl CoA dehydrogenases (see p. 192). CoQ transfers electrons to Complex III (cytochrome bc1). Thus, a function of CoQ is to link the flavoprotein dehydrogenases to the cytochromes. 5. Cytochromes: The remaining members of the ETC are cytochrome proteins. Each contains a heme group (a porphyrin ring plus iron). Unlike the heme groups of hemoglobin, the cytochrome iron is reversibly converted from its ferric (Fe3+) to its ferrous (Fe2+) form as a normal part of its function as an acceptor and donor of electrons. Electrons are passed along the chain from cytochrome bc1 (Complex III), to cytochrome c, and then to cytochromes a + a3 ([Complex IV] see Fig. 6.8). As electrons flow, four H+ are pumped across the inner mitochondrial membrane at Complex III and two at Complex IV. [Note: Cytochrome c is located in the intermembrane space, loosely associated with the outer face of the inner membrane. As seen with CoQ, cytochrome c is a mobile electron carrier.] 6.

1	Cytochrome a + a3: Because this cytochrome complex (Complex IV) is the only electron carrier in which the heme iron has an available coordination site that can react directly with O2, it also is called cytochrome c oxidase. At Complex IV, the transported electrons, O2, and free H+ are brought together, and O2 is reduced to H2O (see Fig. 6.8). [Note: Four electrons are required to reduce one molecule of O2 to two molecules of H2O.] Cytochrome c oxidase contains Cu atoms that are required for this complicated reaction to occur. Electrons move from CuA to cytochrome a to cytochrome a3 (in association with CuB) to O2. 7.

1	7. Site-specific inhibitors: Inhibitors of specific sites in the ETC have been identified and are illustrated in Figure 6.10. These respiratory inhibitors prevent the passage of electrons by binding to a component of the chain, blocking the oxidation-reduction reaction. Therefore, all electron carriers before the block are fully reduced, whereas those located after the block are oxidized. [Note: Inhibition of the ETC inhibits ATP synthesis because these processes are tightly coupled (see p. 78).] NaN3 = sodium azide. Leakage of electrons from the ETC produces reactive oxygen species (ROS), such as superoxide (O2−·), hydrogen peroxide (H2O2), and hydroxyl radicals (OH·). ROS damage DNA and proteins and cause lipid peroxidation. Enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase are cellular defenses against ROS (see p. 148). D. Free energy release during electron transport

1	D. Free energy release during electron transport The free energy released as electrons are transferred along the ETC from an electron donor (reducing agent or reductant) to an electron acceptor (oxidizing agent or oxidant) is used to pump H+ at Complexes I, III, and IV. [Note: The electrons can be transferred as hydride ions to NAD+; as hydrogen atoms to FMN, CoQ, and FAD; or as electrons to cytochromes.] 1.

1	Redox pairs: Oxidation (loss of electrons) of one substance is always accompanied by reduction (gain of electrons) of a second. For example, Figure 6.11 shows the oxidation of NADH to NAD+ by NADH dehydrogenase at Complex I, accompanied by the reduction of FMN, the prosthetic group, to FMNH2. Such redox reactions can be written as the sum of two separate half reactions, one an oxidation and the other a reduction (see Fig. 6.11). NAD+ and NADH form a redox pair, as do FMN and FMNH2. Redox pairs differ in their tendency to lose electrons. This tendency is a characteristic of a particular redox pair and can be quantitatively specified by a constant, E0 (the standard reduction potential), with units in volts. 2.

1	2. Standard reduction potential: The E0 of various redox pairs can be ordered from the most negative E0 to the most positive. The more negative the E0 of a redox pair, the greater the tendency of the reductant member of that pair to lose electrons. The more positive the E0, the greater the tendency of the oxidant member of that pair to accept electrons. Therefore, electrons flow from the pair with the more negative E0 to that with the more positive E0. The E0 values for some members of the ETC are shown in Figure 6.12. [Note: The components of the chain are arranged in order of increasingly positive E0 values.] 3. Relationship of ∆G0 to ∆E0: The ∆G0 is related directly to the magnitude of the change in E0: where n = number of electrons transferred (1 for a cytochrome, 2 for NADH, FADH2, and CoQ)

1	F = Faraday constant (23.1 kcal/volt mol) ∆E0 = E0 of the electron-accepting pair minus the E0 of the electron-donating pair ∆G0 = change in the standard free energy 4. ∆G0 of ATP: The ∆G0 for the phosphorylation of ADP to ATP is +7.3 kcal/mol. The transport of a pair of electrons from NADH to O2 through the ETC releases 52.6 kcal. Therefore, more than sufficient energy is available to produce three ATP from three ADP and three Pi (3 × 7.3 = 21.9 kcal/mol), sometimes expressed as a P/O ratio (ATP made per O atom reduced) of 3:1. The remaining calories are used for ancillary reactions or released as heat. [Note: The P:O for FADH2 is 2:1 because Complex I is bypassed.] VI. PHOSPHORYLATION OF ADP TO ATP The transfer of electrons down the ETC is energetically favored because NADH is a strong electron donor and O2 is an avid electron acceptor. However, the flow of electrons does not directly result in ATP synthesis. A. Chemiosmotic hypothesis

1	A. Chemiosmotic hypothesis The chemiosmotic hypothesis (also known as the Mitchell hypothesis) explains how the free energy generated by the transport of electrons by the ETC is used to produce ATP from ADP + Pi. 1. Proton pump: Electron transport is coupled to ADP phosphorylation by the pumping of H+ across the inner mitochondrial membrane, from the matrix to the intermembrane space, at Complexes I, III, and IV. For each pair of electrons transferred from NADH to O2, 10 H+ are pumped. This creates an electrical gradient (with more positive charges on the cytosolic side of the membrane than on the matrix side) and a pH (chemical) gradient (the cytosolic side of the membrane is at a lower pH than the matrix side), as shown in Figure 6.13. The energy (proton-motive force) generated by these gradients is sufficient to drive ATP synthesis. Thus, the H+ gradient serves as the common intermediate that couples oxidation to phosphorylation. 2.

1	ATP synthase: The multisubunit enzyme ATP synthase ([Complex V] Fig. 6.14) synthesizes ATP using the energy of the H+ gradient. It contains a membrane domain (Fo) that spans the inner mitochondrial membrane and an extramembranous domain (F1) that appears as a sphere that protrudes into the mitochondrial matrix (see Fig. 6.13). The chemiosmotic hypothesis proposes that after H+ have been pumped to the cytosolic side of the inner mitochondrial membrane, they reenter the matrix by passing through a H+ channel in the Fo domain, driving rotation of the c ring of Fo and, at the same time, dissipating the pH and electrical gradients. Rotation in Fo causes conformational changes in the three β subunits of F1 that allow them to bind ADP + Pi, phosphorylate ADP to ATP, and release ATP. One complete rotation of the c ring produces three ATP. [Note: ATP synthase is also called F1/Fo-ATPase because the enzyme can also catalyze the hydrolysis of ATP to ADP and Pi.] contains eight subunits. One

1	rotation of the c ring produces three ATP. [Note: ATP synthase is also called F1/Fo-ATPase because the enzyme can also catalyze the hydrolysis of ATP to ADP and Pi.] contains eight subunits. One complete turn of the ring is driven by eight H+ (protons) moving through the Fo domain. The resulting conformational changes in the three β subunits of the F1 domain allow phosphorylation of three adenosine diphosphates (ADP) to three ATP.] Pi = inorganic phosphate.

1	a. Coupling in oxidative phosphorylation: In normal mitochondria, ATP synthesis is coupled to electron transport through the H+ gradient. Increasing (or decreasing) one process has the same effect on the other. For example, hydrolysis of ATP to ADP and Pi in energy-requiring reactions increases the availability of substrates for ATP synthase and, thus, increases H+ flow through the enzyme. Electron transport and H+ pumping by the ETC increase to maintain the H+ gradient and allow ATP synthesis.

1	b. Oligomycin: This drug binds to the Fo (hence the letter “o”) domain of ATP synthase, closing the H+ channel and preventing reentry of H+ into the matrix, thereby inhibiting phosphorylation of ADP to ATP. Because the pH and electrical gradients cannot be dissipated in the presence of this phosphorylation inhibitor, electron transport stops because of the difficulty of pumping any more H+ against the steep gradient. This dependency of cellular respiration on the ability to phosphorylate ADP to ATP is known as respiratory control and is the consequence of the tight coupling of these processes. c.

1	c. Uncoupling proteins: Uncoupling proteins (UCP) occur in the inner mitochondrial membrane of mammals, including humans. These proteins form channels that allow H+ to reenter the mitochondrial matrix without energy being captured as ATP (Fig. 6.15). The energy is released as heat, and the process is called nonshivering thermogenesis. UCP1, also called thermogenin, is responsible for heat production in the mitochondria-rich brown adipocytes of mammals. [Note: Cold causes catecholamine-dependent activation of UCP1 expression.] In brown fat, unlike the more abundant white fat, ~90% of its respiratory energy is used for thermogenesis in infants in response to cold. Thus, brown fat is involved in energy expenditure, whereas white fat is involved in energy storage. [Note: Brown fat depots have recently been shown to be present in adults.] d.

1	Synthetic uncouplers: Electron transport and phosphorylation of ADP can also be uncoupled by compounds that shuttle H+ across the inner mitochondrial membrane, dissipating the gradient. The classic example is 2,4-dinitrophenol, a lipophilic H+ carrier (ionophore) that readily diffuses through the mitochondrial membrane (Fig. 6.16). This uncoupler causes electron transport to proceed at a rapid rate without establishing a H+ gradient, much as do the UCP. Again, energy is released as heat rather than being used to synthesize ATP. [Note: In high doses, aspirin and other salicylates uncouple oxidative phosphorylation. This explains the fever that accompanies toxic overdoses of these drugs.] B. Membrane transport systems The inner mitochondrial membrane is impermeable to most charged or hydrophilic substances. However, it contains numerous transport proteins that permit passage of certain molecules from the cytosol to the mitochondrial matrix. 1.

1	1. ATP and ADP transport: The inner membrane requires specialized carriers to transport ADP and Pi from the cytosol (where ATP is hydrolyzed to ADP in many energy-requiring reactions) into mitochondria, where ATP can be resynthesized. An adenine nucleotide antiporter imports one ADP from the cytosol into the matrix, while exporting one ATP from the matrix into the cytosol (see Fig. 6.13). A symporter cotransports Pi and H+ from the cytosol into the matrix. 2.

1	Reducing equivalent transport: The inner mitochondrial membrane lacks an NADH transporter, and NADH produced in the cytosol (for example, in glycolysis; see p. 101) cannot directly enter the mitochondrial matrix. However, reducing equivalents of NADH are transported from the cytosol into the matrix using substrate shuttles. In the glycerol 3phosphate shuttle (Fig. 6.17A), two electrons are transferred from NADH to dihydroxyacetone phosphate by cytosolic glycerol 3-phosphate dehydrogenase. The glycerol 3-phosphate produced is oxidized by the mitochondrial isozyme as FAD is reduced to FADH2. CoQ of the ETC oxidizes the FADH2. Therefore, the glycerol 3-phosphate shuttle results in the synthesis of two ATP for each cytosolic NADH oxidized. This contrasts with the malate-aspartate shuttle (Fig. 6.17B), which produces NADH (rather than FADH2) in the mitochondrial matrix, thereby yielding three ATP for each cytosolic NADH oxidized by malate dehydrogenase as oxaloacetate is reduced to malate.

1	6.17B), which produces NADH (rather than FADH2) in the mitochondrial matrix, thereby yielding three ATP for each cytosolic NADH oxidized by malate dehydrogenase as oxaloacetate is reduced to malate. A transport protein moves malate into the mitochondrial matrix.

1	inner mitochondrial membrane. A. Glycerol 3-phosphate shuttle. B. Malate aspartate shuttle. DHAP = dihydroxyacetone phosphate; NAD(H) = dinucleotide; CoQ = coenzyme Q. C. Inherited defects in oxidative phosphorylation

1	Thirteen of the ~90 polypeptides required for oxidative phosphorylation are encoded by mtDNA and synthesized in mitochondria, whereas the remaining proteins are encoded by nuclear DNA, synthesized in the cytosol, and then transported into mitochondria. Defects in oxidative phosphorylation are more likely a result of alterations in mtDNA, which has a mutation rate about 10 times greater than that of nuclear DNA. Tissues with the greatest ATP requirement (for example, the central nervous system, skeletal and heart muscle, and the liver) are most affected by defects in oxidative phosphorylation. Mutations in mtDNA are responsible for several diseases, including some cases of mitochondrial myopathies, and Leber hereditary optic neuropathy, a disease in which bilateral loss of central vision occurs as a result of neuroretinal degeneration, including damage to the optic nerve. [Note: mtDNA is maternally inherited because mitochondria from the sperm cell do not enter the fertilized egg.]

1	D. Mitochondria and apoptosis The process of apoptosis (programmed cell death) may be initiated through the intrinsic (mitochondrial-mediated) pathway by the formation of pores in the outer mitochondrial membrane. These pores allow cytochrome c to leave the intermembrane space and enter the cytosol. There, cytochrome c, in association with proapoptotic factors, activates a family of proteolytic enzymes (the caspases), causing cleavage of key proteins and resulting in the morphologic and biochemical changes characteristic of apoptosis. VII. CHAPTER SUMMARY

1	The change in free energy (∆G) occurring during a reaction predicts the direction in which that reaction will spontaneously proceed. If ∆G is negative (that is, the product has a lower free energy than the substrate), then the reaction is spontaneous as written. If ∆G is positive, then the reaction is not spontaneous. If ∆G = 0, then the reaction is in equilibrium. The ∆G of the forward reaction is equal in magnitude but opposite in sign to that of the back reaction. The ∆G are additive in any sequence of consecutive reactions, as are the standard free energy changes (∆G0). Therefore, reactions or processes that have a large, positive ∆G are made possible by coupling with those that have a large, negative ∆G such as ATP hydrolysis. The reduced coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) each donate a pair of electrons to a specialized set of electron carriers, consisting of flavin mononucleotide (FMN), iron-sulfur centers, coenzyme Q, and

1	and flavin adenine dinucleotide (FADH2) each donate a pair of electrons to a specialized set of electron carriers, consisting of flavin mononucleotide (FMN), iron-sulfur centers, coenzyme Q, and a series of heme-containing cytochromes, collectively called the electron transport chain. This pathway is present in the inner mitochondrial membrane (impermeable to most substances) and is the final common pathway by which electrons derived from different fuels of the body flow to oxygen (O2), which has a large, positive reduction potential (E0), reducing it to water. The terminal cytochrome, cytochrome c oxidase, is the only cytochrome able to bind O2. Electron transport results in the pumping of protons (H+) across the inner mitochondrial membrane from the matrix to the intermembrane space, 10 H+ per NADH oxidized. This process creates electrical and pH gradients across the inner mitochondrial membrane. After H+ have been transferred to the cytosolic side of the membrane, they reenter the

1	H+ per NADH oxidized. This process creates electrical and pH gradients across the inner mitochondrial membrane. After H+ have been transferred to the cytosolic side of the membrane, they reenter the matrix by passing through the Fo H+ channel in ATP synthase (Complex V), dissipating the pH and electrical gradients and causing conformational changes in the F1 β subunits of the synthase that result in the synthesis of ATP from ADP + inorganic phosphate. Electron transport and phosphorylation are tightly coupled in oxidative phosphorylation ([OXPHOS] Fig. 6.18). Inhibition of one process inhibits the other. These processes can be uncoupled by uncoupling protein-1 of the inner mitochondrial membrane of brown adipocytes and by synthetic compounds such as 2,4-dinitrophenol and aspirin, all of which dissipate the H+ gradient. In uncoupled mitochondria, the energy produced by electron transport is released as heat rather than being used to synthesize ATP. Mutations in mitochondrial DNA, which

1	dissipate the H+ gradient. In uncoupled mitochondria, the energy produced by electron transport is released as heat rather than being used to synthesize ATP. Mutations in mitochondrial DNA, which is maternally inherited, are responsible for some cases of mitochondrial diseases such as Leber hereditary optic neuropathy. The release of cytochrome c into the cytoplasm and subsequent activation of proteolytic caspases results in apoptotic cell death.

1	Choose the ONE best answer. .1. 2,4-Dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, was used as a weight-loss agent in the 1930s. Reports of fatal overdoses led to its discontinuation in 1939. Which of the following would most likely be true concerning individuals taking 2,4-DNP? A. ATP levels in the mitochondria are greater than normal. B. Body temperature is elevated as a result of hypermetabolism. C. Cyanide has no effect on electron flow. D. The proton gradient across the inner mitochondrial membrane is greater than normal. E. The rate of electron transport is abnormally low.

1	C. Cyanide has no effect on electron flow. D. The proton gradient across the inner mitochondrial membrane is greater than normal. E. The rate of electron transport is abnormally low. Correct answer = B. When phosphorylation is uncoupled from electron flow, a decrease in the proton gradient across the inner mitochondrial membrane and, therefore, impaired ATP synthesis are expected. In an attempt to compensate for this defect in energy capture, metabolism and electron flow to oxygen are increased. This hypermetabolism will be accompanied by elevated body temperature because the energy in fuels is largely wasted, appearing as heat. The electron transport chain will still be inhibited by cyanide. .2. Which of the following has the strongest tendency to gain electrons? A. Coenzyme Q B. Cytochrome c C. Flavin adenine dinucleotide D. Nicotinamide adenine dinucleotide E. Oxygen

1	.2. Which of the following has the strongest tendency to gain electrons? A. Coenzyme Q B. Cytochrome c C. Flavin adenine dinucleotide D. Nicotinamide adenine dinucleotide E. Oxygen Correct answer = E. Oxygen is the terminal acceptor of electrons in the electron transport chain (ETC). Electrons flow down the ETC to oxygen because it has the highest (most positive) reduction potential (E0). The other choices precede oxygen in the ETC and have lower E0 values. .3. Explain why and how the malate-aspartate shuttle moves nicotinamide adenine dinucleotide reducing equivalents from the cytosol to the mitochondrial matrix.

1	.3. Explain why and how the malate-aspartate shuttle moves nicotinamide adenine dinucleotide reducing equivalents from the cytosol to the mitochondrial matrix. There is no transporter for nicotinamide adenine dinucleotide (NADH) in the inner mitochondrial membrane. However, cytoplasmic NADH can be oxidized to NAD+ by malate dehydrogenase as oxaloacetate (OAA) is reduced to malate. The malate is transported across the inner membrane to the matrix where the mitochondrial isozyme of malate dehydrogenase oxidizes it to OAA as mitochondrial NAD+ is reduced to NADH. This NADH can be oxidized by Complex I of the electron transport chain, generating three ATP through the coupled processes of oxidative phosphorylation. .4. Carbon monoxide (CO) binds to and inhibits Complex IV of the electron transport chain. What effect, if any, should this respiratory inhibitor have on phosphorylation of adenosine diphosphate (ADP) to ATP?

1	Inhibition of electron transport by respiratory inhibitors such as CO results in an inability to maintain the proton (H+) gradient. Therefore, phosphorylation of ADP to ATP is inhibited, as are ancillary reactions such as calcium uptake by mitochondria, because they also require the H+ gradient. Introduction to Carbohydrates 7 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Introduction to Carbohydrates 7 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW Carbohydrates (saccharides) are the most abundant organic molecules in nature. They have a wide range of functions, including providing a significant fraction of the dietary calories for most organisms, acting as a storage form of energy in the body, and serving as cell membrane components that mediate some forms of intercellular communication. Carbohydrates also serve as a structural component of many organisms, including the cell walls of bacteria, the exoskeleton of insects, and the fibrous cellulose of plants. [Note: The full set of carbohydrates produced by an organism is its glycome.] The empiric formula for many of the simpler carbohydrates is (CH2O)n, where n ≥3, hence the name “hydrate of carbon.” II. CLASSIFICATION AND STRUCTURE

1	Monosaccharides (simple sugars) can be classified according to the number of carbon atoms they contain. Examples of some monosaccharides commonly found in humans are listed in Figure 7.1. They can also be classified by the type of carbonyl group they contain. Carbohydrates with an aldehyde as their carbonyl group are called aldoses, whereas those with a keto as their carbonyl group are called ketoses (Fig. 7.2). For example, glyceraldehyde is an aldose, whereas dihydroxyacetone is a ketose. Carbohydrates that have a free carbonyl group have the suffix -ose. [Note: Ketoses have an additional “ul” in their suffix such as xylulose. There are exceptions, such as fructose, to this rule.] Monosaccharides can be linked by glycosidic bonds to create larger structures (Fig. 7.3). Disaccharides contain two monosaccharide units, oligosaccharides contain three to ten monosaccharide units, and polysaccharides contain more than ten monosaccharide units and can be hundreds of sugar units in length.

1	A. Isomers and epimers Compounds that have the same chemical formula but have different structures are called isomers. For example, fructose, glucose, mannose, and galactose are all isomers of each other, having the same chemical formula, C6H12O6. Carbohydrate isomers that differ in configuration around only one specific carbon atom (with the exception of the carbonyl carbon, see C. 1. below) are defined as epimers of each other. For example, glucose and galactose are C-4 epimers because their structures differ only in the position of the –OH (hydroxyl) group at carbon 4. [Note: The carbons in sugars are numbered beginning at the end that contains the carbonyl carbon (that is, the aldehyde or keto group), as shown in Fig. 7.4.] Glucose and mannose are C-2 epimers. However, because galactose and mannose differ in the position of –OH groups at two carbons (carbons 2 and 4), they are isomers rather than epimers (see Fig. 7.4). B. Enantiomers

1	B. Enantiomers A special type of isomerism is found in the pairs of structures that are mirror images of each other. These mirror images are called enantiomers, and the two members of the pair are designated as a D-and an L-sugar (Fig. 7.5). The vast majority of the sugars in humans are D-isomers. In the D-isomeric form, the –OH group on the asymmetric carbon (a carbon linked to four different atoms or groups) farthest from the carbonyl carbon is on the right, whereas in the L-isomer, it is on the left. Most enzymes are specific for either the D or the L form, but enzymes known as isomerases are able to interconvert D-and L-isomers. comparison to a triose, glyceraldehyde. [Note: The asymmetric carbons are shown in green.] C. Monosaccharide cyclization

1	comparison to a triose, glyceraldehyde. [Note: The asymmetric carbons are shown in green.] C. Monosaccharide cyclization Less than 1% of each of the monosaccharides with five or more carbons exists in the open-chain (acyclic) form in solution. Rather, they are predominantly found in a ring (cyclic) form, in which the aldehyde (or keto) group has reacted with a hydroxyl group on the same sugar, making the carbonyl carbon (carbon 1 for an aldose, carbon 2 for a ketose) asymmetric. This asymmetric carbon is referred to as the anomeric carbon.

1	1. Anomers: Creation of an anomeric carbon (the former carbonyl carbon) generates a new pair of isomers, the α and β configurations of the sugar (for example, α-D-glucopyranose and β-D-glucopyranose), as shown in Figure 7.6, that are anomers of each other. [Note: In the α configuration, the –OH group on the anomeric carbon projects to the same side as the ring in a modified Fischer projection formula (see Fig. 7.6A) and is trans to the CH2OH group in a Haworth projection formula (see Fig. 7.6B).

1	The α and β forms are not mirror images, and they are referred to as diastereomers.] Enzymes are able to distinguish between these two structures and use one or the other preferentially. For example, glycogen is synthesized from α-D-glucopyranose, whereas cellulose is synthesized from β-D-glucopyranose. The cyclic α and β anomers of a sugar in solution spontaneously (but slowly) form an equilibrium mixture, a process known as mutarotation (see Fig. 7.6). [Note: For glucose, the α form makes up 36% of the mixture.] six-membered ring (5 C + 1 O) is termed a pyranose, whereas one with a five-membered ring (4 C + 1 O) is a furanose. Virtually all glucose in solution is in the pyranose form.] 2. Reducing sugars: If the hydroxyl group on the anomeric carbon of a cyclized sugar is not linked to another compound by a glycosidic bond (see E. below), the ring can open. The sugar can act as a reducing agent and is termed a reducing sugar. Such sugars can react with chromogenic agents (for

1	to another compound by a glycosidic bond (see E. below), the ring can open. The sugar can act as a reducing agent and is termed a reducing sugar. Such sugars can react with chromogenic agents (for example, the Benedict reagent) causing the reagent to be reduced and colored as the aldehyde group of the acyclic sugar is oxidized to a carboxyl group. All monosaccharides, but not all disaccharides, are reducing sugars. [Note: Fructose, a ketose, is a reducing sugar because it can be isomerized to an aldose.]

1	A colorimetric test can detect a reducing sugar in urine. A positive result is indicative of an underlying pathology (because sugars are not normally present in urine) and can be followed up by more specific tests to identify the reducing sugar. D. Monosaccharide joining Monosaccharides can be joined to form disaccharides, oligosaccharides, and polysaccharides. Important disaccharides include lactose (galactose + glucose), sucrose (glucose + fructose), and maltose (glucose + glucose). Important polysaccharides include branched glycogen (from animal sources) and starch (plant sources) and unbranched cellulose (plant sources). Each is a polymer of glucose. E. Glycosidic bonds

1	E. Glycosidic bonds The bonds that link sugars are called glycosidic bonds. They are formed by enzymes known as glycosyltransferases that use nucleotide sugars (activated sugars) such as uridine diphosphate glucose as substrates. Glycosidic bonds between sugars are named according to the numbers of the connected carbons and with regard to the position of the anomeric hydroxyl group of the first sugar involved in the bond. If this anomeric hydroxyl is in the α configuration, then the linkage is an α-bond. If it is in the β configuration, then the linkage is a β-bond. Lactose, for example, is synthesized by forming a glycosidic bond between carbon 1 of β-galactose and carbon 4 of glucose. Therefore, the linkage is a β(1→4) glycosidic bond (see Fig. 7.3). [Note: Because the anomeric end of the glucose residue is not involved in the glycosidic linkage, it (and, therefore, lactose) remains a reducing sugar.] F. Carbohydrate linkage to noncarbohydrates

1	F. Carbohydrate linkage to noncarbohydrates Carbohydrates can be attached by glycosidic bonds to noncarbohydrate structures, including purine and pyrimidine bases (found in nucleic acids), aromatic rings (such as those found in steroids and bilirubin), proteins (found in glycoproteins and proteoglycans), and lipids (found in glycolipids). If the group on the noncarbohydrate molecule to which the sugar is attached is an –NH2 group, then the bond is called an N-glycosidic link. If the group is an –OH, then the bond is an O-glycosidic link (Fig. 7.7). [Note: All sugar-sugar glycosidic bonds are O-type linkages.] III. DIETARY CARBOHYDRATE DIGESTION

1	III. DIETARY CARBOHYDRATE DIGESTION The principal sites of dietary carbohydrate digestion are the mouth and intestinal lumen. This digestion is rapid and is catalyzed by enzymes known as glycoside hydrolases (glycosidases) that hydrolyze glycosidic bonds (Fig. 7.8). Because little monosaccharide is present in diets of mixed animal and plant origin, the enzymes are primarily endoglycosidases that hydrolyze polysaccharides and oligosaccharides and disaccharidases that hydrolyze tri-and disaccharides into their reducing sugar components. Glycosidases are usually specific for the structure and configuration of the glycosyl residue to be removed as well as for the type of bond to be broken. The final products of carbohydrate digestion are the monosaccharides glucose, galactose, and fructose that are absorbed by cells (enterocytes) of the small intestine. A. Salivary α-amylase

1	A. Salivary α-amylase The major dietary polysaccharides are of plant (starch, composed of amylose and amylopectin) and animal (glycogen) origin. During mastication (chewing), salivary α-amylase acts briefly on dietary starch and glycogen, hydrolyzing random α(1→4) bonds. [Note: There are both α(1→4)-and β(1→4)-endoglucosidases in nature, but humans do not produce the latter. Therefore, we are unable to digest cellulose, a carbohydrate of plant origin containing β(1→4) glycosidic bonds between glucose residues.] Because branched amylopectin and glycogen also contain α(1→6) bonds, which α-amylase cannot hydrolyze, the digest resulting from its action contains a mixture of short, branched and unbranched oligosaccharides known as dextrins (Fig. 7.9). [Note: Disaccharides are also present as they, too, are resistant to amylase.] Carbohydrate digestion halts temporarily in the stomach, because the high acidity inactivates salivary α-amylase. B. Pancreatic α-amylase

1	Carbohydrate digestion halts temporarily in the stomach, because the high acidity inactivates salivary α-amylase. B. Pancreatic α-amylase When the acidic stomach contents reach the small intestine, they are neutralized by bicarbonate secreted by the pancreas, and pancreatic αamylase continues the process of starch digestion. C. Intestinal disaccharidases

1	C. Intestinal disaccharidases The final digestive processes occur primarily at the mucosal lining of the duodenum and upper jejunum and include the action of several disaccharidases (see Fig. 7.9). For example, isomaltase cleaves the α(1→6) bond in isomaltose, and maltase cleaves the α(1→4) bond in maltose and maltotriose, each producing glucose. Sucrase cleaves the α(1→2) bond in sucrose, producing glucose and fructose, and lactase (β-galactosidase) cleaves the β(1→4) bond in lactose, producing galactose and glucose. [Note: The substrates for isomaltase are broader than its name suggests, and it hydrolyzes the majority of maltose.] Trehalose, an α(1→1) disaccharide of glucose found in mushrooms and other fungi, is cleaved by trehalase. These enzymes are transmembrane proteins of the brush border on the luminal (apical) surface of the enterocytes.

1	Sucrase and isomaltase are enzymic activities of a single protein that is cleaved into two functional subunits, which remain associated in the cell membrane and form the sucrase-isomaltase (SI) complex. In contrast, maltase is one of two enzymic activities of the single membrane protein maltase-glucoamylase (MGA) that does not get cleaved. Its second enzymic activity, glucoamylase, cleaves α(1→4) glycosidic bonds in dextrins. D. Intestinal absorption of monosaccharides

1	D. Intestinal absorption of monosaccharides The upper jejunum absorbs the bulk of the monosaccharide products of digestion. However, different sugars have different mechanisms of absorption (Fig. 7.10). For example, galactose and glucose are taken into enterocytes by secondary active transport that requires a concurrent uptake (symport) of sodium (Na+) ions. The transport protein is the sodium-dependent glucose cotransporter 1 (SGLT-1). [Note: Sugar transport is driven by the Na+ gradient created by the Na+-potassium (K+) ATPase that moves Na+ out of the enterocyte and K+ in (see Fig. 7.10).] Fructose absorption utilizes an energy-and Na+-independent monosaccharide transporter (GLUT-5). All three monosaccharides are transported from the enterocytes into the portal circulation by yet another transporter, GLUT-2. [Note: See p. 97 for a discussion of these transporters.] E. Abnormal degradation of disaccharides

1	E. Abnormal degradation of disaccharides The overall process of carbohydrate digestion and absorption is so efficient in healthy individuals that ordinarily all digestible dietary carbohydrate is absorbed by the time the ingested material reaches the lower jejunum. However, because only monosaccharides are absorbed, any deficiency (genetic or acquired) in a specific disaccharidase activity of the intestinal mucosa causes the passage of undigested carbohydrate into the large intestine. As a consequence of the presence of this osmotically active material, water is drawn from the mucosa into the large intestine, causing osmotic diarrhea. This is reinforced by the bacterial fermentation of the remaining carbohydrate to two-and three-carbon compounds (which are also osmotically active) plus large volumes of carbon dioxide and hydrogen gas (H2), causing abdominal cramps, diarrhea, and flatulence. 1.

1	1. Digestive enzyme deficiencies: Genetic deficiencies of the individual disaccharidases result in disaccharide intolerance. Alterations in disaccharide degradation can also be caused by a variety of intestinal diseases, malnutrition, and drugs that injure the mucosa of the small intestine. For example, brush border enzymes are rapidly lost in normal individuals with severe diarrhea, causing a temporary, acquired enzyme deficiency. Therefore, patients suffering or recovering from such a disorder cannot drink or eat significant amounts of dairy products or sucrose without exacerbating the diarrhea. 2.

1	Lactose intolerance: Over 60% of the world’s adults are lactose intolerant (Fig. 7.11). This is particularly manifested in certain populations. For example, up to 90% of adults of African or Asian descent are lactase deficient. Consequently, they are less able to metabolize lactose than are individuals of Northern European origin. The age-dependent loss of lactase activity starting at approximately age 2 years represents a reduction in the amount of enzyme produced. It is thought to be caused by small variations in the DNA sequence of a region on chromosome 2 that controls expression of the gene for lactase, also on chromosome 2. Treatment for this disorder is to reduce consumption of milk; eat yogurts and some cheeses (bacterial action and aging process decrease lactose content) as well as green vegetables, such as broccoli, to ensure adequate calcium intake; use lactase-treated products; or take lactase in pill form prior to eating. [Note: Because the loss of lactase is the norm for

1	green vegetables, such as broccoli, to ensure adequate calcium intake; use lactase-treated products; or take lactase in pill form prior to eating. [Note: Because the loss of lactase is the norm for most of the world’s adults, use of the terms adult-type hypolactasia or lactase nonpersistence rather than lactose intolerance is becoming more common.] Rare cases of congenital lactase deficiency are known.

1	3. Congenital sucrase-isomaltase deficiency: This autosomal-recessive disorder results in an intolerance of ingested sucrose. Congenital SI deficiency has a prevalence of 1:5,000 in individuals of European descent and appears to be much more common (up to 1:20) in the Inuit people of Greenland and Canada. Treatment includes the dietary restriction of sucrose and enzyme replacement therapy. 4. Diagnosis: Identification of a specific enzyme deficiency can be obtained by performing oral tolerance tests with the individual disaccharides. Measurement of H2 in the breath is a reliable test for determining the amount of ingested carbohydrate not absorbed by the body, but which is metabolized instead by the intestinal flora (see Fig. 7.11). IV. CHAPTER SUMMARY

1	IV. CHAPTER SUMMARY Monosaccharides (Fig. 7.12) containing an aldehyde group are called aldoses, and those with a keto group are called ketoses. Disaccharides, oligosaccharides, and polysaccharides consist of monosaccharides linked by glycosidic bonds. Compounds with the same chemical formula but different structures are called isomers. Two monosaccharide isomers differing in configuration around one specific carbon atom (not the carbonyl carbon) are defined as epimers. In enantiomers (mirror images), the members of the sugar pair are designated as D-and L-isomers. When the aldehyde group on an acyclic sugar gets oxidized as a chromogenic agent gets reduced, that sugar is a reducing sugar. When a sugar cyclizes, an anomeric carbon is created from the carbonyl carbon of the aldehyde or keto group. The sugar can have two configurations, forming α or β anomers. A sugar can have its anomeric carbon linked to an –NH2 or an –

1	OH group on another structure through N-and O-glycosidic bonds, respectively. Salivary α-amylase initiates digestion of dietary polysaccharides (for example, starch or glycogen), producing oligosaccharides. Pancreatic α-amylase continues the process. The final digestive processes occur at the mucosal lining of the small intestine. Several disaccharidases (for example, lactase [β-galactosidase], sucrase, isomaltase, and maltase) produce monosaccharides (glucose, galactose, and fructose). These enzymes are transmembrane proteins of the luminal brush border of intestinal mucosal cells (enterocytes). Absorption of the monosaccharides requires specific transporters. If carbohydrate degradation is deficient (as a result of heredity, disease, or drugs that injure the intestinal mucosa), undigested carbohydrate will pass into the large intestine, where it can cause osmotic diarrhea. Bacterial fermentation of the material produces large volumes of carbon dioxide and hydrogen gas, causing

1	carbohydrate will pass into the large intestine, where it can cause osmotic diarrhea. Bacterial fermentation of the material produces large volumes of carbon dioxide and hydrogen gas, causing abdominal cramps, diarrhea, and flatulence. Lactose intolerance, primarily caused by the age-dependent loss of lactase (adult-type hypolactasia), is by far the most common of these deficiencies.

1	Choose the ONE best answer. .1. Which of the following statements best describes glucose? A. It is a C-4 epimer of galactose. B. It is a ketose and usually exists as a furanose ring in solution. C. It is produced from dietary starch by the action of α-amylase. D. It is utilized in biological systems only in the L-isomeric form. Correct answer = A. Because glucose and galactose differ only in configuration around carbon 4, they are C-4 epimers that are interconvertible by the action of an epimerase. Glucose is an aldose sugar that typically exists as a pyranose ring in solution. Fructose, however, is a ketose with a furanose ring. α-Amylase does not produce monosaccharides. The D-isomeric form of carbohydrates is the form typically found in biologic systems, in contrast to amino acids that typically are found in the L-isomeric form.

1	.2. A young man entered his physician’s office complaining of bloating and diarrhea. His eyes were sunken, and the physician noted additional signs of dehydration. The patient’s temperature was normal. He explained that the episode had occurred following a birthday party at which he had participated in an ice cream–eating contest. The patient reported prior episodes of a similar nature following ingestion of a significant amount of dairy products. This clinical picture is most probably due to a deficiency in the activity of: A. isomaltase. B. lactase. C. pancreatic α-amylase. D. salivary α-amylase. E. sucrase. Correct answer = B. The physical symptoms suggest a deficiency in an enzyme responsible for carbohydrate degradation. The symptoms observed following the ingestion of dairy products suggest that the patient is deficient in lactase as a result of the age-dependent reduction in expression of the enzyme.

1	.3. Routine examination of the urine of an asymptomatic pediatric patient showed a positive reaction with Clinitest (a copper reduction method of detecting reducing sugars) but a negative reaction with the glucose oxidase test for detecting glucose. Using these data, show on the chart below which of the sugars could (YES) or could not (NO) be present in the urine of this individual. Each of the listed sugars, except for sucrose and glucose, could be present in the urine of this individual. Clinitest is a nonspecific test that produces a change in color if urine is positive for reducing substances such as reducing sugars (fructose, galactose, glucose, lactose, xylulose). Because sucrose is not a reducing sugar, it is not detected by Clinitest. The glucose oxidase test will detect only glucose, and it cannot detect other sugars. The negative glucose oxidase test coupled with a positive reducing sugar test means that glucose cannot be the reducing sugar in the patient’s urine.

1	.4. Why are α-glucosidase inhibitors that are taken with meals, such as acarbose and miglitol, used in the treatment of diabetes? What effect should these drugs have on the digestion of lactose? α-Glucosidase inhibitors slow the production of glucose from dietary carbohydrates, thereby reducing the postprandial rise in blood glucose and facilitating better blood glucose control in diabetic patients. These drugs have no effect on lactose digestion because the disaccharide lactose contains a βglycosidic bond, not an α-glycosidic bond. Introduction to Metabolism and Glycolysis 8 For additional ancillary materials related to this chapter, please visit thePoint. I. METABOLISM OVERVIEW

1	In Chapter 5, individual enzymic reactions were analyzed in an effort to explain the mechanisms of catalysis. However, in cells, these reactions rarely occur in isolation. Instead, they are organized into multistep sequences called pathways, such as that of glycolysis (Fig. 8.1). In a pathway, the product of one reaction serves as the substrate of the subsequent reaction. Most pathways can be classified as either catabolic (degradative) or anabolic (synthetic). Catabolic pathways break down complex molecules, such as proteins, polysaccharides, and lipids, to a few simple molecules (for example, carbon dioxide, ammonia, and water). Anabolic pathways form complex end products from simple precursors, for example, the synthesis of the polysaccharide glycogen from glucose. [Note: Pathways that regenerate a component are called cycles.] Different pathways can intersect, forming an integrated and purposeful network of chemical reactions. Metabolism is the sum of all the chemical changes

1	that regenerate a component are called cycles.] Different pathways can intersect, forming an integrated and purposeful network of chemical reactions. Metabolism is the sum of all the chemical changes occurring in a cell, a tissue, or the body. The next several chapters focus on the central metabolic pathways that are involved in synthesizing and degrading carbohydrates, lipids, and amino acids.

1	A. Metabolic map Metabolism is best understood by examining its component pathways. Each pathway is composed of multienzyme sequences, and each enzyme, in turn, may exhibit important catalytic or regulatory features. A metabolic map containing the important central pathways of energy metabolism is presented in Figure 8.2. This “big picture” view of metabolism is useful in tracing connections between pathways, visualizing the purposeful movement of metabolic intermediates (metabolites), and depicting the effect on the flow of intermediates if a pathway is blocked (for example, by a drug or an inherited deficiency of an enzyme). [Note: The metabolome is the full complement of metabolites in an organism.] Throughout the next three units of this book, each pathway under discussion will be repeatedly featured as part of the major metabolic map shown in Figure 8.2. intermediates of protein metabolism. UDP = uridine diphosphate; P = phosphate; -

1	intermediates of protein metabolism. UDP = uridine diphosphate; P = phosphate; - CoA = coenzyme A; CO2 = carbon dioxide; HCO3 = bicarbonate; NH3 = ammonia. B. Catabolic pathways Catabolic reactions serve to capture chemical energy in the form of ATP from the degradation of energy-rich fuel molecules. ATP generation by degradation of complex molecules occurs in three stages, as shown in Figure 8.3. [Note: Catabolic pathways are typically oxidative and require oxidized coenzymes such as nicotinamide adenine dinucleotide (NAD+).] Catabolism also allows molecules in the diet (or nutrient molecules stored in cells) to be converted into basic building blocks needed for the synthesis of complex molecules. Catabolism, then, is a convergent process (that is, a wide variety of molecules are transformed into a few common end products). 1.

1	1. Hydrolysis of complex molecules: In the first stage, complex molecules are broken down into their component building blocks. For example, proteins are degraded to amino acids, polysaccharides to monosaccharides, and fats (triacylglycerols) to free fatty acids and glycerol. 2. Conversion of building blocks to simple intermediates: In the second stage, these diverse building blocks are further degraded to acetyl coenzyme A (CoA) and a few other simple molecules. Some energy is captured as ATP, but the amount is small compared with the energy produced during the third stage of catabolism. 3. Oxidation of acetyl coenzyme A: The tricarboxylic acid (TCA) cycle (see p. 109) is the final common pathway in the oxidation of fuel molecules that produce acetyl CoA. Oxidation of acetyl CoA generates large amounts of ATP via oxidative phosphorylation as electrons flow from NADH and flavin adenine dinucleotide (FADH2) to oxygen ([O2] see p. 73). C. Anabolic pathways

1	73). C. Anabolic pathways In contrast to catabolism, anabolism is a divergent process in which a few biosynthetic precursors (such as amino acids) form a wide variety of polymeric, or complex, products (such as proteins [Fig. 8.4]). Anabolic reactions require energy (are endergonic), which is generally provided by the hydrolysis of ATP to adenosine diphosphate (ADP) and inorganic phosphate (Pi). [Note: Catabolic reactions generate energy (are exergonic).] Anabolic reactions often involve chemical reductions in which the reducing power is most frequently provided by the electron donor NADPH (phosphorylated NADH, see p. 147). II. METABOLISM REGULATION

1	Anabolic reactions often involve chemical reductions in which the reducing power is most frequently provided by the electron donor NADPH (phosphorylated NADH, see p. 147). II. METABOLISM REGULATION The pathways of metabolism must be coordinated so that the production of energy or the synthesis of end products meets the needs of the cell. Furthermore, individual cells function as part of a community of interacting tissues, not in isolation. Thus, a sophisticated communication system has evolved to coordinate the functions of the body. Regulatory signals that inform an individual cell of the metabolic state of the body as a whole include hormones, neurotransmitters, and the availability of nutrients. These, in turn, influence signals generated within the cell (Fig. 8.5). A. Intracellular communication

1	A. Intracellular communication The rate of a metabolic pathway can respond to regulatory signals that arise from within the cell. For example, the rate may be influenced by the availability of substrates, product inhibition, or alterations in the levels of allosteric activators or inhibitors. These intracellular signals typically elicit rapid responses and are important for the moment-to-moment regulation of metabolism. B. Intercellular communication

1	B. Intercellular communication The ability to respond to intercellular signals is essential for the development and survival of organisms. Signaling between cells provides for long-range integration of metabolism and usually results in a response, such as a change in gene expression, that is slower than is seen with intracellular signals. Communication between cells can be mediated, for example, by surface-to-surface contact and, in some tissues, by formation of gap junctions, allowing direct communication between the cytoplasms of adjacent cells. However, for energy metabolism, the most important route of communication is chemical signaling between cells by blood-borne hormones or by neurotransmitters. C. Second messenger systems

1	Hormones and neurotransmitters can be thought of as signals and their receptors as signal detectors. Receptors respond to a bound ligand by initiating a series of reactions that ultimately result in specific intracellular responses. Second messenger molecules, so named because they intervene between the original extracellular messenger (the neurotransmitter or hormone) and the ultimate intracellular effect, are part of the cascade of events that converts (transduces) ligand binding into a response. Two of the most widely recognized second messenger systems are the calcium/phosphatidylinositol system (see p. 205) and the adenylyl cyclase (adenylate cyclase) system, which is particularly important in regulating the pathways of intermediary metabolism. Both involve the binding of ligands, such as epinephrine or glucagon, to specific G protein–coupled receptors (GPCR) on the cell (plasma) membrane. GPCR are characterized by an extracellular ligand-binding domain, seven transmembrane α

1	such as epinephrine or glucagon, to specific G protein–coupled receptors (GPCR) on the cell (plasma) membrane. GPCR are characterized by an extracellular ligand-binding domain, seven transmembrane α helices, and an intracellular domain that interacts with trimeric G proteins (Fig. 8.6). [Note: Insulin, another key regulator of metabolism, binds a membrane tyrosine kinase receptor (see p. 311) and not a GPCR.]

1	D. Adenylyl cyclase The recognition of a chemical signal by some GPCR, such as the β-and α2adrenergic receptors, triggers either an increase or a decrease in the activity of adenylyl cyclase (AC). This is a membrane-bound enzyme that converts ATP to 3ʹ,5ʹ-adenosine monophosphate (cyclic AMP, or cAMP). The chemical signals are most often hormones or neurotransmitters, each of which binds to a unique type of GPCR. Therefore, tissues that respond to more than one signal must have several different GPCR, each of which can be linked to AC.

1	1. Guanosine triphosphate–dependent regulatory proteins: The effect of the activated, occupied GPCR on second messenger formation is indirect, mediated by specialized trimeric proteins (α, β, and γ subunits) of the cell membrane. These proteins, referred to as G proteins because the α subunit binds guanosine di-or triphosphates (GDP or GTP), form a link in the chain of communication between the receptor and AC. In the inactive form of a G protein, the α subunit is bound to GDP (Fig. 8.7). Ligand binding causes a conformational change in the receptor, triggering replacement of this GDP with GTP. The GTP-bound form of the α subunit dissociates from the βγ subunits and moves to AC, affecting enzyme activity. Many molecules of active Gα protein are formed by one activated receptor. [Note: The ability of a hormone or neurotransmitter to stimulate or inhibit AC depends on the type of Gα protein that is linked to the receptor. One type, designated Gs, stimulates AC (see Fig. 8.7), whereas

1	ability of a hormone or neurotransmitter to stimulate or inhibit AC depends on the type of Gα protein that is linked to the receptor. One type, designated Gs, stimulates AC (see Fig. 8.7), whereas another type, designated Gi, inhibits the enzyme (not shown).] The actions of the Gα–GTP complex are short-lived because Gα has an inherent GTPase activity, resulting in the rapid hydrolysis of GTP to GDP. This causes inactivation of Gα, its dissociation from AC, and its reassociation with the βγ dimer.

1	Toxins from Vibrio cholerae (cholera) and Bordetella pertussis (whooping cough) cause inappropriate activation of AC through covalent modification (ADP-ribosylation) of different G proteins. With cholera, the GTPase activity of Gαs is inhibited in intestinal cells. With whooping cough, Gαi is inactivated in respiratory tract cells.

1	2. Protein kinases: The next step in the cAMP second messenger system is the activation of a family of enzymes called cAMP-dependent protein kinases such as protein kinase A (PKA), as shown in Figure 8.8. cAMP activates PKA by binding to its two regulatory subunits, causing the release of its two catalytically active subunits. These subunits transfer phosphate from ATP to specific serine or threonine residues of protein substrates. The phosphorylated proteins may act directly on the cell’s ion channels or, if enzymes, may become activated or inhibited. [Note: Several types of protein kinases are not cAMP dependent, for example, protein kinase C, described on p. 205.] 3. Protein phosphatases: The phosphate groups added to proteins by protein kinases are removed by protein phosphatases, enzymes that hydrolytically cleave phosphate esters (see Fig. 8.8). This insures that changes in protein activity induced by phosphorylation are not permanent. 4.

1	4. cAMP hydrolysis: cAMP is rapidly hydrolyzed to 5ʹ-AMP by cAMP phosphodiesterase that cleaves the cyclic 3ʹ,5ʹ-phosphodiester bond. 5ʹAMP is not an intracellular signaling molecule. Therefore, the effects of neurotransmitter-or hormone-mediated increases of cAMP are rapidly terminated if the extracellular signal is removed. [Note: cAMP phosphodiesterase is inhibited by caffeine, a methylxanthine derivative.] III. GLYCOLYSIS OVERVIEW

1	The glycolytic pathway is used by all tissues for the oxidation of glucose to provide energy (as ATP) and intermediates for other metabolic pathways. Glycolysis is at the hub of carbohydrate metabolism because virtually all sugars, whether arising from the diet or from catabolic reactions in the body, can ultimately be converted to glucose (Fig. 8.9A). Pyruvate is the end product of glycolysis in cells with mitochondria and an adequate supply of O2. This series of ten reactions is called aerobic glycolysis because O2 is required to reoxidize the NADH formed during the oxidation of glyceraldehyde 3-phosphate (Fig. 8.9B). Aerobic glycolysis sets the stage for the oxidative decarboxylation of pyruvate to acetyl CoA, a major fuel of the TCA cycle. Alternatively, pyruvate is reduced to lactate as NADH is oxidized to NAD+ (Fig. 8.9C). This conversion of glucose to lactate is called anaerobic glycolysis because it can occur without the participation of O2. Anaerobic glycolysis allows the

1	as NADH is oxidized to NAD+ (Fig. 8.9C). This conversion of glucose to lactate is called anaerobic glycolysis because it can occur without the participation of O2. Anaerobic glycolysis allows the production of ATP in tissues that lack mitochondria (for example, red blood cells [RBC] and parts of the eye) or in cells deprived of sufficient O2 (hypoxia).

1	IV. GLUCOSE TRANSPORT INTO CELLS Glucose cannot diffuse directly into cells but enters by one of two transport systems: a sodium (Na+)-and ATP-independent transport system or a Na+-and ATP-dependent cotransport system. A. Sodium-and ATP-independent transport system This passive system is mediated by a family of 14 glucose transporter (GLUT) isoforms found in cell membranes. They are designated GLUT-1 to GLUT-14. These monomeric protein transporters exist in the membrane in two conformational states (Fig. 8.10). Extracellular glucose binds to the transporter, which then alters its conformation, transporting glucose across the cell membrane via facilitated diffusion. Because GLUT transport one molecule at a time, they are uniporters. 1. Tissue specificity: GLUT display a tissue-specific pattern of expression.

1	1. Tissue specificity: GLUT display a tissue-specific pattern of expression. For example, GLUT-3 is the primary isoform in neurons. GLUT-1 is abundant in RBC and the blood–brain barrier but is low in adult muscle, whereas GLUT-4 is abundant in muscle and adipose tissue. [Note: The number of GLUT-4 transporters active in these tissues is increased by insulin. (See p. 311 for a discussion of insulin and glucose transport.)] GLUT-2 is abundant in the liver, kidneys, and pancreatic β cells. The other GLUT isoforms also have tissue-specific distributions. 2.

1	2. Specialized functions: In facilitated diffusion, transporter-mediated glucose movement is down a concentration gradient (that is, from a high concentration to a lower one, therefore requiring no energy). For example, GLUT-1, GLUT-3, and GLUT-4 are primarily involved in glucose uptake from the blood. In contrast, GLUT-2, in the liver and kidneys, can either transport glucose into these cells when blood glucose levels are high or transport glucose from these cells when blood glucose levels are low (for example, during fasting). GLUT-5 is unusual in that it is the primary transporter for fructose (not glucose) in the small intestine and the testes (see p. 87). B. Sodium-and ATP-dependent cotransport system

1	B. Sodium-and ATP-dependent cotransport system This energy-requiring process transports glucose against (up) its concentration gradient (that is, from low extracellular concentrations to higher intracellular concentrations) as Na+ is transported down its electrochemical gradient. [Note: The gradient is created by the Na+potassium (K+) ATPase (see Fig. 7.10, p. 87).] Because this secondary active transport process requires the concurrent uptake (symport) of Na+, the transporter is a sodium-dependent glucose cotransporter (SGLT). This type of cotransport occurs in the epithelial cells of the intestine (see p. 87), renal tubules, and choroid plexus. [Note: The choroid plexus, part of the blood–brain barrier, also contains GLUT-1.] V. GLYCOLYSIS REACTIONS

1	V. GLYCOLYSIS REACTIONS The conversion of glucose to pyruvate occurs in two stages (Fig. 8.11). The first five reactions of glycolysis correspond to an energy-investment phase in which the phosphorylated forms of intermediates are synthesized at the expense of ATP. The subsequent reactions of glycolysis constitute an energy-generation phase in which a net of two molecules of ATP are formed by substrate-level phosphorylation (see p. 102) per glucose molecule metabolized. A. Glucose phosphorylation Phosphorylated sugar molecules do not readily penetrate cell membranes because there are no specific transmembrane carriers for these compounds and because they are too polar to diffuse through the lipid core of membranes. Therefore, the irreversible phosphorylation of glucose (Fig.

1	8.12) effectively traps the sugar as cytosolic glucose 6-phosphate and commits it to further metabolism in the cell. Mammals have four isozymes (I–IV) of the enzyme hexokinase that catalyze the phosphorylation of glucose to glucose 6-phosphate.

1	1. Hexokinases I–III: In most tissues, glucose phosphorylation is catalyzed by one of these isozymes of hexokinase, which is one of three regulatory enzymes of glycolysis (along with phosphofructokinase and pyruvate kinase). They are inhibited by the reaction product glucose 6-phosphate, which accumulates when further metabolism of this hexose phosphate is reduced. Hexokinases I–III have a low Michaelis constant (Km) and, therefore, a high affinity (see p. 59) for glucose. This permits the efficient phosphorylation and subsequent metabolism of glucose even when tissue concentrations of glucose are low (Fig. 8.13). However, because these isozymes have a low maximal velocity ([Vmax] see p. 57) for glucose, they do not sequester (trap) cellular phosphate in the form of phosphorylated glucose or phosphorylate more glucose than the cell can use. [Note: These isozymes have broad substrate specificity and are able to phosphorylate several hexoses in addition to glucose.] 2. Hexokinase IV: In

1	or phosphorylate more glucose than the cell can use. [Note: These isozymes have broad substrate specificity and are able to phosphorylate several hexoses in addition to glucose.] 2. Hexokinase IV: In liver parenchymal cells and pancreatic β cells, glucokinase (the hexokinase IV isozyme) is the predominant enzyme responsible for glucose phosphorylation. In β cells, glucokinase functions as a glucose sensor, determining the threshold for insulin secretion (see p. 309). [Note: Hexokinase IV also serves as a glucose sensor in hypothalamic neurons, playing a key role in the adrenergic response to hypoglycemia (see p. 315).] In the liver, the enzyme facilitates glucose phosphorylation during hyperglycemia. Despite the popular but misleading name glucokinase, the sugar specificity of the enzyme is similar to that of other hexokinase isozymes.

1	a. Kinetics: Glucokinase differs from hexokinases I–III in several important properties. For example, it has a much higher Km, requiring a higher glucose concentration for half-saturation (see Fig. 8.13). Thus, glucokinase functions only when the intracellular concentration of glucose in the hepatocyte is elevated such as during the brief period following consumption of a carbohydrate-rich meal, when high levels of glucose are delivered to the liver via the portal vein. Glucokinase has a high Vmax, allowing the liver to effectively remove the flood of glucose delivered by the portal blood. This prevents large amounts of glucose from entering the systemic circulation following such a meal, thereby minimizing hyperglycemia during the absorptive period. [Note: GLUT-2 insures that blood glucose equilibrates rapidly across the hepatocyte membrane.] b. Regulation: Glucokinase activity is not directly inhibited by glucose 6phosphate as are the other hexokinases. Instead, it is indirectly

1	equilibrates rapidly across the hepatocyte membrane.] b. Regulation: Glucokinase activity is not directly inhibited by glucose 6phosphate as are the other hexokinases. Instead, it is indirectly inhibited by fructose 6-phosphate (which is in equilibrium with glucose 6-phosphate, a product of glucokinase) and is indirectly stimulated by glucose (a substrate of glucokinase). Regulation is achieved by reversible binding to the hepatic protein glucokinase regulatory protein (GKRP). In the presence of fructose 6-phosphate, glucokinase binds tightly to GKRP and is translocated to the nucleus, thereby rendering the enzyme inactive (Fig. 8.14). When glucose levels in the blood (and also in the hepatocyte, as a result of GLUT-2) increase, glucokinase is released from GKRP, and the enzyme reenters the cytosol where it phosphorylates glucose to glucose 6-phosphate. [Note: GKRP is a competitive inhibitor of glucose use by glucokinase.]

1	Glucokinase functions as a glucose sensor in blood glucose homeostasis. Inactivating mutations of glucokinase are the cause of a rare form of diabetes, maturity onset diabetes of the young type 2 (MODY 2) that is characterized by impaired insulin secretion and hyperglycemia. B. Glucose 6-phosphate isomerization The isomerization of glucose 6-phosphate to fructose 6-phosphate is catalyzed by phosphoglucose isomerase (Fig. 8.15). The reaction is readily reversible and is not a rate-limiting or regulated step. C. Fructose 6-phosphate phosphorylation The irreversible phosphorylation reaction catalyzed by phosphofructokinase-1 (PFK-1) is the most important control point and the rate-limiting and committed step of glycolysis (Fig. 8.16). PFK-1 is controlled by the available concentrations of the substrates ATP and fructose 6-phosphate as well as by other regulatory molecules. 1.

1	1. Regulation by intracellular energy levels: PFK-1 is inhibited allosterically by elevated levels of ATP, which act as an energy-rich signal indicating an abundance of high-energy compounds. Elevated levels of citrate, an intermediate in the TCA cycle (see p. 111), also inhibit PFK-1. [Note: Inhibition by citrate favors the use of glucose for glycogen synthesis (see p. 126).] Conversely, PFK-1 is activated allosterically by high concentrations of AMP, which signal that the cell’s energy stores are depleted. 2.

1	Regulation by fructose 2,6-bisphosphate: Fructose 2,6-bisphosphate is the most potent activator of PFK-1 (see Fig. 8.16) and is able to activate the enzyme even when ATP levels are high. It is formed from fructose 6phosphate by phosphofructokinase-2 (PFK-2). Unlike PFK-1, PFK-2 is a bifunctional protein that has both the kinase activity that produces fructose 2,6-bisphosphate and the phosphatase activity that dephosphorylates fructose 2,6-bisphosphate to fructose 6-phosphate. In the liver isozyme, phosphorylation of PFK-2 inactivates the kinase domain and activates the phosphatase domain (Fig. 8.17). The opposite is seen in the cardiac isozyme. Skeletal PFK-2 is not covalently regulated. [Note: Fructose 2,6-bisphosphate is an inhibitor of fructose 1,6-bisphosphatase, an enzyme of gluconeogenesis (see p. 121). The reciprocal actions of fructose 2,6-bisphosphate on glycolysis (activation) and gluconeogenesis (inhibition) insure that both pathways are not fully active at the same time,

1	(see p. 121). The reciprocal actions of fructose 2,6-bisphosphate on glycolysis (activation) and gluconeogenesis (inhibition) insure that both pathways are not fully active at the same time, preventing a futile cycle of glucose oxidation to pyruvate followed by glucose resynthesis from pyruvate.] a.

1	During the well-fed state: Decreased levels of glucagon and elevated levels of insulin (such as occur following a carbohydrate-rich meal) cause an increase in hepatic fructose 2,6-bisphos-phate (PFK-2 is dephosphorylated) and, thus, in the rate of glycolysis (see Fig. 8.17). Therefore, fructose 2,6-bisphosphate acts as an intracellular signal of glucose abundance. b. During fasting: By contrast, the elevated levels of glucagon and low levels of insulin that occur during fasting (see p. 327) cause a decrease in hepatic fructose 2,6-bisphosphate (PFK-2 is phosphorylated). This results in inhibition of glycolysis and activation of gluconeogenesis. D. Fructose 1,6-bisphosphate cleavage

1	D. Fructose 1,6-bisphosphate cleavage Aldolase cleaves fructose 1,6-bisphosphate to dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (see Fig. 8.16). The reaction is reversible and not regulated. [Note: Aldolase B, the hepatic isoform, also cleaves fructose 1-phosphate and functions in dietary fructose metabolism (see p. 138).] E. Dihydroxyacetone phosphate isomerization Triose phosphate isomerase interconverts DHAP and glyceraldehyde 3phosphate (see Fig. 8.16). DHAP must be isomerized to glyceraldehyde 3phosphate for further metabolism by the glycolytic pathway. This isomerization results in the net production of two molecules of glyceraldehyde 3-phosphate from the cleavage products of fructose 1,6bisphosphate. [Note: DHAP is utilized in triacylglycerol synthesis (see p. 188).] F. Glyceraldehyde 3-phosphate oxidation

1	F. Glyceraldehyde 3-phosphate oxidation The conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate (1,3-BPG) by glyceraldehyde 3-phosphate dehydrogenase is the first oxidation-reduction reaction of glycolysis (Fig. 8.18). [Note: Because there is a limited amount of NAD+ in the cell, the NADH formed by the dehydrogenase reaction must be oxidized for glycolysis to continue. Two major mechanisms for oxidizing NADH to NAD+ are the reduction of pyruvate to lactate by lactate dehydrogenase (LDH) (anaerobic, see p. 96) and the electron transport chain ([ETC] aerobic, see p. 74). Because NADH cannot cross the inner mitochondrial membrane, the ETC requires the malate-aspartate and glycerol 3-phosphate substrate shuttles to move NADH reducing equivalents into the mitochondrial matrix (see p. 79).] 1.

1	1,3-Bisphosphoglycerate synthesis: The oxidation of the aldehyde group of glyceraldehyde 3-phosphate to a carboxyl group is coupled to the attachment of Pi to the carboxyl group. This phosphate group, linked to carbon 1 of the 1,3-BPG product by a high-energy bond (see p. 73), conserves much of the free energy (see p. 69) produced by the oxidation of glyceraldehyde 3-phosphate. This high-energy phosphate drives ATP synthesis in the next reaction of glycolysis. 2.

1	Arsenic poisoning: The toxicity of arsenic is due primarily to the inhibition by trivalent arsenic (arsenite) of enzymes such as the pyruvate dehydrogenase complex (PDHC), which require lipoic acid as a coenzyme (see p. 110). However, pentavalent arsenic (arsenate) can prevent net ATP and NADH production by glycolysis without inhibiting the pathway itself. It does so by competing with Pi as a substrate for glyceraldehyde 3-phosphate dehydrogenase, forming a complex that spontaneously hydrolyzes to form 3-phosphoglycerate (see Fig. 8.18). By bypassing the synthesis of and phosphate transfer from 1,3-BPG, the cell is deprived of energy usually obtained from the glycolytic pathway. [Note: Arsenate also competes with Pi binding to the F1 domain of ATP synthase (see p. 78), resulting in formation of ADP-arsenate that is rapidly hydrolyzed.] 3. 2,3-Bisphosphoglycerate synthesis in RBC: Some of the 1,3-BPG is converted to 2,3-BPG by the action of bisphosphoglycerate mutase (see Fig. 8.18).

1	of ADP-arsenate that is rapidly hydrolyzed.] 3. 2,3-Bisphosphoglycerate synthesis in RBC: Some of the 1,3-BPG is converted to 2,3-BPG by the action of bisphosphoglycerate mutase (see Fig. 8.18). 2,3-BPG, which is found in only trace amounts in most cells, is present at high concentration in RBC and serves to increase O2 delivery (see p. 31). 2,3-BPG is hydrolyzed by a phosphatase to 3phosphoglycerate, which is also an intermediate in glycolysis (see Fig. 8.18). In the RBC, glycolysis is modified by inclusion of these shunt reactions.

1	G. 3-Phosphoglycerate synthesis and ATP production When 1,3-BPG is converted to 3-phosphoglycerate, the high-energy phosphate group of 1,3-BPG is used to synthesize ATP from ADP (see Fig. 8.18). This reaction is catalyzed by phosphoglycerate kinase, which, unlike most other kinases, is physiologically reversible. Because two molecules of 1,3-BPG are formed from each glucose molecule, this kinase reaction replaces the two ATP molecules consumed by the earlier formation of glucose 6-phosphate and fructose 1,6-bisphosphate. [Note: This reaction is an example of substrate-level phosphorylation, in which the energy needed for the production of a high-energy phosphate comes from a substrate rather than from the ETC (see J. below and p. 113 for other examples).] H. Phosphate group shift The shift of the phosphate group from carbon 3 to carbon 2 of phosphoglycerate by phosphoglycerate mutase is freely reversible. I. 2-Phosphoglycerate dehydration

1	H. Phosphate group shift The shift of the phosphate group from carbon 3 to carbon 2 of phosphoglycerate by phosphoglycerate mutase is freely reversible. I. 2-Phosphoglycerate dehydration The dehydration of 2-phosphoglycerate by enolase redistributes the energy within the substrate, forming phosphoenolpyruvate (PEP), which contains a high-energy enol phosphate (see Fig. 8.18). The reaction is reversible, despite the high-energy nature of the product. [Note: Fluoride inhibits enolase, and water fluoridation reduces lactate production by mouth bacteria, decreasing dental caries (see p. 405).] J. Pyruvate synthesis and ATP production The conversion of PEP to pyruvate, catalyzed by pyruvate kinase (PK), is the third irreversible reaction of glycolysis. The high-energy enol phosphate in PEP is used to synthesize ATP from ADP and is another example of substrate-level phosphorylation (see Fig. 8.18). 1.

1	1. Feedforward regulation: PK is activated by fructose 1,6-bisphosphate, the product of the PFK-1 reaction. This feedforward (instead of the more usual feedback) regulation has the effect of linking the two kinase activities: increased PFK-1 activity results in elevated levels of fructose 1,6-bisphosphate, which activates PK. [Note: PK is inhibited by ATP.] 2. Covalent regulation in the liver: Phosphorylation by cAMP-dependent PKA leads to inactivation of the hepatic isozyme of PK (Fig. 8.19). When blood glucose levels are low, elevated glucagon increases the intracellular level of cAMP, which causes the phosphorylation and inactivation of PK in the liver only. Therefore, PEP is unable to continue in glycolysis and, instead, enters the gluconeogenesis pathway. This partly explains the observed inhibition of hepatic glycolysis and stimulation of gluconeogenesis by glucagon. Dephosphorylation of PK by a phosphatase results in reactivation of the enzyme. diphosphate.

1	3. Pyruvate kinase deficiency: Because mature RBC lack mitochondria, they are completely dependent on glycolysis for ATP production. ATP is required to meet the metabolic needs of RBC and to fuel the ion pumps necessary for the maintenance of the flexible, biconcave shape that allows them to squeeze through narrow capillaries. The anemia observed in glycolytic enzyme deficiencies is a consequence of the reduced rate of glycolysis, leading to decreased ATP production by substrate-level phosphorylation. The resulting alterations in the RBC membrane lead to changes in cell shape and, ultimately, to phagocytosis by cells of the mononuclear phagocyte system, particularly splenic macrophages. The premature death and lysis of RBC result in mild-to-severe nonspherocytic hemolytic anemia, with the severe form requiring regular transfusions. Among patients with rare genetic defects of glycolytic enzymes, the majority has a deficiency in PK. [Note: Liver PK is encoded by the same gene as the RBC

1	severe form requiring regular transfusions. Among patients with rare genetic defects of glycolytic enzymes, the majority has a deficiency in PK. [Note: Liver PK is encoded by the same gene as the RBC isozyme. However, liver cells show no effect because they can synthesize more PK and can also generate ATP by oxidative phosphorylation.] Severity depends both on the degree of enzyme deficiency (generally 5%–35% of normal levels) and on the extent to which RBC compensate by synthesizing increased levels of 2,3-BPG (see p. 31). Almost all individuals with PK deficiency have a mutant enzyme that shows altered kinetics or decreased stability (Fig. 8.20). Individuals heterozygous for PK deficiency have resistance to the most severe forms of malaria.

1	The tissue-specific expression of PK in RBC and the liver results from the use of different start sites in transcription (see p. 473) of the gene that encodes the enzyme. K. Pyruvate reduction to lactate Lactate, formed from pyruvate by LDH, is the final product of anaerobic glycolysis in eukaryotic cells (Fig. 8.21). Reduction to lactate is the major fate for pyruvate in tissues that are poorly vascularized (for example, the lens and cornea of the eye and the kidney medulla) or in RBC that lack mitochondria. 1.

1	1. Lactate formation in muscle: In exercising skeletal muscle, NADH production (by glyceraldehyde 3-phosphate dehydrogenase and by the three NAD+-linked dehydrogenases of the TCA cycle, see p. 113) exceeds the oxidative capacity of the ETC. This results in an elevated NADH/NAD+ ratio, favoring reduction of pyruvate to lactate by LDH. Therefore, during intense exercise, lactate accumulates in muscle, causing a drop in the intracellular pH, potentially resulting in cramps. Much of this lactate eventually diffuses into the bloodstream and can be used by the liver to make glucose (see p. 118). 2.

1	2. Lactate utilization: The direction of the LDH reaction depends on the relative intracellular concentrations of pyruvate and lactate and on the ratio of NADH/NAD+. For example, in the liver and heart, this ratio is lower than in exercising muscle. Consequently, the liver and heart oxidize lactate (obtained from the blood) to pyruvate. In the liver, pyruvate is either converted to glucose by gluconeogenesis or converted to acetyl CoA that is oxidized in the TCA cycle. Heart muscle exclusively oxidizes lactate to carbon dioxide and water via the TCA cycle. 3.

1	Lactic acidosis: Elevated concentrations of lactate in the plasma, termed lactic acidosis (a type of metabolic acidosis), occur when there is a collapse of the circulatory system, such as with myocardial infarction, pulmonary embolism, and uncontrolled hemorrhage, or when an individual is in shock. The failure to bring adequate amounts of O2 to the tissues results in impaired oxidative phosphorylation and decreased ATP synthesis. To survive, the cells rely on anaerobic glycolysis for generating ATP, producing lactic acid as the end product. [Note: Production of even meager amounts of ATP may be lifesaving during the period required to reestablish adequate blood flow to the tissues.] The additional O2 required to recover from a period when O2 availability has been inadequate is termed the O2 debt. [Note: The O2 debt is often related to patient morbidity or mortality. In many clinical situations, measuring the blood levels of lactic acid allows the rapid, early detection of O2 debt in

1	O2 debt. [Note: The O2 debt is often related to patient morbidity or mortality. In many clinical situations, measuring the blood levels of lactic acid allows the rapid, early detection of O2 debt in patients and the monitoring of their recovery.]

1	L. Energy yield from glycolysis Despite the production of some ATP by substrate-level phosphorylation during glycolysis, the end product, pyruvate or lactate, still contains most of the energy originally contained in glucose. The TCA cycle is required to release that energy completely (see p. 109). 1. Anaerobic glycolysis: A net of two molecules of ATP are generated for each molecule of glucose converted to two molecules of lactate (Fig. 8.22). There is no net production or consumption of NADH. 2. Aerobic glycolysis: The generation of ATP is the same as in anaerobic glycolysis (that is, a net gain of two ATP per molecule of glucose). Two molecules of NADH are also produced per molecule of glucose. Ongoing aerobic glycolysis requires the oxidation of most of this NADH by the ETC, producing three ATP for each NADH molecule entering the chain (see p. 77). [Note: NADH cannot cross the inner mitochondrial membrane, and substrate shuttles are required (see p. 79).]

1	VI. HORMONAL REGULATION

1	Regulation of the activity of the irreversible glycolytic enzymes by allosteric activation/inhibition or covalent phosphorylation/dephosphorylation is short term (that is, the effects occur over minutes or hours). Superimposed on these effects on the activity of preexisting enzyme molecules are the long-term hormonal effects on the number of new enzyme molecules. These hormonal effects can result in 10-to 20-fold increases in enzyme synthesis that typically occur over hours to days. Regular consumption of meals rich in carbohydrate or administration of insulin initiates an increase in the amount of glucokinase, PFK-1, and PK in the liver (Fig. 8.23). The change reflects an increase in gene transcription, resulting in increased enzyme synthesis. Increased availability of these three enzymes favors the conversion of glucose to pyruvate, a characteristic of the absorptive state (see p. 321). [Note: The transcriptional effects of insulin and carbohydrate (specifically glucose) are

1	enzymes favors the conversion of glucose to pyruvate, a characteristic of the absorptive state (see p. 321). [Note: The transcriptional effects of insulin and carbohydrate (specifically glucose) are mediated by the transcription factors sterol regulatory element–binding protein-1c and carbohydrate response element–binding protein, respectively. These factors also regulate transcription of genes involved in fatty acid synthesis (see p. 184).] Conversely, gene expression of the three enzymes is decreased when plasma glucagon is high and insulin is low (for example, as seen in fasting or diabetes).

1	VII. ALTERNATE FATES OF PYRUVATE Pyruvate can be metabolized to products other than lactate. A. Oxidative decarboxylation to acetyl CoA Oxidative decarboxylation of pyruvate by the PDHC is an important pathway in tissues with a high oxidative capacity such as cardiac muscle (Fig. 8.24). PDHC irreversibly converts pyruvate, the end product of aerobic glycolysis, into acetyl CoA, a TCA cycle substrate (see p. 109) and the carbon source for fatty acid synthesis (see p. 183). B. Carboxylation to oxaloacetate Carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase is a biotin-dependent reaction (see Fig. 8.24). This irreversible reaction is important because it replenishes the TCA cycle intermediate and provides substrate for gluconeogenesis (see p. 118). C. Reduction to ethanol (microorganisms)

1	C. Reduction to ethanol (microorganisms) The reduction of pyruvate to ethanol occurs by the two reactions summarized in Figure 8.24. The decarboxylation of pyruvate to acetaldehyde by thiamine-requiring pyruvate decarboxylase occurs in yeast and certain other microorganisms but not in humans. VIII. CHAPTER SUMMARY

1	Most pathways can be classified as either catabolic (degrade complex molecules to a few simple products with ATP production) or anabolic (synthesize complex end products from simple precursors with ATP hydrolysis). The rate of a metabolic pathway can respond to regulatory signals such as intracellular allosteric activators or inhibitors. Intercellular signaling provides for the integration of metabolism. The primary route of this communication is chemical signaling (for example, by hormones or neurotransmitters). Second messenger molecules transduce a chemical signal (hormone or neurotransmitter binding) to appropriate intracellular responders. Adenylyl cyclase (AC) is a cell membrane enzyme that synthesizes cyclic adenosine monophosphate (cAMP) in response to chemical signals, such as the hormones glucagon and epinephrine. Following binding of a hormone to its cell-surface G protein–coupled receptor, a guanosine triphosphate–dependent regulatory protein (G protein) is activated that,

1	hormones glucagon and epinephrine. Following binding of a hormone to its cell-surface G protein–coupled receptor, a guanosine triphosphate–dependent regulatory protein (G protein) is activated that, in turn, activates AC. The cAMP produced activates protein kinase A, which phosphorylates a variety of enzymes, causing their activation or deactivation. Phosphorylation is reversed by phosphatases. Aerobic glycolysis, in which pyruvate is the end product, occurs in cells with mitochondria and an adequate supply of oxygen ([O2],

1	Fig. 8.25). Anaerobic glycolysis, in which lactic acid is the end product, occurs in cells that lack mitochondria and in cells deprived of sufficient O2. Glucose is passively transported across membranes by 1 of 14 glucose transporter (GLUT) isoforms. GLUT-1 is abundant in RBC and the brain, GLUT-4 (which is insulin dependent) in muscle and adipose tissue, and GLUT-2 in the liver, kidneys, and pancreatic β cells. The oxidation of glucose to pyruvate (glycolysis, see Fig. 8.25) occurs through an energy-investment phase in which phosphorylated intermediates are synthesized at the expense of ATP and an energy-generation phase in which ATP is produced by substrate-level phosphorylation. In the energy-investment phase, glucose is phosphorylated by hexokinase (found in most tissues) or glucokinase (a hexokinase found in liver cells and pancreatic β cells). Hexokinase has a high affinity (low Km) and a low maximal velocity (Vmax) for glucose and is inhibited by glucose 6-phosphate.

1	glucokinase (a hexokinase found in liver cells and pancreatic β cells). Hexokinase has a high affinity (low Km) and a low maximal velocity (Vmax) for glucose and is inhibited by glucose 6-phosphate. Glucokinase has a high Km and a high Vmax for glucose. It is regulated indirectly by fructose 6-phosphate (inhibits) and glucose (activates) via glucokinase regulatory protein. Glucose 6-phosphate is isomerized to fructose 6phosphate, which is phosphorylated to fructose 1,6-bisphosphate by phosphofructokinase-1 (PFK-1). This enzyme is allosterically inhibited by ATP and citrate and activated by AMP. Fructose 2,6-bisphosphate, whose synthesis by bifunctional phosphofructokinase-2 (PFK-2) is increased in the liver by insulin and decreased by glucagon, is the most potent allosteric activator of PFK-1. A total of two ATP are used during this phase of glycolysis. Fructose 1,6-bisphosphate is cleaved to form two trioses that are further metabolized by the glycolytic pathway, forming pyruvate.

1	PFK-1. A total of two ATP are used during this phase of glycolysis. Fructose 1,6-bisphosphate is cleaved to form two trioses that are further metabolized by the glycolytic pathway, forming pyruvate. During this phase, four ATP and two nicotinamide adenine dinucleotide (NADH) are produced per glucose molecule. The final step in pyruvate synthesis from phosphoenolpyruvate is catalyzed by pyruvate kinase (PK). This enzyme is allosterically activated by fructose 1,6-bisphosphate, and the hepatic isozyme is inhibited covalently by glucagon via the cAMP pathway. PK deficiency accounts for the majority of all inherited defects in glycolytic enzymes. Effects are restricted to RBC and present as mild-tosevere chronic, nonspherocytic hemolytic anemia. Glycolytic gene transcription is enhanced by insulin and glucose. In anaerobic glycolysis, NADH is reoxidized to NAD+ by the reduction of pyruvate to lactate via lactate dehydrogenase. This occurs in cells such as RBC that lack mitochondria and in

1	and glucose. In anaerobic glycolysis, NADH is reoxidized to NAD+ by the reduction of pyruvate to lactate via lactate dehydrogenase. This occurs in cells such as RBC that lack mitochondria and in tissues such as exercising muscle, where production of NADH exceeds the oxidative capacity of the respiratory chain. Elevated concentrations of lactate in the plasma (lactic acidosis) occur with circulatory system collapse or shock. Pyruvate also can be 1) oxidatively decarboxylated to acetyl CoA by pyruvate dehydrogenase, 2) carboxylated to oxaloacetate (a TCA cycle intermediate) by pyruvate carboxylase, or 3) reduced to ethanol by microbial pyruvate decarboxylase.

1	Choose the ONE best answer. .1. Which of the following best describes the activity level and phosphorylation state of the listed hepatic enzymes in an individual who consumed a carbohydrate-rich meal about an hour ago? PFK-1 = phosphofructokinase1; PFK-2 = phosphofructokinase-2; P = phosphorylated. Correct answer = C. Immediately following a meal, blood glucose levels and hepatic uptake of glucose increase. The glucose is phosphorylated to glucose 6-phosphate and used in glycolysis. In response to the rise in blood glucose, the insulin/glucagon ratio increases. As a result, the kinase domain of PFK-2 is dephosphorylated and active. Its product, fructose 2,6-bisphosphate, allosterically activates PFK-1. (PFK-1 is not covalently regulated.) Active PFK-1 produces fructose 1,6-bisphosphate that is a feedforward activator of pyruvate kinase. Hepatic pyruvate kinase is covalently regulated, and the rise in insulin favors dephosphorylation and activation.

1	.2. Which of the following statements is true for anabolic pathways only? A. Their irreversible (nonequilibrium) reactions are regulated. B. They are called cycles if they regenerate an intermediate. C. They are convergent and generate a few simple products. D. They are synthetic and require energy. E. They typically require oxidized coenzymes. Correct answer = D. Anabolic processes are synthetic and energy requiring (endergonic). Statements A and B apply to both anabolic and catabolic processes, whereas C and E apply only to catabolic processes. .3. Compared with the resting state, vigorously contracting skeletal muscle shows: A. decreased AMP/ATP ratio. B. decreased levels of fructose 2,6-bisphosphate. C. decreased NADH/NAD+ ratio. D. increased oxygen availability. E. increased reduction of pyruvate to lactate.

1	A. decreased AMP/ATP ratio. B. decreased levels of fructose 2,6-bisphosphate. C. decreased NADH/NAD+ ratio. D. increased oxygen availability. E. increased reduction of pyruvate to lactate. Correct answer = E. Vigorously contracting skeletal muscle shows an increase in the reduction of pyruvate to lactate compared with resting muscle. The levels of reduced nicotinamide adenine dinucleotide (NADH) increase and exceed the oxidative capacity of the electron transport chain. Consequently, the levels of adenosine monophosphate (AMP) increase. The concentration of fructose 2,6-bisphosphate is not a key regulatory factor in skeletal muscle. .4. Glucose uptake by: A. brain cells is through energy-requiring (active) transport. B. intestinal mucosal cells requires insulin. C. liver cells is through facilitated diffusion involving a glucose transporter. D. most cells is through simple diffusion up a concentration gradient.

1	C. liver cells is through facilitated diffusion involving a glucose transporter. D. most cells is through simple diffusion up a concentration gradient. Correct answer = C. Glucose uptake in the liver, brain, muscle, and adipose tissue is down a concentration gradient, and the diffusion is facilitated by tissue-specific glucose transporters (GLUT). In adipose and muscle tissues, insulin is required for glucose uptake. Moving glucose against a concentration gradient requires energy and is seen with the sodium-dependent glucose cotransporter 1 (SGLT1) of intestinal mucosal cells. .5. Given that the Km of glucokinase for glucose is 10 mM, whereas that of hexokinase is 0.1 mM, which isozyme will more closely approach Vmax at the normal blood glucose concentration of 5 mM?

1	.5. Given that the Km of glucokinase for glucose is 10 mM, whereas that of hexokinase is 0.1 mM, which isozyme will more closely approach Vmax at the normal blood glucose concentration of 5 mM? Correct answer = Hexokinase. Km (Michaelis constant) is that substrate concentration that gives one half Vmax (maximal velocity). When blood glucose concentration is 5 mM, hexokinase (Km = 0.1 mM) will be saturated, but glucokinase (Km = 10 mM) will not. .6. In patients with whooping cough, Gαi is inhibited. How does this lead to a rise in cyclic adenosine monophosphate (cAMP)? G proteins of the Gαi type inhibit adenylyl cyclase (AC) when their associated G protein–coupled receptor is bound by ligand. If Gαi is inhibited by pertussis toxin, AC production of cAMP is inappropriately activated. For additional ancillary materials related to this chapter, please visit thePoint. I. CYCLE OVERVIEW

1	I. CYCLE OVERVIEW The tricarboxylic acid cycle ([TCA cycle] also called the citric acid cycle, or the Krebs cycle) plays several roles in metabolism. It is the final pathway where the oxidative catabolism of carbohydrates, amino acids, and fatty acids converge, their carbon skeletons being converted to carbon dioxide (CO2), as shown in cycle. The TCA cycle is an aerobic pathway, because oxygen (O2) is required as the final electron acceptor. Reactions such as the catabolism of some amino acids generate intermediates of the cycle and are called anaplerotic (from the Greek for “filling up”) reactions. The TCA cycle also provides intermediates for a number of important anabolic reactions, such as glucose formation from the carbon skeletons of some amino acids and the synthesis of some amino acids (see p. 267) and heme (see p. 278). Therefore, this cycle should not be viewed as a closed system but, instead, as an open one with compounds entering and leaving as required.

1	II. CYCLE REACTIONS In the TCA cycle, oxaloacetate (OAA) is first condensed with an acetyl group from acetyl coenzyme A (CoA) and then is regenerated as the cycle is completed (see Fig. 9.1). Two carbons enter the cycle as acetyl CoA and two leave as CO2. Therefore, the entry of one acetyl CoA into one round of the TCA cycle does not lead to the net production or consumption of intermediates. A. Acetyl CoA production

1	The major source of acetyl CoA for the TCA cycle is the oxidative decarboxylation of pyruvate by the multienzyme pyruvate dehydrogenase complex (PDH complex, or PDHC). However, the PDHC (described below) is not a component of the TCA cycle. Pyruvate, the end product of aerobic glycolysis, is transported from the cytosol into the mitochondrial matrix by the pyruvate mitochondrial carrier of the inner mitochondrial membrane. In the matrix, the PDHC converts pyruvate to acetyl CoA. [Note: Fatty acid oxidation is another source of acetyl CoA (see p. 192).] 1. PDHC component enzymes: The PDHC is a protein aggregate of multiple copies of three enzymes, pyruvate decarboxylase ([E1] sometimes called pyruvate dehydrogenase), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). Each catalyzes a part of the overall reaction (Fig. 9.2). Their physical association links the reactions in proper sequence without the release of intermediates. In addition to the enzymes

1	(E3). Each catalyzes a part of the overall reaction (Fig. 9.2). Their physical association links the reactions in proper sequence without the release of intermediates. In addition to the enzymes participating in the conversion of pyruvate to acetyl CoA, the PDHC also contains two regulatory enzymes, pyruvate dehydrogenase kinase (PDH kinase) and pyruvate dehydrogenase phosphatase (PDH phosphatase).

1	dioxide; TPP = thiamine pyrophosphate; L = lipoic acid; CoA = coenzyme A; FAD(H2) and NAD(H) = flavin and nicotinamide adenine dinucleotides; ~ = high-energy bond. 2. Coenzymes: The PDHC contains five coenzymes that act as carriers or oxidants for the intermediates of the reactions shown in Figure 9.2. E1 requires thiamine pyrophosphate (TPP), E2 requires lipoic acid and CoA, and E3 requires FAD and NAD+. [Note: TPP, lipoic acid, and FAD are tightly bound to the enzymes and function as coenzymes–prosthetic groups (see p. 54).] Deficiencies of thiamine or niacin can cause serious central nervous system problems. This is because brain cells are unable to produce sufficient ATP (via the TCA cycle) if the PDHC is inactive. Wernicke-Korsakoff, an encephalopathy-psychosis syndrome due to thiamine deficiency, may be seen with alcohol abuse (see p. 383).

1	3. Regulation: Covalent modifications by the two regulatory enzymes of the PDHC alternately activate and inactivate E1. PDH kinase phosphorylates and inactivates E1, whereas PDH phosphatase dephosphorylates and activates E1 (Fig. 9.3). The kinase itself is allosterically activated by ATP, acetyl CoA, and NADH. Therefore, in the presence of these high-energy products, the PDHC is turned off. [Note: It is actually the rise in the ATP/ADP (adenosine diphosphate), NADH/NAD+, or acetyl CoA/CoA ratios that affects enzymic activity.] Pyruvate is a potent inhibitor of PDH kinase. Therefore, if pyruvate concentrations are elevated, E1 will be maximally active. Calcium (Ca2+) is a strong activator of PDH phosphatase, stimulating E1 activity. This is particularly important in skeletal muscle, where Ca2+ release during contraction stimulates the PDHC and, thus, energy production. [Note: Although covalent regulation by the kinase and phosphatase is primary, the PDHC is also subject to product

1	Ca2+ release during contraction stimulates the PDHC and, thus, energy production. [Note: Although covalent regulation by the kinase and phosphatase is primary, the PDHC is also subject to product (NADH and acetyl CoA) inhibition.] 4. Deficiency: A deficiency of the α subunits of the tetrameric E1 component of the PDHC, although very rare, is the most common biochemical cause of congenital lactic acidosis. The deficiency results in a decreased ability to convert pyruvate to acetyl CoA, causing pyruvate to be shunted to lactate via lactate dehydrogenase (see p. 103). This creates particular problems for the brain, which relies on the TCA cycle for most of its energy and is particularly sensitive to acidosis. Symptoms are variable and include neurodegeneration, muscle spasticity, and, in the neonatal-onset form, early death. The gene for the α subunit is X linked, and because both males and females may be affected, the deficiency is classified as X-linked dominant. Although there is no

1	neonatal-onset form, early death. The gene for the α subunit is X linked, and because both males and females may be affected, the deficiency is classified as X-linked dominant. Although there is no proven treatment for PDHC deficiency, dietary restriction of carbohydrate and supplementation with thiamine may reduce symptoms in select patients.

1	Leigh syndrome (subacute necrotizing encephalomyelopathy) is a rare, progressive, neurodegenerative disorder caused by defects in mitochondrial ATP production, primarily as a result of mutations in genes that encode proteins of the PDHC, the ETC, or ATP synthase. Both nuclear and mitochondrial DNA can be affected.

1	5. Arsenic poisoning: As previously described (see p. 101), pentavalent arsenic (arsenate) can interfere with glycolysis at the glyceraldehyde 3phosphate step, thereby decreasing ATP production. However, arsenic poisoning is due primarily to inhibition of enzyme complexes that require lipoic acid as a coenzyme, including PDH, α-ketoglutarate dehydrogenase (see E. below), and branched-chain α-keto acid dehydrogenase (see p. 266). Arsenite (the trivalent form of arsenic) forms a stable complex with the thiol (−SH) groups of lipoic acid, making that compound unavailable to serve as a coenzyme. When it binds to lipoic acid in the PDHC, pyruvate (and, consequently, lactate) accumulates. As with PDHC deficiency, this particularly affects the brain, causing neurologic disturbances and death. B. Citrate synthesis

1	B. Citrate synthesis The irreversible condensation of acetyl CoA and OAA to form citrate (a tricarboxylic acid) is catalyzed by citrate synthase, the initiating enzyme of the TCA cycle (Fig. 9.4). This aldol condensation has a highly negative change in standard free energy ([∆G0] see p. 70), which strongly favors citrate formation. The enzyme is inhibited by citrate (product inhibition). Substrate availability is another means of regulation for citrate synthase. The binding of OAA greatly increases the enzyme’s affinity for acetyl CoA. [Note: Citrate, in addition to being an intermediate in the TCA cycle, is a source of acetyl CoA for the cytosolic synthesis of fatty acids (see p. 183) and cholesterol (see p. 220). Citrate also inhibits phosphofructokinase-1 (PFK-1), the rate-limiting enzyme of glycolysis (see p. 99), and activates acetyl CoA carboxylase (the rate-limiting enzyme of fatty acid synthesis, see p. 183).] C. Citrate isomerization

1	C. Citrate isomerization Citrate is isomerized to isocitrate through hydroxyl group migration catalyzed by aconitase (aconitate hydratase), an iron-sulfur protein (see Fig. 9.4). [Note: Aconitase is inhibited by fluoroacetate, a plant toxin that is used as a pesticide. Fluoroacetate is converted to fluoroacetyl CoA that condenses with OAA to form fluorocitrate, a potent inhibitor of aconitase.] D. Oxidative decarboxylation of isocitrate Isocitrate dehydrogenase catalyzes the irreversible oxidative decarboxylation of isocitrate to α-ketoglutarate, yielding the first of three NADH molecules produced by the cycle and the first release of CO2 (see Fig. 9.4). This is one of the rate-limiting steps of the TCA cycle. The enzyme is allosterically activated by ADP (a low-energy signal) and Ca2+ and is inhibited by ATP and NADH, levels of which are elevated when the cell has abundant energy stores. E. Oxidative decarboxylation of α-ketoglutarate

1	The irreversible conversion of α-ketoglutarate to succinyl CoA is catalyzed by the α-ketoglutarate dehydrogenase complex, a protein aggregate of multiple copies of three enzymes (Fig. 9.5). The mechanism of this oxidative decarboxylation is very similar to that used for the conversion of pyruvate to acetyl CoA by the PDHC. The reaction releases the second CO2 and produces the second NADH of the cycle. The coenzymes required are TPP, lipoic acid, FAD, NAD+, and CoA. Each functions as part of the catalytic mechanism in a way analogous to that described for the PDHC (see p. 110). The large negative ∆G0 of the reaction favors formation of succinyl CoA, a high-energy thioester similar to acetyl CoA. The αketoglutarate dehydrogenase complex is inhibited by its products, NADH and succinyl CoA, and activated by Ca2+ . However, it is not regulated by phosphorylation/dephosphorylation reactions as described for the PDHC. [Note: α-Ketoglutarate is also produced by the oxidative deamination (see

1	activated by Ca2+ . However, it is not regulated by phosphorylation/dephosphorylation reactions as described for the PDHC. [Note: α-Ketoglutarate is also produced by the oxidative deamination (see p. 252) and transamination of the amino acid glutamate (see p. 250).]

1	F. Succinyl coenzyme A cleavage Succinate thiokinase (also called succinyl CoA synthetase, named for the reverse reaction) cleaves the high-energy thioester bond of succinyl CoA (see Fig. 9.5). This reaction is coupled to phosphorylation of guanosine diphosphate (GDP) to guanosine triphosphate (GTP). GTP and ATP are energetically interconvertible by the nucleoside diphosphate kinase reaction: The generation of GTP by succinate thiokinase is another example of substrate-level phosphorylation (see p. 102). [Note: Succinyl CoA is also produced from propionyl CoA derived from the metabolism of fatty acids with an odd number of carbon atoms (see p. 193) and from the metabolism of several amino acids (see pp. 265–266). It can be converted to pyruvate for gluconeogenesis (see p. 118) or used in heme synthesis (see p. 278).] G. Succinate oxidation

1	G. Succinate oxidation Succinate is oxidized to fumarate by succinate dehydrogenase, as its coenzyme FAD is reduced to FADH2 (see Fig. 9.5). Succinate dehydrogenase is the only enzyme of the TCA cycle that is embedded in the inner mitochondrial membrane. As such, it functions as Complex II of the ETC (see p. 75). [Note: FAD, rather than NAD+, is the electron acceptor because the reducing power of succinate is not sufficient to reduce NAD+.] H. Fumarate hydration Fumarate is hydrated to malate in a freely reversible reaction catalyzed by fumarase (fumarate hydratase, see Fig. 9.5). [Note: Fumarate is also produced by the urea cycle (see p. 255), in purine synthesis (see Fig. 22.7 on p. 294), and during catabolism of the amino acids phenylalanine and tyrosine (see p. 263).] I. Malate oxidation

1	I. Malate oxidation Malate is oxidized to OAA by malate dehydrogenase (Fig. 9.6). This reaction produces the third and final NADH of the cycle. The ∆G0 of the reaction is positive, but the reaction is driven in the direction of OAA by the highly exergonic citrate synthase reaction. [Note: OAA is also produced by the transamination of the amino acid aspartic acid (see p. 250).] III. ENERGY PRODUCED BY THE CYCLE

1	III. ENERGY PRODUCED BY THE CYCLE Four pairs of electrons are transferred during one turn of the TCA cycle: three pairs reducing three NAD+ to NADH and one pair reducing FAD to FADH2. Oxidation of one NADH by the ETC leads to formation of three ATP, whereas oxidation of FADH2 produces two ATP (see p. 77). The total yield of ATP from the oxidation of one acetyl CoA is shown in Figure 9.7. Figure 9.8 summarizes the reactions of the TCA cycle. [Note: The cycle does not involve the net consumption or production of intermediates. Two carbons entering as acetyl CoA are balanced by two CO2 exiting.] dinucleotides; GDP and GTP = guanosine di-and triphosphates; Pi = inorganic phosphate. IV. CYCLE REGULATION

1	IV. CYCLE REGULATION In contrast to glycolysis, which is regulated primarily by PFK-1, the TCA cycle is controlled by the regulation of several enzymes (see Fig. 9.8). The most important of these regulated enzymes are those that catalyze reactions with highly negative ∆G0: citrate synthase, isocitrate dehydrogenase, and the αketoglutarate dehydrogenase complex. Reducing equivalents needed for oxidative phosphorylation are generated by the PDHC and the TCA cycle, and both processes are upregulated in response to a decrease in the ATP/ADP ratio. V. CHAPTER SUMMARY

1	Pyruvate is oxidatively decarboxylated by the pyruvate dehydrogenase complex (PDHC), producing acetyl coenzyme A (CoA), which is the major fuel for the tricarboxylic acid (TCA) cycle (Fig. 9.9). The multienzyme PDHC requires five coenzymes: thiamine pyrophosphate, lipoic acid, flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD+), and CoA. The PDHC is regulated by covalent modification of E1 (pyruvate decarboxylase) by PDH kinase and PDH phosphatase: Phosphorylation inhibits E1. PDH kinase is allosterically activated by ATP, acetyl CoA, and NADH and inhibited by pyruvate. The phosphatase is activated by calcium (Ca2+). E1 deficiency is the most common biochemical cause of congenital lactic acidosis. The brain is particularly affected in this X-linked dominant disorder. Arsenic poisoning causes inactivation of the PDHC by binding to lipoic acid. In the TCA cycle, citrate is synthesized from oxaloacetate (OAA) and acetyl CoA by citrate synthase, which is inhibited

1	Arsenic poisoning causes inactivation of the PDHC by binding to lipoic acid. In the TCA cycle, citrate is synthesized from oxaloacetate (OAA) and acetyl CoA by citrate synthase, which is inhibited by product. Citrate is isomerized to isocitrate by aconitase (aconitate hydratase). Isocitrate is oxidatively decarboxylated by isocitrate dehydrogenase to α-ketoglutarate, producing carbon dioxide (CO2) and NADH. The enzyme is inhibited by ATP and NADH and activated by adenosine diphosphate (ADP) and Ca2+ . α-Ketoglutarate is oxidatively decarboxylated to succinyl CoA by the α-ketoglutarate dehydrogenase complex, producing CO2 and NADH. The enzyme is very similar to the PDHC and uses the same coenzymes. The α-ketoglutarate dehydrogenase complex is activated by Ca2+ and inhibited by NADH and succinyl CoA but is not covalently regulated. Succinyl CoA is cleaved by succinate thiokinase (also called succinyl CoA synthetase), producing succinate and guanosine triphosphate (GTP). This is an

1	succinyl CoA but is not covalently regulated. Succinyl CoA is cleaved by succinate thiokinase (also called succinyl CoA synthetase), producing succinate and guanosine triphosphate (GTP). This is an example of substrate-level phosphorylation. Succinate is oxidized to fumarate by succinate dehydrogenase, producing FADH2. Fumarate is hydrated to malate by fumarase (fumarate hydratase), and malate is oxidized to OAA by malate dehydrogenase, producing NADH. Three NADH and one FADH2 are produced by one round of the TCA cycle. The generation of acetyl CoA by the oxidation of pyruvate via the PDHC also produces an NADH. Oxidation of the NADH and FADH2 by the ETC yields 14 ATP. The terminal phosphate of the GTP produced by substrate-level phosphorylation in the TCA cycle can be transferred to ADP by nucleoside diphosphate kinase, yielding another ATP. Therefore, a total of 15 ATP are produced from the complete mitochondrial oxidation of pyruvate to CO2.

1	NAD(H) = nicotinamide adenine dinucleotide; FAD(H2) = flavin adenine dinucleotide; GDP and GTP = guanosine di-and triphosphates; ADP = adenosine diphosphate; Pi = inorganic phosphate. Choose the ONE best answer. .1. The conversion of pyruvate to acetyl coenzyme A and carbon dioxide: A. involves the participation of lipoic acid. B. is activated when pyruvate decarboxylase of the pyruvate dehydrogenase complex (PDHC) is phosphorylated by PDH kinase in the presence of ATP. C. is reversible. D. occurs in the cytosol. E. requires the coenzyme biotin.

1	C. is reversible. D. occurs in the cytosol. E. requires the coenzyme biotin. Correct answer = A. Lipoic acid is an intermediate acceptor of the acetyl group formed in the reaction. [Note: Lipoic acid linked to a lysine residue in E2 functions as a “swinging arm” that allows interaction with E1 and E3.] The PDHC catalyzes an irreversible reaction that is inhibited when the decarboxylase component (E1) is phosphorylated. The PDHC is located in the mitochondrial matrix. Biotin is utilized by carboxylases, not decarboxylases. .2. Which one of the following conditions decreases the oxidation of acetyl coenzyme A by the citric acid cycle? A. A high availability of calcium B. A high acetyl CoA/CoA ratio C. A low ATP/ADP ratio D. A low NAD+/NADH ratio Correct answer = D. A low NAD+/NADH (oxidized to reduced nicotinamide adenine dinucleotide) ratio limits the rates of the NAD+-requiring dehydrogenases. High availability of calcium and substrate (acetyl coenzyme

1	A) and a low ATP/ADP (adenosine tri-to diphosphate) ratio stimulate the cycle. .3. The following is the sum of three steps in the citric acid cycle. A + B + FAD + H2O → C + FADH2 + NADH Choose the lettered answer that corresponds to the missing “A,” “B,” and “C” in the equation. Correct answer = B. Succinate + NAD+ + FAD + H2O → oxaloacetate + NADH + FADH2. .4. A 1-month-old male shows neurologic problems and lactic acidosis. Enzyme assay for pyruvate dehydrogenase complex (PDHC) activity on extracts of cultured skin fibroblasts showed 5% of normal activity with a low concentration of thiamine pyrophosphate (TPP) but 80% of normal activity when the assay contained a thousand-fold higher concentration of TPP. Which one of the following statements concerning this patient is correct? A. Administration of thiamine is expected to reduce his serum lactate level and improve his clinical symptoms. B. A high-carbohydrate diet would be expected to be beneficial for this patient.

1	A. Administration of thiamine is expected to reduce his serum lactate level and improve his clinical symptoms. B. A high-carbohydrate diet would be expected to be beneficial for this patient. C. Citrate production from aerobic glycolysis is expected to be increased. D. PDH kinase, a regulatory enzyme of the PDHC, is expected to be active.

1	C. Citrate production from aerobic glycolysis is expected to be increased. D. PDH kinase, a regulatory enzyme of the PDHC, is expected to be active. Correct answer = A. The patient appears to have a thiamine-responsive PDHC deficiency. The pyruvate decarboxylase (E1) component of the PDHC fails to bind thiamine pyrophosphate at low concentration but shows significant activity at a high concentration of the coenzyme. This mutation, which affects the Km (Michaelis constant) of the enzyme for the coenzyme, is present in some, but not all, cases of PDHC deficiency. Because the PDHC is an integral part of carbohydrate metabolism, a diet low in carbohydrates would be expected to blunt the effects of the enzyme deficiency. Aerobic glycolysis generates pyruvate, the substrate of the PDHC. Decreased activity of the complex decreases production of acetyl coenzyme A, a substrate for citrate synthase. Because PDH kinase is allosterically inhibited by pyruvate, it is inactive.

1	.5. Which coenzyme–cosubstrate is used by dehydrogenases in both glycolysis and the tricarboxylic acid cycle? Oxidized nicotinamide adenine dinucleotide (NAD+) is used by glyceraldehyde 3-phosphate dehydrogenase of glycolysis and by isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase of the tricarboxylic acid cycle. [Note: E3 of the pyruvate dehydrogenase complex requires oxidized flavin adenine dinucleotide (FAD) and NAD+.] For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Some tissues, such as the brain, red blood cells (RBC), kidney medulla, lens and cornea of the eye, testes, and exercising muscle, require a continuous supply of glucose as a metabolic fuel. Liver glycogen, an essential postprandial source of glucose, can meet these needs for <24 hours in the absence of dietary intake of carbohydrate (see p. 125). During a prolonged fast, however, hepatic glycogen stores are depleted, and glucose is made from noncarbohydrate precursors. The formation of glucose does not occur by a simple reversal of glycolysis, because the overall equilibrium of glycolysis strongly favors pyruvate formation (that is, the change in standard free energy [∆G0] is negative). Instead, glucose is synthesized de novo by a special pathway, gluconeogenesis, which requires both mitochondrial and cytosolic enzymes. [Note: Deficiencies of gluconeogenic enzymes cause hypoglycemia.] During an overnight fast, ~90% of gluconeogenesis occurs in the liver, with the remaining ~10%

1	mitochondrial and cytosolic enzymes. [Note: Deficiencies of gluconeogenic enzymes cause hypoglycemia.] During an overnight fast, ~90% of gluconeogenesis occurs in the liver, with the remaining ~10% occurring in the kidneys. However, during prolonged fasting, the kidneys become major glucose-producing organs, contributing ~40% of the total glucose production. [Note: The small intestine can also make glucose.] Figure 10.1 shows the relationship of gluconeogenesis to other essential pathways of energy metabolism.

1	carbon dioxide. II. SUBSTRATES Gluconeogenic precursors are molecules that can be used to produce a net synthesis of glucose. The most important gluconeogenic precursors are glycerol, lactate, and α-keto acids obtained from the metabolism of glucogenic amino acids. [Note: All but two amino acids (leucine and lysine) are glucogenic (see p. 262).] A. Glycerol Glycerol is released during the hydrolysis of triacylglycerols (TAG) in adipose tissue (see p. 190) and is delivered by the blood to the liver. Glycerol is phosphorylated by glycerol kinase to glycerol 3-phosphate, B. Lactate Lactate from anaerobic glycolysis is released into the blood by exercising skeletal muscle and by cells that lack mitochondria such as RBC. In the Cori cycle, this lactate is taken up by the liver and oxidized to pyruvate that is converted to glucose, which is released back into the circulation (Fig. 10.2). C. Amino acids

1	C. Amino acids Amino acids produced by hydrolysis of tissue proteins are the major sources of glucose during a fast. Their metabolism generates α-keto acids, such as pyruvate that is converted to glucose, or α-ketoglutarate that can enter the tricarboxylic acid (TCA) cycle and form oxaloacetate (OAA), a direct precursor of phosphoenolpyruvate (PEP). [Note: Acetyl coenzyme A (CoA) and compounds that give rise only to acetyl CoA (for example, acetoacetate, lysine, and leucine) cannot give rise to a net synthesis of glucose. This is because of the irreversible nature of the pyruvate dehydrogenase complex (PDHC), which converts pyruvate to acetyl CoA (see p. 109). These compounds give rise instead to ketone bodies (see p. 195) and are termed ketogenic.] III. REACTIONS

1	195) and are termed ketogenic.] III. REACTIONS Seven glycolytic reactions are reversible and are used in the synthesis of glucose from lactate or pyruvate. However, three glycolytic reactions are irreversible and must be circumvented by four alternate reactions that energetically favor the synthesis of glucose. These irreversible reactions, which together are unique to gluconeogenesis, are described below. A. Pyruvate carboxylation The first roadblock to overcome in the synthesis of glucose from pyruvate is the irreversible conversion in glycolysis of PEP to pyruvate by pyruvate kinase (PK). In gluconeogenesis, pyruvate is carboxylated by pyruvate carboxylase (PC) to OAA, which is converted to PEP by PEPcarboxykinase (PEPCK) (Fig. 10.3). mitochondrial and cytosolic isozymes of malate dehydrogenase; GTP and GDP = guanosine tri-and diphosphates; ADP = adenosine diphosphate.

1	mitochondrial and cytosolic isozymes of malate dehydrogenase; GTP and GDP = guanosine tri-and diphosphates; ADP = adenosine diphosphate. 1. Biotin: PC requires the coenzyme biotin (see p. 385) covalently bound to the ε-amino group of a lysine residue in the enzyme (see Fig. 10.3). ATP hydrolysis drives formation of an enzyme–biotin–carbon dioxide (CO2) intermediate, which then carboxylates pyruvate to form OAA. [Note: HCO3− provides the CO2.] The PC reaction occurs in the mitochondria of liver and kidney cells and has two purposes: to allow production of PEP, an important substrate for gluconeogenesis, and to provide OAA that can replenish the TCA cycle intermediates that may become depleted. Muscle cells also contain PC but use the OAA product only for the replenishment (anaplerotic) purpose and do not synthesize glucose. [Note: Pyruvate carrier protein moves pyruvate from the cytosol into mitochondria.]

1	PC is one of several carboxylases that require biotin. Others include acetyl CoA carboxylase (p. 183), propionyl CoA carboxylase (p. 194), and methylcrotonyl CoA carboxylase (p. 266). 2. Allosteric regulation: PC is allosterically activated by acetyl CoA. Elevated levels of acetyl CoA in mitochondria signal a metabolic state in which increased synthesis of OAA is required. For example, this occurs during fasting, when OAA is used for gluconeogenesis in the liver and kidneys. Conversely, at low levels of acetyl CoA, PC is largely inactive, and pyruvate is primarily oxidized by the PDHC to acetyl CoA that can be further oxidized by the TCA cycle (see p. 109). B. Oxaloacetate transport to the cytosol

1	For gluconeogenesis to continue, OAA must be converted to PEP by PEPCK. PEP production in the cytosol requires transport of OAA out of mitochondria. However, there is no OAA transporter in the inner mitochondrial membrane, and OAA is first reduced to malate by mitochondrial malate dehydrogenase (MD). Malate is transported into the cytosol and reoxidized to OAA by cytosolic MD as nicotinamide adenine dinucleotide (NAD+) is reduced to NADH (see Fig. 10.3). The NADH is used in the reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3phosphate by glyceraldehyde 3-phosphate dehydrogenase (see p. 101), a reaction common to glycolysis and gluconeogenesis. [Note: When abundant, lactate is oxidized to pyruvate as NAD+ is reduced. The pyruvate is transported into mitochondria and carboxylated by PC to OAA, which can be converted to PEP by the mitochondrial isozyme of PEPCK. PEP is transported to the cytosol. OAA can also be converted to aspartate that is transported into the cytosol.]

1	C. Cytosolic oxaloacetate decarboxylation OAA is decarboxylated and phosphorylated to PEP in the cytosol by PEPCK. The reaction is driven by hydrolysis of guanosine triphosphate ([GTP] see Fig. 10.3). The combined actions of PC and PEPCK provide an energetically favorable pathway from pyruvate to PEP. PEP is then acted on by the reactions of glycolysis running in the reverse direction until it becomes fructose 1,6-bisphosphate. The pairing of carboxylation with decarboxylation drives reactions that would otherwise be energetically unfavorable. This strategy is also used in fatty acid (FA) synthesis (see p. 184). D. Fructose 1,6-bisphosphate dephosphorylation

1	D. Fructose 1,6-bisphosphate dephosphorylation Hydrolysis of fructose 1,6-bisphosphate by fructose 1,6-bisphosphatase, found in the liver and kidneys, bypasses the irreversible phosphofructokinase-1 (PFK-1) reaction of glycolysis and provides an energetically favorable pathway for the formation of fructose 6-phosphate (Fig. 10.4). This reaction is an important regulatory site of gluconeogenesis. 1. Regulation by intracellular energy levels: Fructose 1,6-bisphosphatase is inhibited by a rise in the adenosine monophosphate (AMP)/ATP ratio, which signals a low-energy state in the cell. Conversely, low AMP and high ATP levels stimulate gluconeogenesis, an energy-requiring pathway. 2.

1	2. Regulation by fructose 2,6-bisphosphate: Fructose 1,6-bisphosphatase is inhibited by fructose 2,6-bisphosphate, an allosteric effector whose concentration is influenced by the insulin/glucagon ratio. When glucagon is high, the effector is not made by hepatic PFK-2 (see p. 99), and thus, the phosphatase is active (Fig. 10.5). [Note: The signals that inhibit (low energy, high fructose 2,6-bisphosphate) or activate (high energy, low fructose 2,6-bisphosphate) gluconeogenesis have the opposite effect on glycolysis, providing reciprocal control of the pathways that synthesize and oxidize glucose (see p. 100).] E. Glucose 6-phosphate dephosphorylation

1	Glucose 6-phosphate hydrolysis by glucose 6-phosphatase bypasses the irreversible hexokinase/glucokinase reaction and provides an energetically favorable pathway for the formation of free glucose (Fig. 10.6). The liver is the primary organ that produces free glucose from glucose 6-phosphate. This process requires a complex of two proteins found only in gluconeogenic tissue: glucose 6-phosphate translocase, which transports glucose 6-phosphate across the endoplasmic reticular (ER) membrane, and glucose 6-phosphatase, which removes the phosphate, producing free glucose (see Fig. 10.6). [Note: These ER membrane proteins are also required for the final step of glycogen degradation (see p. 130). Glycogen storage diseases Ia and Ib, caused by deficiencies in the phosphatase and the translocase, respectively, are characterized by severe fasting hypoglycemia, because free glucose is unable to be produced from either gluconeogenesis or glycogenolysis.] Specific transporters are responsible for

1	respectively, are characterized by severe fasting hypoglycemia, because free glucose is unable to be produced from either gluconeogenesis or glycogenolysis.] Specific transporters are responsible for moving the free glucose into the cytosol and then into blood.

1	F. Summary of the reactions of glycolysis and gluconeogenesis Of the 11 reactions required to convert pyruvate to free glucose, 7 are catalyzed by reversible glycolytic enzymes (Fig. 10.7). The 3 irreversible reactions (catalyzed by hexokinase/glucokinase, PFK-1, and PK) are circumvented by reactions catalyzed by glucose 6-phosphatase, fructose 1,6-bisphosphatase, PC, and PEPCK. In gluconeogenesis, the equilibria of the reversible glycolytic reactions are pushed toward glucose synthesis as a result of the essentially irreversible formation of PEP, fructose 6-phosphate, and glucose by the gluconeogenic enzymes. [Note: The stoichiometry of gluconeogenesis from two pyruvate molecules couples the cleavage of six high-energy phosphate bonds and the oxidation of two NADH with the formation of one glucose molecule (see Fig. 10.7).] IV. REGULATION

1	IV. REGULATION The moment-to-moment regulation of gluconeogenesis is determined primarily by the circulating level of glucagon and by the availability of gluconeogenic substrates. In addition, slow adaptive changes in enzyme amount result from an alteration in the rate of enzyme synthesis or degradation or both. [Note: Hormonal control of the glucoregulatory system is presented in Chapter 23.] This peptide hormone from pancreatic islet α cells (see p. 313) stimulates gluconeogenesis by three mechanisms. 1. Changes in allosteric effectors: Glucagon lowers hepatic fructose 2,6bisphosphate, resulting in fructose 1,6-bisphosphatase activation and PFK-1 inhibition, thereby favoring gluconeogenesis over glycolysis (see Fig. 10.5). [Note: See pp. 99–100 for the role of fructose 2,6bisphosphate in glycolysis regulation.] 2.

1	Covalent modification of enzyme activity: Glucagon binds its G protein– coupled receptor (see p. 95) and, via an elevation in cyclic AMP (cAMP) level and cAMP-dependent protein kinase A activity, stimulates the conversion of hepatic PK to its inactive (phosphorylated) form. This decreases PEP conversion to pyruvate, which has the effect of diverting PEP to gluconeogenesis (Fig. 10.8). the enzyme. [Note: Only the hepatic isozyme is subject to covalent regulation.] AMP. 3. Induction of enzyme synthesis: Glucagon increases transcription of the gene for PEPCK via the transcription factor cAMP response element– binding (CREB) protein, thereby increasing the availability of this enzyme as levels of its substrate rise during fasting. [Note: Cortisol (a glucocorticoid) also increases expression of the gene, whereas insulin decreases expression.] B. Substrate availability

1	B. Substrate availability The availability of gluconeogenic precursors, particularly glucogenic amino acids, significantly influences the rate of glucose synthesis. Decreased insulin levels favor mobilization of amino acids from muscle protein to provide the carbon skeletons for gluconeogenesis. The ATP and NADH coenzymes required for gluconeogenesis are primarily provided by FA oxidation. C. Allosteric activation by acetyl CoA Allosteric activation of hepatic PC by acetyl CoA occurs during fasting. As a result of increased TAG hydrolysis in adipose tissue, the liver is flooded with FA (see p. 330). The rate of formation of acetyl CoA by β-oxidation of these FA exceeds the capacity of the liver to oxidize it to CO2 and water. As a result, acetyl CoA accumulates and activates PC. [Note: Acetyl CoA inhibits the PDHC (by activating PDH kinase; see p. 111). Thus, this single compound can divert pyruvate toward gluconeogenesis and away from the TCA cycle (Fig. 10.9).]

1	D. Allosteric inhibition by AMP Fructose 1,6-bisphosphatase is inhibited by AMP, a compound that activates PFK-1. This results in reciprocal regulation of glycolysis and gluconeogenesis seen previously with fructose 2,6-bisphosphate (see p. 121). [Note: Thus, elevated AMP stimulates energy-producing pathways and inhibits energy-requiring ones.] V. CHAPTER SUMMARY

1	Gluconeogenic precursors include glycerol released during triacylglycerol hydrolysis in adipose tissue, lactate released by cells that lack mitochondria and by exercising skeletal muscle, and α-keto acids (for example, αketoglutarate and pyruvate) derived from glucogenic amino acid metabolism (Fig. 10.10). Seven of the reactions of glycolysis are reversible and are used for gluconeogenesis in the liver and kidneys. Three reactions, catalyzed by pyruvate kinase, phosphofructokinase-1, and glucokinase/hexokinase, are physiologically irreversible and must be circumvented. Pyruvate is converted to oxaloacetate and then to phosphoenolpyruvate (PEP) by pyruvate carboxylase (PC) and PEPcarboxykinase (PEPCK ). PC requires biotin and ATP and is allosterically activated by acetyl coenzyme A. PEPCK requires guanosine triphosphate. Transcription of its gene is increased by glucagon and cortisol and decreased by insulin. Fructose 1,6-bisphosphate is converted to fructose 6phosphate by fructose

1	requires guanosine triphosphate. Transcription of its gene is increased by glucagon and cortisol and decreased by insulin. Fructose 1,6-bisphosphate is converted to fructose 6phosphate by fructose 1,6-bisphosphatase. This enzyme is inhibited by a high adenosine monophosphate (AMP)/ATP ratio. It is also inhibited by fructose 2,6-bisphosphate, the primary allosteric activator of glycolysis. Glucose 6-phosphate is dephosphorylated to glucose by glucose 6phosphatase. This enzyme of the endoplasmic reticular membrane catalyzes the final step in gluconeogenesis and in glycogen degradation. Its deficiency results in severe, fasting hypoglycemia.

1	dioxide. Choose the ONE best answer. 0.1. Which one of the following statements concerning gluconeogenesis is correct? A. It is an energy-producing (exergonic) process. B. It is important in maintaining blood glucose during a 2-day fast. C. It is inhibited by a fall in the insulin/glucagon ratio. D. It occurs in the cytosol of muscle cells. E. It uses carbon skeletons provided by fatty acid degradation.

1	C. It is inhibited by a fall in the insulin/glucagon ratio. D. It occurs in the cytosol of muscle cells. E. It uses carbon skeletons provided by fatty acid degradation. Correct answer = B. During a 2-day fast, glycogen stores are depleted, and gluconeogenesis maintains blood glucose. This is an energy-requiring (endergonic) pathway (both ATP and GTP get hydrolyzed) that occurs primarily in the liver, with the kidneys becoming major glucose producers in prolonged fasting. Gluconeogenesis uses both mitochondrial and cytosolic enzymes and is stimulated by a fall in the insulin/glucagon ratio. Fatty acid degradation yields acetyl coenzyme A (CoA), which cannot be converted to glucose. This is because there is no net gain of carbons from acetyl CoA in the tricarboxylic acid cycle, and the pyruvate dehydrogenase complex is physiologically irreversible. It is the carbon skeletons of most amino acids that are glucogenic.

1	0.2. Which reaction in the diagram below would be inhibited in the presence of large amounts of avidin, an egg white protein that binds and sequesters biotin? Correct answer = C. Pyruvate is carboxylated to oxaloacetate by pyruvate carboxylase, a biotin-requiring enzyme. B (pyruvate dehydrogenase complex) requires thiamine pyrophosphate, lipoic acid, flavin and nicotinamide adenine dinucleotides (FAD and NAD+), and coenzyme A; D (transaminase) requires pyridoxal phosphate; E (lactate dehydrogenase) requires NADH. 0.3. Which one of the following reactions is unique to gluconeogenesis? A. 1,3-Bisphosphoglycerate → 3-phosphoglycerate B. Lactate → pyruvate C. Oxaloacetate → phosphoenolpyruvate D. Phosphoenolpyruvate → pyruvate Correct answer = C. The other reactions are common to both gluconeogenesis and glycolysis. 0.4. Use the chart below to show the effect of adenosine monophosphate (AMP) and fructose 2,6-bisphosphate on the listed enzymes of gluconeogenesis and glycolysis.

1	0.4. Use the chart below to show the effect of adenosine monophosphate (AMP) and fructose 2,6-bisphosphate on the listed enzymes of gluconeogenesis and glycolysis. Both fructose 2,6-bisphosphate and adenosine monophosphate inhibit fructose 1,6-bisphosphatase of gluconeogenesis and activate phosphofructokinase-1 of glycolysis. This results in reciprocal regulation of the two pathways. 0.5. The metabolism of ethanol by alcohol dehydrogenase produces reduced nicotinamide adenine dinucleotide (NADH) from the oxidized (NAD+) form. What effect is the fall in the NAD+/NADH ratio expected to have on gluconeogenesis? Explain.

1	The increase in NADH as ethanol is oxidized decreases the availability of oxaloacetate (OAA) because the reversible oxidation of malate to OAA by malate dehydrogenase of the tricarboxylic acid cycle is driven in the reverse direction by NADH. Additionally, the reversible reduction of pyruvate to lactate by lactate dehydrogenase is driven to lactate by NADH. Thus, two important gluconeogenic substrates, OAA and pyruvate, decrease as a result of the increase in NADH during ethanol metabolism. Consequently, gluconeogenesis decreases. 0.6. Given that acetyl coenzyme A cannot be a substrate for gluconeogenesis, why is its production in fatty acid oxidation essential for gluconeogenesis? Acetyl coenzyme A inhibits the pyruvate dehydrogenase complex and activates pyruvate carboxylase, pushing pyruvate to gluconeogenesis and away from oxidation. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	A constant source of blood glucose is an absolute requirement for human life. Glucose is the greatly preferred energy source for the brain and the required energy source for cells with few or no mitochondria such as mature red blood cells. Glucose is also essential as an energy source for exercising muscle, where it is the substrate for anaerobic glycolysis. Blood glucose can be obtained from three primary sources: the diet, glycogen degradation, and gluconeogenesis. Dietary intake of glucose and glucose precursors, such as starch (a polysaccharide), disaccharides, and monosaccharides, is sporadic and, depending on the diet, is not always a reliable source of blood glucose. In contrast, gluconeogenesis (see p. 117) can provide sustained synthesis of glucose, but it is somewhat slow in responding to a falling blood glucose level. Therefore, the body has developed mechanisms for storing a supply of glucose in a rapidly mobilized form, namely, glycogen. In the absence of a dietary source

1	to a falling blood glucose level. Therefore, the body has developed mechanisms for storing a supply of glucose in a rapidly mobilized form, namely, glycogen. In the absence of a dietary source of glucose, this sugar is rapidly released into the blood from liver glycogen. Similarly, muscle glycogen is extensively degraded in exercising muscle to provide that tissue with an important energy source. When glycogen stores are depleted, specific tissues synthesize glucose de novo, using glycerol, lactate, pyruvate, and amino acids as carbon sources for gluconeogenesis (see Chapter 10). Figure 11.1 shows the reactions of glycogen synthesis and degradation as part of the essential pathways of energy metabolism.

1	II. STRUCTURE AND FUNCTION The main stores of glycogen are found in skeletal muscle and liver, although most other cells store small amounts of glycogen for their own use. The function of muscle glycogen is to serve as a fuel reserve for the synthesis of ATP during muscle contraction. That of liver glycogen is to maintain the blood glucose concentration, particularly during the early stages of a fast (Fig. 11.2; also see p. 329). [Note: Liver glycogen can maintain blood glucose for <24 hours.] A. Amounts in liver and muscle

1	A. Amounts in liver and muscle Approximately 400 g of glycogen make up 1%–2% of the fresh weight of resting muscle, and ~100 g of glycogen make up to 10% of the fresh weight of a well-fed adult liver. What limits the production of glycogen at these levels is not clear. However, in some glycogen storage diseases (GSD) (see Fig. 11.8), the amount of glycogen in the liver and/or muscle can be significantly higher. [Note: In the body, muscle mass is greater than liver mass. Consequently, most of the body’s glycogen is found in skeletal muscle.] B. Structure

1	B. Structure Glycogen is a branched-chain polysaccharide made exclusively from α-Dglucose. The primary glycosidic bond is an α(1→4) linkage. After an average of 8–14 glucosyl residues, there is a branch containing an α(1→6) linkage (Fig. 11.3). A single glycogen molecule can contain up to 55,000 glucosyl residues. These polymers of glucose exist as large, spherical, cytoplasmic granules (particles) that also contain most of the enzymes necessary for glycogen synthesis and degradation. C. Glycogen store fluctuation

1	C. Glycogen store fluctuation Liver glycogen stores increase during the well-fed state (see p. 323) and are depleted during a fast (see p. 329). Muscle glycogen is not affected by short periods of fasting (a few days) and is only moderately decreased in prolonged fasting (weeks). Muscle glycogen is synthesized to replenish muscle stores after they have been depleted following strenuous exercise. [Note: Glycogen synthesis and degradation go on continuously. The difference between the rates of these two processes determines the levels of stored glycogen during specific physiologic states.] III. SYNTHESIS (GLYCOGENESIS) Glycogen is synthesized from molecules of α-D-glucose. The process occurs in the cytosol and requires energy supplied by ATP (for the phosphorylation of glucose) and uridine triphosphate (UTP).

1	Glycogen is synthesized from molecules of α-D-glucose. The process occurs in the cytosol and requires energy supplied by ATP (for the phosphorylation of glucose) and uridine triphosphate (UTP). A. Uridine diphosphate glucose synthesis α-D-Glucose attached to uridine diphosphate (UDP) is the source of all the glucosyl residues that are added to the growing glycogen molecule. UDP-glucose (Fig. 11.4) is synthesized from glucose 1-phosphate and UTP by UDP–glucose pyrophosphorylase (Fig. 11.5). Pyrophosphate (PPi), the second product of the reaction, is hydrolyzed to two inorganic phosphates (Pi) by pyrophosphatase. The hydrolysis is exergonic, insuring that the UDP–glucose pyrophosphorylase reaction proceeds in the direction of UDP-glucose production. [Note: Glucose 1-phosphate is generated from glucose 6-phosphate by phosphoglucomutase. Glucose 1,6-bisphosphate is an obligatory intermediate in this reversible reaction (Fig. 11.6).] B. Primer requirement and synthesis

1	Glycogen synthase makes the α(1→4) linkages in glycogen. This enzyme cannot initiate chain synthesis using free glucose as an acceptor of a molecule of glucose from UDP-glucose. Instead, it can only elongate already existing chains of glucose and, therefore, requires a primer. A fragment of glycogen can serve as a primer. In the absence of a fragment, the homodimeric protein glycogenin can serve as an acceptor of glucose from UDP-glucose (see Fig. 11.5). The side-chain hydroxyl group of tyrosine-194 in the protein is the site at which the initial glucosyl unit is attached. Because the reaction is catalyzed by glycogenin itself via autoglucosylation, glycogenin is an enzyme. Glycogenin then catalyzes the transfer of at least four molecules of glucose from UDP-glucose, producing a short, α(1→4)-linked glucosyl chain. This short chain serves as a primer that is able to be elongated by glycogen synthase, which is recruited by glycogenin, as described in C. below. [Note: Glycogenin stays

1	glucosyl chain. This short chain serves as a primer that is able to be elongated by glycogen synthase, which is recruited by glycogenin, as described in C. below. [Note: Glycogenin stays associated with and forms the core of a glycogen granule.]

1	C. Elongation by glycogen synthase Elongation of a glycogen chain involves the transfer of glucose from UDP-glucose to the nonreducing end of the growing chain, forming a new glycosidic bond between the anomeric hydroxyl group of carbon 1 of the activated glucose and carbon 4 of the accepting glucosyl residue (see Fig. 11.5). [Note: The nonreducing end of a carbohydrate chain is one in which the anomeric carbon of the terminal sugar is linked by a glycosidic bond to another molecule, making the terminal sugar nonreducing (see p. 84).] The enzyme responsible for making the α(1→4) linkages in glycogen is glycogen synthase. [Note: The UDP released when the new α(1→4) glycosidic bond is made can be phosphorylated to UTP by nucleoside diphosphate kinase (UDP + ATP ⇄ UTP + ADP; see p. 296).] D. Branch formation

1	D. Branch formation If no other synthetic enzyme acted on the chain, the resulting structure would be a linear (unbranched) chain of glucosyl residues attached by α(1→4) linkages. Such a compound is found in plant tissues and is called amylose. In contrast, glycogen has branches located, on average, eight glucosyl residues apart, resulting in a highly branched, tree-like structure (see Fig. 11.3) that is far more soluble than the unbranched amylose. Branching also increases the number of nonreducing ends to which new glucosyl residues can be added (and also, as described in IV. below, from which these residues can be removed), thereby greatly accelerating the rate at which glycogen synthesis can occur and dramatically increasing the size of the glycogen molecule. 1.

1	1. Branch synthesis: Branches are made by the action of the branching enzyme, amylo-α(1→4)→α(1→6)-transglycosylase. This enzyme removes a set of six to eight glucosyl residues from the nonreducing end of the glycogen chain, breaking an α(1→4) bond to another residue on the chain, and attaches it to a nonterminal glucosyl residue by an α(1→6) linkage, thus functioning as a 4:6 transferase. The resulting new, nonreducing end (see “i” in Fig. 11.5), as well as the old nonreducing end from which the six to eight residues were removed (see “o” in Fig. 11.5), can now be further elongated by glycogen synthase. 2. Additional branch synthesis: After elongation of these two ends has been accomplished, their terminal six to eight glucosyl residues can be removed and used to make additional branches. IV. DEGRADATION (GLYCOGENOLYSIS)

1	IV. DEGRADATION (GLYCOGENOLYSIS) The degradative pathway that mobilizes stored glycogen in liver and skeletal muscle is not a reversal of the synthetic reactions. Instead, a separate set of cytosolic enzymes is required. When glycogen is degraded, the primary product is glucose 1-phosphate, obtained by breaking α(1→4) glycosidic bonds. In addition, free glucose is released from each α(1→6)–linked glucosyl residue (branch point). A. Chain shortening Glycogen phosphorylase sequentially cleaves the α(1→4) glycosidic bonds between the glucosyl residues at the nonreducing ends of the glycogen chains by simple phosphorolysis (producing glucose 1-phosphate) until four glucosyl units remain on each chain at a branch point (Fig. 11.7). The resulting structure is called a limit dextrin, and phosphorylase cannot degrade it any further (Fig. 11.8). [Note: Phosphorylase requires pyridoxal phosphate (a derivative of vitamin B6; see p. 382) as a coenzyme.] B. Branch removal

1	B. Branch removal Branches are removed by the two enzymic activities of a single bifunctional protein, the debranching enzyme (see Fig. 11.8). First, oligoα(1→4)→α(1→4)-glucantransferase activity removes the outer three of the four glucosyl residues remaining at a branch. It next transfers them to the nonreducing end of another chain, lengthening it accordingly. Thus, an α(1→4) bond is broken and an α(1→4) bond is made, and the enzyme functions as a 4:4 transferase. Next, the remaining glucose residue attached in an α(1→6) linkage is removed hydrolytically by amylo-α(1→6)glucosidase activity, releasing free (nonphosphorylated) glucose. The glucosyl chain is now available again for degradation by glycogen phosphorylase until four glucosyl units in the next branch are reached. C. Glucose 1-phosphate isomerization to glucose 6phosphate

1	C. Glucose 1-phosphate isomerization to glucose 6phosphate Glucose 1-phosphate, produced by glycogen phosphorylase, is isomerized in the cytosol to glucose 6-phosphate by phosphoglucomutase (see Fig. 11.6). In the liver, glucose 6-phosphate is transported into the endoplasmic reticulum (ER) by glucose 6-phosphate translocase. There, it is dephosphorylated to glucose by glucose 6-phosphatase (the same enzyme used in the last step of gluconeogenesis; see p. 121). The glucose is then transported from the ER to the cytosol. Hepatocytes release glycogen-derived glucose into the blood to help maintain blood glucose levels until the gluconeogenic pathway is actively producing glucose. [Note: Muscle lacks glucose 6-phosphatase. Consequently, glucose 6-phosphate cannot be dephosphorylated and sent into the blood. Instead, it enters glycolysis, providing energy needed for muscle contraction.] D. Lysosomal degradation

1	D. Lysosomal degradation A small amount (1%–3%) of glycogen is degraded by the lysosomal enzyme, acid α(1→4)-glucosidase (acid maltase). The purpose of this autophagic pathway is unknown. However, a deficiency of this enzyme causes accumulation of glycogen in vacuoles in the lysosomes, resulting in the serious GSD type II: Pompe disease (see Fig. 11.8). [Note: Pompe disease is the only GSD that is a lysosomal storage disease.] Lysosomal storage diseases are genetic disorders characterized by the accumulation of abnormal amounts of carbohydrates or lipids primarily due to their decreased lysosomal degradation resulting from decreased activity or amount of lysosomal acid hydrolases. V. GLYCOGENESIS AND GLYCOGENOLYSIS REGULATION

1	V. GLYCOGENESIS AND GLYCOGENOLYSIS REGULATION Because of the importance of maintaining blood glucose levels, the synthesis and degradation of its glycogen storage form are tightly regulated. In the liver, glycogenesis accelerates during periods when the body has been well fed, whereas glycogenolysis accelerates during periods of fasting. In skeletal muscle, glycogenolysis occurs during active exercise, and glycogenesis begins as soon as the muscle is again at rest. Regulation of synthesis and degradation is accomplished on two levels. First, glycogen synthase and glycogen phosphorylase are hormonally regulated (by covalent phosphorylation/dephosphorylation) to meet the needs of the body as a whole. Second, these same enzymes are allosterically regulated (by effector molecules) to meet the needs of a particular tissue. A. Covalent activation of glycogenolysis

1	A. Covalent activation of glycogenolysis The binding of hormones, such as glucagon or epinephrine, to plasma membrane G protein–coupled receptors ([GPCR] see p. 94) signals the need for glycogen to be degraded, either to elevate blood glucose levels or to provide energy for exercising muscle. 1. Protein kinase A activation: Binding of glucagon or epinephrine to their specific hepatocyte GPCR, or of epinephrine to a specific myocyte GPCR, results in the G protein–mediated activation of adenylyl cyclase. This enzyme catalyzes the synthesis of cyclic adenosine monophosphate (cAMP), which activates cAMP-dependent protein kinase A (PKA). cAMP binds the two regulatory subunits of tetrameric PKA, releasing two individual catalytic subunits that are active (Fig. 11.9; also see p. 95). PKA then phosphorylates several enzymes of glycogen metabolism, affecting their activity. [Note: When cAMP is removed, the inactive tetramer reforms.] on next page.) catalytic subunit. 2.

1	2. Phosphorylase kinase activation: Phosphorylase kinase exists in two forms: an inactive “b” form and an active “a” form. Active PKA phosphorylates the inactive “b” form of phosphorylase kinase, producing the active “a” form (see Fig. 11.9). 3. Glycogen phosphorylase activation: Glycogen phosphorylase also exists in a dephosphorylated, inactive “b” form and a phosphorylated, active “a” form. Phosphorylase kinase a is the only enzyme that phosphorylates glycogen phosphorylase b to its active “a” form, which then begins glycogenolysis (see Fig. 11.9). 4.

1	4. Signal amplification: The cascade of reactions described above activates glycogenolysis. The large number of sequential steps serves to amplify the effect of the hormonal signal (that is, a few hormone molecules binding to their GPCR result in a number of PKA molecules being activated that can each activate many phosphorylase kinase molecules). This causes the production of many active glycogen phosphorylase a molecules that can degrade glycogen. 5.

1	5. Phosphorylated state maintenance: The phosphate groups added to phosphorylase kinase and phosphorylase in response to cAMP are maintained because the enzyme that hydrolytically removes the phosphate, protein phosphatase-1 (PP1), is inactivated by inhibitor proteins that are also phosphorylated and activated in response to cAMP (see Fig. 11.9). [Note: PP1 is activated by a signal cascade initiated by insulin (see Fig. 27.7 on p. 311). Because insulin also activates the phosphodiesterase that degrades cAMP, it opposes the effects of glucagon and epinephrine.] B. Covalent inhibition of glycogenesis

1	B. Covalent inhibition of glycogenesis The regulated enzyme in glycogenesis, glycogen synthase, also exists in two forms, the active “a” form and the inactive “b” form. However, in contrast to phosphorylase kinase and phosphorylase, the active form of glycogen synthase is dephosphorylated, whereas the inactive form is phosphorylated at several sites on the enzyme, with the level of inactivation proportional to the degree of phosphorylation (Fig. 11.10). Phosphorylation is catalyzed by several different protein kinases in response to cAMP (for example, PKA and phosphorylase kinase) or other signaling mechanisms (see C. below). Glycogen synthase b can be reconverted to the “a” form by PP1. Figure 11.11 summarizes the covalent regulation of glycogen metabolism. = regulatory subunit; C = catalytic subunit; ADP = adenosine diphosphate. C. Allosteric regulation of glycogenesis and glycogenolysis

1	In addition to hormonal signals, glycogen synthase and glycogen phosphorylase respond to the levels of metabolites and energy needs of the cell. Glycogenesis is stimulated when glucose and energy levels are high, whereas glycogenolysis is increased when glucose and energy levels are low. This allosteric regulation allows a rapid response to the needs of a cell and can override the effects of hormone-mediated covalent regulation. [Note: The “a” and “b” forms of the allosteric enzymes of glycogen metabolism are each in an equilibrium between the R (relaxed, more active) and T (tense, less active) conformations (see p. 28). The binding of effectors shifts the equilibrium and alters enzymic activity without directly altering the covalent modification.] 1. Regulation in the well-fed state: In the well-fed state, glycogen synthase b in both liver and muscle is allosterically activated by glucose 6phosphate, which is present in elevated concentrations (Fig. 11.12). In contrast, glycogen

1	In the well-fed state, glycogen synthase b in both liver and muscle is allosterically activated by glucose 6phosphate, which is present in elevated concentrations (Fig. 11.12). In contrast, glycogen phosphorylase a is allosterically inhibited by glucose 6-phosphate, as well as by ATP, a high-energy signal. [Note: In liver, but not muscle, free glucose is also an allosteric inhibitor of glycogen phosphorylase a.] (A) and muscle (B). P = phosphate; AMP = adenosine monophosphate.

1	2. Glycogenolysis activation by AMP: Muscle glycogen phosphorylase (myophosphorylase), but not the liver isozyme, is active in the presence of the high AMP concentrations that occur under extreme conditions of anoxia and ATP depletion. AMP binds to glycogen phosphorylase b, causing its activation without phosphorylation (see Fig. 11.9). [Note: Recall that AMP also activates phosphofructokinase-1 of glycolysis (see p. 99), allowing glucose from glycogenolysis to be oxidized.] 3. Glycogenolysis activation by calcium: Calcium (Ca2+) is released into the sarcoplasm in muscle cells (myocytes) in response to neural stimulation and in the liver in response to epinephrine binding to α1 adrenergic receptors. The Ca2+ binds to calmodulin (CaM), the most widely distributed member of a family of small, Ca2+-binding proteins. The binding of four molecules of Ca2+ to CaM triggers a conformational change such that the activated Ca2+–CaM complex binds to and activates protein molecules, often

1	of small, Ca2+-binding proteins. The binding of four molecules of Ca2+ to CaM triggers a conformational change such that the activated Ca2+–CaM complex binds to and activates protein molecules, often enzymes, that are inactive in the absence of this complex (Fig. 11.13). Thus, CaM functions as an essential subunit of many complex proteins. One such protein is the tetrameric phosphorylase kinase, whose “b” form is activated by the binding of Ca2+ to its δ subunit (CaM) without the need for the kinase to be phosphorylated by PKA. [Note: Epinephrine at β-adrenergic receptors signals through a rise in cAMP, not Ca2+ (see p. 131).] a.

1	Muscle phosphorylase kinase activation: During muscle contraction, there is a rapid and urgent need for ATP. It is supplied by the degradation of muscle glycogen to glucose 6-phosphate, which enters glycolysis. Nerve impulses cause membrane depolarization, which promotes Ca2+ release from the sarcoplasmic reticulum into the sarcoplasm of myocytes. The Ca2+ binds the CaM subunit, and the complex activates muscle phosphorylase kinase b (see Fig. 11.9). b. Liver phosphorylase kinase activation: During physiologic stress, epinephrine is released from the adrenal medulla and signals the need for blood glucose. This glucose initially comes from hepatic glycogenolysis. Binding of epinephrine to hepatocyte α1-adrenergic

1	GPCR activates a phospholipid-dependent cascade (see p. 205) that results in movement of Ca2+ from the ER into the cytoplasm. A Ca2+– CaM complex forms and activates hepatic phosphorylase kinase b. [Note: The released Ca2+ also helps to activate protein kinase C that can phosphorylate (therefore, inactivate) glycogen synthase a.] VI. GLYCOGEN STORAGE DISEASES

1	VI. GLYCOGEN STORAGE DISEASES GSD are a group of genetic diseases caused by defects in enzymes required for glycogen degradation or, more rarely, glycogen synthesis. They result either in formation of glycogen that has an abnormal structure or in the accumulation of excessive amounts of normal glycogen in specific tissues as a result of impaired degradation. A particular enzyme may be defective in a single tissue, such as the liver (resulting in hypoglycemia) or muscle (causing muscle weakness), or the defect may be more generalized, affecting a variety of tissues, such as the heart and kidneys. Severity ranges from fatal in early childhood to mild disorders that are not life threatening. Some of the more prevalent GSD are illustrated in Figure 11.8. [Note: Only GSD II is lysosomal because glycogen metabolism occurs primarily in the cytosol.] VII. CHAPTER SUMMARY

1	The main stores of glycogen in the body are found in skeletal muscle, where they serve as a fuel reserve for the synthesis of ATP during muscle contraction, and in the liver, where they are used to maintain the blood glucose concentration, particularly during the early stages of a fast. Glycogen is a highly branched polymer of α-D-glucose. The primary glycosidic bond is an α(1→4) linkage. After about 8–14 glucosyl residues, there is a branch containing an α(1→6) linkage. Uridine diphosphate (UDP)-glucose, the building block of glycogen, is synthesized from glucose 1-phosphate and UTP by UDP–glucose pyrophosphorylase (Fig. 11.14). Glucose from UDP-glucose is transferred to the nonreducing ends of glycogen chains by primer-requiring glycogen synthase, which makes the α(1→4) linkages. The primer is made by glycogenin. Branches are formed by amylo-α(1→4)→α(1→6)-transglycosylase (a 4:6 transferase), which transfers a set of six to eight glucosyl residues from the nonreducing end of the

1	primer is made by glycogenin. Branches are formed by amylo-α(1→4)→α(1→6)-transglycosylase (a 4:6 transferase), which transfers a set of six to eight glucosyl residues from the nonreducing end of the glycogen chain (breaking an α(1→4) linkage), and making an α(1→6) linkage to another residue in the chain. Pyridoxal phosphate–requiring glycogen phosphorylase cleaves the α(1→4) bonds between glucosyl residues at the nonreducing ends of the glycogen chains, producing glucose 1-phosphate. This sequential degradation continues until four glucosyl units remain before a branch point. The resulting structure is called a limit dextrin that is degraded by the bifunctional debranching enzyme. Oligo-α(1→4)→α(1→4)-glucantransferase (a 4:4 transferase) activity removes the outer three of the four glucosyl residues at a branch and transfers them to the nonreducing end of another chain, where they can be released as glucose 1-phosphate by glycogen phosphorylase. The remaining single glucose residue

1	residues at a branch and transfers them to the nonreducing end of another chain, where they can be released as glucose 1-phosphate by glycogen phosphorylase. The remaining single glucose residue attached in an α(1→6) linkage is removed hydrolytically by the amylo-α(1→6) glucosidase activity of debranching enzyme, releasing free glucose. Glucose 1-phosphate is converted to glucose 6-phosphate by phosphoglucomutase. In muscle, glucose 6phosphate enters glycolysis. In liver, the phosphate is removed by glucose 6-phosphatase (an enzyme of the endoplasmic reticular membrane), releasing free glucose that can be used to maintain blood glucose levels at the beginning of a fast. A deficiency of the phosphatase causes glycogen storage disease Ia (von Gierke disease) and results in an inability of the liver to provide free glucose to the body during a fast. It affects both glycogen degradation and gluconeogenesis. Glycogen synthesis and degradation are reciprocally regulated to meet whole-body

1	the liver to provide free glucose to the body during a fast. It affects both glycogen degradation and gluconeogenesis. Glycogen synthesis and degradation are reciprocally regulated to meet whole-body needs by the same hormonal signals (namely, an elevated insulin level results in overall increased glycogenesis and decreased glycogenolysis, whereas an elevated glucagon, or epinephrine, level causes the opposite effects). Key enzymes are phosphorylated by a family of protein kinases, some of which are dependent on cyclic adenosine monophosphate (cAMP), a compound increased by glucagon and epinephrine. Phosphate groups are removed by protein phosphatase-1 (active when its inhibitor is inactive in response to elevated insulin levels). In addition to this covalent regulation, glycogen synthase, phosphorylase kinase, and phosphorylase are allosterically regulated to meet tissues’ needs. In the well-fed state, glycogen synthase is activated by glucose 6-phosphate, but glycogen phosphorylase

1	phosphorylase kinase, and phosphorylase are allosterically regulated to meet tissues’ needs. In the well-fed state, glycogen synthase is activated by glucose 6-phosphate, but glycogen phosphorylase is inhibited by glucose 6-phosphate as well as by ATP. In the liver, free glucose also serves as an allosteric inhibitor of glycogen phosphorylase. The rise in calcium in muscle during exercise and in liver in response to epinephrine activates phosphorylase kinase by binding to the enzyme’s calmodulin subunit. This allows the enzyme to activate glycogen phosphorylase, thereby causing glycogen degradation. AMP activates glycogen phosphorylase (myophosphorylase) in muscle.

1	Choose the ONE best answer. For Questions 11.1–11.4, match the deficient enzyme to the clinical finding in selected glycogen storage diseases (GSD). 1.1. Exercise intolerance, with no rise in blood lactate during exercise Correct answer = E. Myophosphorylase (the muscle isozyme of glycogen phosphorylase) deficiency (or, McArdle disease) prevents glycogen degradation in muscle, depriving muscle of glycogen-derived glucose, resulting in decreased glycolysis and its anaerobic product, lactate. Correct answer = D. 4:6 Transferase (branching enzyme) deficiency (or, Andersen disease), a defect in glycogen synthesis, results in glycogen with fewer branches and decreased solubility. Correct answer = B. Acid maltase [acid α(1→4)-glucosidase] deficiency (or, Pompe disease) prevents degradation of any glycogen brought into lysosomes. A variety of tissues are affected, with the most severe pathology resulting from heart damage.

1	Correct answer = A. Glucose 6-phosphatase deficiency (or, von Gierke disease) prevents the liver from releasing free glucose into the blood, causing severe fasting hypoglycemia, lactic acidemia, hyperuricemia, and hyperlipidemia. 1.2. Fatal, progressive cirrhosis and glycogen with longer-than-normal outer chains 1.3. Generalized accumulation of glycogen, severe hypotonia, and death from heart failure 1.4. Severe fasting hypoglycemia, lactic acidemia, hyperuricemia, and hyperlipidemia 1.5. Epinephrine and glucagon have which one of the following effects on hepatic glycogen metabolism? A. Both glycogen phosphorylase and glycogen synthase are activated by phosphorylation but at significantly different rates. B. Glycogen phosphorylase is inactivated by the resulting rise in calcium, whereas glycogen synthase is activated. C. Glycogen phosphorylase is phosphorylated and active, whereas glycogen synthase is phosphorylated and inactive. D. The net synthesis of glycogen is increased.

1	C. Glycogen phosphorylase is phosphorylated and active, whereas glycogen synthase is phosphorylated and inactive. D. The net synthesis of glycogen is increased. Correct answer = C. Epinephrine and glucagon both cause increased glycogen degradation and decreased synthesis in the liver through covalent modification (phosphorylation) of key enzymes of glycogen metabolism. Glycogen phosphorylase is phosphorylated and active (“a” form), whereas glycogen synthase is phosphorylated and inactive (“b” form). Glucagon does not cause a rise in calcium. 1.6. In contracting skeletal muscle, a sudden elevation of the sarcoplasmic calcium concentration will result in: A. activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A. B. conversion of cAMP to AMP by phosphodiesterase. C. direct activation of glycogen synthase b. D. direct activation of phosphorylase kinase b. E. inactivation of phosphorylase kinase a by the action of protein phosphatase-1.

1	C. direct activation of glycogen synthase b. D. direct activation of phosphorylase kinase b. E. inactivation of phosphorylase kinase a by the action of protein phosphatase-1. Correct answer = D. Calcium (Ca2+) released from the sarcoplasmic reticulum during exercise binds to the calmodulin subunit of phosphorylase kinase, thereby allosterically activating the dephosphorylated “b” form of this enzyme. The other choices are not caused by an elevation of cytosolic Ca2+ . [Note: Ca2+ also activates hepatic phosphorylase kinase b.] 1.7. Explain why the hypoglycemia seen with type Ia glycogen storage disease (glucose 6-phosphatase deficiency) is severe, whereas that seen with type VI (liver phosphorylase deficiency) is mild.

1	With type Ia, the liver is unable to generate free glucose either from glycogenolysis or gluconeogenesis because both processes produce glucose 6phosphate. With type VI, the liver is still able to produce free glucose from gluconeogenesis, but glycogenolysis is inhibited. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW Glucose is the most common monosaccharide consumed by humans, and its metabolism has already been discussed. Two other monosaccharides, fructose and galactose, also occur in significant amounts in the diet (primarily in disaccharides) and make important contributions to energy metabolism. In addition, galactose is an important component of glycosylated proteins. Figure 12.1 shows the metabolism of fructose and galactose as part of the essential pathways of energy metabolism. II. FRUCTOSE METABOLISM

1	II. FRUCTOSE METABOLISM About 10% of the calories in the Western diet are supplied by fructose (~55 g/day). The major source of fructose is the disaccharide sucrose, which, when cleaved in the intestine, releases equimolar amounts of fructose and glucose. Fructose is also found as a free monosaccharide in many fruits, in honey, and in high-fructose corn syrup (typically, 55% fructose and 45% glucose), which is used to sweeten soft drinks and many foods (see p. 364). Fructose transport into cells is not insulin dependent (unlike that of glucose into certain tissues; see p. 97), and, in contrast to glucose, fructose does not promote the secretion of insulin. A. Phosphorylation

1	For fructose to enter the pathways of intermediary metabolism, it must first be phosphorylated (Fig. 12.2). This can be accomplished by either hexokinase or fructokinase. Hexokinase phosphorylates glucose in most cells of the body (see p. 98), and several additional hexoses can serve as substrates for this enzyme. However, it has a low affinity (that is, a high Michaelis constant [Km]; see p. 59) for fructose. Therefore, unless the intracellular concentration of fructose becomes unusually high, the normal presence of saturating concentrations of glucose means that little fructose is phosphorylated by hexokinase. Fructokinase provides the primary mechanism for fructose phosphorylation (see Fig. 12.2). The enzyme has a low Km for fructose and a high Vmax ([maximal velocity] see p. 57). It is found in the liver (which processes most of the dietary fructose), kidneys, and the small intestine and converts fructose to fructose 1-phosphate, using ATP as the phosphate donor. [Note: These

1	It is found in the liver (which processes most of the dietary fructose), kidneys, and the small intestine and converts fructose to fructose 1-phosphate, using ATP as the phosphate donor. [Note: These three tissues also contain aldolase B, discussed in section B.]

1	B. Fructose 1-phosphate cleavage Fructose 1-phosphate is not phosphorylated to fructose 1,6-bisphosphate as is fructose 6-phosphate (see p. 99) but is cleaved by aldolase B (also called fructose 1-phosphate aldolase) to two trioses, dihydroxyacetone phosphate (DHAP) and glyceraldehyde. [Note: Humans express three aldolase isozymes (the products of three different genes): aldolase A in most tissues; aldolase B in the liver, kidneys, and small intestine; and aldolase C in the brain. All cleave fructose 1,6-bisphosphate produced during glycolysis to DHAP and glyceraldehyde 3-phosphate (see p. 101), but only aldolase B cleaves fructose 1-phosphate.] DHAP can be used in glycolysis or gluconeogenesis, whereas glyceraldehyde can be metabolized by a number of pathways, as illustrated in Figure 12.3. C. Kinetics

1	C. Kinetics The rate of fructose metabolism is more rapid than that of glucose because triose production from fructose 1-phosphate bypasses phosphofructokinase-1, the major rate-limiting step in glycolysis (see p. 99). D. Disorders

1	A deficiency of one of the key enzymes required for the entry of fructose into metabolic pathways can result in either a benign condition as a result of fructokinase deficiency (essential fructosuria) or a severe disturbance of liver and kidney metabolism as a result of aldolase B deficiency (hereditary fructose intolerance [HFI]), which occurs in ~1:20,000 live births (see Fig. 12.3). The first symptoms of HFI appear when a baby is weaned from lactose-containing milk and begins ingesting food containing sucrose or fructose. Fructose 1-phosphate accumulates, resulting in a drop in the level of inorganic phosphate (Pi) and, therefore, of ATP production. As ATP falls, adenosine monophosphate (AMP) rises. The AMP is degraded, causing hyperuricemia (and lactic acidemia; see p. 299). The decreased availability of hepatic ATP decreases gluconeogenesis (causing hypoglycemia with vomiting) and protein synthesis (causing a decrease in blood-clotting factors and other essential proteins). Renal

1	availability of hepatic ATP decreases gluconeogenesis (causing hypoglycemia with vomiting) and protein synthesis (causing a decrease in blood-clotting factors and other essential proteins). Renal reabsorption of Pi is also decreased. [Note: The drop in Pi also inhibits glycogenolysis (see p. 128).] Diagnosis of HFI can be made on the basis of fructose in the urine, enzyme assay using liver cells, or by DNA-based testing (see Chapter 34). With HFI, sucrose, as well as fructose, must be removed from the diet to prevent liver failure and possible death. [Note: Individuals with HFI display an aversion to sweets and, consequently, have an absence of dental caries.]

1	E. Mannose conversion to fructose 6-phosphate Mannose, the C-2 epimer of glucose (see p. 84), is an important component of glycoproteins (see p. 166). Hexokinase phosphorylates mannose, producing mannose 6-phosphate, which, in turn, is reversibly isomerized to fructose 6-phosphate by phosphomannose isomerase. [Note: Most intracellular mannose is synthesized from fructose or is preexisting mannose produced by the degradation of glycoproteins and salvaged by hexokinase. Dietary carbohydrates contain little mannose.] F. Glucose conversion to fructose via sorbitol Most sugars are rapidly phosphorylated following their entry into cells. Therefore, they are trapped within the cells, because organic phosphates cannot freely cross membranes without specific transporters. An alternate mechanism for metabolizing a monosaccharide is to convert it to a polyol (sugar alcohol) by the reduction of an aldehyde group, thereby producing an additional hydroxyl group.

1	1. Sorbitol synthesis: Aldose reductase reduces glucose, producing sorbitol (or, glucitol; Fig. 12.4), but the Km is high. This enzyme is found in many tissues, including the retina, lens, kidneys, peripheral nerves, ovaries, and seminal vesicles. A second enzyme, sorbitol dehydrogenase, can oxidize sorbitol to fructose in cells of the liver, ovaries, and seminal vesicles (see Fig. 12.4). The two-reaction pathway from glucose to fructose in the seminal vesicles benefits sperm cells, which use fructose as a major carbohydrate energy source. The pathway from sorbitol to fructose in the liver provides a mechanism by which any available sorbitol is converted into a substrate that can enter glycolysis.

1	2. Hyperglycemia and sorbitol metabolism: Because insulin is not required for the entry of glucose into cells of the retina, lens, kidneys, and peripheral nerves, large amounts of glucose may enter these cells during times of hyperglycemia (for example, in uncontrolled diabetes). Elevated intracellular glucose concentrations and an adequate supply of reduced nicotinamide adenine dinucleotide phosphate (NADPH) cause aldose reductase to produce a significant increase in the amount of sorbitol, which cannot pass efficiently through cell membranes and, therefore, remains trapped inside the cell (see Fig. 12.4). This is exacerbated when sorbitol dehydrogenase is low or absent (for example, in cells of the retina, lens, kidneys, and peripheral nerves). As a result, sorbitol accumulates in these cells, causing strong osmotic effects and cell swelling due to water influx and retention. Some of the pathologic alterations associated with diabetes can be partly attributed to this osmotic stress,

1	cells, causing strong osmotic effects and cell swelling due to water influx and retention. Some of the pathologic alterations associated with diabetes can be partly attributed to this osmotic stress, including cataract formation, peripheral neuropathy, and microvascular problems leading to nephropathy and retinopathy (see p. 345). [Note: Use of NADPH in the aldose reductase reaction decreases the generation of reduced glutathione, an important antioxidant (see p. 148), and may be related to diabetic complications.]

1	III. GALACTOSE METABOLISM The major dietary source of galactose is lactose (galactosyl β-1,4-glucose) obtained from milk and milk products. [Note: The digestion of lactose by βgalactosidase (lactase) of the intestinal mucosal cell membrane was discussed on p. 87.] Some galactose can also be obtained by lysosomal degradation of glycoproteins and glycolipids. Like fructose (and mannose), the transport of galactose into cells is not insulin dependent. A. Phosphorylation Like fructose, galactose must be phosphorylated before it can be further metabolized. Most tissues have a specific enzyme for this purpose, galactokinase, which produces galactose 1-phosphate (Fig. 12.5). As with other kinases, ATP is the phosphate donor. B. Uridine diphosphate–galactose formation

1	B. Uridine diphosphate–galactose formation Galactose 1-phosphate cannot enter the glycolytic pathway unless it is first converted to uridine diphosphate (UDP)-galactose (Fig. 12.6). This occurs in an exchange reaction, in which UDP-glucose reacts with galactose 1phosphate, producing UDP-galactose and glucose 1-phosphate (see Fig. 12.5). The reaction is catalyzed by galactose 1-phosphate uridylyltransferase (GALT). [Note: The glucose 1-phosphate product can be isomerized to glucose 6-phosphate, which can enter glycolysis or be dephosphorylated.] C. UDP-galactose conversion to UDP-glucose For UDP-galactose to enter the mainstream of glucose metabolism, it must first be isomerized to its C-4 epimer, UDP-glucose, by UDP-hexose 4epimerase. This “new” UDP-glucose (produced from the original UDPgalactose) can participate in biosynthetic reactions (for example, glycogenesis) as well as in the GALT reaction. [Note: See Fig. 12.5 for a summary of the interconversions.]

1	D. UDP-galactose in biosynthetic reactions UDP-galactose can serve as the donor of galactose units in a number of synthetic pathways, including synthesis of lactose (see IV. below), glycoproteins (see p. 166), glycolipids (see p. 210), and glycosaminoglycans (see p. 158). [Note: If galactose is not provided by the diet (for example, when it cannot be released from lactose owing to a lack of β-galactosidase in people who are lactose intolerant), all tissue requirements for UDP-galactose can be met by the action of UDP-hexose 4-epimerase on UDP-glucose, which is efficiently produced from glucose 1-phosphate and uridine triphosphate (see Fig. 12.5).] E. Disorders

1	E. Disorders GALT is severely deficient in individuals with classic galactosemia (see Fig. 12.5). In this disorder, galactose 1-phosphate and, therefore, galactose accumulate. Physiologic consequences are similar to those found in HFI (see p. 138), but a broader spectrum of tissues is affected. The accumulated galactose is shunted into side pathways such as that of galactitol production. This reaction is catalyzed by aldose reductase, the same enzyme that reduces glucose to sorbitol (see p. 139). GALT deficiency is part of the newborn screening panel. Treatment of galactosemia requires removal of galactose and lactose from the diet. [Note: Deficiencies in galactokinase and the epimerase result in less severe disorders of galactose metabolism, although cataracts are common (see Fig. 12.5).] IV. LACTOSE SYNTHESIS

1	Lactose is a disaccharide that consists of a molecule of β-galactose attached by a β(1→4) linkage to glucose. Therefore, lactose is galactosyl β(1→4)-glucose. Because lactose (milk sugar) is made by lactating (milk-producing) mammary glands, milk and other dairy products are the dietary sources of lactose. Lactose is synthesized in the Golgi by lactose synthase (UDP-galactose:glucose galactosyltransferase), which transfers galactose from UDP-galactose to glucose, releasing UDP (Fig. 12.7). This enzyme is composed of two proteins, A and B. Protein A is a β-D-galactosyltransferase and is found in a number of body tissues. In tissues other than the lactating mammary gland, this enzyme transfers galactose from UDP-galactose to N-acetyl-D-glucosamine, forming the same β(1→4) linkage found in lactose, and producing N-acetyllactosamine, a component of the structurally important N-linked glycoproteins (see p. 167). In contrast, protein B is found only in lactating mammary glands. It is

1	in lactose, and producing N-acetyllactosamine, a component of the structurally important N-linked glycoproteins (see p. 167). In contrast, protein B is found only in lactating mammary glands. It is αlactalbumin, and its synthesis is stimulated by the peptide hormone prolactin. Protein B forms a complex with the enzyme, protein A, changing the specificity of that transferase (by decreasing the Km for glucose) so that lactose, rather than

1	N-acetyllactosamine, is produced (see Fig. 12.7). V. CHAPTER SUMMARY

1	The major source of fructose is the disaccharide sucrose, which, when cleaved, releases equimolar amounts of fructose and glucose (Fig. 12.8). Transport of fructose into cells is insulin independent. Fructose is first phosphorylated to fructose 1-phosphate by fructokinase and then cleaved by aldolase B to dihydroxyacetone phosphate and glyceraldehyde. These enzymes are found in the liver, kidneys, and small intestine. A deficiency of fructokinase causes a benign condition (essential fructosuria), whereas a deficiency of aldolase B causes hereditary fructose intolerance (HFI), in which severe hypoglycemia and liver failure lead to death if fructose (and sucrose) is not removed from the diet. Mannose, an important component of glycoproteins, is phosphorylated by hexokinase to mannose 6phosphate, which is reversibly isomerized to fructose 6-phosphate by phosphomannose isomerase. Glucose can be reduced to sorbitol (glucitol) by aldose reductase in many tissues, including the lens, retina,

1	which is reversibly isomerized to fructose 6-phosphate by phosphomannose isomerase. Glucose can be reduced to sorbitol (glucitol) by aldose reductase in many tissues, including the lens, retina, peripheral nerves, kidneys, ovaries, and seminal vesicles. In the liver, ovaries, and seminal vesicles, a second enzyme, sorbitol dehydrogenase, can oxidize sorbitol to produce fructose. Hyperglycemia results in the accumulation of sorbitol in those cells lacking sorbitol dehydrogenase. The resulting osmotic events cause cell swelling and may contribute to the cataract formation, peripheral neuropathy, nephropathy, and retinopathy seen in diabetes. The major dietary source of galactose is lactose. The transport of galactose into cells is insulin independent. Galactose is first phosphorylated by galactokinase (a deficiency results in cataracts) to galactose 1phosphate. This compound is converted to uridine diphosphate (UDP)galactose by galactose 1-phosphate uridylyltransferase (GALT), with the

1	(a deficiency results in cataracts) to galactose 1phosphate. This compound is converted to uridine diphosphate (UDP)galactose by galactose 1-phosphate uridylyltransferase (GALT), with the nucleotide supplied by UDP-glucose. A deficiency of this enzyme causes classic galactosemia. Galactose 1-phosphate accumulates, and excess galactose is converted to galactitol by aldose reductase. This causes liver damage, brain damage, and cataracts. Treatment requires removal of galactose (and lactose) from the diet. For UDP-galactose to enter the mainstream of glucose metabolism, it must first be isomerized to UDP-glucose by UDP-hexose 4-epimerase. This enzyme can also be used to produce UDP-galactose from UDP-glucose when the former is required for glycoprotein and glycolipid synthesis. Lactose is a disaccharide of galactose and glucose. Milk and other dairy products are the dietary sources of lactose. Lactose is synthesized by lactose synthase from UDP-galactose and glucose in the lactating

1	a disaccharide of galactose and glucose. Milk and other dairy products are the dietary sources of lactose. Lactose is synthesized by lactose synthase from UDP-galactose and glucose in the lactating mammary gland. The enzyme has two subunits, protein A (which is a galactosyltransferase found in most cells where it synthesizes N-acetyllactosamine) and protein B (α-lactalbumin, which is found only in lactating mammary glands, and whose synthesis is stimulated by the peptide hormone prolactin). When both subunits are present, the transferase produces lactose.

1	Choose the ONE best answer. 2.1. A nursing female with classic galactosemia is on a galactose-free diet. She is able to produce lactose in breast milk because: A. galactose can be produced from fructose by isomerization. B. galactose can be produced from a glucose metabolite by epimerization. C. hexokinase can efficiently phosphorylate galactose to galactose 1phosphate. D. the enzyme affected in galactosemia is activated by a hormone produced in the mammary gland. Correct answer = B. Uridine diphosphate (UDP)-glucose is converted to UDPgalactose by UDP-hexose 4-epimerase, thereby providing the appropriate form of galactose for lactose synthesis. Isomerization of fructose to galactose does not occur in the human body. Galactose is not converted to galactose 1phosphate by hexokinase. A galactose-free diet provides no galactose. Galactosemia is the result of an enzyme (galactose 1-phosphate uridylyltransferase) deficiency.

1	2.2. A 5-month-old boy is brought to his physician because of vomiting, night sweats, and tremors. History revealed that these symptoms began after fruit juices were introduced to his diet as he was being weaned off breast milk. The physical examination was remarkable for hepatomegaly. Tests on the baby’s urine were positive for reducing sugar but negative for glucose. The infant most likely suffers from a deficiency of: A. aldolase B. B. fructokinase. C. galactokinase. D. β-galactosidase.

1	A. aldolase B. B. fructokinase. C. galactokinase. D. β-galactosidase. Correct answer = A. The symptoms suggest hereditary fructose intolerance, a deficiency in aldolase B. Deficiencies in fructokinase or galactokinase result in relatively benign conditions characterized by elevated levels of fructose or galactose in the blood and urine. Deficiency in β-galactosidase (lactase) results in a decreased ability to degrade lactose (milk sugar). Congenital lactase deficiency is quite rare and would have presented much earlier in this baby (and with different symptoms). Typical lactase deficiency (adult hypolactasia) presents at a later age. 2.3. Lactose synthesis is essential in the production of milk by mammary glands. In lactose synthesis: A. galactose from galactose 1-phosphate is transferred to glucose by galactosyltransferase (protein A), generating lactose. B. protein A is used exclusively in lactose synthesis.

1	A. galactose from galactose 1-phosphate is transferred to glucose by galactosyltransferase (protein A), generating lactose. B. protein A is used exclusively in lactose synthesis. C. α-lactalbumin (protein B) regulates the specificity of protein A by decreasing its affinity for glucose. D. protein B expression is stimulated by prolactin. Correct answer = D. α-Lactalbumin (protein B) expression is increased by the hormone prolactin. Uridine diphosphate–galactose is the form used by the galactosyltransferase (protein A). Protein A is also involved in the synthesis of the amino sugar N-acetyllactosamine. Protein B decreases the Michaelis constant (Km) and, so, increases the affinity of protein A for glucose.

1	2.4. A 3-month-old girl is developing cataracts. Other than not having a social smile or being able to track objects visually, all other aspects of the girl’s examination are normal. Tests on the baby’s urine are positive for reducing sugar but negative for glucose. Which enzyme is most likely deficient in this girl? A. Aldolase B B. Fructokinase C. Galactokinase D. Galactose 1-phosphate uridylyltransferase

1	A. Aldolase B B. Fructokinase C. Galactokinase D. Galactose 1-phosphate uridylyltransferase Correct answer = C. The girl is deficient in galactokinase and is unable to appropriately phosphorylate galactose. Galactose accumulates in the blood (and urine). In the lens of the eye, galactose is reduced by aldose reductase to galactitol, a sugar alcohol, which causes osmotic effects that result in cataract formation. Deficiency of galactose 1-phosphate uridylyltransferase also results in cataracts but is characterized by liver damage and neurologic effects. Fructokinase deficiency is a benign condition. Aldolase B deficiency is severe, with effects on several tissues. Cataracts are not typically seen. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	I. OVERVIEW The pentose phosphate pathway (or, hexose monophosphate shunt) occurs in the cytosol. It includes an irreversible oxidative phase, followed by a series of reversible sugar–phosphate interconversions (Fig. 13.1). In the oxidative phase, carbon 1 of a glucose 6-phosphate molecule is released as carbon dioxide (CO2), and one pentose sugar-phosphate plus two reduced nicotinamide adenine dinucleotide phosphates (NADPH) are produced. The rate and direction of the reversible reactions are determined by the supply of and demand for intermediates of the pathway. The pentose phosphate pathway provides a major portion of the body’s NADPH, which functions as a biochemical reductant. It also produces ribose 5-phosphate, required for nucleotide biosynthesis (see p. 293), and provides a mechanism for the conversion of pentose sugars to triose and hexose intermediates of glycolysis. No ATP is directly consumed or produced in the pathway. II. IRREVERSIBLE OXIDATIVE REACTIONS

1	II. IRREVERSIBLE OXIDATIVE REACTIONS The oxidative portion of the pentose phosphate pathway consists of three irreversible reactions that lead to the formation of ribulose 5-phosphate, CO2, and two molecules of NADPH for each molecule of glucose 6-phosphate oxidized (Fig. 13.2). This portion of the pathway is particularly important in the liver, lactating mammary glands, and adipose tissue for the NADPH-dependent biosynthesis of fatty acids (see p. 186); in the testes, ovaries, placenta, and adrenal cortex for the NADPH-dependent biosynthesis of steroid hormones (see p. 237); and in red blood cells (RBC) for the NADPH-dependent reduction of glutathione (see p. 148). A. Glucose 6-phosphate dehydrogenation

1	A. Glucose 6-phosphate dehydrogenation Glucose 6-phosphate dehydrogenase (G6PD) catalyzes the oxidation of glucose 6-phosphate to 6-phosphogluconolactone as the coenzyme NADP+ gets reduced to NADPH. This initial reaction is the committed, rate-limiting, and regulated step of the pathway. NADPH is a potent competitive inhibitor of G6PD, and the ratio of NADPH/NADP+ is sufficiently high to substantially inhibit the enzyme under most metabolic conditions. However, with increased demand for NADPH, the ratio of NADPH/NADP+ decreases, and flux through the pathway increases in response to the enhanced activity of G6PD. [Note: Insulin upregulates expression of the gene for G6PD, and flux through the pathway increases in the absorptive state (see p. 323).]

1	B. Ribulose 5-phosphate formation 6-Phosphogluconolactone is hydrolyzed by 6-phosphogluconolactone hydrolase in the second step. The oxidative decarboxylation of the product, 6-phosphogluconate, is catalyzed by 6-phosphogluconate dehydrogenase. This third irreversible step produces ribulose 5-phosphate (a pentose sugar– phosphate), CO2 (from carbon 1 of glucose), and a second molecule of NADPH (see Fig. 13.2). III. REVERSIBLE NONOXIDATIVE REACTIONS

1	The nonoxidative reactions of the pentose phosphate pathway occur in all cell types synthesizing nucleotides and nucleic acids. These reactions catalyze the interconversion of sugars containing three to seven carbons (see Fig. 13.2). These reversible reactions permit ribulose 5-phosphate (produced by the oxidative portion of the pathway) to be converted either to ribose 5-phosphate (needed for nucleotide synthesis; see p. 293) or to intermediates of glycolysis (that is, fructose 6-phosphate and glyceraldehyde 3-phosphate). For example, many cells that carry out reductive biosynthetic reactions have a greater need for NADPH than for ribose 5-phosphate. In this case, transketolase (which transfers two-carbon units in a thiamine pyrophosphate [TPP]-requiring reaction) and transaldolase (which transfers three-carbon units) convert the ribulose 5phosphate produced as an end product of the oxidative phase to glyceraldehyde 3-phosphate and fructose 6-phosphate, which are glycolytic

1	(which transfers three-carbon units) convert the ribulose 5phosphate produced as an end product of the oxidative phase to glyceraldehyde 3-phosphate and fructose 6-phosphate, which are glycolytic intermediates. In contrast, when the demand for ribose for nucleotides and nucleic acids is greater than the need for NADPH, the nonoxidative reactions can provide the ribose 5phosphate from glyceraldehyde 3-phosphate and fructose 6-phosphate in the absence of the oxidative steps (Fig. 13.3).

1	In addition to transketolase, TPP is required by the multienzyme complexes pyruvate dehydrogenase (see p. 110), α-ketoglutarate dehydrogenase of the tricarboxylic acid cycle (see p. 112), and branched-chain α-keto acid dehydrogenase of branched-chain amino acid catabolism (see p. 266). IV. NADPH USES The coenzyme NADPH differs from nicotinamide adenine dinucleotide (NADH) only by the presence of a phosphate group on one of the ribose units (Fig. 13.4). This seemingly small change in structure allows NADPH to interact with NADPH-specific enzymes that have unique roles in the cell. For example, in the cytosol of hepatocytes, the steady-state NADP+/NADPH ratio is ~0.1, which favors the use of NADPH in reductive biosynthetic reactions. This contrasts with the high NAD+/NADH ratio (~1,000), which favors an oxidative role for NAD+. This section summarizes some important NADPH-specific functions in reductive biosynthesis and detoxification reactions. A. Reductive biosynthesis

1	A. Reductive biosynthesis Like NADH, NADPH can be thought of as a high-energy molecule. However, the electrons of NADPH are used for reductive biosynthesis, rather than for transfer to the electron transport chain as is seen with NADH (see p. 74). Thus, in the metabolic transformations of the pentose phosphate pathway, part of the energy of glucose 6-phosphate is conserved in NADPH, a molecule with a negative reduction potential (see p. 76), that, therefore, can be used in reactions requiring an electron donor, such as fatty acid (see p. 186), cholesterol (see p. 221), and steroid hormone (see p. 237) synthesis. B. Hydrogen peroxide reduction

1	B. Hydrogen peroxide reduction Hydrogen peroxide (H2O2) is one of a family of reactive oxygen species (ROS) that are formed from the partial reduction of molecular oxygen ([O2], Fig. 13.5A). These compounds are formed continuously as byproducts of aerobic metabolism, through reactions with drugs and environmental toxins, or when the level of antioxidants is diminished, all creating the condition of oxidative stress. These highly reactive oxygen intermediates can cause serious chemical damage to DNA, proteins, and unsaturated lipids and can lead to cell death. ROS have been implicated in a number of pathologic processes, including reperfusion injury, cancer, inflammatory disease, and aging. The cell has several protective mechanisms that minimize the toxic potential of these compounds. [Note: ROS can also be generated in the killing of microbes by white blood cells (WBC; see D. below).]

1	B. Actions of antioxidant enzymes. G-SH = reduced glutathione; G-S-S-G = oxidized glutathione. [Note: See Fig. 13.6B for the regeneration of G-SH.] 1. Enzymes that catalyze antioxidant reactions Reduced glutathione (GSH), a tripeptide-thiol (γ-glutamylcysteinylglycine) present in most cells, can chemically detoxify H2O2 (Fig. 13.5B). This reaction, catalyzed by the selenoprotein (see p. 407) glutathione peroxidase, forms oxidized glutathione (G-S-S-G), which no longer has protective properties. The cell regenerates G-SH in a reaction catalyzed by glutathione reductase, using NADPH as a source of reducing equivalents. Thus, NADPH indirectly provides electrons for the reduction of H2O2 (Fig. 13.6). Additional enzymes, such as superoxide dismutase and catalase, catalyze the conversion of other ROS to harmless products (see Fig. 13.5B). As a group, these enzymes serve as a defense system to guard against the toxic effects of ROS. glutathione.

1	2. Antioxidant chemicals A number of intracellular reducing agents, such as ascorbate (see p. 381), vitamin E (see p. 395), and β-carotene (see p. 386), are able to reduce and, thereby, detoxify ROS in the laboratory. Consumption of foods rich in these antioxidant compounds has been correlated with a reduced risk for certain types of cancers as well as decreased frequency of certain other chronic health problems. Therefore, it is tempting to speculate that the effects of these compounds are, in part, an expression of their ability to quench the toxic effect of ROS. However, clinical trials with antioxidants as dietary supplements have failed to show clear beneficial effects. In the case of dietary supplementation with β-carotene, the rate of lung cancer in smokers increased rather than decreased. Thus, the health-promoting effects of dietary fruits and vegetables likely reflect a complex interaction among many naturally occurring compounds, which has not been duplicated by consumption

1	Thus, the health-promoting effects of dietary fruits and vegetables likely reflect a complex interaction among many naturally occurring compounds, which has not been duplicated by consumption of isolated antioxidant compounds.

1	C. Cytochrome P450 monooxygenase system

1	Monooxygenases (mixed-function oxidases) incorporate one atom from O2 into a substrate (creating a hydroxyl group), with the other atom being reduced to water (H2O). In the cytochrome P450 (CYP) monooxygenase system, NADPH provides the reducing equivalents required by this series of reactions (Fig. 13.7). This system performs different functions in two separate locations in cells. The overall reaction catalyzed by a CYP enzyme is where R may be a steroid, drug, or other chemical. [Note: CYP enzymes are actually a superfamily of related, heme-containing monooxygenases that participate in a broad variety of reactions. The P450 in the name reflects the absorbance at 450 nm by the protein.] 1. Mitochondrial system An important function of the CYP monooxygenase system found associated with the inner mitochondrial membrane is the biosynthesis of steroid hormones. In steroidogenic tissues, such as the placenta, ovaries, testes, and adrenal cortex, it is used to hydroxylate intermediates in

1	inner mitochondrial membrane is the biosynthesis of steroid hormones. In steroidogenic tissues, such as the placenta, ovaries, testes, and adrenal cortex, it is used to hydroxylate intermediates in the conversion of cholesterol to steroid hormones, a process that makes these hydrophobic compounds more water soluble (see p. 237). The liver uses this same system in bile acid synthesis (see p.

1	224) and the hydroxylation of cholecalciferol to 25hydroxycholecalciferol ([vitamin D3] see p. 390), and the kidney uses it to hydroxylate vitamin D3 to its biologically active 1,25-dihydroxylated form.

1	2. Microsomal system The microsomal CYP monooxygenase system found associated with the membrane of the smooth endoplasmic reticulum (particularly in the liver) functions primarily in the detoxification of foreign compounds (xenobiotics). These include numerous drugs and such varied pollutants as petroleum products and pesticides. CYP enzymes of the microsomal system (for example, CYP3A4) can be used to hydroxylate these toxins (phase I). The purpose of these modifications is two-fold. First, it may itself activate or inactivate a drug and second, make a toxic compound more soluble, thereby facilitating its excretion in the urine or feces. Frequently, however, the new hydroxyl group will serve as a site for conjugation with a polar molecule, such as glucuronic acid (see p. 161), which will significantly increase the compound’s solubility (phase II). [Note: Polymorphisms (see p. 491) in the genes for CYP enzymes can lead to differences in drug metabolism.]

1	D. White blood cell phagocytosis and microbe killing Phagocytosis is the ingestion by receptor-mediated endocytosis of microorganisms, foreign particles, and cellular debris by WBC (leukocytes) such as neutrophils and macrophages (monocytes). It is an important defense mechanism, particularly in bacterial infections. Neutrophils and monocytes are armed with both oxygen-independent and oxygen-dependent mechanisms for killing bacteria. 1. Oxygen-independent Oxygen-independent mechanisms use pH changes in phagolysosomes and lysosomal enzymes to destroy pathogens. 2.

1	Oxygen-dependent Oxygen-dependent mechanisms include the enzymes NADPH oxidase and myeloperoxidase (MPO) that work together in killing bacteria (Fig. 13.8). Overall, the MPO system is the most potent of the bactericidal mechanisms. An invading bacterium is recognized by the immune system and attacked by antibodies that bind it to a receptor on a phagocytic cell. After internalization of the microorganism has occurred, NADPH oxidase, located in the leukocyte cell membrane, activated and reduces O2 from the surrounding tissue to superoxide ( ), a free radical ROS, as NADPH is oxidized. The rapid consumption of O2 that accompanies formation of is referred to as the respiratory burst. [Note: Active NADPH oxidase is a membrane-associated complex containing a flavocytochrome plus additional peptides that translocate from the cytoplasm upon activation of the leukocyte. Electrons move from NADPH to O2 via flavin adenine nucleotide (FAD) and heme, . Rare genetic deficiencies in NADPH oxidase

1	that translocate from the cytoplasm upon activation of the leukocyte. Electrons move from NADPH to O2 via flavin adenine nucleotide (FAD) and heme, . Rare genetic deficiencies in NADPH oxidase cause chronic granulomatous disease (CGD) characterized by severe, persistent infections and the formation of granulomas (nodular areas of inflammation) that sequester the bacteria that were not destroyed.] Next, is converted to H2O2 (also a ROS), either spontaneously or catalyzed by superoxide dismutase. In the presence of MPO, a heme-containing lysosomal enzyme present within the phagolysosome, peroxide plus chloride ions are converted to hypochlorous acid ([HOCl] the major component of household bleach), which kills the bacteria. The peroxide can also be partially reduced to the hydroxyl radical (OH•), a

1	ROS, or be fully reduced to H2O by catalase or glutathione peroxidase. [Note: Deficiencies in MPO do not confer increased susceptibility to infection because peroxide from NADPH oxidase is bactericidal.] G; NADP(H) = nicotinamide adenine dinucleotide phosphate; = superoxide; H2O2 = hydrogen peroxide; HOCl = hypochlorous acid; OH• = hydroxyl radical. E. Nitric oxide synthesis

1	Nitric oxide (NO) is recognized as a mediator in a broad array of biologic systems. NO is the endothelium-derived relaxing factor that causes vasodilation by relaxing vascular smooth muscle. It also acts as a neurotransmitter, prevents platelet aggregation, and plays an essential role in macrophage function. It has a very short half-life in tissues (3–10 seconds) because it reacts with O2 and and is converted into nitrates and nitrites including peroxynitrite (O=NOO−), a reactive nitrogen species (RNS). [Note: NO is a free radical gas that is often confused with nitrous oxide (N2O), the “laughing gas” that is used as an anesthetic and is chemically stable.] 1. Nitric oxide synthase Arginine, O2, and NADPH are substrates for cytosolic NO synthase ([NOS], Fig. 13.9). Flavin mononucleotide (FMN), FAD, heme, and tetrahydrobiopterin (see p. 268) are coenzymes, and NO and citrulline are products of the reaction. Three NOS isozymes, each the product of a different gene, have been identified.

1	FAD, heme, and tetrahydrobiopterin (see p. 268) are coenzymes, and NO and citrulline are products of the reaction. Three NOS isozymes, each the product of a different gene, have been identified. Two are constitutive (synthesized at a constant rate), calcium (Ca2+)–calmodulin (CaM)-dependent enzymes (see p. 133). They are found primarily in endothelium (eNOS) and neural tissue (nNOS) and constantly produce very low levels of NO for vasodilation and neurotransmission. An inducible, Ca2+-independent enzyme (iNOS) can be expressed in many cells, including macrophages and neutrophils, as an early defense against pathogens. The specific inducers for iNOS vary with cell type and include proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), and bacterial endotoxins such as lipopolysaccharide (LPS). These compounds promote synthesis of iNOS, which can result in large amounts of NO being produced over hours or even days.

1	2.

1	Nitric oxide and vascular endothelium NO is an important mediator in the control of vascular smooth muscle tone. NO is synthesized by eNOS in endothelial cells and diffuses to vascular smooth muscle, where it activates the cytosolic form of guanylyl cyclase (or, guanylate cyclase) to form cyclic guanosine monophosphate (cGMP). [Note: This reaction is analogous to the formation of cyclic adenosine monophosphate (cAMP) by adenylyl cyclase (see p. 95).] The resultant rise in cGMP causes activation of protein kinase G, which phosphorylates Ca2+ channels, causing decreased entry of Ca2+ into smooth muscle cells. This decreases the Ca2+–CaM activation of myosin light-chain kinase, thereby decreasing smooth muscle contraction and favoring relaxation. Vasodilator nitrates, such as nitroglycerin, are metabolized to NO, which causes relaxation of vascular smooth muscle and, therefore, lowers blood pressure. Thus, NO can be envisioned as an endogenous nitrovasodilator. [Note: Under hypoxic

1	are metabolized to NO, which causes relaxation of vascular smooth muscle and, therefore, lowers blood pressure. Thus, NO can be envisioned as an endogenous nitrovasodilator. [Note: Under hypoxic conditions, nitrite (NO2−) can be reduced to NO, which binds to deoxyhemoglobin. The NO is released into the blood, causing vasodilation and increasing blood flow.] 3.

1	Nitric oxide and macrophage bactericidal activity In macrophages, iNOS activity is normally low, but synthesis of the enzyme is significantly stimulated by bacterial LPS and by release of IFN-γ response to infection. Activated macrophages form radicals that combine with NO to form intermediates that decompose, producing the highly bactericidal OH• radical. 4. Additional functions NO is a potent inhibitor of platelet adhesion and aggregation (by activating the cGMP pathway). It is also characterized as a neurotransmitter in the central and peripheral nervous systems. V. G6PD DEFICIENCY

1	G6PD deficiency is a hereditary condition characterized by hemolytic anemia caused by the inability to detoxify oxidizing agents. G6PD deficiency is the most common disease-producing enzyme abnormality in humans, affecting >400 million individuals worldwide. This deficiency has the highest prevalence in the Middle East, tropical Africa and Asia, and parts of the Mediterranean. G6PD deficiency is X linked and is, in fact, a family of deficiencies caused by a number of different mutations in the gene encoding G6PD. Only some of the resulting protein variants cause clinical symptoms. [Note: In addition to hemolytic anemia, a clinical manifestation of G6PD deficiency is neonatal jaundice appearing 1–4 days after birth. The jaundice, which may be severe, typically results from increased production of unconjugated bilirubin (see p. 285).] The life span of individuals with a severe form of G6PD deficiency may be somewhat shortened as a result of complications arising from chronic hemolysis.

1	of unconjugated bilirubin (see p. 285).] The life span of individuals with a severe form of G6PD deficiency may be somewhat shortened as a result of complications arising from chronic hemolysis. This negative effect of G6PD deficiency has been balanced in evolution by an advantage in survival, an increased resistance to Plasmodium falciparum malaria. [Note: Sickle cell trait and the thalassemias also confer resistance to malaria.]

1	A. G6PD role in red blood cells

1	Diminished G6PD activity impairs the ability of the cell to form the NADPH that is essential for the maintenance of the G-SH pool. This results in a decrease in the detoxification of free radicals and peroxides formed within the cell (Fig. 13.10). G-SH also helps maintain the reduced states of sulfhydryl groups in proteins, including hemoglobin. Oxidation of those sulfhydryl groups leads to the formation of denatured proteins that form insoluble masses (called Heinz bodies) that attach to RBC membranes (Fig. 13.11). Additional oxidation of membrane proteins causes RBC to be rigid (less deformable), and they are removed from the circulation by macrophages in the spleen and liver. Although G6PD deficiency occurs in all cells of the affected individual, it is most severe in RBC, where the pentose phosphate pathway provides the only means of generating NADPH. Additionally, the RBC has no nucleus or ribosomes and cannot renew its supply of the enzyme. Thus, RBC are particularly vulnerable

1	phosphate pathway provides the only means of generating NADPH. Additionally, the RBC has no nucleus or ribosomes and cannot renew its supply of the enzyme. Thus, RBC are particularly vulnerable to enzyme variants with diminished stability. [Note: Other tissues have an alternative source of NADPH (NADP+-dependent malate dehydrogenase [malic enzyme]; see p. 186) that can keep G-SH reduced.] pentose phosphate pathway.

1	B. Precipitating factors in G6PD deficiency Most individuals who have inherited one of the G6PD mutations do not show clinical manifestations (that is, they are asymptomatic). However, some patients with G6PD deficiency develop hemolytic anemia if they are treated with an oxidant drug, ingest fava beans, or contract a severe infection. 1. Oxidant drugs Commonly used drugs that produce hemolytic anemia in patients with G6PD deficiency are best remembered from the mnemonic AAA: antibiotics (for example, sulfamethoxazole and chloramphenicol), antimalarials (for example, primaquine but not chloroquine or quinine), and antipyretics (for example, acetanilide but not acetaminophen). 2.

1	2. Favism Some forms of G6PD deficiency, for example, the Mediterranean variant, are particularly susceptible to the hemolytic effect of the fava (broad) bean, a dietary staple in the Mediterranean region. Favism, the hemolytic effect of ingesting fava beans, is not observed in all individuals with G6PD deficiency, but all patients with favism have G6PD deficiency. 3. Infection Infection is the most common precipitating factor of hemolysis in G6PD deficiency. The inflammatory response to infection results in the generation of free radicals in macrophages. The radicals can diffuse into the RBC and cause oxidative damage. C. Variant G6PD properties

1	Almost all G6PD variants are caused by point mutations (see p. 449) in the gene for G6PD. Some mutations do not affect enzymic activity. However, other mutations result in decreased catalytic activity, decreased stability, or an alteration of binding affinity for NADP+ or glucose 6-phosphate. [Note: Active G6PD exists as a homodimer or tetramer. Mutations at the interface between subunits can affect stability.] The severity of the disease usually correlates with the amount of residual enzyme activity in the patient’s RBC. For example, variants can be classified as shown in Figure 13.12. G6PD A– is the prototype of the moderate (class III) form of the disease. The RBC contain an unstable but kinetically normal G6PD, with most of the enzyme activity present in the reticulocytes and younger RBC (Fig. 13.13). Therefore, the oldest RBC have the lowest level of enzyme activity and are preferentially removed in a hemolytic episode. Because hemolysis does not affect younger cells, the

1	RBC (Fig. 13.13). Therefore, the oldest RBC have the lowest level of enzyme activity and are preferentially removed in a hemolytic episode. Because hemolysis does not affect younger cells, the episodes are self-limiting. G6PD Mediterranean is the prototype of a more severe (class II) deficiency. Class I mutations (rare) are the most severe and are associated with chronic nonspherocytic hemolytic anemia, which occurs even in the absence of oxidative stress.

1	D. G6PD molecular biology The cloning of the gene for G6PD and the sequencing of its DNA (see Chapter 34) have permitted the identification of mutations that cause G6PD deficiency. More than 400 G6PD variants have been identified, a finding that explains the numerous biochemical and clinical phenotypes that have been described. Most mutations that result in enzymic deficiency are missense mutations (see p. 449) in the coding region. Both G6PD A− and G6PD Mediterranean represent mutant enzymes that differ from the respective normal variants by a single amino acid. Large deletions or frameshift mutations have not been identified, suggesting that complete absence of G6PD activity is likely lethal. VI. CHAPTER SUMMARY

1	The pentose phosphate pathway includes an irreversible oxidative phase followed by a series of reversible sugar–phosphate interconversions (Fig. 13.14). No ATP is directly consumed or produced in the pathway. The reduced nicotinamide adenine dinucleotide phosphate (NADPH)-producing oxidative portion of the pathway is important in providing reducing equivalents for reductive biosynthesis and detoxification reactions. In this part of the pathway, glucose 6-phosphate is irreversibly converted to ribulose 5-phosphate, and two NADPH are produced. The regulated step is catalyzed by glucose 6-phosphate dehydrogenase (G6PD), which is strongly inhibited by a rise in the NADPH/NADP+ ratio. Reversible nonoxidative reactions interconvert sugars. This part of the pathway converts ribulose 5-phosphate to ribose 5-phosphate, required for nucleotide and nucleic acid synthesis, or to fructose 6-phosphate and glyceraldehyde 3-phosphate (glycolytic intermediates). NADPH is a source of reducing

1	to ribose 5-phosphate, required for nucleotide and nucleic acid synthesis, or to fructose 6-phosphate and glyceraldehyde 3-phosphate (glycolytic intermediates). NADPH is a source of reducing equivalents in reductive biosynthesis, such as the production of fatty acids in liver, adipose tissue, and the mammary gland; cholesterol in the liver; and steroid hormones in the placenta, ovaries, testes, and adrenal cortex. It is also required by red blood cells (RBC) for hydrogen peroxide reduction. Reduced glutathione (G-SH) is used by glutathione peroxidase to reduce the peroxide to water. The oxidized glutathione (G-S-S-G) produced is reduced by glutathione reductase, using NADPH as the source of electrons. NADPH provides reducing equivalents for the mitochondrial cytochrome P450 monooxygenase system, which is used in steroid hormone synthesis in steroidogenic tissue, bile acid synthesis in the liver, and vitamin D activation in the liver and kidneys. The microsomal system uses NADPH to

1	system, which is used in steroid hormone synthesis in steroidogenic tissue, bile acid synthesis in the liver, and vitamin D activation in the liver and kidneys. The microsomal system uses NADPH to detoxify foreign compounds (xenobiotics), such as drugs and a variety of pollutants. NADPH provides the reducing equivalents for phagocytes in the process of eliminating invading microorganisms. NADPH oxidase uses molecular oxygen (O2) and electrons from NADPH to produce superoxide radicals, which, in turn, can be converted to peroxide by superoxide dismutase. Myeloperoxidase catalyzes the formation of bactericidal hypochlorous acid from peroxide and chloride ions. Rare genetic defects in NADPH oxidase cause chronic granulomatous disease characterized by severe, persistent, infections and granuloma formation. NADPH is required for the synthesis of nitric oxide (NO), an important free radical gas that causes vasodilation by relaxing vascular smooth muscle, acts as a neurotransmitter, prevents

1	formation. NADPH is required for the synthesis of nitric oxide (NO), an important free radical gas that causes vasodilation by relaxing vascular smooth muscle, acts as a neurotransmitter, prevents platelet aggregation, and helps mediate macrophage bactericidal activity. NO is made from arginine and O2 by three different NADPH-dependent NO synthases (NOS). The endothelial (eNOS) and neuronal (nNOS) isozymes constantly produce very low levels of NO for vasodilation and neurotransmission, respectively. The inducible isozyme (iNOS) produces large amounts of NO for defense against pathogens. G6PD deficiency impairs the ability of the cell to form the NADPH that is essential for the maintenance of the G-SH pool. The cells most affected are RBC because they do not have additional sources of NADPH. G6PD deficiency is an X-linked disease characterized by hemolytic anemia caused by the production of free radicals and peroxides following administration of oxidant drugs, ingestion of fava beans,

1	G6PD deficiency is an X-linked disease characterized by hemolytic anemia caused by the production of free radicals and peroxides following administration of oxidant drugs, ingestion of fava beans, or severe infections. The extent of the anemia depends on the amount of residual enzyme. Class I variants, the most severe (and least common), are associated with chronic nonspherocytic hemolytic anemia. Babies with G6PD deficiency may experience neonatal jaundice.

1	Choose the ONE best answer. 3.1. In preparation for a trip to an area of India where chloroquine-resistant malaria is endemic, a young man is given primaquine prophylactically. Soon thereafter, he develops a hemolytic condition due to a deficiency in glucose 6-phosphate dehydrogenase. A less-than-normal level of which of the following is a consequence of the enzyme deficiency and the underlying cause of the hemolysis? A. Glucose 6-phosphate B. Oxidized form of nicotinamide adenine dinucleotide C. Reduced form of glutathione D. Ribose 5-phosphate

1	A. Glucose 6-phosphate B. Oxidized form of nicotinamide adenine dinucleotide C. Reduced form of glutathione D. Ribose 5-phosphate Correct answer = C. Glutathione (G-SH) is essential for red cell integrity and is maintained in this reduced (functional) form by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent glutathione reductase. The NADPH is from the oxidative portion of the pentose phosphate pathway. Individuals with a deficiency of the regulated enzyme of this pathway, glucose 6-phosphate dehydrogenase (G6PD), have a decreased ability to generate NADPH and, therefore, a decreased ability to keep G-SH reduced. When treated with an oxidant drug such as primaquine, some patients with G6PD deficiency develop a hemolytic anemia. Primaquine does not affect glucose 6phosphate levels. Nicotinamide adenine dinucleotide (NAD[H]) is neither produced by the pathway nor used as a coenzyme by G-SH reductase. A decrease in ribose 5-phosphate does not cause hemolysis.

1	3.2. Septic shock, a state of acute circulatory failure characterized by persistent arterial hypotension (low blood pressure) and inadequate organ perfusion refractory to fluid resuscitation, results from a severe inflammatory response to bacterial infection. It has a high mortality rate and is associated with changes in the level of nitric oxide. Which statement concerning septic shock is most likely correct? A. Activation of endothelial nitric oxide synthase causes an increase in nitric oxide. B. High mortality is the result of the long half-life of nitric oxide. C. Lysine, the nitrogen source for nitric oxide synthesis, is deaminated by bacteria. D. Overproduction of nitric oxide by a calcium-independent enzyme is the cause of the hypotension.

1	C. Lysine, the nitrogen source for nitric oxide synthesis, is deaminated by bacteria. D. Overproduction of nitric oxide by a calcium-independent enzyme is the cause of the hypotension. Correct answer = D. Overproduction of short-lived (not long-lived) nitric oxide (NO) by calcium-independent, inducible nitric oxide synthase (iNOS) results in excessive vasodilation leading to hypotension. The endothelial enzyme (eNOS) is constitutive and produces low levels of NO at a consistent rate. NOS use arginine, not lysine, as the source of the nitrogen.

1	3.3. An individual who has recently been prescribed a drug (atorvastatin) to lower cholesterol levels is advised to limit consumption of grapefruit juice, because high intake of the juice reportedly results in an increased level of the drug in the blood, increasing the risk of side effects. Atorvastatin is a substrate for the cytochrome P450 enzyme CYP3A4, and grapefruit juice inhibits the enzyme. Which statement concerning CYP enzymes is most likely correct? They: A. accept electrons from reduced nicotinamide adenine dinucleotide. B. catalyze the hydroxylation of hydrophobic molecules. C. differ from nitric oxide synthase in that they contain heme. D. function in association with an oxidase.

1	B. catalyze the hydroxylation of hydrophobic molecules. C. differ from nitric oxide synthase in that they contain heme. D. function in association with an oxidase. Correct answer = B. The CYP enzymes hydroxylate hydrophobic compounds, making them more water soluble. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) from the pentose phosphate pathway is the electron donor. Both the CYP enzymes and the nitric oxide synthase isozymes contain heme. 3.4. In male patients who are hemizygous for X-linked glucose 6-phosphate dehydrogenase deficiency, pathophysiologic consequences are more apparent in red blood cells (RBC) than in other cells such as in the liver. Which one of the following provides the most reasonable explanation for this different response? A. Excess glucose 6-phosphate in the liver, but not in RBC, can be channeled to glycogen, thereby averting cellular damage.

1	A. Excess glucose 6-phosphate in the liver, but not in RBC, can be channeled to glycogen, thereby averting cellular damage. B. Liver cells, in contrast to RBC, have alternative mechanisms for supplying the reduced nicotinamide adenine dinucleotide phosphate required for maintaining cell integrity. C. Because RBC do not have mitochondria, production of ATP required to maintain cell integrity depends exclusively on the shunting of glucose 6-phosphate to the pentose phosphate pathway. D. In RBC, in contrast to liver cells, glucose 6-phosphatase activity decreases the level of glucose 6-phosphate, resulting in cell damage.

1	D. In RBC, in contrast to liver cells, glucose 6-phosphatase activity decreases the level of glucose 6-phosphate, resulting in cell damage. Correct answer = B. Cellular damage is directly related to decreased ability of the cell to regenerate reduced glutathione, for which large amounts of reduced nicotinamide adenine dinucleotide phosphate (NADPH) are needed, and RBC have no means other than the pentose phosphate pathway of generating NADPH. It is decreased product (NADPH), not increased substrate (glucose 6phosphate), that is the problem. RBC do not have glucose 6-phosphatase. The pentose phosphate pathway does not generate ATP. 3.5. An essential coenzyme for several enzymes of metabolism is derived from the vitamin thiamine. Measurement of the activity of what enzyme in red blood cells could be used to determine thiamine status in the body?

1	Red blood cells do not have mitochondria and, so, do not contain mitochondrial enzymes such as pyruvate dehydrogenase that require the thiamine-derived coenzyme thiamine pyrophosphate (TPP). However, they do contain the cytosolic TPP-requiring transketolase, whose activity is used clinically to assess thiamine status. Glycosaminoglycans, Proteoglycans, and Glycoproteins 14 For additional ancillary materials related to this chapter, please visit thePoint. I. GLYCOSAMINOGLYCAN OVERVIEW

1	I. GLYCOSAMINOGLYCAN OVERVIEW Glycosaminoglycans (GAG) are large complexes of negatively charged heteropolysaccharide chains. They are generally associated with a small amount of protein (core protein), forming proteoglycans, which typically consist of up to 95% carbohydrate. GAG have the special ability to bind large amounts of water, thereby producing the gel-like matrix that forms the basis of the body’s ground substance, which, along with fibrous structural proteins such as collagen, elastin, and fibrillin-1, and adhesive proteins such as fibronectin, makes up the extracellular matrix (ECM). The hydrated GAG serve as a flexible support for the ECM, interacting with the structural and adhesive proteins, and as a molecular sieve, influencing movement of materials through the ECM. The viscous, lubricating properties of mucous secretions also result from the presence of GAG, which led to the original naming of these compounds as mucopolysaccharides. II. STRUCTURE

1	II. STRUCTURE GAG are long, unbranched, heteropolysaccharide chains composed of a repeating disaccharide unit [acidic sugar–amino sugar]n (Fig. 14.1). [Note: A single exception is keratan sulfate, which contains galactose rather than an acidic sugar.] The amino sugar is either D-glucosamine or D-galactosamine, in which the amino group is usually acetylated, thus eliminating its positive charge. The amino sugar may also be sulfated on carbon 4 or 6 or on a nonacetylated nitrogen. The acidic sugar is either D-glucuronic acid or its C-5 epimer Liduronic acid (Fig. 14.2). These uronic sugars contain carboxyl groups that are negatively charged at physiologic pH and, together with the sulfate groups (−SO42−), give GAG their strongly negative nature. A. Structure–function relationship

1	A. Structure–function relationship Because of the high concentration of negative charges, these heteropolysaccharide chains tend to be extended in solution. They repel each other and are surrounded by a shell of water molecules. When brought together, they slide past each other, much as two magnets with the same polarity seem to slide past each other. This produces the slippery consistency of mucous secretions and synovial fluid. When a solution of GAG is compressed, the water is squeezed out, and the GAG are forced to occupy a smaller volume. When the compression is released, the GAG spring back to their original, hydrated volume because of the repulsion of their negative charges. This property contributes to the resilience of synovial fluid and the vitreous humor of the eye (Fig. 14.3). B. Classification

1	B. Classification The six major types of GAG are divided according to monomeric composition, type of glycosidic linkages, and degree and location of sulfate units. The structure of the GAG and their distribution in the body is illustrated in Figure 14.4. All GAG, except for hyaluronic acid, are sulfated and are found covalently attached to protein, forming proteoglycan monomers. C. Proteoglycans Proteoglycans are found in the ECM and on the outer surface of cells. 1.

1	C. Proteoglycans Proteoglycans are found in the ECM and on the outer surface of cells. 1. Monomer structure: A proteoglycan monomer found in cartilage consists of a core protein to which up to 100 linear chains of GAG are covalently attached. These chains, which may each be composed of up to 200 disaccharide units, extend out from the core protein and remain separated from each other because of charge repulsion. The resulting structure resembles a bottle brush (Fig. 14.5). In cartilage proteoglycans, the species of GAG include chondroitin sulfate and keratan sulfate. [Note: Proteoglycans are grouped into gene families that encode core proteins with common structural features. The aggrecan family (aggrecan, versican, neurocan, and brevican), abundant in cartilage, is an example.] 2.

1	GAG–protein linkage: This covalent linkage is most commonly through a trihexoside (galactose-galactose-xylose) and a serine residue in the protein. An O-glycosidic bond (see p. 86) is formed between the xylose and the hydroxyl group of the serine (Fig. 14.6). 3. Aggregate formation: Many proteoglycan monomers can associate with one molecule of hyaluronic acid to form proteoglycan aggregates. The association is not covalent and occurs primarily through ionic interactions between the core protein and the hyaluronic acid. The association is stabilized by additional small proteins called link proteins (Fig. 14.7). III. SYNTHESIS

1	III. SYNTHESIS The heteropolysaccharide chains are elongated by the sequential addition of alternating acidic and amino sugars donated primarily by their uridine diphosphate (UDP) derivatives. The reactions are catalyzed by a family of specific glycosyltransferases. Because GAG are produced for export from the cell, their synthesis occurs primarily in the Golgi and not in the cytosol. A. Amino sugar synthesis Amino sugars are essential components of glycoconjugates such as proteoglycans, glycoproteins, and glycolipids. The synthetic pathway of amino sugars (hexosamines) is very active in connective tissues, where as much as 20% of glucose flows through this pathway.

1	1. N-Acetylglucosamine and N-acetylgalactosamine: The monosaccharide fructose 6-phosphate is the precursor of N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). A hydroxyl group on the fructose is replaced by the amide nitrogen of a glutamine, and the glucosamine 6phosphate product gets acetylated, isomerized, and activated, producing the nucleotide sugar UDP-GlcNAc (Fig. 14.8). UDP-GalNAc is generated by the epimerization of UDP-GlcNAc. It is these nucleotide sugar forms of the amino sugars that are used to elongate the carbohydrate chains. pyrophosphate.

1	pyrophosphate. 2. N-Acetylneuraminic acid: NANA, a nine-carbon, acidic monosaccharide (see Fig. 17.15, p. 209), is a member of the family of sialic acids, each of which is acylated at a different site. These compounds are usually found as terminal carbohydrate residues of oligosaccharide side chains of glycoproteins, of glycolipids, or, less frequently, of GAG. N-Acetylmannosamine 6-phosphate (derived from fructose 6-phosphate) and phosphoenolpyruvate (an intermediate in glycolysis; see p. 102) are the immediate sources of the carbons and nitrogens for NANA synthesis (see Fig. 14.8). Before NANA can be added to a growing oligosaccharide, it must be activated to cytidine monophosphate (CMP)NANA by reacting with cytidine triphosphate (CTP). CMP-NANA synthetase catalyzes the reaction. [Note: CMP-NANA is the only nucleotide sugar in human metabolism in which the carrier nucleotide is a monophosphate rather than a diphosphate.] B. Acidic sugar synthesis

1	B. Acidic sugar synthesis D-Glucuronic acid, whose structure is that of glucose with an oxidized carbon 6 (−CH2OH → −COOH), and its C-5 epimer, L-iduronic acid, are essential components of GAG. Glucuronic acid is also required for the detoxification of lipophilic compounds, such as bilirubin (see p. 282), steroids (see p. 240), and many drugs, including the statins (see p. 224), because conjugation with glucuronate (glucuronidation) increases water solubility. In plants and mammals (other than guinea pigs and primates, including humans), glucuronic acid is a precursor of ascorbic acid (vitamin C) as shown in Figure 14.9. This uronic acid pathway also provides a mechanism by which dietary D-xylulose can enter the central metabolic pathways.

1	C) as shown in Figure 14.9. This uronic acid pathway also provides a mechanism by which dietary D-xylulose can enter the central metabolic pathways. 1. Glucuronic acid: Glucuronic acid can be obtained in small amounts from the diet and from the lysosomal degradation of GAG. It also can be synthesized by the uronic acid pathway, in which glucose 1-phosphate reacts with uridine triphosphate (UTP) and is converted to UDP-glucose. Oxidation of UDP-glucose produces UDP-glucuronic acid, the form that supplies glucuronic acid for GAG synthesis and glucuronidation (Fig. 14.10). The end product of glucuronic acid metabolism in humans is Dxylulose 5-phosphate, which can enter the pentose phosphate pathway and produce the glycolytic intermediates glyceraldehyde 3-phosphate and fructose 6-phosphate (see Fig. 14.9; see also Fig. 13.2, p. 146).

1	2. l-Iduronic acid: Synthesis of L-iduronic acid occurs after D-glucuronic acid has been incorporated into the carbohydrate chain. Uronosyl 5epimerase causes epimerization of the D-to the L-sugar. C. Core protein synthesis The core protein is made by ribosomes on the rough endoplasmic reticulum (RER), enters the RER lumen, and then moves to the Golgi, where it is glycosylated by membrane-bound glycosyltransferases. D. Carbohydrate chain synthesis Carbohydrate chain formation is initiated by synthesis of a short linker on the core protein on which carbohydrate chain synthesis will occur. The most common linker is a trihexoside formed by the transfer of a xylose from UDP-xylose to the hydroxyl group of a serine (or threonine) catalyzed by xylosyltransferase. Two galactose molecules are then added, completing the trihexoside. This is followed by sequential addition of alternating acidic and amino sugars (Fig. 14.11) and epimerization of some D-glucuronyl to Liduronyl residues.

1	E. Sulfate group addition Sulfation of a GAG occurs after the monosaccharide to be sulfated has been incorporated into the growing carbohydrate chain. The source of the sulfate is 3´-phosphoadenosyl-5´-phosphosulfate ([PAPS] a molecule of adenosine monophosphate with a sulfate group attached to the 5´-phosphate; see Fig. 17.16, p. 210). The sulfation reaction is catalyzed by sulfotransferases. Synthesis of the sulfated GAG chondroitin sulfate is shown in Figure 14.11. [Note: PAPS is also the sulfur donor in glycosphingolipid synthesis (see p. 210).] A defect in the sulfation of the growing GAG chains results in one of several autosomal-recessive disorders, the chondrodystrophies, which affect the proper development and maintenance of the skeletal system. IV. DEGRADATION

1	IV. DEGRADATION GAG are degraded in lysosomes, which contain hydrolytic enzymes that are most active at a pH of ~5. Therefore, as a group, these enzymes are called acid hydrolases. [Note: The low pH optimum is a protective mechanism that prevents the enzymes from destroying the cell should leakage occur into the cytosol where the pH is neutral.] The half-lives of GAG vary from minutes to months and are influenced by the type of GAG and its location in the body. A. GAG phagocytosis Because GAG are extracellular or cell-surface compounds, they must first be engulfed by invagination of the cell membrane (phagocytosis), forming a vesicle inside of which are the GAG to be degraded. This vesicle then fuses with a lysosome, forming a single digestive vesicle in which the GAG are efficiently degraded (see p. 150 for a discussion of phagocytosis). B. Lysosomal degradation

1	B. Lysosomal degradation The lysosomal degradation of GAG requires a large number of acid hydrolases for complete digestion. First, the polysaccharide chains are cleaved by endoglycosidases, producing oligosaccharides. Further degradation of the oligosaccharides occurs sequentially from the nonreducing end (see p. 127) of each chain, the last group (sulfate or sugar) added during synthesis being the first group removed (by sulfatases or exoglycosidases). Examples of some of these enzymes and the bonds they hydrolyze are shown in Figure 14.12. [Note: Endo-and exoglycosidases are also involved in the lysosomal degradation of glycoproteins (see p. 170) and glycolipids (see p. 210). Deficiencies in these enzymes result in the accumulation of partially degraded carbohydrates, causing tissue damage.] glucosamine; S = sulfate.

1	Multiple sulfatase deficiency (Austin disease) is a rare lysosomal storage disease in which all sulfatases are nonfunctional because of a defect in the formation of formylglycine, an amino acid derivative required at the active site for enzymic activity to occur. V. MUCOPOLYSACCHARIDOSES

1	The mucopolysaccharidoses are hereditary diseases (approximately 1:25,000 live births) caused by a deficiency of any one of the lysosomal hydrolases normally involved in the degradation of heparan sulfate and/or dermatan sulfate (see Fig. 14.12). They are progressive disorders characterized by lysosomal accumulation of GAG in various tissues, causing a range of symptoms, such as skeletal and ECM deformities, and intellectual disability. All are autosomal-recessive disorders except Hunter syndrome, which is X linked. Children who are homozygous for any one of these diseases are apparently normal at birth and then gradually deteriorate. In severe deficiencies, death occurs in childhood. There currently is no cure. Incomplete lysosomal degradation of GAG results in the presence of oligosaccharides in the urine. These fragments can be used to diagnose the specific mucopolysaccharidosis by identifying the structure present on the nonreducing end of the oligosaccharide, because that residue

1	in the urine. These fragments can be used to diagnose the specific mucopolysaccharidosis by identifying the structure present on the nonreducing end of the oligosaccharide, because that residue would have been the substrate for the missing enzyme. Diagnosis is confirmed by measuring the patient’s cellular level of the lysosomal hydrolases. Bone marrow and cord blood transplants, in which transplanted macrophages produce the enzymes that degrade GAG, have been used to treat Hurler and Hunter syndromes, with limited success. Enzyme replacement therapy is available for both syndromes but does not prevent neurologic damage.

1	VI. GLYCOPROTEIN OVERVIEW

1	Glycoproteins are proteins to which oligosaccharides (glycans) are covalently attached. [Note: Glycosylation is the most common posttranslational modification of proteins.] They differ from the proteoglycans in several important ways. Glycoproteins contain highly variable amounts of carbohydrate but typically less than that of proteoglycans. For example, the glycoprotein immunoglobulin G (IgG) contains <4% of its mass as carbohydrate, whereas the proteoglycan aggrecan contains >80%. In glycoproteins, the glycan is relatively short (usually two to ten sugar residues in length, although it can be longer); does not contain repeating disaccharide units and, consequently, is structurally diverse; is often branched instead of linear; and may or may not be negatively charged. Membrane-bound glycoproteins participate in a broad range of cellular phenomena, including cell-surface recognition (by other cells, hormones, and viruses), cell-surface antigenicity (such as the blood group antigens),

1	participate in a broad range of cellular phenomena, including cell-surface recognition (by other cells, hormones, and viruses), cell-surface antigenicity (such as the blood group antigens), and as components of the ECM and of the mucins of the gastrointestinal and urogenital tracts, where they act as protective biologic lubricants. In addition, almost all of the globular proteins present in human plasma are glycoproteins, although albumin is an exception. Figure 14.13 summarizes some glycoprotein functions.

1	VII. OLIGOSACCHARIDE STRUCTURE The oligosaccharide (glycan) components of glycoproteins are generally branched heteropolymers composed primarily of D-hexoses, with the addition in some cases of neuraminic acid (a nonose) and of L-fucose, a 6-deoxyhexose. A. Carbohydrate–protein linkage The glycan may be attached to the protein through an N-or an O-glycosidic link (see p. 86). In the former case, the sugar chain is attached to the amide group of an asparagine side chain and, in the latter case, to the hydroxyl group of either a serine or threonine side chain. [Note: In the case of collagen, there is an O-glycosidic linkage between galactose or glucose and the hydroxyl group of hydroxylysine (see p. 47).] B. N-and O-Linked oligosaccharides A glycoprotein may contain only one type of glycosidic linkage (N or O linked) or may have both types within the same molecule. 1.

1	B. N-and O-Linked oligosaccharides A glycoprotein may contain only one type of glycosidic linkage (N or O linked) or may have both types within the same molecule. 1. O-Linked: The O-linked glycans may have one or more of a wide variety of sugars arranged in either a linear or a branched pattern. Many are found in extracellular glycoproteins or as membrane glycoprotein components. For example, O-linked oligosaccharides on the surface of red blood cells help provide the ABO blood group determinants. If the terminal sugar on the glycan is GalNAc, the blood group is A. If it is galactose, the blood group is B. If neither GalNAc nor galactose is present, the blood group is O. 2.

1	2. N-Linked: The N-linked glycans fall into two broad classes: complex oligosaccharides and high-mannose oligosaccharides. Both contain the same pentasaccharide core shown in Figure 14.14, but the complex oligosaccharides contain a diverse group of additional sugars, for example, GlcNAc, GalNAc, L-fucose, and NANA, whereas the high mannose oligosaccharides contain primarily mannose. VIII. GLYCOPROTEIN SYNTHESIS Proteins destined to function in the cytoplasm are synthesized on free cytosolic ribosomes. However, proteins, including glycoproteins, that are destined for cellular membranes, lysosomes, or to be exported from the cell, are synthesized on ribosomes attached to the RER. These proteins contain specific signal sequences that act as molecular addresses, targeting the proteins to their proper destinations. An N-terminal hydrophobic sequence initially directs these proteins to the RER, allowing the growing polypeptide to be extruded into the lumen (see p.

1	459). The proteins are then transported via secretory vesicles to the Golgi, which acts as a sorting center (Fig. 14.15). In the Golgi, those glycoproteins that are to be secreted from the cell (or are targeted for lysosomes) are packaged into vesicles that fuse with the cell (or lysosomal) membrane and release their contents. Those that are destined to become components of the cell membrane are integrated into the Golgi membrane, which buds off, forming vesicles that add their membrane-bound glycoproteins to the cell membrane. [Note: Therefore, the membrane glycoproteins are oriented with the carbohydrate portion on the outside of the cell (see Fig. 14.15).] A.

1	A. The precursors of the carbohydrate components of glycoproteins are nucleotide sugars, which include UDP-glucose, UDP-galactose, UDP-GlcNAc, and UDP-GalNAc. In addition, guanosine diphosphate (GDP)mannose, GDP-L-fucose (which is synthesized from GDP-mannose), and CMP-NANA may donate sugars to the growing chain. [Note: When the acidic NANA is present, the oligosaccharide has a negative charge at physiologic pH.] The oligosaccharides are covalently attached to the side chains of specific amino acids in the protein, where the three-dimensional structure of the protein determines whether or not a specific amino acid is glycosylated. B. O-Linked glycoprotein synthesis

1	B. O-Linked glycoprotein synthesis Synthesis of the O-linked glycoproteins is very similar to that of the GAG (see p. 158). First, the protein to which sugars are to be attached is synthesized on the RER and extruded into its lumen. Glycosylation begins with the transfer of GalNAc (from UDP-GalNAc) to the hydroxyl group of a specific serine or threonine residues. The glycosyltransferases responsible for the stepwise synthesis (from individual sugars) of the oligosaccharides are bound to the membranes of the Golgi. They act in a specific order, without using a template as is required for DNA, ribonucleic acid (RNA), and protein synthesis (see Unit VII), but instead by recognizing the actual structure of the growing oligosaccharide as the appropriate substrate. C. N-Linked glycoprotein synthesis

1	C. N-Linked glycoprotein synthesis Synthesis of N-linked glycoproteins occurs in the lumen of the RER and requires the participation of the phosphorylated form of dolichol (dolichol pyrophosphate), a lipid of the RER membrane (Fig. 14.16). The initial product is processed in the RER and Golgi. = terminal group (fucose or Nacetylneuraminic acid); mRNA = messenger RNA; Asn = asparagine.

1	1. Dolichol-linked oligosaccharide synthesis: As with the O-linked glycoproteins, the protein is synthesized on the RER and enters its lumen. However, it does not become glycosylated with individual sugars. Instead, a lipid-linked oligosaccharide is first constructed. This consists of dolichol (an RER membrane lipid made from an intermediate of cholesterol synthesis; see p. 221) attached through a pyrophosphate linkage to an oligosaccharide containing GlcNAc, mannose, and glucose. The sugars to be added sequentially to the dolichol by membrane-bound glycosyltransferases are first GlcNAc, followed by mannose and glucose (see Fig. 14.16). The entire 14-sugar oligosaccharide is then transferred from dolichol to the amide nitrogen of an asparagine residue in the protein to be glycosylated by a protein–oligosaccharide transferase present in the RER. [Note: Tunicamycin inhibits N-linked glycosylation.]

1	Congenital disorders of glycosylation (CDG) are syndromes caused primarily by defects in the N-linked glycosylation of proteins, either oligosaccharide assembly (type I) or processing (type II). 2.

1	2. N-Linked oligosaccharide processing: After addition to the protein, the N-linked oligosaccharide is processed by the removal of specific mannosyl and glucosyl residues as the glycoprotein moves through the RER. Finally, the oligosaccharide chains are completed in the Golgi by addition of a variety of sugars (for example, GlcNAc, GalNAc, and additional mannoses and then fucose or NANA as terminal groups) to produce a complex glycoprotein. Alternatively, they are not processed further, leaving branched, mannose-containing chains in a high-mannose glycoprotein (see Fig. 14.16). The ultimate fate of N-linked glycoproteins is the same as that of the O-linked glycoproteins (for example, they can be released by the cell or become part of a cell membrane). In addition, N-linked glycoproteins can be targeted to the lysosomes. [Note: Nonenzymatic glycosylation of proteins is known as glycation (see p. 33).] 3.

1	Lysosomal enzymes: N-Linked glycoproteins being processed in the Golgi can be phosphorylated on carbon 6 of one or more mannosyl residues. UDP-GlcNAc provides the phosphate in a reaction catalyzed by a phosphotransferase. Receptors, located in the Golgi membrane, bind the mannose 6-phosphate residues of these proteins, which are then packaged into vesicles and sent to the lysosomes (Fig. 14.17). I-Cell disease is a rare lysosomal storage disease in which the phosphotransferase is deficient. This causes the proteins to be secreted, rather than being targeted to lysosomes. Consequently, the acid hydrolases normally found in the lysosomal matrix are absent, resulting in an accumulation of the substrates for these missing enzymes. [Note: I-Cell disease is so named because of the large inclusion bodies seen in cells of patients with this disease.] In addition, high amounts of lysosomal enzymes are found in the patient’s plasma and urine, indicating that the targeting process to lysosomes

1	bodies seen in cells of patients with this disease.] In addition, high amounts of lysosomal enzymes are found in the patient’s plasma and urine, indicating that the targeting process to lysosomes (rather than the synthetic pathway of these enzymes) is deficient. I-Cell disease is characterized by skeletal abnormalities, restricted joint movement, coarse (dysmorphic) facial features, and severe psychomotor impairment. [Note:

1	Because I-cell disease has features in common with the mucopolysaccharidoses and sphingolipidoses (see p. 210), it is termed a mucolipidosis (type II).] Currently, there is no cure, and death from cardiopulmonary complications usually occurs in early childhood. IX. LYSOSOMAL GLYCOPROTEIN DEGRADATION

1	IX. LYSOSOMAL GLYCOPROTEIN DEGRADATION Degradation of glycoproteins is similar to that of the GAG (see p. 163). The lysosomal acid hydrolases are each generally specific for the removal of one component of the glycoprotein. They are primarily exoenzymes that remove their respective groups in the reverse order of their incorporation (last on, first off). If any one degradative enzyme is missing, degradation by the other exoenzymes cannot continue. A group of very rare autosomal-recessive diseases called the glycoprotein storage diseases (oligosaccharidoses), caused by a deficiency of any one of the degradative enzymes, results in accumulation of partially degraded structures in the lysosomes. For example, α-mannosidosis type 3 is a severe, progressive, fatal deficiency of the enzyme α-mannosidase. Presentation is similar to Hurler syndrome, but immune deficiency is also seen. Mannose-rich oligosaccharide fragments appear in the urine. Diagnosis is by enzyme assay. X. CHAPTER SUMMARY

1	Glycosaminoglycans (GAG) are long, negatively charged, unbranched, heteropolysaccharide chains generally composed of a repeating disaccharide unit [acidic sugar–amino sugar]n (Fig. 14.18). The amino sugar is either D-glucosamine or D-galactosamine in which the amino group is usually acetylated, thus eliminating its positive charge. The amino sugar may also be sulfated on carbon 4 or 6 or on a nonacetylated nitrogen. The acidic sugar is either D-glucuronic acid or its C-5 epimer L-iduronic acid. GAG bind large amounts of water, thereby producing the gel-like matrix that forms the basis of the body’s ground substance. The viscous, lubricating properties of mucous secretions are also caused by the presence of GAG, which led to the original naming of these compounds as mucopolysaccharides. There are six major types of GAG, including chondroitin 4-and 6-sulfates, keratan sulfate, dermatan sulfate, heparin, heparan sulfate, and hyaluronic acid. All GAG, except hyaluronic acid, are found

1	There are six major types of GAG, including chondroitin 4-and 6-sulfates, keratan sulfate, dermatan sulfate, heparin, heparan sulfate, and hyaluronic acid. All GAG, except hyaluronic acid, are found covalently attached to a core protein, forming proteoglycan monomers. Many proteoglycan monomers associate with a molecule of hyaluronic acid to form proteoglycan aggregates. GAG are synthesized in the Golgi. The polysaccharide chains are elongated by the sequential addition of alternating acidic and amino sugars, donated by their UDP derivatives. D-Glucuronate may be epimerized to L-iduronate. The last step in synthesis is sulfation of some of the amino sugars. The source of the sulfate is 3′-phosphoadenosyl-5′-phosphosulfate (PAPS). The completed proteoglycans are secreted into the extracellular matrix (ECM) or remain associated with the outer surface of cells. GAG are degraded by lysosomal acid hydrolases. They are first broken down to oligosaccharides, which are degraded sequentially

1	matrix (ECM) or remain associated with the outer surface of cells. GAG are degraded by lysosomal acid hydrolases. They are first broken down to oligosaccharides, which are degraded sequentially from the nonreducing end of each chain. A deficiency of any one of the hydrolases results in a mucopolysaccharidosis. These are hereditary disorders in which GAG accumulate in tissues, causing symptoms such as skeletal and ECM deformities and intellectual disability. Examples of these genetic diseases include Hunter (X-linked) and Hurler syndromes. Glycoproteins are proteins to which oligosaccharides (glycans) are covalently attached. They differ from the proteoglycans in that the length of the glycoprotein’s carbohydrate chain is relatively short (usually two to ten sugar residues long, although it can be longer), may be branched, and does not contain serial disaccharide units. Membrane-bound glycoproteins participate in a broad range of cellular phenomena, including cell-surface recognition

1	it can be longer), may be branched, and does not contain serial disaccharide units. Membrane-bound glycoproteins participate in a broad range of cellular phenomena, including cell-surface recognition (by other cells, hormones, and viruses), cell-surface antigenicity (such as the blood group antigens), and as components of the ECM and of the mucins of the gastrointestinal and urogenital tracts, where they act as protective biologic lubricants. In addition, almost all of the globular proteins present in human plasma are glycoproteins. Glycoproteins are synthesized in the rough endoplasmic reticulum (RER) and the Golgi. The precursors of the carbohydrate components of glycoproteins are nucleotide sugars. O-Linked glycoproteins are synthesized in the Golgi by the sequential transfer of sugars from their nucleotide carriers to the hydroxyl group of a serine or threonine residue in the protein. N-Linked glycoproteins are synthesized by the transfer of a preformed oligosaccharide from its

1	from their nucleotide carriers to the hydroxyl group of a serine or threonine residue in the protein. N-Linked glycoproteins are synthesized by the transfer of a preformed oligosaccharide from its RER membrane lipid carrier, dolichol pyrophosphate, to the amide N of an asparagine residue in the protein. They contain varying amounts of mannose. A deficiency in the phosphotransferase that phosphorylates mannose residues at carbon 6 in N-linked glycoprotein enzymes destined for the lysosomes results in I-cell disease. Glycoproteins are degraded in lysosomes by acid hydrolases. A deficiency of any one of these enzymes results in a lysosomal glycoprotein storage disease (oligosaccharidosis), resulting in accumulation of partially degraded structures in the lysosome.

1	Choose the ONE best answer. 4.1. Mucopolysaccharidoses are hereditary lysosomal storage diseases. They are caused by: A. defects in the degradation of glycosaminoglycans. B. defects in the targeting of enzymes to lysosomes. C. an increased rate of synthesis of the carbohydrate component of proteoglycans. D. an insufficient rate of synthesis of proteolytic enzymes. E. the synthesis of abnormally small amounts of core proteins. F. the synthesis of heteropolysaccharides with an altered structure. Correct answer = A. The mucopolysaccharidoses are caused by deficiencies in any one of the lysosomal acid hydrolases responsible for the degradation of glycosaminoglycans (not proteins). The enzyme is correctly targeted to the lysosome, so blood levels of the enzyme do not increase, but it is nonfunctional. In these diseases, synthesis of the protein and carbohydrate components of proteoglycans is unaffected, in terms of both structure and amount.

1	4.2. The presence of the following compound in the urine of a patient suggests a deficiency in which one of the enzymes listed below? A. Galactosidase B. Glucuronidase C. Iduronidase D. Mannosidase E. Sulfatase Correct answer = E. Degradation of glycoproteins follows the rule: last on, first off. Because sulfation is the last step in the synthesis of this sequence, a sulfatase is required for the next step in the degradation of the compound shown. 4.3. An 8-month-old boy with coarse facial features, skeletal abnormalities, and delays in both growth and development is diagnosed with I-cell disease based on his presentation and on histologic and biochemical testing. I-Cell disease is characterized by: A. decreased production of cell surface O-linked glycoproteins. B. elevated levels of acid hydrolases in the blood. C. inability to N-glycosylate proteins. D. increased synthesis of proteoglycans. E. oligosaccharides in the urine.

1	B. elevated levels of acid hydrolases in the blood. C. inability to N-glycosylate proteins. D. increased synthesis of proteoglycans. E. oligosaccharides in the urine. Correct answer = B. I-Cell disease is a lysosomal storage disease caused by deficiency of the phosphotransferase needed for synthesis of the mannose 6phosphate signal that targets acid hydrolases to the lysosomal matrix. This results in secretion of these enzymes from the cell and accumulation of materials within the lysosome because of impaired degradation. None of the other choices relates to I-cell disease or lysosomal function. Oligosaccharides in the urine are characteristic of the muco-and polysaccharidoses but not I-cell disease (a type II mucolipidosis). 4.4. An infant with corneal clouding has dermatan sulfate and heparan sulfate in his urine. Decreased activity of which of the enzymes listed below would confirm the suspected diagnosis of Hurler syndrome? A. α-L-Iduronidase B. α-Glucuronidase

1	A. α-L-Iduronidase B. α-Glucuronidase C. Glycosyltransferase D. Iduronate sulfatase Correct answer = A. Hurler syndrome, a defect in the lysosomal degradation of glycosaminoglycans (GAG) with corneal clouding, is due to a deficiency in αL-iduronidase. β-Glucuronidase is deficient in Sly syndrome, and iduronate sulfatase is deficient in Hunter syndrome. Glycosyltransferases are enzymes of GAG synthesis. 4.5. Distinguish between glycoproteins and proteoglycans. Glycoproteins are proteins to which short, branched, structurally diverse oligosaccharide chains (glycans) are attached. Proteoglycans consist of a core protein to which long, unbranched, glycosaminoglycan (GAG) chains are attached. GAG are large complexes of negatively charged heteropolysaccharides composed of repeating [acidic sugar-amino sugar]n disaccharide units. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	I. OVERVIEW Lipids are a heterogeneous group of water-insoluble (hydrophobic) organic molecules (Fig. 15.1). Because of their insolubility in aqueous solutions, body lipids are generally found compartmentalized, as in the case of membrane-associated lipids or droplets of triacylglycerol in adipocytes, or transported in blood in association with protein, as in lipoprotein particles (see p. 227) or on albumin. Lipids are a major source of energy for the body, and they also provide the hydrophobic barrier that permits partitioning of the aqueous contents of cells and subcellular structures. Lipids serve additional functions in the body (for example, some fat-soluble vitamins have regulatory or coenzyme functions, and the prostaglandins and steroid hormones play major roles in the control of the body’s homeostasis). Deficiencies or imbalances of lipid metabolism can lead to some of the major clinical problems encountered by physicians, such as atherosclerosis, diabetes, and obesity.

1	II. DIGESTION, ABSORPTION, SECRETION, AND UTILIZATION The average daily intake of lipids by U.S. adults is ~78 g, of which >90% is triacylglycerol ([TAG], formerly called triglyceride [TG]), that consists of three fatty acids (FA) esterified to a glycerol backbone (see Fig. 15.1). The remainder of the dietary lipids consists primarily of cholesterol, cholesteryl esters, phospholipids, and nonesterified (free) FA (FFA). The digestion of dietary lipids begins in the stomach and is completed in the small intestine. The process is summarized in Figure 15.2. A. Digestion in the stomach

1	A. Digestion in the stomach Lipid digestion in the stomach is limited. It is catalyzed by lingual lipase that originates from glands at the back of the tongue and gastric lipase that is secreted by the gastric mucosa. Both enzymes are relatively acid stable, with optimal pH values of 4 to 6. These acid lipases hydrolyze FA from TAG molecules, particularly those containing short-or medium-chainlength (≤12 carbons) FA such as are found in milk fat. Consequently, these lipases play a particularly important role in lipid digestion in infants for whom milk fat is the primary source of calories. They also become important digestive enzymes in individuals with pancreatic insufficiency such as those with cystic fibrosis (CF). Lingual and gastric lipases aid these patients in degrading TAG molecules (especially those with short-to medium-chain FA) despite a near or complete absence of pancreatic lipase (see Section D.1. below). B. Cystic fibrosis

1	B. Cystic fibrosis CF is the most common lethal genetic disease in Caucasians of Northern European ancestry and has a prevalence of ~1:3,300 births in the United States. CF is an autosomal-recessive disorder caused by mutations to the gene for the CF transmembrane conductance regulator (CFTR) protein that functions as a chloride channel on epithelium in the pancreas, lungs, testes, and sweat glands. Defective CFTR results in decreased secretion of chloride and increased uptake of sodium and water. In the pancreas, the depletion of water on the cell surface results in thickened mucus that clogs the pancreatic ducts, preventing pancreatic enzymes from reaching the intestine, thereby leading to pancreatic insufficiency. Treatment includes replacement of these enzymes and supplementation with fat-soluble vitamins. [Note: CF also causes chronic lung infections with progressive pulmonary disease and male infertility.] C. Emulsification in the small intestine

1	C. Emulsification in the small intestine The critical process of dietary lipid emulsification occurs in the duodenum.

1	Emulsification increases the surface area of the hydrophobic lipid droplets so that the digestive enzymes, which work at the interface of the droplet and the surrounding aqueous solution, can act effectively. Emulsification is accomplished by two complementary mechanisms, namely, use of the detergent properties of the conjugated bile salts and mechanical mixing due to peristalsis. Bile salts, made in the liver and stored in the gallbladder, are amphipathic derivatives of cholesterol (see p. 224). Conjugated bile salts consist of a hydroxylated sterol ring structure with a side chain to which a molecule of glycine or taurine is covalently attached by an amide linkage (Fig. 15.3). These emulsifying agents interact with the dietary lipid droplets and the aqueous duodenal contents, thereby stabilizing the droplets as they become smaller from peristalsis and preventing them from coalescing. [Note: See p. 225 for a more complete discussion of bile salt metabolism.]

1	D. Degradation by pancreatic enzymes The dietary TAG, cholesteryl esters, and phospholipids are enzymatically degraded (digested) in the small intestine by pancreatic enzymes, whose secretion is hormonally controlled. 1.

1	Triacylglycerol degradation: TAG molecules are too large to be taken up efficiently by the mucosal cells (enterocytes) of the intestinal villi. Therefore, they are hydrolyzed by an esterase, pancreatic lipase, which preferentially removes the FA at carbons 1 and 3. The primary products of hydrolysis are, thus, a mixture of 2-monoacylglycerol (2-MAG) and FFA (see Fig. 15.2). [Note: Pancreatic lipase is found in high concentrations in pancreatic secretions (2%–3% of the total protein present), and it is highly efficient catalytically, thus insuring that only severe pancreatic deficiency, such as that seen in CF, results in significant malabsorption of fat.] A second protein, colipase, also secreted by the pancreas, binds the lipase at a ratio of 1:1 and anchors it at the lipid–aqueous interface. Colipase restores activity to lipase in the presence of inhibitory substances like bile salts that bind the micelles. [Note: Colipase is secreted as the zymogen, procolipase, which is activated

1	Colipase restores activity to lipase in the presence of inhibitory substances like bile salts that bind the micelles. [Note: Colipase is secreted as the zymogen, procolipase, which is activated in the intestine by trypsin.] Orlistat, an antiobesity drug, inhibits gastric and pancreatic lipases, thereby decreasing fat absorption, resulting in weight loss.

1	2. Cholesteryl ester degradation: Most dietary cholesterol is present in the free (nonesterified) form, with 10%–15% present in the esterified form. Cholesteryl esters are hydrolyzed by pancreatic cholesteryl ester hydrolase (cholesterol esterase), which produces cholesterol plus FFA (see Fig. 15.2). Activity of this enzyme is greatly increased in the presence of bile salts. 3.

1	3. Phospholipid degradation: Pancreatic juice is rich in the proenzyme of phospholipase A2 that, like procolipase, is activated by trypsin and, like cholesteryl ester hydrolase, requires bile salts for optimum activity. Phospholipase A2 removes one FA from carbon 2 of a phospholipid, leaving a lysophospholipid. For example, phosphatidylcholine (the predominant phospholipid of digestion) becomes lysophosphatidylcholine. The remaining FA at carbon 1 can be removed by lysophospholipase, leaving a glycerylphosphoryl base (for example, glycerylphosphorylcholine, see Fig. 15.2) that may be excreted in the feces, further degraded, or absorbed.

1	4. Control: Pancreatic secretion of the hydrolytic enzymes that degrade dietary lipids in the small intestine is hormonally controlled (Fig. 15.4). Cells in the mucosa of the lower duodenum and jejunum produce the peptide hormone cholecystokinin (CCK), in response to the presence of lipids and partially digested proteins entering these regions of the upper small intestine. CCK acts on the gallbladder (causing it to contract and release bile, a mixture of bile salts, phospholipids, and free cholesterol) and on the exocrine cells of the pancreas (causing them to release digestive enzymes). It also decreases gastric motility, resulting in a slower release of gastric contents into the small intestine (see p. 353). Other intestinal cells produce another peptide hormone, secretin, in response to the low pH of the chyme entering the intestine from the stomach. Secretin causes the pancreas to release a solution rich in bicarbonate that helps neutralize the pH of the intestinal contents,

1	to the low pH of the chyme entering the intestine from the stomach. Secretin causes the pancreas to release a solution rich in bicarbonate that helps neutralize the pH of the intestinal contents, bringing them to the appropriate pH for digestive activity by pancreatic enzymes.

1	E. Absorption by enterocytes

1	FFA, free cholesterol, and 2-MAG are the primary products of lipid digestion in the jejunum. These, plus bile salts and fat-soluble vitamins (A, D, E, and K), form mixed micelles (that is, disc-shaped clusters of a mixture of amphipathic lipids that coalesce with their hydrophobic groups on the inside and their hydrophilic groups on the outside). Therefore, mixed micelles are soluble in the aqueous environment of the intestinal lumen (Fig. 15.5). These particles approach the primary site of lipid absorption, the brush border membrane of the enterocytes. This microvilli-rich apical membrane is separated from the liquid contents of the intestinal lumen by an unstirred water layer that mixes poorly with the bulk fluid. The hydrophilic surface of the micelles facilitates the transport of the hydrophobic lipids through the unstirred water layer to the brush border membrane where they are absorbed. Bile salts are absorbed in the terminal ileum, with <5% being lost in the feces. [Note:

1	the hydrophobic lipids through the unstirred water layer to the brush border membrane where they are absorbed. Bile salts are absorbed in the terminal ileum, with <5% being lost in the feces. [Note: Relative to other dietary lipids, cholesterol is only poorly absorbed by the enterocytes. Drug therapy (for example, with ezetimibe) can further reduce cholesterol absorption in the small intestine.] Because short-and medium-chain FA are water soluble, they do not require the assistance of mixed micelles for absorption by the intestinal mucosa.

1	F. Triacylglycerol and cholesteryl ester resynthesis

1	The mixture of lipids absorbed by the enterocytes migrates to the smooth endoplasmic reticulum (SER) where biosynthesis of complex lipids takes place. The long-chain FA are first converted into their activated form by fatty acyl coenzyme A (CoA) synthetase (thiokinase), as shown in Figure 15.6. Using the fatty acyl CoA derivatives, the 2-MAG absorbed by the enterocytes are converted to TAG through sequential reacylations by two acyltransferases, acyl CoA:monoacylglycerol acyltransferase and acyl CoA:diacylglycerol acyltransferase. Lysophospholipids are reacylated to form phospholipids by a family of acyltransferases, and cholesterol is acylated primarily by acyl CoA:cholesterol acyltransferase (see p. 232). [Note: Virtually all long-chain FA entering the enterocytes are used in this fashion to form TAG, phospholipids, and cholesteryl esters. Short-and medium-chain FA are not converted to their CoA derivatives and are not reesterified to 2-MAG. Instead, they are released into the

1	fashion to form TAG, phospholipids, and cholesteryl esters. Short-and medium-chain FA are not converted to their CoA derivatives and are not reesterified to 2-MAG. Instead, they are released into the portal circulation, where they are carried by serum albumin to the liver.]

1	G. Lipid malabsorption Lipid malabsorption, resulting in increased lipid (including the fat-soluble vitamins and essential FA, see p. 182) in the feces, a condition known as steatorrhea, can be caused by disturbances in lipid digestion and/or absorption (Fig. 15.7). Such disturbances can result from several conditions, including CF (causing poor digestion) and short bowel syndrome (causing decreased absorption). The ability of short-and medium-chain FA to be taken up by enterocytes without the aid of mixed micelles has made them important in medical nutrition therapy for individuals with malabsorption disorders. H. Secretion from enterocytes

1	The newly resynthesized TAG and cholesteryl esters are very hydrophobic and aggregate in an aqueous environment. Therefore, they must be packaged as particles of lipid droplets surrounded by a thin layer composed of phospholipids, nonesterified cholesterol, and a molecule of the protein apolipoprotein (apo) B-48 (see p. 228). This layer stabilizes the particle and increases its solubility, thereby preventing multiple particles from coalescing. [Note: Microsomal triglyceride transfer protein is essential for the assembly of all TAG-rich apo B–containing particles in the ER (see p. 228).] The lipoprotein particles are released by exocytosis from enterocytes into the lacteals (lymphatic vessels in the villi of the small intestine). The presence of these particles in the lymph after a lipid-rich meal gives it a milky appearance. This lymph is called chyle (as opposed to chyme, the name given to the semifluid mass of partially digested food that passes from the stomach to the duodenum),

1	meal gives it a milky appearance. This lymph is called chyle (as opposed to chyme, the name given to the semifluid mass of partially digested food that passes from the stomach to the duodenum), and the particles are named chylomicrons. Chylomicrons follow the lymphatic system to the thoracic duct and are then conveyed to the left subclavian vein, where they enter the blood. The steps in the production of chylomicrons are summarized in Figure 15.6. [Note: Once released into blood, the nascent (immature) chylomicrons pick up apolipoproteins E and C-II from high-density lipoproteins and mature. (For a more detailed description of chylomicron structure and metabolism, see p. 227.)]

1	I. Use by the tissues Most of the TAG contained in chylomicrons is broken down in the capillary beds of skeletal and cardiac muscle and adipose tissue. The TAG is degraded to FFA and glycerol by lipoprotein lipase (LPL). This enzyme is synthesized and secreted primarily by adipocytes and muscle cells. Secreted LPL is anchored to the luminal surface of endothelial cells in the capillaries of muscle and adipose tissues. [Note: Familial chylomicronemia (type I hyperlipoproteinemia) is a rare, autosomal-recessive disorder caused by a deficiency of LPL or its coenzyme apo C-II (see p. 228). The result is fasting chylomicronemia and severe hypertriacylglycerolemia, which can cause pancreatitis.] 1.

1	Fate of free fatty acids: The FFA derived from the hydrolysis of TAG may either directly enter adjacent muscle cells and adipocytes or be transported in the blood in association with serum albumin until they are taken up by cells. [Note: Human serum albumin is a large protein secreted by the liver. It transports a number of primarily hydrophobic compounds in the circulation, including FFA and some drugs.] Most cells can oxidize FA to produce energy (see p. 190). Adipocytes can also reesterify FFA to produce TAG molecules, which are stored until the FA are needed by the body (see p. 188). 2. Fate of glycerol: Glycerol released from TAG is taken up from the blood and phosphorylated by hepatic glycerol kinase to produce glycerol 3phosphate, which can enter either glycolysis or gluconeogenesis by oxidation to dihydroxyacetone phosphate (see p. 101) or be used in TAG synthesis (see p. 189). 3.

1	3. Fate of chylomicron remnants: After most of the TAG has been removed, the chylomicron remnants (which contain cholesteryl esters, phospholipids, apolipoproteins, fat-soluble vitamins, and a small amount of TAG) bind to receptors on the liver (apo E is the ligand; see p. 229) and are endocytosed. The intracellular remnants are hydrolyzed to their component parts. Cholesterol and the nitrogenous bases of phospholipids (for example, choline) can be recycled by the body. [Note: If removal of remnants by the liver is decreased because of impaired binding to their receptor, they accumulate in the plasma. This is seen in the rare type III hyperlipoproteinemia (also called familial dysbetalipoproteinemia or broad beta disease, see p. 231).] III. CHAPTER SUMMARY

1	III. CHAPTER SUMMARY Dietary lipid digestion begins in the stomach and continues in the small intestine (Fig. 15.8). Cholesteryl esters, phospholipids, and triacylglycerols (TAG) containing long-chain-length fatty acids (FA) are degraded in the small intestine by pancreatic enzymes. The most important of these enzymes are cholesterol esterase, phospholipase A2, and pancreatic lipase.

1	In cystic fibrosis, thickened mucus prevents these enzymes reaching the intestine. In contrast, TAG in milk fat contain short-to medium-chainlength FA and are degraded in the stomach by acid lipases (lingual lipase and gastric lipase). The hydrophobic nature of lipids requires that dietary lipids be emulsified for efficient degradation. Emulsification occurs in the small intestine using peristaltic action (mechanical mixing) and bile salts (detergents). The primary products of dietary lipid degradation are 2monoacylglycerol, nonesterified (free) cholesterol, and free FA. These compounds, plus the fat-soluble vitamins, form mixed micelles that facilitate dietary lipid absorption by intestinal mucosal cells (enterocytes). These cells use activated long-chain FA to regenerate TAG and cholesteryl esters and also synthesize protein (apolipoprotein [apo] B-48), all of which are then assembled with the fat-soluble vitamins into lipoprotein particles called chylomicrons. Short-and

1	and cholesteryl esters and also synthesize protein (apolipoprotein [apo] B-48), all of which are then assembled with the fat-soluble vitamins into lipoprotein particles called chylomicrons. Short-and medium-chain FA enter blood directly. Chylomicrons are first released into the lymph and then enter the blood, where their lipid core is degraded by lipoprotein lipase (with apo C-II as the coenzyme) in the capillaries of muscle and adipose tissues. Thus, dietary lipids are made available to the peripheral tissues. Fat maldigestion or malabsorption causes steatorrhea (lipid in the feces). A deficiency in the ability to degrade chylomicron components, or remove chylomicron remnants after TAG has been degraded, results in accumulation of these particles in blood.

1	Choose the ONE best answer. 5.1. Which one of the following statements about lipid digestion is correct? A. Large lipid droplets are emulsified (have their surface area increased) in the mouth through the act of chewing (mastication). B. The enzyme colipase facilitates the binding of bile salts to mixed micelles, maximizing the activity of pancreatic lipase. C. The peptide hormone secretin causes the gallbladder to contract and release bile. D. Patients with cystic fibrosis have difficulties with digestion because their pancreatic secretions are less able to reach the small intestine, the primary site of lipid digestion. E. Formation of triacylglycerol-rich chylomicrons is independent of protein synthesis in the intestinal mucosa.

1	E. Formation of triacylglycerol-rich chylomicrons is independent of protein synthesis in the intestinal mucosa. Correct answer = D. Patients with cystic fibrosis, a genetic disease resulting in a deficiency of a functional chloride transporter, have thickened mucus that impedes the flow of pancreatic enzymes into the duodenum. Emulsification occurs through peristalsis, which provides mechanical mixing, and bile salts that function as detergents. Colipase restores activity to pancreatic lipase in the presence of inhibitory bile salts that bind the micelles. Cholecystokinin is the hormone that causes contraction of the gallbladder and release of stored bile, and secretin causes release of bicarbonate. Chylomicron formation requires synthesis of apolipoprotein B-48. 5.2. Which one of the following statements about lipid absorption from the intestine is correct? A. Dietary triacylglycerol must be completely hydrolyzed to free fatty acids and glycerol before absorption.

1	A. Dietary triacylglycerol must be completely hydrolyzed to free fatty acids and glycerol before absorption. B. The triacylglycerol carried by chylomicrons is degraded by lipoprotein lipase, producing fatty acids that are taken up by muscle and adipose tissues and glycerol that is taken up by the liver. C. Fatty acids that contain ≤12 carbon atoms are absorbed and enter the circulation primarily via the lymphatic system. D. Deficiencies in the ability to absorb fat result in excessive amounts of chylomicrons in the blood.

1	D. Deficiencies in the ability to absorb fat result in excessive amounts of chylomicrons in the blood. Correct answer = B. The triacylglycerols (TAG) in chylomicrons are degraded to fatty acids (FA) and glycerol by lipoprotein lipase on capillary endothelial surfaces in muscle and adipose tissue, thus providing a source of FA to these tissues for degradation or storage and providing glycerol for hepatic metabolism. In the duodenum, TAG are degraded to one 2-monoacylglycerol + two free FA that get absorbed. Medium-and short-chain FA enter directly into blood (not lymph), and they neither require micelles nor get packaged into chylomicrons. Because chylomicrons contain dietary lipids that were digested and absorbed, a defect in fat absorption would result in decreased production of chylomicrons. Fatty Acid, Triacylglycerol, and Ketone Body Metabolism 16 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Fatty acids exist free in the body (that is, they are nonesterified) and as fatty acyl esters in more complex molecules such as triacylglycerols (TAG). Low levels of free fatty acids (FFA) occur in all tissues, but substantial amounts can sometimes be found in the plasma, particularly during fasting. Plasma FFA (transported on serum albumin) are in route from their point of origin (TAG of adipose tissue or circulating lipoproteins) to their site of consumption (most tissues). FFA can be oxidized by many tissues, particularly liver and muscle, to provide energy and, in the liver, to provide the substrate for ketone body synthesis. Fatty acids are also structural components of membrane lipids, such as phospholipids and glycolipids (see p. 201). Fatty acids attached to certain proteins enhance the ability of those proteins to associate with membranes (see p. 206). Fatty acids are also precursors of the hormone-like prostaglandins (see p. 213). Esterified fatty acids, in the form of TAG

1	the ability of those proteins to associate with membranes (see p. 206). Fatty acids are also precursors of the hormone-like prostaglandins (see p. 213). Esterified fatty acids, in the form of TAG stored in white adipose tissue (WAT), serve as the major energy reserve of the body. Alterations in fatty acid metabolism are associated with obesity and diabetes. Figure 16.1 illustrates the metabolic pathways of fatty acid synthesis and degradation and their relationship to carbohydrate metabolism.

1	II. FATTY ACID STRUCTURE A fatty acid consists of a hydrophobic hydrocarbon chain with a terminal carboxyl group that has a pKa (see p. 6) of ~4.8 (Fig. 16.2). At physiologic pH, the terminal carboxyl group (–COOH) ionizes, becoming –COO−. [Note: When the pH is above the pK, the deprotonated form predominates (see p. 7).] This anionic group has an affinity for water, giving the fatty acid its amphipathic nature (having both a hydrophilic and a hydrophobic region). However, for longchain-length fatty acids (LCFA), the hydrophobic portion is predominant. These molecules are highly water insoluble and must be transported in the circulation in association with protein. More than 90% of the fatty acids found in plasma are in the form of fatty acid esters (primarily TAG, cholesteryl esters, and phospholipids) contained in circulating lipoprotein particles (see p. 227). FFA are transported in the circulation in association with albumin, the most abundant protein in serum.

1	A. Fatty acid saturation Fatty acid chains may contain no double bonds (that is, be saturated) or contain one or more double bonds (that is, be mono-or polyunsaturated). In humans, the majority are saturated or monounsaturated. When double bonds are present, they are nearly always in the cis rather than in the trans configuration. The introduction of a cis double bond causes the fatty acid to bend or kink at that position (Fig. 16.3). If the fatty acid has two or more double bonds, they are always spaced at three-carbon intervals. [Note: In general, addition of double bonds decreases the melting temperature (Tm) of a fatty acid, whereas increasing the chain length increases the Tm. Because membrane lipids typically contain LCFA, the presence of double bonds in some fatty acids helps maintain the fluid nature of those lipids. See p. 363 for a discussion of the dietary occurrence of cis and trans unsaturated fatty acids.] B. Fatty acid chain length and double bond positions

1	The common names and structures of some fatty acids of physiologic importance are listed in Figure 16.4. In humans, fatty acids with an even number of carbon atoms (16, 18, or 20) predominate, with longer fatty acids (>22 carbons) being found in the brain. The carbon atoms are numbered, beginning with the carbonyl carbon as carbon 1. The number before the colon indicates the number of carbons in the chain, and those after the colon indicate the numbers and positions (relative to the carboxyl end) of double bonds. For example, as denoted in Figure 16.4, arachidonic acid, 20:4(5,8,11,14), is 20 carbons long and has four double bonds (between carbons 5–6, 8–9, 11–12, and 14–15). [Note: Carbon 2, the carbon to which the carboxyl group is attached, is also called the α-carbon, carbon 3 is the βcarbon, and carbon 4 is the γ-carbon. The carbon of the terminal methyl group is called the ω-carbon regardless of the chain length.] The double bonds in a fatty acid can also be referenced relative

1	βcarbon, and carbon 4 is the γ-carbon. The carbon of the terminal methyl group is called the ω-carbon regardless of the chain length.] The double bonds in a fatty acid can also be referenced relative to the ω (methyl) end of the chain. Arachidonic acid is referred to as an ω-6 fatty acid because the terminal double bond is six bonds from the ω end (Fig. 16.5A). [Note: The equivalent designation of n-6 may also be used (Fig. 16.5B).] Another ω-6 fatty acid is the essential linoleic acid 18:2(9,12). In contrast, α-linolenic acid, 18:3(9,12,15), is an essential ω-3 fatty acid.

1	C. Essential fatty acids Linoleic acid, the precursor of ω-6 arachidonic acid that is the substrate for prostaglandin synthesis (see p. 213), and α-linolenic acid, the precursor of ω-3 fatty acids that are important for growth and development, are dietary essentials in humans because we lack the enzymes needed to synthesize them. Plants provide us with these essential fatty acids. [Note: Arachidonic acid becomes essential if linoleic acid is deficient in the diet. See p. 362 for a discussion of the nutritional significance of ω-3 and ω-6 fatty acids.] Essential fatty acid deficiency (rare) can result in a dry, scaly dermatitis as a result of an inability to synthesize molecules that provide the water barrier in skin (see p. 206). III. FATTY ACID DE NOVO SYNTHESIS

1	III. FATTY ACID DE NOVO SYNTHESIS Carbohydrates and proteins obtained from the diet in excess of the body’s needs for these nutrients can be converted to fatty acids. In adults, de novo fatty acid synthesis occurs primarily in the liver and lactating mammary glands and, to a lesser extent, in adipose tissue. This cytosolic process is endergonic (see p. 70) and reductive. It incorporates carbons from acetyl coenzyme A (CoA) into the growing fatty acid chain, using ATP and reduced nicotinamide adenine dinucleotide phosphate (NADPH). [Note: Dietary TAG also supply fatty acids. See p. 321 for a discussion of the metabolism of dietary nutrients in the well-fed state.] A. Cytosolic acetyl CoA production

1	The first step in fatty acid synthesis is the transfer of acetate units from mitochondrial acetyl CoA to the cytosol. Mitochondrial acetyl CoA is produced by the oxidation of pyruvate (see p. 109) and by the catabolism of certain amino acids (see p. 266). However, the CoA portion of acetyl CoA cannot cross the inner mitochondrial membrane, and only the acetyl portion enters the cytosol. It does so as part of citrate produced by the condensation of acetyl CoA with oxaloacetate (OAA) by citrate synthase (Fig. 16.6). [Note: The transport of citrate to the cytosol occurs when the mitochondrial citrate concentration is high. This is observed when isocitrate dehydrogenase of the tricarboxylic acid (TCA) cycle is inhibited by the presence of large amounts of ATP, causing citrate and isocitrate to accumulate (see p. 112). Therefore, cytosolic citrate may be viewed as a high-energy signal. Because a large amount of ATP is needed for fatty acid synthesis, the increase in both ATP and citrate

1	to accumulate (see p. 112). Therefore, cytosolic citrate may be viewed as a high-energy signal. Because a large amount of ATP is needed for fatty acid synthesis, the increase in both ATP and citrate enhances this pathway.] In the cytosol, citrate is cleaved to OAA and acetyl CoA by ATP citrate lyase.

1	B. Acetyl CoA carboxylation to malonyl CoA The energy for the carbon-to-carbon condensations in fatty acid synthesis is supplied by the carboxylation and then decarboxylation of acyl groups in the cytosol. The carboxylation of acetyl CoA to malonyl CoA is catalyzed by acetyl CoA carboxylase (ACC ) (Fig. 16.7). ACC transfers carbon dioxide (CO2) from bicarbonate ( ) in an ATP-requiring reaction. The coenzyme is biotin (vitamin B7), which is covalently bound to a lysyl residue of the carboxylase (see Fig. 28.16, p. 385). ACC carboxylates the bound biotin, which transfers the activated carboxyl group to acetyl CoA. acetylCoAcarboxylase. diphosphate.

1	1. Acetyl CoA carboxylase short-term regulation: This carboxylation is both the rate-limiting and the regulated step in fatty acid synthesis (see Fig. 16.7). The inactive form of ACC is a protomer (complex of ≥2 polypeptides). The enzyme is allosterically activated by citrate, which causes protomers to polymerize, and allosterically inactivated by palmitoyl CoA (the end product of the pathway), which causes depolymerization. A second mechanism of short-term regulation is by reversible phosphorylation. Adenosine monophosphate–activated protein kinase (AMPK) phosphorylates and inactivates ACC. AMPK itself is activated allosterically by AMP and covalently by phosphorylation via several kinases. At least one of these AMPK kinases is activated by cyclic AMP (cAMP)–dependent protein kinase A (PKA). Thus, in the presence of counterregulatory hormones, such as epinephrine and glucagon, ACC is phosphorylated and inactive (Fig. 16.8). In the presence of insulin, ACC is dephosphorylated and

1	A (PKA). Thus, in the presence of counterregulatory hormones, such as epinephrine and glucagon, ACC is phosphorylated and inactive (Fig. 16.8). In the presence of insulin, ACC is dephosphorylated and active. [Note: This is analogous to the regulation of glycogen synthase (see p. 131).] itself is regulated both covalently and allosterically. CoA = coenzyme A; ADP phosphate.

1	2. Acetyl CoA carboxylase long-term regulation: Prolonged consumption of a diet containing excess calories (particularly high-carbohydrate, low-fat diets) causes an increase in ACC synthesis, thereby increasing fatty acid synthesis. A low-calorie or a high-fat, low-carbohydrate diet has the opposite effect. [Note: ACC synthesis is upregulated by carbohydrate (specifically glucose) via the transcription factor carbohydrate response element–binding protein (ChREBP) and by insulin via the transcription factor sterol regulatory element–binding protein-1c (SREBP-1c). Fatty acid synthase (see C. below) is similarly regulated. The function and regulation of SREBP are described on p. 222.] Metformin, used in the treatment of type 2 diabetes, lowers plasma TAG through activation of AMPK, resulting in inhibition of ACC activity (by phosphorylation) and inhibition of ACC and fatty acid synthase expression (by decreasing SREBP-1c). Metformin lowers blood glucose by increasing AMPK-mediated

1	in inhibition of ACC activity (by phosphorylation) and inhibition of ACC and fatty acid synthase expression (by decreasing SREBP-1c). Metformin lowers blood glucose by increasing AMPK-mediated glucose uptake by muscle.

1	C. Eukaryotic fatty acid synthase The remaining series of reactions of fatty acid synthesis in eukaryotes is catalyzed by the multifunctional, homodimeric enzyme fatty acid synthase (FAS). The process involves the addition of two carbons from malonyl CoA to the carboxyl end of a series of acyl acceptors. Each FAS monomer is a multicatalytic polypeptide with six different enzymic domains plus a 4ʹphosphopantetheine-containing acyl carrier protein (ACP) domain. 4ʹ-Phosphopantetheine, a derivative of pantothenic acid (vitamin B5, see p. 385), carries acyl units on its terminal thiol (–SH) group and presents them to the catalytic domains of FAS during fatty acid synthesis. It also is a component of CoA. [Note: In prokaryotes, FAS is a multienzyme complex.] The reaction numbers in brackets below refer to Figure 16.9. adenine dinucleotide phosphate. 1. An acetyl group is transferred from acetyl CoA to the –SH group of the ACP. Domain: Malonyl/acetyl CoA–ACP transacylase. 2.

1	adenine dinucleotide phosphate. 1. An acetyl group is transferred from acetyl CoA to the –SH group of the ACP. Domain: Malonyl/acetyl CoA–ACP transacylase. 2. Next, this two-carbon fragment is transferred to a temporary holding site, the –SH group of a cysteine residue on the condensing enzyme domain (see [4] below). 3. The now-vacant ACP accepts a three-carbon malonyl group from malonyl CoA. Domain: Malonyl/acetyl CoA–ACP transacylase. 4. The acetyl group on the cysteine residue condenses with the malonyl group on ACP as the CO2 originally added by ACC is released. The result is a four-carbon unit attached to the ACP domain. The loss of free energy from the decarboxylation drives the reaction. Domain: 3Ketoacyl–ACP synthase, also known as condensing enzyme. The next three reactions convert the 3-ketoacyl group to the corresponding saturated acyl group by a pair of NADPH-requiring reductions and a dehydration step. 1.

1	The next three reactions convert the 3-ketoacyl group to the corresponding saturated acyl group by a pair of NADPH-requiring reductions and a dehydration step. 1. The keto group is reduced to an alcohol. Domain: 3-Ketoacyl–ACP reductase. 2. A molecule of water is removed, creating a trans double bond between carbons 2 and 3 (the α-and β-carbons). Domain: 3-Hydroxyacyl–ACP dehydratase. 3. The double bond is reduced. Domain: Enoyl–ACP reductase.

1	This sequence of steps results in the production of a four-carbon group (butyryl) whose three terminal carbons are fully saturated and which remains attached to the ACP domain. The steps are repeated (indicated by an asterisk), beginning with the transfer of the butyryl unit from the ACP to the cysteine residue [2*], the attachment of a malonyl group to the ACP [3*], and the condensation of the two groups liberating CO2 [4*]. The carbonyl group at the β-carbon (carbon 3, the third carbon from the sulfur) is then reduced [5*], dehydrated [6*], and reduced [7*], generating hexanoyl-ACP. This cycle of reactions is repeated five more times, each time incorporating a two-carbon unit (derived from malonyl CoA) into the growing fatty acid chain at the carboxyl end. When the fatty acid reaches a length of 16 carbons, the synthetic process is terminated with palmitoyl-S-ACP. [Note: Shorter-length fatty acids are produced in the lactating mammary gland.] Palmitoyl thioesterase, the final

1	a length of 16 carbons, the synthetic process is terminated with palmitoyl-S-ACP. [Note: Shorter-length fatty acids are produced in the lactating mammary gland.] Palmitoyl thioesterase, the final catalytic activity of FAS, cleaves the thioester bond, releasing a fully saturated molecule of palmitate (16:0). [Note: All the carbons in palmitic acid have passed through malonyl CoA except the two donated by the original acetyl CoA (the first acyl acceptor), which are found at the methyl (ω) end of the fatty acid. This underscores the rate-limiting nature of the ACC reaction.]

1	D. Reductant sources The synthesis of one palmitate requires 14 NADPH, a reductant (reducing agent). The pentose phosphate pathway (see p. 145) is a major supplier of the NADPH. Two NADPH are produced for each molecule of glucose 6phosphate that enters this pathway. The cytosolic conversion of malate to pyruvate, in which malate is oxidized and decarboxylated by cytosolic malic enzyme (NADP+-dependent malate dehydrogenase), also produces cytosolic NADPH (and CO2), as shown in Figure 16.10. [Note: Malate can arise from the reduction of OAA by cytosolic NADH-dependent malate dehydrogenase (see Fig. 16.10). One source of the cytosolic NADH required for this reaction is glycolysis (see p. 101). OAA, in turn, can arise from citrate cleavage by ATP citrate lyase.] A summary of the interrelationship between glucose metabolism and palmitate synthesis is shown in Figure 16.11. tricarboxylic acid; PC = pyruvate carboxylase; PDH = pyruvate dehydrogenase. E. Further elongation

1	tricarboxylic acid; PC = pyruvate carboxylase; PDH = pyruvate dehydrogenase. E. Further elongation Although palmitate, a 16-carbon, fully saturated LCFA (16:0), is the primary end product of FAS activity, it can be further elongated by the addition of two-carbon units to the carboxylate end primarily in the smooth endoplasmic reticulum (SER). Elongation requires a system of separate enzymes rather than a multifunctional enzyme. Malonyl CoA is the two-carbon donor, and NADPH supplies the electrons. The brain has additional elongation capabilities, allowing it to produce the very-long-chain fatty acids ([VLCFA] over 22 carbons) that are required for synthesis of brain lipids. F. Chain desaturation Enzymes (fatty acyl CoA desaturases) also present in the SER are responsible for desaturating LCFA (that is, adding cis double bonds). The desaturation reactions require oxygen (O2), NADH, cytochrome b5, and its flavin adenine dinucleotide (FAD)-linked reductase. The fatty acid and the

1	NADH get oxidized as the O2 gets reduced to H2O. The first double bond is typically inserted between carbons 9 and 10, producing primarily oleic acid, 18:1(9), and small amounts of palmitoleic acid, 16:1(9). A variety of polyunsaturated fatty acids can be made through additional desaturation combined with elongation. Humans have carbon 9, 6, 5, and 4 desaturases but lack the ability to introduce double bonds from carbon 10 to the ω end of the chain. This is the basis for the nutritional essentiality of the polyunsaturated ω-6 linoleic acid and ω-3 linolenic acid. G. Storage as triacylglycerol components

1	G. Storage as triacylglycerol components Mono-, di-, and triacylglycerols consist of one, two, or three molecules of fatty acid esterified to a molecule of glycerol. Fatty acids are esterified through their carboxyl groups, resulting in a loss of negative charge and formation of neutral fat. [Note: An acylglycerol that is solid at room temperature is called a fat. If liquid, it is an oil.] 1. Arrangement: The three fatty acids esterified to a glycerol molecule to form a TAG are usually not of the same type. The fatty acid on carbon 1 is typically saturated, that on carbon 2 is typically unsaturated, and that on carbon 3 can be either. Recall that the presence of the unsaturated fatty acid(s) decrease(s) the Tm of the lipid. An example of a TAG molecule is shown in Figure 16.12. 2.

1	2. Triacylglycerol storage and function: Because TAG are only slightly soluble in water and cannot form stable micelles by themselves, they coalesce within white adipocytes to form large oily droplets that are nearly anhydrous. These cytosolic lipid droplets are the major energy reserve of the body. [Note: TAG stored in brown adipocytes serve as a source of heat through nonshivering thermogenesis (see p. 79).] 3.

1	Glycerol 3-phosphate synthesis: Glycerol 3-phosphate is the initial acceptor of fatty acids during TAG synthesis. There are two major pathways for its production (Fig. 16.13). [Note: A third process (glyceroneogenesis) is described on p. 190.] In both liver (the primary site of TAG synthesis) and adipose tissue, glycerol 3-phosphate can be produced from glucose, first using the reactions of the glycolytic pathway to produce dihydroxyacetone phosphate ([DHAP], see p. 101). DHAP is reduced by glycerol 3-phosphate dehydrogenase to glycerol 3phosphate. A second pathway found in the liver, but not in adipose tissue, uses glycerol kinase to convert free glycerol to glycerol 3phosphate (see Fig. 16.13). [Note: The glucose transporter in adipocytes (GLUT-4) is insulin dependent (see p. 312). Thus, when plasma glucose levels are low, adipocytes have only a limited ability to synthesize glycerol phosphate and cannot produce TAG de novo.] 4.

1	Fatty acid activation: A free fatty acid must be converted to its activated form (bound to CoA through a thioester link) before it can participate in metabolic processes such as TAG synthesis. This reaction, illustrated in Figure 15.6 on p. 177, is catalyzed by a family of fatty acyl CoA synthetases (thiokinases). 5. Triacylglycerol synthesis: This pathway from glycerol 3-phosphate involves four reactions, shown in Figure 16.14. These include the sequential addition of two fatty acids from fatty acyl CoA, the removal of phosphate, and the addition of the third fatty acid. H. Triacylglycerol fate in liver and adipose tissue

1	H. Triacylglycerol fate in liver and adipose tissue In WAT, TAG is stored in a nearly anhydrous form as fat droplets in the cytosol of the cells. It serves as “depot fat,” ready for mobilization when the body requires it for fuel. Little TAG is stored in healthy liver. Instead, most is exported, packaged with other lipids and apolipoproteins to form lipoprotein particles called very-low-density lipoproteins (VLDL). Nascent VLDL are secreted directly into the blood where they mature and function to deliver the endogenously derived lipids to the peripheral tissues. [Note: Recall from Chapter 15 that chylomicrons carry dietary (exogenously derived) lipids. Plasma lipoproteins are discussed in Chapter 18.] IV. FAT MOBILIZATION AND FATTY ACID OXIDATION

1	IV. FAT MOBILIZATION AND FATTY ACID OXIDATION Fatty acids stored in WAT, in the form of neutral TAG, serve as the body’s major fuel storage reserve. TAG provide concentrated stores of metabolic energy because they are highly reduced and largely anhydrous. The yield from the complete oxidation of fatty acids to CO2 and H2O is 9 kcal/g fat (as compared to 4 kcal/g protein or carbohydrate, see Fig. 27.5 on p. 359). A. Fatty acid release from fat The mobilization of stored fat requires the hydrolytic release of FFA and glycerol from their TAG form. This process of lipolysis is achieved by lipases. It is initiated by adipose triglyceride lipase (ATGL), which generates a diacylglycerol that is the preferred substrate for hormone-sensitive lipase (HSL). The monoacylglycerol (MAG) product of HSL is acted upon by MAG lipase.

1	1. Hormone-sensitive lipase regulation: HSL is active when phosphorylated by PKA, a cAMP-dependent protein kinase. cAMP is produced in the adipocyte when catecholamines (such as epinephrine) bind to cell membrane β-adrenergic receptors and activate adenylyl cyclase (Fig. 16.15). The process is similar to that of the activation of glycogen phosphorylase (see Fig. 11.9, p. 131). [Note: Because ACC is inhibited by hormone-directed phosphorylation, when the cAMP-mediated cascade is activated (see Fig. 16.8), fatty acid synthesis is turned off and TAG degradation is turned on.] In the presence of high plasma levels of insulin, HSL is dephosphorylated and inactivated. Insulin also suppresses expression of ATGL. [Note: Fat droplets are coated by a protein (perilipin) that limits access of HSL. Phosphorylation of perilipin by PKA allows translocation and binding of phosphorylated HSL to the droplet.] pyrophosphate; ADP = adenosine diphosphate; = phosphate. 2.

1	2. Fate of glycerol: The glycerol released during TAG degradation cannot be metabolized by adipocytes because they lack glycerol kinase. Rather, glycerol is transported through the blood to the liver, which has the kinase. The resulting glycerol 3-phosphate can be used to form TAG in the liver or can be converted to DHAP by reversal of the glycerol 3phosphate dehydrogenase reaction illustrated in Figure 16.13. DHAP can participate in glycolysis or gluconeogenesis. 3.

1	Fate of fatty acids: The FFA move through the cell membrane of the adipocyte and bind to serum albumin. They are transported to tissues such as muscle, enter cells, get activated to their CoA derivatives, and are oxidized for energy in mitochondria. Regardless of their levels, plasma FFA cannot be used for fuel by red blood cells (RBC), which have no mitochondria. The brain does not use fatty acids for energy to any appreciable extent, but the reasons are less clear. [Note: Over 50% of the fatty acids released from adipose TAG are reesterified to glycerol 3phosphate. WAT does not express glycerol kinase, and the glycerol 3phosphate is produced by glyceroneogenesis, an incomplete version of gluconeogenesis: pyruvate to OAA via pyruvate carboxylase and OAA to phosphoenolpyruvate (PEP) via phosphoenolpyruvate carboxykinase. The PEP is converted (by reactions common to glycolysis and gluconeogenesis) to DHAP, which is reduced to glycerol 3-phosphate. The process decreases plasma FFA,

1	phosphoenolpyruvate carboxykinase. The PEP is converted (by reactions common to glycolysis and gluconeogenesis) to DHAP, which is reduced to glycerol 3-phosphate. The process decreases plasma FFA, molecules associated with insulin resistance in type 2 diabetes and obesity (see p. 343).]

1	B. Fatty acid β-oxidation The major pathway for catabolism of fatty acids is a mitochondrial pathway called β-oxidation, in which two-carbon fragments are successively removed from the carboxyl end of the fatty acyl CoA, producing acetyl CoA, NADH, and FADH2. 1. Long-chain fatty acid transport into mitochondria: After a LCFA enters a cell, it is converted in the cytosol to its CoA derivative by long-chain fatty acyl CoA synthetase (thiokinase), an enzyme of the outer mitochondrial membrane. Because β-oxidation occurs in the mitochondrial matrix, the fatty acid must be transported across the inner mitochondrial membrane that is impermeable to CoA. Therefore, a specialized carrier transports the long-chain acyl group from the cytosol into the mitochondrial matrix. This carrier is carnitine, and this rate-limiting transport process is called the carnitine shuttle (Fig. 16.16). a.

1	a. Translocation steps: First, the acyl group is transferred from CoA to carnitine by carnitine palmitoyltransferase I (CPT-I), an enzyme of the outer mitochondrial membrane. [Note: CPT-I is also known as CAT-I for carnitine acyltransferase I.] This reaction forms an acylcarnitine and regenerates free CoA. Second, the acylcarnitine is transported into the mitochondrial matrix in exchange for free carnitine by carnitine–acylcarnitine translocase. Carnitine palmitoyltransferase 2 (CPT-II, or CAT-II), an enzyme of the inner mitochondrial membrane, catalyzes the transfer of the acyl group from carnitine to CoA in the mitochondrial matrix, thus regenerating free carnitine. b.

1	b. Carnitine shuttle inhibitor: Malonyl CoA inhibits CPT-I, thus preventing the entry of long-chain acyl groups into the mitochondrial matrix. Therefore, when fatty acid synthesis is occurring in the cytosol (as indicated by the presence of malonyl CoA), the newly made palmitate cannot be transferred into mitochondria and degraded. [Note: Muscle tissue, although it does not synthesize fatty acids, contains the mitochondrial isozyme of ACC (ACC2), allowing regulation of βoxidation. The liver contains both isozymes.] Fatty acid oxidation is also regulated by the acetyl CoA/CoA ratio: As the ratio increases, the CoA-requiring thiolase reaction decreases (Fig. 16.17). nicotinamide adenine dinucleotide. c.

1	nicotinamide adenine dinucleotide. c. Carnitine sources: Carnitine can be obtained from the diet, where it is found primarily in meat products. It can also be synthesized from the amino acids lysine and methionine by an enzymatic pathway found in the liver and kidneys but not in skeletal or cardiac muscle. Therefore, these latter tissues are totally dependent on uptake of carnitine provided by endogenous synthesis or the diet and distributed by the blood. [Note: Skeletal muscle contains ~97% of all carnitine in the body.] d.

1	Carnitine deficiencies: Such deficiencies result in decreased ability of tissues to use LCFA as a fuel. Primary carnitine deficiency is caused by defects in a membrane transporter that prevent uptake of carnitine by cardiac and skeletal muscle and the kidneys, causing carnitine to be excreted. Treatment includes carnitine supplementation. Secondary carnitine deficiency occurs primarily as a result of defects in fatty acid oxidation leading to the accumulation of acylcarnitines that are excreted in the urine, decreasing carnitine availability. Acquired secondary carnitine deficiency can be seen, for example, in patients with liver disease (decreased carnitine synthesis) or those taking the antiseizure drug valproic acid (decreased renal reabsorption). [Note: Defects in mitochondrial oxidation can also be caused by deficiencies in CPT-I and CPT-II. CPT-I deficiency affects the liver, where an inability to use LCFA for fuel greatly impairs that tissue’s ability to synthesize glucose (an

1	can also be caused by deficiencies in CPT-I and CPT-II. CPT-I deficiency affects the liver, where an inability to use LCFA for fuel greatly impairs that tissue’s ability to synthesize glucose (an endergonic process) during a fast. This can lead to severe hypoglycemia, coma, and death. CPT-II deficiency can affect the liver and cardiac and skeletal muscle. The most common (and least severe) form affects skeletal muscle. It presents as muscle weakness with myoglobinemia following prolonged exercise. Treatment includes avoidance of fasting and adopting a diet high in carbohydrates and low in fat but supplemented with medium-chain TAG.] 2.

1	Shorter-chain fatty acid entry into mitochondria: Fatty acids ≤12 carbons can cross the inner mitochondrial membrane without the aid of carnitine or the CPT system. Once inside the mitochondria, they are activated to their CoA derivatives by matrix enzymes and are oxidized. [Note: Medium-chain fatty acids are plentiful in human milk. Because their oxidation is not dependent on CPT-I, malonyl CoA is not inhibitory.] 3.

1	β-Oxidation reactions: The first cycle of β-oxidation is shown in Figure 16.17. It consists of a sequence of four reactions involving the β-carbon (carbon 3) that results in shortening the fatty acid by two carbons at the carboxylate end. The steps include an oxidation that produces FADH2, a hydration, a second oxidation that produces NADH, and a CoAdependent thiolytic cleavage that releases a molecule of acetyl CoA. Each step is catalyzed by enzymes with chain-length specificity. [Note: For LCFA, the last three steps are catalyzed by a trifunctional protein.] These four steps are repeated for saturated fatty acids of even-numbered carbon chains (n/2) − 1 times (where n is the number of carbons), each cycle producing one acetyl CoA plus one NADH and one FADH2. The final cycle produces two acetyl CoA. The acetyl CoA can be oxidized or used in hepatic ketogenesis (see V. below). The reduced coenzymes are oxidized by the electron transport chain, NADH by Complex I, and FADH2 by coenzyme

1	acetyl CoA. The acetyl CoA can be oxidized or used in hepatic ketogenesis (see V. below). The reduced coenzymes are oxidized by the electron transport chain, NADH by Complex I, and FADH2 by coenzyme Q (see p. 75). [Note: Acetyl CoA is a positive allosteric effector of pyruvate carboxylase (see p. 119), thus linking fatty acid oxidation and gluconeogenesis.] 4. β-Oxidation energy yield: The energy yield from fatty acid β-oxidation is high. For example, the oxidation of a molecule of palmitoyl CoA to CO2 and H2O produces 8 acetyl CoA, 7 NADH, and 7 FADH2, from which 131 ATP can be generated. However, activation of the fatty acid requires two ATP. Therefore, the net yield from palmitate is 129 ATP (Fig. 16.18). A comparison of the processes of synthesis and degradation of long-chain saturated fatty acids with an even number of carbon atoms is provided in Figure 16.19.

1	adenine dinucleotide; NADH = nicotinamide adenine dinucleotide; TCA = tricarboxylic acid; CoQ = coenzyme Q; CO2 = carbon dioxide. 5. Medium-chain fatty acyl CoA dehydrogenase deficiency: In mitochondria, there are four fatty acyl CoA dehydrogenase species, each with distinct but overlapping specificity for either short-, medium-, long-, or very-long-chain fatty acids. Medium-chain fatty acyl CoA dehydrogenase (MCAD) deficiency, an autosomal-recessive disorder, is the most common inborn error of β-oxidation, being found in 1:14,000 births worldwide, with a higher incidence in Caucasians of Northern European descent. It results in decreased ability to oxidize fatty acids with six to ten carbons (which accumulate and can be measured in urine), severe hypoglycemia (because the tissues must increase their reliance on glucose), and hypoketonemia (because of decreased production of acetyl CoA; see p. 195). Treatment includes avoidance of fasting. 6.

1	6. Oxidation of fatty acids with an odd number of carbons: This process proceeds by the same reaction steps as that of fatty acids with an even number of carbons, until the final three carbons are reached. This product, propionyl CoA, is metabolized by a three-step pathway (Fig. 16.20). [Note: Propionyl CoA is also produced during the metabolism of certain amino acids (see Fig. 20.11, p. 266).] a. d-Methylmalonyl CoA synthesis: First, propionyl CoA is carboxylated, forming D-methylmalonyl CoA. The enzyme propionyl CoA carboxylase has an absolute requirement for the coenzymes biotin and ATP, as do ACC and most other carboxylases. b. l-Methylmalonyl CoA formation: Next, the D-isomer is converted to the L-form by the enzyme methylmalonyl CoA racemase. c.

1	c. Succinyl CoA synthesis: Finally, the carbons of L-methylmalonyl CoA are rearranged, forming succinyl CoA, which can enter the TCA cycle (see p. 113). [Note: This is the only example of a glucogenic precursor generated from fatty acid oxidation.] The enzyme methylmalonyl CoA mutase requires a coenzyme form of vitamin B12 (deoxyadenosylcobalamin). The mutase reaction is one of only two reactions in the body that require vitamin B12 (see p. 379). [Note: In patients with vitamin B12 deficiency, both propionate and methylmalonate are excreted in the urine. Two types of heritable methylmalonic acidemia and aciduria have been described: one in which the mutase is missing or deficient (or has reduced affinity for the coenzyme) and one in which the patient is unable to convert vitamin B12 into its coenzyme form. Either type results in metabolic acidosis and neurologic manifestations.] 7.

1	Unsaturated fatty acid β-oxidation: The oxidation of unsaturated fatty acids generates intermediates that cannot serve as substrates for 2,3-enoyl CoA hydratase (see Fig. 16.17). Consequently, additional enzymes are required. Oxidation of a double bond at an odd-numbered carbon, such as 18:1(9) (oleic acid), requires one additional enzyme, 3,2-enoyl CoA isomerase, which converts the 3-cis derivative obtained after three rounds of β-oxidation to the 2-trans derivative required by the hydratase. Oxidation of a double bond at an even-numbered carbon, such as 18:2(9,12) (linoleic acid), requires an NADPH-dependent 2,4-dienoyl CoA reductase in addition to the isomerase. [Note: Because unsaturated fatty acids are less reduced than saturated fatty acids, fewer reducing equivalents are produced by their oxidation.] 8.

1	Peroxisomal β-oxidation: VLCFA ≥22 carbons in length undergo a preliminary β-oxidation in peroxisomes, because peroxisomes and not mitochondria are the primary site of the synthetase that activates fatty acids of this length. The shortened fatty acid (linked to carnitine) diffuses to a mitochondrion for further oxidation. In contrast to mitochondrial βoxidation, the initial dehydrogenation in peroxisomes is catalyzed by a FAD-containing acyl CoA oxidase. The FADH2 produced is oxidized by O2, which is reduced to hydrogen peroxide (H2O2). Therefore, no ATP is generated from this step. The H2O2 is reduced to H2O by catalase (see p. 148). [Note: Genetic defects in the ability either to target matrix proteins to peroxisomes (resulting in Zellweger syndrome, a peroxisomal biogenesis disorder) or to transport VLCFA across the peroxisomal membrane (resulting in X-linked adrenoleukodystrophy) lead to accumulation of VLCFA in the blood and tissues.] C. Peroxisomal α-oxidation

1	C. Peroxisomal α-oxidation Branched-chain phytanic acid, a product of chlorophyll metabolism, is not a substrate for acyl CoA dehydrogenase because of the methyl group on its βcarbon (Fig. 16.21). Instead, it is hydroxylated at the α-carbon by phytanoyl CoA α-hydroxylase (PhyH); carbon 1 is released as CO2; and the product, 15-carbon-long pristanal, is oxidized to pristanic acid, which is activated to its CoA derivative and undergoes β-oxidation. Refsum disease is a rare, autosomal-recessive disorder caused by a deficiency of peroxisomal PhyH. This results in the accumulation of phytanic acid in the plasma and tissues. The symptoms are primarily neurologic, and the treatment involves dietary restriction to halt disease progression. [Note: ω-Oxidation (at the methyl terminus) also is known and generates dicarboxylic acids. Normally a minor pathway of the SER, its upregulation is seen with conditions such as MCAD deficiency that limit fatty acid β-oxidation.]

1	V. KETONE BODIES: ALTERNATIVE FUEL FOR CELLS

1	Liver mitochondria have the capacity to convert acetyl CoA derived from fatty acid oxidation into ketone bodies. The compounds categorized as ketone bodies are acetoacetate, 3-hydroxybutyrate (also called β-hydroxybutyrate), and acetone (a nonmetabolized side product, Fig. 16.22). [Note: The two functional ketone bodies are organic acids.] Acetoacetate and 3-hydroxybutyrate are transported in the blood to the peripheral tissues. There they can be reconverted to acetyl CoA, which can be oxidized by the TCA cycle. Ketone bodies are important sources of energy for the peripheral tissues because they 1) are soluble in aqueous solution and, therefore, do not need to be incorporated into lipoproteins or carried by albumin as do the other lipids; 2) are produced in the liver during periods when the amount of acetyl CoA present exceeds the oxidative capacity of the liver; and 3) are used in proportion to their concentration in the blood by extrahepatic tissues, such as skeletal and cardiac

1	the amount of acetyl CoA present exceeds the oxidative capacity of the liver; and 3) are used in proportion to their concentration in the blood by extrahepatic tissues, such as skeletal and cardiac muscle, the intestinal mucosa, and the renal cortex. Even the brain can use ketone bodies to help meet its energy needs if the blood levels rise sufficiently. Thus, ketone bodies spare glucose, which is particularly important during prolonged periods of fasting (see p. 332). [Note: Disorders of fatty acid oxidation present with the general picture of hypoketosis (because of decreased availability of acetyl CoA) and hypoglycemia (because of increased reliance on glucose for energy).] carbon dioxide.

1	A. Ketone body synthesis by the liver: Ketogenesis During a fast, the liver is flooded with fatty acids mobilized from adipose tissue. The resulting elevated hepatic acetyl CoA produced by fatty acid oxidation inhibits pyruvate dehydrogenase (see p. 111) and activates pyruvate carboxylase ([PC] see p. 119). The OAA produced by PC is used by the liver for gluconeogenesis rather than for the TCA cycle. Additionally, fatty acid oxidation decreases the NAD+/NADH ratio, and the rise in NADH shifts OAA to malate (see p. 113). The decreased availability of OAA for condensation with acetyl CoA results in the increased use of acetyl CoA for ketone body synthesis. [Note: Acetyl CoA for ketogenesis is also generated by the catabolism of ketogenic amino acids (see p. 262).] 1.

1	3-Hydroxy-3-methylglutaryl CoA synthesis: The first step, formation of acetoacetyl CoA, occurs by reversal of the final thiolase reaction of fatty acid oxidation (see Fig. 16.17). Mitochondrial 3-hydroxy-3methylglutaryl (HMG) CoA synthase combines a third molecule of acetyl CoA with acetoacetyl CoA to produce HMG CoA. HMG CoA synthase is the rate-limiting step in the synthesis of ketone bodies and is present in significant quantities only in the liver. [Note: HMG CoA is also an intermediate in cytosolic cholesterol synthesis (see p. 220). The two pathways are separated by location in, and conditions of, the cell.] 2.

1	Ketone body synthesis: HMG CoA is cleaved by HMG CoA lyase to produce acetoacetate and acetyl CoA, as shown in Figure 16.22. Acetoacetate can be reduced to form 3-hydroxybutyrate with NADH as the electron donor. [Note: Because ketone bodies are not linked to CoA, they can cross the inner mitochondrial membrane.] Acetoacetate can also spontaneously decarboxylate in the blood to form acetone, a volatile, biologically nonmetabolized compound that can be detected in the breath. The equilibrium between acetoacetate and 3-hydroxybutyrate is determined by the NAD+/NADH ratio. Because this ratio is low during fatty acid oxidation, 3-hydroxybutyrate synthesis is favored. B. Ketone body use by the peripheral tissues: Ketolysis

1	B. Ketone body use by the peripheral tissues: Ketolysis Although the liver constantly synthesizes low levels of ketone bodies, their production increases during fasting when ketone bodies are needed to provide energy to the peripheral tissues. 3-Hydroxybutyrate is oxidized to acetoacetate by 3-hydroxybutyrate dehydrogenase, producing NADH (Fig. 16.23). Acetoacetate is then provided with a CoA molecule taken from succinyl CoA by succinyl CoA:acetoacetate CoA transferase (thiophorase). This reaction is reversible, but the product, acetoacetyl CoA, is actively removed by its cleavage to two acetyl CoA by thiolase. This pulls the reaction forward. Extrahepatic tissues, including the brain but excluding cells lacking mitochondria (for example, RBC), efficiently oxidize acetoacetate and 3-hydroxybutyrate in this manner. In contrast, although the liver actively produces ketone bodies, it lacks thiophorase and, therefore, is unable to use ketone bodies as fuel.

1	C. Excessive ketone body production in diabetes mellitus When the rate of formation of ketone bodies is greater than the rate of their use, their levels begin to rise in the blood (ketonemia) and, eventually, in the urine (ketonuria). This is seen most often in cases of uncontrolled type 1 diabetes mellitus (T1D), where the blood concentration of ketone bodies may reach 90 mg/dl (versus <3 mg/dl in normal individuals), and urinary excretion of ketone bodies may be as high as 5,000 mg/24 hour. The elevation of the ketone body concentration in the blood can result in acidemia. [Note: The carboxyl group of a ketone body has a pKa of ~4.

1	Therefore, each ketone body loses a proton (H+) as it circulates in the blood, which lowers the pH.] Also, in uncontrolled T1D, urinary loss of glucose and ketone bodies results in dehydration. Therefore, the increased number of H+ circulating in a decreased volume of plasma can cause a severe acidosis (ketoacidosis, Fig. 16.24) known as diabetic ketoacidosis (DKA).] A frequent symptom of DKA is a fruity odor on the breath, which results from increased production of acetone. Ketoacidosis may also be seen in cases of prolonged fasting (see p. 330) and excessive ethanol consumption (see p. 318). VI. CHAPTER SUMMARY

1	A fatty acid, generally a linear hydrocarbon chain with a terminal carboxyl group, can be saturated or unsaturated. Two unsaturated fatty acids are dietary essentials: linoleic and α-linolenic acids. Fatty acids are synthesized in the liver cytosol following a meal containing excess carbohydrate and protein. Carbons used to synthesize fatty acids are provided by acetyl coenzyme A (CoA), energy by ATP, and reducing equivalents by nicotinamide adenine dinucleotide phosphate ([NADPH], Fig. 16.25) provided by the pentose phosphate pathway and malic enzyme. Citrate carries two-carbon acetyl units from the mitochondrial matrix to the cytosol. The regulated step in fatty acid synthesis is the carboxylation of acetyl CoA to malonyl CoA by biotin-and ATP-requiring acetyl CoA carboxylase (ACC). Citrate allosterically activates ACC, and palmitoyl CoA inhibits it. ACC can also be activated by insulin and inactivated by adenosine monophosphate–activated protein kinase (AMPK) in response to

1	Citrate allosterically activates ACC, and palmitoyl CoA inhibits it. ACC can also be activated by insulin and inactivated by adenosine monophosphate–activated protein kinase (AMPK) in response to epinephrine, glucagon, or a rise in AMP. The remaining steps in fatty acid synthesis are catalyzed by the multifunctional enzyme, fatty acid synthase, which produces palmitoyl CoA by adding two-carbon units from malonyl CoA to a series of acyl acceptors. Fatty acids can be elongated and desaturated in the smooth endoplasmic reticulum (SER). When fatty acids are required for energy, hormone-sensitive lipase (activated by epinephrine, and inhibited by insulin), along with other lipases, degrades triacylglycerol (TAG) stored in adipocytes. The fatty acid products are carried by serum albumin to the liver and peripheral tissues, where their oxidation provides energy. The glycerol backbone of the degraded TAG is carried by the blood to the liver, where it serves as a gluconeogenic precursor. Fatty

1	and peripheral tissues, where their oxidation provides energy. The glycerol backbone of the degraded TAG is carried by the blood to the liver, where it serves as a gluconeogenic precursor. Fatty acid degradation (β-oxidation) occurs in mitochondria. The carnitine shuttle is required to transport long-chain fatty acids from the cytosol to the mitochondrial matrix. A translocase and the enzymes carnitine palmitoyltransferases (CPT) I and II are required. CPT-I is inhibited by malonyl CoA, thereby preventing simultaneous synthesis and degradation of fatty acids. Mitochondrial fatty acid β-oxidation produces acetyl CoA, nicotinamide adenine dinucleotide (NADH), and flavin adenine dinucleotide (FADH2). The first step in β-oxidation is catalyzed by one of four acyl CoA dehydrogenases, each with chain-length specificity. Medium-chain fatty acyl CoA dehydrogenase (MCAD) deficiency causes a decrease in fatty acid oxidation (process stops once a medium-chain fatty acid is produced), resulting

1	chain-length specificity. Medium-chain fatty acyl CoA dehydrogenase (MCAD) deficiency causes a decrease in fatty acid oxidation (process stops once a medium-chain fatty acid is produced), resulting in hypoketonemia and severe hypoglycemia. Oxidation of fatty acids with an odd number of carbons proceeds two carbons at a time (producing acetyl CoA) until three-carbon propionyl CoA remains. This compound is carboxylated to methylmalonyl CoA (by biotin-and ATP-requiring propionyl CoA carboxylase), which is then converted to succinyl CoA (a gluconeogenic precursor) by vitamin B12-requiring methylmalonyl CoA mutase. A genetic error in the mutase or vitamin B12 deficiency causes methylmalonic acidemia and aciduria. β-Oxidation of unsaturated fatty acids requires additional enzymes. β-Oxidation of verylong-chain fatty acids and α-oxidation of branched-chain fatty acids occur in the peroxisome. Deficiencies result in X-linked adrenoleukodystrophy and Refsum disease, respectively. ω-Oxidation,

1	verylong-chain fatty acids and α-oxidation of branched-chain fatty acids occur in the peroxisome. Deficiencies result in X-linked adrenoleukodystrophy and Refsum disease, respectively. ω-Oxidation, normally a minor pathway, occurs in the SER. Liver mitochondria can convert acetyl CoA derived from fatty acid oxidation into acetoacetate and 3-hydroxybutyrate (ketone bodies). Peripheral tissues possessing mitochondria can oxidize 3hydroxybutyrate to acetoacetate, which can be cleaved to two acetyl CoA, thereby producing energy for the cell. Unlike fatty acids, ketone bodies are utilized by the brain and, therefore, are important fuels during a fast. Because the liver lacks thiophorase required to degrade ketone bodies, it synthesizes them specifically for the peripheral tissues. Ketoacidosis occurs when the rate of ketone body formation is greater than the rate of use, as is seen in cases of uncontrolled type 1 diabetes mellitus.

1	tricarboxylic acid; VLDL = very-low-density lipoprotein. Choose the ONE best answer. 6.1. When oleic acid, 18:1(9), is desaturated at carbon 6 and then elongated, what is the correct representation of the product? A. 19:2(7,9) B. 20:2 (ω-6) C. 20:2(6,9) D. 20:2(8,11) Correct answer = D. Fatty acids are elongated in the smooth endoplasmic reticulum by adding two carbons at a time to the carboxylate end (carbon 1) of the molecule. This pushes the double bonds at carbon 6 and carbon 9 farther away from carbon 1. The 20:2(8,11) product is an ω-9 (n-9) fatty acid. 6.2. A 4-month-old child is being evaluated for fasting hypoglycemia. Laboratory tests at admission reveal low levels of ketone bodies (hypoketonemia), free carnitine, and long-chain acylcarnitines in the blood. Free fatty acid levels in the blood were elevated. Deficiency of which of the following would best explain these findings? A. Adipose triglyceride lipase B. Carnitine transporter

1	A. Adipose triglyceride lipase B. Carnitine transporter C. Carnitine palmitoyltransferase-I D. Long-chain fatty acid dehydrogenase Correct answer = B. A defect in the carnitine transporter (primary carnitine deficiency) would result in low levels of carnitine in the blood (as a result of increased urinary loss) and low levels in the tissues. In the liver, this decreases fatty acid oxidation and ketogenesis. Consequently, blood levels of free fatty acids rise. Deficiencies of adipose triglyceride lipase would decrease fatty acid availability. Deficiency of carnitine palmitoyltransferase I would result in elevated blood carnitine. Defects in any of the enzymes of β-oxidation would result in secondary carnitine deficiency, with a rise in acylcarnitines.

1	6.3. A teenager, concerned about his weight, attempts to maintain a fat-free diet for a period of several weeks. If his ability to synthesize various lipids were examined, he would be found to be most deficient in his ability to synthesize: A. cholesterol. B. glycolipids. C. phospholipids. D. prostaglandins. E. triacylglycerol. Correct answer = D. Prostaglandins are synthesized from arachidonic acid. Arachidonic acid is synthesized from linoleic acid, an essential fatty acid obtained by humans from dietary lipids. The teenager would be able to synthesize all other compounds but, presumably, in somewhat decreased amounts.

1	6.4. A 6-month-old boy was hospitalized following a seizure. History revealed that for several days prior, his appetite was decreased owing to a stomach virus. At admission, his blood glucose was 24 mg/dl (age-referenced normal is 60–100). His urine was negative for ketone bodies and positive for a variety of dicarboxylic acids. Blood carnitine levels (free and acyl bound) were normal. A tentative diagnosis of medium-chain fatty acyl coenzyme A dehydrogenase (MCAD) deficiency is made. In patients with MCAD deficiency, the fasting hypoglycemia is a consequence of: A. decreased acetyl coenzyme A production. B. decreased ability to convert acetyl coenzyme A to glucose. C. increased conversion of acetyl coenzyme A to acetoacetate. D. increased production of ATP and nicotinamide adenine dinucleotide.

1	B. decreased ability to convert acetyl coenzyme A to glucose. C. increased conversion of acetyl coenzyme A to acetoacetate. D. increased production of ATP and nicotinamide adenine dinucleotide. Correct answer = A. Impaired oxidation of fatty acids <12 carbons in length results in decreased production of acetyl-coenzyme A (CoA), the allosteric activator of pyruvate carboxylase, a gluconeogenic enzyme, and, thus, glucose levels fall. Acetyl CoA can never be used for the net synthesis of glucose. Acetoacetate is a ketone body, and with medium-chain fatty acyl CoA dehydrogenase deficiency, ketogenesis is decreased as a result of decreased production of the substrate, acetyl CoA. Impaired fatty acid oxidation means that less ATP and nicotinamide adenine dinucleotide are made, and both are needed for gluconeogenesis.

1	6.5. Explain why with Zellweger syndrome both very-long-chain fatty acids (VLCFA) and long-chain phytanic acid accumulate, whereas with X-linked adrenoleukodystrophy, only VLCFA accumulate. Zellweger syndrome is caused by an inability to target matrix proteins to the peroxisome. Therefore, all peroxisomal activities are affected because functional peroxisomes are unable to be formed. In X-linked adrenoleukodystrophy, the defect is an inability to transport VLCFA into the peroxisome, but other peroxisomal functions, such as α-oxidation, are normal. Phospholipid, Glycosphingolipid, and Eicosanoid Metabolism 17 For additional ancillary materials related to this chapter, please visit thePoint. I. PHOSPHOLIPID OVERVIEW

1	Phospholipids are polar, ionic compounds composed of an alcohol that is attached by a phosphodiester bond to either diacylglycerol (DAG) or sphingosine. Like fatty acids (FA), phospholipids are amphipathic in nature. That is, each has a hydrophilic head, which is the phosphate group plus whatever alcohol is attached to it (for example, serine, ethanolamine, and choline; highlighted in blue in Fig. 17.1A), and a long, hydrophobic tail containing FA or FA-derived hydrocarbons (shown in orange in Fig. 17.1A). Phospholipids are the predominant lipids of cell membranes. In membranes, the hydrophobic portion of a phospholipid molecule is associated with the nonpolar portions of other membrane constituents, such as glycolipids, proteins, and cholesterol. The hydrophilic (polar) head of the phospholipid extends outward, interacting with the intracellular or extracellular aqueous environment (see Fig. 17.1A). Membrane phospholipids also function as a reservoir for intracellular messengers,

1	extends outward, interacting with the intracellular or extracellular aqueous environment (see Fig. 17.1A). Membrane phospholipids also function as a reservoir for intracellular messengers, and, for some proteins, phospholipids serve as anchors to cell membranes. Nonmembrane phospholipids serve additional functions in the body, for example, as components of lung surfactant and essential components of bile, where their detergent properties aid cholesterol solubilization.

1	II. PHOSPHOLIPID STRUCTURE There are two classes of phospholipids: those that have glycerol (from glucose) as a backbone and those that have sphingosine (from serine and palmitate). Both classes are found as structural components of membranes, and both play a role in the generation of lipid signaling molecules. A. Glycerophospholipids Phospholipids that contain glycerol are called glycerophospholipids (or phosphoglycerides). Glycerophospholipids constitute the major class of phospholipids and are the predominant lipids in membranes. All contain (or are derivatives of) phosphatidic acid (PA), which is DAG with a phosphate group on carbon 3 (Fig. 17.1B). PA is the simplest phosphoglyceride and is the precursor of the other members of this group. 1. From phosphatidic acid and an alcohol: The phosphate group on PA can be esterified to a compound containing an alcohol group (see Fig. 17.1). For example: 2.

1	1. From phosphatidic acid and an alcohol: The phosphate group on PA can be esterified to a compound containing an alcohol group (see Fig. 17.1). For example: 2. Cardiolipin: Two molecules of PA esterified through their phosphate groups to an additional molecule of glycerol form cardiolipin, or diphosphatidylglycerol (Fig. 17.2). Cardiolipin is found in membranes in bacteria and eukaryotes. In eukaryotes, cardiolipin is virtually exclusive to the inner mitochondrial membrane, where it maintains the structure and function of certain respiratory complexes of the electron transport chain. [Note: Cardiolipin is antigenic and is recognized by antibodies 3.

1	Plasmalogens: When the FA at carbon 1 of a glycerophospholipid is replaced by an unsaturated alkyl group attached by an ether (rather than by an ester) linkage to the core glycerol molecule, an ether phosphoglyceride known as a plasmalogen is produced. For example, phosphatidalethanolamine, which is abundant in nerve tissue (Fig. 17.3A), is the plasmalogen that is similar in structure to (Ab) raised against Treponema pallidum, the bacterium that causes syphilis. The Wasserman test for syphilis detects these Ab.] phosphatidylethanolamine. Phosphatidalcholine (abundant in heart muscle) is the other quantitatively significant ether lipid in mammals. [Note: Plasmalogens have “al” rather than “yl” in their names.] plasmalogen phosphatidalethanolamine. B. Platelet-activating factor. ( is a long, hydrophobic hydrocarbon chain.) 4. Platelet-activating factor: A second example of an ether glycerophospholipid is platelet-activating factor (PAF), which has a saturated alkyl group in an ether

1	hydrophobic hydrocarbon chain.) 4. Platelet-activating factor: A second example of an ether glycerophospholipid is platelet-activating factor (PAF), which has a saturated alkyl group in an ether link to carbon 1 and an acetyl residue (rather than a FA) at carbon 2 of the glycerol backbone (Fig. 17.3B). PAF is synthesized and released by a variety of cell types. It binds to surface receptors, triggering potent thrombotic and acute inflammatory events. For example, PAF activates inflammatory cells and mediates hypersensitivity, acute inflammatory, and anaphylactic reactions. It causes platelets to aggregate and activate and neutrophils and alveolar macrophages to generate superoxide radicals to kill bacteria (see p. 150). It also lowers blood pressure. [Note: PAF is one of the most potent bioactive molecules known, causing effects at concentrations as low as 10−11 mol/l.]

1	B. Sphingophospholipids: Sphingomyelin The backbone of sphingomyelin is the amino alcohol sphingosine, rather than glycerol (Fig. 17.4). A long-chain-length FA (LCFA) is attached to the amino group of sphingosine through an amide linkage, producing a ceramide, which can also serve as a precursor of glycolipids (see p. 209). The alcohol group at carbon 1 of sphingosine is esterified to phosphorylcholine, producing sphingomyelin, the only significant sphingophospholipid in humans. Sphingomyelin is an important constituent of the myelin sheath of nerve fibers. [Note: The myelin sheath is a layered, membranous structure that insulates and protects neuronal axons of the central nervous system (CNS).] III. PHOSPHOLIPID SYNTHESIS

1	Glycerophospholipid synthesis involves either the donation of PA from cytidine diphosphate (CDP)-DAG to an alcohol or the donation of the phosphomonoester of the alcohol from CDP-alcohol to DAG (Fig. 17.5). In both cases, the CDP-bound structure is considered an activated intermediate, and cytidine monophosphate (CMP) is released as a side product. Therefore, a key concept in glycerophospholipid synthesis is activation, of either DAG or the alcohol to be added, by linkage with CDP. [Note: This is similar in principle to the activation of sugars by their attachment to uridine diphosphate (UDP) (see p. 126).] The FA esterified to the glycerol alcohol groups can vary widely, contributing to the heterogeneity of this group of compounds, with saturated FA typically found at carbon 1 and unsaturated ones at carbon 2. Most phospholipids are synthesized in the smooth endoplasmic reticulum (SER). From there, they are transported to the Golgi and then to membranes of organelles or the plasma

1	ones at carbon 2. Most phospholipids are synthesized in the smooth endoplasmic reticulum (SER). From there, they are transported to the Golgi and then to membranes of organelles or the plasma membrane or are secreted from the cell by exocytosis. [Note: Ether lipid synthesis from dihydroxyacetone phosphate begins in peroxisomes.] pyrophosphate. ( is a fatty acid hydrocarbon chain.)

1	A. Phosphatidic acid PA is the precursor of other glycerophospholipids. The steps in its synthesis from glycerol 3-phosphate and two fatty acyl coenzyme A (CoA) molecules were illustrated in Figure 16.14, p. 189, in which PA is shown as a precursor of triacylglycerol (TAG). Essentially all cells except mature erythrocytes can synthesize phospholipids, whereas TAG synthesis occurs essentially only in the liver, adipose tissue, lactating mammary glands, and intestinal mucosal cells. B. Phosphatidylcholine and phosphatidylethanolamine

1	B. Phosphatidylcholine and phosphatidylethanolamine The neutral phospholipids PC and PE are the most abundant phospholipids in most eukaryotic cells. The primary route of their synthesis uses choline and ethanolamine obtained either from the diet or from the turnover of the body’s phospholipids. [Note: In the liver, PC also can be synthesized from PS and PE (see 2. below).] 1. Synthesis from preexisting choline and ethanolamine: These synthetic pathways involve the phosphorylation of choline or ethanolamine by kinases, followed by conversion to the activated form, CDP-choline or CDP-ethanolamine. Finally, choline phosphate or ethanolamine phosphate is transferred from the nucleotide (leaving CMP) to a molecule of DAG (see Fig. 17.5). a.

1	a. Significance of choline reutilization: The reutilization of choline is important because, although humans can synthesize choline de novo, the amount made is insufficient for our needs. Thus, choline is an essential dietary nutrient with an adequate intake (see p. 358) of 550 mg for men and 425 mg for women. [Note: Choline is also used for the synthesis of acetylcholine, a neurotransmitter.] b.

1	Phosphatidylcholine in lung surfactant: The pathway described above is the principal pathway for the synthesis of dipalmitoylphosphatidylcholine (DPPC or, dipalmitoyl lecithin). In DPPC, positions 1 and 2 on the glycerol are occupied by palmitate, a saturated LCFA. DPPC, made and secreted by type II pneumocytes, is a major lipid component of lung surfactant, which is the extracellular fluid layer lining the alveoli. Surfactant serves to decrease the surface tension of this fluid layer, reducing the pressure needed to reinflate alveoli, thereby preventing alveolar collapse (atelectasis). [Note: Surfactant is a complex mixture of lipids (90%) and proteins (10%), with DPPC being the major component for reducing surface tension.]

1	Fetal lung maturity can be gauged by determining the DPPC/sphingomyelin ratio, usually written as L (for lecithin)/S, in amniotic fluid. A value ≥2 is evidence of maturity, because it reflects the shift from sphingomyelin to DPPC synthesis that occurs in pneumocytes at ~32 weeks’ gestation.

1	c. Lung maturity: Respiratory distress syndrome (RDS) in preterm infants is associated with insufficient surfactant production and/or secretion and is a significant cause of all neonatal deaths in Western countries. Lung maturation can be accelerated by giving the mother glucocorticoids shortly before delivery to induce expression of specific genes. Postnatal administration of natural or synthetic surfactant (by intratracheal instillation) is also used. [Note: Acute RDS, seen in all age groups, is the result of alveolar damage (due to infection, injury, or aspiration) that causes fluid to accumulate in the alveoli, impeding the exchange of oxygen (O2) and carbon dioxide (CO2).] 2. Phosphatidylcholine synthesis from phosphatidylserine: The liver requires a mechanism for producing PC, even when free choline levels are low, because it exports significant amounts of PC in the bile and as a component of plasma lipoproteins. To provide the needed PC, PS is decarboxylated to PE by PS

1	even when free choline levels are low, because it exports significant amounts of PC in the bile and as a component of plasma lipoproteins. To provide the needed PC, PS is decarboxylated to PE by PS decarboxylase. PE then undergoes three methylation steps to produce PC, as illustrated in Figure 17.6. Sadenosylmethionine is the methyl group donor (see p. 264).

1	C. Phosphatidylserine PS synthesis in mammalian tissues is provided by the base exchange reaction, in which the ethanolamine of PE is exchanged for free serine (see Fig. 17.6). This reaction, although reversible, is used primarily to produce the PS required for membrane synthesis. PS has a net negative charge. (See online Chapter 35 for the role of PS in clotting.) D. Phosphatidylinositol

1	PI is synthesized from free inositol and CDP-DAG, as shown in Figure 17.5. PI is an unusual phospholipid in that it most frequently contains stearic acid on carbon 1 and arachidonic acid on carbon 2 of the glycerol. Therefore, PI serves as a reservoir of arachidonic acid in membranes and, thus, provides the substrate for prostaglandin (see p. 213) synthesis when required. Like PS, PI has a net negative charge. [Note: There is asymmetry in the phospholipid composition of the cell membrane. PS and PI, for example, are found primarily on the inner leaflet. Asymmetry is achieved by ATP-dependent enzymes known as “flippases” and “floppases.”] 1. Role in signal transduction across membranes: The phosphorylation of membrane-bound PI produces polyphosphoinositides such as phosphatidylinositol 4,5-bisphosphate ([PIP2]; Fig. 17.7). The cleavage of PIP2 by phospholipase C occurs in response to the binding of various neurotransmitters, hormones, and growth factors to G protein–coupled receptors

1	([PIP2]; Fig. 17.7). The cleavage of PIP2 by phospholipase C occurs in response to the binding of various neurotransmitters, hormones, and growth factors to G protein–coupled receptors (GPCR), such as the α1 adrenergic receptor, on the cell membrane and activation of the Gq α-subunit (Fig. 17.8). The products of this cleavage, inositol 1,4,5-trisphosphate (IP3) and DAG, mediate the mobilization of intracellular calcium and the activation of protein kinase

1	C, which act synergistically to evoke specific cellular responses. Signal transduction across the membrane is, thus, accomplished.

1	2. Role in membrane protein anchoring: Specific proteins can be covalently attached through a carbohydrate bridge to membrane-bound PI (Fig. 17.9). For example, lipoprotein lipase, an enzyme that degrades triacylglycerol in lipoprotein particles (see p. 228), is attached to capillary endothelial cells by a glycosyl phosphatidylinositol (GPI) anchor. [Note: GPI-linked proteins are also found in a variety of parasitic protozoans, such as trypanosomes and leishmania.] Being attached to a membrane lipid (rather than being an integral part of the membrane) allows GPI-anchored proteins increased lateral mobility on the extracellular surface of the plasma membrane. The protein can be cleaved from its anchor by the action of phospholipase C (see Fig. 17.9). [Note: A deficiency in the synthesis of GPI in hematopoietic cells results in the hemolytic disease paroxysmal nocturnal hemoglobinuria, because GPI-anchored proteins protect blood cells from complement-mediated lysis.]

1	E. Phosphatidylglycerol and cardiolipin Phosphatidylglycerol occurs in relatively large amounts in mitochondrial membranes and is a precursor of cardiolipin (diphosphatidyglycerol). It is synthesized from CDP-DAG and glycerol 3-phosphate. Cardiolipin (see Fig. 17.2) is synthesized by the transfer of DAG 3-phosphate from CDPDAG to a pre-existing molecule of phosphatidylglycerol. F. Sphingomyelin

1	F. Sphingomyelin Sphingomyelin, a sphingosine-based phospholipid, is found in cell membranes and in the myelin sheath. The synthesis of sphingomyelin is shown in Figure 17.10. Briefly, palmitoyl CoA condenses with serine, as CoA and the carboxyl group (as CO2) of serine are lost. [Note: This reaction, like the decarboxylation reactions involved in the synthesis of PE from PS and of regulators from amino acids (for example, the catecholamines from tyrosine; see p. 286), requires pyridoxal phosphate (a derivative of vitamin B6) as a coenzyme.] The product is reduced in a nicotinamide adenine dinucleotide phosphate (NADPH)-requiring reaction to sphinganine (dihydrosphingosine). The sphinganine is acylated at the amino group with one of a variety of LCFA and then desaturated to produce a ceramide, the immediate precursor of sphingomyelin (and other sphingolipids, as described on p. 208).

1	Ceramides play a key role in maintaining the skin’s water-permeability barrier. Decreased ceramide levels are associated with a number of skin diseases. Phosphorylcholine from PC is transferred to the ceramide, producing sphingomyelin and DAG. [Note: Sphingomyelin of the myelin sheath contains predominantly longer-chain FA such as lignoceric acid and nervonic acid, whereas gray matter of the brain has sphingomyelin that contains primarily stearic acid.] IV. PHOSPHOLIPID DEGRADATION The degradation of phosphoglycerides is performed by phospholipases found in all tissues and pancreatic juice. [Note: For a discussion of phospholipid digestion, see p. 175.] A number of toxins and venoms have phospholipase activity, and several pathogenic bacteria produce phospholipases that dissolve cell membranes and allow the spread of infection. Sphingomyelin is degraded by the lysosomal phospholipase, sphingomyelinase (see B. below). A. Phosphoglycerides

1	Phospholipases hydrolyze the phosphodiester bonds of phosphoglycerides, with each enzyme cleaving the phospholipid at a specific site. The major phospholipases are shown in Figure 17.11. [Note: Removal of the FA from carbon 1 or 2 of a phosphoglyceride produces a lysophosphoglyceride, which is the substrate for lysophospholipases.] Phospholipases release molecules that can serve as second messengers (for example, DAG and IP3) or that are the substrates for synthesis of messengers (for example, arachidonic acid). Phospholipases are responsible not only for degrading phospholipids but also for remodeling them. For example, phospholipases A1 and A2 remove specific FA from membrane-bound phospholipids, which can be replaced with different FA using fatty acyl CoA transferase. This mechanism is used as one way to create the unique lung surfactant DPCC (see p. 204) and to insure that carbon 2 of PI (and sometimes of PC) is bound to arachidonic acid. [Note: Barth syndrome, a rare X-linked

1	is used as one way to create the unique lung surfactant DPCC (see p. 204) and to insure that carbon 2 of PI (and sometimes of PC) is bound to arachidonic acid. [Note: Barth syndrome, a rare X-linked disorder characterized by cardiomyopathy, muscle weakness, and neutropenia, is the result of defects in cardiolipin remodeling.] phosphatidylinositol 4,5-bisphosphate; R1 and R2 = fatty acids; X = an alcohol.

1	B. Sphingomyelin

1	Sphingomyelin is degraded by sphingomyelinase, a lysosomal enzyme that removes phosphorylcholine, leaving a ceramide. The ceramide is, in turn, cleaved by ceramidase into sphingosine and a free FA (Fig. 17.12). [Note: The released ceramide and sphingosine regulate signal transduction pathways, in part by influencing the activity of protein kinase C and, thus, the phosphorylation of its protein substrates. They also promote apoptosis.] Niemann-Pick disease (types A and B) is an autosomal-recessive disorder caused by the inability to degrade sphingomyelin due to a deficiency of sphingomyelinase, a type of phospholipase C. In the severe infantile form (type A, which shows <1% of normal enzymic activity), the liver and spleen are the primary sites of lipid deposits and are, therefore, greatly enlarged. The lipid consists primarily of the sphingomyelin that cannot be degraded (Fig. 17.13). Infants with this lysosomal storage disease experience rapid and progressive neurodegeneration as a

1	enlarged. The lipid consists primarily of the sphingomyelin that cannot be degraded (Fig. 17.13). Infants with this lysosomal storage disease experience rapid and progressive neurodegeneration as a result of deposition of sphingomyelin in the CNS, and they die in early childhood. A less severe variant (type B, which shows up to 10% of normal activity) with a later age of onset and a longer survival time causes little to no damage to neural tissue, but lungs, spleen, liver, and bone marrow are affected, resulting in a chronic form of the disease. Although Niemann-Pick disease occurs in all ethnic groups, type A occurs with greater frequency in the Ashkenazi Jewish population.

1	type A.] V. GLYCOLIPID OVERVIEW Glycolipids are molecules that contain both carbohydrate and lipid components. Like the phospholipid sphingomyelin, glycolipids are derivatives of ceramides in which a LCFA is attached to the amino alcohol sphingosine. Therefore, they are more precisely called glycosphingolipids. [Note: Thus, ceramides are the precursors of both phosphorylated and glycosylated sphingolipids.] Like the phospholipids, glycosphingolipids are essential components of all membranes in the body, but they are found in greatest amounts in nerve tissue. They are located in the outer leaflet of the plasma membrane, where they interact with the extracellular environment. As such, they play a role in the regulation of cellular interactions (for example, adhesion and recognition), growth, and development.

1	Membrane glycosphingolipids associate with cholesterol and GPI-anchored proteins to form lipid rafts, laterally mobile microdomains of the plasma membrane that function to organize and regulate membrane signaling and trafficking functions. Glycosphingolipids are antigenic and are the source of ABO blood group antigens (see p. 165), various embryonic antigens specific for particular stages of fetal development, and some tumor antigens. [Note: The carbohydrate portion of a glycolipid is the antigenic determinant.] They have been coopted for use as cell surface receptors for cholera and tetanus toxins as well as for certain viruses and microbes. Genetic disorders associated with an inability to properly degrade the glycosphingolipids result in lysosomal accumulation of these compounds. [Note: Changes in the carbohydrate portion of glycosphingolipids (and glycoproteins) are characteristic of transformed cells (cells with dysregulated growth).] VI. GLYCOSPHINGOLIPID STRUCTURE

1	VI. GLYCOSPHINGOLIPID STRUCTURE The glycosphingolipids differ from sphingomyelin in that they do not contain phosphate, and the polar head function is provided by a monosaccharide or oligosaccharide attached directly to the ceramide by an O-glycosidic bond (Fig. 17.14). The number and type of carbohydrate moieties present determine the type of glycosphingolipid. A. Neutral glycosphingolipids

1	The simplest neutral glycosphingolipids are the cerebrosides. These are ceramide monosaccharides that contain either a molecule of galactose (forming ceramide-galactose or galactocerebroside, the most common cerebroside found in myelin, as shown in Fig. 17.14) or glucose (forming ceramide-glucose or glucocerebroside, an intermediate in the synthesis and degradation of the more complex glycosphingolipids). [Note: Members of a group of galacto-or glucocerebrosides may also differ from each other in the type of FA attached to the sphingosine.] As their name implies, cerebrosides are found predominantly in the brain and peripheral nerves, with high concentrations in the myelin sheath. Ceramide oligosaccharides (or globosides) are produced by attaching additional monosaccharides to a glucocerebroside, for example, ceramide-glucose-galactose (also known as lactosylceramide). The additional monosaccharides can include substituted sugars such as N-acetylgalactosamine.

1	B. Acidic glycosphingolipids Acidic glycosphingolipids are negatively charged at physiologic pH. The negative charge is provided by N-acetylneuraminic acid ([NANA], a sialic acid, as shown in Fig. 17.15) in gangliosides or by sulfate groups in sulfatides. 1.

1	1. Gangliosides: These are the most complex glycosphingolipids and are found primarily in the ganglion cells of the CNS, particularly at the nerve endings. They are derivatives of ceramide oligosaccharides and contain one or more molecules of NANA (from CMP-NANA). The notation for these compounds is G (for ganglioside) plus a subscript M, D, T, or Q to indicate whether there is one (mono), two (di), three (tri), or four (quatro) molecules of NANA in the ganglioside, respectively. Additional numbers and letters in the subscript designate the monomeric sequence of the carbohydrate attached to the ceramide. (See Fig. 17.15 for the structure of GM2.) Gangliosides are of medical interest because several lipid storage disorders involve the accumulation of NANA-containing glycosphingolipids in cells (see Fig. 17.20, p. 212). 2.

1	2. Sulfatides: These sulfoglycosphingolipids are sulfated galactocerebrosides that are negatively charged at physiologic pH. Sulfatides are found predominantly in the brain and kidneys. VII. GLYCOSPHINGOLIPID SYNTHESIS AND DEGRADATION Synthesis of glycosphingolipids occurs primarily in the Golgi by sequential addition of glycosyl monomers transferred from UDP-sugar donors to the acceptor molecule. The mechanism is similar to that used in glycoprotein synthesis (see p. 166). A. Enzymes involved in synthesis The enzymes involved in the synthesis of glycosphingolipids are glycosyltransferases that are specific for the type and location of the glycosidic bond formed. [Note: These enzymes can recognize both glycosphingolipids and glycoproteins as substrates.] B. Sulfate group addition

1	B. Sulfate group addition A sulfate group from the sulfate carrier 3ʹ-phosphoadenosine-5ʹphosphosulfate ([PAPS], Fig. 17.16) is added by a sulfotransferase to the 3ʹ-hydroxyl group of the galactose in a galactocerebroside, forming the sulfatide galactocerebroside 3-sulfate (Fig. 17.17). [Note: PAPS is also the sulfur donor in glycosaminoglycan synthesis (see p. 162) and steroid hormone catabolism (see p. 240).] An overview of the synthesis of sphingolipids is shown in Figure 17.18. C. Glycosphingolipid degradation

1	C. Glycosphingolipid degradation Glycosphingolipids are internalized by phagocytosis as described for the glycosaminoglycans (see p. 163). All of the enzymes required for the degradative process are present in lysosomes, which fuse with the phagosomes. The lysosomal enzymes hydrolytically and irreversibly cleave specific bonds in the glycosphingolipid. As seen with the glycosaminoglycans and glycoproteins (see p. 170), degradation is a sequential process following the rule “last on, first off,” in which the last group added during synthesis is the first group removed in degradation. Therefore, defects in the degradation of the polysaccharide chains in these three glycoconjugates result in lysosomal storage diseases. D. Sphingolipidoses

1	D. Sphingolipidoses In a normal individual, synthesis and degradation of glycosphingolipids are balanced, so that the amount of these compounds present in membranes is constant. If a specific lysosomal acid hydrolase required for degradation is partially or totally missing, a sphingolipid accumulates. Lysosomal lipid storage diseases caused by these deficiencies are called sphingolipidoses. The result of a specific acid hydrolase deficiency may be seen dramatically in nerve tissue, where neurologic deterioration can lead to early death. Figure 17.20 provides an outline of the pathway of sphingolipid degradation and descriptions of some sphingolipidoses. [Note: Some sphingolipidoses can also result from defects in lysosomal activator proteins (for example, the saposins) that facilitate access of the hydrolases to short carbohydrate chains as degradation proceeds.] 1.

1	Common properties: A specific lysosomal hydrolytic enzyme is deficient in the classic form of each disorder. Therefore, usually, only a single sphingolipid (the substrate for the deficient enzyme) accumulates in the involved organs in each disease. [Note: The rate of biosynthesis of the accumulating lipid is normal.] The disorders are progressive and, although many are fatal in childhood, extensive phenotypic variability is seen leading to the designation of different clinical types, such as types A and B in Niemann-Pick disease. Genetic variability is also seen because a given disorder can be caused by any one of a variety of mutations within a single gene. The sphingolipidoses are autosomal-recessive disorders, except for Fabry disease, which is X linked. The incidence of the sphingolipidoses is low in most populations, except for Gaucher and Tay-Sachs diseases, which, like Niemann-Pick disease, show a high frequency in the Ashkenazi Jewish population. [Note: Tay-Sachs also has a

1	is low in most populations, except for Gaucher and Tay-Sachs diseases, which, like Niemann-Pick disease, show a high frequency in the Ashkenazi Jewish population. [Note: Tay-Sachs also has a high frequency in Irish American, French Canadian, and Louisiana Cajun populations.] 2.

1	Diagnosis and treatment: A specific sphingolipidosis can be diagnosed by measuring enzyme activity in cultured fibroblasts or peripheral leukocytes or by analyzing DNA (see Chapter 34). Histologic examination of the affected tissue is also useful. [Note: Shell-like inclusion bodies are seen in Tay-Sachs, and a crumpled tissue paper appearance of the cytosol is seen in Gaucher disease (Fig. 17.19).] Prenatal diagnosis, using cultured amniocytes or chorionic villi, is available. Gaucher disease, in which macrophages become engorged with glucocerebroside, and Fabry disease, in which globosides accumulate in the vascular endothelial lysosomes of the brain, heart, kidneys, and skin, are treated by recombinant human enzyme replacement therapy, but the monetary cost is extremely high. Gaucher has also been treated by bone marrow transplantation (because macrophages are derived from hematopoietic stem cells) and by substrate reduction therapy through pharmacologic reduction of

1	Gaucher has also been treated by bone marrow transplantation (because macrophages are derived from hematopoietic stem cells) and by substrate reduction therapy through pharmacologic reduction of glucosylceramide, the substrate for the deficient enzyme.

1	VIII. EICOSANOIDS: PROSTAGLANDINS, THROMBOXANES, AND LEUKOTRIENES

1	Prostaglandins (PG), thromboxanes (TX), and leukotrienes (LT) are collectively known as eicosanoids to reflect their origin from ω-3 and ω-6 polyunsaturated FA with 20 carbons (eicosa = 20). They are extremely potent compounds that elicit a wide range of responses, both physiologic (inflammatory response) and pathologic (hypersensitivity). They insure gastric integrity and renal function, regulate smooth muscle contraction (the intestine and uterus are key sites) and blood vessel diameter, and maintain platelet homeostasis. Although they have been compared to hormones in terms of their actions, eicosanoids differ from endocrine hormones in that they are produced in very small amounts in almost all tissues rather than in specialized glands and act locally rather than after transport in the blood to distant sites. Eicosanoids are not stored, and they have an extremely short half-life, being rapidly metabolized to inactive products. Their biologic actions are mediated by plasma membrane

1	blood to distant sites. Eicosanoids are not stored, and they have an extremely short half-life, being rapidly metabolized to inactive products. Their biologic actions are mediated by plasma membrane GPCR (see p. 94), which are different in different organ systems and typically result in changes in cyclic adenosine monophosphate production. Examples of eicosanoid structures are shown in Figure 17.21.

1	life. Cer = ceramide; Gal = galactose; Glc = glucose; GalNAc = Nacetylgalactosamine; NANA = N-acetylneuraminic acid; CNS = central nervous system. = sulfate; ERT = enzyme replacement therapy. known as prostacyclin. Thromboxanes are designated by TX and leukotrienes by LT.] A. Prostaglandin and thromboxane synthesis

1	A. Prostaglandin and thromboxane synthesis Arachidonic acid, an ω-6 FA containing 20 carbons and four double bonds (an eicosatetraenoic FA), is the immediate precursor of the predominant type of human PG (series 2 or those with two double bonds, as shown in Fig. 17.22). It is derived by the elongation and desaturation of the essential FA linoleic acid, also an ω-6 FA. Arachidonic acid is incorporated into membrane phospholipids (typically PI) at carbon 2, from which it is released by phospholipase A2 (Fig. 17.23) in response to a variety of signals, such as a rise in calcium. [Note: Series 1 PG contain one double bond and are derived from an ω-6 eicosatrienoic FA, dihomo-γ-linolenic acid, whereas series 3 PG contain three double bonds and are derived from eicosapentaenoic acid (EPA), an ω-3 FA. See p. 363.] activities (cyclooxygenase and peroxidase) of PGH2 synthase (prostaglandin endoperoxide synthase). G-SH = reduced glutathione; G-S-S-G = oxidized glutathione; PG = prostaglandin.

1	1.

1	Prostaglandin H2 synthase: The first step in PG and TX synthesis is the oxidative cyclization of free arachidonic acid to yield PGH2 by PGH2 synthase (or, prostaglandin endoperoxide synthase). This enzyme is an ER membrane-bound protein that has two catalytic activities: fatty acid cyclooxygenase (COX), which requires two molecules of O2, and peroxidase, which requires reduced glutathione (see p. 148). PGH2 is converted to a variety of PG and TX, as shown in Figure 17.23, by cell-specific synthases. [Note: PG contain a five-carbon ring, whereas TX contain a heterocyclic six-membered oxane ring (see Fig. 17.21).] Two isozymes of PGH2 synthase, usually denoted as COX-1 and COX-2, are known. COX-1 is made constitutively in most tissues and is required for maintenance of healthy gastric tissue, renal homeostasis, and platelet aggregation. COX-2 is inducible in a limited number of tissues in response to products of activated immune and inflammatory cells. [Note: The increase in PG

1	tissue, renal homeostasis, and platelet aggregation. COX-2 is inducible in a limited number of tissues in response to products of activated immune and inflammatory cells. [Note: The increase in PG synthesis subsequent to the induction of COX-2 mediates the pain, heat, redness, and swelling of inflammation and the fever of infection.] 2.

1	Synthesis inhibition: The synthesis of PG and TX can be inhibited by unrelated compounds. For example, cortisol (a steroidal anti-inflammatory agent) inhibits phospholipase A2 activity (see Fig. 17.23) and, therefore, arachidonic acid, the substrate for PG and TX synthesis, is not released from membrane phospholipids. Aspirin, indomethacin, and phenylbutazone (all nonsteroidal anti-inflammatory drugs [NSAID]) inhibit both COX-1 and COX-2 and, thus, prevent the synthesis of the parent molecule, PGH2. [Note: Systemic inhibition of COX-1, with subsequent damage to the stomach and the kidneys and impaired clotting of blood, is the basis of aspirin’s toxicity.] Aspirin (but not other NSAID) also induces synthesis of lipoxins (anti-inflammatory mediators made from arachidonic acid) and resolvins and protectins (inflammationresolving mediators made from EPA). Inhibitors specific for COX-2 (the coxibs, for example, celecoxib) were designed to reduce pathologic inflammatory processes mediated

1	and protectins (inflammationresolving mediators made from EPA). Inhibitors specific for COX-2 (the coxibs, for example, celecoxib) were designed to reduce pathologic inflammatory processes mediated by COX-2 while maintaining the physiologic functions of COX-1. However, their use has been associated with increased risk of heart attacks, likely as a result of decreased PGI2 synthesis (see B. below), and some have been withdrawn from the market.

1	B. Thromboxanes and prostaglandins in platelet homeostasis

1	Thromboxane A2 (TXA2) is produced by COX-1 in activated platelets. It promotes platelet adhesion and aggregation and contraction of vascular smooth muscle, thereby promoting formation of blood clots (thrombi). (See online Chapter 35.) Prostacyclin (PGI2), produced by COX-2 in vascular endothelial cells, inhibits platelet aggregation and stimulates vasodilation and, so, impedes thrombogenesis. The opposing effects of TXA2 and PGI2 limit thrombi formation to sites of vascular injury. [Note: Aspirin has an antithrombogenic effect. It inhibits TXA2 synthesis by COX-1 in platelets and PGI2 synthesis by COX-2 in endothelial cells through irreversible acetylation of these isozymes (Fig. 17.24). COX-1 inhibition cannot be overcome in platelets, which lack nuclei. However, COX-2 inhibition can be overcome in endothelial cells because they have a nucleus and, therefore, can generate more of the enzyme. This difference is the basis of low-dose aspirin therapy used to lower the risk of stroke and

1	in endothelial cells because they have a nucleus and, therefore, can generate more of the enzyme. This difference is the basis of low-dose aspirin therapy used to lower the risk of stroke and heart attacks by decreasing formation of thrombi.]

1	C. Leukotriene synthesis Arachidonic acid is converted to a variety of linear hydroperoxy (–OOH) acids by a separate pathway involving a family of lipoxygenases (LOX). For example, 5-LOX converts arachidonic acid to 5-hydroperoxy-6,8,11,14 eicosatetraenoic acid ([5-HPETE]; see Fig. 17.23). 5-HPETE is converted to a series of LT containing four double bonds, the nature of the final products varying according to the tissue. LT are mediators of allergic response and inflammation. Inhibitors of 5-LOX and LT-receptor antagonists are used in the treatment of asthma. [Note: LT synthesis is inhibited by cortisol and not by NSAID. Aspirin-exacerbated respiratory disease is a response to LT overproduction with NSAID use in ~10% of individuals with asthma.] IX. CHAPTER SUMMARY

1	Phospholipids are polar, ionic compounds composed of an alcohol (for example, choline or ethanolamine) attached by a phosphodiester bond either to diacylglycerol (DAG), producing phosphatidylcholine or phosphatidylethanolamine, or to the amino alcohol sphingosine (Fig. 17.25). Addition of a long-chain fatty acid to sphingosine produces a ceramide. Addition of phosphorylcholine produces the phospholipid sphingomyelin. Phospholipids are the predominant lipids of cell membranes. Nonmembrane phospholipids serve as components of lung surfactant and bile. Dipalmitoylphosphatidylcholine, also called dipalmitoyl lecithin, is the major lipid component of lung surfactant. Insufficient surfactant production causes respiratory distress syndrome. Phosphatidylinositol (PI) serves as a reservoir for arachidonic acid in membranes. The phosphorylation of membrane-bound PI produces phosphatidylinositol 4,5-bisphosphate (PIP2). This compound is degraded by phospholipase C in response to the binding of

1	acid in membranes. The phosphorylation of membrane-bound PI produces phosphatidylinositol 4,5-bisphosphate (PIP2). This compound is degraded by phospholipase C in response to the binding of various neurotransmitters, hormones, and growth factors to membrane G protein– coupled receptors. The products of this degradation, inositol 1,4,5trisphosphate (IP3) and DAG, mediate the mobilization of intracellular calcium and the activation of protein kinase C, which act synergistically to evoke cellular responses. Specific proteins can be covalently attached via a carbohydrate bridge to membrane-bound PI, forming a glycosyl phosphatidylinositol (GPI) anchor. A deficiency in GPI synthesis in hematopoietic cells results in the hemolytic disease paroxysmal nocturnal hemoglobinuria. The degradation of phosphoglycerides is performed by phospholipases found in all tissues and pancreatic juice. Sphingomyelin is degraded to a ceramide plus phosphorylcholine by the lysosomal enzyme sphingomyelinase, a

1	phosphoglycerides is performed by phospholipases found in all tissues and pancreatic juice. Sphingomyelin is degraded to a ceramide plus phosphorylcholine by the lysosomal enzyme sphingomyelinase, a deficiency of which causes Niemann-Pick (A and B) disease. Glycosphingolipids are derivatives of ceramides to which carbohydrates have been attached. Adding one sugar molecule to the ceramide produces a cerebroside, adding an oligosaccharide produces a globoside, and adding an acidic N-acetylneuraminic acid molecule produces a ganglioside. Glycosphingolipids are found predominantly in cell membranes of the brain and peripheral nervous tissue, with high concentrations in the myelin sheath. They are antigenic. Glycolipids are degraded in the lysosomes by acid hydrolases. A deficiency of any one of these enzymes causes a sphingolipidosis, in which a characteristic sphingolipid accumulates. Prostaglandins (PG), thromboxanes (TX), and leukotrienes (LT), the eicosanoids, are produced in very

1	of these enzymes causes a sphingolipidosis, in which a characteristic sphingolipid accumulates. Prostaglandins (PG), thromboxanes (TX), and leukotrienes (LT), the eicosanoids, are produced in very small amounts in almost all tissues, act locally, and have an extremely short half-life. They serve as mediators of the inflammatory response. Arachidonic acid is the immediate precursor of the predominant class of human PG (those with two double bonds). It is derived by the elongation and desaturation of the essential fatty acid linoleic acid and is stored in the membrane as a component of a phospholipid, generally PI. Arachidonic acid is released from the phospholipid by phospholipase A2 (inhibited by cortisol). Synthesis of the PG and TX begins with the oxidative cyclization of free arachidonic acid to yield PGH2 by PGH2 synthase (or, prostaglandin endoperoxide synthase), an endoplasmic reticular membrane protein that has two catalytic activities: fatty acid cyclooxygenase (COX) and

1	acid to yield PGH2 by PGH2 synthase (or, prostaglandin endoperoxide synthase), an endoplasmic reticular membrane protein that has two catalytic activities: fatty acid cyclooxygenase (COX) and peroxidase. There are two isozymes of PGH2 synthase: COX-1 (constitutive) and COX-2 (inducible). Aspirin irreversibly inhibits both.

1	Opposing effects of PGI2 and TXA2 limit clot formation. LT are linear molecules produced from arachidonic acid by the 5-lipoxygenase (5-LOX) pathway. They mediate allergic response. Their synthesis is inhibited by cortisol and not by aspirin. glycosphingolipids, and eicosanoids. PLA2 = phospholipase A2; = sulfate ion; NSAID = nonsteroidal anti-inflammatory drugs. Choose the ONE best answer. 7.1. Aspirin-exacerbated respiratory disease (AERD) is a severe reaction to nonsteroidal anti-inflammatory drugs (NSAID) characterized by bronchoconstriction 30 minutes to several hours after ingestion. Which of the following statements best explains the symptoms seen in patients with AERD? NSAID: A. inhibit the activity of the cystic fibrosis transmembrane conductance regulator protein, resulting in thickened mucus that block airways. B. inhibit cyclooxygenase but not lipoxygenase, resulting in the flow of arachidonic acid to leukotriene synthesis.

1	B. inhibit cyclooxygenase but not lipoxygenase, resulting in the flow of arachidonic acid to leukotriene synthesis. C. activate the cyclooxygenase activity of prostaglandin H2 synthase, resulting in increased synthesis of prostaglandins that promote vasodilation. D. activate phospholipases, resulting in decreased amounts of dipalmitoylphosphatidylcholine and alveolar collapse (atelectasis). Correct answer = B. NSAID inhibit cyclooxygenase but not lipoxygenase, so any arachidonic acid available is used for the synthesis of bronchoconstricting leukotrienes. NSAID have no effect on the cystic fibrosis (CF) transmembrane conductance regulator protein, defects in which are the cause of CF. Steroids, not NSAID, inhibit phospholipase A2. Cyclooxygenase is inhibited by NSAID, not activated. NSAID have no effect on phospholipases.

1	7.2. An infant, born at 28 weeks’ gestation, rapidly gave evidence of respiratory distress. Clinical laboratory and imaging results supported the diagnosis of infant respiratory distress syndrome. Which of the following statements about this syndrome is true? A. It is unrelated to the baby’s premature birth. B. It is a consequence of too few type II pneumocytes. C. The lecithin/sphingomyelin ratio in the amniotic fluid is likely to be high (>2). D. The concentration of dipalmitoylphosphatidylcholine in the amniotic fluid would be expected to be lower than that of a full-term baby. E. It is an easily treated disorder with low mortality. F. It is treated by administering surfactant to the mother just before she gives birth.

1	E. It is an easily treated disorder with low mortality. F. It is treated by administering surfactant to the mother just before she gives birth. Correct answer = D. Dipalmitoylphosphatidylcholine (DPPC or, dipalmitoyl lecithin) is the lung surfactant found in mature, healthy lungs. Respiratory distress syndrome (RDS) can occur in lungs that make too little of this compound. If the lecithin/sphingomyelin (L/S) ratio in amniotic fluid is ≥2, a newborn’s lungs are considered to be sufficiently mature (premature lungs would be expected to have a ratio <2). The RDS would not be due to too few type II pneumocytes, which would simply be secreting sphingomyelin rather than DPPC at 28 weeks’ gestation. The mother is given a glucocorticoid, not surfactant, prior to giving birth (antenatally). Surfactant would be administered to the baby postnatally to reduce surface tension.

1	7.3. A 10-year-old boy was evaluated for burning sensations in his feet and clusters of small, red-purple spots on his skin. Laboratory studies revealed protein in his urine. Enzymatic analysis revealed a deficiency of αgalactosidase, and enzyme replacement therapy was recommended. The most likely diagnosis is: A. Fabry disease. B. Farber disease. C. Gaucher disease. D. Krabbe disease. E. Niemann-Pick disease. Correct answer = A. Fabry disease, a deficiency of α-galactosidase, is the only X-linked sphingolipidosis. It is characterized by pain in the extremities, a red-purple skin rash (generalized angiokeratomas), and kidney and cardiac complications. Protein in his urine indicates kidney damage. Enzyme replacement therapy is available. 7.4. Current medical advice for individuals experiencing chest pain is to call emergency medical services and chew a regular strength, noncoated aspirin. What is the basis for recommending aspirin?

1	7.4. Current medical advice for individuals experiencing chest pain is to call emergency medical services and chew a regular strength, noncoated aspirin. What is the basis for recommending aspirin? Aspirin has an antithrombogenic effect: It prevents formation of blood clots that could occlude heart vessels. Aspirin inhibits thromboxane A2 synthesis by cyclooxygenase-1 in platelets through irreversible acetylation, thereby inhibiting platelet activation and vasoconstriction. Chewing a noncoated aspirin increases the rate of its absorption. Cholesterol, Lipoprotein, and Steroid Metabolism 18 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Cholesterol, the characteristic steroid alcohol of animal tissues, performs a number of essential functions in the body. For example, cholesterol is a structural component of all cell membranes, modulating their fluidity, and, in specialized tissues, cholesterol is a precursor of bile acids, steroid hormones, and vitamin D. Therefore, it is critically important that the cells of the body be assured an appropriate supply of cholesterol. To meet this need, a complex series of transport, biosynthetic, and regulatory mechanisms has evolved. The liver plays a central role in the regulation of the body’s cholesterol homeostasis. For example, cholesterol enters the hepatic cholesterol pool from a number of sources including dietary cholesterol as well as that synthesized de novo by extrahepatic tissues and by the liver itself. Cholesterol is eliminated from the liver as unmodified cholesterol in the bile, or it can be converted to bile salts that are secreted into the intestinal lumen. It

1	tissues and by the liver itself. Cholesterol is eliminated from the liver as unmodified cholesterol in the bile, or it can be converted to bile salts that are secreted into the intestinal lumen. It can also serve as a component of plasma lipoproteins that carry lipids to the peripheral tissues. In humans, the balance between cholesterol influx and efflux is not precise, resulting in a gradual deposition of cholesterol in the tissues, particularly in the endothelial linings of blood vessels. This is a potentially life-threatening occurrence when the lipid deposition leads to plaque formation, causing the narrowing of blood vessels (atherosclerosis) and increased risk of cardio-, cerebro-, and peripheral vascular disease. Figure 18.1 summarizes the major sources of liver cholesterol and the routes by which cholesterol leaves the liver.

1	II. CHOLESTEROL STRUCTURE Cholesterol is a very hydrophobic compound. It consists of four fused hydrocarbon rings (A–D) called the steroid nucleus, and it has an eight-carbon, branched hydrocarbon chain attached to carbon 17 of the D ring. Ring A has a hydroxyl group at carbon 3, and ring B has a double bond between carbon 5 and carbon 6 (Fig. 18.2). A. Sterols

1	Steroids with 8 to 10 carbon atoms in the side chain at carbon 17 and a hydroxyl group at carbon 3 are classified as sterols. Cholesterol is the major sterol in animal tissues. It arises from de novo synthesis and absorption of dietary cholesterol. Intestinal uptake of cholesterol is mediated by the Niemann-Pick C1-like 1 protein, the target of the drug ezetimibe that reduces absorption of dietary cholesterol (see p. 176). [Note: Plant sterols (phytosterols), such as β-sitosterol, are poorly absorbed by humans (5% absorbed as compared to 40% for cholesterol). After entering the enterocytes, they are actively transported back into the intestinal lumen. Defects in the efflux transporter (ABCG5/8) result in the rare condition of sitosterolemia. Because some cholesterol is transported back as well, plant sterols reduce the absorption of dietary cholesterol. Daily ingestion of plant sterol esters supplied, for example, in spreads, is one of a number of dietary strategies to reduce plasma

1	well, plant sterols reduce the absorption of dietary cholesterol. Daily ingestion of plant sterol esters supplied, for example, in spreads, is one of a number of dietary strategies to reduce plasma cholesterol levels (see p. 363).]

1	B. Cholesteryl esters Most plasma cholesterol is in an esterified form (with a fatty acid [FA] attached at carbon 3, as shown in Fig. 18.2), which makes the structure even more hydrophobic than free (nonesterified) cholesterol. Cholesteryl esters are not found in membranes and are normally present only in low levels in most cells. Because of their hydrophobicity, cholesterol and its esters must be transported in association with protein as a component of a lipoprotein particle (see p. 227) or be solubilized by phospholipids and bile salts in the bile (see p. 226). III. CHOLESTEROL SYNTHESIS

1	Cholesterol is synthesized by virtually all tissues in humans, although liver, intestine, adrenal cortex, and reproductive tissues, including ovaries, testes, and placenta, make the largest contributions to the cholesterol pool. As with FA, all the carbon atoms in cholesterol are provided by acetyl coenzyme A (CoA), and nicotinamide adenine dinucleotide phosphate (NADPH) provides the reducing equivalents. The pathway is endergonic, being driven by hydrolysis of the high-energy thioester bond of acetyl CoA and the terminal phosphate bond of ATP. Synthesis requires enzymes in the cytosol, the membrane of the smooth endoplasmic reticulum (SER), and the peroxisome. The pathway is responsive to changes in cholesterol concentration, and regulatory mechanisms exist to balance the rate of cholesterol synthesis against the rate of cholesterol excretion. An imbalance in this regulation can lead to an elevation in circulating levels of plasma cholesterol, with the potential for vascular disease.

1	A. 3-Hydroxy-3-methylglutaryl coenzyme A synthesis The first two reactions in the cholesterol biosynthetic pathway are similar to those in the pathway that produces ketone bodies (see Fig. 16.22, p. 196). They result in the production of 3-hydroxy-3-methylglutaryl CoA ([HMG CoA], Fig. 18.3). First, two acetyl CoA molecules condense to form acetoacetyl CoA. Next, a third molecule of acetyl CoA is added by HMG CoA synthase, producing HMG CoA, a six-carbon compound. [Note: Liver parenchymal cells contain two isoenzymes of the synthase. The cytosolic enzyme participates in cholesterol synthesis, whereas the mitochondrial enzyme functions in the pathway for ketone body synthesis.] B. Mevalonate synthesis

1	B. Mevalonate synthesis HMG CoA is reduced to mevalonate by HMG CoA reductase. This is the rate-limiting and key regulated step in cholesterol synthesis. It occurs in the cytosol, uses two molecules of NADPH as the reducing agent, and releases CoA, making the reaction irreversible (Fig. 18.4). [Note: HMG CoA reductase is an integral membrane protein of the SER, with its catalytic domain projecting into the cytosol. Regulation of reductase activity is discussed in D. below.] C. Cholesterol synthesis from mevalonate The reactions and enzymes involved in the synthesis of cholesterol from mevalonate are illustrated in Figure 18.5. [Note: The numbers shown in brackets below correspond to numbered reactions shown in this figure.] [1] Mevalonate is converted to 5-pyrophosphomevalonate in two steps, each of which transfers a phosphate group from ATP.

1	[2] A five-carbon isoprene unit, isopentenyl pyrophosphate (IPP), is formed by the decarboxylation of 5-pyrophosphomevalonate. The reaction requires ATP. [Note: IPP is the precursor of a family of molecules with diverse functions, the isoprenoids. Cholesterol is a sterol isoprenoid. Nonsterol isoprenoids include dolichol (see p. 167) and ubiquinone (or, coenzyme Q; see p. 75).] [3] IPP is isomerized to 3,3-dimethylallyl pyrophosphate (DPP). [4] IPP and DPP condense to form 10-carbon geranyl pyrophosphate (GPP).

1	[4] IPP and DPP condense to form 10-carbon geranyl pyrophosphate (GPP). [5] A second molecule of IPP then condenses with GPP to form 15-carbon farnesyl pyrophosphate (FPP). [Note: Covalent attachment of farnesyl to proteins, a process known as prenylation, is one mechanism for anchoring proteins (for example, ras) to the inner face of plasma membranes.] [6] Two molecules of FPP combine, releasing pyrophosphate, and are reduced, forming the 30-carbon compound squalene. [Note: Squalene is formed from six isoprenoid units. Because 3 ATP are hydrolyzed per mevalonate residue converted to IPP, a total of 18 ATP are required to make the polyisoprenoid squalene.] [7] Squalene is converted to the sterol lanosterol by a sequence of two reactions catalyzed by SER-associated enzymes that use molecular oxygen (O2) and NADPH. The hydroxylation of linear squalene triggers the cyclization of the structure to lanosterol.

1	The conversion of lanosterol to cholesterol is a multistep process involving shortening of the side chain, oxidative removal of methyl groups, reduction of double bonds, and migration of a double bond. Smith-Lemli-Opitz syndrome (SLOS), an autosomal-recessive disorder of cholesterol biosynthesis, is caused by a partial deficiency in 7-dehydrocholesterol-7reductase, the enzyme that reduces the double bond in 7dehydrocholesterol (7-DHC), thereby converting it to cholesterol. SLOS is one of several multisystem, embryonic malformation syndromes associated with impaired cholesterol synthesis. [Note: 7-DHC is converted to vitamin D3 in the skin (see p. 390).] D. HMG CoA reductase is the major control point for cholesterol biosynthesis and is subject to different kinds of metabolic control. 1.

1	Sterol-dependent regulation of gene expression: Expression of the gene for HMG CoA reductase is controlled by the trans-acting factor, sterol regulatory element–binding protein-2 (SREBP-2), which binds DNA at the cis-acting sterol regulatory element (SRE) upstream of the reductase gene. Inactive SREBP-2 is an integral protein of the SER membrane and associates with a second SER membrane protein, SREBP cleavage– activating protein (SCAP). When sterol levels in the SER are low, the SREBP-2–SCAP complex moves from the ER to the Golgi. In the Golgi membrane, SREBP-2 is sequentially acted upon by two proteases, which generate a soluble fragment that enters the nucleus, binds the SRE, and functions as a transcription factor. This results in increased synthesis of HMG CoA reductase and, therefore, increased cholesterol synthesis (Fig. 18.6). However, if sterols are abundant, they bind SCAP at its sterolsensing domain and induce the binding of SCAP to yet other ER membrane proteins, the

1	increased cholesterol synthesis (Fig. 18.6). However, if sterols are abundant, they bind SCAP at its sterolsensing domain and induce the binding of SCAP to yet other ER membrane proteins, the insulin-induced gene proteins (INSIG). This results in the retention of the SCAP–SREBP complex in the SER, thereby preventing the activation of SREBP-2 and leading to downregulation of cholesterol synthesis. [Note: SREBP-1c upregulates expression of enzymes involved in FA synthesis in response to insulin (see p. 184).] 2.

1	Sterol-accelerated enzyme degradation: The reductase itself is a sterolsensing integral protein of the SER membrane. When sterol levels in the SER are high, the enzyme binds to INSIG proteins. Binding leads to cytosolic transfer, ubiquitination, and proteasomal degradation of the reductase (see p. 247). 3. Sterol-independent phosphorylation/dephosphorylation: HMG CoA reductase activity is controlled covalently through the actions of adenosine monophosphate (AMP)–activated protein kinase ([AMPK] see p. 183) and a phosphoprotein phosphatase (see Fig. 18.6). The phosphorylated form of the enzyme is inactive, whereas the dephosphorylated form is active. [Note: Because AMPK is activated by AMP, cholesterol synthesis, like FA synthesis, is decreased when ATP availability is decreased.] 4.

1	Hormonal regulation: The activity of HMG CoA reductase is controlled hormonally. An increase in insulin favors dephosphorylation (activation) of the reductase, whereas an increase in glucagon and epinephrine has the opposite effect. 5. Drug inhibition: The statin drugs (atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, and simvastatin) are structural analogs of HMG CoA and are (or are metabolized to) reversible, competitive inhibitors of HMG CoA reductase (Fig. 18.7). They are used to decrease plasma cholesterol levels in patients with hypercholesterolemia. IV. CHOLESTEROL DEGRADATION

1	IV. CHOLESTEROL DEGRADATION Humans cannot metabolize the cholesterol ring structure to carbon dioxide and water. Rather, the intact steroid nucleus is eliminated from the body by conversion to bile acids and bile salts, a small percentage of which is excreted in the feces, and by secretion of cholesterol into the bile, which transports it to the intestine for elimination. [Note: Some of the cholesterol in the intestine is modified by bacteria before excretion. The primary compounds made are the isomers coprostanol and cholestanol, which are reduced derivatives of cholesterol. Together with cholesterol, these compounds make up the bulk of neutral fecal sterols.] V. BILE ACIDS AND BILE SALTS

1	V. BILE ACIDS AND BILE SALTS Bile consists of a watery mixture of organic and inorganic compounds. Phosphatidylcholine (PC), or lecithin (see p. 202), and conjugated bile salts are quantitatively the most important organic components of bile. Bile can either pass directly from the liver, where it is synthesized, into the duodenum through the common bile duct, or be stored in the gallbladder when not immediately needed for digestion. A. Structure

1	A. Structure The bile acids contain 24 carbons, with two or three hydroxyl groups and a side chain that terminates in a carboxyl group. The carboxyl group has a pKa (see p. 6) of ~6. In the duodenum (pH ~6), this group will be protonated in half of the molecules (the bile acids) and deprotonated in the rest (the bile salts). The terms bile acid and bile salt are frequently used interchangeably, however. Both forms have hydroxyl groups that are α in orientation (they lie below the plane of the rings) and methyl groups that are β (they lie above the plane of the rings). Therefore, the molecules have both a polar and a nonpolar surface and can act as emulsifying agents in the intestine, helping prepare dietary fat (triacylglycerol [TAG]) and other complex lipids for degradation by pancreatic digestive enzymes. B. Synthesis

1	B. Synthesis Bile acids are synthesized in the liver by a multistep, multiorganelle pathway in which hydroxyl groups are inserted at specific positions on the steroid structure; the double bond of the cholesterol B ring is reduced; and the hydrocarbon chain is shortened by three carbons, introducing a carboxyl group at the end of the chain. The most common resulting compounds, cholic acid (a triol) and chenodeoxycholic acid (a diol), as shown in Figure 18.8, are called primary bile acids. [Note: The rate-limiting step in bile acid synthesis is the introduction of a hydroxyl group at carbon 7 of the steroid nucleus by 7-α-hydroxylase, a SER-associated cytochrome P450 monooxygenase found only in liver. Expression of the enzyme is downregulated by bile acids (Fig. 18.9)]. C. Conjugation

1	Before the bile acids leave the liver, they are conjugated to a molecule of either glycine or taurine (an end product of cysteine metabolism) by an amide bond between the carboxyl group of the bile acid and the amino group of the added compound. These new structures include glycocholic and glycochenodeoxycholic acids and taurocholic and taurochenodeoxycholic acids (Fig. 18.10). The ratio of glycine to taurine forms in the bile is ~3/1. Addition of glycine or taurine results in the presence of a carboxyl group with a lower pKa (from glycine) or a sulfonate group (from taurine), both of which are fully ionized (negatively charged) at the alkaline pH of bile. The conjugated, ionized bile salts are more effective detergents than the unconjugated ones because of their enhanced amphipathic nature. Therefore, only the conjugated forms are found in the bile. Individuals with genetic deficiencies in the conversion of cholesterol to bile acids are treated with exogenously supplied

1	nature. Therefore, only the conjugated forms are found in the bile. Individuals with genetic deficiencies in the conversion of cholesterol to bile acids are treated with exogenously supplied chenodeoxycholic acid.

1	Bile salts provide the only significant mechanism for cholesterol excretion, both as a metabolic product of cholesterol and as a solubilizer of cholesterol in bile. D. Bacterial action on bile salts Bacteria of the intestinal microbiota (see p. 372) can deconjugate (remove glycine and taurine) bile salts. They can also dehydroxylate carbon 7, producing secondary bile salts such as deoxycholic acid from cholic acid and lithocholic acid from chenodeoxycholic acid. E. Enterohepatic circulation

1	Bile salts secreted into the intestine are efficiently reabsorbed (>95%) and reused. The liver actively secretes bile salts via the bile salt export pump. In the intestine, they are reabsorbed in the terminal ileum via the apical sodium (Na+)–bile salt cotransporter and returned to the blood via a separate transport system. [Note: Lithocholic acid is only poorly absorbed.] They are efficiently taken up from blood by the hepatocytes via an isoform of the cotransporter and reused. [Note: Albumin binds bile salts and transports them through the blood as was seen with FA (see p. 181).] The continuous process of secretion of bile salts into the bile, their passage through the duodenum where some are deconjugated then dehydroxylated to secondary bile salts, their uptake in the ileum, and their subsequent return to the liver as a mixture of primary and secondary forms is termed the enterohepatic circulation (Fig. 18.11). Between 15 and 30 g of bile salts are secreted from the liver into the

1	subsequent return to the liver as a mixture of primary and secondary forms is termed the enterohepatic circulation (Fig. 18.11). Between 15 and 30 g of bile salts are secreted from the liver into the duodenum each day, yet only ~0.5 g (<3%) is lost daily in the feces. Approximately 0.5 g/day is synthesized from cholesterol in the liver to replace the amount lost. Bile acid sequestrants, such as cholestyramine, bind bile salts in the gut and prevent their reabsorption, thereby promoting their excretion. They are used in the treatment of hypercholesterolemia because the removal of bile salts relieves the inhibition on bile acid synthesis in the liver, thereby diverting additional cholesterol into that pathway. [Note: Dietary fiber also binds bile salts and increases their excretion (see p. 365).]

1	F. Bile salt deficiency: Cholelithiasis

1	The movement of cholesterol from the liver into the bile must be accompanied by the simultaneous secretion of phospholipid and bile salts. If this dual process is disrupted and more cholesterol is present than can be solubilized by the bile salts and PC present, the cholesterol may precipitate in the gallbladder, leading to cholesterol gallstone disease or cholelithiasis (Fig. 18.12). This disorder is typically caused by a decrease of bile acids in the bile. Cholelithiasis also may result from increased secretion of cholesterol into bile, as seen with the use of fibrates (for example, gemfibrozil) to reduce cholesterol (and TAG) in the blood. Laparoscopic cholecystectomy (surgical removal of the gallbladder through a small incision) is currently the treatment of choice. However, for patients who are unable to undergo surgery, oral administration of chenodeoxycholic acid to supplement the body’s supply of bile acids results in a gradual (months to years) dissolution of the gallstones.

1	who are unable to undergo surgery, oral administration of chenodeoxycholic acid to supplement the body’s supply of bile acids results in a gradual (months to years) dissolution of the gallstones. [Note: Cholesterol stones account for >85% of cases of cholelithiasis, with bilirubin and mixed stones accounting for the rest].

1	VI. PLASMA LIPOPROTEINS The plasma lipoproteins are spherical macromolecular complexes of lipids and proteins (apolipoproteins). The lipoprotein particles include chylomicrons, verylow-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). They differ in lipid and protein composition, size, density (Fig. 18.13), and site of origin. [Note: Because lipoprotein particles constantly interchange lipids and apolipoproteins, the actual apolipoprotein and lipid content of each class of particles is somewhat variable.] Lipoproteins function both to keep their component lipids soluble as they transport them in the plasma and to provide an efficient mechanism for transporting their lipid contents to (and from) the tissues. In humans, the transport system is less perfect than in other animals and, as a result, humans experience a gradual deposition of lipid (especially cholesterol) in tissues. A. Composition

1	A. Composition Lipoproteins are composed of a neutral lipid core (containing TAG and cholesteryl esters) surrounded by a shell of amphipathic apolipoproteins, phospholipid, and nonesterified (free) cholesterol (Fig. 18.14). These amphipathic compounds are oriented such that their polar portions are exposed on the surface of the lipoprotein, thereby rendering the particle soluble in aqueous solution. The TAG and cholesterol carried by the lipoproteins are obtained either from the diet (exogenous source) or from de novo synthesis (endogenous source). [Note: The cholesterol (C) content of plasma lipoproteins is now routinely measured in fasting blood. Total C = LDL-C + HDL-C + VLDL-C, where VLDL-C is calculated by dividing TAG by 5 because the TAG/cholesterol ratio is 5/1 in VLDL. The goal value for total cholesterol is <200 mg/dl.] 1.

1	Size and density: Chylomicrons are the lipoprotein particles lowest in density and largest in size and that contain the highest percentage of lipid (as TAG) and the lowest percentage of protein. VLDL and LDL are successively denser, having higher ratios of protein to lipid. HDL particles are the smallest and densest. Plasma lipoproteins can be separated on the basis of their electrophoretic mobility, as shown in Figure 18.15, or on the basis of their density by ultracentrifugation. 2.

1	2. Apolipoproteins: The apolipoproteins associated with lipoprotein particles have a number of diverse functions, such as providing recognition sites for cell-surface receptors and serving as activators or coenzymes for enzymes involved in lipoprotein metabolism. Some of the apolipoproteins are required as essential structural components of the particles and cannot be removed (in fact, the particles cannot be produced without them), whereas others are transferred freely between lipoproteins. Apolipoproteins are divided by structure and function into several major classes, denoted by letters, with each class having subclasses (for example, apolipoprotein [apo] C-I, apo C-II, and apo CIII). [Note: The functions of all the apolipoproteins are not yet known.] B. Chylomicron metabolism

1	B. Chylomicron metabolism Chylomicrons are assembled in intestinal mucosal cells and carry dietary (exogenous) TAG, cholesterol, fat-soluble vitamins, and cholesteryl esters to the peripheral tissues (Fig. 18.16). [Note: TAG account for close to 90% of the lipids in a chylomicron.] 1. Apolipoprotein synthesis: Apo B-48 is unique to chylomicrons. Its synthesis begins on the rough ER (RER), and it is glycosylated as it moves through the RER and Golgi. [Note: Apo B-48 is so named because it constitutes the N-terminal 48% of the protein encoded by the gene for apo B. Apo B-100, which is synthesized by the liver and found in VLDL and LDL, represents the entire protein encoded by this gene. Posttranscriptional editing (see p. 474) of a cytosine to a uracil in intestinal apo B-100 messenger RNA (mRNA) creates a nonsense (stop) codon (see p. 449), allowing translation of only 48% of the mRNA.] 2.

1	Chylomicron assembly: Many enzymes involved in TAG, cholesterol, and phospholipid synthesis are located in the SER. Assembly of the apolipoprotein and lipid into chylomicrons requires microsomal triglyceride transfer protein ([MTP] see p. 177), which loads apo B-48 with lipid. This occurs before transition from the ER to the Golgi, where the particles are packaged in secretory vesicles. These fuse with the plasma membrane releasing the lipoproteins, which then enter the lymphatic system and, ultimately, the blood. [Note: Chylomicrons leave the lymphatic system via the thoracic duct that empties into the left subclavian vein.] 3.

1	Nascent chylomicron modification: The particle released by the intestinal mucosal cell is called a nascent chylomicron because it is functionally incomplete. When it reaches the plasma, the particle is rapidly modified, receiving apo E (which is recognized by hepatic receptors) and apo C. The latter includes apo C-II, which is necessary for the activation of lipoprotein lipase (LPL), the enzyme that degrades the TAG contained in the chylomicron. The source of these apolipoproteins is circulating HDL (see Fig. 18.16). [Note: Apo C-III on TAG-rich lipoproteins inhibits LPL.] 4. Triacylglycerol degradation by lipoprotein lipase: LPL is an extracellular enzyme that is anchored to the capillary walls of most tissues but predominantly those of adipose tissue and cardiac and skeletal muscle.

1	The adult liver does not express this enzyme. [Note: A hepatic lipase is found on the surface of endothelial cells of the liver. It plays a role in TAG degradation in chylomicrons and VLDL and is important in HDL metabolism (see p. 234).] LPL, activated by apo C-II on circulating chylomicrons, hydrolyzes the TAG in these particles to FA and glycerol. The FA are stored (in adipose) or used for energy (in muscle). The glycerol is taken up by the liver, converted to dihydroxyacetone phosphate (an intermediate of glycolysis), and used in lipid synthesis or gluconeogenesis. [Note: Patients with a deficiency of LPL or apo C-II (type I hyperlipoproteinemia or familial chylomicronemia) show a dramatic accumulation (≥1,000 mg/dl) of chylomicron-TAG in the plasma (hypertriacylglycerolemia) even in the fasted state. They are at increased risk for acute pancreatitis. Treatment is the reduction of dietary fat.] 5.

1	Lipoprotein lipase expression: LPL is synthesized by adipose tissue and by cardiac and skeletal muscle. Expression of the tissue-specific isozymes is regulated by nutritional state and hormonal level. For example, in the fed state (elevated insulin levels), LPL synthesis is increased in adipose but decreased in muscle tissue. Fasting (decreased insulin) favors LPL synthesis in muscle. [Note: The highest concentration of LPL is in cardiac muscle, reflecting the use of FA to provide much of the energy needed for cardiac function.] 6.

1	Chylomicron remnant formation: As the chylomicron circulates, and >90% of the TAG in its core is degraded by LPL, the particle decreases in size and increases in density. In addition, the C apolipoproteins (but not apo B or E) are returned to HDL. The remaining particle, called a remnant, is rapidly removed from the circulation by the liver, whose cell membranes contain lipoprotein receptors that recognize apo E (see Fig. 18.16). Chylomicron remnants bind to these receptors and are taken into the hepatocytes by endocytosis. The endocytosed vesicle then fuses with a lysosome, and the apolipoproteins, cholesteryl esters, and other components of the remnant are hydrolytically degraded, releasing amino acids, free cholesterol, and FA. The receptor is recycled. [Note: The mechanism of receptor-mediated endocytosis is illustrated for LDL in Fig. 18.20.] C. Very-low-density lipoprotein metabolism

1	C. Very-low-density lipoprotein metabolism VLDL are produced in the liver (Fig. 18.17). They are composed predominantly of endogenous TAG (~60%), and their function is to carry this lipid from the liver (site of synthesis) to the peripheral tissues. There, the TAG is degraded by LPL, as discussed for chylomicrons (see p. 228). [Note: Nonalcoholic fatty liver (hepatic steatosis) occurs in conditions in which there is an imbalance between hepatic TAG synthesis and the secretion of VLDL. Such conditions include obesity and type 2 diabetes mellitus (see p. 343).] 1.

1	Release from the liver: VLDL are secreted directly into the blood by the liver as nascent particles containing apo B-100. They must obtain apo CII and apo E from circulating HDL (see Fig. 18.17). As with chylomicrons, apo C-II is required for activation of LPL. [Note: Abetalipoproteinemia is a rare hypolipoproteinemia caused by a defect in MTP, leading to an inability to load apo B with lipid. Consequently, few VLDL or chylomicrons are formed, and TAG accumulates in the liver and intestine. Absorption of fat-soluble vitamins is decreased. LDL are low.] 2.

1	Modification in the circulation: As VLDL pass through the circulation, TAG is degraded by LPL, causing the VLDL to decrease in size and become denser. Surface components, including the C and E apolipoproteins, are returned to HDL, but the particles retain apo B-100. Additionally, some TAG are transferred from VLDL to HDL in an exchange reaction that concomitantly transfers cholesteryl esters from HDL to VLDL. This exchange is accomplished by cholesteryl ester transfer protein (CETP), as shown in Figure 18.18.

1	3. Conversion to low-density lipoproteins: With these modifications, the VLDL is converted in the plasma to LDL. Intermediate-density lipoproteins (IDL) of varying sizes are formed during this transition. IDL can also be taken up by liver cells through receptor-mediated endocytosis that uses apo E as the ligand. Apo E is normally present in three isoforms, E-2 (the least common), E-3 (the most common), and E-4. Apo E-2 binds poorly to receptors, and patients who are homozygotic for apo E-2 are deficient in the clearance of IDL and chylomicron remnants. These individuals have familial type III hyperlipoproteinemia (familial dysbetalipoproteinemia or broad beta disease), with hypercholesterolemia and premature atherosclerosis. [Note: The apo E-4 isoform confers increased susceptibility to an earlier age of onset of the late-onset form of Alzheimer disease. The effect is dose dependent, with homozygotes being at greatest risk. Estimates of the risk vary.]

1	D. Low-density lipoprotein metabolism LDL particles contain much less TAG than their VLDL predecessors and have a high concentration of cholesterol and cholesteryl esters (Fig. 18.19). About 70% of plasma cholesterol is in LDL. 1. Receptor-mediated endocytosis: The primary function of LDL particles is to provide cholesterol to the peripheral tissues (or return it to the liver). They do so by binding to plasma membrane LDL receptors that recognize apo B-100 (but not apo B-48). Because these LDL receptors can also bind apo E, they are known as apo B-100/apo E receptors. A summary of the uptake and degradation of LDL particles is presented in Figure 18.20. [Note: The numbers in brackets below refer to corresponding numbers on that figure.] A similar mechanism of receptor-mediated endocytosis is used for the uptake and degradation of chylomicron remnants and IDL by the liver.

1	[1] LDL receptors are negatively charged glycoproteins that are clustered in pits on cell membranes. The cytosolic side of the pit is coated with the protein clathrin, which stabilizes the pit.

1	[2] After binding, the LDL–receptor complex is endocytosed. [Note: Defects in the synthesis of functional LDL receptors causes a significant elevation in plasma LDL-C. Patients with such deficiencies have type IIa hyperlipidemia (familial hypercholesterolemia [FH]) and premature atherosclerosis. Autosomal dominant hypercholesterolemia can also be caused by defects in apo B-100 that reduce its binding to the receptor and by increased activity of a protease, proprotein convertase subtilisin/kexin type 9 (PCSK9), which promotes internalization and lysosomal degradation of the receptor. PCSK9 inhibitors are now available for the treatment of hypercholesterolemia.] [3] The vesicle containing LDL loses its clathrin coat and fuses with other similar vesicles, forming larger vesicles called endosomes.

1	[4] The pH of the endosome falls (due to the proton-pumping activity of endosomal ATPase), which allows separation of the LDL from its receptor. The receptors then migrate to one side of the endosome, whereas the LDL stay free within the lumen of the vesicle.

1	[5] The receptors can be recycled, whereas the lipoprotein remnants in the vesicle are transferred to lysosomes and degraded by lysosomal acid hydrolases, releasing free cholesterol, amino acids, FA, and phospholipids. These compounds can be reutilized by the cell. [Note: Lysosomal storage diseases result from rare autosomal-recessive deficiencies in the ability to hydrolyze lysosomal cholesteryl esters (Wolman disease) or to transport free cholesterol out of the lysosome (Niemann-Pick disease, type C).] 2. Endocytosed cholesterol and cholesterol homeostasis: The chylomicron remnant–, IDL-, and LDL-derived cholesterol affects cellular cholesterol content in several ways (see Fig. 18.20). First, expression of the gene for HMG CoA reductase is inhibited by high cholesterol, and de novo cholesterol synthesis decreases as a result. Additionally, degradation of the reductase is accelerated. Second, synthesis of new LDL receptor protein is reduced by decreasing the expression of the LDL

1	synthesis decreases as a result. Additionally, degradation of the reductase is accelerated. Second, synthesis of new LDL receptor protein is reduced by decreasing the expression of the LDL receptor gene, thus limiting further entry of LDL-C into cells. [Note: As was seen with the reductase gene (see p. 222), transcriptional regulation of the LDL receptor gene involves an SRE and SREBP-2. This allows coordinate regulation of the expression of these proteins.] Third, if the cholesterol is not required immediately for some structural or synthetic purpose, it is esterified by acyl CoA:cholesterol acyltransferase (ACAT). ACAT transfers a FA from a fatty acyl CoA to cholesterol, producing a cholesteryl ester that can be stored in the cell (Fig. 18.21). The activity of ACAT is enhanced in the presence of increased intracellular cholesterol.

1	3. Uptake by macrophage scavenger receptors: In addition to the highly specific and regulated receptor-mediated pathway for LDL uptake described above, macrophages possess high levels of scavenger receptor activity. These receptors, known as scavenger receptor class A (SR-A), can bind a broad range of ligands and mediate the endocytosis of chemically modified LDL in which the lipid or apo B component has been oxidized. Unlike the LDL receptor, the scavenger receptor is not downregulated in response to increased intracellular cholesterol. Cholesteryl esters accumulate in macrophages and cause their transformation into “foam” cells, which participate in the formation of atherosclerotic plaque (Fig. 18.22). LDL-C is the primary cause of atherosclerosis. E. High-density lipoprotein metabolism

1	E. High-density lipoprotein metabolism HDL comprise a heterogeneous family of lipoproteins with a complex metabolism that is not yet completely understood. HDL particles are formed in the blood by the addition of lipid to apo A-1, an apolipoprotein made and secreted by the liver and intestine. Apo A-1 accounts for ~70% of the apolipoproteins in HDL. HDL perform a number of important functions, including the following. 1. Apolipoprotein supply: HDL particles serve as a circulating reservoir of apo C-II (the apolipoprotein that is transferred to VLDL and chylomicrons and is an activator of LPL) and apo E (the apolipoprotein required for the receptor-mediated endocytosis of IDL and chylomicron remnants). 2.

1	2. Nonesterified cholesterol uptake: Nascent HDL are disc-shaped particles containing primarily phospholipid (largely PC) and apo A, C, and E. They take up cholesterol from nonhepatic (peripheral) tissues and return it to the liver as cholesteryl esters (Fig. 18.23). [Note: HDL particles are excellent acceptors of nonesterified cholesterol as a result of their high concentration of phospholipids, which are important solubilizers of cholesterol.] 3.

1	Cholesterol esterification: The cholesterol taken up by HDL is immediately esterified by the plasma enzyme lecithin:cholesterol acyltransferase (LCAT, also known as PCAT, in which P stands for phosphatidylcholine, the source of the FA). This enzyme is synthesized and secreted by the liver. LCAT binds to nascent HDL and is activated by apo A-I. LCAT transfers the FA from carbon 2 of PC to cholesterol. This produces a hydrophobic cholesteryl ester, which is sequestered in the core of the HDL, and lysophosphatidylcholine, which binds to albumin. [Note: Esterification maintains the cholesterol concentration gradient, allowing continued efflux of cholesterol to HDL.] As the discoidal nascent HDL accumulates cholesteryl esters, it first becomes a spherical, relatively cholesteryl ester–poor HDL3 and, eventually, a cholesteryl ester–rich HDL2 particle that carries these esters to the liver. Hepatic lipase, which degrades TAG and phospholipids, participates in the conversion of HDL2 to HDL3

1	and, eventually, a cholesteryl ester–rich HDL2 particle that carries these esters to the liver. Hepatic lipase, which degrades TAG and phospholipids, participates in the conversion of HDL2 to HDL3 (see Fig. 18.23). CETP (see p. 231) transfers some of the cholesteryl esters from HDL to VLDL in exchange for TAG, relieving product inhibition of LCAT. Because VLDL are catabolized to LDL, the cholesteryl esters transferred by CETP are ultimately taken up by the liver (see p. 231).

1	4.

1	Reverse cholesterol transport: The selective transfer of cholesterol from peripheral cells to HDL and from HDL to the liver for bile acid synthesis or disposal via the bile is a key component of cholesterol homeostasis. This process of reverse cholesterol transport (RCT) is, in part, the basis for the inverse relationship seen between plasma HDL concentration and atherosclerosis and for the designation of HDL as the “good” cholesterol carrier. [Note: Exercise and estrogen raise HDL levels.] RCT involves efflux of cholesterol from peripheral cells to HDL, esterification of the cholesterol by LCAT, binding of the cholesteryl ester–rich HDL (HDL2) to liver (and, perhaps, steroidogenic cells), selective transfer of the cholesteryl esters into these cells, and release of lipid-depleted HDL (HDL3). The efflux of cholesterol from peripheral cells is mediated primarily by the transport protein ABCA1. [Note: Tangier disease is a very rare deficiency of ABCA1 and is characterized by the virtual

1	The efflux of cholesterol from peripheral cells is mediated primarily by the transport protein ABCA1. [Note: Tangier disease is a very rare deficiency of ABCA1 and is characterized by the virtual absence of HDL particles due to degradation of lipid-poor apo A-1.] Cholesteryl ester uptake by the liver is mediated by the cell-surface receptor SR-B1 (scavenger receptor class B type 1) that binds HDL (see p. 232 for SR-A receptors). The HDL particle itself is not taken up. Instead, there is selective uptake of the cholesteryl ester from the HDL particle. [Note: Low HDL-C is a risk factor for atherosclerosis.]

1	ABCA1 is an ATP-binding cassette (ABC) protein. ABC proteins use energy from ATP hydrolysis to transport materials, including lipids, in and out of cells and across intracellular compartments. In addition to Tangier disease, defects in specific ABC proteins result in sitosterolemia, cystic fibrosis, X-linked adrenoleukodystrophy, respiratory distress syndrome due to decreased surfactant secretion, and liver disease due to decreased bile salt secretion. F. Lipoprotein (a) and heart disease

1	Lipoprotein (a), or Lp(a), is nearly identical in structure to an LDL particle. Its distinguishing feature is the presence of an additional apolipoprotein molecule, apo(a), which is covalently linked at a single site to apo B-100. Circulating levels of Lp(a) are determined primarily by genetics. However, factors such as diet may play some role, as trans FA have been reported to increase it. The physiologic function of Lp(a) is unknown. When present in large quantities in the plasma, Lp(a) is associated with an increased risk of coronary heart disease. [Note: Apo(a) is structurally homologous to plasminogen, the precursor of a blood protease whose target is fibrin, the main protein component of blood clots (see Chapter 35 online). It is hypothesized that elevated Lp(a) slows the breakdown of blood clots that trigger heart attacks because it competes with plasminogen for binding to fibrin.] Niacin reduces Lp(a), as well as LDL-C and TAG, and raises HDLC. VII. STEROID HORMONES

1	Cholesterol is the precursor of all classes of steroid hormones: glucocorticoids (for example, cortisol), mineralocorticoids (for example, aldosterone), and the sex hormones (that is, androgens, estrogens, and progestins), as shown in Figure 18.24. [Note: Glucocorticoids and mineralocorticoids are collectively called corticosteroids.] Synthesis and secretion occur in the adrenal cortex (cortisol, aldosterone, and androgens), ovaries and placenta (estrogens and progestins), and testes (testosterone). Steroid hormones are transported by the blood from their sites of synthesis to their target organs. Because of their hydrophobicity, they must be complexed with a plasma protein. Albumin can act as a nonspecific carrier and does carry aldosterone. However, specific steroid-carrier plasma proteins bind the steroid hormones more tightly than does albumin (for example, corticosteroid-binding globulin, or transcortin, is responsible for transporting cortisol). A number of genetic diseases are

1	bind the steroid hormones more tightly than does albumin (for example, corticosteroid-binding globulin, or transcortin, is responsible for transporting cortisol). A number of genetic diseases are caused by deficiencies in specific steps in the biosynthesis of steroid hormones. Some representative diseases are described in Figure 18.25.

1	A. Synthesis

1	Synthesis involves shortening the hydrocarbon chain of cholesterol and hydroxylating the steroid nucleus. The initial and rate-limiting reaction converts cholesterol to the 21-carbon pregnenolone. It is catalyzed by the cholesterol side-chain cleavage enzyme, a cytochrome P450 (CYP) mixed function oxidase of the inner mitochondrial membrane (see p. 149) that is also known as P450scc and desmolase. NADPH and O2 are required for the reaction. The cholesterol substrate can be newly synthesized, taken up from lipoproteins, or released by an esterase from cholesteryl esters stored in the cytosol of steroidogenic tissues. The cholesterol moves to the outer mitochondrial membrane. An important control point is the subsequent movement from the outer to the inner mitochondrial membrane. This process is mediated by StAR (steroidogenic acute regulatory) protein. Pregnenolone is the parent compound for all steroid hormones (see Fig. 18.25). It is oxidized and then isomerized to progesterone,

1	is mediated by StAR (steroidogenic acute regulatory) protein. Pregnenolone is the parent compound for all steroid hormones (see Fig. 18.25). It is oxidized and then isomerized to progesterone, which is further modified to the other steroid hormones by CYP protein–catalyzed hydroxylation reactions in the SER and mitochondria. A defect in the activity or amount of an enzyme in this pathway can lead to a deficiency in the synthesis of hormones beyond the affected step and to an excess in the hormones or metabolites before that step. Because all members of the pathway have potent biologic activity, serious metabolic imbalances occur with enzyme deficiencies (see Fig. 18.25). Collectively, these disorders are known as the congenital adrenal hyperplasias (CAH), because they result in enlarged adrenals. [Note: Addison disease, due to autoimmune destruction of the adrenal cortex, is characterized by adrenocortical insufficiency.]

1	B. Adrenal cortical steroid hormones

1	Steroid hormones are synthesized and secreted in response to hormonal signals. The corticosteroids and androgens are made in different regions of the adrenal cortex and are secreted into blood in response to different signals. [Note: The adrenal medulla makes catecholamines (see p. 285).] 1. Cortisol: Its production in the middle layer (zona fasciculata) of the adrenal cortex is controlled by the hypothalamus, to which the pituitary gland is attached (Fig. 18.26). In response to severe stress (for example, infection), corticotropin-releasing hormone (CRH), produced by the hypothalamus, travels through capillaries to the anterior lobe of the pituitary, where it induces the production and secretion of adrenocorticotropic hormone (ACTH), a peptide. ACTH stimulates the adrenal cortex to synthesize and secrete the glucocorticoid cortisol, the stress hormone. [Note: ACTH binds to a membrane G protein–coupled receptor, resulting in cyclic AMP (cAMP) production and activation of protein

1	synthesize and secrete the glucocorticoid cortisol, the stress hormone. [Note: ACTH binds to a membrane G protein–coupled receptor, resulting in cyclic AMP (cAMP) production and activation of protein kinase A ([PKA] see p. 94). PKA phosphorylates and activates both the esterase that converts cholesteryl ester to free cholesterol and StAR protein.] Cortisol allows the body to respond to stress through its effects on intermediary metabolism (for example, increased gluconeogenesis) and the inflammatory and immune responses (which are decreased). As cortisol levels rise, the release of CRH and ACTH is inhibited. [Note: The reduction of cortisol in CAH results in a rise in ACTH that causes adrenal hyperplasia.] 2. Aldosterone: Its production in the outer layer (zona glomerulosa) of the adrenal cortex is induced by a decrease in the plasma Na+/potassium (K+) ratio and by the hormone angiotensin II (Ang-II). Ang-II (an octapeptide) is produced from angiotensin I ([Ang-I] a decapeptide) by

1	cortex is induced by a decrease in the plasma Na+/potassium (K+) ratio and by the hormone angiotensin II (Ang-II). Ang-II (an octapeptide) is produced from angiotensin I ([Ang-I] a decapeptide) by angiotensinconverting enzyme (ACE), an enzyme found predominantly in the lungs but also distributed widely in the body. [Note: Ang-I is produced in the blood by cleavage of an inactive precursor, angiotensinogen, secreted by the liver. Cleavage is catalyzed by renin, made and secreted by the kidneys.] Ang-II binds to cell surface receptors. However, in contrast to ACTH, its effects are mediated through the phosphatidylinositol 4,5bisphosphate pathway (see p. 205) and not by cAMP. Aldosterone’s primary effect is on the kidney tubules, where it stimulates Na+ and water uptake and K+ excretion (Fig. 18.27). [Note: An effect of aldosterone is an increase in blood pressure. Competitive inhibitors of ACE are used to treat renin-dependent hypertension.] 3. Androgens: Both the inner (zona

1	(Fig. 18.27). [Note: An effect of aldosterone is an increase in blood pressure. Competitive inhibitors of ACE are used to treat renin-dependent hypertension.] 3. Androgens: Both the inner (zona reticularis) and middle layers of the adrenal cortex produce androgens, primarily dehydroepiandrosterone and androstenedione. Although adrenal androgens themselves are weak, they are converted by aromatase (CYP19) to testosterone, a stronger androgen, in the testes and to estrogens in the ovaries (primarily) of premenopausal women. [Note: Postmenopausal women produce estrogen at extragonadal sites such as the breast. Aromatase inhibitors are used in the treatment of estrogen-responsive breast cancer in these women.]

1	C. Gonadal steroid hormones The testes and ovaries (gonads) synthesize hormones necessary for sexual differentiation and reproduction. A single hypothalamic-releasing factor, gonadotropin-releasing hormone, stimulates the anterior pituitary to release the glycoproteins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Like ACTH, LH and FSH bind to surface receptors and cause an increase in cAMP. LH stimulates the testes to produce testosterone and the ovaries to produce estrogens and progesterone (see Fig. 18.27). FSH regulates the growth of ovarian follicles and stimulates testicular spermatogenesis. D. Mechanism

1	Each steroid hormone diffuses across the plasma membrane of its target cell and binds to a specific cytosolic or nuclear receptor. These receptor–ligand complexes accumulate in the nucleus, dimerize, and bind to specific regulatory DNA sequences (hormone response elements [HRE]) in association with coactivator proteins, thereby causing increased transcription of targeted genes (Fig. 18.28). An HRE is found in the promoter or an enhancer element (see p. 440) for genes that respond to a specific steroid hormone, thus insuring coordinated regulation of these genes. Hormone–receptor complexes can also inhibit transcription in association with corepressors. [Note: The binding of a hormone to its receptor causes a conformational change in the receptor that uncovers its DNA-binding domain, allowing the complex to interact through a zinc finger motif with the appropriate DNA sequence. Receptors for the steroid hormones, plus those for thyroid hormone, retinoic acid (see p. 386), and

1	allowing the complex to interact through a zinc finger motif with the appropriate DNA sequence. Receptors for the steroid hormones, plus those for thyroid hormone, retinoic acid (see p. 386), and 1,25-dihydroxycholecalciferol (vitamin D; see p. 390), are members of a superfamily of structurally related gene regulators that function in a similar way.]

1	E. Further metabolism Steroid hormones are generally converted into inactive metabolic excretion products in the liver. Reactions include reduction of unsaturated bonds and the introduction of additional hydroxyl groups. The resulting structures are made more soluble by conjugation with glucuronic acid or sulfate (from 3′phosphoadenosyl-5′-phosphosulfate; see p. 162). These conjugated metabolites are fairly water soluble and do not need protein carriers. They are eliminated in feces and urine. VIII. CHAPTER SUMMARY

1	Cholesterol is a hydrophobic compound, with a single hydroxyl group located at carbon 3 of the A ring, to which a fatty acid (FA) can be attached, producing an even more hydrophobic cholesteryl ester. Cholesterol is synthesized by virtually all human tissues, although primarily by the liver, intestine, adrenal cortex, and reproductive tissues (Fig. 18.29). All the carbon atoms are provided by acetyl coenzyme A (CoA), and nicotinamide adenine dinucleotide phosphate provides the reducing equivalents. The pathway is driven by hydrolysis of the high-energy thioester bond of acetyl CoA and the terminal phosphate bond of ATP. Synthesis requires enzymes of the cytosol, smooth endoplasmic reticulum (SER), and peroxisomes. The rate-limiting and regulated step in cholesterol synthesis is catalyzed by the SER-membrane protein hydroxymethylglutaryl coenzyme A (HMG CoA) reductase, which produces mevalonate from HMG CoA. The enzyme is regulated by a number of mechanisms: 1) increased expression of

1	by the SER-membrane protein hydroxymethylglutaryl coenzyme A (HMG CoA) reductase, which produces mevalonate from HMG CoA. The enzyme is regulated by a number of mechanisms: 1) increased expression of the reductase gene when cholesterol levels are low, via the transcription factor, sterol regulatory element–binding protein-2 (SREBP-2), bound to a sterol regulatory element (SRE), resulting in increased enzyme and, therefore, cholesterol, synthesis; 2) accelerated degradation of the reductase protein when cholesterol levels are high; 3) phosphorylation (causing inactivation of reductase activity) by adenosine monophosphate–activated protein kinase [AMPK] and dephosphorylation (activation) by a phosphoprotein phosphatase; and 4) hormonal regulation by insulin and glucagon. Statins are competitive inhibitors of HMG CoA reductase. These drugs are used to decrease plasma cholesterol in patients with hypercholesterolemia. The ring structure of cholesterol cannot be degraded in humans.

1	Cholesterol is eliminated from the body either by conversion to bile salts or by secretion into the bile. Bile salts and phosphatidylcholine (PC) are quantitatively the most important organic components of bile. The rate-limiting step in bile acid synthesis is catalyzed by cholesterol-7-αhydroxylase, which is inhibited by bile acids. Before the bile acids leave the liver, they are conjugated to a molecule of either glycine or taurine, producing the conjugated bile salts glycocholic or taurocholic acid and glycochenodeoxycholic or taurochenodeoxycholic acid. Bile salts (deprotonated) are more amphipathic than bile acids (protonated) and, therefore, are more effective emulsifiers of dietary fat. Intestinal bacteria can remove the glycine and taurine as well as a hydroxyl group from the steroid nucleus, producing the secondary bile salts, deoxycholic and lithocholic acids. Bile salts are efficiently reabsorbed (>95%) in the intestinal ileum by a sodium–bile salt cotransporter, returned

1	nucleus, producing the secondary bile salts, deoxycholic and lithocholic acids. Bile salts are efficiently reabsorbed (>95%) in the intestinal ileum by a sodium–bile salt cotransporter, returned to the blood, and carried by albumin back to the liver where they are taken up by the hepatic isoform of the cotransporter and reused (enterohepatic circulation, which bile acid sequestrants reduce). If more cholesterol enters the bile than can be solubilized by the available bile salts and PC, cholesterol gallstone disease (cholelithiasis) can occur.

1	The plasma lipoproteins (see Fig. 18.29) include chylomicrons, verylow-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). They function to keep lipids (primarily triacylglycerol [TAG] and cholesteryl esters) soluble as they transport them between tissues. Lipoproteins are composed of a neutral lipid (TAG, cholesteryl esters, or both) core surrounded by a shell of amphipathic apolipoproteins, phospholipid, and nonesterified cholesterol. Chylomicrons are assembled in intestinal mucosal cells from dietary lipids (primarily TAG). Each nascent chylomicron particle has one molecule of apolipoprotein (apo) B-48. They are released from the cells into the lymphatic system and travel to the blood, where they receive apo C-II and apo E from HDL. Apo C-II activates endothelial lipoprotein lipase (LPL), which degrades the TAG in chylomicrons to FA and glycerol. The FA that are released are stored (in adipose

1	apo C-II and apo E from HDL. Apo C-II activates endothelial lipoprotein lipase (LPL), which degrades the TAG in chylomicrons to FA and glycerol. The FA that are released are stored (in adipose tissue) or used for energy (in muscle). The glycerol is metabolized by the liver. Patients with a deficiency of LPL or apo C-II show a dramatic accumulation of chylomicrons in the plasma (type I hyperlipoproteinemia or familial chylomicronemia) even if fasted. After most of the TAG is removed, apo C-II is returned to HDL, and the chylomicron remnant, carrying most of the dietary cholesterol, binds to a liver receptor that recognizes apo E. The particle is endocytosed, and its contents degraded by lysosomal enzymes. Defective uptake of these remnants (and IDL) causes type III hyperlipoproteinemia or dysbetalipoproteinemia. Nascent VLDL are produced in the liver and are composed predominantly of TAG. They contain a single molecule of apo B-100. Like chylomicrons, VLDL receive apo C-II and apo E

1	dysbetalipoproteinemia. Nascent VLDL are produced in the liver and are composed predominantly of TAG. They contain a single molecule of apo B-100. Like chylomicrons, VLDL receive apo C-II and apo E from HDL in the plasma. VLDL carry hepatic TAG to the peripheral tissues where LPL degrades the lipid. Additionally, the VLDL particle receives cholesteryl esters from HDL in exchange for TAG. This process is accomplished by cholesteryl ester transfer protein (CETP). VLDL in the plasma is first converted to IDL and then to LDL, a much smaller, denser particle. Apo C-II and apo E are returned to HDL, but the LDL retains apo B-100, which is recognized by receptors on peripheral tissues and the liver. LDL undergo receptor-mediated endocytosis, and their contents are degraded in the lysosomes. The protease proprotein convertase subtilisin/kexin type 9 (PCSK9) prevents receptor recycling. Defects in the synthesis of functional LDL receptors causes type IIa hyperlipoproteinemia (familial

1	The protease proprotein convertase subtilisin/kexin type 9 (PCSK9) prevents receptor recycling. Defects in the synthesis of functional LDL receptors causes type IIa hyperlipoproteinemia (familial hypercholesterolemia [FH]). The endocytosed cholesterol decreases expression of HMG CoA reductase (and LDL receptors) through prevention of SREBP-2 binding to the SRE. Some of it can be esterified by acyl CoA:cholesterol acyltransferase (ACAT) and stored. HDL are created by lipidation of apo A-1 synthesized in the liver and intestine. They have a number of functions, including 1) serving as a circulating reservoir of apo C-II and apo E for chylomicrons and VLDL; 2) removing cholesterol from peripheral tissues via ABCA1 and esterifying it using lecithin:cholesterol acyl transferase (LCAT), a liver-synthesized plasma enzyme that is activated by apo A-1; and 3) delivering these cholesteryl esters to the liver (reverse cholesterol transport) for uptake via scavenger receptor-B1 (SR-B1).

1	Cholesterol is the precursor of all classes of steroid hormones, which include glucocorticoids, mineralocorticoids, and the sex hormones (androgens, estrogens, and progestins). Synthesis, using primarily cytochrome P450 mixed function oxidases, occurs in the adrenal cortex (cortisol in the zona fasciculata, aldosterone in the zona glomerulosa, and androgens in the zona reticularis), ovaries and placenta (estrogens and progestins), and testes (testosterone). The initial and rate-limiting step is the conversion of cholesterol to pregnenolone by the side-chain cleavage enzyme P450scc. Deficiencies in synthesis lead to congenital adrenal hyperplasia (CAH). Each steroid hormone diffuses across the plasma membrane of its target cell and binds to a specific intracellular receptor. These receptor–hormone complexes accumulate in the nucleus, dimerize, and bind to specific regulatory DNA sequences (hormone response elements) in association with coactivator proteins, thereby causing increased

1	complexes accumulate in the nucleus, dimerize, and bind to specific regulatory DNA sequences (hormone response elements) in association with coactivator proteins, thereby causing increased transcription of targeted genes. In association with corepressors, transcription is decreased.

1	lipoproteins; TAG = triacylglycerol; NADPH = nicotinamide adenine dinucleotide phosphate; C = carbon. Choose the ONE best answer. 8.1. Mice were genetically engineered to contain hydroxymethylglutaryl coenzyme A reductase in which serine 871, a phosphorylation site, was replaced by alanine. Which of the following statements concerning the modified form of the enzyme is most likely to be correct? A. The enzyme is nonresponsive to ATP depletion. B. The enzyme is nonresponsive to statin drugs. C. The enzyme is nonresponsive to the sterol response element–sterol response element–binding protein system. D. The enzyme is unable to be degraded by the ubiquitin–proteasome system.

1	C. The enzyme is nonresponsive to the sterol response element–sterol response element–binding protein system. D. The enzyme is unable to be degraded by the ubiquitin–proteasome system. Correct answer = A. The reductase is regulated by covalent phosphorylation and dephosphorylation. Depletion of ATP results in a rise in adenosine monophosphate (AMP), which activates AMP kinase (AMPK), thereby phosphorylating and inactivating the reductase. In the absence of the serine, a common phosphorylation site, the enzyme cannot be phosphorylated by AMPK. The enzyme is also regulated physiologically through changes in transcription and degradation and pharmacologically by statin drugs (competitive inhibitors), but none of these depends on serine phosphorylation.

1	8.2. Calculate the amount of cholesterol in the low-density lipoproteins in an individual whose fasting blood gave the following lipid-panel test results: total cholesterol = 300 mg/dl, high-density lipoprotein cholesterol = 25 mg/dl, triglycerides = 150 mg/dl. A. 55 mg/dl B. 95 mg/dl C. 125 mg/dl D. 245 mg/dl Correct answer = D. The total cholesterol in the blood of a fasted individual is equal to the sum of the cholesterol in low-density lipoproteins plus the cholesterol in high-density lipoproteins plus the cholesterol in very-lowdensity lipoproteins (VLDL). This last term is calculated by dividing the triacylglycerol value by 5 because cholesterol accounts for about 1/5 of the volume of VLDL in fasted blood. For Questions 18.3 and 18.4, use the following scenario.

1	For Questions 18.3 and 18.4, use the following scenario. A young girl with a history of severe abdominal pain was taken to her local hospital at 5 a.m. in severe distress. Blood was drawn, and the plasma appeared milky, with the triacylglycerol level >2,000 mg/dl (normal = 4–150 mg/dl). The patient was placed on a diet extremely limited in fat but supplemented with medium-chain triglycerides. 8.3. Which of the following lipoprotein particles are most likely responsible for the appearance of the patient’s plasma? A. Chylomicrons B. High-density lipoproteins C. Intermediate-density lipoproteins D. Low-density lipoproteins E. Very-low-density lipoproteins

1	A. Chylomicrons B. High-density lipoproteins C. Intermediate-density lipoproteins D. Low-density lipoproteins E. Very-low-density lipoproteins Correct answer = A. The milky appearance of her plasma was a result of triacylglycerol-rich chylomicrons. Because 5 a.m. is presumably several hours after her evening meal, the patient must have difficulty degrading these lipoprotein particles. Intermediate-, low-, and high-density lipoproteins contain primarily cholesteryl esters, and, if one or more of these particles was elevated, it would cause hypercholesterolemia. Very-low-density lipoproteins do not cause the described milky appearance of plasma. 8.4. Which one of the following proteins is most likely to be deficient in this patient? A. Apolipoprotein A-I B. Apolipoprotein B-48 C. Apolipoprotein C-II D. Cholesteryl ester transfer protein E. Microsomal triglyceride transfer protein

1	A. Apolipoprotein A-I B. Apolipoprotein B-48 C. Apolipoprotein C-II D. Cholesteryl ester transfer protein E. Microsomal triglyceride transfer protein Correct answer = C. The triacylglycerol (TAG) in chylomicrons is degraded by endothelial lipoprotein lipase (LPL), which requires apolipoprotein (apo) C-II as a coenzyme. Deficiency of LPL or apo C-II results in decreased ability to degrade chylomicrons to their remnants, which get cleared (via apo E) by liver receptors. Apo A-I is the coenzyme for lecithin:cholesterol acyltransferase; apo B-48 is the characteristic structural protein of chylomicrons; cholesteryl ester transfer protein catalyzes the cholesteryl ester–TAG exchange between high-density and very-low-density lipoproteins (VLDL); and microsomal triglyceride transfer protein is involved in the formation, not degradation, of chylomicrons (and VLDL). 8.5. Complete the table below for an individual with classic 21-α-hydroxylase deficiency relative to a normal individual.

1	8.5. Complete the table below for an individual with classic 21-α-hydroxylase deficiency relative to a normal individual. How might the results be changed if this individual were deficient in 17-αhydroxylase, rather than 21-α-hydroxylase? Classic 21-α-hydroxylase deficiency causes mineralocorticoids (aldosterone) and glucocorticoids (cortisol) to be virtually absent. Because aldosterone increases blood pressure, and cortisol increases blood glucose, their deficiencies result in a decrease in blood pressure and blood glucose, respectively. Cortisol normally feeds back to inhibit adrenocorticotropic hormone (ACTH) release by the pituitary, and, so, its absence results in an elevation in ACTH. The loss of 21-α-hydroxylase pushes progesterone and pregnenolonetoandrogensynthesisand,therefore,causesandrostenedionelevelstorise.With17-α-hydroxylasedeficiency,sexhormonesynthesiswouldbedecreased.Mineralocorticoidproductionwouldbeincreased,leadingtohypertension.

1	UNIT IV Nitrogen Metabolism Amino Acids: Nitrogen Disposal 19 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Unlike fats and carbohydrates, amino acids are not stored by the body. That is, no protein exists whose sole function is to maintain a supply of amino acids for future use. Therefore, amino acids must be obtained from the diet, synthesized de novo, or produced from the degradation of body protein. Any amino acids in excess of the biosynthetic needs of the cell are rapidly degraded. The first phase of catabolism involves the removal of the α-amino groups (usually by transamination and subsequent oxidative deamination), forming ammonia and the corresponding α-keto acids, the carbon skeletons of amino acids. A portion of the free ammonia is excreted in the urine, but most is used in the synthesis of urea (Fig. 19.1), which is quantitatively the most important route for disposing of nitrogen from the body. In the second phase of amino acid catabolism, described in Chapter 20, the carbon skeletons of the α-keto acids are converted to common intermediates of energy-producing metabolic

1	from the body. In the second phase of amino acid catabolism, described in Chapter 20, the carbon skeletons of the α-keto acids are converted to common intermediates of energy-producing metabolic pathways. These compounds can be metabolized to carbon dioxide (CO2) and water (H2O), glucose, fatty acids, or ketone bodies by the central pathways of metabolism described in Chapters 8–13 and 16.

1	II. OVERALL NITROGEN METABOLISM Amino acid catabolism is part of the larger process of the metabolism of nitrogen-containing molecules. Nitrogen enters the body in a variety of compounds present in food, the most important being amino acids contained in dietary protein. Nitrogen leaves the body as urea, ammonia, and other products derived from amino acid metabolism (such as creatinine, see p. 287). The role of body proteins in these transformations involves two important concepts: the amino acid pool and protein turnover. A. Amino acid pool

1	Free amino acids are present throughout the body, such as in cells, blood, and the extracellular fluids. For the purpose of this discussion, envision all of these amino acids as if they belonged to a single entity, called the amino acid pool. This pool is supplied by three sources: 1) amino acids provided by the degradation of endogenous (body) proteins, most of which are reutilized; 2) amino acids derived from exogenous (dietary) protein; and 3) nonessential amino acids synthesized from simple intermediates of metabolism (Fig. 19.2). Conversely, the amino acid pool is depleted by three routes: 1) synthesis of body protein, 2) consumption of amino acids as precursors of essential nitrogen-containing small molecules, and 3) conversion of amino acids to glucose, glycogen, fatty acids, and ketone bodies or oxidation to CO2 + H2O (see Fig. 19.2). Although the amino acid pool is small (comprising ~90–100 g of amino acids) in comparison with the amount of protein in the body (~12 kg in a

1	ketone bodies or oxidation to CO2 + H2O (see Fig. 19.2). Although the amino acid pool is small (comprising ~90–100 g of amino acids) in comparison with the amount of protein in the body (~12 kg in a 70-kg man), it is conceptually at the center of whole-body nitrogen metabolism.

1	In healthy, well-fed individuals, the input to the amino acid pool is balanced by the output. That is, the amount of amino acids contained in the pool is constant. The amino acid pool is said to be in a steady state, and the individual is said to be in nitrogen balance (see p. 367). B. Protein turnover Most proteins in the body are constantly being synthesized and then degraded (turned over), permitting the removal of abnormal or unneeded proteins. For many proteins, regulation of synthesis determines the concentration of protein in the cell, with protein degradation assuming a minor role. For other proteins, the rate of synthesis is constitutive (that is, essentially constant), and cellular levels of the protein are controlled by selective degradation. 1.

1	1. Rate: In healthy adults, the total amount of protein in the body remains constant because the rate of protein synthesis is just sufficient to replace the protein that is degraded. This process, called protein turnover, leads to the hydrolysis and resynthesis of 300–400 g of body protein each day. The rate of protein turnover varies widely for individual proteins. Short-lived proteins (for example, many regulatory proteins and misfolded proteins) are rapidly degraded, having half-lives measured in minutes or hours. Long-lived proteins, with half-lives of days to weeks, constitute the majority of proteins in the cell. Structural proteins, such as collagen, are metabolically stable and have half-lives measured in months or years. 2.

1	2. Protein degradation: There are two major enzyme systems responsible for degrading proteins: the ATP-dependent ubiquitin (Ub)–proteasome system of the cytosol and the ATP-independent degradative enzyme system of the lysosomes. Proteasomes selectively degrade damaged or short-lived proteins. Lysosomes use acid hydrolases (see p. 162) to nonselectively degrade intracellular proteins (autophagy) and extracellular proteins (heterophagy), such as plasma proteins, that are taken into the cell by endocytosis.

1	a. Ubiquitin–proteasome system: Proteins selected for degradation by the cytosolic ubiquitin–proteasome system are first modified by the covalent attachment of Ub, a small, globular, nonenzymic protein that is highly conserved across eukaryotic species. Ubiquitination of the target substrate occurs through isopeptide linkage of the α-carboxyl group of the C-terminal glycine of Ub to the ε-amino group of a lysine in the protein substrate by a three-step, enzyme-catalyzed, ATP-dependent process. [Note: Enzyme 1 (E1, an activating enzyme) activates Ub, which is then transferred to E2 (a conjugating enzyme). E3 (a ligase) identifies the protein to be degraded and interacts with E2-Ub. There are many more E3 proteins than there are E1 or E2.] The consecutive addition of four or more Ub molecules to the target protein generates a polyubiquitin chain. Proteins tagged with Ub chains are recognized by a large, barrel-shaped, macromolecular, proteolytic complex called a proteasome (Fig. 19.3).

1	to the target protein generates a polyubiquitin chain. Proteins tagged with Ub chains are recognized by a large, barrel-shaped, macromolecular, proteolytic complex called a proteasome (Fig. 19.3). The proteasome unfolds, deubiquitinates, and cuts the target protein into fragments that are then further degraded by cytosolic proteases to amino acids, which enter the amino acid pool. The Ub is recycled. It is noteworthy that the selective degradation of proteins by the ubiquitin–proteosome complex (unlike simple hydrolysis by proteolytic enzymes) requires ATP hydrolysis.

1	b. Degradation signals: Because proteins have different half-lives, it is clear that protein degradation cannot be random but, rather, is influenced by some structural aspect of the protein that serves as a degradation signal, which is recognized and bound by an E3. The half-life of a protein is also influenced by the amino (N)-terminal residue, the so-called N-end rule, and ranges from minutes to hours. Destabilizing N-terminal amino acids include arginine and posttranslationally modified amino acids such as acetylated alanine. In contrast, serine is a stabilizing amino acid. Additionally, proteins rich in sequences containing proline, glutamate, serine, and threonine (called PEST sequences after the one-letter designations for these amino acids) are rapidly ubiquitinated and degraded and, therefore, have short half-lives. III. DIETARY PROTEIN DIGESTION

1	III. DIETARY PROTEIN DIGESTION Most of the nitrogen in the diet is consumed in the form of protein, typically amounting to 70–100 g/day in the American diet (see Fig. 19.2). Proteins are generally too large to be absorbed by the intestine. [Note: An example of an exception to this rule is that newborns can take up maternal antibodies in breast milk.] Therefore, proteins must be hydrolyzed to yield di-and tripeptides as well as individual amino acids, which can be absorbed. Proteolytic enzymes responsible for degrading proteins are produced by three different organs: the stomach, the pancreas, and the small intestine (Fig. 19.4). A. Digestion by gastric secretion The digestion of proteins begins in the stomach, which secretes gastric juice, a unique solution containing hydrochloric acid (HCl) and the proenzyme pepsinogen. 1.

1	The digestion of proteins begins in the stomach, which secretes gastric juice, a unique solution containing hydrochloric acid (HCl) and the proenzyme pepsinogen. 1. Hydrochloric acid: Stomach HCl is too dilute (pH 2–3) to hydrolyze proteins. The acid, secreted by the parietal cells of the stomach, functions instead to kill some bacteria and to denature proteins, thereby making them more susceptible to subsequent hydrolysis by proteases. 2.

1	2. Pepsin: This acid-stable endopeptidase is secreted by the chief cells of the stomach as an inactive zymogen (or proenzyme), pepsinogen. [Note: In general, zymogens contain extra amino acids in their sequences that prevent them from being catalytically active. Removal of these amino acids permits the proper folding required for an active enzyme.] In the presence of HCl, pepsinogen undergoes a conformational change that allows it to cleave itself (autocatalysis) to the active form, pepsin, which releases polypeptides and a few free amino acids from dietary proteins. B. Digestion by pancreatic enzymes

1	B. Digestion by pancreatic enzymes On entering the small intestine, the polypeptides produced in the stomach by the action of pepsin are further cleaved to oligopeptides and amino acids by a group of pancreatic proteases that include both endopeptidases (that cleave within) and exopeptidases (that cut at an end). [Note: Bicarbonate (HCO3−), secreted by the pancreas in response to the intestinal hormone secretin, raises the intestinal pH.] 1. Specificity: Each of these enzymes has a different specificity for the amino acid R-groups adjacent to the susceptible peptide bond (Fig. 19.5). For example, trypsin cleaves only when the carbonyl group of the peptide bond is contributed by arginine or lysine. These enzymes, like pepsin described above, are synthesized and secreted as inactive zymogens. 2. Zymogen release: The release and activation of the pancreatic zymogens are mediated by the secretion of cholecystokinin, a polypeptide hormone of the small intestine (see p. 176). 3.

1	2. Zymogen release: The release and activation of the pancreatic zymogens are mediated by the secretion of cholecystokinin, a polypeptide hormone of the small intestine (see p. 176). 3. Zymogen activation: Enteropeptidase (also called enterokinase), a serine protease synthesized by and present on the luminal (apical) surface of intestinal mucosal cells (enterocytes) of the brush border, converts the pancreatic zymogen trypsinogen to trypsin by removal of a hexapeptide from the N-terminus of trypsinogen. Trypsin subsequently converts other trypsinogen molecules to trypsin by cleaving a limited number of specific peptide bonds in the zymogen. Thus, enteropeptidase unleashes a cascade of proteolytic activity because trypsin is the common activator of all the pancreatic zymogens (see Fig. 19.5). 4.

1	4. Digestion abnormalities: In individuals with a deficiency in pancreatic secretion (for example, because of chronic pancreatitis, cystic fibrosis, or surgical removal of the pancreas), the digestion and absorption of fat and protein are incomplete. This results in the abnormal appearance of lipids in the feces (a condition called steatorrhea; see p. 177) as well as undigested protein. Celiac disease (celiac sprue) is a disease of malabsorption resulting from immune-mediated damage to the small intestine in response to ingestion of gluten (or gliadin produced from gluten), a protein found in wheat, barley, and rye. C. Digestion of oligopeptides by small intestine enzymes The luminal surface of the enterocytes contains aminopeptidase, an exopeptidase that repeatedly cleaves the N-terminal residue from oligopeptides to produce even smaller peptides and free amino acids. D. Amino acid and small peptide intestinal absorption

1	D. Amino acid and small peptide intestinal absorption Most free amino acids are taken into enterocytes via sodium-dependent secondary active transport by solute carrier (SLC) proteins of the apical membrane. At least seven different transport systems with overlapping amino acid specificities are known. Di-and tripeptides, however, are taken up by a proton-linked peptide transporter (PepT1). The peptides are then hydrolyzed to free amino acids. Regardless of their source, free amino acids are released from enterocytes into the portal system by sodium-independent transporters of the basolateral membrane. Therefore, only free amino acids are found in the portal vein after a meal containing protein. These amino acids are either metabolized by the liver or released into the general circulation. [Note: Branched-chain amino acids (BCAA) are not metabolized by the liver but, instead, are sent from the liver to muscle via the blood.] E. Absorption abnormalities

1	The small intestine and the proximal tubules of the kidneys have common transport systems for amino acid uptake. Consequently, a defect in any one of these systems results in an inability to absorb particular amino acids into the intestine and into the kidney tubules. For example, one system is responsible for the uptake of cystine and the dibasic amino acids ornithine, arginine, and lysine (represented as COAL). In the inherited disorder cystinuria, this carrier system is defective, and all four amino acids appear in the urine (Fig. 19.6). Cystinuria occurs at a frequency of 1 in 7,000 individuals, making it one of the most common inherited diseases and the most common genetic error of amino acid transport. The disease expresses itself clinically by the precipitation of cystine to form kidney stones (calculi), which can block the urinary tract. Oral hydration is an important part of treatment for this disorder. [Note: Defects in the uptake of tryptophan by a neutral amino acid

1	form kidney stones (calculi), which can block the urinary tract. Oral hydration is an important part of treatment for this disorder. [Note: Defects in the uptake of tryptophan by a neutral amino acid transporter can result in Hartnup disorder and pellagra-like (see p. 384) dermatologic and neurologic symptoms.]

1	IV. NITROGEN REMOVAL FROM AMINO ACIDS The presence of the α-amino group keeps amino acids safely locked away from oxidative breakdown. Removing the α-amino group is essential for producing energy from any amino acid and is an obligatory step in the catabolism of all amino acids. Once removed, this nitrogen can be incorporated into other compounds or excreted as urea, with the carbon skeletons being metabolized. This section describes transamination and oxidative deamination, reactions that ultimately provide ammonia and aspartate, the two sources of urea nitrogen (see p. 253). A. Transamination: Funneling amino groups to glutamate

1	The first step in the catabolism of most amino acids is the transfer of their α-amino group to α-ketoglutarate (Fig. 19.7), producing an α-keto acid (derived from the original amino acid) and glutamate. α-Ketoglutarate plays a pivotal role in amino acid metabolism by accepting the amino groups from most amino acids, thereby becoming glutamate. Glutamate produced by transamination can be oxidatively deaminated (see B. below) or used as an amino group donor in the synthesis of nonessential amino acids. This transfer of amino groups from one carbon skeleton to another is catalyzed by a family of enzymes called aminotransferases (also called transaminases). These enzymes are found in the cytosol and mitochondria of cells throughout the body. All amino acids, with the exception of lysine and threonine, participate in transamination at some point in their catabolism. [Note: These two amino acids lose their α-amino groups by deamination (see pp. 265–266).] 1. Substrate specificity: Each

1	threonine, participate in transamination at some point in their catabolism. [Note: These two amino acids lose their α-amino groups by deamination (see pp. 265–266).] 1. Substrate specificity: Each aminotransferase is specific for one or, at most, a few amino group donors. Aminotransferases are named after the specific amino group donor, because the acceptor of the amino group is almost always α-ketoglutarate. Two important aminotransferase reactions are catalyzed by alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as shown in Figure 19.8.

1	a. Alanine aminotransferase: ALT is present in many tissues. The enzyme catalyzes the transfer of the amino group of alanine to α-ketoglutarate, resulting in the formation of pyruvate and glutamate. The reaction is readily reversible. However, during amino acid catabolism, this enzyme (like most aminotransferases) functions in the direction of glutamate synthesis. [Note: In effect, glutamate acts as a collector of nitrogen from most amino acids.] b. Aspartate aminotransferase: AST is an exception to the rule that aminotransferases funnel amino groups to form glutamate. During amino acid catabolism, AST primarily transfers amino groups from glutamate to oxaloacetate, forming aspartate, which is used as a source of nitrogen in the urea cycle (see p. 255). Like other transaminations, the AST reaction is reversible.

1	2. Mechanism: All aminotransferases require the coenzyme pyridoxal phosphate (a derivative of vitamin B6; see p. 382), which is covalently linked to the ε-amino group of a specific lysine residue at the active site of the enzyme. Aminotransferases act by transferring the amino group of an amino acid to the pyridoxal part of the coenzyme to generate pyridoxamine phosphate. The pyridoxamine form of the coenzyme then reacts with an α-keto acid to form an amino acid, at the same time regenerating the original aldehyde form of the coenzyme. Figure 19.9 shows these two component reactions for the transamination catalyzed by AST. 3.

1	3. Equilibrium: For most transamination reactions, the equilibrium constant is near 1. This allows the reaction to function in both amino acid degradation through removal of α-amino groups (for example, after consumption of a protein-rich meal) and biosynthesis of nonessential amino acids through addition of amino groups to the carbon skeletons of α-keto acids (for example, when the supply of amino acids from the diet is not adequate to meet the synthetic needs of cells). 4.

1	4. Diagnostic value: Aminotransferases are normally intracellular enzymes, with the low levels found in the plasma representing the release of cellular contents during normal cell turnover. Elevated plasma levels of aminotransferases indicate damage to cells rich in these enzymes. For example, physical trauma or a disease process can cause cell lysis, resulting in release of intracellular enzymes into the blood. Two aminotransferases, AST and ALT, are of particular diagnostic value when they are found in the plasma. a.

1	a. Hepatic disease: Plasma AST and ALT are elevated in nearly all hepatic diseases but are particularly high in conditions that cause extensive cell necrosis, such as severe viral hepatitis, toxic injury, and prolonged circulatory collapse. ALT is more specific than AST for liver disease, but the latter is more sensitive because the liver contains larger amounts of AST. Serial measurements of AST and ALT (liver function tests) are often useful in determining the course of liver damage. Figure 19.10 shows the early release of ALT into the blood, following ingestion of a liver toxin. [Note: The elevation in bilirubin results from hepatocellular damage that decreases the hepatic conjugation and excretion of bilirubin (see p. 282).] b. Nonhepatic disease: Aminotransferases may be elevated in nonhepatic diseases such as those that cause damage to cardiac or skeletal muscle. However, these disorders can usually be distinguished clinically from liver disease.

1	B. Oxidative deamination: Amino group removal

1	In contrast to transamination reactions that transfer amino groups, oxidative deamination reactions result in the liberation of the amino group as free ammonia (Fig. 19.11). These reactions occur primarily in the liver and kidney. They provide α-keto acids that can enter the central pathways of energy metabolism and ammonia, which is a source of nitrogen in hepatic urea synthesis. [Note: Ammonia exists primarily as ammonium (NH4+) in aqueous solution, but it is the unionized form (NH3) that crosses membranes.] 1. Glutamate dehydrogenase: As described above, the amino groups of most amino acids are ultimately funneled to glutamate by means of transamination with α-ketoglutarate. Glutamate is unique in that it is the only amino acid that undergoes rapid oxidative deamination, a reaction catalyzed by glutamate dehydrogenase ([GDH], see Fig. 19.11). Therefore, the sequential action of transamination (resulting in the transfer of amino groups from most amino acids to α-ketoglutarate to

1	catalyzed by glutamate dehydrogenase ([GDH], see Fig. 19.11). Therefore, the sequential action of transamination (resulting in the transfer of amino groups from most amino acids to α-ketoglutarate to produce glutamate) and the oxidative deamination of that glutamate (regenerating α-ketoglutarate) provide a pathway whereby the amino groups of most amino acids can be released as ammonia.

1	a. Coenzymes: GDH, a mitochondrial enzyme, is unusual in that it can use either nicotinamide adenine dinucleotide (NAD+) or its phosphorylated reduced form (NADPH) as a coenzyme (see Fig. 19.11). NAD+ is used primarily in oxidative deamination (the simultaneous loss of ammonia coupled with the oxidation of the carbon skeleton, as shown in Fig. 19.12A), whereas NADPH is used in reductive amination (the simultaneous gain of ammonia coupled with the reduction of the carbon skeleton, as shown in Fig. 19.12B). NADP(H) = nicotinamide adenine dinucleotide phosphate. b.

1	NADP(H) = nicotinamide adenine dinucleotide phosphate. b. Reaction direction: The direction of the reaction depends on the relative concentrations of glutamate, α-ketoglutarate, and ammonia and the ratio of oxidized to reduced coenzymes. For example, after ingestion of a meal containing protein, glutamate levels in the liver are elevated, and the reaction proceeds in the direction of amino acid degradation and the formation of ammonia (see Fig. 19.12A). High ammonia levels are required to drive the reaction to glutamate synthesis. c. Allosteric regulators: Guanosine triphosphate is an allosteric inhibitor of GDH, whereas adenosine diphosphate is an activator. Therefore, when energy levels are low in the cell, amino acid degradation by GDH is high, facilitating energy production from the carbon skeletons derived from amino acids.

1	2. d-Amino acid oxidase: D-Amino acids (see p. 5) are supplied by the diet but are not used in the synthesis of mammalian proteins. They are, however, efficiently metabolized to α-keto acids, ammonia, and hydrogen peroxide in the peroxisomes of liver and kidney cells by flavin adenine dinucleotide–dependent D-amino acid oxidase (DAO). The αketo acids can enter the general pathways of amino acid metabolism and be reaminated to L-isomers or catabolized for energy. [Note: DAO degrades D-serine, the isomeric form of serine that modulates N-methylD-aspartate (NMDA)-type glutamate receptors. Increased DAO activity has been linked to increased susceptibility to schizophrenia. DAO also converts glycine to glyoxylate (see p. 263).] L-Amino acid oxidases are found in snake venom. C. Ammonia transport to the liver

1	Two mechanisms are available in humans for the transport of ammonia from peripheral tissues to the liver for conversion to urea. Both are important in, but not exclusive to, skeletal muscle. The first uses glutamine synthetase to combine ammonia with glutamate to form glutamine, a nontoxic transport form of ammonia (Fig. 19.13). The glutamine is transported in the blood to the liver where it is cleaved by glutaminase to glutamate and ammonia (see p. 256). The glutamate is oxidatively deaminated to ammonia and α-ketoglutarate by GDH. The ammonia is converted to urea. The second transport mechanism involves the formation of alanine by the transamination of pyruvate produced from both aerobic glycolysis and metabolism of the succinyl coenzyme A (CoA) generated by the catabolism of the BCAA isoleucine and valine. Alanine is transported in the blood to the liver, where it is transaminated by ALT to pyruvate. The pyruvate is used to synthesize glucose, which can enter the blood and be used

1	and valine. Alanine is transported in the blood to the liver, where it is transaminated by ALT to pyruvate. The pyruvate is used to synthesize glucose, which can enter the blood and be used by muscle, a pathway called the glucose–alanine cycle. The glutamate product of ALT can be deaminated by GDH, generating ammonia. Thus, both alanine and glutamine carry ammonia to the liver.

1	V. UREA CYCLE is the major disposal form of amino groups derived from amino acids and accounts for ~90% of the nitrogen-containing components of urine. One nitrogen of the urea molecule is supplied by free ammonia and the other nitrogen by aspartate. [Note: Glutamate is the immediate precursor of both ammonia (through oxidative deamination by GDH) and aspartate nitrogen (through transamination of oxaloacetate by AST).] The carbon and oxygen of urea are derived from CO2 (as HCO3−). Urea is produced by the liver and then is transported in the blood to the kidneys for excretion in the urine. A. Reactions The first two reactions leading to the synthesis of urea occur in the mitochondrial matrix, whereas the remaining cycle enzymes are located in the cytosol (Fig. 19.14). [Note: Gluconeogenesis (see p. 117) and heme synthesis (see p. 278) also involve both the mitochondrial matrix and the cytosol.]

1	Pi = inorganic phosphate; NAD(H) = nicotinamide adenine dinucleotide; MD = malate dehydrogenase. 1. Carbamoyl phosphate formation: Formation of carbamoyl phosphate by carbamoyl phosphate synthetase I (CPS I) is driven by cleavage of two molecules of ATP. Ammonia incorporated into carbamoyl phosphate is provided primarily by the oxidative deamination of glutamate by mitochondrial GDH (see Fig. 19.11). Ultimately, the nitrogen atom derived from this ammonia becomes one of the nitrogens of urea. CPS I requires N-acetylglutamate (NAG) as a positive allosteric activator (see Fig. 19.14). [Note: Carbamoyl phosphate synthetase II participates in the biosynthesis of pyrimidines (see p. 302). It does not require NAG, uses glutamine as the nitrogen source, and occurs in the cytosol.] 2.

1	Citrulline formation: The carbamoyl portion of carbamoyl phosphate is transferred to ornithine by ornithine transcarbamylase (OTC) as the phosphate is released as inorganic phosphate. The reaction product, citrulline, is transported to the cytosol. [Note: Ornithine and citrulline move across the inner mitochondrial membrane via an antiporter. These basic amino acids are not incorporated into cellular proteins because there are no codons for them (see p. 447).] Ornithine is regenerated with each turn of the urea cycle, much in the same way that oxaloacetate is regenerated by the reactions of the tricarboxylic acid (TCA) cycle (see p. 109). 3.

1	3. Argininosuccinate formation: Argininosuccinate synthetase combines citrulline with aspartate to form argininosuccinate. The α-amino group of aspartate provides the second nitrogen that is ultimately incorporated into urea. The formation of argininosuccinate is driven by the cleavage of ATP to adenosine monophosphate and pyrophosphate. This is the third and final molecule of ATP consumed in the formation of urea. 4.

1	4. Argininosuccinate cleavage: Argininosuccinate is cleaved by argininosuccinate lyase to yield arginine and fumarate. The arginine serves as the immediate precursor of urea. The fumarate is hydrated to malate, providing a link with several metabolic pathways. Malate can be oxidized by malate dehydrogenase to oxaloacetate, which can be transaminated to aspartate (see Fig. 19.8) and enter the urea cycle (see Fig. 19.14). Alternatively, malate can be transported into mitochondria via the malate–aspartate shuttle (see p. 80), reenter the TCA cycle, and get oxidized to oxaloacetate, which can be used for gluconeogenesis (see p. 120). [Note: Malate oxidation generates NADH for oxidative phosphorylation (see p. 77), thereby reducing the energy cost of the urea cycle.] 5.

1	Arginine cleavage to ornithine and urea: Arginase-I hydrolyzes arginine to ornithine and urea and is virtually exclusive to the liver. Therefore, only the liver can cleave arginine, thereby synthesizing urea, whereas other tissues, such as the kidney, can synthesize arginine from citrulline. [Note: Arginase-II in kidneys controls arginine availability for nitric oxide synthesis (see p. 150).] 6.

1	Fate of urea: Urea diffuses from the liver and is transported in the blood to the kidneys, where it is filtered and excreted in the urine (see Fig. 19.19). A portion of the urea diffuses from the blood into the intestine and is cleaved to CO2 and ammonia by bacterial urease. The ammonia is partly lost in the feces and is partly reabsorbed into the blood. In patients with kidney failure, plasma urea levels are elevated, promoting a greater transfer of urea from blood into the gut. The intestinal action of urease on this urea becomes a clinically important source of ammonia, contributing to the hyperammonemia often seen in these patients. Oral administration of antibiotics reduces the number of intestinal bacteria responsible for this ammonia production. B. Overall stoichiometry

1	B. Overall stoichiometry Because four high-energy phosphate bonds are consumed in the synthesis of each molecule of urea, the synthesis of urea is irreversible, with a large, negative ∆G (see p. 70). One nitrogen of the urea molecule is supplied by free ammonia and the other nitrogen by aspartate. Glutamate is the immediate precursor of both ammonia (through oxidative deamination by GDH) and aspartate nitrogen (through transamination of oxaloacetate by AST). In effect, both nitrogen atoms of urea arise from glutamate, which, in turn, gathers nitrogen from other amino acids (Fig. 19.15). C. Regulation

1	C. Regulation NAG is an essential activator for CPS I, the rate-limiting step in the urea cycle. It increases the affinity of CPS I for ATP. NAG is synthesized from acetyl CoA and glutamate by N-acetylglutamate synthase (NAGS), as shown in Figure 19.16, in a reaction for which arginine is an activator. The cycle is also regulated by substrate availability (short-term regulation) and enzyme induction (long term). activator of carbamoyl phosphate synthetase I. CoA = coenzyme A. VI. AMMONIA METABOLISM

1	activator of carbamoyl phosphate synthetase I. CoA = coenzyme A. VI. AMMONIA METABOLISM Ammonia is produced by all tissues during the metabolism of a variety of compounds, and it is disposed of primarily by formation of urea in the liver. However, the blood ammonia level must be kept very low, because even slightly elevated concentrations (hyperammonemia) are toxic to the central nervous system (CNS). Therefore, a mechanism is required for the transport of nitrogen from the peripheral tissues to the liver for ultimate disposal as urea while keeping circulating levels of free ammonia low. A. Sources

1	A. Sources Amino acids are quantitatively the most important source of ammonia because most Western diets are high in protein and provide excess amino acids, which travel to the liver and undergo transdeamination (that is, the linking of the aminotransferase and GDH reactions), producing ammonia. [Note: The liver catabolizes straight-chain amino acids, primarily.] However, substantial amounts of ammonia can be obtained from other sources. 1. Glutamine: An important source of plasma glutamine is from the catabolism of BCAA in skeletal muscle. This glutamine is taken up by cells of the intestine, the liver, and the kidneys. The liver and kidneys generate ammonia from glutamine by the actions of glutaminase (Fig.

1	19.17) and GDH. In the kidneys, most of this ammonia is excreted into + the urine as NH4 , which provides an important mechanism for maintaining the body’s acid–base balance through the excretion of protons. In the liver, the ammonia is detoxified to urea and excreted. [Note: α-Ketoglutarate, the second product of GDH, is a glucogenic precursor in the liver and kidneys.] Ammonia is also generated by intestinal glutaminase. Enterocytes obtain glutamine either from the blood or from digestion of dietary protein. [Note: Intestinal glutamine metabolism also produces alanine, which is used by the liver for gluconeogenesis, and citrulline, which is used by the kidneys to synthesize arginine.] 2. Intestinal bacteria: Ammonia is formed from urea by the action of bacterial urease in the lumen of the intestine. This ammonia is absorbed from the intestine by way of the portal vein, and virtually all is removed by the liver via conversion to urea. 3.

1	3. Amines: Amines obtained from the diet and monoamines that serve as hormones or neurotransmitters give rise to ammonia by the action of monoamine oxidase (see p. 286). 4. Purines and pyrimidines: In the catabolism of purines and pyrimidines, amino groups attached to the ring atoms are released as ammonia (see Fig. 22.15 on p. 300). B. Transport in the circulation Although ammonia is constantly produced in the tissues, it is present at very low levels in blood. This is due both to the rapid removal of blood ammonia by the liver and to the fact that several tissues, particularly muscle, release amino acid nitrogen in the form of glutamine and alanine, rather than as free ammonia (see Fig. 19.13). 1. Urea: Formation of urea in the liver is quantitatively the most important disposal route for ammonia. Urea travels in the blood from the liver to the kidneys, where it passes into the glomerular filtrate. 2.

1	2. Glutamine: This amide of glutamate provides a nontoxic storage and transport form of ammonia (Fig. 19.18). The ATP-requiring formation of glutamine from glutamate and ammonia by glutamine synthetase occurs primarily in skeletal muscle and the liver but is also important in the CNS, where it is the major mechanism for the removal of ammonia in the brain. Glutamine is found in plasma at concentrations higher than other amino acids, a finding consistent with its transport function. [Note: The liver keeps blood ammonia levels low through glutaminase, GDH, and the urea cycle in periportal (close to inflow of blood) hepatocytes and through glutamine synthetase as an ammonia scavenger in the perivenous hepatocytes.] Ammonia metabolism is summarized in Figure 19.19. C. Hyperammonemia

1	C. Hyperammonemia The capacity of the hepatic urea cycle exceeds the normal rates of ammonia generation, and the levels of blood ammonia are normally low (5–35 µmol/l). However, when liver function is compromised, due either to genetic defects of the urea cycle or liver disease, blood levels can be >1,000 µmol/l. Such hyperammonemia is a medical emergency, because ammonia has a direct neurotoxic effect on the CNS. For example, elevated concentrations of ammonia in the blood cause the symptoms of ammonia intoxication, which include tremors, slurring of speech, somnolence (drowsiness), vomiting, cerebral edema, and blurring of vision. At high concentrations, ammonia can cause coma and death. There are two major types of hyperammonemia. 1.

1	1. Acquired: Liver disease is a common cause of acquired hyperammonemia in adults and may be due, for example, to viral hepatitis or to hepatotoxins such as alcohol. Cirrhosis of the liver may result in formation of collateral circulation around the liver. As a result, portal blood is shunted directly into the systemic circulation and does not have access to the liver. Therefore, the conversion of ammonia to urea is severely impaired, leading to elevated levels of ammonia. 2.

1	Congenital: Genetic deficiencies of each of the five enzymes of the urea cycle (and of NAGS) have been described, with an overall incidence of ~1:25,000 live births. X-linked OTC deficiency is the most common of these disorders, predominantly affecting males, although female carriers may become symptomatic. All of the other urea cycle disorders follow an autosomal-recessive inheritance pattern. In each case, the failure to synthesize urea leads to hyperammonemia during the first weeks following birth. [Note: The hyperammonemia seen with arginase deficiency is less severe because arginine contains two waste nitrogens and can be excreted in the urine.] Historically, urea cycle defects had high morbidity (neurologic manifestations) and mortality. Treatment included restriction of dietary protein in the presence of sufficient calories to prevent protein catabolism. Administration of compounds that bind covalently to nonessential amino acids, producing nitrogen-containing molecules that

1	in the presence of sufficient calories to prevent protein catabolism. Administration of compounds that bind covalently to nonessential amino acids, producing nitrogen-containing molecules that are excreted in the urine, has improved survival. For example, phenylbutyrate given orally is converted to phenylacetate. This condenses with glutamine to form phenylacetylglutamine, which is excreted (Fig. 19.20).

1	VII. CHAPTER SUMMARY

1	Nitrogen enters the body in a variety of compounds present in food, the most important being amino acids contained in dietary protein. Nitrogen leaves the body as urea, ammonia, and other products derived from amino acid metabolism (Fig. 19.21). Free amino acids in the body are produced by hydrolysis of dietary protein by proteases activated from their zymogen form in the stomach and intestine, degradation of tissue proteins, and de novo synthesis. This amino acid pool is consumed in the synthesis of body protein, metabolized for energy, or its members used as precursors for other nitrogen-containing compounds. Free amino acids from digestion are taken up by intestinal enterocytes via sodium-dependent secondary active transport. Small peptides are taken up via proton-linked transport. Note that body protein is simultaneously degraded and resynthesized, a process known as protein turnover. The concentration of a cellular protein may be determined by regulation of its synthesis or

1	Note that body protein is simultaneously degraded and resynthesized, a process known as protein turnover. The concentration of a cellular protein may be determined by regulation of its synthesis or degradation. The ATP-dependent, cytosolic, selective ubiquitin–proteasome and ATP-independent, relatively nonselective lysosomal acid hydrolases are the two major enzyme systems that are responsible for degrading proteins. Nitrogen cannot be stored, and amino acids in excess of the biosynthetic needs of the cell are quickly degraded. The first phase of catabolism involves the transfer of the α-amino groups through transamination by pyridoxal phosphate–dependent aminotransferases (transaminases), followed by oxidative deamination of glutamate by glutamate dehydrogenase, forming ammonia and the corresponding α-keto acids. A portion of the free ammonia is excreted in the urine. Some ammonia is used in converting glutamate to glutamine for safe transport, but most is used in the hepatic

1	the corresponding α-keto acids. A portion of the free ammonia is excreted in the urine. Some ammonia is used in converting glutamate to glutamine for safe transport, but most is used in the hepatic synthesis of urea, which is quantitatively the most important route for disposing of nitrogen from the body. Alanine also carries nitrogen to the liver for disposal as urea. The two major causes of hyperammonemia (with its neurologic effects) are acquired liver disease and congenital deficiencies of urea cycle enzymes such as X-linked ornithine transcarbamylase.

1	Choose the ONE best answer. 9.1. In this transamination reaction (right), which of the following are the products X and Y? A. Alanine, α-ketoglutarate B. Aspartate, α-ketoglutarate C. Glutamate, alanine D. Pyruvate, aspartate Correct answer = B. Transamination reactions always have an amino acid and an α-keto acid as substrates. The products of the reaction are also an amino acid (corresponding to the α-keto substrate) and an α-keto acid (corresponding to the amino acid substrate). Three amino acid α-keto acid pairs commonly encountered in metabolism are alanine/pyruvate, aspartate/oxaloacetate, and glutamate/α-ketoglutarate. In this question, glutamate is deaminated to form αketoglutarate, and oxaloacetate is aminated to form aspartate. 9.2. Which one of the following statements about amino acids and their metabolism is correct? A. Free amino acids are taken into the enterocytes by a single proton-linked transport system.

1	9.2. Which one of the following statements about amino acids and their metabolism is correct? A. Free amino acids are taken into the enterocytes by a single proton-linked transport system. B. In healthy, well-fed individuals, the input to the amino acid pool exceeds the output. C. The liver uses ammonia to buffer protons. D. Muscle-derived glutamine is metabolized in liver and kidney tissue to ammonia + a gluconeogenic precursor. E. The first step in the catabolism of most amino acids is their oxidative deamination. F. The toxic ammonia generated from the amide nitrogen of amino acids is transported through blood as arginine.

1	F. The toxic ammonia generated from the amide nitrogen of amino acids is transported through blood as arginine. Correct answer = D. Glutamine, produced by the catabolism of branched-chain amino acids in muscle, is deaminated by glutaminase to ammonia + glutamate. The glutamate is deaminated by glutamate dehydrogenase to ammonia + αketoglutarate, which can be used for gluconeogenesis. Free amino acids are taken into enterocytes by several different sodium-linked transport systems. Healthy, well-fed individuals are in nitrogen balance, in which nitrogen input equals output. The liver converts ammonia to urea, and the kidneys use ammonia to buffer protons. Amino acid catabolism begins with transamination that generates glutamate. The glutamate undergoes oxidative deamination. Toxic ammonia is transported as glutamine and alanine. Arginine is synthesized and hydrolyzed in the hepatic urea cycle. For Questions 19.3–19.5, use the following scenario.

1	For Questions 19.3–19.5, use the following scenario. A female neonate appeared healthy until age ~24 hours, when she became lethargic. A sepsis workup proved negative. At 56 hours, she started showing focal seizure activity. The plasma ammonia level was found to be 887 µmol/l (normal 5–35 µmol/l). Quantitative plasma amino acid levels revealed a marked elevation of citrulline but not argininosuccinate. 9.3. Which one of the following enzymic activities is most likely to be deficient in this patient? A. Arginase B. Argininosuccinate lyase C. Argininosuccinate synthetase D. Carbamoyl phosphate synthetase I

1	A. Arginase B. Argininosuccinate lyase C. Argininosuccinate synthetase D. Carbamoyl phosphate synthetase I E. Ornithine transcarbamylase Correct answer = C. Genetic deficiencies of each of the five enzymes of the urea cycle, as well as deficiencies in N-acetylglutamate synthase, have been described. The accumulation of citrulline (but not argininosuccinate) in the plasma of this patient means that the enzyme required for the conversion of citrulline to argininosuccinate (argininosuccinate synthetase) is defective, whereas the enzyme that cleaves argininosuccinate (argininosuccinate lyase) is functional. 9.4. Which one of the following would also be elevated in the blood of this patient? A. Asparagine B. Glutamine C. Lysine D. Urea

1	9.4. Which one of the following would also be elevated in the blood of this patient? A. Asparagine B. Glutamine C. Lysine D. Urea Correct answer = B. Deficiencies of the enzymes of the urea cycle result in the failure to synthesize urea and lead to hyperammonemia in the first few weeks after birth. Glutamine will also be elevated because it acts as a nontoxic storage and transport form of ammonia. Therefore, elevated glutamine accompanies hyperammonemia. Asparagine and lysine do not serve this sequestering role. Urea would be decreased because of impaired activity of the urea cycle. [Note: Alanine would also be elevated in this patient.] 9.5. Why might supplementation with arginine be of benefit to this patient? The arginine will be cleaved by arginase to urea and ornithine. Ornithine will be combined with carbamoyl phosphate by ornithine transcarbamylase to form citrulline. Citrulline, containing one waste nitrogen, will be excreted. Amino Acids: Degradation and Synthesis 20

1	Amino Acids: Degradation and Synthesis 20 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Amino acid degradation involves removal of the α-amino group, followed by the catabolism of the resulting α-keto acids (carbon skeletons). These pathways converge to form seven intermediate products: oxaloacetate, pyruvate, αketoglutarate, fumarate, succinyl coenzyme A (CoA), acetyl CoA, and acetoacetate. The products directly enter the pathways of intermediary metabolism, resulting either in the synthesis of glucose, ketone bodies, or lipids or in the production of energy through their oxidation to carbon dioxide (CO2) by the tricarboxylic acid (TCA) cycle. Figure 20.1 provides an overview of these pathways, with a more detailed summary presented in Figure 20.15 (see p. 269). Nonessential amino acids (Fig. 20.2) can be synthesized in sufficient amounts from the intermediates of metabolism or, as in the case of cysteine and tyrosine, from essential amino acids. In contrast, because the essential amino acids cannot be synthesized (or synthesized in sufficient amounts) by humans, they

1	or, as in the case of cysteine and tyrosine, from essential amino acids. In contrast, because the essential amino acids cannot be synthesized (or synthesized in sufficient amounts) by humans, they must be obtained from the diet in order for normal protein synthesis to occur. Genetic defects in the pathways of amino acid metabolism can cause serious disease.

1	postoperative infections, and immunosuppression.] II. GLUCOGENIC AND KETOGENIC AMINO ACIDS Amino acids can be classified as glucogenic, ketogenic, or both, based on which of the seven intermediates are produced during their catabolism (see Fig. 20.2). A. Glucogenic amino acids Amino acids whose catabolism yields pyruvate or one of the intermediates of the TCA cycle are termed glucogenic. Because these intermediates are substrates for gluconeogenesis (see p. 118), they can give rise to the net synthesis of glucose in the liver and kidney. B. Ketogenic amino acids

1	B. Ketogenic amino acids Amino acids whose catabolism yields either acetoacetate or one of its precursors (acetyl CoA or acetoacetyl CoA) are termed ketogenic (see Fig. 20.2). Acetoacetate is one of the ketone bodies, which also include 3hydroxybutyrate and acetone (see p. 195). Leucine and lysine are the only exclusively ketogenic amino acids found in proteins. Their carbon skeletons are not substrates for gluconeogenesis and, therefore, cannot give rise to the net synthesis of glucose. III. AMINO ACID CARBON SKELETON The pathways by which amino acids are catabolized are conveniently organized according to which one (or more) of the seven intermediates listed above is produced from a particular amino acid. A. Amino acids that form oxaloacetate

1	A. Amino acids that form oxaloacetate Asparagine is hydrolyzed by asparaginase, liberating ammonia and aspartate (Fig. 20.3). Aspartate loses its amino group by transamination to form oxaloacetate (see Fig. 20.3). [Note: Some rapidly dividing leukemic cells are unable to synthesize sufficient asparagine to support their growth. This makes asparagine an essential amino acid for these cells, which, therefore, require asparagine from the blood. Asparaginase, which hydrolyzes asparagine to aspartate, can be administered systemically to treat leukemia. Asparaginase lowers the level of asparagine in the plasma, thereby depriving cancer cells of a required nutrient.] B. Amino acids that form α-ketoglutarate via glutamate 1. Glutamine: This amino acid is hydrolyzed to glutamate and ammonia by the enzyme glutaminase (see p. 256). Glutamate is converted to αketoglutarate by transamination or through oxidative deamination by glutamate dehydrogenase (see p. 252). 2.

1	2. Proline: This amino acid is oxidized to glutamate. Glutamate is transaminated or oxidatively deaminated to form α-ketoglutarate. 3. Arginine: This amino acid is hydrolyzed by arginase to produce ornithine (and urea). [Note: The reaction occurs primarily in the liver as part of the urea cycle (see p. 255).] Ornithine is subsequently converted to α-ketoglutarate, with glutamate semialdehyde as an intermediate. 4.

1	4. Histidine: This amino acid is oxidatively deaminated by histidase to urocanic acid, which subsequently forms N-formiminoglutamate ([FIGlu], Fig. 20.4). FIGlu donates its formimino group to tetrahydrofolate (THF), leaving glutamate, which is degraded as described above. [Note: Individuals deficient in folic acid excrete increased amounts of FIGlu in the urine, particularly after ingestion of a large dose of histidine. The FIGlu excretion test has been used in diagnosing a deficiency of folic acid. See p. 267 for a discussion of folic acid, THF, and one-carbon metabolism.] C. Amino acids that form pyruvate 1. Alanine: This amino acid loses its amino group by transamination to 2. Serine: This amino acid can be converted to glycine as THF becomes N5,N10-methylenetetrahydrofolate (N5,N10-MTHF), as shown in Figure 20.6A. Serine can also be converted to pyruvate (see Fig. 20.6B). 3.

1	3. Glycine: This amino acid can be converted to serine by the reversible addition of a methylene group from N5,N10-MTHF (see Fig. 20.6A) or oxidized to CO2 and ammonia by the glycine cleavage system. [Note: Glycine can be deaminated to glyoxylate (by a d-amino acid oxidase; see form pyruvate (Fig. 20.5). [Note: Tryptophan catabolism produces alanine and, therefore, pyruvate (see Fig. 20.10 on p. 265).] p. 253), which can be oxidized to oxalate or transaminated to glycine. Deficiency of the transaminase in liver peroxisomes causes overproduction of oxalate, the formation of oxalate stones, and kidney damage (primary oxaluria type 1).] 4. Cysteine: This sulfur-containing amino acid undergoes desulfurization to yield pyruvate. [Note: The sulfate released can be used to synthesize 3′phosphoadenosine-5′-phosphosulfate (PAPS), an activated sulfate donor to a variety of acceptors.] Cysteine can also be oxidized to its disulfide derivative, cystine. 5.

1	5. Threonine: This amino acid is converted to pyruvate in most organisms but is a minor pathway (at best) in humans. D. Amino acids that form fumarate 1. Phenylalanine and tyrosine: Hydroxylation of phenylalanine produces tyrosine (Fig. 20.7). This irreversible reaction, catalyzed by tetrahydrobiopterin-requiring phenylalanine hydroxylase (PAH), initiates the catabolism of phenylalanine. Thus, phenylalanine metabolism and tyrosine metabolism merge, leading ultimately to fumarate and acetoacetate formation. Therefore, phenylalanine and tyrosine are both glucogenic and ketogenic. 2. Inherited deficiencies: Inherited deficiencies in the enzymes of phenylalanine and tyrosine metabolism lead to the diseases phenylketonuria (PKU) (see p. 270), tyrosinemia (see p. 274), and alkaptonuria (see p. 274) as well as the condition of albinism (see p. 273). E. Amino acids that form succinyl CoA: Methionine

1	E. Amino acids that form succinyl CoA: Methionine Methionine is one of four amino acids that form succinyl CoA. This sulfur-containing amino acid deserves special attention because it is converted to S-adenosylmethionine (SAM), the major methyl group donor in one-carbon metabolism (Fig. 20.8). Methionine is also the source of homocysteine (Hcy), a metabolite associated with atherosclerotic vascular disease and thrombosis (see p. 265). the methyl group carrier and donor.] PPi = pyrophosphate; Pi = inorganic phosphate; NH3 = ammonia. 1. S-Adenosylmethionine synthesis: Methionine condenses with ATP, forming SAM, a high-energy compound that is unusual in that it contains no phosphate. The formation of SAM is driven by hydrolysis of all three phosphate bonds in ATP (see Fig. 20.8). 2.

1	2. Activated methyl group: The methyl group attached to the sulfur in SAM is activated and can be transferred by methyltransferases to a variety of acceptors such as norepinephrine in the synthesis of epinephrine. The methyl group is usually transferred to nitrogen or oxygen atoms (as with epinephrine synthesis and degradation, respectively; see p. 286) and sometimes to carbon atoms (as with cytosine). The reaction product, Sadenosylhomocysteine (SAH), is a simple thioether, analogous to methionine. The resulting loss of free energy makes methyl transfer essentially irreversible. 3. S-Adenosylhomocysteine hydrolysis: After donation of the methyl group, SAH is hydrolyzed to Hcy and adenosine. Hcy has two fates. If there is a deficiency of methionine, Hcy may be remethylated to methionine (see Fig. 20.8). If methionine stores are adequate, Hcy may enter the transsulfuration pathway, where it is converted to cysteine.

1	a. Methionine resynthesis: Hcy accepts a methyl group from N5methyltetrahydrofolate (N5-methyl-THF) in a reaction requiring methylcobalamin, a coenzyme derived from vitamin B12 (see p. 379). [Note: The methyl group is transferred by methionine synthase from the B12 derivative to Hcy, regenerating methionine. Cobalamin is remethylated from N5-methyl-THF.] b. Cysteine synthesis: Hcy condenses with serine, forming cystathionine, which is hydrolyzed to α-ketobutyrate and cysteine (see Fig. 20.8). This vitamin B6–requiring sequence has the net effect of converting serine to cysteine and Hcy to α-ketobutyrate, which is oxidatively decarboxylated to form propionyl CoA. Propionyl CoA is converted to succinyl CoA (see Fig. 16.20 on p. 195). Because Hcy is synthesized from the essential amino acid methionine, cysteine is not an essential amino acid as long as sufficient methionine is available.

1	4. Relationship of homocysteine to vascular disease: Elevations in plasma Hcy levels promote oxidative damage, inflammation, and endothelial dysfunction and are an independent risk factor for occlusive vascular diseases such as cardiovascular disease (CVD) and stroke (Fig. 20.9). Mild elevations (hyperhomocysteinemia) are seen in ~7% of the population. Epidemiologic studies have shown that plasma Hcy levels are inversely related to plasma levels of folate, B12, and B6, the three vitamins involved in the conversion of Hcy to methionine and cysteine. Supplementation with these vitamins has been shown to reduce circulating levels of Hcy. However, in patients with established CVD, vitamin therapy does not decrease cardiovascular events or death. This raises the question as to whether Hcy is a cause of the vascular damage or merely a marker of such damage. [Note: Large elevations in plasma Hcy as a result of rare deficiencies in cystathionine β-synthase of the transsulfuration pathway are

1	cause of the vascular damage or merely a marker of such damage. [Note: Large elevations in plasma Hcy as a result of rare deficiencies in cystathionine β-synthase of the transsulfuration pathway are seen in patients with classic homocystinuria (resulting from severe hyperhomocysteinemia [>100 µmol/l], see p. 273).] Deficiencies in the remethylation reaction also result in a rise in Hcy.

1	Elevated homocysteine and decreased folic acid levels in pregnant women are associated with increased incidence of neural tube defects (improper closure, as in spina bifida) in the fetus. Periconceptual supplementation with folate reduces the risk of such defects. F. Other amino acids that form succinyl CoA Degradation of valine, isoleucine, and threonine also results in the production of succinyl CoA, a TCA cycle intermediate and gluconeogenic compound. [Note: It is metabolized to pyruvate.] 1. Valine and isoleucine: These amino acids are branched-chain amino acids (BCAA) that generate propionyl CoA, which is converted to methylmalonyl CoA and then succinyl CoA by biotin-and vitamin B12 – requiring reactions. 2.

1	2. Threonine: This amino acid is dehydrated to α-ketobutyrate, which is converted to propionyl CoA and then to succinyl CoA. Propionyl CoA, then, is generated by the catabolism of the amino acids methionine, valine, isoleucine, and threonine. [Note: Propionyl CoA also is generated by the oxidation of odd-numbered fatty acids (see p. 193).] G. Amino acids that form acetyl CoA or acetoacetyl CoA Tryptophan, leucine, isoleucine, and lysine form acetyl CoA or acetoacetyl CoA directly, without pyruvate serving as an intermediate. As noted earlier, phenylalanine and tyrosine also give rise to acetoacetate during their catabolism (see Fig. 20.7). Therefore, there are a total of six partly or wholly ketogenic amino acids. 1. Tryptophan: This amino acid is both glucogenic and ketogenic, because its catabolism yields alanine and acetoacetyl CoA (Fig. 20.10). [Note: Quinolinate from tryptophan catabolism is used in the synthesis of nicotinamide adenine dinucleotide ([NAD], see p. 383).] 2.

1	Leucine: This amino acid is exclusively ketogenic, because its catabolism yields acetyl CoA and acetoacetate (Fig. 20.11). The first two reactions in the catabolism of leucine and the other BCAA, isoleucine and valine, are catalyzed by enzymes that use all three BCAA (or their derivatives) as substrates (see H. below). 3. Isoleucine: This amino acid is both ketogenic and glucogenic, because its metabolism yields acetyl CoA and propionyl CoA. 4. Lysine: This amino acid is exclusively ketogenic and is unusual in that neither of its amino groups undergoes transamination as the first step in catabolism. Lysine is ultimately converted to acetoacetyl CoA. H. Branched-chain amino acid degradation

1	H. Branched-chain amino acid degradation The BCAA isoleucine, leucine, and valine are essential amino acids. In contrast to other amino acids, they are catabolized primarily by the peripheral tissues (particularly muscle), rather than by the liver. Because these three amino acids have a similar route of degradation, it is convenient to describe them as a group (see Fig. 20.11). 1. Transamination: Transfer of the amino groups of all three BCAA to α ketoglutarate is catalyzed by a single, vitamin B6–requiring enzyme, branched-chain amino acid aminotransferase, that is expressed primarily in skeletal muscle. 2.

1	2. Oxidative decarboxylation: Removal of the carboxyl group of the α-keto acids derived from leucine, valine, and isoleucine is catalyzed by a single multienzyme complex, branched-chain α-keto acid dehydrogenase (BCKD) complex. This complex uses thiamine pyrophosphate, lipoic acid, oxidized flavin adenine dinucleotide (FAD), NAD+, and CoA as its coenzymes and produces NADH. [Note: This reaction is similar to the conversion of pyruvate to acetyl CoA by the pyruvate dehydrogenase (PDH) complex (see p. 109) and α-ketoglutarate to succinyl CoA by the α-ketoglutarate dehydrogenase complex (see p. 112). The dihydrolipoyl dehydrogenase (Enzyme 3, or E3) component is identical in all three complexes.] 3.

1	Dehydrogenations: Oxidation of the products formed in the BCKD reaction produces α-β-unsaturated acyl CoA derivatives and FADH2. These reactions are analogous to the FAD-linked dehydrogenation in the β-oxidation of fatty acids (see p. 192). [Note: Deficiency in the dehydrogenase specific for isovaleryl CoA causes neurologic problems and is associated with a “sweaty feet” odor in body fluids.] 4. End products: The catabolism of isoleucine ultimately yields acetyl CoA and succinyl CoA, rendering it both ketogenic and glucogenic. Valine yields succinyl CoA and is glucogenic. Leucine is ketogenic, being metabolized to acetoacetate and acetyl CoA. In addition, NADH and FADH2 are produced in the decarboxylation and dehydrogenation reactions, respectively. [Note: BCAA catabolism also results in glutamine and alanine being synthesized and sent out into the blood from muscle (see p. 253).] IV. FOLIC ACID AND AMINO ACID METABOLISM

1	IV. FOLIC ACID AND AMINO ACID METABOLISM Some synthetic pathways require the addition of single-carbon groups that exist in a variety of oxidation states, including formyl, methenyl, methylene, and methyl. These single-carbon groups can be transferred from carrier compounds such as THF and SAM to specific structures that are being synthesized or modified. The “one-carbon pool” refers to the single-carbon units attached to this group of carriers. [Note: CO2, coming from bicarbonate (HCO3–), is carried by the vitamin biotin (see p. 385), which is a prosthetic group for most carboxylation reactions but is not considered a member of the one-carbon pool. Defects in the ability to add or remove biotin from carboxylases result in multiple carboxylase deficiency. Treatment is supplementation with biotin.] A. Folic acid and one-carbon metabolism

1	A. Folic acid and one-carbon metabolism The active form of folic acid, THF, is produced from folate by dihydrofolate reductase in a two-step reaction requiring two nicotinamide adenine dinucleotide phosphate (NADPH). The one-carbon unit carried by THF is bound to N5 or N10 or to both N5 and N10 . Figure 20.12 shows the structures of the various members of the THF family and their interconversions and indicates the sources of the one-carbon units and the synthetic reactions in which the specific members participate. [Note: Folate deficiency presents as a megaloblastic anemia because of decreased availability of the purines and of the thymidine monophosphate needed for DNA synthesis (see p. 303).] V. BIOSYNTHESIS OF NONESSENTIAL AMINO ACIDS

1	V. BIOSYNTHESIS OF NONESSENTIAL AMINO ACIDS Nonessential amino acids are synthesized from intermediates of metabolism or, as in the case of tyrosine and cysteine, from the essential amino acids phenylalanine and methionine, respectively. The synthetic reactions for the nonessential amino acids are described below and are summarized in Figure 20.15. [Note: Some amino acids found in proteins, such as hydroxyproline and hydroxylysine (see p. 45), are produced by posttranslational modification (after incorporation into a protein) of their precursor (parent) amino acids.] A. Synthesis from α-keto acids

1	A. Synthesis from α-keto acids Alanine, aspartate, and glutamate are synthesized by transfer of an amino group to the α-keto acids pyruvate, oxaloacetate, and α-ketoglutarate, respectively. These transamination reactions (Fig. 20.13; also see p. 250) are the most direct of the biosynthetic pathways. Glutamate is unusual in that it can also be synthesized by reversal of oxidative deamination, catalyzed by glutamate dehydrogenase, when ammonia levels are high (see p. 252). B. Synthesis by amidation 1. Glutamine: This amino acid, which contains an amide linkage with ammonia at the γ-carboxyl, is formed from glutamate by glutamine synthetase (see Fig. 19.18, p. 256). The reaction is driven by the hydrolysis of ATP. In addition to producing glutamine for protein synthesis, the reaction also serves as a major mechanism for the transport of ammonia in a nontoxic form. (See p. 256 for a discussion of ammonia metabolism.) 2.

1	Asparagine: This amino acid, which contains an amide linkage with ammonia at the β-carboxyl, is formed from aspartate by asparagine synthetase, using glutamine as the amide donor. Like the synthesis of glutamine, the reaction requires ATP and has an equilibrium far in the direction of amide synthesis. C. Proline Glutamate via glutamate semialdehyde is converted to proline by cyclization and reduction reactions. [Note: The semialdehyde can also be transaminated to ornithine.] D. Serine, glycine, and cysteineThe pathways of synthesis for these amino acids are interconnected. 1.

1	D. Serine, glycine, and cysteineThe pathways of synthesis for these amino acids are interconnected. 1. Serine: This amino acid arises from 3-phosphoglycerate, a glycolytic intermediate (see Fig. 8.18, p. 101), which is first oxidized to 3phosphopyruvate and then transaminated to 3-phosphoserine. Serine is formed by hydrolysis of the phosphate ester. Serine can also be formed from glycine through transfer of a hydroxymethyl group by serine hydroxymethyltransferase using N5,N10-MTHF as the one-carbon donor (see Fig. 20.6A). [Note: Selenocysteine (Sec), the 21st genetically encoded amino acid, is synthesized from serine and selenium (see p. 407), while serine is attached to transfer RNA. Sec is found in ~25 human proteins including glutathione peroxidase (see p. 148) and thioredoxin reductase (see p. 297).] 2.

1	Glycine: This amino acid is synthesized from serine by removal of a hydroxymethyl group, also by serine hydroxymethyltransferase (see Fig. 20.6A). THF is the one-carbon acceptor. 3. Cysteine: This amino acid is synthesized by two consecutive reactions in which Hcy combines with serine, forming cystathionine, which, in turn, is hydrolyzed to α-ketobutyrate and cysteine (see Fig. 20.8). [Note: Hcy is derived from methionine, as described on p. 264. Because methionine is an essential amino acid, cysteine synthesis requires adequate dietary intake of methionine.] E. Tyrosine

1	E. Tyrosine Tyrosine is formed from phenylalanine by PAH (see p. 263). The reaction requires molecular oxygen and the coenzyme tetrahydrobiopterin (BH4), which is synthesized from guanosine triphosphate. One atom of molecular oxygen becomes the hydroxyl group of tyrosine, and the other atom is reduced to water. During the reaction, BH4 is oxidized to dihydrobiopterin (BH2). BH4 is regenerated from BH2 by NADH-requiring dihydropteridine reductase. Tyrosine, like cysteine, is formed from an essential amino acid and is, therefore, nonessential only in the presence of adequate dietary phenylalanine. VI. AMINO ACID METABOLISM DISORDERS

1	VI. AMINO ACID METABOLISM DISORDERS These single gene disorders, a subset of the inborn errors of metabolism, are caused by mutations that generally result in abnormal proteins, most often enzymes. The inherited defects may be expressed as a total loss of enzyme activity or, more frequently, as a partial deficiency in catalytic activity. Without treatment, the amino acid disorders almost invariably result in intellectual disability or other developmental abnormalities as a consequence of harmful accumulation of metabolites. Although >50 of these disorders have been described, many are rare, occurring in <1 per 250,000 in most populations (Fig. 20.14). Collectively, however, they constitute a very significant portion of pediatric genetic diseases (Fig. 20.15).

1	boxes. Classification of amino acids is color coded: Red = glucogenic; brown = glucogenic and ketogenic; green = ketogenic. Compounds in BLUE ALL CAPS are the seven metabolites to which all amino acid metabolism converges. CoA = coenzyme A; NAD(H) = nicotinamide adenine dinucleotide. A. Phenylketonuria

1	PKU is the most common clinically encountered inborn error of amino acid metabolism (incidence 1:15,000). It is caused by a deficiency of PAH (Fig. 20.16). Biochemically, PKU is characterized by hyperphenylalaninemia. Phenylalanine is present in high concentrations (ten times normal) not only in plasma but also in urine and body tissues. Tyrosine, which normally is formed from phenylalanine by PAH, is deficient. Treatment includes dietary restriction of phenylalanine and supplementation with tyrosine. [Note: Hyperphenylalaninemia may also be caused by rare deficiencies in any of the several enzymes required to synthesize BH4 or in dihydropteridine reductase, which regenerates BH4 from BH2 (Fig. 20.17). Such deficiencies indirectly raise phenylalanine concentrations, because PAH requires BH4 as a coenzyme. BH4 is also required for tyrosine hydroxylase and tryptophan hydroxylase, which catalyze reactions leading to the synthesis of neurotransmitters, such as serotonin and the

1	requires BH4 as a coenzyme. BH4 is also required for tyrosine hydroxylase and tryptophan hydroxylase, which catalyze reactions leading to the synthesis of neurotransmitters, such as serotonin and the catecholamines. Simply restricting dietary phenylalanine does not reverse the central nervous system effects due to deficiencies in neurotransmitters. Supplementation with BH4 and replacement therapy with L-3,4 dihydroxyphenylalanine and 5-hydroxytryptophan (products of the affected tyrosine hydroxylase– and tryptophan hydroxylase–catalyzed reactions) improves the clinical outcome in these variant forms of hyperphenylalaninemia, although the response is unpredictable.]

1	Screening of newborns for a number of treatable disorders, including inborn errors of amino acid metabolism, is done by tandem mass spectrometry of blood obtained from a heel prick. By law, all states must screen for >20 disorders, with some screening for >50. All states screen for PKU. 1. Additional characteristics: As the name suggests, PKU is also characterized by elevated levels of a phenylketone in the urine. a. Elevated phenylalanine metabolites: Phenylpyruvate (a phenylketone), phenylacetate, and phenyllactate, which are not normally produced in significant amounts in the presence of functional PAH, are elevated in PKU (Fig. 20.18). These metabolites give urine a characteristic musty (“mousy”) odor. b.

1	b. Central nervous system effects: Severe intellectual disability, developmental delay, microcephaly, and seizures are characteristic findings in untreated PKU. The affected individual typically shows symptoms of intellectual disability by age 1 year and rarely achieves an intelligence quotient (IQ) >50 (Fig. 20.19). [Note: These clinical manifestations are now rarely seen as a result of newborn screening programs, which allow early diagnosis and treatment.] c. Hypopigmentation: Patients with untreated PKU may show a deficiency of pigmentation (fair hair, light skin color, and blue eyes). The hydroxylation of tyrosine by copper-requiring tyrosinase, which is the first step in the formation of the pigment melanin, is decreased in PKU because tyrosine is decreased. 2.

1	2. Newborn screening and diagnosis: Early diagnosis of PKU is important because the disease is treatable by dietary means. Because of the lack of neonatal symptoms, laboratory testing for elevated blood levels of phenylalanine is mandatory for detection. However, the infant with PKU frequently has normal blood levels of phenylalanine at birth because the mother clears increased blood phenylalanine in her affected fetus through the placenta. Normal levels of phenylalanine may persist until the newborn is exposed to 24–48 hours of protein feeding. Thus, screening tests are typically done after this time to avoid false negatives. For newborns with a positive screening test, diagnosis is confirmed through quantitative determination of phenylalanine levels. 3.

1	3. Prenatal diagnosis: Classic PKU is caused by any of 100 or more different mutations in the gene that encodes PAH. The frequency of any given mutation varies among populations, and the disease is often doubly heterozygous (that is, the PAH gene has a different mutation in each allele). Despite this complexity, prenatal diagnosis is possible (see p. 493). 4.

1	Treatment: Because most natural protein contains phenylalanine, an essential amino acid, it is impossible to satisfy the body’s protein requirement without exceeding the phenylalanine limit when ingesting a normal diet. Therefore, in PKU, blood phenylalanine level is maintained close to the normal range by feeding synthetic amino acid preparations free of phenylalanine, supplemented with some natural foods (such as fruits, vegetables, and certain cereals) selected for their low phenylalanine content. The amount is adjusted according to the tolerance of the individual as measured by blood phenylalanine levels. The earlier treatment is started, the more completely neurologic damage can be prevented. Individuals who are appropriately treated can have normal intelligence. [Note: Treatment must begin during the first 7–10 days of life to prevent cognitive impairment.] Because phenylalanine is an essential amino acid, overzealous treatment that results in blood phenylalanine levels below

1	begin during the first 7–10 days of life to prevent cognitive impairment.] Because phenylalanine is an essential amino acid, overzealous treatment that results in blood phenylalanine levels below normal is avoided. In patients with PKU, tyrosine cannot be synthesized from phenylalanine, and, therefore, it becomes an essential amino acid and must be supplied in the diet. Discontinuance of the phenylalanine-restricted diet in early childhood is associated with poor performance on IQ tests. Adult PKU patients show deterioration of IQ scores after discontinuation of the diet (Fig. 20.20). Therefore, lifelong restriction of dietary phenylalanine is recommended. [Note: Individuals with PKU are advised to avoid aspartame, an artificial sweetener that contains phenylalanine.] 5. Maternal phenylketonuria: If women with PKU who are not on a low-phenylalanine diet become pregnant, the offspring can be affected with maternal PKU syndrome. High blood phenylalanine in the mother has a teratogenic

1	If women with PKU who are not on a low-phenylalanine diet become pregnant, the offspring can be affected with maternal PKU syndrome. High blood phenylalanine in the mother has a teratogenic effect, causing microcephaly and congenital heart abnormalities in the fetus. Because these developmental responses to high phenylalanine occur during the first months of pregnancy, dietary control of blood phenylalanine must begin prior to conception and be maintained throughout the pregnancy.

1	B. Maple syrup urine disease Maple syrup urine disease (MSUD) is a rare (1:185,000), autosomalrecessive disorder in which there is a partial or complete deficiency in BCKD, the mitochondrial enzyme complex that oxidatively decarboxylates leucine, isoleucine, and valine (see Fig. 20.11). These BCAA and their corresponding α-keto acids accumulate in the blood, causing a toxic effect that interferes with brain functions. The disease is characterized by feeding problems, vomiting, ketoacidosis, changes in muscle tone, neurologic problems that can result in coma (primarily because of the rise in leucine), and a characteristic maple syrup–like odor of the urine because of the rise in isoleucine. If untreated, the disease is fatal. If treatment is delayed, intellectual disability results. 1.

1	1. Classification: MSUD includes a classic type and several variant forms. The classic, neonatal-onset form is the most common type of MSUD. Leukocytes or cultured skin fibroblasts from these patients show little or no BCKD activity. Infants with classic MSUD show symptoms within the first several days of life. If not diagnosed and treated, classic MSUD is lethal in the first weeks of life. Patients with intermediate forms have a higher level of enzyme activity (up to 30% of normal). The symptoms are milder and show an onset from infancy to adolescence. Patients with the rare thiamine-dependent variant of MSUD respond to large doses of this vitamin. 2. Screening and diagnosis: As with PKU, prenatal diagnosis and newborn screening are available, and most affected individuals are compound heterozygotes. 3.

1	2. Screening and diagnosis: As with PKU, prenatal diagnosis and newborn screening are available, and most affected individuals are compound heterozygotes. 3. Treatment: MSUD is treated with a synthetic formula that is free of BCAA, supplemented with limited amounts of leucine, isoleucine, and valine to allow for normal growth and development without producing toxic levels. [Note: Elevated leucine is the cause of the neurologic damage in MSUD, and its level is carefully monitored.] Early diagnosis and lifelong dietary treatment are essential if the child with MSUD is to develop normally. [Note: BCAA are an important energy source in times of metabolic need, and individuals with MSUD are at risk of decompensation during periods of increased protein catabolism.] C. Albinism

1	C. Albinism Albinism refers to a group of conditions in which a defect in tyrosine metabolism results in a deficiency in the production of melanin. These defects result in the partial or full absence of pigment from the skin, hair, and eyes. Albinism appears in different forms, and it may be inherited by one of several modes: autosomal recessive (primary mode), autosomal dominant, or X linked. Total absence of pigment from the hair, eyes, and skin (Fig. 20.21), tyrosinase-negative oculocutaneous albinism (type 1 albinism), results from an absent or defective copper-requiring tyrosinase. It is the most severe form of the condition. In addition to hypopigmentation, affected individuals have vision defects and photophobia (sunlight hurts their eyes). They are at increased risk for skin cancer. D. Homocystinuria

1	The homocystinurias are a group of disorders involving defects in the metabolism of Hcy. These autosomal-recessive diseases are characterized by high urinary levels of Hcy, high plasma levels of Hcy and methionine, and low plasma levels of cysteine. The most common cause of homocystinuria is a defect in the enzyme cystathionine β-synthase, which converts Hcy to cystathionine (Fig. 20.22). Individuals homozygous for cystathionine β-synthase deficiency exhibit dislocation of the lens (ectopia lentis), skeletal anomalies (long limbs and fingers), intellectual disability, and an increased risk for developing thrombi (blood clots). Thrombosis is the major cause of early death in these individuals. Treatment includes restriction of methionine and supplementation with vitamin B12 and folate. Additionally, some patients are responsive to oral administration of pyridoxine (vitamin B6), which is converted to pyridoxal phosphate, the coenzyme of cystathionine β-synthase. These patients usually

1	some patients are responsive to oral administration of pyridoxine (vitamin B6), which is converted to pyridoxal phosphate, the coenzyme of cystathionine β-synthase. These patients usually have a milder and later onset of clinical symptoms compared with B6nonresponsive patients. [Note: Deficiencies in methylcobalamin (see Fig.

1	20.8) or N5,N10-MTHF reductase ([MTHFR]; see Fig. 20.12) also result in elevated Hcy.] E. Alkaptonuria Alkaptonuria is a rare organic aciduria involving a deficiency in homogentisic acid oxidase, resulting in the accumulation of homogentisic acid (HA), an intermediate in the degradative pathway of tyrosine (see Fig.

1	20.15 on p. 269). The condition has three characteristic symptoms: homogentisic aciduria (the urine contains elevated levels of HA, which is oxidized to a dark pigment on standing, as shown in Fig. 20.23A), early onset of arthritis in the large joints, and deposition of black pigment (ochronosis) in cartilage and collagenous tissue (see Fig. 20.23B). Dark staining of diapers can indicate the disease in infants, but usually no symptoms are present until about age 40 years. Treatment includes dietary restriction of phenylalanine and tyrosine to reduce HA levels. Although alkaptonuria is not life threatening, the associated arthritis may be severely crippling. [Note: Deficiencies in fumarylacetoacetate hydrolase, the terminal enzyme of tyrosine metabolism, result in tyrosinemia type I (see Fig. 20.15) and a characteristic cabbage-like odor to urine.] VII. CHAPTER SUMMARY

1	Amino acids whose catabolism yields pyruvate or an intermediate of the tricarboxylic acid cycle are termed glucogenic (Fig. 20.24). They can give rise to the net formation of glucose in the liver and kidneys. The solely glucogenic amino acids are glutamine, glutamate, proline, arginine, histidine, alanine, serine, glycine, cysteine, methionine, valine, threonine, aspartate, and asparagine. Amino acids whose catabolism yields either acetoacetate or one of its precursors, acetyl coenzyme A (CoA) or acetoacetyl CoA, are termed ketogenic. Leucine and lysine are solely ketogenic. Tyrosine, phenylalanine, tryptophan, and isoleucine are both ketogenic and glucogenic. Nonessential amino acids can be synthesized from metabolic intermediates or from the carbon skeletons of essential amino acids. Essential amino acids need to be obtained from the diet. They include histidine, methionine, threonine, valine, isoleucine, phenylalanine, tryptophan, leucine, and lysine. Phenylketonuria (PKU) is

1	Essential amino acids need to be obtained from the diet. They include histidine, methionine, threonine, valine, isoleucine, phenylalanine, tryptophan, leucine, and lysine. Phenylketonuria (PKU) is caused by a deficiency of phenylalanine hydroxylase (PAH), which converts phenylalanine to tyrosine. Hyperphenylalaninemia may also be caused by deficiencies in the enzymes that synthesize or regenerate the coenzyme for PAH, tetrahydrobiopterin. Untreated individuals with PKU suffer from severe intellectual disability, developmental delay, microcephaly, seizures, and a characteristic musty (mousy) smell of the urine. Treatment involves controlling dietary phenylalanine. Tyrosine becomes an essential dietary component for people with PKU. Maple syrup urine disease (MSUD) is caused by a partial or complete deficiency in branched-chain a-keto acid dehydrogenase, the enzyme that decarboxylates the branched-chain amino acids (BCAA) leucine, isoleucine, and valine. Symptoms include feeding

1	or complete deficiency in branched-chain a-keto acid dehydrogenase, the enzyme that decarboxylates the branched-chain amino acids (BCAA) leucine, isoleucine, and valine. Symptoms include feeding problems, vomiting, ketoacidosis, changes in muscle tone, and a characteristic sweet smell of the urine. If untreated, the disease leads to neurologic problems that result in death. Treatment involves controlling BCAA intake. Other important genetic diseases associated with amino acid metabolism include albinism, homocystinuria, methylmalonic acidemia, alkaptonuria, histidinemia, tyrosinemia, and cystathioninuria.

1	Choose the ONE best answer. For Questions 20.1–20.3, match the deficient enzyme with the associated clinical sign or laboratory finding in urine. 0.1. Cystathionine β-synthase 0.2. Homogentisic acid oxidase 0.3. Tyrosinase

1	0.1. Cystathionine β-synthase 0.2. Homogentisic acid oxidase 0.3. Tyrosinase Correct answers = F, A, D. A deficiency in cystathionine β-synthase of methionine degradation results in a rise in homocysteine. A deficiency in homogentisic acid oxidase of tyrosine degradation results in a rise in homogentisic acid, which forms a black pigment that is deposited in connective tissue (ochronosis). A deficiency in tyrosinase results in decreased formation of melanin from tyrosine in the skin, hair, and eyes. A sweaty feet– like odor is characteristic of isovaleryl coenzyme A dehydrogenase deficiency. Cystine crystals in urine are seen with cystinuria, a defect in intestinal and renal cystine absorption. Increased branched-chain amino acids are seen in maple syrup urine disease, increased methionine is seen in defects in homocysteine metabolism, and increased phenylalanine is seen in phenylketonuria.

1	0.4. A 1-week-old infant, who was born at home in a rural, medicallyunderserved area, has undetected classic phenylketonuria. Which statement about this baby and/or her treatment is correct? A. A diet devoid of phenylalanine should be initiated immediately. B. Dietary treatment will be discontinued in adulthood. C. Supplementation with vitamin B6 is required. D. Tyrosine is an essential amino acid. Correct answer = D. In patients with phenylketonuria, tyrosine cannot be synthesized from phenylalanine and, hence, becomes essential and must be supplied in the diet. Phenylalanine in the diet must be controlled but cannot be eliminated entirely because it is an essential amino acid. Dietary treatment must begin during the first 7–10 days of life to prevent intellectual disability, and lifelong restriction of phenylalanine is recommended to prevent cognitive decline. Additionally, elevated levels of phenylalanine are teratogenic to a developing fetus.

1	0.5. Which one of the following statements concerning amino acids is correct? A. Alanine is ketogenic. B. Amino acids that are catabolized to acetyl coenzyme A are glucogenic. C. Branched-chain amino acids are catabolized primarily in the liver. D. Cysteine is essential for individuals consuming a diet severely limited in methionine. Correct answer = D. Methionine is the precursor of cysteine, which becomes essential if methionine is severely restricted. Alanine is a key glucogenic amino acid. Acetyl coenzyme A (CoA) cannot be used for the net synthesis of glucose. Amino acids catabolized to acetyl CoA are ketogenic. Branched-chain amino acids are catabolized primarily in skeletal muscle. 0.6. In an individual with the dihydrolipoyl dehydrogenase (E3)-deficient form of maple syrup urine disease, why would lactic acidosis be an expected finding?

1	0.6. In an individual with the dihydrolipoyl dehydrogenase (E3)-deficient form of maple syrup urine disease, why would lactic acidosis be an expected finding? The three α-keto acid dehydrogenase complexes (pyruvate dehydrogenase [PDH], α-ketoglutarate dehydrogenase, and branched-chain α-keto acid dehydrogenase [BCKD]) have dihydrolipoyl dehydrogenase (Enzyme 3, or E3) in common. In E3-deficient maple syrup urine disease, in addition to the branched-chain amino acids and their α-keto acid derivatives accumulating as a result of decreased activity of BCKD, lactate will also be increased because of decreased activity of PDH. 0.7. In contrast to the vitamin B6–derived pyridoxal phosphate required in most enzymic reactions involving amino acids, what coenzyme is required by the aromatic amino acid hydroxylases? Tetrahydrobiopterin, made from guanosine triphosphate, is the required coenzyme. Amino Acids: Conversion to Specialized Products 21

1	Tetrahydrobiopterin, made from guanosine triphosphate, is the required coenzyme. Amino Acids: Conversion to Specialized Products 21 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW In addition to serving as building blocks for proteins, amino acids are precursors of many nitrogen-containing compounds that have important physiologic functions (Fig. 21.1). These molecules include porphyrins, neurotransmitters, hormones, purines, and pyrimidines. [Note: See p. 151 for the synthesis of nitric oxide from arginine.] II. PORPHYRIN METABOLISM

1	II. PORPHYRIN METABOLISM Porphyrins are cyclic compounds that readily bind metal ions, usually ferrous (Fe2+) or ferric (Fe3+) iron. The most prevalent metalloporphyrin in humans is heme, which consists of one Fe2+ coordinated in the center of the tetrapyrrole ring of protoporphyrin IX (see p. 279). Heme is the prosthetic group for hemoglobin (Hb), myoglobin, the cytochromes, the cytochrome P450 (CYP) monooxygenase system, catalase, nitric oxide synthase, and peroxidase. These hemeproteins are rapidly synthesized and degraded. For example, 6–7 g of Hb is synthesized each day to replace heme lost through the normal turnover of erythrocytes. The synthesis and degradation of the associated porphyrins and recycling of the iron are coordinated with the turnover of hemeproteins. A. Structure

1	A. Structure Porphyrins are cyclic planar molecules formed by the linkage of four pyrrole rings through methenyl bridges (Fig. 21.2). Three structural features of these molecules are relevant to understanding their medical significance. 1. Side chains: Different porphyrins vary in the nature of the side chains attached to each of the four pyrrole rings. Uroporphyrin contains acetate (−CH2–COO–) and propionate (−CH2–CH2–COO–) side chains; coproporphyrin contains methyl (−CH3) and propionate groups; and protoporphyrin IX (and heme b, the most common heme) contains vinyl (−CH=CH2), methyl, and propionate groups. [Note: The methyl and vinyl groups are produced by decarboxylation of acetate and propionate side chains, respectively.] 2.

1	Side chain distribution: The side chains of porphyrins can be ordered around the tetrapyrrole nucleus in four different ways, designated by Roman numerals I to IV. Only type III porphyrins, which contain an asymmetric substitution on ring D (see Fig. 21.2), are physiologically important in humans. [Note: Protoporphyrin IX is a member of the type III series.] 3. Porphyrinogens: These porphyrin precursors (for example, uroporphyrinogen) exist in a chemically reduced, colorless form and serve as intermediates between porphobilinogen (PBG) and the oxidized, colored protoporphyrins in heme biosynthesis. B. Heme biosynthesis

1	The major sites of heme biosynthesis are the liver, which synthesizes a number of heme proteins (particularly the CYP proteins), and the erythrocyte-producing cells of the bone marrow, which are active in Hb synthesis. In the liver, the rate of heme synthesis is highly variable, responding to alterations in the cellular heme pool caused by fluctuating demands for hemeproteins. In contrast, heme synthesis in erythroid cells is relatively constant and is matched to the rate of globin synthesis. [Note: Over 85% of all heme synthesis occurs in erythroid tissue. Mature red blood cells (RBC) lack mitochondria and are unable to synthesize heme.] The initial reaction and the last three steps in the formation of porphyrins occur in mitochondria, whereas the intermediate steps of the biosynthetic pathway occur in the cytosol. [Note: Fig. 21.8 summarizes heme synthesis.] 1. δ-Aminolevulinic acid formation: All the carbon and nitrogen atoms of the porphyrin molecule are provided by glycine (a

1	pathway occur in the cytosol. [Note: Fig. 21.8 summarizes heme synthesis.] 1. δ-Aminolevulinic acid formation: All the carbon and nitrogen atoms of the porphyrin molecule are provided by glycine (a nonessential amino acid) and succinyl coenzyme A (a tricarboxylic acid cycle intermediate) that condense to form δ-aminolevulinic acid (ALA) in a reaction catalyzed by ALA synthase ([ALAS], Fig. 21.3). This reaction requires pyridoxal phosphate ([PLP] see p. 382) as a coenzyme and is the committed and rate-limiting step in porphyrin biosynthesis. [Note: There are two ALAS isoforms, each produced by different genes and controlled by different mechanisms. ALAS1 is found in all tissues, whereas ALAS2 is erythroid specific. Loss-of-function mutations in ALAS2 result in X-linked sideroblastic anemia and iron overload.] (Continued in Figs. 21.4 and 21.5.) a.

1	Heme (hemin) effects: When porphyrin production exceeds the availability of the apoproteins that require it, heme accumulates and is converted to hemin by the oxidation of Fe2+ to Fe3+ . Hemin decreases the amount (and, thus, the activity) of ALAS1 by repressing transcription of its gene, increasing degradation of its messenger RNA, and decreasing import of the enzyme into mitochondria. [Note: In erythroid cells, ALAS2 is controlled by the availability of intracellular iron (see p. 475).] b.

1	Drug effects: Administration of any of a large number of drugs results in a significant increase in hepatic ALAS1 activity. These drugs are metabolized by the microsomal CYP monooxygenase system, a hemeprotein oxidase system found in the liver (see p. 149). In response to these drugs, the synthesis of CYP proteins increases, leading to an enhanced consumption of heme, a component of these proteins. This, in turn, causes a decrease in the concentration of heme in liver cells. The lower intracellular heme concentration leads to an increase in the synthesis of ALAS1 and prompts a corresponding increase in the synthesis of ALA. 2. Porphobilinogen formation: The cytosolic condensation of two ALA to form PBG by zinc-containing ALA dehydratase (PBG synthase) is extremely sensitive to inhibition by heavy metal ions (for example, lead) that replace the zinc (see Fig. 21.3). This inhibition is, in part, responsible for the elevation in ALA and the anemia seen in lead poisoning. 3.

1	3. Uroporphyrinogen formation: The condensation of four PBG produces the linear tetrapyrrole hydroxymethylbilane, which is cyclized and isomerized by uroporphyrinogen III synthase to produce the asymmetric uroporphyrinogen III. This cyclic tetrapyrrole undergoes decarboxylation of its acetate groups by uroporphyrinogen III decarboxylase (UROD), generating coproporphyrinogen III (Fig. 21.4). The reactions occur in the cytosol. 4. Heme formation: Coproporphyrinogen III enters the mitochondrion, and two propionate side chains are decarboxylated by coproporphyrinogen III oxidase to vinyl groups generating protoporphyrinogen IX, which is oxidized to protoporphyrin IX. The introduction of iron (as Fe2+) into protoporphyrin IX produces heme. This step can occur spontaneously, but the rate is enhanced by ferrochelatase, an enzyme that, like ALA dehydratase, is inhibited by lead (Fig. 21.5). C. Porphyrias

1	Porphyrias are rare, inherited (or sometimes acquired) defects in heme synthesis, resulting in the accumulation and increased excretion of porphyrins or porphyrin precursors (see Fig. 21.8). [Note: Inherited porphyrias are autosominal-dominant (AD) or autosomal-recessive (AR) disorders.] Each porphyria results in the accumulation of a unique pattern of intermediates caused by the deficiency of an enzyme in the heme synthetic pathway. [Note: Porphyria, derived from the Greek for purple, refers to the red-blue color caused by pigment-like porphyrins in the urine of some patients with defects in heme synthesis.] 1. Clinical manifestations: The porphyrias are classified as erythropoietic or hepatic, depending on whether the enzyme deficiency occurs in the erythropoietic cells of the bone marrow or in the liver. Hepatic porphyrias can be further classified as chronic or acute. In general, individuals with an enzyme defect prior to the synthesis of the tetrapyrroles manifest abdominal and

1	or in the liver. Hepatic porphyrias can be further classified as chronic or acute. In general, individuals with an enzyme defect prior to the synthesis of the tetrapyrroles manifest abdominal and neuropsychiatric signs, whereas those with enzyme defects leading to the accumulation of tetrapyrrole intermediates show photosensitivity (that is, their skin itches and burns [pruritus] when exposed to sunlight). [Note: Photosensitivity is a result of the oxidation of colorless porphyrinogens to colored porphyrins, which are photosensitizing molecules thought to participate in the formation of superoxide radicals from oxygen. These radicals can oxidatively damage membranes and cause the release of destructive enzymes from lysosomes.] a. Chronic hepatic porphyria: Porphyria cutanea tarda, the most common porphyria, is a chronic disease of the liver. The disease is associated with severe deficiency of UROD, but clinical expression of the deficiency is influenced by various factors, such as

1	most common porphyria, is a chronic disease of the liver. The disease is associated with severe deficiency of UROD, but clinical expression of the deficiency is influenced by various factors, such as hepatic iron overload, exposure to sunlight, alcohol ingestion, estrogen therapy, and the presence of hepatitis B or C or HIV infections. [Note: Mutations to UROD are found in only 20% of affected individuals. Inheritance is AD.] Clinical onset is typically during the fourth or fifth decade of life. Porphyrin accumulation leads to cutaneous symptoms (Fig. 21.6) as well as urine that is red to brown in natural light (Fig.

1	21.7) and pink to red in fluorescent light. b. Acute hepatic porphyrias: Acute hepatic porphyrias (ALA dehydratase–deficiency porphyria, acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria) are characterized by acute attacks of gastrointestinal (GI), neuropsychiatric, and motor symptoms that may be accompanied by photosensitivity (Fig. 21.8). Porphyrias leading to accumulation of ALA and PBG, such as acute intermittent porphyria, cause abdominal pain and neuropsychiatric disturbances, ranging from anxiety to delirium. Symptoms of the acute hepatic porphyrias are often precipitated by use of drugs, such as barbiturates and ethanol, which induce the synthesis of the heme-containing CYP microsomal drug-oxidation system. This further decreases the amount of available heme, which, in turn, promotes increased synthesis of ALAS1. c.

1	c. Erythropoietic porphyrias: The chronic erythropoietic porphyrias (congenital erythropoietic porphyria and erythropoietic protoporphyria) cause photosensitivity characterized by skin rashes and blisters that appear in early childhood (see Fig. 21.8). 2. Increased δ-aminolevulinic acid synthase activity: One common feature of the hepatic porphyrias is decreased synthesis of heme. In the liver, heme normally functions as a repressor of the ALAS1 gene. Therefore, the absence of this end product results in an increase in the synthesis of ALAS1 (derepression). This causes an increased synthesis of intermediates that occur prior to the genetic block. The accumulation of these toxic intermediates is the major pathophysiology of the porphyrias. 3.

1	3. Treatment: During acute porphyria attacks, patients require medical support, particularly treatment for pain and vomiting. The severity of acute symptoms of the porphyrias can be diminished by intravenous injection of hemin and glucose, which decreases the synthesis of ALAS1. Protection from sunlight, ingestion of β-carotene (provitamin A; see p. 386) that scavenges free radicals, and phlebotomy (removes porphyrins) are helpful in porphyrias with photosensitivity. D. Heme degradation After ~120 days in the circulation, RBC are taken up and degraded by the mononuclear phagocyte system (MPS), particularly in the liver and spleen (Fig. 21.9). Approximately 85% of heme destined for degradation comes from senescent RBC. The remainder is from the degradation of hemeproteins other than Hb.

1	1. Bilirubin formation: The first step in the degradation of heme is catalyzed by microsomal heme oxygenase in macrophages of the MPS. In the presence of nicotinamide adenine dinucleotide phosphate and oxygen, the enzyme catalyzes three successive oxygenations that result in opening of the porphyrin ring (converting cyclic heme to linear biliverdin), production of carbon monoxide (CO), and release of Fe2+ (see Fig. 21.9). [Note: The CO has biologic function, acting as a signaling molecule and anti-inflammatory. Iron is discussed in Chapter 29.] Biliverdin, a green pigment, is reduced, forming the red-orange bilirubin. Bilirubin and its derivatives are collectively termed bile pigments. [Note: The changing colors of a bruise reflect the varying pattern of intermediates that occurs during heme degradation.]

1	Bilirubin, unique to mammals, appears to function at low levels as an antioxidant. In this role, it is oxidized to biliverdin, which is then reduced by biliverdin reductase, regenerating bilirubin. 2. Bilirubin uptake by the liver: Because bilirubin is only slightly soluble in plasma, it is transported through blood to the liver by binding noncovalently to albumin. [Note: Certain anionic drugs, such as salicylates and sulfonamides, can displace bilirubin from albumin, permitting bilirubin to enter the central nervous system (CNS). This causes the potential for neural damage in infants (see p. 285).] Bilirubin dissociates from the carrier albumin molecule, enters a hepatocyte via facilitated diffusion, and binds to intracellular proteins, particularly the protein ligandin. 3.

1	3. Bilirubin diglucuronide formation: In the hepatocyte, bilirubin solubility is increased by the sequential addition of two molecules of glucuronic acid in a process called conjugation. The reactions are catalyzed by microsomal bilirubin UDP-glucuronosyltransferase (bilirubin UGT) using uridine diphosphate (UDP)-glucuronic acid as the glucuronate donor. The bilirubin diglucuronide product is referred to as conjugated bilirubin (CB). [Note: Varying degrees of deficiency of bilirubin UGT result in Crigler-Najjar I and II and Gilbert syndrome, with Crigler-Najjar I being the most severe.] 4.

1	Bilirubin secretion into bile: CB is actively transported against a concentration gradient into the bile canaliculi and then into the bile. This energy-dependent, rate-limiting step is susceptible to impairment in liver disease. [Note: A rare deficiency in the protein required for transport of CB out of the liver results in Dubin-Johnson syndrome.] Unconjugated bilirubin (UCB) is normally not secreted into bile. 5.

1	5. Urobilin formation in the intestine: CB is hydrolyzed and reduced by gut bacteria to yield urobilinogen, a colorless compound. Most of the urobilinogen is further oxidized by bacteria to stercobilin, which gives feces the characteristic brown color. However, some is reabsorbed from the gut and enters the portal blood. A portion of this urobilinogen participates in the enterohepatic urobilinogen cycle in which it is taken up by the liver and then resecreted into the bile. The remainder of the urobilinogen is transported by the blood to the kidney, where it is converted to yellow urobilin and excreted, giving urine its characteristic color. The metabolism of bilirubin is summarized in Figure 21.10. E. Jaundice

1	E. Jaundice Jaundice (or, icterus) refers to the yellow color of skin, nail beds, and sclerae (whites of the eyes) caused by bilirubin deposition, secondary to increased bilirubin levels in the blood (hyperbilirubinemia) as shown in Figure 21.11. Although not a disease, jaundice is usually a symptom of an underlying disorder. [Note: Blood bilirubin levels are normally ≤1 mg/dl. Jaundice is seen at 2–3 mg/dl.] 1. Types: Jaundice can be classified into three major types described below. However, in clinical practice, jaundice is often more complex than indicated in this simple classification. For example, the accumulation of bilirubin may be a result of defects at more than one step in its metabolism.

1	a. Hemolytic (prehepatic): The liver has the capacity to conjugate and excrete >3,000 mg of bilirubin/day, whereas the normal production of bilirubin is only 300 mg/day. This excess capacity allows the liver to respond to increased heme degradation with a corresponding increase in conjugation and secretion of CB. However, extensive hemolysis (for example, in patients with sickle cell anemia or deficiency of pyruvate kinase or glucose 6-phosphate dehydrogenase) may produce bilirubin faster than it can be conjugated. UCB levels in the blood become elevated (unconjugated hyperbilirubinemia), causing jaundice (Fig. 21.12A). [Note: With hemolysis, more CB is made and excreted into the bile, the amount of urobilinogen entering the enterohepatic circulation is increased, and urinary urobilinogen is increased.] b.

1	Hepatocellular (hepatic): Damage to liver cells (for example, in patients with cirrhosis or hepatitis) can cause unconjugated hyperbilirubinemia as a result of decreased conjugation. Urobilinogen is increased in the urine because hepatic damage decreases the enterohepatic circulation of this compound, allowing more to enter the blood, from which it is filtered into the urine. The urine consequently darkens, whereas stools may be a pale, clay color. Plasma levels of alanine and aspartate transaminases (ALT and AST, respectively; see p. 251) are elevated. If CB is made but is not efficiently secreted from the liver into bile (intrahepatic cholestasis), it can leak into the blood (regurgitation), causing a conjugated hyperbilirubinemia. c.

1	c. Obstructive (posthepatic): In this instance, jaundice is not caused by overproduction of bilirubin or decreased conjugation but, instead, results from obstruction of the common bile duct (extrahepatic cholestasis). For example, the presence of a tumor or bile stones may block the duct, preventing passage of CB into the intestine. Patients with obstructive jaundice experience GI pain and nausea and produce stools that are a pale, clay color. The CB regurgitates into the blood (conjugated hyperbilirubinemia). The CB is eventually excreted in the urine (which darkens over time) and is referred to as urinary bilirubin. Urinary urobilinogen is absent.

1	2. Jaundice in newborns: Most newborn infants (60% of full term and 80% of preterm) show a rise in UCB in the first postnatal week (and a transient, physiologic jaundice) because the activity of hepatic bilirubin UGT is low at birth (it reaches adult levels in about 4 weeks), as shown in Figures 21.12B and 21.13. Elevated UCB, in excess of the binding capacity of albumin (20–25 mg/dl), can diffuse into the basal ganglia, causing toxic encephalopathy (kernicterus) and a pathologic jaundice. Therefore, newborns with significantly elevated bilirubin levels are treated with blue fluorescent light (phototherapy), as shown in Figure 21.14, which converts bilirubin to more polar and, therefore, water-soluble isomers. These photoisomers can be excreted into the bile without conjugation to glucuronic acid. [Note: Because of solubility differences, only UCB crosses the blood–brain barrier, and only CB appears in urine.] 3. Bilirubin measurement: Bilirubin is commonly measured by the van den

1	acid. [Note: Because of solubility differences, only UCB crosses the blood–brain barrier, and only CB appears in urine.] 3. Bilirubin measurement: Bilirubin is commonly measured by the van den Bergh reaction, in which diazotized sulfanilic acid reacts with bilirubin to form red azodipyrroles that are measured colorimetrically. In aqueous solution, the water-soluble CB reacts rapidly with the reagent (within 1 minute) and is said to be direct reacting. The UCB, which is much less soluble in aqueous solution, reacts more slowly. However, when the reaction is carried out in methanol, both CB and UCB are soluble and react with the reagent, providing the total bilirubin value. The indirect-reacting bilirubin, which corresponds to the UCB, is obtained by subtracting the direct-reacting bilirubin from the total bilirubin. [Note: In normal plasma, only ~4% of the total bilirubin is conjugated, or direct reacting, because most is secreted into bile.]

1	III. A. Dopamine, norepinephrine (NE), and epinephrine (or, adrenaline) are biologically active (biogenic) amines that are collectively termed catecholamines. Dopamine and NE are synthesized in the brain and function as neurotransmitters. Epinephrine is synthesized from NE in the adrenal medulla. 1. Function: Outside the CNS, NE and its methylated derivative, epinephrine, are hormone regulators of carbohydrate and lipid metabolism. NE and epinephrine are released from storage vesicles in the adrenal medulla in response to fright, exercise, cold, and low levels of blood glucose. They increase the degradation of glycogen and triacylglycerol as well as increase blood pressure and the output of the heart. These effects are part of a coordinated response to prepare the individual for stress and are often called the “fight-or-flight” reactions. 2.

1	2. Synthesis: The catecholamines are synthesized from tyrosine, as shown in Figure 21.15. Tyrosine is first hydroxylated by tyrosine hydroxylase to form L-3,4-dihydroxyphenylalanine (DOPA) in a reaction analogous to that described for the hydroxylation of phenylalanine (see p. 263). The tetrahydrobiopterin (BH4)-requiring enzyme is abundant in the CNS, the sympathetic ganglia, and the adrenal medulla, and it catalyzes the rate-limiting step of the pathway. DOPA is decarboxylated in a reaction requiring PLP to form dopamine, which is hydroxylated by dopamine βhydroxylase to yield NE in a reaction that requires ascorbic acid (vitamin C) and copper. Epinephrine is formed from NE by an N-methylation reaction using S-adenosylmethionine (SAM) as the methyl donor (see p. 264).

1	Parkinson disease, a neurodegenerative movement disorder, is due to insufficient dopamine production as a result of the idiopathic loss of dopamine-producing cells in the brain. Administration of L-DOPA (levodopa) is the most common treatment, because dopamine cannot cross the blood–brain barrier.

1	3. Degradation: The catecholamines are inactivated by oxidative deamination catalyzed by monoamine oxidase (MAO) and by Omethylation catalyzed by catechol-O-methyltransferase (COMT) using SAM as the methyl donor (Fig. 21.16). The reactions can occur in either order. The aldehyde products of the MAO reaction are oxidized to the corresponding acids. The products of these reactions are excreted in the urine as vanillylmandelic acid (VMA) from epinephrine and NE and homovanillic acid (HVA) from dopamine. [Note: VMA and the metanephrines are increased with pheochromocytomas, rare tumors of the adrenal gland characterized by excessive production of catecholamines.] 4. Monoamine oxidase inhibitors: MAO is found in neural and other tissues, such as the intestine and liver. In the neuron, this enzyme oxidatively deaminates and inactivates any excess neurotransmitter molecules (NE, dopamine, or serotonin) that may leak out of synaptic vesicles when the neuron is at rest. MAO inhibitors (MAOI)

1	oxidatively deaminates and inactivates any excess neurotransmitter molecules (NE, dopamine, or serotonin) that may leak out of synaptic vesicles when the neuron is at rest. MAO inhibitors (MAOI) may irreversibly or reversibly inactivate the enzyme, permitting neurotransmitter molecules to escape degradation and, therefore, both to accumulate within the presynaptic neuron and to leak into the synaptic space. This causes activation of NE and serotonin receptors and may be responsible for the antidepressant action of MAOI. [Note: The interaction of MAOI with tyraminecontaining foods is discussed on p. 373.]

1	B. Histamine Histamine is a chemical messenger that mediates a wide range of cellular responses, including allergic and inflammatory reactions and gastric acid secretion. A powerful vasodilator, histamine is formed by decarboxylation of histidine in a reaction requiring PLP (Fig. 21.17). It is secreted by mast cells as a result of allergic reactions or trauma. Histamine has no clinical applications, but agents that interfere with the action of histamine have important therapeutic applications. C. Serotonin

1	C. Serotonin Serotonin, also called 5-hydroxytryptamine (5-HT), is synthesized and/or stored at several sites in the body (Fig. 21.18). The largest amount by far is found in the intestinal mucosa. Smaller amounts occur in the CNS, where it functions as a neurotransmitter, and in platelets (see online Chapter 35). Serotonin is synthesized from tryptophan, which is hydroxylated in a BH4 requiring reaction analogous to that catalyzed by phenylalanine hydroxylase. The product, 5-hydroxytryptophan, is decarboxylated to 5HT. Serotonin has multiple physiologic roles including pain perception and regulation of sleep, appetite, temperature, blood pressure, cognitive functions, and mood (causes a feeling of well-being). [Note: Selective serotonin reuptake inhibitors (SSRI) maintain serotonin levels, thereby functioning as antidepressants.] Serotonin is degraded by MAO to 5hydroxy-3-indoleacetic acid (5-HIAA). D. Creatine

1	D. Creatine Creatine phosphate (also called phosphocreatine), the phosphorylated derivative of creatine found in muscle, is a high-energy compound that provides a small but rapidly mobilized reserve of high-energy phosphates that can be reversibly transferred to adenosine diphosphate (Fig. 21.19) to maintain the intracellular level of ATP during the first few minutes of intense muscular contraction. [Note: The amount of creatine phosphate in the body is proportional to the muscle mass.] 1. Synthesis: Creatine is synthesized in the liver and kidneys from glycine and the guanidino group of arginine, plus a methyl group from SAM (see Fig. 21.19). Animal products are dietary sources. Creatine is reversibly phosphorylated to creatine phosphate by creatine kinase, using ATP as the phosphate donor. [Note: The presence of creatine kinase (MB isozyme) in the plasma is indicative of heart damage and is used in the diagnosis of myocardial infarction (see p. 65).] 2.

1	Degradation: Creatine and creatine phosphate spontaneously cyclize at a slow but constant rate to form creatinine, which is excreted in the urine. The amount excreted is proportional to the total creatine phosphate content of the body and, therefore, can be used to estimate muscle mass. When muscle mass decreases for any reason (for example, from paralysis or muscular dystrophy), the creatinine content of the urine falls. In addition, a rise in blood creatinine is a sensitive indicator of kidney malfunction, because creatinine normally is rapidly cleared from the blood and excreted. A typical adult male excretes ~1–2 g of creatinine/day. E. Melanin

1	E. Melanin Melanin is a pigment that occurs in several tissues, particularly the eye, hair, and skin. It is synthesized from tyrosine in melanocytes (pigmentforming cells) of the epidermis. It functions to protect underlying cells from the harmful effects of sunlight. [Note: A defect in melanin production results in oculocutaneous albinism, the most common type being due to defects in copper-containing tyrosinase (see p. 273).] IV. CHAPTER SUMMARY

1	Amino acids are precursors of many nitrogen (N)-containing compounds including porphyrins, which, in combination with ferrous (Fe2+) iron, form heme (Fig. 21.20). The major sites of heme biosynthesis are the liver, which synthesizes a number of hemeproteins (particularly cytochrome P450 enzymes), and the erythrocyte-producing cells of the bone marrow, which are active in hemoglobin synthesis. In the liver, the rate of heme synthesis is highly variable, responding to alterations in the cellular heme pool caused by fluctuating demands for hemeproteins. In contrast, heme synthesis in erythroid cells is relatively constant and is matched to the rate of globin synthesis. Heme synthesis starts with glycine and succinyl coenzyme A. The committed step is the formation of δ-aminolevulinic acid (ALA). This mitochondrial reaction is catalyzed by ALA synthase-1 (ALAS1) in the liver (inhibited by hemin, the oxidized form of heme that accumulates when heme is being underutilized) and ALAS2 in

1	(ALA). This mitochondrial reaction is catalyzed by ALA synthase-1 (ALAS1) in the liver (inhibited by hemin, the oxidized form of heme that accumulates when heme is being underutilized) and ALAS2 in erythroid tissues (regulated by iron). Porphyrias are caused by inherited or acquired (lead poisoning) defects in heme synthesis, resulting in the accumulation and increased excretion of porphyrins or porphyrin precursors. Enzymic defects early in the pathway cause abdominal pain and neuropsychiatric symptoms, whereas later defects cause photosensitivity. Degradation of heme occurs in the mononuclear phagocyte system, particularly in the liver and spleen. The first step is the production by heme oxygenase of biliverdin, which is subsequently reduced to bilirubin. Bilirubin is transported by albumin to the liver, where its solubility is increased by the addition of two molecules of glucuronic acid by bilirubin uridine diphosphate-glucuronosyltransferase (bilirubin UGT). Bilirubin

1	by albumin to the liver, where its solubility is increased by the addition of two molecules of glucuronic acid by bilirubin uridine diphosphate-glucuronosyltransferase (bilirubin UGT). Bilirubin diglucuronide (conjugated bilirubin) is transported into the bile canaliculi, where it is first hydrolyzed and reduced by gut bacteria to yield urobilinogen, which is further oxidized by bacteria to stercobilin. Jaundice (icterus) refers to the yellow color of the skin and sclerae that is caused by deposition of bilirubin, secondary to increased bilirubin levels in the blood. Three commonly encountered types of jaundice are hemolytic (prehepatic), obstructive (posthepatic), and hepatocellular (hepatic) (see Fig. 21.20). Other important N-containing compounds derived from amino acids include the catecholamines (dopamine, norepinephrine, and epinephrine), creatine, histamine, serotonin, melanin, and nitric oxide.

1	bilirubin or decreased secretion of conjugated bilirubin from the liver into bile.] CoA = coenzyme A; CO = carbon monoxide; Fe = iron. Choose the ONE best answer. 1.1. δ-Aminolevulinic acid synthase activity: A. catalyzes the committed step in porphyrin biosynthesis. B. is decreased by iron in erythrocytes. C. is decreased in the liver in individuals treated with certain drugs such as the barbiturate phenobarbital. D. occurs in the cytosol. E. requires tetrahydrobiopterin as a coenzyme. Correct answer = A. δ-Aminolevulinic acid synthase is mitochondrial and catalyzes the rate-limiting and regulated step of porphyrin synthesis. It requires pyridoxal phosphate as a coenzyme. Iron increases production of the erythroid isozyme. The hepatic isozyme is increased in patients treated with certain drugs.

1	1.2. A 50-year-old man presented with painful blisters on the backs of his hands. He was a golf instructor and indicated that the blisters had erupted shortly after the golfing season began. He did not have recent exposure to common skin irritants. He had partial complex seizure disorder that had begun ~3 years earlier after a head injury. The patient had been taking phenytoin (his only medication) since the onset of the seizure disorder. He admitted to an average weekly ethanol intake of ~18 12-oz cans of beer. The patient’s urine was reddish orange. Cultures obtained from skin lesions failed to grow organisms. A 24-hour urine collection showed elevated uroporphyrin (1,000 mg; normal, <27 mg). The most likely diagnosis is: A. acute intermittent porphyria. B. congenital erythropoietic porphyria. C. erythropoietic protoporphyria. D. hereditary coproporphyria. E. porphyria cutanea tarda.

1	A. acute intermittent porphyria. B. congenital erythropoietic porphyria. C. erythropoietic protoporphyria. D. hereditary coproporphyria. E. porphyria cutanea tarda. Correct answer = E. The disease is associated with a deficiency in uroporphyrinogen III decarboxylase (UROD), but clinical expression of the enzyme deficiency is influenced by hepatic injury caused by environmental (for example, ethanol) and infectious (for example, hepatitis B virus) agents. Exposure to sunlight can also be a precipitating factor. Clinical onset is typically during the fourth or fifth decade of life. Porphyrin accumulation leads to cutaneous symptoms and urine that is red to brown. Treatment of the patient’s seizure disorder with phenytoin caused increased synthesis of δaminolevulinic acid synthase and, therefore, of uroporphyrinogen, the substrate of the deficient UROD. The laboratory and clinical findings are inconsistent with other porphyrias.

1	21.3. A patient presents with jaundice, abdominal pain, and nausea. Clinical laboratory results are shown below. What is the most likely cause of the jaundice? A. Decreased hepatic conjugation of bilirubin B. Decreased hepatic uptake of bilirubin C. Decreased secretion of bile into the intestine D. Increased hemolysis Correct answer = C. The data are consistent with an obstructive jaundice in which a block in the common bile duct decreases the secretion of bile containing conjugated bilirubin (CB) into the intestine (stool will be pale in color). The CB regurgitates into the blood (conjugated hyperbilirubinemia). The CB is excreted in the urine (which darkens) and is referred to as urinary bilirubin. Urinary urobilinogen is not present because its source is intestinal urobilinogen, which is low. The other choices do not match the data.

1	1.4. A 2-year-old child was brought to his pediatrician for evaluation of gastrointestinal problems. The parents report that the boy has been listless for the last few weeks. Lab tests reveal a microcytic, hypochromic anemia. Blood lead levels are elevated. Which of the enzymes listed below is most likely to have higher-than-normal activity in the liver of this child? A. δ-Aminolevulinic acid synthase B. Bilirubin UDP glucuronosyltransferase C. Ferrochelatase D. Heme oxygenase E. Porphobilinogen synthase

1	A. δ-Aminolevulinic acid synthase B. Bilirubin UDP glucuronosyltransferase C. Ferrochelatase D. Heme oxygenase E. Porphobilinogen synthase Correct answer = A. This child has the acquired porphyria of lead poisoning. Lead inhibits both δ-aminolevulinic acid dehydratase and ferrochelatase and, consequently, heme synthesis. The decrease in heme derepresses δaminolevulinic acid synthase-1 (the hepatic isozyme), resulting in an increase in its activity. The decrease in heme also results in decreased hemoglobin synthesis, and anemia is seen. Ferrochelatase is directly inhibited by lead. The other choices are enzymes of heme degradation. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Ribonucleoside and deoxyribonucleoside phosphates (nucleotides) are essential for all cells. Without them, neither ribonucleic acid (RNA) nor deoxyribonucleic acid (DNA) can be produced, and, therefore, proteins cannot be synthesized or cells proliferate. Nucleotides also serve as carriers of activated intermediates in the synthesis of some carbohydrates, lipids, and conjugated proteins (for example, uridine diphosphate [UDP]-glucose and cytidine diphosphate [CDP]choline) and are structural components of several essential coenzymes, such as coenzyme A, flavin adenine dinucleotide (FAD[H2]), nicotinamide adenine dinucleotide (NAD[H]), and nicotinamide adenine dinucleotide phosphate (NADP[H]). Nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), serve as second messengers in signal transduction pathways. In addition, nucleotides play an important role as energy sources in the cell. Finally, nucleotides are important regulatory compounds

1	serve as second messengers in signal transduction pathways. In addition, nucleotides play an important role as energy sources in the cell. Finally, nucleotides are important regulatory compounds for many of the pathways of intermediary metabolism, inhibiting or activating key enzymes. The purine and pyrimidine bases found in nucleotides can be synthesized de novo or can be obtained through salvage pathways that allow the reuse of the preformed bases resulting from normal cell turnover. [Note: Little of the purines and pyrimidines supplied by diet is utilized and is degraded instead.]

1	II. STRUCTURE Nucleotides are composed of a nitrogenous base; a pentose monosaccharide; and one, two, or three phosphate groups. The nitrogen-containing bases belong to two families of compounds: the purines and the pyrimidines. A. Purine and pyrimidine bases

1	A. Purine and pyrimidine bases Both DNA and RNA contain the same purine bases: adenine (A) and guanine (G). Both DNA and RNA contain the pyrimidine cytosine (C), but they differ in their second pyrimidine base: DNA contains thymine (T), whereas RNA contains uracil (U). T and U differ in that only T has a methyl group (Fig. 22.1). Unusual (modified) bases are occasionally found in some species of DNA (for example, in some viral DNA) and RNA (for example, in transfer RNA [tRNA]). Base modifications include methylation, glycosylation, acetylation, and reduction. Some examples of unusual bases are shown in Figure 22.2. [Note: The presence of an unusual base in a nucleotide sequence may aid in its recognition by specific enzymes or protect it from being degraded by nucleases.] B. Nucleosides

1	B. Nucleosides The addition of a pentose sugar to a base through an N-glycosidic bond (see p. 86) produces a nucleoside. If the sugar is ribose, a ribonucleoside is produced, and if the sugar is 2-deoxyribose, a deoxyribonucleoside is produced (Fig. 22.3A). The ribonucleosides of A, G, C, and U are named adenosine, guanosine, cytidine, and uridine, respectively. The deoxyribonucleosides of A, G, C, and T have the added prefix deoxy-(for example, deoxyadenosine). [Note: The compound deoxythymidine is often simply called thymidine, with the deoxy-prefix being understood, because it is incorporated into DNA only.] The carbon and nitrogen atoms in the rings of the base and the sugar are numbered separately (see Fig. 22.3B). [Note: Carbons in the pentose are numbered 1′ to 5′. Thus, when the 5′carbon of a nucleoside (or nucleotide) is referred to, a carbon atom in the pentose, rather than an atom in the base, is being specified.] C. Nucleotides

1	The addition of one or more phosphate groups to a nucleoside produces a nucleotide. The first phosphate group is attached by an ester linkage to the 5′-OH of the pentose, forming a nucleoside 5′-phosphate or a 5′-nucleotide. The type of pentose is denoted by the prefix in the names 5′-ribonucleotide and 5′-deoxyribonucleotide. If one phosphate group is attached to the 5′carbon of the pentose, the structure is a nucleoside monophosphate, like adenosine monophosphate (AMP, or adenylate). If a second or third phosphate is added to the nucleoside, a nucleoside diphosphate (for example, adenosine diphosphate [ADP] or triphosphate, for example, ATP) results (Fig. 22.4). The second and third phosphates are each connected to the nucleotide by a “high-energy bond” (a bond with a large, negative change in free energy [–∆G, see p. 70] of hydrolysis). [Note: The phosphate groups are responsible for the negative charges associated with nucleotides and cause DNA and RNA to be referred to as nucleic

1	in free energy [–∆G, see p. 70] of hydrolysis). [Note: The phosphate groups are responsible for the negative charges associated with nucleotides and cause DNA and RNA to be referred to as nucleic acids.]

1	III. PURINE NUCLEOTIDE SYNTHESIS The atoms of the purine ring are contributed by a number of compounds, including amino acids (aspartate, glycine, and glutamine), carbon dioxide (CO2), and N10-formyltetrahydrofolate (N10-formyl-THF), as shown in Figure 22.5. The purine ring is constructed primarily in the liver by a series of reactions that add the donated carbons and nitrogens to a preformed ribose 5-phosphate. [Note: Synthesis of ribose 5-phosphate from glucose 6-phosphate by the pentose phosphate pathway is discussed on p. 147.]

1	A. 5-Phosphoribosyl-1-pyrophosphate synthesis 5-Phosphoribosyl-1-pyrophosphate (PRPP) is an activated pentose that participates in the synthesis and salvage of purines and pyrimidines. Synthesis of PRPP from ATP and ribose 5-phosphate is catalyzed by PRPP synthetase (Fig. 22.6). This X-linked enzyme is activated by inorganic phosphate and inhibited by purine nucleotides (end-product inhibition). [Note: Because the sugar moiety of PRPP is ribose, ribonucleotides are the end products of de novo purine synthesis. When deoxyribonucleotides are required for DNA synthesis, the ribose sugar moiety is reduced (see p. 297).] reaction. [Note: This is not the committed step of purine synthesis because PRPP is used in other pathways such as salvage (see p. 296).] = phosphate; Pi = inorganic phosphate; AMP = adenosine monophosphate; Mg = magnesium. B. 5-Phosphoribosylamine synthesis

1	B. 5-Phosphoribosylamine synthesis Synthesis of 5-phosphoribosylamine from PRPP and glutamine is shown in Figure 22.7. The amide group of glutamine replaces the pyrophosphate group attached to carbon 1 of PRPP. This is the committed step in purine nucleotide biosynthesis. The enzyme that catalyzes the reaction, glutamine:phosphoribosylpyrophosphate amidotransferase (GPAT), is inhibited by the purine 5′-nucleotides AMP and guanosine monophosphate (GMP, or guanylate), the end products of the pathway. The rate of the reaction is also controlled by the intracellular concentration of PRPP. [Note: The concentration of PRPP is normally far below the Michaelis constant (Km) for the GPAT. Therefore, any small change in the PRPP concentration causes a proportional change in rate of the reaction (see p. 59).] dioxide. C. Inosine monophosphate synthesis

1	C. Inosine monophosphate synthesis The next nine steps in purine nucleotide biosynthesis leading to the synthesis of inosine monophosphate ([IMP] whose base is hypoxanthine) are illustrated in Figure 22.7. IMP is the parent purine nucleotide for AMP and GMP. Four steps in this pathway require ATP as an energy source, and two steps in the pathway require N10-formyl-THF as a one-carbon donor (see p. 267). [Note: Hypoxanthine is found in tRNA (see Fig. 32.9 on p. 453).] D. Synthetic inhibitors Some synthetic inhibitors of purine synthesis (for example, the sulfonamides) are designed to inhibit the growth of rapidly dividing microorganisms without interfering with human cell functions (see Fig. 22.7). Other purine synthesis inhibitors, such as structural analogs of folic acid (for example, methotrexate), are used pharmacologically to control the spread of cancer by interfering with the synthesis of nucleotides and, therefore, of DNA and RNA (see Fig. 22.7).

1	Inhibitors of human purine synthesis are extremely toxic to tissues, especially to developing structures such as in a fetus, or to cell types that normally replicate rapidly, including those of bone marrow, skin, gastrointestinal (GI) tract, immune system, or hair follicles. As a result, individuals taking such anticancer drugs can experience adverse effects, including anemia, scaly skin, GI tract disturbance, immunodeficiency, and hair loss. E. Adenosine and guanosine monophosphate synthesis

1	E. Adenosine and guanosine monophosphate synthesis The conversion of IMP to either AMP or GMP uses a two-step, energy-and nitrogen-requiring pathway (Fig. 22.8). [Note: AMP synthesis requires guanosine triphosphate (GTP) as an energy source and aspartate as a nitrogen source, whereas GMP synthesis requires ATP and glutamine.] Also, the first reaction in each pathway is inhibited by the end product of that pathway. This provides a mechanism for diverting IMP to the synthesis of the purine present in lesser amounts. If both AMP and GMP are present in adequate amounts, the de novo pathway of purine nucleotide synthesis is inhibited at the GPAT step. showing feedback inhibition. NAD(H) = nicotinamide adenine dinucleotide; = pyrophosphate. F. Nucleoside di-and triphosphate synthesis

1	showing feedback inhibition. NAD(H) = nicotinamide adenine dinucleotide; = pyrophosphate. F. Nucleoside di-and triphosphate synthesis Nucleoside diphosphates are synthesized from the corresponding nucleoside monophosphates by base-specific nucleoside monophosphate kinases (Fig. 22.9). [Note: These kinases do not discriminate between ribose or deoxyribose in the substrate.] ATP is generally the source of the transferred phosphate because it is present in higher concentrations than the other nucleoside triphosphates. Adenylate kinase is particularly active in the liver and in muscle, where the turnover of energy from ATP is high. Its function is to maintain equilibrium among the adenine nucleotides (AMP, ADP, and ATP). Nucleoside diphosphates and triphosphates are interconverted by nucleoside diphosphate kinase, an enzyme that, unlike the monophosphate kinases, has broad substrate specificity. G. Purine salvage pathway

1	Purines that result from the normal turnover of cellular nucleic acids, or the small amount that is obtained from the diet and not degraded, can be converted to nucleoside triphosphates and used by the body. This is referred to as the salvage pathway for purines. [Note: Salvage is particularly important in the brain.] 1. purine base salvage to nucleotides: Two enzymes are involved: adenine phosphoribosyltransferase (APRT) and X-linked hypoxanthine– guanine phosphoribosyltransferase (HGPRT). Both use PRPP as the source of the ribose 5-phosphate group (Fig. 22.10). The release of pyrophosphate and its subsequent hydrolysis by pyrophosphatase makes these reactions irreversible. [Note: Adenosine is the only purine nucleoside to be salvaged. It is phosphorylated to AMP by adenosine kinase.] deficiencies of HGPRT are known. As the amount of functional enzyme increases, the severity of the symptoms decreases.] IMP, GMP, and AMP = inosine, guanosine, and adenosine monophosphates; PRPP =

1	deficiencies of HGPRT are known. As the amount of functional enzyme increases, the severity of the symptoms decreases.] IMP, GMP, and AMP = inosine, guanosine, and adenosine monophosphates; PRPP = 5-phosphoribosyl1-pyrophosphate; PPi = pyrophosphate.

1	2. Lesch-Nyhan syndrome: This is a rare, X-linked recessive disorder associated with a virtually complete deficiency of HGPRT. The deficiency results in an inability to salvage hypoxanthine or guanine, from which excessive amounts of uric acid, the end product of purine degradation, are then produced (see p. 298). In addition, the lack of this salvage pathway causes increased PRPP levels and decreased IMP and GMP levels. As a result, GPAT (the regulated step in purine synthesis) has excess substrate and decreased inhibitors available, and de novo purine synthesis is increased. The combination of decreased purine reutilization and increased purine synthesis results in increased degradation of purines and the production of large amounts of uric acid, making HGPRT deficiency an inherited cause of hyperuricemia. In patients with Lesch-Nyhan syndrome, the hyperuricemia frequently results in the formation of uric acid stones in the kidneys (urolithiasis) and the deposition of urate crystals

1	of hyperuricemia. In patients with Lesch-Nyhan syndrome, the hyperuricemia frequently results in the formation of uric acid stones in the kidneys (urolithiasis) and the deposition of urate crystals in the joints (gouty arthritis) and soft tissues. In addition, the syndrome is characterized by motor dysfunction, cognitive deficits, and behavioral disturbances that include self-mutilation (for example, biting of lips and fingers), as shown in Figure 22.11.

1	IV. DEOXYRIBONUCLEOTIDE SYNTHESIS The nucleotides described thus far all contain ribose (ribonucleotides). DNA synthesis, however, requires 2′-deoxyribonucleotides, which are produced from ribonucleoside diphosphates by the enzyme ribonucleotide reductase during the S-phase of the cell cycle (see p. 423). [Note: The same enzyme acts on pyrimidine ribonucleotides.] A. Ribonucleotide reductase

1	A. Ribonucleotide reductase Ribonucleotide reductase (ribonucleoside diphosphate reductase) is a dimer composed of two nonidentical subunits, R1 (or, α) and the smaller R2 (or, β), and is specific for the reduction of purine nucleoside diphosphates (ADP and GDP) and pyrimidine nucleoside diphosphates (CDP and UDP) to their deoxy forms (dADP, dGDP, dCDP, and dUDP). The immediate donors of the hydrogen atoms needed for the reduction of the 2′-hydroxyl group are two sulfhydryl (–SH) groups on the enzyme itself (R1 subunit), which form a disulfide bond during the reaction (see p. 19). [Note: R2 contains the stable tyrosyl radical required for catalysis at R1.] 1.

1	Reduced enzyme regeneration: In order for ribonucleotide reductase to continue to produce deoxyribonucleotides at R1, the disulfide bond created during the production of the 2′-deoxy carbon must be reduced. The source of the reducing equivalents is thioredoxin, a protein coenzyme of ribonucleotide reductase. Thioredoxin contains two cysteine residues separated by two amino acids in the peptide chain. The two –SH groups of thioredoxin donate their hydrogen atoms to ribonucleotide reductase, forming a disulfide bond in the process (Fig. 22.12). 2. Reduced thioredoxin regeneration: Thioredoxin must be converted back to its reduced form in order to continue performing its function. The reducing equivalents are provided by NADPH + H+, and the reaction is catalyzed by thioredoxin reductase, a selenoprotein (see p. 268). B. Deoxyribonucleotide synthesis regulation

1	B. Deoxyribonucleotide synthesis regulation Ribonucleotide reductase is responsible for maintaining a balanced supply of the deoxyribonucleotides required for DNA synthesis. Consequently, the regulation of the enzyme is complex. In addition to the catalytic site, R1 contains two distinct allosteric sites involved in regulating enzymic activity (Fig. 22.13). 1. Activity sites: The binding of dATP to allosteric sites (known as activity sites) on R1 inhibits the overall catalytic activity of the enzyme and, therefore, prevents the reduction of any of the four nucleoside diphosphates. This effectively prevents DNA synthesis and explains the toxicity of increased levels of dATP seen in conditions such as adenosine deaminase (ADA) deficiency (see p. 301). In contrast, ATP bound to these sites activates the enzyme. 2.

1	2. Substrate specificity sites: The binding of nucleoside triphosphates to additional allosteric sites (known as substrate specificity sites) on R1 regulates substrate specificity, causing an increase in the conversion of different species of ribonucleotides to deoxyribonucleotides as they are required for DNA synthesis. For example, deoxythymidine triphosphate binding at the specificity site causes a conformational change that allows reduction of GDP to dGDP at the catalytic site when ATP is at the activity site. The drug hydroxyurea (hydroxycarbamide) inhibits ribonucleotide reductase, thereby inhibiting the generation of substrates for DNA synthesis. The drug is an antineoplastic agent and is used in the treatment of cancers such as melanoma. Hydroxyurea is also used in the treatment of sickle cell anemia (see p. 36). However, the increase in fetal hemoglobin seen with hydroxyurea is because of changes in gene expression and not to ribonucleotide reductase inhibition.

1	V. PURINE NUCLEOTIDE DEGRADATION Degradation of dietary nucleic acids occurs in the small intestine, where pancreatic nucleases hydrolyze them to nucleotides. The nucleotides are sequentially degraded by intestinal enzymes to nucleosides, phosphorylated sugars, and free bases. Uric acid is the end product of intestinal purine degradation. [Note: Purine nucleotides from de novo synthesis are degraded in the liver primarily. The free bases are sent out from the liver and salvaged by peripheral tissues.] A. Degradation in the small intestine

1	Ribonucleases and deoxyribonucleases, secreted by the pancreas, hydrolyze dietary RNA and DNA to oligonucleotides that are further hydrolyzed by pancreatic phosphodiesterases, producing a mixture of 3′and 5′-mononucleotides. At the intestinal mucosal surface, nucleotidases remove the phosphate groups hydrolytically, releasing nucleosides that are taken into enterocytes by sodium-dependent transporters and degraded by nucleosidases (nucleoside phosphorylases) to free bases plus (deoxy) ribose 1-phosphate. Dietary purine bases are not used to any appreciable extent for the synthesis of tissue nucleic acids. Instead, they are degraded to uric acid in the enterocytes. Most of the uric acid enters the blood and is eventually excreted in the urine. A summary of this pathway is shown in Figure 22.14. [Note: Mammals other than primates express urate oxidase (uricase), which cleaves the purine ring, generating allantoin. Modified recombinant urate oxidase is now used clinically to lower urate

1	[Note: Mammals other than primates express urate oxidase (uricase), which cleaves the purine ring, generating allantoin. Modified recombinant urate oxidase is now used clinically to lower urate levels.]

1	B. Uric acid formation A summary of the steps in the production of uric acid and the genetic diseases associated with deficiencies of specific degradative enzymes are shown in Figure 22.15. [Note: The bracketed numbers refer to specific reactions in the figure.] some of the genetic diseases associated with this pathway. [Note: The numbers in brackets refer to the corresponding numbered citations in the text.] BMT = bone marrow transplantation; ERT = enzyme replacement therapy; Pi = inorganic phosphate; H2O2 = hydrogen peroxide; NH3 = ammonia. [1] An amino group is removed from AMP to produce IMP by AMP (adenylate) deaminase or from adenosine to produce inosine (hypoxanthine-ribose) by adenosine deaminase. [2] IMP and GMP are converted into their respective nucleoside forms, inosine and guanosine, by the action of 5′-nucleotidase.

1	[2] IMP and GMP are converted into their respective nucleoside forms, inosine and guanosine, by the action of 5′-nucleotidase. [3] Purine nucleoside phosphorylase converts inosine and guanosine into their respective purine bases, hypoxanthine and guanine. [Note: A mutase interconverts ribose 1-and ribose 5-phosphate.] [4] Guanine is deaminated to form xanthine. [5] Hypoxanthine is oxidized by molybdenum-containing xanthine oxidase (XO) to xanthine, which is further oxidized by XO to uric acid, the final product of human purine degradation. Uric acid is excreted primarily in the urine.

1	C. Diseases associated with purine degradation 1. Gout: Gout is a disorder initiated by high levels of uric acid (the end product of purine catabolism) in blood (hyperuricemia), as a result of either the overproduction or underexcretion of uric acid. The hyperuricemia can lead to the deposition of monosodium urate (MSU) crystals in the joints and an inflammatory response to the crystals, causing first acute and then progressing to chronic gouty arthritis. Nodular masses of MSU crystals (tophi) may be deposited in the soft tissues, resulting in chronic tophaceous gout (Fig. 22.16). Formation of uric acid stones in the kidney (urolithiasis) may also be seen. [Note: Hyperuricemia is not sufficient to cause gout, but gout is always preceded by hyperuricemia. Hyperuricemia is typically asymptomatic but may be indicative of comorbid conditions such as hypertension.] The definitive diagnosis of gout requires aspiration and examination of synovial fluid (Fig. 22.17) from an affected joint (or

1	but may be indicative of comorbid conditions such as hypertension.] The definitive diagnosis of gout requires aspiration and examination of synovial fluid (Fig. 22.17) from an affected joint (or material from a tophus) using polarized light microscopy to confirm the presence of needle-shaped MSU crystals (Fig. 22.18).

1	a. Uric acid underexcretion: In >90% of individuals with hyperuricemia, the cause is underexcretion of uric acid. Underexcretion can be primary, because of as-yet-unidentified inherent excretory defects, or secondary to known disease processes that affect how the kidney handles urate (for example, in lactic acidosis, lactate increases renal urate reabsorption, thereby decreasing its excretion) and to environmental factors such as the use of drugs (for example, thiazide diuretics) or exposure to lead (saturnine gout). b.

1	Uric acid overproduction: A less common cause of hyperuricemia is from the overproduction of uric acid. Primary hyperuricemia is, for the most part, idiopathic (having no known cause). However, several identified mutations in the gene for X-linked PRPP synthetase result in the enzyme having an increased maximal velocity ([Vmax] see p. 57) for the production of PRPP, a lower Km (see p. 59) for ribose 5phosphate, or a decreased sensitivity to purine nucleotides, its allosteric inhibitors (see p. 62). In each case, increased availability of PRPP increases purine production, resulting in elevated levels of plasma uric acid. Lesch-Nyhan syndrome (see p. 296) also causes hyperuricemia as a result of the decreased salvage of hypoxanthine and guanine and the subsequent increased availability of PRPP. Secondary hyperuricemia is typically the consequence of increased availability of purines (for example, in patients with myeloproliferative disorders or who are undergoing chemotherapy and so

1	PRPP. Secondary hyperuricemia is typically the consequence of increased availability of purines (for example, in patients with myeloproliferative disorders or who are undergoing chemotherapy and so have a high rate of cell turnover). Hyperuricemia can also be the result of seemingly unrelated metabolic diseases, such as von Gierke disease (see Fig. 11.8 on p.

1	130) or hereditary fructose intolerance (see p. 138). A diet rich in meat, seafood (particularly shellfish), and ethanol is associated with increased risk of gout, whereas a diet rich in low-fat dairy products is associated with a decreased risk.

1	c. Treatment: Acute attacks of gout are treated with anti-inflammatory agents. Colchicine, steroidal drugs such as prednisone, and nonsteroidal drugs such as indomethacin are used. [Note: Colchicine prevents formation of microtubules, thereby decreasing the movement of neutrophils into the affected area. Like the other anti-inflammatory drugs, it has no effect on uric acid levels.] Long-term therapeutic strategies for gout involve lowering the uric acid level below its saturation point (6.5 mg/dl), thereby preventing the deposition of MSU crystals. Uricosuric agents, such as probenecid or sulfinpyrazone, that increase renal excretion of uric acid, are used in patients who are underexcretors of uric acid. Allopurinol, a structural analog of hypoxanthine, inhibits uric acid synthesis and is used in patients who are overproducers of uric acid. Allopurinol is oxidized to oxypurinol, a long-lived inhibitor of XO. This results in an accumulation of hypoxanthine and xanthine (see Fig.

1	and is used in patients who are overproducers of uric acid. Allopurinol is oxidized to oxypurinol, a long-lived inhibitor of XO. This results in an accumulation of hypoxanthine and xanthine (see Fig. 22.15), compounds more soluble than uric acid and, therefore, less likely to initiate an inflammatory response. In patients with normal levels of HGPRT, the hypoxanthine can be salvaged, reducing the levels of PRPP and, therefore, de novo purine synthesis. Febuxostat, a nonpurine inhibitor of XO, is also available. [Note: Uric acid levels in the blood normally are close to the saturation point. One reason for this may be the strong antioxidant effects of uric acid.] 2. Adenosine deaminase deficiency

1	ADA is expressed in a variety of tissues, but, in humans, lymphocytes have the highest activity of this cytoplasmic enzyme. A deficiency of ADA results in an accumulation of adenosine, which is converted to its ribonucleotide or deoxyribonucleotide forms by cellular kinases. As dATP levels rise, ribonucleotide reductase is inhibited, thereby preventing the production of all deoxyribose-containing nucleotides (see p. 297). Consequently, cells cannot make DNA and divide. [Note: The dATP and adenosine that accumulate in ADA deficiency lead to developmental arrest and apoptosis of lymphocytes.] In its most severe form, this autosomal-recessive disorder causes a type of severe combined immunodeficiency disease (SCID), involving a decrease in T cells, B cells, and natural killer cells. ADA deficiency accounts for ~14% of cases of SCID in the United States. Treatments include bone marrow transplantation, enzyme replacement therapy, and gene therapy (see p. 501). Without appropriate

1	ADA deficiency accounts for ~14% of cases of SCID in the United States. Treatments include bone marrow transplantation, enzyme replacement therapy, and gene therapy (see p. 501). Without appropriate treatment, children with this disorder usually die from infection by age 2 years. [Note: Purine nucleoside phosphorylase deficiency results in a less severe immunodeficiency primarily involving T cells.]

1	VI. PYRIMIDINE SYNTHESIS AND DEGRADATION Unlike the synthesis of the purine ring, which is constructed on a pre-existing ribose 5-phosphate, the pyrimidine ring is synthesized before being attached to ribose 5-phosphate, which is donated by PRPP. The sources of the atoms in the pyrimidine ring are glutamine, CO2, and aspartate (Fig. 22.19). A. Carbamoyl phosphate synthesis

1	A. Carbamoyl phosphate synthesis The regulated step of this pathway in mammalian cells is the synthesis of carbamoyl phosphate from glutamine and CO2, catalyzed by carbamoyl phosphate synthetase (CPS) II. CPS II is inhibited by uridine triphosphate (the end product of this pathway, which can be converted into the other pyrimidine nucleotides) and is activated by PRPP. [Note: Carbamoyl phosphate, synthesized by CPS I, is also a precursor of urea (see p. 253). Defects in ornithine transcarbamylase of the urea cycle promote pyrimidine synthesis because of increased availability of carbamoyl phosphate. A comparison of the two enzymes is presented in Figure 22.20.] B. Orotic acid synthesis

1	The second step in pyrimidine synthesis is the formation of carbamoylaspartate, catalyzed by aspartate transcarbamoylase. The pyrimidine ring is then closed by dihydroorotase. The resulting dihydroorotate is oxidized to produce orotic acid (orotate), as shown in Figure 22.21. The human enzyme that produces orotate, dihydroorotate dehydrogenase, is a flavin mononucleotide-containing protein of the inner mitochondrial membrane. All other enzymes in pyrimidine biosynthesis are cytosolic. [Note: The first three enzymic activities in this pathway (CPS II, aspartate transcarbamoylase, and dihydroorotase) are actually three different catalytic domains of a single polypeptide known as CAD from the first letter in the name of each domain. (See p. 18 for a discussion of domains.) This is an example of a multifunctional or multicatalytic polypeptide that facilitates the ordered synthesis of an important compound. Synthesis of the purine nucleotide IMP also involves multifunctional proteins.]

1	C. Pyrimidine nucleotide synthesis

1	The completed pyrimidine ring is converted to the nucleotide orotidine monophosphate (OMP) in the second stage of pyrimidine nucleotide synthesis (see Fig. 22.21). As seen with the purines, PRPP is the ribose 5phosphate donor. The enzyme orotate phosphoribosyltransferase produces OMP and releases pyrophosphate, thereby making the reaction biologically irreversible. [Note: Both purine and pyrimidine synthesis require glutamine, aspartic acid, and PRPP as essential precursors.] OMP (orotidylate) is decarboxylated to uridine monophosphate (UMP) by orotidylate decarboxylase. The phosphoribosyltransferase and decarboxylase activities are separate catalytic domains of a single polypeptide called UMP synthase. Hereditary orotic aciduria (a very rare disorder) may be caused by a deficiency of one or both activities of this bifunctional enzyme, resulting in orotic acid in the urine (see Fig. 22.21). UMP is sequentially phosphorylated to UDP and UTP. [Note: The UDP is a substrate for

1	of one or both activities of this bifunctional enzyme, resulting in orotic acid in the urine (see Fig. 22.21). UMP is sequentially phosphorylated to UDP and UTP. [Note: The UDP is a substrate for ribonucleotide reductase, which generates dUDP. The dUDP is phosphorylated to dUTP, which is rapidly hydrolyzed to dUMP by UTP diphosphatase (dUTPase). Thus, dUTPase plays an important role in reducing availability of dUTP as a substrate for DNA synthesis, thereby preventing erroneous incorporation of uracil into DNA.]

1	D. Cytidine triphosphate synthesis Cytidine triphosphate (CTP) is produced by amination of UTP by CTP synthetase (Fig. 22.22), with glutamine providing the nitrogen. Some of this CTP is dephosphorylated to CDP, which is a substrate for ribonucleotide reductase. The dCDP product can be phosphorylated to dCTP for DNA synthesis or dephosphorylated to dCMP that is deaminated to dUMP.

1	E. Deoxythymidine monophosphate synthesis dUMP is converted to deoxythymidine monophosphate (dTMP) by thymidylate synthase, which uses N5,N10-methylene-THF as the source of the methyl group (see p. 267). This is an unusual reaction in that THF contributes not only a one-carbon unit but also two hydrogen atoms from the pteridine ring, resulting in the oxidation of THF to dihydrofolate ([DHF], Fig. 22.23). Inhibitors of thymidylate synthase include thymine analogs such as 5-fluorouracil, which serve as antitumor agents. 5Fluorouracil is metabolically converted to 5-fluorodeoxyuridine monophosphate (5-FdUMP), which becomes permanently bound to the inactivated thymidylate synthase, making the drug a suicide inhibitor (see p. 60). DHF can be reduced to THF by dihydrofolate reductase (see Fig.

1	28.2, p. 378), an enzyme that is inhibited by folate analogs such as methotrexate. By decreasing the supply of THF, these drugs not only inhibit purine synthesis (see Fig. 22.7), but, by preventing methylation of dUMP to dTMP, they also decrease the availability of this essential component of DNA. DNA synthesis is inhibited and cell growth slowed. Thus, these drugs are used to treat cancer. [Note: Acyclovir (a purine analog) and AZT (3′-azido-3′-deoxythymidine, a pyrimidine analog) are used to treat infections of herpes simplex virus and human immunodeficiency virus, respectively. Each inhibits the viral DNA polymerase.] F. Pyrimidine salvage and degradation

1	F. Pyrimidine salvage and degradation Unlike the purine ring, which is not cleaved in humans and is excreted as poorly soluble uric acid, the pyrimidine ring is opened and degraded to highly soluble products, β-alanine (from the degradation of CMP and UMP) and β-aminoisobutyrate (from TMP degradation), with the production of ammonia and CO2. Pyrimidine bases can be salvaged to nucleosides, which are phosphorylated to nucleotides. However, their high solubility makes pyrimidine salvage less significant clinically than purine salvage. [Note: The salvage of pyrimidine nucleosides is the basis for using uridine in the treatment of hereditary orotic aciduria (see p. 302).] VII. CHAPTER SUMMARY

1	Nucleotides are composed of a nitrogenous base (adenine = A, guanine = G, cytosine = C, uracil = U, and thymine = T); a pentose sugar; and one, two, or three phosphate groups (Fig. 22.24). A and G are purines, and C, U, and T are pyrimidines. If the sugar is ribose, the nucleotide is a ribonucleoside phosphate (for example, adenosine monophosphate [AMP]), and it can have several functions in the cell, including being a component of RNA. If the sugar is deoxyribose, the nucleotide is a deoxyribonucleoside phosphate (for example, deoxyAMP) and will be found almost exclusively as a component of DNA. The committed step in purine synthesis uses 5-phosphoribosyl-1-pyrophosphate ([PRPP], an activated pentose that provides the ribose 5-phosphate for de novo purine and pyrimidine synthesis and salvage) and nitrogen from glutamine to produce phosphoribosylamine. The enzyme is glutamine:phosphoribosylpyrophosphate amidotransferase and is inhibited by AMP and guanosine monophosphate (the end

1	salvage) and nitrogen from glutamine to produce phosphoribosylamine. The enzyme is glutamine:phosphoribosylpyrophosphate amidotransferase and is inhibited by AMP and guanosine monophosphate (the end products of the pathway) and activated by PRPP. Purine nucleotides can also be produced from preformed purine bases by using salvage reactions catalyzed by adenine phosphoribosyltransferase (APRT) and hypoxanthine–guanine phosphoribosyltransferase (HGPRT). A near-total deficiency of HGPRT causes Lesch-Nyhan syndrome, a severe, inherited form of hyperuricemia accompanied by compulsive self-mutilation. All deoxyribonucleotides are synthesized from ribonucleotides by the enzyme ribonucleotide reductase. This enzyme is highly regulated (for example, it is strongly inhibited by deoxyadenosine triphosphate [dATP], a compound that is overproduced in bone marrow cells in individuals with adenosine deaminase [ADA] deficiency). ADA deficiency causes severe combined immunodeficiency disease. The end

1	[dATP], a compound that is overproduced in bone marrow cells in individuals with adenosine deaminase [ADA] deficiency). ADA deficiency causes severe combined immunodeficiency disease. The end product of purine degradation is uric acid, a compound of low solubility whose overproduction or undersecretion causes hyperuricemia that, if accompanied by the deposition of monosodium urate crystals in joints and soft tissues and an inflammatory response to those crystals, results in gout. The first step in pyrimidine synthesis, the production of carbamoyl phosphate by carbamoyl phosphate synthetase II, is the regulated step in this pathway (it is inhibited by uridine triphosphate [UTP] and activated by PRPP). The UTP produced by this pathway can be converted to cytidine triphosphate. Deoxyuridine monophosphate can be converted to deoxythymidine monophosphate by thymidylate synthase, an enzyme targeted by anticancer drugs such as 5-fluorouracil. The regeneration of tetrahydrofolate from

1	monophosphate can be converted to deoxythymidine monophosphate by thymidylate synthase, an enzyme targeted by anticancer drugs such as 5-fluorouracil. The regeneration of tetrahydrofolate from dihydrofolate produced in the thymidylate synthase reaction requires dihydrofolate reductase, an enzyme targeted by the drug methotrexate. Pyrimidine degradation results in soluble products.

1	guanosine, cytidine, thymidine, and inosine monophosphates; d = deoxy; PPi = pyrophosphate; PRPP = 5-phosphoribosyl-1-pyrophosphate. Choose the ONE best answer. 2.1. Azaserine, a drug with research applications, inhibits glutamine-dependent enzymes. Incorporation of which of the ring nitrogens (N) in the generic purine structure shown would most likely be affected by azaserine? A. 1 B. 3 C. 7 D. 9 Correct answer = D. The N at position 9 is supplied by glutamine in the first step of purine de novo synthesis, and its incorporation would be affected by azaserine. The N at position 1 is supplied by aspartate and at position 7 by glycine. The N at position 3 is also supplied by glutamine, but azaserine would have inhibited purine synthesis prior to this step.

1	2.2. A 42-year-old male patient undergoing radiation therapy for prostate cancer develops severe pain in the metatarsal phalangeal joint of his right big toe. Monosodium urate crystals are detected by polarized light microscopy in fluid obtained from this joint by arthrocentesis. This patient’s pain is directly caused by the overproduction of the end product of which of the following metabolic pathways? A. De novo pyrimidine biosynthesis B. Pyrimidine degradation C. De novo purine biosynthesis D. Purine salvage E. Purine degradation

1	A. De novo pyrimidine biosynthesis B. Pyrimidine degradation C. De novo purine biosynthesis D. Purine salvage E. Purine degradation Correct answer = E. The patient’s pain is caused by gout, resulting from an inflammatory response to the crystallization of excess urate (as monosodium urate) in his joints. Radiation therapy caused cell death, with degradation of nucleic acids and their constituent purines. Uric acid, the end product of purine degradation, is a relatively insoluble compound that can cause gout (and kidney stones). Pyrimidine metabolism is not associated with uric acid production. Overproduction of purines can indirectly result in hyperuricemia. Purine salvage decreases uric acid production. 2.3. Which one of the following enzymes of nucleotide metabolism is correctly paired with its pharmacologic inhibitor? A. Dihydrofolate reductase—methotrexate B. Inosine monophosphate dehydrogenase—hydroxyurea C. Ribonucleotide reductase—5-fluorouracil

1	A. Dihydrofolate reductase—methotrexate B. Inosine monophosphate dehydrogenase—hydroxyurea C. Ribonucleotide reductase—5-fluorouracil D. Thymidylate synthase—allopurinol E. Xanthine oxidase—probenecid Correct answer = A. Methotrexate interferes with folate metabolism by acting as a competitive inhibitor of the enzyme dihydrofolate reductase. This starves cells for tetrahydrofolate and makes them unable to synthesize purines and thymidine monophosphate. Inosine monophosphate dehydrogenase is inhibited by mycophenolic acid. Ribonucleotide reductase is inhibited by hydroxyurea. Thymidylate synthase is inhibited by 5-fluorouracil. Xanthine oxidase is inhibited by allopurinol. Probenecid increases renal excretion of urate but does not inhibit its production.

1	2.4. A 1-year-old female patient is lethargic, weak, and anemic. Her height and weight are low for her age. Her urine contains an elevated level of orotic acid. Activity of uridine monophosphate synthase is low. Administration of which of the following is most likely to alleviate her symptoms? A. Adenine B. Guanine C. Hypoxanthine D. Thymidine E. Uridine

1	A. Adenine B. Guanine C. Hypoxanthine D. Thymidine E. Uridine Correct answer = E. The elevated excretion of orotic acid and low activity of uridine monophosphate (UMP) synthase indicate that the patient has orotic aciduria, a very rare genetic disorder affecting de novo pyrimidine synthesis. Deficiencies in one or both catalytic domains of UMP synthase leave the patient unable to synthesize pyrimidines. Uridine, a pyrimidine nucleoside, is a useful treatment because it bypasses the missing activities and can be salvaged to UMP, which can be converted to all the other pyrimidines. Although thymidine is a pyrimidine nucleoside, it cannot be converted to other pyrimidines. Hypoxanthine, guanine, and adenine are all purine bases and cannot be converted to pyrimidines. 2.5. What laboratory test would help in distinguishing an orotic aciduria caused by ornithine transcarbamylase deficiency from that caused by uridine monophosphate synthase deficiency?

1	2.5. What laboratory test would help in distinguishing an orotic aciduria caused by ornithine transcarbamylase deficiency from that caused by uridine monophosphate synthase deficiency? Blood ammonia level would be expected to be elevated in ornithine transcarbamylase deficiency that affects the urea cycle but not in uridine monophosphate synthase deficiency. UNIT V Integration of Metabolism Metabolic Effects of Insulin and Glucagon 23 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Four major tissues play a dominant role in fuel metabolism: liver, adipose, muscle, and brain. These tissues contain unique sets of enzymes, such that each tissue is specialized for the storage, use, or generation of specific fuels. These tissues do not function in isolation but rather form part of a network in which one tissue may provide substrates to another or process compounds produced by other tissues. Communication between tissues is mediated by the nervous system, by the availability of circulating substrates, and by variation in the levels of plasma hormones (Fig. 23.1). The integration of energy metabolism is controlled primarily by the actions of two peptide hormones, insulin and glucagon (secreted in response to changing substrate levels in the blood), with the catecholamines epinephrine and norepinephrine (secreted in response to neural signals) playing a supporting role. Changes in the circulating levels of these hormones allow the body to store energy when food is

1	epinephrine and norepinephrine (secreted in response to neural signals) playing a supporting role. Changes in the circulating levels of these hormones allow the body to store energy when food is abundant or to make stored energy available such as during survival crises (for example, famine, severe injury, and “fight-or-flight” situations). This chapter describes the structure, secretion, and metabolic effects of the two hormones that most profoundly affect energy metabolism.

1	II. INSULIN Insulin is a peptide hormone produced by the β cells of the islets of Langerhans, which are clusters of cells embedded in the endocrine portion of the pancreas (Fig. 23.2). [Note: “Insulin” is from the Latin for island.] The islets make up only about 1%–2% of the total cells of the pancreas. Insulin is the most important hormone coordinating the use of fuels by tissues. Its metabolic effects are anabolic, favoring, for example, synthesis of glycogen, triacylglycerol (TAG), and protein. A. Structure Insulin is composed of 51 amino acids arranged in two polypeptide chains, designated A (21 amino acids) and B, which are linked together by two disulfide bonds (Fig. 23.3A). The insulin molecule also contains an intramolecular disulfide bond between amino acid residues of the A chain. [Note: Insulin was the first peptide for which the primary structure was determined and the first therapeutic molecule made by recombinant DNA technology (see p. 486).] B. Synthesis

1	The processing and transport of intermediates that occur during the synthesis of insulin are shown in Figures 23.3B and 23.4. Biosynthesis involves production of two inactive precursors, preproinsulin and proinsulin, which are sequentially cleaved to form the active hormone plus the connecting or C-peptide in a 1:1 ratio (see Fig. 23.4). [Note: The C-peptide is essential for proper insulin folding. Also, because its half-life in plasma is longer than that of insulin, the C-peptide level is a good indicator of insulin production and secretion.] Insulin is stored in cytosolic granules that, given the proper stimulus (see C.1. below), are released by exocytosis. (See p. 459 for a discussion of the synthesis of secreted proteins.) Insulin is degraded by insulin-degrading enzyme, which is present in the liver and, to a lesser extent, in the kidneys. Insulin has a plasma half-life of ~6 minutes. This short duration of action permits rapid changes in circulating levels of the hormone.

1	C. Secretion regulation Secretion of insulin is regulated by bloodborne fuels and hormones. 1. Increased secretion: Insulin secretion by the pancreatic β cells is closely coordinated with the secretion of glucagon by pancreatic α cells (Fig. 23.5). The relative amounts of glucagon and insulin released are normally regulated such that the rate of hepatic glucose production is kept equal to the use of glucose by peripheral tissues. This maintains blood glucose between 70 and 140 mg/dl. In view of its coordinating role, it is not surprising that the β cell responds to a variety of stimuli. In particular, insulin secretion is increased by glucose, amino acids, and gastrointestinal peptide hormones. a.

1	a. Glucose: Ingestion of a carbohydrate-rich meal leads to a rise in blood glucose, the primary stimulus for insulin secretion (see Fig. 23.5). The β cells are the most important glucose-sensing cells in the body. Like the liver, β cells contain GLUT-2 transporters and express glucokinase (hexokinase IV; see p. 98). At blood glucose levels >45 mg/dl, glucokinase phosphorylates glucose in amounts proportional to the glucose concentration. Proportionality results from the lack of direct inhibition of glucokinase by glucose 6-phosphate, its product. Additionally, the sigmoidal relationship between the velocity of the reaction and substrate concentration (see p. 98) maximizes the enzyme’s responsiveness to changes in blood glucose level. Metabolism of glucose 6-phosphate generates ATP, leading to insulin secretion (see blue box below). b.

1	b. Amino acids: Ingestion of protein causes a transient rise in plasma amino acid levels (for example, arginine) that enhances the glucose-stimulated secretion of insulin. [Note: Fatty acids have a similar effect.] c. Gastrointestinal peptide hormones: The intestinal peptides glucagonlike peptide-1 (GLP-1) and gastric inhibitory polypeptide ([GIP] also called glucose-dependent insulinotropic peptide) increase the sensitivity of β cells to glucose. They are released from the small intestine after the ingestion of food, causing an anticipatory rise in insulin levels and, thus, are referred to as incretins. Their action may account for the fact that the same amount of glucose given orally induces a much greater secretion of insulin than if given intravenously (IV).

1	Glucose-dependent release of insulin into blood is mediated through a rise in calcium (Ca2+) concentration in the β cell. Glucose taken into β cells by GLUT-2 is phosphorylated and metabolized, with subsequent production of ATP. ATP-sensitive potassium (K+) channels close, causing depolarization of the plasma membrane, opening of voltage-gated Ca2+ channels, and influx of Ca2+ into the cell. Ca2+ causes vesicles containing insulin to be exocytosed from the β cell. Sulfonylureas, oral agents used to treat type 2 diabetes, increase insulin secretion by closing ATP-sensitive K+ channels.

1	2. Decreased secretion: The synthesis and release of insulin are decreased when there is a scarcity of dietary fuels and also during periods of physiologic stress (for example, infection, hypoxia, and vigorous exercise), thereby preventing hypoglycemia. These effects are mediated primarily by the catecholamines norepinephrine and epinephrine, which are made from tyrosine in the sympathetic nervous system (SNS) and the adrenal medulla and then secreted. Secretion is largely controlled by neural signals. The catecholamines (primarily epinephrine) have a direct effect on energy metabolism, causing a rapid mobilization of energy-yielding fuels, including glucose from the liver (produced by glycogenolysis or gluconeogenesis; see p. 121) and fatty acids (FA) from adipose tissue (produced by lipolysis; see p. 189). In addition, these biogenic amines can override the normal glucose-stimulated release of insulin. Thus, in emergency situations, the SNS largely replaces the plasma glucose

1	by lipolysis; see p. 189). In addition, these biogenic amines can override the normal glucose-stimulated release of insulin. Thus, in emergency situations, the SNS largely replaces the plasma glucose concentration as the controlling influence over β-cell secretion. The regulation of insulin secretion is summarized in Figure 23.6.

1	D. Metabolic effects Insulin promotes the storage of nutrients as glycogen, TAG, and protein and inhibits their mobilization. 1. Effects on carbohydrate metabolism: The effects of insulin on glucose metabolism promote its storage and are most prominent in three tissues: liver, muscle, and adipose. In liver and muscle, insulin increases glycogen synthesis. In muscle and adipose, insulin increases glucose uptake by increasing the number of glucose transporters (GLUT-4; see p.

1	97) in the cell membrane. Thus, the IV administration of insulin causes an immediate decrease in blood glucose level. In the liver, insulin decreases the production of glucose through the inhibition of glycogenolysis and gluconeogenesis. [Note: The effects of insulin are due not just to changes in enzyme activity but also in enzyme amount insofar as insulin alters gene transcription.] 2. Effects on lipid metabolism: A rise in insulin rapidly causes a significant reduction in the release of FA from adipose tissue by inhibiting the activity of hormone-sensitive lipase, a key enzyme of TAG degradation in adipocytes. Insulin acts by promoting the dephosphorylation and, hence, inactivation of the enzyme (see p. 190). Insulin also increases the transport and metabolism of glucose into adipocytes, providing the glycerol 3-phosphate substrate for TAG synthesis (see p. 188). Expression of the gene for lipoprotein lipase, which degrades TAG in circulating chylomicrons and very-low-density

1	providing the glycerol 3-phosphate substrate for TAG synthesis (see p. 188). Expression of the gene for lipoprotein lipase, which degrades TAG in circulating chylomicrons and very-low-density lipoproteins ([VLDL] see p. 229), is increased by insulin in adipose, thereby providing FA for esterification to the glycerol. [Note: Insulin also promotes the conversion of glucose to TAG in the liver. The TAG are secreted in VLDL.] 3. Effects on protein synthesis: In most tissues, insulin stimulates both the entry of amino acids into cells and protein synthesis (translation). [Note: Insulin stimulates protein synthesis through covalent activation of factors required for translation initiation.]

1	E. Mechanism Insulin binds to specific, high-affinity receptors in the cell membrane of most tissues, including liver, muscle, and adipose. This is the first step in a cascade of reactions ultimately leading to a diverse array of biologic actions (Fig. 23.7). 1. Insulin receptor: The insulin receptor is synthesized as a single polypeptide that is glycosylated and cleaved into α and β subunits, which are then assembled into a tetramer linked by disulfide bonds (see Fig. 23.7). The extracellular α subunits contain the insulin-binding site. A hydrophobic domain in each β subunit spans the plasma membrane. The cytosolic domain of the β subunit is a tyrosine kinase, which is activated by insulin. As a result, the insulin receptor is classified as a tyrosine kinase receptor. 2.

1	2. Signal transduction: The binding of insulin to the α subunits of the insulin receptor induces conformational changes that are transmitted to the β subunits. This promotes a rapid autophosphorylation of specific tyrosine residues on each β subunit (see Fig. 23.7). Autophosphorylation initiates a cascade of cell-signaling responses, including phosphorylation of a family of proteins called insulin receptor substrates (IRS). At least four IRS have been identified that show similar structures but different tissue distributions. Phosphorylated IRS proteins interact with other signaling molecules through specific domains (known as SH2), activating a number of pathways that affect gene expression, cell metabolism, and growth. The actions of insulin are terminated by dephosphorylation of the receptor. 3.

1	3. Membrane effects: Glucose transport in some tissues, such as muscle and adipose, increases in the presence of insulin (Fig. 23.8). Insulin promotes movement of insulin-sensitive glucose transporters (GLUT-4) from a pool located in intracellular vesicles to the cell membrane. [Note: Movement is the result of a signaling cascade in which an IRS binds to and activates a kinase (phosphoinositide 3-kinase), leading to phosphorylation of the membrane phospholipid phosphatidylinositol 4,5bisphosphate (PIP2) to the 3,4,5-trisphosphate form (PIP3) that binds to and activates phosphoinositide-dependent kinase 1. This kinase, in turn, activates Akt (or protein kinase B), resulting in GLUT-4 movement.] In contrast, other tissues have insulin-insensitive systems for glucose transport (Fig. 23.9). For example, hepatocytes, erythrocytes, and cells of the nervous system, intestinal mucosa, renal tubules, and cornea do not require insulin for glucose uptake. 4.

1	4. Receptor regulation: Binding of insulin is followed by internalization of the hormone–receptor complex. Once inside the cell, insulin is degraded in the lysosomes. The receptors may be degraded, but most are recycled to the cell surface. [Note: Elevated levels of insulin promote the degradation of receptors, thereby decreasing the number of surface receptors. This is one type of downregulation.] 5.

1	Time course: The binding of insulin provokes a wide range of actions. The most immediate response is an increase in glucose transport into adipocytes and skeletal and cardiac muscle cells that occurs within seconds of insulin binding to its membrane receptor. Insulin-induced changes in enzymic activity in many cell types occur over minutes to hours and reflect changes in the phosphorylation states of existing proteins. Insulin-induced increase in the amount of many enzymes, such as glucokinase, liver pyruvate kinase, acetyl coenzyme A (CoA) carboxylase (ACC), and fatty acid synthase, requires hours to days. These changes reflect an increase in gene expression through increased transcription (mediated by sterol regulatory element–binding protein-1c; see p. 184) and translation. III. GLUCAGON

1	Glucagon is a peptide hormone secreted by the α cells of the pancreatic islets of Langerhans. Glucagon, along with epinephrine, norepinephrine, cortisol, and growth hormone (the counterregulatory hormones), opposes many of the actions of insulin (Fig. 23.10). Most importantly, glucagon acts to maintain blood glucose levels by activation of hepatic glycogenolysis and gluconeogenesis. Glucagon is composed of 29 amino acids arranged in a single polypeptide chain. [Note: Unlike insulin, the amino acid sequence of glucagon is the same in all mammalian species examined to date.] Glucagon is synthesized as a large precursor molecule (preproglucagon) that is converted to glucagon through a series of selective proteolytic cleavages, similar to those described for insulin biosynthesis (see Fig. 23.3). In contrast to insulin, preproglucagon is processed to different products in different tissues, for example, GLP-1 in intestinal L cells. Like insulin, glucagon has a short half-life.

1	A. Increased secretion The α cell is responsive to a variety of stimuli that signal actual or potential hypoglycemia (Fig. 23.11). Specifically, glucagon secretion is increased by low blood glucose, amino acids, and catecholamines. 1. Low blood glucose: A decrease in plasma glucose concentration is the primary stimulus for glucagon release. During an overnight or prolonged fast, elevated glucagon levels prevent hypoglycemia (see Section IV below for a discussion of hypoglycemia). 2. Amino acids: Amino acids (for example, arginine) derived from a meal containing protein stimulate the release of glucagon. The glucagon effectively prevents the hypoglycemia that would otherwise occur as a result of the increased insulin secretion that also occurs after a protein meal. 3.

1	3. Catecholamines: Elevated levels of circulating epinephrine (from the adrenal medulla), norepinephrine (from sympathetic innervation of the pancreas), or both stimulate the release of glucagon. Thus, during periods of physiologic stress, the elevated catecholamine levels can override the effect on the α cell of circulating substrates. In these situations, regardless of the concentration of blood glucose, glucagon levels are elevated in anticipation of increased glucose use. In contrast, insulin levels are depressed. B. Decreased secretion Glucagon secretion is significantly decreased by elevated blood glucose and by insulin. Both substances are increased following ingestion of glucose or a carbohydrate-rich meal (see Fig. 23.5). The regulation of glucagon secretion is summarized in Figure 23.11. C. Metabolic effects Glucagon is a catabolic hormone that promotes the maintenance of blood glucose levels. Its primary target is the liver. 1.

1	C. Metabolic effects Glucagon is a catabolic hormone that promotes the maintenance of blood glucose levels. Its primary target is the liver. 1. Effects on carbohydrate metabolism: The IV administration of glucagon leads to an immediate rise in blood glucose. This results from an increase in the degradation of liver glycogen and an increase in hepatic gluconeogenesis. 2.

1	2. Effects on lipid metabolism: The primary effect of glucagon on lipid metabolism is inhibition of FA synthesis through phosphorylation and subsequent inactivation of ACC by adenosine monophosphate (AMP)– activated protein kinase (see p. 184). The resulting decrease in malonyl CoA production removes the inhibition on long-chain FA β-oxidation (see p. 191). Glucagon also plays a role in lipolysis in adipocytes, but the major activators of hormone-sensitive lipase (via phosphorylation by protein kinase A) are the catecholamines. The free FA released are taken up by liver and oxidized to acetyl CoA, which is used in ketone body synthesis. 3. Effects on protein metabolism: Glucagon increases uptake by the liver of amino acids supplied by muscle, resulting in increased availability of carbon skeletons for gluconeogenesis. As a consequence, plasma levels of amino acids are decreased. D. Mechanism

1	D. Mechanism Glucagon binds to high-affinity G protein–coupled receptors (GPCR) on the cell membrane of hepatocytes. The GPCR for glucagon is distinct from the GPCR that bind epinephrine. [Note: Glucagon receptors are not found on skeletal muscle.] Glucagon binding results in activation of adenylyl cyclase in the plasma membrane (Fig. 23.12; also see p. 94). This causes a rise in cyclic AMP (cAMP), which, in turn, activates cAMP-dependent protein kinase A and increases the phosphorylation of specific enzymes or other proteins. This cascade of increasing enzymic activities results in the phosphorylation-mediated activation or inhibition of key regulatory enzymes involved in carbohydrate and lipid metabolism. An example of such a cascade in glycogen degradation is shown in Figure 11.9 on p. 131. [Note: Glucagon, like insulin, affects gene transcription. For example, glucagon induces expression of phosphoenolpyruvate carboxykinase (see p. 122).] IV. HYPOGLYCEMIA

1	IV. HYPOGLYCEMIA Hypoglycemia is characterized by 1) central nervous system (CNS) symptoms, including confusion, aberrant behavior, or coma; 2) a simultaneous blood glucose level ≤50 mg/dl; and 3) symptoms being resolved within minutes following glucose administration (Fig. 23.13). Hypoglycemia is a medical emergency because the CNS has an absolute requirement for a continuous supply of bloodborne glucose to serve as a metabolic fuel. Transient hypoglycemia can cause cerebral dysfunction, whereas severe, prolonged hypoglycemia causes brain damage. Therefore, it is not surprising that the body has multiple overlapping mechanisms to prevent or correct hypoglycemia. The most important hormone changes in combating hypoglycemia are increased secretion of glucagon and the catecholamines, combined with decreased insulin secretion. A. Symptoms

1	A. Symptoms The symptoms of hypoglycemia can be divided into two categories. Adrenergic (neurogenic, autonomic) symptoms, such as anxiety, palpitation, tremor, and sweating, are mediated by catecholamine release (primarily epinephrine) regulated by the hypothalamus in response to hypoglycemia. Adrenergic symptoms typically occur when blood glucose levels fall abruptly. The second category of hypoglycemic symptoms is neuroglycopenic. The impaired delivery of glucose to the brain (neuroglycopenia) results in impairment of brain function, causing headache, confusion, slurred speech, seizures, coma, and death. Neuroglycopenic symptoms often result from a gradual decline in blood glucose, often to levels <50 mg/dl. The slow decline in glucose deprives the CNS of fuel but fails to trigger an adequate adrenergic response. B. Glucoregulatory systems

1	B. Glucoregulatory systems Humans have two overlapping glucose-regulating systems that are activated by hypoglycemia: 1) the pancreatic α cells, which release glucagon, and 2) receptors in the hypothalamus, which respond to abnormally low concentrations of blood glucose. The hypothalamic glucoreceptors can trigger both the secretion of catecholamines (mediated by the sympathetic division of the autonomic nervous system) and release of adrenocorticotropic hormone (ACTH) and growth hormone by the anterior pituitary (see Fig. 23.13). [Note: ACTH increases cortisol synthesis and secretion in the adrenal cortex (see p. 239).] Glucagon, the catecholamines, cortisol, and growth hormone are sometimes called the counterregulatory hormones because each opposes the action of insulin on glucose use. 1.

1	1. Glucagon and epinephrine: Secretion of these counterregulatory hormones is most important in the acute, short-term regulation of blood glucose levels. Glucagon stimulates hepatic glycogenolysis and gluconeogenesis. Epinephrine promotes glycogenolysis and lipolysis. It inhibits insulin secretion, thereby preventing GLUT-4–mediated uptake of glucose by muscle and adipose tissues. Epinephrine assumes a critical role in hypoglycemia when glucagon secretion is deficient, for example, in the late stages of type 1 diabetes mellitus (see p. 340). The prevention or correction of hypoglycemia fails when the secretion of both glucagon and epinephrine is deficient. 2. Cortisol and growth hormone: These counterregulatory hormones are less important in the short-term maintenance of blood glucose concentrations. They do, however, play a role in the long-term (transcriptional) management of glucose metabolism. C. Types

1	C. Types Hypoglycemia may be divided into four types: 1) insulin induced, 2) postprandial (sometimes called reactive hypoglycemia), 3) fasting hypoglycemia, and 4) alcohol related. 1. Insulin-induced hypoglycemia: Hypoglycemia occurs frequently in patients with diabetes who are receiving insulin treatment, particularly those striving to achieve tight control of blood glucose levels. Mild hypoglycemia in fully conscious patients is treated by oral administration of carbohydrate. Unconscious patients are typically given glucagon subcutaneously or intramuscularly (Fig. 23.14). 2. Postprandial hypoglycemia: This is the second most common form of hypoglycemia. It is caused by an exaggerated insulin release following a meal, prompting transient hypoglycemia with mild adrenergic symptoms. The plasma glucose level returns to normal even if the patient is not fed. The only treatment usually required is that the patient eats frequent small meals rather than the usual three large meals. 3.

1	3. Fasting hypoglycemia: Low blood glucose during fasting is rare but is more likely to present as a serious medical problem. Fasting hypoglycemia, which tends to produce neuroglycopenic symptoms, may result from a reduction in the rate of glucose production by hepatic glycogenolysis or gluconeogenesis. Thus, low blood glucose levels are often seen in patients with hepatocellular damage or adrenal insufficiency or in fasting individuals who have consumed large quantities of ethanol (see 4. below). Alternately, fasting hypoglycemia may be the result of an increased rate of glucose use by the peripheral tissues because of overproduction of insulin by rare pancreatic tumors. If left untreated, a patient with fasting hypoglycemia may lose consciousness and experience convulsions and coma. [Note: Certain inborn errors of metabolism, for example, defects in FA oxidation, result in fasting hypoglycemia.] 4.

1	Alcohol-related hypoglycemia: Alcohol (ethanol) is metabolized in the liver by two oxidation reactions (Fig. 23.15). Ethanol is first converted to acetaldehyde by zinc-containing alcohol dehydrogenase. Acetaldehyde is subsequently oxidized to acetate by aldehyde dehydrogenase (ALDH). [Note: ALDH is inhibited by disulfiram, a drug that is used in the treatment of chronic alcoholism. The resulting rise in acetaldehyde results in flushing, tachycardia, hyperventilation, and nausea.] In each reaction, electrons are transferred to oxidized nicotinamide adenine dinucleotide (NAD+), resulting in an increase in the ratio of the reduced form (NADH) to NAD+. The abundance of NADH favors the reduction of pyruvate to lactate and of oxaloacetate (OAA) to malate. Recall from p. 118 that pyruvate and OAA are substrates in the synthesis of glucose. Thus, the ethanol-mediated increase in NADH causes these gluconeogenic precursors to be diverted into alternate pathways, resulting in the decreased

1	OAA are substrates in the synthesis of glucose. Thus, the ethanol-mediated increase in NADH causes these gluconeogenic precursors to be diverted into alternate pathways, resulting in the decreased synthesis of glucose. This can precipitate hypoglycemia, particularly in individuals who have depleted their stores of liver glycogen. [Note: Decreased availability of OAA allows acetyl CoA to be diverted to ketone body synthesis in the liver (see p. 195) and can result in alcoholic ketosis that may result in ketoacidosis.] Hypoglycemia can produce many of the behaviors associated with alcohol intoxication, such as agitation, impaired judgment, and combativeness. Therefore, alcohol consumption in vulnerable individuals (such as those who are fasted or have engaged in prolonged, strenuous exercise) can produce hypoglycemia that may contribute to the behavioral effects of alcohol. Because alcohol consumption can also increase the risk for hypoglycemia in patients using insulin, those in an

1	can produce hypoglycemia that may contribute to the behavioral effects of alcohol. Because alcohol consumption can also increase the risk for hypoglycemia in patients using insulin, those in an intensive insulin treatment protocol (see p. 340) are counseled about the increased risk of hypoglycemia that generally occurs many hours after alcohol ingestion.

1	B. Inhibition of gluconeogenesis resulting from hepatic metabolism of ethanol. NAD(H) = nicotinamide adenine dinucleotide. Chronic alcohol consumption can also result in alcoholic fatty liver because of increased hepatic synthesis of TAG coupled with impaired formation or release of VLDL. This occurs as a result of decreased FA oxidation because of a fall in the NAD+/NADH ratio and increased lipogenesis because of the increased availability of FA (decreased catabolism) and of glyceraldehyde 3-phosphate (the dehydrogenase is inhibited by the low NAD+/NADH ratio; see p. 101). With continued alcohol consumption, alcoholic fatty liver can progress first to alcoholic hepatitis and then to alcoholic cirrhosis. V. CHAPTER SUMMARY

1	The integration of energy metabolism is controlled primarily by insulin and the opposing actions of glucagon and the catecholamines, particularly epinephrine (Fig. 23.16). Changes in the circulating levels of these hormones allow the body to store energy when food is abundant or to make stored energy available in times of physiologic stress (for example, during survival crises, such as famine). Insulin is a peptide hormone produced by the β cells of the islets of Langerhans of the pancreas. It consists of disulfide-linked A and B chains. A rise in blood glucose is the most important signal for insulin secretion. The catecholamines, secreted in response to stress, trauma, or extreme exercise, inhibit insulin secretion. Insulin increases glucose uptake (by glucose transporters (GLUT-4) in muscle and adipose tissue) and the synthesis of glycogen, protein, and triacylglycerol: It is an anabolic hormone. These actions are mediated by binding to its membrane tyrosine kinase receptor.

1	in muscle and adipose tissue) and the synthesis of glycogen, protein, and triacylglycerol: It is an anabolic hormone. These actions are mediated by binding to its membrane tyrosine kinase receptor. Binding initiates a cascade of cell-signaling responses, including phosphorylation of a family of proteins called insulin receptor substrate proteins. Glucagon is a monomeric peptide hormone produced by the α cells of the pancreatic islets (both insulin and glucagon synthesis involve formation of inactive precursors that are cleaved to form the active hormones). Glucagon, along with epinephrine, norepinephrine, cortisol, and growth hormone (the counterregulatory hormones), opposes many of the actions of insulin. Glucagon acts to maintain blood glucose during periods of potential hypoglycemia. Glucagon increases glycogenolysis, gluconeogenesis, fatty acid oxidation, ketogenesis, and amino acid uptake: It is a catabolic hormone. Glucagon secretion is stimulated by low blood glucose, amino

1	Glucagon increases glycogenolysis, gluconeogenesis, fatty acid oxidation, ketogenesis, and amino acid uptake: It is a catabolic hormone. Glucagon secretion is stimulated by low blood glucose, amino acids, and the catecholamines. Its secretion is inhibited by elevated blood glucose and by insulin. Glucagon binds to high-affinity G protein–coupled receptors on the cell membrane of hepatocytes. Binding results in the activation of adenylyl cyclase, which produces the second messenger cyclic adenosine monophosphate (cAMP). Subsequent activation of cAMPdependent protein kinase A results in the phosphorylation-mediated activation or inhibition of key regulatory enzymes involved in carbohydrate and lipid metabolism. Both insulin and glucagon affect gene transcription.

1	Hypoglycemia is characterized by low blood glucose accompanied by adrenergic and neuroglycopenic symptoms that are rapidly resolved by the administration of glucose. Insulin-induced, postprandial, and fasting hypoglycemia result in release of glucagon and epinephrine. The rise in nicotinamide adenine dinucleotide (NADH) that accompanies ethanol metabolism inhibits gluconeogenesis, leading to hypoglycemia in individuals with depleted stores. Alcohol consumption also increases the risk for hypoglycemia in patients using insulin. Chronic alcohol consumption can cause fatty liver disease. Choose the ONE best answer. 3.1. Which of the following statements is true for insulin but not for glucagon? A. It is a peptide hormone secreted by pancreatic cells. B. Its actions are mediated by binding to a receptor found on the cell membrane of liver cells. C. Its effects include alterations in gene expression. D. Its secretion is decreased by the catecholamines.

1	C. Its effects include alterations in gene expression. D. Its secretion is decreased by the catecholamines. E. Its secretion is increased by amino acids. F. Its synthesis involves a nonfunctional precursor that gets cleaved to yield a functional molecule. Correct answer = D. Secretion of insulin by pancreatic β cells is inhibited by the catecholamines, whereas glucagon secretion by the α cells is stimulated by them. All of the other statements are true for both insulin and glucagon. 3.2. In which one of the following tissues is glucose transport into the cell insulin dependent? A. Adipose B. Brain C. Liver D. Red blood cells Correct answer = A. The glucose transporter (GLUT-4) in adipose (and muscle) tissue is dependent on insulin. Insulin results in movement of GLUT-4 from intracellular vesicles to the cell membrane. The other tissues in the list contain GLUT that are independent of insulin because they are always located on the cell membrane.

1	3.3. A 39-year-old woman is brought to the emergency room complaining of weakness and dizziness. She recalls getting up early that morning to do her weekly errands and had skipped breakfast. She drank a cup of coffee for lunch and had nothing to eat during the day. She met with friends at 8 p.m. and had a few drinks. As the evening progressed, she soon became weak and dizzy and was taken to the hospital. Laboratory tests revealed her blood glucose to be 45 mg/dl (normal = 70–99). She was given orange juice and immediately felt better. The biochemical basis of her alcohol-induced hypoglycemia is an increase in: A. fatty acid oxidation. B. the ratio of the reduced oxidized forms of nicotinamide adenine dinucleotide. C. oxaloacetate and pyruvate. D. use of acetyl coenzyme A in fatty acid synthesis.

1	A. fatty acid oxidation. B. the ratio of the reduced oxidized forms of nicotinamide adenine dinucleotide. C. oxaloacetate and pyruvate. D. use of acetyl coenzyme A in fatty acid synthesis. Correct answer = B. The oxidation of ethanol to acetate by dehydrogenases is accompanied by the reduction of nicotinamide adenine dinucleotide (NAD+) to NADH. The rise in the NADH/NAD+ ratio shifts pyruvate to lactate and oxaloacetate (OAA) to malate, decreasing the availability of substrates for gluconeogenesis and resulting in hypoglycemia. The rise in NADH also reduces the NAD+ needed for fatty acid (FA) oxidation. The decrease in OAA shunts any acetyl coenzyme A produced to ketogenesis. Note that the inhibition of FA degradation results in their reesterification into triacylglycerol that can result in fatty liver.

1	3.4. A patient is diagnosed with an insulinoma, a rare neuroendocrine tumor, the cells of which are derived primarily from pancreatic β cells. Which of the following would logically be characteristic of an insulinoma? A. Decreased body weight B. Decreased connecting peptide in the blood C. Decreased glucose in the blood D. Decreased insulin in the blood Correct answer = C. Insulinomas are characterized by constant production of insulin (and, therefore, of C-peptide) by the tumor cells. The increase in insulin drives glucose uptake by tissues such as muscle and adipose that have insulin-dependent glucose transporters, resulting in hypoglycemia. However, the hypoglycemia is insufficient to suppress insulin production and secretion. Insulinomas, then, are characterized by increased blood insulin and decreased blood glucose. Insulin, as an anabolic hormone, results in weight gain.

1	3.5. In a patient with an even rarer glucagon-secreting tumor derived from the α cells of the pancreas, how would the presentation be expected to differ relative to the patient in Question 23.4? A glucagon-secreting tumor of the pancreas (glucagonoma) would result in hyperglycemia, not hypoglycemia. The constant production of glucagon would result in constant gluconeogenesis, using amino acids from proteolysis as substrates. This results in loss of body weight. The Feed–Fast Cycle 24

1	The absorptive (well-fed) state is the 2-to 4-hour period after ingestion of a normal meal. During this interval, transient increases in plasma glucose, amino acids, and triacylglycerols (TAG) occur, the latter primarily as components of chylomicrons synthesized and secreted by the intestinal mucosal cells (see p. 228). Islet tissue of the pancreas responds to the elevated level of glucose with increased secretion of insulin and decreased secretion of glucagon. The elevated insulin/glucagon ratio and the ready availability of circulating substrates make the absorptive state an anabolic period characterized by increased synthesis of TAG and glycogen to replenish fuel stores as well as increased synthesis of protein. During this absorptive period, virtually all tissues use glucose as a fuel, and the metabolic response of the body is dominated by alterations in the metabolism of liver, adipose tissue, skeletal muscle, and brain. In this chapter, an “organ map” is introduced that traces

1	and the metabolic response of the body is dominated by alterations in the metabolism of liver, adipose tissue, skeletal muscle, and brain. In this chapter, an “organ map” is introduced that traces the movement of metabolites between tissues. The goal is to create an expanded and clinically useful vision of whole-body metabolism.

1	II. REGULATORY MECHANISMS The flow of intermediates through metabolic pathways is controlled by four mechanisms: 1) the availability of substrates, 2) allosteric regulation of enzymes, 3) covalent modification of enzymes, and 4) induction-repression of enzyme synthesis, primarily through regulation of transcription. Although this scheme may at first seem redundant, each mechanism operates on a different timescale (Fig. 24.1) and allows the body to adapt to a wide variety of physiologic situations. In the absorptive state, these regulatory mechanisms insure that available nutrients are captured as glycogen, TAG, and protein. A. Allosteric effectors

1	A. Allosteric effectors Allosteric changes usually involve rate-determining reactions. For example, glycolysis in the liver is stimulated following a meal by an increase in fructose 2,6-bisphosphate, an allosteric activator of phosphofructokinase-1 ([PFK-1] see p. 99). In contrast, gluconeogenesis is decreased by fructose 2,6-bisphosphate, an allosteric inhibitor of fructose 1,6-bisphosphatase (see p. 122). B. Covalent modification

1	B. Covalent modification The activity of many enzymes is regulated by the addition (via kinases, such as cyclic adenosine monophosphate [cAMP ]–dependent protein kinase A [PKA] and adenosine monophosphate–activated protein kinase [AMPK]) or removal (via phosphatases) of phosphate groups from specific serine, threonine, or tyrosine residues of the protein. In the absorptive state, most of the covalently regulated enzymes are in the dephosphorylated form and are active (Fig. 24.2). Three exceptions are glycogen phosphorylase kinase (see p. 132), glycogen phosphorylase (see p. 132), and hormone-sensitive lipase (HSL) (see p. 189), which are inactive in their dephosphorylated form. [Note: In the liver, the phosphatase domain of bifunctional phosphofructokinase-2 (PFK-2) is inactive when the protein is dephosphorylated (see p. 100).] C. Induction and repression of enzyme synthesis

1	C. Induction and repression of enzyme synthesis Increased (induction of) or decreased (repression of) enzyme synthesis leads to changes in the number of enzyme molecules, rather than changing the activity of existing enzyme molecules. Enzymes subject to synthesis regulation are often those that are needed under specific physiologic conditions. For example, in the well-fed state, elevated insulin levels result in an increase in the synthesis of key enzymes, such as acetyl coenzyme A (CoA) carboxylase (ACC) and fatty acid synthase (see p. 313), involved in anabolic metabolism. In the fasted state, glucagon induces expression of phosphoenolpyruvate carboxykinase (PEPCK) of gluconeogenesis (see p. 314). [Note: Both hormones affect gene transcription.] III. LIVER: NUTRIENT DISTRIBUTION CENTER

1	III. LIVER: NUTRIENT DISTRIBUTION CENTER The liver is uniquely situated to process and distribute dietary nutrients because the venous drainage of the gut and pancreas passes through the hepatic portal vein before entry into the general circulation. Thus, after a meal, the liver is bathed in blood containing absorbed nutrients and elevated levels of insulin secreted by the pancreas. During the absorptive period, the liver takes up carbohydrates, lipids, and most amino acids. These nutrients are then metabolized, stored, or routed to other tissues. In this way, the liver smooths out potentially broad fluctuations in the availability of nutrients for the peripheral tissues. A. Carbohydrate metabolism

1	A. Carbohydrate metabolism The liver is normally a glucose-producing rather than a glucose-using organ. However, after a meal containing carbohydrate, the liver becomes a net consumer, retaining roughly 60 g of every 100 g of glucose presented by the portal system. This increased use reflects increased glucose uptake by the hepatocytes. Their insulin-independent glucose transporter (GLUT 2) has a low affinity (high Km [Michaelis constant]) for glucose and, therefore, takes up glucose only when blood glucose is high (see p. 98). Processes that are upregulated when hepatic glucose is increased include the following. 1. Increased glucose phosphorylation: The elevated levels of glucose within the hepatocyte (as a result of elevated extracellular levels) allow glucokinase to phosphorylate glucose to glucose 6-phosphate (Fig. 24.3, ). [Note: Glucokinase has a high Km for glucose, is not subject to direct product inhibition, and has a sigmoidal reaction curve (see p. 98).] 2.

1	Increased glycogenesis: The conversion of glucose 6-phosphate to glycogen is favored by the activation of glycogen synthase, both by dephosphorylation and by increased availability of glucose 6-phosphate, its positive allosteric effector (see Fig. 24.3, ). 3. Increased pentose phosphate pathway activity: The increased availability of glucose 6-phosphate, combined with the active use of nicotinamide adenine dinucleotide phosphate (NADPH) in hepatic lipogenesis, stimulates the pentose phosphate pathway (see p. 145). This pathway typically accounts for 5%–10% of the glucose metabolized by the liver (see Fig. 24.3, ). 4.

1	4. Increased glycolysis: In the liver, glycolysis is significant only during the absorptive period following a carbohydrate-rich meal. The conversion of glucose to pyruvate is stimulated by the elevated insulin/glucagon ratio that results in increased amounts of the regulated enzymes of glycolysis: glucokinase, PFK-1, and pyruvate kinase ([PK] see p. 105). Additionally, PFK-1 is allosterically activated by fructose 2,6bisphosphate generated by the active (dephosphorylated) kinase domain of bifunctional PFK-2. PK is dephosphorylated and active. Pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl CoA, is active (dephosphorylated) because pyruvate inhibits

1	PDH kinase (see Fig. 24.3, ). The acetyl CoA either is used as a substrate for fatty acid (FA) synthesis or is oxidized for energy in the tricarboxylic acid (TCA) cycle. (See Fig. 24.4 for the central role of glucose 6-phosphate.) 5. Decreased glucose production: While glycolysis and glycogenesis (pathways that promote glucose storage) are being stimulated in the liver in the absorptive state, gluconeogenesis and glycogenolysis (pathways that generate glucose) are being inhibited. Pyruvate carboxylase (PC), which catalyzes the first step in gluconeogenesis, is largely inactive because of low levels of acetyl CoA, its allosteric activator (see p. 119). [Note: The acetyl CoA is being used for FA synthesis.] The high insulin/glucagon ratio also favors inactivation of other gluconeogenic enzymes such as fructose 1,6-bisphosphatase (see Fig. 8.17, p. 100). Glycogenolysis is inhibited by dephosphorylation of glycogen phosphorylase and phosphorylase kinase. [Note: The increased uptake and

1	such as fructose 1,6-bisphosphatase (see Fig. 8.17, p. 100). Glycogenolysis is inhibited by dephosphorylation of glycogen phosphorylase and phosphorylase kinase. [Note: The increased uptake and decreased production of blood glucose in the absorptive period prevents hyperglycemia.]

1	B. Fat metabolism 1. Increased fatty acid synthesis: Liver is the primary site of de novo synthesis of FA (see Fig. 24.3, ). FA synthesis, a cytosolic process, is favored in the absorptive period by availability of the substrates acetyl CoA (from glucose and amino acid metabolism) and NADPH (from glucose metabolism in the pentose phosphate pathway) and by the activation of ACC, both by dephosphorylation and by the presence of its allosteric activator, citrate. [Note: Inactivity of AMPK favors dephosphorylation.] ACC catalyzes the formation of malonyl CoA from acetyl CoA, the rate-limiting reaction for FA synthesis (see p. 183). [Note: Malonyl CoA inhibits carnitine palmitoyltransferase-I (CPT-I) of FA oxidation (see p. 191). Thus, citrate directly activates FA synthesis and indirectly inhibits FA degradation.] a.

1	Source of cytosolic acetyl coenzyme A: Pyruvate from aerobic glycolysis enters mitochondria and is decarboxylated by PDH. The acetyl CoA product is combined with oxaloacetate (OAA) to form citrate via citrate synthase of the TCA cycle. Citrate leaves the mitochondria (as a result of the inhibition of isocitrate dehydrogenase by ATP) and enters the cytosol. Citrate is cleaved by ATP citrate lyase (induced by insulin), producing the acetyl CoA substrate of ACC plus OAA. b. Additional source of NADPH: The OAA is reduced to malate, which is oxidatively decarboxylated to pyruvate by malic enzyme as NADPH is formed (see Fig. 16.11 on p. 187).

1	b. Additional source of NADPH: The OAA is reduced to malate, which is oxidatively decarboxylated to pyruvate by malic enzyme as NADPH is formed (see Fig. 16.11 on p. 187). 2. Increased triacylglycerol synthesis: TAG synthesis is favored because fatty acyl CoA are available both from de novo synthesis and from hydrolysis of the TAG component of chylomicron remnants removed from the blood by hepatocytes (see p. 178). Glycerol 3-phosphate, the backbone for TAG synthesis, is provided by glycolysis (see p. 189). The liver packages these endogenous TAG into very-low-density lipoprotein (VLDL) particles that are secreted into the blood for use by extrahepatic tissues, particularly adipose and muscle tissues (see Fig. 24.3, ). C. Amino acid metabolism 1.

1	C. Amino acid metabolism 1. Increased amino acid degradation: In the absorptive period, more amino acids are present than the liver can use in the synthesis of proteins and other nitrogen-containing molecules. The surplus amino acids are not stored but are either released into the blood for other tissues to use in protein synthesis or deaminated, with the resulting carbon skeletons being degraded by the liver to pyruvate, acetyl CoA, or TCA cycle intermediates. These metabolites can be oxidized for energy or used in FA synthesis (see Fig. 24.3, ). The liver has limited capacity to initiate degradation of the branched-chain amino acids (BCAA) leucine, isoleucine, and valine. They pass through the liver essentially unchanged and are metabolized in muscle (see p. 266). 2.

1	2. Increased protein synthesis: The body does not store protein for energy in the same way that it maintains glycogen or TAG reserves. However, a transient increase in the synthesis of hepatic proteins does occur in the absorptive state, resulting in replacement of any proteins that may have been degraded during the previous period of fasting (see Fig. 24.3, ). IV. ADIPOSE TISSUE: ENERGY STORAGE DEPOT Adipose tissue is second only to the liver in its ability to distribute fuel molecules. In a 70-kg man, white adipose tissue (WAT) weighs ~14 kg, or about half as much as the total muscle mass. Nearly the entire volume of each adipocyte in WAT can be occupied by a droplet of anhydrous, calorically dense TAG (Fig. 24.5).

1	A. Carbohydrate metabolism 1. Increased glucose transport: Circulating insulin levels are elevated in the absorptive state, resulting in an influx of glucose into adipocytes via insulin-sensitive GLUT-4 recruited to the cell surface from intracellular vesicles (Fig. 24.6, ). The glucose is phosphorylated by hexokinase. 2. Increased glycolysis: The increased intracellular availability of glucose results in an enhanced rate of glycolysis (see Fig. 24.6, ). In adipose tissue, glycolysis serves a synthetic function by supplying glycerol 3phosphate for TAG synthesis (see p. 188). [Note: Adipose tissue lacks glycerol kinase.] 3.

1	Increased pentose phosphate pathway activity: Adipose tissue can metabolize glucose by means of the pentose phosphate pathway, thereby producing NADPH, which is essential for FA synthesis (see p. 186 and Fig. 24.6, ). However, in humans, de novo synthesis is not a major source of FA in adipose tissue, except when refeeding a previously fasted individual (see Fig. 24.6, ). B. Fat metabolism

1	B. Fat metabolism Most of the FA added to the TAG stores of adipocytes after consumption of a lipid-containing meal are provided by the degradation of exogenous (dietary) TAG in chylomicrons sent out by the intestine and endogenous TAG in VLDL sent out by the liver (see Fig. 24.6, ). The FA are released from the lipoproteins by lipoprotein lipase (LPL), an extracellular enzyme attached to the endothelial cells of capillary walls in many tissues, particularly adipose and muscle (see p. 228). In adipose tissue, LPL is upregulated by insulin. Thus, in the fed state, elevated levels of glucose and insulin favor storage of TAG (see Fig. 24.6, ), all the carbons of which are supplied by glucose. [Note: Elevated insulin favors the dephosphorylated (inactive) form of HSL (see p. 189), thereby inhibiting lipolysis in the well-fed state.] V. RESTING SKELETAL MUSCLE

1	V. RESTING SKELETAL MUSCLE Skeletal muscle accounts for ~40% of the body mass in individuals of healthy weight, and it can use glucose, amino acids, FA, and ketone bodies as fuel. In the well-fed state, muscle takes up glucose via GLUT-4 (for energy and glycogen synthesis) and amino acids (for energy and protein synthesis). In contrast to liver, there is no covalent regulation of PFK-2 in skeletal muscle. However, in the cardiac isozyme, the kinase domain is activated by epinephrine-mediated phosphorylation (see p. 100). Skeletal muscle is unique in being able to respond to substantial changes in the demand for ATP that accompanies contraction. At rest, muscle accounts for ~25% of the oxygen (O2) consumption of the body, whereas during vigorous exercise, it is responsible for up to 90%. This underscores the fact that skeletal muscle, despite its potential for transient periods of anaerobic glycolysis, is an oxidative tissue.

1	A. Carbohydrate metabolism 1. Increased glucose transport: The transient increase in plasma glucose and insulin after a carbohydrate-rich meal leads to an increase in glucose transport into muscle cells (myocytes) by GLUT-4 (see p. 97 and Fig. 24.7, ), thereby reducing blood glucose. Glucose is phosphorylated to glucose 6-phosphate by hexokinase and metabolized to meet the energy needs of myocytes. 2. Increased glycogenesis: The increased insulin/glucagon ratio and the availability of glucose 6-phosphate favor glycogen synthesis, particularly if glycogen stores have been depleted as a result of exercise (see p. 126 and Fig. 24.7, ). B. Fat metabolism FA are released from chylomicrons and VLDL by the action of LPL (see pp. 228 and 231). However, FA are of secondary importance as a fuel for resting muscle during the well-fed state, in which glucose is the primary source of energy. C. Amino acid metabolism 1.

1	C. Amino acid metabolism 1. Increased protein synthesis: An increase in amino acid uptake and protein synthesis occurs in the absorptive period after ingestion of a meal containing protein (see Fig. 24.7, and ). This synthesis replaces protein degraded since the previous meal. 2. Increased branched-chain amino acid uptake: Muscle is the principal site for degradation of the BCAA because it contains the required transaminase (see p. 266). The dietary BCAA escape metabolism by the liver and are taken up by muscle, where they are used for protein synthesis (see Fig. 24.7, ) and as energy sources. VI. BRAIN

1	Although contributing only 2% of the adult weight, the brain accounts for a consistent 20% of the basal O2 consumption of the body at rest. Because the brain is vital to the proper functioning of all organs of the body, special priority is given to its fuel needs. To provide energy, substrates must be able to cross the endothelial cells that line the blood vessels in the brain (the blood–brain barrier [BBB]). In the fed state, the brain exclusively uses glucose as a fuel (GLUT-1 of the BBB is insulin independent), completely oxidizing ~140 g/day to carbon dioxide and water. Because the brain contains no significant stores of glycogen, it is completely dependent on the availability of blood glucose (Fig. 24.8, ). [Note: If blood glucose levels fall to <50 mg/dl (normal fasted blood glucose is 70–99 mg/dl), cerebral function is impaired (see p. 315).] The brain also lacks significant stores of TAG, and the FA circulating in the blood make little contribution to energy production for

1	is 70–99 mg/dl), cerebral function is impaired (see p. 315).] The brain also lacks significant stores of TAG, and the FA circulating in the blood make little contribution to energy production for reasons that are unclear. The intertissue exchanges characteristic of the absorptive period are summarized in Figure 24.9.

1	VII. OVERVIEW OF THE FASTED STATE

1	Fasting begins if no food is ingested after the absorptive period. It may result from an inability to obtain food, the desire to lose weight rapidly, or clinical situations in which an individual cannot eat (for example, because of trauma, surgery, cancer, or burns). In the absence of food, plasma levels of glucose, amino acids, and TAG fall, triggering a decline in insulin secretion and an increase in glucagon, epinephrine, and cortisol secretion. The decreased insulin/counterregulatory hormone ratio and the decreased availability of circulating substrates make the postabsorptive period of nutrient deprivation a catabolic period characterized by degradation of TAG, glycogen, and protein. This sets into motion an exchange of substrates among the liver, adipose tissue, skeletal muscle, and brain that is guided by two priorities: 1) the need to maintain adequate plasma levels of glucose to sustain energy metabolism in the brain, red blood cells, and other glucose-requiring tissues and

1	brain that is guided by two priorities: 1) the need to maintain adequate plasma levels of glucose to sustain energy metabolism in the brain, red blood cells, and other glucose-requiring tissues and 2) the need to mobilize FA from TAG in WAT for the synthesis and release of ketone bodies by the liver to supply energy to other tissues and spare body protein. As a result, blood glucose levels are maintained within a narrow range in fasting, while FA and ketone body levels increase. [Note: Maintaining glucose requires that the substrates for gluconeogenesis (such as pyruvate, alanine, and glycerol) be available.]

1	A. Fuel stores The metabolic fuels available in a normal 70-kg man at the beginning of a fast are shown in Figure 24.10. Observe the enormous caloric stores available in the form of TAG compared with those contained in glycogen. [Note: Although protein is listed as an energy source, each protein also has a function unrelated to energy metabolism (for example, as a structural component of the body or as an enzyme). Therefore, only about one third of the body’s protein can be used for energy production without fatally compromising vital functions.] B. Enzymic changes

1	In fasting (as in the well-fed state), the flow of intermediates through the pathways of energy metabolism is controlled by four mechanisms: 1) the availability of substrates, 2) allosteric regulation of enzymes, 3) covalent modification of enzymes, and 4) induction-repression of enzyme synthesis. The metabolic changes observed in fasting are generally opposite those described for the absorptive state (see Fig. 24.9). For example, although most of the enzymes regulated by covalent modification are dephosphorylated and active in the well-fed state, they are phosphorylated and inactive in the fasted state. Three exceptions are glycogen phosphorylase (see p. 132), glycogen phosphorylase kinase (see p. 132), and HSL (see p. 189), which are active in their phosphorylated states. In fasting, substrates are not provided by the diet but are available from the breakdown of stores and/or tissues, such as glycogenolysis with release of glucose from the liver, lipolysis with release of FA and

1	substrates are not provided by the diet but are available from the breakdown of stores and/or tissues, such as glycogenolysis with release of glucose from the liver, lipolysis with release of FA and glycerol from TAG in adipose tissue, and proteolysis with release of amino acids from muscle. Recognition that the changes in fasting are the reciprocal of those in the fed state is helpful in understanding the ebb and flow of metabolism.

1	VIII. LIVER IN FASTING The primary role of the liver in fasting is maintenance of blood glucose through the production of glucose (from glycogenolysis and gluconeogenesis) for glucose-requiring tissues and the synthesis and distribution of ketone bodies for use by other tissues. Therefore, hepatic metabolism is distinguished from peripheral (or extrahepatic) metabolism. A. Carbohydrate metabolism

1	The liver first uses glycogen degradation and then gluconeogenesis to maintain blood glucose levels to sustain energy metabolism of the brain and other glucose-requiring tissues in the fasted state. [Note: Recall that the presence of glucose-6-phosphatase in the liver allows the production of free glucose both from glycogenolysis and from gluconeogenesis (see Fig. 24.4).] 1. Increased glycogenolysis: Figure 24.11 shows the sources of blood glucose after ingestion of 100 g of glucose. During the brief absorptive period, ingested glucose is the major source of blood glucose. Several hours later, blood glucose levels have declined sufficiently to cause increased secretion of glucagon and decreased secretion of insulin. The increased glucagon/insulin ratio causes a rapid mobilization of liver glycogen stores (which contain ~80 g of glycogen in the fed state) because of PKA-mediated phosphorylation (and activation) of glycogen phosphorylase kinase that phosphorylates (and activates)

1	liver glycogen stores (which contain ~80 g of glycogen in the fed state) because of PKA-mediated phosphorylation (and activation) of glycogen phosphorylase kinase that phosphorylates (and activates) glycogen phosphorylase (see p. 132). Figure 24.11 shows that because liver glycogen is exhausted by 24 hours of fasting, hepatic glycogenolysis is a transient response to early fasting. Figure 24.12, , shows glycogen degradation as part of the overall metabolic response of the liver during fasting. [Note: Phosphorylation of glycogen synthase simultaneously inhibits glycogenesis.]

1	See Section B.2 for an explanation of the decline in gluconeogenesis.] 2. Increased gluconeogenesis: The synthesis of glucose and its release into the circulation are vital hepatic functions during short-and long-term fasting (see Fig. 24.12, ). The carbon skeletons for gluconeogenesis are derived primarily from glucogenic amino acids and lactate from muscle and glycerol from adipose tissue. Gluconeogenesis, favored by activation of fructose 1,6-bisphosphatase (because of decreased availability of its inhibitor fructose 2,6-bisphosphate; see p. 121) and by induction of PEPCK by glucagon (see p. 122), begins 4–6 hours after the last meal and becomes fully active as stores of liver glycogen are depleted (see Fig. 24.11). [Note: The decrease in fructose 2,6-bisphosphate simultaneously inhibits glycolysis at PFK-1 (see p. 99).]

1	B. Fat metabolism 1. Increased fatty acid oxidation: The oxidation of FA obtained from TAG hydrolysis in adipose tissue is the major source of energy in hepatic tissue in the fasted state (see Fig. 24.12, ). The fall in malonyl CoA because of phosphorylation (inactivation) of ACC by AMPK removes the brake on CPT-I, allowing β-oxidation to occur (see p. 191). FA oxidation generates NADH, flavin adenine dinucleotide (FADH2), and acetyl CoA. The NADH inhibits the TCA cycle and shifts OAA to malate. This results in acetyl CoA being available for ketogenesis. The acetyl CoA is also an allosteric activator of PC and an allosteric inhibitor of PDH, thereby favoring use of pyruvate in gluconeogenesis (see Fig. 10.9, p. 122). [Note: Acetyl CoA cannot be used as a substrate for gluconeogenesis, in part because the PDH reaction is irreversible.] Oxidation of NADH and FADH2 coupled with oxidative phosphorylation supplies the energy required by the PC and PEPCK reactions of gluconeogenesis.

1	2. Increased ketogenesis: The liver is unique in being able to synthesize and release ketone bodies, primarily 3-hydroxybutyrate but also acetoacetate, for use as fuel by peripheral tissues but not by the liver itself because liver lacks thiophorase (see p. 197). Ketogenesis, which starts during the first days of fasting (Fig. 24.13), is favored when the concentration of acetyl CoA from FA oxidation exceeds the oxidative capacity of the TCA cycle. [Note: Ketogenesis releases CoA, insuring its availability for continued FA oxidation.] The availability of circulating water-soluble ketone bodies is important in fasting because they can be used for fuel by most tissues, including the brain, once their blood level is high enough. Ketone body concentration in blood increases from ~50 µM to ~6 mM in fasting. This reduces the need for gluconeogenesis from amino acid carbon skeletons, thus preserving essential protein (see Fig. 24.11). Ketogenesis as part of the overall hepatic response to

1	mM in fasting. This reduces the need for gluconeogenesis from amino acid carbon skeletons, thus preserving essential protein (see Fig. 24.11). Ketogenesis as part of the overall hepatic response to fasting is shown in Figure 24.12, . [Note: Ketone bodies are organic acids and, when present at high concentrations, can cause ketoacidosis.]

1	IX. A. Glucose transport by insulin-sensitive GLUT-4 into the adipocyte and its subsequent metabolism are decreased because of low levels of circulating insulin. This results in decreased TAG synthesis. B. Fat metabolism 1. Increased fat degradation: The PKA-mediated phosphorylation and activation of HSL (see p. 189) and subsequent hydrolysis of stored fat (TAG) are enhanced by the elevated catecholamines norepinephrine and epinephrine. These hormones, which are secreted from the sympathetic nerve endings in adipose tissue and/or from the adrenal medulla, are physiologically important activators of HSL (Fig. 24.14, ). 2.

1	2. Increased fatty acid release: FA obtained from hydrolysis of TAG stored in adipocytes are primarily released into the blood (see Fig. 24.14, ). Bound to albumin, they are transported to a variety of tissues for use as fuel. The glycerol produced from TAG degradation is used as a gluconeogenic precursor by the liver, which contains glycerol kinase. [Note: FA can also be oxidized to acetyl CoA, which can enter the TCA cycle, thereby producing energy for the adipocyte. They also can be reesterified to glycerol 3-phosphate (from glyceroneogenesis, see p. 190), generating TAG and reducing plasma FA concentration.] 3. Decreased fatty acid uptake: In fasting, LPL activity of adipose tissue is low. Consequently, FA in circulating TAG of lipoproteins are less available to adipose tissue than to muscle. X. RESTING SKELETAL MUSCLE IN FASTING

1	X. RESTING SKELETAL MUSCLE IN FASTING Resting muscle switches from glucose to FA as its major fuel source in fasting. [Note: By contrast, exercising muscle initially uses creatine phosphate and its glycogen stores. During intense exercise, glucose 6-phosphate from glycogenolysis is converted to lactate by anaerobic glycolysis (see p. 118). The lactate is used by the liver for gluconeogenesis (Cori cycle; see p. 118). As these glycogen reserves are depleted, free FA provided by the degradation of TAG in adipose tissue become the dominant energy source. The contraction-based rise in AMP activates AMPK that phosphorylates and inactivates the muscle isozyme of ACC, decreasing malonyl CoA and allowing FA oxidation (see p. 183).] A. Carbohydrate metabolism p. 97) and subsequent glucose metabolism are decreased because circulating insulin levels are low. Therefore, the glucose from hepatic gluconeogenesis is unavailable to muscle and adipose. B. Lipid metabolism

1	B. Lipid metabolism Early in fasting, muscle uses FA from adipose tissue and ketone bodies from the liver as fuels (Fig. 24.15, and ). In prolonged fasting, muscle decreases its use of ketone bodies (thus sparing them for the brain) and oxidizes FA almost exclusively. [Note: The acetyl CoA from FA oxidation indirectly inhibits PDH (by activation of PDH kinase) and spares pyruvate, which is transaminated to alanine and used by the liver for gluconeogenesis (glucose–alanine cycle; see p. 253).] C. Protein metabolism

1	C. Protein metabolism During the first few days of fasting, there is a rapid breakdown of muscle protein (for example, glycolytic enzymes), providing amino acids that are used by the liver for gluconeogenesis (see Fig. 24.15, ). Because muscle does not have glucagon receptors, muscle proteolysis is initiated by a fall in insulin and sustained by a rise in glucocorticoids. [Note: Alanine and glutamine are quantitatively the most important glucogenic amino acids released from muscle. They are produced by the catabolism of BCAA (see p. 267). The glutamine is used as a fuel by enterocytes, for example, which send out alanine that is used in hepatic gluconeogenesis (glucose–alanine cycle)]. In the second week of fasting, the rate of muscle proteolysis decreases, paralleling a decline in the need for glucose as a fuel for the brain, which has begun using ketone bodies as a source of energy. XI. BRAIN IN FASTING

1	XI. BRAIN IN FASTING During the early days of fasting, the brain continues to use only glucose as a fuel (Fig. 24.16, ). Blood glucose is maintained by hepatic gluconeogenesis from glucogenic precursors, such as amino acids from proteolysis and glycerol from lipolysis. In prolonged fasting (beyond 2–3 weeks), plasma ketone bodies (see Fig. 24.12) reach significantly elevated levels and replace glucose as the primary fuel for the brain (see Figs. 24.16, and 24.17). This reduces the need for protein catabolism for gluconeogenesis: Ketone bodies spare glucose and, thus, muscle protein. [Note: As the duration of a fast extends from overnight to days to weeks, blood glucose levels initially drop and then are maintained at the lower level (65–70 mg/dl).] The metabolic changes that occur during fasting insure that all tissues have an adequate supply of fuel molecules. The response of the major tissues involved in energy metabolism during fasting is summarized in Figure 24.18.

1	XII. KIDNEY IN LONG-TERM FASTING

1	As fasting continues into early starvation and beyond, the kidney plays important roles. The renal cortex expresses the enzymes of gluconeogenesis, including glucose 6-phosphatase, and, in late fasting, ~50% of gluconeogenesis occurs here. [Note: A portion of this glucose is used by the kidney itself.] The kidney also provides compensation for the acidosis that accompanies the increased production of ketone bodies (organic acids). The glutamine released from the muscle’s metabolism of BCAA is taken up by the kidney and acted upon by renal glutaminase and glutamate dehydrogenase (see p. 256), producing αketoglutarate, which can be used as a substrate for gluconeogenesis, plus ammonia (NH3). The NH3 picks up protons from ketone body dissociation and is excreted in the urine as ammonium (NH4+), thereby decreasing the acid load in the body (Fig. 24.19). Therefore, in long-term fasting, there is a switch from nitrogen disposal in the form of urea to disposal in the form of NH4 . [Note: As

1	thereby decreasing the acid load in the body (Fig. 24.19). Therefore, in long-term fasting, there is a switch from nitrogen disposal in the form of urea to disposal in the form of NH4 . [Note: As ketone body concentration rises, enterocytes, typically consumers of glutamine, become consumers of ketone bodies. This allows more glutamine to be available to the kidney.]

1	XIII. CHAPTER SUMMARY

1	The flow of intermediates through metabolic pathways is controlled by four regulatory mechanisms: 1) the availability of substrates, 2) allosteric activation and inhibition of enzymes, 3) covalent modification of enzymes, and 4) induction-repression of enzyme synthesis. In the absorptive state, the 2-to 4-hour period after ingestion of a meal, these mechanisms insure that available nutrients are captured as glycogen, triacylglycerol (TAG), and protein (Fig. 24.20). During this interval, transient increases in plasma glucose, amino acids, and TAG occur, the last primarily as components of chylomicrons synthesized by the intestinal mucosal cells. The pancreas responds to the elevated levels of glucose with an increased secretion of insulin and a decreased secretion of glucagon. The elevated insulin/glucagon ratio and the ready availability of circulating substrates make the absorptive state an anabolic period during which virtually all tissues use glucose as a fuel. In addition, the

1	insulin/glucagon ratio and the ready availability of circulating substrates make the absorptive state an anabolic period during which virtually all tissues use glucose as a fuel. In addition, the liver replenishes its glycogen stores, replaces any needed hepatic proteins, and increases TAG synthesis. The latter are packaged in very-low-density lipoproteins, which are exported to the peripheral tissues. Adipose tissue increases TAG synthesis and storage, whereas muscle increases protein synthesis to replace protein degraded since the previous meal. In the fed state, the brain uses glucose exclusively as a fuel. In fasting, plasma levels of glucose, amino acids, and TAG fall, triggering a decline in insulin secretion and an increase in glucagon and epinephrine secretion. The decreased insulin/counterregulatory hormone ratio and the decreased availability of circulating substrates make the fasting state a catabolic period. This sets into motion an exchange of substrates among the liver,

1	hormone ratio and the decreased availability of circulating substrates make the fasting state a catabolic period. This sets into motion an exchange of substrates among the liver, adipose tissue, skeletal muscle, and brain that is guided by two priorities: 1) the need to maintain adequate plasma levels of glucose to sustain energy metabolism of the brain and other glucose-requiring tissues and 2) the need to mobilize fatty acids (FA) from adipose tissue and release ketone bodies from liver to supply energy to other tissues. To accomplish these goals, the liver degrades glycogen and initiates gluconeogenesis, using increased FA oxidation to supply the energy and reducing equivalents needed for gluconeogenesis and the acetyl coenzyme A building blocks for ketogenesis. The adipose tissue degrades stored TAG, thus providing FA and glycerol to the liver. The muscle can also use FA as fuel as well as ketone bodies supplied by the liver. The liver uses the glycerol for gluconeogenesis. Muscle

1	stored TAG, thus providing FA and glycerol to the liver. The muscle can also use FA as fuel as well as ketone bodies supplied by the liver. The liver uses the glycerol for gluconeogenesis. Muscle protein is degraded to supply amino acids for the liver to use in gluconeogenesis but decreases as ketone bodies increase. The brain can use both glucose and ketone bodies as fuels. From late fasting into starvation, the kidneys play important roles by synthesizing glucose and excreting the protons from ketone body dissociation as ammonium (NH4+).

1	Choose the ONE best answer. 4.1. Which one of the following is elevated in plasma during the absorptive (well-fed) state as compared with the postabsorptive (fasted) state? A. Acetoacetate B. Chylomicrons C. Free fatty acids D. Glucagon Correct answer = B. Triacylglycerol-rich chylomicrons are synthesized in (and released from) the intestine following ingestion of a meal. Acetoacetate, free fatty acids, and glucagon are elevated in the fasted state, not the absorptive state. 4.2. Which one of the following statements concerning liver in the absorptive state is correct? A. Fructose 2,6-bisphosphate is elevated. B. Insulin stimulates the uptake of glucose. C. Most enzymes that are regulated by covalent modification are in the phosphorylated state. D. The oxidation of acetyl coenzyme A is increased. E. The synthesis of glucokinase is repressed.

1	C. Most enzymes that are regulated by covalent modification are in the phosphorylated state. D. The oxidation of acetyl coenzyme A is increased. E. The synthesis of glucokinase is repressed. Correct answer = A. The increased insulin and decreased glucagon levels characteristic of the absorptive state promote the synthesis of fructose 2,6bisphosphate, which allosterically activates phosphofructokinase-1 of glycolysis. Most covalently modified enzymes are in the dephosphorylated state and are active. Acetyl coenzyme A is not oxidized in the well-fed state because it is being used in fatty acid synthesis. Uptake of glucose (by glucose transporter-2) into the liver is insulin independent. Synthesis of glucokinase is induced by insulin in the well-fed state. 4.3. Which one of the following enzymes is phosphorylated and active in an individual who has been fasting for 12 hours? A. Arginase B. Carnitine palmitoyltransferase-I C. Fatty acid synthase D. Glycogen synthase

1	A. Arginase B. Carnitine palmitoyltransferase-I C. Fatty acid synthase D. Glycogen synthase E. Hormone-sensitive lipase F. Phosphofructokinase-1 G. Pyruvate dehydrogenase Correct answer = E. Hormone-sensitive lipase of adipocytes is phosphorylated and activated by protein kinase A in response to epinephrine. Choices A, B, C, and F are not regulated covalently. Choices D and G are regulated covalently but are inactive if phosphorylated. 4.4. For a 70-kg man, in which one of the periods listed below do ketone bodies supply the major portion of the caloric needs of brain? A. Absorptive period B. Overnight fast C. Three-day fast D. Four-week fast E. Five-month fast

1	A. Absorptive period B. Overnight fast C. Three-day fast D. Four-week fast E. Five-month fast Correct answer = D. Ketone bodies, made from the acetyl coenzyme A product of fatty acid oxidation, increase in the blood in fasting but must reach a critical level to cross the blood–brain barrier. Typically, this occurs in the second to third week of a fast. Fat stores in a 70-kg (~154-lb) man would not be able to supply his energy needs for 5 months. 24.5. The diagram below shows inputs to and outputs from pyruvate, a central molecule in energy metabolism. Which letter on the diagram represents a reaction that requires biotin and is activated by acetyl coenzyme A?

1	Which letter on the diagram represents a reaction that requires biotin and is activated by acetyl coenzyme A? Correct answer = C. Pyruvate carboxylase, a mitochondrial enzyme of gluconeogenesis, requires biotin (and ATP) and is allosterically activated by acetyl coenzyme A from fatty acid oxidation. None of the other choices meets these criteria. A = pyruvate kinase; B = pyruvate dehydrogenase complex; D = aspartate aminotransferase; E = alanine aminotransferase; F = lactate dehydrogenase. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	I. OVERVIEW Diabetes mellitus (diabetes) is not one disease but rather is a heterogeneous group of multifactorial, primarily polygenic syndromes characterized by an elevated fasting blood glucose (FBG) caused by a relative or absolute deficiency in insulin. Over 29 million people in the United States (~9% of the population) have diabetes. Of this number, ~8 million are as yet undiagnosed. Diabetes is the leading cause of adult blindness and amputation and a major cause of renal failure, nerve damage, heart attacks, and strokes. Most cases of diabetes mellitus can be separated into two groups (Fig. 25.1), type 1 ([T1D] formerly called insulin-dependent diabetes mellitus) and type 2 ([T2D] formerly called non– insulin-dependent diabetes mellitus). The incidence and prevalence of T2D is increasing because of the aging of the U.S. population and the increasing prevalence of obesity and sedentary lifestyles (see p. 349). The increase in children with T2D is particularly disturbing.

1	II. TYPE 1

1	T1D constitutes <10% of the ~21 million known cases of diabetes in the United States. The disease is characterized by an absolute deficiency of insulin caused by an autoimmune attack on the islet β cells of the pancreas. In T1D, the islets of Langerhans become infiltrated with activated T lymphocytes, leading to a condition called insulitis. Over a period of years, this autoimmune attack on the β cells leads to gradual depletion of the β-cell population (Fig. 25.2). However, symptoms appear abruptly when 80%–90% of the β cells have been destroyed. At this point, the pancreas fails to respond adequately to ingestion of glucose, and insulin therapy is required to restore metabolic control and prevent life-threatening ketoacidosis. β-Cell destruction requires both a stimulus from the environment (such as a viral infection) and a genetic determinant that causes the β cells to be mistakenly identified as “nonself.” [Note: Among monozygotic (identical) twins, if one sibling develops T1D,

1	(such as a viral infection) and a genetic determinant that causes the β cells to be mistakenly identified as “nonself.” [Note: Among monozygotic (identical) twins, if one sibling develops T1D, the other twin has only a 30%– 50% chance of developing the disease. This contrasts with T2D (see p. 341), in which the genetic influence is stronger and, in virtually all monozygotic twinships, the disease eventually develops in both individuals.]

1	A. Diagnosis

1	The onset of T1D is typically during childhood or puberty, and symptoms develop suddenly. Individuals with T1D can usually be recognized by the abrupt appearance of polyuria (frequent urination), polydipsia (excessive thirst), and polyphagia (excessive hunger), often triggered by physiologic stress such as an infection. These symptoms are usually accompanied by fatigue and weight loss. The diagnosis is confirmed by a FBG ≥126 mg/dl (normal is 70–99). [Note: Fasting is defined as no caloric intake for at least 8 hours.] A FBG of 100–125 mg/dl is categorized as an impaired FBG. Individuals with impaired FBG are considered prediabetic and are at increased risk for developing T2D. Diagnosis can also be made on the basis of a nonfasting (random) blood glucose level >200 mg/dl or a glycated hemoglobin (see p. 340) concentration ≥6.5 mg/dl (normal is <5.7) in an individual with symptoms of hyperglycemia. [Note: The oral glucose tolerance test, in which blood glucose is measured 2 hours after

1	(see p. 340) concentration ≥6.5 mg/dl (normal is <5.7) in an individual with symptoms of hyperglycemia. [Note: The oral glucose tolerance test, in which blood glucose is measured 2 hours after ingestion of a solution containing 75 g of glucose, also is used but is less convenient. It is most typically used to screen pregnant women for gestational diabetes (see p. 342).]

1	When blood glucose is >180 mg/dl, the ability of renal sodium-dependent glucose transporters (SGLT) to reclaim glucose is impaired, and glucose “spills” into urine. The loss of glucose is accompanied by the loss of water, resulting in the characteristic polyuria (with dehydration) and polydipsia of diabetes. B. Metabolic changes The metabolic abnormalities of T1D result from a deficiency of insulin that profoundly affects metabolism in three tissues: liver, skeletal muscle, and white adipose (Fig. 25.3). 1.

1	Hyperglycemia and ketonemia: Elevated levels of blood glucose and ketone bodies are the hallmarks of untreated T1D (see Fig. 25.3). Hyperglycemia is caused by increased hepatic production of glucose via gluconeogenesis, combined with diminished peripheral utilization (muscle and adipose tissue have the insulin-regulated glucose transporter GLUT-4; see p. 97). Ketonemia results from increased mobilization of fatty acids (FA) from triacylglycerol (TAG) in adipose tissue, combined with accelerated hepatic FA β-oxidation and synthesis of 3hydroxybutyrate and acetoacetate (ketone bodies; see p. 196). [Note: Acetyl coenzyme A from β-oxidation is the substrate for ketogenesis and the allosteric activator of pyruvate carboxylase, a gluconeogenic enzyme.] Diabetic ketoacidosis (DKA), a type of metabolic acidosis caused by an imbalance between ketone body production and use, occurs in 25%–40% of those newly diagnosed with T1D and may recur if the patient becomes ill (most commonly with an

1	metabolic acidosis caused by an imbalance between ketone body production and use, occurs in 25%–40% of those newly diagnosed with T1D and may recur if the patient becomes ill (most commonly with an infection) or does not comply with therapy. DKA is treated by replacing fluid and electrolytes and administering short-acting insulin to gradually correct hyperglycemia without precipitating hypoglycemia.

1	2. Hypertriacylglycerolemia: Not all of the FA flooding the liver can be disposed of through oxidation and ketone body synthesis. These excess FA are converted to TAG, which are packaged and secreted in very-lowdensity lipoproteins ([VLDL] see p. 230). Chylomicrons rich in dietary TAG are secreted by the intestinal mucosal cells following a meal (see p. 227). Because lipoprotein TAG degradation catalyzed by lipoprotein lipase in the capillary beds of adipose tissue (see p. 228) is low in diabetes (synthesis of the enzyme is decreased when insulin levels are low), the plasma chylomicron and VLDL levels are elevated, resulting in hypertriacylglycerolemia (see Fig. 25.3). C. Treatment

1	Individuals with T1D must rely on exogenous insulin delivered subcutaneously (subq) either by periodic injection or by continuous pump-assisted infusion to control the hyperglycemia and ketonemia. Two types of therapeutic injection regimens are currently used, standard and intensive. [Note: Pump delivery is also considered intensive therapy.] 1. Standard versus intensive treatment: Standard treatment is typically two to three daily injections of recombinant human insulin. Mean blood glucose levels obtained are typically 225–275 mg/dl, with a glycated hemoglobin (HbA1c) level (see p. 33) of 8%–9% of the total hemoglobin (blue arrow in Fig. 25.4). [Note: The rate of formation of HbA1c is proportional to the average blood glucose concentration over the previous 3 months. Thus, HbA1c provides a measure of how well treatment has normalized blood glucose over that time in a patient with diabetes.] In contrast to standard therapy, intensive treatment seeks to more closely normalize blood

1	a measure of how well treatment has normalized blood glucose over that time in a patient with diabetes.] In contrast to standard therapy, intensive treatment seeks to more closely normalize blood glucose through more frequent monitoring and subsequent injections of insulin, typically ≥4 times a day. Mean blood glucose levels of 150 mg/dl can be achieved, with HbA1c ~7% of the total hemoglobin (see red arrow in Fig. 25.4). [Note: Normal mean blood glucose is ~100 mg/dl, and HbA1c is ≤6% (see black arrow in Fig. 25.4).] Therefore, normalization of glucose values (euglycemia) is not achieved even in intensively treated patients. Nonetheless, patients on intensive therapy show a ≥50% reduction in the long-term microvascular complications of diabetes (that is, retinopathy, nephropathy, and neuropathy) compared with patients receiving standard care. This confirms that the complications of diabetes are related to an elevation of plasma glucose.

1	therapy. [Note: Nondiabetic individuals are included for comparison.] 2. Hypoglycemia: One of the therapeutic goals in cases of diabetes is to decrease blood glucose levels in an effort to minimize the development of long-term complications of the disease (see p. 345 for a discussion of the chronic complications of diabetes). However, appropriate dosage of insulin is difficult to achieve. Hypoglycemia caused by excess insulin is the most common complication of insulin therapy, occurring in >90% of patients. The frequency of hypoglycemic episodes, seizures, and coma is particularly high with intensive treatment regimens designed to achieve tight control of blood glucose (Fig. 25.5). In normal individuals, hypoglycemia triggers a compensatory secretion of counterregulatory hormones, most notably glucagon and epinephrine, which promote hepatic production of glucose (see p. 315). However, patients with T1D also develop a deficiency of glucagon secretion. This defect occurs early in the

1	notably glucagon and epinephrine, which promote hepatic production of glucose (see p. 315). However, patients with T1D also develop a deficiency of glucagon secretion. This defect occurs early in the disease and is almost universally present 4 years after diagnosis. Therefore, these patients rely on epinephrine secretion to prevent severe hypoglycemia. However, as the disease progresses, T1D patients show diabetic autonomic neuropathy and impaired ability to secrete epinephrine in response to hypoglycemia. The combined deficiency of glucagon and epinephrine secretion creates a symptom-free condition sometimes called “hypoglycemia unawareness.” Thus, patients with long-standing T1D are particularly vulnerable to hypoglycemia. Hypoglycemia can also be caused by strenuous exercise. Because exercise promotes glucose uptake into muscle and decreases the need for exogenous insulin, patients are advised to check blood glucose levels before or after intensive exercise to prevent or abort

1	exercise promotes glucose uptake into muscle and decreases the need for exogenous insulin, patients are advised to check blood glucose levels before or after intensive exercise to prevent or abort hypoglycemia.

1	3. Contraindications for tight control: Children are not put on a program of tight control of blood glucose before age 8 years because of the risk that episodes of hypoglycemia may adversely affect brain development. Elderly people typically do not go on tight control because hypoglycemia can cause strokes and heart attacks in this population. Also, the major goal of tight control is to prevent complications many years later. Tight control, then, is most worthwhile for otherwise healthy people who can expect to live at least 10 more years. [Note: For most nonpregnant adults with diabetes, the individual treatment strategies and goals are based on the duration of diabetes, age/life expectancy, and known comorbid conditions.] III. TYPE 2

1	III. TYPE 2 T2D is the most common form of the disease, afflicting >90% of the U.S. population with diabetes. [Note: American Indians, Alaskan Natives, Hispanic and Latino Americans, African Americans, and Asian Americans have the highest prevalence.] Typically, T2D develops gradually without obvious symptoms. The disease is often detected by routine screening tests. However, many individuals with T2D have symptoms of polyuria and polydipsia of several weeks’ duration. Polyphagia may be present but is less common. Patients with T2D have a combination of insulin resistance and dysfunctional β cells (Fig.

1	25.6) but do not require insulin to sustain life. However, in >90% of these patients, insulin eventually will be required to control hyperglycemia and keep HbA1c <7%. The metabolic alterations observed in T2D are milder than those described for type 1, in part because insulin secretion in T2D, although inadequate, does restrain ketogenesis and blunts the development of DKA. [Note: Insulin suppresses the release of glucagon (see p. 314).] Diagnosis is based on the presence of hyperglycemia as described above. The pathogenesis does not involve viruses or autoimmune antibodies and is not completely understood. [Note: An acute complication of T2D in the elderly is a hyperosmolar hyperglycemic state characterized by severe hyperglycemia and dehydration and altered mental status.]

1	T2D is characterized by hyperglycemia, insulin resistance, impaired insulin secretion, and, ultimately, β-cell failure. The eventual need for insulin therapy has eliminated the designation of T2D as non–insulin-dependent diabetes. A. Insulin resistance Insulin resistance is the decreased ability of target tissues, such as the liver, white adipose, and skeletal muscle, to respond properly to normal (or elevated) circulating concentrations of insulin. For example, insulin resistance is characterized by increased hepatic glucose production, decreased glucose uptake by muscle and adipose tissue, and increased adipose lipolysis with production of free fatty acids (FFA).

1	1. Insulin resistance and obesity: Although obesity is the most common cause of insulin resistance and increases the risk of T2D, most people with obesity and insulin resistance do not develop diabetes. In the absence of a defect in β-cell function, obese individuals can compensate for insulin resistance with elevated levels of insulin. For example, Figure 25.7A shows that insulin secretion is two to three times higher in obese subjects than it is in lean individuals. This higher insulin concentration compensates for the diminished effect of the hormone (as a result of insulin resistance) and produces blood glucose levels similar to those observed in lean individuals (Fig. 25.7B). 2.

1	2. Insulin resistance and type 2 diabetes: Insulin resistance alone will not lead to T2D. Rather, T2D develops in insulin-resistant individuals who also show impaired β-cell function. Insulin resistance and subsequent risk for the development of T2D is commonly observed in individuals who are obese, physically inactive, or elderly and in the 3%–5% of pregnant women who develop gestational diabetes. These patients are unable to sufficiently compensate for insulin resistance with increased insulin release. Figure 25.8 shows the time course for the development of hyperglycemia and the loss of β-cell function. 3.

1	Causes of insulin resistance: Insulin resistance increases with weight gain and decreases with weight loss, and excess adipose tissue (particularly in the abdomen) is key in the development of insulin resistance. Adipose is not simply an energy storage tissue, but also a secretory tissue. With obesity, there are changes in adipose secretions that result in insulin resistance (Fig. 25.9). These include secretion of proinflammatory cytokines such as interleukin 6 and tumor necrosis factor-α by activated macrophages (inflammation is associated with insulin resistance); increased synthesis of leptin, a protein with proinflammatory effects (see p. 353 for additional effects of leptin); and decreased secretion of adiponectin (see p. 350), a protein with anti-inflammatory effects. The net result is chronic, low-grade inflammation. One effect of insulin resistance is increased lipolysis and production of FFA (see Fig. 25.9). FFA availability decreases use of glucose, contributing to

1	result is chronic, low-grade inflammation. One effect of insulin resistance is increased lipolysis and production of FFA (see Fig. 25.9). FFA availability decreases use of glucose, contributing to hyperglycemia, and increases ectopic deposition of TAG in liver (hepatic steatosis). [Note: Steatosis results in nonalcoholic fatty liver disease (NAFLD). If accompanied by inflammation, a more serious condition, nonalcoholic steatohepatitis (NASH), can develop.] FFA also have a proinflammatory effect. In the long term, FFA impair insulin signaling. [Note: Adiponectin increases FA β-oxidation (see p. 349). Consequently, a decrease in this adipocyte protein contributes to FFA availability.]

1	B. Dysfunctional β cells In T2D, the pancreas initially retains β-cell capacity, resulting in insulin levels that vary from above normal to below normal. However, with time, the β cell becomes increasingly dysfunctional and fails to secrete enough insulin to correct the prevailing hyperglycemia. For example, insulin levels are high in typical, obese, T2D patients but not as high as in similarly obese individuals who do not have diabetes. Thus, the natural progression of the disease results in a declining ability to control hyperglycemia with endogenous secretion of insulin (Fig. 25.10). Deterioration of β-cell function may be accelerated by the toxic effects of sustained hyperglycemia and elevated FFA and a proinflammatory environment. C. Metabolic changes The abnormalities of glucose and TAG metabolism in T2D are the result of insulin resistance expressed primarily in liver, skeletal muscle, and white adipose tissue (Fig. 25.11). 1.

1	The abnormalities of glucose and TAG metabolism in T2D are the result of insulin resistance expressed primarily in liver, skeletal muscle, and white adipose tissue (Fig. 25.11). 1. Hyperglycemia: Hyperglycemia is caused by increased hepatic production of glucose, combined with diminished use of glucose by muscle and adipose tissues. Ketonemia is usually minimal or absent in patients with T2D because the presence of insulin, even in the presence of insulin resistance, restrains hepatic ketogenesis. 2.

1	2. Dyslipidemia: In the liver, FFA are converted to TAG, which are packaged and secreted in VLDL. Dietary TAG–rich chylomicrons are synthesized and secreted by the intestinal mucosal cells following a meal. Because lipoprotein TAG degradation catalyzed by lipoprotein lipase in adipose tissue is low in diabetes, the plasma chylomicron and VLDL levels are elevated, resulting in hypertriacylglycerolemia (see Fig. 25.10). Low levels of high-density lipoproteins are also associated with T2D, likely as a result of increased degradation. D. Treatment

1	D. Treatment The goal in treating T2D is to maintain blood glucose concentrations within normal limits and to prevent the development of long-term complications. Weight reduction, exercise, and medical nutrition therapy (dietary modifications) often correct the hyperglycemia of newly diagnosed T2D. Oral hypoglycemic agents, such as metformin (decreases hepatic gluconeogenesis), sulfonylureas (increase insulin secretion; see p. 310), thiazolidinediones (decrease FFA levels and increase peripheral insulin sensitivity), α-glucosidase inhibitors (decrease absorption of dietary carbohydrate), and SGLT inhibitors (decrease renal reabsorption of glucose), or subq insulin therapy may be required to achieve satisfactory plasma glucose levels. [Note: Bariatric surgery in morbidly obese individuals with T2D has been shown to result in disease remission in most patients. Remission may not be permanent.] IV. CHRONIC EFFECTS AND PREVENTION

1	As noted previously, available therapies moderate the hyperglycemia of diabetes but fail to completely normalize metabolism. The long-standing elevation of blood glucose is associated with the chronic vascular complications of diabetes including cardiovascular disease (CVD) and stroke (macrovascular complications) as well as retinopathy, nephropathy, and neuropathy (microvascular). Intensive insulin treatment (see p. 340) delays the onset and slows the progression of some long-term complications. For example, the incidence of retinopathy decreases as control of blood glucose improves and HbA1c levels decrease (Fig. 25.12). [Note: Data concerning the effect of tight control on CVD in T2D are less clear.] The benefits of tight control of blood glucose outweigh the increased risk of severe hypoglycemia in most patients. How hyperglycemia causes the chronic complications of diabetes is unclear. In cells in which glucose uptake is not dependent on insulin, elevated blood glucose leads to

1	hypoglycemia in most patients. How hyperglycemia causes the chronic complications of diabetes is unclear. In cells in which glucose uptake is not dependent on insulin, elevated blood glucose leads to increased intracellular glucose and its metabolites. For example, increased intracellular sorbitol contributes to cataract formation (see p. 140) in diabetes. Additionally, hyperglycemia promotes glycation of cellular proteins in a reaction analogous to the formation of HbA1c. These glycated proteins undergo additional reactions and become advanced glycation end products (AGE) that mediate some of the early microvascular changes of diabetes and can reduce wound healing. Some AGE bind to a membrane receptor (RAGE), causing the release of proinflammatory molecules. There is currently no preventative treatment for T1D. The risk for T2D can be significantly decreased by a combined regimen of medical nutrition therapy, weight loss, exercise, and aggressive control of hypertension and

1	preventative treatment for T1D. The risk for T2D can be significantly decreased by a combined regimen of medical nutrition therapy, weight loss, exercise, and aggressive control of hypertension and dyslipidemias. For example, Figure 25.13 shows the incidence of disease in normal and overweight individuals with varying degrees of exercise.

1	V. CHAPTER SUMMARY

1	Diabetes mellitus is a heterogeneous group of syndromes characterized by an elevation of fasting blood glucose that is caused by a relative or absolute deficiency of insulin (Fig. 25.14). Diabetes is the leading cause of adult blindness and amputation and a major cause of renal failure, nerve damage, heart attacks, and stroke. Diabetes can be classified into two groups, type 1 (T1D) and type 2 (T2D). T1D constitutes ~10% of >29 million cases of diabetes in the United States. The disease is characterized by an absolute deficiency of insulin caused by an autoimmune attack on the pancreatic β cells. This destruction requires an environmental stimulus (such as a viral infection) and a genetic determinant that causes the β cell to be mistakenly identified as “nonself.” The metabolic abnormalities of T1D include hyperglycemia, diabetic ketoacidosis (DKA), and hypertriacylglycerolemia that result from a deficiency of insulin. Those with T1D must rely on exogenous insulin delivered

1	abnormalities of T1D include hyperglycemia, diabetic ketoacidosis (DKA), and hypertriacylglycerolemia that result from a deficiency of insulin. Those with T1D must rely on exogenous insulin delivered subcutaneously to control hyperglycemia and ketoacidosis. T2D has a strong genetic component. It results from a combination of insulin resistance and dysfunctional β cells. Insulin resistance is the decreased ability of target tissues, such as liver, white adipose, and skeletal muscle, to respond properly to normal (or elevated) circulating concentrations of insulin. Obesity is the most common cause of insulin resistance. However, most people with obesity and insulin resistance do not develop diabetes. In the absence of a defect in β-cell function, obese individuals without diabetes can compensate for insulin resistance with elevated levels of insulin. Insulin resistance alone will not lead to T2D. Rather, T2D develops in insulin-resistant individuals who also show impaired β-cell

1	compensate for insulin resistance with elevated levels of insulin. Insulin resistance alone will not lead to T2D. Rather, T2D develops in insulin-resistant individuals who also show impaired β-cell function. The acute metabolic alterations observed in T2D are milder than those described for the insulin-dependent form of the disease, in part because insulin secretion in T2D, although inadequate, does restrain ketogenesis and blunts the development of DKA. Available treatments for diabetes moderate the hyperglycemia but fail to completely normalize metabolism. The long-standing elevation of blood glucose is associated with the chronic complications of diabetes including cardiovascular disease and stroke (macrovascular) as well as retinopathy, nephropathy, and neuropathy (microvascular).

1	Choose the ONE best answer. 5.1. Three patients being evaluated for gestational diabetes are given an oral glucose tolerance test. Based on the data shown below, which patient is prediabetic? A. Patient #1 B. Patient #2 C. Patient #3 D. None Correct answer = B. Patient #2 has a normal fasting blood glucose (FBG) but an impaired glucose tolerance (GT) as reflected in her blood glucose level at 2 hours and, so, is described as prediabetic. Patient #1 has a normal FBG and GT, whereas patient #3 has diabetes. 5.2. Relative or absolute lack of insulin in humans would result in which one of the following reactions in the liver? A. Decreased activity of hormone-sensitive lipase B. Decreased gluconeogenesis from lactate C. Decreased glycogenolysis D. Increased formation of 3-hydroxybutyrate E. Increased glycogenesis

1	A. Decreased activity of hormone-sensitive lipase B. Decreased gluconeogenesis from lactate C. Decreased glycogenolysis D. Increased formation of 3-hydroxybutyrate E. Increased glycogenesis Correct answer = D. Low insulin levels favor the liver producing ketone bodies, using acetyl coenzyme A generated by β-oxidation of the fatty acids provided by hormone-sensitive lipase (HSL) in adipose tissue (not liver). Low insulin also causes activation of HSL, decreased glycogen synthesis, and increased gluconeogenesis and glycogenolysis. 5.3. Which one of the following is characteristic of untreated diabetes regardless of the type? A. Hyperglycemia B. Ketoacidosis C. Low levels of hemoglobin A1c D. Normal levels of C-peptide E. Obesity F. Simple inheritance pattern

1	A. Hyperglycemia B. Ketoacidosis C. Low levels of hemoglobin A1c D. Normal levels of C-peptide E. Obesity F. Simple inheritance pattern Correct answer = A. Elevated blood glucose occurs in type 1 diabetes (T1D) as a result of a lack of insulin. In type 2 diabetes (T2D), hyperglycemia is due to a defect in β-cell function and insulin resistance. The hyperglycemia results in elevated hemoglobin A1c levels. Ketoacidosis is rare in T2D, whereas obesity is rare in T1D. C (connecting)-peptide is a measure of insulin synthesis. It would be virtually absent in T1D and initially increased then decreased in T2D. Both forms of the disease show complex genetics. 5.4. An obese individual with type 2 diabetes typically: A. benefits from receiving insulin about 6 hours after a meal. B. has a lower plasma level of glucagon than does a normal individual. C. has a lower plasma level of insulin than does a normal individual early in the disease process.

1	B. has a lower plasma level of glucagon than does a normal individual. C. has a lower plasma level of insulin than does a normal individual early in the disease process. D. shows improvement in glucose tolerance if body weight is reduced. E. shows sudden onset of symptoms. Correct answer = D. Many individuals with type 2 diabetes are obese, and almost all show some improvement in blood glucose with weight reduction. Symptoms usually develop gradually. These patients have elevated insulin levels and usually do not require insulin (certainly not 6 hours after a meal) until late in the disease. Glucagon levels are typically normal. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Obesity is a disorder of body weight regulatory systems characterized by an accumulation of excess body fat. In primitive societies, in which daily life required a high level of physical activity and food was only available intermittently, a genetic tendency favoring storage of excess calories as fat may have had a survival value. Today, however, the sedentary lifestyle and abundance and wide variety of palatable, inexpensive foods in industrialized societies has undoubtedly contributed to an obesity epidemic. As adiposity has increased, so has the risk of developing associated diseases, such as type 2 diabetes (T2D), cardiovascular disease (CVD), hypertension, cancer, and arthritis. Particularly alarming is the explosion of obesity in children and adolescents, which has shown a threefold increase in prevalence over the last four decades. [Note: Approximately 17% of those age 2–19 years are obese.] In the United States, the lifetime risk of becoming overweight or obese is ~50% and

1	increase in prevalence over the last four decades. [Note: Approximately 17% of those age 2–19 years are obese.] In the United States, the lifetime risk of becoming overweight or obese is ~50% and 25%, respectively. Obesity has increased globally, and, by some estimates, there are more obese than undernourished individuals worldwide.

1	II. ASSESSMENT Because the amount of body fat is difficult to measure directly, it is usually determined from an indirect measure, the body mass index (BMI), which has been shown to correlate with the amount of body fat in most individuals. [Note: Exceptions are athletes who have large amounts of lean muscle mass.] Measuring the waist size with a tape measure is also used to screen for obesity, because this measurement reflects the amount of fat in the central abdominal area of the body. The presence of excess central fat is associated with an increased risk for morbidity and mortality, independent of the BMI. [Note: A waist size ≥40 in (men) and ≥35 in (women) is considered a risk factor.] A. Body mass index

1	A. Body mass index The BMI (defined as weight in kg/[height in m]2) provides a measure of relative weight, adjusted for height. This allows comparisons within and between populations. The healthy range for the BMI is between 18.5 and 24.9. Individuals with a BMI between 25 and 29.9 are considered overweight, those with a BMI ≥30 are defined as obese, and a BMI >40 is considered severely (morbidly) obese (Fig. 26.1). These cutoffs are based on studies examining the relationship of BMI to premature death and are similar in men and women. Nearly two thirds of U.S. adults are overweight, and more than one third of those are obese. Children with a BMI-for-age above the 95th percentile are considered obese. B. Anatomic differences in fat deposition

1	B. Anatomic differences in fat deposition The anatomic distribution of body fat has a major influence on associated health risks. A waist/hip ratio (WHR) >0.8 for women and >1.0 for men is defined as android, apple-shaped, or upper-body obesity and is associated with more fat deposition in the trunk (Fig. 26.2A). In contrast, a lower WHR reflects a preponderance of fat distributed in the hips and thighs and is called gynoid, pear-shaped, or lower-body obesity. It is defined as a WHR of <0.8 for women and <1.0 for men. The pear shape, more commonly found in women, presents a much lower risk of metabolic disease, and some studies indicate that it may actually be protective. Thus, the clinician can use simple indices of body shape to identify those who may be at higher risk for metabolic diseases associated with obesity.

1	About 80%–90% of human body fat is stored in subcutaneous (subq) depots in the abdominal (upper body) and the gluteal-femoral (lower body) regions. The remaining 10%–20% is in visceral depots located deep within the abdominal cavity (Fig. 26.2B). Excess fat in visceral and abdominal subq stores increases health risks associated with obesity. C. Biochemical differences in regional fat depots

1	C. Biochemical differences in regional fat depots The regional types of fat described above are biochemically different. Subq adipocytes from the lower body, particularly in women, are larger, very efficient at fat (triacylglycerol [TAG]) deposition, and tend to mobilize fatty acids (FA) more slowly than subq adipocytes from the upper body. Visceral adipocytes are the most metabolically active. In obese individuals, both abdominal subcutaneous and visceral depots have high rates of lipolysis and contribute to increased availability of free fatty acids (FFA). These metabolic differences may contribute to the higher health risk found in individuals with upper body (abdominal) obesity. [Note: FFA impair insulin signaling and are proinflammatory (see p. 343).] 1.

1	Endocrine function: White adipose tissue, once thought to be a passive reservoir of TAG, is now known to play an active role in body weight regulatory systems. For example, the adipocyte is an endocrine cell that secretes a number of protein regulators (adipokines), such as the hormones leptin and adiponectin. Leptin regulates appetite as well as metabolism (see p. 352). Adiponectin reduces FFA levels in the blood (by increasing FA oxidation in muscles) and has been associated with improved lipid profiles, increased insulin sensitivity resulting in better glycemic control, and reduced inflammation in patients with diabetes. [Note: Adiponectin levels decrease as body weight increases, whereas leptin levels increase.] 2.

1	Importance of portal circulation: With obesity, there is increased release of FFA and secretion of proinflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α), from adipose tissue. [Note: Cytokines are small proteins that regulate the immune system.] One hypothesis for why abdominal adipose depots have such a large influence on metabolic dysfunction in obesity is that the FFA and cytokines released from these depots enter the portal vein and, therefore, have direct access to the liver. In the liver, they may lead to insulin resistance (see p. 343) and increased hepatic synthesis of TAG, which are released as components of very-low-density lipoprotein particles and contribute to the hypertriacylglycerolemia associated with obesity. By contrast, FFA from lower body subq adipose depots enter the general circulation, where they can be oxidized in muscle and, therefore, reach the liver in lower concentration. D. Adipocyte size and number

1	As TAG are stored, adipocytes can expand to an average of two to three times their normal volume (Fig. 26.3). However, the ability of fat cells to expand is limited. With prolonged overnutrition, preadipocytes within adipose tissue are stimulated to proliferate and differentiate into mature fat cells, increasing the number of adipocytes. Thus, most obesity is due to a combination of increased fat cell size (hypertrophy) and number (hyperplasia). Obese individuals can have up to five times the normal number of adipocytes. [Note: Like other tissues, the adipose tissue undergoes continuous remodeling. Contrary to early dogma, we now know that adipocytes can die. The estimated average lifespan of an adipocyte is 10 years.] If excess calories cannot be accommodated within adipose tissue, the excess FA “spill over” into other tissues, such as muscle and the liver. The amount of this ectopic fat is strongly associated with insulin resistance. With weight loss in an obese individual, the size

1	FA “spill over” into other tissues, such as muscle and the liver. The amount of this ectopic fat is strongly associated with insulin resistance. With weight loss in an obese individual, the size of the fat cells is reduced, but the number is not usually affected. Thus, a normal amount of body fat is achieved by decreasing the size of the fat cell below normal. However, small fat cells are very efficient at reaccumulating fat, and this may drive appetite and weight regain.

1	III. BODY WEIGHT REGULATION The body weight of most individuals tends to be relatively stable over time. This observation prompted the hypothesis that each individual has a biologically predetermined “set point” for body weight. The body attempts to add to adipose stores when the body weight falls below the set point and to lose adipose from stores when the body weight rises above the set point. Thus, the body defends the set point. For example, with weight loss, appetite increases and energy expenditure falls, whereas with overfeeding, appetite falls and energy expenditure may slightly increase (Fig. 26.4). However, a strict set point model explains neither why some individuals fail to revert to their starting weight after a period of overeating nor the current epidemic of obesity. A. Genetic contributions It is now evident that genetic mechanisms play a major role in determining body weight. 1.

1	A. Genetic contributions It is now evident that genetic mechanisms play a major role in determining body weight. 1. Biologic origin: The importance of genetics as a determinant of obesity is indicated by the observation that children who are adopted usually show a body weight that correlates with their biologic rather than adoptive parents. Furthermore, identical twins have very similar BMI (Fig. 26.5), whether reared together or apart, and their BMI are more similar than those of nonidentical, dizygotic twins. 2.

1	2. Mutations: Rare, single gene mutations can cause human obesity. For example, mutations in the gene for leptin (causing decreased production) or its receptor (decreased function) result in hyperphagia (increased appetite for and consumption of food) and severe obesity (Fig. 26.6), underscoring the importance of the leptin system in regulating human body weight (see IV below). [Note: Most obese humans have elevated leptin levels but are resistant to the appetite-regulating effects of this hormone.] B. Environmental and behavioral contributions

1	The epidemic of obesity occurring over the last several decades cannot be simply explained by changes in genetic factors, which are stable on this short time scale. Clearly, environmental factors, such as the ready availability of palatable, energy-dense foods, play a role. Furthermore, sedentary lifestyles decrease physical activity and enhance the tendency to gain weight. Eating behaviors, such as portion size, variety of foods consumed, an individual’s food preferences, and the number of people present during eating, also influence food consumption. However, it is important to note that many individuals in this same environment do not become obese. The susceptibility to obesity appears to be explained, at least in part, by an interaction of an individual’s genes and his or her environment and can be influenced by additional factors such as maternal under-or overnutrition that may “set” the body regulatory systems to defend a higher or lower level of body fat. Thus, epigenetic

1	and can be influenced by additional factors such as maternal under-or overnutrition that may “set” the body regulatory systems to defend a higher or lower level of body fat. Thus, epigenetic changes (see p.

1	476) likely influence the risk for obesity. IV. MOLECULAR INFLUENCES The cause of obesity can be summarized in a deceptively simple application of the first law of thermodynamics: Obesity results when energy (caloric) intake exceeds energy expenditure. However, the mechanism underlying this imbalance involves a complex interaction of biochemical, neurologic, environmental, and psychologic factors. The basic neural and humoral pathways that regulate appetite, energy expenditure, and body weight involve systems that regulate short-term food intake (meal to meal), and signals for the long-term (day to day, week to week, year to year) regulation of body weight (Fig. 26.7). and overnourished (B) states. CCK = cholecystokinin; PYY = peptide YY. A. Long-term signals reflect the status of fat (TAG) stores. 1.

1	and overnourished (B) states. CCK = cholecystokinin; PYY = peptide YY. A. Long-term signals reflect the status of fat (TAG) stores. 1. Leptin: Leptin is an adipocyte peptide hormone that is made and secreted in proportion to the size of fat stores. It acts on the brain to regulate food intake and energy expenditure. When we consume more calories than we need, body fat increases, and leptin production by adipocytes increases. The body adapts by increasing energy use (increasing activity) and decreasing appetite (an anorexigenic effect). When body fat decreases, the opposite effects occur. Unfortunately, most obese individuals are leptin resistant, and the leptin system may be better at preventing weight loss than preventing weight gain. [Note: Leptin’s effects are mediated through binding to receptors in the arcuate nucleus of the hypothalamus.] 2.

1	Insulin: Obese individuals are also hyperinsulinemic. Like leptin, insulin acts on hypothalamic neurons to dampen appetite. (See Chapter 23 for the effects of insulin on metabolism.) [Note: Obesity is associated with insulin resistance (see p. 342).] B. Short-term signals

1	Short-term signals from the gastrointestinal (GI) tract control hunger and satiety, which affect the size and number of meals over a time course of minutes to hours. In the absence of food intake (between meals), the stomach produces ghrelin, an orexigenic (appetite-stimulating) hormone that drives hunger. As food is consumed, GI hormones, including cholecystokinin and peptide YY, among others, induce satiety (an anorexigenic effect), thereby terminating eating, through actions on gastric emptying and neural signals to the hypothalamus. Within the hypothalamus, neuropeptides (such as orexigenic neuropeptide Y [NPY] and anorexigenic α-melanocyte–stimulating hormone [α-MSH]) and neurotransmitters (such as anorexigenic serotonin and dopamine) are important in regulating hunger and satiety. Long-term and short-term signals interact, insofar as leptin increases secretion of α-MSH and decreases secretion of NPY. Thus, there are many complex regulatory loops that control the size and number

1	and short-term signals interact, insofar as leptin increases secretion of α-MSH and decreases secretion of NPY. Thus, there are many complex regulatory loops that control the size and number of meals in relationship to the status of body fat stores. [Note: α-MSH, a cleavage product of proopiomelanocortin, binds to the melanocortin-4 receptor (MC4R). Loss-of-function mutations to MC4R are associated with early-onset obesity.]

1	V. METABOLIC EFFECTS The primary metabolic effects of obesity include dyslipidemias, glucose intolerance, and insulin resistance expressed primarily in the liver, skeletal muscle, and adipose tissue. These metabolic abnormalities reflect molecular signals originating from the increased mass of adipocytes (see Fig. 25.9, p. 343, and Fig. 26.7). [Note: About 30% of obese individuals do not show these metabolic abnormalities.] A. Metabolic syndrome

1	A. Metabolic syndrome Abdominal obesity is associated with a cluster of metabolic abnormalities (hyperglycemia, insulin resistance, hyperinsulinemia, dyslipidemia [low levels of high-density lipoprotein (HDL) and elevated TAG], and hypertension) that is referred to as the metabolic syndrome (Fig. 26.8). It is a risk factor for CVD and T2D. The low-grade, chronic, systemic inflammation seen with obesity contributes to the pathogenesis of insulin resistance and T2D and likely plays a role in metabolic syndrome. In obesity, adipocytes release proinflammatory mediators such as IL-6 and TNF-α. Additionally, levels of adiponectin, which normally dampens inflammation and sensitizes tissues to insulin, are low. B. Nonalcoholic liver disease Obesity is associated with ectopic deposition of TAG in the liver (hepatic steatosis) and results in increased risk for nonalcoholic fatty liver disease ([NAFLD], see p. 343). VI. OBESITY AND HEALTH

1	VI. OBESITY AND HEALTH Obesity is correlated with an increased risk of death (Fig. 26.9) and is a risk factor for a number of chronic conditions, including T2D, dyslipidemias, hypertension, CVD, some cancers, gallstones, arthritis, gout, pelvic floor disorders (for example, urinary incontinence), NAFLD, and sleep apnea. The relationship between obesity and associated morbidities is stronger among individuals age <55 years. After age 74 years, there is no longer an association between increased BMI and mortality. [Note: Obesity also has social consequences (for example, stigmatization and discrimination).] Weight loss in obese individuals leads to decreased blood pressure, plasma TAG, and blood glucose levels. HDL increase. VII. WEIGHT REDUCTION

1	VII. WEIGHT REDUCTION Weight reduction can help reduce the complications of obesity. To achieve weight reduction, the obese patient must decrease energy intake or increase energy expenditure, although decreasing energy intake is thought to contribute more to inducing weight loss. Typically, a plan for weight reduction combines dietary change; increased physical activity; and behavioral modification, which can include nutrition education and meal planning, recording food intake through food diaries, modifying factors that lead to overeating, and relearning cues to satiety. Medications or surgery may be recommended. Once weight loss is achieved, weight maintenance is a separate process that requires vigilance because the majority of patients regain weight after they stop their weight-loss efforts. B. Caloric restriction

1	B. Caloric restriction Dieting is the most commonly practiced approach to weight control. Because 1 lb of adipose tissue corresponds to ~3,500 kcal, the effect that caloric restriction will have on the amount of adipose tissue can be estimated. Weight loss on calorie-restricted diets is determined primarily by caloric intake and not nutrient composition. [Note: However, compositional aspects can affect glycemic control and the blood lipid profile.] Caloric restriction is ineffective over the long term for many individuals. Over 90% of people who attempt to lose weight regain the lost weight when dietary intervention is suspended. Nonetheless, although few individuals will reach their ideal weight with treatment, weight losses of 10% of body weight over a 6-month period often reduce blood pressure and lipid levels and enhance control of T2D. A. Physical activity

1	A. Physical activity An increase in physical activity can create an energy deficit. Although adding exercise to a hypocaloric regimen may not produce a greater weight loss initially, exercise is a key component of programs directed at maintaining weight loss. In addition, physical activity increases cardiopulmonary fitness and reduces the risk of CVD, independent of weight loss. Persons who combine caloric restriction and exercise with behavioral treatment may expect to lose ~5%–10% of initial body weight over a period of 4–6 months. Studies show that individuals who maintain their exercise program regain less weight after their initial weight loss. C. Pharmacologic treatment

1	C. Pharmacologic treatment The U.S. Food and Drug Administration has approved several weight-loss medications for use in adults. They include orlistat (decreases absorption of dietary fat), lorcaserin and phentermine in combination with topiramate (promote satiety through serotonin signaling), liraglutide (decreases appetite by activating the glucagon-like peptide 1 receptor), and buproprion in combination with naltrexone (increase metabolism by increasing norepinephrine). Their effects on weight reduction tend to be modest. [Note: Pharmacologic activation of brown adipocytes (see p. 79) is being explored.] D. Surgical treatment

1	D. Surgical treatment Gastric bypass and restriction surgeries are effective in causing weight loss in severely obese individuals. Through mechanisms that remain poorly understood, these operations greatly improve glycemic control in morbidly obese diabetic individuals. [Note: Implantation of a device that electrically stimulates the vagus nerve to decrease food intake has been approved.] VIII. CHAPTER SUMMARY

1	Obesity, the accumulation of excess body fat, results when energy (caloric) intake exceeds energy expenditure (Fig. 26.10). Obesity is increasing in industrialized countries because of a reduction in daily energy expenditure and an increase in energy intake resulting from the increasing availability of palatable, inexpensive foods. The body mass index (BMI) is easy to determine and highly correlated to body fat. Nearly 69% of U.S. adults are overweight (BMI ≥25), and >33% of this group are obese (BMI ≥30). The anatomic distribution of body fat has a major influence on associated health risks. Excess fat located in the abdomen (upper body, apple shape), as reflected in waist size, is associated with greater risk for hypertension, insulin resistance, diabetes, dyslipidemia, and coronary heart disease as compared to fat located in the hips and thighs (lower body, pear shape). A person’s weight is determined by genetic and environmental factors. Appetite is influenced by afferent, or

1	heart disease as compared to fat located in the hips and thighs (lower body, pear shape). A person’s weight is determined by genetic and environmental factors. Appetite is influenced by afferent, or incoming, signals (that is, neural signals, circulating hormones such as leptin, and metabolites) that are integrated by the hypothalamus. These diverse signals prompt release of hypothalamic peptides (such as neuropeptide Y and α-melanocyte– stimulating hormone) and activate outgoing, efferent neural signals. Obesity is correlated with an increased risk of death and is also a risk factor for a number of chronic conditions. Weight reduction is achieved best with negative energy balance, that is, by decreasing caloric intake and increasing physical activity. Virtually all diets that limit particular groups of foods or macronutrients lead to short-term weight loss. Long-term maintenance of weight loss is difficult to achieve. Modest reduction in food intake occurs with pharmacologic

1	particular groups of foods or macronutrients lead to short-term weight loss. Long-term maintenance of weight loss is difficult to achieve. Modest reduction in food intake occurs with pharmacologic treatment. Surgical procedures, such as gastric bypass, designed to limit food intake are an option for the severely obese patient who has not responded to other treatments.

1	Choose the ONE best answer. For Questions 26.1 and 26.2, use the following scenario. A 40-year-old woman, 5 ft, 1 in (155 cm) tall and weighing 188 lb (85.5 kg), seeks your advice on how to lose weight. Her waist measured 41 in and her hips 39 in. The remainder of the physical examination and the blood laboratory data were all within the normal range. Her only child (who is age 14 years), her sister, and both of her parents are overweight. The patient recalls being overweight throughout her childhood and adolescence. Over the past 15 years, she had been on seven different diets for periods of 2 weeks to 3 months, losing from 5 to 25 lb each time. On discontinuation of the diets, she regained weight, returning to 185–190 lb. 6.1. Calculate and interpret the body mass index for the patient. Body mass index (BMI) = weight in kg/(height in m)2 = 85.5/1.552 = 35.6. Because her BMI is >30, the patient is classified as obese.

1	Body mass index (BMI) = weight in kg/(height in m)2 = 85.5/1.552 = 35.6. Because her BMI is >30, the patient is classified as obese. 6.2. Which one of the following statements best describes the patient? A. She has approximately the same number of adipocytes as an individual of normal weight, but each adipocyte is larger. B. She shows an apple pattern of fat distribution. C. She would be expected to show higher-than-normal levels of adiponectin. D. She would be expected to show lower-than-normal levels of circulating leptin. E. She would be expected to show lower-than-normal levels of circulating triacylglycerols.

1	Correct answer = B. Her waist/hip ratio (WHR) is 1.05 (41/39). Apple shape is defined as a WHR of >0.8 for women and >1.0 for men. Therefore, she has an apple pattern of fat distribution, more commonly seen in males. Compared with other women of the same body weight who have a gynoid (pear-shaped) fat pattern, her android fat pattern places her at greater risk for diabetes, hypertension, dyslipidemia, and coronary heart disease. Individuals with marked obesity and a history dating to early childhood have a fat depot made up of too many adipocytes, each fully loaded with triacylglycerol (TAG). Plasma leptin levels are proportional to fat mass, suggesting that resistance to leptin, rather than its deficiency, occurs in human obesity. Adiponectin levels decrease with increasing fat mass. The elevated circulating free fatty acids characteristic of obesity are carried to the liver and converted to TAG. The TAG are released as components of very-low-density lipoproteins, resulting in

1	The elevated circulating free fatty acids characteristic of obesity are carried to the liver and converted to TAG. The TAG are released as components of very-low-density lipoproteins, resulting in elevated plasma TAG levels, or are stored in the liver, resulting in hepatic steatosis.

1	Nutrition: Overview and Macronutrients For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW Nutrients are the constituents of food necessary to sustain the normal functions of the body. All energy (calories) is provided by three classes of nutrients: fats, carbohydrates, and protein (Fig. 27.1). Because the intake of these energy-rich molecules is larger (g amounts) than that of the other dietary nutrients, they are called macronutrients. This chapter focuses on the kinds and amounts of macronutrients that are needed to maintain optimal health and prevent chronic disease. Those nutrients needed in lesser amounts (mg or µg), vitamins and minerals, are called micronutrients and are considered in Chapters 28 and 29. II. DIETARY REFERENCE INTAKES

1	II. DIETARY REFERENCE INTAKES Committees of U.S. and Canadian experts organized by the Food and Nutrition Board of the Institute of Medicine of the National Academy of Sciences have compiled Dietary Reference Intakes (DRI), which are estimates of the amounts of nutrients required to prevent deficiencies and maintain optimal health and growth. The DRI expands on the Recommended Dietary Allowances (RDA), which have been published with periodic revisions since 1941. Unlike the RDA, the DRI establishes upper limits on the consumption of some nutrients and incorporates the role of nutrients in lifelong health, going beyond deficiency diseases. Both the DRI and the RDA refer to long-term average daily nutrient intakes, because it is not necessary to consume the full RDA every day. A. Definition The DRI consists of four dietary reference standards for the intake of nutrients designated for specific life stage (age) groups, physiologic states, and gender (Fig. 27.2). 1.

1	A. Definition The DRI consists of four dietary reference standards for the intake of nutrients designated for specific life stage (age) groups, physiologic states, and gender (Fig. 27.2). 1. Estimated average requirement: The average daily nutrient intake level estimated to meet the requirement of one half of the healthy individuals in a particular life stage and gender group is the Estimated Average Requirement (EAR). It is useful in estimating the actual requirements in groups and individuals. 2.

1	2. Recommended dietary allowance: The RDA is the average daily nutrient intake level that is sufficient to meet the requirements of nearly all (97%– 98%) individuals in a particular life stage and gender group. The RDA is not the minimal requirement for healthy individuals, but it is intentionally set to provide a margin of safety for most individuals. The EAR serves as the foundation for setting the RDA. If the standard deviation (SD) of the EAR is available and the requirement for the nutrient is normally distributed, the RDA is set at 2 SD above the EAR (that is, RDA = EAR + 2 SDEAR). 3.

1	3. Adequate intake: An Adequate Intake (AI) is set instead of an RDA if sufficient scientific evidence is not available to calculate an EAR or RDA. The AI is based on estimates of nutrient intake by a group (or groups) of apparently healthy people. For example, the AI for young infants, for whom human milk is the recommended sole source of food for the first 6 months, is based on the estimated daily mean nutrient intake supplied by human milk for healthy, full-term infants who are exclusively breast-fed. 4.

1	4. Tolerable upper intake level: The highest average daily nutrient intake level that is likely to pose no risk of adverse health effects to almost all individuals in the general population is the Tolerable Upper Intake Level (UL, or TUL). As intake increases above the UL, the potential risk of adverse effects may increase. The UL is useful because of the increased availability of fortified foods and the increased use of dietary supplements. For some nutrients, there may be insufficient data on which to develop a UL. B. Using the dietary reference intakes

1	B. Using the dietary reference intakes Most nutrients have a set of DRI (Fig. 27.3). Usually a nutrient has an EAR and a corresponding RDA. Most are set by age and gender and may be influenced by special factors, such as pregnancy and lactation in women (see p. 372). When the data are not sufficient to estimate an EAR (or an RDA), an AI is designated. Intakes below the EAR need to be improved because the probability of adequacy is ≤50% (Fig. 27.4). Intakes between the EAR and RDA likely need to be improved because the probability of adequacy is <98%, and intakes at or above the RDA can be considered adequate. Intakes above the AI can be considered adequate. Intakes between the UL and the RDA can be considered to have no risk for adverse effects. [Note: Because the DRI is designed to meet the nutritional needs of the healthy, it does not include any special needs of the sick.] III. ENERGY REQUIREMENT IN HUMANS

1	III. ENERGY REQUIREMENT IN HUMANS The Estimated Energy Requirement (EER) is the average dietary energy intake predicted to maintain an energy balance (that is, the calories consumed are equal to the energy expended) in a healthy adult of a defined age, gender, and height whose weight and level of physical activity are consistent with good health. Differences in the genetics, body composition, metabolism, and behavior of individuals make it difficult to accurately predict a person’s caloric requirements. However, some simple approximations can provide useful estimates. For example, sedentary adults require ~30 kcal/kg/day to maintain body weight, moderately active adults require 35 kcal/kg/day, and very active adults require 40 kcal/kg/day. A. Energy content of food

1	A. Energy content of food The energy content of food is calculated from the heat released by the total combustion of food in a calorimeter. It is expressed in kilocalories (kcal, or Cal). The standard conversion factors for determining the metabolic caloric value of fat, protein, and carbohydrate are shown in Figure 27.5. Note that the energy content of fat is more than twice that of carbohydrate or protein, whereas the energy content of ethanol is intermediate between those of fat and carbohydrate. [Note: The joule (J) is a unit of energy widely used in countries other than the United States. One cal = 4.2 J; 1 kcal (1 Cal, 1 food calorie) = 4.2 kJ. For uniformity, many scientists are promoting the use of joules rather than calories in the United States. However, kcal still predominates and is used throughout this text.] Figure27.5Averageenergyavailablefromthemacronutrientsandalcohol. B. Use of food energy in the body

1	Figure27.5Averageenergyavailablefromthemacronutrientsandalcohol. B. Use of food energy in the body The energy generated by metabolism of the macronutrients is used for three energy-requiring processes that occur in the body: resting metabolic rate (RMR), physical activity, and the thermic effect of food. The number of kcal expended by these processes in a 24-hour period is the total energy expenditure (TEE).

1	1. Resting metabolic rate: RMR is the energy expended by an individual in a resting, postabsorptive state. It represents the energy required to carry out the normal body functions, such as respiration, blood flow, and ion transport. RMR can be determined by measuring oxygen (O2) consumed or carbon dioxide (CO2) produced (indirect calorimetry). [Note: The ratio of CO2 to O2 is the respiratory quotient (RQ). It reflects the substrate being oxidized for energy (Fig. 27.6).] RMR also can be estimated using equations that include sex and age (RMR reflects lean muscle mass, which is highest in men and the young) as well as height and weight. A commonly used rough estimate is 1 kcal/kg/hour for men and 0.9 kcal/kg/hour for women. [Note: A basal metabolic rate (BMR) can be determined if more stringent environmental conditions are used, but it is not routinely done. RMR is ~10% higher than the BMR.] In an adult, the 24-hour RMR, known as the resting energy expenditure (REE), is ~1,800 kcal for

1	environmental conditions are used, but it is not routinely done. RMR is ~10% higher than the BMR.] In an adult, the 24-hour RMR, known as the resting energy expenditure (REE), is ~1,800 kcal for men (70 kg) and 1,300 kcal for women (50 kg). From 60%–75% of the TEE in sedentary individuals is attributable to the REE (Fig. 27.7). [Note: Hospitalized individuals are commonly hypercatabolic, and the RMR is multiplied by an injury factor that ranges from 1.0 (mild infection) to 2.0 (severe burns) in calculating their TEE.] 2.

1	Physical activity: Muscular activity provides the greatest variation in the TEE. The amount of energy consumed depends on the duration and intensity of the exercise. This energy cost is expressed as a multiple of the RMR (range is 1.1 to >8.0) that is referred to as the physical activity ratio (PAR) or the metabolic equivalent of the task (MET). In general, a lightly active person requires ~30%–50% more calories than the RMR (see Fig. 27.7), whereas a highly active individual may require ≥100% calories above the RMR. 3. Thermic effect of food: The production of heat by the body increases as much as 30% above the resting level during the digestion and absorption of food. This is called the thermic effect of food, or diet-induced thermogenesis. The thermic response to food intake may amount to 5%– 10% of the TEE. IV. ACCEPTABLE MACRONUTRIENT DISTRIBUTION RANGES

1	IV. ACCEPTABLE MACRONUTRIENT DISTRIBUTION RANGES Acceptable Macronutrient Distribution Ranges (AMDR) are defined as a range of intakes for a particular macronutrient that is associated with reduced risk of chronic disease while providing adequate amounts of essential nutrients. The AMDR for adults is 45%–65% of their total calories from carbohydrates, 20%– 35% from fat, and 10%–35% from protein (Fig. 27.8). The biologic properties of dietary fat, carbohydrate, and protein are described below. V. DIETARY FATS The incidence of a number of chronic diseases is significantly influenced by the kinds and amounts of nutrients consumed (Fig. 27.9). Dietary fats most strongly influence the incidence of coronary heart disease (CHD), but evidence linking dietary fat and the risk for cancer or obesity is much weaker.

1	Earlier recommendations emphasized decreasing the total amount of dietary fat. Unfortunately, this resulted in increased consumption of refined grains and added sugars. Data now show that the type of fat is a more important risk factor than the total amount of fat. A. Plasma lipids and coronary heart disease Plasma cholesterol may arise from the diet or from endogenous biosynthesis. In either case, cholesterol is transported between the tissues in combination with protein and phospholipids as lipoproteins.

1	1. Low-density and high-density lipoproteins: The level of plasma cholesterol is not precisely regulated but, rather, varies in response to diet. Elevated levels of total cholesterol (hypercholesterolemia) result in an increased risk for CHD (Fig. 27.10). A much stronger correlation exists between CHD and the level of cholesterol in low-density lipoproteins ([LDL-C] see p. 234). As LDL-C increases, CHD increases. In contrast, elevated levels of high-density lipoprotein cholesterol (HDL C) have been associated with a decreased risk for heart disease (see p. 235). [Note: Elevated plasma triacylglycerol (TAG) is associated with CHD, but a causative relationship has yet to be demonstrated.] Abnormal levels of plasma lipids (dyslipidemias) act in combination with smoking, obesity, sedentary lifestyle, insulin resistance, and other risk factors to increase the risk of CHD.

1	2. Benefits of lowering plasma cholesterol: Dietary or drug treatment of hypercholesterolemia has been shown to be effective in decreasing LDLC, increasing HDL-C, and reducing the risk for cardiovascular events. The diet-induced changes in plasma cholesterol concentrations are modest, typically 10%–20%, whereas treatment with statin drugs decreases plasma cholesterol by 30%–60% (see p. 224). [Note: Dietary and drug treatment can also lower TAG.] B. Dietary fats and plasma lipids TAG are quantitatively the most important class of dietary fats. The influence of TAG on blood lipids is determined by the chemical nature of their constituent fatty acids. The absence or presence and number of double bonds (saturated versus mono-and polyunsaturated), the location of the double bonds (ω-6 versus ω-3), and the cis versus trans configuration of the unsaturated fatty acids are the most important structural features that influence blood lipids.

1	1. Saturated fats: TAG composed primarily of fatty acids whose hydrocarbon chains do not contain any double bonds are referred to as saturated fats. Consumption of saturated fats is positively associated with high levels of total plasma cholesterol and LDL-C and an increased risk of CHD. The main sources of saturated fatty acids are dairy and meat products and some vegetable oils, such as coconut and palm oils (a major source of fat in Latin America and Asia, although not in the United States). Many experts strongly advise limiting intake of saturated fats to <10% of total caloric intake and replacing them with unsaturated fats (and whole grains). Saturated fatty acids with carbon chain lengths of 14 (myristic) and 16 (palmitic) are most potent in increasing the plasma cholesterol level. Stearic acid (18 carbons, found in many foods including chocolate) has little effect on blood cholesterol.

1	2. Monounsaturated fats: TAG containing primarily fatty acids with one double bond are referred to as monounsaturated fats. Monounsaturated fatty acids (MUFA) are generally obtained from plant-based oils. When substituted for saturated fatty acids in the diet, MUFA lower both total plasma cholesterol and LDL-C and maintain or increase HDL-C. This ability of MUFA to favorably modify lipoprotein levels may explain, in part, the observation that Mediterranean cultures, with diets rich in olive oil (high in monounsaturated oleic acid), show a low incidence of CHD. [Note: Although there is no AMDR for MUFA, a common recommendation is 10%–20% of caloric intake.] a. The Mediterranean diet: The Mediterranean diet is an example of a diet rich in MUFA (from olive oil) and polyunsaturated fatty acids or PUFA (from fish oils, plant oils, and some nuts) but low in saturated fat. For example, Figure 27.11 shows the composition of the Mediterranean diet in comparison with both a Western diet similar

1	or PUFA (from fish oils, plant oils, and some nuts) but low in saturated fat. For example, Figure 27.11 shows the composition of the Mediterranean diet in comparison with both a Western diet similar to that consumed in the United States and a typical low-fat diet. The Mediterranean diet contains seasonally fresh food, with an abundance of plant material, low amounts of red meat, and olive oil as the principal source of fat. The Mediterranean diet is associated with decreased plasma total cholesterol and LDL-C, decreased TAG, and increased HDL-C when compared with a typical Western diet higher in saturated fats.

1	3. Polyunsaturated fats: TAG containing primarily fatty acids with more than one double bond are referred to as polyunsaturated fats. The effects of PUFA on cardiovascular disease are influenced by the location of the double bonds within the molecule.

1	a. ω-6 Fatty acids: These are long-chain PUFA, with the first double bond beginning at the sixth bond position when starting from the methyl (ω) end of the fatty acid molecule. [Note: They are also called n-6 fatty acids (see p. 182).] Consumption of fats containing ω-6 PUFA, principally linoleic acid (18:2 [9,12]), obtained from vegetable oils, lowers plasma cholesterol when substituted for saturated fats. Plasma LDL-C is lowered, but HDL-C, which protects against CHD, is also lowered, partially offsetting the benefits of lowering LDL-C. Nuts, avocados, olives, soybeans, and various oils, including sunflower and corn oil, are common sources of these fatty acids. The AMDR for linoleic acid is 5%–10%. [Note: The lower recommendation for intake of PUFA relative to MUFA is because of concern that free radical– mediated oxidation (peroxidation) of PUFA may lead to deleterious products.] b. ω-3 Fatty acids: These are long-chain PUFA, with the first double bond beginning at the third bond

1	that free radical– mediated oxidation (peroxidation) of PUFA may lead to deleterious products.] b. ω-3 Fatty acids: These are long-chain PUFA, with the first double bond beginning at the third bond position from the methyl (ω) end. Dietary ω-3 PUFA suppress cardiac arrhythmias, reduce plasma TAG, decrease the tendency for thrombosis, lower blood pressure, and substantially reduce risk of cardiovascular mortality (Fig. 27.12), but they have little effect on LDL-C or HDL-C levels. Evidence suggests that they have anti-inflammatory effects. The ω-3 PUFA, principally α-linolenic acid, 18:3(9,12,15), are found in plant oils, such as flaxseed and canola, and some nuts, such as walnuts. The AMDR for αlinolenic acid is 0.6%–1.2%. Fish oil contains the long-chain ω-3 docosahexaenoic acid (DHA, 22:6) and eicosapentaenoic acid (EPA, 20:5). Two fatty fish (for example, salmon) meals per week are recommended. For patients with documented CHD, 1 g/day of fish oils is recommended, while 2–4 g/day is

1	and eicosapentaenoic acid (EPA, 20:5). Two fatty fish (for example, salmon) meals per week are recommended. For patients with documented CHD, 1 g/day of fish oils is recommended, while 2–4 g/day is prescribed to lower TAG. [Note: DHA is included in infant formulas to promote brain development.] Linoleic and α-linolenic acids are essential fatty acids (EFA) required for membrane fluidity and synthesis of eicosanoids (see p. 213). EFA deficiency, caused primarily by fat malabsorption, is characterized by scaly dermatitis as a result of the depletion of skin ceramides with long-chain fatty acids (see p. 206).

1	= eicosapentaenoic acid (20:5); DHA = docosahexaenoic acid (22:6). 4. Trans fatty acids: Trans fatty acids (Fig. 27.13) are chemically classified as unsaturated fatty acids but behave more like saturated fatty acids in the body because they elevate LDL-C and lower HDL-C, thereby increasing the risk of CHD. Trans fatty acids do not occur naturally in plants but occur in small amounts in animals. However, trans fatty acids are formed during the hydrogenation of vegetable oils (for example, in the manufacture of margarine and partially hydrogenated vegetable oil). Trans fatty acids are a major component of many commercial baked goods, such as cookies, and most deep-fried foods. Many manufacturers have reformulated their products to be free of trans fats. In 2006, the U.S. Food and Drug Administration required that Nutrition Facts labels (see p. 370) portray the trans fat content of packaged food. By 2018, virtually no industrial trans fatty acids will be permitted in food.

1	370) portray the trans fat content of packaged food. By 2018, virtually no industrial trans fatty acids will be permitted in food. 5. Dietary cholesterol: Cholesterol is found only in animal products. The effect of dietary cholesterol on plasma cholesterol (Fig. 27.14) is less important than the amount and types of fatty acids consumed. Many experts recommend ≤300 mg/day. However, having an upper limit has become controversial. concentrations to an increase in dietary cholesterol intake. C. Other dietary factors affecting coronary heart disease

1	concentrations to an increase in dietary cholesterol intake. C. Other dietary factors affecting coronary heart disease Moderate consumption of alcohol (up to 1 drink/day for women and up to 2 drinks/day for men) decreases the risk of CHD, because there is a positive correlation between moderate alcohol (ethanol) consumption and the plasma concentration of HDL-C. However, because of the potential dangers of alcohol abuse, health professionals are reluctant to recommend increased alcohol consumption to their patients. Red wine may provide cardioprotective benefits in addition to those resulting from its alcohol content (for example, red wine contains phenolic compounds that inhibit lipoprotein oxidation; see p. 235). [Note: These antioxidants are also present in raisins and grape juice.] Figure 27.15 summarizes the effects of dietary fats. [Note: Recent studies (including meta-analyses) have raised questions concerning the current guidelines for dietary fat in the prevention of CHD.]

1	VI. DIETARY CARBOHYDRATES The primary role of dietary carbohydrates is to provide energy. Although self-reported caloric intake in the United States peaked in 2003 and is now declining, the incidence of obesity has dramatically increased (see p. 349). During this same period, carbohydrate consumption has significantly increased (as fat consumption decreased), leading some observers to link obesity with carbohydrate consumption. However, obesity has also been related to increasingly inactive lifestyles and to calorie-dense foods served in expanded portion size. Carbohydrates are not inherently fattening. A. Classification Dietary carbohydrates are classified as simple sugars (monosaccharides and disaccharides), complex sugars (polysaccharides), and fiber.

1	A. Classification Dietary carbohydrates are classified as simple sugars (monosaccharides and disaccharides), complex sugars (polysaccharides), and fiber. 1. Monosaccharides: Glucose and fructose are the principal monosaccharides found in food. Glucose is abundant in fruits, sweet corn, corn syrup, and honey. Free fructose is found together with free glucose in honey and fruits (for example, apples).

1	a. High-fructose corn syrup: High-fructose corn syrups (HFCS) are corn syrups that have undergone enzymatic processing to convert their glucose into fructose and have then been mixed with pure corn syrup (100% glucose) to produce a desired sweetness. In the United States, HFCS 55 (containing 55% fructose and 42% glucose) is commonly used as a substitute for sucrose in beverages, including soft drinks, with HFCS 42 used in processed foods. The composition and metabolism of HFCS and sucrose are similar, the major difference being that HFCS is ingested as a mixture of monosaccharides (Fig. 27.16). Most studies have shown no significant difference between sucrose and HFCS meals in either postprandial glucose or insulin responses. [Note: The rise in the use of HFCS parallels the rise in obesity, but a causal relationship has not been demonstrated.] (B) leads to absorption of glucose plus fructose. 2.

1	2. Disaccharides: The most abundant disaccharides are sucrose (glucose + fructose), lactose (glucose + galactose), and maltose (glucose + glucose). Sucrose is ordinary table sugar and is abundant in molasses and maple syrup. Lactose is the principal sugar found in milk. Maltose is a product of enzymic digestion of polysaccharides. It is also found in significant quantities in beer and malt liquors. The term “sugar” refers to monosaccharides and disaccharides. “Added sugars” are those sugars and syrups (such as HFCS) added to foods during processing or preparation. 3. Polysaccharides: Complex carbohydrates are polysaccharides (most often polymers of glucose) that do not have a sweet taste. Starch is an example of a complex carbohydrate that is found in abundance in plants. Common sources include wheat and other grains, potatoes, dried peas and beans (legumes), and vegetables. 4.

1	Fiber: Dietary fiber is defined as the nondigestible, nonstarch carbohydrates and lignin (a noncarbohydrate polymer of aromatic alcohols) present intact in plants. Soluble fiber is the edible part of plants that is resistant to digestion and absorption in the human small intestine but is completely or partially fermented by bacteria to short-chain fatty acids in the large intestine. Insoluble fiber passes through the digestive track largely unchanged. Dietary fiber provides little energy but has several beneficial effects. First, it adds bulk to the diet (Fig. 27.17). Fiber can absorb 10–15 times its own weight in water, drawing fluid into the lumen of the intestine and increasing bowel motility and promoting bowel movements (laxation). Soluble fiber delays gastric emptying and can result in a sensation of fullness (satiety). This delayed emptying also results in reduced spikes in blood glucose following a meal. Second, consumption of soluble fiber has been shown to lower LDL-C levels

1	in a sensation of fullness (satiety). This delayed emptying also results in reduced spikes in blood glucose following a meal. Second, consumption of soluble fiber has been shown to lower LDL-C levels by increasing fecal bile acid excretion and interfering with bile acid reabsorption (see p. 225). For example, diets rich (25–50 g/day) in the soluble fiber oat bran are associated with a modest, but significant, reduction in risk for CHD by lowering total cholesterol and LDL-C levels. Also, fiber-rich diets decrease the risk for constipation, hemorrhoids, and diverticulosis. The AI for dietary fiber is 25 g/day for women and 38 g/day for men. However, most American diets are far lower in fiber at ~15 g/day. [Note: “Functional fiber” is the term used for isolated fiber that has proven health benefits such as commercially available fiber supplements.]

1	B. Dietary carbohydrate and blood glucose Some carbohydrate-containing foods produce a rapid rise followed by a steep fall in blood glucose concentration, whereas others result in a gradual rise followed by a slow decline (Fig. 27.18). Thus, they differ in their glycemic response (GR). [Note: Fiber blunts the GR.] The glycemic index (GI) ranks carbohydrate-rich foods on a scale of 0–100 based on the GR they cause relative to the GR caused by the same amount (50 g) of carbohydrate eaten in the form of white bread or glucose. A low GI is <55, whereas a high GI is ≥70. Evidence suggests that a low-GI diet improves glycemic control in diabetic individuals. Food with a low GI tends to create a sense of satiety over a longer period of time and may be helpful in limiting caloric intake. [Note: How much a typical serving size of a food raises blood glucose is referred to as the glycemic load (GL). A food (for example, carrots) can have a high GI and a low GL.] C. Carbohydrate requirements

1	C. Carbohydrate requirements Carbohydrates are not essential nutrients, because the carbon skeletons of most amino acids can be converted into glucose (see p. 261). However, the absence of dietary carbohydrate leads to ketogenesis (see p. 195) and degradation of body protein whose constituent amino acids provide carbon skeletons for gluconeogenesis (see p. 118). The RDA for carbohydrate is set at 130 g/day for adults and children, based on the amount of glucose used by carbohydrate-dependent tissues, such as the brain and erythrocytes. However, this level of intake is usually exceeded. Adults should consume 45%–65% of their total calories from carbohydrates. It is now recommended that added sugars represent no more than 10% of total energy intake because of concerns that they may displace nutrient-rich foods from the diet. [Note: Added sugars are associated with increased body weight and type 2 diabetes.] D. Simple sugars and disease

1	D. Simple sugars and disease There is no direct evidence that the consumption of simple sugars naturally present in food is harmful. Contrary to folklore, diets high in sucrose do not lead to diabetes or hypoglycemia. Also contrary to popular belief, carbohydrates are not inherently fattening. They yield 4 kcal/g (the same as protein and less than one half that of fat; see Fig. 27.5) and result in fat synthesis only when consumed in excess of the body’s energy needs. However, there is an association between sucrose consumption and dental caries, particularly in the absence of fluoride treatment (see p. 405). VII. DIETARY PROTEIN The AMDR for protein is 10%–35%. Dietary protein provides the essential amino acids (see Fig. 20.2, p. 262). Nine of the 20 amino acids needed for the synthesis of body proteins are essential (that is, they cannot be synthesized in humans). A. Protein quality

1	A. Protein quality The quality of a dietary protein is a measure of its ability to provide the essential amino acids (EAA) required for tissue maintenance. Most government agencies have adopted the Protein Digestibility–Corrected Amino Acid Score (PDCAAS) as the standard by which to evaluate protein quality. PDCAAS is based on the profile of EAA after correcting for the digestibility of the protein. The highest possible score under these guidelines is 1.00. This amino acid score provides a method to balance intakes of poorer-quality proteins with high-quality dietary proteins. 1.

1	1. Proteins from animal sources: Proteins from animal sources (meat, poultry, milk, and fish) have a high quality because they contain all the EAA in proportions similar to those required for synthesis of human tissue proteins (Fig. 27.19), and they are more readily digested. [Note: Gelatin prepared from animal collagen is an exception. It has a low biologic value as a result of deficiencies in several EAA.] 2. Proteins from plant sources: Plant proteins have a lower quality than do animal proteins. However, proteins from different plant sources may be combined in such a way that the result is equivalent in nutritional value to animal protein. For example, wheat (lysine deficient but methionine rich) may be combined with kidney beans (methionine poor but lysine rich) to produce a higher biologic value than either of the component proteins (Fig. 27.20). [Note: Animal proteins can also complement the biologic value of plant proteins.] B. Nitrogen balance

1	B. Nitrogen balance Nitrogen balance occurs when the amount of nitrogen consumed equals that of the nitrogen excreted in the urine (primarily as urinary urea nitrogen, or UUN), sweat, and feces. Most healthy adults are normally in nitrogen balance. [Note: There is, on average, 1 g nitrogen in 6.25 g protein.] 1. Positive nitrogen balance: This occurs when nitrogen intake exceeds nitrogen excretion. It is observed during situations in which tissue growth occurs, for example, in childhood, pregnancy, or during recovery from an emaciating illness. 2. Negative nitrogen balance: This occurs when nitrogen loss is greater than nitrogen intake. It is associated with inadequate dietary protein; lack of an essential amino acid; or during physiologic stresses, such as trauma, burns, illness, or surgery.

1	Nitrogen (N) balance (g Nin – g Nout) in a 24-hour period can be determined by the formula, N balance = protein intake in g/6.25 – (UUN + 4 g), where 4 g accounts for urinary loss in forms other than UUN plus loss in skin and feces. C. Protein requirements

1	C. Protein requirements The amount of dietary protein required in the diet varies with its biologic value. The greater the proportion of animal protein in the diet, the less protein is required. The RDA for protein is computed for proteins of mixed biologic value at 0.8 g/kg of body weight for adults, or ~56 g of protein for a 70-kg individual. People who exercise strenuously on a regular basis may benefit from extra protein to maintain muscle mass, and a daily intake of ~1 g/kg has been recommended for athletes. Women who are pregnant or lactating require up to 30 g/day in addition to their basal requirements. To support growth, infants should consume 2 g/kg/day. [Note: Disease states influence protein needs. Protein restriction may be needed in kidney disease, whereas burns require increased protein intake.] 1.

1	Consumption of excess protein: There is no physiologic advantage to the consumption of more protein than the RDA. Protein consumed in excess of the body’s needs is deaminated, and the resulting carbon skeletons are metabolized to provide energy or acetyl coenzyme A for fatty acid synthesis. When excess protein is eliminated from the body as urinary nitrogen, it is often accompanied by increased urinary calcium, thereby increasing the risk of nephrolithiasis (kidney stones) and osteoporosis. 2.

1	2. The protein-sparing effect of carbohydrates: The dietary protein requirement is influenced by the carbohydrate content of the diet. When the intake of carbohydrates is low, amino acids are deaminated to provide carbon skeletons for the synthesis of glucose that is needed as a fuel by the central nervous system. If carbohydrate intake is <130 g/day, substantial amounts of protein are metabolized to provide precursors for gluconeogenesis. Therefore, carbohydrate is considered to be “protein-sparing,” because it allows amino acids to be used for repair and maintenance of tissue protein rather than for gluconeogenesis. D. Protein-energy (calorie) malnutrition

1	D. Protein-energy (calorie) malnutrition In developed countries, protein-energy malnutrition (PEM), also known as protein-energy undernutrition (PEU), is most commonly seen in patients with medical conditions that decrease appetite or alter how nutrients are digested or absorbed or in hospitalized patients with major trauma or infections. [Note: Such highly catabolic patients frequently require intravenous (IV, or parenteral) or tube-based (enteral) administration of nutrients.] PEM may also be seen in children or the elderly who are malnourished. In developing countries, an inadequate intake of protein and/or calories is the primary cause of PEM. Affected individuals show a variety of symptoms, including a depressed immune system with a reduced ability to resist infection. Death from secondary infection is common. PEM is a spectrum of degrees of malnutrition, and two extreme forms are kwashiorkor and marasmus (Fig. 27.21). [Note: Marasmic kwashiorkor has features of both forms.] 1.

1	Kwashiorkor: Kwashiorkor occurs when protein deprivation is relatively greater than the reduction in total calories. Protein deprivation is associated with severely decreased synthesis of visceral protein. Kwashiorkor is commonly seen in developing countries in children after weaning at about age 1 year, when their diet consists predominantly of carbohydrates. Typical symptoms include stunted growth, skin lesions, depigmented hair, anorexia, fatty liver, bilateral pitting edema, and decreased serum albumin concentration. Edema results from the lack of adequate blood proteins, primarily albumin, to maintain the distribution of water between blood and tissues. It may mask muscle and fat loss. Therefore, chronic malnutrition is reflected in the level of serum albumin. [Note: Because caloric intake from carbohydrates may be adequate, insulin levels suppress lipolysis and proteolysis. Kwashiorkor is, therefore, nonadapted malnutrition.] 2.

1	Marasmus: Marasmus occurs when calorie deprivation is relatively greater than the reduction in protein. It usually occurs in developing countries in children younger than age 1 year when breast milk is supplemented or replaced with watery gruels of native cereals that are usually deficient in both protein and calories. Typical symptoms include arrested growth, extreme muscle wasting and depletion of subcutaneous fat (emaciation), weakness, and anemia (Fig. 27.22). Individuals with marasmus do not show the edema observed in kwashiorkor. [Note: Refeeding severely malnourished individuals can result in hypophosphatemia (see p. 400), because any available phosphate is used to phosphorylate carbohydrate intermediates. Milk is frequently given because it is rich in phosphate.]

1	Cachexia, a wasting disorder characterized by loss of appetite and muscle atrophy (with or without increased lipolysis) that cannot be reversed by conventional nutritional support, is seen with a number of chronic diseases, such as cancer and chronic pulmonary and renal disease. It is associated with decreased treatment tolerance and response and decreased survival time. VIII. NUTRITION TOOLS A set of tools has been developed that gives consumers information about what (and how much) they should eat as well as the nutritional content of the foods they do eat. Additional tools allow medical professionals to assess whether or not the nutritional needs of an individual are being met. A. MyPlate

1	A. MyPlate MyPlate was designed by the U.S. Department of Agriculture (USDA) to graphically illustrate its recommendations as to what food groups and how much of each should be consumed daily. In MyPlate, the relative amounts of each of five food groups (vegetables, grains, protein, fruit, and dairy) are represented by the relative size of their section on the plate (Fig. 27.23). The number of servings depends on variables that include age and sex. [Note: MyPlate replaced MyPyramid in 2011.] B. Nutrition facts label Most types of packaged goods are required to have a Nutrition Facts label, or “food label” (Fig. 27.24), that includes the size of a single serving, the Cal it provides, and the number of servings per container. In addition, a percent daily value (%DV) is shown for most nutrients listed. [Note: The %DV is based on a 2,000-Cal diet for healthy adults.] 1.

1	Percent daily value: The %DV compares the amount of a given nutrient in a single serving of a product to the recommended daily intake for that nutrient. For example, the %DV for the micronutrients listed, as well as for total carbohydrates and fiber, are based on their recommended minimum daily intake. Thus, if the label lists 20% for calcium, one serving provides 20% of the minimum recommended amount of calcium needed each day. In contrast, the %DV for saturated fat, cholesterol, and sodium are based on their recommended maximum daily intake, and the %DV reflects what percentage of this maximum a serving provides. There is no %DV for protein because the recommended intake depends on body weight (see p. 367). [Note: “Sugars” represents mono-and disaccharides. The remainder of the carbohydrate (total carbohydrate – [fiber + sugars]) is the oligo-and polysaccharides.] 2.

1	Proposed revisions: In 2014, the USDA proposed the following changes to the Nutrition Facts label for implementation by 2018: Added sugars, vitamin D, and potassium are to be included; vitamins A and C, total fat, and Cal from fat are to be removed; and serving size is to be adjusted to reflect the amounts people are now consuming. Additionally, design changes to highlight key parts of the label were proposed (Fig. 27.25). [Note: The proposed addition/removal of certain micronutrients is based on newer data on the risk for underingestion.] C. Nutrition assessment

1	C. Nutrition assessment Nutrition assessment evaluates nutritional status based on clinical information. It includes (but is not limited to) dietary history, anthropometric measures, and laboratory data. [Note: Assessment findings may result in medical nutrition therapy, which is the treatment of medical conditions through changes in diet (for example, replacement of long-chain TAG with medium-chain TAG in malabsorption disorders) and/or the method of intake (for example, enteral [tube] or parenteral [IV] feeding).] 1.

1	Dietary history: This is a record of food intake over a period of time. For a food diary, the specific types and exact amounts of food eaten are recorded in “real time” (as soon as possible after eating) for a period of 3–7 days. Retrospective approaches include a food frequency questionnaire (for example, what fruits were eaten and how often they were eaten in a typical day, week, or month) and a 24-hour recall of the specific foods and the amounts eaten in the last 24 hours. 2.

1	2. Anthropometric measures: These are physical measures of the body. They include (but are not limited to) weight, height, body mass index (an indicator of obesity, see p. 349), skin-fold thickness (an indicator of subcutaneous fat), and waist circumference (an indicator of abdominal fat, see p. 349). [Note: Ideal body weight can be calculated using the Hamwi method: 106 lb (for males) or 100 lb (for females) for the first 5 ft of height + 5 lb for every inch over 5 ft, with an adjustment of −10% for a small frame and + 10% for a large one.] 3.

1	Laboratory data: These are obtained by tests performed on body fluids, tissues, and waste. They can include plasma LDL-C (for cardiovascular risk), fecal fat (for malabsorption), red cell indices (for vitamin deficiencies), and N balance and serum proteins (such as albumin and transthyretin [prealbumin]) for protein–energy status. [Note: These proteins are made in the liver and transport molecules such as fatty acids and thyroxine (see p. 406) through blood. Low albumin levels correlate with increased morbidity and mortality in hospitalized patients. The short half-life (2–3 days) of transthyretin as compared to that of albumin (20 days) has led to its use in monitoring the progress of hospitalized patients.] Nutritional insufficiency can be the result of inadequate nutrient intake (caused, for example, by an inability to eat, loss of appetite, or decreased availability), inadequate absorption, decreased utilization, increased excretion, or increased requirements.

1	IX. NUTRITION AND THE LIFE STAGES Macronutrient energy sources, micronutrients, EFA, and EAA are required at every life stage. Additionally, each stage has specific nutrition needs. A. Infancy, childhood, and adolescence

1	The rapid growth and development in infancy (birth to age 1 year) and childhood (age 1 year to adolescence) necessitate higher energy and protein needs relative to body size than are required in subsequent life stages. In adolescence, the marked increases in height and weight that occur increase nutritional needs. Growth charts (Fig. 27.26) are used to compare an individual’s stature (height) and/or weight to the expected values for others of the same age (≤20 years) and sex. They are based on data from large numbers of normal individuals over time. [Note: Deviations from the expected growth curve, as reflected in the crossing of two or more percentile lines, raise concern.] 1. Infants: Ideal infant nutrition is based on human breast milk because it provides calories and most micronutrients in amounts appropriate for the human infant. Carbohydrates, protein, and fat are present in a 7:3:1 ratio. [Note: In addition to the disaccharide lactose, human milk contains nearly 200 unique

1	in amounts appropriate for the human infant. Carbohydrates, protein, and fat are present in a 7:3:1 ratio. [Note: In addition to the disaccharide lactose, human milk contains nearly 200 unique oligosaccharides. About 90% of the microbiota (the population of microbes) in the breast-fed infant’s intestine is represented by one type, Bifidobacterium infantis, which expresses all the enzymes needed to degrade these complex sugars. The sugars, in turn, act as prebiotics that support the growth of B. infantis, a probiotic (helpful bacteria).] Breast milk is low in vitamin D, however, and exclusively breast-fed babies require vitamin D supplementation. [Note: Human milk provides antibodies and other proteins that reduce the risk of infection.]

1	The microbiota in and on the human body plus their genomes are referred to as the microbiome. It is acquired at birth from the environment and changes with the life stages. The gut microbiome influences host nutrition by facilitating processing of food consumed and is itself influenced by that food. Its relationship with undernutrition, obesity, and diabetes is under investigation. 2. Children: As with infants, children have increased need for calories and nutrients. The primary concerns in this stage, however, are deficiencies of iron and calcium. 3. Adolescents: In the teen years, the increases in height and weight increase the need for calories, protein, calcium, iron, and phosphorus. Eating patterns in this stage can result in overconsumption of fat, sodium, and sugar and underconsumption of vitamin A, thiamine, and folic acid. [Note: Eating disorders and obesity are concerns in this age group.] B. Adulthood

1	B. Adulthood Overnutrition is a concern in young adults, whereas malnutrition is a concern in older adults. 1. Young adults: Nutrition in young adults focuses on the maintenance of good health and the prevention of disease. The goal is a diet rich in plant-based foods (with a focus on fiber and whole grains), limited intake of saturated fat and trans fatty acids, and balanced intake of ω-3 and ω-6 PUFA. 2. Pregnant or lactating women: The requirements for calories, protein, and virtually all micronutrients increase in pregnancy and lactation. Supplementation with folic acid (to prevent neural tube defects [see p. 379]), vitamin D, calcium, iron, iodine, and DHA is typically recommended. 3.

1	3. Older adults: Aging increases the risk of malnutrition. Decreased appetite resulting from a reduced sense of taste (dysgeusia) and smell (hyposmia) decreases nutrient intake. [Note: Physical limitations, including problems with dentition, and psychosocial factors, such as isolation, may also play a role in reduced intake.] Inadequate intake of protein, calcium, and vitamins D and B12 is common. B12 deficiency can result from decreased absorption caused by achlorhydria (reduced stomach acid, see p. 381). In aging, lean muscle mass decreases and fat increases, resulting in decreased RMR. [Note: Drug–nutrient interactions can occur at any life stage but are more common as the number of medications increases as in aging.] Monoamine oxidase inhibitors (MAOI), used to treat depression (see p.

1	Monoamine oxidase inhibitors (MAOI), used to treat depression (see p. 287) and early Parkinson disease, can interact with tyramine-containing foods. Tyramine is a monoamine derived from the decarboxylation of tyrosine during the curing, aging, or fermentation of food (Fig. 27.27). It causes the release of norepinephrine, increasing blood pressure and heart rate. Patients who take an MAOI and consume such foods are at risk for a hypertensive crisis. X. CHAPTER SUMMARY

1	The Dietary Reference Intakes (DRI) provide estimates of the amounts of nutrients required to prevent deficiencies and maintain optimal health and growth. They consist of the Estimated Average Requirement (EAR), the average daily nutrient intake level estimated to meet the requirement of 50% of the healthy individuals in a particular life stage (age) and gender group; the Recommended Dietary Allowance (RDA), the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97%–98%) individuals in a life stage and gender group; the Adequate Intake (AI), which is set instead of an RDA if sufficient scientific evidence is not available to calculate the RDA; and the Tolerable Upper Intake Level (UL), the highest average daily nutrient intake level that is likely to pose no risk of adverse health effects to almost all individuals in the general population. The energy generated by the metabolism of the macronutrients (9 kcal/g of fat and 4 kcal/g of

1	is likely to pose no risk of adverse health effects to almost all individuals in the general population. The energy generated by the metabolism of the macronutrients (9 kcal/g of fat and 4 kcal/g of protein or carbohydrate) is used for three energy-requiring processes that occur in the body: resting metabolic rate, physical activity, and the thermic effect of food. Acceptable Macronutrient Distribution Ranges (AMDR) are defined as the ranges of intake for a particular macronutrient that are associated with reduced risk of chronic disease while providing adequate amounts of essential nutrients. Adults should consume 45%–65% of their total calories from carbohydrates, 20%–35% from fat, and 10%–35% from protein (Fig. 27.28). Elevated levels of cholesterol in low-density lipoproteins (LDL-C) result in increased risk for coronary heart disease (CHD). In contrast, elevated levels of cholesterol in high-density lipoproteins (HDL-C) have been associated with a decreased risk for CHD. Dietary

1	result in increased risk for coronary heart disease (CHD). In contrast, elevated levels of cholesterol in high-density lipoproteins (HDL-C) have been associated with a decreased risk for CHD. Dietary or drug treatment of hypercholesterolemia is effective in decreasing LDL-C, increasing HDLC, and reducing the risk for CHD. Consumption of saturated fats is strongly associated with high levels of total plasma and LDL-C. When substituted for saturated fatty acids in the diet, monounsaturated fats lower both total plasma cholesterol and LDL-C but maintain or increase HDL-C. Consumption of fats containing ω-6 polyunsaturated fatty acids lowers plasma LDL-C, but HDL-C, which protects against CHD, is also lowered. Dietary ω-3 polyunsaturated fats suppress cardiac arrhythmias and reduce plasma triacylglycerols, decrease the tendency for thrombosis, and substantially reduce the risk of cardiovascular mortality. Carbohydrates provide energy and fiber to the diet. When they are consumed as part

1	decrease the tendency for thrombosis, and substantially reduce the risk of cardiovascular mortality. Carbohydrates provide energy and fiber to the diet. When they are consumed as part of a diet in which caloric intake is equal to energy expenditure, they do not promote obesity. Dietary protein provides essential amino acids. Protein quality is a measure of its ability to provide the essential amino acids required for tissue maintenance. Proteins from animal sources, in general, have a higher-quality protein than that derived from plants. However, proteins from different plant sources may be combined in such a way that the result is equivalent in nutritional value to animal protein. Positive nitrogen (N) balance occurs when N intake exceeds N excretion. It is observed in situations in which tissue growth occurs, for example, in childhood, pregnancy, or during recovery from an emaciating illness. Negative N balance occurs when N losses are greater than N intake. It is associated with

1	tissue growth occurs, for example, in childhood, pregnancy, or during recovery from an emaciating illness. Negative N balance occurs when N losses are greater than N intake. It is associated with inadequate dietary protein; lack of an essential amino acid; or during physiologic stresses such as trauma, burns, illness, or surgery. Kwashiorkor occurs when protein deprivation is relatively greater than the reduction in total calories. It is characterized by edema. Marasmus occurs when calorie deprivation is relatively greater than the reduction in protein. No edema is seen. Both are extreme forms of protein-energy malnutrition (PEM). Nutrition Facts labels give consumers information about the nutritional content of packaged foods. Medical assessment of nutritional status includes dietary history, anthropometric measures, and laboratory data. Each life stage has specific nutrition needs. Growth charts are used to monitor the growth pattern of an individual from birth through adolescence.

1	anthropometric measures, and laboratory data. Each life stage has specific nutrition needs. Growth charts are used to monitor the growth pattern of an individual from birth through adolescence. Drug–nutrient interactions are of concern, especially in older adults.

1	Choose the ONE best answer. 7.1. For the child shown at right, which of the statements would support a diagnosis of kwashiorkor? The child: A. appears plump due to increased deposition of fat in adipose tissue. B. displays abdominal and peripheral edema. C. has a serum albumin level above normal. D. has markedly decreased weight for height. The correct answer = B. Kwashiorkor is caused by inadequate protein intake in the presence of fair to good energy (calorie) intake. Typical findings in a patient with kwashiorkor include abdominal and peripheral edema (note the swollen belly and legs) caused largely by a decreased serum albumin concentration. Body fat stores are depleted, but weight for height can be normal because of edema. Treatment includes a diet adequate in calories and protein. 7.2. Which one of the following statements concerning dietary fat is correct? A. Coconut oil is rich in monounsaturated fats, and olive oil is rich in saturated fats.

1	7.2. Which one of the following statements concerning dietary fat is correct? A. Coconut oil is rich in monounsaturated fats, and olive oil is rich in saturated fats. B. Fatty acids containing trans double bonds, unlike the naturally occurring cis isomers, raise high-density lipoprotein cholesterol levels. C. The polyunsaturated fatty acids linoleic and linolenic acids are required components. D. Triacylglycerols obtained from plants generally contain less unsaturated fatty acids than do those from animals. Correct answer = C. Humans are unable to make linoleic and linolenic fatty acids. Consequently, these fatty acids are essential in the diet. Coconut oil is rich in saturated fats, and olive oil is rich in monounsaturated fats. Trans fatty acids raise plasma levels of low-density lipoprotein cholesterol, not high-density lipoprotein cholesterol. Triacylglycerols obtained from plants generally contain more unsaturated fatty acids than do those from animals.

1	7.3. Given the information that a 70-kg man is consuming a daily average of 275 g of carbohydrate, 75 g of protein, and 65 g of fat, which one of the following conclusions can reasonably be drawn? A. About 20% of calories are derived from fats. B. The diet contains a sufficient amount of fiber. C. The individual is in nitrogen balance. D. The proportions of carbohydrate, protein, and fat in the diet conform to current recommendations. E. The total energy intake per day is about 3,000 kcal.

1	D. The proportions of carbohydrate, protein, and fat in the diet conform to current recommendations. E. The total energy intake per day is about 3,000 kcal. Correct answer = D. The total energy intake is (275 g carbohydrate × 4 kcal/g) + (75 g protein × 4 kcal/g) + (65 g fat × 9 kcal/g) = 1,100 + 300 + 585 = 1,985 total kcal/day. The percentage of calories derived from carbohydrate is 1,100/1,985 = 55, from protein is 300/1,985 = 15, and from fat is 585/1,985 = 30. These are very close to current recommendations. The amount of fiber or nitrogen balance cannot be deduced from the data presented. If the protein is of low biologic value, a negative nitrogen balance is possible.

1	7.6. In chronic bronchitis, excessive mucus production causes airway obstruction that results in hypoxemia (low blood oxygen level), impaired expiration, and hypercapnia (carbon dioxide retention). Why might a high-fat, low-carbohydrate diet be recommended for a patient with chronic obstructive pulmonary disease caused by chronic bronchitis? A. Fat contains more oxygen atoms relative to carbon or hydrogen atoms than do carbohydrates. B. Fat is calorically less dense than carbohydrates. C. Fat metabolism generates less carbon dioxide. D. The respiratory quotient (RQ) for fat is higher than the RQ for carbohydrates.

1	C. Fat metabolism generates less carbon dioxide. D. The respiratory quotient (RQ) for fat is higher than the RQ for carbohydrates. Correct answer = C. A treatment goal for the chronic obstructive pulmonary disease (COPD) caused by acute bronchitis is to insure appropriate nutrition without increasing the respiratory quotient (RQ), which is the ratio of carbon dioxide (CO2) produced to oxygen consumed, thereby minimizing the production of CO2. Less CO2 is produced from the metabolism of fat (RQ = 0.7) than from the catabolism of carbohydrate (RQ =1.0). Fat contains fewer oxygen atoms. Fat is calorically denser than is carbohydrate. [Note: RQ is determined by indirect calorimetry.] 7.7. A 32-year-old man who was rescued from a house fire was admitted to the hospital with burns over 45% of his body (severe burns). The man weighs 154 lb (70 kg) and is 72 in (183 cm) tall. Which one of the following is the best rapid estimate of the immediate daily caloric needs of this patient?

1	A. 1,345 kcal B. 1,680 kcal C. 2,690 kcal D. 3,360 kcal Correct answer = D. A commonly used rough estimate of the total energy expenditure (TEE) for men is 1 kcal/1 kg body weight/24 hours. [Note: It is 0.8 kcal for women.] For this patient, that value is 1,680 kcal (1 kcal/kg/hour × 24 hours × 70 kg). In addition, an injury factor of 2 for severe burns must be included in the calculation: 1,680 kcal × 2 = 3,360 kcal. 7.8. Which one of the following is the best advice to give a patient who asks about the notation “%DV” (percent daily value) on the Nutrition Facts label? A. Achieve 100% daily value for each nutrient each day. B. Select foods that have the highest percent daily value for all nutrients. C. Select foods with a low percent daily value for the micronutrients. D. Select foods with a low percent daily value for saturated fat.

1	C. Select foods with a low percent daily value for the micronutrients. D. Select foods with a low percent daily value for saturated fat. Correct answer = D. The percent daily value (%DV) compares the amount of a given nutrient in a single serving of a product to the recommended daily intake for that nutrient. The %DV for the micronutrients listed on the label, as well as for total carbohydrates and fiber, are based on their recommended minimum daily intake, whereas the %DV for saturated fat, cholesterol, and sodium are based on their recommended maximum daily intake. For Questions 27.7 and 27.8, use the following case.

1	For Questions 27.7 and 27.8, use the following case. A sedentary 50-year-old man weighing 176 lb (80 kg) requests a physical. He denies any health problems. Routine blood analysis is unremarkable except for plasma total cholesterol of 295 mg/dl. (Reference value is <200 mg.) The man refuses drug therapy for his hypercholesterolemia. Analysis of a 1-day dietary recall showed the following: 7.4. Decreasing which one of the following dietary components would have the greatest effect in lowering the patient’s plasma cholesterol? A. Carbohydrates B. Cholesterol C. Fiber D. Monounsaturated fat E. Polyunsaturated fat F. Saturated fat

1	A. Carbohydrates B. Cholesterol C. Fiber D. Monounsaturated fat E. Polyunsaturated fat F. Saturated fat Correct answer = F. The intake of saturated fat most strongly influences plasma cholesterol in this diet. The patient is consuming a high-calorie, high-fat diet with 42% of the fat as saturated fat. The most important dietary recommendations are to lower total caloric intake, substitute monounsaturated and polyunsaturated fats for saturated fats, and increase dietary fiber. A decrease in dietary cholesterol would be helpful but is not a primary objective. 7.5. What information would be necessary to estimate the patient’s total energy expenditure?

1	7.5. What information would be necessary to estimate the patient’s total energy expenditure? The daily basal energy expenditure (estimated resting metabolic rate/hour × 24 hours) and a physical activity ratio (PAR) based on the type and duration of physical activities are needed variables. An additional 10% would be added to account for the thermic effect of food. Note that if the patient were hospitalized, an injury factor (IF) would be included in the calculation, and the PAR would be modified. Tables of PAR and IF are available. Micronutrients: Vitamins 28 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Vitamins are chemically unrelated organic compounds that cannot be synthesized in adequate quantities by humans and, therefore, must be supplied by the diet. Nine vitamins (folic acid, cobalamin, ascorbic acid, pyridoxine, thiamine, niacin, riboflavin, biotin, and pantothenic acid) are classified as water soluble. Because they are readily excreted in the urine, toxicity is rare. However, deficiencies can occur quickly. Four vitamins (A, D, K, and E) are termed fat soluble (Fig. 28.1). They are released, absorbed, and transported (in chylomicrons, see p. 227) with dietary fat. They are not readily excreted, and significant quantities are stored in the liver and adipose tissue. In fact, consumption of vitamins A and D in excess of the Dietary Reference Intakes (see Chapter 27) can lead to accumulation of toxic quantities of these compounds. Vitamins are required to perform specific cellular functions. For example, many of the water-soluble vitamins are precursors of coenzymes for the

1	accumulation of toxic quantities of these compounds. Vitamins are required to perform specific cellular functions. For example, many of the water-soluble vitamins are precursors of coenzymes for the enzymes of intermediary metabolism. In contrast to the water-soluble vitamins, only one fat-soluble vitamin (vitamin K) has a coenzyme function.

1	II. FOLIC ACID (VITAMIN B9) Folic acid (or, folate), which plays a key role in one-carbon metabolism, is essential for the biosynthesis of several compounds. Folic acid deficiency is probably the most common vitamin deficiency in the United States, particularly among pregnant women and individuals with alcoholism. [Note: Leafy, dark-green vegetables are a good source of folic acid.] A. Function Tetrahydrofolate (THF), the reduced, coenzyme form of folate, receives one-carbon fragments from donors such as serine, glycine, and histidine and transfers them to intermediates in the synthesis of amino acids, purine nucleotides, and thymidine monophosphate (TMP), a pyrimidine nucleotide incorporated into DNA (Fig. 28.2). B. Nutritional anemias

1	B. Nutritional anemias Anemia is a condition in which the blood has a lower than normal concentration of hemoglobin, which results in a reduced ability to transport oxygen (O2). Nutritional anemias (that is, those caused by inadequate intake of one or more essential nutrients) can be classified according to the size of the red blood cells (RBC), or mean corpuscular volume (MCV), observed in the blood (Fig. 28.3). Microcytic anemia (MCV below normal), caused by lack of iron, is the most common form of nutritional anemia. The second major category of nutritional anemia, macrocytic (MCV above normal), results from a deficiency in folic acid or vitamin B12. [Note: These macrocytic anemias are commonly called megaloblastic because a deficiency of either vitamin (or both) causes accumulation of large, immature RBC precursors, known as megaloblasts, in the bone marrow and the blood (Fig. 28.4). Hypersegmented neutrophils are also seen.] 1.

1	Folate and anemia: Inadequate serum levels of folate can be caused by increased demand (for example, pregnancy and lactation; see p. 372), poor absorption caused by pathology of the small intestine, alcoholism, or treatment with drugs (for example, methotrexate) that are dihydrofolate reductase inhibitors (see Fig. 28.2). A folate-free diet can cause a deficiency within a few weeks. A primary result of folic acid deficiency is megaloblastic anemia (see Fig. 28.4), caused by diminished synthesis of purine nucleotides and TMP, which leads to an inability of cells (including RBC precursors) to make DNA and, therefore, an inability to divide. 2.

1	Folate and neural tube defects: Spina bifida and anencephaly, the most common neural tube defects (NTD), affect ~3,000 pregnancies in the United States annually. Folic acid supplementation before conception and during the first trimester has been shown to significantly reduce NTD. Therefore, all women of childbearing age are advised to consume 0.4 mg/day (400 µg/day) of folic acid to reduce the risk of having a pregnancy affected by NTD and ten times that amount if a previous pregnancy was affected. Adequate folate nutrition must occur at the time of conception because critical folate-dependent development occurs in the first weeks of fetal life, at a time when many women are not yet aware of their pregnancy. In 1998, the U.S. Food and Drug Administration authorized the addition of folic acid to wheat flour and enriched grain products, resulting in a dietary supplementation of ~0.1 mg/day. This supplementation allows ~50% of all reproductive-aged women to receive 0.4 mg of folate from

1	to wheat flour and enriched grain products, resulting in a dietary supplementation of ~0.1 mg/day. This supplementation allows ~50% of all reproductive-aged women to receive 0.4 mg of folate from all sources.

1	III. COBALAMIN (VITAMIN B12) Vitamin B12 is required in humans for two essential enzymatic reactions: the remethylation of homocysteine (Hcy) to methionine and the isomerization of methylmalonyl coenzyme A (CoA), which is produced during the degradation of some amino acids (isoleucine, valine, threonine, and methionine) and fatty acids (FA) with odd numbers of carbon atoms (Fig. 28.5). When cobalamin is deficient, unusual (branched) FA accumulate and become incorporated into cell membranes, including those of the central nervous system (CNS). This may account for some of the neurologic manifestations of vitamin B12 deficiency. [Note: Folic acid (as N5-methyl THF) is also required in the remethylation of Hcy. Therefore, deficiency of B12 or folate results in elevated Hcy levels.] A. Structure and coenzyme forms

1	Hcy. Therefore, deficiency of B12 or folate results in elevated Hcy levels.] A. Structure and coenzyme forms Cobalamin contains a corrin ring system that resembles the porphyrin ring of heme (see p. 279), but differs in that two of the pyrrole rings are linked directly rather than through a methene bridge. Cobalt (see p. 407) is held in the center of the corrin ring by four coordination bonds with the nitrogens of the pyrrole groups. The remaining coordination bonds of the cobalt are with the nitrogen of 5,6-dimethylbenzimidazole and with cyanide in commercial preparations of the vitamin in the form of cyanocobalamin (Fig. 28.6). The physiologic coenzyme forms of cobalamin are 5′deoxyadenosylcobalamin and methylcobalamin, in which cyanide is replaced with 5′-deoxyadenosine or a methyl group, respectively (see Fig. 28.6). B. Distribution

1	B. Distribution Vitamin B12 is synthesized only by microorganisms, and it is not present in plants. Animals obtain the vitamin preformed from their intestinal microbiota (see p. 371) or by eating foods derived from other animals. Cobalamin is present in appreciable amounts in liver, red meat, fish, eggs, dairy products, and fortified cereals. C. Folate trap hypothesis

1	C. Folate trap hypothesis The effects of cobalamin deficiency are most pronounced in rapidly dividing cells, such as the erythropoietic tissue of bone marrow and the mucosal cells of the intestine. Such tissues need both the N5,N10-methylene and N10-formyl forms of THF for the synthesis of nucleotides required for DNA replication (see pp. 292 and 303). However, in vitamin B12 deficiency, the utilization of the N5-methyl form of THF in the B12 dependent methylation of Hcy to methionine is impaired. Because the methylated form cannot be converted directly to other forms of THF, folate is trapped in the N5-methyl form, which accumulates. The levels of the other forms decrease. Thus, cobalamin deficiency leads to a deficiency of the THF forms needed in purine and TMP synthesis, resulting in the symptoms of megaloblastic anemia. D. Clinical indications for cobalamin

1	D. Clinical indications for cobalamin In contrast to other water-soluble vitamins, significant amounts (2–5 mg) of vitamin B12 are stored in the body. As a result, it may take several years for the clinical symptoms of B12 deficiency to develop as a result of decreased intake of the vitamin. [Note: Deficiency happens much more quickly (in months) if absorption is impaired (see below). The Schilling test evaluates B12 absorption.] B12 deficiency can be determined by the level of methylmalonic acid in blood, which is elevated in individuals with low intake or decreased absorption of the vitamin. 1. Pernicious anemia: Vitamin B12 deficiency is most commonly seen in patients who fail to absorb the vitamin from the intestine (Fig. 28.7). B12 is released from food in the acidic environment of the stomach. [Note:

1	Malabsorption of cobalamin in the elderly is most often due to reduced secretion of gastric acid (achlorhydria).] Free B12 then binds a glycoprotein (R-protein or haptocorrin), and the complex moves into the intestine. B12 is released from the R-protein by pancreatic enzymes and binds another glycoprotein, intrinsic factor (IF). The cobalamin–IF complex travels through the intestine and binds to a receptor (cubilin) on the surface of mucosal cells in the ileum. The cobalamin is transported into the mucosal cell and, subsequently, into the general circulation, where it is carried by its binding protein (transcobalamin). B12 is taken up and stored in the liver, primarily. It is released into bile and efficiently reabsorbed in the ileum. Severe malabsorption of vitamin B12 leads to pernicious anemia. This disease is most commonly a result of an autoimmune destruction of the gastric parietal cells that are responsible for the synthesis of IF (lack of IF prevents B12 absorption). [Note:

1	anemia. This disease is most commonly a result of an autoimmune destruction of the gastric parietal cells that are responsible for the synthesis of IF (lack of IF prevents B12 absorption). [Note: Patients who have had a partial or total gastrectomy become IF deficient and, therefore, B12 deficient.] Individuals with cobalamin deficiency are usually anemic (folate recycling is impaired), and they show neuropsychiatric symptoms as the disease develops. The CNS effects are irreversible. Pernicious anemia requires lifelong treatment with either high-dose oral B12 or intramuscular injection of cyanocobalamin. [Note: Supplementation works even in the absence of IF because ~1% of B12 uptake is by IF-independent diffusion.]

1	Folic acid supplementation can partially reverse the hematologic abnormalities of B12 deficiency and, therefore, can mask a cobalamin deficiency. Thus, to prevent the later CNS effects of B12 deficiency, therapy for megaloblastic anemia is initiated with both vitamin B12 and folic acid until the cause of the anemia can be determined. IV. ASCORBIC ACID (VITAMIN C) The active form of vitamin C is ascorbic acid (Fig. 28.8). Its main function is as a reducing agent. Vitamin C is a coenzyme in hydroxylation reactions (for example, hydroxylation of prolyl and lysyl residues in collagen; see p. 47), where its role is to keep the iron (Fe) of hydroxylases in the reduced, ferrous (Fe+2) form. Thus, vitamin C is required for the maintenance of normal connective tissue as well as for wound healing. Vitamin C also facilitates the absorption of dietary nonheme iron from the intestine by reduction of the ferric form (Fe+3) to Fe+2 (see p. 403). A. Deficiency

1	A. Deficiency Ascorbic acid deficiency results in scurvy, a disease characterized by sore and spongy gums, loose teeth, fragile blood vessels, hemorrhage, swollen joints, bone changes, and fatigue (Fig. 28.9). Many of the deficiency symptoms can be explained by the decreased hydroxylation of collagen, resulting in defective connective tissue. A microcytic anemia caused by decreased absorption of iron may also be seen. B. Chronic disease prevention Vitamin C is one of a group of nutrients that includes vitamin E (see p. 395) and β-carotene (see p. 386), which are known as antioxidants. [Note: Vitamin C regenerates the functional, reduced form of vitamin E.] Consumption of diets rich in these compounds is associated with a decreased incidence of some chronic diseases, such as cardiovascular disease (CVD) and certain cancers. However, clinical trials involving supplementation with the isolated antioxidants have failed to demonstrate any convincing preventive effects.

1	V. PYRIDOXINE (VITAMIN B6) Vitamin B6 is a collective term for pyridoxine, pyridoxal, and pyridoxamine, all derivatives of pyridine. They differ only in the nature of the functional group attached to the ring (Fig. 28.10). Pyridoxine occurs primarily in plants, whereas pyridoxal and pyridoxamine are found in foods obtained from animals. All three compounds can serve as precursors of the biologically active coenzyme, pyridoxal phosphate (PLP). PLP functions as a coenzyme for a large number of enzymes, particularly those that catalyze reactions involving amino acids, for example, in the transsulfuration of Hcy to cysteine (see p. 264). [Note: PLP is also required by glycogen phosphorylase (see p. 128).] A. Clinical indications for pyridoxine

1	A. Clinical indications for pyridoxine Isoniazid, a drug commonly used to treat tuberculosis, can induce a vitamin B6 deficiency by forming an inactive derivative with PLP. Thus, dietary supplementation with B6 is an adjunct to isoniazid treatment. Otherwise, dietary deficiencies in pyridoxine are rare but have been observed in newborn infants fed formulas low in B6, in women taking oral contraceptives, and in those with alcoholism. B. Toxicity Vitamin B6 is the only water-soluble vitamin with significant toxicity. Neurologic symptoms (sensory neuropathy) occur at intakes above 500 mg/day, an amount nearly 400 times the recommended dietary allowance (RDA) and over 5 times the tolerable upper limit (UL). (See Chapter 27 for a discussion of RDA and UL.) Substantial improvement, but not complete recovery, occurs when the vitamin is discontinued. VI. THIAMINE (VITAMIN B1)

1	VI. THIAMINE (VITAMIN B1) Thiamine pyrophosphate (TPP) is the biologically active form of the vitamin, formed by the transfer of a pyrophosphate group from ATP to thiamine (Fig. 28.11). TPP serves as a coenzyme in the formation or degradation of α-ketols by transketolase (Fig. 28.12A) and in the oxidative decarboxylation of α-keto acids (Fig. 28.12B). A. Clinical indications for thiamine

1	A. Clinical indications for thiamine The oxidative decarboxylation of pyruvate and α-ketoglutarate, which plays a key role in energy metabolism of most cells, is particularly important in tissues of the CNS. In thiamine deficiency, the activity of these two dehydrogenase-catalyzed reactions is decreased, resulting in decreased production of ATP and, therefore, impaired cellular function. TPP is also required by branched-chain α-keto acid dehydrogenase of muscle (see p. 266). [Note: It is the decarboxylase of each of these α-keto acid dehydrogenase multienzyme complexes that requires TPP.] Thiamine deficiency is diagnosed by an increase in erythrocyte transketolase activity observed with addition of TPP. 1.

1	1. Beriberi: This severe thiamine-deficiency syndrome is found in areas where polished rice is the major component of the diet. Adult beriberi is classified as dry (characterized by peripheral neuropathy, especially in the legs) or wet (characterized by edema because of dilated cardiomyopathy). 2. Wernicke-Korsakoff syndrome: In the United States, thiamine deficiency, which is seen primarily in association with chronic alcoholism, is due to dietary insufficiency or impaired intestinal absorption of the vitamin. Some individuals with alcoholism develop Wernicke-Korsakoff syndrome, a thiamine-deficiency state characterized by mental confusion, gait ataxia, nystagmus (a to-and-fro motion of the eyeballs), and ophthalmoplegia (weakness of eye muscles) with Wernicke encephalopathy as well as memory problems and hallucinations with Korsakoff dementia. The syndrome is treatable with thiamine supplementation, but recovery of memory is typically incomplete. VII. NIACIN (VITAMIN B3)

1	Niacin, or nicotinic acid, is a substituted pyridine derivative. The biologically active coenzyme forms are nicotinamide adenine dinucleotide (NAD+) and its phosphorylated derivative, nicotinamide adenine dinucleotide phosphate (NADP+), as shown in Figure 28.13. Nicotinamide, a derivative of nicotinic acid that contains an amide instead of a carboxyl group, also occurs in the diet. Nicotinamide is readily deaminated in the body and, therefore, is nutritionally equivalent to nicotinic acid. NAD+ and NADP+ serve as coenzymes in oxidation–reduction reactions in which the coenzyme undergoes reduction of the pyridine ring by accepting two electrons from a hydride ion, as shown in Figure 28.14. The reduced forms of NAD+ and NADP+ are NADH and NADPH, respectively. [Note: A metabolite of tryptophan, quinolinate, can be converted to NAD(P). In comparison, 60 mg of tryptophan = 1 mg of niacin.] to NADH. [Note: The hydride ion consists of a hydrogen (H) atom plus an electron.] = phosphate.

1	A. Distribution Niacin is found in unrefined and enriched grains and cereal, milk, and lean meats (especially liver). B. Clinical indications for niacin 1. Deficiency: A deficiency of niacin causes pellagra, a disease involving the skin, gastrointestinal tract, and CNS. The symptoms of pellagra progress through the three Ds: dermatitis (photosensitive), diarrhea, and dementia. If untreated, death (a fourth D) occurs. Hartnup disorder, characterized by defective absorption of tryptophan, can result in pellagra-like symptoms. [Note: Corn is low in both niacin and tryptophan. Corn-based diets can cause pellagra.] 2.

1	Hyperlipidemia treatment: Niacin at doses of 1.5 g/day, or 100 times the RDA, strongly inhibits lipolysis in adipose tissue, the primary producer of circulating free fatty acids (FFA). The liver normally uses these circulating FFA as a major precursor for triacylglycerol (TAG) synthesis. Thus, niacin causes a decrease in liver TAG synthesis, which is required for very-low-density lipoprotein ([VLDL] see p. 230) production. Low-density lipoprotein (LDL, the cholesterol-rich lipoprotein) is derived from VLDL in the plasma. Thus, both plasma TAG (in VLDL) and cholesterol (in LDL) are lowered. Therefore, niacin is particularly useful in the treatment of type IIb hyperlipoproteinemia, in which both VLDL and LDL are elevated. The high doses of niacin required can cause acute, prostaglandin-mediated flushing. Aspirin can reduce this side effect by inhibiting prostaglandin synthesis (see p. 214). Itching may also occur. [Note: Niacin raises high-density lipoprotein and lowers Lp(a) levels

1	flushing. Aspirin can reduce this side effect by inhibiting prostaglandin synthesis (see p. 214). Itching may also occur. [Note: Niacin raises high-density lipoprotein and lowers Lp(a) levels (see p. 237).]

1	VIII. RIBOFLAVIN (VITAMIN B2) The two biologically active forms of B2 are flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), formed by the transfer of an adenosine monophosphate moiety from ATP to FMN (Fig. 28.15). FMN and FAD are each capable of reversibly accepting two hydrogen atoms, forming FMNH2 or FADH2, respectively. FMN and FAD are bound tightly, sometimes covalently, to flavoenzymes (for example, NADH dehydrogenase [FMN] and succinate dehydrogenase [FAD]) that catalyze the oxidation or reduction of a substrate. Riboflavin deficiency is not associated with a major human disease, although it frequently accompanies other vitamin deficiencies. Deficiency symptoms include dermatitis, cheilosis (fissuring at the corners of the mouth), and glossitis (the tongue appearing smooth and dark). [Note: Because riboflavin is light sensitive, phototherapy for hyperbilirubinemia (see p. 285) may require supplementation with the vitamin.] IX. BIOTIN (VITAMIN B7)

1	Biotin is a coenzyme in carboxylation reactions, in which it serves as a carrier of activated carbon dioxide (CO2) (see Fig. 10.3, p. 119, for the mechanism of biotin-dependent carboxylations). Biotin is covalently bound to the ε-amino group of lysine residues in biotin-dependent enzymes (Fig. 28.16). Biotin deficiency does not occur naturally because the vitamin is widely distributed in food. Also, a large percentage of the biotin requirement in humans is supplied by intestinal bacteria. However, the addition of raw egg white to the diet as a source of protein can induce symptoms of biotin deficiency, namely, dermatitis, hair loss, loss of appetite, and nausea. Raw egg white contains the glycoprotein avidin, which tightly binds biotin and prevents its absorption from the intestine. With a normal diet, however, it has been estimated that 20 eggs/day would be required to induce a deficiency syndrome. [Note: Inclusion of raw eggs in the diet is not recommended because of the possibility

1	normal diet, however, it has been estimated that 20 eggs/day would be required to induce a deficiency syndrome. [Note: Inclusion of raw eggs in the diet is not recommended because of the possibility of salmonellosis caused by infection with Salmonella enterica.]

1	Multiple carboxylase deficiency results from decreased ability to add biotin to carboxylases during their synthesis or to remove it during their degradation. Treatment is biotin supplementation. X. PANTOTHENIC ACID (VITAMIN B5) Pantothenic acid is a component of CoA, which functions in the transfer of acyl groups (Fig. 28.17). CoA contains a thiol group that carries acyl compounds as activated thiol esters. Examples of such structures are succinyl CoA, fatty acyl CoA, and acetyl CoA. Pantothenic acid is also a component of the acyl carrier protein domain of fatty acid synthase (see p. 184). Eggs, liver, and yeast are the most important sources of pantothenic acid, although the vitamin is widely distributed. Pantothenic acid deficiency is not well characterized in humans, and no RDA has been established. XI. VITAMIN A

1	XI. VITAMIN A Vitamin A is a fat-soluble vitamin that comes primarily from animal sources as retinol (preformed vitamin A), a retinoid. The retinoids, a family of structurally related molecules, are essential for vision, reproduction, growth, and maintenance of epithelial tissues. They also play a role in immune function. Retinoic acid, derived from oxidation of retinol, mediates most of the actions of the retinoids, except for vision, which depends on retinal, the aldehyde derivative of retinol. A. Structure The retinoids include the natural forms of vitamin A, retinol and its metabolites (Fig. 28.18), and synthetic forms (drugs). 1. Retinol: A primary alcohol containing a β-ionone ring with an unsaturated side chain, retinol is found in animal tissues as a retinyl ester with long-chain FA. It is the storage form of vitamin A. 2. Retinal: This is the aldehyde derived from the oxidation of retinol. Retinal and retinol can readily be interconverted. 3.

1	2. Retinal: This is the aldehyde derived from the oxidation of retinol. Retinal and retinol can readily be interconverted. 3. Retinoic acid: This is the acid derived from the oxidation of retinal. Retinoic acid cannot be reduced in the body and, therefore, cannot give rise to either retinal or retinol. 4. β-Carotene: Plant foods contain β-carotene (provitamin A), which can be oxidatively and symmetrically cleaved in the intestine to yield two molecules of retinal. In humans, the conversion is inefficient, and the vitamin A activity of β-carotene is only about 1/12 that of retinol. B. Absorption and transport to the liver

1	B. Absorption and transport to the liver Retinyl esters from the diet are hydrolyzed in the intestinal mucosa, releasing retinol and FFA (Fig. 28.19). Retinol derived from esters and from the reduction of retinal from β-carotene cleavage is reesterified to long-chain FA within the enterocytes and secreted as a component of chylomicrons into the lymphatic system. Retinyl esters contained in chylomicron remnants are taken up by, and stored in, the liver. [Note: All fat-soluble vitamins are carried in chylomicrons.] C. Release from the liver

1	C. Release from the liver When needed, retinol is released from the liver and transported through the blood to extrahepatic tissues by retinol-binding protein complexed with transthyretin (see Fig. 28.19). The ternary complex binds to a transport protein on the surface of the cells of peripheral tissues, permitting retinol to enter. An intracellular retinol-binding protein carries retinol to sites in the nucleus where the vitamin regulates transcription in a manner analogous to that of steroid hormones. D. Retinoic acid mechanism of action

1	D. Retinoic acid mechanism of action Retinol is oxidized to retinoic acid. Retinoic acid binds with high affinity to specific receptor proteins (retinoic acid receptors [RAR]) present in the nucleus of target tissues such as epithelial cells (Fig. 28.20). The activated retinoic acid–RAR complex binds to response elements on DNA and recruits activators or repressors to regulate retinoid-specific RNA synthesis, resulting in control of the production of specific proteins that mediate several physiologic functions. For example, retinoids control the expression of the gene for keratin in most epithelial tissues of the body. [Note: The RAR proteins are part of the superfamily of transcriptional regulators that includes the nuclear receptors for steroid and thyroid hormones and vitamin D, all of which function in a similar way (see p. 240).] E. Functions 1.

1	E. Functions 1. Visual cycle: Vitamin A is a component of the visual pigments of rod and cone cells. Rhodopsin, the visual pigment of the rod cells in the retina, consists of 11-cis retinal bound to the protein opsin (see Fig. 28.19). When rhodopsin, a G protein–coupled receptor, is exposed to light, a series of photochemical isomerizations occurs, which results in the bleaching of rhodopsin and release of all-trans retinal and opsin. This process activates the G protein transducin, triggering a nerve impulse that is transmitted by the optic nerve to the brain. Regeneration of rhodopsin requires isomerization of all-trans retinal back to 11-cis retinal. All-trans retinal is reduced to all-trans retinol, esterified, and isomerized to 11-cis retinol that is oxidized to 11-cis retinal. The latter combines with opsin to form rhodopsin, thus completing the cycle. Similar reactions are responsible for color vision in the cone cells. 2.

1	2. Epithelial cell maintenance: Vitamin A is essential for normal differentiation of epithelial tissues and mucus secretion and, thus, supports the body’s barrier-based defense against pathogens. 3. Reproduction: Retinol and retinal are essential for normal reproduction, supporting spermatogenesis in the male and preventing fetal resorption in the female. Retinoic acid is inactive in maintaining reproduction and in the visual cycle but promotes growth and differentiation of epithelial cells. F. Distribution Liver, kidney, cream, butter, and egg yolk are good sources of preformed vitamin A. Yellow, orange, and dark-green vegetables and fruits are good sources of the carotenes (provitamin A). G. Requirement The RDA for adults is 900 retinol activity equivalents (RAE) for males and 700 RAE for females. In comparison, 1 RAE = 1 µg of retinol, 12 µg of β carotene, or 24 µg of other carotenoids. H. Clinical indications for vitamin A

1	H. Clinical indications for vitamin A Although chemically related, retinoic acid and retinol have distinctly different therapeutic applications. Retinol and its carotenoid precursor are used as dietary supplements, whereas various forms of retinoic acid are useful in dermatology (Fig. 28.21). 1.

1	1. Deficiency: Vitamin A, administered as retinol or retinyl esters, is used to treat patients who are deficient in the vitamin. Night blindness (nyctalopia) is one of the earliest signs of vitamin A deficiency. The visual threshold is increased, making it difficult to see in dim light. Prolonged deficiency leads to an irreversible loss in the number of visual cells. Severe deficiency leads to xerophthalmia, a pathologic dryness of the conjunctiva and cornea, caused, in part, by increased keratin synthesis. If untreated, xerophthalmia results in corneal ulceration and, ultimately, in blindness because of the formation of opaque scar tissue. The condition is most commonly seen in children in developing tropical countries. Over 500,000 children worldwide are blinded each year by xerophthalmia caused by insufficient vitamin A in the diet. 2.

1	2. Skin conditions: Dermatologic problems such as acne are effectively treated with retinoic acid or its derivatives (see Fig. 28.21). Mild cases of acne and skin aging are treated with tretinoin (all-trans retinoic acid). Tretinoin is too toxic for systemic (oral) administration in treating skin conditions and is confined to topical application. [Note: Oral tretinoin is used in treating acute promyelocytic leukemia.] In patients with severe cystic acne unresponsive to conventional therapies, isotretinoin (13-cis retinoic acid) is administered orally. An oral synthetic retinoid is used to treat psoriasis. I. Retinoid toxicity 1.

1	I. Retinoid toxicity 1. Vitamin A: Excessive intake of vitamin A (but not carotene) produces a toxic syndrome called hypervitaminosis A. Amounts exceeding 7.5 mg/day of retinol should be avoided. Early signs of chronic hypervitaminosis A are reflected in the skin, which becomes dry and pruritic (because of decreased keratin synthesis); in the liver, which becomes enlarged and can become cirrhotic; and in the CNS, where a rise in intracranial pressure may mimic the symptoms of a brain tumor. Pregnant women, in particular, should not ingest excessive quantities of vitamin A because of its potential for teratogenesis (causing congenital malformations in the developing fetus). UL is 3,000 µg preformed vitamin A/day. [Note: Vitamin A promotes bone growth. In excess, however, it is associated with decreased bone mineral density and increased risk of fractures.] 2.

1	Isotretinoin: The drug, an isomer of retinoic acid, is teratogenic and absolutely contraindicated in women with childbearing potential unless they have severe, disfiguring cystic acne that is unresponsive to standard therapies. Pregnancy must be excluded before treatment begins, and birth control must be used. Prolonged treatment with isotretinoin can result in an increase in TAG and cholesterol, providing some concern for an increased risk of CVD. XII. VITAMIN D The D vitamins are a group of sterols that have a hormone-like function. The active molecule, 1,25-dihydroxycholecalciferol ([1,25-diOH-D3], or calcitriol), binds to intracellular receptor proteins. The 1,25-diOH-D3–receptor complex interacts with response elements in the nuclear DNA of target cells in a manner similar to that of vitamin A (see Fig. 28.20) and either selectively stimulates or represses gene transcription. The most prominent actions of calcitriol are to regulate the serum levels of calcium and phosphorus.

1	A. Distribution 1. Endogenous vitamin precursor: 7-Dehydrocholesterol, an intermediate in cholesterol synthesis, is converted to cholecalciferol in the dermis and epidermis of humans exposed to sunlight and transported to liver bound to vitamin D–binding protein. 2. Diet: Ergocalciferol (vitamin D2), found in plants, and cholecalciferol (vitamin D3), found in animal tissues, are sources of preformed vitamin D activity (Fig. 28.22). Vitamin D2 and vitamin D3 differ chemically only in the presence of an additional double-bond and methyl group in the plant sterol. Dietary vitamin D is packaged into chylomicrons. [Note: Preformed vitamin D is a dietary requirement only in individuals with limited exposure to sunlight.]

1	B. Metabolism 1. 1,25-Dihydroxycholecalciferol formation: Vitamins D2 and D3 are not biologically active but are converted in vivo to calcitriol, the active form of the D vitamin, by two sequential hydroxylation reactions (Fig. 28.23). The first hydroxylation occurs at the 25 position and is catalyzed by a specific 25-hydroxylase in the liver. The product of the reaction, 25hydroxycholecalciferol ([25-OH-D3], calcidiol), is the predominant form of vitamin D in the serum and the major storage form. 25-OH-D3 is further hydroxylated at the 1 position by 25-hydroxycholecalciferol 1 hydroxylase found primarily in the kidney, resulting in the formation of 1,25-diOH-D3 (calcitriol). [Note: Both hydroxylases are cytochrome P450 proteins (see p. 149).] 2. Hydroxylation regulation: Calcitriol is the most potent vitamin D metabolite. Its formation is tightly regulated by the level of serum phosphate (PO43−) and calcium ions (Ca2+) as shown in Figure 28.24. 25

1	Hydroxycholecalciferol 1-hydroxylase activity is increased directly by low serum PO43− or indirectly by low serum Ca2+ , which triggers the secretion of parathyroid hormone (PTH) from the chief cells of the parathyroid gland. PTH upregulates the 1-hydroxylase. Thus, hypocalcemia caused by insufficient dietary Ca2+ results in elevated levels of serum 1,25-diOH-D3. [Note: 1,25-diOH-D3 inhibits expression of PTH, forming a negative feedback loop. It also inhibits activity of the 1-hydroxylase.] C. Function The overall function of calcitriol is to maintain adequate serum levels of Ca2+ . It performs this function by 1) increasing uptake of Ca2+ by the intestine, 2) minimizing loss of Ca2+ by the kidney by increasing reabsorption, and 3) stimulating resorption (demineralization) of bone when blood Ca2+ is low (see Fig. 28.23). 1.

1	1. Effect on the intestine: Calcitriol stimulates intestinal absorption of Ca2+ by first entering the intestinal cell and binding to a cytosolic receptor. The 1,25-diOH-D3–receptor complex then moves to the nucleus where it selectively interacts with response elements on the DNA. As a result, Ca2+ uptake is enhanced by increased expression of the calcium-binding protein calbindin. Thus, the mechanism of action of 1,25-diOH-D3 is typical of steroid hormones (see p. 240). 2. Effect on bone: Bone is composed of collagen and crystals of Ca5(PO4)3OH (hydroxylapatite). When blood Ca2+ is low, 1,25-diOH-D3 stimulates bone resorption by a process that is enhanced by PTH. The result is an increase in serum Ca2+ . Therefore, bone is an important reservoir of Ca2+ that can be mobilized to maintain serum levels. [Note: PTH and calcitriol also work together to prevent renal loss of Ca2+.] D. Distribution and requirement

1	D. Distribution and requirement Vitamin D occurs naturally in fatty fish, liver, and egg yolk. Milk, unless it is artificially fortified, is not a good source. The RDA for individuals ages 1–70 years is 15 µg/day and 20 µg/day if over age 70 years. Experts disagree, however, on the optimal level of vitamin D needed to maintain health. [Note: 1 µg vitamin D = 40 international units (IU).] Because breast milk is a poor source of vitamin D, supplementation is recommended for breastfed babies.

1	E. Clinical indications for vitamin D 1. Nutritional rickets: Vitamin D deficiency causes a net demineralization of bone, resulting in rickets in children and osteomalacia in adults (Fig. 28.25). Rickets is characterized by the continued formation of the collagen matrix of bone, but incomplete mineralization results in soft, pliable bones. In osteomalacia, demineralization of preexisting bones increases their susceptibility to fracture. Insufficient exposure to daylight and/or deficiencies in vitamin D consumption occur predominantly in infants and the elderly. Vitamin D deficiency is more common in the northern latitudes, because less vitamin D synthesis occurs in the skin as a result of reduced exposure to ultraviolet light. [Note: Loss-of-function mutations in the vitamin D receptor result in hereditary vitamin D– deficient rickets.] 2.

1	Renal osteodystrophy: Chronic kidney disease causes decreased ability to form active vitamin D as well as increased retention of PO43− , resulting in hyperphosphatemia and hypocalcemia. The low blood Ca2+ causes a rise in PTH and associated bone demineralization with release of Ca2+ and PO43− . Supplementation with vitamin D is an effective therapy. However, supplementation must be accompanied by PO43− reduction therapy to prevent further bone loss and precipitation of calcium phosphate crystals. 3. Hypoparathyroidism: Lack of PTH causes hypocalcemia and hyperphosphatemia. [Note: PTH increases phosphate excretion.] Patients may be treated with vitamin D and calcium supplementation. F. Toxicity

1	Hypoparathyroidism: Lack of PTH causes hypocalcemia and hyperphosphatemia. [Note: PTH increases phosphate excretion.] Patients may be treated with vitamin D and calcium supplementation. F. Toxicity Like all fat-soluble vitamins, vitamin D can be stored in the body and is only slowly metabolized. High doses (100,000 IU for weeks or months) can cause loss of appetite, nausea, thirst, and weakness. Enhanced Ca2+ absorption and bone resorption results in hypercalcemia, which can lead to deposition of calcium salts in soft tissue (metastatic calcification). The UL is 100 µg/day (4,000 IU/day) for individuals ages 9 years or older, with a lower level for those under age 9 years. [Note: Toxicity is only seen with use of supplements. Excess vitamin D produced in the skin is converted to inactive forms.] XIII. VITAMIN K

1	XIII. VITAMIN K The principal role of vitamin K is in the posttranslational modification of a number of proteins (most of which are involved with blood clotting), in which it serves as a coenzyme in the carboxylation of certain glutamic acid residues in these proteins. Vitamin K exists in several active forms, for example, in plants as phylloquinone (or vitamin K1), and in intestinal bacteria as menaquinone (or vitamin K2). A synthetic form of vitamin K, menadione, is able to be converted to K2. A. Function 1. γ-Carboxyglutamate formation: Vitamin K is required in the hepatic synthesis of the blood clotting proteins, prothrombin (factor [F]II) and FVII, FIX, and FX. (See online Chapter 35.) Formation of the functional clotting factors requires the vitamin K–dependent carboxylation of several glutamic acid residues to γ-carboxyglutamate (Gla) residues (Fig. 28.26). The carboxylation reaction requires γ-glutamyl carboxylase, O2,

1	CO2, and the hydroquinone form of vitamin K (which gets oxidized to the epoxide form). The formation of Gla residues is sensitive to inhibition by warfarin, a synthetic analog of vitamin K that inhibits vitamin K epoxide reductase (VKOR), the enzyme required to regenerate the functional hydroquinone form of vitamin K. 2. Prothrombin interaction with membranes: The Gla residues are good chelators of positively charged calcium ions, because of their two adjacent, negatively charged carboxylate groups. With prothrombin, for example, the prothrombin–calcium complex is able to bind to negatively charged membrane phospholipids on the surface of damaged endothelium and platelets. Attachment to membrane increases the rate at which the proteolytic conversion of prothrombin to thrombin can occur (Fig. 28.27). 3.

1	3. γ-Carboxyglutamate residues in other proteins: Gla residues are also present in proteins other than those involved in forming a blood clot. For example, osteocalcin and matrix Gla protein of bone and proteins C and S (involved in limiting the formation of blood clots) also undergo γcarboxylation. Figure28.27RoleofvitaminKinbloodcoagulation.CO2=carbondioxide. B. Distribution and requirement Vitamin K is found in cabbage, kale, spinach, egg yolk, and liver. The adequate intake for vitamin K is 120 µg/day for adult males and 90 µg for adult females. There is also synthesis of the vitamin by the gut microbiota. C. Clinical indications for vitamin K 1.

1	C. Clinical indications for vitamin K 1. Deficiency: A true vitamin K deficiency is unusual because adequate amounts are generally obtained from the diet and produced by intestinal bacteria. If the bacterial population in the gut is decreased (for example, by antibiotics), the amount of endogenously formed vitamin is decreased, and this can lead to hypoprothrombinemia in the marginally malnourished individual (for example, a debilitated geriatric patient). This condition may require supplementation with vitamin K to correct the bleeding tendency. In addition, certain cephalosporin antibiotics (for example, cefamandole) cause hypoprothrombinemia, apparently by a warfarin-like mechanism that inhibits VKOR. Consequently, their use in treatment is usually supplemented with vitamin K. Deficiency can also affect bone health. 2.

1	2. Deficiency in the newborn: Because newborns have sterile intestines, they initially lack the bacteria that synthesize vitamin K. Because human milk provides only about one fifth of the daily requirement for vitamin K, it is recommended that all newborns receive a single intramuscular dose of vitamin K as prophylaxis against hemorrhagic disease of the newborn. D. Toxicity Prolonged administration of large doses of menadione can produce hemolytic anemia and jaundice in the infant, because of toxic effects on the RBC membrane. Therefore, it is no longer used to treat vitamin K deficiency. No UL for the natural form has been set. XIV. VITAMIN E The E vitamins consist of eight naturally occurring tocopherols, of which αtocopherol is the most active (Fig. 28.28). Vitamin E functions as an antioxidant in prevention of nonenzymic oxidations (for example, oxidation of LDL (see p.

1	232) and peroxidation of polyunsaturated FA by O2 and free radicals). [Note: Vitamin C regenerates active vitamin E.] A. Distribution and requirements Vegetable oils are rich sources of vitamin E, whereas liver and eggs contain moderate amounts. The RDA for α-tocopherol is 15 mg/day for adults. The vitamin E requirement increases as the intake of polyunsaturated FA increases to limit FA peroxidation. B. Deficiency Newborns have low reserves of vitamin E, but breast milk (and formulas) contain the vitamin. Very-low-birth-weight infants may be given supplements to prevent the hemolysis and retinopathy associated with vitamin E deficiency. When observed in adults, deficiency is usually associated with defective lipid absorption or transport. [Note: Abetalipoproteinemia, caused by a defect in the formation of chylomicrons (and VLDL), results in vitamin E deficiency (see p. 231).] C. Clinical indications for vitamin E

1	C. Clinical indications for vitamin E Vitamin E is not recommended for the prevention of chronic disease, such as CVD or cancer. Clinical trials using vitamin E supplementation have been uniformly disappointing. For example, subjects in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study trial who received high doses of vitamin E not only lacked cardiovascular benefit but also had an increased incidence of stroke. [Note: Vitamins E and C are used to slow the progression of age-related macular degeneration.] D. Toxicity Vitamin E is the least toxic of the fat-soluble vitamins, and no toxicity has been observed at doses of 300 mg/day (UL = 1,000 mg/day). Populations consuming diets high in fruits and vegetables show decreased incidence of some chronic diseases. However, clinical trials have failed to show a definitive benefit from supplements of folic acid; vitamins A, C, or E; or antioxidant combinations for the prevention of cancer or CVD. XV. CHAPTER SUMMARY

1	XV. CHAPTER SUMMARY The vitamins are summarized in Figure 28.29 on pp. 396–397. Choose the ONE best answer. For Questions 28.1–28.5, match the vitamin deficiency to the clinical consequence. 8.1. Bleeding 8.2. Diarrhea and dermatitis 8.3. Neural tube defects 8.4. Night blindness (nyctalopia) 8.5. Sore, spongy gums and loose teeth

1	8.1. Bleeding 8.2. Diarrhea and dermatitis 8.3. Neural tube defects 8.4. Night blindness (nyctalopia) 8.5. Sore, spongy gums and loose teeth Correct answers = H, B, A, C, E. Vitamin K is required for formation of the γcarboxyglutamate residues in several proteins required for blood clotting. Consequently, a deficiency of vitamin K results in a tendency to bleed. Niacin deficiency is characterized by the three Ds: diarrhea, dermatitis, and dementia (and death, a fourth D, if untreated). Folic acid deficiency can result in neural tube defects in the developing fetus. Night blindness is one of the first signs of vitamin A deficiency. Rod cells in the retina detect white and black images and work best in low light, for example, at night. Rhodopsin, the visual pigment of the rod cells, consists of 11-cis retinal bound to the protein opsin. Vitamin C is required for the hydroxylation of proline and lysine during collagen synthesis.

1	Severe vitamin C deficiency (scurvy) results in defective connective tissue, characterized by sore and spongy gums, loose teeth, capillary fragility, anemia, and fatigue. 8.6. A 52-year-old woman presents with fatigue of several months’ duration. Blood studies reveal a macrocytic anemia, reduced levels of hemoglobin, elevated levels of homocysteine, and normal levels of methylmalonic acid. Which of the following is most likely deficient in this woman? A. Folic acid B. Folic acid and vitamin B12 C. Iron D. Vitamin C

1	A. Folic acid B. Folic acid and vitamin B12 C. Iron D. Vitamin C Correct answer = A. Macrocytic anemia is seen with deficiencies of folic acid, vitamin B12, or both. Vitamin B12 is utilized in only two reactions in the body: the remethylation of homocysteine (Hcy) to methionine, which also requires folic acid (as tetrahydrofolate [THF]), and the isomerization of methylmalonyl coenzyme A to succinyl coenzyme A, which does not require THF. The elevated Hcy and normal methylmalonic acid levels in the patient’s blood reflect a deficiency of folic acid as the cause of the macrocytic anemia. Iron deficiency causes microcytic anemia, as can vitamin C deficiency.

1	8.7. A 10-month-old African American girl, whose family recently located from Maine to Virginia, is being evaluated for the bowed appearance of her legs. The parents report that the baby is still being breastfed and takes no supplements. Radiologic studies confirm the suspicion of rickets caused by vitamin D deficiency. Which one of the following statements concerning vitamin D is correct? A. A deficiency results in an increased secretion of calbindin. B. Chronic kidney disease results in overproduction of 1,25dihydroxycholecalciferol (calcitriol). C. 25-Hydroxycholecalciferol (calcidiol) is the active form of the vitamin. D. It is required in the diet of individuals with limited exposure to sunlight. E. Its actions are mediated through binding to G protein–coupled receptors. F. It opposes the effect of parathyroid hormone.

1	E. Its actions are mediated through binding to G protein–coupled receptors. F. It opposes the effect of parathyroid hormone. Correct answer = D. Vitamin D is required in the diet of individuals with limited exposure to sunlight, such as those living at northern latitudes like Maine and those with dark skin. Note that breast milk is low in vitamin D, and the lack of supplementation increases the risk of a deficiency. Vitamin D deficiency results in decreased synthesis of calbindin. Chronic kidney disease decreases production of calcitriol (1,25-dihydroxycholecalciferol), the active form of the vitamin. Vitamin D binds to nuclear receptors and alters gene transcription. Its effects are synergistic with parathyroid hormone. 8.8. Why might a deficiency of vitamin B6 result in a fasting hypoglycemia? Deficiency of what other vitamin could also result in hypoglycemia?

1	8.8. Why might a deficiency of vitamin B6 result in a fasting hypoglycemia? Deficiency of what other vitamin could also result in hypoglycemia? Vitamin B6 is required for glycogen degradation by glycogen phosphorylase. A deficiency would result in fasting hypoglycemia. Additionally, a deficiency of biotin (required by pyruvate carboxylase of gluconeogenesis) would also result in fasting hypoglycemia. Micronutrients: Minerals 29 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Micronutrients: Minerals 29 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW Minerals are inorganic substances (elements) required in small amounts by the body. They function in a number of processes including formation of bones and teeth, fluid balance, nerve conduction, muscle contraction, signaling, and catalysis. [Note: Several minerals are essential enzyme cofactors.] Like the organic vitamins (see Chapter 28), minerals are micronutrients required in mg or µg amounts. Those required by adults in the largest amounts (>100 mg/day) are referred to as the macrominerals. Minerals required in amounts between 1 and 100 mg/day are the microminerals (trace minerals). Ultratrace minerals are required in amounts <1 mg/day (Fig. 29.1). [Note: The classification of specific minerals into these categories can vary among sources.] Mineral concentrations in the body are influenced by their rates of absorption and excretion. II. MACROMINERALS

1	II. MACROMINERALS The macrominerals include calcium (Ca2+), phosphorus ([P] as inorganic phosphate [Pi, or PO43−]), magnesium (Mg2+), sodium (Na+), chloride (Cl−), and potassium (K+). [Note: The free ionic forms are electrolytes.] A. Calcium and phosphorus These macrominerals are considered together because they are components of hydroxylapatite (Ca5[PO4]3OH), which makes up bones and teeth.

1	1. Calcium: Ca2+ is the most abundant mineral in the body, with ~98% being found in bones. The remainder is involved in a number of processes such as signaling, muscle contraction, and blood clotting. Ca2+ binds to a variety of proteins including calmodulin (see p. 133), phospholipase A2 (see p. 213), and protein kinase C (see p. 205) and alters their activity. [Note: Calbindin is a vitamin D–induced intracellular Ca2+-binding protein involved in Ca2+ absorption in the intestine (see p. 392).] Dairy products, many green vegetables (for example, broccoli, but not spinach), and fortified orange juice are good dietary sources. Although dietary deficiency syndromes are unknown, average Ca2+ intake in the United States is insufficient for optimal bone health. Toxicity is seen only with supplements (tolerable upper limit [UL] = 2,500 mg/day for adults). Hypercalcemia (elevated serum Ca2+) can result from overproduction of parathyroid hormone (PTH). This may cause constipation and kidney

1	(tolerable upper limit [UL] = 2,500 mg/day for adults). Hypercalcemia (elevated serum Ca2+) can result from overproduction of parathyroid hormone (PTH). This may cause constipation and kidney stones. Hypocalcemia (low serum Ca2+) can result from a deficiency of PTH or vitamin D. It can lead to bone demineralization (resorption). [Note: The hormonal regulation of serum Ca2+ levels was presented in the vitamin D section of Chapter 28 and is reviewed in 3. below.]

1	Bone mass increases from infancy through the early reproductive years and then shows an age-related loss in both men and women that increases the risk for fracture. This loss is greatest in postmenopausal Caucasian women. Some studies have shown that supplementation with Ca2+ and vitamin D decreases this risk.

1	2. Phosphorus: Free phosphate (Pi) is the most abundant intracellular anion. However, 85% of the body’s phosphorus is in the form of inorganic hydroxylapatite, with most of the remainder in intracellular organic compounds such as phospholipids, nucleic acids, ATP, and creatine phosphate. Phosphate is supplied as ATP for kinases and as Pi for phosphorylases (for example, glycogen phosphorylase, see p. 128). [Note: Its addition (by kinases) or removal (by phosphatases) is an important means of covalent regulation of enzymes (see Chapter 24).] Phosphorus is widely distributed in food (milk is a good source), and dietary deficiency is rare. Hypophosphatemia can be caused by refeeding carbohydrates to malnourished patients (refeeding syndrome, see p. 369), overuse of aluminum-containing antacids (aluminum chelates Pi), and increased urinary loss in response to increased production of PTH (see below). Muscle weakness is a common symptom. Hyperphosphatemia is caused primarily by decreased

1	(aluminum chelates Pi), and increased urinary loss in response to increased production of PTH (see below). Muscle weakness is a common symptom. Hyperphosphatemia is caused primarily by decreased PTH levels. The excess Pi can combine with Ca2+ and form crystals that deposit in soft tissue (metastatic calcification). [Note: The Ca2+/Pi ratio is important for bone formation (the ratio is ~2/1 in bone), and some experts are concerned that replacement of Ca2+-rich milk by Ca2+-poor, Pi-rich soft drinks can affect bone health.] 3. Hormonal regulation: Serum levels of Ca2+ and Pi are primarily controlled by calcitriol (1,25-dihydroxycholecalciferol, the active form of vitamin D) and PTH, both of which respond to a decrease in serum Ca2+ . Calcitriol, produced by the kidneys, increases serum Ca2+ and Pi by increasing bone resorption and intestinal absorption and renal reabsorption of Ca2+ and Pi (Fig. 29.2). PTH (from the parathyroid glands) increases serum Ca2+ by increasing bone resorption,

1	and Pi by increasing bone resorption and intestinal absorption and renal reabsorption of Ca2+ and Pi (Fig. 29.2). PTH (from the parathyroid glands) increases serum Ca2+ by increasing bone resorption, increasing renal reabsorption of Ca2+ , and activating the renal 1-hydroxylase that produces calcitriol from calcidiol (see p. 390) (Fig. 29.3). In contrast to calcitriol, PTH decreases Pi reabsorption in the kidneys, lowering serum Pi. [Note: High serum Pi increases PTH and decreases calcitriol.] A third hormone, calcitonin (from the C cells of the thyroid gland), responds to elevated serum Ca2+ levels by promoting bone mineralization and increasing renal excretion of Ca2+ (and Pi).

1	B. Magnesium About 60% of the body’s Mg2+ is in bone, but it accounts for just 1% of the bone mass. The mineral is required by a variety of enzymatic reactions, including phosphorylation by kinases (Mg2+ binds the ATP cosubstrate) and phosphodiester bond formation by DNA and RNA polymerases. Mg2+ is widely distributed in foods, but the average intake in the United States is below the recommended level. Hypomagnesemia can result from decreased absorption or increased excretion of Mg2+ . Symptoms include hyperexcitability of skeletal muscles and nerves and cardiac arrhythmias. With hypermagnesemia, hypotension is seen. [Note: Magnesium sulfate is used in the treatment of preeclampsia, a hypertensive disorder of pregnancy.] C. Sodium, chloride, and potassium

1	These macrominerals are considered together because they play important roles in several physiologic processes. For example, they maintain water balance, osmotic equilibrium, acid–base balance (pH), and the electrical gradients across cell membranes (membrane potential) that are essential for the functioning of neurons and myocytes. [Note: These processes are discussed in Lippincott’s Illustrated Reviews: Physiology.] 1. Sodium and chloride: Na+ and Cl− are primarily extracellular electrolytes. They are readily absorbed from foods containing salt (NaCl), much of which comes from processed foods. [Note: Na+ is required for the intestinal absorption (and renal reabsorption) of glucose and galactose (see p. 87) and free amino acids (see p. 249) by Na+-linked transporters. Cl− is used to form hydrochloric acid required for digestion (see p. 248).] In the United States, the average daily consumption of NaCl is 1.5–3 times the adequate intake (AI) of 3.8 mg/day (UL = 5.8 g/day). Dietary

1	hydrochloric acid required for digestion (see p. 248).] In the United States, the average daily consumption of NaCl is 1.5–3 times the adequate intake (AI) of 3.8 mg/day (UL = 5.8 g/day). Dietary deficiency is rare.

1	a. Hypertension: Na+ intake is related to blood pressure (BP). Ingestion of Na+ stimulates thirst centers in the brain and secretion of antidiuretic hormone from the pituitary, leading to water retention. This results in an increase in plasma volume and, consequently, an increase in BP. Chronic hypertension can damage the heart, kidneys, and blood vessels. Modest reductions in Na+ intake have been shown to result in modest reductions in BP. [Note: Some populations (for example, African Americans) are “salt sensitive” and have larger responses to Na+.] b.

1	Hyper-and hyponatremia: Hypernatremia, typically caused by excess water loss, and hyponatremia, typically caused by decreased ability to excrete water, can result in severe brain damage. [Note: Chronic hyponatremia increases Ca2+ excretion and can result in osteoporosis (low bone mass).] 2. Potassium: In contrast to Na+, K+ is primarily an intracellular electrolyte. [Note: The concentration differential of Na+ and K+ across the cell membrane is maintained by the Na+/K+ ATPase (Fig. 29.4).] In contrast to Na+ and Cl−, K+ (like Mg2+) is underingested in Western diets because its primary sources, fruits and vegetables, are underingested. [Note: Increasing dietary K+ decreases BP by increasing Na+ excretion.] There is a narrow range for normal serum K+ levels, and even modest changes (up or down, resulting in hyper-or hypokalemia) can result in cardiac arrhythmias and skeletal muscle weakness. [Note: Hypokalemia can result from the inappropriate use of laxatives to lose weight.] No UL for

1	resulting in hyper-or hypokalemia) can result in cardiac arrhythmias and skeletal muscle weakness. [Note: Hypokalemia can result from the inappropriate use of laxatives to lose weight.] No UL for K+ has been established.

1	III. MICROMINERALS (TRACE MINERALS) The trace minerals include copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn). They are required by adults in amounts between 1 and 100 mg/day. A. Copper Cu is a key component of several enzymes that play critical functions in the body (Fig. 29.5). These include ferroxidases such as the ceruloplasmin and hephaestin involved in the oxidation of ferrous iron (Fe2+) to the ferric form (Fe3+) that is required for its intracellular storage or transport through blood (see B.1. below). Meat, shellfish, nuts, and whole grains are good dietary sources of Cu. Dietary deficiency is uncommon. If a deficiency does develop, anemia may be seen because of the effect on Fe metabolism. Toxicity from dietary sources is rare (UL = 10 mg/day). Menkes syndrome and Wilson disease are genetic causes of Cu deficiency and Cu overload, respectively. 1.

1	1. Menkes syndrome: In Menkes syndrome (“kinky hair” disease), a rare X-linked (1:140,000 males) disorder, efflux of dietary Cu out of intestinal enterocytes into the circulation by a Cu-transporting ATPase (ATP7A) is impaired. This results in systemic Cu deficiency. Consequently, urinary and serum free (unbound) Cu are low, as is the concentration of ceruloplasmin, which carries over 90% of the Cu in the circulation (Fig. 29.6). Progressive neurologic degeneration and connective tissue disorders are seen, as are changes to hair. Parenteral administration of Cu has been used as a treatment with varying success. [Note: The mildest form of Menkes syndrome is called occipital horn syndrome.] 2.

1	Wilson disease: In Wilson disease, an autosomal-recessive (AR) disorder affecting 1:35,000 live births, efflux of excess Cu from the liver by ATP7B is impaired. Cu accumulates in the liver; leaks into the blood; and is deposited in the brain, eyes, kidneys, and skin. In contrast to Menkes syndrome, urinary and serum free Cu are high (see Fig. 29.6). Hepatic dysfunction and neurologic and psychiatric symptoms are seen. Kayser-Fleischer rings (corneal deposits of Cu) may be present (Fig. 29.7). Life-long use of Cu-chelating agents, such as penicillamine, is the treatment. The bioavailability (percent of the amount ingested that is able to be absorbed) of a mineral can be influenced by other minerals. For example, excess Zn decreases the absorption of Cu, and Cu is needed for the absorption of Fe. B. Iron

1	B. Iron The adult body typically contains 3–4 g of Fe. It is a component of many proteins, both catalytic (for example, hydroxylases such as prolyl hydroxylase, see p. 47) and noncatalytic. Iron can be linked to sulfur (S) as seen in the Fe–S proteins of the electron transport chain (see p. 75), or it can be part of the heme prosthetic group (see p. 25) in proteins such as hemoglobin (~70% of all Fe), myoglobin, and the cytochromes. [Note: Free ionic Fe is toxic because it can cause production of the hydroxyl radical, a reactive oxygen species (ROS).] Dietary Fe is available as Fe2+ in heme (animal sources) and Fe3+ in nonheme sources (plants). Heme iron is less abundant, but it is better absorbed. Meat, poultry, some shellfish, ready-toeat cereals, lentils, and molasses are good dietary sources of Fe. About 10% of ingested Fe is absorbed. This amount, ~1−2 mg/day, is sufficient to replace Fe lost from the body primarily by the sloughing of cells. 1.

1	Absorption, storage, and transport: Intestinal uptake of heme is by a heme carrier protein (Fig. 29.8). Within the enterocytes, heme oxygenase releases Fe2+ from heme (see p. 282). Nonheme Fe is taken up via the apical membrane protein divalent metal ion transporter-1 (DMT-1). [Note: Vitamin C enhances absorption of nonheme Fe because it is the coenzyme for duodenal cytochrome b (Dcytb), a ferrireductase that reduces Fe3+ to Fe2+.] Absorbed Fe2+ from heme and nonheme sources has two possible fates: It can be 1) oxidized to Fe3+ and stored by the intracellular protein ferritin (up to 4,500 Fe3+/ferritin) or 2) transported out of the enterocyte by the basolateral membrane protein ferroportin, oxidized by the Cu-containing membrane protein hephaestin, and taken up by the plasma transport protein transferrin (2 Fe3+/transferrin), as shown in Figure 29.8. [Note: Cells other than enterocytes use the Cu-containing plasma protein ceruloplasmin in place of hephaestin.] In normal individuals,

1	transferrin (2 Fe3+/transferrin), as shown in Figure 29.8. [Note: Cells other than enterocytes use the Cu-containing plasma protein ceruloplasmin in place of hephaestin.] In normal individuals, transferrin (Tf) is about one third saturated with Fe3+ . Ferroportin, the only known exporter of Fe from cells to the blood in humans, is regulated by the hepatic peptide hepcidin that induces internalization and lysosomal degradation of ferroportin. Therefore, hepcidin is the central molecule in Fe homeostasis. [Note: Transcription of hepcidin is suppressed when Fe is deficient.] 2.

1	Recycling: Macrophages phagocytose old and/or damaged red blood cells (RBC), freeing heme Fe that is sent out of the cells via ferroportin, oxidized by ceruloplasmin, and transported by Tf as described above. This recycled Fe meets ~90% of our daily need, which is predominantly for erythropoiesis. 3. Uptake: Tf-bound Fe3+ from enterocytes and macrophages binds to receptors (TfR) on erythroblasts and other Fe-requiring cells and is taken up by receptor-mediated endocytosis. The Fe3+ is released from Tf for use (or stored on ferritin), and the TfR (and Tf) is recycled in a process similar to the receptor-mediated endocytosis seen with low-density lipoprotein particles (see p. 231). [Note: Regulation of the translation of the messenger RNA for ferritin and the TfR by iron regulatory proteins and iron-responsive elements is discussed on p. 474.] 4.

1	Deficiency: Fe deficiency can result in a microcytic, hypochromic anemia (Fig. 29.9), the most common anemia in the United States, as a result of decreased hemoglobin synthesis and, consequently, decreased RBC size. Treatment is the administration of Fe.

1	5. Excess: Fe overload can occur with accidental ingestion. [Note: Acute Fe poisoning is the most common cause of poisoning deaths of children age <6 years (UL = 40 mg/day for children, 45 mg/day for adults).] Treatment is use of an Fe chelator. Overload can also occur with genetic defects. An example is hereditary hemochromatosis (HH), an AR disorder of Fe overload found primarily in those of Northern European ancestry. It is most commonly caused by mutations to the HFE (high iron) gene. Hyperpigmentation with hyperglycemia (“bronze diabetes”) and damage to the liver (a major storage site for Fe), pancreas, and heart may be seen. In HH, serum Fe and Tf saturation are elevated. Treatment is phlebotomy or use of Fe chelators. [Note: Fe overload is seen with mutations to proteins of Fe metabolism that result in inappropriately low levels of hepcidin. It can result in hemosiderosis (the deposition of hemosiderin, an intracellular, insoluble storage form of Fe).] C. Manganese

1	C. Manganese Mn is important for the function of several enzymes (Fig. 29.10). Whole grains, legumes (for example, beans and peas), nuts, and tea (especially green tea) are good sources of the mineral. Consequently, Mn deficiency in humans is rare. Toxicity from foods and/or supplements is also rare (UL = 11 mg/day for adults). D. Zinc

1	Zn plays important structural and catalytic functions in the body. Zinc fingers are supersecondary structures (motifs, see p. 18) in proteins (for example, transcription factors) that bind to DNA and regulate gene expression (Fig. 29.11). Hundreds of enzymes require Zn for activity. Examples include alcohol dehydrogenase, which oxidizes ethanol to acetaldehyde (see p. 317); carbonic anhydrase, which is important in the bicarbonate buffer system (see p. 30); porphobilinogen synthase of heme synthesis, which is inhibited by lead (lead replaces the zinc; see p. 279); and the nonmitochondrial isoform of superoxide dismutase (SOD), which also requires Cu (see Fig. 29.5). Dietary sources of Zn include meat, fish, eggs, and dairy products. Phytates (phosphate storage molecules in some plant products) irreversibly bind Zn in the intestine, decreasing its absorption, and can result in a deficiency. [Note: Phytates may also bind Ca2+ and nonheme Fe.] Several drugs (for example, penicillamine)

1	irreversibly bind Zn in the intestine, decreasing its absorption, and can result in a deficiency. [Note: Phytates may also bind Ca2+ and nonheme Fe.] Several drugs (for example, penicillamine) chelate metals, and their use may cause Zn deficiency. [Note: Severe deficiency is seen with a defect in the intestinal transporter for Zn that results in the malabsorption disorder acrodermatitis enteropathica. Symptoms include rashes, slowed growth and development, diarrhea, and immune deficiencies. Vision problems may also occur because Zn is needed in the metabolism of vitamin A.]

1	Eukaryotic cells infected with bacteria can restrict availability of the essential micronutrients Fe, Mn, and Zn to the pathogens. This decreases the intracellular survival of the pathogen and is known as “nutritional immunity.” E. Other microminerals Chromium (Cr) and fluorine (F) also play roles in the body. Cr potentiates the action of insulin by an unknown mechanism. It is found in fruits, vegetables, dairy products, and meat. F (as fluoride [F−]) is added to water in many parts of the world to reduce the incidence of dental caries (Fig. 29.12). F− replaces the hydroxyl group of hydroxylapatite, forming fluoroapatite that is more resistant to the enamel-dissolving acid produced by mouth bacteria. IV. ULTRATRACE MINERALS The ultratrace minerals include iodine (I), selenium (Se), and molybdenum (Mo). They are required by adults in amounts <1 mg/day. A. Iodine

1	A. Iodine I is utilized in the synthesis of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) that are required for development, growth, and metabolism. Circulating iodide (I−) is taken up (“trapped”) and concentrated in the epithelial follicular cells of the thyroid gland. It then is sent into the colloid of the follicular lumen where it is oxidized to iodine (I2) by thyroperoxidase (TPO), as shown in Figure 29.13. TPO then uses I2 to iodinate selected tyrosine residues in thyroglobulin (Tg), forming monoiodinated tyrosine (MIT) and diiodinated tyrosine (DIT), as shown in Figure 29.14. [Note: Tg is synthesized and secreted into colloid by follicular cells.] The coupling of two DIT on Tg gives T4, whereas coupling one MIT and one DIT gives T3. The iodinated Tg is endocytosed and stored in follicular cells until needed, at which time it is proteolytically digested to release T3 and T4, which are secreted into the circulation (see Fig. 29.13).

1	Under normal conditions, ~90% of secreted thyroid hormone is T4 that is carried by transthyretin. In target tissues (for example, the liver and developing brain), T4 is converted to T3 (the more active form) by Se-containing deiodinases. T3 binds to a nuclear receptor that binds DNA at thyroid response elements and functions as a transcription factor. [Note: Thyroid hormone production is controlled by thyrotropin (thyroid-stimulating hormone ([TSH]) from the anterior pituitary. TSH secretion is itself controlled by thyrotropin-releasing hormone (TRH) from the hypothalamus.] 1.

1	Hypothyroidism: Underingestion of I can result in goiter (enlargement of the thyroid in response to excessive stimulation by TSH), as shown in Figure 29.15. More severe deficiency results in hypothyroidism that is characterized by fatigue, weight gain, decreased thermogenesis, and decreased metabolic rate (see p. 359). If hormone deficiency occurs during fetal and infant development (congenital hypothyroidism), irreversible intellectual disability (formerly called “cretinism”), hearing loss, spasticity, and short stature can result. In the United States, dairy products, seafood, and meat are the primary sources of I. The use of iodized salt has greatly reduced dietary I deficiency. [Note: Autoimmune destruction of TPO is a cause of Hashimoto thyroiditis (a primary hypothyroidism).] 2.

1	Hyperthyroidism: This condition is the result of overproduction of thyroid hormone. Although it can be caused by overingestion of I-containing supplements (UL = 1.1 g/day for adults), the most common cause of hyperthyroidism is Graves disease, in which an antibody that mimics the effect of TSH is produced, resulting in dysregulated production of thyroid hormone. This can cause nervousness, weight loss, increased perspiration and heart rate, protruding eyes (exophthalmos, Fig. 29.16), and goiter. B. Selenium

1	B. Selenium Se is present in ~25 human proteins (selenoproteins) as a constituent of the amino acid selenocysteine, which is derived from serine (see p. 268). Selenoproteins include glutathione peroxidase that oxidizes glutathione in the reduction of hydrogen peroxide, a ROS, to water (see p. 148); thioredoxin reductase that reduces thioredoxin, a coenzyme of ribonucleotide reductase (see p. 297); and deiodinases that remove I from thyroid hormones. Meat, dairy products, and grains are important dietary sources. Keshan disease, first identified in China, is a cardiomyopathy caused by eating foods produced from Se-deficient soil. Toxicity (selenosis) caused by overingestion of supplements causes brittle nails and hair. Cutaneous and neurologic effects may also be seen (UL = 400 µg in adults). C. Molybdenum

1	C. Molybdenum Mo functions as a cofactor for a small number of mammalian oxidases (Fig. 29.17). Legumes are important dietary sources. No dietary deficiency syndromes are known. Mo has low toxicity in humans (UL = 2 mg/day in adults). Cobalt (Co), an ultratrace mineral, is a component of vitamin B12 (cobalamin, see p. 379), which is required as methylcobalamin in the remethylation of homocysteine to methionine (see p. 264) or adenosylcobalamin in the isomerization of methylmalonyl coenzyme A (CoA) to succinyl CoA (see p. 194). No Recommended Dietary Allowance or Daily Reference Intake (see p. 358) has been established for Co. V. CHAPTER SUMMARY The minerals are summarized in Figure 29.18 on p. 408. For Questions 29.1–29.7, match the mineral to the most appropriate description. A. Calcium B. Chloride C. Copper D. Iodine E. Iron F. Magnesium G. Manganese H. Molybdenum I. Phosphorus J. Potassium K. Selenium L. Sodium

1	A. Calcium B. Chloride C. Copper D. Iodine E. Iron F. Magnesium G. Manganese H. Molybdenum I. Phosphorus J. Potassium K. Selenium L. Sodium M. Zinc 9.1. Elevated levels of which mineral may result in hypertension in certain populations? 9.2. Which mineral is the major extracellular anion? 9.3. A decrease of which mineral is seen in refeeding syndrome and with overuse of aluminum-containing antacids? 9.4. Which mineral is a constituent of some amino acids found in proteins involved in antioxidant defense, thyroid hormone metabolism, and redox reactions? 9.5. Which mineral is required for the formation of a supersecondary protein structure that allows binding to DNA? (Its deficiency can result in a dermatitis.) 9.6. Deficiency of which mineral can cause bone pain, tetany (intermittent muscle spasms), paresthesia (a “pins and needles” sensation), and an increased tendency to bleed? 9.7. Deficiency of which mineral can result in goiter and a decreased metabolic rate?

1	Correct answers = L, B, I, K, M, A, D. Hypernatremia (elevation of serum sodium) can lead to water retention that can cause hypertension in salt-sensitive populations (for example, African Americans). Chloride is the major extracellular anion. [Note: Sodium is the major extracellular cation, potassium is the major intracellular cation, and phosphate is the major intracellular anion. The concentration differential across the membrane is maintained by active transport.] Carbohydrate metabolism involves the generation of phosphorylated intermediates. Refeeding severely malnourished individuals traps phosphate and results in hypophosphatemia. Muscle weakness is a common symptom. Selenocysteine, an amino acid formed from serine and selenium, is found in proteins (selenoproteins) such as glutathione peroxidase, deiodinases, and thioredoxin reductase. Zinc fingers are a type of structural motif found in proteins (for example, transcription factors) that bind to DNA. Severe deficiency of zinc

1	peroxidase, deiodinases, and thioredoxin reductase. Zinc fingers are a type of structural motif found in proteins (for example, transcription factors) that bind to DNA. Severe deficiency of zinc as a result of mutations to its intestinal transporter can result in acrodermatitis enteropathica, which is characterized by dermatitis, diarrhea, and alopecia. Calcium is required for bone mineralization, muscle contraction, nerve conduction, and blood clotting. Its deficiency will affect all of these processes. Thyroid hormones are iodinated tyrosines released by proteolytic digestion of thyroglobulin. Underingestion of iodine causes enlargement of the thyroid in an attempt to increase hormone synthesis. [Note: Goiter can also result if too much hormone is made, as in Graves disease, or if too little is made, as in Hashimoto disease. Both are autoimmune diseases.] Thyroid hormone increases the resting metabolic rate.

1	9.8. DiGeorge syndrome is a congenital condition that results in structural anomalies and failure of the thymus and parathyroid glands to develop. Clinical manifestations include recurrent infections as a consequence of a deficiency in T cells. Which one of the following is an expected clinical consequence of the deficiency in parathyroid hormone? A. Increased bone resorption B. Increased calcium reabsorption in the kidney C. Increased serum calcitriol D. Increased serum phosphate Correct answer = D. Parathyroid hormone (PTH) increases bone resorption (demineralization) resulting in the release of calcium and phosphate. It also increases the renal reabsorption of calcium, because PTH activates the renal hydroxylase that converts calcidiol to calcitriol. PTH also increases the renal excretion of phosphate. With the hypoparathyroidism of DiGeorge syndrome, all of these activities of PTH are impaired. Consequently, hypocalcemia and hyperphosphatemia are seen.

1	For questions 29.9 and 29.10, match the signs and symptoms to the pathology. A. Graves disease B. Hereditary hemochromatosis C. Hypercalcemia D. Hyperphosphatemia E. Keshan disease F. Menkes syndrome G. Selenosis H. Wilson disease 9.9. A 28-year-old male is seen for complaints of recent, severe, upper-rightquadrant pain. He also reports some difficulty with fine motor tasks. No jaundice is observed on physical examination. Laboratory tests were remarkable for elevated liver function tests (serum aspartate and alanine aminotransferases) and elevated urinary calcium and phosphate. Ophthalmology consult revealed Kayser-Fleischer rings in the cornea. The patient was started on penicillamine and zinc.

1	Correct answer = H. The patient has Wilson disease, an autosomal-recessive disorder that decreases copper efflux from the liver because of mutations to the hepatic copper transport protein ATP7B. Some copper leaks into the blood and is deposited in the brain, eyes, kidney, and skin. This results in liver and kidney damage, neurologic effects, and corneal changes caused by the excess copper. Administration of the metal chelator penicillamine is the treatment. [Note: Because zinc is also chelated, supplementation with zinc is common.] Graves disease results in hyperthyroidism. Hereditary hemochromatosis is a disorder of iron overload. Keshan disease is the result of selenium deficiency, whereas selenosis is caused by selenium excess. Menkes syndrome is the result of a systemic deficiency in copper as a result of mutations to ATP7A, an intestinal copper transport protein.

1	9.10. A 52-year-old female is seen because of unplanned changes in the pigmentation of her skin that give her a tanned appearance. Physical examination shows hyperpigmentation, hepatomegaly, and mild scleral icterus. Laboratory tests are remarkable for elevated serum transaminases (liver function tests) and fasting blood glucose. Results of other tests are pending. Correct answer = B. The patient has hereditary hemochromatosis, a disease of iron overload that results from inappropriately low levels of hepcidin caused primarily by mutations to the HFE (high iron) gene. Hepcidin regulates ferroportin, the only known iron export protein in humans, by increasing its degradation. The increase in iron with hepcidin deficiency causes hyperpigmentation and hyperglycemia (“bronze diabetes”). Phlebotomy or use of iron chelators is the treatment. [Note: Pending lab tests would show an increase in serum iron and transferrin saturation.] UNIT VII Storage and Expression of Genetic Information

1	UNIT VII Storage and Expression of Genetic Information DNA Structure, Replication, and Repair For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Nucleic acids are required for the storage and expression of genetic information. There are two chemically distinct types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid ([RNA] see Chapter 31). DNA, the repository of genetic information (or, genome), is present not only in chromosomes in the nucleus of eukaryotic organisms, but also in mitochondria and the chloroplasts of plants. Prokaryotic cells, which lack nuclei, have a single chromosome but may also contain nonchromosomal DNA in the form of plasmids. The genetic information found in DNA is copied and transmitted to daughter cells through DNA replication. The DNA contained in a fertilized egg encodes the information that directs the development of an organism. This development may involve the production of billions of cells. Each cell is specialized, expressing only those functions that are required for it to perform its role in maintaining the organism. Therefore, DNA must be able not only to replicate

1	of cells. Each cell is specialized, expressing only those functions that are required for it to perform its role in maintaining the organism. Therefore, DNA must be able not only to replicate precisely each time a cell divides, but also to have the information that it contains be selectively expressed. Transcription (RNA synthesis) is the first stage in the expression of genetic information (see Chapter 31). Next, the code contained in the nucleotide sequence of messenger RNA molecules is translated (protein synthesis; see Chapter 32), thus completing gene expression. The regulation of gene expression is discussed in Chapter 33.

1	The flow of information from DNA to RNA to protein is termed the “central dogma” of molecular biology (Fig. 30.1) and is descriptive of all organisms, with the exception of some viruses that have RNA as the repository of their genetic information. II. DNA STRUCTURE DNA is a polymer of deoxyribonucleoside monophosphates (dNMP) covalently linked by 3′→5′-phosphodiester bonds. With the exception of a few viruses that contain single-stranded DNA (ssDNA), DNA exists as a double-stranded molecule (dsDNA), in which the two strands wind around each other, forming a double helix. [Note: The sequence of the linked dNMP is primary structure, whereas the double helix is secondary structure.] In eukaryotic cells, DNA is found associated with various types of proteins (known collectively as nucleoprotein) present in the nucleus, whereas the protein–DNA complex is present in a non–membrane-bound region known as the nucleoid in prokaryotes. A. 3′→5′-Phosphodiester bonds

1	A. 3′→5′-Phosphodiester bonds Phosphodiester bonds join the 3′-hydroxyl group of the deoxypentose of one nucleotide to the 5′-hydroxyl group of the deoxypentose of an adjacent nucleotide through a phosphoryl group (Fig. 30.2). The resulting long, unbranched chain has polarity, with both a 5′-end (the end with the free phosphate) and a 3′-end (the end with the free hydroxyl) that are not attached to other nucleotides. By convention, the bases located along the resulting deoxyribose-phosphate backbone are always written in sequence from the 5′-end of the chain to the 3′-end. For example, the sequence of bases in the DNA shown in Figure 30.2D (5′-TACG-3′) is read “thymine, adenine, cytosine, guanine.” Phosphodiester linkages between nucleotides can be hydrolyzed enzymatically by a family of nucleases, deoxyribonucleases for DNA and ribonucleases for RNA, or cleaved hydrolytically by chemicals. [Note: Only RNA is cleaved by alkali.] B. Double helix

1	In the double helix, the two chains are coiled around a common axis called the helical axis. The chains are paired in an antiparallel manner (that is, the 5′-end of one strand is paired with the 3′-end of the other strand), as shown in Figure 30.3. In the DNA helix, the hydrophilic deoxyribose-phosphate backbone of each chain is on the outside of the molecule, whereas the hydrophobic bases are stacked inside. The overall structure resembles a twisted ladder. The spatial relationship between the two strands in the helix creates a major (wide) groove and a minor (narrow) groove. These grooves provide access for the binding of regulatory proteins to their specific recognition sequences along the DNA chain. [Note: Certain anticancer drugs, such as dactinomycin (actinomycin D), exert their cytotoxic effect by intercalating into the narrow groove of the DNA double helix, thereby interfering with DNA (and RNA) synthesis.] 1. Base-pairing: The bases of one strand of DNA are paired with the

1	effect by intercalating into the narrow groove of the DNA double helix, thereby interfering with DNA (and RNA) synthesis.] 1. Base-pairing: The bases of one strand of DNA are paired with the bases of the second strand, so that an adenine (A) is always paired with a thymine (T), and a cytosine (C) is always paired with a guanine (G). [Note: The base pairs are perpendicular to the helical axis (see Fig. 30.3).] Therefore, one polynucleotide chain of the DNA double helix is always the complement of the other. Given the sequence of bases on one chain, the sequence of bases on the complementary chain can be determined (Fig. 30.4). [Note: The specific base-pairing in DNA leads to the Chargaff rule, which states that in any sample of dsDNA, the amount of A equals the amount of T, the amount of G equals the amount of C, and the total amount of purines (A + G) equals the total amount of pyrimidines (T + C).] The base pairs are held together by hydrogen bonds: two between A and T and three

1	of G equals the amount of C, and the total amount of purines (A + G) equals the total amount of pyrimidines (T + C).] The base pairs are held together by hydrogen bonds: two between A and T and three between G and C (Fig. 30.5). These hydrogen bonds, plus the hydrophobic interactions between the stacked bases, stabilize the structure of the double helix.

1	2. DNA strand separation: The two strands of the double helix separate when hydrogen bonds between the paired bases are disrupted. Disruption can occur in the laboratory if the pH of the DNA solution is altered so that the nucleotide bases ionize, or if the solution is heated. [Note: Covalent phosphodiester bonds are not broken by such treatment.] When DNA is heated, the temperature at which one half of the helical structure is lost is defined as the melting temperature (Tm). The loss of helical structure in DNA, called denaturation, can be monitored by measuring its absorbance at 260 nm. [Note: ssDNA has a higher relative absorbance at this wavelength than does dsDNA.] Because there are three hydrogen bonds between G and C but only two between A and T, DNA that contains high concentrations of A and T denatures at a lower temperature than does G-and C-rich DNA (Fig. 30.6). Under appropriate conditions, complementary DNA strands can reform the double helix by the process called

1	of A and T denatures at a lower temperature than does G-and C-rich DNA (Fig. 30.6). Under appropriate conditions, complementary DNA strands can reform the double helix by the process called renaturation (or, reannealing). [Note: Separation of the two strands over short regions occurs during both DNA and RNA synthesis.] 3. Structural forms: There are three major structural forms of DNA: the B form (described by Watson and Crick in 1953), the A form, and the Z form. The B form is a right-handed helix with 10 base pairs (bp) per 360° turn (or twist) of the helix, and with the planes of the bases perpendicular to the helical axis. Chromosomal DNA is thought to consist primarily of B-DNA (Fig. 30.7 shows a space-filling model of B-DNA). The A form is produced by moderately dehydrating the B form. It is also a right-handed helix, but there are 11 bp per turn, and the planes of the base pairs are tilted 20° away from the perpendicular to the helical axis. The conformation found in DNA–RNA

1	form. It is also a right-handed helix, but there are 11 bp per turn, and the planes of the base pairs are tilted 20° away from the perpendicular to the helical axis. The conformation found in DNA–RNA hybrids (see p. 418) or RNA–RNA double-stranded regions is probably very close to the A form. Z-DNA is a left-handed helix that contains 12 bp per turn (see Fig. 30.7). [Note: The deoxyribose-phosphate backbone zigzags, hence, the name Z-DNA.] Stretches of Z-DNA can occur naturally in regions of DNA that have a sequence of alternating purines and pyrimidines (for example, poly GC). Transitions between the B and Z helical forms of DNA may play a role in regulating gene expression.

1	C. Linear and circular DNA molecules

1	Each chromosome in the nucleus of a eukaryote consists of one long, linear molecule of dsDNA, which is bound by a complex mixture of proteins (histone and nonhistone, see p. 425) to form chromatin. Eukaryotes have closed, circular, dsDNA molecules in their mitochondria, as do plant chloroplasts. A prokaryotic organism typically contains a single, circular, dsDNA molecule. [Note: Circular DNA is “supercoiled,” that is, the double helix crosses over on itself one or more times. Supercoiling can result in overwinding (positive supercoiling) or underwinding (negative supercoiling) of DNA. Supercoiling, a type of tertiary structure, compacts DNA.] Each prokaryotic chromosome is associated with nonhistone proteins that help compact the DNA to form a nucleoid. In addition, most species of bacteria also contain small, circular, extrachromosomal DNA molecules called plasmids. Plasmid DNA carries genetic information and undergoes replication that may or may not be synchronized to chromosomal

1	also contain small, circular, extrachromosomal DNA molecules called plasmids. Plasmid DNA carries genetic information and undergoes replication that may or may not be synchronized to chromosomal division. [Note: The use of plasmids as vectors in recombinant DNA technology is described in Chapter 34.]

1	Plasmids may carry genes that convey antibiotic resistance to the host bacterium and may facilitate the transfer of genetic information from one bacterium to another. III. STEPS IN PROKARYOTIC DNA REPLICATION

1	When the two strands of dsDNA are separated, each can serve as a template for the replication (synthesis) of a new complementary strand. This produces two daughter molecules, each of which contains two DNA strands (one old, one new) in an antiparallel orientation (see Fig. 30.3). This process is called semiconservative replication because, although the parental duplex is separated into two halves (and, therefore, is not conserved as an entity), each of the parental strands remains intact in one of the two new duplexes (Fig. 30.8). The enzymes involved in DNA replication are template-directed, magnesium (Mg2+)requiring polymerases that can synthesize the complementary sequence of each strand with extraordinary fidelity. The reactions described in this section were first known from studies of the bacterium Escherichia coli (E. coli), and the description given below refers to the process in prokaryotes. DNA synthesis in higher organisms is more complex but involves the same types of

1	of the bacterium Escherichia coli (E. coli), and the description given below refers to the process in prokaryotes. DNA synthesis in higher organisms is more complex but involves the same types of mechanisms. In either case, initiation of DNA replication commits the cell to continue the process until the entire genome has been replicated.

1	A. Complementary strand separation In order for the two complementary strands of the parental dsDNA to be replicated, they must first separate (or “melt”) over a small region, because the polymerases use only ssDNA as a template. In prokaryotic organisms, DNA replication begins at a single, unique nucleotide sequence, a site called the origin of replication, or ori (oriC in E. coli), as shown in Figure 30.9A. [Note: This sequence is referred to as a consensus sequence, because the order of nucleotides is essentially the same at each site.] The ori includes short, AT-rich segments that facilitate melting. In eukaryotes, replication begins at multiple sites along the DNA helix (Fig. 30.9B). Having multiple origins of replication provides a mechanism for rapidly replicating the great length of eukaryotic DNA molecules. B. Replication fork formation

1	B. Replication fork formation As the two strands unwind and separate, synthesis occurs at two replication forks that move away from the origin in opposite directions (bidirectionally), generating a replication bubble (see Fig. 30.9). [Note: The term “replication fork” derives from the Y-shaped structure in which the tines of the fork represent the separated strands (Fig. 30.10).] 1. Required proteins: Initiation of DNA replication requires the recognition of the origin (start site) by a group of proteins that form the prepriming complex. These proteins are responsible for melting at the ori, maintaining the separation of the parental strands, and unwinding the double helix ahead of the advancing replication fork. In E. coli, these proteins include the following. a.

1	a. DnaA protein: DnaA protein initiates replication by binding to specific nucleotide sequences (DnaA boxes) within oriC. Binding causes an AT-rich region (the DNA unwinding element) in the origin to melt. Melting (strand separation) results in a short, localized region of ssDNA. b. DNA helicases: These enzymes bind to ssDNA near the replication fork and then move into the neighboring double-stranded region, forcing the strands apart (in effect, unwinding the double helix). Helicases require energy provided by ATP hydrolysis (see Fig. 30.10). Unwinding at the replication fork causes supercoiling in other regions of the DNA molecule. [Note: DnaB is the principal helicase of replication in E. coli. Binding of this hexameric protein to DNA requires DnaC.] c.

1	Single-stranded DNA–binding protein: This protein binds to the ssDNA generated by helicases (see Fig. 30.10). Binding is cooperative (that is, the binding of one molecule of single-stranded binding [SSB] protein makes it easier for additional molecules of SSB protein to bind tightly to the DNA strand). The SSB proteins are not enzymes, but rather serve to shift the equilibrium between dsDNA and ssDNA in the direction of the single-stranded forms. These proteins not only keep the two strands of DNA separated in the area of the replication origin, thus providing the single-stranded template required by polymerases, but also protect the DNA from nucleases that degrade ssDNA.

1	2. Solving the problem of supercoils: As the two strands of the double helix are separated, a problem is encountered, namely, the appearance of positive supercoils in the region of DNA ahead of the replication fork as a result of overwinding (Fig. 30.11) and negative supercoils in the region behind the fork. The accumulating positive supercoils interfere with further unwinding of the double helix. [Note: Supercoiling can be demonstrated by tightly grasping one end of a helical telephone cord while twisting the other end. If the cord is twisted in the direction of tightening the coils, the cord will wrap around itself in space to form positive supercoils. If the cord is twisted in the direction of loosening the coils, the cord will wrap around itself in the opposite direction to form negative supercoils.] To solve this problem, there is a group of enzymes called DNA topoisomerases, which are responsible for removing supercoils in the helix by transiently cleaving one or both of the DNA

1	supercoils.] To solve this problem, there is a group of enzymes called DNA topoisomerases, which are responsible for removing supercoils in the helix by transiently cleaving one or both of the DNA strands.

1	a. Type I DNA topoisomerases: These enzymes reversibly cleave one strand of the double helix. They have both strand-cutting and strand-resealing activities. They do not require ATP, but rather appear to store the energy from the phosphodiester bond they cleave, reusing the energy to reseal the strand (Fig. 30.12). Each time a transient nick is created in one DNA strand, the intact DNA strand is passed through the break before it is resealed, thus relieving (relaxing) accumulated supercoils. Type I topoisomerases relax negative supercoils (that is, those that contain fewer turns of the helix than does relaxed DNA) in E. coli and both negative and positive supercoils (that is, those that contain fewer or more turns of the helix than does relaxed DNA) in many prokaryotic cells (but not E. coli) and in eukaryotic cells. b.

1	b. Type II DNA topoisomerases: These enzymes bind tightly to the DNA double helix and make transient breaks in both strands. The enzyme then causes a second stretch of the DNA double helix to pass through the break and, finally, reseals the break (Fig. 30.13). As a result, both negative and positive supercoils can be relieved by this ATP-requiring process. DNA gyrase, a type II topoisomerase found in bacteria and plants, has the unusual property of being able to introduce negative supercoils into circular DNA using energy from the hydrolysis of ATP. This facilitates the replication of DNA because the negative supercoils neutralize the positive supercoils introduced during opening of the double helix. It also aids in the transient strand separation required during transcription (see p. 436).

1	Anticancer agents, such as the camptothecins, target human type I topoisomerases, whereas etoposide targets human type II topoisomerases. Bacterial DNA gyrase is a unique target of a group of antimicrobial agents called fluoroquinolones (for example, ciprofloxacin). C. Direction of DNA replication The DNA polymerases (DNA pols) responsible for copying the DNA templates are only able to read the parental nucleotide sequences in the 3′→5′ direction, and they synthesize the new DNA strands only in the 5′→3′ (antiparallel) direction. Therefore, beginning with one parental double helix, the two newly synthesized stretches of nucleotide chains must grow in opposite directions, one in the 5′→3′ direction toward the replication fork and one in the 5′→3′ direction away from the replication fork (Fig. 30.14). This feat is accomplished by a slightly different mechanism on each strand. 1.

1	1. Leading strand: The strand that is being copied in the direction of the advancing replication fork is synthesized continuously and is called the leading strand. 2. Lagging strand: The strand that is being copied in the direction away from the replication fork is synthesized discontinuously, with small fragments of DNA being copied near the replication fork. These short stretches of discontinuous DNA, termed Okazaki fragments, are eventually joined (ligated) by ligase to become a single, continuous strand. The new strand of DNA produced by this mechanism is termed the lagging strand. D. RNA primer

1	D. RNA primer DNA pols cannot initiate synthesis of a complementary strand of DNA on a totally single-stranded template. Rather, they require an RNA primer, which is a short piece of RNA base-paired to the DNA template, thereby forming a double-stranded DNA–RNA hybrid. The free hydroxyl group on the 3′end of the RNA primer serves as the first acceptor of a deoxynucleotide by action of a DNA pol (Fig. 30.15). [Note: Recall that glycogen synthase also requires a primer (see p. 126).] 1.

1	Primase: A specific RNA polymerase, called primase (DnaG), synthesizes the short stretches of RNA (~10 nucleotides long) that are complementary and antiparallel to the DNA template. In the resulting hybrid duplex, the U (uracil) in RNA pairs with A in DNA. As shown in Figure 30.16, these short RNA sequences are constantly being synthesized at the replication fork on the lagging strand, but only one RNA sequence at the origin of replication is required on the leading strand. The substrates for this process are 5′-ribonucleoside triphosphates, and pyrophosphate is released as each ribonucleoside monophosphate is added through formation of a 3′→5′-phosphodiester bond. [Note: The RNA primer is later removed, as described in F. below.] 2.

1	Primosome: The addition of primase converts the prepriming complex of proteins required for DNA strand separation (see p. 415) to a primosome. The primosome makes the RNA primer required for leading-strand synthesis and initiates Okazaki fragment formation in discontinuous lagging-strand synthesis. As with DNA synthesis, the direction of synthesis of the primer is 5′→3′. E. Chain elongation Prokaryotic (and eukaryotic) DNA pols elongate a new DNA strand by adding deoxyribonucleotides, one at a time, to the 3′-end of the growing chain (see Fig. 30.16). The sequence of nucleotides that are added is dictated by the base sequence of the template strand with which the incoming nucleotides are paired.

1	1. DNA polymerase III: DNA chain elongation is catalyzed by the multisubunit enzyme, DNA pol III. Using the 3′-hydroxyl group of the RNA primer as the acceptor of the first deoxyribonucleotide, DNA pol III begins to add nucleotides along the single-stranded template that specifies the sequence of bases in the newly synthesized chain. DNA pol III is a highly processive enzyme (that is, it remains bound to the template strand as it moves along and does not diffuse away and then rebind before adding each new nucleotide). The processivity of DNA pol III is the result of the β subunits of the holoenzyme forming a ring that encircles and moves along the template strand of the DNA, thus serving as a sliding DNA clamp. [Note: Clamp formation is facilitated by a protein complex, the clamp loader, and ATP hydrolysis.] The new (daughter) strand grows in the 5′→3′ direction, antiparallel to the parental strand (see Fig. 30.16). The nucleotide substrates are 5′deoxyribonucleoside triphosphates.

1	and ATP hydrolysis.] The new (daughter) strand grows in the 5′→3′ direction, antiparallel to the parental strand (see Fig. 30.16). The nucleotide substrates are 5′deoxyribonucleoside triphosphates. Pyrophosphate (PPi) is released when each new deoxynucleoside monophosphate is added to the free 3′hydroxyl group of the growing chain through a 3′→5′-phosphodiester bond (see Fig. 30.15). Hydrolysis of PPi to 2 Pi by pyrophosphatase means that a total of two high-energy bonds are used to drive the addition of each deoxynucleotide.

1	The production of PPi with subsequent hydrolysis to 2 Pi is a common theme in biochemistry. Removal of the PPi product drives a reaction in the forward direction, making it essentially irreversible. All four substrates (deoxyadenosine triphosphate [dATP], deoxythymidine triphosphate [dTTP], deoxycytidine triphosphate [dCTP], and deoxyguanosine triphosphate [dGTP]) must be present for DNA elongation to occur. If one of the four is in short supply, DNA synthesis stops when that nucleotide is depleted.

1	2. Proofreading newly synthesized DNA: It is highly important for the survival of an organism that the nucleotide sequence of DNA be replicated with as few errors as possible. Misreading of the template sequence could result in deleterious, perhaps lethal, mutations. To insure replication fidelity, DNA pol III has a proofreading activity (3′→5′ exonuclease, Fig. 30.17) in addition to its 5′→3′ polymerase activity. As each nucleotide is added to the chain, DNA pol III checks to make certain the base of the newly added nucleotide is, in fact, the complement of the base on the template strand. If it is not, the 3′→5′ exonuclease activity removes the error in the direction opposite to polymerization. [Note: Because the enzyme requires an improperly base-paired 3′hydroxy terminus, it does not degrade correctly paired nucleotide sequences.] For example, if the template base is C and the enzyme inserts an A instead of a G into the new chain, the 3′→5′ exonuclease activity hydrolytically

1	not degrade correctly paired nucleotide sequences.] For example, if the template base is C and the enzyme inserts an A instead of a G into the new chain, the 3′→5′ exonuclease activity hydrolytically removes the misplaced nucleotide. The 5′→3′ polymerase activity then replaces it with the correct nucleotide containing G (see Fig. 30.17). [Note: The 5′→3′ polymerase and 3′→5′ exonuclease domains are located on different subunits of DNA pol III.]

1	F. RNA primer excision and replacement by DNA DNA pol III continues to synthesize DNA on the lagging strand until it is blocked by proximity to an RNA primer. When this occurs, the RNA is excised and the gap filled by DNA pol I. 1.

1	5′→3′ Exonuclease activity: In addition to having the 5′→3′ polymerase activity that synthesizes DNA and the 3′→5′ exonuclease activity that proofreads the newly synthesized DNA like DNA pol III, monomeric DNA pol I also has a 5′→3′ exonuclease activity that is able to hydrolytically remove the RNA primer. [Note: Exonucleases remove nucleotides from the end of the DNA chain, rather than cleaving the chain internally as do endonucleases (Fig. 30.18).] First, DNA pol I locates the space (nick) between the 3′-end of the DNA newly synthesized by DNA pol III and the 5′-end of the adjacent RNA primer. Next, DNA pol I hydrolytically removes the RNA nucleotides ahead of itself, moving in the 5′→3′ direction (5′→3′ exonuclease activity). As it removes ribonucleotides, DNA pol I replaces them with deoxyribonucleotides, synthesizing DNA in the 5′→3′ direction (5′→3′ polymerase activity). As it synthesizes the DNA, it also proofreads using its 3′→5′ exonuclease activity to remove errors. This

1	deoxyribonucleotides, synthesizing DNA in the 5′→3′ direction (5′→3′ polymerase activity). As it synthesizes the DNA, it also proofreads using its 3′→5′ exonuclease activity to remove errors. This removal/synthesis/proofreading continues until the RNA primer is totally degraded, and the gap is filled with DNA (Fig. 30.19). [Note: DNA pol I uses its 5′→3′ polymerase activity to fill in gaps generated during most types of DNA repair (see p. 428).] 2.

1	Comparison of 5′→3′ and 3′→5′ exonuclease activities: The 5′→3′ exonuclease activity of DNA pol I allows the polymerase, moving 5′→3′, to hydrolytically remove one or more nucleotides at a time from the 5′-end of the ~10 nucleotide–long RNA primer. In contrast, the 3′→5′ exonuclease activity of DNA pol I and pol III allows these polymerases, moving 3′→5′, to hydrolytically remove one misplaced nucleotide at a time from the 3′-end of a growing DNA strand, increasing the fidelity of replication such that newly replicated DNA has one error per 107 nucleotides. G. DNA ligase The final phosphodiester linkage between the 5′-phosphate group on the DNA synthesized by DNA pol III and the 3′-hydroxyl group on the DNA made by DNA pol I is catalyzed by DNA ligase (Fig. 30.20). The joining of these two stretches of DNA requires energy, which in most organisms is provided by the cleavage of ATP to adenosine monophosphate + PPi. H. Termination

1	H. Termination Replication termination in E. coli is mediated by sequence-specific binding of the protein Tus (terminus utilization substance) to replication termination (ter) sites on the DNA, stopping the movement of the replication fork. IV. EUKARYOTIC DNA REPLICATION The process of eukaryotic DNA replication closely follows that of prokaryotic DNA synthesis. Some differences, such as the multiple origins of replication in eukaryotic cells versus single origins of replication in prokaryotes, have already been noted. Eukaryotic origin recognition proteins, ssDNA-binding proteins, and ATP-dependent DNA helicases have been identified, and their functions are analogous to those of the prokaryotic proteins previously discussed. In contrast, RNA primers are removed by RNase H and flap endonuclease 1 (FEN1) rather than by a DNA pol (Fig. 30.21). A. Eukaryotic cell cycle

1	A. Eukaryotic cell cycle The events surrounding eukaryotic DNA replication and cell division (mitosis) are coordinated to produce the cell cycle (Fig. 30.22). The period preceding replication is called the G1 phase (Gap 1). DNA replication occurs during the S (synthesis) phase. Following DNA synthesis, there is another phase (G2, or Gap 2) before mitosis (M). Cells that have stopped dividing, such as mature T lymphocytes, are said to have gone out of the cell cycle into the G0 phase. Such quiescent cells can be stimulated to reenter the G1 phase to resume division. [Note: The cell cycle is controlled at a series of checkpoints that prevent entry into the next phase of the cycle until the preceding phase has been completed. Two key classes of proteins that control the progress of a cell through the cell cycle are the cyclins and cyclin-dependent kinases (Cdk).] B. Eukaryotic DNA polymerases

1	B. Eukaryotic DNA polymerases At least five high-fidelity eukaryotic DNA pols have been identified and categorized on the basis of molecular weight, cellular location, sensitivity to inhibitors, and the templates or substrates on which they act. They are designated by Greek letters rather than by Roman numerals (Fig. 30.23). 1. Pol α: Pol α is a multisubunit enzyme. One subunit has primase activity, which initiates strand synthesis on the leading strand and at the beginning of each Okazaki fragment on the lagging strand. The primase subunit synthesizes a short RNA primer that is extended by the 5′→3′ polymerase activity of pol α, generating a short piece of DNA. [Note: Pol α is also referred to as pol α/primase.] 2.

1	Pol ε and pol δ: Pol ε is recruited to complete DNA synthesis on the leading strand, whereas pol δ elongates the Okazaki fragments of the lagging strand, each using 3′→5′ exonuclease activity to proofread the newly synthesized DNA. [Note: DNA pol ε associates with proliferating cell nuclear antigen (PCNA), a protein that serves as a sliding DNA clamp in much the same way the β subunits of DNA pol III do in E. coli, thus insuring high processivity.] 3. Pol β and pol γ: Pol β is involved in gap filling in DNA repair. Pol γ replicates mitochondrial DNA. C. Telomeres

1	Pol β and pol γ: Pol β is involved in gap filling in DNA repair. Pol γ replicates mitochondrial DNA. C. Telomeres Telomeres are complexes of DNA plus proteins (collectively known as shelterin) located at the ends of linear chromosomes. They maintain the structural integrity of the chromosome, preventing attack by nucleases, and allow repair systems to distinguish a true end from a break in dsDNA. In humans, telomeric DNA consists of several thousand tandem repeats of a noncoding hexameric sequence, AGGGTT, base-paired to a complementary region containing C and A. The G-rich strand is longer than its C-rich complement, leaving ssDNA a few hundred nucleotides in length at the 3′end. The single-stranded region is thought to fold back on itself, forming a loop structure that is stabilized by protein. 1.

1	1. Telomere shortening: Eukaryotic cells face a special problem in replicating the ends of their linear DNA molecules. Following removal of the RNA primer from the extreme 5′-end of the lagging strand, there is no way to fill in the remaining gap with DNA. Consequently, in most normal human somatic cells, telomeres shorten with each successive cell division. Once telomeres are shortened beyond some critical length, the cell is no longer able to divide and is said to be senescent. In germ cells and stem cells, as well as in cancer cells, telomeres do not shorten and the cells do not senesce. This is a result of the ribonucleoprotein telomerase, which maintains telomeric length in these cells. 2.

1	2. Telomerase: This complex contains a protein (Tert) that acts as a reverse transcriptase and a short piece of RNA (Terc) that acts as a template. The C-rich RNA template base-pairs with the G-rich, single-stranded 3′end of telomeric DNA (Fig. 30.24). The reverse transcriptase uses the RNA template to synthesize DNA in the usual 5′→3′ direction, extending the already longer 3′-end. Telomerase then translocates to the newly synthesized end, and the process is repeated. Once the G-rich strand has been lengthened, primase activity of DNA pol α can use it as a template to synthesize an RNA primer. The primer is extended by DNA pol α and then removed by nucleases. Telomeres may be viewed as mitotic clocks in that their length in most cells is inversely related to the number of times the cells have divided. The study of telomeres provides insight into the biology of normal aging, diseases of premature aging (the progerias), and cancer. D. Reverse transcriptases

1	D. Reverse transcriptases As seen with telomerase, reverse transcriptases are RNA-directed DNA pols. A reverse transcriptase is involved in the replication of retroviruses, such as human immunodeficiency virus (HIV). These viruses carry their genome in the form of ssRNA molecules. Following infection of a host cell, the viral enzyme reverse transcriptase uses the viral RNA as a template for the 5′→3′ synthesis of viral DNA, which then becomes integrated into host chromosomes. Reverse transcriptase activity is also seen with transposons, DNA elements that can move about the genome (see p. 477). In eukaryotes, most transposons are transcribed to RNA, the RNA is used as a template for DNA synthesis by a reverse transcriptase encoded by the transposon, and the DNA is randomly inserted into the genome. [Note: Transposons that involve an RNA intermediate are called retrotransposons or retroposons.] E. DNA replication inhibition by nucleoside analogs

1	E. DNA replication inhibition by nucleoside analogs DNA chain growth can be blocked by the incorporation of certain nucleoside analogs that have been modified on the sugar portion (Fig. 30.25). For example, removal of the hydroxyl group from the 3′-carbon of the deoxyribose ring as in 2′,3′-dideoxyinosine ([ddI] also known as didanosine), or conversion of the deoxyribose to another sugar, such as arabinose, prevents further chain elongation. By blocking DNA replication, these compounds slow the division of rapidly growing cells and viruses. Cytosine arabinoside (cytarabine, or araC) has been used in anticancer chemotherapy, whereas adenine arabinoside (vidarabine, or araA) is an antiviral agent. Substitution on the sugar moiety, as seen in azidothymidine (AZT), also called zidovudine (ZDV), also terminates DNA chain elongation. [Note: These drugs are generally supplied as nucleosides, which are then converted to nucleotides by cellular kinases.] V. EUKARYOTIC DNA ORGANIZATION

1	A typical (diploid) human somatic cell contains 46 chromosomes, whose total DNA is ~2 m long! It is difficult to imagine how such a large amount of genetic material can be effectively packaged into a volume the size of a cell nucleus so that it can be efficiently replicated and its genetic information expressed. To do so requires the interaction of DNA with a large number of proteins, each of which performs a specific function in the ordered packaging of these long molecules of DNA. Eukaryotic DNA is associated with tightly bound basic proteins, called histones. These serve to order the DNA into fundamental structural units, called nucleosomes, which resemble beads on a string. Nucleosomes are further arranged into increasingly more complex structures that organize and condense the long DNA molecules into chromosomes that can be segregated during cell division. [Note: The complex of DNA and protein found inside the nuclei of eukaryotic cells is called chromatin.]

1	A. Histones and nucleosome formation There are five classes of histones, designated H1, H2A, H2B, H3, and H4. These small, evolutionally conserved proteins are positively charged at physiologic pH as a result of their high content of lysine and arginine. Because of their positive charge, they form ionic bonds with negatively charged DNA. Histones, along with ions such as Mg2+ , help neutralize the negatively charged DNA phosphate groups.

1	1. Nucleosomes: Two molecules each of H2A, H2B, H3, and H4 form the octameric core of the individual nucleosome “beads.” Around this structural core, a segment of dsDNA is wound nearly twice (Fig. 30.26). Winding eliminates a helical turn, causing negative supercoiling. [Note: The N-terminal ends of these histones can be acetylated, methylated, or phosphorylated. These reversible covalent modifications influence how tightly the histones bind to the DNA, thereby affecting the expression of specific genes. Histone modification is an example of epigenetics, or heritable changes in gene expression caused without alteration of the nucleotide sequence.] Neighboring nucleosomes are joined by linker DNA ~50 bp long. H1 is not found in the nucleosome core, but instead binds to the linker DNA chain between the nucleosome beads. H1 is the most tissue specific and species specific of the histones. It facilitates the packing of nucleosomes into more compact structures.

1	2. Higher levels of organization: Nucleosomes can be packed more tightly (stacked) to form a nucleofilament. This structure assumes the shape of a coil, often referred to as a 30-nm fiber. The fiber is organized into loops that are anchored by a nuclear scaffold containing several proteins. Additional levels of organization lead to the final chromosomal structure (Fig. 30.27). B. Nucleosome fate during DNA replication Parental nucleosomes are disassembled to allow access to DNA during replication. Once DNA is synthesized, nucleosomes form rapidly. Their histone proteins come both from de novo synthesis and from the transfer of parental histones. VI. DNA REPAIR

1	Despite the elaborate proofreading system employed during DNA synthesis, errors (including incorrect base-pairing or insertion of one to a few extra nucleotides) can occur. In addition, DNA is constantly being subjected to environmental insults that cause the alteration or removal of nucleotide bases. The damaging agents can be either chemicals (for example, nitrous acid, which can deaminate bases) or radiation (for example, nonionizing ultraviolet [UV] radiation, which can fuse two pyrimidines adjacent to each other in the DNA, and high-energy ionizing radiation, which can cause double-strand breaks). Bases are also altered or lost spontaneously from mammalian DNA at a rate of many thousands per cell per day. If the damage is not repaired, a permanent change (mutation) is introduced that can result in any of a number of deleterious effects, including loss of control over the proliferation of the mutated cell, leading to cancer. Luckily, cells are remarkably efficient at repairing

1	can result in any of a number of deleterious effects, including loss of control over the proliferation of the mutated cell, leading to cancer. Luckily, cells are remarkably efficient at repairing damage done to their DNA. Most of the repair systems involve recognition of the damage (lesion) on the DNA, removal or excision of the damage, replacement or filling the gap left by excision using the sister strand as a template for DNA synthesis, and ligation. These excision repair systems remove one to tens of nucleotides. [Note: Repair synthesis of DNA can occur outside of the S phase.]

1	A. Mismatch repair Sometimes replication errors escape the proofreading activity during DNA synthesis, causing a mismatch of one to several bases. In E. coli, mismatch repair (MMR) is mediated by a group of proteins known as the Mut proteins (Fig. 30.28). Homologous proteins are present in humans. [Note: MMR occurs within minutes of replication and reduces the error rate of replication from 1 in 107 to 1 in 109 nucleotides.] 1.

1	Mismatched strand identification: When a mismatch occurs, the Mut proteins that identify the mispaired nucleotide(s) must be able to discriminate between the correct strand and the strand with the mismatch. In prokaryotes, discrimination is based on the degree of methylation. GATC sequences, which are found once every thousand nucleotides, are methylated on the adenine (A) residue by DNA adenine methylase (DAM). This methylation is not done immediately after synthesis, so the DNA is hemimethylated (that is, the parental strand is methylated, but the daughter strand is not). The methylated parental strand is assumed to be correct, and it is the daughter strand that gets repaired. [Note: The exact mechanism by which the daughter strand is identified in eukaryotes is not yet known, but likely involves recognition of nicks in the newly synthesized strand.] 2.

1	Repair procedure: When the strand containing the mismatch is identified, an endonuclease nicks the strand, and the mismatched nucleotide(s) is/are removed by an exonuclease. Additional nucleotides at the 5′-and 3′-ends of the mismatch are also removed. The gap left by removal of the nucleotides is filled, using the sister strand as a template, by a DNA pol, typically DNA pol III. The 3′-hydroxyl of the newly synthesized DNA is joined to the 5′-phosphate of the remaining stretch of the original DNA strand by DNA ligase. Mutation to the proteins involved in MMR in humans is associated with hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch syndrome. Although HNPCC confers an increased risk for developing colon cancer (as well as other cancers), only about 5% of all colon cancer is the result of mutations in MMR. B. Nucleotide excision repair

1	B. Nucleotide excision repair Exposure of a cell to UV radiation can result in the covalent joining of two adjacent pyrimidines (usually thymines), producing a dimer. These intrastrand cross-links prevent DNA pol from replicating the DNA strand beyond the site of dimer formation. Thymine dimers are excised in bacteria by UvrABC proteins in a process known as nucleotide excision repair (NER), as illustrated in Figure 30.29. A related pathway is present in humans (see 2. below). [Note: Transcription-coupled repair, a type of NER, fixes DNA lesions encountered during RNA synthesis.] 1. Recognition and excision of UV-induced dimers: A UV-specific endonuclease (called uvrABC excinuclease) recognizes the bulky dimer and cleaves the damaged strand on both the 5′-side and 3′-side of the lesion. A short oligonucleotide containing the dimer is excised, leaving a gap in the DNA strand. This gap is filled in using a DNA pol I and DNA ligase. NER occurs throughout the cell cycle. 2.

1	2. UV radiation and cancer: Pyrimidine dimers can be formed in the skin cells of humans exposed to UV radiation in unfiltered sunlight. In the rare genetic disease xeroderma pigmentosum (XP), the cells cannot repair the damaged DNA, resulting in extensive accumulation of mutations and, consequently, early and numerous skin cancers (Fig. 30.30). XP can be caused by defects in any of the several genes that code for the XP proteins required for NER of UV damage in humans. C. Base excision repair

1	C. Base excision repair DNA bases can be altered, either spontaneously, as is the case with cytosine, which slowly undergoes deamination (the loss of its amino group) to form uracil, or by the action of deaminating or alkylating compounds. For example, nitrous acid, which is formed by the cell from precursors such as the nitrates, deaminates cytosine, adenine (to hypoxanthine), and guanine (to xanthine). Dimethyl sulfate can alkylate (methylate) adenine. Bases can also be lost spontaneously. For example, ~10,000 purine bases are lost this way per cell per day. Lesions involving base alterations or loss can be corrected by base excision repair ([BER], Fig. 30.31). 1.

1	1. Abnormal base removal: In BER, abnormal bases, such as uracil, which can occur in DNA by either deamination of cytosine or improper use of dUTP instead of dTTP during DNA synthesis, are recognized by specific DNA glycosylases that hydrolytically cleave them from the deoxyribosephosphate backbone of the strand. This leaves an apyrimidinic site, or apurinic if a purine was removed, both referred to as AP sites. 2. AP site recognition and repair: Specific AP endonucleases recognize that a base is missing and initiate the process of excision and gap filling by making an endonucleolytic cut just to the 5′-side of the AP site. A deoxyribose phosphate lyase removes the single, base-free, sugar phosphate residue. DNA pol I and DNA ligase complete the repair process. D. Double-strand break repair

1	Ionizing radiation, chemotherapeutic agents such as doxorubicin, and oxidative free radicals (see p. 148) can cause double-strand breaks in DNA that can be lethal to the cell. [Note: Such breaks also occur naturally during genetic recombination.] dsDNA breaks cannot be corrected by the previously described strategy of excising the damage on one strand and using the undamaged strand as a template for replacing the missing nucleotide(s). Instead, they are repaired by one of two systems. The first is nonhomologous end joining (NHEJ), in which a group of proteins mediates the recognition, processing, and ligation of the ends of two DNA fragments. However, some DNA is lost in the process. Consequently, NHEJ is error prone and mutagenic. Defects in NHEJ are associated with a predisposition to cancer and immunodeficiency syndromes. The second repair system, homologous recombination (HR), uses the enzymes that normally perform genetic recombination between homologous chromosomes during

1	to cancer and immunodeficiency syndromes. The second repair system, homologous recombination (HR), uses the enzymes that normally perform genetic recombination between homologous chromosomes during meiosis. This system is much less error prone (“error-free”) than NHEJ because any DNA that was lost is replaced using homologous DNA as a template. HR occurs in late S and G2 of the cell cycle, whereas NHEJ can occur anytime.

1	[Note: Mutations to the proteins BRCA1 or BRCA2 (breast cancer 1 or 2), which are involved in HR, increase the risk for developing breast and ovarian cancer.] VII. CHAPTER SUMMARY

1	DNA is a polymer of deoxynucleoside monophosphates covalently linked by 3′→5′-phosphodiester bonds (Fig. 30.32). The resulting long, unbranched chain has polarity, with both a 5′-end (free phosphate) and a 3′end (free hydroxyl). The sequence of nucleotides is read 5′→3′. DNA exists as a double-stranded molecule, in which the two chains are paired in an antiparallel manner and wind around each other, forming a double helix. Adenine pairs with thymine, and cytosine pairs with guanine. Each strand of the double helix serves as a template for constructing a complementary daughter strand (semiconservative replication). DNA replication occurs in the S phase of the cell cycle and begins at an origin of replication. As the two strands unwind and separate, synthesis occurs at two replication forks that move away from the origin in opposite directions (bidirectionally). Helicase unwinds the double helix. As the two strands of the double helix are separated, positive supercoils are produced in

1	that move away from the origin in opposite directions (bidirectionally). Helicase unwinds the double helix. As the two strands of the double helix are separated, positive supercoils are produced in the region of DNA ahead of the replication fork and negative supercoils behind the fork. DNA topoisomerases types I and II remove supercoils. DNA polymerases (pols) synthesize new DNA strands only in the 5′→3′ direction. Therefore, one of the newly synthesized stretches of nucleotide chains must grow in the 5′→3′ direction toward the replication fork (leading strand) and one in the 5′→3′ direction away from the replication fork (lagging strand). DNA pols require a primer, a short stretch of RNA synthesized by primase. Leading-strand synthesis needs only one RNA primer (continuous synthesis), whereas the lagging strand needs many (discontinuous synthesis involving Okazaki fragments). In Escherichia coli (E. coli), DNA chain elongation is catalyzed by DNA pol III, using 5′-deoxyribonucleoside

1	the lagging strand needs many (discontinuous synthesis involving Okazaki fragments). In Escherichia coli (E. coli), DNA chain elongation is catalyzed by DNA pol III, using 5′-deoxyribonucleoside triphosphates as substrates. The enzyme proofreads the newly synthesized DNA, removing terminal mismatched nucleotides with its 3′→5′ exonuclease activity. RNA primers are removed by DNA pol I, using its 5′→3′ exonuclease activity. This enzyme fills the gaps with DNA, proofreading as it synthesizes. The final phosphodiester linkage is catalyzed by DNA ligase. There are at least five high-fidelity eukaryotic DNA pols. Pol α is a multisubunit enzyme, one subunit of which is a primase. Pol α 5′→3′ polymerase activity adds a short piece of DNA to the RNA primer. Pol ε completes DNA synthesis on the leading strand, whereas pol δ elongates each lagging strand fragment. Pol β is involved with DNA repair, and pol γ replicates mitochondrial DNA. Pols ε, δ, and γ use 3′→5′ exonuclease activity to

1	the leading strand, whereas pol δ elongates each lagging strand fragment. Pol β is involved with DNA repair, and pol γ replicates mitochondrial DNA. Pols ε, δ, and γ use 3′→5′ exonuclease activity to proofread. Nucleoside analogs containing modified sugars can be used to block DNA chain growth. They are useful in anticancer and antiviral chemotherapy. Telomeres are stretches of highly repetitive DNA complexed with protein that protect the ends of linear chromosomes. As most cells divide and age, these sequences are shortened, contributing to senescence. In cells that do not senesce (for example, germline and cancer cells), the ribonucleoprotein telomerase employs its protein component reverse transcriptase to extend the telomeres, using its RNA component as a template. There are five classes of positively charged histone (H) proteins. Two of each of histones H2A, H2B, H3, and H4 form an octameric structural core around which DNA is wrapped, creating a nucleosome. The DNA connecting

1	of positively charged histone (H) proteins. Two of each of histones H2A, H2B, H3, and H4 form an octameric structural core around which DNA is wrapped, creating a nucleosome. The DNA connecting the nucleosomes, called linker DNA, is bound to H1. Nucleosomes can be packed more tightly to form a nucleofilament. Additional levels of organization create a chromosome. Most DNA damage can be corrected by excision repair involving recognition and removal of the damage by repair proteins, followed by replacement by DNA pols and joining by ligase. Ultraviolet radiation can cause thymine dimers that are recognized and removed in E. coli by uvrABC proteins of nucleotide excision repair. Defects in the XP proteins needed for nucleotide excision repair of thymine dimers in humans result in xeroderma pigmentosum. Mismatched bases are repaired by a similar process of recognition and removal by Mut proteins in E. coli. The extent of methylation is used for strand identification in prokaryotes.

1	pigmentosum. Mismatched bases are repaired by a similar process of recognition and removal by Mut proteins in E. coli. The extent of methylation is used for strand identification in prokaryotes. Defective mismatch repair by homologous proteins in humans is associated with hereditary nonpolyposis colorectal cancer. Abnormal bases (such as uracil) are removed by DNA N-glycosylases in base excision repair, and the sugar phosphate at the apyrimidinic or apurinic site is cut out. Double-strand breaks in DNA are repaired by nonhomologous end joining (error prone) and template-requiring homologous recombination (“error-free”).

1	Choose the ONE best answer. 0.1. A 10-year-old girl is brought by her parents to the dermatologist. She has many freckles on her face, neck, arms, and hands, and the parents report that she is unusually sensitive to sunlight. Two basal cell carcinomas are identified on her face. Based on the clinical picture, which of the following processes is most likely to be defective in this patient? A. Repair of double-strand breaks by error-prone homologous recombination B. Removal of mismatched bases from the 3′-end of Okazaki fragments by a methyl-directed process C. Removal of pyrimidine dimers from DNA by nucleotide excision repair D. Removal of uracil from DNA by base excision repair

1	C. Removal of pyrimidine dimers from DNA by nucleotide excision repair D. Removal of uracil from DNA by base excision repair Correct answer = C. The sensitivity to sunlight, extensive freckling on parts of the body exposed to the sun, and presence of skin cancer at a young age indicate that the patient most likely suffers from xeroderma pigmentosum (XP). These patients are deficient in any one of several XP proteins required for nucleotide excision repair of pyrimidine dimers in ultraviolet radiation– damaged DNA. Double-strand breaks are repaired by nonhomologous end joining (error prone) or homologous recombination (“error free”). Methylation is not used for strand discrimination in eukaryotic mismatch repair. Uracil is removed from DNA molecules by a specific glycosylase in base excision repair, but a defect in this process does not cause XP.

1	0.2. Telomeres are complexes of DNA and protein that protect the ends of linear chromosomes. In most normal human somatic cells, telomeres shorten with each division. In stem cells and in cancer cells, however, telomeric length is maintained. In the synthesis of telomeres: A. telomerase, a ribonucleoprotein, provides both the RNA and the protein needed for synthesis. B. the RNA of telomerase serves as a primer. C. the RNA of telomerase is a ribozyme. D. the protein of telomerase is a DNA-directed DNA polymerase. E. the shorter 3′→5′ strand gets extended. F. the direction of synthesis is 3′→5′.

1	C. the RNA of telomerase is a ribozyme. D. the protein of telomerase is a DNA-directed DNA polymerase. E. the shorter 3′→5′ strand gets extended. F. the direction of synthesis is 3′→5′. Correct answer = A. Telomerase is a ribonucleoprotein particle required for telomere maintenance. Telomerase contains an RNA that serves as the template, not the primer, for the synthesis of telomeric DNA by the reverse transcriptase of telomerase. Telomeric RNA has no catalytic activity. As a reverse transcriptase, telomerase synthesizes DNA using its RNA template and so is an RNA-directed DNA polymerase. The direction of synthesis, as with all DNA synthesis, is 5′→3′, and it is the 3′-end of the already longer 5′→3′ strand that gets extended.

1	0.3. While studying the structure of a small gene that was sequenced during the Human Genome Project, an investigator notices that one strand of the DNA molecule contains 20 A, 25 G, 30 C, and 22 T. How many of each base is found in the complete double-stranded molecule? A. A=40,G =50,C =60,T =44 E. A=42,G =55,C =55,T =42 B. A=44,G =60,C =50,T =40 C. A=45,G =45,C =52,T =52 D. A=50,G =47,C =50,T =47 Correct answer = B. The two DNA strands are complementary to each other, with A base-paired with T and G base-paired with C. So, for example, the 20 A on the first strand would be paired with 20 T on the second strand, the 25 G on the first strand would be paired with 25 C on the second strand, and so forth. When these are all added together, the correct numbers of each base are indicated in choice B. Notice that, in the correct answer, A = T and G = C. 0.4. List the order in which the following enzymes participate in prokaryotic replication. A. Ligase

1	0.4. List the order in which the following enzymes participate in prokaryotic replication. A. Ligase B. Polymerase I (3′→5′ exonuclease activity) C. Polymerase I (5′→3′ exonuclease activity) D. Polymerase I (5′→3′ polymerase activity) E. Polymerase III F. Primase Correct answer: F, E, C, D, B, A. Primase makes the RNA primer; polymerase (pol) III extends the primer with DNA (and proofreads); pol I removes the primer with its 5′→3′ exonuclease activity, fills in the gap with its 5′→3′ polymerase activity, and removes errors with its 3′→5′ exonuclease activity; and ligase makes the 5′→3′-phosphodiester bond that links the DNA made by pols I and III. 0.5. Dideoxynucleotides lack a 3′-hydroxyl group. Why would incorporation of a dideoxynucleotide into DNA stop replication? The lack of the 3′-OH group prevents formation of the 3′-hydroxyl → 5′phosphate bond that links one nucleotide to the next in DNA. RNA Structure, Synthesis, and Processing 31

1	The lack of the 3′-OH group prevents formation of the 3′-hydroxyl → 5′phosphate bond that links one nucleotide to the next in DNA. RNA Structure, Synthesis, and Processing 31 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	The genetic master plan of an organism is contained in the sequence of deoxyribonucleotides in its DNA. However, it is through ribonucleic acid (RNA), the “working copies” of DNA, that the master plan is expressed (Fig. 31.1). The copying process, during which a DNA strand serves as a template for the synthesis of RNA, is called transcription. Transcription produces messenger RNA (mRNA), which are translated into sequences of amino acids (proteins), and ribosomal RNA (rRNA), transfer RNA (tRNA), and additional RNA molecules that perform specialized structural, catalytic, and regulatory functions and are not translated. That is, they are noncoding RNA (ncRNA). Therefore, the final product of gene expression can be RNA or protein, depending upon the gene. [Note: Only ~2% of the genome encodes proteins.] A central feature of transcription is that it is highly selective. For example, many transcripts are made of some regions of the DNA. In other regions, few or no transcripts are made.

1	proteins.] A central feature of transcription is that it is highly selective. For example, many transcripts are made of some regions of the DNA. In other regions, few or no transcripts are made. This selectivity is due, at least in part, to signals embedded in the nucleotide sequence of the DNA. These signals instruct the RNA polymerase where to start, how often to start, and where to stop transcription. Several regulatory proteins are also involved in this selection process. The biochemical differentiation of an organism’s tissues is ultimately a result of the selectivity of the transcription process. [Note: This selectivity of transcription is in contrast to the “all-or-none” nature of genomic replication.] Another important feature of transcription is that many RNA transcripts that initially are faithful copies of one of the two DNA strands may undergo various modifications, such as terminal additions, base modifications, trimming, and internal segment removal, which convert the

1	are faithful copies of one of the two DNA strands may undergo various modifications, such as terminal additions, base modifications, trimming, and internal segment removal, which convert the inactive primary transcript into a functional molecule. The transcriptome is the complete set of RNA transcripts expressed by a genome.

1	II. RNA STRUCTURE There are three major types of RNA that participate in the process of protein synthesis: rRNA, tRNA, and mRNA. Like DNA, these RNA are unbranched polymeric molecules composed of nucleoside monophosphates joined together by 3′→5′-phosphodiester bonds (see p. 412). However, they differ from DNA in several ways. For example, they are considerably smaller than DNA, contain ribose instead of deoxyribose and uracil instead of thymine, and exist as single strands that are capable of folding into complex structures. The three major types of RNA also differ from each other in size, function, and special structural modifications. [Note: In eukaryotes, additional small ncRNA molecules found in the nucleolus (snoRNA), nucleus (snRNA), and cytoplasm (microRNA [miRNA]) perform specialized functions as described on pp. 441, 442, and 475.]

1	A. Ribosomal RNA rRNA are found in association with several proteins as components of the ribosomes, the complex structures that serve as the sites for protein synthesis (see p. 451). Prokaryotic cells contain three distinct size species of rRNA (23S, 16S, and 5S, where S is the Svedberg unit for sedimentation rate that is determined by the size and shape of the particle), as shown in Figure 31.2. Eukaryotic cells contain four rRNA species (28S, 18S, 5.8S, and 5S). Together, rRNA make up ~80% of the total RNA in the cell. [Note: Some RNA function as catalysts, for example, an rRNA in protein synthesis (see p. 455). RNA with catalytic activity is termed a ribozyme.]

1	B. Transfer RNA tRNA are the smallest (4S) of the three major types of RNA molecules. There is at least one specific type of tRNA molecule for each of the 20 amino acids commonly found in proteins. Together, tRNA make up ~15% of the total RNA in the cell. The tRNA molecules contain a high percentage of unusual (modified) bases, for example, dihydrouracil (see Fig. 22.2, p. 292), and have extensive intrachain base-pairing (Fig. 31.3) that leads to characteristic secondary and tertiary structure. Each tRNA serves as an adaptor molecule that carries its specific amino acid, covalently attached to its 3′-end, to the site of protein synthesis. There, it recognizes the genetic code sequence on an mRNA, which specifies the addition of that amino acid to the growing peptide chain (see p. 447).

1	C. Messenger RNA mRNA comprises only ~5% of the RNA in a cell, yet is by far the most heterogeneous type of RNA in size and base sequence. mRNA is coding RNA in that it carries genetic information from DNA for use in protein synthesis. In eukaryotes, this involves transport of mRNA out of the nucleus and into the cytosol. An mRNA carrying information from more than one gene is polycistronic (cistron = gene). Polycistronic mRNA is characteristic of prokaryotes. An mRNA carrying information from only one gene is monocistronic and is characteristic of eukaryotes. In addition to the protein-coding regions that can be translated, mRNA contains untranslated regions at its 5′-and 3′-ends (Fig. 31.4). Special structural characteristics of eukaryotic (but not prokaryotic) mRNA include a long sequence of adenine nucleotides (a poly-A tail) on the 3′-end of the RNA, plus a cap on the 5′-end consisting of a molecule of 7-methylguanosine attached through an unusual (5′→5′) triphosphate linkage.

1	of adenine nucleotides (a poly-A tail) on the 3′-end of the RNA, plus a cap on the 5′-end consisting of a molecule of 7-methylguanosine attached through an unusual (5′→5′) triphosphate linkage. The mechanisms for modifying mRNA to create these special structural characteristics are discussed on pp. 441–442.

1	III. PROKARYOTIC GENE TRANSCRIPTION The structure of magnesium-requiring RNA polymerase (RNA pol), the signals that control transcription, and the varieties of modification that RNA transcripts can undergo differ among organisms, particularly from prokaryotes to eukaryotes. Therefore, the discussions of prokaryotic and eukaryotic transcription are presented separately. A. Prokaryotic RNA polymerase

1	In bacteria, one species of RNA pol synthesizes all of the RNA except for the short RNA primers needed for DNA replication [Note: RNA primers are synthesized by the specialized, monomeric enzyme primase (see p. 418).] RNA pol is a multisubunit enzyme that recognizes a nucleotide sequence (the promoter region) at the beginning of a length of DNA that is to be transcribed. It next makes a complementary RNA copy of the DNA template strand and then recognizes the end of the DNA sequence to be transcribed (the termination region). RNA is synthesized from its 5′-end to its 3′-end, antiparallel to its DNA template strand (see p. 415). The template is copied as it is in DNA synthesis, in which a guanine (G) on the DNA specifies a cytosine (C) in the RNA, a C specifies a G, a thymine (T) specifies an adenine (A), but an A specifies a uracil (U) instead of a T (Fig. 31.5). The RNA, then, is complementary to the DNA template (antisense, minus) strand and identical to the coding (sense, plus)

1	an adenine (A), but an A specifies a uracil (U) instead of a T (Fig. 31.5). The RNA, then, is complementary to the DNA template (antisense, minus) strand and identical to the coding (sense, plus) strand, with U replacing T. Within the DNA molecule, regions of both strands can serve as templates for transcription. For a given gene, however, only one of the two DNA strands can be the template. Which strand is used is determined by the location of the promoter for that gene. Transcription by RNA pol involves a core enzyme and several auxiliary proteins.

1	1. Core enzyme: Five of the enzyme’s peptide subunits, 2 α, 1 β, 1 β′, and 1 Ω, are required for enzyme assembly (α, Ω), template binding (β′), and the 5′→3′ polymerase activity (β) and together are referred to as the core enzyme (Fig. 31.6). However, this enzyme lacks specificity (that is, it cannot recognize the promoter region on the DNA template). 2. Holoenzyme: The σ subunit (sigma factor) enables RNA pol to recognize promoter regions on the DNA. The σ subunit plus the core enzyme make up the holoenzyme. [Note: Different σ factors recognize different groups of genes, with σ70 predominating.] B. Steps in RNA synthesis The process of transcription of a typical gene of Escherichia coli (E. coli) can be divided into three phases: initiation, elongation, and termination. A transcription unit extends from the promoter to the termination region, and the initial product of transcription by RNA pol is termed the primary transcript.

1	1. Initiation: Transcription begins with the binding of the RNA pol holoenzyme to a region of the DNA known as the promoter, which is not transcribed. The prokaryotic promoter contains characteristic consensus sequences (Fig. 31.7). [Note: Consensus sequences are idealized sequences in which the base shown at each position is the base most frequently (but not necessarily always) encountered at that position.] Those that are recognized by prokaryotic RNA pol σ factors include the following. a.

1	a. –35 Sequence: A consensus sequence (5′-TTGACA-3′), centered about 35 bases to the left of the transcription start site (see Fig. 31.7), is the initial point of contact for the holoenzyme, and a closed complex is formed. [Note: By convention, the regulatory sequences that control transcription are designated by the 5′→3′ nucleotide sequence on the coding strand. A base in the promoter region is assigned a negative number if it occurs prior to (to the left of, toward the 5′-end of, or “upstream” of) the transcription start site. Therefore, the TTGACA sequence is centered at approximately base −35. The first base at the transcription start site is assigned a position of +1. There is no base designated “0”.] b.

1	Pribnow box: The holoenzyme moves and covers a second consensus sequence (5′-TATAAT-3′), centered at about −10 (see Fig. 31.7), which is the site of melting (unwinding) of a short stretch (~14 base pairs) of DNA. This initial melting converts the closed initiation complex to an open complex known as a transcription bubble. [Note: A mutation in either the −10 or the −35 sequence can affect the transcription of the gene controlled by the mutant promoter.] 2.

1	Elongation: Once the promoter has been recognized and bound by the holoenzyme, local unwinding of the DNA helix continues (Fig. 31.8), mediated by the polymerase. [Note: Unwinding generates supercoils in the DNA that can be relieved by DNA topoisomerases (see p. 417).] RNA pol begins to synthesize a transcript of the DNA sequence, and several short pieces of RNA are made and discarded. The elongation phase begins when the transcript (typically starting with a purine) exceeds 10 nucleotides in length. Sigma is then released, and the core enzyme is able to leave (clear) the promoter and move along the template strand in a processive manner, serving as its own sliding clamp. During transcription, a short DNA–RNA hybrid helix is formed (see Fig. 31.8). Like DNA pol, RNA pol uses nucleoside triphosphates as substrates and releases pyrophosphate each time a nucleoside monophosphate is added to the growing chain. As with replication, transcription is always in the 5′→3′ direction. In

1	triphosphates as substrates and releases pyrophosphate each time a nucleoside monophosphate is added to the growing chain. As with replication, transcription is always in the 5′→3′ direction. In contrast to DNA pol, RNA pol does not require a primer and does not have a 3′→5′ exonuclease domain for proofreading. [Note: Misincorporation of a ribonucleotide causes RNA pol to pause, backtrack, cleave the transcript, and restart. Nonetheless, transcription has a higher error rate than does replication.] 3.

1	Termination: The elongation of the single-stranded RNA chain continues until a termination signal is reached. Termination can be intrinsic (occur without additional proteins) or dependent upon the participation of a protein known as the ρ (rho) factor. a. ρ-Independent: Seen with most prokaryotic genes, this requires that a sequence in the DNA template generates a sequence in the nascent (newly made) RNA that is self-complementary (Fig. 31.9). This allows the RNA to fold back on itself, forming a GC-rich stem (stabilized by hydrogen bonds) plus a loop. This structure is known as a “hairpin.” Additionally, just beyond the hairpin, the RNA transcript contains a string of Us at the 3′-end. The bonding of these Us to the complementary As of the DNA template is weak. This facilitates the separation of the newly synthesized RNA from its DNA template, as the double helix “zips up” behind the RNA pol.

1	b. ρ-Dependent: This requires the participation of the additional protein rho, which is a hexameric ATPase with helicase activity. Rho binds a C-rich rho utilization (rut) site near the 5′-end of the nascent RNA and, using its ATPase activity, moves along the RNA until it reaches the RNA pol paused at the termination site. The ATP-dependent helicase activity of rho separates the RNA–DNA hybrid helix, causing the release of the RNA. 4. Antibiotics: Some antibiotics prevent bacterial cell growth by inhibiting RNA synthesis. For example, rifampin (rifampicin) inhibits transcription initiation by binding to the β subunit of prokaryotic RNA pol and preventing chain growth beyond three nucleotides (Fig. 31.10). Rifampin is important in the treatment of tuberculosis. Dactinomycin (actinomycin D) was the first antibiotic to find therapeutic application in tumor chemotherapy. It inserts (intercalates) between the DNA bases and inhibits transcription initiation and elongation.

1	D) was the first antibiotic to find therapeutic application in tumor chemotherapy. It inserts (intercalates) between the DNA bases and inhibits transcription initiation and elongation. IV. EUKARYOTIC GENE TRANSCRIPTION

1	IV. EUKARYOTIC GENE TRANSCRIPTION The transcription of eukaryotic genes is a far more complicated process than transcription in prokaryotes. Eukaryotic transcription involves separate polymerases for the synthesis of rRNA, tRNA, and mRNA. In addition, a large number of proteins called transcription factors (TF) are involved. TF bind to distinct sites on the DNA within the core promoter region, close (proximal) to it, or some distance away (distal). They are required for both the assembly of a transcription initiation complex at the promoter and the determination of which genes are to be transcribed. [Note: Each eukaryotic RNA pol has its own promoters and TF that bind core promoter sequences.] For TF to recognize and bind to their specific DNA sequences, the chromatin structure in that region must be decondensed (relaxed) to allow access to the DNA. The role of transcription in the regulation of gene expression is discussed in Chapter 33. A. Chromatin structure and gene expression

1	The association of DNA with histones to form nucleosomes (see p. 425) affects the ability of the transcription machinery to access the DNA to be transcribed. Most actively transcribed genes are found in a relatively decondensed form of chromatin called euchromatin, whereas most inactive segments of DNA are found in highly condensed heterochromatin. The interconversion of these forms is called chromatin remodeling. A major component of chromatin remodeling is the covalent modification of histones (for example, the acetylation of lysine residues at the amino terminus of histone proteins), as shown in Figure 31.11. Acetylation, mediated by histone acetyltransferases (HAT), eliminates the positive charge on the lysine, thereby decreasing the interaction of the histone with the negatively charged DNA. Removal of the acetyl group by histone deacetylases (HDAC) restores the positive charge and fosters stronger interactions between histones and DNA. [Note: The ATP-dependent repositioning of

1	DNA. Removal of the acetyl group by histone deacetylases (HDAC) restores the positive charge and fosters stronger interactions between histones and DNA. [Note: The ATP-dependent repositioning of nucleosomes is also required to access DNA.]

1	B. Nuclear RNA polymerases There are three distinct types of RNA pol in the nucleus of eukaryotic cells. All are large enzymes with multiple subunits. Each type of RNA pol recognizes particular genes. [Note: Mitochondria contain a single RNA pol that resembles the bacterial enzyme.] 1. RNA polymerase I: This enzyme synthesizes the precursor of the 28S, 18S, and 5.8S rRNA in the nucleolus. 2. RNA polymerase II: This enzyme synthesizes the nuclear precursors of mRNA that are processed and then translated to proteins. RNA pol II also synthesizes certain small ncRNA, such as snoRNA, snRNA, and miRNA. a.

1	a. Promoters for RNA polymerase II: In some genes transcribed by RNA pol II, a sequence of nucleotides (TATAAA) that is nearly identical to that of the Pribnow box (see p. 436) is found centered ~25 nucleotides upstream of the transcription start site. This core promoter consensus sequence is called the TATA, or Hogness, box. In the majority of genes, however, no TATA box is present. Instead, different core promoter elements such as Inr (initiator) or DPE (downstream promoter element) are present (Fig. 31.12). [Note: No one consensus sequence is found in all core promoters.] Because these sequences are on the same molecule of DNA as the gene being transcribed, they are cis-acting. The sequences serve as binding sites for proteins known as general transcription factors (GTF), which in turn interact with each other and with RNA pol II. b.

1	General transcription factors: GTF are the minimal requirements for recognition of the promoter, recruitment of RNA pol II to the promoter, formation of the preinitiation complex, and initiation of transcription at a basal level (Fig. 31.13A). GTF are encoded by different genes, synthesized in the cytosol, and diffuse (transit) to their sites of action, and so are trans-acting. [Note: In contrast to the prokaryotic holoenzyme, eukaryotic RNA pol II does not itself recognize and bind the promoter. Instead, TFIID, a GTF containing TATA-binding protein and TATA-associated factors, recognizes and binds the TATA box (and other core promoter elements). TFIIF, another GTF, brings the polymerase to the promoter. The helicase activity of TFIIH melts the DNA, and its kinase activity phosphorylates polymerase, allowing it to clear the promoter.] c. Regulatory elements and transcriptional activators: Additional consensus sequences lie upstream of the core promoter (see Fig. 31.12). Those close to

1	allowing it to clear the promoter.] c. Regulatory elements and transcriptional activators: Additional consensus sequences lie upstream of the core promoter (see Fig. 31.12). Those close to the core promoter (within ~200 nucleotides) are the proximal regulatory elements, such as the CAAT and GC boxes. Those farther away are the distal regulatory elements such as enhancers (see d. below). Proteins known as transcriptional activators or specific transcription factors (STF) bind these regulatory elements. STF bind to promoter proximal elements to regulate the frequency of transcription initiation and to distal elements to mediate the response to signals such as hormones (see p. 472) and regulate which genes are expressed at a given point in time. A typical protein-coding eukaryotic gene has binding sites for many such factors. STF have two binding domains. One is a DNA-binding domain, the other is a transcription activation domain that recruits the GTF to the core promoter as well as

1	binding sites for many such factors. STF have two binding domains. One is a DNA-binding domain, the other is a transcription activation domain that recruits the GTF to the core promoter as well as coactivator proteins such as the HAT enzymes involved in chromatin modification. [Note: Mediator, a multisubunit coactivator of RNA pol II–catalyzed transcription, binds the polymerase, the GTF, and the STF and regulates transcription initiation.]

1	Transcriptional activators bind DNA through a variety of motifs, such as the helix-loop-helix, zinc finger, and leucine zipper (see p. 18).

1	d. Role of enhancers: Enhancers are special DNA sequences that increase the rate of initiation of transcription by RNA pol II. Enhancers are typically on the same chromosome as the gene whose transcription they stimulate (Fig. 31.13B). However, they can 1) be located upstream (to the 5′-side) or downstream (to the 3′-side) of the transcription start site, 2) be close to or thousands of base pairs away from the promoter (Fig. 31.14), and 3) occur on either strand of the DNA. Enhancers contain DNA sequences called response elements that bind STF. By bending or looping the DNA, STF can interact with other TF bound to a promoter and with RNA pol II, thereby stimulating transcription (see Fig. 31.13B). Mediator also binds enhancers. [Note: Although silencers are similar to enhancers in that they also can act over long distances, they reduce gene expression.] e. RNA polymerase II inhibitor: α-Amanitin, a potent toxin produced by the poisonous mushroom Amanita phalloides (sometimes called

1	they also can act over long distances, they reduce gene expression.] e. RNA polymerase II inhibitor: α-Amanitin, a potent toxin produced by the poisonous mushroom Amanita phalloides (sometimes called the “death cap”), binds RNA pol II tightly and slows its movement, thereby inhibiting mRNA synthesis.

1	3. RNA polymerase III: This enzyme synthesizes tRNA, 5S rRNA, and some snRNA and snoRNA. V. POSTTRANSCRIPTIONAL MODIFICATION OF RNA A primary transcript is the initial, linear, RNA copy of a transcription unit (the segment of DNA between specific initiation and termination sequences). The primary transcripts of both prokaryotic and eukaryotic tRNA and rRNA are posttranscriptionally modified by cleavage of the original transcripts by ribonucleases. tRNA are further modified to help give each species its unique identity. In contrast, prokaryotic mRNA is generally identical to its primary transcript, whereas eukaryotic mRNA is extensively modified both co-and posttranscriptionally.

1	A. Ribosomal RNA rRNA of both prokaryotic and eukaryotic cells are generated from long precursor molecules called pre-rRNA. The 23S, 16S, and 5S rRNA of prokaryotes are produced from a single pre-rRNA molecule, as are the 28S, 18S, and 5.8S rRNA of eukaryotes (Fig. 31.15). [Note: Eukaryotic 5S rRNA is synthesized by RNA pol III and modified separately.] The prerRNA are cleaved by ribonucleases to yield intermediate-sized pieces of rRNA, which are further processed (trimmed by exonucleases and modified at some bases and riboses) to produce the required RNA species. [Note: In eukaryotes, rRNA genes are found in long, tandem arrays. rRNA synthesis and processing occur in the nucleolus, with base and sugar modifications facilitated by snoRNA.] B. Transfer RNA

1	B. Transfer RNA Both eukaryotic and prokaryotic tRNA are also made from longer precursor molecules that must be modified (Fig. 31.16). Sequences at both ends of the molecule are removed, and, if present, an intron is removed from the anticodon loop by nucleases. Other posttranscriptional modifications include addition of a –CCA sequence by nucleotidyltransferase to the 3′terminal end of tRNA and modification of bases at specific positions to produce the unusual bases characteristic of tRNA (see p. 291). C. Eukaryotic messenger RNA The collection of all the primary transcripts synthesized in the nucleus by RNA pol II is known as heterogeneous nuclear RNA (hnRNA). The premRNA components of hnRNA undergo extensive co-and posttranscriptional modification in the nucleus and become mature mRNA. These modifications usually include the following. [Note: Pol II itself recruits the proteins required for the modifications.] 1.

1	Addition of a 5′-cap: This is the first of the processing reactions for premRNA (Fig. 31.17). The cap is a 7-methylguanosine attached to the 5′terminal end of the mRNA through an unusual 5′→5′-triphosphate linkage that is resistant to most nucleases. Creation of the cap requires removal of the γ phosphoryl group from the 5′-triphosphate of the premRNA, followed by addition of guanosine monophosphate (from guanosine triphosphate) by the nuclear enzyme guanylyltransferase. Methylation of this terminal guanine occurs in the cytosol and is catalyzed by guanine-7-methyltransferase. S-Adenosylmethionine is the source of the methyl group (see p. 263). Additional methylation steps may occur. The addition of this 7-methylguanosine cap helps stabilize the mRNA and permits efficient initiation of translation (see p. 455). 2.

1	2. Addition of a 3′-poly-A tail: Most eukaryotic mRNA (with several exceptions, including those for the histones) have a chain of 40–250 adenylates (adenosine monophosphates) attached to the 3′-end (see Fig. 31.17). This poly-A tail is not transcribed from the DNA but rather is added by the nuclear enzyme, polyadenylate polymerase, using ATP as the substrate. The pre-mRNA is cleaved downstream of a consensus sequence, called the polyadenylation signal sequence (AAUAAA), found near the 3′-end of the RNA, and the poly-A tail is added to the new 3′end. Tailing terminates eukaryotic transcription. Tails help stabilize the mRNA, facilitate its exit from the nucleus, and aid in translation. After the mRNA enters the cytosol, the poly-A tail is gradually shortened. 3.

1	3. Splicing: Maturation of eukaryotic mRNA usually involves removal from the primary transcript of RNA sequences (introns or intervening sequences) that do not code for protein. The remaining coding (expressed) sequences, the exons, are joined together to form the mature mRNA. The process of removing introns and joining exons is called splicing. The molecular complex that accomplishes these tasks is known as the spliceosome. A few eukaryotic primary transcripts contain no introns (for example, those from histone genes). Others contain a few introns, whereas some, such as the primary transcripts for the α chains of collagen, contain >50 introns that must be removed.

1	a. Role of small nuclear RNA: In association with multiple proteins, uracil-rich snRNA form five small nuclear ribonucleoprotein particles (snRNP, or “snurp”) designated as U1, U2, U4, U5, and U6 that mediate splicing. They facilitate the removal of introns by forming base pairs with the consensus sequences at each end of the intron (Fig. 31.18). [Note: In systemic lupus erythematosus, an autoimmune disease, patients produce antibodies against their own nuclear proteins such as snRNP.] b.

1	Mechanism: The binding of snRNP brings the sequences of neighboring exons into the correct alignment for splicing, allowing two transesterification reactions (catalyzed by the RNA of U2, U5, and U6) to occur. The 2′-OH group of an adenine nucleotide (known as the branch site A) in the intron attacks the phosphate at the 5′-end of the intron (splice-donor site), forming an unusual 2′→5′-phosphodiester bond and creating a “lariat” structure (see Fig. 31.18). The newly freed 3′-OH of exon 1 attacks the 5′-phosphate at the spliceacceptor site, forming a phosphodiester bond that joins exons 1 and 2. The excised intron is released as a lariat, which is typically degraded but may be a precursor for ncRNA such as snoRNA. [Note: The GU and AG sequences at the beginning and end, respectively, of introns are invariant. However, additional sequences are critical for splice-site recognition.] After introns have been removed and exons joined, the mature mRNA molecules pass into the cytosol through

1	are invariant. However, additional sequences are critical for splice-site recognition.] After introns have been removed and exons joined, the mature mRNA molecules pass into the cytosol through pores in the nuclear membrane. [Note: The introns in tRNA (see Fig. 31.16) are removed by a different mechanism.] c.

1	Effect of splice site mutations: Mutations at splice sites can lead to improper splicing and the production of aberrant proteins. It is estimated that at least 20% of all genetic diseases are a result of mutations that affect RNA splicing. For example, mutations that cause the incorrect splicing of β-globin mRNA are responsible for some cases of β-thalassemia, a disease in which the production of the βglobin protein is defective (see p. 38). Splice site mutations can result in exons being skipped (removed) or introns retained. They can also activate cryptic splice sites, which are sites that contain the 5′ or 3′ consensus sequence but are not normally used.

1	4. Alternative splicing: The pre-mRNA molecules from >90% of human genes can be spliced in alternative ways in different tissues. Because this produces multiple variations of the mRNA and, therefore, of its protein product (Fig. 31.19), it is a mechanism for producing a large, diverse set of proteins from a limited set of genes. For example, the mRNA for tropomyosin (TM), an actin filament–binding protein of the cytoskeleton (and of the contractile apparatus in muscle cells), undergoes extensive tissue-specific alternative splicing with production of multiple isoforms of the TM protein. VI. CHAPTER SUMMARY

1	Three major types of RNA participate in the process of protein synthesis: ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA), as shown in Figure 31.20. They are unbranched polymers of nucleotides but differ from DNA by containing ribose instead of deoxyribose and uracil instead of thymine. rRNA is a component of the ribosomes. tRNA serves as an adaptor molecule that carries a specific amino acid to the site of protein synthesis. mRNA (coding RNA) carries genetic information from DNA for use in protein synthesis. The process of RNA synthesis is called transcription, and the substrates are ribonucleoside triphosphates. The enzyme that synthesizes RNA is RNA polymerase (RNA pol). In prokaryotic cells, the core enzyme has five subunits (2 α, 1 β, 1 β′, and 1 Ω) and possesses 5′→3′ polymerase activity needed for transcription. The core enzyme requires an additional subunit, sigma (σ) factor, to recognize the nucleotide sequence (promoter region) at the beginning of the DNA

1	polymerase activity needed for transcription. The core enzyme requires an additional subunit, sigma (σ) factor, to recognize the nucleotide sequence (promoter region) at the beginning of the DNA to be transcribed. This region contains consensus sequences that are highly conserved and include the −10 Pribnow box and the −35 sequence. Another protein, rho (ρ), is required for termination of transcription of some genes. There are three distinct types of RNA pol in the nucleus of eukaryotic cells. RNA pol I synthesizes the precursor of rRNA in the nucleolus. In the nucleoplasm, RNA pol II synthesizes the precursors for mRNA and some noncoding RNA, and RNA pol III synthesizes the precursors of tRNA and 5S rRNA. In both prokaryotes and eukaryotes, RNA pol does not require a primer. Proofreading involves the polymerase backtracking and cleaving the transcript. Core promoters for genes transcribed by RNA pol II contain cis-acting consensus sequences, such as the TATA (Hogness) box, which

1	involves the polymerase backtracking and cleaving the transcript. Core promoters for genes transcribed by RNA pol II contain cis-acting consensus sequences, such as the TATA (Hogness) box, which serve as binding sites for transacting general transcription factors. Upstream of these are proximal regulatory elements, such as the CAAT and GC boxes, and distal regulatory elements, such as enhancers. Specific transcription factors (transcriptional activators) and Mediator complex bind these elements and regulate the frequency of transcription initiation, the response to signals such as hormones, and which genes are expressed at any given time. Eukaryotic transcription requires that the chromatin be relaxed (decondensed) in a process known as chromatin remodeling. A primary transcript is a linear copy of a transcription unit, the segment of DNA between specific initiation and termination sequences. The primary transcripts of both prokaryotic and eukaryotic tRNA and rRNA are

1	is a linear copy of a transcription unit, the segment of DNA between specific initiation and termination sequences. The primary transcripts of both prokaryotic and eukaryotic tRNA and rRNA are posttranscriptionally modified. The rRNA are synthesized from long precursor molecules called pre-rRNA. These precursors are cleaved and trimmed by ribonucleases, producing the three largest rRNA, and bases and sugars are modified. Eukaryotic 5S rRNA is synthesized by RNA pol III and is modified separately. Prokaryotic and eukaryotic tRNA are also made from longer precursor molecules (pre-tRNA). If present, an intron is removed by nucleases, and both ends of the molecule are trimmed by ribonucleases. A 3′-CCA sequence is added, and bases at specific positions are modified. Prokaryotic mRNA is generally identical to its primary transcript, whereas eukaryotic pre-mRNA is extensively modified co-and posttranscriptionally. For example, a 7-methylguanosine cap is attached to the 5′-end of the mRNA

1	identical to its primary transcript, whereas eukaryotic pre-mRNA is extensively modified co-and posttranscriptionally. For example, a 7-methylguanosine cap is attached to the 5′-end of the mRNA through a 5′→5′ linkage. A long poly-A tail, not transcribed from the DNA, is attached by polyadenylate polymerase to the 3′-end of most mRNA. Most eukaryotic mRNA also contains intervening sequences (introns) that must be removed for the mRNA to be functional. Their removal, as well as the joining of expressed sequences (exons), requires a spliceosome composed of small nuclear ribonucleoprotein particles (“snurps”) that mediate the process of splicing. Eukaryotic mRNA is monocistronic, containing information from just one gene, whereas prokaryotic mRNA is polycistronic.

1	Choose the ONE best answer. 1.1. An 8-month-old male with severe anemia is found to have β-thalassemia. Genetic analysis shows that one of his β-globin genes has a mutation that creates a new splice-acceptor site 19 nucleotides upstream of the normal splice-acceptor site of the first intron. Which of the following best describes the new messenger RNA molecule that can be produced from this mutant gene? A. Exon 1 will be too short. B. Exon 1 will be too long. C. Exon 2 will be too short. D. Exon 2 will be too long. E. Exon 2 will be missing.

1	A. Exon 1 will be too short. B. Exon 1 will be too long. C. Exon 2 will be too short. D. Exon 2 will be too long. E. Exon 2 will be missing. Correct answer = D. Because the mutation creates an additional splice-acceptor site (the 3′-end) upstream of the normal acceptor site of intron 1, the 19 nucleotides that are usually found at the 3′-end of the excised intron 1 lariat can remain behind as part of exon 2. The presence of these extra nucleotides in the coding region of the mutant messenger RNA (mRNA) molecule will prevent the ribosome from translating the message into a normal β-globin protein molecule. Those mRNA for which the normal splice site is used to remove the first intron will be normal, and their translation will produce normal β-globin protein.

1	1.2. A 4-year-old child who easily tires and has trouble walking is diagnosed with Duchenne muscular dystrophy, an X-linked recessive disorder. Genetic analysis shows that the patient’s gene for the muscle protein dystrophin contains a mutation in its promoter region. Of the choices listed, which would be the most likely effect of this mutation? A. Initiation of dystrophin transcription will be defective. B. Termination of dystrophin transcription will be defective. C. Capping of dystrophin messenger RNA will be defective. D. Splicing of dystrophin messenger RNA will be defective. E. Tailing of dystrophin messenger RNA will be defective.

1	C. Capping of dystrophin messenger RNA will be defective. D. Splicing of dystrophin messenger RNA will be defective. E. Tailing of dystrophin messenger RNA will be defective. Correct answer = A. Mutations in the promoter typically prevent formation of the RNA polymerase II transcription initiation complex, resulting in a decrease in the initiation of messenger RNA (mRNA) synthesis. A deficiency of dystrophin mRNA will result in a deficiency in the production of the dystrophin protein. Capping, splicing, and tailing defects are not a consequence of promoter mutations. They can, however, result in mRNA with decreased stability (capping and tailing defects) or an mRNA in which exons have been skipped (lost) or introns retained (splicing defects). 1.3. A mutation to this sequence in eukaryotic messenger RNA (mRNA) will affect the process by which the 3′-end polyadenylate (poly-A) tail is added to the mRNA. A. AAUAAA B. CAAT C. CCA D. GU… A…AG E. TATAAA

1	A. AAUAAA B. CAAT C. CCA D. GU… A…AG E. TATAAA Correct answer = A. An endonuclease cleaves mRNA just downstream of this polyadenylation signal, creating a new 3′-end to which polyadenylate polymerase adds the poly-A tail using ATP as the substrate in a template-independent process. CAAT and TATAAA are sequences found in promoters for RNA polymerase II. CCA is added to the 3′-end of pre-transfer RNA by nucleotidyltransferase. GU…A…AG denotes an intron in eukaryotic premRNA. 1.4. This protein factor identifies the promoter of protein-coding genes in eukaryotes. A. Pribnow box B. Rho C. Sigma D. TFIID E. U1 Correct answer = D. The general transcription factor TFIID recognizes and binds core promoter elements such as the TATA-like box in eukaryotic protein-coding genes. These genes are transcribed by RNA polymerase II. The

1	Pribnow box is a cis-acting element in prokaryotic promoters. Rho is involved in the termination of prokaryotic transcription. Sigma is the subunit of prokaryotic RNA polymerase that recognizes and binds the prokaryotic promoter. U1 is a ribonucleoprotein involved in splicing of eukaryotic premRNA. 1.5. What is the sequence (conventionally written) of the RNA product of the DNA template sequence, GATCTAC, also conventionally written? Correct answer = 5′-GUAGAUC-3′. Nucleic acid sequences are conventionally written 5′ to 3′. The template strand (5′-GATCTAC-3′) is used as 3′CATCTAG-5′. The RNA product is complementary to the template strand (and identical to the coding strand), with U replacing T. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Genetic information, stored in the chromosomes and transmitted to daughter cells through DNA replication, is expressed through transcription to RNA and, in the case of messenger RNA (mRNA), subsequent translation into proteins (polypeptides) as shown in Figure 32.1. [Note: The proteome is the complete set of proteins expressed in a cell.] The process of protein synthesis is called translation because the “language” of the nucleotide sequence on the mRNA is translated into the language of an amino acid sequence. Translation requires a genetic code, through which the information contained in the nucleotide sequence is expressed to produce a specific amino acid sequence. Any alteration in the nucleotide sequence may result in an incorrect amino acid being inserted into the protein, potentially causing disease or even death of the organism. Newly made immature (nascent) proteins undergo a number of processes to achieve their functional form. They must fold properly, and misfolding can

1	causing disease or even death of the organism. Newly made immature (nascent) proteins undergo a number of processes to achieve their functional form. They must fold properly, and misfolding can result in aggregation or degradation of the protein. Many proteins are covalently modified to alter their activities. Lastly, proteins are targeted to their final intra-or extracellular destinations by signals present in the proteins themselves.

1	II. THE GENETIC CODE The genetic code is a “dictionary” that identifies the correspondence between a sequence of nucleotide bases and a sequence of amino acids. Each individual “word” in the code is composed of three nucleotide bases. These genetic words are called codons. A. Codons Codons are presented in the mRNA language of adenine (A), guanine (G), cytosine (C), and uracil (U). Their nucleotide sequences are always written from the 5′-end to the 3′-end. The four nucleotide bases are used to produce the three-base codons. Therefore, 64 different combinations of bases exist, taken three at a time (a triplet code), as shown in the table in Figure 32.2. many common amino acids are shown as examples. 1.

1	many common amino acids are shown as examples. 1. How to translate a codon: This table can be used to translate any codon and, thus, to determine which amino acids are coded for by an mRNA sequence. For example, the codon AUG codes for methionine ([Met] see Fig. 32.2). [Note: AUG is the initiation (start) codon for translation.] Sixty-one of the 64 codons code for the 20 standard amino acids (see p. 1). 2. Termination codons: Three of the codons, UAA, UAG, and UGA, do not code for amino acids but, rather, are termination (also called stop, or nonsense) codons. When one of these codons appears in an mRNA sequence, synthesis of the polypeptide coded for by that mRNA stops. B. Characteristics

1	B. Characteristics Usage of the genetic code is remarkably consistent throughout all living organisms. It is assumed that once the standard genetic code evolved in primitive organisms, any mutation (a permanent change in DNA sequence) that altered its meaning would have caused the alteration of most, if not all, protein sequences, resulting in lethality. Characteristics of the genetic code include the following. 1. Specificity: The genetic code is specific (unambiguous), because a particular codon always codes for the same amino acid. 2. Universality: The genetic code is virtually universal insofar as its specificity has been conserved from very early stages of evolution, with only slight differences in the manner in which the code is translated. [Note: An exception occurs in mitochondria, in which a few codons have meanings different than those shown in Figure 32.2. For example, UGA codes for tryptophan (Trp).] 3.

1	Degeneracy: The genetic code is degenerate (sometimes called redundant). Although each codon corresponds to a single amino acid, a given amino acid may have more than one triplet coding for it. For example, arginine (Arg) is specified by six different codons (see Fig. 32.2). Only Met and Trp have just one coding triplet. 4. Nonoverlapping and commaless: The genetic code is nonoverlapping and commaless, meaning that the code is read from a fixed starting point as a continuous sequence of bases, taken three at a time without any punctuation between codons. For example, AGCUGGAUACAU is read as AGC UGG AUA CAU. C. Consequences of altering the nucleotide sequence Changing a single nucleotide base (a point mutation) in the coding region of an mRNA can lead to any one of three results (Fig. 32.3). 1.

1	C. Consequences of altering the nucleotide sequence Changing a single nucleotide base (a point mutation) in the coding region of an mRNA can lead to any one of three results (Fig. 32.3). 1. Silent mutation: The codon containing the changed base may code for the same amino acid. For example, if the serine (Ser) codon UCA is changed at the third base and becomes UCU, it still codes for Ser. This is termed a silent mutation. 2. Missense mutation: The codon containing the changed base may code for a different amino acid. For example, if the Ser codon UCA is changed at the first base and becomes CCA, it will code for a different amino acid (in this case, proline [Pro]). This is termed a missense mutation. 3.

1	3. Nonsense mutation: The codon containing the changed base may become a termination codon. For example, if the Ser codon UCA is changed at the second base and becomes UAA, the new codon causes premature termination of translation at that point and the production of a shortened (truncated) protein. This is termed a nonsense mutation. [Note: The nonsense-mediated degradation pathway can degrade mRNA containing premature stops.] 4. Other mutations: These can alter the amount or structure of the protein produced by translation.

1	a. Trinucleotide repeat expansion: Occasionally, a sequence of three bases that is repeated in tandem will become amplified in number so that too many copies of the triplet occur. If this happens within the coding region of a gene, the protein will contain many extra copies of one amino acid. For example, expansion of the CAG codon in exon 1 of the gene for huntingtin protein leads to the insertion of many extra glutamine residues in the protein, causing the neurodegenerative disorder Huntington disease (Fig. 32.4). The additional glutamines result in an abnormally long protein that is cleaved, producing toxic fragments that aggregate in neurons. If the trinucleotide repeat expansion occurs in an untranslated region (UTR) of a gene, the result can be a decrease in the amount of protein produced, as seen in fragile X syndrome and myotonic dystrophy. Over 20 triplet expansion diseases are known. [Note: In fragile X syndrome, the most common cause of intellectual disability in males, the

1	as seen in fragile X syndrome and myotonic dystrophy. Over 20 triplet expansion diseases are known. [Note: In fragile X syndrome, the most common cause of intellectual disability in males, the expansion results in gene silencing through DNA hypermethylation (see p. 476).] b.

1	Splice site mutations: Mutations at splice sites (see p. 443) can alter the way in which introns are removed from pre-mRNA molecules, producing aberrant proteins. [Note: In myotonic dystrophy, a muscle disorder, gene silencing is the result of splicing alterations due to triplet expansion.] c.

1	Frameshift mutations: If one or two nucleotides are either deleted from or added to the coding region of an mRNA, a frameshift mutation occurs, altering the reading frame. This can result in a product with a radically different amino acid sequence or a truncated product due to the eventual creation of a termination codon (Fig. 32.5). If three nucleotides are added, a new amino acid is added to the peptide. If three are deleted, an amino acid is lost. Loss of three nucleotides maintains the reading frame but can result in serious pathology. For example, cystic fibrosis (CF), a chronic, progressive, inherited disease that primarily affects the pulmonary and digestive systems, is most commonly caused by deletion of three nucleotides from the coding region of a gene, resulting in the loss of phenylalanine (Phe, or F; see p. 5) at the 508th position (∆F508) in the CF transmembrane conductance regulator (CFTR) protein encoded by that gene. This ∆F508 mutation prevents normal folding of

1	of phenylalanine (Phe, or F; see p. 5) at the 508th position (∆F508) in the CF transmembrane conductance regulator (CFTR) protein encoded by that gene. This ∆F508 mutation prevents normal folding of CFTR, leading to its destruction by the proteasome (see p. 247). CFTR normally functions as a chloride channel in epithelial cells, and its loss results in the production of thick, sticky secretions in the lungs and pancreas, leading to lung damage and digestive deficiencies (see p. 174). The incidence of CF is highest (1 in 3,300) in those of Northern European origin. In >70% of individuals with CF, the ∆F508 mutation is the cause of the disease.

1	= guanine; U = uracil. III. COMPONENTS REQUIRED FOR TRANSLATION A large number of components are required for the synthesis of a protein. These include all the amino acids that are found in the finished product, the mRNA to be translated, transfer RNA (tRNA) for each of the amino acids, functional ribosomes, energy sources, and enzymes as well as noncatalytic protein factors needed for the initiation, elongation, and termination steps of polypeptide chain synthesis. A. Amino acids All the amino acids that eventually appear in the finished protein must be present at the time of protein synthesis. If one amino acid is missing, translation stops at the codon specifying that amino acid. [Note: This demonstrates the importance of having all the essential amino acids (see p. 262) in sufficient quantities in the diet to insure continued protein synthesis.] B. Transfer RNA

1	262) in sufficient quantities in the diet to insure continued protein synthesis.] B. Transfer RNA At least one specific type of tRNA is required for each amino acid. In humans, there are at least 50 species of tRNA, whereas bacteria contain at least 30 species. Because there are only 20 different amino acids commonly carried by tRNA, some amino acids have more than one specific tRNA molecule. This is particularly true of those amino acids that are coded for by several codons.

1	1. Amino acid attachment site: Each tRNA molecule has an attachment site for a specific (cognate) amino acid at its 3′-end (Fig. 32.6). The carboxyl group of the amino acid is in an ester linkage with the 3′-hydroxyl of the ribose portion of the A nucleotide in the –CCA sequence at the 3′-end of the tRNA. [Note: A tRNA with a covalently attached (activated) amino acid is charged. Without an attached amino acid, it is uncharged.] 2. Anticodon: Each tRNA molecule also contains a three-base nucleotide sequence, the anticodon, which pairs with a specific codon on the mRNA (see Fig. 32.6). This codon specifies the insertion into the growing polypeptide chain of the amino acid carried by that tRNA. C. Aminoacyl-tRNA synthetases

1	This family of 20 different enzymes is required for attachment of amino acids to their corresponding tRNA. Each member of this family recognizes a specific amino acid and all the tRNA that correspond to that amino acid (isoaccepting tRNA, up to five per amino acid). Aminoacyl-tRNA synthetases catalyze a two-step reaction that results in the covalent attachment of the α-carboxyl group of an amino acid to the A in the –CCA sequence at the 3′-end of its corresponding tRNA. The overall reaction requires ATP, which is cleaved to adenosine monophosphate and inorganic pyrophosphate (PPi), as shown in Figure 32.7. The extreme specificity of the synthetases in recognizing both the amino acid and its cognate tRNA contributes to the high fidelity of translation of the genetic message. In addition to their synthetic activity, the aminoacyl-tRNA synthetases have a proofreading, or editing activity that can remove an incorrect amino acid from the enzyme or the tRNA molecule.

1	RNA (tRNA) by an aminoacyl-tRNA synthetase. PPi = pyrophosphate; Pi = monophosphate; ~ = high-energy bond. D. Messenger RNA The specific mRNA required as a template for the synthesis of the desired polypeptide must be present. [Note: In eukaryotes, mRNA is circularized for use in translation.] E. Functionally competent ribosomes

1	E. Functionally competent ribosomes As shown in Figure 32.8, ribosomes are large complexes of protein and ribosomal RNA (rRNA), in which rRNA predominates. They consist of two subunits (one large and one small) whose relative sizes are given in terms of their sedimentation coefficients, or S (Svedberg) values. [Note: Because the S values are determined by both shape and size, their numeric values are not strictly additive. For example, the prokaryotic 50S and 30S ribosomal subunits together form a 70S ribosome. The eukaryotic 60S and 40S subunits form an 80S ribosome.] Prokaryotic and eukaryotic ribosomes are similar in structure and serve the same function, namely, as the macromolecular complexes in which the synthesis of proteins occurs.

1	The small ribosomal subunit binds mRNA and determines the accuracy of translation by insuring correct base-pairing between the mRNA codon and the tRNA anticodon. The large ribosomal subunit catalyzes formation of the peptide bonds that link amino acid residues in a protein. 1. Ribosomal RNA: As discussed on p. 434, prokaryotic ribosomes contain three size species of rRNA, whereas eukaryotic ribosomes contain four (see Fig. 32.8). The rRNA are generated from a single pre-rRNA by the action of ribonucleases, and some bases and riboses are modified. 2. Ribosomal proteins: Ribosomal proteins are present in greater numbers in eukaryotic ribosomes than in prokaryotic ribosomes. These proteins play a variety of roles in the structure and function of the ribosome and its interactions with other components of the translation system. 3.

1	3. A, P, and E sites: The ribosome has three binding sites for tRNA molecules: the A, P, and E sites, each of which extends over both subunits. Together, they cover three neighboring codons. During translation, the A site binds an incoming aminoacyl-tRNA as directed by the codon currently occupying this site. This codon specifies the next amino acid to be added to the growing peptide chain. The P site is occupied by peptidyl-tRNA. This tRNA carries the chain of amino acids that has already been synthesized. The E site is occupied by the empty tRNA as it is about to exit the ribosome. (See Fig. 32.13 for an illustration of the role of the A, P, and E sites in translation.) 4.

1	Cellular location: In eukaryotic cells, the ribosomes either are free in the cytosol or are in close association with the endoplasmic reticulum (which is then known as the rough endoplasmic reticulum, or RER). RER-associated ribosomes are responsible for synthesizing proteins (including glycoproteins; see p. 166) that are to be exported from the cell, incorporated into membranes, or imported into lysosomes (see p. 169 for an overview of the latter process). Cytosolic ribosomes synthesize proteins required in the cytosol itself or destined for the nucleus, mitochondria, or peroxisomes. [Note: Mitochondria contain their own ribosomes (55S) and their own unique, circular DNA. Most mitochondrial proteins, however, are encoded by nuclear DNA, synthesized completely in the cytosol, and then targeted to mitochondria.] F. Protein factors

1	F. Protein factors Initiation, elongation, and termination (or, release) factors are required for polypeptide synthesis. Some of these protein factors perform a catalytic function, whereas others appear to stabilize the synthetic machinery. [Note: A number of the factors are small, cytosolic G proteins and thus are active when bound to guanosine triphosphate (GTP) and inactive when bound to guanosine diphosphate (GDP). See p. 95 for a discussion of the membrane-associated G proteins.] G. Energy sources

1	G. Energy sources Cleavage of four high-energy bonds (see p. 73) is required for the addition of one amino acid to the growing polypeptide chain: two from ATP in the aminoacyl-tRNA synthetase reaction, one in the removal of PPi and one in the subsequent hydrolysis of the PPi, to two Pi by pyrophosphatase, and two from GTP, one for binding the aminoacyl-tRNA to the A site and one for the translocation step (see Fig. 32.13, p. 457). [Note: Additional ATP and GTP molecules are required for initiation in eukaryotes, whereas an additional GTP molecule is required for termination in both eukaryotes and prokaryotes.] Translation, then, is a major consumer of energy. IV. CODON RECOGNITION BY TRANSFER RNA Correct pairing of the codon in the mRNA with the anticodon of the tRNA is essential for accurate translation (see Fig. 32.6). Most tRNA (isoaccepting tRNA) recognize more than one codon for a given amino acid. A. Antiparallel binding between codon and anticodon

1	A. Antiparallel binding between codon and anticodon Binding of the tRNA anticodon to the mRNA codon follows the rules of complementary and antiparallel binding, that is, the mRNA codon is read 5′→3′ by an anticodon pairing in the opposite (3′→5′) orientation (Fig. 32.9). [Note: Nucleotide sequences are always written in the 5′ to 3′ direction unless otherwise noted. Two nucleotide sequences orient in an antiparallel manner.] B. Wobble hypothesis

1	B. Wobble hypothesis The mechanism by which a tRNA can recognize more than one codon for a specific amino acid is described by the wobble hypothesis, which states that codon–anticodon pairing follows the traditional Watson-Crick rules (G pairs with C and A pairs with U) for the first two bases of the codon but can be less stringent for the last base. The base at the 5′-end of the anticodon (the first base of the anticodon) is not as spatially defined as the other two bases. Movement of that first base allows nontraditional base-pairing with the 3′-base of the codon (the last base of the codon). This movement is called wobble and allows a single tRNA to recognize more than one codon. Examples of these flexible pairings are shown in Figure 32.9. The result of wobble is that 61 tRNA species are not required to read the 61 codons that code for amino acids. V. STEPS IN TRANSLATION

1	V. STEPS IN TRANSLATION The process of protein synthesis translates the 3-letter alphabet of nucleotide sequences on mRNA into the 20-letter alphabet of amino acids that constitute proteins. The mRNA is translated from its 5′-end to its 3′-end, producing a protein synthesized from its amino (N)-terminal end to its carboxyl (C)-terminal end. Prokaryotic mRNA often have several coding regions (that is, they are polycistronic; see p. 434). Each coding region has its own initiation and termination codon and produces a separate species of polypeptide. In contrast, each eukaryotic mRNA has only one coding region (that is, it is monocistronic). The process of translation is divided into three separate steps: initiation, elongation, and termination. Eukaryotic translation resembles that of prokaryotes in most aspects. Individual differences are noted in the text.

1	One important difference is that translation and transcription are temporally linked in prokaryotes, with translation starting before transcription is completed as a consequence of the lack of a nuclear membrane in prokaryotes. A. Initiation Initiation of protein synthesis involves the assembly of the components of the translation system before peptide-bond formation occurs. These components include the two ribosomal subunits, the mRNA to be translated, the aminoacyl-tRNA specified by the first codon in the message, GTP, and initiation factors that facilitate the assembly of this initiation complex (see Fig. 32.13). [Note: In prokaryotes, three initiation factors are known (IF-1, IF-2, and IF-3), whereas in eukaryotes, there are many (designated eIF to indicate eukaryotic origin). Eukaryotes also require ATP for initiation.] The following are two mechanisms by which the ribosome recognizes the nucleotide sequence (AUG) that initiates translation.

1	1. Shine-Dalgarno sequence: In Escherichia coli (E. coli), a purine-rich sequence of nucleotide bases, known as the Shine-Dalgarno (SD) sequence, is located six to ten bases upstream of the initiating AUG codon on the mRNA molecule (that is, near its 5′-end). The 16S rRNA component of the small (30S) ribosomal subunit has a nucleotide sequence near its 3′-end that is complementary to all or part of the SD sequence. Therefore, the 5′-end of the mRNA and the 3′-end of the 16S rRNA can form complementary base pairs, facilitating the positioning of the 30S subunit on the mRNA in close proximity to the initiating AUG codon (Fig. 32.10). 2.

1	2. 5′-Cap: Eukaryotic mRNA do not have SD sequences. In eukaryotes, the small (40S) ribosomal subunit (aided by members of the eIF-4 family of proteins) binds close to the cap structure at the 5′-end of the mRNA and moves 5′→3′ along the mRNA until it encounters the initiator AUG. This scanning process requires ATP. Cap-independent initiation can occur if the 40S subunit binds to an internal ribosome entry site close to the start codon. [Note: Interactions between the cap-binding eIF-4 proteins and the poly-A tail–binding proteins on eukaryotic mRNA mediate circularization of the mRNA and likely prevent the use of incompletely processed mRNA in translation.] 3.

1	Initiation codon: The initiating AUG is recognized by a special initiator tRNA (tRNAi). Recognition is facilitated by IF-2-GTP in prokaryotes and eIF-2-GTP (plus additional eIF) in eukaryotes. The charged tRNAi is the only tRNA recognized by (e)IF-2 and the only tRNA to go directly to the P site on the small subunit. [Note: Base modifications distinguish tRNAi from the tRNA used for internal AUG codons.] In bacteria and mitochondria, tRNAi carries an N-formylated methionine (fMet), as shown in Figure 32.11. After Met is attached to tRNAi, the formyl group is added by the enzyme transformylase, which uses N10-formyl tetrahydrofolate (see p. 267) as the carbon donor. In eukaryotes, tRNAi carries a Met that is not formylated. In both prokaryotic and eukaryotic cells, this N-terminal Met is usually removed before translation is completed. The large ribosomal subunit then joins the complex, and a functional ribosome is formed with the charged tRNAi in the P site. The A site is empty.

1	is usually removed before translation is completed. The large ribosomal subunit then joins the complex, and a functional ribosome is formed with the charged tRNAi in the P site. The A site is empty. [Note: Specific (e)IF function as anti-association factors and prevent premature addition of the large subunit.] The GTP on (e)IF-2 gets hydrolyzed to GDP. In eukaryotes, the guanine nucleotide exchange factor eIF-2B facilitates the reactivation of eIF-2-GDP through replacement of GDP by GTP.

1	B. Elongation

1	Elongation of the polypeptide involves the addition of amino acids to the carboxyl end of the growing chain. Delivery of the aminoacyl-tRNA whose codon appears next on the mRNA template in the ribosomal A site (a process known as decoding) is facilitated in E. coli by elongation factors EF-Tu-GTP and EF-Ts and requires GTP hydrolysis. [Note: In eukaryotes, comparable elongation factors are EF-1α-GTP and EF-1βγ. Both EF-Ts and EF-1βγ function in guanine nucleotide exchange.] Peptide-bond formation between the α-carboxyl group of the amino acid in the P site and the αamino group of the amino acid in the A site is catalyzed by peptidyltransferase, an activity intrinsic to an rRNA of the large subunit (Fig. 32.12). [Note: Because this rRNA catalyzes the reaction, it is a ribozyme (see p. 54).] After the peptide bond has been formed, the peptide on the tRNA at the P site is transferred to the amino acid on the tRNA at the A site, a process known as transpeptidation. The ribosome then

1	54).] After the peptide bond has been formed, the peptide on the tRNA at the P site is transferred to the amino acid on the tRNA at the A site, a process known as transpeptidation. The ribosome then advances three nucleotides toward the 3′-end of the mRNA. This process is known as translocation and, in prokaryotes, requires the participation of EF-G-GTP (eukaryotes use EF-2-GTP) and GTP hydrolysis. Translocation causes movement of the uncharged tRNA from the P to the E site for release and movement of the peptidyl-tRNA from the A to the P site. The process is repeated until a termination codon is encountered. [Note: Because of the length of most mRNA, more than one ribosome at a time can translate a message. Such a complex of one mRNA and a number of ribosomes is called a polysome, or polyribosome.]

1	C. Termination

1	Termination occurs when one of the three termination codons moves into the A site. These codons are recognized in E. coli by release factors: RF-1, which recognizes UAA and UAG, and RF-2, which recognizes UGA and UAA. The binding of these release factors results in hydrolysis of the bond linking the peptide to the tRNA at the P site, causing the nascent protein to be released from the ribosome. A third release factor, RF-3-GTP, then causes the release of RF-1 or RF-2 as GTP is hydrolyzed (see Fig. 32.13). [Note: Eukaryotes have a single release factor, eRF, which recognizes all three termination codons. A second factor, eRF-3, functions like the prokaryotic RF-3. See Figure 32.14 for a summary of the factors used in translation.] The steps in prokaryotic protein synthesis, as well as some antibiotic inhibitors of the process, are summarized in Figure 32.13. The newly synthesized polypeptide may undergo further modification as described below, and the ribosomal subunits, mRNA, tRNA,

1	antibiotic inhibitors of the process, are summarized in Figure 32.13. The newly synthesized polypeptide may undergo further modification as described below, and the ribosomal subunits, mRNA, tRNA, and protein factors can be recycled and used to synthesize another polypeptide. [Note: In prokaryotes, ribosome recycling factors mediate separation of the subunits. In eukaryotes, eRF and ATP hydrolysis are required.]

1	D. Translation regulation Gene expression is most commonly regulated at the transcriptional level, but translation may also be regulated. An important mechanism by which this is achieved in eukaryotes is by covalent modification of eIF-2: Phosphorylated eIF-2 is inactive (see p. 476). In both eukaryotes and prokaryotes, regulation can also be achieved through proteins that bind mRNA and inhibit its use by blocking translation. E. Protein folding Proteins must fold to assume their functional, native state. Folding can be spontaneous (as a result of the primary structure) or facilitated by proteins known as chaperones (see p. 20). F. Protein targeting

1	Although most protein synthesis in eukaryotes is initiated in the cytoplasm, many proteins perform their functions within subcellular organelles or outside of the cell. Such proteins normally contain amino acid sequences that direct the proteins to their final locations. For example, secreted proteins are targeted during synthesis (cotranslational targeting) to the RER by the presence of an N-terminal hydrophobic signal sequence. The sequence is recognized by the signal recognition particle (SRP), a ribonucleoprotein that binds the ribosome, halts elongation, and delivers the ribosome–peptide complex to an RER membrane channel (the translocon) via interaction with the SRP receptor. Translation resumes, the protein enters the RER lumen, and its signal sequence is cleaved (Fig. 32.15). The protein moves through the RER and the Golgi, is processed, packaged into vesicles, and secreted. Proteins targeted after synthesis (posttranslational) include nuclear proteins that contain an

1	The protein moves through the RER and the Golgi, is processed, packaged into vesicles, and secreted. Proteins targeted after synthesis (posttranslational) include nuclear proteins that contain an internal, short, basic nuclear localization signal; mitochondrial matrix proteins that contain an N-terminal, amphipathic, α-helical mitochondrial entry sequence; and peroxisomal proteins that contain a C-terminal tripeptide signal.

1	VI. CO-AND POSTTRANSLATIONAL MODIFICATIONS Many polypeptides are covalently modified, either while they are still attached to the ribosome (cotranslational) or after their synthesis has been completed (posttranslational). These modifications may include removal of part of the translated sequence or the covalent addition of one or more chemical groups required for protein activity. A. Trimming Many proteins destined for secretion are initially made as large, precursor molecules that are not functionally active. Portions of the protein must be removed by specialized endoproteases, resulting in the release of an active molecule. The cellular site of the cleavage reaction depends on the protein to be modified. Some precursor proteins are cleaved in the RER or the Golgi; others are cleaved in developing secretory vesicles (for example, insulin; see Fig. 23.4, p. 309); and still others, such as collagen (see p. 47), are cleaved after secretion. B. Covalent attachments

1	B. Covalent attachments Protein function can be affected by the covalent attachment of a variety of chemical groups (Fig. 32.16). Examples include the following. 1. Phosphorylation: Phosphorylation occurs on the hydroxyl groups of serine, threonine, or, less frequently, tyrosine residues in a protein. It is catalyzed by one of a family of protein kinases and may be reversed by the action of protein phosphatases. The phosphorylation may increase or decrease the functional activity of the protein. Several examples of phosphorylation reactions have been previously discussed (for example, see Chapter 11, p. 132, for the regulation of glycogen synthesis and degradation). 2.

1	2. Glycosylation: Many of the proteins that are destined to become part of a membrane or to be secreted from a cell have carbohydrate chains added en bloc to the amide nitrogen of an asparagine (N-linked) or built sequentially on the hydroxyl groups of a serine, threonine, or hydroxylysine (O-linked). N-glycosylation occurs in the RER and Oglycosylation in the Golgi. (The process of producing such glycoproteins was discussed on p. 165.) N-glycosylated acid hydrolases are targeted to the matrix of lysosomes by the phosphorylation of mannose residues at carbon 6 (see p. 169). 3. Hydroxylation: Proline and lysine residues of the α chains of collagen are extensively hydroxylated by vitamin C–dependent hydroxylases in the RER (see p. 47). 4.

1	3. Hydroxylation: Proline and lysine residues of the α chains of collagen are extensively hydroxylated by vitamin C–dependent hydroxylases in the RER (see p. 47). 4. Other covalent modifications: These may be required for the functional activity of a protein. For example, additional carboxyl groups can be added to glutamate residues by vitamin K–dependent carboxylation (see p. 393). The resulting γ-carboxyglutamate (Gla) residues are essential for the activity of several of the blood-clotting proteins. (See online Chapter 35.) Biotin is covalently bound to the ε-amino groups of lysine residues of biotin-dependent enzymes that catalyze carboxylation reactions such as pyruvate carboxylase (see Fig. 10.3 on p. 119). Attachment of lipids, such as farnesyl groups, can help anchor proteins to membranes (see p. 221). Many eukaryotic proteins are cotranslationally acetylated at the N-end. [Note: Reversible acetylation of histone proteins influences gene expression (see p. 476).]

1	C. Protein degradation Proteins that are defective (for example, misfolded) or destined for rapid turnover are often marked for destruction by ubiquitination, the covalent attachment of chains of a small, highly conserved protein called ubiquitin (see Fig. 19.3 on p. 247). Proteins marked in this way are rapidly degraded by the proteasome, which is a macromolecular, ATP-dependent, proteolytic system located in the cytosol. For example, misfolding of the CFTR protein (see p. 450) results in its proteasomal degradation. [Note: If folding is impeded, unfolded proteins accumulate in the RER causing stress that triggers the unfolded protein response, in which the expression of chaperones is increased; global translation is decreased by eIF-2 phosphorylation; and the unfolded proteins are sent to the cytosol, ubiquitinated, and degraded in the proteasome by a process called ER-associated degradation.] VII. CHAPTER SUMMARY

1	Codons are composed of three nucleotide bases presented in the messenger RNA (mRNA) language of adenine (A), guanine (G), cytosine (C), and uracil (U). They are always written 5′→3′. Of the 64 possible three-base combinations, 61 code for the 20 standard amino acids and 3 signal termination of protein synthesis (translation). Altering the nucleotide sequence in a codon can cause silent mutations (the altered codon codes for the original amino acid), missense mutations (the altered codon codes for a different amino acid), or nonsense mutations (the altered codon is a termination codon). Characteristics of the genetic code include specificity, universality, and degeneracy, and it is nonoverlapping and commaless (Fig. 32.17). Requirements for protein synthesis include all the amino acids that eventually appear in the finished protein; at least one specific type of transfer RNA (tRNA) for each amino acid; one aminoacyl-tRNA synthetase for each amino acid; the mRNA coding for the protein

1	eventually appear in the finished protein; at least one specific type of transfer RNA (tRNA) for each amino acid; one aminoacyl-tRNA synthetase for each amino acid; the mRNA coding for the protein to be synthesized; fully competent ribosomes (70S in prokaryotes, 80S in eukaryotes); protein factors needed for initiation, elongation, and termination of protein synthesis; and ATP and guanosine triphosphate (GTP) as energy sources. tRNA has an attachment site for a specific amino acid at its 3′-end and an anticodon region that can recognize the codon specifying the amino acid the tRNA is carrying. Ribosomes are large complexes of protein and ribosomal RNA (rRNA). They consist of two subunits, 30S and 50S in prokaryotes and 40S and 60S in eukaryotes. Each ribosome has three binding sites for tRNA molecules: the A, P, and E sites that cover three neighboring codons. The A site binds an incoming aminoacyl-tRNA, the P site is occupied by peptidyl-tRNA, and the E site is occupied by the empty

1	molecules: the A, P, and E sites that cover three neighboring codons. The A site binds an incoming aminoacyl-tRNA, the P site is occupied by peptidyl-tRNA, and the E site is occupied by the empty tRNA as it is about to exit the ribosome. Recognition of an mRNA codon is accomplished by the tRNA anticodon, which binds to the codon following the rules of complementarity and antiparallel binding. The wobble hypothesis states that the first (5′) base of the anticodon is not as spatially defined as the other two bases. Movement of that first base allows nontraditional base-pairing with the last (3′) base of the codon, thus allowing a single tRNA to recognize more than one codon for a specific amino acid. For initiation of protein synthesis, the components of the translation system are assembled, and mRNA associates with the small ribosomal subunit. The process requires initiation factors (IF). In prokaryotes, a purine-rich region of the mRNA (the Shine-Dalgarno sequence) base-pairs with a

1	and mRNA associates with the small ribosomal subunit. The process requires initiation factors (IF). In prokaryotes, a purine-rich region of the mRNA (the Shine-Dalgarno sequence) base-pairs with a complementary sequence on 16S rRNA, resulting in the positioning of the small subunit on the mRNA so that translation can begin. The 5′-cap (bound by proteins of the eIF-4 family) on eukaryotic mRNA is used to position the small subunit on the mRNA. The initiation codon is AUG, and N-formylmethionine is the initiating amino acid in prokaryotes, whereas methionine is used in eukaryotes. The charged initiating tRNA (tRNAi) is brought to the P site by (e)IF-2. In elongation, the polypeptide chain is lengthened by the addition of amino acids to the carboxyl end of its growing chain. The process requires elongation factors that facilitate the binding of the aminoacyl-tRNA to the A site as well as the movement of the ribosome along the mRNA. The formation of the peptide bond is catalyzed by

1	requires elongation factors that facilitate the binding of the aminoacyl-tRNA to the A site as well as the movement of the ribosome along the mRNA. The formation of the peptide bond is catalyzed by peptidyltransferase, which is an activity intrinsic to the rRNA of the large subunit and, therefore, is a ribozyme. Following peptide-bond formation, the ribosome advances along the mRNA in the 5′→3′ direction to the next codon (translocation). Because of the length of most mRNA, more than one ribosome at a time can translate a message, forming a polysome. Termination begins when one of the three termination codons moves into the A site. These codons are recognized by release factors. The newly synthesized protein is released from the ribosomal complex, and the ribosome is dissociated from the mRNA. Initiation, elongation, and termination are driven by the hydrolysis of GTP. Initiation in eukaryotes also requires ATP for scanning. Numerous antibiotics interfere with the process of protein

1	mRNA. Initiation, elongation, and termination are driven by the hydrolysis of GTP. Initiation in eukaryotes also requires ATP for scanning. Numerous antibiotics interfere with the process of protein synthesis. Many polypeptide chains are covalently modified during or after translation. Such modifications include amino acid removal; phosphorylation, which may activate or inactivate the protein; glycosylation, which plays a role in protein targeting; and hydroxylation such as that seen in collagen. Protein targeting can be either cotranslational (as with secreted proteins) or posttranslational (as with mitochondrial matrix proteins). Proteins must fold to achieve their functional form. Folding can be spontaneous or facilitated by chaperones. Proteins that are defective (for example, misfolded) or destined for rapid turnover are marked for destruction by the attachment of chains of a small, highly conserved protein called ubiquitin. Ubiquitinated proteins are rapidly degraded by a

1	or destined for rapid turnover are marked for destruction by the attachment of chains of a small, highly conserved protein called ubiquitin. Ubiquitinated proteins are rapidly degraded by a cytosolic complex known as the proteasome.

1	Choose the ONE best answer. 2.1. A 20-year-old man with a microcytic anemia is found to have an abnormal form of β-globin (Hemoglobin Constant Spring) that is 172 amino acids long, rather than the 141 found in the normal protein. Which of the following point mutations is consistent with this abnormality? Use Figure 32.2 to answer the question. A. CGA→UGA B. GAU→GAC C. GCA→GAA D. UAA→CAA D. UAA→UAG

1	A. CGA→UGA B. GAU→GAC C. GCA→GAA D. UAA→CAA D. UAA→UAG Correct answer = D. Mutating the normal termination (stop) codon from UAA to CAA in β-globin messenger RNA causes the ribosome to insert a glutamine at that point. It will continue extending the protein chain until it comes upon the next stop codon farther down the message, resulting in an abnormally long protein. The replacement of CGA (arginine) with UGA (stop) would cause the protein to be too short. GAU and GAC both code for aspartate and would cause no change in the protein. Changing GCA (alanine) to GAA (glutamate) would not change the size of the protein product. A change from UAA to UAG would simply change one termination codon for another and would have no effect on the protein.

1	2.2. A pharmaceutical company is studying a new antibiotic that inhibits bacterial protein synthesis. When this antibiotic is added to an in vitro protein synthesis system that is translating the messenger RNA sequence AUGUUUUUUUAG, the only product formed is the dipeptide fMet-Phe. What step in protein synthesis is most likely inhibited by the antibiotic? A. Initiation B. Binding of a charged transfer RNA to the ribosomal A site C. Peptidyltransferase activity D. Ribosomal translocation E. Termination Correct answer = D. Because fMet-Phe (formylated methionyl-phenylalanine) is made, the ribosomes must be able to complete initiation, bind Phe-tRNA to the A site, and use peptidyltransferase activity to form the first peptide bond. Because the ribosome is not able to proceed any further, ribosomal movement (translocation) is most likely the inhibited step. Therefore, the ribosome is stopped before it reaches the termination codon of this message.

1	2.3. A transfer RNA (tRNA) molecule that is supposed to carry cysteine (tRNAcys) is mischarged, so that it actually carries alanine (ala-tRNAcys). Assuming no correction occurs, what will be the fate of this alanine residue during protein synthesis? It will: A. be incorporated into a protein in response to a codon for alanine. B. be incorporated into a protein in response to a codon for cysteine. C. be incorporated randomly at any codon. D. remain attached to the tRNA because it cannot be used for protein synthesis. E. be chemically converted to cysteine by cellular enzymes. Correct answer = B. Once an amino acid is attached to a tRNA molecule, only the anticodon of that tRNA determines the specificity of incorporation. Therefore, the incorrectly activated alanine will be incorporated into the protein at a position determined by a cysteine codon.

1	2.4. In a patient with cystic fibrosis (CF) caused by the ∆F508 mutation, the mutant CF transmembrane conductance regulator (CFTR) protein folds incorrectly. The patient’s cells modify this abnormal protein by attaching ubiquitin molecules to it. What is the fate of this modified CFTR protein? A. It performs its normal function because the ubiquitin largely corrects for the effect of the mutation. B. It is degraded by the proteasome. C. It is placed into storage vesicles. D. It is repaired by cellular enzymes. E. It is secreted from the cell. Correct answer = B. Ubiquitination usually marks old, damaged, or misfolded proteins for destruction by the cytosolic proteasome. There is no known cellular mechanism for repair of damaged proteins. 2.5. Many antimicrobials inhibit translation. Which of the following antimicrobials is correctly paired with its mechanism of action? A. Erythromycin binds to the 60S ribosomal subunit. B. Puromycin inactivates elongation factor-2.

1	A. Erythromycin binds to the 60S ribosomal subunit. B. Puromycin inactivates elongation factor-2. C. Streptomycin binds to the 30S ribosomal subunit. D. Tetracyclines inhibit peptidyltransferase. Correct answer = C. Streptomycin binds the 30S subunit and inhibits translation initiation. Erythromycin binds the 50S ribosomal subunit (60S denotes a eukaryote) and blocks the tunnel through which the peptide leaves the ribosome. Puromycin has structural similarity to aminoacyl-transfer RNA. It is incorporated into the growing chain, inhibits elongation, and results in premature termination in both prokaryotes and eukaryotes. Tetracyclines bind the 30S ribosomal subunit and block access to the A site, inhibiting elongation.

1	2.6. Translation of a synthetic polyribonucleotide containing the repeating sequence CAA in a cell-free protein-synthesizing system produces three homopolypeptides: polyglutamine, polyasparagine, and polythreonine. If the codons for glutamine and asparagine are CAA and AAC, respectively, which of the following triplets is the codon for threonine? A. AAC B. ACA C. CAA D. CAC E. CCA Correct answer = B. The synthetic polynucleotide sequence of CAACAACAACAA … could be read by the in vitro protein-synthesizing system starting at the first C, the first A, or the second A (that is, in any one of three reading frames). In the first case, the first triplet codon would be CAA, which codes glutamine; in the second case, the first triplet codon would be AAC, which codes for asparagine; in the last case, the first triplet codon would be ACA, which codes for threonine. 2.7. Which of the following is required for both prokaryotic and eukaryotic protein synthesis?

1	2.7. Which of the following is required for both prokaryotic and eukaryotic protein synthesis? A. Binding of the small ribosomal subunit to the Shine-Dalgarno sequence B. Formylated methionyl-transfer (t)RNA C. Movement of the messenger RNA out of the nucleus and into the cytoplasm D. Recognition of the 5′-cap by initiation factors E. Translocation of the peptidyl-tRNA from the A site to the P site Correct answer = E. In both prokaryotes and eukaryotes, continued translation (elongation) requires movement of the peptidyl-tRNA from the A to the P site to allow the next aminoacyl-tRNA to enter the A site. Only prokaryotes have a Shine-Dalgarno sequence and use formylated methionine and only eukaryotes have a nucleus and co-and posttranscriptionally process their mRNA.

1	2.8. α1-Antitrypsin (AAT) deficiency can result in emphysema, a lung pathology, because the action of elastase, a serine protease, is unopposed. Deficiency of AAT in the lungs is the consequence of impaired secretion from the liver, the site of its synthesis. Proteins such as AAT that are destined to be secreted are best characterized by which of the following statements? A. Their synthesis is initiated on the smooth endoplasmic reticulum. B. They contain a mannose 6-phosphate targeting signal. C. They always contain methionine as the N-terminal amino acid. D. They are produced from translation products that have an N-terminal hydrophobic signal sequence. E. They contain no sugars with O-glycosidic linkages because their synthesis does not involve the Golgi.

1	E. They contain no sugars with O-glycosidic linkages because their synthesis does not involve the Golgi. Correct answer = D. Synthesis of secreted proteins is begun on free (cytosolic) ribosomes. As the N-terminal signal sequence of the peptide emerges from the ribosome, it is bound by the signal recognition particle, taken to the rough endoplasmic reticulum (RER), threaded into the lumen, and cleaved as translation continues. The proteins move through the RER and the Golgi and undergo processing such as N-glycosylation (RER) and O-glycosylation (Golgi). In the Golgi, they are packaged in secretory vesicles and released from the cell. The smooth endoplasmic reticulum is associated with synthesis of lipids, not proteins, and has no ribosomes attached. Phosphorylation at carbon 6 of terminal mannose residues in glycoproteins targets these proteins (acid hydrolases) to lysosomes. The N-terminal methionine is removed from most proteins during processing.

1	2.9. Why is the genetic code described as both degenerate and unambiguous? A given amino acid can be coded for by more than one codon (degenerate code), but a given codon codes for just one particular amino acid (unambiguous code). Regulation of Gene Expression 33 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Regulation of Gene Expression 33 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW Gene expression refers to the multistep process that ultimately results in the production of a functional gene product, either ribonucleic acid (RNA) or protein. The first step in gene expression, the use of deoxyribonucleic acid (DNA) for the synthesis of RNA (transcription), is the primary site of regulation in both prokaryotes and eukaryotes. In eukaryotes, however, gene expression also involves extensive posttranscriptional and posttranslational processes as well as actions that influence access to particular regions of the DNA. Each of these steps can be regulated to provide additional control over the kinds and amounts of functional products that are produced.

1	Not all genes are tightly regulated. For example, genes described as constitutive encode products required for basic cellular functions and so are expressed at essentially a constant level. They are also known as “housekeeping” genes. Regulated genes, however, are expressed only under certain conditions. They may be expressed in all cells or in only a subset of cells, for example, hepatocytes. The ability to regulate gene expression (that is, to determine if, how much, and when particular gene products will be made) gives the cell control over structure and function. It is the basis for cellular differentiation, morphogenesis, and adaptability of any organism. Control of gene expression is best understood in prokaryotes, but many themes are repeated in eukaryotes. Figure 33.1 shows some of the sites where gene expression can be controlled. II. REGULATORY SEQUENCES AND MOLECULES

1	Regulation of transcription, the initial step in all gene expression, is controlled by regulatory sequences of DNA that are usually embedded in the noncoding regions of the genome. The interaction between these DNA sequences and regulatory molecules, such as transcription factors, can induce or repress the transcriptional machinery, influencing the kinds and amounts of products that are produced. The regulatory DNA sequences are called cis-acting because they influence expression of genes on the same chromosome as the regulatory sequence (see p. 439). The regulatory molecules are called trans-acting because they can diffuse (transit) through the cell from their site of synthesis to their DNA-binding sites (Fig. 33.2). For example, a protein transcription factor (a trans-acting molecule) that regulates a gene on chromosome 6 might itself have been produced from a gene on chromosome 11. The binding of proteins to DNA is through structural motifs such as the zinc finger (Fig. 33.3),

1	that regulates a gene on chromosome 6 might itself have been produced from a gene on chromosome 11. The binding of proteins to DNA is through structural motifs such as the zinc finger (Fig. 33.3), leucine zipper, or helix-turn-helix in the protein.

1	III. REGULATION OF PROKARYOTIC GENE EXPRESSION In prokaryotes such as the bacterium Escherichia coli (E. coli), regulation of gene expression occurs primarily at the level of transcription and, in general, is mediated by the binding of trans-acting proteins to cis-acting regulatory elements on their single DNA molecule (chromosome). [Note: Regulating the first step in the expression of a gene is an efficient approach, insofar as energy is not wasted making unneeded gene products.] Transcriptional control in prokaryotes can involve the initiation or premature termination of transcription. A. Messenger RNA transcription from bacterial operons

1	A. Messenger RNA transcription from bacterial operons In bacteria, the structural genes that encode proteins involved in a particular metabolic pathway are often found sequentially grouped on the chromosome along with the cis-acting elements that regulate the transcription of these genes. The transcription product is a single polycistronic messenger RNA ([mRNA] see p. 434). The genes are, thus, coordinately regulated (that is, turned on or off as a unit). This entire package is referred to as an operon. B. Operators in bacterial operons

1	B. Operators in bacterial operons Bacterial operons contain an operator, a segment of DNA that regulates the activity of the structural genes of the operon by reversibly binding a protein known as the repressor. If the operator is not bound by the repressor, RNA polymerase (RNA pol) binds the promoter, passes over the operator, and reaches the protein-coding genes that it transcribes to mRNA. If the repressor is bound to the operator, the polymerase is blocked and does not produce mRNA. As long as the repressor is bound to the operator, no mRNA (and, therefore, no proteins) are made. However, when an inducer molecule is present, it binds to the repressor, causing the repressor to change shape so that it no longer binds the operator. When this happens, RNA pol can initiate transcription. One of the best-understood examples is the inducible lactose (lac) operon of E. coli that illustrates both positive and negative regulation (Fig. 33.4). C. Lactose operon

1	The lac operon contains the genes that code for three proteins involved in the catabolism of the disaccharide lactose: the lacZ gene codes for βgalactosidase, which hydrolyzes lactose to galactose and glucose; the lacY gene codes for a permease, which facilitates the movement of lactose into the cell; and the lacA gene codes for thiogalactoside transacetylase, which acetylates lactose. [Note: The physiologic function of this acetylation is unknown.] All of these proteins are maximally produced only when lactose is available to the cell and glucose is not. [Note: Bacteria use glucose, if available, as a fuel in preference to any other sugar.] The regulatory portion of the operon is upstream of the three structural genes and consists of the promoter region where RNA pol binds and two additional sites, the operator (O) and the catabolite activator protein (CAP) sites, where regulatory proteins bind. The lacZ, lacY, and lacA genes are maximally expressed only when the O site is empty and

1	sites, the operator (O) and the catabolite activator protein (CAP) sites, where regulatory proteins bind. The lacZ, lacY, and lacA genes are maximally expressed only when the O site is empty and the CAP site is bound by a complex of cyclic adenosine monophosphate ([cAMP] see p. 94) and the CAP, sometimes called the cAMP regulatory protein (CRP). A regulatory gene, the lacI gene, codes for the repressor protein (a trans-acting factor) that binds to the O site with high affinity. [Note: The lacI gene has its own promoter and is not part of the lac operon.] 1.

1	When only glucose is available: In this case, the lac operon is repressed (turned off). Repression is mediated by the repressor protein binding via a helix-turn-helix motif (Fig. 33.5) to the O site, which is downstream of the promoter (see Fig. 33.4A). Binding of the repressor interferes with the binding of RNA pol to the promoter, thereby inhibiting transcription of the structural genes. This is an example of negative regulation. 2.

1	When only lactose is available: In this case, the lac operon is induced (maximally expressed, or turned on). A small amount of lactose is converted to an isomer, allolactose. This compound is an inducer that binds to the repressor protein, changing its conformation so that it can no longer bind to the O site. In the absence of glucose, adenylyl cyclase is active, and cAMP is made and binds to the CAP. The cAMP–CAP transacting complex binds to the CAP site, causing RNA pol to initiate transcription with high efficiency at the promoter site (see Fig. 33.4B). This is an example of positive regulation. The transcript is a single polycistronic mRNA molecule that contains three sets of start and stop codons. Translation of the mRNA produces the three proteins that allow lactose to be used for energy production by the cell. [Note: In contrast to the inducible lacZ, lacY, and lacA genes, whose expression is regulated, the lacI gene is constitutive. Its gene product, the repressor protein, is

1	production by the cell. [Note: In contrast to the inducible lacZ, lacY, and lacA genes, whose expression is regulated, the lacI gene is constitutive. Its gene product, the repressor protein, is always made and is active unless the inducer is present.] 3.

1	When both glucose and lactose are available: In this case, the lac operon is uninduced, and transcription is negligible, even if lactose is present at a high concentration. Adenylyl cyclase is inhibited in the presence of glucose (a process known as catabolite repression) so no cAMP–CAP complex forms, and the CAP site remains empty. Therefore, the RNA pol is unable to effectively initiate transcription, even though the repressor is not bound to the O site. Consequently, the three structural genes of the operon are expressed only at a very low (basal) level (see Fig. 33.4C). [Note: Induction causes a 50-fold enhancement over basal expression.] D. Tryptophan operon

1	The tryptophan (trp) operon contains five structural genes that code for enzymes required for the synthesis of the amino acid tryptophan. As with the lac operon, the trp operon is subject to negative control. However, for the repressible trp operon, negative control includes Trp itself binding to a repressor protein and facilitating the binding of the repressor to the operator: Trp is a corepressor. Because repression by Trp is not always complete, the trp operon, unlike the lac operon, is also regulated by a process known as attenuation. With attenuation, transcription is initiated but is terminated well before completion (Fig. 33.6). If Trp is plentiful, transcription initiation that escaped repression by Trp is attenuated (stopped) by the formation of an attenuator, a hairpin (stem-loop) structure in the mRNA similar to that seen in rho-independent termination (see p. 437). [Note: Because transcription and translation are temporally linked in prokaryotes (see p. 454), attenuation

1	structure in the mRNA similar to that seen in rho-independent termination (see p. 437). [Note: Because transcription and translation are temporally linked in prokaryotes (see p. 454), attenuation also results in the formation of a truncated, nonfunctional peptide product that is rapidly degraded.] If Trp becomes scarce, the operon is expressed. The 5′-end of the mRNA contains two adjacent codons for Trp. The lack of Trp causes ribosomes to stall at these codons, covering regions of the mRNA required for formation of the attenuation hairpin. This prevents attenuation and allows transcription to continue.

1	Transcriptional attenuation can occur in prokaryotes because translation of an mRNA begins before its synthesis is complete. This does not occur in eukaryotes because the presence of a membrane-bound nucleus spatially and temporally separates transcription and translation. E. Coordination of transcription and translation Although transcriptional regulation of mRNA production is primary in bacteria, regulation of ribosomal RNA (rRNA) and protein synthesis plays important roles in adaptation to environmental stress. 1.

1	1. Stringent response: E. coli has seven operons that synthesize the rRNA needed for ribosome assembly, and each is regulated in response to changes in environmental conditions. Regulation in response to amino acid starvation is known as the stringent response. The binding of an uncharged transfer RNA (tRNA) to the A site of a ribosome (see p. 452) triggers a series of events that leads to the production of the alarmone guanosine tetraphosphate (ppGpp). The synthesis of this unusual derivative of guanosine diphosphate (GDP) is catalyzed by stringent factor (RelA), an enzyme physically associated with ribosomes. Elevated levels of ppGpp result in inhibition of rRNA synthesis (Fig. 33.7). [Note: In addition to rRNA synthesis, tRNA synthesis and some mRNA synthesis (for example, for ribosomal proteins) are also inhibited. However, synthesis of mRNA for enzymes required for amino acid biosynthesis is not inhibited. ppGpp binds RNA pol and alters promoter 2.

1	Regulatory ribosomal proteins: Operons for ribosomal proteins (rproteins) can be inhibited by an excess of their own protein products. For each operon, one specific r-protein functions in the repression of selection through use of different sigma factors for the polymerase (see p. 435).] translation of the polycistronic mRNA from that operon (Fig. 33.8). The r-protein does so by binding to the Shine-Dalgarno (SD) sequence located on the mRNA just upstream of the first initiating AUG codon (see p. 448) and acting as a physical impediment to the binding of the small ribosomal subunit to the SD sequence. Thus, one r-protein inhibits synthesis of all the r-proteins of the operon. This same r-protein also binds to rRNA and with a higher affinity than for mRNA. If the concentration of rRNA falls, the r-protein then is available to bind its own mRNA and inhibit its translation. This coordinated regulation keeps the synthesis of r-proteins in balance with the transcription of rRNA, so that

1	the r-protein then is available to bind its own mRNA and inhibit its translation. This coordinated regulation keeps the synthesis of r-proteins in balance with the transcription of rRNA, so that each is present in appropriate amounts for the formation of ribosomes.

1	IV. REGULATION OF EUKARYOTIC GENE EXPRESSION The higher degree of complexity of eukaryotic genomes, as well as the presence of a nuclear membrane, necessitates a wider range of regulatory processes. As with the prokaryotes, transcription is the primary site of regulation. Again, the theme of trans-acting factors binding to cis-acting elements is seen. Operons, however, are not found in eukaryotes, which must use alternate strategies to solve the problem of how to coordinately regulate all the genes required for a specific response. In eukaryotes, gene expression is also regulated at multiple levels other than transcription. For example, the major modes of posttranscriptional regulation at the mRNA level are alternative mRNA splicing and polyadenylation, control of mRNA stability, and control of translational efficiency. Additional regulation at the protein level occurs by mechanisms that modulate stability, processing, or targeting of the protein. A. Coordinate regulation

1	The need to coordinately regulate a group of genes to cause a particular response is of key importance in organisms with more than one chromosome. An underlying theme occurs repeatedly: A trans-acting protein functions as a specific transcription factor (STF) that binds to a cisacting regulatory consensus sequence (see p. 415) on each of the genes in the group even if they are on different chromosomes. [Note: The STF has a DNA-binding domain (DBD) and a transcription activation domain (TAD). The TAD recruits coactivators, such as histone acetyltransferases (see p. 438), and the general transcription factors (see p. 439) that, along with RNA pol, are required for formation of the transcription initiation complex at the promoter. Although the TAD recruits a variety of proteins, the specific effect of any one of them is dependent upon the protein composition of the complex. This is known as combinatorial control.] Examples of coordinate regulation in eukaryotes include the galactose

1	effect of any one of them is dependent upon the protein composition of the complex. This is known as combinatorial control.] Examples of coordinate regulation in eukaryotes include the galactose circuit and the hormone response system.

1	1. Galactose circuit: This regulatory scheme allows for the use of galactose when glucose is not available. In yeast, a unicellular organism, the genes required to metabolize galactose are on different chromosomes. Coordinated expression is mediated by the protein Gal4 (Gal = galactose), a STF that binds to a short regulatory DNA sequence upstream of each of the genes. The sequence is called the upstream activating sequence Gal (UASGal). Binding of Gal4 to UASGal through zinc fingers in its DBD occurs in both the absence and presence of galactose. When the sugar is absent, the regulatory protein Gal80 binds Gal4 at its TAD, thereby inhibiting gene transcription (Fig. 33.9A). When present, galactose activates the Gal3 protein. Gal3 binds Gal80, thereby allowing Gal4 to activate transcription (Fig. 33.9B). [Note: Glucose prevents the use of galactose by inhibting expression of Gal4 protein.]

1	B. presence of galactose. [Note: Target genes, whether on the same or a different chromosome, each have an upstream activating sequence galactose (UASGal).] TAD = transcription activation domain; DBD = DNA-binding domain; mRNA = messenger RNA. 2. Hormone response system: Hormone response elements (HRE) are DNA sequences that bind trans-acting proteins and regulate gene expression in response to hormonal signals in multicellular organisms. Hormones bind to either intracellular (nuclear) receptors (for example, steroid hormones; see p. 240) or cell-surface receptors (for example, the peptide hormone glucagon; see p. 314). a.

1	Intracellular receptors: Members of the nuclear receptor superfamily, which includes the steroid hormone (glucocorticoids, mineralocorticoids, androgens, and estrogens), vitamin D, retinoic acid, and thyroid hormone receptors, function as STF. In addition to domains for DNA-binding and transcriptional activation, these receptors also contain a ligand-binding domain. For example, the steroid hormone cortisol (a glucocorticoid) binds intracellular receptors at the ligand-binding domain (Fig. 33.10). Binding causes a conformational change in the receptor that activates it. The receptor– hormone complex enters the nucleus, dimerizes, and binds via a zinc finger motif to DNA at a regulatory element, the glucocorticoid response element (GRE) that is an example of a HRE. Binding allows recruitment of coactivators to the TAD and results in expression of cortisol-responsive genes, each of which is under the control of its own GRE. Binding of the receptor–hormone complex to the GRE allows

1	of coactivators to the TAD and results in expression of cortisol-responsive genes, each of which is under the control of its own GRE. Binding of the receptor–hormone complex to the GRE allows coordinate expression of a group of target genes, even though these genes are on different chromosomes. The GRE can be located upstream or downstream of the genes it regulates and at great distances from them. The GRE, then, can function as a true enhancer (see p. 440). [Note: If associated with repressors, hormone–receptor complexes inhibit transcription.] b.

1	Cell-surface receptors: These receptors include those for insulin, epinephrine, and glucagon. Glucagon, for example, is a peptide hormone that binds its G protein–coupled plasma membrane receptor on glucagon-responsive cells. This extracellular signal is then transduced to intracellular cAMP, a second messenger (Fig. 33.11; also see Fig. 8.7 on p. 95), which can affect protein expression (and activity) through protein kinase A–mediated phosphorylation. In response to a rise in cAMP, a trans-acting factor (cAMP response element–binding [CREB] protein) is phosphorylated and activated. Active CREB protein binds via a leucine zipper motif to a cis-acting regulatory element, the cAMP response element (CRE), resulting in transcription of target genes with CRE in their promoters. [Note: The genes for phosphoenolpyruvate carboxykinase and glucose 6phosphatase, key enzymes of gluconeogenesis (see p. 122), are examples of genes upregulated by the cAMP/CRE/CREB system.]

1	B. Messenger RNA processing and use Eukaryotic mRNA undergoes several processing events before it is exported from the nucleus to the cytoplasm for use in protein synthesis. Capping at the 5′-end (see p. 441), polyadenylation at the 3′-end (see p. 442), and splicing (see p. 442) are essential for the production of a functional eukaryotic messenger from most pre-mRNA. Variations in splicing and polyadenylation can affect gene expression. In addition, messenger stability also affects gene expression. 1. Alternative splicing: Tissue-specific protein isoforms can be made from the same pre-mRNA through alternative splicing, which can involve exon skipping (loss), intron retention, and use of alternative splice-donor or -acceptor sites (Fig. 33.12). For example, the pre-mRNA for tropomyosin (TM) undergoes tissue-specific alternative splicing to yield a number of TM isoforms (see p. 443). [Note: Over 90% of all human genes undergo alternative splicing.] 2.

1	Alternative polyadenylation: Some pre-mRNA transcripts have more than one site for cleavage and polyadenylation. Alternative polyadenylation (APA) generates mRNA with different 3′-ends, altering the untranslated region (UTR) or the coding (translated) sequence. [Note: APA is involved in the production of the membrane-bound and secreted forms of immunoglobulin M.] The use of alternative splicing and polyadenylation sites, as well as alternative transcription start sites explains, at least in part, how the ~20,000 to 25,000 genes in the human genome can give rise to well over 100,000 proteins. 3. Messenger RNA editing: Even after mRNA has been fully processed, it may undergo an additional posttranscriptional modification in which a base in the mRNA is altered. This is known as RNA editing. An important example in humans occurs with the transcript for apolipoprotein (apo) B, an essential component of chylomicrons (see p.

1	228) and very-low-density lipoproteins ([VLDL] see p. 230). Apo B mRNA is made in the liver and the small intestine. However, in the intestine only, the cytosine (C) base in the CAA codon for glutamine is enzymatically deaminated to uracil (U), changing the sense codon to the nonsense or stop codon UAA, as shown in Figure 33.13. This results in a shorter protein (apo B-48, representing 48% of the message) being made in the intestine (and incorporated into chylomicrons) than is made in the liver (apo B-100, full-length, incorporated into VLDL). U = uracil. 4. Messenger RNA stability: How long an mRNA remains in the cytosol before it is degraded influences how much protein product can be produced from it. Regulation of iron metabolism and the gene-silencing process of RNA interference (RNAi) illustrate the importance of mRNA stability in the regulation of gene expression.

1	a. Iron metabolism: Transferrin (Tf) is a plasma protein that transports iron. Tf binds to cell-surface receptors (transferrin receptors [TfR]) that get internalized and provide cells, such as erythroblasts, with iron. The mRNA for the TfR has several cis-acting iron-responsive elements (IRE) in its 3′-UTR. IRE have a short stem-loop structure that can be bound by trans-acting iron regulatory proteins (IRP), as shown in Figure 33.14. When the iron concentration in the cell is low, the IRP bind to the 3′-IRE and stabilize the mRNA for TfR, allowing TfR synthesis. When intracellular iron levels are high, the IRP dissociate. The lack of IRP bound to the mRNA hastens its destruction, resulting in decreased TfR synthesis. [Note: The mRNA for ferritin, an intracellular protein of iron storage, has a single IRE in its 5′-UTR. When iron levels in the cell are low, IRP bind the 5′-IRE and prevent the use of the mRNA, and less ferritin is made. When iron accumulates in the cell, the IRP

1	has a single IRE in its 5′-UTR. When iron levels in the cell are low, IRP bind the 5′-IRE and prevent the use of the mRNA, and less ferritin is made. When iron accumulates in the cell, the IRP dissociate, allowing synthesis of ferritin molecules to store the excess iron. Aminolevulinic acid synthase 2, the regulated enzyme of heme synthesis (see p. 278) in erythroblasts, also contains a 5′-IRE.] (See Chapter 29 for a discussion of iron metabolism.) b. RNA interference: RNAi is a mechanism of gene silencing through decreased expression of mRNA, either by repression of translation or by increased degradation. It plays a key role in such fundamental processes as cell proliferation, differentiation, and apoptosis. RNAi is mediated by short (~22 nucleotides), noncoding RNA called microRNA (miRNA). The miRNA arise from far longer, genomically encoded nuclear transcripts, primary miRNA (pri-miRNA), that are partially processed in the nucleus to pre-miRNA by an endonuclease (Drosha) then

1	(miRNA). The miRNA arise from far longer, genomically encoded nuclear transcripts, primary miRNA (pri-miRNA), that are partially processed in the nucleus to pre-miRNA by an endonuclease (Drosha) then transported to the cytoplasm. There, an endonuclease (Dicer) completes the processing and generates short, double-stranded miRNA. A single strand (the guide or antisense strand) of the miRNA associates with a cytosolic protein complex known as the RNA-induced silencing complex (RISC). The guide strand hybridizes with a complementary sequence in the 3′-UTR of a full-length target mRNA, bringing RISC to the mRNA. This can result in repression of translation of the mRNA or its degradation by an endonuclease (Argonaute/Ago/Slicer) of the RISC. The extent of complementarity appears to be the determining factor (Fig. 33.15). RNAi can also be triggered by the introduction of exogenous double-stranded short interfering RNA (siRNA) into a cell, a process that has enormous therapeutic potential.

1	The first clinical trial of RNAi-based therapy involved the neovascular form of age-related macular degeneration (AMD), which is triggered by overproduction of vascular endothelial growth factor (VEGF), leading to the sprouting of excess blood vessels behind the retina. The vessels leak, clouding and often entirely destroying vision (therefore, neovascular AMD is also referred to as wet AMD). An siRNA was designed to target the mRNA of VEGF and promote its degradation. Although considerable effort and resources have been expended to develop RNAi-based therapeutics, especially for the treatment of cancer, no products have gone from trials to the market. The research applications of RNAi, however, have grown rapidly.

1	5. Messenger RNA translation: Regulation of gene expression can also occur at the level of mRNA translation. One mechanism by which translation is regulated is through phosphorylation of the eukaryotic translation initiation factor, eIF-2 (Fig. 33.16). Phosphorylation of eIF-2 inhibits its function and so inhibits translation at the initiation step (see p. 459). [Note: Phosphorylation of eIF-2 prevents its reactivation by inhibiting GDP-GTP exchange.] Phosphorylation is catalyzed by kinases that are activated in response to environmental conditions, such as amino acid starvation, heme deficiency in erythroblasts, the presence of double-stranded RNA (signaling viral infection), and the accumulation of misfolded proteins in the rough endoplasmic reticulum (see p. 460). phosphate; = phosphate. C. Regulation through variations in DNA

1	phosphate; = phosphate. C. Regulation through variations in DNA Gene expression in eukaryotes is also influenced by the accessibility of DNA to the transcriptional apparatus, the amount of DNA, and the arrangement of DNA. [Note: Localized transitions between the B and Z forms of DNA (see p. 414) can also affect gene expression.] 1.

1	Access to DNA: In eukaryotes, DNA is found complexed with histone and nonhistone proteins to form chromatin (see p. 425). Transcriptionally active, decondensed chromatin (euchromatin) differs from the more condensed, inactive form (heterochromatin) in a number of ways. Active chromatin contains histone proteins that have been covalently modified at their amino terminal ends by reversible methylation, acetylation, or phosphorylation (see p. 438 for a discussion of histone acetylation/deacetylation by histone acetyltransferase and histone deacetylase). Such modifications decrease the positive charge of these basic proteins, thereby decreasing the strength of their association with negatively charged DNA. This relaxes the nucleosome (see p. 425), allowing transcription factors access to specific regions on the DNA. Nucleosomes can also be repositioned, an ATP-requiring process that is part of chromatin remodeling. Another difference between transcriptionally active and inactive chromatin

1	regions on the DNA. Nucleosomes can also be repositioned, an ATP-requiring process that is part of chromatin remodeling. Another difference between transcriptionally active and inactive chromatin is the extent of methylation of cytosine bases in CG-rich regions (CpG islands) in the promoter region of many genes. Methylation is by methyltransferases that use S-adenosylmethionine as the methyl donor (Fig. 33.17). Transcriptionally active genes are less methylated (hypomethylated) than their inactive counterparts, suggesting that DNA hypermethylation silences gene expression. Modification of histones and methylation of DNA are epigenetic in that they are heritable changes in DNA that alter gene expression without altering the base sequence.

1	2. Amount of DNA: A change up or down in the number of copies of a gene can affect the amount of gene product produced. An increase in copy number (gene amplification) has contributed to increased genomic complexity and is still a normal developmental process in certain nonmammalian species. In mammals, however, gene amplification is seen with some diseases and in response to particular chemotherapeutic drugs such as methotrexate, an inhibitor of the enzyme dihydrofolate reductase (DHFR), required for the synthesis of thymidine triphosphate (TTP) in the pyrimidine biosynthetic pathway (see p. 303). TTP is essential for DNA synthesis. Gene amplification results in an increase in the number of DHFR genes and resistance to the drug, allowing TTP to be made. 3.

1	3. Arrangement of DNA: The process by which immunoglobulins (antibodies) are produced by B lymphocytes involves permanent rearrangements of the DNA in these cells. The immunoglobulins (for example, IgG) consist of two light and two heavy chains, with each chain containing regions of variable and constant amino acid sequence. The variable region is the result of somatic recombination of segments within both the light-and the heavy-chain genes. During B-lymphocyte development, single variable (V), diversity (D), and joining (J) gene segments are brought together through gene rearrangement to form a unique variable region (Fig. 33.18). This process allows the generation of 109−1011 different immunoglobulins from a single gene, providing the diversity needed for the recognition of an enormous number of antigens. [Note: Pathologic DNA rearrangement is seen with translocation, a process by which two different chromosomes exchange DNA segments.] 4.

1	Mobile DNA elements: Transposons (Tn) are mobile segments of DNA that move in an essentially random manner from one site to another on the same or a different chromosome. Movement is mediated by transposase, an enzyme encoded by the Tn itself. Movement can be direct, in which transposase cuts out and then inserts the Tn at a new site, or replicative, in which the Tn is copied and the copy inserted elsewhere while the original remains in place. In eukaryotes, including humans, replicative transposition frequently involves an RNA intermediate made by a reverse transcriptase (see p. 424), in which case the Tn is called a retrotransposon. Transposition has contributed to structural variation in the genome but also has the potential to alter gene expression and even to cause disease. Tn comprise ~50% of the human genome, with retrotransposons accounting for 90% of Tn. Although the vast majority of these retrotransposons have lost the ability to move, some are still active. Their

1	Tn comprise ~50% of the human genome, with retrotransposons accounting for 90% of Tn. Although the vast majority of these retrotransposons have lost the ability to move, some are still active. Their transposition is thought to be the basis for some rare cases of hemophilia A and Duchenne muscular dystrophy. [Note: The growing problem of antibiotic-resistant bacteria is a consequence, at least in part, of the exchange of plasmids among bacterial cells. If the plasmids contain Tn-carrying antibiotic resistance genes, the recipient bacteria gain resistance to one or more antimicrobial drugs.]

1	V. CHAPTER SUMMARY

1	Gene expression results in the production of a functional gene product (either RNA or protein) through the processes of transcription and translation (Fig. 33.19). Genes can be either constitutive (always expressed, housekeeping genes) or regulated (expressed only under certain conditions in all cells or in a subset of cells). The ability to appropriately induce (positively regulate) or repress (negatively regulate) genes is essential in all organisms. Regulation of gene expression occurs primarily at transcription in both prokaryotes and eukaryotes and is mediated through trans-acting proteins binding to cis-acting regulatory DNA elements. In eukaryotes, regulation also occurs through DNA modifications and through posttranscriptional and posttranslational processing. In prokaryotes, such as Escherichia coli, the coordinate regulation of genes whose protein products are required for a particular process is achieved through operons (groups of genes sequentially arranged on the

1	such as Escherichia coli, the coordinate regulation of genes whose protein products are required for a particular process is achieved through operons (groups of genes sequentially arranged on the chromosome along with the regulatory elements that determine their transcription). The lac operon contains the Z, Y, and A structural genes, the protein products of which are needed for the catabolism of lactose. It is subject to negative and positive regulation. When glucose is available, the operon is repressed by the binding of the repressor protein (the product of the lacI gene) to the operator, thus preventing transcription. When only lactose is present, the operon is induced by an isomer of lactose (allolactose) that binds the repressor protein, preventing it from binding to the operator. In addition, cyclic adenosine monophosphate (cAMP) binds the catabolite activator protein (CAP), and the complex binds the DNA at the CAP site. This increases promoter efficiency and results in the

1	In addition, cyclic adenosine monophosphate (cAMP) binds the catabolite activator protein (CAP), and the complex binds the DNA at the CAP site. This increases promoter efficiency and results in the expression of the structural genes through the production of a polycistronic messenger RNA (mRNA). When both glucose and lactose are present, glucose prevents formation of cAMP, and transcription of these genes is negligible. The trp operon contains genes needed for the synthesis of tryptophan (Trp), and, like the lac operon, it is regulated by negative control. Unlike the lac operon, it is also regulated by attenuation, in which mRNA synthesis that escaped repression by Trp is terminated before completion. Transcription of ribosomal RNA and transfer RNA is selectively inhibited in prokaryotes by the stringent response to amino acid starvation. Translation is also a site of prokaryotic gene regulation: Excess ribosomal proteins bind the Shine-Dalgarno sequence on their own polycistronic

1	by the stringent response to amino acid starvation. Translation is also a site of prokaryotic gene regulation: Excess ribosomal proteins bind the Shine-Dalgarno sequence on their own polycistronic mRNA, preventing ribosomes from binding. Gene regulation is more complex in eukaryotes. Operons are not present, but coordinate regulation of the transcription of genes located on different chromosomes can be achieved through the binding of trans-acting proteins to cis-acting elements as seen in the galactose circuit in unicellular yeast. In multicellular organisms, hormones can cause coordinated regulation, either through the binding of the hormone receptor–hormone complex to the DNA (as with steroid hormones) or through the binding of a protein that is activated in response to a second messenger (as with glucagon). In each case, binding to DNA is mediated through structural motifs such as the zinc finger. Co-and posttranscriptional regulation is also seen in eukaryotes and includes

1	messenger (as with glucagon). In each case, binding to DNA is mediated through structural motifs such as the zinc finger. Co-and posttranscriptional regulation is also seen in eukaryotes and includes alternative mRNA splicing and polyadenylation, mRNA editing, and variations in mRNA stability as seen with transferrin receptor synthesis and with RNA interference. Regulation at the translational level can be caused by the phosphorylation and inhibition of eukaryotic initiation factor 2. Gene expression in eukaryotes is also influenced by accessibility of DNA to the transcriptional apparatus (as seen with epigenetic changes to histone proteins), the amount of DNA, and the arrangement of the DNA.

1	reticulum. Choose the ONE best answer. 3.1. Which of the following mutations is most likely to result in reduced expression of the lac operon? A. cya− (no adenylyl cyclase made) B. i− (no repressor protein made) C. Oc (operator cannot bind repressor protein) D. One resulting in impaired glucose uptake Correct answer = A. In the absence of glucose, adenylyl cyclase makes cyclic adenosine monophosphate (cAMP), which forms a complex with the catabolite activator protein (CAP). The cAMP–CAP complex binds the CAP site on the DNA, causing RNA polymerase to bind more efficiently to the lac operon promoter, thereby increasing expression of the operon. With cya− mutations, adenylyl cyclase is not made, and so the operon is unable to be maximally expressed even when glucose is absent and lactose is present. The absence of a repressor protein or decreased ability of the repressor to bind the operator results in constitutive (essentially constant) expression of the lac operon.

1	3.2. Which of the following is best described as cis-acting? A. Cyclic adenosine monophosphate response element–binding protein B. Operator C. Repressor protein D. Thyroid hormone nuclear receptor Correct answer = B. The operator is part of the DNA itself, and so is cis-acting. The cyclic adenosine monophosphate response element–binding protein, repressor protein, and thyroid hormone nuclear receptor protein are molecules that diffuse (transit) to the DNA, bind, and affect the expression of that DNA and so are trans-acting. 3.3. Which of the following is the basis for the intestine-specific expression of apolipoprotein B-48? A. DNA rearrangement and loss B. DNA transposition C. RNA alternative splicing D. RNA editing E. RNA interference

1	A. DNA rearrangement and loss B. DNA transposition C. RNA alternative splicing D. RNA editing E. RNA interference Correct answer = D. The production of apolipoprotein (apo) B-48 in the intestine and apo B-100 in liver is the result of RNA editing in the intestine, where a sense codon is changed to a nonsense codon by posttranscriptional deamination of cytosine to uracil. DNA rearrangement and transposition, as well as RNA interference and alternative splicing, do alter gene expression but are not the basis of apo B-48 tissue-specific production. 3.4. Which of the following is most likely to be true in hemochromatosis, a disease of iron accumulation? A. The messenger RNA for the transferrin receptor is stabilized by the binding of iron regulatory proteins to its 3′-iron-responsive elements. B. The messenger RNA for the transferrin receptor is not bound by iron regulatory proteins and is degraded.

1	B. The messenger RNA for the transferrin receptor is not bound by iron regulatory proteins and is degraded. C. The messenger RNA for ferritin is not bound by iron regulatory proteins at its 5′-iron-responsive element and is translated. D. The messenger RNA for ferritin is bound by iron regulatory proteins and is not translated. E. Both B and C are correct. Correct answer = E. When iron levels in the body are high, as is seen with hemochromatosis, there is increased synthesis of the iron-storage molecule, ferritin, and decreased synthesis of the transferrin receptor (TfR) that mediates iron uptake by cells. These effects are the result of cis-acting iron-responsive elements not being bound by trans-acting iron regulatory proteins, resulting in degradation of the messenger RNA (mRNA) for TfR and increased translation of the mRNA for ferritin.

1	3.5. Patients with estrogen receptor–positive (hormone responsive) breast cancer may be treated with the drug tamoxifen, which binds the estrogen nuclear receptor without activating it. Which of the following is the most logical outcome of tamoxifen use? A. Increased acetylation of estrogen-responsive genes B. Increased growth of estrogen receptor–positive breast cancer cells C. Increased production of cyclic adenosine monophosphate D. Inhibition of the estrogen operon E. Inhibition of transcription of estrogen-responsive genes

1	C. Increased production of cyclic adenosine monophosphate D. Inhibition of the estrogen operon E. Inhibition of transcription of estrogen-responsive genes Correct answer = E. Tamoxifen competes with estrogen for binding to the estrogen nuclear receptor. Tamoxifen fails to activate the receptor, preventing its binding to DNA sequences that upregulate expression of estrogen-responsive genes. Tamoxifen, then, blocks the growth-promoting effects of these genes and results in growth inhibition of estrogen-dependent breast cancer cells. Acetylation increases transcription by relaxing the nucleosome. Cyclic adenosine monophosphate is a regulatory signal mediated by cell-surface rather than nuclear receptors. Mammalian cells do not have operons. 3.6. The ZYA region of the lac operon will be maximally expressed if: A. cyclic adenosine monophosphate levels are low. B. glucose and lactose are both available. C. the attenuation stem-loop is able to form. D. the CAP site is occupied.

1	A. cyclic adenosine monophosphate levels are low. B. glucose and lactose are both available. C. the attenuation stem-loop is able to form. D. the CAP site is occupied. Correct answer = D. It is only when glucose is gone, cyclic adenosine monophosphate (cAMP) levels are increased, the cAMP–catabolite activator protein (CAP) complex is bound to the CAP site, and lactose is available that the operon is maximally expressed (induced). If glucose is present, the operon is off as a result of catabolite repression. The lac operon is not regulated by attenuation, a mechanism for stopping transcription in some operons such as the trp operon. 3.7. X chromosome inactivation is a process by which one of two X chromosomes in mammalian females is condensed and inactivated to prevent overexpression of X-linked genes. What would most likely be true about the degree of DNA methylation and histone acetylation on the inactivated X chromosome?

1	Cytosines in CpG islands would be hypermethylated, and histone proteins would be deacetylated. Both conditions are associated with decreased gene expression, and both are important in maintaining X inactivation. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	In the past, efforts to understand genes and their expression have been confounded by the immense size and complexity of human deoxyribonucleic acid (DNA). The human genome contains ~3 billion (109) base pairs (bp) that encode 20,000–25,000 protein-coding genes located on 23 chromosomes in the haploid genome. It is now possible to determine the nucleotide sequence of long stretches of DNA, and the entire human genome has been sequenced. This effort (called the Human Genome Project and completed in 2003) was made possible by several tools that have already contributed to our understanding of many genetic diseases (Fig. 34.1). These include 1) the discovery of restriction endonucleases that permit the cleavage of huge DNA molecules into defined fragments, 2) the development of cloning techniques that provide a mechanism for amplification of specific nucleotide sequences, and 3) the ability to synthesize specific probes, which has allowed the identification and manipulation of nucleotide

1	that provide a mechanism for amplification of specific nucleotide sequences, and 3) the ability to synthesize specific probes, which has allowed the identification and manipulation of nucleotide sequences of interest. These and other experimental approaches have permitted the identification of both normal and mutant nucleotide sequences in DNA. This knowledge has led to the development of methods for the diagnosis of genetic diseases and some successes in the treatment of patients by gene therapy. [Note: The genomes of several viruses, prokaryotes, and nonhuman eukaryotes have also been sequenced.]

1	II. RESTRICTION ENDONUCLEASES One of the major obstacles to molecular analysis of genomic DNA is the immense size of the molecules involved. The discovery of a special group of bacterial enzymes, called restriction endonucleases (restriction enzymes), which cleave double-stranded DNA (dsDNA) into smaller, more manageable fragments, opened the way for DNA analysis. Because each enzyme cleaves dsDNA at a specific nucleotide sequence (restriction site), restriction enzymes are used experimentally to obtain precisely defined DNA segments called restriction fragments. A. Specificity

1	A. Specificity Restriction endonucleases recognize short stretches of dsDNA (4–8 bp) that contain specific nucleotide sequences. These sequences, which differ for each restriction enzyme, are palindromes, that is, they exhibit twofold rotational symmetry (Fig. 34.2). This means that, within a short region of the dsDNA, the nucleotide sequence on the two strands is identical if each is read in the 5′→3′ direction. Therefore, if you turn the page upside down (that is, rotate it 180° around its axis of symmetry) the sequence remains the same. In bacteria, restriction endonucleases limit (restrict) the expression of nonbacterial (foreign) DNA through cleavage. Bacterial DNA is protected from cleavage by methylation of adenine at the restriction site. B. Nomenclature

1	B. Nomenclature A restriction enzyme is named according to the organism from which it was isolated. The first letter of the name is from the genus of the bacterium. The next two letters are from the name of the species. An additional letter indicates the type or strain (as needed), and a number (Roman numeral) is appended to indicate the order in which the enzyme was discovered in that particular organism. For example, HaeIII is the third restriction endonuclease isolated from the bacterium Haemophilus aegyptius. C. Sticky and blunt ends

1	C. Sticky and blunt ends Restriction enzymes cleave dsDNA so as to produce a 3′-hydroxyl group on one end and a 5′-phosphate group on the other. Some restriction endonucleases, such as TaqI, form staggered cuts that produce sticky or cohesive ends (that is, the resulting DNA fragments have single-stranded regions that are complementary to each other), as shown in Figure 34.3. Other restriction endonucleases, such as HaeIII, produce fragments that have blunt ends that are entirely double stranded and, therefore, do not form hydrogen bonds with each other. Using the enzyme DNA ligase (see p. 418), sticky ends of a DNA fragment of interest can be covalently joined with other DNA fragments that have sticky ends produced by cleavage with the same restriction endonuclease (Fig. 34.4). [Note: A ligase encoded by bacteriophage T4 can covalently join blunt-ended fragments.] D. Restriction sites

1	D. Restriction sites A DNA sequence that is recognized and cut by a restriction enzyme is called a restriction site. Restriction endonucleases cleave dsDNA into fragments of different sizes depending upon the size of the sequence recognized. For example, an enzyme that recognizes a specific 4-bp sequence produces many cuts in the DNA molecule, one every 44 bp. In contrast, an enzyme requiring a unique sequence of 6 bp produces fewer cuts (one every 46 bp) and, therefore, longer pieces. Hundreds of these enzymes, each having different cleavage specificities (varying in both nucleotide sequences and length of recognition sites), are commercially available. III. DNA CLONING

1	Introduction of a foreign DNA molecule into a replicating cell permits the cloning or, amplification (that is, the production of many identical copies) of that DNA. [Note: Human DNA for cloning can be obtained from blood, saliva, and solid tissue.] In some cases, a single DNA fragment can be isolated and purified prior to cloning. More commonly, to clone a nucleotide sequence of interest, the total cellular DNA is first cleaved with a specific restriction enzyme, creating hundreds of thousands of fragments. Each of the resulting DNA fragments is joined to a DNA vector molecule (referred to as a cloning vector) to form a hybrid, or recombinant, DNA molecule. Each recombinant molecule carries its inserted DNA fragment into a single host cell (for example, a bacterium), where it is replicated. [Note: The process of introducing foreign DNA into a cell is called transformation for bacteria and yeast and transfection for higher eukaryotes.] As the host cell multiplies, it forms a clone in

1	[Note: The process of introducing foreign DNA into a cell is called transformation for bacteria and yeast and transfection for higher eukaryotes.] As the host cell multiplies, it forms a clone in which every bacterium contains copies of the same inserted DNA fragment, hence the name “cloning.” The cloned DNA can be released from its vector by cleavage (using the appropriate restriction endonuclease) and isolated. By this mechanism, many identical copies of the DNA of interest can be produced. [Note: An alternative to amplification by biologic cloning, the polymerase chain reaction (PCR), is described on p. 495.]

1	A. Vectors A vector is a molecule of DNA to which the fragment of DNA to be cloned is joined. Essential properties of a vector include the 1) capacity for autonomous replication within a host cell, 2) presence of at least one specific nucleotide sequence recognized by a restriction endonuclease, and 3) presence of at least one gene (such as an antibiotic resistance gene) that confers the ability to select for the vector. Commonly used vectors include plasmids and viruses. 1.

1	Prokaryotic plasmids: Prokaryotic organisms typically contain single, large, circular chromosomes. In addition, most species of bacteria also normally contain small, circular, extrachromosomal DNA molecules called plasmids (Fig. 34.5). Plasmid DNA undergoes replication that may or may not be synchronized to chromosomal division. Plasmids may carry genes that convey antibiotic resistance to the host bacterium and may facilitate the transfer of genetic information from one bacterium to another. They can be readily isolated from bacterial cells, their circular DNA cleaved at specific sites by restriction endonucleases, and up to 15 kb (kilobases) of foreign DNA (cut with the same restriction enzyme) inserted. The recombinant plasmid vector can be introduced into a bacterium, producing large numbers of copies of the plasmid. The bacteria are grown in the presence of antibiotics, thus selecting for cells containing the hybrid plasmids, which provide antibiotic resistance (Fig. 34.6).

1	numbers of copies of the plasmid. The bacteria are grown in the presence of antibiotics, thus selecting for cells containing the hybrid plasmids, which provide antibiotic resistance (Fig. 34.6). Artificial plasmids are routinely constructed. An example is the classic pBR322 (see Fig. 34.5), which contains an origin of replication, two antibiotic resistance genes, and >40 unique restriction sites. Use of plasmids is limited by the size of the DNA that can be inserted.

1	2. Other vectors: The development of improved vectors that can more efficiently accommodate larger DNA segments, or express the passenger genes in different cell types, has aided molecular genetics research and therapeutics. In addition to the prokaryotic plasmids described above, naturally occurring viruses that infect bacteria (bacteriophage λ, for example) or mammalian cells (retroviruses, for example), as well as artificial constructs such as cosmids and bacterial or yeast artificial chromosomes (BAC or YAC, respectively), are currently used as cloning vectors. [Note: BAC and YAC can accept DNA inserts of 100–300 kb and 250–1,000 kb, respectively.] B. DNA libraries

1	B. DNA libraries A DNA library is a collection of cloned restriction fragments of the DNA of an organism. Two kinds of libraries are commonly used: genomic libraries and complementary DNA (cDNA) libraries. Genomic libraries ideally contain a copy of every DNA nucleotide sequence in the genome. In contrast, cDNA libraries contain those DNA sequences that only appear as processed messenger RNA (mRNA) molecules, and these differ according to cell type and environmental conditions. [Note: cDNA lacks introns and the control regions of the genes, whereas these are present in genomic DNA.] 1.

1	Genomic DNA libraries: A genomic library is created by digestion of the total DNA of an organism with a restriction endonuclease and subsequent ligation to an appropriate vector. The recombinant DNA molecules replicate within host bacteria. Thus, the amplified DNA fragments collectively represent the entire genome of the organism and are called a genomic library. Regardless of the restriction enzyme used, the chances are good that the gene of interest contains more than one restriction site recognized by that enzyme. If this is the case, and if the digestion is allowed to go to completion, the gene of interest is fragmented (that is, it is not contained in any one clone in the library). To avoid this usually undesirable result, a partial digestion is performed in which either the amount or the time of action of the enzyme is limited. This results in cleavage occurring at only a fraction of the restriction sites on any one DNA molecule, thus producing fragments of ~20 kb. Enzymes that

1	the time of action of the enzyme is limited. This results in cleavage occurring at only a fraction of the restriction sites on any one DNA molecule, thus producing fragments of ~20 kb. Enzymes that cut very frequently (that is, those that recognize 4-bp sequences) are generally used for this purpose so that the result is an almost random collection of fragments. This insures a high degree of probability that the gene of interest is contained, intact, in some fragment.

1	2. Complementary DNA libraries: If a protein-coding gene of interest is expressed at a high level in a particular tissue, the mRNA transcribed from that gene is likely also present at high concentrations in the cells of that tissue. For example, reticulocyte mRNA is composed largely of molecules that code for the α-globin and β-globin chains of hemoglobin

1	A (HbA). This mRNA can be used as a template to make a cDNA molecule using the enzyme reverse transcriptase (Fig. 34.7). Therefore, the resulting cDNA is a double-stranded copy of mRNA. [Note: The template mRNA is isolated from transfer RNA and ribosomal RNA by the presence of its poly-A tail.] cDNA can be amplified by biologic cloning or by PCR. It can be used as a probe to locate the gene that encodes the original mRNA (or fragments of the gene) in mixtures containing many unrelated DNA fragments. If the mRNA used as a template is a mixture of many different size species, the resulting cDNA is heterogeneous. These mixtures can be cloned to form a cDNA library. Because cDNA has no introns, it can be cloned into an expression vector for the synthesis of eukaryotic proteins by bacteria (Fig. 34.8). These special plasmids contain a bacterial promoter for transcription of the cDNA and a Shine-Dalgarno (SD) sequence (see p. 454) that allows the bacterial ribosome to initiate translation

1	34.8). These special plasmids contain a bacterial promoter for transcription of the cDNA and a Shine-Dalgarno (SD) sequence (see p. 454) that allows the bacterial ribosome to initiate translation of the resulting mRNA molecule. The cDNA is inserted downstream of the promoter and within a gene for a protein that is expressed in the bacterium (for example, lacZ; see p. 466), such that the mRNA produced contains an SD sequence, a few codons for the bacterial protein, and all the codons for the eukaryotic protein. This allows for more efficient expression and results in the production of a fusion protein. [Note: Therapeutic human insulin is made in bacteria through this technology. However, the extensive co-and posttranslational modifications required for most other human proteins (for example, blood clotting factors) necessitates the use of eukaryotic, even mammalian, hosts.]

1	C. Sequencing cloned DNA fragments

1	The base sequence of DNA fragments that have been cloned can be determined. The original procedure for this purpose was the Sanger dideoxy chain termination method illustrated in Figure 34.9. In this method, the single-stranded DNA (ssDNA) to be sequenced is used as the template for DNA synthesis by DNA polymerase (DNA pol). A radiolabeled primer complementary to the 3′-end of the target DNA is added, along with the four deoxyribonucleoside triphosphates (dNTP). The sample is divided into four reaction tubes, and a small amount of one of the four dideoxyribonucleoside triphosphates (ddNTP) is added to each tube. Because it contains no 3′-hydroxyl group, incorporation of a ddNMP terminates elongation at that point. The products of this reaction, then, consist of a mixture of DNA strands of different lengths, each terminating at a specific base. Separation of the various DNA products by size in an electric field using polyacrylamide gel electrophoresis, followed by autoradiography,

1	of different lengths, each terminating at a specific base. Separation of the various DNA products by size in an electric field using polyacrylamide gel electrophoresis, followed by autoradiography, yields a pattern of bands from which the DNA base sequence can be read. [Note: The shorter the fragment, the farther it travels on the gel, with the shortest fragment representing that which was made first (that is, the 5′-end).] In place of a labeled primer, a mixture of the four ddNTP linked to different fluorescent dyes and in a single reaction tube is now commonly used. The mixture is separated by capillary electrophoresis, the fluorescent labels are detected, and a color readout of the sequence is generated (Fig. 34.10). [Note: The Human Genome Project used variations of this technique to sequence the human genome.] Advances in sequencing technology, so-called next generation, or high-throughput sequencing, now allow the rapid sequencing of an entire genome with increased fidelity and

1	sequence the human genome.] Advances in sequencing technology, so-called next generation, or high-throughput sequencing, now allow the rapid sequencing of an entire genome with increased fidelity and decreased cost through the simultaneous (parallel) sequencing of many DNA pieces. [Note: Sequencing of the exome, that portion of the genome that encodes proteins, is now possible.]

1	IV. PROBES Cleavage of large DNA molecules by restriction enzymes produces an enormous array of fragments. How can the DNA sequence of interest be picked out of such a mixture? The answer lies in the use of a probe, a short piece of ssDNA or RNA, labeled with a radioisotope, such as 32P, or with a nonradioactive molecule, such as biotin or a fluorescent dye. The sequence of a probe is complementary to a sequence in the DNA of interest, called the target DNA. Probes are used to identify which band on a gel or which clone in a library contains the target DNA, a process called screening. A. Hybridization to DNA

1	A. Hybridization to DNA The utility of probes hinges on the process of hybridization (or annealing) in which a probe containing a complementary sequence binds a single-stranded sequence of a target DNA. ssDNA, produced by alkaline denaturation of dsDNA, is first bound to a solid support, such as a nitrocellulose membrane. The immobilized DNA strands are prevented from self-annealing but are available for hybridization to the exogenous, radiolabeled, single-stranded probe. The extent of hybridization is measured by the retention of radioactivity on the membrane. Excess probe molecules that do not hybridize are removed by washing the membrane. B. Synthetic oligonucleotide probes

1	If the sequence of all or part of the target DNA is known, short, single-stranded oligonucleotide probes can be synthesized that are complementary to a small region of the gene of interest. If the sequence of the gene is unknown, the amino acid sequence of the protein, the final gene product, may be used to construct a nucleic acid probe using the genetic code as a guide. Because of the degeneracy of the genetic code (see p. 449), it is necessary to synthesize several oligonucleotides. [Note: Oligonucleotides can be used to detect single-base changes in the sequence to which they are complementary. In contrast, cDNA probes contain many thousands of bases, and their binding to a target DNA with a single-base change is unaffected.] 1. Detecting the βS-globin mutation: A synthetic allele-specific oligonucleotide (ASO) probe can be used to detect the presence of the sickle cell mutation in the β-globin gene (Fig. 34.11). DNA, isolated from white blood cells (WBC) and amplified, is

1	oligonucleotide (ASO) probe can be used to detect the presence of the sickle cell mutation in the β-globin gene (Fig. 34.11). DNA, isolated from white blood cells (WBC) and amplified, is denatured and applied to a membrane. A radiolabeled oligonucleotide probe, complementary to the point mutation (GAG → GTG, glutamate → valine) at codon 6 in patients with the βS gene, is applied to the membrane. DNA isolated from a heterozygous individual (sickle cell trait) or a homozygous patient (sickle cell anemia) contains a sequence that is complementary to the probe and a double-stranded hybrid form can be detected. In contrast, DNA obtained from normal individuals is not complementary at this position and, therefore, does not form a hybrid (see Fig. 34.11). Use of a pair of such ASO probes (one specific for the normal allele and one specific for the mutant allele) allows all three possible genotypes (homozygous normal, heterozygous, and homozygous mutant) to be distinguished (Fig. 34.12).

1	specific for the normal allele and one specific for the mutant allele) allows all three possible genotypes (homozygous normal, heterozygous, and homozygous mutant) to be distinguished (Fig. 34.12). [Note: ASO probes are useful only if the mutation and its location are known.]

1	C. Biotinylated probes

1	Because the disposal of radioactive waste is becoming increasingly expensive, nonradiolabeled probes have been developed. One of the most successful is based on the vitamin biotin (see p. 385), which can be chemically linked to the nucleotides used to synthesize the probe. Biotin was chosen because it binds very tenaciously to avidin, a readily available protein contained in chicken egg whites. Avidin can be attached to a fluorescent dye detectable optically with great sensitivity. Thus, a DNA fragment (displayed, for example, by gel electrophoresis) that hybridizes with the biotinylated probe can be made visible by immersing the gel in a solution of dye-coupled avidin. After washing away the excess avidin, the DNA fragment that binds the probe is fluorescent. [Note: Labeled probes can allow detection and localization of DNA or RNA sequences in cell or tissue preparations, a process called in situ hybridization (ISH). If the probe is fluorescent (F), the technique is called FISH.]

1	D. Antibodies If no amino acid sequence information is available to guide the synthesis of a probe for direct detection of the DNA of interest, a gene can be identified indirectly by cloning cDNA in an expression vector that allows the cloned cDNA to be transcribed and translated. A labeled antibody is used to identify which bacterial colony produces the protein and, therefore, contains the cDNA of interest. V. SOUTHERN BLOTTING Southern blotting is a technique that combines the use of restriction enzymes, electrophoresis, and DNA probes to generate, separate, and detect pieces of DNA. A. Procedure

1	This method, named after its inventor, Edward Southern, involves the following steps (Fig. 34.13). First, DNA is extracted from cells, for example, a patient’s WBC. Second, the DNA is cleaved into many fragments using a restriction enzyme. Third, the resulting fragments (all of which are negatively charged) are separated on the basis of size by electrophoresis. [Note: Because the large fragments move more slowly than the smaller ones, the lengths of the fragments, usually expressed as the number of base pairs, can be calculated from comparison of the position of the band relative to standard fragments of known size.] The DNA fragments in the gel are denatured and transferred (blotted) to a nitrocellulose membrane for analysis. If the original DNA represents the individual’s entire genome, the enzymic digest contains ≥106 fragments. The gene of interest is on only one (or a few if the gene itself was fragmented) of these pieces of DNA. If all the DNA fragments were visualized by a

1	the enzymic digest contains ≥106 fragments. The gene of interest is on only one (or a few if the gene itself was fragmented) of these pieces of DNA. If all the DNA fragments were visualized by a nonspecific technique, they would appear as an unresolved blur of overlapping bands. To avoid this, the last step in Southern blotting uses a probe to identify the DNA fragments of interest. The patterns observed on Southern blot analysis depend both on the specific restriction endonuclease and on the probe used to visualize the restriction fragments. [Note: Variants of the Southern blot have been facetiously named northern if RNA is being studied (see p. 499) and western if protein is being studied (see p. 500), neither of which relates to anyone’s name or to points of the compass.]

1	B. Mutation detection Southern blotting can detect DNA mutations such as large insertions or deletions, trinucleotide repeat expansions, and rearrangements of nucleotides. It can also detect point mutations (replacement of one nucleotide by another; see p. 449) that cause the loss or gain of restriction sites. Such mutations cause the pattern of bands to differ from those seen with a normal gene. Longer fragments are generated if a restriction site is lost. For example, in Figure 34.13, person 2 lacks a restriction site present in person 1. Alternatively, the point mutation may create a new cleavage site with the production of shorter fragments. [Note: Most sequence differences at restriction sites are harmless variations in the DNA.] VI. RESTRICTION FRAGMENT LENGTH POLYMORPHISM

1	It has been estimated that the genomes of any two unrelated people are 99.5% identical. With 6 billion bp in the diploid human genome, that represents variation in ~30 million bp. These genome variations are the result of mutations that lead to polymorphisms. A polymorphism is a change in genotype that can result in no change in phenotype or a change in phenotype that is harmless, causes increased susceptibility to a disease, or, rarely, causes the disease. It is traditionally defined as a sequence variation at a given locus (allele) in >1% of a population. Polymorphisms primarily occur in the 98% of the genome that does not encode proteins (that is, in introns and intergenic regions). A restriction fragment length polymorphism (RFLP) is a genetic variant that can be observed by cleaving the DNA into fragments (restriction fragments) with a restriction endonuclease. The length of the restriction fragments is altered if the variant alters the DNA so as to create or abolish a

1	cleaving the DNA into fragments (restriction fragments) with a restriction endonuclease. The length of the restriction fragments is altered if the variant alters the DNA so as to create or abolish a restriction site. RFLP can be used to detect human genetic variations, for example, in prospective parents or in fetal tissue.

1	A. DNA variations resulting in RFLP Two types of DNA variations commonly result in RFLP: single-base changes in the DNA sequence and tandem repeats of DNA sequences.

1	1. Single-base changes: About 90% of human genome variation comes in the form of single nucleotide polymorphisms (SNPs, pronounced “snips”), that is, variations that involve just one base (Fig. 34.14). The substitution of one nucleotide at a restriction site can render the site unrecognizable by a particular restriction endonuclease. A new restriction site can also be created by the same mechanism. In either case, cleavage with an endonuclease results in fragments of lengths that differ from the normal and can be detected by DNA hybridization (see Fig. 34.13). The altered restriction site can be either at the site of a disease-causing mutation (rare) or at a site some distance from the mutation. [Note: The HapMap, developed by The International Haplotype Map Project, is a catalog of common SNP in the human genome. The data are being used in genome-wide association studies (GWAS) to identify those alleles that affect health and disease.] 2. Tandem repeats: Polymorphisms in chromosomal

1	SNP in the human genome. The data are being used in genome-wide association studies (GWAS) to identify those alleles that affect health and disease.] 2. Tandem repeats: Polymorphisms in chromosomal DNA can also arise from the presence of a variable number of tandem repeats (VNTR), as shown in Figure 34.15. These are short sequences of DNA at scattered locations in the genome, repeated in tandem (one after another). The number of these repeat units varies from person to person but is unique for any given individual and, therefore, serves as a molecular “fingerprint.” Cleavage by restriction enzymes yields fragments that vary in length depending on how many repeated segments are contained in the fragment (see Fig. 34.15). Many different VNTR loci have been identified and are extremely useful for DNA fingerprint analysis, such as in forensic and paternity cases. It is important to emphasize that these polymorphisms, whether SNP or VNTR, are simply markers, which, in most cases, have no

1	for DNA fingerprint analysis, such as in forensic and paternity cases. It is important to emphasize that these polymorphisms, whether SNP or VNTR, are simply markers, which, in most cases, have no known effect on the structure, function, or rate of production of any particular protein.

1	B. Tracing chromosomes from parent to offspring If the DNA of an individual has gained a restriction site by base substitution, then enzymic cleavage yields at least one additional fragment. Conversely, if a mutation results in loss of a restriction site, fewer fragments are produced by enzymic cleavage. An individual who is heterozygous for a polymorphism has a sequence variation in the DNA of one chromosome and not in the homologous chromosome. In such individuals, each chromosome can be traced from parent to offspring by determining the presence or absence of the polymorphism. C. Prenatal diagnosis Families with a history of severe genetic disease, such as an affected previous child or near relative, may wish to determine the presence of the disorder in a developing fetus. Prenatal diagnosis, in association with genetic counseling, allows for an informed reproductive decision if the fetus is affected. 1.

1	1. Methods available: The available diagnostic methods vary in sensitivity and specificity. Visualization of the fetus, for example, by ultrasound or fiberoptic devices (fetoscopy), is useful only if the genetic abnormality results in gross anatomic defects (for example, neural tube defects [NTD]). The chemical composition of the amniotic fluid can also provide diagnostic clues. For example, the presence of high levels of αfetoprotein is associated with NTD. Fetal cells obtained from amniotic fluid or from biopsy of the chorionic villi can be used for karyotyping, which assesses the morphology of metaphase chromosomes. Staining and cell sorting techniques permit the rapid identification of trisomies and translocations that produce an extra chromosome or chromosomes of abnormal lengths. However, molecular analysis of fetal DNA provides the most detailed genetic picture. 2.

1	2. DNA sources: DNA may be obtained from blood cells, amniotic fluid, or chorionic villi (Fig. 34.16). For amniotic fluid, it was formerly necessary to grow cells in culture for 2–3 weeks in order to have sufficient DNA for analysis. The ability to amplify DNA by PCR has dramatically shortened the time needed for a DNA analysis. 3. Direct diagnosis of sickle cell anemia using RFLP: The genetic disorders of Hb are the most common genetic diseases in humans. In the case of sickle cell anemia (Fig. 34.17), the point mutation that gives rise to the disease (see p. 35) is actually one and the same mutation that gives rise to the polymorphism. However, direct detection by RFLP of diseases that result from point mutations is limited to only a few genetic diseases. a. Early diagnostic efforts: In the past, prenatal diagnosis of sickle cell anemia involved the determination of the amount and kinds of Hb synthesized in the nucleated red cells obtained from fetal blood.

1	However, the invasive procedures to obtain fetal blood have a high mortality rate (~5%), and analysis cannot be carried out until late in the second trimester of pregnancy when HbA (and its HbS variant) begins to be produced.

1	b. RFLP analysis: In sickle cell anemia, the sequence alteration caused by the point mutation abolishes the recognition site of the restriction endonuclease MstII: CCTNAGG (where N is any nucleotide; see Fig. 34.17). Thus, the A-to-T mutation in codon 6 of the βS-globin gene eliminates a cleavage site for the enzyme. Normal DNA digested with MstII yields a 1.15-kb fragment, whereas a 1.35-kb fragment is generated from the βS gene as a result of the loss of one MstII cleavage site. Diagnostic techniques that allow analysis of fetal DNA from amniotic cells or chorionic villus sampling rather than fetal blood have proved valuable because they provide safe, early detection of sickle cell anemia as well as other genetic diseases. [Note: Genetic disorders caused by insertions or deletions between two restriction sites, rather than by the creation or loss of cleavage sites, will also display RFLP.] 4. Indirect diagnosis of phenylketonuria using RFLP: The gene for phenylalanine hydroxylase

1	two restriction sites, rather than by the creation or loss of cleavage sites, will also display RFLP.] 4. Indirect diagnosis of phenylketonuria using RFLP: The gene for phenylalanine hydroxylase (PAH), the enzyme deficient in phenylketonuria ([PKU] see p. 270), is located on chromosome 12. It spans ~90 kb of genomic DNA and contains 13 exons separated by introns (Fig. 34.18; see p. 442 for a description of exons and introns). Mutations in the PAH gene usually do not directly affect any restriction endonuclease recognition site. To establish a diagnostic protocol for PKU, DNA from family members of the affected individual must be analyzed. The goal is to identify genetic markers (RFLP) that are tightly linked to the disease trait. Once these markers are identified, RFLP analysis can be used to carry out prenatal diagnosis.

1	a.

1	Mutant gene identification: Determining the presence of the mutant gene by identifying the polymorphism marker can be done if two conditions are satisfied. First, if the polymorphism is closely linked to a disease-producing mutation, the defective gene can be traced by detection of the RFLP. For example, if DNA from a family carrying a disease-causing gene is examined by restriction enzyme cleavage and Southern blotting, it is sometimes possible to find an RFLP that is consistently associated with that gene (that is, they show close linkage and are coinherited). It is then possible to trace the inheritance of the gene within a family without knowledge of the nature of the genetic defect or its precise location in the genome. [Note: The polymorphism may be known from the study of other families with the disorder or may be discovered to be unique in the family under investigation.] Second, for autosomal-recessive disorders, such as PKU, the presence of an affected individual in the

1	families with the disorder or may be discovered to be unique in the family under investigation.] Second, for autosomal-recessive disorders, such as PKU, the presence of an affected individual in the family would aid in the diagnosis. This individual would have the mutation present on both chromosomes, allowing identification of the RFLP associated with the genetic disorder.

1	b.

1	RFLP analysis: The presence of abnormal genes for PAH can be shown using DNA polymorphisms as markers to distinguish between normal and mutant genes. For example, Figure 34.19 shows a typical pattern obtained when DNA from members of an affected family is cleaved with an appropriate restriction enzyme and subjected to electrophoresis. The vertical arrows represent the cleavage sites for the restriction enzyme used. The presence of a polymorphic site creates fragment “b” in the autoradiogram (after hybridization with a labeled PAH-cDNA probe), whereas the absence of this site yields only fragment “a.” Note that subject II-2 demonstrates that the polymorphism, as shown by the presence of fragment “b,” is associated with the mutant gene. Therefore, in this particular family, the appearance of fragment “b” corresponds to the presence of a polymorphic site that marks the abnormal gene for PAH. The absence of fragment “b” corresponds to having only the normal gene. In Figure 34.19,

1	of fragment “b” corresponds to the presence of a polymorphic site that marks the abnormal gene for PAH. The absence of fragment “b” corresponds to having only the normal gene. In Figure 34.19, examination of fetal DNA shows that the fetus inherited two abnormal genes from the parents and, therefore, has PKU.

1	c. Value of DNA testing: DNA-based testing is useful not only in determining if an unborn fetus is affected by PKU but also in detecting unaffected carriers of the mutated gene to aid in family planning. [Note: PKU is treatable by dietary restriction of phenylalanine. Early diagnosis and treatment are essential in preventing severe neurologic damage in affected individuals.] VII. POLYMERASE CHAIN REACTION PCR is an in vitro method for amplifying a selected DNA sequence that does not rely on the biologic (in vivo) cloning method described on p. 483. PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. The method can be used to amplify DNA sequences from any source, including viral, bacterial, plant, or animal. The steps in PCR are summarized in Figures 34.20 and 34.21. A. Procedure

1	A. Procedure PCR uses DNA pol to repetitively amplify targeted portions of genomic or cDNA. Each cycle of amplification doubles the amount of DNA in the sample, leading to an exponential increase (2n, where n = cycle number) in DNA with repeated cycles of amplification. The amplified DNA products can then be separated by gel electrophoresis, detected by Southern blotting and hybridization, and sequenced. 1.

1	1. Constructing primer: It is not necessary to know the nucleotide sequence of the target DNA in the PCR method. However, it is necessary to know the nucleotide sequence of short segments on each side of the target DNA. These stretches, called flanking sequences, bracket the DNA sequence of interest. The nucleotide sequences of the flanking regions are used to construct two, single-stranded oligonucleotides, usually 20– 35 nucleotides long, which are complementary to the respective flanking sequences. The 3′-hydroxyl end of each oligonucleotide points toward the target sequence (see Fig. 34.20). These synthetic oligonucleotides function as primers in PCR. 2. Denaturing DNA: The target DNA to be amplified is heated to ~95°C to separate the dsDNA into single strands. 3. Annealing primers: The separated strands are cooled to ~50°C and the two primers (one for each strand) anneal to a complementary sequence on the ssDNA. 4.

1	Extending primers: DNA pol and dNTP (in excess) are added to the mixture (~72°C) to initiate the synthesis of two new strands complementary to the original DNA strands. DNA pol adds nucleotides to the 3′-hydroxyl end of the primer, and strand growth extends in the 5′→3′ direction across the target DNA, making complementary copies of the target. [Note: PCR products can be several thousand base pairs long.] At the completion of one cycle of replication, the reaction mixture is heated again to separate the strands (of which there are now four). Each strand binds a complementary primer, and the step of primer extension is repeated. By using a heat-stable DNA pol (for example, Taq from the bacterium Thermus aquaticus that normally lives at high temperatures), the polymerase is not denatured and, therefore, does not have to be added at each successive cycle. However, Taq lacks proofreading activity. Typically, 20–30 cycles are run during this process, amplifying the DNA by a million-fold

1	and, therefore, does not have to be added at each successive cycle. However, Taq lacks proofreading activity. Typically, 20–30 cycles are run during this process, amplifying the DNA by a million-fold (220) to a billion-fold (230). [Note: Each extension product includes a sequence at its 5′-end that is complementary to the primer (see Fig. 34.20). Thus, each newly synthesized strand can act as a template for the successive cycles (see Fig. 34.21). This leads to an exponential increase in the amount of target DNA with each cycle, hence, the name “polymerase chain reaction.”] Probes can be made during PCR by adding labeled nucleotides to the last few cycles.

1	B. Advantages The major advantages of PCR over biologic cloning as a mechanism for amplifying a specific DNA sequence are sensitivity and speed. DNA sequences present in only trace amounts can be amplified to become the predominant sequence. PCR is so sensitive that DNA sequences present in an individual cell can be amplified and studied. Isolating and amplifying a specific DNA sequence by PCR is faster and less technically difficult than traditional cloning methods using recombinant DNA techniques. C. Applications PCR has become a very common tool in research, forensics, and clinical diagnostics. 1. Comparison of a normal gene to its mutant form: PCR allows the synthesis of mutant DNA in sufficient quantities for a sequencing protocol without laborious biologic cloning of the DNA. 2.

1	1. Comparison of a normal gene to its mutant form: PCR allows the synthesis of mutant DNA in sufficient quantities for a sequencing protocol without laborious biologic cloning of the DNA. 2. Forensic analysis of DNA samples: DNA fingerprinting by means of PCR has revolutionized the analysis of evidence from crime scenes. DNA isolated from a single human hair, a tiny spot of blood, or a sample of semen is sufficient to determine whether the sample comes from a specific individual. The DNA markers analyzed for such fingerprinting are most commonly a type of polymorphism known as short tandem repeats. These are very similar to the VNTR described previously (see p. 491) but are smaller in size. [Note: Paternity testing uses the same techniques.] 3.

1	491) but are smaller in size. [Note: Paternity testing uses the same techniques.] 3. Detection of low-abundance nucleic acid sequences: Viruses that have a long latency period, such as human immunodeficiency virus (HIV), are difficult to detect at the early stage of infection using conventional methods. PCR offers a rapid and sensitive method for detecting viral DNA sequences even when only a small proportion of cells harbors the virus. [Note: Quantitative PCR (qPCR), also known as real-time PCR, allows quantification of the amount (copy number) of the target nucleic acid after each cycle of amplification (that is, in real time) rather than at the end and is useful in determining viral load (the amount of virus).] 4.

1	Prenatal diagnosis and carrier detection of cystic fibrosis: Cystic fibrosis is an autosomal-recessive genetic disease resulting from mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most common mutation is a three-base deletion that results in the loss of a phenylalanine residue from the CFTR protein (see p. 450). Because the mutant allele is three bases shorter than the normal allele, it is possible to distinguish them from each other by the size of the PCR products obtained by amplifying that portion of the DNA. Figure 34.22 illustrates how the results of such a PCR test can distinguish between homozygous normal, heterozygous (carriers), and homozygous mutant (affected) individuals. The simultaneous amplification of multiple regions of a target DNA using multiple primer pairs is known as multiplex PCR. It allows detection of the loss of ≥1 exons in a gene with many exons such as the gene for CFTR, which has 27 exons.

1	VIII. GENE EXPRESSION ANALYSIS The tools of biotechnology not only allow the study of gene structure, but also provide ways of analyzing the mRNA and protein products of gene expression. A. Determining messenger RNA levels mRNA levels are usually determined by the hybridization of labeled probes to either mRNA itself or to cDNA produced from mRNA. [Note: Amplification by PCR of cDNA made from mRNA by retroviral reverse transcriptase (RT) is referred to as RT-PCR.] 1. Northern blots Northern blots are similar to Southern blots (see Fig. 34.13), except that the sample contains a mixture of mRNA molecules that are separated by electrophoresis, then transferred to a membrane and hybridized with a radiolabeled probe. The bands obtained by autoradiography give a measure of the amount and size of the mRNA molecules in the sample.

1	2. Microarrays: DNA microarrays contain thousands of immobilized ssDNA sequences organized in an area no larger than a microscope slide. These microarrays are used to analyze a sample for the presence of gene variations or mutations (genotyping) or to determine the patterns of mRNA production (gene expression analysis), analyzing thousands of genes at the same time. For genotyping analysis, the sample is from genomic DNA. For expression analysis, the population of mRNA molecules from a particular cell type is converted to cDNA and labeled with a fluorescent tag (Fig. 34.23). This mixture is then exposed to a gene (or, DNA) chip, which is a glass slide or membrane containing thousands of tiny spots of DNA, each corresponding to a different gene. The amount of fluorescence bound to each spot is a measure of the amount of that particular mRNA in the sample. DNA microarrays are used to determine the differing patterns of gene expression in two different types of cell (for example, normal

1	is a measure of the amount of that particular mRNA in the sample. DNA microarrays are used to determine the differing patterns of gene expression in two different types of cell (for example, normal and cancer cells; see Fig. 34.23). They can also be used to subclassify cancers, such as breast cancer, to optimize treatment. [Note: Microarrays involving proteins and the antibodies or other proteins that recognize them are being used to identify biomarkers to aid in the diagnosis, prognosis, and treatment of disease based on a patient’s protein expression profile. Protein (and DNA) microarrays are important tools in the development of personalized (precision) medicine in which the treatment and/or prevention strategies consider the genetic, environmental, and lifestyle variations among individuals.]

1	B. Protein analysis The kinds and amounts of proteins in cells do not always directly correspond to the amounts of mRNA present. Some mRNA are translated more efficiently than others, and some proteins undergo posttranslational modification. When analyzing the abundance and interactions of a large number of proteins, automated methods involving a variety of techniques, such as mass spectrometry and two-dimensional electrophoresis, are used. When investigating one, or a limited number of proteins, labeled antibodies (Ab) are used to detect and quantify specific proteins and to determine posttranslational modifications. 1.

1	1. Enzyme-linked immunosorbent assays: These assays (known as ELISA) are performed in the wells of a microtiter dish. The antigen (protein) is bound to the plastic of the dish. The probe used consists of an Ab specific for the protein (such as troponin, see p. 66) to be measured. The Ab is covalently bound to an enzyme, which will produce a colored product when exposed to its substrate. The amount of color produced is proportional to the amount of Ab present and, indirectly, to the amount of protein in a test sample. 2. Western blots: Western blots (also called immunoblots) are similar to Southern blots, except that it is protein molecules in the sample that are separated by electrophoresis and blotted (transferred) to a membrane. The probe is a labeled Ab, which produces a band at the location of its antigen. 3.

1	3. Detecting exposure to human immunodeficiency virus: ELISA and western blots are commonly used to detect exposure to HIV by measuring the amount of anti-HIV Ab present in a patient’s blood sample. ELISA are used as the primary screening tool because they are very sensitive. Because these assays sometimes give false positives, however, western blots, which are more specific, are often used as a confirmatory test (Fig. 34.24). [Note: ELISA and western blots can only detect HIV exposure after anti-HIV Ab appear in the bloodstream. PCR based testing for HIV is more useful in the first few months after exposure.] C. Proteomics

1	C. Proteomics The study of the proteome, or all the proteins expressed by a genome, including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other macromolecules, is known as proteomics. The 20,000–25,000 protein-coding genes of the human genome translate into well over 100,000 proteins when posttranscriptional and posttranslational modifications are considered. Although a genome remains essentially unchanged, the amounts and types of proteins in any particular cell change dramatically as genes are turned on and off. [Note: Proteomics (and genomics) required the parallel development of bioinformatics, the computer-based organization, storage, and analysis of biologic data.] Figure 34.25 compares some of the analytic techniques discussed in this chapter. IX. GENE THERAPY

1	The goal of gene therapy is to treat disease through delivery of the normal, cloned DNA for a gene into the somatic cells of a patient who has a defect in that gene as a result of a disease-causing mutation. Because somatic gene therapy changes only the targeted somatic cells, the change is not passed on to the next generation. [Note: In germline gene therapy, the germ cells are modified, and so the change is passed on. A long-standing moratorium on germline gene therapy is in effect worldwide.] There are two types of gene transfer: 1) ex vivo, in which cells from the patient are removed, transduced, and returned, and 2) in vivo, in which the cells are directly transduced. Both types require use of a viral vector to deliver the DNA. Challenges of gene therapy include development of vectors, achievement of long-lived expression, and prevention of side effects such as an immune response. The first successful gene therapy involved two patients with severe combined immunodeficiency

1	vectors, achievement of long-lived expression, and prevention of side effects such as an immune response. The first successful gene therapy involved two patients with severe combined immunodeficiency disease (SCID) caused by mutations to the gene for adenosine deaminase (ADA, see p. 301). It utilized mature T lymphocytes transduced ex vivo with a retroviral vector (Fig. 34.26). [Note: Human ADA cDNA is now used.] Since 1990, only a small number of patients (with a variety of disorders, such as hemophilia, cancers, and certain types of blindness) have been treated with gene therapy, with varying degrees of success.

1	Gene editing, as opposed to gene addition, allows a mutated gene to be repaired. Combinations of DNA-binding molecules (proteins or RNA) and endonucleases are used to identify and cleave the mutated sequence. Cleavage activates homologous recombination repair of dsDNA breaks (see p. 429) that integrates DNA containing the correct sequence into the gene. [Note: An endonuclease guided to a specific DNA sequence by a custom-designed RNA has been used in gene editing in human cell lines. The technique is based on (and named for) the prokaryotic CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats [CRISPR]-associated protein) system that identifies and cleaves foreign DNA in bacterial cells. CRISPR is currently used in the laboratory but not in the clinic.] X. TRANSGENIC ANIMALS

1	Transgenic animals can be produced by injecting a cloned foreign gene (a transgene) into a fertilized egg. If the gene randomly and stably integrates into a chromosome, it will be present in the germline of the resulting animal and can be passed from generation to generation. A giant mouse called “Supermouse” was produced in this way by injecting the gene for rat growth hormone into a fertilized mouse egg. [Note: Transgenic animals have been designed that produce therapeutic human proteins in their milk, a process called “pharming.” Antithrombin, an anticlotting protein (see online Chapter 35), was produced by transgenic goats and approved for clinical use in 2009.] If the functional transgene undergoes targeted (not random) insertion, a knockin (KI) mouse that expresses the gene is created. Targeted insertion of a nonfunctional version of the transgene creates a knockout (KO) mouse that does not express the gene. Such genetically engineered animals can serve as models for the study

1	Targeted insertion of a nonfunctional version of the transgene creates a knockout (KO) mouse that does not express the gene. Such genetically engineered animals can serve as models for the study of a corresponding human disease.

1	XI. CHAPTER SUMMARY

1	Restriction endonucleases are bacterial enzymes that cleave double-stranded DNA (dsDNA) into smaller fragments. Each enzyme cleaves at a specific 4–8 base-pair sequence (a restriction site), producing DNA segments called restriction fragments. The sequences that are recognized are palindromic. Restriction enzymes form either staggered cuts (sticky ends) or blunt-end cuts on the DNA. Bacterial DNA ligases can join two DNA fragments from different sources if they have been cut by the same restriction endonuclease. This hybrid combination of two fragments is called a recombinant DNA molecule. Introduction of a foreign DNA molecule into a replicating cell permits the amplification (production of many copies) of the DNA, a process called cloning. A vector is a molecule of DNA to which the fragment of DNA to be cloned is joined. Vectors must be capable of autonomous replication within the host cell, must contain at least one specific nucleotide sequence recognized by a restriction

1	the fragment of DNA to be cloned is joined. Vectors must be capable of autonomous replication within the host cell, must contain at least one specific nucleotide sequence recognized by a restriction endonuclease, and must carry at least one gene that confers the ability to select for the vector such as an antibiotic resistance gene. Prokaryotic organisms normally contain small, circular, extrachromosomal DNA molecules called plasmids that can serve as vectors. They can be readily isolated from the bacterium (or artificially constructed), joined with the DNA of interest, and reintroduced into the bacterium, which will replicate, thus making multiple copies of the hybrid plasmid. A DNA library is a collection of cloned restriction fragments of the DNA of an organism. A genomic library is a collection of fragments of dsDNA obtained by digestion of the total DNA of the organism with a restriction endonuclease and subsequent ligation to an appropriate vector. It ideally contains a copy of

1	collection of fragments of dsDNA obtained by digestion of the total DNA of the organism with a restriction endonuclease and subsequent ligation to an appropriate vector. It ideally contains a copy of every DNA nucleotide sequence in the genome. In contrast, complementary DNA (cDNA) libraries contain only those DNA sequences that are complementary to processed messenger RNA (mRNA) molecules present in a cell and differ according to cell type and environmental conditions. Because cDNA has no introns, it can be cloned into an expression vector for the synthesis of human proteins by bacteria or eukaryotes. Cloned, then purified, fragments of DNA can be sequenced, for example, using the Sanger dideoxy chain termination method. A probe is a small piece of RNA or single-stranded DNA (usually labeled with a radioisotope, such as 32P, or another identifiable compound, such as biotin or a fluorescent dye) that has a nucleotide sequence complementary to the DNA molecule of interest (target DNA).

1	with a radioisotope, such as 32P, or another identifiable compound, such as biotin or a fluorescent dye) that has a nucleotide sequence complementary to the DNA molecule of interest (target DNA). Probes can be used to identify which clone of a library or which band on a gel contains the target DNA. Southern blotting is a technique that can be used to detect specific sequences present in DNA. The DNA is cleaved using a restriction endonuclease, after which the pieces are separated by gel electrophoresis and are denatured and transferred (blotted) to a nitrocellulose membrane for analysis. The fragment of interest is detected using a probe. The human genome contains many thousands of polymorphisms (DNA sequence variations at a given locus). Polymorphisms can arise from single-base changes and from tandem repeats. A polymorphism can serve as a genetic marker that can be followed through families. A restriction fragment length polymorphism (RFLP) is a genetic variant that can be observed

1	from tandem repeats. A polymorphism can serve as a genetic marker that can be followed through families. A restriction fragment length polymorphism (RFLP) is a genetic variant that can be observed by cleaving the DNA into restriction fragments using a restriction enzyme. A base substitution in one or more nucleotides at a restriction site can render the site unrecognizable by a particular restriction endonuclease. A new restriction site also can be created by the same mechanism. In either case, cleavage with the endonuclease results in fragments of lengths differing from the normal that can be detected by hybridization with a probe. RFLP analysis can be used to diagnose genetic diseases early in the gestation of a fetus. The polymerase chain reaction (PCR), another method for amplifying a selected DNA sequence, does not rely on the biologic cloning method. PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence,

1	a selected DNA sequence, does not rely on the biologic cloning method. PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. The method can be used to amplify DNA sequences from any source. Applications of the PCR technique include 1) efficient comparison of a normal gene with a mutant form of the gene, 2) forensic analysis of DNA samples, 3) detection of low-abundance nucleic acid sequences, and 4) prenatal diagnosis and carrier detection (for example, of cystic fibrosis). The products of gene expression (mRNA and proteins) can be measured by techniques such as northern blots, which are like Southern blots except that the sample contains a mixture of mRNA molecules that are separated by electrophoresis, then hybridized to a radiolabeled probe; microarrays are used to determine the differing patterns of gene

1	that the sample contains a mixture of mRNA molecules that are separated by electrophoresis, then hybridized to a radiolabeled probe; microarrays are used to determine the differing patterns of gene expression in two different types of cells (for example, normal and cancer cells); enzyme-linked immunosorbent assays (ELISA); and western blots (immunoblots) are used to detect specific proteins. Proteomics is the study of all the proteins expressed by a genome. The goal of gene therapy is the insertion of a normal cloned gene to replace a defective gene in a somatic cell, whereas the goal of gene editing is the repair of a mutated gene. Insertion of a foreign gene (transgene) into the germline of an animal creates a transgenic animal that can produce therapeutic proteins or serve as gene knockin or knockout models for human diseases.

1	Choose the ONE best answer. 4.1. HindIII is a restriction endonuclease. Which of the following is most likely to be the recognition sequence for this enzyme? A. AAGAAG B. AAGAGA C. AAGCTT D. AAGGAA E. AAGTTC Correct answer = C. The vast majority of restriction endonucleases recognize palindromes in double-stranded DNA, and AAGCTT is the only palindrome among the choices. Because the sequence of only one DNA strand is given, the base sequence of the complementary strand must be determined. To be a palindrome, both strands must have the same sequence when read in the 5′→3′ direction. Thus, the complement of 5′-AAGCTT-3′ is also 5′-AAGCTT-3′.

1	4.2. An Ashkenazi Jewish couple has their 6-month-old son evaluated for listlessness, poor head control, and a fixed gaze. Tay-Sachs disease, an autosomal-recessive disease of lipid degradation, is diagnosed. The couple also has a daughter. The family’s pedigree is shown to the right, along with Southern blots of a restriction fragment length polymorphism very closely linked to the gene for hexosaminidase A, which is defective in Tay-Sachs disease. Which of the statements below is most accurate with respect to the daughter? A. She has a 25% chance of having Tay-Sachs disease. B. She has a 50% chance of having Tay-Sachs disease. C. She has Tay-Sachs disease. D. She is a carrier for Tay-Sachs disease. E. She is homozygous normal.

1	B. She has a 50% chance of having Tay-Sachs disease. C. She has Tay-Sachs disease. D. She is a carrier for Tay-Sachs disease. E. She is homozygous normal. Correct answer = E. Because they have an affected son, both the biological father and mother must be carriers for this disease. The affected son must have inherited a mutant allele from each parent. Because he shows only the 3kilobase (kb) band on the Southern blot, the mutant allele for this disease must be linked to the 3-kb band. The normal allele must be linked to the 4-kb band, and because the daughter inherited only the 4-kb band, she must be homozygous normal for the hexosaminidase A gene. 4.3. A physician would like to determine the global patterns of gene expression in two different types of tumor cells in order to develop the most appropriate form of chemotherapy for each patient. Which of the following techniques would be most appropriate for this purpose? A. Enzyme-linked immunosorbent assay B. Microarray

1	A. Enzyme-linked immunosorbent assay B. Microarray C. Northern blot D. Southern blot E. Western blot Correct answer = B. Microarray analysis allows the determination of messenger RNA (mRNA) production (gene expression) from thousands of genes at once. A northern blot only measures mRNA production from one gene at a time. Western blots and enzyme-linked immunosorbent assay measure protein production (also gene expression) but only from one gene at a time. Southern blots are used to analyze DNA, not the products of DNA expression. 4.4. A 2-week-old infant is diagnosed with a urea cycle defect. Enzymic analysis showed no activity for ornithine transcarbamoylase (OTC), an enzyme of the cycle. Molecular analysis revealed that the messenger RNA (mRNA) product of the gene for OTC was identical to that of a control. Which of the techniques listed below was most likely used to analyze mRNA? A. Dideoxy chain termination B. Northern blot C. Polymerase chain reaction D. Southern blot

1	A. Dideoxy chain termination B. Northern blot C. Polymerase chain reaction D. Southern blot E. Western blot Correct answer = B. Northern blot allows analysis of the messenger RNA present (expressed) in a particular cell or tissue. Southern blot is used for DNA analysis, whereas western blot is used for protein analysis. Dideoxy chain termination is used to sequence DNA. Polymerase chain reaction is used to generate multiple, identical copies of a DNA sequence in vitro. 4.5. For the patient above, which phase of the central dogma was most likely affected? Correct answer = Translation. The gene is present and is able to be expressed as evidenced by normal production of messenger RNA. The lack of enzymic activity means that some aspect of protein synthesis is affected. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW

1	Blood clotting (coagulation) is designed to rapidly stop bleeding from a damaged blood vessel in order to maintain a constant blood volume (hemostasis). Coagulation is accomplished through vasoconstriction and the formation of a clot (thrombus) that consists of a plug of platelets (primary hemostasis) and a meshwork of the protein fibrin (secondary hemostasis) that stabilizes the platelet plug. Clotting occurs in association with membranes on the surface of platelets and damaged blood vessels (Fig. 35.1). [Note: If clotting occurs within an intact vessel such that the lumen is occluded and blood flow is impeded, a condition known as thrombosis, serious tissue damage, and even death can occur. This is what happens, for example, during a myocardial infarction (MI).] Processes to limit clot formation to the area of damage and remove the clot once vessel repair is underway also play essential roles in hemostasis. [Note: Separate discussions of the formation of the platelet plug and the

1	formation to the area of damage and remove the clot once vessel repair is underway also play essential roles in hemostasis. [Note: Separate discussions of the formation of the platelet plug and the fibrin meshwork facilitate presentation of these multistep, multicomponent processes. However, the two work together to maintain hemostasis.]

1	II. FIBRIN MESHWORK FORMATION The formation of the fibrin meshwork involves two unique pathways that converge to form a common pathway (Fig. 35.2). In each pathway, the major components are proteins (called factors [F]) designated by Roman numerals. The factors are glycoproteins that are synthesized and secreted by the liver, primarily. [Note: Several factors are also denoted by alternative names. For example, factor X (FX), the point of pathway convergence, is also known as Stuart factor.] A. Proteolytic cascade

1	Within the pathways, a cascade is set up in which proteins are converted from an inactive form, or zymogen, to an active form by proteolytic cleavage in which the protein product of one activation reaction initiates another. The active form of a factor is denoted by a lowercase “a” after the numeral. The active proteins FIIa, FVIIa, FIXa, FXa, FXIa, and FXIIa are enzymes that function as serine proteases with trypsin-like specificity and, therefore, cleave a peptide bond on the carboxyl side of an arginine or lysine residue in a polypeptide. For example, FIX (Christmas factor) is activated through cleavage at arginine 145 and arginine 180 by FXIa (Fig. 35.3). The proteolytic cascade results in enormous rate acceleration, because one active protease can produce many molecules of active product each of which, in turn, can activate many molecules of the next protein in the cascade. In some cases, activation can be caused by a conformational change in the protein in the absence of

1	product each of which, in turn, can activate many molecules of the next protein in the cascade. In some cases, activation can be caused by a conformational change in the protein in the absence of proteolysis. [Note: Nonproteolytic proteins play a role as accessory proteins (cofactors) in the pathways. FIII, FV, and FVIII are the accessory proteins.]

1	B. Role of phosphatidylserine and calcium The presence of the negatively charged phospholipid phosphatidylserine (PS) and positively charged calcium ions (Ca2+) accelerates the rate of some steps in the clotting cascade. 1. Phosphatidylserine: PS is located primarily on the intracellular (cytosolic) face of the plasma membrane. [Note: Flippases create the asymmetry (see p. 205).] Its exposure signals injury to the endothelial cells that line blood vessels. PS is also exposed on the surface of activated platelets. 2.

1	2. Calcium ions: Ca2+ binds the negatively charged γ-carboxyglutamate (Gla) residues present in four of the serine proteases of clotting (FII, FVII, FIX, and FX), facilitating the binding of these proteins to exposed phospholipids (Fig. 35.4). The Gla residues are good chelators of Ca2+ because of their two adjacent negatively charged carboxylate groups (Fig. 35.5). [Note: The use of chelating agents such as sodium citrate to bind Ca2+ in blood-collecting tubes or bags prevents the blood from clotting.] C. Formation of γ-carboxyglutamate residues γ-Carboxylation is a posttranslational modification in which 9–12 glutamate residues (at the amino [N]-terminus of the target protein) get carboxylated at the γ carbon, thereby forming Gla residues. The process occurs in the rough endoplasmic reticulum (RER) of the liver.

1	1. γ-Carboxylation: This carboxylation reaction requires a protein substrate, oxygen (O2), carbon dioxide (CO2), γ-glutamyl carboxylase, and the hydroquinone form of vitamin K as a coenzyme (Fig. 35.6). In the reaction, the hydroquinone form of vitamin K gets oxidized to its epoxide form as O2 is reduced to water. [Note: Dietary vitamin K, a fat-soluble vitamin (see p. 393), is reduced from the quinone form to the hydroquinone coenzyme form by vitamin K reductase (Fig. 35.7).] 2. Inhibition by warfarin: The formation of Gla residues is sensitive to inhibition by warfarin, a synthetic analog of vitamin K that inhibits the enzyme vitamin K epoxide reductase (VKOR). The reductase, an integral protein of the RER membrane, is required to regenerate the functional hydroquinone form of vitamin K from the epoxide form generated in the γ-carboxylation reaction. Thus, warfarin is an anticoagulant that inhibits clotting by functioning as a vitamin K antagonist. Warfarin salts are used

1	vitamin K from the epoxide form generated in the γ-carboxylation reaction. Thus, warfarin is an anticoagulant that inhibits clotting by functioning as a vitamin K antagonist. Warfarin salts are used therapeutically to limit clot formation. [Note: Warfarin is used commercially as a pest control agent such as in rat poison. It was developed by the Wisconsin Alumni Research Foundation, hence the name.]

1	Genetic differences (genotypes) in the gene for catalytic subunit 1 of VKOR (VKORC1) influence patient response to warfarin. For example, a polymorphism (see p. 491) in the promoter region of the gene decreases gene expression, resulting in less VKOR being made, thereby necessitating a lower dose of warfarin to achieve a therapeutic level. Polymorphisms in the cytochrome P450 enzyme (CYP2C9) that metabolizes warfarin are also known. In 2010, the U.S. Food and Drug Administration added a genotype-based dose table to the warfarin label (package insert). The influence of genetics on an individual’s response to drugs is known as pharmacogenetics. D. Pathways Three distinct pathways are involved in formation of the fibrin meshwork: the extrinsic pathway, the intrinsic pathway, and the common pathway. Production of FXa by the extrinsic and intrinsic pathways initiates the common pathway (see Fig. 35.2).

1	1. Extrinsic: This pathway involves a protein, tissue factor (TF), that is not in the blood but becomes exposed when blood vessels get injured. TF (or, FIII) is a transmembrane glycoprotein abundant in vascular subendothelium. It is an extravascular accessory protein and not a protease. Any injury that exposes FIII to blood rapidly (within seconds) initiates the extrinsic (or, TF) pathway. Once exposed, TF binds a circulating Gla-containing protein, FVII, activating it through conformational change. [Note: FVII can also be activated proteolytically by thrombin (see 3. below).] Binding of FVII to TF requires the presence of Ca2+ and phospholipids. The TF–FVIIa complex then binds and activates FX by proteolysis (Fig. 35.8). Therefore, activation of FX by the extrinsic pathway occurs in association with the cell membrane. The extrinsic pathway is quickly inactivated by tissue factor pathway inhibitor (TFPI) that, in a FXa-dependent process, binds to the TF–FVIIa complex and prevents

1	association with the cell membrane. The extrinsic pathway is quickly inactivated by tissue factor pathway inhibitor (TFPI) that, in a FXa-dependent process, binds to the TF–FVIIa complex and prevents further production of FXa. [Note: TF and FVII are unique to the extrinsic pathway.] 2. Intrinsic: All of the protein factors involved in the intrinsic pathway are present in the blood and are, therefore, intravascular. The intrinsic pathway involves two phases: the contact phase and the FX-activation phase, each with known deficiencies.

1	a. Contact phase: This phase results in the activation of FXII (Hageman factor) by conformational change through binding to a negative surface. Deficiencies in FXII (or in the other proteins of this phase, high molecular weight kininogen and prekallikrein) do not result in bleeding, calling into question the importance of this phase in coagulation. However, the contact phase does play a role in inflammation. [Note: FXII can be activated proteolytically by thrombin (see 3. below)]. b.

1	b. Factor X–activation phase: The sequence of events leading to the activation of FX to FXa by the intrinsic pathway is initiated by FXIIa (Fig. 35.9). FXIIa activates FXI, and FXIa activates FIX, a Glacontaining serine protease. FIXa combines with FVIIIa (a bloodborne accessory protein), and the complex activates FX, a Gla-containing serine protease. [Note: The complex containing FIXa, FVIIIa, and FX forms on exposed negatively charged membrane regions, and FX gets activated to FXa. This complex is sometimes referred to as Xase. Binding of the complex to membrane phospholipids requires Ca2+.] c. Factor XII deficiency: A deficiency in FXII does not lead to a bleeding disorder. This is because FXI, the next protein in the cascade, can be activated proteolytically by thrombin (see 3. below). d.

1	d. Hemophilia: Hemophilia is a coagulopathy, a defect in the ability to clot. Hemophilia A, which accounts for 80% of all hemophilia, results from deficiency of FVIII, whereas deficiency of FIX results in hemophilia B. Each deficiency is characterized by decreased and delayed ability to clot and/or formation of abnormally friable (easily disrupted) clots. This can be manifested, for example, by bleeding into the joints (Fig. 35.10). The extent of the factor deficiency determines the severity of the disease. Current treatment for hemophilia is factor replacement therapy using FVIII or FIX obtained from pooled human blood or from recombinant DNA technology. However, antibodies to the factors can develop. Gene therapy is a goal. Because the genes for both proteins are on the X chromosome, hemophilia is an X-linked disorder. [Note: Deficiency of FXI results in a bleeding disorder that sometimes is referred to as hemophilia C.] with hemophilia.

1	The inactivation of the extrinsic pathway by TFPI results in dependence on the intrinsic pathway for continued production of FXa. This explains why individuals with hemophilia bleed even though they have an intact extrinsic pathway.

1	3. Common: FXa produced by both the intrinsic and the extrinsic paths initiates the common pathway, a sequence of reactions that results in the generation of fibrin (FIa), as shown in Figure 35.11. FXa associates with FVa (a bloodborne accessory protein) and, in the presence of Ca2+ and phospholipids, forms a membrane-bound complex referred to as prothrombinase. The complex cleaves prothrombin (FII) to thrombin (FIIa). [Note: FVa potentiates the proteolytic activity of FXa.] The binding of Ca2+ to the Gla residues in FII facilitates the binding of FII to the membrane and to the prothrombinase complex, with subsequent cleavage to FIIa. Cleavage excises the Gla-containing region, releasing FIIa from the membrane and, thereby, freeing it to activate fibrinogen (FI) in the blood. [Note: This is the only example of cleavage of a Gla protein that results in the release of a Gla-containing peptide. The peptide travels to the liver where it is thought to act as a signal for increased

1	This is the only example of cleavage of a Gla protein that results in the release of a Gla-containing peptide. The peptide travels to the liver where it is thought to act as a signal for increased production of clotting proteins.] Oral, direct inhibitors of FXa have been approved for clinical use as anticoagulants. In contrast to warfarin, they have a more rapid onset and shorter half-life and do not require routine monitoring.

1	A common point mutation (G20210A) in which an adenine (A) replaces a guanine (G) at nucleotide 20210 in the 3′ untranslated region of the gene for FII leads to increased levels of FII in the blood. This results in thrombophilia, a condition characterized by an increased tendency for blood to clot. a. Fibrinogen cleavage to fibrin: FI is a soluble glycoprotein made by the liver. It consists of dimers of three different polypeptide chains [(αβγ)2] held together at the N termini by disulfide bonds. The N termini of the α and β chains form “tufts” on the central of three globular domains (Fig. 35.12). The tufts are negatively charged and result in repulsion between FI molecules. Thrombin (FIIa) cleaves the charged tufts (releasing fibrinopeptides A and B), and FI becomes FIa. As a result of the loss of charge, the FIa monomers are able to noncovalently associate in a staggered array, and a soft (soluble) fibrin clot is formed. b.

1	b. Fibrin cross-linking: The associated FIa molecules get covalently cross-linked. This converts the soft clot to a hard (insoluble) clot. FXIIIa, a transglutaminase, covalently links the γ-carboxamide of a glutamine residue in one FIa molecule to the ε-amino of a lysine residue in another through formation of an isopeptide bond and release of ammonia (Fig. 35.13). [Note: FXIII is also activated by thrombin.] c.

1	Importance of thrombin: The activation of FX by the extrinsic pathway provides the “spark” of FXa that results in the initial activation of thrombin. FIIa then activates factors of the common (FV, FI, FXIII), intrinsic (FXI, FVIII), and extrinsic (FVII) pathways (Fig. 35.14). It also activates FXII of the contact phase. The extrinsic pathway, then, initiates clotting by the generation of FXa, and the intrinsic pathway amplifies and sustains clotting after the extrinsic pathway has been inhibited by TFPI. [Note: Hirudin, a peptide secreted from the salivary gland of medicinal leeches, is a potent direct thrombin inhibitor (DTI).

1	Injectable recombinant hirudin has been approved for clinical use. Dabigatran is an oral DTI.] Additional crosstalk between the pathways of clotting is achieved by the FVIIa–TF-mediated activation of the intrinsic pathway and the FXIIa-mediated activation of the extrinsic pathway. The complete picture of physiologic blood clotting via the formation of a hard fibrin clot is shown in Figure 35.15. The factors of the clotting cascade are shown organized by function in Figure 35.16. activated form would be denoted by an “a” after the numeral. [Note: Calcium is IV. There is no VI. I (fibrin) is neither a protease nor an accessory protein. XIII is a transglutaminase.] Gla = γ-carboxyglutamate.

1	IV. There is no VI. I (fibrin) is neither a protease nor an accessory protein. XIII is a transglutaminase.] Gla = γ-carboxyglutamate. Clinical laboratory tests are available to evaluate the extrinsic through common pathways (prothrombin time [PT] using thromboplastin and expressed as the international normalized ratio [INR]) and the intrinsic through common pathways (activated partial thromboplastin time [aPTT]). Thromboplastin is a combination of phospholipids + FIII. A derivative, partial thromboplastin contains just the phospholipid portion because FIII is not needed to activate the intrinsic pathway. III. LIMITING CLOTTING

1	III. LIMITING CLOTTING The ability to limit clotting to areas of damage (anticoagulation) and to remove clots once repair processes are underway (fibrinolysis) are exceedingly important aspects of hemostasis. These actions are performed by proteins that inactivate clotting factors either by binding to them and removing them from the blood or by degrading them and also by proteins that degrade the fibrin meshwork. A. Inactivating proteins Proteins synthesized by the liver and by the blood vessels themselves balance the need to form clots at sites of vessel injury with the need to limit their formation beyond the injured area.

1	1. Antithrombin: Antithrombin III (ATIII), also referred to simply as antithrombin (AT), is a hepatic protein that circulates in the blood. It inactivates free FIIa by binding to it and carrying it to the liver (Fig. 35.17). Thus, ATIII removes FIIa from the blood, preventing it from participating in coagulation. [Note: ATIII is a serine protease inhibitor, or “serpin.” A serpin contains a reactive loop to which a specific protease binds. Once bound, the protease cleaves a peptide bond in the serpin causing a conformational change that traps the enzyme in a covalent complex. α1-Antitrypsin (see p. 50) is also a serpin.] The affinity of ATIII for FIIA is greatly increased when ATIII is bound to heparin, an intracellular glycosaminogly-can (see p. 159) released in response to injury by mast cells associated with blood vessels. Heparin, an anticoagulant, is used therapeutically to limit clot formation. [Note: In contrast to the anticoagulant warfarin, which has a slow onset and a long

1	cells associated with blood vessels. Heparin, an anticoagulant, is used therapeutically to limit clot formation. [Note: In contrast to the anticoagulant warfarin, which has a slow onset and a long half-life and is administered orally, heparin has a rapid onset and a short half-life and requires intravenous administration. The two drugs are commonly used in an overlapping manner in the treatment (and prevention) of thrombosis.] ATIII also inactivates FXa and the other serine proteases of clotting, FIXa, FXIa, FXIIa, and the FVIIa–TF complex. [Note: ATIII binds to a specific pentasaccharide within the oligosaccharide form of heparin. Inhibition of FIIa requires the oligosaccharide form, whereas inhibition of FXa requires only the pentasaccharide form. Fondaparinux, a synthetic version of the pentasaccharide, is used clinically to inhibit FXa.] 2. Protein C–protein S complex: Protein C, a circulating Gla-containing protein made in the liver, is activated by FIIa complexed with

1	of the pentasaccharide, is used clinically to inhibit FXa.] 2. Protein C–protein S complex: Protein C, a circulating Gla-containing protein made in the liver, is activated by FIIa complexed with thrombomodulin. Thrombomodulin, an integral membrane glycoprotein of endothelial cells, binds FIIa, thereby decreasing FIIa’s affinity for fibrinogen and increasing its affinity for protein C. Protein C in complex with protein S, also a Gla-containing protein, forms the activated protein C (APC) complex that cleaves the accessory proteins FVa and FVIIIa, which are required for maximal activity of FXa (Fig. 35.18). Protein S helps anchor APC to the clot. Thrombomodulin, then, modulates the activity of thrombin, converting it from a protein of coagulation to a protein of anticoagulation, thereby limiting the extent of clotting. Factor V Leiden is a mutant form of FV (glutamine is substituted for arginine at position 506) that is resistant to APC. It is the most common inherited cause of

1	limiting the extent of clotting. Factor V Leiden is a mutant form of FV (glutamine is substituted for arginine at position 506) that is resistant to APC. It is the most common inherited cause of thrombophilia in the United States, with highest frequency in the Caucasian population. Heterozygotes have a 7-fold increase in the risk for venous thrombosis, and homozygotes have up to a 50-fold increase. [Note: Women with FV Leiden are at even greater risk of thrombosis during pregnancy or when taking estrogen.]

1	Thrombophilia (hypercoagulability) can result from deficiencies of proteins C, S, and ATIII; from the presence of FV Leiden and antiphospholipid antibodies; and from excess production of FII (G20210A mutation). [Note: A thrombus that forms in the deep veins of the leg (deep venous thrombosis, or DVT) can cause a pulmonary embolism (PE) if the clot (or a piece of it) breaks off, travels to the lungs, and blocks circulation.] B. Fibrinolysis

1	Clots are temporary patches that must be removed once wound repair has begun. The fibrin clot is cleaved by the protein plasmin to fibrin degradation products (Fig. 35.19). [Note: Measurement of D-dimer, a fibrin degradation product containing two cross-linked D domains released by the action of plasmin, can be used to assess the extent of clotting (see Fig. 35.12).] Plasmin is a serine protease that is generated from plasminogen by plasminogen activators. Plasminogen, secreted by the liver into the circulation, binds to FIa and is incorporated into clots as they form. Tissue plasminogen activator (TPA, t-PA), made by vascular endothelial cells and secreted in an inactive form in response to FIIa, becomes active when bound to FIa–plasminogen. Bound plasmin and TPAa are protected from their inhibitors, α2-antiplasmin and plasminogen activator inhibitors, respectively. Once the fibrin clot is dissolved, plasmin and TPAa become available to their inhibitors. Therapeutic fibrinolysis in

1	inhibitors, α2-antiplasmin and plasminogen activator inhibitors, respectively. Once the fibrin clot is dissolved, plasmin and TPAa become available to their inhibitors. Therapeutic fibrinolysis in patients with an MI or an ischemic stroke can be achieved by treatment with commercially available TPA made by recombinant DNA techniques. Mechanical clot removal (thrombectomy) is also possible. [Note: Urokinase is a plasminogen activator (u-PA) made in a variety of tissues and originally isolated from urine. Streptokinase (from bacteria) activates both free and fibrin-bound plasminogen.]

1	Plasminogen contains structural motifs known as “kringle domains” that mediate protein–protein interactions. Because lipoprotein (a) [Lp(a)] also contains kringle domains, it competes with plasminogen for binding to FIa. The potential to inhibit fibrinolysis may be the basis for the association of elevated Lp(a) with increased risk for cardiovascular disease (see p. 236). IV. PLATELET PLUG FORMATION Platelets (thrombocytes) are small, anucleate fragments of megakaryocytes that adhere to exposed collagen of damaged endothelium, get activated, and aggregate to form a platelet plug (Fig. 35.20; also see Fig. 35.1). Formation of the platelet plug is referred to as primary hemostasis because it is the first response to bleeding. In a normal adult, there are 150,000–450,000 platelets per µl of blood. They have a life span of up to 10 days, after which they are taken up by the liver and spleen and destroyed. Clinical laboratory tests to measure platelet number and activity are available.

1	A. Adhesion

1	Adhesion of platelets to exposed collagen at the site of vessel injury is mediated by the protein von Willebrand factor (VWF). VWF binds to collagen, and platelets bind to VWF via glycoprotein Ib (GPIb), a component of a membrane receptor complex (GPIb–V–IX) on the platelet surface (Fig. 35.21). Binding to VWF stops the forward movement of platelets. [Note: Deficiency in the receptor for VWF results in Bernard-Soulier syndrome, a disorder of decreased platelet adhesion.] VWF is a glycoprotein that is released from platelets. It also is made and secreted by endothelial cells. In addition to mediating the binding of platelets to collagen, VWF also binds to and stabilizes FVIII in the blood. Deficiency of VWF results in von Willebrand disease (VWD), the most common inherited coagulopathy. VWD results from decreased binding of platelets to collagen and a deficiency in FVIII (due to increased degradation). Platelets can also bind directly to collagen via the membrane receptor glycoprotein

1	results from decreased binding of platelets to collagen and a deficiency in FVIII (due to increased degradation). Platelets can also bind directly to collagen via the membrane receptor glycoprotein VI (GPVI). Once adhered, platelets get activated. [Note: Damage to the endothelium also exposes FIII, initiating the extrinsic pathway of blood clotting and activation of FX (see Fig. 35.8).]

1	B. Activation

1	Once adhered to areas of injury, platelets get activated. Platelet activation involves morphologic (shape) changes and degranulation, the process by which platelets secrete the contents of their α and δ (or, dense) storage granules. Activated platelets also expose PS on their surface. The externalization of PS is mediated by a Ca2+-activated enzyme known as scramblase that disrupts the membrane asymmetry created by flippases (see p. 205). Thrombin is the most potent platelet activator. FIIa binds to and activates protease-activated receptors, a type of G protein–coupled receptor (GPCR), on the surface of platelets (Fig. 35.22). FIIa is primarily associated with Gq proteins (see p. 205), resulting in activation of phospholipase C and a rise in diacylglycerol (DAG) and inositol trisphosphate (IP3). [Note: Thrombomodulin, through its binding of FIIa, decreases the availability of FIIa for platelet activation (see Fig. 35.18).] 1. Degranulation: DAG activates protein kinase C, a key event

1	(IP3). [Note: Thrombomodulin, through its binding of FIIa, decreases the availability of FIIa for platelet activation (see Fig. 35.18).] 1. Degranulation: DAG activates protein kinase C, a key event for degranulation. IP3 causes the release of Ca2+ (from dense granules). The Ca2+ activates phospholipase A2, which cleaves membrane phospholipids to release arachidonic acid, the substrate for the synthesis of thromboxane A2 (TXA2) in activated platelets by cyclooxygenase-1 (COX-1) (see p. 214). TXA2 causes vasoconstriction, augments degranulation, and binds to platelet GPCR, causing activation of additional platelets. Recall that aspirin irreversibly inhibits COX and, consequently, TXA2 synthesis and is referred to as an antiplatelet drug.

1	Degranulation also results in release of serotonin and adenosine diphosphate (ADP) from dense granules. Serotonin causes vasoconstriction. ADP binds to GPCR on the surface of platelets, activating additional platelets. [Note: Some antiplatelet drugs, such as clopidogrel, are ADP-receptor antagonists.] Platelet-derived growth factor (involved in wound healing), VWF, FV, FXIII, and FI are among other proteins released from α granules. [Note: Platelet-activating factor (PAF), an ether phospholipid (see p. 202) synthesized by a variety of cell types including endothelial cells and platelets, binds PAF receptors (GPCR) on the surface of platelets and activates them.] 2. Morphologic change: The change in shape of activated platelets from discoidal to spherical with pseudopod-like processes that facilitate platelet–platelet and platelet–surface interactions (Fig. 35.23) is initiated by the release of Ca2+ from dense granules. Ca2+ bound to calmodulin (see p. 133) mediates the activation of

1	facilitate platelet–platelet and platelet–surface interactions (Fig. 35.23) is initiated by the release of Ca2+ from dense granules. Ca2+ bound to calmodulin (see p. 133) mediates the activation of myosin light chain kinase that phosphorylates the myosin light chain, resulting in a major reorganization of the platelet cytoskeleton.

1	C. Aggregation Activation causes dramatic changes in platelets that lead to their aggregation. Structural changes in a surface receptor (GPIIb/IIIa) expose binding sites for fibrinogen. Bound FI molecules link activated platelets to one another (Fig. 35.24), with a single FI able to bind two platelets. FI is converted to FIa by FIIa and then covalently cross-linked by FXIIIa coming from both the blood and the platelets. [Note: The exposure of PS on the surface of activated platelets allows formation of the Xase complex (VIIIa, IXa, X, and Ca2+) with subsequent formation of FXa and generation of FIIa.] Fibrin formation (secondary hemostasis) strengthens the platelet plug. [Note: Rare defects in the platelet receptor for FI result in Glanzmann thrombasthenia (decreased platelet function), whereas autoantibodies to this receptor are a cause of immune thrombocytopenia (decreased platelet number).]

1	Unnecessary activation of platelets is prevented because 1) an intact vascular wall is separated from the blood by a monolayer of endothelial cells, preventing the contact of platelets with collagen; 2) endothelial cells synthesize prostaglandin I2 (PGI2, or prostacyclin) and nitric oxide, each of which causes vasodilation; and 3) endothelial cells have a cell surface ADPase that converts ADP to adenosine monophosphate. V. CHAPTER SUMMARY Blood clotting (coagulation) is designed to rapidly stop bleeding from a damaged blood vessel in order to maintain a constant blood volume (hemostasis). Coagulation is accomplished through formation of a clot (thrombus) consisting of a plug of platelets and a meshwork of the protein fibrin (Fig. 35.25).

1	The formation of the fibrin meshwork by the clotting cascade involves the extrinsic and intrinsic pathways (and their associated protein factors [F]) that converge at FXa to form the common pathway. Many of the protein factors are serine proteases with trypsin-like specificity. Calcium binds the negatively charged γ-carboxyglutamate (Gla) residues present in certain of the clotting proteases (FII, FVII, FIX, and FX), facilitating the binding of these proteins to exposed negatively charged phosphatidylserine at the site of injury and on the surface of platelets. γ-Glutamyl carboxylase and its coenzyme, the hydroquinone form of vitamin K, are required for formation of Gla residues. In the reaction, vitamin K gets oxidized to the nonfunctional epoxide form. Warfarin, a synthetic analog of vitamin K used clinically to reduce clotting, inhibits the enzyme vitamin K epoxide reductase that regenerates the functional reduced form. The extrinsic pathway is initiated by exposure of FIII (tissue

1	K used clinically to reduce clotting, inhibits the enzyme vitamin K epoxide reductase that regenerates the functional reduced form. The extrinsic pathway is initiated by exposure of FIII (tissue factor [TF]), an accessory protein, in vascular subendothelium. Exposed TF binds a circulating Glacontaining protein, FVII, activating it through conformational change. The TF–FVIIa complex then binds and activates FX by proteolysis. FXa from the extrinsic pathway allows thrombin production by the common pathway. Thrombin then activates components of the intrinsic pathway. The extrinsic pathway is rapidly inhibited by tissue factor pathway inhibitor. The intrinsic pathway is initiated by FXIIa. FXIIa activates FXI, and FXIa activates FIX. FIXa combines with FVIIIa (an accessory protein), and the complex activates FX. FVIII deficiency results in hemophilia A, whereas FIX deficiency results in the less common hemophilia B. FXa associates with FVa (an accessory protein), forming prothrombinase

1	complex activates FX. FVIII deficiency results in hemophilia A, whereas FIX deficiency results in the less common hemophilia B. FXa associates with FVa (an accessory protein), forming prothrombinase that cleaves prothrombin (FII) to thrombin (FIIa). Thrombin then cleaves fibrinogen to fibrin (FIa). Fibrin monomers associate, forming a soluble (soft) fibrin clot. The fibrin molecules get cross-linked by FXIIIa, a transglutaminase, forming an insoluble (hard) fibrin clot. Proteins synthesized by the liver and by blood vessels themselves balance coagulation with anticoagulation. Antithrombin III, a serine protease inhibitor, or serpin, binds to and removes thrombin from the blood. Its affinity for thrombin is increased by heparin, which is used therapeutically to limit clot formation. Protein C, a Gla-containing protein, is activated by the thrombin–thrombomodulin complex. Thrombomodulin decreases thrombin’s affinity for fibrinogen, converting it from a protein of coagulation to a

1	Protein C, a Gla-containing protein, is activated by the thrombin–thrombomodulin complex. Thrombomodulin decreases thrombin’s affinity for fibrinogen, converting it from a protein of coagulation to a protein of anticoagulation. Protein C in complex with protein S (a Gla-containing protein) forms the activated protein C (APC) complex that cleaves the accessory proteins FVa and FVIIIa. FV Leiden is resistant to APC. It is the most common inherited thrombophilic condition in the United States. The fibrin clot is cleaved (fibrinolysis) by the protein plasmin, a serine protease that is generated from plasminogen by plasminogen activators such as tissue plasminogen activator (TPA, t-PA).

1	Recombinant TPA is used clinically.

1	Wound to a tissue damages blood vessels and exposes collagen. Platelets (thrombocytes) adhere to the exposed collagen, get activated, and aggregate to form a platelet plug. Adhesion is mediated by von Willebrand factor (VWF). VWF binds collagen, and platelets bind VWF via glycoprotein Ib (GPIb) within a receptor complex on the platelet surface. Deficiency of VWF results in von Willebrand disease, the most common inherited coagulopathy. Once adhered, platelets get activated. Platelet activation involves changes in shape (discoidal to spherical with pseudopodia) and degranulation, the process by which platelets release the contents of their storage granules. Thrombin is the most potent activator of platelets. Thrombin binds to protease-activated G protein–coupled receptors on the surface of platelets. Activated platelets release substances that cause vasoconstriction (serotonin and thromboxane A2 [TXA2]), recruit and activate other platelets (adenosine diphosphate and TXA2), and support

1	platelets. Activated platelets release substances that cause vasoconstriction (serotonin and thromboxane A2 [TXA2]), recruit and activate other platelets (adenosine diphosphate and TXA2), and support the formation of a fibrin clot (FV, FXIII, and fibrinogen). Activation causes changes in platelets that lead to their aggregation. Structural changes in a surface receptor (GPIIb/IIIa) expose binding sites for fibrinogen. Fibrinogen molecules link activated platelets to one another. The fibrinogen is activated to fibrin by thrombin and then cross-linked by FXIIIa coming both from the blood and from platelets. The initial loose plug of platelets (primary hemostasis) is strengthened by the fibrin meshwork (secondary hemostasis). Disorders of platelets and coagulation proteins can result in deviations in the ability to clot. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are clinical laboratory tests used to evaluate the clotting cascade.

1	Choose the ONE best answer. For Questions 31.1–31.5, match the most appropriate protein factors (F) of clotting to the description. 5.1. This factor activates components of the intrinsic, extrinsic, and common pathways. 5.2. This factor converts the soluble clot to an insoluble clot. 5.3. This factor initiates the common pathway. 5.4. This factor is an accessory protein that potentiates the activity of factor Xa. 5.5. This factor is a γ-carboxyglutamate–containing serine protease of the extrinsic pathway.

1	5.4. This factor is an accessory protein that potentiates the activity of factor Xa. 5.5. This factor is a γ-carboxyglutamate–containing serine protease of the extrinsic pathway. Correct answers = B, J, H, D, E. Thrombin (FII) is formed in the common pathway and activates components in each of the three pathways of the clotting cascade. FXIII, a transglutaminase, covalently cross-links associated fibrin monomers, thereby converting a soluble clot to an insoluble one. The generation of FXa by the intrinsic and extrinsic pathways initiates the common pathway. FV increases the activity of FXa. It is one of three accessory (nonprotease) proteins. The others are FIII (tissue factor) and FVIII (complexes with FIX to activate FX). FVII is a γ-carboxyglutamate–containing serine protease that complexes with FIII in the extrinsic pathway. 5.6. In which patient would prothrombin time be unaffected and activated partial thromboplastin time be prolonged? A. A patient on aspirin therapy

1	5.6. In which patient would prothrombin time be unaffected and activated partial thromboplastin time be prolonged? A. A patient on aspirin therapy B. A patient with end-stage liver disease C. A patient with hemophilia D. A patient with thrombocytopenia Correct answer = C. Prothrombin time (PT) measures the activity of the extrinsic through the common pathways, and activated partial thromboplastin time (aPTT) measures the activity of the intrinsic through the common pathways. Patients with hemophilia are deficient in either FVIII (hemophilia

1	A) or FIX (hemophilia B), components of the common pathway. They have an intact extrinsic pathway. Therefore, the PT is unaffected, and the aPTT is prolonged. Patients on aspirin therapy and those with thrombocytopenia have alterations in platelet function and number, respectively, and not in the proteins of the clotting cascade. Therefore, both the PT and the aPTT are unaffected. Patients with end-stage liver disease have decreased ability to synthesize clotting proteins. They show prolonged PT and aPTT. 5.7. Which one of the following can be ruled out in a patient with thrombophilia? A. A deficiency of antithrombin III B. A deficiency of FIX C. A deficiency of protein C D. An excess of prothrombin E. Expression of FV Leiden Correct answer = B. Symptomatic deficiencies in clotting factors will present with a decreased ability to clot (coagulopathy). Thrombophilia, however, is characterized by an increased tendency to clot. Choices A, C, D, and E result in thrombophilia.

1	5.8. Current guidelines for the treatment of patients with acute ischemic stroke (a stroke caused by a blood clot obstructing a vessel that supplies blood to the brain) include the recommendation that tissue plasminogen activator (TPA) be used shortly after the onset of symptoms. The basis of the recommendation for TPA is that it activates: A. antithrombin III. B. the activated protein C complex. C. the receptor for von Willebrand factor. D. the serine protease that degrades fibrin. E. thrombomodulin.

1	A. antithrombin III. B. the activated protein C complex. C. the receptor for von Willebrand factor. D. the serine protease that degrades fibrin. E. thrombomodulin. Correct answer = D. TPA converts plasminogen to plasmin. Plasmin (a serine protease) degrades the fibrin meshwork, removing the obstruction to blood flow. Antithrombin III in association with heparin binds thrombin and carries it to the liver, decreasing thrombin's availability in the blood. The activated protein C complex degrades the accessory proteins FV and FVIII. The platelet receptor for von Willebrand factor is not affected by TPA. Thrombomodulin binds thrombin and converts it from a protein of coagulation to one of anticoagulation by decreasing its activation of fibrinogen and increasing its activation of protein C.

1	5.9. The adhesion, activation, and aggregation of platelets provide the initial plug at the site of vessel injury. Which of the following statements concerning the formation of this platelet plug is correct? A. Activated platelets undergo a shape change that decreases their surface area. B. Formation of a platelet plug is prevented in intact vessels by the production of thromboxane A2 by endothelial cells. C. The activation phase requires production of cyclic adenosine monophosphate. D. The adhesion phase is mediated by the binding of platelets to von Willebrand factor via glycoprotein Ib. E. Thrombin activates platelets by binding to a protease-activated G protein–coupled receptor and causing activation of protein kinase A.

1	E. Thrombin activates platelets by binding to a protease-activated G protein–coupled receptor and causing activation of protein kinase A. Correct answer = D. The adhesion phase of platelet plug formation is initiated by the binding of von Willebrand factor to a receptor (glycoprotein Ib) on the surface of platelets. Shape change from discoidal to spherical with pseudopodia increases the surface area of platelets. Thromboxane A2 is made by platelets. It causes platelet activation and vasoconstriction. Adenosine diphosphate is released from activated platelets, and it itself activates platelets. Thrombin works primarily through receptors coupled to Gq proteins causing activation of phospholipase C. 5.11. Nephrotic syndrome is a kidney disease characterized by protein loss in the urine (≥3 g/day) that is accompanied by edema. The loss of protein results in a hypercoagulable state. Excretion of which of the following proteins would explain the thrombophilia seen in the syndrome?

1	A. Antithrombin III B. FV C. FVIII D. Prothrombin Correct answer = A. Antithrombin III (ATIII) inhibits the action of thrombin (FIIa), a Gla-containing protein of clotting that activates the extrinsic, intrinsic, and common pathways. Excretion of ATIII in nephrotic syndrome allows the actions of FIIa to continue, resulting in a hypercoagulable state. The other choices are proteins required for clotting. Their excretion in the urine would decrease clotting. 5.12. Blocking the action of which of the following proteins would be a rational therapy for hemophilia B? A. FIX B. FXIII C. Protein C D. Tissue factor pathway inhibitor

1	5.12. Blocking the action of which of the following proteins would be a rational therapy for hemophilia B? A. FIX B. FXIII C. Protein C D. Tissue factor pathway inhibitor Correct answer = D. Hemophilia B is a coagulopathy caused by decreased thrombin production by the common pathway as a result of a deficiency in FIX of the intrinsic pathway. Because the extrinsic pathway also can result in thrombin production, blocking the inhibitor of this pathway (tissue factor pathway inhibitor) should, in principle, increase thrombin production. 5.13. The parents of a newborn baby girl refuse to allow the baby to be given the injection of vitamin K that is recommended shortly after birth to prevent vitamin K deficiency bleeding, which is caused by the low levels of the vitamin in newborns. The activity of which one of the following protein factors involved in clotting would be decreased with vitamin K deficiency? A. FV B. FVII C. FXI D. FXII E. FXIII

1	A. FV B. FVII C. FXI D. FXII E. FXIII Correct answer = B. FVII is a γ-carboxyglutamate (Gla)-containing protein of clotting. The creation of Gla residues by γ-glutamyl carboxylase requires vitamin K as a coenzyme. FII, FIX, and FX, as well as proteins C and S that limit clotting, also contain Gla residues. The other choices do not contain Gla residues. 5.14. Thrombin, produced in the common pathway of clotting, has both procoagulant and anticoagulant activities. Which one of the following is an anticoagulant activity of thrombin? A. Activating FXIII B. Binding to thrombomodulin C. Increasing nitric oxide production D. Inhibiting FV and FVIII E. Inhibiting platelet activation F. Inhibiting tissue factor pathway inhibitor

1	A. Activating FXIII B. Binding to thrombomodulin C. Increasing nitric oxide production D. Inhibiting FV and FVIII E. Inhibiting platelet activation F. Inhibiting tissue factor pathway inhibitor Correct answer = B. Thrombin bound to thrombomodulin activates protein C that degrades the accessory proteins FV and FVIII, thereby inhibiting clotting. Activation of FXIII by thrombin strengthens the fibrin clot. Nitric oxide, a vasodilator made by endothelial cells, decreases clot formation. It is not affected by thrombin. Thrombin is a powerful activator of platelets. Inhibition of tissue factor pathway inhibitor would increase clotting. 5.15. A student is reviewing the use of prothrombin time (PT) and activated partial thromboplastin time (aPTT) in evaluating a suspected deficiency of a clotting protein. Which one of the following results would be correct for a deficiency in FXIII? A. Both prothrombin time and activated partial thromboplastin time are decreased.

1	A. Both prothrombin time and activated partial thromboplastin time are decreased. B. Both prothrombin time and activated partial thromboplastin time are increased. C. Both prothrombin time and activated partial thromboplastin time are unchanged. D. Only prothrombin time is affected. E. Only activated partial thromboplastin time is affected. Correct answer = C. FXIII is a transglutaminase that cross-links fibrin molecules in a soft clot to form a hard clot. Its deficiency does not affect the PT or aPTT tests. [Note: It is evaluated by a clot solubility test.] 5.16. Why do individuals with Scott syndrome, a rare disorder caused by mutations to scramblase in platelets, have a tendency to bleed?

1	Scramblase moves phosphatidylserine (PS) from the cytosolic leaflet to the extracellular leaflet in the plasma membrane of platelets. This disrupts the asymmetrical localization of membrane phospholipids created by ATP-dependent flippases (move PS from extracellular to cytosolic leaflet) and floppases (move phosphatidylcholine [PC] in the opposite direction). Having PS on the outer face of platelet membranes provides a site for protein clotting factors to interact and activate thrombin. If scramblase is inactive, PS is not available to these factors, and bleeding results.

1	5.10. Several days after having had their home treated for an infestation of rats, the parents of a 3-year-old girl become concerned that she might be ingesting the poison-containing pellets. After calling the Poison Hotline, they take her to the emergency department. Blood studies reveal a prolonged prothrombin and activated partial thromboplastin time and a decreased concentration of FII, FVII, FIX, and FX. Why might administration of vitamin K be a rational approach to the treatment of this patient? Many rodent poisons are super warfarins, drugs that have a long half-life in the body. Warfarin inhibits γ-carboxylation (production of γ-carboxyglutamate, or Gla, residues), and the clotting proteins reported as decreased are the Glacontaining proteases of the clotting cascade. [Note: Proteins C and S of anticlotting are also Gla-containing proteins.] Because warfarin functions as a vitamin K antagonist, administration of vitamin K is a rational approach to treatment.

1	I. I. Integrative Cases Metabolic pathways, initially presented in isolation, are, in fact, linked to form an interconnected network. The following four integrative case studies illustrate how a perturbation in one process can result in perturbations in other processes of the network. Case 1: Chest Pain Patient Presentation: BJ, a 35-year-old man with severe substernal chest pain of ~2 hours’ duration, is brought by ambulance to his local hospital at 5 AM. The pain is accompanied by dyspnea (shortness of breath), diaphoresis (sweating), and nausea.

1	Focused History: BJ reports episodes of exertional chest pain in the last few months, but they were less severe and of short duration. He smokes (2–3 packs per day), drinks alcohol only rarely, eats a “typical” diet, and walks with his wife most weekends. His blood pressure has been normal. Family history reveals that his father and paternal aunt died of heart disease at age 45 and 39 years, respectively. His mother and younger (age 31 years) brother are said to be in good health. Physical Examination (Pertinent Findings): BJ is pale and clammy and is in distress due to chest pain. Blood pressure and respiratory rate are elevated. Lipid deposits are noted on the periphery of his corneas (corneal arcus; see left image) and under the skin on and around his eyelids (xanthelasmas; see right image). No deposits on his tendons (xanthomas) are detected.

1	Pertinent Test Results: BJ’s electrocardiogram is consistent with a myocardial infarction (MI). Angiography reveals areas of severe stenosis (narrowing) of several coronary arteries. Initial results from the clinical laboratory include the following: H = High; L = Low. [Note: BJ had not eaten for ~8 hours prior to the blood draw.] Diagnosis: MI, the irreversible necrosis (death) of heart muscle secondary to ischemia (decreased blood supply), is caused by the occlusion (blockage) of a blood vessel most commonly by a blood clot (thrombus). BJ subsequently is determined to have heterozygous familial hypercholesterolemia (FH), also known as type IIa hyperlipidemia. Immediate Treatment: BJ is given O2, a vasodilator, pain medication, and drugs to dissolve blood clots (thrombolytics) and reduce clotting (antithrombotics).

1	Immediate Treatment: BJ is given O2, a vasodilator, pain medication, and drugs to dissolve blood clots (thrombolytics) and reduce clotting (antithrombotics). Long-Term Treatment: Lipid-lowering drugs (for example, high-potency statins, bile acid [BA] sequestrants, and niacin); daily aspirin; β-blockers; and counseling on nutrition, exercise, and smoking cessation would be part of the long-term treatment plan. Prognosis: Patients with heterozygous FH have ~50% of the normal number of functional LDL receptors and a hypercholesterolemia (two to three times normal) that puts them at high risk (>50% risk) for premature coronary heart disease (CHD). However, <5% of patients with hypercholesterolemia have FH.

1	Nutrition Nugget: Dietary recommendations for individuals with heterozygous FH include limiting saturated fats to <7% of total calories and cholesterol to <200 mg/day, substituting unsaturated fats for saturated fats, and adding soluble fiber (10–20 g/day) and plant sterols (2 g/day) for their hypocholesterolemic effects. Fiber increases BA excretion. This results in increased hepatic uptake of cholesterol-rich LDL to supply the substrate for BA synthesis. Plant sterols decrease cholesterol absorption in the intestine.

1	Genetics Gem: FH is caused by hundreds of different mutations in the gene for the LDL receptor (on chromosome 19) that affect receptor amount and/or function. FH is an autosomal-dominant disease in which homozygotes are more seriously affected than heterozygotes. Heterozygous FH has an incidence of ~1:500 in the general population. It is associated with increased risk of cardiovascular disease. Genetic screening of the first-degree relatives of BJ would identify affected individuals for treatment. Review Questions: Choose the ONE best answer. RQ1. Triacylglycerols are glycerol-based lipids. Which of the following is also a glycerol-based lipid? A. Ganglioside GM2 B. Phosphatidylcholine C. Prostaglandin PGI2 D. Sphingomyelin E. Vitamin D RQ2. Statins are of benefit to patients with hypercholesterolemia because they: A. decrease a rate-limiting and regulated step of de novo cholesterol biosynthesis by inhibiting hydroxymethylglutaryl coenzyme A (HMG CoA) reductase.

1	A. decrease a rate-limiting and regulated step of de novo cholesterol biosynthesis by inhibiting hydroxymethylglutaryl coenzyme A (HMG CoA) reductase. B. decrease expression of the gene for the LDL receptor by preventing the movement of the sterol regulatory element–binding protein-2 (SREBP 2) in complex with SREBP cleavage–activating protein (SCAP) from the membrane of the endoplasmic reticulum to the membrane of the Golgi. C. increase the oxidation of cholesterol to CO2 + H2O. D. interfere with the absorption of bile salts in the enterohepatic circulation, thereby causing the liver to take up cholesterol from the blood for use in BA synthesis. E. reduce cholesterol by increasing steroid hormone and vitamin D synthesis. RQ3. Statins are competitive inhibitors of HMG CoA reductase. Which of the following statements about competitive inhibitors is correct? A. Competitive inhibitors are examples of irreversible inhibitors.

1	A. Competitive inhibitors are examples of irreversible inhibitors. B. Competitive inhibitors increase both the apparent Michaelis constant (Km) and the apparent maximal velocity (Vmax). C. Competitive inhibitors increase the apparent Km and have no effect on the Vmax. D. Competitive inhibitors decrease both the apparent Km and the apparent Vmax. E. Competitive inhibitors have no effect on the Km and decrease the apparent Vmax. RQ4. In an MI, a blood clot forms as a result of injury to a blood vessel that leads to production of a platelet plug and a fibrin meshwork. The clot occludes the blood vessel, preventing blood flow and, therefore, delivery of O2. Destruction of the clot (thrombolysis) restores blood flow. Which one of the following is an example of a thrombolytic agent? A. Activated protein C complex B. Antithrombin III C. Aspirin D. Factor XIII E. Heparin F. Tissue plasminogen activator G. Vitamin K H. Warfarin

1	A. Activated protein C complex B. Antithrombin III C. Aspirin D. Factor XIII E. Heparin F. Tissue plasminogen activator G. Vitamin K H. Warfarin RQ5. Decreased tissue perfusion results in hypoxia (decreased O2 availability). Relative to normoxia, in hypoxia the: A. electron transport chain will be upregulated to provide protons for ATP synthesis. B. ratio of the oxidized form of nicotinamide adenine dinucleotide (NAD+) to the reduced form (NADH) will increase. C. pyruvate dehydrogenase complex will be active. D. process of substrate-level phosphorylation will be increased in the cytosol. E. tricarboxylic acid cycle will be upregulated to provide the reducing equivalents needed for oxidative phosphorylation to occur.

1	E. tricarboxylic acid cycle will be upregulated to provide the reducing equivalents needed for oxidative phosphorylation to occur. RQ6. Genetic screening of BJ’s first-degree relatives would be accomplished by mutation analysis via polymerase chain reaction–based amplification followed by automated sequencing of the promoter region and the 18 exons of the LDL receptor gene. This process would involve the: A. generation and use of complementary DNA (cDNA). B. initiation of DNA synthesis with dideoxynucleotides. C. isolation of genomic DNA from germ cells. D. use of fluorescently labeled nucleotides. TQ1. Relative to an individual with familial defective LDL receptors, what would be the expected phenotype in an individual with familial defective apolipoprotein B-100? With apolipoprotein E-2, the isoform that only poorly binds its receptor? TQ2. Why was aspirin prescribed? Hint: What pathway of lipid metabolism is affected by aspirin?

1	TQ2. Why was aspirin prescribed? Hint: What pathway of lipid metabolism is affected by aspirin? TQ3. Heart muscle normally uses aerobic metabolism to meet its energy needs. However, in hypoxia, anaerobic glycolysis is increased. What allosteric activator of glycolysis is responsible for this effect? With hypoxia, what will be the end product of glycolysis? TQ4. One of the reasons for encouraging smoking cessation and exercise for BJ is that these changes raise the level of HDL, and elevated HDL reduces the risk for CHD. How does a rise in HDL reduce the risk for CHD? Case 2: Severe Fasting Hypoglycemia Patient Presentation: JS is a 4-month-old boy whose mother is concerned about the “twitching” movements he makes just before feedings. She tells the pediatrician that the movements started ~1 week ago, are most apparent in the morning, and disappear shortly after eating.

1	Focused History: JS is the product of a normal pregnancy and delivery. He appeared normal at birth. On his growth charts, he has been at the 30th percentile for both weight and length since birth. His immunizations are up to date. JS last ate a few hours ago. Physical Examination (Pertinent Findings): JS appears sleepy and feels clammy to the touch. His respiratory rate is elevated. His temperature is normal. JS has a protuberant, firm abdomen that appears to be nontender. His liver is palpable 4 cm below the right costal margin and is smooth. His kidneys are enlarged and symmetrical. Pertinent Test Results: H = High; L = Low.

1	Pertinent Test Results: H = High; L = Low. JS is sent to the regional children’s hospital for further evaluation. Ultrasound studies confirm hepatomegaly and renomegaly and show no evidence of tumors. A liver biopsy is performed. The hepatocytes are distended. Staining reveals large amounts of lipid (primarily triacylglycerol) and carbohydrate. Liver glycogen is elevated in amount and normal in structure. Enzyme assay using liver homogenate treated with detergent reveals <10% of the normal activity of glucose 6-phosphatase, an enzyme of the endoplasmic reticular (ER) membrane in the liver and the kidneys. Diagnosis: JS has glucose 6-phosphatase deficiency (glycogen storage disease [GSD] type Ia, von Gierke disease).

1	Diagnosis: JS has glucose 6-phosphatase deficiency (glycogen storage disease [GSD] type Ia, von Gierke disease). Treatment (Immediate): JS was given glucose intravenously, and his blood glucose level rose into the normal range. However, as the day progressed, it fell to well below normal. Administration of glucagon had no effect on blood glucose levels but increased blood lactate. JS’s blood glucose levels were able to be maintained by constant infusion of glucose. Prognosis: Individuals with glucose 6-phosphatase deficiency develop hepatic adenomas starting in the second decade of life and are at increased risk for hepatic carcinoma. Kidney glomerular function is impaired and can result in kidney failure. Patients are at increased risk for developing gout, but this rarely occurs before puberty.

1	Nutrition Nugget: Long-term medical nutrition therapy for JS is designed to maintain his blood glucose levels in the normal range. Frequent (every 2–3 hours) daytime feedings rich in carbohydrate (provided by uncooked cornstarch that is slowly hydrolyzed) and nighttime nasogastric infusion (pump assisted) of glucose are advised. Avoidance of fructose and galactose is recommended because they are metabolized to glycolytic intermediates and lactate, which can exacerbate the metabolic problems. Calcium and vitamin D supplements are prescribed.

1	Genetics Gem: GSD Ia is an autosomal-recessive disorder caused by >100 known mutations to the gene for glucose 6-phosphatase located on chromosome 17. It has an incidence of 1:100,000 and accounts for ~25% of all cases of GSD in the United States. It is one of the few genetic causes of hypoglycemia in newborns. GSD Ia is not routinely screened for in newborns. [Note: Deficiency of the translocase that moves glucose 6phosphate into the ER is the cause of GSD Ib. Hypoglycemia and neutropenia are seen.] Review Questions: Choose the ONE best answer. RQ1. JS is hypoglycemic because: A. free (nonphosphorylated) glucose cannot be produced from either glycogenolysis or gluconeogenesis as a result of the deficiency in glucose 6-phosphatase. B. glycogen phosphorylase is dephosphorylated and inactive, and glycogen cannot be degraded. C. hormone-sensitive lipase is dephosphorylated and inactive, and fatty acid substrates for gluconeogenesis cannot be generated.

1	C. hormone-sensitive lipase is dephosphorylated and inactive, and fatty acid substrates for gluconeogenesis cannot be generated. D. the decrease in the insulin/glucagon ratio upregulates glucose transporters in the liver and kidneys, resulting in increased uptake of blood glucose. RQ2. JS was prescribed calcium supplements because chronic acidosis can cause bone demineralization, resulting in osteopenia. Vitamin D (1,25-diOH-D3) was also prescribed because vitamin D: A. binds Gq protein–coupled membrane receptors and causes a rise in inositol trisphosphate with release of calcium from intracellular stores. B. cannot be synthesized by humans and, therefore, must be supplied in the diet. C. is a fat-soluble vitamin that increases intestinal absorption of calcium. D. is the coenzyme-prosthetic group for calbindin, a calcium transporter in the intestine.

1	C. is a fat-soluble vitamin that increases intestinal absorption of calcium. D. is the coenzyme-prosthetic group for calbindin, a calcium transporter in the intestine. RQ3. The hepatomegaly and renomegaly seen in JS are primarily the result of an increase in the amount of glycogen stored in these organs. What is the basis for glycogen accumulation in these organs? A. Glycolysis is downregulated, which pushes glucose to glycogenesis. B. Increased oxidation of fatty acids spares glucose for glycogenesis. C. Glucose 6-phosphate is an allosteric activator of glycogen synthase b. D. The rise in the insulin/glucagon ratio favors glycogenesis. RQ4. Glucose 6-phosphatase is an integral protein of the ER membrane. Which of the following statements about such proteins is correct? A. If glycosylated, the carbohydrate is on the portion of the protein that extends into the cytosol. B. They are synthesized on ribosomes that are free in the cytosol.

1	A. If glycosylated, the carbohydrate is on the portion of the protein that extends into the cytosol. B. They are synthesized on ribosomes that are free in the cytosol. C. The membrane-spanning domain consists of hydrophilic amino acids. D. The initial targeting signal is an amino terminal hydrophobic signal sequence. TQ1. What is the likely reason for JS’s twitching movements? TQ2. Why was the liver homogenate treated with detergent? Hint: Think about where the enzyme is located. TQ3. Why is JS’s blood glucose level unaffected by glucagon? Hint: What is the role of glucagon in normal individuals who experience a drop in blood glucose? TQ4. Why are urate and lactate elevated in a disorder of glycogen metabolism? Hint: It is the result of a decrease in inorganic phosphate (Pi), but why is Pi decreased? TQ5. A.Why are triacylglycerols and cholesterol elevated? Hint: Glucose is the primary carbon source for their synthesis. B.Why are ketone bodies not elevated?

1	TQ5. A.Why are triacylglycerols and cholesterol elevated? Hint: Glucose is the primary carbon source for their synthesis. B.Why are ketone bodies not elevated? Case 3: Hyperglycemia and Hyperketonemia Patient Presentation: MW, a 40-year-old woman, was brought to the hospital in a disoriented, confused state by her husband. Focused History: As noted on her medical alert bracelet, MW has had type 1 diabetes (T1D) for the last 24 years. Her husband reports that this is her first medical emergency in 2 years. Physical Examination (Pertinent Findings): MW displayed signs of dehydration (such as dry mucous membranes and skin, poor skin turgor, and low blood pressure) and acidosis (such as deep, rapid breathing [Kussmaul respiration]). Her breath had a faintly fruity odor. Her temperature was normal.

1	Pertinent Test Results: Rapid, bedside tests were strongly positive for glucose and acetoacetate and negative for protein. Results on blood tests performed by the clinical laboratory are shown below: H=High;L=Low. Microscopic examination of her urine revealed a urinary tract infection (UTI). Diagnosis: MW is in diabetic ketoacidosis (DKA) that was precipitated by a UTI. [Note: Diabetes increases the risk for infections such as UTI.] Immediate Treatment: MW was rehydrated with normal saline given intravenously (IV). She also was given insulin IV. Blood glucose, ketone bodies, and electrolytes were measured periodically. Antibiotic treatment of her UTI was started. Long-Term Treatment: Diabetes increases the risk for macrovascular complications (such as coronary artery disease and stroke) and microvascular complications (such as retinopathy, nephropathy, and neuropathy). Ongoing monitoring for these complications will be continued.

1	Prognosis: Diabetes is the seventh leading cause of death by disease in the United States. Individuals with diabetes have a reduced life expectancy relative to those without diabetes. Nutrition Nugget: Monitoring total intake of carbohydrates is primary in blood glucose control. Carbohydrates should come from whole grains, vegetables, legumes, and fruits. Low-fat dairy products and nuts and fish rich in ω-3 fatty acids are encouraged. Intake of saturated and trans fats should be minimized. Genetics Gem: Autoimmune destruction of pancreatic β cells is characteristic of T1D. Of the genetic loci that confer risk for T1D, the human-leukocyte antigen (HLA) region on chromosome 6 has the strongest association. The majority of genes in the HLA region are involved in the immune response. Review Questions: Choose the ONE best answer. RQ1. Which of the following statements concerning T1D is correct?

1	Review Questions: Choose the ONE best answer. RQ1. Which of the following statements concerning T1D is correct? A. Diagnosis can be made by measuring the level of glucose or glycated hemoglobin (HbA1c) in the blood. B. During periods of physiologic stress, the urine of an individual with T1D would likely test negative for reducing sugars. C. T1D is associated with obesity and a sedentary lifestyle. D. The characteristic metabolic abnormalities seen in T1D result from insensitivity to both insulin and glucagon. E. Treatment with exogenous insulin allows normalization of blood glucose (euglycemia). RQ2. DKA occurs when the rate of ketone body production is greater than the rate of utilization. Which of the following statements concerning ketone body metabolism is correct? Ketone bodies: A. are made in mitochondria from acetyl coenzyme A (CoA) primarily produced by the oxidation of glucose. B. are utilized by many tissues, particularly the liver, after conversion to acetyl CoA.

1	A. are made in mitochondria from acetyl coenzyme A (CoA) primarily produced by the oxidation of glucose. B. are utilized by many tissues, particularly the liver, after conversion to acetyl CoA. C. include acetoacetate, which can impart a fruity odor to the breath. D. require albumin for transport through the blood. E. utilized in energy metabolism are organic acids that can add to the proton load of the body. RQ3. Adipose lipolysis followed by β-oxidation of the fatty acid (FA) products is required for the generation of ketone bodies. Which of the following statements concerning the generation and use of FA is correct? A. Mitochondrial β-oxidation of FA is inhibited by malonyl CoA. B. Production of FA from adipose lipolysis is upregulated by insulin. C. The acetyl CoA product of FA β-oxidation favors the use of pyruvate for gluconeogenesis by activating the pyruvate dehydrogenase complex. D. The β-oxidation of FA utilizes reducing equivalents generated by gluconeogenesis.

1	D. The β-oxidation of FA utilizes reducing equivalents generated by gluconeogenesis. E. The FA produced by lipolysis are taken up by the brain and oxidized for energy. TQ1. At admission, MW was hypoinsulinemic, and she was given insulin. Why did MW’s hypoinsulinemia result in hyperglycemia? Hint: What is the role of insulin in glucose metabolism? TQ2. Why is there glucose in MW’s urine (glucosuria)? How is the glucosuria related to her dehydrated state? TQ3. Why is the majority of the acetyl CoA from FA β-oxidation being used for ketogenesis rather than being oxidized in the tricarboxylic acid cycle? TQ4. Was MW in positive or negative nitrogen balance when she was brought to the hospital? TQ5. What response to the DKA is apparent in MW? What response is likely occurring in the kidney? Hint: In addition to conversion to urea, how is toxic ammonia removed from the body?

1	TQ5. What response to the DKA is apparent in MW? What response is likely occurring in the kidney? Hint: In addition to conversion to urea, how is toxic ammonia removed from the body? TQ6. What would be true about the levels of ketone bodies and glucose during periods of physiologic stress in individuals with impaired FA oxidation? Case 4: Hypoglycemia, Hyperketonemia, and Liver Dysfunction Patient Presentation: AK, a 59-year-old male with slurred speech, ataxia (loss of skeletal muscle coordination), and abdominal pain, was dropped off at the Emergency Department (ED). Focused History: AK is known to the ED staff from previous visits. He has a 6year history of chronic, excessive alcohol consumption. He is not known to take illicit drugs. At this ED visit, AK reports that he has been drinking heavily in the past day or so. He cannot recall having eaten anything in that time. There is evidence of recent vomiting, but no blood is apparent.

1	Physical Examination (Pertinent Findings): The physical examination was remarkable for AK’s emaciated appearance. (His body mass index was later determined to be 17.5, which put him in the underweight category.) His facial cheeks were erythematous (red in color) due to dilated blood vessels in the skin (telangiectasia). Eye movement was normal. Neither icterus (jaundice) nor edema (swelling due to fluid retention) was seen. The liver was slightly enlarged. Bedside tests revealed hypoglycemia and hyperketonemia (as acetoacetate). Blood was drawn and sent to the clinical laboratory. Pertinent Test Results: DUI = driving under the influence; H = High; L = Low. Additional Tests: Complete blood count (CBC) and blood smear revealed a macrocytic anemia (see right image). Folate and B12 levels were ordered. Diagnosis: AK is diagnosed with alcoholism. Treatment (Immediate): Thiamine and glucose were given intravenously.

1	Diagnosis: AK is diagnosed with alcoholism. Treatment (Immediate): Thiamine and glucose were given intravenously. Prognosis: Alcoholism (alcohol dependence) is the third most common cause of preventable death in the United States. People with alcoholism are at increased risk for liver cirrhosis, pancreatitis, gastrointestinal bleeding, and some cancers. Nutrition Nugget: Those with alcoholism are at risk for vitamin deficiencies as a result of decreased intake and absorption. Thiamine (vitamin B1) deficiency is common and can have serious consequences such as Wernicke-Korsakoff syndrome with its neurologic effects. Thiamine pyrophosphate (TPP), the coenzyme form, is required for the dehydrogenase-mediated oxidation of α-keto acids (such as pyruvate) as well as the transfer of two-carbon ketol groups by transketolase in the reversible sugar interconversions in the pentose phosphate pathway.

1	Genetics Gem: Acetaldehyde, the product of ethanol oxidation by the hepatic, cytosolic, nicotinamide adenine dinucleotide (NAD+)-requiring enzyme alcohol dehydrogenase (ADH), is oxidized to acetate by the mitochondrial, NAD+-requiring aldehyde dehydrogenase (ALDH2). The majority of individuals of East Asian (but not European or African) heritage have a single nucleotide polymorphism (SNP) that renders ALDH2 essentially inactive. This results in aldehyde-induced facial flushing and mild to moderate intoxication after consumption of small amounts of ethanol. Review Questions: Choose the ONE best answer. RQ1. Many of the metabolic consequences of chronic excessive alcohol consumption seen in AK are the result of an increase in the ratio of reduced nicotinamide adenine dinucleotide (NADH) to its oxidized form (NAD+) in both the cytoplasm and mitochondria. Which of the following statements concerning the effects of the rise in mitochondrial NADH is correct?

1	A. Fatty acid oxidation is increased. B. Gluconeogenesis is increased. C. Lipolysis is inhibited. D. The tricarboxylic acid cycle is inhibited. E. The reduction of malate to oxaloacetate in the malate–aspartate shuttle is increased. RQ2. Ethanol can also be oxidized by cytochrome P450 (CYP) enzymes, and CYP2E1 is an important example. CYP2E1, which is ethanol inducible, generates reactive oxygen species (ROS) in its metabolism of ethanol. Which of the following statements concerning the CYP proteins is correct? A. CYP proteins are heme-containing dioxygenases. B. CYP proteins of the inner mitochondrial membrane are involved in detoxification reactions. C. CYP proteins of the smooth endoplasmic reticular membrane are involved in the synthesis of steroid hormones, bile acids, and calcitriol. D. ROS such as hydrogen peroxide generated by CYP2E1 can be oxidized by glutathione peroxidase.

1	D. ROS such as hydrogen peroxide generated by CYP2E1 can be oxidized by glutathione peroxidase. E. The pentose phosphate pathway is an important source of the nicotinamide adenine dinucleotide phosphate (NADPH) that provides the reducing equivalents needed for activity of CYP proteins and the regeneration of functional glutathione. RQ3. Alcohol is known to modulate the levels of serotonin in the central nervous system, where the monoamine functions as a neurotransmitter. Which of the following statements about serotonin is correct? Serotonin is: A. associated with anxiety and depression. B. degraded via methylation by monoamine oxidase, which also degrades the catecholamines. C. released by activated platelets. D. synthesized from tyrosine in a two-step process that utilizes a tetrahydrobiopterin-requiring hydroxylase and a pyridoxal phosphate– requiring carboxylase.

1	C. released by activated platelets. D. synthesized from tyrosine in a two-step process that utilizes a tetrahydrobiopterin-requiring hydroxylase and a pyridoxal phosphate– requiring carboxylase. RQ4. Chronic, excessive consumption of alcohol is a leading cause of acute pancreatitis, a painful inflammatory condition that results from autodigestion of the gland by premature activation of pancreatic enzymes. Which of the following statements concerning the pancreas is correct? A. Autodigestion of the pancreas would be expected to result in a decrease in pancreatic proteins in the blood. B. In individuals who progress from acute to chronic pancreatitis, with the characteristic structural changes that result in decreased pancreatic function, diabetes and steatorrhea are expected findings. C. In response to secretin, the exocrine pancreas secretes protons to lower the pH in the intestinal lumen. D. Pancreatitis may also be seen in individuals with hypercholesterolemia.

1	C. In response to secretin, the exocrine pancreas secretes protons to lower the pH in the intestinal lumen. D. Pancreatitis may also be seen in individuals with hypercholesterolemia. TQ1. A. What effect does the rise in cytosolic NADH seen with ethanol metabolism have on glycolysis? Hint: What coenzyme is required in glycolysis? B. How does this relate to the fatty liver (hepatic steatosis) commonly seen in alcohol-dependent individuals? TQ2. Why might individuals with a history of gouty attacks be advised to reduce their consumption of ethanol? TQ3. Why might prothrombin time be affected in alcohol-dependent individuals? TQ4. Folate and vitamin B12 deficiencies cause a macrocytic anemia that may be seen in those with alcoholism. Why is it advisable to measure vitamin B12 levels before supplementing with folate in an individual with macrocytic anemia? Case 1: Answers to Review Questions

1	RQ1.Answer = B. Phosphatidylcholine is a glycerol-based phospholipid derived from diacylglycerol phosphate (phosphatidic acid) and cytidine diphosphatecholine. Gangliosides are derived from ceramides, lipids with a sphingosine backbone. Prostaglandins of the 2 series (such as PGI2) are derived from the 20 carbon polyunsaturated fatty acid arachidonic acid. Sphingomyelin is a sphingophospholipid derived from ceramide. Vitamin D is derived from an intermediate in the biosynthetic pathway for the sterol cholesterol. RQ2.Answer = A. Statins inhibit hydroxymethylglutaryl coenzyme A (HMG CoA) reductase, thereby preventing the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of HMG CoA to mevalonate and decreasing cholesterol biosynthesis (see figure below). The decrease in cholesterol content caused by statins results in movement of the sterol regulatory element–binding protein-2 (SREBP-2) in complex with SREBP cleavage– activating protein (SCAP) from the endoplasmic

1	cholesterol content caused by statins results in movement of the sterol regulatory element–binding protein-2 (SREBP-2) in complex with SREBP cleavage– activating protein (SCAP) from the endoplasmic reticular membrane to the Golgi membrane. SREBP-2 is cleaved, generating a transcription factor that moves to the nucleus and binds to the sterol regulatory element upstream of the genes for HMG CoA reductase and the low-density lipoprotein (LDL) receptor, increasing their expression. Humans are unable to degrade the steroid nucleus to CO2 +

1	H2O. Bile acid (BA) sequestrants, such as cholestyramine, prevent the absorption of bile salts by the liver, thereby increasing their excretion. The liver then takes up cholesterol via the LDL receptor and uses it to make BA, thereby reducing blood cholesterol levels. Steroid hormones are synthesized from cholesterol, and vitamin D is synthesized in skin from an intermediate (7dehydrocholesterol) in the cholesterol biosynthetic pathway. Therefore, inhibition of cholesterol synthesis would be expected to decrease their production as well.

1	RQ3.Answer = C. Competitive inhibitors bind to the same site as the substrate (S) and prevent the S from binding. This results in an increase in the apparent Km (Michaelis constant, or that S concentration that gives one half of the maximal velocity [Vmax]). However, because the inhibition can be reversed by adding additional substrate, the Vmax is unchanged (see figure at right). It is noncompetitive inhibitors that decrease the apparent Vmax and have no effect on Km.

1	RQ4.Answer = F. Tissue plasminogen activator (TPA) converts plasminogen to plasmin that degrades fibrin (fibrinolysis), thereby degrading the clot (thrombolysis). Aspirin, an inhibitor of cyclooxygenase, is an antiplatelet drug. Antithrombin III (ATIII) removes thrombin from the blood, and its action is potentiated by heparin. Activated protein C (APC) complex cleaves the accessory proteins factor (F)Va and FVIIIa. ATIII and APC are involved in anticoagulation. FXIII is a transglutaminase that cross-links the fibrin meshwork. Vitamin K is a fat-soluble vitamin required for the γ-carboxylation of FII, FVII, FIX, and FX. Warfarin prevents regeneration of the functional, reduced form of vitamin K. RQ5.Answer = D. In hypoxia, substrate-level phosphorylation in glycolysis provides ATP. Oxidative phosphorylation is inhibited by the lack of O2. Because the rate of ATP synthesis by oxidative phosphorylation controls the rate of cellular respiration, electron transport is inhibited. The

1	phosphorylation is inhibited by the lack of O2. Because the rate of ATP synthesis by oxidative phosphorylation controls the rate of cellular respiration, electron transport is inhibited. The resulting rise in the ratio of the reduced form of nicotinamide adenine dinucleotide (NADH) to the oxidized form (NAD+) inhibits the tricarboxylic acid cycle and the pyruvate dehydrogenase complex. RQ6.Answer = D. Fluorescently labeled nucleotides allow the base sequence of the DNA of interest to be determined. Complementary DNA (cDNA) is generated from processed messenger RNA and would not contain the promoter. Dideoxynucleotides lack the 3′-OH needed to form the 3′→5′-phosphodiester bond that joins the nucleotides and, thus, will terminate DNA synthesis. Genomic DNA obtained from white cells isolated from a blood sample would be the source of the DNA.

1	Case 1: Answers to Thought Questions

1	TQ1.The phenotype would be the same. In familial defective apolipoprotein (apo) B-100, LDL receptors are normal in number and function, but the ligand for the receptor is altered such that binding to the receptor is decreased. Decreased ligand–receptor binding results in increased levels of LDL in the blood with hypercholesterolemia. [Note: The phenotype would be the same in individuals with a gain-of-function mutation to PCSK9, the protease that decreases recycling of the LDL receptor, thereby increasing its degradation.] With the apo E-2 isoform, cholesterol-rich chylomicron remnants and intermediate-density lipoproteins would accumulate in blood. TQ2.Aspirin irreversibly inhibits cyclooxygenase (COX) and, therefore, the synthesis of prostaglandins (PG), such as PGI2 in vascular endothelial cells, and thromboxanes (TX), such as TXA2 in activated platelets. TXA2 promotes vasoconstriction and formation of a platelet plug, whereas PGI2 inhibits these events. Because platelets are

1	cells, and thromboxanes (TX), such as TXA2 in activated platelets. TXA2 promotes vasoconstriction and formation of a platelet plug, whereas PGI2 inhibits these events. Because platelets are anucleate, they cannot overcome this inhibition by synthesizing more COX. However, endothelial cells have a nucleus. Aspirin, then, inhibits formation of blood clots by preventing production of TXA2 for the life of the platelet. TQ3.The decrease in ATP (as the result of a decrease in O2 and, thus, a decrease in oxidative phosphorylation) causes an increase in adenosine monophosphate (AMP). AMP allosterically activates phosphofructokinase-1, the key regulated enzyme of glycolysis. The rise in glycolysis increases the production of ATP by substrate-level phosphorylation. It also increases the ratio of the reduced to oxidized forms of NAD. Under anaerobic conditions, pyruvate produced in glycolysis is reduced to lactate by lactate dehydrogenase as NADH is oxidized to NAD+. NAD+ is required for

1	of the reduced to oxidized forms of NAD. Under anaerobic conditions, pyruvate produced in glycolysis is reduced to lactate by lactate dehydrogenase as NADH is oxidized to NAD+. NAD+ is required for continued glycolysis. Because fewer ATP molecules are produced per molecule of substrate in substrate-level phosphorylation relative to oxidative phosphorylation, there is a compensatory increase in the rate of glycolysis under anaerobic conditions.

1	TQ4.High-density lipoprotein (HDL) functions in reverse cholesterol transport. It takes cholesterol from nonhepatic (peripheral) tissues (for example, the endothelial layer of arteries) and brings it to the liver (see figure on the next page). The ABCA1 transporter mediates the efflux of cholesterol to HDL. The cholesterol is esterified by extracellular lecithin–cholesterol acyltransferase (LCAT) that requires apo A-1 as a coenzyme. Some cholesteryl ester is transferred to very-low-density lipoproteins (VLDL) by cholesteryl ester transfer protein (CETP) in exchange for triacylglycerol. The remainder is taken up by a scavenger receptor (SR-B1) on the surface of hepatocytes. The liver can use the cholesterol from HDL in the synthesis of bile acids. Removal of cholesterol from endothelial cells prevents its accumulation (as cholesterol or cholesteryl ester), decreasing the risk of heart disease. [Note: In contrast, LDL carries cholesterol from the liver to peripheral tissues or back to

1	prevents its accumulation (as cholesterol or cholesteryl ester), decreasing the risk of heart disease. [Note: In contrast, LDL carries cholesterol from the liver to peripheral tissues or back to the liver.]

1	Case 2: Answers to Review Questions RQ1.Answer = A. Deficiency of glucose 6-phosphatase prevents the glucose 6phosphate generated by glycogenolysis and gluconeogenesis from being dephosphorylated and released into the blood (see figure below). Blood glucose levels fall, and a severe, fasting hypoglycemia results. [Note: JS’s symptoms appeared only recently because, at age 4 months, his feedings are less frequent.] Hypoglycemia stimulates release of glucagon, which leads to phosphorylation and activation of glycogen phosphorylase kinase that phosphorylates and activates glycogen phosphorylase. Epinephrine is also released and leads to phosphorylation and activation of hormone-sensitive lipase. However, typical fatty acids (FA) cannot serve as substrates for gluconeogenesis. The glucose transporters in the liver and kidneys are insulin insensitive.

1	RQ2.Answer = C. Vitamin D is a fat-soluble vitamin that functions as a steroid hormone. In complex with its intracellular nuclear receptor, it increases transcription of the gene for calbindin, a calcium (Ca2+) transporter protein in the intestine (see figure at right). Vitamin D does not bind to a membrane receptor and does not produce second messengers. It can be synthesized in the skin by the action of ultraviolet light on an intermediate of cholesterol synthesis, 7dehydrocholesterol. Of the fat-soluble vitamins (A, D, E, and K), only K functions as a coenzyme.

1	RQ3.Answer = C. Glucose 6-phosphate is a positive allosteric effector of the covalently inhibited (phosphorylated) glycogen synthase b. With the rise in glucose 6-phosphate, glycogen synthesis is activated, and glycogen stores are increased in both the liver and the kidneys. The increased availability of glucose 6-phosphate also drives glycolysis. The increase in glycolysis provides substrates for lipogenesis, thereby increasing synthesis of FA and triacylglycerols (TAG). In hypoglycemia, the insulin/glucagon ratio is low, not high.

1	RQ4.Answer = D. Membrane proteins are initially targeted to the endoplasmic reticulum (ER) by an amino terminal hydrophobic signal sequence. Glycosylation is the most common posttranslational modification found in proteins. The glycosylated portion of membrane proteins is found on the extracellular face of the membrane. The membrane-spanning domain consists of ~22 hydrophobic amino acids. Proteins destined for secretion or for membranes, the ER lumen, Golgi, or lysosomes are synthesized on ribosomes associated with the ER. Case 2: Answers to Thought Questions

1	TQ1.The twitching is the result of the adrenergic response to hypoglycemia and is mediated by the rise in epinephrine. The adrenergic response includes tremor and sweating. Neuroglycopenia (impaired delivery of glucose to the brain) results in impairment of brain function that can lead to seizures, coma, and death. Neuroglycopenic symptoms develop if the hyperglycemia persists. TQ2.Detergents are amphipathic molecules (that is, they have both hydrophilic [polar] and hydrophobic [nonpolar] regions). Detergents solubilize membranes, thereby disrupting membrane structure. If the problem were the translocase needed to move the glucose 6-phosphate substrate into the ER, rather than the phosphatase, disruption of the ER membrane would allow the substrate access to the phosphatase. TQ3.Glucagon, a peptide hormone released from pancreatic α cells in hypoglycemia, binds its plasma membrane G protein–coupled receptor on hepatocytes. The αs subunit of the associated trimeric G protein is

1	a peptide hormone released from pancreatic α cells in hypoglycemia, binds its plasma membrane G protein–coupled receptor on hepatocytes. The αs subunit of the associated trimeric G protein is activated (guanosine diphosphate is replaced by guanosine triphosphate), separates from the β and γ subunits, and activates adenylyl cyclase that generates cyclic adenosine monophosphate (cAMP) from ATP. cAMP activates protein kinase A (PKA) that phosphorylates and activates glycogen phosphorylase kinase, which phosphorylates and activates glycogen phosphorylase. The phosphorylase degrades glycogen, generating glucose 1-phosphate that is converted to glucose 6-phosphate. With glucose 6-phosphatase deficiency, the degradative process stops here (see figure below). Consequently, administration of glucagon is unable to cause a rise in blood glucose. [Note: Epinephrine would be similarly ineffective.]

1	TQ4.The availability of inorganic phosphate (Pi) is decreased because it is trapped as phosphorylated glycolytic intermediates as a result of the upregulation of glycolysis by the rise in glucose 6-phosphate. Urate is elevated because the trapping of Pi decreases the ability to phosphorylate adenosine diphosphate (ADP) to ATP, and the fall in ATP causes a rise in adenosine monophosphate (AMP). The AMP is degraded to urate. Additionally, the availability of glucose 6-phosphate drives the pentose phosphate pathway, resulting in a rise in ribose 5-phosphate (from ribulose 5-phosphate) and, consequently, a rise in purine synthesis. Nicotinamide adenine dinucleotide phosphate (NADPH) also rises. Purines made beyond need are degraded to urate (see figure on the next page). [Note: The decrease in Pi reduces the activity of glycogen phosphorylase, resulting in increased storage of glycogen with a normal structure.] Lactate is elevated because the decrease in phosphorylation of ADP to ATP

1	in Pi reduces the activity of glycogen phosphorylase, resulting in increased storage of glycogen with a normal structure.] Lactate is elevated because the decrease in phosphorylation of ADP to ATP results in a decrease in cellular respiration (respiratory control) as a result of these processes being coupled. As a consequence, reduced nicotinamide adenine dinucleotide (NADH) from glycolysis cannot be oxidized by Complex I of the electron transport chain. Instead, it is oxidized by cytosolic lactate dehydrogenase with its coenzyme NADH as pyruvate is reduced to lactate. [Note: Pyruvate is increased as a result of the increase in glycolysis.] The lactate ionizes, releasing protons (H+) and leading to a metabolic acidosis (low pH caused here by increased production of acid). Respiratory compensation causes an increased respiratory rate.

1	TQ5.Increased glycolysis results in increased availability of glycerol 3phosphate for hepatic TAG synthesis. Additionally, some of the pyruvate generated in glycolysis will be oxidatively decarboxylated to acetyl coenzyme A (CoA). However, the tricarboxylic acid cycle is inhibited by the rise in NADH, and the acetyl CoA is transported to the cytosol as citrate. The rise of acetyl CoA in the cytosol results in increased fatty acid (FA) synthesis. Recall that citrate is an allosteric activator of acetyl CoA carboxylase (ACC). The malonyl product of ACC inhibits FA oxidation at the carnitine palmitoyltransferase I step. Because mitochondrial FA oxidation generates the acetyl CoA substrate for hepatic ketogenesis, ketone body levels do not rise. The FA gets esterified to the glycerol backbone, resulting in an increase in TAG that gets sent out of the liver as components of very-low-density lipoproteins (VLDL). [Note: The hypoglycemia results in release of epinephrine and the activation of

1	resulting in an increase in TAG that gets sent out of the liver as components of very-low-density lipoproteins (VLDL). [Note: The hypoglycemia results in release of epinephrine and the activation of TAG lipolysis with release of free FA into the blood. The FA are oxidized, with the excess used in hepatic TAG synthesis.] The acetyl CoA is also a substrate for cholesterol synthesis. Thus, the increase in glycolysis results in the hyperlipidemia seen in JS (see figure below).

1	Case 3: Answers to Review Questions RQ1.Correct answer = A. Diabetes is characterized by hyperglycemia. Chronic hyperglycemia can result in the nonenzymatic glycosylation (glycation) of hemoglobin (Hb), producing HbA1c. Therefore, measurement of glucose or

1	HbA1c in the blood is used to diagnose diabetes. In response to physiologic stress (for example, a urinary tract infection), secretion of counterregulatory hormones (such as the catecholamines) results in a rise in blood glucose. Glucose is a reducing sugar. It is type 2 diabetes (T2D) that is associated with obesity and a sedentary lifestyle and is caused by insensitivity to insulin (insulin resistance). T1D is caused by lack of insulin as a result of the autoimmune destruction of pancreatic β cells. Even individuals on a program of tight glycemic control do not achieve euglycemia. RQ2.Correct answer = E. The ketone bodies 3-hydroxybutyrate and acetoacetate are organic acids, and their ionization contributes to the proton load of the body. Ketone bodies are made in the mitochondria of liver cells using acetyl coenzyme A (CoA) generated primarily from the β-oxidation of fatty acids ([FA]; see figure on the next page). Because they are water soluble, they do not require a transporter.

1	cells using acetyl coenzyme A (CoA) generated primarily from the β-oxidation of fatty acids ([FA]; see figure on the next page). Because they are water soluble, they do not require a transporter. The liver cannot use them because it lacks the enzyme thiophorase, which moves CoA from succinyl CoA to acetoacetate for conversion to two molecules of acetyl CoA. It is the acetone released in the breath that can impart a fruity odor.

1	RQ3.Correct answer = A. Malonyl CoA, an intermediate of FA synthesis, inhibits FA β-oxidation through inhibition of carnitine palmitoyltransferase I. Lipolysis occurs when the insulin/counterregulatory hormone ratio decreases. Acetyl CoA, the product of FA β-oxidation, inhibits the pyruvate dehydrogenase (PDH) complex through activation of PDH kinase and activates pyruvate carboxylase. Acetyl CoA, then, pushes pyruvate to gluconeogenesis. β-Oxidation generates reduced nicotinamide adenine dinucleotide (NADH), the reducing equivalent required for gluconeogenesis. FA are not readily catabolized for energy by the brain. Case 3: Answers to Thought Questions

1	Case 3: Answers to Thought Questions TQ1.Hypoinsulinemia results in hyperglycemia because insulin is required for the uptake of blood glucose by muscle and adipose tissue. Their glucose transporter (GLUT-4) is insulin dependent in that insulin is required for movement of the transporter to the cell surface from intracellular storage sites. Insulin is also required to suppress hepatic gluconeogenesis. Insulin suppresses the release of glucagon from pancreatic α cells. The resulting rise in the insulin/glucagon ratio results in the dephosphorylation and activation of the kinase domain of bifunctional phosphofructokinase-2 (PFK-2). The fructose 2,6bisphosphate produced by PFK-2 activates phosphofructokinase-1 of glycolysis (see figure below). It also inhibits fructose 1,6-bisphosphatase (FBP-2), thereby inhibiting gluconeogenesis. With hypoinsulinemia, the failure to take up glucose from the blood while simultaneously sending it out into the blood results in hyperglycemia.

1	TQ2.The blood glucose level has exceeded the capacity of the kidney to reabsorb glucose (via a sodium-dependent glucose transporter [SGLT]). The high concentration of glucose in the urine osmotically draws water from the body. This causes increased urination (polyuria) with loss of water that results in dehydration. TQ3.The NADH generated in FA β-oxidation inhibits the tricarboxylic acid (TCA) cycle at the three NADH-producing dehydrogenase steps. This shifts acetyl CoA away from oxidation in the TCA cycle and toward use as a substrate in hepatic ketogenesis.

1	TQ4.MW was in negative nitrogen balance: More nitrogen was going out than coming in. This is reflected in the elevated blood urea nitrogen (BUN) level seen in the patient (see figure at top right). [Note: The BUN value also reflects dehydration.] Muscle proteolysis and amino acid catabolism are occurring as a result of the fall in insulin. (Recall that skeletal muscle does not express the glucagon receptor.) Amino acid catabolism produces ammonia (NH3), which is converted to urea by the hepatic urea cycle and sent into the blood. [Note: Urea in the urine is reported as urinary urea nitrogen.] TQ5.The Kussmaul respiration seen in MW is a respiratory response to the metabolic acidosis. Hyperventilation blows off CO2 and water, reducing the concentration of protons (H+) and bicarbonate (HCO3−) as reflected in the following equation: The renal response includes, in part, the excretion of H+ as ammonium (NH4+). Degradation of branched-chain amino acids in skeletal muscle results in the

1	as reflected in the following equation: The renal response includes, in part, the excretion of H+ as ammonium (NH4+). Degradation of branched-chain amino acids in skeletal muscle results in the release of large amounts of glutamine (Gln) into the blood. The kidneys take up and catabolize the Gln, generating NH3 in the process. The NH3 is converted to NH4+ by secreted H+ and is excreted (see figure at middle right). [Note: When ketone bodies are plentiful, enterocytes shift to using them as a fuel instead of

1	Gln. This increases the amount of Gln going to the kidney.] TQ6.Because FA β-oxidation supplies the acetyl CoA substrate for ketogenesis, impaired β-oxidation decreases the ability to make ketone bodies. Ketone bodies are an alternate to the use of glucose, and, thus, dependence on glucose increases. Because FA β-oxidation supplies the NADH and the nucleoside triphosphates needed for gluconeogenesis, glucose production decreases. The result is a hypoketotic hypoglycemia. Recall that this was seen with medium-chain acyl CoA dehydrogenase (MCAD) deficiency. Case 4: Answers to Review Questions

1	RQ1.Answer = D. The rise in reduced nicotinamide adenine dinucleotide (NADH) in the mitochondria decreases the tricarboxylic acid (TCA) cycle, fatty acid (FA) oxidation, and gluconeogenesis. NADH inhibits the isocitrate dehydrogenase reaction, the key regulated step of the TCA cycle, and the αketoglutarate dehydrogenase reaction (see figure at bottom right). It also favors the reduction of oxaloacetate (OAA) to malate (not malate to OAA), decreasing the availability of OAA for condensation with acetyl coenzyme A (CoA) in the TCA cycle and for gluconeogenesis. FA oxidation requires the oxidized form of nicotinamide adenine dinucleotide (NAD+) for the 3-hydroxyacyl CoA dehydrogenase step and, thus, is inhibited by the rise in NADH. The decrease in FA oxidation decreases the production of ATP and acetyl CoA (the allosteric activator of pyruvate carboxylase) needed for gluconeogenesis. Lipolysis is activated in fasting as a consequence of the fall in insulin and the rise in catecholamines

1	and acetyl CoA (the allosteric activator of pyruvate carboxylase) needed for gluconeogenesis. Lipolysis is activated in fasting as a consequence of the fall in insulin and the rise in catecholamines that result in activation of hormone-sensitive lipase.

1	RQ2.Answer = E. The irreversible, oxidative portion of the pentose phosphate pathway provides the nicotinamide adenine dinucleotide phosphate (NADPH) that supplies the reducing equivalents needed for activity of cytochrome P450 (CYP) proteins and for the regeneration of functional (reduced) glutathione. It is also an important source of NADPH for reductive biosynthetic processes in the cytosol, such as FA and cholesterol synthesis. [Note: Malic enzyme is another source.] CYP proteins are monooxygenases (mixed-function oxidases). They incorporate one O atom from O2 into the substrate as the other is reduced to water. It is the CYP proteins of the smooth endoplasmic reticular membrane that are involved in detoxification reactions. Those of the inner mitochondrial membrane are involved in the synthesis of steroid hormones, bile acids, and vitamin D. Reactive oxygen species are reduced by glutathione peroxidase as glutathione is oxidized. RQ3.Answer = C. Serotonin is released by activated

1	synthesis of steroid hormones, bile acids, and vitamin D. Reactive oxygen species are reduced by glutathione peroxidase as glutathione is oxidized. RQ3.Answer = C. Serotonin is released by activated platelets and causes vasoconstriction and platelet aggregation. [Note: Platelets do not synthesize serotonin, but they take up that which was made in the intestine and secreted into the blood.] Serotonin is associated with a feeling of well-being. It is degraded to 5-hydroxyindoleacetic acid by monoamine oxidase that catalyzes oxidative deamination. It is catechol-O-methyltransferase that catalyzes the methylation step in the degradation of the catecholamines. Serotonin is synthesized from tryptophan in a two-step process that utilizes tetrahydrobiopterin (BH4) requiring tryptophan hydroxylase and a pyridoxal phosphate (PLP)-requiring decarboxylase (see figure at right). RQ4.Answer = B. The exocrine pancreas secretes enzymes required for the digestion of dietary carbohydrate, protein, and

1	and a pyridoxal phosphate (PLP)-requiring decarboxylase (see figure at right). RQ4.Answer = B. The exocrine pancreas secretes enzymes required for the digestion of dietary carbohydrate, protein, and fat. The endocrine pancreas secretes the peptide hormones insulin and glucagon. Damage that affects the functions of the pancreas would lead to diabetes (decreased insulin) and steatorrhea (fatty stool), with the latter the consequence of maldigestion of dietary fat. As was seen with the rise of troponins in a myocardial infarction and transaminases in liver damage, loss of cellular integrity (as would be seen in autodigestion of the pancreas) results in proteins that normally are intracellular being found in higher-than-normal concentrations in the blood. Secretin causes the pancreas to release bicarbonate to raise the pH of the chyme coming to the intestine from the stomach. Pancreatic enzymes work best at neutral or slightly alkaline pH. Pancreatitis is seen in individuals with

1	to release bicarbonate to raise the pH of the chyme coming to the intestine from the stomach. Pancreatic enzymes work best at neutral or slightly alkaline pH. Pancreatitis is seen in individuals with hypertriglyceridemia as a result of a deficiency in lipoprotein lipase or its coenzyme, apolipoprotein C-II.

1	Case 4: Answers to Thought Questions TQ1.A. The rise in cytosolic NADH seen with ethanol metabolism inhibits glycolysis. The glyceraldehyde 3-phosphate dehydrogenase step requires NAD+, which gets reduced as glyceraldehyde 3-phosphate gets oxidized. With the rise in NADH, glyceraldehyde 3-phosphate accumulates.

1	B. Glyceraldehyde 3-phosphate from glycolysis is converted to glycerol 3phosphate, the initial acceptor of FA in triacylglycerol (TAG) synthesis (see figure at right). FA are available because of increased synthesis (from acetyl CoA, which is increased as a result of both increased production from the acetate product of acetaldehyde oxidation and decreased use in the TCA cycle), increased availability from lipolysis in adipose tissue, and decreased degradation. The TAG produced in the liver accumulate (due, in part, to decreased production of very-low-density lipoproteins) and cause fatty liver (steatosis). Hepatic steatosis is an early (and reversible) stage in alcohol-related liver disease. Subsequent stages are alcohol-related hepatitis (sometimes reversible) and cirrhosis (irreversible).

1	TQ2.The rise in NADH favors the reduction of pyruvate to lactate by lactate dehydrogenase. Lactate decreases the renal excretion of uric acid, thereby causing hyperuricemia, a necessary step in an acute gouty attack. [Note: The shift from pyruvate to lactate decreases the availability of pyruvate, a substrate for gluconeogenesis. This contributes to the hypoglycemia seen in AK.] TQ3.Prothrombin time (PT) measures the time it takes for plasma to clot after the addition of tissue factor (FIII), thereby allowing evaluation of the extrinsic (and common) pathways of coagulation. In the extrinsic pathway, FIII activates FVII in a calcium (Ca2+)-and phospholipid (PL)-dependent process (see figure at bottom right). FVII, like most of the proteins of clotting, is made by the liver. Alcohol-induced liver damage can decrease its synthesis. Additionally, FVII has a short half-life, and, as a γ-carboxyglutamate (Gla)-containing protein, its synthesis requires vitamin K. Poor nutrition can result

1	liver damage can decrease its synthesis. Additionally, FVII has a short half-life, and, as a γ-carboxyglutamate (Gla)-containing protein, its synthesis requires vitamin K. Poor nutrition can result in decreased availability of vitamin K and, therefore, decreased ability to clot. [Note: Severe liver disease results in prolonged PT and activated partial thromboplastin time, or aPPt.]

1	TQ4.Administration of folate can mask a deficiency in vitamin B12 by reversing the hematologic manifestation (macrocytic anemia) of the deficiency. However, folate has no effect on the neurologic damage caused by B12 deficiency. Over time, then, the neurologic effects can become severe and irreversible. Thus, folate can mask a deficiency of B12 and prevent treatment until the neuropathy is apparent. III. Focused Cases Case 1: Microcytic Anemia Patient Presentation: ME is a 24-year-old man who is being evaluated as a follow-up to a preplacement medical evaluation he had prior to starting his new job. Focused History: ME has no significant medical issues. His family history is unremarkable, but he knows little of the health status of those family members who remain in Greece. Pertinent Findings: The physical examination was normal. Routine analysis of his blood included the following results:

1	Pertinent Findings: The physical examination was normal. Routine analysis of his blood included the following results: Based on the data, hemoglobin (Hb) electrophoresis was performed. The results are as follows: H = High; L = Low. [Note: HbA includes HbA1c.] Diagnosis: ME has β-thalassemia trait (β-thalassemia minor) that is causing a microcytic anemia (see image at right). Ethnicity (such as being of Mediterranean origin) influences the risk for thalassemia. Treatment: None is required at this time. Patients are advised that iron supplements will not prevent their anemia. Prognosis: β-Thalassemia trait does not cause mortality or significant morbidity. Patients should be informed of the genetic nature of their autosomal-recessive condition for family planning considerations because homozygous β-thalassemia (Cooley anemia) is a serious disorder. Case-Related Questions: Choose the ONE best answer.

1	Case-Related Questions: Choose the ONE best answer. Q1. Mutations to the gene for β globin that result in decreased production of the protein are the cause of β-thalassemia. The mutations primarily affect gene transcription or posttranscriptional processing of the messenger RNA (mRNA) product. Which of the following statements concerning mRNA is correct? A. Eukaryotic mRNA is polycistronic. B. mRNA synthesis involves trans-acting factors binding to cis-acting elements. C. mRNA synthesis is terminated at the DNA base sequence thymine adenine guanine (TAG). D. Polyadenylation of the 5′-end of eukaryotic mRNA requires a methyl donor.

1	C. mRNA synthesis is terminated at the DNA base sequence thymine adenine guanine (TAG). D. Polyadenylation of the 5′-end of eukaryotic mRNA requires a methyl donor. E. Splicing of eukaryotic mRNA involves removal of exons and joining of introns by the proteasome. Q2. HbA, a tetramer of 2 α and 2 β globin chains, delivers O2 from the lungs to the tissues and protons and CO2 from the tissues to the lungs. Increased concentration of which of the following will result in decreased O2 delivery by HbA? A. 2,3-Bisphosphoglycerate B. Carbon dioxide C. Carbon monoxide D. Protons Q3. What is the basis for the increase in HbA2 and HbF (fetal Hb) in the βthalassemias? Q4. Why is the allele-specific oligonucleotide (ASO) hybridization technique useful in the diagnosis of all cases of sickle cell anemia but not all cases of β-thalassemia? Case 2: Skin Rash Patient Presentation: KL is a 34-year-old woman who presents with a red, nonitchy rash on her left thigh and flu-like symptoms.

1	Case 2: Skin Rash Patient Presentation: KL is a 34-year-old woman who presents with a red, nonitchy rash on her left thigh and flu-like symptoms. Focused History: KL reports that the rash first appeared a little over 2 weeks ago. It started out small but has gotten larger. She also thinks she is getting the flu because her muscles and joints ache (myalgia and arthralgia, respectively), and she has had a headache for the last few days. Upon questioning, KL reports that she and her husband took a camping trip through New England last month. Pertinent Findings: The physical examination is remarkable for the presence of a red, circular, flat lesion ~11 cm in size that resembles a bullseye (erythema migrans) (see image at right). KL also has a low-grade fever. Diagnosis: KL has Lyme disease caused by the bacterium Borrelia burgdorferi, which is transmitted by the bite of a tick in the genus Ixodes. Infected ticks are endemic in the Northeast region of the United States.

1	Treatment: KL is prescribed doxycycline, an antibiotic in the tetracycline family. Monitoring of KL will continue until all symptoms have completely resolved. Blood is drawn for clinical laboratory tests. Prognosis: Patients treated with the appropriate antibiotic in the early stages of Lyme disease typically recover quickly and completely. Case-Related Questions: Choose the ONE best answer. Q1. Antibiotics in the tetracycline class inhibit protein synthesis (translation) at the initiation step. Which of the following statements about translation is correct? A. In eukaryotic translation, the initiating amino acid is formylated methionine. B. Only the charged initiating transfer RNA goes directly to the ribosomal A site. C. Peptidyltransferase is a ribozyme that forms the peptide bond between two amino acids. D. Prokaryotic translation can be inhibited by the phosphorylation of initiation factor 2.

1	C. Peptidyltransferase is a ribozyme that forms the peptide bond between two amino acids. D. Prokaryotic translation can be inhibited by the phosphorylation of initiation factor 2. E. Termination of translation is independent of guanosine triphosphate hydrolysis. F. The Shine-Dalgarno sequence facilitates the binding of the large ribosomal subunit to eukaryotic messenger RNA (mRNA). Q2. The Centers for Disease Control and Prevention recommends a two-tier testing procedure for Lyme disease that involves a screening enzyme-linked immunosorbent assay (ELISA) followed by a confirmatory western blot analysis on any sample with a positive or equivocal ELISA result. Which of the following statements about these testing procedures is correct? A. Both techniques are used to detect specific mRNA. B. Both techniques involve the use of antibodies. C. ELISA requires the use of electrophoresis.

1	A. Both techniques are used to detect specific mRNA. B. Both techniques involve the use of antibodies. C. ELISA requires the use of electrophoresis. D. Western blots require use of the polymerase chain reaction. Q3. Why are eukaryotic cells unaffected by antibiotics in the tetracycline class? Case 3: Blood on the Toothbrush Patient Presentation: LT is an 84-year-old man whose gums have been bleeding for several months. Focused History: LT is a widower and lives alone in a suburban community on the East Coast. He no longer drives. His two children live on the West Coast and come east infrequently. Since the death of his wife 11 months ago, he has been isolated and finds it hard to get out of the house. His appetite has changed, and he is content with cereal, coffee, and packaged snacks. Chewing is difficult.

1	Pertinent Findings: The physical examination was remarkable for the presence of swollen dark-colored gums (see image at right). Several of LT’s teeth were loose, including one that anchors his dental bridge. Several black and blue marks (ecchymoses) were noted on the legs, and an unhealed sore was present on the right wrist. Inspection of his scalp revealed tiny red spots (petechiae) around some of the hair follicles. Blood was drawn for testing. Results of tests on LT’s blood: The test for blood in his stool (occult blood test) was negative. Results of follow-up tests (obtained several days after the appointment) included the following: H = High; L = Low. Diagnosis: LT has vitamin C deficiency with a microcytic, hypochromic anemia secondary to the deficiency. Treatment: LT was prescribed vitamin C (as oral ascorbic acid) and iron (as oral ferrous sulfate) supplements. He will also be referred to social services. Prognosis: The prognosis for recovery is excellent.

1	Prognosis: The prognosis for recovery is excellent. Case-Related Questions: Choose the ONE best answer. Q1. Which of the following statements about vitamin C is correct? Vitamin C is: A. a competitor of iron absorption in the intestine. B. a fat-soluble vitamin with a 3-month supply typically stored in adipose tissue. C. a coenzyme in several enzymic reactions such as the hydroxylation of proline. D. required for the cross-linking of collagen. Q2. In contrast to the microcytic anemia characteristic of iron deficiency (common in older adults), a macrocytic anemia is seen with deficiencies of vitamin B12 and/or folic acid. These vitamin deficiencies are also common in older adults. Which of the following statements concerning these vitamins is correct? A. An inability to absorb B12 results in pernicious anemia. B. Both vitamins cause changes in gene expression. C. Folic acid plays a key role in energy metabolism in most cells.

1	A. An inability to absorb B12 results in pernicious anemia. B. Both vitamins cause changes in gene expression. C. Folic acid plays a key role in energy metabolism in most cells. D. Treatment with methotrexate can result in toxic levels of the coenzyme form of folic acid. E. Vitamin B12 is the coenzyme for enzymes catalyzing amino acid deaminations, decarboxylations, and transaminations. Q3. How do hemolytic anemias differ from nutritional anemias? Case 4: Rapid Heart Rate, Headache, and Sweating Patient Presentation: BE is a 45-year-old woman who presents with concerns about sudden (paroxysmal), intense, brief episodes of headache, sweating (diaphoresis), and a racing heart (palpitations).

1	Patient Presentation: BE is a 45-year-old woman who presents with concerns about sudden (paroxysmal), intense, brief episodes of headache, sweating (diaphoresis), and a racing heart (palpitations). Focused History: BE reports that the attacks started ~3 weeks ago. They last from 2 to 10 minutes, during which time she feels quite anxious. During the attacks, it feels as though her heart is skipping beats (arrhythmia). At first, she thought the attacks were related to recent stress at work and maybe even menopause. The last time it happened, she was in a pharmacy and had her blood pressure taken. She was told it was 165/110 mm Hg. BE notes that she has lost weight (~8 lbs) in this period even though her appetite has been good.

1	Pertinent Findings: The physical examination was remarkable for BE’s thin, pale appearance. Blood pressure was elevated (150/100 mm Hg), as was the heart rate (110–120 beats/minute). Based on BE’s history, blood levels of normetanephrine and metanephrine were ordered. They were found to be elevated. Diagnosis: BE has a pheochromocytoma, a rare catecholamine-secreting tumor of the adrenal medulla. Treatment: Imaging studies of the abdomen were done to locate the tumor. Surgical resection of the tumor was performed. The tumor was found to be nonmalignant. Follow-up measurement of plasma metanephrines was performed 2 weeks later and was in the normal range. Prognosis: The 5-year survival rate for nonmalignant pheochromocytomas is >95%. Case-Related Questions: Choose the ONE best answer. Q1. Pheochromocytomas secrete norepinephrine (NE) and epinephrine. Which of the following statements concerning the synthesis and degradation of these two biogenic amines is correct?

1	Q1. Pheochromocytomas secrete norepinephrine (NE) and epinephrine. Which of the following statements concerning the synthesis and degradation of these two biogenic amines is correct? A. The substrate for their synthesis is tryptophan, which is hydroxylated to 3,4-dihydroxyphenylalanine (DOPA) by tetrahydrobiopterin-requiring tryptophan hydroxylase. B. The conversion of DOPA to dopamine utilizes a pyridoxal phosphate– requiring carboxylase. C. The conversion of NE to epinephrine requires vitamin C. D. Degradation involves methylation by catechol-O-methyltransferase and produces normetanephrine from NE and metanephrine from epinephrine. E. Normetanephrine and metanephrine are oxidatively deaminated to homovanillic acid by monoamine oxidase. Q2. Which of the following statements concerning the actions of epinephrine and/or NE are correct? A. NE functions as a neurotransmitter and a hormone.

1	Q2. Which of the following statements concerning the actions of epinephrine and/or NE are correct? A. NE functions as a neurotransmitter and a hormone. B. They are initiated by autophosphorylation of select tyrosine residues in their receptors. C. They are mediated by binding to adrenergic receptors, which are a class of nuclear receptors. D. They result in the activation of glycogen and triacylglycerol synthesis. Q3. NE bound to certain receptors causes vasoconstriction and an increase in blood pressure. Why might NE be used clinically in the treatment of septic shock? Case 5: Sun Sensitivity Patient Presentation: AZ is a 6-year-old boy who is being evaluated for freckle-like areas of hyperpigmentation on his face, neck, forearms, and lower legs. Focused History: AZ’s father reports that the boy has always been quite sensitive to the sun. His skin turns red (erythema) and his eyes hurt (photophobia) if he is exposed to the sun for any period of time.

1	Pertinent Findings: The physical examination was remarkable for the presence of thickened, scaly areas (actinic keratosis) and hyperpigmented areas on skin exposed to ultraviolet (UV) radiation from the sun. Small dilated blood vessels (telangiectasia) were also seen. Tissue from several sites on his face was biopsied, and two were later determined to be squamous cell carcinomas. Diagnosis: AZ has xeroderma pigmentosum, a rare defect in nucleotide excision repair of DNA. Treatment: Protection from sunlight through use of sunscreens such as protective clothing that reflect UV radiation and chemicals that absorb it is essential. Frequent skin and eye examinations are recommended. Prognosis: Most patients with xeroderma pigmentosum die at an early age from skin cancers. However, survival beyond middle age is possible. Case-Related Questions: Choose the ONE best answer. Q1. Which of the following statements about DNA repair mechanisms is correct? DNA repair:

1	Case-Related Questions: Choose the ONE best answer. Q1. Which of the following statements about DNA repair mechanisms is correct? DNA repair: A. is performed only by eukaryotes. B. of double-strand breaks is error free. C. of mismatched bases involves repair of the parental strand. D. of UV radiation–induced pyrimidine dimers involves removal of a short oligonucleotide containing the dimer. E. of uracil produced by the deamination of cytosine requires the actions of endo-and exonucleases to remove the uracil base. Q2. Which one of the following statements about DNA synthesis (replication) is correct? Replication: A. in both eukaryotes and prokaryotes requires an RNA primer. B. in eukaryotes requires condensation of chromatin. C. in prokaryotes is accomplished by a single DNA polymerase. D. is initiated at random sites in the genome.

1	B. in eukaryotes requires condensation of chromatin. C. in prokaryotes is accomplished by a single DNA polymerase. D. is initiated at random sites in the genome. E. produces a polymer of deoxyribonucleoside monophosphates linked by 5′→3′-phosphodiester bonds. . What is the difference between DNA proofreading and repair? Case 6: Dark Urine and Yellow Sclerae Patient Presentation: JF is a 13-year-old boy who presents with fatigue and yellow sclerae. Focused History: JF began treatment ~4 days ago with a sulfonamide antibiotic and a urinary analgesic for a urinary tract infection. He had been told that his urine would change color (become reddish) with the analgesic, but he reports that it has gotten darker (more brownish) over the last 2 days. Last night, his mother noticed that his eyes had a yellow tint. JF says he feels as though he has no energy.

1	Pertinent Findings: The physical examination was remarkable for JF’s pale appearance, mild scleral icterus (jaundice), mild splenomegaly, and increased heart rate (tachycardia). JF’s urine tested positive for hemoglobin (hemoglobinuria). A peripheral blood smear reveals a lower-than-normal number of red blood cells (RBC), with some containing precipitated hemoglobin (Heinz bodies; see image at right), and a higher-than-normal number of reticulocytes (immature RBC). Results of the complete blood count (CBC) and blood chemistry tests are pending. Diagnosis: JF has glucose 6-phosphate dehydrogenase (G6PD) deficiency, an X-linked disorder that causes hemolysis (RBC lysis).

1	Diagnosis: JF has glucose 6-phosphate dehydrogenase (G6PD) deficiency, an X-linked disorder that causes hemolysis (RBC lysis). Treatment: G6PD deficiency can result in a hemolytic anemia in affected individuals exposed to oxidative agents. JF will be switched to a different antibiotic. He will be advised that he is susceptible to certain drugs (for example, sulfa drugs), foods (fava or broad beans), and certain chemicals (for example, naphthalene), and must avoid exposure to them. Prognosis: In the absence of exposure to oxidative agents, G6PD deficiency does not cause mortality or significant morbidity. Case-Related Questions: Choose the ONE best answer. Q1. G6PD catalyzes the regulated step in the pentose phosphate pathway. Which of the following statements concerning G6PD and the pentose phosphate pathway is correct? A. Deficiency of G6PD occurs only in RBC. B. Deficiency of G6PD results in an inability to keep glutathione in its functional, reduced form.

1	A. Deficiency of G6PD occurs only in RBC. B. Deficiency of G6PD results in an inability to keep glutathione in its functional, reduced form. C. The pentose phosphate pathway includes one reversible reductive reaction followed by a series of phosphorylated sugar interconversions. D. The reduced nicotinamide adenine dinucleotide phosphate (NADPH) product of the pentose phosphate pathway is utilized in processes such as fatty acid oxidation. Q2. The results of JF's CBC were consistent with a hemolytic anemia. Blood chemistry tests revealed an elevation in the bilirubin level. Which of the following statements concerning bilirubin is correct? A. Hyperbilirubinemia can cause deposition of bilirubin in the skin and sclerae resulting in jaundice. B. The solubility of bilirubin is increased by conjugating it with two molecules of ascorbic acid in the liver. C. The conjugated form of bilirubin increases in the blood with a hemolytic anemia.

1	B. The solubility of bilirubin is increased by conjugating it with two molecules of ascorbic acid in the liver. C. The conjugated form of bilirubin increases in the blood with a hemolytic anemia. D. Phototherapy can increase the solubility of the excess bilirubin generated in the porphyrias. Q3. Why is urinary urobilinogen increased relative to normal in hemolytic jaundice and absent in obstructive jaundice? Case 7: Joint Pain Patient Presentation: IR is a 22-year-old male who presents for follow-up 10 days after having been treated in the Emergency Department (ED) for severe inflammation at the base of his thumb.

1	Patient Presentation: IR is a 22-year-old male who presents for follow-up 10 days after having been treated in the Emergency Department (ED) for severe inflammation at the base of his thumb. Focused History: This was IR’s first occurrence of severe joint pain. In the ED, he was given an anti-inflammatory medication. Fluid aspirated from the carpometacarpal joint of the thumb was negative for organisms but positive for needle-shaped monosodium urate (MSU) crystals (see image at right). The inflammatory symptoms have since resolved. IR reports he is in good health otherwise, with no significant past medical history. His body mass index (BMI) is 31. No tophi (deposits of MSU crystals under the skin) were detected in the physical examination.

1	Pertinent Findings: Results on a 24-hour urine specimen and blood tests requested in advance of this visit reveal that IR is not an undersecretor of uric acid. His blood urate was 8.5 mg/dl (reference = 2.5–8.0). The unusually young age of presentation is suggestive of an enzymopathy of purine metabolism, and additional blood tests are ordered. Diagnosis: IR has gout (MSU crystal deposition disease), a type of inflammatory arthritis. Treatment: IR was given prescriptions for allopurinol and colchicine. The treatment goals are to reduce his blood urate levels to <6.0 mg/dl and prevent additional attacks. He was advised to lose weight because being overweight or obese is a risk factor for gout. His BMI of 31 puts him in the obese category. He was also given written information on the association between diet and gout. Prognosis: Gout increases the risk of developing renal stones. It is also associated with hypertension, diabetes, and heart disease.

1	Prognosis: Gout increases the risk of developing renal stones. It is also associated with hypertension, diabetes, and heart disease. Case-Related Questions: Choose the ONE best answer. Q1. Allopurinol is converted in the body to oxypurinol, which functions as a noncompetitive inhibitor of an enzyme in purine metabolism. Which of the following statements concerning purine metabolism and its regulation is correct? A. As a noncompetitive inhibitor, oxypurinol increases the apparent Michaelis constant (Km) of the target enzyme. B. Colchicine inhibits xanthine oxidase, an enzyme of purine degradation. C. Glutamate provides two of the nitrogen atoms of the purine ring. D. In purine nucleotide synthesis, the ring system is first constructed and then attached to ribose 5-phosphate. E. Oxypurinol inhibits the amidotransferase that initiates degradation of the purine ring system.

1	E. Oxypurinol inhibits the amidotransferase that initiates degradation of the purine ring system. F. Partial or complete enzymic deficiencies in the salvage of purine bases are characterized by hyperuricemia. Q2. Purines are one type of nitrogenous base found in nucleotides. Pyrimidines are the other. Which of the following statements is true of the pyrimidines? A. Carbamoyl phosphate synthetase I is the regulated enzymic activity in pyrimidine ring synthesis. B. Methotrexate decreases synthesis of the pyrimidine nucleotide thymidine monophosphate. C. Orotic aciduria is a pathology of pyrimidine degradation. D. Pyrimidine nucleotide synthesis is independent of 5-phosphoribosyl-1pyrophosphate (PRPP). Q3. IR is subsequently shown to have a form of PRPP synthetase that shows increased enzymic activity. Why does this result in hyperuricemia? Case 8: No Bowel Movement Patient Presentation: DW is a 48-hour-old female who has not yet had a bowel movement.

1	Case 8: No Bowel Movement Patient Presentation: DW is a 48-hour-old female who has not yet had a bowel movement. Focused History: DW is the full-term product of a normal pregnancy and delivery. She appeared normal at birth. DW is the first child of parents of Northern European ethnicity. The parents are both in good health, and their family histories are unremarkable. Pertinent Findings: DW has a distended abdomen. She recently vomited small amounts of bilious (green-colored) material. Diagnosis: Meconium ileus (obstruction of the ileum by meconium, the first stool produced by newborns) was confirmed by abdominal x-rays. About 98% of full-term newborns with meconium ileus have cystic fibrosis (CF). Diagnosis of CF was subsequently confirmed with a chloride sweat test. Treatment: The ileus was successfully treated nonsurgically. For management of the CF, the family was referred to the CF center at the regional children’s hospital.

1	Treatment: The ileus was successfully treated nonsurgically. For management of the CF, the family was referred to the CF center at the regional children’s hospital. Prognosis: CF is the most common life-limiting autosomal-recessive disease in Caucasians. Case-Related Questions: Choose the ONE best answer. Q1. CF is the result of mutations to the gene that encodes the CF transmembrane conductance regulator (CFTR) protein that functions as a chloride channel in the apical membrane of epithelial cells on a mucosal surface. Which of the following statements concerning CF is correct? A. Clinical manifestations of CF are the consequence of chloride retention with increased water reabsorption that causes mucus on the epithelial surface to be excessively thick and sticky. B. Excessive pancreatic secretion of insulin in CF commonly results in hypoglycemia.

1	B. Excessive pancreatic secretion of insulin in CF commonly results in hypoglycemia. C. Genetic testing for CF may involve the use of a set of probes for the most common mutations, a technique known as restriction fragment length polymorphism analysis. D. Some mutations result in premature degradation of the CFTR protein through tagging with ubiquinone followed by proteasome-mediated proteolysis. E. The most common mutation, ∆F508, results in the loss of a codon for phenylalanine (F) and is classified as a frameshift mutation. Q2. The CFTR protein is an intrinsic plasma membrane glycoprotein. Targeting of proteins destined to function as components of membranes: A. includes transport to and through the Golgi. B. involves an amino-terminal signal sequence that is retained in the functional protein. C. occurs after the protein has been completely synthesized (that is, posttranslationally).

1	B. involves an amino-terminal signal sequence that is retained in the functional protein. C. occurs after the protein has been completely synthesized (that is, posttranslationally). D. requires the presence of mannose 6-phosphate residues on the protein. Q3. Why might steatorrhea be seen with CF? Case 9: Elevated Ammonia Patient Presentation: RL is a 40-hour-old male with signs of cerebral edema. Focused History: RL is the full-term product of a normal pregnancy and delivery. He appeared normal at birth. At age 36 hours, he became irritable, lethargic, and hypothermic. He fed only poorly and vomited. He also displayed tachypneic (rapid) breathing and neurologic posturing. At age 38 hours, he had a seizure.

1	Pertinent Findings: Respiratory alkalosis (increased pH, decreased CO2 [hypocapnia]), increased ammonia, and decreased blood urea nitrogen were found. An amino acid screen revealed that argininosuccinate was increased >60fold over baseline, and citrulline was increased 4-fold. Glutamine was elevated, and arginine (Arg) was decreased relative to normal. Diagnosis: RL has a urea cycle enzyme defect with neonatal onset. Treatment: Hemodialysis was performed to remove ammonia. Sodium phenylacetate and sodium benzoate were administered to aid in excretion of waste nitrogen, as was Arg. Long-term treatment will include lifelong limitation of dietary protein; supplementation with essential amino acids; and administration of Arg, sodium phenylacetate, and sodium phenylbutyrate. Prognosis: Survival into adulthood is possible. The degree of neurologic impairment is related to the degree and extent of the hyperammonemia. Case-Related Questions: Choose the ONE best answer.

1	Prognosis: Survival into adulthood is possible. The degree of neurologic impairment is related to the degree and extent of the hyperammonemia. Case-Related Questions: Choose the ONE best answer. Q1. Based on the findings, which enzyme of the urea cycle is most likely to be deficient in this patient? A. Arginase B. Argininosuccinate lyase C. Argininosuccinate synthetase D. Carbamoyl phosphate synthetase I E. Ornithine transcarbamoylase Q2. Why is Arg supplementation helpful in this case? Q3. In individuals with partial (milder) deficiency of urea cycle enzymes, the level of which one of the following would be expected to be decreased during periods of physiologic stress? A. Alanine B. Ammonia C. Glutamine D. Insulin E. pH Case 10: Calf Pain Patient Presentation: CR is a 19-year-old female who is being evaluated for pain and swelling in her right calf.

1	A. Alanine B. Ammonia C. Glutamine D. Insulin E. pH Case 10: Calf Pain Patient Presentation: CR is a 19-year-old female who is being evaluated for pain and swelling in her right calf. Focused History: Ten days ago, CR had her spleen removed following a bicycle accident in which she fractured her tibial eminence, necessitating immobilization of the right knee. She has had a good recovery from the surgery. CR is no longer taking pain medication but has continued her oral contraceptives (OCP). Pertinent Findings: CR’s right calf is reddish in color (erythematous) and warm to the touch. It is visibly swollen. The left calf is normal in appearance and is without pain. An ultrasound is ordered. Diagnosis: CR has a deep venous thrombosis (DVT). OCP are a risk factor for DVT, as are surgery and immobilization. Treatment (Immediate): Heparin and warfarin are administered. Prognosis: In the 10 years following a DVT, about one third of individuals have a recurrence.

1	Treatment (Immediate): Heparin and warfarin are administered. Prognosis: In the 10 years following a DVT, about one third of individuals have a recurrence. Case-Related Questions: Choose the ONE best answer. Q1. A DVT is a blood clot that occludes the lumen of a deep vein, most commonly in the leg. Which of the following statements about the clotting cascade is correct? A. A deficiency in factor (F)IX of the intrinsic pathway results in hemophilia A. B. FIII of the extrinsic pathway is a serine protease. C. Formation of the fibrin meshwork is referred to as primary hemostasis. D. Thrombin proteolytically activates components of the extrinsic, intrinsic, and common pathways. E. Vitamin K is required for the activation of fibrinogen. Q2. Which one of the following would increase the risk of thrombosis? A. Excess production of antithrombin B. Excess production of protein S C. Expression of FV Leiden D. Hypoprothrombinemia

1	A. Excess production of antithrombin B. Excess production of protein S C. Expression of FV Leiden D. Hypoprothrombinemia E. von Willebrand disease Q3. Compare and contrast the actions of heparin and warfarin. Focused Cases: Answers to Case-Based Questions Case 1: Anemia with β-Thalassemia Minor

1	E. von Willebrand disease Q3. Compare and contrast the actions of heparin and warfarin. Focused Cases: Answers to Case-Based Questions Case 1: Anemia with β-Thalassemia Minor Answer = B. Transcription (synthesis of single-stranded RNA from the template strand of double-stranded DNA) requires the binding of proteins (trans-acting factors) to sequences on the DNA (cis-acting elements). Eukaryotic messenger RNA (mRNA) is monocistronic because it contains information from just one gene (cistron). The base sequence TAG (thymine adenine guanine) in the coding strand of DNA is U(uracil)AG in the mRNA. UAG is a signal that terminates translation (protein synthesis), not transcription. It is formation of the 5′-cap of eukaryotic mRNA that requires methylation (using S-adenosylmethionine), not 3′-end polyadenylation. Splicing is the spliceosome-mediated process by which introns are removed from eukaryotic mRNA and exons joined.

1	Answer = C. Carbon monoxide (CO) increases the affinity of hemoglobin (Hb)A for O2, thereby decreasing the ability of HbA to offload O2 in the tissues. CO stabilizes the R (relaxed) or oxygenated form and shifts the O2 dissociation curve to the left, decreasing O2 delivery (see figure at top right). The other choices decrease the affinity for O2, stabilize the T (tense) or deoxygenated form, and cause a right shift in the curve. HbA2 and fetal Hb (HbF) do not contain β globin. As β globin production decreases, synthesis of HbA2 (α2δ2) and HbF (α2γ2) increases.

1	Sickle cell anemia is caused by a single point mutation (A→T) in the gene for β globin that results in the replacement of glutamate by valine at the sixth amino acid position in the protein. Mutational analysis using allele-specific oligonucleotide (ASO) probes for that mutation (βS) and for the normal sequence (βA) is used in diagnosis (see figure at lower right). β-Thalassemia, in contrast, is caused by hundreds of different mutations. Mutational analysis using ASO probes can assess common mutations, including point mutations, in at-risk populations (for example, those of Greek ancestry). However, less common mutations are often not included in the panel and can be detected only by DNA sequencing. Case 2: Skin Rash with Lyme Disease

1	Case 2: Skin Rash with Lyme Disease Answer = C. Peptide-bond formation between the amino acid in the A site of the ribosome and the amino acid last added to the growing peptide in the P site is catalyzed by an RNA of the large ribosomal subunit. Any RNA with catalytic activity is referred to as a ribozyme (see figure on the next page). Formylated methionine is used to initiate prokaryotic translation. The charged initiating transfer RNA (tRNAi) is the only tRNA that goes directly to the P site, leaving the A site available for the tRNA carrying the next amino acid of the protein being made. Eukaryotic translation is inhibited by the phosphorylation of initiation factor 2 (eIF-2). The Shine-Dalgarno sequence is found in prokaryotic messenger RNA (mRNA) and facilitates the interaction of the mRNA with the small ribosomal subunit. In eukaryotes, the cap-binding proteins perform that task.

1	Answer = B. The enzyme-linked immunosorbent assay (ELISA) and western blot are used to analyze proteins. Each makes use of antibodies to detect and quantify the protein of interest. It is western blots that utilize electrophoresis. The polymerase chain reaction (PCR) is used to amplify DNA. Antibiotics in the tetracycline family inhibit protein synthesis by binding to and blocking the A site of the small (30S) ribosomal subunit in prokaryotes. Tetracycline specifically interacts with the 16S ribosomal RNA (rRNA) component of the 30S subunit, inhibiting translation initiation. Eukaryotes do not contain 16S rRNA. Their small (40S) subunit contains 18S rRNA, which does not bind tetracycline. Case 3: Blood on the Toothbrush with Vitamin C Deficiency

1	Case 3: Blood on the Toothbrush with Vitamin C Deficiency Answer = C. Vitamin C (ascorbic acid) functions as a coenzyme in the hydroxylation of proline and lysine in the synthesis of collagen, a fibrous protein of the extracellular matrix. Vitamin C is also the coenzyme for duodenal cytochrome b (Dcytb) that reduces dietary iron from the ferric (Fe3+) to the ferrous (Fe2+) form that is required for absorption via the divalent metal transporter (DMT) of enterocytes (see figure below). With a deficiency of vitamin C, uptake of dietary iron is impaired and results in a microcytic, hypochromic anemia. As a water-soluble vitamin, vitamin C is not stored. Cross-linking of collagen by lysyl oxidase requires copper, not vitamin C.

1	Answer = A. An inability to absorb vitamin B12 leads to pernicious anemia and is most commonly caused by decreased production of intrinsic factor (IF) by the parietal cells of the stomach (see figure at right). Vitamins D and A, in complex with their receptors, bind to DNA and alter gene expression. Thiamine (vitamin B1) is a coenzyme in the oxidative decarboxylation of pyruvate and αketoglutarate and, therefore, is important in energy metabolism in most cells. Methotrexate inhibits dihydrofolate reductase, the enzyme that reduces dihydrofolate to tetrahydrofolate (THF), the functional coenzyme form of folate. This results in decreased availability of THF. It is pyridoxine (vitamin B6) as pyridoxal phosphate that is the coenzyme for most reactions involving amino acids. [Note: Tetrahydrobiopterin is required by aromatic amino acid hydroxylases and nitric oxide synthases.] Nutritional anemias are characterized by either increased red blood cell (RBC) size (folate and B12 deficiencies)

1	is required by aromatic amino acid hydroxylases and nitric oxide synthases.] Nutritional anemias are characterized by either increased red blood cell (RBC) size (folate and B12 deficiencies) or decreased RBC size (iron and vitamin C deficiencies). In hemolytic anemias, such as is seen in glucose 6-phosphate dehydrogenase and pyruvate kinase deficiencies and in sickle cell anemia, RBC size typically is normal, and RBC number is decreased.

1	Case 4: Rapid Heart Rate, Headache, and Sweating with a Pheochromocytoma Answer = D. Degradation of both epinephrine and norepinephrine (NE) involves methylation by catechol-O-methyltransferase (COMT) that produces normetanephrine from NE and metanephrine from epinephrine (see figure at right). Both of these products are deaminated to vanillylmandelic acid by monoamine oxidase (MAO). The substrate for the synthesis of the catecholamines is tyrosine, which gets hydroxylated to 3,4dihydroxyphenylalanine (DOPA) by tetrahydrobiopterin-requiring tyrosine hydroxylase. DOPA is converted to dopamine by a pyridoxal phosphate– requiring decarboxylase. [Note: Most carboxylases require biotin.] NE is converted to epinephrine by methylation, and S-adenosylmethionine provides the methyl group.

1	Answer = A. NE released from the sympathetic nervous system functions as a neurotransmitter that acts on postsynaptic neurons and causes, for example, increased heart rate. It is also released from the adrenal medulla and, along with epinephrine, functions as a counterregulatory hormone that results in mobilization of stored fuels (for example, glucose and triacylglycerols). These actions are mediated by the binding of NE to adrenergic receptors, which are G protein–coupled receptors of the plasma membrane, and not to nuclear receptors like those of steroid hormones or membrane tyrosine kinase receptors like that of insulin. Septic shock is vasodilatory hypotension (low blood pressure caused by blood vessel dilation) resulting from the production of large amounts of nitric oxide by inducible nitric oxide synthase in response to infection. NE bound to receptors on smooth muscle cells causes vasoconstriction and, thus, raises blood pressure.

1	Case 5: Sun Sensitivity with Xeroderma Pigmentosum

1	Answer = D. Pyrimidine dimers are the characteristic DNA lesions caused by ultraviolet (UV) radiation. Their repair involves the excision of an oligonucleotide containing the dimer and replacement of that oligonucleotide, a process known as nucleotide excision repair (NER). (See figure at right for a representation of the process in prokaryotes.) DNA repair systems are found in prokaryotes and eukaryotes. Nothing is error free, but the homologous recombination (HR) method of double-strand break repair is much less prone to error than is the nonhomologous end joining (NHEJ) method because any DNA that was lost is replaced. Mismatched-base repair (MMR) involves identification and repair of the newly synthesized (daughter) strand. In prokaryotes, the extent of strand methylation is used to discriminate between the strands. Base excision repair (BER), the mechanism by which uracil is removed from DNA, utilizes a glycosylase to remove the base, creating an apyrimidinic or apurinic (AP)

1	discriminate between the strands. Base excision repair (BER), the mechanism by which uracil is removed from DNA, utilizes a glycosylase to remove the base, creating an apyrimidinic or apurinic (AP) site. The sugar-phosphate is then removed by the actions of an endo-and exonuclease.

1	Answer = A. All replication requires an RNA primer because DNA polymerases (pol) cannot initiate DNA synthesis. The chromatin of eukaryotes gets decondensed (relaxed) for replication. Relaxation can be accomplished, for example, by acetylation via histone acetyltransferases. Prokaryotes have more than one DNA pol. For example, pol III extends the RNA primer with DNA, and pol I removes the primer and replaces it with DNA. Replication is initiated at specific locations (one in prokaryotes, many in eukaryotes) that are recognized by proteins (for example, DnaA in prokaryotes). Deoxynucleoside monophosphates (dNMP) are joined by a phosphodiester bond that links the 3′hydroxyl group of the last dNMP added with the 5′-phosphate group of the incoming nucleotide, thereby forming a 3′→5′-phosphodiester bond as pyrophosphate is released. Proofreading occurs during replication in the S (synthesis of DNA) phase of the cell cycle and involves the 3′→5′ exonuclease activity possessed by some DNA

1	bond as pyrophosphate is released. Proofreading occurs during replication in the S (synthesis of DNA) phase of the cell cycle and involves the 3′→5′ exonuclease activity possessed by some DNA pol (see figure below). Because repair can occur independently of replication, it can be performed outside of the S phase.

1	Case 6: Dark Urine and Yellow Sclerae with

1	Answer = B. Glutathione in its reduced form (G-SH) is an important antioxidant. The selenium-containing enzyme glutathione peroxidase reduces hydrogen peroxide (H2O2, a reactive oxygen species) to water as glutathionine is oxidized (G-S-S-G). Reduced nicotinamide adenine dinucleotide phosphate (NADPH)requiring glutathionine reductase regenerates G-SH from G-S-S-G (see Figure A). The NADPH is supplied by the oxidative reactions of the pentose phosphate pathway (see Figure B), which is regulated by the availability of NADPH at the glucose 6-phosphate dehydrogenase (G6PD)-catalyzed step (the first step). Deficiency of G6PD occurs in all cells, but the effects are seen in red blood cells where the pentose phosphate pathway is the only source of NADPH. The pathway involves two irreversible oxidative reactions, each of which generates NADPH. The NADPH is used in reductive processes such as fatty acid synthesis (not oxidation) as well as steroid hormone and cholesterol synthesis.

1	Answer = A. Jaundice (icterus) refers to the yellow color of the skin, nail beds, and sclerae that results from bilirubin deposition when the bilirubin level in the blood is elevated (hyperbilirubinemia; see Image C). Bilirubin has low solubility in aqueous solutions, and its solubility is increased by conjugation with uridine diphosphate–glucuronic acid in the liver, forming bilirubin diglucuronide or conjugated bilirubin (CB). In hemolytic conditions, such as G6PD deficiency, both CB and unconjugated bilirubin (UCB) are increased, but it is UCB that is found in the blood. CB is sent into the intestine. Phototherapy is used to treat unconjugated hyperbilirubinemia because it converts bilirubin to isomeric forms that are more water soluble. Bilirubin is the product of heme degradation in cells of the mononuclear phagocyte system, particularly in the liver and the spleen. The porphyrias are pathologies of heme synthesis and, therefore, are not characterized by hyperbilirubinemia.

1	With hemolysis, more bilirubin is produced and conjugated. CB is sent to the intestine where it is converted to urobilinogen, some of which is reabsorbed, enters the portal blood, and travels to the kidney. Because the source of urinary urobilinogen is intestinal urobilinogen, urinary urobilinogen will be low in obstructive jaundice because intestinal urobilinogen will be low as a result of the obstruction of the common bile duct (see Figure D). Case 7: Joint Pain with Gout

1	Case 7: Joint Pain with Gout Answer = F. Salvage of the purine bases hypoxanthine and guanine to the purine nucleotides inosine monophosphate (IMP) and guanosine monophosphate (GMP) by hypoxanthine-guanine phosphoribosyltransferase (HGPRT) requires 5-phosphoribosyl-1-pyrophosphate (PRPP) as the source of the ribose 1phosphate. Salvage decreases the amount of substrate available for degradation to uric acid. Therefore, a deficiency in salvage results in hyperuricemia (see figure at right). Noncompetitive inhibitors such as oxypurinol have no effect on the Michaelis constant (Km) but decrease the apparent maximal velocity (Vmax).

1	Colchicine is an anti-inflammatory drug. It has no effect on the enzymes of purine synthesis or degradation. Glutamine (not glutamate) is a nitrogen source for purine ring synthesis. In purine nucleotide synthesis, the purine ring system is constructed on the ribose 5-phosphate provided by PRPP. Allopurinol and its metabolite, oxypurinol, inhibit xanthine oxidase of purine degradation. The amidotransferase is the regulated enzyme of purine synthesis. Its activity is decreased by purine nucleotides and increased by PRPP.

1	Answer = B. Methotrexate inhibits dihydrofolate reductase, decreasing the availability of N5,N10-methylene tetrahydrofolate needed for synthesis of deoxythymidine monophosphate (dTMP) from deoxyuridine monophosphate (dUMP) by thymidylate synthase (see figure at right). Carbamoyl phosphate synthetase (CPS) II is the regulated enzymic activity of pyrimidine biosynthesis in humans. CPS I is an enzyme of the urea cycle. Orotic aciduria is a rare pathology of pyrimidine synthesis caused by a deficiency in one or both enzymic activities of bifunctional uridine monophosphate synthase. Pyrimidine nucleotide synthesis, like purine synthesis and salvage, requires PRPP. Increased activity of PRPP synthetase results in increased synthesis of PRPP. This results in an increase in purine nucleotide synthesis beyond need. The excess purine nucleotides get degraded to uric acid, thereby causing hyperuricemia. Case 8: No Bowel Movement with Cystic Fibrosis

1	Answer = A. The clinical manifestations of cystic fibrosis (CF) are the consequence of chloride retention with increased water absorption that causes mucus on an epithelial surface to be excessively thick and sticky. The result is pulmonary and gastrointestinal problems such as respiratory infection and impaired exocrine and endocrine pancreatic functions (pancreatic insufficiency). Impaired endocrine pancreatic function can result in diabetes with associated hyperglycemia. The genetic testing technique described, and one used in the diagnosis of CF, is the use of allele-specific oligonucleotides (ASO). Some mutations do result in increased degradation of the CF transmembrane conductance regulator (CFTR) protein, but degradation is initiated by tagging the protein with ubiquitin. Frameshift mutations alter the reading frame through the addition or deletion of nucleotides by a number not divisible by three. Because the ∆F509 mutation is caused by the loss of three nucleotides that code

1	mutations alter the reading frame through the addition or deletion of nucleotides by a number not divisible by three. Because the ∆F509 mutation is caused by the loss of three nucleotides that code for phenylalanine (F) at position 509 in the CFTR protein, it is not a frameshift mutation. Answer = A. Targeting of proteins destined to function as components of the plasma membrane is an example of cotranslational targeting. It involves the initiation of translation on cytosolic ribosomes; recognition of the amino (N)terminal signal sequence in the protein by the signal recognition particle; movement of the protein-synthesizing complex to the outer face of the membrane of the endoplasmic reticulum (ER); and continuation of protein synthesis, such that the protein is threaded into the lumen of the ER and packaged into vesicles that travel to and through the Golgi and eventually fuse with the plasma membrane. The N-terminal signal sequence is removed by a peptidase in the lumen of the ER.

1	the ER and packaged into vesicles that travel to and through the Golgi and eventually fuse with the plasma membrane. The N-terminal signal sequence is removed by a peptidase in the lumen of the ER. Mannose 6-phosphate is the signal that cotranslationally targets proteins to the matrix of the lysosome where they function as acid hydrolases.

1	The pancreatic insufficiency seen in some patients with CF results in a decreased ability to digest food, and digestion is required for absorption. Dietary fats move through the intestine and are excreted in the stool (see figure at right), which is foul-smelling and bulky and may float. Patients are at risk for malnutrition and deficiencies in fat-soluble vitamins. Oral supplementation of pancreatic enzymes is the treatment. Case 9: Hyperammonemia with a Urea Cycle Defect

1	Answer = B. Argininosuccinate lyase (ASL) cleaves argininosuccinate to arginine (Arg) and fumarate. The increase in argininosuccinate and citrulline and the decrease in Arg seen in RL indicate a deficiency in ASL (see figure below). With arginase deficiency, Arg would be increased, not decreased. Additionally, with arginase deficiency, the hyperammonemia would be less severe because two nitrogens are excreted. Deficiency of argininosuccinate synthetase (ASS) would also cause an increase in citrulline, but argininosuccinate would be low to absent. Deficiency of carbamoyl phosphate synthetase (CPS) I is characterized by low levels of Arg and citrulline. Deficiency of ornithine transcarbamoylase (OTC), the only X-linked enzyme of the urea cycle, would result in low levels of Arg and citrulline and elevated levels of urinary orotic acid. [Note: The orotic acid is elevated because the carbamoyl phosphate (CP) substrate of OTC is being used in the cytosol as a substrate for pyrimidine

1	and elevated levels of urinary orotic acid. [Note: The orotic acid is elevated because the carbamoyl phosphate (CP) substrate of OTC is being used in the cytosol as a substrate for pyrimidine synthesis.]

1	Arg supplementation is helpful because the Arg will be hydrolyzed to urea + ornithine by arginase. The ornithine will be combined with CP to form citrulline (see figure above). With ASL (and ASS) deficiency, citrulline accumulates and is excreted, thereby carrying waste nitrogen out of the body. Answer = D. In individuals with milder (partial) deficiencies in the enzymes of the urea cycle, hyperammonemia may be triggered by physiologic stress (for example, an illness or prolonged fasting) that decreases the insulin/counterregulatory hormone ratio. [Note: The degree of the hyperammonemia is usually less severe than that seen in the neonatal onset forms.] The shift in the ratio results, in part, in skeletal muscle proteolysis, and the amino acids that are released get degraded. Degradation involves transamination by pyridoxal phosphate–requiring aminotransferases that generate the α-keto acid derivative of the amino acid + glutamate. The glutamate undergoes oxidative deamination to

1	involves transamination by pyridoxal phosphate–requiring aminotransferases that generate the α-keto acid derivative of the amino acid + glutamate. The glutamate undergoes oxidative deamination to α-ketoglutarate and ammonia (NH3) by glutamate dehydrogenase (GDH; see figure at right). [Note: GDH is unusual in that it uses both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzymes.]

1	The NH3, which is toxic, can be transported to the liver as glutamine (Gln) and alanine (Ala). The Gln is generated by the amination of glutamate by ATP-requiring glutamine synthetase. In the liver, the enzyme glutaminase removes the NH3, which can be converted to urea by the urea cycle or excreted as ammonium (NH4+) (see figure at right). Gln, then, is a nontoxic vehicle of NH3 transport in the blood. Ala is generated in skeletal muscle from the catabolism of the branched-chain amino acids (BCAA). In the liver, Ala is transaminated by alanine transaminase (ALT) to pyruvate (used in gluconeogenesis) and glutamate. Thus, Ala carries nitrogen to the liver for conversion to urea (see figure below). Therefore, defects in the urea cycle would result in an elevation in NH3, Gln, and Ala. The elevated NH3 drives respiration, and the hyperventilation causes a rise in pH (respiratory alkalosis). [Note: Hyperammonemia is toxic to the nervous system. Although the exact mechanisms are not

1	The elevated NH3 drives respiration, and the hyperventilation causes a rise in pH (respiratory alkalosis). [Note: Hyperammonemia is toxic to the nervous system. Although the exact mechanisms are not completely understood, it is known that the metabolism of large amounts of NH3 to Gln (in the astrocytes of the brain) results in osmotic effects that cause the brain to swell. Additionally, the rise in Gln decreases the availability of glutamate, an excitatory neurotransmitter.]

1	Case 10: Swollen, Painful Calf with Deep Venous Thrombosis Answer = D. Thrombin, a serine protease, is activated by the prothrombinase complex of factor (F)Xa + FVa. Once formed, activated thrombin (FIIa) proteolytically activates components of the extrinsic (FVII) and intrinsic (FXI, FVIII) pathways, generating FXa. Thrombin can also activate FV, FI, and FXIII of the common pathway (see figure below). Hemophilia A is caused by a deficiency in FVIII. FIX deficiency results in hemophilia B. FIII, also known as tissue factor (TF), is a transmembrane glycoprotein of the vascular endothelium. It functions as an accessory protein and not a protease. Formation of the platelet plug is primary hemostasis, and formation of the fibrin meshwork is secondary hemostasis. Vitamin K is required for the activation (γ-carboxylation) of FII, FVII, FIX, and FX (proteases that require calcium [Ca2+] and phospholipids [PL]) but not for FI (fibrinogen).

1	Answer = C. FV Leiden is a mutant form of FV that is resistant to proteolysis by the activated protein C complex. Decreased ability to degrade FV allows continued production of activated thrombin and leads to an increased risk of clot formation or thrombophilia. Antithrombin III (ATIII) and protein S are proteins of anticoagulation. Increased, not decreased, production of prothrombin would result in thrombophilia. Deficiency of von Willebrand factor causes a coagulopathy or a deficiency in clotting through effects on FVIII and platelets. Heparin and warfarin are anticoagulants. Heparin, a glycosaminoglycan, increases the affinity of ATIII for thrombin. Binding of ATIII removes thrombin from the blood and prevents it from converting fibrinogen to fibrin. Warfarin, a synthetic analog of vitamin K, inhibits vitamin K epoxide reductase and prevents the regeneration of the functional hydroquinone form of the vitamin that is required for the γ-carboxylation of glutamate residues to

1	of vitamin K, inhibits vitamin K epoxide reductase and prevents the regeneration of the functional hydroquinone form of the vitamin that is required for the γ-carboxylation of glutamate residues to γ-carboxyglutamate (Gla) residues in FII, FVII, FIX, and FX (see figures below).

1	Database of High-Yield Facts The seventh edition of First Aid for the USMLE Step 2 CK contains a revised and expanded database of clinical material that student authors and faculty have identified as high yield for boards review. The facts are organized according to subject matter, whether medical specialty (e.g., Cardiovascular, Renal) or high-yield topic (e.g., Ethics) in medicine. Each subject is then divided into smaller subsections of related facts. Individual facts are generally presented in a logical approach, from basic definitions and epidemiology to History/Physical Exam, Diagnosis, and Treatment. Lists, mnemonics, and tables are used when helpful in forming key associations.

1	The content is mostly useful for reviewing material already learned. This section is not ideal for learning complex or highly conceptual material for the first time. Black-and-white images appear throughout the text. In some cases, reference is made to the “clinical image” section at the end of Section 2, which contains full-color glossy plates of histology and patient pathology by topic. At the end of Section 2, we also feature a Rapid Review chapter of key facts and classic associations to cram a day or two before the exam. The Database of High-Yield Facts is not comprehensive. Use it to complement your core study material and not as your primary study source. The facts and notes have been condensed and edited to emphasize the essential material. Work with the material, add your own notes and mnemonics, and recognize that not all memory techniques work for all students.

1	We update Section 2 biannually to keep current with new trends in boards content as well as to expand our database of high-yield information. However, we must note that inevitably many other high-yield entries and topics are not yet included in our database. We actively encourage medical students and faculty to submit entries and mnemonics so that we may enhance the database for future students. We also solicit recommendations of additional tools for study that may be useful in preparing for the examination, such as diagrams, charts, and computer-based tutorials (see How to Contribute, p. xv).

1	The entries in this section reﬂect student opinions of what is high yield. Owing to the diverse sources of material, no attempt has been made to trace or reference the origins of entries individually. We have regarded mnemonics as essentially in the public domain. All errors and omissions will be gladly corrected if brought to the attention of the authors, either through the publisher or directly by e-mail. To evaluate patients for cardiac abnormalities, methodically assess the ECG for rate, rhythm, axis, intervals, waveforms, and chamber enlargement (see Figures 2.1-1 and 2.1-2). The normal heart rate is 60–100 bpm. A rate < 60 bpm is bradycardia; > 100 bpm is tachycardia. Look for sinus rhythm (P before every QRS and QRS after every P), irregular rhythms, junctional or ventricular rhythms (no P before a QRS), and ectopic beats. Normal: An upright (positive) QRS in leads I and II (–30 degrees to +105 degrees).

1	Normal: An upright (positive) QRS in leads I and II (–30 degrees to +105 degrees). Left-axis deviation: An upright QRS in lead I and a downward (negative) QRS in lead II (< –30 degrees). Right-axis deviation: A downward QRS in lead I and an upright QRS in lead II (> +105 degrees). II aVRI III aVL aVF V1 V2 V3 V4 V5 V6 F IGU R E 2.1 -1. Normal electrocardiogram from a healthy subject. Quickly estimate heart rate by counting the number of large boxes subtended by two consecutive QRS complexes, as follows: 300-150-100-75-6050-30 bpm. An upright QRS in leads I and ll—the “double thumbs-up” sign—signif es a normal axis.

1	An upright QRS in leads I and ll—the “double thumbs-up” sign—signif es a normal axis. Sinus rhythm is present with a heart rate of 75 bpm. The PR interval is 0.16 sec; the QRS interval (duration) is 0.08 sec; the QT interval is 0.36 sec; QTc is 0.40 sec; and the mean QRS axis is about +70 degrees. The precordial leads show normal R-wave progression with the transition zone (R wave = S wave) in lead V3. (Reproduced, with permission, from Fauci AS et al. Harrison’s Principles of Internal Medicine, 17th ed. New York: McGraw-Hill, 2008: Fig. 221-7.) Time (s) 1.0 0.5 0 -0.5 0 0.2 0.4 0.6 P T S Q U Isoelectric line ST segment PR interval QT interval Potential (mV) Action potential QRS P T 0.2 0.4 0.6 Time (s) SA node "pacemaker" inherent dominance with slow phase of upstroke AV node 100-msec delay atrioventricular delay F IGU R E 2.1 -2. Electrocardiogram measurements. WiLLiaM MaRRoW: W pattern of QRS in V1–V2 and M pattern of QRS in V3–V6 for LBBB

1	F IGU R E 2.1 -2. Electrocardiogram measurements. WiLLiaM MaRRoW: W pattern of QRS in V1–V2 and M pattern of QRS in V3–V6 for LBBB M pattern of QRS in V1–V2 and W pattern of QRS in V3–V6 for RBBB Normal: PR interval between 120 and 200 msec and QRS < 100 msec. Atrioventricular (AV) block: PR interval > 200 msec, or P with no QRS afterward. Left bundle branch block (LBBB): QRS duration > 120 msec; no R wave in V1; wide, tall R waves in I, V5, and V6. Right bundle branch block (RBBB): QRS duration > 120 msec; RSR′ complex (“rabbit ears”); qR or R morphology with a wide R wave in V1; QRS pattern with a wide S wave in I, V5, and V6. Long QT syndrome: QTc > 440 msec. An underdiagnosed congenital disorder that predisposes to ventricular tachyarrhythmias. Ischemia: Inverted T waves; poor R-wave progression in precordial leads; ST-segment changes (elevation or depression).

1	Ischemia: Inverted T waves; poor R-wave progression in precordial leads; ST-segment changes (elevation or depression). Transmural infarct: Significant Q waves (> 40 msec or more than one-third of the QRS amplitude). ST elevation; T-wave inversion; the presence of possible impending infarction based on plaque instability. ■Atrial hypertrophy: Right atrial abnormality if P-wave amplitude in lead II is < 2.5 mm; left atrial abnormality if P-wave width in lead II is > 120 msec, or if terminal negative deﬂ ection in V1 is > 1 mm in amplitude and > 40 msec in duration. F IGU R E 2.1 -3. Left ventricular hypertrophy. Shown are leads V1, V2, V5, and V6. S wave in V2 + R wave in V5 = 55 mm. Note ST changes and T-wave inversion in V5 and V6, suggesting strain. (Reproduced, with permission, from Gomella LG et al. Clinician’s Pocket Reference, 11th ed. New York: McGraw-Hill, 2006: Fig. 19-27.) Left ventricular hypertrophy (LVH; see Figure 2.1-3):

1	Left ventricular hypertrophy (LVH; see Figure 2.1-3): Cornell criteria: Amplitude of R in aVL + S in V3 > 28 mm in men or > 20 mm in women. Sokolow-Lyon criteria: S in V1 + R in V5 or V6 > 35 mm. Right ventricular hypertrophy (RVH): Right-axis deviation and an R wave in V1 > 7 mm. Key exam findings that can narrow the differential include the following: Jugular venous distention (JVD, > 7 cm above sternal angle): Suggests right heart failure, pulmonary hypertension, volume overload, tricuspid regurgitation, or pericardial disease. Hepatojugular reﬂ ux: Fluid overload; impaired right ventricular compliance. Kussmaul’s sign (↑ in JVP with inspiration): Right ventricular infarction, postoperative cardiac tamponade, tricuspid regurgitation, constrictive pericarditis. Systolic murmurs: Aortic stenosis: Harsh systolic ejection murmur; radiation to carotids. Mitral regurgitation: Holosystolic murmur; radiation to axillae or to carotids.

1	Systolic murmurs: Aortic stenosis: Harsh systolic ejection murmur; radiation to carotids. Mitral regurgitation: Holosystolic murmur; radiation to axillae or to carotids. Mitral valve prolapse: Midsystolic or late-systolic click. Flow murmur: Very common, and does not imply cardiac disease. Diastolic murmurs: Always abnormal. Aortic regurgitation: Early decrescendo murmur. Mitral stenosis: Midto late, low-pitched murmur. Heart auscultation locations from the upper right sternal border, upper left sternal border, lower left sternal border, and apex: All (Aortic) People (Pulmonic) Try (Tricuspid) McDonald’s (Mitral). Causes of CHF— Hypertension Endocrine Anemia Rheumatic heart disease Toxins Failure to take meds Arrhythmia Infection Lung (pulmonary Gallops: S3 gallop: Dilated cardiomyopathy (ﬂoppy ventricle), mitral valve disease; often normal in younger patients and in high-output states (e.g., pregnancy).

1	Gallops: S3 gallop: Dilated cardiomyopathy (ﬂoppy ventricle), mitral valve disease; often normal in younger patients and in high-output states (e.g., pregnancy). S4 gallop: Hypertension, diastolic dysfunction (stiff ventricle), aortic stenosis; often normal in younger patients and athletes. Edema: Pulmonary: Left heart failure. Peripheral: Right heart failure and biventricular failure, peripheral venous disease, constrictive pericarditis, tricuspid regurgitation, hepatic disease, lymphedema; also nephrotic syndrome, hypoalbuminemia, and drugs. Peripheral pulses: ■↑: Compensated aortic regurgitation, coarctation (arms > legs), patent ductus arteriosus. ■↓: Peripheral arterial disease. Pulsus paradoxus (↓ systolic BP with inspiration): Pericardial tamponade; also asthma and COPD, tension pneumothorax, foreign body in airway. Pulsus alternans (alternating weak and strong pulses): Cardiac tamponade, impaired left ventricular systolic function; poor prognosis.

1	Pulsus alternans (alternating weak and strong pulses): Cardiac tamponade, impaired left ventricular systolic function; poor prognosis. Pulsus parvus et tardus (weak and delayed pulse): Aortic stenosis. Table 2.1-1 outlines the etiologies, clinical presentation, and treatment of common bradyarrhythmias and conduction abnormalities. Tables 2.1-2 and 2.1-3 outline the etiologies, clinical presentation, and treatment of common supraventricular and ventricular tachyarrhythmias.

1	Tables 2.1-2 and 2.1-3 outline the etiologies, clinical presentation, and treatment of common supraventricular and ventricular tachyarrhythmias. Defined as a clinical syndrome caused by the inability of the heart to pump enough blood to maintain ﬂuid and metabolic homeostasis. Risk factors include coronary artery disease (CAD), hypertension, cardiomyopathy, valvular heart disease, and diabetes. The American Heart Association/American College of Cardiology (AHA/ACC) guidelines classify heart failure according to clinical syndromes, but alternative classification systems include functional severity, left-sided vs. right-sided failure, and systolic vs. diastolic failure (see Tables 2.1-4 through 2.1-7). T AB LE 2.1 -1. Bradyarrhythmias and Conduction Abnormalities

1	Sinus bradycardia Normal response to cardiovascular conditioning; can also result from sinus node dysfunction or from β-blocker or calcium channel blocker (CCB) excess. May be asymptomatic, but may also present with lightheadedness, syncope, chest pain, or hypotension. Ventricular rate < 60 bpm; normal P wave before every QRS complex. None necessary if asymptomatic; atropine may be used to ↑ heart rate; pacemaker placement is the def nitive treatment in severe cases. First-degree AV block Can occur in normal individuals; associated with ↑ vagal tone and with β-blocker or CCB use. Asymptomatic. PR interval > 200 msec. None necessary. Second-degree AV block (Mobitz I/ Wenckebach) Drug effects (digoxin, β-blockers, CCBs) or ↑ vagal tone; sinoatrial conduction disease; right coronary ischemia or infarction. Usually asymptomatic. Progressive PR lengthening until a dropped beat occurs; PR interval then resets. Stop the offending drug. Atropine or pacemaker placement as clinically indicated.

1	or infarction. Usually asymptomatic. Progressive PR lengthening until a dropped beat occurs; PR interval then resets. Stop the offending drug. Atropine or pacemaker placement as clinically indicated. Second-degree AV block (Mobitz II) Results from f brotic disease of the conduction system or from acute, subacute, or prior myocardial infarction (MI). Occasionally syncope; frequent progression to third-degree AV block. Unexpected dropped beat(s) without a change in PR interval. Pacemaker placement. Third-degree AV block (complete) No electrical communication between the atria and ventricles. Syncope, dizziness, acute heart failure, hypotension, cannon A waves. No relationship between P waves and QRS complexes. Pacemaker placement. Sick sinus syndrome (SSS)/ tachycardia-bradycardia syndrome A heterogeneous disorder consisting of abnormalities in supraventricular impulse generation and conduction that lead to intermittent supraventricular tachy-and bradyarrhythmias. 2° to tachycardia or

1	A heterogeneous disorder consisting of abnormalities in supraventricular impulse generation and conduction that lead to intermittent supraventricular tachy-and bradyarrhythmias. 2° to tachycardia or bradycardia; may include syncope, palpitations, dyspnea, chest pain, TIA, and stroke. The most common indication for pacemaker placement.

1	T AB LE 2.1 -2. Supraventricular Tachyarrhythmias Atrial Sinus tachycardia Normal physiologic response to fear, pain, and exercise. Can also be 2° to hyperthyroidism, volume contraction, infection, or pulmonary embolism. Palpitations, shortness of breath. Ventricular rate > 100 bpm; normal P waves before every QRS complex. Treat the underlying cause. Atrial f brillation (AF) Acute AF— PIRATES: Pulmonary disease Ischemia Rheumatic heart disease Anemia/Atrial myxoma Thyrotoxicosis Ethanol Sepsis Chronic AF— hypertension, CHF. Often asymptomatic, but may present with shortness of breath, chest pain, or palpitations. Physical exam reveals irregularly irregular pulse. No discernible P waves, with variable and irregular QRS response. Estimate risk of stroke using CHAD2 score. Anticoagulation if > 48 hours (to prevent CVA); rate control (CCBs, β-blockers, digoxin, amiodarone).

1	Estimate risk of stroke using CHAD2 score. Anticoagulation if > 48 hours (to prevent CVA); rate control (CCBs, β-blockers, digoxin, amiodarone). Initiate cardioversion only if new onset (< 48 hours) or if transesophageal echocardiogram (TEE) shows no left atrial clot, or after 3–6 weeks of warfarin treatment with satisfactory INR (2–3). See the Hematology chapter for more information on warfarin. T AB LE 2.1 -2. Supraventricular Tachyarrhythmias (continued) Atrioventricular Circular movement Palpitations, shortness of breath, angina, A retrograde P Same as that for reciprocating of an impulse syncope, lightheadedness. wave is often AVNRT. tachycardia between the AV

1	Atrial f utter Circular movement of electrical activity around the atrium at a rate of 300 times per minute. Usually asymptomatic, but can present with palpitations, syncope, and lightheadedness. Regular rhythm; “sawtooth” appearance of P waves can be seen. Atrial rate is usually 240–320 bpm with varying degrees of blockade. Anticoagulation and rate control. Cardiovert according to AF criteria. Multifocal atrial tachycardia Multiple atrial pacemakers or reentrant pathways; COPD, hypoxemia. May be asymptomatic. Three or more unique P-wave Treat the underlying morphologies; or β-blockers for rate > 100 bpm. rate control and disorder; verapamil suppression of atrial pacemakers (not very effective). AV junction reentry tachycardia depolarizes Atrioventricular A reentry circuit Palpitations, shortness of breath, angina, Rate 150–250 Carotid massage, nodal (AVNRT) in the AV node the atrium and ventricle nearly simultaneously. syncope, lightheadedness. bpm; P wave is often buried Valsalva,

1	shortness of breath, angina, Rate 150–250 Carotid massage, nodal (AVNRT) in the AV node the atrium and ventricle nearly simultaneously. syncope, lightheadedness. bpm; P wave is often buried Valsalva, or adenosine in QRS or shortly after. arrhythmia. Cardiovert can stop the if hemodynamically unstable.

1	seen after a (AVRT) node and the normal QRS. atrium through a bypass tract. Seen in Wolff-Parkinson-White syndrome. Paroxysmal atrial tachycardia Rapid ectopic pacemaker in the atrium (not sinus node). Palpitations, shortness of breath, angina, syncope, lightheadedness. Rate > 100 bpm; P wave with an unusual axis before each normal QRS. Adenosine can be used to unmask underlying atrial activity. T AB LE 2.1 -3. Ventricular Tachyarrhythmias

1	Premature ventricular contraction (PVC) Ectopic beats arise from ventricular foci. Associated with hypoxia, electrolyte abnormalities, and hyperthyroidism. Usually asymptomatic, but may lead to palpitations. Early, wide QRS not preceded by a P wave. PVCs are usually followed by a compensatory pause. Treat the underlying cause. If symptomatic, give β-blockers or occasionally other antiarrhythmics. Ventricular tachycardia (VT) Can be associated with CAD, MI, and structural heart disease. Nonsustained VT is often asymptomatic; sustained ventricular tachycardia can lead to palpitations, hypotension, angina, and syncope. Can progress to VF. Three or more consecutive PVCs; wide QRS complexes in a regular rapid rhythm; AV dissociation. Cardioversion and antiarrhythmics (e.g., amiodarone, lidocaine, procainamide). Ventricular f brillation (VF) Associated with CAD and structural heart disease. Also associated with cardiac arrest (together with asystole). Syncope, absence of blood pressure,

1	procainamide). Ventricular f brillation (VF) Associated with CAD and structural heart disease. Also associated with cardiac arrest (together with asystole). Syncope, absence of blood pressure, pulselessness. Totally erratic wide-complex tracing. Immediate electrical cardioversion and ACLS protocol. Torsades de pointes Associated with long QT syndrome, proarrhythmic response to medications, hypokalemia, and congenital deafness. Can present with sudden cardiac death; typically associated with palpitations, dizziness, and syncope. Polymorphous QRS; VT with rates between 150 and 250 bpm. Correct hypokalemia; withdraw offending drugs. Give magnesium initially and cardiovert if unstable.

1	T AB LE 2.1 -4. AHA/ACC Classifcation and Treatment of CHF A B Patients with structural heart disease (e.g., a history of MI, left ventricular systolic dysfunction, or valvular disease) who have never had symptoms of CHF. ACEIs, β-blockers. Patients with structural heart disease who have prior or current symptoms of CHF (shortness of breath, fatigue, ↓ exercise tolerance). Treatment includes diuretics, ACEIs, β-blockers, digitalis, and dietary salt restriction. Patients with marked symptoms of CHF at rest despite maximal medical therapy. C D Patients who are at high risk of developing CHF because of the presence of risk factors, but who have no identifed structural or functional abnormalities and no signs or symptoms of CHF. Manage treatable risk factors (hypertension, smoking, hyperlipidemia, obesity, exercise, alcohol abuse). ACEIs can be used in patients with atherosclerotic vascular disease, DM, or hypertension.

1	Manage treatable risk factors (hypertension, smoking, hyperlipidemia, obesity, exercise, alcohol abuse). ACEIs can be used in patients with atherosclerotic vascular disease, DM, or hypertension. Treatment options include mechanical assist devices, heart transplantation, continuous IV inotropic drugs, and hospice care for end-stage patients. T AB LE 2.1 -5. NYHA Functional Classifcation of CHF T AB LE 2.1 -6. Left-Sided vs. Right-Sided Heart Failure TAB LE 2.1 -7. Comparison of Systolic and Diastolic Dysfunction Patient age Often < 65 years of age. Often > 65 years of age. Comorbidities Dilated cardiomyopathy, valvular heart disease. Restrictive or hypertrophic cardiomyopathy; renal disease or hypertension. Physical exam Displaced PMI, S3 gallop. Sustained PMI, S4 gallop. CXR Pulmonary congestion, cardiomegaly. Pulmonary congestion, normal heart size. ECG/echocardiography Q waves, ↓ EF (< 40%). LVH, normal EF (> 55%).

1	The most common cause of right-sided heart failure is left-sided heart failure. Diuretics are for symptomatic relief only and confer no mortality benef t. Heart failure caused by systolic dysfunction is defined as a ↓ EF (< 50%) and left ventricular end-diastolic volumes. It is caused by inadequate left ventricular contractility or ↑ afterload. The heart compensates for low EF and preload through hypertrophy and ventricular dilation (Frank-Starling law), but the compensation ultimately fails, leading to ↑ myocardial work and worsening systolic function. Exertional dyspnea is the earliest and most common presenting symptom and progresses to orthopnea, paroxysmal nocturnal dyspnea (PND), and finally rest dyspnea. Chronic cough, fatigue, lower extremity edema, nocturia, Cheyne-Stokes respirations, and/or abdominal fullness may be seen. Look for signs to distinguish leftfrom right-sided heart failure (see Table 2.1-6).

1	Look for signs to distinguish leftfrom right-sided heart failure (see Table 2.1-6). CHF is a clinical syndrome whose diagnosis is based on signs and symptoms. Exam reveals parasternal lift, an elevated and sustained left ventricular impulse, and an S3/S4 gallop. CXR: Cardiomegaly, cephalization of pulmonary vessels, pleural effusions, vascular plumpness, and prominent hila. Echocardiogram: ↓ EF and ventricular dilation. Lab abnormalities: BNP > 500, ↑ creatinine, ↓ sodium. ECG: Usually nondiagnostic, but MI or AF may precipitate acute exacerbations. ■Acute: Correct underlying causes such as arrhythmias, myocardial ischemia, and drugs (e.g., CCBs, antiarrhythmics, NSAIDs, alcohol, thyroid and valvular disease, high-output states). Diurese aggressively with loop and thiazide diuretics (see Table 2.1-8). TABLE 2.1-8. Types of Diuretics

1	Loop diuretics Furosemide, ethacrynic acid, bumetanide, torsemide Loop of Henle ↓ Na+/K+/2Cl− cotransporter; ↓ urine concentration; ↑ Ca2+ excretion. Ototoxicity, hypokalemia, hypocalcemia, dehydration, gout. Thiazide diuretics HCTZ, chlorothiazide, chlorthalidone Early distal tubule ↓ NaCl reabsorption leading to ↓ diluting capacity of nephron; ↓ Ca2+ excretion. Hypokalemic metabolic alkalosis, hyponatremia, hyperglycemia, hyperlipidemia, hyperuricemia, hypercalcemia. K+-sparing agents Spironolactone, triamterene, amiloride Cortical collecting tubule Spironolactone is an aldosterone receptor antagonist; triamterene and amiloride block Na+ channels. Hyperkalemia, gynecomastia, hirsutism, sexual dysfunction. Carbonic anhydrase inhibitors Acetazolamide Proximal convoluted tubule NaHCO3 diuresis ↓ total body NaHCO3. Hyperchloremic metabolic acidosis, neuropathy, NH3 toxicity, sulfa allergy. Osmotic agents Mannitol Proximal tubule Creates ↑ tubular f uid osmolarity, leading to ↑ urine f

1	↓ total body NaHCO3. Hyperchloremic metabolic acidosis, neuropathy, NH3 toxicity, sulfa allergy. Osmotic agents Mannitol Proximal tubule Creates ↑ tubular f uid osmolarity, leading to ↑ urine f ow. Pulmonary edema, dehydration. Contraindicated in anuria and CHF.

1	Give ACEIs to all patients who can tolerate them. If a patient cannot tolerate ACEIs, consider an angiotensin receptor blocker (ARB). β-blockers should not be used during decompensated CHF but should be started once the patient is euvolemic. Treat acute pulmonary congestion with LMNOP (see mnemonic). Chronic: Control comorbid conditions (e.g., diabetes, hypertension, obesity) and limit dietary sodium and ﬂ uid intake. Long-term β-blockers and ACEIs/ARBs together help prevent neurohormonal remodeling of the heart. All of these agents ↓ mortality for New York Heart Association (NYHA) class II–IV patients. Daily ASA and a statin are recommended for ischemic heart disease to prevent further ischemic events. Chronic diuretic therapy (loop diuretics +/– thiazide) can prevent volume overload.

1	Daily ASA and a statin are recommended for ischemic heart disease to prevent further ischemic events. Chronic diuretic therapy (loop diuretics +/– thiazide) can prevent volume overload. Low-dose spironolactone ↓ mortality risk when given with ACEIs and loop diuretics in patients with left ventricular systolic dysfunction and NYHA class III–IV heart failure. Monitor for hyperkalemia. Anticoagulate patients with AF and those with a history of previous embolic events or a mobile left ventricular thrombus. Consider an implantable biventricular cardiac defibrillator (ICD) in patients with both an EF < 30% and CAD. Loops lose calcium, whereas thiazides save it. ■CHF that is unresponsive to maximal medical therapy may require a mechanical left ventricular assist device or cardiac transplantation.

1	Loops lose calcium, whereas thiazides save it. ■CHF that is unresponsive to maximal medical therapy may require a mechanical left ventricular assist device or cardiac transplantation. Defined by ↓ ventricular compliance with normal systolic function. The ventricle has either impaired active relaxation (2° to ischemia, aging, and/or hypertrophy) or impaired passive filling (scarring from prior MI; restrictive cardiomyopathy). Left ventricular end-diastolic pressure ↑, cardiac output remains essentially normal, and EF is normal or ↑. Associated with stable and unstable angina, shortness of breath, dyspnea on exertion, arrhythmias, MI, heart failure, and sudden death. Diuretics are first-line therapy (see Table 2.1-8). Maintain rate and BP control via β-blockers, ACEIs, ARBs, or CCBs. Digoxin is not useful in these patients. Myocardial disease; categorized as dilated, hypertrophic, or restrictive (see Table 2.1-9).

1	Digoxin is not useful in these patients. Myocardial disease; categorized as dilated, hypertrophic, or restrictive (see Table 2.1-9). The most common cardiomyopathy. Left ventricular dilation and systolic dysfunction (low EF) must be present for diagnosis. Most cases are idiopathic, but known 2° causes include alcohol, myocarditis, postpartum status, drugs (doxorubicin, AZT, cocaine), endocrinopathies (thyroid dysfunction, acromegaly, pheochromocytoma), infection (coxsackievirus, HIV, Chagas’ disease, parasites), genetic factors, and nutritional disorders (wet beriberi). The two most common causes of 2° dilated cardiomyopathy are ischemia and long-standing hypertension. T AB LE 2.1 -9. Differential Diagnosis of Cardiomyopathies

1	T AB LE 2.1 -9. Differential Diagnosis of Cardiomyopathies Major abnormality Impaired contractility Impaired relaxation Impaired elasticity Left ventricular cavity size (end diastole) ↑↑ ↓ ↑ Left ventricular cavity size (end systole) ↑↑ ↓↓ ↑ Ejection fraction (EF) ↓↓ ↑ or ↔ ↓ or ↔ Wall thickness ↓, variable ↑↑ ↑, variable Often presents with gradual development of CHF symptoms. Exam often reveals displacement of the left venticular impulse, JVD, an S3/S4 gallop, or mitral/tricuspid regurgitation. Echocardiography is diagnostic. ECG may show nonspecific ST-T changes, low-voltage QRS, sinus tachycardia, and ectopy. LBBB is common. CXR shows an enlarged, balloon-like heart and pulmonary congestion. Address the underlying etiology (e.g., stop all alcohol use, treat endocrine disorders).

1	CXR shows an enlarged, balloon-like heart and pulmonary congestion. Address the underlying etiology (e.g., stop all alcohol use, treat endocrine disorders). Treat symptoms of CHF with diuretics, and prevent disease progression with ACEIs, β-blockers, and aldosterone antagonists. Consider anticoagulation to ↓ thrombus risk only if AF or an intraventricular thrombus is present. Digoxin is a second-line agent; avoid CCBs in CHF. Consider an ICD if EF < 35%.

1	Consider an ICD if EF < 35%. LVH results in impaired left ventricular relaxation and filling (diastolic dysfunction). Hypertrophy frequently involves the interventricular septum, leading to left ventricular outﬂow tract obstruction and impaired ejection of blood. The congenital form, hypertrophic obstructive cardiomyopathy (HOCM), is inherited as an autosomal-dominant trait in 50% of HOCM patients and is the most common cause of sudden death in young, healthy athletes in the United States. Other causes of marked hypertrophy include hypertension and aortic stenosis. Patients may be asymptomatic but may also present with syncope, dyspnea, palpitations, angina, or sudden cardiac death. Exam often reveals a sustained apical impulse, an S4 gallop, and a systolic ejection crescendo-decrescendo murmur that ↑ with ↓ preload (e.g., Valsalva maneuver, squatting).

1	Exam often reveals a sustained apical impulse, an S4 gallop, and a systolic ejection crescendo-decrescendo murmur that ↑ with ↓ preload (e.g., Valsalva maneuver, squatting). Obstruction is worsened by ↑ myocardial contractility or by ↓ left ventricular filling (e.g., exercise, Valsalva maneuvers, vasodilators, dehydration). Echocardiography is diagnostic and shows an asymmetrically thickened left ventricular wall and dynamic obstruction of blood ﬂ ow. ECG may show signs of LVH. CXR may reveal left atrial enlargement (LAE) 2° to mitral regurgitation. ■β-blockers are initial therapy for symptomatic relief; CCBs are second-line agents. An S3 gallop signif es the end of rapid ventricular f lling in the setting of f uid overload and is associated with dilated cardiomyopathy. Hypertrophic cardiomyopathy is the most common cause of sudden death in young, healthy athletes in the United States.

1	Hypertrophic cardiomyopathy is the most common cause of sudden death in young, healthy athletes in the United States. An S4 gallop signif es a stiff, noncompliant ventricle and associated with hypertrophic cardiomyopathy. Surgical options for HOCM include dual-chamber pacing, partial excision or catheter ablation of the myocardial septum, ICD placement, and mitral valve replacement. Patients should avoid intense athletic competition and training. Defined as ↓ elasticity of myocardium leading to impaired diastolic filling without significant systolic dysfunction (a normal or near-normal EF). It is caused by infiltrative disease (amyloidosis, sarcoidosis, hemochromatosis) or by scarring and fibrosis (2° to radiation or doxorubicin). Signs and symptoms of left-sided and right-sided heart failure occur, but symptoms of right-sided heart failure (JVD, peripheral edema) often predominate.

1	Signs and symptoms of left-sided and right-sided heart failure occur, but symptoms of right-sided heart failure (JVD, peripheral edema) often predominate. CXR, MRI, and cardiac catheterization may be helpful, but echocardiography is key to diagnosis and reveals rapid early filling with a normal or near-normal EF. Cardiac biopsy may reveal fibrosis or evidence of infiltration. ECG frequently shows LBBB. Therapeutic options are limited and generally are palliative only. Medical treatment includes cautious use of diuretics for ﬂuid overload, vasodilators to ↓ filling pressure, and anticoagulation if not contraindicated. Major risk factors for CAD include age, male gender, hyperlipidemia, DM, hypertension, obesity, a family history, and smoking. The classic triad of angina consists of substernal chest pain that is provoked by exertion and relieved by rest or nitrates.

1	The classic triad of angina consists of substernal chest pain that is provoked by exertion and relieved by rest or nitrates. Clinical manifestations of CAD include stable and unstable angina, shortness of breath, dyspnea on exertion, arrhythmias, MI, heart failure, and sudden death. Risk factors include DM, a family history of premature CAD, smoking, dyslipidemia, abdominal obesity, hypertension, and male gender. Substernal chest pain 2° to myocardial ischemia (O2 supply and demand mismatch). Prinzmetal’s (variant) angina mimics angina pectoris but is caused by vasospasm of coronary vessels. It classically affects young women at rest in the early morning and is associated with ST-segment elevation in the absence of cardiac enzyme elevation. The classic triad consists of substernal chest pain or pressure (often described as a heaviness or pressure without pain), usually precipitated by stress or exertion and relieved by rest or nitrates.

1	Pain can radiate to the left arm, jaw, and neck and may be associated with shortness of breath, nausea/vomiting, diaphoresis, or lightheadedness. Examination of patients experiencing stable angina is generally unremarkable. Look for carotid and peripheral bruits suggesting atherosclerosis and hypertension. Rule out pulmonary, GI, or other cardiac causes of chest pain. Angina may be diagnosed from the history alone, but significant ST-segment changes on exercise stress test with ECG monitoring is diagnostic of CAD. Women and diabetics classically experience “silent” ischemic events and present “atypically”; for this reason, it is necessary to maintain a high index of suspicion in this patient population. Treat acute symptoms with ASA, O2 and/or IV nitroglycerin, and IV morphine, and consider IV β-blockers. The efficacy of nondihydropyridine CCBs (diltiazem, verapamil) and ACEIs has also been validated.

1	Patients with a suspected MI must be admitted and monitored until acute MI is ruled out by serial cardiac enzymes. Treat chronic symptoms with nitrates, ASA, and β-blockers; CCBs are second-line agents for symptomatic control only. Initiate risk factor reduction (e.g., smoking, cholesterol, hypertension). Hormone replacement therapy is not protective in postmenopausal women. Women, diabetics, the elderly, and post–heart transplant patients may have atypical, clinically silent MIs. Only ASA and β-blockers have been shown to have a mortality benef t in the treatment of angina. A spectrum of clinical syndromes caused by plaque disruption or vasospasm that leads to acute myocardial ischemia.

1	A spectrum of clinical syndromes caused by plaque disruption or vasospasm that leads to acute myocardial ischemia. Unstable angina describes chest pain that is new onset, is accelerating (i.e., occurs with less exertion, lasts longer, or is less responsive to medications), or occurs at rest; it is distinguished from stable angina by patient history. It signals the presence of possible impending infarction based upon plaque instability. In contrast, NSTEMI indicates myocardial necrosis marked by elevations in troponin I, troponin T, or CK-MB. Patients should be risk stratified according to the TIMI (Thrombolysis in Myocardial Infarction study) criteria to determine the likelihood of adverse cardiac events (see Table 2.1-10). Unstable angina is not associated with elevated cardiac markers, but ST changes may be seen on ECG and are indicative of high-risk occlusions. NSTEMI is diagnosed by serial cardiac enzymes and ECG.

1	Unstable angina is not associated with elevated cardiac markers, but ST changes may be seen on ECG and are indicative of high-risk occlusions. NSTEMI is diagnosed by serial cardiac enzymes and ECG. Acute treatment of symptoms is the same as that for stable angina. Clopidogrel, unfractionated heparin or enoxaparin, and glycoprotein IIb/IIIa inhibitors (e.g., eptifibatide, tirofiban, abciximab) should also be considered. Patients with chest pain refractory to medical therapy, a TIMI score of ≥ 3, a troponin elevation, or ST changes > 1 mm should be given heparin and scheduled for angiography and possible revascularization (percutaneous coronary intervention [PCI] or CABG). Think unstable angina if chest pain is new onset, accelerating, or occurring at rest. T AB LE 2.1 -1 0. TIMI Risk Score for Unstable Angina/NSTEMI DEATH, MI, OR URGENT CHARACTERISTICS POINT RISK SCORE DEATH OR MI REVASCULARIZATION

1	T AB LE 2.1 -1 0. TIMI Risk Score for Unstable Angina/NSTEMI DEATH, MI, OR URGENT CHARACTERISTICS POINT RISK SCORE DEATH OR MI REVASCULARIZATION History Age ≥ 65 years 1 0/1 3 5 ≥ 3 CAD risk factors (family history, DM, tobacco, hypertension, ↑ cholesterol) 1 2 3 8 Known CAD (stenosis > 50%) 1 3 5 13 ASA use in past seven days 1 4 7 20 Presentation 5 12 26 Severe angina (≥ 2 episodes within 24 hours) 1 6/7 19 41 ST deviation ≥ 0.5 mm 1 + cardiac marker 1 Risk score—total points (0–7) Higher-risk patients (risk score ≥ 3) beneft more from enoxaparin (vs. unfractionated heparin), glycoprotein IIb/IIIa inhibitors, and early angiography. Defined as ST-segment elevations and cardiac enzyme release 2° to prolonged cardiac ischemia and necrosis. Presents with acute-onset substernal chest pain, commonly described as a pressure or tightness that can radiate to the left arm, neck, or jaw.

1	Presents with acute-onset substernal chest pain, commonly described as a pressure or tightness that can radiate to the left arm, neck, or jaw. Associated symptoms may include diaphoresis, shortness of breath, lightheadedness, anxiety, nausea/vomiting, and syncope. Physical exam may reveal arrhythmias, new mitral regurgitation (ruptured papillary muscle), hypotension (cardiogenic shock), and evidence of new CHF (rales, peripheral edema, S3 gallop). The best predictor of survival is left ventricular EF. ■ECG: Look for ST-segment elevations or new LBBB. ST-segment depressions in leads V1–V2 can also be reciprocal change indicating infarction in the posterior wall. Sequence of ECG changes: Peaked T waves → ST-segment elevation → Q waves → T-wave inversion → ST-segment normalization → T-wave normalization over several hours to days.

1	Sequence of ECG changes: Peaked T waves → ST-segment elevation → Q waves → T-wave inversion → ST-segment normalization → T-wave normalization over several hours to days. Cardiac enzymes: Troponin I is most sensitive; CK-MB is more specific. Both can take up to six hours to rise after the onset of chest pain (see Figure 2.1-4). ST-segment abnormalities: ST-segment elevation in leads II, III, and aVF is consistent with an inferior MI involving the RCA/PDA and LCA (see Figure 2.1-5). ST-segment elevation in leads V1–V4 usually indicates an anterior MI involving the LAD and diagonal branches (see Figure 2.1-6). ST-segment elevation in leads I, aVL, and V5–V6 points to a lateral MI involving the LCA. ST-segment depression in leads V1–V2 can be “reciprocal change” indicative of an acute infarct in the posterior wall. Six key medications should be considered: ASA, β-blockers, clopidogrel, morphine, nitrates, and O2.

1	Six key medications should be considered: ASA, β-blockers, clopidogrel, morphine, nitrates, and O2. If the patient is in heart failure or in cardiogenic shock, do not give β-blockers; instead, give ACEIs, provided that the patient is not hypotensive. Emergent angiography and PCI should be performed; if possible, the patient should undergo PCI for the lesion thought to be responsible for STEMI. Common causes of chest pain include GERD, angina, esophageal pain, musculoskeletal disorders (costochondritis, trauma), and pneumonia. Other causes of ST-segment elevation (often diffuse across multiple leads) include acute pericarditis, LVH, LBBB, and a normal variant (e.g., “early repolarization”). F IGU R E 2.1 -4. Typical pattern of serum marker elevation after an acute MI.

1	F IGU R E 2.1 -4. Typical pattern of serum marker elevation after an acute MI. CK-MB = creatine kinase, MB isoenzyme; cTnI = cardiac troponin I; cTnT = cardiac troponin T; LD1 = lactate dehydrogenase isoenzyme 1; MLC = myosin light chain. (Reproduced, with permission, from Tintinalli JE et al. Tintinalli’s Emergency Medicine: A Comprehensive Study Guide, 6th ed. New York: McGraw-Hill, 2004: Fig. 49-1.) FIGURE 2.1-5. Inferior wall MI. In this patient with acute chest pain, the ECG demonstrated acute ST-segment elevation in leads II, III, and aVF with reciprocal ST-segment depression and T-wave ﬂattening in leads I, aVL, and V4–V6. (Reproduced, with permission, from Stobo J et al. The Principles and Practice of Medicine, 23rd ed. Stamford, CT: Appleton & Lange, 1996: 20.) FIGURE 2.1-6. Anterior wall MI.

1	FIGURE 2.1-6. Anterior wall MI. This patient presented with acute chest pain. The ECG showed acute ST-segment elevation in leads aVL and V1–V6, and hyperacute T waves. (Reproduced, with permission, from Stobo J et al. The Principles and Practice of Medicine, 23rd ed. Stamford, CT: Appleton & Lange, 1996: 19.) If the patient presents within three hours, PCI cannot be performed within 90 minutes, and there are no contraindications to thrombolysis (e.g., a history of hemorrhagic stroke or recent ischemic stroke, severe heart failure, or cardiogenic shock), thrombolysis with tPA, reteplase, or streptokinase should be performed instead of PCI. In the setting of three-vessel disease, left main coronary artery disease, discrete lesions not amenable to PCI, or diffuse disease with good target vessels, PCI should be attempted immediately for the lesion thought to be responsible for STEMI; the patient is a candidate for CABG afterward.

1	Long-term treatment includes ASA, ACEIs, β-blockers, high-dose statins, and clopidogrel (if PCI was performed). Modify risk factors with dietary changes, exercise, and tobacco cessation. Arrhythmia is the most common complication and cause of death following acute MI; lethal arrhythmia is the most common cause of death following acute MI. Less common complications include reinfarction, left ventricular wall rupture, VSD, pericarditis, papillary muscle rupture (with mitral regurgitation), left ventricular aneurysm or pseudoaneurysm, and mural thrombi. Dressler’s syndrome, an autoimmune process occurring 2–10 weeks post-MI, presents with fever, pericarditis, pleural effusion, leukocytosis, and ↑ ESR. A timeline of common post-MI complications is as follows: First day: Heart failure (treat with nitroglycerin and diuretics). 2–4 days: Arrhythmia, pericarditis (diffuse ST elevation with PR depression).

1	First day: Heart failure (treat with nitroglycerin and diuretics). 2–4 days: Arrhythmia, pericarditis (diffuse ST elevation with PR depression). 5–10 days: Left ventricular wall rupture (acute pericardial tamponade causing electrical alternans, pulseless electrical activity), papillary muscle rupture (severe mitral regurgitation). Weeks to months: Ventricular aneurysm (CHF, arrhythmia, persistent ST elevation, mitral regurgitation, thrombus formation). Unable to perform PCI (diffuse disease) Stenosis of left main coronary artery Triple-vessel disease Total cholesterol > 200 mg/dL, LDL > 130 mg/dL, triglycerides > 500 mg/ dL, and HDL < 40 mg/dL are risk factors for CAD. Etiologies include obesity, DM, alcoholism, hypothyroidism, nephrotic syndrome, hepatic disease, Cushing’s disease, OCP use, high-dose diuretic use, and familial hypercholesterolemia. Most patients have no specific signs or symptoms.

1	Most patients have no specific signs or symptoms. Patients with extremely high triglyceride or LDL levels may have xanthomas (eruptive nodules in the skin over the tendons), xanthelasmas (yellow fatty deposits in the skin around the eyes), and lipemia retinalis (creamy appearance of retinal vessels). Dyslipidemia: ■Conduct a fasting lipid profile for patients > 20 years of age and repeat ev ery five years or sooner if elevated. ■Total serum cholesterol > 200 mg/dL on two different occasions is diagnostic of hypercholesterolemia. T AB LE 2.1 -1 1. ATP III Guidelines for Risk Stratifcation of Hypercholesterolemia

1	■Total serum cholesterol > 200 mg/dL on two different occasions is diagnostic of hypercholesterolemia. T AB LE 2.1 -1 1. ATP III Guidelines for Risk Stratifcation of Hypercholesterolemia CAD or CAD risk equivalentsa < 100 mg/dL (or < 70) > 100 mg/dL > 130 mg/dL 2+ risk factorsb < 130 mg/dL > 130 mg/dL > 160 mg/dL 0–1 risk factorb < 160 mg/dL > 160 mg/dL > 190 mg/dL a CAD risk equivalents = symptomatic carotid artery disease, peripheral arterial disease, abdominal aortic aneurysm, diabetes. b Risk factors = cigarette smoking, hypertension, low HDL (< 40 mg/dL), a family history of premature CAD, and age (men > 45 years; women > 55 years). An HDL > 60 mg/dL counts as a “negative” risk factor and removes one risk factor from the total score. ■LDL > 130 mg/dL or HDL < 40 mg/dL, even if total serum cholesterol is < 200 mg/dL, is diagnostic of dyslipidemia.

1	■LDL > 130 mg/dL or HDL < 40 mg/dL, even if total serum cholesterol is < 200 mg/dL, is diagnostic of dyslipidemia. Based on risk stratification (see Table 2.1-11). Risk factors include diabetes (considered a CAD risk equivalent), smoking, hypertension, HDL < 40 mg/dL, age > 45 (males), age > 55 (females), and early CAD in first-degree relatives (males < 55 and females < 65). The first intervention should be a 12-week trial of diet and exercise in a patient with no known atherosclerotic vascular disease. Commonly used lipid-lowering agents are listed in Table 2.1-12. The BP goal in uncomplicated hypertension is < 140/< 90. For diabetics or patients with renal disease, the goal is < 130/< 80. Defined as a systolic BP > 140 mmHg and/or a diastolic BP > 90 based on three measurements separated in time (see Table 2.1-13). Classified as 1° or 2°.

1	Defined as a systolic BP > 140 mmHg and/or a diastolic BP > 90 based on three measurements separated in time (see Table 2.1-13). Classified as 1° or 2°. Hypertension with no identifiable cause. Represents 95% of cases of hypertension. Risk factors include a family history of hypertension or heart disease, a high-sodium diet, smoking, obesity, race (blacks > whites), and advanced age. Hypertension is asymptomatic until complications develop. Patients should be evaluated for end-organ damage to the brain (stroke, dementia), eye (cotton-wool exudates, hemorrhage), heart (LVH), and kidney (proteinuria, chronic kidney disease). Renal bruits may signify renal artery stenosis as the cause of hypertension. Conduct cardiovascular, neurologic, ophthalmologic, and abdominal exams. Obtain a UA, BUN/creatinine, CBC, and electrolytes to assess the extent of end-organ damage. T AB LE 2.1 -1 2. Lipid-Lowering Agents

1	HMG-CoA reductase inhibitors (statins) Atorvastatin, simvastatin, lovastatin, pravastatin, rosuvastatin Inhibit the rate-limiting step in cholesterol synthesis. ↓ LDL, ↓ triglycerides ↑ LFTs, myositis, warfarin potentiation. Lipoprotein lipase stimulators (f brates) Gemf brozil ↑ lipoprotein lipase, leading to ↑ VLDL and triglyceride catabolism. ↓ triglycerides, ↑ HDL GI upset, cholelithiasis, myositis, ↑ LFTs. Cholesterol absorption inhibitors Ezetimibe (Zetia) ↓ absorption of cholesterol at the small intestine brush border. ↓ LDL Diarrhea, abdominal pain. Can cause angioedema. Niacin Niaspan ↓ fatty acid release from adipose tissue; ↓ hepatic synthesis of LDL. ↑ HDL, ↓ LDL Skin fushing (can be prevented with ASA), paresthesias, pruritus, GI upset, ↑ LFTs. Bile acid resins Cholestyramine, colestipol, colesevelam Bind intestinal bile acids lead to ↓ bile acid stores and ↑ catabolism of LDL from plasma. ↓ LDL Constipation, GI upset, LFT abnormalities, myalgias. Can ↓ absorption of

1	colestipol, colesevelam Bind intestinal bile acids lead to ↓ bile acid stores and ↑ catabolism of LDL from plasma. ↓ LDL Constipation, GI upset, LFT abnormalities, myalgias. Can ↓ absorption of other drugs from the small intestine.

1	T AB LE 2.1 -1 3. JNC-7 Classifcation and Management of Hypertension Normal < 120 and < 80 Encourage Prehypertension 120–139 or 80–89 Yes No antihypertensive drug indicated. Stage 1 hypertension 140–159 or 90–99 Yes Thiazide diuretics for most patients; ACEIs, ARBs, β-blockers, CCBs, or a combination may be considered. Stage 2 hypertension ≥ 160 or ≥ 100 Yes Two-drug combination for most patients (usually a thiazide diuretic plus an ACEI, an ARB, a β-blocker, or a CCB). Hypertensive crises are diagnosed on the basis of the extent of end-organ damage, not BP measurement. Rule out 2° causes of hypertension, particularly in younger patients. Begin with lifestyle modifications (e.g., weight loss, smoking cessation, salt reduction). Weight loss is the single most effective lifestyle modification. The BP goal in otherwise healthy patients is < 140/< 90. The goal in diabetics or patients with renal disease with proteinuria is < 130/< 80.

1	Diuretics (which are inexpensive and particularly effective in African-Americans), ACEIs, and β-blockers (which are beneficial for patients with CAD) have been shown to ↓ mortality in uncomplicated hypertension. They are first-line agents unless a comorbid condition requires another medication (see Table 2.1-14). Periodically test for end-organ complications, including renal (BUN, creatinine, urine protein-to-creatinine ratio) and cardiac (ECG evidence of hypertrophy) complications. Hypertension 2° to an identifiable organic cause. See Table 2.1-15 for the diagnosis and treatment of common causes. A spectrum of clinical presentations in which elevated BPs lead to end-organ damage. Presents with end-organ damage revealed by chest pain (ischemia or MI), back pain (aortic dissection), or changes in mental status (hypertensive encephalopathy).

1	Presents with end-organ damage revealed by chest pain (ischemia or MI), back pain (aortic dissection), or changes in mental status (hypertensive encephalopathy). Hypertensive urgency: Diagnosed on the basis of an elevated BP with only mild to moderate symptoms (headache, chest pain, syncope) and without end-organ damage. Hypertensive emergency: Diagnosed by a significantly elevated BP with signs or symptoms of impending end-organ damage such as ARF, intracra- T AB LE 2.1 -1 4. Treatment of 1° Hypertension with Comorbid Conditions Heart failure Thiazide diuretics, β-blockers, ACEIs, ARBs, aldosterone antagonists. Post-MI β-blockers, ACEIs, aldosterone antagonists. High CVD risk Thiazide diuretics, β-blockers, ACEIs, CCBs. Diabetes Thiazide diuretics, β-blockers, ACEIs, ARBs, CCBs. Chronic kidney disease ACEIs, ARBs. Recurrent stroke prevention Thiazide diuretics, ACEIs.

1	T AB LE 2.1 -1 5. Common Causes of 2° Hypertension 1° renal disease Often unilateral renal parenchymal disease. Treat with ACEIs, which slow the progression of renal disease. Renal artery stenosis Especially common in patients < 25 and > 50 years of age with recent-onset hypertension. Etiologies include f bromuscular dysplasia (usually in younger patients) and atherosclerosis (usually in older patients). Diagnose with MRA or renal artery Doppler ultrasound. May be treated with angioplasty or stenting. Consider ACEIs as adjunctive or temporary therapy in unilateral disease. (In bilateral disease, ACEIs can accelerate kidney failure by preferential vasodilation of the efferent arteriole.) Open surgery is a second option if angioplasty is not effective or feasible. OCP use Common in women > 35 years of age, obese women, and those with long-standing use. Discontinue OCPs (effect may be delayed). Pheochromocytoma An adrenal gland tumor that secretes epinephrine and norepinephrine, leading

1	35 years of age, obese women, and those with long-standing use. Discontinue OCPs (effect may be delayed). Pheochromocytoma An adrenal gland tumor that secretes epinephrine and norepinephrine, leading to episodic headache, sweating, and tachycardia. Diagnose with urinary metanephrines and catecholamine levels or plasma metanephrine. Surgical removal of tumor after treatment with both α-blockers and β-blockers. Conn’s syndrome (hyperaldosteronism) Most often 2° to an aldosterone-producing adrenal adenoma. Causes the triad of hypertension, unexplained hypokalemia, and metabolic alkalosis. Surgical removal of tumor. Cushing’s syndrome Due to an ACTH-producing pituitary tumor, an ectopic ACTH-secreting tumor, or cortisol secretion by an adrenal adenoma or carcinoma. (See the Endocrinology chapter for more details.) Surgical removal of tumor. Coarctation of the aorta See the Pediatrics chapter. Surgical repair.

1	nial hemorrhage, papilledema, or ECG changes suggestive of ischemia or pulmonary edema. ■Malignant hypertension: Diagnosed on the basis of progressive renal failure and/or encephalopathy with papilledema. Hypertensive urgencies: Can be treated with oral antihypertensives (e.g., β-blockers, clonidine, ACEIs) with the goal of gradually lowering BP over 24–48 hours (see Tables 2.1-16 and 2.1-17). Hypertensive emergencies: Treat with IV medications (labetalol, nitroprusside, nicardipine) with the goal of lowering mean arterial pressure by no more than 25% over the first two hours to prevent cerebral hypoperfusion or coronary insufficiency. T AB LE 2.1 -1 6. Major Classes of Antihypertensive Agents

1	Diuretics Thiazide, loop, K+ sparing ↓ extracellular fuid volume and thereby ↓ vascular resistance. Hypokalemia (not with K+ sparing), hyperglycemia, hyperlipidemia, hyperuricemia, azotemia. β-adrenergic blockers (β-blockers) Propranolol, metoprolol, nadolol, atenolol, timolol, carvedilol, labetalol ↓ cardiac contractility and renin release. Bronchospasm (in severe active asthma), bradycardia, CHF exacerbation, impotence, fatigue, depression. Centrally acting adrenergic agonists Methyldopa, clonidine Inhibit the sympathetic nervous system via central α2-adrenergic receptors. Somnolence, orthostatic hypotension, impotence, rebound hypertension. α1-adrenergic blockers Prazosin, terazosin, phenoxybenzamine Cause vasodilation by blocking actions of norepinephrine on vascular smooth muscle. Orthostatic hypotension. CCBs Dihydropyridines (nifedipine, felodipine, amlodipine), nondihydropyridines (diltiazem, verapamil) ↓ smooth muscle tone and cause vasodilation; may also ↓ cardiac output.

1	Orthostatic hypotension. CCBs Dihydropyridines (nifedipine, felodipine, amlodipine), nondihydropyridines (diltiazem, verapamil) ↓ smooth muscle tone and cause vasodilation; may also ↓ cardiac output. Dihydropyridines: Headache, fushing, peripheral edema. Nondihydropyridines: ↓ contractility. Vasodilators Hydralazine, minoxidil ↓ peripheral resistance by dilating arteries/arterioles. Hydralazine: Headache, lupus-like syndrome. Minoxidil: Orthostasis, hirsutism. Captopril, enalapril, fosinopril, benazepril, lisinopril Block aldosterone formation, reducing peripheral resistance and salt/water retention. Cough, rashes, leukopenia, hyperkalemia. Losartan, valsartan, irbesartan Block aldosterone effects, reducing peripheral resistance and salt/water retention. Rashes, leukopenia, and hyperkalemia but no cough. ACEIs ARBs

1	Results from acute or chronic pericardial insults; may lead to pericardial effusion. Inﬂammation of the pericardial sac. Can compromise cardiac output via tamponade or constrictive pericarditis. Most commonly idiopathic, although known etiologies include viral infection, TB, SLE, uremia, drugs, radiation, and neoplasms. May also occur after MI (either within days after MI or as a delayed phenomenon, i.e., Dressler’s syndrome) or open heart surgery. TABLE 2.1-17. Antihypertensive Agents for Specifc Patient Populations Diabetes with proteinuria ACEIs or ARBs. CHF β-blockers, ACEIs or ARBs, diuretics (including spironolactone). Isolated systolic hypertension Diuretics are preferred; long-acting dihydropyridines. MI β-blockers without intrinsic sympathomimetic activity; ACEIs. Osteoporosis Thiazide diuretics. BPH α1-adrenergic blockers. May present with pleuritic chest pain, dyspnea, cough, and fever. Chest pain tends to worsen in the supine position and with inspiration.

1	May present with pleuritic chest pain, dyspnea, cough, and fever. Chest pain tends to worsen in the supine position and with inspiration. Exam may reveal a pericardial friction rub, elevated JVP, and pulsus paradoxus (a ↓ in systolic BP > 10 mmHg on inspiration). CXR, ECG, and echocardiogram to rule out MI and pneumonia. ECG changes include diffuse ST-segment elevation and PR-segment depressions followed by T-wave inversions (see Figure 2.1-7). Pericardial thickening or effusion may be evident on echocardiography. Address the underlying cause (e.g., corticosteroids/immunosuppressants for SLE, dialysis for uremia) or symptoms (e.g., ASA for post-MI pericarditis, ASA/NSAIDs for viral pericarditis). Avoid corticosteroids within a few days after MI, as they can predispose to ventricular wall rupture. Pericardial effusions without symptoms can be followed, but evidence of tamponade requires pericardiocentesis, with continuous drainage as needed.

1	Pericardial effusions without symptoms can be followed, but evidence of tamponade requires pericardiocentesis, with continuous drainage as needed. Excess ﬂuid in the pericardial sac, leading to compromised ventricular filling and ↓ cardiac output. The condition is more closely related to the rate of ﬂ uid formation than to the size of the effusion. Risk factors include pericarditis, malignancy, SLE, TB, and trauma (commonly stab wounds medial to the left nipple). ■Presents with fatigue, dyspnea, anxiety, tachycardia, and tachypnea that can rapidly progress to shock and death. Causes of pericarditis— Beck’s triad can diagnose acute cardiac tamponade: F IGU R E 2.1 -7. Acute pericarditis.

1	Causes of pericarditis— Beck’s triad can diagnose acute cardiac tamponade: F IGU R E 2.1 -7. Acute pericarditis. Diffuse ST-segment elevations in multiple leads not consistent with any discrete coronary vas cular territory and PR-segment depressions. (Reproduced, with permission, from Gomella LG et al. Clinician’s Pocket Reference, 11th ed. New York: McGraw-Hill, 2006: Fig. 19-34.) ■Examination of a patient with acute tamponade may reveal Beck’s triad (hypotension, distant heart sounds, and JVD), a narrow pulse pressure, pulsus paradoxus, and Kussmaul’s sign (JVD on inspiration). Echocardiogram shows right atrial and right ventricular diastolic collapse. CXR shows an enlarged, globular heart. If present on ECG, electrical alternans is diagnostic. Aggressive volume expansion with IV ﬂ uids. Urgent pericardiocentesis (aspirate will be nonclotting blood). Decompensation may warrant balloon pericardiotomy and pericardial window.

1	Aggressive volume expansion with IV ﬂ uids. Urgent pericardiocentesis (aspirate will be nonclotting blood). Decompensation may warrant balloon pericardiotomy and pericardial window. Until recently, rheumatic fever (which affects the mitral valve more often than the aortic valve) was the most common cause of valvular heart disease in U.S. adults; the leading cause is now mechanical degeneration. Subtypes are listed in Table 2.1-18 along with their etiologies, presentation, diagnosis, and treatment. Aortic aneurysms are most commonly associated with atherosclerosis. Most are abdominal, and > 90% originate below the renal arteries. Usually asymptomatic and discovered incidentally on exam or radiologic study. Risk factors include hypertension, high cholesterol, other vascular disease, a family history, smoking, gender (males > females), and age. Exam demonstrates a pulsatile abdominal mass or abdominal bruits.

1	Exam demonstrates a pulsatile abdominal mass or abdominal bruits. Ruptured aneurysm leads to hypotension and severe, tearing abdominal pain that radiates to the back. Abdominal ultrasound for diagnosis or to follow an aneurysm over time. CT may be useful to determine the precise anatomy. In asymptomatic patients, monitoring is appropriate for lesions < 5 cm. Surgical repair is indicated if the lesion is > 5.5 cm (abdominal), > 6 cm (thoracic), or smaller but rapidly enlarging. Emergent surgery for symptomatic or ruptured aneurysms. A transverse tear in the intima of a vessel that results in blood entering the media, creating a false lumen and leading to a hematoma that propagates longitudinally. Most commonly 2° to hypertension. The most common sites of origin are above the aortic valve and distal to the left subclavian artery. Most often occurs at 40–60 years of age, with a greater frequency in males than in females.

1	Aortic aneurysm is most often associated with atherosclerosis, while aortic dissection is commonly linked to hypertension. TABLE 2.1-18. Types of Valvular Heart Disease

1	Aortic stenosis Most often seen in the elderly. Unicuspid and bicuspid valves can lead to symptoms in childhood and adolescence. May be asymptomatic for years despite signif cant stenosis. Once symptomatic, usually progresses from angina to syncope to CHF to death within f ve years. Cx (also indications for valve replacements): ACS—Angina, CHF, Syncope. PE: Pulsus parvus et tardus (weak, delated carotid upstroke) and a single or paradoxically split S2 sound; systolic murmur radiating to the carotids. Dx: Echocardiography. Valve replacement. Balloon valvuloplasty can bridge patients to aortic valve replacement but is not def nitive treatment. Aortic regurgitation Acute: Infective endocarditis, aortic dissection, chest trauma. Chronic: Valve malformations, rheumatic fever, connective tissue disorders. Causes: CREAM— Congenital Rheumatic damage, Endocarditis, Aortic dissection/ Aortic root dilatation, Marfan’s syndrome. Acute: Rapid onset of pulmonary congestion, cardiogenic shock, and

1	disorders. Causes: CREAM— Congenital Rheumatic damage, Endocarditis, Aortic dissection/ Aortic root dilatation, Marfan’s syndrome. Acute: Rapid onset of pulmonary congestion, cardiogenic shock, and severe dyspnea. Chronic: Slowly progressive onset of dyspnea on exertion, orthopnea, and PND. PE: Blowing diastolic murmur at the left sternal border, mid-diastolic rumble (Austin Flint murmur), and midsystolic apical murmur. Widened pulse pressure causes de Musset’s sign (head bob with heartbeat), Corrigan’s sign (water-hammer pulse), and Duroziez’s sign (femoral bruit). Dx: Echocardiography. Vasodilator therapy (dihydropyridines or ACEIs) for isolated aortic regurgitation until symptoms become severe enough to warrant valve replacement. Mitral valve stenosis The most common etiology continues to be rheumatic fever. Symptoms range from dyspnea, orthopnea, and PND to infective endocarditis and arrhythmias. PE: Opening snap and mid-diastolic murmur at apex; pulmonary edema. Dx:

1	continues to be rheumatic fever. Symptoms range from dyspnea, orthopnea, and PND to infective endocarditis and arrhythmias. PE: Opening snap and mid-diastolic murmur at apex; pulmonary edema. Dx: Echocardiography. Antiarrhythmics (digoxin, β-blockers) for symptomatic relief; mitral balloon valvotomy and valve replacement are effective for severe cases. Mitral valve regurgitation Primarily 2° to rheumatic fever or chordae tendineae rupture after MI. Patients present with dyspnea, orthopnea, and fatigue. PE: Holosystolic murmur radiating to axillae. Dx: Echocardiography will demonstrate regurgitant f ow; angiography can assess the severity of disease. Antiarrhythmics if necessary (AF is common with LAE; nitrates and diuretics to ↓ preload).

1	Sudden tearing/ripping pain in the anterior chest in ascending dissection; interscapular back pain in descending dissection. The patient is typically hypertensive. If a patient is hypotensive, consider pericardial tamponade, hypovolemia from blood loss, or acute MI from involvement of the coronary arteries. Asymmetric pulses and BP measurements are indicative of aortic dissection. Signs of pericarditis or pericardial tamponade may be seen; a murmur of aortic regurgitation may be heard if the aortic valve is involved with a proximal dissection. Neurologic deficits may be seen if the aortic arch or spinal arteries are involved. ECG, CXR (shows widening of the mediastinum, cardiomegaly, or new left pleural effusion). CT angiography is the gold standard of imaging. TEE can provide details of the thoracic aorta, the proximal coronary arteries, the origins of arch vessels, the presence of a pericardial effusion, and aortic valve integrity.

1	TEE can provide details of the thoracic aorta, the proximal coronary arteries, the origins of arch vessels, the presence of a pericardial effusion, and aortic valve integrity. There are two systems of classification for aortic dissection: DeBakey system: Classifies dissections as involving both the ascending and descending aorta (type I), confined to the ascending aorta (type II), or confined to the descending aorta (type III). Stanford system: Classifies dissection of the ascending aorta as type A and all others as type B. Monitor and medically manage BP and heart rate as necessary. Do not give thrombolytics. If the dissection involves the ascending aorta, it is a surgical emergency; descending dissections can often be managed with BP and heart rate control. Ascending aortic dissections are surgical emergencies, but descending dissections can often be treated medically.

1	Ascending aortic dissections are surgical emergencies, but descending dissections can often be treated medically. Clot formation in the large veins of the extremities or pelvis. The classic Virchow’s triad of risk factors includes venous stasis (e.g., from plane ﬂ ights, bed rest, or incompetent venous valves in the lower extremities), endothelial trauma (injury to the lower extremities), and hypercoagulable states (e.g., malignancy, pregnancy, OCP use). Presents with unilateral lower extremity pain, erythema, and swelling. Homans’ sign is calf tenderness with passive foot dorsiﬂexion (poor sensitivity and specificity for DVT). Doppler ultrasound; spiral CT or V/Q scan may be used to evaluate for pulmonary embolism (see Figure 2.1-8). Virchow’s triad: hemostasis, trauma (endothelial damage), hypercoagulability. A negative D-dimer test can be used to rule out the possibility of pulmonary embolism in low-risk patients.

1	Virchow’s triad: hemostasis, trauma (endothelial damage), hypercoagulability. A negative D-dimer test can be used to rule out the possibility of pulmonary embolism in low-risk patients. F IGU R E 2.1 -8. Algorithm for diagnostic imaging of DVT and PE. (Reproduced, with permission, from Fauci AS et al. Harrison’s Principles of Internal Medicine, 17th ed. New York: McGraw-Hill, 2008: Fig. 256-1.) Initial anticoagulation with IV unfractionated heparin or SQ lowmolecular-weight heparin followed by PO warfarin for a total of 3–6 months. Consider an IVC filter in patients with contraindications to anticoagulation. Hospitalized patients should receive DVT prophylaxis consisting of exercise as tolerated, anti-thromboembolic stockings, and SQ unfractionated heparin or low-molecular-weight heparin.

1	Hospitalized patients should receive DVT prophylaxis consisting of exercise as tolerated, anti-thromboembolic stockings, and SQ unfractionated heparin or low-molecular-weight heparin. Occlusion of the blood supply to the extremities by atherosclerotic plaque. The lower extremities are most commonly affected. Clinical manifestations depend on the vessels involved, the extent and rate of obstruction, and the presence of collateral blood ﬂ ow. Presents with intermittent claudication (reproducible leg pain that occurs with walking and is always relieved with rest). As the disease progresses, pain occurs at rest and affects the distal extremities. Dorsal foot ulcerations may develop 2° to poor perfusion. A painful, cold, numb foot is characteristic of critical limb ischemia. Aortoiliac disease: Associated with Leriche’s syndrome (buttock claudication, ↓ femoral pulses, male impotence). Femoropopliteal disease: Calf claudication; pulses below the femoral artery are absent.

1	Femoropopliteal disease: Calf claudication; pulses below the femoral artery are absent. Acute ischemia: Most often caused by embolization from the heart; acute occlusions commonly occur at bifurcations distal to the last palpable pulse. May also be 2° to cholesterol atheroembolism (“blue toe syndrome”). ■Severe chronic ischemia: Lack of blood perfusion leads to muscle atrophy, pallor, cyanosis, hair loss, and gangrene/necrosis. Carefully palpate pulses and auscultate for bruits. Measurement of ankle and brachial systolic BP (ankle-brachial index, or ABI) can provide objective evidence of atherosclerosis (rest pain usually occurs with an ABI < 0.4). A high ABI can indicate calcification of the arteries. Doppler ultrasound helps identify stenosis and occlusion. Doppler ankle systolic pressure readings that are > 90% of brachial readings are normal. Arteriography and digital subtraction angiography are necessary for surgical evaluation.

1	Arteriography and digital subtraction angiography are necessary for surgical evaluation. Control underlying conditions (e.g., DM, other cardiac risk factors); eliminate tobacco and institute careful hygiene and foot care. Exercise helps develop collateral circulation. ASA, cilostazol, and thromboxane inhibitors may improve symptoms; anticoagulants may prevent clot formation. Angioplasty and stenting have a variable success rate that is dependent on the area of occlusion. Surgery (arterial bypass) or amputation can be employed when conservation treatment fails. Avoid β-blockers in peripheral arterial disease 2° to B2-mediated peripheral vasoconstriction.

1	Surgery (arterial bypass) or amputation can be employed when conservation treatment fails. Avoid β-blockers in peripheral arterial disease 2° to B2-mediated peripheral vasoconstriction. Disruption of the lymphatic circulation that results in peripheral edema and chronic infection of the extremities. Often a complication of surgery involving lymph node dissection. In underdeveloped countries, parasitic infection can lead to lymphatic obstruction, resulting in edema. Congenital malformations of the lymphatic system, such as Milroy’s disease, can present with lymphedema in childhood. Postmastectomy patients present with unexplained swelling of the upper extremity. Immigrants present with progressive swelling of the lower extremities bilaterally with no cardiac abnormalities (i.e., filariasis). Children present with progressive, bilateral swelling of the extremities. Diagnosis is clinical. Rule out other causes of edema, such as cardiac and metabolic disorders.

1	Children present with progressive, bilateral swelling of the extremities. Diagnosis is clinical. Rule out other causes of edema, such as cardiac and metabolic disorders. Directed at symptom management, as no curative treatment exists. Agents such as diuretics are ineffective and relatively contraindicated. Exercise, massage therapy, and pressure garments to mobilize and limit ﬂuid accumulation may be of help. ■Maintain vigilance for cellulitis with prompt gram-antibiotic coverage for infection. Cardiac syncope is associated with one-year sudden cardiac death rates of up to 40%. A sudden, temporary loss of consciousness and postural tone 2° to cerebral hypoperfusion. Etiologies are either cardiac or noncardiac: Cardiac: Valvular lesions, arrhythmias, pulmonary embolism, cardiac tamponade, aortic dissection.

1	Cardiac: Valvular lesions, arrhythmias, pulmonary embolism, cardiac tamponade, aortic dissection. Noncardiac: Orthostatic/hypovolemic hypotension, neurologic (TIA, stroke), metabolic abnormalities, neurocardiogenic syndromes (e.g., vasovagal/micturition syncope), psychiatric. Rule out many potential etiologies. Triggers, prodromal symptoms, and associated symptoms should be investigated. Cardiac causes of syncope are typically associated with very brief or absent prodromal symptoms, a history of exertion, lack of association with changes in position, and/or a history of cardiac disease. Depending on the suspected etiology, Holter monitors or event recorders (arrhythmias), echocardiograms (structural abnormalities), and stress tests (ischemia) can be useful diagnostic tools. Tailored to the etiology. Layers of the Skin 75 Common Terminology 75 Allergic and Immune-Mediated Disorders 75

1	Tailored to the etiology. Layers of the Skin 75 Common Terminology 75 Allergic and Immune-Mediated Disorders 75 The skin consists of three layers: the epidermis, the dermis, and subcutaneous tissue (see Figure 2.2-1). Table 2.2-1 describes pertinent components of the epidermis, the dermis, and the various skin appendages. Table 2.2-2 outlines terms frequently used to describe common manifestations of dermatologic disease. Table 2.2-3 outlines the types and mechanisms of hypersensitivity reactions. Descriptions of common allergic and immune-mediated disorders follow. A relapsing inﬂammatory skin disorder that is common in infancy and presents differently in different age groups. It is characterized by pruritus that leads to lichenification (see Figure 2.2-2). Eczema is the “itch that rashes.” Atopic dermatitis is commonly associated with asthma and allergic rhinitis. Patients are at ↑ risk of 2° bacterial and viral infection.

1	Eczema is the “itch that rashes.” Atopic dermatitis is commonly associated with asthma and allergic rhinitis. Patients are at ↑ risk of 2° bacterial and viral infection. Triggers include climate, food, contact with allergens or physical or chemical irritants, and emotional factors. FIGURE 2.2-1. Layers of the skin. (Adapted, with permission, from Hardman JG et al. Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10th ed. New York: McGraw-Hill, 2001: 1805.) TABLE 2.2-1. Components of Skin Layers Keratinocytes Melanocytes Langerhans cells Merkel cells Fibroblasts (synthesize collagen, elastin, and ground substance) Mast cells Monocytes/macrophages Vessels/lymphatics Nerves Smooth muscle Nails (nail matrix, nail fold, nail plate, nail bed) Hair complex (hair follicles, sebaceous glands, apocrine glands) T AB LE 2.2-2. Common Terms Used to Describe Skin Lesions

1	Macule A fat lesion that differs in color from surrounding skin (< 1 cm in diameter). Papule An elevated solid lesion that is generally small (< 5 mm in diameter). Patch A small, circumscribed area differing in color from the surrounding surface (> 1 cm in diameter). Plaque An elevated solid lesion (> 5 mm in diameter). Cyst An epithelial-lined sac containing fuid or semisolid material. Vesicle A f uid-flled, very small (< 0.5-mm), elevated lesion. Bulla A large vesicle (> 5 mm). Wheal (or hive) An area of localized edema that follows vascular leakage and usually disappears within hours. Erosion A circumscribed, superfcial depression resulting from the loss of some or all of the epidermis. Ulcer A deeper depression resulting from destruction of the epidermis and upper dermis. Scale Abnormal shedding or accumulation of stratum corneum in f akes. Crust A hardened deposit of dried serum, blood, or purulent exudates. Lichenifcation Thickening of the epidermis. Scar A healing defect of the

1	shedding or accumulation of stratum corneum in f akes. Crust A hardened deposit of dried serum, blood, or purulent exudates. Lichenifcation Thickening of the epidermis. Scar A healing defect of the dermis (the epidermis alone heals without a scar).

1	T AB LE 2.2-3. Types and Mechanisms of Hypersensitivity Reactions

1	Type I Anaphylactic and atopic: Antigen cross-links IgE on presensitized mast cells and basophils, triggering the release of vasoactive amines (i.e., histamine). Reaction develops rapidly after antigen exposure as a result of preformed antibody. Examples include anaphylaxis, asthma, urticarial drug reactions, and local wheal and f are. First and Fast (anaphylaxis). Types I, II, and III are all antibody, or B-cell, mediated. Type II Cytotoxic: IgM and IgG bind to antigen on an “enemy” cell, leading to lysis (by complement) or phagocytosis. Examples include autoimmune hemolytic anemia, Rh disease (erythroblastosis fetalis), Goodpasture’s syndrome, and rheumatic fever. Cy-2-toxic. Antibody and complement lead to membrane attack complex (MAC). Type III Immune complex: Antigen-antibody complexes activate complement, which attracts neutrophils; neutrophils release lysosomal enzymes. Examples include polyarteritis nodosa, immune complex glomerulonephritis, SLE, and rheumatoid arthritis.

1	activate complement, which attracts neutrophils; neutrophils release lysosomal enzymes. Examples include polyarteritis nodosa, immune complex glomerulonephritis, SLE, and rheumatoid arthritis. Serum sickness: An immune complex disease (type III) in which antibodies to the foreign proteins are produced (takes f ve days). Immune complexes form and are deposited in membranes, where they fx complement (leading to tissue damage). More common than Arthus reaction. Arthus reaction: A local subacute antibody-mediated hypersensitivity (type III) reaction. Intradermal injection of antigen induces antibodies, which form antigen-antibody complexes in the skin. Characterized by edema, necrosis, and activation of complement. Examples include hypersensitivity pneumonitis and thermophilic actinomycetes. Imagine an immune complex as three things stuck together: antigen-antibody complement. Includes many glomerulonephritides and vasculitides. Most serum sickness is now caused by drugs (not serum).

1	Imagine an immune complex as three things stuck together: antigen-antibody complement. Includes many glomerulonephritides and vasculitides. Most serum sickness is now caused by drugs (not serum). Fever, urticaria, arthralgias, proteinuria, and lymphadenopathy occur 5–10 days after antigen exposure. Antigen-antibody complexes cause the Arthus reaction. Type IV Delayed (cell-mediated) type: Sensitized T lymphocytes encounter antigen and then release lymphokines (leading to macrophage activation). Examples include TB skin tests, transplant rejection, and contact dermatitis (e.g., poison ivy, poison oak). 4th and last—delayed. Cell mediated, not antibody mediated; therefore, it is not transferable by serum.

1	Clinical manifestations by age group are as follows: Infants: Erythematous, weeping, pruritic patches on the face, scalp, and diaper area. Children: Dry, scaly, pruritic, excoriated patches in the ﬂ exural areas and neck. Adults: Lichenification and dry, fissured skin, often limited to the hands. FIGURE 2.2-2. Atopic dermatitis. Erythema toxicum of the newborn resembles eczema, presenting with red papules/ vesicles with surrounding erythema. ↑ eosinophils will be seen on biopsy. This typically benign rash rarely appears after f ve days of age and is usually gone in 7–14 days; treatment is typically observation. Hyperpigmentation, lichenification, and scaling are seen in the antecubital fossae. (Courtesy of Robert Swerlick, MD, as published in Fauci AS, Braunwald E, Kasper DL, et al. Harrison’s Principles of Internal Medicine, 17th ed. New York: McGraw-Hill, 2008.)

1	Principles of Internal Medicine, 17th ed. New York: McGraw-Hill, 2008.) Diagnosis is made clinically. Patients may have mild eosinophilia and ↑ IgE. Rule out seborrheic dermatitis, contact dermatitis, pityriasis rosea, drug eruption, and cutaneous T-cell lymphoma. Prophylactic measures include use of nondrying soaps, application of moisturizers, and avoidance of known triggers. Treat with topical corticosteroids (avoid systemic steroids in light of their side effect profile), PUVA, and topical immunomodulators (e.g., tacrolimus, pimecrolimus). Topical corticosteroids should not be used for longer than 2–3 weeks. A type IV hypersensitivity reaction that results from contact with an allergen to which the patient has previously been exposed and sensitized. Dermatitis develops when the patient is reexposed to the allergen or to a cross-reactive compound. More common in adults than in children.

1	Commonly presents with pruritus and rash, but can also present with edema, fever, and lymphadenopathy. Frequently implicated allergens include poison ivy, poison oak, nickel, soaps, detergents, cosmetics, and rubber products containing latex (e.g., gloves and elastic bands in clothing). Characteristic distributions involve areas where makeup, clothing, perfume, nickel jewelry, and plants come into contact with the skin. The dermatitis begins in the area of contact with the antigen, with its appearance varying with the acuity of the lesion. Acute: Approximately 24–48 hours after an allergic contact, the skin becomes erythematous, presenting with tiny blisters followed by scale and crusts. Lesions are intensely pruritic. Subacute: Results from episodic exposure or a weak allergen. Lesions are less “angry appearing” than those of an acute inﬂ ammatory rash, and some lichenification is seen.

1	Subacute: Results from episodic exposure or a weak allergen. Lesions are less “angry appearing” than those of an acute inﬂ ammatory rash, and some lichenification is seen. Chronic: Results from extended exposure to an allergen. Characterized by erythema and lichenification with fissuring, often with superimposed acute dermatitis. The overall shape of the rash often mimics that of the exposing object (see Figure 2.2-3), but it can also spread over the body via transfer of allergen by the hands or via circulating T lymphocytes. Patients are at ↑ risk of 2° infection. Diagnosed by clinical impression. A patch test can be used to establish the causative allergen after the acute-phase rash has been treated. The differential includes atopic dermatitis, seborrheic dermatitis, impetigo, HSV, herpes zoster, and fungal infection. Prophylaxis consists of avoidance of the offending allergen.

1	Prophylaxis consists of avoidance of the offending allergen. Treat with topical or systemic corticosteroids as needed and with cool, wet compresses to relieve and debride the skin. The pathogenesis of contact dermatitis involves allergenic molecules that are passed through the epidermis and taken up by Langerhans cells, which carry them to the lymph nodes and expose them to T lymphocytes. FIGURE 2.2-3. Contact dermatitis. Shown above are erythematous papules and vesicles with serous weeping localized to areas of contact with the offending agent. (Reproduced, with permission, from Hurwitz RM. Pathology of the Skin: Atlas of Clinical-Pathological Correlation, 2nd ed. Stamford, CT: Appleton & Lange, 1998: 3.) Suspect HIV in a young person with severe seborrheic dermatitis.

1	Suspect HIV in a young person with severe seborrheic dermatitis. A common disease that may be caused by Pityrosporum ovale, a generally harmless yeast found in sebum and hair follicles. It has a predilection for areas with oily skin such as the scalp, eyebrows, nasolabial folds, and midchest. The appearance of rash varies with age: Infants: Presents as a severe, red diaper rash with yellow scale, erosions, and blisters. A thick crust (“cradle cap”) may be seen on the scalp. Children/adults: Red, scaly patches are seen around the ears, eyebrows, nasolabial fold, midchest, and scalp. The rash is more localized and less dramatic than that seen in infants. Patients with HIV/AIDS can develop severe seborrheic dermatitis or an overlapping syndrome of severe seborrheic dermatitis, psoriasis, psoriatic arthritis, and even Reiter’s syndrome. Diagnosed by clinical impression. Rule out contact dermatitis and psoriasis.

1	Diagnosed by clinical impression. Rule out contact dermatitis and psoriasis. Treatment consists of selenium sulfide or zinc pyrithione shampoos for the scalp, and topical antifungals and/or topical corticosteroids for other areas. Low-concentration coal tar shampoos are still available despite concerns about carcinogenicity. A T-cell-mediated inﬂammatory dermatosis characterized by erythematous patches and silvery scales due to dermal inﬂammation and epidermal hyperplasia. Five percent of patients also have a seronegative arthritis. The condition usually starts in puberty or young adulthood, and its incidence is 2–4%. The typical lesion is a round, sharply bordered erythematous patch with silvery scales (see Figure 2.2-4A). Lesions are classically found on the extensor surfaces, including the elbows, knees, scalp, and lumbosacral regions.

1	Lesions are classically found on the extensor surfaces, including the elbows, knees, scalp, and lumbosacral regions. Lesions may initially appear very small (guttate) but may slowly enlarge and become conﬂuent. Psoriatic nails feature pitting, “oil spots,” and onycholysis, or lifting of the nail plate (see Figure 2.2-4B). Psoriatic lesions can be provoked by local irritation or by trauma (Koebner’s phenomenon). Streptococcal infection can lead to cutaneous immune complex deposition, which triggers guttate psoriasis. Some medications, such as β-blockers, lithium, and ACEIs, can also induce psoriasis. Psoriatic arthritis usually begins in the hands with “sausage digits,” but the knees, wrists and ankles as well as the lumbosacral region may also be affected. Arthritic patients with spinal involvement are usually HLAB27 .

1	When pustular psoriasis, a less common form, is generalized, it can be life threatening, presenting with fever, electrolyte abnormalities, and loss of serum proteins. AB FIGURE 2.2-4. Psoriasis. (A) Skin changes. The classic sharply demarcated plaques with silvery scales are commonly located on the extensor surfaces (e.g., elbows, knees). (B) Nail changes. Note the pitting, onycholysis, and “oil spots.” (Reproduced, with permission, from Hur witz RM. Pathology of the Skin: Atlas of Clinical-Pathological Correlation, 2nd ed. Stamford, CT: Appleton & Lange, 1998: 15, 18.) Clinical impression is usually sufficient for diagnosis. Classically presents with the Auspitz sign (bleeding when scale is scraped), but biopsy can be useful. Histology classically shows a thickened epidermis, elongated rete ridges, an absent granular cell layer, preservation of nuclei, and a sterile neutrophilic infiltrate (Munro’s microabscess) in the stratum corneum.

1	Treat with topical steroids combined with keratolytic agents, tar, or anthralin along with UV therapy, including PUVA. Methotrexate may be used for severe cases. Retinoids (vitamin A derivatives) may also be used. Arthritis should be treated first with NSAIDs and then with methotrexate if necessary. Systemic corticosteroids should be avoided, as tapering can induce psoriatic ﬂ ares. Recently, biologic agents such as TNF-α inhibitors have proven effective in severe psoriatic arthritis and psoriasis. Urticaria is characterized by superficial, intense edema in a localized area. It is usually acute but can also be chronic (lasting > 6 weeks). The condition results from the release of vasoactive substances (histamine, prostaglandins) from mast cells in a type I hypersensitivity response. ■Hives can range in severity from a few itchy bumps to life-threatening anaphylaxis.

1	■Hives can range in severity from a few itchy bumps to life-threatening anaphylaxis. The typical lesion is an elevated papule or plaque that is reddish or white and variable in size. Lesions are widespread and last a few hours. In severe allergic reactions, extracutaneous manifestations can include tongue swelling, angioedema (deeper, more diffuse swelling), asthma, GI symptoms, joint swelling, and fever. Acute urticaria is a response to a trigger that may be a food, drug, virus, insect bite, or physical stimulus. Chronic urticaria is usually idiopathic. Diagnosed by clinical impression and patient report. Biopsy demonstrates perivascular edema. It can often be difficult to determine the cause. Treat with systemic antihistamines. Topical medications are of no benefit.

1	Treat with systemic antihistamines. Topical medications are of no benefit. Maintain a high suspicion for a cutaneous drug reaction in patients who are hospitalized and develop rashes. Such reactions can take many forms, including urticarial, lupus-like, vasculitic, purpuric, lichenoid, and vesicular. Drugs can cause all four types of hypersensitivity reactions, and sometimes the same drug may cause different types of reactions in different patients. Eruptions occur 7–14 days after exposure, so if a patient reacts within a day or two of starting a new drug (i.e., a drug they have never taken before), that drug is probably not the causative agent. Eruptions are generally widespread, relatively symmetrical, and pruritic. Most are relatively short-lived, disappearing within 1–2 weeks following removal of the offending agent.

1	Eruptions are generally widespread, relatively symmetrical, and pruritic. Most are relatively short-lived, disappearing within 1–2 weeks following removal of the offending agent. The exception is fixed drug eruption, which consists of reddish macules or papules that develop in the same area (usually the genitalia, face, or extremities) each time the patient is exposed to the triggering agent. After these lesions resolve, there is often a persistent brown pigmentation. Extreme complications of drug eruptions include erythroderma and toxic epidermal necrolysis (TEN). Diagnosed by clinical impression. Patients may have eosinophilia and eosinophils on histopathology. Discontinue the offending agent; treat symptoms with antihistamines.

1	Diagnosed by clinical impression. Patients may have eosinophilia and eosinophils on histopathology. Discontinue the offending agent; treat symptoms with antihistamines. A cutaneous reaction pattern with classic targetoid lesions that has many triggers and is often recurrent. Although some cases are idiopathic, many are triggered by recurrent HSV infection of the lip. Other common triggers are drugs (e.g., sulfa drugs, anticonvulsants, barbiturates, penicillin, NSAIDs) and mycoplasmal infections. The characteristic lesion has a target appearance (see Figure 2.2-5), but other types of lesions may be seen as well. The disease can occur on mucous membranes, where erosions are seen. Typically, lesions start as erythematous macules that become centrally clear and then develop a blister. The palms and soles are often affected. May be associated with systemic symptoms, including fever, myalgias, headache, and arthralgias (these symptoms may precede eruption).

1	May be associated with systemic symptoms, including fever, myalgias, headache, and arthralgias (these symptoms may precede eruption). In its minor form, the disease is uncomplicated and localized to the skin. However, severe erythema multiforme can lead to TEN or Stevens-Johnson syndrome, in which patients are very ill with involvement of at least two mucosal surfaces. Diagnosed by clinical impression. A history of recurrent labial herpes should be sought in all cases with multiple recurrences. Symptomatic treatment is all that is necessary; systemic corticosteroids are of no benefit. Minor cases can be treated with antipruritics; major cases should be treated as burns. In patients with HSV, suppressive acyclovir may ↓ the frequency of rashes. FIGURE 2.2-5. Erythema multiforme.

1	Minor cases can be treated with antipruritics; major cases should be treated as burns. In patients with HSV, suppressive acyclovir may ↓ the frequency of rashes. FIGURE 2.2-5. Erythema multiforme. Evolving erythematous plaques and papules are seen with a target appearance consisting of a dull red center, a pale zone, and a darker outer ring. (Reproduced, with permission, from Hurwitz RM. Pathology of the Skin: Atlas of Clinical-Pathological Correlation, 2nd ed. Stamford, CT: Appleton & Lange, 1998: 24.) SJS and TEN may be confused with staphylococcal scalded-skin syndrome (SSSS), another condition in which patients lose widespread sheets of skin. SSSS is usually seen in children < 6 years of age and has an infectious etiology. SJS/TEN is generally seen in adults and is usually caused by a drug reaction. Patients with erythema nodosum may have a falseVDRL (as in SLE).

1	Patients with erythema nodosum may have a falseVDRL (as in SLE). SJS and TEN constitute two different points on the spectrum of life-threatening exfoliative mucocutaneous diseases that are often caused by a drug-induced immunologic reaction. The epidermal separation of SJS involves < 10% of body surface area (BSA), whereas TEN involves > 30% of BSA. Involvement of 10–30% of BSA is often considered SJS/TEN overlap. May be preceded by erythema multiforme, a ﬂulike prodrome, skin tenderness, a maculopapular drug rash, or painful mouth lesions. Often associated with a history of exposure to new drugs, such as penicillin, sulfonamides, seizure medications (e.g., phenytoin, carbamazepine), quinolones, cephalosporins, allopurinol, corticosteroids, or NSAIDs. Exam reveals severe mucosal erosions with widespread erythematous, cutaneous macules or atypical targetoid lesions. The epidermal lesions often become conﬂuent and show a Nikolsky’s sign and epidermal detachment.

1	Mucous membranes of the eyes, mouth, and genitals often become eroded and hemorrhagic as well. SJS: Biopsy shows degeneration of the basal layer of the epidermis and perivascular mononuclear infiltrate with some eosinophils in the papillary dermis. Subepidermal blisters may also be seen. TEN: Biopsy shows full-thickness eosinophilic epidermal necrosis with cell-poor infiltrate; there is a sparse perivascular lymphocytic infiltrate. TEN vs. SSSS: On biopsy, TEN demonstrates full-thickness epidermal damage, whereas SSSS shows only superficial damage. The differential also includes graft-versus-host reaction (usually after bone marrow transplant), radiation therapy, and burns.

1	The differential also includes graft-versus-host reaction (usually after bone marrow transplant), radiation therapy, and burns. Patients have the same complications as burn victims, including thermoregulatory difficulties, electrolyte disturbances, and 2° infections. Treatment includes skin coverage and maintenance of ﬂuid and electrolyte balance. Controversial treatments include systemic corticosteroids in the early stages of SJS/ TEN or IVIG. There is a high risk of mortality. A panniculitis whose triggers include infection (e.g., Streptococcus, Coccidioides, Yersinia, TB), drug reactions (e.g., sulfonamides, various antibiotics, OCPs), and chronic inﬂ ammatory diseases (e.g., sarcoidosis, Crohn’s disease, ulcerative colitis, Behçet’s disease). Painful, erythematous nodules appear on the patient’s lower legs (see Figure 2.2-6) and slowly spread, turning brown or gray. Patients may present with fever and joint pain. FIGURE 2.2-6. Erythema nodosum.

1	FIGURE 2.2-6. Erythema nodosum. Erythematous plaques and nodules are commonly located on pretibial areas. Lesions are pain ful and indurated but heal spontaneously without ulceration. (Reproduced, with permission, from Hurwitz RM. Pathology of the Skin: Atlas of Clinical-Pathological Correlation, 2nd ed. Stamford, CT: Appleton & Lange, 1998: 132.) Diagnosed by clinical impression. Histology shows nonspecific septal panniculitis. Workup should include an ASO titer, a PPD test in patients who are high risk, a CXR to rule out sarcoid, and a small bowel series to rule out IBD in patients with GI symptoms. Remove the triggering factor and treat the underlying disease where possible. NSAIDs can be used but may lead to erythema multiforme.

1	Remove the triggering factor and treat the underlying disease where possible. NSAIDs can be used but may lead to erythema multiforme. A life-threatening autoimmune condition characterized by an intraepidermal blister leading to widespread painful erosions of the skin and mucous membranes. Antibodies are directed against desmoglein molecules responsible for keratinocyte adherence, leading to loss of cellular attachment. Patients are generally middle-aged (40–60). Initial presentation is with mucous membrane involvement, typically mouth ulcers, with progression to skin involvement. Rarely, an intact blister may be seen, but generally presents only with erosions, often accompanied by crusting, weeping, and 2° infections. Along with the clinical picture, a Nikolsky’s sign (the ability to produce a blister by rubbing skin adjacent to a natural blister) and skin biopsy with immunoﬂ uorescence confirm the diagnosis.

1	Along with the clinical picture, a Nikolsky’s sign (the ability to produce a blister by rubbing skin adjacent to a natural blister) and skin biopsy with immunoﬂ uorescence confirm the diagnosis. Biopsy shows acantholysis (intraepidermal split with free-ﬂ oating keratinocytes in the blister). Immunoﬂ uorescence and ELISA are confirmatory for antidesmoglein antibodies. Long-term treatment is generally required. Initially, systemic corticosteroids are used at high doses in combination with steroid-sparing agents introduced early to ↓ corticosteroid side effects. Steroid-sparing agents include mycophenolate mofetil and azathioprine. Recently, rituximab and IVIG have been successfully used for recalcitrant disease.

1	Steroid-sparing agents include mycophenolate mofetil and azathioprine. Recently, rituximab and IVIG have been successfully used for recalcitrant disease. An acquired blistering disease that leads to separation at the epidermal basement membrane. It is most commonly seen in patients 60–80 years of age. Its pathogenesis involves antibodies that are developed against the bullous pemphigoid antigen, which lies superficially in the basement membrane zone (BMZ). Antigen-antibody complexes activate complement and eosinophil degranulation that provoke an inﬂammatory reaction and lead to F IGU R E 2.2-7. Bullous pemphigoid. Multiple tense serous and partially hemorrhagic bullae can be seen. (Reproduced, with permission, from Fitzpatrick TB. Color Atlas & Synopsis of Clinical Dermatology, 4th ed. New York: McGraw-Hill, 2001: 100.) separation at the BMZ. The blisters are stable because their roof consists of nearly normal epidermis.

1	Presents with firm, stable blisters that arise on erythematous skin, often preceded by urticarial lesions. Nikolsky’s sign is . The blisters form crusts and erosions (see Figure 2.2-7). Mucous membranes are less commonly involved than is the case in pemphigus. Diagnosed according to the clinical picture. Skin biopsy shows a subepidermal blister, often with an eosinophil-rich infiltrate. Immunoﬂ uorescence demonstrates linear IgG and C3 immunoglobulin and complement at the dermal-epidermal junction. Systemic corticosteroids. Topical corticosteroids can help prevent blister formation when applied to early lesions. A painful, recurrent vesicular eruption of the mucocutaneous surfaces due to infection with HSV. HSV-1 usually produces oral-labial lesions, whereas HSV-2 usually causes genital lesions. The virus spreads through epidermal cells, causing them to fuse into giant cells. The local host inﬂ ammatory response leads to erythema and swelling.

1	The initial infection is passed by direct contact, after which the herpesvirus remains dormant in local nerve ganglia. 1° episodes are generally longer and more severe than recurrences. Onset is preceded by prodromal tingling, burning, or pain but can also present with lymphadenopathy, fever, discomfort, malaise, and edema of involved tissue. Recurrences are limited to mucocutaneous areas innervated by the involved nerve. Recurrent oral herpes (HSV-1): Typically consists of the common “cold sore,” which presents as a cluster of crusted vesicles on an erythematous base (see Figure 2.2-8A). It is often triggered by sun and fever. Recurrent genital herpes (HSV-2): Unilateral and characterized by a cluster of blisters on an erythematous base, but with less pain and systemic involvement than the 1° infection. Diagnosed primarily by the clinical picture. Multinucleated giant cells on Tzanck smear (see Figure 2.2-8B) yield a presumptive diagnosis.

1	Diagnosed primarily by the clinical picture. Multinucleated giant cells on Tzanck smear (see Figure 2.2-8B) yield a presumptive diagnosis. VZV has the same appearance on Tzanck, so culture or direct ﬂ uorescent antibody staining is needed for definitive diagnosis. Dermatitis herpetiformis differs from HSV, consisting of pruritic papules and vesicles on the elbows, knees, buttocks, neck, and scalp. Granular IgA is seen on dermal papillae. The condition is associated with celiac disease (15–25% of celiac patients may have it). Treat with dapsone and a gluten-free diet. AB FIGURE 2.2-8. Herpes simplex. (A) 1° infection. Grouped vesicles on an erythematous base on the patient’s lips and oral mucosa may progress to pustules before resolving. (B) Tzanck smear. The multinucleated giant cells from vesicular ﬂuid provide a presumptive diagnosis of HSV infec tion. The Tzanck smear cannot distinguish between HSV and VZV infection. (Reproduced, with permission, from Hurwitz

1	RM. Pathology of the Skin: Atlas of Clinical-Pathological Correlation, 2nd ed. Stamford, CT: Appleton & Lange, 1998: 145.) Oral or IV acyclovir (IV for severe cases or for immunocompromised patients) ↓ both the frequency and the severity of recurrences. Daily acyclovir, valacyclovir, or famciclovir suppressive therapy may be used in patients with > 6 outbreaks per year or for those with erythema multiforme. Acyclovir ointment is somewhat effective in reducing the duration of viral shedding but does not prevent recurrence. In AIDS patients, HSV can persist, with ulcers remaining resistant to antiviral therapy. Symptomatic HSV infection lasting > 1 month can be considered an AIDS-defining illness. VZV causes two different diseases, varicella and herpes zoster—with transmission occurring via respiratory droplet or by direct contact. VZV has an incubation period of 10–20 days, with contagion beginning 24 hours before the eruption appears and lasting until lesions have crusted.

1	■Varicella: A prodrome consisting of malaise, fever, headache, and myalgia occurs 24 hours before the onset of the rash. Pruritic lesions appear in crops over a period of 2–3 days, evolving from red macules to grouped central vesicles (“dewdrop on a rose petal”) and then crusting over. At any given time, patients have all stages of lesions over their entire body. The trunk, face, scalp, and mucous membranes are involved, but the palms and soles are spared. In adults, chickenpox is often more severe, with systemic complications such as pneumonia and encephalitis. Zoster: Herpes zoster represents the recurrence of VZV in a specific nerve, with lesions cropping up along the nerve’s dermatomal distribution. Outbreaks are usually preceded by intense local pain and then arise as grouped blisters on an erythematous base (see Figure 2.2-9).

1	In immunocompromised patients, zoster can lead to severe local disease, disseminated cutaneous disease, and systemic diseases that mimic varicella. Older patients with severe zoster may develop postherpetic neuralgia. Diagnosed by the clinical picture. Varicella is self-limited in healthy children. A vaccine is available for infants, children, and adults and is routinely used for disease prevention. Adults should be treated with systemic acyclovir. Although acyclovir may speed the cutaneous course of zoster, pain control is most important for patients with this disease. A poxvirus infection that is most common in young children and in AIDS patients. It is spread by physical contact. ■The rash is composed of tiny waxy papules, frequently with central umbilication. In children, lesions are found on the trunk, extremities, or face If you see giant molluscum (see Figure 2.2-10). In adults, they are commonly found on the genitalia contagiosum, think HIV. and in the perineal region.

1	and in the perineal region. FIGURE 2.2-9. Varicella zoster. The unilateral dermatomal distribution of the grouped vesicles on an erythematous base is characteristic. FIGURE 2.2-10. Molluscum contagiosum. The dome-shaped, ﬂeshy, umbilicated papule on the child’s eyelid is characteristic. (Repro duced, with permission, from Hurwitz RM. Pathology of the Skin: Atlas of Clinical-Pathological Correlation, 2nd ed. Stamford, CT: Appleton & Lange, 1998: 149.) In AIDS patients, lesions often appear on the face and can become quite large. Lesions are asymptomatic unless they become inﬂamed or irritated. Diagnosed by the clinical picture, and confirmed by expressing and staining the contents of the papules. Giemsa or Wright’s stain allows for the identification of large inclusion or molluscum bodies.

1	Any local destructive method is effective, including curetting, freezing, or applying trichloroacetic acid to the lesions. Lesions resolve spontaneously over months to years and are often left untreated in children. Warts are caused by many different types of HPV and can occur on skin, mucous membranes, and other epithelia. Although usually benign, some subtypes of HPV (especially 16 and 18) lead to squamous malignancies. Spread is by direct contact. HPV leads to hyperproliferation of infected cells. Common warts are the most prevalent HPV infection. Although most often seen on the hands, they can occur anywhere. On plantar and palmar surfaces, warts tend to grow downward into the skin rather than outward, giving them a ﬂ atter appearance.

1	The classic genital wart is a cauliﬂower-like papule or nodule appearing on the glans penis, the vulva, or the perianal region. Warts on mucous membranes are generally velvety and white, appearing on oral, genital, and even laryngeal mucosa. Laryngeal warts are transmitted to infants by mothers with genital HPV. Diagnosed by the clinical picture. Acetowhitening can be helpful in visualizing mucosal lesions. There is a long latency period, with children sometimes acquiring HPV at birth and not manifesting any lesions until years later. Treatment centers on destruction of the tissue by curettage, cryotherapy, or acid keratolytics. Genital warts are treated locally with podophyllin, trichloroacetic acid, imiquimod, or 5-FU. HPV lesions on the cervix must be monitored cytologically and histologically for evidence of malignancy.

1	A superficial, weeping local infection that primarily occurs in children and is caused by both group A streptococcal and staphylococcal organisms. It is transmitted by direct contact. There are two types: Common type: Characterized by pustules and honey-colored crusts on an erythematous base; generally appears on the face (see Figure 2.211). Bullous type: Usually acral; characterized by large stable blisters. Bullous impetigo is almost always caused by S. aureus and can evolve into SSSS. Streptococcal impetigo can be complicated by acute streptococcal glomerulonephritis. FIGURE 2.2-11. Impetigo. What is another skin condition caused by group A strep? Erysipelas, which presents as a small red patch on the cheek that turns into a painful raised, shiny red plaque. Patients often have a history of trauma or pharyngitis. Treat with penicillin.

1	Dried pustules with a superficial golden-brown crust are most commonly found around the nose and mouth. (Reproduced, with permission, from Hurwitz RM. Pathology of the Skin: Atlas of Clinical-Pathological Correlation, 2nd ed. Stamford, CT: Appleton & Lange, 1998: 165.) Scarlet fever: “Sunburn with goosebumps” appearance; strawberry tongue. Caused by S. pyogenes. Treatment: Penicillin. Salmonella typhi: Small pink papules on the trunk (“rose spots”) in groups of 10–20 plus gallbladder disease. Treatment: Cholecystectomy for chronic carrier state. Ludwig’s angina is a bilateral cellulitis of the submaxillary/ sublingual spaces that usually results from an infected tooth. It presents with dysphagia, drooling, fever, and a red, warm mouth and can lead to death from asphyxiation. Diagnosed by the clinical picture.

1	Diagnosed by the clinical picture. Treat with antibiotics with antistaphylococcal activity. Topical antibiotics are often sufficient, but systemic agents can hasten recovery and prevent spread to other patients. An infection caused by Corynebacterium, presenting as brownish-red patches with fine scales that characteristically appear along major skin folds. It is more common among diabetics. Wood’s light exam reveals coral-red ﬂ uorescence of lesions. Gram staining reveals gram-, filamentous rods. Treat with erythromycin. (Remember: erythrasma is treated with erythromycin.) A deep, local infection involving the connective tissue, subcutaneous tissue, or muscle in addition to the skin. It is commonly caused by staphylococci or group A streptococci originating from an area of damaged skin or from a systemic source of infection. Community-acquired MRSA is an increasingly common cause. Risk factors include diabetes, IV drug use, venous stasis, and immune compromise.

1	Presents with red, hot, swollen, tender skin. Fever and chills are also common. Diagnosed by the clinical picture; wound culture may aid in diagnosis and help determine antibiotic sensitivities for treatment. Blood cultures should be obtained when bacteremia is suspected. Culture and sensitivities are important in case of MRSA. Rule out abscess, urticaria, contact dermatitis, osteomyelitis, and necrotizing fasciitis. Treat with 7–10 days of oral antibiotics for mild cases or with IV antibiotics if there is evidence of systemic toxicity, comorbid conditions, DM, extremes of age, hand or orbital involvement, or other concerns. Deep infection along a fascial plane causing severe pain followed by anesthesia. Infection is caused by S. pyogenes or Clostridium perfringens. A history of trauma or a recent surgery to the affected area is often but not always elicited.

1	Presents with sudden onset of pain and swelling at the site of trauma or recent surgery. Pain often progresses to anesthesia. An area of erythema quickly spreads over the course of hours to days. Margins move out into normal skin, and skin becomes dusky or purplish near the site of insult, ultimately leading to necrosis. Necrosis can initially have the appearance of undermining of the skin and subcutaneous layer; if the skin is open, gloved fingers can easily pass between the two layers to reveal yellow-green necrotic fascia (infection spreads quickly in deep fascia). The most important signs are tissue necrosis, a putrid discharge, bullae, severe pain, gas production, rapid burrowing through fascial planes, lack of classical tissue inﬂammatory signs, and intravascular volume loss. Local radiographs or CT scans show air in tissue. Biopsy from the edge of the lesion can be diagnostic. A surgical emergency. Early and aggressive surgical debridement is critical.

1	Local radiographs or CT scans show air in tissue. Biopsy from the edge of the lesion can be diagnostic. A surgical emergency. Early and aggressive surgical debridement is critical. If Streptococcus is the principal organism involved, penicillin G is the drug of choice. Clindamycin is second line. For anaerobic coverage, give metronidazole or a third-generation cephalosporin. Inﬂammation of the hair follicle. Although typically caused by infection with Staphylococcus, Streptococcus, and gram-bacteria, folliculitis may occasionally be caused by yeast such as Candida albicans or Pityrosporum ovale. It may also be mechanical, arising from ingrown hairs (most common in patients with curly hair). Presents as a tiny pustule that appears at the opening of a hair follicle and usually has a hair penetrating it. When the infection is deeper, a furuncle, or hair follicle abscess, develops.

1	Presents as a tiny pustule that appears at the opening of a hair follicle and usually has a hair penetrating it. When the infection is deeper, a furuncle, or hair follicle abscess, develops. Furuncles are larger and more painful than folliculitic lesions and may disseminate to adjacent follicles to form a carbuncle. Patients with diabetes or immunosuppression are at ↑ risk. Folliculitis can be a critical problem in AIDS patients, in whom the disease is intensely pruritic and resistant to therapy. Diagnosed by the clinical picture. Fournier gangrene is a form of necrotizing fasciitis that is localized to the scrotum and perineal area. leads to “hot tub folliculitis.” Ironically, erythromycin does not cause erythema with sun exposure. It is tetracycline and doxycycline that can cause serious photosensitivity!

1	leads to “hot tub folliculitis.” Ironically, erythromycin does not cause erythema with sun exposure. It is tetracycline and doxycycline that can cause serious photosensitivity! Topical antibiotics can be used to treat mild disease, but severe cases require systemic antibiotics. Large lesions must be incised, drained, and cultured to rule out MRSA. Patients who are prone to ingrown hairs should be advised not to shave. An endogenous skin disease that is common among adolescents. The pathogenesis involves hormonal activation of sebaceous glands, the development of the comedo or plugged sebaceous follicle, and involvement of Propionibacterium acnes in the follicle, causing inﬂammation. Comedones may be caused by medications (e.g., lithium, corticosteroids) or by topical occlusion (e.g., cosmetics). There are three stages of acne lesions: Comedo: May be open (“blackheads”) or closed (“whiteheads”); present in large quantities but with little inﬂ ammation.

1	There are three stages of acne lesions: Comedo: May be open (“blackheads”) or closed (“whiteheads”); present in large quantities but with little inﬂ ammation. Inﬂ ammatory: The comedo ruptures, creating a pustule that can be large and nodular. Scar: As the inﬂ ammation heals, scars may develop. Picking at papules exacerbates scarring. Two types of cysts can occur in acne: inﬂammatory cysts, which are large, ﬂuctuant pustules, and epidermoid cysts, which develop along the eyebrows and behind the ears. Acne first develops at puberty and typically persists for several years. Males are more likely to have severe, cystic acne than are females. Women in their 20s tend to have a variant that ﬂares cyclically with menstruation, featuring fewer comedones and more painful lesions on the chin. Androgenic stimulation may contribute to these lesions. Diagnosed by the clinical picture. Treat comedones with topical tretinoin (Retin-A) and benzoyl peroxide.

1	Diagnosed by the clinical picture. Treat comedones with topical tretinoin (Retin-A) and benzoyl peroxide. Inﬂammatory lesions should be treated with topical antibiotics (e.g., erythromycin, clindamycin) or systemic agents (e.g., tetracycline, erythromycin). Isotretinoin (Accutane) leads to marked improvement in > 90% of acne patients and has greatly improved the treatment of severe acne. Isotretinoin is, however, a teratogen and may cause transient elevations in cholesterol, triglycerides, and LFTs, and it may also be associated with depression. Patients on isotretinoin are thus carefully monitored and are required to get monthly blood tests to check quantitative serum β-hCG (to rule out pregnancy), LFTs, cholesterol, and triglycerides. Monthly refills are contingent on completion of blood testing and evaluation by a dermatologist.

1	Abscesses in the sacrococcygeal region that usually occur near the top of the natal cleft. Their name may not be appropriate, as not all such cysts contain hair, and not all are true cysts. Repetitive trauma to the region plays a role. The condition is thought to start as a folliculitis that becomes an abscess complicated by perineal microbes, especially Bacteroides. It most commonly occurs between the ages of 20 and 40, affecting men more often than women. Patients present with an abscess at the natal cleft that can be tender, ﬂ uctuant, warm, and indurated and is sometimes associated with purulent drainage or cellulitis. Systemic symptoms are uncommon, but cysts may develop into perianal fistulas. Risk factors include deep and hairy natal clefts, obesity, and a sedentary lifestyle. Diagnosed by the clinical picture. Rule out perirectal and anal abscess.

1	Risk factors include deep and hairy natal clefts, obesity, and a sedentary lifestyle. Diagnosed by the clinical picture. Rule out perirectal and anal abscess. Treatment consists of incision and drainage of the abscess under local anesthesia followed by sterile packing of the wound. Abscesses should be allowed to heal by 2° intention. Antibiotics are not needed unless cellulitis is present; if they are prescribed, both aerobic and anaerobic coverage is required. Good local hygiene and shaving of the sacrococcygeal skin can help prevent recurrence. Patients should follow up with a surgeon. Caused by Malassezia furfur, a yeast that is part of the normal skin ﬂ ora (morphologic variants are Pityrosporum ovale and Pityrosporum orbiculare). It is unclear what leads the organism to overgrow on the skin surface and become a pathogen, but humid and sweaty conditions as well as host factors such as oily skin can contribute. Cushing’s syndrome and immunosuppression are also risk factors.

1	Patients present with small, scaly patches of varying color, usually on the chest or back. Lesions may be hypopigmented as a result of interference with melanin production, or they may be hyperpigmented by virtue of thickened scale. Diagnosed by clinical impression, and confirmed by potassium hydroxide (KOH) preparation of scale that reveals a “spaghetti and meatballs” pattern of hyphae and spores. Treat lesions with topical selenium sulfide daily for one week, followed by application once weekly for prophylaxis. Commonly called “yeast infection” or “thrush,” candidiasis can be caused by any Candida species but is most commonly caused by C. albicans. In immune-competent patients, it typically presents as a superficial infection of the skin or mucous membranes in moist areas such as skin folds, armpits, the vagina, and below the breasts. Oral thrush is not uncommon among children, but in adults it is often a sign of a weakened immune system.

1	Patients often have a history of antibiotic use, steroid use, or diabetes. Symptoms vary according to the site affected: Oral candidiasis: Presents with painless white plaques that cannot easily be scraped off. Candidiasis of the skin: Presents as pink, circular, erythematous macules that converge, with smaller satellite lesions seen nearby, often in skin folds. In infants, infection can often be seen in the diaper area and along the inguinal folds. Diagnosed by the clinical picture. Confirmed by KOH preparation of a scraping or swab of the affected area. KOH dissolves the skin cells but leaves the Candida untouched such that candidal hyphae and pseudospores become visible. Oral candidiasis: Oral ﬂuconazole; nystatin swish and swallow. Superficial (skin) candidiasis: Topical antifungals; keep skin clean and dry. Diaper rash: Topical nystatin.

1	Oral candidiasis: Oral ﬂuconazole; nystatin swish and swallow. Superficial (skin) candidiasis: Topical antifungals; keep skin clean and dry. Diaper rash: Topical nystatin. Dermatophytes live only in tissues with keratin (i.e., the skin, nails, and hair) and are a common cause of infection. Causative organisms include Microsporum, Trichophyton, and Epidermophyton. The immune response to the dermatophyte, rather than the organism itself, is responsible for many of the symptoms. Pets are a reservoir for Microsporum. Other risk factors include diabetes, ↓ peripheral circulation, immune compromise, and chronic maceration of skin (e.g., from athletic activities). Presentation varies according to subtype: ■ Tinea corporis: Presents as a scaly, pruritic eruption with a sharp, irregular border, often with central clearing. May be seen in immunocompromised patients or in children following contact with infected pets.

1	Tinea pedis/manuum: Presents as chronic interdigital scaling with erosions between the toes (“athlete’s foot”) or as a thickened, scaly skin on the soles. Asymmetric involvement of the hands is typical. Tinea cruris (“jock itch”): A chronic infection of the groin (typically sparing the scrotum) that is usually associated with tinea pedis. Tinea capitis (“ringworm”): A diffuse, scaly scalp eruption similar to seborrheic dermatitis. Diagnosed by the clinical picture; confirmed by scales prepared in KOH showing hyphae. Patients can be treated with topical or systemic antifungals. Tinea capitis must be treated with systemic drugs.

1	Patients can be treated with topical or systemic antifungals. Tinea capitis must be treated with systemic drugs. Lice live off blood and on specific parts of the body, depending on their species. The head louse lives on the scalp and lays its eggs as nits attached to hair; the body louse lives in clothing and bites only the body. The pubic louse lives on pubic hair. Lice are spread through body contact or by the sharing of bedclothes and other garments. They secrete local toxins that lead to pruritus. Patients with lice often experience severe pruritus, and 2° bacterial infection of the excoriations is a risk. Classroom epidemics of head lice are common. Body lice are seen in people with inadequate hygiene or in those with crowded living conditions. Pubic lice (called “crabs” because of their squat, crablike body shape) contain anticoagulant in their saliva, so their bites often turn blue. Lice can be seen on hairs or in clothes.

1	Lice can be seen on hairs or in clothes. Head lice: Treat with OTC pyrethrin (RID) and mechanical removal of nits. Body lice: Wash body, clothes, and bedding thoroughly. Treating the body with topical permethrin or pyrethrin may also be required. Pubic lice: Treat with RID. Caused by Sarcoptes scabiei, a tiny arthropod that mates on the skin surface, after which the female digs a passage into the stratum corneum and lays her eggs. The burrowing leads to pruritus that ↑ in intensity once an allergy to Other ulcers (all can be treated with specialized wound dressings): ■Venous stasis ulcers: Found near the lateral or medial malleolus, often in association with lower extremity edema. Treat with compression (Unna boots or compression stockings) and elevation. ■Arterial insufficiency ulcers: Found on the heel and tips of toes. Typically very painful. ■Neuropathic ulcers: Found on the underside of the foot and toes, usually at pressure points. Typically painless.

1	■Neuropathic ulcers: Found on the underside of the foot and toes, usually at pressure points. Typically painless. the mite or its products develops. Scabies mites are spread through close contact. Patients present with intense pruritus, especially at night and after hot showers. The most commonly affected sites are the hands, axillae, and genitals. On exam, the mite’s track can sometimes be seen along with erythematous, excoriated papules. 2° bacterial infection is common. A history of pruritus in several family members is suggestive. The mite may be identifiable by scraping an intact tunnel and looking under the microscope, but this is often difficult. Patients should be treated overnight with 1–2 applications of 5% permethrin from the neck down, and their contacts should be treated as well. Oral ivermectin is also effective. Pruritus may persist for two weeks after treatment, so symptomatic treatment should be provided.

1	Result from ischemic necrosis following continuous pressure on an area of skin that restricts microcirculation to the area. Ulcers are most commonly seen in bedridden patients who lie in one spot for too long. An underlying bony prominence or lack of fat ↑ the likelihood of ulcer formation. Patients who lack mobility or cutaneous sensation are also at ↑ risk. Incontinence of urine or stool may macerate the skin, facilitating ulceration. Ulcers are graded by degree of damage: Grade I: Characterized by persistent redness. Grade II: Marked by ulceration. Grade III: Involves destruction of structures beneath the skin such as muscle or fat. Diagnosed by the history and clinical appearance. Prevention is key and involves routinely moving bedridden patients and using special beds that distribute pressure. Once an ulcer has developed, low-grade lesions can be treated with routine wound care, including hydrocolloid dressings. High-grade lesions require surgical debridement.

1	Defined as necrosis of body tissue. There are three subtypes: dry, wet, and gas. The presence of one subtype does not exclude the others. Etiologies are as follows: Dry gangrene: Due to insufficient blood ﬂow to tissue, typically from atherosclerosis. Wet gangrene: Involves bacterial infection, usually with skin ﬂ ora. Gas gangrene: Due to Clostridium perfringens infection. Dry gangrene: Early signs are a dull ache, cold, and pallor of the ﬂ esh. As necrosis sets in, the tissue (usually a toe) becomes bluish-black, dry, and shriveled. Diabetes, vasculopathy, and smoking are risk factors. Wet gangrene: The tissue appears bruised, swollen, or blistered with pus. Gas gangrene: Typically occurs at a site of recent injury or surgery, presenting with swelling around the injury and with skin that turns pale and then dark red. Bacteria are rapidly destructive of tissue, producing gas that separates healthy tissue and exposes it to infection. A medical emergency.

1	Diagnosed by clinical impression. Surgical debridement, with amputation if necessary, is the mainstay of treatment. Antibiotics alone do not suffice by virtue of inadequate blood ﬂow, but they should be given as an adjuvant to surgery. Gas gangrene can be treated with hyperbaric oxygen, which is toxic to the anaerobic C. perfringens. Susceptible patients should maintain careful foot care and should avoid trauma. A condition in which the skin in the intertriginous zones (genital and axillary regions and especially the nape of the neck) is hyperkeratotic and hyperpigmented with a velvety appearance (see Figure 2.2-12). Associated with DM, Cushing’s disease, HAIR-AN syndrome, and obesity. May also be a paraneoplastic sign of underlying adenocarcinoma (usually GI). Dx: Clinical appearance. Tx: May be treated with topical retinoids, but typically not treated. Patients should be encouraged to lose weight.

1	Dx: Clinical appearance. Tx: May be treated with topical retinoids, but typically not treated. Patients should be encouraged to lose weight. A chronic inﬂammatory dermatosis involving the skin and mucous membranes. The condition is intensely pruritic, can be induced by drugs, and can be associated with HCV infection. Hx/PE: Presents with violaceous, ﬂat-topped, polygonal papules. Lesions may have Wickham’s striae (white stripes), especially on the mucous mem- Lichen planus is the “P” disease: Planar, Purple, Pruritic, Persistent, Polygonal, Penile, Perioral, Puzzling, and Koebner’s Phenomenon. FIGURE 2.2-12. Acanthosis nigricans. Velvety, dark brown epidermal thickening of the armpit is seen with prominent skin fold and feathered edges. (Reproduced, with permission, from Wolff K et al. Fitzpatrick’s Color Atlas &

1	Velvety, dark brown epidermal thickening of the armpit is seen with prominent skin fold and feathered edges. (Reproduced, with permission, from Wolff K et al. Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York: McGraw-Hill, 2005: 87.) branes (see Figure 2.2-13), as well as prominent Koebner’s phenomena (lesions that appear at the site of trauma). The initial lesions often appear on the genitalia, where they are ulcerated. ■Although most cases resolve spontaneously over 6–18 months, those with oral involvement have a more chronic course. Dx: Histology reveals a “lichenoid pattern”—i.e., a band of T lymphocytes at the epidermal-dermal junction with damage to the basal layer. Tx: Mild cases can be treated with topical corticosteroids. For severe disease, systemic corticosteroids may be used. Tretinoin gel may be helpful on oral mucosa.

1	Tx: Mild cases can be treated with topical corticosteroids. For severe disease, systemic corticosteroids may be used. Tretinoin gel may be helpful on oral mucosa. A chronic disorder of pilosebaceous units. The disorder has a female predominance and is more common among those with fair skin. Its etiology is unclear. Patients are generally middle-aged and often have an abnormal ﬂ ushing response to various substances. Early in the disease, central facial erythema is seen with telangiectasias. Later, papules and pustules may develop. Associated findings include ocular keratitis and rhinophyma (sebaceous gland hyperplasia of the nose). FIGURE 2.2-13. Lichen planus. Flat-topped, polygonal, sharply defined papules of violaceous color are grouped and conﬂ uent. The surface is shiny and reveals fine white lines (Wickham’s striae). (Reproduced, with permis sion, from Wolff K et al. Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed.

1	The surface is shiny and reveals fine white lines (Wickham’s striae). (Reproduced, with permis sion, from Wolff K et al. Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York: McGraw-Hill, 2005: 125.) Diagnosed by the clinical picture. Rosacea can be confused with acne but is not follicular in origin and involves an older age group. In a high percentage of patients, ↑ numbers of Demodex mites (which normally live harmlessly in hair follicles, especially in the facial area) are found on the facial skin and can be seen by microscopic examination of skin scrapings. Treat with low-potency topical corticosteroids or topical metronidazole. In more severe disease, systemic antibiotics may be used. Extremely severe cases can be treated with short-term oral metronidazole.

1	An acute dermatitis that is pink and scaly. Its etiology is unknown, but it has been hypothesized to represent a reaction to a viral infection with human herpesvirus (HHV) 6 or 7 because it tends to occur in mini-epidemics among young adults. ■The initial lesion is a herald patch that is several centimeters in diameter and erythematous with a peripheral scale. Days to weeks later, a 2° exanthem appears, presenting with multiple tiny, symmetric papules with a fine “cigarette paper” scale (see Figure 2.2-14). Papules are arranged along skin lines, giving a classic “Christmas tree pattern” on the patient’s back. Patients are generally asymptomatic, although the disease may be more extensive, pruritic, and chronic among African-Americans. Diagnosed by clinical impression and confirmed by KOH exam to rule out fungus (the herald patch may be mistaken for tinea corporis). The differential also includes 2° syphilis (RPR should be ordered), guttate psoriasis, and drug eruptions.

1	Patients usually heal without treatment in 2–3 weeks, but skin lubrication, topical antipruritics, and systemic antihistamines may occasionally be necessary. Severe cases can be treated with a short course of systemic corticosteroids. A disease of depigmentation whose pathogenesis is unknown. The mechanism may be autoimmune, neurologic, or both. ■ Patients develop small, sharply demarcated, depigmented macules or patches on otherwise normal skin, often on the hands, face, or genitalia. FIGURE 2.2-14. Pityriasis rosea. The round to oval erythematous plaques are often covered with a fine white scale (“cigarette paper”) and are often found on the trunk and proximal extremities. Plaques are often preceded by a larger herald patch (arrow). (Reproduced, with permission, from Hurwitz RM. Pathology of the Skin: Atlas of Clinical-Pathological Correlation, 2nd ed. Stamford, CT: Appleton & Lange, 1998: 13.)

1	These spots then expand, sometimes in dermatomal patterns, to include large segments of skin. The disease is usually chronic and progressive, with some patients becoming completely depigmented. Many patients have serologic markers of autoimmune disease (e.g., antithyroid antibodies, DM, pernicious anemia) but seldom present with these diseases. Patients with malignant melanoma may develop an antimelanocyte immune response that leads to vitiligo. Diagnosed by the history and clinical picture, with histology demonstrating total absence of melanocytes. Conditions to rule out include postinﬂ ammatory hypopigmentation, scleroderma, piebaldism, and toxic exposure (pheno-lated cleansers are toxic to melanocytes).

1	Topical or systemic psoralens and exposure to sunlight or PUVA may be helpful. Patients must wear sunscreen because depigmented skin lacks inherent sun protection. Dyes and makeup may be used to color the skin, or the skin may be chemically bleached to produce a uniformly white color. A very common skin tumor, appearing in almost all patients after age 40. The etiology is unknown. When many seborrheic keratoses erupt suddenly, they may be part of a paraneoplastic syndrome due to tumor production of epidermal growth factors. Lesions have no malignant potential but may be a cosmetic problem. Present as exophytic, waxy brown papules and plaques with prominent follicle openings (see Figure 2.2-15). Lesions often appear in great numbers and have a “stuck-on” appearance. Lesions may become irritated either spontaneously or by external trauma, “Seborrheic keratoses, or especially in the groin, breast, or axillae. Irritated lesions are smoother and SKs, look StucK on.” redder.

1	Diagnosed by the clinical picture; can be confirmed by histology showing hyperplasia of benign, basaloid epidermal cells with horn pseudocysts (prominent follicular openings). Rule out actinic keratosis, lentigo (focal ↑ in melanocytes), squamous cell carcinoma (SCC), and basal cell carcinoma (BCC). Cryotherapy or curettage is curative. A precursor of SCC in situ. Lesions are caused by exposure to sunlight. FIGURE 2.2-15. Seborrheic keratoses. Multiple brown, warty papules and nodules are seen on the back, characterized by a “stuck-on” appearance. (Reproduced, with permission, from Fitzpatrick TB. Color Atlas & Synopsis of Clinical Dermatology, 4th ed. New York: McGraw-Hill, 2001: 195.)

1	Clinical Dermatology, 4th ed. New York: McGraw-Hill, 2001: 195.) Lesions appear on sun-exposed areas (especially the face and arms) and primarily affect older patients, who rarely have a solitary lesion. They are erythematous with a light scale that can become thick and crusted (see Figure 2.2-16). Early lesions may be difficult to visualize and may be easier to find by palpation. Diagnosed by clinical impression. Biopsy is seldom necessary but shows intraepidermal atypia over a sun-damaged dermis. The differential includes Bowen’s disease, a form of squamous cell carcinoma in situ. Cryosurgery, topical 5-FU, or topical imiquimod can be used to destroy the lesion. If carcinoma is suspected, biopsy followed by excision or curettage is appropriate. Patients should be advised to use sun protection. FIGURE 2.2-16. Actinic keratosis.

1	FIGURE 2.2-16. Actinic keratosis. The discrete patch above has an erythematous base and a rough white scale. (Reproduced, with permission, from Hurwitz RM. Pathology of the Skin: Atlas of Clinical-Pathological Correlation, 2nd ed. Stamford, CT: Appleton & Lange, 1998: 359.) The second most common skin tumor, with locally destructive effects as well as the potential for metastasis and death. UV light is the most common causative factor, but exposure to chemical carcinogens, prior radiation therapy, and the presence of chronically draining infectious sinuses (as in osteomyelitis) also predispose patients to developing SCC. Most SCCs occur in older adults with sun-damaged skin, arising from actinic keratoses. ■SCCs have a variety of forms, and a single patient will often have multiple variants (see Figure 2.2-17). FIGURE 2.2-17. Squamous cell carcinoma.

1	■SCCs have a variety of forms, and a single patient will often have multiple variants (see Figure 2.2-17). FIGURE 2.2-17. Squamous cell carcinoma. Note the crusting and ulceration of this erythematous plaque. Most lesions are exophytic nodules with erosion or ulceration. (Reproduced, with permission, from Hurwitz RM. Pathology of the Skin: Atlas of Clinical-Pathological Correlation, 2nd ed. Stamford, CT: Appleton & Lange, 1998: 360.) Keratoacanthoma, a benign epithelial tumor, can look like SCC but develops more rapidly and may regress spontaneously. Treatment is usually similar to that of SCCs. ■SCCs that arise from actinic keratoses rarely metastasize, but those that arise on the lips and on ulcers are more likely to do so. SCC occurs on the lip far more commonly than does BCC. Diagnosed by clinical suspicion and confirmed by biopsy, which is necessary for accurate diagnosis and appropriate therapeutic planning.

1	Diagnosed by clinical suspicion and confirmed by biopsy, which is necessary for accurate diagnosis and appropriate therapeutic planning. Histology shows intraepidermal atypical keratinocytes, with penetration of the basement membrane by malignant epidermal cells growing into the dermis. SCCs are graded histologically. Surgical excision. Lesions with high metastatic potential may require additional radiation or chemotherapy. The most common malignant skin tumor, BCC is slow growing and locally destructive but has virtually no metastatic potential. Chronic UV light exposure is the main risk factor. Multiple lesions on non-sun-exposed areas are suggestive of arsenic exposure or inherited basal cell nevus syndrome. Most lesions appear on the face and on other sun-exposed areas. There are many types of BCC with varying degrees of pigmentation, ulceration, and depth of growth (see Figure 2.2-18). FIGURE 2.2-18. Basal cell carcinoma.

1	There are many types of BCC with varying degrees of pigmentation, ulceration, and depth of growth (see Figure 2.2-18). FIGURE 2.2-18. Basal cell carcinoma. Seen above is an erythematous, ﬂeshy, telangiectatic nodule with a translucent surface. (Reproduced, with permission, from Hurwitz RM. Pathology of the Skin: Atlas of Clinical-Pathological Correlation, 2nd ed. Stamford, CT: Appleton & Lange, 1998: 362.) Diagnosed by clinical impression; confirmed by biopsy showing islands of proliferating epithelium resembling the basal layer of the epidermis. The differential includes benign tumors, hypopigmented melanocytic nevi, melanoma, dermatitis, psoriasis, and Paget’s disease. Options include excision, curettage and electrodesiccation/cautery, deep cryotherapy, superficial radiation therapy, and Mohs’ surgery. Cure rates are > 95%.

1	Options include excision, curettage and electrodesiccation/cautery, deep cryotherapy, superficial radiation therapy, and Mohs’ surgery. Cure rates are > 95%. The most common life-threatening dermatologic disease; incidence has been increasing throughout the world. Risk factors include short, intense bursts of sun exposure (especially in childhood and with intermittent exposure) and the presence of congenital melanocytic nevi, an ↑ number of nevi, or dysplastic nevi. Immunosuppression also ↑ risk. Some patients inherit a predisposition to melanoma with the familial atypical mole and melanoma (FAM-M) syndrome. There are several subtypes (see Table 2.2-4). Malignant melanomas usually begin in the epidermal basal layer, where melanocytes are normally found. The first growth phase is horizontal-intraepidermal, presenting with a lesion that is ﬂat but increasing in diameter (typical of lentigo maligna or melanoma in situ). Later, there is a vertical growth phase with dermal invasion.

1	Lesion characteristics that are suggestive of melanoma include irregular pigment, irregular contour and border, nodule and ulcer formation, and changes in size/shape/color/contour/surface noted by the patient (see Figure 2.2-19). TABLE 2.2-4. Types of Melanoma Lentigo maligna Arises in a lentigo. Usually found on sun-damaged skin of the face. Superfcial spreading Typically affects younger adults, presenting on the trunk in men and on the legs in women. A relatively prolonged horizontal growth phase helps identify the disease early, when it is still conf ned to the epidermis. Nodular Lesions have a rapid vertical growth phase and appear as a rapidly growing reddish-brown nodule with ulceration or hemorrhage. Acral lentiginous Begins on the hands and feet as a slowly spreading, pigmented patch. Type most commonly seen in Asians and African-Americans. Amelanotic Presents as a lesion without clinical pigmentation. Extremely diffcult to identify. FIGURE 2.2-19. Melanoma.

1	FIGURE 2.2-19. Melanoma. Note the asymmetry, border irregularity, color variation, and large diameter of this plaque. (Reproduced, with permission, from Hurwitz RM. Pathology of the Skin: Atlas of Clinical- Pathological Correlation, 2nd ed. Stamford, CT: Appleton & Lange, 1998: 432.) ■Malignant melanoma may metastasize, and 10% of patients with metastatic melanoma have no known 1° lesion. Metastasis may be local (to nearby skin), regional (between the original lesion and its regional lymph nodes), or distant (via lymphatic or hematogenous spread to almost every organ in the body). Early recognition and treatment are essential. All adults should be examined for lesions that are suspicious for melanoma according to the ABCDE criteria, which identify dysplastic nevi and superficial spreading melanoma (see mnemonic).

1	The onset of pruritus is also an early sign of malignant change. An excisional biopsy should be performed on any suspicious lesion. Malignancy is determined histologically. Malignant melanomas are staged by Breslow’s thickness (depth of invasion measured in millimeters) and by tumor-node-metastasis (TNM) staging (see Table 2.2-5). Clark’s level is another classification system linking melanoma depth to prognosis (see Table 2.2-6). TABLE 2.2-5. TNM Staging of Melanoma T AB LE 2.2-6. Characterization of Melanoma by Clark’s Level Lesions confined to the skin are treated by excision with margins. Lymph node dissection is useful for staging but does not ↑ survival. Chemotherapy and radiation therapy may be used but are not likely to be successful. Malignant melanoma has the potential to relapse after several years; patients with early melanoma are at low risk for relapse but are at high risk for the development of subsequent melanomas. Patient surveillance is thus essential.

1	A vascular proliferative disease that has been attributed to a herpesvirus, HHV8, which is also called Kaposi’s sarcoma–associated herpesvirus (KSHV). There are several types of KS: The classic variant is characterized by multicentric vascular macules and coalescent papules and plaques on the lower extremities. It usually occurs in the elderly, with a preponderance of cases in patients of Ashkenazi Jewish or Mediterranean descent. More disseminated cases occur in African KS (endemic KS) and in immunocompromised patients. Epidemic HIV-associated KS is an aggressive form of the disease, and although less common since the advent of HAART, it remains the most common HIV-associated malignancy. Diagnosed by history and clinical impression, which are confirmed by biopsy showing spindle cells (elongated tumor cells) with HHV-8 staining. The presence of the viral protein LANA in tumor cells can also be detected for diagnostic confirmation.

1	Treatment is technically palliative. Local lesions may be treated with radiation or cryotherapy; surgery is not recommended. Widespread or internal disease is treated with systemic chemotherapy (anthracyclines, paclitaxil, or IFN-α). Not a fungus, but rather a slow, progressive neoplastic proliferation of T cells. Its pathogenesis is thought to be related to chronic immunostimulation that leads helper T cells to gather in the epidermis. Industrial exposure to irritating chemicals appears to ↑ risk. The disease is chronic and is more common in men than in women. The early lesion is a nonspecific, psoriatic-appearing plaque that is palpable and often pruritic. Stage I involves limited plaques, papules, and patches affecting < 10% BSA with no nodal involvement.

1	Stage I involves limited plaques, papules, and patches affecting < 10% BSA with no nodal involvement. Stage II is characterized by limited or generalized skin involvement with palpable lymph nodes or one or more skin tumors with multicentric, often conﬂuent reddish-brown nodules (see Figure 2.2-20). Rarely, patients skip the plaque stage and present directly with tumors. Stage III is characterized by generalized erythroderma. FIGURE 2.2-20. Mycosis fungoides. Massive nodular infiltration of the face leads to a leonine facies. (Reproduced, with permission, from Fitzpatrick TB. Color Atlas & Synopsis of Clinical Dermatology, 4th ed. New York: McGraw-Hill, 2001: 541.) Stage IV is characterized by biopsy-lymph nodes or internal organ spread. Patients may have dermatopathic lymphadenopathy without actual tumor involvement of the node. However, the internal organs can be involved, including the nodes, liver, and spleen.

1	Patients may have dermatopathic lymphadenopathy without actual tumor involvement of the node. However, the internal organs can be involved, including the nodes, liver, and spleen. Sézary’s syndrome is the leukemic phase of cutaneous T-cell lymphoma and is characterized by circulating Sézary cells in the peripheral blood, erythroderma, and lymphadenopathy. Diagnosed by clinical features and histology, with immunologic characterization and electron microscopy showing the typical Sézary or Lutzner cells (cerebriform lymphocytes). The early lesion is clinically indistinguishable from dermatitis, so histologic diagnosis is indicated for any dermatitis that is chronic and resistant to treatment. Once clinical tumors have evolved, histology is useful for showing the type of cells present. Stage I disease: Treat topically with steroids, retinoids, chemotherapy, or PUVA. Stage II disease: Treat systemically with retinoids, interferon, monoclonal antibodies, or chemotherapy.

1	Stage I disease: Treat topically with steroids, retinoids, chemotherapy, or PUVA. Stage II disease: Treat systemically with retinoids, interferon, monoclonal antibodies, or chemotherapy. Photopheresis is the mainstay of treatment for many patients. For more extensive or advanced disease, radiation therapy is an effective option. Treatment modalities are often combined. Disorders of Glucose Metabolism 114 TYPE 1 DIABETES MELLITUS 114 TYPE 2 DIABETES MELLITUS 115 METABOLIC SYNDROME 117 Due to autoimmune pancreatic β-cell destruction leading to insulin deficiency and abnormal fuel metabolism. Classically presents with polyuria (especially nocturia), polydipsia, polyphagia, and rapid, unexplained weight loss. Patients may also present with ketoacidosis. Usually affects nonobese children or young adults. Associated with HLA-DR3 and -DR4. At least one of the following is required to make the diagnosis: A fasting (> 8-hour) plasma glucose of ≥ 126 mg/dL on two separate occasions.

1	Associated with HLA-DR3 and -DR4. At least one of the following is required to make the diagnosis: A fasting (> 8-hour) plasma glucose of ≥ 126 mg/dL on two separate occasions. A random plasma glucose of ≥ 200 mg/dL plus symptoms. A two-hour postprandial glucose of ≥ 200 mg/dL after a glucose tolerance test on two separate occasions if the results of initial testing are equivocal. Insulin (see Table 2.3-1) and self-monitoring of blood glucose in the normal range (80–120 mg/dL). Higher blood glucose levels (≥ 200 mg/dL) can be tolerated, particularly in the very young, in light of the ↑ risk of hypoglycemia. Routine HbA1c testing (with a goal HbA1c < 8 in children), frequent BP checks, foot checks, annual dilated-eye exams, annual microalbuminuria screening, and a lipid profile every 2–5 years. TABLE 2.3-1. Types of Insulin

1	TABLE 2.3-1. Types of Insulin Regular 30–60 minutes 2–4 hours 5–8 hours Humalog (lispro) 5–10 minutes 0.5–1.5 hours 6–8 hours NovoLog (aspart) 10–20 minutes 1–3 hours 3–5 hours Apidra (glulisine) 5–15 minutes 1.0–1.5 hours 1.0–2.5 hours NPH 2–4 hours 6–10 hours 18–28 hours Levemir (detemir) 2 hours No discernible peak 20 hours Lantus (glargine) 1–4 hours No discernible peak 20–24 hours a Combination preparations mix longer-acting and shorter-acting types of insulin together to provide immediate and extended coverage in the same injection (e.g., 70 NPH/30 regular = 70% NPH + 30% regular). Table 2.3-2 outlines the acute, chronic, and treatment-related complications of DM. A dysfunction in glucose metabolism that is best characterized as varying degrees of insulin resistance that may lead to β-cell burnout and insulin dependence. TABLE 2.3-2. Complications of DM

1	Complications of treatment Dawn phenomenon Early-morning hyperglycemia caused by ↓ effectiveness of insulin and ↑ secretion of growth hormone (GH) and other hormones overnight. Move P.M. insulin closer to bedtime to treat. Acute complications Diabetic ketoacidosis (DKA) Hyperosmolar hyperglycemic state (HHS) Hyperglycemia-induced crisis that most commonly occurs in type 1 DM. Often precipitated by stress (including infections, MI, trauma, or alcohol) or by noncompliance with insulin therapy. May present with abdominal pain, vomiting, Kussmaul respirations, and a fruity, acetone breath odor. Patients are severely dehydrated with many electrolyte abnormalities and may also develop mental status changes. Treatment includes f uids, potassium, insulin, bicarbonate (if pH is < 7), and treatment of the initiating event or underlying disease process. Presents as profound dehydration, mental status changes, hyperosmolarity, and extremely high plasma glucose (> 600 mg/dL) without acidosis and

1	of the initiating event or underlying disease process. Presents as profound dehydration, mental status changes, hyperosmolarity, and extremely high plasma glucose (> 600 mg/dL) without acidosis and with small or absent ketones. Occurs in type 2 DM; precipitated by acute stress (dehydration, infections) and often can be fatal. Treatment includes aggressive ﬂuid and electrolyte replacement and insulin. Treat the initiating event. Chronic complications Retinopathy (nonproliferative, proliferative) Diabetic nephropathy Neuropathy Macrovascular complications Appears when diabetes has been present for at least 3–5 years. Preventive measures include control of hyperglycemia and hypertension, annual eye exams, and laser photocoagulation therapy for retinal neovascularization. Characterized by glomerular hyperfltration followed by microalbuminuria. Preventive measures include ACEIs or ARBs and BP/glucose control. Peripheral, symmetric, sensorimotor neuropathy leading to burning pain, foot

1	glomerular hyperfltration followed by microalbuminuria. Preventive measures include ACEIs or ARBs and BP/glucose control. Peripheral, symmetric, sensorimotor neuropathy leading to burning pain, foot trauma, infections, and diabetic ulcers. Treat with preventive foot care and analgesics. Late complications due to autonomic dysfunction include delayed gastric emptying, esophageal dysmotility, impotence, and orthostatic hypotension. Cardiovascular, cerebrovascular, and peripheral vascular disease. Cardiovascular disease is the most common cause of death in diabetic patients. The goal BP is < 130/< 75; ↓ LDL to < 100 mg/dL; ↓ triglycerides to < 150 mg/dL. Patients should also be started on low-dose ASA.

1	■Somogyi effect: Nocturnal hypoglycemia leading to a surge of counterregulatory hormones, leading in turn to hyperglycemia in the morning. ■Dawn phenomenon: Nocturnal secretion of GH leading to early-morning hyperglycemia. Patients typically present with symptoms of hyperglycemia. Onset is more insidious than that of type 1 DM, and patients often present with complications. Nonketotic hyperglycemia may be seen in the setting of poor glycemic control. Usually occurs in older adults with obesity (often truncal); has a strong genetic predisposition. Diagnostic criteria are the same as those for type 1 DM. Follow-up testing: Patients with no risk factors: Test at 45 years of age; retest every three years. Patients with impaired fasting glucose (> 110 mg/dL but < 126 mg/ dL): Follow up with frequent retesting. The goal of treatment is tight glucose control—i.e., blood glucose levels ranging from 80 to 120 mg/dL and HbA1c levels < 7. Treatment measures include the following:

1	The goal of treatment is tight glucose control—i.e., blood glucose levels ranging from 80 to 120 mg/dL and HbA1c levels < 7. Treatment measures include the following: Diet, weight loss, and exercise. Oral agents (monotherapy or combination if uncontrolled): Sulfonylureas (glipizide, glyburide, and glimepiride): Insulin secretagogues. Hypoglycemia and weight gain are side effects. Meglitinides (repaglinide and nateglinide): Short-acting agents whose mechanism of action is similar to that of sulfonylureas. Metformin: Inhibits hepatic gluconeogenesis; ↑ peripheral sensitivity to insulin. Side effects include weight loss, GI upset, and, rarely, lactic acidosis. Contraindicated in the elderly (those > 80 years of age) and in patients with renal disease. Thiazolidinediones (the “glitazones”): ↑ insulin sensitivity. Side effects include weight gain, edema, and potential hepatotoxicity.

1	Thiazolidinediones (the “glitazones”): ↑ insulin sensitivity. Side effects include weight gain, edema, and potential hepatotoxicity. α-glucosidase inhibitors: ↓ intestinal absorption of carbohydrates. Rarely used owing to the side effect of ﬂ atulence. DDP-4 inhibitors (sitagliptin): Inhibit the degradation of the endogenous enzyme that breaks down glucagon-like peptide 1 (GLP-1). Insulin (alone or in conjunction with oral agents). Incretins (exenatide): GLP-1 agonists. Injected subcutaneously. Delay absorption of food; ↑ insulin secretion and ↓ glucagon secretion. Side effects include nausea and (rarely) pancreatitis. Statins for hypercholesterolemia (goal LDL < 100); glucose control and fibric acid derivatives for hypertriglyceridemia. Strict BP control to < 130/80; ACEIs/ARBs are usually first-line agents. Antiplatelet agents (ASA) for patients at risk of cardiovascular disease or for those > 40 years of age.

1	Strict BP control to < 130/80; ACEIs/ARBs are usually first-line agents. Antiplatelet agents (ASA) for patients at risk of cardiovascular disease or for those > 40 years of age. Regular screening for cardiovascular disease, nephropathy, retinopathy, neuropathy, and smoking cessation. See Table 2.3-2 for an outline of the complications of DM. Note that the presence of diabetes is equivalent to the highest risk for cardiovascular disease regardless of all other risk factors. Also known as insulin resistance syndrome or syndrome X. Associated with an ↑ risk of CAD and mortality from a cardiovascular event. Presents with abdominal obesity, high BP, impaired glycemic control, and dyslipidemia. Three out of five of the following criteria must be met: Abdominal obesity (↑ waist girth): > 40 inches in men and > 35 inches in women. Triglycerides ≥ 150 mg/dL. HDL < 40 mg/dL in men and < 50 mg/dL in women. BP ≥ 130/85 mmHg or administration of antihypertensive drugs.

1	Triglycerides ≥ 150 mg/dL. HDL < 40 mg/dL in men and < 50 mg/dL in women. BP ≥ 130/85 mmHg or administration of antihypertensive drugs. Fasting glucose ≥ 100 mg/dL. Intensive weight loss, aggressive cholesterol lowering, and BP control. Metformin has been shown to slow the onset of diabetes in this high-risk population. Testing of Thyroid Function TFTs include the following (see also Table 2.3-3): ■TSH measurement: The single best test for assessing thyroid function. High TSH levels lead to 1° hypothyroidism; low TSH levels lead to thyrotoxicosis. ■Radioactive iodine uptake (RAIU) and scan: Determines the level of iodine uptake by the thyroid. Useful in differentiating thyrotoxic states, but has a limited role in determining malignancy. T AB LE 2.3-3. Common Thyroid Function Abnormalities In 1° endocrine disturbances, the gland itself is abnormal. In 2° endocrine disturbances, the HPA malfunctions.

1	T AB LE 2.3-3. Common Thyroid Function Abnormalities In 1° endocrine disturbances, the gland itself is abnormal. In 2° endocrine disturbances, the HPA malfunctions. 1° hyperthyroidism ↓↑↑Graves’ disease, toxic multinodular goiter, toxic adenoma, amiodarone, molar pregnancy, postpartum thyrotoxicosis, postviral thyroiditis. 1° hypothyroidism ↑↓↓Hashimoto’s thyroiditis, hypothyroid phase of thyroiditis, iatrogenic factors (radioactive iodine thyroid ablation, excision with inadequate supplementation, external radiation, lithium, or amiodarone), iodine def ciency, inf ltrative disease. Thyroid storm is an acute, life-threatening form of thyrotoxicosis that may present with atrial f brillation, fever, and delirium. Admit to intensive care. TBG-elevating states that should not be treated include pregnancy, estrogen administration, infections, nephrotic syndrome, and anabolic steroid use.

1	TBG-elevating states that should not be treated include pregnancy, estrogen administration, infections, nephrotic syndrome, and anabolic steroid use. Total T4 measurement: Not an adequate screening test. Ninety-nine percent of circulating T4 is bound to thyroxine-binding globulin (TBG). Total T4 levels can be altered by changes in levels of binding proteins. T3 resin uptake (T3RU): Used with total T4 or T3 to correct for changes in TBG levels (e.g., the free thyroxine index = total T4 × T3RU). Free T4 measurement: The preferred screening test for thyroid hormone levels; more useful for unstable thyroid states. Refers to causes of thyrotoxicosis (↑ levels of T3/T4 due to any cause) in which the thyroid overproduces thyroid hormone, including Graves’ disease, toxic multinodular goiter (also called Plummer’s disease), and toxic adenomas. Presents with weight loss, heat intolerance, nervousness, palpitations, ↑bowel frequency, insomnia, and menstrual abnormalities.

1	Presents with weight loss, heat intolerance, nervousness, palpitations, ↑bowel frequency, insomnia, and menstrual abnormalities. Exam reveals warm, moist skin, goiter, sinus tachycardia or atrial f brillation, fine tremor, lid lag, and hyperactive reﬂ exes. Exophthalmos, pretibial myxedema, and thyroid bruits are seen only in Graves’ disease (see Figure 2.3-1). The initial test of choice is serum TSH level, followed by T4 levels and, rarely, T3 (unless TSH is low and free T4 is not elevated). See Table 2.3-3. AB F IGU R E 2.3-1. Physical signs of Graves’ disease. (A) Graves’ ophthalmopathy. (B) Pretibial myxedema. (Figure 2.3-1A reproduced, with permission, from the Pathology Education Instructional Resource [PEIR] digital library [ http://peir.

1	(A) Graves’ ophthalmopathy. (B) Pretibial myxedema. (Figure 2.3-1A reproduced, with permission, from the Pathology Education Instructional Resource [PEIR] digital library [ http://peir. at the University of Alabama, Birmingham. Figure 2.3-1B reproduced, with permission, from Greenspan FS, Strewler GJ. Basic and Clinical Endocrinology, 5th ed. Stamford, CT: Appleton & Lange, 1997.) 1° therapy is radioactive 131I thyroid ablation; antithyroid drugs (methimazole or propylthiouracil) may also be used if radioactive iodine is not indicated. Thyroidectomy is rarely indicated. Give propranolol for adrenergic symptoms while awaiting the resolution of hyperthyroidism. Administer levothyroxine to prevent hypothyroidism in patients who have undergone ablation or surgery.

1	Give propranolol for adrenergic symptoms while awaiting the resolution of hyperthyroidism. Administer levothyroxine to prevent hypothyroidism in patients who have undergone ablation or surgery. Hashimoto’s thyroiditis is the most common cause of hypothyroidism (see Table 2.3-3). Anti-TPO antibodies are . The second most common cause is iatrogenic. Myxedema coma refers to severe hypothyroidism with ↓ mental status, hypothermia, and other parasympathetic symptoms. Mortality is 30–60%. Presents with weakness, fatigue, cold intolerance, constipation, weight gain, depression, menstrual irregularities, and hoarseness. Exam may reveal dry, cold, puffy skin accompanied by edema, bradycardia, and delayed relaxation of DTRs. See Table 2.3-3. Uncomplicated hypothyroidism (e.g., Hashimoto’s disease): Administer levothyroxine. Myxedema coma: Treat with IV levothyroxine and IV hydrocortisone (if adrenal insufficiency has not been excluded).

1	Myxedema coma: Treat with IV levothyroxine and IV hydrocortisone (if adrenal insufficiency has not been excluded). Inﬂammation of the thyroid gland. Common subtypes include subacute granulomatous, radiation-induced, autoimmune (lymphocytic, chronic, or Hashimoto’s), postpartum, and drug-induced (e.g., amiodarone) thyroiditis. The subacute form presents with a tender thyroid accompanied by malaise and URI symptoms. Other forms are associated with painless goiter. Thyroid dysfunction (typically thyrotoxicosis followed by hypothyroidism), with ↓ uptake on RAIU during the thyrotoxic phase. β-blockers for hyperthyroidism; levothyroxine for hypothyroidism. Subacute thyroiditis is usually self-limited; treat with NSAIDs or with oral corticosteroids for severe cases. TSH receptor antibodies are seen in patients with Graves’ disease. The most Popular is Papillary: Check calcitonin levels if medullary cancer is suspected.

1	TSH receptor antibodies are seen in patients with Graves’ disease. The most Popular is Papillary: Check calcitonin levels if medullary cancer is suspected. Thyroid nodules are very common and show an ↑ incidence with age. Most are benign. Usually asymptomatic on initial presentation. Hyperfunctioning nodules present with hyperthyroidism and local symptoms (dysphagia, dyspnea, cough, choking sensation) and are associated with a family history (especially medullary thyroid cancer). An ↑ risk of malignancy is associated with a history of neck irradiation, “cold” nodules on radionuclide scan, male sex, age < 20 or > 70, firm and fixed solitary nodules, a family history (especially medullary thyroid cancer), and rapidly growing nodules with hoarseness. Check for anterior cervical lymphadenopathy. Carcinoma (see Table 2.3 4) may be frm and f xed. Medullary thyroid carcinoma is associated with multiple endocrine neoplasia (MEN) type 2 and familial medullary thyroid cancer.

1	Medullary thyroid carcinoma is associated with multiple endocrine neoplasia (MEN) type 2 and familial medullary thyroid cancer. The best method of assessing a nodule for malignancy is f ne-needle aspiration (FNA), which has high sensitivity and moderate specificity. TFTs (TSH to exclude hyperfunction). Ultrasound determines if the nodule is solid or cystic; a radioactive scan determines whether it is hot or cold (cancers are usually cold and solid). Hot nodules are never cancerous and should not be biopsied. TABLE 2.3-4. Types of Thyroid Carcinoma

1	TABLE 2.3-4. Types of Thyroid Carcinoma Papillary Represents 75–80% of thyroid cancers. The female-to-male ratio is 3:1. Slow growing; found in thyroid hormone–producing cells. Ninety percent of patients survive 10 years or more after diagnosis; the prognosis is worse in elderly patients or those with large tumors. Follicular Accounts for 17% of thyroid cancers; found in thyroid hormone– producing cells. Ninety percent of patients survive 10 years or longer after diagnosis; the prognosis is worse in elderly patients or those with large tumors. Medullary Responsible for 6–8% of thyroid cancers; found in calcitonin-producing C cells; the prognosis is related to degree of vascular invasion. Eighty percent of patients survive at least 10 years after surgery. Anaplastic Accounts for < 2% of thyroid cancers; rapidly enlarges and metastasizes. Ten percent of patients survive for > 3 years. a Tumors may contain mixed papillary and follicular pathologies.

1	Benign FNA: Follow with physical exam/ultrasound or a trial of levothyroxine suppression treatment. Malignant FNA: Surgical resection is first-line treatment; adjunctive radioiodine ablation following excision is appropriate for follicular lesions. Indeterminate FNA: Remove the nodule by surgical excision and wait for final pathology. Medullary thyroid cancer has a poorer prognosis than papillary and follicular types. Anaplastic thyroid cancer has an extremely poor prognosis. Associated with autosomal-dominant inheritance. Subtypes are as follows: MEN type 1 (Wermer’s syndrome): Pancreatic islet cell tumors (e.g., Zollinger-Ellison syndrome, insulinomas, VIPomas), parathyroid hyperplasia, and pituitary adenomas. MEN type 2A (Sipple’s syndrome): Medullary carcinoma of the thyroid, pheochromocytoma or adrenal hyperplasia, parathyroid gland hyperplasia.

1	MEN type 2A (Sipple’s syndrome): Medullary carcinoma of the thyroid, pheochromocytoma or adrenal hyperplasia, parathyroid gland hyperplasia. MEN type 2B: Medullary carcinoma of the thyroid, pheochromocytoma, oral and intestinal ganglioneuromatosis (mucosal neuromas), marfanoid habitus. A common metabolic bone disease characterized by low bone mass and microarchitectural disruption, with bone mineral density > 2.5 SDs below normal peak bone mass. Most often affects thin, postmenopausal women (17%), especially Caucasians and Asians, with risk doubling (30%) after age 65. Commonly asymptomatic even in the presence of a vertebral fracture. Exam may reveal hip fractures, vertebral compression fractures (loss of height and progressive thoracic kyphosis), and/or distal radius fractures following minimal trauma (see Figure 2.3-2). Bone pain unrelated to fracture is most likely osteomalacia rather than osteoporosis.

1	Bone pain unrelated to fracture is most likely osteomalacia rather than osteoporosis. Smoking, excessive caffeine or alcohol intake, a history of estrogen-depleting conditions (e.g., amenorrhea, eating disorders), thyroid dysfunction, and steroid use are all associated with an ↑ risk. DEXA: The gold standard; reveals significant osteopenia (bone mineral density < 2.5 SDs from normal peak level), most commonly in the vertebral bodies, proximal femur, and distal radius. Labs: Markers of bone turnover (↑ urinary N-telopeptides and deoxypyridinoline) can facilitate diagnosis in equivocal cases but are not routinely used; rule out 2° causes with TFTs, CMP, serum 25-hydroxyvitamin D, CBC, and testosterone (in men). X-rays: Global demineralization is apparent only after > 30% of bone density is lost. Osteoporosis is the most common cause of pathologic fractures in elderly, thin women. F IGU R E 2.3-2. Radiographic findings in osteoporosis.

1	Osteoporosis is the most common cause of pathologic fractures in elderly, thin women. F IGU R E 2.3-2. Radiographic findings in osteoporosis. compression). (Reproduced, with permission, from Kasper DL et al. Harrison’s Principles of Internal Medicine, 16th ed. New York: McGraw-Hill, 2005: 2269.) Prevention with calcium supplementation and vitamin D. Smoking cessation and weight-bearing exercises help maintain bone density. Bisphosphonates (e.g., alendronate, risedronate, ibandronate, zoledronic acid), selective estrogen receptor modulators (e.g., raloxifene), and intranasal calcitonin may be used to prevent resorption and stabilize bone mineral density. Estrogen replacement therapy may be indicated for short-term treatment in the symptomatic perimenopausal period. Recombinant PTH may be used in patients with the highest level of risk.

1	Estrogen replacement therapy may be indicated for short-term treatment in the symptomatic perimenopausal period. Recombinant PTH may be used in patients with the highest level of risk. Characterized by an ↑ rate of bone turnover. Causes both excessive resorption and excessive formation of bone, leading to a “mosaic” lamellar bone pattern. Suspected to be due to latent viral infection in genetically susceptible individuals. Found in roughly 4% of men and women > 40 years of age, and associated with 1° hyperparathyroidism in up to one-fifth of patients. Usually asymptomatic, but may present with aching bone or joint pain, headaches, skull deformities, fractures, or nerve entrapment (leads to loss of hearing in 30–40% of cases). Based on clinical history, characteristic radiographic changes (see Figure 2.33), and lab findings. F IGU R E 2.3-3. Radiographic findings in Paget’s disease. Skull of a 58-year-old woman with Paget’s disease of bone. (Reproduced, with permission, from

1	F IGU R E 2.3-3. Radiographic findings in Paget’s disease. Skull of a 58-year-old woman with Paget’s disease of bone. (Reproduced, with permission, from Kasper DL et al. Harrison’s Principles of Internal Medicine, 16th ed. New York: McGraw-Hill, 2005: 2280.) Imaging: Radionuclide bone scan is the most sensitive test. Labs: Abnormalities include ↑ serum alkaline phosphatase with normal calcium and phosphate levels; urinary pyridinolines may be helpful. Must be differentiated from metastatic bone disease. The majority of patients are asymptomatic and require no treatment. There is no cure for Paget’s disease. Bisphosphonates and calcitonin are used to slow osteoclastic bone resorption; NSAIDs and acetaminophen can be given for arthritis pain. Pathologic fractures, cardiac complications, osteosarcoma (up to 1%).

1	Pathologic fractures, cardiac complications, osteosarcoma (up to 1%). Eighty percent of 1° cases are due to a single adenoma and 15% to parathyroid hyperplasia. The most common 2° cause is phosphate retention in chronic kidney disease, which leads to renal osteodystrophy. 3° hyperparathyroidism occurs when chronic 2° hyperparathyroidism progresses to an unregulated state, resulting in hypercalcemia. Most cases are asymptomatic, but signs and symptoms may be mild and include stones (nephrolithiasis), bones (bone pain, myalgias, arthralgias, fractures), abdominal groans (abdominal pain, nausea, vomiting, PUD, pancreatitis), and psychiatric overtones (fatigue, depression, anxiety, sleep disturbances). Chronic renal insufficiency and gout are also associated with 1° hyperparathyroidism.

1	Chronic renal insufficiency and gout are also associated with 1° hyperparathyroidism. Relative surgical criteria for hyperparathyroidism include age < 50, markedly ↓ bone mass, nephrolithiasis/renal insuff ciency, profoundly elevated serum calcium or life-threatening hypercalcemia, and urine calcium > 400 mg in 24 hours. Labs reveal hypercalcemia, hypophosphatemia, and hypercalciuria. Intact PTH is inappropriately ↑ relative to ionized calcium (see Table 2.3-5). Vitamin D deficiency may obscure mild cases. A radionuclide parathyroid scan may help with localization of an adenoma, although localization is not always necessary. Cancer is the most likely alternative diagnosis and must be ruled out, but with carcinoma PTH is usually < 25 pg/mL unless hyperparathyroidism is also present. Lithium and thiazides may exacerbate hyperparathyroidism.

1	Parathyroidectomy if the patient is symptomatic or if certain criteria are met. For acute hypercalcemia, give IV fuids (with a loop diuretic in the setting of renal or heart failure), IV bisphosphonate, and calcitonin. Oral phosphate binders (aluminum hydroxide, calcium salts, sevelamer hydrochloride, and lanthanum carbonate) and dietary phosphate restriction are used in patients with 2° hyperparathyroidism regardless of dialysis status. Cinacalcet ↓ PTH as well as calcium and phosphate and may be a helpful adjunct in end-stage renal disease. Hypercalcemia is the most severe complication, presenting acutely with coma or altered mental status, bone disease, nephrolithiasis, and abdominal pain with nausea and vomiting.

1	Hypercalcemia is the most severe complication, presenting acutely with coma or altered mental status, bone disease, nephrolithiasis, and abdominal pain with nausea and vomiting. The most common endogenous cause of Cushing’s syndrome is hypersecretion of ACTH from a pituitary adenoma (known as Cushing’s disease, or central hypercortisolism). Other endogenous causes include ectopic ACTH secretion from neoplasia (e.g., carcinoid tumor, small cell lung cancer) and excess adrenal secretion of cortisol (e.g., bilateral adrenal hyperplasia, adenoma, adrenal cancer). The condition is most commonly iatrogenic, resulting from treatment with exogenous corticosteroids. T AB LE 2.3-5. Functions and Mechanisms of PTH

1	T AB LE 2.3-5. Functions and Mechanisms of PTH Chief cells of parathyroid ↑ bone resorption of calcium and phosphate. ↑ kidney resorption of calcium in the distal convoluted tubule. ↓ kidney resorption of phosphate. ↑ 1,25-(OH)2 vitamin D (cholecalciferol) production by stimulating kidney 1α-hydroxylase. PTH ↑ serum Ca2+, ↓ serum (PO4)3–, and ↑urine (PO4)3–. PTH stimulates both osteoclasts and osteoblasts. ↓ in free serum Ca2+ ↑ PTH secretion. Presents with hypertension, central obesity, muscle wasting, thin skin with purple striae, psychological disturbances, hirsutism, moon facies, and “buffalo hump.” Exam reveals depression, oligomenorrhea, growth retardation, proximal weakness, acne, excessive hair growth, symptoms of diabetes (2° to glucose intolerance), and ↑ susceptibility to infection. Headache or cranial nerve deficits are also seen with increasing size of the pituitary mass. Diagnosis is as follows (see also Table 2.3-6):

1	Diagnosis is as follows (see also Table 2.3-6): Begin with a screen: An elevated 24-hour free urine cortisol or evening salivary cortisol or a overnight low-dose dexamethasone suppression test is considered abnormal if A.M. cortisol is persistently elevated following overnight suppression. Distinguish ACTH-dependent from ACTH-independent causes: If late-afternoon ACTH levels are elevated, Cushing’s disease or ectopic ACTH is likely. Hyperglycemia, glycosuria, and hypokalemia may also be present. Surgical resection of the hypersecretory source (pituitary, adrenal). Pituitary radiotherapy may also be considered. Blockers of adrenal steroidogenesis (e.g., ketoconazole, aminoglutethimide) may be of benefit. Permanent hormone replacement therapy to correct deficiencies after treatment of the 1° lesion. An adult condition due to a benign pituitary GH adenoma. Children with excess GH production present with gigantism.

1	An adult condition due to a benign pituitary GH adenoma. Children with excess GH production present with gigantism. ■ Presents with enlargement of the jaw, hands, and feet and coarsening of facial features. May lead to carpal tunnel syndrome, diastolic dysfunction, hypertension, and arthritis. T AB LE 2.3-6. Laboratory Findings in Cushing’s Syndrome For unknown reasons, patients with DI prefer ice-cold beverages. Bitemporal hemianopia may result from compression of the optic chiasm by a pituitary adenoma. Excess GH may also lead to glucose intolerance or diabetes. Imaging: MRI of the pituitary shows a sellar lesion. Labs: Screen by measuring insulin-like growth factor 1 (IGF-1) levels (↑with acromegaly); confirm the diagnosis with an oral glucose suppression test (GH levels will remain elevated despite glucose suppression; baseline GH is not a reliable test). Transsphenoidal surgical resection or external beam radiation of the tumor.

1	Transsphenoidal surgical resection or external beam radiation of the tumor. Octreotide can be used to suppress GH secretion. Pegvisomant can be used to block GH receptors in refractory cases. Prolactinoma is the most common functioning pituitary tumor. Other pathologic causes include craniopharyngioma, irradiation, drugs (e.g., dopamine), and cirrhosis. Hx/PE: Hypogonadism is manifested by infertility, oligomenorrhea, or amenorrhea. Galactorrhea, gynecomastia, or bitemporal hemianopia may be prominent. Dx: The serum prolactin level is typically > 200 mg/mL. Tx: Dopamine agonists (f rst-line therapy): Cabergoline or bromocriptine. Surgery: Should be considered when medical treatment has failed, when the patient desires future pregnancies, or in the setting of visual field defects. Failure to concentrate urine as a result of central or nephrogenic ADH dysfunction. Subtypes are as follows:

1	Failure to concentrate urine as a result of central or nephrogenic ADH dysfunction. Subtypes are as follows: Central DI: The posterior pituitary fails to secrete ADH. Causes include tumor, ischemia (Sheehan’s syndrome), traumatic cerebral injury, infection, and autoimmune disorders. Nephrogenic DI: The kidneys fail to respond to circulating ADH. Causes include renal diseases and drugs (e.g., lithium, demeclocycline). Presents with polydipsia, polyuria, and persistent thirst with dilute urine. Patients may present with hypernatremia and dehydration, but if given unlimited access to water, they are typically normonatremic. ■ During a water deprivation test, patients excrete a high volume of dilute urine. Desmopressin acetate (DDAVP), a synthetic analog of ADH, can be used to distinguish central from nephrogenic DI. Central DI: DDAVP challenge will ↓urine output and ↑urine osmolarity. Nephrogenic DI: DDAVP challenge will not significantly ↓ urine output.

1	Central DI: DDAVP challenge will ↓urine output and ↑urine osmolarity. Nephrogenic DI: DDAVP challenge will not significantly ↓ urine output. MRI may show a pituitary or hypothalamic mass in central DI. Treat the underlying cause. Central DI: Administer DDAVP intranasally or orally. Nephrogenic DI: Salt restriction and water intake are the 1° treatment. Thiazide diuretics are used to promote mild volume depletion and to stimulate proximal reabsorption of salt and water. A common cause of euvolemic hyponatremia that results from stimulated ADH release independent of serum osmolality. Associated with CNS disease (e.g., head injury, tumor), pulmonary disease (e.g., sarcoid, pneumonia), ectopic tumor production/paraneoplastic syndrome (e.g., small cell carcinoma), drugs (e.g., antipsychotics, antidepressants), or surgery.

1	Diagnose on the basis of a urine osmolality > 50–100 mOsm/kg with concurrent serum hyposmolarity in the absence of a physiologic reason for ↑ADH (e.g., CHF, cirrhosis, hypovolemia). Urinary sodium ≥ 20 mEq/L demonstrates that the patient is not hypo-Fluid restriction is the volemic. cornerstone of SIADH treatment. Restrict f uid and address the underlying cause. If hyponatremia is severe (< 110 mEq/L) or if the patient is significantly symptomatic (e.g., comatose, seizing), cautiously give hypertonic saline. Demeclocycline can help normalize serum sodium by antagonizing the action of ADH in the collecting duct. Chronic correction depends on treatment of the underlying disorder. Tolvaptan (a vasopressin agonist) can be used to treat refractory chronic cases.

1	Chronic correction depends on treatment of the underlying disorder. Tolvaptan (a vasopressin agonist) can be used to treat refractory chronic cases. May be 1° or 2°. Etiologies are as follows: ■ 1°: ■Most commonly caused by autoimmune adrenal cortical destruction (Addison’s disease), leading to deficiencies of mineralocorticoids and glucocorticoids. Autoimmune destruction may occur as part of a The 4 S’s of adrenal crisis management: Salt: 0.9% saline Steroids: IV hydrocortisone 100 mg q 8 h Search for the underlying illness Do not delay in giving steroids when diagnosing a patient with AI. polyglandular autoimmune syndrome (hypothyroidism, type 1 DM, vitiligo, premature ovarian failure, testicular failure, pernicious anemia). ■Other causes of 1° AI include congenital enzyme deficiencies, adrenal hemorrhage, TB, and other infections. ■ 2°: Caused by ↓ ACTH production by the pituitary; most often due to cessation of long-term glucocorticoid treatment.

1	■ 2°: Caused by ↓ ACTH production by the pituitary; most often due to cessation of long-term glucocorticoid treatment. Most symptoms are nonspecific. Weakness, fatigue, and anorexia with weight loss are common. GI manifestations, hypotension, and salt craving are also seen. Hyperpigmentation (due to ↑ ACTH secretion) is seen in Addison’s disease, especially in areas of sun exposure or friction. Labs show hyponatremia and eosinophilia (1° or 2°). Hyperkalemia is specific to 1° AI. Hypercalcemia is seen in up to one-third of cases. Diagnosis is confirmed with plasma cortisol levels: Low plasma cortisol levels (< 20 μg/dL) during a period of high physiologic stress is confirmatory. A random plasma cortisol level > 20 μg/dL excludes the diagnosis. Confrmatory test with synthetic ACTH stimulation test: A plasma cortisol level > 20 μg/dL excludes the diagnosis. Glucocorticoid replacement, with mineralocorticoid replacement if 1°.

1	Confrmatory test with synthetic ACTH stimulation test: A plasma cortisol level > 20 μg/dL excludes the diagnosis. Glucocorticoid replacement, with mineralocorticoid replacement if 1°. In adrenal crisis, correct electrolyte abnormalities as needed; provide 50% dextrose to correct hypoglycemia; and initiate volume resuscitation. ■↑ steroids during periods of stress (e.g., major surgery, trauma, infection). Avoid 2° AI by tapering steroids slowly. A tumor of chromaffin tissue found either in the adrenal medulla or in extra-adrenal sites that secrete catecholamines. May be associated with von Hippel–Lindau syndrome, neurofibromatosis, or MEN 2 syndromes. Hx/PE: Presents with intermittent tachycardia, palpitations, chest pain, diaphoresis, hypertension, headache, tremor, and anxiety. Crises may be precipitated by anesthesia.

1	Hx/PE: Presents with intermittent tachycardia, palpitations, chest pain, diaphoresis, hypertension, headache, tremor, and anxiety. Crises may be precipitated by anesthesia. Dx: CT or MRI often demonstrates a suprarenal mass. Screen with plasma-free metanephrines (metanephrine and normetanephrine) or 24hour urine metanephrines. MIBG scan is sometimes helpful. Tx: Surgical resection. Preoperatively, use α-adrenergic blockade f rst to control hypertension, followed by β-blockade to control tachycardia. Never give β-blockade first; otherwise, unopposed α-adrenergic stimulation will lead to refractory hypertension. Results from excessive secretion of aldosterone from the zona glomerulosa of the adrenal cortex. It is usually due to adrenocortical hyperplasia (70%) but can also result from unilateral adrenal adenoma (Conn’s syndrome). Presents with hypertension, headache, polyuria, and muscle weakness. Tetany, paresthesias, and peripheral edema are seen in severe cases.

1	Presents with hypertension, headache, polyuria, and muscle weakness. Tetany, paresthesias, and peripheral edema are seen in severe cases. Patients have diastolic hypertension without edema. Labs show hypokalemia, mild hypernatremia, metabolic alkalosis, hypomagnesemia, and an ↑aldosterone/plasma renin activity ratio (from hyposecretion of renin that fails to ↑ appropriately with volume depletion as well as from hypersecretion that does not suppress with volume expansion). CT or MRI may reveal an adrenal mass. Laparoscopic or open adrenalectomy for adrenal tumors (after correcting BP and potassium). Treat bilateral hyperplasia with spironolactone, an aldosterone receptor antagonist.

1	Laparoscopic or open adrenalectomy for adrenal tumors (after correcting BP and potassium). Treat bilateral hyperplasia with spironolactone, an aldosterone receptor antagonist. A family of inherited disorders that lead to cortisol def ciency. Most cases are due to 21-hydroxylase defciency (autosomal recessive) that leads to ↓ cortisol production with elevated cortisol precursors (e.g., 17-OH progesterone). In severe cases, mineralocorticoid deficiency with salt wasting may develop. Other causes include 11and 17-hydroxylase deficiencies. Cortisol deficiency stimulates ACTH synthesis, leading to overproduction of adrenal androgens. Presents with ambiguous genitalia in female infants and virilization when manifested later in life. Also characterized by macrogenitosomia in male infants; precocious puberty (if manifested later in life); and hypertension (with 11and 17-hydroxylase deficiencies). Diagnosed by high levels of cortisol precursors and androgens found in blood and urine.

1	Diagnosed by high levels of cortisol precursors and androgens found in blood and urine. Medical: Immediate fuid resuscitation and salt repletion. Administer cortisol to ↓ ACTH and adrenal androgens. Fludrocortisone is appropriate for severe 21-hydroxylase deficiency. Surgical: May be required in the case of ambiguous genitalia in female infants. Refer to the Gynecology chapter for information on the diagnosis and treatment of late-onset congenital adrenal hyperplasia. Assessment of Disease Frequency 132 Assessment of Diagnostic Studies 132 Assessment of Risk 134 Measures of Effect 135 Survival Curves 136 Treatment 136 Screening Recommendations 141 Causes of Death 143 Reportable Diseases 144 The prevalence of a disease is the number of existing cases in the population at a specifc moment in time. The incidence of a disease is the number of new cases in the disease-free population that develop over a period of time.

1	The incidence of a disease is the number of new cases in the disease-free population that develop over a period of time. Prevalent cases are incident cases that have persisted in a population for various reasons: Prevalence = Incidence × Average duration of disease A prevalence study is one in which people in a population are examined for the presence of a disease of interest at a given point in time. The advantages of prevalence studies are as follows: They provide an effcient means of examining a population, allowing cases and noncases to be assessed all at once. They can be used as a basis for diagnostic testing. They can be used to plan which health services to offer and where. Their disadvantages include the following: One cannot determine causal relationships because information is obtained only at a single point in time. The risk or incidence of disease cannot be directly measured.

1	One cannot determine causal relationships because information is obtained only at a single point in time. The risk or incidence of disease cannot be directly measured. Physicians often use tests to try to ascertain a diagnosis, but because no test is perfect, a given result may be falsely or (see Figure 2.4-1). When deciding whether to administer a test, one should thus consider both its sensitivity and its specif city. ■Sensitivity is the probability that a patient with a disease will have a test result. A sensitive test will rarely miss people with the disease and is therefore good at ruling people out. Incidence is measured with a cohort study; prevalence is measured with a prevalence study. As the mortality of a disease decreases, the prevalence of that disease increases (e.g., HIV infection). Another name for a prevalence study is a cross-sectional study. F IGU R E 2.4-1. Sensitivity, specificity, PPV, and NPV.

1	Another name for a prevalence study is a cross-sectional study. F IGU R E 2.4-1. Sensitivity, specificity, PPV, and NPV. ■Specificity is the probability that a patient without a disease will have a test result. A specific test will rarely determine that someone has the disease when in fact they do not and is therefore good at ruling people in. The ideal test is both sensitive and specifc, but a trade-off must often be made between sensitivity and specif city. High sensitivity is particularly desirable when there is a signif cant penalty for missing a disease. It is also desirable early in a diagnostic workup, when it is necessary to reduce a broad differential. Example: An initial ELISA test for HIV infection. High specificity is useful for confrming a likely diagnosis or for situations in which false-results may prove harmful. Example: A Western blot confrmatory HIV test. Example: You search for your physician, Mary Adel, MD, in the local phone book.

1	Example: You search for your physician, Mary Adel, MD, in the local phone book. Identifying all individuals named “Mary” would be a sensitive test, as anyone named Mary Adel would likely be included in the results. However, it is not specif c, as numerous other physicians would also be included. Remember the mnemonic “Snout”: High SENsitivity would allow one to rule out physicians not listed with this f rst name. Identifying only individuals with the full name “Mary Lucy Adel IV” is very specifc, since individuals not named Mary Adel are especially likely to be declined. However, it is not sensitive, as she may be overlooked if she is not listed by her complete name. Remember the mnemonic “SPin”: High SPecifcity would allow one to rule in physicians listed with this full name. Once a test has been administered and a patient’s result has been made available, that result must be interpreted through use of predictive values (or post-test probabilities):

1	Once a test has been administered and a patient’s result has been made available, that result must be interpreted through use of predictive values (or post-test probabilities): The positive predictive value (PPV) is the probability that a patient with a test result truly has the disease. The more specifc a test, the higher its PPV. The higher the disease prevalence, the higher the PPV of the test for that disease. The negative predictive value (NPV) is the probability that a patient with a test result truly does not have the disease. The more sensitive a test, the higher its NPV. The lower the disease prevalence, the higher the NPV of the test for that disease. Another way to describe the performance of a diagnostic test involves the use of likelihood ratios (LRs), which express how much more or less likely a given test result is in diseased as opposed to nondiseased people: Diseased people with a test result Sensitivity Nondiseased people with a test result 1 − specif city

1	Diseased people with a test result Sensitivity Nondiseased people with a test result 1 − specif city SnOUT: Sensitive tests rule OUT disease. SpIN: Specific tests rule IN disease. Because the predictive value of a test is affected by disease prevalence, it is advantageous to apply diagnostic tests to patients with an ↑ likelihood of having the disease being sought (i.e., an at-risk population). If a test has an LR of 1, it does not change the pretest probability of disease. If the LR is 10, it makes disease 45% more likely. If the LR is 0.1, disease is 45% less likely. Diseased people with a test result 1 − sensitivity Nondiseased people with a test result Specif city If you see a set of COworkers being followed over time, think COhort study. Cohort studies are also known as longitudinal studies or incidence studies.

1	If you see a set of COworkers being followed over time, think COhort study. Cohort studies are also known as longitudinal studies or incidence studies. Knowledge of risk factors may be used to predict future disease in a given patient. Risk factors may be causal (immediate or distant causes of disease) or disease markers (in which removing the risk factor does not necessarily ↓ the likelihood of disease). Risk factors can be diffcult to discover for a variety of reasons, including the following: A disease may have a long latency period, with risky exposures remote and forgotten. A risky exposure may be so common that its impact is hard to discern. Disease incidence may be low, and it is hard to draw conclusions from infrequent events. Risk associated with any individual exposure is small and hard to parse out. There is seldom a close, constant relationship between a risk factor and a disease.

1	Risk associated with any individual exposure is small and hard to parse out. There is seldom a close, constant relationship between a risk factor and a disease. The best way to determine whether an exposure actually ↑ disease risk is with a prospective study. The ideal study for risk factor assessment would be an experiment in which the researcher controls risk exposure and then relates it to disease incidence. Doing so, however, may be unethical as well as prohibitively intrusive, time consuming, and expensive. Instead, observational studies such as cohort or case-control studies are used to determine risk.

1	In a cohort study, a group of people is assembled, none of whom have the outcome of interest (i.e., the disease), but all of whom could potentially experience that outcome. For each possible risk factor, the members of the cohort are classifed as either exposed or unexposed. All the cohort members are then followed over time, and rates of outcome events are compared in the two exposure groups. Advantages of cohort studies are as follows: They follow the same logic as the clinical question (if people are exposed, will they get the disease?). They are the only way to directly determine incidence. They can be used to assess the relationship of a given exposure to many diseases. In prospective studies, exposure is elicited without bias from a known outcome. The disadvantages of such studies include the following: They can be time consuming and expensive. Studies assess only the relationship of the disease to the few exposure factors recorded at the start of the study.

1	They can be time consuming and expensive. Studies assess only the relationship of the disease to the few exposure factors recorded at the start of the study. They require many subjects and are thus ineffcient and cannot be used to study rare diseases. Cohort studies may be prospective—in which a cohort is assembled in the present and followed into the future—or they may be retrospective, in which a cohort is identifed from past records and followed to the present. Case-control studies are essentially cohort studies in reverse. In such studies, a researcher selects two groups—one with disease (cases) and one without (controls)—and then looks back in time to measure the comparative frequency of exposure to a possible risk factor in the two groups. The validity of a case-control study depends on appropriate selection of cases and controls, the manner in which exposure is measured, and the manner in which extraneous variables (confounders) are dealt with.

1	Cases and controls should be comparable in terms of opportunity for exposure (i.e., they should be members of the same base population with an equal opportunity of risk factor exposure). Cases should be newly diagnosed using explicit criteria for diagnosis. Exposures should be assessed in as unbiased a fashion as possible. Confounding may be ↓ by matching subjects or through the stratif cation of subjects. Multivariable analysis may be helpful in this context. Advantages of such studies are as follows: Studies use small groups, thereby reducing expense. They can be used to study rare diseases and can easily examine multiple risk factors. Disadvantages include the following: Studies cannot calculate disease prevalence, incidence, or relative risk, because the numbers of subjects with and without a disease are determined artifcially by the investigator rather than by nature (an odds ratio can be used to estimate relative risk).

1	Retrospective data may be inaccurate owing to recall or survivorship biases. If alcohol intake among individuals with breast cancer is compared with that of individuals without breast cancer, think case-control study. Absolute risk: The number of finger injuries among hospital staff this week. There are several ways to express and compare risk. These include the following: Absolute risk: Defned as the incidence of disease. Attributable risk (or risk difference): The additional incidence of disease that is due to a risky exposure, on top of the background incidence from other causes. Attributable risk = Incidence of disease in exposed – Incidence in unexposed Relative risk (or risk ratio): Expresses how much more likely an exposed person is to get disease in comparison to an unexposed person. This indicates the strength of the association between exposure and disease, making it useful when one is considering disease etiology.

1	■ Odds ratio: An estimate of relative risk that is used in case-control studies. The odds ratio tells how much more likely it is that a person with a disease has been exposed to a risk factor than someone without the disease. The Attributable risk: (number of injuries among staff who used scissors) – (number of injuries among staff who did not use scissors). Relative risk: (number of injuries among staff who used scissors) ÷ (number of injuries among staff who did not use scissors). F IGU R E 2.4-2. Relative risk (RR) vs. odds ratio (OR). lower the disease incidence, the more closely it approximates relative risk (see Figure 2.4-2). Odds that a diseased person is exposed Odds ratio = Odds that a nondiseased person is exposed Odds ratio: (odds that injured staff used scissors) ÷ (odds that uninjured staff used scissors).

1	Odds that a diseased person is exposed Odds ratio = Odds that a nondiseased person is exposed Odds ratio: (odds that injured staff used scissors) ÷ (odds that uninjured staff used scissors). Once a diagnosis has been established, it is important to be able to describe the associated prognosis. Survival analysis is used to summarize the average time from one event (e.g., presentation, diagnosis, or start of treatment) to any outcome that can occur only once during follow-up (e.g., death or recurrence of cancer). The usual method is with a Kaplan-Meier curve (see Figure 2.4-3) describing the survival (or time-to-event if the measured outcome is not death) in a cohort of patients, with the probability of survival de Probability of event creasing over time as patients die or drop out (are censored) from the study. Odds = 1 − probability of

1	Probability of event creasing over time as patients die or drop out (are censored) from the study. Odds = 1 − probability of Studies are typically used to judge the best treatment for a disease. Although the gold standard for such evaluation is a randomized, double-masked controlled trial, other types of studies may be used as well (e.g., an observational F IGU R E 2.4-3. Example of a Kaplan-Meier curve. study, in which the exposure in question is a therapeutic intervention). In descending order of quality, published studies regarding treatment options include meta-analyses, randomized controlled trials, and case series/case reports.

1	A randomized controlled trial is an experimental, prospective study in which subjects are randomly assigned to a treatment or control group. Random assignment helps ensure that the two groups are truly comparable. The control group may be treated with a placebo or with the accepted standard of care. The study may be masked in one of two ways: single-masked, in which patients do not know which treatment group they are in, or double-masked, in which neither the patients nor their physicians know who is in which group. Double-masked studies are the gold standard for studying treatment effects. Advantages of randomized controlled trials are as follows: They involve minimal bias. They have the potential to demonstrate causal relationships. Disadvantages include the following: They are costly and time intensive. Informed consent may be diffcult to obtain. Some interventions (e.g., surgery) are not amenable to masking. Ethical standards cannot allow all variables to be controlled.

1	Informed consent may be diffcult to obtain. Some interventions (e.g., surgery) are not amenable to masking. Ethical standards cannot allow all variables to be controlled. Def ned as any process that causes results to systematically differ from the truth. Good studies and data analyses seek to minimize potential bias. Types of bias include the following: Selection bias: Occurs when samples or participants are selected that differ from other groups in additional determinants of outcome. Examples: A surgeon may select patients without signifcant comorbid conditions to receive surgery, resulting in unexpectedly positive outcomes compared to previous studies. Similarly, individuals concerned about a family history of breast cancer may be more likely to self-select in entering a mammography program, giving the impression of a prevalence that is higher than reality.

1	Measurement bias: Occurs when measurement or data-gathering methods differ between groups. Example: One group is assessed by CT while another group is assessed by MRI. Confounding bias: Occurs when a third variable is associated with both the dependent variable and the independent variable (either positively or negatively), inducing a falseassociation (type I bias). Example: Fishermen in an area may experience a higher incidence of cataracts than the general population. However, becoming a fsherman will not in itself give you cataracts; rather, it is the sunlight to which those fshermen are exposed in their outdoor work. Recall bias: Results from a difference between two groups in the retrospective recall of past factors or outcomes. Example: A patient with cancer may be more motivated than a healthy individual to recall past episodes of chemical exposure.

1	Lead-time bias: Results from earlier detection of disease, giving an appearance of prolonged survival when in fact the natural course is not altered. Example: A new and widely used screening test that detects cancer f ve Randomization minimizes bias and confounding; double-blinded studies prevent observation bias. Studies that are masked and randomized are better protected from the effects of bias, whereas observational studies are particularly susceptible to bias. Confounding variables reduce the internal validity of a study. years earlier may give the impression that patients are living longer with the disease.

1	Confounding variables reduce the internal validity of a study. years earlier may give the impression that patients are living longer with the disease. ■ Length bias: Occurs when screening tests detect a disproportionate number of slowly progressive diseases but miss rapidly progressive ones, leading to overestimation of the beneft of the screen. Example: A better prognosis for patients with cancer is celebrated after implementation of a new screening program. However, this test disproportionally detects slow-growing tumors, which generally tend to be less aggressive. Even with bias reduction, unsystematic random error is unavoidable owing to chance variation in studied data. Types of errors are as follows: Type I (α) error: Defned as the probability of saying that there is a difference in treatment effects between groups when in fact there is not (i.e., a false-conclusion).

1	Type I (α) error: Defned as the probability of saying that there is a difference in treatment effects between groups when in fact there is not (i.e., a false-conclusion). The p-value is an estimate of the probability that differences in treatment effects in a study could have happened by chance alone. Classically, differences associated with a p < 0.05 are statistically signif cant. Type II (β) error: Defned as the probability of saying that there is no difference in treatment effects (i.e., a falseconclusion) when in fact a difference exists. Power is the probability that a study will fnd a statistically signif cant difference when one is truly there. It relates directly to the number of subjects. Power (β) = 1 – type II error. The confidence interval (CI) is a way of expressing statistical signif cance (p-value) that shows the size of the effect and the statistical power. CIs are interpreted as follows:

1	The confidence interval (CI) is a way of expressing statistical signif cance (p-value) that shows the size of the effect and the statistical power. CIs are interpreted as follows: If one is using a 95% CI, there is a 95% chance that the interval contains the true effect size, which is likely to be closest to the point estimate near the center of the interval. Example: You would like to estimate the percentage of women with a specifc disease. A 10% result from a sample of 3000 women would provide a 95% CI of 9%–11%, whereas a 10% f nding from a sample of 30 women would yield a CI of –1% of 21%. The frst case has more power because the sample size is larger, producing a narrow interval. In the latter case, you cannot state with 95% certainty that women in general even have the disease! If the CI includes the value corresponding to a relative risk of 1.0 (i.e., zero treatment difference), the results are not statistically signif cant. If the CI is wide, power is low.

1	If the CI includes the value corresponding to a relative risk of 1.0 (i.e., zero treatment difference), the results are not statistically signif cant. If the CI is wide, power is low. ■There are three levels of prevention: 1° prevention: Includes measures to ↓ the incidence of disease. 2° prevention: Focuses on identifying the disease early, when it is asymptomatic or mild, and implementing measures that can halt or slow disease progression. 3° prevention: Includes measures that ↓ morbidity or mortality resulting from the presence of disease. TABLE 2.4-1. Types of Vaccinations Live attenuated Measles, mumps, rubella, polio (Sabin), yellow fever, inﬂuenza (nasal spray). Inactivated (killed) Cholera, inﬂuenza, HAV, polio (Salk), rabies, inﬂuenza (injection). Toxoid Diphtheria, tetanus. Subunit HBV, pertussis, Streptococcus pneumoniae, HPV, meningococcus. Conjugate Hib, S. pneumoniae.

1	Prevention may be accomplished by a combination of immunization, chemoprevention, behavioral counseling, and screening. A good screening test has the following characteristics: It has high sensitivity and specifcity. It has a high PPV. It is inexpensive, easy to administer, and safe. Treatment after screening is more effective than subsequent treatment without screening. Vaccines work by mimicking infections and triggering an immune response in which memory cells are formed to recognize and fght any future infection. There are several different vaccine formulations, as indicated in Table 2.4-1. Recommended vaccination schedules for children and adults are outlined in Figures 2.4-4 through 2.4-6. 1° prevention: A woman reduces dietary intake of fat or alcohol to reduce her risk of developing breast cancer. 2° prevention: A woman obtains a mammogram to screen for breast cancer. 3° prevention: A woman undergoes surgical intervention for breast cancer.

1	2° prevention: A woman obtains a mammogram to screen for breast cancer. 3° prevention: A woman undergoes surgical intervention for breast cancer. Diphtheria, Tetanus, Pertussis Range of recommended Measles, Mumps, Rubella Varicella Hepatitis A Meningococcal F IGU R E 2.4-4. Recommended vaccinations for children 0–6 years of age. (Reproduced from the Centers for Disease Control and Prevention, Atlanta, Georgia.) F IGU R E 2.4-5. Recommended vaccinations for children 7–18 years of age. Range of recommended ages (Reproduced from the Centers for Disease Control and Prevention, Atlanta, Georgia.)

1	Range of recommended ages (Reproduced from the Centers for Disease Control and Prevention, Atlanta, Georgia.) VACCINE AGE GROUP 19–26 years 27–49 years 50–59 years 60–64 years >65 years Tetanus, diphtheria, pertussis (Td/Tdap)* Human papillomavirus (HPV)* Measles, mumps, rubella (MMR) * Influenza* Pneumococcal (polysaccharide) Hepatitis A* Hepatitis B* Meningococcal* Zoster Varicella * 3 doses 1 or more doses 2 doses 1 or 2 doses 1 dose 3 doses (females) Substitute 1-time dose ofTdap forTd booster; then boost withTd every 10 yrs 2 doses 1 or 2 doses 1 dose 1 dose *Covered by the Vaccine Injury Compensation Program. For all persons in this category who meet the age requirements and who lack evidence of immunity No recommendationRecommended if some other risk factor is present (e.g., on the basis of medical, 1 dose ann u all y Td booster every 10 yrs (e.g., lack documentation of vaccination or have occupational, lifestyle, or other indications) no evidence of prior infection)

1	F IGU R E 2.4-6. Recommended vaccinations for adults. (Reproduced from the Centers for Disease Control and Prevention, Atlanta, Georgia.) T AB LE 2.4-2. Stages of Change in Behavioral Counseling

1	Precontemplation Denial or ignorance of the problem. A 51-year-old smoker has not even thought about cessation. Contemplation Ambivalence or conﬂicted emotions; assessing benefits and barriers to change. A 43-year-old crack cocaine addict considers treatment for her addiction. Preparation Experimenting with small changes; collecting information about change. A 28-year-old heroin addict visits his doctor to ask questions about quitting. Taking direct action toward achieving a goal. A 33-year-old enters a rehabilitation facility for treatment of addiction to prescription narcotics. Maintaining a new behavior; avoiding temptation. A 41-year-old continues to visit Alcoholics Anonymous meetings to gain support and reinforcement against relapse. Action Maintenance ■Live vaccines should not be administered to immunosuppressed patients. Such vaccines are also contraindicated in pregnant women owing to a theoretical risk of maternal-fetal transmission.

1	Defned as the use of drugs to prevent disease. Examples include supplementation of water and food with healthful vitamins and minerals; aspirin prophylaxis for heart disease; statin treatment for hypercholesterolemia; and perioperative β-blockade. In offering counsel, physicians should tailor their education and suggestions to the individual patient as well as to his or her stage of change (see Table 2.4-2). Counseling is most likely to be successful when it accommodates the patient’s willingness to change or lack thereof. Tables 2.4-3 and 2.4-4 outline recommended health care screening measures by age group and modality. T AB LE 2.4-3. Health Screening Methods by Age

1	T AB LE 2.4-3. Health Screening Methods by Age Birth–10 years Height and weight, BP, vision and hearing screening, hemoglobinopathy screen (at birth), phenylalanine level (at birth), TSH and/or T4 (at birth), lead level (at least one time before six years of age). 11–24 years Height and weight, BP, Pap test, gonorrhea and chlamydia (GC) screen (if sexually active), rubella serology or vaccination (women only); screen for risky behaviors, including substance abuse. 25–64 years Height and weight, BP (every two years), cholesterol (every five years), Pap test and bimanual pelvic exam, fecal occult blood test (FOBT) or fecal immunochemical test (FIT), sigmoidoscopy or colonoscopy, mammography, rubella serology or vaccination (women only); screen for alcohol abuse and depression. ≥ 65 years Height and weight, BP, FOBT or FIT, sigmoidoscopy or colonoscopy, Pap test, vision and hearing screening, osteoporosis (DEXA) scans for women; screen for alcohol abuse and depression.

1	T AB LE 2.4-4. Health Screening Methods by Modality Once every 10 years in patients ≥ 50 years of age (or ≥ 40 years of age for high-risk patients). The preferred modality if there is a known history of dysplasia. CT virtual colonoscopy is comparable to standard colonoscopy and can be used every five years as an initial screening exam. Once every year within three years after onset of vaginal intercourse, but no later than age 21 up to age 30; once every two years with liquid-based Pap test. The interval can be ↑ to once every 2–3 years for women > 30 years of age with three cytology tests. Offer every three years to female patients in 20s and 30s, and every year to females 40 or more years of age (controversial). a Different medical societies have various recommendations regarding cancer screening. Refer to the National Cancer Institute’s Web site for a recent summary of recommendations: www.nci.nih.gov/cancer_information/testing.

1	Colonoscopy Flexible sigmoidoscopy Once every five years in patients ≥ 50 years of age (or ≥ 40 years of age for high-risk patients). FOBT or FIT Once every year in patients ≥ 50 years of age (or ≥ 40 years of age for high-risk patients). Bimanual pelvic exam Once every 1–3 years in patients 20–40 years of age; once yearly in patients ≥ 40 years of age. Pap test Mammography Once every 1–2 years in patients ≥ 40 years of age; once yearly in patients ≥ 50 years of age (controversial). Endometrial tissue sampling Not recommended as a screening test; indicated for postmenopausal bleeding. CXR Not recommended as a screening test. Skin exam Insufficient evidence. DRE Offer every year to high-risk patients at 40 years and to others at 50 years of age (controversial). PSA Offer every year to high-risk patients at 40 years and to others at 50 years of age (controversial). Clinical breast exam

1	The leading cause of cancer mortality in the United States is lung cancer. Prostate and breast cancers are the most prevalent cancers in men and women, respectively, with lung and colorectal cancers ranking second and third most common in both sexes. Table 2.4-5 lists the principal causes of death in the United States by age group. TABLE 2.4-5. Leading Causes of Death by Age Group

1	TABLE 2.4-5. Leading Causes of Death by Age Group All ages Heart disease, cancer, stroke, chronic lower respiratory disease, injuries, diabetes. < 1 year Congenital anomalies, disorders related to low birth weight, SIDS, maternal complications. 1–4 years Injuries, congenital anomalies, cancer, homicide, heart disease. 5–14 years Injuries, cancer, congenital anomalies, homicide, suicide, heart disease. 15–24 years Injuries, homicide, suicide, cancer, heart disease. 25–44 years Injuries, cancer, heart disease, suicide, HIV, homicide. 45–64 years Cancer, heart disease, injuries, stroke, diabetes, chronic lower respiratory disease. ≥ 65 years Heart disease, cancer, stroke, chronic lower respiratory disease, Alzheimer’s disease, diabetes. Adapted, with permission, from the National Center for Health Statistics, Department of Health and Human Services, www.cdc.gov/nchs.

1	By law, disease reporting is mandated at the state level, and the list of diseases that must be reported to public health authorities varies slightly by state. The CDC has a list of nationally notifable diseases that states voluntarily report to the CDC. These diseases include but are not limited to those listed in Table 2.4-6. T AB LE 2.4-6. Common Reportable Diseases

1	T AB LE 2.4-6. Common Reportable Diseases STDs HIV, AIDS, syphilis, gonorrhea, chlamydia, chancroid, HCV. Tick-borne disease Lyme disease, ehrlichiosis, Rocky Mountain spotted fever. Potential bioweapons Anthrax, smallpox, plague. Vaccine-preventable disease Diphtheria, tetanus, pertussis, measles, mumps, rubella, polio, varicella, HAV, HBV, H. inﬂ uenzae (invasive), meningococcal disease. Water-/food-borne disease Cholera, giardiasis, legionella, listeriosis, botulism, shigellosis, shiga toxin–producing E. coli, salmonellosis, trichinellosis, typhoid. Zoonoses Tularemia, psittacosis, brucellosis, rabies. Miscellaneous TB, leprosy, toxic shock syndrome, SARS, West Nile virus, VRSA, coccidioidomycosis, cryptosporidiosis. Confidentiality 149 Conﬂict of Interest 149 Malpractice 150

1	Confidentiality 149 Conﬂict of Interest 149 Malpractice 150 Respect for autonomy: Clinicians are obligated to respect patients as individuals and to honor their preferences in medical care. Example: A surgeon presents the risks and benefits of tumor resection to her patient before consent is given to proceed with the procedure. Beneficence: Physicians have a responsibility to act in the patient’s best interest. As a fiduciary, the physician stands in a special relationship of trust and responsibility to patients. Respect for patient autonomy may conﬂ ict with beneficence. Example: An elderly woman is adamant that she does not want to go to a rehabilitation facility and thus refuses amputation of a potentially gangrenous foot. The procedure is necessary to prevent life-threatening complications. The physician has a responsibility to act in the patient’s best interest.

1	Nonmaleficence: “Do no harm.” The responsibility to avert avoidable harms is a strong duty. However, if the potential benefits of an intervention outweigh the risks, an autonomous patient may make an informed decision to consent and proceed. Example: A patient may choose to undergo dialysis, accepting that his discomfort would be outweighed by the greater good of sustaining life. Justice: With regard to individual patients, fairness is expressed as the notion that equal persons should be treated equally. Health care is an important resource, and access to quality health care gives individuals a greater chance for a healthy and productive life. Fair distribution of this resource is an ongoing challenge for health policy and in the clinical arena.

1	Defined as willing acceptance (without coercion) of a medical intervention by a patient after adequate discussion with a physician about the nature of the intervention along with its indications, risks, benefits, and potential alternatives (including no treatment). Patients may change their minds at any time. Informed consent is required for significant procedures unless: Emergency treatment is required. Examples: An unconscious patient presents with cerebral edema after a motor vehicle collision, or a patient without previously indicated DNR/DNI status undergoes cardiac arrest. Patients lack decision-making capacity (consent can be obtained from a surrogate decision maker). Examples: Patients may present with dementia or significant psychiatric disturbances. Minors generally require surrogate decision makers until they demonstrate adequate decision-making capacity or are of legal age.

1	Consent for treatment is implied in life-threatening situations when parents cannot be contacted. Emancipated minors do not require parental consent for medical care. Minors are emancipated if they are married, in the armed services, or are financially independent of their parents and have sought legal emancipation.

1	Minors may consent to care for sexually transmitted infections without parental consent or knowledge. Rules concerning contraception, preg nancy, and abortion services and treatment for drug and alcohol dependency vary across the United States. (Some states leave the decision of informing parents about adolescent use of confidential services to the physician, based on the best interest of the patient. Other states limit disclosure.) ■Refusal of treatment: A parent has the right to refuse treatment for his/her child as long as those decisions do not pose a serious threat to the child’s well-being (e.g., refusing immunizations is not considered a serious threat). If a decision is not in the best interest of the child, a physician may seek a court order to provide treatment against parental wishes. In emergent situations, if withholding treatment jeopardizes the child’s safety, treatment can be initiated on the basis of legal precedent. Example: A physician provides blood transfusion to

1	wishes. In emergent situations, if withholding treatment jeopardizes the child’s safety, treatment can be initiated on the basis of legal precedent. Example: A physician provides blood transfusion to save the life of a six-year-old child seriously injured in a motor vehicle collision despite parental requests to withhold such a measure.

1	Minors may consent to care for sexually transmitted infections without parental consent or knowledge. Competence: Refers to a person’s legal capacity to make decisions and be held accountable in a court of law. Competence is assessed by the courts and is often used interchangeably with the term decision-making capacity (see below). Decision-making capacity: A medical term that refers to the ability of a patient to understand relevant information, appreciate the severity of the medical situation and its consequences, communicate a choice, and deliberate rationally about one’s values in relation to the decision being made. This can be assessed by the physician. Decision-making capacity is best understood as varying with the complexity of the decision involved. Example: The level of capacity needed for a decision about liver transplantation is different from that needed to choose between two types of pain medication for fracture-related pain.

1	Incompetent patients, as assessed by the courts, or temporarily incapacitated patients cannot decide to accept or refuse treatment. Example: An intoxicated patient with altered mental status cannot refuse thiamine supplementation. In general, patients who have decision-making capacity have the right to refuse or discontinue treatment. Example: Jehovah’s Witnesses can refuse blood products. Living will: Addresses a patient’s wishes to maintain, withhold, or withdraw life-sustaining treatment in the event of terminal disease or a persistent vegetative state. Examples include DNR (do not resuscitate) and DNI (do not intubate) orders. Durable power of attorney for health care (DPOAHC): Legally designates a surrogate health care decision maker if a patient lacks decision-making capacity. More ﬂ exible than a living will. Surrogates should make decisions consistent with the person’s stated wishes.

1	If no living will or DPOAHC exists, decisions should be made by close family members (spouse, adult children, parents, and adult siblings), friends, or personal physicians, in that order. In the absence of a living will or DPOA, the Spouse CHIPS in For the Person: Spouse, CHIldren, Parent, Sibling, Friend, Personal physician. DNR/DNI orders do not mean “do not treat.” Patients cannot demand futile treatment from their physicians. A patient’s family cannot require that a doctor withhold information from the patient. Withdrawal of Care Patients and their decision makers have the right to forgo or withdraw life-sustaining treatment. No ethical distinction is considered to exist between withdrawing a treatment that offers no benefit and withholding one that is not indicated. This may include ventilation, ﬂuids, nutrition, and medications such as antibiotics.

1	If the intent is to relieve suffering and medications administered are titrated for that purpose, then it is considered ethical to provide palliative treatment to relieve pain and suffering, even if it may hasten a patient’s death. Example: A physician prescribes midazolam to a patient who is expected to die within a day in order to offer relief from the acute distress of grand mal seizures and pain. Euthanasia is the administration of a lethal agent with the intent to end life. It is opposed by the AMA Code of Medical Ethics and is illegal in all states. Patients who request euthanasia should be evaluated for inadequate pain control and comorbid depression. Physician-assisted suicide is prescribing a lethal agent to a patient who will self-administer it to end his/her own life. This is currently illegal except in the state of Oregon.

1	Physician-assisted suicide is prescribing a lethal agent to a patient who will self-administer it to end his/her own life. This is currently illegal except in the state of Oregon. Physicians are not ethically obligated to provide treatment and may refuse a family member’s request for further intervention on the grounds of futility under the following circumstances: There is no pathophysiologic rationale for treatment. A given intervention has already failed. Maximal intervention is currently failing. Treatment will not achieve the goals of care. Patients have a right to know about their medical status, prognosis, and treatment options (full disclosure). A patient’s family cannot require that a doctor withhold information from the patient.

1	Patients have a right to know about their medical status, prognosis, and treatment options (full disclosure). A patient’s family cannot require that a doctor withhold information from the patient. A doctor may withhold information only if the patient requests not to be told or in the rare case when a physician determines that disclosure would severely harm the patient or undermine their informed decision-making capacity (therapeutic privilege). Physicians are obligated to inform patients of mistakes made in their medical treatment. If the specific error or series of errors is not known, the physician should communicate this with the family promptly and maintain contact with the patient as investigations reveal more facts.

1	If the specific error or series of errors is not known, the physician should communicate this with the family promptly and maintain contact with the patient as investigations reveal more facts. Physicians are obligated to inform patients considering involvement in a clinical research protocol about the purpose of the research study and the entire study design as it will affect the patient’s treatment. This includes the possible risks, benefits, and alternatives to the research protocol. An informed consent form approved by the overseeing research institutional review board (IRB) must be completed for participation in any clinical research protocol, describing the possible risks and benefits of involvement in the research study. Information disclosed by a patient to his/her physician and information about a patient’s medical condition are confidential and cannot be divulged without expressed patient consent.

1	Information disclosed by a patient to his/her physician and information about a patient’s medical condition are confidential and cannot be divulged without expressed patient consent. A patient may waive the right to confidentiality (e.g., with insurance companies). It is ethically and legally necessary to override confidentiality in the following situations: Patient intent to commit a violent crime (Tarasoff decision): Physicians have a duty to protect the intended victim through reasonable means (e.g., warn the victim, notify police). Suicidal patients. Child and elder abuse. Reportable infectious diseases (duty to warn public officials and identifiable people at risk). Gunshot and knife wounds (duty to notify the police). Impaired automobile drivers. Example: A patient begins to drive one week after hospitalization for seizures, although the department of motor vehicles in his state requires that licensed drivers be seizure free for at least three months.

1	Occurs when physicians find themselves having two interests in a given situation and their professional obligations are inﬂuenced by personal interest in another matter. Example: A physician may own stock in a pharmaceutical company (financial interest) that produces a drug he is prescribing to his patient (patient care interest). Physicians should disclose existing conﬂicts of interest to affected parties (e.g., patients, institutions, audiences of journal articles or scientific meetings). The essential elements of a civil suit under negligence include the four D’s: The physician has a Duty to the patient. Dereliction of duty occurs. There is Damage to the patient. Dereliction is the Direct cause of damage. Unlike a criminal suit, in which the burden of proof is “beyond a reasonable doubt,” the burden of proof in a malpractice suit is “a preponderance of the evidence.”

1	Unlike a criminal suit, in which the burden of proof is “beyond a reasonable doubt,” the burden of proof in a malpractice suit is “a preponderance of the evidence.” Disorders of the Stomach and Duodenum 156 GASTRITIS 156 GASTRIC CANCER 157 PEPTIC ULCER DISEASE 157 ZOLLINGER-ELLISON SYNDROME 158 Disorders of the Small Bowel 159 DIARRHEA 159 MALABSORPTION 161 LACTOSE INTOLERANCE 162 CARCINOID SYNDROME 162 IRRITABLE BOWEL SYNDROME 162 SMALL BOWEL OBSTRUCTION 163 ILEUS 164 MESENTERIC ISCHEMIA 165 APPENDICITIS 166 Disorders of the Large Bowel 166 DIVERTICULAR DISEASE 166 LARGE BOWEL OBSTRUCTION 167 COLON AND RECTAL CANCER 167 ISCHEMIC COLITIS 170 Difficulty swallowing (dysphagia) or pain with swallowing (odynophagia) due to abnormalities of the oropharynx or esophagus. Presentation varies according to the location:

1	Difficulty swallowing (dysphagia) or pain with swallowing (odynophagia) due to abnormalities of the oropharynx or esophagus. Presentation varies according to the location: Oropharyngeal dysphagia: Presents with difficulty passing material from the oropharynx to the esophagus. Usually involves liquids more than solids. Causes can be neurologic or muscular and include stroke, Parkinson’s disease, myasthenia gravis, prolonged intubation, and Zenker’s diverticula. Esophageal dysphagia: Usually involves solids more than liquids for most obstructive causes (strictures, Schatzki rings, webs, carcinoma) and is generally progressive. Motility disorders (achalasia, scleroderma, esophageal spasm) present with both liquid and solid dysphagia. Examine for masses (e.g., goiter, tumor) and anatomic defects. Oropharyngeal dysphagia: Cine-esophagram.

1	Examine for masses (e.g., goiter, tumor) and anatomic defects. Oropharyngeal dysphagia: Cine-esophagram. Esophageal dysphagia: Barium swallow followed by endoscopy, manometry, and/or pH monitoring. If an obstructive lesion is suspected, proceed directly to endoscopy with biopsy. Odynophagia: Upper endoscopy. Etiology dependent. Table 2.6-1 outlines the etiology, diagnosis, and treatment of infectious esophagitis. ■A motility disorder in which normal peristalsis is periodically interrupted by high-amplitude nonperistaltic contractions of unknown etiology. Also known as nutcracker esophagus. T AB LE 2.6-1. Causes of Infectious Esophagitis Esophageal webs are associated with iron deficiency anemia and glossitis (Plummer-Vinson syndrome). Candidal esophagitis is an AIDS-defining illness.

1	Esophageal webs are associated with iron deficiency anemia and glossitis (Plummer-Vinson syndrome). Candidal esophagitis is an AIDS-defining illness. Candida albicans Oral thrush Yellow-white plaques adherent to the mucosa. Fluconazole PO HSV Oral ulcers Small, deep ulcerations; multinucleated giant cells with nuclear inclusions on biopsy. Acyclovir IV CMV Retinitis, colitis Large, superfcial ulcerations; intranuclear inclusions on biopsy. Ganciclovir IV The musculature of the upper one-third of the esophagus is skeletal, whereas that of the lower two-thirds is smooth muscle. Malignancy may mimic achalasia (pseudoachalasia). Hx/PE: Presents with chest pain, dysphagia, and odynophagia. Often precipitated by ingestion of hot or cold liquids; relieved by nitroglycerin. Dx: Barium swallow may show a corkscrew-shaped esophagus. Esophageal manometry reveals high-amplitude, simultaneous contractions.

1	Dx: Barium swallow may show a corkscrew-shaped esophagus. Esophageal manometry reveals high-amplitude, simultaneous contractions. Tx: Nitrates and calcium channel blockers (CCBs) for symptomatic relief; surgery (esophageal myotomy) for severe, incapacitating symptoms. A motor disorder of the esophagus characterized by impaired relaxation of the lower esophageal sphincter (LES) and loss of peristalsis in the distal two-thirds of the esophagus. Thought to result from the loss or absence of inhibitory ganglion cells in the myenteric (Auerbach’s) plexus. Hx/PE: Progressive dysphagia, chest pain, regurgitation of undigested food, weight loss, and nocturnal cough are common symptoms. Dx: Barium swallow reveals esophageal dilation with a “bird’s beak” tapering of the distal esophagus (see Figure 2.6-1). Manometry shows ↑ resting LES pressure, incomplete LES relaxation upon swallowing, and ↓ peristalsis in the body of the esophagus.

1	Manometry shows ↑ resting LES pressure, incomplete LES relaxation upon swallowing, and ↓ peristalsis in the body of the esophagus. Mechanical causes of obstruction must be ruled out by endoscopy. Tx: Nitrates, CCBs, or endoscopic injection of botulinum toxin into the LES may provide short-term relief of symptoms. Pneumatic balloon dilation or surgical (Heller) myotomy are definitive treatment options. FIGURE 2.6-1. Achalasia. Barium esophagram reveals esophageal dilation with a “bird’s beak” tapering of the distal esophagus. (Reproduced from Waters PF, DeMeester TR. Foregut motor disorders and their surgical management. Med Clin North Am 65: 1235, © 1981, with permission from Elsevier.) The second most common esophageal motility disorder (after achalasia). Cervical outpouching through the cricopharyngeal muscle is called Zenker’s diverticulum. Diverticula can also occur in the middle and distal third of the esophagus.

1	Cervical outpouching through the cricopharyngeal muscle is called Zenker’s diverticulum. Diverticula can also occur in the middle and distal third of the esophagus. Hx/PE: Patients complain of chest pain, dysphagia, halitosis, and regurgitation of undigested food. Dx: Barium swallow will demonstrate outpouchings. Tx: If symptomatic, treat with surgical excision of the diverticulum. For Zenker’s diverticulum, myotomy of the cricopharyngeus is required to relieve the high-pressure zone. Squamous cell carcinoma (SCC) is the most common type of esophageal cancer worldwide, while adenocarcinoma is most prevalent in the United States, Europe, and Australia. The latter is associated with Barrett’s esophagus (columnar metaplasia of the distal esophagus 2° to chronic GERD). Alcohol use and smoking have been identified as major risk factors for SCC.

1	Alcohol use and smoking have been identified as major risk factors for SCC. Hx/PE: Progressive dysphagia, initially to solids and later to liquids, is common. Weight loss, odynophagia, GERD, GI bleeding, and vomiting are also seen. Dx: Barium study shows narrowing of the esophagus with an irregular border protruding into the lumen. EGD and biopsy confirm the diagnosis. CT and endoscopic ultrasound are used for staging. Tx: Chemoradiation and surgical resection is the definitive treatment, but the prognosis is poor. Surgery is also offered for high-grade Barrett’s dysplasia. Patients who are not candidates for surgery may require an endoscopically placed esophageal stent for palliation and improved quality of life. Symptomatic reﬂux of gastric contents into the esophagus, most commonly as a result of transient LES relaxation. Can be due to an incompetent LES, gastroparesis, or hiatal hernia.

1	Symptomatic reﬂux of gastric contents into the esophagus, most commonly as a result of transient LES relaxation. Can be due to an incompetent LES, gastroparesis, or hiatal hernia. Patients present with heartburn that commonly occurs 30–90 minutes after a meal, worsens with reclining, and often improves with antacids, sitting, or standing. Substernal chest pain can be difficult to distinguish from other causes. Sour taste (“water brash”), globus, unexplained cough, and morning hoarseness can be clues. Exam is usually normal unless a systemic disease (e.g., scleroderma) is present. The history and clinical impression are important. An empiric trial of lifestyle modification and medical treatment is often attempted first. Studies may include barium swallow (to look for hiatal hernia), esophageal manometry, and 24-hour pH monitoring. Squamous cell esophageal cancer is associated with tobacco and alcohol use.

1	Squamous cell esophageal cancer is associated with tobacco and alcohol use. Esophageal cancer metastasizes early because the esophagus lacks a serosa. Risk factors for GERD include hiatal hernia and ↑ intra-abdominal pressure (e.g., obesity, pregnancy). GERD can mimic cough-variant asthma. Patients with GERD should avoid caffeine, alcohol, chocolate, garlic, onions, mints, and nicotine. Type A gastritis is associated with pernicious anemia due to the lack of intrinsic factor necessary for the absorption of vitamin B12. ■ EGD with biopsies should be performed in patients whose symptoms are unresponsive to initial empiric therapy, long-standing (to rule out Barrett’s esophagus and adenocarcinoma), or suggestive of complicated disease (e.g., anorexia, weight loss, dysphagia/odynophagia). Lifestyle: Weight loss, head-of-bed elevation, reduction of meal size, and avoidance of nocturnal meals and substances that ↓ LES tone.

1	Lifestyle: Weight loss, head-of-bed elevation, reduction of meal size, and avoidance of nocturnal meals and substances that ↓ LES tone. Pharmacologic: Start with antacids in patients with mild, intermittent symptoms; use H2 receptor antagonists (cimetidine, ranitidine) or PPIs (omeprazole, lansoprazole) in patients with chronic and frequent symptoms. PPIs are preferred for severe or erosive disease. Surgical: For refractory or severe disease, Nissen fundoplication may offer significant relief. Esophagitis, esophageal stricture, aspiration of gastric contents, upper GI bleeding, Barrett’s esophagus. Herniation of a portion of the stomach upward into the chest through a diaphragmatic opening. There are two common types: Sliding hiatal hernias (95%): The gastroesophageal junction and a portion of the stomach are displaced above the diaphragm.

1	Sliding hiatal hernias (95%): The gastroesophageal junction and a portion of the stomach are displaced above the diaphragm. Paraesophageal hiatal hernias (5%): The gastroesophageal junction remains below the diaphragm, while a neighboring portion of the fundus herniates into the mediastinum. Hx/PE: May be asymptomatic. Those with sliding hernias may present with GERD. Dx: Commonly an incidental finding on CXR; also frequently diagnosed by barium swallow or EGD. Tx: Sliding hernias: Medical therapy and lifestyle modifications to ↓ GERD symptoms. Paraesophageal hernias: Surgical gastropexy (attachment of the stomach to the rectus sheath and closure of the hiatus) is recommended to prevent gastric volvulus. Inﬂammation of the stomach lining. Subtypes are as follows: Acute gastritis: Rapidly developing, superficial lesions that are often due to NSAID use, alcohol, H. pylori infection, and stress from severe illness (e.g., burns, CNS injury).

1	Acute gastritis: Rapidly developing, superficial lesions that are often due to NSAID use, alcohol, H. pylori infection, and stress from severe illness (e.g., burns, CNS injury). Chronic gastritis: ■Type A (10%): Occurs in the fundus and is due to autoantibodies to parietal cells. Causes pernicious anemia and is associated with other autoimmune disorders, such as thyroiditis. Also ↑ the risk of gastric adenocarcinoma. ■Type B (90%): Occurs in the antrum and may be caused by NSAID use or H. pylori infection. Often asymptomatic, but associated with ↑ risk of PUD and gastric cancer. Patients may be asymptomatic or may complain of epigastric pain, nausea, vomiting, hematemesis, or melena. Upper endoscopy can visualize the gastric lining. H. pylori infection can be detected by urease breath test, serum IgG antibodies (which indicate exposure, not current infection), H. pylori stool antigen, or endoscopic biopsy.

1	H. pylori infection can be detected by urease breath test, serum IgG antibodies (which indicate exposure, not current infection), H. pylori stool antigen, or endoscopic biopsy. ■↓ intake of offending agents. Antacids, sucralfate, H2 blockers, and/or PPIs may help. Triple therapy (amoxicillin, clarithromycin, omeprazole) to treat H. pylori infection. Give prophylactic H2 blockers or PPIs to patients at risk for stress ulcers (e.g., ICU patients). This malignant tumor is the second most common cause of cancer-related death worldwide and is particularly common in Korea and Japan. Tumors are generally adenocarcinomas, which exhibit two morphologic types: Intestinal type: Thought to arise from intestinal metaplasia of gastric mucosal cells. Risk factors include a diet high in nitrites and salt and low in fresh vegetables (antioxidants), H. pylori colonization, and chronic gastritis. Diffuse type: Tends to be poorly differentiated and not associated with

1	Diffuse type: Tends to be poorly differentiated and not associated with H. pylori infection or chronic gastritis. Risk factors are largely unknown; signet ring cells are seen on pathology. Hx/PE: Early signs are indigestion and loss of appetite. Advanced cases generally present with abdominal pain, weight loss, or upper GI bleeding. Dx: Early gastric carcinoma is largely asymptomatic and is discovered serendipitously with endoscopic examination of high-risk individuals. Tx: Successful treatment rests entirely on early detection and surgical removal of the tumor. Five-year survival is < 10% for advanced disease. Damage to the gastric or duodenal mucosa caused by impaired mucosal defense and/or ↑ acidic gastric contents. H. pylori plays a causative role in > 90% of duodenal ulcers and 70% of gastric ulcers. Other risk factors include corticosteroid, NSAID, alcohol, and tobacco use. Males are affected more often than females.

1	■Classically presents with chronic or periodic dull, burning epigastric pain that improves with meals (especially duodenal ulcers), worsens 2–3 hours after eating, and can radiate to the back. Gastric adenocarcinoma that metastasizes to the ovary is called a Krukenberg tumor. Gastric cancer may present with Virchow’s node (an enlarged left supraclavicular lymph node). After a meal, pain from a Gastric ulcer is Greater, whereas Duodenal pain Decreases. Roughly 5–7% of lower GI bleeds are from an upper GI source. Rule out Zollinger-Ellison syndrome with serum gastrin levels in cases of GERD and PUD that are refractory to medical management. Misoprostol can help patients with PUD who require NSAID therapy (e.g., for arthritis). Patients may also complain of nausea, hematemesis (“coffee-ground” emesis), or blood in the stool (melena or hematochezia). Exam may reveal varying degrees of epigastric tenderness and, if there is active bleeding, a stool guaiac.

1	Exam may reveal varying degrees of epigastric tenderness and, if there is active bleeding, a stool guaiac. An acute perforation can present with a rigid abdomen, rebound tenderness, guarding, or other signs of peritoneal irritation. AXR to rule out perforation (free air under the diaphragm); CBC to assess for GI bleeding (low or ↓ hematocrit). Upper endoscopy with biopsy to confirm PUD and to rule out active bleeding or gastric adenocarcinoma (10% of gastric ulcers); barium swallow is an alternative. H. pylori testing. In recurrent or refractory cases, serum gastrin can be used to screen for Zollinger-Ellison syndrome (patients must discontinue PPI use prior to testing). Acute: Rule out active bleeding with serial hematocrits, a rectal exam with stool guaiac, and NG lavage. Monitor the patient’s hematocrit and BP and treat with IV hydration, transfusion, IV PPIs, endoscopy, and surgery as needed for complications. If perforation is likely, emergent surgery is indicated.

1	If perforation is likely, emergent surgery is indicated. Pharmacologic: Involves protecting the mucosa, decreasing acid production, and eradicating H. pylori infection. Treat mild disease with antacids or with sucralfate, bismuth, and misoprostol (a prostaglandin analog) for mucosal protection. PPIs or H2 receptor antagonists may be used to ↓ acid secretion. Patients with confirmed H. pylori infection should receive triple therapy (amoxicillin, clarithromycin, and omeprazole). Discontinue use of exacerbating agents. Patients with recurrent or severe disease may require chronic symptomatic therapy. Endoscopy and surgery: Patients with symptomatic gastric ulcers for > 2 months that are refractory to medical therapy should have either endoscopy or an upper GI series with barium to rule out gastric adenocarcinoma. Refractory cases may require a surgical procedure such as parietal cell vagotomy (the most selective and preferred surgical approach).

1	Refractory cases may require a surgical procedure such as parietal cell vagotomy (the most selective and preferred surgical approach). Hemorrhage (posterior ulcers that erode into the gastroduodenal artery), gastric outlet obstruction, perforation (usually anterior ulcers), intractable pain. ■A rare condition characterized by gastrin-producing tumors in the duodenum and/or pancreas that lead to oversecretion of gastrin. ■↑ gastrin results in the production of high levels of gastric acid by the gastric mucosa, leading to recurrent/intractable ulcers in the stomach and duodenum (may occur more distally). In 20% of cases, gastrinomas are associated with MEN 1 (pancreas, pituitary, and parathyroid tumors). Hx/PE: Patients may present with unresponsive, recurrent gnawing, burning abdominal pain as well as with diarrhea, nausea, vomiting, fatigue, weakness, weight loss, and GI bleeding.

1	Hx/PE: Patients may present with unresponsive, recurrent gnawing, burning abdominal pain as well as with diarrhea, nausea, vomiting, fatigue, weakness, weight loss, and GI bleeding. Dx: ↑ fasting serum gastrin levels are characteristic, as is ↑ gastrin following a secretin stimulation test. Octreotide scan can localize the tumor. Tx: Requires ↓ acid production. H2 blockers are typically ineffective, but a moderateto high-dose PPI often controls symptoms. In light of the malignant potential of the tumor, surgical resection should be attempted where possible. Zollinger-Ellison syndrome is associated with MEN 1 syndrome in roughly 20% of cases. Defined as the production of > 200 g of feces per day along with ↑ frequency or ↓ consistency of stool. Broadly, the four etiologic mechanisms are ↑ motility, ↑ secretion, ↑ luminal osmolarity, and inﬂammation (see also Table 2.6-2). Acute diarrhea: Acute onset with < 2 weeks of symptoms; usually infectious and self-limited.

1	Acute diarrhea: Acute onset with < 2 weeks of symptoms; usually infectious and self-limited. Causes include bacteria with preformed toxins (e.g., S. aureus, Bacillus cereus), noninvasive bacteria (e.g., enterotoxigenic E. coli, Vibrio cholerae, C. difficile), invasive bacteria (e.g., enteroinvasive E. coli, Salmonella, Shigella, Campylobacter, Yersinia), parasites (e.g., Giardia, Entamoeba histolytica), and opportunistic organisms (e.g., Cryptosporidium, Isospora, Microsporidium, CMV). One of the most common causes of pediatric diarrhea is rotavirus infection (most common during winter). Chronic diarrhea: Insidious onset with > 4 weeks of symptoms.

1	One of the most common causes of pediatric diarrhea is rotavirus infection (most common during winter). Chronic diarrhea: Insidious onset with > 4 weeks of symptoms. Can be due to ↑ intestinal secretion (e.g., carcinoid, VIPomas), malabsorption/osmotic diarrhea (e.g., bacterial overgrowth, pancreatic insufficiency, mucosal abnormalities, lactose intolerance), inf ammatory bowel disease (IBD), or altered motility (e.g., IBS). ↑ stool osmotic gap and resolution of diarrhea with fasting suggest osmotic diarrhea. See Figure 2.6-2 for a diagnostic algorithm. Acute diarrhea usually does not require laboratory investigation unless the patient has a high fever, bloody diarrhea, or diarrhea lasting > 4–5 days. Send stool for fecal leukocytes, bacterial culture, C. difficile toxin, and O&P. Consider sigmoidoscopy in patients with bloody diarrhea. Acute diarrhea:

1	Send stool for fecal leukocytes, bacterial culture, C. difficile toxin, and O&P. Consider sigmoidoscopy in patients with bloody diarrhea. Acute diarrhea: When bacterial infection is not suspected, treat with antidiarrheals (e.g., loperamide, bismuth salicylate) and oral rehydration solutions. Acute diarrhea is generally infectious and self-limited. Organisms that cause bloody diarrhea include Salmonella, Shigella, E. coli, and Campylobacter. Diarrhea after ingestion of raw eggs or dairy: think Salmonella. T AB LE 2.6-2. Causes of Infectious Diarrhea

1	Campylobacter The most common etiology of infectious diarrhea. Ingestion of contaminated food or water. Affects young children and young adults. Generally lasts 7–10 days. Fecal RBCs and WBCs. Frequently bloody diarrhea. Rule out appendicitis and IBD. Erythromycin. Clostridium difficile Recent treatment with antibiotics (penicillins, cephalosporins, clindamycin). Affects hospitalized adult patients. Watch for toxic megacolon. Fever, abdominal pain, possible systemic toxicity. Fecal RBCs and WBCs. Most commonly causes colitis, but can involve the small bowel. Identify C. difficile toxin in the stool. Sigmoidoscopy shows pseudomembranes. Cessation of the inciting antibiotic. PO metronidazole or vancomycin; IV metronidazole if the patient cannot tolerate PO. Entamoeba histolytica Ingestion of contaminated food or water; history of travel in developing countries. Incubation period can last up to three months. Severe abdominal pain, fever. Fecal RBCs and WBCs. Chronic amebic colitis mimics

1	contaminated food or water; history of travel in developing countries. Incubation period can last up to three months. Severe abdominal pain, fever. Fecal RBCs and WBCs. Chronic amebic colitis mimics IBD. Steroids can lead to fatal perforation. Treat with metronidazole. E. coli O157:H7 Ingestion of contaminated food (raw meat). Affects children and the elderly. Generally lasts 5–10 days. Severe abdominal pain, low-grade fever, vomiting. Fecal RBCs and WBCs. It is important to rule out GI bleed and ischemic colitis. HUS is a possible complication. Avoid antibiotic or antidiarrheal therapy, which ↑ HUS risk. Ingestion of contaminated poultry or eggs. Affects young children and elderly patients. Generally lasts 2–5 days. Prodromal headache, fever, myalgia, abdominal pain. Fecal WBCs. Sepsis is a concern, as 5–10% of patients become bacteremic. Sickle cell patients are susceptible to invasive disease leading to osteomyelitis. Treat bacteremia or at-risk patients (e.g., sickle cell

1	Sepsis is a concern, as 5–10% of patients become bacteremic. Sickle cell patients are susceptible to invasive disease leading to osteomyelitis. Treat bacteremia or at-risk patients (e.g., sickle cell patients) with oral quinolone or TMP-SMX. Extremely contagious; transmitted between people by the fecal-oral route. Affects young children and institutionalized patients. Fecal RBCs and WBCs. May lead to severe dehydration. Can also cause febrile seizures in the very young. Treat with TMP-SMX to ↓ person-to-person spread. Salmonella Shigella

1	Exclude: 1. Causes of acute diarrhea 2. 3. 4. 5. 6. Fecal leukocytesand occult blood Flexible sigmoidoscopy with biopsy Upper GI series, barium enema HIGH-YIELD FACTS Inflammatory bowel disease Cancer Stool electrolytes, osmolality, weight/24 h, quantitative fat Increased osmotic gap Normal osmotic gap (Reproduced, with permission, from McPhee SJ et al. Current Medical Diagnosis & Treatment, 48th ed. New York: McGraw- Hill, 2009: Fig. 15-2.) If the patient has evidence of systemic infection (e.g., fever, chills, malaise), avoid antimotility agents and consider antibiotics after stool studies have been sent. Chronic diarrhea: Identify the underlying cause and treat symptoms with loperamide, opioids, octreotide, or cholestyramine. Pediatric diarrhea: For children who cannot take medication or PO ﬂ uids—hospitalize, give IV ﬂuids, replete electrolytes, and treat the underlying cause.

1	Pediatric diarrhea: For children who cannot take medication or PO ﬂ uids—hospitalize, give IV ﬂuids, replete electrolytes, and treat the underlying cause. ■Inability to absorb nutrients as a result of an underlying condition such as bile salt def ciency (e.g., bacterial overgrowth, ileal disease), short bowel syndrome, mucosal abnormalities (e.g., celiac disease, Whipple’s disease, tropical sprue), and pancreatic insuff ciency. The small bowel is most commonly involved. Cutaneous ﬂ ushing, diarrhea, wheezing, and cardiac valvular lesions are the most common manifestations of carcinoid tumors. Half of all patients with IBS have comorbid psychiatric disturbances. May result in ↓ absorption of protein, fat, carbohydrates, and micronutrients (folate/B12/iron). Hx/PE: Presents with frequent, loose, watery stools and/or pale, foul-smelling, bulky stools associated with abdominal pain, f atus, bloating, weight loss, nutritional def ciencies, and fatigue.

1	Hx/PE: Presents with frequent, loose, watery stools and/or pale, foul-smelling, bulky stools associated with abdominal pain, f atus, bloating, weight loss, nutritional def ciencies, and fatigue. Tx: Etiology dependent. Institute a gluten-free diet for patients with celiac sprue. Severely affected patients may receive TPN, immunosuppressants, and anti-inﬂ ammatory medications. Results from a defciency of lactase, a brush-border enzyme that hydrolyzes the disaccharide lactose into glucose and galactose. Lactase deficiency is common among populations of African, Asian, and Native American descent. It may also occur transiently 2° to an acute episode of gastroenteritis or other disorders affecting the proximal small intestinal mucosa. Hx/PE: Presents with abdominal bloating, f atulence, cramping, and watery diarrhea following milk ingestion.

1	Hx/PE: Presents with abdominal bloating, f atulence, cramping, and watery diarrhea following milk ingestion. Dx: Hydrogen breath test reveals ↑ breath hydrogen following ingestion of a lactose load (indicates metabolism of lactose by colonic bacteria). An empiric lactose-free diet that results in symptom resolution is highly suggestive of the diagnosis. Tx: Avoidance of dairy products; lactase enzyme replacement. Due to liver metastasis of carcinoid tumors (hormone-producing enterochromaffin cells) that most commonly arise from the ileum and appendix and produce vasoactive substances such as serotonin and substance P. Prior to metastasis, most secreted hormones undergo first-pass metabolism by the liver and do not reach systemic circulation. Hx/PE: Cutaneous fushing, diarrhea, abdominal cramps, wheezing, and right-sided cardiac valvular lesions are the most common manifestations. Symptoms usually follow eating, exertion, or excitement.

1	Hx/PE: Cutaneous fushing, diarrhea, abdominal cramps, wheezing, and right-sided cardiac valvular lesions are the most common manifestations. Symptoms usually follow eating, exertion, or excitement. Dx: High urine levels of the serotonin metabolite 5-HIAA are diagnostic. Octreotide scan can localize the tumor. Tx: Treatment includes octreotide (for symptoms) and debulking of tumor mass. An idiopathic functional disorder that is characterized by changes in bowel habits that ↑ with stress as well as by abdominal pain that is relieved by bowel movements. It is most common in the second and third decades, but since the syndrome is chronic, patients may present at any age. Half of all IBS patients who seek medical care have comorbid psychiatric disorders (e.g., depression, anxiety, fibromyalgia). ■Patients present with abdominal pain, a change in bowel habits (diarrhea and/or constipation), abdominal distention, mucous stools, and relief of pain with a bowel movement.

1	■Patients present with abdominal pain, a change in bowel habits (diarrhea and/or constipation), abdominal distention, mucous stools, and relief of pain with a bowel movement. IBS rarely awakens patients from sleep; vomiting, significant weight loss, and constitutional symptoms are also uncommon. Exam is usually unremarkable except for mild abdominal tenderness. A diagnosis of exclusion based on clinical history. Tests to rule out other GI causes include CBC, TSH, electrolytes, stool cultures, abdominal films, and barium contrast studies. Manometry can assess sphincter function. Psychological: Patients need reassurance from their physicians. They should not be told that their symptoms are “all in their head.” Dietary: Fiber supplements (psyllium) may help. Pharmacologic: Treat with TCAs, antidiarrheals (loperamide), and antispasmodics (anticholinergics such as dicyclomine).

1	Dietary: Fiber supplements (psyllium) may help. Pharmacologic: Treat with TCAs, antidiarrheals (loperamide), and antispasmodics (anticholinergics such as dicyclomine). Defined as blocked passage of bowel contents through the small bowel. Fluid and gas can build up proximal to the obstruction, leading to ﬂuid and electrolyte imbalances and significant abdominal discomfort. The obstruction can be complete or partial, and ischemia or necrosis of the bowel may occur. SBO may arise from adhesions from a prior abdominal surgery (60% of cases), hernias (10–20%), neoplasms (10–20%), intussusception, gallstone ileus, stricture due to IBD, or volvulus. Patients typically experience cramping abdominal pain with a recurrent crescendo-decrescendo pattern at 5to 10-minute intervals. Vomiting typically follows the pain; early emesis is bilious and nonfeculent if the obstruction is proximal but feculent if it is distal.

1	Vomiting typically follows the pain; early emesis is bilious and nonfeculent if the obstruction is proximal but feculent if it is distal. In partial obstruction, there is continued passage of ﬂatus but no stool, whereas in complete obstruction, no ﬂatus or stool is passed (obstipation). Abdominal exam often reveals distention, tenderness, prior surgical scars, or hernias. Bowel sounds are characterized by high-pitched tinkles and peristaltic rushes. Peristalsis may disappear later in disease progression. Fever, hypotension, rebound tenderness, and tachycardia suggest peritonitis, a surgical emergency. CBC may demonstrate leukocytosis if there is ischemia or necrosis of bowel. Labs often reﬂ ect dehydration and metabolic alkalosis due to vomiting. Lactic acidosis is particularly worrisome, as it suggests necrotic bowel and the need for emergent surgical intervention.

1	Labs often reﬂ ect dehydration and metabolic alkalosis due to vomiting. Lactic acidosis is particularly worrisome, as it suggests necrotic bowel and the need for emergent surgical intervention. Abdominal films often demonstrate a stepladder pattern of dilated small-bowel loops, air-f uid levels (see Figure 2.6-3), and a paucity of gas in the colon. The presence of radiopaque material at the cecum is suggestive of gallstone ileus. IBS is a diagnosis of exclusion. The leading cause of SBO in children is hernias. The leading cause of SBO in adults is adhesions. Never let the sun rise or set on a complete SBO. Anticholinergics, opioids, and hypokalemia slow GI motility. F IGU R E 2.6-3. Acute mechanical obstruction of the small intestine (upright film). Note the air-ﬂuid levels, marked distention of bowel loops, and absence of colonic gas. (Repro duced, with permission, from Kasper DL et al. Harrison’s Principles of Internal Medicine, 16th ed. New York: McGraw-Hill, 2005: 1804.)

1	For partial obstruction, supportive care may be sufficient and should include NPO status, NG suction, IV hydration, correction of electrolyte abnormalities, and Foley catheterization to monitor ﬂ uid status. Surgery is required in cases of complete SBO, vascular compromise (necrotic bowel), or symptoms lasting > 3 days without resolution. Exploratory laparotomy may be performed with lysis of adhesions, resection of necrotic bowel, and evaluation for stricture, IBD, and hernias. There is a 2% mortality risk for a nonstrangulated SBO; strangulated SBO is associated with up to a 25% mortality rate depending on the time between diagnosis and treatment. A second-look laparotomy or laparoscopy may be performed 18–36 hours after initial surgical treatment to reevaluate bowel viability if ischemia is a concern.

1	A second-look laparotomy or laparoscopy may be performed 18–36 hours after initial surgical treatment to reevaluate bowel viability if ischemia is a concern. Loss of peristalsis without structural obstruction. Risk factors include recent surgery/GI procedures, severe medical illness, immobility, hypokalemia or other electrolyte imbalances, hypothyroidism, DM, and medications that slow GI motility (e.g., anticholinergics, opioids). Presenting symptoms include diffuse, constant, moderate abdominal discomfort; nausea and vomiting (especially with eating); and an absence of fatulence or bowel movements. Exam may reveal diffuse tenderness and abdominal distention, no peritoneal signs, and ↓ or absent bowel sounds. A rectal exam is required to rule out fecal impaction in elderly patients. Diffusely distended loops of small and large bowel are seen on supine AXR with air-ﬂuid levels on upright view.

1	A rectal exam is required to rule out fecal impaction in elderly patients. Diffusely distended loops of small and large bowel are seen on supine AXR with air-ﬂuid levels on upright view. A Gastrografin study can rule out partial obstruction; CT can rule out neo-In diagnosing ileus, look for plasms. air throughout the small and large bowel on AXR. ■↓ or discontinue the use of narcotics and any other drugs that reduce bowel motility. Temporarily ↓ or discontinue oral feeds. Initiate NG suction/parenteral feeds as necessary. Replete electrolytes as needed. ↓ mesenteric blood supply leading to insufficient perfusion to intestinal tissue and ischemic injury. Causes include acute arterial occlusion (usually involving the SMA) from thrombosis (due to atherosclerosis) or embolism (due to atrial fibrillation or ↓ ejection fraction); nonocclusive arterial disease (low cardiac output, arteriolar vasospasm); and venous thrombosis (due to hypercoagulable states).

1	Patients present with sudden onset of severe abdominal pain out of proportion to the exam. A history of prior episodes of similar abdominal pain after eating (“intestinal angina”) may be present. Other symptoms may include nausea, vomiting, diarrhea, and bloody stools. Early abdominal exam is often unremarkable; later findings may include peritoneal signs (suggest bowel infarction). Lab tests may show leukocytosis, metabolic acidosis with ↑ lactate, ↑ amylase, ↑ LDH, and ↑ CK. AXR and CT may reveal bowel wall edema (“thumbprinting”) and air within the bowel wall (pneumatosis intestinalis). Mesenteric angiography is the gold standard for arterial occlusive disease. Volume resuscitation, broad-spectrum antibiotics, optimization of hemodynamics, and avoidance of vasoconstrictors. Anticoagulation for arterial or venous thrombosis or embolism. The mortality rate for acute mesenteric ischemia is > 50%.

1	Anticoagulation for arterial or venous thrombosis or embolism. The mortality rate for acute mesenteric ischemia is > 50%. Diverticulosis is the most common cause of acute lower GI bleeding in patients > 40 years of age. Diverticular disease must be distinguished from colon cancer with perforation. Avoid ﬂ exible sigmoidoscopy and barium enemas in the initial stages of diverticulitis because of perforation risk. Early laparotomy for acute arterial occlusive disease or if evidence of peritonitis or clinical deterioration is present. Angioplasty and thrombectomy +/– endovascular stenting for acute arterial thrombosis. Embolectomy for acute arterial embolism. Resection of infarcted bowel. Sepsis/septic shock, multisystem organ failure, death. See the Emergency Medicine chapter.

1	Embolectomy for acute arterial embolism. Resection of infarcted bowel. Sepsis/septic shock, multisystem organ failure, death. See the Emergency Medicine chapter. Outpouchings of mucosa and submucosa (false diverticula) that herniate through the colonic muscle layers in areas of high intraluminal pressure; most commonly found in the sigmoid colon. Diverticulosis is the most common cause of acute lower GI bleeding in patients > 40 years of age. Risk factors include a low-fber and high-fat diet, advanced age (65% occur in those > 80 years of age), and connective tissue disorders (e.g., Ehlers-Danlos syndrome). Diverticulitis is due to inﬂammation and, potentially, perforation of a diverticulum 2° to fecalith impaction. Diverticulosis is often asymptomatic. Bleeding is painless and sudden, generally presenting as hematochezia with symptoms of anemia (fatigue, lightheadedness, dyspnea on exertion).

1	Diverticulosis is often asymptomatic. Bleeding is painless and sudden, generally presenting as hematochezia with symptoms of anemia (fatigue, lightheadedness, dyspnea on exertion). Diverticulitis presents with LLQ abdominal pain, fever, nausea, vomiting, and constipation. Perforation is a serious complication that leads to peritonitis and shock. CBC may show leukocytosis. Diagnosis is based on AXR (to rule out free air, ileus, or obstruction), colonoscopy, or barium enema. Sigmoidoscopy/colonoscopy must be avoided in those with early diverticulitis due to the risk of perforation. In patients with severe disease or in those who show lack of improvement, abdominal CT may reveal abscess or free air. Uncomplicated diverticulosis: Patients can be followed and placed on a high-f ber diet or fiber supplements. Diverticular bleeding: Bleeding usually stops spontaneously; transfuse and hydrate as needed. If bleeding does not stop, angiography with embolization or surgery is indicated.

1	Diverticular bleeding: Bleeding usually stops spontaneously; transfuse and hydrate as needed. If bleeding does not stop, angiography with embolization or surgery is indicated. Diverticulitis: Treat with bowel rest (NPO), NG tube placement, and broad-spectrum antibiotics (metronidazole and a ﬂuoroquinolone or a secondor third-generation cephalosporin) if the patient is stable. Avoid barium enema and ﬂexible sigmoidoscopy if diverticulitis is suspected. ■For perforation, perform immediate surgical resection of diseased bowel via a Hartmann’s procedure with a temporary colostomy. Table 2.6-3 describes features that distinguish SBO from LBO. Figure 2.6-4 demonstrates the classic radiographic findings of LBO. The second leading cause of cancer mortality in the United States after lung cancer. There is an ↑ incidence with age, with a peak incidence at 70– 80 years. There has also been an observed association between colon cancer

1	T AB LE 2.6-3. Characteristics of Small and Large Bowel Obstruction

1	History Moderate to severe acute abdominal pain; copious emesis. Cramping pain with distal SBO. Fever, signs of dehydration, and hypotension may be seen. Constipation/obstipation, deep and cramping abdominal pain (less intense than SBO), nausea/ vomiting (less than SBO but more commonly feculent). PE Abdominal distention (distal SBO), abdominal tenderness, visible peristaltic waves, fever, hypovolemia. Look for surgical scars/hernias; perform a rectal exam. High-pitched “tinkly” bowel sounds; later, absence of bowel sounds. Signif cant distention, tympany, and tenderness; examine for peritoneal irritation or mass; fever or signs of shock suggest perforation/peritonitis or ischemia/necrosis. High-pitched “tinkly” bowel sounds; later, absence of bowel sounds. Etiologies Adhesions (postsurgery), hernias, neoplasm, volvulus, intussusception, gallstone ileus, foreign body, Crohn’s disease, CF, stricture, hematoma. Colon cancer, diverticulitis, volvulus, fecal impaction, benign tumors.

1	hernias, neoplasm, volvulus, intussusception, gallstone ileus, foreign body, Crohn’s disease, CF, stricture, hematoma. Colon cancer, diverticulitis, volvulus, fecal impaction, benign tumors. Assume colon cancer until proven otherwise. Differential LBO, paralytic ileus, gastroenteritis. SBO, paralytic ileus, appendicitis, IBD, Ogilvie’s syndrome (pseudo-obstruction). CBC, electrolytes, lactic acid, AXR (see Figure 2.6-3); contrast studies (determine if it is partial or complete), CT scan. CBC, electrolytes, lactic acid, AXR (see Figure 2.6-4), CT scan; water contrast enema (if perforation is suspected); sigmoidoscopy/colonoscopy if stable.

1	Hospitalize. Partial SBO can be treated conservatively with NG decompression and NPO status. Patients with complete SBO should be managed aggressively with NPO status, NG decompression, IV fuids, electrolyte replacement, and surgical correction. Hospitalize. Obstruction can be relieved with a Gastrografn enema, colonoscopy, or a rectal tube; however, surgery is usually required. Ischemic colon usually requires partial colectomy with a diverting colostomy. Treat the underlying cause (e.g., neoplasm). TABLE 2.6-4. FIGURE 2.6-4. Large bowel obstruction. Barium study shows the “bird-beak” sign, with juxtaposed adjacent bowel walls in the dilated loop pointing toward the site of obstruction. (Reproduced, with permission, from Way LW. Current Surgical Diagnosis & Treatment, 10th ed. Stamford, CT: Appleton & Lange, 1994: 676.) and Streptococcus bovis bacteremia. Risk factors and screening protocols are summarized in Table 2.6-4.

1	■In the absence of screening, colon and rectal cancer typically present with symptoms only after a prolonged period of silent growth. Age. Hereditary syndromes—familial adenomatous polyposis (100% risk by age 40), Gardner’s disease, hereditary nonpolyposis colorectal cancer (HNPCC). Family history. IBD—ulcerative colitis carries a higher risk than does Crohn’s disease. Adenomatous polyps—villous polyps progress more often than tubular polyps and sessile more than pedunculated polyps. Lesions > 2 cm carry an ↑ risk. Past history of colorectal cancer. High-fat, low-f ber diet. A DRE should be performed yearly for patients ≥ 50 years of age. Up to 10% of all lesions are palpable with DRE. Stool guaiac should be performed every year for patients ≥ 50 years of age. Up to 50% of guaiac tests are due to colorectal cancer. Colonoscopy every 10 years in those ≥ 50 years of age OR yearly FOBT with sigmoidoscopy every 5 years.

1	Colonoscopy every 10 years in those ≥ 50 years of age OR yearly FOBT with sigmoidoscopy every 5 years. Colonoscopy should be performed every 10 years in patients ≥ 40 years of age who have a frst-degree relative with colorectal cancer or adenomatous polyps, or 10 years prior to the age at diagnosis of the youngest family member. Although often asymptomatic, patients may present with unexplained anemia or vague abdominal pain. Other features depend on location: Right-sided lesions: Often bulky, ulcerating masses that lead to anemia from chronic occult blood loss. Patients may complain of weight loss, anorexia, diarrhea, weakness, or vague abdominal pain. Obstruction is rare. Left-sided lesions: Typically “apple-core” obstructing masses (see Figure 2.6-5). Patients complain of a change in bowel habits (e.g., ↓ stool caliber, constipation, obstipation), colicky abdominal pain, and/or blood-streaked stools. Obstruction is common.

1	Rectal lesions: Usually present with bright red blood per rectum, often with tenesmus and/or rectal pain. Can coexist with hemorrhoids, so rectal cancer must be ruled out in all patients with rectal bleeding. Order a CBC (often shows microcytic anemia) and stool occult blood. Perform sigmoidoscopy to evaluate rectal bleeding and all suspicious left-sided lesions. Rule out synchronous right-sided lesions with colonoscopy. If colonoscopy is incomplete, rule out additional lesions with an air-contrast barium enema. Determine the degree of invasion in rectal cancer with endorectal ultrasound. Order a CXR, LFTs, and an abdominal/pelvic CT for metastatic workup. Metastases may arise from direct extension to local viscera, hematogenous spread (40–50% go to the liver, but spread may also occur to bone, lungs, and brain), or lymphatic spread (to pelvic lymph nodes). Iron deficiency anemia in an elderly male is colorectal cancer until proven otherwise. FIGURE 2.6-5. Colon carcinoma.

1	Iron deficiency anemia in an elderly male is colorectal cancer until proven otherwise. FIGURE 2.6-5. Colon carcinoma. The encircling carcinoma appears as an “apple-core” filling defect in the descending colon on barium enema x-ray. (Reproduced, with permission, from Way LW. Current Surgical Diagnosis & Treatment, 10th ed. Stamford, CT: Appleton & Lange, 1994: 658.) ■Staging is based on the depth of tumor penetration into the bowel wall and the presence of lymph node involvement and distant metastases. Ischemic colitis is a known complication of AAA repair due to sacrifice of the inferior mesenteric artery. One unit of PRBCs should ↑ hemoglobin by 1 unit and hematocrit by 3–4 units. Surgical resection of the 1° cancer is the treatment of choice. Regional lymph node dissection should be performed for staging purposes. For rectal lesions, the resection technique depends on the proximity of the lesion to the anal verge (the junction between the anal canal and the anal skin).

1	For rectal lesions, the resection technique depends on the proximity of the lesion to the anal verge (the junction between the anal canal and the anal skin). Abdominoperineal resection: For distal lesions < 10 cm from the anal verge, when the sphincter cannot be preserved, the rectum and anus are resected and a permanent colostomy is placed. Low anterior resection: For proximal lesions > 10 cm from the anal verge, a 1° anastomosis is created between the colon and rectum. Wide local excision: For small, low-grade, well-differentiated tumors in the lower third of the rectum. Adjuvant chemotherapy: Used in cases of colon cancer with lymph nodes. Can be considered for rectal cancers. Follow with serial CEA levels (diagnostically nonspecific, but useful for monitoring recurrence), colonoscopy, LFTs, CXR, and abdominal CT (for metastasis). Due to lack of arterial blood supply to the colon. Severity ranges from superficial mucosal involvement to full-thickness necrosis.

1	Due to lack of arterial blood supply to the colon. Severity ranges from superficial mucosal involvement to full-thickness necrosis. The most commonly affected site is the left colon, particularly the “watershed area” at the splenic ﬂ exure. Incidence ↑ with age. Hx/PE: Presents with crampy lower abdominal pain associated with bloody diarrhea. Fever and peritoneal signs suggest infarction. Dx: CBC may reveal leukocytosis. Flexible sigmoidoscopy or colonoscopy to assess colonic mucosa. Tx: Supportive therapy with bowel rest, IV ﬂuids, and broad-spectrum antibiotics. Surgery with resection is indicated for infarction, fulminant colitis, or obstruction 2° to ischemic stricture. Bleeding from the GI tract may present as hematemesis, hematochezia, and/or melena. Upper GI tract bleeding is defined as bleeding from lesions proximal to the ligament of Treitz (the anatomic boundary between the duodenum and jejunum). Table 2.6-5 presents the features of upper and lower GI bleeding.

1	Comprises Crohn’s disease and ulcerative colitis (see Figure 2.6-6). Most common in Caucasians and Ashkenazi Jews, appearing most frequently during the teens to early 30s or in the 50s. Table 2.6-6 summarizes the features of IBD. T AB LE 2.6-5. Features of Upper and Lower GI Bleeding

1	HIGH-YIELD FACTS History/PE Hematemesis (”coffee-ground” emesis), melena > hematochezia, depleted volume status (e.g., tachycardia, lightheadedness, hypotension). Hematochezia > melena, but can be either. Diagnosis NG tube and NG lavage; endoscopy if stable. Rule out upper GI bleed with NG lavage. Anoscopy/sigmoidoscopy for patients < 45 years of age with small-volume bleeding. Colonoscopy if stable; arteriography or exploratory laparotomy if unstable. Etiologies PUD, esophagitis/gastritis, Mallory-Weiss tear, esophageal varices. Diverticulosis (60%), IBD, hemorrhoids/f ssures, neoplasm, AVM. Initial management Protect the airway (may need intubation). Stabilize the patient with IV fuids and PRBCs (hematocrit may be normal early in acute blood loss). Similar to upper GI bleed. Long-term management Endoscopy followed by therapy directed at the underlying cause (e.g., high-dose PPIs for PUD; octreotide and/or banding for varices). Depends on the underlying etiology. Endoscopic therapy

1	management Endoscopy followed by therapy directed at the underlying cause (e.g., high-dose PPIs for PUD; octreotide and/or banding for varices). Depends on the underlying etiology. Endoscopic therapy (e.g., epinephrine injection), intra-arterial vasopressin infusion or embolization, or surgery for diverticular disease or angiodysplasia.

1	F IGU R E 2.6-6. Inﬂammatory bowel disease. (A) Crohn’s disease. Barium enema x-ray reveals deep transverse fissures, ulcers, and edema of the bowel. (B) Ulcerative colitis. Barium enema x-ray demonstrates shortening of the colon, loss of haustra (“lead pipe” appearance), and fine serrations of the bowel edges from small ulcers. (Reproduced, with permission, from Stobo J et al. The Principles and Practice of Medicine, 23rd ed. Stamford, CT: Appleton & Lange, 1996: 135.) T AB LE 2.6-6. Features of Ulcerative Colitis and Crohn’s Disease

1	Site of involvement The rectum is always involved. May extend proximally in a continuous fashion. Infammation and ulceration are limited to the mucosa and submucosa. May involve any portion of the GI tract, particularly the ileocecal region, in a discontinuous pattern (“skip lesions”). The rectum is often spared. Transmural inﬂ ammation is seen. History/PE Bloody diarrhea, lower abdominal cramps, tenesmus, urgency. Exam may reveal orthostatic hypotension, tachycardia, abdominal tenderness, frank blood on rectal exam, and extraintestinal manifestations. Abdominal pain, abdominal mass, low-grade fever, weight loss, watery diarrhea. Exam may reveal fever, abdominal tenderness or mass, perianal fissures, fistulas, and extraintestinal manifestations. Extraintestinal manifestations Aphthous stomatitis, episcleritis/uveitis, arthritis, primary sclerosing cholangitis, erythema nodosum, and pyoderma gangrenosum. The same as ulcerative colitis, as well as gallstones, nephrolithiasis, and

1	stomatitis, episcleritis/uveitis, arthritis, primary sclerosing cholangitis, erythema nodosum, and pyoderma gangrenosum. The same as ulcerative colitis, as well as gallstones, nephrolithiasis, and fstulas to the skin, bladder, or between bowel loops. Diagnosis CBC, AXR, stool cultures, O&P, stool assay for The same lab workup as ulcerative colitis. Upper GI

1	C. difficile. Colonoscopy can show diffuse and continuous rectal involvement, friability, edema, and pseudopolyps. Defnitive diagnosis can be made with biopsy. 5-ASA agents (e.g., sulfasalazine, mesalamine), topical or oral; corticosteroids and immunomodulating agents (e.g., azathioprine) for refractory disease. Total proctocolectomy is curative for longstanding or fulminant colitis or toxic megacolon; it also ↓ cancer risk. Markedly ↑ risk of colorectal cancer in longstanding cases (monitor with frequent fecal occult blood screening and yearly colonoscopy with multiple biopsies after eight years of disease). series with small bowel follow-through. Colonoscopy may show aphthoid, linear, or stellate ulcers, strictures, “cobblestoning,” and “skip lesions.” “Creeping fat” may also be present during laparotomy. Defnitive diagnosis can be made with biopsy.

1	Defnitive diagnosis can be made with biopsy. 5-ASA agents; corticosteroids and immunomodulating agents (e.g., azathioprine, infiximab) are indicated if no improvement is seen. Surgical resection may be necessary for suspected perforation, stricture, fstula, or abscess; may recur anywhere in the GI tract. Incidence of 2° malignancy is lower than in ulcerative colitis, but greater than the general population. Abnormal protrusions of abdominal contents (usually the small intestine) into the inguinal region through a weakness or defect in the abdominal wall. Defined as direct or indirect on the basis of their relationship to the inguinal canal. ■ Indirect: Herniation of abdominal contents through the internal and then external inguinal rings and eventually into the scrotum (in males). The most common hernia in both genders. Due to a congenital patent processus vaginalis. Protrudes lateral to the inferior epigastric vessels.

1	The most common hernia in both genders. Due to a congenital patent processus vaginalis. Protrudes lateral to the inferior epigastric vessels. Direct: Herniation of abdominal contents through the ﬂ oor of Hesselbach’s triangle. Protrudes medial to the epigastric vessels. Hernial sac contents do not traverse the internal inguinal ring; they herniate directly through the abdominal wall and are contained within the aponeurosis of the external oblique muscle. Most often due to an acquired defect in the transversalis fascia from mechanical breakdown that ↑ with age. Because of the risk of incarceration and strangulation, surgical management (open or laparoscopic) is indicated unless specific contraindications are present. Repair of a direct inguinal hernia involves correcting the defect in the transversalis fascia.

1	Repair of a direct inguinal hernia involves correcting the defect in the transversalis fascia. Indirect inguinal hernias are repaired by isolating and ligating the hernial sac and reducing the size of the internal inguinal ring to allow only the spermatic cord structures in males to pass through. Hesselbach’s triangle is an area bounded by the inguinal ligament, the inferior epigastric artery, and the rectus abdominis. Colic results from transient cystic duct blockage from impacted stones. Although risk factors include the 4 F’s—Female, Fat, Fertile, and Forty—the disorder is common and can occur in any patient. Flatulence can be thought of as a “5th F.” Other risk factors include OCP use, rapid weight loss, a family history, chronic hemolysis (pigment stones in sickle cell disease), small bowel resection, and TPN. Patients present with postprandial abdominal pain (usually in the RUQ) that radiates to the right subscapular area or the epigastrium.

1	Patients present with postprandial abdominal pain (usually in the RUQ) that radiates to the right subscapular area or the epigastrium. Pain is abrupt; is followed by gradual relief; and is often associated with nausea and vomiting, fatty food intolerance, dyspepsia, and ﬂ atulence. Gallstones may be asymptomatic in up to 80% of patients. Exam may reveal RUQ tenderness and a palpable gallbladder. Plain x-rays are rarely diagnostic; only 10–15% of stones are radiopaque. RUQ ultrasound is the imaging modality of choice (85–90% sensitive). Table 2.6-7 contrasts lab findings with those of other forms of biliary disease. Cholecystectomy is curative and can be performed electively for symptomatic gallstones. It is generally performed laparoscopically. Asymptomatic gallstones do not require any intervention. Patients may require preoperative endoscopic retrograde cholangiopancreatography (ERCP) for common bile duct stones.

1	Patients may require preoperative endoscopic retrograde cholangiopancreatography (ERCP) for common bile duct stones. Treat nonsurgical candidates with dietary modif cation (avoid triggers such as fatty foods). Pigmented gallstones result from hemolysis (black) or infection (brown). Most gallstones are precipitations of cholesterol and are not radiopaque. TAB LE 2.6-7. Differential Diagnosis of Biliary Disease The cystic artery usually passes through the anatomic triangle of Calot, comprising the common hepatic duct, the cystic duct, and the inferior border of the liver. In patients with significant medical problems (including DM), delay cholecystectomy until acute inﬂ ammation resolves. Recurrent biliary colic, acute cholecystitis, choledocholithiasis, ascending cholangitis, gallstone ileus, gallstone pancreatitis.

1	Recurrent biliary colic, acute cholecystitis, choledocholithiasis, ascending cholangitis, gallstone ileus, gallstone pancreatitis. Prolonged blockage of the cystic duct, usually by an impacted stone, that leads to obstructive distention, inﬂammation, superinfection, and possibly gangrene of the gallbladder (acute gangrenous cholecystitis). Acalculous cholecystitis occurs in the absence of cholelithiasis in patients who are chronically debilitated, those who are critically ill in the ICU or on TPN, and trauma or burn victims. Patients present with RUQ pain, nausea, vomiting, and fever. Symptoms are typically more severe and of longer duration than those of biliary colic. RUQ tenderness, inspiratory arrest during deep palpation of the RUQ (Murphy’s sign), low-grade fever, mild icterus, and possibly guarding or rebound tenderness may be present on exam. Fever is often present, and CBC shows leukocytosis (see Table 2.6-7).

1	Fever is often present, and CBC shows leukocytosis (see Table 2.6-7). Ultrasound may demonstrate stones, bile sludge, pericholecystic ﬂ uid, a thickened gallbladder wall, gas in the gallbladder, and an ultrasonic Murphy’s sign (see Figure 2.6-7). Obtain a HIDA scan when ultrasound is equivocal (see Figure 2.6-8); nonvisualization of the gallbladder on HIDA scan suggests acute cholecystitis. Hospitalize patients, administer broad-spectrum IV antibiotics and IV f u-ids, and replete electrolytes. If diagnosed soon after onset, early cholecystectomy is indicated. For stable patients or those with significant medical problems, surgery can be delayed for 4–6 weeks. F IGU R E 2.6-7. Acute cholecystitis, ultrasound. Note the sludge-filled, thick-walled gallbladder with a hyperechoic stone and acoustic shadow (arrow). (B) This patient exhibits sludge and pericholecystic ﬂuid (arrow) but no gallstones. (Reproduced, with permission, from Grendell J.

1	J. Current Diagnosis & Treatment in Gastroenterology, 1st ed. Stamford, CT: Appleton & Lange, 1996: 212.) Gangrene, empyema, perforation, emphysematous gallbladder (due to infection by gas-forming organisms), fistulization, gallstone ileus, sepsis, abscess formation. ■Gallstones in the common bile duct. Symptoms vary according to the degree of obstruction, the duration of the obstruction, and the extent of bacterial infection. F IGU R E 2.6-8. Acute cholecystitis, HIDA scan. IV dye is taken up by hepatocytes and is conjugated and excreted into the common bile duct. The gallbladder is not visualized, although activity is present in the liver, common duct, and small bowel, suggesting cystic duct obstruction due to acute cholecystitis. (Reproduced, with permission, from Grendell J. Current Diagnosis & Treatment in Gastroenterology, 1st ed. Stamford, CT: Appleton & Lange, 1996: 217.) Gallstone pancreatitis occurs when stones in the ampulla also obstruct the pancreatic duct.

1	Gallstone pancreatitis occurs when stones in the ampulla also obstruct the pancreatic duct. Charcot’s triad consists of RUQ pain, jaundice, and fever/chills. Reynolds’ pentad consists of RUQ pain, jaundice, fever/chills, shock, and altered mental status. Hx/PE: Although sometimes asymptomatic, it often presents with biliary colic, jaundice, fever, and pancreatitis. Dx: The hallmark is ↑ alkaline phosphatase and total bilirubin, which may be the only abnormal lab values (see Table 2.6-7). Tx: Management generally consists of ERCP with sphincterotomy followed by semielective cholecystectomy. Common bile duct exploration may be necessary.

1	Tx: Management generally consists of ERCP with sphincterotomy followed by semielective cholecystectomy. Common bile duct exploration may be necessary. An acute bacterial infection of the biliary tree that commonly occurs 2° to obstruction, usually from gallstones (choledocholithiasis) or primary sclerosing cholangitis (progressive inﬂammation of the biliary tree associated with ulcerative colitis). Other etiologies include bile duct stricture and malignancy (biliary or pancreatic). Gram-enterics (e.g., E. coli, Enterobacter, Pseudomonas) are commonly identified pathogens. Charcot’s triad—RUQ pain, jaundice, and fever/chills—is classic and seen in 50–70% of cases. Reynolds’ pentad—Charcot’s triad plus septic shock and altered mental status—may be present in acute suppurative cholangitis and suggests sepsis. Look for leukocytosis, ↑ bilirubin, and ↑ alkaline phosphatase (see Table 2.6-7).

1	Look for leukocytosis, ↑ bilirubin, and ↑ alkaline phosphatase (see Table 2.6-7). Obtain blood cultures to rule out sepsis. Ultrasound or CT may be a useful adjunct, but diagnosis is often clinical. Magnetic resonance cholangiopancreatography (MRCP) is a noninvasive test that may also be useful. ERCP is both diagnostic and therapeutic (biliary drainage). Patients often require ICU admission for monitoring, hydration, BP support, and broad-spectrum IV antibiotic treatment. Patients with acute suppurative cholangitis require emergent bile duct decompression via ERCP/sphincterotomy, percutaneous transhepatic drainage, or open decompression. A mechanical obstruction resulting from the passage of a large (> 2.5-cm) stone into the bowel through a cholecystoduodenal fistula. Obstruction is often at the terminal ileum (TI)/ileocecal valve.

1	A mechanical obstruction resulting from the passage of a large (> 2.5-cm) stone into the bowel through a cholecystoduodenal fistula. Obstruction is often at the terminal ileum (TI)/ileocecal valve. Hx/PE: Many patients have no previous history of biliary symptoms and present as an SBO. The classic presentation is that of a subacute SBO in an elderly woman. Dx: Pneumobilia (gas in the biliary tree) seen on imaging can confirm, in addition to an upper GI series with small bowel follow-through showing a TI obstruction. Tx: Laparotomy with stone extraction (enterolithotomy) or manipulation into the colon. ■An idiopathic disorder characterized by inﬂ ammation, fibrosis, and strictures of extraand intrahepatic bile ducts. The disease usually presents in young men with IBD (most often ulcerative colitis). Hx/PE: Presents with progressive jaundice, pruritus, and fatigue. Dx: is significantly associated with ■ Laboratory findings include ↑ alkaline phosphatase and ↑ bilirubin.

1	Hx/PE: Presents with progressive jaundice, pruritus, and fatigue. Dx: is significantly associated with ■ Laboratory findings include ↑ alkaline phosphatase and ↑ bilirubin. ulcerative colitis. MRCP/ERCP show multiple bile duct strictures with dilatations between strictures. Liver biopsy reveals periductal sclerosis (“onion skinning”). Tx: High-dose ursodeoxycholic acid; endoscopic dilation and short-term stenting of bile duct strictures; liver transplantation. Patients are at ↑ risk for cholangiocarcinoma. Liver diseases can be divided into distinct patterns based on LFT results: Hepatocellular injury: ↑ AST and ALT. Cholestasis: ↑ alkaline phosphatase and bilirubin. Isolated hyperbilirubinemia: ↑ bilirubin; normal aminotransferases and alkaline phosphatase.

1	Hepatocellular injury: ↑ AST and ALT. Cholestasis: ↑ alkaline phosphatase and bilirubin. Isolated hyperbilirubinemia: ↑ bilirubin; normal aminotransferases and alkaline phosphatase. Jaundice, which can be seen in any of the patterns outlined above, is a clinical sign that arises when excess bilirubin (> 2.5 mg/dL) is circulating in the blood. Figures 2.6-9 and 2.6-10 summarize the clinical approaches toward cholestasis and isolated hyperbilirubinemia. Hepatocellular injury is described in the section that follows. Inﬂammation of the liver leading to liver cell injury and necrosis. The causes of acute hepatitis include viruses (e.g., HAV, HBV, HCV, HDV, HEV) and drug-induced disease (e.g., alcohol, acetaminophen, INH, methyldopa). The Ductal dilation? FIGURE 2.6-9. Approach to cholestasis. HCV is Chronic. Eighty percent of patients with HCV infection will develop chronic hepatitis. An AST/ALT ratio > 2 suggests alcoholic hepatitis—you’re toASTed.

1	HCV is Chronic. Eighty percent of patients with HCV infection will develop chronic hepatitis. An AST/ALT ratio > 2 suggests alcoholic hepatitis—you’re toASTed. causes of chronic hepatitis include viruses (e.g., HBV, HCV, HDV), alcoholic hepatitis, autoimmune hepatitis, ischemic hepatitis, and hereditary etiologies (e.g., Wilson’s disease, hemochromatosis, α1-antitrypsin deficiency). HAV and HEV are transmitted by the fecal-oral route. HBV and HCV are transmitted by bodily ﬂuids, although the risk of acquiring HCV sexually is very low. Acute hepatitis often starts with a viral prodrome of nonspecific symptoms (e.g., malaise, fever, joint pain, fatigue, URI symptoms, nausea, vomiting, changes in bowel habits) followed by jaundice and RUQ tenderness. Exam often reveals jaundice, scleral icterus, tender hepatomegaly, possible splenomegaly, and lymphadenopathy.

1	Exam often reveals jaundice, scleral icterus, tender hepatomegaly, possible splenomegaly, and lymphadenopathy. Chronic hepatitis usually gives rise to symptoms indicative of chronic liver disease (jaundice, fatigue, hepatosplenomegaly). At least 80% of those infected with HCV and 10% of those with HBV will develop chronic hepatitis. Dramatically ↑ ALT and AST and ↑ bilirubin/alkaline phosphatase are present in the acute form. In chronic hepatitis, ALT and AST are ↑ for > 6 months with a concurrent ↑ in alkaline phosphatase/bilirubin and hypoalbuminemia. In severe cases, PT will be prolonged, as all clotting factors except factor VIII are produced by the liver. The diagnosis of viral hepatitis is made by hepatitis serology (see Table 2.6-8 and Figure 2.6-11 for a description and timing of serologic markers) and by liver biopsy in chronic or severe cases.

1	The diagnosis of viral hepatitis is made by hepatitis serology (see Table 2.6-8 and Figure 2.6-11 for a description and timing of serologic markers) and by liver biopsy in chronic or severe cases. ANA, anti–smooth muscle antibody, and antimitochondrial antibody point to autoimmune hepatitis. Iron saturation (hemochromatosis) and ceruloplasmin (Wilson’s disease) can identify other causes. Treatment is etiology specific; monitor for resolution of symptoms over time. Steroids for severe alcoholic hepatitis. Immunosuppression with steroids and other agents (azathioprine) for autoimmune hepatitis. T AB LE 2.6-8. Key Hepatitis Serologic Markers

1	Steroids for severe alcoholic hepatitis. Immunosuppression with steroids and other agents (azathioprine) for autoimmune hepatitis. T AB LE 2.6-8. Key Hepatitis Serologic Markers HBsAg Antigen found on the surface of HBV; continued presence indicates carrier state. HBsAb Antibody to HBsAg; provides immunity to HBV. HBcAg Antigen associated with core of HBV. HBcAb Antibody to HBcAg; during the window period. IgM HBcAb is an indicator of recent disease. HBeAg A second, different antigenic determinant in the HBV core. An important indicator of transmissibility (BEware!). IgM HAVAb IgM antibody to HAV; the best test to detect active hepatitis A. HBeAb Antibody to e antigen; indicates low transmissibility. IFN-α, lamivudine (3TC), or adefovir for chronic HBV infection; peginterferon and ribavirin for chronic HCV infection. Liver transplantation is the treatment of choice for patients with end-stage liver failure.

1	Liver transplantation is the treatment of choice for patients with end-stage liver failure. ICU management and emergent transplant for fulminant hepatic failure. Important diagnostic tests Incubation Prodrome, The sequelae of chronic hepatitis include cirrhosis, liver failure, and hepatocellular carcinoma. Level of detection F IGU R E 2.6-1 1. Time course of hepatitis B with serologic markers. Alcoholism, chronic hepatitis, and other chronic liver diseases lead to cirrhosis. Gut, butt, and caput—the three anastomoses commonly seen in cirrhosis. Cirrhosis, liver failure, hepatocellular carcinoma (3–5%). Defined as f brosis and nodular regeneration resulting from hepatocellular injury. Etiologies include causes of chronic hepatitis, biliary tract disease (e.g., primary biliary cirrhosis, primary sclerosing cholangitis), right-sided heart failure, constrictive pericarditis, and Budd-Chiari syndrome (hepatic vein thrombosis 2° to hypercoagulability).

1	Presents with jaundice, ascites, spontaneous bacterial peritonitis, hepatic encephalopathy (e.g., asterixis, altered mental status), gastroesophageal varices, coagulopathy, and renal dysfunction. Weakness, anorexia, and weight loss are also seen in advanced disease. Exam may reveal an enlarged, palpable, or firm liver. Stigmata of portal hypertension and signs of liver failure may be present (see Figures 2.6-12 and 2.6-13). Lab studies show ↓ albumin, ↑ PT/PTT, and ↑ bilirubin. Anemia or thrombocytopenia (2° to hypersplenism) may also be seen. Abdominal ultrasound with Doppler can assess liver size, the presence of ascites, and the patency of splenic and hepatic veins. The etiology of ascites can be established through measurement of the serum-ascites albumin gradient (SAAG = serum albumin – ascites albumin); see Table 2.6-9. Obtain hepatitis serologies and autoimmune hepatitis studies.

1	Obtain hepatitis serologies and autoimmune hepatitis studies. Serum ferritin, ceruloplasmin, and α1-antitrypsin may help identify additional causes, such as hemochromatosis, Wilson’s disease, and α1antitrypsin deficiency, respectively. Liver biopsy showing bridging fibrosis and nodular regeneration. Effects of portal hypertension Effects of liver cell failure Loss of sexual hair F IGU R E 2.6-1 2. Presentation of cirrhosis/portal hypertension. (Adapted, with permission, from Chandrasoma P, Taylor CE. Concise Pathology, 3rd ed. Stamford, CT: Appleton & Lange, 1998: 654.) Superior mesenteric v. Gastroesophageal vv. Superior-middle/inferior rectal vv. Gastrorenal-splenorenal vv. Paraumbilical v. IVC Systemic venousPortal venousPortal v. L. gastric v. Retro-peritoneal paravertebral v Splenic v. 1 2 3 4 5 v. F IGU R E 2.6-1 3. Portosystemic anastomoses. 1. Left gastric–azygos → esophageal varices. 2. Superior–middle/inferior rectal → hemorrhoids.

1	F IGU R E 2.6-1 3. Portosystemic anastomoses. 1. Left gastric–azygos → esophageal varices. 2. Superior–middle/inferior rectal → hemorrhoids. 3. Paraumbilical–inferior epigastric → caput medusae (navel). 4. Gastrorenal-splenorenal. 5. Retroperitoneal paravertebral. Aimed at ameliorating the complications of cirrhosis/portal hypertension. Ascites: Sodium restriction and diuretics (furosemide and spironolactone). Noncirrhotic causes of Rule out infectious and neoplastic causes; perform paracentesis to ob- tain SAAG, cell count with differential, and cultures. If possible, treat underlying liver disease. right heart failure, splenic Spontaneous bacterial peritonitis: vein thrombosis, and Presents with fever, abdominal pain, and altered mental status. schistosomiasis. Check peritoneal ﬂuid if there is a possibility of infection. The ﬂ uid is if there are > 250 PMNs/mL or > 500 WBCs.

1	schistosomiasis. Check peritoneal ﬂuid if there is a possibility of infection. The ﬂ uid is if there are > 250 PMNs/mL or > 500 WBCs. Treat with IV antibiotics (e.g., third-generation cephalosporin) to cover both gram-(Enterococcus) and gram-(E. coli, Klebsiella) organisms until a causative organism is identified. T AB LE 2.6-9. Serum-Ascites Albumin Gradient SAAG > 1.1 SAAG < 1.1 Ascites is due to protein leakage: ■Nephrotic syndrome ■Tuberculosis ■Malignancy (e.g., ovarian cancer) Ascites is related to portal hypertension: Presinusoidal: Splenic or portal vein thrombosis, schistosomiasis Sinusoidal: Cirrhosis, massive hepatic metastases Postsinusoidal: Right heart failure, constrictive pericarditis, Budd-Chiari syndrome Spontaneous bacterial peritonitis is diagnosed by > 250 PMNs/mL or > 500 WBCs in the ascitic ﬂ uid. Primary biliary cirrhosis is an autoimmune disease that presents with jaundice and pruritus in middle-aged women.

1	Primary biliary cirrhosis is an autoimmune disease that presents with jaundice and pruritus in middle-aged women. Complications of hepatocellular carcinoma include GI bleeding, liver failure, and metastasis. Hepatorenal syndrome: A diagnosis of exclusion; difficult to treat and often requires dialysis. Hepatic encephalopathy: Due to ↓ clearance of ammonia. Often precipitated by dehydration, infection, electrolyte abnormalities, and GI bleeding. Treat with dietary protein restriction, lactulose, and rifaximin. Esophageal varices: Monitor for GI bleeding; treat medically (β-blockers, octreotide), endoscopically (band ligation), or surgically (portocaval shunt). Consider liver transplantation for patients with advanced disease. An autoimmune disorder characterized by destruction of intrahepatic bile ducts. The disease most commonly presents in middle-aged women with other autoimmune conditions.

1	An autoimmune disorder characterized by destruction of intrahepatic bile ducts. The disease most commonly presents in middle-aged women with other autoimmune conditions. Hx/PE: Presents with progressive jaundice, pruritus, and malabsorption of the fat-soluble vitamins (A, D, E, K). Dx: Laboratory findings include ↑ alkaline phosphatase, ↑ bilirubin, antimitochondrial antibody, and ↑ cholesterol. Tx: Ursodeoxycholic acid (slows progression of disease); cholestyramine for pruritus; liver transplantation. One of the most common cancers worldwide despite its relatively low incidence in the United States. 1° risk factors for the development of hepatocellular carcinoma in the United States are cirrhosis and chronic hepatitis (HCV). In developing countries, af atoxins (in various food sources) and HBV infection are also major risk factors.

1	Patients commonly present with RUQ tenderness, abdominal distention, and signs of chronic liver disease such as jaundice, easy bruisability, and coagulopathy. Cachexia and weakness may be present. Exam may reveal tender enlargement of the liver. Often suggested by the presence of a mass on ultrasound or CT as well as by abnormal LFTs and significantly elevated α-fetoprotein (AFP) levels. Liver biopsy for definitive diagnosis. For small tumors that are detected early, aggressive tumor resection or orthotopic liver transplantation may be successful. Chemotherapy and radiation are generally not effective, although they may be used to shrink large tumors prior to surgery (neoadjuvant therapy). Monitor tumor recurrence with serial AFP levels. Prevent exposure to hepatic carcinogens and vaccinate against hepatitis in high-risk individuals.

1	Monitor tumor recurrence with serial AFP levels. Prevent exposure to hepatic carcinogens and vaccinate against hepatitis in high-risk individuals. Caused by hyperabsorption of iron with parenchymal hemosiderin accumulation in the liver, pancreas, heart, adrenals, testes, pituitary, and kidneys. It is an autosomal-recessive disease that usually occurs in males of northern European descent and is rarely recognized before the fifth decade. 2° hemochromatosis may occur with iron overload and is common in patients receiving chronic transfusion therapy (e.g., for α-thalassemia) as well as in alcoholics (alcohol ↑ iron absorption). Patients may present with abdominal pain or symptoms of DM, hypogonadism, arthropathy of the MCP joints, heart failure, or cirrhosis. Exam may reveal bronze skin pigmentation, pancreatic dysfunction, cardiac dysfunction (CHF), hepatomegaly, and testicular atrophy. ■↑ serum iron, percent saturation of iron, and ferritin with ↓ serum transferrin.

1	■↑ serum iron, percent saturation of iron, and ferritin with ↓ serum transferrin. Fasting transferrin saturation (serum iron divided by transferrin level) > 45% is the most sensitive diagnostic test. Glucose intolerance and mildly elevated AST and alkaline phosphatase can be present. Perform a liver biopsy (to determine hepatic iron index), hepatic MRI, or HFE gene mutation screen. Weekly phlebotomy; when serum iron levels ↓, perform maintenance phlebotomy every 2–4 months. Deferoxamine can be used for maintenance therapy. Cirrhosis, hepatocellular carcinoma, cardiomegaly leading to CHF and/or conduction defects, DM, impotence, arthropathy, hypopituitarism. ■↓ ceruloplasmin and excessive deposition of copper in the liver and brain due to a deficient copper-transporting protein. Linked to an autosomalrecessive defect on chromosome 13. Usually occurs in patients < 30 years of age; 50% of patients are symptomatic by age 15.

1	Hx: Patients with present hemolytic anemia, liver abnormalities (jaundice 2° to hepatitis/cirrhosis), and neurologic (loss of coordination, tremor, dysphagia) as well as psychiatric (psychosis, anxiety, mania, depression) abnormalities. PE: May reveal Kayser-Fleischer rings in the cornea (green-to-brown deposits of copper in Descemet’s membrane) as well as jaundice, hepatomegaly, asterixis, choreiform movements, and rigidity. Dx: ↓ serum ceruloplasmin, ↑ urinary copper excretion, ↑ hepatic copper. Tx: Dietary copper restriction (avoid shellfish, liver, legumes), penicillamine (a copper chelator that ↑ urinary copper excretion; administer with pyridoxine), and possibly oral zinc (↑ fecal excretion). Wilson’s disease— ABCD Asterixis Basal ganglia deterioration Ceruloplasmin ↓, Cirrhosis, Copper ↑, Carcinoma (hepatocellular), Choreiform movements Dementia

1	Wilson’s disease— ABCD Asterixis Basal ganglia deterioration Ceruloplasmin ↓, Cirrhosis, Copper ↑, Carcinoma (hepatocellular), Choreiform movements Dementia Table 2.6-10 outlines the important features of acute and chronic pancreatitis. Table 2.6-11 lists Ranson’s criteria for predicting mortality associated with acute pancreatitis. T AB LE 2.6-1 0. Features of Acute and Chronic Pancreatitis Treatment Prognosis Complications Can have chronic pain and pancreatic exocrine and endocrine dysfunction. Chronic pain, malnutrition/weight loss, pancreatic cancer.

1	Pathophysiology Leakage of pancreatic enzymes into pancreatic and peripancreatic tissue, often 2° to gallstone disease or alcoholism. Irreversible parenchymal destruction leading to pancreatic dysfunction. Time course Abrupt onset of severe pain. Persistent, recurrent episodes of severe pain. Risk factors Gallstones, alcoholism, hypercalcemia, hypertriglyceridemia, trauma, drug side effects (thiazide diuretics), viral infections, post-ERCP, scorpion bites. Alcoholism (90%), gallstones, hyperparathyroidism, hypercholesterolemia, cystic f brosis. May also be idiopathic. History/PE Severe epigastric pain (radiating to the back); nausea, vomiting, weakness, fever, shock. Flank discoloration (Grey Turner’s sign) and periumbilical discoloration (Cullen’s sign) may be evident on exam. Recurrent episodes of persistent epigastric pain; anorexia, nausea, constipation, f atulence, steatorrhea, weight loss, DM. Diagnosis ↑ amylase, ↑ lipase, ↓ calcium if severe; “sentinel ↑ or normal amylase and

1	episodes of persistent epigastric pain; anorexia, nausea, constipation, f atulence, steatorrhea, weight loss, DM. Diagnosis ↑ amylase, ↑ lipase, ↓ calcium if severe; “sentinel ↑ or normal amylase and lipase, ↓ stool elastase, Removal of the offending agent if possible. Standard supportive measures: IV f uids/electrolyte replacement, analgesia, bowel rest, NG suction, nutritional support, O2, “tincture of time.” IV antibiotics, respiratory support, and surgical debridement if necrotizing pancreatitis is present.

1	loop” or “colon cutoff sign” on AXR. Abdominal ultrasound or CT may show an enlarged pancreas with stranding, abscess, hemorrhage, necrosis, or pseudocyst. Roughly 85–90% are mild and self-limited; 10– 15% are severe, requiring ICU admission. Mortality may approach 50% in severe cases. Pancreatic pseudocyst, fistula formation, hypocalcemia, renal failure, pleural effusion, chronic pancreatitis, sepsis. Mortality 2° to acute pancreatitis can be predicted with Ranson’s criteria (see Table 2.6-11). glycosuria, pancreatic calcifications, and mild ileus on AXR and CT (“chain of lakes”). Analgesia, exogenous lipase/trypsin and medium-chain fatty-acid diet, avoidance of causative agents (EtOH), celiac nerve block, surgery for intractable pain or structural causes. TABLE 2.6-11. Ranson’s Criteria for Acute Pancreatitisa a The risk of mortality is 20% with 3–4 signs, 40% with 5–6 signs, and 100% with ≥ 7 signs.

1	TABLE 2.6-11. Ranson’s Criteria for Acute Pancreatitisa a The risk of mortality is 20% with 3–4 signs, 40% with 5–6 signs, and 100% with ≥ 7 signs. Roughly 75% are adenocarcinomas in the head of the pancreas. Risk factors include smoking, chronic pancreatitis, a first-degree relative with pancreatic cancer, and a high-fat diet. Incidence rises after age 45; slightly more common in men. Presents with abdominal pain radiating toward the back, as well as with obstructive jaundice, loss of appetite, nausea, vomiting, weight loss, weakness, fatigue, and indigestion. Often asymptomatic, and thus presents late in the disease course. Exam may reveal a palpable, nontender gallbladder (Courvoisier’s sign) or migratory thrombophlebitis (Trousseau’s sign). Use CT to detect a pancreatic mass, dilated pancreatic and bile ducts, the extent of vascular involvement (particularly the SMA, SMV, and portal vein), and metastases (hepatic).

1	Use CT to detect a pancreatic mass, dilated pancreatic and bile ducts, the extent of vascular involvement (particularly the SMA, SMV, and portal vein), and metastases (hepatic). If a mass is not visualized, use ERCP or endoscopic ultrasound for better visualization. CA-19-9 is often elevated, but this measure lacks sensitivity and specificity. Most patients present with metastatic disease, and treatment is palliative. Some 10–20% of pancreatic head tumors have no evidence of metastasis or major vessel involvement and may be resected using the Whipple procedure (pancreaticoduodenectomy). Chemotherapy with 5-FU and gemcitabine may improve short-term survival, but long-term prognosis is poor (5–10% five-year survival). The classic presentation of pancreatic cancer is painless, progressive obstructive jaundice. Neutropenia 210 Eosinophilia 211 Transplant Medicine 212 Diseases Associated with Neoplasms 213

1	The classic presentation of pancreatic cancer is painless, progressive obstructive jaundice. Neutropenia 210 Eosinophilia 211 Transplant Medicine 212 Diseases Associated with Neoplasms 213 Bleeding disorders due to platelet dysfunction usually manifest as petechiae, whereas disorders of coagulation factors cause other symptoms, such as hemarthroses. Hemostasis requires the interaction of blood vessels, platelets, monocytes, and coagulation factors. This activates the clotting cascade, as shown in Figure 2.7-1. Heparin: ↑ PTT, activates antithrombin III and affects the intrinsic pathway, and ↓ fibrinogen levels; protamine sulfate is the antidote. Warfarin: ↑ PT, inhibits vitamin K and affects the extrinsic pathway, and is teratogenic, since its small size allows it to cross the placenta. Vitamin K is the antidote. Goal INR of 2.0–3.0 (2.5–3.5 in patients with mechanical valves).

1	Enoxaparin (low-molecular-weight heparin [LMWH]): Inhibits factor Xa and does not have to be monitored; dosing is once or twice daily. Heparin-to-warfarin conversion is necessary because warfarin inhibits proteins C and S before other vitamin K–dependent factors (II, VII, IX, and X), leading to a transient period of paradoxical hypercoagulability before proper anticoagulation. Intrinsic pathway—measured by PTT (initiated by exposure of collagen following vascular trauma) Hemophilia B is characterized by factor IX deficiency. 2 Hemophilia A is characterized by factor VIII deficiency. 3 Thrombin is inactivated by antithrombin III. The rate of inactivation increases in the presence of heparin. * Vitamin K–dependent clotting factors (II, VII, IX, X). Their synthesis is inhibited by warfarin. FIGURE 2.7-1. Coagulation cascade.

1	* Vitamin K–dependent clotting factors (II, VII, IX, X). Their synthesis is inhibited by warfarin. FIGURE 2.7-1. Coagulation cascade. A defciency of a clotting factor that leads to a bleeding diathesis. Subtypes are distinguished on the basis of which factor is lacking (see Table 2.7-1). The condition is usually hereditary but may be acquired through the development of an antibody to a clotting factor. This may occur in patients with autoimmune or lymphoproliferative disease, postpartum, or following a blood transfusion. Patients are nearly always male and may have a family history. Presents with spontaneous hemorrhage into the tissues and joints that, if left untreated, can lead to arthropathy and joint destruction. Spontaneous intracerebral hemorrhages, renal and retroperitoneal bleeding, and GI bleeding may also be seen. Mild cases may have major hemorrhage after surgery or trauma but are otherwise asymptomatic. Evaluate for suspected clotting factor def ciency:

1	Mild cases may have major hemorrhage after surgery or trauma but are otherwise asymptomatic. Evaluate for suspected clotting factor def ciency: PT: Usually normal, but isolated elevations are seen in congenital factor VII deficiency. aPTT: Prolonged (the more prolonged, the more severe the hemophilia). Thrombin time, fbrinogen, bleeding time: Usually normal. Conduct a mixing study: Mix the patient’s plasma with normal plasma; if this corrects the aPTT, a factor deficiency is likely. If the aPTT does not correct, the patient may have a clotting factor inhibitor. Obtain factor assays: Specific factor assays should then be performed for factors VII, VIII, IX, XI, and XII. Hemophilia is characterized according to factor level as follows: Mild: > 5% of normal. Moderate: 1–3% of normal. Severe: ≤ 1% of normal.

1	Mild: > 5% of normal. Moderate: 1–3% of normal. Severe: ≤ 1% of normal. ■Treat bleeding episodes with immediate transfusion of clotting factors (or cryoprecipitate) to at least 40% of normal concentration. Factor VIII has a half-life of 12 hours, so patients should be dosed BID to maintain ad- TABLE 2.7-1. Types of Hemophilia The classic case of hemophilia is the boy (X-linked) from the Imperial Russian family (Recessive) who presents with hemarthroses following minimal or no trauma. Cryoprecipitate consists of factors VIII and XIII, vWF, fibrinogen, and fibronectin. Hemophilia B (factor IX defciency) (9%) X-linked inheritance. Hemophilia C (factor XI defciency) (< 1%) Most common in Ashkenazi Jews. Hemophilia A (factor VIII defciency) (90%) X-linked inheritance; the most common severe congenital clotting def ciency. Factor VII defciency (< 1%) Presents in a milder, likely heterozygous form. DDAVP helps the body release extra factor VIII.

1	DDAVP helps the body release extra factor VIII. ASA ↑ the risk of bleeding in patients with von Willebrand’s disease. Ristocetin cofactor assay measures the ability of vWF to agglutinate platelets in vitro in the presence of ristocetin. Suspect pulmonary embolism in a patient with rapid onset of hypoxia, hypercapnia, tachycardia, and an ↑ alveolar-arterial oxygen gradient without another obvious explanation. equate levels. Factor IX has a half-life of 24 hours, so daily transfusion is needed. The length of treatment varies with the lesion, extending up to several weeks after orthopedic surgery. Mild hemophiliacs may be treated with desmopressin (DDAVP); if so, they should be f uid restricted to prevent the side effect of hyponatremia. It may be necessary to transfuse RBCs, depending on the degree of blood loss.

1	It may be necessary to transfuse RBCs, depending on the degree of blood loss. Fifteen percent of patients who are treated for hemophilia A develop neutralizing IgG antibodies to factor VIII, which precludes further treatment with replacement factor. An autosomal-dominant condition in which patients have deficient or defective von Willebrand’s factor (vWF) with low levels of factor VIII, which is carried by vWF. Symptoms are due to platelet dysfunction and to deficient factor VIII. The disease is milder than hemophilia. vWD is the most common inherited bleeding disorder (1% of the population is affected). Presents with easy bruising, mucosal bleeding (e.g., epistaxis, oral bleeding), menorrhagia, and postincisional bleeding. Platelet dysfunction is not severe enough to produce petechiae. Symptoms worsen with ASA use. Look for a family history of bleeding disorders. Platelet count and PT are normal, but a prolonged aPTT may be seen as a result of factor VIII deficiency.

1	Look for a family history of bleeding disorders. Platelet count and PT are normal, but a prolonged aPTT may be seen as a result of factor VIII deficiency. A ristocetin cofactor assay of patient plasma can measure the capacity of vWF to agglutinate platelets. Bleeding episodes can be treated with DDAVP; menorrhagia can be controlled with OCPs. Avoid ASA and other inhibitors of platelet function. Also called thrombophilias or prothrombotic states, hypercoagulable states is an all-inclusive term describing conditions that ↑ a patient’s risk of developing thromboembolic disease. Causes are multiple and may be genetic, acquired, or physiologic (see Table 2.7-2). Acquired causes are usually 2° to an underlying clinical condition, disease process, or lifestyle. Inherited causes are collectively called hereditary thrombotic disease, of which factor V Leiden (a polymorphism in factor V, rendering it resistant to inactivity by activated protein C, or APC) is the most common.

1	■ Presents with recurrent thrombotic complications, including DVT, pulmonary embolism, arterial thrombosis, MI, and stroke. Women may have recurrent miscarriages. T AB LE 2.7 -2. Causes of Hypercoagulable States ■Although patients may have no recognizable predisposing factors, they usually have one or more of the causative factors outlined in Table 2.7-2. They may also have a family history. Under ideal circumstances, patients should be diagnosed before they are symptomatic, but this rarely occurs. Prior to workup for hereditary causes, acquired causes of abnormal coagulation values should be ruled out. Confrmation of a hereditary abnormality requires two abnormal values that are obtained while the patient is asymptomatic and untreated, with similar values obtained in two other family members.

1	Workup for hypercoagulability includes lupus antigen/antiphospholipid syndrome, antithrombin III deficiency, protein C and S deficiencies, APC resistance, homocysteine elevation, and prothrombin G20210A mutation. Treatment should address the type of thrombotic event as well as the area of thrombosis. Treat DVT and pulmonary embolism with heparin (unfractionated or LMWH) followed by 3–6 months of oral warfarin anticoagulation for the first event, 6–12 months for the second, and lifelong anticoagulation for subsequent events. Heparin-to-warfarin conversion is necessary (see the “Coagulation Cascade” discussion above). A common disorder among hospitalized patients, second only to liver disease as a cause of acquired coagulopathy. It is caused by deposition of f brin in small blood vessels, leading to thrombosis and end-organ damage. Depletion of clotting factors and platelets leads to a bleeding diathesis. May be associated with almost any severe illness.

1	■Disorders commonly associated with DIC include obstetric complications, infections with septicemia, neoplasms, acute promyelocytic leukemia, pan- DIC is characterized by both thrombosis and hemorrhage. Petechiae suggest Platelet deficiency. Bleeding into body Cavities or joints suggests Clotting factor deficiency. The three causes of microangiopathic hemolytic anemia are HUS, TTP, and DIC. creatitis, intravascular hemolysis, vascular disorders (e.g., aortic aneurysm), massive tissue injury and trauma, drug reactions, acidosis, and ARDS. ■Presentation varies according to whether the disease is acute or chronic: Acute: Presents with generalized bleeding out of venipuncture sites into organs, with ecchymoses and petechiae. Patients who are in shock may have acral cyanosis. Chronic: Presents with bruising and mucosal bleeding, thrombophlebitis, renal dysfunction, and transient neurologic syndromes. Diagnosed as outlined in Table 2.7-3.

1	Chronic: Presents with bruising and mucosal bleeding, thrombophlebitis, renal dysfunction, and transient neurologic syndromes. Diagnosed as outlined in Table 2.7-3. DIC may be confused with severe liver disease, but unlike liver disease, factor VIII is depressed. Treatment of the underlying illness often results in spontaneous reversal. Patients often require RBC transfusion and shock management. Platelets should be transfused in the event of hemorrhage with a platelet count < 20,000. Part of a spectrum of diseases that includes hemolytic-uremic syndrome (HUS) and HELLP syndrome (see the Obstetrics chapter); thought to be due to platelet microthrombi that block off small blood vessels, leading to end-organ ischemia and dysfunction. RBCs are fragmented by contact with the microthrombi, leading to hemolysis (microangiopathic hemolytic anemia). The cause of initial microthrombus formation is unknown but may be infectious (bacterial toxins), drug related, autoimmune, or idiopathic.

1	A clinical syndrome characterized by f ve signs/symptoms: low platelet count, microangiopathic hemolytic anemia, neurologic changes (delirium, seizure, stroke), impaired renal function, and fever. Diagnosis is largely clinical. It is rare for all signs to be present, but the presence of schistocytes (broken RBCs) on peripheral smear with low platelets and rising creatinine is highly suggestive. Nucleated RBCs are also often seen in the peripheral smear. Hemolytic anemia labs include elevated indirect bilirubin, LDH, and AST along with low haptoglobin. Coagulation factors are normal. TABLE 2.7-3. Laboratory Values in DIC Overlapping conditions are HUS, HELLP syndrome, and DIC. HUS: Characterized by renal failure, hemolytic anemia, and low platelets. Severe elevations in creatinine are more typical of HUS than of TTP. HELLP syndrome: Affects pregnant women, often occurring in conjunction with preeclampsia (see the Obstetrics chapter).

1	HELLP syndrome: Affects pregnant women, often occurring in conjunction with preeclampsia (see the Obstetrics chapter). DIC: Distinguished from TTP by prolonged PT and aPTT. Treat with corticosteroids to ↓ the formation of microthrombi along with plasma replacement and plasmapheresis (the mainstay of severe cases). Rarely, splenectomy is performed with mixed results. Platelet transfusions are contraindicated. A relatively common cause of thrombocytopenia. IgG antibodies are formed against the patient’s platelets. Bone marrow production of platelets is ↑, with ↑ megakaryocytes in the marrow. The most common immunologic disorder in women of childbearing age. May be acute or chronic. Patients often feel well and present with no systemic symptoms. They may have minor bleeding, easy bruising, petechiae, hematuria, hematemesis, or melena. Bleeding is mucocutaneous. Usually there is no splenomegaly.

1	ITP is associated with a range of conditions, including lymphoma, leukemia, SLE, HIV, and HCV. The clinical presentation is as follows: Acute: Abrupt onset of hemorrhagic complications following a viral illness. Commonly affects children 2–6 years of age, with males and females affected equally. Chronic: Insidious onset that is unrelated to infection. Most often affects adults 20–40 years of age; women are three times more likely to be affected than men. A diagnosis of exclusion, as the test for platelet-associated antibodies is a poor one. Once other causes of thrombocytopenia have been ruled out, a diagnosis can be made on the basis of the history and physical, a CBC, and a peripheral blood smear showing normal RBC morphology. Most patients do not require bone marrow biopsy, which would show ↑ megakaryocytes as the only abnormality. Most patients with acute childhood ITP spontaneously remit, but this is rarely the case in chronic ITP.

1	Most patients with acute childhood ITP spontaneously remit, but this is rarely the case in chronic ITP. Treatment is reserved for patients with symptomatic bleeding. Those with platelet counts > 20,000 are generally asymptomatic. Platelet transfusions are of no benefit, as patients’ IgG levels will also lead to destruction of platelets. The main therapies are corticosteroids, high-dose gamma globulin (IVIG), and splenectomy. Most patients respond to corticosteroids, but if Anti-D (Rh) immunoglobulin and rituximab are emerging therapies for ITP. Anti-D (Rh) immunoglobulin and IVIG act as “decoys” so that WBCs will recognize them they cannot be tapered after 3–6 months, splenectomy should be considered. An emerging therapy is anti-D (Rh) immunoglobulin, which binds to RBCs so that they are destroyed instead of the patient’s platelets. Patients will have a drop in their hematocrit. Rhand splenectomized patients should not receive this treatment.

1	In pregnant patients, severe thrombocytopenia may occur in the fetus. instead of IgG on platelets. Figure 2.7-2 illustrates the various blood cell categories and lineages. Anemia is a disorder of low hematocrit and hemoglobin. There are several subtypes, which are classified according to red cell morphology (MCV, RDW, color, shape) and reticulocyte count (see Figure 2.7-3). Once anemia has been diagnosed by a low hemoglobin/hematocrit, the approach starts with the MCV. An MCV < 80 fL indicates microcytic anemia; between 80 fL and 100 fL is normocytic; and > 100 fL is macrocytic. A condition in which iron loss exceeds intake. May occur when dietary intake is insuff cient for the patient’s needs (e.g., when needs are ↑ by growth or pregnancy) or in the setting of chronic blood loss, usually 2° to menstruation or GI bleeding. Toddlers, adolescent girls, and women of childbearing age are most commonly affected. F IGU R E 2.7 -2. Blood cell differentiation. CBC, reticulocyte count

1	F IGU R E 2.7 -2. Blood cell differentiation. CBC, reticulocyte count Retic < 2.5 Retic > 2.5 Iron deficiency anemia in an elderly patient could be due to colorectal cancer and needs to be evaluated to rule out malignancy. Iron deficiency anemia can be due to a hookworm infection. FIGURE 2.7-3. Anemia algorithm. Symptoms include fatigue, weakness, brittle nails, and pica. If the anemia develops slowly, patients are generally asymptomatic. Physical findings include glossitis, angular cheilitis, and koilonychia (“spoon nails”). Bone marrow biopsy looking for evidence of iron stores is the gold standard but is seldom performed. Iron deficiency is often confused with anemia of chronic disease, in which iron use by the body is impaired. Labs can help differentiate the two conditions (see Table 2.7-4). Peripheral blood smear shows hypochromic and microcytic RBCs with a low reticulocyte count.

1	Peripheral blood smear shows hypochromic and microcytic RBCs with a low reticulocyte count. Low serum ferritin reﬂects low body stores of iron and confirms the diagnosis. However, ferritin is also an acute-phase reactant and may thus obscure evidence of iron deficiency. Treat with replacement iron for 4–6 months. Oral iron sulfate may lead to nausea, constipation, diarrhea, and abdominal pain. Antacids may interfere with iron absorption. If necessary, IV iron dextran can be administered but is associated with a 10% risk of serious side effects, including anaphylaxis. Hence, this is usually done only by a hematologist. T AB LE 2.7 -4. Iron Defciency Anemia vs. Anemia of Chronic Disease Most macrocytic anemias are caused by processes that interfere with normal DNA synthesis and replication. B12 deficiency can be due to infection by a tapeworm, Diphyllobothrium latum.

1	Most macrocytic anemias are caused by processes that interfere with normal DNA synthesis and replication. B12 deficiency can be due to infection by a tapeworm, Diphyllobothrium latum. Causes of oxidative stress in G6PD deficiency include infection, metabolic acidosis, fava beans, antimalarials, dapsone, sulfonamides, and nitrofurantoin. Vitamin B12 (cobalamin) and folate def ciency interfere with DNA synthesis, leading to a delay in blood cell maturation. Cobalamin deficiency is due to malabsorption, usually from pernicious anemia (destruction of parietal cells, which produce the intrinsic factor needed for cobalamin absorption). Folate deficiency results from insufficient dietary folate, malabsorption, alcoholism, or use of certain drugs. Drugs that interfere with DNA synthesis, including many chemotherapeutic agents, may lead to megaloblastic anemia. Presents with fatigue, pallor, diarrhea, loss of appetite, headaches, and tingling/numbness of the hands and feet.

1	Presents with fatigue, pallor, diarrhea, loss of appetite, headaches, and tingling/numbness of the hands and feet. Cobalamin deficiency affects the nervous system, so patients lacking that vitamin may develop a demyelinating disorder and may present with symptoms of motor, sensory, autonomic, and/or neuropsychiatric dysfunction, known as subacute combined degeneration of the cord. Peripheral smear shows RBCs with an elevated MCV. Hypersegmented (> 5) granulocytes can also be seen. Bone marrow sample reveals giant neutrophils and hypersegmented mature granulocytes. The Schilling test (ingestion of radiolabeled cobalamin both with and without added intrinsic factor) is classic for measuring absorption of cobalamin but is rarely performed. Serum vitamin levels are poorly diagnostic of deficiencies and are thus used with adjunctive tests, including methylmalonic acid (MMA) and homocysteine levels: B12 def ciency: Elevated MMA and homocysteine.

1	B12 def ciency: Elevated MMA and homocysteine. Folate def ciency: Normal MMA; elevated homocysteine. Address the cause of the anemia, and correct the underlying cause. Occurs when bone marrow production is unable to compensate for ↑ destruction of circulating blood cells. Etiologies include the following: G6PD def ciency: An X-linked recessive disease that ↑ RBC sensitivity to oxidative stress. Paroxysmal nocturnal hemoglobinuria: A disorder in which blood cell sensitivity to complement activation is ↑. Hereditary spherocytosis: An abnormality of the RBC membrane. Autoimmune RBC destruction: Occurs 2º to EBV infection, mycoplasmal infection, CLL, rheumatoid disease, or medications. Sickle cell disease: A recessive β-globin mutation (see the following section). Microangiopathic hemolytic anemia: TTP, HUS, DIC. Mechanical hemolysis: Associated with mechanical heart valves. Other: Malaria, hypersplenism. Presents with pallor, fatigue, tachycardia, and tachypnea.

1	Mechanical hemolysis: Associated with mechanical heart valves. Other: Malaria, hypersplenism. Presents with pallor, fatigue, tachycardia, and tachypnea. Patients are typically jaundiced, with low haptoglobin and elevated indirect bilirubin and LDH. Urine is dark with hemoglobinuria, and there is ↑ excretion of urinary and fecal urobilinogen. Reticulocyte count is elevated. Diagnosed by the history and clinical presentation. High LDH, elevated indirect bilirubin, and ↓ haptoglobin levels are consistent with a diagnosis of hemolytic anemia. Also obtain a reticulocyte count. A Coombs’ test is used to detect autoimmune hemolysis. Treatment varies with the cause of hemolysis but typically includes corticosteroids to address immunologic causes and iron supplementation to replace urinary losses. Splenectomy may be helpful, and transfusion may be necessary to treat severe anemia.

1	A rare condition caused by failure of blood cell production due to destruction of bone marrow cells. It may be hereditary, as in Fanconi’s anemia; may have an autoimmune or a viral etiology (e.g., HIV, parvovirus B19); or may result from exposure to toxins (e.g., drugs, cleaning solvents) or radiation. Patients are typically pancytopenic, with symptoms resulting from a lack of RBCs, WBCs, and platelets—e.g., pallor, weakness, a tendency to infection, petechiae, bruising, and bleeding. The disease may be of sudden or sustained onset and may be of variable severity, depending on the patient’s blood counts. Diagnosed by clinical presentation and CBC; verifed by a bone marrow biopsy revealing hypocellularity and space occupied by fat. The differential includes megaloblastic anemia, as both diseases feature an elevated MCV. The classic case of G6PD deficiency is an African-American male soldier in Vietnam who took quinine.

1	The differential includes megaloblastic anemia, as both diseases feature an elevated MCV. The classic case of G6PD deficiency is an African-American male soldier in Vietnam who took quinine. Indirect Coombs’ tests detect antibodies to RBCs in the patient’s serum. Direct Coombs’ tests detect sensitized erythrocytes. Patients with Fanconi’s anemia may be identified on physical exam by café au lait spots, short stature, and radial/thumb hypoplasia/ aplasia. SCD represents a qualitative defect in the β-globin chain. Patients with SCD classically get osteomyelitis with Salmonella. They are also at ↑ risk of avascular necrosis of the hip. Blood transfusion and stem cell transplantation to replace absent cells; immunosuppression with cyclosporin A and antithymocyte globulin to prevent autoimmune destruction of marrow. Infections are a major cause of mortality and should be treated aggressively.

1	An autosomal-recessive disorder most commonly caused by a mutation of adult hemoglobin (the β chain has glu replaced by val). Signs and symptoms are due to ↓ red cell survival and a tendency of sickled cells to lead to vasoocclusion. Asymptomatic during the first year or two of life; may first present with dactylitis in childhood. Later, hemolysis results in anemia, jaundice, cholelithiasis, ↑ cardiac output (murmur and cardiomegaly), and delayed growth. Vaso-occlusion leads to ischemic organ damage, especially splenic infarction, which predisposes to pneumococcal sepsis, and acute chest syndrome (pneumonia and/or pulmonary infarction). Patients also experience painful crises of unknown etiology. Common triggers for a vaso-occlusive crisis (VOC) include cold temperatures, dehydration, and infection.

1	Other potential complications include splenic sequestration, which occurs in patients who have not infarcted their spleens, and aplastic crisis, which is usually 2° to infection with parvovirus B19. The sickle cell screen is based on a blood smear with sickle cells and target cells. The gold standard is quantitative hemoglobin electrophoresis. Treat with hydroxyurea, which stimulates the production of fetal hemoglobin (hydroxyurea is teratogenic, so it is contraindicated in pregnancy). If hydroxyurea does not prove effective, chronic transfusion therapy, which carries the risk of iron overload, can be tried. Treat cholelithiasis with cholecystectomy. VOCs must be treated with adequate pain management, O2 therapy, IV ﬂuid rehydration, and antibiotics (if infection is suspected to be the trigger).

1	Treat cholelithiasis with cholecystectomy. VOCs must be treated with adequate pain management, O2 therapy, IV ﬂuid rehydration, and antibiotics (if infection is suspected to be the trigger). To prevent VOCs from progressing to acute chest syndrome, initiate aggressive hydration and incentive spirometry, and keep the sickle variant < 40%. This can be done with simple transfusions or, if necessary, exchange transfusion in an ICU setting. Hereditary disorders involving ↓ or absent production of normal globin chains of hemoglobin. α-thalassemia is caused by a mutation of one or more of the four genes for α-hemoglobin; β-thalassemia results from a mutation of one or both of the two genes for β-hemoglobin. Thalassemia is most common among people of African, Middle Eastern, and Asian descent. Disease presentation and prognosis vary with the number of genes missing (see Table 2.7-5).

1	Thalassemia is most common among people of African, Middle Eastern, and Asian descent. Disease presentation and prognosis vary with the number of genes missing (see Table 2.7-5). Diagnosed by hemoglobin electrophoresis evaluation (but note that this is normal in α-thalassemia) and DNA studies. Most patients do not require treatment, but those with β-thalassemia major and hemoglobin H disease are commonly transfusion dependent and should be given iron chelators (deferoxamine) to prevent overload. Erythrocytosis (an abnormal elevation of hematocrit) may be either 1° (due to ↑ RBC production) or 2° (due to ↓ plasma volume and hemoconcentration).

1	Erythrocytosis (an abnormal elevation of hematocrit) may be either 1° (due to ↑ RBC production) or 2° (due to ↓ plasma volume and hemoconcentration). T AB LE 2.7 -5. Differential Diagnosis of Thalassemias β-thalassemia major 0/2 β Patients develop severe microcytic anemia in the frst year of life and need chronic transfusions or marrow transplant to survive. β-thalassemia minor 1/2 β Patients are asymptomatic, but their cells are microcytic and hypochromic on peripheral smear. Hydrops fetalis 0/4 α Patients die in utero. Hemoglobin H disease 1/4 α Patients have severe hypochromic, microcytic anemia with chronic hemolysis, splenomegaly, jaundice, and cholelithiasis. The reticulocyte count elevates to compensate, and one-third of patients have skeletal changes due to expanded erythropoiesis. α-thalassemia trait 2/4 α Patients have low MCV but are usually asymptomatic. Silent carrier 3/4 α Patients have no signs or symptoms of disease.

1	True polycythemia vera is characterized by a high red cell mass. Premedication with acetaminophen and diphenhydramine is sometimes used to prevent transfusion reactions. Hemoglobinuria in hemolytic transfusion reaction may lead to acute tubular necrosis and subsequent renal failure. Characterized by ↑ hematocrit, ↓ tissue blood ﬂow and oxygenation, and ↑ cardiac work. Patients present with “hyperviscosity syndrome,” which consists of easy bleeding/bruising, blurred vision, neurologic abnormalities, plethora, pruritus (especially after a warm bath), hepatomegaly, splenomegaly, and CHF. 1° erythrocytosis is associated with hypoxia (from lung disease, smoking, high altitudes, or a poor intrauterine environment), neoplasia (erythropoietin-producing tumors), or polycythemia vera (PCV), in which there is clonal proliferation of a pluripotent marrow stem cell. 2° erythrocytosis is associated with excessive diuresis, severe gastroenteritis, and burns.

1	2° erythrocytosis is associated with excessive diuresis, severe gastroenteritis, and burns. Erythrocytosis is diagnosed clinically and by cell counts, with ABGs used to assess hypoxia or imaging to demonstrate neoplasia. Patients with PCV have excess RBCs, WBCs, and platelets. Levels of erythropoietin may be useful in distinguishing PCV, in which levels are low, from other causes of polycythemia. Phlebotomy relieves symptoms of erythrocytosis, but treatment should also address the underlying cause. PCV can be treated with cytoreductive drugs such as hydroxyurea or interferon. Because PCV is prothrombotic, ASA should also be used. With treatment, survival is 7–10 years. Blood transfusion is generally safe but may result in a variety of adverse reactions. Nonhemolytic febrile reactions and minor allergic reactions are the most common, each occurring in 3–4% of all transfusions. Etiologies are as follows:

1	Nonhemolytic febrile reactions: Involve cytokine formation during the storage of blood, and WBC antibodies. Minor allergic reactions: Involve antibody formation (usually IgA) against donor proteins. Usually occur following transfusion of plasma-containing product. Hemolytic transfusion reactions: Entail the development of antibodies against donor erythrocytes. Usually result from ABO incompatibility or from antibody against minor antigens. Nonhemolytic febrile reactions: Present with fever, chills, rigors, and malaise. Symptom onset is 1–6 hours following transfusion. Minor allergic reactions: Characterized by urticaria. Hemolytic transfusion reactions: Present with fever, chills, nausea, ﬂ ushing, apprehension, back pain, burning at the IV site, tachycardia, tachypnea, and hypotension. Symptoms begin following the transfusion of only a small amount of blood. Diagnosed by clinical impression.

1	Diagnosed by clinical impression. Nonhemolytic febrile reactions: Stop the transfusion and control fever with acetaminophen. Rule out infection. Minor allergic reactions: Administer antihistamines. In the setting of a severe reaction, stop the transfusion and give epinephrine +/– steroids. Hemolytic transfusion reactions: Stop the transfusion immediately. Replace donor blood with normal saline; administer vigorous IV ﬂ uids; and maintain good urine output (diuretics and pressors may be used to ↑ renal blood ﬂ ow). The porphyrias are a group of inherited disorders that include acute intermittent porphyria, porphyria cutanea tarda, and erythropoietic porphyria. Some porphyrias are autosomal dominant (e.g., acute intermittent porphyria) and others autosomal recessive (e.g., erythropoietic porphyria). All involve abnormalities of heme production that lead to an accumulation of porphyrins.

1	Signs and symptoms vary with the type of porphyria. In general, however, porphyrias are characterized by a combination of photodermatitis, neuropsychiatric complaints, and visceral complaints that typically take the form of a colicky abdominal pain and seizures. Physical exam reveals tachycardia, skin erythema and blisters, aref exia, and a nonspecifc abdominal exam. Patients with the erythropoietic form present with hemolytic anemia. Acute attacks are associated with stimulants of ↑ heme synthesis such as fasting or chemical exposures; well-known triggers are alcohol, barbiturates, and OCPs. Urine may appear red or brown after an acute attack. Patients may have a family history. Diagnosed by a combination of the history and physical along with labs showing elevated blood, urine, and stool porphyrins. Enzyme assays may also be helpful. Avoidance of triggers of acute attacks; symptomatic treatment during acute episodes.

1	Avoidance of triggers of acute attacks; symptomatic treatment during acute episodes. High doses of glucose may be administered to ↓ heme synthesis during mild attacks, and IV hematin can be given for severe attacks (provides feedback to the heme synthetic pathway). Heme is necessary for the production of hemoglobin, myoglobin, and cytochrome molecules. The telltale sign of porphyria is pink urine. The classic case of porphyria involves a college student who consumes alcohol and barbiturates at a party, and then has an acute episode of abdominal pain and brown urine the next day. Malignant proliferations of hematopoietic cells, categorized by the type of cell involved and their level of differentiation. Leukemias may be acute or chronic, lymphocytic or myelogenous. The four oncologic emergencies: hypercalcemia, spinal cord compression, superior vena cava syndrome, and leukostasis.

1	The four oncologic emergencies: hypercalcemia, spinal cord compression, superior vena cava syndrome, and leukostasis. Acute myelogenous and lymphocytic leukemias are clonal disorders of early hematopoietic stem cells. They are characterized by rapid growth of immature blood cells (blasts), which overwhelms the ability of bone marrow to produce normal cells. Acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL) affect children as well as adults. ALL is the most common childhood malignancy. Disease onset and progression are rapid; patients present with signs and symptoms of anemia (pallor, fatigue) and thrombocytopenia (petechiae, purpura, bleeding). Medullary expansion and periosteal involvement may lead to bone pain (common in ALL). On exam, patients may have hepatosplenomegaly and swollen/bleeding gums from leukemic infiltration and ↓ platelets. Leukemic cells also infiltrate the skin and CNS.

1	On exam, patients may have hepatosplenomegaly and swollen/bleeding gums from leukemic infiltration and ↓ platelets. Leukemic cells also infiltrate the skin and CNS. Based on examination of the patient’s bone marrow, obtained by biopsy and aspiration, or peripheral blood if circulating blasts are present. Marrow that is infiltrated with blast cells (> 20–30%) is consistent with a leukemic process. In AML, the leukemic cells are myeloblasts; in ALL they are lymphoblasts. These cells may be distinguished by examination of morphology, cytogenetics, cytochemistry, and immunophenotyping (see Table 2.7-6). The WBC count is usually elevated, but the cells are dysfunctional, and patients may be neutropenic with a history of frequent infection. If the WBC is very high (> 100,000), there is a risk of leukostasis (blasts occluding the microcirculation, leading to pulmonary edema, CNS symptoms, ischemic injury, and DIC).

1	The type of acute leukemia is further classified according to the FAB system (ALL: L1–L3; AML: M0–M7) and karyotype analysis. Prognosis varies with leukemic cytogenetics. T AB LE 2.7 -6. Myeloblasts vs. Lymphoblasts Size Larger (2–4 times RBC) Smaller (1.5–3.0 times RBC) Amount of cytoplasm More Less Nucleoli Conspicuous Inconspicuous Granules Common, f ne Uncommon, coarse Auer rods Present in 50% of cases Absent Myeloperoxidase ALL and AML are treated primarily with chemotherapeutic agents, although transfusions, antibiotics, and colony-stimulating factors are also used. Patients with unfavorable genetics or those who do not achieve remission may be candidates for bone marrow transplantation. Prior to therapy, patients should be well hydrated, and if their WBC counts are high, they may be started on allopurinol to prevent hyperuricemia and renal insufficiency resulting from blast lysis (tumor lysis syndrome).

1	Leukostasis syndrome may be treated with hydroxyurea +/– leukapheresis to rapidly ↓ WBC count. Indicators of a poor prognosis are as follows: ALL: Age < 1 year or > 10 years; an ↑ in WBC count to > 50,000; the presence of the Philadelphia chromosome t(9,22) (associated with B-cell cancer); CNS involvement at diagnosis; and L3 morphology (associated with Burkitt’s lymphoma). AML: Age > 60 years, CD34+ or MDR1+ phenotype, elevated LDH, mutations in chromosomes 5 or 7, t(6,9), trisomy 8 or a more complex karyotype, FLT3 gene mutation, and FAB M7 (acute megakaryocytic variant). AML type (FAB) M3—acute promyelocytic leukemia (APL)—has a good prognosis because it is responsive to all-trans-retinoic acid (ATRA) therapy. The mutation that causes this form of AML contains the retinoic acid receptor. ATRA is less toxic than conventional chemotherapy.

1	A malignant, clonal proliferation of functionally incompetent lymphocytes that accumulate in the bone marrow, peripheral blood, lymph nodes, spleen, and liver. The most common type of leukemia. A rare form of T-cell CLL exists, but almost all cases involve well-differentiated B lymphocytes. The etiology is unknown, although there is some genetic contribution, as first-degree relatives of patients with CLL are three times more likely than others to develop a lymphoid malignancy. Primarily affects older adults (median age 65); the male-to-female ratio is 2:1. Often asymptomatic, but many patients present with fatigue, malaise, and infection. Common physical findings are lymphadenopathy and splenomegaly. Diagnosed by the clinical picture; may be confirmed by f ow cytometry demonstrating the presence of CD5—normally found only on T cells—on leukemic cells with the characteristic B-cell antigens CD20 and CD21.

1	CBC shows lymphocytosis (lymphocyte count > 5000) with an abundance of small, normal-appearing lymphocytes and ruptured smudge cells on peripheral smear. Granulocytopenia, anemia, and thrombocytopenia are common owing to marrow infiltration with leukemic cells. Abnormal function by the leukemic cells leads to hypogammaglobulinemia. Bone marrow biopsy is rarely required for diagnosis or staging but may provide prognostic information and may help assess response to therapy. A characteristic sign for AML type M3 (APL) is the Auer rod. Eighty-five percent of children with ALL achieve complete remission with chemotherapy. CLL may be complicated by autoimmune hemolytic anemia. The lymphoid equivalent of CLL is small lymphocytic lymphoma. The clinical stage correlates with expected survival (see Table 2.7-7).

1	CLL may be complicated by autoimmune hemolytic anemia. The lymphoid equivalent of CLL is small lymphocytic lymphoma. The clinical stage correlates with expected survival (see Table 2.7-7). Treatment is palliative. The degree of peripheral lymphocytosis does not correlate with prognosis, nor does it dictate when treatment should be initiated. Treatment is often withheld until patients are symptomatic—e.g., when they present with recurrent infection, severe lymphadenopathy or splenomegaly, anemia, and thrombocytopenia. Treatment consists primarily of chemotherapy, especially with alkylating agents, although radiation may be useful for localized lymphadenopathy. Although CLL is not curable, long disease-free intervals may be achieved with adequate treatment of symptoms.

1	Although CLL is not curable, long disease-free intervals may be achieved with adequate treatment of symptoms. Involves clonal expansion of myeloid progenitor cells, leading to leukocytosis with excess granulocytes and basophils and sometimes ↑ erythrocytes and platelets as well. To truly be CML, the BCR-ABL translocation must be present. In > 90% of patients, this is reﬂected by the Philadelphia chromosome t(9,22). CML primarily affects middle-aged patients. With routine blood testing, many patients are diagnosed while asymptomatic. However, typical signs and symptoms are those of anemia. Patients frequently have splenomegaly with LUQ pain and early satiety. Hepatomegaly may be present as well. Constitutional symptoms of weight loss, anorexia, fever, and chills may also be seen. Patients with CML go through three disease phases: Chronic: Without treatment, typically lasts 3.5–5.0 years. Signs and symptoms are as described above. Infection and bleeding complications are rare.

1	Chronic: Without treatment, typically lasts 3.5–5.0 years. Signs and symptoms are as described above. Infection and bleeding complications are rare. Accelerated: A transition toward blast crisis, with an ↑ in peripheral and bone marrow blood counts. Should be suspected when the differential shows an abrupt ↑ in basophils and thrombocytopenia < 100,000. Blast: Resembles acute leukemia; survival is 3–6 months. ■Diagnosed by the clinical picture, including labs; cytogenetic analysis usually reveals the Philadelphia chromosome. TAB LE 2.7 -7. Clinical Staging of CLL (Rai Staging) CBwC shows a very high WBC—often > 100,000 at diagnosis, and sometimes reaching > 500,000. Differential shows granulocytes in all stages of maturation. Rarely, the WBC count will be so elevated as to cause a hyperviscosity syndrome. Leukocyte alkaline phosphatase is low; LDH, uric acid, and B12 levels are elevated.

1	Leukocyte alkaline phosphatase is low; LDH, uric acid, and B12 levels are elevated. Varies with disease phase and is undergoing rapid change, particularly since the introduction of targeted therapies: Chronic: Imatinib. Younger patients can be treated with allogeneic stem cell transplantation if a suitable matched sibling donor is available. Blast: Therapy as for acute leukemia or dasatinib plus hematopoietic stem cell transplant or clinical trial. A malignant disorder of well-differentiated B lymphocytes with an unclear cause. HCL is a rare disease that accounts for 2% of adult leukemia cases and most commonly affects older males. Typically presents with pancytopenia, bone marrow infiltration, and splenomegaly.

1	Typically presents with pancytopenia, bone marrow infiltration, and splenomegaly. Patients complain of weakness, fatigue, petechiae and bruising, infection (especially with atypical mycobacteria such as Mycobacterium avium– intracellulare), abdominal pain, early satiety, and weight loss. Symptoms are similar to those of CLL except that patients rarely have lymphadenopathy. Diagnosed by the history, physical exam, and labs; confirmed through the identification of hairy cells in the blood, marrow, or spleen. Tartrate-resistant acid phosphatase (TRAP) staining of hairy cells, electron microscopy, and f ow cytometry (quantification of ﬂ uorescence on cells with ﬂuorescent-labeled monoclonal antibodies bound to cell-specific antigens) are helpful in distinguishing the pathognomonic hairy cells.

1	CBC usually demonstrates leukopenia (making the name leukemia a misnomer); roughly 85% of the time, a peripheral smear shows hairy cells, or mononuclear cells with abundant pale cytoplasm and cytoplasmic projections. Ten percent of patients have a benign course and never require therapy, but the remainder develop progressive pancytopenia and splenomegaly. The median survival without treatment is five years. Treatment begins when patients are symptomatic or extremely cytopenic. Nucleoside analogs are currently the initial treatment of choice, and effectively induce remission. Other treatment options include splenectomy, which may improve blood counts, and IFN-α. Imatinib (Gleevec) is a selective inhibitor of the BCR-ABL tyrosine kinase. The treatment of high-grade NHL may be complicated by tumor lysis syndrome, in which rapid cell death releases intracellular contents and leads to hyperkalemia, hyperphosphatemia, hyperuricemia, and hypocalcemia.

1	Malignant transformations of lymphoid cells residing primarily in lymphoid tissues, especially the lymph nodes. Classically organized into Hodgkin’s and non-Hodgkin’s varieties. NHL represents a progressive clonal expansion of B cells, T cells, and/or natural killer (NK) cells stimulated by chromosomal translocations, most commonly t(14,18); by the inactivation of tumor suppressor genes; or by the introduction of exogenous genes by oncogenic viruses (e.g., EBV, HTLV-1, HCV). There is a strong association between H. pylori infection and MALT gastric lymphoma. Most NHLs (almost 85%) are of B-cell origin. NHL is the most common hematopoietic neoplasm and is five times more common than Hodgkin’s lymphoma. The median patient age is > 50 years, but NHL may also be found in children, who tend to have more aggressive, higher-grade disease. Patient presentation varies with disease grade (see Table 2.7-8).

1	The median patient age is > 50 years, but NHL may also be found in children, who tend to have more aggressive, higher-grade disease. Patient presentation varies with disease grade (see Table 2.7-8). Excisional lymph node biopsy is necessary for diagnosis; the disease may first present at an extranodal site, which should be biopsied for diagnosis as well. A CSF exam should be done in patients with HIV, neurologic signs or symptoms, or 1° CNS lymphoma. Disease staging (Ann Arbor classif cation) is based on the number of nodes and on whether the disease crosses the diaphragm. ■Treatment is based on histopathologic classification rather than on stage. Symptomatic patients are treated with radiation and chemotherapy T AB LE 2.7 -8. Presentation of Non-Hodgkin’s Lymphoma

1	■Treatment is based on histopathologic classification rather than on stage. Symptomatic patients are treated with radiation and chemotherapy T AB LE 2.7 -8. Presentation of Non-Hodgkin’s Lymphoma Painless peripheral adenopathy. Cytopenia from bone marrow involvement. Fatigue and weakness. Peripheral adenopathy, splenomegaly, hepatomegaly. Adenopathy. Extranodal disease (GI, GU, skin, thyroid, CNS). B symptoms (temperature > 38.5°C, night sweats, weight loss). Mass formation (e.g., abdominal mass with bowel obstruction in Burkitt’s lymphoma; mediastinal mass and SVC syndrome in lymphoblastic lymphoma). Bulky adenopathy, splenomegaly, hepatomegaly. Masses (abdominal, testicular, mediastinal). Skin f ndings. Low Intermediate to high (CHOP: cyclophosphamide [Cytoxan], Adriamycin, Oncovin [vincristine], and prednisone).

1	■The rule of thumb is for low-grade, indolent NHL to be treated with a palliative approach in symptomatic patients, and for high-grade, aggressive NHL to be treated aggressively with a curative approach. A predominantly B-cell malignancy with an unclear etiology. There is a possible association with EBV. HD has a bimodal age distribution, peaking first in the third decade (primarily the nodular sclerosing type) and then in the elderly at around age 60 (mainly the lymphocyte-depleted type). It has a male predominance in childhood. HD commonly presents as cervical adenopathy, although it may also present as a mediastinal mass; it is usually found above the diaphragm, with infradiaphragmatic involvement suggesting more widely disseminated disease. Patients also have systemic B symptoms, pruritus, and hepatosplenomegaly. Pel-Ebstein fevers (1–2 weeks of high fever alternating with 1–2 afebrile weeks) and alcohol-induced pain at nodal sites are rare signs that are specific for HD.

1	Fine-needle biopsy is usually nondiagnostic, so diagnosis is usually made by excisional lymph node biopsy, which is examined for the classic Reed-Sternberg (RS) cells (giant abnormal B cells with bilobar nuclei and huge, eosinophilic nucleoli, which create an “owl’s-eye” appearance) and for abnormal nodal morphology. Histologic types, in descending order of frequency, include nodular sclerosing, mixed cellularity, lymphocyte predominant, and lymphocyte depleted. Staging is based on the number of nodes, the presence of B symptoms, and whether the disease crosses the diaphragm; staging laparotomy is not recommended. Treatment is stage dependent, involving chemotherapy and/or radiation. Radiation is directed toward the involved lymph node area plus the next contiguous region. Chemotherapy regimens used include ABVD (Adriamycin, bleomycin, vinblastine, dacarbazine) and MOPP (mechlorethamine, Oncovin [vincristine], procarbazine, and prednisone).

1	Five-year survival rates are very good and are 90% for stage I and II disease (nodal disease limited to one side of the diaphragm), 84% for stage III, and 65% for stage IV. Chemotherapy and radiation can lead to 2° neoplasms such as AML, NHL, breast cancer, and thyroid cancer. Clonal proliferation of malignant plasma cells at varying stages of differentiation, with excessive production of monoclonal immunoglobulins or immunoglobulin fragments (kappa/lambda light chains). It is commonly believed The combination of anemia and bone pain must always raise suspicion of multiple myeloma. Bone pain at rest should raise concern for malignancy. Hypercalcemia manifests in symptoms of polyuria, constipation, confusion, nausea, vomiting, and lethargy. Because multiple myeloma is an osteoclastic process, a bone scan, which detects osteoblastic activity, may be .

1	Because multiple myeloma is an osteoclastic process, a bone scan, which detects osteoblastic activity, may be . to be a disease of the elderly, with a peak incidence in the seventh decade. Risk factors for disease development include radiation; monoclonal gammopathy of undetermined significance (MGUS); and, possibly, petroleum, pesticides, and other chemicals. Patients present with anemia, plasmacytosis of the bone marrow, lytic bone lesions, hypercalcemia, and renal abnormalities. They are prone to infection and have elevated monoclonal (M) proteins in the serum and/or urine. The classic triad of diagnostic criteria are > 10% plasma cells in the bone marrow and/or histologically proven plasma cell infiltration, M protein in serum or urine, and evidence of lytic bone lesions.

1	The presence of M proteins alone is insuffcient for the diagnosis of multiple myeloma; MGUS is relatively common. Other lymphoproliferative diseases may also result in M proteins, including CLL, lymphoma, Waldenström’s macroglobulinemia, and amyloidosis. Patients should be evaluated with a skeletal survey, a bone marrow biopsy, serum and urine protein electrophoresis, and CBC. Treat with chemotherapy. Common initial treatment involves a combination of melphalan (an oral alkylating agent) and prednisone and other agents. Myeloma cells tend to become resistant to drugs by an MDR gene mechanism, and autologous stem cell transplantation may be used to support more intensive doses of chemotherapy.

1	A clonal disorder of B cells that leads to a malignant monoclonal gammopathy. Elevated levels of IgM result in hyperviscosity syndrome, coagulation abnormalities, cryoglobulinemia, cold agglutinin disease (leading to autoimmune hemolytic anemia), and amyloidosis. Tissue is infiltrated by IgM and neoplastic plasma cells. A chronic, indolent disease of the elderly. Presents with nonspecific symptoms of lethargy and weight loss along with Raynaud’s phenomenon from cryoglobulinemia. Patients complain of neurologic problems ranging from mental status changes to sensorimotor peripheral neuropathy and blurry vision. Organomegaly and organ dysfunction affecting the skin, GI tract, kidneys, and lungs are also seen. As with multiple myeloma, MGUS is a precursor to disease. Labs show elevated ESR, uric acid, LDH, and alkaline phosphatase.

1	As with multiple myeloma, MGUS is a precursor to disease. Labs show elevated ESR, uric acid, LDH, and alkaline phosphatase. Bone marrow biopsy and aspirate are required to establish the diagnosis. Marrow shows abnormal plasma cells, classically with Dutcher bodies (PASIgM deposits around the nucleus). Serum and urine protein electrophoresis and immunofixation are also used. Excess immunoglobulin is removed with plasmapheresis; underlying lymphoma is treated with chemotherapy. A generic term referring to extracellular deposition of protein fibrils. There are many different kinds of amyloidosis, involving different types of deposited fibrils with varying etiologies (see Table 2.7-9). Classically a disease of the elderly.

1	The clinical presentation depends on the type of precursor protein, tissue distribution, and the amount of amyloid deposition. In the two most common forms of systemic amyloidosis, 1° (AL) and 2° (AA), the major sites of clinically important amyloid deposition are in the kidneys, heart, and liver. In some disorders, clinically important amyloid deposition is limited to one organ (e.g., the brain in Alzheimer’s disease). Diagnosed by the clinical picture; confirmed by tissue biopsy with Congo red staining showing apple-green birefringence under polarized light. 1° amyloidosis is treated with experimental chemotherapy to reduce protein burden; in 2° amyloidosis, the underlying condition should be addressed. Transplantation is also used; kidney transplantation may cure dialysis-related amyloid, and liver transplantation may cure heritable amyloid. TABLE 2.7-9. Types of Amyloidosis

1	TABLE 2.7-9. Types of Amyloidosis AL A plasma cell dyscrasia, with deposition of monoclonal light-chain fragments. Associated with multiple myeloma and Waldenstr’s macroglobulinemia. AA Deposition of the acute-phase reactant serum amyloid A. Associated with chronic infammatory diseases (e.g., rheumatoid arthritis), infections, and neoplasms. Dialysis-related Deposition of β2 microglobulin, which accumulates in patients on long-term dialysis. Heritable Deposition of abnormal gene products (e.g., transthyretin, aka prealbumin). A heterogeneous group of disorders. Senile-systemic Deposition of otherwise normal transthyretin. An absolute neutrophil count (ANC) < 1500, where ANC = (WBC count) (% bands + % segmented neutrophils) (0.01). Neutropenia may be due to a combination of ↓ production, sequestration to marginated or tissue pools, and ↑ destruction or utilization. It may be acquired or intrinsic (see Table 2.7-10).

1	Patients are at ↑ risk of infection, which varies inversely with neutrophil count. Acute neutropenia: Associated with S. aureus, Pseudomonas, E. coli, Proteus, and Klebsiella sepsis. Chronic and autoimmune neutropenia: Presents with recurrent sinusitis, stomatitis, gingivitis, and perirectal infections rather than sepsis. Some chronic neutropenias are accompanied by splenomegaly (e.g., Felty’s syndrome, Gaucher’s disease, sarcoidosis). The history and physical exam are the cornerstones of diagnosis. CBC with ANC may be used to follow neutropenia. If thrombocytopenia or anemia is present, bone marrow biopsy and aspirate should be performed. Serum immunologic evaluation, ANA levels, and a workup for collagen vascular disease may be merited. T AB LE 2.7 -1 0. Acquired vs. Intrinsic Neutropenia

1	HEMATOLOGY/ONCOLOGY Acquired Drug induced (e.g., chemotherapy, ethanol, antibiotics, NSAIDs), usually by marrow suppression. Marrow-inf ltrating disorders. Postinfectious. HIV infection. Benign familial leukopenia (seen in Yemenite Jews, West Indians, and people of African descent; due to genetic variation). Chronic idiopathic neutropenia (occurs in infancy and childhood; thought to be due to production of antineutrophil IgG). Autoimmune neutropenia (isolated or 2° to rheumatoid arthritis or SLE). Nutritional def ciency (B12/folate or thiamine def ciency). Metabolic disease (ketoacidosis, hyperglycinuria, orotic aciduria, MMA, hypothyroidism, Gaucher’s disease). Intrinsic Dyskeratosis congenita (X-linked, with integument abnormalities and hypocellular marrow). Kostmann’s syndrome (aka infantile agranulocytosis). Shwachman-Diamond-Oski syndrome (neutropenia, metaphyseal dysplasia, and pancreatic insuff ciency). Chédiak-Higashi syndrome (oculocutaneous albinism, neurologic impairment,

1	agranulocytosis). Shwachman-Diamond-Oski syndrome (neutropenia, metaphyseal dysplasia, and pancreatic insuff ciency). Chédiak-Higashi syndrome (oculocutaneous albinism, neurologic impairment, and giant granules in cells). Fanconi’s anemia.

1	■Infection management is most important; patients may not be able to mount an inﬂammatory response to infection owing to their lack of neutrophils. Fever in the context of neutropenia should be treated immediately with broad-spectrum antibiotics. Suspected fungal infections should be treated by fungemia. appropriately as well. Hematopoietic stem cell factors such as G-CSF can be used to shorten the duration of neutropenia. In some instances, IVIG and allogeneic bone marrow transplantation may be used. Absolute eosinophil count = (WBC) (% eosinophils) (0.01). Normal levels do not exceed 350. Eosinophilia can be triggered by the overproduction of one or more of three eosinophilopoietic cytokines (IL-3, IL-5, and GM-CSF) or by chemokines that stimulate the migration of eosinophils into peripheral blood and tissues. Eosinophilia may be a 1° disorder, but it usually occurs 2° to another cause (see Table 2.7-11).

1	A travel, medication, atopic, and diet history should be elicited along with a history of symptoms relating to lymphoma/leukemia. Physical exam findings vary. Patients with hypereosinophilic syndrome (HES) may present with fever, anemia, and prominent cardiac findings (emboli from mural thrombi, abnormal ECGs, CHF, murmurs). Other affected organs include the lung, liver, spleen, skin, and nervous system (due to eosinophilic infiltration). ■In addition to a history and physical, a CBC and differential should be obtained, and CSF should be analyzed for eosinophilia, which is suggestive T AB LE 2.7 -1 1. Etiologies of Eosinophilia 1° Hypereosinophilic syndrome: Unknown etiology. Hereditary eosinophilia: Autosomal-dominant inheritance; rare. Eosinophilia-myalgia syndrome: Results from abnormal tryptophan metabolism. 2° Allergic states with elevated serum IgE: The most common cause in the United States. Parasitic diseases: The most common cause worldwide. Other:

1	Allergic states with elevated serum IgE: The most common cause in the United States. Parasitic diseases: The most common cause worldwide. Other: Coccidioidomycosis. Vasculitis (e.g., Churg-Strauss syndrome). Benign or malignant hematologic disorders; also solid tumors. Collagen vascular diseases (e.g., dermatomyositis, PAN). Drug induced (e.g., sulfonamides, iodides, ASA, phenytoin). of a drug reaction or infection with a coccidioidomycosis or a helminth. Hematuria with eosinophilia may be a sign of schistosomiasis. ■Imaging of the lungs, abdomen, pelvis, and brain may demonstrate a focal defect that may be helpful in narrowing down the potential causes. Medication should be tailored to the cause of the eosinophilia. HES is treated with corticosteroid and cytotoxic agents to ↓ the eosinophilia. Tissue transplantation is increasingly used to treat a variety of diseases. Types of transplantation include the following: Autologous: Transplantation from the patient to him/herself.

1	Tissue transplantation is increasingly used to treat a variety of diseases. Types of transplantation include the following: Autologous: Transplantation from the patient to him/herself. Allogeneic: Transplantation from a donor to a genetically different patient. Syngeneic: Transplantation between identical twins (i.e., from a donor to a genetically identical patient). With allogeneic donation, efforts are made to ABO and HLA match the donor and recipient. Even with antigenic matching and immunosuppression, however, transplants may be rejected. There are three types of rejection: hyperacute, acute, and chronic (see Table 2.7-12). Graft-versus-host disease (GVHD) is a complication specific to allogeneic bone marrow transplantation in which donated T cells attack host tissues. It may be acute (occurring < 100 days post-transplant) or chronic (occurring > 100 days afterward).

1	Minor histocompatibility antigens are thought to be responsible for GVHD, which typically presents with skin changes, cholestatic liver dysfunction, obstructive lung disease, or GI problems. Patients are treated with high-dose corticosteroids. T AB LE 2.7 -1 2. Types of Transplant Rejection Timing after transplant Within minutes. Five days to three months. Months to years. Pathomechanism Preformed antibodies. T-cell mediated. Chronic immune reaction causing f brosis. Tissue fndings Vascular thrombi; tissue ischemia. Laboratory evidence of tissue destruction such as ↑ GGT, alkaline phosphatase, LDH, BUN, or creatinine. Gradual loss of organ function. Prevention Check ABO compatibility. N/A N/A Treatment Cytotoxic agents. Confrm with sampling of transplanted tissue; treat with corticosteroids, antilymphocyte antibodies (OKT3), tacrolimus, or mycophenolate mofetil (MMF). No treatment; biopsy to rule out treatable acute reaction.

1	The typical regimen after transplant can include these commonly used drugs: prednisone, MMF, FK506 (tacrolimus), TMP-SMX, ganciclovir, and ketoconazole. A variant of GVHD is the graft-versus-leukemia effect, in which leukemia patients who are treated with an allogeneic bone marrow transplant have significantly lower relapse rates than those treated with an autologous transplant. This difference is thought to be due to a reaction of donated T cells against leukemic cells. Table 2.7-13 outlines conditions that are commonly associated with neoplasms. T AB LE 2.7 -1 3. Disorders Associated with Neoplasms

1	Down syndrome ALL (“We will ALL go Down together”). Xeroderma pigmentosum Squamous cell and basal cell carcinomas of the skin. Chronic atrophic gastritis, pernicious anemia, postsurgical gastric remnants Gastric adenocarcinoma. Tuberous sclerosis (facial angiofbroma, seizures, mental retardation) Astrocytoma and cardiac rhabdomyoma. Actinic keratosis Squamous cell carcinoma of the skin. Barrett’s esophagus (chronic GI ref ux) Esophageal adenocarcinoma. Plummer-Vinson syndrome (atrophic glossitis, esophageal webs, anemia; all due to iron def ciency) Squamous cell carcinoma of the esophagus. Cirrhosis (alcoholic, HBV or HCV) Hepatocellular carcinoma. Ulcerative colitis Colonic adenocarcinoma. Paget’s disease of bone 2° osteosarcoma and f brosarcoma. AIDS Aggressive malignant NHLs and Kaposi’s sarcoma. Autoimmune diseases (e.g., myasthenia gravis) Benign and malignant thymomas. Acanthosis nigricans (hyperpigmentation and epidermal thickening) Visceral malignancy (stomach, lung, breast,

1	sarcoma. Autoimmune diseases (e.g., myasthenia gravis) Benign and malignant thymomas. Acanthosis nigricans (hyperpigmentation and epidermal thickening) Visceral malignancy (stomach, lung, breast, uterus). Dysplastic nevus Malignant melanoma. Immunodefciency states Malignant lymphomas.

1	Infections of the Eyes and Ears 248 INFECTIOUS CONJUNCTIVITIS 248 ORBITAL CELLULITIS 248 OTITIS EXTERNA 249 Some common causes of pneumonia are outlined in Table 2.8-1. May present classically or atypically. Classic symptoms: Sudden onset, fever, productive cough (purulent yellow-green sputum or hemoptysis), dyspnea, night sweats, pleuritic chest pain. Atypical symptoms: Gradual onset, dry cough, headaches, myalgias, sore throat. Lung exam may show ↓ or bronchial breath sounds, rales, wheezing, dullness to percussion, egophony, and tactile fremitus. Elderly patients as well as those with COPD, diabetes, or immunocompromised status may have minimal or atypical signs on physical exam. Workup includes physical exam, CXR, CBC, sputum Gram stain and culture (see Figures 2.8-1 and 2.8-2), nasopharyngeal aspirate, blood culture, and ABG. Tests for specifc pathogens include the following:

1	Tests for specifc pathogens include the following: Legionella: Urine Legionella antigen test (detects only serogroup 1), sputum staining with direct fuorescent antibody (DFA), culture. Chlamydia pneumoniae: Serologic testing, culture, PCR. TABLE 2.8-1. Common Causes of Pneumonia

1	TABLE 2.8-1. Common Causes of Pneumonia Atypical Nosocomial (hospital acquired) Immunocompromised Mycoplasma, Legionella, Chlamydia Gramrods (GNRs), Staphylococcus, anaerobes Staphylococcus, gramrods, fungi, viruses, Pneumocystis jiroveci (with HIV), mycobacteria Aspiration Anaerobes Alcoholics/IV drug users S. pneumoniae, Klebsiella, Staphylococcus Cystic fbrosis (CF) Pseudomonas, Burkholderia, S. aureus, mycobacteria COPD H. inﬂ uenzae, Moraxella catarrhalis, S. pneumoniae Postviral Staphylococcus, H. inﬂuenzae Neonate Group B streptococci (GBS), E. coli Recurrent Obstruction, bronchogenic carcinoma, lymphoma, Wegener’s granulomatosis, immunodefciency, unusual organisms (e.g., Nocardia, Coxiella burnetii, Aspergillus, Pseudomonas) Viruses (RSV) Mycoplasma Chlamydia pneumoniae Streptococcus pneumoniae Mycoplasma C. pneumoniae S. pneumoniae S. pneumoniae H. inﬂ uenzae Anaerobes Viruses Mycoplasma S. pneumoniae Viruses Anaerobes H. inﬂ uenzae Gram-rods Special groups:

1	An adequate sputum Gram stain sample has many PMNs (> 25 cells/hpf) and few epithelial cells (< 25 cells/hpf). FIGURE 2.8-1. S. aureus. These clusters of gramcocci were isolated from the sputum of a patient who developed pneumonia while hospitalized. Mycoplasma: Usually clinical. Serum cold agglutinins and serum Mycoplasma antigen may also be used. Streptococcus pneumoniae: Urine pneumococcal antigen test, culture. Viral: Nasopharyngeal aspirate, rapid tests for pathogens (e.g., inf uenza, RSV), DFA, viral culture. FIGURE 2.8-2. S. pneumoniae. Sputum sample from a patient with pneumonia. Note the characteristic lancet-shaped gramdiplococci. Table 2.8-2 summarizes the recommended initial treatment for pneumonia. Outpatient treatment with oral antibiotics is recommended only in uncomplicated cases.

1	Table 2.8-2 summarizes the recommended initial treatment for pneumonia. Outpatient treatment with oral antibiotics is recommended only in uncomplicated cases. In-hospital treatment with IV antibiotics is recommended for patients > 65 years of age and in those with comorbidity (alcoholism, COPD, diabetes, malnutrition), immunosuppression, unstable vitals or signs of respiratory failure, altered mental status, and/or multilobar involvement. For patients with obstructive diseases (e.g., CF or bronchiectasis), consider adding pseudomonal, staphylococcal, or anaerobic coverage. Infection due to Mycobacterium tuberculosis. Initial infection usually leads to latent TB infection (LTBI) that is asymptomatic. Most symptomatic cases (i.e., cases of active disease) are due to reactivation of latent infection rather TABLE 2.8-2. Treatment of Pneumonia

1	TABLE 2.8-2. Treatment of Pneumonia The PORT criteria (i.e., pneumonia severity index) risk-stratifies patients with pneumonia on the basis of age, comorbidity, and presentation. Note: The PORT criteria do not apply to AIDS patients.

1	Outpatient community-acquired pneumonia, patients ≤ 65 years of age, otherwise healthy S. pneumoniae, Mycoplasma pneumoniae, C. pneumoniae, H. inﬂ uenzae, viral. Macrolide (azithromycin), doxycycline, or f uoroquinolone. Patients > 65 years of age or with comorbidity (COPD, heart failure, renal failure, diabetes, liver disease, EtOH abuse) S. pneumoniae, H. inﬂ uenzae, aerobic GNRs (E. coli, Enterobacter, Klebsiella), S. aureus, Legionella, viruses. Macrolide or f uoroquinolone. Consider adding a second-generation cephalosporin or β-lactam to the macrolide. Community-acquired pneumonia requiring hospitalization S. pneumoniae, H. inﬂ uenzae, anaerobes, aerobic GNRs, Legionella, Chlamydia. Extended-spectrum cephalosporin, β-lactam/β-lactamase inhibitor, or fuoroquinolone. Add a macrolide if atypical organisms are suspected. Community-acquired pneumonia requiring ICU care S. pneumoniae, H. inﬂ uenzae, anaerobes, aerobic GNRs, Mycoplasma, Legionella, Pseudomonas. Fluoroquinolone or

1	if atypical organisms are suspected. Community-acquired pneumonia requiring ICU care S. pneumoniae, H. inﬂ uenzae, anaerobes, aerobic GNRs, Mycoplasma, Legionella, Pseudomonas. Fluoroquinolone or extended-spectrum cephalosporin or β-lactam/β-lactamase inhibitor + macrolide. Institution-/hospital-acquired pneumonia—patients hospitalized > 48 hours or in a long-term care facility > 14 days GNRs (including Pseudomonas and Acinetobacter), S. aureus, Legionella, mixed f ora. Extended-spectrum cephalosporin or β-lactam with antipseudomonal activity or carbapenem. Consider adding an aminoglycoside or a f uoroquinolone for coverage of resistant organisms (Pseudomonas) until lab sensitivities identify the best single agent. Patients who are critically ill or worsening over 24–48 hours on initial antibiotic therapy Methicillin-resistant S. aureus (MRSA). Add vancomycin or linezolid; broader gram-coverage.

1	TB almost always presents with an extended duration (> 3 weeks) of symptoms. than to 1° exposure. Pulmonary TB is most common, but disseminated or extrapulmonary TB should be considered as well. TB can infect almost any organ system, including the lungs, CNS, GU tract, bone, and GI tract. Risk factors for active disease (i.e., reactivation) include immunosuppression (HIV), alcoholism, preexisting lung disease, diabetes, and advancing age. Risk factors for TB exposure include homelessness and crowded living conditions (e.g., prison), immigration/travel from developing nations, working in an allied health profession, and interacting with known TB contacts.

1	Presents with cough, hemoptysis, dyspnea, weight loss, fatigue, night sweats, fever, cachexia, hypoxia, tachycardia, lymphadenopathy, an abnormal lung exam, and a prolonged (> 3-week) symptom duration. TB is a common cause of fever of unknown origin (FUO). HIV patients can present with atypical signs and symptoms and have higher rates of extrapulmonary TB. ■ Active disease: Mycobacterial culture of sputum (or blood/tissue for extrapulmonary disease) is the gold standard but can take weeks to obtain. A sputum acid-fast stain (see Figure 2.8-3) can yield rapid preliminary results but lacks sensitivity. Three A.M. sputum samples for AFB stain and a mycobacterial culture are advised. If the results of the stain are but there is a high degree of clinical suspicion, proceed to bronchoscopy with bronchoalveolar lavage or biopsy. HIV patients have a high rate of sputum stains (i.e., a AFB smear accompanied by a culture).

1	AFB smear accompanied by a culture). The most common fnding among typical hosts is a cavitary inf ltrate in the upper lobe on CXR. HIV patients or those with 1° TB may show lower lobe infltrates with or without cavitation. Multiple f ne nodular F IGU R E 2.8-3. TB organisms are identifed by their red color (“red snappers”) on acid-fast staining. (Reproduced, with permission, from the Pathology Education Instructional Resource Digital Library [ http://peir.net] at the University of Alabama, Birmingham.) densities distributed throughout both lungs are typical of miliary TB, which represents hematologic or lymphatic dissemination. ■LTBI (i.e., previous exposure): Diagnose with a PPD test (see Figure 2.8-4). Immunocompromised individuals with LTBI may not mount a PPD (anergy). Interferon-gamma release assays are now available to diagnose LTBI as well. All cases of LTBI should be evaluated (e.g., with a CXR) to rule out active disease.

1	All cases should be reported to local and state health departments. Respiratory isolation should be instituted if TB is suspected. Treatment is as follows: Active TB: Directly observed multidrug therapy with a four-drug regimen (INH, pyrazinamide, rifampin, ethambutol) × 2 months, followed by four months with INH and rifampin. Administer vitamin B6 (pyridoxine) with INH to prevent peripheral neuritis. Latent TB: For conversion of PPD without signs/symptoms of active disease, initiate therapy with INH × 9 months. Alternative regimens include INH × 6 months or rifampin × 4 months. Viral causes are more common (90% in adults), but it is important to identify streptococcal pharyngitis (group A β-hemolytic Streptococcus pyogenes). Etiologies are as follows: Bacterial: Group A streptococci (GAS), Neisseria gonorrhoeae, Corynebacterium diphtheriae, M. pneumoniae. Viral: Rhinovirus, coronavirus, adenovirus, HSV, EBV, CMV, inf uenza virus, coxsackievirus, acute HIV infection.

1	Viral: Rhinovirus, coronavirus, adenovirus, HSV, EBV, CMV, inf uenza virus, coxsackievirus, acute HIV infection. Typical of streptococcal pharyngitis: Fever, sore throat, pharyngeal erythema, tonsillar exudate, cervical lymphadenopathy, soft palate petechiae, headache, vomiting, scarlatiniform rash (indicates scarlet fever). Atypical of streptococcal pharyngitis: Coryza, hoarseness, rhinorrhea, cough, conjunctivitis, anterior stomatitis, ulcerative lesions, GI symptoms. Rifampin turns body ﬂ uids orange. Ethambutol can cause optic neuritis. INH causes peripheral neuritis and hepatitis.

1	Rifampin turns body ﬂ uids orange. Ethambutol can cause optic neuritis. INH causes peripheral neuritis and hepatitis. PPD is injected intradermally on the volar surface of the forearm. The diameter of induration is measured at 48–72 hours. BCG vaccination typically renders a patient PPD but should not preclude prophylaxis as recommended for unvaccinated individuals. The size of induration that indicates a test is interpreted as follows: ≥ 5 mm: HIV or risk factors, close TB contacts, CXR evidence of TB. ≥ 10 mm: Indigent/homeless, residents of developing nations, IV drug use, chronic illness, residents of health and correctional institutions, and health care workers. ≥ 15 mm: Everyone else, including those with no known risk factors. A reaction with controls implies anergy from immunosuppression, old age, or malnutrition and thus does not rule out TB. FIGURE 2.8-4. PPD interpretation.

1	A reaction with controls implies anergy from immunosuppression, old age, or malnutrition and thus does not rule out TB. FIGURE 2.8-4. PPD interpretation. The Centor criteria for identifying streptococcal pharyngitis are fever, tonsillar exudate, tender anterior cervical lymphadenopathy, and lack of cough (three of four are required). It is important to identify and treat streptococcal pharyngitis in order to prevent rheumatic fever, but overdiagnosis and overtreatment may lead to ↑ cost and antibiotic resistance. Always consider occult sinusitis in febrile ICU patients. Diagnosed by clinical evaluation, rapid GAS antigen detection, and throat culture. With three out of four of the Centor criteria, the sensitivity of rapid antigen testing is > 90%.

1	Diagnosed by clinical evaluation, rapid GAS antigen detection, and throat culture. With three out of four of the Centor criteria, the sensitivity of rapid antigen testing is > 90%. If GAS is suspected, begin empiric antibiotic therapy with penicillin × 10 days. Cephalosporins, amoxicillin, and azithromycin are alternative options. Symptom relief can be attained with fuids, rest, antipyretics, and salt-water gargles. Nonsuppurative: Acute rheumatic fever (see the Cardiovascular chapter), poststreptococcal glomerulonephritis. Suppurative: Cervical lymphadenitis, mastoiditis, sinusitis, otitis media, retropharyngeal or peritonsillar abscess, and, rarely, thrombophlebitis of the jugular vein (Lemierre’s syndrome) due to Fusobacterium, an oral anaerobe.

1	Peritonsillar abscess may present with odynophagia, trismus (“lockjaw”), a muffed voice, unilateral tonsillar enlargement, and erythema, with the uvula and soft palate deviated away from the affected side. Culture abscess fuid and localize the abscess via intraoral ultrasound or CT. Treat with antibiotics and surgical drainage. Refers to infammation of the paranasal sinuses. The maxillary sinuses are most commonly affected. Can be classifed by site, organism, or chronicity. Subtypes include the following: Acute sinusitis (symptoms lasting < 1 month): Most commonly associated with viruses, S. pneumoniae, H. inﬂ uenzae, and M. catarrhalis. Bacterial causes are rare and are characterized by symptoms lasting < 1 week. Chronic sinusitis (symptoms persisting > 3 months): Represents a chronic infammatory process. Often due to obstruction of sinus drainage and ongoing low-grade anaerobic infections. In diabetic patients, mucormycosis should be considered.

1	Presents with fever, facial pain/pressure, headache, nasal congestion, and discharge. Exam may reveal tenderness, erythema, and swelling over the affected area. High fever, leukocytosis, and a purulent nasal discharge are suggestive of acute bacterial sinusitis. A clinical diagnosis. Culture and radiography are generally not required for acute sinusitis but may guide the management of chronic cases. Transillumination shows opacifcation of the sinuses (low sensitivity). CT is the test of choice for sinus imaging (see Figure 2.8-5) but is usually necessary only if symptoms persist after treatment. MRI is useful for differentiating soft tissue (as in a tumor) from mucus. FIGURE 2.8-5. Sinusitis.

1	FIGURE 2.8-5. Sinusitis. Compare the opacifed left maxillary sinus and normal air-flled right sinus on this coronal CT scan. (Reproduced, with permission, from Lalwani AK. Current Diagnosis & Treatment in Oto laryngology: Head and Neck Surgery, 2nd ed. New York: McGraw-Hill, 2008: Fig. 14-2.) ■Bacterial culture by sinus tap is the gold standard for diagnosis but is not routinely performed because of discomfort. Endoscopically guided cultures from the middle meatus are gaining popularity. Most cases of acute sinusitis are viral and/or self-limited and can thus be treated with symptomatic therapy (decongestants, antihistamines, pain relief). Acute bacterial sinusitis (usually < 7 days): Consider amoxicillin/clavulanate 500 mg PO TID × 10 days or, alternatively, clarithromycin, azithromycin, TMP-SMX, fuoroquinolone, or a second-generation cephalosporin × 10 days.

1	Chronic sinusitis (4–12 weeks): Adjuvant therapy with intranasal corticosteroids, decongestants, and antihistamines may be useful in combating the allergic/infammatory component of the disease. Antibiotics given are similar to those used for acute disease, although a longer course (3–6 weeks) may be necessary. Surgical intervention may also be required. A pulmonary fungal infection endemic to the southwestern United States (e.g., San Joaquin Valley, California). Can present as an acute or subacute pneumonia or as a fulike illness, and may involve extrapulmonary sites, including bone, CNS, and skin. The incubation period is 1–4 weeks after exposure. Filipino, African-American, pregnant, and HIV-patients are at ↑ risk. Potential complications of sinusitis include meningitis, frontal bone osteomyelitis, cavernous sinus thrombosis, and abscess formation.

1	Potential complications of sinusitis include meningitis, frontal bone osteomyelitis, cavernous sinus thrombosis, and abscess formation. Consider coccidioidomycosis in the HIV-, Filipino, African-American, or pregnant patient from the southwestern United States with respiratory infection. Patients present with fever, anorexia, headache, chest pain, cough, dyspnea, arthralgias, and night sweats. Disseminated infection can present with meningitis, bone lesions, and soft tissue abscesses. Obtain bronchoalveolar lavage along with fungal cultures of sputum, wound exudate, or other affected tissue. Identify Coccidioides immitis spherules on H&E or other special stains of sputum or tissue. Precipitin antibodies (IgM) ↑ within two weeks and disappear after two months; complement fxation antibodies (IgG) ↑ at 1–3 months. Titers > 1:16 indicate disseminated infection. CXR fndings may be normal or may show infltrates, nodules, cavity, mediastinal or hilar adenopathy, or pleural effusion.

1	CXR fndings may be normal or may show infltrates, nodules, cavity, mediastinal or hilar adenopathy, or pleural effusion. Consider bronchoscopy, fne-needle biopsy, open lung biopsy, or pleural biopsy if serology is indeterminate. Acute: IV therapy is rarely necessary; however, consider IV amphotericin B for severe or protracted 1° pulmonary infection and disseminated disease. PO fuconazole or itraconazole may be used for mild infection or continuation therapy once the patient is stable. Chronic: No treatment is needed for asymptomatic chronic pulmonary nodules or cavities. Progressive cavitary or symptomatic disease usually requires surgery plus long-term azole therapy for 8–12 months. A highly contagious orthomyxovirus transmitted by droplet nuclei. There are three types of infuenza: A, B, and C. Subtypes of infuenza A (e.g., H5N1, H1N1) are classifed on the basis of glycoproteins (hemagglutinin and neuraminidase). Relevant terms are as follows:

1	Antigenic drift: Refers to small, gradual changes in surface proteins through point mutations. These small changes are suffcient to allow the virus to escape immune recognition, accounting for the fact that individuals can be infected with infuenza multiple times. Antigenic shift: Refers to an acute, major change in the infuenza A subtype (signifcant genetic reassortment) circulating among humans; leads to pandemics. In the United States, the typical infuenza season begins in November and lasts until March. Vaccination with inactivated infuenza virus is recommended for patients > 50 years of age, children 6 months to 19 years of age, patients of any age with chronic medical problems (e.g., diabetes, heart disease, renal failure, HIV), pregnant women, nursing home residents, and contacts of high-risk groups (e.g., health care workers).

1	Patients typically present with abrupt onset of fevers, myalgias, chills, cough, coryza, and weakness. Elderly patients may have atypical presentations characterized only by confusion. Leukopenia is a common fnding. Rapid infuenza tests of viral antigens from nasopharyngeal swabs are available. More defnitive diagnosis can be made with DFA tests or viral culture. Symptomatic care with analgesics and cough medicine. Antivirals such as oseltamivir or zanamivir are most effective when used within two days of onset and may shorten the course of infection by 1–2 days. Severe 1° viral pneumonia, 2° bacterial pneumonia, sinusitis, bronchitis, and exacerbation of COPD and asthma can occur. Reye’s syndrome, or fatty liver encephalopathy, has been associated with ASA use in children with viral infections, including inf uenza. A live attenuated, nasally delivered inﬂ uenza vaccine is available for healthy people 2–49 years of age who are not pregnant or severely immunocompromised.

1	A live attenuated, nasally delivered inﬂ uenza vaccine is available for healthy people 2–49 years of age who are not pregnant or severely immunocompromised. Risk factors for meningitis include recent ear infection, sinusitis, immunodef ciencies, recent neurosurgical procedures, and sick contacts. Causes are listed in Table 2.8-3. Patients present with fever, malaise, headache, neck stiffness, photophobia, altered mental status, nausea/vomiting, seizures, or signs of meningeal irritation ( Kernig’s and Brudzinski’s signs). Obtain blood cultures. LP for CSF Gram stain and culture; obtain glucose, protein, WBC count plus differential, RBC count, and opening pressure (in the absence of papilledema or focal neurologic def cits). Viral PCRs (e.g., HSV); cryptococcal antigen (for HIV patients). CT or MRI to rule out other diagnoses. CBC may reveal leukocytosis; CSF fndings vary (see Table 2.8-4). TAB LE 2.8-3. Causes of Meningitisa,b

1	CT or MRI to rule out other diagnoses. CBC may reveal leukocytosis; CSF fndings vary (see Table 2.8-4). TAB LE 2.8-3. Causes of Meningitisa,b GBS E. coli/GNRs Listeria S. pneumoniae Neisseria meningitidis H. inﬂ uenzae type b Enteroviruses N. meningitidis Enteroviruses S. pneumoniae HSV S. pneumoniae GNRs Listeria N. meningitidis a Causes in HIV include Cryptococcus, CMV, HSV, VZV, TB, toxoplasmosis (brain abscess), and JC virus (PML). b Note: The incidence of H. inﬂ uenzae meningitis has ↓ greatly with the introduction of the H. inﬂ uenzae vaccine in the last 10–15 years. TABLE 2.8-4. CSF Prof les

1	TABLE 2.8-4. CSF Prof les Normal < 10 < 5 ∼2/3 of serum 15–45 10–20 Clear 3–12 Bacterial meningitis ↔ ↑ (> 1000 PMNs) ↓ ↑ ↑ Cloudy ↔ or ↑ Viral meningitis ↔ ↑ (monos/ lymphs) ↔ ↔ or ↑ ↔ or ↑ Most often clear ↔ or ↑ Aseptic meningitis ↔ ↑ ↔ ↔ or ↑ ↔ Clear ↔ SAH ↑↑ ↑ ↔ ↑ ↔ or ↑ Yellow/red ↔ or ↑ Guillain-Barré syndrome ↔ ↔ ↔ or ↑ ↑↑ ↔ Clear or yellow (high protein) ↔ MS ↔ ↔ or ↑ ↔ ↔ ↔ Clear ↑↑ Pseudotumor cerebri ↔ ↔ ↔ ↔ ↑↑↑ Clear ↔ Antibiotics should be administered rapidly (see Table 2.8-5) and may be given empirically up to two hours before an LP is performed. Some cases of viral meningitis can be treated with supportive care and close follow-up. Close contacts of patients with meningococcal meningitis should receive rifampin, ciprofoxacin, or ceftriaxone prophylaxis.

1	Close contacts of patients with meningococcal meningitis should receive rifampin, ciprofoxacin, or ceftriaxone prophylaxis. T AB LE 2.8-5. Empiric Treatment of Bacterial Meningitis < 1 month GBS, E. coli/GNRs, Listeria. Ampicillin + cefotaxime or gentamicin. 1–3 months Pneumococci, meningococci, H. inﬂ uenzae. Vancomycin IV + ceftriaxone or cefotaxime. 3 months – adulthood Pneumococci, meningococci. Vancomycin IV + ceftriaxone or cefotaxime. > 60 years/alcoholism/ chronic illness Pneumococci, gram-bacilli, Listeria, meningococci. Ampicillin + vancomycin + cefotaxime or ceftriaxone. ■ Dexamethasone may be benefcial in bacterial meningitis, especially S. pneumoniae, if given 15–20 minutes before antibiotics. Cerebral edema: Visible on CT/MRI. Presents with loss of oculocephalic refex. Treat with IV mannitol. Subdural effusions: May be seen on CT scan. Occur in 50% of infants with H. inﬂ uenzae meningitis. No treatment is necessary.

1	Subdural effusions: May be seen on CT scan. Occur in 50% of infants with H. inﬂ uenzae meningitis. No treatment is necessary. Ventriculitis/hydrocephalus: Presents as a worsening clinical picture with improved CSF fndings. Requires ventriculostomy and possibly intraventricular antibiotics. Seizures: Treat with benzodiazepines and phenytoin. Hyponatremia: Administer fuids and monitor sodium concentration. Subdural empyema: Presents with intractable seizures. Requires surgical evacuation. Other: Cranial nerve palsies, sensorineural hearing loss, coma, death. HSV and arboviruses are the most common causes of encephalitis. Rarer etiologies include CMV, toxoplasmosis, West Nile virus, VZV, Borrelia, Rickettsia, Legionella, enterovirus, Mycoplasma, and cerebral malaria. Children and the elderly are the most vulnerable.

1	Presents with altered consciousness, headache, fever, and seizures. Lethargy, confusion, coma, and focal neurologic defcits (cranial nerve def cits, accentuated DTRs) may also be present. The differential includes brain abscess or malignancy, toxic-metabolic encephalopathy, subdural hematoma, and SAH. CSF shows lymphocytic pleocytosis and moderately ↑ protein. RBCs without evidence of trauma suggest HSV encephalitis. The glucose level is low in tuberculous, fungal, bacterial, and amebic infections. Obtain a CSF Gram stain (bacteria), acid-fast stain (mycobacteria), India ink (Cryptococcus), wet preparation (free-living amebae), and Giemsa stain (trypanosomes). PCR for HSV, CMV, EBV, VZV, and enterovirus. MRI may demonstrate a contrast-enhancing lesion in the temporal lobe (in HSV).

1	MRI may demonstrate a contrast-enhancing lesion in the temporal lobe (in HSV). HSV encephalitis requires immediate IV acyclovir. CMV encephalitis is treated with IV ganciclovir +/– foscarnet. Give doxycycline for suspected Rocky Mountain spotted fever, Lyme disease, or ehrlichiosis. A focal, suppurative infection of the brain parenchyma, usually with a “ringenhancing” appearance due to fbrous capsule. The most common infective organisms are streptococci, staphylococci, and anaerobes; multiple organisms are often implicated (80–90% of cases are polymicrobial). Nonbacterial Although other medications may be used, rifampin is the frequently tested prophylaxis of choice for close contacts of patients with meningococcal meningitis. The presence of RBCs in CSF without a history of trauma indicates HSV encephalitis. HSV encephalitis is associated with high morbidity. PCR is highly sensitive and specific. A full course of IV acyclovir is mandatory.

1	HSV encephalitis is associated with high morbidity. PCR is highly sensitive and specific. A full course of IV acyclovir is mandatory. The classic clinical triad of headache, fever, and a focal neurologic deficit is present in 50% of cases of brain abscess. When fever is absent, 1° and metastatic brain tumors become the major differential diagnoses. In general, do not LP a patient with a mass lesion in the brain. Some commonly tested AIDS-defining illnesses: causes include Toxoplasma, Aspergillus, and Candida; zygomycosis should be contemplated in immunocompromised hosts, and neurocysticercosis should be considered in relevant epidemiologic settings. Modes of transmission include the following: Direct spread: Due to paranasal sinusitis (10% of cases; frequently affects young males; often due to Streptococcus milleri), otitis media or mastoiditis (33%), or dental infection (2%). Direct inoculation: Affects patients with a history of head trauma or neurosurgical procedures.

1	Direct inoculation: Affects patients with a history of head trauma or neurosurgical procedures. Hematogenous spread (25% of cases): Often shows an MCA distribution with multiple abscesses that are poorly encapsulated and located at the gray-white junction. Headache, drowsiness, inattention, confusion, and seizures are early symptoms, followed by signs of increasing ICP and then a focal neurologic def cit. Headache is the most common symptom and is often dull, constant, and refractory to treatment. ↑ ICP leads to CN III and CN VI def cits. CT scan will show a ring-enhancing lesion with a low-density core. MRI has higher sensitivity for early abscesses and posterior fossa lesions. CSF analysis is not necessary and may precipitate a herniation syndrome. Lab values may show peripheral leukocytosis, ↑ ESR, and ↑ CRP.

1	CSF analysis is not necessary and may precipitate a herniation syndrome. Lab values may show peripheral leukocytosis, ↑ ESR, and ↑ CRP. Initiate broad-spectrum IV antibiotics and surgical drainage (aspiration or excision) if necessary for diagnostic and/or therapeutic purposes. Lesions < 2 cm can often be treated medically. Administer a third-generation cephalosporin + metronidazole +/– vancomycin; give IV therapy for 6–8 weeks followed by 2–3 weeks PO. Obtain serial CT/MRIs to follow resolution. Dexamethasone with taper may be used in severe cases to ↓ cerebral edema; IV mannitol may be used to ↓ ICP. CNS lymphoma, toxoplasmosis, or PML P. jiroveci pneumonia or recurrent bacterial pneumonia A retrovirus that targets and destroys CD4+ T lymphocytes. Infection is characterized by a progressively high rate of viral replication that leads to a progressive decline in CD4+ count (see Figure 2.8-6).

1	CD4+ count: Indicates the degree of immunosuppression; guides therapy and prophylaxis and helps determine prognosis. Viral load: May predict the rate of disease progression; provides indications for treatment and gauges response to antiretroviral therapy. ■In acute HIV (acute infection/seroconversion, acute retroviral syndrome), the initial infection is often asymptomatic, but patients may also present with mononucleosis-like or f ulike symptoms (e.g., fever, lymphadenop ACUTE LATENT IMMUNODEFICIENCY Acute symptoms Opportunistic infections and malignancies CD4 lymphocytes Anti-p24 antibodies Anti-gp120 antibodies Virus, p24 antigen FIGURE 2.8-6. Time course of HIV infection. (Adapted, with permission, from Levinson W, Jawetz E. Medical Microbiology and Immunol ogy: Examination & Board Review, 6th ed. New York: McGraw-Hill, 2000: 276.) athy, maculopapular rash, pharyngitis, diarrhea, nausea/vomiting, weight loss, headache).

1	■HIV may later present as night sweats, weight loss, thrush, recurrent infections, or opportunistic infections. Complications are inversely correlated with CD4+ count (see Figure 2.8-7). F IGU R E 2.8-7. Relationship of CD4+ count to development of opportunistic infections. (Reproduced, with permission, from McPhee SJ et al. Current Medical Diagnosis & Treatment, 48th ed. New York: McGraw-Hill, 2009: Fig. 31-1.) The homozygous CCR5 mutation may confer resistance to HIV infection. HIVpatients who are pregnant should avoid efavirenz. If the patient is not on antiretroviral therapy at the time of delivery, she should be treated with zidovudine (AZT) intrapartum. Infants should receive AZT for six weeks after birth. MMR is the only live vaccine that should be given to HIV patients. Do not give oral polio vaccine to HIV-patients or their contacts. The Major Pathogens Concerning Complete T-Cell Collapse

1	MMR is the only live vaccine that should be given to HIV patients. Do not give oral polio vaccine to HIV-patients or their contacts. The Major Pathogens Concerning Complete T-Cell Collapse ELISA test (high sensitivity, moderate specifcity): Detects anti-HIV antibodies in the bloodstream (can take up to six months to appear after exposure). Western blot (low sensitivity, high specif city): Conf rmatory. Rapid HIV tests are now available. Baseline evaluation should include HIV RNA PCR (viral load), CD4+ cell count, CXR, PPD skin testing, Pap smear, VDRL/RPR, and serologies for CMV, hepatitis, toxoplasmosis, and VZV. Evaluation for acute retroviral syndrome (acute HIV) should include HIV RNA PCR (viral load); ELISA may be .

1	Evaluation for acute retroviral syndrome (acute HIV) should include HIV RNA PCR (viral load); ELISA may be . Initiate antiretroviral therapy for the following: (1) symptomatic patients (i.e., those with AIDS-defning illness) regardless of CD4+ count or viral load; (2) asymptomatic patients with a CD4+ count < 350; (3) pregnant patients; and (4) those with specifc HIV-related conditions (e.g., HIV-associated nephropathy). The initial regimen should generally consist of some combination of two nucleoside/nucleotide reverse transcriptase inhibitors (RTIs) plus either one non-nucleoside RTI (NNRTI) or one protease inhibitor. The most important principle is to select multiple medications (usually at least three) in order to achieve durable treatment response and limit the emergence of resistance. The choice of regimen depends on drug-drug interactions, drug tolerance, patient adherence, and comorbid conditions (e.g., hyperlipidemia, refux). Monotherapy/dual therapy should not be used.

1	The choice of regimen depends on drug-drug interactions, drug tolerance, patient adherence, and comorbid conditions (e.g., hyperlipidemia, refux). Monotherapy/dual therapy should not be used. The goal of therapy is viral suppression (< 50 copies), which usually occurs more rapidly than immune reconstitution. CD4+ count and viral load should thus be carefully monitored. An HIV genotype should be obtained before the initiation of therapy and when resistance is suspected, as such testing can provide mutation information and identify resistance to specif c antiretrovirals. Table 2.8-6 outlines prophylactic measures against opportunistic infections. Figure 2.8-8 illustrates the microscopic appearance of some common opportunistic organisms. Risk factors include xerostomia, antibiotic use, denture use, and immunosuppressed states (e.g., HIV, leukemias, lymphomas, cancer, diabetes, corticosteroid inhaler use, immunosuppressive treatment).

1	Hx/PE: Presents with soft white plaques that can be rubbed off, with an erythematous base and possible mucosal burning. The differential includes oral hairy leukoplakia (lateral borders of the tongue; not easily rubbed off). Odynophagia is characteristic of candidal esophagitis. Dx: Usually clinical. KOH or Gram stain shows budding yeast and/or pseudohyphae. Tx: Treat thrush with local therapy (e.g., nystatin suspension or clotrimazole tablets, or a PO azole such as fuconazole). Treat candidal esophagitis with PO azole therapy. T AB LE 2.8-6. Prophylaxis for HIV-Related Opportunistic Infections

1	P. jiroveci pneumonia CD4+ < 200/mm3, prior P. jiroveci infection, unexplained fever × 2 weeks, or HIV-related oral candidiasis. Single-strength TMP-SMX or dapsone +/– pyrimethamine. Discontinue prophylaxis when CD4+ is > 200 for ≥ 3 months. Mycobacterium avium complex (MAC) CD4+ < 50–100/mm3. Weekly azithromycin or daily clarithromycin. Discontinue prophylaxis when CD4+ is > 100/mm3 for > 6 months. Toxoplasma gondii CD4+ < 100/mm3 + IgG serologies. Double-strength TMP-SMX. — M. tuberculosis PPD > 5 mm or “high risk” (see TB section). Sensitive: INH × 9 months (+ pyridoxine) or rifampin +/– pyrazinamide × 2 months. Include pyridoxine with INH-containing regimens. Candida Multiple recurrences. Esophagitis: Fluconazole. Oral: Nystatin swish and swallow. — HSV Multiple recurrences. Acyclovir, famciclovir, or valacyclovir. — All patients. Pneumovax. Give every fve years or when CD4+ is < 200. All patients. Infuenza vaccine annually. — S. pneumoniae Inf uenza

1	Pseudohyphae + budding yeasts Rare fruiting bodies45° angle branching septate hyphae 5–10 μm yeasts with wide capsular halo Narrow-based unequal budding Germ tubes at 37°C Irregular broad (empty-looking) nonseptate hyphae, wide-angle branching F IGU R E 2.8-8. Common opportunistic organisms. The CSF antigen test for cryptococcal meningitis is highly sensitive and specific. Risk factors include AIDS and exposure to pigeon droppings. Hx/PE: Presents with headache, fever, impaired mentation, and absent meningismus. The differential includes toxoplasmosis, lymphoma, TB meningitis, AIDS dementia complex, PML, HSV encephalitis, and other fungal disease. Dx: LP (↓ CSF glucose; ↑ protein; ↑ leukocyte count with monocytic predominance, ↑↑ opening pressure); CSF cryptococcal antigen test, India ink stain, and fungal culture.

1	Dx: LP (↓ CSF glucose; ↑ protein; ↑ leukocyte count with monocytic predominance, ↑↑ opening pressure); CSF cryptococcal antigen test, India ink stain, and fungal culture. Tx: ■IV amphotericin B + f ucytosine × 2 weeks; then give f uconazole 400 mg × 8 weeks. Lifelong maintenance therapy should be administered with fuconazole 200 mg QD, or until CD4+ is > 200 for > 6 months. ■↑ opening pressure may require serial LPs or VP shunt for management. Risk factors include AIDS, spelunking, and exposure to bird or bat excrement, especially in the Ohio and Mississippi river valleys. 1° exposure is often asymptomatic or causes a f ulike illness. Presentation may range from no symptoms to fulminant disease with pulmonary or extrapulmonary manifestations. Fever, weight loss, hepatosplenomegaly, lymphadenopathy, nonproductive cough, and pancytopenia indicate disseminated infection (most often within 14 days).

1	Fever, weight loss, hepatosplenomegaly, lymphadenopathy, nonproductive cough, and pancytopenia indicate disseminated infection (most often within 14 days). The differential includes atypical bacterial pneumonias, blastomycosis, coccidioidomycosis, TB, sarcoidosis, pneumoconiosis, and lymphoma. CXR shows diffuse nodular densities, focal infltrate, cavity, or hilar lymphadenopathy (chronic infection is usually cavitary). The urine and serum polysaccharide antigen test is the most sensitive test for making the initial diagnosis, monitoring response to therapy, and diagnosing relapse. Culture is also diagnostic (blood, sputum, bone marrow, CSF). The yeast form is seen with silver stain on biopsy (bone marrow, lymph node, liver) or bronchoalveolar lavage. Depends on the severity of disease and the host: Mild pulmonary disease or stable nodules: Treat supportively in the immunocompromised host. Chronic cavitary lesions: Give itraconazole for > 1 year.

1	Mild pulmonary disease or stable nodules: Treat supportively in the immunocompromised host. Chronic cavitary lesions: Give itraconazole for > 1 year. Severe acute pulmonary disease or disseminated disease: Amphotericin B or amphotericin B liposomal × 3–10 days followed by itraconazole × 12 weeks or longer. Maintenance therapy with daily itraconazole. Formerly known as Pneumocystis carinii pneumonia, or PCP. Risk factors include impaired cellular immunity and AIDS. Presents with dyspnea on exertion, fever, nonproductive cough, tachypnea, weight loss, fatigue, and impaired oxygenation. Can also present as disseminated disease or as local disease in other organ systems. The differential includes TB, histoplasmosis, and coccidioidomycosis. Diagnosed by cytology of induced sputum or bronchoscopy specimen with silver stain and immunofuorescence. Obtain an ABG to check PaO2.

1	Diagnosed by cytology of induced sputum or bronchoscopy specimen with silver stain and immunofuorescence. Obtain an ABG to check PaO2. CXR may show diffuse, bilateral interstitial infltrates with a ground-glass appearance, but any presentation is possible. Treat with high-dose TMP-SMX × 21 days. Clindamycin and primaquine constitute an alternative regimen for patients with sulfa allergy. A prednisone taper should be used in patients with moderate to severe hypoxemia (PaO2 < 70 mm or an arterial-alveolar oxygen gradient > 35).

1	A prednisone taper should be used in patients with moderate to severe hypoxemia (PaO2 < 70 mm or an arterial-alveolar oxygen gradient > 35). Most 1° CMV infections are asymptomatic; serious reactivation generally occurs only in immunocompromised patients. Seventy percent of adults in the United States have been infected. Transmission occurs via sexual contact, in breast milk, via respiratory droplets in nursery or day care facilities, and through blood transfusions. Risk factors for reactivation include the f rst 100 days status post tissue or bone marrow transplant and HIV positivity with a CD4+ < 100 or a viral load > 10,000. Systemic infection may resemble EBV mononucleosis (see the discussion of infectious mononucleosis). Specifc manifestations are as follows: CMV retinitis: Has a high rate of retinal detachment (“pizza pie” retinopathy), and presents with foaters and visual feld changes (CD4+ < 50).

1	Specifc manifestations are as follows: CMV retinitis: Has a high rate of retinal detachment (“pizza pie” retinopathy), and presents with foaters and visual feld changes (CD4+ < 50). GI and hepatobiliary involvement: Can present with multiple nonspecifc GI symptoms, including bloody diarrhea. CMV, microsporidia, and cryptosporidia have been implicated in the development of AIDS cholangiopathy. CMV pneumonitis: Presents with cough, fever, and sparse sputum production; associated with a high mortality rate. Much more common in patients with hematologic malignancies and transplant patients than in those with AIDS. CNS involvement: Can include polyradiculopathy, transverse myelitis, and subacute encephalitis (CD4+ < 50; periventricular calcif cations). Suspect P. jiroveci pneumonia in any HIV patient who presents with nonproductive cough and dyspnea. Treat CMV infection with ganciclovir. Virus isolation, culture, tissue examination, serum PCR.

1	Suspect P. jiroveci pneumonia in any HIV patient who presents with nonproductive cough and dyspnea. Treat CMV infection with ganciclovir. Virus isolation, culture, tissue examination, serum PCR. Treat with ganciclovir or foscarnet. Treat underlying disease if the patient is immunocompromised. Ubiquitous organisms causing pulmonary and disseminated infection in several demographic groups. The 1° form occurs in apparently healthy nonsmokers (Lady Windermere syndrome); a 2° pulmonary form affects patients with preexisting pulmonary disease such as COPD, TB, or CF. Disseminated infection occurs in AIDS patients with a CD4+ < 50. There is no evidence that behavioral change affects exposure. Disseminated M. avium infection in AIDS is associated with fever, weakness, and weight loss in patients who are not on HAART or chemoprophylaxis for MAC. Hepatosplenomegaly and lymphadenopathy are occasionally seen.

1	Hepatosplenomegaly and lymphadenopathy are occasionally seen. Adrenal insuffciency is possible in the setting of infltration into the adrenals. Obtain mycobacterial blood cultures ( in 2–3 weeks). Labs show anemia, hypoalbuminemia, and ↑ serum alkaline phosphatase and LDH. Biopsy of bone marrow, intestine, or liver reveals foamy macrophages with acid-fast bacilli. Typical granulomas may be absent in immunocompromised patients. Treat with clarithromycin and ethambutol +/– rifabutin and HAART. Continue for > 12 months and until CD4+ is > 100 for > 6 months. Weekly azithromycin for those with a CD4+ < 50 or AIDS-def ning opportunistic infection. Risk factors include ingesting raw or undercooked meat and changing cat litter. Worldwide, exposure is highest in France. 1° infection is usually asymptomatic. Reactivated toxoplasmosis occurs in immunosuppressed patients and may present in specif c organs (brain, lung, and eye > heart, skin, GI tract, and liver).

1	1° infection is usually asymptomatic. Reactivated toxoplasmosis occurs in immunosuppressed patients and may present in specif c organs (brain, lung, and eye > heart, skin, GI tract, and liver). ■Encephalitis is common in seropositive AIDS patients. Classically, CNS lesions present with fever, headache, altered mental status, seizures, and focal neurologic def cits. Serology, PCR (indicates exposure and risk for reactivation); tissue examination for histology, isolation of the organism in mice, or tissue culture. In the setting of CNS involvement, obtain a CT scan (can show multiple isodense or hypodense, ring-enhancing mass lesions) or an MRI (predilection for basal ganglia; more sensitive). Induction with high-dose PO pyrimethamine + sulfadiazine and leukovorin × 4–8 weeks; maintenance with low-dose pyrimethamine + sulfadiazine and leukovorin until the disease has resolved clinically and radio-graphically.

1	TMP-SMX (Bactrim DS) or pyrimethamine + dapsone can be used for prophylaxis in patients with a CD4+ < 100 and a toxoplasmosis IgG. The two most likely differential diagnoses of ring-enhancing lesions in AIDS patients are toxoplasmosis and CNS lymphoma. The most common bacterial STD in the United States. Caused by Chlamydia trachomatis, which can infect the genital tract, urethra, anus, and eye. Risk factors include unprotected sexual intercourse, new or multiple partners, and frequent douching. Often coexists with or mimics N. gonorrhoeae infection (known as nongonococcal urethritis when gonorrhea is absent). LGV serovars of Chlamydia cause lymphogranuloma venereum, an emerging cause of proctocolitis. Infection is often asymptomatic but may present with urethritis, mucopurulent cervicitis, or PID. Exam may reveal cervical/adnexal tenderness in women or penile discharge and testicular tenderness in men.

1	Exam may reveal cervical/adnexal tenderness in women or penile discharge and testicular tenderness in men. The differential includes gonorrhea, endometriosis, PID, orchitis, vaginitis, and UTI. Lymphogranuloma venereum presents in its 1° form as a painless, transient papule or shallow ulcer. In its 2° form, it presents as painful swelling of the inguinal nodes, and in its 3° form it can present as an “anogenital syndrome” (anal pruritus with discharge, rectal strictures, rectovaginal f stula, and elephantiasis). Diagnosis is usually clinical. Culture is the gold standard. Urine tests (PCR or ligase chain reaction) are a rapid means of detection, while DNA probes and immunofuorescence (for gonorrhea/chlamydia) take 48–72 hours. Gram stain of urethral or genital discharge may show PMNs but no bacteria (intracellular). Chlamydia infection is a common cause of nongonococcal urethritis in men.

1	Gram stain of urethral or genital discharge may show PMNs but no bacteria (intracellular). Chlamydia infection is a common cause of nongonococcal urethritis in men. Chlamydia species cause arthritis, neonatal conjunctivitis, pneumonia, nongonococcal urethritis/ PID, and lymphogranuloma venereum. Treat for gonorrhea and chlamydia in light of the high prevalence of coinfection. Syphilis is the “great imitator” because its dermatologic findings resemble those of many other diseases. Doxycycline 100 mg PO BID × 7 days or azithromycin 1 g PO × 1 day. Use erythromycin in pregnant patients. Treat sexual partners, and maintain a low threshold to treat for N. gonorrhoeae. LGV serovars require prolonged therapy for 21 days. Chronic infection and pelvic pain, Reiter’s syndrome (urethritis, conjunctivitis, arthritis), Fitz-Hugh–Curtis syndrome (perihepatic inf ammation and f brosis). Ectopic pregnancy/infertility can result from PID (in women) and epididymitis (in men).

1	Ectopic pregnancy/infertility can result from PID (in women) and epididymitis (in men). A gramintracellular diplococcus that can infect almost any site in the female reproductive tract. Infection in men tends to be limited to the urethra. Presents with a greenish-yellow discharge, pelvic or adnexal pain, and swollen Bartholin’s glands. Men experience a purulent urethral discharge, dysuria, and erythema of the urethral meatus. The differential includes chlamydia, endometriosis, pharyngitis, PID, vaginitis, UTI, salpingitis, and tubo-ovarian abscess. Gram stain and culture is the gold standard for any site (i.e., pharynx, cervix, urethra, or anus). Nucleic acid amplif cation tests can be sent on penile/vaginal tissue or from urine. Disseminated disease may present with monoarticular septic arthritis, rash, and/or tenosynovitis.

1	Disseminated disease may present with monoarticular septic arthritis, rash, and/or tenosynovitis. Ceftriaxone IM or cefepime PO × 1 dose. Also treat for presumptive chlamydia coinfection (doxycycline × 7 days or macrolide × 1 dose). Condoms are effective prophylaxis. Treat the sexual partner or partners if possible. Fluoroquinolones should not be used because of emerging resistance. Disseminated disease requires IV ceftriaxone for at least 24 hours. Persistent infection with pain; infertility; tubo-ovarian abscess with rupture; disseminated gonococcal infection (see Figure 2.8-9). Caused by Treponema pallidum, a spirochete. AIDS can accelerate the course of disease progression. ■1° (10–90 days after infection): Presents with a painless ulcer (chancre; see Figure 2.8-10). F IGU R E 2.8-9. Disseminated gonococcal infection. Hemorrhagic, painful pustules on erythematous bases. (Reproduced, with permission, from

1	F IGU R E 2.8-9. Disseminated gonococcal infection. Hemorrhagic, painful pustules on erythematous bases. (Reproduced, with permission, from Wolff K et al. Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York: McGraw-Hill, 2005: Fig. 27-17.) 2° (4–8 weeks after chancre): Presents with low-grade fever, headache, malaise, and generalized lymphadenopathy with a diffuse, symmetric, asymptomatic (nonpruritic) maculopapular rash on the soles and palms. Highly infective 2° eruptions include mucous patches or condylomata lata (see Figure 2.8-11). Meningitis, hepatitis, nephropathy, and eye involvement may also be seen. Early latent (period from resolution of 1° or 2° syphilis to end of f rst year of infection): No symptoms; serology. Late latent (period of asymptomatic infection beyond the f rst year): No symptoms; or serology. One-third progress to 3° syphilis. FIGURE 2.8-10. 1° syphilis.

1	Late latent (period of asymptomatic infection beyond the f rst year): No symptoms; or serology. One-third progress to 3° syphilis. FIGURE 2.8-10. 1° syphilis. The chancre is an ulcerated papule with a smooth, clean base; raised, indurated borders; and scant discharge. (Reproduced, with permission, from Bondi EE. Dermatology: Diagnosis and Therapy, 1st ed. Stamford, CT: Appleton & Lange, 1991: 394.) FIGURE 2.8-11. Condylomata lata.

1	FIGURE 2.8-11. Condylomata lata. Typical appearance of the verrucous heaped-up lesions of condylomata lata. (Reproduced, with permission, from Wolff K et al. Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York: McGraw-Hill, 2005: Fig. 27-27.) ■ 3° (late manifestations appearing 1–20 years after initial infection): Presents with destructive, granulomatous gummas. Neurosyphilis includes tabes dorsalis (posterior column degeneration), meningitis, and Argyll Robertson pupil (constricts with accommodation but not reactive to light). Cardiovascular fndings include dilated aortic root, aortitis, aortic root aneurysms, and aortic regurgitation. ■See Table 2.8-7. VDRL false positives are seen with Viruses (mononucleosis, HSV, HIV, hepatitis), Drugs/IV drug use, Rheumatic fever/Rheumatoid arthritis, and SLE/Leprosy. TABLE 2.8-7.

1	■See Table 2.8-7. VDRL false positives are seen with Viruses (mononucleosis, HSV, HIV, hepatitis), Drugs/IV drug use, Rheumatic fever/Rheumatoid arthritis, and SLE/Leprosy. TABLE 2.8-7. Dark-f eld microscopy Identifes motile spirochetes (only 1° and 2° lesions). VDRL/RPR Rapid and cheap, but sensitivity is only 60–75% in 1° disease. Many false positives. FTA-ABS Sensitive and specifc. Used as a 2° diagnostic test. TPPAa Sensitivity and specifcity similar to FTA-ABS but easier to use. Becoming the 2° test of choice. a TPPA = T. pallidum particle agglutination test. ■Neurosyphilis should be suspected and ruled out in any AIDS patient with neurologic symptoms and a RPR. 1°/2°: Benzathine penicillin IM × 1 day. Tetracycline or doxycycline × 14 days may be used for patients with penicillin allergies. Latent infection: Treat with benzathine penicillin once weekly × 3 weeks. Neurosyphilis: Treat with penicillin IV; penicillin-allergic patients should be desensitized prior to therapy.

1	Latent infection: Treat with benzathine penicillin once weekly × 3 weeks. Neurosyphilis: Treat with penicillin IV; penicillin-allergic patients should be desensitized prior to therapy. See Table 2.8-8 for a description of common sexually transmitted genital lesions along with an outline of their diagnosis and treatment. Remember that treatment of syphilis can result in an acute ﬂ ulike illness known as the Jarisch-Herxheimer reaction. Affect females more frequently than males, and E. coli cultures are obtained in 80% of cases. See the mnemonic SEEKS PP for other pathogens. Risk factors include the presence of catheters or other urologic instrumentation, anatomic abnormalities (e.g., BPH, vesicoureteral ref ux), previous UTIs or pyelonephritis, diabetes mellitus (DM), recent antibiotic use, immunosuppression, and pregnancy.

1	Present with dysuria, urgency, frequency, suprapubic pain, and possibly hematuria. Children may present with bed-wetting, poor feeding, recurrent fevers, and foul-smelling urine. The differential includes vaginitis, STDs, urethritis or acute urethral syndrome, and prostatitis. Diagnosed by clinical symptoms. In the absence of symptoms, treatment is warranted only for children, those with anatomical GU tract anomalies, pregnant women, those with instrumented urinary tracts, patients scheduled for GU surgery, and renal transplant patients. Urine dipstick/UA: ↑ leukocyte esterase (a marker of WBCs) is 75% sensitive and up to 95% specif c. ↑ nitrites (a marker of bacteria), ↑ urine pH (Proteus infections), and hematuria (seen with cystitis) are also commonly seen. Microscopic analysis: Pyuria (> 5 WBCs/hpf) and bacteriuria (1 organism/hpf = 106 organisms/mL) are suggestive. Urine culture: The gold standard is > 105 CFU/mL.

1	Microscopic analysis: Pyuria (> 5 WBCs/hpf) and bacteriuria (1 organism/hpf = 106 organisms/mL) are suggestive. Urine culture: The gold standard is > 105 CFU/mL. Uncomplicated UTI: Treat on an outpatient basis with PO TMP-SMX or a f uoroquinolone × 3 days, or nitrofurantoin × 7 days. Note that resistance to TMP-SMX and fuoroquinolones has been increasing. Complicated UTI (urinary obstruction, men, renal transplant, catheters, instrumentation): Administer the same antibiotics as above, but for 7–14 days. Common UTI bugs— SEEKS PP Serratia E. coli Enterobacter Klebsiella pneumoniae S. saprophyticus Pseudomonas Proteus mirabilis T AB LE 2.8-8. Sexually Transmitted Genital Lesions

1	Lesion Papule becomes a beefy-red ulcer with a characteristic rolled edge of granulation tissue Papule or pustule (chancroid; see Figure 2.8-12) Vesicle (3–7 days postexposure) Papule (condylomata acuminata; warts) Papule (chancre) Appearance Raised red lesions with a white border Irregular, deep, well demarcated, necrotic Regular, red, shallow ulcer Irregular, pink or white, raised; caulif ower Regular, red, round, raised Number 1 or multiple 1–3 Multiple Multiple Single Size 5–10 mm 10–20 mm 1–3 mm 1–5 mm 1 cm Pain No Yes Yes No No Concurrent signs and symptoms Granulomatous ulcers Inguinal lymphadenopathy Malaise, myalgias, and fever with vulvar burning and pruritus Pruritus Regional adenopathy Clinical exam, biopsy (Donovan bodies) Diffcult to culture; diagnosis is made on clinical grounds Tzanck smear shows multinucleated giant cells; viral cultures; DFA or serology Clinical exam; biopsy for conf rmation Spirochetes seen under dark-f eld microscopy; T. pallidum identifed by serum

1	Tzanck smear shows multinucleated giant cells; viral cultures; DFA or serology Clinical exam; biopsy for conf rmation Spirochetes seen under dark-f eld microscopy; T. pallidum identifed by serum antibody test Doxycycline (100 mg BID) or azithromycin (1 g weekly) × 3 weeks Doxycycline (100 mg BID) or azithromycin (1 g weekly) × 3 weeks Acyclovir or valacyclovir for 1° infection Cryotherapy; topical agents such as podophyllin, trichloroacetic acid, or 5-FU cream Penicillin IM Diagnosis Treatmentd a Previously known as Calymmatobacterium granulomatis. b Some 85% of genital herpes lesions are caused by HSV-2. c HPV serotypes 6 and 11 are associated with genital warts; types 16, 18, and 31 are associated with cervical cancer. d For all, treat sexual partners.

1	FIGURE 2.8-12. Chancroid. Multiple, painful ulcers. (Reproduced, with permission, from Wolff K et al. Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York: McGraw-Hill, 2005: Fig. 27-31.) Pregnant patients: Treat asymptomatic bacteruria or symptomatic UTI with nitrofurantoin or cephalosporin × 3–7 days. Avoid f uoroquinolones. Confrm clearance with a repeat culture post-treatment. Patients with urosepsis should be hospitalized and initially treated with IV antibiotics. Consider broader coverage to include resistant GNRs or enterococcus. Prophylactic antibiotics may be given to women with uncomplicated recurrent UTIs. Check for prostatitis in men. Nearly 85% of community-acquired cases of pyelonephritis result from the same pathogens that cause cystitis. Cystitis and pyelonephritis have similar risk factors. ■Signs and symptoms are similar to those of cystitis but show evidence of upper urinary tract disease.

1	■Signs and symptoms are similar to those of cystitis but show evidence of upper urinary tract disease. ■ Symptoms include f ank pain, fever/chills, and nausea/vomiting. Dysuria, frequency, and urgency are also possible. ■UA and culture: Results are similar to those of cystitis, but with WBC casts. Send blood cultures to rule out urosepsis. Pyelonephritis is the most common serious medical complication of pregnancy. Twenty to thirty percent of patients with untreated bacteriuria will develop pyelonephritis. When in doubt, admit a patient with pyelonephritis and administer IV antibiotics. Urosepsis must be considered in any elderly patient with altered mental status. SIRS = two or more of the following: 1. Temperature: Either < 36°C or > 38°C (i.e., hypothermia or fever). 2. Tachypnea: > 20 breaths per minute or PaCO2 < 32 mmHg on ABG. 3. Tachycardia: HR > 90 bpm. 4. Leukocytosis/leukopenia: WBC < 4000 cells/mm3 or > 12,000 cells/mm3. CBC: Reveals leukocytosis.

1	2. Tachypnea: > 20 breaths per minute or PaCO2 < 32 mmHg on ABG. 3. Tachycardia: HR > 90 bpm. 4. Leukocytosis/leukopenia: WBC < 4000 cells/mm3 or > 12,000 cells/mm3. CBC: Reveals leukocytosis. Imaging: In general, imaging is not necessary. Ultrasound can be used to rule out obstruction and calculi and can often confrm the diagnosis noninvasively, but CT is becoming the test of choice in patients with adequate renal function who are not responding to therapy. In recurrent cases, IVP may demonstrate renal scarring. For mild cases, patients may be treated on an outpatient basis for 10–14 days. Fluoroquinolones are frst-line therapy. Encourage ↑ PO f uids and monitor closely.

1	For mild cases, patients may be treated on an outpatient basis for 10–14 days. Fluoroquinolones are frst-line therapy. Encourage ↑ PO f uids and monitor closely. Admit and administer IV antibiotics to patients who have serious medical complications or systemic symptoms, are pregnant, present with severe nausea and vomiting, or have suspected bacteremia. Fluoroquinolones, thirdor fourth-generation cephalosporins, β-lactam/β-lactamase inhibitors, and carbapenem may be used depending on disease severity. Defned as the presence of systemic infammatory response syndrome (SIRS) with a documented infection, induced by microbial invasion or toxins in the bloodstream. Severe sepsis refers to sepsis with end-organ dysfunction due to poor perfusion. Septic shock refers to sepsis with hypotension and organ dysfunction from vasodilation. Examples include the following: Gram-shock (e.g., staphylococci and streptococci) 2° to f uid loss caused by exotoxins.

1	Gram-shock (e.g., staphylococci and streptococci) 2° to f uid loss caused by exotoxins. Gram-shock (e.g., E. coli, Klebsiella, Proteus, and Pseudomonas) 2° to vasodilation caused by endotoxins (lipopolysaccharide). Neonates: GBS, E. coli, Listeria monocytogenes, H. inﬂ uenzae. Children: H. inﬂ uenzae, pneumococcus, meningococcus. Adults: Gramcocci, aerobic grambacilli, anaerobes (dependent on the presumed site of infection). IV drug users/indwelling lines: S. aureus, coagulase-Staphylococcus species. Asplenic patients: Pneumococcus, H. inﬂ uenzae, meningococcus (encapsulated organisms). Presents with abrupt onset of fever and chills, altered mental status, tachycardia, and tachypnea. Severe sepsis is seen in end-organ dysfunction. Hypotension occurs in cases of septic shock. Septic shock is typically a warm shock with warm skin and extremities. This contrasts with cardiogenic shock, which typically presents with cool skin and extremities.

1	Septic shock is typically a warm shock with warm skin and extremities. This contrasts with cardiogenic shock, which typically presents with cool skin and extremities. Petechiae, ecchymoses, or abnormal coagulation tests suggest DIC (2–3% of cases). A clinical diagnosis. Labs show leukocytosis or leukopenia with ↑ bands, thrombocytopenia (50% of cases), evidence of ↓ tissue perfusion (↑ creatinine, ↑ LFTs), and abnormal coagulation studies (↑ INR). It is critical to obtain cultures of all appropriate sites (e.g., blood, sputum, CSF, wound, urine). Imaging (CXR, CT) may aid in establishing the etiology or site of infection. ICU admission may be required. Treat aggressively with IV f uids, pressors, and empiric antibiotics (based on the likely source of infection). Treat underlying factors (e.g., remove Foley catheter or infected lines). The 1° goal is to maintain BP and perfusion to end organs.

1	Treat underlying factors (e.g., remove Foley catheter or infected lines). The 1° goal is to maintain BP and perfusion to end organs. A protozoal disease caused by four strains of the genus Plasmodium (P. falciparum, P. vivax, P. ovale, and P. malariae) and transmitted by the bite of an infected female Anopheles mosquito. P. falciparum has the highest morbidity and causes the largest number of deaths, occasionally within 24 hours of symptom onset. Recent outbreaks have occurred in parts of the southern and eastern United States and in Europe, mainly through the arrival of infected travelers and immigrants from endemic areas. Travelers to endemic areas should take chemoprophylaxis and use mosquito repellent and bed nets to minimize exposure.

1	Patients have a history of exposure in a malaria-endemic area, with periodic attacks of sequential chills, fever (> 41°C), and diaphoresis occurring over 4–6 hours. Splenomegaly often appears four or more days after onset of symptoms. Patients are often asymptomatic between attacks, which recur every 2–3 days, depending on the Plasmodium strain. Severely ill patients may present with hyperpyrexia, prostration, impaired consciousness, agitation, hyperventilation, and bleeding. The presence of rash, lymphadenopathy, neck stiffness, or photophobia suggests a different or additional diagnosis. Timely diagnosis of the correct strain is essential because P. falciparum can be fatal and is often resistant to standard chloroquine treatment.

1	Timely diagnosis of the correct strain is essential because P. falciparum can be fatal and is often resistant to standard chloroquine treatment. Giemsa-or Wright-stained thick and thin blood f lms should be sent for expert microscopic evaluation to determine the strain as well as the degree of parasitemia. Specimens should be obtained at eight-hour intervals for three days, including during and between febrile periods. CBC usually demonstrates normochromic, normocytic anemia with reticulocytosis. If resources allow, more sensitive serologic tests are available, including rapid antigen detection methods, fuorescent antibody methods, and PCR. Malaria should be considered in the differential for any patient who has emigrated from or recently traveled to tropical locations and presents with fever. P. vivax, P. ovale, and P. malariae can all cause symptoms months to years after initial infection.

1	P. vivax, P. ovale, and P. malariae can all cause symptoms months to years after initial infection. Obtain a finger stick in a patient with malaria and mental status changes to rule out hypoglycemia. ■Uncomplicated malarial infection can be treated orally, with the choice of medication determined by the Plasmodium strain. Chloroquine has been the standard antimalarial medication, but increasing resistance has led to A young adult who presents with the triad of fever, sore throat, and lymphadenopathy may have infectious mononucleosis. The lymphocytosis in EBV infection is predominantly due to B-cell proliferation, but the atypical cells are T lymphocytes. the use of other medications, including quinine with clindamycin or doxycycline, atovaquone, mefoquine, artesunate, and halofantrine.

1	the use of other medications, including quinine with clindamycin or doxycycline, atovaquone, mefoquine, artesunate, and halofantrine. The life cycles of P. vivax and P. ovale strains include dormant liver hypnozoite forms, which are resistant to treatment with chloroquine. Thus, in cases of P. vivax, P. ovale, or an unknown strain, primaquine is added to eradicate the hypnozoites in the liver. Severe infections can be treated with parenteral antimalarial medications (IV quinidine) with transition to oral regimens as tolerated. Newer combinations such as proguanil/atovaquone (Malarone) eliminate the need for multiple medications. Symptoms can be treated with supportive care. Cerebral malaria: Headache, change in mental status, neurologic signs, retinal hemorrhages, convulsions, delirium, coma. Severe hemolytic anemia: Usually associated with P. falciparum infection. Acute tubular necrosis and renal failure: Associated with blackwater fever (dark urine due to hemoglobinuria).

1	Severe hemolytic anemia: Usually associated with P. falciparum infection. Acute tubular necrosis and renal failure: Associated with blackwater fever (dark urine due to hemoglobinuria). Noncardiogenic pulmonary edema: Often precipitated by overly rapid re-hydration. Other: Additional complications include grambacteremia, acute hepatopathy, hypoglycemia, cardiac dysrhythmias, secretory diarrhea, lactic acidosis, DIC, and a low birth rate in children of infected mothers. Most commonly occurs in young adult patients; usually due to acute EBV infection. Transmission most often occurs through exchange of body f uids, including saliva. Presents with fever and pharyngitis. Fatigue invariably accompanies initial illness and may persist for 3–6 months. Exam may reveal low-grade fever, generalized lymphadenopathy (especially posterior cervical), tonsillar exudate and enlargement, palatal petechiae, a generalized maculopapular rash, splenomegaly, and bilateral upper eyelid edema.

1	Patients who present with pharyngitis as their 1° symptom may be misdiagnosed as having streptococcal pharyngitis (30% of patients with infectious mononucleosis are asymptomatic carriers of group A strep in their oropharynx). Treatment of patients with ampicillin (for streptococcal pharyngitis) during acute EBV infection can cause a prolonged, pruritic, drug-related maculopapular rash. This rash does not portend future sensitivity to β-lactams and will remit with discontinuation of ampicillin. The differential also includes CMV, toxoplasmosis, HIV, HHV-6, other causes of viral hepatitis, and lymphoma. Diagnosed by the heterophil antibody (Monospot) test (may be in the frst few weeks after symptoms begin). The EBV-infected proliferating B cells produce a characteristic antibody that agglutinates the horse and sheep RBCs that are the basis for the Monospot test.

1	EBV-specif c antibodies can be ordered in patients with suspected mono nucleosis and a Monospot test. Infectious mononucleosis syndromes that are Monospot and EBV-antibody are most often due to CMV infection. Acute HIV and other viral etiologies should be considered. CBC with differential often reveals mild thrombocytopenia with relative lymphocytosis and > 10% atypical T lymphocytes. CMP usually reveals mildly elevated transaminases, alkaline phosphatase, and total bilirubin. Treatment is mostly supportive, as there is no effective antiviral therapy. Corticosteroids are indicated for airway compromise due to tonsillar enlargement, severe thrombocytopenia, or severe autoimmune hemolytic anemia. CNS infection: Can present as aseptic meningitis, encephalitis, meningoencephalitis, cranial nerve palsies (particularly CN VII), optic and peripheral neuritis, transverse myelitis, or Guillain-Barré syndrome.

1	Splenic rupture: Occurs in < 0.5% of cases. More common in males, and presents with abdominal pain, referred shoulder pain, or hemodynamic compromise. Upper airway obstruction: Treat with steroids. Bacterial superinfection: Ten percent of patients develop streptococcal pharyngitis secondarily. Fulminant hepatic necrosis: More common in males; the most common cause of death in affected males. Autoimmune hemolytic anemia: Occurs in 2% of patients during the f rst two weeks. Coombs . Mild anemia lasts 1–2 months. Treat with corticosteroids if severe. Other: Rare complications associated with acute EBV infection include hepatitis (which can be fulminant), myocarditis or pericarditis with electrocardiographic changes, pneumonia with pleural effusion, interstitial nephritis, genital ulcerations, and vasculitis. About one-third of patients with infectious mononucleosis have coexisting streptococcal pharyngitis that requires treatment. Fever of Unknown Origin (FUO)

1	About one-third of patients with infectious mononucleosis have coexisting streptococcal pharyngitis that requires treatment. Fever of Unknown Origin (FUO) A temperature of > 38.3°C of at least three weeks’ duration that remains undiagnosed following three outpatient visits or three days of hospitalization. Presents with fever, headache, myalgia, and malaise. The differential includes the following: Infectious: TB, endocarditis (e.g., HACEK organisms; see the discussion of infective endocarditis), occult abscess, osteomyelitis, catheter infections. In HIV patients, consider MAC, histoplasmosis, CMV, or lymphoma. Neoplastic: Lymphomas, leukemias, hepatic and renal cell carcinomas. Autoimmune: Still’s disease, SLE, cryoglobulinemia, polyarteritis nodosa, connective tissue disease, granulomatous disease (including sarcoidosis). Miscellaneous: Pulmonary emboli, alcoholic hepatitis, drug fever, familial Mediterranean fever, factitious fever. ■ Undiagnosed (10–15%).

1	Miscellaneous: Pulmonary emboli, alcoholic hepatitis, drug fever, familial Mediterranean fever, factitious fever. ■ Undiagnosed (10–15%). Overall, infections and cancer account for the majority of cases of FUO (> 60%). Autoimmune diseases account for ~15%. In the elderly, rheumatic diseases account for one-third of cases. FUO patients without other symptoms do not require empiric antibiotic therapy. With fever and rash, think— Lyme disease is the most common vector-borne disease in North America. Labs: Confrm the presence of fever and take a detailed history (including family, social, sexual, occupational, dietary, exposures [pets/animals], and travel); obtain a CXR, CBC with differential, ESR, multiple blood cultures, sputum Gram stain and culture, UA and culture, and PPD. Specifc tests (ANA, RF, viral cultures, viral serologies/antigen tests) can be obtained if an infectious or autoimmune etiology is suspected.

1	Imaging: CT of the chest and abdomen should be done early in the workup of a true FUO. Rule out drug fever. Invasive testing (marrow/liver biopsy) is generally low yield. Laparoscopy and colonoscopy are higher yield as second-line tests (after CT). Stop unnecessary medications. Give empiric antibiotics to severely ill patients until the etiology has been determined. Stop antibiotics if there is no response. Defned as a single oral temperature of ≥ 38.3°C (101°F) or a temperature of ≥ 38.0°C (100.4°F) for ≥ 1 hour in a neutropenic patient (i.e., an absolute neutrophil count of < 500 cells/mm3). Hx/PE: Common in cancer patients undergoing chemotherapy (neutropenic nadir 7–10 days postchemotherapy). Infammation may be minimal or absent. Dx: Send appropriate cultures, including blood, urine, sputum, and wound. Consider testing for viruses, fungi, and mycobacteria.

1	Dx: Send appropriate cultures, including blood, urine, sputum, and wound. Consider testing for viruses, fungi, and mycobacteria. Conduct a thorough physical exam, but avoid a rectal exam in light of the bleeding risk if the patient is thrombocytopenic. Obtain a CBC with differential, serum creatinine, BUN, and transaminases; order blood, urine, lesion, and stool cultures. CXR for patients with respiratory symptoms; CT scan to evaluate for abscesses or other occult infection. Tx: Empiric antibiotic therapy (see Figure 2.8-13). Routine use of colony-stimulating factors is not indicated. If fevers persist after 72 hours despite antibiotic therapy, start antifungal treatment. A tick-borne disease caused by the spirochete Borrelia burgdorferi. Usually seen during the summer months, and carried by Ixodes ticks on white-tailed deer and white-footed mice. Endemic to the Northeast, northern Midwest, and Pacif c coast.

1	Hx/PE: Presents with the onset of rash with fever, malaise, fatigue, headache, myalgias, and/or arthralgias. Infection usually occurs after a tick feeds for > 18 hours. ■1° (early localized disease): Erythema migrans begins as a small erythematous macule or papule that is found at the tick-feeding site and expands slowly over days to weeks. The border may be macular or raised, often with central clearing (“bull’s eye”).

1	Fever (≥ 38.3°C) + Neutropenia (< 500 neutrophils/mm3) PO IV (outpatient) Vancomycin not needed Reassess after 3–5 days Vancomycin needed Intermediate/High Risk High-dose chemo? Hematologic malignancy? Neutropenia > 14 days? Combination Therapy: Aminoglycosides + antipseudomonal, penicillin, cefepime, ceftazidime, or carbapenem Vancomycin + cefepime, ceftazidime, or carbapenem ± aminoglycoside Monotherapy: Cefepime, ceftazidine, or carbapenem Ciprofloxacin + amoxicillin/clavulanate (adults only) Low Risk Solid tumors treated as outpatient with conventional chemotherapy? Minimal comorbidity? Neutropenia < 7 days? F IGU R E 2.8-1 3. Empiric treatment algorithm for a neutropenic fever patient. 2° (early disseminated disease): Presents with migratory polyarthropathies, neurologic phenomena (e.g., Bell’s palsy), meningitis and/or myocarditis, and conduction abnormalities (third-degree heart block). 3° (late disease): Arthritis and subacute encephalitis (memory loss and mood change).

1	3° (late disease): Arthritis and subacute encephalitis (memory loss and mood change). Dx: Clinical diagnosis of erythema migrans is as follows: ELISA and Western blot: Use the Western blot to conf rm a or indeterminate ELISA. A ELISA denotes exposure but is not specif c for active disease. Western blots sent without ELISA have high rates of false positives. Tissue culture/PCR: Extremely diffcult to obtain; not routinely done. Tx: Treat early disease with doxycycline and more advanced disease (e.g., CNS or arthritic disease) with ceftriaxone. Consider empiric therapy for patients with the characteristic rash, arthralgias, or a tick bite acquired in an endemic area. Prevent with tick bite avoidance. A disease caused by Rickettsia rickettsii and carried by the American dog tick (Dermacentor variabilis). The organism invades the endothelial lining of capillaries and causes small vessel vasculitis.

1	Hx/PE: Presents with headache, fever, malaise, and rash. The characteristic rash is initially macular (beginning on the wrists and ankles) but becomes petechial/purpuric as it spreads centrally (see Figure 2.8-14). Altered mental status or DIC may develop in severe cases. Dx: Clinical diagnosis should be confrmed with indirect immunof uorescence of rash biopsy. Tx: Doxycycline or chloramphenicol (for multidrug-resistant organisms). Lyme arthritis can be very subtle and minimally inﬂ ammatory and can wax and wane. Rocky Mountain spotted fever starts on the wrists and ankles and then spreads centrally. FIGURE 2.8-14. Rocky Mountain spotted fever. These erythematous macular lesions will evolve into a petechial rash that will spread centrally. (Courtesy of Daniel Noltkamper, MD, as published in Knoop KJ et al. Atlas of Emergency Medicine, 2nd ed. New York: McGraw-Hill, 2002: 382.)

1	The condition can be rapidly fatal if left untreated. If clinical suspicion is high, begin treatment while awaiting testing. Prevent by avoiding tick bites. Causes of red eye: A common complaint in the emergency room setting, inf ammation of the conjunctiva is most often bacterial or viral but can also be fungal, parasitic, allergic, or chemical. It is essential to differentiate potentially vision-threatening infectious etiologies from allergic or other causes of conjunctivitis, as well as to identify other vision-threatening conditions that may mimic conjunctivitis. See Table 2.8-9 for the common etiologies of infectious conjunctivitis. Commonly due to infection of the paranasal sinuses; can lead to endophthalmitis and blindness. Usually caused by streptococci, staphylococci (including MRSA), and H. inﬂ uenzae (in children). In diabetic and immunocompromised patients, mucormycosis and Rhizopus are in the differential.

1	Hx/PE: Presents with acute-onset fever, proptosis, ↓ EOM, ocular pain, and ↓ visual acuity. Look for a history of ocular trauma or sinusitis. Pala T AB LE 2.8-9. Common Causes of Infectious Conjunctivitis

1	T AB LE 2.8-9. Common Causes of Infectious Conjunctivitis Bacterial Staphylococci, streptococci, Haemophilus, Pseudomonas, Moraxella Foreign body sensation, purulent discharge. Gram stain and culture if severe. Antibiotic drops/ ointment. N. gonorrhoeae An emergency! Corneal involvement can lead to perforation and blindness. Gram stain shows gram-intracellular diplococci. IM ceftriaxone, PO ciprofoxacin or of oxacin. Inpatient treatment if complicated. C. trachomatis A–C Recurrent epithelial keratitis in childhood, trichiasis, corneal scarring, and entropion. The leading cause of preventable blindness worldwide. Giemsa stain, chlamydial cultures. Azithromycin, tetracycline, or erythromycin × 3–4 weeks. Viral Adenovirus (most common) Copious watery discharge, severe ocular irritation, preauricular lymphadenopathy. Occurs in epidemics. Contagious; self-limited. Topical corticosteroids with supervision of an ophthalmologist.

1	tal or nasal mucosal ulceration with coexisting maxillary and/or ethmoid sinusitis suggests mucormycosis or Rhizopus. Dx: Mostly clinical. Blood and tissue fuid culture; CT scan (to rule out orbital abscess and intracranial involvement). Tx: Admit. Immediate IV antibiotics; request an ophthalmologic/ENT consult. Abscess formation may necessitate surgery. Diabetic and immunocompromised patients should be treated with amphotericin B and surgical debride ment (often associated with cavernous sinus thrombosis) if Mucor or Rhizopus is diagnosed. An inf ammation of the external auditory canal, also known as “swimmer’s ear.” Pseudomonas (from poorly chlorinated pools) and Enterobacteriaceae are the most common etiologic agents. Both grow in the presence of excess moisture.

1	Hx/PE: Presents with pain, pruritus, and possible purulent discharge. Exam reveals pain with movement of the tragus/pinna (unlike otitis media) and an edematous and erythematous ear canal. See the Pediatrics chapter for a discussion of otitis media. Dx: A clinical diagnosis. Gram stain and culture are helpful if a fungal etiology is suspected. CT scan if the patient is toxic appearing. Neisseria conjunctivitis is an ocular emergency often requiring inpatient parenteral antibiotic therapy. Diabetics are at risk for malignant otitis externa. ■ Tx: Antibiotic and steroid eardrops. Use systemic antibiotics in patients with severe disease. Diabetics are at risk for malignant otitis externa and osteomyelitis of the skull base and thus require hospitalization and IV antibiotics. pain with movement of the tragus/pinna.

1	pain with movement of the tragus/pinna. Infection of the endocardium, usually 2° to bacterial or other infectious causes. Most commonly affects the heart valves, especially the mitral valve. Risk factors include rheumatic, congenital, or valvular heart disease; prosthetic heart valves; IV drug abuse; and immunosuppression. Etiologies are as follows: S. aureus (both methicillin-sensitive and methicillin-resistant): The causative agent in > 80% of cases of acute bacterial endocarditis in patients with a history of IV drug abuse. Viridans streptococci: The most common pathogens for left-sided sub-acute bacterial endocarditis. Coagulase-Staphylococcus: The most common infecting organism in prosthetic valve endocarditis. Streptococcus bovis: S. bovis endocarditis is associated with coexisting GI malignancy.

1	Coagulase-Staphylococcus: The most common infecting organism in prosthetic valve endocarditis. Streptococcus bovis: S. bovis endocarditis is associated with coexisting GI malignancy. Candida and Aspergillus species: Account for most cases of fungal endocarditis. Predisposing factors include long-term indwelling IV catheters, malignancy, AIDS, organ transplantation, and IV drug use. Table 2.8-10 lists the causes of endocarditis. Constitutional symptoms are common (fever/FUO, weight loss, fatigue). Exam reveals a heart murmur. The mitral valve is more commonly affected than the aortic valve in non–IV drug users; more right-sided involvement is found in IV drug users (tricuspid valve > mitral valve > aortic valve).

1	Osler’s nodes (small, tender nodules on the f nger and toe pads), Janeway lesions (small peripheral hemorrhages; see Figure 2.8-15), splinter hemorrhages (subungual petechiae; see Figure 2.8-16), Roth’s spots (retinal hemorrhages), focal neurologic defcits from embolic stroke, and other embolic phenomena are also seen. TABLE 2.8-10. Causes of Endocarditis S. aureus (IV drug abuse) S. pneumoniae N. gonorrhoeae Viridans streptococci (native valve) Enterococcus S. epidermidis (prosthetic valve) S. bovis (GI insult) Fungi Cancer (poor prognosis). Mets seed valves; emboli can cause cerebral infarcts. Haemophilus parainﬂ uenzae Actinobacillus Cardiobacterium Eikenella Kingella Coxiella burnetii Brucella Bartonella Libman-Sacks endocarditis (autoantibody to valve) FIGURE 2.8-15. Janeway lesions.

1	FIGURE 2.8-15. Janeway lesions. Peripheral embolization to the sole leads to a cluster of erythematous macules known as Janeway lesions. (Courtesy of the Department of Dermatology, Wilford Hall USAF Medical Center and Brooke Army Medical Center, San Antonio, TX, as published in Knoop KJ et al. Atlas of Emergency Medicine, 2nd ed. New York: McGraw-Hill, 2002: 384.) Diagnosis is guided by risk factors, clinical symptoms, and the Duke criteria (see Table 2.8-11). CBC with leukocytosis and left shift; ↑ ESR and CRP. ■Early empiric IV antibiotic treatment includes vancomycin or nafcillin + gentamicin. Ampicillin/sulbactam or ceftriaxone are alternative choices depending on the suspected organism. Tailor antibiotics once the causative agent is known. Acute valve replacement is sometimes necessary. The FIGURE 2.8-16. Splinter hemorrhages.

1	FIGURE 2.8-16. Splinter hemorrhages. Note the splinter hemorrhages along the distal aspect of the nail plate, due to emboli from sub-acute bacterial endocarditis. (Courtesy of the Armed Forces Institute of Pathology, Bethesda, MD, as published in Knoop KJ et al. Atlas of Emergency Medicine, 2nd ed. New York: McGraw-Hill, 2002: 384.) T AB LE 2.8-1 1. Duke Criteria for the Diagnosis of Endocarditis Endocarditis: indications for surgery— Suppurative local complications with conduction abnormalities Resection of mycotic aneurysm Ineffective antimicrobial therapy (e.g., vs. fungi) The anthrax-associated pruritic papule forms an ulcer with an edematous halo and then a black eschar. At least two separate blood cultures for a typical organism, persistent bacteremia with any organism, or a single culture of Coxiella burnetii. Evidence of endocardial involvement (via transesophageal echocardiography or new murmur).

1	Predisposing risk factors. Fever ≥ 38.3°C. Vascular phenomena: Septic emboli, septic infarcts, mycotic aneurysm, Janeway lesions. Immunologic phenomena: Glomerulonephritis, Osler’s nodes. Roth’s spots. Microbiological evidence that does not meet major criteria. prognosis for prosthetic valve endocarditis is poor. See the mnemonic PUS RIVER for indications for surgery. ■Give antibiotic prophylaxis before dental work in patients with high-risk valvular disease (e.g., those with previous endocarditis or a prosthetic valve). Caused by the spore-forming gram-bacterium Bacillus anthracis. Its natural incidence is rare, but infection is an occupational hazard for veterinarians, farmers, and individuals who handle animal wool, hair, hides, or bone meal products. Also a biological weapon. B. anthracis can cause cutaneous (most common), inhalation (most deadly), or GI anthrax.

1	Cutaneous: Presents 1–7 days after skin exposure and penetration of spores. The lesion begins as a pruritic papule that enlarges to form an ulcer surrounded by a satellite bulbus/lesion with an edematous halo and a round, regular, and raised edge. Regional lymphadenopathy is also characteristic. The lesion evolves into a black eschar within 7–10 days. Inhalational: Presents with fever, dyspnea, hypoxia, hypotension, or symptoms of pneumonia (1–3 days after exposure), classically due to hemorrhagic mediastinitis. GI: Occurs after the ingestion of poorly cooked, contaminated meat; can present with dysphagia, nausea/vomiting, bloody diarrhea, and abdominal pain. CXR is the most sensitive test for inhalational disease (widened mediastinum, pleural effusions, infltrates). Aerobic culture and Gram stain of ulcer exudate show nonmotile short chains of bacilli. Antibody tests are also useful in confrming the diagnosis.

1	■ Ciprof oxacin or doxycycline plus one or two additional antibiotics for at least 14 days are frst-line therapy for inhalational disease or cutaneous disease of the face, head, or neck. Many strains express β-lactamases that confer resistance to penicillin; therefore, penicillin and amoxicillin are no longer recommended as single agents for inhalational disease. ■For cutaneous disease, treat for 7–10 days. Postexposure prophylaxis (ciprof oxacin) to prevent inhalation anthrax should be continued for 60 days. Bone or bone marrow infection 2° to direct spread from a soft tissue infection (80% of cases) is most common in adults, whereas infection due to hematogenous seeding (20% of cases) is more common in children (metaphyses of the long bones) and IV drug users (vertebral bodies). Common pathogens are outlined in Table 2.8-12.

1	Presents with localized bone pain and tenderness along with warmth, swelling, erythema, and limited motion of the adjacent joint. Systemic symptoms (fevers, chills) and purulent drainage may be present. Labs: ↑ WBC count, ESR (> 100), and CRP levels. Blood cultures may be . Imaging: X-rays are often initially but may show periosteal elevation within 10–14 days. Bone scans are also used and are sensitive for osteomyelitis but lack specif city. MRI (test of choice) will show ↑ signal in the bone marrow and associated soft tissue infection. Defnitive diagnosis is made by bone aspiration with Gram stain and culture. Clinical diagnosis made by probing through the soft tissue to bone is usually suffcient, as aspiration carries a risk of infection. T AB LE 2.8-1 2. Common Pathogens in Osteomyelitis Penicillin and amoxicillin are no longer recommended as single agents for the treatment of disseminated anthrax. Treat with ciproﬂ oxacin plus one or two other antibiotics.

1	Penicillin and amoxicillin are no longer recommended as single agents for the treatment of disseminated anthrax. Treat with ciproﬂ oxacin plus one or two other antibiotics. Osteomyelitis is associated with peripheral vascular disease, diabetes, penetrating soft tissue injuries, chronic decubitus ulcers, and IV drug abuse. Most people S. aureus IV drug user S. aureus or Pseudomonas Sickle cell disease Salmonella Hip replacement S. epidermidis Foot puncture wound Pseudomonas Chronic S. aureus, Pseudomonas, Enterobacteriaceae Diabetic Polymicrobial, Pseudomonas, S. aureus, streptococci, anaerobes Diabetic osteomyelitis should be treated with antibiotics targeting gramorganisms and anaerobes. Surgical debridement of necrotic, infected bone followed by IV antibiotics × 4–6 weeks. Empiric antibiotic selection is based on the suspected organism and Gram stain.

1	Surgical debridement of necrotic, infected bone followed by IV antibiotics × 4–6 weeks. Empiric antibiotic selection is based on the suspected organism and Gram stain. Consider clindamycin + ciprofoxacin, ampicillin/sulbactam, or oxacillin/ nafcillin (for methicillin-sensitive S. aureus); vancomycin (for MRSA); or ceftriaxone or ciprofoxacin (for gram-bacteria). Chronic osteomyelitis, sepsis, septic arthritis. Long-standing chronic osteomyelitis with a draining sinus tract may eventually lead to squamous cell carcinoma (Marjolin’s ulcer). Table 2.9-1 outlines the presentation and treatment of orthopedic injuries that commonly affect adults. Volkmann’s contracture of the wrist and f ngers is caused by compartment syndrome due to supracondylar fractures. Phalen’s maneuver: placing the wrists in f exion reproduces aching and numbness in < 60 seconds. Tinel’s sign: tapping over the median nerve at the wrist elicits tingling in the median nerve distribution.

1	Tinel’s sign: tapping over the median nerve at the wrist elicits tingling in the median nerve distribution. ■↑pressure within a confined space that compromises nerve, muscle, and soft tissue perfusion. Occurs primarily in the anterior compartment of the lower leg and forearm 2° to trauma (fracture or muscle injury) to the affected compartment. Hx/PE: Presents with pain out of proportion to physical findings; pain with passive motion of the fingers and toes; and paresthesias, pallor, poikilothermia, pulselessness, and paralysis. Pulselessness occurs late. Dx: Measure compartment pressures (usually ≥ 30 mmHg); measure delta pressures (diastolic pressure – compartment pressure). Tx: Immediate fasciotomy to ↓ pressures and ↑ tissue perfusion.

1	Dx: Measure compartment pressures (usually ≥ 30 mmHg); measure delta pressures (diastolic pressure – compartment pressure). Tx: Immediate fasciotomy to ↓ pressures and ↑ tissue perfusion. Entrapment of the median nerve at the wrist caused by ↓ size or space of the carpal tunnel, leading to paresthesias, pain, and occasionally paralysis. Can be precipitated by overuse of wrist ﬂexors, diabetes mellitus, or thyroid dysfunction. Commonly occurs in pregnant and middle-aged women. Presents with aching over the thenar area of the hand and proximal forearm. Pain may extend to the shoulder. Paresthesia or numbness is seen in a median nerve distribution. Symptoms worsen at night or when the wrists are held in ﬂexion or extension. Patients may report frequently dropping objects or inability to open jars. Exam shows thenar atrophy (if CTS is long-standing). Phalen’s maneuver and Tinel’s sign are . A clinical diagnosis, although EMG testing can be used to confirm.

1	Exam shows thenar atrophy (if CTS is long-standing). Phalen’s maneuver and Tinel’s sign are . A clinical diagnosis, although EMG testing can be used to confirm. Splint the wrist in a neutral position at night and during the day if possible. Administer NSAIDs. Conservative treatment can include corticosteroid injection of the carpal canal. Work-related CTS may benefit from ergonomic aids. CTS of pregnancy usually resolves after delivery. Surgical release of the carpal tunnel is a widely accepted treatment, particularly for fixed sensory loss, thenar weakness, or intolerable symptoms. Permanent loss of sensation, hand strength, and fine motor skills. T AB LE 2.9-1. Common Adult Orthopedic Injuries

1	Shoulder dislocation Anterior dislocation: Most common; the axillary artery and nerve are at risk. Patients hold the arm in slight abduction and external rotation. Posterior dislocation: Rare; associated with seizure and electrocutions; can injure the radial artery. Patients hold the arm in adduction and internal rotation. Reduction followed by a sling and swath. Recurrent dislocations may need surgical repair. Hip dislocation Posterior dislocation: Most common (> 90%); occurs via a posteriorly directed force on an internally rotated, fexed, adducted hip (“dashboard injury”). Associated with a risk of sciatic nerve injury and avascular necrosis (AVN). Anterior dislocation: Can injure the obturator nerve. Closed reduction followed by abduction pillow/ bracing. Evaluate with CT scan after reduction. Colles’ fracture Involves the distal radius. Often results from a fall onto an outstretched hand, leading to a dorsally displaced, dorsally angulated fracture. Commonly seen in the elderly

1	reduction. Colles’ fracture Involves the distal radius. Often results from a fall onto an outstretched hand, leading to a dorsally displaced, dorsally angulated fracture. Commonly seen in the elderly (osteoporosis) and children. Closed reduction followed by application of a long-arm cast; open reduction if the fracture is intra-articular. Scaphoid fracture The most commonly fractured carpal bone. May take two weeks for radiographs to show the fracture. Assume a fracture if there is tenderness in the anatomical snuff box. Thumb spica cast. If displacement or navicular nonunion is present, treat with open reduction. With proximal third scaphoid fractures, AVN may result from disruption of blood f ow. Boxer’s fracture Fracture of the ffth metacarpal neck. Due to forward trauma of a closed fst (e.g., punching a wall). Closed reduction and ulnar gutter splint; percutaneous pinning if the fracture is excessively angulated. If skin is broken, assume infection by human oral pathogens and

1	fst (e.g., punching a wall). Closed reduction and ulnar gutter splint; percutaneous pinning if the fracture is excessively angulated. If skin is broken, assume infection by human oral pathogens and treat with surgical irrigation, debridement, and IV antibiotics (covering Eikenella). Humerus fracture Direct trauma. May have radial nerve palsy leading to wrist drop and loss of thumb abduction (see Figure 2.9-1). Hanging-arm cast vs. coaptation splint and sling. Functional bracing. “Nightstick fracture” Ulnar shaft fracture resulting from self-defense with the arm against a blunt object. Open reduction and internal fxation (ORIF) if signif cantly displaced. Monteggia’s fracture Diaphyseal fracture of the proximal ulna with subluxation of the radial head. ORIF of the shaft fracture (due to poor fracture diaphyseal blood supply) and closed reduction of the radial head.

1	T AB LE 2.9-1. Common Adult Orthopedic Injuries (continued) Femoral fracture Direct trauma. Beware of fat emboli, which present with fever, change in mental status, dyspnea, hypoxia, petechiae, and ↓ platelets. Intramedullary nailing of the femur. Irrigate and debride open fractures. Tibial fracture Direct trauma. Watch for compartment syndrome. Casting vs. intramedullary nailing. Open fractures An orthopedic emergency; patients must be taken to the OR in < 6 hours owing to ↑ infection risk. OR emergently to repair fracture. Treat with antibiotics and tetanus prophylaxis. Achilles tendon rupture Presents with a sudden “pop” like a rif e shot. More likely with ↓ physical conditioning. Exam shows limited plantar f exion and a Thompson’s test (pressure on the gastrocnemius leading to absent foot plantar f exion). Treat surgically followed by long leg cast for six weeks. Knee injuries

1	Diaphyseal fracture of the radius with dislocation of the distal radioulnar joint. Results from a direct blow to the radius. ORIF of the radius and casting of the fractured forearm in supination to reduce the distal radioulnar joint. ↑ risk with osteoporosis. Presents with a shortened and externally rotated leg. Displaced femoral neck fractures: Associated with an ↑ risk of AVN, nonunion, and DVTs. Present with knee instability, edema, and hematoma. ACL: Results from a noncontact twisting mechanism, forced hyperextension, or impact to an extended knee. anterior drawer and Lachman tests. Rule out a meniscal or MCL injury. PCL: Results from forced hyperextension. posterior drawer test. Meniscal tears: Result from an acute twisting injury or a degenerative tear in elderly patients. Clicking or locking may be present. Exam shows joint line tenderness and a McMurray’s test.

1	Meniscal tears: Result from an acute twisting injury or a degenerative tear in elderly patients. Clicking or locking may be present. Exam shows joint line tenderness and a McMurray’s test. ORIF with parallel pinning of the femoral neck. Displaced fractures in elderly patients may require a hip hemiarthroplasty. Anticoagulate to ↓ the likelihood of DVTs. Treatment of MCL/LCL and meniscal tears is usually conservative. Treatment of ACL injuries is generally surgical with graft from the patellar or hamstring tendons. Operative PCL repair is reserved for highly competitive athletes. Operative meniscal repair is for younger patients with signifcant tears or older patients whose symptoms do not respond to conservative treatment. F IGU R E 2.9-1. Lateral condyle fracture of the humerus. (Reproduced, with permission, from Skinner HB. Current Diagnosis & Treatment in Orthopedics, 2nd ed. Stamford, CT: Appleton & Lange, 2000: 572.)

1	(Reproduced, with permission, from Skinner HB. Current Diagnosis & Treatment in Orthopedics, 2nd ed. Stamford, CT: Appleton & Lange, 2000: 572.) Inf ammation of the bursa by repetitive use, trauma, infection, or systemic inf ammatory disease. A bursa is a ﬂ attened sac filled with a small amount of synovial ﬂuid that serves as a protective buffer between bones and overlapping muscles. Common sites of bursitis include subacromial, olecranon, trochanteric, prepatellar, and infrapatellar bursae. Septic bursitis is more common in superficial bursae (olecranon, prepatellar, and infrapatellar bursae). Presents with localized tenderness, ↓ range of motion (ROM), edema, and erythema; patients may have a history of trauma or inﬂ ammatory disease. Needle aspiration is indicated if septic bursitis is suspected; no labs or imaging is needed. Conservative treatment includes rest, heat and ice, elevation, and NSAIDs.

1	Needle aspiration is indicated if septic bursitis is suspected; no labs or imaging is needed. Conservative treatment includes rest, heat and ice, elevation, and NSAIDs. Intrabursal corticosteroid injection can be considered (contraindicated if septic bursitis is suspected). Septic bursitis should be treated with 7–10 days of antibiotics. Oral f uoroquinolones are An inf ammatory condition characterized by pain at tendinous insertions associated with an ↑ risk of into bone associated with swelling or impaired function. It commonly occurs tendon rupture and tendinitis. Most lower back pain is mechanical; bed rest is contraindicated. Red f ags for lower back pain include age > 50, > 6 weeks of pain, previous cancer history, severe pain, constitutional symptoms, and loss of anal sphincter tone.

1	Red f ags for lower back pain include age > 50, > 6 weeks of pain, previous cancer history, severe pain, constitutional symptoms, and loss of anal sphincter tone. in the supraspinatus, biceps, wrist extensor, patellar, iliotibial band, posterior tibial, and Achilles tendons. Overuse is the most common cause and includes work-related activities or an ↑ in activity level. Presents with pain at a tendinous insertion that worsens with repetitive stress and resisted strength testing of the affected muscle group. Wrist ﬂexor tendinitis (lateral epicondylitis, or tennis elbow) worsens with resisted dorsiﬂexion of the wrist. A clinical diagnosis. Consider a radiograph if there is a history of trauma. Treat with rest and NSAIDs; apply ice for the first 24–48 hours. Consider splinting or immobilization. Begin strengthening exercises once pain has subsided.

1	Treat with rest and NSAIDs; apply ice for the first 24–48 hours. Consider splinting or immobilization. Begin strengthening exercises once pain has subsided. If conservative treatment fails, consider peritendinous injection of lidocaine and corticosteroids. Never inject the Achilles tendon in view of the ↑ risk of rupture. Avoid repetitive injection. Table 2.9-2 outlines the motor, reﬂex, and sensory deficits with which low back pain is associated. Causes include degenerative changes, trauma, or neck/back strain or sprain. Most common (95%) in the lumbar region, especially at L4–L5 and L5–S1. Presents with sudden onset of severe, electricity-like LBP, usually preceded by several months of aching, “discogenic” pain. Common among middle-aged and older men. Exacerbated by ↑ intra-abdominal pressure or Valsalva (e.g., coughing). Associated with sciatica, paresthesias, muscle weakness, atrophy, contractions, or spasms. T AB LE 2.9-2. Motor and Sensory Deficits in Back Pain

1	Associated with sciatica, paresthesias, muscle weakness, atrophy, contractions, or spasms. T AB LE 2.9-2. Motor and Sensory Deficits in Back Pain L4 L5 S1 Foot dorsifexion (tibialis anterior) Patellar Medial aspect of the lower leg. Big toe dorsifexion (extensor hallucis longus), foot eversion (peroneus muscles) None Dorsum of the foot and lateral aspect of the lower leg. Plantar fexion (gastrocnemius/soleus), hip extension (gluteus maximus). Achilles Plantar and lateral aspects of the foot. A passive straight leg raise ↑pain (highly sensitive but not specific). A crossed straight leg raise ↑ pain (highly specific but not sensitive). Large midline herniations can cause cauda equina syndrome. Obtain an ESR and a plain radiograph if other causes of back pain are suspected (e.g., infection, trauma, compression fracture). Order a stat MRI for cauda equina syndrome or for a severe or rapidly progressing neurologic deficit.

1	Order a stat MRI for cauda equina syndrome or for a severe or rapidly progressing neurologic deficit. Order an MRI if symptoms are refractory to conservative management. MRI may show disk herniation (see Figure 2.9-2). NSAIDs in scheduled doses, physical therapy, and local heat lead to resolution within four weeks in 80% of cases. Epidural or nerve block may be of benefit. Severe or rapidly evolving neurologic deficits and cauda equina syndrome are indications for discectomy. Narrowing of the lumbar or cervical spinal canal, leading to compression of the nerve roots. Most commonly due to degenerative joint disease; typically occurs in middle-aged or elderly patients. ■Presents with neck pain, back pain that radiates to the buttocks and legs, and leg numbness/weakness. Lung, breast, and prostate cancer can metastasize to the vertebrae and cause back pain.

1	■Presents with neck pain, back pain that radiates to the buttocks and legs, and leg numbness/weakness. Lung, breast, and prostate cancer can metastasize to the vertebrae and cause back pain. Bowel or bladder dysfunction (urinary overf ow incontinence), impotence, and saddle-area anesthesia are consistent with cauda equina syndrome, which is a surgical emergency. FIGURE 2.9-2. Disk herniation. MRI reveals herniations of L4–L5 and L5–S1. (Reproduced, with permission, from Skinner HB. Current Diagnosis & Treatment in Orthopedics, 1st ed. Stamford, CT: Appleton & Lange, 1995: 186.) The most common benign bone tumor is osteochondroma. Leg cramping is worse at rest, with standing, and with walking (pseudoor neurogenic claudication). Symptoms improve with fexion at the hips and bending forward. Radiographs show degenerative changes that include disk space narrowing, facet hypertrophy, and spondylolisthesis, leading to a narrowed spinal canal. MRI or CT shows spinal stenosis.

1	Radiographs show degenerative changes that include disk space narrowing, facet hypertrophy, and spondylolisthesis, leading to a narrowed spinal canal. MRI or CT shows spinal stenosis. Mild to moderate: NSAIDs and abdominal muscle strengthening. Advanced: Epidural corticosteroid injections can provide relief. Refractory: Surgical laminectomy may achieve significant short-term success, but many patients will have a recurrence of symptoms. The second most common 1° malignant tumor of bone (after multiple myeloma). Tends to occur in the metaphyseal regions of the distal femur, proximal tibia, and proximal humerus; often metastasizes to the lungs. Some cases are preceded by Paget’s disease. Risk factors include male gender and age 20– 30. Presents as progressive and eventually intractable pain that is worse at night. Constitutional symptoms such as fever, weight loss, and night sweats may be present. Erythema and enlargement over the site of the tumor may be seen.

1	Constitutional symptoms such as fever, weight loss, and night sweats may be present. Erythema and enlargement over the site of the tumor may be seen. See the Endocrinology chapter for a discussion of osteosarcoma vs. Paget’s disease. Radiographs show Codman’s triangle (periosteal new bone formation at the diaphyseal end of the lesion) or a “sunburst pattern” of the osteosarcoma (see Figure 2.9-3)—in contrast to multilayered “onion skinning,” which is classic for Ewing’s sarcoma. MRI and CT facilitate staging (soft tissue and bony invasion) and planning for surgery. Limb-sparing surgical procedures and preand postoperative chemotherapy (e.g., methotrexate, doxorubicin, cisplatin, ifosfamide). Amputation may be necessary. A common, chronic, noninﬂ ammatory arthritis of the synovial joints. Characterized by deterioration of the articular cartilage and osteophyte bone formation at the joint surfaces. Risk factors include a family history, obesity, and a history of joint trauma.

1	FIGURE 2.9-3. Osteosarcoma. “Sunburst” appearance of neoplastic bone formation in the femur of a 15-year-old girl. Amputation was required owing to the size of the tumor. (Reproduced, with permission, from Skinner HB. Current Diagnosis & Treatment in Orthopedics, 2nd ed. Stamford, CT: Appleton & Lange, 2000: 272.) Presents with crepitus, ↓ ROM, and initially pain that worsens with activity and weight bearing but improves with rest. Morning stiffness lasts for < 30 minutes. Stiffness is also experienced after periods of rest (“gelling”). Radiographs show joint space narrowing, osteophytes, subchondral sclerosis, and subchondral bone cysts (see Figure 2.9-4). Radiograph severity does not correlate with symptomatology. Synovial ﬂuid shows straw-colored ﬂuid, normal viscosity, and a WBC count < 2000 cells/μL.

1	Synovial ﬂuid shows straw-colored ﬂuid, normal viscosity, and a WBC count < 2000 cells/μL. Physical therapy, weight reduction, and NSAIDs. Intra-articular corticosteroid injections may provide temporary relief. Consider joint replacement (e.g., total hip/knee arthroplasty) in advanced cases. FIGURE 2.9-4. Osteoarthritis. Plain radiographs show joint space narrowing, osteophytes, and subchondral degenerative cysts involving the DIP and PIP joints, with sparing of the MCP. (Reproduced, with permission, from USMLERx.com.) A pain syndrome accompanied by loss of function and autonomic dysfunction, usually occurring after trauma. Formerly known as reﬂ ex sympathetic dystrophy. The disease has three phases: acute sympathetic denervation and underactivity → dystrophic phase → atrophic phase. Diffuse pain occurs out of proportion to the initial injury, often in a nonanatomic distribution. Pain can occur at any time relative to the initial injury.

1	Diffuse pain occurs out of proportion to the initial injury, often in a nonanatomic distribution. Pain can occur at any time relative to the initial injury. Loss of function of the affected limb is seen. Sympathetic dysfunction occurs and may be documented by skin, soft tissue, or blood ﬂ ow changes. Skin temperature, hair growth, and nail growth may ↑ or ↓. Edema may be present. A clinical diagnosis, but objective evidence of changes in skin temperature, hair growth, or nail growth may be present. Medications include NSAIDs, corticosteroids, low-dose TCAs, gabapentin, pregabalin, and calcitonin (no oral medications are consistently effective). Physical therapy modalities such as heat, ice, desensitization techniques, and gentle ROM exercises may be helpful. Chemical sympathetic blockade may relieve symptoms. Referral to a chronic pain specialist is appropriate for complicated cases.

1	Chemical sympathetic blockade may relieve symptoms. Referral to a chronic pain specialist is appropriate for complicated cases. A centrally mediated chronic pain disorder characterized by soft tissue and axial skeletal pain in the absence of joint pain. Inﬂammation is notably absent. Hx/PE: Most common in women 30–50 years of age; associated with depression, anxiety, sleep disorders, IBS, and cognitive disorders (“fibro fog”). Dx: Multiple (≥ 11 of 18), diffuse tender points over all four body quadrants and the axial skeleton must be present for diagnosis (see Figure 2.9-5). The presence of < 11 of 18 tender points or non-fibromyalgiaassociated tender points is known as myofascial pain syndrome. Tx: Antidepressants (an SSRI/TCA combination has proven efficacy), gabapentin, pregabalin, muscle relaxants, and physical therapy (stretching, heat application, hydrotherapy). Avoid narcotics.

1	Tx: Antidepressants (an SSRI/TCA combination has proven efficacy), gabapentin, pregabalin, muscle relaxants, and physical therapy (stretching, heat application, hydrotherapy). Avoid narcotics. Recurrent attacks of acute monoarticular arthritis resulting from intra-articular deposition of monosodium urate crystals due to disorders of urate metabolism. Risk factors include male gender, obesity, and postmenopausal status in females. ■Presents with excruciating joint pain of sudden onset that can awaken the patient from sleep. F IGU R E 2.9-5. Tender points characteristic of fibromyalgia. Gout crystals appear yeLLow when paraLLel to the condenser. Causes of hyperuricemia: ↑ cell turnover (hemolysis, blast crisis, tumor lysis, myelodysplasia, psoriasis) Cyclosporine Dehydration Diabetes insipidus Diet (e.g., ↑ red meat, Colchicine inhibits neutrophil chemotaxis and is most effective when used early during a gout f are (use is limited by a narrow therapeutic window).

1	Colchicine inhibits neutrophil chemotaxis and is most effective when used early during a gout f are (use is limited by a narrow therapeutic window). Most commonly affects the frst MTP joint (podagra) and the midfoot, knees, ankles, and wrists; the hips and shoulders are generally spared. Joints are erythematous, swollen, and exquisitely tender. Tophi (urate crystal deposits in soft tissue) may be seen with chronic disease. Joint ﬂuid aspirate shows needle-shaped, negatively birefringent crystals (see Table 2.9-3 and Clinical Images). An elevated WBC count in the joint aspirate or peripheral blood may be seen during ﬂ ares. Serum uric acid is usually ↑ (≥ 7.5), but patients may have normal levels. Punched-out erosions with overhanging cortical bone (“rat-bite” erosions) are seen in advanced gout. Acute attacks: High-dose NSAIDs (e.g., indomethacin), colchicine, and/ or steroids.

1	Punched-out erosions with overhanging cortical bone (“rat-bite” erosions) are seen in advanced gout. Acute attacks: High-dose NSAIDs (e.g., indomethacin), colchicine, and/ or steroids. Maintenance therapy: Allopurinol for overproducers, those with contraindications to probenecid treatment (tophi, renal stones, chronic renal failure), and refractory cases; probenecid for undersecretors. Weight loss and avoidance of triggers of hyperuricemia will prevent recurrent attacks in many patients. A chronic inﬂammatory disease of the spine and pelvis that causes sacroiliitis leading to fusion of the affected joints. Strongly associated with HLA-B27. Risk factors include male gender and a family history. Typical onset is in the late teens and early 20s. Presents with fatigue, intermittent hip pain, and LBP that worsens with inactivity and in the mornings.

1	Typical onset is in the late teens and early 20s. Presents with fatigue, intermittent hip pain, and LBP that worsens with inactivity and in the mornings. ■↓ spine ﬂ exion ( Schober test), loss of lumbar lordosis, hip pain and stiffness, and ↓ chest expansion are seen as the disease progresses. Anterior uveitis and heart block may occur. Other forms of seronegative spondyloarthropathy must be ruled out, including the following: Reactive arthritis (formerly known as Reiter’s syndrome): A disease of young men. The characteristic arthritis, uveitis, conjunctivitis, and urethritis usually follow an infection with Campylobacter, Shigella, Salmonella, Chlamydia, or Ureaplasma. Psoriatic arthritis: An oligoarthritis that can include the DIP joints. TABLE 2.9-3. Gout vs. Pseudogout Associated with psoriatic skin changes and sausage-shaped digits (dactylitis).

1	Psoriatic arthritis: An oligoarthritis that can include the DIP joints. TABLE 2.9-3. Gout vs. Pseudogout Associated with psoriatic skin changes and sausage-shaped digits (dactylitis). ■Enteropathic spondylitis: An ankylosing spondylitis–like disease characterized by sacroiliitis that is usually asymmetric and is associated with IBD. HLA-B27 is found in 85–95% of cases. Radiographs may show fused sacroiliac joints, squaring of the lumbar vertebrae, development of vertical syndesmophytes, and bamboo spine. ESR or CRP is ↑ in 75% of cases. RF; ANA. NSAIDs (e.g., indomethacin) for pain; exercise to improve posture and breathing. Tumor necrosis factor (TNF) inhibitors or sulfasalazine can be used in refractory cases.

1	RF; ANA. NSAIDs (e.g., indomethacin) for pain; exercise to improve posture and breathing. Tumor necrosis factor (TNF) inhibitors or sulfasalazine can be used in refractory cases. Polymyositis is a progressive, systemic connective tissue disease characterized by immune-mediated striated muscle inﬂ ammation. Dermatomyositis presents with symptoms of polymyositis plus cutaneous involvement, although the pathogenesis is different. Most often affect patients 50–70 years of age; the male-to-female ratio is 1:2. African-Americans are affected more often than Caucasians. Distinguished as follows: Polymyositis: Presents with symmetric, progressive proximal muscle weakness, pain, and difficulty breathing or swallowing (advanced disease).

1	Distinguished as follows: Polymyositis: Presents with symmetric, progressive proximal muscle weakness, pain, and difficulty breathing or swallowing (advanced disease). Dermatomyositis: Patients may have heliotrope rash (a violaceous periorbital rash), “shawl sign” (a rash involving the shoulders, upper chest, and back), and/or Gottron’s papules (a papular rash with scales located on the dorsa of the hands, over bony prominences). Patients may also develop myocarditis and cardiac conduction deficits. Can be associated with an underlying malignancy, especially lung and breast carcinoma. ■↑serum CK and anti-Jo-1 antibodies are seen (see Table 2.9-4). ■Muscle biopsy reveals inﬂammation and muscle fibers in varying stages of necrosis and regeneration. High-dose corticosteroids with taper after 4–6 weeks to ↓ the maintenance dose. Azathioprine and/or methotrexate can be used as steroid-sparing agents. T AB LE 2.9-4. Common Antibodies and Their Disease Associations

1	Azathioprine and/or methotrexate can be used as steroid-sparing agents. T AB LE 2.9-4. Common Antibodies and Their Disease Associations ANA SLE Anti-CCP RA Anticentromere CREST syndrome Anti-dsDNA SLE Antihistone Drug-induced SLE Anti-Jo-1 Polymyositis/dermatomyositis Antimitochondrial Primary biliary cirrhosis Anti-Scl-70 Scleroderma Anti-Sm SLE Anti-TSHR Graves’ disease c-ANCA Vasculitis, especially Wegener’s p-ANCA Vasculitis, microscopic polyangiitis Rheumatoid factor RA U1RNP antibody Mixed connective tissue disease Sjren’s syndrome (keratoconjunctivitis sicca) is a common ocular manifestation of rheumatoid arthritis. Felty’s syndrome is characterized by rheumatoid arthritis, splenomegaly, and neutropenia.

1	Sjren’s syndrome (keratoconjunctivitis sicca) is a common ocular manifestation of rheumatoid arthritis. Felty’s syndrome is characterized by rheumatoid arthritis, splenomegaly, and neutropenia. A systemic autoimmune disorder characterized by chronic, destructive, inﬂammatory arthritis with symmetric involvement of both large and small joints that results in synovial hypertrophy and pannus formation, ultimately leading to erosion of adjacent cartilage, bone, and tendons. Risk factors include female gender, age 35–50, and HLA-DR4. Presents with insidious onset of morning stiffness for > 1 hour along with painful, warm swelling of multiple symmetric joints (wrists, MCP joints, ankles, knees, shoulders, hips, and elbows) for > 6 weeks. Fever, fatigue, malaise, anorexia, and weight loss may also be seen. Ulnar deviation of the fingers is seen with MCP joint hypertrophy (see Figure 2.9-6).

1	Fever, fatigue, malaise, anorexia, and weight loss may also be seen. Ulnar deviation of the fingers is seen with MCP joint hypertrophy (see Figure 2.9-6). Also presents with ligament and tendon deformations (e.g., swan-neck and boutonnière deformities), vasculitis, atlantoaxial subluxation (intubation risk), and keratoconjunctivitis sicca. F IGU R E 2.9-6. Rheumatoid arthritis. Note the boutonnière deformities of the digits, ulnar deviation of the fingers, MCP joint hyper trophy, and severe involvement of the PIP joints. (Reproduced, with permission, from Chan drasoma P. Concise Pathology, 3rd ed. Stamford, CT: Appleton & Lange, 1998: 978.) Labs: ■↑RF (IgM antibodies against Fc IgG) is seen in > 75% of cases. The presence of anti-CCP (cyclic citrullinated peptide) is more spe cific than RF. ■↑ ESR may also be seen. Anemia of chronic disease. Synovial ﬂuid aspirate shows turbid ﬂ uid, ↓ viscosity, and an ↑ WBC count (3000–50,000 cells/μL). Radiographs:

1	Anemia of chronic disease. Synovial ﬂuid aspirate shows turbid ﬂ uid, ↓ viscosity, and an ↑ WBC count (3000–50,000 cells/μL). Radiographs: Early: Soft tissue swelling and juxta-articular demineralization. Late: Joint space narrowing and erosions. NSAIDs (can be reduced or discontinued following successful treatment with disease-modifying antirheumatic drugs [DMARDs]). DMARDs should be started early and include hydroxychloroquine, sulfasalazine, and methotrexate. Second-line agents include TNF inhibitors, rituximab (anti-CD20), and leﬂ unomide. Also called systemic sclerosis; characterized by inﬂammation that leads to progressive tissue fibrosis through excessive deposition of type I and type III collagen. Commonly manifests as CREST syndrome (limited form), but can also occur in a diffuse form involving the skin as well as the GI, GU, renal, pulmonary, and cardiovascular systems. Risk factors include female gender and age 35–50.

1	Exam may reveal symmetric thickening of the skin of face and/or distal extremities. CREST syndrome involves Calcinosis, Raynaud’s phenomenon, Esophageal dysmotility, Sclerodactyly, and Telangiectasias. (lupus cerebritis, Libman-Sacks endocarditis: noninfectious vegetations often seen on the mitral valve in association with SLE and antiphospholipid syndrome. ■The diffuse form can lead to pulmonary f brosis, cor pulmonale, acute renal failure, and malignant hypertension. RF and ANA may be . Anticentromere antibodies are specific for CREST syndrome (see Table 2.9-4). Anti-Scl-70 (antitopoisomerase 1) antibodies are associated with diffuse disease and a poor prognosis (see Table 2.9-4). Eosinophilia may be seen. Corticosteroids for acute ﬂares; penicillamine can be used for skin changes. Calcium channel blockers for Raynaud’s phenomenon. ACEIs for renal disease and prevention of a scleroderma renal crisis.

1	Calcium channel blockers for Raynaud’s phenomenon. ACEIs for renal disease and prevention of a scleroderma renal crisis. Mortality is due to pulmonary hypertension and complications of pulmonary hypertension. A multisystem autoimmune disorder related to antibody-mediated cellular attack and deposition of antigen-antibody complexes. African-American women are at highest risk. Usually affects women of childbearing age. Presents with nonspecific symptoms such as fever, anorexia, weight loss, and symmetric joint pain. The mnemonic DOPAMINE RASH summarizes the criteria for diagnosing SLE (see also Figure 2.9-7 and Clinical Images). Patients with four of the criteria are likely to have SLE (96% sensitive and specific). A ANA is highly sensitive but not specific. Anti-dsDNA and anti-Sm antibodies are highly specific but not as sensitive (see Table 2.9-4). Drug-induced SLE: antihistone antibodies are seen in 100% of cases but are nonspecific.

1	Drug-induced SLE: antihistone antibodies are seen in 100% of cases but are nonspecific. Neonatal SLE: Associated with anti-Ro antibodies transmitted from mother to neonate. The following may also be seen: Antiphospholipid antibodies. Anemia, leukopenia, and/or thrombocytopenia. Proteinuria and/or casts. NSAIDs for mild joint symptoms. Corticosteroids for acute exacerbations. Corticosteroids, hydroxychloroquine, cyclophosphamide, and azathioprine for progressive or refractory cases. F IGU R E 2.9-7. Systemic lupus erythematosus. Erythematous patches and plaques of SLE, predominantly in sun-exposed areas. Note the malar rash across the bridge of the nose. (Reproduced, with permission, from Hurwitz RM. Pathology of the Skin: Atlas of Clinical-Pathological Correlation, 2nd ed. Stamford, CT: Appleton & Lange, 1998: 39.)

1	Also called giant cell arteritis; due to subacute granulomatous inﬂ ammation of the large vessels, including the aorta, external carotid (especially the temporal branch), and vertebral arteries. The most feared manifestation is blindness 2° to occlusion of the central retinal artery (a branch of the internal carotid artery). Risk factors include polymyalgia rheumatica (affects almost half of TA patients), age > 50, and female gender. Presents with new headache (unilateral or bilateral); scalp pain and temporal tenderness; and jaw claudication. Fever, permanent monocular blindness, weight loss, and myalgias/arthralgias (especially of the shoulders and hips) are also seen. ESR > 50 (usually > 100). Ophthalmologic evaluation. Temporal artery biopsy: Look for thrombosis; necrosis of the media; and lymphocytes, plasma cells, and giant cells.

1	ESR > 50 (usually > 100). Ophthalmologic evaluation. Temporal artery biopsy: Look for thrombosis; necrosis of the media; and lymphocytes, plasma cells, and giant cells. High-dose prednisone begun immediately to prevent ocular involvement (or involvement of the remaining eye after onset of monocular blindness). Obtain a biopsy, but do not delay treatment. Conduct a follow-up eye exam. Risk factors include female gender and age > 50. Hx/PE: Presents with pain and stiffness of the shoulder and pelvic girdle musculature with difficulty getting out of a chair or lifting the arms above the head. Other symptoms include fever, malaise, and weight loss. Weakness is generally not appreciated on exam. Dx: Labs reveal a markedly ↑ESR, often associated with anemia. Tx: Low-dose prednisone (10–20 mg/day). Table 2.9-5 outlines the presentation and treatment of common pediatric orthopedic injuries.

1	Dx: Labs reveal a markedly ↑ESR, often associated with anemia. Tx: Low-dose prednisone (10–20 mg/day). Table 2.9-5 outlines the presentation and treatment of common pediatric orthopedic injuries. An X-linked recessive disorder resulting from a deficiency of dystrophin, a cytoskeletal protein. Onset is usually at 3–5 years of age. Affects axial and proximal muscles more than distal muscles. May present with progressive clumsiness, fatigability, difficulty standing or walking, difficulty walking on toes (gastrocnemius shortening), Gowers’ maneuver (using the hands to push off the thighs when rising from the ﬂoor), and waddling gait. Pseudohypertrophy of the gastrocnemius muscles is also seen. Mental retardation is common. Table 2.9-6 outlines the differential diagnosis of DMD and Becker muscular dystrophy. dystrophin immunostain; ↑ CK. EMG shows polyphasic potentials and ↑ recruitment.

1	Table 2.9-6 outlines the differential diagnosis of DMD and Becker muscular dystrophy. dystrophin immunostain; ↑ CK. EMG shows polyphasic potentials and ↑ recruitment. Muscle biopsy shows necrotic muscle fibers from degeneration and variation in fiber size with fibrosis from regeneration. Physical therapy is necessary to maintain ambulation and to prevent contractures. Liberal use of tendon release surgery may prolong ambulation. Mortality is due to pulmonary congestion caused by high-output cardiac failure; cardiac fibrosis → arrhythmias and weak skeletal muscles → cardiopulmonary complications → pneumonia and respiratory failure. T AB LE 2.9-5. Orthopedic Injuries in Children

1	Clavicular fracture The most commonly fractured long bone in children. May be birth related (especially in large infants) and can be associated with brachial nerve palsies. Usually involves the middle third of the clavicle, with the proximal fracture end displaced superiorly owing to the pull of the sternocleidomastoid. Figure-of-eight sling vs. arm sling. Greenstick fracture Incomplete fracture involving the cortex of only one side of the bone. Reduction with casting. Order flms at 10–14 days. Nursemaid’s elbow Radial head subluxation that typically occurs as a result of being pulled or lifted by the hand. Presents with pain and refusal to bend the elbow. Manual reduction by gentle supination of the elbow at 90 degrees of f exion. No immobilization. Torus fracture Buckling of the cortex of a long bone 2° to trauma. Usually occurs in the distal radius or ulna. Cast immobilization for 3–5 weeks. Supracondylar humerus fracture Tends to occur at 5–8 years of age. Proximity to the

1	cortex of a long bone 2° to trauma. Usually occurs in the distal radius or ulna. Cast immobilization for 3–5 weeks. Supracondylar humerus fracture Tends to occur at 5–8 years of age. Proximity to the brachial artery ↑ the risk of Volkmann’s contracture (results from compartment syndrome of the forearm). Cast immobilization; closed reduction with percutaneous pinning if signif cantly displaced. Osgood-Schlatter disease Overuse apophysitis of the tibial tubercle. Causes localized pain, especially with quadriceps contraction, in active young boys. ↓ activity for 2–3 months or until asymptomatic. A neoprene brace may provide symptomatic relief. Salter-Harris fracture Fractures of the growth plate in children. Classifed by fracture location: ■ I: Physis (growth plate). ■ II: Metaphysis and physis. ■ III: Epiphysis and physis. ■ IV: Epiphysis, metaphysis, and physis. ■ V: Crush injury of the physis. Types I and II: Conservative. Types III–V: Surgical repair to prevent complications such as

1	■ III: Epiphysis and physis. ■ IV: Epiphysis, metaphysis, and physis. ■ V: Crush injury of the physis. Types I and II: Conservative. Types III–V: Surgical repair to prevent complications such as leg length inequality.

1	Developmental Dysplasia of the Hip Also called congenital hip dislocation; can result in subluxed, dislocatable, or dislocated femoral heads, leading to early degenerative joint disease of the hips. Dislocations result from poor development of the acetabulum and hip due to lax musculature and from excessive uterine packing in the ﬂ exed and adducted position (e.g., breech presentation), leading to excessive stretching of the posterior hip capsule and adductor muscle contracture. T AB LE 2.9-6. DMD vs. Becker Muscular Dystrophy Onset 3–5 years. 5–15 years and beyond. Life expectancy Teens. 30s–40s. Mental retardation Common. Uncommon. Western blot Dystrophin is markedly ↓ or absent. Dystrophin levels are normal, but protein is abnormal. Most commonly found in f rst-born females born in the breech position.

1	Most commonly found in f rst-born females born in the breech position. Barlow’s maneuver: Pressure is placed on the inner aspect of the abducted thigh, and the hip is then adducted, leading to an audible “clunk” as the femoral head dislocates posteriorly. Ortolani’s maneuver: The thighs are gently abducted from the midline with anterior pressure on the greater trochanter. A soft click signifies reduction of the femoral head into the acetabulum. Allis’ (Galeazzi’s) sign: The knees are at unequal heights when the hips and knees are ﬂexed (the dislocated side is lower). Asymmetric skin folds and limited abduction of the affected hip are also seen. Early detection is critical to allow for proper hip development. Ultrasound may be helpful, especially after 10 weeks of age. Radiographs are unreliable until patients are > 4 months of age because of the radiolucency of the neonatal femoral head. Begin treatment early.

1	Radiographs are unreliable until patients are > 4 months of age because of the radiolucency of the neonatal femoral head. Begin treatment early. < 6 months: Splint with a Pavlik harness (maintains the hip ﬂ exed and abducted). To prevent AVN, do not ﬂex the hips > 60 degrees. 6–15 months: Spica cast. 15–24 months: Open reduction followed by spica cast. Joint contractures and AVN of the femoral head. Without treatment, a significant defect is likely in patients < 2 years of age. Idiopathic AVN of the femoral head (see Figure 2.9-8). Most commonly found in boys 4–10 years of age. Usually a self-limited disease, with symptoms lasting < 18 months. F IGU R E 2.9-8 Legg-Calvé-Perthes disease. AVN of the femoral head. (Reproduced, with permission, from Skinner HB. Current Diagnosis & Treatment in Orthopedics, 2nd ed. Stamford, CT: Appleton & Lange, 2000: 543.) Generally asymptomatic at first, but patients can develop a painless limp.

1	Generally asymptomatic at first, but patients can develop a painless limp. If pain is present, it can be in the groin or anterior thigh, or it may be referred to the knee. Limited abduction and internal rotation; atrophy of the affected leg. Usually unilateral (85–90%). Observation is sufficient if there is limited femoral head involvement or if full ROM is present. If extensive or if there is ↓ ROM, consider bracing, hip abduction with a Petrie cast, or an osteotomy. The prognosis is good if the patient is < 5 years of age and has full ROM, ↓ femoral head involvement, and a stable joint. Separation of the proximal femoral epiphysis through the growth plate, leading to medial and posterior displacement of the femoral head (relative to the femoral neck). May be due to an imbalance between growth hormone and sex hormones. Risk factors include obesity, age 11–13, male gender, and African-American ethnicity. Associated with hypothyroidism and other endocrinopathies.

1	■Typically presents with acute or insidious thigh or knee pain and a painful limp. Differential diagnosis of pediatric limp— Acute cases present with restricted ROM and, commonly, inability to bear weight. Bilateral in 40–50% of cases. Characterized by limited internal rotation and abduction of the hip. Flex-ion of the hip results in an obligatory external rotation 2° to physical displacement that is observed as further loss of internal rotation with hip ﬂ ex-ion. Radiographs of both hips in AP and frog-leg lateral views reveal posterior and medial displacement of the femoral head (see Figure 2.9-9). Rule out hypothyroidism with TSH. The disease is progressive, so treatment should begin promptly. No weight bearing should be allowed until the defect is surgically stabilized. Gentle closed reduction is appropriate only in acute slips. F IGU R E 2.9-9. Slipped capital femoral epiphysis.

1	No weight bearing should be allowed until the defect is surgically stabilized. Gentle closed reduction is appropriate only in acute slips. F IGU R E 2.9-9. Slipped capital femoral epiphysis. AP x-ray. The medial displacement of the left femoral epiphysis is best seen with a line drawn up the lateral femoral neck. The abnormal epiphysis does not protrude beyond this line. Frog-leg lateral x-ray. Posterior displacement of the femoral epiphysis is characteristic. (Reproduced, with permission, from Skinner HB. Current Diagnosis & Treatment in Orthopedics, 2nd ed. Stamford, CT: Appleton & Lange, 2000: 546.) Chondrolysis, AVN of the femoral head, and premature hip osteoarthritis leading to hip arthroplasty.

1	Chondrolysis, AVN of the femoral head, and premature hip osteoarthritis leading to hip arthroplasty. A lateral curvature of the spine of > 10 degrees occurring in the thoracic and/or lumbar spine and associated with rotation of the vertebrae and sometimes excessive kyphosis or lordosis. Most commonly idiopathic, developing in early adolescence. Other etiologies are congenital or associated with neuromuscular, vertebral, or spinal cord disease. The male-to-female ratio is 1:7 for curves that progress and require treatment. Idiopathic disease is usually identified during school physical screening. Vertebral and rib rotation deformities are accentuated by a forward bending test. Radiographs of the spine (posterior, anterior, and full-length views). Close observation for < 20 degrees of curvature. Spinal bracing for 20–49 degrees of curvature. Curvature may progress even with bracing. Surgical correction for > 50 degrees of curvature.

1	Close observation for < 20 degrees of curvature. Spinal bracing for 20–49 degrees of curvature. Curvature may progress even with bracing. Surgical correction for > 50 degrees of curvature. Severe scoliosis can create restrictive lung disease. A nonmigratory, nonsuppurative monoand polyarthritis with bony destruction that occurs in patients ≤ 16 years of age and lasts > 6 weeks. Formerly known as juvenile rheumatoid arthritis. Approximately 95% of cases resolve by puberty. More common in girls than in boys. Can be accompanied by fever, nodules, erythematous rashes, pericarditis, and fatigue. Subtypes are as follows: Pauciarticular: An asymmetric arthritis that involves weight-bearing joints. Associated with an ↑ risk of iridocyclitis that leads to blindness if left untreated. Polyarticular: Resembles RA with symmetric involvement of multiple (≥ 5) small joints. Systemic features are less prominent; carries a ↓ risk of iridocyclitis.

1	Polyarticular: Resembles RA with symmetric involvement of multiple (≥ 5) small joints. Systemic features are less prominent; carries a ↓ risk of iridocyclitis. Acute febrile: The least common subtype; manifests as arthritis with daily high, spiking fevers and a maculopapular, evanescent, salmon-colored rash. Hepatosplenomegaly and serositis may also be seen. No iridocyclitis is present; remission may occur within one year. Occurs equally in girls and boys. There is no diagnostic test for JIA. Labs: A RF is found in 15% of cases. ANA may be , especially in the pauciarticular subtype. ■↑ ESR, WBC count, and platelets. Imaging: Soft tissue swelling and osteoporosis may be seen. NSAIDs or corticosteroids; methotrexate is second-line therapy. Disorders of the Neuromuscular Junction 300 MYASTHENIA GRAVIS 300 LAMBERT-EATON MYASTHENIC SYNDROME 301 MULTIPLE SCLEROSIS 301 GUILLAIN-BARRÉ SYNDROME 302 AMYOTROPHIC LATERAL SCLEROSIS 303

1	Disorders of the Neuromuscular Junction 300 MYASTHENIA GRAVIS 300 LAMBERT-EATON MYASTHENIC SYNDROME 301 MULTIPLE SCLEROSIS 301 GUILLAIN-BARRÉ SYNDROME 302 AMYOTROPHIC LATERAL SCLEROSIS 303 Tables 2.10-1 through 2.10-5 and Figure 2.10-1 outline critical aspects of clinical neuroanatomy, including cranial nerve functions; the clinical presentation of common facial nerve lesions; spinal cord anatomy and functions; UMN and LMN signs; and pertinent clinical reﬂ exes. Acute onset of focal neurologic deficits resulting from disruption of cerebral circulation. Many classifications exist, but the most common comparison involves TABLE 2.10-1. Cranial Nerve Functions Stroke is the third most common cause of death and the leading cause of major disability in the United States.

1	Olfactory I Smell Sensory Some Optic II Sight Sensory Say Oculomotor III Eye movement, pupillary constriction, lens accommodation, eyelid opening Motor Marry Trochlear IV Eye movement Motor Money Trigeminal V Mastication, facial sensation (including orbits, sinuses, tongue, teeth, and buccal mucosa), intracranial sensation (including meninges and blood vessels) Both But Abducens VI Eye movement Motor My Facial VII Facial movement, taste from the anterior two-thirds of the tongue, lacrimation, salivation (submandibular and sublingual glands), eyelid closing Both Brother Vestibulocochlear VIII Hearing, balance Sensory Says Glossopharyngeal IX Taste from the posterior third of the tongue, oropharyngeal sensation, swallowing (stylopharyngeus), salivation (parotid gland), monitoring carotid body and sinus chemoand baroreceptors Both Big Vagus X Taste from the epiglottic region, swallowing, palatal elevation, talking, thoracoabdominal viscera, monitoring aortic arch chemoand baroreceptors.

1	and sinus chemoand baroreceptors Both Big Vagus X Taste from the epiglottic region, swallowing, palatal elevation, talking, thoracoabdominal viscera, monitoring aortic arch chemoand baroreceptors. Both Brains Accessory XI Head turning, shoulder shrugging Motor Matter Hypoglossal XII Tongue movement Motor Most Adapted, with permission, from Le T, Bhushan V et al. First Aid for the USMLE Step 1 2009. New York: McGraw-Hill, 2009: 394.

1	TABLE 2.10-2. Facial Nerve Lesions UMN lesion LMN lesion Bell’s palsy Lesion of the motor cortex or the connection between the cortex and the facial nucleus. Contralateral paralysis of the lower face only. Ipsilateral paralysis of the upper and lower face. Complete destruction of the facial nucleus itself or its branchial efferent fbers (facial nerve proper). Peripheral ipsilateral facial paralysis with inability to close the eye on the involved side. Can occur idiopathically; gradual recovery is seen in most cases. Seen as a complication in AIDS, Lyme disease, Sarcoidosis, Tumors, and Diabetes. ALexander Bell with STD: AIDS, Lyme, Sarcoid, Tumors, Diabetes. Adapted, with permission, from Le T, Bhushan V et al. First Aid for the USMLE Step 1 2009. New York: McGraw-Hill, 2009: 397. Upperdivision Lower division Face area of motor cortex Cortico-bulbar tract (UMN lesion = central facial) Facial nucleus LMN lesion CN VII (LMN lesion = Bell´s palsy) TABLE 2.10-3.

1	TABLE 2.10-3. ischemic (80%) vs. hemorrhagic (20%). Table 2.10-6 contrasts modifiable and nonmodifiable risk factors associated with stroke. Etiologies are as follows: Atherosclerosis of the extracranial vessels (internal/common carotid, basilar, and vertebral arteries). Lacunar infarcts in regions supplied by perforating vessels (result from hypertension, hypercholesterolemia, or diabetes). Cardiac or aortic emboli: Thromboemboli (AF, ventricular hypokinesis, prosthetic valves, marantic endocarditis), atheroemboli (aortic arch atherosclerosis), infectious emboli (bacterial endocarditis), paradoxical emboli (via patent foramen ovale). Hypercoagulable states: Include those associated with antiphospholipid antibodies, activated protein C resistance, malignancy, and OCPs in the context of smoking. Craniocervical dissection: Trauma, fibromuscular dysplasia (young females), inﬂ ammatory/infectious diseases.

1	Craniocervical dissection: Trauma, fibromuscular dysplasia (young females), inﬂ ammatory/infectious diseases. Lateral corticospinal Movement of contralateral limbs Pyramidal, at the cervicomedullary junction 1° motor cortex Fine touch, vibration, conscious proprioception Arcuate fbers at the medulla Pacini’s and Meissner’s tactile disks, muscle spindles, and Golgi tendon organs Pain, temperature Ventral white commissure at spinal cord level Free nerve endings, pain f bers Dorsal column medial lemniscus Spinothalamic Adapted, with permission, from Le T, Bhushan V et al. First Aid for the USMLE Step 1 2009. New York: McGraw-Hill, 2009: 105. TABLE 2.10-4. UMN vs. LMN Signs

1	Adapted, with permission, from Le T, Bhushan V et al. First Aid for the USMLE Step 1 2009. New York: McGraw-Hill, 2009: 105. TABLE 2.10-4. UMN vs. LMN Signs Pattern of weakness Pyramidal (arm extensors, leg f exors) Variable Tone Spastic (↑); initially f accid (↓) Flaccid (↓) DTRs ↑ (initially ↓ or normal) ↓Miscellaneous signs Babinski’s, other CNS signs Atrophy, fasciculations ■ Other causes: Venous sinus thrombosis, sickle cell anemia, vasculitis (e.g., giant cell arteritis). Symptoms are dependent on the vascular territory affected: Middle cerebral artery (MCA): Aphasia (dominant hemisphere), neglect (nondominant hemisphere), contralateral paresis and sensory loss in the face and arm, gaze preference toward the side of the lesion, homonymous hemianopia. Anterior cerebral artery (ACA): Contralateral paresis and sensory loss in the leg; cognitive or personality changes. Posterior cerebral artery (PCA): Homonymous hemianopia, memory deficits, dyslexia/alexia.

1	Posterior cerebral artery (PCA): Homonymous hemianopia, memory deficits, dyslexia/alexia. Basilar artery: Coma, “locked-in” syndrome, cranial nerve palsies (e.g., diplopia), apnea, visual symptoms, drop attacks, dysphagia, dysarthria, vertigo, “crossed” weakness and sensory loss affecting the ipsilateral face and contralateral body. Lacunar: Pure motor or sensory stroke, dysarthria–clumsy hand syndrome, ataxic hemiparesis. TIA: A transient neurologic deficit that lasts < 24 hours (most last < 1 hour) and is determined to be of ischemic etiology. Many TIAs (~30–50%) are associated with small, asymptomatic strokes on diffusion-weighted MRI. ■Emergent head CT without contrast (see Figure 2.10-2) to differentiate ischemic from hemorrhagic stroke and to identify potential candidates for thrombolytic therapy. TABLE 2.10-5. Clinical Reﬂ exes MCA stroke can cause CHANGes: Contralateral paresis and sensory loss in the face and arm the side of the lesion

1	TABLE 2.10-5. Clinical Reﬂ exes MCA stroke can cause CHANGes: Contralateral paresis and sensory loss in the face and arm the side of the lesion Biceps = C5 nerve root. Triceps = C7 nerve root. Patella = L4 nerve root. Achilles = S1 nerve root. Babinski––dorsifexion of the big toe and fanning of other toes; sign of UMN lesion, but normal refex in the frst year of life. Refexes count up in order. S1, 2 L3, 4 C5, 6 C7, 8 C5, 6 L3, 4 C7, 8 S1, 2 Adapted, with permission, from Le T, Bhushan V et al. First Aid for the USMLE Step 1 2009. New York: McGraw-Hill, 2009: 392. FIGURE 2.10-1. Spinal cord lesions. (Reproduced, with permission, from Le T, Bhushan V et al. First Aid for the USMLE Step 1 2009. New York: McGraw-Hill, 2009: 389.) MRI to identify early ischemic changes (e.g., diffusion-weighted MRI is sensitive for acute stroke). ECG and an echocardiogram if embolic stroke is suspected.

1	MRI to identify early ischemic changes (e.g., diffusion-weighted MRI is sensitive for acute stroke). ECG and an echocardiogram if embolic stroke is suspected. Vascular studies of intracranial and extracranial disease include carotid ultrasound, transcranial Doppler, MRA, and angiography (see Figure 2.10-3). T AB LE 2.1 0-6. Modifiable and Nonmodifiable Risk Factors for Stroke FAME: Family history of MI or stroke Age > 60 Male gender Ethnicity (African-American, Hispanic, Asian) “Live the way a COACH SHoulDD”: CAD Obesity Atrial f brillation Carotid stenosis Hypercholesterolemia Smoking Hypertension Diabetes Drug use (cocaine or IV drugs) F IGU R E 2.1 0-2. CT/MRI findings in ischemic stroke in the right MCA territory.

1	F IGU R E 2.1 0-2. CT/MRI findings in ischemic stroke in the right MCA territory. (A) CT shows low density and effacement of cortical sulci (between arrowheads) and compression of the anterior horn of the lateral ventricle (arrow). (B) T1-weighted MRI shows loss of sulcal markings (between arrowheads) and compression of the anterior horn of the lateral ventricle (arrow). (C) T2-weighted MRI scan shows increased signal intensity (between arrowheads) and ventricular compression (arrow). (Reproduced, with permission, from Aminoff MJ. Clinical Neurology, 3rd ed. Stamford, CT: Appleton & Lange, 1996: 275.) F IGU R E 2.1 0-3. Vascular studies preand postendarterectomy. Contraindications to tPA therapy— Stroke or head trauma within the last three months Anticoagulation with INR > 1.7 or prolonged PTT Low platelet count (< 100,000/mm3) Elevated BP: Systolic > 185 or diastolic BP > 110 Surgery in the past 14 days TIA (mild symptoms or rapid improvement of symptoms)

1	Low platelet count (< 100,000/mm3) Elevated BP: Systolic > 185 or diastolic BP > 110 Surgery in the past 14 days TIA (mild symptoms or rapid improvement of symptoms) GI or urinary bleeding in the past 21 days Seizures present at the onset of stroke (A) Carotid arteriogram showing stenosis of the proximal internal carotid artery. (B) Postop erative arteriogram with restoration of the normal luminal size following endarterectomy. (Reproduced, with permission, from Way LW. Current Surgical Diagnosis & Treatment, 10th ed. Stamford, CT: Appleton & Lange, 1994: 763.) ■Screen for hypercoagulable states with a history of thrombosis, in the setting of a first stroke, or in patients < 50 years of age. ■ Acute:

1	■ Acute: Ischemic stroke: tPA is indicated if administered within three hours of symptom onset, but first rule out contraindications. Be aware of potential bleeding or angioedema. Intra-arterial thrombolysis can be used for select patients within six hours of major stroke from MCA occlusion if such patients are not suitable candidates for IV tPA. Hemorrhagic stroke: See the discussion of parenchymal hemorrhage. ICU admission should be considered, especially for comatose patients or for those who are unable to protect their airways for possible intubation. Monitor for signs and symptoms of brain swelling, ↑ ICP, and herniation. Serial CTs are helpful in the evaluation of deteriorating patients. As a temporizing measure, treat with mannitol and hyperventilation. ASA is associated with ↓ morbidity and mortality in acute ischemic stroke presenting ≤ 48 hours from onset.

1	ASA is associated with ↓ morbidity and mortality in acute ischemic stroke presenting ≤ 48 hours from onset. Allow permissive hypertension and hypoxemia to maintain perfusion of ischemic cerebral tissue. However, in the setting of severe hypertension (SBP > 220 or DBP >120) or hemorrhagic stroke, treat with IV labetalol or nicardipine infusion. Additionally, to administer tPA, SBP must be < 185 and DBP < 110. Treat fever and hyperglycemia, as both are associated with worse prognoses in the setting of acute stroke. Prevent and treat post-stroke complications such as aspiration pneumonia, UTI, and DVT. Immediate labs to be obtained include CBC with platelets, cardiac enzymes and troponin, electrolytes, BUN, creatinine, serum glucose, PTT, PT, INR, lipid profile, and oxygen saturation. Prevention and long-term treatment: ASA, clopidogrel: If stroke is 2° to small vessel disease or thrombosis, or if anticoagulation is contraindicated.

1	Prevention and long-term treatment: ASA, clopidogrel: If stroke is 2° to small vessel disease or thrombosis, or if anticoagulation is contraindicated. Carotid endarterectomy: If stenosis is > 70% in symptomatic patients or > 60% in asymptomatic patients (contraindicated in 100% occlusion). Anticoagulation: In new AF or hypercoagulable states, the target INR is 2–3. In cases involving a prosthetic valve, the target INR is 3–4 or add an antiplatelet agent. Management of hypertension, hypercholesterolemia, and diabetes (hypertension is the single greatest risk factor for stroke). Etiologies of SAH include trauma, berry aneurysms, AVM, and trauma to the circle of Willis.

1	Etiologies of SAH include trauma, berry aneurysms, AVM, and trauma to the circle of Willis. Aneurysmal SAH presents with an abrupt-onset, intensely painful “thunderclap” headache, often followed by neck stiffness and other signs of meningeal irritation, including photophobia, nausea/vomiting, and meningeal stretch signs. Rapid development of obstructive hydrocephalus or seizures often leads to ↓ arousal or frank coma and death in the absence of neurosurgical intervention. More than one-third of patients will give a history of a “sentinel bleed” days to weeks earlier marked by an abrupt-onset headache, often with nausea/vomiting, or transient diplopia that completely resolved in a matter of minutes to hours. Immediate head CT without contrast (see Figure 2.10-4) to look for blood in the subarachnoid space. Sensitivity is > 95% in those with severe SAH but is much lower in patients with normal mental status.

1	Immediate LP if CT is to look for RBCs, xanthochromia (yellowish CSF due to breakdown of RBCs), ↑ protein (from the RBCs), and ↑ ICP. Note that LP results can be falsely in the first 6–12 hours (because xanthochromia has not yet developed) and after the first 24–28 hours (because xanthochromia has resolved). Four-vessel angiography (or equivalent noninvasive angiography such as CT angiography with 3-D reconstructions) should be performed once SAH is confirmed. Noninvasive angiography is warranted in high-risk cases and in those with high clinical suspicion even if CT and LP are unrevealing. Call neurosurgery. Prevent rebleeding (most likely to occur in the first 48 hours) by maintaining systolic BP < 150 until the aneurysm is clipped or coiled. Prevent vasospasm and associated neurologic deterioration (most likely to occur 5–7 days after SAH) by administering calcium channel blockers (CCBs), IV ﬂuids, and pressors to maintain BP. Give phenytoin for seizure prophylaxis.

1	■↓ ICP by raising the head of the bed and instituting hyperventilation. Treat hydrocephalus through a lumbar drain or serial LPs. CN III palsy with pupillary involvement is associated with berry aneurysms. Conditions associated with berry aneurysms that can MAKE an SAH more likely: dominant, polycystic) Ehlers-Danlos syndrome Sickle cell anemia Atherosclerosis History (familial) The characteristic “worst headache of my life” of SAH comes on quickly, in contrast to migraine (peak intensity > 30 minutes). F IGU R E 2.1 0-4. Subarachnoid hemorrhage. Mental status changes associated with an expanding epidural hematoma occur within minutes to hours and classically have a lucid interval. With a subdural hematoma, mental status changes can occur within days to weeks. (A) CT scan without contrast reveals blood in the subarachnoid space at the base of the brain (arrows). (B) A normal CT scan without contrast shows no density in this region (arrows).

1	(A) CT scan without contrast reveals blood in the subarachnoid space at the base of the brain (arrows). (B) A normal CT scan without contrast shows no density in this region (arrows). (Reproduced, with permission, from Aminoff MJ. Clinical Neurology, 3rd ed. Stamford, CT: Appleton & Lange, 1996: 78.) ■ Surgical clipping is the defnitive treatment for aneurysms. Endovascular coiling is an option for poor surgical candidates. Risk factors include hypertension, tumor, amyloid angiopathy (in the elderly), anticoagulation, and vascular malformations (AVMs, cavernous hemangiomas). Hx/PE: Presents with focal motor and sensory defcits that often worsen as the hematoma expands. Severe headache of sudden onset, nausea/ vomiting, seizures, lethargy, or obtundation may also be seen. Dx: Immediate noncontrast head CT (see Figure 2.10-5). Look for mass effect or edema that may predict herniation.

1	Dx: Immediate noncontrast head CT (see Figure 2.10-5). Look for mass effect or edema that may predict herniation. Tx: Similar to that of SAH. Elevate the head of the bed and institute anti-seizure prophylaxis. Surgical evacuation may be necessary if mass effect is present. Several types of herniation may occur, including central, uncal, subfalcine, and tonsillar (see Figure 2.10-6 and Table 2.10-7). Typically occurs following head trauma (usually falls or assaults), leading to rupture of bridging veins and accumulation of blood between the dura and arachnoid membranes. Common in the elderly and alcoholics. Hx/PE: Presents with headache, changes in mental status, and contra-lateral hemiparesis. Changes may be subacute or chronic. May present as pseudodementia in the elderly. F IGU R E 2.1 0-5. Intraparenchymal hematoma.

1	F IGU R E 2.1 0-5. Intraparenchymal hematoma. Head CT without contrast reveals the irregularly shaped hyperdensity with midline shift of the choroid plexus. (Reproduced, with permission, from Saunders CE. Current Emergency Diagno sis & Treatment, 4th ed. Stamford, CT: Appleton & Lange, 1992: 248.) Dx: CT demonstrates a crescent-shaped, concave hyperdensity acutely (isodense subacutely, hypodense chronically) that does not cross the mid-line (see Figure 2.10-7A). Tx: Surgical evacuation if symptomatic. Subdural blood may regress spontaneously if it is chronic. 1. 2. 3. Uncal herniation 4. Cerebellar tonsillar herniation into the Tentorium foramen magnum Coma and death result when these herniations compress the brain stem F IGU R E 2.1 0-6. Sites of herniation syndromes. (Adapted, with permission, from Simon RP et al. Clinical Neurology, 4th ed. Stamford, CT: Appleton & Lange, 1999: 314.) TAB LE 2.1 0-7. Clinical Presentation of Herniation Syndromes

1	TAB LE 2.1 0-7. Clinical Presentation of Herniation Syndromes Cingulate herniation Occurs 2° to mass lesions of the frontal lobes. No specifc signs or symptoms; frequently seen on head CT. Downward transtentorial (central) herniation Occurs when large supratentorial mass lesions push the midbrain inferiorly. Presents with a rapid change in mental status; bilaterally small and reactive pupils; Cheyne-Stokes respirations; and fexor or extensor posturing. Uncal herniation Occurs 2° to mass lesions of the middle fossa. CN III becomes entrapped, leading to a fxed and dilated ipsilateral pupil followed by an eye that is deviated “down and out.” Ipsilesional hemiparesis (“false localizing”) results from compression of the cerebral peduncle (opposite the mass lesion) against the tentorial edge. Cerebellar tonsillar herniation into the foramen magnum Occurs 2° to posterior fossa mass lesions. Tonsillar herniation → medullary compression → respiratory arrest. Usually rapidly fatal.

1	Usually a result of a lateral skull fracture leading to a tear of the middle meningeal artery. Hx/PE: Obvious, severe trauma induces an immediate loss of consciousness followed by a lucid interval (minutes to hours). Uncal herniation F IGU R E 2.1 0-7. Subdural vs. epidural hematoma. (A) Subdural hematoma. Note the crescent shape and the mass effect with midline shift. (B) Epidural hematoma with classic biconvex lens shape. (Reproduced, with permission, from Aminoff MJ. Clinical Neurology, 3rd ed. Stamford, CT: Appleton & Lange, 1996: 296.) leads to coma with a “blown pupil” (fixed and dilated ipsilateral pupil) and ultimately ipsilateral hemiparesis. ■Dx: CT shows a lens-shaped, convex hyperdensity limited by the sutures (see Figure 2.10-7B). ■ Tx: Emergent neurosurgical evacuation. May quickly evolve to brain her- niation and death 2° to the arterial source of bleeding. compression. Causes of headache include the following:

1	■ Tx: Emergent neurosurgical evacuation. May quickly evolve to brain her- niation and death 2° to the arterial source of bleeding. compression. Causes of headache include the following: Acute (new; onset seconds to minutes): New migraine/cluster headache, aneurysmal SAH, acutely ↑ ICP (e.g., colloid cyst, obstructive hydrocephalus), acute ocular disease (e.g., angle-closure glaucoma), cerebral venous thrombosis, cavernous sinus thrombosis, craniocervical dissection, pituitary apoplexy, acute severe hypertension (e.g., pheochromocytoma), angina (rare), ischemic stroke/intraparenchymal hemorrhage (headache is usually not the presenting manifestation).

1	Subacute (new; onset hours to days): New migraine/tension-type headache or viral syndrome vs. meningitis, other cranial infections (acute sinusitis, dental infections, orbital infections, cavernous sinus infections, otitis media/mastoiditis), temporal arteritis, subacutely ↑ ICP (e.g., large tumor, progressive hydrocephalus, altitude sickness, pseudotumor cerebri), sub-acute ocular disease (e.g., keratitis, iritis, scleritis, orbital infection), sub-acute severe hypertension (e.g., hypertensive encephalopathy, eclampsia), intracranial hypotension (e.g., spontaneous, post-LP), subdural hematoma, carbon monoxide, lead poisoning in children (rare), encephalitis. Chronic/episodic: Migraine, cluster headache, tension-type headache, medication overuse headaches (e.g., caffeine withdrawal, “rebound” headaches from NSAID or analgesic overuse), trigeminal or other neuralgias (e.g., glossopharyngeal, postherpetic), TMJ disease, cervical arthritis, chronic sinusitis.

1	Conduct full general and neurologic exams, including a funduscopic exam. Evaluate the following: Chronicity: Recent or recently changed headaches warrant immediate workup if they are not clearly migraines or other 1° headache disorders. Characteristics: Intensity: Severe headaches are more likely to be dangerous, but not all dangerous headaches are severely intense (e.g., temporal arteritis). Location: Posterior headaches are less likely to be benign, especially in children. Duration: Headaches lasting > 72 hours are not migraines. Diurnal variation: Cluster headaches and those from elevated ICP usually occur or worsen at night. Triggers: Examples include chocolate and red wine. Provocative factors: Lying down may make high-ICP headaches worse; standing up may make low-ICP headaches worse. Palliative factors: Sleep clears migraines.

1	Provocative factors: Lying down may make high-ICP headaches worse; standing up may make low-ICP headaches worse. Palliative factors: Sleep clears migraines. Recent-onset headaches warrant immediate workup! If headache is associated with focal neurologic def cits, rule out more serious etiologies with CT or MRI. Rule out SAH with a CT and LP if symptoms are abrupt in onset; then consider other causes typically missed by CT (e.g., dural thrombosis, dissection, or apoplexy). If a 20-year-old female develops headaches after drinking red wine, think migraine.

1	If a 20-year-old female develops headaches after drinking red wine, think migraine. Associated symptoms/signs: Significant findings include fever or rash (consider meningitis or other infectious causes), jaw claudication (specific for temporal arteritis), or constitutional symptoms such as weight loss (associated with neoplastic, inﬂammatory, or infectious conditions). Photophobia, nausea, and vomiting are associated with migraine, aneurysmal SAH, and meningitis, but neck stiffness is more likely to accompany the latter two. Neurologic sequelae: Look for diplopia, mental status changes or associated symptoms (numbness, weakness, dizziness, ataxia, visual disturbances), papilledema, or pupillary abnormalities (partial CN III palsy or Horner’s syndrome). Patient risk factors: High-risk patients are > 50 years of age, immunocompromised, or with preexisting malignancy. If SAH is suspected, obtain a head CT without contrast. If CT is , LP is mandatory. Obtain a CBC.

1	If SAH is suspected, obtain a head CT without contrast. If CT is , LP is mandatory. Obtain a CBC. If temporal arteritis is suspected, obtain ESR. A CT/MRI is needed for suspected SAH, ↑ ICP, or focal neurologic findings. Use CT without contrast to evaluate acute hemorrhage. Affects females more often than males; may be familial. Associated with vascular and brain neurotransmitter (serotonin) changes. Pain is ultimately linked to trigeminal nucleus activation in the brain stem. Auras appear to result from a different pathomechanism (electrical spreading depression in the brain, possibly linked to ion channel dysfunction) and may occur with or without the pain of migraine headache. Onset usually occurs by the early 20s. Triggers include certain foods (e.g., red wine), fasting, stress, menses, OCPs, bright light, and disruptions in normal sleep patterns. Hx/PE:

1	Triggers include certain foods (e.g., red wine), fasting, stress, menses, OCPs, bright light, and disruptions in normal sleep patterns. Hx/PE: Presents with a throbbing headache (> 2 hours but usually < 24 hours, and almost always < 72 hours in duration) that is associated with nausea, vomiting, photophobia, and noise sensitivity. Headache is usually relieved by sleep and darkness. Classic migraines: Often unilateral and preceded by a visual aura in the form of either scintillating scotomas (bright or ﬂashing lights) or visual field cuts. Common migraines: May be bilateral and periorbital without preceding auras. Dx: Based on history. Tx: Avoid known triggers. Abortive therapy includes triptans (first line after OTC NSAIDs have failed), metoclopramide, and various analgesics. Consider symptomatic treatment for nausea.

1	Tx: Avoid known triggers. Abortive therapy includes triptans (first line after OTC NSAIDs have failed), metoclopramide, and various analgesics. Consider symptomatic treatment for nausea. Prophylaxis for frequent or severe migraines includes anticonvulsants (e.g., gabapentin, topiramate), TCAs (e.g., amitriptyline), β-blockers (propranolol), and CCBs. Males are affected more often than females; average age of onset is 25. Hx/PE: Presents as a brief, excruciating, unilateral periorbital headache that lasts 30 minutes to three hours, during which the patient tends to be extremely restless. Attacks tend to occur in clusters, affecting the same part of the head at the same time of day (commonly during sleep) and in a certain season of the year. Associated symptoms include ipsilateral lacrimation of the eye, conjunctival injection, Horner’s syndrome, and nasal stuffiness.

1	Associated symptoms include ipsilateral lacrimation of the eye, conjunctival injection, Horner’s syndrome, and nasal stuffiness. Dx: Classic presentations with a history of repeated attacks over an extended period of time require no evaluation. First episodes require a workup to exclude disorders associated with Horner’s syndrome (e.g., carotid artery dissection or cavernous sinus infection). Tx: Acute therapy: High-ﬂ ow O2 (100% non-rebreather), dihydroergotamine, octreotide, sumatriptan or zolmitriptan. Prophylactic therapy: Transitional (prednisone, ergotamine), maintenance (verapamil, methysergide, lithium, valproic acid, topiramate). Considered by some to be a milder form of migraine headache. More common in females than in males.

1	Considered by some to be a milder form of migraine headache. More common in females than in males. Hx/PE: Presents with tight, bandlike pain that is not associated with sensory phobia, nausea/vomiting, or auras and is brought on by fatigue or stress. Nonspecific symptoms (e.g., anxiety, poor concentration, difficulty sleeping) may also be seen. May be generalized or most intense in the frontal, occipital, and neck regions. Usually occurs at the end of the day. Dx: A diagnosis of exclusion. Be particularly aware of giant cell arteritis in patients > 50 years of age with new headaches; always obtain an ESR even if headaches are mild and unassociated with constitutional or vascular symptoms. There are no focal neurologic signs. Tx: Relaxation, massage, hot baths, and avoidance of exacerbating factors. NSAIDs and acetaminophen are first-line abortive therapy, but trip-tans may also be considered.

1	Tx: Relaxation, massage, hot baths, and avoidance of exacerbating factors. NSAIDs and acetaminophen are first-line abortive therapy, but trip-tans may also be considered. The usual etiology involves a suppurative process of the orbit, nasal sinuses, or central face that leads to septic thrombosis of the cavernous sinus. Non-septic thrombosis is rare; S. aureus is the most common causative agent. The syndrome can also be seen with nonbacterial agents, particularly fungi (Mucor or Aspergillus species). Current antibiotics have greatly ↓ both incidence and mortality. Headache is the most common presenting symptom. Patients may present with orbital pain, edema, diplopia (2° to oculomotor, abducens, or trochlear nerve involvement), or visual disturbances and may describe a recent history of sinusitis or facial infection. On exam, they typically appear ill and have a fever.

1	If a 25-year-old male wakes up repeatedly during the night with unilateral periorbital pain associated with ipsilateral lacrimation, think cluster headache. If a 30-year-old female complains of headaches at the end of the day that worsen with stress and improve with relaxation or massage, think tension-type headache. Tension-type headaches are the most common type of headache diagnosed in adults. Additional signs may include red eye, proptosis, ptosis, or ophthalmoplegia of the affected eye (partial or complete). Diminished pupillary constriction (CN III) and/or dilation (Horner’s) may also be present. Changes in mental status such as confusion, drowsiness, or coma suggest spread to the CNS or sepsis. Late findings include meningismus or systemic signs of sepsis. Lab studies show an ↑ WBC count. Blood cultures reveal the causative agent in up to 50% of cases. CSF exam may reveal ↑ protein consistent with a parameningeal reaction unless there is frank meningitis.

1	Blood cultures reveal the causative agent in up to 50% of cases. CSF exam may reveal ↑ protein consistent with a parameningeal reaction unless there is frank meningitis. MRI (with gadolinium, pituitary protocol) is the principal means of confirming the anatomic diagnosis; MR or CT venography may be important adjuncts. Biopsy of paranasal sinuses or other affected tissue is often necessary in fungal cases for organism identification by histology and culture. Treat aggressively and empirically with a penicillinase-resistant penicillin (nafcillin or oxacillin) plus a thirdor fourth-generation cephalosporin (e.g., ceftriaxone or cefepime) to provide broad-spectrum coverage pending blood culture results. Potential anaerobic infection from sinus or dental sources should be covered with metronidazole. Vancomycin can be added to address potential MRSA involvement. Antifungal therapy is required for fungal cases. IV antibiotics are recommended for at least 3–4 weeks.

1	IV antibiotics are recommended for at least 3–4 weeks. Paroxysmal events associated with aberrant electrical activity in the brain detectable by EEG, leading to changes in neurologic perception or behavior. An aura is experienced by 50–60% of patients with epilepsy. See Table 2.10-8 for common etiologies by age. To further narrow down the etiology of a seizure, assess the following: Determine whether the patient has a history of epilepsy (i.e., a history of unprovoked and recurrent seizures). Other seizures may be self-limited and may resolve once an underlying medical condition has been treated. Elevated serum prolactin levels are consistent with an epileptic seizure in the immediate postictal period. TABLE 2.10-8. Causes of Seizure by Age Group

1	TABLE 2.10-8. Causes of Seizure by Age Group Non-neurologic etiologies include hypoglycemia, hyponatremia, hypocalcemia, hyperosmolar states, hepatic encephalopathy, uremia, porphyria, drug overdose (cocaine, antidepressants, neuroleptics, methylxanthines, lidocaine), drug withdrawal (alcohol and other sedatives), eclampsia, hyperthermia, hypertensive encephalopathy, head trauma, and cerebral hypoperfusion. Seizures with a focal onset (or focal postictal deficit) suggest focal CNS pathology. They may be the presenting sign of a tumor, stroke, AVM, infection, hemorrhage, or developmental abnormality. First seizures that resolve after a single episode are frequently left untreated when the underlying cause is unknown. Arise from a discrete region, or an “epileptogenic focus,” in one cerebral hemisphere and do not lead to loss of consciousness unless they secondarily generalize.

1	Arise from a discrete region, or an “epileptogenic focus,” in one cerebral hemisphere and do not lead to loss of consciousness unless they secondarily generalize. Simple partial seizures: May include motor features (e.g., jacksonian march, or the progressive jerking of successive body regions) as well as sensory, autonomic, or psychic features (e.g., fear, déjà vu, hallucinations) without alteration of consciousness. A postictal focal neurologic deficit (e.g., hemiplegia/hemiparesis, or Todd’s paralysis) is possible and usually resolves within 24 hours. Often confused with acute stroke (ruled out by MRI). Complex partial seizures: Typically involve the temporal lobe (70–80%) with bilateral spread of the aberrant electrical discharge. Characterized by an impaired level of consciousness, auditory or visual hallucinations, déjà vu, automatisms (e.g., lip smacking, chewing, or even walking), and postictal confusion/disorientation and amnesia. Obtain an EEG.

1	Obtain an EEG. Rule out systemic causes with a CBC, electrolytes, calcium, fasting glucose, LFTs, a renal panel, RPR, ESR, and a toxicology screen. A focal seizure implies a focal brain lesion. Rule out a mass by MRI or CT with contrast. Treat the underlying cause. Recurrent partial seizures: Phenytoin, oxcarbazepine, carbamazepine (Tegretol), phenobarbital, and valproic acid can be administered as mono-therapy. In children, phenobarbital is the first-line anticonvulsant. Intractable temporal lobe seizures: Consider anterior temporal lobectomy. Primarily idiopathic. Partial seizures can evolve into secondarily generalized tonic-clonic seizures. If a patient presents wth progressive jerking of successive body regions and hallucinations but without loss of consciousness, think simple partial seizures. If a patient presents with an episode of lip smacking associated with an impaired level of consciousness and followed by confusion, think complex partial seizures.

1	If a patient presents with an episode of lip smacking associated with an impaired level of consciousness and followed by confusion, think complex partial seizures. Both simple partial and complex partial seizures may evolve into 2° generalized tonic-clonic (grand mal) seizures. If a patient presents with clonic movements associated with loss of consciousness and incontinence, think tonicclonic (grand mal) seizures. If a child is brought from school to her pediatrician after experiencing f ve-second episodes of staring into space, think absence (petit mal) seizures. Presents with sudden onset of loss of consciousness with tonic extension of the back and extremities, continuing with 1–2 minutes of repetitive, symmetric clonic movements. Marked by incontinence and tongue biting. Patients may appear cyanotic during the ictal period. Consciousness is slowly regained in the postictal period, but patients are confused and may prefer to sleep; muscle aches and headaches may be present.

1	EEG typically shows 10-Hz activity during the tonic phase and slow waves during the clonic phase. Protect the airway. Treat the underlying cause if known. 1° generalized tonic-clonic seizures: Phenytoin, fosphenytoin, or valproate constitutes first-line therapy. Lamotrigine or topiramate may be used as adjunctive therapy. Secondarily generalized tonic-clonic seizures: Treatment is the same as that for partial seizures. Begin in childhood; subside before adulthood. Often familial. Hx/PE: Present with brief (5to 10-second), often unnoticeable episodes of impaired consciousness occurring up to hundreds of times per day. Patients are amnestic during and immediately after seizures and may appear to be daydreaming or staring. Eye ﬂuttering or lip smacking is common. Dx: EEG shows classic three-per-second spike-and-wave discharges. Tx: Ethosuximide is the first-line agent.

1	Dx: EEG shows classic three-per-second spike-and-wave discharges. Tx: Ethosuximide is the first-line agent. A medical emergency consisting of prolonged (> 10-minute) or repetitive seizures that occur without a return to baseline consciousness. May be either convulsive (the more medically urgent form) or nonconvulsive. Common causes include anticonvulsant withdrawal/noncompliance, anoxic brain injury, EtOH/sedative withdrawal or other drug intoxication, metabolic disturbances (e.g., hyponatremia), head trauma, and infection. Death usually results from the underlying medical condition and may occur in 10% of cases of status epilepticus. Determine the underlying cause with pulse oximetry, CBC, electrolytes, calcium, glucose, ABGs, LFTs, BUN/creatinine, ESR, antiepileptic drug levels, and a toxicology screen. Obtain an EEG and brain imaging, but defer testing until the patient is stabilized. Obtain a stat head CT to evaluate for intracranial hemorrhage.

1	Obtain an EEG and brain imaging, but defer testing until the patient is stabilized. Obtain a stat head CT to evaluate for intracranial hemorrhage. Obtain an LP in the setting of fever or meningeal signs, but only after having done a CT scan to assess the safety of the LP. Maintain ABCs; consider rapid intubation for airway protection. Administer thiamine, glucose, and naloxone to presumptively treat potential etiologies. Give IV benzodiazepine (lorazepam or diazepam) plus a loading dose of fosphenytoin. If seizures continue, intubate and load with phenobarbital. Consider an IV sedative (midazolam or pentobarbital) and initiate continuous EEG monitoring. Initiate a meticulous search for the underlying cause. A form of generalized epilepsy that typically begins within six months of birth. May be idiopathic or 2° to a variety of conditions, including PKU, perinatal infections, hypoxic-ischemic injury, and tuberous sclerosis.

1	Affects males more often than females; associated with a family history. Hx/PE: Presents with tonic, bilateral, symmetric jerks of the head, trunk, and extremities that tend to occur in clusters of 5–10; arrest of psycho-motor development occurs at the age of seizure onset. The majority of patients have mental retardation. Dx: Look for an abnormal interictal EEG characterized by hypsarrhythmia. Tx: Hormonal therapy with ACTH, prednisone, and clonazepam or valproic acid. Medications may treat the spasms but have little impact on patients’ long-term prognosis. Treatment of status epilepticus: First ABCs, Then Begin Giving Naloxone: Fosphenytoin, ABCs, Thiamine, Benzodiazepine, Glucose, Naloxone. If a male infant is brought to the hospital with muscular jerks and an uncle who had the same problem, think infantile spasms (West syndrome).

1	If a male infant is brought to the hospital with muscular jerks and an uncle who had the same problem, think infantile spasms (West syndrome). A common cause of recurrent peripheral vertigo resulting from a dislodged otolith that leads to disturbances in the semicircular canals (95% posterior, 5% horizontal). Hx/PE: Patients with posterior canal BPPV present with transient, episodic vertigo (lasting < 1 minute) and mixed upbeat-torsional nystagmus triggered by changes in head position (classically while turning in bed, getting in/out of bed, or reaching overhead). Some patients complain of nonvertiginous feelings of dizziness or lightheadedness. Nausea and vomiting are uncommon owing to the short-lived stimulus. Dx:

1	Dx: Have the patient turn his or her head 45 degrees right or left and go from a sitting to a supine position while quickly turning the head to the side (Dix-Hallpike maneuver). If vertigo and the typical nystagmus (upbeat and toward the affected shoulder) are reproduced, BPPV is the likely diagnosis. Nystagmus that persists for > 1 minute, gait disturbance, or nausea/ vomiting that is out of proportion to nystagmus should raise concern for a central lesion. Tx: Some 80% of cases can be resolved at the bedside using the modified Epley maneuver (a 270-degree head rotation from the Dix-Hallpike testing position). With BPPV, it’s all in the name: Benign = otolith (not a tumor). Paroxysmal = sudden, temporary episodes lasting < 1 minute. Positional = starts while turning in bed or reaching overhead. Vertigo = vertigo or dizziness is the main symptom.

1	Paroxysmal = sudden, temporary episodes lasting < 1 minute. Positional = starts while turning in bed or reaching overhead. Vertigo = vertigo or dizziness is the main symptom. If you see a 27-year-old male who presents with vertigo and vomiting for one week after having been diagnosed with a viral infection, think acute vestibular neuritis. ■The condition usually subsides spontaneously in weeks to months but often recurs months or years later. Antivertigo medications (e.g., meclizine) are generally contraindicated, as they tend to inhibit central compensation, which may lead to chronic unsteadiness and falls in the elderly. Typically a diagnosis of exclusion once the more serious causes of vertigo (e.g., cerebellar stroke) have been ruled out.

1	Typically a diagnosis of exclusion once the more serious causes of vertigo (e.g., cerebellar stroke) have been ruled out. Hx/PE: Presents with acute onset of severe vertigo, head-motion intolerance, and gait unsteadiness accompanied by nausea, vomiting, and nystagmus. Auditory or aural symptoms (“labyrinthitis”) may include unilateral tinnitus, ear fullness, or hearing loss. Without auditory or aural symptoms (more common), the condition is known as “vestibular neuritis.” Dx: Acute peripheral vestibulopathy demonstrates the following: An abnormal vestibulo-ocular reﬂex (VOR) as determined by a bedside head impulse test (i.e., rapid head rotation from lateral to center while staring at the examiner’s nose). A predominantly horizontal nystagmus that always beats in one direction, opposite the lesion. No vertical eye misalignment by alternate cover testing.

1	A predominantly horizontal nystagmus that always beats in one direction, opposite the lesion. No vertical eye misalignment by alternate cover testing. If patients are “high risk” (i.e., if they have atypical eye findings or any neurologic symptoms or signs, cannot stand independently, have head or neck pain, are > 50 years of age, or have one or more stroke risk factors), they should be imaged by MRI with diffusion-weighted imaging. For low-risk patients, consider caloric testing as ancillary support for the diagnosis. Labyrinthitis (with auditory symptoms) is mimicked by lateral pontine/ cerebellar stroke (AICA arterial territory). Vestibular neuritis (without auditory symptoms) is mimicked by lateral medullary/cerebellar stroke (PICA arterial territory).

1	Tx: Acute treatment consists of corticosteroids given < 72 hours after symptom onset and vestibular sedatives (e.g., meclizine). The condition usually subsides spontaneously within weeks to months. Once the hyperacute stage has passed, cautious engagement in normal physical activities and exercise should be encouraged. A cause of recurrent vertigo with auditory symptoms that affects at least 1 in 500 in the United States. More common among females. Hx/PE: Presents with recurrent episodes of severe vertigo, hearing loss, tinnitus, or ear fullness, often lasting hours to days. Nausea and vomiting are typical. Patients progressively lose low-frequency hearing over years and may become deaf on the affected side.

1	Dx: Diagnosis is made clinically and is based on a thorough history and physical exam. Two episodes (lasting 20 minutes or more) with remission of symptoms between episodes, hearing loss documented at least once with audiometry, and tinnitus or aural fullness are needed to make the diagnosis once other causes (e.g., TIA, otosyphilis) have been ruled out. A brain MRI with vascular imaging (e.g., MRA) may help assess potential intracranial pathology, particularly cerebrovascular disease. ■ Tx: Classically, a low-sodium diet and diuretic therapy were first-line treatments. As theories of pathogenesis have shifted, many clinicians have begun to treat patients with “migraine” diets, other lifestyle changes, prophylactic antimigraine medications, and occasionally benzodiazepines or antiemetics. For severe unilateral cases, ablative therapies (e.g., intratympanic gentamicin to damage the labyrinth, vestibular nerve section) have been used with some success.

1	A cause of recurrent vertigo (usually without auditory symptoms) that affects roughly 10% of migraine sufferers. More common among females. Its pathogenesis may be related to intermittent electrical (ion channel) dysfunction in the cerebellum. Hx/PE: Presents with recurrent episodes of mild dizziness to severe vertigo lasting minutes to days. Nausea, vomiting, and photophobia are common; headaches are variably present and may be mild or severe. Patients are left with no substantial deficits in between spells, although balance may deteriorate over decades. Dx: The diagnosis is usually made clinically on the basis of a thorough history and physical exam to exclude other causes.

1	Dx: The diagnosis is usually made clinically on the basis of a thorough history and physical exam to exclude other causes. Patients who would otherwise qualify for a diagnosis of Ménière’s save the absence of auditory symptoms and documented hearing loss are likely to have vestibular migraine. A history of photoor phonophobia during the episode, particularly if dizziness is associated with headache, is highly suggestive in such patients. The diagnosis is one of exclusion, and care should be taken to ensure that patients do not have intermittent dizziness due to TIA. In patients < 50 years of age, a history of recent trauma or of severe, abrupt-onset, or persistent pain (> 72 hours) should raise concern for vertebral artery dissection with TIAs. A brain MRI with vascular imaging (e.g., MRA) is sometimes indicated to assess potential intracranial pathology, particularly cerebrovascular disease. Episodic ataxia type 2 can be corroborated by genetic testing.

1	Episodic ataxia type 2 can be corroborated by genetic testing. Tx: Can usually be prevented through migraine medication, diet, or lifestyle changes. Benzodiazepines or antiemetics may be tried. Surgical therapies are not indicated. One of the most common causes of loss of consciousness 2° to an abrupt drop in cerebral perfusion. Etiologies include cardiac arrhythmias and cardiac outﬂow obstruction, vasovagal syncope, orthostatic hypotension, micturition-related syncope, basilar TIAs, and idiopathic causes. Presyncope is described as a feeling of imminent loss of consciousness without actual fainting. Commonly confused with seizures. Hx/PE: Patients may report a trigger (e.g., standing for long period of time, fear/sight of blood, Valsalva maneuver). Ménière’s disease consists of recurrent episodes, but unlike BPPV, these usually last hours to days. Unlike Ménière’s, vestibular migraine usually has no associated auditory or aural symptoms.

1	Ménière’s disease consists of recurrent episodes, but unlike BPPV, these usually last hours to days. Unlike Ménière’s, vestibular migraine usually has no associated auditory or aural symptoms. Rule out vertebral artery dissection in those with persistent head or neck pain and intermittent isolated dizziness or vertigo. ■Typically follows a prodrome of lightheadedness or dizziness, muff ed sounds, constricting vision, diffuse weakness, diaphoresis, or pallor. Leads to loss of consciousness and muscle tone for < 30 seconds and recovery within seconds. Dx: Structural CNS causes (e.g., basilar TIA, intermittent obstructive hydrocephalus) are rare among patients who return to normal mental status and have a normal neurologic examination after a brief loss of consciousness. Seizures are more likely if limb jerking is unilateral or lasts > 30 seconds; if there is prolonged confusion after the episode; or if the patient bites the lateral aspect of their tongue.

1	Seizures are more likely if limb jerking is unilateral or lasts > 30 seconds; if there is prolonged confusion after the episode; or if the patient bites the lateral aspect of their tongue. Unless there is a clear vasovagal faint in a young patient without cardiac disease or risk factors, place all patients on telemetry or Holter monitoring to evaluate for arrhythmia, and rule out myocardial is chemia with an ECG and cardiac enzymes. Obtain an EEG to rule out seizures. Consider an echocardiogram, a tilt-table test, or neuroimaging, especially vascular. Tx: Treat the underlying cause; avoid triggers. An autoimmune disorder caused by antibodies that bind to postsynaptic acetylcholine (ACh) receptors located at the neuromuscular junction. Most often affects young adult females, and can be associated with thyrotoxicosis, thymoma, and other autoimmune disorders.

1	Presents with ﬂ uctuating fatigable ptosis or double vision, bulbar symptoms (e.g., dysarthria, dysphagia), and proximal muscle weakness. Symptoms typically worsen as the day progresses but f uctuate dramatically. Patients may report difficulty climbing stairs, rising from a chair, brushing their hair, and swallowing. Myasthenic crisis is rare but includes the potentially lethal complications of respiratory compromise and aspiration. Edrophonium (Tensilon test): Anticholinesterase leads to rapid amelioration of symptoms. Rarely used today owing to the risk of bradycardia. Ice test: Place a pack of ice on one eye for five minutes; ptosis resolves transiently. An abnormal single-f ber EMG and/or a decremental response to repetitive nerve stimulation can yield additional confirmation. ACh antibodies are in 80% of patients; anti–muscle-specific kinase (anti-MuSK) antibodies are in 5%.

1	ACh antibodies are in 80% of patients; anti–muscle-specific kinase (anti-MuSK) antibodies are in 5%. Chest CT is used to evaluate for thymoma. Eighty-five percent of patients with thymoma have antibodies against striated muscle. Follow serial FVCs to determine the need to intubate. Anticholinesterases (pyridostigmine) are used for symptomatic treatment. Prednisone and other immunosuppressants (e.g., azathioprine, cyclosporine, mycophenolate) are the mainstays of treatment. In severe cases, plasmapheresis or IVIG may provide temporary relief (days to weeks). Resection of thymoma can be curative. Avoid giving certain antibiotics (e.g., aminoglycosides) and drugs (e.g., β-blockers) to patients with myasthenia gravis. An autoimmune disorder caused by antibodies directed toward presynaptic calcium channels in the neuromuscular junction. Small cell lung carcinoma is a significant risk factor (60% of cases).

1	An autoimmune disorder caused by antibodies directed toward presynaptic calcium channels in the neuromuscular junction. Small cell lung carcinoma is a significant risk factor (60% of cases). Hx/PE: Presents with weakness and fatigability of proximal muscles along with depressed or absent DTRs. Extraocular, respiratory, and bulbar muscles are typically spared. Dx: Repetitive nerve stimulation reveals a characteristic incremental response. Also diagnosed by autoantibodies to presynaptic calcium channels and a chest CT indicative of a lung neoplasm. Tx: Treat small cell lung carcinoma; tumor resection may reverse symptoms. 3,4-diaminopyridine or guanidine can be given; acetylcholinesterase inhibitors (pyridostigmine) can be added to either regimen. Corticosteroids and azathioprine can be combined or used alone for immunosuppression.

1	Although the pathogenesis of MS is unclear, there is evidence of an autoimmune etiology in genetically susceptible individuals who are exposed to environmental triggers such as viral infections. Such potential etiologies are thought to be T-cell mediated. The female-to-male ratio is 3:2, and onset is typically between 20 and 40 years of age. MS becomes more common as one moves farther away from the equator. Subtypes are relapsing-remitting, primary progressive, secondary progressive, and progressive relapsing (see Table 2.10-9). Presents with multiple neurologic complaints that are separated in time and space and are not explained by a single lesion. As the disease progresses, permanent deficits may accumulate. Limb weakness, optic neuritis, paresthesias, diplopia, vertigo, nystagmus, gait unsteadiness, urinary retention, sexual and bowel dysfunction, depression, and cognitive impairment are also seen. Symptoms classically worsen transiently with hot showers.

1	Attacks are unpredictable but on average occur every 1.5 years, lasting for 6–8 weeks. Neurologic symptoms can come and go or be progressive. The prognosis is best with a relapsing and remitting history. Lhermitte’s sign, demonstrated by sharp pain traveling up or down the neck and back with ﬂexion, generally suggests the presence of cervical myelitis. Repetitive nerve stimulation reveals a characteristic incremental response in Lambert-Eaton myasthenic syndrome, and a decremental response in myasthenia gravis. The classic triad in MS is scanning speech, intranuclear ophthalmoplegia, and nystagmus. Pregnancy may be associated with a ↓ in symptoms of MS. T AB LE 2.1 0-9. Subtypes of Multiple Sclerosis

1	The classic triad in MS is scanning speech, intranuclear ophthalmoplegia, and nystagmus. Pregnancy may be associated with a ↓ in symptoms of MS. T AB LE 2.1 0-9. Subtypes of Multiple Sclerosis Relapses Yes. No acute episodes. Yes. Yes. Progression None. From onset. Not at onset; begins to progress later. From onset. Course of symptoms Full recovery, or def cits may remain after each episode. Minor remissions and plateaus may take place during progression. Relapses, minor remissions, and plateaus may take place during progression. Full recovery, or progressive def cits may remain after each episode. 66% 19% Develops after relapsing-remitting type. 15% Best. Worse. Worse. Worse. Percentage of cases at onset Prognosis For optic neuritis, give IV, not oral, corticosteroids. MRI shows multiple, asymmetric, often periventricular white matter lesions (Dawson’s fingers), especially in the corpus callosum. Active lesions enhance with gadolinium.

1	MRI shows multiple, asymmetric, often periventricular white matter lesions (Dawson’s fingers), especially in the corpus callosum. Active lesions enhance with gadolinium. CSF reveals mononuclear pleocytosis (> 5 cells/μL), an ↑ IgG index, or oligoclonal bands (nonspecific). Abnormal somatosensory or visual evoked potentials may also be present. Corticosteroids should be given during acute exacerbations. Immunomodulators alter relapse rates in relapsing-remitting MS and include interferon-β1a (Avonex/Rebif), interferon-β1b (Betaseron), and copolymer-1 (Copaxone). Mitoxantrone can be given for worsening relapsing/remitting or progressive MS. Alternative treatments include cyclophosphamide, IVIG, and plasmapheresis. Symptomatic therapy is crucial and includes baclofen for spasticity; cholinergics for urinary retention; anticholinergics for urinary incontinence; carbamazepine or amitriptyline for painful paresthesias; and antidepressants for clinical depression.

1	An acute, rapidly progressive, acquired demyelinating autoimmune disorder of the peripheral nerves that results in weakness. Also known as acute inﬂ ammatory demyelinating polyneuropathy. Associated with recent Campylobacter jejuni infection, viral infection, or inﬂuenza vaccination. Approximately 85% of patients make a complete or near-complete recovery (may take up to one year). The mortality rate is < 5%. Classically presents with progressive (over days), symmetric, ascending paralysis (distal to proximal) involving the trunk, diaphragm, and cranial nerves. Atypical presentations are common, including variants that begin with cranial involvement or progress unpredictably to involve the respiratory muscles. Autonomic dysregulation, areﬂexia, and dysesthesias may be present. Evidence of diffuse demyelination is seen on EMG and nerve conduction studies, which show ↓ nerve conduction velocity.

1	Autonomic dysregulation, areﬂexia, and dysesthesias may be present. Evidence of diffuse demyelination is seen on EMG and nerve conduction studies, which show ↓ nerve conduction velocity. Supported by a CSF protein level > 55 mg/dL with little or no pleocytosis (albuminocytologic dissociation). Admit to the ICU for impending respiratory failure. Plasmapheresis and IVIG are first-line treatments. Corticosteroids are not indicated. Aggressive physical rehabilitation is imperative. A chronic, progressive degenerative disease of unknown etiology characterized by loss of upper and lower motor neurons. Also known as Lou Gehrig’s disease. ALS has an unrelenting course and almost always progresses to respiratory failure and death, usually within five years of diagnosis. Males are more commonly affected than females, and onset is generally between ages 40 and 80.

1	Presents with asymmetric, slowly progressive weakness (over months to years) affecting the arms, legs, diaphragm, and lower cranial nerves. Some patients initially present with fasciculations. Weight loss is common. Associated with UMN and/or LMN signs (see Table 2.10-4). Eye movements and sphincter tone are generally spared. Tongue atrophy and fasciculation may be apparent. Emotional lability is a common feature. The clinical presentation is usually diagnostic. Involvement of the tongue (CN XII) or oropharyngeal muscles (CN IX, X), known as “bulbar” involvement, suggests pathology above the foramen magnum and generally excludes the most common differential, cervical spondylosis with compressive myelopathy, as a cause. EMG/nerve conduction studies reveal widespread denervation and fibrillation potentials. Such studies are principally performed to exclude other demyelinating motor neuropathies.

1	EMG/nerve conduction studies reveal widespread denervation and fibrillation potentials. Such studies are principally performed to exclude other demyelinating motor neuropathies. CT/MRI of the cervical spine is done to exclude structural lesions, particularly in those without bulbar involvement. A 55-year-old male presents with slowly progressive weakness in his left upper extremity and later his right, associated with fasciculations but without bladder disturbance and with a normal cervical MRI. Think ALS. Supportive measures and patient education. A chronic, progressive, global decline in multiple cognitive areas (see the mnemonic the 5 A’s of dementia). Alzheimer’s disease accounts for 60–80% of cases. The differential diagnosis is described in the mnemonic DEMENTIAS. Take care not to confuse delirium and dementia (see the Psychiatry chapter).

1	Risk factors for AD include age, female gender, a family history, Down syndrome, and low educational status. Pathology involves neurof brillary tangles, neuritic plaques with amyloid deposition, amyloid angiopathy, and neuronal loss. Amnesia for newly acquired information is usually the first presenting sign, followed by language deficits, acalculia, depression, agitation, psychosis, and apraxia (inability to perform skilled movements). Mild cognitive impairment may precede AD by 10 years. Survival is 5–10 years from the onset of symptoms, with death usually occurring 2° to aspiration pneumonia or other infections. Except for the mental state, the physical exam is generally normal. A diagnosis of exclusion that can be defnitively diagnosed only on autopsy; suggested by clinical features and by an insidiously progressive cognitive course without substantial motor impairment.

1	A diagnosis of exclusion that can be defnitively diagnosed only on autopsy; suggested by clinical features and by an insidiously progressive cognitive course without substantial motor impairment. MRI or CT may show atrophy and can rule out other causes, particularly vascular dementia, normal pressure hydrocephalus, and chronic subdural hematoma. PET imaging shows nonspecific bilateral temporoparietal hypometabolism. CSF is normal. Neuropsychological testing can help distinguish dementia from depression. Hypothyroidism, vitamin B12 deficiency, and neurosyphilis should be ruled out in atypical cases. Prevention of associated symptoms: Provide supportive therapy for the patient and family. Treat depression, agitation, sleep disorders, hallucinations, and delusions.

1	Prevention of associated symptoms: Provide supportive therapy for the patient and family. Treat depression, agitation, sleep disorders, hallucinations, and delusions. Prevention of disease progression: Cholinesterase inhibitors (donepezil, rivastigmine, galantamine, and tacrine) are first-line therapy. Tacrine is associated with hepatotoxicity and is less often used. Memantine, an NMDA receptor antagonist, may slow decline in moderate to severe disease. Dementia associated with a history of stroke and cerebrovascular disease is the second most common type of dementia. Risk factors include age, hypertension, diabetes, embolic sources, and a history of stroke. Criteria for the diagnosis of vascular dementia include the presence of dementia and two or more of the following: Focal neurologic signs on exam. Symptom onset that was abrupt, stepwise, or related to stroke. Brain imaging showing evidence of old infarctions or extensive deep white matter changes 2° to chronic ischemia.

1	Symptom onset that was abrupt, stepwise, or related to stroke. Brain imaging showing evidence of old infarctions or extensive deep white matter changes 2° to chronic ischemia. Protocols for the prevention and treatment of vascular dementia are the same as those for stroke. A rare, progressive form of dementia characterized by atrophy of the frontal and temporal lobes. Round intraneuronal inclusions known as Pick bodies are the classic pathologic finding. Patients present with signifcant changes in behavior and personality early in the disease. Other symptoms include speech disturbance, inattentiveness, and occasionally extrapyramidal signs. Unlike Parkinson’s disease, frontotemporal dementia rarely begins after age 75. The diagnosis is suggested by clinical features and by evidence of circumscribed frontotemporal atrophy revealed by MRI or CT. Treatment is symptomatic only; no curative therapy has yet been made available.

1	Treatment is symptomatic only; no curative therapy has yet been made available. A potentially treatable form of dementia that is thought to arise from impaired CSF outﬂow from the brain. Symptoms include the classic triad of dementia, gait apraxia, and urinary incontinence. Headaches and other signs of ↑ ICP (e.g., papilledema) typically do not appear, although continuous ICP monitoring may reveal spikes of elevated pressure. If a patient shows abrupt changes in symptoms over time rather than a steady decline, think vascular dementia. If a patient presents with rapid cognitive decline over the course of weeks to months, think CJD. The diagnosis is suggested by clinical features. The gait is classically described as “magnetic” or with “feet glued to the ﬂoor,” as the forefoot is not completely dorsally extended. LP or continuous lumbar CSF drainage for several days reveals normal pressure but may cause clinically significant improvement of the patient’s symptoms.

1	LP or continuous lumbar CSF drainage for several days reveals normal pressure but may cause clinically significant improvement of the patient’s symptoms. CT or MRI shows ventricular enlargement out of proportion to sulcal atrophy. Surgical CSF shunting is the treatment of choice. Although it is the most common prion disease, CJD remains an extremely rare form of dementia. CJD is a member of the transmissible spongiform encephalopathies, all of which are characterized by spongy degeneration, neuronal loss, and astrocytic proliferation. In CJD, an abnormal protease-resistant prion protein accumulates in the brain. CJD causes a subacute dementia with ataxia or myoclonic jerks with rapid clinical decline that is noted weeks to months after symptom onset. Suggested by clinical features. The differential diagnosis often includes viral encephalitis, Hashimoto’s (steroid-responsive) encephalopathy, and toxic encephalopathy (e.g., lithium or bismuth).

1	Suggested by clinical features. The differential diagnosis often includes viral encephalitis, Hashimoto’s (steroid-responsive) encephalopathy, and toxic encephalopathy (e.g., lithium or bismuth). EEG shows pyramidal signs and periodic sharp waves. MRI with diffusion-weighted imaging may show ↑ T2 and FLAIR intensity in the putamen and the head of the caudate and is also used to exclude structural brain lesions. CSF is usually normal. Definitive diagnosis can be made only by brain biopsy or autopsy. Specimens must be handled with special precautions to avoid transmission. Currently, there is no effective treatment. Most patients die within one year of symptom onset.

1	Currently, there is no effective treatment. Most patients die within one year of symptom onset. A rare, hyperkinetic, autosomal-dominant disease involving multiple abnormal CAG triplet repeats (< 29 is normal) within the HD gene on chromosome 4. The number of repeats typically expands in subsequent generations, leading to earlier expression and more severe disease (anticipation). Life expectancy is 20 years from the time of diagnosis. Presents at 30–50 years of age with gradual onset of chorea (sudden onset of purposeless, involuntary dancelike movements), altered behavior, and dementia (begins as irritability, clumsiness, fidgetiness, moodiness, and antisocial behavior). Weight loss and depression may also be seen. A clinical diagnosis confirmed by genetic testing. CT/MRI show cerebral atrophy (especially of the caudate and putamen). Molecular genetic testing is conducted to determine the number of CAG repeats.

1	CT/MRI show cerebral atrophy (especially of the caudate and putamen). Molecular genetic testing is conducted to determine the number of CAG repeats. There is no cure, and disease progression cannot be halted. Treat symptomatically. Reserpine or tetrabenazine can be given to minimize unwanted movements. Psychosis should preferably be treated with atypical antipsychotics to reduce the risk of extrapyramidal side effects or tardive dyskinesia. SSRIs are first-line therapy for depression. Genetic counseling should be offered to offspring. An idiopathic hypokinetic disorder that usually begins after age 50–60 and is attributable to dopamine depletion in the substantia nigra. It is characterized pathologically by Lewy bodies, which are intraneuronal eosinophilic inclusions. The “Parkinson’s tetrad” consists of the following: Resting tremor (e.g., “pill rolling”). Rigidity: “Cogwheeling” due to the combined effects of rigidity and tremor.

1	The “Parkinson’s tetrad” consists of the following: Resting tremor (e.g., “pill rolling”). Rigidity: “Cogwheeling” due to the combined effects of rigidity and tremor. Bradykinesia: Slowed movements and difficulty initiating movements. Festinating gait (a wide leg stance with short accelerating steps) without arm swing is also seen. Postural instability: Stooped posture, impaired righting reﬂ exes, freezing, falls. Other manifestations include masked facies, memory loss, and micrographia. Parkinsonism is the broader clinical phenotype of bradykinesia and rigidity (with or without substantial tremor). It is often caused by disorders other than idiopathic Parkinson’s disease, most commonly multiple subcortical infarcts (“vascular parkinsonism”).

1	Nonidiopathic causes of parkinsonism include viral encephalitis (postencephalitic parkinsonism), trauma (dementia pugilistica), and numerous toxins (e.g., manganese, MPTP [designer drugs], and, iatrogenically, neuroleptics [tardive dyskinesia]). Some of these disorders are diagnosed on the basis of failure of levodopa/carbidopa to produce a clinical response. Other idiopathic dementias may mimic Parkinson’s disease and include progressive supranuclear palsy and multiple system atrophy. If a 43-year-old male patient presents with sudden onset of chorea, irritability, and antisocial behavior and his father experienced these symptoms at a slightly older age, think Huntington’s disease. A signif cant difference between gait abnormalities in Parkinson’s and that of NPH is preservation of arm swing in NPH. There are four PaRTS to Parkinson’s: Postural instability, Rigidity (cogwheeling), Tremor (“pill rolling”), and Slowed movements (bradykinesia).

1	There are four PaRTS to Parkinson’s: Postural instability, Rigidity (cogwheeling), Tremor (“pill rolling”), and Slowed movements (bradykinesia). A side effect associated with pramipexole is uncontrolled gambling. Most CNS tumors are metastatic. The most common 1° CNS tumors in adults are glioblastoma multiforme and meningioma. The most common 1° CNS tumors in children are medulloblastomas and astrocytomas. Liver and Skin Go to the BRain: Liver, Skin, GI, Breast, Renal. Two-thirds of 1° brain tumors in adults are supratentorial. One-third of those in children are supratentorial. Levodopa and carbidopa, both of which are dopamine precursors, are the mainstays of therapy. Dopamine agonists (ropinirole, pramipexole, bromocriptine) can be used for treatment in early disease. Apomorphine is another dopamine agonist that can be used for rescue therapy if a sudden additional dose is needed. Selegiline (an MAO-B inhibitor) may be neuroprotective and may ↓ the need for levodopa.

1	Selegiline (an MAO-B inhibitor) may be neuroprotective and may ↓ the need for levodopa. Catechol-O-methyltransferase (COMT) inhibitors (entacapone or tolcapone) are not given alone but ↑ the availability of levodopa to the brain and may ↓ motor ﬂ uctuations. Amantadine has mild antiparkinsonian activity and may improve akinesia, rigidity, and tremor. It can be used for temporary, short-term monotherapy early in the course of the disease. If medical therapy fails, surgical pallidotomy or chronic deep brain stimulation may produce clinical benefit. Intracranial neoplasms may be 1° (30%) or metastatic (70%). Of all 1° brain tumors, 40% are benign, and these rarely spread beyond the CNS. Metastatic tumors are most often from 1° lung, breast, kidney, and GI tract neoplasms and melanoma. They occur at the gray-white junction; may be multiple discrete nodules; and are characterized by rapid growth, invasiveness, necrosis, and neovascularization.

1	More common in males than in females, except for meningiomas. Symptoms depend on tumor type and location (see Table 2.10-10), local growth and resulting mass effect, cerebral edema, or elevated ICP 2° to ventricular obstruction. Although headaches are often thought of as the main presenting symptom, only 31% of patients present with headache at diagnosis and only 8% have headache as the sole presenting feature. Seizures or slowly progressive focal motor deficits are the most common presenting features. When ↑ ICP is the presenting feature, symptoms include headache, nausea/vomiting, and diplopia (false localizing CN VI palsies). However, in the era of neuroimaging, it is relatively rare for patients to present with ↑ ICP.

1	Hemispheric tumors often produce visual field abnormalities and neuropsychiatric symptoms, including personality changes, lethargy, syncope, cognitive decline, aphasia, apraxia, and depression. Parasellar lesions usually present with visual loss and/or diplopia. Posterior fossa lesions tend to present with gait ataxia or cranial nerve deficits and/or ↑ ICP from obstructive hydrocephalus. Metastases that tend to present with intracranial hemorrhage include renal cell carcinoma, thyroid cancer, choriocarcinoma, and melanoma. ■Contrast CT and MRI with and without gadolinium to localize and determine the extent of the lesion. Gadolinium-enhanced MRI is generally better for visualizing soft tissue tumors and vascularity, but CT is better for TABLE 2.10-10. Common 1° Neoplasms

1	Astrocytoma Arises in brain parenchyma. Low-grade astrocytomas are relatively uncommon. Presents with seizures, focal defcits, or headache. Has a protracted course. Has a better prognosis than glioblastoma multiforme (see below). Resection if possible; radiation. Glioblastoma multiforme (grade IV astrocytoma) High mitotic activity and either endothelial proliferation or necrosis in tumor, leading to ring-enhancing lesions on MRI. There is a major difference between grade III and grade IV. The most common 1° brain tumor. Presents with headache and ↑ seizures, focal def cits, or headache. Progresses rapidly and has a poor prognosis (< 1 year from the time of prognosis). Surgical removal/resection. Radiation and chemotherapy have variable results. Meningioma Originates from the dura mater or arachnoid. Presentation depends on location; often related to cranial neuropathy or is an incidental f nding. Good prognosis. Incidence ↑ with age. Imaging may reveal dural tail. Surgical resection;

1	arachnoid. Presentation depends on location; often related to cranial neuropathy or is an incidental f nding. Good prognosis. Incidence ↑ with age. Imaging may reveal dural tail. Surgical resection; radiation for unresectable tumors. Acoustic neuroma (schwannoma) Derived from Schwann cells. Presents with ipsilateral tinnitus, hearing loss, vertigo, and late signs of CN V–VII or brain stem compression. Surgical removal. A primitive neuroectodermal tumor. Arises from the fourth ventricle and causes ↑ ICP. Common in children. Highly malignant; may seed the subarachnoid space. May cause obstructive hydrocephalus. Surgical resection coupled with radiation and chemotherapy. May arise from the ependyma of a ventricle (commonly the fourth) or the spinal cord. Common in children; low grade. May cause obstructive hydrocephalus. Surgical resection; radiation. Medulloblastoma Ependymoma evaluating skull base lesions and for emergencies (e.g., obstructive hydrocephalus) when an MRI cannot be

1	cause obstructive hydrocephalus. Surgical resection; radiation. Medulloblastoma Ependymoma evaluating skull base lesions and for emergencies (e.g., obstructive hydrocephalus) when an MRI cannot be rapidly acquired.

1	■Histologic diagnosis via CT-guided biopsy or surgical tumor debulking/ removal. Resection (if possible), radiation, and chemotherapy. Therapy is highly dependent on tumor type, histology, progression, and site (see Table 2.10-10). Corticosteroids can be used to ↓ vasogenic edema and ↓ ICP. Management is often palliative. Seizure prophylaxis can be used in patients who have had a seizure. Symptoms of ↑ ICP: Headache that is worse in the morning or with recumbency In NF1, you— spots) NN (2 Neurof bromas) OT (OpTic glioma) FAI (Freckling, Axillary relative with NF1) NF1 and NF2 are clinically evident by ages 15 and 20, respectively. If you see infantile spasms in the setting of a hypopigmented lesion on the child’s trunk, consider tuberous sclerosis.

1	NF1 and NF2 are clinically evident by ages 15 and 20, respectively. If you see infantile spasms in the setting of a hypopigmented lesion on the child’s trunk, consider tuberous sclerosis. The most common neurocutaneous disorder. There are two major types: neurofibromatosis 1 (NF1, or von Recklinghausen’s syndrome) and neurofibromatosis 2 (NF2). Both obey autosomal-dominant inheritance. The NF genes are located on chromosome 17 and 22, respectively, for NF1 and NF2. Diagnostic criteria for NF1 include two or more of the following: Six café au lait spots (each ≥ 5 mm in children or ≥ 15 mm in adults). Two neurofibromas of any type. Freckling in the axillary or inguinal area. Optic glioma. Two Lisch nodules (pigmented iris hamartomas). Bone abnormality (e.g., kyphoscoliosis). A f rst-degree relative with NF1. Diagnostic criteria for NF2 are as follows:

1	Optic glioma. Two Lisch nodules (pigmented iris hamartomas). Bone abnormality (e.g., kyphoscoliosis). A f rst-degree relative with NF1. Diagnostic criteria for NF2 are as follows: Bilateral acoustic neuromas or a first-degree relative with NF2 and either unilateral acoustic neuromas or two of any of the following: neurofibromas, meningiomas, gliomas, or schwannoma. Other features include seizures, skin nodules, and café au lait spots. MRI of the brain, brain stem, and spine with gadolinium. Obtain a complete dermatologic exam, ophthalmologic exam, and family history. Auditory testing is recommended. There is no cure; treatment is symptomatic (e.g., surgery for kyphoscoliosis or debulking of tumors). Acoustic neuromas and optic gliomas can be treated with surgery or radio-surgery. Meningiomas may be resected. Affects many organ systems, including the CNS, skin, heart, retina, and kidneys. Obeys autosomal-dominant inheritance.

1	Affects many organ systems, including the CNS, skin, heart, retina, and kidneys. Obeys autosomal-dominant inheritance. Presents with convulsive seizures (infantile spasms in infants),“ash-leaf” hypopigmented lesions on the trunk and extremities, and mental retardation (↑ likelihood with early age of onset). Other skin manifestations include sebaceous adenomas (small red nodules on the nose and cheeks in the shape of a butterﬂy) and a shagreen patch (a rough papule in the lumbosacral region with an orange-peel consistency). Two retinal lesions are recognized: (1) mulberry tumors, which arise from the nerve head; and (2) phakomas, which are round, ﬂat, gray lesions located peripherally in the retina. Symptoms are 2° to small benign tumors that grow on the face, eyes, brain, kidney, and other organs. Mental retardation and CHF from cardiac rhabdomyoma may also be seen. Renal involvement may include hamartomas, angiomyolipomas, or, rarely, renal cell carcinoma.

1	Mental retardation and CHF from cardiac rhabdomyoma may also be seen. Renal involvement may include hamartomas, angiomyolipomas, or, rarely, renal cell carcinoma. Diagnosis is usually clinical. Skin lesions are enhanced by a Wood’s UV lamp. Imaging: Head CT: Reveals calcified tubers within the cerebrum in the periventricular area. Lesions may on rare occasion transform into malignant astrocytomas. ECG: Evaluate for rhabdomyoma of the heart, especially in the apex of the left ventricle (affects > 50% of patients). Renal ultrasound: May reveal renal hamartomas, masses, or polycystic disease. Renal CT: May show angiomyolipomas (causing cystic or fibrous pulmonary changes). CXR: May reveal pulmonary lesions or cardiomegaly 2° to rhabdomyoma. Treatment should be based on symptoms (e.g., cosmetic surgery for adenoma sebaceum).

1	CXR: May reveal pulmonary lesions or cardiomegaly 2° to rhabdomyoma. Treatment should be based on symptoms (e.g., cosmetic surgery for adenoma sebaceum). Simple partial or complex partial seizures can be controlled with oxcarbazepine or carbamazepine; lamotrigine can be given for generalized seizures. Treat infantile spasms with ACTH or vigabatrin. Surgical intervention may be indicated in the setting of ↑ ICP or for seizures associated with an epileptogenic focus or severe developmental delay. A general term for speech and language disorders. Usually results from insults (e.g., strokes, tumors, abscesses) to the “dominant hemisphere” (the left hemisphere in > 95% of right-handed people and 60–80% of left-handed people). ■ A disorder of language production, including writing, with intact comprehension. Due to an insult to Broca’s area in the posterior inferior fron Broca’s aphasia is also known tal gyrus. Often 2° to a left superior MCA stroke. Also known as motor aphasia.

1	Broca’s aphasia is also known tal gyrus. Often 2° to a left superior MCA stroke. Also known as motor aphasia. Hx/PE: Presents with impaired repetition, frustration with awareness of aphasia. deficits, arm and facial hemiparesis, hemisensory loss, and apraxia of the oral muscles. Speech is described as “telegraphic” and agrammatical with frequent pauses. Tx: Speech therapy (varying outcomes with intermediate prognosis). Wernicke’s Aphasia Wernicke’s aphasia is also known as f uent or receptive ■ A disorder of language comprehension with intact yet nonsensical production. Also known as sensory aphasia. aphasia. BROca’s is BROken and Wernicke’s is Wordy. Due to an insult to Wernicke’s area in the left posterior superior temporal (perisylvian) gyrus. Often 2° to left inferior/posterior MCA embolic stroke.

1	BROca’s is BROken and Wernicke’s is Wordy. Due to an insult to Wernicke’s area in the left posterior superior temporal (perisylvian) gyrus. Often 2° to left inferior/posterior MCA embolic stroke. Hx/PE: Presents with preserved f uency of language with impaired repetition and comprehension, leading to “word salad.” Patients are unable to follow commands; make frequent use of neologisms (made-up words) and paraphasic errors (word substitutions); show lack of awareness of deficits; and exhibit right upper homonymous quadrantanopia 2° to involvement of Meyer’s loop. Tx: Treat the underlying etiology and institute speech therapy.

1	Tx: Treat the underlying etiology and institute speech therapy. A state of unconsciousness marked by a profound suppression of responses to external and internal stimuli (i.e., a state of unarousable unresponsiveness). Lesser states of impaired arousal are known as “obtundation” or “stupor.” Coma is due to either catastrophic structural CNS injury or diffuse metabolic dysfunction. Coma indicates bilateral dysfunction of both cerebral hemispheres or the brain stem (pons or higher); when structural, coma usually results from bilateral pathology. Causes include the following: Diffuse hypoxic/ischemic encephalopathy (e.g., postcardiac arrest). Diffuse axonal injury from high-acceleration trauma (e.g., MVA). Brain herniation (e.g., cerebral mass lesion, SAH with obstructive hydrocephalus). Widespread infection (e.g., viral encephalitis or advanced bacterial meningitis). Massive brain stem hemorrhage or infarction (e.g., pontine myelinolysis).

1	Widespread infection (e.g., viral encephalitis or advanced bacterial meningitis). Massive brain stem hemorrhage or infarction (e.g., pontine myelinolysis). Electrolyte disturbances (e.g., hypoglycemia). Exogenous toxins (e.g., opiates, benzodiazepines, EtOH, other drugs). Generalized seizure activity or postictal states. Endocrine (e.g., severe hypothyroidism) or metabolic dysfunction (e.g., thiamine deficiency). Obtain a complete medical history from witnesses, including current medications (e.g., sedatives). Conduct thorough medical and neurologic exams, including assessments of mental status, spontaneous motor activity, muscular tone, breathing pattern, funduscopy, pupillary response, eye movements (including the doll’seye maneuver if the neck has been cleared from fracture), corneal reﬂ ex, cold-water caloric testing, gag reﬂex, and motor or autonomic responses to noxious stimuli applied to limbs, trunk and face (e.g., retromandibular pressure, nasal tickle).

1	Typically made by a combination of the history/physical and laboratory tests or neuroimaging. Check glucose, electrolytes, calcium, a renal panel, LFTs, ABG, a toxicology screen, and blood and CSF cultures. Other metabolic tests (e.g., TSH) may be performed based on the clinical index of suspicion. Obtain a head CT without contrast before other imaging to evaluate for hemorrhage or structural changes. Imaging should precede LP in light of the risk of herniation. Obtain an MRI to exclude structural changes and ischemia (e.g., brain stem). EEG may be both diagnostic and prognostic. Rule out catatonia, hysterical or conversion unresponsiveness, “locked-in” syndrome, or persistent vegetative state (PVS), all of which can be confused with true coma (see Table 2.10-11). “Locked-in” syndrome: Patients are awake and alert but can move only their eyes and eyelids. Associated with central pontine myelinolysis, brain stem stroke, and advanced ALS.

1	“Locked-in” syndrome: Patients are awake and alert but can move only their eyes and eyelids. Associated with central pontine myelinolysis, brain stem stroke, and advanced ALS. PVS: Characterized by normal wake-sleep cycles but lack of awareness of self or the environment. The most common causes are trauma with diffuse cortical injury or hypoxic ischemic injury. Initial treatment should consist of the following measures: Stabilize the patient: Attend to ABCs. Reverse the reversible: Administer DONT—Dextrose, Oxygen, Naloxone, and Thiamine. Identify and treat the underlying cause and associated complications. Prevent further damage. Table 2.10-12 describes the neurologic symptoms commonly associated with nutritional deficiencies. Figure 2.10-8 illustrates common visual field defects and the anatomic areas with which they are associated. T AB LE 2.1 0-1 1. Differential Diagnosis of Coma

1	Figure 2.10-8 illustrates common visual field defects and the anatomic areas with which they are associated. T AB LE 2.1 0-1 1. Differential Diagnosis of Coma Alertness Wakeful and alert with retained cognitive abilities. Wakefulness without awareness. Unconscious; no sleep-wake cycles. Unconscious; no sleep-wake cycles. Most common causes Central pontine myelinolysis, brain stem stroke, advanced ALS. Diffuse cortical injury or hypoxic ischemic injury. See above. Same as coma. Voluntary motor ability Eyes and eyelids. None. None. None. Respiratory drive Yes. Yes. Yes. None. T AB LE 2.1 0-1 2. Neurologic Syndromes Associated with Nutritional Deficiencies

1	Thiamine (vitamin B1) Wernicke’s encephalopathy Korsakoff’s dementia The classic triad consists of encephalopathy (disorientation, inattentiveness, confusion, coma), ophthalmoplegia (nystagmus, lateral rectus palsy, conjugate gaze palsy, vertical gaze palsy), and ataxia (polyneuropathy; cerebellar and vestibular dysfunction leading to problems standing or walking). Above, plus anterograde and retrograde amnesia, horizontal nystagmus, and confabulations. Alcoholics, hyperemesis, starvation, renal dialysis, AIDS. Can be brought on or exacerbated by high-dose glucose administration. Same as above. Usually occurs in the “resolution” phase of Wernicke’s syndrome that was treated late or inadequately. Reversible almost immediately with thiamine administration. Always give thiamine before glucose. Irreversible. Cyanocobalamin (vitamin B12)a Combined system disease (CSD) or subacute combined degeneration of the posterior and lateral columns of the spinal cord (see the discussion of clinical

1	Irreversible. Cyanocobalamin (vitamin B12)a Combined system disease (CSD) or subacute combined degeneration of the posterior and lateral columns of the spinal cord (see the discussion of clinical neuroanatomy); peripheral neuropathy. Gradual, progressive onset. Symmetric paresthesias, stocking-glove sensory neuropathy, leg stiffness, spasticity, paraplegia, bowel and bladder dysfunction, sore tongue. Dementia. Patients with pernicious anemia; strict vegetarians; status post gastric or ileal resection; ileal disease (e.g., Crohn’s); alcoholics or others with malnutrition. B12 injections or large oral doses. Folatea Folate def ciency Irritability; personality changes without the neurologic symptoms of CSD. Alcoholics; patients with pernicious anemia. Reversible if corrected early. a Associated with ↑ homocysteine and ↑ risk of vascular events.

1	In the eye, aqueous humor is produced by the ciliary body on the iris, travels through the pupil into the anterior chamber, and is then drained via the trabecular meshwork in the angle of the anterior chamber. ■Any process that disrupts this natural ﬂ ow can ↑ intraocular pressure (IOP), damaging the optic nerve and causing visual field deficits. Glaucoma is the result of such damage to the nerve. 1. 2. 3. 4. 5. 6. Left hemianopia with macular sparing FIGURE 2.10-8. Visual field defects. Defect in visual field of Lt. eye Rt. eye 1 2 3 4 Optic chiasm Optic nerve Optic tract loop Calcarine fissure Visual cortex 1 5 3 2 Dorsal optic radiation Lt. Rt. Lateral geniculatebody ■Open-angle glaucoma is much more common in the United States than closed-angle glaucoma.

1	Occurs when the iris dilates and pushes against the lens of the eye, disrupting ﬂow of aqueous humor into the anterior chamber. Pressure in the posterior chamber then pushes the peripheral iris forward and blocks the angle. Risk factors include family history, older age, Asian ethnicity, hyperopia, prolonged pupillary dilation (prolonged time in a dark area, stress, medications), anterior uveitis, and lens dislocation. Hx/PE: Classically presents with extreme eye pain, blurred vision, headache, nausea, and vomiting. A hard, red eye is seen (from acute closure of a narrow anterior chamber angle); the pupil is dilated and nonreactive to light. IOP is ↑. If it resolves spontaneously prior to presentation (e.g., with pupillary constriction in sunlight), ophthalmologic examination may reveal narrow angles in one or both eyes.

1	Dx: Diagnosis is based on clinical history and examination. Those that resolve may mimic a migraine headache with blurred vision; the distinction is that the headaches and blurred vision are more likely to be triggered by darkness (due to pupillary dilation) rather than by bright lights (migraine). Tx: This is a medical emergency that can cause blindness. Treatment to ↓ IOP may include eyedrops (timolol, pilocarpine, apraclonidine) or systemic medications (oral or IV acetazolamide, IV mannitol). Laser peripheral iridotomy, which creates a hole in the peripheral iris, is curative and may be performed prophylactically. generally occurs bilaterally, ■Flow of aqueous humor through the trabecular meshwork is limited, in- creasing IOP. A diseased trabecular meshwork obstructs proper drainage of the eye, leading to a gradual ↑ in pressure and progressive vision loss. occurs unilaterally.

1	In the United States, macular degeneration is the leading cause of permanent bilateral visual loss in the elderly. Risk factors include age > 40 years, African-American ethnicity, diabetes, and myopia. Hx/PE: Usually asymptomatic until late in the clinical course, when patients may begin to notice visual deficits. Should be suspected in patients > 35 years of age who need frequent lens changes and have mild headaches, visual disturbances, and impaired adaptation to darkness. The earliest visual defect is seen in the peripheral nasal fields. Cupping of the optic nerve head is seen on funduscopic exam. Dx: Tonometry, ophthalmoscopic visualization of the optic nerve, and visual field testing are most important. A diseased trabecular meshwork obstructs proper drainage of the eye, gradually increasing pressure and leading to progressive vision loss.

1	Tx: Treat with topical β-blockers (timolol, betaxolol) to ↓ aqueous humor production or with pilocarpine to ↑ aqueous outﬂow. Carbonic anhydrase inhibitors may also be used. If medication fails, laser trabeculoplasty or a trabeculectomy can improve aqueous drainage. More common among Caucasians, females, smokers, and those with a family history. Presents with painless loss of central vision. Atrophic (“dry”) macular degeneration: Responsible for 80% of cases. Causes gradual vision loss. Exudative or neovascular (“wet”) macular degeneration: Much less common, but associated with more rapid and severe vision damage. Funduscopy by an ophthalmologist reveals drusen and/or pigmentary changes in patients with atrophic AMD. Hemorrhage and subretinal ﬂuid are suggestive of exudative AMD. Atrophic AMD: No treatment is currently available, although a combination of vitamins (vitamin C, vitamin E, beta-carotene, and zinc) has been found to slow disease progression.

1	Atrophic AMD: No treatment is currently available, although a combination of vitamins (vitamin C, vitamin E, beta-carotene, and zinc) has been found to slow disease progression. An ↑ mortality rate from high doses of vitamin E and an elevated lung cancer incidence among individuals on beta-carotene supplementation may require modification of this regimen for smokers. Exudative AMD: VEGF inhibitors have been shown to improve vision (ranibizumab, bevacizumab) or slow visual loss (pegaptanib) in patients with exudative AMD. Photodynamic therapy with verteporfin, which involves use of a laser to selectively target retinal vessels, may be useful in conjunction with VEGF inhibitors. Occurs in elderly patients and is often idiopathic. Hx/PE:

1	Occurs in elderly patients and is often idiopathic. Hx/PE: Central retinal artery occlusion: Presents with sudden, painless, unilateral blindness. The pupil reacts to a near stimulus but is sluggishly reactive to direct light. Patients present with a cherry-red spot on the fovea, retinal swelling (whitish appearance to the nerve fiber layer), and retinal arteries that may appear bloodless. Central retinal vein occlusion: Characterized by rapid, painless vision loss of variable severity. A choked, swollen optic disk with hemorrhages, venous stasis retinal hemorrhages, cotton-wool spots, and edema of the macula may be seen on funduscopic exam. Tx:

1	Tx: Central retinal artery occlusion: Intra-arterial thrombolysis of the ophthalmic artery within eight hours of onset of symptoms may produce benefit in some patients, although evidence remains controversial. Other treatments applied but of unclear benefit include increasing IOP through drainage of the anterior chamber or IV acetazolamide. If implemented, treatments should be applied immediately before irreversible retinal infarction and permanent blindness ensue. Central retinal vein occlusion: Laser photocoagulation has variable results. Physiology of Normal Pregnancy 321 THE BASICS OF PREGNANCY 321 NORMAL PHYSIOLOGY OF PREGNANCY 321 Teratology 325 Maternal-Fetal Infections 326 Spontaneous Abortion 328 Elective Termination of Pregnancy 329 Normal Labor and Delivery 329 Medical Complications of Pregnancy 336 HYPEREMESIS GRAVIDARUM 336 DIABETES IN PREGNANCY 338 GESTATIONAL AND CHRONIC HYPERTENSION 340 PREECLAMPSIA AND ECLAMPSIA 340 ANTEPARTUM HEMORRHAGE 342

1	Medical Complications of Pregnancy 336 HYPEREMESIS GRAVIDARUM 336 DIABETES IN PREGNANCY 338 GESTATIONAL AND CHRONIC HYPERTENSION 340 PREECLAMPSIA AND ECLAMPSIA 340 ANTEPARTUM HEMORRHAGE 342 Obstetric Complications of Pregnancy 342 ECTOPIC PREGNANCY 342 INTRAUTERINE GROWTH RESTRICTION 344 FETAL MACROSOMIA 345 POLYHYDRAMNIOS 345 OLIGOHYDRAMNIOS 345 Rh ISOIMMUNIZATION 345 GESTATIONAL TROPHOBLASTIC DISEASE 346 MULTIPLE GESTATIONS 348 Abnormal Labor and Delivery 348 SHOULDER DYSTOCIA 348 FAILURE TO PROGRESS 348 RUPTURE OF MEMBRANES 349 PRETERM LABOR 350 FETAL MALPRESENTATION 351 INDICATIONS FOR CESAREAN SECTION 351 EPISIOTOMY 353 The Basics of Pregnancy The following terms and concepts are central to an understanding of the physiologic processes of pregnancy (see also Figure 2.11-1). Gravidity: The number of times a woman has been pregnant. Parity: The number of pregnancies that led to a birth beyond 20 weeks’ gestational age or an infant weighing > 500 g.

1	Gravidity: The number of times a woman has been pregnant. Parity: The number of pregnancies that led to a birth beyond 20 weeks’ gestational age or an infant weighing > 500 g. Developmental age (DA): The number of weeks and days since fertilization. Typically used only in research, as the exact date of fertilization is not commonly known. Gestational age (GA): The number of weeks and days measured from the frst day of the last menstrual period (LMP). GA can also be determined by: Fundal height: At 20 weeks, the uterus is at the umbilicus and grows approximately 1 cm/week. Quickening, or appreciation of fetal movement: Typically occurs at 17–18 weeks. Fetal heart tones: Can be heard at 10–12 weeks by Doppler. Ultrasound: Measures fetal crown-rump length (CRL) at 5–12 weeks and measures biparietal diameter (BPD), femur length (FL), and abdominal circumference (AC) from 13 weeks. Ultrasound measurement of GA is most reliable during the f rst trimester.

1	Normal Physiology of Pregnancy The normal physiologic changes that occur during pregnancy are graphically illustrated according to system in Figures 2.11-2 and 2.11-3. Nägele’s rule: due date = last menstrual period + nine months + seven days. F IGU R E 2.1 1 -1. Perinatal nomenclature by date. (Reproduced, with permission, from DeCherney AH, Nathan L. Current Diagnosis & Treatment: Obstetrics & Gynecology, 10th ed. New York: McGraw-Hill, 2007: Fig. 9-1.) (Reproduced, with permission, from Gardner DG, Shoback D. Greenspan’s Basic & Clinical Endocrinology, 8th ed. New York: McGraw-Hill, 2007: Fig. 17-2A.) The goal of prenatal care is to prevent, diagnose, and treat conditions that can lead to adverse outcomes in pregnancy. Expected weight gain, nutrition, and exercise recommendations are outlined in Table 2.11-1. Get a quantitative β-hCG: To diagnose and follow ectopic pregnancy. To monitor trophoblastic disease. To screen for fetal aneuploidy.

1	Get a quantitative β-hCG: To diagnose and follow ectopic pregnancy. To monitor trophoblastic disease. To screen for fetal aneuploidy. Still UNDERage at 18: trisomy 18 = ↓ AFP, ↓ estriol, ↓β-hCG, ↓ inhibin A. Table 2.11-2 outlines a typical prenatal diagnostic testing schedule by week. The sections that follow describe each recommended screening modality in further detail. The standard for diagnosing pregnancy. Produced by the placenta; peaks at 100,000 mIU/mL by 10 weeks of gesta tion. ■↓ throughout the second trimester; levels off in the third trimester. hCG levels double approximately every 48 hours during early pregnancy. This is often used to diagnose ectopic pregnancy when doubling is abnormal. Maternal serum α-fetoprotein (MSAFP), which is produced by the fetus, crosses the placenta in small amounts and enters the maternal circulation. Measurement results are reported as multiples of the median (MoMs) and depend on accurate gestational dating.

1	Elevated MSAFP (> 2.5 MoMs): Associated with open neural tube defects (anencephaly, spina bifda), abdominal wall defects (gastroschisis, omphalocele), multiple gestation, incorrect gestational dating, fetal death, and placental abnormalities (e.g., placental abruption). Decreased MSAFP (< 0.5 MoM): Associated with trisomy 21 and 18, fetal demise, and inaccurate gestational dating. MSAFP is now rarely performed alone because its sensitivity for detecting chromosomal abnormalities is ↑ by adding inhibin A to estriol, β-hCG, and MSAFP (quad screening). Results of quad screening are as follows: gradually increases an addi tional 10% by 28 weeks. Peripheral venous Progressive increase to term. distention Peripheral vascular Progressive decrease to term. +40 resistance +30 Pulmonary Respiratory rate Unchanged. +20 Respiratory rate, vital capacity Tidal volume Increases by 30–40%. Expiratory reserve Gradual decrease. Vital capacity Unchanged. +10

1	Pulmonary Respiratory rate Unchanged. +20 Respiratory rate, vital capacity Tidal volume Increases by 30–40%. Expiratory reserve Gradual decrease. Vital capacity Unchanged. +10 Respiratory minute Increases by 40%. volume ElectrolytesElectrolytes Unchanged. 0 second trimester. +20Hematocrit Decreases slightly. +10Fibrinogen Increases. Gastrointestinal Sphincter tone Decreases. 0 Gastric emptying Increases. time (Reproduced, with permission, from Gardner DG, Shoback D. Greenspan’s Basic & Clinical Endocrinology, 8th ed. New York: McGraw-Hill, 2007: Fig. 17-2B.) TABLE 2.11-1. Standard Prenatal Care

1	Weight gain Guidelines for weight gain in pregnancy: ■Recommended gain: An additional 100–300 kcal/day; 500 kcal/day during breastfeeding. ■Excessive gain: > 1.5 kg/mo. ■Inadequate gain: < 1 kg/mo. Guidelines according to prepregnancy body mass index (BMI): ■Underweight (BMI < 19.8): 12–18 kg gain. ■Acceptable (BMI 19.8–26.0): 11–16 kg gain. ■Overweight (BMI 26.1–29.0): 7–11 kg gain. ■Severely overweight (BMI > 29.0): 7 kg gain. Nutrition Guidelines for nutritional supplementation: ■Folic acid supplements (↓ neural tube defects for all reproductive-age women): 0.4 mg/day, or 4 mg/day for women with a history of neural tube defects in prior pregnancies. ■Iron: Starting at the frst visit, 30 mg/day of elemental iron (or 150 mg of iron sulfate). ■Calcium: 1300 mg/day for women < 19 years of age; 1000 mg/day for those > 19 years of age. Additional guidelines for complete vegetarians: ■Vitamin D: 10 μg or 400 IU/day. ■Vitamin B12: 2 μg/day. Exercise Thirty minutes of moderate exercise

1	of age; 1000 mg/day for those > 19 years of age. Additional guidelines for complete vegetarians: ■Vitamin D: 10 μg or 400 IU/day. ■Vitamin B12: 2 μg/day. Exercise Thirty minutes of moderate exercise daily is recommended.

1	Trisomy 18: All four are ↓. Trisomy 21: ↓ AFP and estriol; ↑ β-hCG and inhibin A. 2 up, 2 down: trisomy 21 = ↓ AFP, ↓ estriol, ↑β-hCG, ↑ inhibin A. Recommended at weeks 9–14. Pregnancy-associated plasma protein A (PAPP-A) + ultrasound-determined nuchal transparency (a measure of fuid in the fetal neck) + free β-hCG can detect ~85% of cases of Down syndrome and ~97% of cases of trisomy 18. Advantages: Screen of low-risk pregnant women (< 35 years of age); available earlier and less invasive than chorionic villus sampling (see below). Recommended at weeks 10–12. Involves transcervical or transabdominal aspiration of placental (chorionic villi) tissue. Advantages: Has a diagnostic accuracy comparable to that of amniocentesis; available at 10–12 weeks’ gestation. Disadvantages: Carries a risk of fetal loss (1–2%); cannot detect open neural tube defects. Limb defects have been associated with CVS performed at ≤ 9 weeks. T AB LE 2.1 1 -2. Prenatal Diagnostic Testing Schedule

1	Limb defects have been associated with CVS performed at ≤ 9 weeks. T AB LE 2.1 1 -2. Prenatal Diagnostic Testing Schedule Prenatal visits Weeks 0–28: Every four weeks. Weeks 29–35: Every two weeks. Weeks 36–birth: Every week. Initial visit 9–14 weeks Offer PAPP-A + nuchal transparency + free β-hCG +/– CVS. 15–20 weeks Offer MSAFP or quad screen (AFP, estriol, β-hCG, and inhibin A) +/– amniocentesis. 18–20 weeks Ultrasound for full anatomic screen. 24–28 weeks One-hour glucose challenge test for gestational diabetes screen. Heme: CBC, Rh factor, type and screen. Infectious disease: UA and culture, rubella antibody titer, HBsAg, RPR/VDRL, cervical gonorrhea and chlamydia, PPD, HIV, Pap smear (to check for dysplasia). If indicated: HbA1c, sickle cell screening. Discuss genetic screening: Tay-Sachs, CF. Recommended at 15–20 weeks. Consists of transabdominal aspiration of amniotic fuid using an ultrasound-guided needle and evaluation of fetal cells for genetic studies.

1	Recommended at 15–20 weeks. Consists of transabdominal aspiration of amniotic fuid using an ultrasound-guided needle and evaluation of fetal cells for genetic studies. Advantages: Detects ~80% of open neural tube defects, ~85% of cases of Down syndrome, and ~60% of cases of trisomy 18. Disadvantages: Risks include premature rupture of membranes (PROM), chorioamnionitis, and fetal-maternal hemorrhage, which can result in fetal loss (0.5%). Indicated for the following: In women who will be > 35 years of age at the time of delivery. In conjunction with an abnormal quad screen. In Rh-sensitized pregnancy to obtain fetal blood type or to detect fetal hemolysis. To evaluate fetal lung maturity via a lecithin-to-sphingomyelin ratio ≥ 2.5 or to detect the presence of phosphatidylglycerol (done during the third trimester).

1	To evaluate fetal lung maturity via a lecithin-to-sphingomyelin ratio ≥ 2.5 or to detect the presence of phosphatidylglycerol (done during the third trimester). Major defects are apparent in about 3% of births and in roughly 4.5% of children by f ve years of age. Table 2.11-3 lists FDA risk classif cations of pharmaceutical products for use during pregnancy. Table 2.11-4 outlines common teratogenic agents. T AB LE 2.1 1 -3. FDA Risk Classifcation of Drugs for Use During Pregnancy

1	Category A Adequate and well-controlled studies in women fail to demonstrate a risk to the fetus in the frst trimester (and there is no risk in later trimesters). The possibility of fetal harm seems remote. Vitamin B6, vitamin E, folic acid (within the recommended daily allowances). Category B Either animal reproduction studies have not demonstrated risk to the fetus but no adequate and well-controlled studies in pregnant women have been reported, or animal reproduction studies have shown an adverse effect that was not confrmed in controlled studies in women in the frst trimester (and there is no evidence of risk in later trimesters). Ampicillin, acetaminophen, bupropion. Category C Either studies in animals have revealed adverse effects on the fetus but no controlled studies in women have been reported, or studies in women and animals are not available. Drugs should be given only if the potential benef t justifes the potential risk to the fetus. Diphenhydramine, rifampin, zidovudine

1	been reported, or studies in women and animals are not available. Drugs should be given only if the potential benef t justifes the potential risk to the fetus. Diphenhydramine, rifampin, zidovudine (AZT). Category D Positive evidence of human fetal risk exists, but the benefts from use in pregnant women may be acceptable despite the risk. Alcohol, phenytoin, tetracycline. Category X Studies in animals or humans have demonstrated fetal abnormalities, or evidence exists of fetal risk based on human experience, or both, and the risk in pregnant women clearly outweighs any possible benef t. Isotretinoin, thalidomide, warfarin.

1	Pregnant women should not change the cat’s litterbox. First-trimester toxoplasmosis infection is less common but more severe. Third-trimester infection is more common but less severe. May occur at any time during pregnancy, labor, and delivery. Common sequelae include premature delivery, CNS abnormalities, anemia, jaundice, hepatosplenomegaly, and growth retardation. The most common pathogens can be remembered through use of the mnemonic TORCHeS (see also Table 2.11-5): Toxoplasmosis: Transplacental transmission, with 1° infection occurring via consumption of raw meat or contact with cat feces. Specif c f ndings include hydrocephalus, intracranial calcifications, chorioretinitis, and ring-enhancing lesions on head CT. Other: Parvovirus, varicella, Listeria, TB, malaria, fungi. Rubella: Transplacental transmission in the frst trimester. Specif c f ndings include a purpuric “blueberry muffin” rash, cataracts, mental retardation, hearing loss, and patent ductus arteriosus (PDA).

1	CMV: The most common congenital infection; primarily transmitted transplacentally. Specif c fndings include a petechial rash (similar to “blueberry muffn” rash) and periventricular calcifications. Herpes: Intrapartum transmission if the mother has active lesions. Can cause skin, eye, and mouth infections or life-threatening CNS/systemic infection. HIV: Transmission can occur in utero, at the time of delivery, or via breast milk. Occurs in 13–39% of births to infected mothers. The combination of AZT treatment (prenatally, intrapartum, and neonatally for the f rst six weeks of life) and cesarean delivery can lower transmission to 2%. Newborns with congenitally acquired HIV are often asymptomatic. Failure to T AB LE 2.1 1 -4. Common Teratogenic Agents and Their Associated Defects

1	ACEIs Fetal renal tubular dysplasia and neonatal renal failure, oligohydramnios, intrauterine growth restriction (IUGR), lack of cranial ossif cation. Alcohol Fetal alcohol syndrome (growth restriction before and after birth, mental retardation, midfacial hypoplasia, renal and cardiac defects). Consumption of > 6 drinks per day is associated with a 40% risk of fetal alcohol syndrome. Androgens Virilization of females; advanced genital development in males. Carbamazepine Neural tube defects, fngernail hypoplasia, microcephaly, developmental delay, IUGR. Cocaine Bowel atresias; congenital malformations of the heart, limbs, face, and GU tract; microcephaly; IUGR; cerebral infarctions. Diethylstilbestrol (DES) Clear cell adenocarcinoma of the vagina or cervix, vaginal adenosis, abnormalities of the cervix and uterus or testes, possible infertility. Lead ↑ spontaneous abortion (SAB) rate; stillbirths. Lithium Congenital heart disease (Ebstein’s anomaly). Methotrexate ↑ SAB rate. Organic

1	of the cervix and uterus or testes, possible infertility. Lead ↑ spontaneous abortion (SAB) rate; stillbirths. Lithium Congenital heart disease (Ebstein’s anomaly). Methotrexate ↑ SAB rate. Organic mercury Cerebral atrophy, microcephaly, mental retardation, spasticity, seizures, blindness. Phenytoin IUGR, mental retardation, microcephaly, dysmorphic craniofacial features, cardiac defects, f ngernail hypoplasia. Radiation Microcephaly, mental retardation. Medical diagnostic radiation delivering < 0.05 Gy to the fetus has no teratogenic risk. Streptomycin and kanamycin Hearing loss; CN VIII damage. Tetracycline Permanent yellow-brown discoloration of deciduous teeth; hypoplasia of tooth enamel. Thalidomide Bilateral limb defciencies, anotia and microtia, cardiac and GI anomalies. Trimethadione and paramethadione Cleft lip or cleft palate, cardiac defects, microcephaly, mental retardation. Valproic acid Neural tube defects (spina bifda); minor craniofacial defects. Vitamin A and

1	and paramethadione Cleft lip or cleft palate, cardiac defects, microcephaly, mental retardation. Valproic acid Neural tube defects (spina bifda); minor craniofacial defects. Vitamin A and derivatives ↑ SAB rate, microtia, thymic agenesis, cardiovascular defects, craniofacial dysmorphism, microphthalmia, cleft lip or cleft palate, mental retardation. Warfarin (wages war on the fetus) Nasal hypoplasia and stippled bone epiphyses, developmental delay, IUGR, ophthalmologic abnormalities.

1	T AB LE 2.1 1 -5. Diagnosis and Treatment of Common Congenital Infections

1	Toxoplasmosis Serologic testing Pyrimethamine + sulfadiazine Avoid exposure to cat feces during pregnancy. Spiramycin prophylaxis for the third trimester. Rubella Serologic testing Immunize before pregnancy; vaccinate the mother after delivery if serologic titers remain . CMV Urine culture, PCR of amniotic f uid Postpartum ganciclovir HSV Serologic testing Acyclovir Perform a C-section if lesions are present at delivery. ELISA, Western blot AZT or nevirapine in pregnant women with HIV; perform elective C-section if viral load is > 1000; treat infants with prophylactic AZT; avoid breastfeeding. Dark-f eld microscopy, VDRL/RPR, FTA-ABS Penicillin Penicillin in pregnant women who test . HIV Syphilis thrive, bacterial infections with common organisms, and an ↑ incidence of upper and lower respiratory diseases may appear early or may be delayed for months to years. HIV-mothers should be counseled not to breastfeed their infants.

1	Syphilis: Primarily intrapartum transmission. Specif c fndings include a maculopapular skin rash, lymphadenopathy, hepatomegaly, “snufﬂ es” (mucopurulent rhinitis), and osteitis. In childhood, late congenital syphilis is characterized by saber shins, saddle nose, CNS involvement, and Hutchinson’s triad: peg-shaped upper central incisors, deafness, and interstitial keratitis (photophobia, lacrimation). Defned as loss of products of conception (POC) prior to the 20th week of pregnancy. Approximately 60% of chemically evident pregnancies and 15– 20% of clinically diagnosed pregnancies terminate in a SAB. More than 80% will occur in the frst trimester. Risk factors are as follows: Chromosomal abnormalities: A factor in approximately 50% of SABs in the frst trimester, 20–30% in second-trimester losses, and 5–10% in third-trimester losses. Maternal factors: Maternal trauma, ↑ maternal age, infection, dietary def ciencies.

1	Maternal factors: Maternal trauma, ↑ maternal age, infection, dietary def ciencies. Inherited thrombophilias: Factor V Leiden, prothrombin, antithrombin, proteins C and S, methylene tetrahydrofolate reductase (hyperhomocysteinemia). Immunologic issues: Antiphospholipid antibodies, alloimmune factors. Anatomic issues: Uterine abnormalities, incompetent cervix, cervical conization or loop electrosurgical excision procedure (LEEP), cervical injury, DES exposure, anatomical abnormalities of the cervix. Endocrinologic issues: Diabetes mellitus (DM), hypothyroidism, progeste rone def ciency. Environmental factors: Tobacco (with > 14 cigarettes/day, the risk is twofold), alcohol, caffeine (> 500 mg caffeine/day), toxins, drugs, radiation. Fetal factors: Anatomic malformation. See Table 2.11-6 for types of SAB. ■↓ levels of hCG. Ultrasound: Can identify the gestational sac 5–6 weeks from the LMP, a fetal pole at six weeks, and fetal cardiac activity at 6–7 weeks.

1	See Table 2.11-6 for types of SAB. ■↓ levels of hCG. Ultrasound: Can identify the gestational sac 5–6 weeks from the LMP, a fetal pole at six weeks, and fetal cardiac activity at 6–7 weeks. With accurate dating, a small, irregular intrauterine sac without a fetal pole on transvaginal ultrasound is diagnostic of an abnormal pregnancy. Maternal Rh type should be determined and RhoGAM given if the type is Rh . It has been estimated that 50% of all pregnancies in the United States are unintended. Some 25% of all pregnancies end in elective abortion. Options for elective abortion depend on GA and patient preferences (see Table 2.11-7). Leopold’s maneuvers are used to determine fetal lie (longitudinal or transverse) and, if possible, fetal presentation (breech or cephalic). Cervical examination:

1	Leopold’s maneuvers are used to determine fetal lie (longitudinal or transverse) and, if possible, fetal presentation (breech or cephalic). Cervical examination: Evaluate dilation, effacement, station, cervical position, and cervical consistency. Use the Bishop score (see Table 2.11-8) to evaluate the favorability of delivery and the probability of succeeding with an induction. Scoring is interpreted as follows: 0–4: Indicates a 45–50% chance of failure. Give prostaglandins for induction. 5–9: Points to a 10% chance of failure. Give pitocin for induction. 10–13: Associated with a very high probability of success. There is no need for intervention for induction. Confrm or determine fetal presentation. Determine fetal position through palpation of the fetal sutures and fontanelles. Conduct a sterile speculum exam if rupture of membranes (ROM) is suspected. Table 2.11-9 and Figure 2.11-4 depict the normal stages of labor.

1	Conduct a sterile speculum exam if rupture of membranes (ROM) is suspected. Table 2.11-9 and Figure 2.11-4 depict the normal stages of labor. FHR monitoring is the most common obstetric procedure and is used in 85% of live births in the United States. It may be performed with an electrode at T AB LE 2.1 1 -6. Types of Spontaneous Abortion

1	Complete POC is expelled. Pain ceases, but spotting may persist. Closed os. Ultrasound shows an empty uterus. POC should be sent to pathology to conf rm fetal tissue. None. Incomplete Some POC is expelled. Bleeding/mild cramping. Visible tissue on exam. Open os. Ultrasound shows retained fetal tissue. Manual uterine aspiration (MUA) or D&C. Threatened No POC is expelled. Uterine bleeding +/– abdominal pain. Closed os + intact membranes + fetal cardiac motion on ultrasound. Pelvic rest for 24–48 hours and follow-up ultrasound to assess the viability of conceptus. Inevitable No POC is expelled. Uterine bleeding and cramps. Open os +/− ROM. MUA, D&C, misoprostol, or expectant management. Missed No POC is expelled. No fetal cardiac motion. No uterine bleeding. Brownish vaginal discharge. Closed os. No fetal cardiac activity; retained fetal tissue on ultrasound. MUA, D&C, or misoprostol. Septic Endometritis leading to septicemia. Maternal mortality is 10–15%. Hypotension, hypothermia, ↑

1	os. No fetal cardiac activity; retained fetal tissue on ultrasound. MUA, D&C, or misoprostol. Septic Endometritis leading to septicemia. Maternal mortality is 10–15%. Hypotension, hypothermia, ↑ WBC count. MUA, D&C, and IV antibiotics. Intrauterine fetal demise Absence of fetal cardiac activity. Uterus small for GA; no fetal heart tones or movement on ultrasound. Induce labor; evacuate the uterus (D&E) to prevent DIC at GA > 16 weeks. Recurrenta If early in pregnancy, often due to chromosomal abnormalities. If later in pregnancy, often due to hypercoagulable states (e.g., SLE, factor V Leiden, protein S def ciency). Incompetent cervix should be suspected with a history of painless dilation of the cervix and delivery of a normal fetus between 18 and 32 weeks. Karyotyping of both parents. Hypercoagulability workup of mother. Evaluate for uterine abnormalities. Surgical cerclage procedures to suture the cervix closed until labor or ROM occurs with subsequent removal prior to delivery.

1	Hypercoagulability workup of mother. Evaluate for uterine abnormalities. Surgical cerclage procedures to suture the cervix closed until labor or ROM occurs with subsequent removal prior to delivery. Restriction of activities. a Defned as two or more consecutive SABs or a total of three SABs in one year.

1	tached to the fetal scalp (a method that yields more precise results), or external monitoring can be conducted using Doppler ultrasound (a less invasive option). However, continuous electronic FHR monitoring has not been shown to be more effective than appropriate intermittent monitoring. TABLE 2.11-7. Elective Termination of Pregnancy First trimester (90% therapeutic abortions [TABs]) Medical management: ■Oral mifepristone (low dose) + oral/vaginal misoprostol ■IM/oral methotrexate + oral/vaginal misoprostol ■Vaginal or sublingual or buccal misoprostol (high dose), repeated up to three times Surgical management: ■ Manual aspiration ■D&C with vacuum aspiration Up to: 49 days’ GA 49 days’ GA 56 days’ GA Up to 13 weeks’ GA Second trimester (10% TABs) Obstetric management: Induction of labor (typically with prostaglandins, amniotomy, and oxytocin) Surgical management: D&E 13–24 weeks’ GA (depending on state laws) Same as above

1	Patients without complications: Review FHR tracings every 30 minutes in the frst stage of labor and every 15 minutes in the second stage of labor. Patients with complications: Review FHR tracings every 15 minutes in the frst stage of labor and every 5 minutes in the second stage of labor. Rate (normal = 110–160 bpm): FHR < 110 bpm: Bradycardia. Can be caused by congenital heart malformations or severe hypoxia (2° to uterine hyperstimulation, cord prolapse, or rapid fetal descent). FHR > 160 bpm: Tachycardia. Can be caused by hypoxia, maternal fever, or fetal anemia. Variability (normal beat-to-beat variability = 6–25 bpm): See Figures 2.11-5 and 2.11-6. Undetectable variability: Indicates severe fetal distress. TABLE 2.11-8. Bishop Score TABLE 2.11-9. Stages of Labor

1	Undetectable variability: Indicates severe fetal distress. TABLE 2.11-8. Bishop Score TABLE 2.11-9. Stages of Labor First Latent Active Onset of labor to 3–4 cm dilation 4 cm to complete cervical dilation (10 cm) 6–11 hrs 4–6 hrs (1.2 cm/hr) 4–8 hrs 2–3 hrs (1.5 cm/hr) Prolongation seen with excessive sedation and hypertonic uterine contractions. Prolongation seen with cephalopelvic disproportion. Second Complete cervical dilation to delivery of infant 0.5–3.0 hrs 5–30 min Baby goes through all cardinal movements of delivery. Third Delivery of infant to delivery of placenta 0–0.5 hr 0–0.5 hr Uterus contracts and placenta separates to establish hemostasis. Minimal variability: < 6 bpm. Indicates fetal distress or the effects of opioids or magnesium. Normal variability: 6–25 bpm. Marked variability: > 25 bpm. May indicate fetal distress; may occur before a ↓ in variability. Sinusoidal variability: Points to serious fetal anemia; may also occur during maternal meperidine use.

1	Sinusoidal variability: Points to serious fetal anemia; may also occur during maternal meperidine use. FIGURE 2.11-4. Stages of labor. Cervical dilation, level of descent, and orientation of occipitoanterior presentation during various stages of labor. (Reproduced, with permission, from DeCherney AH. Current Obstetric & Gynecologic Diagnosis & Treatment, 8th ed. Stamford, CT: Appleton & Lange, 1994: 211.) F IGU R E 2.1 1 -5. Varying (variable) fetal heart rate decelerations. Deceleration B exhibits “shoulders” of acceleration compared with deceleration A. (Reproduced, with permission, from Cunningham FG et al. Williams Obstetrics, 22nd ed. New York: McGraw-Hill, 2005: Fig. 18-21.) F IGU R E 2.1 1 -6. Late fetal heart rate decelerations.

1	F IGU R E 2.1 1 -6. Late fetal heart rate decelerations. Late decelerations due to uteroplacental insuffciency resulting from placental abruption. Immediate cesarean delivery was performed. Umbilical artery pH was 7.05 and PO2 was 11 mmHg. (Reproduced, with permission, from Cunningham FG et al. Williams Obstetrics, 22nd ed. New York: McGraw-Hill, 2005: Fig. 18-17.) Accelerations: Onset of an ↑ in FHR to a peak in < 30 seconds. Reassuring because they indicate fetal ability to appropriately respond to the environment. Decelerations: See Table 2.11-10. In general, antepartum fetal surveillance is used in pregnancies in which the risk of antepartum fetal demise is ↑. Testing is initiated in most at-risk patients TABLE 2.11-10. Types of Fetal Deceleration

1	TABLE 2.11-10. Types of Fetal Deceleration Early A visually apparent, gradual (onset to nadir in > 30 sec) ↓ in FHR with a return to baseline that mirrors the uterine contraction. Head compression from the uterine contraction (normal). Late A visually apparent, gradual (onset to nadir in > 30 sec) ↓ in FHR with return to baseline whose onset, nadir, and recovery occur after the beginning, peak, and end of uterine contraction, respectively. Uteroplacental insuff ciency and fetal hypoxemia. Variable An abrupt (onset to nadir in < 30 sec), visually apparent ↓ in FHR below baseline lasting ≥ 15 sec but < 2 min. Umbilical cord compression. Illustrations reproduced, with permission, from Cunningham FG et al. Williams Obstetrics, 22nd ed. New York: McGraw-Hill, 2005: Figs.18-14, 18-16, and 18-18. at 32–34 weeks (or 26–28 weeks if there are multiple worrisome risk factors present). The following assessments are made:

1	at 32–34 weeks (or 26–28 weeks if there are multiple worrisome risk factors present). The following assessments are made: Fetal movement assessment: Assessed by the mother as the number of fetal movements over one hour. The average time to obtain 10 movements is 20 minutes. Maternal reports of ↓ fetal movements should be evaluated by means of the tests described below. Nonstress test (NST): Performed with the mother resting in the lateral tilt position (to prevent supine hypotension). FHR is monitored externally by Doppler along with a tocodynamometer to detect uterine contractions. Acoustic stimulation may be used. “Reactive” (normal response): Two accelerations of ≥ 15 bpm above baseline lasting for at least 15 seconds over a 20-minute period (see Figure 2.11-7).

1	“Reactive” (normal response): Two accelerations of ≥ 15 bpm above baseline lasting for at least 15 seconds over a 20-minute period (see Figure 2.11-7). “Nonreactive”: Fewer than two accelerations over a 20-minute period. Perform further tests (e.g., a biophysical profle). Lack of FHR accelerations may occur with any of the following: GA < 32 weeks, fetal sleeping, fetal CNS anomalies, and maternal sedative or narcotic administration. Contraction stress test (CST): Performed in the lateral recumbent position. FHR is monitored during spontaneous or induced (via nipple stimulation or oxytocin) contractions. Reactivity is determined from fetal heart monitoring, as with the NST. The procedure is contraindicated in women with preterm membrane rupture or known placenta previa; women with a history of uterine surgery; and women who are at high risk for preterm labor. FIGURE 2.11-7. Reactive nonstress test.

1	FIGURE 2.11-7. Reactive nonstress test. (Reproduced, with permission, from Cunningham FG et al. Williams Obstetrics, 22nd ed. New York: McGraw-Hill, 2005: Fig. 15-7.) A negative CST is good; a positive one is bad. When performing a BPP, remember to– Test the Baby, MAN! “Positive” CST: Defned by late decelerations following 50% or more of contractions in a 10-minute window; raises concerns about fetal compromise. Delivery is usually warranted. “Negative” CST: Defned as no late or signifcant variable decelerations within 10 minutes and at least three contractions. Highly predictive of fetal well-being in conjunction with a normal NST. “Equivocal” CST: Def ned by intermittent late decelerations or significant variable decelerations. Biophysical profile (BPP): Uses real-time ultrasound to assign a score of 2 (normal) or 0 (abnormal) to fve parameters: fetal tone, breathing, movement, amniotic fuid volume, and NST. Scoring is as follows: 8–10: Reassuring for fetal well-being.

1	6: Considered equivocal. Term pregnancies are usually delivered with this prof le. 0–4: Extremely worrisome for fetal asphyxia; strong consideration should be given to immediate delivery if no other explanation is found. Modified biophysical profile (mBPP): Combines the NST with the amniotic fuid index (AFI, or the sum of the measurements of the deepest cord-free amniotic fuid measured in each of the abdominal quadrants). The test is considered normal with a reactive NST and an AFI > 5 cm. Umbilical artery Doppler velocimetry: With IUGR, there is reduction and even reversal of umbilical artery diastolic fow. The test is of benef t only when IUGR is suspected. Oligohydramnios (AFI < 5 cm) always warrants further workup.

1	Oligohydramnios (AFI < 5 cm) always warrants further workup. Uterine contractions and cervical dilation result in visceral pain (T10–L1). Descent of the fetal head and pressure on the vagina and perineum result in somatic pain (pudendal nerve, S2–S4). In the absence of a medical contraindication, maternal request is a suffcient medical indication for pain relief during labor. Absolute contraindications to regional anesthesia (epidural, spinal, or combination) include the following (see also Table 2.11-11): Maternal use of a once-daily dose of low-molecular-weight heparin (LMWH) within 12 hours Skin infection over the site of needle placement ■↑ ICP caused by a mass lesion If “morning sickness” persists after the f rst trimester, think hyperemesis gravidarum.

1	Skin infection over the site of needle placement ■↑ ICP caused by a mass lesion If “morning sickness” persists after the f rst trimester, think hyperemesis gravidarum. Persistent vomiting not related to other causes, acute starvation (usually large ketonuria), and weight loss (usually at least a 5% ↓ from prepregnancy weight). Occurs in 0.5–2.0% of pregnancies. More common in f rst pregnancies, multiple gestations, and molar pregnancies. ↑β-hCG and ↑ estradiol have been implicated in its pathophysiology. T AB LE 2.1 1 -1 1. Available Methods of Anesthesia and Analgesia

1	Nonpharmacologic (social support, massages, breathing, aromatherapy, ambulation and repositioning) No known side effects. Works by increasing coping with pain rather than eliminating pain. Limited pain relief. Opioids Provide an adequate level of pain relief for some women without the risks associated with regional anesthesia. Local block (lidocaine) Excellent anesthesia before episiotomy and during repair of lacerations; can be used to perform a pudendal block. Rarely, may cause seizures, hypotension, and cardiac arrhythmias. Epidural The most effective form of pain relief; can also be used for cesarean delivery or postpartum tubal ligation. Can result in pruritus, fever, hypotension, and transient FHR deceleration. Spinal Rapid-onset analgesia that provides excellent pain relief for procedures of limited duration (30–250 minutes). Limited duration; puts patients at risk for hypotension, postdural puncture headache, and transient neurologic symptoms. Combined spinal epidural General

1	Offers rapid onset of spinal analgesia combined with the ability to prolong the duration of analgesia with continuous epidural infusion. Used in emergent cesarean delivery and indicated in some cases of FHR abnormality; can be useful in cases where regional anesthesia is absolutely contraindicated or fails. The sedative effect of opioids ↓ FHR variability and ↑ the possible need for neonatal naloxone administration and f ve-minute Apgar scores < 7. Carries the risks of both procedures; may ↑ the risk of bradycardia and emergent cesarean delivery over epidural analgesia alone. Requires airway control; carries a signif cant risk of maternal aspiration and neonatal depression (inhaled anesthetic agents readily cross the placenta); associated with higher maternal morbidity rates than epidural anesthesia. Distinguish from “morning sickness,” acid refux, gastroenteritis, hyperthyroidism, and neurologic conditions. Rule out molar pregnancy: Check β-hCG level and ultrasound.

1	Distinguish from “morning sickness,” acid refux, gastroenteritis, hyperthyroidism, and neurologic conditions. Rule out molar pregnancy: Check β-hCG level and ultrasound. Determine severity: Evaluate for ketonemia, ketonuria, hyponatremia, and hypokalemic, hypochloremic metabolic alkalosis. Measure liver enzymes, serum bilirubin, and serum amylase/lipase. The f rst step in the diagnosis of hyperemesis gravidarum is to rule out molar pregnancy with ultrasound +/– β-hCG. The f rst step in the management of hyperemesis gravidarum is vitamin B6 and doxylamine if not severe or antiemetics and IV f uids if severe. Gestational diabetes is typically asymptomatic, so the f rst step in diagnosis is a one-hour glucose challenge at 24–28 weeks. If ≥ 140 mg/dL, perform a three-hour glucose challenge test. Keys to the management of gestational diabetes: (1) the ADA diet; (2) insulin if needed; (3) ultrasound for fetal growth; and (4) NST beginning at 30–32 weeks.

1	Keys to the management of gestational diabetes: (1) the ADA diet; (2) insulin if needed; (3) ultrasound for fetal growth; and (4) NST beginning at 30–32 weeks. First step: Administer vitamin B6. Second step: Doxylamine (an antihistamine) PO. Third step: Promethazine or dimenhydrinate PO/PR. If severe: Metoclopramide, ondansetron, prochlorperazine, or promethazine IM/PO. If dehydrated: IV fuids, IV nutritional supplementation, and dimenhydrinate IV. Diabetes in pregnancy is divided into two categories: gestational diabetes, in which onset occurs during pregnancy, and pregestational diabetes. The White classif cation (see Table 2.11-12) is commonly used to classify women according to the nature of their diabetes and the associated risk. Carbohydrate intolerance of variable severity that is frst diagnosed during pregnancy. Occurs in 3–5% of all pregnancies, usually in late pregnancy.

1	Carbohydrate intolerance of variable severity that is frst diagnosed during pregnancy. Occurs in 3–5% of all pregnancies, usually in late pregnancy. Typically asymptomatic. Edema, polyhydramnios, or a large-for-GA infant (> 90th percentile) may be warning signs. First step: One-hour 50-g glucose challenge test; venous plasma glucose is measured one hour later (at 24–28 weeks). Values ≥ 140 mg/dL are considered abnormal. Next step: Confrm with an oral three-hour (100-g) glucose tolerance test showing any two of the following: fasting > 95 mg/dL; one hour > 180 mg/ dL; two hours > 155 mg/dL; three hours > 140 mg/dL. Mother: First step: Start with the ADA diet, regular exercise, and strict glucose monitoring (four times a day). Tight maternal glucose control (fasting glucose < 100; oneto two-hour postprandial glucose < 150) improves outcomes. Next step: Add insulin if dietary control is insuff cient. Give intrapartum insulin and dextrose to maintain tight control during delivery.

1	Next step: Add insulin if dietary control is insuff cient. Give intrapartum insulin and dextrose to maintain tight control during delivery. Fetus: Obtain periodic ultrasound and NSTs to assess fetal growth and wellbeing. It may be necessary to induce labor at 39–40 weeks. More than 50% of patients go on to develop glucose intolerance and/or type 2 DM later in life. T AB LE 2.1 1 -1 2. White Classifcation of Diabetes in Pregnancy A1 Gestational diabetes; insulin not required. A2 Gestational diabetes; insulin required. B Age of onset 20 years or older or duration < 10 years. C Age of onset 10–19 years or duration 10–19 years. D Age of onset < 10 years or duration > 20 years. F Nephropathy. H Cardiomyopathy. R Proliferating retinopathy. RF Retinopathy and nephropathy. T Renal transplant.

1	Observed in 1% of all pregnancies. Insulin requirements may ↑ as much as threefold. Poorly controlled DM is associated with an ↑ risk of congenital malformations, fetal loss, and maternal/fetal morbidity during labor and delivery. Mother: Renal, ophthalmologic, and cardiac evaluation to assess for end-organ damage. Strict glucose control (with diet, exercise, insulin therapy, and frequent self-monitoring for type 1 and type 2 DM) to minimize fetal defects. Fasting morning: ≤ 90 mg/dL. Two-hour postprandial: < 120 mg/dL. Fetus: 18–20 weeks: Ultrasound to determine fetal age and growth; evaluate for cardiac anomalies and polyhydramnios; quad screen to screen for developmental anomalies. 32–34 weeks: Close fetal surveillance (e.g., NST, CST, BPP). Admit if maternal DM has been poorly controlled or fetal parameters are a concern. Serial ultrasounds for fetal growth. Delivery and postpartum:

1	Delivery and postpartum: Maintain normoglycemia (80–100 mg/dL) during labor with an IV insulin drip and hourly glucose measurements. Consider early delivery in the setting of poor maternal glucose control, preeclampsia, macrosomia, or evidence of fetal lung maturity. Cesarean delivery should be considered for an estimated fetal weight (EFW) > 4500 g. Greater than eight, investigate! If HbA1c is > 8%, look for congenital abnormalities. If UA before 20 weeks reveals glycosuria, think pregestational diabetes. Hyperglycemia in the f rst trimester suggests preexisting diabetes and should be managed as pregestational diabetes. Encourage breastfeeding with an appropriate ↑ in caloric intake. Continue glucose monitoring postpartum. Insulin needs rapidly ↓ after delivery. See Table 2.11-13. Distinguished as follows:

1	Encourage breastfeeding with an appropriate ↑ in caloric intake. Continue glucose monitoring postpartum. Insulin needs rapidly ↓ after delivery. See Table 2.11-13. Distinguished as follows: Gestational hypertension (formerly known as pregnancy-induced hypertension): Idiopathic hypertension without signif cant proteinuria (< 300 mg/L) that develops at > 20 weeks’ gestation. As many as 25% of patients may go on to develop preeclampsia. Chronic hypertension: Present before conception and at < 20 weeks’ gestation, or may persist for > 12 weeks postpartum. Up to one-third of patients may develop superimposed preeclampsia. Tx: Monitor BP closely and treat with appropriate antihypertensives (e.g., methyldopa, labetalol, nifedipine). Do not give ACEIs or diuretics, as ACEIs are known to lead to uterine ischemia, and diuretics can aggravate low plasma volume to the point of uterine ischemia. Cx: Complications are similar to those of preeclampsia (see below). Distinguished as follows:

1	Cx: Complications are similar to those of preeclampsia (see below). Distinguished as follows: Preeclampsia: New-onset hypertension (SBP ≥ 140 mmHg or DBP ≥ 90 mmHg) and proteinuria (> 300 mg of protein in a 24-hour period) occurring at > 20 weeks’ gestation. Eclampsia: New-onset grand mal seizures in women with preeclampsia. HELLP syndrome (hemolytic anemia, elevated liver enzymes, and low platelets): A variant of preeclampsia with a poor prognosis. ■The etiology is unknown, but clinical manifestations are explained by vasospasm leading to hemorrhage and organ necrosis.

1	■The etiology is unknown, but clinical manifestations are explained by vasospasm leading to hemorrhage and organ necrosis. T AB LE 2.1 1 -1 3. Complications of Pregestational Diabetes Mellitus macrosomia) and need for C-section Preterm labor Infection Polyhydramnios Postpartum hemorrhage Maternal mortality Macrosomia or IUGR Cardiac and renal defects Neural tube defects (e.g., sacral agenesis) Hypocalcemia Polycythemia Hyperbilirubinemia IUGR Hypoglycemia from hyperinsulinemia Respiratory distress syndrome (RDS) Birth injury (e.g., shoulder dystocia) Perinatal mortality ■Risk factors include nulliparity, African-American ethnicity, extremes of age (< 20 or > 35), multiple gestation, molar pregnancy, renal disease (due to SLE or type 1 DM), a family history of preeclampsia, and chronic hypertension. See Table 2.11-14 for the signs and symptoms of preeclampsia and eclampsia. The only cure for preeclampsia/eclampsia is delivery of the fetus. Preeclampsia:

1	See Table 2.11-14 for the signs and symptoms of preeclampsia and eclampsia. The only cure for preeclampsia/eclampsia is delivery of the fetus. Preeclampsia: If the patient is close to term or preeclampsia worsens, induce delivery with IV oxytocin, prostaglandin, or amniotomy. If far from term, treat with modifed bed rest and expectant management. Severe preeclampsia: First step: Control BP with labetalol and/or hydralazine (goal < 160/110 with a DBP of 90–100 to maintain fetal blood f ow). Second step: Prevent seizures with continuous magnesium sulfate drip. Watch for signs of magnesium toxicity (loss of DTRs, respiratory paralysis, coma). Continue seizure prophylaxis for 24 hours postpartum. Treat magnesium toxicity with IV calcium gluconate. Third step: Deliver by induction or C-section when mother is stable. Eclampsia: First step: ABCs with supplemental O2.

1	Third step: Deliver by induction or C-section when mother is stable. Eclampsia: First step: ABCs with supplemental O2. Second step: Seizure control/prophylaxis with magnesium. If seizures recur, give IV diazepam. Monitor magnesium blood levels and magnesium toxicity; monitor fetal status. Control BP (labetalol and/or hydralazine). Limit fuids; Foley for strict I/Os. T AB LE 2.1 1 -1 4. Presentation of Preeclampsia and Eclampsia Signs of severe preeclampsia are persistent headache or other cerebral or visual disturbances, persistent epigastric pain, and hyperreactive ref exes.

1	Signs of severe preeclampsia are persistent headache or other cerebral or visual disturbances, persistent epigastric pain, and hyperreactive ref exes. Mild preeclampsia Usually asymptomatic. BP ≥ 140/90 on two occasions > 6 hours apart. Proteinuria (> 300 mg/24 hrs or 1–2 urine dipsticks). Edema. Severe preeclampsia BP > 160/110 on two occasions > 6 hours apart. Renal: Proteinuria (> 5 g/24 hrs or 3–4 urine dipsticks) or oliguria (< 500 mL/24 hrs). Cerebral changes: Headache, somnolence. Visual changes: Blurred vision, scotomata. Hyperactive ref exes/clonus. RUQ pain. Hemolysis, elevated liver enzymes, thrombocytopenia (HELLP syndrome). Eclampsia The most common signs preceding an eclamptic attack are headache, visual changes, and RUQ/epigastric pain. Seizures are severe if not controlled with anticonvulsant therapy. With third-trimester bleeding, think anatomically: Vagina: bloody show, trauma Cervix: cervical cancer, cervical/vaginal lesion

1	With third-trimester bleeding, think anatomically: Vagina: bloody show, trauma Cervix: cervical cancer, cervical/vaginal lesion Placenta: placental abruption, placenta previa Fetus: fetal bleeding The classic triad of ectopic pregnancy PAVEs the way for diagnosis: ■Third step: Initiate delivery if the patient is stable and convulsions are controlled. Postpartum management is the same as that for preeclampsia. Seizures may occur antepartum (25%), intrapartum (50%), or postpartum (25%); most occur within 48 hours after delivery. Preeclampsia: Prematurity, fetal distress, stillbirth, placental abruption, seizure, DIC, cerebral hemorrhage, serous retinal detachment, fetal/ maternal death. Eclampsia: Cerebral hemorrhage, aspiration pneumonia, hypoxic encephalopathy, thromboembolic events, fetal/maternal death. Defned as any bleeding that occurs after 20 weeks’ gestation. Complicates 3–5% of pregnancies (prior to 20 weeks, bleeding is referred to as threatened abortion).

1	Defned as any bleeding that occurs after 20 weeks’ gestation. Complicates 3–5% of pregnancies (prior to 20 weeks, bleeding is referred to as threatened abortion). The most common causes are placental abruption and placenta previa (see Table 2.11-15 and Figure 2.11-8). Other causes include other forms of abnormal placentation (e.g., placenta accreta), ruptured uterus, genital tract lesions, and trauma. Most often tubal, but can be abdominal, ovarian, or cervical. Presents with abdominal pain and vaginal spotting/bleeding, although some patients are asymptomatic. Associated with etiologies that cause scarring to the fallopian tubes, including a history of PID, pelvic surgery, DES use, or endometriosis. The differential includes surgical abdomen, abortion, ovarian torsion, PID, and ruptured ovarian cyst. Approach a woman of reproductive age presenting with abdominal pain as a ruptured ectopic pregnancy until proven otherwise. Proceed as follows:

1	Approach a woman of reproductive age presenting with abdominal pain as a ruptured ectopic pregnancy until proven otherwise. Proceed as follows: First step: pregnancy test and a transvaginal ultrasound showing an empty uterus. Second step: Confrm with a serial hCG without appropriate hCG doubling. Medical treatment (methotrexate) is suffcient for small, unruptured tubal pregnancies. Surgical options for salpingectomy or salpingostomy with evacuation (laparoscopy vs. laparotomy). Tubal rupture and hemoperitoneum (an obstetric emergency). T AB LE 2.1 1 -1 5. Placental Abruption vs. Placenta Previa

1	Premature (before delivery) separation of normally implanted placenta. Abnormal placental implantation: ■ Total: Placenta covers the cervical os. ■ Marginal: Placenta extends to the margin of the os. ■ Low-lying: Placenta is in close proximity to the os. 1 in 100. 1 in 200. Risk factors Hypertension, abdominal/pelvic trauma, tobacco or cocaine use, previous abruption, rapid decompression of an overdistended uterus, excessive stimulation. Prior C-sections, grand multiparity, advanced maternal age, multiple gestation, prior placenta previa. Symptoms Painful, dark vaginal bleeding that does not spontaneously cease. Abdominal pain, uterine hypertonicity. Fetal distress. Painless, bright red bleeding that often ceases in 1–2 hours with or without uterine contractions. Usually no fetal distress. Diagnosis Primarily clinical. Transabdominal/transvaginal ultrasound sensitivity is only 50%; look for retroplacental clot; most useful for ruling out previa. Transabdominal/transvaginal ultrasound

1	Diagnosis Primarily clinical. Transabdominal/transvaginal ultrasound sensitivity is only 50%; look for retroplacental clot; most useful for ruling out previa. Transabdominal/transvaginal ultrasound sensitivity is > 95%; look for an abnormally positioned placenta. Pathophysiology Incidence

1	Stabilize patients with mild abruption and a premature fetus; manage expectantly (hospitalize; start IV and fetal monitoring; type and cross blood; bed rest). Moderate to severe abruption: Immediate delivery (vaginal delivery with amniotomy if mother and fetus are stable and delivery is expected soon; C-section for maternal or fetal distress). No vaginal exam! Stabilize patients with a premature fetus; manage expectantly. Give tocolytics. Serial ultrasound to assess fetal growth; resolution of partial previa. Give betamethasone to help with fetal lung maturity. Deliver by C-section. Indications for delivery include labor, life-threatening bleeding, fetal distress, documented fetal lung maturity, and 36 weeks’ GA.

1	Deliver by C-section. Indications for delivery include labor, life-threatening bleeding, fetal distress, documented fetal lung maturity, and 36 weeks’ GA. Hemorrhagic shock. ↑ risk of placenta accreta. Coagulopathy: DIC in 10%. Vasa previa (fetal vessels crossing the internal os). Recurrence risk is 5–16% and rises to 25% after Preterm delivery, PROM, IUGR, congenital two previous abruptions. anomalies. Fetal hypoxia. Recurrence risk is 4–8%. F IGU R E 2.1 1 -8. Placental implantation. (A) Normal placenta. (B) Low implantation. (C) Partial placenta previa. (D) Complete placenta previa. (Adapted, with permis sion, from DeCherney AH. Current Obstetric & Gynecologic Diagnosis & Treatment, 8th ed. Stamford, CT: Appleton & Lange, 1994: 404.) Defned as an EFW less than the 10th percentile for GA. Affected infants are commonly born to women with systemic diseases that lead to uteroplacental insuffciency (intrauterine infection, hypertension, anemia).

1	Affected infants are commonly born to women with systemic diseases that lead to uteroplacental insuffciency (intrauterine infection, hypertension, anemia). Other risk factors include maternal substance abuse, placenta previa, and multiple gestations. First step: Diagnose by confrming serial fundal height measurements with ultrasound. Second step: Ultrasound the fetus for EFW (although as pregnancy advances, ultrasound fetal weight estimates become increasingly unreliable). First step: Explore the underlying etiology and correct if possible. If near due date: Administer steroids (e.g., betamethasone) to accelerate fetal lung maturity. Then: Perform fetal monitoring with NST, CST, BPP, and umbilical artery Doppler velocimetry. A nonreassuring status near term may prompt delivery. ↑ perinatal morbidity and mortality. Defned as a birth weight > 90th percentile. A common sequela of gestational diabetes.

1	↑ perinatal morbidity and mortality. Defned as a birth weight > 90th percentile. A common sequela of gestational diabetes. Dx: Diagnose by weighing the newborn at birth (prenatal diagnosis is imprecise). Tx: Planned cesarean delivery may be considered for an EFW > 5000 g in women without diabetes and for an EFW > 4500 g in women with diabetes. Cx: ↑ risk of shoulder dystocia (leading to brachial plexus injury and Erb-Duchenne palsy) as birth weight ↑. Defned as an AFI > 20 on ultrasound. May be present in normal pregnancies, but fetal chromosomal developmental abnormalities must be considered. Etiologies include maternal DM, multiple gestation, isoimmunization, pulmonary abnormalities (e.g., cystic lung malformations), fetal anomalies (e.g., duodenal atresia, tracheoesophageal fstula, anencephaly), and twin-twin transfusion syndrome. Hx/PE: Usually asymptomatic.

1	Hx/PE: Usually asymptomatic. Dx: Fundal height greater than expected. Evaluation includes ultrasound for fetal anomalies, glucose testing for DM, and Rh screen. Tx: Etiology specif c. Cx: Preterm labor, fetal malpresentation, cord prolapse. An AFI < 5 cm on ultrasound. Usually asymptomatic, but IUGR or fetal distress may be present. Etiologies include fetal urinary tract abnormalities (e.g., renal agenesis, GU obstruction), chronic uteroplacental insuff ciency, and ROM. Dx: The sum of the deepest amniotic fuid pocket in all four abdominal quadrants on ultrasound. Tx: Rule out inaccurate gestational dates. Treat the underlying cause if possible. Cx: Associated with a 40-fold ↑ in perinatal mortality. Other complications include musculoskeletal abnormalities (e.g., clubfoot, facial distortion), pulmonary hypoplasia, umbilical cord compression, and IUGR.

1	In this condition, fetal RBCs leak into the maternal circulation, and maternal anti-Rh IgG antibodies form that can cross the placenta, leading to hemolysis of fetal Rh RBCs (erythroblastosis fetalis). There is an ↑ risk among an Rh-women who have had a previous SAB or TAB as well as among those who have undergone a previous delivery with no RhoGAM given. Sensitized Rhmothers with titers > 1:16 should be closely monitored with serial ultrasound and amniocentesis for evidence of fetal hemolysis. In severe cases, initiate preterm delivery when fetal lungs are mature. Prior to delivery, intrauterine blood transfusions may be given to correct a low fetal hematocrit. If the mother is Rh at 28 weeks and the father is Rh or unknown, give RhoGAM (Rh immune globulin). If the baby is Rh , give RhoGAM postpartum.

1	If the mother is Rh at 28 weeks and the father is Rh or unknown, give RhoGAM (Rh immune globulin). If the baby is Rh , give RhoGAM postpartum. Give RhoGAM to Rh-mothers who undergo abortion or who have had an ectopic pregnancy, amniocentesis, vaginal bleeding, or placenta previa/ placental abruption. Type and screen is critical; follow β-hCG closely and prevent pregnancy for one year. Hydrops fetalis occurs when fetal hemoglobin drops to < 7 g/dL. Other complications include fetal hypoxia and acidosis, kernicterus, prematurity, and death. A range of proliferative trophoblastic abnormalities that can be benign or malignant. Complete moles: Usually result from sperm fertilization of an empty ovum; 46,XX (paternally derived). Incomplete (partial) moles: Occur when a normal ovum is fertilized by two sperm (or a haploid sperm that duplicates its chromosomes); usually 69,XXY and contain fetal tissue.

1	Incomplete (partial) moles: Occur when a normal ovum is fertilized by two sperm (or a haploid sperm that duplicates its chromosomes); usually 69,XXY and contain fetal tissue. Presents with f rst-trimester uterine bleeding (most common), hyperemesis gravidarum, preeclampsia/eclampsia at < 24 weeks, and uterine size greater than dates. Risk factors include extremes of age (< 20 or > 40 years) and a diet def cient in folate or beta-carotene. No fetal heartbeat is detected. Pelvic exam may reveal enlarged ovaries (bilateral theca-lutein cysts) or expulsion of grapelike molar clusters into the vagina. Labs show markedly ↑ serum β-hCG (usually > 100,000 mIU/mL), and pelvic ultrasound reveals a “snowstorm” appearance with no gestational sac or fetus present (see Figure 2.11-9). CXR may show lung metastases; D&C reveals “cluster-of-grapes” tissue (see Figure 2.11-10). FIGURE 2.11-9. Molar pregnancy.

1	CXR may show lung metastases; D&C reveals “cluster-of-grapes” tissue (see Figure 2.11-10). FIGURE 2.11-9. Molar pregnancy. Transvaginal ultrasound shows a large, complex intrauterine mass with cystic regions that have the characteristic appearance of grapes.(Reproduced, with permission, from Tintinalli JE et al. Tintinalli’s Emergency Medicine: A Comprehensive Study Guide, 6th ed. New York: McGraw-Hill, 2004: Fig. 113-27.) Evacuate the uterus and follow with weekly β-hCG. Treat malignant disease with chemotherapy (methotrexate or dactinomycin) and residual uterine disease with hysterectomy; chemotherapy and irradiation are highly effective for metastases. F IGU R E 2.1 1 -1 0. Gross specimen of hydatidiform mole.

1	F IGU R E 2.1 1 -1 0. Gross specimen of hydatidiform mole. A complete or classical hydatidiform mole is characterized grossly by an abundance of edematous enlarged chorionic villi but no fetus or fetal membranes. There are theca-lutein cysts in both ovaries (arrows). (Reproduced, with permission, from Cunningham FG et al. Williams Obstetrics, 22nd ed. New York: McGraw-Hill, 2005: Fig.11-1.) Molar pregnancy may progress to malignant GTD, including invasive moles (10–15%) and choriocarcinoma (2–5%) with pulmonary or CNS metastases. Trophoblastic pulmonary emboli may also be seen. Affect 3% of all live births. Since 1980, there has been a 65% ↑ in the frequency of dizygotic (fraternal) twins and a 500% ↑ in triplet and high-order births (most due to the advent of assisted reproductive technology). The incidence of monozygotic (identical) twins has remained steady.

1	Hx/PE: Characterized by rapid uterine growth, excessive maternal weight gain, and palpation of three or more large fetal parts on Leopold’s maneuvers. Dx: Ultrasound; hCG, HPL, and MSAFP are elevated for GA. Tx: Multifetal reduction and selective fetal termination is an option for higher-order multiple pregnancies; antepartum fetal surveillance for IUGR. Management by a high-risk specialist is recommended. Cx: Maternal: Patients are six times more likely to be hospitalized with complications—e.g., preeclampsia, preterm labor, preterm PROM (PPROM), placental abruption, pyelonephritis, and postpartum hemorrhage. Fetal: Complications include twin-to-twin transfusion syndrome, IUGR, and preterm labor. Affects 0.6–1.4% of all deliveries in the United States. Risk factors include obesity, diabetes, a history of a macrosomic infant, and a history of prior shoulder dystocia.

1	Affects 0.6–1.4% of all deliveries in the United States. Risk factors include obesity, diabetes, a history of a macrosomic infant, and a history of prior shoulder dystocia. Diagnosed by a prolonged second stage of labor, recoil of the perineum (“turtle sign”), and lack of spontaneous restitution. In the event of dystocia, be the mother’s HELPER: Help reposition. Episiotomy. Leg elevated (McRoberts’ maneuver). Pressure (suprapubic). Enter the vagina and attempt rotation (Wood’s screw). Reach for the fetal arm. Failure to Progress Associated with chorioamnionitis, occiput posterior position, nulliparity, and elevated birth weight. First-stage protraction or arrest: Labor that fails to produce adequate rates of progressive cervical change. Prolonged second-stage arrest: Nulliparous: Inadequate cervical dilation after > 3 hours with regional anesthesia; > 2 hours without. Multiparous: Inadequate cervical dilation after > 2 hours with regional anesthesia; > 1 hour without.

1	Multiparous: Inadequate cervical dilation after > 2 hours with regional anesthesia; > 1 hour without. See Table 2.11-16. Chorioamnionitis leads to fetal infection, pneumonia, and bacteremia. Some 10% of those affected have permanent injury, 11% have a risk of postpartum hemorrhage, and 3.8% are at risk of fourth-degree laceration. Rupture of Membranes (ROM) Distinguished as follows: Spontaneous ROM: Occurs > 1 hour before onset of labor. May be precipitated by vaginal or cervical infections, abnormal membrane physiology, or cervical incompetence. PPROM: Rupture of membranes occurring at < 37 weeks’ gestation. Prolonged ROM: Defned as rupture > 18 hours prior to delivery. Risk factors include low socioeconomic status (SES), young maternal age, smoking, and STDs. Patients often report a “gush” of clear or blood-tinged amniotic ﬂ uid. Uterine contractions may be present. TABLE 2.11-16. Failure to Progress

1	Patients often report a “gush” of clear or blood-tinged amniotic ﬂ uid. Uterine contractions may be present. TABLE 2.11-16. Failure to Progress First Latent Active Failure to have progressive cervical change: ■Prima: > 20 hours ■Multi: > 14 hours ■Prima: > 2 hrs ■Multi: > 2 hrs after reaching 3–4 cm Second Arrest of fetal descent: ■Prima: > 2 hrs; > 3 hours with epidural ■Multi: > 1 hr; > 2 hrs with epidural Close observation with ↓ in epidural rate and continued oxytocin. Assisted vaginal delivery (forceps or vacuum). C-section. Therapeutic rest via parenteral analgesia; oxytocin; amniotomy; cervical ripening. Amniotomy; oxytocin; C-section if the previous interventions are ineffective. a Augmentation with oxytocin should be considered when contraction frequency is < 3 in a 10-minute period or intensity of contraction is < 25 mmHg above baseline. To minimize the risk of infection, do not perform digital vaginal exams on women with PROM.

1	To minimize the risk of infection, do not perform digital vaginal exams on women with PROM. Preterm labor = regular uterine contractions + concurrent cervical change at < 37 weeks’ gestation. First step: A sterile speculum exam reveals pooling of amniotic fuid in the vaginal vault. Nitrazine paper test is (paper turns blue, indicating alkaline pH of amniotic f uid). Fern test is (a ferning pattern is seen under a microscope after amniotic fuid dries on a glass slide). Second step: Ultrasound to assess amniotic f uid volume. If unsure: Ultrasound-guided transabdominal instillation of indigo carmine dye to check for leakage (unequivocal test). Minimize infection risk; do not perform digital vaginal exams on women who are not in labor or for whom labor is not planned immediately. Check fetal heart tracing, maternal temperature, WBC count, and uterine tenderness for evidence of chorioamnionitis. Depends on GA and fetal lung maturity.

1	Check fetal heart tracing, maternal temperature, WBC count, and uterine tenderness for evidence of chorioamnionitis. Depends on GA and fetal lung maturity. Term: First check GBS status and fetal presentation; then labor may be induced or the patient can be observed for 24–72 hours. > 34–36 weeks’ gestation: Labor induction may be considered. < 32 weeks’ gestation: Expectant management with bed rest and pelvic rest. Antibiotics: Given to prevent infection and to prolong the latency period in the absence of infection. Antenatal corticosteroids (e.g., betamethasone or dexamethasone × 48 hours): Can be given to promote fetal lung maturity in the absence of intra-amniotic infection prior to 32 weeks’ GA. If signs of infection or fetal distress develop, give antibiotics (ampicillin and gentamicin) and induce labor. Preterm labor and delivery, chorioamnionitis, placental abruption, cord prolapse. Defned as onset of labor between 20 and 37 weeks’ gestation.

1	Preterm labor and delivery, chorioamnionitis, placental abruption, cord prolapse. Defned as onset of labor between 20 and 37 weeks’ gestation. Occurs in > 10% of all U.S. pregnancies and is the 1° cause of neonatal morbidity and mortality. Risk factors include multiple gestation, infection, PROM, uterine anomalies, previous preterm labor or delivery, polyhydramnios, placental abruption, poor maternal nutrition, and low SES. Most patients have no identifiable risk factors. Patients may have menstrual-like cramps, onset of low back pain, pelvic pressure, and new vaginal discharge or bleeding. ■ Requires regular uterine contractions (≥ 3 contractions of 30 seconds each over a 30-minute period) and concurrent cervical change at < 37 weeks’ gestation. Assess for contraindications to tocolysis (e.g., infection, nonreassuring fetal testing, placental abruption). Perform a sterile speculum exam to rule out PROM.

1	Assess for contraindications to tocolysis (e.g., infection, nonreassuring fetal testing, placental abruption). Perform a sterile speculum exam to rule out PROM. Obtain an ultrasound to rule out fetal or uterine anomalies, verify GA, and assess fetal presentation and amniotic f uid volume. Obtain cultures for chlamydia, gonorrhea, and GBS. Obtain a UA and urine culture. Hydration and bed rest. Unless contraindicated, begin tocolytic therapy (β-mimetics, MgSO4, CCBs, PGIs) and give steroids to accelerate fetal lung maturation. Give penicillin or ampicillin for GBS prophylaxis if preterm delivery is likely. RDS, intraventricular hemorrhage, PDA, necrotizing enterocolitis, retinopathy of prematurity, bronchopulmonary dysplasia, death.

1	RDS, intraventricular hemorrhage, PDA, necrotizing enterocolitis, retinopathy of prematurity, bronchopulmonary dysplasia, death. Defned as any presentation other than vertex (i.e., head closest to birth canal, chin to chest, occiput anterior). Risk factors include prematurity, prior breech delivery, uterine anomalies, polyor oligohydramnios, multiple gestations, PPROM, hydrocephalus, anencephaly, and placenta previa. Breech presentations are the most common form (affect 3% of all deliveries) and involve presentation of the fetal lower extremities or buttocks into the maternal pelvis (see Figure 2.11-11). Subtypes include the following: Frank breech (50–75%): The thighs are fexed and the knees are extended. Footling breech (20%): One or both legs are extended below the buttocks. Complete breech (5–10%): The thighs and knees are f exed. Follow: Up to 75% spontaneously change to vertex by week 38.

1	Footling breech (20%): One or both legs are extended below the buttocks. Complete breech (5–10%): The thighs and knees are f exed. Follow: Up to 75% spontaneously change to vertex by week 38. External version: If the fetus has not reverted spontaneously, a version may be attempted by applying directed pressure to the maternal abdomen to turn the infant to vertex. The success rate is roughly 50%. Risks of version are placental abruption and cord compression, so be prepared for an emergency C-section if needed. Trial of breech vaginal delivery: Attempt only if delivery is imminent; otherwise contraindicated. Complications include cord prolapse and/or head entrapment. Elective C-section: Recommended given the lower risk of fetal morbidity. Breech presentation is the most common fetal malpresentation. See Table 2.11-17 for indications. For both elective and indicated cesarean delivery, sodium citrate should be used to ↓ gastric acidity and prevent acid aspiration syndrome.

1	See Table 2.11-17 for indications. For both elective and indicated cesarean delivery, sodium citrate should be used to ↓ gastric acidity and prevent acid aspiration syndrome. FIGURE 2.11-11. Types of breech presentations. (Reproduced, with permission, from DeCherney AH. Current Obstetric & Gynecologic Diagnosis & Treatment, 8th ed. Stamford, CT: Appleton & Lange, 1994: 411.) TABLE 2.11-17. Indications for Cesarean Section Surgical extension of the vaginal opening into the perineum. There are two types: median (midline) and mediolateral. Complications include extension to the anal sphincter (third degree) or rectum (fourth degree), which is more common with midline episiotomy, as well as bleeding, infection, dyspareunia, and, in rare cases, rectovaginal fstula formation or maternal death. Routine use of episiotomy is not recommended.

1	Routine use of episiotomy is not recommended. Defned as a loss of > 500 mL of blood for vaginal delivery or > 1000 mL for C-section occurring before, during, or after delivery of the placenta. Table 2.11-18 summarizes common causes. Complications include acute blood loss (potentially fatal), anemia due to chronic blood loss (predisposes to puerperal infection), and Sheehan’s syndrome (pituitary ischemia and necrosis; the 1° cause of anterior pituitary insuffciency in adult females, most commonly presenting as failure to lactate). T AB LE 2.1 1 -1 8. Common Causes of Postpartum Hemorrhage

1	Risk factors Uterine overdistention (multiple gestation, macrosomia, polyhydramnios). Exhausted myometrium (rapid or prolonged labor, oxytocin stimulation). Uterine infection. Conditions interfering with contractions (anesthesia, myomas, MgSO4). Precipitous labor. Operative vaginal delivery (forceps, vacuum extraction). Large infant. Inadequate episiotomy repair. Placenta accreta/increta/percreta. Placenta previa. Uterine leiomyomas. Preterm delivery. Previous C-section/ curettage. Diagnosis Palpation of a soft, enlarged, “boggy” uterus. The most common cause of postpartum hemorrhage (90%). Manual and visual inspection of the lower genital tract for any laceration > 2 cm long. Manual and visual inspection of the placenta and uterine cavity for missing cotyledons. Ultrasound may also be used to inspect the uterus. Treatmenta Bimanual uterine massage (usually successful). Oxytocin infusion. Methergine (methylergonovine) if not hypertensive. Prostaglandin (PGF2a). Surgical repair of the

1	to inspect the uterus. Treatmenta Bimanual uterine massage (usually successful). Oxytocin infusion. Methergine (methylergonovine) if not hypertensive. Prostaglandin (PGF2a). Surgical repair of the physical defect. Manual removal of remaining placental tissue. Curettage with suctioning (take care not to perforate the uterine fundus). a For all uterine causes, when bleeding persists after conventional therapy, uterine/internal iliac artery ligation or hysterectomy can be lifesaving.

1	Postpartum endometritis: The 7 W’s of postpartum fever (10 days postdelivery): Womb (endomyometritis) Wind (atelectasis, pneumonia) Water (UTI) Walk (DVT, pulmonary embolism) Wound (incision, episiotomy) Weaning (breast engorgement, abscess, mastitis) Breastfeeding is contraindicated in maternal HIV infection, active hepatitis, and use of certain medications. Characterized by a temperature ≥ 38°C for at least two of the first ten postpartum days (not including the first 24 hours). Risk factors for postpartum endometritis include emergent C-section, PROM, prolonged labor, multiple intrapartum vaginal exams, intrauterine manipulations, delivery, low SES, young age, prolonged ruptured membranes, bacterial colonization, and corticosteroid use. For endometritis, hospitalize and give broad-spectrum empiric IV antibiotics (e.g., clindamycin and gentamicin) until patients have been afebrile for 48 hours (24 hours for chorioamnionitis). Add ampicillin for complicated cases.

1	For persistent postpartum fever that is not responsive to broad-spectrum antibiotics, think septic pelvic thrombophlebitis, in which pelvic infection leads to infection of the vein wall and intimal damage, leading in turn to thrombogenesis. The clot is then invaded by microorganisms. Suppuration follows, with liquefaction, fragmentation, and, finally, septic embolization. Presents with abdominal and back pain and a picket-fence fever curve (“hectic” fevers) with wide swings from normal to as high as 41°C (105.8°F). Diagnose with blood cultures and CT looking for a pelvic abscess. Treat with broad-spectrum antibiotics and anticoagulation with heparin × 7–10 days. Defned as pituitary ischemia and necrosis that leads to anterior pituitary insuffciency 2° to massive obstetric hemorrhage and shock. Hx/PE: The 1° cause of anterior pituitary insuffciency in adult females. The most common presenting syndrome is failure to lactate (due to ↓ prolactin levels).

1	Hx/PE: The 1° cause of anterior pituitary insuffciency in adult females. The most common presenting syndrome is failure to lactate (due to ↓ prolactin levels). Other symptoms include weakness, lethargy, cold insensitivity, genital atrophy, and menstrual disorders. Dx: The diagnosis is established with provocative hormonal testing and MRI of the pituitary and hypothalamus to rule out tumor or other pathology. Tx: Treatment consists of the replacement of all defcient hormones. However, some patients may recover TSH and even gonadotropin function after cortisol replacement alone. During pregnancy, ↑ estrogen and progesterone result in breast hypertrophy and inhibition of prolactin release. After delivery of the placenta, hormone levels ↓ markedly and prolactin is released, stimulating milk production. Periodic infant suckling leads to further release of prolactin and oxytocin, which stimulate myoepithelial cell contraction and milk ejection (“let-down ref ex”).

1	Colostrum (“early breast milk”) contains protein, fat, secretory IgA, and minerals. Within one week postpartum, mature milk with protein, fat, lactose, and water is produced. High IgA levels in colostrum provide passive immunity for the infant and protect against enteric bacteria. Other benef ts include ↓ incidence of infant allergies, early upper respiratory tract infections, and GI infections; facilitation of mother-child bonding; and maternal weight loss. Contraindications to breastfeeding include HIV infection, active HBV and HCV infection, and use of certain medications (e.g., tetracycline, chloramphenicol, warfarin). Cellulitis of the periglandular tissue caused by nipple trauma from breastfeeding coupled with the introduction of bacteria, usually S. aureus, from the infant’s pharynx into the nipple ducts. Affects 2–3% of nursing women. Symptoms often begin 2–4 weeks postpartum.

1	Symptoms often begin 2–4 weeks postpartum. Breast symptoms are usually unilateral and include breast tenderness, a palpable mass, erythema, edema, warmth, and possible purulent nipple drainage. Signifcant fever, chills, and malaise may also be seen. Differentiate from simple breast swelling. Infection is suggested by focal symptoms, a breast milk culture, an ↑ WBC count, and fever. ■ First step: Continued breastfeeding to prevent the accumulation of infected material (or use of a breast pump in patients who are no longer The treatment of mastitis breastfeeding) and PO antibiotics (dicloxacillin, cephalexin, amoxicillin/ clavulanate, azithromycin, clindamycin). ■ Next step: If abscess is present, treat with incision and drainage. continued breastfeeding. Menarche and Normal Female Development 359 Normal Menstrual Cycle 359 Menopause 360 Contraception 361 Sexual Assault 361 Abnormalities of the Menstrual Cycle 364

1	Menarche and Normal Female Development 359 Normal Menstrual Cycle 359 Menopause 360 Contraception 361 Sexual Assault 361 Abnormalities of the Menstrual Cycle 364 Breast development (thelarche) precedes menarche and usually begins between the ages of 8 and 11. Menarche usually occurs between the ages of 10 and 16. Figure 2.12-1 graphically illustrates the stages of normal female development. The progression of a normal menstrual cycle is as follows (see also Figure 2.12-2): Follicular phase (days 1–13): Typically lasts about 13 days but may vary. ↑ FSH → growth of follicles → ↑ estrogen production. Results in the development of straight glands and thin secretions of the uterine lining (proliferative phase). Ovulation (day 14): LH and FSH spike results in rupture of the ovarian follicle and release of a mature ovum. Ruptured follicular cells involute and create the corpus luteum.

1	Ovulation (day 14): LH and FSH spike results in rupture of the ovarian follicle and release of a mature ovum. Ruptured follicular cells involute and create the corpus luteum. Luteal phase (days 15–28): This phase is the length of time (14 days) that the corpus luteum can survive without further LH stimulation. HEIGHT SPURT GROWTH RATE AGE RANGE PEAK Height 3 in/y Weight 17.5 lb/y 11.5–16.5 yWeight 6 lb/y AGE RANGE 10-16.5 y MENARCHE Average Height 62.5 in (158.5 cm) Average Weight 106 lb (48 kg) RATING PUBIC HAIR Initial hair is straight and fine. AGE RANGE 8–14 years Pubic hair becomes coarse, darkens, and spreads. Hair looks like an adult’s but limited in area. Inverted triangular pattern is established. AGE RANGE 12.5–16.5 years F IGU R E 2.1 2-1. Normal female development. (Reproduced, with permission, from Hay WW Jr et al. Current Diagnosis & Treatment: Pediatrics, 19th ed. New York: McGraw-Hill, 2008: Fig. 3-4.)

1	F IGU R E 2.1 2-1. Normal female development. (Reproduced, with permission, from Hay WW Jr et al. Current Diagnosis & Treatment: Pediatrics, 19th ed. New York: McGraw-Hill, 2008: Fig. 3-4.) Currently, HRT is not recommended as f rst-line treatment for menopausal symptoms. (Reproduced, with permission, from Fauci AS et al. Harrison’s Principles of Internal Medicine, 17th ed. New York: McGraw-Hill, 2008: Fig. 341-8.) The corpus luteum produces estrogen and progesterone, allowing the endometrial lining to develop thick endometrial glands with thick secretions (secretory phase). In the absence of implantation, the corpus luteum cannot be sustained, and the endometrial lining sloughs off. Cessation of menses for a minimum of 12 months as a result of cessation of follicular development. Average age of onset is 51. “Premature menopause” is defned as ovarian failure and menstrual cessation before age 40. The mnemonic HAVOC lists prominent features of menopause.

1	The mnemonic HAVOC lists prominent features of menopause. Other symptoms include insomnia, anxiety/irritability, vaginal bleeding, poor concentration, mood changes, dyspareunia, and loss of libido. Labs f rst show ↑ FSH and then show ↑ LH. DEXA scan to follow bone density for osteoporosis. Lipid prof le (↑ total cholesterol, ↓ HDL). ■ Vasomotor symptoms: ■HRT (combination estrogen and progestin): HRT has been shown to ↑ cardiovascular morbidity and mortality and may ↑ the incidence of breast and endometrial cancers. For this reason, clinicians should thoroughly review the risks and benef ts of HRT before initiating treatment. Posthysterectomy patients do not need progestin. Unopposed estrogen in patients with a uterus predisposes to endometrial cancer. Contraindications to HRT include vaginal bleeding, suspected or known breast cancer, endometrial cancer, and a history of thromboembolism, chronic liver disease, or hypertriglyceridemia.

1	Contraindications to HRT include vaginal bleeding, suspected or known breast cancer, endometrial cancer, and a history of thromboembolism, chronic liver disease, or hypertriglyceridemia. Non-HRT: Venlafaxine and, less commonly, clonidine can be given to ↓ the frequency of hot f ashes. Vaginal atrophy: Long term: Estradiol vaginal ring. Short term: Estrogen vaginal cream will relieve symptoms. Once a woman is Osteoporosis: Treat with daily calcium supplementation and exercise; pos postmenopausal, she should sibly bisphosphonates. osteoporosis. ■Of women who are sexually active and do not use a contraceptive method, 85% will become pregnant within one year. Absolute contraindications to various methods are as follows: Estrogen-containing hormonal methods (OCPs, NuvaRing, “the patch”): A history of CAD or DVT Mirena and Copper T IUDs: PID that is active (within three months) or recurrent A Pap smear with squamous intraepithelial lesions or two atypical Pap smears

1	A history of CAD or DVT Mirena and Copper T IUDs: PID that is active (within three months) or recurrent A Pap smear with squamous intraepithelial lesions or two atypical Pap smears A history of heart valve replacement or artif cial joints Copper T alone: Copper intolerance (allergy to copper, Wilson’s disease); severe dysmenorrhea and/or menorrhagia. Mirena alone: Levonorgestrel allergy, breast cancer, acute liver disease or liver tumor, or a history of BRCA. Table 2.12-1 describes the effectiveness of contraceptive methods along with their relative advantages and disadvantages. Emergency contraception (EC) methods prevent pregnancy after unprotected sex or contraceptive failure. Table 2.12-2 describes the various methods of EC. More than one sexual partner and nulliparity are not absolute contraindications to IUD use. The most effective methods of contraception are longer-term methods that ↓ user error.

1	More than one sexual partner and nulliparity are not absolute contraindications to IUD use. The most effective methods of contraception are longer-term methods that ↓ user error. Combined hormonal methods of contraception protect against endometrial, ovarian, and breast cancer. Sexual assault is the most frequently unreported crime in the United States. Physicians are often required to evaluate rape victims and collect evidence. Most rape victims are women; however, men may also be victims of rape. ■Take a full history, including contraceptive use, last time of coitus, condom use prior to the assault, drug or alcohol use, history of STDs, history of mental illness or defciency, description of the assailant, location and T AB LE 2.1 2-1. Contraceptive Methods

1	Most effective: > 99% (1 in 100 women will become pregnant using these methods) Implanon (“the implant”): Progestin-only implant Lasts up to three years. Immediate fertility with removal. Safe with breastfeeding. Associated with weight gain, depression, irregular bleeding (no period; spotting; heavier, more frequent periods). Intrauterine device with progestin (Mirena): Inf ammation from foreign body; cervical thickening and endometrial decidualization with progesterone Effective for up to f ve years. Lighter periods; less cramping. Immediate fertility with removal. Safe with breastfeeding. Spotting (up to six months). Associated with acne. Risk of uterine puncture (1/1000). Intrauterine device— ParaGard (Copper T): Infammation from foreign body; copper exerts a spermicidal effect Effective for up to 10 years. Immediate fertility with removal. Safe with breastfeeding. ↑ cramping and bleeding (5–10%). Risk of uterine puncture (1/1000). Surgical sterilization (vasectomy, tubal ligation)

1	for up to 10 years. Immediate fertility with removal. Safe with breastfeeding. ↑ cramping and bleeding (5–10%). Risk of uterine puncture (1/1000). Surgical sterilization (vasectomy, tubal ligation) Permanently effective. Safe with breastfeeding. Tubal ligation: Irreversible; ↑ ectopic pregnancy. Vasectomy: Most failures are due to not waiting for two semen samples. Very effective: 90–99% (1–10 in 100 women will become pregnant using these methods) Depo-Provera (medroxy-progesterone): IM injection every three months Lighter or no periods. Each shot works for three months. Safe with breastfeeding. Associated with irregular bleeding and weight gain. Decreases in bone mineral density (reversible). Delayed fertility after discontinuation (one shot can last 10 months). Ortho Evra (“the patch”): Combined weekly estrogen and progestin dermal patch Can make periods more regular. Not administered daily. Risk of thromboembolism (especially for smokers and those > 35 years of age). NuvaRing

1	Combined weekly estrogen and progestin dermal patch Can make periods more regular. Not administered daily. Risk of thromboembolism (especially for smokers and those > 35 years of age). NuvaRing (“the ring”): Combined low-dose progestin and an estrogen vaginal ring Can make periods more regular. Can improve acne. Used continuously for three weeks; then one week without the ring. Safe to use continuously to skip periods. May ↑ vaginal discharge. Spotting occurs in the frst 1–2 months.

1	T AB LE 2.1 2-1. Contraceptive Methods (continued)

1	Oral contraceptive pills (combination estrogen and progestin): Inhibit FSH/LH, suppressing ovulation; thicken cervical mucus; decidualize endometrium ↓ risk of ovarian and endometrial cancers.a Predictable, lighter, less painful menses. Often improve acne. Immediate fertility upon cessation. Require daily compliance. Breakthrough bleeding (10–30%). Risk of thromboembolism (especially for smokers and those > 35 years of age). Progestin-only “minipills”: Thicken cervical mucus Safe with breastfeeding. Requires strict compliance (taking pill at the same time each day). Moderately effective: 75–90% (up to 10–25 women in 100 will become pregnant using these methods) Male condoms: A latex sheath covers the penis The only method that effectively protects against pregnancy and STDs, including HIV. Possible allergy to latex or spermicides. Diaphragm with spermicide Some protection against STDs. Must be ftted by provider. Female condom Some protection against STDs. Can be clumsy and diff cult.

1	Possible allergy to latex or spermicides. Diaphragm with spermicide Some protection against STDs. Must be ftted by provider. Female condom Some protection against STDs. Can be clumsy and diff cult. Fertility awareness methods No side effects. Often used by women who have a cultural, religious, or personal preference for “natural” contraception. Requires partner’s participation. No STD/HIV protection. Less effective: 68–74% (26–32 women in 100 will become pregnant using these methods) Withdrawal No side effects. No STD/HIV protection. Spermicide Frequent use may protect against HIV. May be used as a 2° method. Not recommended as a 1° method. a Other combined hormonal methods (e.g., patch, ring) may also protect against endometrial, breast, and ovarian cancer; however, data are still lacking given their relatively recent introduction.

1	time of the assault, circumstances of the assault (e.g., penile penetration, use of condoms, extragenital acts, use or display of weapons), and the patient’s actions since the assault (e.g., douching, bathing, brushing teeth, urination/defecation, changing clothes). ■Conduct a complete physical exam, making note of any signs of trauma, along with a detailed pelvic exam, including a survey of the external genitals, vagina, cervix, and anus. Saline prep for sperm. Gonorrhea and chlamydia smear/culture (including rectal if appropriate).

1	Saline prep for sperm. Gonorrhea and chlamydia smear/culture (including rectal if appropriate). T AB LE 2.1 2-2. Emergency Contraceptive Methods “Morning-After Pill”—used within 120 hours of unprotected sex Combined estrogen/progestin (75% effective) Progestin only (80% effective) Available over the counter. Does not disrupt embryo postimplantation. Can be used as bridge contraception. Safe for all women. Same as above. Fewer nausea/vomiting side effects than combined EC. Nausea, vomiting, fatigue, headache, dizziness, breast tenderness. No protection against STDs. Same as above. Copper T IUD—used within 7 days of unprotected sex (99% effective) Can be used as EC and continued for up to 10 years of contraception. High initial cost of insertion. Must be inserted by provider. No protection against STDs. Serologic testing for HIV, syphilis, HSV, HBV, and CMV. Serum pregnancy test. Blood alcohol level; urine toxicology screen. STD treatment (ceftriaxone plus doxycycline).

1	Serologic testing for HIV, syphilis, HSV, HBV, and CMV. Serum pregnancy test. Blood alcohol level; urine toxicology screen. STD treatment (ceftriaxone plus doxycycline). HIV risk assessment and possible postexposure prophylaxis. EC for pregnancy prevention. Refer for psychological counseling. Arrange for follow-up with the same physician or with another provider if more appropriate. Follow-up should include repeat screening for STDs, repeat screening for pregnancy, and a discussion of coping methods with appropriate referrals for psychiatric care if needed. Pediatric vaginal discharge is caused by a variety of factors and may be normal, but STDs resulting from sexual abuse must be ruled out. Etiologies of vaginal discharge in pediatric patients include the following: Infectious vulvovaginitis: May present with a malodorous, yellow-green, purulent discharge; most often caused by group A streptococcus.

1	Infectious vulvovaginitis: May present with a malodorous, yellow-green, purulent discharge; most often caused by group A streptococcus. May also be caused by any STD resulting from sexual abuse (STDs must be ruled out and, if found, reported to child protective services). Foreign objects. Candidal infection: May be associated with diabetes; measure glucose and/or check for glycosuria. Sarcoma botryoides (rhabdomyosarcoma): A malignancy with lesions that have the appearance of “bunches of grapes” within the vagina. Onset of 2° sexual characteristics before the age of eight. Subtypes are as follows (see also Table 2.12-3): Central precocious puberty: Results from early activation of hypothalamic GnRH production. Most commonly idiopathic (also known as constitutional or true); may be related to obesity. May also be caused by CNS tumors. Peripheral precocious puberty: Also called pseudo-precocious puberty. Results from nonhypothalamic GnRH production.

1	Peripheral precocious puberty: Also called pseudo-precocious puberty. Results from nonhypothalamic GnRH production. Signs of estrogen excess (breast development and possibly vaginal bleeding) point to ovarian cysts or tumors. Signs of androgen excess (pubic and/or axillary hair, enlarged clitoris, acne, and/or ↑ body odor) suggest adrenal tumors or congenital adrenal hyperplasia (CAH). First step: Obtain a radiograph of the wrist and hand to determine bone age. If bone age is within one year of chronological age, puberty has not started or has just recently begun. If bone age exceeds chronological age by > 2 years, puberty has been present for at least one year or is progressing rapidly. Next step: Conduct a GnRH agonist (leuprolide) stimulation test. Central precocious puberty: If LH response is , obtain a cranial MRI to look for CNS tumors. In girls 6–8 years of age with signs of precocious puberty, the incidence of CNS tumor is 2% in the absence of other CNS signs.

1	In girls 6–8 years of age with signs of precocious puberty, the incidence of CNS tumor is 2% in the absence of other CNS signs. If CNS tumors are ruled out, constitutional precocious puberty is the likely etiology. Peripheral precocious puberty: If LH response is , order the following: Ultrasound of the ovaries and/or adrenals: To look for ovarian or adrenal cysts/tumors. Estradiol: Levels will be ↑ in ovarian cysts or tumors. T AB LE 2.1 2-3. Causes of Precocious Pubertal Development If onset of 2° sexual characteristics is seen by age eight, work up for precocious puberty by determining bone age and conducting a GnRH stimulation test to distinguish central from peripheral precocious puberty.

1	Constitutional (idiopathic) Hypothalamic lesions (hamartomas, tumors, congenital malformations) Dysgerminomas Hydrocephalus CNS infections CNS trauma/irradiation Pineal tumors (rare) Neurofbromatosis with CNS involvement Tuberous sclerosis Congenital adrenal hyperplasia Adrenal tumors McCune-Albright syndrome (polyostotic f brous dysplasia) Gonadal tumors Exogenous estrogen, oral (OCPs) or topical Ovarian cysts (females) Surgery is not required for the treatment of ambiguous genitalia in congenital adrenal hyperplasia; it is always cosmetic and may be deferred. Androgen (DHEA, DHEAS): Especially critical in the setting of advanced bone age or signs of adrenarche. 17-OH progesterone: To screen for advanced bone age or adrenarche. Central precocious puberty: Leuprolide is frst-line therapy. With treatment, physical changes regress or cease to progress. Peripheral precocious puberty: Treat the cause.

1	Central precocious puberty: Leuprolide is frst-line therapy. With treatment, physical changes regress or cease to progress. Peripheral precocious puberty: Treat the cause. Ovarian cysts: No intervention is necessary, as cysts will usually regress spontaneously. CAH: Treat with glucocorticoids. Surgery is not required for the treatment of ambiguous genitalia. Adrenal or ovarian tumors: Require surgical resection. McCune-Albright syndrome: Antiestrogens (tamoxifen) or estrogen synthesis blockers (ketoconazole or testolactone) may be effective. 1° amenorrhea is def ned as the absence of menses by age 16 with 2° sexual development present, or the absence of 2° sexual characteristics by age 14. Absence of 2° sexual characteristics (no estrogen production): Etiologies are as follows: Constitutional growth delay: The most common cause. 1° ovarian insuff ciency: Most commonly Turner’s syndrome. Look for a history of radiation and chemotherapy.

1	Constitutional growth delay: The most common cause. 1° ovarian insuff ciency: Most commonly Turner’s syndrome. Look for a history of radiation and chemotherapy. Central hypogonadism: May be caused by a variety of factors, including the following: Undernourishment, stress, prolactinemia, or exercise. CNS tumor or cranial irradiation. Kallmann’s syndrome (isolated gonadotropin def ciency) associated with anosmia. Presence of 2° sexual characteristics (evidence of estrogen production but other anatomic or genetic problems): Etiologies include the following: Mlerian agenesis: Absence of two-thirds of the vagina; uterine abnormalities. Imperforate hymen: Presents with hematocolpos (blood in the vagina) that cannot escape, along with a bulging hymen. Requires surgical opening. Complete androgen insensitivity: Patients present with breast development (aromatization of testosterone to estrogen) but are amenorrheic and lack pubic hair. First step: Get a pregnancy test.

1	Complete androgen insensitivity: Patients present with breast development (aromatization of testosterone to estrogen) but are amenorrheic and lack pubic hair. First step: Get a pregnancy test. Next step: Obtain a radiograph to determine if bone age is consistent with pubertal onset (> 12 years in girls). ■If the patient is of short stature (bone age < 12 years) with normal growth velocity, constitutional growth delay (the most common cause of 1° amenorrhea) is the probable cause. ■If bone age is > 12 years but there are no signs of puberty, obtain LH/ FSH and consider where the problem is on the HPA axis (see Figure 2.12-3). ■↓ GnRH, ↓ LH/FSH, ↓ estrogen/progesterone at prepuberty levels: Points to constitutional growth delay (puberty has not yet started). ■↓ GnRH, ↓ LH/FSH, ↓ estrogen/progesterone: Hypogonadotropic hypogonadism. Suggests a hypothalamic or pituitary problem.

1	■↓ GnRH, ↓ LH/FSH, ↓ estrogen/progesterone: Hypogonadotropic hypogonadism. Suggests a hypothalamic or pituitary problem. ■↑ GnRH, ↑ LH/FSH, ↓ estrogen/progesterone: Hypergonadotropic hypogonadism. Points to a condition in which the ovaries fail to produce estrogen. ■↑ GnRH, ↑ LH/FSH, high estrogen or testosterone: Suggests PCOS or a problem with estrogen receptors. ■Normal pubertal hormone levels: Indicates an anatomic problem (menstrual blood can’t get out). Constitutional growth delay: ↓ GnRH, ↓ LH/FSH, ↓ E/P at prepuberty levels (↓ GnRH, ↓ LH/FSH, ↓ E/P): (↓ GnRH, ↓ LH/FSH, ↓ E/P): Gonadotropin-releasing hormone (GnRH) Anorexia, excess exercise, weight loss, stress (GnRH deficiency) Sheehan’s syndrome Hypothalamus -Tumors, infection, trauma, Anorexia, excess exercise, Pituitary gland Hyperprolactinemia weight loss, stress Hypothyroidism

1	Sheehan’s syndrome Hypothalamus -Tumors, infection, trauma, Anorexia, excess exercise, Pituitary gland Hyperprolactinemia weight loss, stress Hypothyroidism Luteinizing hormone (LH) Follicle-stimulating hormone (FSH) Adrenal—CAH, Cushing’s Adrenal syndrome, Addison’s disease Pancreas gland Thyroid Other endocrine glands (↑ GnRH, ↑ LH/FSH, ↓ E/P): (chemotherapy, radiation, idiopathic) (gonadotropin-resistant (↑ GnRH, ↑ LH/FSH, ↓ E/P): ovary syndrome) Premature ovarian failure; 40(↑ GnRH, ↑ LH/FSH): -Menopause-Androgen insensitivity Chemotherapy with (↑ testosterone, ↑ estrogen) alkylating agents-PCOS (↑ estrogen, androgen) hydroxylase or aromatase) Primary anatomic: Secondary anatomic: Anovulatory problem: Normal hormone levels Normal hormone levels PCOS -Transverse vaginal septum to endometritis, scarring -Vaginal and/or cervical after delivery, or D&C agenesis Cervical stenosis F IGU R E 2.1 2-3. Causes of primary and secondary amenorrhea.

1	F IGU R E 2.1 2-3. Causes of primary and secondary amenorrhea. Ultrasound may be needed to evaluate the ovaries. Normal breast development and no uterus: Obtain a karyotype to evalu- The f rst step in the workup of 1° or 2° amenorrhea is a pregnancy test! ate for androgen insensitivity syndrome. Stigmata of Turner’s syndrome: Obtain a karyotype. Normal breast development and uterus: Measure prolactin and obtain a cranial MRI. Constitutional growth delay: No treatment is needed. Hypogonadism: Begin HRT with estrogen alone at the lowest dose. Twelve to eighteen months later, begin cyclic estrogen/progesterone therapy (if the uterus is present). Anatomic: Generally requires surgical intervention. Defned as the absence of menses for six consecutive months in women who have passed menarche. First step: Get a pregnancy test.

1	Anatomic: Generally requires surgical intervention. Defned as the absence of menses for six consecutive months in women who have passed menarche. First step: Get a pregnancy test. Second step: β-hCG: Measure TSH and prolactin. ■↑ TSH: Indicates hypothyroidism. ■↑ prolactin (inhibits the release of LH and FSH): Points to a thy roid pathology. Order an MRI of the pituitary to rule out tumor. ■↑↑ prolactin: Suggests a prolactin-secreting pituitary adenoma. Normal β-hCG: Initiate a progestin challenge (10 days of progestin). progestin challenge (withdrawal bleed): Indicates anovulation that is likely due to noncyclic gonadotropin secretion, pointing to PCOS or idiopathic anovulation. Check LH levels, and if LH is moderately high, the etiology is likely PCOS. Marked elevation of LH can indicate premature menopause. progestin challenge (no bleed): Indicates uterine abnormality or estrogen defciency. Check FSH levels. ■↑ FSH: Indicates hypergonadotropic hypogonadism/ovarian fail ure.

1	progestin challenge (no bleed): Indicates uterine abnormality or estrogen defciency. Check FSH levels. ■↑ FSH: Indicates hypergonadotropic hypogonadism/ovarian fail ure. ■↓ FSH: Obtain a cyclic estrogen/progesterone test. A withdrawal bleed points to hypogonadotropic hypogonadism; a withdrawal bleed suggests an endometrial or anatomic problem. Signs of hyperglycemia (polydipsia, polyuria) or hypotension: Conduct a 1-mg overnight dexamethasone suppression test to distinguish CAH, Cushing’s syndrome, and Addison’s syndrome. If clinical virilization is present: Measure testosterone, DHEAS, and 17–hydroxyprogesterone. Mild pattern: PCOS, CAH, or Cushing’s syndrome. Moderate to severe pattern: Look for an ovarian or adrenal tumor. ■ Hypothalamic: Reverse the underlying cause and induce ovulation with gonadotropins. Tumors: Excision; medical therapy for prolactinomas (e.g., bromocriptine, cabergoline).

1	■ Hypothalamic: Reverse the underlying cause and induce ovulation with gonadotropins. Tumors: Excision; medical therapy for prolactinomas (e.g., bromocriptine, cabergoline). Premature ovarian failure (age < 40 years): If the uterus is present, treat with estrogen plus progestin replacement therapy. Menstrual pain associated with ovulatory cycles in the absence of pathologic f ndings. Caused by uterine vasoconstriction, anoxia, and sustained contractions mediated by an excess of prostaglandin (PGF2α). Presents with low, midline, spasmodic pelvic pain that often radiates to the back or inner thighs. Cramps occur in the frst 1–3 days of menstruation and may be associated with nausea, diarrhea, headache, and f ushing. There are no pathologic fndings on pelvic exam. A diagnosis of exclusion. Rule out 2° dysmenorrhea (see workup below). NSAIDs; topical heat therapy; combined OCPs, Mirena IUD.

1	There are no pathologic fndings on pelvic exam. A diagnosis of exclusion. Rule out 2° dysmenorrhea (see workup below). NSAIDs; topical heat therapy; combined OCPs, Mirena IUD. Menstrual pain for which an organic cause exists. Common causes include endometriosis and adenomyosis, tumors, fbroids, adhesions, polyps, and PID. Look for pathology. Patients may have a palpable uterine mass, cervical motion tenderness, adnexal tenderness, a vaginal or cervical discharge, or visible vaginal pathology (mucosal tears, masses, prolapse). However, normal abdominal and pelvic exams do not rule out pathology. See Table 2.12-4 for distinguishing features of endometriosis vs. adenomyosis. First step: Obtain a β-hCG to exclude ectopic pregnancy. Second step: Order the following: A CBC with differential to rule out infection or neoplasm. UA to rule out UTI. Gonococcal/chlamydial swabs to rule out STDs/PID. Stool guaiac to rule out GI pathology.

1	A CBC with differential to rule out infection or neoplasm. UA to rule out UTI. Gonococcal/chlamydial swabs to rule out STDs/PID. Stool guaiac to rule out GI pathology. Third step: Look for pelvic pathology causing pain (see Table 2.12-4). Treatment is etiology specif c. T AB LE 2.1 2-4. Endometriosis vs. Adenomyosis

1	Def nition Functional endometrial glands and stroma outside the uterus. Endometrial tissue in the myometrium of the uterus. History/PE Presents with cyclical pelvic and/or rectal pain and dyspareunia (painful intercourse). Presents with the classic triad of noncyclical pain, menorrhagia, and an enlarged uterus. Diagnosis Requires direct visualization by laparoscopy or laparotomy. Classic lesions have a blue-black (“raspberry”) or dark brown (“powder-burned”) appearance. Ovaries may have endometriomas (the characteristic “chocolate cysts”). Ultrasound is useful but cannot distinguish between leiomyoma and adenomyosis. MRI can aid in diagnosis but is costly. Treatment Pharmacologic: Inhibit ovulation. Combination OCPs are frst line; other options include GnRH analogs (leuprolide) and danazol. Conservative surgical treatment: Excision, cauterization, or ablation of the lesions and lysis of adhesions. Twenty percent of patients can become pregnant subsequent to treatment. Defnitive

1	danazol. Conservative surgical treatment: Excision, cauterization, or ablation of the lesions and lysis of adhesions. Twenty percent of patients can become pregnant subsequent to treatment. Defnitive surgical treatment: TAH/BSO +/– lysis of adhesions. Pharmacologic: Largely symptomatic relief. NSAIDs (frst line) plus OCPs or progestins. Conservative surgical treatment: Endometrial ablation or resection using hysteroscopy. Complete eradication of deep adenomyosis is diffcult and results in high treatment failure. Defnitive surgical treatment: Hysterectomy is the only def nitive treatment. Complications Infertility (the most common cause among menstruating women > 30 years of age). Rarely, can progress to endometrial carcinoma.

1	Pregnancy is the most common cause of abnormal uterine bleeding and amenorrhea. Always check a pregnancy test! Normal menstrual bleeding ranges from two to seven days. Vaginal bleeding that occurs six or more months following the cessation of menstrual function is cancer related until proven otherwise. Assess the extent of bleeding: Menorrhagia: ↑ amount of fow (> 80 mL of blood loss per cycle) or prolonged bleeding (fow lasting > 8 days); may lead to anemia. Oligomenorrhea: An ↑ length of time between menses (35–90 days between cycles). Polymenorrhea: Frequent menstruation (< 21-day cycle); anovular. Metrorrhagia: Bleeding between periods. Menometrorrhagia: Excessive and irregular bleeding. Pelvic exam: Look for an enlarged uterus, a cervical mass, or polyps to assess for myomas, pregnancy, or cervical cancer. First step: Obtain a β-hCG to rule out ectopic pregnancy. Second step: Order a CBC to rule out anemia. Third step:

1	First step: Obtain a β-hCG to rule out ectopic pregnancy. Second step: Order a CBC to rule out anemia. Third step: Pap smear to rule out cervical cancer (which can present with bleeding). TFTs to rule out hyper-/hypothyroidism and hyperprolactinemia. Obtain platelet count, bleeding time, and PT/PTT to rule out von Willebrand’s disease and factor XI def ciency. Order an ultrasound to evaluate the ovaries, uterus, and endometrium. Look for uterine masses, polycystic ovaries, and thickness of the endometrium. If the endometrium is ≥ 4 mm in a postmenopausal woman, obtain an endometrial biopsy. An endometrial biopsy should also be obtained if the patient is > 35 years of age, obese (BMI > 35), and diabetic. Heavy bleeding: Since heavy or prolonged uterine bleeding has likely denuded the endometrial cavity, estrogen is needed to rapidly promote endometrial growth.

1	Heavy bleeding: Since heavy or prolonged uterine bleeding has likely denuded the endometrial cavity, estrogen is needed to rapidly promote endometrial growth. First step: For hemorrhage, high-dose estrogen IV stabilizes the endometrial lining and stops bleeding within one hour. Next step: If bleeding is not controlled within 12–24 hours, a D&C is indicated. Ovulatory bleeding: The goal is to ↓ blood loss. First step: NSAIDs to ↓ blood loss. Next step: If the patient is hemodynamically stable, treat with OCPs or a Mirena IUD to thicken the endometrium and control the bleeding. If this is not effective within 24 hours, look for an alternative diagnosis. Anovulatory bleeding: The goal is to convert proliferative endometrium to secretory endometrium. Give progestins × 10 days to stimulate withdrawal bleeding.

1	Anovulatory bleeding: The goal is to convert proliferative endometrium to secretory endometrium. Give progestins × 10 days to stimulate withdrawal bleeding. For young patients with anovulatory bleeding who may also have a bleeding disorder, give desmopressin followed by a rapid ↑ in von Willebrand’s factor and factor VIII (lasts roughly six hours). If medical management fails, options include the following: D&C: An appropriate diagnostic/therapeutic option. Hysteroscopy: Can help identify endometrial polyps as well as aid in the performance of directed uterine biopsies. Hysterectomy or endometrial ablation: Appropriate in women who fail or do not want hormonal treatment, have symptomatic anemia, and/or experience a disruption in their quality of life from persistent, unscheduled bleeding. First-line treatment of abnormal uterine bleeding consists of NSAIDs to ↓ blood loss! OCPs and the Mirena IUD are highly effective treatment options for menorrhagia.

1	First-line treatment of abnormal uterine bleeding consists of NSAIDs to ↓ blood loss! OCPs and the Mirena IUD are highly effective treatment options for menorrhagia. Complications of abnormal uterine bleeding are anemia and endometrial hyperplasia +/− carcinoma. A 21-hydroxylase defciency that can present in its most severe, classic form as a newborn female infant with ambiguous genitalia and life-threatening salt wasting (see the Endocrinology chapter for a discussion of early-onset CAH). Milder forms present later in life. 11β-hydroxylase defciency is a less common cause of adrenal hyperplasia. Presents with excessive hirsutism, acne, amenorrhea and/or abnormal uterine bleeding, infertility, and, rarely, a palpable pelvic mass. Hirsutism = male hair pattern. Virilization = frontal balding, muscularity, clitoromegaly, and deepening of the voice. Defeminization = ↓ breast size; loss of feminine adipose tissue.

1	Hirsutism = male hair pattern. Virilization = frontal balding, muscularity, clitoromegaly, and deepening of the voice. Defeminization = ↓ breast size; loss of feminine adipose tissue. The most severe form of PCOS is HAIR-AN syndrome: HyperAndrogenism, Insulin Resistance, and Acanthosis Nigricans. ■↑ androgens (testosterone > 2 ng; DHEAS > 7 μg/mL): Rule out adrenal or ovarian neoplasm. ■↑ serum testosterone: Suspect an ovarian tumor. ■↑ DHEAS: Suspect an adrenal source (adrenal tumor, Cushing’s syn drome, CAH). ■↑ 17-OH progesterone levels (either basally or in response to ACTH stimulation). ■Table 2.12-5 outlines the differential diagnosis of hyperandrogenism. Glucocorticoids (e.g., prednisone). Medical therapy for adrenal and ovarian disorders prevents new terminal hair growth but does not resolve hirsutism. Laser ablation, electrolysis, or conventional hair removal techniques must be used to remove unwanted hair.

1	One of the most common endocrine disorders in reproductive women, with a prevalence of 6–10% among U.S. women of reproductive age. Also known as Stein-Leventhal syndrome. Diagnosis requires two of the following three criteria: 1. 2. 3. Clinical or biochemical evidence of hyperandrogenism T AB LE 2.1 2-5. Differential Diagnosis of Hyperandrogenism

1	PCOS Irregular menses, slow-onset hirsutism, obesity, infertility, hypertension, a family history of PCOS or DM. Fasting glucose, insulin/lipid prof le, BP, ultrasound for ovarian cysts. 21-hydroxylase def ciency—nonclassic (late-onset) CAH Severe hirsutism or virilization, a strong family history of CAH, short stature, signs of defeminization. More common among Ashkenazi Jews. 17-hydroxyprogesterone (17-HP) levels before and after ACTH stimulation test > 10 ng/dL; CYP21 genotype. 21-hydroxylase def ciency—classic (congenital) CAH Same as above but with congenital virilization. 17-HP levels > 30 ng/dL. Hypothyroidism Fatigue, weight gain, amenorrhea. TSH. Hyperprolactinemia Amenorrhea, galactorrhea, infertility. Prolactin. Androgen-secreting neoplasm Pelvic mass, rapid-onset hirsutism or virilization, > 30 with onset of symptoms. Pelvic ultrasound or abdominal/pelvic CT. Cushing’s syndrome Hypertension, buffalo hump, purple striae, truncal obesity. Elevated BP plus dexamethasone

1	or virilization, > 30 with onset of symptoms. Pelvic ultrasound or abdominal/pelvic CT. Cushing’s syndrome Hypertension, buffalo hump, purple striae, truncal obesity. Elevated BP plus dexamethasone suppression test.

1	High BP. BMI > 30 (obesity). Stigmata of hyperandrogenism or insulin resistance (menstrual cycle disturbances, hirsutism, obesity, acne, androgenic alopecia, acanthosis nigricans). Women with PCOS are also at ↑ risk for the following: Metabolic syndrome—insulin resistance, obesity, atherogenic dyslipidemia, and hypertension Biochemical hyperandrogenemia: ↑ testosterone (total +/− free); DHEAS, DHEA. Exclude other causes of hyperandrogenism: TSH, prolactin. 17-OH progesterone to rule out nonclassical CAH. Consider screening in the setting of clinical signs of Cushing’s syndrome (e.g., moon facies, buffalo hump, abdominal striae) or acromegaly (e.g., ↑ head size). Evaluate for metabolic abnormalities: Two-hour oral glucose tolerance test. Fasting lipid and lipoprotein levels (total cholesterol, HDL, LDL, triglycerides). Optional tests:

1	Evaluate for metabolic abnormalities: Two-hour oral glucose tolerance test. Fasting lipid and lipoprotein levels (total cholesterol, HDL, LDL, triglycerides). Optional tests: Ultrasound: Look for > 8 small, subcapsular follicles forming a “pearl necklace” sign (see Figure 2.12-4). Seen in roughly two-thirds of women with PCOS. Gonadotropins: ↑ LH/FSH ratio (> 2:1). Fasting insulin levels. 24-hour urine for free cortisol: Adult-onset CAH or Cushing’s syndrome. F IGU R E 2.1 2-4. Polycystic ovary with prominent multiple cysts. (Reproduced, with permission, from DeCherney AH. Current Obstetric & Gynecologic Diagnosis & Treatment, 8th ed. Stamford, CT: Appleton & Lange, 1994: 747.) ↓ the risk of endometrial hyperplasia/carcinoma among women with PCOS. Women who are not attempting to conceive: Treat with a combination of OCPs, progestin, and metformin (or other insulin-sensitizing agents).

1	Women who are not attempting to conceive: Treat with a combination of OCPs, progestin, and metformin (or other insulin-sensitizing agents). Women who are attempting to conceive: Clomiphene +/− metformin is frst-line treatment for ovulatory stimulation. Symptom-specif c treatment: Hirsutism: Combination OCPs are frst line; antiandrogens (spironolactone, f nasteride) and metformin may also be used. Cardiovascular risk factors and lipid levels: Diet, weight loss, and exercise plus potentially lipid-controlling medication (e.g., statins). ↑ risk of early-onset type 2 DM; ↑ risk of miscarriages; ↑ long-term risk of breast and endometrial cancer due to unopposed estrogen secretion. Defned as inability to conceive after 12 months of normal, regular, unprotected sexual activity. 1° infertility is characterized by no prior pregnancies; 2° infertility occurs in the setting of at least one prior pregnancy. Etiologies are shown in Figure 2.12-5 and Table 2.11-6.

1	Cyst and Abscess of Bartholin’s Duct Obstruction of the gland leads to pain, swelling, and abscess formation. Infertility14% of reproductive aged women 5 million couples in the U.S. Female causes 58% Male causes 25% Unexplained 17% Amenorrhea/ ovulatory dysfunction 46% Hypothalamic/ pituitary causes 51% Polycystic ovary syndrome 30% Premature ovarian failure 12% Tubal defect 38% Primary hypogonadism ( FSH) 30–40% Secondary hypogonadism ( FSH, LH) 2% Disordered sperm transport 10–20% Unknown 40–50% Endometriosis 9% Other 7% Uterine or outflow tract disorders 7% FIGURE 2.12-5. Causes of infertility. (Reproduced, with permission, from Fauci AS et al. Harrison’s Principles of Internal Medicine, 17th ed. New York: McGraw-Hill, 2008: Fig. 341-9.) TABLE 2.12-6. Infertility Workup

1	Male causes Testicular injury or infection Medications (corticosteroids, cimetidine, spironolactone) Thyroid or liver disease Signs of hypogonadism Varicocele TSH Prolactin Karyotype (to rule out Klinefelter’s syndrome) Semen analysis Treatment of hormonal def ciency Intrauterine insemination (IUI) Donor insemination In vitro fertilization (IVF) Intracytoplasmic sperm injection Ovulatory factors Age (incidence ↑ with age) Symptoms of hyper-/ hypothyroidism Galactorrhea Menstrual cycle abnormalities Basal body temperature Ovulation predictor Midluteal progesterone Early follicular FSH +/– estradiol level (ovarian reserve) TSH, prolactin, androgens Ovarian sonography (antral follicle count) Endometrial biopsy (luteal phase defect) Treatment depends on the etiology (e.g., levothyroxine, dopamine) Induction of ovulation with clomiphene, gonadotropins, and pulsatile GnRH IUI IVF Tubal/ pelvic factors History of PID, appendicitis, endometriosis, pelvic adhesions, tubal surgery

1	dopamine) Induction of ovulation with clomiphene, gonadotropins, and pulsatile GnRH IUI IVF Tubal/ pelvic factors History of PID, appendicitis, endometriosis, pelvic adhesions, tubal surgery Hysterosalpingogram, endometrial biopsy Laparoscopic resection or ablation of endometriomas or f broids IVF Cervical factors Abnormal Pap smears, postcoital bleeding, cryotherapy, conization, or DES exposure in utero Pap smear Physical exam Antisperm antibodies IUI with washed sperm IVF

1	Presents with periodic painful swelling on either side of the introitus and dyspareunia. A fuctuant swelling 1–4 cm in diameter is seen in the inferior portion of either labium minus. Tenderness is evidence of active infection. Asymptomatic cysts do not require therapy. Frequent warm soaks may be helpful. If an abscess develops, treat with aspiration or incision and drainage. Culture for Chlamydia and other pathogens. Antibiotics are unnecessary unless cellulitis is present. A spectrum of conditions that cause vulvovaginal symptoms such as itching, burning, irritation, and abnormal discharge. The most common causes are bacterial vaginosis, vulvovaginal candidiasis, and trichomoniasis (see Table 2.12-7). Presents with a change in discharge, malodor, pruritus, irritation, burning, swelling, dyspareunia, and dysuria. Normal secretions are as follows: Midcycle estrogen surge: Clear, elastic, mucoid secretions.

1	Normal secretions are as follows: Midcycle estrogen surge: Clear, elastic, mucoid secretions. Luteal phase/pregnancy: Thick and white secretions; adhere to the vaginal wall.

1	TABLE 2.12-7. Causes of Vaginitis 15–50% (most common). 5–50%. 15–30%. Refects a shift in vaginal f ora. Protozoal f agellates (an STD). Usually Candida albicans. Risk factors Pregnancy, > 1 sexual partner, female sexual partner, frequent douching. An STD. Unprotected sex with multiple partners. DM, broad-spectrum antibiotic use, pregnancy, corticosteroids, HIV, OCP use, IUD use, young age at f rst intercourse, ↑ frequency of intercourse. History Odor, ↑ discharge. ↑ discharge, odor, pruritus, dysuria. Pruritus, dysuria, burning, ↑ discharge. Exam Mild vulvar irritation. “Strawberry petechiae” in the upper vagina/cervix (rare). Erythematous, excoriated vulva/ vagina. Discharge Homogenous, grayish-white, f shy/stale odor. Profuse, malodorous, yellow-green, frothy. Thick, white, curdy texture without odor. Wet mounta “Clue cells” (epithelial cells coated with bacteria; see Figure 2.12-6); shift in vaginal f ora (↑ cocci, ↓ lactobacilli). Motile trichomonads (fagellated organisms that

1	without odor. Wet mounta “Clue cells” (epithelial cells coated with bacteria; see Figure 2.12-6); shift in vaginal f ora (↑ cocci, ↓ lactobacilli). Motile trichomonads (fagellated organisms that are slightly larger than WBCs). — KOH prep whiff test (fshy smell). — Hyphae (see Figure 2.12–6). Treatment PO or vaginal metronidazole or clindamycin. Single-dose PO metronidazole or tinidazole. Treat partners; test for other STDs. Topical azole or PO f uconazole. Complications Chorioamnionitis/endometritis, infection, preterm delivery, miscarriage, PID. Same as for bacterial vaginosis. Oral azoles should be avoided in pregnancy. a If there are many WBCs and no organism on saline smear, suspect Chlamydia. Incidence Etiology

1	AB FIGURE 2.12-6. Causes of vaginitis. (A) Candidal vaginitis. Candida albicans organisms are evident on KOH wet mount. (B) Gardnerella vaginalis. Note the granular epithelial cells (“clue cells”) and indistinct cell margins. (Image A reproduced, with permission, from Wolff K et al. Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York: McGraw-Hill, 2005: 717. Image B reproduced, with permission, from Kasper DL et al. Harrison’s Principles of Internal Medicine, 16th ed. New York: McGraw-Hill, 2005: 767.) Conduct a thorough examination of the vulva, vaginal walls, and cervix. If there are many WBCs and no organism on saline smear, suspect Chlamydia. Samples from the speculum exam should be obtained for vaginal pH, amine (“whiff”) test, wet mount (with saline), and 10% hydroxide (KOH) microscopy.

1	Samples from the speculum exam should be obtained for vaginal pH, amine (“whiff”) test, wet mount (with saline), and 10% hydroxide (KOH) microscopy. To rule out cervicitis, DNA tests or cultures for Neisseria gonorrhoeae or Chlamydia trachomatis should be obtained in patients with a purulent discharge, numerous leukocytes on wet prep, cervical friability, and any symptoms of PID. Treatment is etiology specifc (see Table 2.12-7). Infammation of the uterine cervix. Because the female genital tract is contiguous from the vulva to the fallopian tubes, there is some overlap between vulvovaginitis and cervicitis. Etiologies are as follows: Infectious (most common): Chlamydia, gonococcus, Trichomonas, HSV, HPV. Noninfectious: Trauma, radiation exposure, malignancy. Hx/PE: Yellow-green mucopurulent discharge; cervical motion tenderness; absence of other signs of PID. Dx/Tx: See the discussion of STDs in the Infectious Disease chapter.

1	Hx/PE: Yellow-green mucopurulent discharge; cervical motion tenderness; absence of other signs of PID. Dx/Tx: See the discussion of STDs in the Infectious Disease chapter. Criteria for the clinical diagnosis of bacterial vaginosis (three of four are required): Vaginal pH > 4.5 Clue cells comprise > 20% of epithelial cells on wet mount A polymicrobial infection of the upper genital tract that is associated with Neisseria gonorrhoeae (one-third of cases), Chlamydia trachomatis (one-third of cases), and endogenous aerobes/anaerobes. The lifetime risk is 1–3%. Risk IUDs do not ↑ PID risk. The chandelier sign is def ned as severe cervical motion tenderness that makes the patient “jump for the chandelier” on exam. Mild and subclinical PID is a major cause of tubal factor infertility, ectopic pregnancy, and chronic pelvic pain due to pelvic scarring. factors include non-Caucasian ethnicity, douching, smoking, multiple sex partners, and prior STDs and/or PID.

1	factors include non-Caucasian ethnicity, douching, smoking, multiple sex partners, and prior STDs and/or PID. Presents with lower abdominal pain, fever and chills, menstrual disturbances, and a purulent cervical discharge. are also seen. Diagnosed by the presence of acute lower abdominal or pelvic pain plus one of the following: A WBC count > 10,000 has poor positive and negative predictive value for PID. Order a β-hCG and ultrasound to rule out pregnancy and to evaluate for the possibility of tubo-ovarian abscess. Ultrasound is a noninvasive means of diagnosing PID. Look for: Thickening or dilation of the fallopian tubes Fluid in the cul-de-sac Antibiotic treatment should not be delayed while awaiting culture results. All sexual partners should be examined and treated appropriately. Outpatient regimens: Regimen A: Of oxacin or levof oxacin × 14 days +/– metronidazole × 14 days.

1	Outpatient regimens: Regimen A: Of oxacin or levof oxacin × 14 days +/– metronidazole × 14 days. Regimen B: Ceftriaxone IM × 1 dose or cefoxitin plus probenecid plus doxycycline × 14 days +/– metronidazole × 14 days. Inpatient antibiotic regimens: Cefoxitin or cefotetan plus doxycycline × 14 days. Clindamycin plus gentamicin × 14 days. Surgery: Drainage of a tubo-ovarian/pelvic abscess is appropriate if the mass persists after antibiotic treatment; the abscess is > 4–6 cm; or the mass is in the cul-de-sac in the midline and drainable through the vagina. If the abscess is dissecting the rectovaginal septum and is fxed to the vaginal membrane, colpotomy drainage is appropriate. If the patient’s condition deteriorates, perform exploratory laparotomy. Surgery may range from TAH/BSO with lysis of adhesions in severe cases to conservative surgery for women who desire to maintain fertility.

1	Surgery may range from TAH/BSO with lysis of adhesions in severe cases to conservative surgery for women who desire to maintain fertility. Some 25% of women with acute disease develop repeated episodes of infection, chronic pelvic pain, dyspareunia, ectopic pregnancy, or infertility. RUQ pain (Fitz-Hugh–Curtis syndrome) may indicate an associated perihepatitis (abnormal liver function, shoulder pain). The risk of infertility ↑ with repeated episodes of salpingitis and is esti mated to approach 10% after the frst episode, 25% after the second episode, and 50% after a third episode. Caused by preformed S. aureus toxin (TSST-1); often occurs within f ve days of the onset of a menstrual period in women who have used tampons. The incidence in menstruating women is now 6–7:100,000 annually. Nonmenstrual cases are nearly as common as menstrual cases. Presents with abrupt onset of fever, vomiting, and watery diarrhea, with fever 38.9°C (102°F) or higher.

1	Presents with abrupt onset of fever, vomiting, and watery diarrhea, with fever 38.9°C (102°F) or higher. A diffuse macular erythematous rash is also seen. Nonpurulent conjunctivitis is common. Desquamation, especially of the palms and soles, generally occurs during recovery within 1–2 weeks of illness. Blood cultures are because symptoms result from preformed toxin and are not due to the invasive properties of the organism. Rapid rehydration. Antistaphylococcal drugs (nafcillin, oxacillin); vancomycin for women with penicillin allergy. Corticosteroids can reduce the severity of illness and ↓ fever. Manage renal or cardiac failure. The mortality rate associated with TSS is 3–6%. Three major causes of death are ARDS, intractable hypotension, and hemorrhage 2° to DIC.

1	Manage renal or cardiac failure. The mortality rate associated with TSS is 3–6%. Three major causes of death are ARDS, intractable hypotension, and hemorrhage 2° to DIC. TSS is a rare but potentially fatal reaction to S. aureus toxin. Diagnosis is clinical because reaction is to the toxin produced by the bacteria, not to the bacterium itself. The f rst steps in treatment are rapid rehydration and antibiotic treatment. Gynecologic cancers include uterine, endometrial, ovarian, cervical, and vulvar neoplasms. Ovarian cancer carries the highest mortality. The most common benign neoplasm of the female genital tract. The tumor is discrete, round, frm, and often multiple and is composed of smooth muscle and connective tissue. Tumors are estrogen and progesterone sensitive, so they often ↑ in size during pregnancy and ↓ after menopause. Malignant transformation to leiomyosarcoma is rare (0.1–0.5%). Prevalence is 25% among Caucasian women and 50% among African-American women.

1	HISTORY/PE Uterine myomas are benign The majority of cases are asymptomatic. Symptomatic patients may present with the following: menorrhagia. ■Bleeding: Longer, heavier periods; anemia. If a uterine mass continues to grow after menopause, malignancy must be ruled out with a biopsy. Eighty percent of women with endometrial carcinoma have vaginal bleeding, but only 5–10% of women with abnormal vaginal bleeding have endometrial cancer. Pressure: Pelvic pressure and bloating; constipation and rectal pressure; urinary frequency or retention. Pain: 2° dysmenorrhea, dyspareunia. Pelvic symptoms: A frm, nontender, irregular enlarged (“lumpybumpy”), or cobblestone uterus may be seen. CBC: To look for anemia. Ultrasound: To look for uterine myomas; can also exclude ovarian masses. MRI: Can delineate intramural and submucous myomas. Pharmacologic: NSAIDs. Combined hormonal contraception. Medroxyprogesterone acetate or danazol to slow or stop bleeding.

1	MRI: Can delineate intramural and submucous myomas. Pharmacologic: NSAIDs. Combined hormonal contraception. Medroxyprogesterone acetate or danazol to slow or stop bleeding. GnRH analogs (leuprolide or nafarelin) to ↓ the size of myomas, suppress further growth, and ↓ surrounding vascularity. Also used prior to surgery. Surgery: Emergent surgery is indicated for torsion of a pedunculated myoma. Women of childbearing years: Myomectomy or hysteroscopy with leiomyoma resection. Women who have completed childbearing: Total or subtotal abdominal or vaginal hysterectomy. Uterine artery embolization (~ 25% will need further invasive treatment). Infertility may be due to a myoma that distorts the uterine cavity and plays a role similar to that of an IUD.

1	Uterine artery embolization (~ 25% will need further invasive treatment). Infertility may be due to a myoma that distorts the uterine cavity and plays a role similar to that of an IUD. Type I endometrioid adenocarcinomas derive from atypical endometrial hyperplasia and are the most common female reproductive cancer in the United States (~35,000 cases/year). Type II cancers derive from serous or clear cell histology (see Table 2.12-8). Type I: Vaginal bleeding (early fnding); pain (late fnding); metabolic syndrome. Type II: No vaginal bleeding. Endometrial/endocervical biopsy. Vaginal ultrasound shows a thickened endometrium leading to hypertrophy and neoplastic change. TYPE I: ENDOMETRIOID TYPE II: SEROUS TABLE 2.12-8. Types of Endometrial Cancer

1	Vaginal ultrasound shows a thickened endometrium leading to hypertrophy and neoplastic change. TYPE I: ENDOMETRIOID TYPE II: SEROUS TABLE 2.12-8. Types of Endometrial Cancer Epidemiology 75% of endometrial cancers. 25% of endometrial cancers. Etiology Unopposed estrogen stimulation (e.g., tamoxifen use, exogenous estrogen-only therapy). Unrelated to estrogen; the p53 mutation is present in 90% of cases. Precursor lesion Hyperplasia and atypical hyperplasia. None. Mean age at diagnosis 55 years. 67 years. Prognosis Favorable. Poor. Type I: High-dose progestins for women of childbearing age; TAH/BSO +/– radiation for postmenopausal women. Type II: TAH/BSO with adjuvant chemotherapy for advanced-stage cancer.

1	Type I: High-dose progestins for women of childbearing age; TAH/BSO +/– radiation for postmenopausal women. Type II: TAH/BSO with adjuvant chemotherapy for advanced-stage cancer. The upper third of the cervix is made up of columnar cells (similar to the lower uterine segment). The lower two-thirds of the cervix is made up of squamous cells (similar to the vagina). The exposure of columnar cells to an acidic vaginal pH results in metaplasia to squamous cells. The normal squamocolumnar junction (transformation zone) is located in the ectocervix and can be exposed to carcinogens, resulting in cervical intraepithelial neoplasia (CIN), an abnormal proliferation or overgrowth of the basal cell layer. HPV DNA is found in 99.7% of all cervical carcinomas. HPV 16 is the most prevalent type in squamous cell carcinoma; HPV 18 is most prevalent in adenocarcinoma.

1	HPV DNA is found in 99.7% of all cervical carcinomas. HPV 16 is the most prevalent type in squamous cell carcinoma; HPV 18 is most prevalent in adenocarcinoma. Additional risk factors for cervical cancer include immunosuppression, infection with HIV or a history of STDs, tobacco use, high parity, and OCPs. The Gardasil vaccine may protect against HPV types 6, 11, 16, and 18 and may also prevent the development of cervical cancer. Metrorrhagia, postcoital spotting, and cervical ulceration are the most common signs. A bloody or purulent, malodorous, nonpruritic discharge may appear after invasion. ■Starting at age 21 or no more than three years after becoming sexually active, women should have a Pap smear with conventional cervical cytology on a yearly basis or liquid-based cervical cytology once every two years. Table 2.12-9 outlines the classifcation of Pap smear results. Hormonal contraceptives are protective against endometrial cancer.

1	Hormonal contraceptives are protective against endometrial cancer. Screening of asymptomatic women for endometrial cancer is not recommended. Fifty percent of women with cervical cancer had not had a Pap smear in the three years preceding their diagnosis, and another 10% had not been screened in f ve years. T AB LE 2.1 2-9. Classifcation Systems for Pap Smears 1 Benign Benign Normal 2 Benign with inf ammation Benign with inf ammation Normal, atypical squamous cells (ASC) 3 Mild dysplasia CIN I LSIL 3 Moderate dysplasia CIN II HSIL 3 Severe dysplasia CIN II HSIL 4 Carcinoma in situ CIN II HSIL 5 Invasive cancer Invasive cancer Invasive cancer Women ≥ 30 years of age who have had three consecutive normal tests may ↑ their screening interval to once every three years. For women ≥ 30 years of age, HPV DNA testing for high-risk strands may be used for screening as well.

1	For women ≥ 30 years of age, HPV DNA testing for high-risk strands may be used for screening as well. Screening should be discontinued for women ≥ 70 years of age who have had three or more normal Pap smears. Women with DES exposure and/or immunocompromised status (including HIV positivity) should continue as long as they do not have a life-limiting condition. Women who have had the HPV vaccine should continue cervical cancer screening according to established guidelines. Recent guidelines for the diagnosis and follow-up of cervical cancer distinguish women ≤ 21 from those > 21 years of age for all subtypes of cervical lesions except atypical glandular cells. The diagnosis and follow-up of specifc subtypes of cervical lesions should thus proceed as follows: Atypical glandular cells (AGC): < 35 years of age with no endometrial cancer risk factors: Proceed to colposcopy, endocervical curettage (ECC), and HPV DNA testing.

1	Atypical glandular cells (AGC): < 35 years of age with no endometrial cancer risk factors: Proceed to colposcopy, endocervical curettage (ECC), and HPV DNA testing. ≥ 35 years of age, endometrial cancer factors, or abnormal bleeding: Add an endometrial biopsy. Atypical squamous cells of undetermined signif cance (ASC-US): ≤ 21 years of age: Repeat Pap smear at 12 months. If Pap smear is or reveals ASC-US or LSIL, repeat at 12 months. > 21 years of age: Immediate colposcopy, HPV DNA testing, and repeat Pap smear at 6 months. Low-grade squamous intraepithelial lesions (LSIL): ≤ 21 years of age: Same as ASC-US. > 21 years of age: Immediate colposcopy. High-grade squamous intraepithelial lesions (HSIL) or atypical squamous cells suspicious of high-grade dysplasia (ASC-H): Immediate colposcopy is indicated for all age groups. ■Treatment is based on fndings of colposcopy: If colposcopy is satisfactory, proceed to treatment based on f ndings.

1	■Treatment is based on fndings of colposcopy: If colposcopy is satisfactory, proceed to treatment based on f ndings. If colposcopy is unsatisfactory, perform ECC and cervical biopsy and proceed to treatment based on f ndings. Noninvasive disease: Treatment based on biopsy results for noninvasive lesions (stage 0 disease) is as follows (see Figure 2.12-7): CIN I: ■Untreated CIN I will regress in 60% of patients, progress in 10%, and persist in 30%. Thus, the mainstay of treatment for CIN I is close observation. For women > 21 years of age, Pap smear screening at 6 and 12 months and/or HPV DNA testing at 12 months is indicated. For women ≤ 21 years of age, HPV testing is not recommended. After two Pap smears or a DNA test, patients can be managed with routine annual follow-up. Persistent CIN I can be treated with ablative (cryotherapy or laser ablation) or excisional therapy (loop electrosurgical excision procedure [LEEP]; laser and cold-knife conization).

1	Persistent CIN I can be treated with ablative (cryotherapy or laser ablation) or excisional therapy (loop electrosurgical excision procedure [LEEP]; laser and cold-knife conization). CD F IGU R E 2.1 2-7. Cervical intraepithelial neoplasia. (A) Colpophotograph illustrating a low-grade cervical intraepithelial neoplasia (CIN) in the transformation zone. (B)–(D).Histopathology of CIN I, II, and III. (Reproduced, with permission, from Kantarjian HM et al. MD Anderson Manual of Medical Oncology, 1st ed. New York: McGraw-Hill: Fig. 24-4.) CIN II and III: Untreated CIN II will regress in 43% of patients, progress in 22%, and persist in 35%. Untreated CIN III will regress in 32% of patients, progress in 14%, and persist in 56%. CIN II and III should be treated with ablative (cryotherapy or laser ablation) or excisional therapy (LEEP; laser and cold-knife conization). Hysterectomy is a treatment option for recurrent CIN II or III.

1	Hysterectomy is a treatment option for recurrent CIN II or III. Postablative or excisional therapy follow-up is as follows: CIN I, II, or III with margins: Pap smear at 12 months and/or HPV testing. CIN II or III with margins: Pap smear at 6 months; consider repeat ECC. If margins are unknown, obtain a Pap smear at 6 months and HPV DNA testing at 12 months. ■ Invasive disease: Treatment based on biopsy results for invasive carcinoma is as follows (for staging, see Figure 2.12-8): Microinvasive carcinoma (stage IA1): Treat with cone biopsy and close follow-up or simple hysterectomy. Stages IA2, IB1, and IIA: May be treated either with radical hysterectomy with concomitant radiation and chemotherapy or with radiation plus chemotherapy alone. Stages IB2, IIB, III, and IV: Treat with radiation therapy plus concurrent cisplatin-based chemotherapy. ■The overall fve-year relative survival rate for carcinoma of the cervix is 68% in Caucasian women and 55% in African-American women.

1	■The overall fve-year relative survival rate for carcinoma of the cervix is 68% in Caucasian women and 55% in African-American women. FIGURE 2.12-8. Staging of cervical cancer. Anatomic display of the stages of cervix cancer, defned by location, extent of tumor, frequency of presentation, and f ve-year survival. (Reproduced, with permission, from Fauci AS et al. Harrison’s Principles of Internal Medicine, 17th ed. New York: McGraw-Hill, 2008: Fig. 93-1.) Survival rates are inversely proportionate to the stage of cancer: ■Stage 0: 99–100%. Stage IA: > 95%. Stage IB-IIA: 80–90%. Stage IIB: 65%. Stage III: 40%. Stage IV: < 20%. Almost two-thirds of patients with untreated carcinoma of the cervix die of uremia when ureteral obstruction is bilateral.

1	Stage IB-IIA: 80–90%. Stage IIB: 65%. Stage III: 40%. Stage IV: < 20%. Almost two-thirds of patients with untreated carcinoma of the cervix die of uremia when ureteral obstruction is bilateral. Responsible for 1–4% of gynecologic malignancies. Some 90–95% are squamous lesions occurring in women > 50 years of age, followed by melanoma, basal cell carcinoma, adenocarcinoma, sarcoma, Bartholin’s gland tumors, and metastatic disease. Risk factors include HPV (types 16, 18, and 31), lichen sclerosus, infrequent medical exams, diabetes, obesity, hypertension, cardiovascular disease, and immunosuppression. Presents with pruritus, pain, or ulceration of the mass. Early: Lesions may appear white, pigmented, raised, thickened, nodular, or ulcerative. Late: Presents with a large, caulifower-like or hard ulcerated area in the vulva. The frst step is vulvar punch biopsy for any suspicious lesions.

1	Late: Presents with a large, caulifower-like or hard ulcerated area in the vulva. The frst step is vulvar punch biopsy for any suspicious lesions. Vulvar intraepithelial neoplasia (VIN) is considered precancerous and more commonly occurs in premenopausal women. VIN I and II: Associated with mild and moderate dysplasia. VIN III: Carcinoma in situ. Precancerous lesions: Reduce irritative or other predisposing causes. Topical corticosteroids (e.g., betamethasone, clobetasol) and crotamiton are particularly effective for pruritus. High-grade VIN: Topical chemotherapy, laser ablation, wide local excision, skinning vulvectomy, and simple vulvectomy. Invasive: Treated with (1) radical vulvectomy and regional lymphadenectomy or (2) wide local excision of the 1° tumor with inguinal lymph node dissection +/– preoperative radiation, chemotherapy, or both.

1	Accounts for 1–2% of all gynecologic malignancies. Squamous cell carcinoma usually occurs in postmenopausal woman, whereas other histologic types (rhabdosarcoma, endodermal sinus tumor, adenocarcinoma, and clear cell adenocarcinoma from DES) usually affect younger women. Risk factors include immunosuppression, chronic irritation (e.g., long-term pessary use or prolapse of female organs), low socioeconomic status, radiation for cervical cancer, hysterectomy for dysplasia, multiple sexual partners, and DES exposure. Frequency of female genital tract cancers: endometrial > ovarian > cervical. Number of deaths: ovarian > endometrial > cervical. Any palpable ovarian or adnexal mass in a premenarchal or postmenopausal patient is suggestive of an ovarian neoplasm. Characterized by abnormal vaginal bleeding, an abnormal discharge, or postcoital bleeding. Presents in the upper third of the vagina in 75% of patients. Cytology, colposcopy, and biopsy.

1	Characterized by abnormal vaginal bleeding, an abnormal discharge, or postcoital bleeding. Presents in the upper third of the vagina in 75% of patients. Cytology, colposcopy, and biopsy. Local excision of involved areas when they are few and small. Extensive involvement of the vaginal mucosa may require partial or complete vaginectomy. Invasive disease requires radiation therapy or radical surgery. Most ovarian tumors are benign, but malignant tumors are the leading cause of death from reproductive tract cancer. Risk factors include the following: Age, low parity, ↓ fertility, or delayed childbearing. A family history. Patients with one affected frst-degree relative have a 5% lifetime risk. With two or more affected frst-degree relatives, the risk is 7%. The BRCA1 mutation carries a 45% lifetime risk of ovarian cancer. The BRCA2 mutation is associated with a 25% lifetime risk.

1	The BRCA1 mutation carries a 45% lifetime risk of ovarian cancer. The BRCA2 mutation is associated with a 25% lifetime risk. Lynch II syndrome, or hereditary nonpolyposis colorectal cancer (HNPCC), is associated with an ↑ risk of colon, ovarian, endometrial, and breast cancer. OCPs taken for fve years or more ↓ risk by 29%. Both benign and malignant ovarian neoplasms are generally asymptomatic. Mild, nonspecifc GI symptoms or pelvic pressure/pain may be seen. Early disease is typically not detected on routine pelvic exam. Some 75% of woman present with advanced malignant disease, as evidenced by abdominal pain and bloating, a palpable abdominal mass, and ascites. Table 2.12-10 differentiates the benign and malignant characteristics of pelvic masses. Tumor markers (see Table 2.12-11): ↑ CA-125 is associated with epithelial cell cancer (90% of ovarian cancers) but is used only as a marker for progression and recurrence.

1	Tumor markers (see Table 2.12-11): ↑ CA-125 is associated with epithelial cell cancer (90% of ovarian cancers) but is used only as a marker for progression and recurrence. Premenopausal women: ↑ CA-125 may point to benign disease such as endometriosis. Postmenopausal women: ↑ CA-125 (> 35 units) indicates an ↑ likelihood that the ovarian tumor is malignant. Transvaginal ultrasound: Used to screen high-risk women. T AB LE 2.1 2-1 0. Benign vs. Malignant Pelvic Masses Treatment of ovarian masses is as follows: Premenarchal women: Masses > 2 cm require exploratory laparotomy. Premenopausal women: Observation for 4–6 weeks is suffcient for asymptomatic, mobile, unilateral, simple cystic masses < 8–10 cm. Most resolve spontaneously. Surgical evaluation is warranted for masses > 8–10 cm as well as for those that are unchanged on repeat pelvic exam and ultrasound. Postmenopausal women:

1	Surgical evaluation is warranted for masses > 8–10 cm as well as for those that are unchanged on repeat pelvic exam and ultrasound. Postmenopausal women: Asymptomatic, unilateral simple cysts < 5 cm in diameter with a normal CA-125 should be closely followed with ultrasound. Palpable masses warrant surgical evaluation by exploratory laparotomy. Treatment of ovarian cancer is as follows: Surgery: Surgical staging followed by TAH/BSO with omentectomy and pelvic and para-aortic lymphadenectomy. TABLE 2.12-11. Ovarian Tumor Markers Epithelial CA-125 Endodermal sinus AFP Embryonal carcinoma AFP, hCG Choriocarcinoma hCG Dysgerminoma LDH Granulosa cell Inhibin ■Benign neoplasms warrant tumor removal or unilateral oophorectomy. Postoperative chemotherapy is routine except for women with early-stage or low-grade ovarian cancer. Radiation therapy is effective for dysgerminomas.

1	Postoperative chemotherapy is routine except for women with early-stage or low-grade ovarian cancer. Radiation therapy is effective for dysgerminomas. Women with the BRCA1 gene mutation should be screened annually with ultrasound and CA-125 testing. Prophylactic oophorectomy is recommended by age 35 or whenever childbearing is completed. OCP use ↓ risk. Risk factors for pelvic organ prolapse include vaginal birth (particularly with use of forceps), genetic predisposition, advancing age, prior pelvic surgery, connective tissue disorders, and ↑ intra-abdominal pressure associated with obesity or straining with chronic constipation. Presents with the sensation of a bulge or protrusion in the vagina. Urinary or fecal incontinence, a sense of incomplete bladder emptying, and dyspareunia are also seen. The degree of prolapse can be evaluated by having the woman perform the Valsalva maneuver while in the lithotomy position.

1	The degree of prolapse can be evaluated by having the woman perform the Valsalva maneuver while in the lithotomy position. Supportive measures include a high-fber diet and weight reduction in obese patients and limitation of straining and lifting. Pessaries may temporarily reduce prolapse and are helpful in women who do not wish to undergo surgery or who are chronically ill. The most common surgical procedure is vaginal or abdominal hysterectomy with vaginal vault suspension. Defned as the involuntary loss of urine due to either bladder or sphincteric dysfunction. Table 2.12-12 outlines the types of incontinence along with their distinguishing features and treatment (see also the mnemonic DIAPPERS). Exclude fstula in cases of total incontinence. Look for neurologic abnormalities in cases of urge incontinence (spasticity, faccidity, rectal sphincter tone) or distended bladder in overf ow incontinence. First step: Obtain a UA and urine culture to exclude UTI.

1	First step: Obtain a UA and urine culture to exclude UTI. Next step: ■Voiding diary; possible urodynamic testing. Causes of urinary incontinence without specif c urogenital pathology— Excessive urinary output (hyperglycemia, hypercalcemia, CHF) TABLE 2.12-12. Types of Incontinence

1	Total Uncontrolled loss at all times and in all positions. Loss of sphincteric eff ciency (previous surgery, nerve damage, cancer inf ltration). Abnormal connection between the urinary tract and the skin (f stula). Surgery. Stress After ↑ intra-abdominal pressure (coughing, sneezing, lifting). Urethral sphincteric insuff ciency due to laxity of pelvic f oor musculature; common in multiparous women or after pelvic surgery. Kegel exercises and pessary. Vaginal vault suspension surgery. Strong, unexpected urge to void that is unrelated to position or activity. Detrusor hyperrefexia or sphincter dysfunction due to inf ammatory conditions or neurogenic disorders of the bladder. Anticholinergic medications or TCAs; behavioral training (biofeedback). Chronic urinary retention. Chronically distended bladder with ↑ intravesical pressure that just exceeds the outlet resistance, allowing a small amount of urine to dribble out. Placement of urethral catheter in acute settings. Treat underlying

1	bladder with ↑ intravesical pressure that just exceeds the outlet resistance, allowing a small amount of urine to dribble out. Placement of urethral catheter in acute settings. Treat underlying diseases. Timed voiding. a Etiologies include inhibited contractions, local irritation (cystitis, stone, tumor), and CNS causes. b Etiologies include physical agents (tumor, stricture), neurologic factors (lesions), and medications.

1	Serum creatinine to exclude renal dysfunction. Cystogram to demonstrate fstula sites and descensus of the bladder neck. Table 2.12-12 outlines treatment options according to subtype. The most common of all benign breast conditions. Involves exaggerated stromal tissue response to hormones and growth factors. Findings include cysts (gross and microscopic), papillomatosis, adenosis, fbrosis, and ductal epithelial hyperplasia. Primarily affects women 30–50 years of age; rarely found in postmenopausal woman. Associated with trauma and caffeine use. Presents with cyclic bilateral mastalgia and swelling, with symptoms most prominent just before menstruation. Rapid f uctuation in the size of the masses is common. Other symptoms include an irregular, bumpy consistency to the breast tissue (“oatmeal with raisins”). The differential diagnosis of a breast mass includes f brocystic disease, f broadenoma, mastitis/ abscess, fat necrosis, and breast cancer.

1	The differential diagnosis of a breast mass includes f brocystic disease, f broadenoma, mastitis/ abscess, fat necrosis, and breast cancer. Intraductal papilloma is a common cause of bloody nipple discharge. See Figure 2.12-9 for an algorithm of a breast mass workup. Mammography is of limited use. Ultrasound can help differentiate a cystic from a solid mass. Fine-needle aspiration (FNA) of a discrete mass that is suggestive of a cyst is indicated to alleviate pain as well as to confrm the cystic nature of the mass. Perform an excisional biopsy if no fuid is obtained or if the fuid is bloody on aspiration. There is an ↑ risk of breast cancer if ductal epithelial hyperplasia or cellular atypia is present. Dietary modifcations (e.g., caffeine restriction). Danazol may be given for severe pain but is rarely used in view of its side effects (acne, hirsutism, edema). Consider use of OCPs, which ↓ hormonal f uctuations.

1	Danazol may be given for severe pain but is rarely used in view of its side effects (acne, hirsutism, edema). Consider use of OCPs, which ↓ hormonal f uctuations. A benign, slow-growing breast tumor with epithelial and stromal components. The most common breast lesion in women < 30 years of age. Cystosarcoma phyllodes is a large f broadenoma. Suspicious mass: -Age > 35 -Family history -Firm, rigid -Axillary adenopathy -Skin changes FNA Excisional biopsy Excisional biopsy Follow-up monthly × 3 Clear fluid, mass disappears Bloody fluid Residual mass or thickening DCIS/cancer: Treat as indicated Mammography Core or excisional biopsy Negative: Reassure, routine follow-up Nonsuspicious mass: -Age < 35 -No family history -Movable, fluctuant -Size change w/cycle CystSolid Cytology Malignant Treatment Repeat FNA or open surgical biopsy Benign or inconclusive FIGURE 2.12-9. Workup of a breast mass.

1	FIGURE 2.12-9. Workup of a breast mass. Presents as a round or ovoid, rubbery, discrete, relatively mobile, non-tender mass 1–3 cm in diameter. Usually solitary, although up to 20% of patients develop multiple f broadenomas. Tumors do not change during the menstrual cycle. Does not occur after menopause unless the patient is on HRT. Breast ultrasound can differentiate cystic from solid masses. Needle biopsy or FNA. Excision with pathologic exam if the diagnosis remains uncertain. Excision is curative, but recurrence is common. The most common cancer (affects one in eight women) and the second most common cause of cancer death in women (after lung cancer). Sixty percent occur in the upper outer quadrant. Risk factors include the following (most women have no risk factors): Female gender, older age. A personal history of breast cancer. Breast cancer in a f rst-degree relative. BRCA1 and BRCA2 mutations (associated with early onset). A high-fat and low-f ber diet.

1	Female gender, older age. A personal history of breast cancer. Breast cancer in a f rst-degree relative. BRCA1 and BRCA2 mutations (associated with early onset). A high-fat and low-f ber diet. A history of fbrocystic change with cellular atypia. ■↑ exposure to estrogen (nulliparity, early menarche, late menopause, f rst full-term pregnancy after age 35). Ninety percent of breast cancers are found by the patient. Clinical manifestations include the following: Early f ndings: May present as a single, nontender, frm-to-hard mass with ill-defned margins or as mammographic abnormalities with no palpable mass. Later f ndings: Skin or nipple retraction, axillary lymphadenopathy, breast enlargement, redness, edema, pain, fxation of the mass to the skin or chest wall. Late f ndings: Ulceration; supraclavicular lymphadenopathy; edema of the arm; metastases to the bone, lung, and liver.

1	Late f ndings: Ulceration; supraclavicular lymphadenopathy; edema of the arm; metastases to the bone, lung, and liver. Prolonged unilateral scaling erosion of the nipple with or without discharge (Paget’s disease of the nipple). Metastatic disease: Back or bone pain, jaundice, weight loss. A frm or hard axillary node > 1 cm. Axillary nodes that are matted or fxed to the skin (stage III); ipsilateral supraclavicular or infraclavicular nodes (stage IV). ↑ exposure to estrogen (early menarche, late menopause, nulliparity) ↑ the risk of breast cancer. In a postmenopausal woman with a new breast lesion, maintain a high degree of clinical suspicion for breast cancer. The f rst step in the workup of a suspicious mass in postmenopausal women and in those > 30 years of age is a mammogram. For premenopausal women < 30 years of age, get an ultrasound. Breast cancer stages: Stage I: Tumor size < 2 cm. Stage II: Tumor size 2–5 cm. Stage III: Axillary node involvement.

1	Breast cancer stages: Stage I: Tumor size < 2 cm. Stage II: Tumor size 2–5 cm. Stage III: Axillary node involvement. Stage IV: Distant metastasis. Diagnostic measures differ for postmenopausal and premenopausal women. Postmenopausal women: The frst step is mammography. Look for ↑ density with microcalcifcations and irregular borders. Mammography can detect lesions roughly two years before they become clinically palpable (see Figure 2.12-10A). Premenopausal women: The frst step for women < 30 years of age is ultrasound, which can distinguish a solid mass from a benign cyst (see Figure 2.12-10B). Additional measures include the following: Tumor markers for recurrent breast cancer: Include CEA and CA 15-3 or CA 27-29. Receptor status of tumor: Determine estrogen receptor (ER), progesterone receptor (PR), and HER2/neu status. Metastatic disease: Labs: ↑ ESR, ↑ alkaline phosphatase (liver and bone metastases), ↑ calcium.

1	Metastatic disease: Labs: ↑ ESR, ↑ alkaline phosphatase (liver and bone metastases), ↑ calcium. Imaging: CXR; CT of the chest, abdomen, pelvis, and brain; bone scan. Pharmacologic: All hormone receptor– patients should receive tamoxifen. ER-patients should receive chemotherapy. Trastuzumab, a monoclonal antibody that binds to HER2/neu receptors on the cancer cell, is highly effective in HER2/neu-expressive cancers. Surgical options include the following: Partial mastectomy (lumpectomy) plus axillary dissection followed by radiation therapy. Modifed radical mastectomy (total mastectomy plus axillary dissection). Contraindications to breast-conserving therapy include large tumor size, subareolar location, multifocal tumors, fxation to the chest wall, or involvement of the nipple or overlying skin. Stage IV disease should be treated with radiotherapy and hormonal therapy; mastectomy may be required for local symptom control.

1	Stage IV disease should be treated with radiotherapy and hormonal therapy; mastectomy may be required for local symptom control. TNM staging (I–IV) is the most reliable indicator of prognosis. ERand PRstatus is associated with a favorable course. Cancer localized to the breast has a 75–90% cure rate. With spread to the axilla, the fve-year survival is 40–50%. Aneuploidy is associated with a poor prognosis. Pleural effusion occurs in 50% of patients with metastatic breast cancer; edema of the arm is common. B F IGU R E 2.1 2-1 0. Palpable breast mass on mammography and ultrasound. (A) Craniocaudal mammography of a palpable mass (arrows). (B) Ultrasound image demonstrating a solid mass with irregular borders (arrows) consistent with cancer. (Reproduced, with permission, from Brunicardi FC et al. Schwartz’s Principles of Surgery, 8th ed. New York: McGraw-Hill, 2005: Fig. 16-26.)

1	Also known as nonaccidental trauma; includes neglect as well as physical, sexual, and psychological maltreatment of children. Suspect abuse if the history is discordant with physical findings or if there is a delay in obtaining appropriate medical care. Certain injuries in children such as retinal hemorrhages and specific fracture types are pathognomonic for abuse. Abuse or neglect in infants may present as apnea, seizures, feeding intolerance, excessive irritability or somnolence, or failure to thrive (FTT). Neglect in older children may present as poor hygiene or behavioral abnormalities. Exam findings may include the following: Injuries in atypical places (e.g., the face or thighs) or patterns (stockingglove burns, cigarette burns, belt marks). Spiral fractures of the humerus and femur (strongly suggest abuse in children < 3 years of age) or epiphyseal/metaphyseal “bucket fractures,” which suggest shaking or jerking of the child’s limbs. Posterior rib fractures.

1	Posterior rib fractures. Genital trauma, bleeding, or discharge. Rule out conditions that mimic abuse—e.g., bleeding disorders or Mongolian spots (bruises), osteogenesis imperfecta (fractures), bullous impetigo (cigarette burns), and “coining” (an alternative treatment in certain cultures). A skeletal survey and bone scan can show fractures in various stages of healing. Test for gonorrhea, syphilis, chlamydia, and HIV if sexual abuse is suspected. Rule out shaken baby syndrome (SBS) by performing an ophthalmologic exam for retinal hemorrhages and a noncontrast CT for subdural hematomas. Infants with SBS often do not exhibit external signs of abuse. Consider an MRI to visualize white matter changes (diffuse axonal injury associated with violent shaking) and the extent of intraand extracranial bleeds. MRI often requires the young patient to be sedated and/or intubated, while CT usually does not.

1	Document injuries, including location, size, shape, color, and the nature of all lesions, bruises, or burns. Notify child protective services (CPS) for evaluation and possible removal of the child from the home. Hospitalize if necessary to stabilize injuries or to protect the child. Consider abuse if the caretaker’s story and the child’s injuries don’t match. Reporting any suspicion of child abuse is mandatory; you cannot be sued for doing so. Intrauterine risk factors for congenital heart disease include maternal drug use (alcohol, lithium, thalidomide, phenytoin), maternal infections (rubella), and maternal illness (DM, PKU). Disease is classified by the presence or absence of cyanosis: Transposition of the great vessels = two arteries switched Tetralogy of Fallot (four) VSD is the most common congenital heart defect. Acyanotic conditions (“pink babies”): Have left-to-right shunts in which oxygenated blood from the lungs is shunted back into the pulmonary circulation.

1	Acyanotic conditions (“pink babies”): Have left-to-right shunts in which oxygenated blood from the lungs is shunted back into the pulmonary circulation. Cyanotic conditions (“blue babies”): Have right-to-left shunts in which deoxygenated blood is shunted into the systemic circulation. A condition in which an opening in the ventricular septum allows blood to ﬂow between ventricles. VSD is the most common congenital heart defect. It is more common among patients with Apert’s syndrome (cranial deformities, fusion of the fingers and toes), Down syndrome, fetal alcohol syndrome, TORCH syndrome (toxoplasmosis, other agents, rubella, CMV, HSV), cri du chat syndrome, and trisomies 13 and 18. Small defects are usually asymptomatic at birth, but exam reveals a harsh holosystolic murmur heard best at the lower left sternal border.

1	Small defects are usually asymptomatic at birth, but exam reveals a harsh holosystolic murmur heard best at the lower left sternal border. Large defects can present with frequent respiratory infections, dyspnea, FTT, and CHF. If present, the holosystolic murmur is softer and more blowing but can be accompanied by a systolic thrill, crackles, a narrow S2 with an ↑ P1, and a mid-diastolic apical rumble reﬂ ecting ↑ ﬂow across the mitral valve. Echocardiogram is diagnostic. ECG and CXR can demonstrate LVH with small defects and show both LVH and RVH with larger VSDs. CXR may show ↑ pulmonary vascular markings. Most small VSDs close spontaneously; patients should be monitored via echocardiography. Surgical repair is indicated in symptomatic patients who fail medical management, children < 1 year of age with signs of pulmonary hypertension, and older children with large VSDs that have not ↓ in size over time.

1	Treat existing CHF with diuretics, inotropes, and ACEIs; treat respiratory infections as needed. A condition in which an opening in the atrial septum allows blood to ﬂ ow between the atria, leading to left-to-right shunting. Associated with Holt-Oram syndrome (absent radii, ASD, first-degree heart block), fetal alcohol syndrome, and Down syndrome. Ostium primum defects present in early childhood with findings of a murmur or fatigue with exertion. Ostium secundum defects (most common) tend to present in late childhood or early adulthood. Symptom onset and severity depend on the size of the defect. Symptoms of easy fatigability, frequent respiratory infections, and FTT can be observed, but patients are frequently asymptomatic. ASD has a f xed, widely split S2.

1	Symptoms of easy fatigability, frequent respiratory infections, and FTT can be observed, but patients are frequently asymptomatic. ASD has a f xed, widely split S2. ■Exam reveals a right ventricular heave; a wide and fxed, split S2; and a systolic ejection murmur at the upper left sternal border (from ↑ ﬂ ow across the pulmonary valve). There may also be a mid-diastolic rumble at the left lower sternal border. Echocardiogram with color ﬂow Doppler reveals blood ﬂow between the atria (diagnostic), paradoxical ventricular wall motion, and a dilated right ventricle. ECG most commonly shows right axis deviation and RVH, although other patterns are possible depending on the type of defect. PR prolongation is common. CXR reveals cardiomegaly and ↑ pulmonary vascular markings. Most lesions are small defects that may close spontaneously and do not require treatment.

1	CXR reveals cardiomegaly and ↑ pulmonary vascular markings. Most lesions are small defects that may close spontaneously and do not require treatment. Surgical closure is indicated in infants with CHF and in patients with more than a 2:1 ratio of pulmonary to systemic blood ﬂow. Early correction prevents complications such as arrhythmias, right ventricular dysfunction, and Eisenmenger’s syndrome. Failure of the ductus arteriosus to close in the first few days of life, leading to a left-to-right shunt from the aorta to the pulmonary artery. Risk factors include maternal first-trimester rubella infection, prematurity, and female gender. Typically asymptomatic; patients with large defects may present with FTT, recurrent lower respiratory tract infections, lower extremity clubbing, and CHF. Exam reveals a wide pulse pressure; a continuous “machinery murmur” at the second left intercostal space at the sternal border; a loud S2; and bounding peripheral pulses.

1	Exam reveals a wide pulse pressure; a continuous “machinery murmur” at the second left intercostal space at the sternal border; a loud S2; and bounding peripheral pulses. A color ﬂow Doppler demonstrating blood ﬂow from the aorta into the pulmonary artery is diagnostic. With larger PDAs, echocardiography shows left atrial and left ventricular enlargement. ECG may show LVH, and CXR may show cardiomegaly if large lesions. Give indomethacin unless the PDA is needed for survival (e.g., transposition of the great vessels, tetralogy of Fallot, hypoplastic left heart) or if indomethacin is contraindicated (e.g., intraventricular hemorrhage). If indomethacin fails or if the child is > 6–8 months of age, surgical closure is required. In Eisenmenger’s syndrome, left-to-right shunt leads to pulmonary hypertension and shunt reversal. In infants presenting in a shocklike state within the f rst few weeks of life, look for: 1. 2. Inborn error of metabolism 3.

1	In infants presenting in a shocklike state within the f rst few weeks of life, look for: 1. 2. Inborn error of metabolism 3. Ductal-dependent congenital heart disease, usually left-sided lesions (as the ductus is closing) 4. Come IN and CLOSE the door: give INdomethacin to CLOSE a PDA. What maternally ingested drug is associated with Ebstein’s anomaly (tricuspid valve displacement into the right ventricle)? Lithium. Coarctation is a cause of 2° hypertension in children. Coarctation of the Aorta Constriction of a portion of the aorta, leading to ↑ ﬂow proximal to and ↓ ﬂow distal to the coarctation. Occurs just below the left subclavian artery in 98% of patients. The condition is associated with Turner’s syndrome, berry aneurysms, and male gender. More than two-thirds of patients have a bicuspid aortic valve. Often presents in childhood with asymptomatic hypertension. Lower extremity claudication, syncope, epistaxis, and headache may be present.

1	Often presents in childhood with asymptomatic hypertension. Lower extremity claudication, syncope, epistaxis, and headache may be present. The classic physical exam finding is a systolic BP that is higher in the upper extremities; the difference in BP between the left and right arm can indicate the point of coarctation. Additional findings include weak femoral pulses, radiofemoral delay, a short systolic murmur in the left axilla, and a forceful apical impulse. In infancy, critical coarctation requires a patent PDA for survival. Such infants may present in the first few weeks of life in a shocklike state when the PDA closes. Differential cyanosis may be seen with lower oxygen saturation in the left arm and lower extremities (postductal areas) as compared to the right arm (preductal area). Echocardiography and color ﬂow Doppler are diagnostic. CXR in young children may demonstrate cardiomegaly and pulmonary congestion.

1	Echocardiography and color ﬂow Doppler are diagnostic. CXR in young children may demonstrate cardiomegaly and pulmonary congestion. In older children, the following compensatory changes may be seen: LVH on ECG; the “3” sign on CXR due to preand postdilatation of the coarctation segment with aortic wall indentation; and “rib notching” due to collateral circulation through the intercostal arteries. If severe coarctation presents in infancy, the ductus arteriosus should be kept open with prostaglandin E1 (PGE1). Surgical correction or balloon angioplasty (controversial). Monitor for restenosis, aneurysm development, and aortic dissection. Transposition of the Great Vessels

1	Surgical correction or balloon angioplasty (controversial). Monitor for restenosis, aneurysm development, and aortic dissection. Transposition of the Great Vessels The most common cyanotic congenital heart lesion in the newborn. In this condition, the aorta is connected to the right ventricle and the pulmonary artery to the left ventricle, creating parallel pulmonary and systemic circulations. Without a septal defect or a PDA, it is incompatible with life. Risk factors include diabetic mothers and, rarely, DiGeorge syndrome (see the mnemonic CATCH 22). ■Critical illness and cyanosis typically occur immediately after birth. Reverse differential cyanosis may be seen if left ventricular outﬂow tract obstruction (e.g., coarctation, aortic stenosis) is also present. ■Exam reveals tachypnea, progressive hypoxemia, and extreme cyanosis. Some patients have signs of CHF, and a single loud S2 is often present. There may not be a murmur if no VSD is present. Echocardiography.

1	Echocardiography. CXR may show a narrow heart base, absence of the main pulmonary artery segment (“egg-shaped silhouette”), and ↑ pulmonary vascular markings. Start IV PGE1 to maintain or open the PDA. If surgery is not feasible within the first few days of life or if the PDA cannot be maintained with prostaglandin, perform balloon atrial septostomy to create or enlarge an ASD. Surgical correction (arterial or atrial switch). Tetralogy of Fallot Consists of pulmonary stenosis, overriding aorta, RVH, and VSD. The most common cyanotic congenital heart disease in children. Early cyanosis results from right-to-left shunting across the VSD. As right-sided pressures ↓ in the weeks after birth, the shunt direction reverses and cyanosis may ↓. If the degree of pulmonary stenosis is severe, the right-sided pressures may remain high and cyanosis may worsen over time. Risk factors include maternal PKU and DiGeorge syndrome.

1	Presents in infancy or early childhood with dyspnea and fatigability. Cyanosis is often not present at birth but develops over the first two years of life; the degree of cyanosis often reﬂects the extent of pulmonary stenosis. Infants are often asymptomatic until 4–6 months of age, when CHF may develop and may manifest as diaphoresis with feeding or tachypnea. Children often squat for relief (↑ systemic vascular resistance) during hypoxemic episodes (“tet spells”). Hypoxemia may lead to FTT or mental status changes. Exam reveals a systolic ejection murmur at the left upper sternal border (right ventricular outﬂow obstruction), a right ventricular heave, and a single S2. Echocardiography and catheterization. CXR shows a “boot-shaped” heart with ↓ pulmonary vascular markings. Remember that a VSD may result in ↑ pulmonary vascular markings. ECG shows right-axis deviation and RVH.

1	CXR shows a “boot-shaped” heart with ↓ pulmonary vascular markings. Remember that a VSD may result in ↑ pulmonary vascular markings. ECG shows right-axis deviation and RVH. Lesions with severe pulmonary stenosis or atresia require immediate PGE1 to keep the PDA open along with urgent surgical consultation. Treat hypercyanotic “tet spells” with O2, propranolol, phenylephrine, knee-chest position, ﬂuids, and morphine. Temporary palliation can be achieved through the creation of an artificial shunt (e.g., balloon atrial septostomy) before definitive surgical correction (Blalock-Taussig shunt). Coarctation in infancy may present with differential cyanosis, while transposition of the great arteries may present with reverse differential cyanosis. Transposition of the great vessels is the most common cyanotic heart disease of newborns. Tetralogy of Fallot is the most common cyanotic heart disease of childhood.

1	Transposition of the great vessels is the most common cyanotic heart disease of newborns. Tetralogy of Fallot is the most common cyanotic heart disease of childhood. Both transposition of the great vessels and tetralogy of Fallot are initially treated with PGE1 and are def nitively treated with surgical correction. Table 2.13-1 highlights major developmental milestones. Commonly tested milestones appear in bold.

1	T AB LE 2.1 3-1. Developmental Milestones 2 months Lifts head/chest when prone. Tracks past midline. Alerts to sound; coos. Recognizes parent; social smile. 4–5 months Rolls front to back, back to front (5 months). Grasps rattle. Orients to voice; begins to make consonant sounds; razzes. Enjoys looking around; laughs. 6 months Sits unassisted. Transfers objects; raking grasp. Babbles. Stranger anxiety. 9–10 months Crawls; pulls to stand. Uses three-finger (immature) pincer grasp. Says “mama/dada” (nonspecif c). Waves bye-bye; plays pat-a-cake. 12 months Cruises (11 months); walks alone. Uses two-finger (mature) pincer grasp. Says “mama/dada” (specif c). Imitates actions; separation anxiety. 15 months Walks backward. Uses cup. Uses 4–6 words. Temper tantrums. 18 months Runs; kicks a ball. Builds tower of 2–4 cubes. Names common objects. May start toilet training. 2 years Walks up/down steps with help; jumps. Builds tower of six cubes. Uses two-word phrases. Follows two-step commands;

1	Builds tower of 2–4 cubes. Names common objects. May start toilet training. 2 years Walks up/down steps with help; jumps. Builds tower of six cubes. Uses two-word phrases. Follows two-step commands; removes clothes. 3 years Rides tricycle; climbs stairs with alternating feet (3–4 years). Copies a circle; uses utensils. Uses three-word sentences. Brushes teeth with help; washes/dries hands. Hops. Copies a square. Knows colors and some numbers. Cooperative play; plays board games. Skips; walks backward for long distances. Ties shoelaces; knows left and right; prints letters. Uses f ve-word sentences. Domestic role playing; plays dress-up. 4 years 5 years a For premature infants < 2 years of age, chronological age must be adjusted for gestational age. For example, an infant born at seven months’ gestation (two months early) would be expected to perform at the four-month level at the chronological age of six months.

1	At each well-child check, height, weight, and head circumference are plotted on growth charts specific for gender and age: Head circumference: Measured routinely in the first two years. ↑ head circumference can indicate hydrocephalus or tumor; ↓ head circumference can point to microcephaly (e.g., TORCH infections). Height and weight: Measured routinely until adulthood. The pattern of growth is more important than the raw numbers. Infants may lose 5–10% of birth weight (BW) over the first few days but should return to their BW by 14 days. Infants can be expected to double their BW by 4–5 months, triple by one year, and quadruple by two years. Failure to thrive: Persistent weight less than the 5th percentile for age or “falling off the growth curve” (i.e., crossing two major percentile lines on a growth chart). Classified as follows:

1	Failure to thrive: Persistent weight less than the 5th percentile for age or “falling off the growth curve” (i.e., crossing two major percentile lines on a growth chart). Classified as follows: Organic: Due to an underlying medical condition such as cystic fibro-sis, congenital heart disease, celiac sprue, pyloric stenosis, chronic infection (e.g., HIV), and GERD. Nonorganic: Primarily due to psychosocial factors such as maternal depression, neglect, or abuse. A careful dietary history and close observation of maternal-infant interactions (especially preparation of formula and feeding) are critical to diagnosis. Children should be hospitalized if there is evidence of neglect or severe malnourishment. Calorie counts and supplemental nutrition (if breastfeeding is inadequate) are mainstays of treatment.

1	Children should be hospitalized if there is evidence of neglect or severe malnourishment. Calorie counts and supplemental nutrition (if breastfeeding is inadequate) are mainstays of treatment. Tanner staging: Performed to assess physical development in boys and girls. Stage 1 is preadolescent; stage 5 is adult. Increasing stages are assigned for testicular and penile growth in boys and breast growth in girls; pubic hair development is used for both stages. Girls: The average age of puberty is 10.5 years. Generally the order of progression is thelarche (breast bud development) → pubarche (pubic hair development) → growth spurt → menarche (first menstrual bleeding). The average age of menarche in United States girls is 12.5 years. Boys: The average age of puberty is 11.5 years. Generally the order of progression is gonadarche (testicular enlargement) → pubarche → adrenarche (axillary hair, facial hair, vocal changes) → growth spurt.

1	Variants of normal sexual development are as follows: Delayed puberty: No testicular enlargement in boys by age 14, or no breast development or pubic hair in girls by age 13. Constitutional growth delay: A normal variant, and the most common cause of delayed puberty. The growth curve lags behind others of the same age but is consistent. There is often a family history, and children ultimately achieve target height potential. Pathological puberty delay: Rarely, due to systemic disease (e.g., IBD), malnutrition (e.g., anorexia nervosa), gonadal dysgenesis (e.g., Klinefelter’s syndrome, Turner’s syndrome), or endocrine abnormalities (e.g., hypopituitarism, hypothyroidism, Kallmann’s syndrome, androgen insensitivity syndrome, Prader-Willi syndrome) Precocious puberty: Any sign of 2° sexual maturation in girls < 8 years or boys < 9 years of age. Often idiopathic; may be central or peripheral (see the Gynecology chapter).

1	Precocious puberty: Any sign of 2° sexual maturation in girls < 8 years or boys < 9 years of age. Often idiopathic; may be central or peripheral (see the Gynecology chapter). Signs of autism include no babbling and/or gesturing by 12 months, no single words by 16 months, no two-word phrases by 24 months, failure to make eye contact, or any loss of language or social skills. Infants with FTT will f rst fall off of the weight curve, then the height curve, and f nally the head circumference curve. Tables 2.13-2 and 2.13-3 outline common genetic diseases and their associated abnormalities. TABLE 2.13-2. Genetic Diseases

1	Down syndrome Trisomy 21 (most common) or robertsonian translocation (higher risk of recurrence) The most common chromosomal disorder and cause of mental retardation. Associated with advanced maternal age. Presents with mental retardation, a fat facial profle, prominent epicanthal folds, and a simian crease. Associated with duodenal atresia, Hirschsprung’s disease, and congenital heart disease (the most common malformation is atrioventricular canal, which includes an ASD and VSD with mitral and tricuspid valve abnormalities due to endocardial cushion defects). Associated with an ↑ risk of acute lymphocytic leukemia (ALL), hypothyroidism, and early-onset Alzheimer’s. Edwards’ syndrome Trisomy 18 Presents with severe mental retardation, rocker-bottom feet, low-set ears, micrognathia, clenched hands, and a prominent occiput. Associated with congenital heart disease. May have horseshoe kidneys. Death usually occurs within one year of birth. Patau’s syndrome Trisomy 13 Presents with severe

1	hands, and a prominent occiput. Associated with congenital heart disease. May have horseshoe kidneys. Death usually occurs within one year of birth. Patau’s syndrome Trisomy 13 Presents with severe mental retardation, microphthalmia, microcephaly, cleft lip/palate, abnormal forebrain structures (holoprosencephaly), “punched-out” scalp lesions, and polydactyly. Associated with congenital heart disease. Death usually occurs within one year of birth. Klinefelter’s syndrome (male) 45,XXY Presence of an inactivated X chromosome (Barr body). One of the most common causes of hypogonadism in males. Presents with testicular atrophy, a eunuchoid body shape, tall stature, long extremities, and gynecomastia. Treat with testosterone (prevents gynecomastia; improves 2° sexual characteristics). Turner’s syndrome (female) 45,XO The most common cause of 1° amenorrhea; due to gonadal dysgenesis. No Barr body. Features include short stature, shield chest, widely spaced nipples, webbed neck, coarctation

1	syndrome (female) 45,XO The most common cause of 1° amenorrhea; due to gonadal dysgenesis. No Barr body. Features include short stature, shield chest, widely spaced nipples, webbed neck, coarctation of the aorta, and/or bicuspid aortic valve. May present with lymphedema of the hands and feet in the neonatal period. May have horseshoe kidney. Double Y males 47,XYY Observed with ↑ frequency among inmates of penal institutions. Phenotypically normal; patients are very tall with severe acne and antisocial behavior (seen in 1–2% of XYY males).

1	TABLE 2.13-2. Genetic Diseases (continued) Phenylketonuria (PKU) ↓ phenylalanine hydroxylase or ↓ tetrahydrobiopterin cofactor Screened for at birth; screening is valid only after the baby has had a protein meal (i.e., a normal breast or formula feed). Tyrosine becomes essential and phenylalanine builds up excess phenyl ketones. Presents with mental retardation, fair skin, eczema, and a musty or mousy urine odor. Blond-haired, blue-eyed infants. Associated with an ↑ risk of heart disease. Treat with ↓ phenylalanine and ↑ tyrosine in diet. A mother with PKU who wants to become pregnant must restrict her diet as above before conception. Fragile X syndrome An X-linked defect affecting the methylation and expression of the FMR1 gene The second most common cause of genetic mental retardation. Presents with macro-orchidism; a long face with a large jaw; large, everted ears; and autism. A triplet repeat disorder that may show genetic anticipation.

1	An autosomal-recessive disorder caused by mutations in the CFTR gene (chloride channel) on chromosome 7 and characterized by widespread exocrine gland dysfunction. CF is the most common severe genetic disease in the United States and is most frequently found in Caucasians. Fifty percent of patients present with FTT or chronic sinopulmonary disease. Characterized by recurrent pulmonary infections (especially with Pseudomonas and S. aureus) with subsequent cyanosis, digital clubbing, cough, dyspnea, bronchiectasis, hemoptysis, chronic sinusitis, rhonchi, rales, hyperresonance to percussion, and nasal polyposis. Fifteen percent of infants present with meconium ileus. Patients usually have greasy stools and ﬂatulence; other prominent GI symptoms include pancreatitis, rectal prolapse, hypoproteinemia, biliary cirrhosis, jaundice, and esophageal varices. GI symptoms are more prominent in infancy, while pulmonary manifestations predominate thereafter.

1	GI symptoms are more prominent in infancy, while pulmonary manifestations predominate thereafter. Additional symptoms include type 2 DM, a “salty taste,” male infertility (agenesis of the vas deferens), and unexplained hyponatremia. Patients are at risk for fat-soluble vitamin deficiency (vitamins A, D, E, and K) 2° to malabsorption and may present with manifestations of these deficiencies. Sweat chloride test > 60 mEq/L for those < 20 years of age and > 80 mEq/L in adults; DNA probe test. Most states now perform mandatory newborn screening, but occasional false positives do occur, so children must be brought in for a sweat test to distinguish disease from a carrier state. The sweat chloride test has traditionally been considered the gold standard for the diagnosis of CF, but conf rmatory genetic analysis is now routinely done. T AB LE 2.1 3-3. Lysosomal Storage Diseases

1	Fabry’s disease Caused by a def ciency of α-galactosidase A that leads to accumulation of ceramide trihexoside in the heart, brain, and kidneys. Findings include renal failure and an ↑ risk of stroke and MI. X-linked recessive. Krabbe’s disease Absence of galactosylceramide and galactoside (due to galactosylceramidase defciency), leading to the accumulation of galactocerebroside in the brain. Characterized by optic atrophy, spasticity, and early death. Autosomal recessive. Gaucher’s disease Caused by a defciency of glucocerebrosidase that leads to the accumulation of glucocerebroside in the brain, liver, spleen, and bone marrow (Gaucher’s cells with characteristic “crinkled paper,” enlarged cytoplasm). May present with hepatosplenomegaly, anemia, and thrombocytopenia. Type 1, the more common form, is compatible with a normal life span and does not affect the brain. Autosomal recessive. Niemann-Pick disease A defciency of sphingomyelinase that leads to the buildup of sphingomyelin

1	common form, is compatible with a normal life span and does not affect the brain. Autosomal recessive. Niemann-Pick disease A defciency of sphingomyelinase that leads to the buildup of sphingomyelin cholesterol in reticuloendothelial and parenchymal cells and tissues. Patients with type A die by the age of three. Autosomal recessive. No man PICKs (Niemann-PICK) his nose with his sphinger. Tay-Sachs disease An absence of hexosaminidase that leads to GM2 ganglioside accumulation. Infants may appear normal until 3–6 months of age, when weakness begins and development slows and regresses. Exaggerated startle response. Death occurs by the age of three. A cherry-red spot is visible on the macula. The carrier rate is 1 in 30 Jews of European descent (1 in 300 for others). Tay-SaX lacks heXosaminidase. Metachromatic leukodystrophy A defciency of arylsulfatase A that leads to the accumulation of sulfatide in the brain, kidney, liver, and peripheral nerves. Autosomal recessive. Hurler’s

1	Metachromatic leukodystrophy A defciency of arylsulfatase A that leads to the accumulation of sulfatide in the brain, kidney, liver, and peripheral nerves. Autosomal recessive. Hurler’s syndrome A def ciency of α-L-iduronidase that leads to corneal clouding and mental retardation. Autosomal recessive. Hunter’s syndrome A defciency of iduronate sulfatase. A mild form of Hurler’s with no corneal clouding and mild mental retardation. X-linked recessive. Hunters need to see (no corneal clouding) to aim for the X.

1	Pulmonary manifestations are managed with chest physical therapy, bronchodilators, corticosteroids, antibiotics, and DNase. Administer pancreatic enzymes and fat-soluble vitamins A, D, E, and K for malabsorption. Nutritional counseling and support with a high-calorie and high-protein diet are essential for health maintenance. ■Patients who have severe disease (but who can tolerate surgery) may be candidates for lung or pancreas transplants. Life expectancy was once around 20 years, but with newer treatments it is increasing to past age 30.

1	A condition in which one portion of the bowel invaginates or “telescopes” into an adjacent segment, usually proximal to the ileocecal valve (see Figure 2.131). The most common cause of bowel obstruction in the first two years of life (males > females); usually seen between three months and three years of age. The cause is often unknown. Risk factors include conditions with potential lead points, including Meckel’s diverticulum, intestinal lymphoma (> 6 years of age), Henoch-Schönlein purpura, parasites, polyps, adenovirus or rotavirus infection, celiac disease, and CF. Presents with abrupt-onset, colicky abdominal pain in apparently healthy children, often accompanied by ﬂexed knees and vomiting. The child may appear well in between episodes if intussusception is released. The classic triad is abdominal pain, vomiting, and blood per rectum (affects only one in three patients).

1	The classic triad is abdominal pain, vomiting, and blood per rectum (affects only one in three patients). Late signs include bloody mucus in stools (red “currant jelly” stool), lethargy, and fever. The condition may progress to shock as blood ﬂow to the affected segment is compromised. On exam, look for abdominal tenderness, a stool guaiac, and a palpable “sausage-shaped” RUQ abdominal mass. FIGURE 2.13-1. Intussusception. A segment of bowel telescopes into an adjacent segment, causing obstruction. (Reproduced, with permission, from Way LW. Current Surgical Diagnosis & Treatment, 10th ed. Stamford, CT: Appleton & Lange, 1994: 1222.) enema is both diagnostic and therapeutic in most cases of intussusception. The classic metabolic derangement in pyloric stenosis is hypochloremic, hypokalemic metabolic alkalosis (due to persistent emesis of HCl). Meckel’s rule of 2’s— Most common in children under 2 2 times as common in males

1	Meckel’s rule of 2’s— Most common in children under 2 2 times as common in males Contains 2 types of tissue (pancreatic and gastric) 2 inches long Found within 2 feet of the ileocecal valve Occurs in 2% of the population Correct any volume or electrolyte abnormalities and check CBC (for leukocytosis). Abdominal plain films are often normal early in the disease, but later they may show small bowel obstruction, perforation, or a soft tissue mass. Ultrasound may show a “target sign.” In the setting of high clinical suspicion, an air-contrast barium enema should be performed without delay, as it is diagnostic in > 95% of cases and curative in > 80%. If the child is unstable or has peritoneal signs or if enema reduction is unsuccessful, perform surgical reduction and resection of gangrenous bowel.

1	Hypertrophy of the pyloric sphincter, leading to gastric outlet obstruction. More common in firstborn males; associated with tracheoesophageal fistula and with a maternal history of pyloric stenosis. Nonbilious emesis typically begins around three weeks of age and progresses to projectile emesis after most to all feedings. Babies initially feed well but eventually suffer from malnutrition and dehydration. Exam may reveal a palpable olive-shaped, mobile, nontender epigastric mass and visible gastric peristaltic waves. Abdominal ultrasound is the imaging modality of choice and reveals a hypertrophic pylorus. Barium studies reveal a narrow pyloric channel (“string sign”) or a pyloric beak. Correct existing dehydration and acid-base/electrolyte abnormalities. Surgical correction with pyloromyotomy.

1	Barium studies reveal a narrow pyloric channel (“string sign”) or a pyloric beak. Correct existing dehydration and acid-base/electrolyte abnormalities. Surgical correction with pyloromyotomy. Caused by failure of the omphalomesenteric (or vitelline) duct to obliterate. The most common congenital abnormality of the small intestine, affecting up to 2% of children. Most frequently occurs in children < 2 years of age. Typically asymptomatic, and often discovered incidentally. Classically presents with sudden, painless rectal bleeding. Abdominal pain typically signifies complications such as diverticulitis, volvulus, and intussusception. A Meckel scintigraphy scan (technetium-99m pertechnetate) is diagnostic; plain films have limited value but can be useful in diagnosing obstruction or perforation. In the presence of active bleeding, treatment is excision of the diverticulum together with the adjacent ileal segment (ulcers frequently develop in adjacent ileum).

1	In the presence of active bleeding, treatment is excision of the diverticulum together with the adjacent ileal segment (ulcers frequently develop in adjacent ileum). If the condition is asymptomatic but discovered intraoperatively, treatment is controversial but often involves excision. Congenital lack of ganglion cells in the distal colon, leading to uncoordinated peristalsis and ↓ motility. Associated with male gender, Down syndrome, Waardenburg’s syndrome, and multiple endocrine neoplasia (MEN) type 2. Neonates present with failure to pass meconium within 48 hours of birth, accompanied by bilious vomiting and FTT; children with less severe lesions may present later in life with chronic constipation. Physical exam may reveal abdominal distention and explosive discharge of stool following rectal exam.

1	Physical exam may reveal abdominal distention and explosive discharge of stool following rectal exam. Barium enema is the imaging study of choice and reveals a narrowed distal colon with proximal dilation. Plain films reveal distended bowel loops with a paucity of air in the rectum. Anorectal manometry detects a failure of the internal sphincter to relax after distention of the rectal lumen. It is typically used in atypical presentations or older children. Full-thickness rectal biopsy confirms the diagnosis and reveals absence of the myenteric (Auerbach’s) plexus and submucosal (Meissner’s) plexus along with hypertrophied nerve trunks enhanced with acetylcholinesterase stain. Traditionally a two-stage surgical repair is used involving the creation of a diverting colostomy at the time of diagnosis, followed several weeks later by a definitive “pull-through” procedure connecting the remaining colon to the rectum. Malrotation with Volvulus

1	Malrotation with Volvulus Congenital malrotation of the midgut results in abnormal positioning of the small intestine (cecum in the right hypochondrium) and formation of fibrous bands (Ladd’s bands). Bands predispose to obstruction and constriction of blood ﬂ ow. Often presents in the newborn period with bilious emesis, crampy abdominal pain, distention, and the passage of blood or mucus in stool. Postsurgical adhesions can lead to obstruction and volvulus at any point in life. The def nitive diagnosis of Hirschsprung’s disease requires a full-thickness rectal biopsy. Pneumatosis intestinalis on plain f lms is pathognomonic for necrotizing enterocolitis in neonates. AXR may reveal the absence of intestinal gas but may also be normal. If the patient is stable, an upper GI is the study of choice and shows an abnormal location of the ligament of Treitz. Ultrasound may be used, but sensitivity is determined by the experience of the ultrasonographer.

1	NG tube insertion to decompress the intestine. IV ﬂ uid hydration. Surgical repair (emergent when volvulus is present). A condition in which a portion of the bowel undergoes necrosis. The most common GI emergency in neonates; most commonly occurs in premature infants but can occur in full-term infants as well. Symptoms are nonspecific and include feeding intolerance, delayed gastric emptying, abdominal distention, and bloody stools. Symptoms may rapidly progress to intestinal perforation, peritonitis, abdominal erythema, and shock. Maintain a high index of suspicion. Lab findings are nonspecific and may show hyponatremia, metabolic acidosis, leukopenia or leukocytosis with left shift, thrombocytopenia, and coagulopathy (DIC with prolonged PT, aPTT, and a D-dimer).

1	Plain abdominal radiographs may show dilated bowel loops, pneumatosis intestinalis (intramural air bubbles representing gas produced by bacteria within the bowel wall), portal venous gas, or abdominal free air. Serial abdominal plain films should be taken every six hours. Ultrasound may also be helpful in discerning free air, areas of loculation or walled-off abscesses, and bowel necrosis. Initiate supportive measures, including NPO, an orogastric tube for gastric decompression, correction of dehydration and electrolyte abnormalities, TPN, and IV antibiotics. Indications for surgery are perforation or radiographic worsening on serial abdominal plain films. An ileostomy with mucous fistula is typically performed, with a reanastomosis later. Complications include formation of intestinal strictures and short-bowel syndrome.

1	Complications include formation of intestinal strictures and short-bowel syndrome. Congenital immunodeficiencies are rare and often present with chronic or recurrent infections (e.g., chronic thrush), unusual or opportunistic organisms, incomplete treatment response, or FTT. Categorization is based on the one immune system component that is abnormal (see also Table 2.13-4). T AB LE 2.1 3-4. Pediatric Immunodeficiencies

1	B cell X-linked agamma-globulinemia (Bruton’s) Common variable immunodef ciency IgA def ciency A B-cell def ciency in boys only. Immunoglobulin level drops in the 20s and 30s; usually a combined Band T-cell defect. Mild; the most common immunodef ciency. Life threatening; encapsulated Pseudomonas, Streptococcus pneumoniae, and Haemophilus infections after six months (passive immunity through maternal antibodies wanes). ↑ pyogenic upper and lower respiratory infections; ↑ risk of lymphoma and autoimmune disease. Usually asymptomatic; patients may develop recurrent respiratory or GI infections. Anaphylactic transfusion reaction due to anti-IgA antibodies is a common presentation. Quantitative immunoglobulin levels. If low, conf rm diagnosis with Band T-cell subsets (absent B cells; T cells are often high); absent tonsils and other lymphoid tissue may be a clue. Treat with prophylactic antibiotics and IVIG. Quantitative immunoglobulin levels; confrm with Band T-cell subsets; treat with

1	are often high); absent tonsils and other lymphoid tissue may be a clue. Treat with prophylactic antibiotics and IVIG. Quantitative immunoglobulin levels; confrm with Band T-cell subsets; treat with IVIG. Quantitative IgA levels; treat infections. Do not give immunoglobulins (can lead to the production of anti-IgA antibodies). T cell Thymic aplasia (DiGeorge syndrome) See mnemonic. Presents with tetany (2° to hypocalcemia) in the f rst days of life. Variable risk of infection. ↑↑↑ infections with fungi and Pneumocystis jiroveci pneumonia (formerly P. carinii). Absolute lymphocyte count; mitogen stimulation response; delayed hypersensitivity skin testing. Treat with bone marrow transplantation and IVIG for antibody def ciency; PCP prophylaxis. Thymus transplantation is an alternative. Combined Ataxia-telangiectasia Severe combined immunodef ciency (SCID) Oculocutaneous telangiectasias and progressive cerebellar ataxia. Caused by a DNA repair defect. Severe lack of B and T cells. ↑

1	Combined Ataxia-telangiectasia Severe combined immunodef ciency (SCID) Oculocutaneous telangiectasias and progressive cerebellar ataxia. Caused by a DNA repair defect. Severe lack of B and T cells. ↑ incidence of non-Hodgkin’s lymphoma, leukemia, and gastric carcinoma. Severe, frequent bacterial infections; chronic candidiasis; and opportunistic organisms. No specifc treatment; may require IVIG depending on the severity of the Ig def ciency. Treat with bone marrow transplant or stem cell transplant and IVIG for antibody def ciency. Needs PCP prophylaxis.

1	T AB LE 2.1 3-4. Pediatric Immunodeficiencies (continued)

1	Combined (continued) Wiskott-Aldrich syndrome An X-linked disorder with less severe Band T-cell dysfunction. Patients have eczema, ↑ IgE/IgA, ↓ IgM, and thrombocytopenia. The classic presentation involves bleeding, eczema, and recurrent otitis media. ↑↑ risk of atopic disorders, lymphoma/leukemia, and infection from S. pneumoniae, S. aureus, and H. inﬂ uenzae type b. Phagocytic Chronic granulomatous disease (CGD) Leukocyte adhesion def ciency Chédiak-Higashi syndrome An X-linked (2/3) or autosomal-recessive (1/3) disease with def cient superoxide production by PMNs and macrophages. Anemia, lymphadenopathy, and hypergamma-globulinemia may be present. A defect in the chemotaxis of leukocytes. An autosomal-recessive disorder that leads to Chronic skin, pulmonary, GI, and urinary tract infections; osteomyelitis and hepatitis. Infecting organisms are catalase . ↑ risk of infection with Aspergillus. May have granulomas of the skin and GI/GU tracts. Recurrent skin, mucosal, and pulmonary

1	osteomyelitis and hepatitis. Infecting organisms are catalase . ↑ risk of infection with Aspergillus. May have granulomas of the skin and GI/GU tracts. Recurrent skin, mucosal, and pulmonary infections. May present as omphalitis in the newborn period with delayed separation of the umbilical cord. ↑↑ incidence of overwhelming infections with S. pyogenes, a defect in neutrophil S. aureus, and Pseudomonas chemotaxis/microtubule species. polymerization.

1	The syndrome includes oculocutaneous albinism, neuropathy, and neutropenia. Treatment is supportive (IVIG and antibiotics). Patients rarely survive to adulthood. Patients with severe infections may be treated with a bone marrow transplant. Absolute neutrophil count with neutrophil assays. The nitroblue tetrazolium test is diagnostic for CGD. Treat with daily TMP-SMX; judicious use of antibiotics during infections. IFN-γ can ↓ the incidence of serious infection. Bone marrow transplantation and gene therapy are new therapies. No pus with minimal infammation in wounds (due to a chemotaxis defect). High WBCs in blood. Bone marrow transplantation is curative. Look for giant granules in neutrophils. Bone marrow transplant is the treatment of choice. T AB LE 2.1 3-4. Pediatric Immunodeficiencies (continued)

1	Look for giant granules in neutrophils. Bone marrow transplant is the treatment of choice. T AB LE 2.1 3-4. Pediatric Immunodeficiencies (continued) Complement C1 esterase def ciency (hereditary angioedema) Terminal complement def ciency (C5–C9) An autosomal-dominant disorder with recurrent episodes of angioedema lasting 2–72 hours and provoked by stress or trauma. Inability to form membrane attack complex (MAC). Can lead to life-threatening airway edema. Recurrent meningococcal or gonococcal infections. Rarely, lupus or glomerulonephritis. Total hemolytic complement (CH50) to assess the quantity and function of complement. Purif ed C1 esterase and FFP can be used prior to surgery. Meningococcal vaccine and appropriate antibiotics.

1	Total hemolytic complement (CH50) to assess the quantity and function of complement. Purif ed C1 esterase and FFP can be used prior to surgery. Meningococcal vaccine and appropriate antibiotics. B-cell def ciencies: Most common (50%). Typically present after six months of age with recurrent sinopulmonary, GI, and urinary tract infections with encapsulated organisms (H. inﬂ uenzae, S. pneumoniae, Neisseria meningitidis). Treated with IVIF (except for IgA deficiencies). T-cell def ciencies: Tend to present earlier (1–3 months) with opportunistic and low-grade fungal, viral, and intracellular bacterial infections (e.g., mycobacteria). 2° B-cell dysfunction may also be seen. Phagocyte def ciencies: Characterized by mucous membrane infections, abscesses, and poor wound healing. Infections with catalaseorganisms (e.g., S. aureus), fungi, and gram-enteric organisms are common.

1	Complement def ciencies: Present in children with congenital asplenia or splenic dysfunction (sickle cell disease). Characterized by recurrent bacterial infections with encapsulated organisms. A multisystemic acute vasculitis that primarily affects young children (80% are < 5 years of age), particularly those of Asian ancestry. Divided into acute, sub-acute, and chronic phases. Acute phase: Lasts 1–2 weeks and presents with the following symptoms (fever plus four or more of the criteria below are required for diagnosis): Fever (usually > 40°C) for at least f ve days. Bilateral, nonexudative, painless conjunctivitis sparing the limbic area. Polymorphous rash (primarily truncal). Cervical lymphadenopathy (often painful and unilateral, with at least one node > 1.5 cm). Diffuse mucous membrane erythema (e.g., “strawberry tongue”); dry, red, chapped lips.

1	Cervical lymphadenopathy (often painful and unilateral, with at least one node > 1.5 cm). Diffuse mucous membrane erythema (e.g., “strawberry tongue”); dry, red, chapped lips. Erythema of the palms and soles; indurative edema of the hands and feet; late desquamation of the fingertips (in the subacute phase). Other manifestations include sterile pyuria, gallbladder hydrops, hepatitis, and arthritis. Untreated Kawasaki disease can lead to coronary aneurysms and even myocardial infarction! Conjunctivitis Rash Adenopathy Strawberry tongue Hands and feet (red, swollen, f aky skin) BURN (fever > 40°C for ≥ 5 days) Subacute phase: Begins after the abatement of fever and typically lasts for an additional 2–3 weeks. Manifestations are thrombocytosis and elevated ESR. Untreated children may begin to develop coronary artery aneurysms (40%); all patients should be assessed by echocardiography at diagnosis.

1	Chronic phase: Begins when all clinical symptoms have disappeared; lasts until ESR returns to baseline. Untreated children are at risk of aneurysmal expansion and MI. High-dose ASA (for inﬂammation and fever) and IVIG (to prevent aneurysms). Low-dose ASA is then continued, usually for six weeks. Children who develop coronary aneurysms may require chronic anticoagulation with ASA or other antiplatelet medications. Corticosteroids may be used in IVIG-refractory cases, but routine use is not recommended. Referral to a pediatric cardiologist and routine follow-up echocardiograms to assess for progression of coronary artery aneurysms are important parts of ongoing management. An autoimmune disorder manifesting as arthritis with “morning stiffness” and gradual loss of motion that is present for at least six weeks in a patient < 16 years of age. Formerly known as juvenile rheumatoid arthritis (JRA).

1	Pauciarticular (oligoarthritis): Most common; four or fewer joints are involved (usually weight-bearing); usually ANA and RF . Involves young females; uveitis is common and requires slit-lamp exam for evaluation. No systemic symptoms. Polyarthritis: Involves five or more joints; symmetric. RF positivity is rare and indicates severe disease; younger children may be ANA with milder disease. Systemic symptoms are rare. Systemic-onset (Still’s disease): May present with recurrent high fever (usually > 39°C), hepatosplenomegaly, and a salmon-colored macular rash; usually RF and ANA . NSAIDs and strengthening exercises. Corticosteroids and immunosuppressive medications (methotrexate; anti-TNF agents such as etanercept) are second-line agents. Corticosteroids are used in the presence of carditis.

1	Corticosteroids and immunosuppressive medications (methotrexate; anti-TNF agents such as etanercept) are second-line agents. Corticosteroids are used in the presence of carditis. A suppurative infection of the middle ear cavity that is common in children. Up to 75% of children have at least three episodes by the age of two. Common pathogens include S. pneumoniae, nontypable H. inﬂ uenzae, Moraxella catarrhalis, and viruses such as inﬂuenza A, RSV, and parainﬂ uenza virus. Symptoms include ear pain, fever, crying, irritability, difficulty feeding or sleeping, vomiting, and diarrhea. Young children may tug on their ears. Signs on otoscopic exam reveal an erythematous tympanic membrane (TM), bulging or retraction of the TM, loss of TM light reﬂ ex, and ↓ TM mobility (test with an insufﬂ ator bulb). High-dose amoxicillin (80–90 mg/kg/day) × 10 days for empiric therapy. Resistant cases may require amoxicillin/clavulanic acid.

1	High-dose amoxicillin (80–90 mg/kg/day) × 10 days for empiric therapy. Resistant cases may require amoxicillin/clavulanic acid. Complications include TM perforation, mastoiditis, meningitis, cholesteatomas, and chronic otitis media. Recurrent otitis media can cause hearing loss with resultant speech and language delay. Chronic otitis media may require tympanostomy tubes. An acute inﬂammatory illness of the small airways that primarily affects infants and children < 2 years of age, often in the fall or winter months. RSV is the most common cause; others include parainﬂ uenza, inﬂ uenza, and metapneumovirus. Progression to respiratory failure is a potentially fatal complication. For severe RSV, risk factors include age < 6 months, male gender, prematurity, heart or lung disease, and immunodeficiency. Presents with low-grade fever, rhinorrhea, cough, and apnea (in young infants).

1	Presents with low-grade fever, rhinorrhea, cough, and apnea (in young infants). Exam reveals tachypnea, wheezing, intercostal retractions, crackles, prolonged expiration, expiratory wheezing, and hyperresonance to percussion. Predominantly a clinical diagnosis; routine cases do not need blood work or a CXR. CXR may be obtained to rule out pneumonia and may show hyperinﬂ ation of the lungs with ﬂattened diaphragms, interstitial infiltrates, and atelectasis. Nasopharyngeal aspirate to test for RSV is highly sensitive and specific but has little effect on management (infants should be treated for bronchiolitis regardless of whether RSV is or not). Treatment is primarily supportive; treat mild disease with outpatient management using ﬂuids and nebulizers if needed.

1	Treatment is primarily supportive; treat mild disease with outpatient management using ﬂuids and nebulizers if needed. Hospitalize in the setting of marked respiratory distress, O2 saturation of < 92%, toxic appearance, dehydration/poor oral feeding, a history of prematurity (< 34 weeks), age < 3 months, underlying cardiopulmonary disease, or unreliable parents. Treat inpatients with contact isolation, hydration, and O2. A trial of aerosolized albuterol may be attempted; continue albuterol therapy only if effective. RSV is the most common cause of bronchiolitis. Ribavirin is an antiviral drug that has a controversial role in bronchiolitis treatment. It is sometimes used in high-risk infants with underlying heart, lung, or immune disease. RSV prophylaxis with injectable polyor monoclonal antibodies (RespiGam or Synagis) is recommended in winter for high-risk patients ≤ 2 years of age (e.g., those with a history of prematurity, chronic lung disease, or congenital heart disease).

1	An acute viral inﬂammatory disease of the larynx, primarily within the subglottic space. Pathogens include parainf uenza virus type 1 (most common), 2, and 3; RSV; inﬂuenza; and adenovirus. Bacterial superinfection may progress to tracheitis. Prodromal URI symptoms are typically followed by low-grade fever, mild dyspnea, inspiratory stridor that worsens with agitation, a hoarse voice, and a characteristic barking cough (usually at night). Diagnosed by clinical impression; often based on the degree of stridor and respiratory distress. AP neck film may show the classic “steeple sign” from subglottic narrowing (see Figure 2.13-2), but the finding is neither sensitive nor specif c. Table 2.13-5 differentiates croup from epiglottitis and tracheitis. FIGURE 2.13-2. Croup.

1	Table 2.13-5 differentiates croup from epiglottitis and tracheitis. FIGURE 2.13-2. Croup. The x-ray shows marked subglottic narrowing of the airway (“steeple sign”). (Reproduced, with permission, from Saunders CE. Current Emergency Diagnosis & Treatment, 4th ed. Stamford, CT: Appleton & Lange, 1992: 448.) T AB LE 2.1 3-5. Characteristics of Croup, Epiglottitis, and Tracheitis

1	T AB LE 2.1 3-5. Characteristics of Croup, Epiglottitis, and Tracheitis Age group affected 3 months to 3 years 3–7 years 3 months to 2 years Incidence in children presenting with stridor 88% 8% 2% Pathogen Parainf uenza virus H. inﬂ uenzae Often S. aureus Onset Prodrome (1–7 days) Rapid (4–12 hours) Prodrome (3 days) leading to acute decompensation (10 hours) Fever severity Low grade High grade Intermediate grade Associated symptoms Barking cough, hoarseness Muffed voice, drooling Variable respiratory distress Position preference None Seated, neck extended None Response to racemic epinephrine Stridor improves None None CXR fndings “Steeple sign” on AP flm “Thumbprint sign”’ on lateral f lm Subglottic narrowing Mild cases: Outpatient management with cool mist therapy and ﬂ uids. Moderate cases: May require supplemental O2, oral and IM corticosteroids, and nebulized racemic epinephrine.

1	Mild cases: Outpatient management with cool mist therapy and ﬂ uids. Moderate cases: May require supplemental O2, oral and IM corticosteroids, and nebulized racemic epinephrine. Severe cases (e.g., respiratory distress at rest, inspiratory stridor): Hospitalize and give nebulized racemic epinephrine. A serious and rapidly progressive infection of supraglottic structures (e.g., the epiglottis and aryepiglottic folds). Prior to immunization, H. inﬂ uenzae type b was the 1° pathogen. Common causes now include Streptococcus species, Epiglottitis can lead to nontypable H. inﬂ uenzae, and viral agents. obstruction. Presents with acute-onset high fever (39–40°C), dysphagia, drooling, a mufﬂed voice, inspiratory retractions, cyanosis, and soft stridor. Patients sit with the neck hyperextended and the chin protruding (“sniffing dog” position) and lean forward in a “tripod” position to maximize air entry.

1	Patients sit with the neck hyperextended and the chin protruding (“sniffing dog” position) and lean forward in a “tripod” position to maximize air entry. Untreated infection can lead to life-threatening airway obstruction and respiratory arrest. Diagnosed by clinical impression. The differential diagnosis must include diffuse and localized causes of airway obstruction (see Tables 2.13-5 and 2.13-6). The airway must be secured before def nitive diagnosis. In light of potential laryngospasm and airway compromise, do not examine the throat unless an anesthesiologist or otolaryngologist is present. Definitive diagnosis is made via direct fiberoptic visualization of a cherry-red, swollen epiglottis and arytenoids. Lateral x-ray shows a swollen epiglottis obliterating the valleculae (“thumbprint sign”; see Figure 2.13-3). This disease is a true emergency. Keep the patient (and parents) calm, call anesthesia, and transfer the patient to the OR.

1	This disease is a true emergency. Keep the patient (and parents) calm, call anesthesia, and transfer the patient to the OR. Treat with endotracheal intubation or tracheostomy and IV antibiotics (ceftriaxone or cefuroxime). Bacterial meningitis most often occurs in children < 3 years of age; common organisms include S. pneumoniae, N. meningitidis, and E. coli. Enteroviruses are the most common agents of viral meningitis and occur in children of all ages. Risk factors include sinofacial infections, trauma, and sepsis. ■Bacterial meningitis classically presents with the triad of headache, high fever, and nuchal rigidity. T AB LE 2.1 3-6. Comparison of Retropharyngeal Abscess and Peritonsillar Abscess

1	Age group affected Six months to six years. Usually > 10 years of age. History/PE Muffed “hot potato” voice; trismus; drooling; cervical lymphadenopathy. Usually unilateral; may see mass in the posterior pharyngeal wall on visual inspection. Muffed “hot potato” voice; trismus; drooling; displacement of the affected tonsil medially and laterally. Pathogen Group A streptococcus (most common); S. aureus; Bacteroides. Group A streptococcus (most common); S. aureus; S. pneumoniae; anaerobes. Preferred position Supine with the neck extended (sitting up or fexing the neck worsens symptoms). None. Diagnosis On lateral neck x-ray, the soft tissue plane should be ≤ 50% of the width of the corresponding vertebral body. Contrast CT of the neck helps differentiate abscess from cellulitis. Usually clinical. Treatment Aspiration or incision and drainage of abscess; antibiotics. Incision and drainage +/− tonsillectomy; antibiotics. FIGURE 2.13-3. Epiglottitis.

1	FIGURE 2.13-3. Epiglottitis. The classic swollen epiglottis (“thumbprint sign”; arrow) and obstructed airway are seen on lateral neck x-ray. (Reproduced, with permission, from Saunders CE. Current Emergency Diag nosis & Treatment, 4th ed. Stamford, CT: Appleton & Lange, 1992: 447.) Viral meningitis is typically preceded by a prodromal illness that includes fever, sore throat, and fatigue. Kernig’s sign (pain on knee extension when the hip is ﬂexed) and Brudzinski’s sign (pain with passive neck ﬂexion) are nonspecific signs of meningeal irritation. Additional physical exam findings may include signs of ↑ ICP (papilledema, cranial nerve palsies) or petechial rash (N. meningitidis). Signs in neonates include lethargy, hyperor hypothermia, poor tone, a bulging fontanelle, and vomiting. Head CT to rule out ↑ ICP (risk of brain stem herniation). Perform an LP; send cell count with differential, glucose and protein levels, Gram stain, and culture.

1	Head CT to rule out ↑ ICP (risk of brain stem herniation). Perform an LP; send cell count with differential, glucose and protein levels, Gram stain, and culture. Empiric antibiotic therapy regimens (ceftriaxone, vancomycin, ampicillin) should be administered until bacterial meningitis can be excluded. Neonates should receive ampicillin and cefotaxime or gentamicin. Consider acyclovir if there is concern for herpes encephalitis (e.g., if the mother had HSV lesions at the time of the infant’s birth). Older children should receive ceftriaxone and vancomycin. A bacterial infection caused by Bordetella pertussis (whooping cough), a grambacillus. The DTaP vaccine (given in five doses in early childhood) is protective, but immunity wanes by adolescence. Adolescents and young adults serve as the primary reservoir for pertussis. Pertussis can be life threatening for young infants but is generally a milder infection in older children and adults.

1	Has three stages: (1) catarrhal (mild URI symptoms; lasts 1–2 weeks), (2) paroxysmal (paroxysms of cough with inspiratory whoop and post-tussive emesis; lasts 2–3 months), and (3) convalescent (symptoms wane). Patients most often present in the paroxysmal stage but are most contagious in the catarrhal stage. The classic presentation is an infant < 6 months of age with post-tussive emesis and apnea. Labs show an elevated WBC count with lymphocytosis (often ≥ 70%). Culture is the gold standard. Hospitalize infants < 6 months of age. Give erythromycin × 14 days to patients and close contacts (including day care contacts). Patients should not return to school or day care until five days of antibiotics have been administered or until three weeks have elapsed if no therapy has been initiated. Table 2.13-7 outlines the clinical presentation of common viral exanthems. TABLE 2.13-7. Viral Exanthems

1	Erythema infectiosum (f fth disease) Parvovirus B19 Prodrome: None; fever is often absent or low grade. Rash: “Slapped-cheek,” erythematous rash. An erythematous, pruritic, maculopapular rash starts on the arms and spreads to the trunk and legs. Worsens with fever and sun exposure. Arthritis, hemolytic anemia, encephalopathy. Congenital infection is associated with fetal hydrops and death. Aplastic crisis may be precipitated in children with ↑ RBC turnover (e.g., sickle cell anemia, hereditary spherocytosis) or in those with ↓ RBC production (e.g., severe iron def ciency anemia). Measles Paramyxovirus Prodrome: Low-grade fever with Cough, Coryza, and Conjunctivitis (the “3 C’s”); Koplik’s spots (small irregular red spots with central gray specks) appear on the buccal mucosa after 1–2 days. Rash: An erythematous maculopapular rash spreads from the head toward the feet. Common: Otitis media, pneumonia, laryngotracheitis. Rare: Subacute sclerosing panencephalitis.

1	TABLE 2.13-7. Viral Exanthems (continued)

1	Rubella Rubella virus Prodrome: Asymptomatic or tender, generalized lymphadenopathy. Rash: Presents with an erythematous, tender maculopapular rash that also starts on the face and spreads distally. In contrast to measles, children with rubella often have only a low-grade fever and do not appear as ill. Polyarthritis may be seen in adolescents. Encephalitis, thrombocytopenia (a rare complication of postnatal infection). Congenital infection is associated with congenital anomalies. Roseola infantum HHV-6 Prodrome: Acute onset of high fever (> 40°C); no other symptoms for 3–4 days. Rash: A maculopapular rash appears as fever breaks (begins on the trunk and quickly spreads to the face and extremities) and often lasts < 24 hours. Febrile seizures may occur as a result of rapid fever onset. Varicella Varicella-zoster virus (VZV) Prodrome: Mild fever, anorexia, and malaise precede the rash by 24 hours. Rash: Generalized, pruritic, “teardrop” vesicular periphery; lesions are often at

1	Varicella Varicella-zoster virus (VZV) Prodrome: Mild fever, anorexia, and malaise precede the rash by 24 hours. Rash: Generalized, pruritic, “teardrop” vesicular periphery; lesions are often at different stages of healing. Infectious from 24 hours before eruption until lesions crust over. Progressive varicella with meningoencephalitis and hepatitis occurs in immunocompromised children. Congenital infection is associated with congenital anomalies. Varicella zoster VZV Prodrome: Reactivation of varicella infection; starts as pain along an affected sensory nerve. Rash: Pruritic “teardrop” vesicular rash in a dermatomal distribution. Uncommon unless the patient is immunocompromised. Encephalopathy, aseptic meningitis, pneumonitis, TTP, Guillain-Barré syndrome, cellulitis, arthritis. Hand-foot-and-mouth disease Coxsackie A Prodrome: Fever, anorexia, oral pain. Rash: Oral ulcers; maculopapular vesicular rash on the hands and feet and sometimes on the buttocks. None (self-limited).

1	APGAR (0, 1, 2 in each category) Appearance (blue/pale, pink trunk, all pink) Pulse (0, < 100, > 100) Grimace with stimulation (0, grimace, grimace and cough) Activity (limp, some, active) Respiratory effort (0, irregular, regular) Direct hyperbilirubinemia is always pathologic. A rapid scoring system that helps evaluate the need for neonatal resuscitation. Each of five parameters (see the mnemonic APGAR) is assigned a score of 0–2 at one and five minutes after birth. Scores of 8–10: Typically reﬂect good cardiopulmonary adaptation. Scores of 4–7: Indicate the possible need for resuscitation. Infants should be observed, stimulated, and possibly given ventilatory support. Scores of 0–3: Indicate the need for immediate resuscitation. Table 2.13-8 describes selected congenital malformations. An elevated serum bilirubin concentration (> 5 mg/dL) due to ↑ hemolysis or ↓ excretion. Subtypes are as follows: Conjugated (direct) hyperbilirubinemia: Always pathologic.

1	An elevated serum bilirubin concentration (> 5 mg/dL) due to ↑ hemolysis or ↓ excretion. Subtypes are as follows: Conjugated (direct) hyperbilirubinemia: Always pathologic. Unconjugated (indirect) hyperbilirubinemia: May be physiologic or pathologic. See Table 2.13-9 for differentiating characteristics. Kernicterus: A complication of unconjugated hyperbilirubinemia that results from irreversible bilirubin deposition in the basal ganglia, pons, and cerebellum. It typically occurs at levels of > 25–30 mg/dL and can be fatal. Risk factors include prematurity, asphyxia, and sepsis. The differential includes the following: Conjugated: Extrahepatic cholestasis (biliary atresia, choledochal cysts), intrahepatic cholestasis (neonatal hepatitis, inborn errors of metabolism, TPN cholestasis), Dubin-Johnson syndrome, Rotor’s syndrome, TORCH infections (see the Infectious Disease chapter).

1	Unconjugated: Physiologic jaundice, hemolysis, breast milk jaundice, ↑ enterohepatic circulation (e.g., GI obstruction), disorders of bilirubin metabolism, hemolysis, sepsis, Crigler-Najjar syndrome, Gilbert’s syndrome. The history should focus on diet (breast milk or formula), intrauterine drug exposure, and family history (hemoglobinopathies, enzyme deficiencies, RBC defects). Physical exam may reveal signs of hepatic or GI dysfunction (abdominal distention, delayed passage of meconium, light-colored stools, dark urine), infection, or hemoglobinopathies (cephalohematomas, bruising, pallor, petechiae, and hepatomegaly). Kernicterus presents with lethargy, poor feeding, a high-pitched cry, hypertonicity, and seizures; jaundice may follow a cephalopedal progression as bilirubin concentrations ↑. CBC with peripheral blood smear; blood typing of mother and infant (for ABO or Rh incompatibility); Coombs’ test and bilirubin levels.

1	CBC with peripheral blood smear; blood typing of mother and infant (for ABO or Rh incompatibility); Coombs’ test and bilirubin levels. Ultrasound and/or HIDA scan can confirm suspected cholestatic disease. T AB LE 2.1 3-8. Selected Congenital Malformations

1	Tracheoesophageal f stula Tract between the trachea and esophagus. Associated with defects such as esophageal atresia and VACTERL (Vertebral, Anal, Cardiac, Tracheal, Esophageal, Renal, Limb) anomalies. Presentation: Polyhydramnios in utero, ↑ oral secretions, inability to feed, gagging, aspiration pneumonia, respiratory distress. Diagnosis: CXR showing an NG tube coiled in the esophagus identifes esophageal atresia. The presence of air in the GI tract is suggestive; confrm with bronchoscopy. Treatment: Surgical repair. Congenital diaphragmatic hernia GI tract segments protrude through the diaphragm into the thorax; 90% are posterior left (Bochdalek). Presentation: Respiratory distress (from pulmonary hypoplasia and pulmonary hypertension); sunken abdomen; bowel sounds over the left hemithorax. Diagnosis: Ultrasound in utero; confrmed by postnatal CXR. Treatment: High-frequency ventilation or extracorporeal membrane oxygenation to manage pulmonary hypertension; surgical repair.

1	hemithorax. Diagnosis: Ultrasound in utero; confrmed by postnatal CXR. Treatment: High-frequency ventilation or extracorporeal membrane oxygenation to manage pulmonary hypertension; surgical repair. Gastroschisis Herniation of the intestine only through the abdominal wall next to the umbilicus (usually on the right) with no sac. Presentation: Polyhydramnios in utero; often premature; associated with GI stenoses or atresia. Treatment: A surgical emergency! Single-stage closure is possible in only 10% of cases. Omphalocele Herniation of abdominal viscera through the abdominal wall at the umbilicus into a sac covered by peritoneum and amniotic membrane. Presentation/diagnosis: Polyhydramnios in utero; often premature; associated with other GI and cardiac defects. Seen in Beckwith-Wiedemann syndrome and trisomies. Treatment: C-section can prevent sac rupture; if the sac is intact, postpone surgical correction until the patient is fully resuscitated. Keep the sac covered/stable with

1	syndrome and trisomies. Treatment: C-section can prevent sac rupture; if the sac is intact, postpone surgical correction until the patient is fully resuscitated. Keep the sac covered/stable with petroleum and gauze. Intermittent NG suction to prevent abdominal distention. Duodenal atresia Complete or partial failure of the duodenal lumen to recanalize during gestational weeks 8–10. Presentation: Polyhydramnios in utero; bilious emesis within hours after frst feeding; associated with Down syndrome and other cardiac/GI anomalies (e.g., annular pancreas, malrotation, imperforate anus). Diagnosis: Abdominal radiographs show the “double-bubble” sign (air bubbles in the stomach and duodenum) proximal to the site of the atresia. Treatment: Surgical repair.

1	For direct hyperbilirubinemia, check LFTs, bile acids, blood cultures, sweat test, and tests for aminoacidopathies and α1-antitrypsin deficiency. A jaundiced neonate who is febrile, hypotensive, and/or tachypneic needs a full sepsis workup and ICU monitoring. Treat underlying causes (e.g., infection). Treat unconjugated hyperbilirubinemia with phototherapy (for mild elevations) or exchange transfusion (for severe elevations > 20 mg/dL). Start T AB LE 2.1 3-9. Physiologic vs. Pathologic Jaundice RDS is the most common cause of respiratory failure in preterm infants. An L/S ratio < 2:1 indicates a need for maternal glucocorticoid administration.

1	RDS is the most common cause of respiratory failure in preterm infants. An L/S ratio < 2:1 indicates a need for maternal glucocorticoid administration. Not present until 72 hours after birth. Present in the frst 24 hours of life. Bilirubin ↑ < 5 mg/dL/day. Bilirubin ↑ > 0.5 mg/dL/hour. Bilirubin peaks at < 14–15 mg/dL. Bilirubin ↑ to > 15 mg/dL. Direct bilirubin is < 10% of total. Direct bilirubin is > 10% of total. Resolves by one week in term infants and two weeks in preterm infants. Persists beyond one week in term infants and two weeks in preterm infants. phototherapy earlier (10–15 mg/dL) for preterm infants. Phototherapy with conjugated hyperbilirubinemia can lead to skin bronzing.

1	phototherapy earlier (10–15 mg/dL) for preterm infants. Phototherapy with conjugated hyperbilirubinemia can lead to skin bronzing. The most common cause of respiratory failure in preterm infants (affects > 70% of infants born at 28–30 weeks’ gestation); formerly known as hyaline membrane disease. Surfactant def ciency leads to poor lung compliance, alveolar collapse, and atelectasis. Risk factors include maternal DM, male gender, and the second born of twins. Presents in the first 48–72 hours of life with a respiratory rate > 60/min, progressive hypoxemia, cyanosis, nasal ﬂaring, intercostal retractions, and expiratory grunting. Check ABGs, CBC, and blood cultures to rule out infection. Diagnosis is based mainly on characteristic CXR findings: RDS: “Ground-glass” appearance, diffuse atelectasis, and air bronchograms on CXR. Transient tachypnea of the newborn: Retained amniotic ﬂ uid results in prominent perihilar streaking in interlobular fissures.

1	Transient tachypnea of the newborn: Retained amniotic ﬂ uid results in prominent perihilar streaking in interlobular fissures. Meconium aspiration: Coarse, irregular infiltrates; hyperexpansion and pneumothoraces. Congenital pneumonia: Nonspecific patchy infiltrates; neutropenia, tracheal aspirate, and Gram stain suggest the diagnosis. Continuous positive airway pressure (CPAP) or intubation and mechanical ventilation. Artificial surfactant administration ↓ mortality. Pretreat mothers at risk for preterm delivery (< 30 weeks’ gestation) with corticosteroids; if > 30 weeks, monitor fetal lung maturity via a lecithin-to-sphingomyelin (L/S) ratio and the presence of phosphatidylglycerol in amniotic ﬂ uid. Persistent PDA, bronchopulmonary dysplasia, retinopathy of prematurity, barotrauma from positive pressure ventilation, intraventricular hemorrhage, and NEC are complications of treatment.

1	Persistent PDA, bronchopulmonary dysplasia, retinopathy of prematurity, barotrauma from positive pressure ventilation, intraventricular hemorrhage, and NEC are complications of treatment. A range of nonhereditary, nonprogressive disorders of movement and posture; the most common movement disorder in children. Often results from perinatal neurologic insult, but in most cases the cause is unknown. Risk factors include low birth weight, intrauterine exposure to maternal infection, prematurity, perinatal asphyxia, trauma, brain malformation, and neonatal cerebral hemorrhage. Categories include the following: Pyramidal (spastic): Spastic paresis of any or all limbs. Accounts for 75% of cases. Mental retardation is present in up to 90% of cases.

1	Pyramidal (spastic): Spastic paresis of any or all limbs. Accounts for 75% of cases. Mental retardation is present in up to 90% of cases. Extrapyramidal (dyskinetic): A result of damage to extrapyramidal tracts. Subtypes are ataxic (difficulty coordinating purposeful movements), choreoathetoid, and dystonic (uncontrollable jerking, writhing, or posturing). Abnormal movements worsen with stress and disappear during sleep. May be associated with seizure disorders, behavioral disorders, hearing or vision impairment, learning disabilities, and speech deficits. Affected limbs may show hyperreﬂexia, pathologic reﬂexes (e.g., Babinski), ↑ tone/contractures, weakness, and/or underdevelopment. ■Toe walking and scissor gait are common. Hip dislocations and scoliosis may be seen.

1	■Toe walking and scissor gait are common. Hip dislocations and scoliosis may be seen. Diagnosed by clinical impression. Ultrasound or CT may be useful in infants to identify intracranial hemorrhage or structural malformations. MRI is diagnostic in older children. EEG may be useful in patients with seizures. There is no cure for cerebral palsy. Special education, physical therapy, braces, and surgical release of contractures may help. Treat spasticity with diazepam, dantrolene, or baclofen. Baclofen pumps and posterior rhizotomy may alleviate severe contractures. Usually occur in children between six months and five years of age who have no evidence of intracranial infection or other causes. Risk factors include a rapid ↑ in temperature and a history of febrile seizures in a close relative. Febrile seizures recur in 30% of patients. ■Seizures usually occur during the onset of fever and may be the first sign of an underlying illness (e.g., otitis media, roseola).

1	■Seizures usually occur during the onset of fever and may be the first sign of an underlying illness (e.g., otitis media, roseola). The most common presenting symptom of cerebral palsy is delayed motor development. Perform an LP if CNS infection is suspected in a patient with a febrile seizure. Simple febrile seizures do not cause brain damage, usually do not recur, and do not lead to an ↑ risk of epilepsy. ALL is the most common childhood malignancy, followed by CNS tumors and lymphomas. ■ Classified as simple or complex: Simple: A short-duration (< 15-minute), generalized seizure with one seizure in a 24-hour period. High fever (> 39°C) and fever onset within hours of the seizure are typical. Complex: A long-duration (> 15-minute) or focal seizure, or multiple seizures in a 24-hour period. Low-grade fever for several days before seizure onset may be present.

1	Complex: A long-duration (> 15-minute) or focal seizure, or multiple seizures in a 24-hour period. Low-grade fever for several days before seizure onset may be present. Focus on finding a source of infection. LP is indicated if there are clinical signs of CNS infection (e.g., altered consciousness, meningismus, a tense/ bulging anterior fontanelle) after ruling out ↑ ICP. No lab studies are needed if presentation is consistent with febrile seizures in children > 18 months of age. Infants < 12 months of age need a sepsis workup (CBC, UA, and blood, urine, and CSF culture). For atypical presentations, obtain electrolytes, serum glucose, blood cultures, UA, and CBC with differential. Use antipyretic therapy (acetaminophen; avoid ASA in light of the risk of Reye’s syndrome) and treat any underlying illness. Note that antipyretic therapy does not ↓ the recurrence of febrile seizures.

1	For complex seizures, perform a thorough neurologic evaluation, including an EEG and MRI. Chronic anticonvulsant therapy (e.g., diazepam or phenobarbital) may be necessary. The risk of recurrence is < 30% and is highest within one year of the initial episode. For simple febrile seizures, there is no ↑ risk of developmental abnormalities or epilepsy. Risk factors for the development of epilepsy include complex febrile seizures (~10% risk), a family history of epilepsy, and an abnormal neurologic exam or developmental delay. A hematopoietic malignancy of lymphocytic or myeloblastic origin. The most common childhood malignancy; 97% of cases are acute leukemias (ALL > AML). ALL is most common in male Caucasian children between two and five years of age; AML is seen most frequently in male African-American children throughout childhood. Associated with trisomy 21, Fanconi’s anemia, prior radiation, severe combined immunodeficiency, and congenital bone marrow failure states.

1	Symptoms are abrupt in onset. They are initially nonspecific (anorexia, fatigue) and are followed by bone pain with refusal to bear weight, fever (from neutropenia), anemia, ecchymoses, petechiae, and/or hepatosplenomegaly. CNS metastases may be associated with headache, vomiting, and papilledema. ■AML can present with a chloroma, a greenish soft-tissue tumor on the skin or spinal cord. CBC, coagulation studies, and peripheral blood smear (high numbers of blast cells). Peripheral smears show lymphoblasts in 90% of cases. WBC counts can be low, normal, or high. Obtain a bone marrow aspirate for immunophenotyping (TdT assay and a panel of monoclonal antibodies to Tand B-cell antigens) and genetic analysis, which help confirm the diagnosis. CXR to rule out a mediastinal mass. Chemotherapy based, including induction, consolidation, and maintenance phases.

1	CXR to rule out a mediastinal mass. Chemotherapy based, including induction, consolidation, and maintenance phases. Tumor lysis syndrome (hyperkalemia, hyperphosphatemia, hyperuricemia) is common prior to and during the initiation of treatment. Treat with ﬂ u-ids, diuretics, allopurinol, urine alkalinization, and reduction of phosphate syndrome at the onset of any intake. chemotherapy regimen. An embryonal tumor of neural crest origin. More than half of patients are < 2 years of age, and 70% of patients have distant metastases at presentation. Associated with neurofibromatosis, Hirschsprung’s disease, and the N-myc oncogene. Lesion sites are most commonly abdominal, thoracic, and cervical (in descending order). Symptoms may vary with location and may include a nontender abdominal mass (may cross the midline), Horner’s syndrome, hypertension, or cord compression (from a paraspinal tumor). Patients may have anemia, FTT, and fever.

1	Patients may have anemia, FTT, and fever. More than 50% of patients will have metastases at diagnosis. Signs include bone marrow suppression, proptosis, hepatomegaly, subcutaneous nodules, and opsoclonus/myoclonus. CT scan; fine-needle aspirate of tumor. Histologically appears as small, round, blue tumor cells with a characteristic rosette pattern. Elevated 24-hour urinary catecholamines (VMA and HVA). Bone scan and bone marrow aspirate. CBC, LFTs, coagulation panel, BUN/creatinine. Local excision plus postsurgical chemotherapy and/or radiation. Wilms’ tumor is associated A renal tumor of embryonal origin that is most commonly seen in children 2–5 years of age. Associated with Beckwith-Wiedemann syndrome (hemihy-hemihypertrophy. pertrophy, macroglossia, visceromegaly), neurofibromatosis, and WAGR syndrome (Wilms’, Aniridia, Genitourinary abnormalities, mental Retardation). Presents as an asymptomatic, nontender, smooth abdominal mass.

1	Presents as an asymptomatic, nontender, smooth abdominal mass. Abdominal pain, fever, hypertension, and microscopic or gross hematuria are seen. CBC, BUN, creatinine, and UA. Abdominal ultrasound. CT scans of the chest and abdomen are used to detect metastases. Local resection and nephrectomy with postsurgical chemotherapy and radiation depending on stage and histology. It is critical to distinguish between Ewing’s sarcoma and osteosarcoma (see Table 2.13-10). An important aspect of every well-child visit. Commonly tested advice includes the following: T AB LE 2.1 3-1 0. Ewing’s Sarcoma vs. Osteosarcoma

1	An important aspect of every well-child visit. Commonly tested advice includes the following: T AB LE 2.1 3-1 0. Ewing’s Sarcoma vs. Osteosarcoma Sarcoma (neuroectoderm); associated with chromosome 11:22 translocation. Osteoblasts (mesenchyme). Commonly seen in Caucasian male adolescents. Commonly seen in male adolescents. History/PE Local pain and swelling. Systemic symptoms (fever, anorexia, fatigue) are common. Local pain and swelling. Systemic symptoms are rare. Location Midshaft of long bones (femur, pelvis, f bula, humerus). Metaphyses of long bones (distal femur, proximal tibia, proximal humerus). Metastases to lungs in 20%. Leukocytosis, ↑ ESR. Lytic bone lesion with “onion skin” periosteal reaction on plain x-ray. ↑ alkaline phosphatase. “Sunburst” lytic bone lesions. Chest CT to rule out pulmonary metastases. Local excision, chemotherapy, and radiation. Local excision, chemotherapy. Origin Epidemiology Diagnosis Treatment Keep the water heater at < 48.8°C (< 120ºF).

1	Keep the water heater at < 48.8°C (< 120ºF). Babies should sleep on their backs without any stuffed animals or other toys in the crib (to ↓ the risk of SIDS). Car safety seats should be rear facing and should be placed in the back of the car (seats can face forward if the child is > 1 year of age and weighs > 20 lbs). No solid foods should be given prior to six months; they should then be introduced gradually and one at a time. Do not give cow’s milk prior to 12 months. Syrup of ipecac (an emetic) is no longer routinely recommended for accidental poisoning. Poison control should be contacted immediately for assistance. Objective hearing screening (otoacoustic emissions and/or auditory brain stem response) for newborns prior to discharge is common. Objective hearing screening is indicated for children with a history of meningitis, TORCH infections, measles and mumps, and recurrent otitis media or chronic middle ear infections.

1	Objective hearing screening is indicated for children with a history of meningitis, TORCH infections, measles and mumps, and recurrent otitis media or chronic middle ear infections. The red reﬂex should be checked at birth. Leukocoria is the lack of a red reﬂ ex. Strabismus (ocular misalignment) is normal until three months of age; beyond three months, children should be evaluated by a pediatric ophthalmologist and may require corrective lenses, occlusion, and/or surgery to prevent amblyopia (suppression of retinal images in a misaligned eye, leading to permanent vision loss). The Epidemiology chapter summarizes CDC-recommended vaccinations for the pediatric population. Contraindications and precautions in this population are as follows: Contraindications: Severe allergy to a vaccine component or a prior dose of vaccine. Patients who are allergic to eggs may not receive MMR or inﬂ uenza vaccine. Encephalopathy within seven days of prior pertussis vaccination.

1	Encephalopathy within seven days of prior pertussis vaccination. Avoid live vaccines (oral polio vaccine, varicella, MMR) in immunocompromised and pregnant patients (exception: HIV patients may receive MMR and varicella). Precautions: Current moderate to severe illness (with or without fever). Prior reactions to pertussis vaccine (fever > 40.5°C, shocklike state, persistent crying for > 3 hours within 48 hours of vaccination, or seizure within three days of vaccination). A history of receiving IVIG in the past year. The following are not contraindications to vaccination: Mild illness and/or low-grade fever. Current antibiotic therapy. Prematurity. Pneumococcal polysaccharide vaccine (PPV) should be administered to high-risk groups (sickle cell disease or splenectomy, immunodeficient). Leukocoria indicates retinoblastoma, congenital cataracts, or retinopathy of prematurity.

1	Leukocoria indicates retinoblastoma, congenital cataracts, or retinopathy of prematurity. New evidence has shown impaired intelligence and neurodevelopmental outcomes among children exposed to lead levels as low as 10 μg/dL. Most exposure in children is due to lead-contaminated household dust from leaded paint. Screening should be routinely performed at 12 and 24 months for patients living in high-risk areas (pre-1950s homes or zip codes with high percentages of elevated blood lead levels). Presents with irritability, hyperactivity or apathy, anorexia, intermittent abdominal pain, constipation, intermittent vomiting, and peripheral neuropathy (wrist or foot drop). Acute encephalopathy (usually with levels > 70 μg/dL) is characterized by ↑ ICP, vomiting, confusion, seizures, and coma. Blood lead level. CBC and peripheral blood smear show microcytic, hypochromic anemia and basophilic stippling. < 45 μg/dL and asymptomatic: Retest at 1–3 months; remove sources of lead exposure.

1	Blood lead level. CBC and peripheral blood smear show microcytic, hypochromic anemia and basophilic stippling. < 45 μg/dL and asymptomatic: Retest at 1–3 months; remove sources of lead exposure. 45–69 μg/dL: Chelation therapy (inpatient EDTA or outpatient oral succimer [DMSA]). ≥ 70 μg/dL: Chelation therapy (inpatient EDTA + BAL [IM dimercaprol]). Buspirone is another drug, in addition to SSRIs, that should not be used with MAOIs. Many OCD patients initially present to a nonpsychiatrist— e.g., they may consult a dermatologist with a skin complaint 2° to overwashing hands. Uncontrollable, excessive anxiety or worry about activities or events in life that leads to signifcant impairment or distress. The male-to-female ratio is 1:2; clinical onset is usually in the early 20s. Hx/PE: Presents with anxiety on most days (six or more months) and with three or more somatic symptoms (restlessness, fatigue, diff culty concentrating, irritability, muscle tension, disturbed sleep). Tx:

1	Tx: Lifestyle changes, psychotherapy, medication. SSRIs, venlafaxine, and buspirone are most often used (see Table 2.14-1). Benzodiazepines may be used for immediate symptom relief. Taper benzodiazepines as soon as long-term treatment is initiated (e.g., with SSRIs) in light of the high risk of tolerance and dependence. Do not stop benzodiazepines “cold turkey,” as patients may develop potentially lethal withdrawal symptoms similar to those of alcohol withdrawal. Patient education is essential. Characterized by obsessions and/or compulsions that lead to signif cant distress and dysfunction in social or personal areas. Typically presents in late adolescence or early adulthood; prevalence is equal in males and females. Often a chronic condition that is diff cult to treat.

1	Typically presents in late adolescence or early adulthood; prevalence is equal in males and females. Often a chronic condition that is diff cult to treat. Hx/PE: ■Obsessions: Persistent, unwanted, and intrusive ideas, thoughts, impulses, or images that lead to marked anxiety or distress (e.g., fear of contamination, fear of harm to oneself or to loved ones) and occur despite the patient’s attempts to prevent them. T AB LE 2.1 4-1. Anxiolytic Medications

1	T AB LE 2.1 4-1. Anxiolytic Medications SSRIs (f uoxetine, sertraline, paroxetine, citalopram, escitalopram) First-line treatment for generalized anxiety disorder, OCD, and PTSD. Nausea, GI upset, somnolence, sexual dysfunction, agitation. Buspirone Generalized anxiety disorder, OCD, PTSD. Seizures with chronic use. No tolerance, dependence, or withdrawal. β-blockers Performance anxiety, PTSD. Bradycardia, hypotension. Benzodiazepines Anxiety, insomnia, alcohol withdrawal, muscle spasm, night terrors, sleepwalking. ↓ sleep duration; risk of abuse, tolerance, and dependence; disinhibition in young or old patients; confusion. Flumazenil (competitive antagonist at GABA receptor) Antidote to benzodiazepine intoxication. Resedation; nausea, dizziness, vomiting, and pain at the injection site. Compulsions: Repeated mental acts or behaviors that neutralize anxiety from obsessions (e.g., hand washing, elaborate rituals for ordinary tasks, counting, excessive checking).

1	Compulsions: Repeated mental acts or behaviors that neutralize anxiety from obsessions (e.g., hand washing, elaborate rituals for ordinary tasks, counting, excessive checking). Patients recognize these behaviors as excessive and irrational products of their own minds (vs. obsessive-compulsive personality disorder, or OCPD; see Table 2.14-2). Patients wish they could get rid of the obsessions and/or compulsions. Tx: Pharmacotherapy (SSRIs are frst-line pharmacologic treatment; see Table 2.14-1); cognitive-behavioral therapy (CBT) using exposure and desensitization relaxation techniques. Patient education is imperative. Characterized by recurrent, unexpected panic attacks. Two to three times more common in females than in males. Agoraphobia is present in 30–50% of cases. The average age of onset is 25, but may occur at any age. Hx/PE:

1	Two to three times more common in females than in males. Agoraphobia is present in 30–50% of cases. The average age of onset is 25, but may occur at any age. Hx/PE: Panic attacks are def ned as discrete periods of intense fear or discomfort in which at least four of the following symptoms develop abruptly and peak within 10 minutes: tachypnea, chest pain, palpitations, diaphoresis, nausea, trembling, dizziness, fear of dying or “going crazy,” depersonalization, or hot f ashes. Perioral and/or acral paresthesias, when present, are fairly specif c to panic attacks, which produce hyperventilation and low oxygen saturation. Patients present with one or more months of concern about having additional attacks or signifcant behavior change as a result of the attacks—e.g., avoiding situations that may precipitate attacks. Elucidate if a patient has panic disorder with or without agoraphobia so that agoraphobia can also be addressed in the treatment plan.

1	Elucidate if a patient has panic disorder with or without agoraphobia so that agoraphobia can also be addressed in the treatment plan. The differential should include the following: Medical conditions: Angina, MI, arrhythmias, hyperthyroidism, vitamin B12 def ciency, pheochromocytoma. Psychiatric conditions: Substance-induced anxiety, generalized anxiety disorder, PTSD. Tx: CBT, pharmacotherapy (e.g., SSRIs, TCAs). Benzodiazepines (e.g., clonazepam) may be used for immediate relief, but long-term use should be avoided in light of the potential for addiction and tolerance (see Table 2.14-1). Taper benzodiazepines as soon as long-term treatment is initiated (e.g., SSRIs). Alprazolam (Xanax) is an SSRI that is sometimes used to treat panic disorder, but it has such a short half-life that patients can go into mild withdrawal within a day. TABLE 2.14-2. OCD vs. OCPD

1	Alprazolam (Xanax) is an SSRI that is sometimes used to treat panic disorder, but it has such a short half-life that patients can go into mild withdrawal within a day. TABLE 2.14-2. OCD vs. OCPD Characterized by obsessions and/or compulsions. Patients are excessively conscientious and inf exible. Patients recognize the obsessions/ compulsions and want to be rid of them (ego-dystonic). Patients do not recognize their behavior as problematic (ego-syntonic). Agoraphobia is def ned as fear of being alone in public places. Literally translated, it means “fear of the marketplace.” Top causes of PTSD in males are (1) rape and (2) combat. Top causes in females are (1) childhood abuse and (2) rape. In patients with a history of substance abuse, benzodiazepines should be avoided in view of their high addictive potential. Defned as follows:

1	In patients with a history of substance abuse, benzodiazepines should be avoided in view of their high addictive potential. Defned as follows: Social phobia: Characterized by marked fear provoked by social or performance situations in which embarrassment may occur. It may be specifc (e.g., public speaking, urinating in public) or general (e.g., social interaction) and often begins in adolescence. Specific phobia: Anxiety is provoked by exposure to a feared object or situation (e.g., animals, heights, airplanes). Most cases begin in childhood. Hx/PE: Presents with excessive or unreasonable fear and/or avoidance of an object or situation that is persistent and leads to signifcant distress or impairment in function. A related history of traumatic events or panic attacks may be present. Patients recognize that their fear is excessive. Tx:

1	Tx: Specific phobias: CBT involving desensitization through incremental exposure to the feared object or situation and relaxation techniques. Other options include supportive, family, and insight-oriented psychotherapy. Social phobias: CBT, SSRIs, low-dose benzodiazepines, or β-blockers (for performance anxiety) may be used (see Table 2.14-1). Follows exposure to an extreme, life-threatening traumatic event (e.g., assault, combat, witnessing a violent crime) that evoked intense fear, helplessness, or horror. Hx/PE: Characterized by reexperiencing of the event (e.g., nightmares), avoidance of stimuli associated with the trauma, numbed responsiveness (e.g., detachment, anhedonia), and ↑ arousal (e.g., hypervigilance, exaggerated startle) that lead to signifcant distress or impairment in functioning. Symptoms must persist for > 1 month.

1	Symptoms must persist for > 1 month. Survivor guilt, irritability, poor concentration, amnesia, personality change, sleep disturbance, substance abuse, depression, and suicidality may be present. Some controversy exists regarding the prevalence of PTSD, which some clinicians feel is overdiagnosed. It is therefore prudent to carefully consider other diagnoses in the differential—e.g., major depressive disorder, personality disorder, or adjustment disorder. Tx: SSRIs are frst line; buspirone, TCAs, and MAOIs may be helpful (see Table 2.14-1). Short-term agents targeting anxiety include β-blockers and α2-agonists (e.g., clonidine). Benzodiazepines are also used but should be avoided in light of their addictive potential, as there is a high incidence of substance abuse among individuals with PTSD. Psychotherapy and support groups are useful. Affect memory, orientation, judgment, and attention.

1	Psychotherapy and support groups are useful. Affect memory, orientation, judgment, and attention. An impairment in cognitive functioning with global deficits. Level of consciousness is stable. Prevalence is highest among those > 85 years of age. The course is persistent and progressive. The most common causes are Alzheimer’s disease (50%) and multi-infarct dementia (25%). Other causes are outlined in the mnemonic DEMENTIAS. Diagnostic criteria include memory impairment and one or more of the following: Aphasia: Language impairment. Apraxia: Inability to perform motor activities. Agnosia: Inability to recognize previously known objects. Impaired executive function (problems with planning, organizing, and abstracting) in the presence of a clear sensorium. Personality, mood, and behavior changes are common (e.g., wandering and aggression). A careful history and physical are critical. Serial mini-mental status exams should be performed.

1	Personality, mood, and behavior changes are common (e.g., wandering and aggression). A careful history and physical are critical. Serial mini-mental status exams should be performed. Rule out treatable causes of dementia; obtain CBC, RPR, CMP, TFTs, HIV, B12/folate, ESR, UA, and a head CT or MRI. n Table 2.14-3 outlines key characteristics distinguishing dementia from delirium. TABLE 2.14-3. Delirium vs. Dementia Causes of dementia— Degenerative diseases (Parkinson’s, Huntington’s) Endocrine (thyroid, parathyroid, pituitary, adrenal) Metabolic (alcohol, electrolytes, vitamin B12 def ciency, glucose, hepatic, renal, Wilson’s disease) Exogenous (heavy metals, carbon monoxide, drugs) Infection (meningitis, encephalitis, endocarditis, syphilis, HIV, prion diseases, Lyme disease) Stroke/Structure (vascular dementia, ischemia, vasculitis, normal pressure hydrocephalus)

1	Infection (meningitis, encephalitis, endocarditis, syphilis, HIV, prion diseases, Lyme disease) Stroke/Structure (vascular dementia, ischemia, vasculitis, normal pressure hydrocephalus) Level of attention Impaired (f uctuating). Usually alert. Onset Acute. Gradual. Course Fluctuating from hour to hour. Progressive deterioration. Consciousness Clouded. Intact. Hallucinations Present (often visual or tactile). Occur in approximately 30% of patients in highly advanced disease. Prognosis Reversible. Largely irreversible, but up to 15% of cases are due to treatable causes and are reversible. Major causes of delirium— It is common for delirium to be superimposed on dementia. MDEs can be present in major depressive disorder or in bipolar disorder types I and II. Provide environmental cues and a rigid structure for the patient’s daily life.

1	MDEs can be present in major depressive disorder or in bipolar disorder types I and II. Provide environmental cues and a rigid structure for the patient’s daily life. Cholinesterase inhibitors are used to treat. Low-dose antipsychotics may be used for agitation. Avoid benzodiazepines, which may worsen disinhibition and confusion. Family, caregiver, and patient education and support are imperative. An acute disturbance of consciousness with altered cognition that develops over a short period of time (usually hours to days). Children, the elderly, and hospitalized patients (e.g., ICU psychosis) are particularly susceptible. Major causes are outlined in the mnemonic I WATCH DEATH. Symptoms are potentially reversible if the underlying cause can be treated. Presents with acute onset of waxing and waning consciousness with lucid intervals and perceptual disturbances (hallucinations, illusions, delusions). Patients may be combative, anxious, paranoid, or stuporous.

1	Patients may be combative, anxious, paranoid, or stuporous. Also characterized by a ↓ attention span and short-term memory, a reversed sleep-wake cycle, and ↑ symptoms at night (sundowning). Check vitals, pulse oximetry, and glucose; perform physical and neurologic exams. Note recent medications (narcotics, anticholinergics, steroids, or benzodiazepines), substance use, prior episodes, medical problems, signs of organ failure (kidney, liver), and infection (occult UTI is common in the elderly; check UA). Order lab and radiologic studies to identify a possible underlying cause. Treat underlying causes (delirium is often reversible). Normalize fuids and electrolytes. Optimize the sensory environment. Use low-dose antipsychotics (e.g., haloperidol) for agitation and psychotic symptoms. Conservative use of physical restraints may be necessary to prevent harm to the patient or others. Also known as affective disorders.

1	Conservative use of physical restraints may be necessary to prevent harm to the patient or others. Also known as affective disorders. A mood disorder characterized by one or more major depressive episodes (MDEs). The male-to-female ratio is 1:2; lifetime prevalence ranges from 15% to 25%. Onset is usually in the mid-20s; in the elderly, prevalence ↑ with age. Chronic illness and stress ↑ risk. Up to 15% of patients die by suicide. Diagnosis requires depressed mood or anhedonia (loss of interest/pleasure) and five or more signs/symptoms from the SIG E CAPS mnemonic for a two-week period. Table 2.14-4 outlines the differential diagnosis of conditions that may be mistaken for depression. Selected depression subtypes include the following: Psychotic features: Typically mood-congruent delusions/hallucinations. Postpartum: Occurs within one month postpartum; has a 10% incidence and a high risk of recurrence. Psychotic symptoms are common.

1	Postpartum: Occurs within one month postpartum; has a 10% incidence and a high risk of recurrence. Psychotic symptoms are common. Atypical: Characterized by weight gain, hypersomnia, and rejection sensitivity. Seasonal: Depressive episodes tend to occur during a particular season, most commonly winter. Responds well to light therapy +/– antidepressants. Double depression: MDE in a patient with dysthymia. Has a poorer prognosis than MDE alone. Pharmacotherapy: Effective in 50–70% of patients. Allow 2–6 weeks to take effect; treat for ≥ 6 months (see Table 2.14-5). Psychotherapy: Psychotherapy combined with antidepressants is more effective than either treatment alone. Electroconvulsive therapy (ECT): Safe, highly effective, often lifesaving therapy that is reserved for refractory depression or psychotic depression, or if rapid improvement in mood is needed. T AB LE 2.1 4-4. Differential Diagnosis of Major Depression Symptoms of a depressive episode—

1	T AB LE 2.1 4-4. Differential Diagnosis of Major Depression Symptoms of a depressive episode— Interest (loss of interest or pleasure in activities) Guilt (feelings of worthlessness or inappropriate guilt)

1	Guilt (feelings of worthlessness or inappropriate guilt) Mood disorder due to a medical condition Hypothyroidism, Parkinson’s disease, CNS neoplasm, other neoplasm (e.g., pancreatic cancer), stroke (especially ACA stroke), dementias, parathyroid disorders. Substance-induced mood disorder Illicit drugs, alcohol, antihypertensives, corticosteroids, OCPs. Adjustment disorder with depressed mood A constellation of symptoms resembling an MDE but not meeting the criteria for MDE. Occurs within three months of an identif able stressor. Normal bereavement Occurs after the loss of a loved one. No severe impairment/suicidality; usually resolves in one year, but varies with cultural norms. May lead to major depressive disorder requiring treatment. Illusions/hallucinations of the deceased can be normal as long as the person recognizes them as such. Dysthymia Milder, chronic depression with depressed mood present most of the time for at least two years; often treatment resistant.

1	T AB LE 2.1 4-5. Indications and Side Effects of Common Antidepressants

1	SSRIs Fluoxetine, sertraline, paroxetine, citalopram, f uvoxamine Depression and anxiety. Sexual side effects, GI distress, agitation, insomnia, tremor, diarrhea. Serotonin syndrome (fever, myoclonus, mental status changes, cardiovascular collapse) can occur if SSRIs are used with MAOIs. Paroxetine can cause pulmonary hypertension in the fetus. Atypicals Bupropion, venlafaxine, mirtazapine, trazodone Depression, anxiety, and chronic pain. Bupropion: ↓ seizure threshold; minimal sexual side effects. Contraindicated in patients with eating disorders as well as in seizure patients. Venlafaxine: Diastolic hypertension. Mirtazapine: Weight gain, sedation. Trazodone: Highly sedating; priapism. Nortriptyline, desipramine, amitriptyline, imipramine Depression, anxiety disorder, chronic pain, migraine headaches, enuresis (imipramine). Lethal with overdose owing to cardiac conduction arrhythmias (e.g., long QRS). Monitor in the ICU for 3–4 days following an OD. Anticholinergic effects (dry

1	migraine headaches, enuresis (imipramine). Lethal with overdose owing to cardiac conduction arrhythmias (e.g., long QRS). Monitor in the ICU for 3–4 days following an OD. Anticholinergic effects (dry mouth, constipation, urinary retention, sedation). Phenelzine, tranylcypromine, selegiline (patch form available) Depression, especially atypical. Hypertensive crisis if taken with high-tyramine foods (cheese, red wine). Sexual side effects, orthostatic hypotension, weight gain. TCAs MAOIs

1	Discontinue SSRIs at least two weeks before starting an MAOI. Wait f ve weeks if the patient was on f uoxetine. Mixed states and mania are psychiatric emergencies 2° to impaired judgment and great risk of harm to self and others. May also be used for mania and psychosis. Usually requires 6–12 treatments. Adverse effects include postictal confusion, arrhythmias, headache, and anterograde amnesia. ■Contraindications include recent MI/stroke, intracranial mass, and high anesthetic risk (a relative contraindication). Phototherapy: Effective for patients whose depression has a seasonal pattern. Transcranial magnetic stimulation (TMS): Now approved for the treatment of major depression. TMS is about as effective as medications but is not as effective as ECT.

1	Transcranial magnetic stimulation (TMS): Now approved for the treatment of major depression. TMS is about as effective as medications but is not as effective as ECT. Prevalence is approximately 1% for type I and an additional 3% for type II; males and females are affected equally. A family history of bipolar illness signif cantly ↑ risk. Average age of onset is 20, and the frequency of mood episodes tends to ↑ with age. Up to 10–15% of those affected die by suicide. Subtypes are as follows: ■Bipolar I: At least one manic or mixed episode (usually requiring hospitalization). Bipolar II: At least one MDE and one hypomanic episode (less intense than mania). Patients do not meet the criteria for full manic or mixed episodes. Rapid cycling: Four or more episodes (MDE, manic, mixed, or hypo-manic) in one year. Cyclothymic: Chronic and less severe, with alternating periods of hypo-mania and moderate depression for > 2 years.

1	Cyclothymic: Chronic and less severe, with alternating periods of hypo-mania and moderate depression for > 2 years. The mnemonic DIG FAST outlines the clinical presentation of mania. Patients may report excessive engagement in pleasurable activities (e.g., excessive spending or sexual activity), reckless behaviors, and/or psychotic features. Antidepressant use may trigger manic episodes. A manic episode is one week or more of persistently elevated, expansive, or irritable mood plus three DIG FAST symptoms. Psychotic symptoms are common in mania. Symptoms are not due to a substance or medical condition and lead to signifcant impairment socially, occupationally, or familially. Hypomania is similar but does not involve marked functional impairment or psychotic symptoms and does not require hospitalization. ■ Mania: Give mood stabilizers for maintenance therapy (see Table 2.14-6) and antipsychotics in the acute phase (see the discussion of psychotic dis-

1	■ Mania: Give mood stabilizers for maintenance therapy (see Table 2.14-6) and antipsychotics in the acute phase (see the discussion of psychotic dis- TABLE 2.14-6. Mood Stabilizers Symptoms of mania—

1	Flight of ideas (or racing thoughts) Activities/psychomotor Agitation 439 Lithium First-line mood stabilizer. Used for acute mania (in combination with antipsychotics), for prophylaxis in bipolar disorders, and for augmentation in depression treatment. Thirst, polyuria, diabetes insipidus, tremor, weight gain, hypothyroidism, nausea, diarrhea, seizures, teratogenicity (if used in the frst trimester), acne, vomiting. Narrow therapeutic window (but blood level can be monitored). Lithium toxicity: > 1.5 mEq/L; presents with ataxia, dysarthria, delirium, and acute renal failure. Avoid lithium in patients with ↓ renal function. Carbamazepine Second-line mood stabilizer; anticonvulsant; trigeminal neuralgia. Nausea, skin rash, leukopenia, AV block. Rarely, aplastic anemia (monitor CBC biweekly). Stevens-Johnson syndrome. Bipolar disorder; anticonvulsant. GI (nausea, vomiting), tremor, sedation, alopecia, weight gain. Rarely, pancreatitis, thrombocytopenia, fatal hepatotoxicity, and

1	Stevens-Johnson syndrome. Bipolar disorder; anticonvulsant. GI (nausea, vomiting), tremor, sedation, alopecia, weight gain. Rarely, pancreatitis, thrombocytopenia, fatal hepatotoxicity, and agranulocytosis. Second-line mood stabilizer; anticonvulsant. Blurred vision, GI distress, Stevens-Johnson syndrome. ↑ dose slowly to monitor for rashes.

1	Characteristics of personality disorders— orders below). Benzodiazepines may be of beneft in refractory agitation. Bipolar depression: Mood stabilizers +/– antidepressants. Start mood stabilizers first (see Table 2.14-6) to avoid inducing mania. ECT may be used to treat refractory cases. In patients with severe depression or bipolar II with predominantly depressive features, antidepressant treatment can be augmented with low-dose lithium—e.g., at blood levels of 0.4–0.6 mEq/L. Personality can be defned as an individual’s set of emotional and behavioral traits, which are generally stable and predictable. Personality disorders are defned when one’s traits become chronically rigid and maladaptive and affect most aspects of one’s life (see the mnemonic MEDIC). Onset occurs by early adulthood. Personality disorders are defned under Axis II in the DSM-IV (see defnition of axes on the following page). Specifc disorders are outlined in Table 2.14-7.

1	TAB LE 2.1 4-7. Signs and Symptoms of Personality Disorders Distrustful, suspicious; interpret others’ motives as malevolent. Isolated, detached “loners.” Restricted emotional expression. Odd behavior, perceptions, and appearance. Magical thinking; ideas of reference. Patients are suspicious and distrustful of psychiatrists, making it diffcult to form therapeutic relationships between patient and psychiatrist. Be clear, honest, noncontrolling, and nondefensive. Unstable mood, relationships, and self-image; feelings of emptiness. Impulsive. History of suicidal ideation or self-harm. Excessively emotional and attention seeking. Sexually provocative; theatrical. Grandiose; need admiration; have sense of entitlement. Lack empathy. Violate rights of others, social norms, and laws. Impulsive; lack remorse. Begins in childhood as conduct disorder. Patients change the rules and demand attention. They are manipulative and demanding and will split staff members.

1	Patients change the rules and demand attention. They are manipulative and demanding and will split staff members. Be frm. Stick to the treatment plan. Be fair. Do not be punitive or derogatory. Be consistent. Do not change rules. Preoccupied with perfectionism, order, and control at the expense of eff ciency. Infexible morals, values. Socially inhibited; rejection sensitive. Fear being disliked or ridiculed. Submissive, clingy; need to be taken care of. Diffculty making decisions. Feel helpless. Patients are controlling and may sabotage their treatment. Words may be inconsistent with actions. Avoid power struggles. Give clear recommendations, but do not push patients into decisions. Ask about attitudes, mood variability, activities, and reaction to stress.

1	Avoid power struggles. Give clear recommendations, but do not push patients into decisions. Ask about attitudes, mood variability, activities, and reaction to stress. Patients have chronic problems dealing with responsibilities, roles, and stressors. They may also deny their behavior, have diff culty understanding the cause of their problems, have diffculty changing their behavior patterns, and frequently refuse psychiatric care. Psychotherapy is the mainstay of therapy. Pharmacotherapy is reserved for cases with comorbid mood, anxiety, or psychotic signs/symptoms. Characterized by hallucinations, delusions, disordered thoughts, behavioral disturbances, and disrupted social functioning with a clear sensorium.

1	Characterized by hallucinations, delusions, disordered thoughts, behavioral disturbances, and disrupted social functioning with a clear sensorium. Epidemiology: Prevalence is approximately 1%; males and females are affected equally. Peak onset is earlier in males (ages 18–25) than in females (ages 25–35), and has an ↑ incidence in those born in winter or early spring. Schizophrenia in frst-degree relatives also ↑ risk. Ten percent of those affected commit suicide. Etiology: Etiologic theories focus on neurotransmitter abnormalities such as dopamine dysregulation (frontal hypoactivity; limbic hyperactivity; effcacy of dopamine antagonists) and brain abnormalities on CT and MRI (enlarged ventricles and ↓ cortical volume). Subtypes are as follows: Paranoid: Delusions (often of persecution of the patient) and/or hallucinations are present. Cognitive function is usually preserved. Associated with the best overall prognosis.

1	Paranoid: Delusions (often of persecution of the patient) and/or hallucinations are present. Cognitive function is usually preserved. Associated with the best overall prognosis. Disorganized: Speech and behavior patterns are highly disordered and disinhibited with fat affect. The thought disorder is pronounced, and the patient has poor contact with reality. Carries the worst prognosis. Catatonic: A rare form characterized by psychomotor disturbance with two or more of the following: excessive motor activity, immobility, extreme negativism, mutism, waxy fexibility, echolalia, or echopraxia. Two or more of the following are present continuously for six or more months with social or occupational dysfunction: symptoms: Hallucinations (most often auditory), delusions, disorganized speech, bizarre behavior, and thought disorder. symptoms: Flat affect, ↓ emotional reactivity, poverty of speech, lack of purposeful actions, and anhedonia. The differential includes the following:

1	symptoms: Flat affect, ↓ emotional reactivity, poverty of speech, lack of purposeful actions, and anhedonia. The differential includes the following: Schizophreniform disorder: Symptoms of schizophrenia with a duration of < 6 months. Schizoaffective disorder: Combines the symptoms of schizophrenia with a major affective disorder (major depressive disorder or bipolar disorder). Five categories are used by the DSM-IV to classify the factors contributing to an individual’s mental state: Axis I: Psychiatric disorders. Axis II: Personality disorders and mental retardation. Axis III: Physical and medical problems. Axis IV: Social and environmental problems/ stressors. Axis V: The Global Assessment of Functioning (GAF), which rates a patient’s overall level of social, occupational, and psychological functioning on a scale of 1 (nonfunctional) to 100 (extremely high functioning across several areas). Antipsychotics (see Table 2.14-8); long-term follow-up.

1	Antipsychotics (see Table 2.14-8); long-term follow-up. Supportive psychotherapy, training in social skills, vocational rehabilita tion, and illness education may help. Terms used to describe ■ symptoms may be more diffcult to treat. components of psychosis: Delusion: A f xed false idiosyncratic belief. Hallucination: Perception of an object or event without an existing external stimulus. Illusion: False perception of an actual external stimulus. A persistent pattern of excessive inattention and/or hyperactivity/impulsivity. More common in males; typically presents between ages 3 and 13. Often shows a familial pattern. Diagnosis requires six or more symptoms from each category listed below for six or more months in at least two settings, leading to signifcant social and academic impairment. Some symptoms must be present in patients before age seven.

1	■Inattention: Poor attention span in schoolwork/play; poor attention to detail or careless mistakes; does not listen when spoken to; has difficulty T AB LE 2.1 4-8. Antipsychotic Medications Typical antipsychotics Haloperidol, droperidol, f uphenazine, thioridazine, chlorpromazine Atypical antipsychotics Clozapine, risperidone (also available in long-acting depot injection), quetiapine, olanzapine, ziprasidone, aripiprazole Currently frst-line treatment for schizophrenia given fewer EPS and anticholinergic effects. Clozapine is reserved for severe treatment resistance and severe tardive dyskinesia. Weight gain, type 2 DM, somnolence, sedation, and QTc prolongation. Agranulocytosis requiring weekly CBC monitoring (clozapine). Psychotic disorders, acute agitation, acute mania, Tourette’s syndrome. Thought to be more effective for symptoms of schizophrenia; primarily block D2 dopamine receptors.

1	Psychotic disorders, acute agitation, acute mania, Tourette’s syndrome. Thought to be more effective for symptoms of schizophrenia; primarily block D2 dopamine receptors. For patients in whom compliance is a major issue, consider antipsychotics that come in depot forms (haloperidol, f uphenazine). (EPS; see Table 2.14-9), hyperprolactinemia. Anticholinergic effects (dry mouth, urinary retention, constipation). Seizures, hypotension, sedation, and QTc prolongation. (thioridazine). Neuroleptic malignant syndrome: Fever, muscle rigidity, autonomic instability, elevated CK, clouded consciousness. Stop medication; provide supportive care in the ICU; administer dantrolene or bromocriptine (see Table 2.14-9). T AB LE 2.1 4-9. Extrapyramidal Symptoms and Treatment

1	Acute dystonia Involuntary muscle contraction or spasm (e.g., torticollis, oculogyric crisis). Anticholinergics (benztropine or diphenhydramine); some patients on antipsychotics who are prone to dystonic reactions may need regular prophylactic dosing of these. Akathisia Subjective/objective restlessness, which is perceived as being distressing. ↓ neuroleptic and try β-blockers (propranolol). Benzodiazepines or anticholinergics may help. Pseudoparkinsonism (e.g., shuffing gait, cogwheel rigidity). Give an anticholinergic (benztropine) or a dopamine agonist (amantadine). ↓ the dose of neuroleptic or discontinue (if tolerated). Stereotypic oral-facial movements. Likely from dopamine receptor sensitization. Often irreversible (50%). Discontinue or ↓ the dose of neuroleptic; attempt treatment with more appropriate drugs; and consider changing neuroleptic (e.g., to clozapine or risperidone). Giving anticholinergics or decreasing neuroleptics may initially worsen tardive dyskinesia.

1	following instructions or finishing tasks; loses items needed to complete tasks; is forgetful and easily distracted. ■ Hyperactivity/impulsivity: Fidgets; leaves seat in classroom; runs around inappropriately; cannot play quietly; talks excessively; does not wait turn; interrupts others. Initial treatment may be nonpharmacologic (e.g., behavior modif cation). Sugar and food additives are not considered etiologic factors. Pharmacologic treatment includes the following: Psychostimulants: Methylphenidate (Ritalin), dextroamphetamine (Dexedrine), mixed salts of dextroamphetamine and amphetamine (Adderall), atomoxetine (Strattera), pemoline (Cylert). Adverse effects include insomnia, irritability, ↓ appetite, tic exacerbation, and ↓ growth velocity (normalizes when medication is stopped). Antidepressants (e.g., SSRIs, nortriptyline, bupropion) and α2-agonists (e.g., clonidine).

1	Antidepressants (e.g., SSRIs, nortriptyline, bupropion) and α2-agonists (e.g., clonidine). More common in males. May be associated with tuberous sclerosis and fragile X syndrome. Symptom severity and IQ vary widely. ■Characterized by abnormal or impaired social interaction and communication together with restricted activities and interests, evident before age three. Children must exhibit ADHD symptoms in two or more settings (e.g., home and school). Conduct disorder is seen in Children. Antisocial personality disorder is seen in Adults. Patients fail to develop normal social behaviors (e.g., social smile, eye contact) and lack interest in relationships. The development of spoken language is delayed or absent. Children show stereotyped speech and behavior (e.g., hand f apping) and restricted interests (e.g., preoccupation with parts of objects). Other pervasive developmental disorders include the following:

1	Other pervasive developmental disorders include the following: Asperger’s syndrome: An autism-like disorder of social impairment and repetitive activities, behaviors, and interests without marked language or cognitive delays. Rett’s disorder: A genetic neurodegenerative disorder of females with progressive impairment (e.g., language, head growth, coordination) after five months of normal development. Childhood disintegrative disorder: Severe developmental regression after > 2 years of normal development (e.g., language, motor skills, social skills, bladder/bowel control, play). Intensive special education, behavioral management, and symptom-targeted medications (e.g., neuroleptics for aggression; SSRIs for stereotyped behavior). Family support and counseling are crucial. Include conduct disorder and oppositional def ant disorder. More common among males and in patients with a history of abuse. Hx/PE:

1	Family support and counseling are crucial. Include conduct disorder and oppositional def ant disorder. More common among males and in patients with a history of abuse. Hx/PE: Conduct disorder: A repetitive, persistent pattern of violating the basic rights of others or age-appropriate societal norms or rules for one year or more. Behaviors may be aggressive (e.g., rape, robbery, animal cruelty) or nonaggressive (e.g., stealing, lying, deliberately annoying people). May progress to antisocial personality disorder in adulthood. Oppositional defiant disorder: A pattern of negativistic, defiant, disobedient, and hostile behavior toward authority fgures (e.g., losing temper, arguing) for six or more months. May progress to conduct disorder. Tx: Individual and family therapy. Occur more frequently in males and in those of low socioeconomic status (SES); often exhibit a familial pattern. Hx/PE:

1	Tx: Individual and family therapy. Occur more frequently in males and in those of low socioeconomic status (SES); often exhibit a familial pattern. Hx/PE: Academic functioning is substantially lower than expected for age, intelligence, and education as measured by standardized test achievement in reading, mathematics, or written expression. Learning problems signifcantly interfere with schooling and daily activities. Always rule out physical disorders (e.g., deafness) and social factors (e.g., non–English speakers). Tx: Interventions include remedial classes or individualized learning strategies. Associated with male gender, chromosomal abnormalities, congenital infections, teratogens, inborn errors of metabolism, and alcohol/illicit substances during pregnancy. Hx/PE: Patients have signifcantly subaverage intellectual functioning (an IQ of < 70) with deficits in adaptive functioning (e.g., hygiene, social skills); onset is before the age of 18.

1	Hx/PE: Patients have signifcantly subaverage intellectual functioning (an IQ of < 70) with deficits in adaptive functioning (e.g., hygiene, social skills); onset is before the age of 18. Levels of severity are mild (IQ 50–70; 85% of cases), moderate (IQ 35–49), severe (IQ 20–34), and profound (IQ < 20). Tx: 1° prevention consists of educating the general public about possible causes of mental retardation and providing optimal prenatal screening and health care to mothers and their children. Treatment measures include family counseling and support; speech and language therapy; occupational/physical therapy; behavioral intervention; educational assistance; and social skills training. More common in males; shows a genetic predisposition. Associated with ADHD, learning disorders, and OCD. Hx/PE: Begins prior to age 18.

1	More common in males; shows a genetic predisposition. Associated with ADHD, learning disorders, and OCD. Hx/PE: Begins prior to age 18. Characterized by multiple motor (e.g., blinking, grimacing) and vocal (e.g., grunting, coprolalia) tics occurring many times per day, recurrently, for > 1 year with social or occupational impairment. Tx: Treatments include dopamine receptor antagonists (haloperidol, pimozide) or clonidine. Behavioral therapy may be of beneft, and counseling can aid in social adjustment and coping. Stimulants can worsen or precipitate tics. Fetal alcohol syndrome is the number one avoidable cause of mental retardation. Coprolalia = repetition of obscene words. Features of substance dependence— Both substance abuse and substance dependence are maladaptive patterns of substance use that lead to clinically signifcant impairment. Substance abuse is distinguished from substance dependence as follows:

1	Substance abuse: Requires one or more of the following in one year: Failure to fulfill responsibilities at work, school, or home. Use in physically hazardous situations (e.g., driving while intoxicated). Legal problems during the time of substance use. Continued substance use despite recurrent social or interpersonal problems 2° to the effects of such use (e.g., frequent arguments with spouse over the substance use). Substance dependence: Requires three or more of the following in one year: Tolerance and use of progressively larger amounts to obtain the same desired effect. Withdrawal symptoms when not taking the substance. Failed attempts to cut down use or abstain from the substance. Three or more of seven within a 12-month period: Harm (physical and psychosocial) with continued use Desire to cut down/ control Time spent obtaining/ using the substance is ↑ A diagnosis of substance dependence trumps a diagnosis of substance abuse.

1	Desire to cut down/ control Time spent obtaining/ using the substance is ↑ A diagnosis of substance dependence trumps a diagnosis of substance abuse. Signifcant time spent obtaining the substance (e.g., visiting many doctors to obtain a prescription for pain pills). Isolation from life activities. Taking greater amounts of the substance than intended. Continued substance abuse despite recurrent physical or psychological problems 2° to the effect of the substance use. Substance use is often denied or underreported, so seek out collateral information from family and friends. Check urine and blood toxicology screens, LFTs, and serum EtOH level. The management of intoxication for selected drugs is described in Table 2.14-10. T AB LE 2.1 4-1 0. Signs and Symptoms of Substance Abuse

1	Alcohol Disinhibition, emotional lability, slurred speech, ataxia, aggression, blackouts, hallucinations, memory impairment, impaired judgment, coma. Tremor, tachycardia, hypertension, malaise, nausea, seizures, DTs, agitation. Opioids Euphoria leading to apathy, CNS depression, constipation, pupillary constriction, and respiratory depression (life threatening in overdose). Naloxone/naltrexone will block opioid receptors and reverse effects (beware of antagonist clearing before opioids, particularly with long-acting opioids such as methadone). Dysphoria, insomnia, anorexia, myalgias, fever, lacrimation, diaphoresis, dilated pupils, rhinorrhea, piloerection, nausea, vomiting, stomach cramps, diarrhea, yawning. Opioid withdrawal is not life threatening, “hurts all over,” and does not cause seizures. Amphetamines Psychomotor agitation, impaired judgment, hypertension, pupillary dilation, tachycardia, fever, diaphoresis, anxiety, angina, euphoria, prolonged wakefulness/attention,

1	cause seizures. Amphetamines Psychomotor agitation, impaired judgment, hypertension, pupillary dilation, tachycardia, fever, diaphoresis, anxiety, angina, euphoria, prolonged wakefulness/attention, arrhythmias, delusions, seizures, hallucinations. Haloperidol can be given for severe agitation and symptom-targeted medications (e.g., antiemetics, NSAIDs). Postuse “crash” with anxiety, lethargy, headache, stomach cramps, hunger, fatigue, depression/dysphoria, sleep disturbance, nightmares. Cocaine Psychomotor agitation, euphoria, impaired judgment, tachycardia, pupillary dilation, hypertension, paranoia, hallucinations, sudden death. ECG changes from ischemia are often seen (“cocaine chest pain”). Treat with haloperidol for severe agitation along with symptom-specifc medications (e.g., to control hypertension). Postuse “crash” with hypersomnolence, depression, malaise, severe craving, angina, suicidality, ↑ appetite, nightmares, “cocaine bugs.”

1	T AB LE 2.1 4-1 0. Signs and Symptoms of Substance Abuse (continued)

1	Phencyclidine hydrochloride (PCP) Assaultiveness, belligerence, psychosis, violence, impulsiveness, psychomotor agitation, fever, tachycardia, vertical/horizontal nystagmus, hypertension, impaired judgment, ataxia, seizures, delirium. Give benzodiazepines or haloperidol for severe symptoms; otherwise reassure. Acidifcation of urine or gastric lavage can help eliminate the drug. Recurrence of intoxication symptoms due to reabsorption in the GI tract; sudden onset of severe, random violence. LSD Marked anxiety or depression, delusions, visual hallucinations, fashbacks, pupillary dilation, impaired judgment, diaphoresis, tachycardia, hypertension, heightened senses (e.g., colors become more intense). Supportive counseling; traditional antipsychotics for psychotic symptoms; benzodiazepines for anxiety. Marijuana Euphoria, slowed sense of time, impaired judgment, social withdrawal, ↑ appetite, dry mouth, conjunctival injection, hallucinations, anxiety, paranoia, amotivational syndrome.

1	for anxiety. Marijuana Euphoria, slowed sense of time, impaired judgment, social withdrawal, ↑ appetite, dry mouth, conjunctival injection, hallucinations, anxiety, paranoia, amotivational syndrome. Barbiturates Low safety margin; respiratory depression. Anxiety, seizures, delirium, life-threatening cardiovascular collapse. Benzodiazepines Interactions with alcohol, amnesia, ataxia, somnolence, mild respiratory depression. (Avoid using for insomnia in the elderly; can cause paradoxical agitation even in relatively low doses.) Rebound anxiety, seizures, tremor, insomnia, hypertension, tachycardia, death. Restlessness, insomnia, diuresis, muscle twitching, arrhythmias, tachycardia, fushed face, psychomotor agitation. Headache, lethargy, depression, weight gain, irritability, craving. Restlessness, insomnia, anxiety, arrhythmias. Irritability, headache, anxiety, weight gain, craving, bradycardia, diff culty concentrating, insomnia. Caffeine Nicotine

1	Occurs more often in males (4:1) and in those 21–34 years of age, although the incidence in females is rising. Also associated with a family history. Hx/PE: See Table 2.14-10 for the symptoms of intoxication and withdrawal. Look for palmar erythema or telangiectasias as well as for other signs and symptoms of end-organ complications. Dx: Screen with the CAGE questionnaire. Monitor vital signs for evidence of withdrawal. Labs may reveal ↑ LFTs, LDH, and MCV. Tx: Rule out medical complications; correct electrolyte abnormalities. Start a benzodiazepine taper for withdrawal symptoms. Add haloperidol for hallucinations and psychotic symptoms. Pinpoint pupils are not always a reliable sign of opioid ingestion because coingestions can lead to normal or enlarged pupils. Also look for a ↓ respiratory rate, track marks, and ↓breath sounds. CAGE questionnaire: 1. Have you ever felt the need to Cut down on your drinking? 2. Have you ever felt Annoyed by criticism of your drinking? 3.

1	CAGE questionnaire: 1. Have you ever felt the need to Cut down on your drinking? 2. Have you ever felt Annoyed by criticism of your drinking? 3. Have you ever felt Guilty about drinking? 4. Have you ever had to take a morning Eye opener? More than one “yes” answer makes alcoholism likely. There are two types of anorexia nervosa: Give multivitamins and folic acid; administer thiamine before glucose (which depletes thiamine) to prevent Wernicke’s encephalopathy. Give anticonvulsants to patients with a seizure history. Group therapy, disulfram, or naltrexone can aid patients with dependence. Long-term rehabilitative therapy (e.g., Alcoholic Anonymous). ■ Cx: GI bleeding from gastritis, ulcers, varices, or Mallory-Weiss tears.

1	Long-term rehabilitative therapy (e.g., Alcoholic Anonymous). ■ Cx: GI bleeding from gastritis, ulcers, varices, or Mallory-Weiss tears. Pancreatitis, liver disease, DTs, alcoholic hallucinosis, peripheral neuropathy, Wernicke’s encephalopathy, Korsakoff’s psychosis, fetal alcohol syndrome, cardiomyopathy, anemia, aspiration pneumonia, ↑ risk of sustaining trauma (e.g., subdural hematoma). Risk factors include female gender, low self-esteem, and high SES. Also associated with OCD, major depressive disorder, anxiety, and careers such as modeling, gymnastics, ballet, and running. Hx/PE: Diagnosed as follows (see also Table 2.14-11): Body weight is < 85% of that expected. Patients present with refusal to maintain normal body weight, an intense fear of weight gain, a distorted body image (patients perceive themselves as fat), and amenorrhea. Patients restrict (e.g., fasting, excessive exercise) or binge and purge (through vomiting, laxatives, and diuretics).

1	Patients restrict (e.g., fasting, excessive exercise) or binge and purge (through vomiting, laxatives, and diuretics). Signs and symptoms include lanugo, dry skin, bradycardia, lethargy, hypotension, cold intolerance, and hypothermia (as low as 35°C). Dx: Measure height and weight; check CBC, electrolytes, endocrine levels, and ECG. Perform a psychiatric evaluation to screen patients for co-morbid conditions. Tx: Initially, monitor caloric intake to restore nutritional status and to stabilize weight; then focus on weight gain. Hospitalize if necessary to restore nutritional status, rehydrate, and correct electrolyte imbalances. Once the patient is medically stable, initiate individual, family, and group psychotherapy. Treat comorbid depression and anxiety. Cx: Mitral valve prolapse, arrhythmias, hypotension, bradycardia, amenor- TABLE 2.14-11. Anorexia vs. Bulimia

1	Cx: Mitral valve prolapse, arrhythmias, hypotension, bradycardia, amenor- TABLE 2.14-11. Anorexia vs. Bulimia Body image Disturbed body image; use extensive measures to avoid weight gain (e.g., purging, excess exercise). Same. Binge eating May occur. Same. Patients are underweight (≥ 15% below expected weight). Patients are of normal weight or are overweight. Patients are typically not distressed by their illness and may thus be resistant to treatment. Patients are typically distressed about their symptoms and are thus easier to treat. Weight Attitude toward illness rhea (missing three consecutive cycles), nephrolithiasis, osteoporosis, multiple stress fractures, pancytopenia, thyroid abnormalities (see Table 2.1412). Mortality from suicide or medical complications is > 10%. More common among women; associated with low self-esteem, mood disorders, and OCD. Hx/PE: Diagnostic criteria are as follows (see also Table 2.14-11):

1	More common among women; associated with low self-esteem, mood disorders, and OCD. Hx/PE: Diagnostic criteria are as follows (see also Table 2.14-11): Patients have normal weight or are overweight. For at least two times a week for three or more months, patients have episodes of binge eating and compensatory behaviors that include purging or fasting. Patients are usually ashamed and conceal their behaviors. Signs include dental enamel erosion, enlarged parotid glands, and scars on the dorsal hand surfaces (from inducing vomiting). Patients usually have normal body weight. Tx: Psychotherapy focuses on behavior modifcation and body image. Antidepressants may be effective for both depressed and nondepressed patients. Cx: See Table 2.14-12. Interest in sexual activity usually does not ↓ with aging. Men usually require ↑ stimulation of the genitalia for longer periods of time to reach orgasm; intensity of orgasm ↓, and the length of the refractory period before the next orgasm ↑.

1	Men usually require ↑ stimulation of the genitalia for longer periods of time to reach orgasm; intensity of orgasm ↓, and the length of the refractory period before the next orgasm ↑. In women, estrogen levels ↓ after menopause, leading to vaginal dryness and thinning, which may result in discomfort during coitus. May be treated with HRT, estrogen vaginal suppositories, or other vaginal creams. Preoccupation with or engagement in unusual sexual fantasies, urges, or behaviors for > 6 months with clinically signifcant impairment in one’s life. Includes criminal sex offenders (e.g., pedophilia); see Table 2.14-13. Found almost exclusively in men, and usually begins before or during puberty. Sexual excitement is derived from unique exposures to certain situations, individuals, or objects.

1	Sexual excitement is derived from unique exposures to certain situations, individuals, or objects. T AB LE 2.1 4-1 2. Medical Complications of Eating Disorders be more disturbed by their behavior than anorexics and are more easily engaged in therapy. Anorexic patients deny health risks associated with their behavior, making them resistant to treatment. Bupropion should be avoided in the treatment of patients with eating disorders, as it is associated with a ↓ seizure threshold. Cachexia Hypothermia Fatigue Electrolyte abnormalities Arrhythmias Sudden death Hypotension Bradycardia Prolonged QT interval Dental erosions and decay Abdominal pain Delayed gastric emptying Amenorrhea Nephrolithiasis Dermatologic: Lanugo Hematologic: Leukopenia Neurologic: Seizures Musculoskeletal: Osteoporosis, stress fractures T AB LE 2.1 4-1 3. Features of Common Paraphilias

1	T AB LE 2.1 4-1 3. Features of Common Paraphilias Exhibitionism Sexual arousal from exposing one’s genitals to a stranger. Pedophilia Urges or behaviors involving sexual activities with children. Voyeurism Observing unsuspecting persons unclothed or involved in sex. Fetishism Use of nonliving objects (often clothing) for sexual arousal. Transvestic fetishism Cross-dressing for sexual arousal. Frotteurism Touching or rubbing one’s genitalia against a nonconsenting person (common in subways). Sexual sadism Sexual arousal from inficting suffering on sexual partner. Sexual masochism Sexual arousal from being hurt, humiliated, bound, or threatened. ■ Tx: Treatment includes insight-oriented psychotherapy and behavioral therapy. Antiandrogens (e.g., Depo-Provera) have been used for hypersexual paraphilic activity.

1	■ Tx: Treatment includes insight-oriented psychotherapy and behavioral therapy. Antiandrogens (e.g., Depo-Provera) have been used for hypersexual paraphilic activity. Strong, persistent cross-gender identification and discomfort with one’s assigned sex or gender role of the assigned sex in the absence of intersexual disorders. Patients may have a history of dressing like the opposite sex, taking sex hormones, or pursuing surgeries to reassign their sex. More common in males than in females. Associated with depression, anxiety, substance abuse, and personality disorders, which may be addressed and treated. Tx: Treatment is complex and includes educating the patient about culturally acceptable behavior patterns. Other options include sex-reassignment surgery or hormonal treatment (e.g., estrogen for males, testosterone for females). Supportive psychotherapy is helpful. Problems in sexual arousal, desire, or orgasm or pain with sexual intercourse.

1	Problems in sexual arousal, desire, or orgasm or pain with sexual intercourse. Prevalence is 30%; one-third of cases are attributable to biological factors and another third to psychological factors. Tx: Treatment depends on the particular condition. Pharmacologic strategies include sildenafl (Viagra) and bupropion (Wellbutrin). Psychotherapeutic strategies include sensate focusing. Up to one-third of all American adults suffer from some type of sleep disorder during their lives. The term dyssomnia describes any condition that leads to a disturbance in the normal rhythm or pattern of sleep. Insomnia is the most common example. Risk factors include female gender, the presence of mental and medical disorders, substance abuse, and advanced age. Affects up to 30% of the general population; causes sleep disturbance that is not attributable to physical or mental conditions. Often exacerbated by anxiety, and patients may become preoccupied with getting enough sleep.

1	Dx: Patients present with a history of nonrestorative sleep or difficulty initiating or maintaining sleep that is present at least three times a week for one month. Tx: First-line therapy includes the initiation of good sleep hygiene measures, which include the following: Establishment of a regular sleep schedule Limiting of caffeine intake Avoidance of daytime naps Warm baths in the evening Use of the bedroom for sleep and sexual activity only Exercising early in the day Avoidance of large meals near bedtime Pharmacotherapy is considered second-line therapy and should be initiated with care for short periods of time (< 2 weeks). Pharmacologic agents include diphenhydramine (Benadryl), zolpidem (Ambien), zaleplon (Sonata), and trazodone (Desyrel).

1	Dx: Diagnosed when a patient complains of excessive daytime sleepiness or nighttime sleep that occurs for > 1 month. The excessive somnolence cannot be attributable to medical or mental illness, medications, poor sleep hygiene, insuffcient sleep, or narcolepsy. Tx: First-line therapy includes stimulant drugs such as amphetamines. Antidepressants such as SSRIs may be useful in some patients. May affect up to 0.16% of the population. Onset typically occurs by young adulthood, generally before the age of 30. Some forms of narcolepsy may have a genetic component. Dx: Manifestations include excessive daytime somnolence and ↓REM sleep latency on a daily basis for at least three months. Sleep attacks are the classic symptom; patients cannot avoid falling asleep. Factitious disorders and malingering are distinct from somatoform disorders in that they involve conscious and intentional processes. ■The characteristic excessive sleepiness may be associated with the following:

1	■The characteristic excessive sleepiness may be associated with the following: Cataplexy: Sudden loss of muscle tone that leads to collapse. Hypnagogic hallucinations: Occur as the patient is falling asleep. Hypnopompic hallucinations: Occur as the patient awakens. Sleep paralysis: Brief paralysis upon awakening. ■ Tx: Treat with a regimen of scheduled daily naps plus stimulant drugs such as amphetamines; give SSRIs for cataplexy. Occurs 2° to disturbances in breathing during sleep that lead to excessive daytime somnolence and sleep disruption. Etiologies can be either central or peripheral. Central sleep apnea (CSA): A condition in which both airf ow and respiratory effort cease. CSA is linked to morning headaches, mood changes, and repeated awakenings during the night.

1	Central sleep apnea (CSA): A condition in which both airf ow and respiratory effort cease. CSA is linked to morning headaches, mood changes, and repeated awakenings during the night. Obstructive sleep apnea (OSA): A condition in which airf ow ceases as a result of obstruction along the respiratory passages. OSA is strongly associated with snoring. Risk factors include male gender, obesity, prior upper airway surgeries, a deviated nasal septum, a large uvula or tongue, and retrognathia (recession of the mandible). In both forms, arousal results in cessation of the apneic event. Associated with sudden death in infants and elderly, headaches, depression, ↑ systolic blood pressure, and pulmonary hypertension. Dx: Sleep studies (polysomnography) document the number of arousals, obstructions, and episodes of ↓ O2 saturation; distinguish OSA from CSA; and identify possible movement disorders, seizures, or other sleep disorders. Tx:

1	Tx: OSA: Nasal continuous positive airway pressure (CPAP). Weight loss if obese. In children, most cases are due to tonsillar/adenoidal hypertrophy, which is corrected surgically. CSA: Mechanical ventilation (e.g., BPAP) with a backup rate for severe cases. A spectrum of disorders characterized by a misalignment between desired and actual sleep periods. Subtypes include jet-lag type, shift-work type, delayed sleep-phase type, and unspecif ed. Tx: Jet-lag type usually resolves within 2–7 days without specific treatment. Shift-work type may respond to light therapy. Oral melatonin may be useful if given 5½ hours prior to the desired bedtime. Patients often present with medically unexplained somatic symptoms, generally with varying etiologies. Somatoform disorders: Patients have no conscious control over symptoms. The fve main categories are outlined in Table 2.14-14.

1	Somatoform disorders: Patients have no conscious control over symptoms. The fve main categories are outlined in Table 2.14-14. Factitious disorders: Patients fabricate symptoms or cause self-injury to assume the sick role (1° gain). More common in males. Munchausen’s syndrome: Common among health care workers. Munchausen’s syndrome by proxy: A “caregiver” makes someone else ill and enjoys taking on the role of concerned onlooker. Malingering: Patients intentionally cause or feign symptoms for 2° gain of financial benefit or housing. Most frequently affects women < 35 years of age who f ll the following criteria: Are experiencing marital discord and are substance abusers or have a partner who is a substance abuser; or Are pregnant, are of low SES, or have obtained a restraining order. In malingering, patients intentionally simulate illness for personal gain. Sexual abusers are usually male and are often known to the victim (and are often family members). TABLE 2.14-14.

1	Somatization disorder Multiple, chronic somatic symptoms from different organ systems with multiple GI, sexual, neurologic, and pain complaints. Frequent clinical contacts and/or surgeries; signif cant functional impairment. The male-to-female ratio is 1:20; onset is usually before age of 30. Schedule regular appointments with the identifed 1° caregiver who maintains communication with consultants and specialists; psychotherapy. Conversion disorder Symptoms or defcits of voluntary motor or sensory function (e.g., blindness, seizure-like movements, paralysis) incompatible with medical processes. Close temporal relationship to stress or intense emotion. More common in young females and in lower socioeconomic and less educated groups. Usually resolves spontaneously, but psychotherapy may help. Hypochondriasis Preoccupation with or fear of having a serious disease despite medical reassurance, leading to signifcant distress/impairment. Often involves a history of prior physical disease.

1	Hypochondriasis Preoccupation with or fear of having a serious disease despite medical reassurance, leading to signifcant distress/impairment. Often involves a history of prior physical disease. Men and women are equally affected. Onset is in adulthood. Manage with group therapy and schedule regular appointments with the patient’s 1° caregiver. Body dysmorphic disorder Preoccupation with an imagined physical defect or abnormality that leads to signif cant distress/impairment. Patients often present to dermatologists or plastic surgeons. Has a slight female predominance. May be associated with depression. SSRIs may be of benef t. Somatoform pain disorder Intensity or profle of pain symptoms is inconsistent with physiologic processes. Close temporal relationship with psychological factors. More common in females; peak onset is at 40–50 years of age. May be associated with depression. Treatment includes rehabilitation (e.g., physical therapy), psychotherapy, and behavioral therapy.

1	More common in females; peak onset is at 40–50 years of age. May be associated with depression. Treatment includes rehabilitation (e.g., physical therapy), psychotherapy, and behavioral therapy. Analgesia is usually not helpful. TCAs and SNRIs (venlafaxine and duloxetine) may be therapeutic.

1	Suicide is the third leading cause of death (after homicide and accidents) among 15to 24-year-olds in the United States. Emergent inpatient hospitalization is required for patients with suicidal intentions. Victims of childhood abuse are more likely to become adult victims of abuse. Hx/PE: Patients typically have multiple somatic complaints, frequent ER visits, and unexplained injuries with delayed medical treatment. They may also avoid eye contact or act afraid or hostile. Children may exhibit precocious sexual behavior, genital or anal trauma, STDs, UTIs, and psychiatric problems. Other clues include a partner who answers questions for the patient or refuses to leave the exam room. Tx: Perform a screening assessment of the patient’s safety domestically and in their close personal relationships. Provide medical care, emotional support, and counseling; educate the patient about support services and refer appropriately. Documentation is crucial.

1	Accounts for 30,000 deaths per year in the United States; the eighth overall cause of death in the United States. One suicide occurs every 20 minutes. Risk factors include male gender, age greater than 45 years, psychiatric disorders (major depression, presence of psychotic symptoms), a history of an admission to a psychiatric institution, a previous suicide attempt, a history of violent behavior, ethanol or substance abuse, recent severe stressors, and a family suicide history (see the mnemonic SAD PERSONS). Women are more likely to attempt suicide, whereas men are more likely to succeed by virtue of their ↑ use of more lethal methods. Dx: Perform a comprehensive psychiatric evaluation. Ask about family history, previous attempts, ambivalence toward death, and hopelessness. Ask directly about suicidal ideation, intent, and plan, and look for available means.

1	Ask about family history, previous attempts, ambivalence toward death, and hopelessness. Ask directly about suicidal ideation, intent, and plan, and look for available means. Tx: A patient who endorses suicidality requires emergent inpatient hospitalization even against his will. Suicide risk may ↑ after antidepressant therapy is initiated because a patient’s energy to act on suicidal thoughts can return before the depressed mood lifts. Neoplasms of the Lungs 469 LUNG NODULES 469 LUNG CANCER 470 Beware—all that wheezes is not asthma! Asthma should be suspected in children with multiple episodes of croup and URIs associated with dyspnea.

1	Beware—all that wheezes is not asthma! Asthma should be suspected in children with multiple episodes of croup and URIs associated with dyspnea. Characterized by airway narrowing, obstructive lung diseases restrict air movement and often cause air trapping. The etiologies of obstructive lung disease are described in the mnemonic ABCT. Figure 2.15-1 illustrates the role of lung volume measurements in the diagnosis of lung disease; Table 2.15-1 and Figure 2.15-2 contrast obstructive with restrictive lung disease. Reversible airway obstruction 2° to bronchial hyperreactivity, airway inﬂ ammation, mucous plugging, and smooth muscle hypertrophy. Presents with cough, episodic wheezing, dyspnea, and/or chest tightness. Symptoms often worsen at night or early in the morning.

1	Presents with cough, episodic wheezing, dyspnea, and/or chest tightness. Symptoms often worsen at night or early in the morning. Exam reveals wheezing, prolonged expiratory duration (↓ I/E ratio), accessory muscle use, tachypnea, tachycardia, ↓ breath sounds (late sign), ↓O2 saturation (late sign), hyperresonance, and possible pulsus paradoxus. ■ABGs: Mild hypoxia and respiratory alkalosis. Normalizing PCO2, respiratory acidosis, and more severe hypoxia in an acute exacerbation warrant close observation, as they may indicate fatigue and impending respiratory failure. F IGU R E 2.1 5-1. Lung volumes in the interpretation of pulmonary function tests. (Reproduced, with permission, from Gomella LG et al. Clinician’s Pocket Reference, 11th ed. New York: McGraw-Hill, 2006: Fig. 18-1.) T AB LE 2.1 5-1. Obstructive vs. Restrictive Lung Diseasea a FEV1 = forced expiratory volume in one second; FVC = forced vital capacity.

1	T AB LE 2.1 5-1. Obstructive vs. Restrictive Lung Diseasea a FEV1 = forced expiratory volume in one second; FVC = forced vital capacity. Spirometry/PFTs: ↓ FEV1/FVC; peak fow is diminished acutely; ↑ RV and total lung capacity (TLC). CBC: Possible eosinophilia. CXR: Hyperinf ation. Methacholine challenge: Tests for bronchial hyperresponsiveness; useful when PFTs are normal but asthma is still suspected. In general, avoid allergens or any potential triggers. Management is as follows (see also Tables 2.15-2 and 2.15-3): Asthma triggers include allergens, URIs, cold air, exercise, drugs, and stress. F IGU R E 2.1 5-2. Obstructive vs. restrictive lung disease. Note: Obstructive lung volumes > normal (↑ TLC, ↑ FRC, ↑ RV); restrictive lung volumes < normal. In both obstructive and restrictive disease, FEV1 and FVC are reduced, but in obstructive disease, FEV1 is more dramatically reduced, resulting in a ↓FEV1/FVC ratio.

1	T AB LE 2.1 5-2. Common Asthma Medications and Their Mechanisms β2-agonists Albuterol: Relaxes bronchial smooth muscle (β2-adrenoceptors). Give during acute exacerbations. Salmeterol: Long-acting agent for prophylaxis. Corticosteroids Beclomethasone, prednisone: Inhibit the synthesis of virtually all cytokines; inactivate NF-κB, a transcription factor for TNF-α, among other inﬂ ammatory agents. Inhaled corticosteroids are the first-line treatment for long-term control of asthma. Muscarinic antagonists Ipratropium: Competitively blocks muscarinic receptors, preventing bronchoconstriction. Methylxanthines Theophylline: Likely causes bronchodilation by inhibiting phosphodiesterase, thereby ↓ cAMP hydrolysis and ↑ cAMP levels. Usage is limited because of its narrow therapeutic-toxic index (cardiotoxicity, neurotoxicity). Cromolyn Prevents release of vasoactive mediators from mast cells. Useful for exercise-induced bronchospasm. Effective only for the prophylaxis of asthma; not effective

1	neurotoxicity). Cromolyn Prevents release of vasoactive mediators from mast cells. Useful for exercise-induced bronchospasm. Effective only for the prophylaxis of asthma; not effective during an acute asthmatic attack. Toxicity is rare. Antileukotrienes Zileuton: A 5-lipoxygenase pathway inhibitor. Blocks conversion of arachidonic acid to leukotrienes. Montelukast, zafirlukast: Blocks leukotriene receptors.

1	TABLE 2.15-3. Acute: O2, bronchodilating agents (short-acting inhaled β2-agonists are f rst-line therapy), ipratropium (never use alone for asthma), systemic corticosteroids, magnesium (for severe exacerbations). Maintain a low threshold for intubation in severe cases or acutely in patients with PCO2 > 50 mmHg or PO2 < 50 mmHg. Chronic: Measure lung function (FEV1, peak fow, and sometimes ABGs) to guide management. Administer long-acting inhaled bronchodilators and/ or inhaled corticosteroids, systemic corticosteroids, cromolyn, or, rarely, Medications for Chronic Treatment of Asthma

1	Medications for Chronic Treatment of Asthma Mild intermittent ≤ 2 days/week ≤ 2 nights/month ≥ 80% No daily medications. PRN short-acting bronchodilator. Mild persistent > 2/week but < 1/day > 2 nights/month ≥ 80% Daily low-dose inhaled corticosteroids. PRN short-acting bronchodilator. Moderate persistent Daily > 1 night/week 60–80% Lowto medium-dose inhaled corticosteroids + long-acting inhaled β2-agonists. Severe persistent Continual, frequent ≤ 60% High-dose inhaled corticosteroids + long-acting inhaled β2-agonists. Possible PO corticosteroids. PRN short-acting bronchodilator. theophylline. Montelukast and other leukotriene antagonists are oral adjuncts to inhalant therapy. A disease caused by cycles of infection and infammation in the bronchi/bronchioles that lead to permanent fbrosis, remodeling, and dilation of bronchi. Presents with chronic cough accompanied by frequent bouts of yellow or green sputum production, dyspnea, and possible hemoptysis and halitosis.

1	Presents with chronic cough accompanied by frequent bouts of yellow or green sputum production, dyspnea, and possible hemoptysis and halitosis. Associated with a history of pulmonary infections (e.g., Pseudomonas, Haemophilus, TB), hypersensitivity (allergic bronchopulmonary aspergillosis), cystic f brosis, immunodefciency, localized airway obstruction (foreign body, tumor), aspiration, autoimmune disease (e.g., rheumatoid arthritis, SLE), or IBD. Exam reveals rales, wheezes, rhonchi, purulent mucus, and occasional hemoptysis. CXR: ↑ bronchovascular markings; tram lines (parallel lines outlining dilated bronchi as a result of peribronchial inf ammation and f brosis); areas of honeycombing. High-resolution CT: Dilated airways and ballooned cysts at the end of the bronchus (mostly lower lobes). Spirometry shows a ↓ FEV1/FVC ratio. Antibiotics for bacterial infections; consider inhaled corticosteroids.

1	Antibiotics for bacterial infections; consider inhaled corticosteroids. Maintain bronchopulmonary hygiene (cough control, postural drainage, chest physiotherapy). Consider lobectomy for localized disease or lung transplantation for severe disease. Characterized by ↓ lung function with airfow obstruction. Generally 2° to chronic bronchitis or emphysema, which are distinguished as follows: Chronic bronchitis: Productive cough for > 3 months per year for two consecutive years. Emphysema: Terminal airway destruction and dilation that may be 2° to smoking (centrilobular) or to α1-antitrypsin deficiency (panlobular). Symptoms are minimal or nonspecifc until the disease is advanced. The clinical spectrum includes the following (most patients are a combination of the two phenotypes):

1	Symptoms are minimal or nonspecifc until the disease is advanced. The clinical spectrum includes the following (most patients are a combination of the two phenotypes): Emphysema (“pink puffer”): Dyspnea, pursed lips, minimal cough, ↓ breath sounds, late hypercarbia/hypoxia. Patients often have a thin, wasted appearance. Pure emphysematous patients tend to have few reactive episodes between exacerbations. aspiration Or 2° to foreign bodies Necrotizing pneumonia Chemical corrosive smoking cessation, pneumococcal and inﬂ uenza vaccines) Dilators (β2-agonists, anticholinergics) In COPD patients with chronic hypercapnia, high concentrations of O2 may suppress patients’ hypoxic respiratory drive. Supplemental oxygen titrated to > 90% SaO2 for > 15 hours a day and smoking cessation are the only interventions proven to improve survival in patients with COPD.

1	Supplemental oxygen titrated to > 90% SaO2 for > 15 hours a day and smoking cessation are the only interventions proven to improve survival in patients with COPD. ■Chronic bronchitis (“blue bloater”): Cyanosis with mild dyspnea; productive cough. Patients are often overweight with peripheral edema, rhonchi, and early signs of hypercarbia/hypoxia. Look for the classic barrel chest, use of accessory chest muscles, JVD, end-expiratory wheezing, and muffed breath sounds. CXR: Hyperinﬂ ated lungs, ↓ lung markings with fat diaphragms, and a thin-appearing heart and mediastinum. Parenchymal bullae or subpleural blebs (pathognomonic of emphysema) are also seen (see Figure 2.15-3). PFTs: ↓ FEV1/FVC; normal or ↓ FVC; normal or ↑ TLC (emphysema, asthma); ↓ DLCO (in emphysema). ABGs: Hypoxemia with acute or chronic respiratory acidosis (↑ PCO2). Blood cultures: Obtain if the patient is febrile.

1	ABGs: Hypoxemia with acute or chronic respiratory acidosis (↑ PCO2). Blood cultures: Obtain if the patient is febrile. Gram stain and sputum culture: Consider in the setting of fever or productive cough, especially if infltrate is seen on CXR. Acute exacerbations: O2, inhaled β-agonists (albuterol) and anticholinergics (ipratropium, tiotropium), IV +/− inhaled corticosteroids, antibiotics. Severe cases may beneft from noninvasive ventilation. Consider intubation in the setting of severe hypoxemia or hypercapnia, impending respiratory fatigue, or changes in mental status. Chronic: Smoking cessation, supplemental O2 (if resting PaO2 is ≤ 55 mmHg or SaO2 is ≤ 89%, or in the setting of cor pulmonale, pulmonary hypertension, a hematocrit > 55%, or nocturnal hypoxia), inhaled β-agonists, AB F IGU R E 2.1 5-3. Chronic obstructive pulmonary disease.

1	AB F IGU R E 2.1 5-3. Chronic obstructive pulmonary disease. Note the hyperinfated and hyperlucent lungs, f at diaphragms, ↑ AP diameter, narrow mediastinum, and large upper bullae on (A) AP and (B) lateral CXRs. (Reproduced, with permission, from Stobo J et al. The Principles and Practice of Medicine, 23rd ed. Stamford, CT: Appleton & Lange, 1996: 135.) anticholinergics (tiotropium), systemic or inhaled corticosteroids. Give pneumococcal and ﬂ u vaccines. Characterized by a loss of lung compliance, restrictive lung diseases result in ↑ lung stiffness and ↓ lung expansion. Table 2.15-1 and Figure 2.15-2 contrast obstructive with restrictive lung disease. The etiologies of restrictive lung disease are shown in the mnemonic PAINT.

1	A heterogeneous group of disorders characterized by inﬂ ammation and/or fibrosis of the interalveolar septum. In advanced disease, cystic spaces develop in the lung periphery (“honeycombing”). Causes include idiopathic interstitial pneumonias, collagen vascular disease, granulomatous disorders, drugs, hypersensitivity disorders, pneumoconiosis, and eosinophilic pulmonary syndromes. Presents with shallow, rapid breathing; dyspnea with exercise; and a nonproductive cough. Patients may have cyanosis, inspiratory squeaks, fne or “Velcro-like” crackles, fnger clubbing, or right heart failure. ■ CXR: Reticular, nodular, or ground-glass pattern; “honeycomb” pattern (severe disease). ■↓TLC, ↓FVC, ↓DLCO (may be normal if the cause is extrapulmonary), normal FEV1/FVC. Serum markers of connective tissue diseases should be obtained if clinically indicated.

1	■↓TLC, ↓FVC, ↓DLCO (may be normal if the cause is extrapulmonary), normal FEV1/FVC. Serum markers of connective tissue diseases should be obtained if clinically indicated. Supportive. Avoid exposure to causative agents. Some inf ammatory diseases respond to corticosteroids or other anti-inf ammatory/immunosuppressive agents. A multisystem disease of unknown etiology characterized by noncaseating granulomas. Most commonly found in African-American females and northern European Caucasians; most often arises in the third or fourth decade of life. Presents with fever, cough, malaise, weight loss, dyspnea, and arthritis. The lungs, liver, eyes, skin (erythema nodosum, violaceous skin plaques), nervous system, heart, and kidney may be affected. Symptoms may be GRUELING (see mnemonic). Causes of restrictive lung disease— Pleural (fibrosis, effusions, empyema, pneumothorax) Alveolar (edema, hemorrhage, pus)

1	Causes of restrictive lung disease— Pleural (fibrosis, effusions, empyema, pneumothorax) Alveolar (edema, hemorrhage, pus) Interstitial lung disease (idiopathic interstitial pneumonias), Inﬂ ammatory (sarcoid, cryptogenic organizing pneumonitis), Idiopathic Neuromuscular (myasthenia, phrenic nerve palsy, myopathy) Thoracic wall (kyphoscoliosis, obesity, ascites, pregnancy, ankylosing spondylitis) Medications that can cause or contribute to interstitial lung disease include busulfan, nitrofurantoin, amiodarone, bleomycin, radiation, and long-term high O2 concentration (e.g., ventilators). CXR: Radiographic fndings are used to stage the disease. Biopsy: Lymph node biopsy or transbronchial/video-assisted thoracoscopic lung biopsy reveals noncaseating granulomas. PFTs: Restrictive or obstructive pattern and ↓ diffusion capacity.

1	Biopsy: Lymph node biopsy or transbronchial/video-assisted thoracoscopic lung biopsy reveals noncaseating granulomas. PFTs: Restrictive or obstructive pattern and ↓ diffusion capacity. Other findings: ↑serum ACE levels (neither sensitive nor specif c), hypercalcemia, hypercalciuria, ↑ alkaline phosphatase (with liver involvement), lymphopenia, cranial nerve defects, arrhythmias. Systemic corticosteroids are indicated for constitutional symptoms, hypercalcemia, or extrathoracic organ involvement. Risk factors include environmental exposure to antigens leading to alveolar thickening and granulomas. Types and etiologies are listed in Table 2.15-4. Acute: Dyspnea, fever, malaise, shivering, and cough starting 4–6 hours after exposure. Chronic: Patients present with progressive dyspnea; exam reveals f ne bilateral rales. CXR is normal or shows miliary nodular infltrate (acute); fbrosis is seen in the upper lobes (chronic).

1	Chronic: Patients present with progressive dyspnea; exam reveals f ne bilateral rales. CXR is normal or shows miliary nodular infltrate (acute); fbrosis is seen in the upper lobes (chronic). Avoid ongoing exposure to inciting agents; give corticosteroids to ↓ chronic inf ammation. T AB LE 2.1 5-4. Antigens of Hypersensitivity Pneumonitis Farmer’s lung Spores of actinomycetes from moldy hay. Bird fancier’s lung Antigens from feathers, excreta, serum. Mushroom worker’s lung Spores of actinomycetes from compost. Malt worker’s lung Spores of Aspergillus clavatus in grain. Grain handler’s lung Grain weevil dust. Bagassosis Spores of actinomycetes from sugarcane. Air conditioner lung Spores of actinomycetes from air conditioners. Risk factors include prolonged occupational exposure and inhalation of small inorganic dust particles. Table 2.15-5 outlines the f ndings and diagnostic criteria associated with common pneumoconioses. Avoid triggers; supportive therapy and supplemental O2.

1	Table 2.15-5 outlines the f ndings and diagnostic criteria associated with common pneumoconioses. Avoid triggers; supportive therapy and supplemental O2. The most common form of idiopathic interstitial pneumonia. Has an unrelenting progression, with death usually occurring within 5–10 years. Exertional dyspnea and a nonproductive cough. Inspiratory crackles and/or clubbing on exam. T AB LE 2.1 5-5. Diagnosis of Pneumoconioses Usual interstitial pneumonia is one of the most common forms of interstitial pneumonia.

1	Asbestosis Work involving manufacture of tile or brake linings, insulation, construction, demolition, or shipbuilding. Presents 15–20 years after initial exposure. CXR: Linear opacities at lung bases and interstitial fibrosis; calcified pleural plaques are indicative of benign pleural disease. ↑ risk of mesothelioma (rare) and lung cancer; risk of lung cancer is higher in smokers. Coal miner’s disease Work in underground coal mines. CXR: Small nodular opacities (< 1 cm) in upper lung zones. Spirometry: Consistent with restrictive disease. Progressive massive fibrosis. Work in mines or quarries or with glass, pottery, or silica. CXR: Small (< 1-cm) nodular opacities in upper lung zones. Eggshell calcifications. Spirometry: Consistent with restrictive disease. ↑ risk of TB; need annual TB skin test. Progressive massive fibrosis. Work in high-technology fields such as aerospace, nuclear, and electronics plants; ceramics industries; foundries; plating facilities; dental material sites;

1	skin test. Progressive massive fibrosis. Work in high-technology fields such as aerospace, nuclear, and electronics plants; ceramics industries; foundries; plating facilities; dental material sites; and dye manufacturing. CXR: Diffuse infiltrates; hilar adenopathy. Requires chronic corticosteroid treatment. Silicosis Berylliosis

1	High-resolution CT: Patchy opacities at the lung bases, often with honeycombing. PFTs: Restrictive pattern. Surgical biopsy (usually required to confrm the diagnosis): Interstitial in-f ammation, fbrosis, and honeycombing. Options include corticosteroids, cytotoxic agents (azathioprine, cyclophosphamide), antifbrotic agents (have not been shown to improve survival), and lung transplantation. A diverse group of disorders characterized by eosinophilic pulmonary infiltrates and peripheral blood eosinophilia. Includes allergic bronchopulmonary aspergillosis, Löfﬂ er’s syndrome, and acute eosinophilic pneumonia. Presents with dyspnea, cough, and/or fever. CBC reveals peripheral eosinophilia; CXR shows pulmonary inf ltrates. Removal of the extrinsic cause or treatment of underlying infection if identifed. Corticosteroid treatment may be used if no cause is identif ed.

1	Removal of the extrinsic cause or treatment of underlying infection if identifed. Corticosteroid treatment may be used if no cause is identif ed. Causes include ventilation-perfusion (V/Q) mismatch, right-to-left shunt, hypoventilation, low inspired O2 content (important at altitudes), and diffusion impairment. Findings depend on the etiology. ↓HbO2 saturation, cyanosis, tachypnea, shortness of breath, pleuritic chest pain, and altered mental status may be seen. Pulse oximetry: Demonstrates ↓ HbO2 saturation. CXR: To rule out ARDS, atelectasis, or an infltrative process (e.g., pneumonia) and to look for signs of pulmonary embolism. ABGs: To evaluate PaO2 and to calculate the alveolar-arterial (A-a) oxygen gradient ([(Patm − 47) × FiO2 − (PaCO2/0.8)] − PaO2). An ↑ A-a gradient suggests a V/Q mismatch or a diffusion impairment. Figure 2.15-4 summarizes the approach toward hypoxemic patients. Is PaCO2 increased? Hypoventilation Yes Yes No No Is PAO2 − PaO2 increased?

1	Is PaCO2 increased? Hypoventilation Yes Yes No No Is PAO2 − PaO2 increased? Is PAO2 − PaO2 increased? Yes Yes NoNo Is low PO2 correctable with O2? 1. High altitude 2. ↓ FiO2 HIGH-YIELD FACTS Hypoventilation plus another mechanism Shunt 1. Alveolar collapse (asthma, COPD) 2. Intra-alveolar filling (pneumonia, pulmonary edema) 3. Intracardiac shunt 4. Vascular shunt within lungs 1. Airways disease (asthma, COPD) 2. Interstitial lung disease 3. Alveolar disease 4. Pulmonary vascular disease V/Q mismatch 1. ↓ respiratory drive 2. Neuromuscular disease Hypoventilation alone F IGU R E 2.1 5-4. Determination of the mechanism of hypoxia. (Reproduced, with permission, from Kasper DL et al. Harrison’s Principles of Internal Medicine, 16th ed. New York: McGraw- Hill, 2005.) Address the underlying etiology. Administer O2 before initiating evaluation.

1	Hill, 2005.) Address the underlying etiology. Administer O2 before initiating evaluation. If the patient is on a ventilator, ↑ O2 saturation by increasing FiO2, positive end-expiratory pressure (PEEP), or the I/E ratio. Hypercapnic patients: ↑ minute ventilation. Acute respiratory failure with refractory hypoxemia, ↓lung compliance, and noncardiogenic pulmonary edema. The pathogenesis is thought to be endothelial injury. Common triggers include sepsis, pneumonia, aspiration, multiple blood transfusions, inhaled/ingested toxins, and trauma. Overall mortality is 30–40%. Presents with acute-onset (12–48 hours) tachypnea, dyspnea, and tachycardia +/− fever, cyanosis, labored breathing, diffuse high-pitched rales, and hypoxemia in the setting of one of the systemic infammatory causes or exposure. Additional fndings are as follows: Phase 1 (acute injury): Normal physical exam; possible respiratory alkalosis.

1	Phase 1 (acute injury): Normal physical exam; possible respiratory alkalosis. Phase 2 (6–48 hours): Hyperventilation, hypocapnia, widening A-a oxygen gradient. Phase 3: Acute respiratory failure, tachypnea, dyspnea, ↓ lung compliance, scattered rales, diffuse chest infltrates on CXR (see Figure 2.15-5). F IGU R E 2.1 5-5. AP CXR showing a diffuse alveolar filling pattern 2° to ARDS. (Reproduced, with permission, from Kasper DL et al. Harrison’s Principles of Internal Medicine, 16th ed. New York: McGraw-Hill, 2005: 1497.) ■ Phase 4: Severe hypoxemia unresponsive to therapy; ↑ intrapulmonary shunting; metabolic and respiratory acidosis. The criteria for ARDS diagnosis (according to the American-European Consensus Conference defnition) are as follows: Acute onset of respiratory distress. PaO2/FiO2 ratio ≤ 200 mmHg. Bilateral pulmonary infltrates on CXR. No evidence of cardiac origin (capillary wedge pressure < 18 mmHg or no clinical evidence of elevated left atrial pressure).

1	PaO2/FiO2 ratio ≤ 200 mmHg. Bilateral pulmonary infltrates on CXR. No evidence of cardiac origin (capillary wedge pressure < 18 mmHg or no clinical evidence of elevated left atrial pressure). There is no standard treatment. Treat the underlying disease and maintain adequate perfusion to prevent end organ damage. Minimize injury induced by mechanical ventilation by ventilating with low tidal volumes. Use PEEP to recruit collapsed alveoli and titrate PEEP and FiO2 to achieve adequate oxygenation. Goal oxygenation is PaO2 > 60 mmHg or SaO2 > 90% on FiO2 ≤ 0.6. Slowly wean patients from ventilation, and follow with extubation trials (see Table 2.15-6). Pulmonary hypertension is defned as a mean pulmonary arterial pressure of > 25 mmHg (normal = 15 mmHg). It is classifed as either 1° (if the etiology

1	Pulmonary hypertension is defned as a mean pulmonary arterial pressure of > 25 mmHg (normal = 15 mmHg). It is classifed as either 1° (if the etiology T AB LE 2.1 5-6. Criteria for Extubation from Mechanical Ventilationa a Patients who meet these criteria are typically given a weaning (T-piece) trial to determine if they are ready for extubation. is unknown) or 2°. 1° pulmonary hypertension most often occurs in young or middle-aged women. The main causes of 2° pulmonary hypertension include the following: ■↑ pulmonary venous pressure from left-sided heart failure or mitral valve disease. ■↑ pulmonary blood fow 2° to congenital heart disease with left-to-right shunt. Hypoxic vasoconstriction 2° to chronic lung disease (e.g., COPD). Thromboembolic disease. Remodeling of pulmonary vessels 2° to structural lung disease. Presents with dyspnea on exertion, fatigue, lethargy, syncope with exertion, chest pain, and symptoms of right-sided CHF (edema, abdominal distention, JVD).

1	Presents with dyspnea on exertion, fatigue, lethargy, syncope with exertion, chest pain, and symptoms of right-sided CHF (edema, abdominal distention, JVD). Inquire about a history of COPD, interstitial lung disease, heart disease, sickle cell anemia, emphysema, and pulmonary emboli. Exam reveals a loud, palpable S2 (often split), a systolic ejection murmur, an S4, or a parasternal heave. CXR shows enlargement of central pulmonary arteries. ECG demonstrates RVH. Echocardiogram and right heart catheterization may show signs of right ventricular overload and may aid in the diagnosis of the underlying cause. Supplemental O2, anticoagulation, vasodilators, and diuretics if symptoms of right-sided CHF are present. Treat underlying causes of 2° pulmonary hypertension. Causes of pulmonary hypertension include left heart failure, mitral valve disease, and ↑ resistance in the pulmonary veins, including hypoxic vasoconstriction.

1	Causes of pulmonary hypertension include left heart failure, mitral valve disease, and ↑ resistance in the pulmonary veins, including hypoxic vasoconstriction. Other etiologies of embolic disease include postpartum status (amniotic f uid emboli), fracture (fat emboli), and cardiac surgery (air emboli). Dyspnea, tachycardia, and a normal CXR in a hospitalized and/or bedridden patient should raise suspicion of pulmonary embolism. Occlusion of the pulmonary vasculature by a blood clot. Ninety-f ve percent of emboli originate from DVTs in the deep leg veins. Often leads to pulmonary infarction, right heart failure, and hypoxemia. Virchow’s triad consists of the following: Stasis: Immobility, CHF, obesity, surgery, ↑ central venous pressure. Endothelial injury: Trauma, surgery, recent fracture, previous DVT. Hypercoagulable states: Pregnancy/postpartum, OCP use, coagulation disorders (e.g., protein C/protein S defciency, factor V Leiden), malignancy, severe burns.

1	Hypercoagulable states: Pregnancy/postpartum, OCP use, coagulation disorders (e.g., protein C/protein S defciency, factor V Leiden), malignancy, severe burns. Presents with sudden-onset dyspnea, pleuritic chest pain, low-grade fever, cough, and, rarely, hemoptysis. Pulmonary embolism: Hypoxia and hypocarbia with resulting respiratory alkalosis. Tachypnea, tachycardia, and fever. Exam may reveal a loud P2 and prominent jugular A waves with right heart failure. Venous thrombosis: Unilateral swelling; cords on the calf. ABGs: Respiratory alkalosis (2° hyperventilation) with PO2 < 80 mmHg. CXR: Usually normal, but may show atelectasis, pleural effusion, Hampton’s hump (a wedge-shaped infarct), or Westermark’s sign (oligemia in the affected lung zone). ECG: Not diagnostic; most commonly reveals sinus tachycardia. The classic triad of S1Q3T3—acute right heart strain with an S wave in lead I, a Q wave in lead III, and an inverted T wave in lead III—is uncommon.

1	Helical (spiral) CT with IV contrast: Sensitive for pulmonary embolism. V/Q scan: May reveal segmental areas of mismatch. Results are reported with a designated probability of pulmonary embolism (low, indeterminate, or high) and are interpreted in combination with clinical suspicion. Angiogram: The gold standard, but more invasive and rarely done today (see Figure 2.15-6). D-dimer: Sensitive but not specifc in patients at risk for DVT or pulmonary embolism (a useful “rule-out” test in patients with a low clinical suspicion, but it cannot rule out DVT or pulmonary embolism alone in patients with an intermediate or high clinical suspicion of clot). Venous ultrasound of the lower extremity: Can detect a clot that may have given off the pulmonary embolism. Serial ultrasounds have high diagnostic specif city. Heparin: Bolus followed by weight-based continuous infusion of unfractioned heparin or low-molecular-weight heparin (LMWH) SQ.

1	Heparin: Bolus followed by weight-based continuous infusion of unfractioned heparin or low-molecular-weight heparin (LMWH) SQ. Warfarin: For long-term anticoagulation, usually given for at least six months unless the underlying predisposing factor persists (and is then given indefnitely). Follow INR (goal = 2–3). IVC filter: Indicated for patients with documented DVT in a lower extremity if anticoagulation is contraindicated or if patients experience recurrent emboli while anticoagulated. FIGURE 2.15-6. Pulmonary embolus. A large flling defect in the pulmonary artery is evident on pulmonary angiogram. Thrombolysis: Indicated only in cases of massive DVT or pulmonary embolism causing right heart failure and hemodynamic instability (contraindicated in patients with recent surgery or bleeding). DVT prophylaxis: Treat all immobile patients; give SQ heparin, LMWH, intermittent pneumatic compression of the lower extremities (less effective), and early ambulation (most effective).

1	DVT prophylaxis: Treat all immobile patients; give SQ heparin, LMWH, intermittent pneumatic compression of the lower extremities (less effective), and early ambulation (most effective). Often asymptomatic, or patients may present with chronic cough, dyspnea, and shortness of breath. Always inquire about smoking and exposure history. Serial CXRs: Determine the location, progression, and extent of the nodule. Chest CT: Determine the nature, extent, and infltrating nature of the nodule. Characteristics favoring carcinoma: Age > 45–50; smoking history; history of malignancy; new or enlarging (unless very rapidly enlarging) lesions; absence of calcifcation or irregular calcifcation; size > 2 cm; irregular margins. Lung nodule clues based on the history: Recent immigrant: Think TB. From the southwestern United States: Think coccidioidomycosis. From the Ohio River Valley: Think histoplasmosis. Squamous and Small cell cancers are Central lesions.

1	Recent immigrant: Think TB. From the southwestern United States: Think coccidioidomycosis. From the Ohio River Valley: Think histoplasmosis. Squamous and Small cell cancers are Central lesions. ■Characteristics favoring a benign lesion: Age < 35; no change from old flms; central/uniform/laminated/popcorn calcifcation; size < 2 cm; smooth margins. Surgical resection is indicated for nodules at high risk for malignancy. Low-risk nodules can be followed with CXR or CT every three months for one year and then every six months for another year. An invasive diagnostic procedure is indicated if the size of the nodule ↑. The leading cause of cancer death in the United States. Risk factors include tobacco smoke (except for bronchoalveolar carcinoma) and radon or asbestos exposure. Types are as follows: Small cell lung cancer (SCLC): Highly correlated with cigarette exposure. Central location. Neuroendocrine origin; associated with paraneoplastic syndromes (see Table 2.15-7).

1	Small cell lung cancer (SCLC): Highly correlated with cigarette exposure. Central location. Neuroendocrine origin; associated with paraneoplastic syndromes (see Table 2.15-7). Metastases are often found on presentation (intrathoracic and extrathoracic sites such as brain, liver, and bone). Non–small cell lung cancer (NSCLC): Less likely to metastasize. TAB LE 2.1 5-7. Paraneoplastic Syndromes of Lung Cancer Adenocarcinoma: The most common lung cancer; peripheral location. Includes bronchoalveolar carcinoma, which is associated with multiple nodules, interstitial infltration, and prolifc sputum production but is not associated with smoking. Squamous cell carcinoma: Central location; 98% are seen in smokers. Large cell/neuroendocrine carcinomas: Least common; associated with a poor prognosis. Presents with cough, hemoptysis, dyspnea, wheezing, postobstructive pneumonia, chest pain, weight loss, and possible abnormalities on respiratory exam (crackles, atelectasis).

1	Presents with cough, hemoptysis, dyspnea, wheezing, postobstructive pneumonia, chest pain, weight loss, and possible abnormalities on respiratory exam (crackles, atelectasis). Other f ndings include Horner’s syndrome (miosis, ptosis, anhidrosis) in patients with Pancoast’s tumor at the apex of the lung; superior vena cava syndrome (obstruction of the SVC with supraclavicular venous engorgement); hoarseness (2° to recurrent laryngeal nerve involvement); and many paraneoplastic syndromes (see Table 2.15-7). CXR or chest CT. Fine-needle aspiration (CT guided) for peripheral lesions and bronchoscopy (biopsy or brushing) for central lesions. Thoracoscopic biopsy may be performed, with conversion to open thoracotomy if the lesion is found to be malignant. ■ SCLC: Unresectable. Often responds to radiation and chemotherapy initially but always recurs; has a lower median survival rate than NSCLC. Has usually metastasized at the time of diagnosis.

1	■ SCLC: Unresectable. Often responds to radiation and chemotherapy initially but always recurs; has a lower median survival rate than NSCLC. Has usually metastasized at the time of diagnosis. Chemotherapy is the mainstay ■NSCLC: Surgical resection in early stages (IA, IB, IIA, IIB, and possibly IIIA). The extent of resection is based on lesion size; the presence of me-of treatment for small cell tastases; and the patient’s age, general health, and lung function. Supple- lung cancer. ment surgery with radiation or chemotherapy (depending on the stage). Palliative radiation and/or chemotherapy is appropriate for symptomatic but unresectable disease. An abnormal accumulation of ﬂuid in the pleural space. Classifed as follows: Transudate: 2° to ↑ pulmonary capillary wedge pressure (PCWP) or ↓ oncotic pressure. Exudate: 2° to ↑ pleural vascular permeability. Table 2.15-8 lists the possible causes of both transudates and exudates.

1	Exudate: 2° to ↑ pleural vascular permeability. Table 2.15-8 lists the possible causes of both transudates and exudates. Presents with dyspnea, pleuritic chest pain, and/or cough. Exam reveals dullness to percussion and ↓breath sounds over the effusion. A pleural friction rub may be present. TABLE 2.15-8. Causes of Pleural Effusions CHF Cirrhosis Nephrotic syndrome Pneumonia (parapneumonic effusion) TB Malignancy Pulmonary embolism Collagen vascular disease (rheumatoid arthritis, SLE) Pancreatitis Trauma Complicated parapneumonic effusions necessitate chest tube drainage. Presentation of pneumothorax: CXR shows costophrenic angle blunting. Thoracentesis is indicated for new effusions > 1 cm in decubitus view, except with bilateral effusions and other clinical evidence of CHF. The effusion is an exudate if it meets Light’s criteria: The ratio of pleural to serum protein is > 0.5 or The ratio of pleural to serum LDH is > 0.6 or

1	The effusion is an exudate if it meets Light’s criteria: The ratio of pleural to serum protein is > 0.5 or The ratio of pleural to serum LDH is > 0.6 or Pleural fuid LDH is more than two-thirds the upper normal limit of serum LDH A parapneumonic effusion is classifed as complicated in the setting of a Gram stain or culture or a pH < 7.2 (normal is 7.6) or a glucose level of < 60. The presence of pus indicates an empyema. Treatment is directed toward the underlying condition causing the effusion. Complicated parapneumonic effusions and empyemas require chest tube drainage in addition to antibiotic therapy. A collection of air in the pleural space that can lead to pulmonary collapse. Subtypes are as follows: 1° spontaneous pneumothorax: 2° to rupture of subpleural apical blebs (usually found in tall, thin young males).

1	2° pneumothorax: 2° to COPD, TB, trauma, Pneumocystis jiroveci (formerly P. carinii) pneumonia, and iatrogenic factors (thoracentesis, subclavian line placement, positive-pressure mechanical ventilation, bronchoscopy). Tension pneumothorax: A pulmonary or chest wall defect acts as a one-way valve, drawing air into the pleural space during inspiration but trapping air during expiration. Etiologies include penetrating trauma, infection, and positive-pressure mechanical ventilation. Shock and death result unless the condition is immediately recognized and treated. Presents with acute onset of unilateral pleuritic chest pain and dyspnea. Exam reveals tachypnea, diminished or absent breath sounds, hyperreso F IGU R E 2.1 5-7. Tension pneumothorax. Note the hyperlucent lung feld, hyperexpanded lower diaphragm, collapsed lung, tracheal de viation, mediastinal shift, and compression of the opposite lung on AP CXR.

1	Note the hyperlucent lung feld, hyperexpanded lower diaphragm, collapsed lung, tracheal de viation, mediastinal shift, and compression of the opposite lung on AP CXR. nance, ↓tactile fremitus, and JVD 2° to compression of the superior vena cava. ■Suspect tension pneumothorax in the presence of tracheal deviation, respiratory distress, falling O2 saturation, hypotension, and distended neck veins. CXR shows the presence of a visceral pleural line and/or lung retraction from the chest wall (best seen in end-expiratory flms; see Figure 2.15-7). Small pneumothoraces may resorb spontaneously. Supplemental O2 therapy is helpful. Large, symptomatic pneumothoraces require chest tube placement. Tension pneumothorax requires immediate needle decompression (second intercostal space at the midclavicular line) followed by chest tube placement.

1	Tension pneumothorax requires immediate needle decompression (second intercostal space at the midclavicular line) followed by chest tube placement. In hypernatremia, certain patients (e.g., infants, intubated patients, those with a change in mental status) may not drink enough free water to replace insensible losses. Hypernatremia causes— The 6 D’s Diuresis Dehydration Diabetes insipidus Docs (iatrogenic) Diarrhea Disease (e.g., kidney, sickle cell) Serum sodium > 145 mEq/L. Usually due to water loss rather than sodium gain. Presents with thirst (due to hypertonicity) as well as with oliguria or polyuria (depending on the etiology). Neurologic symptoms include mental status changes, weakness, focal neurologic deficits, and seizures. Exam reveals “doughy” skin and signs of volume depletion. Assess volume status by conducting a clinical exam and measuring urine volume and osmolality. Hypertonic Na+ gain: Due to hypertonic saline/tube feeds or ↑aldosterone (suppresses ADH).

1	Assess volume status by conducting a clinical exam and measuring urine volume and osmolality. Hypertonic Na+ gain: Due to hypertonic saline/tube feeds or ↑aldosterone (suppresses ADH). Pure water loss: Due to central or nephrogenic diabetes insipidus; characterized by large volumes of dilute urine. Do not neglect dermal and respiratory insensible losses. Hypotonic f uid loss: Due to ↓ intake, diuretics, intrinsic renal disease, GI losses (diarrhea), burns, and osmotic diuresis (mannitol, glucose in DKA, urea with high protein feeds). A minimal volume (approximately 500 ml/day) of maximally concentrated urine (> 800 mOsm/kg) suggests adequate renal response without adequate free-water replacement. Treat the underlying causes and replace free-water deficit with hypotonic saline, D5W, or oral water, depending on volume status.

1	Treat the underlying causes and replace free-water deficit with hypotonic saline, D5W, or oral water, depending on volume status. Correction of chronic hypernatremia (> 36–48 hours) should be accomplished gradually over 48–72 hours (≤ 0.5 mEq/L/hr) to prevent neurologic damage 2° to cerebral edema. Serum sodium < 135 mEq/L. Almost always due to ↑ ADH. May be asymptomatic or may present with confusion, lethargy, muscle cramps, hyporeﬂexia, and nausea. Can progress to seizures, coma, or brain stem herniation. Hyponatremia can be categorized according to serum and urine osmolality as well as by volume status (i.e., by clinical exam). Osmolality is classified as follows: High (> 295 mEq/L): Hyperglycemia, hypertonic infusion (e.g., mannitol). Normal (280–295 mEq/L): Hypertriglyceridemia, paraproteinemia (pseudohyponatremia).

1	High (> 295 mEq/L): Hyperglycemia, hypertonic infusion (e.g., mannitol). Normal (280–295 mEq/L): Hypertriglyceridemia, paraproteinemia (pseudohyponatremia). Hypervolemic hyponatremia is caused by “nephrOSIS, cirrhOSIS, and cardiOSIS.” ■ Low (< 280 mEq/L): Applies to the majority of cases. Hypotonic etiolog ies are listed in Table 2.16-1. Specific treatments are outlined in Table 2.16-1. Chronic hyponatremia (> 72 hours’ duration) should be corrected slowly (no more than 0.5 mEq/L/hr) in order to prevent central pontine myelinolysis (symptoms include paraparesis/quadriparesis, dysarthria, and coma). Serum potassium > 5 mEq/L. Etiologies are as follows: Spurious: Hemolysis of blood samples, fist clenching during blood draws, delays in sample analysis, extreme leukocytosis or thrombocytosis. ■↓excretion: Renal insufficiency, drugs (e.g., spironolactone, triamterene, ACEIs, trimethoprim, NSAIDs), hypoaldosteronism, type IV renal tubular acidosis (RTA).

1	■↓excretion: Renal insufficiency, drugs (e.g., spironolactone, triamterene, ACEIs, trimethoprim, NSAIDs), hypoaldosteronism, type IV renal tubular acidosis (RTA). Cellular shifts: Cell lysis, tissue injury (rhabdomyolysis), insulin deficiency, acidosis, drugs (e.g., succinylcholine, digitalis, arginine, β-blockers), exercise. Iatrogenic. May be asymptomatic or may present with nausea, vomiting, intestinal colic, aref exia, weakness, ﬂaccid paralysis, and paresthesias. Confirm hyperkalemia with a repeat blood draw. In the setting of extreme leukocytosis or thrombocytosis, check plasma potassium. ECG findings include tall, peaked T waves; a wide QRS; PR prolongation; and loss of P waves (see Figure 2.16-1). Can progress to sine waves, ventricular fibrillation, and cardiac arrest. T AB LE 2.1 6-1. Evaluation and Treatment of Hypotonic Hyponatremia

1	T AB LE 2.1 6-1. Evaluation and Treatment of Hypotonic Hyponatremia Consider using hypertonic saline only if a patient has seizures due to hyponatremia, and use it cautiously and brief y. In most instances, normal saline is the best replacement f uid. What dreaded complication can arise from treating hyponatremia too rapidly? Central pontine myelinolysis. Hypervolemic Renal failure, nephrotic syndrome, cirrhosis, CHF, hypothyroidism, 2° or 3° adrenal insuff ciency. Water restriction; consider diuretics; cortisol replacement with adrenal insuff ciency; thyroid replacement with hypothyroidism. Euvolemic SIADH, renal failure, drugs, psychogenic polydipsia, oxytocin use. Water restriction. Hypovolemic Diuretics (especially thiazides), vomiting, diarrhea, bleeding, third spacing, dehydration, DKA, 1° adrenal insuff ciency. Replete volume with normal saline. FIGURE 2.16-1. Hyperkalemia on ECG. Hypokalemia is usually due to renal or GI losses

1	FIGURE 2.16-1. Hyperkalemia on ECG. Hypokalemia is usually due to renal or GI losses Hypokalemia sensitizes the heart to digitalis toxicity because K+ and digitalis compete for the same sites on the Na+/K+ pump, so if a patient is on digitalis, potassium levels must be carefully monitored. Electrocardiographic manifestations include peaked T waves, PR prolongation, and a widened QRS complex. (Reproduced, with permission, from Cogan MG. Fluid and Electrolytes, 1st ed. Stamford, CT: Appleton & Lange, 1991: 170.) Values of > 6.5 mEq/L or ECG changes (especially PR prolongation or wide QRS) require emergent treatment. The mnemonic C BIG K summarizes the treatment of hyperkalemia. First give calcium gluconate for cardiac cell membrane stabilization. Give bicarbonate and/or insulin and glucose to temporarily shift potassium into cells. β-agonists (e.g., albuterol) promote cellular reuptake of potassium. Eliminate potassium from diet and IV ﬂ uids.

1	β-agonists (e.g., albuterol) promote cellular reuptake of potassium. Eliminate potassium from diet and IV ﬂ uids. Kayexalate to remove potassium from the body. Dialysis is appropriate for patients with renal failure or for severe, refractory cases. Serum potassium < 3.6 mEq/L. Etiologies are as follows: Transcellular shifts: Insulin, β2-agonists, alkalosis, familial hypokalemic periodic paralysis. GI losses: Diarrhea, chronic laxative abuse, vomiting, NG suction. Renal losses: Diuretics (e.g., loop or thiazide), 1° mineralocorticoid excess or 2° hyperaldosteronism, ↓ circulating volume, Bartter’s and Gitelman’s syndromes, drugs (e.g., gentamicin, amphotericin), DKA, hypomagnesemia, type I RTA (defective distal H+ secretion), polyuria. Presents with fatigue, muscle weakness or cramps, ileus, hypotension, hyporeﬂexia, paresthesias, rhabdomyolysis, and ascending paralysis. Twenty-four-hour or spot urine potassium may distinguish renal from GI losses.

1	Twenty-four-hour or spot urine potassium may distinguish renal from GI losses. ECG may show T-wave fattening, U waves (an additional wave after the T wave), and ST-segment depression, leading to AV block and subsequent cardiac arrest. Consider RTA in the setting of metabolic acidosis. Treat the underlying disorder. Oral and/or IV potassium repletion. Do not exceed 20 mEq/L/hr. Replace magnesium, as this deficiency complicates potassium repletion. Monitor ECG and plasma potassium levels frequently during replacement. Serum calcium > 10.2 mg/dL. The most common causes are hyperparathyroidism and malignancy (e.g., breast cancer, squamous cell carcinoma, multiple myeloma). Other causes are summarized in the mnemonic CHIMPANZEES. Usually asymptomatic and discovered by routine labs, but may present with bones (osteopenia, fractures), stones (kidney stones), abdominal groans (anorexia, constipation), and psychiatric overtones (weakness, fatigue, altered mental status).

1	Order a total/ionized calcium, albumin, phosphate, PTH, parathyroid hormone–related peptide (PTHrP), vitamin D, and ECG (may show a short QT interval). IV hydration followed by furosemide to ↑ calcium excretion. Calcitonin, bisphosphonates (e.g., pamidronate), glucocorticoids, calcimimetics, and dialysis are used for severe or refractory cases. Avoid thiazide diuretics, which ↑ tubular reabsorption of calcium. Serum calcium < 8.5 mg/dL. Etiologies include hypoparathyroidism (postsurgical, idiopathic), malnutrition, hypomagnesemia, acute pancreatitis, vitamin D deficiency, and pseudohypoparathyroidism. In infants, consider DiGeorge’s syndrome (tetany shortly after birth; absence of thymic shadow). Presents with abdominal muscle cramps, dyspnea, tetany, perioral and acral paresthesias, and convulsions.

1	Presents with abdominal muscle cramps, dyspnea, tetany, perioral and acral paresthesias, and convulsions. Facial spasm elicited from tapping of the facial nerve (Chvostek’s sign) and carpal spasm after arterial occlusion by a BP cuff (Trousseau’s sign) are classic findings that are most commonly seen in severe hypocalcemia. Order an ionized Ca2+, Mg+, PTH, albumin, and possibly calcitonin. If the patient is post-thyroidectomy, review the operative note to determine the number of parathyroid glands removed. ECG may show a prolonged QT interval. Causes of hypercalcemia— Hyperthyroidism Iatrogenic (e.g., thiazides, parenteral (e.g., MEN 1) Excess vitamin A Excess vitamin D Sarcoidosis and other Loops (furosemide) Lose calcium. A classic case of hypocalcemia is a patient who develops cramps and tetany following thyroidectomy. Serum calcium may be falsely low in hypoalbuminemia; check ionized calcium. Treat the underlying disorder. Magnesium repletion.

1	Serum calcium may be falsely low in hypoalbuminemia; check ionized calcium. Treat the underlying disorder. Magnesium repletion. Administer oral calcium supplements; give IV calcium for severe symptoms. Serum magnesium < 1.5 mEq/L. Etiologies are as follows: ■↓intake: Malnutrition, malabsorption, short bowel syndrome, TPN. ■↑loss: Diuretics, diarrhea, vomiting, hypercalcemia, drugs (e.g., aminoglycosides, amphotericin), alcoholism, kidney losses (e.g., recovering ATN, postobstructive diuresis). ■ Miscellaneous: DKA, pancreatitis, extracellular ﬂuid volume expansion. Symptoms are generally related to concurrent hypocalcemia and hypokalemia; they include anorexia, nausea, vomiting, muscle cramps, and weakness. In severe cases, symptoms may also include hyperactive reﬂ exes, tetany, paresthesias, irritability, confusion, lethargy, seizures, and arrhythmias. Labs may show concurrent hypocalcemia and hypokalemia. ECG may reveal prolonged PR and QT intervals.

1	Labs may show concurrent hypocalcemia and hypokalemia. ECG may reveal prolonged PR and QT intervals. IV and oral supplements. Hypokalemia and hypocalcemia will not correct without magnesium correction. See Figure 2.16-2 for a diagnostic algorithm of acid-base disorders. A net ↓ in either tubular H+ secretion or HCO3 reabsorption that leads to a non–anion gap metabolic acidosis. There are three main types of RTA; type IV (distal) is the most common form (see Table 2.16-2). An abrupt ↓ in renal function leading to the retention of creatinine and BUN. ↓ urine output (oliguria, defined as < 500 cc/day) is not required for ARF. ARF is categorized as follows (see also Table 2.16-3): Prerenal: ↓ renal perfusion. Intrinsic: Injury within the nephron unit. Postrenal: Urinary outﬂow obstruction. Generally, both kidneys must be obstructed before one can see a significant ↑ in BUN and creatinine. Alcoholics are the most common patient population with hypomagnesemia.

1	Alcoholics are the most common patient population with hypomagnesemia. Aspirin (salicylate) overdose can cause both a metabolic acidosis and a respiratory alkalosis. If an asthmatic patient’s blood gas goes from alkalotic to normal, it can be a sign of respiratory muscle fatigue, which requires urgent intubation. pH < 7.4 pH > 7.4 Acidosis Alkalosis Respiratory Metabolic Respiratory alkalosis acidosis acidosis with –Hyperventilation compensation –Aspirin ingestion (early) –Chronic lung disease –Opioids, narcotics, sedatives –Weakening of F IGU R E 2.1 6-2. Diagnostic algorithm for acid-base disorders. Metabolic alkalosis with compensation –Vomiting –Diuretic use –Antacid use –Hyperaldosteronism Symptoms of uremia include malaise, fatigue, confusion, oliguria, anorexia, and nausea. Exam may show a pericardial rub, asterixis, hypertension, ↓ urine output, and an ↑ respiratory rate (compensation of metabolic acidosis or from pulmonary edema 2° to volume overload)

1	Exam may show a pericardial rub, asterixis, hypertension, ↓ urine output, and an ↑ respiratory rate (compensation of metabolic acidosis or from pulmonary edema 2° to volume overload) Category-specific symptoms are as follows: Prerenal: Thirst, orthostatic hypotension, tachycardia, ↓ skin turgor, dry mucous membranes, reduced axillary sweating, stigmata of comorbid conditions. Intrinsic: Associated with a history of drug exposure (aminoglycosides, NSAIDs), infection, or exposure to contrast media or toxins (e.g., myoglobin, myeloma protein). Hematuria or tea-colored urine, foamy urine (from proteinuria), hypertension, and/or edema may also be present. Atheroemboli: Subcutaneous nodules, livedo reticularis, digital ischemia. Postrenal: Prostatic disease, ↓ urine output leading to suprapubic pain, distended bladder and ﬂ ank pain. Check serum electrolytes. Examine the urine for RBCs, WBCs, casts (see Table 2.16-4), and urine eosinophils.

1	Check serum electrolytes. Examine the urine for RBCs, WBCs, casts (see Table 2.16-4), and urine eosinophils. An FeNa < 1%, a UNa < 20, a urine specifc gravity > 1.020, or a BUN/Cr ratio > 20 suggests a prerenal etiology. A urinary catheter and renal ultrasound can help rule out obstruction. Ultrasound can also identify kidneys that are ↓ in size, as occurs with chronic kidney disease. Patients with ARF may have a normal urine volume. An FeNa < 1% indicates that the kidneys are trying to conserve sodium, suggesting a prerenal etiology. TABLE 2.16-2. Types of Renal Tubular Acidosis

1	Defect H+ secretion HCO3 – reabsorption Aldosterone def ciency or resistance, leading to defects in Na+ reabsorption and H+ and K+ excretion Serum K+ Low Low High Urinary pH > 5.3 5.3 initially; < 5.3 once serum is acidic < 5.3 Etiologies (most common) Hereditary, cirrhosis, autoimmune disorders (Sjren’s syndrome, SLE), hypercalciuria, sickle cell disease, drugs (lithium, amphotericin) Hereditary (idiopathic or part of syndromes such as Fanconi’s syndrome or cystinosis), drugs (carbonic anhydrase inhibitors), multiple myeloma, amyloidosis, heavy metal poisoning, vitamin D def ciency 1° aldosterone def ciency, hyporeninemic hypoaldosteronism (e.g., from kidney disease, ACEIs, NSAIDs), drugs (e.g., amiloride, spironolactone, heparin), pseudohypoaldosteronism Potassium citrate Potassium citrate Furosemide, mineralocorticoid +/– glucocorticoid replacement, and low-potassium diet in patients with aldosterone def ciency Nephrolithiasis Rickets, osteomalacia Hyperkalemia Treatment

1	citrate Furosemide, mineralocorticoid +/– glucocorticoid replacement, and low-potassium diet in patients with aldosterone def ciency Nephrolithiasis Rickets, osteomalacia Hyperkalemia Treatment Complications

1	TABLE 2.16-3. Causes of Acute Renal Failure Acute tubular necrosis (ATN) Acute/allergic interstitial nephritis Glomerulonephritis Thromboembolism Renovascular disease (HUS/TTP, scleroderma) Prostatic disease Nephrolithiasis Pelvic tumors Recent pelvic surgery Retroperitoneal f brosis Hypovolemia (hemorrhage, dehydration, burns) Cardiogenic shock (↓ CO) Systemic vasodilation (sepsis, burns) Anaphylaxis Drugs (ACEIs, ARBs, NSAIDs) Renal artery stenosis Cirrhosis with ascites T AB LE 2.1 6-4. Findings on Microscopic Urine Examination in Acute Renal Failure Hyaline casts Normal fnding, but an ↑ amount suggests volume depletion Prerenal Red cell casts, dysmorphic red cells Glomerulonephritis Intrinsic White cells, eosinophils Allergic interstitial nephritis, atheroembolic disease Intrinsic Granular casts, renal tubular cells, “muddy-brown cast” ATN Intrinsic White cells, white cell casts Pyelonephritis Postrenal

1	In patients with oliguria, the FeNa can help identify prerenal failure and distinguish it from intrinsic renal disease. Obtain a renal biopsy only when the cause of intrinsic renal disease is unclear. Balance ﬂuids and electrolytes; avoid nephrotoxic drugs. In acute or allergic interstitial nephritis, adjust or discontinue offending medications. Dialyze if indicated (see the mnemonic AEIOU) using hemodialysis. Peritoneal dialysis should be considered only for long-term dialysis patients or for patients who are not hemodynamically stable. Metabolic acidosis; hyperkalemia leading to arrhythmias. Hypertension (from renin hypersecretion). Volume overload leading to CHF and pulmonary edema. Chronic kidney disease may result, requiring dialysis to prevent the buildup of K+, H+, phosphate, and toxic metabolites. Ingestions (salicylates, theophylline, methanol, barbiturates, lithium, ethylene glycol)

1	Ingestions (salicylates, theophylline, methanol, barbiturates, lithium, ethylene glycol) Uremic symptoms (pericarditis, encephalopathy, bleeding, nausea, pruritus, myoclonus) Most commonly due to diabetes mellitus (DM), hypertension, and glomerulonephritis. Another commonly tested etiology is polycystic kidney disease (the autosomal-dominant form is more common and is adult onset; the autosomalrecessive form is seen in children). Generally asymptomatic until GFR < 30, but patients gradually experience the signs and symptoms of uremia (anorexia, nausea, vomiting, uremic pericarditis, “uremic frost,” delirium, seizures, coma). Common metabolic derangements include the following: Azotemia (↑ BUN and creatinine). Fluid retention (hypertension, edema, CHF, pulmonary edema). Metabolic acidosis. Hyperkalemia. Anemia of chronic disease (↓ erythropoietin production).

1	Azotemia (↑ BUN and creatinine). Fluid retention (hypertension, edema, CHF, pulmonary edema). Metabolic acidosis. Hyperkalemia. Anemia of chronic disease (↓ erythropoietin production). Hypocalcemia, hyperphosphatemia (↓ phosphate excretion; impaired vitamin D production leading to renal osteodystrophy). ACEIs/ARBs and hypertension control have been shown to ↓ the progression of CKD. Additional pharmacotherapy is as follows: Erythropoietin analogs for anemia of chronic disease. Fluid restriction; low Na+/K+/phosphate intake. Oral phosphate binders and calcitriol (1,25-OH vitamin D) for renal osteodystrophy. Renal replacement therapy includes hemodialysis, peritoneal dialysis, and renal transplantation. Table 2.16-5 and Figure 2.16-3 summarize the mechanisms of action and side effects of commonly used diuretics. T AB LE 2.1 6-5. Mechanism of Action and Side Effects of Diuretics

1	Carbonic anhydrase inhibitors Acetazolamide Proximal convoluted tubule. Inhibit carbonic anhydrase, ↑ H+ reabsorption, block Na+/ H+ exchange. Hyperchloremic metabolic acidosis, sulfa allergy. Osmotic agents Mannitol, urea Entire tubule. ↑ tubular fuid osmolarity. Pulmonary edema due to CHF and anuria. Loop agents Furosemide, ethacrynic acid, bumetanide, torsemide Ascending loop of Henle. Inhibit Na+/K+/2Cl– transporter. Water loss, metabolic alkalosis, ↓ K+, ↓Ca2+, ototoxicity, sulfa allergy (except ethacrynic acid, hyperuricemia). Thiazide agents HCTZ, chlorothiazide Distal convoluted tubule. Inhibit Na+/Cl– transporter. Water loss, metabolic alkalosis, ↓Na+, ↓ K+, ↑ glucose, ↑Ca2+, ↑ uric acid, sulfa allergy, pancreatitis. K+-sparing agents Spironolactone, triamterene, amiloride Cortical collecting tubule. Aldosterone receptor antagonist (spironolactone); block sodium channel (triamterene, amiloride). Metabolic acidosis; ↑ K+; antiandrogenic effects, including gynecomastia

1	Cortical collecting tubule. Aldosterone receptor antagonist (spironolactone); block sodium channel (triamterene, amiloride). Metabolic acidosis; ↑ K+; antiandrogenic effects, including gynecomastia (spironolactone).

1	Glomerulusconvoluted tubule Proximal straight tubule 1 Cortex Outer medulla Diuretics 1 Acetazolamide 2 Osmotic agents (mannitol) 3 Loop agents (e.g., furosemide) 4 Thiazides5 Aldosterone antagonists6 ADH antagonists2 2 2 3 4 5 6 Thin descending limbH2O H2O H2O (+ADH) Inner medulla Loop of Henle Thin ascending limbThick ascending limbNa+ K+ K+ 2Cl−Ca2+ Mg2+ tubule(+PTH) (+aldosterone) ? 4 F IGU R E 2.1 6-3. Overview of diuretics. (Reproduced, with permission, from Katzung BG. Basic & Clinical Pharmacology, 10th ed. New York: McGraw-Hill, 2007: Fig. 15-1.) A disorder of glomerular inﬂammation, also called glomerulonephritis. Proteinuria may be present but is usually < 1.5 g/day. Causes are summarized in Table 2.16-6. The classic findings are oliguria, macroscopic/microscopic hematuria (teaor cola-colored urine), hypertension, and edema. the patient has hematuria, hypertension, and oliguria. UA shows hematuria and possibly mild proteinuria.

1	UA shows hematuria and possibly mild proteinuria. Patients have a ↓ GFR with elevated BUN and creatinine. Complement, ANA, ANCA, and anti-GBM antibody levels should be measured to determine the underlying etiology. Renal biopsy may be useful for histologic evaluation. T AB LE 2.1 6-6. Causes of Nephritic Syndrome

1	Immune complex Postinfectious glomerulonephritis IgA nephropathy (Berger’s disease) Classically associated with recent group A β-hemolytic streptococcal infection, but can be seen with any infection (usually 2–6 weeks prior). The most common type; associated with upper respiratory or GI infections. Commonly seen in young males; may be seen in Henoch-Schlein purpura. Oliguria, edema, hypertension, teaor cola-colored urine. Episodic gross hematuria or persistent microscopic hematuria. Low serum C3 that normalizes 6–8 weeks after presentation; ↑ASO titer; lumpy-bumpy immunoﬂ uorescence. Normal C3. Supportive. Almost all children and most adults have a complete recovery. Glucocorticoids for select patients; ACEIs in patients with proteinuria. Some 20% of cases progress to end-stage renal disease (ESRD). Pauci-immune Wegener’s granulomatosis Granulomatous inf ammation of the respiratory tract and kidney with necrotizing vasculitis. Fever, weight loss, hematuria, hearing disturbances,

1	disease (ESRD). Pauci-immune Wegener’s granulomatosis Granulomatous inf ammation of the respiratory tract and kidney with necrotizing vasculitis. Fever, weight loss, hematuria, hearing disturbances, respiratory and sinus symptoms. Cavitary pulmonary lesions bleed and lead to hemoptysis. Presence of c-ANCA (cell-mediated immune response). Renal biopsy shows segmental necrotizing glomerulonephritis with few immunoglobulin deposits on immunof uorescence. High-dose corticosteroids and cytotoxic agents. Patients tend to have frequent relapses. Anti-GBH disease Goodpasture’s syndrome Alport’s syndrome Rapidly progressing glomerulonephritis with pulmonary hemorrhage; peak incidence is in males in their mid-20s. Hereditary glomerulonephritis; presents in boys 5–20 years of age. Hemoptysis, dyspnea, possible respiratory failure. Asymptomatic hematuria associated with nerve deafness and eye disorders. Linear anti-GBM deposits on immunof uorescence; iron def ciency anemia; hemosiderin-f lled

1	possible respiratory failure. Asymptomatic hematuria associated with nerve deafness and eye disorders. Linear anti-GBM deposits on immunof uorescence; iron def ciency anemia; hemosiderin-f lled macrophages in sputum; pulmonary infltrates on CXR. GBM splitting on electron microscopy. Plasma exchange therapy; pulsed steroids. May progress to ESRD. Progresses to renal failure. Anti-GBM nephritis may recur after transplant.

1	Treat hypertension, ﬂuid overload, and uremia with salt and water restriction, diuretics, and, if necessary, dialysis. In some cases, corticosteroids are useful in reducing glomerular inﬂ ammation. Defined as proteinuria (≥ 3.5 g/day), generalized edema, hypoalbuminemia, and hyperlipidemia. Approximately one-third of all cases result from systemic diseases such as DM, SLE, or amyloidosis. Causes are summarized in Table 2.16-7. TAB LE 2.1 6-7. Causes of Nephrotic Syndrome

1	Minimal change disease The most common cause of nephrotic syndrome in children. Idiopathic etiology; 2° causes include NSAIDs and hematologic malignancies. Tendency toward infections and thrombotic events. Light microscopy appears normal; electron microscopy shows fusion of epithelial foot processes with lipid-laden renal cortices. Steroids; excellent prognosis. Focal segmental glomerulosclerosis Idiopathic, IV drug use, HIV infection, obesity. The typical patient is a young African-American male with uncontrolled hypertension. Microscopic hematuria; biopsy shows sclerosis in capillary tufts. Prednisone, cytotoxic therapy, ACEIs/ARBs to ↓ proteinuria. Membranous nephropathy The most common nephropathy in Caucasian adults. 2° causes includes solid tumor malignancies (especially in patients > 60 years of age) and immune complex disease. Associated with HBV, syphilis, malaria, and gold. “Spike-and-dome” appearance due to granular deposits of IgG and C3 at the basement membrane.

1	patients > 60 years of age) and immune complex disease. Associated with HBV, syphilis, malaria, and gold. “Spike-and-dome” appearance due to granular deposits of IgG and C3 at the basement membrane. Prednisone and cytotoxic therapy for severe disease. Diabetic nephropathy Two characteristic forms: diffuse hyalinization and nodular glomerulosclerosis (Kimmelstiel-Wilson lesions). Generally have long-standing, poorly controlled DM with evidence of retinopathy or neuropathy. Thickened GBM; ↑mesangial matrix. Tight control of blood sugar; ACEIs for type 1 DM and ARBs for type 2 DM.

1	TAB LE 2.1 6-7. Causes of Nephrotic Syndrome (continued)

1	Lupus nephritis Classifed as WHO types I–VI. Both nephrotic and nephritic. The severity of renal disease often determines overall prognosis. Proteinuria or RBCs on UA may be found during evaluation of SLE patients. Mesangial proliferation; subendothelial and/or subepithelial immune complex deposition. Prednisone and cytotoxic therapy may slow disease progression. Renal amyloidosis 1° (plasma cell dyscrasia) and 2° (infectious or infammatory) are the most common. Patients may have multiple myeloma or a chronic inf ammatory disease (e.g., rheumatoid arthritis, TB). Nodular glomerulosclerosis; EM reveals amyloid f brils; apple-green birefringence with Congo red stain. Prednisone and melphalan. Bone marrow transplant may be used for multiple myeloma. Membrano-proliferative nephropathy Can also be nephritic syndrome. Type I is associated with HCV, cryoglobulinemia, SLE, and subacute bacterial endocarditis. Idiopathic form is present at 8–30 years of age. Slow progression to renal failure.

1	be nephritic syndrome. Type I is associated with HCV, cryoglobulinemia, SLE, and subacute bacterial endocarditis. Idiopathic form is present at 8–30 years of age. Slow progression to renal failure. “Tram-track,” double-layered basement membrane. Type I has subendothelial deposits and mesangial deposits; all three types have low serum C3; type II by way of C3 nephritic factor. Corticosteroids and cytotoxic agents may help.

1	Proteinuria, hypoalbuminemia, edema, hyperlipidemia, and hyperlipiduria in nephrotic syndrome are due to the initial ↑ in permeability of the glomerulus to protein. Presents with generalized edema and foamy urine. In severe cases, dyspnea and ascites may develop. Patients have ↑ susceptibility to infection as well as a predisposition to hypercoagulable states with an ↑ risk for venous thrombosis and pulmonary embolism. UA shows proteinuria (≥ 3.5 g/day) and lipiduria. Blood chemistry shows ↓ albumin (< 3 g/dL) and hyperlipidemia. Evaluation should include workup for 2° causes. Renal biopsy is used to definitively diagnose the underlying etiology. ■ Treat with protein and salt restriction, judicious diuretic therapy, and antihyperlipidemics. Immunosuppressant medications may be useful for certain etiologies. ACEIs ↓ proteinuria and diminish the progression of renal disease in patients with renal scarring.

1	Immunosuppressant medications may be useful for certain etiologies. ACEIs ↓ proteinuria and diminish the progression of renal disease in patients with renal scarring. Vaccinate with 23-polyvalent pneumococcus vaccine (PPV23), as patients are at ↑ risk of Streptococcus pneumoniae infection. Renal calculi. Stones are most commonly calcium oxalate but may also be calcium phosphate, struvite, uric acid, or cystine (see Table 2.16-8 and Figure 2.16-4). Risk factors include a family history, low f uid intake, gout, medications (allopurinol, chemotherapy, loop diuretics), postcolectomy/ postileostomy, specific enzyme deficiencies, type I RTA (due to alkaline urinary pH and associated hypocitruria), and hyperparathyroidism. Most common in older males. Presents with acute onset of severe, colicky f ank pain that may radiate to the testes or vulva and is associated with nausea and vomiting.

1	Presents with acute onset of severe, colicky f ank pain that may radiate to the testes or vulva and is associated with nausea and vomiting. Patients are unable to get comfortable and shift position frequently (as opposed to those with peritonitis, who lie still). UA may show gross or microscopic hematuria (85%) and an altered urine pH. KUB (kidney/ureter/bladder radiography) identifies radiopaque stones but will miss the 10% of stones that are radiolucent. TABLE 2.16-8. Types of Nephrolithiasis Which bacteria are associated with “staghorn calculi”? Urease-producing organisms such as Proteus.

1	Calcium oxalate/ calcium phosphate 83% The most common causes are idiopathic hypercalciuria and 1° hyperparathyroidism. Alkaline urine. Radiopaque. Hydration, dietary sodium and protein restriction, thiazide diuretic. Avoid ↓calcium intake (can lead to hyperoxaluria and an ↑ risk of osteoporosis). Struvite (Mg-NH4 -PO4) or “triple phosphate” 9% Associated with urease-producing organisms (e.g., Proteus). Form staghorn calculi. Alkaline urine. Radiopaque. Hydration; treat UTI if present; surgical removal of staghorn stone. Uric acid 7% Associated with gout, xanthine oxidase defciency, and high purine turnover states (e.g., chemotherapy). Acidic urine (pH < 5.5). Radiolucent. Hydration; alkalinize urine with citrate, which is converted to HCO3 – in the liver; dietary purine restriction and allopurinol. Cystine 1% Due to a defect in renal transport of certain amino acids (COLA—cystine, ornithine, lysine, and arginine). Hexagonal crystals. Radiopaque. Hydration, dietary sodium restriction,

1	Cystine 1% Due to a defect in renal transport of certain amino acids (COLA—cystine, ornithine, lysine, and arginine). Hexagonal crystals. Radiopaque. Hydration, dietary sodium restriction, alkalinization of urine, penicillamine.

1	FIGURE 2.16-4. Nephrolithiasis. KUB shows two dense 1-cm calcifications (arrows) projecting over the midportion of the left kidney, consistent with nephrolithiasis. (Reproduced, with permission, from Chen MY, Pope TL Jr., Ott DJ. Basic Radiology, 1st ed. New York: McGraw-Hill, 2004: 243.) Noncontrast abdominal CT scans are the gold standard for the diagnosis of kidney stones. Consider a renal ultrasound to look for obstruction (ultrasound is also preferred for pregnant patients, in whom radiation from CT should be avoided). An IVP can be used to confirm the diagnosis if there is a lack of contrast filling below the stone. Hydration and analgesia are the initial treatment. Kidney stones < 5 mm in diameter can pass through the urethra; stones < 3 cm in diameter can be treated with extracorporeal shock-wave lithotripsy (ESWL), percutaneous nephrolithotomy, or retrograde ureteroscopy. Preventive measures include hydration; additional prophylaxis is dependent on stone composition.

1	Preventive measures include hydration; additional prophylaxis is dependent on stone composition. Characterized by the presence of progressive cystic dilation of the renal tubules, as well as by cysts in the spleen, liver, and pancreas. The two major forms are as follows: ■Autosomal dominant (ADPKD): Most common. Usually asymptomatic until patients are > 30 years of age. One-half of ADPKD patients will have ESRD requiring dialysis by age 60. Associated with an ↑ risk of cerebral aneurysm, especially in patients with a family history. Autosomal recessive (ARPKD): Less common but more severe. Presents in infants and young children with renal failure, liver fibrosis, and portal hypertension; may lead to death in the first few years of life. Pain and hematuria are the most common presenting symptoms. Sharp, localized pain may result from cyst rupture, infection, or passage of renal calculi.

1	Pain and hematuria are the most common presenting symptoms. Sharp, localized pain may result from cyst rupture, infection, or passage of renal calculi. Additional findings include hypertension, hepatic cysts, cerebral berry aneurysms, diverticulosis, and mitral valve prolapse. Patients may have large, palpable kidneys on abdominal exam. Based on ultrasound (most common) or CT scan. Multiple bilateral cysts will be present throughout the renal parenchyma, and renal enlargement will be visualized. Genetic testing by DNA linkage analysis for ADPKD1 and ADPKD2 is available. Prevent complications and ↓ the rate of progression to ESRD. Early management of UTIs is critical to prevent renal cyst infection. BP control (ACEIs, ARBs) is necessary to ↓ hypertension-induced renal damage. Dialysis and renal transplantation are used to manage patients with ESRD.

1	Dialysis and renal transplantation are used to manage patients with ESRD. If a patient with known ADPKD develops a sudden-onset, severe headache, you must rule out subarachnoid hemorrhage from a ruptured berry aneurysm! Dilation of renal calyces. Usually occurs 2° to obstruction of the urinary tract. In pediatric patients, the obstruction is often at the ureteropelvic junction. In adults, it may be due to BPH, tumors, aortic aneurysms, or renal calculi. Can also be caused by high-output urinary ﬂow and vesicoureteral reﬂ ux. May be asymptomatic, or may present with ﬂ ank/back pain, ↓ urine output, abdominal pain, and UTIs. ■ Ultrasound or CT scan to detect dilation of the renal calyces and/or ureter. ■↑ BUN and creatinine provide evidence of 2° renal failure. Surgically correct any anatomic obstruction; use laser or sound wave lithotripsy if calculi are causing obstruction.

1	Surgically correct any anatomic obstruction; use laser or sound wave lithotripsy if calculi are causing obstruction. Ureteral stent placement across the obstructed area of the urinary tract and/or percutaneous nephrostomy tube placement to relieve pressure may be appropriate if the urinary outﬂow tract is not sufficiently cleared of obstruction. Foley or suprapubic catheters may be required for lower urinary tract obstruction (e.g., BPH). Left untreated, hydronephrosis resulting from urinary obstruction leads to hypertension, acute or chronic renal failure, or sepsis, and has a very poor prognosis. A VCUG should be obtained in all boys presenting with their f rst UTI, girls < 3 years of age with their f rst UTI or < 5 years of age with febrile UTI, and older girls with pyelonephritis or recurrent UTIs. Bringing the testes into the scrotum does not ↓ the risk of testicular cancer.

1	Bringing the testes into the scrotum does not ↓ the risk of testicular cancer. Retrograde projection of urine from the bladder to the ureters and kidneys. Often caused by insufficient tunneling of the ureters into submucosal bladder tissue, leading to ineffective restriction of retrograde urine ﬂow during bladder contraction. May also be due to posterior urethral valves, urethral or meatal stenosis, or a neurogenic bladder. Classified as follows: Mild refux (grades I–II): No ureteral or renal pelvic dilation. Often resolves spontaneously. Moderate to severe refux (grade III–V): Ureteral dilation with associated caliceal blunting in severe cases. Patients present with recurrent UTIs, typically in childhood. Prenatal ultrasound may identify hydronephrosis. Obtain a voiding cystourethrogram (VCUG) to detect abnormalities at ureteral insertion sites and to classify the grade of reﬂ ux. Nuclear renal scan (DMSA or MAG-3) can be used to evaluate for renal function.

1	Nuclear renal scan (DMSA or MAG-3) can be used to evaluate for renal function. Treat infections aggressively. Treat mild reﬂux with daily prophylactic antibiotics (amoxicillin if < 2 months of age; otherwise TMP-SMX or nitrofurantoin) until reﬂ ux resolves. Surgery (ureteral reimplantation) is generally reserved for children with persistent high-grade (III to V) reﬂux or for those with breakthrough pyelonephritis while on prophylaxis. Inadequate treatment can lead to progressive renal scarring and ESRD. Failure of one or both of the testes to fully descend into the scrotum. Low birth weight is a risk factor. Bilateral cryptorchidism is associated with prematurity, oligospermia, congenital malformation syndromes (Prader-Willi, Noonan syndromes), and infertility. Associated with an ↑ risk of testicular malignancy.

1	The testes cannot be manipulated into the scrotal sac with gentle pressure (vs. retractile testes) and may be palpated anywhere along the inguinal canal or in the abdomen. Orchiopexy by 6–12 months of age (most testes will spontaneously descend by 3 months). If discovered later, treat with orchiectomy to avoid the risk of testicular cancer. Table 2.16-9 outlines the etiologies, presentation, diagnosis, and treatment of scrotal swelling. Found in 10–25% of middle-aged and elderly men. Classified as failure to initiate (e.g., psychological, endocrinologic, neurologic), failure to fill (e.g., arteriogenic), or failure to store (e.g., veno-occlusive dysfunction). Risk factors include DM, atherosclerosis, medications (e.g., β-blockers, SSRIs, TCAs, diuretics), hypertension, heart disease, surgery or radiation for prostate cancer, and spinal cord injury. Because patients rarely volunteer this complaint, physicians should make a specific inquiry.

1	Because patients rarely volunteer this complaint, physicians should make a specific inquiry. Ask about risk factors (diabetes, peripheral vascular disease), medication use, recent life changes, and psychological stressors. The distinction between psychological and organic ED is based on the presence of nocturnal or early-morning erections (if present, it is nonorganic) and on situation dependence (i.e., occurring with only one partner). T AB LE 2.1 6-9. Differential Diagnosis of Scrotal Swelling Painless causes Hydrocele Varicocele Remnant of the processus vaginalis. Dilation of the pampiniform venous plexus (“bag of worms”). Usually asymptomatic; transilluminates. Asymptomatic or presents with vague, aching scrotal pain. Affects the left testicle more often than the right. May disappear in the supine position. Does not transilluminate. Lab and radiologic workups are rarely indicated. Obtain an ultrasound if there is concern for inguinal hernia or testicular cancer.

1	May disappear in the supine position. Does not transilluminate. Lab and radiologic workups are rarely indicated. Obtain an ultrasound if there is concern for inguinal hernia or testicular cancer. Ultrasound. Typically none unless hernia is present or hydrocele persists beyond 12–18 months of age (indicates patent processus vaginalis, which leads to an ↑ risk for inguinal hernia). If symptomatic or if testis makes up < 40% of total volume, may be treated surgically with a varicocelectomy or ligation, or through embolization via interventional radiology. T AB LE 2.1 6-9. Differential Diagnosis of Scrotal Swelling (continued ) Infection of the epididymis, usually from STIs, prostatitis, and/or ref ux. Twisting of the spermatic cord, leading to ischemia and possible testicular infarction. Typically affects those > 30 years of age; presents with epididymal tenderness, tender/ enlarged testicle(s), fever, scrotal thickening, erythema, and pyuria.

1	Typically affects those > 30 years of age; presents with epididymal tenderness, tender/ enlarged testicle(s), fever, scrotal thickening, erythema, and pyuria. Pain may ↓ with scrotal elevation ( Prehn’s sign). Typically affects those < 30 years of age; presents with intense, acute-onset scrotal pain that remains the same or ↑ with scrotal elevation ( Prehn’s sign). Pain is often accompanied by nausea/vomiting and/or dizziness. Loss of cremasteric refex is also seen. UA, culture (pyuria). Culture often shows Neisseria gonorrhoeae, E. coli, or Chlamydia. Doppler ultrasound shows normal to ↑blood fow to testes. Doppler ultrasound shows ↓blood f ow to testes. (If there is a high clinical suspicion for testicular torsion, do not wait for ultrasound and proceed immediately to surgery!) Antibiotics (tetracycline, f uoroquinolones); NSAIDs; scrotal support for pain. Attempt manual detorsion.

1	Antibiotics (tetracycline, f uoroquinolones); NSAIDs; scrotal support for pain. Attempt manual detorsion. Immediate surgery to salvage testis (the testicle is often unsalvageable after six hours of ischemia). Orchiopexy of both testes to prevent future torsion. “Point and Shoot”: The Parasympathetic nervous system mediates erection; the Sympathetic nervous system mediates ejaculation. ■ Evaluate for neurologic dysfunction (e.g., anal tone, lower extremity sensation) and for hypogonadism (e.g., small testes, loss of 2° sexual characteristics). Testosterone and gonadotropin levels may be abnormal. Check prolactin levels, as elevated prolactin can result in ↓ androgen activity. Patients with psychological ED may benefit from psychotherapy or sex therapy involving discussion and exercises with the appropriate partner.

1	Patients with psychological ED may benefit from psychotherapy or sex therapy involving discussion and exercises with the appropriate partner. Oral sildenafl (Viagra), vardenafl (Levitra), and tadalaf l (Cialis) are phosphodiesterase-5 (PDE5) inhibitors that result in prolonged action of cGMP-mediated smooth muscle relaxation and ↑ blood ﬂow in the corpora cavernosa. Testosterone is a useful therapy for patients with hypogonadism of testicular or pituitary origin; it is discouraged for patients with normal testosterone levels. Vacuum pumps, intracavernosal prostaglandin injections, and surgical implantation of semirigid or inﬂatable penile prostheses are alternatives for patients who fail PDE5 therapy. Enlargement of the prostate that is a normal part of the aging process and is seen in > 80% of men by age 80. Most commonly presents in men > 50 years of age. BPH can result in urinary retention, recurrent UTIs, bladder and renal calculi, hydronephrosis, and kidney damage over time.

1	Obstructive symptoms: Hesitancy, weak stream, intermittent stream, incomplete emptying, urinary retention, bladder fullness. Irritative symptoms: Nocturia, daytime frequency, urge incontinence, opening hematuria. On DRE, the prostate is uniformly enlarged with a rubbery texture. If the prostate is hard or has irregular lesions, cancer should be suspected. Conduct a DRE to screen for masses; if findings are suspicious, evaluate for prostate cancer. Obtain a UA and urine culture to rule out infection and hematuria. Measure creatinine levels to rule out obstructive uropathy and renal insufficiency. PSA testing and cystoscopy are not recommended for longitudinal BPH monitoring. Medical therapy includes α-blockers (e.g., terazosin), which relax smooth muscle in the prostate and bladder neck, as well as 5α-reductase inhibitors (e.g., finasteride), which inhibit the production of dihydrotestosterone.

1	Transurethral resection of the prostate (TURP) or open prostatectomy is appropriate for patients with moderate to severe symptoms. The most common cancer in men and the second leading cause of cancer death in men (after lung cancer). Risk factors include advanced age and a family history. Usually asymptomatic, but may present with obstructive urinary symptoms (e.g., urinary retention, a ↓ in the force of the urine stream) as well as with lymphedema due to obstructing metastases, constitutional symptoms, and back pain due to bone metastases. DRE may reveal a palpable nodule or an area of induration. Early carcinoma is usually not detectable on exam. A tender prostate suggests prostatitis. What drugs are an absolute contraindication to sildenaf l? Nitrates (the combined effect of ↓ BP can lead to myocardial ischemia). BPH most commonly occurs in the central (periurethral) zone of the prostate and may not be detected on DRE.

1	BPH most commonly occurs in the central (periurethral) zone of the prostate and may not be detected on DRE. The major side effect of α-blockers is orthostatic hypotension. Leading causes of cancer death in men: 1. Lung cancer 2. Prostate cancer 3. Colorectal cancer 4. Pancreatic cancer 5. Leukemia An elevated PSA may be due to BPH, prostatitis, UTI, prostatic trauma, or carcinoma. An annual DRE after age 50 is the recommended screening method for prostate cancer. Suggested by clinical findings and/or a markedly ↑PSA (> 4 ng/mL). Definitive diagnosis is made with ultrasound-guided transrectal biopsy, which typically shows adenocarcinoma. Tumors are graded by the Gleason histologic system, which sums the scores (from 1 to 5) of the two most dysplastic samples (10 is the highest grade). Look for metastases with CXR and bone scan (metastatic lesions show an osteoblastic or ↑ bone density). Fully 40% of patients present with metastatic disease at diagnosis.

1	Look for metastases with CXR and bone scan (metastatic lesions show an osteoblastic or ↑ bone density). Fully 40% of patients present with metastatic disease at diagnosis. Treatment is controversial, as many cases of prostate cancer are slow to progress. Treatment choice is based on the aggressiveness of the tumor and the patient’s mortality risk. Watchful waiting may be the best approach for elderly patients with low-grade tumors. Radical prostatectomy and radiation therapy (e.g., brachytherapy or external beam) are associated with an ↑ risk of incontinence and/or impotence. PSA, while controversial as a screening test, is used to follow patients post-treatment to evaluate for disease recurrence. Treat metastatic disease with androgen ablation (e.g., GnRH agonists, orchiectomy, ﬂutamide) and chemotherapy.

1	Treat metastatic disease with androgen ablation (e.g., GnRH agonists, orchiectomy, ﬂutamide) and chemotherapy. All males > 50 years of age should have an annual DRE. Screening should begin earlier in African-American males and in those with a first-degree relative with prostate cancer. Screening with PSA is common, but its utility remains controversial. The second most common urologic cancer and the most frequent malignant tumor of the urinary tract; usually a transitional cell carcinoma. Most prevalent in males during the sixth and seventh decades. Risk factors include smoking, diets rich in meat and fat, schistosomiasis, chronic treatment with cyclophosphamide, and occupational exposure to aniline dye (a benzene derivative). Gross hematuria is the most common presenting symptom. Other urinary symptoms, such as frequency, urgency, and dysuria, may also be seen, but most patients are asymptomatic in the early stages of disease. Cystoscopy with biopsy is diagnostic.

1	Other urinary symptoms, such as frequency, urgency, and dysuria, may also be seen, but most patients are asymptomatic in the early stages of disease. Cystoscopy with biopsy is diagnostic. UA often shows hematuria (macroor microscopic); cytology may show dysplastic cells. IVP can examine the upper urinary tract as well as defects in bladder filling. ■MRI, CT, and bone scan are important tools with which to define invasion and metastases. Treatment depends on the extent of spread beyond the bladder mucosa. Carcinoma in situ: Intravesicular chemotherapy. Superf cial cancers: Complete transurethral resection or intravesicular chemotherapy with mitomycin-C or BCG (the TB vaccine). Large, high-grade recurrent lesions: Intravesicular chemotherapy. Invasive cancers without metastases: Radical cystectomy or radiotherapy for patients who are deemed poor candidates for radical cystectomy as well as for those with unresectable local disease.

1	Invasive cancers without metastases: Radical cystectomy or radiotherapy for patients who are deemed poor candidates for radical cystectomy as well as for those with unresectable local disease. Invasive cancers with distant metastases: Chemotherapy alone. An adenocarcinoma from tubular epithelial cells (~80–90% of all malignant tumors of the kidney). Tumors can spread along the renal vein to the IVC and can metastasize to lung and bone. Risk factors include male gender, smoking, obesity, acquired cystic kidney disease in ESRD, and von Hippel– Lindau disease. Presenting signs include hematuria, f ank pain, and a palpable f ank mass. Metastatic disease can present with weight loss and malaise. Many patients have fever or other constitutional symptoms. Left-sided varicocele may seen in males (due to tumor blockage of the left gonadal vein, which empties into the left renal vein; the right gonadal vein empties directly into the IVC).

1	Anemia is common at presentation, but polycythemia due to ↑ erythropoietin production may be seen in 5–10% of patients. Ultrasound and/or CT to characterize the renal mass (usually complex cysts or solid tumor). Surgical resection may be curative in localized disease. Response rates from radiation or chemotherapy are only 15–30%. Newer tyrosine kinase inhibitors (sorafenib, sunitinib), which ↓ tumor angiogenesis and cell proliferation, have shown promising results and have recently been approved by the FDA for the treatment of renal cell carcinoma. The classic triad of renal cell carcinoma is hematuria, f ank pain, and a palpable f ank mass, but only 5–10% present with all three components of the triad.

1	The classic triad of renal cell carcinoma is hematuria, f ank pain, and a palpable f ank mass, but only 5–10% present with all three components of the triad. A heterogeneous group of neoplasms. Some 95% of testicular tumors derive from germ cells, and virtually all are malignant. Cryptorchidism is associated with an ↑ risk of neoplasia in both testes. Klinefelter’s syndrome is also a risk factor. Testicular cancer is the most common malignancy in males 15–34 years of age. β-hCG in men = choriocarcinoma. Patients most often present with painless enlargement of the testes. Most testicular cancers occur between ages 15 and 30, but seminomas have a peak incidence between 40 and 50 years of age. Testicular ultrasound. CXR and abdominal/pelvic CT to evaluate for metastasis. Tumor markers are useful for diagnosis and in monitoring treatment response. β-hCG is always elevated in choriocarcinoma and is elevated in 10% of seminomas.

1	Tumor markers are useful for diagnosis and in monitoring treatment response. β-hCG is always elevated in choriocarcinoma and is elevated in 10% of seminomas. α-fetoprotein (AFP) is often elevated in nonseminomatous germ cell tumors, particularly endodermal sinus (yolk sac) tumors. It is also elevated in hepatocellular carcinoma, hepatoblastoma, and neuroblastoma. Radical orchiectomy. Seminomas are exquisitely radiosensitive and also respond to chemotherapy. Platinum-based chemotherapy is used for nonseminomatous germ cell tumors. The steps underlying the acute management of a trauma patient can be remembered with the mnemonic ABCDE. Establishing airway patency takes precedence over all other treatment, followed by providing respiratory support and treating conditions that impair respiration, followed in turn by providing circulatory support and treating conditions that impair circulation. Airway:

1	Airway: Start with supplemental O2 by nasal cannula or face mask for conscious patients. Use a jaw-thrust maneuver to reposition the tongue in an unconscious patient. A chin-lift maneuver can be used as a last resort but should be avoided where possible, as it requires movement of the potentially unstable C-spine. An oropharyngeal or nasopharyngeal airway may facilitate bag-mask ventilation. Perform intubation in patients with apnea, significantly depressed mental status (Glasgow Coma Scale [GCS] < 8), or impending airway compromise (e.g., significant maxillofacial trauma or inhalation injury in fires). Perform a surgical airway (cricothyroidotomy) in patients who cannot be intubated or in whom there is significant maxillofacial trauma, making intubation impractical. Maintain cervical spine stabilization/immobilization in trauma patients until the spine is appropriately cleared through exam and radiographic studies. However, never allow this concern to delay airway management.

1	Breathing: Thorough cardiac and pulmonary exams will identify the five thoracic causes of immediate death: tension pneumothorax, cardiac tamponade, open pneumothorax, massive hemothorax, and airway obstruction. If tension pneumothorax (absent breath sounds on the affected side in combination with shock and hypoxemia) is identified, immediate needle decompression is needed. This is accomplished by placing a 16to 18-gauge angiocatheter into the second intercostal space at the midclavicular line, followed by placement of a chest tube. If open pneumothorax is identified, an occlusive dressing must be applied immediately. This must be secured on three sides only to prevent the development of tension pneumothorax.

1	If open pneumothorax is identified, an occlusive dressing must be applied immediately. This must be secured on three sides only to prevent the development of tension pneumothorax. Massive hemothorax is diagnosed through chest tube placement and is defined as > 1000 cc of immediate blood return or > 200/hour for > 2–4 hours. The treatment for massive hemothorax is volume resuscitation followed by surgery to repair the site of bleeding. The treatment for ﬂail chest is supportive, followed by surgical fixation of the chest wall. Circulation: Apply direct pressure to any actively bleeding wounds. Place a 16-gauge IV in each antecubital fossa. Isotonic ﬂuids (LR or NS) are repleted in a 3:1 ratio (ﬂuid to blood loss). Start with a ﬂuid bolus of 1–2 L in adults; then recheck vitals and continue repletion as indicated. If the patient remains tachycardic or hypotensive after the first 2 L of isotonic ﬂuid, transfusion with packed RBCs may be indicated.

1	If the patient remains tachycardic or hypotensive after the first 2 L of isotonic ﬂuid, transfusion with packed RBCs may be indicated. To remember the Glasgow Coma Scale, think 4-eyes, Jackson-5, V6 engine. Four points can be assigned for eye response, five points for verbal response, and six points for motor response. Patients with chest trauma and shock may have cardiac tamponade. The relevant signs are JVD, hypotension, and mufﬂed heart sounds. This can be diagnosed with bedside ultrasound. If tamponade is diagnosed, an immediate pericardiocentesis is necessary. Disability/Exposure: Disability (CNS dysfunction) is assessed and quantified with the GCS. Exposure requires that the patient be completely disrobed and assessed for injury and temperature status on both the front and back of the body. Hypothermia is a common problem in trauma and can worsen bleeding; once the exam is done, the patient should be covered with warm blankets.

1	Once the patient is stable, conduct a full examination. For unstable patients with suspected hemoperitoneum or tamponade, do a focused abdominal sonography for trauma (FAST) scan. Four spaces are checked for blood: between the right kidney and liver (Morrison’s pouch), between the left kidney and spleen (the splenorenal recess), posterior to the bladder (the pouch of Douglas), and in the pericardium. Hemoperitoneum requires immediate surgical consultation for operative management; hemopericardium requires immediate pericardiocentesis. Radiology studies should be ordered on the basis of the patient assessment. A CXR is needed for all patients with thoracic trauma, and a head CT should be ordered for all patients with loss of consciousness or depressed mental status. A C-spine CT is needed for all patients with neck pain or tenderness, neurologic findings, or depressed mental status.

1	After urethral injury has been ruled out, place a Foley catheter if it is necessary to monitor urine output (e.g., in hemodynamically unstable patients, patients receiving ﬂuid resuscitation, or patients undergoing major surgery). Pertinent labs should be ordered based on the mechanism of injury, suspicion of intoxication or OD, and past medical history. A rough estimate of SBP can be made on the basis of palpated pulses. Carotids correspond roughly to an SBP of 60 mmHg, femorals to an SBP of 70 mmHg, and radials to an SBP of 80 mmHg. The evaluation and treatment of penetrating trauma depend on the location and extent of the injury. Intubate early. Immediate surgical exploration is mandatory for patients with shock and active ongoing hemorrhage from neck wounds. All wounds that violate the platysma are considered true penetrating neck trauma. The neck is divided into three zones, and treatment varies according to which zone is injured (see Figure 2.17-1).

1	All wounds that violate the platysma are considered true penetrating neck trauma. The neck is divided into three zones, and treatment varies according to which zone is injured (see Figure 2.17-1). Diagnostic workup is individualized based on the location of the wound, suspected injuries, and the preference of the trauma surgeon. Appropriate tests may include angiography of the aorta and carotid/cerebral arteries, CT scan of the neck with or without CT angiography, Doppler ultrasound, contrast esophagography, esophagoscopy, or bronchoscopy. Immediately evaluate trauma patients for tension pneumothorax, cardiac tamponade, open pneumothorax, massive hemothorax, ﬂ ail chest, and airway obstruction. FIGURE 2.17-1. Zones of the neck. (Reproduced, with permission, from Way LW. Current Surgical Diagnosis & Treatment, 10th ed. Stamford, CT: Appleton & Lange, 1994: 223.)

1	FIGURE 2.17-1. Zones of the neck. (Reproduced, with permission, from Way LW. Current Surgical Diagnosis & Treatment, 10th ed. Stamford, CT: Appleton & Lange, 1994: 223.) Unstable patients with penetrating thoracic injuries require immediate intubation followed by assessment and treatment of the life-threatening injuries described above. Empiric placement of bilateral chest tubes may be needed if the precise nature of injury is unclear. Open thoracotomy may be indicated for patients with penetrating chest trauma leading to cardiac arrest, provided that the patient arrested in the ED or shortly before arrival. Leave any impaled objects in place until the patient is taken to the OR, as such objects may tamponade further blood loss. Beware of tension pneumothorax, open pneumothorax, massive hemothorax, ﬂail chest, cardiac tamponade, aortic disruption, diaphragmatic tear, and esophageal injury. If a previously stable chest trauma patient suddenly dies, suspect air embolism.

1	If a previously stable chest trauma patient suddenly dies, suspect air embolism. A new diastolic murmur after chest trauma suggests aortic dissection. The absence of pain does not rule out an abdominal injury. Gunshot wounds usually require immediate exploratory laparotomy, although stable patients can be managed conservatively in select cases. Stab wounds in a hemodynamically unstable patient or in a patient with peritoneal signs or evisceration require immediate exploratory laparotomy. Stab wounds in a hemodynamically stable patient warrant a CT or FAST scan followed by close inpatient observation. ■Complete neurovascular assessment is critical; check pulses, motor function, and sensory function. Arteriography and surgical management are required for patients with suspected vascular injuries. Nerve injuries generally require surgical repair.

1	Arteriography and surgical management are required for patients with suspected vascular injuries. Nerve injuries generally require surgical repair. Early wound irrigation and tissue debridement, not antibiotic therapy, are the most important steps in the treatment of contaminated wounds. However, do administer antibiotics and tetanus prophylaxis. A rapid deceleration head injury causes coup-contrecoup injuries, in which a bleed is noted both at the site of impact and across from the point of impact. Epidural hematomas: Lenticular in shape on head CT. Bleed is from the medial meningeal artery (the higher arterial pressure is able to push the dura away from the skull, causing the lens shape on imaging). These bleeds cannot cross suture lines but can expand rapidly and cause herniation and death. Patients classically have loss of consciousness immediately after the injury and then have a “lucid interval” after which they become comatose.

1	Subdural hematomas: Follow the curve of the skull and result from shearing of the dural bridging veins. These bleeds can cross suture lines. In a pediatric patient, a subdural hematoma coupled with retinal hemorrhages constitutes child abuse until proven otherwise. These bleeds accumulate more slowly than epidural hematomas but can still cause death. They may present acutely (immediate), subacutely (days), or chronically (weeks). The classic cause is a rapid deceleration injury (e.g., high-speed motor vehicle accidents, ejection from vehicles, and falls from heights). Since complete aortic rupture is rapidly fatal (85% die at the scene), patients with aortic disruption who are seen in the ED usually have a contained hematoma within the adventitia. Laceration is most common just proximal to the ligamentum arteriosum.

1	Immediate CXR reveals a widened mediastinum (> 8 cm), loss of aortic knob, pleural cap, deviation of the trachea and esophagus to the right, and depression of the left main stem bronchus. CT evaluation and/or transesophageal echocardiography (TEE) prior to surgery. Aortography is the gold standard for evaluation. Basic trauma management (ABCs); emergent surgery for defect repair. ■Three or more adjacent ribs fractured at two points, causing paradoxical inward movement of the ﬂail segment with inspiration. Other signs of suspected child abuse are spiral fractures in the limbs, bucket-handle fractures, bruises, and rib fractures. Marfan’s syndrome, syphilis, and Ehlers-Danlos syndrome all predispose to a weak aortic wall. Aortic disruption is often associated with first and second rib, scapular, and sternal fractures. Kehr’s sign: Referred shoulder pain due to diaphragmatic irritation (classically on the left due to spleen rupture).

1	Kehr’s sign: Referred shoulder pain due to diaphragmatic irritation (classically on the left due to spleen rupture). Hx/PE: Presents with crepitus and abnormal chest wall movement. Abnormal chest wall movement may not be appreciated if the patient is splinting because of pain. Dx: Primarily clinical, although CXR, O2 saturation, and blood gases may help. Tx: O2, narcotic analgesia. Respiratory support, including intubation and mechanical ventilation, may be needed to treat hypoxemia in severe cases. Surgical fixation of the chest wall is generally needed. Cx: Respiratory compromise is a complication due to underlying pulmonary contusion. The spleen and liver are the most commonly injured organs following blunt abdominal trauma. Symptoms are consistent with signs of blood loss and include hypotension, tachycardia, and peritonitis. Suspect spleen or liver injury when lower rib fractures are present.

1	Pancreatic rupture should be suspected after a direct epigastric blow (handlebar injury). Diaphragmatic rupture may occur with blunt or penetrating trauma. It is difficult to diagnose and often missed. Kehr’s sign may be present; radio-graphs may demonstrate abdominal viscera in the thorax. The kidneys are the most commonly injured GU organ in trauma, with injuries including renal contusion, laceration, fracture, and pedicle injury. In hemodynamically stable patients, abdominal blunt trauma can be diagnosed with FAST scan (see the 2° Survey section), CT scan, and serial abdominal exams. In hemodynamically unstable patients, abdominal blunt trauma should be treated with immediate exploratory laparotomy to look for organ injury or intra-abdominal bleeding. Most commonly occur after high-speed traumas such as motor vehicle accidents or falls from heights. Require immediate attention by the orthopedist owing to their life-threatening potential.

1	Most commonly occur after high-speed traumas such as motor vehicle accidents or falls from heights. Require immediate attention by the orthopedist owing to their life-threatening potential. May present with an unstable pelvis upon compression. Pelvic x-rays may confirm the fracture; in a stable patient, a CT scan of the pelvis will better define the extent of injury. If hypotension and shock are present, an exsanguinating hemorrhage is likely. In the field, MAST (military antishock trousers; rarely used today) can be used to maintain adequate BP and organ perfusion. Consider embolization of bleeding vessels, emergent external pelvic fixation, or, in a hemodynamically stable patient, internal fixation. Give blood early. Hemorrhage results in death in 50% of patients. Pelvic injuries can be associated with urethral injury. It is suggested by blood at the urethral meatus; a high-riding, “ballotable” prostate; or lack of a prostate.

1	Pelvic injuries can be associated with urethral injury. It is suggested by blood at the urethral meatus; a high-riding, “ballotable” prostate; or lack of a prostate. If present, a retrograde urethrogram must be performed to rule out injury before a Foley catheter is placed. Never explore a pelvic or retroperitoneal hematoma. Follow with serial hemoglobin and hematocrit. Table 2.17-1 summarizes the basic management of cardiac arrhythmias in an acute setting. T AB LE 2.1 7 -1. Management of Cardiac Arrhythmiasa,b Updated in 2005, CPR is 30 compressions to every two breaths for all arrest victims after puberty. Unsynchronized shock with 360 J → 360-J shock → epinephrine → 360-J shock →amiodarone or lidocaine → 360-J shock → epinephrine. Vasopressin may be given in place of the frst or second dose of epinephrine. Amiodarone, lidocaine, procainamide, or sotalol may be used for stable ventricular tachycardia.

1	Vasopressin may be given in place of the frst or second dose of epinephrine. Amiodarone, lidocaine, procainamide, or sotalol may be used for stable ventricular tachycardia. Epinephrine or vasopressin; simultaneously search for the underlying cause (see the 5 H’s and 5 T’s mnemonic) and provide empiric treatment. Give atropine for bradycardic PEA only. If unstable, perform synchronized electrical cardioversion. If stable, control rate with vagal maneuvers (Valsalva maneuver, carotid sinus massage, or cold stimulus). If resistant to maneuvers, give up to three doses of adenosine followed by other AV-nodal blocking agents (calcium channel blockers [CCBs] or β-blockers). If unstable, perform synchronized electrical cardioversion starting at 100 J. If stable, control rate with diltiazem or β-blockers and anticoagulate if duration is > 48 hours.

1	If unstable, perform synchronized electrical cardioversion starting at 100 J. If stable, control rate with diltiazem or β-blockers and anticoagulate if duration is > 48 hours. Elective cardioversion may be performed if duration is < 48 hours; otherwise, the clinician must anticoagulate or perform TEE prior to conversion. Do not give nodal blockers if there is evidence of Wolff-Parkinson-White syndrome (δ waves) on ECG. If symptomatic, give atropine and consider dopamine, epinephrine, and glucagon. If Mobitz II or third-degree heart block is present, place transcutaneous pacemaker pads, and have atropine at the bedside. A temporary transvenous pacemaker may be required for hemodynamically unstable patients. a In all cases, disruptions of CPR should be minimized. After a shock or administration of a drug, CPR should be resumed immedi ately, and fve cycles of CPR should be given before checking for a pulse or rhythm.

1	b Doses of electricity listed above assume a monophasic defbrillator. Maximum energy output on more modern biphasic def brilla tors is usually 150–200 J. Asystole Epinephrine and atropine. Ventricular fbrillation or pulseless ventricular tachycardia Pulseless electrical activity (PEA) Supraventricular tachycardia (SVT) Atrial f brillation/f utter Bradycardia Possible causes of PEA— The 5 H’s and 5 T’s Hypovolemia Hypoxia Hydrogen ion: Acidosis Hyper/Hypo: K+, other metabolic Hypothermia Tablets: Drug OD, ingestion Tamponade: Cardiac Tension pneumothorax Thrombosis: Coronary Thrombosis: Pulmonary Acute-onset abdominal pain has many potential etiologies and may require immediate medical or surgical intervention. Sharp, focal pain generally implies a parietal (peritoneal) etiology; dull, diffuse pain is commonly of visceral (organ) origin. Figure 2.17-2 identifies the common causes of acute abdomen.

1	Obtain a complete history, including the elements indicated in the mnemonic OPQRST. Obtain a full gynecologic history for females (including last menstrual period, pregnancy, and any STD symptoms). If a female has abdominal pain with cervical motion tenderness, there should be a low threshold to treat for pelvic inﬂammatory disease (PID). Perforation leads to sudden onset of diffuse, severe pain, usually with abdominal rigidity on exam. Obstruction leads to acute onset of severe, radiating, colicky pain. Patients may complain of obstipation or bilious emesis. Inﬂ ammation leads to gradual onset (over 10–12 hours) of constant, illdefined pain. Associated symptoms include the following: ■Anorexia, nausea, vomiting, changes in bowel habits, hematochezia, and melena suggest GI etiologies. Rapid onset of severe,Abrupt, excruciating pain ulcer Mesenteric thrombosis, Intermittent, colicky pain,Gradual, steady pain crescendo with free intervals Acute cholecystitis, acute cholangitis,

1	Rapid onset of severe,Abrupt, excruciating pain ulcer Mesenteric thrombosis, Intermittent, colicky pain,Gradual, steady pain crescendo with free intervals Acute cholecystitis, acute cholangitis, Small bowelAppendicitis, FIGURE 2.17-2. Acute abdomen. The location and character of pain are helpful in the differential diagnosis of the acute abdomen. (Reproduced, with permission, from Way LW. Current Surgical Diagnosis & Treatment, 10th ed. Stamford, CT: Appleton & Lange, 1994: 444.) Fever and cough suggest pneumonia. Hematuria, pyuria, and costovertebral angle tenderness point to a GU etiology. If associated with meals, consider mesenteric ischemia, PUD, biliary disease, pancreatitis, or bowel pathology. A family history of abdominal pain may indicate familial Mediterranean fever or acute intermittent porphyria.

1	A family history of abdominal pain may indicate familial Mediterranean fever or acute intermittent porphyria. If peritoneal signs, shock, or impending shock is present, emergent exploratory laparotomy is necessary. A positive β-hCG in the setting of shock is a ruptured ectopic pregnancy until proven otherwise. Abdominal pain plus syncope or shock in an older patient is a ruptured abdominal aortic aneurysm (AAA) until proven otherwise. If the patient is stable, a complete physical exam—including a rectal exam and, in women, a pelvic exam—is mandatory. Obtain electrolytes, LFTs, amylase, lipase, urine or serum β-hCG, UA, and a CBC with differential.

1	Obtain electrolytes, LFTs, amylase, lipase, urine or serum β-hCG, UA, and a CBC with differential. Consider a CXR for suspected perforation or pulmonary pathology. AXR may be useful for obstruction, but CT is more sensitive and specific. CT is used to diagnose appendicitis, diverticulitis, abscess, renal stones, AAA, obstruction, and other pathology. Ultrasound is the best tool for diagnosing gallstones and gynecologic pathology. Bedside ultrasound (FAST and aortic views) can diagnose hemoperitoneum and AAA in unstable patients. Hemodynamically unstable patients must have emergent surgical management. In stable patients, expectant management may include NPO status, NG tube placement, IV ﬂuids, placement of a Foley catheter (to monitor urine output and ﬂuid status), and vital sign monitoring with serial abdominal exams and serial labs.

1	Give broad-spectrum antibiotics to all patients with perforation or signs of sepsis. Antibiotics may also be indicated for patients with infectious processes such as cholecystitis, diverticulitis, and pyelonephritis. Type and cross all unstable patients. The inciting event is obstruction of the appendiceal lumen with subsequent inﬂammation and infection. Rising intraluminal pressure leads to vascular compromise of the appendix, ischemia, necrosis, and possible perforation. Etiologies include hypertrophied lymphoid tissue (55–65%), fecalith (35%), foreign body, tumor (e.g., carcinoid tumor), and parasites. Incidence peaks in the early teens (most patients are between 10 and 30 years of age), and the male-to-female ratio is 2:1. Presents with dull periumbilical pain lasting 1–12 hours that leads to sharp RLQ pain at McBurney’s point. Also presents with nausea, vomiting, anorexia (“hamburger sign”), and low-grade fever.

1	Presents with dull periumbilical pain lasting 1–12 hours that leads to sharp RLQ pain at McBurney’s point. Also presents with nausea, vomiting, anorexia (“hamburger sign”), and low-grade fever. Psoas, obturator, and Rovsing’s signs are insensitive tests that may be . If the patient remembers the exact moment of pain onset, think perforation. Pneumonia can present as right or left upper quadrant abdominal pain. All female patients with an acute abdomen require a pelvic exam and a pregnancy test to rule out PID, ectopic pregnancy, and ovarian torsion. McBurney’s point is located one-third of the distance from the anterior superior iliac spine to the umbilicus. “Hamburger sign”: If a patient wants to eat, consider a diagnosis other than appendicitis. Anorexia is 80% specific for appendicitis. Psoas sign: Passive extension of the hip leading to RLQ pain. Obturator sign: Passive internal rotation of the ﬂ exed hip leading to RLQ pain.

1	Psoas sign: Passive extension of the hip leading to RLQ pain. Obturator sign: Passive internal rotation of the ﬂ exed hip leading to RLQ pain. Rovsing’s sign: Deep palpation of the LLQ leading to RLQ pain. In perforated appendix, partial pain relief is possible, but peritoneal signs (e.g., rebound, guarding, hypotension, ↑ WBC count, fever) will ultimately develop. Children, the elderly, pregnant women, and those with retrocecal appendices may have atypical presentations that may result in misdiagnosis and ↑ mortality. Diagnosed by clinical impression. Look for fever, mild leukocytosis (11,000–15,000 cells/μL) with left shift, and UA with a few RBCs and/or WBCs. If the clinical diagnosis is unequivocal, no imaging studies are necessary. Otherwise, studies include the following: KUB: Fecalith or loss of psoas shadow. Ultrasound: Enlarged, noncompressible appendix. CT scan with contrast (95–98% sensitive): Periappendiceal stranding or ﬂuid; enlarged appendix.

1	KUB: Fecalith or loss of psoas shadow. Ultrasound: Enlarged, noncompressible appendix. CT scan with contrast (95–98% sensitive): Periappendiceal stranding or ﬂuid; enlarged appendix. The patient should be NPO and should receive IV hydration and antibiotics with anaerobic and gram-coverage. Immediate open or laparoscopic appendectomy is the definitive treatment. If appendicitis is not found, complete exploration of the abdomen is performed. Perforation: Administer antibiotics until the patient is afebrile with a normalized WBC count; the wound should be closed by delayed 1° closure. Abscess: Treat with broad-spectrum antibiotics and percutaneous drainage; an elective appendectomy should be performed 6–8 weeks later. The second leading cause of death in children. Serious burn patients should be treated in an ICU setting. Burns can be chemical, electrical, or thermal and are categorized by depth of tissue destruction:

1	First degree: Only the epidermis is involved. The area is painful and erythematous, but blisters are not present, and capillary refill is intact. Looks like a sunburn. Second degree: The epidermis and partial thickness of the dermis are involved. The area is painful, and blisters are present. Third degree: The epidermis, the full thickness of the dermis, and potentially deeper tissues are involved. The area is painless, white, and charred. Patients may present with obvious skin wounds, but significant deep destruction may not be visible, especially with electrical burns. Conduct a thorough airway and lung exam to assess for inhalation injury. Always assume carbon monoxide poisoning in patients with inhalation injury. Consider cyanide poisoning in closed-space fires with burning carpets and textiles. Assess the ABCs. If airway compromise is impending, intubate. Be vigilant for shock, inhalation injury, and carbon monoxide poisoning. Obtain a CXR and a carboxyhemoglobin level.

1	Assess the ABCs. If airway compromise is impending, intubate. Be vigilant for shock, inhalation injury, and carbon monoxide poisoning. Obtain a CXR and a carboxyhemoglobin level. Evaluate the percentage of body surface area (% BSA) involved. Supportive measures; tetanus, stress ulcer prophylaxis, and IV narcotic analgesia. For secondand third-degree burns, ﬂuid repletion using the Parkland formula is critical; adjust repletion on the basis of additional insensible losses to maintain at least 1 cc/kg/hr of urine output. Topical silver sulfadiazine and mafenide may be used prophylactically; however, there is no proven benefit associated with the use of PO/IV antibiotics or corticosteroids. Shock and superinfection, with the latter most likely due to Pseudomonas or gramcocci. Criteria for transfer to a burn center include the following: Full-thickness burn > 5% of BSA. Partial-thickness burn > 10% BSA.

1	Criteria for transfer to a burn center include the following: Full-thickness burn > 5% of BSA. Partial-thickness burn > 10% BSA. Any fullor partial-thickness burn over critical areas (face, hands, feet, genitals, perineum, major joints). Circumferential burns; chemical, electrical, or lightning injury; inhalation injury. Any special psychosocial or rehabilitative care needs. Use the “rule of 9’s” to estimate % BSA in adults: Parkland formula: Fluids for the first 24 hours = 4 ×patient’s weight in kg × % BSA. Give 50% of ﬂ uids over the first eight hours and the remaining 50% over the following 16 hours. The 5 W’s of postoperative fever: Wind: Atelectasis,

1	The 5 W’s of postoperative fever: Wind: Atelectasis, Occurs in 40% of all postoperative patients. Remember the mnemonic “Wind, Water, Wounds, Walking, and Wonder drugs.” ■↓ the risk of postoperative fever with incentive spirometry, preand postoperative antibiotics when indicated, short-term Foley catheter use, early ambulation, and DVT prophylaxis (e.g., anticoagulation, compression stockings). Fevers before postoperative day 3 are unlikely to be infectious unless Clostridium or β-hemolytic streptococci are involved. pneumonia Water: UTI Wounds: Wound infection, abscess Walking: DVT Wonder drugs: Drug ■A body temperature < 95° F (35° C) accompanied by mental status and neurologic deficits. TABLE 2.17-2. Types of Shock

1	Hypovolemic Trauma, blood loss, dehydration with inadequate f uid repletion, third spacing, burns. ↓↓↑Replete with isotonic solution (e.g., LR or NS) and blood in a 3:1 (f uid-to-blood) ratio. Cardiogenic CHF, arrhythmia, structural heart disease (severe mitral regurgitation, VSD), Ml (> 40% of left ventricular function). ↓↑↑Identify the cause and treat if possible. Give inotropic support with pressors such as dopamine (if hypotensive) or dobutamine (if not hypotensive). Obstructive Cardiac tamponade, tension pneumothorax, massive pulmonary embolism. ↓↑↑Treat the underlying cause: pericardiocentesis, decompression of pneumothorax, thrombolysis. Bacteremia, especially gram-organisms. ↑↓↓Administer broad-spectrum antibiotics. Measure central venous pressure (CVP) and give fuid until CVP = 8. Then give pressors (dopamine or norepinephrine). Obtain cultures prior to administration of antibiotics if possible. Bee stings, medication, food allergies. ↑↓↓Give diphenhydramine. If severe,

1	= 8. Then give pressors (dopamine or norepinephrine). Obtain cultures prior to administration of antibiotics if possible. Bee stings, medication, food allergies. ↑↓↓Give diphenhydramine. If severe, administer 1:1000 epinephrine. a PCWP = pulmonary capillary wedge pressure. b PVR = peripheral vascular resistance.

1	Remove the patient from the cold or windy environment and remove wet clothing. In mild cases, rewarm the patient with blankets or warm water. In more severe cases, the patient may need a heating blanket, warmed oxygen, or warm IV ﬂuids. In unstable patients, invasive rewarming may be necessary (e.g., NG or bladder lavage, pleural lavage, or cardiac bypass). If the patient has frostbite, thaw the affected areas with the same methods. Patients will need narcotic analgesia for thawing. Monitor the ECG for arrhythmias such as bradycardia and slow atrial fibrillation. The stereotypic sign is the J wave (aka Osborn wave). Also monitor electrolytes and acid-base balance. Do not stop resuscitation efforts until the patient has been warmed. A body temperature > 104° F (40° C), possibly from heat stroke. Cool the patient with cold water, wet blankets, and ice. Consider a benzodiazepine to prevent convulsions. Rule out causes of fever such as infection or drug reaction.

1	Cool the patient with cold water, wet blankets, and ice. Consider a benzodiazepine to prevent convulsions. Rule out causes of fever such as infection or drug reaction. A hypoxemic poisoning syndrome seen in patients who have been exposed to automobile exhaust, smoke inhalation, barbecues, or old appliances in poorly ventilated locations. Presents with hypoxemia, cherry-red skin (rare), confusion, and headaches. Coma or seizures occur in severe cases. Chronic low-level exposure may cause ﬂ ulike symptoms with generalized myalgias, nausea, and headaches. Ask about symptoms in others living in the same house. Suspect smoke inhalation in the presence of singed nose hairs, facial burns, hoarseness, wheezing, or carbonaceous sputum. Check an ABG and serum carboxyhemoglobin level (normal is < 5% in nonsmokers and < 10% in smokers). Check an ECG in the elderly and in patients with a history of cardiac disease.

1	Check an ABG and serum carboxyhemoglobin level (normal is < 5% in nonsmokers and < 10% in smokers). Check an ECG in the elderly and in patients with a history of cardiac disease. Treat with 100% O2 until asymptomatic and carboxyhemoglobin falls to normal levels. Use hyperbaric O2 for pregnant patients, nonresponders, those with signs of CNS or cardiac ischemia, or those with severely ↑ carboxyhemoglobin to facilitate displacement of carbon monoxide from hemoglobin. Patients with airway burns or smoke inhalation may require early intubation, since upper airway edema can rapidly lead to complete obstruction. Table 2.17-3 outlines drug interactions and reactions that are commonly encountered in a clinical setting. Table 2.17-4 summarizes antidotes and treatments for substances commonly encountered in overdoses and intoxications. If a patient is unresponsive, it is common to empirically treat with a dose of Narcan.

1	Malignant hyperthermia and neuroleptic malignant syndrome (NMS) should be ruled out in any suspected case of hyperthermia. Malignant hyperthermia would be seen after halothane exposure, and NMS after a neuroleptic. Both conditions are treated with dantrolene. In carbon monoxide poisoning, the patient’s oxygen saturation is usually normal. This is because carboxyhemoglobin is read by the pulse oximeter as a normal saturated hemoglobin molecule. T AB LE 2.1 7 -3. Drug Interactions and Reactions

1	Induction of P-450 enzymes Barbiturates, phenytoin, carbamazepine, rifampin, quinidine, griseofulvin. Inhibition of P-450 enzymes Cimetidine, ketoconazole, INH, grapefruit, erythromycin, sulfonamides. Metabolism by P-450 enzymes Benzodiazepines, amide anesthetics, metoprolol, propranolol, nifedipine, phenytoin, quinidine, theophylline, warfarin, barbiturates. ↑ risk of digoxin toxicity Quinidine, cimetidine, amiodarone, CCBs. Competition for albumin-binding sites Warfarin, ASA, phenytoin. Blood dyscrasias Ibuprofen, quinidine, methyldopa, chemotherapeutic agents. Hemolysis in G6PD-defcient patients Sulfonamides, isoniazid (INH), ASA, ibuprofen, nitrofurantoin, primaquine, pyrimethamine, chloramphenicol. Gynecomastia Spironolactone, Estrogens, Digitalis, Cimetidine, chronic Alcohol use, Ketoconazole: “Some Excellent Drugs Create Awesome Knockers.” Stevens-Johnson syndrome Ethosuximide, sulfonamides. Photosensitivity Tetracycline, amiodarone, sulfonamides. Drug-induced SLE Procainamide,

1	“Some Excellent Drugs Create Awesome Knockers.” Stevens-Johnson syndrome Ethosuximide, sulfonamides. Photosensitivity Tetracycline, amiodarone, sulfonamides. Drug-induced SLE Procainamide, hydralazine, INH, penicillamine, chlorpromazine, methyldopa, quinidine.

1	TABLE 2.17-4. Specif c Antidotes Acetaminophen N-acetylcysteine. Acid/alkali ingestion Upper endoscopy to evaluate for stricture. Anticholinesterases, organophosphates Atropine, pralidoxime. Antimuscarinic/anticholinergic agents Physostigmine. Arsenic, mercury, gold Succimer, dimercaprol. β-blockers Glucagon. Barbiturates (phenobarbital) Urine alkalinization, dialysis, activated charcoal, supportive care. Benzodiazepines Flumazenil (can precipitate withdrawal). TABLE 2.17-4. Specifc Antidotes (continued)

1	TABLE 2.17-4. Specifc Antidotes (continued) Black widow bite Calcium gluconate, methocarbamol. Carbon monoxide 100% O2, hyperbaric O2. Copper, arsenic, lead, gold Penicillamine. Cyanide Amyl nitrate, sodium nitrate, sodium thiosulfate. Digitalis Normalize K+ but avoid giving Ca++, Mg++, or lidocaine (for torsades), antidigitalis Fab. Heparin Protamine sulfate. INH Pyridoxine. Iron salts Deferoxamine. Lead Succimer, CaEDTA, dimercaprol. Methanol, ethylene glycol (antifreeze) EtOH, fomepizole, dialysis, calcium gluconate for ethylene glycol. Methemoglobin Methylene blue. Opioids Naloxone. Salicylates Urine alkalinization, dialysis, activated charcoal. TCAs Sodium bicarbonate for QRS prolongation; diazepam or lorazepam for seizures; cardiac monitor for arrhythmias. Theophylline Activated charcoal. Consider repeat doses. tPA, streptokinase Aminocaproic acid. Warfarin Vitamin K, FFP. Table 2.17-5 outlines the major side effects of select drugs. Management of Drug Withdrawal

1	Table 2.17-5 outlines the major side effects of select drugs. Management of Drug Withdrawal Table 2.17-6 summarizes common drug withdrawal symptoms and treatment. Table 2.17-7 summarizes the signs and symptoms of key vitamin deficiencies. TABLE 2.17-5. Drug Side Effects

1	ACEIs Cough, rash, proteinuria, angioedema, taste changes, teratogenic effects. Amantadine Ataxia, livedo reticularis. Aminoglycosides Ototoxicity, nephrotoxicity (acute tubular necrosis). Amiodarone Acute: AV block, hypotension, bradycardia. Chronic: pulmonary f brosis, peripheral deposition leading to bluish discoloration, arrhythmias, hypo-/hyperthyroidism, corneal deposition. Amphotericin Fever/rigors, nephrotoxicity, bone marrow suppression, anemia. Antipsychotics Sedation, acute dystonic reaction, akathisia, parkinsonism, tardive dyskinesia, NMS. Azoles (e.g., fuconazole) Inhibition of P-450 enzymes. AZT Thrombocytopenia, megaloblastic anemia. β-blockers Asthma exacerbation, masking of hypoglycemia, impotence, bradycardia, AV block, CHF. Benzodiazepines Sedation, dependence, respiratory depression. Bile acid resins Gl upset, malabsorption of vitamins and medications. CCBs Peripheral edema, constipation, cardiac depression. Carbamazepine Induction of P-450 enzymes,

1	respiratory depression. Bile acid resins Gl upset, malabsorption of vitamins and medications. CCBs Peripheral edema, constipation, cardiac depression. Carbamazepine Induction of P-450 enzymes, agranulocytosis, aplastic anemia, liver toxicity. Chloramphenicol Gray baby syndrome, aplastic anemia. Cisplatin Nephrotoxicity, acoustic nerve damage. Clonidine Dry mouth; severe rebound headache and hypertension. Clozapine Agranulocytosis. Corticosteroids Mania, hyperglycemia (acute), immunosuppression, bone mineral loss, thinning of skin, easy bruising, myopathy, cataracts (chronic). Cyclophosphamide Myelosuppression, hemorrhagic cystitis. Digoxin Gl disturbance, yellow visual changes, arrhythmias (e.g., junctional tachycardia or SVT). Doxorubicin Cardiotoxicity (cardiomyopathy). Ethyl alcohol Renal dysfunction, CNS depression. Fluoroquinolones Cartilage damage in children; Achilles tendon rupture in adults. Furosemide Ototoxicity, hypokalemia, nephritis, gout. Gemf brozil Myositis,

1	Renal dysfunction, CNS depression. Fluoroquinolones Cartilage damage in children; Achilles tendon rupture in adults. Furosemide Ototoxicity, hypokalemia, nephritis, gout. Gemf brozil Myositis, reversible ↑ in LFTs.

1	TABLE 2.17-5. Drug Side Effects (continued)

1	Halothane Hepatotoxicity, malignant hyperthermia. HCTZ Hypokalemia, hyponatremia, hyperuricemia, hyperglycemia, hypercalcemia. HMG-CoA reductase inhibitors Myositis, reversible ↑ in LFTs. Hydralazine Drug-induced SLE. Hydroxychloroquine Retinopathy. INH Peripheral neuropathy (prevent with pyridoxine/vitamin B6), hepatotoxicity, inhibition of P-450 enzymes, seizures with overdose, hemolysis in G6PD def ciency. MAOIs Hypertensive tyramine reaction, serotonin syndrome (with meperidine). Methanol Blindness. Methotrexate Hepatic fbrosis, pneumonitis, anemia. Methyldopa Coombs’ test, drug-induced SLE. Metronidazole Disulfram reaction, vestibular dysfunction, metallic taste. Niacin Cutaneous f ushing. Nitroglycerin Hypotension, tachycardia, headache, tolerance. Penicillamine Drug-induced SLE. Penicillin/β-lactams Hypersensitivity reactions. Phenytoin Nystagmus, diplopia, ataxia, arrhythmia (in toxic doses), gingival hyperplasia, hirsutism, teratogenic effects. Prazosin First-dose

1	SLE. Penicillin/β-lactams Hypersensitivity reactions. Phenytoin Nystagmus, diplopia, ataxia, arrhythmia (in toxic doses), gingival hyperplasia, hirsutism, teratogenic effects. Prazosin First-dose hypotension. Procainamide Drug-induced SLE. Propylthiouracil Agranulocytosis, aplastic anemia. Quinidine Cinchonism (headache, tinnitus), thrombocytopenia, arrhythmias (e.g., torsades de pointes). Reserpine Depression. Rifampin Induction of P-450 enzymes; orange-red body secretions. Salicylates Fever; hyperventilation with respiratory alkalosis and metabolic acidosis; dehydration, diaphoresis, hemorrhagic gastritis. SSRIs Anxiety, sexual dysfunction, serotonin syndrome if taken with MAOIs.

1	TABLE 2.17-5. Drug Side Effects (continued) Succinylcholine Malignant hyperthermia, hyperkalemia. TCAs Sedation, coma, anticholinergic effects, seizures and arrhythmias. Tetracyclines Tooth discoloration, photosensitivity, Fanconi’s syndrome, Gl distress. Trimethoprim Megaloblastic anemia, leukopenia, granulocytopenia. Valproic acid Teratogenicity leads to neural tube defects; rare fatal hepatotoxicity. Vancomycin Nephrotoxicity, ototoxicity, “red man syndrome” (histamine release; not an allergy). Vinblastine Severe myelosuppression. Vincristine Peripheral neuropathy, paralytic ileus. T AB LE 2.1 7 -6. Symptoms and Treatment of Drug Withdrawal

1	Alcohol Tremor (6–12 hours). Tachycardia, hypertension, agitation, seizures (within 48 hours). Hallucinations, DTs—severe autonomic instability leading to tachycardia, hypertension, delirium, and possibly death (within 2–7 days). Mortality is 15–20%. Benzodiazepines (can require massive doses); haloperidol for hallucinations; thiamine, folate, and multivitamin replacement (do not affect withdrawal, but most alcoholics are def cient). Barbiturates Anxiety, seizures, delirium, tremor; cardiac and respiratory depression. Benzodiazepines. Benzodiazepines Rebound anxiety, seizures, tremor, insomnia. Benzodiazepines. Monitor for DTs. Cocaine/ amphetamines Depression, hyperphagia, hypersomnolence. Supportive treatment. Avoid pure β-blockers (may lead to unopposed α activity, causing hypertension). Opioids Anxiety, insomnia, fulike symptoms, piloerection, fever, rhinorrhea, lacrimation, yawning, nausea, stomach cramps, diarrhea, mydriasis. Antiemetics, muscle relaxers, and NSAIDs for mild

1	Opioids Anxiety, insomnia, fulike symptoms, piloerection, fever, rhinorrhea, lacrimation, yawning, nausea, stomach cramps, diarrhea, mydriasis. Antiemetics, muscle relaxers, and NSAIDs for mild symptoms; clonidine, buprenorphine, or methadone for moderate to severe symptoms.

1	TABLE 2.17-7. Vitamin Functions and Def ciencies

1	Vitamin A Night blindness, dry skin. Vitamin B1 (thiamine) Beriberi (polyneuritis, dilated cardiomyopathy, high-output CHF, edema), Wernicke-Korsakoff syndrome. Vitamin B2 (ribofavin) Angular stomatitis, cheilosis, corneal vascularization. Vitamin B3 (niacin) Pellagra (diarrhea, dermatitis, dementia). Vitamin B5 (pantothenate) Dermatitis, enteritis, alopecia, adrenal insuff ciency. Vitamin B6 (pyridoxine) Convulsions, hyperirritability; required during administration of INH. Vitamin B12 (cobalamin) Macrocytic, megaloblastic anemia; neurologic symptoms (e.g., optic neuropathy, subacute combined degeneration, paresthesias); glossitis. Vitamin C Scurvy (e.g., swollen gums, bruising, anemia, poor wound healing). Vitamin D Rickets in children (bending bones), osteomalacia in adults (soft bones), hypocalcemic tetany. Vitamin E ↑ fragility of RBCs. Vitamin K Neonatal hemorrhage; ↑ PT and aPTT, normal BT. Biotin Dermatitis, enteritis. Can be caused by ingestion of raw eggs or antibiotic use.

1	hypocalcemic tetany. Vitamin E ↑ fragility of RBCs. Vitamin K Neonatal hemorrhage; ↑ PT and aPTT, normal BT. Biotin Dermatitis, enteritis. Can be caused by ingestion of raw eggs or antibiotic use. Folic acid The most common vitamin defciency in the United States. Sprue; macrocytic, megaloblastic anemia without neurologic symptoms. Magnesium Weakness, muscle cramps, exacerbation of hypocalcemic tetany, CNS hyperirritability leading to tremors, choreoathetoid movement. Selenium Keshan disease (cardiomyopathy).

1	Classic ECG finding in atrial ﬂutter. Definition of unstable angina. Antihypertensive for a diabetic patient with proteinuria. Beck’s triad for cardiac tamponade. Drugs that slow AV node transmission. Hypercholesterolemia treatment that leads to ﬂ ushing and pruritus. Murmur—hypertrophic obstructive cardiomyopathy (HOCM). Murmur—aortic insufficiency. Murmur—aortic stenosis. Murmur—mitral regurgitation. Murmur—mitral stenosis. Treatment for atrial fibrillation and atrial ﬂutter. Treatment for ventricular fibrillation. Autoimmune complication occurring 2–4 weeks post-MI. IV drug use with JVD and holosystolic murmur at the left sternal border. Treatment? Diagnostic test for hypertrophic cardiomyopathy. A fall in systolic BP of > 10 mmHg with inspiration. Classic ECG findings in pericarditis. Definition of hypertension. Eight surgically correctable causes of hypertension.

1	A fall in systolic BP of > 10 mmHg with inspiration. Classic ECG findings in pericarditis. Definition of hypertension. Eight surgically correctable causes of hypertension. “Sawtooth” P waves. Angina is new, is worsening, or occurs at rest. ACEI. Hypotension, distant heart sounds, and JVD. β-blockers, digoxin, calcium channel blockers. Niacin. Systolic ejection murmur heard along the lateral sternal border that ↑ with Valsalva maneuver and standing. Diastolic, decrescendo, high-pitched, blowing murmur that is best heard sitting up; ↑ with ↓ preload (handgrip maneuver). Systolic crescendo/decrescendo murmur that radiates to the neck; ↑ with ↑ preload (Valsalva maneuver). Holosystolic murmur that radiates to the axillae or carotids. Diastolic, midto late, low-pitched murmur. If unstable, cardiovert. If stable or chronic, rate control with calcium channel blockers or β-blockers.

1	Diastolic, midto late, low-pitched murmur. If unstable, cardiovert. If stable or chronic, rate control with calcium channel blockers or β-blockers. Immediate cardioversion. Dressler’s syndrome: fever, pericarditis, ↑ ESR. Treat existing heart failure and replace the tricuspid valve. Echocardiogram (showing thickened left ventricular wall and outﬂ ow obstruction). Pulsus paradoxus (seen in cardiac tamponade). Low-voltage, diffuse ST-segment elevation. BP > 140/90 on three separate occasions two weeks apart. Renal artery stenosis, coarctation of the aorta, pheochromocytoma, Conn’s syndrome, Cushing’s syndrome, unilateral renal parenchymal disease, hyperthyroidism, hyperparathyroidism. Evaluation of a pulsatile abdominal mass and bruit. Indications for surgical repair of abdominal aortic aneurysm. Treatment for acute coronary syndrome. What is metabolic syndrome? Appropriate diagnostic test? A 50-year-old man with angina can exercise to 85% of maximum predicted heart rate.

1	What is metabolic syndrome? Appropriate diagnostic test? A 50-year-old man with angina can exercise to 85% of maximum predicted heart rate. A 65-year-old woman with left bundle branch block and severe osteoarthritis has unstable angina. Target LDL in a patient with diabetes. Signs of active ischemia during stress testing. ECG findings suggesting MI. Coronary territories in MI. A young patient has angina at rest with ST-segment elevation. Cardiac enzymes are normal. Common symptoms associated with silent Mls. The diagnostic test for pulmonary embolism. An agent that reverses the effects of heparin. The coagulation parameter affected by warfarin. A young patient with a family history of sudden death collapses and dies while exercising. Endocarditis prophylaxis regimens. The 6 P’s of ischemia due to peripheral vascular disease. Virchow’s triad. The most common cause of hypertension in young women. The most common cause of hypertension in young men.

1	The 6 P’s of ischemia due to peripheral vascular disease. Virchow’s triad. The most common cause of hypertension in young women. The most common cause of hypertension in young men. Abdominal ultrasound and CT. > 5.5 cm, rapidly enlarging, symptomatic, or ruptured. Morphine, O2, sublingual nitroglycerin, ASA, IV β-blockers, heparin. Abdominal obesity, high triglycerides, low HDL, hypertension, insulin resistance, prothrombotic or proinﬂ ammatory states. Exercise stress treadmill with ECG. Pharmacologic stress test (e.g., dobutamine echo). < 70. Angina, ST-segment changes on ECG, or ↓ BP. ST-segment elevation (depression means ischemia), ﬂattened T waves, and Q waves. Anterior wall (LAD/diagonal), inferior (PDA), posterior (left circumﬂex/oblique, RCA/marginal), septum (LAD/diagonal). Prinzmetal’s angina. CHF, shock, and altered mental status. V/Q scan. Protamine. PT. Hypertrophic cardiomyopathy.

1	CHF, shock, and altered mental status. V/Q scan. Protamine. PT. Hypertrophic cardiomyopathy. and gentamicin before and amoxicillin after. Pain, pallor, pulselessness, paralysis, paresthesia, poikilothermia. Stasis, hypercoagulability, endothelial damage. OCPs. Excessive EtOH. “Stuck-on” appearance. Red plaques with silvery-white scales and sharp margins. The most common type of skin cancer; the lesion is a pearly-colored papule with a translucent surface and telangiectasias. Honey-crusted lesions. A febrile patient with a history of diabetes presents with a red, swollen, painful lower extremity. Nikolsky’s sign. Nikolsky’s sign. A 55-year-old obese patient presents with dirty, velvety patches on the back of the neck. Dermatomal distribution. Flat-topped papules. Iris-like target lesions. A lesion characteristically occurring in a linear pattern in areas where skin comes into contact with clothing or jewelry.

1	Presents with a herald patch, Christmas-tree pattern. A 16-year-old presents with an annular patch of alopecia with broken-off, stubby hairs. Pinkish, scaling, ﬂat lesions on the chest and back; KOH prep has a “spaghetti-and-meatballs” appearance. Four characteristics of a nevus suggestive of melanoma. A premalignant lesion from sun exposure that can lead to squamous cell carcinoma. “Dewdrops on a rose petal.” “Cradle cap.” Associated with Propionibacterium acnes and changes in androgen levels. Seborrheic keratosis. Psoriasis. Basal cell carcinoma. Impetigo. Cellulitis. Pemphigus vulgaris. Bullous pemphigoid. Acanthosis nigricans. Check fasting blood glucose to rule out diabetes. Varicella zoster. Lichen planus. Erythema multiforme. Contact dermatitis. Pityriasis rosea. Alopecia areata (an autoimmune process). Pityriasis versicolor. Asymmetry, border irregularity, color variation, and large diameter. Actinic keratosis.

1	Pityriasis rosea. Alopecia areata (an autoimmune process). Pityriasis versicolor. Asymmetry, border irregularity, color variation, and large diameter. Actinic keratosis. Lesions of 1° varicella. Seborrheic dermatitis. Treat with antifungals. Acne vulgaris. A painful, recurrent vesicular eruption of mucocutaneous surfaces. Inﬂammation and epithelial thinning of the anogenital area, predominantly in postmenopausal women. Exophytic nodules on the skin with varying degrees of scaling or ulceration; the second most common type of skin cancer. Herpes simplex. Lichen sclerosus. Squamous cell carcinoma. Bias introduced into a study when a clinician is aware of the patient’s treatment type. Bias introduced when screening detects a disease earlier and thus lengthens the time from diagnosis to death.

1	Bias introduced into a study when a clinician is aware of the patient’s treatment type. Bias introduced when screening detects a disease earlier and thus lengthens the time from diagnosis to death. If you want to know if geographical location affects infant mortality rate but most variation in infant mortality is predicted by socioeconomic status, then socioeconomic status is a _____. The number of true positives divided by the number of patients with the disease is _____. Sensitive tests have few false negatives and are used to rule _____ a disease. PPD reactivity is used as a screening test because most people with TB (except those who are anergic) will have a PPD. Highly sensitive or specific? Chronic diseases such as SLE—higher prevalence or incidence? Epidemics such as inﬂuenza—higher prevalence or incidence? Cross-sectional survey—incidence or prevalence? Cohort study—incidence or prevalence? Case-control study—incidence or prevalence?

1	Epidemics such as inﬂuenza—higher prevalence or incidence? Cross-sectional survey—incidence or prevalence? Cohort study—incidence or prevalence? Case-control study—incidence or prevalence? Describe a test that consistently gives identical results, but the results are wrong. Difference between a cohort and a case-control study. Observational bias. Lead-time bias. Confounding variable. Sensitivity. Out. Highly sensitive for TB. Higher prevalence. Higher incidence. Prevalence. Incidence and prevalence. Neither. High reliability, low validity. Cohort studies can be used to calculate relative risk (RR), incidence, and/or odds ratio (OR). Case-control studies can be used to calculate an OR. Attributable risk? Relative risk? Odds ratio? Number needed to treat? In which patients do you initiate colorectal cancer screening early?

1	Attributable risk? Relative risk? Odds ratio? Number needed to treat? In which patients do you initiate colorectal cancer screening early? The most common cancer in men and the most common cause of death from cancer in men. The percentage of cases within one SD of the mean? Two SDs? Three SDs? Birth rate? Fertility rate? Mortality rate? Neonatal mortality rate? Postnatal mortality rate? Infant mortality rate? Fetal mortality rate? Perinatal mortality rate? Maternal mortality rate? The incidence rate (IR) of a disease in exposed – the IR of a disease in unexposed. The IR of a disease in a population exposed to a particular factor ÷ the IR of those not exposed. The likelihood of a disease among individuals exposed to a risk factor compared to those who have not been exposed. 1 ÷ (rate in untreated group – rate in treated group).

1	The likelihood of a disease among individuals exposed to a risk factor compared to those who have not been exposed. 1 ÷ (rate in untreated group – rate in treated group). Patients with IBD; those with familial adenomatous polyposis (FAP)/hereditary nonpolyposis colorectal cancer (HNPCC); and those who have first-degree relatives with adenomatous polyps (< 60 years of age) or colorectal cancer. Prostate cancer is the most common cancer in men, but lung cancer causes more deaths. 68%, 95.4%, 99.7%. Number of live births per 1000 population in one year. Number of live births per 1000 females (15–44 years of age) in one year. Number of deaths per 1000 population in one year. Number of deaths from birth to 28 days per 1000 live births in one year. Number of deaths from 28 days to one year per 1000 live births in one year. Number of deaths from birth to one year of age per 1000 live births (neonatal + postnatal mortality) in one year.

1	Number of deaths from 28 days to one year per 1000 live births in one year. Number of deaths from birth to one year of age per 1000 live births (neonatal + postnatal mortality) in one year. Number of deaths from 20 weeks’ gestation to birth per 1000 total births in one year. Number of deaths from 20 weeks’ gestation to one month of life per 1000 total births in one year. Number of deaths during pregnancy to 90 days postpartum per 100,000 live births in one year. True or false: Once patients sign a statement giving consent, they must continue treatment. A 15-year-old pregnant girl requires hospitalization for preeclampsia. Is parental consent required? A doctor refers a patient for an MRI at a facility he/she owns. Involuntary psychiatric hospitalization can be undertaken for which three reasons? True or false: Withdrawing a nonbeneficial treatment is ethically similar to withholding a nonindicated one.

1	Involuntary psychiatric hospitalization can be undertaken for which three reasons? True or false: Withdrawing a nonbeneficial treatment is ethically similar to withholding a nonindicated one. When can a physician refuse to continue treating a patient on the grounds of futility? An eight-year-old child is in a serious accident. She requires emergent transfusion, but her parents are not present. Conditions in which confidentiality must be overridden. Involuntary commitment or isolation for medical treatment may be undertaken for what reason? A 10-year-old child presents in status epilepticus, but her parents refuse treatment on religious grounds. A son asks that his mother not be told about her recently discovered cancer. False. Patients may change their minds at any time. Exceptions to the requirement of informed consent include emergency situations and patients without decision-making capacity.

1	False. Patients may change their minds at any time. Exceptions to the requirement of informed consent include emergency situations and patients without decision-making capacity. No. Parental consent is not necessary for the medical treatment of pregnant minors. Conﬂict of interest. The patient is a danger to self, a danger to others, or gravely disabled (unable to provide for basic needs). True. When there is no rationale for treatment, maximal intervention is failing, a given intervention has already failed, and treatment will not achieve the goals of care. Treat immediately. Consent is implied in emergency situations. Real threat of harm to third parties; suicidal intentions; certain contagious diseases; elder and child abuse. When treatment noncompliance represents a serious danger to public health (e.g., active TB). Treat because the disease represents an immediate threat to the child’s life. Then seek a court order.

1	When treatment noncompliance represents a serious danger to public health (e.g., active TB). Treat because the disease represents an immediate threat to the child’s life. Then seek a court order. A physician can withhold information from the patient only in the rare case of therapeutic privilege or if the patient requests not to be told. A patient’s family cannot require the physician to withhold information from the patient. Diagnostic modality used when ultrasound is equivocal for cholecystitis. Risk factors for cholelithiasis. Inspiratory arrest during palpation of the RUQ. The most common cause of SBO in patients with no history of abdominal surgery. The most common cause of SBO in patients with a history of abdominal surgery. Identify key organisms causing diarrhea: A 25-year-old Jewish man presents with pain and watery diarrhea after meals. Exam shows fistulas between the bowel and skin and nodular lesions on his tibias.

1	A 25-year-old Jewish man presents with pain and watery diarrhea after meals. Exam shows fistulas between the bowel and skin and nodular lesions on his tibias. Inﬂammatory disease of the colon with ↑ risk of colon cancer. Extraintestinal manifestations of IBD. Medical treatment for IBD. Difference between Mallory-Weiss and Boerhaave tears. Charcot’s triad. Reynolds’ pentad. Medical treatment for hepatic encephalopathy. HIDA scan. Fat, female, fertile, forty, ﬂ atulent. Murphy’s sign, seen in acute cholecystitis. Hernia. Adhesions. S. aureus E. coli O157:H7 Bacillus cereus Salmonella Vibrio, HAV Isospora, Cryptosporidium, Mycobacterium avium complex Crohn’s disease. Ulcerative colitis (greater risk than Crohn’s). Uveitis, ankylosing spondylitis, pyoderma gangrenosum, erythema nodosum, 1° sclerosing cholangitis. 5-ASA agents and steroids during acute exacerbations. Mallory-Weiss—superficial tear in the esophageal mucosa; Boerhaave—full-thickness esophageal rupture.

1	5-ASA agents and steroids during acute exacerbations. Mallory-Weiss—superficial tear in the esophageal mucosa; Boerhaave—full-thickness esophageal rupture. RUQ pain, jaundice, and fever/chills in the setting of ascending cholangitis. Charcot’s triad plus shock and mental status changes, with suppurative ascending cholangitis. ↓ protein intake, lactulose, rifaximin. First step in the management of a patient with an acute GI bleed. A four-year-old child presents with oliguria, petechiae, and jaundice following an illness with bloody diarrhea. Most likely diagnosis and cause? Post-HBV exposure treatment. Classic causes of drug-induced hepatitis. A 40-year-old obese woman with elevated alkaline phosphatase, elevated bilirubin, pruritus, dark urine, and clay-colored stools. Hernia with highest risk of incarceration—indirect, direct, or femoral?

1	Hernia with highest risk of incarceration—indirect, direct, or femoral? A 50-year-old man with a history of alcohol abuse presents with boring epigastric pain that radiates to the back and is relieved by sitting forward. Management? Establish the ABCs. Hemolytic-uremic syndrome (HUS) due to E. coli O157: H7. HBV immunoglobulin. TB medications (INH, rifampin, pyrazinamide), acetaminophen, and tetracycline. Biliary tract obstruction. Femoral hernia. Confirm the diagnosis of acute pancreatitis with elevated amylase and lipase. Make the patient NPO and give IV ﬂ uids, O2, analgesia, and “tincture of time.” Four causes of microcytic anemia. An elderly man with hypochromic, microcytic anemia is asymptomatic. Diagnostic tests? Precipitants of hemolytic crisis in patients with G6PD deficiency.

1	Four causes of microcytic anemia. An elderly man with hypochromic, microcytic anemia is asymptomatic. Diagnostic tests? Precipitants of hemolytic crisis in patients with G6PD deficiency. The most common inherited cause of hypercoagulability. The most common inherited bleeding disorder. The most common inherited hemolytic anemia. Diagnostic test for hereditary spherocytosis. Pure RBC aplasia. Anemia associated with absent radii and thumbs, diffuse hyperpigmentation, café au lait spots, microcephaly, and pancytopenia. Medications and viruses that lead to aplastic anemia. TICS—Thalassemia, Iron deficiency, anemia of Chronic disease, and Sideroblastic anemia. Fecal occult blood test and sigmoidoscopy; suspect colorectal cancer. Sulfonamides, antimalarial drugs, fava beans. Factor V Leiden mutation. von Willebrand’s disease. Hereditary spherocytosis. Osmotic fragility test. Diamond-Blackfan anemia. Fanconi’s anemia.

1	Sulfonamides, antimalarial drugs, fava beans. Factor V Leiden mutation. von Willebrand’s disease. Hereditary spherocytosis. Osmotic fragility test. Diamond-Blackfan anemia. Fanconi’s anemia. Chloramphenicol, sulfonamides, radiation, HIV, chemotherapeutic agents, hepatitis, parvovirus B19, EBV. How to distinguish polycythemia vera from 2° polycythemia. Thrombotic thrombocytopenic purpura (TTP) pentad? HUS triad? Treatment for TTP. Treatment for idiopathic thrombocytopenic purpura (ITP) in children. Which of the following are ↑ in DIC: fibrin split products, D-dimer, fibrinogen, platelets, and hematocrit. An eight-year-old boy presents with hemarthrosis and ↑ PTT with normal PT and bleeding time. Diagnosis? Treatment? A 14-year-old girl presents with prolonged bleeding after dental surgery and with menses, normal PT, normal or ↑ PTT, and ↑ bleeding time. Diagnosis? Treatment?

1	A 14-year-old girl presents with prolonged bleeding after dental surgery and with menses, normal PT, normal or ↑ PTT, and ↑ bleeding time. Diagnosis? Treatment? A 60-year-old African-American man presents with bone pain. Workup for multiple myeloma might reveal? Reed-Sternberg cells. A 10-year-old boy presents with fever, weight loss, and night sweats. Exam shows an anterior mediastinal mass. Suspected diagnosis? Microcytic anemia with ↓ serum iron, ↓ total iron-binding capacity (TIBC), and normal or ↑ ferritin. Microcytic anemia with ↓ serum iron, ↓ ferritin, and ↑ TIBC. An 80-year-old man presents with fatigue, lymphadenopathy, splenomegaly, and isolated lymphocytosis. Suspected diagnosis? The lymphoma equivalent of CLL. A late, life-threatening complication of chronic myelogenous leukemia (CML). Auer rods on blood smear. AML subtype associated with DIC. Both have ↑ hematocrit and RBC mass, but polycythemia vera should have normal O2 saturation and low erythropoietin levels.

1	Both have ↑ hematocrit and RBC mass, but polycythemia vera should have normal O2 saturation and low erythropoietin levels. “FAT RN”: Fever, Anemia, Thrombocytopenia, Renal dysfunction, Neurologic abnormalities. Anemia, thrombocytopenia, and acute renal failure. Emergent large-volume plasmapheresis, corticosteroids, antiplatelet drugs. Usually resolves spontaneously; may require IVIG and/or corticosteroids. Fibrin split products and D-dimer are elevated; platelets, fibrinogen, and hematocrit are ↓. A) or factor VIII or IX supplements. von Willebrand’s disease; treat with desmopressin, FFP, or cryoprecipitate. Monoclonal gammopathy, Bence Jones proteinuria, “punched-out” lesions on x-ray of the skull and long bones. Hodgkin’s lymphoma. Non-Hodgkin’s lymphoma. Anemia of chronic disease. Iron deficiency anemia. Chronic lymphocytic leukemia (CLL). Small lymphocytic lymphoma. Blast crisis (fever, bone pain, splenomegaly, pancytopenia). Acute myelogenous leukemia (AML). M3.

1	Iron deficiency anemia. Chronic lymphocytic leukemia (CLL). Small lymphocytic lymphoma. Blast crisis (fever, bone pain, splenomegaly, pancytopenia). Acute myelogenous leukemia (AML). M3. Electrolyte changes in tumor lysis syndrome. Treatment for AML M3. A 50-year-old man presents with early satiety, splenomegaly, and bleeding. Cytogenetics show t(9,22). Diagnosis? Heinz bodies? An autosomal-recessive disorder with a defect in the GPIIbIIIa platelet receptor and ↓ platelet aggregation. Virus associated with aplastic anemia in patients with sickle cell anemia. A 25-year-old African-American man with sickle cell anemia has sudden onset of bone pain. Management of pain crisis? A significant cause of morbidity in thalassemia patients. Treatment? ↓ Ca2+, ↑ K+, ↑ phosphate, ↑ uric acid. Retinoic acid. CML. Intracellular inclusions seen in thalassemia, G6PD deficiency, and postsplenectomy. Glanzmann’s thrombasthenia. Parvovirus B19.

1	↓ Ca2+, ↑ K+, ↑ phosphate, ↑ uric acid. Retinoic acid. CML. Intracellular inclusions seen in thalassemia, G6PD deficiency, and postsplenectomy. Glanzmann’s thrombasthenia. Parvovirus B19. O2, analgesia, hydration, and, if severe, transfusion. Iron overload; use deferoxamine. The three most common causes of fever of unknown origin (FUO). Four signs and symptoms of streptococcal pharyngitis. A nonsuppurative complication of streptococcal infection that is not altered by treatment of 1° infection. Asplenic patients are particularly susceptible to these organisms. The number of bacteria on a clean-catch specimen to diagnose a UTI. Which healthy population is susceptible to UTIs? A patient from California or Arizona presents with fever, malaise, cough, and night sweats. Diagnosis? Treatment? Nonpainful chancre. A “blueberry muffin” rash is characteristic of what congenital infection? Infection, cancer, and autoimmune disease.

1	Nonpainful chancre. A “blueberry muffin” rash is characteristic of what congenital infection? Infection, cancer, and autoimmune disease. Fever, pharyngeal erythema, tonsillar exudate, lack of cough. Postinfectious glomerulonephritis. Encapsulated organisms—pneumococcus, meningococcus, Haemophilus inﬂ uenzae, Klebsiella. 105 bacteria/mL. Pregnant women. Treat this group aggressively because of potential complications. Coccidioidomycosis. Amphotericin B. 1° syphilis. Rubella. Meningitis in neonates. Causes? Treatment? Meningitis in infants. Causes? Treatment? What should always be done prior to LP? CSF findings: Low glucose, PMN predominance Normal glucose, lymphocytic predominance Initially presents with a pruritic papule with regional lymphadenopathy; evolves into a black eschar after 7–10 days. Treatment? Findings in 3° syphilis. Characteristics of 2° Lyme disease. Cold agglutinins.

1	Findings in 3° syphilis. Characteristics of 2° Lyme disease. Cold agglutinins. A 24-year-old man presents with soft white plaques on his tongue and the back of his throat. Diagnosis? Workup? Treatment? Begin Pneumocystis jiroveci (formerly P. carinii) pneumonia prophylaxis in an HIV-positive patient at what CD4 count? Mycobacterium avium–intracellulare (MAI) prophylaxis? Risk factors for pyelonephritis. Neutropenic nadir postchemotherapy. Erythema migrans. Classic physical findings for endocarditis. Aplastic crisis in sickle cell disease. Ring-enhancing brain lesion on CT with seizures. Group B strep, E. coli, Listeria. Treat with gentamicin and ampicillin. Pneumococcus, meningococcus, H. inﬂ uenzae. Treat with cefotaxime and vancomycin. Check for ↑ ICP; look for papilledema. Cutaneous anthrax. Treat with penicillin G or ciproﬂ oxacin. Tabes dorsalis, general paresis, gummas, Argyll Robertson pupil, aortitis, aortic root aneurysms.

1	Cutaneous anthrax. Treat with penicillin G or ciproﬂ oxacin. Tabes dorsalis, general paresis, gummas, Argyll Robertson pupil, aortitis, aortic root aneurysms. Arthralgias, migratory polyarthropathies, Bell’s palsy, myocarditis. Mycoplasma. Candidal thrush. Workup should include an HIV test. Treat with nystatin oral suspension. ≤ 200 for P. jiroveci (with TMP-SMX); ≤ 50–100 for MAI (with clarithromycin/azithromycin). Pregnancy, vesicoureteral reﬂux, anatomic anomalies, indwelling catheters, kidney stones. 7–10 days. Lesion of 1° Lyme disease. Fever, heart murmur, Osler’s nodes, splinter hemorrhages, Janeway lesions, Roth’s spots. Parvovirus B19. Taenia solium (cysticercosis). Name the organism: Pregnant women with pets Alcoholic with pneumonia A 55-year-old man who is a smoker and a heavy drinker presents with a new cough and ﬂulike symptoms. Gram stain shows no organisms; silver stain of sputum shows gram-negative rods. What is the diagnosis?

1	A middle-aged man presents with acute-onset monoarticular joint pain and bilateral Bell’s palsy. What is the likely diagnosis, and how did he get it? Treatment? A patient develops endocarditis three weeks after receiving a prosthetic heart valve. What organism is suspected? Legionella pneumonia. Lyme disease, Ixodes tick, doxycycline. S. aureus or S. epidermidis. Back pain that is exacerbated by standing and walking and relieved with sitting and hyperﬂexion of the hips. Joints in the hand affected in rheumatoid arthritis. Joint pain and stiffness that worsen over the course of the day and are relieved by rest. Genetic disorder associated with multiple fractures and commonly mistaken for child abuse. Hip and back pain along with stiffness that improves with activity over the course of the day and worsens at rest. Diagnostic test? Arthritis, conjunctivitis, and urethritis in young men. Associated organisms?

1	Arthritis, conjunctivitis, and urethritis in young men. Associated organisms? A 55-year-old man has sudden, excruciating first MTP joint pain after a night of drinking red wine. Diagnosis, workup, and chronic treatment? Spinal stenosis. MCP and PIP joints; DIP joints are spared. Osteoarthritis. Osteogenesis imperfecta. Suspect ankylosing spondylitis. Check HLA-B27. Reactive (Reiter’s) arthritis. Associated with Campylobacter, Shigella, Salmonella, Chlamydia, and Ureaplasma. Gout. Needle-shaped, negatively birefringent crystals are seen on joint ﬂuid aspirate. Chronic treatment with allopurinol or probenecid. Rhomboid-shaped, positively birefringent crystals on joint ﬂ uid aspirate. An elderly woman presents with pain and stiffness of the shoulders and hips; she cannot lift her arms above her head. Labs show anemia and ↑ ESR.

1	An active 13-year-old boy has anterior knee pain. Diagnosis? Bone is fractured in a fall on an outstretched hand. Complication of scaphoid fracture. Signs suggesting radial nerve damage with humeral fracture. A young child presents with proximal muscle weakness, waddling gait, and pronounced calf muscles. A first-born female who was born in breech position is found to have asymmetric skin folds on her newborn exam. Diagnosis? Treatment? An 11-year-old obese African-American boy presents with sudden onset of limp. Diagnosis? Workup? The most common 1° malignant tumor of bone. Pseudogout. Polymyalgia rheumatica. Osgood-Schlatter disease. Distal radius (Colles’ fracture). Avascular necrosis. Wrist drop, loss of thumb abduction. Duchenne muscular dystrophy. Developmental dysplasia of the hip. If severe, consider a Pavlik harness to maintain abduction. Slipped capital femoral epiphysis. AP and frog-leg lateral view. Multiple myeloma.

1	Developmental dysplasia of the hip. If severe, consider a Pavlik harness to maintain abduction. Slipped capital femoral epiphysis. AP and frog-leg lateral view. Multiple myeloma. Unilateral, severe periorbital headache with tearing and conjunctival erythema. Prophylactic treatment for migraine. The most common pituitary tumor. Treatment? A 55-year-old patient presents with acute “broken speech.” What type of aphasia? What lobe and vascular distribution? The most common cause of SAH. A crescent-shaped hyperdensity on CT that does not cross the midline. A history significant for initial altered mental status with an intervening lucid interval. Diagnosis? Most likely source? Treatment? CSF findings with SAH. Cluster headache. Antihypertensives, antidepressants, anticonvulsants. Prolactinoma. Dopamine agonists (e.g., bromocriptine). Broca’s aphasia. Frontal lobe, left MCA distribution. Trauma; the second most common is berry aneurysm. Subdural hematoma—bridging veins torn.

1	Trauma; the second most common is berry aneurysm. Subdural hematoma—bridging veins torn. Epidural hematoma. Middle meningeal artery. Neurosurgical evacuation. Elevated ICP, RBCs, xanthochromia. Albuminocytologic dissociation. Cold water is ﬂushed into a patient’s ear, and the fast phase of the nystagmus is toward the opposite side. Normal or pathologic? The most common 1° sources of metastases to the brain. May be seen in children who are accused of inattention in class and confused with ADHD. The most frequent presentation of intracranial neoplasm. The most common cause of seizures in children (2–10 years).

1	The most common cause of seizures in young adults (18–35 years). First-line medication for status epilepticus. Confusion, confabulation, ophthalmoplegia, ataxia. What % lesion is an indication for carotid endarterectomy? The most common causes of dementia. Combined UMN and LMN disorder. Rigidity and stiffness with resting tremor and masked facies. The mainstay of Parkinson’s therapy. Treatment for Guillain-Barré syndrome. Rigidity and stiffness that progress to choreiform movements, accompanied by moodiness and altered behavior. A six-year-old girl presents with a port-wine stain in the V2 distribution as well as with mental retardation, seizures, and ipsilateral leptomeningeal angioma. Café au lait spots on skin. Hyperphagia, hypersexuality, hyperorality, and hyperdocility. May be administered to a symptomatic patient to diagnose myasthenia gravis. Guillain-Barré syndrome (↑ protein in CSF without a significant ↑ in cell count). Normal.

1	Guillain-Barré syndrome (↑ protein in CSF without a significant ↑ in cell count). Normal. Lung, breast, skin (melanoma), kidney, GI tract. Absence seizures. Headache. Infection, febrile seizures, trauma, idiopathic. Trauma, alcohol withdrawal, brain tumor. IV benzodiazepine. Wernicke’s encephalopathy due to a deficiency of thiamine. Seventy percent if the stenosis is symptomatic. Alzheimer’s and multi-infarct. ALS. Parkinson’s disease. Levodopa/carbidopa. IVIG or plasmapheresis. Huntington’s disease. Sturge-Weber syndrome. Treat symptomatically. Possible focal cerebral resection of the affected lobe. Neurofibromatosis type 1. Klüver-Bucy syndrome (amygdala). Edrophonium. 1° causes of third-trimester bleeding.

1	Neurofibromatosis type 1. Klüver-Bucy syndrome (amygdala). Edrophonium. 1° causes of third-trimester bleeding. Classic ultrasound and gross appearance of complete hydatidiform mole. Chromosomal pattern of a complete mole. Molar pregnancy containing fetal tissue. Symptoms of placental abruption. Symptoms of placenta previa. When should a vaginal exam be performed with suspected placenta previa? Antibiotics with teratogenic effects. Shortest AP diameter of the pelvis. Medication given to accelerate fetal lung maturity. The most common cause of postpartum hemorrhage. Treatment for postpartum hemorrhage. Typical antibiotics for group B streptococcus (GBS) prophylaxis. A patient fails to lactate after an emergency C-section with marked blood loss. Uterine bleeding at 18 weeks’ gestation; no products expelled; membranes ruptured; cervical os open. Uterine bleeding at 18 weeks’ gestation; no products expelled; cervical os closed. Placental abruption and placenta previa.

1	Placental abruption and placenta previa. Snowstorm on ultrasound. “Cluster-of-grapes” appearance on gross examination. 46,XX. Partial mole. Continuous, painful vaginal bleeding. Self-limited, painless vaginal bleeding. Never. Tetracycline, ﬂ uoroquinolones, aminoglycosides, sulfonamides. Obstetric conjugate: between the sacral promontory and the midpoint of the symphysis pubis. Betamethasone or dexamethasone × 48 hours. Uterine atony. Uterine massage; if that fails, give oxytocin. IV penicillin or ampicillin. Sheehan’s syndrome (postpartum pituitary necrosis). Inevitable abortion. Threatened abortion. Cause of amenorrhea with normal prolactin, no response to estrogen-progesterone challenge, and a history of D&C. Therapy for polycystic ovarian syndrome. Medication used to induce ovulation. Diagnostic step required in a postmenopausal woman who presents with vaginal bleeding. Indications for medical treatment of ectopic pregnancy.

1	Medication used to induce ovulation. Diagnostic step required in a postmenopausal woman who presents with vaginal bleeding. Indications for medical treatment of ectopic pregnancy. Medical options for endometriosis. Laparoscopic findings in endometriosis. The most common location for an ectopic pregnancy. How to diagnose and follow a leiomyoma. Natural history of a leiomyoma. A patient has ↑ vaginal discharge and petechial patches in the upper vagina and cervix. Treatment for bacterial vaginosis. The most common cause of bloody nipple discharge. Contraceptive methods that protect against PID. Unopposed estrogen is contraindicated in which cancers? A patient presents with recent PID with RUQ pain. Breast malignancy presenting as itching, burning, and erosion of the nipple. Annual screening for women with a strong family history of ovarian cancer. A 50-year-old woman leaks urine when laughing or coughing. Nonsurgical options?

1	Annual screening for women with a strong family history of ovarian cancer. A 50-year-old woman leaks urine when laughing or coughing. Nonsurgical options? A 30-year-old woman has unpredictable urine loss. Examination is normal. Medical options? Lab values suggestive of menopause. The most common cause of female infertility. Asherman’s syndrome. Weight loss and OCPs. Clomiphene citrate. Endometrial biopsy. Stable, unruptured ectopic pregnancy of < 3.5 cm at < 6 weeks’ gestation. OCPs, danazol, GnRH agonists. “Chocolate cysts,” powder burns. Ampulla of the oviduct. Ultrasound. Regresses after menopause. Trichomonas vaginitis. Oral or topical metronidazole. Intraductal papilloma. OCPs and barrier contraception. Endometrial or estrogen receptor– breast cancer. Consider Fitz-Hugh–Curtis syndrome. Paget’s disease. CA-125 and transvaginal ultrasound. Kegel exercises, estrogen, pessaries for stress incontinence.

1	CA-125 and transvaginal ultrasound. Kegel exercises, estrogen, pessaries for stress incontinence. Anticholinergics (oxybutynin) or β-adrenergics (metaproterenol) for urge incontinence. ↑ serum FSH. Endometriosis. Nontender abdominal mass associated with elevated VMA and HVA. The most common type of tracheoesophageal fistula (TEF). Diagnosis? Not contraindications to vaccination. Tests to rule out shaken baby syndrome. A neonate has meconium ileus. Bilious emesis within hours after the first feeding. A two-month-old baby presents with nonbilious projectile emesis. What are the appropriate steps in management? The most common 1° immunodeficiency. An infant has a high fever and onset of rash as fever breaks. What is he at risk for? What is the immunodeficiency? A boy has chronic respiratory infections. Nitroblue tetrazolium test is . A child has eczema, thrombocytopenia, and high levels of IgA. A four-month-old boy has life-threatening Pseudomonas infection.

1	A child has eczema, thrombocytopenia, and high levels of IgA. A four-month-old boy has life-threatening Pseudomonas infection. Acute-phase treatment for Kawasaki disease. Treatment for mild and severe unconjugated hyperbilirubinemia. Sudden onset of mental status changes, emesis, and liver dysfunction after taking aspirin. A child has loss of red light reﬂex. Diagnosis? Neuroblastoma. Esophageal atresia with distal TEF (85%). Unable to pass NG tube. Mild illness and/or low-grade fever, current antibiotic therapy, and prematurity. Ophthalmologic exam, CT, and MRI. CF or Hirschsprung’s disease. Duodenal atresia. Correct metabolic abnormalities. Then correct pyloric stenosis with pyloromyotomy. Selective IgA deficiency. Febrile seizures (roseola infantum). High-dose aspirin for inﬂammation and fever; IVIG to prevent coronary artery aneurysms. Phototherapy (mild) or exchange transfusion (severe). Reye’s syndrome. Suspect retinoblastoma.

1	High-dose aspirin for inﬂammation and fever; IVIG to prevent coronary artery aneurysms. Phototherapy (mild) or exchange transfusion (severe). Reye’s syndrome. Suspect retinoblastoma. Vaccinations at a six-month well-child visit. Tanner stage 3 in a six-year-old girl. Infection of small airways with epidemics in winter and spring. Cause of neonatal RDS. A condition associated with red “currant-jelly” stools. A congenital heart disease that causes 2° hypertension. First-line treatment for otitis media. The most common pathogen causing croup. A homeless child is small for his age and has peeling skin and a swollen belly. Defect in an X-linked syndrome with mental retardation, gout, self-mutilation, and choreoathetosis. A newborn girl has a continuous “machinery murmur.” HBV, DTaP, Hib, IPV, PCV. Precocious puberty. RSV bronchiolitis.

1	HBV, DTaP, Hib, IPV, PCV. Precocious puberty. RSV bronchiolitis. Surfactant deficiency. Intussusception. Coarctation of the aorta. Amoxicillin × 10 days. Parainﬂuenza virus type 1. Kwashiorkor (protein malnutrition). Lesch-Nyhan syndrome (purine salvage problem with HGPRTase deficiency). Patent ductus arteriosus (PDA). First-line pharmacotherapy for depression. Antidepressants associated with hypertensive crisis. Galactorrhea, impotence, menstrual dysfunction, and ↓ libido. A 17-year-old girl has left arm paralysis after her boyfriend dies in a car crash. No medical cause is found. Name the defense mechanism: A mother who is angry at her husband yells at her child. A pedophile enters a monastery. A woman calmly describes a grisly murder. A hospitalized 10-year-old begins to wet his bed. Life-threatening muscle rigidity, fever, and rhabdomyolysis. Amenorrhea, bradycardia, and abnormal body image in a young female. SSRIs. MAOIs. Patient on dopamine antagonist.

1	Life-threatening muscle rigidity, fever, and rhabdomyolysis. Amenorrhea, bradycardia, and abnormal body image in a young female. SSRIs. MAOIs. Patient on dopamine antagonist. Conversion disorder. Neuroleptic malignant syndrome. Anorexia. A 35-year-old man has recurrent episodes of palpitations, diaphoresis, and fear of going crazy. The most serious side effect of clozapine. A 21-year-old man has three months of social withdrawal, worsening grades, ﬂattened affect, and concrete thinking. Key side effects of atypical antipsychotics. A young weight lifter receives IV haloperidol and complains that his eyes are deviated sideways. Diagnosis? Treatment? Medication to avoid in patients with a history of alcohol withdrawal seizures. A 13-year-old boy has a history of theft, vandalism, and violence toward family pets. A five-month-old girl has ↓ head growth, truncal dyscoordination, and ↓ social interaction.

1	A 13-year-old boy has a history of theft, vandalism, and violence toward family pets. A five-month-old girl has ↓ head growth, truncal dyscoordination, and ↓ social interaction. A patient hasn’t slept for days, lost $20,000 gambling, is agitated, and has pressured speech. Diagnosis? Treatment? After a minor fender bender, a man wears a neck brace and requests permanent disability. A nurse presents with severe hypoglycemia; blood analysis reveals no elevation in C-peptide. A patient continues to use cocaine after being in jail, losing his job, and not paying child support. A violent patient has vertical and horizontal nystagmus. A woman who was abused as a child frequently feels outside of or detached from her body. A man has repeated, intense urges to rub his body against unsuspecting passengers on a bus. A schizophrenic patient takes haloperidol for one year and develops uncontrollable tongue movements. Diagnosis? Treatment?

1	A schizophrenic patient takes haloperidol for one year and develops uncontrollable tongue movements. Diagnosis? Treatment? A man unexpectedly ﬂies across the country, takes a new name, and has no memory of his prior life. Panic disorder. Agranulocytosis. Schizophreniform disorder (diagnosis of schizophrenia requires ≥ 6 months of symptoms). Weight gain, type 2 DM, QT prolongation. Acute dystonia (oculogyric crisis). Treat with benztropine or diphenhydramine. Neuroleptics. Conduct disorder. Rett’s disorder. Acute mania. Start a mood stabilizer (e.g., lithium). Malingering. Factitious disorder (Munchausen syndrome). Substance abuse. Phencyclidine hydrochloride (PCP) intoxication. Depersonalization disorder. Frotteurism (a paraphilia). Tardive dyskinesia. ↓ or discontinue haloperidol and consider another antipsychotic (e.g., risperidone, clozapine). Dissociative fugue. Risk factors for DVT. Criteria for exudative effusion. Causes of exudative effusion.

1	Dissociative fugue. Risk factors for DVT. Criteria for exudative effusion. Causes of exudative effusion. Causes of transudative effusion. may indicate? Dyspnea, lateral hilar lymphadenopathy on CXR, noncaseating granulomas, ↑ ACE, and hypercalcemia. PFTs showing ↓ FEV1/FVC. PFTs showing ↑ FEV1/FVC. Honeycomb pattern on CXR. Diagnosis? Treatment? Treatment for SVC syndrome. Treatment for mild, persistent asthma. Treatment for COPD exacerbation. Treatment for chronic COPD. Acid-base disorder in pulmonary embolism. Non–small cell lung cancer (NSCLC) associated with hypercalcemia. Lung cancer associated with SIADH. Lung cancer highly related to cigarette exposure. A tall white male presents with acute shortness of breath. Diagnosis? Treatment? Treatment of tension pneumothorax. Stasis, endothelial injury, and hypercoagulability (Virchow’s triad).

1	Diagnosis? Treatment? Treatment of tension pneumothorax. Stasis, endothelial injury, and hypercoagulability (Virchow’s triad). Pleural/serum protein > 0.5; pleural/serum LDH > 0.6. Think of leaky capillaries. Malignancy, TB, bacterial or viral infection, pulmonary embolism with infarct, and pancreatitis. Think of intact capillaries. CHF, liver or kidney disease, and protein-losing enteropathy. Fatigue and impending respiratory failure. Sarcoidosis. Obstructive pulmonary disease (e.g., asthma). Restrictive pulmonary disease. Diffuse interstitial pulmonary fibrosis. Supportive care. Steroids may help. Radiation. Inhaled β-agonists and inhaled corticosteroids. O2, bronchodilators, antibiotics, corticosteroids with taper, smoking cessation. Smoking cessation, home O2, β-agonists, anticholinergics, systemic or inhaled corticosteroids, ﬂu and pneumococcal vaccines. Hypoxia and hypocarbia (respiratory alkalosis). Squamous cell carcinoma.

1	Small cell lung cancer (SCLC). SCLC. Spontaneous pneumothorax. Spontaneous regression. Supplemental O2 may be helpful. Immediate needle thoracostomy. Characteristics favoring carcinoma in an isolated pulmonary nodule. Hypoxemia and pulmonary edema with normal pulmonary capillary wedge pressure. Sequelae of asbestos exposure. ↑ risk of what infection with silicosis? Causes of hypoxemia. Classic CXR findings for pulmonary edema. Age > 45–50 years; lesions new or larger in comparison to old films; absence of calcification or irregular calcification; size > 2 cm; irregular margins. ARDS. Pulmonary fibrosis, pleural plaques, bronchogenic carcinoma (mass in lung field), mesothelioma (pleural mass). Mycobacterium tuberculosis. Right-to-left shunt, hypoventilation, low inspired O2 tension, diffusion defect, V/Q mismatch. Cardiomegaly, prominent pulmonary vessels, Kerley B lines, “bat’s-wing” appearance of hilar shadows, and perivascular and peribronchial cuffing.

1	Cardiomegaly, prominent pulmonary vessels, Kerley B lines, “bat’s-wing” appearance of hilar shadows, and perivascular and peribronchial cuffing. Renal tubular acidosis (RTA) associated with abnormal H+ secretion and nephrolithiasis. RTA associated with abnormal HCO3 and rickets. RTA associated with aldosterone defect. “Doughy” skin Differential of hypervolemic hyponatremia. Chvostek’s and Trousseau’s signs. The most common causes of hypercalcemia. T-wave ﬂattening and U waves. Peaked T waves and widened QRS. First-line treatment for moderate hypercalcemia. Type of ARF in a patient with FeNa < 1%. A 49-year-old man presents with acute-onset ﬂank pain and hematuria. The most common type of nephrolithiasis. A 20-year-old man presents with a palpable ﬂank mass and hematuria. Ultrasound shows bilateral enlarged kidneys with cysts. Associated brain anomaly? Type I (distal) RTA.

1	Type I (distal) RTA. Type II (proximal) RTA. Type IV (distal) RTA. Hypernatremia. Cirrhosis, CHF, nephritic syndrome. Hypocalcemia. Malignancy and hyperparathyroidism. Hypokalemia. Hyperkalemia. IV hydration and loop diuretics (furosemide). Prerenal. Nephrolithiasis. Calcium oxalate. Cerebral berry aneurysms (autosomal-dominant PCKD). Hematuria, hypertension, and oliguria. Proteinuria, hypoalbuminemia, hyperlipidemia, hyperlipiduria, and edema. The most common form of nephritic syndrome. The most common form of glomerulonephritis. Glomerulonephritis with deafness. Glomerulonephritis with hemoptysis. Presence of red cell casts in urine sediment. Eosinophils in urine sediment. Waxy casts in urine sediment and Maltese crosses (seen with lipiduria). Drowsiness, asterixis, nausea, and a pericardial friction rub. A 55-year-old man is diagnosed with prostate cancer. Treatment options?

1	Treatment options? Low urine specific gravity in the presence of high serum osmolality. Treatment of SIADH? Hematuria, ﬂank pain, and palpable ﬂank mass. Testicular cancer associated with β-hCG, AFP. The most common type of testicular cancer. The most common histology of bladder cancer. Complication of overly rapid correction of hyponatremia. Salicylate ingestion occurs in what type of acid-base disorder? Acid-base disturbance commonly seen in pregnant women. Three systemic diseases that lead to nephrotic syndrome. Elevated erythropoietin level, elevated hematocrit, and normal O2 saturation suggest? A 55-year-old male presents with irritative and obstructive urinary symptoms. Treatment options? Nephritic syndrome. Nephrotic syndrome. Membranous glomerulonephritis. IgA nephropathy (Berger’s disease). Alport’s syndrome. Wegener’s granulomatosis and Goodpasture’s syndrome. Glomerulonephritis/nephritic syndrome. Allergic interstitial nephritis. Nephrotic syndrome.

1	Uremic syndrome seen in patients with renal failure. Wait, surgical resection, radiation and/or androgen suppression. Diabetes insipidus. Fluid restriction, demeclocycline. Renal cell carcinoma (RCC). Choriocarcinoma. Seminoma, a type of germ cell tumor. Transitional cell carcinoma. Central pontine myelinolysis. Anion gap acidosis and 1° respiratory alkalosis due to central respiratory stimulation. Respiratory alkalosis. DM, SLE, and amyloidosis. RCC or other erythropoietin-producing tumor; evaluate with CT scan. Likely BPH. Options include no treatment, terazosin, finasteride, or surgical intervention (TURP). Class of drugs that may cause syndrome of muscle rigidity, hyperthermia, autonomic instability, and extrapyramidal symptoms. Side effects of corticosteroids.

1	Class of drugs that may cause syndrome of muscle rigidity, hyperthermia, autonomic instability, and extrapyramidal symptoms. Side effects of corticosteroids. Treatment for DTs. Treatment for acetaminophen overdose. Treatment for opioid overdose. Treatment for benzodiazepine overdose. Treatment for neuroleptic malignant syndrome and malignant hyperthermia. Treatment for malignant hypertension. Treatment of atrial fibrillation. Treatment of supraventricular tachycardia. Causes of drug-induced SLE. Macrocytic, megaloblastic anemia with neurologic symptoms. Macrocytic, megaloblastic anemia without neurologic symptoms. A burn patient presents with cherry-red ﬂushed skin and coma. SaO2 is normal, but carboxyhemoglobin is elevated. Treatment? Blood in the urethral meatus or high-riding prostate. Test to rule out urethral injury. Radiographic evidence of aortic disruption or dissection. Radiographic indications for surgery in patients with acute abdomen.

1	Radiographic indications for surgery in patients with acute abdomen. Antipsychotics (neuroleptic malignant syndrome). Acute mania, immunosuppression, thin skin, osteoporosis, easy bruising, myopathies. Benzodiazepines. N-acetylcysteine. Naloxone. Flumazenil. Dantrolene. Nitroprusside. Rate control, rhythm conversion, and anticoagulation. If stable, rate control with carotid massage or other vagal stimulation; if unsuccessful, consider adenosine. INH, penicillamine, hydralazine, procainamide, chlorpromazine, methyldopa, quinidine. B12 deficiency. Folate deficiency. Treat CO poisoning with 100% O2 or with hyperbaric O2 if poisoning is severe or the patient is pregnant. Bladder rupture or urethral injury. Retrograde cystourethrogram. Widened mediastinum (> 8 cm), loss of aortic knob, pleural cap, tracheal deviation to the right, depression of left main stem bronchus.

1	Retrograde cystourethrogram. Widened mediastinum (> 8 cm), loss of aortic knob, pleural cap, tracheal deviation to the right, depression of left main stem bronchus. Free air under the diaphragm, extravasation of contrast, severe bowel distention, space-occupying lesion (CT), mesenteric occlusion (angiography). The most common organism in burn-related infections. Method of calculating ﬂuid repletion in burn patients. Acceptable urine output in a trauma patient. Acceptable urine output in a stable patient. Cannon “a” waves. Signs of neurogenic shock. Signs of ↑ ICP (Cushing’s triad). CO, ↓ pulmonary capillary wedge pressure (PCWP), peripheral vascular resistance (PVR). CO, ↑ PCWP, ↑ PVR. CO, ↓ PCWP, ↓ PVR. Treatment of septic shock. Treatment of cardiogenic shock. Treatment of hypovolemic shock. Treatment of anaphylactic shock. Supportive treatment for ARDS. Signs of air embolism. Trauma series. Pseudomonas.

1	Trauma series. Pseudomonas. Parkland formula. 50 cc/hr. 30 cc/hr. Third-degree heart block. Hypotension and bradycardia. Hypertension, bradycardia, and abnormal respirations. Hypovolemic shock. Cardiogenic (or obstructive) shock. Septic or anaphylactic shock. Fluids and antibiotics. Identify cause; pressors (e.g., dopamine). Identify cause; ﬂuid and blood repletion. Diphenhydramine or epinephrine 1:1000. Continuous positive airway pressure. A patient with chest trauma who was previously stable suddenly dies. AP chest, AP/lateral C-spine, AP pelvis.

1	This section is a database of recommended clinical science review books, sample examination books, and commercial review courses marketed to medical students studying for the USMLE Step 2 CK. For each book, we list the Title of the book, the First Author (or editor), the Current Publisher, the Copyright Year, the Edition, the Number of Pages, the ISBN Code, the Approximate List Price, the Format of the book, and the Number of Test Questions. Most entries also include Summary Comments that describe the style and utility of each book for studying. Finally, each book receives a Rating. The books are sorted into a comprehensive section as well as into sections corresponding to the six clinical disciplines (internal medicine, neurology, OB/GYN, pediatrics, psychiatry, and surgery). Within each section, books are arranged first by Rating, then by Author, and finally by Title.

1	For this seventh edition of First Aid for the USMLE Step 2 CK, the database of review books has been completely revised, with in-depth summary comments on more than 100 books and software. A letter rating scale with six different grades reﬂects the detailed student evaluations. Each book receives a rating as follows: The Rating is meant to reﬂect the overall usefulness of the book in preparing for the USMLE Step 2 CK exam. This is based on a number of factors, including: The cost of the book The readability of the text The appropriateness and accuracy of the book The quality and number of sample questions The quality of written answers to sample questions The quality and appropriateness of the illustrations (e.g., graphs, diagrams, photographs) The length of the text (longer is not necessarily better) The quality and number of other books available in the same discipline The importance of the discipline on the USMLE Step 2 CK exam

1	The length of the text (longer is not necessarily better) The quality and number of other books available in the same discipline The importance of the discipline on the USMLE Step 2 CK exam Please note that the rating does not reﬂect the quality of the book for purposes other than reviewing for the USMLE Step 2 CK exam. Many books with low ratings are well written and informative but are not ideal for boards preparation. We have also avoided listing or commenting on the wide variety of general textbooks available in the clinical sciences. Evaluations are based on the cumulative results of formal and informal surveys of hundreds of medical students from medical schools across the country. The summary comments and overall ratings represent a consensus opinion, but there may have been a large range of opinions or limited student feedback on any particular book. Please note that the data listed are subject to change because: Publishers’ prices change frequently.

1	Please note that the data listed are subject to change because: Publishers’ prices change frequently. Individual bookstores often charge an additional markup. New editions come out frequently, and the quality of updating varies. The same book may be reissued through another publisher. We actively encourage medical students and faculty to submit their opinions and ratings of these clinical science review books so that we may update our database (see “How to Contribute,” p. xv). In addition, we ask that publishers and authors submit review copies of clinical science review books, including new editions and books not included in our database, for evaluation. We also solicit reviews of new books or suggestions for alternate modes of study that may be useful in preparing for the examination, such as ﬂash cards, computer-based tutorials, commercial review courses, and Internet Web sites.

1	No material in this book, including the ratings, reﬂects the opinion or inﬂuence of the publisher. All errors and omissions will gladly be corrected if brought to the attention of the authors through the publisher. Lippincott Williams & Wilkins, 2006, 3rd ed., 546 pages, ISBN 9781405105095 A concise book presented in outline format, packed with key information across the various fields of medicine. Pros: Very high yield. Makes nice use of tables and charts. Good for quick study and last-minute review. Useful on the wards as well, and fits in a pocket. Cons: Small print. Not tremendously detailed, but covers many topics. More in-depth books are required for further explanation. Summary: A good, comprehensive review, but lacks some detail.

1	Hanley & Belfus, 2004, 2nd ed., 350 pages, ISBN 9781560536109 Includes 750 vignette-style questions in 15 test blocks. Pros: Questions are case based and offer a good approximation of real boards questions. Questions cover high-yield topics, and explanations are terse but adequate. Many questions also include images and associated laboratory findings. Cons: Explanations may not be adequate for those who require an in-depth review of certain topics. Summary: Excellent vignette-type questions in mock exam format. Hanley & Belfus, 2004, 2nd ed., 250 pages, ISBN 9781560536086

1	Hanley & Belfus, 2004, 2nd ed., 250 pages, ISBN 9781560536086 Typical Secrets-series format, with questions and answers organized by specialty. Pros: A concise review of many high-yield topics. Makes good use of clinical images, including patient photos, blood smears, and radiographs. Gives clinical pearls that help differentiate and diagnose common clinical presentations. Cons: Contains no clinical vignettes; simply lists questions that might be posed on the wards. Does not follow Step 2 CK format. Expensive. Content overlaps with that of other books by Brochert. Summary: An excellent book to page through during brief periods of “downtime.” Does not substitute for a formal review or practice tests. A portable book that stresses relevant topics in a quick, easy format. $39.95 Review $35.95 Test/750 q $39.95 Review First Aid Cases for the USMLE Step 2 CK $41.95 Review

1	$39.95 Review $35.95 Test/750 q $39.95 Review First Aid Cases for the USMLE Step 2 CK $41.95 Review LE McGraw-Hill, 2006, 1st ed., 272 pages, ISBN 9780071464116 A review of high-yield clinical vignettes for the Step 2 CK exam. Pros: Cases provide detailed answers to high-yield topics and are arranged in an easy-to-follow format. Emphasizes the most likely diagnosis, the next step, and initial management answers. Cons: Some topics are either not covered or given only brief treatment. Summary: A good review with emphasis on vignette-style case presentations and boardsrelevant answers, but not sufficient as a stand-alone text for review.

1	B+Crush Step 2 BROCHERT Mosby, 2007, 3rd ed., 352 pages, ISBN 9781416029762 A comprehensive review of many high-yield topics, organized by spe-cialty. Pros: Places good emphasis on key points. The conversational style is easy to read. Makes good use of charts and diagrams. Covers surgical topics in more depth than similar books. Cons: Not compre-hensive. Contains no practice questions or vignettes. Not sufficiently detailed to be used alone for Step 2 CK preparation. Summary: A solid review of key points and frequently tested topics. Should probably be supplemented with other review material and practice tests. $39.95 Review B+Lange Q&A: USMLE Step 2 CHAN McGraw-Hill, 2008, 6th ed., 440 pages, ISBN 9780071494007 Review questions organized by specialty along with two comprehen-sive practice exams. Pros: Overall question content is good, with broad coverage of high-yield topics. Well illustrated. Cons: Vignettes are brief, with short explanations. Some questions are more detailed than

1	exams. Pros: Overall question content is good, with broad coverage of high-yield topics. Well illustrated. Cons: Vignettes are brief, with short explanations. Some questions are more detailed than actual Step 2 CK questions. Summary: Well suited to focused spe-cialty review by virtue of overall review questions on high-yield topics. A good buy for the number of questions. $44.95 Test/1000+ q B+Lange Practice Tests: USMLE Step 2 GOLDBERG McGraw-Hill, 2005, 3rd ed., 288 pages, ISBN 9780071446167 Comprehensive test questions. Pros: A great source of high-yield ques-tions covering all topics. Many questions include clinically relevant ra-diographs and photographs of pathology. Offers adequate explanations. Cons: Some questions are not vignette style and do not reﬂ ect boards format. Summary: A good compilation of test questions that focus on high-yield material, but some questions still do not reﬂect boards style. A good source of supplemental questions. $44.95 Test/900 q

1	NMS Review for USMLE Step 2 CK $46.95 Test/900 q Lippincott Williams & Wilkins, 2006, 3rd ed., 654 pages, ISBN 9780781765220 A comprehensive review book in question-and-answer format. Pros: Offers clear, concise, and broad coverage of high-yield topics, presented in a format similar to that of the actual Step 2 CK exam. Complete explanations. Cons: Questions are more detailed than needed for the boards. Lacks illustrations or images. Summary: A good source of Step 2 CK–style questions with appropriate format and content, but questions may be more detailed than those on the actual exam.

1	B+Underground Clinical Vignettes: Step 2 Bundle KIM Lippincott Williams & Wilkins; 2007, 4th ed., 256 pages, ISBN 9780781763639 A set containing clinical case scenarios of the various specialties, in-cluding OB/GYN, neurology, internal medicine, surgery, emergency medicine, psychiatry, and pediatrics, along with an extensive color at-las supplement. Pros: Well organized by focus points: pathogenesis, epidemiology, management, complications, and associated diseases. Small, portable texts. Cons: Not comprehensive; best used as a supple-ment for review. Summary: Organized and easy-to-read clinical vi-gnettes. Excellent as a supplement to studying, but not sufficient by it-self. More economical to purchase the nine-volume set than individual volumes. $159.95 Review B+Step-Up to USMLE Step 2 VAN KLEUNEN Lippincott Williams & Wilkins, 2007, 2nd ed., 352 pages, ISBN 9780781771566 A Step 2 CK test review typical of the Step-Up series format, organized by system. Pros: A concise boards review

1	KLEUNEN Lippincott Williams & Wilkins, 2007, 2nd ed., 352 pages, ISBN 9780781771566 A Step 2 CK test review typical of the Step-Up series format, organized by system. Pros: A concise boards review resource with many tables or-ganizing the information and quick facts isolated in the page margins. Cons: Not very detailed, but covers most boards exam topics and serves as a good source of study organization. Summary: A good, com-prehensive review for the Step 2 CK exam with many quick-study fea-tures. $39.95 Review

1	B Lange Outline Review: USMLE Step 2 GOLDBERG McGraw-Hill, 2006, 5th ed., 650 pages, ISBN 9780071451925 A comprehensive boards review book with chapters organized by clini-cal discipline. Pros: A comprehensive review source with extensive coverage of clinical topics and an organized, in-depth review of each. Cons: Covers some low-yield topics. Includes relatively few images, figures, or tables. Summary: A solid, single-source, comprehensive re-view for the Step 2 CK exam. $41.95 Review B−USMLE Step 2 Made Ridiculously Simple CARL MedMaster, 2006, 3rd ed., 356 pages, ISBN 9780940780729 A general review of topics for the Step 2 CK exam. Presented in out-line format with tables and brief discussions. Pros: A quick review. Useful in areas that might otherwise be overlooked—e.g., ophthalmol-ogy, dermatology, and ENT. Helpful for last-minute review. Cons: The tabular format does not provide substantive details but aids in the memorization of lists. Some have found it too simple. Summary:

1	dermatology, and ENT. Helpful for last-minute review. Cons: The tabular format does not provide substantive details but aids in the memorization of lists. Some have found it too simple. Summary: Highlights most high-yield topics, but should not be used alone for re-view. $29.95 Review

1	Kaplanmedical.com A compilation of online programs for Step 2 CK review, including a large test bank. Pros: Questions can be arranged by topic or randomly to simulate the real exam. Tests are timed to simulate boards conditions. Includes an extensive number of questions in vignette format. The content level of questions reﬂects the boards test. Explanations are thorough, and the text now reports the national average for each question. Allows students to identify strong and weak points. Cons: Very expensive. Online lectures can be difficult to watch for extensive periods of time. Summary: A good source of questions with thorough explanations, but the price may be prohibitive for many. USMLERx.com

1	USMLERx.com Step 2 CK Qmax A test bank with more than 2600 questions. Pros: Written by students who have done well on the Step 2 CK exam; then reviewed by faculty experts. Offers highly comprehensive explanations. Cons: A newer resource that is still evolving. Summary: A great question bank source. New, but feedback from students has been excellent thus far. USMLEWorld.com A test bank with more than 2000 questions. Similar to the Kaplan test bank. Pros: Features well-written questions with explanations. Cheaper than Kaplan. Questions tend to be more difficult than those on the actual exam, but many students find this an advantage during preparation. Cons: Some questions are overly picky. Summary: An excellent source of questions that is cheaper than Kaplan. USMLEasy.com

1	USMLEasy.com A comprehensive test bank with more than 3300 questions and explanations. Similar in style to the Kaplan question bank described above. Pros: Features a large number of questions. Mimics the CBT format. Cheaper than Kaplan. Access is often free through medical libraries. Cons: Questions can be more obscure than those appearing on the actual exam. Questions overlap with those of the PreTest series of review books. Summary: A fair source of questions that may be good for supplemental review, especially in preparation for clerkship shelf exams.

1	A−Step-Up to Medicine AGABEGI Lippincott Williams & Wilkins, 2008, 2nd ed., 560 pages, ISBN 9780781771535 A comprehensive review of commonly tested diseases and topics in in-ternal medicine, organized in an outline format. Includes a color atlas and an appendix on interpreting x-rays, ECGs, and physical exam find-ings. Pros: Very comprehensive, with informative tables and diagrams to help synthesize information. Includes occasional clinical vignettes that correlate with the topic being discussed. Quick facts to remember are included in the margins of each page. Cons: Very lengthy. Geared more toward clerkship preparation than Step 2 CK review. Summary: A good book that is packed with useful information for the wards, but may be too lengthy and detailed for the Step 2 CK exam. $42.95 Review A−High-Yield Internal Medicine NIRULA Lippincott Williams & Wilkins, 2006, 3rd ed., 128 pages, ISBN 9780781781695 A core review of internal medicine in outline format. Pros: Focuses on high-yield

1	A−High-Yield Internal Medicine NIRULA Lippincott Williams & Wilkins, 2006, 3rd ed., 128 pages, ISBN 9780781781695 A core review of internal medicine in outline format. Pros: Focuses on high-yield diseases and symptoms. A quick and easy read. Cons: Not comprehensive. Lacks many illustrations and has no index. Summary: A good, fast review presented in a format that allows for quick and re-petitive reading. Best used as a supplement, not as a primary study source. $26.95 Review

1	First Aid for the Medicine Clerkship $42.95 Review McGraw-Hill, 2005, 2nd ed., 416 pages, ISBN 9780071448758 A high-yield review of symptoms and diseases. Pros: A comprehensive review that is well organized by symptom with good illustrations, scenarios, diagrams, algorithms, and mnemonics. Cons: May not be suited to readers who prefer information arranged in text form. May be too basic for certain topics. Summary: An excellent, concise review of medicine for those who prefer its format. Underground Clinical Vignettes: Emergency Medicine $22.95 Review

1	Underground Clinical Vignettes: Emergency Medicine $22.95 Review Lippincott Williams & Wilkins, 2007, 4th ed., 256 pages, ISBN 9780781768344 A clinical vignette–based review of emergency medicine topics. Pros: Well organized by focus points: pathogenesis, epidemiology, management, complications, and associated diseases. Well illustrated, and includes high-yield “minicases” and links to the Underground Clinical Vignettes’ Clinical and Basic Science Color Atlases. Cons: Not comprehensive; best used as a supplement. Summary: An organized and easy-to-read supplement to studying. Underground Clinical Vignettes: Internal Medicine, Vols. I and II Lippincott Williams & Wilkins, 2007, 4th ed., 256 pages each, ISBN 9780781768351, 9780781768368

1	Underground Clinical Vignettes: Internal Medicine, Vols. I and II Lippincott Williams & Wilkins, 2007, 4th ed., 256 pages each, ISBN 9780781768351, 9780781768368 A clinical vignette–based review of common topics in internal medicine. Pros: Well organized by focus points: pathogenesis, epidemiology, management, complications, and associated diseases. Vignettes mirror the boards-style presentation of questions. Cons: Not comprehensive; best used as a supplement. Summary: An organized and easy-to-read supplement to studying. Lippincott Williams & Wilkins, 2006, 2nd ed., 425 pages, ISBN 9781405104913

1	Lippincott Williams & Wilkins, 2006, 2nd ed., 425 pages, ISBN 9781405104913 A compendium of vignette-type cases arranged by symptom, followed by related questions and answers. Pros: An excellent companion to the Blueprints series. Focuses on high-yield cases. Easy to read with nice illustrations and a good review of management. Cons: Not comprehensive; use as a supplement for review. Better suited to clerkship preparation than to the Step 2 CK exam. Summary: An organized and easy-to-read supplement. Adds clinical correlates to the Blueprints series. Best used with the Blueprints text. Hanley & Belfus, 2004, 4th ed., 608 pages, ISBN 9781560533870 A question-and-answer text typical of the Secrets series. Pros: Covers a great deal of clinically relevant information. Concise answers are given with pearls, tips, and memory aids. Cons: Too lengthy and detailed for USMLE review. Summary: Not a focused review. May be more appropriate for wards use.

1	$22.95 Review each $29.95 Test/200 q $39.95 Review

1	B Medicine Recall BERGIN Lippincott Williams & Wilkins, 2007, 3rd ed., 832 pages, ISBN 9780781794145 A review book presented in standard Recall-series question-and-answer format, organized by medical specialty. Pros: Addresses a broad range of high-yield clinical topics. Presented in a format that is good for self-quizzing. Appropriate level of detail. Cons: Contains no vignettes or images; requires significant time commitment. Style simulates ques-tions asked on rounds, not those on the Step 2 CK exam. Summary: Written in a style that may be more conducive to wards than to boards preparation. Best used as a supplement to other resources. $39.95 Review B In A Page Emergency Medicine CATERINO Blackwell Publishing, 2003, 1st ed., 344 pages, ISBN 9781405103572 A collection of short, one-page summaries of 250 medical emergencies discussed in terms of etiology, differential diagnosis, presentation, diag-nostic tests, treatment, and disposition. Pros: Concise and high yield. Covers a wide

1	summaries of 250 medical emergencies discussed in terms of etiology, differential diagnosis, presentation, diag-nostic tests, treatment, and disposition. Pros: Concise and high yield. Covers a wide variety of emergencies seen in the ER. Cons: The text is crowded and somewhat confusing. Contains no images or diagrams. Summary: Good for use during the emergency medicine clerkship, but may not be appropriate for Step 2 CK review. $34.95 Review B Blueprints Clinical Cases in Family Medicine CHANG Lippincott Williams & Wilkins, 2006, 2nd ed., 437 pages, ISBN 9781405104951 A compendium of vignette-type cases arranged by symptom, followed by related questions and answers. Pros: An excellent companion to the Blueprints series. Focuses on high-yield cases. Easy to read with nice illustrations and a good review of management. Cons: Not compre-hensive; best used as a supplement for review. Summary: An organized and easy-to-read supplement. Adds clinical correlates to the Blueprints series. Best

1	a good review of management. Cons: Not compre-hensive; best used as a supplement for review. Summary: An organized and easy-to-read supplement. Adds clinical correlates to the Blueprints series. Best if used with the Blueprints text. $34.95 Test/200 q

1	B Internal Medicine Pearls HEFFNER Elsevier, 2001, 2nd ed., 249 pages, ISBN 9781560534044 One-page reviews of more than 200 diseases discussed by etiology, epi-demiology, signs/symptoms, differential diagnosis, diagnostic tests, treatment, and prognosis. Pros: A fast and concise review of high-yield information on common diseases. Cons: Text is crowded onto one page without any images or diagrams. Summary: Useful for quick study on the wards, but may not be comprehensive enough for the Step 2 CK exam. $41.95 Review B Blueprints Q & A Step 2 Medicine SHINAR Lippincott Williams & Wilkins, 2004, 2nd ed., 276 pages, ISBN 9781405103893 Two hundred vignette-style questions. Pros: A nice companion to the Blueprints series. Focuses on high-yield topics. Explanations are easy to follow. Cons: Not comprehensive; best used as a supplement for re-view. Expensive; includes few questions given the cost of the book. Summary: An organized and easy-to-read supplement. Adds clinical correlates to the

1	comprehensive; best used as a supplement for re-view. Expensive; includes few questions given the cost of the book. Summary: An organized and easy-to-read supplement. Adds clinical correlates to the Blueprints series. $19.95 Test/200 q B First Aid for the Emergency Medicine Clerkship STEAD McGraw-Hill, 2006, 2nd ed., 416 pages, ISBN 9780071448734 A high-yield review of symptoms and diseases. Pros: A comprehensive review; well organized by symptom with good illustrations, scenarios, diagrams, algorithms, and mnemonics. Cons: May not be suited to the reader who prefers information arranged in text form. Summary: An excellent review of emergency medicine and a nice presentation of high-yield topics for Step 2 CK preparation, but not sufficient for stand-alone review for the Step 2 CK exam. $42.95 Review B Blueprints in Medicine YOUNG Lippincott Williams & Wilkins, 2009, 5th ed., 448 pages, ISBN 9780781788700 A text review of internal medicine, organized by common diseases and common

1	$42.95 Review B Blueprints in Medicine YOUNG Lippincott Williams & Wilkins, 2009, 5th ed., 448 pages, ISBN 9780781788700 A text review of internal medicine, organized by common diseases and common symptoms. Includes a question-and-answer section with ex-planations. Pros: A well-organized, concise review that makes for easy reading. Differential diagnoses for symptoms are helpful. Contains good charts and diagrams. Cons: Offers few illustrations. Contains some superﬂuous details, and some areas are too broad and simplistic to be useful for testing purposes. Summary: Poorly illustrated, but a good primary boards review for internal medicine. $38.95 Review/Test/100 q

1	PreTest Medicine $26.95 Test/500 q McGraw-Hill, 2006, 11th ed., 356 pages, ISBN 9780071455534 A question-and-answer format organized by medical subspecialty. Pros: Organization by subspecialty helps readers pinpoint weak areas. Offers a substantial number of vignette-style questions with detailed explanations. Cons: Many questions are more detailed than needed for the boards and are geared more toward the shelf exam. Few illustrations. Summary: A solid source of challenging review questions.

1	A−Blueprints in Neurology DRISLANE Lippincott Williams & Wilkins, 2009, 3rd ed., 256 pages, ISBN 9780781796859 A review of neurology by disease and symptom with a brief exam. Pros: Reviews high-yield topics of a complex discipline while remaining easy to follow. Makes good use of tables, images, and diagrams. Questions in the exam are similar to those found on both the shelf exam and Step 2 CK. Cons: Lengthy. Summary: An excellent review of high-yield material for the wards and the Step 2 CK exam. $37.95 Review B+PreTest Neurology ANSCHEL McGraw-Hill, 2009, 7th ed., 352 pages, ISBN 9780071597920 A question-and-answer review of neurology. Pros: Offers thorough cov-erage of neurology topics with a good number of clinical vignettes. Places appropriate emphasis on common topics and thorough explana-tions of answers. Good practice for interpreting common head CTs/ MRIs that might be tested. Cons: Some questions may be more de-tailed than needed for the boards. Summary: A good source of

1	explana-tions of answers. Good practice for interpreting common head CTs/ MRIs that might be tested. Cons: Some questions may be more de-tailed than needed for the boards. Summary: A good source of test questions for rapid review of neurology, but may be too detailed for boards review. $26.95 Test/500 q

1	B+Underground Clinical Vignettes: Neurology KIM Lippincott Williams & Wilkins, 2007, 4th ed., 256 pages, ISBN 9780781768375 A clinical vignette–based review of high-yield topics in neurology. Pros: Well organized by focus points: pathogenesis, epidemiology, management, complications, and associated diseases. Well illustrated, and includes “minicases,” links to a color atlas supplement, and up-dated treatments. Cons: Not comprehensive; best used as a supple-ment for review. Summary: An organized and easy-to-read supplement to studying. Lengthy for a dedicated review of neurology. $22.95 Review B Neurology Recall MILLER Lippincott Williams & Wilkins, 2003, 2nd ed., 377 pages, ISBN 9780781745888 Brief question-and-answer format. Pros: Reviews many important facts, making it useful for self-quizzing. Cons: Not a comprehensive review. Lengthy and lacks illustrations. Concepts are not integrated. Sum-mary: Good for review of some high-yield concepts, but not a stand-alone resource for this

1	Cons: Not a comprehensive review. Lengthy and lacks illustrations. Concepts are not integrated. Sum-mary: Good for review of some high-yield concepts, but not a stand-alone resource for this topic. $36.95 Review

1	B Neurology Secrets ROLAK Elsevier, 2004, 4th ed., 480 pages, ISBN 9781560536215 A review book presented in Secrets-series question-and-answer format. Pros: A concise review of many high-yield topics. Makes good use of clinical images. Cons: Contains no clinical vignettes; instead, offers lists of questions that might be posed on the wards. Lacks a structured format, and leaves out important information. Relatively expensive and lengthy. Not a reference book. Summary: Overall, good content for self-quizzing and study, but does not substitute for a formal review or practice tests. More appropriate for clerkship than for boards review. $45.95 Review B Blueprints Clinical Cases in Neurology SHETH Lippincott Williams & Wilkins, 2006, 2nd ed., 390 pages, ISBN 9781405104944 Vignette-type cases organized by symptom, followed by related ques-tion and answers. Pros: An excellent companion to the Blueprints sub-specialty series. Focuses on high-yield cases. Easy to read, with nice

1	cases organized by symptom, followed by related ques-tion and answers. Pros: An excellent companion to the Blueprints sub-specialty series. Focuses on high-yield cases. Easy to read, with nice il-lustrations and a good review of management. Cons: Not comprehensive; best used as a supplement. Offers few illustrations. Summary: Organized and easy to read. Adds clinical correlates to the Blueprints series. $29.95 Review

1	A−Blueprints in Obstetrics and Gynecology CALLAHAN Lippincott Williams & Wilkins, 2008, 5th ed., 368 pages, ISBN 9780781782494 A text review with tables and illustrations. Includes a short exam with explanations. Pros: Places strong emphasis on high-yield topics with concise text, clear diagrams, and many classic illustrations. Makes for easy reading. Appropriate for both clinical clerkship and Step 2 CK preparation. Cons: Some topics are overly detailed, while some are not detailed enough. Summary: Overall, a good choice for boards and wards preparation. $38.95 Review/Test/100 q A−Blueprints Clinical Cases in Obstetrics and Gynecology CAUGHEY Lippincott Williams & Wilkins, 2006, 2nd ed., 418 pages, ISBN 9781405104906 Vignette-type cases arranged by symptom, followed by related ques-tions and answers. Pros: An excellent companion to the Blueprints se-ries. Focuses on high-yield cases. Easy to read, with nice illustrations and a good review of management. Cons: Not comprehensive; best

1	and answers. Pros: An excellent companion to the Blueprints se-ries. Focuses on high-yield cases. Easy to read, with nice illustrations and a good review of management. Cons: Not comprehensive; best used as a supplement. Summary: An organized and easy-to-read sup-plement. Adds clinical correlates to the Blueprints series. $29.95 Test/200 q A−

1	Underground Clinical Vignettes: OB/GYN $22.95 Review Lippincott Williams & Wilkins, 2007, 4th ed., 256 pages, ISBN 9780781768405 A clinical vignette–style review of frequently tested diseases in obstetrics and gynecology. Pros: Well organized by focus points: pathogenesis, epidemiology, management, complications, and associated diseases. Well illustrated. An easy read that stresses high-yield facts. Cons: Not comprehensive; best used as a supplement. Summary: Wellorganized and easy-to-read practice vignettes.

1	B+NMS Obstetrics and Gynecology PFEIFER Lippincott Williams & Wilkins, 2007, 6th ed., 496 pages, ISBN 9780781770712 A detailed outline of OB/GYN with few tables and diagrams. Pros: A comprehensive review for both wards and boards. The final exam is relatively good and offers complete explanations. Cons: The OB/GYN review is dense and lengthy. Many questions do not reﬂect the boards format. Lacks illustrations. Summary: A complete review with ques-tions and discussions. Better for clerkship studying than for boards re-view. $48.95 Review/Test/500 q B+High-Yield Obstetrics and Gynecology SAKALA Lippincott Williams & Wilkins, 2005, 2nd ed., 208 pages, ISBN 9780781796309 A review of high-yield topics in outline format. Clinical scenarios at the end of each chapter highlight key points. Pros: Easy to read, with a good discussion of high-yield topics. Cons: Lacks depth and contains no practice questions. Summary: A quick but superficial review. $26.95 Review B+B+

1	First Aid for the OB/GYN Clerkship McGraw-Hill, 2006, 2nd ed., 304 pages, ISBN 9780071448741 A high-yield review of symptoms and diseases. Pros: A comprehensive review with nice diagrams, images, charts, algorithms, and mnemonics. Cons: Lengthy review. Summary: An excellent review of OB/ GYN, but too lengthy for boards review. Case Files: Obstetrics and Gynecology McGraw-Hill, 2009, 3rd ed., 456 pages, ISBN 9780071605809 A review of OB/GYN in case format with questions and answers following each vignette. Pros: Cases reﬂect high-yield topics and are arranged in an easy-to-follow format. Cons: Some topics are either not covered or given only brief treatment. Contains few diagrams and images. Lengthy and time-consuming for one topic. Explanations are terse. Summary: A good review of the subject in clinical vignette format, but may be too detailed for Step 2 CK review. $42.95 Review $32.95 Review

1	B+Blueprints Q & A Step 2 Obstetrics & Gynecology TRAN Lippincott Williams & Wilkins, 2004, 2nd ed., 168 pages, ISBN 9781405103909 One hundred vignette-style questions. Pros: A nice companion to the Blueprints series. Focuses on high-yield topics. Explanations are easy to follow. Cons: Not comprehensive; best used as a supplement for re-view. Sparse images. Some questions are esoteric and not boards-like. Summary: A well-organized and easy-to-read supplement. Adds clini-cal correlates to the Blueprints series. $19.95 Test/200 q B Obstetrics and Gynecology Secrets BADER Mosby, 2004, 3rd ed., 448 pages, ISBN 9780323034159 A review book presented in Secrets-series question-and-answer format, organized by topic within OB/GYN. Pros: Offers good coverage of many high-yield, clinically relevant topics. Cons: Too detailed to be useful for rapid review. Contains no vignettes and few illustrations or images. Summary: Good clinical content, but does not serve as a for-mal topic review. Better

1	topics. Cons: Too detailed to be useful for rapid review. Contains no vignettes and few illustrations or images. Summary: Good clinical content, but does not serve as a for-mal topic review. Better for use during clerkship than for Step 2 CK preparation. $31.95 Review B PreTest Obstetrics and Gynecology SCHNEIDER McGraw-Hill, 2009, 12th ed., 355 pages, ISBN 9780071599795 A question-and-answer review with detailed explanations for OB/GYN. Pros: Organization by subtopic may be useful for studying weak areas. Good content emphasis. Generally well illustrated. Cons: Some ques-tions are too difficult or detailed. Vignette-based questions are shorter and more simplistic than those on the Step 2 CK. Summary: A decent source of questions to supplement topic study, especially for addressing specific areas of weakness. $26.95 Test/500 q B−Obstetrics and Gynecology Recall BOURGEOIS Lippincott Williams & Wilkins, 2007, 3rd ed., 608 pages, ISBN 9780781770699 A review book presented in standard

1	areas of weakness. $26.95 Test/500 q B−Obstetrics and Gynecology Recall BOURGEOIS Lippincott Williams & Wilkins, 2007, 3rd ed., 608 pages, ISBN 9780781770699 A review book presented in standard Recall-series question-and-answer style. Pros: The two-column format makes the text useful for self-quiz-zing. Reviews many high-yield concepts and facts. Cons: Questions emphasize individual facts but do not integrate concepts. Contains no vignettes or images. Coverage of some topics is spotty. Summary: Use-ful for review of selected concepts, but not a comprehensive source for Step 2 CK preparation. More appropriate for clerkship than for boards. $36.95 Review/Test/350 q

1	A−Platinum Vignettes: Pediatrics BROCHERT Hanley & Belfus, 2002, 1st ed., 104 pages, ISBN 9781560535331 A clinical vignette–based review of common topics in pediatrics. Pros: Well-written cases are similar to boards-type vignettes. Well illustrated. The discussion is organized by pathophysiology, diagnosis and treat-ment, and more high-yield facts. Cons: Expensive for the amount of material. Not comprehensive; best used as a supplement. Has not been updated since 2002. Summary: An organized and easy-to-read supplement to studying. $29.95 Review A−Underground Clinical Vignettes: Pediatrics KIM Lippincott Williams & Wilkins, 2007, 4th ed., 256 pages, ISBN 9780781768443 A clinical vignette review of frequently tested topics in pediatrics. Pros: Well organized by focus points: pathogenesis, epidemiology, manage-ment, complications, and associated diseases. Well illustrated, and the new edition includes “minicases” to broaden subject material and present more high-yield information. Cons:

1	manage-ment, complications, and associated diseases. Well illustrated, and the new edition includes “minicases” to broaden subject material and present more high-yield information. Cons: Not comprehensive; best used as a supplement to text review. Summary: Well organized and easy to read, but intended as a supplement for review. $22.95 Review A−

1	PreTest Pediatrics $26.95 Test/500 q McGraw-Hill, 2006, 11th ed., 464 pages, ISBN 9780071455527 A question-and-answer review with detailed discussion. Pros: Organization by organ system is useful for pinpointing weaknesses. Gives strong, thorough explanations. Includes a fair number of vignette-style questions. Well illustrated. Cons: Some questions are too detailed or emphasize low-yield topics. Summary: A good source of questions and review for pediatrics. Solid content with good illustrations, although not entirely in Step 2 CK format.

1	B+Blueprints Q & A Step 2 Pediatrics FOTI Lippincott Williams & Wilkins, 2004, 2nd ed., 176 pages, ISBN 9781405103916 Vignette-style questions. Pros: A nice companion to the Blueprints se-ries. Focuses on high-yield topics. Explanations are easy to follow. Cons: Not comprehensive; best used as a supplement for review. Sparse images. Summary: An organized and easy-to-read supplement. Adds clinical correlates to the Blueprints series. $17.95 Test/100 q B+In A Page Pediatrics KAHAN Lippincott Williams & Wilkins, 2008, 2nd ed., 384 pages, ISBN 9780781770453 One-page reviews of 228 diseases/topics discussed by etiology, epide-miology, signs/symptoms, differential diagnosis, diagnostic tests, treat-ment, and prognosis. Pros: A fast and concise review of high-yield in-formation on common diseases. Cons: Each topic is crowded onto one page without any images or diagrams. Includes low-yield topics. Sum-mary: Useful for quick study on the wards, but too time intensive for Step 2 CK review.

1	Cons: Each topic is crowded onto one page without any images or diagrams. Includes low-yield topics. Sum-mary: Useful for quick study on the wards, but too time intensive for Step 2 CK review. $34.95 Review B+Blueprints Clinical Cases in Pediatrics LONDHE Lippincott Williams & Wilkins, 2006, 2nd ed., 426 pages, ISBN 9781405104920 Vignette-type cases arranged by symptom followed by related questions and answers. Pros: An excellent companion to the Blueprints series. Focuses on high-yield cases. Easy to read with nice illustrations and a good review of management. Cons: Not comprehensive; best used as a supplement for review. Summary: An organized and easy-to-read supplement. Adds clinical correlates to the Blueprints series. $29.95 Test/200 q B+Blueprints in Pediatrics MARINO Lippincott Williams & Wilkins, 2008, 5th ed., 384 pages, ISBN 9780781782517 A text review of pediatrics with tables and diagrams. Includes a ques-tion-and-answer section with explanations. Pros: Appropriate focus

1	& Wilkins, 2008, 5th ed., 384 pages, ISBN 9780781782517 A text review of pediatrics with tables and diagrams. Includes a ques-tion-and-answer section with explanations. Pros: Appropriate focus on high-yield topics. Cons: A relatively dense text with few illustrations. Overly detailed. Summary: Good for a more comprehensive review. $38.95 Review/Test/100 q

1	B+Case Files: Pediatrics TOY McGraw-Hill, 2006, 2nd ed., 576 pages, ISBN 9780071463027 A review of pediatrics in case format with questions and answers fol-lowing each vignette. Pros: Cases reﬂect high-yield topics and are ar-ranged in an easy-to-follow format. Emphasizes the next step and the most likely diagnosis. Cons: Not suited for high-yield rapid review. Summary: An excellent review with emphasis on vignette-style case presentation and important boards-type answers, but may be too de-tailed for a stand-alone boards review book. Excellent for clerkship preparation. $29.95 Review B+A&L’s Review of Pediatrics VIESSMAN McGraw-Hill, 2004, 6th ed., 250 pages, ISBN 9780838503034 A question-and-answer review of pediatrics with detailed explanations. Pros: Questions focus on boards-relevant content. The last chapter in-cludes excellent vignette-based questions. Offers thorough, wellwritten explanations. Includes a nice primer on test-taking strategies. Cons: Non-vignette-based questions

1	content. The last chapter in-cludes excellent vignette-based questions. Offers thorough, wellwritten explanations. Includes a nice primer on test-taking strategies. Cons: Non-vignette-based questions are shorter and more straightfor-ward than those on the Step 2 CK exam. Some questions may be too detailed for Step 2 CK preparation. Poorly illustrated. Summary: An excellent, concise review with appropriate content and good discus-sions, but the majority of questions do not reﬂect Step 2 CK style. $41.95 Test/1000+ q B−NMS Pediatrics DWORKIN Lippincott Williams & Wilkins, 2008, 5th ed., 480 pages, ISBN 9780781770750 A general review of pediatrics in outline format. Includes questions at the end of each chapter. Pros: A thorough, detailed review of pediat-rics. Boldfacing highlights key points. Case studies and a comprehen-sive exam at the end of the book (also provided on CD-ROM) are helpful. Includes a good discussion. Cons: A dense, lengthy text. Lacks good illustrations of any kind.

1	Case studies and a comprehen-sive exam at the end of the book (also provided on CD-ROM) are helpful. Includes a good discussion. Cons: A dense, lengthy text. Lacks good illustrations of any kind. Summary: A thorough review, but more appropriate for clerkships than for Step 2 CK review, in large part be-cause of its depth. $42.95 Review/Test/100+ q

1	Pediatrics Recall $36.95 Review Lippincott Williams & Wilkins, 2007, 3rd ed., 508 pages, ISBN 9780781771184 A review book presented in a concise question-and-answer format typical of the Recall series. Pros: The two-column format makes self-quizzing easy. Emphasis is placed on diagnosis and management. Cons: Requires time commitment, and not all topics are covered thoroughly. Contains no vignettes. Summary: Useful material, but does not provide a systematic review or a substitute for practice tests. Better suited to clerkship review. Pediatric Secrets $39.95 Review Elsevier, 2005, 4th ed., 688 pages, ISBN 9781560536277 A question-and-answer text typical of the Secrets series, organized by pediatric subspecialty. Pros: Includes a thorough discussion of a wide variety of clinical topics. Cons: Detailed content is geared toward the wards and requires significant time investment. Contains no images or illustrations. Summary: Too detailed for USMLE review; better suited to clerkship.

1	A Blueprints in Psychiatry MURPHY Lippincott Williams & Wilkins, 2007, 2nd ed., 304 pages, ISBN 9781405104968 A brief text review of psychiatry with DSM-IV criteria. Includes a brief question-and-answer section at the end of the book. Pros: A clear, con-cise review of psychiatry with helpful tables. Offers good coverage of high-yield topics, including pharmacology. A quick read. Cons: Too general in certain areas; requires some supplementation. Summary: A rapid review with appropriate coverage of high-yield topics. $29.95 Review/Test/74 q A−Blueprints Clinical Cases in Psychiatry HOBLYN Lippincott Williams & Wilkins, 2007, 2nd ed., 304 pages, ISBN 9781405104968 Vignette-type cases arranged by symptom, followed by related ques-tions and answers. Pros: An excellent companion to the Blueprints se-ries. Focuses on high-yield cases. Easy to read with nice illustrations and a good review of management. Cons: Not comprehensive; best used as a supplement for review. Summary: An organized and

1	se-ries. Focuses on high-yield cases. Easy to read with nice illustrations and a good review of management. Cons: Not comprehensive; best used as a supplement for review. Summary: An organized and easy-to-read supplement. Adds clinical correlates to the Blueprints series. $29.95 Test/200 q

1	Underground Clinical Vignettes: Psychiatry $22.95 Review Lippincott Williams & Wilkins, 2007, 4th ed., 256 pages, ISBN 9780781768467 A clinical vignette–based review of frequently tested topics in psychiatry. Pros: Well organized by focus points: pathogenesis, epidemiology, management, and associated diseases. Well illustrated, and includes “minicases” that present high-yield information. Cons: Not comprehensive; best used as a supplement. Summary: Offers organized and easy-to-read practice vignettes. PreTest Psychiatry $26.95 Test/500 q

1	PreTest Psychiatry $26.95 Test/500 q McGraw-Hill, 2006, 11th ed., 355 pages, ISBN 9780071455541 A question-and-answer review of topics in psychiatry. Pros: Questions are well written and organized. Most questions have appropriate content level. Offers good explanations. Cons: Includes too few vignette-type questions. Some questions are too detailed. Summary: A good source of questions and review for psychiatry and the Step 2 CK exam, although the format may not reﬂect the actual test. Lippincott Williams & Wilkins, 2003, 2nd ed., 150 pages, ISBN 9780781742689 A brief outline-format review of psychiatry. Pros: A quick read, with clinical vignettes scattered throughout. Offers concise tables. Cons: Not sufficiently detailed for in-depth review. Summary: An excellent, quick review of psychiatry for use as an additional study source. Similar to High-Yield Behavioral Sciences by the same author. A&L’s Review of Psychiatry

1	A&L’s Review of Psychiatry McGraw-Hill, 2003, 7th ed., 304 pages, ISBN 9780071402538 A general review of psychiatry with questions and answers. Pros: Includes 114 vignette-style questions appropriate for boards review. Appropriate content emphasis; thorough explanations. The new edition features updated treatment and management sections. Cons: Questions are shorter and more straightforward than those of the boards. Summary: A decent source of boards review for psychiatry, but does not reﬂect boards format. Case Files: Psychiatry McGraw-Hill, 2006, 2nd ed., 408 pages, ISBN 9780071462822

1	Case Files: Psychiatry McGraw-Hill, 2006, 2nd ed., 408 pages, ISBN 9780071462822 A review of psychology in case format with questions and answers following each vignette. Pros: Cases reﬂect high-yield topics and are arranged in an easy-to-follow format. Emphasizes the next step, the most likely diagnosis, and the best initial treatment. Cons: Not suited to high-yield rapid review, and may be too detailed for Step 2 CK review. Summary: An excellent subject review that places emphasis on vignette-style case presentation and important boards-type answers. Great for the wards, and a good supplement for the boards. $28.95 Review $34.95 Test/900+ q $29.95 Review

1	REVIEW RESOURCESB Platinum Vignettes: Psychiatry BROCHERT Hanley & Belfus, 2002, 1st ed., 102 pages, ISBN 9781560535348 A clinical vignette review of common topics in psychiatry. Pros: Well-written cases are similar to boards-type vignettes. Well illustrated. Dis-cussion is organized by pathophysiology, diagnosis and treatment, and more high-yield facts. Cons: Not comprehensive; best used as a sup-plement. Summary: An organized and easy-to-read supplement to studying. Needs updating. $29.95 Review B Blueprints Q & A Step 2 Psychiatry MCLOONE Lippincott Williams & Wilkins, 2004, 2nd ed., 96 pages, ISBN 9781405103923 Vignette-style questions. Pros: A nice companion to the Blueprints se-ries. Focuses on high-yield topics. Explanations are easy to follow. Cons: Not comprehensive; best used as a supplement for review. Sparse images. Some questions are esoteric and not boards-like. Sum-mary: An organized and easy-to-read supplement. Adds clinical corre-lates to the Blueprints series. $17.95

1	a supplement for review. Sparse images. Some questions are esoteric and not boards-like. Sum-mary: An organized and easy-to-read supplement. Adds clinical corre-lates to the Blueprints series. $17.95 Test/200 q B NMS Psychiatry SCULLY Lippincott Williams & Wilkins, 2007, 5th ed., 300 pages, ISBN 9780781765145 A general review of topics in outline format with questions at the end of each chapter and a comprehensive final exam. Pros: A well-written text with concise disease discussions. Includes an expanded pharma-cology section. Questions test appropriate content and have complete explanations, and the new edition offers more vignette-style questions. A good companion text for the clerkship. Cons: Does not contain enough vignette-style questions. Lengthy for purposes of boards review. Summary: A detailed review that requires time commitment. A good single choice for clerkship study, but may be too long for Step 2 CK re-view. $42.95 Review/Test/500 q

1	First Aid for the Psychiatry Clerkship $42.95 Review McGraw-Hill, 2005, 2nd ed., 208 pages, ISBN 9780071448727 A high-yield review of symptoms and diseases. Pros: A comprehensive review that includes DSM-IV criteria with nice mnemonics and scenarios. Includes high-yield tear-out cards. Cons: May not appeal to readers who prefer information in text format. Summary: A good review of high-yield topics in psychiatry, but better suited to clerkship than to Step 2 CK study.

1	B−Psychiatry Recall FADEM Lippincott Williams & Wilkins, 2009, 3rd ed., 210 pages, ISBN 9780781776981 A review book presented in the quick question-and-answer format typi-cal of the Recall series. Pros: The two-column format is conducive to self-quizzing. Covers many high-yield facts and concepts necessary for the USMLE. Cons: Lacks vignettes, so does not substitute for practice tests. Summary: Requires time commitment. Some topics are glossed over. Best used as a supplement to other resources. $36.95 Review B−Psychiatry Made Ridiculously Simple GOOD MedMaster, 2005, 4th ed., 98 pages, ISBN 9780940780682 Part of the “Made Ridiculously Simple” series. Pros: A comprehensive, fast read with nice tables and entertaining illustrations to highlight key points. Cons: Some areas are not detailed enough, while others are too verbose. Not boards oriented. Summary: A good, fast review, but more helpful for clerkship than for the boards. $13.95 Review B−Psychiatric Secrets JACOBSON Hanley &

1	enough, while others are too verbose. Not boards oriented. Summary: A good, fast review, but more helpful for clerkship than for the boards. $13.95 Review B−Psychiatric Secrets JACOBSON Hanley & Belfus, 2000, 2nd ed., 536 pages, ISBN 9781560534181 A review book presented in the question-and-discussion format typical of the Secrets series, organized by topic. Pros: Offers clear explanations of important concepts in psychiatry. Makes for good wards reading. Cons: Too detailed and lengthy for review purposes. Lacks vignettes. Summary: Requires significant time commitment; not for rapid, fo-cused review. Best suited to clerkship review. $39.95 Review B−BRS Psychiatry SHANER Lippincott Williams & Wilkins, 2000, 2nd ed., 419 pages, ISBN 9780683307665 A comprehensive review of psychiatry in outline format. Vignette-style questions follow each chapter. Pros: A thorough, systematic review of clinical psychiatry. Clear, concise definitions are provided. Cons: Great deal of information for

1	outline format. Vignette-style questions follow each chapter. Pros: A thorough, systematic review of clinical psychiatry. Clear, concise definitions are provided. Cons: Great deal of information for single-topic review. The pharmacology section is out of date. Summary: Good material, but requires a large time investment; needs updating. $37.95 Review/400 q

1	A−Underground Clinical Vignettes: Surgery KIM Lippincott Williams & Wilkins, 2007, 4th ed., 256 pages, ISBN 9780781768474 A clinical vignette–based review of frequently tested surgical topics. Pros: Well organized by focus points: pathogenesis, epidemiology, management, complications, and associated diseases. Well illustrated and includes “minicases” that present high-yield information. Cons: Not comprehensive; best used as a supplement to review. Summary: Well-organized and easy-to-read practice vignettes. $22.95 Review A−Case Files: Surgery TOY McGraw-Hill, 2006, 2nd ed., 504 pages, ISBN 9780071463041 A review of surgery in case format with questions and answers follow-ing each vignette. Pros: Cases reﬂect high-yield topics and are ar-ranged in an easy-to-follow format. Emphasizes the next step and the most likely diagnosis. Cons: Not suited to high-yield rapid review, and may be too detailed for Step 2 CK preparation. Summary: An excel-lent review with emphasis on vignette-style

1	next step and the most likely diagnosis. Cons: Not suited to high-yield rapid review, and may be too detailed for Step 2 CK preparation. Summary: An excel-lent review with emphasis on vignette-style case presentation and im-portant boards-type answers. Great for clerkship study, and a good sup-plement for boards study. $29.95 Review

1	B+Platinum Vignettes: Surgery and Trauma BROCHERT Hanley & Belfus, 2002, 1st ed., 102 pages, ISBN 9781560535355 A clinical vignette–based review of common topics in surgery and trauma medicine. Pros: Well-written cases are similar to boards-type vi-gnettes. Well illustrated. Discussion is organized by pathophysiology, diagnosis and treatment, and more high-yield facts. Cons: Expensive for the amount of material. Not comprehensive; best used as a supple-ment. Summary: An organized and easy-to-read supplement to study-ing. $29.95 Review B+PreTest Surgery KAO McGraw-Hill, 2009, 12th ed., 373 pages, ISBN 9780071598637 A review of topics in general surgery in question-and-answer format. Pros: Predominantly case based. Well organized by subspecialty. Cons: Many questions are too detailed or esoteric and do not reﬂ ect boards style. Some explanations are overly detailed. Summary: A thorough re-view, but questions may be beyond the level needed for Step 2 CK preparation. $26.95 Test/500 q

1	B NMS Surgery JARRELL Lippincott Williams & Wilkins, 2007, 5th ed., 645 pages, ISBN 9780781759014 An outline review of general surgery and surgical subspecialties. Pros: Well organized and thorough. Vignette-style questions are included af-ter each chapter with good explanations. Cons: Dense, detailed text. Few tables or illustrations. Summary: A comprehensive surgery review, but very time consuming. More appropriate for clerkship than for boards review. $42.95 Review/Test/350 q B In A Page Surgery KAHAN Lippincott Williams & Wilkins, 2003, 1st ed., 288 pages, ISBN 9781405103657 One-page reviews of diseases/topics discussed by etiology, epidemiol-ogy, signs/symptoms, differential diagnosis, diagnostic tests, treatment, and prognosis. Pros: A fast and concise review of high-yield informa-tion on common diseases. Cons: Text is crowded onto one page with-out any images or diagrams. Includes low-yield topics. Summary: Use-ful for quick study on the wards, but too time intensive for Step 2

1	on common diseases. Cons: Text is crowded onto one page with-out any images or diagrams. Includes low-yield topics. Summary: Use-ful for quick study on the wards, but too time intensive for Step 2 CK review. $34.95 Review

1	Blueprints in Surgery $38.95 Review/Test/100 q Lippincott Williams & Wilkins, 2009, 5th ed., 320 pages, ISBN 9780781788687 A short text review of general surgery with tables and diagrams. A brief question-and-answer section is included. Pros: Well organized. Easy to read, with a strong focus on high-yield topics. Includes clear diagrams. Cons: Some sections are overly detailed (e.g., anatomy), while others are occasionally too simplistic. Too few illustrations. Summary: A good review of surgery, but not ideal for Step 2 CK preparation. Blueprints Clinical Cases in Surgery $29.95 Test/200 q Lippincott Williams & Wilkins, 2006, 2nd ed., 304 pages, ISBN 9781405104937

1	Blueprints Clinical Cases in Surgery $29.95 Test/200 q Lippincott Williams & Wilkins, 2006, 2nd ed., 304 pages, ISBN 9781405104937 Vignette-type cases arranged by symptom followed by related questions and answers. Pros: A excellent companion to the Blueprints series. Focuses on high-yield cases. Easy to read with nice illustrations and a good review of management. Cons: Not comprehensive; best used as a supplement for review. Summary: An organized and easy-to-read supplement. Adds clinical correlates to the Blueprints series.

1	B High-Yield Surgery NIRULA Lippincott Williams & Wilkins, 2005, 2nd ed., 160 pages, ISBN 9780781776561 An outline review of most common general surgery topics. Pros: Con-cise; useful for quick topic review. Well organized. Cons: Information can be superficial. Some topics are omitted. Offers no practice ques-tions. Summary: A lean text for rapid review. $26.95 Review B A&L’s Review of Surgery WAPNICK McGraw-Hill, 2003, 4th ed., 320 pages, ISBN 9780071378147 A general review of surgery with questions and answers. Pros: Good clinical emphasis. Includes many vignette-style questions. Explana-tions are thorough. Cons: Some questions are too short, and the style does not reﬂect that of the Step 2 CK exam. Questions are highly vari-able in difficulty and are often far too detailed. Offers few illustrations. Summary: Good content for the exam, but much too detailed for clerkship and Step 2 CK review. $34.95 Test/1000+ q B−Surgical Recall BLACKBOURNE Lippincott Williams & Wilkins, 2008, 5th

1	illustrations. Summary: Good content for the exam, but much too detailed for clerkship and Step 2 CK review. $34.95 Test/1000+ q B−Surgical Recall BLACKBOURNE Lippincott Williams & Wilkins, 2008, 5th ed., 800 pages, ISBN 9780781770767 A review book presented in standard Recall-series question-and-answer format. Pros: Questions emphasize important, high-yield clinical con-cepts. Columns allow for self-testing. Fast review. Good preparation for “pimping” on rounds. Cons: Does not feature boards-type questions. Poorly organized. Coverage of some topics is spotty. Summary: A use-ful adjunct to a more organized topic review. Much more appropriate for clerkship than for boards review. $44.95 Review BAbernathy’s Surgical Secrets HARKEN Elsevier, 2008, 6th ed., 534 pages, ISBN 9780323057110 A review book presented in a question-and-answer format typical of the Secrets series. Pros: Discussions are up to date and thorough. Cons: Too detailed for the purposes of the Step 2 CK, yet not

1	A review book presented in a question-and-answer format typical of the Secrets series. Pros: Discussions are up to date and thorough. Cons: Too detailed for the purposes of the Step 2 CK, yet not comprehen-sive. Summary: Not a well-organized review. Better suited to clerkship than to boards preparation. $47.95 Review−

1	Pocket Surgery $36.95 Review Lippincott Williams & Wilkins, 2001, 1st ed., 160 pages, ISBN 9780781735797 A review of high-yield surgical material in outline format. Pros: A fast, easy read. Portable. Highlights high-yield information in “fact boxes.” Cons: Some material is not detailed enough. Offers no illustrations. Summary: Good for rapid review during clerkship. Does not contain enough detailed information to be used as a single study source for the boards. First Aid for the International Medical Graduate $39.95 Review McGraw-Hill, 2002, 2nd ed., 313 pages, ISBN 9780071385329 A high-yield review for the IMG on how to pass the USMLE boards and adapt to medical culture in the United States. Pros: A comprehensive, well-organized review. Cons: Some readers may need to obtain additional information from other sources. Immigration and visa information is outdated. Summary: An excellent review of material for the IMG. Best used as a primer for boards review.

1	Commercial preparation courses can be helpful for some students, but such courses are typically expensive and require significant time commitment. They are usually effective in organizing study material for students who feel overwhelmed by the sheer volume of material involved in Step 2 CK preparation. Note that multiweek courses may be quite intense and may thus leave limited time for independent study. Also note that some commercial courses are designed for first-time test takers while others focus on students who are repeating the exam. In addition, some courses are geared toward IMGs who want to take all three Steps in a limited amount of time.

1	Student experience and satisfaction with review courses are highly variable. We suggest that you discuss options with recent graduates of the review courses you are considering. In addition, course content and structure can change rapidly. Some student opinions can be found in discussion groups on the World Wide Web. Listed below is contact information for some Step 2 CK commercial review courses. 440 Wrangler Drive, Suite 100 Coppell, TX 75019 (214) 632-5466 Fax: (214) 292.8568 info@falconreviews.com www.falconreviews.com 700 South Flower Street Los Angeles, CA 90017 (800) KAP-TEST (800-527-8378) www.kaptest.com P.O. Box 22174 East Lansing, MI 48909-2174 (866) MedPass (866-633-7277) Fax: (517) 347-7005 contactus@northwesternmedicalreview.com http://northwesternmedicalreview.com 1909 Tyler Street, Suite 305 Hollywood, FL 33020 (877) 662-2005 Fax: (954) 926-3333 sales@pmre.com www.pmre.com Youel’s Prep, Inc.

1	Youel’s Prep, Inc. P.O. Box 31479 Palm Beach Gardens, FL 33420 (800) 645-3985 Fax: (561) 622-4858 info@youelsprep.com www.youelsprep.com A-a alveolar-arterial (oxygen gradient) AAA abdominal aortic aneurysm AAMC Association of American Medical Colleges ABG arterial blood gas ABI ankle-brachial index ABVD Adriamycin (doxorubicin), bleomycin, vinblastine, dacarbazine AC abdominal circumference ACA anterior cerebral artery ACC American College of Cardiology ACE angiotensin-converting enzyme ACEI angiotensin-converting enzyme (protocol) ACTH adrenocorticotropic hormone AD Alzheimer’s disease ADA American Diabetes Association,

1	Americans with Disabilities Act ADH antidiuretic hormone ADHD attention-defcit hyperactivity disorder ADPKD autosomal-dominant polycystic disease 5-aminosalicylic acid acetylsalicylic acid atypical squamous cells atypical squamous cells suspicious of high-grade dysplasia atypical squamous cells of tachycardia abdominal x-ray British anti-Lewisite (dimercaprol), cytoplasmic antibody CBC complete blood count CBT cognitive-behavioral therapy, computer-based testing CCB calcium channel blocker CCS computer-based case simulation CD cluster of differentiation CEA carcinoembryonic antigen CF cystic f brosis CFU colony-forming unit CGD chronic granulomatous disease cGMP cyclic guanosine monophosphate CHF congestive heart failure CHOP cyclophosphamide (Cytoxan), hydroxydaunorubicin (Adriamycin), Oncovin (vincristine), prednisone

1	CI conf dence interval CIN candidate identif cation number, cervical intraepithelial neoplasia CJD Creutzfeldt-Jakob disease CK clinical knowledge, creatine kinase CKD chronic kidney disease CK-MB creatine kinase, MB fraction CLL chronic lymphocytic leukemia CML chronic myelogenous leukemia CMP comprehensive metabolic panel CMV cytomegalovirus CN cranial nerve CNS central nervous system CO carbon monoxide COGME Council on Graduate Medical disease continuous positive airway pressure cardiopulmonary resuscitation child protective services creatinine crown-rump length C-reactive protein clinical skills central sleep apnea combined system disease cerebrospinal f uid contraction stress test computed tomography carpal tunnel syndrome cerebrovascular accident central venous pressure chorionic villus sampling chest x-ray dilation and curettage dilation and evacuation developmental age diastolic blood pressure desmopressin acetate diethylstilbestrol dual-energy x-ray absorptiometry direct f

1	villus sampling chest x-ray dilation and curettage dilation and evacuation developmental age diastolic blood pressure desmopressin acetate diethylstilbestrol dual-energy x-ray absorptiometry direct f uorescent antibody dehydroepiandrosterone dehydroepiandrosterone sulfate Department of Homeland Security diabetes insipidus disseminated intravascular coagulation distal interphalangeal (joint) diabetic ketoacidosis diffusion capacity of carbon monoxide diabetes mellitus disease-modifying antirheumatic drug Duchenne muscular dystrophy dimercaptosuccinic acid deoxyribonucleic acid do not intubate do not resuscitate durable power of attorney for health acid Diagnostic and Statistical Manual (of mental disorders) diphtheria, tetanus, acellular pertussis (vaccine) deep tendon ref exes

1	ENT ear, nose, and throat ER emergency room, estrogen receptor FeNa fractional excretion of sodium FiO2 fraction of inspired oxygen FSMB Federation of State Medical Boards FTT failure to thrive

1	ER emergency room, estrogen receptor FeNa fractional excretion of sodium FiO2 fraction of inspired oxygen FSMB Federation of State Medical Boards FTT failure to thrive HHV 5-HIAA Hib 5-f uorouracil fever of unknown origin forced vital capacity glucose-6-phosphate dehydrogenase gestational age gamma-aminobutyric acid global assessment of functioning group A streptococcus glioblastoma multiforme, glomerular basement membrane group B streptococcus, Guillain- stimulating factor gram-negative rod gonadotropin-releasing hormone gestational trophoblastic disease genitourinary graft-versus-host disease hematoxylin and eosin highly active antiretroviral therapy hepatitis A virus hemoglobin A1c hepatitis B core antibody hepatitis B core antigen hepatitis B surface antibody hepatitis B surface antigen hepatitis B virus human chorionic gonadotropin hairy cell leukemia hydrochlorothiazide hepatitis C virus Huntington’s disease, Hodgkin’s nonketotic (coma) hyperosmolar hyperglycemic state,

1	Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure juvenile rheumatoid arthritis jugular venous distention jugular venous pulse potassium hydroxide Kaposi’s sarcoma Kaposi’s sarcoma–related herpesvirus kidney, ureter, bladder left anterior descending (artery) left atrial enlargement left bundle branch block large bowel obstruction low back pain lateral collateral ligament lactate dehydrogenase low-density lipoprotein loop electrosurgical excision procedure lower esophageal sphincter liver function test lymphogranuloma venereum luteinizing hormone left lower quadrant lower motor neuron last menstrual period low-molecular-weight heparin lumbar puncture lactated Ringer’s, likelihood ratio low-grade squamous intraepithelial lesion latent tuberculosis infection left upper quadrant left ventricular hypertrophy membrane attack complex, concentration medial collateral ligament metacarpophalangeal (joint) mean corpuscular volume,

1	MgSO4 magnesium sulfate MGUS monoclonal gammopathy of undetermined signif cance MHPSA Mental Health Professional Shortage Area MI myocardial infarction MIBG metaiodobenzylguanidine (scan) MMA methylmalonic acid MMF mycophenolate mofetil MMR measles, mumps, rubella (vaccine) MoM multiple of the median MOPP mechlorethamine, Oncovin (vincristine), procarbazine, prednisone MPTP 1-methyl-4-phenyl-1,2,3,6 cholangiopancreatography MRI magnetic resonance imaging MRSA methicillin-resistant S. aureus MS multiple sclerosis MSAFP maternal serum α-fetoprotein MSSA methicillin-susceptible S. aureus MTP metatarsophalangeal (joint) MUA manual uterine aspiration MUA/P Medically Underserved Area and

1	Population MuSK muscle-specif c kinase MVA motor vehicle accident NaHCO3 sodium bicarbonate NBME National Board of Medical disorder oral contraceptive pills overdose operating room open reduction and internal f xation obstructive sleep apnea over the counter partial pressure of carbon dioxide in cytoplasmic antibody partial pressure of oxygen in arterial blood pregnancy-associated plasma protein A period acid–Schiff posterior cerebral artery percutaneous coronary intervention polycystic kidney disease posterior cruciate ligament partial pressure of carbon dioxide polycystic ovarian syndrome phencyclidine hydrochloride polymerase chain reaction polycythemia vera, pneumococcal vaccine pulmonary capillary wedge pressure patent ductus arteriosus phosphodiesterase pulseless electrical activity positive end-expiratory pressure positron emission tomography pulmonary function test prostaglandin protease inhibitor posterior inferior cerebellar artery pelvic inf ammatory disease proximal

1	positive end-expiratory pressure positron emission tomography pulmonary function test prostaglandin protease inhibitor posterior inferior cerebellar artery pelvic inf ammatory disease proximal interphalangeal (joint) polycystic kidney disease phenylketonuria point of maximal impulse promyelocytic leukemia polymorphonuclear (leukocyte) paroxysmal nocturnal dyspnea per os (by mouth) partial pressure of oxygen product of conception tuberculin) PPI proton pump inhibitor PPROM preterm premature rupture of vaccine, positive predictive value PR per rectum, progesterone receptor PRBC packed red blood cell PRN pro re nata (as needed) PROM premature rupture of membranes PSA prostate-specif c antigen PT prothrombin time PTCA percutaneous transluminal coronary angioplasty PTH parathyroid hormone PTHrP parathyroid hormone–related protein PTSD post-traumatic stress disorder PTT partial thromboplastin time PUD peptic ulcer disease PUVA psoralen plus ultraviolet A PVC premature ventricular

1	PTHrP parathyroid hormone–related protein PTSD post-traumatic stress disorder PTT partial thromboplastin time PUD peptic ulcer disease PUVA psoralen plus ultraviolet A PVC premature ventricular contraction PVR peripheral vascular resistance PVS persistent vegetative state QD once a day RA rheumatoid arthritis RAIU radioactive iodine uptake RBBB right bundle branch block RBC red blood cell RDS respiratory distress syndrome RDW red cell distribution width REM rapid eye movement RF rheumatoid factor RLQ right lower quadrant ROM range of motion, rupture of

1	Information System Student and Exchange Visitor Program syndrome of inappropriate secretion TMJ temporomandibular joint TMP-SMX trimethoprim-sulfamethoxazole TMS transcranial magnetic stimulation TNF tumor necrosis factor TNM tumor, node, metastasis (staging) TOEFL Test of English as a Foreign Language tPA tissue plasminogen activator TPN total parenteral nutrition TPO thyroid peroxidase TPPA T. pallidum particle agglutination purpura TURP transurethral resection of the prostate TV tidal volume UA urinalysis UMN upper motor neuron URI upper respiratory infection USDA United States Department of Examination urinary tract infection ultraviolet Department of Veterans Affairs vital capacity voiding cystourethrography Venereal Disease Research

1	Examination urinary tract infection ultraviolet Department of Veterans Affairs vital capacity voiding cystourethrography Venereal Disease Research Laboratory vascular endothelial growth factor ventricular f brillation vulvar intraepithelial neoplasia very low density lipoprotein vanillylmandelic acid vaso-occlusive crisis vestibulo-ocular ref ex ventriculoperitoneal ventilation/perfusion (scan) vancomycin-resistant S. aureus ventricular septal defect ventricular tachycardia von Willebrand’s disease von Willebrand factor varicella-zoster virus white blood cell World Health Organization Hemoglobin, plasma

1	Hemoglobin, plasma Mean corpuscular hemoglobin Platelet count Prothrombin time Reticulocyte count Sedimentation rate, erythrocyte (Westergren) Proteins, total 1–4 mg/dL 0.16–0.62 μmol/L 4500–11,0 00/mm3 4.5–11.0 109/L 54–62% 0.54–0.62 3–5% 0.03–0.05 1–3% 0.01–0.03 0–0.75% 0–0.0075 25–33% 0.25–0.33 3–7% 0.03–0.07 25.4–34.6 pg/cell 0.39–0.54 fmol/cell 150,000–400,000/mm3 150–400 109/L 11–15 seconds 11–15 seconds 0.5–1.5% of red cells 0.005–0.015 Male: 0–15 mm/h 0–15 mm/h Female: 0–20 mm/h 0–20 mm/h < 150 mg/24 h < 0.15 g/24 h

1	PRESIDENT DILIP V. IESTE, M.D. PRESIDENT-ELECT JEFFREY A. LIEBERMAN, M.D. TREASURER DAVID FASSLER, M.D. SECRETARY ROGER PEELE, M.D. SPEAKER R. SCOTT BENSON, MD. SPEAKER-ELECT MELINDA L. YOUNG, MD. Board of Trustees JEFFREY AKAKA, MD. CAROL A. BERNSTEIN, M.D. BRIAN CROWLEY, MD. ANITA S. EVERETT, MD. JEFFREY GELLER, MD, MPH. Mac DAVID GBAFF, MD. \TAMESA:GREENE7M:D. JUDITH F. KASHTAN, M.D. MOLLY K. MCVOY, M.D. JAMES E. NlNINGER, MD. JOHN M. OLDHAM, MD. ALAN F. SCHATZBERG, MD. ALIK S. WIDGE, M.D., PHD. ERIK R. VANDERLIP, M.D., MENTAL DISORDERS, DSM and DSM-5 are trademarks of the American Psychiatric Association. Use of these terms is prohibited without permission of the American Psychiatric Association.

1	MENTAL DISORDERS, DSM and DSM-5 are trademarks of the American Psychiatric Association. Use of these terms is prohibited without permission of the American Psychiatric Association. ALL RIGHTS RESERVED. Unless authorized in writing by the APA, no part of this book may be reproduced or used in a manner inconsistent with the APA’s copyright. This prohibition applies to unauthorized uses or reproductions in any form, including electronic applications. Correspondence regarding copyright permissions should be directed to DSM Permissions, American Psychiatric Publishing, 1000 Wilson Boulevard, Suite 1825, Arlington, VA 22209- 3901. Manufactured in the United States of America on acid-free paper. Arlington, VA 22209-3901 www.psych.org The correct citation for this book is American Psychiatric Association: Diagnostic and Statisti- cal Manual of Mental Disorders, Fifth Edition. Arlington, VA, American Psychiatric Associa- tion, 2013. Library of Congress Cataloging-in-Publication Data

1	Library of Congress Cataloging-in-Publication Data Diagnostic and statistical manual of mental disorders : DSM-5. — 5th ed. p. ; cm. Includes index. ISBN 978- 0- 89042 ”554 1 (hardcover: alk. paper) — ISBN 978- 0- 89042-555- 8 (pbk.: alk. paper) I. American Psychiatric Association; 1.1 American Psychiatric Association. DSM—S Task Force. III Title: DSM- 5. IV. Title: DSM- V. [DNLMz 1. Diagnostic and statistical manual of mental disorders. 5th ed. 2. Mental Disorders— classification. 3. Mental Disorders—diagnosis. WM 15] RC455.2.C4 616.89‘075—dc23 British Library Cataloguing in Publication Data K C. A CIP record is available from the British Library. Text Design—Tammy I. Cordova H— 5 :5) . C Li DSM-5 Classification ................................... xiii Preface ............................................... xli Introduction ............................................ 5 Use of the Manual ...................................... 19

1	Preface ............................................... xli Introduction ............................................ 5 Use of the Manual ...................................... 19 Cautionary Statement for Forensic Use of DSM-5 ............ 25 Neurodevelopmental Disorders ........................... 31 Schizophrenia Spectrum and Other Psychotic Disorders ...... 87 Bipolar and Related Disorders ........................... 123 Depressive Disorders .................................. 155 Anxiety Disorders ...................................... 189 Obsessive-Compulsive and Related Disorders ............. 235 Trauma- and Stressor-Related Disorders .................. 265 Dissociative Disorders ................................. 291 Somatic Symptom and Related Disorders ................. 309 Feeding and Eating Disorders ........................... 329 Elimination Disorders .................................. 355 Sleep-Wake Disorders .................................. 361

1	Feeding and Eating Disorders ........................... 329 Elimination Disorders .................................. 355 Sleep-Wake Disorders .................................. 361 Sexual Dysfunctions ................................... 423 Gender Dysphoria ..................................... 451 Disruptive, Impulse-Control, and Conduct Disorders ........ 461 Substance-Related and Addictive Disorders ............... 481 Neurocognitive Disorders ............................... 591 Personality Disorders .................................. 645 Paraphilic Disorders ................................... 685 Other Mental Disorders ................................ 707 and Other Adverse Effects of Medication ................ 709 Other Conditions That May Be a Focus of Clinical Attention . .715 Assessment Measures ................................. 733 Cultural Formulation ................................... 749 Alternative DSM-5 Model for Personality Disorders ......... 761

1	Assessment Measures ................................. 733 Cultural Formulation ................................... 749 Alternative DSM-5 Model for Personality Disorders ......... 761 Conditions for Further Study ............................ 783 Highlights of Changes From DSM-IV to DSM-5 ............. 809 Glossary of Technical Terms ............................ 817 Glossary of Cultural Concepts of Distress ................. 833 Alphabetical Listing of DSM-5 Diagnoses and Codes (ICD-9-CM and lCD-10—CM) ............................ 839 Numerical Listing of DSM-5 Diagnoses and Codes (lCD-9-CM) ......................................... 863 Numerical Listing of DSM-5 Diagnoses and Codes (lCD-10-CM) ........................................ 877 DSM-5 Advisors and Other Contributors .................. 897 Index ................................................ 917 DAVID J. KUPFER, M.D. DARREL A. REGIER, M.D., M.P.H. William E. Narrow, M.D., M.P.H.,

1	Index ................................................ 917 DAVID J. KUPFER, M.D. DARREL A. REGIER, M.D., M.P.H. William E. Narrow, M.D., M.P.H., Dan C. Blazer, M.D., Ph.D., M.P.H. Jack D. Burke Jr., M.D., M.P.H. William T. Carpenter Ir., M.D. F. Xavier Castellanos, M.D. Wilson M. Compton, M.D., M.P.E. Joel E. Dimsdale, M.D. Javier I. Escobar, M.D., M.Sc. Ian A. Fawcett, M.D. Bridget F. Grant, Ph.D., PhD. (2009—) Steven E. Hyman, M.D. (2007—2012) Dilip V. Ieste, M.D. (2007—2011) Helena C. Kraemer, PhD. Daniel T. Mamah, M.D., M.P.E. James P. McNulty, A.B., Sc.B. Howard B. Moss, M.D. (2007—2009) Susan K. Schultz, M.D., Text Editor Emily A. Kuhl, Ph.D., APA Text Editor Charles P. O’Brien, M.D., PhD. Roger Peele, M.D. Katharine A. Phillips, M.D. Daniel S. Pine, M.D. Charles F. Reynolds III, M.D. Maritza Rubio-Stipec, Sc.D. David Shaffer, M.D. Andrew E. Skodol II, M.D. Susan E. Swedo, M.D. B. Timothy Walsh, M.D. Philip Wang, M.D., Dr.PH. (2007-2012)

1	Charles F. Reynolds III, M.D. Maritza Rubio-Stipec, Sc.D. David Shaffer, M.D. Andrew E. Skodol II, M.D. Susan E. Swedo, M.D. B. Timothy Walsh, M.D. Philip Wang, M.D., Dr.PH. (2007-2012) William M. Womack, M.D. Kimberly A. Yonkers, M.D. Kenneth J. Zucker, PhD. Norman Sartorius, M.D., Ph.D., Consultant APA Division of Research Staff on DSM-5 Darrel A. Regier, M.D., M.P.H., Director, Division of Research William E. Narrow, M.D., M.P.H., Emily A. Kuhl, Ph.D., Senior Science Diana E. Clarke, Ph.D., M.Sc., Research Lisa H. Greiner, M.S.S.A., DSM-5 Field Eve K. Moscicki, Sc.B., M.P.H., Director, Practice Research Network S. Janet Kuramoto, PhD. M.H.S., Senior Scientific Research Associate, Amy Porfiri, M.B.A. Jennifer J. Shupinka, Assistant Director, Seung-Hee Hong, DSM Senior Research Anne R. Hiller, DSM Research Associate Alison S. Beale, DSM Research Associate Spencer R. Case, DSM Research Associate Joyce C. West, Ph.D., M.P.P.,

1	Seung-Hee Hong, DSM Senior Research Anne R. Hiller, DSM Research Associate Alison S. Beale, DSM Research Associate Spencer R. Case, DSM Research Associate Joyce C. West, Ph.D., M.P.P., Health Policy Research Director, Practice Farifteh F. Duffy, Ph.D., Quality Care Research Director, Practice Lisa M. Countis, Field Operations Manager, Practice Research Network Christopher M. Reynolds, APA Office of the Medical Director JAMES H. SCULLY JR., M.D. Michael B. First, M.D. Maria N. Ward, M.Ed., RHIT, CCS-P DAVID SHAFFER, M.D. F. XAVIER CASTELLANOS, M.D. Paul I. Frick, Ph.D., Text Coordinator Luis Augusto Rohde, M.D., Sc.D. Glorisa Canino, PhD. Rosemary Tannock, PhD. Terrie E. Moffitt, PhD. Joel T. Nigg, PhD. Eric A. Taylor, M.B. Richard Todd, Ph.D., M.D. (d. 2008) Anxiety, Obsessive-Compulsive Spectrum, Posttraumatic, KATHARINE A. PHILLIPS, M.D. Michelle G. Craske, Ph.D., Text I. Gavin Andrews, M.D. Susan M. Bbgels, PhD. Matthew J. Friedman, M.D., PhD.

1	Anxiety, Obsessive-Compulsive Spectrum, Posttraumatic, KATHARINE A. PHILLIPS, M.D. Michelle G. Craske, Ph.D., Text I. Gavin Andrews, M.D. Susan M. Bbgels, PhD. Matthew J. Friedman, M.D., PhD. Eric Hollander, M.D. (2007—2009) Roberto Lewis—Fernéndez, M.D., M.T.S. Robert S. Pynoos, M.D., M.P.H. Scott L. Rauch, M.D. H. Blair Simpson, M.D., PhD. David Spiegel, M.D. Dan J. Stein, M.D., PhD. Murray B. Stein, M.D. Robert I. Ursano, M.D. Hans-Ulrich Wittchen, PhD. DANIEL S. PINE, M.D. Ronald E. Dahl, M.D. E. Jane Costello, PhD. (2007—2009) Regina Smith James, M.D. Rachel G. Klein, PhD. James F. Leckman, M.D. Ellen Leibenluft, M.D. Judith H. L. Rapoport, M.D. Charles H. Zeanah, M.D. B. TIMOTHY WALSH, M.D. Stephen A. Wonderlich, Ph.D., Evelyn Attia, M.D. Anne E. Becker, M.D., Ph.D., Sc.M. Rachel Bryant-Waugh, M.D. Hans W. Hoek, M.D., PhD. Richard E. Kreipe, M.D. Marsha D. Marcus, PhD. James E. Mitchell, M.D. Ruth H. StriegeI-Moore, PhD.

1	Anne E. Becker, M.D., Ph.D., Sc.M. Rachel Bryant-Waugh, M.D. Hans W. Hoek, M.D., PhD. Richard E. Kreipe, M.D. Marsha D. Marcus, PhD. James E. Mitchell, M.D. Ruth H. StriegeI-Moore, PhD. G. Terence Wilson, PhD. Barbara E. Wolfe, Ph.D. A.P.R.N. \ JAN A. FAWCETT, M.D. Ellen Frank, Ph.D., Text Coordinator Kenneth S. Kendler, M.D., PhD. Jules Angst, M.D. (2007—2008) (2007—2010) William H. Coryell, M.D. Mario Maj, M.D., PhD. Lori L. Davis, M.D. Husseini K. Manji, M.D. (2007—2008) Raymond J. DePaulo, M.D. Michael R. Phillips, M.D. Sir David Goldberg, M.D. Trisha Suppes, M.D., PhD. James S. Jackson, PhD. Carlos A. Zarate, M.D. DILIP V. JESTE, M.D. (2007—2011) DAN G. BLAZER, M.D., PH.D., M.P.H. RONALD C. PETERSEN, M.D., PHD. Mary Ganguli, M.D., M.P.H., Igor Grant, M.D. Text Coordinator Eric J. Lenze, M.D. Deborah Blacker, M.D., Sc.D. Jane S. Paulsen, PhD. Warachal Faison, M.D. (2007—2008) Perminder S. Sachdev, M.D., PhD. SUSAN E. SWEDO, M.D.

1	Text Coordinator Eric J. Lenze, M.D. Deborah Blacker, M.D., Sc.D. Jane S. Paulsen, PhD. Warachal Faison, M.D. (2007—2008) Perminder S. Sachdev, M.D., PhD. SUSAN E. SWEDO, M.D. Gillian Baird, M.A., M.B., B.Chir., Catherine E. Lord, PhD. Text Coordinator Joseph Piven, M.D. Edwin H. Cook ]1'., M.D. Sally I. Rogers, PhD. Francesca G. Happé, PhD. Sarah J. Spence, M.D., PhD. James C. Harris, M.D. Fred Volkmar, M.D. (2007—2009) Walter E. Kaufmann, M.D. Amy M. Wetherby, PhD. Bryan H. King, M.D. Harry H. Wright, M.D. ANDREW E. SKODOL, M.D. JOHN M. OLDI-IAM, M.D. Robert F. Krueger, Ph.D., Text Lee Anna Clark, PhD. Coordinator W. John Livesley, M.D., PhD. (2007—2012) Renato D. Alarcon, M.D., M.P.H. Leslie C. Morey, PhD. Carl C. Bell, M.D. Larry J. Siever, M.D.

1	Coordinator W. John Livesley, M.D., PhD. (2007—2012) Renato D. Alarcon, M.D., M.P.H. Leslie C. Morey, PhD. Carl C. Bell, M.D. Larry J. Siever, M.D. Donna S. Bender, PhD. Roel Verheul, PhD. (2008—2012) 1The members of the Personality and Personality Disorders Work Group are responsible for the alternative DSM-S model for personality disorders that is included in Section III. The Section II personality disorders criteria and text (with updating of the text) are retained from DSM-IV—TR. WILLIAM T. CARPENTER JR., M.D. Deanna M. Barch, Ph.D., Text Juan R. Bustillo, M.D. Wolfgang Gaebel, M.D. Raquel E. Gur, M.D., PhD. Stephan H. Heckers, M.D. Dolores Malaspina, M.D., M.S.P.I—I. Michael I. Owen, M.D., PhD. Susan K. Schultz, M.D. Rajiv Tandon, M.D. Ming T. Tsuang, M.D., PhD. Jim van Os, M.D. KENNETH J. ZUCKER, PHD. Lori Brotto, Ph.D., Text Coordinator Irving M. Binik, PhD. Ray M. Blanchard, PhD. Peggy T. Cohen-Kettenis, PhD. Jack Drescher, M.D. Cynthia A. Graham, PhD.

1	KENNETH J. ZUCKER, PHD. Lori Brotto, Ph.D., Text Coordinator Irving M. Binik, PhD. Ray M. Blanchard, PhD. Peggy T. Cohen-Kettenis, PhD. Jack Drescher, M.D. Cynthia A. Graham, PhD. Martin P. Kaﬂ<a, M.D. Richard B. Krueger, M.D. Niklas Langstrém, M.D., PhD. Heino F.L. Meyer-Bahlburg, Dr. rer. nat. Friedemann Pféifﬂin, M.D. Robert Taylor Segraves, M.D., PhD. CHARLES F. REYNOLDS HI, M.D. Ruth M. O’Hara, Ph.D., Text Coordinator Charles M. Morin, PhD. Allan 1. Pack, PhD. Kathy P. Parker, Ph.D., R.N. Susan Redline, M.D., M.P.H. Dieter Riemann, PhD. JOEL E. DIMSDALE, M.D. James L. Levenson, M.D., Text Arthur J. Barsky III, M.D. Francis Creed, M.D. Nancy Frasure-Smith, PhD. (2007—201 1) Michael R. Irwin, M.D. Francis J. Keefe, PhD. (2007—2011) Sing Lee, M.D. Michael Sharpe, M.D. Lawson R. Wulsin, M.D. CHARLES P. O'BRIEN, M.D., PHD. THOMAS J. CROWLEY, M.D. Wilson M. Compton, M.D., M.P.E., Marc Auriacombe, M.D. Guilherme L. G. Borges, M.D., Dr.Sc.

1	Michael Sharpe, M.D. Lawson R. Wulsin, M.D. CHARLES P. O'BRIEN, M.D., PHD. THOMAS J. CROWLEY, M.D. Wilson M. Compton, M.D., M.P.E., Marc Auriacombe, M.D. Guilherme L. G. Borges, M.D., Dr.Sc. Kathleen K. Bucholz, PhD. Alan I. Budney, PhD. Bridget F. Grant, Ph.D., PhD. Deborah S. Hasin, PhD. Thomas R. Kosten, M.D. (2007—2008) Walter Ling, M.D. Spero M. Manson, PhD. (2007—2008) A. Thomas McLellan, PhD. (2007—2008) N ancy M. Petry, PhD. Marc A. Schuckit, M.D. Wim van den Brink, M.D., PhD. STEVEN E. HYMAN, M.D. William T. Carpenter Ir., M.D. William E. Narrow, M.D., M.P.H. Wilson M. Compton, M.D., M.P.E. Charles P. O’Brien, M.D., PhD. Jan A. Fawcett, M.D. John M. Oldham, M.D. Helena C. Kraemer, PhD. Katharine A. Phillips, M.D. David I. Kupfer, M.D. Darrel A. Regier, M.D., M.P.H. ERIC 1. LENZE, M.D. SUSAN K. SCHULTZ, M.D. DANIEL S. PINE, M.D. Dan G. Blazer, M.D., Ph.D., M.P.H. Daniel T. Mamah, M.D., M.P.E. F. Xavier Castellanos, M.D. Andrew E. Skodol II, M.D.

1	ERIC 1. LENZE, M.D. SUSAN K. SCHULTZ, M.D. DANIEL S. PINE, M.D. Dan G. Blazer, M.D., Ph.D., M.P.H. Daniel T. Mamah, M.D., M.P.E. F. Xavier Castellanos, M.D. Andrew E. Skodol II, M.D. Wilson M. Compton, M.D., M.P.E. Susan E. Swedo, M.D. KIMBERLY A. YONKERS, M.D. ROBERTO LEWIs-FERNANDEZ, M.D., M.T.S. Co-Chair, Cross-Culturul Issues Renato D. Alarcon, M.D., M.P.H. Leslie C. Morey, PhD. Diana E. Clarke, Ph.D., M.Sc. William E. Narrow, M.D., M.P.H. Javier I. Escobar, M.D., M.Sc. Roger Peele, M.D. Ellen Frank, PhD. Philip Wang, M.D., Dr.P.H. (2007—2012) James S. Jackson, PhD. William M. Womack, M.D. Spiro M. Manson, PhD. (2007—2008) Kenneth J. Zucker, PhD. James P. McNulty, A.B., Sc.B. LAWSON R. WULSIN, M.D. Ronald E. Dahl, M.D. Richard E. Kreipe, M.D. Joel E. Dimsdale, M.D. Ronald C. Petersen, Ph.D., M.D. Javier I. Escobar, M.D., M.SC. Charles F. Reynolds III, M.D. Dilip V. Jeste, M.D. (2007—2011) Robert Taylor Segraves, M.D., PhD.

1	Joel E. Dimsdale, M.D. Ronald C. Petersen, Ph.D., M.D. Javier I. Escobar, M.D., M.SC. Charles F. Reynolds III, M.D. Dilip V. Jeste, M.D. (2007—2011) Robert Taylor Segraves, M.D., PhD. Walter E. Kaufmann, M.D. B. Timothy Walsh, M.D. JANE S. PAULSEN, PHD. I. Gavin Andrews, M.D. Hans W. Hoek, M.D., PhD. Glorisa Canino, PhD. Helena C. Kraemer, PhD. Lee Anna Clark, PhD. William E. Narrow, M.D., M.P.H. Diana E. Clarke, Ph.D., M.Sc. David Shaffer, M.D. Michelle G. Craske, PhD. JACK D. BURKE ]R., M.D., M.P.H. Lee Anna Clark, PhD. Helena C. Kraemer, PhD. Diana E. Clarke, Ph.D., M.Sc. William E. Narrow, M.D., M.P.H. Bridget F. Grant, Ph.D., PhD. David Shaffer, M.D. WILLIAM E. NARROW, M.D., M.P.H. Jack D. Burke Ir., M.D., M.P.H. David ]. Kupfer, M.D. Diana E. Clarke, Ph.D., M.Sc. Darrel A. Regier, M.D., M.P.H. Helena C. Kraemer, PhD. David Shaffer, M.D. WOLFGANG GAEBEL, M.D. Ellen Frank, Ph.D. Dan J. Stein, M.D., PhD. Charles P. O’Brien, M.D., PhD. Eric A. Taylor, M.B.

1	Helena C. Kraemer, PhD. David Shaffer, M.D. WOLFGANG GAEBEL, M.D. Ellen Frank, Ph.D. Dan J. Stein, M.D., PhD. Charles P. O’Brien, M.D., PhD. Eric A. Taylor, M.B. Norman Sartorius, M.D., Ph.D., David I. Kupfer, M.D. Consultant Darrel A. Regier, M.D., M.P.H. Susan K. Schultz, M.D. 1. :5. Glassmcatloni Before each disorder name, ICD-9-CM codes are provided, followed by ICD-10—CM codes in parentheses. Blank lines indicate that either the ICD-9-CM or the ICD-lO-CM code is not applicable. For some disorders, the code can be indicated only according to the subtype or specifier. ICD-9-CM codes are to be used for coding purposes in the United States through Sep- tember 30, 2014. ICD-10-CM codes are to be used starting October 1, 2014. Following chapter titles and disorder names, page numbers for the corresponding text or criteria are included in parentheses.

1	Following chapter titles and disorder names, page numbers for the corresponding text or criteria are included in parentheses. Note for all mental disorders due to another medical condition: Indicate the name of the other medical condition in the name of the mental disorder due to [the medical condi- tion]. The code and name for the other medical condition should be listed first immedi- ately before the mental disorder due to the medical condition. 319 L_.__) Intellectual Disability (Intellectual Developmental Disorder) (33) Specify current severity: 315.8 (F88) Global Developmental Delay (41) 315.39 (F80.9) Language Disorder (42) 315.39 (F80.0) Speech Sound Disorder (44) 315.35 (F80.81) Childhood-Onset Fluency Disorder (Stuttering) (45) Note: Later—onset cases are diagnosed as 307.0 (F985) adult-onset ﬂuency disorder. 315.39 (F80.89) Social (Pragmatic) Communication Disorder (47) 307.9 (F80.9) Unspecified Communication Disorder (49) 299.00 (F84.0) Autism Spectrum Disorder (50)

1	315.39 (F80.89) Social (Pragmatic) Communication Disorder (47) 307.9 (F80.9) Unspecified Communication Disorder (49) 299.00 (F84.0) Autism Spectrum Disorder (50) Specify if: Associated with a known medical or genetic condition or envi— ronmental factor; Associated with another neurodevelopmental, men- tal, or behavioral disorder Specify current severity for Criterion A and Criterion B: Requiring very substantial support, Requiring substantial support, Requiring support Specify if: With or without accompanying intellectual impairment, With or without accompanying language impairment, With catatonia (use additional code 293.89 [F06.1]) _._ (_._) Attention-Deficit/Hyperactivity Disorder (59) Specify whether: 314.01 (F90.2) Combined presentation 314.00 (F90.0) Predominantly inattentive presentation 314.01 (F90.1) Predominantly hyperactive/ impulsive presentation Specify if: In partial remission

1	Specify if: In partial remission Specify current severity: Mild, Moderate, Severe 314.01 (F90.8) Other Specified Attention-Deficit/ Hyperactivity Disorder (65) 314.01 (F90.9) Unspecified Attention-Deficit/Hyperactivity Disorder (66) _._ (_._) Specific Learning Disorder (66) Specify if: 315.00 (F81.0) With impairment in reading (specify if with word reading accuracy, reading rate or ﬂuency, reading comprehension) 315.2 (F81.81) With impairment in written expression (specify if with spelling accuracy, grammar and punctuation accuracy, clarity or organization of written expression) 315.1 (F81 .2) With impairment in mathematics (specify if with number sense, memorization of arithmetic facts, accurate or ﬂuent calculation, accurate math reasoning) Specify current severity: Mild, Moderate, Severe 315.4 (F82) DevelopmentalCoordination Disorder (74) 307.3 (F98.4) Stereotypic Movement Disorder (77) Specify if: With self-injurious behavior, Without self—injurious behavior

1	Specify if: With self-injurious behavior, Without self—injurious behavior Specify if: Associated with a known medical or genetic condition, neuro— developmental disorder, or environmental factor Specify current severity: Mild, Moderate, Severe 307.23 (F952) Tourette's Disorder (81) 307.22 (F95.1) Persistent (Chronic) Motor 0r Vocal Tic Disorder (81) Specify if: With motor tics only, With vocal tics only 307.21 (F95.0) Provisional Tic Disorder (81) 307.20 (F95.8) \ Other Specified Tic Disorder (85) 307.20 (F959) Unspecified Tic Disorder (85) 315.8 (F88) Other Specified Neurodevelopmental Disorder (86) 315.9 (F89) Unspecified Neurodevelopmental Disorder (86) The following specifiers apply to Schizophrenia Spectrum and Other Psychotic Disorders where indicated: aSpecify if: The following course specifiers are only to be used after a 1-year duration of the dis— order: First episode, currently in acute episode; First episode, currently in partial remission;

1	First episode, currently in full remission; Multiple episodes, currently in acute episode; Mul- tiple episodes, currently in partial remission; Multiple episodes, currently in full remission; bSpeczfy if: With catatonia (use additional code 293.89 [F06.1]) CSpecify current severity of delusions, hallucinations, disorganized speech, abnormal psycho- motor behavior, negative symptoms, impaired cognition, depression, and mania symptoms 301.22 (F21) Schizotypal (Personality) Disorder (90) 297.1 (F22) Delusional Disordera' c (90) Specify whether: Erotomanic type, Grandiose type, Jealous type, Persecu- tory type, Somatic type, Mixed type, Unspecified type Specify if: With bizarre content 298.8 (F23) Brief Psychotic Disorderb' C (94) Specify it: With marked stressor(s), Without marked stressor(s), With 295.40 (F2031) Schizophreniform Disorderb' c (96)

1	Specify it: With marked stressor(s), Without marked stressor(s), With 295.40 (F2031) Schizophreniform Disorderb' c (96) Specify if: With good prognostic features, Without good prognostic fea- 295.90 (F20.9) Schizophreniaa' b C (99) _._ (_.7) Schizoaffective Disordera' 1" c (105) Specify whether: 295.70 (F25.0) Bipolar type 295.70 (F25.1) Depressive type _._ (_._) Substance/Medication-Induced Psychotic Disorderc (110) Note: See the criteria set and corresponding recording procedures for substance-specific codes and lCD-9-CM and ICD-lO-CM coding. Specify if: With onset during intoxication, With onset during withdrawal _._ (7i.i) Psychotic Disorder Due to Another Medical ConditionC (115) Specify whether: 293.81 (F06.2) With delusions 293.82 (F06.0) With hallucinations 293.39 (F06.1) 293.89 (F06.1) 293.89 (F06.1) 298.8 (F28) 298.9 (F29) Catatonic Disorder Due to Another Medical Condition (120)

1	Catatonic Disorder Due to Another Medical Condition (120) Note: Code first 781.99 (R29.818) other symptoms involving nervous and musculoskeletal systems. The following specifiers apply to Bipolar and Related Disorders where indicated: aSpecify: With anxious distress (specify current severity: mild, moderate, moderatesevere, severe); catatonia (use additional code 293.89 [F06.1]); With peripartum onset; With seasonal pattern 296.41 (1:31.11) 296.42 (F31.12) 296.43 (1:31.13) 296.44 (F31.2) 296.45 (F31.73) 296.46 (1:31.74) 296.40 (F31.9) 296.40 (F31.0) 296.45 (F31.73) 296.46 (1:31.74) 296.40 (F31.9) _._ (2.2) 296.51 (1:31.31) 296.52 (1:31.32) 296.53 (F31.4) 296.54 (F31.5) 296.55 (1:31.75) 296.56 (F31.76) 296.50 (F31.9) 296.7 (F31.9) 296.89 (F31.81) Specify current or most recent episode: Hypomanic, Depressed Specify course if full criteria for a mood episode are not currently met: In partial remission, In full remission

1	Specify current or most recent episode: Hypomanic, Depressed Specify course if full criteria for a mood episode are not currently met: In partial remission, In full remission Specify severity if full criteria for a mood episode are not currently met: Mild, Moderate, Severe 301.13 (F34.0) Cyclothymic Disorder (139) Specify if: With anxious distress _._ (7.4 Substance/ Medication-Induced Bipolar and Related Disorder (142) Note: See the criteria set and corresponding recording procedures for substance-speciﬂc codes and ICD-9—CM and ICD-lO-CM coding. Specify if: With onset during intoxication, With onset during withdrawal 293.83 (_._) Bipolar and Related Disorder Due to Another Medical Condition Specify if: 296.89 (F3139) Other Specified Bipolar and Related Disorder (148) 296.80 (F31.9) Unspecified Bipolar and Related Disorder (149)

1	The following specifiers apply to Depressive Disorders where indicated: aSpeczfy: With anxious distress (specify current severity: mild, moderate, moderate-severe, (use additional code 293.89 [F06.1]); With peripartum onset; With seasonal pattern 296.99 (F34.8) Disruptive Mood Dysregulation Disorder (156) _._ (_-2) Major Depressive Disorder3 (160) _._ L.i) Single episode 296.21 (F320) Mild 296.22 (F32.1) Moderate 296.23 (F322) Severe 296.24 (F32.3) With psychotic features 296.25 (F32.4) In partial remission 296.26 (F325) In full remission 296.20 (F32.9) Unspecified _._ (_._) Recurrent episode 296.31 (F33.0) Mild 296.32 (F33.1) Moderate 296.33 (F332) Severe 296.34 (F333) With psychotic features 296.35 (F33.41) In partial remission 296.36 (F33.42) In full remission 296.30 (F33.9) Unspecified 300.4 (F34.1) Persistent Depressive Disorder (Dysthymia)a (168) Specify if: In partial remission, In full remission Specify if: Early onset, Late onset

1	Specify if: In partial remission, In full remission Specify if: Early onset, Late onset Specify if: With pure dysthymic syndrome; With persistent major depres- sive episode; With intermittent major depressive episodes, with current 625.4 (N94.3) 293.83 (_._) (F06.34) 311 (F32.8) 311 (F32.9) episode; With intermittent major depressive episodes, without current Specify current severity: Mild, Moderate, Severe Note: See the criteria set and corresponding recording procedures for substance-specific codes and ICD-9-CM and ICD-IO-CM coding. Specify if: With onset during intoxication, With onset during withdrawal Depressive Disorder Due to Another Medical Condition (180) Specify if: 309.21 (F93.0) 312.23 (F94.0) 300.29 (_._) (F40.21 9) (F40.228) (F40.230) (F40.231 ) (F4D.232) (F40.233) (F40.248) (F40.298) 300.23 (F40.10) 300.01 (F41.0) 300.22 (1:40.00) 300.02 (F41.1) Specify if: Fear of blood Fear of injections and transfusions Fear of other medical care Fear of injury

1	Specify if: Fear of blood Fear of injections and transfusions Fear of other medical care Fear of injury Specify if: Performance only Note: See the criteria set and corresponding recording procedures for substance-specific codes and ICD-9-CM and ICD-lO-CM coding. Specify if: With onset during intoxication, With onset during withdrawal, 293.84 (F06.4) Anxiety Disorder Due to Another Medical Condition (230) 300.09 (F41.8) \ Other Specified Anxiety Disorder (233) 300.00 (F41.9) Unspecified Anxiety Disorder (233) The following specifier applies to Obsessive-Compulsive and Related Disorders where indicated: aSpecz'fy if: With good or fair insight, With poor insight, With absent insight/delusional beliefs 300.3 (F42) Obsessive-Compulsive Disordera (237) Specify if: Tic-related 300.7 (F4522) Body Dysmorphic Disordera (242) Specify if: With muscle dysmorphia 300.3 (F42) Hoarding Disordera (247)

1	Specify if: Tic-related 300.7 (F4522) Body Dysmorphic Disordera (242) Specify if: With muscle dysmorphia 300.3 (F42) Hoarding Disordera (247) Specify if: With excessive acquisition 312.39 (F632) Trichotillomania (Hair-Pulling Disorder) (251) 698.4 (L98.1) Excoriation (Skin-Picking) Disorder (254) Note: See the criteria set and corresponding recording procedures for substance-specific codes and ICD—9—CM and ICD—lO-CM coding. Specify it: With onset during intoxication, With onset during withdrawal, 294.8 (F06.8) Obsessive-Compulsive and Related Disorder Due to Another Specify if: With obsessive-compulsive disorder—like symptoms, With appearance preoccupations, With hoarding symptoms, With hair- pulling symptoms, With skin-picking symptoms 300.3 (F42) Other Specified Obsessive-Compulsive and Related Disorder 300.3 (F42) Unspecified Obsessive-Compulsive and Related Disorder (264) 313.89 (F94.1) Reactive Attachment Disorder (265) Specify if: Persistent

1	Specify if: Persistent Specify current severity: Severe 313.89 (F94.2) Disinhibited Social Engagement Disorder (268) Specify if: Persistent Specify current severity: Severe 309.81 (F43.10) Posttraumatic Stress Disorder (includes Posttraumatic Stress Specify whether: With dissociative symptoms Specify if: With delayed expression 308.3 (F43.0) Acute Stress Disorder (280) _-2 (#2.) 309.0 (F4321) 309.24 (F4322) 309.28 (F4323) 309.3 (F43.24) 309.4 (F43.25) 309.9 (F43.20) 309.89 (F43.8) 309.9 (F43.9) Specify whether: With disturbance of conduct With mixed disturbance of emotions and conduct 300.14 (F44.81) 300.12 (F44.0) 300.13 (F44.1) 300.6 (F48.1) 300.15 (F44.89) 300.15 (F44.9) Specify if: 300.82 (F45.1) 300.7 (F4521) 300.11 (_._) (F44.4) (F44.4) (F44.4) (F44.4) (F44.5) (F44.6) (F44.6) (F44.7) Specify if: With predominant pain Specify if: Persistent Specify current severity: Mild, Moderate, Severe Specify whether: Care seeking type, Care avoidant type Specify symptom type:

1	Specify if: With predominant pain Specify if: Persistent Specify current severity: Mild, Moderate, Severe Specify whether: Care seeking type, Care avoidant type Specify symptom type: Specify if: Acute episode, Persistent Specify if: With psychological stressor (specify stressor), Without psycho- Specify current severity: Mild, Moderate, Severe, Extreme 300.19 (F68.10) Factitious Disorder (includes Factitious Disorder Imposed 011 Self, Specify Single episode, Recurrent episodes 300.89 (F458) Other Specified Somatic Symptom and Related Disorder (327) 300.82 (F45.9) Unspecified Somatic Symptom and Related Disorder (327) The following specifiers apply to Feeding and Eating Disorders where indicated: aSpecify if: In remission bSpeczfy if: In partial remission, In full remission

1	The following specifiers apply to Feeding and Eating Disorders where indicated: aSpecify if: In remission bSpeczfy if: In partial remission, In full remission CSpecify current severity: Mild, Moderate, Severe, Extreme 307.52 (i.i) Picaa (329) (F50.8) In adults 307.53 (F9821) Rumination Disordera (332) 307.59 (F50.8) Avoidant/ Restrictive Food Intake Disordera (334) 307.1 (_._) Anorexia Nervosab' c (338) Specify whether: (F50.01) Restricting type (F50.02) Binge-eating /purging type 307.51 (F502) Bulimia Nervosab' C (345) 307.51 (F50.8) Binge-Eating Disorderb' c (350) 307.59 (F50.8) Other Specified Feeding or Eating Disorder (353) 307.50 (F503) Unspecified Feeding 01' Eating Disorder (354) 307.6 (F98.0) Enuresis (355) Specify whether: Nocturnal only, Diurnal only, Nocturnal and diurnal 307.7 (F98.1) Encopresis (357)

1	Specify whether: Nocturnal only, Diurnal only, Nocturnal and diurnal 307.7 (F98.1) Encopresis (357) Specify whether: With constipation and overﬂow incontinence, Without _._ (_._) Other Specified Elimination Disorder (359) 788.39 (N39.498) With urinary symptoms 787.60 (R15.9) With fecal symptoms _._ (_.;) Unspecified Elimination Disorder (360) 788.30 (R32) With urinary symptoms 787.60 (R159) With fecal symptoms The following specifiers apply to Sleep—Wake Disorders where indicated: aSpecify if: Episodic, Persistent, Recurrent bSpecify if: Acute, Subacute, Persistent CSpeczfy current severity: Mild, Moderate, Severe 780.52 (G47.00) 780.54 (647.10) 347.00 (647.419) 347.01 (G47.411) 347.00 (647.419) 347.00 (647.419) 347.10 (647.429) Specify if: With non—sleep disorder mental comorbidity, With other medical comorbidity, With other sleep disorder Specify if: With mental disorder, With medical condition, With another Specify whether:

1	Specify if: With mental disorder, With medical condition, With another Specify whether: Narcolepsy without cataplexy but with hypocretin deficiency Narcolepsy with cataplexy but without hypocretin deficiency Autosomal dominant cerebellar ataxia, deafness, and Autosomal dominant narcolepsy, obesity, and type 2 diabetes Narcolepsy secondary to another medical condition 327.23 (647.33) 327.21 (647.31) 786.04 (R063) 780.57 (647.37) 327.24 (647.34) 327.25 (647.35) 327.26 (647.36) 307.45 (G47.21) 307.45 (647.22) 307.45 (647.23) 307.45 (647.24) Specify whether: Central sleep apnea comorbid with opioid use Note: First code opioid use disorder, if present. Specify whether: Specify whether: Specify if: Familial, Overlapping with n0n-24-hour sleep-wake type Specify if: Familial 307.45 (647.26) Shift work type (397) 307.45 (647.20) Unspecified type _._ (i.J Non—Rapid Eye Movement Sleep Arousal Disorders (399) Specify whether: 307.46 (F51.3) Sleepwalking type

1	Specify whether: 307.46 (F51.3) Sleepwalking type Specify if: With sleep-related eating, With sleep-related sexual 307.46 (F51.4) Sleep terror type 307.47 (F515) Nightmare Disorderb'c(404) Specify if: During sleep onset Specify it: With associated non—sleep disorder, With associated other medical condition, With associated other sleep disorder 327.42 (647.52) Rapid Eye Movement Sleep Behavior Disorder (407) 333.94 (625.81) Restless Legs Syndrome (410) _._ (i.J Substance/Medication-Induced Sleep Disorder (413) Note: See the criteria set and corresponding recording procedures for substance-specific codes and ICD-9-CM and ICD—lO-CM coding. Specify whether: Insomnia type, Daytime sleepiness type, Parasomnia type, Mixed type

1	Specify whether: Insomnia type, Daytime sleepiness type, Parasomnia type, Mixed type Specify it: With onset during intoxication, With onset during discontinua- 780.52 (647.09) Other Specified Insomnia Disorder (420) 780.52 (647.00) Unspecified Insomnia Disorder (420) 780.54 (647.19) Other Specified Hypersomnolence Disorder (421) 780.54 (647.10) Unspecified Hypersomnolence Disorder (421) 780.59 (647.8) Other Specified Sleep—Wake Disorder (421) 780.59 (647.9) Unspecified Sleep-Wake Disorder (422) The following specifiers apply to Sexual Dysfunctions where indicated: aSpecify whether: Lifelong, Acquired bSpecify whether: Generalized, Situational “Specify current severity: Mild, Moderate, Severe 302.74 (F5232) Delayed Ejaculationa' bl C (424) 302.72 (F52.21) Erectile Disordera'b’ C (426) 302.73 (F5231) Female Orgasmic Disordera' b’ c (429)

1	Specify if: Never experienced an orgasm under any situation 302.72 (F5222) Female Sexual Interest/Arousal Disordera'b'C (433) 302.76 (F526) Genito—Pelvic Pain/ Penetration Disordera' c (437) 302.71 (F52.0) 302.75 (F52.4) _._ (-2) 302.79 (F52.8) 302.70 (F52.9) Note: See the criteria set and corresponding recording procedures for substance-specific codes and ICD-9-CM and ICD-lO-CM coding. Specify if: With onset during intoxication, With onset during withdrawal, _._ (2-_) 302.6 (F642) 302.85 (F64.1) 302.6 (F64.8) 302.6 (F64.9) Specify if: With a disorder of sex development Specify if: With a disorder of sex development Specify if: Posttransition Note: Code the disorder of sex development if present, in addition to gender dysphoria. Disruptive, lmpulse-Control, and Conduct Disorders (461) 31 3.81 (F913) 312.34 (F63.81) _._ (_-J 312.81 (F91.1) 312.32 (F91.2) 312.89 (F91.9) 301.7 (F602) 312.33 (F63.1) 312.32 (F63.3) 312.89 (F91.8) 312.9 (F91.9)

1	Specify current severity: Mild, Moderate, Severe Specify whether: Specify it: With limited prosocial emotions Specify current severity: Mild, Moderate, Severe Other Specified Disruptive, Impulse-Control, and Conduct Unspecified Disruptive, Impulse-Control, and Conduct Disorder The following specifiers and note apply to Substance-Related and Addictive Disorders where indicated: aSpecify if: In early remission, In sustained remission bSpecify if: In a controlled environment CSpecify if: With perceptual disturbances dThe ICD-lO-CM code indicates the comorbid presence of a moderate or severe substance use disorder, which must be present in order to apply the code for substance withdrawal. _._ (#._) Alcohol Use Disordera' b (490)

1	_._ (#._) Alcohol Use Disordera' b (490) Specify current severity: 305.00 (F10.10) Mild 303.90 (F10.20) Moderate 303.90 (F10.20) Severe 303.00 (_._) Alcohol Intoxication (497) (F10.129) With use disorder, mild (F10.229) With use disorder, moderate or severe (F10.929) Without use disorder 291.81 (_._) Alcohol Withdrawal“ d (499) (F10.239) Without perceptual disturbances (F10.232) With perceptual disturbances _._ (_._) Other Alcohol-Induced Disorders (502) 291.9 (F10.99) Unspecified Alcohol-Related Disorder (503) 305.90 (F15.929) Caffeine Intoxication (503) 292.0 (F1593) Caffeine Withdrawal (506) _._ (_._) Other Caffeine—Induced Disorders (508) 292.9 (F1599) Unspecified Caffeine-Related Disorder (509) _._ (_._) Cannabis Use Disordera’ b (509) Specify current severity: 305.20 (F1210) Mild 304.30 (F12.20) Moderate 304.30 (F1220) Severe 292.89 (_._) (F12.129) (F12.229) (F12.929) (F12.122) (1:12.222) (F12.922) 292.0 (F12.288) 292.9 (F1299) With use disorder, mild

1	With use disorder, mild With use disorder, moderate or severe With use disorder, mild With use disorder, moderate or severe _._ (7.4 305.90 (F16.10) 304.60 (F1620) 304.60 (F16.20) 305.30 (F16.10) 304.50 (F16.20) 304.50 (F16.20) 292.89 (_._) (F16.129) (F16.229) (F16.929) 292.89 (_._) (F16.129) (F16.229) (F16.929) 292.89 (F16.983) 292.9 (F16.99) 292.9 (F16.99) Specify current severity: Specify the particular hallucinogen Specify current severity: With use disorder, mild With use disorder, moderate or severe With use disorder, mild With use disorder, moderate or severe 305.90 (F18.10) Specify the particular inhalant

1	With use disorder, mild With use disorder, moderate or severe With use disorder, mild With use disorder, moderate or severe 305.90 (F18.10) Specify the particular inhalant Specify current severity: 304.60 (F1820) Moderate 304.60 (F1820) Severe 292.89 (7.i) Inhalant Intoxication (538) (F18.129) With use disorder, mild (F18.229) With use disorder, moderate or severe (F18.929) Without use disorder _._ (_._) Other Inhalant-Induced Disorders (540) 292.9 (F18.99) Unspecified Inhalant-Related Disorder (540) _._ (_._) Opioid Use Disordera (541) Specify if: On maintenance therapy, In a controlled environment

1	Specify if: On maintenance therapy, In a controlled environment Specify current severity: 305.50 (F11.10) Mild 304.00 (F1 1.20) Moderate 304.00 (F1 1.20) Severe 292.89 (_._) Opioid IntoxicationC (546) (F11.129) With use disorder, mild (F11.229) With use disorder, moderate or severe (F11.929) Without use disorder (F11.122) With use disorder, mild (F11.222) With use disorder, moderate or severe (F11.922) Without use disorder 292.0 (F11.23) Opioid Withdrawald (547) _._ (_._) Other Opioid-Induced Disorders (549) 292.9 (F11.99) Unspecified Opioid-Related Disorder (550) Sedative-, Hypnotic-, or Anxiolytic-Related Disorders (550) _._ (_._) Sedative, Hypnotic, or Anxiolytic Use Disordera' b (550)

1	Sedative-, Hypnotic-, or Anxiolytic-Related Disorders (550) _._ (_._) Sedative, Hypnotic, or Anxiolytic Use Disordera' b (550) Specify current severity: 305.40 (F13.10) Mild 304.10 (F1320) Moderate 304.10 (F1320) Severe 292.89 (_._) Sedative, Hypnotic, or Anxiolytic Intoxication (556) (F13.129) With use disorder, mild (F13.229) With use disorder, moderate or severe (F13.929) Without use disorder 292.0 (~42) Sedative, Hypnotic, 0r Anxiolytic Withdrawal“ d (557) (F13.239) Without perceptual disturbances (F13.232) With perceptual disturbances (_._) Other Sedative-, Hypnotic-, or Anxiolytic-Induced Disorders 292.9 (F1399) Unspecified Sedative-, Hypnotic—, or AnxiolytiC-Related Disorder _._ (_._) Stimulant Use Disordera' b (561)

1	Specify current severity: _-_ (A._) Mild 305.70 (F1510) Amphetamine-type substance 305.60 (F14.10) Cocaine 305.70 (F15.10) Other or unspecified stimulant _._ (_._) Moderate 304.40 (F1520) Amphetamine-type substance 304.20 (F1420) Cocaine 304.40 (F15.20) Other or unspecified stimulant __._ L_._) Severe 304.40 (F1520) Amphetamine-type substance 304.20 (F14.20) Cocaine 304.40 (F1520) Other or unspecified stimulant 292.89 (_._) Stimulant IntoxicationC (567)

1	Specify the specific intoxicant 292.89 (_._) Amphetamine or other stimulant, Without perceptual (F15.129) With use disorder, mild (F15.229) With use disorder, moderate or severe (F15.929) Without use disorder 292.89 (i._) Cocaine, Without perceptual disturbances (F14.129) With use disorder, Inild (F14.229) With use disorder, moderate or severe (F14.929) Without use disorder 292.89 (7.#) Amphetamine or other stimulant, With perceptual (F15.122) With use disorder, mild (F15.222) With use disorder, moderate or severe (F15.922) Without use disorder 292.89 (_._) Cocaine, With perceptual disturbances (F14.122) With use disorder, mild (F14.222) With use disorder, moderate or severe (F14.922) Without use disorder 292.0 (_._) Stimulant Withdrawald (569)

1	Specify the specific substance causing the withdrawal syndrome (F15.23) Amphetamine or other stimulant _._ (7.2) Other Stimulant-Induced Disorders (570) 292.9 (7.7) Unspecified Stimulant—Related Disorder (570) (F14.99) Cocaine _._ (7.7 Tobacco Use Disordera (571) Specify if: On maintenance therapy, In a controlled environment Specify current severity: 305.1 (272.0) Mild 305.1 (F17.200) Moderate 305.1 (F17.200) Severe 292.0 (F17.203) Tobacco Withdrawald (575) __._ (_.7) Other Tobacco-Induced Disorders (576) 292.9 (F17.209) Unspecified Tobacco-Related Disorder (577) _._ (__._) Other (or Unknown) Substance Use Disordera' b (577)

1	Specify current severity: 305.90 (F19.10) Mild 304.90 (F1920) Moderate 304.90 (F1920) Severe 292.89 7.7) Other (or Unknown) Substance Intoxication (581) (F19.129) With use disorder, mild (F19.229) With use disorder, moderate or severe (F19.929) Without use disorder 292.0 (F19.239) Other (or Unknown) Substance Withdrawald (583) _._ (7.7) Other (or Unknown) Substance—Induced Disorders (584) 292.9 (F19.99) Unspecified Other (or Unknown) Substance—Related Disorder (585) 312.31 (F63.0) Gambling Disordera (585) Specify if: Episodic, Persistent Specify Current severity: Mild, Moderate, Severe _-_ (_._) Delirium (596) 3Note: See the criteria set and corresponding recording procedures for substance-specific codes and ICD-9-CM and ICD-lO-CM coding. Specify whether: _._ (7.7) Substance intoxication deliriuma _._ (7.7) Substance withdrawal deliriuma 292.81 (7.7 Medication-induced deliriuma 293.0 (F05) Delirium due to another medical condition 293.0 (F05) Delirium due to multiple etiologies

1	Specify if: Acute, Persistent Specify if: Hyperactive, Hypoactive, Mixed level of activity 780.09 (R410) Other Specified Delirium (602) 780.09 (R41.0) Unspecified Delirium (602) Specify whether due to: Alzheimer’s disease, Frontotemporal lobar degeneration, Lewy body disease, Vascular disease, Traumatic brain injury, Substance/medication use, HIV infection, Prion disease, Parkinson’s disease, Huntington’s disease, Another medical condition, Multi- ple etiologies, Unspecified aSpecify Without behavioral disturbance, With behavioral disturbance. For possible major neuro- cognitive disorder and for mild neurocognitive disorder, behavioral disturbance cannot be coded but should still be indicated in writing. bSpecify current severity: Mild, Moderate, Severe. This specifier applies only to major neurocogni— tive disorders (including probable and possible).

1	bSpecify current severity: Mild, Moderate, Severe. This specifier applies only to major neurocogni— tive disorders (including probable and possible). Note: As indicated for each subtype, an additional medical code is needed for probable major neurocognitive disorder or major neurocognitive disorder. An additional medical code should not be used for possible major neurocognitive disorder or mild neurocognitive disorder. Major or Mild Neurocognitive Disorder Due to Alzheimer’s Disease (611) _._ (7.7) Probable Major Neurocognitive Disorder Due to Alzheimer's Note: Code first 331.0 (G303) Alzheimer’s disease. 294.11 (F02.81) With behavioral disturbance 294.10 (F02.80) Without behavioral disturbance 331.9 (631.9) Possible Major Neurocognitive Disorder Due to Alzheimer’s 331 .83 (631.84) Mild Neurocognitive Disorder Due to Alzheimer’s Diseasea 7.7 (7.7) Probable Major Neurocognitive Disorder Due to Frontotemporal Note: Code first 331.19 (G31.09) frontotemporal disease.

1	Note: Code first 331.19 (G31.09) frontotemporal disease. 294.1 1 (F02.81) With behavioral disturbance 294.10 (F02.80) Without behavioral disturbance 331 .9 (631.9) Possible Major Neurocognitive Disorder Due to Frontotemporal 331.83 (631.84) Mild Neurocognitive Disorder Due to Frontotemporal Lobar _._ (7.7) Probable Major Neurocognitive Disorder With Lewy Bodiesb Note: Code first 331.82 (631.83) Lewy body disease. 294.1 1 (F02.81) With behavioral disturbance 294.10 (F02.80) Without behavioral disturbance 331.9 (631.9) Possible Major Neurocognitive Disorder With Lewy Bodiesa' b 331.83 (631.84) Mild Neurocognitive Disorder With Lewy Bodiesa 7.7 (7.7 Probable Major Vascular Neurocognitive Disorderb Note: No additional medical code for vascular disease. 290.40 (F01.51) With behavioral disturbance 290.40 (F01.50) Without behavioral disturbance 331.9 (631.9) Possible Major Vascular Neurocognitive Disordera' b 331.83 (631.84) Mild Vascular Neurocognitive Disordera

1	Major or Mild Neurocognitive Disorder Due to Traumatic Brain Injury (624) 7.7 (7.7) Major Neurocognitive Disorder Due to Traumatic Brain Injuryb Note: For ICD—9-CM, code first 907.0 late effect of intracranial injury without skull fracture. For ICD-10«CM, code first $06.2X9S diffuse traumatic brain injury with loss of consciousness of unspecified duration, sequela. 294.11 (F02.81) With behavioral disturbance 294.10 (F02.80) Without behavioral disturbance 331.83 (631.84) Mild Neurocognitive Disorder Due to Traumatic Brain Injury8 Note: No additional medical code. See the criteria set and corresponding recording procedures for substance-specific codes and ICD—9-CM and ICD-IO-CM coding. Specify if: Persistent Major or Mild Neurocognitive Disorder Due to HIV Infection (632) .7) Major Neurocognitive Disorder Due to HIV Infectionb Note: Code first 042 (320) HIV infection.

1	Specify if: Persistent Major or Mild Neurocognitive Disorder Due to HIV Infection (632) .7) Major Neurocognitive Disorder Due to HIV Infectionb Note: Code first 042 (320) HIV infection. 294.11 (F02.81) With behavioral disturbance 294.10 (F02.80) Without behavioral disturbance 331.83 (631.84) Mild Neurocognitive Disorder Due to HIV Infectiona Major or Mild Neurocognitive Disorder Due to Prion Disease (634) 7.7 ( .7) Major Neurocognitive Disorder Due to Prion Diseaseb Note: Code first 046.79 (A813) prion disease. 294.1 1 (F02.81) With behavioral disturbance 294.10 (F02.80) Without behavioral disturbance 331.83 (631.84) Mild Neurocognitive Disorder Due to Prion Diseasea Major or Mild Neurocognitive Disorder Due to Parkinson’s Disease (636) 7.7 (7.7) Major Neurocognitive Disorder Probably Due to Parkinson’s Note: Code first 332.0 (020) Parkinson’s disease.

1	Major or Mild Neurocognitive Disorder Due to Parkinson’s Disease (636) 7.7 (7.7) Major Neurocognitive Disorder Probably Due to Parkinson’s Note: Code first 332.0 (020) Parkinson’s disease. 294.11 (F02.81) With behavioral disturbance 294.10 (F02.80) Without behavioral disturbance 331.9 (631.9) Major Neurocognitive Disorder Possibly Due to Parkinson’s 331.83 (631.84) Mild Neurocognitive Disorder Due to Parkinson’s Diseasea Major or Mild Neurocognitive Disorder Due to Huntington’s Disease (638) .7) Major Neurocognitive Disorder Due to Huntington's Diseaseb Note: Code first 333.4 (610) Huntington’s disease. 294.11 (F02.81) With behavioral disturbance 294.10 (F02.80) Without behavioral disturbance 331.83 (631.84) Mild Neurocognitive Disorder Due to Huntington’s Diseaseal _._ (2 Major or Mild Neurocognitive Disorder Due to Another Medical Condition (641) .7) Major Neurocognitive Disorder Due to Another Medical Note: Code first the other medical condition.

1	Major or Mild Neurocognitive Disorder Due to Another Medical Condition (641) .7) Major Neurocognitive Disorder Due to Another Medical Note: Code first the other medical condition. 294.11 (F02.81) With behavioral disturbance 294.10 (F02.80) Without behavioral disturbance 331.83 (631.84) Mild Neurocognitive Disorder Due to Another Medical Major or Mild Neurocognitive Disorder Due to Multiple Etiologies (642) .7) Major Neurocognitive Disorder Due to Multiple Etiologiesb Note: Code first all the etiological medical conditions (with the exception of vascular disease).

1	Note: Code first all the etiological medical conditions (with the exception of vascular disease). 294.11 (F02.81) With behavioral disturbance 294.10 (F02.80) Without behavioral disturbance 331.83 (631.84) Mild Neurocognitive Disorder Due to Multiple Etiologiesa 799.59 (R419) Unspecified Neurocognitive Disordera 301.0 (F60.0) Paranoid Personality Disorder (649) 301.20 (F60.1) Schizoid Personality Disorder (652) 301.22 (F21) Schizotypal Personality Disorder (655) 301.7 (F602) Antisocial Personality Disorder (659) 301.83 (F603) Borderline Personality Disorder (663) 301.50 (F60.4) Histrionic Personality Disorder (667) 301.81 (F60.81) Narcissistic Personality Disorder (669) 301.82 (F606) 301.6 (F60.7) 301.4 (F60.5) 310.1 (F07.0) 301.89 (F60.89) 301.9 (F609) Personality Change Due to Another Medical Condition (682) Specify whether: Labile type, Disinhibited type, Aggressive type, Apathetic type, Paranoid type, Other type, Combined type, Unspecified type

1	Specify whether: Labile type, Disinhibited type, Aggressive type, Apathetic type, Paranoid type, Other type, Combined type, Unspecified type The following specifier applies to Paraphilic Disorders where indicated: aSpeczfy if: In a controlled environment, In full remission 302.82 (F65.3) 302.4 (F652) 302.89 (F65.81) 302.83 (F65.51) 302.84 (F6552) 302.2 (F65.4) 302.81 (F65.0) 302.3 (F65.1) 302.89 (F6589) Specify whether: Sexually aroused by exposing genitals to prepubertal children, Sexually aroused by exposing genitals to physically mature individuals, Sexually aroused by exposing genitals to prepubertal chil- dren and to physically mature individuals Specify if: With asphyxiophilia Specify whether: Exclusive type, Nonexclusive type Specify if: Sexually attracted to males, Sexually attracted to females, Sexu- ally attracted to both Specify if: Limited to incest Specify: Body part(s), Nonliving object(s), Other

1	Specify if: Sexually attracted to males, Sexually attracted to females, Sexu- ally attracted to both Specify if: Limited to incest Specify: Body part(s), Nonliving object(s), Other Specify if: With fetishism, With autogynephilia 302.9 (F65.9) Unspecified Paraphilic Disorder (705) 294.8 (F06.8) Other Specified Mental Disorder Due to Another Medical 294.9 (F09) Unspecified Mental Disorder Due to Another Medical Condition 300.9 (F99) Other Specified Mental Disorder (708) 300.9 (F99) Unspecified Mental Disorder (708) Other Adverse Effects of Medication (709) 332.1 (621.11) 332.1 (621.19) 333.92 (621.0) 333.72 (624.02) 333.99 (625.71) 333.85 (624.01) 333.72 (624.09) 333.99 (625.71) 333.1 (625.1) 333.99 (625.79) 995.29 (T43.205A) 995.29 (T43.205D) 995.29 (T432058) _._ (7._) 995.20 (T50.905A) 995.20 (T50.905D) 995.20 (T509055) Other Adverse Effect of Medication (714) Problems Related to Family Upbringing (715) V61.20 (262.820) V61.8 (262.891) V61.8 (262.29) V61.29 (262.898)

1	Other Adverse Effect of Medication (714) Problems Related to Family Upbringing (715) V61.20 (262.820) V61.8 (262.891) V61.8 (262.29) V61.29 (262.898) Other Problems Related to Primary Support Group (716) V61.10 (263.0) V61.03 (263.5) V61.8 (263.8) V6282 (263.4) Disruption of Family by Separation or Divorce (716) Child Physical Abuse, Confirmed (717) 995.54 (T 74.12XA) Initial encounter 995.54 (T 74.12XD) Subsequent encounter Child Physical Abuse, Suspected (717) 995.54 (T 76.12XA) Initial encounter 995.54 (T 76.1 2XD) Subsequent encounter Other Circumstances Related to Child Physical Abuse (718) V61.21 (269.010) Encounter for mental health services for victim of child abuse V61.21 (269.020) Encounter for mental health services for Victim of nonparental V15.41 (262.810) Personal history (past history) of physical abuse in childhood V61 .22 (269.011) Encounter for mental health services for perpetrator of parental

1	V15.41 (262.810) Personal history (past history) of physical abuse in childhood V61 .22 (269.011) Encounter for mental health services for perpetrator of parental V62.83 (269.021) Encounter for mental health services for perpetrator of Child Sexual Abuse, Confirmed (718) 995.53 (T74.22XA) Initial encounter 995.53 (T 74.22XD) Subsequent encounter Child Sexual Abuse, Suspected (718) 995.53 (T76.22XA) Initial encounter 995.53 (T76.22XD) Subsequent encounter Other Circumstances Related to Child Sexual Abuse (718) V61.21 (269.010) Encounter for mental health services for victim of child sexual V61.21 (269.020) Encounter for mental health services for victim of nonparental V15.41 (262.810) Personal history (past history) of sexual abuse in childhood V61 .22 (269.01 1) Encounter for mental health services for perpetrator of parental V62.83 (269.021) Encounter for mental health services for perpetrator of

1	V61 .22 (269.01 1) Encounter for mental health services for perpetrator of parental V62.83 (269.021) Encounter for mental health services for perpetrator of Child Neglect, Confirmed (718) 995.52 (T74.02XA) Initialencounter 995.52 (T74.02XD) Subsequent encounter Child Neglect, Suspected (719) 995.52 (T 76.02XA) Initial encounter 995.52 (T 76.02XD) Subsequent encounter Other Circumstances Related to Child Neglect (719) V61.21 (269.010) Encounter for mental health services for victim of child neglect V61.21 (269.020) Encounter for mental health services for victim of nonparental V15.42 (262.812) Personal history (past history) of neglect in childhood V61.22 (269.011) Encounter for mental health services for perpetrator of parental V62.83 (269.021) Encounter for mental health services for perpetrator of Child Psychological Abuse, Confirmed (719) 995.51 (T 74.32XA) Initial encounter 995.51 (T 74.32XD) Subsequent encounter

1	V62.83 (269.021) Encounter for mental health services for perpetrator of Child Psychological Abuse, Confirmed (719) 995.51 (T 74.32XA) Initial encounter 995.51 (T 74.32XD) Subsequent encounter Child Psychological Abuse, Suspected (719) 995.51 (T 76.32XA) Initial encounter 995.51 (T 76.32XD) Subsequent encounter Other Circumstances Related to Child Psychological Abuse (719) V61.21 (269.010) Encounter for mental health services for victim of child V61.21 (269.020) Encounter for mental health services for victim of nonparental V15.42 (262.811) Personal history (past history) of psychological abuse in V61 .22 (269.011) Encounter for mental health services for perpetrator of parental V62.83 (269.021) Encounter for mental health services for perpetrator of Spouse or Partner Violence, Physical (720) Spouse or Partner Violence, Physical, Confirmed (720) 995.81 (T 74.1 1 XA) Initial encounter 995.81 (T 74.11XD) Subsequent encounter

1	Spouse or Partner Violence, Physical (720) Spouse or Partner Violence, Physical, Confirmed (720) 995.81 (T 74.1 1 XA) Initial encounter 995.81 (T 74.11XD) Subsequent encounter Spouse or Partner Violence, Physical, Suspected (720) 995.81 (T 76.1 1 XA) Initial encounter 995.81 (T76.11XD) Subsequent encounter Other Circumstances Related to Spouse or Partner Violence, Physical (720) V61.11 (269.11) Encounter for mental health services for victim of spouse or partner violence, physical V15.41 (291.410) Personal history (past history) of spouse or partner violence, V61.12 (269.12) Encounter for mental health services for perpetrator of spouse or partner violence, physical Spouse or Partner Violence, Sexual (720) Spouse or Partner Violence, Sexual, Confirmed (720) 995.83 (T 74.21 XA) Initial encounter 995.83 (T 74.21XD) Subsequent encounter Spouse or Partner Violence, Sexual, Suspected (720) 995.83 (T 76.21 XA) Initial encounter 995.83 (T 76.21 XD) Subsequent encounter

1	Spouse or Partner Violence, Sexual, Suspected (720) 995.83 (T 76.21 XA) Initial encounter 995.83 (T 76.21 XD) Subsequent encounter Other Circumstances Related to Spouse 0r Partner Violence, Sexual (720) V61.11 (269.81) Encounter for mental health services for Victim of spouse or partner violence, sexual V15.41 (291.410) Personal history (past history) of spouse or partner violence, V61.12 (269.12) Encounter for mental health services for perpetrator of spouse or partner violence, sexual Spouse or Partner, Neglect (721) Spouse or Partner Neglect, Confirmed (721) 995.85 (T74.01XA) Initial encounter 995.85 (T 74.01 XD) Subsequent encounter Spouse or Partner Neglect, Suspected (721) 995.85 (T 76.01 XA) Initial encounter 995.85 (T76.01XD) Subsequent encounter Other Circumstances Related to Spouse or Partner Neglect (721) V61.11 (269.11) Encounter for mental health services for victim of spouse or V15.42 (291.412) Personal history (past history) of spouse or partner neglect

1	V61.11 (269.11) Encounter for mental health services for victim of spouse or V15.42 (291.412) Personal history (past history) of spouse or partner neglect V61.12 (269.12) Encounter for mental health services for perpetrator of spouse Spouse or Partner Abuse, Psychological (721) Spouse or Partner Abuse, Psychological, Confirmed (721) 995.82 (T 74.31XA) Initial encounter 995.82 (T 74.31XD) Subsequent encounter Spouse or Partner Abuse, Psychological, Suspected (721) 995.82 (T 76.31XA) Initial encounter 995.82 (T76.31XD) Subsequent encounter Other Circumstances Related to Spouse or Partner Abuse, Psychological (721) V61.11 (269.11) Encounter for mental health services for victim of spouse or V15.42 (291.411) Personal history (past history) of spouse or partner V61.12 (269.12) Encounter for mental health services for perpetrator of spouse Adult Physical Abuse by Nonspouse or Nonpartner, Confirmed (722) 995.81 (T74.11XA) Initial encounter 995.81 (T74.11XD) Subsequent encounter

1	Adult Physical Abuse by Nonspouse or Nonpartner, Confirmed (722) 995.81 (T74.11XA) Initial encounter 995.81 (T74.11XD) Subsequent encounter Adult Physical Abuse by Nonspouse or Nonpartner, Suspected (722) 995.81 (T76.1 1 XA) Initial encounter 995.81 (T76.11XD) Subsequent encounter Adult Sexual Abuse by Nonspouse or Nonpartner, Confirmed (722) 995.83 (T74.21XA) Initial encounter 995.83 (T 74.21 XD) Subsequent encounter Adult Sexual Abuse by Nonspouse or Nonpartner, Suspected (722) 995.83 (T 76.21 XA) Initial encounter 995.83 (T76.21XD) Subsequent encounter Adult Psychological Abuse by Nonspouse or Nonpartner, Confirmed (722) 995.82 (T 74.31 XA) Initial encounter 995.82 (T 74.31 XD) Subsequent encounter Adult Psychological Abuse by Nonspouse or Nonpartner, Suspected (722) 995.82 (T 76.31 XA) Initial encounter 995.82 (T 76.31 XD) Subsequent encounter Other Circumstances Related to Adult Abuse by Nonspouse or Nonpartner (722)

1	Other Circumstances Related to Adult Abuse by Nonspouse or Nonpartner (722) V65.49 (269.81) Encounter for mental health services for victim of nonspousal V62.83 (269.82) Encounter for mental health services for perpetrator of V62.3 (255.9) Academic or Educational Problem (723) V62.21 (256.82) Problem Related to Current Military Deployment Status (723) V62.29 (256.9) Other Problem Related to Employment (723) V60.0 (259.0) Homelessness (723) V60.1 (259.1) Inadequate Housing (723) V60.89 (259.2) Discord With Neighbor, Lodger, or Landlord (723) V60.6 (259.3) Problem Related to Living in a Residential Institution (724) V60.2 (259.4) Lack of Adequate Food or Safe Drinking Water (724) V60.2 (259.5) Extreme Poverty (724) V60.2 (259.6) Low Income (724) V60.2 (259.7) Insufficient Social Insurance or Welfare Support (724) V60.9 (259.9) Unspecified Housing or Economic Problem (724) Other Problems Related to the Social Environment (724) V62.89 (260.0) Phase of Life Problem (724)

1	V60.9 (259.9) Unspecified Housing or Economic Problem (724) Other Problems Related to the Social Environment (724) V62.89 (260.0) Phase of Life Problem (724) V60.3 (260.2) Problem Related to Living Alone (724) V62.4 (260.3) Acculturation Difficulty (724) V62.4 (260.4) Social Exclusion or Rejection (724) V62.4 (260.5) Target of (Perceived) Adverse Discrimination or Persecution (724) V62.9 (260.9) Unspecified Problem Related to Social Environment (725) Problems Related to Crime or Interaction With the Legal System (725) V62.89 (265.4) Victim of Crime (725) V62.5 (265.0) Conviction in Civil or Criminal Proceedings Without V62.5 (265.1) Imprisonment or Other Incarceration (725) V62.5 (265.2) Problems Related to Release From Prison (725) V62.5 (265.3) Problems Related to Other Legal Circumstances (725) V65.49 (270.9) Sex Counseling (725) V65.40 (271.9) Other Counseling or Consultation (725) Problems Related to Other Psychosocial, Personal, and Environmental

1	V65.49 (270.9) Sex Counseling (725) V65.40 (271.9) Other Counseling or Consultation (725) Problems Related to Other Psychosocial, Personal, and Environmental V62.89 (265.8) Religious or Spiritual Problem (725) V61.7 (264.0) Problems Related to Unwanted Pregnancy (725) V61.5 (264.1) Problems Related to Multiparity (725) V62.89 (264.4) Discord With Social Service Provider, Including Probation Officer, Case Manager, or Social Services Worker (725) V62.89 (265.4) Victim of Terrorism or Torture (725) V62.22 (265.5) Exposure to Disaster, War, or Other Hostilities (725) V62.89 (265.8) Other Problem Related to Psychosocial Circumstances (725) V62.9 (265.9) Unspecified Problem Related to Unspecified Psychosocial Other Circumstances of Personal History (726) v15.49 (291.49) v15.59 (291.5) V62.22 (291.82) v15.89 (291.89) ve9.9 (272.9) v71.o1 (272.811) V71.02 (272.810) Other Personal History of Psychological Trauma (726) Personal History of Self-Harm (726)

1	V62.22 (291.82) v15.89 (291.89) ve9.9 (272.9) v71.o1 (272.811) V71.02 (272.810) Other Personal History of Psychological Trauma (726) Personal History of Self-Harm (726) Personal History of Military Deployment (726) Problem Related to Lifestyle (726) Problems Related to Access to Medical and Other Health Care (726) V63.9 (275.3) V63.8 (275.4) Unavailability or Inaccessibility of Health Care Facilities (726) Unavailability or Inaccessibility of Other Helping Agencies (726) Nonadherence to Medical Treatment (726) V15.81 (291.19) 278.00 (E669) V65.2 (276.5) V40.31 (291.83) V62.89 (R4133) Nonadherence to Medical Treatment (726) The American Psychiatric Association’s Diagnostic and Statistical Manual of

1	The American Psychiatric Association’s Diagnostic and Statistical Manual of Mental Disorders (DSM) is a classification of mental disorders with associated criteria de- signed to facilitate more reliable diagnoses of these disorders. With successive editions over the past 60 years, it has become a standard reference for clinical practice in the mental health field. Since a complete description of the underlying pathological processes is not possible for most mental disorders, it is important to emphasize that the current diagnos- tic criteria are the best available description of how mental disorders are expressed and can be recognized by trained clinicians. DSM is intended to serve as a practical, functional, and ﬂexible guide for organizing information that can aid in the accurate diagnosis and treatment of mental disorders. It is a tool for clinicians, an essential educational resource for students and practitioners, and a reference for researchers in the field.

1	Although this edition of DSM was designed first and foremost to be a useful guide to clinical practice, as an official nomenclature it must be applicable in a wide diversity of contexts. DSM has been used by clinicians and researchers from different orientations (bi- ological, psychodynamic, cognitive, behavioral, interpersonal, family/systems), all of whom strive for a common language to communicate the essential characteristics of men- tal disorders presented by their patients. The information is of value to all professionals associated with various aspects of mental health care, including psychiatrists, other physicians, psychologists, social workers, nurses, counselors, forensic and legal special- ists, occupational and rehabilitation therapists, and other health professionals. The criteria are concise and explicit and intended to facilitate an objective assessment of symptom pre- sentations in a variety of clinical settings—inpatient, outpatient, partial hospital, consul-

1	The criteria are concise and explicit and intended to facilitate an objective assessment of symptom pre- sentations in a variety of clinical settings—inpatient, outpatient, partial hospital, consul- tation-liaison, clinical, private practice, and primary care—as well in general community epidemiological studies of mental disorders. DSM-5 is also a tool for collecting and com- rates. Finally, the criteria and corresponding text serve as a textbook for students early in their profession who need a structured way to understand and diagnose mental disorders as well as for seasoned professionals encountering rare disorders for the first time. Fortu- nately, all of these uses are mutually compatible.

1	These diverse needs and interests were taken into consideration in planning DSM-S. The classification of disorders is harmonized with the World Health Organization’s Inter- national Classification ofDiseuses (ICD), the official coding system used in the United States, so that the DSM criteria define disorders identified by ICD diagnostic names and code numbers. In DSM-5, both ICD—9-CM and ICD-lO-CM codes (the latter scheduled for adop- tion in October 2014) are attached to the relevant disorders in the classification.

1	Although DSM-S remains a categorical classification of separate disorders, we recog— nize that mental disorders do not always fit completely within the boundaries of a single disorder. Some symptom domains, such as depression and anxiety, involve multiple di- of disorders. In recognition of this reality, the disorders included in DSM-S were reordered into a revised organizational structure meant to stimulate new clinical perspectives. This new structure corresponds with the organizational arrangement of disorders planned for

1	ICD-11 scheduled for release in 2015. Other enhancements have been introduced to pro- mote ease of use across all settings: 0 Representation of developmental issues related to diagnosis. The change in chapter organization better reﬂects a lifespan approach, with disorders more frequently diag- nosed in childhood (e.g., neurodevelopmental disorders) at the beginning of the man- ual and disorders more applicable to older adulthood (e.g., neurocognitive disorders) at the end of the manual. Also, within the text, subheadings on development and course provide descriptions of how disorder presentations may change across the lifespan.

1	Age-related factors specific to diagnosis (e.g., symptom presentation and prevalence differences in certain age groups) are also included in the text. For added emphasis, these age-related factors have been added to the criteria themselves where applicable (e.g., in the criteria sets for insomnia disorder and posttraumatic stress disorder, spe- cific criteria describe how symptoms might be expressed in children). Likewise, gender and cultural issues have been integrated into the disorders where applicable.

1	0 Integration of scientific findings from the latest research in genetics and neuroimag- ing. The revised chapter structure was informed by recent research in neuroscience and by emerging genetic linkages between diagnostic groups. Genetic and physiological risk factors, prognostic indicators, and some putative diagnostic markers are high- lighted in the text. This new structure should improve clinicians’ ability to identify di- agnoses in a disorder spectrum based on common neurocircuitry, genetic vulnerability, and environmental exposures.

1	0 Consolidation of autistic disorder, Asperger’s disorder, and pervasive developmen- tal disorder into autism spectrum disorder. Symptoms of these disorders represent a single continuum of mild to severe impairments in the two domains of social commu- ders. This change is designed to improve the sensitivity and specificity of the criteria for the diagnosis of autism spectrum disorder and to identify more focused treatment tar- gets for the specific impairments identified.

1	' Streamlined classification of bipolar and depressive disorders. Bipolar and depres— sive disorders are the most commonly diagnosed conditions in psychiatry. It was there- fore important to streamline the presentation of these disorders to enhance both clinical and educational use. Rather than separating the definition of manic, hypomanic, and major depressive episodes from the definition of bipolar I disorder, bipolar II disorder, and major depressive disorder as in the previous edition, we included all of the com- ponent criteria within the respective criteria for each disorder. This approach will facil- itate bedside diagnosis and treatment of these important disorders. Likewise, the provide far greater clinical guidance than was previously provided in the simple be- reavement exclusion criterion. The new specifiers of anxious distress and mixed fea- tures are now fully described in the narrative on specifier variations that accompanies the criteria for these disorders.

1	' Restructuring of substance use disorders for consistency and clarity. The categories an overarching new category of substance use disorders—with the specific substance used defining the specific disorders. "Dependence” has been easily confused with the term ”addiction” when, in fact, the tolerance and withdrawal that previously defined dependence are actually very normal responses to prescribed medications that affect the central nervous system and do not necessarily indicate the presence of an addiction. By revising and clarifying these criteria in DSM-S, we hope to alleviate some of the widespread misunderstanding about these issues.

1	By revising and clarifying these criteria in DSM-S, we hope to alleviate some of the widespread misunderstanding about these issues. 0 Enhanced specificity for major and mild neurocognitive disorders. Given the explo- sion in neuroscience, neuropsychology, and brain imaging over the past 20 years, it was critical to convey the current state-of-the-art in the diagnosis of specific types of disor- ders that were previously referred to as the "dementias” or organic brain diseases. Bi- specific molecular genetic findings for rare variants of Alzheimer’s disease and Hun- tington’s disease have greatly advanced clinical diagnoses, and these disorders and others have now been separated into specific subtypes.

1	9 Transition in conceptualizing personality disorders. Although the benefits of a more dimensional approach to personality disorders have been identified in previous edi- tions, the transition from a categorical diagnostic system of individual disorders to one based on the relative distribution of personality traits has not been widely accepted. In DSM-S, the categorical personality disorders are virtually unchanged from the previous edition. However, an alternative "hybrid" model has been proposed in Section III to guide future research that separates interpersonal functioning assessments and the ex- pression of pathological personality traits for six specific disorders. A more dimensional profile of personality trait expression is also proposed for a trait-specified approach.

1	0 Section 111: new disorders and features. A new section (Section III) has been added to highlight disorders that require further study but are not sufficiently well established to be a part of the official classification of mental disorders for routine clinical use. Dimen- sional measures of symptom severity in 13 symptom domains have also been incorpo- rated to allow for the measurement of symptom levels of varying severity across all diagnostic groups. Likewise, the WHO Disability Assessment Schedule (WHODAS), a standard method for assessing global disability levels for mental disorders that is based on the International Classification of Functioning, Disability and Health (ICF) and is ap- plicable in all of medicine, has been provided to replace the more limited Global As- sessment of Functioning scale. It is our hope that as these measures are implemented over time, they will provide greater accuracy and ﬂexibility in the clinical description of sessments.

1	0 Online enhancements. DSM-5 features online supplemental information. Additional cross-cutting and diagnostic severity measures are available online (www.psychiatry.org/ dsm5), linked to the relevant disorders. In addition, the Cul- tural Formulation Interview, Cultural Formulation Interview—Informant Version, and supplementary modules to the core Cultural Formulation Interview are also included online at www.psychiatry.org/dsm5. These innovations were designed by the leading authorities on mental disorders in the world and were implemented on the basis of their expert review, public commentary, and independent peer review. The 13 work groups, under the direction of the DSM-S Task

1	Force, in conjunction with other review bodies and, eventually, the APA Board of Trust- ees, collectively represent the global expertise of the specialty. This effort was supported by an extensive base of advisors and by the professional staff of the APA Division of Re- search; the names of everyone involved are too numerous to mention here but are listed in the Appendix. We owe tremendous thanks to those who devoted countless hours and in- valuable expertise to this effort to improve the diagnosis of mental disorders. We would especially like to acknowledge the chairs, text coordinators, and members of the 13 work groups, listed in the front of the manual, who spent many hours in this vol- unteer effort to improve the scientific basis of clinical practice over a sustained 6-year pe— riod. Susan K. Schultz, M.D., who served as text editor, worked tirelessly with Emily A.

1	Kuhl, Ph.D., senior science writer and DSM-5 staff text editor, to coordinate the efforts of the work groups into a cohesive whole. William E. Narrow, M.D., M.P.H., led the research group that developed the overall research strategy for DSM-5, including the field trials, that greatly enhanced the evidence base for this revision. In addition, we are grateful to those who contributed so much time to the independent review of the revision proposals, including Kenneth S. Kendler, M.D., and Robert Freedman, M.D., co-chairs of the Scien- tific Review Committee; John S. McIntyre, M.D., and Joel Yager, M.D., co-chairs of the Clinical and Public Health Committee; and Glenn Martin, M.D., chair of the APA Assem- bly review process. Special thanks go to Helena C. Kraemer, Ph.D., for her expert statistical consultation; Michael B. First, M.D., for his valuable input on the coding and review of cri- teria; and Paul S. Appelbaum, M.D., for feedback on forensic issues. Maria N. Ward,

1	M.Ed., RHIT, CCS—P, also helped in verifying all ICD coding. The Summit Group, which included these consultants, the chairs of all review groups, the task force chairs, and the APA executive officers, chaired by Dilip V. Jeste, M.D., provided leadership and vision in helping to achieve compromise and consensus. This level of commitment has contributed to the balance and objectivity that we feel are hallmarks of DSM-5. We especially wish to recognize the outstanding APA Division of Research staff— identified in the Task Force and Work Group listing at the front of this manual—who worked tirelessly to interact with the task force, work groups, advisors, and reviewers to resolve issues, serve as liaisons between the groups, direct and manage the academic and routine clinical practice field trials, and record decisions in this important process. In par- ticular, we appreciate the support and guidance provided by James H. Scully Ir., M.D.,

1	Medical Director and CEO of the APA, through the years and travails of the development process. Finally, we thank the editorial and production staff of American Psychiatric Pub- lishing—specifically, Rebecca Rinehart, Publisher; John McDuffie, Editorial Director; Ann Eng, Senior Editor; Greg Kuny, Managing Editor; and Tammy Cordova, Graphics Design Manager—for their guidance in bringing this all together and creating the final product. It is the culmination of efforts of many talented individuals who dedicated their time, exper- tise, and passion that made DSM-S possible. David I. Kupfer, M.D. Darrel A. Regier, M.D., M.P.H. December 19, 2012 Introduction..................................................5 Cautionary Statement for Forensic Use of DSM-5..................25

1	Darrel A. Regier, M.D., M.P.H. December 19, 2012 Introduction..................................................5 Cautionary Statement for Forensic Use of DSM-5..................25 This section is a basic orientation to the purpose, structure, content, and use of DSM-S. It is not intended to provide an exhaustive account of the evo- lution of DSM-5, but rather to give readers a succinct overview of its key ele- ments. The introductory section describes the public, professional, and expert review process that was used to extensively evaluate the diagnostic criteria presented in Section II. A summary of the DSM-5 structure, harmonization with ICD-11, and the transition to a non-axial system with a new approach to as- sessing disability is also presented. “Use of the Manual" includes “Definition of a Mental Disorder,” forensic considerations, and a brief overview of the diag- nostic process and use of coding and recording procedures.

1	The creation of the fifth edition of Diagnostic and Statistical Manual ofMental Disorders (DSM-S) was a massive undertaking that involved hundreds of people working toward a common goal over a 12-year process. Much thought and deliberation were involved in evaluating the diagnostic criteria, considering the organization of every aspect of the man- ual, and creating new features believed to be most useful to clinicians. All of these efforts were directed toward the goal of enhancing the Clinical usefulness of DSM-5 as a guide in the diagnosis of mental disorders.

1	Reliable diagnoses are essential for guiding treatment recommendations, identifying prevalence rates for mental health service planning, identifying patient groups for clinical and basic research, and documenting important public health information such as mor— bidity and mortality rates. As the understanding of mental disorders and their treatments has evolved, medical, scientific, and clinical professionals have focused on the character- istics of specific disorders and their implications for treatment and research.

1	While DSM has been the cornerstone of substantial progress in reliability, it has been well recognized by both the American Psychiatric Association (APA) and the broad scientific com- munity working on mental disorders that past science was not mature enough to yield fully validated diagnoses—that is, to provide consistent, strong, and objective scientific validators of individual DSM disorders. The science of mental disorders continues to evolve. However, the last two decades since DSM-IV was released have seen real and durable progress in such areas as cognitive neuroscience, brain imaging, epidemiology, and genetics. The DSM-S Task

1	Force overseeing the new edition recognized that research advances will require careful, iter— ative changes if DSM is to maintain its place as the touchstone classification of mental disor— ders. Finding the right balance is critical. Speculative results do not belong in an official nosology, but at the same time, DSM must evolve in the context of other clinical research ini- tiatives in the field. One important aspect of this transition derives from the broad recognition observations. The results of numerous studies of comorbidity and disease transmission in farn— ilies, including twin studies and molecular genetic studies, make strong arguments for what many astute clinicians have long observed: the boundaries between many disorder "catego- ries" are more ﬂuid over the life course than DSM-IV recognized, and many symptoms as- signed to a single disorder may occur, at varying levels of severity, in many other disorders.

1	These findings mean that DSM, like other medical disease classifications, should accomo- date ways to introduce dimensional approaches to mental disorders, including dimensions that cut across current categories. Such an approach should permit a more accurate description of patient presentations and increase the validity of a diagnosis (i.e., the degree to which diag- nostic criteria reﬂect the comprehensive manifestation of an underlying psychopathological disorder). DSM—S is designed to better fill the need of clinicians, patients, families, and re- searchers for a clear and concise description of each mental disorder organized by explicit di— agnostic criteria, supplemented, when appropriate, by dimensional measures that cross diagnostic boundaries, and a brief digest of information about the diagnosis, risk factors, as- sociated features, research advances, and various expressions of the disorder.

1	Clinical training and experience are needed to use DSM for determining a diagnosis. The diagnostic criteria identify symptoms, behaviors, cognitive functions, personality traits, phys- ical signs, syndrome combinations, and durations that require clinical expertise to differenti- ate from normal life variation and transient responses to stress. To facilitate a thorough examination of the range of symptoms present, DSM can serve clinicians as a guide to identify the most prominent symptoms that should be assessed when diagnosing a disorder. Although some mental disorders may have well—defined boundaries around symptom clusters, scien- tific evidence now places many, if not most, disorders on a spectrum with closely related dis- orders that have shared symptoms, shared genetic and environmental risk factors, and possibly shared neural substrates (perhaps most strongly established for a subset of anxiety disorders by neuroimaging and animal models). In short, we have come to recognize

1	risk factors, and possibly shared neural substrates (perhaps most strongly established for a subset of anxiety disorders by neuroimaging and animal models). In short, we have come to recognize that the boundaries between disorders are more porous than originally perceived.

1	Many health profession and educational groups have been involved in the development and testing of DSM-S, including physicians, psychologists, social workers, nurses, counselors, epidemiologists, statisticians, neuroscientists, and neuropsychologists. Finally, patients, fam- ilies, lawyers, consumer organizations, and advocacy groups have all participated in revising DSM-5 by providing feedback on the mental disorders described in this volume. Their moni- toring of the descriptions and explanatory text is essential to improve understanding, reduce stigma, and advance the treatment and eventual cures for these conditions.

1	The APA first published a predecessor of DSM in 1844, as a statistical classification of in- stitutionalized mental patients. It was designed to improve communication about the types of patients cared for in these hospitals. This forerunner to DSM also was used as a component of the full U.S. census. After World War II, DSM evolved through four major editions into a diagnostic classification system for psychiatrists, other physicians, and other mental health professionals that described the essential features of the full range of mental disorders. The current edition, DSM-5, builds on the goal of its predecessors (most recently, DSM-IV-TR, or Text Revision, published in 2000) of providing guidelines for di- agnoses that can inform treatment and management decisions.

1	In 1999, the APA launched an evaluation of the strengths and weaknesses of DSM based on emerging research that did not support the boundaries established for some mental disor- ders. This effort was coordinated with the World Health Organization (WHO) Division of Mental Health, the World Psychiatric Association, and the National Institute of Mental

1	Mental Health, the World Psychiatric Association, and the National Institute of Mental Health (NIMH) in the form of several conferences, the proceedings of which were published in 2002 in a monograph entitled A Research Agenda for DSM—V. Thereafter, from 2003 to 2008, a cooperative agreement with the APA and the WHO was supported by the NIMH, the Na- tional Institute on Drug Abuse (NTDA), and the National Institute on Alcoholism and Alco- hol Abuse (NIAAA) to convene 13 international DSM-S research planning conferences, involving 400 participants from 39 countries, to review the world literature in specific diag- nostic areas to prepare for revisions in developing both DSM-S and the International Classi— fication of Diseases, 11th Revision (ICD—11). Reports from these conferences formed the basis for future DSM-5 Task Force reviews and set the stage for the new edition of DSM. In 2006, the APA named David I. Kupfer, M.D., as Chair and Darrel A. Regier, M.D.,

1	M.P.H., as Vice-Chair of the DSM-S Task Force. They were charged with recommending chairs for the 13 diagnostic work groups and additional task force members with a multi- disciplinary range of expertise who would oversee the development of DSM-5. An addi- tional vetting process was initiated by the APA Board of Trustees to disclose sources of income and thus avoid conﬂicts of interest by task force and work group members. The full disclosure of all income and research grants from commercial sources, including the phar- maceutical industry, in the previous 3 years, the imposition of an income cap from all com- mercial sources, and the publication of disclosures on a Web site set a new standard for the field. Thereafter, the task force of 28 members was approved in 2007, and appointments of work group advisors with no voting authority were also approved to participate in the pro- cess. A clear concept of the next evolutionary stage for the classification of mental disorders was central

1	work group advisors with no voting authority were also approved to participate in the pro- cess. A clear concept of the next evolutionary stage for the classification of mental disorders was central to the efforts of the task force and the work groups. This vision emerged as the task force and work groups recounted the history of DSM-IV’s classification, its current strengths and limitations, and strategic directions for its revision. An intensive 6—year pro- cess involved conducting literature reviews and secondary analyses, publishing research reports in scientific journals, developing draft diagnostic Criteria, posting preliminary drafts on the DSM-S Web site for public comment, presenting preliminary findings at pro- fessional meetings, performing field trials, and revising criteria and text.

1	Proposals for the revision of DSM-5 diagnostic criteria were developed by members of the work groups on the basis of rationale, scope of change, expected impact on clinical man- agement and public health, strength of the supporting research evidence, overall clarity, and clinical utility. Proposals encompassed changes to diagnostic criteria; the addition of new disorders, subtypes, and specifiers; and the deletion of existing disorders.

1	In the proposals for revisions, strengths and weaknesses in the current criteria and no- sology were first identified. Novel scientific findings over the previous two decades were considered, leading to the creation of a research plan to assess potential changes through literature reviews and secondary data analyses. Four principles guided the draft revisions: 1) DSM-S is primarily intended to be a manual to be used by clinicians, and revisions must by research evidence; 3) where possible, continuity should be maintained with previous editions of DSM; and 4) no a priori constraints should be placed on the degree of change between DSM-IV and DSM-5.

1	Building on the initial literature reviews, work groups identified key issues within their diagnostic areas. Work groups also examined broader methodological concerns, such as the presence of contradictory findings within the literature; development of a re- fined definition of mental disorder; cross-cutting issues relevant to all disorders; and the revision of disorders categorized in DSM-IV as ”not otherwise specified.” Inclusion of a proposal for revision in Section II was informed by consideration of its advantages and disadvantages for public health and clinical utility, the strength of the evidence, and the magnitude of the Change. New diagnoses and disorder subtypes and specifiers were sub- ject to additional stipulations, such as demonstration of reliability (i.e., the degree to which two clinicians could independently arrive at the same diagnosis for a given patient). Dis- orders with low clinical utility and weak validity were considered for deletion. Placement amount of

1	which two clinicians could independently arrive at the same diagnosis for a given patient). Dis- orders with low clinical utility and weak validity were considered for deletion. Placement amount of empirical evidence generated on the diagnosis, diagnostic reliability or valid- ity, presence of clear clinical need, and potential benefit in advancing research.

1	The use of field trials to empirically demonstrate reliability was a noteworthy improvement in- troduced in DSM-III. The design and implementation strategy of the DSM-5 Field Trials rep- resent several changes over approaches used for DSM-III and DSM-IV, particularly in obtaining data on the precision of kappa reliability estimates (a statistical measure that assesses level of agreement between raters that corrects for chance agreement due to prevalence rates) in the context of clinical settings with high levels of diagnostic comorbidity. For DSM-S, field trials were extended by using two distinctive designs: one in large, diverse medical-academic settings, and the other in routine clinical practices. The former capitalized on the need for large sample sizes to test hypotheses on reliability and clinical utility of a range of diagnoses in a variety of patient populations; the latter supplied valuable information about how proposed revisions performed in everyday clinical settings

1	and clinical utility of a range of diagnoses in a variety of patient populations; the latter supplied valuable information about how proposed revisions performed in everyday clinical settings among a diverse sample of DSM users. It is anticipated that future clinical and basic research studies will focus on the validity of the re- vised categorical diagnostic criteria and the underlying dimensional features of these disor- ders (including those now being explored by the NIMI-I Research Domain Criteria initiative).

1	The medical-academic field trials were conducted at 11 North American medical-academic sites and assessed the reliability, feasibility, and clinical utility of select revisions, with priority given to those that represented the greatest degree of change from DSM-IV or those potentially having the greatest public health impact. The full clinical patient populations coming to each site were screened for DSM—IV diagnoses or qualifying symptoms likely to predict several spe- cific DSM-S disorders of interest. Stratified samples of four to seven specific disorders, plus a stratum containing a representative sample of all other diagnoses, were identified for each site.

1	Patients consented to the study and were randomly assigned for a clinical interview by a cli- nician blind to the diagnosis, followed by a second interview with a clinician blind to previous diagnoses. Patients first filled out a computer-assisted inventory of cross-cutting symptoms in more than a dozen psychological domains. These inventories were scored by a central server, and results were provided to Clinicians before they conducted a typical clinical interview (with no structured protocol). Clinicians were required to score the presence of qualifying criteria on a computer-assisted DSM-S diagnostic checklist, determine diagnoses, score the severity of the diagnosis, and submit all data to the central Web-based server. This study design allowed the calculation of the degree to which two independent clinicians could agree on a diagnosis (us- ing the intraclass kappa statistic) and the agreement of a single patient or two different clini- cians on two separate ratings of

1	to which two independent clinicians could agree on a diagnosis (us- ing the intraclass kappa statistic) and the agreement of a single patient or two different clini- cians on two separate ratings of cross-cutting symptoms, personality traits, disability, and diagnostic severity measures (using intraclass correlation coefficients) along with information on the precision of these estimates of reliability. It was also possible to assess the prevalence rates of both DSM-IV and DSM—S conditions in the respective clinical populations.

1	The routine clinical practice field trials involved recruitment of individual psychiatrists and other mental health clinicians. A volunteer sample was recruited that included gener- alist and specialty psychiatrists, psychologists, licensed clinical social workers, counselors, marriage and family therapists, and advanced practice psychiatric mental health nurses. The field trials provided exposure of the proposed DSM-S diagnoses and dimensional mea- sures to a wide range of clinicians to assess their feasibility and clinical utility. In 2010, the APA launched a unique Web site to facilitate public and professional input into

1	DSM-S. All draft diagnostic criteria and proposed changes in organization were posted on www.dsm5.org for a 2-month comment period. Feedback totaled more than 8,000 submis- sions, which were systematically reviewed by each of the 13 work groups, whose members, where appropriate, integrated questions and comments into discussions of draft revisions and plans for field trial testing. After revisions to the initial draft criteria and proposed chapter organization, a second posting occurred in 2011. Work groups considered feedback from both Web postings and the results of the DSM-S Field Trials when drafting proposed final criteria, which were posted on the Web site for a third and final time in 2012. These three iterations of external review produced more than 13,000 individually signed com- ments on the Web site that were received and reviewed by the work groups, plus thousands of organized petition signers for and against some proposed revisions, all of which allowed the task force to

1	ments on the Web site that were received and reviewed by the work groups, plus thousands of organized petition signers for and against some proposed revisions, all of which allowed the task force to actively address concerns of DSM users, as well as patients and advocacy groups, and ensure that clinical utility remained a high priority.

1	The members of the 13 work groups, representing expertise in their respective areas, col- laborated with advisors and reviewers under the overall direction of the DSM-S Task

1	Force to draft the diagnostic criteria and accompanying text. This effort was supported by a team of APA Division of Research staff and developed through a network of text coor- dinators from each work group. The preparation of the text was coordinated by the text editor, working in close collaboration with the work groups and under the direction of the task force chairs. The Scientific Review Committee (SRC) was established to provide a sci- entific peer review process that was external to that of the work groups. The SRC chair, vice-chair, and six committee members were charged with reviewing the degree to which the proposed changes from DSM-IV could be supported with scientific evidence. Each proposal for diagnostic revision required a memorandum of evidence for Change pre- pared by the work group and accompanied by a summary of supportive data organized around validators for the proposed diagnostic criteria (i.e., antecedent validators such as familial aggregation, concurrent

1	the work group and accompanied by a summary of supportive data organized around validators for the proposed diagnostic criteria (i.e., antecedent validators such as familial aggregation, concurrent validators such as biological markers, and prospective validators such as response to treatment or course of illness). The submissions were re- viewed by the SRC and scored according to the strength of the supportive scientific data.

1	Other justifications for change, such as those arising from clinical experience or need or from a conceptual reframing of diagnostic categories, were generally seen as outside the purview of the SRC. The reviewers’ scores, which varied substantially across the different proposals, and an accompanying brief commentary were then returned to the APA Board of Trustees and the work groups for consideration and response.

1	The Clinical and Public Health Committee (CPHC), composed of a chair, vice-chair, and six members, was appointed to consider additional clinical utility, public health, and log- ical clarification issues for criteria that had not yet accumulated the type or level of evi- dence deemed sufficient for change by the SRC. This review process was particularly important for DSM-IV disorders with known deficiencies for which proposed remedies had neither been previously considered in the DSM revision process nor been subjected to replicated research studies. These selected disorders were evaluated by four to five exter- nal reviewers, and the blinded results were reviewed by CPHC members, who in turn made recommendations to the APA Board of Trustees and the work groups.

1	Forensic reviews by the members of the APA Council on Psychiatry and Law were con- ducted for disorders frequently appearing in forensic environments and ones with high potential for inﬂuencing civil and criminal judgments in courtroom settings. Work groups also added forensic experts as advisors in pertinent areas to complement expertise pro- vided by the Council on Psychiatry and Law.

1	The work groups themselves were charged with the responsibility to review the entire re- search literature surrounding a diagnostic area, including old, revised, and new diagnostic cri- teria, in an intensive 6-year review process to assess the pros and cons of making either small iterative changes or major conceptual changes to address the inevitable reification that occurs with diagnostic conceptual approaches that persist over several decades. Such changes in- cluded the merger of previously separate diagnostic areas into more dimensional spectra, such as that which occurred with autism spectrum disorder, substance use disorders, sexual dys- functions, and somatic symptom and related disorders. Other changes included correcting ﬂaws that had become apparent over time in the choice of operational criteria for some disor- ders. These types of changes posed particular challenges to the SRC and CPHC review pro- cesses, which were not constructed to evaluate the validity of DSM-IV

1	of operational criteria for some disor- ders. These types of changes posed particular challenges to the SRC and CPHC review pro- cesses, which were not constructed to evaluate the validity of DSM-IV diagnostic criteria.

1	However, the DSM-S Task Force, which had reviewed proposed changes and had responsi- bility for reviewing the text describing each disorder contemporaneously with the work groups during this period, was in a unique position to render an informed judgment on the sci- entific merits of such revisions. Furthermore, many of these major changes were subject to field trial testing, although comprehensive testing of all proposed changes could not be accommo- dated by such testing because of time limitations and availability of resources. A final recommendation from the task force was then provided to the APA Board of

1	A final recommendation from the task force was then provided to the APA Board of Trustees and the APA Assembly’s Committee on DSM-5 to consider some of the clinical utility and feasibility features of the proposed revisions. The assembly is a deliberative body of the APA representing the district branches and wider membership that is com- posed of psychiatrists from throughout the United States who provide geographic, prac- tice size, and interest-based diversity. The Committee on DSM-5 is a committee made up of a diverse group of assembly leaders.

1	Following all of the preceding review steps, an executive ”summit committee” session was held to consolidate input from review and assembly committee chairs, task force chairs, a forensic advisor, and a statistical advisor, for a preliminary review of each disor- der by the assembly and APA Board of Trustees executive committees. This preceded a preliminary review by the full APA Board of Trustees. The assembly voted, in November 2012, to recommend that the board approve the publication of DSM-5, and the APA Board of Trustees approved its publication in December 2012. The many experts, reviewers, and advisors who contributed to this process are listed in the Appendix.

1	The individual disorder definitions that constitute the operationalized sets of diagnostic criteria provide the core of DSM—5 for clinical and research purposes. These criteria have been subjected to scientific review, albeit to varying degrees, and many disorders have un- dergone field testing for interrater reliability. In contrast, the classification of disorders (the way in which disorders are grouped, which provides a high—level organization for the man- ual) has not generally been thought of as scientifically significant, despite the fact that judg- ments had to be made when disorders were initially divided into chapters for DSM-III.

1	DSM is a medical classification of disorders and as such serves as a historically deter- mined cognitive schema imposed on clinical and scientific information to increase its com- prehensibility and utility. Not surprisingly, as the foundational science that ultimately led to DSM-III has approached a half—century in age, challenges have begun to emerge for cli- nicians and scientists alike that are inherent in the DSM structure rather than in the de- scription of any single disorder. These challenges include high rates of comorbidity within and across DSM chapters, an excessive use of and need to rely on "not otherwise specified” (NOS) criteria, and a growing inability to integrate DSM disorders with the results of ge— netic studies and other scientific findings. As the APA and the WHO began to plan their respective revisions of the DSM and the

1	As the APA and the WHO began to plan their respective revisions of the DSM and the International Classification of Disorders (ICD), both considered the possibility of improving clinical utility (e.g., by helping to explain apparent comorbidity) and facilitating scientific investigation by rethinking the organizational structures of both publications in a linear system designated by alphanumeric codes that sequence chapters according to some ra- tional and relational structure. It was critical to both the DSM-S Task Force and the WHO

1	International Advisory Group on the revision of the ICD—10 Section on Mental and Behav- ioral Disorders that the revisions to the organization enhance clinical utility and remain within the bounds of well-replicated scientific information. Although the need for reform seemed apparent, it was important to respect the state of the science as well as the chal- lenge that overly rapid change would pose for the clinical and research communities. In that spirit, revision of the organization was approached as a conservative, evolutionary di- agnostic reform that would be guided by emerging scientific evidence on the relationships between disorder groups. By reordering and regrouping the existing disorders, the re- vised structure is meant to stimulate new clinical perspectives and to encourage research- ers to identify the psychological and physiological cross-cutting factors that are not bound by strict categorical designations.

1	The use of DSM criteria has the clear virtue of creating a common language for com- munication between clinicians about the diagnosis of disorders. The official criteria and disorders that were determined to have accepted clinical applicability are located in Sec- tion II of the manual. However, it should be noted that these diagnostic criteria and their relationships within the classification are based on current research and may need to be modified as new evidence' is gathered by future research both within and across the do- mains of proposed disorders. “Conditions for Further Study,” described 1n Section III, are those for which we determined that the scientific evidence 15 not yet available to support widespread clinical use. These diagnostic criteria are included to highlight the evolution and direction of scientific advances in these areas to stimulate further research.

1	With any ongoing review process, especially one of this complexity, different viewpoints emerge, and an effort was made to consider various viewpoints and, when warranted, ac- commodate them. For example, personality disorders are included in both Sections II and III. Section II represents an update of the text associated with the same criteria found in DSM-IV-TR, whereas Section 111 includes the proposed research model for personality dis- order diagnosis and conceptualization developed by the DSM-S Personality and Personality Disorders Work Group. As this field evolves, it is hoped that both versions will serve clin- ical practice and research initiatives.

1	Disorders Work Group. As this field evolves, it is hoped that both versions will serve clin- ical practice and research initiatives. The groups tasked with revising the DSM and ICD systems shared the overarching goal of harmonizing the two classifications as much as possible, for the following reasons: . The existence of two major classifications of mental disorders hinders the collection and use of national health statistics, the design of clinical trials aimed at developing new treatments, and the consideration of global applicability of the results by international regulatory agencies. 0 More broadly, the existence of two classifications complicates attempts to replicate sci- entific results across national boundaries. 0 Even when the intention was to identify identical patient populations, DSM-IV and ICD-10 diagnoses did not always agree.

1	0 Even when the intention was to identify identical patient populations, DSM-IV and ICD-10 diagnoses did not always agree. Early in the course of the revisions, it became apparent that a shared organizational structure would help harmonize the classifications. In fact, the use of a shared framework helped to integrate the work of DSM and ICD work groups and to focus on scientific is- sues. The DSM-S organization and the proposed linear structure of the ICD-11 have been endorsed by the leadership of the NIMH Research Domain Criteria (RDoC) project as con- sistent with the initial overall structure of that project.

1	Of course, principled disagreements on the classification of psychopathology and on specific criteria for certain disorders were expected given the current state of scientific knowledge. However, most of the salient differences between the DSM and the ICD classi- fications do not reﬂect real scientific differences, but rather represent historical by-products of independent committee processes.

1	To the surprise of participants in both revision processes, large sections of the content fell relatively easily into place, reﬂecting real strengths in some areas of the scientific lit- erature, such as epidemiology, analyses of comorbidity, twin studies, and certain other ge- netically informed designs. When disparities emerged, they almost always reﬂected the need to make a judgment about where to place a disorder in the face of incomplete—or, more often, conﬂicting—data. Thus, for example, on the basis of patterns of symptoms, co- morbidity, and shared risk factors, attention-deficit/hyperactivity disorder (ADHD) was placed with neurodevelopmental disorders, but the same data also supported strong ar- guments to place ADHD within disruptive, impulse-control, and conduct disorders.

1	These issues were settled with the preponderance of evidence (most notably validators ap- proved by the DSM-5 Task Force). The work groups recognize, however, that future dis- coveries might change the placement as well as the contours of individual disorders and, furthermore, that the simple and linear organization that best supports clinical practice may not fully capture the complexity and heterogeneity of mental disorders. The revised organization is coordinated with the mental and behavioral disorders chapter (Chapter V) of ICD-11, which will utilize an expanded numeric—alphanumeric coding system. How- ever, the official coding system in use in the United States at the time of publication of this manual is that of the International Classification of Diseases, Ninth Revision, Clinical Modifica- tion (ICD-9-CM)—the U.S. adaptation of ICD-9. International Classification of Diseases, Tenth

1	Revision, Clinical Modification (ICD-lO-CM), adapted from ICD-10, is scheduled for imple- mentation in the United States in October 2014. Given the impending release of ICD-ll, it was decided that this iteration, and not ICD—lO, would be the most relevant on which to focus harmonization. However, given that adoption of the ICD-9-CM coding system will remain at the time of the DSM-5 release, it will be necessary to use the ICD-9-CM codes. Further- more, given that DSM-S’s organizational structure reﬂects the anticipated structure of ICD-11, the eventual ICD-11 codes will follow the sequential order of diagnoses in the DSM-5 chapter structure more closely. At present, both the ICD—9-CM and the ICD-10-CM codes have been indicated for each disorder. These codes will not be in sequential order throughout the manual because they were assigned to complement earlier organizational structures. Dimensional Approach to Diagnosis

1	Structural problems rooted in the basic design of the previous DSM Classification, con- structed of a large number of narrow diagnostic categories, have emerged in both clinical practice and research. Relevant evidence comes from diverse sources, including studies of comorbidity and the substantial need for not otherwise specified diagnoses, which repre- sent the majority of diagnoses in areas such as eating disorders, personality disorders, and autism spectrum disorder. Studies of both genetic and environmental risk factors, whether based on twin designs, familial transmission, or molecular analyses, also raise concerns about the categorical structure of the DSM system. Because the previous DSM approach considered each diagnosis as categorically separate from health and from other diagnoses, it did not capture the widespread sharing of symptoms and risk factors across many dis- orders that is apparent in studies of comorbidity. Earlier editions of DSM focused on ex- cluding

1	diagnoses, it did not capture the widespread sharing of symptoms and risk factors across many dis- orders that is apparent in studies of comorbidity. Earlier editions of DSM focused on ex- cluding false-positive results from diagnoses; thus, its categories were overly narrow, as is apparent from the widespread need to use NOS diagnoses. Indeed, the once plausible goal agnostic categories that did not capture clinical reality, symptom heterogeneity within dis- orders, and significant sharing of symptoms across multiple disorders. The historical aspiration of achieving diagnostic homogeneity by progressive subtyping within disorder categories no longer is sensible; like most common human ills, mental disorders are het- erogeneous at many levels, ranging from genetic risk factors to symptoms.

1	Related to recommendations about alterations in the chapter structure of DSM-S, mem- bers of the diagnostic spectra study group examined whether scientific validators could inform possible new groupings of related disorders within the existing categorical frame— work. Eleven such indicators were recommended for this purpose: shared neural sub- strates, family traits, genetic risk factors, specific environmental risk factors, biomarkers, temperamental antecedents, abnormalities of emotional or cognitive processing, symptom similarity, course of illness, high comorbidity, and shared treatment response. These indi- cators served as empirical guidelines to inform decision making by the work groups and the task force about how to cluster disorders to maximize their validity and clinical utility.

1	A series of papers was developed and published in a prominent international journal (Psychological Medicine, Vol. 39, 2009) as part of both the DSM-S and the ICD-ll develop- mental processes to document that such validators were most useful for suggesting large groupings of disorders rather than for ”validating” individual disorder diagnostic criteria. The regrouping of mental disorders in DSM-S is intended to enable future research to en- hance understanding of disease origins and pathophysiological commonalities between time to continuale assess validity. Ongoing revisions of DSM-S will make it a “living doc- ument,” adaptable to future discoveries in neurobiology, genetics, and epidemiology.

1	On the basis of the published findings of this common DSM-5 and ICD-11 analysis, it was demonstrated that clustering of disorders according to what has been termed internal- izing and externalizing factors represents an empirically supported framework. Within both the internalizing group (representing disorders with prominent anxiety, depressive, and somatic symptoms) and the externalizing group (representing disorders with prominent impulsive, disruptive conduct, and substance use symptoms), the sharing of genetic and environmental risk factors, as shown by twin studies, likely explains much of the system— atic comorbidities seen in both clinical and community samples. The adjacent placement of ”intemalizing disorders,” characterized by depressed mood, anxiety, and related physio- logical and cognitive symptoms, should aid in developing new diagnostic approaches, in- cluding dimensional approaches, while facilitating the identification of biological markers.

1	Similarly, adjacencies of the “externalizing group,” including disorders exhibiting antiso- cial behaviors, conduct disturbances, addictions, and impulse-control disorders, should en- courage advances in identifying diagnoses, markers, and underlying mechanisms. Despite the problem posed by categorical diagnoses, the DSM-5 Task Force recognized that it is premature scientifically to propose alternative definitions for most disorders. The organizational structure is meant to serve as a bridge to new diagnostic approaches with- out disrupting current clinical practice or research. With support from DSM-associated training materials, the National Institutes of Health other funding agencies, and scientific publications, the more dimensional DSM-S approach and organizational structure can fa- within the proposed chapters and across adjacent chapters. Such a reformulation of re- search goals should also keep DSM-S central to the development of dimensional approaches coming years.

1	To improve clinical utility, DSM—5 is organized on developmental and lifespan consider- ations. It begins with diagnoses thought to reﬂect developmental processes that manifest early in life (e.g., neurodevelopmental and schizophrenia spectrum and other psychotic disorders), followed by diagnoses that more commonly manifest in adolescence and young adulthood (e.g., bipolar, depressive, and anxiety disorders), and ends with diagno- ses relevant to adulthood and later life (e.g., neurocognitive disorders). A similar approach has been taken, where possible, within each chapter. This organizational structure facili- tates the comprehensive use of lifespan information as a way to assist in diagnostic deci- sion making.

1	The proposed organization of chapters of DSM-5, after the neurodevelopmental disor- ders, is based on groups of internalizing (emotional and somatic) disorders, externalizing disorders, neurocognitive disorders, and other disorders. It is hoped that this organization will encourage further study of underlying pathophysiological processes that give rise to diagnostic comorbidity and symptom heterogeneity. Furthermore, by arranging disorder clusters to mirror clinical reality, DSM-5 should facilitate identification of potential diag- noses by non—mental health specialists, such as primary care physicians. The organizational structure of DSM-5, along with ICD harmonization, is designed to provide better and more ﬂexible diagnostic concepts for the next epoch of research and to serve as a useful guide to clinicians in explaining to patients why they might have received their lifespan. Mental disorders are defined in relation to cultural, social, and familial norms and values.

1	Mental disorders are defined in relation to cultural, social, and familial norms and values. Culture provides interpretive frameworks that shape the experience and expression of the symptoms, signs, and behaviors that are criteria for diagnosis. Culture is transmitted, re- vised, and recreated within the family and other social systems and institutions. Diagnostic assessment must therefore consider whether an individual’s experiences, symptoms, and behaviors differ from sociocultural norms and lead to difficulties in adaptation in the cul- tures of origin and in specific social or familial contexts. Key aspects of culture relevant to di- agnostic classification and assessment have been considered in the development of DSM-S.

1	In Section III, the ”Cultural Formulation” contains a detailed discussion of culture and diagnosis in DSM-5, including tools for in-depth cultural assessment. In the Appendix, the ”Glossary of Cultural Concepts of Distress” provides a description of some common cul- tural syndromes, idioms of distress, and causal explanations relevant to clinical practice.

1	The boundaries between normality and pathology vary across cultures for specific types of behaviors. Thresholds of tolerance for specific symptoms or behaviors differ across cul- tures, social settings, and families. Hence, the level at which an experience becomes prob- lematic or pathological will differ. The judgment that a given behavior is abnormal and requires clinical attention depends on cultural norms that are internalized by the individual and applied by others around them, including family members and clinicians. Awareness of the significance of culture may correct mistaken interpretations of psychopathology, but cul- ture may also contribute to vulnerability and suffering (e.g., by amplifying fears that main- tain panic disorder or health anxiety). Cultural meanings, habits, and traditions can also contribute to either stigma or support in the social and familial response to mental illness.

1	Culture may provide coping strategies that enhance resilience in response to illness, or sug- gest help seeking and options for accessing health care of various types, including alterna- tive and complementary health systems. Culture may inﬂuence acceptance or rejection of a diagnosis and adherence to treatments, affecting the course of illness and recovery. Culture also affects the conduct of the clinical encounter; as a result, cultural differences between the clinician and the patient have implications for the accuracy and acceptance of diagnosis as well as for treatment decisions, prognostic considerations, and clinical outcomes.

1	Historically, the construct of the culture-bound syndrome has been a key interest of cultural psychiatry. In DSM-5, this construct has been replaced by three concepts that offer greater clinical utility: 1. Cultural syndrome is a cluster or group of co-occurring, relatively invariant symptoms found in a specific cultural group, community, or context (e.g., ataque de nervios). The syndrome may or may not be recognized as an illness within the culture (e.g., it might be labeled in various ways), but such cultural patterns of distress and features of illness may nevertheless be recognizable by an outside observer.

1	2. Cultural idiom of distress is a linguistic term, phrase, or way of talking about suffering among individuals of a cultural group (e.g., similar ethnicity and religion) referring to shared concepts of pathology and ways of expressing, communicating, or naming es- sential features of distress (e.g., kufungisisa). An idiom of distress need not be associated with specific symptoms, syndromes, or perceived causes. It may be used to convey a wide range of discomfort, including everyday experiences, subclinical conditions, or suffering due to social circumstances rather than mental disorders. For example, most cultures have common bodily idioms of distress used to express a wide range of suf- fering and concerns.

1	3. Cultural explanation or perceived cause is a label, attribution, or feature of an explanatory model that provides a culturally conceived etiology or cause for symptoms, illness, or distress (e.g., maladi moun). Causal explanations may be salient features of folk classi- fications of disease used by laypersons or healers. These three concepts (for which discussion and examples are provided in Section III and the Appendix) suggest cultural ways of understanding and describing illness experi- ences that can be elicited in the clinical encounter. They inﬂuence symptomatology, help seeking, clinical presentations, expectations of treatment, illness adaptation, and treat- ment response. The same cultural term often serves more than one of these functions. Sex and gender differences as they relate to the causes and expression of medical conditions are established for a number of diseases, including selected mental disorders. Revisions to

1	Sex and gender differences as they relate to the causes and expression of medical conditions are established for a number of diseases, including selected mental disorders. Revisions to DSM—S included review of potential differences between men and women in the expression of mental illness. In terms of nomenclature, sex d(fierences are variations attributable to an individual’s reproductive organs and XX or XY chromosomal complement. Gender diﬂer— ences are variations that result from biological sex as well as an individual’s self—represen- tation that includes the psychological, behavioral, and social consequences of one’s perceived gender. The term gender dz'fi’erences is used in DSM-S because, more commonly, the differences between men and women are a result of both biological sex and individual self-representation. However, some of the differences are based on only biological sex.

1	Gender can inﬂuence illness in a variety of ways. First, it may exclusively determine whether an individual is at risk for a disorder (e.g., as in premenstrual dysphoric disor- der). Second, gender may moderate the overall risk for development of a disorder as shown by marked gender differences in the prevalence and incidence rates for selected mental disorders. Third, gender may inﬂuence the likelihood that particular symptoms of a disorder are experienced by an individual. Attention-deficit/hyperactivity disorder is an example of a disorder with differences in presentation that are most commonly expe- rienced by boys or girls. Gender likely has other effects on the experience of a disorder that are indirectly relevant to psychiatric diagnosis. It may be that certain symptoms are more readily endorsed by men or women, and that this contributes to differences in service pro- vision (e.g., women may be more likely to recognize a depressive, bipolar, or anxiety dis- order and endorse a

1	endorsed by men or women, and that this contributes to differences in service pro- vision (e.g., women may be more likely to recognize a depressive, bipolar, or anxiety dis- order and endorse a more comprehensive list of symptoms than men).

1	Reproductive life cycle events, including estrogen variations, also contribute to gender differences in risk and expression of illness. Thus, a specifier for postpartum onset of mania for the onset of an illness episode. In the case of sleep and energy, alterations are often nor- mative postpartum and thus may have lower diagnostic reliability in postpartum women. The manual is configured to include information on gender at multiple levels. If there are gender-specific symptoms, they have been added to the diagnostic criteria. A gender- related specifier, such as perinatal onset of a mood episode, provides additional informa- tion on gender and diagnosis. Finally, other issues that are pertinent to diagnosis and gen- der considerations can be found in the section ”Gender-Related Diagnostic Issues.” Use of Other Specified and Unspecified Disorders

1	To enhance diagnostic specificity, DSM-5 replaces the previous NOS designation with two options for clinical use: other specified disorder and unspecified disorder. The other specified disorder category is provided to allow the clinician to communicate the specific reason that the presentation does not meet the criteria for any specific category within a diagnos- tic class. This is done by recording the name of the category, followed by the specific rea- son. For example, for an individual with clinically significant depressive symptoms lasting 4 weeks but whose symptomatology falls short of the diagnostic threshold for a major depressive episode, the clinician would record ”other specified depressive disorder, depressive episode with insufficient symptoms." If the clinician chooses not to specify the reason that the criteria are not met for a specific disorder, then ”unspecified depressive disorder” would be diagnosed. Note that the differentiation between other specified and

1	not to specify the reason that the criteria are not met for a specific disorder, then ”unspecified depressive disorder” would be diagnosed. Note that the differentiation between other specified and unspecified disorders is based on the clinician’s decision, providing maximum ﬂexibility for diagnosis. Clinicians do not have to differentiate between other specified and unspec- ified disorders based on some feature of the presentation itself. When the clinician deter- mines that there is evidence to specify the nature of the clinical presentation, the other specified diagnosis can be given. When the clinician is not able to further specify and de- scribe the clinical presentation, the unspecified diagnosis can be given. This is left entirely up to clinical judgment.

1	For a more detailed discussion of how to use other specified and unspecified designa- tions, see ”Use of the Manual" in Section I. The Multiaxial System Despite widespread use and its adoption by certain insurance and governmental agencies, the multiaxial system in DSM-IV was not required to make a mental disorder diagnosis. A nonaxial assessment system was also included that simply listed the appropriate Axis I, II, and III disorders and conditions without axial designations. DSM-S has moved to a nonax- ial documentation of diagnosis (formerly Axes I, II, and III), with separate notations for

1	Axis V). This revision is consistent with the DSM-IV text that states, ”The multiaxial dis- tinction among Axis I, Axis II, and Axis III disorders does not imply that there are funda- mental differences in their conceptualization, that mental disorders are unrelated to physical or biological factors or processes, or that general medical conditions are unrelated to behavioral or psychosocial factors or processes.” The approach of separately noting di- agnosis from psychosocial and contextual factors is also consistent with established WHO and ICD guidance to consider the individual’s functional status separately from his or her diagnoses or symptom status. In DSM-5, Axis III has been combined with Axes I and II. Clinicians should continue to list medical conditions that are important to the understand- ing or management of an individual’s mental disorder(s).

1	Clinicians should continue to list medical conditions that are important to the understand- ing or management of an individual’s mental disorder(s). diagnosis, treatment, and prognosis of mental disorders. Although this axis provided helpful information, even if it was not used as frequently as intended, the DSM-S Task Force recommended that DSM-S should not develop its own classification of psychosocial and environmental problems, but rather use a selected set of the ICD—9—CM V codes and the new Z codes contained in ICD-IO-CM. The ICD-IO Z codes were examined to deter- mine which are most relevant to mental disorders and also to identify gaps.

1	DSM-IV Axis V consisted of the Global Assessment of Functioning (GAF) scale, repre- senting the clinician's judgment of the individual’s overall level of ”functioning on a hy- pothetical continuum of mental health—illness.” It was recommended that the GAP be dropped from DSM-5 for several reasons, including its conceptual lack of clarity (i.e., in- cluding symptoms, suicide risk, and disabilities in its descriptors) and questionable psy- chometrics in routine practice. In order to provide a global measure of disability, the WHO Disability Assessment Schedule (WHODAS) is included, for further study, in Section III of

1	Disability Assessment Schedule (WHODAS) is included, for further study, in Section III of DSM—S (see the chapter ”Assessment Measures”). The WHODAS is based on the Interna- tional Classification of Functioning, Disability and Health (ICF) for use across all of medicine and health care. The WHODAS (version 2.0), and a modification developed for children/ adolescents and their parents by the Impairment and Disability Study Group were in- cluded in the DSM-S field trial. It was challenging to determine what to include in the print version of DSM-S to be most clinically relevant and useful and at the same time maintain a manageable size. For this reason, the inclusion of clinical rating scales and measures in the print edition is limited to those considered most relevant. Additional assessment measures used in the field trials are available online (www.psychiatry.org/dsm5), linked to the relevant disorders. The

1	Cultural Formulation Interview, Cultural Formulation Interview—Informant Version, and supplementary modules to the core Cultural Formulation Interview are also available on- line at www.psychiatry.org/ dsm5. DSM-5 is available as an online subscription at PsychiatryOnlineorg as well as an e— book. The online component contains modules and assessment tools to enhance the diag- nostic criteria and text. Also available online is a complete set of supportive references as well as additional helpful information. The organizational structure of DSM-S, its use of dimensional measures, and compatibility with ICD codes will allow it to be readily adapt- able to future scientific discoveries and refinements in its clinical utility. DSM-5 will be an- alyzed over time to continually assess its validity and enhance its value to clinicians. Use of the Manual The introduction contains much of the history and developmental process of the

1	DSM-S revision. This section is designed to provide a practical guide to using DSM-5, par- ticularly in clinical practice. The primary purpose of DSM-5 is to assist trained clinicians in the diagnosis of their patients’ mental disorders as part of a case formulation assess- ment that leads to a fully informed treatment plan for each individual. The symptoms con- tained in the respective diagnostic criteria sets do not constitute comprehensive definitions of underlying disorders, which encompass cognitive, emotional, behavioral, and physiological processes that are far more complex than can be described in these brief summaries. Rather, they are intended to summarize characteristic syndromes of signs and symptoms that point to an underlying disorder with a characteristic developmental his- tory, biological and environmental risk factors, neuropsychological and physiological cor- relates, and typical clinical course. Approach to Clinical Case Formulation

1	The case formulation for any given patient must involve a careful clinical history and con- cise summary of the social, psychological, and biological factors that may have contrib- uted to developing a given mental disorder. Hence, it is not sufficient to simply check off the symptoms in the diagnostic criteria to make a mental disorder diagnosis. Although a systematic check for the presence of these criteria as they apply to each patient will assure a more reliable assessment, the relative severity and valence of individual criteria and their contribution to a diagnosis require clinical judgment. The symptoms in our diagnos- tic criteria are part of the relatively limited repertoire of human emotional responses to in- ternal and external stresses that are generally maintained in a homeostatic balance without a disruption in normal functioning. It requires clinical training to recognize when the com- bination of predisposing, precipitating, perpetuating, and protective factors has

1	balance without a disruption in normal functioning. It requires clinical training to recognize when the com- bination of predisposing, precipitating, perpetuating, and protective factors has resulted ranges. The ultimate goal of a clinical case formulation is to use the available contextual and diagnostic information in developing a comprehensive treatment plan that is in- formed by the individual’s cultural and social context. However, recommendations for the selection and use of the most appropriate evidence-based treatment options for each dis- order are beyond the scope of this manual.

1	Although decades of scientific effort have gone into developing the diagnostic criteria sets for the disorders included in Section II, it is well recognized that this set of categorical diagnoses does not fully describe the full range of mental disorders that individuals ex- perience and present to clinicians on a daily basis throughout the world. As noted previ- ously in the introduction, the range of genetic/ environmental interactions over the course of human development affecting cognitive, emotional and behavioral function is virtually limitless. As a result, it is impossible to capture the full range of psychopathology in the categorical diagnostic categories that we are now using. Hence, it is also necessary to in- exactly into the diagnostic boundaries of disorders in each chapter. In an emergency de- partment setting, it may be possible to identify only the most prominent symptom ex- pressions associated with a particular chapter—for example, delusions, hallucinations, 20 Use

1	In an emergency de- partment setting, it may be possible to identify only the most prominent symptom ex- pressions associated with a particular chapter—for example, delusions, hallucinations, 20 Use 01 the Manual mania, depression, anxiety, substance intoxication, or neurocognitive symptoms—so that an ”unspecified” disorder in that category is identified until a fuller differential diagnosis is possible.

1	Definition of 8 Mental Disorder Each disorder identified in Section II of the manual (excluding those in the chapters enti- tled ”Medication—Induced Movement Disorders and Other Adverse Effects of Medica— tion” and ”Other Conditions That May Be a Focus of Clinical Attention”) must meet the definition of a mental disorder. Although no definition can capture all aspects of all dis- orders in the range contained in DSM-S, the following elements are required: A mental disorder is a syndrome characterized by clinically significant distur- bance in an individual’s cognition, emotion regulation, or behavior that reﬂects a dysfunction in the psychological, biological, or developmental processes un- derlying mental functioning. Mental disorders are usually associated with signif- icant distress or disability in social, occupational, or other important activities.

1	An expectable or culturally approved response to a common stressor or loss, such as the death of a loved one, is not a mental disorder. Socially deviant be- havior (e.g., political, religious, or sexual) and conflicts that are primarily be- tween the individual and society are not mental disorders unless the deviance or conflict results from a dysfunction in the individual, as described above.

1	The diagnosis of a mental disorder should have clinical utility: it should help clinicians to determine prognosis, treatment plans, and potential treatment outcomes for their pa- tients. However, the diagnosis of a mental disorder is not equivalent to a need for treat- ment. Need for treatment is a complex clinical decision that takes into consideration symptom severity, symptom salience (e.g., the presence of suicidal ideation), the patient’s distress (mental pain) associated with the symptom(s), disability related to the patient’s symptoms, risks and benefits of available treatments, and other factors (e.g., psychiatric symptoms complicating other illness). Clinicians may thus encounter individuals whose for treatment or care. The fact that some individuals do not show all symptoms indicative of a diagnosis should not be used to justify limiting their access to appropriate care.

1	Approaches to validating diagnostic criteria for discrete categorical mental disorders have included the following types of evidence: antecedent validators (similar genetic mark- ers, family traits, temperament, and environmental exposure), concurrent validators (simi— lar neural substrates, biomarkers, emotional and cognitive processing, and symptom similarity), and predictive validators (similar clinical course and treatment response). In

1	DSM-5, we recognize that the current diagnostic criteria for any single disorder will not nec- essarily identify a homogeneous group of patients who can be characterized reliably with all of these validators. Available evidence shows that these validators cross existing diagnostic boundaries but tend to congregate more frequently within and across adjacent DSM-S chap- ter groups. Until incontrovertible etiological or pathophysiological mechanisms are identi- fied to fully validate specific disorders or disorder spectra, the most important standard for the DSM-S disorder criteria will be their clinical utility for the assessment of clinical course and treatment response of individuals grouped by a given set of diagnostic criteria. This definition of mental disorder was developed for clinical, public health, and re- search purposes. Additional information is usually required beyond that contained in the

1	This definition of mental disorder was developed for clinical, public health, and re- search purposes. Additional information is usually required beyond that contained in the DSM-5 diagnostic criteria in order to make legal judgments on such issues as criminal re- sponsibility, eligibility for disability compensation, and competency (see ”Cautionary Statement for Forensic Use of DSM-5” elsewhere in this manual). Use of the Manual 21

1	Statement for Forensic Use of DSM-5” elsewhere in this manual). Use of the Manual 21 There have been substantial efforts by the DSM-5 Task Force and the World Health Orga- nization (WHO) to separate the concepts of mental disorder and disability (impairment in social, occupational, or other important areas of functioning). In the WHO system, the In- ternational Classification of Diseases (ICD) covers all diseases and disorders, while the In- ternational Classification of Functioning, Disability and Health (ICF) provides a separate classification of global disability. The WHO Disability Assessment Schedule (WHODAS) is based on the ICF and has proven useful as a standardized measure of disability for men- tal disorders. However, in the absence of clear biological markers or clinically useful mea- surements of severity for many mental disorders, it has not been possible to completely separate normal and pathological symptom expressions contained in diagnostic criteria.

1	This gap in information is particularly problematic in clinical situations in which the pa- tient’s symptom presentation by itself (particularly in mild forms) is not inherently path- ological and may be encountered in individuals for whom a diagnosis of ”mental disorder" would be inappropriate. Therefore, a generic diagnostic criterion requiring dis- tress or disability has been used to establish disorder thresholds, usually worded ”the dis- turbance causes clinically significant distress or impairment in social, occupational, or other important areas of functioning.” The text following the revised definition of a mental tient’s need for treatment. Use of information from family members and other third parties (in addition to the individual) regarding the individual’s performance is recommended when necessary. Elements of a Diagnosis

1	Elements of a Diagnosis Diagnostic criteria are offered as guidelines for making diagnoses, and their use should be informed by clinical judgment. Text descriptions, including introductory sections of each diagnostic chapter, can help support diagnosis (e.g., providing differential diagnoses; de- scribing the criteria more fully under ”Diagnostic Features”).

1	Following the assessment of diagnostic criteria, clinicians should consider the applica- tion of disorder subtypes and / or specifiers as appropriate. Severity and course specifiers should be applied to denote the individual’s current presentation, but only when the full criteria are met. When full criteria are not met, clinicians should consider whether the tion. Where applicable, specific criteria for defining disorder severity (e.g., mild, moder— ate, severe, extreme), descriptive features (e.g., with good to fair insight; in a controlled environment), and course (e.g., in partial remission, in full remission, recurrent) are pro- vided with each diagnosis. On the basis of the clinical interview, text descriptions, criteria, and clinician judgment, a final diagnosis is made. The general convention in DSM-5 is to allow multiple diagnoses to be assigned for those presentations that meet criteria for more than one DSM-S disorder.

1	Subtypes and specifiers (some of which are coded in the fourth, fifth, or sixth digit) are provided for increased specificity. Subtypes define mutually exclusive and jointly exhaus- tive phenomenological subgroupings within a diagnosis and are indicated by the instruc- tion ”Specify whether” in the criteria set. In contrast, specifiers are not intended to be mutually exclusive or jointly exhaustive, and as a consequence, more than one specifier may be given. Specifiers are indicated by the instruction "Specify" or ”Specify if" in the cri- teria set. Specifiers provide an opportunity to define a more homogeneous subgrouping of 22 Use of the Manual individuals with the disorder who share certain features (e.g., major depressive disorder, with mixed features) and to convey information that is relevant to the management of the individual’s disorder, such as the “with other medical comorbidity” specifier in sleep- wake disorders. Although a fifth digit is sometimes assigned to code a

1	is relevant to the management of the individual’s disorder, such as the “with other medical comorbidity” specifier in sleep- wake disorders. Although a fifth digit is sometimes assigned to code a subtype or specifier (e.g., 294.11 [F0281] major neurocognitive disorder due to Alzheimer’s disease, with be- havioral disturbance) or severity (296.21 [F320] major depressive disorder, single episode, mild), the majority of subtypes and specifiers included in DSM-5 cannot be coded within the ICD-9—CM and ICD-lO-CM systems and are indicated only by including the subtype or specifier after the name of the disorder (e.g., social anxiety disorder [social phobia], per- formance type). Note that in some cases, a specifier or subtype is codable in ICD-10-CM but not in ICD-9-CM. Accordingly, in some cases the 4th or 5th character codes for the sub- types or specifiers are provided only for the ICD-lO-CM coding designations.

1	A DSM-5 diagnosis is usually applied to the individual’s current presentation; previ- ous diagnoses from which the individual has recovered should be clearly noted as such. Specifiers indicating course (e.g., in partial remission, in full remission) may be listed after the diagnosis and are indicated in a number of criteria sets. Where available, severity spec- ifiers are provided to guide clinicians in rating the intensity, frequency, duration, symptom count, or other severity indicator of a disorder. Severity specifiers are indicated by the in- struction ”Specify current severity” in the criteria set and include disorder-specific defini- tions. Descriptivefeatures specifiers have also been provided in the criteria set and convey additional information that can inform treatment planning (e.g., obsessive-compulsive disorder, with poor insight). Not all disorders include course, severity, and / or descriptive features specifiers. Conditions That May Be a Focus of Ciinicai Attention

1	In addition to important psychosocial and environmental factors (see ”The Multiaxial Sys- tem” in the ”Introduction” elsewhere in this manual), these chapters in Section II also con- tain other conditions that are not mental disorders but may be encountered by mental health clinicians. These conditions may be listed as a reason for clinical visit in addition to, or in place of, the mental disorders listed in Section II. A separate chapter is devoted to medication-induced disorders and other adverse effects of medication that may be as— sessed and treated by clinicians in mental health practice such as akathisia, tardive dyski- nesia, and dystonia. The description of neuroleptic malignant syndrome is expanded from that provided in DSM—IV-TR to highlight the emergent and potentially life-threatening na- ture of this condition, and a new entry on antidepressant discontinuation syndrome is pro- vided. An additional chapter discusses other conditions that may be a focus of clinical

1	life-threatening na- ture of this condition, and a new entry on antidepressant discontinuation syndrome is pro- vided. An additional chapter discusses other conditions that may be a focus of clinical attention. These include relational problems, problems related to abuse and neglect, prob- lems with adherence to treatment regimens, obesity, antisocial behavior, and malingering.

1	When more than one diagnosis for an individual is given in an inpatient setting, the prin- cipal diagnosis is the condition established after study to be chieﬂy responsible for occa- sioning the admission of the individual. When more than one diagnosis is given for an individual in an outpatient setting, the reason for visit is the condition that is chieﬂy re- sponsible for the ambulatory care medical services received during the visit. In most cases, the principal diagnosis or the reason for visit is also the main focus of attention or treat- ment. It is often difficult (and somewhat arbitrary) to determine which diagnosis is the principal diagnosis or the reason for visit, especially when, for example, a substance- related diagnosis such as alcohol use disorder is accompanied by a non-substance-related diagnosis such as schizophrenia. For example, it may be unclear which diagnosis should

1	Use of the Manual 23 be considered ”principal” for an individual hospitalized with both schizophrenia and a1- cohol use disorder, because each condition may have contributed equally to the need for admission and treatment. The principal diagnosis is indicated by listing it first, and the re- maining disorders are listed in order of focus of attention and treatment. When the prin- cipal diagnosis or reason for visit is a mental disorder due to another medical condition (e.g., major neurocognitive disorder due to Alzheimer’s disease, psychotic disorder due to malignant lung neoplasm), ICD coding rules require that the etiological medical condition be listed first. In that case, the principal diagnosis or reason for visit would be the mental disorder due to the medical condition, the second listed diagnosis. In most cases, the dis- order listed as the principal diagnosis or the reason for visit is followed by the qualifying

1	The specifier ”provisional" can be used when there is a strong presumption that the full criteria will ultimately be met for a disorder but not enough information is available to make a firm diagnosis. The clinician can indicate the diagnostic uncertainty by recording "(provisional)” following the diagnosis. For example, this diagnosis might be used when an individual who appears to have a major depressive disorder is unable to give an ade- quate history, and thus it cannot be established that the full criteria are met. Another use of the term provisional is for those situations in which differential diagnosis depends exclu- sively on the duration of illness. For example, a diagnosis of schizophreniform disorder re- quires a duration of less than 6 months but of at least 1 month and can only be given provisionally if assigned before remission has occurred.

1	Each disorder is accompanied by an identifying diagnostic and statistical code, which is typically used by institutions and agencies for data collection and billing purposes. There are specific recording protocols for these diagnostic codes (identified as coding notes in the text) that were established by WHO, the US. Centers for Medicare and Medicaid Ser— vices (CMS), and the Centers for Disease Control and Prevention’s National Center for

1	Health Statistics to ensure consistent international recording of prevalence and mortality rates for identified health conditions. For most clinicians, the codes are used to identify the diagnosis or reason for visit for CMS and private insurance service claims. The official coding system in use in the United States as of publication of this manual is ICD-9-CM. Of- ficial adoption of ICD-lO-CM is scheduled to take place on October 1, 2014, and these codes, which are shown parenthetically in this manual, should not be used until the offi- cial implementation occurs. Both ICD-9-CM and ICD-IO—CM codes have been listed 1) pre- ceding the name of the disorder in the classification and 2) accompanying the criteria set for each disorder. For some diagnoses (e.g., neurocognitive and substance/medication- induced disorders), the appropriate code depends on further specification and is listed within the criteria set for the disorder, as coding notes, and, in some cases, further clarified in a

1	induced disorders), the appropriate code depends on further specification and is listed within the criteria set for the disorder, as coding notes, and, in some cases, further clarified in a section on recording procedures. The names of some disorders are followed by alter- native terms enclosed in parentheses, which, in most cases, were the DSM-IV names for the disorders.

1	Looking to the Future: The various components of DSM-S are provided to facilitate patient assessment and to aid in developing a comprehensive case formulation. Whereas the diagnostic criteria in Sec- tion II are well-established measures that have undergone extensive review, the assess- 24 Use of the Manual ment tools, a cultural formulation interview, and conditions for further study included in Section III are those for which we determined that the scientific evidence is not yet avail- able to support widespread clinical use. These diagnostic aids and criteria are included to highlight the evolution and direction of scientific advances in these areas and to stimulate further research.

1	Each of the measures in Section III is provided to aid in a comprehensive assessment of individuals that will contribute to a diagnosis and treatment plan tailored to the individ- ual presentation and clinical context. Where cultural dynamics are particularly important for diagnostic assessment, the cultural formulation interview should be considered as a useful aid to communication with the individual. Cross-cutting symptom and diagnosis- specific severity measures provide quantitative ratings of important clinical areas that are designed to be used at the initial evaluation to establish a baseline for comparison with rat- ings on subsequent encounters to monitor changes and inform treatment planning.

1	The use of such measures will undoubtedly be facilitated by digital applications, and the measures are included in Section III to provide for further evaluation and develop- ment. As with each DSM edition, the diagnostic criteria and the DSM-5 classification of mental disorders reﬂect the current consensus on the evolving knowledge in our field. Forensic Use of DSM-5 Although the DSM-5 diagnostic criteria and text are primarily designed to assist clinicians in conducting clinical assessment, case formulation, and treatment planning,

1	Forensic Use of DSM-5 Although the DSM-5 diagnostic criteria and text are primarily designed to assist clinicians in conducting clinical assessment, case formulation, and treatment planning, DSM-S is also used as a reference for the courts and attorneys in assessing the forensic con- sequences of mental disorders. As a result, it is important to note that the definition of mental disorder included in DSM-S was developed to meet the needs of clinicians, public health professionals, and research investigators rather than all of the technical needs of the courts and legal professionals. It is also important to note that DSM-S does not provide treatment guidelines for any given disorder.

1	When used appropriately, diagnoses and diagnostic information can assist legal deci- sion makers in their determinations. For example, when the presence of a mental disorder is the predicate for a subsequent legal determination (e.g., involuntary civil commitment), the use of an established system of diagnosis enhances the value and reliability of the de- termination. By providing a compendium based on a review of the pertinent clinical and research literature, DSM-S may facilitate legal decision makers’ understanding of the rel- evant characteristics of mental disorders. The literature related to diagnoses also serves as a check on ungrounded speculation about mental disorders and about the functioning of a particular individual. Finally, diagnostic information about longitudinal course may im- prove decision making when the legal issue concerns an individual’s mental functioning at a past or future point in time.

1	However, the use of DSM—S should be informed by an awareness of the risks and lim- itations of its use in forensic settings. When DSM-5 categories, criteria, and textual descrip- tions are employed for forensic purposes, there is a risk that diagnostic information will be misused or misunderstood. These dangers arise because of the imperfect fit between the questions of ultimate concern to the law and the information contained in a clinical diagno- sis. In most situations, the clinical diagnosis of a DSM-5 mental disorder such as intellec- tual disability (intellectual developmental disorder), schizophrenia, major neurocognitive disorder, gambling disorder, or pedophilic disorder does not imply that an individual with such a condition meets legal criteria for the presence of a mental disorder or a speci- fied legal standard (e.g., for competence, criminal responsibility, or disability). For the latter, additional information is usually required beyond that contained in the DSM-5

1	disorder or a speci- fied legal standard (e.g., for competence, criminal responsibility, or disability). For the latter, additional information is usually required beyond that contained in the DSM-5 diagnosis, which might include information about the individual’s functional impairments and how these impairments affect the particular abilities in question. It is precisely because impair- ments, abilities, and disabilities vary widely within each diagnostic category that assign- ment of a particular diagnosis does not imply a specific level of impairment or disability.

1	Use of DSM—S to assess for the presence of a mental disorder by nonclinical, nonmed— ical, or otherwise insufficiently trained individuals is not advised. Nonclinical decision tions regarding the etiology or causes of the individual’s mental disorder or the individ- ual’s degree of control over behaviors that may be associated with the disorder. Even when diminished control over one’s behavior is a feature of the disorder, having the diag- nosis in itself does not demonstrate that a particular individual is (or was) unable to con- trol his or her behavior at a particular time. Neurodevelopmental Disorders ................................. 31 Schizophrenia Spectrum and Other Psychotic Disorders ............ 87 Bipolar and Related Disorders ................................. 1 23 Depressive Disorders ........................................ 155 Anxiety Disorders ........................................... 189 Obsessive-Compulsive and Related Disorders .................... 235

1	Anxiety Disorders ........................................... 189 Obsessive-Compulsive and Related Disorders .................... 235 Trauma- and Stressor-Related Disorders ........................ 265 Dissociative Disorders ....................................... 291 Somatic Symptom and Related Disorders ....................... 309 Feeding and Eating Disorders ................................. 329 Elimination Disorders ........................................ 355 Sleep-Wake Disorders ....................................... 361 Sexual Dysfunctions ......................................... 423 Gender Dysphoria ........................................... 451 Disruptive, Impulse-Control, and Conduct Disorders ............... 461 Substance-Related and Addictive Disorders ..................... 481 Neurocognitive Disorders ..................................... 591 Personality Disorders ........................................ 645

1	Neurocognitive Disorders ..................................... 591 Personality Disorders ........................................ 645 Paraphilic Disorders ......................................... 685 Other Mental Disorders ...................................... 707 Other Adverse Effects of Medication .......................... 709 Other Conditions That May Be a Focus of Clinical Attention ......... 715 | his section contains the diagnostic criteria approved for routine clinical use along with the |CD-9-CM codes (|CD-10 codes are shown parenthetically).

1	For each mental disorder, the diagnostic criteria are followed by descriptive text to assist in diagnostic decision making. Where needed, specific recording procedures are presented with the diagnostic criteria to provide guidance in selecting the most appropriate code. In some cases, separate recording pro- cedures for |CD-9-CM and |CD-10—CM are provided. Although not considered as official DSM-5 disorders, medication-induced movement disorders and other adverse effects of medication, as well as other conditions that may be a focus |CD-10-CM 2 codes), are provided to indicate other reasons for a clinical visit such as environmental factors and relational problems. These codes are adapted official DSM-5 diagnoses, but can provide additional context for a clinical for- mulation and treatment plan. These three components—the criteria and their descriptive text, the medication-induced movement disorders and other ad- verse effects of medication, and the descriptions of other conditions

1	plan. These three components—the criteria and their descriptive text, the medication-induced movement disorders and other ad- verse effects of medication, and the descriptions of other conditions that may be a focus of clinical attention—represent the key elements of the clinical di- agnostic process and thus are presented together.

1	The neurodevelopmental disorders are a group of conditions with onset in the developmental period. The disorders typically manifest early in development, often be- fore the child enters grade school, and are characterized by developmental deficits that produce impairments of personal, social, academic, or occupational functioning. The range of developmental deficits varies from very specific limitations of learning or control of executive functions to global impairments of social skills or intelligence. The neurode- velopmental disorders frequently co—occur; for example, individuals with autism spec- trum disorder often have intellectual disability (intellectual developmental disorder), and many children with attention-deficit/hyperactivity disorder (ADHD) also have a specific learning disorder. For some disorders, the clinical presentation includes symptoms of ex- cess as well as deficits and delays in achieving expected milestones. For example, autism spectrum disorder is diagnosed

1	For some disorders, the clinical presentation includes symptoms of ex- cess as well as deficits and delays in achieving expected milestones. For example, autism spectrum disorder is diagnosed only when the characteristic deficits of social communi- cation are accompanied by excessively repetitive behaviors, restricted interests, and insis- tence 0n sameness.

1	Intellectual disability (intellectual developmental disorder) is characterized by deficits in general mental abilities, such as reasoning, problem solving, planning, abstract thinking, judgment, academic learning, and learning from experience. The deficits result in impair- ments of adaptive functioning, such that the individual fails to meet standards of personal independence and social responsibility in one or more aspects of daily life, including com- munication, social participation, academic or occupational functioning, and personal inde- pendence at home or in community settings. Global developmental delay, as its name implies, is diagnosed when an individual fails to meet expected developmental milestones in several areas of intellectual functioning. The diagnosis is used for individuals who are unable to undergo systematic assessments of intellectual functioning, including children who are too young to participate in standardized testing. Intellectual disability may result

1	who are unable to undergo systematic assessments of intellectual functioning, including children who are too young to participate in standardized testing. Intellectual disability may result from an acquired insult during the developmental period from, for example, a severe head injury, in which case a neurocognitive disorder also may be diagnosed.

1	The communication disorders include language disorder, speech sound disorder, so- cial (pragmatic) communication disorder, and childhood-onset ﬂuency disorder (stutter- ing). The first three disorders are characterized by deficits in the development and use of language, speech, and social communication, respectively. Childhood-onset ﬂuency dis- order is characterized by disturbances of the normal fluency and motor production of speech, including repetitive sounds or syllables, prolongation of consonants or vowel sounds, broken words, blocking, or words produced with an excess of physical tension. Like other neurodevelopmental disorders, communication disorders begin early in life and may produce lifelong functional impairments.

1	Like other neurodevelopmental disorders, communication disorders begin early in life and may produce lifelong functional impairments. Autism spectrum disorder is characterized by persistent deficits in social communica- tion and social interaction across multiple contexts, including deficits in social reciprocity, nonverbal communicative behaviors used for social interaction, and skills in developing, maintaining, and understanding relationships. In addition to the social communication deficits, the diagnosis of autism spectrum disorder requires the presence of restricted, re- petitive patterns of behavior, interests, or activities. Because symptoms Change with de- velopment and may be masked by compensatory mechanisms, the diagnostic criteria may be met based on historical information, although the current presentation must cause sig- nificant impairment.

1	Within the diagnosis of autism spectrum disorder, individual clinical characteristics are noted through the use of specifiers (with or without accompanying intellectual impair- ment; with or without accompanying structural language impairment; associated with a known medical/genetic or environmental/acquired condition; associated with another neurodevelopmental, mental, or behavioral disorder), as well as specifiers that describe the autistic symptoms (age at first concern; with or without loss of established skills; sever- ity). These specifiers provide clinicians with an opportunity to individualize the diagnosis and communicate a richer clinical description of the affected individuals. For example, many individuals previously diagnosed with Asperger’s disorder would now receive a diagnosis of autism spectrum disorder without language or intellectual impairment.

1	ADHD is a neurodevelopmental disorder defined by impairing levels of inattent-ion, dis- organization, and / or hyperactivity—impulsivity. Inattention and disorganization entail inabil- ity to stay on task, seeming not to listen, and losing materials, at levels that are inconsistent with age or developmental level. Hyperactivity—impuisivity entails overactivity, fidgeting, in- ability to stay seated, intruding into other people’s activities, and inability to wait—symptoms that are excessive for age or developmental level. In childhood, ADHD frequently overlaps with disorders that are often considered to be ”externalizing disorders,” such as oppositional defiant disorder and conduct disorder. ADHD often persists into adulthood, with resultant impairments of social, academic and occupational functioning.

1	The neurodevelopmental motor disorders include developmental coordination disor- der, stereotypic movement disorder, and tic disorders. Developmental coordination dis- order is characterized by deficits in the acquisition and execution of coordinated motor skills and is manifested by clumsiness and slowness or inaccuracy of performance of mo- tor skills that cause interference with activities of daily living. Stereotypic movement dis- order is diagnosed when an individual has repetitive, seemingly driven, and apparently purposeless motor behaviors, such as hand ﬂapping, body rocking, head banging, self- biting, or hitting. The movements interfere with social, academic, or other activities. If the behaviors cause self—injury, this should be specified as part of the diagnostic description.

1	Tic disorders are characterized by the presence of motor or vocal tics, which are sudden, rapid, recurrent, nonrhythmic, sterotyped motor movements or vocalizations. The dura- tion, presumed etiology, and clinical presentation define the specific tic disorder that is di- agnosed: Tourette’s disorder, persistent (chronic) motor or vocal tic disorder, provisional tic disorder, other specified tic disorder, and unspecified tic disorder. Tourette’s disorder is diagnosed when the individual has multiple motor and vocal tics that have been present for at least 1 year and that have a waxing—waning symptom course.

1	Specific learning disorder, as the name implies, is diagnosed when there are specific defi- cits in an individual’s ability to perceive or process information efficiently and accurately. This neurodevelopmental disorder first manifests during the years of formal schooling and is characterized by persistent and impairing difficulties with learning foundational academic skills in reading, writing, and / or math. The individual’s performance of the affected academic skills is well below average for age, or acceptable performance levels are achieved only with extraordinary effort. Specific learning disorder may occur in individuals identified as intellec- tually gifted and manifest only when the learning demands or assessment procedures (e.g., tory strategies. For all individuals, specific learning disorder can produce lifelong impairments in activities dependent on the skills, including occupational performance.

1	The use of specifiers for the neurodevelopmental disorder diagnoses enriches the clin- ical description of the individual’s clinical course and current symptomatology. In addi- tion to specifiers that describe the clinical presentation, such as age at onset or severity ratings, the neurodevelopmental disorders may include the specifier "associated with a known medical or genetic condition or environmental factor.” This specifier gives clini- cians an opportunity to document factors that may have played a role in the etiology of the disorder, as well as those that might affect the clinical course. Examples include genetic disorders, such as fragile X syndrome, tuberous sclerosis, and Rett syndrome; medical con- ditions such as epilepsy; and environmental factors, including very low birth weight and fetal alcohol exposure (even in the absence of stigmata of fetal alcohol syndrome).

1	Intellectual disability (intellectual developmental disorder) is a disorder with onset during the developmental period that includes both intellectual and adaptive functioning deficits in conceptual, social, and practical domains. The following three criteria must be met: A. Deficits in intellectual functions, such as reasoning, problem solving, planning, abstract thinking, judgment, academic learning, and learning from experience, confirmed by both clinical assessment and individualized, standardized intelligence testing. B. Deficits in adaptive functioning that result in failure to meet developmental and socio- cultural standards for personal independence and social responsibility. Without ongo- ing support, the adaptive deficits limit functioning in one or more activities of daily life, such as communication, social participation, and independent living, across multiple environments, such as home, school, work, and community.

1	C. Onset of intellectual and adaptive deficits during the developmental period. Note: The diagnostic term intellectual disability is the equivalent term for the |CD-11 diag- nosis of intellectual developmental disorders. Although the term intellectual disability is used throughout this manual, both terms are used in the title to clarify relationships with other classification systems. Moreover, a federal statute in the United States (Public Law 111-256, Rosa's Law) replaces the term mental retardation with intellectual disability, and research journals use the term intellectual disability. Thus, intellectual disability is the term in common use by medical, educational, and other professions and by the lay public and advocacy groups. Coding note: The |CD-9-CM code for intellectual disability (intellectual developmental disorder) is 319, which is assigned regardless of the severity specifier. The |CD-10-CM code depends on the severity specifier (see below).

1	Specify current severity (see Table 1): The various levels of severity are defined on the basis of adaptive functioning, and not IQ scores, because it is adaptive functioning that determines the level of supports required. Moreover, IQ measures are less valid in the lower end of the IQ range.

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1	E0806 0:300:00 0.00528: 060.86 6.553660 638.62: 3:336 6:62.25 3: 0.26. £660 0 0.35.. The essential features of intellectual disability (intellectual developmental disorder) are functioning, in comparison to an individual’s age-, gender-, and socioculturally matched peers (Criterion B). Onset is during the developmental period (Criterion C). The diagnosis of intellectual disability is based on both clinical assessment and standardized testing of intellectual and adaptive functions.

1	Criterion A refers to intellectual functions that involve reasoning, problem solving, planning, abstract thinking, judgment, learning from instruction and experience, and practical understanding. Critical components include verbal comprehension, working memory, perceptual reasoning, quantitative reasoning, abstract thought, and cognitive ef- ficacy. Intellectual functioning is typically measured with individually administered and psychometrically valid, comprehensive, culturally appropriate, psychometrically sound tests of intelligence. Individuals with intellectual disability have scores of approximately two standard deviations or more below the population mean, including a margin for mea— surement error (generally +5 points). On tests with a standard deviation of 15 and a mean of 100, this involves a score of 65—75 (70 1 5). Clinical training and judgment are required to interpret test results and assess intellectual performance.

1	Factors that may affect test scores include practice effects and the ”Flynn effect’ (i.e., overly high scores due to out-of—date test norms). Invalid scores may result from the use of may make an overall IQ score invalid. Instruments must be normed for the individual's so- ciocultural background and native language. Co-occurring disorders that affect communi- cation, language, and / or motor or sensory function may affect test scores. Individual cognitive profiles based on neuropsychological testing are more useful for understanding intellectual abilities than a single IQ score. Such testing may identify areas of relative strengths and weaknesses, an assessment important for academic and vocational planning.

1	IQ test scores are approximations of conceptual functioning but may be insufficient to assess reasoning in real-life situations and mastery of practical tasks. For example, a per- son with an IQ score above 70 may have such severe adaptive behavior problems in social judgment, social understanding, and other areas of adaptive functioning that the person’s actual functioning is comparable to that of individuals with a lower IQ score. Thus, clinical judgment is needed in interpreting the results of IQ tests.

1	Deficits in adaptive functioning (Criterion B) refer to how well a person meets community standards of personal independence and social responsibility, in comparison to others of sim- ilar age and sociocultural background. Adaptive functioning involves adaptive reasoning in three domains: conceptual, social, and practical. The conceptual (academic) domain involves competence in memory, language, reading, writing, math reasoning, acquisition of practical knowledge, problem solving, and judgment in novel situations, among others. The social do- main involves awareness of others’ thoughts, feelings, and experiences; empathy; interper— sonal communication skills; friendship abilities; and social judgment, among others. The practical domain involves learning and self-management across life settings, including personal care, job responsibilities, money management, recreation, self—management of behavior, and school and work task organization, among others. Intellectual capacity, education,

1	including personal care, job responsibilities, money management, recreation, self—management of behavior, and school and work task organization, among others. Intellectual capacity, education, motivation, socialization, personality features, vocational opportunity, cultural experience, and coexisting general medical conditions or mental disorders inﬂuence adaptive functioning.

1	Adaptive functioning is assessed using both clinical evaluation and individualized, culturally appropriate, psychometrically sound measures. Standardized measures are used with knowledgeable informants (e.g., parent or other family member; teacher; coun- selor; care provider) and the individual to the extent possible. Additional sources of infor- mation include educational, developmental, medical, and mental health evaluations. ical judgment. When standardized testing is difficult or impossible, because of a variety of factors (e.g., sensory impairment, severe problem behavior), the individual may be diag- nosed with unspecified intellectual disability. Adaptive functioning may be difficult to assess in a controlled setting (e.g., prisons, detention centers); if possible, corroborative in- formation reﬂecting functioning outside those settings should be obtained.

1	Criterion B is met when at least one domain of adaptive functioning—conceptual, so- cial, or practical—is sufficiently impaired that ongoing support is needed in order for the person to perform adequately in one or more life settings at school, at work, at home, or in the community. To meet diagnostic criteria for intellectual disability, the deficits in adap- tive functioning must be directly related to the intellectual impairments described in Cri- terion A. Criterion C, onset during the developmental period, refers to recognition that intellectual and adaptive deficits are present during childhood or adolescence. Intellectual disability is a heterogeneous condition with multiple causes. There may be associated difficulties with social judgment; assessment of risk; self—management of behav- ior, emotions, or interpersonal relationships; or motivation in school or work environments.

1	Lack of communication skills may predispose to disruptive and aggressive behaviors. Gull- ibility is often a feature, involving naiveté in social situations and a tendency for being easily led by others. Gullibility and lack of awareness of risk may result in exploitation by others and possible victimization, fraud, unintentional criminal involvement, false confessions, and risk for physical and sexual abuse. These associated features can be important in crim- inal cases, including Atkins-type hearings involving the death penalty. Individuals with a diagnosis of intellectual disability with co-occurring mental disor- ders are at risk for suicide. They think about suicide, make suicide attempts, and may die from them. Thus, screening for suicidal thoughts is essential in the assessment process. Be- cause of a lack of awareness of risk and danger, accidental injury rates may be increased.

1	Intellectual disability has an overall general population prevalence of approximately 1%, and prevalence rates vary by age. Prevalence for severe intellectual disability is approxi- mately 6 per 1,000. Onset of intellectual disability is in the developmental period. The age and characteristic features at onset depend on the etiology and severity of brain dysfunction. Delayed motor, language, and social milestones may be identifiable within the first 2 years of life among those with more severe intellectual disability, while mild levels may not be identifiable un- til school age when difficulty with academic learning becomes apparent. All criteria (in- cluding Criterion C) must be fulfilled by history or current presentation. Some children ity have deficits that meet criteria for global developmental delay.

1	When intellectual disability is associated with a genetic syndrome, there may be a char- acteristic physical appearance (as in, e.g., Down syndrome). Some syndromes have a behavioral phenotype, which refers to specific behaviors that are characteristic of particular genetic disorder (e.g., Lesch—Nyhan syndrome). In acquired forms, the onset may be abrupt following an illness such as meningitis or encephalitis or head trauma occurring during the developmental period. When intellectual disability results from a loss of pre- viously acquired cognitive skills, as in severe traumatic brain injury, the diagnoses of in- tellectual disability and of a neurocognitive disorder may both be assigned.

1	Although intellectual disability is generally nonprogressive, in certain genetic disor- ders (e.g., Rett syndrome) there are periods of worsening, followed by stabilization, and in others (e.g., San Phillippo syndrome) progressive worsening of intellectual function. After early childhood, the disorder is generally lifelong, although severity levels may change over time. The course may be inﬂuenced by underlying medical or genetic conditions and co—occurring conditions (e.g., hearing or visual impairments, epilepsy). Early and ongoing in- terventions may improve adaptive functioning throughout childhood and adulthood. In some cases, these result in significant improvement of intellectual functioning, such that the diagnosis of intellectual disability is no longer appropriate. Thus, it is common practice when assessing infants and young children to delay diagnosis of intellectual disability un- til after an appropriate course of intervention is provided. For older children and adults,

1	common practice when assessing infants and young children to delay diagnosis of intellectual disability un- til after an appropriate course of intervention is provided. For older children and adults, the extent of support provided may allow for full participation in all activities of daily liv- ing and improved adaptive function. Diagnostic assessments must determine whether im- proved adaptive skills are the result of a stable, generalized new skill acquisition (in which case the diagnosis of intellectual disability may no longer be appropriate) or whether the improvement is contingent on the presence of supports and ongoing interventions (in which case the diagnosis of intellectual disability may still be appropriate).

1	Genetic and physiological. Prenatal etiologies include genetic syndromes (e.g., se- disorders), inborn errors of metabolism, brain malformations, maternal disease (including placental disease), and environmental inﬂuences (e.g., alcohol, other drugs, toxins, terato— gens). Perinatal causes include a variety of labor and delivery-related events leading to neonatal encephalopathy. Postnatal causes include hypoxic ischemic injury, traumatic brain injury, infections, demyelinating disorders, seizure disorders (e.g., infantile spasms), severe and chronic social deprivation, and toxic metabolic syndromes and intoxications (e.g., lead, mercury). Intellectual disability occurs in all races and cultures. Cultural sensitivity and knowledge are needed during assessment, and the individual’s ethnic, cultural, and linguistic back- ground, available experiences, and adaptive functioning within his or her community and cultural setting must be taken into account.

1	Overall, males are more likely than females to be diagnosed with both mild (average malezfemale ratio 1.6:1) and severe (average malezfemale ratio 12:1) forms of intellectual disability. However, gender ratios vary widely in reported studies. Sex-linked genetic fac- tors and male vulnerability to brain insult may account for some of the gender differences. A comprehensive evaluation includes an assessment of intellectual capacity and adaptive functioning; identification of genetic and nongenetic etiologies; evaluation for associated medical conditions (e.g., cerebral palsy, seizure disorder); and evaluation for co-occurring mental, emotional, and behavioral disorders. Components of the evaluation may include basic pre- and perinatal medical history, three—generational family pedigree, physical exam- ination, genetic evaluation (e.g., karyotype or chromosomal microarray analysis and testing for specific genetic syndromes), and metabolic screening and neuroimaging assessment.

1	The diagnosis of intellectual disability should be made whenever Criteria A, B, and C are met. A diagnosis of intellectual disability should not be assumed because of a particular genetic or medical condition. A genetic syndrome linked to intellectual disability should be noted as a concurrent diagnosis with the intellectual disability. Major and mild neurocognitive disorders. Intellectual disability is categorized as a neu- rodevelopmental disorder and is distinct from the neurocognitive disorders, which are characterized by a loss of cognitive functioning. Major neurocognitive disorder may co- occur with intellectual disability (e.g., an individual with Down syndrome who develops Alzheimer’s disease, or an individual with intellectual disability who loses further cogni- tive capacity following a head injury). In such cases, the diagnoses of intellectual disability and neurocognitive disorder may both be given.

1	Communication disorders and specific learning disorder. These neurodevelopmental disorders are specific to the communication and learning domains and do not show defi- cits in intellectual and adaptive behavior. They may co-occur with intellectual disability. Both diagnoses are made if full criteria are met for intellectual disability and a communi- cation disorder or specific learning disorder. Autism Spectrum disorder. Intellectual disability is common among individuals with autism spectrum disorder. Assessment of intellectual ability may be complicated by so- cial-communication and behavior deficits inherent to autism spectrum disorder, which may interfere with understanding and complying with test procedures. Appropriate as- sessment of intellectual functioning in autism spectrum disorder is essential, with reas- sessment across the developmental period, because IQ scores in autism spectrum disorder may be unstable, particularly in early childhood.

1	Co-occurring mental, neurodevelopmental, medical, and physical conditions are frequent in intellectual disability, with rates of some conditions (e.g., mental disorders, cerebral palsy, and epilepsy) three to four times higher than in the general population. The prognosis and outcome of co-occurring diagnoses may be inﬂuenced by the presence of intellectual disability. Assessment procedures may require modifications because of associated disor- ders, including communication disorders, autism spectrum disorder, and motor, sensory, or other disorders. Knowledgeable informants are essential for identifying symptoms such as irritability, mood dysregulation, aggression, eating problems, and sleep problems, and for assessing adaptive functioning in various community settings.

1	The most common co-occurring mental and neurodevelopmental disorders are atten- behavior); impulse-control disorders; and major neurocognitive disorder. Major depres- sive disorder may occur throughout the range of severity of intellectual disability. Self- agnosis of stereotypic movement disorder. Individuals with intellectual disability, partic- ularly those with more severe intellectual disability, may also exhibit aggression and disruptive behaviors, including harm of others or property destruction. Relationship to Other Classifications ICD-ll (in development at the time of this publication) uses the term intellectual develop- mental disorders to indicate that these are disorders that involve impaired brain functioning early in life. These disorders are described in ICD-11 as a metasyndrome occurring in the developmental period analogous to dementia or neurocognitive disorder in later life. There are four subtypes in ICD-ll: mild, moderate, severe, and profound.

1	There are four subtypes in ICD-ll: mild, moderate, severe, and profound. The American Association on Intellectual and Developmental Disabilities (AAIDD) also uses the term intellectual disability with a similar meaning to the term as used in this manual. The AAIDD’s classification is multidimensional rather than categorical and is based on the disability construct. Rather than listing specifiers as is done in DSM-5, the AAIDD emphasizes a profile of supports based on severity. 315.8 (F88)

1	AAIDD emphasizes a profile of supports based on severity. 315.8 (F88) This diagnosis is reserved for individuals under the age of 5 years when the clinical severity level cannot be reliably assessed during early childhood. This category is diagnosed when an individual fails to meet expected developmental milestones in several areas of intellec- tual functioning, and applies to individuals who are unable to undergo systematic assess- ments of intellectual functioning, including children who are too young to participate in standardized testing. This category requires reassessment after a period of time.

1	This category is reserved for individuals over the age of 5 years when assessment of the degree of intellectual disability (intellectual developmental disorder) by means of locally available procedures is rendered difficult or impossible because of associated sensory or physical impairments, as in blindness or prelingual deafness; locomotor disability; or pres- ence of severe problem behaviors or co-occurring mental disorder. This category should only be used in exceptional circumstances and requires reassessment after a period of time. Disorders of communication include deficits in language, speech, and communication.

1	Speech is the expressive production of sounds and includes an individual’s articulation, fluency, voice, and resonance quality. Language includes the form, function, and use of a conventional system of symbols (i.e., spoken words, sign language, written words, pic- tures) in a rule-governed manner for communication. Communication includes any verbal or nonverbal behavior (whether intentional or unintentional) that inﬂuences the behavior, ideas, or attitudes of another individual. Assessments of speech, language and communi- cation abilities must take into account the individual's cultural and language context, particularly for individuals growing up in bilingual environments. The standardized mea— sures of language development and of nonverbal intellectual capacity must be relevant for the cultural and linguistic group (i.e., tests developed and standardized for one group may not provide appropriate norms for a different group). The diagnostic category of commu- nication disorders

1	the cultural and linguistic group (i.e., tests developed and standardized for one group may not provide appropriate norms for a different group). The diagnostic category of commu- nication disorders includes the following: language disorder, speech sound disorder, childhood-onset ﬂuency disorder (stuttering), social (pragmatic) communication disor- der, and other specified and unspecified communication disorders.

1	Diagnostic Criteria 315.39 (F80.9) A. Persistent difficulties in the acquisition and use of language across modalities (i.e., spoken, written. sign language, or other) due to deficits in comprehension or produc- tion that include the following: 1. Reduced vocabulary (word knowledge and use). 2. Limited sentence structure (ability to put words and word endings together to form sentences based on the rules of grammar and morphology). 3. Impairments in discourse (ability to use vocabulary and connect sentences to ex- plain or describe a topic or series of events or have a conversation). B. Language abilities are substantially and quantitiably below those expected for age, re- sulting in functional limitations in effective communication, social participation, aca- demic achievement. or occupational performance, individually or in any combination. C. Onset of symptoms is in the early developmental period.

1	C. Onset of symptoms is in the early developmental period. D. The difficulties are not attributable to hearing or other sensory impairment. motor dys- function, or another medical or neurological condition and are not better explained by in- tellectual disability (intellectual developmental disorder) or global developmental delay.

1	The core diagnostic features of language disorder are difficulties in the acquisition and use of language due to deficits in the comprehension or production of vocabulary, sentence structure, and discourse. The language deficits are evident in spoken communication, written communication, or sign language. Language learning and use is dependent on both receptive and expressive skills. Expressive ability refers to the production of vocal, ges- tural, or verbal signals, while receptive ability refers to the process of receiving and com- prehending language messages. Language skills need to be assessed in both expressive and receptive modalities as these may differ in severity. For example, an individual’s ex- pressive language may be severely impaired, while his receptive language is hardly im- paired at all.

1	Language disorder usually affects vocabulary and grammar, and these effects then limit the capacity for discourse. The child’s first words and phrases are likely to be delayed in onset; vocabulary size is smaller and less varied than expected; and sentences are shorter and less complex with grammatical errors, especially in past tense. Deficits in com- prehension of language are frequently underestimated, as children may be good at using context to infer meaning. There may be word-finding problems, impoverished verbal def- initions, or poor understanding of synonyms, multiple meanings, or word play appro- priate for age and culture. Problems with remembering new words and sentences are manifested by difficulties following instructions of increasing length, difficulties rehears- ing strings of verbal information (e.g., remembering a phone number or a shopping list), and difficulties remembering novel sound sequences, a skill that may be important for learning new words. Difficulties

1	of verbal information (e.g., remembering a phone number or a shopping list), and difficulties remembering novel sound sequences, a skill that may be important for learning new words. Difficulties with discourse are shown by a reduced ability to provide adequate information about the key events and to narrate a coherent story.

1	The language difficulty is manifest by abilities substantially and quantifiably below that expected for age and significantly interfering with academic achievement, occupa- tional performance, effective communication, or socialization (Criterion B). A diagnosis of language disorder is made based on the synthesis of the individual’s history, direct clinical observation in different contexts (i.e., home, school, or work), and scores from standard- ized tests of language ability that can be used to guide estimates of severity.

1	A positive family history of language disorders is often present. Individuals, even chil- dren, can be adept at accommodating to their limited language. They may appear to be shy or reticent to talk. Affected individuals may prefer to communicate only with family mem- bers or other familiar individuals. Although these social indicators are not diagnostic of a language disorder, if they are notable and persistent, they warrant referral for a full lan- guage assessment. Language disorder, particularly expressive deficits, may co-occur with speech sound disorder.

1	Language acquisition is marked by changes from onset in toddlerhood to the adult level of competency that appears during adolescence. Changes appear across the dimensions of language (sounds, words, grammar, narratives/expository texts, and conversational skills) in age-graded increments and synchronies. Language disorder emerges during the early developmental period; however, there is considerable variation in early vocabulary acquisition and early word combinations, and individual differences are not, as single indicators, highly predictive of later outcomes. By age 4 years, individual differences in language ability are more stable, with better measurement accuracy, and are highly pre- dictive of later outcomes. Language disorder diagnosed from 4 years of age is likely to be stable over time and typically persists into adulthood, although the particular profile of language strengths and deficits is likely to change over the course of development.

1	Children with receptive language impairments have a poorer prognosis than those with predominantly expressive impairments. They are more resistant to treatment, and diffi- culties with reading comprehension are frequently seen. Genetic and physiological. Language disorders are highly heritable, and family mem- bers are more likely to have a history of language impairment. Normal variations in language. Language disorder needs to be distinguished from nor- mal developmental variations, and this distinction may be difficult to make before 4 years of age. Regional, social, or cultural/ethnic variations of language (e.g,, dialects) must be considered when an individual is being assessed for language impairment.

1	Hearing or other sensory impairment. Hearing impairment needs to be excluded as the primary cause of language difficulties. Language deficits may be associated with a hearing impairment, other sensory deficit, or a speech-motor deficit. When language deficits are in excess of those usually associated with these problems, a diagnosis of language disorder may be made. Intellectual disability (intellectual developmental disorder). Language delay is often the presenting feature of intellectual disability, and the definitive diagnosis may not be made until the child is able to complete standardized assessments. A separate diagnosis is not given unless the language deficits are clearly in excess of the intellectual limitations. Neurological disorders. Language disorder can be acquired in association with neuro- logical disorders, including epilepsy (e.gi, acquired aphasia or Landau-Kleffner syndrome).

1	Neurological disorders. Language disorder can be acquired in association with neuro- logical disorders, including epilepsy (e.gi, acquired aphasia or Landau-Kleffner syndrome). Language regression. Loss of speech and language in a child younger than 3 years may be a sign of autism spectrum disorder (with developmental regression) or a specific neuro- logical condition, such as Landau-Kleffner syndrome. Among children older than 3 years, language loss may be a symptom of seizures, and a diagnostic assessment is necessary to exclude the presence of epilepsy (e.g., routine and sleep electroencephalogram).

1	Language disorder is strongly associated with other neurodevelopmental disorders in terms of specific learning disorder (literacy and numeracy), attention-deficit/hyperactiv- ity disorder, autism spectrum disorder, and developmental coordination disorder. It is also associated with social (pragmatic) communication disorder. A positive family history of speech or language disorders is often present. Diagnostic Criteria 315.39 (F80.0) A. Persistent difficulty with speech sound production that interferes with speech intelligi- bility or prevents verbal communication of messages. B. The disturbance causes limitations in effective communication that interfere with social participation, academic achievement. or occupational performance, individually or in any combination. C. Onset of symptoms is in the early developmental period.

1	C. Onset of symptoms is in the early developmental period. D. The difficulties are not attributable to congenital or acquired conditions, such as cere- bral palsy, cleft palate. deafness or hearing loss, traumatic brain injury, or other medi- cal or neurological conditions. Speech sound production describes the clear articulation of the phonemes (i.e., individual sounds) that in combination make up spoken words. Speech sound production requires both the phonological knowledge of speech sounds and the ability to coordinate the movements of the articulators (i.e., the jaw, tongue, and lips,) with breathing and vocalizing for speech. Chil- dren with speech production difficulties may experience difficulty with phonological knowl- edge of speech sounds or the ability to coordinate movements for speech in varying degrees

1	Speech sound disorder is thus heterogeneous in its underlying mechanisms and includes pho- nological disorder and articulation disorder. A speech sound disorder is diagnosed when speech sound production is not what would be expected based on the child’s age and devel- opmental stage and when the deficits are not the result of a physical, structural, neurological, or hearing impairment. Among typically developing children at age 4 years, overall speech should be intelligible, whereas at age 2 years, only 50% may be understandable. Language disorder, particularly expressive deficits, may be found to co-occur with speech sound disorder. A positive family history of speech or language disorders is often present.

1	Language disorder, particularly expressive deficits, may be found to co-occur with speech sound disorder. A positive family history of speech or language disorders is often present. If the ability to rapidly coordinate the articulators is a particular aspect of difficulty, there may be a history of delay or incoordination in acquiring skills that also utilize the articulators and related facial musculature; among others, these skills include chewing, maintaining mouth closure, and blowing the nose. Other areas of motor coordination may be impaired as in developmental coordination disorder. Verbal dyspruxia is a term also used for speech production problems. Speech may be differentially impaired in certain genetic conditions (e.g., Down syn- drome, 22q deletion, FoxPZ gene mutation). If present, these should also be coded.

1	Speech may be differentially impaired in certain genetic conditions (e.g., Down syn- drome, 22q deletion, FoxPZ gene mutation). If present, these should also be coded. Learning to produce speech sounds clearly and accurately and learning to produce con- nected speech fluently are developmental skills. Articulation of speech sounds follows a developmental pattern, which is reﬂected in the age norms of standardized tests. It is not unusual for typically developing children to use developmental processes for shortening words and syllables as they are learning to talk, but their progression in mastering speech sound production should result in mostly intelligible speech by age 3 years. Children with speech sound disorder continue to use immature phonological simplification processes past the age when most children can produce words clearly.

1	accurately according to age and community norms by age 7 years. The most frequently mis- articulated sounds also tend to be learned later, leading them to be called the ”late eight" (I, r, s, 2, th, ch, dzh, and zh). Misarticulation of any of these sounds by itself could be considered within normal limits up to age 8 years. When multiple sounds are involved, it may be appro- priate to target some of those sounds as part of a plan to improve intelligibility prior to the age at which almost all children can produce them accurately. Lisping (i.e., misarticulating sibi- lants) is particularly common and may involve frontal or lateral patterns of airstream direc- tion. It may be associated with an abnormal tongue—thrust swallowing pattern.

1	Most children with speech sound disorder respond well to treatment, and speech dif- ficulties improve over time, and thus the disorder may not be lifelong. However, when a language disorder is also present, the speech disorder has a poorer prognosis and may be associated with specific learning disorders. Normal variations in speech. Regional, social, or cultural/ethnic variations of speech should be considered before making the diagnosis. Hearing or other sensory impairment. Hearing impairment or deafness may result in abnormalities of speech. Deficits of speech sound production may be associated with a hearing impairment, other sensory deficit, or a speech-motor deficit. When speech deficits are in excess of those usually associated with these problems, a diagnosis of speech sound disorder may be made. Structu ral deficits. Speech impairment may be due to structural deficits (e.g., cleft palate).

1	Structu ral deficits. Speech impairment may be due to structural deficits (e.g., cleft palate). Dysarth ria. Speech impairment may be attributable to a motor disorder, such as cerebral palsy. Neurological signs, as well as distinctive features of voice, differentiate dysarthria from speech sound disorder, although in young children (under 3 years) differentiation may be difficult, particularly when there is no or minimal general body motor involve- ment (as in, e.g., Worster-Drought syndrome). Selective mutism. Limited use of speech may be a sign of selective mutism, an anxiety disorder that is characterized by a lack of speech in one or more contexts or settings. Se- lective mutism may develop in children with a speech disorder because of embarassment about their impairments, but many children with selective mutism exhibit normal speech in ”safe” settings, such as at home or with close friends. Diagnostic Criteria 315.35 (F80.81)

1	Diagnostic Criteria 315.35 (F80.81) A. Disturbances in the normal fluency and time patterning of speech that are inappropri- ate for the individual’s age and language skills, persist over time, and are characterized by frequent and marked occurrences of one (or more) of the following: 1. Sound and syllable repetitions. 2. Sound prolongations of consonants as well as vowels. Broken words (e.g., pauses within a word). Audible or silent blocking (filled or unfilled pauses in speech). Circumlocutions (word substitutions to avoid problematic words). Words produced with an excess of physical tension. Monosyllabic whoIe-word repetitions (e.g., “l-l-l-l see him”). >19???” B. The disturbance causes anxiety about speaking or limitations in effective communica- tion, social participation, or academic or occupational performance, individually or in any combination.

1	B. The disturbance causes anxiety about speaking or limitations in effective communica- tion, social participation, or academic or occupational performance, individually or in any combination. C. The onset of symptoms is in the early developmental period. (Note: Later-onset cases are diagnosed as 307.0 [F985] aduIt-onset ﬂuency disorder.) D. The disturbance is not attributable to a speech-motor or sensory deficit, dysfluency as- sociated with neurological insult (e.g., stroke, tumor, trauma). or another medical con- dition and is not better explained by another mental disorder.

1	The essential feature of childhood-onset ﬂuency disorder (stuttering) is a disturbance in the normal ﬂuency and time patterning of speech that is inappropriate for the individual’s age. This disturbance is characterized by frequent repetitions or prolongations of sounds or syllables and by other types of speech dysfluencies, including broken words (e.g., pauses within a word), audible or silent blocking (i.e., filled or unfilled pauses in speech), circumlocutions (i.e., word substitutions to avoid problematic words), words produced with an excess of physical tension, and monosyllabic whole-word repetitions (e.g., ”I-I-I-I see him"). The disturbance in ﬂuency interferes with academic or occupational achieve- ment or with social communication. The extent of the disturbance varies from situation to situation and often is more severe when there is special pressure to communicate (e.g., giv- ing a report at school, interviewing for a job). Dysﬂuency is often absent during oral read- ing,

1	to situation and often is more severe when there is special pressure to communicate (e.g., giv- ing a report at school, interviewing for a job). Dysﬂuency is often absent during oral read- ing, singing, or talking to inanimate objects or to pets.

1	Fearful anticipation of the problem may develop. The speaker may attempt to avoid dys- fluencies by linguistic mechanisms (e.g., altering the rate of speech, avoiding certain words or sounds) or by avoiding certain speech situations, such as telephoning or public speaking. In addition to being features of the condition, stress and anxiety have been shown to exacerbate dysﬂuency. (e.g., eye blinks, tics, tremors of the lips or face, jerking of the head, breathing movements, fist clenching). Children with ﬂuency disorder show a range of language abilities, and the relationship between ﬂuency disorder and language abilities is unclear.

1	Childhood-onset ﬂuency disorder, or developmental stuttering, occurs by age 6 for 80%— 90% of affected individuals, with age at onset ranging from 2 to 7 years. The onset can be insidious or more sudden. Typically, dysﬂuencies start gradually, with repetition of initial consonants, first words of a phrase, or long words. The child may not be aware of dysﬂu- encies. As the disorder progresses, the dysﬂuencies become more frequent and interfering, occurring on the most meaningful words or phrases in the utterance. As the child becomes aware of the speech difficulty, he or she may develop mechanisms for avoiding the dys- ﬂuencies and emotional responses, including avoidance of public speaking and use of short and simple utterances. Longitudinal research shows that 65%—85% of children re- cover from the dysﬂuency, with severity of ﬂuency disorder at age 8 years predicting re- covery or persistence into adolescence and beyond.

1	Genetic and physiological. The risk of stuttering among first-degree biological rela- tives of individuals with childhood-onset ﬂuency disorder is more than three times the risk in the general population. In addition to being features of the condition, stress and anxiety can exacerbate dysﬂu- ency. Impairment of social functioning may result from this anxiety. Sensory deficits. Dysﬂuencies of speech may be associated with a hearing impairment or other sensory deficit or a speech-motor deficit. When the speech dysﬂuencies are in ex- cess of those usually associated with these problems, a diagnosis of childhood-onset ﬂu- ency disorder may be made.

1	Normal speech dysfluencies. The disorder must be distinguished from normal dysﬂu- encies that occur frequently in young children, which include whole-word or phrase rep- etitions (e.g., “I want, I want ice cream”), incomplete phrases, interjections, unfilled pauses, and parenthetical remarks. If these difficulties increase in frequency or complexity as the child grows older, a diagnosis of childhood-onset ﬂuency disorder is appropriate. Medication side effects. Stuttering may occur as a side effect of medication and may be detected by a temporal relationship with exposure to the medication. Adult-onset dysfluencies. If onset of dysﬂuencies is during or after adolescence, it is an ”adult-onset dysﬂuency” rather than a neurodevelopmental disorder. Adult-onset dysﬂu- encies are associated with specific neurological insults and a variety of medical conditions and mental disorders and may be specified with them, but they are not a DSM-S diagnosis.

1	Tourette's disorder. Vocal tics and repetitive vocalizations of Tourette’s disorder should be distinguishable from the repetitive sounds of childhood-onset ﬂuency disorder by their nature and timing. Diagnostic Criteria 315.39 (F80.89) A. Persistent difficulties in the social use of verbal and nonverbal communication as man- ifested by all of the following: 1. Deficits in using communication for social purposes, such as greeting and sharing information, in a manner that is appropriate for the social context. 2. Impairment of the ability to change communication to match context or the needs of the listener. such as speaking differently in a classroom than on a playground. talk- ing differently to a child than to an adult, and avoiding use of overly formal language. 3. Difficulties following rules for conversation and storytelling, such as taking turns in conversation, rephrasing when misunderstood, and knowing how to use verbal and nonverbal signals to regulate interaction.

1	4. Difficulties understanding what is not explicitly stated (e.g., making inferences) and nonliteral or ambiguous meanings of language (e.g., idioms, humor, metaphors, multiple meanings that depend on the context for interpretation). B. The deficits result in functional limitations in effective communication, social participa- tion, social relationships, academic achievement, or occupational performance, indi- vidually or in combination. C. The onset of the symptoms is in the early developmental period (but deficits may not become fully manifest until social communication demands exceed limited capacities). D. The symptoms are not attributable to another medical or neurological condition or to low abilities in the domains of word structure and grammar, and are not better explained by autism spectrum disorder, intellectual disability (intellectual developmental disorder), global developmental delay, or another mental disorder.

1	Social (pragmatic) communication disorder is characterized by a primary difficulty with pragmatics, or the social use of language and communication, as manifested by deficits in understanding and following social rules of verbal and nonverbal communication in nat- uralistic contexts, changing language according to the needs of the listener or situation, and following rules for conversations and storytelling. The deficits in social communica- tion result in functional limitations in effective communication, social participation, devel- opment of social relationships, academic achievement, or occupational performance. The deficits are not better explained by low abilities in the domains of structural language or cognitive ability.

1	The most common associated feature of social (pragmatic) communication disorder is lan- guage impairment, which is characterized by a history of delay in reaching language mile- stones, and historical, if not current, structural language problems (see ”language Disorder” earlier in this chapter). Individuals with social communication deficits may avoid social inter- actions. Attention-deficit/hyperactivity disorder (ADHD), behavioral problems, and specific learning disorders are also more common among affected individuals. in speech and language, diagnosis of social (pragmatic) communication disorder is rare among children younger than 4 years. By age 4 or 5 years, most children should possess adequate speech and language abilities to permit identification of specific deficits in social communication. Milder forms of the disorder may not become apparent until early ado~ lescence, when language and social interactions become more complex.

1	The outcome of social (pragmatic) communication disorder is variable, with some chil- dren improving substantially over time and others continuing to have difficulties persist- ing into adulthood. Even among those who have significant improvements, the early and also in acquisition of other related skills, such as written expression. Genetic and physiological. A family history of autism spectrum disorder, communica- tion disorders, or specific learning disorder appears to increase the risk for social (prag- matic) communication disorder.

1	Autism spectrutn disorder. Autism spectrum disorder is the primary diagnostic con- sideration for individuals presenting with social communication deficits. The two disor- ders can be differentiated by the presence in autism spectrum disorder of restricted/ repetitive patterns of behavior, interests, or activities and their absence in social (prag- matic) communication disorder. Individuals with autism spectrum disorder may only dis- play the restricted / repetitive patterns of behavior, interests, and activities during the early developmental period, so a comprehensive history should be obtained. Current absence of symptoms would not preclude a diagnosis of autism spectrum disorder, if the restricted interests and repetitive behaviors were present in the past. A diagnosis of social (prag- matic) communication disorder should be considered only if the developmental history fails to reveal any evidence of restricted/repetitive patterns of behavior, interests, or ac- tivities.

1	Attention-deficit/hyperactivity disorder. Primary deficits of ADHD may cause impair- ments in social communication and functional limitations of effective communication, so- cial participation, or academic achievement. Social anxiety disorder (social phobia). The symptoms of social communication disor- der overlap with those of social anxiety disorder. The differentiating feature is the timing of the onset of symptoms. In social (pragmatic) communication disorder, the individual has never had effective social communication; in social anxiety disorder, the social com- munication skills developed appropriately but are not utilized because of anxiety, fear, or distress about social interactions. delay. Social communication skills may be deficient among individuals with global de- velopmental delay or intellectual disability, but a separate diagnosis is not given unless the social communication deficits are clearly in excess of the intellectual limitations. 307.9 (F80.9)

1	307.9 (F80.9) This category applies to presentations in which symptoms characteristic of communication disorder that cause clinically significant distress or impairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for com- munication disorder or for any of the disorders in the neurodevelopmental disorders diag- nostic class. The unspecified communication disorder category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for com- munication disorder or for a specific neurodevelopmental disorder, and includes presen- tations in which there is insufficient information to make a more specific diagnosis. Diagnostic Criteria 299.00 (F84.0)

1	Diagnostic Criteria 299.00 (F84.0) A. Persistent deficits in social communication and social interaction across multiple con- texts, as manifested by the following, currently or by history (examples are illustrative, not exhaustive; see text): 1. Deficits in sociaI-emotional reciprocity, ranging, for example, from abnormal social approach and failure of normal back-and-forth conversation; to reduced sharing of interests, emotions, or affect; to failure to initiate or respond to social interactions. 2. Deficits in nonverbal communicative behaviors used for social interaction, ranging, for example, from poorly integrated verbal and nonverbal communication; to abnor- gestures; to a total lack of facial expressions and nonverbal communication.

1	3. Deficits in developing, maintaining, and understanding relationships, ranging, for ex- ample, from difficulties adjusting behavior to suit various social contexts; to difficulties in sharing imaginative play or in making friends; to absence of interest in peers. Specify current severity: Severity is based on social communication impairments and restricted, re- petitive patterns of behavior (seeTable 2). B. Restricted, repetitive patterns of behavior, interests, or activities, as manifested by at least two of the following, currently or by history (examples are illustrative, not exhaus- tive; see text): 1. Stereotyped or repetitive motor movements, use of objects, or speech (e.g., simple motor stereotypies, lining up toys or flipping objects, echolalia, idiosyncratic phrases).

1	2. Insistence on sameness, inflexible adherence to routines, or ritualized patterns of verbal or nonverbal behavior (e.g., extreme distress at small changes, difficulties with transitions, rigid thinking patterns, greeting rituals, need to take same route or eat same food every day). 3. Highly restricted, fixated interests that are abnormal in intensity or focus (e.g., strong attachment to or preoccupation with unusual objects, excessively circum- scribed or perseverative interests). 4. Hyper- or hyporeactivity to sensory input or unusual interest in sensory aspects of the environment (e.g., apparent indifference to pain/temperature, adverse re- sponse to specific sounds or textures, excessive smelling or touching of objects, visual fascination with lights or movement). Specify current severity: Severity is based on social communication impairments and restricted, re- petitive patterns of behavior (see Table 2).

1	Specify current severity: Severity is based on social communication impairments and restricted, re- petitive patterns of behavior (see Table 2). C. Symptoms must be present in the early developmental period (but may not become fully manifest until social demands exceed limited capacities, or may be masked by learned strategies in later life). D. Symptoms cause clinically significant impairment in social, occupational, or other im- portant areas of current functioning. E. These disturbances are not better explained by intellectual disability (intellectual devel- opmental disorder) or global developmental delay. Intellectual disability and autism spectrum disorder frequently co-occur; to make comorbid diagnoses of autism spec- trum disorder and intellectual disability, social communication should be below that ex- pected for general developmental level.

1	Note: Individuals with a well-established DSM-IV diagnosis of autistic disorder, Asperger’s disorder, or pervasive developmental disorder not othenNise specified should be given the diagnosis of autism spectrum disorder. Individuals who have marked deficits in social communication, but whose symptoms do not othenivise meet criteria for autism spectrum disorder, should be evaluated for social (pragmatic) communication disorder. Specify if: Associated with a known medicai or genetic condition or environmental factor (Coding note: Use additional code to identify the associated medical or genetic condition.) Associated with another neurodevelopmental, mental, or behavioral disorder (Coding note: Use additional code[s] to identify the associated neurodevelopmental, mental, or behavioral disorder[s].)

1	Associated with another neurodevelopmental, mental, or behavioral disorder (Coding note: Use additional code[s] to identify the associated neurodevelopmental, mental, or behavioral disorder[s].) With catatonia (refer to the criteria for catatonia associated with another mental dis- order, pp. 119—120, for definition) (Coding note: Use additional code 293.89 [F06.1] catatonia associated with autism spectrum disorder to indicate the presence of the co- morbid catatonia.) For autism spectrum disorder that is associated with a known medical or genetic condition or environmental factor, or with another neurodevelopmental, mental, or behavioral dis- order, record autism spectrum disorder associated with (name of condition, disorder, or factor) (e.g., autism spectrum disorder associated with Rett syndrome). Severity should be recorded as level of support needed for each of the two psychopathological domains in

1	Table 2 (e.g., “requiring very substantial support for deficits in social communication and requiring substantial support for restricted, repetitive behaviors”). Specification of ”with ment" should be recorded next. Language impairment specification should be recorded thereafter. If there is accompanying language impairment, the current level of verbal func— tioning should be recorded (e.g., “with accompanying language impairment—no intelligi- ble speech” or ”with accompanying language impairment—phrase speech"). If catatonia is present, record separately ”catatonia associated with autism spectrum disorder.”

1	The severity specifiers (see Table 2) may be used to describe succinctly the current symp- tomatology (which might fall below level 1), with the recognition that severity may vary by context and fluctuate over time. Severity of social communication difficulties and re— stricted, repetitive behaviors should be separately rated. The descriptive severity categories should not be used to determine eligibility for and provision of services; these can only be developed at an individual level and through discussion of personal priorities and targets. Regarding the specifier ”with or without accompanying intellectual impairment,” un- derstanding the (often uneven) intellectual profile of a child or adult with autism spectrum disorder is necessary for interpreting diagnostic features. Separate estimates of verbal and nonverbal skill are necessary (e.g., using untimed nonverbal tests to assess potential strengths in individuals with limited language).

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1	H :36: 1.855% _mzcfimnfisw wfihswomz N _05: :tommam _mnafimEam b9» wfiumsvomz m .23 m.o_>w:on 02:800.. 092:3: :ozmoisfifioo _m_oow _o>o_ 3:98 aoucoﬂu Eazooaw Emzam .2 203. 3:035 N u..n<._. To use the specifier ”with or without accompanying language impairment,” the cur- rent level of verbal functioning should be assessed and described. Examples of the specific speech (nonverbal), single words only, or phrase speech. Language level in individuals full sentences or has ﬂuent speech. Since receptive language may lag behind expressive language development in autism spectrum disorder, receptive and expressive language skills should be considered separately. The specifier “associated with a known medical or genetic condition or environmental fac- tor" should be used when the individual has a known genetic disorder (e.g., Rett syndrome,

1	The specifier “associated with a known medical or genetic condition or environmental fac- tor" should be used when the individual has a known genetic disorder (e.g., Rett syndrome, Fragile X syndrome, Down syndrome), a medical disorder (e.g. epilepsy), or a history of envi- ronmental exposure (e.g., valproate, fetal alcohol syndrome, very low birth weight). Additional neurodevelopmental, mental or behavioral conditions should also be noted (e.g., attention-deficit/hyperactivity disorder; developmental coordination disorder; dis— ruptive behavior, impulse-control, or conduct disorders; anxiety, depressive, or bipolar disorders; tics or Tourette’s disorder; self—injury; feeding, elimination, or sleep disorders).

1	The essential features of autism spectrum disorder are persistent impairment in reciprocal social communication and social interaction (Criterion A), and restricted, repetitive pat- terns of behavior, interests, or activities (Criterion B). These symptoms are present from early childhood and limit or impair everyday functioning (Criteria C and D). The stage at which functional impairment becomes obvious will vary according to characteristics of the individual and his or her environment. Core diagnostic features are evident in the developmental period, but intervention, compensation, and current supports may mask difficulties in at least some contexts. Manifestations of the disorder also vary greatly de- pending on the severity of the autistic condition, developmental level, and chronological age; hence, the term spectrum. Autism spectrum disorder encompasses disorders previously re- ferred to as early infantile autism, childhood autism, Kanner’s autism, high-functioning autism,

1	age; hence, the term spectrum. Autism spectrum disorder encompasses disorders previously re- ferred to as early infantile autism, childhood autism, Kanner’s autism, high-functioning autism, atypical autism, pervasive developmental disorder not otherwise specified, child- hood disintegrative disorder, and Asperger’s disorder.

1	The impairments in communication and social interaction specified in Criterion A are pervasive and sustained. Diagnoses are most valid and reliable when based on multiple sources of information, including clinician’s observations, caregiver history, and, when possible, self—report. Verbal and nonverbal deficits in social communication have varying manifestations, depending on the individual's age, intellectual level, and language ability, as well as other factors such as treatment history and current support. Many individuals have language deficits, ranging from complete lack of speech through language delays, poor comprehension of speech, echoed speech, or stilted and overly literal language. Even when formal language skills (e.g., vocabulary, grammar) are intact, the use of language for reciprocal social communication is impaired in autism spectrum disorder.

1	Deficits in social-emotional reciprocity (i.e., the ability to engage with others and share thoughts and feelings) are clearly evident in young children with the disorder, who may show little or no initiation of social interaction and no sharing of emotions, along with re- duced or absent imitation of others’ behavior. What language exists is often one-sided, lacking in social reciprocity, and used to request or label rather than to comment, share feelings, or converse. In adults without intellectual disabilities or language delays, deficits sponding to complex social cues (e.g., when and how to join a conversation, what not to say). Adults who have developed compensation strategies for some social challenges still struggle in novel or unsupported situations and suffer from the effort and anxiety of con- sciously calculating what is socially intuitive for most individuals.

1	Deficits in nonverbal communicative behaviors used for social interaction are mani- fested by absent, reduced, or atypical use of eye contact (relative to cultural norms), ges- tures, facial expressions, body orientation, or speech intonation. An early feature of autism spectrum disorder is impaired joint attention as manifested by a lack of pointing, showing, or bringing objects to share interest with others, or failure to follow someone’s pointing or eye gaze. Individuals may learn a few functional gestures, but their repertoire is smaller than that of others, and they often fail to use expressive gestures spontaneously in com- munication. Among adults with ﬂuent language, the difficulty in coordinating nonverbal communication with speech may give the impression of odd, wooden, or exaggerated ”body language" during interactions. Impairment may be relatively subtle within indi- vidual modes (e.g., someone may have relatively good eye contact when speaking) but noticeable in poor

1	”body language" during interactions. Impairment may be relatively subtle within indi- vidual modes (e.g., someone may have relatively good eye contact when speaking) but noticeable in poor integration of eye contact, gesture, body posture, prosody, and facial ex- pression for social communication.

1	Deficits in developing, maintaining, and understanding relationships should be judged against norms for age, gender, and culture. There may be absent, reduced, or atyp- ical social interest, manifested by rejection of others, passivity, or inappropriate ap- proaches that seem aggressive or disruptive. These difficulties are particularly evident in young children, in whom there is often a lack of shared social play and imagination (e.g., age-appropriate ﬂexible pretend play) and, later, insistence on playing by very fixed rules.

1	Older individuals may struggle to understand what behavior is considered appropriate in one situation but not another (e.g., casual behavior during a job interview), or the different ways that language may be used to communicate (e.g., irony, white lies). There may be an apparent preference for solitary activities or for interacting with much younger or older people. Frequently, there is a desire to establish friendships without a complete or realistic idea of what friendship entails (e.g., one-sided friendships or friendships based solely on shared special interests). Relationships with siblings, co-workers, and caregivers are also important to consider (in terms of reciprocity).

1	Autism spectrum disorder is also defined by restricted, repetitive patterns of behavior, interests, or activities (as specified in Criterion B), which show a range of manifestations according to age and ability, intervention, and current supports. Stereotyped or repetitive behaviors include simple motor stereotypies (e.g., hand ﬂapping, finger ﬂicking), repeti- tive use of objects (e.g., spinning coins, lining up toys), and repetitive speech (e.g., echola- lia, the delayed or immediate parroting of heard words; use of "you" when referring to self; stereotyped use of words, phrases, or prosodic patterns). Excessive adherence to rou- tines and restricted patterns of behavior may be manifest in resistance to change (e.g., dis- tress at apparently small changes, such as in packaging of a favorite food; insistence on adherence to rules; rigidity of thinking) or ritualized patterns of verbal or nonverbal be- havior (e.g., repetitive questioning, pacing a perimeter). Highly restricted,

1	favorite food; insistence on adherence to rules; rigidity of thinking) or ritualized patterns of verbal or nonverbal be- havior (e.g., repetitive questioning, pacing a perimeter). Highly restricted, fixated interests in autism spectrum disorder tend to be abnormal in intensity or focus (e.g., a toddler strongly attached to a pan; a child preoccupied with vacuum cleaners; an adult spending hours writing out timetables). Some fascinations and routines may relate to apparent hy- per- or hyporeactivity to sensory input, manifested through extreme responses to specific sounds or textures, excessive smelling or touching of objects, fascination with lights or spinning objects, and sometimes apparent indifference to pain, heat, or cold. Extreme re- action to or rituals involving taste, smell, texture, or appearance of food or excessive food restrictions are common and may be a presenting feature of autism spectrum disorder.

1	Many adults with autism spectrum disorder without intellectual or language disabili- ties learn to suppress repetitive behavior in public. Special interests may be a source of pleasure and motivation and provide avenues for education and employment later in life. Diagnostic criteria may be met when restricted, repetitive patterns of behavior, interests, or activities were clearly present during childhood or at some time in the past, even if symptoms are no longer present. Criterion D requires that the features must cause clinically significant impairment in so- cial, occupational, or other important areas of current functioning. Criterion E specifies that the social communication deficits, although sometimes accompanied by intellectual disabil- ity (intellectual developmental disorder), are not in line with the individual’s developmental level; impairments exceed difficulties expected on the basis of developmental level.

1	Standardized behavioral diagnostic instruments with good psychometric properties, including caregiver interviews, questionnaires and clinician observation measures, are available and can improve reliability of diagnosis over time and across clinicians.

1	Many individuals with autism spectrum disorder also have intellectual impairment and/ or language impairment (e.g., slow to talk, language comprehension behind production). Even those with average or high intelligence have an uneven profile of abilities. The gap between intellectual and adaptive functional skills is often large. Motor deficits are often present, in- cluding odd gait, clumsiness, and other abnormal motor signs (e.g., walking on tiptoes). Self- injury (e.g., head banging, biting the wrist) may occur, and disruptive/challenging behav- iors are more common in children and adolescents with autism spectrum disorder than other disorders, including intellectual disability. Adolescents and adults with autism spec- trum disorder are prone to anxiety and depression. Some individuals develop catatonic—like motor behavior (slowing and ”freezing” mid-action), but these are typically not of the mag- nitude of a catatonic episode. However, it is possible for individuals with autism

1	develop catatonic—like motor behavior (slowing and ”freezing” mid-action), but these are typically not of the mag- nitude of a catatonic episode. However, it is possible for individuals with autism spectrum disorder to experience a marked deterioration in motor symptoms and display a full cata- tonic episode with symptoms such as mutism, posturing, grimacing and waxy ﬂexibility.

1	The risk period for comorbid catatonia appears to be greatest in the adolescent years. In recent years, reported frequencies for autism spectrum disorder across US. and non- U.S. countries have approached 1% of the population, with similar estimates in child and adult samples. It remains unclear whether higher rates reﬂect an expansion of the diag- nostic criteria of DSM-IV to include subthreshold cases, increased awareness, differences in study methodology, or a true increase in the frequency of autism spectrum disorder.

1	The age and pattern of onset also should be noted for autism spectrum disorder. Symptoms are typically recognized during the second year of life (12—24 months of age) but may be seen earlier than 12 months if developmental delays are severe, or noted later than 24 months if symptoms are more subtle. The pattern of onset description might include information about early developmental delays or any losses of social or language skills. In cases where skills have been lost, parents or caregivers may give a history of a gradual or relatively rapid deterioration in social behaviors or language skills. Typically, this would occur be- tween 12 and 24 months of age and is distinguished from the rare instances of developmen- tal regression occurring after at least 2 years of normal development (previously described as childhood disintegrative disorder).

1	The behavioral features of autism spectrum disorder first become evident in early childhood, with some cases presenting a lack of interest in social interaction in the first year of life. Some children with autism spectrum disorder experience developmental pla- teaus or regression, with a gradual or relatively rapid deterioration in social behaviors or use of language, often during the first 2 years of life. Such losses are rare in other disor- ders and may be a useful "red ﬂag" for autism spectrum disorder. Much more unusual and warranting more extensive medical investigation are losses of skills beyond social communication (e.g., loss of self—care, toileting, motor skills) or those occurring after the second birthday (see also Rett syndrome in the section ”Differential Diagnosis” for this disorder).

1	First symptoms of autism spectrum disorder frequently involve delayed language de- velopment, often accompanied by lack of social interest or unusual social interactions (e.g., pulling individuals by the hand without any attempt to look at them), odd play patterns (e.g., carrying toys around but never playing with them), and unusual communication patterns (e.g., knowing the alphabet but not responding to own name). Deafness may be suspected but is typically ruled out. During the second year, odd and repetitive behaviors and the absence of typical play become more apparent. Since many typically developing young children have strong preferences and enjoy repetition (e.g., eating the same foods, watching the same video multiple times), distinguishing restricted and repetitive behav- iors that are diagnostic of autism spectrum disorder can be difficult in preschoolers. The clinical distinction is based on the type, frequency, and intensity of the behavior (e.g., a child who daily lines up

1	are diagnostic of autism spectrum disorder can be difficult in preschoolers. The clinical distinction is based on the type, frequency, and intensity of the behavior (e.g., a child who daily lines up objects for hours and is very distressed if any item is moved).

1	Autism spectrum disorder is not a degenerative disorder, and it is typical for learning and compensation to continue throughout life. Symptoms are often most marked in early childhood and early school years, with developmental gains typical in later childhood in at least some areas (e.g., increased interest in social interaction). A small proportion of in— dividuals deteriorate behaviorally during adolescence, whereas most others improve. Only a minority of individuals with autism spectrum disorder live and work indepen- dently in adulthood; those who do tend to have superior language and intellectual abilities and are able to find a niche that matches their special interests and skills. In general, indi- viduals with lower levels of impairment may be better able to function independently. However, even these individuals may remain socially naive and vulnerable, have difficul- ties organizing practical demands without aid, and are prone to anxiety and depression.

1	However, even these individuals may remain socially naive and vulnerable, have difficul- ties organizing practical demands without aid, and are prone to anxiety and depression. Many adults report using compensation strategies and coping mechanisms to mask their difficulties in public but suffer from the stress and effort of maintaining a socially accept- able facade. Scarcely anything is known about old age in autism spectrum disorder.

1	Some individuals come for first diagnosis in adulthood, perhaps prompted by the diagno- sis of autism in a child in the family or a breakdown of relations at work or home. Obtaining de- tailed developmental history in such cases may be difficult, and it is important to consider self- reported difficulties. Where clinical observation suggests criteria are currently met, autism spectrum disorder may be diagnosed, provided there is no evidence of good social and com- munication skills in childhood. For example, the report (by parents or another relative) that the cation skills throughout childhood would rule out a diagnosis of autism spectrum disorder; however, the absence of developmental information in itself should not do so. Manifestations of the social and communication impairments and restricted/ repeti- tive behaviors that define autism spectrum disorder are clear in the developmental period.

1	Manifestations of the social and communication impairments and restricted/ repeti- tive behaviors that define autism spectrum disorder are clear in the developmental period. In later life, intervention or compensation, as well as current supports, may mask these dif- ficulties in at least some contexts. However, symptoms remain sufficient to cause current impairment in social, occupational, or other important areas of functioning. The best established prognostic factors for individual outcome within autism spectrum disorder are presence or absence of associated intellectual disability and language impair— ment (e.g., functional language by age 5 years is a good prognostic sign) and additional mental health problems. Epilepsy, as a comorbid diagnosis, is associated with greater in- tellectual disability and lower verbal ability.

1	Environmental. A variety of nonspecific risk factors, such as advanced parental age, low birth weight, or fetal exposure to valproate, may contribute to risk of autism spectrum dis- order. Genetic and physiological. Heritability estimates for autism spectrum disorder have ranged from 37% to higher than 90%, based on twin concordance rates. Currently, as many as 15% of cases of autism spectrum disorder appear to be associated with a known genetic mutation, with different de novo copy number variants or de novo mutations in specific genes associated with the disorder in different families. However, even when an autism spectrum disorder is associated with a known genetic mutation, it does not appear to be fully penetrant. Risk for the remainder of cases appears to be polygenic, with perhaps hun- dreds of genetic loci making relatively small contributions.

1	Cultural differences will exist in norms for social interaction, nonverbal communication, and relationships, but individuals with autism spectrum disorder are markedly impaired against the norms for their cultural context. Cultural and socioeconomic factors may affect age at recognition or diagnosis; for example, in the United States, late or underdiagnosis of autism spectrum disorder among African American children may occur. Autism spectrum disorder is diagnosed four times more often in males than in females. In clinic samples, females tend to be more likely to show accompanying intellectual disabil- ity, suggesting that girls without accompanying intellectual impairments or language delays may go unrecognized, perhaps because of subtler manifestation of social and com- munication difficulties. Functional Consequences of Autism Spectrum Disorder

1	Functional Consequences of Autism Spectrum Disorder In young children with autism spectrum disorder, lack of social and communication abil- ities may hamper learning, especially learning through social interaction or in settings with peers. In the home, insistence on routines and aversion to change, as well as sensory sensitivities, may interfere with eating and sleeping and make routine care (e.g., haircuts, dental work) extremely difficult. Adaptive skills are typically below measured IQ. Ex- treme difficulties in planning, organization, and coping with change negatively impact academic achievement, even for students with above-average intelligence. During adult- hood, these individuals may have difficulties establishing independence because of con- tinued rigidity and difficulty with novelty.

1	Many individuals with autism spectrum disorder, even without intellectual disability, have poor adult psychosocial functioning as indexed by measures such as independent living and gainful employment. Functional consequences in old age are unknown, but so- cial isolation and communication problems (e.g., reduced help-seeking) are likely to have consequences for health in older adulthood. Rett syndrome. Disruption of social interaction may be observed during the regressive phase of Rett syndrome (typically between 1—4 years of age); thus, a substantial proportion spectrum disorder. However, after this period, most individuals with Rett syndrome im— prove their social communication skills, and autistic features are no longer a major area of concern. Consequently, autism spectrum disorder should be considered only when all di- agnostic criteria are met. Selective mutism. In selective mutism, early development is not typically disturbed.

1	Selective mutism. In selective mutism, early development is not typically disturbed. The affected child usually exhibits appropriate communication skills in certain contexts and settings. Even in settings where the child is mute, social reciprocity is not impaired, nor are restricted or repetitive patterns of behavior present. Language disorders and social (pragmatic) communication disorder. In some forms of language disorder, there may be problems of communication and some secondary so- cial difficulties. However, specific language disorder is not usually associated with abnor— mal nonverbal communication, nor with the presence of restricted, repetitive patterns of behavior, interests, or activities.

1	but does not show restricted and repetitive behavior or interests, criteria for social (prag- matic) communication disorder, instead of autism spectrum disorder, may be met. The di- agnosis of autism spectrum disorder supersedes that of social (pragmatic) communication disorder whenever the criteria for autism spectrum disorder are met, and care should be taken to enquire carefully regarding past or current restricted/ repetitive behavior.

1	disorder. Intellectual disability without autism spectrum disorder may be difficult to differentiate from autism spectrum disorder in very young children. Individuals with in- challenge for differential diagnosis, since repetitive behavior often occurs in such individ- uals as well. A diagnosis of autism spectrum disorder in an individual with intellectual disability is appropriate when social communication and interaction are significantly im- paired relative to the developmental level of the individual’s nonverbal skills (e.g., fine motor skills, nonverbal problem solving). In contrast, intellectual disability is the appropri- ate diagnosis when there is no apparent discrepancy between the level of social-commu- nicative skills and other intellectual skills.

1	Stereotypic movement disorder. Motor stereotypies are among the diagnostic charac- teristics of autism spectrum disorder, so an additional diagnosis of stereotypic movement disorder is not given when such repetitive behaviors are better explained by the presence of autism spectrum disorder. However, when stereotypies cause self—injury and become a focus of treatment, both diagnoses may be appropriate. Attention-deficit/hyperactivity disorder. Abnormalities of attention (overly focused or easily distracted) are common in individuals with autism spectrum disorder, as is hy- peractivity. A diagnosis of attention-deficit/hyperactivity disorder (ADHD) should be dividuals of comparable mental age.

1	Schizophrenia. Schizophrenia with childhood onset usually develops after a period of normal, or near normal, development. A prodromal state has been described in which so- cial impairment and atypical interests and beliefs occur, which could be confused with the social deficits seen in autism spectrum disorder. Hallucinations and delusions, which are defining features of schizophrenia, are not features of autism spectrum disorder. How- ever, clinicians must take into account the potential for individuals with autism spectrum disorder to be concrete in their interpretation of questions regarding the key features of schizophrenia (e.g., ”Do you hear voices when no one is there?” ”Yes [on the radio]”).

1	Autism spectrum disorder is frequently associated with intellectual impairment and struc- tural language disorder (i.e., an inability to comprehend and construct sentences with proper grammar), which should be noted under the relevant specifiers when applicable. Many in- dividuals with autism spectrum disorder have psychiatric symptoms that do not form part of the diagnostic criteria for the disorder (about 70% of individuals with autism spectrum dis- order may have one comorbid mental disorder, and 40% may have two or more comorbid mental disorders). When criteria for both ADHD and autism spectrum disorder are met, both diagnoses should be given. This same principle applies to concurrent diagnoses of autism spectrum disorder and developmental coordination disorder, anxiety disorders, depressive disorders, and other comorbid diagnoses. Among individuals who are nonverbal or have language deficits, observable signs such as changes in sleep or eating and increases in chal- lenging

1	depressive disorders, and other comorbid diagnoses. Among individuals who are nonverbal or have language deficits, observable signs such as changes in sleep or eating and increases in chal- lenging behavior‘should trigger an evaluation for anxiety or depression. Specific learning dif- ficulties (literacy and numeracy) are common, as is developmental coordination disorder.

1	Medical conditions commonly associated with autism spectrum disorder should be noted under the ”associated with a known medical /genetic or environmental/ acquired condition” specifier. Such medical conditions include epilepsy, sleep problems, and constipation. Avoidant-restrictive food intake disorder is a fairly frequent presenting feature of autism spectrum disorder, and extreme and narrow food preferences may persist. A. A persistent pattern of inattention and/or hyperactivity-impulsivity that interferes with functioning or development, as characterized by (1) and/or (2): 1. Inattention: Six (or more) of the following symptoms have persisted for at least 6 months to a degree that is inconsistent with developmental level and that nega- tively impacts directly on social and academic/occupational activities:

1	Note: The symptoms are not solely a manifestation of oppositional behavior, defi- ance, hostility, or failure to understand tasks or instructions. For older adolescents and adults (age 17 and older), at least five symptoms are required. a. O_ften fails to give close attention to details or makes careless mistakes in schoolwork, at work, or during other activities (e.g., overlooks or misses details, work is inaccurate). b. Often has difficulty sustaining attention in tasks or play activities (e.g., has diffi- culty remaining focused during lectures, conversations, or lengthy reading). 0. Often does not seem to listen when spoken to directly (e.g., mind seems else- where, even in the absence of any obvious distraction). d. Often does not follow through on instructions and fails to finish schoolwork, chores, or duties in the workplace (e.g., starts tasks but quickly loses focus and is easily sidetracked).

1	d. Often does not follow through on instructions and fails to finish schoolwork, chores, or duties in the workplace (e.g., starts tasks but quickly loses focus and is easily sidetracked). e. Often has difficulty organizing tasks and activities (e.g., difficulty managing se- quential tasks; difficulty keeping materials and belongings in order; messy, dis- organized work; has poor time management; fails to meet deadlines). f. Often avoids, dislikes, or is reluctant to engage in tasks that require sustained mental effort (e.g., schoolwork or homework; for older adolescents and adults, preparing reports, completing forms, reviewing lengthy papers). 9. Often loses things necessary for tasks or activities (e.g., school materials, pen- cils, books, tools, wallets, keys, paperwork, eyeglasses, mobile telephones). h. Is often easily distracted by extraneous stimuli (for older adolescents and adults, may include unrelated thoughts).

1	h. Is often easily distracted by extraneous stimuli (for older adolescents and adults, may include unrelated thoughts). i. ls often forgetful in daily activities (e.g., doing chores, running errands; for older adolescents and adults, returning calls, paying bills, keeping appointments). 2. Hyperactivity and impulsivity: Six (or more) of the following symptoms have per- sisted for at least 6 months to a degree that is inconsistent with developmental level and that negatively impacts directly on social and academic/occupational activities: Note: The symptoms are not solely a manifestation of oppositional behavior, defi- ance, hostility, or a failure to understand tasks or instructions. For older adolescents and adults (age 17 and older), at least five symptoms are required. a. Often fidgets with or taps hands or feet or squirms in seat.

1	a. Often fidgets with or taps hands or feet or squirms in seat. b. Often leaves seat in situations when remaining seated is expected (e.g., leaves his or her place in the classroom, in the office or other workplace, or in other situations that require remaining in place). 0. Often runs about or climbs in situations where it is inappropriate. (Note: In ad- olescents or adults, may be limited to feeling restless.) d. Often unable to play or engage in leisure activities quietly. 6. Is often “on the go," acting as if “driven by a motor” (e.g., is unable to be or un- comfortable being still for extended time, as in restaurants, meetings; may be experienced by others as being restless or difficult to keep up with). f. Often talks excessively. g. Often blurts out an answer before a question has been completed (e.g., com- pletes people’s sentences; cannot wait for turn in conversation). h. Often has difficulty waiting his or her turn (e.g., while waiting in line).

1	h. Often has difficulty waiting his or her turn (e.g., while waiting in line). i. Often interrupts or intrudes on others (e.g., butts into conversations. games, or mission; for adolescents and adults, may intrude into or take over what others are doing). . Several inattentive or hyperactive-impulsive symptoms were present prior to age 12 years. . Several inattentive or hyperactive-impulsive symptoms are present in two or more set- tings (e.g., at home, school, or work; with friends or relatives; in other activities). . There is clear evidence that the symptoms interfere with, or reduce the quality of, so- cial, academic, or occupational functioning. . The symptoms do not occur exclusively during the course of schizophrenia or another psychotic disorder and are not better explained by another mental disorder (e.g., mood disorder, anxiety disorder, dissociative disorder, personality disorder, substance intox- ication or withdrawal).

1	Specify whether: 314.01 (F90.2) Combined presentation: If both Criterion A1 (inattention) and Crite- rion A2 (hyperactivity-impulsivity) are met for the past 6 months. 314.00 (F90.0) Predominantly inattentive presentation: If Criterion A1 (inattention) is met but Criterion A2 (hyperactivity-impulsivity) is not met for the past 6 months. 314.01 (F90.1) Predominantly hyperactivefimpulsive presentation: If Criterion A2 (hy- peractivity-impulsivity) is met and Criterion A1 (inattention) is not met for the past 6 months. Specify if: in partial remission: When full criteria were previously met, fewer than the full criteria have been met for the past 6 months, and the symptoms still result in impairment in social, academic, or occupational functioning. Specify current severity: Mild: Few, it any, symptoms in excess of those required to make the diagnosis are present, and symptoms result in no more than minor impairments in social or occupa- tional functioning.

1	Mild: Few, it any, symptoms in excess of those required to make the diagnosis are present, and symptoms result in no more than minor impairments in social or occupa- tional functioning. Moderate: Symptoms or functional impairment between “mild" and “severe" are present. Severe: Many symptoms in excess of those required to make the diagnosis, or several symptoms that are particularly severe, are present, or the symptoms result in marked impairment ih social or occupational functioning.

1	The essential feature of attention-deficit/hyperactivity disorder (ADHD) is a persistent pattern of inattention and / or hyperactivity-impulsivity that interferes with functioning or development. Inattention manifests behaviorally in ADHD as wandering off task, lacking persistence, having difficulty sustaining focus, and being disorganized and is not due to defiance or lack of comprehension. Hyperactivity refers to excessive motor activity (such as a child running about) when it is not appropriate, or excessive fidgeting, tapping, or talk- ativeness. In adults, hyperactivity may manifest as extreme restlessness or wearing others out with their activity. Impulsivity refers to hasty actions that occur in the moment without forethought and that have high potential for harm to the individual (e.g., darting into the street without looking). Impulsivity may reﬂect a desire for immediate rewards or an in- ability to delay gratification. Impulsive behaviors may manifest as social

1	individual (e.g., darting into the street without looking). Impulsivity may reﬂect a desire for immediate rewards or an in- ability to delay gratification. Impulsive behaviors may manifest as social intrusiveness (e.g., interrupting others excessively) and / or as making important decisions without con- sideration of long—term consequences (e.g., taking a job without adequate information).

1	ADHD begins in childhood. The requirement that several symptoms be present before age 12 years conveys the importance of a substantial clinical presentation during child- hood. At the same time, an earlier age at onset is not specified because of difficulties in es- tablishing precise childhood onset retrospectively. Adult recall of childhood symptoms tends to be unreliable, and it is beneficial to obtain ancillary information.

1	Manifestations of the disorder must be present in more than one setting (e.g., home and school, work). Confirmation of substantial symptoms across settings typically cannot be done accurately without consulting informants who have seen the individual in those set- tings. Typically, symptoms vary depending on context within a given setting. Signs of the disorder may be minimal or absent when the individual is receiving frequent rewards for appropriate behavior, is under close supervision, is in a novel setting, is engaged in espe- cially interesting activities, has consistent external stimulation (e.g., via electronic screens), or is interacting in one-on-one situations (e.g., the clinician‘s office). Mild delays in language, motor, or social development are not specific to ADHD but often co- occur. Associated features may include low frustration tolerance, irritability, or mood lability.

1	Mild delays in language, motor, or social development are not specific to ADHD but often co- occur. Associated features may include low frustration tolerance, irritability, or mood lability. Even in the absence of a specific learning disorder, academic or work performance is often im- paired. Inattentive behavior is associated with various underlying cognitive processes, and in- dividuals with ADHD may exhibit cognitive problems on tests of attention, executive function, or memory, although these tests are not sufficiently sensitive or specific to serve as di- agnostic indices. By early adulthood, ADHD is associated with an increased risk of suicide at- tempt, primarily when comorbid with mood, conduct, or substance use disorders.

1	No biological marker is diagnostic for ADHD. As a group, compared with peers, chil— dren with ADHD display increased slow wave electroencephalograms, reduced total brain volume on magnetic resonance imaging, and possibly a delay in posterior to anterior cortical maturation, but these findings are not diagnostic. In the uncommon cases where there is a known genetic cause (e.g., Fragile X syndrome, 22q11 deletion syndrome), the ADHD presentation should still be diagnosed. Population surveys suggest that ADHD occurs in most cultures in about 5% of children and about 2.5% of adults.

1	ADHD presentation should still be diagnosed. Population surveys suggest that ADHD occurs in most cultures in about 5% of children and about 2.5% of adults. Many parents first observe excessive motor activity when the child is a toddler, but symp- toms are difficult to distinguish from highly variable normative behaviors before age 4 years. ADHD is most often identified during elementary school years, and inattention be- comes more prominent and impairing. The disorder is relatively stable through early ad- olescence, but some individuals have a worsened course with development of antisocial behaviors. In most individuals with ADHD, symptoms of motoric hyperactivity become less obvious in adolescence and adulthood, but difficulties with restlessness, inattention, poor planning, and impulsivity persist. A substantial proportion of children with ADHD remain relatively impaired into adulthood.

1	In preschool, the main manifestation is hyperactivity. Inattention becomes more prom- inent during elementary school. During adolescence, signs of hyperactivity (e.g., running and climbing) are less common and may be confined to fidgetiness or an inner feeling of jitteriness, restlessness, or impatience. In adulthood, along with inattention and restless- ness, impulsivity may remain problematic even when hyperactivity has diminished. Temperamental. ADHD is associated with reduced behavioral inhibition, effortful con- trol, or constraint; negative emotionality; and/ or elevated novelty seeking. These traits may predispose some children to ADHD but are not specific to the disorder.

1	Environmental. Very low birth weight (less than 1,500 grams) conveys a two- to three- fold risk for ADHD, but most children with low birth weight do not develop ADHD. Al- though ADHD is correlated with smoking during pregnancy, some of this association reﬂects common genetic risk. A minority of cases may be related to reactions to aspects of diet. There may be a history of child abuse, neglect, multiple foster placements, neurotoxin exposure (e.g., lead), infections (e.g., encephalitis), or alcohol exposure in utero. Exposure to environmental toxicants has been correlated with subsequent ADHD, but it is not known whether these associations are causal. Genetic and physiological. ADHD is elevated in the first-degree biological relatives of individuals with ADHD. The heritability of ADHD is substantial. While specific genes have been correlated with ADHD, they are neither necessary nor sufficient causal factors.

1	Visual and hearing impairments, metabolic abnormalities, sleep disorders, nutritional de- ficiencies, and epilepsy should be considered as possible inﬂuences on ADHD symptoms. ADHD is not associated with specific physical features, although rates of minor phys- ical anomalies (e.g., hypertelorism, highly arched palate, low-set ears) may be relatively elevated. Subtle motor delays and other neurological soft signs may occur. (Note that marked co-occurring clumsiness and motor delays should be coded separately [e.g., de- velopmental coordination disorder].) Course modifiers. Family interaction patterns in early childhood are unlikely to cause ADHD but may inﬂuence its course or contribute to secondary development of conduct problems.

1	Course modifiers. Family interaction patterns in early childhood are unlikely to cause ADHD but may inﬂuence its course or contribute to secondary development of conduct problems. Differences in ADHD prevalence rates across regions appear attributable mainly to differ- ent diagnostic and methodological practices. However, there also may be cultural varia- tion in attitudes toward or interpretations of children’s behaviors. Clinical identification rates in the United States for African American and Latino populations tend to be lower than for Caucasian populations. Informant symptom ratings may be inﬂuenced by cul- tural group of the child and the informant, suggesting that culturally appropriate practices are relevant in assessing ADHD. ADHD is more frequent in males than in females in the general population, with a ratio of approximately 221 in children and 1.621 in adults. Females are more likely than males to present primarily with inattentive features.

1	ADHD is associated with reduced school performance and academic attainment, social re- jection, and, in adults, poorer occupational performance, attainment, attendance, and higher probability of unemployment as well as elevated interpersonal conﬂict. Children with ADHD are significantly more likely than their peers without ADHD to develop con- duct disorder in adolescence and antisocial personality disorder in adulthood, conse— quently increasing the likelihood for substance use disorders and incarceration. The risk of subsequent substance use disorders is elevated, especially when conduct disorder or an- tisocial personality disorder develops. Individuals with ADHD are more likely than peers to be injured. Traffic accidents and violations are more frequent in drivers with ADHD. There may be an elevated likelihood of obesity among individuals with ADHD.

1	There may be an elevated likelihood of obesity among individuals with ADHD. Inadequate or variable self-application to tasks that require sustained effort is often in- terpreted by others as laziness, irresponsibility, or failure to cooperate. Family relation- ships may be characterized by discord and negative interactions. Peer relationships are often disrupted by peer rejection, neglect, or teasing of the individual with ADHD. On av- erage, individuals with ADHD obtain less schooling, have poorer vocational achievement, and have reduced intellectual scores than their peers, although there is great variability. In its severe form, the disorder is markedly impairing, affecting social, familial, and scholas- tic/occupational adjustment.

1	Academic deficits, school-related problems, and peer neglect tend to be most associ— ated with elevated symptoms of inattention, whereas peer rejection and, to a lesser extent, accidental injury are most salient with marked symptoms of hyperactivity or impulsivity. Oppositional defiant disorder. Individuals with oppositional defiant disorder may re- sist work or school tasks that require self—application because they resist conforming to others' demands. Their behavior is characterized by negativity, hostility, and defiance. These symptoms must be differentiated from aversion to school or mentally demanding tasks due to difficulty in sustaining mental effort, forgetting instructions, and impulsivity in individuals with ADHD. Complicating the differential diagnosis is the fact that some individuals with ADHD may develop secondary oppositional attitudes toward such tasks and devalue their importance.

1	Intermittent explosive disorder. ADHD and intermittent explosive disorder share high levels of impulsive behavior. However, individuals with intermittent explosive disorder show serious aggression toward others, which is not characteristic of ADHD, and they do not experience problems with sustaining attention as seen in ADHD. In addition, intermit- tent explosive disorder is rare in childhood. Intermittent explosive disorder may be diag- nosed in the presence of ADHD. Other neurodevelopmental disorders. The increased motoric activity that may occur in

1	Other neurodevelopmental disorders. The increased motoric activity that may occur in ADHD must be distinguished from the repetitive motor behavior that characterizes stereo- typic movement disorder and some cases of autism spectrum disorder. In stereotypic movement disorder, the motoric behavior is generally fixed and repetitive (e.g., body rock- ing, self—biting), whereas the fidgetiness and restlessness in ADHD are typically general- ized and not characterized by repetitive stereotypic movements. In Tourette’s disorder, frequent multiple tics can be mistaken for the generalized fidgetiness of ADHD. Prolonged observation may be needed to differentiate fidgetiness from bouts of multiple tics. Specific learning disorder. Children with specific learning disorder may appear inat- tentive because of frustration, lack of interest, or limited ability. However, inattention in individuals with a specific learning disorder who do not have ADHD is not impairing out- side of academic work.

1	Intellectual disability (intellectual developmental disorder). Symptoms of ADHD are common among children placed in academic settings that are inappropriate to their intel- lectual ability. In such cases, the symptoms are not evident during non-academic tasks. A diagnosis of ADHD in intellectual disability requires that inattention or hyperactivity be excessive for mental age.

1	Autism spectrum disorder. Individuals with ADHD and those with autism spectrum disorder exhibit inattention, social dysfunction, and difficult-to-manage behavior. The so- cial dysfunction and peer rejection seen in individuals with ADHD must be distinguished from the social disengagement, isolation, and indifference to facial and tonal communica- tion cues seen in individuals with autism spectrum disorder. Children with autism spec— trum disorder may display tantrums because of an inability to tolerate a change from their expected course of events. In contrast, children with ADHD may misbehave or have a tan- trum during a major transition because of impulsivity or poor self—control. Reactive attachment disorder. Children with reactive attachment disorder may show social disinhibition, but not the full ADHD symptom cluster, and display other features such as a lack of enduring relationships that are not characteristic of ADHD.

1	Anxiety disorders. ADHD shares symptoms of inattention with anxiety disorders. Indi- viduals with ADHD are inattentive because of their attraction to external stimuli, new activities, or preoccupation with enjoyable activities. This is distinguished from the inat- tention due to worry and rumination seen in anxiety disorders. Restlessness might be seen in anxiety disorders. However, in ADHD, the symptom is not associated with worry and rumination. Depressive disorders. Individuals with depressive disorders may present with inabil- ity to concentrate. However, poor concentration in mood disorders becomes prominent only during a depressive episode.

1	Bipolar disorder. Individuals with bipolar disorder may have increased activity, poor concentration, and increased impulsivity, but these features are episodic, occurring sev- eral days at a time. In bipolar disorder, increased impulsivity or inattention is accompa- nied by elevated mood, grandiosity, and other specific bipolar features. Children with ADHD may show significant changes in mood within the same day; such lability is dis— tinct from a manic episode, which must last 4 or more days to be a clinical indicator of bi- polar disorder, even in children. Bipolar disorder is rare in preadolescents, even when severe irritability and anger are prominent, whereas ADHD is common among children and adolescents who display excessive anger and irritability.

1	Disruptive mood dysregulation disorder. Disruptive mood dysregulation disorder is characterized by pervasive irritability, and intolerance of frustration, but impulsiveness and disorganized attention are not essential features. However, most children and adoles- cents with the disorder have symptoms that also meet criteria for ADHD, which is diag- nosed separately. Substance use disorders. Differentiating ADHD from substance use disorders may be problematic if the first presentation of ADHD symptoms follows the onset of abuse or fre- quent use. Clear evidence of ADHD before substance misuse from informants or previous records may be essential for differential diagnosis.

1	Personality disorders. In adolescents and adults, it may be difficult to distinguish ADHD from borderline, narcissistic, and other personality disorders. All these disorders tend to share the features of disorganization, social intrusiveness, emotional dysregulation, and cognitive dysregulation. However, ADHD is not characterized by fear of abandonment, self—injury, extreme ambivalence, or other features of personality disorder. It may take extended clinical observation, informant interview, or detailed history to distinguish im- pulsive, socially intrusive, or inappropriate behavior from narcissistic, aggressive, or dom- ineering behavior to make this differential diagnosis. Psychotic disorders. ADHD is not diagnosed if the symptoms of inattention and hyperac- tivity occur exclusively during the course of a psychotic disorder.

1	Psychotic disorders. ADHD is not diagnosed if the symptoms of inattention and hyperac- tivity occur exclusively during the course of a psychotic disorder. Medication-induced symptoms of ADHD. Symptoms of inattention, hyperactivity, or impulsivity attributable to the use of medication (e.g., bronchodilators, isoniazid, neuro- leptics [resulting in akathisia], thyroid replacement medication) are diagnosed as other specified or unspecified other (or unknown) substance—related disorders. Neurocognitive disorders. Early major neurocognitive disorder (dementia) and/or mild neurocognitive disorder are not known to be associated with ADHD but may present with similar clinical features. These conditions are distinguished from ADHD by their late onset. In clinical settings, comorbid disorders are frequent in individuals whose symptoms meet criteria for ADHD. In the general population, oppositional defiant disorder co-occurs with

1	ADHD in approximately half of children with the combined presentation and about a quarter with the predominantly inattentive presentation. Conduct disorder co-occurs in about a quarter of children or adolescents with the combined presentation, depending on age and setting. Most children and adolescents with disruptive mood dysregulation dis- order have symptoms that also meet criteria for ADHD; a lesser percentage of children der. Specific learning disorder commonly co-occurs with ADHD. Anxiety disorders and major depressive disorder occur in a minority of individuals with ADHD but more often than in the general population. Intermittent explosive disorder occurs in a minority of adults with ADHD, but at rates above population levels. Although substance use disor— ders are relatively more frequent among adults with ADHD in the general population, the disorders are present in only a minority of adults with ADHD. In adults, antisocial and other personality disorders may co-occur with

1	frequent among adults with ADHD in the general population, the disorders are present in only a minority of adults with ADHD. In adults, antisocial and other personality disorders may co-occur with ADHD. Other disorders that may co-occur with ADHD include obsessive-compulsive disorder, tic disorders, and autism spectrum disorder.

1	314.01 (F90.8) This category applies to presentations in which symptoms characteristic of attention- cial, occupational or other important areas of functioning predominate but do not meet the full criteria for attention-deticit/hyperactivity disorder or any of the disorders in the neuro- developmental disorders diagnostic class. The other specified attention-deficit/hyperactiv- ity disorder category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for attention-deficit/ hyperactivity disorder or any specific neurodevelopmental disorder. This is done by re- cording “other specified attention-deficit/hyperactivity disorder" followed by the specific reason (e.g., ”with insufficient inattention symptoms"). 314.01 (F90.9)

1	314.01 (F90.9) This category applies to presentations in which symptoms characteristic of attention- cial, occupational, or other important areas of functioning predominate but do not meet the fulI criteria for attention-deficit/hyperactivity disorder or any of the disorders in the neuro- developmental disorders diagnostic class. The unspecified attention-deticit/hyperactivity disorder category is used in situations in which the clinician chooses not to specify the rea- son that the criteria are not met for attention-deficit/hyperactivity disorder or for a specific neurodevelopmental disorder, and includes presentations in which there is insufficient in- formation to make a more specific diagnosis.

1	A. Difficulties learning and using academic skills, as indicated by the presence of at least one of the following symptoms that have persisted for at least 6 months, despite the provision of interventions that target those difficulties: 1. Inaccurate or slow and effortful word reading (e.g., reads single words aloud incor- rectly or slowly and hesitantly, frequently guesses words, has difficulty sounding out words). 2. Difficulty understanding the meaning of what is read (e.g., may read text accurately but not understand the sequence, relationships, inferences, or deeper meanings of what is read). 3. Difficulties with spelling (e.g., may add, omit, or substitute vowels or consonants). 4. Difficulties with written expression (e.g., makes multiple grammatical or punctua- sion of ideas lacks clarity).

1	4. Difficulties with written expression (e.g., makes multiple grammatical or punctua- sion of ideas lacks clarity). 5. Difficulties mastering number sense, number facts, or calculation (e.g., has poor understanding of numbers, their magnitude, and relationships; counts on fingers to add singIe-digit numbers instead of recalling the math fact as peers do; gets lost in the midst of arithmetic computation and may switch procedures). 6. Difficulties with mathematical reasoning (e.g., has severe difficulty applying math- ematical concepts, facts, or procedures to solve quantitative problems). B.

1	6. Difficulties with mathematical reasoning (e.g., has severe difficulty applying math- ematical concepts, facts, or procedures to solve quantitative problems). B. The affected academic skills are substantially and quantifiably below those expected for the individual’s chronological age. and cause significant interference with academic or occupatiohal performance, or with activities of daily living, as confirmed by individu- assessment. For individuals age 17 years and older, a documented history of impairing learning difficulties may be substituted for the standardized assessment. . The learning difficulties begin during schooI-age years but may not become fully man- itest until the demands for those affected academic skills exceed the individual’s lim- ited capacities (e.g., as in timed tests. reading or writing lengthy complex reports for a tight deadline, excessively heavy academic loads).

1	. The learning difficulties are not better accounted for by intellectual disabilities, uncor- rected visual or auditory acuity, other mental or neurological disorders, psychosocial adversity, lack of proficiency in the language of academic instruction, or inadequate educational instruction. Note: The four diagnostic criteria are to be met based on a clinical synthesis of the indi- vidual’s history (developmental, medical, family, educational), school reports, and psycho- educational assessment. Coding note: Specify all academic domains and subskills that are impaired. When more than one domain is impaired, each one should be coded individually according to the fol- lowing specifiers. Specify if: 315.00 (F81.0) With impairment in reading:

1	Specify if: 315.00 (F81.0) With impairment in reading: Note: Dyslexia is an alternative term used to refer to a pattern of learning difficulties characterized by problems with accurate or fluent word recognition, poor decoding, and poor spelling abilities. lf dyslexia is used to specify this particular pattern of dif- ficulties, it is important also to specify any additional difficulties that are present, such as difficulties with reading comprehension or math reasoning. 315.2 (F81.81) With impairment in written expression: Clarity or organization of written expression 315.1 (F81.2) With impairment in mathematics: Memorization of arithmetic facts

1	315.2 (F81.81) With impairment in written expression: Clarity or organization of written expression 315.1 (F81.2) With impairment in mathematics: Memorization of arithmetic facts Note: Dyscalculia is an alternative term used to refer to a pattern of difficulties char- acterized by problems processing numerical information, learning arithmetic facts, and performing accurate or fluent calculations. If dyscalculia is used to specify this particular pattern of mathematic difficulties, it is important also to specify any addi- tional difficulties that are present, such as difficulties with math reasoning or word rea- soning accuracy. Specify current severity: Miid: Some difficulties learning skills in one or two academic domains, but of mild enough severity that the individual may be able to compensate or function well when provided with appropriate accommodations or support services, especially during the school years.

1	Moderate: Marked difficulties learning skills in one or more academic domains, so that the individual is unlikely to become proficient without some intervals of intensive and specialized teaching during the school years. Some accommodations or supportive services at least part of the day at school, in the workplace, or at home may be needed to complete activities accurately and efficiently. Severe: Severe difficulties learning skills, affecting several academic domains, so that the individual is unlikely to learn those skills without ongoing intensive individualized and specialized teaching for most of the school years. Even with an array of appropri- ate accommodations or services at home, at school, or in the workplace, the individual may not be able to complete all activities efficiently.

1	Each impaired academic domain and subskill of specific learning disorder should be re- corded. Because of ICD coding requirements, impairments in reading, impairments in writ- ten expression, and impairments in mathematics, with their corresponding impairments in subskills, must be coded separately. For example, impairments in reading and mathematics and impairments in the subskills of reading rate or ﬂuency, reading comprehension, accu- rate or ﬂuent calculation, and accurate math reasoning would be coded and recorded as 315.00 (F810) specific learning disorder with impairment in reading, with impairment in reading rate or ﬂuency and impairment in reading comprehension; 315.1 (F812) specific learning disorder with impairment in mathematics, with impairment in accurate or ﬂuent calculation and impairment in accurate math reasoning.

1	Specific learning disorder is a neurodevelopmental disorder with a biological origin that is the basis for abnormalities at a cognitive level that are associated with the behavioral signs of the disorder. The biological origin includes an interaction of genetic, epigenetic, and en- vironmental factors, which affect the brain’s ability to perceive or process verbal or non- verbal information efficiently and accurately.

1	One essential feature of specific learning disorder is persistent difficulties learning key- stone academic skills (Criterion A), with onset during the years of formal schooling (i.e., the de- velopmental period). Key academic skills include reading of single words accurately and ﬂuently, reading comprehension, written expression and spelling, arithmetic calculation, and mathematical reasoning (solving mathematical problems). In contrast to talking or walking, which are acquired developmental milestones that emerge with brain maturation, academic skills (e.g., reading, spelling, writing, mathematics) have to be taught and learned explicitly.

1	Specific learning disorder disrupts the normal pattern of learning academic skills; it is not sirn- ply a consequence of lack of opportunity of learning or inadequate instruction. Difficulties mastering these key academic skills may also impede learning in other academic subjects (e.g., history, science, social studies), but those problems are attributable to difficulties learning the underlying academic skills. Difficulties learning to map letters with the sounds of one’s lan- guage—to read printed words (often called dyslexia)—is one of the most common manifesta- tions of specific learning disorder. The learning difficulties manifest as a range of observable, descriptive behaviors or symptoms (as listed in Criteria A1—A6). These clinical symptoms may be observed, probed by means of the clinical interview, or ascertained from school reports, rat- ing scales, or descriptions in previous educational or psychological assessments. The learning difficulties are persistent, not transitory.

1	interview, or ascertained from school reports, rat- ing scales, or descriptions in previous educational or psychological assessments. The learning difficulties are persistent, not transitory. In children and adolescents, persistence is defined as restricted progress in learning (i.e., no evidence that the individual is catching up with class- mates) for at least 6 months despite the provision of extra help at home or school. For example, difficulties learning to read single words that do not fully or rapidly remit with the provision of learning disorder. Evidence of persistent learning difficulties may be derived from cumulative school reports, portfolios of the child’s evaluated work, curriculum—based measures, or clinical interview. In adults, persistent difficulty refers to ongoing difficulties in literacy or numeracy skills that manifest during childhood or adolescence, as indicated by cumulative evidence from school reports, evaluated portfolios of work, or previous assessments.

1	A second key feature is that the individual’s performance of the affected academic skills is well below average for age (Criterion B). One robust clinical indicator of difficulties learning academic skills is low academic achievement for age or average achievement that is sustain- able only by extraordinarily high levels of effort or support. In children, the low academic skills teacher’s grades or ratings). Another clinical indicator, particularly in adults, is avoidance of activities that require the academic skills. Also in adulthood, low academic skills interfere with port or report by others). However, this criterion also requires psychometric evidence from an individually administered, psychometrically sound and culturally appropriate test of aca- demic achievement that is norm-referenced or criterion-referenced. Academic skills are dis- tributed along a continuum, so there is no natural cutpoint that can be used to differentiate individuals with and without specific learning

1	or criterion-referenced. Academic skills are dis- tributed along a continuum, so there is no natural cutpoint that can be used to differentiate individuals with and without specific learning disorder. Thus, any threshold used to specify what constitutes significantly low academic achievement (e.g., academic skills well below age expectation) is to a large extent arbitrary. Low achievement scores on one or more standard- ized tests or subtests within an academic domain (i.e., at least 1.5 standard deviations [SD] be- low the population mean for age, which translates to a standard score of 78 or less, which is below the 7th percentile) are needed for the greatest diagnostic certainty. However, precise scores will vary according to the particular standardized tests that are used. On the basis of clinical judgment, a more lenient threshold may be used (e.g., 1.0—2.5 SD below the pop- ulation mean for age), when learning difficulties are supported by converging evidence from clinical

1	basis of clinical judgment, a more lenient threshold may be used (e.g., 1.0—2.5 SD below the pop- ulation mean for age), when learning difficulties are supported by converging evidence from clinical assessment, academic history, school reports, or test scores. Moreover, since standardized tests are not available in all languages, the diagnosis may then be based in part on clinical judgment of scores on available test measures.

1	A third core feature is that the learning difficulties are readily apparent in the early school years in most individuals (Criterion C). However, in others, the learning difficulties may not manifest fully until later school years, by which time learning demands have in- creased and exceed the individual’s limited capacities.

1	Another key diagnostic feature is that the learning difficulties are considered ”spe- cific,” for four reasons. First, they are not attributable to intellectual disabilities (intellec- hearing or vision disorders, or neurological or motor disorders) (Criterion D). Specific els of intellectual functioning (generally estimated by an IQ score of greater than about 70 [:5 points allowing for measurement error]). The phrase ”unexpected academic under- achievement” is often cited as the defining characteristic of specific learning disorder in that the specific learning disabilities are not part of a more general learning difficulty as manifested in intellectual disability or global developmental delay. Specific learning dis- order may also occur in individuals identified as intellectually "gifted." These individuals may be able to sustain apparently adequate academic functioning by using compensatory strategies, extraordinarily high effort, or support, until the learning demands or assess-

1	These individuals may be able to sustain apparently adequate academic functioning by using compensatory strategies, extraordinarily high effort, or support, until the learning demands or assess- ment procedures (e.g., timed tests) pose barriers to their demonstrating their learning or accomplishing required tasks. Second, the learning difficulty cannot be attributed to more general external factors, such as economic or environmental disadvantage, chronic absen- teeism, or lack of education as typically provided in the individual’s community context.

1	Third, the learning difficulty cannot be attributed to a neurological (e.g., pediatric stroke) or motor disorders or to vision or hearing disorders, which are often associated with prob- lems learning academic skills but are distinguishable by presence of neurological signs. Finally, the learning difficulty may be restricted to one academic skill or domain (e.g., read- ing single words, retrieving or calculating number facts).

1	Comprehensive assessment is required. Specific learning disorder can only be diagnosed after formal education starts but can be diagnosed at any point afterward in children, adoles- cents, or adults, providing there is evidence of onset during the years of formal schooling (i.e., the developmental period). No single data source is sufficient for a diagnosis of specific leam- ing disorder. Rather, specific learning disorder is a clinical diagnosis based on a synthesis of the individual’s medical, developmental, educational, and family history; the history of the learning difficulty, including its previous and current manifestation; the impact of the diffi- culty on academic, occupational, or social functioning; previous or current school reports; portfolios of work requiring academic skills; curriculum-based assessments; and previous or current scores from individual standardized tests of academic achievement. If an intellectual, sensory, neurological, or motor disorder is suspected,

1	curriculum-based assessments; and previous or current scores from individual standardized tests of academic achievement. If an intellectual, sensory, neurological, or motor disorder is suspected, then the clinical assessment for specific learning disorder should also include methods appropriate for these disorders. Thus, compre- hensive assessment will involve professionals with expertise in specific learning disorder and psychological/cognitive assessment. Since specific learning disorder typically persists into adulthood, reassessment is rarely necessary, unless indicated by marked changes in the leam- ing difficulties (amelioration or worsening) or requested for specific purposes.

1	Specific learning disorder is frequently but not invariably preceded, in preschool years, by delays in attention, language, or motor skills that may persist and co-occur with specific learning disorder. An uneven profile of abilities is common, such as above-average abili- ties in drawing, design, and other visuospatial abilities, but slow, effortful, and inaccurate reading and poor reading comprehension and written expression. Individuals with spe- logical tests of cognitive processing. However, it remains unclear whether these cognitive abnormalities are the cause, correlate, or consequence of the learning difficulties. Also, al- though cognitive deficits associated with difficulties learning to read words are well doc- umented, those associated with other manifestations of specific learning disorder (e.g., reading comprehension, arithmetic computation, written expression) are underspecified or unknown. Moreover, individuals with similar behavioral symptoms or test scores are found

1	disorder (e.g., reading comprehension, arithmetic computation, written expression) are underspecified or unknown. Moreover, individuals with similar behavioral symptoms or test scores are found to have a variety of cognitive deficits, and many of these processing deficits are also found in other neurodevelopmental disorders (e.g., attention-deficit/hyperactivity disor- der [ADHD], autistic spectrum disorder, communication disorders, developmental coor- dination disorder). Thus, assessment of cognitive processing deficits is not required for diagnostic assessment. Specific learning disorder is associated with increased risk for sui- cidal ideation and suicide attempts in children, adolescents, and adults.

1	There are no known biological markers of specific learning disorder. As a group, indi- viduals with the disorder show circumscribed alterations in cognitive processing and brain structure and function. Genetic differences are also evident at the group level. But cognitive testing, neuroimaging, or genetic testing are not useful for diagnosis at this time. The prevalence of specific learning disorder across the academic domains of reading, writ- ing, and mathematics is 5%—15% among school—age children across different languages and cultures. Prevalence in adults is unknown but appears to be approximately 4%.

1	Onset, recognition, and diagnosis of specific learning disorder usually occurs during the elementary school years when children are required to learn to read, spell, write, and learn mathematics. However, precursors such as language delays or deficits, difficulties in rhyming or couhting, or difficulties with fine motor skills required for writing commonly occur in early childhood before the start of formal schooling. Manifestations may be be- havioral (e.g., a reluctance to engage in learning; oppositional behavior). Specific learning disorder is lifelong, but the course and clinical expression are variable, in part depending on the interactions among the task demands of the environment, the range and severity of the individual’s learning difficulties, the individual’s learning abilities, comorbidity, and the available support systems and intervention. Nonetheless, problems with reading ﬂu- ency and comprehension, spelling, written expression, and numeracy skills in everyday life

1	comorbidity, and the available support systems and intervention. Nonetheless, problems with reading ﬂu- ency and comprehension, spelling, written expression, and numeracy skills in everyday life typically persist into adulthood.

1	Changes in manifestation of symptoms occur with age, so that an individual may have a persistent or shifting array of learning difficulties across the lifespan. Examples of symptoms that may be observed among preschool-age children include a lack of interest in playing games with language sounds (e.g., repetition, rhyming), and they may have trouble learning nursery rhymes. Preschool children with specific learning disorder may frequently use baby talk, rnispronounce words, and have trouble remembering names of let- ters, numbers, or days of the week. They may fail to recognize letters in their own names and have trouble learning to count. Kindergarten—age children with specific learning disorder may be unable to recognize and write letters, may be unable to write their own names, or may use invented spelling. They may have trouble breaking down spoken words into syllables (e.g., ”cowboy” into ”cow" and ”boy”) and trouble recognizing words that rhyme (e.g., cat, bat, hat).

1	Kindergarten-age children also may have trouble connecting letters with their sounds (e.g., let- ter b makes the sound / b / ) and may be unable to recognize phonemes (e.g., do not know which in a set of words [e.g., dog, man, car] starts with the same sound as ”cat”).

1	children), ﬂuent word decoding, spelling, or math facts; reading aloud is slow, inaccurate, and effortful, and some children struggle to understand the magnitude that a spoken or written number represents. Children in primary grades (grades 1—3) may continue to have problems recognizing and manipulating phonemes, be unable to read common one-sylla- ble words (such as mat or top), and be unable recognize common irregularly spelled words (e.g., said, two). They may commit reading errors that indicate problems in con- necting sounds and letters (e.g., ”big” for "got”) and have difficulty sequencing numbers and letters. Children in grades 1-3 also may have difficulty remembering number facts or arithmetic procedures for adding, subtracting, and so forth, and may complain that read- ing or arithmetic is hard and avoid doing it. Children with specific learning disorder in the middle grades (grades 4—6) may rnispronounce or skip parts of long, multisyllable words (e.g., say ”conible” for

1	arithmetic is hard and avoid doing it. Children with specific learning disorder in the middle grades (grades 4—6) may rnispronounce or skip parts of long, multisyllable words (e.g., say ”conible” for ”convertible,” ”aminal” for ”animal”) and confuse words that sound alike (e.g., ”tornado” for ”volcano”). They may have trouble remembering dates, names, and telephone numbers and may have trouble completing homework or tests on time. Children in the middle grades also may have poor comprehension with or without slow, effortful, and inaccurate reading, and they may have trouble reading small function words (e.g., that, the, an, in). They may have very poor spelling and poor written work.

1	They may get the first part of a word correctly, then guess wildly (e.g., read ”clover” as ”clock”), and may express fear of reading aloud or refuse to read aloud.

1	By contrast, adolescents may have mastered word decoding, but reading remains slow and effortful, and they are likely to show marked problems in reading comprehension and written expression (including poor spelling) and poor mastery of math facts or mathemat- ical problem solving. During adolescence and into adulthood, individuals with specific learning disorder may continue to make numerous spelling mistakes and read single words and connected text slowly and with much effort, with trouble pronouncing multi- syllable words. They may frequently need to reread material to understand or get the main point and have trouble making inferences from written text. Adolescents and adults may avoid activities that demand reading or arithmetic (reading for pleasure, reading instruc- tions). Adults with specific learning disorder have ongoing spelling problems, slow and effortful reading, or problems making important inferences from numerical information in work-related written documents. They

1	with specific learning disorder have ongoing spelling problems, slow and effortful reading, or problems making important inferences from numerical information in work-related written documents. They may avoid both leisure and work-related activ- ities that demand reading or writing or use alternative approaches to access print (e.g., text-to-speech/speech-to-text software, audiobooks, audiovisual media).

1	An alternative clinical expression is that of circumscribed learning difficulties that per- sist across the lifespan, such as an inability to master the basic sense of number (e.g., to know which of a pair of numbers or dots represents the larger magnitude), or lack of pro— ficiency in word identification or spelling. Avoidance of or reluctance to engage in activi— ties requiring academic skills is common in children, adolescents, and adults. Episodes of severe anxiety or anxiety disorders, including somatic complaints or panic attacks, are common across the lifespan and accompany both the circumscribed and the broader ex- pression of learning difficulties. Environmental. Prematurity or very low birth weight increases the risk for specific learning disorder, as does prenatal exposure to nicotine.

1	Environmental. Prematurity or very low birth weight increases the risk for specific learning disorder, as does prenatal exposure to nicotine. Genetic and physiological. Specific learning disorder appears to aggregate in families, particularly when affecting reading, mathematics, and spelling. The relative risk of spe- cific learning disorder in reading or mathematics is substantially higher (e.g., 4—8 times and 5—10 times higher, respectively) in first-degree relatives of individuals with these learning difficulties compared with those without them. Family history of reading diffi- disorder in offspring, indicating the combined role of genetic and environmental factors.

1	There is high heritability for both reading ability and reading disability in alphabetic and nonalphabetic languages, including high heritability for most manifestations of learning abil- ities and disabilities (e.g., heritability estimate values greater than 0.6). Covariation between various manifestations of learning difficulties is high, suggesting that genes related to one presentation are highly correlated with genes related to another manifestation.

1	Course modifiers. Marked problems with inattentive behavior in preschool years is pre- dictive of later difficulties in reading and mathematics (but not necessarily specific learn- ing disorder) and nonresponse to effective academic interventions. Delay or disorders in speech or language, or impaired cognitive processing (e.g., phonological awareness, working memory, rapid serial naming) in preschool years, predicts later specific learning disorder in reading and written expression. Comorbidity with ADHD is predictive of worse mental health outcome than that associated with specific learning disorder without ADHD. Systematic, intensive, individualized instruction, using evidence-based interven- tions, may improve or ameliorate the learning difficulties in some individuals or promote the use of compensatory strategies in others, thereby mitigating the otherwise poor out— comes.

1	Specific learning disorder occurs across languages, cultures, races, and socioeconomic conditions but may vary in its manifestation according to the nature of the spoken and written symbol systems and cultural and educational practices. For example, the cognitive processing requirements of reading and of working with numbers vary greatly across or- thographies. In the English language, the observable hallmark clinical symptom of diffi- culties learning to read is inaccurate and slow reading of single words; in other alphabetic languages that have more direct mapping between sounds and letters (e.g., Spanish, Ger— man) and in non-alphabetic languages (e.g., Chinese, Japanese), the hallmark feature is slow but accurate reading. In English-language learners, assessment should include con- sideration of whether the source of reading difficulties is a limited proficiency with Eng- lish or a specific learning disorder. Risk factors for specific learning disorder in English- language

1	con- sideration of whether the source of reading difficulties is a limited proficiency with Eng- lish or a specific learning disorder. Risk factors for specific learning disorder in English- language learners include a family history of specific learning disorder or language delay in the native language, as well as learning difficulties in English and failure to catch up with peers. If there is suspicion of cultural or language differences (e.g., as in an English- language learner), the assessment needs to take into account the individual’s language proficiency in his or her first or native language as well as in the second language (in this example, English). Also, assessment should consider the linguistic and cultural context in which the individual is living, as well as his or her educational and learning history in the original culture and language.

1	Specific learning disorder is more common in males than in females (ratios range from about 2:1 to 3:1) and cannot be attributed to factors such as ascertainment bias, definitional or measurement variation, language, race, or socioeconomic status. Specific learning disorder can have negative functional consequences across the lifespan, including lower academic attainment, higher rates of high school dropout, lower rates of postsecondary education, high levels of psychological distress and poorer overall mental health, higher rates of unemployment and under-employment, and lower incomes. School dropout and co-occurring depressive symptoms increase the risk for poor mental health outcomes, including suicidality, whereas high levels of social or emotional support predict better mental health outcomes.

1	Normal variations in academic attainment. Specific learning disorder is distinguished from normal variations in academic attainment due to external factors (e.g., lack of edu- cational opportunity, consistently poor instruction, learning in a second language), be— cause the learning difficulties persist in the presence of adequate educational opportunity and exposure to the same instruction as the peer group, and competency in the language of instruction, even when it is different from one’s primary spoken language. Intellectual disability (intellectual developmental disorder). Specific learning disorder differs from general learning difficulties associated with intellectual disability, because the learning difficulties occur in the presence of normal levels of intellectual functioning (i.e.,

1	IQ score of at least 70 1 5). If intellectual disability is present, specific learning disorder can be diagnosed only when the learning difficulties are in excess of those usually associated with the intellectual disability. Learning difficulties due to neurological or sensory disorders. Specific learning dis- order is distinguished from learning difficulties due to neurological or sensory disorders (e.g., pediatric stroke, traumatic brain injury, hearing impairment, Vision impairment), be- cause in these cases there are abnormal findings on neurological examination. Neurocognitive disorders. Specific learning disorder is distinguished from learning problems associated with neurodegenerative cognitive disorders, because in specific learning disorder the clinical expression of specific learning difficulties occurs during the developmental period, and the difficulties do not manifest as a marked decline from a for- mer state.

1	Attention-deficit/hyperactivity disorder. Specific learning disorder is distinguished from the poor academic performance associated with ADHD, because in the latter condition the rather may reﬂect difficulties in performing those skills. However, the co-occurrence of specific learning disorder and ADHD is more frequent than expected by chance. If criteria for both disorders are met, both diagnoses can be given. Psychotic disorders. Specific learning disorder is distinguished from the academic and cognitive-processing difficulties associated with schizophrenia or psychosis, because with these disorders there is a decline (often rapid) in these functional domains. Specific learning disorder commonly co-occurs with neurodevelopmental (e.g., ADHD, communication disorders, developmental coordination disorder, autistic spectrum disor- der) or other mental disorders (e.g., anxiety disorders, depressive and bipolar disorders).

1	These comorbidities do not necessarily exclude the diagnosis specific learning disorder but may make testing and differential diagnosis more difficult, because each of the co- occurring disorders independently interferes with the execution of activities of daily liv- ing, including learning. Thus, clinical judgment is required to attribute such impairment to learning difficulties. If there is an indication that another diagnosis could account for the difficulties learning keystone academic skills described in Criterion A, specific learning disorder should not be diagnosed. Motor Disorders, Diagnostic Criteria 315.4 (F82)

1	Motor Disorders, Diagnostic Criteria 315.4 (F82) A. The acquisition and execution of coordinated motor skills is substantially below that ex- pected given the individual’s chronological age and opportunity for skill learning and use. Difficulties are manifested as clumsiness (e.g., dropping or bumping into objects) as well as slowness and inaccuracy of performance of motor skills (e.g., catching an object, using scissors or cutlery, handwriting, riding a bike, or participating in sports). B. The motor skills deficit in Criterion A significantly and persistently interferes with activ- ities of daily living appropriate to chronological age (e.g., self-care and seIf-mainte- nance) and impacts academic/school productivity, prevocational and vocational activities, leisure, and play. C. Onset of symptoms is in the early developmental period.

1	C. Onset of symptoms is in the early developmental period. D. The motor skills deficits are not better explained by intellectual disability (intellectual devel- opmental disorder) or visual impairment and are not attributable to a neurological condi- tion affecting movement (e.g., cerebral palsy, muscular dystrophy, degenerative disorder).

1	The diagnosis of developmental coordination disorder is made by a clinical synthesis of the history (developmental and medical), physical examination, school or workplace report, and tests. The manifestation of impaired skills requiring motor coordination (Criterion A) varies with age. Young children may be delayed in achieving motor milestones (i.e., sitting, crawling, walking), althou h many achieve typical motor milestones. They also may be delayed in de- veloping skills such as negotiating stairs, pedaling, buttoning shirts, completing puzzles, and using zippers. Even when the skill is achieved, movement execution may appear awkward, slow, or less precise than that of peers. Older children and adults may display slow speed or in- accuracy with motor aspects of activities such as assembling puzzles, building models, playing ball games (especially in teams), handwriting, typing, driving, or carrying out self—care skills.

1	Developmental coordination disorder is diagnosed only if the impairment in motor skills significantly interferes with the performance of, or participation in, daily activities in family, social, school, or community life (Criterion B). Examples of such activities include getting dressed, eating meals with age-appropriate utensils and without mess, engaging in physical games with others, using specific tools in class such as rulers and scissors, and participating in team exercise activities at school. Not only is ability to perform these ac- tions impaired, but also marked slowness in execution is common. Handwriting compe- tence is frequently affected, consequently affecting legibility and / or speed of written output and affecting academic achievement (the impact is distinguished from specific learning difficulty by the emphasis on the motoric component of written output skills). In adults, everyday skills in education and work, especially those in which speed and accuracy are

1	specific learning difficulty by the emphasis on the motoric component of written output skills). In adults, everyday skills in education and work, especially those in which speed and accuracy are required, are affected by coordination problems.

1	Criterion C states that the onset of symptoms of developmental coordination disorder must be in the early developmental period. However, developmental coordination disorder is typically not diagnosed before age 5 years because there is considerable variation in the age at acquisition of many motor skills or a lack of stability of measurement in early childhood (e.g., some children catch up) or because other causes of motor delay may not have fully manifested.

1	Criterion D specifies that the diagnosis of developmental coordination disorder is made if the coordination difficulties are not better explained by visual impairment or at- tributable to a neurological condition. Thus, Visual function examination and neurological examination must be included in the diagnostic evaluation. If intellectual disability (intel- lectual developmental disorder) is present, the motor difficulties are in excess of those ex- pected for the mental age; however, no IQ cut-off or discrepancy criterion is specified. Developmental coordination disorder does not have discrete subtypes; however, indi- viduals may be impaired predominantly in gross motor skills or in fine motor skills, in- cluding handwriting skills. Other terms used to describe developmental coordination disorder include childhood dyspraxia, specific developmental disorder of motorfunction, and clumsy child syndrome.

1	Other terms used to describe developmental coordination disorder include childhood dyspraxia, specific developmental disorder of motorfunction, and clumsy child syndrome. Some children with developmental coordination disorder show additional (usually sup- pressed) motor activity, such as choreiform movements of unsupported limbs or mirror movements. These ”overﬂow” movements are referred to as neurodevelopmental immatu rities or neurological soft signs rather than neurological abnormalities. In both current literature and clinical practice, their role in diagnosis is still unclear, requiring further evaluation. The prevalence of developmental coordination disorder in children ages 5—11 years is 5%— 6% (in children age 7 years, 1.8% are diagnosed with severe developmental coordination disorder and 3% with probable developmental coordination disorder). Males are more of- ten affected than females, with a malezfemale ratio between 2:1 and 7:1.

1	The course of developmental coordination disorder is variable but stable at least to 1 year follow-up. Although there may be improvement in the longer term, problems with coor— dinated movements continue through adolescence in an estimated 50%—70% of children.

1	Onset is in early childhood. Delayed motor milestones may be the first signs, or the disor- der is first recognized when the child attempts tasks such as holding a knife and fork, but- toning clothes, or playing ball games. In middle childhood, there are difficulties with motor aspects of assembling puzzles, building models, playing ball, and handwriting, as well as with organizing belongings, when motor sequencing and coordination are re- quired. In early adulthood, there is continuing difficulty in learning new tasks involving complex/automatic motor skills, including driving and using tools. Inability to take notes and handwrite quickly may affect performance in the workplace. Co-occurrence with other disorders (see the section ”Comorbidity” for this disorder) has an additional impact on presentation, course, and outcome. Environmental. Developmental coordination disorder is more common following pre- natal exposure to alcohol and in preterm and low-birth-weight children.

1	Environmental. Developmental coordination disorder is more common following pre- natal exposure to alcohol and in preterm and low-birth-weight children. Genetic and physiological. Impairments in underlying neurodevelopmental processes— particularly in visual-motor skills, both in visual-motor perception and spatial mentalizing— have been found and affect the ability to make rapid motoric adjustments as the complexity of the required movements increases. Cerebellar dysfunction has been proposed, but the neural basis of developmental coordination disorder remains unclear. Because of the co-occurrence of developmental coordination disorder with attention-deficit/hyperactivity disorder (ADHD), specific learning disabilities, and autism spectrum disorder, shared genetic effect has been pro- posed. However, consistent co-occurrence in twins appears only in severe cases.

1	Course modifiers. Individuals with ADHD and with developmental coordination dis- order demonstrate more impairment than individuals with ADHD without developmen- tal coordination disorder. Developmental coordination disorder occurs across cultures, races, and socioeconomic conditions. By definition, "activities of daily living” implies cultural differences necessi— tating consideration of the context in which the individual child is living as well as whether he or she has had appropriate opportunities to learn and practice such activities. Developmental coordination disorder leads to impaired functional performance in activ- ities of daily living (Criterion B), and the impairment is increased with co-occurring con- ditions. Consequences of developmental coordination disorder include reduced participation in team play and sports; poor self—esteem and sense of self—worth; emotional duced physical activity and obesity.

1	Motor impairments due to another medical condition. Problems in coordination may be associated with visual function impairment and specific neurological disorders (e.g., cerebral palsy, progressive lesions of the cerebellum, neuromuscular disorders). In such cases, there are additional findings on neurological examination. Intellectual disability (intellectual developmental disorder). If intellectual disability is present, motor competences may be impaired in accordance with the intellectual disabil- ity. However, if the motor difficulties are in excess of what could be accounted for by the intellectual disability, and criteria for developmental coordination disorder are met, de— velopmental codrdination disorder can be diagnosed as well.

1	Attention-deficit/hyperactivity disorder. Individuals with ADHD may fall, bump into objects, or knock things over. Careful observation across different contexts is required to ascertain if lack of motor competence is attributable to distractibility and impulsiveness rather than to developmental coordination disorder. If criteria for both ADHD and devel- opmental coordination disorder are met, both diagnoses can be given. Autism spectrum disorder. Individuals with autism spectrum disorder may be uninter- ested in participating in tasks requiring complex coordination skills, such as ball sports, which will affect test performance and function but not reﬂect core motor competence. Co- occurrence of developmental coordination disorder and autism spectrum disorder is com- mon. If criteria for both disorders are met, both diagnoses can be given.

1	Joint hypermobility syndrome. Individuals with syndromes causing hyperextensible joints (found on physical examination; often with a complaint of pain) may present with symptoms similar to those of developmental coordination disorder. Disorders that commonly co-occur with developmental coordination disorder include problems of inattention, including ADHD (the most frequent coexisting condition, with problems; and joint hypermobility syndrome. Different clusters of co-occurrence may be present (e.g., a cluster with severe reading disorders, fine motor problems, and handwriting problems; another cluster with impaired movement control and motor planning). Presence testing more difficult and may independently interfere with the execution of activities of daily living, thus requiring examiner judgment in ascribing impairment to motor skills. Diagnostic Criteria 307.3 (F98.4)

1	Diagnostic Criteria 307.3 (F98.4) A. Repetitive, seemingly driven, and apparently purposeless motor behavior (e.g., hand shaking or waving, body rocking, head banging, self-biting, hitting own body). B. The repetitive motor behavior interferes with social, academic, or other activities and may result in seIf-injury. C. Onset is in the early developmental period. D. The repetitive motor behavior is not attributable to the physiological effects of a sub- stance or neurological condition and is not better explained by another neurodevel- opmental or mental disorder (e.g., trichotillomania [hair-pulling disorder], obsessive- compulsive disorder). Specify it: Specify if: Associated with a known medical or genetic condition, neurodevelopmental dis- order, or environmental factor (e.g., Lesch-Nyhan syndrome, intellectual disability [intellectual developmental disorder], intrauterine alcohol exposure)

1	Coding note: Use additional code to identity the associated medical or genetic condition, or neurodevelopmental disorder. Specify current severity: Mild: Symptoms are easily suppressed by sensory stimulus or distraction. Moderate: Symptoms require explicit protective measures and behavioral modification. Severe: Continuous monitoring and protective measures are required to prevent seri- ous injury. For stereotypic movement disorder that is associated with a known medical or genetic condition, neurodevelopmental disorder, or environmental factor, record stereotypic movement disorder associated with (name of condition, disorder, or factor) (e.g., stereo- typic movement disorder associated with Lesch-Nyhan syndrome).

1	The severity of non-self-injurious stereotypic movements ranges from mild presentations that are easily suppressed by a sensory stimulus or distraction to continuous movements that markedly interfere with all activities of daily living. Self—injurious behaviors range in se- verity along various dimensions, including the frequency, impact on adaptive functioning, and severity of bodily injury (from mild bruising or erythema from hitting hand against body, to lacerations or amputation of digits, to retinal detachment from head banging). The essential feature of stereotypic movement disorder is repetitive, seemingly driven, and apparently purposeless motor behavior (Criterion A). These behaviors are often rhythmical movements of the head, hands, or body without obvious adaptive function.

1	The movements may or may not respond to efforts to stop them. Among typically devel- oping children, the repetitive movements may be stopped when attention is directed to them or when the child is distracted from performing them. Among children with neuro- developmental disorders, the behaviors are typically less responsive to such efforts. In other cases, the individual demonstrates self—restraining behaviors (e.g., sitting on hands, wrapping arms in clothing, finding a protective device).

1	The repertoire of behaviors is variable; each individual presents with his or her own in- dividually patterned, ”signature” behavior. Examples of non-self—injurious stereotypic movements include, but are not limited to, body rocking, bilateral ﬂapping or rotating hand movements, ﬂicking or ﬂuttering fingers in front of the face, arm waving or ﬂapping, and head nodding. Stereotyped self—injurious behaviors include, but are not limited to, re- petitive head banging, face slapping, eye poking, and biting of hands, lips, or other body parts. Eye poking is particularly concerning; it occurs more frequently among children with visual impairment. Multiple movements may be combined (e.g., cocking the head, rocking the torso, waving a small string repetitively in front of the face).

1	Stereotypic movements may occur many times during a day, lasting a few seconds to several minutes or longer. Frequency can vary from many occurrences in a single day to several weeks elapsing between episodes. The behaviors vary in context, occurring when the individual is engrossed in other activities, when excited, stressed, fatigued, or bored. Criterion A requires that the movements be “apparently” purposeless. However, some functions may be served by the movements. For example, stereotypic movements might reduce anxiety in response to external stressors.

1	Criterion B states that the stereotypic movements interfere with social, academic, or other activities and, in some children, may result in self—injury (or would if protective mea- sures were not used). If self—injury is present, it should be coded using the specifier. Onset of stereotypic movements is in the early developmental period (Criterion C). Criterion D states that the repetitive, stereotyped behavior in stereotypic movement disorder is not at- tributable to the‘physiological effects of a substance or neurological condition and is not better explained by another neurodevelopmental or mental disorder. The presence of stereotypic movements may indicate an undetected neurodevelopmental problem, espe- cially in children ages 1—3 years.

1	Simple stereotypic movements (e.g., rocking) are common in young typically developing chil- dren. Complex stereotypic movements are much less common (occurring in approximately 3%—4%). Between 4% and 16% of individuals with intellectual disability (intellectual develop- mental disorder) engage in stereotypy and self-injury. The risk is greater in individuals with severe intellectual disability. Among individuals with intellectual disability living in res- idential facilities, 10%—15% may have stereotypic movement disorder with self—injury.

1	Stereotypic movements typically begin within the first 3 years of life. Simple stereotypic move- ments are common in infancy and may be involved in acquisition of motor mastery. In chil- dren who develop complex motor stereotypies, approximately 80% exhibit symptoms before 24 months of age, 12% between 24 and 35 months, and 8% at 36 months or older. In most typ- ically developing children, these movements resolve over time or can be suppressed. Onset of complex motor stereotypies may be in infancy or later in the developmental period. Among individuals with intellectual disability, the stereotyped, self—injurious behaviors may persist for years, even though the typography or pattern of self-injury may change.

1	Environmental. Social isolation is a risk factor for self—stimulation that may progress to stereotypic movements with repetitive self—injury. Environmental stress may also trigger stereotypic behavior. Fear may alter physiological state, resulting in increased frequency of stereotypic behaviors. Genetic and physiological. Lower cognitive functioning is linked to greater risk for stereo- typic behaviors and poorer response to interventions. Stereotypic movements are more fre- quent among individuals with moderate-to-severe/profound intellectual disability, who by

1	Virtue of a particular syndrome (e.g., Rett syndrome) or environmental factor (e.g., an environ- ment with relatively insufficient stimulation) seem to be at higher risk for stereotypies. Repet- itive self-injurious behavior may be a behavioral phenotype in neurogenetic syndromes. For example, in Lesch-Nyhan syndrome, there are both stereotypic dystonic movements and self- mutilation of fingers, lip biting, and other forms of self—injury unless the individual is re- strained, and in Rett syndrome and Cornelia de Lange syndrome, self-injury may result from the hand-to—mouth stereotypies. Stereotypic behaviors may result from a painful medical con- dition (e.g., middle ear infection, dental problems, gastroesophageal reﬂux). Stereotypic movement disorder, with or without self—injury, occurs in all races and culhares.

1	Stereotypic movement disorder, with or without self—injury, occurs in all races and culhares. Cultural attitudes toward unusual behaviors may result in delayed diagnosis. Overall cultural tolerance and attitudes toward stereotypic movement vary and must be considered. Normal development. Simple stereotypic movements are common in infancy and early childhood. Rocking may occur in the transition from sleep to awake, a behavior that usu- ally resolves with age. Complex stereotypies are less common in typically developing children and can usually be suppressed by distraction or sensory stimulation. The indi- vidual’s daily routine is rarely affected, and the movements generally do not cause the child distress. The diagnosis would not be appropriate in these circumstances.

1	Autism spectrum disorder. Stereotypic movements may be a presenting symptom of haviors are being evaluated. Deficits of social communication and reciprocity manifesting in autism spectrum disorder are generally absent in stereotypic movement disorder, and thus social interaction, social communication, and rigid repetitive behaviors and interests are distinguishing features. When autism spectrum disorder is present, stereotypic move- ment disorder is diagnosed only when there is self-injury or when the stereotypic behav- iors are sufficiently severe to become a focus of treatment.

1	Tic disorders. Typically, stereotypies have an earlier age at onset (before 3 years) than do tics, which have a mean age at onset of 5—7 years. They are consistent and fixed in their pattern or topography compared with tics, which are variable in their presentation. Ste— reotypies may involve arms, hands, or the entire body, while tics commonly involve eyes, face, head, and shoulders. Stereotypies are more fixed, rhythmic, and prolonged in dura- tion than tics, which, generally, are brief, rapid, random, and ﬂuctuating. Tics and stereo— typic movements are both reduced by distraction.

1	Obsessive-compulsive and related disorders. Stereotypic movement disorder is dis- tinguished from obsessive-compulsive disorder (OCD) by the absence of obsessions, as well as by the nature of the repetitive behaviors. In 0CD the individual feels driven to per- form repetitive behaviors in response to an obsession or according to rules that must be ap- plied rigidly, whereas in stereotypic movement disorder the behaviors are seemingly driven but apparently purposeless. Trichotillomania (hair-pulling disorder) and excoria- tion (skin-picking) disorder are characterized by body-focused repetitive behaviors (i.e., hair pulling and skin picking) that may be seemingly driven but that are not apparently purposeless, and that may not be patterned or rhythmical. Furthermore, onset in tricho- tillomania and excoriation disorder is not typically in the early developmental period, but rather around puberty or later.

1	Other neurological and medical conditions. The diagnosis of stereotypic movements requires the exclusion of habits, mannerisms, paroxysmal dyskinesias, and benign he- reditary chorea. A neurological history and examination are required to assess features suggestive of other disorders, such as myoclonus, dystonia, tics, and chorea. Involuntary movements associated with a neurological condition may be distinguished by their signs and symptoms. For example, repetitive, stereotypic movements in tardive dyskinesia can be distinguished by a history of chronic neuroleptic use and characteristic oral or facial dyskinesia or irregular trunk or limb movements. These types of movements do not result in self-injury. A diagnosis of stereotypic movement disorder is not appropriate for repet- itive skin picking or scratching associated with amphetamine intoxication or abuse (e.g., patients are diagnosed with substance/medication-induced obsessive-compulsive and re- lated disorder) and repetitive

1	picking or scratching associated with amphetamine intoxication or abuse (e.g., patients are diagnosed with substance/medication-induced obsessive-compulsive and re- lated disorder) and repetitive choreoathetoid movements associated with other neurolog- ical disorders.

1	Stereotypic movement disorder may occur as a primary diagnosis or secondary to another disorder. For example, stereotypies are a common manifestation of a variety of neuro- genetic disorders, Such as Lesch-Nyhan syndrome, Rett syndrome, fragile X syndrome, Cornelia de Lange syndrome, and Smith-Magenis syndrome. When stereotypic move- ment disorder co-occurs with another medical condition, both should be coded. Note: A tic is a sudden, rapid, recurrent, nonrhythmic motor movement or vocalization. Tourette’s Disorder 307.23 (F952) A. Both multiple motor and one or more vocal tics have been present at some time during the illness, although not necessarily concurrently. B. The tics may wax and wane in frequency but have persisted for more than 1 year since first tic onset. C. Onset is before age 18 years.

1	B. The tics may wax and wane in frequency but have persisted for more than 1 year since first tic onset. C. Onset is before age 18 years. D. The disturbance is not attributable to the physiological effects of a substance (e.g., co- caine) or another medical condition (e.g., Huntington‘s disease, postviral encephalitis). Persistent (Chronic) Motor or Vocal Tic Disorder 307.22 (F95.1) A. Single or multiple motor or vocal tics have been present during the illness, but not both motor and vocal. B. The tics may wax and wane in frequency but have persisted for more than 1 year since first tic onset. C. Onset is before age 18 years. D. The disturbance is not attributable to the physiological effects of a substance (e.g., co- caine) or another medical condition (e.g., Huntington's disease, postviral encephalitis). E. Criteria have never been met for Tourette’s disorder. Specify it: Provisional Tic Disorder 307.21 (F95.0) . Single or multiple motor and/or vocal tics.

1	E. Criteria have never been met for Tourette’s disorder. Specify it: Provisional Tic Disorder 307.21 (F95.0) . Single or multiple motor and/or vocal tics. . The tics have been present for less than 1 year since first tic onset. . Onset is before age 18 years. . The disturbance is not attributable to the physiological effects of a substance (e.g., co- caine) or another medical condition (e.g., Huntington's disease, postviral encephalitis). . Criteria have never been met for Tourette‘s disorder or persistent (chronic) motor or vocal tic disorder. The ”motor tics only” or ”vocal tics only” specifier is only required for persistent (chronic) motor or vocal tic disorder.

1	The ”motor tics only” or ”vocal tics only” specifier is only required for persistent (chronic) motor or vocal tic disorder. Tic disorders comprise four diagnostic categories: Tourette’s disorder, persistent (chronic) motor or vocal tic disorder, provisional tic disorder, and the other specified and unspecified tic disorders. Diagnosis for any tic disorder is based on the presence of motor and / or vocal tics (Criterion A), duration of tic symptoms (Criterion B), age at onset (Criterion C), and ab- sence of any known cause such as another medical condition or substance use (Criterion D). The tic disorders are hierarchical in order (i.e., Tourette’s disorder, followed by persistent [Chronic] motor or vocal tic disorder, followed by provisional tic disorder, followed by the other specified and unspecified tic disorders), such that once a tic disorder at one level of the hierarchy is diagnosed, a lower hierarchy diagnosis cannot be made (Criterion E).

1	Tics are sudden, rapid, recurrent, nonrhythmic motor movements or vocalizations. An individual may have various tic symptoms over time, but at any point in time, the tic rep- ertoire recurs in a characteristic fashion. Although tics can include almost any muscle group or vocalization, certain tic symptoms, such as eye blinking or throat clearing, are common across patient populations. Tics are generally experienced as involuntary but can be vol- untarily suppressed for varying lengths of time.

1	Tics can be either simple or complex. Simple motor tics are of short duration (i.e., milli- seconds) and can include eye blinking, shoulder shrugging, and extension of the extrem- ities. Simple vocal tics include throat clearing, sniffing, and grunting often caused by contraction of the diaphragm or muscles of the oropharynx. Complex motor tics are of lon- ger duration (i.e., seconds) and often include a combination of simple tics such as simul- taneous head turning and shoulder shrugging. Complex tics can appear purposeful, such as a tic-like sexual or obscene gesture (copropmxiu) or a tic-like imitation of someone else’s movements (echopraxz'u). Similarly, complex vocal tics include repeating one’s own sounds or words (pulilalia), repeating the last—heard word or phrase (echolalia), or uttering socially unacceptable words, including Obscenities, or ethnic, racial, or religious slurs (coprolalia).

1	Importantly, coprolalia is an abrupt, sharp bark or grunt utterance and lacks the prosody of similar inappropriate speech observed in human interactions. The presence of motor and/ or vocal tics varies across the four tic disorders (Criterion A). For Tourette’s disorder, both motor and vocal tics must be present, whereas for per- sistent (chronic) motor or vocal tic disorder, only motor or only vocal tics are present. For provisional tic disorder, motor and / or vocal tics may be present. For other specified or un- specified tic disorders, the movement disorder symptoms are best characterized as tics but are atypical in presentation or age at onset, or have a known etiology.

1	The 1-year minimum duration criterion (Criterion B) assures that individuals diag— nosed with either Tourette’s disorder or persistent (chronic) motor or vocal tic disorder have had persistent symptoms. Tics wax and wane in severity, and some individuals may have tic-free periods of weeks to months; however, an individual who has had tic symp- toms of greater than 1 year’s duration since first tic onset would be considered to have per- sistent symptoms regardless of duration of tic-free periods. For an individual with motor and / or vocal tics of less than 1 year since first tic onset, a provisional tic disorder diagnosis can be considered. There is no duration specification for other specified and unspecified tic disorders. The onset of tics must occur prior to age 18 years (Criterion C). Tic disorders typically begin in the prepubertal period, with an average age at onset between 4 and 6 years, and with the incidence of new-onset tic disorders decreasing in the teen years. New onset

1	Tic disorders typically begin in the prepubertal period, with an average age at onset between 4 and 6 years, and with the incidence of new-onset tic disorders decreasing in the teen years. New onset of tic symptoms in adulthood is exceedingly rare and is often associated with expo- sures to drugs (e.g., excessive cocaine use) or is a result of a central nervous system insult (e.g., postviral encephalitis). Although tic onset is uncommon in teenagers and adults, it is not uncommon for adolescents and adults to present for an initial diagnostic assessment and, when carefully evaluated, provide a history of milder symptoms dating back to child- hood. New-onset abnormal movements suggestive of tics outside of the usual age range should result in evaluation for other movement disorders or for specific etiologies.

1	Tic symptoms cannot be attributable to the physiological effects of a substance or an- other medical condition (Criterion D). When there is strong evidence from the history, physical examination, and / or laboratory results to suggest a plausible, proximal, and probable cause for a tic disorder, a diagnosis of other specified tic disorder should be used. agnosis of persistent (chronic) motor or vocal tic disorder (Criterion E). Similarly, a previ- ous diagnosis of persistent (chronic) motor or vocal tic disorder negates a diagnosis of provisional tic disorder or other specified or unspecified tic disorder (Criterion E). Tics are common in childhood but transient in most cases. The estimated prevalence of Tourette’s disorder ranges from 3 to 8 per 1,000 in school-age children. Males are more commonly affected than females, with the ratio varying from 2:1 to 4:1. A national survey in the United States estimated 3 per 1,000 for the prevalence of clinically identified cases.

1	The frequency of identified cases was lower among African Americans and Hispanic Americans, which may be related to differences in access to care. Onset of tics is typically between ages 4 and 6 years. Peak severity occurs between ages 10 and 12 years, with a decline in severity during adolescence. Many adults with tic disorders experience diminished symptoms. A small percentage of individuals will have persis- tently severe or worsening symptoms in adulthood.

1	Tic symptoms manifest similarly in all age groups and across the lifespan. Tics wax and wane in severity and change in affected muscle groups and vocalizations over time. As children get older, they begin to report their tics being associated with a premonitory urge—va somatic sensation that precedes the tic—and a feeling of tension reduction follow- ing the expression of the tic. Tics associated with a premonitory urge may be experienced as not completely ”involuntary” in that the urge and the tic can be resisted. An individual may also feel the need to perform a tic in a specific way or repeat it until he or she achieves the feeling that the tic has been done ”just right.”

1	The vulnerability toward developing co-occurring conditions changes as individuals pass through the age of risk for various co-occurring conditions. For example, prepubertal children with tic disorders are more likely to experience attention-deficit/hyperactivity disorder (ADHD), obsessive-compulsive disorder (OCD), and separation anxiety disorder than are teenagers and adults, who are more likely to experience the new onset of major depressive disorder, substance use disorder, or bipolar disorder. Tem peramental. Tics are worsened by anxiety, excitement, and exhaustion and are better during calm, focused activities. Individuals may have fewer tics when engaged in schoolwork or tasks at work than when relaxing at home after school or in the evening. Stressful/exciting events (e.g., taking a test, participating in exciting activities) often make tics worse.

1	Environmental. Observing a gesture or sound in another person may result in an indi- vidual with a tic disorder making a similar gesture or sound, which may be incorrectly perceived by others as purposeful. This can be a particular problem when the individual is interacting with authority figures (e.g., teachers, supervisors, police). Genetic and physiological. Genetic and environmental factors inﬂuence tic symptom expression and severity. Important risk alleles for Tourette’s disorder and rare genetic variants in families with tic disorders have been identified. Obstetrical complications, Older paternal age, lower birth weight, and maternal smoking during pregnancy are as- sociated with worse tic severity.

1	Older paternal age, lower birth weight, and maternal smoking during pregnancy are as- sociated with worse tic severity. Tic disorders do not appear to vary in clinical characteristics, course, or etiology by race, ethnicity, and culture. However, race, ethnicity, and culture may impact how tic disorders are perceived and managed in the family and community, as well as inﬂuencing patterns of help seeking, and choices of treatment. Males are more commonly affected than females, but there are no gender differences in the kinds of tics, age at onset, or course. Women with persistent tic disorders may be more likely to experience anxiety and depression. Functional Consequences of Tic Disorders

1	Functional Consequences of Tic Disorders Many individuals with mild to moderate tic severity experience no distress or impairment in functioning and may even be unaware of their tics. Individuals with more severe symp- toms generally have more impairment in daily living, but even individuals with moderate or even severe tic disorders may function well. The presence of a co-occurring condition, such as ADHD or 0CD, can have greater impact on functioning. Less commonly, tics dis- rupt functioning in daily activities and result in social isolation, interpersonal conflict, peer victimization, inability to work or to go to school, and lower quality of life. The indi- vidual also may experience substantial psychological distress. Rare complications of Tou- rette’s disorder include physical injury, such as eye injury (from hitting oneself in the face), and orthopedic and neurological injury (e.g., disc disease related to forceful head and neck movements).

1	movement disorder. Motor stereotypies are defined as involuntary rhythmic, repetitive, purpose and stop with distraction. Examples include repetitive hand waving/rotating, arm ﬂapping, and finger wiggling. Motor stereotypies can be differentiated from tics based on the former's earlier age at onset (younger than 3 years), prolonged duration (seconds to minutes), constant repetitive fixed form and location, exacerbation when engrossed in ac- tivities, lack of a premonitory urge, and cessation with distraction (e.g., name called or touched). Chorea represents rapid, random, continual, abrupt, irregular, unpredictable, nonstereotyped actions that are usually bilateral and affect all parts of the body (i.e., face, trunk, and limbs). The timing, direction, and distribution of movements vary from mo- ment to moment, and movements usually worsen during attempted voluntary action. Dys- tonia is the simultaneous sustained contracture of both agonist and antagonist muscles, resulting in a

1	from mo- ment to moment, and movements usually worsen during attempted voluntary action. Dys- tonia is the simultaneous sustained contracture of both agonist and antagonist muscles, resulting in a distorted posture or movement of parts of the body. Dystonic postures are of- ten triggered by attempts at voluntary movements and are not seen during sleep.

1	Substance-induced and paroxysmal dyskinesias. Paroxysmal dyskinesias usually oc- cur as dystonic or choreoathetoid movements that are precipitated by voluntary move- ment or exertion and less commonly arise from normal background activity. Myoclonus. Myoclonus is characterized by a sudden unidirectional movement that is often nonrhythmic. It may be worsened by movement and occur during sleep. Myoclonus is differentiated from tics by its rapidity, lack of suppressibility, and absence of a premon- itory urge.

1	Obsessive-compulsive and related disorders. Differentiating obsessive-compulsive behaviors from tics may be difficult. Clues favoring an obsessive-compulsive behavior in- clude a cognitive-based drive (e.g., fear of contamination) and the need to perform the ac- tion in a particular fashion a certain number of times, equally on both sides of the body, or until a ”just right" feeling is achieved. Impulse-control problems and other repetitive be- haviors, including persistent hair pulling, skin picking, and nail biting, appear more goal directed and complex than tics.

1	Many medical and psychiatric conditions have been described as co—occurring with tic disor— ders, with ADHD and obsessive-compulsive and related disorders being particularly com- mon. The obsessive-compulsive symptoms observed in tic disorder tend to be characterized by more aggressive symmetry and order symptoms and poorer response to pharmacotherapy with selective serotonin reuptake inhibitors. Children with ADHD may demonstrate disrup— tive behavior, social immaturity, and learning difficulties that may interfere with academic progress and interpersonal relationships and lead to greater impairment than that caused by a tic disorder. Individuals with tic disorders can also have other movement disorders and other mental disorders, such as depressive, bipolar, or substance use disorders. 307.20 (F95.8)

1	307.20 (F95.8) This category applies to presentations in which symptoms characteristic of a tic disorder that cause clinically significant distress or impairment in social, occupational, or other im- portant areas of functioning predominate but do not meet the full criteria for a tic disorder or any of the disorders in the neurodevelopmental disorders diagnostic class. The other specified tic disorder category is used in situations in which the clinician chooses to com- municate the specific reason that the presentation does not meet the criteria for a tic disor- der or any specific neurodevelopmental disorder. This is done by recording “other specified tic disorder” followed by the specific reason (e.g., ‘With onset after age 18 years"). 307.20 (F959)

1	307.20 (F959) This category applies to presentations in which symptoms characteristic of a tic disorder that cause clinically significant distress or impairment in social, occupational, or other im- portant areas of functioning predominate but do not meet the full criteria for a tic disorder or for any of the disorders in the neurodevelopmental disorders diagnostic class. The un- specified tic disorder category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for a tic disorder or for a specific neurode- velopmental disorder, and includes presentations in which there is insufficient information to make a more specific diagnosis. 315.8 (F88)

1	315.8 (F88) This category applies to presentations in which symptoms characteristic of a neurodevel- opmental disorder that cause impairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the neurodevelopmental disorders diagnostic class. The other specified neurodevelopmental disorder category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for any specific neurode- velopmental disorder. This is done by recording “other specified neurodevelopmental dis- order" followed by the specific reason (e.g., “neurodevelopmental disorder associated with prenatal alcohol exposure"). An example of a presentation that can be specified using the “other specified" desig- nation is the following:

1	An example of a presentation that can be specified using the “other specified" desig- nation is the following: Neurodevelopmental disorder associated with prenatal alcohol exposure: Neu- rodevelopmental disorder associated with prenatal alcohol exposure is characterized by a range of developmental disabilities following exposure to alcohol in utero. 315.9 (F89)

1	315.9 (F89) This category applies to presentations in which symptoms characteristic of a neurodevel- opmental disorder that cause impairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the neurodevelopmental disorders diagnostic class. The unspecified neurodevelopmental dis- order category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for a specific neurodevelopmental disorder, and includes pre- sentations in which there is insufficient information to make a more specific diagnosis (e.g., in emergency room settings).

1	SChlZOph re n | 8. S pectru m and other psychotic disorders include schizophrenia, other psychotic disorders, and schizotypal (personality) disorder. They are defined by ab- normalities in one or more of the following five domains: delusions, hallucinations, disor— ganized thinking (speech), grossly disorganized or abnormal motor behavior (including catatonia), and negative symptoms. Key Features That Define the Psychotic Disorders Delusions are fixed beliefs that are not amenable to change in light of conﬂicting evidence.

1	Key Features That Define the Psychotic Disorders Delusions are fixed beliefs that are not amenable to change in light of conﬂicting evidence. Their content may include a variety of themes (e.g., persecutory, referential, somatic, reli- gious, grandiose). Persecutory delusions (i.e., belief that one is going to be harmed, harassed, and so forth by an individual, organization, or other group) are most common. Referential delusions (i.e., belief that certain gestures, comments, environmental cues, and so forth are directed at oneself) are also common. Grandiose delusions (i.e., when an individual believes that he or she has exceptional abilities, wealth, or fame) and erotomnnic delusions (i.e., when an individual believes falsely that another person is in love with him or her) are also seen. Nihilistic delusions involve the conviction that a major catastrophe will occur, and somatic delusions focus on preoccupations regarding health and organ function.

1	Delusions are deemed bizarre if they are clearly implausible and not understandable to same-culture peers and do not derive from ordinary life experiences. An example of a bi- zarre delusion is the belief that an outside force has removed his or her internal organs and replaced them with someone else’s organs without leaving any wounds or scars. An ex— ample of a nonbizarre delusion is the belief that one is under surveillance by the police, de- spite a lack of convincing evidence. Delusions that express a loss of control over mind or body are generally considered to be bizarre; these include the belief that one’s thoughts have been "removed” by some outside force (thought withdrawal), that alien thoughts have been put into one’s mind (thought insertion), or that one’s body or actions are being acted on or manipulated by some outside force (delusions of control). The distinction between a de- lusion and a strongly held idea is sometimes difficult to make and depends in part on the

1	are being acted on or manipulated by some outside force (delusions of control). The distinction between a de- lusion and a strongly held idea is sometimes difficult to make and depends in part on the degree of conviction with which the belief is held despite clear or reasonable contradictory evidence regarding its veracity.

1	Hallucinations are perception-like experiences that occur without an external stimulus. They are vivid and clear, with the full force and impact of normal perceptions, and not under voluntary control. They may occur in any sensory modality, but auditory halluci- nations are the most common in schizophrenia and related disorders. Auditory hallucina- tions are usually experienced as voices, whether familiar or unfamiliar, that are perceived as distinct from the individual’s own thoughts. The hallucinations must occur in the con- text of a clear sensorium; those that occur while falling asleep (hypnagogic) or waking up (hypnopompic) are considered to be within the range of normal experience. Hallucinations may be a normal part of religious experience in certain cultural contexts.

1	Disorganized thinking (formal thought disorder) is typically inferred from the individual’s speech. The individual may switch from one topic to another (derailment or loose associa- tions). Answers to questions may be obliquely related or completely unrelated (tangential— ity). Rarely, speech may be so severely disorganized that it is nearly incomprehensible and resembles receptive aphasia in its linguistic disorganization (incoherence or ”word salad"). Because mildly disorganized speech is common and nonspecific, the symptom must be se- vere enough to substantially impair effective communication. The severity of the impair- ment may be difficult to evaluate if the person making the diagnosis comes from a different linguistic background than that of the person being examined. Less severe dis- organized thinking or speech may occur during the prodromal and residual periods of schizophrenia.

1	Grossly disorganized or abnormal motor behavior may manifest itself in a variety of ways, ranging from childlike ”silliness” to unpredictable agitation. Problems may be noted in any form of goal-directed behavior, leading to difficulties in performing activities of daily living. Catatonic behavior is a marked decrease in reactivity to the environment. This ranges from resistance to instructions (negativism); to maintaining a rigid, inappropriate or bi- zarre posture; to a complete lack of verbal and motor responses (mutism and stupor). It can excitement). Other features are repeated stereotyped movements, staring, grimacing, mutism, and the echoing of speech. Although catatonia has historically been associated with schizophrenia, catatonic symptoms are nonspecific and may occur in other mental disorders (e.g., bipolar or depressive disorders with catatonia) and in medical conditions (catatonic disorder due to another medical condition).

1	Negative symptoms account for a substantial portion of the morbidity associated with schizophrenia but are less prominent in other psychotic disorders. Two negative symp- toms are particularly prominent in schizophrenia: diminished emotional expression and avolition. Diminished emotional expression includes reductions in the expression of emo- tions in the face, eye contact, intonation of speech (prosody), and movements of the hand, head, and face that normally give an emotional emphasis to speech. Avolition is a decrease in motivated self-initiated purposeful activities. The individual may sit for long periods of time and show little interest in participating in work or social activities. Other negative symptoms include alogia, anhedonia, and asociality. Alogia is manifested by diminished speech output. Anhedonia is the decreased ability to experience pleasure from positive stimuli or a degradation in the recollection of pleasure previously experienced. Asociality refers to the

1	speech output. Anhedonia is the decreased ability to experience pleasure from positive stimuli or a degradation in the recollection of pleasure previously experienced. Asociality refers to the apparent lack of interest in social interactions and may be associated with avo- lition, but it can also be a manifestation of limited opportunities for social interactions.

1	This chapter is organized along a gradient of psychopathology. Clinicians should first con- sider conditions that do not reach full criteria for a psychotic disorder or are limited to one domain of psychopathology. Then they should consider time-limited conditions. Finally, the diagnosis of a schizophrenia spectrum disorder requires the exclusion of another con- dition that may give rise to psychosis. Schizotypal personality disorder is noted within this chapter as it is considered within the schizophrenia spectrum, although its full description is found in the chapter ”Person- ality Disorders.” The diagnosis schizotypal personality disorder captures a pervasive pat- tern of social and interpersonal deficits, including reduced capacity for close relationships; cognitive or perceptual distortions; and eccentricities of behavior, usually beginning by early adulthood but in some cases first becoming apparent in childhood and adolescence.

1	Abnormalities of beliefs, thinking, and perception are below the threshold for the diagno- sis of a psychotic disorder. Two conditions are defined by abnormalities limited to one domain of psychosis: delu- sions or catatonia. Delusional disorder is characterized by at least 1 month of delusions but no other psychotic symptoms. Catatonia is described later in the chapter and further in this discussion. Brief psychotic disorder lasts more than 1 day and remits by 1 month. Schizophreni- form disorder is characterized by a symptomatic presentation equivalent to that of schizo- phrenia except for its duration (less than 6 months) and the absence of a requirement for a decline in functioning.

1	Schizophrenia lasts for at least 6 months and includes at least 1 month of active-phase symptoms. In schizoaffective disorder, a mood episode and the active-phase symptoms of schizophrenia occur together and were preceded or are followed by at least 2 weeks of de- lusions or hallucinations without prominent mood symptoms. Psychotic disorders may be induced by another condition. In substance/medication— induced psychotic disorder, the psychotic symptoms are judged to be a physiological con- sequence of a drug of abuse, a medication, or toxin exposure and cease after removal of the agent. In psychotic disorder due to another medical condition, the psychotic symptoms are judged to be a direct physiological consequence of another medical condition.

1	Catatonia can occur in several disorders, including neurodevelopmental, psychotic, bi— polar, depressive, and other mental disorders. This chapter also includes the diagnoses catatonia associated with another mental disorder (catatonia specifier), catatonic disorder due to another medical condition, and unspecified catatonia, and the diagnostic criteria for all three conditions are described together. ders are included for classifying psychotic presentations that do not meet the criteria for any of the specific psychotic disorders, or psychotic symptomatology about which there is inadequate or contradictory information. Clinician—Rated Assessment of Symptoms and Psychotic disorders are heterogeneous, and the severity of symptoms can predict impor- tant aspects of the illness, such as the degree of cognitive or neurobiological deficits. To move the field forward, a detailed framework for the assessment of severity is included in

1	Section III "Assessment Measures," which may help with treatment planning, prognostic decision making, and research on pathophysiological mechanisms. Section III ”Assess- ment Measures” also contains dimensional assessments of the primary symptoms of psy- chosis, including hallucinations, delusions, disorganized speech (except for substance/ medication-induced psychotic disorder and psychotic disorder due to another medical condition), abnormal psychomotor behavior, and negative symptoms, as well as dimen— sional assessments of depression and mania. The severity of mood symptoms in psychosis has prognostic value and guides treatment. There is growing evidence that schizoaffective disorder is not a distinct nosological category. Thus, dimensional assessments of depres- sion and mania for all psychotic disorders alert clinicians to mood pathology and the need to treat where appropriate. The Section III scale also includes a dimensional assessment of cognitive impairment. Many

1	for all psychotic disorders alert clinicians to mood pathology and the need to treat where appropriate. The Section III scale also includes a dimensional assessment of cognitive impairment. Many individuals with psychotic disorders have impairments in a range of cognitive domains that predict functional status. Clinical neuropsychological as- sessment can help guide diagnosis and treatment, but brief assessments without formal diagnostic purposes. Formal neuropsychological testing, when conducted, should be ad- ministered and scored by personnel trained in the use of testing instruments. If a formal neuropsychological assessment is not conducted, the clinician should use the best avail- able information to make a judgment. Further research on these assessments is necessary in order to determine their clinical utility; thus, the assessments available in Section 111 should serve as a prototype to stimulate such research.

1	Criteria and text for schizotypal personality disorder can be found in the chapter "Person- ality Disorders.” Because this disorder is considered part of the schizophrenia spectrum of disorders, and is labeled in this section of ICD-9 and ICD-10 as schizotypal disorder, it is listed in this chapter and discussed in detail in the DSM-5 chapter ”Personality Disorders." Diagnostic Criteria 297.1 (F22) A. The presence of one (or more) delusions with a duration of 1 month or longer. B. Criterion A for schizophrenia has never been met. Note: Hallucinations, if present, are not prominent and are related to the delusional theme (e.g., the sensation of being infested with insects associated with delusions of infestation). C. Apart from the impact of the delusion(s) or its ramifications, functioning is not markedly impaired, and behavior is not obviously bizarre or odd.

1	C. Apart from the impact of the delusion(s) or its ramifications, functioning is not markedly impaired, and behavior is not obviously bizarre or odd. D. It manic or major depressive episodes have occurred, these have been brief relative to the duration of the delusional periods. E. The disturbance is not attributable to the physiological effects of a substance or an- other medical condition and is not better explained by another mental disorder, such as body dysmorphic disorder or obsessive-compulsive disorder. Specify whether: Erotomanlc type: This subtype applies when the central theme of the delusion is that another person is in love with the individual. Grandiose type: This subtype applies when the central theme of the delusion is the conviction of having some great (but unrecognized) talent or insight or having made some important discovery.

1	Grandiose type: This subtype applies when the central theme of the delusion is the conviction of having some great (but unrecognized) talent or insight or having made some important discovery. Jealous type: This subtype applies when the central theme of the individual's delusion is that his or her spouse or lover is unfaithful. Persecutory type: This subtype applies when the central theme of the delusion in- volves the individual’s belief that he or she is being conspired against, cheated, spied on, followed. poisoned or drugged, maliciously maligned, harassed, or obstructed in the pursuit of long-term goals. Somatic type: This subtype applies when the central theme of the delusion involves bodily functions or sensations. Mixed type: This subtype applies when no one delusional theme predominates.

1	Somatic type: This subtype applies when the central theme of the delusion involves bodily functions or sensations. Mixed type: This subtype applies when no one delusional theme predominates. Unspecified type: This subtype applies when the dominant delusional belief cannot be clearly determined or is not described in the specific types (e.g., referential delu- sions without a prominent persecutory or grandiose component). Specify it: With bizarre content: Delusions are deemed bizarre if they are clearly implausible, not understandable, and not derived from ordinary life experiences (e.g., an individual's be- lief that a stranger has removed his or her internal organs and replaced them with some— one else's organs without leaving any wounds or scars). Specify if: The following course specifiers are only to be used after a 1-year duration of the disorder:

1	Specify if: The following course specifiers are only to be used after a 1-year duration of the disorder: First episode, currently in acute episode: First manifestation of the disorder meet- ing the defining diagnostic symptom and time criteria. An acute episode is a time pe- riod in which the symptom criteria are fulfilled. First episode, currently in partial remission: Partial remission is a time period dur- ing which an improvement after a previous episode is maintained and in which the de- fining criteria of the disorder are only partially fulfilled. First episode, currently in full remission: Full remission is a period of time after a previous episode during which no disorder-specitic symptoms are present. Multiple episodes, currently in acute episode Multiple episodes, currently in partial remission Multiple episodes, currently in full remission

1	Multiple episodes, currently in acute episode Multiple episodes, currently in partial remission Multiple episodes, currently in full remission Continuous: Symptoms fulfilling the diagnostic symptom criteria of the disorder are remaining for the majority of the illness course, with subthreshold symptom periods be- ing very brief relative to the overall course. Specify current severity: Severity is rated by a quantitative assessment of the primary symptoms of psychosis, including delusions, hallucinations, disorganized speech, abnormal psychomotor be- havior, and negative symptoms. Each of these symptoms may be rated for its current severity (most severe in the last 7 days) on a 5-point scale ranging from 0 (not present) to 4 (present and severe). (See CIinician-Ftated Dimensions of Psychosis Symptom Severity in the chapter "Assessment Measures") Note: Diagnosis of delusional disorder can be made without using this severity specifier.

1	In erotomanic type, the central theme of the delusion is that another person is in love with the individual. The person about whom this conviction is held is usually of higher status (e.g., a famous individual or a superior at work) but can be a complete stranger. Efforts to contact the object of the delusion are common. In grandiose type, the central theme of the de- lusion is the conviction of having some great talent or insight or of having made some im- portant discovery. Less commonly, the individual may have the delusion of having a special relationship with a prominent individual or of being a prominent person (in which case the actual individual may be regarded as an impostor). Grandiose delusions may have a religious content. In jealous type, the central theme of the delusion is that of an un- faithful partner. This belief is arrived at without due cause and is based on incorrect infer- ences supported by small bits of "evidence” (e.g., disarrayed clothing). The individual

1	that of an un- faithful partner. This belief is arrived at without due cause and is based on incorrect infer- ences supported by small bits of "evidence” (e.g., disarrayed clothing). The individual with the delusion usually confronts the spouse or lover and attempts to intervene in the imagined infidelity. In persecutory type, the central theme of the delusion involves the in- dividual’s belief of being conspired against, cheated, spied on, followed, poisoned, mali- ciously maligned, harassed, or obstructed in the pursuit of long—term goals. Small slights may be exaggerated and become the focus of a delusional system. The affected individual may engage in repeated attempts to obtain satisfaction by legal or legislative action. Indi- viduals with persecutory delusions are often resentful and angry and may resort to Vio- lence against those they believe are hurting them. In somatic type, the central theme of the delusion involves bodily functions or sensations. Somatic delusions can

1	and angry and may resort to Vio- lence against those they believe are hurting them. In somatic type, the central theme of the delusion involves bodily functions or sensations. Somatic delusions can occur in several forms. Most common is the belief that the individual emits a foul odor; that there is an in- festation of insects on or in the skin; that there is an internal parasite; that certain parts of the body are misshapen or ugly; or that parts of the body are not functioning.

1	The essential feature of delusional disorder is the presence of one or more delusions that persist for at least 1 month (Criterion A). A diagnosis of delusional disorder is not given if the individual has ever had a symptom presentation that met Criterion A for schizophre- nia (Criterion B). Apart from the direct impact of the delusions, impairments in psychoso- such as schizophrenia, and behavior is not obviously bizarre or odd (Criterion C). If mood episodes occur concurrently with the delusions, the total duration of these mood episodes is brief relative to the total duration of the delusional periods (Criterion D). The delusions are not attributable to the physiological effects of a substance (e.g., cocaine) or another medical condition (e.g., Alzheimer’s disease) and are not better explained by another men- tal disorder, such as body dysmorphic disorder or obsessive-compulsive disorder (Crite- rion E).

1	In addition to the five symptom domain areas identified in the diagnostic criteria, the assessment of cognition, depression, and mania symptom domains is vital for making crit- ically important distinctions between the various schizophrenia spectrum and other psy- chotic disorders.

1	Social, marital, or work problems can result from the delusional beliefs of delusional dis- order. Individuals with delusional disorder may be able to factually describe that others view their beliefs as irrational but are unable to accept this themselves (i.e., there may be ”factual insight” but no true insight). Many individuals develop irritable or dysphoric mood, which can usually be understood as a reaction to their delusional beliefs. Anger and violent behavior can occur with persecutory, jealous, and erotomanic types. The individ- ual may engage in litigious or antagonistic behavior (e.g., sending hundreds of letters of protest to the government). Legal difficulties can occur, particularly in jealous and eroto- manic types.

1	The lifetime prevalence of delusional disorder has been estimated at around 0.2%, and the most frequent subtype is persecutory. Delusional disorder, jealous type, is probably more common in males than in females, but there are no major gender differences in the overall frequency of delusional disorder. On average, global function is generally better than that observed in schizophrenia. Al- though the diagnosis is generally stable, a proportion of individuals go on to develop schizophrenia. Delusional disorder has a significant familial relationship with both schizophrenia and schizotypal personality disorder. Although it can occur in younger age groups, the condition may be more prevalent in older individuals. ing the possible presence of delusional disorder. The content of delusions also varies across cultural contexts. Functional Consequences of Delusional Disorder

1	ing the possible presence of delusional disorder. The content of delusions also varies across cultural contexts. Functional Consequences of Delusional Disorder The functional impairment is usually more circumscribed than that seen with other psy- chotic disorders, although in some cases, the impairment may be substantial and include poor occupational functioning and social isolation. When poor psychosocial functioning is present, delusional beliefs themselves often play a significant role. A common character- istic of individuals with delusional disorder is the apparent normality of their behavior and appearance when their delusional ideas are not being discussed or acted on.

1	Obsessive-compulsive and related disorders. If an individual with obsessive—compul— sive disorder is completely convinced that his or her obsessive-compulsive disorder beliefs are true, then the diagnosis of obsessive-compulsive disorder, with absent insight/delu- sional beliefs specifier, should be given rather than a diagnosis of delusional disorder. Similarly, if an individual with body dysmorphic disorder is completely convinced that his or her body dysmorphic disorder beliefs are true, then the diagnosis of body dysmor- phic disorder, with absent insight/ delusional beliefs specifier, should be given rather than a diagnosis of delusional disorder.

1	Delirium, major neurocognitive disorder, psychotic disorder due to another medical con- dition, and substance/medication-induced psychotic disorder. Individuals with these disorders may present with symptoms that suggest delusional disorder. For example, sirn- ple persecutory delusions in the context of major neurocognitive disorder would be di- agnosed as major neurocognitive disorder, with behavioral disturbance. A substance/ atology to delusional disorder but can be distinguished by the chronological relationship of substance use to the onset and remission of the delusional beliefs. Schizophrenia and schizophreniform disorder. Delusional disorder can be distinguished from schizophrenia and schizophreniform disorder by the absence of the other character- istic symptoms of the active phase of schizophrenia.

1	Depressive and bipolar disorders and schizoaffective disorder. These disorders may be distinguished from delusional disorder by the temporal relationship between the mood disturbance and the delusions and by the severity of the mood symptoms. If delusions oc- cur exclusively during mood episodes, the diagnosis is depressive or bipolar disorder with psychotic features. Mood symptoms that meet full criteria for a mood episode can be su- perimposed on delusional disorder. Delusional disorder can be diagnosed only if the total duration of all mood episodes remains brief relative to the total duration of the delusional disturbance. If not, then a diagnosis of other specified or unspecified schizophrenia spec- trum and other psychotic disorder accompanied by other specified depressive disorder, unspecified depressive disorder, other specified bipolar and related disorder, or unspeci- fied bipolar and related disorder is appropriate. Diagnostic Criteria 298.8 (F23)

1	Diagnostic Criteria 298.8 (F23) A. Presence of one (or more) of the following symptoms. At least one of these must be (1). (2). 0r (3): 1. Delusions. 2. Hallucinations. 3. Disorganized speech (e.g., frequent derailment or incoherence). 4. Grossly disorganized or catatonic behavior. Note: Do not include a symptom if it is a culturally sanctioned response. B. Duration of an episode of the disturbance is at least 1 day but less than 1 month, with eventual full return to premorbid level of functioning. C. The disturbance is not better explained by major depressive or bipolar disorder with psychotic features or another psychotic disorder such as schizophrenia or catatonia, and is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication) or another medical condition. Specify it:

1	Specify it: With marked stressor(s) (brief reactive psychosis): If symptoms occur in response to events that, singly or together, would be markedly stressful to almost anyone in similar circumstances in the individual’s culture. Without marked stressor(s): If symptoms do not occur in response to events that, singly or together, would be markedly stressful to almost anyone in similar circum- stances in the individual's culture. With postpartum onset: If onset is during pregnancy or within 4 weeks postpartum. Specify if: With catatonla (refer to the criteria for catatonia associated with another mental dis- order, pp. 119—120, for definition) Coding note: Use additional code 293.89 (F06.1) catatonia associated with brief psychotic disorder to indicate the presence of the comorbid catatonia. Specify current severity:

1	Coding note: Use additional code 293.89 (F06.1) catatonia associated with brief psychotic disorder to indicate the presence of the comorbid catatonia. Specify current severity: Severity is rated by a quantitative assessment of the primary symptoms of psychosis, including delusions, hallucinations, disorganized speech, abnormal psychomotor be- havior, and negative symptoms. Each of these symptoms may be rated for its current severity (most severe in the last 7 days) on a 5-point scale ranging from 0 (not present) to 4 (present and severe). (See CIinician-Rated Dimensions of Psychosis Symptom Severity in the chapter “Assessment Measures”) Note: Diagnosis of brief psychotic disorder can be made without using this severity specifier.

1	The essential feature of brief psychotic disorder is a disturbance that involves the sudden onset of at least one of the following positive psychotic symptoms: delusions, hallucina- tions, disorganized speech (e.g., frequent derailment or incoherence), or grossly abnormal psychomotor behavior, including catatonia (Criterion A). Sudden onset is defined as change from a nonpsychotic state to a clearly psychotic state within 2 weeks, usually with- out a prodrome. An episode of the disturbance lasts at least 1 day but less than 1 month, and the individual eventually has a full return to the premorbid level of functioning (Cri- terion B). The disturbance is not better explained by a depressive or bipolar disorder with psychotic features, by schizoaffective disorder, or by schizophrenia and is not attributable to the physiological effects of a substance (e.g., a hallucinogen) or another medical condi- tion (e.g., subdural hematoma) (Criterion C).

1	In addition to the five symptom domain areas identified in the diagnostic criteria, the assessment of cognition, depression, and mania symptom domains is Vital for making crit- ically important distinctions between the various schizophrenia spectrum and other psy- chotic disorders. Individuals with brief psychotic disorder typically experience emotional turmoil or over- whelming confusion. They may have rapid shifts from one intense affect to another. Although the disturbance is brief, the level of impairment may be severe, and supervision may be required to ensure that nutritional and hygienic needs are met and that the indi- vidual is protected from the consequences of poor judgment, cognitive impairment, or act- ing on the basis of delusions. There appears to be an increased risk of suicidal behavior, particularly during the acute episode.

1	In the United States, brief psychotic disorder may account for 9% of cases of first-onset psychosis. Psychotic disturbances that meet Criteria A and C, but not Criterion B, for brief psychotic disorder (i.e., duration of active symptoms is 1—6 months as opposed to remis- sion within 1 month) are more common in developing countries than in developed coun- tries. Brief psychotic disorder is twofold more common in females than in males. Brief psychotic disorder may appear in adolescence or early adulthood, and onset can oc- cur across the lifespan, with the average age at onset being the mid 305. By definition, a diagnosis of brief psychotic disorder requires a full remission of all symptoms and an eventual full return to the premorbid level of functioning Within 1 month of the onset of the disturbance. In some individuals, the duration of psychotic symptoms may be quite brief (e.g., a few days).

1	Temperamental. Preexisting personality disorders and traits (e.g., schizotypal person- ality disorder; borderline personality disorder; or traits in the psychoticism domain, such as perceptual dysregulation, and the negative affectivity domain, such as suspiciousness) may predispose the individual to the development of the disorder. It is important to distinguish symptoms of brief psychotic disorder from culturally sanc- tioned response patterns. For example, in some religious ceremonies, an individual may report hearing voices, but these do not generally persist and are not perceived as abnormal by most members of the individual’s community. In addition, cultural and religious back- ground must be taken into account when considering whether beliefs are delusional. Functionai Consequences of Brief Psychotic Disorder Despite high rates of relapse, for most individuals, outcome is excellent in terms of social functioning and symptomatology.

1	Functionai Consequences of Brief Psychotic Disorder Despite high rates of relapse, for most individuals, outcome is excellent in terms of social functioning and symptomatology. Other medical conditions. A variety of medical disorders can manifest with psychotic symptoms of short duration. Psychotic disorder due to another medical condition or a de— lirium is diagnosed when there is evidence from the history, physical examination, or lab- oratory tests that the delusions or hallucinations are the direct physiological consequence of a specific medical condition (e.g., Cushing’s syndrome, brain tumor) (see “Psychotic Disorder Due to Another Medical Condition” later in this chapter).

1	Disorder Due to Another Medical Condition” later in this chapter). Substance-related disorders. Substance/medication-induced psychotic disorder, sub- stance-induced delirium, and substance intoxication are distinguished from brief psychotic disorder by the fact that a substance (e.g., a drug of abuse, a medication, exposure to a toxin) is judged to be etiologically related to the psychotic symptoms (see ”Substance /Medication- Induced Psychotic Disorder” later in this chapter). Laboratory tests, such as a urine drug screen or a blood alcohol level, may be helpful in making this determination, as may a care- ful history of substance use with attention to temporal relationships between substance in- take and onset of the symptoms and to the nature of the substance being used.

1	Depressive and bipolar disorders. The diagnosis of brief psychotic disorder cannot be made if the psychotic symptoms are better explained by a mood episode (i.e., the psychotic symptoms occur exclusively during a full major depressive, manic, or mixed episode).

1	Other psychotic disorders. If the psychotic symptoms persist for 1 month or longer, the diagnosis is either schizophreniform disorder, delusional disorder, depressive disorder with psychotic features, bipolar disorder with psychotic features, or other specified or un- specified schizophrenia spectrum and other psychotic disorder, depending on the other symptoms in the presentation. The differential diagnosis between brief psychotic disorder and schizophreniform disorder is difficult when the psychotic symptoms have remitted be- fore 1 month in response to successful treatment with medication. Careful attention should be given to the possibility that a recurrent disorder (e.g., bipolar disorder, recurrent acute ex- acerbations of schizophrenia) may be responsible for any recurring psychotic episodes.

1	Malingering and factitious disorders. An episode of factitious disorder, with predomi- nantly psychological signs and symptoms, may have the appearance of brief psychotic disorder, but in such cases there is evidence that the symptoms are intentionally produced. When malingering involves apparently psychotic symptoms, there is usually evidence that the illness is being feigned for an understandable goal. Personality disorders. In certain individuals with personality disorders, psychosocial stressors may precipitate brief periods of psychotic symptoms. These symptoms are usu- ally transient and do not warrant a separate diagnosis. If psychotic symptoms persist for at least 1 day, an additional diagnosis of brief psychotic disorder may be appropriate. Diagnostic Criteria 295.40 (F20.81)

1	Diagnostic Criteria 295.40 (F20.81) A. Two (or more) of the following, each present for a significant portion of time during a 1-month period (or less it successfully treated). At least one of these must be (1 ). (2), or (3): 1. Delusions. 2. Hallucinations. 3. Disorganized speech (e.g., frequent derailment or incoherence). 4. Grossly disorganized or catatonic behavior. 5. Negative symptoms (i.e., diminished emotional expression or avolition). B. An episode of the disorder lasts at least 1 month but less than 6 months. When the diagnosis must be made without waiting for recovery, it should be qualified as “provi- sional."

1	B. An episode of the disorder lasts at least 1 month but less than 6 months. When the diagnosis must be made without waiting for recovery, it should be qualified as “provi- sional." C. Schizoaffective disorder and depressive or bipolar disorder with psychotic features have been ruled out because either 1) no major depressive or manic episodes have occurred concurrently with the active-phase symptoms, or 2) if mood episodes have occurred dur- ing active-phase symptoms, they have been present for a minority of the total duration of the active and residual periods of the illness. D. The disturbance is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication) or another medical condition. Specify it:

1	D. The disturbance is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication) or another medical condition. Specify it: With good prognostic features: This specifier requires the presence of at least two of the following features: onset of prominent psychotic symptoms within 4 weeks of the premorbid social and occupational functioning; and absence of blunted or flat affect. Without good prognostic features: This specifier is applied if two or more of the above features have not been present. Specify if: With catatonia (refer to the criteria for catatonia associated with another mental disor- der, pp. 119—120, for definition). Coding note: Use additional code 293.89 (F06.1) catatonia associated with schizo- phreniform disorder to indicate the presence of the comorbid catatonia. Specify current severity:

1	Coding note: Use additional code 293.89 (F06.1) catatonia associated with schizo- phreniform disorder to indicate the presence of the comorbid catatonia. Specify current severity: Severity is rated by a quantitative assessment of the primary symptoms of psychosis, including delusions, hallucinations, disorganized speech, abnormal psychomotor be- havior, and negative symptoms. Each of these symptoms may be rated for its current severity (most severe in the last 7 days) on a 5-point scale ranging from 0 (not present) to 4 (present and severe). (See CIinician-Rated Dimensions of Psychosis Symptom Severity in the chapter “Assessment Measures") Note: Diagnosis of schizophreniform disorder can be made without using this severity specifier. Note: For additional information on Associated Features Supporting Diagnosis, Develop- ment and Course (age-related factors), Culture-Related Diagnostic Issues, Gender-Related

1	Note: For additional information on Associated Features Supporting Diagnosis, Develop- ment and Course (age-related factors), Culture-Related Diagnostic Issues, Gender-Related Diagnostic Issues, Differential Diagnosis, and Comorbidity, see the corresponding sec- tions in schizophrenia. The characteristic symptoms of schizophreniform disorder are identical to those of schizo- phrenia (Criterion A). Schizophreniform disorder is distinguished by its difference in du- ration: the total duration of the illness, including prodromal, active, and residual phases, is at least 1 month but less than 6 months (Criterion B). The duration requirement for schizo- phreniform disorder is intermediate between that for brief psychotic disorder, which lasts more than 1 day and remits by 1 month, and schizophrenia, which lasts for at least 6 months.

1	The diagnosis of schizophreniform disorder is made under two conditions. 1) when an ep- isode of illness lasts between 1 and 6 months and the individual has already recovered, and 2) when an individual is symptomatic for less than the 6 months’ duration required for the diagnosis of schizophrenia but has not yet recovered. In this case, the diagnosis should be noted as ”schizophreniform disorder (provisiona1)” because it is uncertain if the indi- vidual will recover from the disturbance within the 6-month period. If the disturbance per- sists beyond 6 months, the diagnosis should be changed to schizophrenia. Another distinguishing feature of schizophreniform disorder is the lack of a criterion requiring impaired social and occupational functioning. While such impairments may po- tentially be present, they are not necessary for a diagnosis of schizophreniform disorder.

1	In addition to the five symptom domain areas identified in the diagnostic criteria, the assessment of cognition, depression, and mania symptom domains is vital for making crit- ically important distinctions between the various schizophrenia spectrum and other psy- chotic disorders. As with schizophrenia, currently there are no laboratory or psychometric tests for schizo— phreniform disorder. There are multiple brain regions where neuroimaging, neuropa- thological, and neurophysiological research has indicated abnormalities, but none are diagnostic.

1	Incidence of schizophreniform disorder across sociocultural settings is likely similar to that observed in schizophrenia. In the United States and other developed countries, the in— cidence is low, possibly fivefold less than that of schizophrenia. In developing countries, the incidence may be higher, especially for the specifier “with good prognostic features"; in some of these settings schizophreniform disorder may be as common as schizophrenia. The development of schizophreniform disorder is similar to that of schizophrenia. About one-third of individuals with an initial diagnosis of schizophreniform disorder (provi— sional) recover within the 6-month period and schizophreniform disorder is their final di- agnosis. The majority of the remaining two-thirds of individuals will eventually receive a diagnosis of schizophrenia or schizoaffective disorder. Genetic and physiological. Relatives of individuals with schizophreniform disorder have an increased risk for schizophrenia.

1	Genetic and physiological. Relatives of individuals with schizophreniform disorder have an increased risk for schizophrenia. For the majority of individuals with schizophreniform disorder who eventually receive a diagnosis of schizophrenia or schizoaffective disorder, the functional consequences are similar to the consequences of those disorders. Most individuals experience dysfunction in several areas of daily functioning, such as school or work, interpersonal relationships, and self—care. Individuals who recover from schizophreniform disorder have better functional outcomes.

1	Other mental disorders and medical conditions. A wide variety of mental and medical conditions can manifest with psychotic symptoms that must be considered in the differ- ential diagnosis of schizophreniform disorder. These include psychotic disorder due to disorder with psychotic features; schizoaffective disorder; other specified or unspecified bi— polar and related disorder; depressive or bipolar disorder with catatonic features; schizophre- phrenia spectrum and other psychotic disorder; schizotypal, schizoid, or paranoid personality disorders,- autism spectrum disorder; disorders presenting in childhood with order; posttraumatic stress disorder; and traumatic brain injury. Since the diagnostic criteria for schizophreniform disorder and schizophrenia differ primarily in duration of illness, the discussion of the differential diagnosis of schizophre- nia also applies to schizophreniform disorder.

1	Brief psychotic disorder. Schizophreniform disorder differs in duration from brief psy- chotic disorder, which has a duration of less than 1 month. Diagnostic Criteria 295.90 (F20.9) A. Two (or more) of the following, each present for a significant portion of time during a 1-month period (or less it successfully treated). At least one of these must be (1 ), (2), or (3): 1. Delusions. Hallucinations. Disorganized speech (e.g., frequent derailment or incoherence). Grossly disorganized or catatonic behavior. Negative symptoms (i.e., diminished emotional expression or avolition). .01wa B. For a significant portion of the time since the onset of the disturbance, level of function- ing in one or more major areas, such as work, interpersonal relations, or self-care, is markedly below the level achieved prior to the onset (or when the onset is in childhood or adolescence, there is failure to achieve expected level of interpersonal, academic, or occupational functioning).

1	C. Continuous signs of the disturbance persist for at least 6 months. This 6-month period must include at least 1 month of symptoms (or less it successfully treated) that meet Cri- terion A (i.e., active-phase symptoms) and may include periods of prodromal or residual symptoms. During these prodromal or residual periods, the signs of the disturbance may A present in an attenuated form (e.g., odd beliefs, unusual perceptual experiences). D. Schizoaffective disorder and depressive or bipolar disorder with psychotic features have been ruled out because either 1) no major depressive or manic episodes have occurred concurrently with the active-phase symptoms, or 2) if mood episodes have occurred during active-phase symptoms, they have been present for a minority of the total duration of the active and residual periods of the illness. E. The disturbance is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication) or another medical condition.

1	E. The disturbance is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication) or another medical condition. F. If there is a history of autism spectrum disorder or a communication disorder of child- hood onset, the additional diagnosis of schizophrenia is made only if prominent delu- sions or hallucinations, in addition to the other required symptoms of schizophrenia, are also present for at least 1 month (or less if successfully treated). Specify if: The following course specifiers are only to be used after a 1-year duration of the disorder and if they are not in contradiction to the diagnostic course criteria. First episode, currently in acute episode: First manifestation of the disorder meet- ing the defining diagnostic symptom and time criteria. An acute episode is a time pe- riod in which the symptom criteria are fulfilled.

1	First episode, currently in partial remission: Partial remission is a period of time during which an improvement after a previous episode is maintained and in which the defining criteria of the disorder are only partially fulfilled. First episode, currently in full remission: Full remission is a period of time after a previous episode during which no disorder-specific symptoms are present. Multiple episodes, currently in acute episode: Multiple episodes may be deter- mined after a minimum of two episodes (i.e., after a first episode, a remission and a minimum of one relapse). Multiple episodes, currently in partial remission Multiple episodes, currently in full remission Continuous: Symptoms fulfilling the diagnostic symptom criteria of the disorder are remaining for the majority of the illness course, with subthreshold symptom periods be- ing very brief relative to the overall course. Specify if:

1	Specify if: With catatonia (refer to the criteria for catatonia associated with another mental disorder, pp. 119—120, for definition). Coding note: Use additional code 293.89 (F061) catatonia associated with schizophrenia to indicate the presence of the comorbid catatonia. Specify current severity: Severity is rated by a quantitative assessment of the primary symptoms of psychosis, including delusions, hallucinations, disorganized speech, abnormal psychomotor be- havior, and negative symptoms. Each of these symptoms may be rated for its current severity (most severe in the last 7 days) on a 5-point scale ranging from 0 (not present) to 4 (present and severe). (See Clinician-Rated Dimensions of Psychosis Symptom Severity in the chapter “Assessment Measures") Note: Diagnosis of schizophrenia can be made without using this severity specifier.

1	Severity in the chapter “Assessment Measures") Note: Diagnosis of schizophrenia can be made without using this severity specifier. The characteristic symptoms of schizophrenia involve a range of cognitive, behavioral, and emotional dysfunctions, but no single symptom is pathognomonic of the disorder. The di- agnosis involves the recognition of a constellation of signs and symptoms associated with impaired occupational or social functioning. Individuals with the disorder will vary sub- stantially on most features, as schizophrenia is a heterogeneous Clinical syndrome.

1	At least two Criterion A symptoms must be present for a significant portion of time during a 1-month period or longer. At least one of these symptoms must be the clear pres- ence of delusions (Criterion A1), hallucinations (Criterion A2), or disorganized speech (Criterion A3). Grossly disorganized or catatonic behavior (Criterion A4) and negative symptoms (Criterion A5) may also be present. In those situations in which the active- phase symptoms remit within a month in response to treatment, Criterion A is still met if the clinician estimates that they would have persisted in the absence of treatment.

1	Schizophrenia involves impairment in one or more major areas of functioning (Crite- rion B). If the disturbance begins in childhood or adolescence, the expected level of func- tion is not attained. Comparing the individual with unaffected siblings may be helpful. The dysfunction persists for a substantial period during the course of the disorder and does not appear to be a direct result of any single feature. Avolition (i.e., reduced drive to pursue goal-directed behavior; Criterion A5) is linked to the social dysfunction described under Criterion B. There is also strong evidence for a relationship between cognitive impairment (see the section "Associated Features Supporting Diagnosis” for this disorder) and func- tional impairment in individuals with schizophrenia.

1	Some signs of the disturbance must persist for a continuous period of at least 6 months (Criterion C). Prodromal symptoms often precede the active phase, and residual symp- toms may follow it, characterized by mild or subthreshold forms of hallucinations or delusions. Individuals may express a variety of unusual or odd beliefs that are not of de- lusional proportions (e.g., ideas of reference or magical thinking); they may have unusual perceptual experiences (e.g., sensing the presence of an unseen person); their speech may disorganized (e.g., mumbling in public). Negative symptoms are common in the pro- dromal and residual phases and can be severe. Individuals who had been socially active may become withdrawn from previous routines. Such behaviors are often the first sign of a disorder.

1	Mood symptoms and full mood episodes are common in schizophrenia and may be con- current with active-phase symptomatology. However, as distinct from a psychotic mood dis- order, a schizophrenia diagnosis requires the presence of delusions or hallucinations in the absence of mood episodes. In addition, mood episodes, taken in total, should be present for only a minority of the total duration of the active and residual periods of the illness. In addition to the five symptom domain areas identified in the diagnostic criteria, the assessment of cognition, depression, and mania symptom domains is vital for making crit- ically important distinctions between the various schizophrenia spectrum and other psy- chotic disorders.

1	Individuals with schizophrenia may display inappropriate affect (e.g., laughing in the ab- sence of an appropriate stimulus); a dysphoric mood that can take the form of depression, anxiety, or anger; a disturbed sleep pattern (e.g., daytime sleeping and nighttime activity); and a lack of interest in eating or food refusal. Depersonalization, derealization, and so- matic concerns may occur and sometimes reach delusional proportions. Anxiety and pho- bias are common. Cognitive deficits in schizophrenia are common and are strongly linked to vocational and functional impairments. These deficits can include decrements in declar- ative memory, working memory, language function, and other executive functions, as well as slower processing speed. Abnormalities in sensory processing and inhibitory capacity, as well as reductions in attention, are also found. Some individuals with schizophrenia show social cognition deficits, including deficits in the ability to infer the intentions of other

1	capacity, as well as reductions in attention, are also found. Some individuals with schizophrenia show social cognition deficits, including deficits in the ability to infer the intentions of other people (theory of mind), and may attend to and then interpret irrelevant events or stimuli as meaningful, perhaps leading to the generation of explanatory delusions. These impairments frequently persist during symptomatic remission.

1	Some individuals with psychosis may lack insight or awareness of their disorder (i.e., anosognosia). This lack of ”insight" includes unawareness of symptoms of schizophrenia and may be present throughout the entire course of the illness. Unawareness of illness is typically a symptom of schizophrenia itself rather than a coping strategy. It is comparable to the lack of awareness of neurological deficits following brain damage, termed anosa— gnosia. This symptom is the most common predictor of non-adherence to treatment, and it predicts higher relapse rates, increased number of involuntary treatments, poorer psycho- social functioning, aggression, and a poorer course of illness.

1	Hostility and aggression can be associated with schizophrenia, although spontaneous or random assault is uncommon. Aggression is more frequent for younger males and for individuals with a past history of violence, non-adherence with treatment, substance abuse, and impulsivity. It should be noted that the vast majority of persons with schizo- phrenia are not aggressive and are more frequently victimized than are individuals in the general population. Currently, there are no radiological, laboratory, or psychometric tests for the disorder. Differences are evident in multiple brain regions between groups of healthy individuals and persons with schizophrenia, including evidence from neuroimaging, neuropatholog- ical, and neurophysiological studies. Differences are also evident in cellular architecture,

1	White matter connectivity, and gray matter volume in a variety of regions such as the pre- frontal and temporal cortices. Reduced overall brain volume has been observed, as well as increased brain volume reduction with age. Brain volume reductions with age are more pronounced in individuals with schizophrenia than in healthy individuals. Finally, indi- viduals with schizophrenia appear to differ from individuals without the disorder in eye- tracking and electrophysiological indices. Neurological soft signs common in individuals with schizophrenia include impairments in motor coordination, sensory integration, and motor sequencing of complex movements; left-right confusion; and disinhibition of associated movements. In addition, minor phys- ical anomalies of the face and limbs may occur.

1	The lifetime prevalence of schizophrenia appears to be approximately 0.3%—0.7%, al- though there is reported variation by race/ethnicity, across countries, and by geographic origin for immigrants and children of immigrants. The sex ratio differs across samples and populations: for example, an emphasis on negative symptoms and longer duration of dis- order (associated with poorer outcome) shows higher incidence rates for males, whereas definitions allowing for the inclusion of more mood symptoms and brief presentations (associated with better outcome) show equivalent risks for both sexes.

1	The psychotic features of schizophrenia typically emerge between the late teens and the mid-30s; onset prior to adolescence is rare. The peak age at onset for the first psychotic ep- isode is in the early- to mid-205 for males and in the late—205 for females. The onset may be abrupt or insidious, but the majority of individuals manifest a slow and gradual develop- ment of a variety of clinically significant signs and symptoms. Half of these individuals complain of depressive symptoms. Earlier age at onset has traditionally been seen as a pre- dictor of worse prognosis. However, the effect of age at onset is likely related to gender, with males having worse premorbid adjustment, lower educational achievement, more prominent negative symptoms and cognitive impairment, and in general a worse out- come. Impaired cognition is common, and alterations in cognition are present during de- velopment and precede the emergence of psychosis, taking the form of stable cognitive impairments during

1	out- come. Impaired cognition is common, and alterations in cognition are present during de- velopment and precede the emergence of psychosis, taking the form of stable cognitive impairments during adulthood. Cognitive impairments may persist when other symptoms are in remission and contribute to the disability of the disease.

1	The predictors of course and outcome are largely unexplained, and course and outcome may not be reliably predicted. The course appears to be favorable in about 20% of those with schizophrenia, and a small number of individuals are reported to recover completely. However, most individuals with schizophrenia still require formal or informal daily living supports, and many remain chronically ill, with exacerbations and remissions of active symptoms, while others have a course of progressive deterioration. Psychotic symptoms tend to diminish over the life course, perhaps in association with normal age-related declines in dopamine activity. Negative symptoms are more closely re- lated to prognosis than are positive symptoms and tend to be the most persistent. Further- more, cognitive deficits associated with the illness may not improve over the course of the illness.

1	The essential features of schizophrenia are the same in childhood, but it is more diffi- cult to make the diagnosis. In children, delusions and hallucinations may be less elaborate than in adults, and visual hallucinations are more common and should be distinguished from normal fantasy play. Disorganized speech occurs in many disorders with childhood onset (e.g., autism spectrum disorder), as does disorganized behavior (e.g., attention-deficit/ hyperactivity disorder). These symptoms should not be attributed to schizophrenia with- out due consideration of the more common disorders of childhood. Childhood-onset cases tend to resemble poor-outcome adult cases, with gradual onset and prominent negative symptoms. Children who later receive the diagnosis of schizophrenia are more likely to have experienced nonspecific emotional-behavioral disturbances and psychopathology, intellectual and language alterations, and subtle motor delays.

1	Late-onset cases (i.e., onset after age 40 years) are overrepresented by females, who may have married. Often, the course is characterized by a predominance of psychotic symptoms with preservation of affect and social functioning. Such late-onset cases can still meet the diagnostic criteria for schizophrenia, but it is not yet clear whether this is the same condition as schizophrenia diagnosed prior to mid-life (e.g., prior to age 55 years). Environmental. Season of birth has been linked to the incidence of schizophrenia, in- cluding late winter/early spring in some locations and summer for the deficit form of the disease. The incidence of schizophrenia and related disorders is higher for children grow- ing up in an urban environment and for some minority ethnic groups.

1	Genetic and physiological. There is a strong contribution for genetic factors in deter- mining risk for schizophrenia, although most individuals who have been diagnosed with schizophrenia have no family history of psychosis. Liability is conferred by a spectrum of risk alleles, common and rare, with each allele contributing only a small fraction to the to- tal population variance. The risk alleles identified to date are also associated with other mental disorders, including bipolar disorder, depression, and autism spectrum disorder. Pregnancy and birth complications with hypoxia and greater paternal age are associated with a higher risk of schizophrenia for the developing fetus. In addition, other prenatal and perinatal adversities, including stress, infection, malnutrition, maternal diabetes, and other medical conditions, have been linked with schizophrenia. However, the vast major- ity of offspring with these risk factors do not develop schizophrenia.

1	Cultural and socioeconomic factors must be considered, particularly when the individual and the clinician do not share the same cultural and socioeconomic background. Ideas that appear to be delusional in one culture (e.g., witchcraft) may be commonly held in another. In some cultures, visual or auditory hallucinations with a religious content (e.g., hearing

1	In some cultures, visual or auditory hallucinations with a religious content (e.g., hearing God’s voice) are a normal part of religious experience. In addition, the assessment of dis- cultures. The assessment of affect requires sensitivity to differences in styles of emotional expression, eye contact, and body language, which vary across cultures. If the assessment is conducted in a language that is different from the individual’s primary language, care must be taken to ensure that alogia is not related to linguistic barriers. In certain cultures, distress may take the form of hallucinations 0r pseudo-hallucinations and overvalued ideas that may present clinically similar to true psychosis but are normative to the pa- tient’s subgroup.

1	A number of features distinguish the clinical expression of schizophrenia in females and males. The general incidence of schizophrenia tends to be slightly lower in females, par- ticularly among treated cases. The age at onset is later in females, with a second mid-life peak as described earlier (see the section ”Development and Course” for this disorder). Symptoms tend to be more affect-laden among females, and there are more psychotic symptoms, as well as a greater propensity for psychotic symptoms to worsen in later life. Other symptom differences include less frequent negative symptoms and disorganization. Finally, social functioning tends to remain better preserved in females. There are, how- ever, frequent exceptions to these general caveats.

1	Finally, social functioning tends to remain better preserved in females. There are, how- ever, frequent exceptions to these general caveats. Approximately 5%—6% of individuals with schizophrenia die by suicide, about 20% attempt suicide on one or more occasions, and many more have significant suicidal ideation. Suicidal behavior is sometimes in response to command hallucinations to harm oneself or others. Suicide risk remains high over the whole lifespan for males and females, although it may be especially high for younger males with comorbid substance use. Other risk factors include having depressive symptoms or feelings of hopelessness and being unemployed, and the risk is higher, also, in the period after a psychotic episode or hospital discharge. Functional Consequences of Schizophrenia

1	Functional Consequences of Schizophrenia Schizophrenia is associated with significant social and occupational dysfunction. Making educational progress and maintaining employment are frequently impaired by avolition or other disorder manifestations, even when the cognitive skills are sufficient for the tasks at hand. Most individuals are employed at a lower level than their parents, and most, par- ticularly men, do not marry or have limited social contacts outside of their family. Major depressive or bipolar disorder with psychotic or catatonic features. The distinc- tion between schizophrenia and major depressive or bipolar disorder with psychotic features or with catatonia depends on the temporal relationship between the mood distur- bance and the psychosis, and on the severity of the depressive or manic symptoms. If de- lusions or hallucinations occur exclusively during a major depressive or manic episode, the diagnosis is depressive or bipolar disorder with psychotic features.

1	Schizoaffective disorder. A diagnosis of schizoaffective disorder requires that a major depressive or manic episode occur concurrently with the active-phase symptoms and that the mood symptoms be present for a majority of the total duration of the active periods. Schizophreniform disorder and brief psychotic disorder. These disorders are of shorter duration than schizophrenia as specified in Criterion C, which requires 6 months of symp- toms. In schizophreniform disorder, the disturbance is present less than 6 months, and in brief psychotic disorder, symptoms are present at least 1 day but less than 1 month. Delusional disorder. Delusional disorder can be distinguished from schizophrenia by the absence of the other symptoms characteristic of schizophrenia (e.g., delusions, prom- inent auditory or visual hallucinations, disorganized speech, grossly disorganized or cata- tonic behavior, negative symptoms).

1	Schizotypal personality disorder. Schizotypal personality disorder may be distinguished from schizophrenia by subthreshold symptoms that are associated with persistent person- ality features. Obsessive-compulsive disorder and body dysmorphic disorder. Individuals with obsessive-compulsive disorder and body dysmorphic disorder may present with poor or absent insight, and the preoccupations may reach delusional proportions. But these disorders are distinguished from schizophrenia by their prominent obsessions, compul- sions, preoccupations with appearance or body odor, hoarding, or body-focused repeti- tive behaviors. Posttraumatic stress disorder. Posttraumatic stress disorder may include ﬂashbacks that have a hallucinatory quality, and hypervigilance may reach paranoid proportions. But a trau- matic event and characteristic symptom features relating to reliving or reacting to the event are required to make the diagnosis.

1	Autism spectrum disorder or communication disorders. These disorders may also have symptoms resembling a psychotic episode but are distinguished by their respective defi- cits in social interaction with repetitive and restricted behaviors and other cognitive and communication deficits. An individual with autism spectrum disorder or communication disorder must have symptoms that meet full criteria for schizophrenia, with prominent hallucinations or delusions for at least 1 month, in order to be diagnosed with schizophre- nia as a comorbid condition.

1	Other mental disorders associated with a psychotic episode. The diagnosis of schizo- phrenia is made only when the psychotic episode is persistent and not attributable to the physiological effects of a substance or another medical condition. Individuals with a de- lirium or major or minor neurocognitive disorder may present with psychotic symptoms, but these would have a temporal relationship to the onset of cognitive changes consistent with those disorders. Individuals with substance/medication-induced psychotic disorder may present with symptoms characteristic of Criterion A for schizophrenia, but the sub- stance/medication-induced psychotic disorder can usually be distinguished by the chron- ological relationship of substance use to the onset and remission of the psychosis in the absence of substance use.

1	Rates of comorbidity with substance-related disorders are high in schizophrenia. Over half of individuals with schizophrenia have tobacco use disorder and smoke cigarettes regularly. Comorbidity with anxiety disorders is increasingly recognized in schizophre- nia. Rates of obsessive-compulsive disorder and panic disorder are elevated in individuals with schizophrenia compared with the general population. Schizotypal or paranoid per- sonality disorder may sometimes precede the onset of schizophrenia. Life expectancy is reduced in individuals with schizophrenia because of associated medical conditions. Weight gain, diabetes, metabolic syndrome, and cardiovascular and pulmonary disease are more common in schizophrenia than in the general population.

1	Poor engagement in health maintenance behaviors (e.g., cancer screening, exercise) in— creases the risk of chronic disease, but other disorder factors, including medications, life- style, cigarette smoking, and diet, may also play a role. A shared vulnerability for psychosis and medical disorders may explain some of the medical comorbidity of schizo- phrenia. A. An uninterrupted period of illness during which there is a major mood episode (major depressive or manic) concurrent with Criterion A of schizophrenia. Note: The major depressive episode must include Criterion A1: Depressed mood. B. Delusions or hallucinations for 2 or more weeks in the absence of a major mood epi- sode (depressive or manic) during the lifetime duration of the illness. C. Symptoms that meet criteria for a major mood episode are present for the majority of the total duration of the active and residual portions of the illness.

1	C. Symptoms that meet criteria for a major mood episode are present for the majority of the total duration of the active and residual portions of the illness. D. The disturbance is not attributable to the effects of a substance (e.g., a drug of abuse, a medication) or another medical condition. Specify whether: 295.70 (F25.0) Bipolar type: This subtype applies if a manic episode is part of the pre- sentation. Major depressive episodes may also occur. 295.70 (F25.1) Depressive type: This subtype applies if only major depressive epi- sodes are part of the presentation. Specify if: With catatonia (refer to the criteria for catatonia associated with another mental disorder, pp. 119—120, for definition). Coding note: Use additional code 293.89 (F06.1) catatonia associated with schizoaffective disorder to indicate the presence of the comorbid catatonia. Specify if:

1	Coding note: Use additional code 293.89 (F06.1) catatonia associated with schizoaffective disorder to indicate the presence of the comorbid catatonia. Specify if: The following course specifiers are only to be used after a 1—year duration of the disorder and if they are not in contradiction to the diagnostic course criteria. First episode, currently in acute episode: First manifestation of the disorder meet- ing the defining diagnostic symptom and time criteria. An acute episode is a time pe- riod in which the symptom criteria are fulfilled. First episode, currently in partial remission: Partial remission is a time period dur- ing which an improvement after a previous episode is maintained and in which the de- fining criteria of the disorder are only partially fulfilled. First episode, currently in full remission: Full remission is a period of time after a previous episode during which no disorder-specific symptoms are present.

1	First episode, currently in full remission: Full remission is a period of time after a previous episode during which no disorder-specific symptoms are present. Multiple episodes, currently in acute episode: Multiple episodes may be deter— mined after a minimum of two episodes (i.e., after a first episode, a remission and a minimum of one relapse). Multiple episodes, currently in partial remission Multiple episodes, currently in full remission Continuous: Symptoms fulfilling the diagnostic symptom criteria of the disorder are remaining for the majority of the illness course, with subthreshold symptom periods be- ing very brief relative to the overall course. Specify current severity:

1	Specify current severity: Severity is rated by a quantitative assessment of the primary symptoms of psychosis, including delusions, hallucinations, disorganized speech, abnormal psychomotor be— havior, and negative symptoms. Each of these symptoms may be rated for its current severity (most severe in the last 7 days) on a 5-point scale ranging from 0 (not present) to 4 (present and severe). (See CIinician-Rated Dimensions of Psychosis Symptom Severity in the chapter “Assessment Measures") Note: Diagnosis of schizoaffective disorder can be made without using this severity specifier. Note: For additional information on Development and Course (age-related factors), Risk and Prognostic Factors (environmental risk factors), Culture-Related Diagnostic Issues, and Gender-Related Diagnostic Issues, see the corresponding sections in schizophrenia, bipolar I and II disorders, and major depressive disorder in their respective chapters.

1	The diagnosis of schizoaffective disorder is based on the assessment of an uninterrupted period of illness during which the individual continues to display active or residual symp- toms of psychotic illness. The diagnosis is usually, but not necessarily, made during the period of psychotic illness. At some time during the period, Criterion A for schizophrenia has to be met. Criteria B (social dysfunction) and F (exclusion of autism spectrum disorder or other communication disorder of childhood onset) for schizophrenia do not have to be met. In addition to meeting Criterion A for schizophrenia, there is a major mood episode (major depressive or manic) (Criterion A for schizoaffective disorder). Because loss of in- terest or pleasure is common in schizophrenia, to meet Criterion A for schizoaffective dis— order, the major depressive episode must include pervasive depressed mood (i.e., the presence of markedly diminished interest or pleasure is not sufficient). Episodes of de- pression

1	dis— order, the major depressive episode must include pervasive depressed mood (i.e., the presence of markedly diminished interest or pleasure is not sufficient). Episodes of de- pression or mania are present for the majority of the total duration of the illness (i.e., after

1	Criterion A has been met) (Criterion C for schizoaffective disorder). To separate schizoaf- fective disorder from a depressive or bipolar disorder with psychotic features, delusions or hallucinations must be present for at least 2 weeks in the absence of a major mood epi— sode (depressive or manic) at some point during the lifetime duration of the illness (Cri- terion B for schizoaffective disorder). The symptoms must not be attributable to the effects of a substance or another medical condition (Criterion D for schizoaffective disorder).

1	for a major mood episode must be present for the majority of the total duration of the ac- tive and residual portion of the illness. Criterion C requires the assessment of mood symp- toms for the entire course of a psychotic illness, which differs from the criterion in DSM-IV, which required only an assessment of the current period of illness. If the mood symptoms are present for only a relatively brief period, the diagnosis is schizophrenia, not schizoaf- fective disorder. When deciding whether an individual’s presentation meets Criterion C, the clinician should review the total duration of psychotic illness (i.e., both active and re- of treatment with antidepressant and / or mood-stabilizing medication) accompanied the psychotic symptoms. This determination requires sufficient historical information and clinical judgment. For example, an individual with a 4-year history of active and residual symptoms of schizophrenia develops depressive and manic episodes that, taken together, do

1	information and clinical judgment. For example, an individual with a 4-year history of active and residual symptoms of schizophrenia develops depressive and manic episodes that, taken together, do not occupy more than 1 year during the 4-year history of psychotic illness. This presen- tation would not meet Criterion C.

1	In addition to the five symptom domain areas identified in the diagnostic criteria, the assessment of cognition, depression, and mania symptom domains is vital for making crit- ically important distinctions between the various schizophrenia spectrum and other psy- chotic disorders.

1	Occupational functioning is frequently impaired, but this is not a defining criterion (in contrast to schizophrenia). Restricted social contact and difficulties with self—care are as- sociated with schizoaffective disorder, but negative symptoms may be less severe and less persistent than those seen in schizophrenia. Anosog-nosia (i.e., poor insight) is also com- mon in schizoaffective disorder, but the deficits in insight may be less severe and perva- sive than those in schizophrenia. Individuals with schizoaffective disorder may be at increased risk for later developing episodes of major depressive disorder or bipolar disor- der if mood symptoms continue following the remission of symptoms meeting Criterion A for schizophrenia. There may be associated alcohol and other substance-related disorders.

1	There are no tests or biological measures that can assist in making the diagnosis of schizoaffective disorder. Whether schizoaffective disorder differs from schizophrenia with regard to associated features such as structural or functional brain abnormalities, cognitive deficits, or genetic risk factors is not clear. Schizoaffective disorder appears to be about one-third as common as schizophrenia. Life- time prevalence of schizoaffective disorder is estimated to be 0.3%. The incidence of schizoaffective disorder is higher in females than in males, mainly due to an increased in- cidence of the depressive type among females.

1	The typical age at onset of schizoaffective disorder is early adulthood, although onset can occur anywhere from adolescence to late in life. A significant number of individuals diag- nosed with another psychotic illness initially will receive the diagnosis schizoaffective dis- order later when the pattern of mood episodes has become more apparent. With the current diagnostic Criterion C, it is expected that the diagnosis for some individuals will convert from schizoaffective disorder to another disorder as mood symptoms become less prominent. The prognosis for schizoaffective disorder is somewhat better than the prog- nosis for schizophrenia but worse than the prognosis for mood disorders. Schizoaffective disorder may occur in a variety of temporal patterns. The following is a typical pattern: An individual may have pronounced auditory hallucinations and per- secutory delusions for 2 months before the onset of a prominent major depressive episode.

1	The psychotic symptoms and the full major depressive episode are then present for 3 months.

1	Then, the individual recovers completely from the major depressive episode, but the psy- chotic symptoms persist for another month before they too disappear. During this period of illness, the individual’s symptoms concurrently met criteria for a major depressive ep- isode and Criterion A for schizophrenia, and during this same period of illness, auditory hallucinations and delusions were present both before and after the depressive phase. The total period of illness lasted for about 6 months, with psychotic symptoms alone present during the initial 2 months, both depressive and psychotic symptoms present during the next 3 months, and psychotic symptoms alone present during the last month. In this in- stance, the duration of the depressive episode was not brief relative to the total duration of the psychotic disturbance, and thus the presentation qualifies for a diagnosis of schizoaf- fective disorder.

1	The expression of psychotic symptoms across the lifespan is variable. Depressive or manic symptoms can occur before the onset of psychosis, during acute psychotic episodes, during residual periods, and after cessation of psychosis. For example, an individual might present with prominent mood symptoms during the prodromal stage of schizo— phrenia. This pattern is not necessarily indicative of schizoaffective disorder, since it is the co—occurrence of psychotic and mood symptoms that is diagnostic. For an individual with symptoms that clearly meet the criteria for schizoaffective disorder but who on further fol- low-up only presents with residual psychotic symptoms (such as subthreshold psychosis and / or prominent negative symptoms), the diagnosis may be changed to schizophrenia, as the total proportion of psychotic illness compared with mood symptoms becomes more prominent. Schizoaffective disorder, bipolar type, may be more common in young adults, whereas schizoaffective disorder,

1	total proportion of psychotic illness compared with mood symptoms becomes more prominent. Schizoaffective disorder, bipolar type, may be more common in young adults, whereas schizoaffective disorder, depressive type, may be more common in older adults.

1	Genetic and physiological. Among individuals with schizophrenia, there may be an in- creased risk for schizoaffective disorder in first-degree relatives. The risk for schizoaffec- schizophrenia, bipolar disorder, or schizoaffective disorder. Cultural and socioeconomic factors must be considered, particularly when the individual and the clinician do not share the same cultural and economic background. Ideas that ap- pear to be delusional in one culture (e.g., witchcraft) may be commonly held in another. There is also some evidence in the literature for the overdiagnosis of schizophrenia com- pared with schizoaffective disorder in African American and Hispanic populations, so care must be talgen to ensure a culturally appropriate evaluation that includes both psy- chotic and affective symptoms.

1	The lifetime risk of suicide for schizophrenia and schizoaffective disorder is 5%, and the presence of depressive symptoms is correlated with a higher risk for suicide. There is ev- idence that suicide rates are higher in North American populations than in European, Eastern European, South American, and Indian populations of individuals with schizo- phrenia or schizoaffective disorder. Functional Consequences of Schizoaffective Disorder Schizoaffective disorder is associated with social and occupational dysfunction, but dys- function is not a diagnostic criterion (as it is for schizophrenia), and there is substantial variability between individuals diagnosed with schizoaffective disorder.

1	Other mental disorders and medical conditions. A wide variety of psychiatric and med- ical conditions can manifest with psychotic and mood symptoms that must be considered in the differential diagnosis of schizoaffective disorder. These include psychotic disorder due to another medical condition; delirium; major neurocognitive disorder; substance/ with psychotic features; major depressive disorder with psychotic features; depressive or bipolar disorders with catatonic features; schizotypal, schizoid, or paranoid personality disorders. Medical conditions and substance use can present with a combination of psy- chotic and mood symptoms, and thus psychotic disorder due to another medical condition needs to be excluded. Distinguishing schizoaffective disorder from schizophrenia and from depressive and bipolar disorders with psychotic features is often difficult. Criterion

1	C is designed to separate schizoaffective disorder from schizophrenia, and Criterion B is designed to distinguish schizoaffective disorder from a depressive or bipolar disorder with psychotic features. More specifically, schizoaffective disorder can be distinguished from a depressive or bipolar disorder with psychotic features due to the presence of prom- inent delusions and / or hallucinations for at least 2 weeks in the absence of a major mood episode. In contrast, in depressive or bipolar disorders with psychotic features, the psy- chotic features primarily occur during the mood episode(s). Because the relative propor- tion of mood to psychotic symptoms may change over time, the appropriate diagnosis may change from and to schizoaffective disorder (e.g., a diagnosis of schizoaffective dis- first 6 months of a persistent psychotic illness would be changed to schizophrenia if active of another mood episode).

1	Psychotic disorder due to another medical condition. Other medical conditions and substance use can manifest with a combination of psychotic and mood symptoms, and thus psychotic disorder due to another medical condition needs to be excluded.

1	Schizophrenia, bipolar, and depressive disorders. Distinguishing schizoaffective dis- order from schizophrenia and from depressive and bipolar disorders with psychotic fea- tures is often difficult. Criterion C is designed to separate schizoaffective disorder from schizophrenia, and Criterion B is designed to distinguish schizoaffective disorder from a depressive or bipolar disorder with psychotic features. More specifically, schizoaffective disorder can be distinguished from a depressive or bipolar disorder with psychotic features based on the presence of prominent delusions and / or hallucinations for at least 2 weeks in the absence of a major mood episode. In contrast, in depressive or bipolar disorder with psychotic features, the psychotic features primarily occur during the mood episode(s). Be- cause the relative proportion of mood to psychotic symptoms may change over time, the appropriate diagnosis may change from and to schizoaffective disorder. (For example, a diagnosis of

1	Be- cause the relative proportion of mood to psychotic symptoms may change over time, the appropriate diagnosis may change from and to schizoaffective disorder. (For example, a diagnosis of schizoaffective disorder for a severe and prominent major depressive episode lasting 3 months during the first 6 months of a chronic psychotic illness would be changed years without a recurrence of another mood episode.)

1	Many individuals diagnosed with schizoaffective disorder are also diagnosed with other mental disorders, especially substance use disorders and anxiety disorders. Similarly, the incidence of medical conditions is increased above base rate for the general population and leads to decreased life expectancy. A. Presence of one or both of the following symptoms: 1. Delusions. 2. Hallucinations. B. There is evidence from the history, physical examination, or laboratory findings of both (1) and (2): 1. The symptoms in Criterion A developed during or soon after substance intoxication or withdrawal or after exposure to a medication. 2. The involved substance/medication is capable of producing the symptoms in Crite- rion A. C. The disturbance is not better explained by a psychotic disorder that is not substance/ medication—induced. Such evidence of an independent psychotic disorder could in- clude the following:

1	C. The disturbance is not better explained by a psychotic disorder that is not substance/ medication—induced. Such evidence of an independent psychotic disorder could in- clude the following: The symptoms preceded the onset of the substance/medication use; the symptoms persist for a substantial period of time (e.g., about 1 month) after the cessation of acute withdrawal or severe intoxication; or there is other evidence of an indepen- dent non-substance/medication-induced psychotic disorder (e.g., a history of recur- rent non-substance/medication-re|ated episodes). D. The disturbance does not occur exclusively during the course of a delirium. E. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning.

1	E. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. Note: This diagnosis should be made instead of a diagnosis of substance intoxication or substance withdrawal only when the symptoms in Criterion A predominate in the clinical picture and when they are sufficiently severe to warrant clinical attention. Coding note: The |CD-9-CM and |CD-10—CM codes for the [specific substance/medica- tion]-induced psychotic disorders are indicated in the table below. Note that the |CD-10-

1	CM code depends on whether or not there is a comorbid substance use disorder present for the same class of substance. If a mild substance use disorder is comorbid with the sub- stance-induced psychotic disorder, the 4th position character is “1 and the clinician should record “mild [substance] use disorder” before the substance—induced psychotic disorder (e.g., “mild cocaine use disorder with cocaine-induced psychotic disorder”). It a moderate or severe substance use disorder is comorbid with the substance-induced psychotic disor- der, the 4th position character is “,"2 and the clinician should record “moderate [substance] use disorder” or “severe [substance] use disorder," depending on the severity of the co- morbid substance use disorder. If there is no comorbid substance use disorder (e.g., after a one-time heavy use of the substance). then the 4th position character is “9“ and the cli- nician should record only the substance-induced psychotic disorder.

1	With use disorder. Without disorder, moderate use Alcohol 291.9 F10.159 F10.259 F10.959 Cannabis 292.9 F12.159 F12.259 F12.959 Phencyclidine 292.9 F16.159 F16.259 F16.959 Other hallucinogen 292.9 F16.159 F16.259 F16.959 Inhalant 292.9 F18.159 F18.259 F18.959 Sedative, hypnotic, or 292.9 F13.159 F13.259 F13.959 Amphetamine (or other 292.9 F15.159 F15.259 F15.959 Cocaine 292.9 F14.159 F14.259 F14.959 Other (or unknown) substance 292.9 F19.159 F19.259 F19.959 Specify it (see Table 1 in the chapter “Substance-Related and Addictive Disorders” for diag- noses associated with substance class): With onset during intoxication: It the criteria are met for intoxication with the sub- stance and the symptoms develop during intoxication. With onset during withdrawal: If the criteria are met for withdrawal from the sub- stance and the symptoms develop during, or shortly after, withdrawal. Specify current severity:

1	With onset during withdrawal: If the criteria are met for withdrawal from the sub- stance and the symptoms develop during, or shortly after, withdrawal. Specify current severity: Severity is rated by a quantitative assessment of the primary symptoms of psychosis, including delusions, hallucinations, abnormal psychomotor behavior. and negative symptoms. Each of these symptoms may be rated for its current severity (most severe in the last 7 days) on a 5-point scale ranging from 0 (not present) to 4 (present and severe). (See CIinician-Rated Dimensions of Psychosis Symptom Severity in the chap- Note: Diagnosis of substance/medication-induced psychotic disorder can be made without using this severity specifier.

1	Note: Diagnosis of substance/medication-induced psychotic disorder can be made without using this severity specifier. lCD-9-CM. The name of the substance/medication-induced psychotic disorder begins with the specific substance (e.g., cocaine, dexamethasone) that is presumed to be causing the delusions or hallucinations. The diagnostic code is selected from the table included in the criteria set, which is based on the drug class. For substances that do not fit into any of the classes (e.g., dexamethasone), the code for ”other substance” should be used; and in cases in which a substance is judged to be an etiological factor but the specific class of sub- stance is unknown, the category "unknown substance” should be used.

1	The name of the disorder is followed by the specification of onset (i.e., onset during in- toxication, onset during withdrawal). Unlike the recording procedures for ICD-lO-CM, which combine the substance-induced disorder and substance use disorder into a single code, for ICD-9-CM a separate diagnostic code is given for the substance use disorder. For example, in the case of delusions occurring during intoxication in a man with a severe co- caine use disorder, the diagnosis is 292.9 cocaine-induced psychotic disorder, with onset during intoxication. An additional diagnosis of 304.20 severe cocaine use disorder is also given. When more than one substance is judged to play a significant role in the development of psychotic symptoms, each should be listed separately (e.g., 292.9 cannabis-induced psy- chotic disorder with onset during intoxication, with severe cannabis use disorder; 292.9 phencyclidine-induced psychotic disorder, with onset during intoxication, with mild phencyclidine use

1	psy- chotic disorder with onset during intoxication, with severe cannabis use disorder; 292.9 phencyclidine-induced psychotic disorder, with onset during intoxication, with mild phencyclidine use disorder).

1	|CD-10-CM. The name of the substance/medicati0n-induced psychotic disorder begins with the specific substance (e.g., cocaine, dexamethasone) that is presumed to be causing the delusions or hallucinations. The diagnostic code is selected from the table included in the criteria set, which is based on the drug class and presence or absence of a comorbid substance use disorder. For substances that do not fit into any of the classes (e.g., dexa- methasone), the code for ”other substance" with no comorbid substance use should be used; and in cases in which a substance is judged to be an etiological factor but the specific class of substance is unknown, the category "unknown substance” with no comorbid sub- stance use should be used.

1	When recording the name of the disorder, the comorbid substance use disorder (if any) is listed first, followed by the word ”with,” followed by the name of the substance—induced psychotic disorder, followed by the specification of onset (i.e., onset during intoxication, onset during withdrawal). For example, in the case of delusions occurring during intoxi- cation in a man with a severe cocaine use disorder, the diagnosis is F14.259 severe cocaine use disorder with cocaine-induced psychotic disorder, with onset during intoxication. A separate diagnosis of the comorbid severe cocaine use disorder is not given. If the sub- (e.g., after a one-time heavy use of the substance), no accompanying substance use disor- der is noted (e.g., F16.959 phencyclidine-induced psychotic disorder, with onset during in- toxication). When more than one substance is judged to play a significant role in the development of psychotic symptoms, each should be listed separately (e.g., F12.259 severe cannabis use

1	during in- toxication). When more than one substance is judged to play a significant role in the development of psychotic symptoms, each should be listed separately (e.g., F12.259 severe cannabis use disorder with cannabis-induced psychotic disorder, with onset during intox- ication; F16.159 mild phencyclidine use disorder with phencyclidine-induced psychotic disorder, with onset during intoxication).

1	The essential features of substance /medication-induced psychotic disorder are prominent delusions and/ or hallucinations (Criterion A) that are judged to be due to the physiolog- ical effects of a substance/medication (i.e., a drug of abuse, a medication, or a toxin expo- sure) (Criterion B). Hallucinations that the individual realizes are substance/medication- induced are not included here and instead would be diagnosed as substance intoxication or substance withdrawal with the accompanying specifier "with perceptual disturbances” (applies to alcohol withdrawal; cannabis intoxication; sedative, hypnotic, or anxiolytic withdrawal; and stimulant intoxication).

1	A substance/medication-induced psychotic disorder is distinguished from a primary psychotic disorder by considering the onset, course, and other factors. For drugs of abuse, there must be evidence from the history, physical examination, or laboratory findings of substance use, intoxication, or withdrawal. Substance/ medication-induced psychotic disorders arise during or soon after exposure to a medication or after substance intoxica- tion or withdrawal but can persist for weeks, whereas primary psychotic disorders may precede the onset of substance/medication use or may occur during times of sustained ab- stinence. Once initiated, the psychotic symptoms may continue as long as the substance/ medication use continues. Another consideration is the presence of features that are atyp- ical of a primary psychotic disorder (e.g., atypical age at onset or course). For example, the appearance of delusions de novo in a person older than 35 years without a known history of a primary psychotic

1	a primary psychotic disorder (e.g., atypical age at onset or course). For example, the appearance of delusions de novo in a person older than 35 years without a known history of a primary psychotic disorder should suggest the possibility of a substance/medication- induced psychotic disorder. Even a prior history of a primary psychotic disorder does not rule out the possibility of a substance /medication-induced psychotic disorder. In contrast, factors that suggest that the psychotic symptoms are better accounted for by a primary psychotic disorder include persistence of psychotic symptoms for a substantial period of time (i.e., a month or more) after the end of substance intoxication or acute substance with- drawal or after cessation of medication use; or a history of prior recurrent primary psy— chotic disorders. Other causes of psychotic symptoms must be considered even in an individual with substance intoxication or withdrawal, because substance use problems are not uncommon among

1	psy— chotic disorders. Other causes of psychotic symptoms must be considered even in an individual with substance intoxication or withdrawal, because substance use problems are not uncommon among individuals with non-substance/medication-induced psychotic disorders.

1	In addition to the four symptom domain areas identified in the diagnostic criteria, the assessment of cognition, depression, and mania symptom domains is vital for making crit- ically important distinctions between the various schizophrenia spectrum and other psy- chotic disorders. Psychotic disorders can occur in association with intoxication with the following classes of substances: alcohol; cannabis; hallucinogens, including phencyclidine and related sub— stances; inhalants; sedatives, hypnotics, and anxiolytics; stimulants (including cocaine); and other (or unknown) substances. Psychotic disorders can occur in association with with- drawal from the following classes of substances: alcohol; sedatives, hypnotics, and anxio- lytics; and other (or unknown) substances.

1	Some of the medications reported to evoke psychotic symptoms include anesthetics and analgesics, anticholinergic agents, anticonvulsants, antihistamines, antihypertensive and cardiovascular medications, antimicrobial medications, antiparkinsonian medica- tions, chemotherapeutic agents (e.g., cyclosporine, procarbazine), corticosteroids, gastro- intestinal medications, muscle relaxants, nonsteroidal anti-inﬂammatory medications, other over-the-counter medications (e.g., phenylephrine, pseudoephedrine), antidepres- sant medication, and disulfiram. Toxins reported to induce psychotic symptoms include anticholinesterase, organophosphate insecticides, sarin and other nerve gases, carbon monoxide, carbon dioxide, and volatile substances such as fuel or paint.

1	Prevalence of substance/medication—induced psychotic disorder in the general popula- tion is unknown. Between 7% and 25% of individuals presenting with a first episode of psychosis in different settings are reported to have substance/medication-induced psy- chotic disorder. The initiation of the disorder may vary considerably with the substance. For example, smoking a high dose of cocaine may produce psychosis within minutes, whereas days or weeks of high-dose alcohol or sedative use may be required to produce psychosis. Alco- hol-induced psychotic disorder, with hallucinations, usually occurs only after prolonged, heavy ingestion of alcohol in individuals who have moderate to severe alcohol use disorder, and the hallucinations are generally auditory in nature.

1	tures. Persecutory delusions may rapidly develop shortly after use of amphetamine or a similarly acting sympathomimetic. The hallucination of bugs or vermin crawling in or un- der the skin (formication) can lead to scratching and extensive skin excoriations. Cannabis- involves persecutory delusions, marked anxiety, emotional lability, and depersonalization. The disorder usually remits within a day but in some cases may persist for a few days.

1	The disorder usually remits within a day but in some cases may persist for a few days. Substance/medication-induced psychotic disorder may at times persist when the offend- ing agent is removed, such that it may be difficult initially to distinguish it from an indepen- dent psychotic disorder. Agents such as amphetamines, phencyclidine, and cocaine have been reported to evoke temporary psychotic states that can sometimes persist for weeks or longer despite removal of the agent and treatment with neuroleptic medication. In later life, poly— pharmacy for medical conditions and exposure to medications for parkinsonism, cardiovas- cular disease, and other medical disorders may be associated with a greater likelihood of psychosis induced by prescription medications as opposed to substances of abuse.

1	With substances for which relevant blood levels are available (e.g., blood alcohol level, other quantifiable blood levels such as digoxin), the presence of a level consistent with tox- icity may increase diagnostic certainty. Substance/medication-induced psychotic disorder is typically severely disabling and consequently is observed most frequently in emergency rooms, as individuals are often brought to the acute-care setting when it occurs. However, the disability is typically self- limited and resolves upon removal of the offending agent.

1	Substance intoxication or substance withdrawal. Individuals intoxicated with stimu- lants, cannabis, the opioid meperidine, or phencyclidine, or those withdrawing from alco- hol or sedatives, may experience altered perceptions that they recognize as drug effects. If reality testing for these experiences remains intact (i.e., the individual recognizes that the perception is substance induced and neither believes in nor acts on it), the diagnosis is not substance/medication-induced psychotic disorder. Instead, substance intoxication or substance withdrawal, with perceptual disturbances, is diagnosed (e.g., cocaine intoxica- tion, with perceptual disturbances). ”Flashback” hallucinations that can occur long after the use of hallucinogens has stopped are diagnosed as hallucinogen persisting perception disorder. If substance/medication-induced psychotic symptoms occur exclusively during the course of a delirium, as in severe forms of alcohol withdrawal, the psychotic symptoms are

1	perception disorder. If substance/medication-induced psychotic symptoms occur exclusively during the course of a delirium, as in severe forms of alcohol withdrawal, the psychotic symptoms are considered to be an associated feature of the delirium and are not diagnosed sepa- rately. Delusions in the context of a major or mild neurocognitive disorder would be di- agnosed as major or mild neurocognitive disorder, with behavioral disturbance.

1	Psychotic Disorder Due to Another Medical Condition 115 Primary psychotic disorder. A substance/medication- induced psychotic disorder is distinguished from a primary psychotic disorder, such as schizophrenia, schizoaffective disorder, delusional disorder, brief psychotic disorder, other specified schizophrenia spectrum and other psychotic disorder, or unspecified schizophrenia spectrum and other psychotic disorder, by the fact that a substance is judged to be etiologically related to the symptoms.

1	Psychotic disorder due to another medical condition. A substance/medication—induced psychotic disorder due to a prescribed treatment for a mental or medical condition must have its onset while the individual is receiving the medication (or during withdrawal, if there is a withdrawal syndrome associated with the medication). Because individuals with medical conditions often take medications for those conditions, the clinician must con- sider the possibility that the psychotic symptoms are caused by the physiological conse- quences of the medical condition rather than the medication, in which case psychotic disorder due to another medical condition is diagnosed. The history often provides the primary basis for such a judgment. At times, a change in the treatment for the medical con- dition (e.g., medication substitution or discontinuation) may be needed to determine em- pirically for that individual whether the medication is the causative agent. If the clinician has ascertained that the

1	medication substitution or discontinuation) may be needed to determine em- pirically for that individual whether the medication is the causative agent. If the clinician has ascertained that the disturbance is attributable to both a medical condition and sub- stance/medication use, both diagnoses (i.e., psychotic disorder due to another medical condition and substance/medication-induced psychotic disorder) may be given.

1	Due to Another Medical Condition A. Prominent hallucinations or delusions. B. There is evidence from the history, physical examination, or laboratory findings that the disturbance is the direct pathophysiological consequence of another medical condi- tion. C. The disturbance is not better explained by another mental disorder. D. The disturbance does not occur exclusively during the course of a delirium. E. The disturbance causes clinically significant distress or impairment in social, occupa- tional. or other important areas of functioning. Specify whether: Code based on predominant symptom: 293.81 (F06.2) With delusions: If delusions are the predominant symptom. 293.82 (F06.0) With hallucinations: If hallucinations are the predominant symptom.

1	Code based on predominant symptom: 293.81 (F06.2) With delusions: If delusions are the predominant symptom. 293.82 (F06.0) With hallucinations: If hallucinations are the predominant symptom. Coding note: Include the name of the other medical condition in the name of the mental disorder (e.g., 293.81 [F06.2] psychotic disorder due to malignant lung neoplasm, with de— lusions). The other medical condition should be coded and listed separately immediately before the psychotic disorder due to the medical condition (e.g., 162.9 [C3490] malignant lung neoplasm; 293.81 [F06.2] psychotic disorder due to malignant lung neoplasm, with delusions). Specify current severity:

1	Specify current severity: Severity is rated by a quantitative assessment of the primary symptoms of psychosis, including delusions, hallucinations, abnormal psychomotor behavior, and negative symptoms. Each of these symptoms may be rated for its current severity (most severe in the last 7 days) on a 5-point scale ranging from 0 (not present) to 4 (present and severe). (See Clinician-Rated Dimensions of Psychosis Symptom Severity in the chap- Note: Diagnosis of psychotic disorder due to another medical condition can be made without using this severity specifier. In addition to the symptom domain areas identified in the diagnostic criteria, the assess- ment of cognition, depression, and mania symptom domains is vital for making critically important distinctions between the various schizophrenia spectrum and other psychotic disorders.

1	The essential features of psychotic disorder due to another medical condition are promi- nent delusions or hallucinations that are judged to be attributable to the physiological ef— fects of another medical condition and are not better explained by another mental disorder (e.g., the symptoms are not a psychologically mediated response to a severe medical con- dition, in which case a diagnosis of brief psychotic disorder, with marked stressor, would be appropriate).

1	Hallucinations can occur in any sensory modality (i.e., visual, olfactory, gustatory, tac- tile, or auditory), but certain etiological factors are likely to evoke specific hallucinatory phenomena. Olfactory hallucinations are suggestive of temporal lobe epilepsy. Hallucina- tions may vary from simple and unformed to highly complex and organized, depending on etiological and environmental factors. Psychotic disorder due to another medical con- dition is generally not diagnosed if the individual maintains reality testing for the hallu- cinations and appreciates that they result from the medical condition. Delusions may have a variety of themes, including somatic, grandiose, religious, and, most commonly, perse- cutory. On the whole, however, associations between delusions and particular medical conditions appear to be less specific than is the case for hallucinations.

1	In determining whether the psychotic disturbance is attributable to another medical condition, the presence of a medical condition must be identified and considered to be the etiology of the psychosis through a physiological mechanism. Although there are no infallible guidelines for determining whether the relationship between the psychotic distur- bance and the medical condition is etiological, several considerations provide some guidance. One consideration is the presence of a temporal association between the onset, exacerba- tion, or remission of the medical condition and that of the psychotic disturbance. A second consideration is the presence of features that are atypical for a psychotic disorder (e.g., atypical age at onset or presence of Visual or olfactory hallucinations). The disturbance must other mental disorder (e.g., an adjustment disorder).

1	The temporal association of the onset or exacerbation of the medical condition offers the greatest diagnostic certainty that the delusions or hallucinations are attributable to a med- ical condition. Additional factors may include concomitant treatments for the underlying medical condition that confer a risk for psychosis independently, such as steroid treatment for autoimmune disorders. Prevalence rates for psychotic disorder due to another medical condition are difficult to es- timate given the wide variety of underlying medical etiologies. Lifetime prevalence has

1	Psychotic Disorder Due to Another Medical Condition 117 been estimated to range from 0.21% to 0.54%. When the prevalence findings are stratified by age group, individuals older than 65 years have a significantly greater prevalence of 0.74% compared with those in younger age groups. Rates of psychosis also vary according to the underlying medical condition; conditions most commonly associated with psy- chosis include untreated endocrine and metabolic disorders, autoimmune disorders (e.g., systemic lupus erythematosus, N—methyl—D—aspartate (NMDA) receptor autoimmune en- cephalitis), or temporal lobe epilepsy. Psychosis due to epilepsy has been further differ— entiated into ictal, postictal, and interictal psychosis. The most common of these is postictal psychosis, observed in 2%—7.8% of epilepsy patients. Among older individuals, there may be a higher prevalence of the disorder in females, although additional gender-related fea- tures are not clear and vary considerably with the gender

1	patients. Among older individuals, there may be a higher prevalence of the disorder in females, although additional gender-related fea- tures are not clear and vary considerably with the gender distributions of the underlying medical conditions.

1	Psychotic disorder due to another medical condition may be a single transient state or it may be recurrent, cycling with exacerbations and remissions of the underlying medical condition. Although treatment of the underlying medical condition often results in a res- olution of the psychosis, this is not always the case, and psychotic symptoms may persist long after the medical event (e.g., psychotic disorder due to focal brain injury). In the con- text of chronic conditions such as multiple sclerosis or chronic interictal psychosis of epi- lepsy, the psychosis may assume a long-term course.

1	The expression of psychotic disorder due to another medical condition does not differ substantially in phenomenology depending on age at occurrence. However, older age groups have a higher prevalence of the disorder, which is most likely due to the increasing medical burden associated with advanced age and the cumulative effects of deleterious exposures and age-related processes (e.g., atherosclerosis). The nature of the underlying medical conditions is likely to change across the lifespan, with younger age groups more affected by epilepsy, head trauma, autoimmune, and neoplastic diseases of early to mid- life, and older age groups more affected by stroke disease, anoxic events, and multiple sys- tem comorbidities. Underlying factors with increasing age, such as preexisting cognitive impairment as well as vision and hearing impairments, may incur a greater risk for psy- chosis, possibly by serving to lower the threshold for experiencing psychosis.

1	Course modifiers. Identification and treatment of the underlying medical condition has the greatest impact on course, although preexisting central nervous system injury may confer a worse course outcome (e.g., head trauma, cerebrovascular disease).

1	The diagnosis of psychotic disorder due to another medical condition depends on the clin- ical condition of each individual, and the diagnostic tests will vary according to that con- dition. A variety of medical conditions may cause psychotic symptoms. These include neurological conditions (e.g., neoplasms, cerebrovascular disease, Huntington‘s disease, multiple sclerosis, epilepsy, auditory or visual nerve injury or impairment, deafness, migraine, central nervous system infections), endocrine conditions (e.g., hyper- and hypo- thyroidism, hyper- and hypoparathyroidism, hyper- and hypoadrenocorticism), metabolic conditions (e.g., hypoxia, hypercarbia, hypoglycemia), ﬂuid or electrolyte imbalances, hepatic or renal diseases, and autoimmune disorders with central nervous system involve- ment (e.g., systemic lupus erythematosus). The associated physical examination findings, laboratory findings, and patterns of prevalence or onset reﬂect the etiological medical condition.

1	Suicide risk in the context of psychotic disorder due to another medical condition is not clearly delineated, although certain conditions such as epilepsy and multiple sclerosis are associated with increased rates of suicide, which may be further increased in the presence of psychosis. Functional Consequences of Psychotic Disorder Due to Another Medical Condition Functional disability is typically severe in the context of psychotic disorder due to another medical condition but will vary considerably by the type of condition and likely improve with successful resolution of the condition.

1	Delirium. Hallucinations and delusions commonly occur in the context of a delirium; however, a separate diagnosis of psychotic disorder due to another medical condition is not given if the disturbance occurs exclusively during the course of a delirium. Delusions in the context of a major or mild neurocognitive disorder would be diagnosed as major or mild neurocognitive disorder, with behavioral disturbance.

1	Substance/medication-induced psychotic disorder. If there is evidence of recent or prolonged substance use (including medications with psychoactive effects), withdrawal from a substance, or exposure to a toxin (e.g., LSD [lysergic acid diethylamide] intoxica- tion, alcohol withdrawal), a substance/medication—induced psychotic disorder should be considered. Symptoms that occur during or shortly after (i.e., within 4 weeks) of substance intoxication or withdrawal or after medication use may be especially indicative of a sub- stance-induced psychotic disorder, depending on the character, duration, or amount of the substance used. If the clinician has ascertained that the disturbance is due to both a medical condition and substance use, both diagnoses (i.e., psychotic disorder due to an- given.

1	Psychotic disorder. Psychotic disorder due to another medical condition must be distin- guished from a psychotic disorder (e.g., schizophrenia, delusional disorder, schizoaffective disorder) or a depressive or bipolar disorder, with psychotic features. In psychotic disor- ders and in depressive or bipolar disorders, with psychotic features, no specific and direct causative physiological mechanisms associated with a medical condition can be demon- strated. Late age at onset and the absence of a personal or family history of schizophrenia or delusional disorder suggest the need for a thorough assessment to rule out the diagno- sis of psychotic disorder due to another medical condition. Auditory hallucinations that involve voices speaking complex sentences are more characteristic of schizophrenia than of psychotic disorder due to a medical condition. Other types of hallucinations (e.g., vi- sual, olfactory) commonly signal a psychotic disorder due to another medical condition or a

1	than of psychotic disorder due to a medical condition. Other types of hallucinations (e.g., vi- sual, olfactory) commonly signal a psychotic disorder due to another medical condition or a substance/medication-induced psychotic disorder.

1	Psychotic disorder due to another medical condition in individuals older than 80 years is associated with concurrent major neurocognitive disorder (dementia). Catatonia can occur in the context of several disorders, including neurodevelopmental, psychotic, bipolar, depressive disorders, and other medical conditions (e.g., cerebral folate deficiency, rare autoimmune and paraneoplastic disorders. The manual does not treat catatonia as an independent class but recognizes a) catatonia associated with another men— tal disorder (i.e., a neurodevelopmental, psychotic disorder, a bipolar disorder, a depres— sive disorder, or other mental disorder), b) catatonic disorder due to another medical condition, and c) unspecified catatonia.

1	Catatonia is defined by the presence of three or more of 12 psychomotor features in the diagnostic criteria for catatonia associated with another mental disorder and catatonic dis- order due to another medical condition. The essential feature of catatonia is a marked psy- chomotor disturbance that may involve decreased motor activity, decreased engagement during interview or physical examination, or excessive and peculiar motor activity. The clinical presentation of catatonia can be puzzling, as the psychomotor disturbance may range from marked unresponsiveness to marked agitation. Motoric immobility may be se- vere (stupor) or moderate (catalepsy and waxy ﬂexibility). Similarly, decreased engage- ment may be severe (mutism) or moderate (negativism). Excessive and peculiar motor behaviors can be complex (e.g., stereotypy) or simple (agitation) and may include echola- lia and echopraxia. In extreme cases, the same individual may wax and wane between de- creased and excessive motor

1	can be complex (e.g., stereotypy) or simple (agitation) and may include echola- lia and echopraxia. In extreme cases, the same individual may wax and wane between de- creased and excessive motor activity. The seemingly opposing clinical features and variable manifestations of the diagnosis contribute to a lack of awareness and decreased recognition of catatonia. During severe stages of catatonia, the individual may need care- ful supervision to avoid self—harm or harming others. There are potential risks from mal- nutrition, exhaustion, hyperpyrexia and self—inﬂicted injury.

1	293.89 (F06.1) A. The clinical picture is dominated by three (or more) of the following symptoms: Stupor (i.e., no psychomotor activity; not actively relating to environment). Catalepsy (i.e., passive induction of a posture held against gravity). Waxy flexibility (i.e., slight, even resistance to positioning by examiner). Mutism (i.e., no, or very little. verbal response [exclude if known aphasial). Negativism (i.e., opposition or no response to instructions or external stimuli). Posturing (i.e., spontaneous and active maintenance of a posture against gravity). Mannerism (i.e., odd, circumstantial caricature of normal actions). Stereotypy (i.e., repetitive, abnormally frequent, non-goal-directed movements). Agitation, not influenced by external stimuli. 10. Grimacing. 11. Echolalia (i.e., mimicking another‘s speech). 12. Echopraxia (i.e., mimicking another’s movements).

1	Agitation, not influenced by external stimuli. 10. Grimacing. 11. Echolalia (i.e., mimicking another‘s speech). 12. Echopraxia (i.e., mimicking another’s movements). Coding note: Indicate the name of the associated mental disorder when recording the name of the condition (i.e., 293.89 [F06.1] catatonia associated with major depressive dis- order). Code first the associated mental disorder (e.g., neurodevelopmental disorder, brief psychotic disorder, schizophreniform disorder. schizophrenia, schizoaffective disorder, bipolar disorder, major depressive disorder, or other mental disorder) (e.g., 295.70 [F251] schizoaffective disorder, depressive type; 293.89 [F06.1] catatonia associated with schizoaffective disorder).

1	Catatonia associated with another mental disorder (catatonia specifier) may be used when criteria are met for catatonia during the course of a neurodevelopmental, psychotic, bipo- lar, depressive, or other mental disorder. The catatonia specifier is appropriate when the clinical picture is characterized by marked psychomotor disturbance and involves at least three of the 12 diagnostic features listed in Criterion A. Catatonia is typically diagnosed in an inpatient setting and occurs in up to 35% of individuals with schizophrenia, but the ma- jority of catatonia cases involve individuals with depressive or bipolar disorders. Before the catatonia specifier is used in neurodevelopmental, psychotic, bipolar, depressive, or other mental disorders, a wide variety of other medical conditions need to be ruled out; these conditions include, but are not limited to, medical conditions due to infectious, met- abolic, or neurological conditions (see ”Catatonic Disorder Due to Another Medical Con-

1	to be ruled out; these conditions include, but are not limited to, medical conditions due to infectious, met- abolic, or neurological conditions (see ”Catatonic Disorder Due to Another Medical Con- dition”). Catatonia can also be a side effect of a medication (see the chapter ”Medication-

1	Induced Movement Disorders and Other Adverse Effects of Medication”). Because of the seriousness of the complications, particular attention should be paid to the possibility that the catatonia is attributable to 333.92 (G210) neuroleptic malignant syndrome. Diagnostic Criteria 293.89 (F06.1) A. The clinical picture is dominated by three (or more) of the following symptoms: 1. Stupor (i.e., no psychomotor activity; not actively relating to environment). Catalepsy (i.e., passive induction of a posture held against gravity). Waxy flexibility (i.e., slight, even resistance to positioning by examiner). Mutism (i.e., no, or very little, verbal response [Note: not applicable if there is an established aphasia]). Negativism (i.e., opposition or no response to instructions or external stimuli). Posturing (i.e., spontaneous and active maintenance of a posture against gravity). Mannerism (i.e., odd, circumstantial caricature of normal actions).

1	Posturing (i.e., spontaneous and active maintenance of a posture against gravity). Mannerism (i.e., odd, circumstantial caricature of normal actions). Stereotypy (i.e., repetitive, abnormally frequent, non-goaI-directed movements). 9. Agitation, not influenced by external stimuli. 10. Grimacing. 11. Echolalia (i.e., mimicking another’s speech). 12. Echopraxia (i.e., mimicking another’s movements). B. There is evidence from the history, physical examination, or laboratory findings that the disturbance is the direct pathophysiological consequence of another medical condition. . The disturbance is not better explained by another mental disorder (e.g., a manic episode). . The disturbance does not occur exclusively during the course of a delirium. . The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning.

1	. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. Coding note: Include the name of the medical condition in the name of the mental disor- der (e.g., 293.89 [F06.1]) catatonic disorder due to hepatic encephalopathy). The other medical condition should be coded and listed separately immediately before the cata- tonic disorder due to the medical condition (e.g., 572.2 [K7190] hepatic encephalopathy; 293.89 [F06.1] catatonic disorder due to hepatic encephalopathy).

1	The essential feature of catatonic disorder due to another medical condition is the presence of catatonia that is judged to be attributed to the physiological effects of another medical condition. Catatonia can be diagnosed by the presence of at least three of the 12 clinical fea- tures in Criterion A. There must be evidence from the history, physical examination, or laboratory findings that the catatonia is attributable to another medical condition (Crite- rion B). The diagnosis is not given if the catatonia is better explained by another mental disorder (e.g., manic episode) (Criterion C) or if it occurs exclusively during the course of a delirium (Criterion D).

1	A variety of medical conditions may cause catatonia, especially neurological conditions (e.g., neoplasms, head trauma, cerebrovascular disease, encephalitis) and metabolic con- ditions (e.g., hypercalcemia, hepatic encephalopathy, homocystinuria, diabetic ketoacido- sis). The associated physical examination findings, laboratory findings, and patterns of prevalence and onset reﬂect those of the etiological medical condition.

1	A separate diagnosis of catatonic disorder due to another medical condition is not given if the catatonia occurs exclusively during the course of a delirium or neuroleptic malignant syndrome. If the individual is currently taking neuroleptic medication, consideration should be given to medication-induced movement disorders (e.g., abnormal positioning may be due to neuroleptic-induced acute dystonia) or neuroleptic malignant syndrome (e.g., catatonic-like features may be present, along with associated vital sign and / or labo- ratory abnormalities). Catatonic symptoms may be present in any of the following five psychotic disorders: brief psychotic disorder, schizophreniform disorder, schizophrenia, schizoaffective disorder, and substance/medication-induced psychotic disorder. It may also be present in some of the neurodevelopmental disorders, in all of the bipolar and de- pressive disorders, and in other mental disorders.

1	This category applies to presentations in which symptoms characteristic of catatonia cause clinically significant distress or impairment in social, occupational, or other impor- tant areas of functioning but either the nature of the underlying mental disorder or other medical condition is unclear, full criteria for catatonia are not met, or there is insufficient information to make a more specific diagnosis (e.g., in emergency room settings). Coding note: Code first 781.99 (R29.818) other symptoms involving nervous and muscu- loskeletal systems, followed by 293.89 (F06.1) unspecified catatonia. 298.8 (F28)

1	Coding note: Code first 781.99 (R29.818) other symptoms involving nervous and muscu- loskeletal systems, followed by 293.89 (F06.1) unspecified catatonia. 298.8 (F28) This category applies to presentations in which symptoms characteristic of a schizophre- pairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the schizophrenia spectrum and other psychotic disorders diagnostic class. The other specified schizophrenia spectrum and oth- er psychotic disorder category is used in situations in which the clinician chooses to com- municate the specific reason that the presentation does not meet the criteria for any specific schizophrenia spectrum and other psychotic disorder. This is done by recording “oth- er specified schizophrenia spectrum and other psychotic disorder“ followed by the specific reason (e.g., “persistent auditory hallucinations").

1	Examples of presentations that can be specified using the “other specified” designation include the following: 1. Perslstent auditory hallucinations occurring in the absence of any other features. 2. Delusions with significant overlapping mood episodes: This includes persistent delusions with periods of overlapping mood episodes that are present for a substantial portion of the delusional disturbance (such that the criterion stipulating only brief mood disturbance in delusional disorder is not met). 3. Attenuated psychosis syndrome: This syndrome is characterized by psychotic-like symptoms that are below a threshold for full psychosis (e.g., the symptoms are less severe and more transient, and insight is relatively maintained).

1	4. Deiusionai symptoms in partner of individual with delusionai disorder: In the context of a relationship, the delusional material from the dominant partner provides content for delusional belief by the individual who may not otherwise entirely meet cri- teria for delusional disorder. 298.9 (F29)

1	298.9 (F29) This category applies to presentations in which symptoms characteristic of a schizophre— pairment in social, occupational. or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the schizophrenia spectrum and oth- er psychotic disorders diagnostic class. The unspecified schizophrenia spectrum and oth- er psychotic disorder category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for a specific schizophrenia spectrum and other psychotic disorder, and includes presentations in which there is insufficient informa- tion to make a more specific diagnosis (e.g., in emergency room settings). Bl polar and related disorders are separated from the depressive disorders in

1	Bl polar and related disorders are separated from the depressive disorders in DSM-5 and placed between the chapters on schizophrenia spectrum and other psychotic disorders and depressive disorders in recognition of their place as a bridge between the two diagnostic classes in terms of symptomatology, family history, and genetics. The di- agnoses included in this chapter are bipolar I disorder, bipolar II disorder, cyclothymic disorder, substance/medication—induced bipolar and related disorder, bipolar and relat- ed disorder dueto another medical condition, other specified bipolar and related disor- der, and unspecified bipolar and related disorder.

1	The bipolar I disorder criteria represent the modern understanding of the classic manic-depressive disorder or affective psychosis described in the nineteenth century, dif- fering from that classic description only to the extent that neither psychosis nor the lifetime experience of a major depressive episode is a requirement. However, the vast majority of individuals whose symptoms meet the criteria for a fully syndromal manic episode also experience major depressive episodes during the course of their lives. Bipolar II disorder, requiring the lifetime experience of at least one episode of major de- pression and at least one hypomanic episode, is no longer thought to be a "milder" condition than bipolar I disorder, largely because of the amount of time individuals with this con- dition spend in depression and because the instability of mood experienced by individuals with bipolar II disorder is typically accompanied by serious impairment in work and social functioning.

1	The diagnosis of cyclothymic disorder is given to adults who experience at least 2 years (for children, a full year) of both hypomanic and depressive periods without ever fulfilling the criteria for an episode of mania, hypomania, or major depression. A large number of substances of abuse, some prescribed medications, and several medical conditions can be associated with manic-like phenomena. This fact is recognized in the diagnoses of substance/medication-induced bipolar and related disorder and bipo- lar and related disorder due to another medical condition.

1	The recognition that many individuals, particularly children and, to a lesser extent, ad- olescents, experience bipolar-like phenomena that do not meet the criteria for bipolar I, bi- polar II, or cyclothymic disorder is reflected in the availability of the other specified bipolar and related disorder category. Indeed, specific criteria for a disorder involving short—duration hypomania are provided in Section III in the hope of encouraging further study of this disorder. For a diagnosis of bipolar l disorder, it is necessary to meet the following criteria for a manic episode. The manic episode may have been preceded by and may be followed by hypo- manic or major depressive episodes. A. A distinct period of abnormally and persistently elevated, expansive, or irritable mood and abnormally and persistently increased goaI-directed activity or energy, lasting at least 1 week and present most of the day, nearly every day (or any duration if hospi- talization is necessary).

1	B. During the period of mood disturbance and increased energy or activity, three (or more) of the following symptoms (four if the mood is only irritable) are present to a sig- nificant degree and represent a noticeable change from usual behavior: Inflated seIf-esteem or grandiosity. Decreased need for sleep (e.g., feels rested after only 3 hours of sleep). More talkative than usual or pressure to keep talking. Flight of ideas or subjective experience that thoughts are racing. Distractibility (i.e., attention too easily drawn to unimportant or irrelevant external stimuli), as reported or observed. Increase in goal-directed activity (either socially, at work or school, or sexually) or psychomotor agitation (i.e., purposeless non-goaI-directed activity). 7. Excessive involvement in activities that have a high potential for painful conse- quences (e.g., engaging in unrestrained buying sprees, sexual indiscretions, or foolish business investments). ?‘PP’NT‘ .0)

1	?‘PP’NT‘ .0) C. The mood disturbance is sufficiently severe to cause marked impairment in social or occupational functioning or to necessitate hospitalization to prevent harm to self or oth- ers. or there are psychotic features. D. The episode is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication, other treatment) or to another medical condition. Note: A full manic episode that emerges during antidepressant treatment (e.g., medi- cation, electroconvulsive therapy) but persists at a fully syndromal level beyond the physiological effect of that treatment is sufficient evidence for a manic episode and, therefore, a bipolar | diagnosis. Note: Criteria A—D constitute a manic episode. At least one lifetime manic episode is re- quired for the diagnosis of bipolar l disorder.

1	Note: Criteria A—D constitute a manic episode. At least one lifetime manic episode is re- quired for the diagnosis of bipolar l disorder. A. A distinct period of abnormally and persistently elevated, expansive, or irritable mood and abnormally and persistently increased activity or energy, lasting at least 4 consec- utive days and present most of the day, nearly every day. B. During the period of mood disturbance and increased energy and activity, three (or more) of the following symptoms (four if the mood is only irritable) have persisted. rep- resent a noticeable change from usual behavior, and have been present to a significant degree: 1. Inflated seli-esteem or grandiosity. Decreased need for sleep (e.g., feels rested after only 3 hours of sleep). More talkative than usual or pressure to keep talking. Flight of ideas or subjective experience that thoughts are racing.

1	Decreased need for sleep (e.g., feels rested after only 3 hours of sleep). More talkative than usual or pressure to keep talking. Flight of ideas or subjective experience that thoughts are racing. Distractibility (i.e., attention too easily drawn to unimportant or irrelevant external stimuli), as reported or observed. 6. increase in goal-directed activity (either socially, at work or school, or sexually) or psychomotor agitation. 7. Excessive involvement in activities that have a high potential for painful conse- quences (e.g., engaging in unrestrained buying sprees, sexual indiscretions, or foolish business investments). . The episode is associated with an unequivocal change in functioning that is uncharac- teristic of the individual when not symptomatic. . The disturbance in mood and the change in functioning are observable by others.

1	. The disturbance in mood and the change in functioning are observable by others. . The episode is not severe enough to cause marked impairment in social or occupa- tional functioning or to necessitate hospitalization. If there are psychotic features, the episode is, by definition, manic. The episode is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication, other treatment). Note: A full hypomanic episode that emerges during antidepressant treatment (e.g., medication, electroconvulsive therapy) but persists at a fully syndromal level beyond the physiological effect of that treatment is sufficient evidence for a hypomanic episode diagnosis. However, caution is indicated so that one or two symptoms (particularly in- creased irritability, edginess, or agitation following antidepressant use) are not taken as sufficient for diagnosis of a hypomanic episode, nor necessarily indicative of a bi- polar diathesis.

1	Note: Criteria A-F constitute a hypomanic episode. Hypomanic episodes are common in bipolar I disorder but are not required for the diagnosis of bipolar | disorder. A. B. C. Five (or more) of the following symptoms have been present during the same 2-week period and represent a change from previous functioning; at least one of the symptoms is either (1) depressed mood or (2) loss of interest or pleasure. Note: Do not include symptoms that are clearly attributable to another medical condi- tion. 1. Depressed mood most of the day, nearly every day, as indicated by either subjec- tive report (e.g., feels sad, empty, or hopeless) or observation made by others (e.g., appears tearful). (Note: In children and adolescents, can be irritable mood.) 2. Markedly diminished interest or pleasure in all, or almost all, activities most of the day, nearly every day (as indicated by either subjective account or observation).

1	3. Significant weight loss when not dieting or weightgain (e.g., a change of more than 5% of body weight in a month), or decrease or increase in appetite nearly every day. (Note: In children, consider failure to make expected weight gain.) 4. Insomnia or hypersomnia nearly every day. 5. Psychomotor agitation or retardation nearly every day (observable by others; not merely subjective feelings of restlessness or being slowed down). 6. Fatigue or loss of energy nearly every day. 7. Feelings of worthlessness or excessive or inappropriate guilt (which may be delu- sional) nearly every day (not merely seIf-reproach or guilt about being sick). 8. Diminished ability to think or concentrate, or indecisiveness, nearly every day (ei- ther by subjective account or as observed by others). 9. Recurrent thoughts of death (not just fear of dying), recurrent suicidal ideation with- out a specific plan, or a suicide attempt or a specific plan for committing suicide.

1	9. Recurrent thoughts of death (not just fear of dying), recurrent suicidal ideation with- out a specific plan, or a suicide attempt or a specific plan for committing suicide. The symptoms cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. The episode is not attributable to the physiological effects of a substance or another medical condition. Note: Criteria A-C constitute a major depressive episode. Major depressive episodes are common in bipolar I disorder but are not required for the diagnosis of bipolar I disorder.

1	Note: Criteria A-C constitute a major depressive episode. Major depressive episodes are common in bipolar I disorder but are not required for the diagnosis of bipolar I disorder. Note: Responses to a significant loss (e.g., bereavement, financial ruin, losses from a natural disaster, a serious medical illness or disability) may include the feelings of intense sadness, rumination about the loss, insomnia, poor appetite, and weight loss noted in Cri- terion A, which may resemble a depressive episode. Although such symptoms may be un- derstandable or considered appropriate to the loss, the presence of a major depressive episode in addition to the normal response to a significant loss should also be carefully considered. This decision inevitably requires the exercise of clinical judgment based on the indiyidual’s history and the cultural norms for the expression of distress in the context of loss.

1	A. Criteria have been met for at least one manic episode (Criteria A—D under "Manic Ep- isode" above). B. The occurrence of the manic and major depressive episode(s) is not better explained by schizoaffective disorder, schizophrenia, schizophreniform disorder, delusional dis- order, or other specified or unspecified schizophrenia spectrum and other psychotic disorder. The diagnostic code for bipolar | disorder is based on type of current or most recent epi- sode and its status with respect to current severity, presence of psychotic features, and remission status. Current severity and psychotic features are only indicated if full criteria are currently met for a manic or major depressive episode. Remission specifiers are only indicated if the full criteria are not currently met for a manic, hypomanic, or major depres- sive episode. Codes are as follows: Mild (p. 154) 296.41 NA 296.51 NA (F31.11) (F3131) Moderate (p. 154) 296.42 NA 296.52 NA (F31.12) (F3132)

1	Mild (p. 154) 296.41 NA 296.51 NA (F31.11) (F3131) Moderate (p. 154) 296.42 NA 296.52 NA (F31.12) (F3132) Severe (p. 154) 296.43 NA 296.53 NA (F31.13) (F31.4) 1In distinguishing grief from a major depressive episode (MDE), it is useful to consider that in grief the predominant affect is feelings of emptiness and loss, while in MDE it is persistent depressed mood and the inability to anticipate happiness or pleasure. The dysphoria in grief is likely to decrease in intensity over days to weeks and occurs in waves, the so-called pangs of grief. These waves tend to be associated with thoughts or reminders of the deceased. The depressed mood of a MDE is more persistent and not tied to specific thoughts or preoccupations.

1	The pain of grief may be accompanied by positive emotions and humor that are uncharacteristic of the pervasive unhappiness and misery characteristic of a major depressive episode. The thought content associated with grief generally features a preoccupation with thoughts and memories of the deceased, rather than the self—critical or pessimistic ruminations seen in a MDE.

1	In grief, self—esteem is generally preserved, whereas in a MDE, feelings of worthlessness and self— loathing are common. If self-derogatory ideation is present in grief, it typically involves per- ceived failings vis-a—vis the deceased (e.g., not visiting frequently enough, not telling the deceased how much he or she was loved). If a bereaved individual thinks about death and dying, such thoughts are generally focused on the deceased and possibly about ”joining" the deceased, whereas in a major depressive episode such thoughts are focused on ending one’s own life because of feeling worthless, undeserving of life, or unable to cope with the pain of depression. With psychotic 296.44 NA 296.54 NA features‘“ (F312) (F31.5) (p. 152) In partial 296.45 296.45 296.55 NA remission (p. 154) (F31.73) (F31.73) (F3175) In full remission 296.46 296.46 296.56 NA (p. 154) (F31.74) (F31.74) (F31.76)

1	In partial 296.45 296.45 296.55 NA remission (p. 154) (F31.73) (F31.73) (F3175) In full remission 296.46 296.46 296.56 NA (p. 154) (F31.74) (F31.74) (F31.76) Unspecified 296.40 296.40 296.50 NA (F31 9.) (F319) (F31. 9) :Severity and psychotic specifiers do not apply; code 296. 40 (F31. 0) for cases not in remission. Severity, psychotic, and remission specifiers do not apply. Code 296. 7 (F31. 9). “*If psychotic features are present, code the’ ’with psychotic features" specifier irrespective of epi- sode severity. In recording the name of a diagnosis, terms should be listed in the following order: bipolar I disorder, type of current or most recent episode, severity/psychotic/remission specifiers, followed by as many specifiers without codes as apply to the current or most recent epi- sode. Specify: Wlth anxious distress (p. 149) With mixed features (pp. 149—150) With rapid cycling (pp. 150—151) With meianchoiic features (p. 151) With atypical features (pp. 151—152)

1	Specify: Wlth anxious distress (p. 149) With mixed features (pp. 149—150) With rapid cycling (pp. 150—151) With meianchoiic features (p. 151) With atypical features (pp. 151—152) With mood—congruent psychotic features (p. 152) With mood-incongruent psychotic features (p. 152) With catatonia (p. 152). Coding note: Use additional code 293.89 (F06.1). With peripartum onset (pp. 152—153) Wlth seasonal pattern (pp. 153—154) The essential feature of a manic episode is a distinct period during which there is an ab- normally, persistently elevated, expansive, or irritable mood and persistently increased activity or energy that is present for most of the day, nearly every day, for a period of at least 1 week (or any duration if hospitalization is necessary), accompanied by at least three additional symptoms from Criterion B. If the mood is irritable rather than elevated or ex- pansive, at least four Criterion B symptoms must be present.

1	Mood in a manic episode is often described as euphoric, excessively cheerful, high, or ”feeling on top of the world.” In some cases, the mood is of such a highly infectious quality that it is easily recognized as excessive and may be characterized by unlimited and hap- hazard enthusiasm for interpersonal, sexual, or occupational interactions. For example, the individual may spontaneously start extensive conversations with strangers in public.

1	Often the predominant mood is irritable rather than elevated, particularly when the indi- vidual’s wishes are denied or if the individual has been using substances. Rapid shifts in mood over brief periods of time may occur and are referred to as lability (i.e., the alterna- tion among euphoria, dysphoria, and irritability). In children, happiness, silliness and "goofiness" are normal in the context of special occasions; however, if these symptoms are recurrent, inappropriate to the context, and beyond what is expected for the developmen- tal level of the child, they may meet Criterion A. If the happiness is unusual for a child (i.e., distinct from baseline), and the mood change occurs at the same time as symptoms that meet Criterion B for mania, diagnostic certainty is increased; however, the mood change must be accompanied by persistently increased activity or energy levels that are obvious to those who know the child well.

1	During the manic episode, the individual may engage in multiple overlapping new projects. The projects are often initiated with little knowledge of the topic, and nothing seems out of the individual’s reach. The increased activity levels may manifest at unusual hours of the day.

1	Inﬂated self-esteem is typically present, ranging from uncritical self-confidence to marked grandiosity, and may reach delusional proportions (Criterion B1). Despite lack of any partic~ ular experience or talent, the individual may embark on complex tasks such as writing a novel or seeking publicity for some impractical invention. Grandiose delusions (e.g., of having a special relationship to a famous person) are common. In children, overestimation of abilities and belief that, for example, they are the best at a sport or the smartest in the class is normal; however, when such beliefs are present despite clear evidence to the contrary or the child at- tempts feats that are clearly dangerous and, most important, represent a change from the child’s normal behavior, the grandiosity criterion should be considered satisfied.

1	One of the most common features is a decreased need for sleep (Criterion B2) and is distinct from insomnia in which the individual wants to sleep or feels the need to sleep but is unable. The individual may sleep little, if at all, or may awaken several hours earlier than usual, feeling rested and full of energy. When the sleep disturbance is severe, the individ- ual may go for days without sleep, yet not feel tired. Often a decreased need for sleep her- alds the onset of a manic episode.

1	Speech can be rapid, pressured, loud, and difficult to interrupt (Criterion 83). Individ- uals may talk continuously and without regard for others’ wishes to communicate, often in an intrusive manner or without concern for the relevance of what is said. Speech is sometimes characterized by jokes, puns, amusing irrelevancies, and theatricality, with dramatic mannerisms, singing, and excessive gesturing. Loudness and forcefulness of speech often become more important than what is conveyed. If the individual’s mood is more irritable than expansive, speech may be marked by complaints, hostile comments, or angry tirades, particularly if attempts are made to interrupt the individual. Both Criterion A and Criterion B symptoms may be accompanied by symptoms of the opposite (i.e., de- pressive) pole (see ”with mixed features" specifier, pp. 149—150).

1	A and Criterion B symptoms may be accompanied by symptoms of the opposite (i.e., de- pressive) pole (see ”with mixed features" specifier, pp. 149—150). Often the individual’s thoughts race at a rate faster than they can be expressed through speech (Criterion B4). Frequently there is ﬂight of ideas evidenced by a nearly continuous ﬂow of accelerated speech, with abrupt shifts from one topic to another. When ﬂight of ideas is se- vere, speech may become disorganized, incoherent, and particularly distressful t0 the individ- ual. Sometimes thoughts are experienced as so crowded that it is very difficult to speak. Distractibility (Criterion BS) is evidenced by an inability to censor immaterial external stimuli (e.g., the interviewer’s attire, background noises or conversations, furnishings in the room) and often prevents individuals experiencing mania from holding a rational con— versation or attending to instructions.

1	The increase in goal-directed activity often consists of excessive planning and partici- pation in multiple activities, including sexual, occupational, political, or religious activi- ties. Increased sexual drive, fantasies, and behavior are often present. Individuals in a manic episode usually show increased sociability (e.g., renewing old acquaintances or calling or contacting friends or even strangers), without regard to the intrusive, domineering, and demanding nature of these interactions. They often display psychomotor agitation or rest- lessness (i.e., purposeless activity) by pacing or by holding multiple conversations simulta- neously. Some individuals write excessive letters, e-mails, text messages, and so forth, on many different topics to friends, public figures, or the media.

1	The increased activity criterion can be difficult to ascertain in children; however, when the child takes on many tasks simultaneously, starts devising elaborate and unrealistic plans for projects, develops previously absent and developmentally inappropriate sexual preoccupations (not accounted for by sexual abuse or exposure to sexually explicit mate— rial), then Criterion B might be met based on clinical judgment. It is essential to determine whether the behavior represents a change from the child’s baseline behavior; occurs most of the day, nearly every day for the requisite time period; and occurs in temporal associa- tion with other symptoms of mania.

1	The expansive mood, excessive optimism, grandiosity, and poor judgment often lead to reckless involvement in activities such as spending sprees, giving away possessions, reckless driving, foolish business investments, and sexual promiscuity that is unusual for the individual, even though these activities are likely to have catastrophic consequences (Criterion B7). The individual may purchase many unneeded items without the money to pay for them and, in some cases, give them away. Sexual behavior may include infidelity or indiscriminate sexual encounters with strangers, often disregarding the risk of sexually transmitted diseases or interpersonal consequences.

1	The manic episode must result in marked impairment in social or occupational func- tioning or require hospitalization to prevent harm to self or others (e.g., financial losses, i1- legal activities, loss of employment, self—injurious behavior). By definition, the presence of psychotic features during a manic episode also satisfies Criterion C.

1	Manic symptoms or syndromes that are attributable to the physiological effects of a drug of abuse (e.g., in the context of cocaine or amphetamine intoxication), the side effects of medications or treatments (e.g., steroids, L-dopa, antidepressants, stimulants), or an- other medical condition do not count toward the diagnosis of bipolar I disorder. However, a fully syndromal manic episode that arises during treatment (e.g., with medications, elec- troconvulsive therapy, light therapy) or drug use and persists beyond the physiological ef- fect of the inducing agent (i.e., after a medication is fully out of the individual’s system or the effects of electroconvulsive therapy would be expected to have dissipated completely) is sufficient evidence for a manic episode diagnosis (Criterion D). Caution is indicated so that one or two symptoms (particularly increased irritability, edginess, or agitation follow- ing antidepressant use) are not taken as sufficient for diagnosis of a manic or

1	Caution is indicated so that one or two symptoms (particularly increased irritability, edginess, or agitation follow- ing antidepressant use) are not taken as sufficient for diagnosis of a manic or hypomanic episode, nor necessarily an indication of a bipolar disorder diathesis. It is necessary to meet criteria for a manic episode to make a diagnosis of bipolar I disorder, but it is not re- quired to have hypomanic or major depressive episodes. However, they may precede or follow a manic episode. Full descriptions of the diagnostic features of a hypomanic epi- sode may be found within the text for bipolar II disorder, and the features of a major de- pressive episode are described within the text for major depressive disorder.

1	During a manic episode, individuals often do not perceive that they are ill or in need of treat- ment and vehemently resist efforts to be treated. Individuals may change their dress, makeup, or personal appearance to a more sexually suggestive or ﬂamboyant style. Some perceive a sharper sense of smell, hearing, or vision. Gambling and antisocial behaviors may accompany the manic episode. Some individuals may become hostile and physically threatening to others and, when delusional, may become physically assaultive or suicidal. Catastrophic conse- quences of a manic episode (e.g., involuntary hospitalization, difficulties with the law, serious financial difficulties) often result from poor judgment, loss of insight, and hyperactivity. Mood may shift very rapidly to anger or depression. Depressive symptoms may occur' during a manic episode and, if present, may last moments, hours, or, more rarely, days (see "with mixed features” specifier, pp. 149—150).

1	The 12-month prevalence estimate in the continental United States was 0.6% for bipolar I disorder as defined in DSM-IV. Twelve-month prevalence of bipolar I disorder across 11 countries ranged from 0.0% to 0.6%. The lifetime male-to-female prevalence ratio is ap- proximately 1.1:1.

1	Mean age at onset of the first manic, hypomanic, or major depressive episode is approxi- mately 18 years for bipolar I disorder. Special considerations are necessary'to detect the di- agnosis in children. Since children of the same chronological age may be at different developmental stages, it is difficult to define with precision what is “normal” or ”ex- pected” at any given point. Therefore, each child should be judged according to his or her own baseline. Onset occurs throughout the life cycle, including first onsets in the 60s or 705. Onset of manic symptoms (e.g., sexual or social disinhibition) in late mid-life or late- life should prompt consideration of medical conditions (e.g., frontotemporal neurocogni- tive disorder) and of substance ingestion or withdrawal.

1	More than 90% of individuals who have a single manic episode go on to have recurrent mood episodes. Approximately 60% of manic episodes occur immediately before a major depressive episode. Individuals with bipolar I disorder who have multiple (four or more) mood episodes (major depressive, manic, or hypomanic) within 1 year receive the speci- fier “with rapid cycling.” Environmental. Bipolar disorder is more common in high-income than in low-income countries (1.4 vs. 0.7%). Separated, divorced, or widowed individuals have higher rates of bipolar I disorder than do individuals who are married or have never been married, but the direction of the association is unclear.

1	Genetic and physiological. A family history of bipolar disorder is one of the strongest and most consistent risk factors for bipolar disorders. There is an average lO-fold increased risk among adult relatives of individuals with bipolar I and bipolar II disorders. Magnitude of risk increases with degree of kinship. Schizophrenia and bipolar disorder likely share a ge- netic origin, reﬂected in familial co-aggregation of schizophrenia and bipolar disorder. Course modifiers. After an individual has a manic episode with psychotic features, subse- quent manic episodes are more likely to include psychotic features. Incomplete inter- episode recovery is more common when the current episode is accompanied by mood— incongruent psychotic features.

1	Little information exists on specific cultural differences in the expression of bipolar I dis- order. One possible explanation for this may be that diagnostic instruments are often translated and applied in different cultures with no transcultural validation. In one US. study, 12-month prevalence of bipolar I disorder was significantly lower for Afro-Carib- beans than for African Americans or whites. Females are more likely to experience rapid cycling and mixed states, and to have patterns of comorbidity that differ from those of males, including higher rates of lifetime eating disor- ders. Females with bipolar I or II disorder are more likely to experience depressive symptoms than males. They also have a higher lifetime risk of alcohol use disorder than are males and a much greater likelihood of alcohol use disorder than do females in the general population.

1	The lifetime risk of suicide in individuals with bipolar disorder is estimated to be at least 15 times that of the general population. In fact, bipolar disorder may account for one-quar- ter of all completed suicides. A past history of suicide attempt and percent days spent de- pressed in the past year are associated with greater risk of suicide attempts or completions. Functional Consequences of Bipoiar I Disorder

1	Functional Consequences of Bipoiar I Disorder Although many individuals with bipolar disorder return to a fully functional level be- tween episodes, approximately 30% show severe impairment in work role function. Func- tional recovery lags substantially behind recovery from symptoms, especially with respect to occupational recovery, resulting in lower socioeconomic status despite equivalent lev- els of education when compared with the general population. Individuals with bipolar I disorder perform more poorly than healthy individuals on cognitive tests. Cognitive im- pairments may contribute to vocational and interpersonal difficulties and persist through the lifespan, even during euthymic periods.

1	Major depressive disorder. Major depressive disorder may also be accompanied by hy- pomanic or manic symptoms (i.e., fewer symptoms or for a shorter duration than required for mania or hypomania). When the individual presents in an episode of major depression, one must depend on corroborating history regarding past episodes of mania or hypoma- nia. Symptoms of irritability may be associated with either major depressive disorder or bipolar disorder, adding to diagnostic complexity. Other bipolar disorders. Diagnosis of bipolar I disorder is differentiated from bipolar II disorder by determining whether there have been any past episodes of mania. Other spec- and II disorders by considering whether either the episodes involving manic or hypo- manic symptoms or the episodes of depressive symptoms fail to meet the full criteria for those conditions. Bipolar disorder due to another medical condition may be distinguished from bipolar

1	Bipolar disorder due to another medical condition may be distinguished from bipolar I and II disorders by identifying, based on best clinical evidence, a causally related medical condition. Generalized anxiety disorder, panic disorder, posttraumatic stress disorder, or other anxiety disorders. These disorders need to be considered in the differential diagnosis as either the primary disorder or, in some cases, a comorbid disorder. A careful history of symptoms is needed to differentiate generalized anxiety disorder from bipolar disorder, as anxious ruminations may be mistaken for racing thoughts, and efforts to minimize anx- ious feelings may be taken as impulsive behavior. Similarly, symptoms of posttraumatic stress disorder need to be differentiated from bipolar disorder. It is helpful to assess the ep- isodic nature of the symptoms described, as well as to consider symptom triggers, in mak- ing this differential diagnosis.

1	Substance/medication-induced bipolar disorder. Substance use disorders may mani- fest with substance.medication-induced manic symptoms that must be distinguished from bipolar I disorder; response to mood stabilizers during a substance/medication- induced mania may not necessarily be diagnostic for bipolar disorder. There may be sub- stantial overlap in view of the tendency for individuals with bipolar I disorder to overuse substances during an episode. A primary diagnosis of bipolar disorder must be estab- lished based on symptoms that remain once substances are no longer being used.

1	Attention-deficitlhyperactivity disorder. This disorder may be misdiagnosed as bipolar disorder, especially in adolescents and children. Many symptoms overlap with the symp- toms of mania, such as rapid speech, racing thoughts, distractibility, and less need for sleep. The "double counting" of symptoms toward both ADHD and bipolar disorder can be avoided if the clinician clarifies whether the symptom(s) represents a distinct episode. Personality disorders. Personality disorders such as borderline personality disorder may have substantial symptomatic overlap with bipolar disorders, since mood lability and impulsivity are common in both conditions. Symptoms must represent a distinct ep- isode, and the noticeable increase over baseline required for the diagnosis of bipolar dis- order must be present. A diagnosis of a personality disorder should not be made during an untreated mood episode.

1	Disorders with prominent irritability. In individuals with severe irritability, particularly children and adolescents, care must be taken to apply the diagnosis of bipolar disorder only to those who have had a clear episode of mania or hypomania—that is, a distinct time period, of the required duration, during which the irritability was clearly different from the individual’s baseline and was accompanied by the onset of Criterion B symptoms. When a child’s irritability is persistent and particularly severe, the diagnosis of disruptive mood dysregulation disorder would be more appropriate. Indeed, when any child is being assessed for mania, it is essential that the symptoms represent a clear change from the child’s typical behavior.

1	Co-occurring mental disorders are common, with the most frequent disorders being any anxiety disorder (e.g., panic attacks, social anxiety disorder [social phobia], specific plio- bia), occurring in approximately three-fourths of individuals; ADHD, any disruptive, im- pulse-control, or conduct disorder (e.g., intermittent explosive disorder, oppositional defiant disorder, conduct disorder), and any substance use disorder (e.g., alcohol use dis- order) occur in over half of individuals with bipolar I disorder. Adults with bipolar I dis- order have high rates of serious and / or untreated co-occurring medical conditions. Metabolic syndrome and migraine are more common among individuals with bipolar dis- order than in the general population. More than half of individuals whose symptoms meet criteria for bipolar disorder have an alcohol use disorder, and those with both disorders are at greater risk for suicide attempt. Diagnostic Criteria 296.89 (F31.81)

1	Diagnostic Criteria 296.89 (F31.81) For a diagnosis of bipolar II disorder, it is necessary to meet the following criteria for a cur- rent or past hypomanic episode and the following criteria for a current or past major de- pressive episode: A. A distinct period of abnormally and persistently elevated, expansive, or irritable mood and abnormally and persistently increased activity or energy, lasting at least 4 consec- utive days and present most of the day, nearly every day. B. During the period of mood disturbance and increased energy and activity, three (or more) of the following symptoms have persisted (four if the mood is only irritable), represent a no- ticeable change from usual behavior, and have been present to a significant degree: 1. Inflated seIf-esteem or grandiosity. 2. Decreased need for sleep (e.g., feels rested after only 3 hours of sleep). 3. More talkative than usual or pressure to keep talking.

1	2. Decreased need for sleep (e.g., feels rested after only 3 hours of sleep). 3. More talkative than usual or pressure to keep talking. 4. Flight of ideas or subjective experience that thoughts are racing. 5. Distractibility (i.e., attention too easily drawn to unimportant or irrelevant external stimuli), as reported or observed. 6. Increase in goaI-directed activity (either socially, at work or school, or sexually) or psychomotor agitation. 7. Excessive involvement in activities that have a high potential for painful conse- quences (e.g., engaging in unrestrained buying sprees, sexual indiscretions, or foolish business investments). C. The episode is associated with an unequivocal change in functioning that is uncharac- teristic of the individual when not symptomatic. D. The disturbance in mood and the change in functioning are observable by others.

1	D. The disturbance in mood and the change in functioning are observable by others. E. The episode is not severe enough to cause marked Impairment in social or occupa- tional functioning or to necessitate hospitalization. If there are psychotic features, the episode is, by definition, manic. F. The episode is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication or other treatment). Note: A full hypomanic episode that emerges during antidepressant treatment (e.g., medication. electroconvulsive therapy) but persists at a fully syndromal level beyond the physiological effect of that treatment is sufficient evidence for a hypomanic episode diagnosis. However, caution is indicated so that one or two symptoms (particularly in- creased irritability, edginess, or agitation following antidepressant use) are not taken as sufficient for diagnosis of a hypomanic episode, nor necessarily indicative of a bi- polar diathesis.

1	A. Five (or more) of the following symptoms have been present during the same 2-week period and represent a change from previous functioning; at least one of the symptoms is either (1) depressed mood or (2) loss of interest or pleasure. Note: Do not include symptoms that are clearly attributable to a medical condition. 1. Depressed mood most of the day, nearly every day, as indicated by either subjec- tive report (e.g., feels sad, empty, or hopeless) or observation made by others (e.g., appears tearful). (Note: In children and adolescents, can be irritable mood.) 2. Markedly diminished interest or pleasure in all, or almost all, activities most of the day, nearly every day (as indicated by either subjective account or observation).

1	3. Significant weight loss when not dieting or weight gain (e.g., a change of more than 5% of body weight in a month), or decrease or increase in appetite nearly every day. (Note: In children, consider failure to make expected weight gain.) 4. Insomnia or hypersomnia nearly every day. 5. Psychomotor agitation or retardation nearly every day (observable by others; not merely subjective feelings of restlessness or being slowed down). 6. Fatigue or loss of energy nearly every day. 7. Feelings of worthlessness or excessive or inappropriate guilt (which may be delu- sional) nearly every day (not merely self-reproach or guilt about being sick). 8. Diminished ability to think or concentrate, or indecisiveness, nearly every day (ei- ther by subjective account or as observed by others). 9. Recurrent thoughts of death (not just fear of dying), recurrent suicidal ideation with- out a specific plan, a suicide attempt, or a specific plan for committing suicide.

1	9. Recurrent thoughts of death (not just fear of dying), recurrent suicidal ideation with- out a specific plan, a suicide attempt, or a specific plan for committing suicide. B. The symptoms cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. C. The episode is not attributable to the physiological effects of a substance or another medical condition. Note: Criteria A—C above constitute a major depressive episode. Note: Responses to a significant loss (e.g., bereavement, financial ruin, losses from a nat- ural disaster, a serious medical illness or disability) may include the feelings of intense sad- ness, rumination about the loss, insomnia, poor appetite, and weight loss noted in Criterion

1	A, which may resemble a depressive episode. Although such symptoms may be under- standable or considered appropriate to the loss, the presence of a major depressive episode in addition to the normal response to a significant loss should be carefully considered. This decision inevitably requires the exercise of clinical judgment based on the individual’s history and the cultural norms for the expression of distress in the context of loss.1 A. Criteria have been met for at least one hypomanic episode (Criteria A—F under “Hypo— “Major Depressive Episode" above). B. There has never been a manic episode. C. The occurrence of the hypomanic episode(s) and major depressive episode(s) is not better explained by schizoaftective disorder, schizophrenia, schizophreniform disor- der, delusional disorder, or other specified or unspecified schizophrenia spectrum and other psychotic disorder.

1	D. The symptoms of depression or the unpredictability caused by frequent alternation be- tween periods of depression and hypomania causes clinically significant distress or im- pairment in social, occupational, or other important areas of functioning. Bipolar |I disorder has one diagnostic code: 296.89 (F31.81). Its status with respect to cur- rent severity, presence of psychotic features, course, and other specifiers cannot be coded but should be indicated in writing (e.g., 296.89 [F3181] bipolar II disorder, current episode depressed, moderate severity, with mixed features; 296.89 [F3181] bipolar II dis- order, most recent episode depressed, in partial remission). Specify current or most recent episode: Specify if: With anxious distress (p. 149)

1	Specify current or most recent episode: Specify if: With anxious distress (p. 149) With mixed features (pp. 149—150) 1 In distinguishing grief from a major depressive episode (MDE), it is useful to consider that in grief the predominant affect is feelings of emptiness and loss, while in a MDE it is persistent depressed mood and the inability to anticipate happiness or pleasure. The dysphoria in grief is likely to decrease in intensity over days to weeks and occurs in waves, the so—called pangs of grief. These waves tend to be associated with thoughts or reminders of the deceased. The depressed mood of a

1	MDE is more persistent and not tied to specific thoughts or preoccupations. The pain of grief may be accompanied by positive emotions and humor that are uncharacteristic of the pervasive unhap- piness and misery characteristic of a MDE. The thought content associated with grief generally fea- tures a preoccupation with thoughts and memories of the deceased, rather than the self—critical or pessimistic ruminations seen in a MDE. In grief, seIf-esteem is generally preserved, whereas in a

1	MDE feelings of worthlessness and self—loathing are common. If self—derogatory ideation is present in grief, it typically involves perceived failings vis-a-vis the deceased (e.g., not visiting frequently enough, not telling the deceased how much he or she was loved). If a bereaved individual thinks about death and dying, such thoughts are generally focused on the deceased and possibly about “joining” the deceased, whereas in a MDE such thoughts are focused on ending one’s own life because of feeling worthless, undeserving of life, or unable to cope with the pain of depression. With rapid cycling (pp. 150—151) With mood-congruent psychotic features (p. 152) With mood-incongruent psychotic features (p. 152) With catatonia (p. 152). Coding note: Use additional code 293.89 (F06.1). With peripartum onset (pp. 152—153) With seasonal pattern (pp. 153-154): Applies only to the pattern of major depressive episodes.

1	With peripartum onset (pp. 152—153) With seasonal pattern (pp. 153-154): Applies only to the pattern of major depressive episodes. Specify course it full criteria for a mood episode are not currently met: In partial remission (p. 154) In full remission (p. 154) Specify severity if full criteria for a mood episode are currently met: Mild (p. 154) Moderate (p. 154) Severe (p. 154)

1	Bipolar II disorder is characterized by a clinical course of recurring mood episodes con- sisting of one or more major depressive episodes (Criteria A—C under “Major Depressive sode”). The major depressive episode must last at least 2 weeks, and the hypomanic epi- sode must last at least 4 days, to meet the diagnostic criteria. During the mood episode(s), the requisite number of symptoms must be present most of the day, nearly every day, and represent a noticeable change from usual behavior and functioning. The presence of a manic episode during the course of illness precludes the diagnosis of bipolar II disorder (Criterion B under ”Bipolar II Disorder"). Episodes of substance/medication-induced de- senting the physiological effects of a medication, other somatic treatments for depression, drugs of abuse, or toxin exposure) or of depressive and related disorder due to another medical condition or bipolar and related disorder due to another medical condition do not count toward a

1	drugs of abuse, or toxin exposure) or of depressive and related disorder due to another medical condition or bipolar and related disorder due to another medical condition do not count toward a diagnosis of bipolar II disorder unless they persist beyond the physiolog- ical effects of the treatment or substance and then meet duration criteria for an episode. In addition, the episodes must not be better accounted for by schizoaffective disorder and are not superimposed on schizophrenia, schizophreniform disorder, delusional disorder, or terion C under ”Bipolar II Disorder”). The depressive episodes or hypomanic ﬂuctuations must cause clinically significant distress or impairment in social, occupational, or other important areas of functioning (Criterion D under ”Bipolar II Disorder”); however, for hy- pomanic episodes, this requirement does not have to be met. A hypomanic episode that causes significant impairment would likely qualify for the diagnosis of manic episode and, therefore,

1	for hy- pomanic episodes, this requirement does not have to be met. A hypomanic episode that causes significant impairment would likely qualify for the diagnosis of manic episode and, therefore, for a lifetime diagnosis of bipolar I disorder. The recurrent major depressive ep- isodes are often more frequent and lengthier than those occurring in bipolar I disorder.

1	Individuals with bipolar II disorder typically present to a clinician during a major de- pressive episode and are unlikely to complain initially of hypomania. Typically, the hy- pomanic episodes themselves do not cause impairment. Instead, the impairment results from the major depressive episodes or from a persistent pattern of unpredictable mood changes and ﬂuctuating, unreliable interpersonal or occupational functioning. Individu- als with bipolar II disorder may not view the hypomanic episodes as pathological or dis- advantageous, although others may be troubled by the individual’s erratic behavior. Clinical information from other informants, such as close friends or relatives, is often use- ful in establishing the diagnosis of bipolar II disorder.

1	A hypomanic episode should not be confused with the several days of euthyrnia and re- stored energy or activity that may follow remission of a major depressive episode. Despite the substantial differences in duration and severity between a manic and hypomanic episode, bi- polar II disorder is not a ”milder form" of bipolar I disorder. Compared with individuals with bipolar I disorder, individuals with bipolar II disorder have greater chronicity of illness and spend, on average, more time in the depressive phase of their illness, which can be severe and / or disabling. Depressive symptoms co-occurring with a hypomanic episode or hypomanic symptoms co-occurring with a depressive episode are common in individuals with bipolar H disorder and are overrepresented in females, particularly hypomania with mixed features. In- dividuals experiencing hypomania with mixed features may not label their symptoms as hy- pomania, but instead experience them as depression with increased energy or

1	hypomania with mixed features. In- dividuals experiencing hypomania with mixed features may not label their symptoms as hy- pomania, but instead experience them as depression with increased energy or irritability.

1	A common feature of bipolar II disorder is impulsivity, which can contribute to suicide at- tempts and substance use disorders. Impulsivity may also stem from a concurrent person- ality disorder, substance use disorder, anxiety disorder, another mental disorder, or a medical condition. There may be heightened levels of creativity in some individuals with a bipolar disorder. However, that relationship may be nonlinear; that is, greater lifetime creative accomplishments have been associated with milder forms of bipolar disorder, and higher creativity has been found in unaffected family members. The individual’s attach- ment to heightened creativity during hypomanic episodes may contribute to ambivalence about seeking treatment or undermine adherence to treatment. The 12-month prevalence of bipolar II disorder, internationally, is 0.3%. In the United

1	The 12-month prevalence of bipolar II disorder, internationally, is 0.3%. In the United States, 12-month prevalence is 0.8%. The prevalence rate of pediatric bipolar II disorder is difficult to establish. DSM-IV bipolar I, bipolar II, and bipolar disorder not otherwise spec- ified yield a combined prevalence rate of 1.8% in US. and non-U.S. community samples, with higher rates (2.7% inclusive) in youths age 12 years or older.

1	Although bipolar II disorder can begin in late adolescence and throughout adulthood, av- erage age at onset is the mid-205, which is slightly later than for bipolar I disorder but ear- lier than for major depressive disorder. The illness most often begins with a depressive episode and is not recognized as bipolar II disorder until a hypomanic episode occurs; this happens in about 12% of individuals with the initial diagnosis of major depressive disor- der. Anxiety, substance use, or eating disorders may also precede the diagnosis, compli- cating its detection. Many individuals experience several episodes of major depression prior to the first recognized hypomanic episode.

1	The number of lifetime episodes (both hypomanic and major depressive episodes) tends to be higher for bipolar II disorder than for major depressive disorder or bipolar I disorder. However, individuals with bipolar I disorder are actually more likely to experi- ence hypomanic symptoms than are individuals with bipolar II disorder.The interval between mood episodes in the course of bipolar II disorder tends to decrease as the indi- vidual ages. While the hypomanic episode is the feature that defines bipolar II disorder, depressive episodes are more enduring and disabling over time. Despite the predomi- nance of depression, once a hypomanic episode has occurred, the diagnosis becomes bi- polar II disorder and never reverts to major depressive disorder.

1	Approximately 5%—15% of individuals with bipolar II disorder have multiple (four or more) mood episodes (hypomanic or major depressive) within the previous 12 months. If this pattern is present, it is noted by the specifier ”with rapid cycling.” By definition, psy- chotic symptoms do not occur in hypomanic episodes, and they appear to be less frequent in the major depressive episodes in bipolar II disorder than in those of bipolar I disorder. Switching from a depressive episode to a manic or hypomanic episode (with or with- out mixed features) may occur, both spontaneously and during treatment for depression. About 5%—15% of individuals with bipolar II disorder will ultimately develop a manic ep- isode, which changes the diagnosis to bipolar I disorder, regardless of subsequent course.

1	Making the diagnosis in children is often a challenge, especially in those with irritabil— ity and hyperarousal that is nonepisodic (i.e., lacks the well-demarcated periods of altered mood). Nonepisodic irritability in youth is associated with an elevated risk for anxiety dis- orders and major depressive disorder, but not bipolar disorder, in adulthood. Persistently irritable youths have lower familial rates of bipolar disorder than do youths who have bi- polar disorder. For a hypomanic episode to be diagnosed, the child’s symptoms must ex- ceed what is expected in a given environment and culture for the child’s developmental stage. Compared with adult onset of bipolar II disorder, childhood or adolescent onset of the disorder may be associated with a more severe lifetime course. The 3-year incidence rate of first-onset bipolar II disorder in adults older than 60 years is 0.34%. However, dis— tinguishing individuals older than 60 years with bipolar II disorder by late versus early age

1	incidence rate of first-onset bipolar II disorder in adults older than 60 years is 0.34%. However, dis— tinguishing individuals older than 60 years with bipolar II disorder by late versus early age at onset does not appear to have any clinical utility.

1	Genetic and physiological. The risk of bipolar II disorder tends to be highest among rel- atives of individuals with bipolar II disorder, as opposed to individuals with bipolar I dis- order or major depressive disorder. There may be genetic factors inﬂuencing the age at onset for bipolar disorders. Course modifiers. A rapid-cycling pattern is associated with a poorer prognosis. Return to previous level of social function for individuals with bipolar II disorder is more likely for individuals of younger age and with less severe depression, suggesting adverse effects of prolonged illness on recovery. More education, fewer years of illness, and being mar- ried are independently associated with functional recovery in individuals with bipolar disorder, even after diagnostic type (I vs. II), current depressive symptoms, and presence of psychiatric comorbidity are taken into account.

1	Whereas the gender ratio for bipolar I disorder is equal, findings on gender differences in bipolar II disorder are mixed, differing by type of sample (i.e., registry, community, or clinical) and country of origin. There is little to no evidence of bipolar gender differences, whereas some, but not all, clinical samples suggest that bipolar II disorder is more com- mon in females than in males, which may reﬂect gender differences in treatment seeking or other factors.

1	Patterns of illness and comorbidity, however, seem to differ by gender, with females being more likely than males to report hypomania with mixed depressive features and a rapid-cycling course. Childbirth may be a specific trigger for a hypomanic episode, which can occur in 10%—20% of females in nonclinical populations and most typically in the early postpartum period. Distinguishing hypomania from the elated mood and reduced sleep that normally accompany the birth of a child may be challenging. Postpartum hypomania may foreshadow the onset of a depression that occurs in about half of females who expe- rience postpartum ”highs.” Accurate detection of bipolar II disorder may help in estab- lishing appropriate treatment of the depression, which may reduce the risk of suicide and infanticide.

1	Suicide risk is high in bipolar II disorder. Approximately one-third of individuals with bi- polar II disorder report a lifetime history of suicide attempt. The prevalence rates of life- time attempted suicide in bipolar II and bipolar I disorder appear to be similar (32.4% and 36.3%, respectively). However, the lethality of attempts, as defined by a lower ratio of at- tempts to completed suicides, may be higher in individuals with bipolar II disorder com- pared with individuals with bipolar I disorder. There may be an association between genetic markers and increased risk for suicidal behavior in individuals with bipolar dis- order, including a 65-fold higher risk of suicide among first-degree relatives of bipolar II probands compared with those with bipolar I disorder. Functional Consequences of Bipolar II Disorder

1	Functional Consequences of Bipolar II Disorder Although many individuals with bipolar II disorder return to a fully functional level be- tween mood episodes, at least 15% continue to have some inter-episode dysfunction, and 20% transition directly into another mood episode without inter-episode recovery. Func- tional recovery lags substantially behind recovery from symptoms of bipolar II disorder, especially in regard to occupational recovery, resulting in lower socioeconomic status de- spite equivalent levels of education with the general population. Individuals with bipolar

1	II disorder perform more poorly than healthy individuals on cognitive tests and, with the exception of memory and semantic ﬂuency, have similar cognitive impairment as do in- dividuals with bipolar I disorder. Cognitive impairments associated with bipolar II disor- der may contribute to vocational difficulties. Prolonged unemployment in individuals with bipolar disorder is associated with more episodes of depression, older age, increased rates of current panic disorder, and lifetime history of alcohol use disorder. Major depressive disorder. Perhaps the most challenging differential diagnosis to con- sider is major depressive disorder, which may be accompanied by hypomanic or manic symptoms that do not meet full criteria (i.e., either fewer symptoms or a shorter duration than required for a hypomanic episode). This is especially true in evaluating individuals with symptoms of irritability, which may be associated with either major depressive dis- order or bipolar II disorder.

1	Cyclothymic disorder. In cyclothymic disorder, there are numerous periods of hypo- manic symptoms and numerous periods of depressive symptoms that do not meet symp— tom or duration criteria for a major depressive episode. Bipolar II disorder is distinguished from cyclothymic disorder by the presence of one or more major depressive episodes. If a major depressive episode occurs after the first 2 years of cyclothymic disorder, the addi- tional diagnosis of bipolar II disorder is given. Schizophrenia spectrum and other related psychotic disorders. Bipolar II disorder must be distinguished from psychotic disorders (e.g., schizoaffective disorder, schizophrenia, and delusional disorder). Schizophrenia, schizoaffective disorder, and delusional disor- der are all characterized by periods of psychotic symptoms that occur in the absence of prominent mood symptoms. Other helpful considerations include the accompanying symptoms, previous course, and family history.

1	Panic disorder or other anxiety disorders. Anxiety disorders need to be considered in the differential diagnosis and may frequently be present as co-occurring disorders. Substance use disorders. Substance use disorders are included in the differential diag~ nosis. Attention-deficit/hyperactivity disorder. Attention-deficit/hyperactivity disorder (ADHD) may be misdiagnosed as bipolar II disorder, especially in adolescents and children. Many symptoms of ADHD, such as rapid speech, racing thoughts, distractibility, and less need for sleep, overlap with the symptoms of hypomania. The double counting of symptoms to- ward both ADHD and bipolar II disorder can be avoided if the clinician clarifies whether the symptoms represent a distinct episode and if the noticeable increase over baseline re- quired for the diagnosis of bipolar II disorder is present.

1	Personality disorders. The same convention as applies for ADHD also applies when evaluating an individual for a personality disorder such as borderline personality disor- der, since mood lability and impulsivity are common in both personality disorders and bi- polar II disorder. Symptoms must represent a distinct episode, and the noticeable increase over baseline required for the diagnosis of bipolar II disorder must be present. A diagnosis the lifetime history supports the presence of a personality disorder. Other bipolar disorders. Diagnosis of bipolar II disorder should be differentiated from ing the presence of fully syndromal hypomania and depression.

1	Bipolar II disorder is more often than not associated with one or more co—occurring mental disorders, with anxiety disorders being the most common. Approximately 60% of individ- uals with bipolar II disorder have three or more co-occurring mental disorders; 75% have an anxiety disorder; and 37% have a substance use disorder. Children and adolescents with bipolar II disorder have a higher rate of co-occurring anxiety disorders compared with those with bipolar I disorder, and the anxiety disorder most often predates the bi- polar disorder. Anxiety and substance use disorders occur in individuals with bipolar II disorder at a higher rate than in the general population. Approximately 14% of individuals with bipolar II disorder have at least one lifetime eating disorder, with binge-eating dis- order being more common than bulimia nervosa and anorexia nervosa.

1	These commonly co-occurring disorders do not seem to follow a course of illness that is truly independent from that of the bipolar disorder, but rather have strong associations with mood states. For example, anxiety and eating disorders tend to associate most with depressive symptoms, and substance use disorders are moderately associated with manic symptoms. Diagnostic Criteria 301.13 (F34.0) A. For at least 2 years (at least 1 year in children and adolescents) there have been nu- merous periods with hypomanic symptoms that do not meet criteria for a hypomanic episode and numerous periods with depressive symptoms that do not meet criteria for a major depressive episode. B. During the above 2-year period (1 year in children and adolescents), the hypomanic and depressive periods have been present for at least half the time and the individual has not been without the symptoms for more than 2 months at a time.

1	C. Criteria for a major depressive, manic, or hypomanic episode have never been met. D. The symptoms in Criterion A are not better explained by schizoaffective disorder. schizophrenia, schizophreniform disorder, delusional disorder. or other specified or un- specified schizophrenia spectrum and other psychotic disorder. E. The symptoms are not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication) or another medical condition (e.g., hyperthyroidism). F. The symptoms cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. Specify it: With anxious distress (see p. 149)

1	Specify it: With anxious distress (see p. 149) The essential feature of cyclothymic disorder is a chronic, ﬂuctuating mood disturbance involving numerous periods of hypomanic symptoms and periods of depressive symp- toms that are distinct from each other (Criterion A). The hypomanic symptoms are of insufficient number, severity, pervasiveness, or duration to meet full criteria for a hypo- manic episode, and the depressive symptoms are of insufficient number, severity, perva— siveness, or duration to meet full criteria for a major depressive episode. During the initial 2-year period (1 year for children or adolescents), the symptoms must be persistent (pres— ent more days than not), and any symptom—free intervals last no longer than 2 months (Criterion B). The diagnosis of cyclothymic disorder is made only if the criteria for a major depressive, manic, or hypomanic episode have never been met (Criterion C).

1	If an individual with cyclothymic disorder subsequently (i.e., after the initial 2 years in adults or 1 year in children or adolescents) experiences a major depressive, manic, or hy- pomanic episode, the diagnosis changes to major depressive disorder, bipolar I disorder, or other specified or unspecified bipolar and related disorder (subclassified as hypomanic episode without prior major depressive episode), respectively, and the cyclothymic disor- der diagnosis is dropped.

1	The cyclothymic disorder diagnosis is not made if the pattern of mood swings is better explained by schizoaffective disorder, schizophrenia, schizophreniform disorder, delu- sional disorder, or other specified and unspecified schizophrenia spectrum and other psychotic disorders (Criterion D), in which case the mood symptoms are considered asso- ciated features of the psychotic disorder. The mood disturbance must also not be attribut- able to the physiological effects of a substance (e.g., a drug of abuse, a medication) or another medical condition (e.g., hyperthyroidism) (Criterion E). Although some individ- uals may function particularly well during some of the periods of hypomania, over the prolonged course of the disorder, there must be clinically significant distress or impair- ment in social, occupational, or other important areas of functioning as a result of the mood disturbance (Criterion F). The impairment may develop as a result of prolonged pe- riods of cyclical, often

1	in social, occupational, or other important areas of functioning as a result of the mood disturbance (Criterion F). The impairment may develop as a result of prolonged pe- riods of cyclical, often unpredictable mood changes (e.g., the individual may be regarded as temperamental, moody, unpredictable, inconsistent, or unreliable).

1	The lifetime prevalence of cyclothymic disorder is approximately 0.4%—1%. Prevalence in mood disorders clinics may range from 3% to 5%. In the general population, cyclothymic disorder is apparently equally common in males and females. In clinical settings, females with cyclothymic disorder may be more likely to present for treatment than males. Cyclothymic disorder usually begins in adolescence or early adult life and is sometimes considered to reﬂect a temperamental predisposition to other disorders in this chapter.

1	Cyclothymic disorder usually begins in adolescence or early adult life and is sometimes considered to reﬂect a temperamental predisposition to other disorders in this chapter. Cyclothymic disorder usually has an insidious onset and a persistent course. There is a 15%—50% risk that an individual with cyclothymic disorder will subsequently develop bi- polar I disorder or bipolar II disorder. Onset of persistent, ﬂuctuating hypomanic and de- pressive symptoms late in adult life needs to be clearly differentiated from bipolar and related disorder due to another medical condition and depressive disorder due to another medical condition (e. g., multiple sclerosis) before the cyclothymic disorder diagnosis IS as- signed Among children with cyclothymic disorder, the mean age at onset of symptoms 15 6. 5 years of age.

1	Genetic and physiological. Major depressive disorder, bipolar I disorder, and bipolar II disorder are more common among first-degree biological relatives of individuals with cyclo- thymic disorder than in the general population. There may also be an increased familial risk of substance-related disorders. Cyclothymic disorder may be more common in the first—degree biological relatives of individuals with bipolar I disorder than in the general population.

1	Bipolar and related disorder due to another medical condition and depressive disorder due to another medical condition. The diagnosis of bipolar and related disorder due to another medical condition or depressive disorder due to another medical condition is made when the mood disturbance is judged to be attributable to the physiological effect of a specific, usually chronic medical condition (e.g., hyperthyroidism). This determination is based on the history, physical examination, or laboratory findings. If it is judged that the hypomanic and depressive symptoms are not the physiological consequence of the med- ical condition, then the primary mental disorder (i.e., cyclothymic disorder) and the med- ical condition are coded. For example, this would be the case if the mood symptoms are considered to be the psychological (not the physiological) consequence of having a chronic medical condition, or if there is no etiological relationship between the hypomanic and de- pressive symptoms

1	to be the psychological (not the physiological) consequence of having a chronic medical condition, or if there is no etiological relationship between the hypomanic and de- pressive symptoms and the medical condition.

1	tion-induced depressive disorder. Substance/medicati0n-induced bipolar and related disorder and substance/medication-induced depressive disorder are distinguished from cyclothymic disorder by the judgment that a substance/ medication (especially stimu- lants) is etiologically related to the mood disturbance. The frequent mood swings in these disorders that are suggestive of cyclothymic disorder usually resolve following cessation of substance/medication use. Bipolar l disorder, with rapid cycling, and bipolar II disorder, with rapid cycling. Both disorders may resemble cyclothymic disorder by virtue of the frequent marked shifts in mood. By definition, in cyclothymic disorder the criteria for a major depressive, manic, or hypomanic episode has never been met, whereas the bipolar I disorder and bipolar II disorder specifier "with rapid cycling” requires that full mood episodes be present.

1	Borderline personality disorder. Borderline personality disorder is associated with marked shifts in mood that may suggest cyclothymic disorder. If the criteria are met for both disorders, both borderline personality disorder and cyclothymic disorder may be di- agnosed. Substance-related disorders and sleep disorders (i.e., difficulties in initiating and main- taining sleep) may be present in individuals with cyclothymic disorder. Most children conditions; they are more likely than other pediatric patients with mental disorders to have comorbid attention-deficit/hyperactivity disorder. A. A prominent and persistent disturbance in mood that predominates in the clinical picture and is characterized by elevated, expansive, or irritable mood, with or without depressed mood, or markedly diminished interest or pleasure in all, or almost all, activities.

1	B. There is evidence from the history, physical examination, or laboratory findings of both (1) and (2): 1. The symptoms in Criterion A developed during or soon after substance intoxication or withdrawal or after exposure to a medication. 2. The involved substance/medication is capable of producing the symptoms in Crite- rion A. C. The disturbance is not better explained by a bipolar or related disorder that is not sub- stance/medication-induced. Such evidence of an independent bipolar or related disor- der could include the following: The symptoms precede the onset of the substance/medication use; the symptoms per- sist for a substantial period of time (e.g., about 1 month) after the cessation of acute withdrawal or severe intoxication; or there is other evidence suggesting the existence (e.g., a history of recurrent non-substance/medication-related episodes). D. The disturbance does not occur exclusively during the course of a delirium.

1	D. The disturbance does not occur exclusively during the course of a delirium. E. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. Coding note: The ICD-Q-CM and ICD-10-CM codes for the [specific substance/medication]- induced bipolar and related disorders are indicated in the table below. Note that the ICD-10-

1	CM code depends on whether or not there is a comorbid substance use disorder present for the same class of substance. If a mild substance use disorder is comorbid with the substance- induced bipolar and related disorder, the 4th position character is “1," and the clinician should record “mild [substance] use disorder’ before the substance-induced bipolar and related dis- order (e.g., “mild cocaine use disorder with cocaine-induced bipolar and related disordef'). If a moderate or severe substance use disorder is comorbid with the substance-induced bipolar and related disorder, the 4th position character is “2," and the clinician should record “moder- ate [substance] use disorder" or “severe [substance] use disorder," depending on the severity of the comorbid substance use disorder. If there is no comorbid substance use disorder (e.g., after a one-time heavy use of the substance), then the 4th position character is ”9," and the clinician should record only the substance-induced bipolar

1	is no comorbid substance use disorder (e.g., after a one-time heavy use of the substance), then the 4th position character is ”9," and the clinician should record only the substance-induced bipolar and related disorder.

1	With use disorder, Without disorder, moderate use Alcohol 291.89 F1014 F1024 F10.94 Phencyclidine 292.84 F1614 F1624 F1694 Other hallucinogen 292.84 F1614 F1624 F1694 With use disorder, Without disorder, moderate use Sedative, hypnotic, or anxiolytic 292.84 F1314 F1324 F1394 Amphetamine (or other 292.84 F1514 F1524 F1594 Cocaine 292.84 F14.14 F1424 F1494 Other (or unknown) substance 292.84 F1914 F1924 F1994 Specify if (see Table 1 in the chapter “Substance-Ftelated and Addictive Disorders" for di- agnoses associated with substance class): With onset during intoxication: If the criteria are met for intoxication with the sub- stance and the symptoms develop during intoxication. With onset during withdrawal: If criteria are met for withdrawal from the substance and the symptoms develop during, or shortly after, withdrawal.

1	With onset during withdrawal: If criteria are met for withdrawal from the substance and the symptoms develop during, or shortly after, withdrawal. |CD-9-CM. The name of the substance/medication-induced bipolar and related disor- der begins with the specific substance (e.g., cocaine, dexamethasone) that is presumed to be causing the bipolar mood symptoms. The diagnostic code is selected from the table in- cluded in the criteria set, which is based on the drug class. For substances that do not fit into any of the classes (e.g., dexamethasone), the code for ”other substance” should be used; and in cases in which a substance is judged to be an etiological factor but the specific class of substance is unknown, the category ”unknown substance” should be used.

1	The name of the disorder is followed by the specification of onset (i.e., onset during in- toxication, onset during withdrawal). Unlike the recording procedures for ICD-lO-CM, which combine the substance-induced disorder and substance use disorder into a single code, for ICD-9-CM a separate diagnostic code is given for the substance use disorder. For example, in the case of irritable symptoms occurring during intoxication in a man with a severe cocaine use disorder, the diagnosis is 292.84 cocaine-induced bipolar and related disorder, with onset during intoxication. An additional diagnosis of 304.20 severe cocaine use disorder is also given. When more than one substance is judged to play a significant role in the development of bipolar mood symptoms, each should be listed separately (e.g., 292.84 methylphenidate-induced bipolar and related disorder, with onset during intoxi- cation; 292.84 dexamethasone-induced bipolar and related disorder, with onset during in- toxication).

1	|CD-10-CM. The name of the substance/medication-induced bipolar and related disor- der begins with the specific substance (e.g., cocaine, dexamethasone) that is presumed to be causing the bipolar mood symptoms. The diagnostic code is selected from the table in- cluded in the criteria set, which is based on the drug class and presence or absence of a co- morbid substance use disorder. For substances that do not fit into any of the classes (e.g., dexamethasone), the code for ”other substance” should be used; and in cases in which a substance is judged to be an etiological factor but the specific class of substance is un- known, the category "unknown substance” should be used.

1	When recording the name of the disorder, the comorbid substance use disorder (if any) is listed first, followed by the word ”with," followed by the name of the substance-induced bipolar and related disorder, followed by the specification of onset (i.e., onset during in— toxication, onset during withdrawal). For example, in the case of irritable symptoms oc- curring during intoxication in a man with a severe cocaine use disorder, the diagnosis is

1	F1424 severe cocaine use disorder with cocaine-induced bipolar and related disorder, with onset during intoxication. A separate diagnosis of the comorbid severe cocaine use disorder is not given. If the substance—induced bipolar and related disorder occurs without a comorbid substance use disorder (e.g., after a one-time heavy use of the substance), no accompanying substance use disorder is noted (e.g., F1594 amphetamine-induced bipolar and related disorder, with onset during intoxication). When more than one substance is judged to play a significant role in the development of bipolar mood symptoms, each should be listed separately (e.g., F15.24 severe methylphenidate use disorder with meth- ylphenidate-induced bipolar and related disorder, with onset during intoxication; F1994 dexamethasone-induced bipolar and related disorder, with onset during intoxication).

1	The diagnostic features of substance/medication—induced bipolar and related disorder are es- sentially the same as those for mania, hypomania, or depression. A key exception to the diag- nosis of substance/medication—induced bipolar and related disorder is the case of hypomania yond the physiological effects of the medication. This condition is considered an indicator of true bipolar disorder, not substance/ medication-induced bipolar and related disorder. Simi- larly, individuals with apparent electroconvulsive therapy—induced manic or hypomanic ep- isodes that persist beyond the physiological effects of the treatment are diagnosed with bipolar disorder, not substance/medication-induced bipolar and related disorder.

1	Side effects of some antidepressants and other psychotropic drugs (e.g., edginess, ag— itation) may resemble the primary symptoms of a manic syndrome, but they are funda- mentally distinct from bipolar symptoms and are insufficient for the diagnosis. That is, the criterion symptoms of mania/hypomania have specificity (simple agitation is not the same as excess involvement in purposeful activities), and a sufficient number of symptoms must be present (not just one or two symptoms) to make these diagnoses. In particular, the appearance of one or two nonspecific symptoms—irritability, edginess, or agitation during antidepressant treatment—in the absence of a full manic or hypomanic syndrome should not be taken to support a diagnosis of a bipolar disorder.

1	Etiology (causally related to the use of psychotropic medications or substances of abuse based on best clinical evidence) is the key variable in this etiologically specified form of bi- polar disorder. Substances/ medications that are typically considered to be associated with substance/medication-induced bipolar and related disorder include the stimulant class of drugs, as well as phencyclidine and steroids; however, a number of potential sub- stances continue to emerge as new compounds are synthesized (e.g., so-called bath salts). A history of such substance use may help increase diagnostic certainty. There are no epidemiological studies of substance/medication-induced mania or bipolar disorder. Each etiological substance may have its own individual risk of inducing a bipo- lar (manic/hypomanic) disorder.

1	In phencyclidine—induced mania, the initial presentation may be one of a delirium with af- fective features, which then becomes an atypically appearing manic or mixed manic state. Bipolar and Related Disorder Due to Another Medical Condition 145 This condition follows the ingestion or inhalation quickly, usually within hours or, at the most, a few days. In stimulant-induced manic or hypomanic states, the response is in min- utes to 1 hour after one or several ingestions or injections. The episode is very brief and typically resolves over 1—2 days. With corticosteroids and some immunosuppressant medications, the mania (or mixed or depressed state) usually follows several days of in- gestion, and the higher doses appear to have a much greater likelihood of producing bi- polar symptoms. Determination of the substance of use can be made through markers in the blood or urine to corroborate diagnosis.

1	Determination of the substance of use can be made through markers in the blood or urine to corroborate diagnosis. from other bipolar disorders, substance intoxication or substance-induced delirium, and medication side effects (as noted earlier). A full manic episode that emerges during anti- depressant treatment (e.g., medication, electroconvulsive therapy) but persists at a fully syndromal level beyond the physiological effect of that treatment is sufficient evidence for a bipolar I diagnosis. A full hypomanic episode that emerges during antidepressant treat- ment (e.g., medication, electroconvulsive therapy) but persists at a fully syndromal level beyond the physiological effect of that treatment is sufficient evidence for a bipolar II di- agnosis only if preceded by a major depressive episode.

1	Comorbidities are those associated with the use of illicit substances (in the case of illegal stimulants or phencyclidine) or diversion of prescribed stimulants. Comorbidities related to steroid or immunosuppressant medications are those medical indications for these preparations. Delirium can occur before or along with manic symptoms in individuals in- gesting phencyclidine or those who are prescribed steroid medications or other immuno- suppressant medications. Due to Another Medical Condition A. A prominent and persistent period of abnormally elevated, expansive, or irritable mood and abnormally increased activity or energy that predominates in the clinical picture. There is evidence from the history. physical examination, or laboratory findings that the dis- turbance is the direct pathophysiological consequence of another medical condition. . The disturbance is not better explained by another mental disorder.

1	. The disturbance is not better explained by another mental disorder. . The disturbance does not occur exclusively during the course of a delirium. . The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning, or necessitates hospitalization to pre- vent harm to self or others, or there are psychotic features. Coding note: The lCD-9-CM code for bipolar and related disorder due to another medical condition is 293.83, which is assigned regardless of the specifier. The |CD-10-CM code depends on the specifier (see below). .03 Specify if: (F0633) With manic features: Full criteria are not met for a manic or hypomanic ep- isode. (F0633) With manic- or hypomanic-like episode: Full criteria are met except Crite- rion D for a manic episode or except Criterion F for a hypomanic episode. (F0634) With mixed features: Symptoms of depression are also present but do not predominate in the clinical picture.

1	(F0634) With mixed features: Symptoms of depression are also present but do not predominate in the clinical picture. Coding note: Include the name of the other medical condition in the name of the mental disorder (e.g., 293.83 [F0633] bipolar disorder due to hyperthyroidism, with manic tea- tures). The other medical condition should also be coded and listed separately immedi- ately before the bipolar and related disorder due to the medical condition (e.g., 242.90 [E0590] hyperthyroidism; 293.83 [F0633] bipolar disorder due to hyperthyroidism, with manic features).

1	The essential features of bipolar and related disorder due to another medical condition are presence of a prominent and persistent period of abnormally elevated, expansive, or irri- table mood and abnormally increased activity or energy predominating in the clinical pic- ture that is attributable to another medical condition (Criterion B). In most cases the manic or hypomanic picture may appear during the initial presentation of the medical condition (i.e., within 1 month); however, there are exceptions, especially in chronic medical condi- tions that might worsen or relapse and herald the appearance of the manic or hypomanic picture. Bipolar and related disorder due to another medical condition would not be diag- nosed when the manic or hypomanic episodes definitely preceded the medical condition, since the proper diagnosis would be bipolar disorder (except in the unusual circumstance in which all preceding manic or hypomanic episodes—or, when only one such episode has occurred, the

1	condition, since the proper diagnosis would be bipolar disorder (except in the unusual circumstance in which all preceding manic or hypomanic episodes—or, when only one such episode has occurred, the preceding manic or hypomanic episode—were associated with ingestion of a substance/medication). The diagnosis of bipolar and related disorder due to another medical condition should not be made during the course of a delirium (Criterion D). The manic or hypomanic episode in bipolar and related disorder due to another medical con- dition must cause clinically significant distress or impairment in social, occupational, or other important areas of functioning to qualify for this diagnosis (Criterion E).

1	Etiology (i.e., a causal relationship to another medical condition based on best clinical ev- idence) is the key variable in this etiologically specified form of bipolar disorder. The list- ing of medical conditions that are said to be able to induce mania is never complete, and the clinician’s best judgment is the essence of this diagnosis. Among the best known of the medical conditions that can cause a bipolar manic or hypomanic condition are Cushing's disease and multiple sclerosis, as well as stroke and traumatic brain injuries.

1	Bipolar and related disorder due to another medical condition usually has its onset acutely or subacutely within the first weeks or month of the onset of the associated medical con- dition. However, this is not always the case, as a worsening or later relapse of the associ- ated medical condition may precede the onset of the manic or hypomanic syndrome. The clinician must make a clinical judgment in these situations about whether the medical con- dition is causative, based on temporal sequence as well as plausibility of a causal relation- Bipolar and Related Disorder Due to Another Medical Condition 147 ship. Finally, the condition may remit before or just after the medical condition remits, particularly when treatment of the manic/hypomanic symptoms is effective.

1	Culture-related differences, to the extent that there is any evidence, pertain to those asso- ciated with the medical condition (e.g., rates of multiple sclerosis and stroke vary around the world based on dietary, genetic factors, and other environmental factors). Gender differences pertain to those associated with the medical condition (e.g., systemic lupus erythematosus is more common in females; stroke is somewhat more common in middle-age males compared with females). Diagnostic markers pertain to those associated with the medical condition (e.g., steroid levels in blood or urine to help corroborate the diagnosis of Cushing’s disease, which can be associated with manic or depressive syndromes; laboratory tests confirming the diag- nosis of multiple sclerosis). Functional Consequences of Bipolar and Related Disorder Due to Another Medical Condition

1	Functional Consequences of Bipolar and Related Disorder Due to Another Medical Condition Functional consequences of the bipolar symptoms may exacerbate impairments associ- ated with the medical condition and may incur worse outcomes due to interference with medical treatment. In general, it is believed, but not established, that the illness, when in— duced by Cushing’s disease, will not recur if the Cushing’s disease is cured or arrested. However, it is also suggested, but not established, that mood syndromes, including de- pressive and manic/hypomanic ones, may be episodic (i.e., recurring) with static brain in- juries and other central nervous system diseases. Symptoms of delirium, catatonia, and acute anxiety. It is important to differentiate symptoms of mania from excited or hypervigilant delirious symptoms; from excited cata- tonic symptoms; and from agitation related to acute anxiety states.

1	Medication-induced depressive or manic symptoms. An important differential diag- nostic observation is that the other medical condition may be treated with medications (e.g., steroids or alpha-interferon) that can induce depressive or manic symptoms. In these cases, clinical judgment using all of the evidence in hand is the best way to try to separate the most likely and/ or the most important of two etiological factors (i.e., association with the medical condition vs. a substance/medication~induced syndrome). The differential di— agnosis of the associated medical conditions is relevant but largely beyond the scope of the present manual. Conditions comorbid with bipolar and related disorder due to another medical condition are those associated with the medical conditions of etiological relevance. Delirium can oc- cur before or along with manic symptoms in individuals with Cushing’s disease. 296.89 (F31.89)

1	296.89 (F31.89) This category applies to presentations in which symptoms characteristic of a bipolar and related disorder that cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the bipolar and related disorders diagnostic class. The other specified bipolar and related disorder category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the cri- teria for any specific bipolar and related disorder. This is done by recording “other speci- fied bipolar and related disorder" followed by the specific reason (e.g., “short-duration cyclothymia").

1	Examples of presentations that can be specified using the “other specified“ designation include the following: 1. Short-duration hypomanic episodes (2-3 days) and major depressive episodes: A lifetime history of one or more major depressive episodes in individuals whose presenta- two or more episodes of short-duration hypomania that meet the full symptomatic criteria for a hypomanic episode but that only last for 2—3 days. The episodes of hypomanic symp- toms do not overlap in time with the major depressive episodes, so the disturbance does not meet criteria for major depressive episode, with mixed features.

1	2. Hypomanic episodes with insufficient symptoms and major depressive epi- sodes: A lifetime history of one or more major depressive episodes in individuals who have experienced one or more episodes of hypomania that do not meet full symp- tomatic criteria (i.e., at least 4 consecutive days of elevated mood and one or two of the other symptoms of a hypomanic episode, or irritable mood and two or three of the other symptoms of a hypomanic episode). The episodes of hypomanic symptoms do not overlap in time with the major depressive episodes, so the disturbance does not meet criteria for major depressive episode, with mixed features. 3. Hypomanic episode without prior major depressive episode: One or more hypo- jor depressive episode or a manic episode. If this occurs in an individual with an established diagnosis of persistent depressive disorder (dysthymia), both diagnoses can be concurrently applied during the periods when the full criteria for a hypomanic episode are met.

1	4. Short-duration cyclothymia (less than 24 months): Multiple episodes of hypomanic symptoms that do not meet criteria for a hypomanic episode and multiple episodes of de- over a period of less than 24 months (less than 12 months for children or adolescents) in an individual whose presentation has never met full criteria for a major depressive, manic, or hypomanic episode and does not meet criteria for any psychotic disorder. Dur- ing the course of the disorder, the hypomanic or depressive symptoms are present for more days than not, the individual has not been without symptoms for more than 2 months at a time, and the symptoms cause clinically significant distress or impairment. 296.80 (F31.9)

1	296.80 (F31.9) This category applies to presentations in which symptoms characteristic of a bipolar and related disorder that cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the bipolar and related disorders diagnostic class. The unspec- ified bipolar and related disorder category is used in situations in which the clinician choos- es not to specify the reason that the criteria are not met for a specific bipolar and related disorder, and includes presentations in which there is insufficient information to make a more specific diagnosis (e.g., in emergency room settings). Specify it: With anxious distress: The presence of at least two of the following symptoms during the majority of days of the current or most recent episode of mania, hypomania, or de— pression: Feeling keyed up or tense. Feeling unusually restless.

1	Feeling keyed up or tense. Feeling unusually restless. Difficulty concentrating because of worry. Fear that something awful may happen. Feeling that the individual might lose control of himself or herself. Specify current severity: Mild: Two symptoms. Moderate: Three symptoms. Moderate-severe: Four or five symptoms. Severe: Four or five symptoms with motor agitation. Note: Anxious distress has been noted as a prominent feature of both bipolar and tings. High levels of anxiety have been associated with higher suicide risk, longer duration of illness, and greater likelihood of treatment nonresponse. As a result, it is clinically useful to specify accurately the presence and severity levels of anxious distress for treatment planning and monitoring of response to treatment. With mixed features: The mixed features specifier can apply to the current manic, hy- pomanic, or depressive episode in bipolar I or bipolar II disorder: 91:59-39)?“

1	With mixed features: The mixed features specifier can apply to the current manic, hy- pomanic, or depressive episode in bipolar I or bipolar II disorder: 91:59-39)?“ Manic or hypomanic episode, with mixed features: A. Full criteria are met for a manic episode or hypomanic episode, and at least three of the following symptoms are present during the majority of days of the current or most recent episode of mania or hypomania: 1. Prominent dysphoria or depressed mood as indicated by either subjective report (e.g., feels sad or empty) or observation made by others (e.g., ap- pears tearful). 2. Diminished interest or pleasure in all, or almost all, activities (as indicated by either subjective account or observation made by others). 3. Psychomotor retardation nearly every day (observable by others; not merely subjective feelings of being slowed down). 4. Fatigue or loss of energy.

1	3. Psychomotor retardation nearly every day (observable by others; not merely subjective feelings of being slowed down). 4. Fatigue or loss of energy. 5. Feelings of worthlessness or excessive or inappropriate guilt (not merely seIf-reproach or guilt about being sick). 6. Recurrent thoughts of death (not just fear of dying), recurrent suicidal ide- ation without a specific plan, or a suicide attempt or a specific plan for com- mitting suicide. B. Mixed symptoms are observable by others and represent a change from the person’s usual behavior. C. For individuals whose symptoms meet full episode criteria for both mania and depression simultaneously, the diagnosis should be manic episode, with mixed features, due to the marked impairment and clinical severity of full mania. D. The mixed symptoms are not attributable to the physiological effects of a sub- stance (e.g., a drug of abuse, a medication, other treatment). Depressive episode, with mixed features:

1	D. The mixed symptoms are not attributable to the physiological effects of a sub- stance (e.g., a drug of abuse, a medication, other treatment). Depressive episode, with mixed features: A. Full criteria are met for a major depressive episode, and at least three of the fol- lowing manic/hypomanic symptoms are present during the majority of days of the current or most recent episode of depression: Elevated, expansive mood. Inflated self-esteem or grandiosity. More talkative than usual or pressure to keep talking. Flight of ideas or subjective experience that thoughts are racing. Increase in energy or goaI-directed activity (either socially, at work or school, or sexually). 6. Increased or excessive involvement in activities that have a high potential for painful consequences (e.g., engaging in unrestrained buying sprees, sexual indiscretions, or foolish business investments).

1	7. Decreased need for sleep (feeling rested despite sleeping less than usual; to be contrasted with insomnia). B. Mixed symptoms are observable by others and represent a change from the person’s usual behavior. C. For individuals whose symptoms meet full episode criteria for both mania and depression simultaneously, the diagnosis should be manic episode, with mixed features. D. The mixed symptoms are not attributable to the physiological effects of a sub- stance (e.g., a drug of abuse, a medication, or other treatment). Note: Mixed features associated with a major depressive episode have been found to be a significant risk factor for the development of bipolar | or bipolar II disorder. As a result. it is clinically useful to note the presence of this specifier for treatment planning and monitoring of response to treatment. 9‘?wa

1	As a result. it is clinically useful to note the presence of this specifier for treatment planning and monitoring of response to treatment. 9‘?wa With rapid cycling (can be applied to bipolar | or bipolar II disorder): Presence of at least four mood episodes in the previous 12 months that meet the criteria for manic, hypomanic, or major depressive episode. Note: Episodes are demarcated by either partial or full remissions of at least 2 months or a switch to an episode of the opposite polarity (e.g., major depressive episode to manic episode).

1	Note: The essential feature of a rapid-cycling bipolar disorder is the occurrence of at least four mood episodes during the previous 12 months. These episodes can occur in any combination and order. The episodes must meet both the duration and symptom number criteria for a major depressive, manic, or hypomanic episode and must be demarcated by either a period of full remission or a switch to an episode of the opposite polarity. Manic and hypomanic episodes are counted as being on the same pole. Except for the fact that they occur more frequently, the episodes that occur in a rapid-cycling pattern are no different from those that occur in a non-rapid- cycling pattern. Mood episodes that count toward defining a rapid-cycling pattern exclude those episodes directly caused by a substance (e.g., cocaine, corticoste- roids) or another medical condition. With melancholic features: A. B.

1	With melancholic features: A. B. One of the following is present during the most severe period of the current episode: 1. Loss of pleasure in all, or almost all, activities. 2. Lack of reactivity to usually pleasurable stimuli (does not feel much better, even temporarily, when something good happens). Three (or more) of the following: 1. A distinct quality of depressed mood characterized by profound despondency, despair, and/or moroseness or by so-called empty mood. Depression that is regularly worse in the morning. EarIy-morning awakening (i.e., at least 2 hours before usual awakening). Marked psychomotor agitation or retardation. Significant anorexia or weight loss. Excessive or inappropriate guilt. 99??!“

1	Significant anorexia or weight loss. Excessive or inappropriate guilt. 99??!“ Note: The specifier “with melancholic features“ is applied if these features are pres- ent at the most severe stage of the episode. There is a near-complete absence of the capacity for pleasure, not merely a diminution. A guideline for evaluating the lack of reactivity of mood is that even highly desired events are not associated with marked brightening of mood. Either mood does not brighten at all, or it brightens only partially (e.g., up to 20%-40% of normal for only minutes at a time). The “dis- tinct quality” of mood that is characteristic of the “with melancholic features" speci- fier is experienced as qualitatively different from that during a nonmelancholic depressive episode. A depressed mood that is described as merely more severe, longer lasting, or present without a reason is not considered distinct in quality. Psy- chomotor changes are nearly always present and are observable by others.

1	Melancholic features exhibit only a modest tendency to repeat across episodes in the same individual. They are more frequent in inpatients, as opposed to outpa- tients; are less likely to occur in milder than in more severe major depressive epi- sodes; and are more likely to occur in those with psychotic features. With atypical features: This specifier can be applied when these features predomi- nate during the majority of days of the current or most recent major depressive epi- sode. A. B. Mood reactivity (i.e., mood brightens in response to actual or potential positive events). Two (or more) of the following features: 1. Significant weight gain or increase in appetite. 2. Hypersomnia. 3. Leaden paralysis (i.e., heavy, leaden feelings in arms or legs). 4. A long-standing pattern of interpersonal rejection sensitivity (not limited to epi- sodes of mood disturbance) that results in significant social or occupational impairment.

1	4. A long-standing pattern of interpersonal rejection sensitivity (not limited to epi- sodes of mood disturbance) that results in significant social or occupational impairment. C. Criteria are not met for “with melancholic features" or “with catatonia“ during the same episode. Note: “Atypical depression” has historical significance (i.e., atypical in contradis- tinction to the more classical agitated, “endogenous" presentations of depression that were the norm when depression was rarely diagnosed in outpatients and al- common or unusual clinical presentation as the term might imply.

1	Mood reactivity is the capacity to be cheered up when presented with positive events (e.g., a visit from children, compliments from others). Mood may become euthymic (not sad) even for extended periods of time if the external circumstances remain favorable. Increased appetite may be manifested by an obvious increase in food intake or by weight gain. Hypersomnia may include either an extended period of nighttime sleep or daytime napping that totals at least 10 hours of sleep per day (or at least 2 hours more than when not depressed). Leaden paralysis is defined as feeling heavy, leaden, or weighted down, usually in the arms or legs. This sensation time. Unlike the other atypical features, pathological sensitivity to perceived inter- personal rejection is a trait that has an early onset and persists throughout most of adult life. Rejection sensitivity occurs both when the person is and is not depressed, though it may be exacerbated during depressive periods.

1	With psychotic features: Delusions or hallucinations are present at any time in the episode. If psychotic features are present, specify if mood-congruent or mood-incon- gruent: With mood-congruent psychotic features: During manic episodes, the con- tent of all delusions and hallucinations is consistent with the typical manic themes of grandiosity, invulnerability, etc., but may also include themes of sus- piciousness or paranoia, especially with respect to others' doubts about the in- dividual’s capacities, accomplishments, and so forth. With mood-incongruent psychotic features: The content of delusions and hallucinations is inconsistent with the episode polarity themes as described above, or the content is a mixture of mood-incongruent and mood-congruent themes.

1	With catatonia: This specifier can apply to an episode of mania or depression if cata- tonic features are present during most of the episode. See criteria for catatonia asso- ciated with a mental disorder in the chapter “Schizophrenia Spectrum and Other Psychotic Disorders.“ With peripartum onset: This specifier can be applied to the current or, if the full crite- ria are not currently met for a mood episode, most recent episode of mania, hypoma- nia, or major depression in bipolar | or bipolar II disorder if onset of mood symptoms occurs during pregnancy or in the 4 weeks following delivery. Note: Mood episodes can have their onset either during pregnancy or postpartum.

1	Note: Mood episodes can have their onset either during pregnancy or postpartum. Although the estimates differ according to the period of Iollow-up after delivery, be- tween 3% and 6% of women will experience the onset of a major depressive epi- sode during pregnancy or in the weeks or months following delivery. Fifty percent of “postpartum" major depressive episodes actually begin prior to delivery. Thus, these episodes are referred to collectively as peripartum episodes. Women with attacks. Prospective studies have demonstrated that mood and anxiety symptoms during pregnancy, as well as the “baby blues," increase the risk for a postpartum major depressive episode.

1	Peripartum-onset mood episodes can present either with or without psychotic featuresx Infanticide is most often associated with postpartum psychotic episodes that are characterized by command hallucinations to kill the infant or delusions that the infant is possessed, but psychotic symptoms can also occur in severe postpar- tum mood episodes without such specific delusions or hallucinations. Postpartum mood (major depressive or manic) episodes with psychotic features appear to occur in from 1 in 500 to 1 in 1,000 deliveries and may be more common in primiparous women. The risk of postpartum episodes with psychotic features is particularly increased for women with prior postpartum mood episodes but is also elevated for those with a prior history of a depressive or bipolar disorder (especially bipolar | disorder) and those with a family history of bipolar disorders.

1	Once a woman has had a postpartum episode with psychotic features, the risk of recurrence with each subsequent delivery is between 30% and 50%. Postpartum episodes must be differentiated from delirium occurring in the postpartum period, which is distinguished by a fluctuating level of awareness or attention. The postpar- tum period is unique with respect to the degree of neuroendocrine alterations and psychosocial adjustments, the potential impact of breast-feeding on treatment plan- ning, and the Iong-term implications of a history of postpartum mood disorder on sub- sequent family planning. With seasonal pattern: This specifier applies to the lifetime pattern of mood episodes.

1	With seasonal pattern: This specifier applies to the lifetime pattern of mood episodes. The essential feature is a regular seasonal pattern of at least one type of episode (i.e., mania. hypomania, or depression). The other types of episodes may not follow this pat- tern. For example, an individual may have seasonal manias, but his or her depressions do not regularly occur at a specific time of year. A. There has been a regular temporal relationship between the onset of manic, hypo- manic, or major depressive episodes and a particular time of the year (e.g., in the fall or winter) in bipolar I or bipolar II disorder. Note: Do not include cases in which there is an obvious effect of seasonally related psychosocial stressors (e.g., regularly being unemployed every winter). B. Full remissions (or a change from major depression to mania or hypomania or vice versa) also occur at a characteristic time of the year (e.g., depression disappears in the spring).

1	B. Full remissions (or a change from major depression to mania or hypomania or vice versa) also occur at a characteristic time of the year (e.g., depression disappears in the spring). C. In the last 2 years, the individual’s manic, hypomanic, or major depressive episodes have demonstrated a temporal seasonal relationship, as defined above, and no non-seasonal episodes of that polarity have occurred during that 2-year period. D. Seasonal manias, hypomanias, or depressions (as described above) substantially outnumber any nonseasonal manias, hypomanias, or depressions that may have occurred over the individual‘s lifetime.

1	D. Seasonal manias, hypomanias, or depressions (as described above) substantially outnumber any nonseasonal manias, hypomanias, or depressions that may have occurred over the individual‘s lifetime. Note: This specifier can be applied to the pattern of major depressive episodes in bipolar | disorder, bipolar II disorder, or major depressive disorder, recurrent. The essential feature is the onset and remission of major depressive episodes at char- acteristic times of the year. In most cases, the episodes begin in fall or winter and remit in spring. Less commonly, there may be recurrent summer depressive epi- sodes. This pattern of onset and remission of episodes must have occurred during at least a 2-year period, without any nonseasonal episodes occurring during this period. In addition, the seasonal depressive episodes must substantially outnum- ber any nonseasonal depressive episodes over the individual‘s lifetime.

1	This specifier does not apply to those situations in which the pattern is better ex- plained by seasonally linked psychosocial stressors (e.g., seasonal unemployment or school schedule). Major depressive episodes that occur in a seasonal pattern are often characterized by prominent energy, hypersomnia, overeating, weight gain, and a craving for carbohydrates. It is unclear whether a seasonal pattern is more likely in recurrent major depressive disorder or in bipolar disorders. However, within the bipolar disorders group, a seasonal pattern appears to be more likely in bipolar II disorder than in bipolar | disorder. In some individuals, the onset of manic or hypomanic episodes may also be linked to a particular season. The prevalence of winter-type seasonal pattern appears to vary with latitude. age, and sex. Prevalence increases with higher latitudes. Age is also a strong pre- dictor of seasonality, with younger persons at higher risk for winter depressive epi- sodes. Specify it:

1	age, and sex. Prevalence increases with higher latitudes. Age is also a strong pre- dictor of seasonality, with younger persons at higher risk for winter depressive epi- sodes. Specify it: In partial remission: Symptoms of the immediately previous manic, hypomanic, or depressive episode are present, but full criteria are not met, or there is a period lasting less than 2 months without any significant symptoms of a manic, hypomanic, or major depressive episode following the end of such an episode. In full remission: During the past 2 months, no significant signs or symptoms of the disturbance were present. Specify current severity: Severity is based on the number of criterion symptoms, the severity of those symptoms, and the degree of functional disability.

1	Specify current severity: Severity is based on the number of criterion symptoms, the severity of those symptoms, and the degree of functional disability. Mild: Few. if any, symptoms in excess of those required to meet the diagnostic criteria are present, the intensity of the symptoms is distressing but manageable, and the symptoms result in minor impairment in social or occupational functioning. Moderate: The number of symptoms, intensity of symptoms, and/or functional impair- ment are between those specified for “mild” and “severe." Severe: The number of symptoms is substantially in excess of those required to make the diagnosis, the intensity of the symptoms is seriously distressing and unmanage- able, and the symptoms markedly interfere with social and occupational functioning.

1	Depressive disorders include disruptive mood dysregulation disorder, major depressive disorder (including major depressive episode), persistent depressive disorder (dysthymia), premenstrual dysphoric disorder, substance/medication-induced depres- sive disorder, depressive disorder due to another medical condition, other specified de- pressive disorder, and unspecified depressive disorder. Unlike in DSM-IV, this chapter ”Depressive Disorders” has been separated from the previous chapter ”Bipolar and Re- lated Disorders.” The common feature of all of these disorders is the presence of sad, empty, or irritable mood, accompanied by somatic and cognitive changes that signifi- cantly affect the individual’s capacity to function. What differs among them are issues of duration, timing, or presumed etiology.

1	In order to address concerns about the potential for the overdiagnosis of and treatment for bipolar disorder in children, a new diagnosis, disruptive mood dysregulation disorder, referring to the presentation of children with persistent irritability and frequent episodes of extreme behavioral dyscontrol, is added to the depressive disorders for children up to 12 years of age. Its placement in this chapter reﬂects the finding that children with this symptom pattern typically develop unipolar depressive disorders or anxiety disorders, rather than bipolar disorders, as they mature into adolescence and adulthood.

1	Major depressive disorder represents the classic condition in this group of disorders. It is characterized by discrete episodes of at least 2 weeks’ duration (although most episodes last considerably longer) involving clear-cut changes in affect, cognition, and neurovege- tative functions and inter-episode remissions. A diagnosis based on a single episode is possible, although the disorder is a recurrent one in the majority of cases. Careful consid- eration is given to the delineation of normal sadness and grief from a major depressive ep- isode. Bereavement may induce great suffering, but it does not typically induce an episode of major depressive disorder. When they do occur together, the depressive symptoms and functional impairment tend to be more severe and the prognosis is worse compared with bereavement that is not accompanied by major depressive disorder. Bereavement—related depression tends to occur in persons with other vulnerabilities to depressive disorders, and recovery

1	with bereavement that is not accompanied by major depressive disorder. Bereavement—related depression tends to occur in persons with other vulnerabilities to depressive disorders, and recovery may be facilitated by antidepressant treatment.

1	A more chronic form of depression, persistent depressive disorder (dysthymia), can be diagnosed when the mood disturbance continues for at least 2 years in adults or 1 year in children. This diagnosis, new in DSM-S, includes both the DSM-IV diagnostic categories of chronic major depression and dysthymia. After careful scientific review of the evidence, premenstrual dysphoric disorder has been moved from an appendix of DSM-IV (“Criteria Sets and Axes Provided for Further Study”) to Section II of DSM-S. Almost 20 years of additional of research on this condition has confirmed a specific and treatment-responsive form of depressive disorder that begins sometime following ovulation and remits within a few days of menses and has a marked impact on functioning.

1	A large number of substances of abuse, some prescribed medications, and several medical conditions can be associated with depression—like phenomena. This fact is recog- nized in the diagnoses of substance/medication-induced depressive disorder and depres- sive disorder due to another medical condition. Diagnostic Criteria 296.99 (F34.8) A. Severe recurrent temper outbursts manifested verbally (e.g., verbal rages) and/or be- haviorally (e.g., physical aggression toward people or property) that are grossly out of proportion in intensity or duration to the situation or provocation. . The temper outbursts are inconsistent with developmental level. . The temper outbursts occur, on average, three or more times per week. . The mood between temper outbursts is persistently irritable or angry most of the day, nearly every day, and is observable by others (e.g., parents, teachers, peers).

1	. The mood between temper outbursts is persistently irritable or angry most of the day, nearly every day, and is observable by others (e.g., parents, teachers, peers). E. Criteria A—D have been present for 12 or more months. Throughout that time, the indi- vidual has not had a period lasting 3 or more consecutive months without all of the symptoms in Criteria A—D. F. Criteria A and D are present in at least two of three settings (i.e., at home, at school, with peers) and are severe in at least one of these. G. The diagnosis should not be made for the first time before age 6 years or after age 18 years. H. By history or observation, the age at onset of Criteria A—E is before 10 years. I. There has never been a distinct period lasting more than 1 day during which the full symptom criteria. except duration, for a manic or hypomanic episode have been met.

1	I. There has never been a distinct period lasting more than 1 day during which the full symptom criteria. except duration, for a manic or hypomanic episode have been met. Note: Developmentally appropriate mood elevation, such as occurs in the context of a highly positive event or its anticipation, should not be considered as a symptom of ma- nia or hypomania. J. The behaviors do not occur exclusively during an episode of major depressive disorder and are not better explained by another mental disorder (e.g., autism spectrum disor- der, posttraumatic stress disorder, separation anxiety disorder, persistent depressive disorder [dysthymia]).

1	Note: This diagnosis cannot coexist with oppositional defiant disorder, intermittent ex- plosive disorder, or bipolar disorder, though it can coexist with others, including major depressive disorder, attention-deficit/hyperactivity disorder, conduct disorder, and substance use disorders. Individuals whose symptoms meet criteria for both disruptive diagnosis of disruptive mood dysregulation disorder. If an individual has ever experi- enced a manic or hypomanic episode, the diagnosis of disruptive mood dysregulation disorder should not be assigned. K. The symptoms are not attributable to the physiological effects of a substance or to an- other medical or neurological condition.

1	K. The symptoms are not attributable to the physiological effects of a substance or to an- other medical or neurological condition. The core feature of disruptive mood dysregulation disorder is chronic, severe persistent ir- ritability. This severe irritability has two prominent clinical manifestations, the first of which is frequent temper outbursts. These outbursts typically occur in response to frus- tration and can be verbal or behavioral (the latter in the form of aggression against prop- erty, self, or others). They must occur frequently (i.e., on average, three or more times per week) (Criterion C) over at least 1 year in at least two settings (Criteria E and F), such as in the home and at school, and they must be developmentally inappropriate (Criterion B).

1	The second manifestation of severe irritability consists of chronic, persistently irritable or angry mood that is present between the severe temper outbursts. This irritable or angry mood must be characteristic of the child, being present most of the day, nearly every day, and noticeable by others in the child’s environment (Criterion D). The clinical presentation of disruptive mood dysregulation disorder must be carefully distinguished from presentations of other, related conditions, particularly pediatric bi- polar disorder. In fact, disruptive mood dysregulation disorder was added to DSM-S to address the considerable concern about the appropriate classification and treatment of children who present with chronic, persistent irritability relative to children who present with classic (i.e., episodic) bipolar disorder.

1	Some researchers view severe, non-episodic irritability as characteristic of bipolar dis- order in children, although both DSM-IV and DSM-S require that both children and adults have distinct episodes of mania or hypomania to qualify for the diagnosis of bipolar I dis- order. During the latter decades of the 20th century, this contention by researchers that severe, nonepisodic irritability is a manifestation of pediatric mania coincided with an up- surge in the rates at which clinicians assigned the diagnosis of bipolar disorder to their pediatric patients. This sharp increase in rates appears to be attributable to clinicians com- bining at least two clinical presentations into a single category. That is, both classic, epi- sodic presentations of mania and non-episodic presentations of severe irritability have been labeled as bipolar disorder in children. In DSM-5, the term bipolar disorder is explicitly reserved for episodic presentations of bipolar symptoms. DSM-IV did not include a

1	irritability have been labeled as bipolar disorder in children. In DSM-5, the term bipolar disorder is explicitly reserved for episodic presentations of bipolar symptoms. DSM-IV did not include a diagno- sis designed to capture youths whose hallmark symptoms consisted of very severe, non- episodic irritability, whereas DSM-S, with the inclusion of disruptive mood dysregulation disorder, provides a distinct category for such presentations.

1	Disruptive mood dysregulation disorder is common among children presenting to pedi- atric mental health clinics. Prevalence estimates of the disorder in the community are un- clear. Based on rates of chronic and severe persistent irritability, which is the core feature of the disorder, the overall 6-month to 1-year period-prevalence of disruptive mood dys- regulation disorder among children and adolescents probably falls in the 2%—5% range. However, rates are expected to be higher in males and school-age children than in females and adolescents. The onset of disruptive mood dysregulation disorder must be before age 10 years, and the diagnosis should not be applied to children with a developmental age of less than 6 years.

1	The onset of disruptive mood dysregulation disorder must be before age 10 years, and the diagnosis should not be applied to children with a developmental age of less than 6 years. It is unknown whether the condition presents only in this age-delimited fashion. Because the symptoms of disruptive mood dysregulation disorder are likely to change as children mature, use of the diagnosis should be restricted to age groups similar to those in which validity has been established (7—18 years). Approximately half of children with severe, chronic irritability will have a presentation that continues to meet criteria for the condition 1 year later. Rates of conversion from severe, nonepisodic irritability to bipolar disorder are very low. Instead, children with chronic irritability are at risk to develop unipolar de- pressive and/ or anxiety disorders in adulthood.

1	dysregulation disorder. Rates of bipolar disorder generally are very low prior to adoles- cence (<1%), with a steady increase into early adulthood (1%—2% prevalence). Disruptive mood dysregulation disorder is more common than bipolar disorder prior to adolescence, and symptoms of the condition generally become less common as children transition into adulthood. Temperamental. Children with chronic irritability typically exhibit complicated psy- chiatric histories. In such children, a relatively extensive history of chronic irritability is common, typically manifesting before full criteria for the syndrome are met. Such predi- agnostic presentations may have qualified for a diagnosis of oppositional defiant disorder. Many children with disruptive mood dysregulation disorder have symptoms that also order, with such diagnoses often being present from a relatively early age. For some chil- dren, the criteria for major depressive disorder may also be met.

1	Genetic and physiological. In terms of familial aggregation and genetics, it has been suggested that children presenting with chronic, non-episodic irritability can be differen- tiated from children with bipolar disorder in their family-based risk. However, these two groups do not differ in familial rates of anxiety disorders, unipolar depressive disorders, or substance abuse. Compared with children with pediatric bipolar disorder or other men- tal illnesses, those with disruptive mood dysregulation disorder exhibit both commonal- ities and differences in information—processing deficits. For example, face-emotion labeling deficits, as well as perturbed decision making and cognitive control, are present in children with bipolar disorder and chronically irritable children, as well as in children with some other psychiatric conditions. There is also evidence for disorder-specific dys- function, such as during tasks assessing attention deployment in response to emotional stimuli, which

1	with some other psychiatric conditions. There is also evidence for disorder-specific dys- function, such as during tasks assessing attention deployment in response to emotional stimuli, which has demonstrated unique signs of dysfunction in children with chronic ir- ritability.

1	Children presenting to clinics with features of disruptive mood dysregulation disorder are predominantly male. Among community samples, a male preponderance appears to be supported. This difference in prevalence between males and females differentiates disrup- tive mood dysregulation disorder from bipolar disorder, in which there is an equal gender prevalence. In general, evidence documenting suicidal behavior and aggression, as well as other se- vere functional consequences, in disruptive mood dysregulation disorder should be noted when evaluating children with chronic irritability.

1	Chronic, severe irritability, such as is seen in disruptive mood dysregulation disorder, is associated with marked disruption in a child’s family and peer relationships, as well as in school performance. Because of their extremely low frustration tolerance, such children generally have difficulty succeeding in school; they are often unable to participate in the activities typically enjoyed by healthy children; their family life is severely disrupted by their outbursts and irritability; and they have trouble initiating or sustaining friendships. Levels of dysfunction in children with bipolar disorder and disruptive mood dysregulation disorder are generally comparable. Both conditions cause severe disruption in the lives of the affected individual and their families. In both disruptive mood dysregulation disorder and pediatric bipolar disorder, dangerous behavior, suicidal ideation or suicide attempts, severe aggression, and psychiatric hospitalization are common.

1	Because chronically irritable children and adolescents typically present with complex histo- ries, the diagnosis of disruptive mood dysregulation disorder must be made while consid- ering the presence or absence of multiple other conditions. Despite the need to consider many other syndromes, differentiation of disruptive mood dysregulation disorder from bi- polar disorder and oppositional defiant disorder requires particularly careful assessment.

1	Bipolar disorders. The central feature differentiating disruptive mood dysregulation disor- der and bipolar disorders in children involves the longitudinal course of the core symptoms. In children, as in adults, bipolar I disorder and bipolar H disorder manifest as an episodic illness with discrete episodes of mood perturbation that can be differentiated from the child’s typical presentation. The mood perturbation that occurs during a manic episode is distinctly different from the child’s usual mood. In addition, during a manic episode, the change in mood must be accompanied by the onset, or worsening, of associated cognitive, behavioral, and physical symptoms (e.g., distractibility, increased goal-directed activity), which are also present to a de- gree that is distinctly different from the child’s usual baseline. Thus, in the case of a manic ep- isode, parents (and, depending on developmental level, children) should be able to identify a distinct time period during which the child’s

1	child’s usual baseline. Thus, in the case of a manic ep- isode, parents (and, depending on developmental level, children) should be able to identify a distinct time period during which the child’s mood and behavior were markedly different from usual. In contrast, the irritability of dismptive mood dysregulation disorder is persistent and is present over many months; while it may wax and wane to a certain degree, severe ini- tability is characteristic of the child with disruptive mood dysregulation disorder. Thus, while bipolar disorders are episodic conditions, disruptive mood dysregulation disorder is not. In fact, the diagnosis of disruptive mood dysregulation disorder cannot be assigned to a child or who has ever had a manic or hypomanic episode lasting more than 1 day. Another central is the presence of elevated or expansive mood and grandiosity. These symptoms are common features of mania but are not characteristic of disruptive mood dysregulation disorder.

1	Oppositional defiant disorder. While symptoms of oppositional defiant disorder typi- cally do occur in children with disruptive mood dysregulation disorder, mood symptoms of disruptive mood dysregulation disorder are relatively rare in children with opposi- tional defiant disorder. The key features that warrant the diagnosis of disruptive mood fiant disorder are the presence of severe and frequently recurrent outbursts and a persis- tent disruption in mood between outbursts. In addition, the diagnosis of disruptive mood dysregulation disorder requires severe impairment in at least one setting (i.e., home, school, or among peers) and mild to moderate impairment in a second setting. For this rea- son, while most children whose symptoms meet criteria for disruptive mood dysregula- disorder, the reverse is not the case. That is, in only approximately 15% of individuals with be met. Moreover, even for children in whom criteria for both disorders are met, only the diagnosis of disruptive

1	the reverse is not the case. That is, in only approximately 15% of individuals with be met. Moreover, even for children in whom criteria for both disorders are met, only the diagnosis of disruptive mood dysregulation disorder should be made. Finally, both the prominent mood symptoms in disruptive mood dysregulation disorder and the high risk for depressive and anxiety disorders in follow-up studies justify placement of disruptive mood dysregulation disorder among the depressive disorders in DSM-5. (Oppositional defiant disorder is included in the chapter "Disruptive, Impulse-Control, and Conduct

1	Disorders") This reﬂects the more prominent mood component among individuals with disruptive mood dysregulation disorder, as compared with individuals with oppositional defiant disorder. Nevertheless, it also should be noted that disruptive mood dysregulation disorder appears to carry a high risk for behavioral problems as well as mood problems.

1	Attention-deficit/hyperactivity disorder, major depressive disorder, anxiety disorders, and autism spectrum disorder. Unlike children diagnosed with bipolar disorder or op- positional defiant disorder, a child whose symptoms meet criteria for disruptive mood dysregulation disorder also can receive a comorbid diagnosis of ADHD, major depressive disorder, and/ or anxiety disorder. However, children whose irritability is present only in the context of a major depressive episode or persistent depressive disorder (dysthymia) should receive one of those diagnoses rather than disruptive mood dysregulation disor- der. Children with disruptive mood dysregulation disorder may have symptoms that also meet criteria for an anxiety disorder and can receive both diagnoses, but children whose ir- ritability is manifest only in the context of exacerbation of an anxiety disorder should re- ceive the relevant anxiety disorder diagnosis rather than disruptive mood dysregulation disorder. In addition,

1	is manifest only in the context of exacerbation of an anxiety disorder should re- ceive the relevant anxiety disorder diagnosis rather than disruptive mood dysregulation disorder. In addition, children with autism spectrum disorders frequently present with temper outbursts when, for example, their routines are disturbed. In that instance, the temper outbursts would be considered secondary to the autism spectrum disorder, and the child should not receive the diagnosis of disruptive mood dysregulation disorder.

1	Intermittent explosive disorder. Children with symptoms suggestive of intermittent explosive disorder present with instances of severe temper outbursts, much like children with disruptive mood dysregulation disorder. However, unlike disruptive mood dysreg- ulation disorder, intermittent explosive disorder does not require persistent disruption in mood between outbursts. In addition, intermittent explosive disorder requires only 3 months of active symptoms, in contrast to the 12-month requirement for disruptive mood dys- regulation disorder. Thus, these two diagnoses should not be made in the same child. For children with outbursts and intercurrent, persistent irritability, only the diagnosis of dis- ruptive mood dysregulation disorder should be made.

1	Rates of comorbidity in disruptive mood dysregulation disorder are extremely high. It is rare to find individuals whose symptoms meet criteria for disruptive mood dysregulation disorder alone. Comorbidity between disruptive mood dysregulation disorder and other the strongest overlap is with oppositional defiant disorder. Not only is the overall rate of comorbidity high in disruptive mood dysregulation disorder, but also the range of comor- bid illnesses appears particularly diverse. These children typically present to the clinic with a wide range of disruptive behavior, mood, anxiety, and even autism spectrum symptoms and diagnoses. However, children with disruptive mood dysregulation disor- der should not have symptoms that meet criteria for bipolar disorder, as in that context, only the bipolar disorder diagnosis should be made. If children have symptoms that meet mood dysregulation disorder, only the diagnosis of disruptive mood dysregulation disor- der should be assigned. Also, as

1	bipolar disorder diagnosis should be made. If children have symptoms that meet mood dysregulation disorder, only the diagnosis of disruptive mood dysregulation disor- der should be assigned. Also, as noted earlier, the diagnosis of disruptive mood dysregu- lation disorder should not be assigned if the symptoms occur only in an anxiety- provoking context, when the routines of a child with autism spectrum disorder or obses- sive-compulsive disorder are disturbed, or in the context of a major depressive episode.

1	A. Five (or more) of the following symptoms have been present during the same 2-week period and represent a change from previous functioning; at least one of the symptoms is either (1) depressed mood or (2) loss of interest or pleasure. Note: Do not include symptoms that are clearly attributable to another medical condition. 1. Depressed mood most of the day, nearly every day, as indicated by either subjec- tive report (e.g., feels sad, empty. hopeless) or observation made by others (e.g., appears tearful). (Note: In children and adolescents. can be irritable mood.) 2. Markedly diminished interest or pleasure in all, or almost all, activities most of the day. nearly every day (as indicated by either subjective account or observation). 3. Significant weight loss when not dieting or weight gain (e.g., a change of more than 5% of body weight in a month), or decrease or increase in appetite nearly every day.

1	3. Significant weight loss when not dieting or weight gain (e.g., a change of more than 5% of body weight in a month), or decrease or increase in appetite nearly every day. (Note: In children, consider failure to make expected weight gain.) 4. Insomnia or hypersomnia nearly every day. 5. Psychomotor agitation or retardation nearly every day (observable by others, not merely subjective feelings of restlessness or being slowed down). 6. Fatigue or loss of energy nearly every day. 7. Feelings of worthlessness or excessive or inappropriate guilt (which may be delu- sional) nearly every day (not merely seIf-reproach or guilt about being sick). 8. Diminished ability to think or concentrate, or indecisiveness, nearly every day (ei- ther by subjective account or as observed by others). 9. Recurrent thoughts of death (not just fear of dying), recurrent suicidal ideation with- out a specific plan, or a suicide attempt or a specific plan for committing suicide.

1	9. Recurrent thoughts of death (not just fear of dying), recurrent suicidal ideation with- out a specific plan, or a suicide attempt or a specific plan for committing suicide. B. The symptoms cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. C. The episode is not attributable to the physiological effects of a substance or to another medical condition. Note: Criteria A—C represent a major depressive episode.

1	C. The episode is not attributable to the physiological effects of a substance or to another medical condition. Note: Criteria A—C represent a major depressive episode. Note: Responses to a significant loss (e.g., bereavement, financial ruin, losses from a nat- ural disaster, a serious medical illness or disability) may include the feelings of intense sad- ness, rumination about the loss, insomnia, poor appetite, and weight loss noted in Criterion A, which may resemble a depressive episode. Although such symptoms may be understand- able or considered appropriate to the loss, the presence of a major depressive episode in addition to the normal response to a significant loss should also be carefully considered. This decision inevitably requires the exercise of clinical judgment based on the individual’s history and the cultural norms for the expression of distress in the context of loss.1

1	D. The occurrence of the major depressive episode is not better explained by schizoaf- fective disorder, schizophrenia, schizophreniform disorder, delusional disorder, or other specified and unspecified schizophrenia spectrum and other psychotic disorders. E. There has never been a manic episode or a hypomanic episode. Note: This exclusion does not apply if all of the manic-Iike or hypomanic-Iike episodes are substance-induced or are attributable to the physiological effects of another med- ical condition.

1	Note: This exclusion does not apply if all of the manic-Iike or hypomanic-Iike episodes are substance-induced or are attributable to the physiological effects of another med- ical condition. 1In distinguishing grief from a major depressive episode (MDE), it is useful to consider that in grief the predominant affect is feelings of emptiness and loss, while in MDE it is persistent depressed mood and the inability to anticipate happiness or pleasure. The dysphoria in grief is likely to decrease in intensity over days to weeks and occurs in waves, the so-Called pangs of grief. These waves tend to be associated with thoughts or reminders of the deceased. The depressed mood of MDE is more persistent and not tied to specific thoughts or preoccupations.

1	The pain of grief may be accompanied by positive emotions and humor that are uncharacteristic of the pervasive unhappiness and misery characteristic of MDE. The thought content associated with grief generally features a preoccupation with thoughts and memories of the deceased, rather than the self—critical or pessimistic ruminations seen in MDE. In grief, self—esteem is gener- ally preserved, whereas in MDE feelings of worthlessness and self—loathing are common. If self- derogatory ideation is present in grief, it typically involves perceived failings vis-a-vis the deceased (e.g., not visiting frequently enough, not telling the deceased how much he or she was loved). If a bereaved individual thinks about death and dying, such thoughts are generally focused on the deceased and possibly about "joining" the deceased, whereas in MDE such thoughts are focused on ending one’s own life because of feeling worthless, undeserving of life, or unable to cope with the pain of depression.

1	The diagnostic code for major depressive disorder is based on whether this is a single or recurrent episode, current severity, presence of psychotic features, and remission status. Current severity and psychotic features are only indicated it full criteria are currently met for a major depressive episode. Remission specifiers are only indicated if the full criteria are not currently met for a major depressive episode. Codes are as follows: Mild (p. 188) 296.21 (F320) 296.31 (F330) Moderate (p. 188) 296.22 (F321) 296.32 (F33.1) Severe (p. 188) 296.23 (F322) 296.33 (F332) With psychotic features“ (p. 186) 296.24 (F323) 296.34 (F333) In partial remission (p. 188) 296.25 (F32.4) 296.35 (F33.41) In full remission (p. 188) 296.26 (F325) 296.36 (F33.42)

1	With psychotic features“ (p. 186) 296.24 (F323) 296.34 (F333) In partial remission (p. 188) 296.25 (F32.4) 296.35 (F33.41) In full remission (p. 188) 296.26 (F325) 296.36 (F33.42) Unspecified 296.20 (F329) 296.30 (F33.9) ‘For an episode to be considered recurrent, there must be an interval of at least 2 consecutive months between separate episodes in which criteria are not met for a major depressive episode. The defini- tions of specifiers are found on the indicated pages. "If psychotic features are present, code the "with psychotic features" specifier irrespective of epi- sode severity. In recording the name of a diagnosis, terms should be listed in the following order: major depressive disorder, single or recurrent episode, severity/psychotic/remission specifiers, followed by as many of the following specifiers without codes that apply to the current episode. Specify: With anxious distress (p. 184) With mixed features (pp. 184—185) With melancholic features (p. 185)

1	Specify: With anxious distress (p. 184) With mixed features (pp. 184—185) With melancholic features (p. 185) With atypical features (pp. 185—186) With mood-congruent psychotic features (p. 186) With mood-incongruent psychotic features (p. 186) With catatonia (p. 186). Coding note: Use additional code 293.89 (F06.1). With peripartum onset (pp. 186—187) With seasonal pattern (recurrent episode only) (pp. 187—188)

1	With peripartum onset (pp. 186—187) With seasonal pattern (recurrent episode only) (pp. 187—188) The criterion symptoms for major depressive disorder must be present nearly every day to be considered present, with the exception of weight change and suicidal ideation. De- pressed mood must be present for most of the day, in addition to being present nearly ev- ery day. Often insomnia or fatigue is the presenting complaint, and failure to probe for accompanying depressive symptoms will result in underdiagnosis. Sadness may be de- demeanor. With individuals who focus on a somatic complaint, clinicians should de- termine whether the distress from that complaint is associated with specific depressive symptoms. Fatigue and sleep disturbance are present in a high proportion of cases; psy- chomotor disturbances are much less common but are indicative of greater overall sever- ity, as is the presence of delusional or near-delusional guilt.

1	The essential feature of a major depressive episode is a period of at least 2 weeks during which there is either depressed mood or the loss of interest or pleasure in nearly all activi- ties (Criterion A). In children and adolescents, the mood may be irritable rather than sad.

1	The individual must also experience at least four additional symptoms drawn from a list that includes changes in appetite or weight, sleep, and psychomotor activity; decreased en- ergy; feelings of worthlessness or guilt; difficulty thinking, concentrating, or making deci- sions; or recurrent thoughts of death or suicidal ideation or suicide plans or attempts. To count toward a major depressive episode, a symptom must either be newly present or must have clearly worsened compared with the person’s pre-episode status. The symptoms must persist for most of the day, nearly every day, for at least 2 consecutive weeks. The ep- isode must be accompanied by clinically significant distress or impairment in social, occu- pational, or other important areas of functioning. For some individuals with milder episodes, functioning may appear to be normal but requires markedly increased effort.

1	The mood in a major depressive episode is often described by the person as depressed, sad, hopeless, discouraged, or "down in the dumps" (Criterion A1). In some cases, sadness may be denied at first but may subsequently be elicited by interview (e.g., by pointing out that the individual looks as if he or she is about to cry). In some individuals who complain of feeling ”blah,” having no feelings, or feeling anxious, the presence of a depressed mood can be inferred from the person’s facial expression and demeanor. Some individuals em— phasize somatic complaints (e.g., bodily aches and pains) rather than reporting feelings of sadness. Many individuals report or exhibit increased irritability (e.g., persistent anger, a tendency to respond to events with angry outbursts or blaming others, an exaggerated sense of frustration over minor matters). In children and adolescents, an irritable or cranky mood may develop rather than a sad or dejected mood. This presentation should be dif-

1	an exaggerated sense of frustration over minor matters). In children and adolescents, an irritable or cranky mood may develop rather than a sad or dejected mood. This presentation should be dif- ferentiated from a pattern of irritability when frustrated.

1	Loss of interest or pleasure is nearly always present, at least to some degree. Individ- uals may report feeling less interested in hobbies, “not caring anymore,” or not feeling any enjoyment in activities that were previously considered pleasurable (Criterion A2). Family members often notice social withdrawal or neglect of pleasurable avocations (e.g., a for- merly avid golfer no longer plays, a child who used to enjoy soccer finds excuses not to practice). In some individuals, there is a significant reduction from previous levels of sex- ual interest or desire. Appetite change may involve either a reduction or increase. Some depressed individ- uals report that they have to force themselves to eat. Others may eat more and may crave specific foods (e.g., sweets or other carbohydrates). When appetite changes are severe (in either direction), there may be a significant loss or gain in weight, or, in children, a failure to make expected weight gains may be noted (Criterion A3).

1	Sleep disturbance may take the form of either difficulty sleeping or sleeping exces- sively (Criterion A4). When insomnia is present, it typically takes the form of middle in- somnia (i.e., waking up during the night and then having difficulty returning to sleep) or terminal insomnia (i.e., waking too early and being unable to return to sleep). Initial in- somnia (i.e., difficulty falling asleep) may also occur. Individuals who present with over- daytime sleep. Sometimes the reason that the individual seeks treatment is for the dis- turbed sleep.

1	Psychomotor changes include agitation (e.g., the inability to sit still, pacing, hand- wringing; or pulling or rubbing of the skin, clothing, or other objects) or retardation (e.g., slowed speech, thinking, and body movements; increased pauses before answering; speech that is decreased in volume, inﬂection, amount, or variety of content, or muteness) (Criterion A5). The psychomotor agitation or retardation must be severe enough to be ob- servable by others and not represent merely subjective feelings. Decreased energy, tiredness, and fatigue are common (Criterion A6). A person may re- port sustained fatigue without physical exertion. Even the smallest tasks seem to require substantial effort. The efficiency with which tasks are accomplished may be reduced. For example, an individual may complain that washing and dressing in the morning are ex- hausting and take twice as long as usual.

1	The sense of worthlessness or guilt associated with a major depressive episode may in- clude unrealistic negative evaluations of one’s worth or guilty preoccupations or rumina- tions over minor past failings (Criterion A7). Such individuals often misinterpret neutral or trivial day-to—day events as evidence of personal defects and have an exaggerated sense of responsibility for untoward events. The sense of worthlessness or guilt may be of delu- sional proportions (e.g., an individual who is convinced that he or she is personally re- sponsible for world poverty). Blaming oneself for being sick and for failing to meet occupational or interpersonal responsibilities as a result of the depression is very common and, unless delusional, is not considered sufficient to meet this criterion.

1	Many individuals report impaired ability to think, concentrate, or make even minor decisions (Criterion A8). They may appear easily distracted or complain of memory diffi— culties. Those engaged in cognitively demanding pursuits are often unable to function. In children, a precipitous drop in grades may reﬂect poor concentration. In elderly individ- uals, memory difficulties may be the chief complaint and may be mistaken for early signs of a dementia (”pseudodementia”). When the major depressive episode is successfully treated, the memory problems often fully abate. However, in some individuals, particu- larly elderly persons, a major depressive episode may sometimes be the initial presenta- tion of an irreversible dementia. Thoughts of death, suicidal ideation, or suicide attempts (Criterion A9) are common.

1	Thoughts of death, suicidal ideation, or suicide attempts (Criterion A9) are common. They may range from a passive wish not to awaken in the morning or a belief that others would be better off if the individual were dead, to transient but recurrent thoughts of com- mitting suicide, to a specific suicide plan. More severely suicidal individuals may have put their affairs in order (e.g., updated wills, settled debts), acquired needed materials (e.g., a rope or a gun), and chosen a location and time to accomplish the suicide. Motivations for suicide may include a desire to give up in the face of perceived insurmountable obstacles, an intense wish to end what is perceived as an unending and excruciatingly painful emo- tional state, an inability to foresee any enjoyment in life, or the wish to not be a burden to others. The resolution of such thinking may be a more meaningful measure of diminished suicide risk than denial of further plans for suicide.

1	The evaluation of the symptoms of a major depressive episode is especially difficult when they occur in an individual who also has a general medical condition (e.g., cancer, stroke, myocardial infarction, diabetes, pregnancy). Some of the criterion signs and symp- toms of a major depressive episode are identical to those of general medical conditions (e.g., weight loss with untreated diabetes; fatigue with cancer; hypersomnia early in preg- nancy; insomnia later in pregnancy or the postpartum). Such symptoms count toward a major depressive diagnosis except when they are clearly and fully attributable to a general medical condition. Nonvegetative symptoms of dysphoria, anhedonia, guilt or worthless- ness, impaired concentration or indecision, and suicidal thoughts should be assessed with particular care in such cases. Definitions of major depressive episodes that have been mod- ified to include only these nonvegetative symptoms appear to identify nearly the same in- dividuals as do the

1	care in such cases. Definitions of major depressive episodes that have been mod- ified to include only these nonvegetative symptoms appear to identify nearly the same in- dividuals as do the full criteria.

1	Major depressive disorder is associated with high mortality, much of which is accounted for by suicide; however, it is not the only cause. For example, depressed individuals ad- mitted to nursing homes have a markedly increased likelihood of death in the first year. In- dividuals frequently present with tearfulness, irritability, brooding, obsessive rumination, anxiety, phobias, excessive worry over physical health, and complaints of pain (e.g., head- aches; joint, abdominal, or other pains). In children, separation anxiety may occur.

1	Although an extensive literature exists describing neuroanatomical, neuroendocrino- logical, and neurophysiological correlates of major depressive disorder, no laboratory test has yielded results of sufficient sensitivity and specificity to be used as a diagnostic tool for this disorder. Until recently, hypothalamic-pituitary-adrenal axis hyperactivity had been the most extensively investigated abnormality associated with major depressive episodes, and it appears to be associated with melancholia, psychotic features, and risks for eventual suicide. Molecular studies have also implicated peripheral factors, including genetic vari- ants in neurotrophic factors and pro-inﬂammatory cytokines. Additionally, functional cific neural systems supporting emotion processing, reward seeking, and emotion regula- tion in adults with major depression.

1	Twelve—month prevalence of major depressive disorder in the United States is approximately 7%, with marked differences by age group such that the prevalence in 18- to 29-year-old indi- viduals is threefold higher than the prevalence in individuals age 60 years or older. Females ex- perience 1.5— to 3-fold higher rates than males beginning in early adolescence. Major depressive disorder may first appear at any age, but the likelihood of onset in- creases markedly with puberty. In the United States, incidence appears to peak in the 205; however, first onset in late life is not uncommon.

1	The course of major depressive disorder is quite variable, such that some individuals rarely, if ever, experience remission (a period of 2 or more months with no symptoms, or only one or two symptoms to no more than a mild degree), while others experience many years with few or no symptoms between discrete episodes. It is important to distinguish individuals who present for treatment during an exacerbation of a chronic depressive ill- ness from those whose symptoms developed recently. Chronicity of depressive symptoms substantially increases the likelihood of underlying personality, anxiety, and substance use disorders and decreases the likelihood that treatment will be followed by full symp- tom resolution. It is therefore useful to ask individuals presenting with depressive symp- toms to identify the last period of at least 2 months during which they were entirely free of depressive symptoms.

1	Recovery typically begins within 3 months of onset for two in five individuals with ma- jor depression and within 1 year for four in five individuals. Recency of onset is a strong determinant of the likelihood of near-term recovery, and many individuals who have been depressed only for several months can be expected to recover spontaneously. Features as- sociated with lower recovery rates, other than current episode duration, include psychotic features, prominent anxiety, personality disorders, and symptom severity. The risk of recurrence becomes progessively lower over time as the duration of re- mission increases. The risk is higher in individuals whose preceding episode was severe, in younger individuals, and in individuals who have already experienced multiple epi- sodes. The persistence of even mild depressive symptoms during remission is a powerful predictor of recurrence.

1	Many bipolar illnesses begin with one or more depressive episodes, and a substantial proportion of individuals who initially appear to have major depressive disorder will prove, in time, to instead have a bipolar disorder. This is more likely in individuals with onset of the illness in adolescence, those with psychotic features, and those with a family history of bipolar illness. The presence of a "with mixed features” specifier also increases the risk for future manic or hypomanic diagnosis. Major depressive disorder, particularly with psychotic features, may also transition into schizophrenia, a change that is much more frequent than the reverse.

1	ders, there appear to be no clear differences by gender in phenomenology, course, or treat- ment response. Similarly, there are no clear effects of current age on the course or treatment response of major depressive disorder. Some symptom differences exist, though, such that hypersomnia and hyperphagia are more likely in younger individuals, and melancholic symptoms, particularly psychomotor disturbances, are more common in older individuals. The likelihood of suicide attempts lessens in middle and late life, although the risk of com- pleted suicide does not. Depressions with earlier ages at onset are more familial and more likely to involve personality disturbances. The course of major depressive disorder within individuals does not generally change with aging. Mean times to recovery appear to be sta— ble over long periods, and the likelihood of being in an episode does not generally increase or decrease with time.

1	Temperamental. Neuroticism (negative affectivity) is a well-established risk factor for the onset of major depressive disorder, and high levels appear to render individuals more likely to develop depressive episodes in response to stressful life events. Environmental. Adverse childhood experiences, particularly when there are multiple experiences of diverse types, constitute a set of potent risk factors for major depressive dis- order. Stressful life events are well recognized as precipitants of major depressive epi— sodes, but the presence or absence of adverse life events near the onset of episodes does not appear to provide a useful guide to prognosis or treatment selection.

1	Genetic and physiological. First—degree family members of individuals with major de~ pressive disorder have a risk for major depressive disorder two- to fourfold higher than that of the general population. Relative risks appear to be higher for early-onset and re- current forms. Heritability is approximately 40%, and the personality trait neuroticism ac- counts for a substantial portion of this genetic liability.

1	Course modifiers. Essentially all major nonmood disorders increase the risk of an indi- vidual developing depression. Major depressive episodes that develop against the back- ground of another disorder often follow a more refractory course. Substance use, anxiety, and borderline personality disorders are among the most common of these, and the pre- senting depressive symptoms may obscure and delay their recognition. However, sus- tained clinical improvement in depressive symptoms may depend on the appropriate treatment of underlying illnesses. Chronic or disabling medical conditions also increase risks for major depressive episodes. Such prevalent illnesses as diabetes, morbid obesity, and cardiovascular disease are often complicated by depressive episodes, and these epi- sodes are more likely to become chronic than are depressive episodes in medically healthy individuals.

1	Surveys of major depressive disorder across diverse cultures have shown sevenfold dif- ferences in 12-month prevalence rates but much more consistency in female-to-male ratio, mean ages at onset, and the degree to which presence of the disorder raises the likelihood of comorbid substance abuse. While these findings suggest substantial cultural differences in the expression of major depressive disorder, they do not permit simple linkages be- tween particular cultures and the likelihood of specific symptoms. Rather, clinicians should be aware that in most countries the majority of cases of depression go unrecog- nized in primary care settings and that in many cultures, somatic symptoms are very likely to constitute the presenting complaint. Among the Criterion A symptoms, insomnia and loss of energy are the most uniformly reported.

1	Although the most reproducible finding in the epidemiology of major depressive disorder has been a higher prevalence in females, there are no clear differences between genders in symptoms, course, treatment response, or functional consequences. In women, the risk for suicide attempts is higher, and the risk for suicide completion is lower. The disparity in suicide rate by gender is not as great among those with depressive disorders as it is in the population as a whole. The possibility of suicidal behavior exists at all times during major depressive episodes.

1	The possibility of suicidal behavior exists at all times during major depressive episodes. The most consistently described risk factor is a past history of suicide attempts or threats, but it should be remembered that most completed suicides are not preceded by unsuccess- ful attempts. Other features associated with an increased risk for completed suicide include male sex, being single or living alone, and having prominent feelings of hopeless- ness. The presence of borderline personality disorder markedly increases risk for future suicide attempts.

1	Many of the functional consequences of major depressive disorder derive from individual symptoms. Impairment can be very mild, such that many of those who interact with the af- fected individual are unaware of depressive symptoms. Impairment may, however, range to complete incapacity such that the depressed individual is unable to attend to basic self- care needs or is mute or catatonic. Among individuals seen in general medical settings, those with major depressive disorder have more pain and physical illness and greater de- creases in physical, social, and role functioning. Manic episodes with irritable mood or mixed episodes. Major depressive episodes with prominent irritable mood may be difficult to distinguish from manic episodes with irritable mood or from mixed episodes. This distinction requires a careful clinical evalua- tion of the presence of manic symptoms.

1	Mood disorder due to another medical condition. A major depressive episode is the appropriate diagnosis if the mood disturbance is not judged, based on individual history, physical examination, and laboratory findings, to be the direct pathophysiological conse- quence of a specific medical condition (e.g., multiple sclerosis, stroke, hypothyroidism). Substance/medication-induced depressive or bipolar disorder. This disorder is distin- guished from major depressive disorder by the fact that a substance (e.g., a drug of abuse, a medication, a toxin) appears to be etiologically related to the mood disturbance. For ex- ample, depressed mood that occurs only in the context of withdrawal from cocaine would be diagnosed as cocaine-induced depressive disorder.

1	Attention-deficit/hyperactivity disorder. Distractibility and low frustration tolerance sode; if the criteria are met for both, attention-deficit/hyperactivity disorder may be diag— nosed in addition to the mood disorder. However, the clinician must be cautious not to overdiagnose a major depressive episode in children with attention-deficit/hyperactivity disorder whose disturbance in mood is characterized by irritability rather than by sadness or loss of interest. Adjustment disorder with depressed mood. A major depressive episode that occurs in response to a psychosocial stressor is distinguished from adjustment disorder with de- pressed mood by the fact that the full criteria for a major depressive episode are not met in adjustment disorder. Sadness. Finally, periods of sadness are inherent aspects of the human experience.

1	Sadness. Finally, periods of sadness are inherent aspects of the human experience. These periods should not be diagnosed as a major depressive episode unless criteria are met for severity (i.e., five out of nine symptoms), duration (i.e., most of the day, nearly ev- ery day for at least 2 weeks), and clinically significant distress or impairment. The diagno- sis other specified depressive disorder may be appropriate for presentations of depressed mood with clinically significant impairment that do not meet criteria for duration or se- verity. Other disorders with which major depressive disorder frequently co-occurs are substance— related disorders, panic disorder, obsessive-compulsive disorder, anorexia nervosa, buli- mia nervosa, and borderline personality disorder. Diagnostic Criteria 300.4 (F34.1) This disorder represents a consolidation of DSM-IV-defined chronic major depressive dis- order and dysthymic disorder.

1	Diagnostic Criteria 300.4 (F34.1) This disorder represents a consolidation of DSM-IV-defined chronic major depressive dis- order and dysthymic disorder. A. Depressed mood for most of the day, for more days than not, as indicated by either subjective account or observation by others, for at least 2 years. Note: In children and adolescents, mood can be irritable and duration must be at least 1 year. B. Presence, while depressed, of two (or more) of the following: Poor appetite or overeating. Insomnia or hypersomnia. Low energy or fatigue. Low self-esteem. Poor concentration or difficulty making decisions. Feelings of hopelessness. C. During the 2-year period (1 year for children or adolescents) of the disturbance, the individ- ual has never been without the symptoms in Criteria A and B for more than 2 months at a time. D. Criteria for a major depressive disorder may be continuously present for 2 years.

1	D. Criteria for a major depressive disorder may be continuously present for 2 years. E. There has never been a manic episode or a hypomanic episode. and criteria have never been met for cyclothymic disorder. F. The disturbance is not better explained by a persistent schizoaffective disorder, schizophrenia, delusional disorder, or other specified or unspecified schizophrenia spectrum and other psychotic disorder. G. The symptoms are not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication) or another medical condition (e.g. hypothyroidism). H. The symptoms cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. P?PF‘JNT‘

1	H. The symptoms cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. P?PF‘JNT‘ Note: Because the criteria for a major depressive episode include four symptoms that are absent from the symptom list for persistent depressive disorder (dysthymia), a very limited number of individuals will have depressive symptoms that have persisted longer than 2 years but will not meet criteria for persistent depressive disorder. If full criteria for a major de— pressive episode have been met at some point during the current episode of illness, they should be given a diagnosis of major depressive disorder. Otherwise, a diagnosis of other specified depressive disorder or unspecified depressive disorder is warranted. Specify it: With anxious distress (p. 184) With mixed features (pp. 184—185) With melancholic features (p. 185) With atypical features (pp. 185—186) With mood-congruent psychotic features (p. 186)

1	With anxious distress (p. 184) With mixed features (pp. 184—185) With melancholic features (p. 185) With atypical features (pp. 185—186) With mood-congruent psychotic features (p. 186) With mood-incongruent psychotic features (p. 186) With peripartum onset (pp. 186—187) Specify it: In partial remission (p. 188) In full remission (p. 188) Specify if: Early onset: If onset is before age 21 years. Late onset: If onset is at age 21 years or older. Specify if (for most recent 2 years of persistent depressive disorder): With pure dysthymic syndrome: Full criteria for a major depressive episode have not been met in at least the preceding 2 years. With persistent major depressive episode: Full criteria for a major depressive epi- sode have been met throughout the preceding 2-year period.

1	With persistent major depressive episode: Full criteria for a major depressive epi- sode have been met throughout the preceding 2-year period. With intermittent major depressive episodes, with current episode: Full criteria for a major depressive episode are currently met. but there have been periods of at least 8 weeks in at least the preceding 2 years with symptoms below the threshold for a full major depressive episode. With intermittent major depressive episodes, without current episode: Full crite- ria for a major depressive episode are not currently met, but there has been one or more major depressive episodes in at least the preceding 2 years. Specify current severity: Mild (p. 188) Moderate (p. 188) Severe (p. 188)

1	Specify current severity: Mild (p. 188) Moderate (p. 188) Severe (p. 188) The essential feature of persistent depressive disorder (dysthymia) is a depressed mood that occurs for most of the day, for more days than not, for at least 2 years, or at least 1 year for children and adolescents (Criterion A). This disorder represents a consolidation of DSM-IV-defined chronic major depressive disorder and dysthymic disorder. Major de- pression may precede persistent depressive disorder, and major depressive episodes may occur during persistent depressive disorder. Individuals whose symptoms meet major de- pressive disorder criteria for 2 years should be given a diagnosis of persistent depressive disorder as well as major depressive disorder. Individuals with persistent depressive disorder describe their mood as sad or "down in the dumps.” During periods of depressed mood, at least two of the six symptoms from

1	Individuals with persistent depressive disorder describe their mood as sad or "down in the dumps.” During periods of depressed mood, at least two of the six symptoms from Criterion B are present. Because these symptoms have become a part of the individual’s day-to-day experience, particularly in the case of early onset (e.g., "I’ve always been this way”), they may not be reported unless the individual is directly prompted. During the 2-year period (1 year for children or adolescents), any symptom-free intervals last no longer than 2 months (Criterion C). Persistent depressive disorder is effectively an amalgam of DSM-IV dysthymic disorder and chronic major depressive episode. The 12—month prevalence in the United States is approxi- mately 0.5% for persistent depressive disorder and 1.5% for chronic major depressive disorder.

1	Persistent depressive disorder often has an early and insidious onset (i.e., in childhood, adolescence, or early adult life) and, by definition, a chronic course. Among individuals with both persistent depressive disorder and borderline personality disorder, the covari- ance of the corresponding features over time suggests the operation of a common mecha- nism. Early onset (i.e., before age 21 years) is associated with a higher likelihood of comorbid personality disorders and substance use disorders. When symptoms rise to the level of a major depressive episode, they are likely to sub- sequently revert to a lower level. However, depressive symptoms are much less likely to resolve in a given period of time in the context of persistent depressive disorder than they are in a major depressive episode.

1	Temperamental. Factors predictive of poorer long—term outcome include higher levels of neuroticism (negative affectivity), greater symptom severity, poorer global functioning, and presence of anxiety disorders or conduct disorder. Environmental. Childhood risk factors include parental loss or separation. Genetic and physiological. There are no clear differences in illness development, course, order. Earlier findings pertaining to either disorder are therefore likely to apply to per- sistent depressive disorder. It is thus likely that individuals with persistent depressive disorder will have a higher proportion of first-degree relatives with persistent depressive disorder than do individuals with major depressive disorder, and more depressive disor- ders in general. A number of brain regions (e.g., prefrontal cortex, anterior cingulate, amygdala, hip- pocampus) have been implicated in persistent depressive disorder. Possible polysomno— graphic abnormalities exist as well.

1	The degree to which persistent depressive disorder impacts social and occupational func- tioning is likely to vary widely, but effects can be as great as or greater than those of major depressive disorder.

1	Major depressive disorder. If there is a depressed mood plus two or more symptoms meeting criteria for a persistent depressive episode for 2 years or more, then the diagnosis of persistent depressive disorder is made. The diagnosis depends on the 2—year duration, which distinguishes it from episodes of depression that do not last 2 years. If the symptom criteria are sufficient for a diagnosis of a major depressive episode at any time during this pe- riod, then the diagnosis of major depression should be noted, but it is coded not as a separate diagnosis but rather as a specifier with the diagnosis of persistent depressive disorder. If the individual’s symptoms currently meet full criteria for a major depressive episode, then the specifier of "with intermittent major depressive episodes, with current episode” would be made. If the major depressive episode has persisted for at least a 2—year duration and re— mains present, then the specifier ”with persistent major depressive episode” is

1	current episode” would be made. If the major depressive episode has persisted for at least a 2—year duration and re— mains present, then the specifier ”with persistent major depressive episode” is used. When full major depressive episode criteria are not currently met but there has been at least one previous episode of major depression in the context of at least 2 years of persistent depres- sive symptoms, then the specifier of ”with intermittent major depressive episodes, without current episode" is used. If the individual has not experienced an episode of major depres- sion in the last 2 years, then the specifier "with pure dysthymic syndrome" is used.

1	Psychotic disorders. Depressive symptoms are a common associated feature of chronic psychotic disorders (e.g., schizoaffective disorder, schizophrenia, delusional disorder). A separate diagnosis of persistent depressive disorder is not made if the symptoms occur only during the course of the psychotic disorder (including residual phases).

1	Depressive or bipolar and related disorder due to another medical condition. Persistent order due to another medical condition. The diagnosis is depressive or bipolar and related disorder due to another medical condition if the mood disturbance is judged, based on his- tory, physical examination, or laboratory findings, to be attributable to the direct patho- physiological effects of a specific, usually chronic, medical condition (e.g., multiple sclerosis). If it is judged that the depressive symptoms are not attributable to the physiolog- ical effects of another medical condition, then the primary mental disorder (e.g., persistent depressive disorder) is recorded, and the medical condition is noted as a concomitant med- ical condition (e.g., diabetes mellitus).

1	Substance/medication-induced depressive or bipolar disorder. A substance/medi— cation-induced depressive or bipolar and related disorder is distinguished from persis- tent depressive disorder when a substance (e.g., a drug of abuse, a medication, a toxin) is judged to be etiologically related to the mood disturbance. Personality disorders. Often, there is evidence of a coexisting personality disturbance. When an individual’s presentation meets the criteria for both persistent depressive disor- der and a personality disorder, both diagnoses are given. In comparison to individuals with major depressive disorder, those with persistent de- pressive disorder are at higher risk for psychiatric comorbidity in general, and for anxiety disorders and substance use disorders in particular. Early-onset persistent depressive dis- order is strongly associated with DSM-IV Cluster B and C personality disorders. Diagnostic Criteria 625.4 (N94.3)

1	Diagnostic Criteria 625.4 (N94.3) A. In the majority of menstrual cycles. at least five symptoms must be present in the final week before the onset of menses, start to improve within a few days after the onset of menses, and become minimal or absent in the week postmenses. B. One (or more) of the following symptoms must be present: 1. Marked affective lability (e.g., mood swings; feeling suddenly sad or tearful, or in- creased sensitivity to rejection). 2. Marked irritability or anger or increased interpersonal conflicts. 3. Marked depressed mood, feelings of hopelessness, or seIf-deprecating thoughts. 4. Marked anxiety, tension, and/or feelings of being keyed up or on edge. C. One (or more) of the following symptoms must additionally be present, to reach a total of five symptoms when combined with symptoms from Criterion B above. . Decreased interest in usual activities (e.g., work, school, friends, hobbies). . Subjective difficulty in concentration.

1	. Decreased interest in usual activities (e.g., work, school, friends, hobbies). . Subjective difficulty in concentration. . Lethargy, easy fatigability, or marked lack of energy. . Marked change in appetite; overeating; or specific food cravings. . Hypersomnia or insomnia. . A sense of being overwhelmed or out of control. . Physical symptoms such as breast tenderness or swelling, joint or muscle pain, a sensation of “bloating,” or weight gain. Note: The symptoms in Criteria A—C must have been met for most menstrual cycles that occurred in the preceding year. D. The symptoms are associated with clinically significant distress or interference with work, school, usual social activities, or relationships with others (e.g., avoidance of so- cial activities; decreased productivity and efficiency at work, school, or home).

1	E. The disturbance is not merely an exacerbation of the symptoms of another disorder, such as major depressive disorder, panic disorder, persistent depressive disorder (dysthymia), or a personality disorder (although it may co-occur with any of these dis- orders). F. Criterion A should be confirmed by prospective daily ratings during at least two symptom- atic cycles. (Note: The diagnosis may be made provisionally prior to this confirmation.) G. The symptoms are not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication, other treatment) or another medical condition (e.g., hy- perthyroidism). If symptoms have not been confirmed by prospective daily ratings of at least two symp- tomatic cycles, "provisional" should be noted after the name of the diagnosis (i.e., "pre- menstrual dysphoric disorder, provisional").

1	The essential features of premenstrual dysphoric disorder are the expression of mood la- bility, irritability, dysphoria, and anxiety symptoms that occur repeatedly during the pre- menstrual phase of the cycle and remit around the onset of menses or shortly thereafter. These symptoms may be accompanied by behavioral and physical symptoms. Symptoms must have occurred in most of the menstrual cycles during the past year and must have an adverse effect on work or social functioning. The intensity and / or expressivity of the ac- companying symptoms may be closely related to social and cultural background charac- teristics of the affected female, family perspectives, and more specific factors such as religious beliefs, social tolerance, and female gender role issues.

1	Typically, symptoms peak around the time of the onset of menses. Although it is not uncommon for symptoms to linger into the first few days of menses, the individual must have a symptom-free period in the follicular phase after the menstrual period begins. While the core symptoms include mood and anxiety symptoms, behavioral and somatic symptoms commonly also occur. However, the presence of physical and/ or behavioral symptoms in the absence of mood and/ or anxious symptoms is not sufficient for a diag- nosis. Symptoms are of comparable severity (but not duration) to those of another mental disorder, such as a major depressive episode or generalized anxiety disorder. In order to confirm a provisional diagnosis, daily prospective symptom ratings are required for at least two symptomatic cycles.

1	Delusions and hallucinations have been described in the late luteal phase of the menstrual cycle but are rare. The premenstrual phase has been considered by some to be a risk period for suicide. Twelve-month prevalence of premenstrual dysphoric disorder is between 1.8% and 5.8% of menstruating women. Estimates are substantially inﬂated if they are based on retro- spective reports rather than prospective daily ratings. However, estimated prevalence based on a daily record of symptoms for 1—2 months may be less representative, as indi- viduals with the most severe symptoms may be unable to sustain the rating process. The most rigorous estimate of premenstrual dysphoric disorder is 1.8% for women whose symptoms meet the full criteria without functional impairment and 1.3% for women whose symptoms meet the current criteria with functional impairment and without co-oc- curring symptoms from another mental disorder.

1	Onset of premenstrual dysphoric disorder can occur at any point after menarche. Inci- dence of new cases over a 40-month follow-up period is 2.5% (95% confidence interval = 1.7—3.7). Anecdotally, many individuals, as they approach menopause, report that symp- toms worsen. Symptoms cease after menopause, although cyclical hormone replacement can trigger the re-expression of symptoms. Environmental. Environmental factors associated with the expression of premenstrual dysphoric disorder include stress, history of interpersonal trauma, seasonal changes, and sociocultural aspects of female sexual behavior in general, and female gender role in par- ticular. Genetic and physiological. Heritability of premenstrual dysphoric disorder is unknown. However, for premenstrual symptoms, estimates for heritability range between 30% and 80%, with the most stable component of premenstrual symptoms estimated to be about 50% heritable.

1	However, for premenstrual symptoms, estimates for heritability range between 30% and 80%, with the most stable component of premenstrual symptoms estimated to be about 50% heritable. Course modifiers. Women who use oral contraceptives may have fewer premenstrual complaints than do women who do not use oral contraceptives. Premenstrual dysphoric disorder is not a culture-bound syndrome and has been observed in individuals in the United States, Europe, India, and Asia. It is unclear as to whether rates differ by race. Nevertheless, frequency, intensity, and expressivity of symptoms and help- seeking patterns may be significantly inﬂuenced by cultural factors. As indicated earlier, the diagnosis of premenstrual dysphoric disorder is appropriately confirmed by 2 months of prospective symptom ratings. A number of scales, including the Daily Rating of Severity of Problems and the Visual Analogue Scales for Premenstrual

1	Daily Rating of Severity of Problems and the Visual Analogue Scales for Premenstrual Mood Symptoms, have undergone validation and are commonly used in clinical trials for premenstrual dysphoric disorder. The Premenstrual Tension Syndrome Rating Scale has a self—report and an observer version, both of which have been validated and used widely to measure illness severity in women who have premenstrual dysphoric disorder. Symptoms must be associated with clinically meaningful distress and/ or an obvious and marked impairment in the ability to function socially or occupationally in the week prior to menses. Impairment in social functioning may be manifested by marital discord and problems with children, other family members, or friends. Chronic marital or job prob- lems should not be confused with dysfunction that occurs only in association with pre- menstrual dysphoric disorder.

1	Premenstrual syndrome. Premenstrual syndrome differs from premenstrual dysphoric disorder in that a minimum of five symptoms is not required, and there is no stipulation of affective symptoms for individuals who have premenstrual syndrome. This condition may be more common than premenstrual dysphoric disorder, although the estimated prevalence of premenstrual syndrome varies. While premenstrual syndrome shares the feature of symptom expression during the premenstrual phase of the menstrual cycle, it is generally considered to be less severe than premenstrual dysphoric disorder. The pres- ence of physical or behavioral symptoms in the premenstruum, without the required affective symptoms, likely meets criteria for premenstrual syndrome and not for premen- strual dysphoric disorder.

1	Dysmenorrhea. Dysmenorrhea is a syndrome of painful menses, but this is distinct from a syndrome characterized by affective changes. Moreover, symptoms of dysmenorrhea begin with the onset of menses, whereas symptoms of premenstrual dysphoric disorder, by defini- tion, begin before the onset of menses, even if they linger into the first few days of menses.

1	Bipolar disorder, major depressive disorder, and persistent depressive disorder (dysthymia). Many women with (either naturally occurring or substance/ medication- that they have premenstrual dysphoric disorder. However, when they chart symptoms, they realize that the symptoms do not follow a premenstrual pattern. Women with an- are unrelated to menstrual cycle phase. However, because the onset of menses constitutes a memorable event, they may report that symptoms occur only during the premenstruum or that symptoms worsen premenstrually. This is one of the rationales for the requirement that symptoms be confirmed by daily prospective ratings. The process of differential di- agnosis, particularly if the clinician relies on retrospective symptoms only, is made more difficult because of the overlap between symptoms of premenstrual dysphoric disorder and some other diagnoses. The overlap of symptoms is particularly salient for differenti- ating premenstrual dysphoric disorder from major

1	overlap between symptoms of premenstrual dysphoric disorder and some other diagnoses. The overlap of symptoms is particularly salient for differenti- ating premenstrual dysphoric disorder from major depressive episodes, persistent de- pressive disorder, bipolar disorders, and borderline personality disorder. However, the rate of personality disorders is no higher in individuals with premenstrual dysphoric dis- order than in those without the disorder.

1	Use of hormonal treatments. Some women who present with moderate to severe pre- menstrual symptoms may be using hormonal treatments, including hormonal contracep- tives. If such symptoms occur after initiation of exogenous hormone use, the symptoms may be due to the use of hormones rather than to the underlying condition of premen- strual dysphoric disorder. If the woman stops hormones and the symptoms disappear, this is consistent with substance/medication-induced depressive disorder.

1	A major depressive episode is the most frequently reported previous disorder in individuals presenting with premenstrual dysphoric disorder. A wide range of medical (e.g., migraine, asthma, allergies, seizure disorders) or other mental disorders (e.g., depressive and bipolar disorders, anxiety disorders, bulimia nervosa, substance use disorders) may worsen in the premenstrual phase; however, the absence of a symptom-free period during the postmen- strual interval obviates a diagnosis of premenstrual dysphoric disorder. These conditions are better considered premenstrual exacerbation of a current mental or medical disorder. Al- though the diagnosis of premenstrual dysphoric disorder should not be assigned in situa— tions in which an individual only experiences a premenstrual exacerbation of another mental or physical disorder, it can be considered in addition to the diagnosis of another men- tal or physical disorder if the individual experiences symptoms and changes in level of func-

1	of another mental or physical disorder, it can be considered in addition to the diagnosis of another men- tal or physical disorder if the individual experiences symptoms and changes in level of func- tioning that are characteristic of premenstrual dysphoric disorder and markedly different from the symptoms experienced as part of the ongoing disorder.

1	A. A prominent and persistent disturbance in mood that predominates in the clinical pic- ture and is characterized by depressed mood or markedly diminished interest or plea- sure in all, or almost all, activities. B. There is evidence from the history, physical examination, or laboratory findings of both (1) and (2): 1. The symptoms in Criterion A developed during or soon after substance intoxication or withdrawal or after exposure to a medication. 2. The involved substance/medication is capable of producing the symptoms in Crite- rion A. C. The disturbance is not better explained by a depressive disorder that is not substance/ medication-induced. Such evidence of an independent depressive disorder could in- clude the following:

1	C. The disturbance is not better explained by a depressive disorder that is not substance/ medication-induced. Such evidence of an independent depressive disorder could in- clude the following: The symptoms preceded the onset of the substance/medication use; the symptoms persist for a substantial period of time (e.g., about 1 month) after the cessation of acute withdrawal or severe intoxication; or there is other evidence suggesting the existence of an independent non-substance/medication-induced depressive disorder (e.g., a his- tory of recurrent non-substance/medication-related episodes). D. The disturbance does not occur exclusively during the course of a delirium. E. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning.

1	E. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. Note: This diagnosis should be made instead of a diagnosis of substance intoxication or substance withdrawal only when the symptoms in Criterion A predominate in the clinical picture and when they are sufficiently severe to warrant clinical attention. Coding note: The |CD-9-CM and |CD-10-CM codes for the [specific substance/medica- tion]-induced depressive disorders are indicated in the table below. Note that the |CD-10-

1	CM code depends on whether or not there is a comorbid substance use disorder present for the same class of substance. If a mild substance use disorder is comorbid with the substance- induced depressive disorder, the 4th position character is “1 and the clinician should record “mild [substance] use disordef’ before the substance-induced depressive disorder (e.g., “mild cocaine use disorder with cocaine-induced depressive disorder”). It a moderate or se- vere substance use disorder is comorbid with the substance-induced depressive disorder, the 4th position character is “,"2 and the clinician should record “moderate [substance] use disorder” or “severe [substance] use disorder," depending on the severity of the comorbid substance use disorder. If there is no comorbid substance use disorder (e.g., after a one- time heavy use of the substance), then the 4th position character is “9," and the clinician should record only the substance-induced depressive disorder.

1	With use disorder, Without disorder, moderate use Alcohol 291.89 F1014 F1024 F10.94 Phencyclidine 292.84 F1614 F1624 F1694 Other hallucinogen 292.84 F1614 F1624 F1694 Inhalant 292.84 F1814 F1824 F1894 Opioid 292.84 F11.14 F1124 F1194 Sedative, hypnotic, or anxiolytic 292.84 F13.14 F1324 F1394 Amphetamine (or other 292.84 F1514 F1524 F1594 Cocaine 292.84 F14.14 F1424 F1494 Other (or unknown) substance 292.84 F1914 F1924 F1994 Specify it (see Table 1 in the chapter “Substance-Related and Addictive Disorders" tor di- agnoses associated with substance class): With onset during intoxication: If criteria are met for intoxication with the substance and the symptoms develop during intoxication. With onset during withdrawal: If criteria are met for withdrawal from the substance and the symptoms develop during, or shortly after, withdrawal.

1	With onset during withdrawal: If criteria are met for withdrawal from the substance and the symptoms develop during, or shortly after, withdrawal. lCD-9-CM. The name of the substance/medication-induced depressive disorder begins with the specific substance (e.g., cocaine, dexamethasone) that is presumed to be causing the depressive symptoms. The diagnostic code is selected from the table included in the criteria set, which is based on the drug class. For substances that do not fit into any of the classes (e.g., dexamethasone), the code for "other substance" should be used; and in cases in which a substance is judged to be an etiological factor but the specific class of substance is unknown, the category ”unknown substance” should be used.

1	The name of the disorder is followed by the specification of onset (i.e., onset during in- toxication, onset during withdrawal). Unlike the recording procedures for ICD-lO-CM, which combine the substance-induced disorder and substance use disorder into a single code, for ICD-9-CM a separate diagnostic code is given for the substance use disorder. For example, in the case of depressive symptoms occurring during withdrawal in a man with a severe cocaine use disorder, the diagnosis is 292.84 cocaine-induced depressive disorder, with onset during withdrawal. An additional diagnosis of 304.20 severe cocaine use dis- order is also given. When more than one substance is judged to play a significant role in the development of depressive mood symptoms, each should be listed separately (e.g., 292.84 methylphenidate-induced depressive disorder, with onset during withdrawal; 292.84 dexamethasone-induced depressive disorder, with onset during intoxication).

1	|CD-10-CM. The name of the substance/medication-induced depressive disorder begins with the specific substance (e.g., cocaine, dexamethasone) that is presumed to be causing the depressive symptoms. The diagnostic code is selected from the table included in the criteria set, which is based on the drug class and presence or absence of a comorbid sub- stance use disorder. For substances that do not fit into any of the classes (e.g., dexameth- asone), the code for “other substance” should be used; and in cases in which a substance is judged to be an etiological factor but the specific class of substance is unknown, the category ”unknown substance” should be used.

1	When recording the name of the disorder, the comorbid substance use disorder (if any) is listed first, followed by the word ”with," followed by the name of the substance-induced de- pressive disorder, followed by the specification of onset (i.e., onset during intoxication, onset during withdrawal). For example, in the case of depressive symptoms occurring during with- drawal in a man with a severe cocaine use disorder, the diagnosis is F1424 severe cocaine use disorder with cocaine-induced depressive disorder, with onset during withdrawal. A separate diagnosis of the comorbid severe cocaine use disorder is not given. If the substance-induced depressive disorder occurs without a comorbid substance use disorder (e.g., after a one—time heavy use of the substance), no accompanying substance use disorder is noted (e.g., F1694 phencyclidine-induced depressive disorder, with onset during intoxication). When more than one substance is judged to play a significant role in the development of

1	use disorder is noted (e.g., F1694 phencyclidine-induced depressive disorder, with onset during intoxication). When more than one substance is judged to play a significant role in the development of depressive mood symptoms, each should be listed separately (e.g., F1524 severe methylphenidate use disorder with methylphenidate-induced depressive disorder, with onset during withdrawal; F1994 dexamethasone-induced depressive disorder, with onset during intoxication).

1	The diagnostic features of substance/medication-induced depressive disorder include the symptoms of a depressive disorder, such as major depressive disorder; however, the de- pressive symptoms are associated with the ingestion, injection, or inhalation of a sub- stance (e.g., drug of abuse, toxin, psychotropic medication, other medication), and the depressive symptoms persist beyond the expected length of physiological effects, intoxi- cation, or withdrawal period. As evidenced by clinical history, physical examination, or laboratory findings, the relevant depressive disorder should have developed during or within 1 month after use of a substance that is capable of producing the depressive disor- der (Criterion B1). In addition, the diagnosis is not better explained by an independent depressive disorder. Evidence of an independent depressive disorder includes the de- pressive disorder preceded the onset of ingestion or withdrawal from the substance; the depressive disorder persists

1	disorder. Evidence of an independent depressive disorder includes the de- pressive disorder preceded the onset of ingestion or withdrawal from the substance; the depressive disorder persists beyond a substantial period of time after the cessation of sub- stance use; or other evidence suggests the existence of an independent non-substance/ medication-induced depressive disorder (Criterion C). This diagnosis should not be made when symptoms occur exclusively during the course of a delirium (Criterion D). The de- pressive disorder associated with the substance use, intoxication, or withdrawal must cause clinically significant distress or impairment in social, occupational, or other impor- tant areas of functioning to qualify for this diagnosis (Criterion E).

1	Some medications (e.g., stimulants, steroids, L-dopa, antibiotics, central nervous system drugs, dermatological agents, chemotherapeutic drugs, immunological agents) can induce depressive mood disturbances. Clinical judgment is essential to determine whether the medication is truly associated with inducing the depressive disorder or whether a primary depressive disorder happened to have its onset while the person was receiving the treatment. For example, a depressive episode that developed within the first several weeks of beginning alpha—methyldopa (an antihypertensive agent) in an individ- ual with no history of major depressive disorder would qualify for the diagnosis of med- ication-induced depressive disorder. In some cases, a previously established condition (e.g., major depressive disorder, recurrent) can recur while the individual is coincidentally taking a medication that has the capacity to cause depressive symptoms (e.g., L-dopa, oral contraceptives). In such cases, the

1	disorder, recurrent) can recur while the individual is coincidentally taking a medication that has the capacity to cause depressive symptoms (e.g., L-dopa, oral contraceptives). In such cases, the clinician must make a judgment as to whether the med- ication is causative in this particular situation.

1	A substance/medication-induced depressive disorder is distinguished from a primary depressive disorder by considering the onset, course, and other factors associated with the substance use. There must be evidence from the history, physical examination, or labora— tory findings of substance use, abuse, intoxication, or withdrawal prior to the onset of the depressive disorder. The withdrawal state for some substances can be relatively pro- tracted, and thus intense depressive symptoms can last for a long period after the cessation of substance use. In a nationally representative U.S. adult population, the lifetime prevalence of substance/ medication-induced depressive disorder is 0.26%.

1	In a nationally representative U.S. adult population, the lifetime prevalence of substance/ medication-induced depressive disorder is 0.26%. A depressive disorder associated with the use of substance (i.e., alcohol, illicit drugs, or a set while the individual is using the substance or during withdrawal, if there is a with— drawal syndrome associated with the substance. Most often, the depressive disorder has its onset within the first few weeks or 1 month of use of the substance. Once the substance is discontinued, the depressive symptoms usually remit within days to several weeks, de- pending on the half—life of the substance/medication and the presence of a withdrawal syndrome. If symptoms persist 4 weeks beyond the expected time course of withdrawal of a particular substance/medication, other causes for the depressive mood symptoms should be considered.

1	Although there are a few prospective controlled trials examining the association of de— pressive symptoms with use of a medication, most reports are from postmarketing sur- veillance studies, retrospective observational studies, or case reports, making evidence of causality difficult to determine. Substances implicated in medication-induced depressive disorder, with varying degrees of evidence, include antiviral agents (efavirenz), cardio- vascular agents (clonidine, guanethidine, methyldopa, reserpine), retinoic acid deriva- tives (isotretinoin), antidepressants, anticonvulsants, anti-migraine agents (triptans), antipsychotics, hormonal agents (corticosteroids, oral contraceptives, gonadotropin- releasing hormone agonists, tamoxifen), smoking cessation agents (varenicline), and im- munological agents (interferon). However, other potential substances continue to emerge as new compounds are synthesized. A history of such substance use may help increase di- agnostic certainty.

1	Temperamental. Factors that appear to increase the risk of substance/medication- induced depressive disorder can be conceptualized as pertaining to the specific type of drug or to a group of individuals with underlying alcohol or drug use disorders. Risk fac- tors common to all drugs include history of major depressive disorder, history of drug- induced depression, and psychosocial stressors. Environmental. There are also risks factors pertaining to a specific type of medication (e.g., increased immune activation prior to treatment for hepatitis C associated with inter- lents) of corticosteroids or high plasma concentrations of efavirenz; and high estrogen/ progesterone content in oral contraceptives.

1	Course modifiers. In a representative US. adult population, compared with individuals with major depressive disorder who did not have a substance use disorder, individuals with substance-induced depressive disorder were more likely to be male, to be black, to have at most a high school diploma, to lack insurance, and to have lower family income. They were also more likely to report higher family history of substance use disorders and antisocial behavior, higher 12-month history of stressful life events, and a greater number of DSM-IV major depressive disorder criteria. They were more likely to report feelings of worthlessness, insomnia/hypersomnia, and thoughts of death and suicide attempts, but less likely to report depressed mood and parental loss by death before age 18 years. Determination of the substance of use can sometimes be made through laboratory assays of the suspected substance in the blood or urine to corroborate the diagnosis.

1	Determination of the substance of use can sometimes be made through laboratory assays of the suspected substance in the blood or urine to corroborate the diagnosis. and behavior from the person’s baseline, is usually temporally associated with initiation of a substance, and must be distinguished from the underlying primary mental disorders. In regard to the treatment-emergent suicidality associated with antidepressants, a US. 99,839 participants enrolled in 372 randomized clinical trials of antidepressants in trials for mental disorders. The analyses showed that when the data were pooled across all adult age groups, there was no perceptible increased risk of suicidal behavior or ideation. How- ever, in age-stratified analyses, the risk for patients ages 18—24 years was elevated, albeit not significantly (odds ratio [OR] = 1.55; 95% confidence interval [CI] = 0.91—2.70). The

1	FDA meta-analyses reveal an absolute risk of suicide in patients taking investigational an- tidepressants of 0.01%. In conclusion, suicide is clearly an extremely rare treatment-emer- gent phenomenon, but the outcome of suicide was serious enough to prompt the FDA to issue an expanded black-box warning in 2007 regarding the importance of careful moni- toring of treatment-emergent suicidal ideation in patients receiving antidepressants.

1	Substance intoxication and withdrawal. Depressive symptoms occur commonly in sub- stance intoxication and substance withdrawal, and the diagnosis of the substance-specific intoxication or withdrawal will usually suffice to categorize the symptom presentation. A diagnosis of substance-induced depressive disorder should be made instead of a diag- nosis of substance intoxication or substance withdrawal when the mood symptoms are sufficiently severe to warrant independent clinical attention. For example, dysphoric mood is a characteristic feature of cocaine withdrawal. Substance/ medication-induced depressive disorder should be diagnosed instead of cocaine withdrawal only if the mood disturbance is substantially more intense or longer lasting than what is usually encountered with cocaine withdrawal and is sufficiently severe to be a separate focus of attention and treatment.

1	Primary depressive disorder. A substance/medication-induced depressive disorder is distinguished from a primary depressive disorder by the fact that a substance is judged to be etiologically related to the symptoms, as described earlier (see section "Development and Course” for this disorder).

1	Depressive disorder due to another medical condition. Because individuals with other medical conditions often take medications for those conditions, the clinician must consider the possibility that the mood symptoms are caused by the physiological consequences of the med- ical condition rather than the medication, in which case depressive disorder due to another medical condition is diagnosed. The history often provides the primary basis for such a judg- ment. At times, a change in the treatment for the other medical condition (e.g., medication sub- stitution or discontinuation) may be needed to determine empirically whether the medication is the causative agent. If the clinician has ascertained that the disturbance is a function of both another medical condition and substance use or withdrawal, both diagnoses (i.e., depressive disorder due to another medical condition and substance/ medication-induced depressive disorder) may be given. When there is insufficient evidence to determine

1	both diagnoses (i.e., depressive disorder due to another medical condition and substance/ medication-induced depressive disorder) may be given. When there is insufficient evidence to determine Whether the depres- sive symptoms are associated with substance (including a medication) ingestion or with- drawal or with another medical condition or are primary (i.e., not a function of either a substance or another medical condition), a diagnosis of other specified depressive disorder or unspecified depressive disorder would be indicated.

1	Compared with individuals with major depressive disorder and no comorbid substance use disorder, those with substance/medication-induced depressive disorder have higher rates of comorbidity with any DSM-IV mental disorder; are more likely to have specific DSM-IV disorders of pathological gambling and paranoid, histrionic, and antisocial per- sonality disorders; and are less likely to have persistent depressive disorder (dysthymia). Compared with individuals with major depressive disorder and a comorbid substance use disorder, individuals with substance/medication-induced depressive disorder are more likely to have alcohol use disorder, any other substance use disorder, and histrionic per- sonality disorder; however, they are less likely to have persistent depressive disorder. Due to Another Medical Condition

1	Due to Another Medical Condition A. A prominent and persistent period of depressed mood or markedly diminished interest or pleasure in all, or almost all, activities that predominates in the clinical picture. 8. There is evidence from the history, physical examination, or laboratory findings that the disturbance is the direct pathophysiological consequence of another medical condi- tion. C. The disturbance is not better explained by another mental disorder (e.g., adjustment disorder, with depressed mood, in which the stressor is a serious medical condition). D. The disturbance does not occur exclusively during the course of a delirium. E. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. Coding note: The |CD-9-CM code for depressive disorder due to another medical condi- tion is 293.83, which is assigned regardless of the specifier. The ICD-10-CM code de- pends on the specifier (see below).

1	Depressive Disorder Due to Another Medical Condition 181 Specify if: (F06.31) With depressive features: Full criteria are not met for a major depressive episode. ‘ (F06.32) With major depressive—llke episode: FuII criteria are met (except Criterion C) for a major depressive episode. (F06.34) With mixed features: Symptoms of mania or hypomania are also present but do not predominate in the clinical picture. Coding note: Include the name of the other medical condition in the name of the mental dis- order (e.g., 293.83 [F0631] depressive disorder due to hypothyroidism, with depressive fea- tures). The other medical condition should also be coded and listed separately immediately before the depressive disorder due to the medical condition (e.g., 244.9 [E039] hypothyroid- ism; 293.83 [F0631] depressive disorder due to hypothyroidism, with depressive features).

1	The essential feature of depressive disorder due to another medical condition is a promi- nent and persistent period of depressed mood or markedly diminished interest or plea- sure in all, or almost all, activities that predominates in the clinical picture (Criterion A) and that is thought to be related to the direct physiological effects of another medical con- dition (Criterion B). In determining whether the mood disturbance is due to a general medical condition, the clinician must first establish the presence of a general medical con— dition. Further, the clinician must establish that the mood disturbance is etiologically re- lated to the general medical condition through a physiological mechanism. A careful and comprehensive assessment of multiple factors is necessary to make this judgment. Al- though there are no infallible guidelines for determining whether the relationship between the mood disturbance and the general medical condition is etiological, several considerations

1	judgment. Al- though there are no infallible guidelines for determining whether the relationship between the mood disturbance and the general medical condition is etiological, several considerations provide some guidance in this area. One consideration is the presence of a temporal association between the onset, exacerbation, or remission of the general medical condition and that of the mood disturbance. A second consideration is the presence of fea— tures that are atypical of primary Mood Disorders (e.g., atypical age at onset or course or absence of family history). Evidence from the literature that suggests that there can be a di- rect association between the general medical condition in question and the development of mood symptoms can provide a useful context in the assessment of a particular situation.

1	Etiology (i.e., a causal relationship to another medical condition based on best clinical ev- idence) is the key variable in depressive disorder due to another medical condition. The listing of the medical conditions that are said to be able to induce major depression is never complete, and the clinician’s best judgment is the essence of this diagnosis.

1	There are clear associations, as well as some neuroanatomical correlates, of depression with stroke, Huntington’s disease, Parkinson’s disease, and traumatic brain injury. Among the neuroendocrine conditions most closely associated with depression are Cushing’s dis- ease and hypothyroidism. There are numerous other conditions thought to be associated with depression, such as multiple sclerosis. However, the literature’s support for a causal association is greater with some conditions, such as Parkinson’s disease and Huntington’s disease, than with others, for which the differential diagnosis may be adjustment disorder, with depressed mood.

1	Following stroke, the onset of depression appears to be very acute, occurring within 1 day or a few days of the cerebrovascular accident (CVA) in the largest case series. However, in some cases, onset of the depression is weeks to months following the CVA. In the largest series, the duration of the major depressive episode following stroke was 9—11 months on average. Similarly, in Huntington’s disease the depressive state comes quite early in the course of the illness. With Parkinson’s disease and Huntington’s disease, it often precedes the major motor impairments and cognitive impairments associated with each condition. This is more prominently the case for Huntington’s disease, in which depression is con- sidered to be the first neuropsychiatric symptom. There is some observational evidence that depression is less common as the dementia of Huntington’s disease progresses.

1	The risk of acute onset of a major depressive disorder following a CVA (within 1 day to a week of the event) appears to be strongly correlated with lesion location, with greatest risk associated with left frontal strokes and least risk apparently associated with right frontal lesions in those individuals who present within days of the stroke. The association with frontal regions and laterality is not observed in depressive states that occur in the 2—6 months following stroke. Gender differences pertain to those associated with the medical condition (e.g., systemic lupus erythematosus is more common in females; stroke is somewhat more common in middle-age males compared with females). Diagnostic markers pertain to those associated with the medical condition (e.g., steroid levels in blood or urine to help corroborate the diagnosis of Cushing’s disease, which can be associated with manic or depressive syndromes).

1	There are no epidemiological studies that provide evidence to differentiate the risk of sui- cide from a major depressive episode due to another medical condition compared with the risk from a major depressive episode in general. There are case reports of suicides in association with major depressive episodes associated with another medical condition. There is a clear association between serious medical illnesses and suicide, particularly shortly after onset or diagnosis of the illness. Thus, it would be prudent to assume that the risk of suicide for major depressive episodes associated with medical conditions is not less than that for other forms of major depressive episode, and might even be greater. Functional Consequences of Depressive Disorder Due to Another Medical Condition

1	Functional Consequences of Depressive Disorder Due to Another Medical Condition Functional consequences pertain to those associated with the medical condition. In gen- eral, it is believed, but not established, that a major depressive episode induced by Cush- ing’s disease will not recur if the Cushing’s disease is cured or arrested. However, it is also suggested, but not established, that mood syndromes, including depressive and manic/ hypomanic ones, may be episodic (i.e., recurring) in some individuals with static brain in- juries and other central nervous system diseases.

1	Depressive disorders not due to another medical condition. Determination of whether a medical condition accompanying a depressive disorder is causing the disorder depends on a) the absence of an episode(s) of depressive episodes prior to the onset of the medical condition, b) the probability that the associated medical condition has a potential to pro- mote or cause a depressive disorder, and c) a course of the depressive symptoms shortly after the onset or worsening of the medical condition, especially if the depressive symp- toms remit near the time that the medical disorder is effectively treated or remits.

1	Medication-induced depressive disorder. An important caveat is that some medical con- ditions are treated with medications (e.g., steroids or alpha-interferon) that can induce depres- sive or manic symptoms. In these cases, clinical judgment, based on all the evidence in hand, is the best way to try to separate the most likely and/ or the most important of two etiological fac- tors (i.e., association with the medical condition vs. a substance-induced syndrome).

1	Adjustment disorders. It is important to differentiate a depressive episode from an ad- justment disorder, as the onset of the medical condition is in itself a life stressor that could bring on either an adjustment disorder or an episode of major depression. The major dif- ferentiating elements are the pervasiveness the depressive picture and the number and quality of the depressive symptoms that the patient reports or demonstrates on the mental status examination. The differential diagnosis of the associated medical conditions is rel- evant but largely beyond the scope of the present manual.

1	Conditions comorbid with depressive disorder due to another medical condition are those associated with the medical conditions of etiological relevance. It has been noted that de- lirium can occur before or along with depressive symptoms in individuals with a variety of medical conditions, such as Cushing’s disease. The association of anxiety symptoms, usually generalized symptoms, is common in depressive disorders, regardless of cause. 311 (F32.8)

1	311 (F32.8) This category applies to presentations in which symptoms characteristic of a depressive disorder that cause clinically significant distress or impairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the depressive disorders diagnostic class. The other specified depressive disorder category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for any specific depressive disorder. This is done by recording “other specified depressive disordef‘ followed by the specific reason (e.g., “short-duration depressive episode").

1	Examples of presentations that can be specified using the “other specified" designation include the following: 1. Recurrent brief depression: Concurrent presence of depressed mood and at least four other symptoms of depression for 2—13 days at least once per month (not associ- ated with the menstrual cycle) for at least 12 consecutive months in an individual and does not currently meet active or residual criteria for any psychotic disorder. 2. Short-duration depressive episode (4—13 days): Depressed affect and at least four of the other eight symptoms of a major depressive episode associated with clinically significant distress or impairment that persists for more than 4 days, but less than 14 days, bipolar disorder, does not currently meet active or residual criteria for any psychotic dis- order, and does not meet criteria for recurrent brief depression.

1	3. Depressive episode with insufficient symptoms: Depressed affect and at least one of the other eight symptoms of a major depressive episode associated with clinically whose presentation has never met criteria for any other depressive or bipolar disorder, does not currently meet active or residual criteria for any psychotic disorder, and does not meet criteria for mixed anxiety and depressive disorder symptoms. 311 (F32.9)

1	311 (F32.9) This category applies to presentations in which symptoms characteristic of a depressive dis- order that cause clinically significant distress or impairment in social, occupational, or other im- portant areas of functioning predominate but do not meet the full criteria for any of the disorders in the depressive disorders diagnostic class. The unspecified depressive disorder category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for a specific depressive disorder, and includes presentations for which there is insut— ficient information to make a more specific diagnosis (e.g., in emergency room settings). Specify it: With anxious distress: Anxious distress is defined as the presence of at least two of the following symptoms during the majority of days of a major depressive episode or persistent depressive disorder (dysthymia): 1. Feeling keyed up or tense. 2. Feeling unusually restless.

1	2. Feeling unusually restless. 3. Difficulty concentrating because of worry. 4. Fear that something awful may happen. 5. Feeling that the individual might lose control of himself or herself. Specify current severity: Mild: Two symptoms. Moderate: Three symptoms. Moderate-severe: Four or five symptoms. Severe: Four or five symptoms and with motor agitation. Note: Anxious distress has been noted as a prominent feature of both bipolar and ma- jor depressive disorder in both primary care and specialty mental health settings. High levels of anxiety have been associated with higher suicide risk, longer duration of ill- ness, and greater likelihood of treatment nonresponse. As a result, it is clinically useful to specify accurately the presence and severity levels of anxious distress for treatment planning and monitoring of response to treatment. With mixed features:

1	With mixed features: A. At least three of the following manic/hypomanic symptoms are present nearly every day during the majority of days of a major depressive episode: 1. Elevated, expansive mood. Inflated self-esteem or grandiosity. More talkative than usual or pressure to keep talking. Flight of ideas or subjective experience that thoughts are racing. Increase in energy or goal-directed activity (either socially, at work or school. or sexually). 6. Increased or excessive involvement in activities that have a high potential for painful consequences (e.g., engaging in unrestrained buying sprees. sexual in- discretions, foolish business investments). 7. Decreased need for sleep (feeling rested despite sleeping less than usual; to be contrasted with insomnia). B. Mixed symptoms are observable by others and represent a change from the per- son’s usual behavior.

1	B. Mixed symptoms are observable by others and represent a change from the per- son’s usual behavior. C. For individuals whose symptoms meet full criteria for either mania or hypomania, the diagnosis should be bipolar | or bipolar II disorder. D. The mixed symptoms are not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication or other treatment). Note: Mixed features associated with a major depressive episode have been found to be a significant risk factor for the development of bipolar I or bipolar II disorder. As a result, it is clinically useful to note the presence of this specifier for treatment planning and monitoring of response to treatment. With melancholic features: A. One of the following is present during the most severe period of the current epi- sode: 1. Loss of pleasure in all, or almost all, activities.

1	With melancholic features: A. One of the following is present during the most severe period of the current epi- sode: 1. Loss of pleasure in all, or almost all, activities. 2. Lack of reactivity to usually pleasurable stimuli (does not feel much better, even temporarily, when something good happens). B. Three (or more) of the following: 1. A distinct quality of depressed mood characterized by profound despondency, despair, and/or moroseness or by so-called empty mood. Depression that is regularly worse in the morning. EarIy-morning awakening (i.e., at least 2 hours before usual awakening). Marked psychomotor agitation or retardation. Significant anorexia or weight loss. 6. Excessive or inappropriate guilt.

1	Significant anorexia or weight loss. 6. Excessive or inappropriate guilt. Note: The specifier “with melancholic features" is applied if these features are present at the most severe stage of the episode. There is a near-complete absence of the ca- pacity for pleasure, not merely a diminution. A guideline for evaluating the lack of reac- tivity of mood is that even highly desired events are not associated with marked brightening of mood. Either mood does not brighten at all, or it brightens only partially (e.g., up to 20%—40% of normal for only minutes at a time). The “distinct quality‘ of mood that is characteristic of the “with melancholic features" specifier is experienced as qual- itatively different from that during a nonmelancholic depressive episode. A depressed mood that is described as merely more severe, longer lasting, or present without a rea- son is not considered distinct in quality. Psychomotor changes are nearly always pres- ent and are observable by others.

1	Melancholic features exhibit only a modest tendency to repeat across episodes in the same individual. They are more frequent in inpatients, as opposed to outpatients; are less likely to occur in milder than in more severe major depressive episodes; and are more likely to occur in those with psychotic features. With atypical features: This specifier can be applied when these features predomi- nate during the majority of days of the current or most recent major depressive episode or persistent depressive disorder. 9135.09!“ A. Mood reactivity (i.e., mood brightens in response to actual or potential positive events). B. Two (or more) of the following: 1. Significant weight gain or increase in appetite. 2. Hypersomnia. 3. Leaden paralysis (i.e., heavy, leaden feelings in arms or legs). 4. A Iong-standing pattern of interpersonal rejection sensitivity (not limited to epi- sodes of mood disturbance) that results in significant social or occupational im- pairment.

1	4. A Iong-standing pattern of interpersonal rejection sensitivity (not limited to epi- sodes of mood disturbance) that results in significant social or occupational im- pairment. C. Criteria are not met for “with melancholic features" or “with catatonia” during the same episode. Note: “Atypical depression” has historical significance (i.e., atypical in contradistinction to the more classical agitated, “endogenous" presentations of depression that were the ical presentation as the term might imply. Mood reactivity is the capacity to be cheered up when presented with positive events (e.g., a visit from children, compliments from others). Mood may become euthymic (not sad) even for extended periods of time if the external circumstances remain favorable.

1	weight gain. Hypersomnia may include either an extended period of nighttime sleep or daytime napping that totals at least 10 hours of sleep per day (or at least 2 hours more than when not depressed). Leaden paralysis is defined as feeling heavy, leaden, or weighted down, usually in the arms or legs. This sensation is generally present for at least an hour a day but often lasts for many hours at a time. Unlike the other atypical features, pathological sensitivity to perceived interpersonal rejection is a trait that has an early onset and persists throughout most of adult life. Rejection sensitivity occurs both when the person is and is not depressed, though it may be exacerbated during depressive periods. With psychotic features: Delusions and/or hallucinations are present.

1	With psychotic features: Delusions and/or hallucinations are present. With mood-congruent psychotic features: The content of all delusions and hal- lucinations is consistent with the typical depressive themes of personal inade- quacy, guilt, disease, death, nihilism. or deserved punishment. With mood-incongruent psychotic features: The content of the delusions or hal- lucinations does not involve typical depressive themes of personal inadequacy, guilt, disease, death, nihilism, or deserved punishment, or the content is a mixture of mood-incongruent and mood-congruent themes. With catatonia: The catatonia specifier can apply to an episode of depression if cata- tonic features are present during most of the episode. See criteria for catatonia asso- ciated with a mental disorder (for a description of catatonia, see the chapter “Schizophrenia Spectrum and Other Psychotic Disorders").

1	With peripartum onset: This specifier can be applied to the current or, it full criteria are not currently met for a major depressive episode, most recent episode of major de- pression if onset of mood symptoms occurs during pregnancy or in the 4 weeks follow- ing delivery. Note: Mood episodes can have their onset either during pregnancy or postpartum. Although the estimates differ according to the period of Iollow-up after delivery, be- tween 3% and 6% of women will experience the onset of a major depressive epi- sode during pregnancy or in the weeks or months following delivery. Fifty percent of “postpartum" major depressive episodes actually begin prior to delivery. Thus, these episodes are referred to collectively as peripartum episodes. Women with attacks. Prospective studies have demonstrated that mood and anxiety symptoms during pregnancy, as well as the “baby blues," increase the risk for a postpartum major depressive episode.

1	Peripartum-onset mood episodes can present either with or without psychotic features. Infanticide is most often associated with postpartum psychotic episodes that are characterized by command hallucinations to kill the infant or delusions that the infant is possessed, but psychotic symptoms can also occur in severe postpar- tum mood episodes without such specific delusions or hallucinations. Postpartum mood (major depressive or manic) episodes with psychotic features appear to occur in from 1 in 500 to 1 in 1,000 deliveries and may be more common in primiparous women. The risk of postpartum episodes with psychotic features is particularly increased for women with prior postpartum mood episodes but is also elevated for those with a prior history of a depressive or bipolar disorder (especially bipolar | disorder) and those with a family history of bipolar disorders.

1	Once a woman has had a postpartum episode with psychotic features, the risk of recurrence with each subsequent delivery is between 30% and 50%. Postpartum episodes must be differentiated from delirium occurring in the postpartum period, which is distinguished by a fluctuating level of awareness or attention. The postpar- tum period is unique with respect to the degree of neuroendocrine alterations and psychosocial adjustments, the potential impact of breast—feeding on treatment planning, and the Iong-term implications of a history of postpartum mood disorder on subsequent family planning. With seasonal pattern: This specifier applies to recurrent major depressive disorder. A. There has been a regular temporal relationship between the onset of major depres- sive episodes in major depressive disorder and a particular time of the year (e.g., in the fall or winter).

1	A. There has been a regular temporal relationship between the onset of major depres- sive episodes in major depressive disorder and a particular time of the year (e.g., in the fall or winter). Note: Do not include cases in which there is an obvious effect of seasonally related psychosocial stressors (e.g., regularly being unemployed every winter). B. Full remissions (or a change from major depression to mania or hypomania) also occur at a characteristic time of the year (e.g., depression disappears in the spring). C. In the last 2 years, two major depressive episodes have occurred that demonstrate the temporal seasonal relationships defined above and no nonseasonal major de- pressive episodes have occurred during that same period. D. Seasonal major depressive episodes (as described above) substantially outnum- ber the nonseasonal major depressive episodes that may have occurred over the individual’s lifetime.

1	D. Seasonal major depressive episodes (as described above) substantially outnum- ber the nonseasonal major depressive episodes that may have occurred over the individual’s lifetime. Note: The specifier “with seasonal pattern" can be applied to the pattern of major de- pressive episodes in major depressive disorder. recurrent. The essential feature is the onset and remission of major depressive episodes at characteristic times of the year. In most cases, the episodes begin in tall or winter and remit in spring. Less commonly, there may be recurrent summer depressive episodes. This pattern of onset and remis- sion of episodes must have occurred during at least a 2-year period. without any non- seasonal episodes occurring during this period. In addition, the seasonal depressive the individual’s lifetime.

1	This specifier does not apply to those situations in which the pattern is better ex- plained by seasonally linked psychosocial stressors (e.g., seasonal unemployment or school schedule). Major depressive episodes that occur in a seasonal pattern are often characterized by prominent energy, hypersomnia, overeating, weight gain, and a crav- ing for carbohydrates. It is unclear whether a seasonal pattern is more likely in recur- rent major depressive disorder or in bipolar disorders. However, within the bipolar disorders group, a seasonal pattern appears to be more likely in bipolar II disorder than in bipolar | disorder. In some individuals, the onset of manic or hypomanic episodes may also be linked to a particular season. The prevalence of winter-type seasonal pattern appears to vary with latitude. age, and sex. Prevalence increases with higher latitudes. Age is also a strong predictor of seasonality, with younger persons at higher risk for winter depressive episodes. Specify it:

1	Specify it: In partial remission: Symptoms of the immediately previous major depressive episode are present, but full criteria are not met, or there is a period lasting less than 2 months without any significant symptoms of a major depressive episode following the end of such an episode. In full remission: During the past 2 months, no significant signs or symptoms of the disturbance were present. Specify current severity: Severity is based on the number of criterion symptoms, the severity of those symptoms, and the degree of functional disability. Mild: Few, if any, symptoms in excess of those required to make the diagnosis are present, the intensity of the symptoms is distressing but manageable, and the symp- toms result in minor impairment in social or occupational functioning. Moderate: The number of symptoms. intensity of symptoms, and/or functional impair- ment are between those specified for “mild” and “severe.”

1	Moderate: The number of symptoms. intensity of symptoms, and/or functional impair- ment are between those specified for “mild” and “severe.” Severe: The number of symptoms is substantially in excess of that required to make the diagnosis, the intensity of the symptoms is seriously distressing and unmanage- able, and the symptoms markedly interfere with social and occupational functioning.

1	Anxiety d isorders include disorders that share features of excessive fear and anxi- ety and related behavioral disturbances. Fear is the emotional response to real or per- ceived imminent threat, whereas anxiety is anticipation of future threat. Obviously, these two states overlap, but they also differ, with fear more often associated with surges of au- tonomic arousal necessary for fight or ﬂight, thoughts of immediate danger, and escape behaviors, and anxiety more often associated with muscle tension and vigilance in prep— aration for future danger and cautious or avoidant behaviors. Sometimes the level of fear or anxiety is reduced by pervasive avoidance behaviors. Panic attacks feature prominently within the anxiety disorders as a particular type of fear response. Panic attacks are not lim- ited to anxiety disorders but rather can be seen in other mental disorders as well.

1	The anxiety disorders differ from one another in the types of objects or situations that induce fear, anxiety, or avoidance behavior, and the associated cognitive ideation. Thus, while the anxiety disorders tend to be highly comorbid with each other, they can be dif- ferentiated by close examination of the types of situations that are feared or avoided and the content of the associated thoughts or beliefs.

1	cessive or persisting beyond developmentally appropriate periods. They differ from tran- sient fear or anxiety, often stress—induced, by being persistent (e.g., typically lasting 6 months or more), although the criterion for duration is intended as a general guide with allowance for some degree of ﬂexibility and is sometimes of shorter duration in children (as in sepa- ration anxiety disorder and selective mutism). Since individuals with anxiety disorders typically overestimate the danger in situations they fear or avoid, the primary determina- tion of whether the fear or anxiety is excessive or out of proportion is made by the clinician, taking cultural contextual factors into account. Many of the anxiety disorders develop in childhood and tend to persist if not treated. Most occur more frequently in females than in males (approximately 2:1 ratio). Each anxiety disorder is diagnosed only when the symp— toms are not attributable to the physiological effects of a substance/medication

1	frequently in females than in males (approximately 2:1 ratio). Each anxiety disorder is diagnosed only when the symp— toms are not attributable to the physiological effects of a substance/medication or to another medical condition or are not better explained by another mental disorder.

1	The chapter is arranged developmentally, with disorders sequenced according to the typical age at onset. The individual with separation anxiety disorder is fearful or anxious about separation from attachment figures to a degree that is developmentally inappro- priate. There is persistent fear or anxiety about harm coming to attachment figures and events that could lead to loss of or separation from attachment figures and reluctance to go away from attachment figures, as well as nightmares and physical symptoms of distress. Al- though the symptoms often develop in childhood, they can be expressed throughout adult— hood as well. Selective mutism is characterized by a consistent failure to speak in social situations in which there is an expectation to speak (e.g., school) even though the individual speaks in other situations. The failure to speak has significant consequences on achievement in aca- demic or occupational settings or otherwise interferes with normal social communication.

1	Individuals with specific phobia are fearful or anxious about or avoidant of circum- scribed objects or situations. A specific cognitive ideation is not featured in this disorder, as it is in other anxiety disorders. The fear, anxiety, or avoidance is almost always imme- diately induced by the phobic situation, to a degree that is persistent and out of proportion to the actual risk posed. There are various types of specific phobias: animal; natural envi- ronment; blood—injection—injury; situational; and other situations.

1	In social anxiety disorder (social phobia), the individual is fearful or anxious about or avoidant of social interactions and situations that involve the possibility of being scruti- nized. These include social interactions such as meeting unfamiliar people, situations in which the individual may be observed eating or drinking, and situations in which the in- dividual performs in front of others. The cognitive ideation is of being negatively evalu- ated by others, by being embarrassed, humiliated, or rejected, or offending others. In panic disorder, the individual experiences recurrent unexpected panic attacks and is behavior in maladaptive ways because of the panic attacks (e.g., avoidance of exercise or of unfamiliar locations). Panic attacks are abrupt surges of intense fear or intense discomfort that reach a peak within minutes, accompanied by physical and / or cognitive symptoms.

1	Limited—symptom panic attacks include fewer than four symptoms. Panic attacks may be expected, such as in response to a typically feared object or situation, or unexpected, meaning that the panic attack occurs for no apparent reason. Panic attacks function as a marker and prognostic factor for severity of diagnosis, course, and comorbidity across an array of dis- orders, including, but not limited to, the anxiety disorders (e.g., substance use, depressive and psychotic disorders). Panic attack may therefore be used as a descriptive specifier for any anxiety disorder as well as other mental disorders. Individuals with agoraphobia are fearful and anxious about two or more of the follow- ing situations: using public transportation; being in open spaces; being in enclosed places; standing in line or being in a crowd; or being outside of the home alone in other situations.

1	The individual fears these situations because of thoughts that escape might be difficult or help might not be available in the event of developing panic-like symptoms or other inca- pacitating or embarrassing symptoms. These situations almost always induce fear or anx- iety and are often avoided and require the presence of a companion. The key features of generalized anxiety disorder are persistent and excessive anxiety and worry about various domains, including work and school performance, that the indi- vidual finds difficult to control. In addition, the individual experiences physical symptoms, centrating or mind going blank; irritability; muscle tension; and sleep disturbance. Substance/ medication-induced anxiety disorder involves anxiety due to substance in- toxication or withdrawal or to a medication treatment. In anxiety disorder due to another medical condition, anxiety symptoms are the physiological consequence of another med- ical condition.

1	Disorder-specific scales are available to better characterize the severity of each anxiety disorder and to capture change in severity over time. For ease of use, particularly for in- dividuals with more than one anxiety disorder, these scales have been developed to have the same format (but different focus) across the anxiety disorders, with ratings of behav- ioral symptoms, cognitive ideation symptoms, and physical symptoms relevant to each disorder. Diagnostic Criteria 309.21 (F93.0) A. Developmentally inappropriate and excessive fear or anxiety concerning separation from those to whom the individual is attached, as evidenced by at least three of the following: 1. Recurrent excessive distress when anticipating or experiencing separation from home or from major attachment figures. 2. Persistent and excessive worry about losing major attachment figures or about pos- sible harm to them, such as illness, injury, disasters, or death.

1	2. Persistent and excessive worry about losing major attachment figures or about pos- sible harm to them, such as illness, injury, disasters, or death. 3. Persistent and excessive worry about experiencing an untoward event (e.g., getting lost, being kidnapped, having an accident, becoming ill) that causes separation from a major attachment figure. 4. Persistent reluctance or refusal to go out, away from home, to school, to work, or elsewhere because of fear of separation. 5. Persistent and excessive tear of or reluctance about being alone or without major attachment figures at home or in other settings. 6. Persistent reluctance or refusal to sleep away from home or to go to sleep without being near a major attachment figure. Repeated nightmares involving the theme of separation. 8. Repeated complaints of physical symptoms (e.g., headaches, stomachaches, nau- sea, vomiting) when separation from major attachment figures occurs or is antici- pated.

1	8. Repeated complaints of physical symptoms (e.g., headaches, stomachaches, nau- sea, vomiting) when separation from major attachment figures occurs or is antici- pated. B. The fear. anxiety, or avoidance is persistent, lasting at least 4 weeks in children and adolescents and typically 6 months or more in adults. C. The disturbance causes clinically significant distress or impairment in social, aca- demic, occupational, or other important areas of functioning. D. The disturbance is not better explained by another mental disorder, such as refusing to leave home because of excessive resistance to change in autism spectrum disorder; delusions or hallucinations concerning separation in psychotic disorders; refusal to go having an illness in illness anxiety disorder.

1	The essential feature of separation anxiety disorder is excessive fear or anxiety concerning separation from home or attachment figures. The anxiety exceeds what may be expected given the person’s developmental level (Criterion A). Individuals with separation anxiety disorder have symptoms that meet at least three of the following criteria: They experience recurrent excessive distress when separation from home or major attachment figures is an— ticipated or occurs (Criterion A1). They worry about the well-being or death of attachment figures, particularly when separated from them, and they need to know the whereabouts of their attachment figures and want to stay in touch with them (Criterion A2). They also worry about untoward events to themselves, such as getting lost, being kidnapped, or having an accident, that would keep them from ever being reunited with their major at- tachment figure (Criterion A3). Individuals with separation anxiety disorder are reluctant or refuse to go out

1	having an accident, that would keep them from ever being reunited with their major at- tachment figure (Criterion A3). Individuals with separation anxiety disorder are reluctant or refuse to go out by themselves because of separation fears (Criterion A4). They have figures at home or in other settings. Children with separation anxiety disorder may be un- able to stay or go in a room by themselves and may display "clinging” behavior, staying close to or ”shadowing" the parent around the house, or requiring someone to be with them when going to another room in the house (Criterion A5). They have persistent reluc- tance or refusal to go to sleep without being near a major attachment figure or to sleep away from home (Criterion A6). Children with this disorder often have difficulty at bed- time and may insist that someone stay with them until they fall asleep. During the night, they may make their way to their parents’ bed (or that of a significant other, such as a sib- ling). Children

1	time and may insist that someone stay with them until they fall asleep. During the night, they may make their way to their parents’ bed (or that of a significant other, such as a sib- ling). Children may be reluctant or refuse to attend camp, to sleep at friends’ homes, or to go on errands. Adults may be uncomfortable when traveling independently (e.g., sleeping in a hotel room). There may be repeated nightmares in which the content expresses the in- dividual’s separation anxiety (e.g., destruction of the family through fire, murder, or other catastrophe) (Criterion A7). Physical symptoms (e.g., headaches, abdominal complaints, nausea, vomiting) are common in children when separation from major attachment fig- ures occurs or is anticipated (Criterion A8). Cardiovascular symptoms such as palpitations, dizziness, and feeling faint are rare in younger children but may occur in adolescents and adults.

1	The disturbance must last for a period of at least 4 weeks in children and adolescents younger than 18 years and is typically 6 months or longer in adults (Criterion B). However, the duration criterion for adults should be used as a general guide, with allowance for some degree of ﬂexibility. The disturbance must cause clinically significant distress or im- pairment in social, academic, occupational, or other important areas of functioning (Cri- terion C).

1	When separated from major attachment figures, children with separation anxiety disorder may exhibit social withdrawal, apathy, sadness, or difficulty concentrating on work or play. Depending on their age, individuals may have fears of animals, monsters, the dark, muggers, burglars, kidnappers, car accidents, plane travel, and other situations that are perceived as presenting danger to the family or themselves. Some individuals become homesick and uncomfortable to the point of misery when away from home. Separation anxiety disorder in children may lead to school refusal, which in turn may lead to academic difficulties and social isolation. When extremely upset at the prospect of separation, chil- dren may show anger or occasionally aggression toward someone who is forcing separa- tion. When alone, especially in the evening or the dark, young children may report unusual perceptual experiences (e.g., seeing people peering into their room, frightening creatures reaching for them, feeling

1	alone, especially in the evening or the dark, young children may report unusual perceptual experiences (e.g., seeing people peering into their room, frightening creatures reaching for them, feeling eyes staring at them). Children with this disorder may be de- scribed as demanding, intrusive, and in need of constant attention, and, as adults, may ap- pear dependent and overprotective. The individual’s excessive demands often become a source of frustration for family members, leading to resentment and conﬂict in the family.

1	The 12-month prevalence of separation anxiety disorder among adults in the United States is 0.9%—1.9%. In children, 6- to 12-month prevalence is estimated to be approximately 4%. In adolescents in the United States, the 12-month prevalence is 1.6%. Separation anxiety is the most prevalent anxiety disorder in children younger than 12 years. In clinical sam- ples of children, the disorder is equally common in males and females. In the community, the disorder is more frequent in females.

1	Periods of heightened separation anxiety from attachment figures are part of normal early development and may indicate the development of secure attachment relationships (e.g., around 1 year of age, when infants may suffer from stranger anxiety). Onset of separation anxiety disorder may be as early as preschool age and may occur at any time during child- hood and more rarely in adolescence. Typically there are periods of exacerbation and re- mission. In some cases, both the anxiety about possible separation and the avoidance of situations involving separation from the home or nuclear family (e.g., going away to col- lege, moving away from attachment figures) may persist through adulthood. However, the majority of children with separation anxiety disorder are free of impairing anxiety dis- orders over their lifetimes. Many adults with separation anxiety disorder do not recall a childhood onset of separation anxiety disorder, although they may recall symptoms.

1	The manifestations of separation anxiety disorder vary with age. Younger children are more reluctant to go to school or may avoid school altogether. Younger children may not express worries or specific fears of definite threats to parents, home, or themselves, and the anxiety is manifested only when separation is experienced. As children age, worries emerge; these are often worries about specific dangers (e.g., accidents, kidnapping, mug- ging, death) or vague concerns about not being reunited with attachment figures. In adults, separation anxiety disorder may limit their ability to cope with changes in circumstances (e.g., moving, getting married). Adults with the disorder are typically overconcemed about their offspring and spouses and experience marked discomfort when separated from them. They may also experience significant disruption in work or social experiences because of needing to continuously check on the whereabouts of a significant other.

1	They may also experience significant disruption in work or social experiences because of needing to continuously check on the whereabouts of a significant other. Environmental. Separation anxiety disorder often develops after life stress, especially a loss (e.g., the death of a relative or pet; an illness of the individual or a relative; a change of schools;parenta1 divorce; a move to a new neighborhood; immigration; a disaster that in- volved periods of separation from attachment figures). In young adults, other examples of life stress include leaving the parental home, entering into a romantic relationship, and be- coming a parent. Parental overprotection and intrusiveness may be associated with sepa- ration anxiety disorder. Genetic and physiological. Separation anxiety disorder in children may be heritable.

1	Genetic and physiological. Separation anxiety disorder in children may be heritable. Heritability was estimated at 73% in a community sample of 6-year-old twins, with higher rates in girls. Children with separation anxiety disorder display particularly enhanced sensitivity to respiratory stimulation using COz-enriched air. There are cultural variations in the degree to which it is considered desirable to tolerate separation, so that demands and opportunities for separation between parents and chil- dren are avoided in some cultures. For example, there is wide variation across countries and cultures with respect to the age at which it is expected that offspring should leave the parental home. It is important to differentiate separation anxiety disorder from the high value some cultures place on strong interdependence among family members.

1	Girls manifest greater reluctance to attend or avoidance of school than boys. Indirect ex- pression of fear of separation may be more common in males than in females, for example, by limited independent activity, reluctance to be away from home alone, or distress when spouse or offspring do things independently or when contact with spouse or offspring is not possible. Separation anxiety disorder in children may be associated with an increased risk for sui- cide. In a community sample, the presence of mood disorders, anxiety disorders, or sub- stance use has been associated with suicidal ideation and attempts. However, this association is not specific to separation anxiety disorder and is found in several anxiety disorders. Functional Consequences of Separatlon Anxiety Disorder

1	Functional Consequences of Separatlon Anxiety Disorder Individuals with separation anxiety disorder often limit independent activities away from home or attachment figures (e.g., in children, avoiding school, not going to camp, having difficulty sleeping alone; in adolescents, not going away to college; in adults, not leaving the parental home, not traveling, not working outside the home). Generalized anxiety disorder. Separation anxiety disorder is distinguished from gener- alized anxiety disorder in that the anxiety predominantly concerns separation from attach- ment figures, and if other worries occur, they do not predominate the clinical picture.

1	Panic disorder. Threats of separation may lead to extreme anxiety and even a panic at- tack. In separation anxiety disorder, in contrast to panic disorder, the anxiety concerns the possibility of being away from attachment figures and worry about untoward events be- falling them, rather than being incapacitated by an unexpected panic attack. Agoraphobia. Unlike individuals with agoraphobia, those with separation anxiety dis- order are not anxious about being trapped or incapacitated in situations from which es- cape is perceived as difficult in the event of panic-like symptoms or other incapacitating symptoms. Conduct disorder. School avoidance (truancy) is common in conduct disorder, but anx- iety about separation is not responsible for school absences, and the child or adolescent usually stays away from, rather than returns to, the home.

1	Social anxiety disorder. School refusal may be due to social anxiety disorder (social pho- bia). In such instances, the school avoidance is due to fear of being judged negatively by oth- ers rather than to worries about being separated from the attachment figures. Posttraumatic stress disorder. Fear of separation from loved ones is common after trau- matic events such as a disasters, particularly when periods of separation from loved ones were experienced during the traumatic event. In posttraumatic stress disorder (PTSD), the central symptoms concern intrusions about, and avoidance of, memories associated with the traumatic event itself, whereas in separation anxiety disorder, the worries and avoid- ance concern the well-being of attachment figures and separation from them.

1	Illness anxiety disorder. Individuals with illness anxiety disorder worry about specific illnesses they may have, but the main concern is about the medical diagnosis itself, not about being separated from attachment figures. Bereavement. Intense yearning or longing for the deceased, intense sorrow and emo- tional pain, and preoccupation with the deceased or the circumstances of the death are ex- pected responses occurring in bereavement, whereas fear of separation from other attachment figures is central in separation anxiety disorder. Depressive and bipolar disorders. These disorders may be associated with reluctance to leave home, but the main concern is not worry or fear of untoward events befalling at- tachment figures, but rather low motivation for engaging with the outside world. How- ever, individuals with separation anxiety disorder may become depressed while being separated or in anticipation of separation.

1	Oppositional defiant disorder. Children and adolescents with separation anxiety disor- der may be oppositional in the context of being forced to separate from attachment figures. Oppositional defiant disorder should be considered only when there is persistent opposi- tional behavior unrelated to the anticipation or occurrence of separation from attachment figures. Psychotic disorders. Unlike the hallucinations in psychotic disorders, the unusual per- ceptual experiences that may occur in separation anxiety disorder are usually based on a misperception of an actual stimulus, occur only in certain situations (e.g., nighttime), and are reversed by the presence of an attachment figure.

1	Personality disorders. Dependent personality disorder is characterized by an indis- criminate tendency to rely on others, whereas separation anxiety disorder involves con- cern about the firoximity and safety of main attachment figures. Borderline personality disorder is characterized by fear of abandonment by loved ones, but problems in identity, self—direction, interpersonal functioning, and impulsivity are additionally central to that disorder, whereas they are not central to separation anxiety disorder. In children, separation anxiety disorder is highly comorbid with generalized anxiety dis- order and specific phobia. In adults, common comorbidities include specific phobia, PTSD, panic disorder, generalized anxiety disorder, social anxiety disorder, agoraphobia, obsessive-compulsive disorder, and personality disorders. Depressive and bipolar disor- ders are also comorbid with separation anxiety disorder in adults. Diagnostic Criteria 312.23 (F94.0)

1	Diagnostic Criteria 312.23 (F94.0) A. Consistent failure to speak in specific social situations in which there is an expectation for speaking (e.g., at school) despite speaking in other situations. B. The disturbance interferes with educational or occupational achievement or with social communication. C. The duration of the disturbance is at least 1 month (not limited to the first month of school). D. The failure to speak is not attributable to a lack of knowledge of, or comfort with, the spoken language required in the social situation. E. The disturbance is not better explained by a communication disorder (e.g., childhood- onset fluency disorder) and does not occur exclusively during the course of autism spectrum disorder, schizophrenia, or another psychotic disorder.

1	When encountering other individuals in social interactions, children with selective mut- ism do not initiate speech or reciprocally respond when spoken to by others. Lack of speech occurs in social interactions with Children or adults. Children with selective mut- ism will speak in their home in the presence of immediate family members but often not even in front of close friends or second-degree relatives, such as grandparents or cousins.

1	The disturbance is often marked by high social anxiety. Children with selective mutism of- ten refuse to speak at school, leading to academic or educational impairment, as teachers often find it difficult to assess skills such as reading. The lack of speech may interfere with social communication, although children with this disorder sometimes use nonspoken or nonverbal means (e.g., grunting, pointing, writing) to communicate and may be willing or eager to perform or engage in social encounters when speech is not required (e.g., nonver- bal parts in school plays).

1	Associated features of selective mutism may include excessive shyness, fear of social em- barrassment, social isolation and withdrawal, clinging, compulsive traits, negativism, temper tantrums, or mild oppositional behavior. Although children with this disorder generally have normal language skills, there may occasionally be an associated commu- nication disorder, although no particular association with a specific communication dis- order has been identified. Even when these disorders are present, anxiety is present as well. In clinical settings, children with selective mutism are almost always given an addi— tional diagnosis of another anxiety disorder—most commonly, social anxiety disorder (so- cial phobia).

1	Selective mutism is a relatively rare disorder and has not been included as a diagnostic cat— egory in epidemiological studies of prevalence of childhood disorders. Point prevalence using various clinic or school samples ranges between 0.03% and 1% depending on the set- ting (e.g., clinic vs. school vs. general population) and ages of the individuals in the sample. The prevalence of the disorder does not seem to vary by sex or race/ethnicity. The disor- der is more likely to manifest in young children than in adolescents and adults.

1	The prevalence of the disorder does not seem to vary by sex or race/ethnicity. The disor- der is more likely to manifest in young children than in adolescents and adults. The onset of selective mutism is usually before age 5 years, but the disturbance may not come to clinical attention until entry into school, where there is an increase in social inter- action and performance tasks, such as reading aloud. The persistence of the disorder is variable. Although clinical reports suggest that many individuals ”outgrow” selective mutism, the longitudinal course of the disorder is unknown. In some cases, particularly in individuals with social anxiety disorder, selective mutism may disappear, but symptoms of social anxiety disorder remain.

1	Temperamental. Temperamental risk factors for selective mutism are not well identi- fied. Negative affectivity (neuroticism) or behavioral inhibition may play a role, as may parental history of shyness, social isolation, and social anxiety. Children with selective mutism may have subtle receptive language difficulties compared with their peers, al- though receptive language is still within the normal range. Environmental. Social inhibition on the part of parents may serve as a model for social reticence and selective mutism in children. Furthermore, parents of children with selective mutism have been described as overprotective or more controlling than parents of chil- dren with other anxiety disorders or no disorder. Genetic and physiological factors. Because of the significant overlap between selective mutism and social anxiety disorder, there may be shared genetic factors between these conditions.

1	Genetic and physiological factors. Because of the significant overlap between selective mutism and social anxiety disorder, there may be shared genetic factors between these conditions. Children in families who have immigrated to a country where a different language is spo- ken may refuse to speak the new language because of lack of knowledge of the language. If comprehension of the new language is adequate but refusal to speak persists, a diagno— sis of selective mutism may be warranted. Functional Consequences of Selective Mutism

1	If comprehension of the new language is adequate but refusal to speak persists, a diagno— sis of selective mutism may be warranted. Functional Consequences of Selective Mutism Selective mutism may result in social impairment, as children may be too anxious to en- gage in reciprocal social interaction with other children. As children with selective mutism mature, they may face increasing social isolation. In school settings, these children may suffer academic impairment, because often they do not communicate with teachers re- garding their academic or personal needs (e.g., not understanding a class assignment, not asking to use the restroom). Severe impairment in school and social functioning, including that resulting from teasing by peers, is common. In certain instances, selective mutism may serve as a compensatory strategy to decrease anxious arousal in social encounters.

1	Communication disorders. Selective mutism should be distinguished from speech dis- turbances that are better explained by a communication disorder, such as language disorder, speech sound disorder (previously phonological disorder), childhood-onset ﬂuency disorder (stuttering), or pragmatic (social) communication disorder. Unlike selec- tive mutism, the speech disturbance in these conditions is not restricted to a specific social situation. Neurodevelopmental disorders and schizophrenia and other psychotic disorders. Individuals with an autism spectrum disorder, schizophrenia or another psychotic disor- der, or severe intellectual disability may have problems in social communication and be unable to speak appropriately in social situations. In contrast, selective mutism should be diagnosed only when a child has an established capacity to speak in some social situations (e.g., typically at home).

1	Social anxiety disorder (social phobia). The social anxiety and social avoidance in so- cial anxiety disorder may be associated with selective mutism. In such cases, both diagno- ses may be given. The most common comorbid conditions are other anxiety disorders, most commonly so- cial anxiety disorder, followed by separation anxiety disorder and specific phobia. Oppo- sitional behaviors have been noted to occur in children with selective mutism, although oppositional behavior may be limited to situations requiring speech. Communication de- lays or disorders also may appear in some children with selective mutism. A. Marked fear or anxiety about a specific object or situation (e.g., ﬂying, heights, animals, receiving an injection, seeing blood). Note: In children, the fear or anxiety may be expressed by crying, tantrums, freezing, or clinging. B. The phobic object or situation almost always provokes immediate fear or anxiety.

1	Note: In children, the fear or anxiety may be expressed by crying, tantrums, freezing, or clinging. B. The phobic object or situation almost always provokes immediate fear or anxiety. C. The phobic object or situation is actively avoided or endured with intense fear or anxiety. D. The fear or anxiety is out of proportion to the actual danger posed by the specific object or situation and to the sociocultural context. E. The fear, anxiety, or avoidance is persistent, typically lasting for 6 months or more. F. The fear, anxiety, or avoidance causes clinically significant distress or impairment in social, occupational, or other important areas of functioning.

1	F. The fear, anxiety, or avoidance causes clinically significant distress or impairment in social, occupational, or other important areas of functioning. G. The disturbance is not better explained by the symptoms of another mental disorder, including tear, anxiety, and avoidance of situations associated with panic-Iike symptoms obsessions (as in obsessive-compulsive disorder); reminders of traumatic events (as in aration anxiety disorder); or social situations (as in social anxiety disorder). Specify if: Code based on the phobic stimulus: 300.29 (F40.218) Animal (e.g.. spiders, insects, dogs). 300.29 (F40.228) Natural environment (e.g., heights, storms, water). 300.29 (F40.23x) Blood-injection-iniury (e.g., needles, invasive medical procedures). Coding note: Select specific tCD-10-CM code as follows: F40.230 tear of blood; F40.231 fear of injections and transfusions; F40.232 tear of other medical care; or F40.233 fear of injury.

1	Coding note: Select specific tCD-10-CM code as follows: F40.230 tear of blood; F40.231 fear of injections and transfusions; F40.232 tear of other medical care; or F40.233 fear of injury. 300.29 (F40.248) Situational (e.g.. airplanes, elevators, enclosed places). 300.29 (F4o.298) Other (e.g., situations that may lead to choking or vomiting; in chil- dren, e.g., loud sounds or costumed characters). Coding note: When more than one phobic stimulus is present, code all |CD-10-CM codes that apply (e.g., for fear of snakes and flying, F40.218 specific phobia, animal, and F40.248 specific phobia, situational).

1	F40.248 specific phobia, situational). It is common for individuals to have multiple specific phobias. The average individual with specific phobia fears three objects or situations, and approximately 75% of individuals with specific phobia fear more than one situation or object. In such cases, multiple specific phobia diagnoses, each with its own diagnostic code reﬂecting the phobic stimulus, would need to be given. For example, if an individual fears thunderstorms and ﬂying, then two diagnoses would be given: specific phobia, natural environment, and specific phobia, situational.

1	A key feature of this disorder is that the fear or anxiety is circumscribed to the presence of a particular situation or object (Criterion A), which may be termed the phobic stimulus. The cat— egories of feared situations or objects are provided as specifiers. Many individuals fear objects or situations from more than one category, or phobic stimulus. For the diagnosis of specific phobia, the response must differ from normal, transient fears that commonly occur in the pop— ulation. To meet the criteria for a diagnosis, the fear or anxiety must be intense or severe (i.e., ”marked") (Criterion A). The amount of fear experienced may vary with proximity to the feared object or situation and may occur in anticipation of or in the actual presence of the object or situation. Also, the fear or anxiety may take the form of a full or limited symptom panic at- tack (i.e., expected panic attack). Another characteristic of specific phobias is that fear or anxi- ety is evoked nearly every time the

1	may take the form of a full or limited symptom panic at- tack (i.e., expected panic attack). Another characteristic of specific phobias is that fear or anxi- ety is evoked nearly every time the individual comes into contact with the phobic stimulus (Criterion B). Thus, an individual who becomes anxious only occasionally upon being con- fronted with the situation or object (e.g., becomes anxious when ﬂying only on one out of every five airplane ﬂights) would not be diagnosed with specific phobia. However, the degree of fear or anxiety expressed may vary (from anticipatory anxiety to a full panic attack) across different occasions of encountering the phobic object or situation because of various contextual factors such as the presence of others, duration of exposure, and other threatening elements such as turbulence on a ﬂight for individuals who fear ﬂying. Fear and anxiety are often expressed dif- ferently between children and adults. Also, the fear or anxiety occurs as soon as the

1	elements such as turbulence on a ﬂight for individuals who fear ﬂying. Fear and anxiety are often expressed dif- ferently between children and adults. Also, the fear or anxiety occurs as soon as the phobic ob- ject or situation is encountered (i.e., immediately rather than being delayed).

1	The individual actively avoids the situation, or if he or she either is unable or decides not to avoid it, the situation or object evokes intense fear or anxiety (Criterion C). Active avoidance means the individual intentionally behaves in ways that are designed to prevent or minimize contact with phobic objects or situations (e.g., takes tunnels instead of bridges on daily commute to work for fear of heights; avoids entering a dark room for fear of spi- ders; avoids accepting a job in a locale where a phobic stimulus is more common). Avoid- ance behaviors are often obvious (e.g., an individual who fears blood refusing to go to the doctor) but are sometimes less obvious (e.g., an individual who fears snakes refusing to look at pictures that resemble the form or shape of snakes). Many individuals with specific ways designed to avoid the phobic object or situation as much as possible (e.g., an indi- vidual diagnosed with specific phobia, animal, who moves to reside in an area devoid of

1	with specific ways designed to avoid the phobic object or situation as much as possible (e.g., an indi- vidual diagnosed with specific phobia, animal, who moves to reside in an area devoid of the particular feared animal). Therefore, they no longer experience fear or anxiety in their daily life. In such instances, avoidance behaviors or ongoing refusal to engage in activities that would involve exposure to the phobic object or situation (e.g., repeated refusal to ac- cept offers for work—related travel because of fear of ﬂying) may be helpful in confirming the diagnosis in the absence of overt anxiety or panic.

1	The fear or anxiety is out of proportion to the actual danger that the object or situation poses, or more intense than is deemed necessary (Criterion D). Although individuals with specific phobia often recognize their reactions as disproportionate, they tend to overesti- mate the danger in their feared situations, and thus the judgment of being out of propor- tion is made by the clinician. The individual’s sociocultural context should also be taken into account. For example, fears of the dark may be reasonable in a context of ongoing violence, and fear of insects may be more disproportionate in settings where insects are consumed in the diet. The fear, anxiety, or avoidance is persistent, typically lasting for 6 months or more (Criterion E), which helps distinguish the disorder from transient fears that are common in the population, particularly among children. However, the duration criterion should be used as a general guide, with allowance for some degree of ﬂexibility.

1	The specific phobia must cause clinically significant distress or impairment in social, oc- cupational, or other important areas of functioning in order for the disorder to be diag- nosed (Criterion F).

1	Individuals with specific phobia typically experience an increase in physiological arousal in anticipation of or during exposure to a phobic object or situation. However, the physi- ological response to the feared situation or object varies. Whereas individuals with situa- tional, natural environment, and animal specific phobias are likely to show sympathetic nervous system arousal, individuals with blood-injection-injury specific phobia often demonstrate a vasovagal fainting or near-fainting response that is marked by initial brief acceleration of heart rate and elevation of blood pressure followed by a deceleration of heart rate and a drop in blood pressure. Current neural systems models for specific phobia emphasize the amygdala and related structures, much as in other anxiety disorders.

1	In the United States, the 12-month community prevalence estimate for specific phobia is approximately 7%—9%. Prevalence rates in European countries are largely similar to those in the United States (e.g., about 6%), but rates are generally lower in Asian, African, and Latin American countries (2%—4%). Prevalence rates are approximately 5% in children and are approximately 16% in 13— to 17-year-olds. Prevalence rates are lower in older individ- uals (about 3%—5%), possibly reﬂecting diminishing severity to subclinical levels. Females are more frequently affected than males, at a rate of approximately 2:1, although rates vary across different phobic stimuli. That is, animal, natural environment, and situational spe- cific phobias are predominantly experienced by females, whereas blood-injection-injury phobia is experienced nearly equally by both genders.

1	Specific phobia sometimes develops following a traumatic event (e.g., being attacked by an animal or stuck in an elevator), observation of others going through a traumatic event (e.g., watching someone drown), an unexpected panic attack in the to be feared situation (e.g., an unexpected panic attack while on the subway), or informational transmission (e.g., ex- tensive media coverage of a plane crash). However, many individuals with specific phobia are unable to recall the specific reason for the onset of their phobias. Specific phobia usu- ally develops in early childhood, with the majority of cases developing prior to age 10 years. The median age at onset is between 7 and 11 years, with the mean at about 10 years.

1	Situational specific phobias tend to have a later age at onset than natural environment, an- imal, or blood-injection-injury specific phobias. Specific phobias that develop in child- hood and adolescence are likely to wax and wane during that period. However, phobias that do persist into adulthood are unlikely to remit for the majority of individuals. When specific phobia is being diagnosed in children, two issues should be considered. First, young children may express their fear and anxiety by crying, tantrums, freezing, or clinging. Second, young children typically are not able to understand the concept of avoidance. Therefore, the clinician should assemble additional information from parents, teachers, or others who know the child well. Excessive fears are quite common in young children but are usually transitory and only mildly impairing and thus considered devel- opmentally appropriate. In such cases a diagnosis of specific phobia would not be made.

1	When the diagnosis of specific phobia is being considered in a child, it is important to assess the degree of impairment and the duration of the fear, anxiety, or avoidance, and whether it is typical for the child’s particular developmental stage.

1	Although the prevalence of specific phobia is lower in older populations, it remains one of the more commonly experienced disorders in late life. Several issues should be con- sidered when diagnosing specific phobia in older populations. First, older individuals may be more likely to endorse natural environment specific phobias, as well as phobias of falling. Second, specific phobia (like all anxiety disorders) tends to co-occur with medical concerns in older individuals, including coronary heart disease and chronic obstructive pulmonary disease. Third, older individuals may be more likely to attribute the symptoms of anxiety to medical conditions. Fourth, older individuals may be more likely to manifest anxiety in an atypical manner (e.g., involving symptoms of both anxiety and depression) and thus be more likely to warrant a diagnosis of unspecified anxiety disorder. Addition— ally, the presence of specific phobia in older adults is associated with decreased quality of life and may

1	and thus be more likely to warrant a diagnosis of unspecified anxiety disorder. Addition— ally, the presence of specific phobia in older adults is associated with decreased quality of life and may serve as a risk factor for major neurocognitive disorder.

1	Although most specific phobias develop in childhood and adolescence, it is possible for a specific phobia to develop at any age, often as the result of experiences that are traumatic. For example, phobias of choking almost always follow a near-choking event at any age. Temperamental. Temperamental risk factors for specific phobia, such as negative affec- tivity (neuroticism) or behavioral inhibition, are risk factors for other anxiety disorders as well. Environmental. Environmental risk factors for specific phobias, such as parental over- protectiveness, parental loss and separation, and physical and sexual abuse, tend to pre- dict other anxiety disorders as well. As noted earlier, negative or traumatic encounters with the feared object or situation sometimes (but not always) precede the development of specific phobia.

1	Genetic and physiological. There may be a genetic susceptibility to a certain category of specific phobia (e.g., an individual with a first-degree relative with a specific phobia of an- imals is significantly more likely to have the same specific phobia than any other category of phobia). Individuals with blood-injection-injury phobia show a unique propensity to vasovagal syncope (fainting) in the presence of the phobic stimulus. In the United States, Asians and Latinos report significantly lower rates of specific phobia than non-Latino whites, African Americans, and Native Americans. In addition to having lower prevalence rates of specific phobia, some countries outside of the United States, par- ticularly Asian and African countries, show differing phobia content, age at onset, and gender ratios.

1	Individuals with specific phobia are up to 60% more likely to make a suicide attempt than are individuals without the diagnosis. However, it is likely that these elevated rates are primarily due to comorbidity with personality disorders and other anxiety disorders. Functional Consequences of Specific Phobia

1	Functional Consequences of Specific Phobia Individuals with specific phobia show similar patterns of impairment in psychosocial functioning and decreased quality of life as individuals with other anxiety disorders and alcohol and substance use disorders, including impairments in occupational and inter- personal functioning. In older adults, impairment may be seen in caregiving duties and volunteer activities. Also, fear of falling in older adults can lead to reduced mobility and reduced physical and social functioning, and may lead to receiving formal or informal home support. The distress and impairment caused by specific phobias tend to increase with the number of feared objects and situations. Thus, an individual who fears four ob- jects or situations is likely to have more impairment in his or her occupational and social roles and a lower quality of life than an individual who fears only one object or situation.

1	Individuals with blood-injection-injury specific phobia are often reluctant to obtain med- ical care even when a medical concern is present. Additionally, fear of vomiting and chok- ing may substantially reduce dietary intake.

1	Agoraphobia. Situational specific phobia may resemble agoraphobia in its clinical pre- sentation, given the overlap in feared situations (e.g., ﬂying, enclosed places, elevators). If an individual fears only one of the agoraphobia situations, then specific phobia, situa— tional, may be diagnosed. If two or more agoraphobic situations are feared, a diagnosis of agoraphobia is likely warranted. For example, an individual who fears airplanes and ele- vators (which overlap with the "public transportation” agoraphobic situation) but does not fear other agoraphobic situations would be diagnosed with specific phobia, situa- tional, whereas an individual who fears airplanes, elevators, and crowds (which overlap with two agoraphobic situations, ”using public transportation" and ”standing in line and or being in a crowd”) would be diagnosed with agoraphobia. Criterion B of agoraphobia (the situations are feared or avoided ”because of thoughts that escape might be difficult or help might not be

1	or being in a crowd”) would be diagnosed with agoraphobia. Criterion B of agoraphobia (the situations are feared or avoided ”because of thoughts that escape might be difficult or help might not be available in the event of developing panic-like symptoms or other inca- from specific phobia. If the situations are feared for other reasons, such as fear of being harmed directly by the object or situations (e.g., fear of the plane crashing, fear of the an- imal biting), a specific phobia diagnosis may be more appropriate.

1	Social anxiety disorder. It the situations are feared because of negative evaluation, so- cial anxiety disorder should be diagnosed instead of specific phobia. Separation anxiety disorder. It the situations are feared because of separation from a primary caregiver or attachment figure, separation anxiety disorder should be diagnosed instead of specific phobia. Panic disorder. Individuals with specific phobia may experience panic attacks when con- fronted with their feared situation or object. A diagnosis of specific phobia would be given if the panic attacks only occurred in response to the specific object or situation, whereas a di- agnosis of panic disorder would be given if the individual also experienced panic attacks that were unexpected (i.e., not in response to the specific phobia object or situation).

1	Obsessive-compulsive disorder. If an individual’s primary fear or anxiety is of an ob- ject or situation as a result of obsessions (e.g., fear of blood due to obsessive thoughts about contamination from blood-borne pathogens [i.e., HIV]; fear of driving due to obsessive im- ages of harming others), and if other diagnostic criteria for obsessive-compulsive disorder are met, then obsessive-compulsive disorder should be diagnosed. Trauma- and stressor-related disorders. If the phobia develops following a traumatic event, posttraumatic stress disorder (PTSD) should be considered as a diagnosis. How- ever, traumatic events can precede the onset of PTSD and specific phobia. In this case, a di- agnosis of specific phobia would be assigned only if all of the criteria for PTSD are not met.

1	Eating disorders. A diagnosis of specific phobia is not given if the avoidance behavior is exclusively limited to avoidance of food and food-related cues, in which case a diagnosis of anorexia nervosa or bulimia nervosa should be considered. Schizophrenia spectmm and other psychotic disorders. When the fear and avoidance are due to delusional thinking (as in schizophrenia or other schizophrenia spectrum and other psychotic disorders), a diagnosis of specific phobia is not warranted. Specific phobia is rarely seen in medical-clinical settings in the absence of other psycho- pathology and is more frequently seen in nonmedical mental health settings. Specific pho- bia is frequently associated with a range of other disorders, especially depression in older adults. Because of early onset, specific phobia is typically the temporally primary disorder.

1	Individuals with specific phobia are at increased risk for the development of other dis- orders, including other anxiety disorders, depressive and bipolar disorders, substance- related disorders, somatic symptom and related disorders, and personality disorders (par- ticularly dependent personality disorder). Diagnostic Criteria 300.23 (F40.10) A. Marked tear or anxiety about one or more social situations in which the individual is exposed to possible scrutiny by others. Examples include social interactions (e.g., hav- ing a conversation, meeting unfamiliar people), being observed (e.g., eating or drink- ing), and performing in front of others (e.g., giving a speech). Note: In children, the anxiety must occur in peer settings and not just during interac- tions with adults. B. The individual fears that he or she will act in a way or show anxiety symptoms that will be negatively evaluated (i.e., will be humiliating or embarrassing; will lead to rejection or offend others).

1	C. The social situations almost always provoke tear or anxiety. Note: In children, the fear or anxiety may be expressed by crying, tantrums, freezing, clinging, shrinking, or failing to speak in social situations. D. The social situations are avoided or endured with intense fear or anxiety. E. The fear or anxiety is out of proportion to the actual threat posed by the social situation and to the sociocultural context. F. The fear, anxiety, or avoidance is persistent, typically lasting for 6 months or more. G. The fear, anxiety, or avoidance causes clinically significant distress or impairment in social, occupational, or other important areas of functioning. H. The fear, anxiety, or avoidance is not attributable to the physiological effects of a sub- stance (e.g., a drug of abuse, a medication) or another medical condition.

1	H. The fear, anxiety, or avoidance is not attributable to the physiological effects of a sub- stance (e.g., a drug of abuse, a medication) or another medical condition. I. The fear. anxiety, or avoidance is not better explained by the symptoms of another mental disorder, such as panic disorder, body dysmorphic disorder, or autism spectrum disorder. J. If another medical condition (e.g., Parkinson’s disease, obesity, disfigurement from bums or injury) is present, the fear, anxiety, or avoidance is clearly unrelated or is excessive. Specify if: Performance only: If the fear is restricted to speaking or performing in public.

1	Specify if: Performance only: If the fear is restricted to speaking or performing in public. Individuals with the performance only type of social anxiety disorder have performance fears that are typically most impairing in their professional lives (e.g., musicians, dancers, performers, athletes) or in roles that require regular public speaking. Performance fears may also manifest in work, school, or academic settings in which regular public presenta- tions are required. Individuals with performance only social anxiety disorder do not fear or avoid nonperformance social situations. The essential feature of social anxiety disorder is a marked, or intense, fear or anxiety of so- cial situations in which the individual may be scrutinized by others. In children the fear or anxiety must occur in peer settings and not just during interactions with adults (Criterion

1	A). When exposed to such social situations, the individual fears that he or she will be neg- atively evaluated. The individual is concerned that he or she will be judged as anxious, weak, crazy, stupid, boring, intimidating, dirty, or unlikable. The individual fears that he or she will act or appear in a certain way or show anxiety symptoms, such as blushing, trembling, sweating, stumbling over one’s words, or staring, that will be negatively eval- uated by others (Criterion B). Some individuals fear offending others or being rejected as a result. Fear of offending others—for example, by a gaze or by showing anxiety symp- toms—may be the predominant fear in individuals from cultures with strong collectivistic orientations. An individual with fear of trembling of the hands may avoid drinking, eat- ing, writing, or pointing in public; an individual with fear of sweating may avoid shaking hands or eating spicy foods; and an individual with fear of blushing may avoid public per- formance,

1	eat- ing, writing, or pointing in public; an individual with fear of sweating may avoid shaking hands or eating spicy foods; and an individual with fear of blushing may avoid public per- formance, bright lights, or discussion about intimate topics. Some individuals fear and avoid urinating in public restrooms when other individuals are present (i.e., paruresis, or "shy bladder syndrome”).

1	The social situations almost always provoke fear or anxiety (Criterion C). Thus, an in- dividual who becomes anxious only occasionally in the social situation(s) would not be di- agnosed with social anxiety disorder. However, the degree and type of fear and anxiety may vary (e.g., anticipatory anxiety, a panic attack) across different occasions. The antici- patory anxiety may occur sometimes far in advance of upcoming situations (e.g., worrying every day for weeks before attending a social event, repeating a speech for days in advance). In children, the fear or anxiety may be expressed by crying, tantrums, freezing, clinging, or shrinking in social situations. The individual will often avoid the feared social situations.

1	In children, the fear or anxiety may be expressed by crying, tantrums, freezing, clinging, or shrinking in social situations. The individual will often avoid the feared social situations. Alternatively, the situations are endured with intense fear or anxiety (Criterion D). Avoid- ance can be extensive (e.g., not going to parties, refusing school) or subtle (e.g., overpre- paring the text of a speech, diverting attention to others, limiting eye contact).

1	The fear or anxiety is judged to be out of proportion to the actual risk of being nega- tively evaluated or to the consequences of such negative evaluation (Criterion E). Some- times, the anxiety may not be judged to be excessive, because it is related to an actual danger (e.g., being bullied or tormented by others). However, individuals with social anx- iety disorder often overestimate the negative consequences of social situations, and thus the judgment of being out of proportion is made by the clinician. The individual’s socio- cultural context needs to be taken into account when this judgment is being made. For ex- ample, in certain cultures, behavior that might otherwise appear socially anxious may be considered appropriate in social situations (e.g., might be seen as a sign of respect).

1	The duration of the disturbance is typically at least 6 months (Criterion F). This dura- tion threshold helps distinguish the disorder from transient social fears that are com— mon, particularly among children and in the community. However, the duration criterion should be used as a general guide, with allowance for some degree of ﬂexibility. The fear, anxiety, and avoidance must interfere significantly with the individual’s normal routine, occupational or academic functioning, or social activities or relationships, or must cause clinically significant distress or impairment in social, occupational, or other important ar- eas of functioning (Criterion G). For example, an individual who is afraid to speak in pub- lic would not receive a diagnosis of social anxiety disorder if this activity is not routinely encountered on the job or in classroom work, and if the individual is not significantly dis- tressed about it. However, if the individual avoids, or is passed over for, the job or

1	is not routinely encountered on the job or in classroom work, and if the individual is not significantly dis- tressed about it. However, if the individual avoids, or is passed over for, the job or educa- tion he or she really wants because of social anxiety symptoms, Criterion G is met.

1	Individuals with social anxiety disorder may be inadequately assertive or excessively sub- missive or, less commonly, highly controlling of the conversation. They may show overly rigid body posture or inadequate eye contact, or speak with an overly soft voice. These in— dividuals may be shy or withdrawn, and they may be less open in conversations and dis- close little about themselves. They may seek employment in jobs that do not require social contact, although this is not the case for individuals with social anxiety disorder, perfor- mance only. They may live at home longer. Men may be delayed in marrying and having a family, Whereas women who would want to work outside the home may live a life as homemaker and mother. Self—medication with substances is common (e.g., drinking be- fore going to a party). Social anxiety among older adults may also include exacerbation of symptoms of medical illnesses, such as increased tremor or tachycardia. Blushing is a hall- mark physical response

1	to a party). Social anxiety among older adults may also include exacerbation of symptoms of medical illnesses, such as increased tremor or tachycardia. Blushing is a hall- mark physical response of social anxiety disorder.

1	The 12-month prevalence estimate of social anxiety disorder for the United States is ap- proximately 7%. Lower 12-month prevalence estimates are seen in much of the world us- ing the same diagnostic instrument, clustering around 0.5%—2.0%,' median prevalence in Europe is 2.3%. The 12-month prevalence rates in children and adolescents are comparable to those in adults. Prevalence rates decrease with age. The 12-month prevalence for older adults ranges from 2% to 5%. In general, higher rates of social anxiety disorder are found in females than in males in the general population (with odds ratios ranging from 1.5 to 2.2), and the gender difference in prevalence is more pronounced in adolescents and young adults. Gender rates are equivalent or slightly higher for males in clinical samples, and it is assumed that gender roles and social expectations play a significant role in ex- plaining the heightened help-seeking behavior in male patients. Prevalence in the United

1	States is higher in American Indians and lower in persons of Asian, Latino, African Amer- ican, and Afro-Caribbean descent compared with non-Hispanic whites.

1	Median age at Onset of social anxiety disorder in the United States is 13 years, and 75% of individuals have an age at onset between 8 and 15 years. The disorder sometimes emerges out of a childhood history of social inhibition or shyness in US. and European studies. On- set can also occur in early childhood. Onset of social anxiety disorder may follow a stress- ful or humiliating experience (e.g., being bullied, vomiting during a public speech), or it may be insidious, developing slowly. First onset in adulthood is relatively rare and is more likely to occur after a stressful or humiliating event or after life changes that require new social roles (e.g., marrying someone from a different social class, receiving a job promo- tion). Social anxiety disorder may diminish after an individual with fear of dating marries and may reemerge after divorce. Among individuals presenting to clinical care, the disor— der tends to be particularly persistent.

1	Adolescents endorse a broader pattern of fear and avoidance, including of dating, compared with younger children. Older adults express social anxiety at lower levels but across a broader range of situations, whereas younger adults express higher levels of so- cial anxiety for specific situations. In older adults, social anxiety may concern disability due to declining sensory functioning (hearing, vision) or embarrassment about one’s ap- pearance (e.g., tremor as a symptom of Parkinson’s disease) or functioning due to medical conditions, incontinence, or cognitive impairment (e.g., forgetting people’s names). In the community approximately 30% of individuals with social anxiety disorder experience re- mission of symptoms within 1 year, and about 50% experience remission within a few years. For approximately 60% of individuals without a specific treatment for social anxiety disorder, the course takes several years or longer.

1	Detection of social anxiety disorder in older adults may be challenging because of sev- eral factors, including a focus on somatic symptoms, comorbid medical illness, limited insight, changes to social environment or roles that may obscure impairment in social functioning, or reticence about describing psychological distress. Temperamental. Underlying traits that predispose individuals to social anxiety disor- der include behavioral inhibition and fear of negative evaluation. Environmental. There is no causative role of increased rates of childhood maltreatment or other early—onset psychosocial adversity in the development of social anxiety disorder. How- ever, childhood maltreatment and adversity are risk factors for social anxiety disorder.

1	Genetic and physiological. Traits predisposing individuals to social anxiety disorder, such as behavioral inhibition, are strongly genetically inﬂuenced. The genetic inﬂuence is subject to gene-environment interaction; that is, children with high behavioral inhibition are more susceptible to environmental inﬂuences, such as socially anxious modeling by parents. Also, social anxiety disorder is heritable (but performance-only anxiety less so). First—degree relatives have a two to six times greater chance of having social anxiety dis- order, and liability to the disorder involves the interplay of disorder-specific (e.g., fear of negative evaluation) and nonspecific (e.g., neuroticism) genetic factors.

1	The syndrome of taijin kyofusho (e.g., in Japan and Korea) is often characterized by social- evaluative concerns, fulfilling criteria for social anxiety disorder, that are associated with the fear that the individual makes other people uncomfortable (e.g., "My gaze upsets peo- ple so they look away and avoid me”), a fear that is at times experienced with delusional intensity. This symptom may also be found in non-Asian settings. Other presentations of taijin kyofusho may fulfill criteria for body dysmorphic disorder or delusional disorder. Immigrant status is associated with significantly lower rates of social anxiety disorder in both Latino and non—Latino white groups. Prevalence rates of social anxiety disorder may not be in line with self—reported social anxiety levels in the same culture—that is, societies with strong collectivistic orientations may report high levels of social anxiety but low prev- alence of social anxiety disorder.

1	Females with social anxiety disorder report a greater number of social fears and comorbid depressive, bipolar, and anxiety disorders, whereas males are more likely to fear dating, have oppositional defiant disorder or conduct disorder, and use alcohol and illicit drugs to relieve symptoms of the disorder. Paruresis is more common in males. Functional Consequences of Social Anxiety Disorder

1	Functional Consequences of Social Anxiety Disorder Social anxiety disorder is associated with elevated rates of school dropout and with de- creased well-being, employment, workplace productivity, socioeconomic status, and quality of life. Social anxiety disorder is also associated with being single, unmarried, or divorced and with not having children, particularly among men. In older adults, there may be impair- ment in caregiving duties and volunteer activities. Social anxiety disorder also impedes lei- sure activities. Despite the extent of distress and social impairment associated with social anxiety disorder, only about half of individuals with the disorder in Western societies ever seek treatment, and they tend to do so only after 15—20 years of experiencing symptoms. Not being employed is a strong predictor for the persistence of social anxiety disorder.

1	Normative shyness. Shyness (i.e., social reticence) is a common personality trait and is not by itself pathological. In some societies, shyness is even evaluated positively. How- ever, when there is a significant adverse impact on social, occupational, and other impor- tant areas of functioning, a diagnosis of social anxiety disorder should be considered, and when full diagnostic criteria for social anxiety disorder are met, the disorder should be di- agnosed. Only a minority (12%) of self—identified shy individuals in the United States have symptoms that meet diagnostic criteria for social anxiety disorder.

1	Agoraphobia. Individuals with agoraphobia may fear and avoid social situations (e.g., go ing to a movie) because escape might be difficult or help might not be available in the event of incapacitation or panic-like symptoms, whereas individuals with social anxiety disorder are most fearful of scrutiny by others. Moreover, individuals with social anxiety disorder are likely to be calm When left entirely alone, which is often not the case in agoraphobia. Panic disorder. Individuals with social anxiety disorder may have panic attacks, but the concern is about fear of negative evaluation, whereas in panic disorder the concern is about the panic attacks themselves.

1	Generalized anxiety disorder. Social worries are common in generalized anxiety disorder, but the focus is more on the nature of ongoing relationships rather than on fear of negative evaluation. Individuals with generalized anxiety disorder, particularly children, may have ex- cessive worries about the quality of their social performance, but these worries also pertain to nonsocial performance and when the individual is not being evaluated by others. In social anx- iety disorder, the worries focus on social performance and others’ evaluation.

1	Separation anxiety disorder. Individuals with separation anxiety disorder may avoid social settings (including school refusal) because of concerns about being separated from attachment figures or, in children, about requiring the presence of a parent when it is not developmentally appropriate. Individuals with separation anxiety disorder are usually comfortable in social settings when their attachment figure is present or when they are at home, whereas those with social anxiety disorder may be uncomfortable when social sit- uations occur at\home or in the presence of attachment figures. Specific phobias. Individuals with specific phobias may fear embarrassment or humil- iation (e.g., embarrassment about fainting when they have their blood drawn), but they do not generally fear negative evaluation in other social situations.

1	Selective mutism. Individuals with selective mutism may fail to speak because of fear of negative evaluation, but they do not fear negative evaluation in social situations where no speaking is required (e.g., nonverbal play). Major depressive disorder. Individuals with major depressive disorder may be con- cerned about being negatively evaluated by others because they feel they are bad or not worthy of being liked. In contrast, individuals with social anxiety disorder are worried about being negatively evaluated because of certain social behaviors or physical symptoms.

1	Body dysmorphic disorder. Individuals with body dysmorphic disorder are preoccu- pied with one or more perceived defects or ﬂaws in their physical appearance that are not observable or appear slight to others; this preoccupation often causes social anxiety and avoidance. If their social fears and avoidance are caused only by their beliefs about their appearance, a separate diagnosis of social anxiety disorder is not warranted. Delusional disorder. Individuals with delusional disorder may have nonbizarre delu- sions and / or hallucinations related to the delusional theme that focus on being rejected by or offending others. Although extent of insight into beliefs about social situations may vary, many individuals with social anxiety disorder have good insight that their beliefs are out of proportion to the actual threat posed by the social situation.

1	Autism spectrum disorder. Social anxiety and social communication deficits are hall- marks of autism spectrum disorder. Individuals with social anxiety disorder typically have adequate age-appropriate social relationships and social communication capacity, although they may appear to have impairment in these areas when first interacting with unfamiliar peers or adults. Personality disorders. Given its frequent onset in childhood and its persistence into and through adulthood, social anxiety disorder may resemble a personality disorder. The most apparent overlap is with avoidant personality disorder. Individuals with avoidant person- ality disorder have a broader avoidance pattern than those with social anxiety disorder. Nonetheless, social anxiety disorder is typically more comorbid with avoidant personality disorder than with other personality disorders, and avoidant personality disorder is more comorbid with social anxiety disorder than with other anxiety disorders.

1	Other mental disorders. Social fears and discomfort can occur as part of schizophrenia, but other evidence for psychotic symptoms is usually present. In individuals with an eat- ing disorder, it is important to determine that fear of negative evaluation about eating disorder symptoms or behaviors (e.g., purging and vomiting) is not the sole source of so- cial anxiety before applying a diagnosis of social anxiety disorder. Similarly, obsessive- compulsive disorder may be associated with social anxiety, but the additional diagnosis of social anxiety disorder is used only when social fears and avoidance are independent of the foci of the obsessions and compulsions. Other medical conditions. Medical conditions may produce symptoms that may be em- barrassing (e.g. trembling in Parkinson’s disease). When the fear of negative evaluation due to other medical conditions is excessive, a diagnosis of social anxiety disorder should be considered.

1	Oppositional defiant disorder. Refusal to speak due to opposition to authority figures should be differentiated from failure to speak due to fear of negative evaluation.

1	Social anxiety disorder is often comorbid with other anxiety disorders, major depressive disorder, and substance use disorders, and the onset of social anxiety disorder generally precedes that of the other disorders, except for specific phobia and separation anxiety dis- order. Chronic social isolation in the course of a social anxiety disorder may result in major depressive disorder. Comorbidity with depression is high also in older adults. Substances may be used as self-medication for social fears, but the symptoms of substance intoxica- tion or withdrawal, such as trembling, may also be a source of (further) social fear. Social anxiety disorder is frequently comorbid with bipolar disorder or body dysmorphic disor- der; for example, an individual has body dysmorphic disorder concerning a preoccupa- tion with a slight irregularity of her nose, as well as social anxiety disorder because of a severe fear of sounding unintelligent. The more generalized form of social anxiety disor- der,

1	preoccupa- tion with a slight irregularity of her nose, as well as social anxiety disorder because of a severe fear of sounding unintelligent. The more generalized form of social anxiety disor- der, but not social anxiety disorder, performance only, is often comorbid with avoidant personality disorder. In children, comorbidities with high-functioning autism and selec- tive mutism are common.

1	Diagnostic Criteria 300.01 (F41.0) A. Recurrent unexpected panic attacks. A panic attack is an abrupt surge of intense fear or intense discomfort that reaches a peak within minutes, and during which time four (or more) of the following symptoms occur: Note: The abrupt surge can occur from a calm state or an anxious state. Palpitations, pounding heart, or accelerated heart rate. Sweating. Trembling or shaking. Sensations of shortness of breath or smothering. Feelings of choking. Chest pain or discomfort. Nausea or abdominal distress. Feeling dizzy, unsteady. light-headed. or faint. Chills or heat sensations. Paresthesias (numbness or tingling sensations). _ Derealization (feelings of unreality) or depersonalization (being detached from one- self). Fear of losing control or “going crazy.” 13. Fear of dying. ????NQS’IPP’NT‘

1	_ Derealization (feelings of unreality) or depersonalization (being detached from one- self). Fear of losing control or “going crazy.” 13. Fear of dying. ????NQS’IPP’NT‘ Note: Culture-specitic symptoms (e.g., tinnitus, neck soreness, headache, uncontrol- lable screaming or crying) may be seen. Such symptoms should not count as one of the four required symptoms. B. At least one of the attacks has been followed by 1 month (or more) of one or both of the following: 1. Persistent concern or worry about additional panic attacks or their consequences (e.g., losing control, having a heart attack, “going crazy”). 2. A significant maladaptive change in behavior related to the attacks (e.g., behaviors designed to avoid having panic attacks, such as avoidance of exercise or unfamiliar situations).

1	2. A significant maladaptive change in behavior related to the attacks (e.g., behaviors designed to avoid having panic attacks, such as avoidance of exercise or unfamiliar situations). C. The disturbance is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication) or another medical condition (e.g., hyperthyroidism, car- diopulmonary disorders). D. The disturbance is not better explained by another mental disorder (e.g., the panic at- tacks do not occur only in response to feared social situations, as in social anxiety dis- order; in response to circumscribed phobic objects or situations, as in specific phobia; in response to obsessions, as in obsessive-compulsive disorder; in response to re- minders of traumatic events, as in posttraumatic stress disorder; or in response to sep- aration from attachment figures, as in separation anxiety disorder).

1	Panic disorder refers to recurrent unexpected panic attacks (Criterion A). A panic attack is an abrupt surge of intense fear or intense discomfort that reaches a peak within minutes, and during which time four or more of a list of 13 physical and cognitive symptoms occur.

1	The term recurrent literally means more than one unexpected panic attack. The term unex— pected refers to a panic attack for which there is no obvious cue or trigger at the time of oc- currence—that is, the attack appears to occur from out of the blue, such as when the individual is relaxing or emerging from sleep (nocturnal panic attack). In contrast, expected panic attacks are attacks for which there is an obvious cue or trigger, such as a situation in which panic attacks typically occur. The determination of whether panic attacks are ex- pected or unexpected is made by the clinician, who makes this judgment based on a com- bination of careful questioning as to the sequence of events preceding or leading up to the attack and the individual’s own judgment of whether or not the attack seemed to occur for no apparent reason. Cultural interpretations may inﬂuence the assignment of panic at- tacks as expected or unexpected (see section ”Culture-Related Diagnostic Issues" for this

1	seemed to occur for no apparent reason. Cultural interpretations may inﬂuence the assignment of panic at- tacks as expected or unexpected (see section ”Culture-Related Diagnostic Issues" for this disorder). In the United States and Europe, approximately one-half of individuals with panic disorder have expected panic attacks as well as unexpected panic attacks. Thus, the presence of expected panic attacks does not rule out the diagnosis of panic disorder. For more details regarding expected versus unexpected panic attacks, see the text accompa- nying panic attacks (pp. 214—217).

1	The frequency and severity of panic attacks vary widely. In terms of frequency, there may be moderately frequent attacks (e.g., one per week) for months at a time, or short bursts of more frequent attacks (e.g., daily) separated by weeks or months without any at- tacks or with less frequent attacks (e.g., two per month) over many years. Persons who have infrequent panic attacks resemble persons with more frequent panic attacks in terms of panic attack symptoms, demographic characteristics, comorbidity with other disorders, family history, and biological data. In terms of severity, individuals with panic disorder four symptoms) attacks, and the number and type of panic attack symptoms frequently differ from one panic attack to the next. However, more than one unexpected full—symp- tom panic attack is required for the diagnosis of panic disorder.

1	The worries about panic attacks or their consequences usually pertain to physical con- cerns, such as worry that panic attacks reﬂect the presence of life-threatening illnesses (e.g., cardiac disease, seizure disorder); social concerns, such as embarrassment or fear of being judged negatively by others because of visible panic symptoms; and concerns about mental functioning, such as "going crazy" or losing control (Criterion B). The maladaptive changes in behavior represent attempts to minimize or avoid panic attacks or their conse- quences. Examples include avoiding physical exertion, reorganizing daily life to ensure that help is available in the event of a panic attack, restricting usual daily activities, and avoiding agoraphobia-type situations, such as leaving home, using public transportation, or shopping. If agoraphobia is present, a separate diagnosis of agoraphobia is given.

1	One type of unexpected panic attack is a nocturnal panic attack (i.e., waking from sleep in a state of panic, which differs from panicking after fully waking from sleep). In the United

1	States, this type of panic attack has been estimated to occur at least one time in roughly one-quarter to one-third of individuals with panic disorder, of whom the majority also have daytime panic attacks. In addition to worry about panic attacks and their conse- quences, many individuals with panic disorder report constant or intermittent feelings of anxiety that are more broadly related to health and mental health concerns. For example, individuals with panic disorder often anticipate a catastrophic outcome from a mild phys- ical symptom or medication side effect (e.g., thinking that they may have heart disease or that a headache means presence of a brain tumor). Such individuals often are relatively in- tolerant of medication side effects. In addition, there may be pervasive concerns about abilities to complete daily tasks or withstand daily stressors, excessive use of drugs (e.g., alcohol, prescribed medications or illicit drugs) to control panic attacks, or extreme behav- iors

1	about abilities to complete daily tasks or withstand daily stressors, excessive use of drugs (e.g., alcohol, prescribed medications or illicit drugs) to control panic attacks, or extreme behav- iors aimed at controlling panic attacks (e.g., severe restrictions on food intake or avoidance of specific foods or medications because of concerns about physical symptoms that pro- voke panic attacks).

1	In the general population, the 12-month prevalence estimate for panic disorder across the United States and several European countries is about 2%—3% in adults and adolescents. In the United States, significantly lower rates of panic disorder are reported among Latinos, African Americans, Caribbean blacks, and Asian Americans, compared with non-Latino whites; American Indians, by contrast, have significantly higher rates. Lower estimates have been reported for Asian, African, and Latin American countries, ranging from 0.1% to 0.8%. Females are more frequently affected than males, at a rate of approximately 2:1. The gender differentiation occurs in adolescence and is already observable before age 14 years.

1	Although panic attacks occur in children, the overall prevalence of panic disorder is low before age 14 years (<0.4%). The rates of panic disorder show a gradual increase during ad- olescence, particularly in females, and possibly following the onset of puberty, and peak dur- ing adulthood. The prevalence rates decline in older individuals (i.e., 0.7% in adults over the age of 64), possibly reﬂecting diminishing severity to subclinical levels. The median age at onset for panic disorder in the United States is 20—24 years. A small number of cases begin in childhood, and onset after age 45 years is unusual but can occur.

1	The median age at onset for panic disorder in the United States is 20—24 years. A small number of cases begin in childhood, and onset after age 45 years is unusual but can occur. The usual course, if the disorder is untreated, is chronic but waxing and waning. Some in- dividuals may have episodic outbreaks with years of remission in between, and others may have continuous severe symptomatology. Only a minority of individuals have full remission without subsequent relapse within a few years. The course of panic disorder typically is complicated by a range of other disorders, in particular other anxiety disor- ders, depressive disorders, and substance use disorders (see section ”Comorbidity" for this disorder).

1	Although panic disorder is very rare in childhood, first occurrence of "fearful spells" is often dated retrospectively back to childhood. As in adults, panic disorder in adolescents tends to have a chronic course and is frequently comorbid with other anxiety, depressive, and bipolar disorders. To date, no differences in the clinical presentation between adoles- cents and adults have been found. However, adolescents may be less worried about addi- tional panic attacks than are young adults. Lower prevalence of panic disorder in older adults appears to be attributable to age-related ”dampening” of the autonomic nervous system response. Many older individuals with "panicky feelings" are observed to have a ”hybrid” of limited-symptom panic attacks and generalized anxiety. Also, older adults tend to attribute their panic attacks to certain stressful situations, such as a medical pro- cedure or social setting. Older individuals may retrospectively endorse explanations for the panic

1	adults tend to attribute their panic attacks to certain stressful situations, such as a medical pro- cedure or social setting. Older individuals may retrospectively endorse explanations for the panic attack\(which would preclude the diagnosis of panic disorder), even if an attack might actually have been unexpected in the moment (and thus qualify as the basis for a panic disorder diagnosis). This may result in under-endorsement of unexpected panic at- tacks in older individuals. Thus, careful questioning of older adults is required to assess whether panic attacks were expected before entering the situation, so that unexpected panic attacks and the diagnosis of panic disorder are not overlooked.

1	While the low rate of panic disorder in children could relate to difficulties in symptom reporting, this seems unlikely given that children are capable of reporting intense fear or panic in relation to separation and to phobic objects or phobic situations. Adolescents might be less willing than adults to openly discuss panic attacks. Therefore, clinicians should be aware that unexpected panic attacks do occur in adolescents, much as they do in adults, and be attuned to this possibility when encountering adolescents presenting with episodes of intense fear or distress.

1	Temperamental. Negative affectivity (neuroticism) (i.e., proneness to experiencing neg- ative emotions) and anxiety sensitivity (i.e., the disposition to believe that symptoms of anxiety are harmful) are risk factors for the onset of panic attacks and, separately, for worry about panic, although their risk status for the diagnosis of panic disorder is un- known. History of "fearful spells" (i.e., limited-symptom attacks that do not meet full cri- teria for a panic attack) may be a risk factor for later panic attacks and panic disorder. Although separation anxiety in childhood, especially when severe, may precede the later development of panic disorder, it is not a consistent risk factor.

1	Although separation anxiety in childhood, especially when severe, may precede the later development of panic disorder, it is not a consistent risk factor. Environmental. Reports of childhood experiences of sexual and physical abuse are more common in panic disorder than in certain other anxiety disorders. Smoking is a risk factor for panic attacks and panic disorder. Most individuals report identifiable stressors in the months before their first panic attack (e.g., interpersonal stressors and stressors related to physical well-being, such as negative experiences with illicit or prescription drugs, dis- ease, or death in the family).

1	Genetic and physiological. It is believed that multiple genes confer vulnerability to panic disorder. However, the exact genes, gene products, or functions related to the genetic re- gions implicated remain unknown. Current neural systems models for panic disorder em— phasize the amygdala and related structures, much as in other anxiety disorders. There is an increased risk for panic disorder among offspring of parents with anxiety, depressive, and bipolar disorders. Respiratory disturbance, such as asthma, is associated with panic disorder, in terms of past history, comorbidity, and family history.

1	The rate of fears about mental and somatic symptoms of anxiety appears to vary across cultures and may inﬂuence the rate of panic attacks and panic disorder. Also, cultural ex- pectations may inﬂuence the classification of panic attacks as expected or unexpected. For example, a Vietnamese individual who has a panic attack after walking out into a windy environment (tru’ng gié; ”hit by the wind") may attribute the panic attack to exposure to wind as a result of the cultural syndrome that links these two experiences, resulting in clas- sification of the panic attack as expected. Various other cultural syndromes are associated with panic disorder, including ataque de nervios (”attack of nerves”) among Latin Ameri- cans and khyfil attacks and ”soul loss” among Cambodians. Atuque de nervios may involve trembling, uncontrollable screaming or crying, aggressive or suicidal behavior, and deper- sonalization or derealization, which may be experienced longer than the few minutes typical of

1	may involve trembling, uncontrollable screaming or crying, aggressive or suicidal behavior, and deper- sonalization or derealization, which may be experienced longer than the few minutes typical of panic attacks. Some clinical presentations of utaque de nervios fulfill criteria for condi- tions other than panic attack (e.g., other specified dissociative disorder). These syndromes impact the symptoms and frequency of panic disorder, including the individual’s attribu- tion of unexpectedness, as cultural syndromes may create fear of certain situations, rang- ing from interpersonal arguments (associated with ataque de nervias), to types of exertion (associated with khyril attacks), to atmospheric wind (associated with trting gié attacks).

1	Clarification of the details of cultural attributions may aid in distinguishing expected and unexpected panic attacks. For more information regarding cultural syndromes, refer to the ”Glossary of Cultural Concepts of Distress" in the Appendix. The specific worries about panic attacks or their consequences are likely to vary from one culture to another (and across different age groups and gender). For panic disorder, U.S. community samples of non—Latino whites have significantly less functional impair- ment than African Americans. There are also higher rates of objectively defined severity in non—Latino Caribbean blacks with panic disorder, and lower rates of panic disorder over- all in both African American and Afro—Caribbean groups, suggesting that among individ- uals of African descent, the criteria for panic disorder may be met only when there is substantial severity and impairment. The clinical features of panic disorder do not appear to differ between males and females.

1	The clinical features of panic disorder do not appear to differ between males and females. There is some evidence for sexual dimorphism, with an association between panic disor- der and the catechol-O-methyltransferase (COMT) gene in females only. Agents with disparate mechanisms of action, such as sodium lactate, caffeine, isoprotere- nol, yohimbine, carbon dioxide, and cholecystokinin, provoke panic attacks in individuals with panic disorder to a much greater extent than in healthy control subjects (and in some cases, than in individuals with other anxiety, depressive, or bipolar disorders without panic attacks). Also, for a proportion of individuals with panic disorder, panic attacks are related to hypersensitive medullary carbon dioxide detectors, resulting in hypocapnia and other respiratory irregularities. However, none of these laboratory findings are consid— ered diagnostic of panic disorder.

1	Panic attacks and a diagnosis of panic disorder in the past 12 months are related to a higher rate of suicide attempts and. suicidal ideation in the past 12 months even when comorbid— ity and a history of childhood abuse and other suicide risk factors are taken into account. Functional Consequences of Panic Disorder Panic disorder is associated with high levels of social, occupational, and physical disabil- ity; considerable economic costs; and the highest number of medical visits among the anx- iety disorders, although the effects are strongest with the presence of agoraphobia. Individuals with panic disorder may be frequently absent from work or school for doctor and emergency room visits, which can lead to unemployment or dropping out of school.

1	Individuals with panic disorder may be frequently absent from work or school for doctor and emergency room visits, which can lead to unemployment or dropping out of school. In older adults, impairment may be seen in caregiving duties or volunteer activities. Full- symptom panic attacks typically are associated with greater morbidity (e.g., greater health care utilization, more disability, poorer quality of life) than limited-symptom attacks. Other specified anxiety disorder or unspecified anxiety disorder. Panic disorder should not be diagnosed if full-symptom (unexpected) panic attacks have never been experienced. In the case of only limited—symptom unexpected panic attacks, an other specified anxiety dis- order or unspecified anxiety disorder diagnosis should be considered.

1	Anxiety disorder due to another medical condition. Panic disorder is not diagnosed if the panic attacks are judged to be a direct physiological consequence of another medical condition. Examples of medical conditions that can cause panic attacks include hyperthy- roidism, hyperparathyroidism, pheochromocytoma, vestibular dysfunctions, seizure dis- orders, and cardiopulmonary conditions (e.g., arrhythmias, supraventricular tachycardia, asthma, chronic obstructive pulmonary disease [COPD]). Appropriate laboratory tests (e.g., serum calcium levels for hyperparathyroidism; Holter monitor for arrhythmias) or physical examinations (e.g., for cardiac conditions) may be helpful in determining the eti- ological role of another medical condition.

1	Substance/medication-induced anxiety disorder. Panic disorder is not diagnosed if the panic attacks are judged to be a direct physiological consequence of a substance. In- toxication with central nervous system stimulants (e.g., cocaine, amphetamines, caffeine) or cannabis and withdrawal from central nervous system depressants (e.g., alcohol, bar- biturates) can precipitate a panic attack. However, if panic attacks continue to occur out- side of the context of substance use (e.g., long after the effects of intoxication or withdrawal have ended), a diagnosis of panic disorder should be considered. In addition, because with increased substance use, especially for purposes of self—medication, a detailed history should be taken to determine if the individual had panic attacks prior to excessive sub- stance use. If this is the case, a diagnosis of panic disorder should be considered in addition to a diagnosis of substance use disorder. Features such as onset after age 45 years or the

1	sub- stance use. If this is the case, a diagnosis of panic disorder should be considered in addition to a diagnosis of substance use disorder. Features such as onset after age 45 years or the presence of atypical symptoms during a panic attack (e.g., vertigo, loss of consciousness, loss of bladder or bowel control, slurred speech, amnesia) suggest the possibility that an- other medical condition or a substance may be causing the panic attack symptoms.

1	Other mental disorders with panic attacks as an associated feature (e.g., other anxiety disorders and psychotic disorders). Panic attacks that occur as a symptom of other anx- iety disorders are expected (e.g., triggered by social situations in social anxiety disorder, by phobic objects or situations in specific phobia or agoraphobia, by worry in generalized anx- iety disorder, by separation from home or attachment figures in separation anxiety disorder) and thus would not meet criteria for panic disorder. (Note: Sometimes an unexpected panic attack is associated with the onset of another anxiety disorder, but then the attacks become expected, whereas panic disorder is characterized by recurrent unexpected panic attacks.) If the panic attacks occur only in response to specific triggers, then only the relevant anxiety disorder is assigned. However, if the individual experiences unexpected panic attacks as well and shows persistent concern and worry or behavioral change because of the

1	then only the relevant anxiety disorder is assigned. However, if the individual experiences unexpected panic attacks as well and shows persistent concern and worry or behavioral change because of the attacks, then an additional diagnosis of panic disorder should be considered.

1	Panic disorder infrequently occurs in clinical settings in the absence of other psychopa- thology. The prevalence of panic disorder is elevated in individuals with other disorders, particularly other anxiety disorders (and especially agoraphobia), major depression, bipo- lar disorder, and possibly mild alcohol use disorder. While panic disorder often has an ear- lier age at onset than the comorbid disorder(s), onset sometimes occurs after the comorbid disorder and may be seen as a severity marker of the comorbid illness.

1	Reported lifetime rates of comorbidity between major depressive disorder and panic disorder vary widely, ranging from 10% to 65% in individuals with panic disorder. In ap- proximately one-third of individuals with both disorders, the depression precedes the on— set of panic disorder. In the remaining two-thirds, depression occurs coincident with or following the onset of panic disorder. A subset of individuals with panic disorder develop a substance-related disorder, which for some represents an attempt to treat their anxiety with alcohol or medications. Comorbidity with other anxiety disorders and illness anxiety disorder is also common.

1	Panic disorder is significantly comorbid with numerous general medical symptoms and conditions, including, but not limited to, dizziness, cardiac arrhythmias, hyperthy- roidism, asthma, COPD, and irritable bowel syndrome. However, the nature of the asso- ciation (e.g., cause and effect) between panic disorder and these conditions remains unclear. Although mitral valve prolapse and thyroid disease are more common among in- dividuals with panic disorder than in the general population, the differences in prevalence are not consistent.

1	Note: Symptoms are presented for the purpose of identifying a panic attack; however, panic attack is not a mental disorder and cannot be coded. Panic attacks can occur in the context of any anxiety disorder as well as other mental disorders (e.g., depressive disor- ders, posttraumatic stress disorder, substance use disorders) and some medical condi- tions (e.g., cardiac, respiratory, vestibular, gastrointestinal). When the presence of a panic attack is identified, it should be noted as a specifier (e.g., “posttraumatic stress disorder with panic attacks”). For panic disorder, the presence of panic attack is contained within the criteria for the disorder and panic attack is not used as a specifier. An abrupt surge of intense fear or intense discomfort that reaches a peak within minutes, and during which time four (or more) of the following symptoms occur: Note: The abrupt surge can occur from a calm state or an anxious state.

1	Note: The abrupt surge can occur from a calm state or an anxious state. 1. Palpitations, pounding heart, or accelerated heart rate. 2. Sweating. 3. Trembling or shaking. 4. Sensations of shortness of breath or smothering. 5. Feelings of choking. 6. Chest pain or discomfort. 7. Nausea or abdominal distress. 8. Feeling dizzy, unsteady, light-headed, or taint. 9. Chilis or heat sensations. 10. Paresthesias (numbness or tingling sensations). 11. Derealization (feelings of unreality) or depersonalization (being detached from oneself). 12. Fear of losing control or "going crazy.” 13. Fear of dying. Note: Culture—specific symptoms (e.g., tinnitus, neck soreness, headache, uncontrollable screaming or crying) may be seen. Such symptoms should not count as one of the four required symptoms.

1	The essential feature of a panic attack is an abrupt surge of intense fear or intense discomfort that reaches a peak within minutes and during which time four or more of 13 physical and cog- nitive symptoms occur. Eleven of these 13 symptoms are physical (e.g., palpitations, sweat- ing), while two are cognitive (i.e., fear of losing control or going crazy, fear of dying). ”Fear of going crazy” is a colloquialism often used by individuals with panic attacks and is not in- tended as a pejorative or diagnostic term. The term within minutes means that the time to peak intensity is literally only a few minutes. A panic attack can arise from either a calm state or an anxious state, and time to peak intensity should be assessed independently of any preceding anxiety. That is, the start of the panic attack is the point at which there is an abrupt increase in discomfort rather than the point at which anxiety first developed. Likewise, a panic attack can return to either an anxious state or a

1	attack is the point at which there is an abrupt increase in discomfort rather than the point at which anxiety first developed. Likewise, a panic attack can return to either an anxious state or a calm state and possibly peak again. A panic attack is dis- tinguished from ongoing anxiety by its time to peak intensity, which occurs within minutes; its discrete nature; and its typically greater severity. Attacks that meet all other criteria but have fewer than four physical and / or cognitive symptoms are referred to as limited—symptom attacks.

1	There are two characteristic types of panic attacks: expected and unexpected. Expected panic attacks are attacks for which there is an obvious cue or trigger, such as situations in which panic attacks have typically occurred. Unexpected panic attacks are those for which there is no obvious cue or trigger at the time of occurrence (e.g., when relaxing or out of sleep [nocturnal panic attack]). The determination of whether panic attacks are expected or unexpected is made by the clinician, who makes this judgment based on a combination of careful questioning as to the sequence of events preceding or leading up to the attack and the individual’s own judgment of whether or not the attack seemed to occur for no ap- parent reason. Cultural interpretations may inﬂuence their determination as expected or unexpected. Culture-specific symptoms (e.g., tinnitus, neck soreness, headache, uncon- trollable screaming or crying) may be seen; however, such symptoms should not count as one of the four

1	or unexpected. Culture-specific symptoms (e.g., tinnitus, neck soreness, headache, uncon- trollable screaming or crying) may be seen; however, such symptoms should not count as one of the four required symptoms. Panic attacks can occur in the context of any mental disorder (e.g., anxiety disorders, depressive disorders, bipolar disorders, eating disorders, obsessive-compulsive and related disorders, personality disorders, psychotic disorders, substance use disorders) and some medical conditions (e.g., cardiac, respiratory, vestibu- lar, gastrointestinal), with the majority never meeting criteria for panic disorder. Recur- rent unexpected panic attacks are required for a diagnosis of panic disorder.

1	One type of unexpected panic attack is a nocturnal panic attack (i.e., waking from sleep in a state of panic), which differs from panicking after fully waking from sleep. Panic attacks are related to a higher rate of suicide attempts and suicidal ideation even when comorbid- ity and other suicide risk factors are taken into account. In the general population, 12-month prevalence estimates for panic attacks in the United

1	In the general population, 12-month prevalence estimates for panic attacks in the United States is 11.2% in adults. Twelve-month prevalence estimates do not appear to differ sig- nificantly among African Americans, Asian Americans, and Latinos. Lower 12-month prevalence estimates for European countries appear to range from 2.7% to 3.3%. Females are more frequently affected than males, although this gender difference is more pro- nounced for panic disorder. Panic attacks can occur in children but are relatively rare until the age of puberty, when the prevalence rates increase. The prevalence rates decline in older individuals, possibly reﬂecting diminishing severity to subclinical levels.

1	The mean age at onset for panic attacks in the United States is approximately 22—23 years among adults. However, the course of panic attacks is likely inﬂuenced by the course of any co-occurring mental disorder(s) and stressful life events. Panic attacks are uncommon, and unexpected panic attacks are rare, in preadolescent children. Adolescents might be less willing than adults to openly discuss panic attacks, even though they present with ep- isodes of intense fear or discomfort. Lower prevalence of panic attacks in older individuals may be related to a weaker autonomic response to emotional states relative to younger in- dividuals. Older individuals may be less inclined to use the word ”fear” and more inclined to use the word ”discomfort” to describe panic attacks. Older individuals with ”panicky feelings” may have a hybrid of limited-symptom attacks and generalized anxiety. In addition, older individuals tend to attribute panic attacks to certain situations that are stressful

1	with ”panicky feelings” may have a hybrid of limited-symptom attacks and generalized anxiety. In addition, older individuals tend to attribute panic attacks to certain situations that are stressful (e.g., medical procedures, social settings) and may retrospectively endorse expla- nations for the panic attack even if it was unexpected in the moment. This may result in un- der-endorsement of unexpected panic attacks in older individuals.

1	Temperamental. Negative affectivity (neuroticism) (i.e., proneness to experiencing neg- ative emotions) and anxiety sensitivity (i.e., the disposition to believe that symptoms of anxiety are harmful) are risk factors for the onset of panic attacks. History of "fearful spells” (i.e., limited-symptom attacks that do not meet full criteria for a panic attack) may be a risk factor for later panic attacks. Environmental. Smoking is a risk factor for panic attacks. Most individuals report iden- tifiable stressors in the months before their first panic attack (e.g., interpersonal stressors and stressors related to physical well-being, such as negative experiences with illicit or prescription drugs, disease, or death in the family).

1	Cultural interpretations may inﬂuence the determination of panic attacks as expected or unexpected. Culture-specific symptoms (e.g., tinnitus, neck soreness, headache, and un- controllable screaming or crying) may be seen; however, such symptoms should not count as one of the four required symptoms. Frequency of each of the 13 symptoms varies cross- culturally (e.g., higher rates of paresthesias in African Americans and of dizziness in sev- eral Asian groups). Cultural syndromes also inﬂuence the cross-cultural presentation of panic attacks, resulting in different symptom profiles across different cultural groups. Ex- amples include khyfil (wind) attacks, a Cambodian cultural syndrome involving dizziness, tinnitus, and neck soreness; and triing gié (wind-related) attacks, a Vietnamese cultural syndrome associated with headaches. Ataque de nervios (attack of nerves) is a cultural syn- drome among Latin Americans that may involve trembling, uncontrollable screaming or crying, aggressive

1	syndrome associated with headaches. Ataque de nervios (attack of nerves) is a cultural syn- drome among Latin Americans that may involve trembling, uncontrollable screaming or crying, aggressive or suicidal behavior, and depersonalization or derealization, and which may be experienced for longer than only a few minutes. Some clinical presentations of ataque de nervios fulfill criteria for conditions other than panic attack (e.g., other specified dissociative disorder). Also, cultural expectations may inﬂuence the classification of panic attacks as expected or unexpected, as cultural syndromes may create fear of certain situa- tions, ranging from interpersonal arguments (associated with atuque de nervios), to types of exertion (associated with khyfil attacks), to atmospheric wind (associated with tning gié at- tacks). Clarification of the details of cultural attributions may aid in distinguishing ex- pected and unexpected panic attacks. For more information about cultural syndromes,

1	with tning gié at- tacks). Clarification of the details of cultural attributions may aid in distinguishing ex- pected and unexpected panic attacks. For more information about cultural syndromes, see ”Glossary of Cultural Concepts of Distress" in the Appendix to this manual.

1	Panic attacks are more common in females than in males, but clinical features or symp- toms of panic attacks do not differ between males and females. Physiological recordings of naturally occurring panic attacks in individuals with panic disorder indicate abrupt surges of arousal, usually of heart rate, that reach a peak within minutes and subside within minutes, and for a proportion of these individuals the panic attack may be preceded by cardiorespiratory instabilities. Functional Consequences of Panic Attacks

1	Functional Consequences of Panic Attacks In the context of\co-occurring mental disorders, including anxiety disorders, depressive disorders, bipolar disorder, substance use disorders, psychotic disorders, and personality disorders, panic attacks are associated with increased symptom severity, higher rates of comorbidity and suicidality, and poorer treatment response. Also, full-symptom panic at- tacks typically are associated with greater morbidity (e.g., greater health care utilization, more disability, poorer quality of life) than limited—symptom attacks. Other paroxysmal episodes (e.g., “anger attacks”). Panic attacks should not be diag- nosed if the episodes do not involve the essential feature of an abrupt surge of intense fear or intense discomfort, but rather other emotional states (e.g., anger, grief).

1	Anxiety disorder due to another medical condition. Medical conditions that can cause or be misdiagnosed as panic attacks include hyperthyroidism, hyperparathyroidism, pheo- chromocytoma, vestibular dysfunctions, seizure disorders, and cardiopulmonary con- ditions (e.g., arrhythmias, supraventricular tachycardia, asthma, chronic obstructive pulmonary disease). Appropriate laboratory tests (e.g., serum calcium levels for hyperpara- thyroidism; Holter monitor for arrhythmias) or physical examinations (e.g., for cardiac con- ditions) may be helpful in determining the etiological role of another medical condition.

1	Substance/medication-induced anxiety disorder. Intoxication with central nervous system stimulants (e.g., cocaine, amphetamines, caffeine) or cannabis and withdrawal from central nervous system depressants (e.g., alcohol, barbiturates) can precipitate a panic attack. A detailed history should be taken to determine if the individual had panic attacks prior to excessive substance use. Features such as onset after age 45 years or the presence of atypical symptoms during a panic attack (e.g., vertigo, loss of consciousness, loss of bladder or bowel control, slurred speech, or amnesia) suggest the possibility that a medical condition or a substance may be causing the panic attack symptoms. Panic disorder. Repeated unexpected panic attacks are required but are not sufficient for the diagnosis of panic disorder (i.e., full diagnostic criteria for panic disorder must be met).

1	Panic disorder. Repeated unexpected panic attacks are required but are not sufficient for the diagnosis of panic disorder (i.e., full diagnostic criteria for panic disorder must be met). Panic attacks are associated with increased likelihood of various comorbid mental dis- orders, including anxiety disorders, depressive disorders, bipolar disorders, impulse- control disorders, and substance use disorders. Panic attacks are associated with increased likelihood of later developing anxiety disorders, depressive disorders, bipolar disorders, and possibly other disorders. Diagnostic Criteria 300.22 (F40.00) A. Marked tear or anxiety about two (or more) of the following five situations: Using public transportation (e.g., automobiles, buses, trains, ships, planes). Being in open spaces (e.g., parking lots, marketplaces, bridges). Being in enclosed places (e.g., shops, theaters, cinemas). Standing in line or being in a crowd. 5. Being outside of the home alone. ?WNT‘

1	Being in enclosed places (e.g., shops, theaters, cinemas). Standing in line or being in a crowd. 5. Being outside of the home alone. ?WNT‘ B. The individual tears or avoids these situations because of thoughts that escape might be difficult or help might not be available in the event of developing panic-like symp- toms or other incapacitating or embarrassing symptoms (e.g., fear of falling in the el- derly; fear of incontinence). C. The agoraphobic situations almost always provoke fear or anxiety. D. The agoraphobic situations are actively avoided, require the presence of a companion, or are endured with intense tear or anxiety. E. The fear or anxiety is out of proportion to the actual danger posed by the agoraphobic situations and to the sociocultural context. F. The fear, anxiety, or avoidance is persistent, typically lasting for 6 months or more.

1	F. The fear, anxiety, or avoidance is persistent, typically lasting for 6 months or more. G. The fear, anxiety. or avoidance causes clinically significant distress or impairment in social, occupational, or other important areas of functioning. H. If another medical condition (e.g., inflammatory bowel disease, Parkinson’s disease) is present, the fear, anxiety, or avoidance is clearly excessive. l. The fear, anxiety, or avoidance is not better explained by the symptoms of another men- tal disorder—for example, the symptoms are not confined to specific phobia, situational type; do not involve only social situations (as in social anxiety disorder); and are not re- lated exclusively to obsessions (as in obsessive-compulsive disorder), perceived defects or flaws in physical appearance (as in body dysmorphic disorder), reminders of traumatic events (as in posttraumatic stress disorder), or fear of separation (as in separation anx- iety disorder).

1	Note: Agoraphobia is diagnosed irrespective of the presence of panic disorder. If an indi- vidual’s presentation meets criteria for panic disorder and agoraphobia, both diagnoses should be assigned.

1	The essential feature of agoraphobia is marked, or intense, fear or anxiety triggered by the real or anticipated exposure to a wide range of situations (Criterion A). The diagnosis re- quires endorsement of symptoms occurring in at least two of the following five situations: 1) using public transporation, such as automobiles, buses, trains, ships, or planes; 2) being in open spaces, such as parking lots, marketplaces, or bridges; 3) being in enclosed spaces, such as shops, theaters, or cinemas; 4) standing in line or being in a crowd; or 5) being out- side of the home alone. The examples for each situation are not exhaustive; other situations may be feared. When experiencing fear and anxiety cued by such situations, individuals typically experience thoughts that something terrible might happen (Criterion B). Individ- uals frequently believe that escape from such situations might be difficult (e.g., ”can’t get out of here”) or that help might be unavailable (e.g., ”there is nobody to

1	happen (Criterion B). Individ- uals frequently believe that escape from such situations might be difficult (e.g., ”can’t get out of here”) or that help might be unavailable (e.g., ”there is nobody to help me") when panic-like symptoms or other incapacitating or embarrassing symptoms occur. ”Panic-like symptoms” refer to any of the 13 symptoms included in the criteria for panic attack, such as dizziness, faintness, and fear of dying. “Other incapacitating or embarrassing symptoms” include symptoms such as vomiting and inﬂammatory bowel symptoms, as well as, in older adults, a fear of falling or, in children, a sense of disorientation and getting lost.

1	The amount of fear experienced may vary with proximity to the feared situation and may occur in anticipation of or in the actual presence of the agoraphobic situation. Also, the fear or anxiety may take the form of a full— or limited-symptom panic attack (i.e., an ex- pected panic attack). Fear or anxiety is evoked nearly every time the individual comes into contact with the feared situation (Criterion C). Thus, an individual who becomes anxious only occasionally in an agoraphobic situation (e.g., becomes anxious when standing in line on only one out of every five occasions) would not be diagnosed with agoraphobia. The in- dividual actively avoids the situation or, if he or she either is unable or decides not to avoid it, the situation evokes intense fear or anxiety (Criterion D). Active avoidance means the in- dividual is currently behaving in ways that are intentionally designed to prevent or min- imize contact with agoraphobic situations. Avoidance can be behavioral (e.g., changing

1	means the in- dividual is currently behaving in ways that are intentionally designed to prevent or min- imize contact with agoraphobic situations. Avoidance can be behavioral (e.g., changing daily routines, choosing a job nearby to avoid using public transportation, arranging for food delivery to avoid entering shops and supermarkets) as well as cognitive (e.g., using distraction to getthrough agoraphobic situations) in nature. The avoidance can become so severe that the person is completely homebound. Often, an individual is better able to con- front a feared situation when accompanied by a companion, such as a partner, friend, or health professional.

1	The fear, anxiety, or avoidance must be out of proportion to the actual danger posed by the agoraphobic situations and to the sociocultural context (Criterion E). Differentiating clinically significant agoraphobic fears from reasonable fears (e.g., leaving the house dur- ing a bad storm) or from situations that are deemed dangerous (e.g., walking in a parking lot or using public transportation in a high-crime area) is important for a number of reasons.

1	First, what constitutes avoidance may be difficult to judge across cultures and sociocultural contexts (e.g., it is socioculturally appropriate for orthodox Muslim women in certain parts of the world to avoid leaving the house alone, and thus such avoidance would not be con- sidered indicative of agoraphobia). Second, older adults are likely to overattribute their fears to age—related constraints and are less likely to judge their fears as being out of pro- portion to the actual risk. Third, individuals with agoraphobia are likely to overestimate danger in relation to panic-like or other bodily symptoms. Agoraphobia should be diag- nosed only if the fear, anxiety, or avoidance persists (Criterion F) and if it causes clinically significant distress or impairment in social, occupational, or other important areas of func- tioning (Criterion G). The duration of "typically lasting for 6 months or more“ is meant to exclude individuals with short-lived, transient problems. However, the

1	or other important areas of func- tioning (Criterion G). The duration of "typically lasting for 6 months or more“ is meant to exclude individuals with short-lived, transient problems. However, the duration criterion should be used as a general guide, with allowance for some degree of ﬂexibility.

1	In its most severe forms, agoraphobia can cause individuals to become completely home- bound, unable to leave their home and dependent on others for services or assistance to pro- vide even for basic needs. Demoralization and depressive symptoms, as well as abuse of alcohol and sedative medication as inappropriate self-medication strategies, are common. Every year approximately 1.7% of adolescents and adults have a diagnosis of agoraphobia. Females are twice as likely as males to experience agoraphobia. Agoraphobia may occur in childhood, but incidence peaks in late adolescence and early adulthood. Twelve-month prevalence in individuals older than 65 years is 0.4%. Prevalence rates do not appear to vary systematically across cultural/racial groups.

1	The percentage of individuals with agoraphobia reporting panic attacks or panic disorder preceding the onset of agoraphobia ranges from 30% in community samples to more than 50% in clinic samples. The majority of individuals with panic disorder show signs of anx- iety and agoraphobia before the onset of panic disorder. In two-thirds of all cases of agoraphobia, initial onset is before age 35 years. There is a substantial incidence risk in late adolescence and early adulthood, with indications for a second high incidence risk phase after age 40 years. First onset in childhood is rare. The overall mean age at onset for agoraphobia is 17 years, although the age at onset without preceding panic attacks or panic disorder is 25—29 years.

1	The course of agoraphobia is typically persistent and chronic. Complete remission is rare (10%), unless the agoraphobia is treated. With more severe agoraphobia, rates of full remission decrease, whereas rates of relapse and chronicity increase. A range of other dis- orders, in particular other anxiety disorders, depressive disorders, substance use disor- ders, and personality disorders, may complicate the course of agoraphobia. The long-term course and outcome of agoraphobia are associated with substantially elevated risk of sec- ondary major depressive disorder, persistent depressive disorder (dysthymia), and sub- stance use disorders.

1	The clinical features of agoraphobia are relatively consistent across the lifespan, although the type of agoraphobic situations triggering fear, anxiety, or avoidance, as well as the type of cognitions, may vary. For example, in children, being outside of the home alone is the most fre- quent situation feared, whereas in older adults, being in shops, standing in line, and being in open spaces are most often feared. Also, cognitions often pertain to becoming lost (in children), to experiencing panic-like symptoms (in adults), to falling (in older adults).

1	The low prevalence of agoraphobia in children could reﬂect difficulties in symptom re— porting, and thus assessments in young children may require solicitation of information from multiple sources, including parents or teachers. Adolescents, particularly males, may be less willing than adults to openly discuss agoraphobic fears and avoidance; how- ever, agoraphobia can occur prior to adulthood and should be assessed in children and adolescents. In older adults, comorbid somatic symptom disorders, as well as motor dis- turbances (e.g., sense of falling or having medical complications), are frequently men- tioned by individuals as the reason for their fear and avoidance. In these instances, care is to be taken in evaluating whether the fear and avoidance are out of proportion to the real danger involved.

1	Temperamental. Behavioral inhibition and neurotic disposition (i.e., negative affectivity [neuroticism] and anxiety sensitivity) are closely associated with agoraphobia but are rel- evant to most anxiety disorders (phobic disorders, panic disorder, generalized anxiety dis- order). Anxiety sensitivity (the disposition to believe that symptoms of anxiety are harmful) is also characteristic of individuals with agoraphobia. Environmental. Negative events in childhood (e.g., separation, death of parent) and other stressful events, such as being attacked or mugged, are associated with the onset of agorapho- bia. Furthermore, individuals with agoraphobia describe the family climate and child-rearing behavior as being characterized by reduced warmth and increased overprotection. Genetic and physiological. Heritability for agoraphobia is 61%. Of the various phobias, agoraphobia has the strongest and most specific association with the genetic factor that represents proneness to phobias.

1	Females have different patterns of comorbid disorders than males. Consistent with gender differences in the prevalence of mental disorders, males have higher rates of comorbid substance use disorders. Functional Consequences of Agoraphobia Agoraphobia is associated with considerable impairment and disability in terms of role functioning, work productivity, and disability days. Agoraphobia severity is a strong de- terminant of the degree of disability, irrespective of the presence of comorbid panic disor- der, panic attacks, and other comorbid conditions. More than one-third of individuals with agoraphobia are completely homebound and unable to work. When diagnostic criteria for agoraphobia and another disorder are fully met, both diagnoses should be assigned, unless the fear, anxiety, or avoidance of agoraphobia is attributable to the other disorder. Weighting of criteria and clinical judgment may be helpful in some cases.

1	Specific phobia, situational type. Differentiating agoraphobia from situational specific phobia can be challenging in some cases, because these conditions share several symptom characteristics and criteria. Specific phobia, situational type, should be diagnosed versus ago- raphobia if the fear, anxiety, or avoidance is limited to one of the agoraphobic situations. Requiring fears from two or more of the agoraphobic situations is a robust means for differen- tiating agoraphobia from specific phobias, particularly the situational subtype. Additional dif- ferentiating features include the cognitive ideation. Thus, if the situation is feared for reasons other than panic-like symptoms or other incapacitating or embarrassing symptoms (e.g., fears of being directly harmed by the situation itself, such as fear of the plane crashing for individ- uals who fear ﬂying), then a diagnosis of specific phobia may be more appropriate.

1	Separation anxiety disorder. Separation anxiety disorder can be best differentiated from agoraphobia by examining cognitive ideation. In separation anxiety disorder, the thoughts are about detachment from significant others and the home environment (i.e., parents or other attachment figures), whereas in agoraphobia the focus is on panic-like symptoms or other incapacitating or embarrassing symptoms in the feared situations. Social anxiety disorder (social phobia). Agoraphobia should be differentiated from so- cial anxiety disorder based primarily on the situational clusters that trigger fear, anxiety, or avoidance and the cognitive ideation. In social anxiety disorder, the focus is on fear of being negatively evaluated. Panic disorder. When criteria for panic disorder are met, agoraphobia should not be di— agnosed if the avoidance behaviors associated with the panic attacks do not extend to avoid- ance of two or more agoraphobic situations.

1	Acute stress disorder and posttraumatic stress disorder. Acute stress disorder and ing whether the fear, anxiety, or avoidance is related only to situations that remind the individual of a traumatic event. If the fear, anxiety, or avoidance is restricted to trauma re- minders, and if the avoidance behavior does not extend to two or more agoraphobic situ- ations, then a diagnosis of agoraphobia is not warranted. Major depressive disorder. In major depressive disorder, the individual may avoid leav- ing home because of apathy, loss of energy, low self—esteem, and anhedonia. If the avoid- ance is unrelated to fears of panic-like or other incapacitating or embarrassing symptoms, then agoraphobia should not be diagnosed.

1	Other medical conditions. Agoraphobia is not diagnosed if the avoidance of situations is judged to be a physiological consequence of a medical condition. This determination is based on history, laboratory findings, and a physical examination. Other relevant medical conditions may include neurodegenerative disorders with associated motor disturbances (e.g., Parkinson’s disease, multiple sclerosis), as well as cardiovascular disorders. Individ- uals with certain medical conditions may avoid situations because of realistic concerns about being incapacitated (e.g., fainting in an individual with transient ischemic attacks) or being embarrassed (e.g., diarrhea in an individual with Crohn’s disease). The diagnosis of agoraphobia should be given only when the fear or avoidance is clearly in excess of that usually associated with these medical conditions.

1	The majority of individuals with agoraphobia also have other mental disorders. The most frequent additional diagnoses are other anxiety disorders (e.g., specific phobias, panic dis- order, social anxiety disorder), depressive disorders (major depressive disorder), PTSD, and alcohol use disorder. Whereas other anxiety disorders (e.g., separation anxiety disor- der, specific phobias, panic disorder) frequently precede onset of agoraphobia, depressive disorders and substance use disorders typically occur secondary to agoraphobia. Diagnostic Criteria 300.02 (F41.1) A. Excessive anxiety and worry (apprehensive expectation), occurring more days than not for at least 6 months, about a number of events or activities (such as work or school performance). B. The individual finds it difficult to control the worry. C. The anxiety and worry are associated with three (or more) of the following six symp- past 6 months): Note: Only one item is required in children.

1	C. The anxiety and worry are associated with three (or more) of the following six symp- past 6 months): Note: Only one item is required in children. Restlessness or feeling keyed up or on edge. Being easily fatigued. Difficulty concentrating or mind going blank. Irritability. Muscle tension. Sleep disturbance (difficulty falling or staying asleep, or restless, unsatisfying sleep). @WPWN.‘ D. The anxiety, worry, or physical symptoms cause clinically significant distress or impair- ment in social, occupational, or other important areas of functioning. E. The disturbance is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication) or another medical condition (e.g., hyperthyroidism).

1	E. The disturbance is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication) or another medical condition (e.g., hyperthyroidism). F. The disturbance is not better explained by another mental disorder (e.g., anxiety or worry about having panic attacks in panic disorder, negative evaluation in social anxi- ety disorder [social phobia], contamination or other obsessions in obsessive-compul- sive disorder, separation from attachment figures in separation anxiety disorder, reminders of traumatic events in posttraumatic stress disorder. gaining weight in an- orexia nervosa, physical complaints in somatic symptom disorder, perceived appear- ance flaws in body dysmorphic disorder, having a serious illness in illness anxiety disorder. or the content of delusional beliefs in schizophrenia or delusional disorder).

1	The essential feature of generalized anxiety disorder is excessive anxiety and worry (ap- prehensive expectation) about a number of events or activities. The intensity, duration, or frequency of the anxiety and worry is out of proportion to the actual likelihood or impact of the anticipated event. The individual finds it difficult to control the worry and to keep worrisome thoughts from interfering with attention to tasks at hand. Adults with gener- alized anxiety disorder often worry about everyday, routine life circumstances, such as possible job responsibilities, health and finances, the health of family members, misfor- tune to their children, or minor matters (e.g., doing household chores or being late for ap- pointments). Children with generalized anxiety disorder tend to worry excessively about their competence or the quality of their performance. During the course of the disorder, the focus of worry may shift from one concern to another.

1	Several features distinguish generalized anxiety disorder from nonpathological anxiety.

1	First, the worries associated with generalized anxiety disorder are excessive and typically in- terfere significantly with psychosocial functioning, whereas the worries of everyday life are not excessive and are perceived as more manageable and may be put off when more pressing matters arise. Second, the worries associated with generalized anxiety disorder are more pervasive, pronounced, and distressing; have longer duration; and frequently occur without precipitants. The greater the range of life circumstances about which a person worries (e.g., finances, children’s safety, job performance), the more likely his or her symp- toms are to meet criteria for generalized anxiety disorder. Third, everyday worries are much less likely to be accompanied by physical symptoms (e.g., restlessness or feeling keyed up or on edge). Individuals with generalized anxiety disorder report subjective distress due to constant worry and related impairment in social, occupational, or other important areas

1	feeling keyed up or on edge). Individuals with generalized anxiety disorder report subjective distress due to constant worry and related impairment in social, occupational, or other important areas of functioning.

1	The anxiety and worry are accompanied by at least three of the following additional symptoms: restlessness or feeling keyed up or on edge, being easily fatigued, difficulty concentrating or mind going blank, irritability, muscle tension, and disturbed sleep, al— though only one additional symptom is required in children. Associated with muscle tension, there may be trembling, twitching, feeling shaky, and muscle aches or soreness. Many individuals with generalized anxiety disorder also expe- rience somatic symptoms (e.g., sweating, nausea, diarrhea) and an exaggerated startle re- sponse. Symptoms of autonomic hyperarousal (e.g., accelerated heart rate, shortness of breath, dizziness) are less prominent in generalized anxiety disorder than in other anxiety disorders, such as panic disorder. Other conditions that may be associated with stress (e.g., irritable bowel syndrome, headaches) frequently accompany generalized anxiety disorder.

1	The 12-month prevalence of generalized anxiety disorder is 0.9% among adolescents and 2.9% among adults in the general community of the United States. The 12-month preva- lence for the disorder in other countries ranges from 0.4% to 3.6%. The lifetime morbid risk is 9.0%. Females are twice as likely as males to experience generalized anxiety disorder. The prevalence of the diagnosis peaks in middle age and declines across the later years of life. Individuals of European descent tend to experience generalized anxiety disorder more frequently than do individuals of non-European descent (i.e., Asian, African, Native American and Pacific Islander). Furthermore, individuals from developed countries are more likely than individuals from nondeveloped countries to report that they have expe- rienced symptoms that meet criteria for generalized anxiety disorder in their lifetime.

1	Many individuals with generalized anxiety disorder report that they have felt anxious and nervous all of their lives. The median age at onset for generalized anxiety disorder is 30 years; however, age at onset is spread over a very broad range. The median age at onset is later than that for the other anxiety disorders. The symptoms of excessive worry and anx- iety may occur early in life but are then manifested as an anxious temperament. Onset of the disorder rarely occurs prior to adolescence. The symptoms of generalized anxiety dis— order tend to be chronic and wax and wane across the lifespan, ﬂuctuating between syn- dromal and subsyndromal forms of the disorder. Rates of full remission are very low.

1	The clinical expression of generalized anxiety disorder is relatively consistent across the lifespan. The primary difference across age groups is in the content of the individual’s worry. Children and adolescents tend to worry more about school and sporting perfor- mance, whereas older adults report greater concern about the well-being of family or their own physical heath. Thus, the content of an individual’s worry tends to be age appropri- ate. Younger adults experience greater severity of symptoms than do older adults.

1	The earlier in life individuals have symptoms that meet criteria for generalized anxiety disorder, the more comorbidity they tend to have and the more impaired they are likely to be. The advent of chronic physical disease can be a potent issue for excessive worry in the elderly. In the frail elderly, worries about safety—and especially about falling—may limit activities. In those with early cognitive impairment, what appears to be excessive worry about, for example, the whereabouts of things is probably better regarded as realistic given the cognitive impairment.

1	In children and adolescents with generalized anxiety disorder) the anxieties and wor- ries often concern the quality of their performance or competence at school or in sporting events, even when their performance is not being evaluated by others. There may be ex- cessive concerns about punctuality. They may also worry about catastrophic events, such as earthquakes or nuclear war. Children with the disorder may be overly conforming, per- fectionist, and unsure of themselves and tend to redo tasks because of excessive dissatis- faction with less-than-perfect performance. They are typically overzealous in seeking other things they are worried about.

1	Generalized anxiety disorder may be overdiagnosed in children. When this diagnosis is being considered in children, a thorough evaluation for the presence of other childhood anxiety disorders and other mental disorders should be done to determine whether the worries may be better explained by one of these disorders. Separation anxiety disorder, so- cial anxiety disorder (social phobia), and obsessive-compulsive disorder are often accom- panied by worries that may mimic those described in generalized anxiety disorder. For example, a child with social anxiety disorder may be concerned about school performance because of fear of humiliation. Worries about illness may also be better explained by sep- aration anxiety disorder or obsessive-compulsive disorder. Temperamental. Behavioral inhibition, negative affectivity (neuroticism), and harm avoidance have been associated with generalized anxiety disorder.

1	Temperamental. Behavioral inhibition, negative affectivity (neuroticism), and harm avoidance have been associated with generalized anxiety disorder. Environmental. Although childhood adversities and parental overprotection have been associated with generalized anxiety disorder, no environmental factors have been identi- fied as specific to generalized anxiety disorder or necessary or sufficient for making the di- agnosis. Genetic and physiological. One-third of the risk of experiencing generalized anxiety disorder is genetic, and these genetic factors overlap with the risk of neuroticism and are shared with other anxiety and mood disorders, particularly major depressive disorder. There is considerable cultural variation in the expression of generalized anxiety disorder.

1	There is considerable cultural variation in the expression of generalized anxiety disorder. For example, in some cultures, somatic symptoms predominate in the expression of the disorder, Whereas in other cultures cognitive symptoms tend to predominate. This differ- ence may be more evident on initial presentation than subsequently, as more symptoms are reported over time. There is no information as to whether the propensity for excessive worrying is related to culture, although the topic being worried about can be culture spe- cific. It is important to consider the social and cultural context when evaluating whether worries about certain situations are excessive.

1	In clinical settings, generalized anxiety disorder is diagnosed somewhat more frequently in females than in males (about 55%—60% of those presenting with the disorder are female). In epidemiological studies, approximately two-thirds are female. Females and males who experience generalized anxiety disorder appear to have similar symptoms but demonstrate different patterns of comorbidity consistent with gender differences in the prevalence of disorders. In females, comorbidity is largely confined to the anxiety disor- ders and unipolar depression, whereas in males, comorbidity is more likely to extend to the substance use disorders as well.

1	Excessive worrying impairs the individual’s capacity to do things quickly and efficiently, whether at home or at work. The worrying takes time and energy; the associated symp- toms of muscle tension and feeling keyed up or on edge, tiredness, difficulty concentrat- ing, and disturbed sleep contribute to the impairment. Importantly the excessive worrying may impair the ability of individuals with generalized anxiety disorder to encourage con- fidence in their children. Generalized anxiety disorder is associated with significant disability and distress that is independent of comorbid disorders, and most non-instit'utionalized adults with the disorder are moderately to seriously disabled. Generalized anxiety disorder accounts for 110 mil- lion disability days per annum in the US. population.

1	Anxiety disorder due to another medical condition. The diagnosis of anxiety disorder associated with another medical condition should be assigned if the individual’s anxiety and worry are judged, based on history, laboratory findings, or physical examination, to be a physiological effect of another specific medical condition (e.g., pheochromocytoma, hyperthyroidism). Substance/medication-induced anxiety disorder. A substance/medication-induced anxiety disorder is distinguished from generalized anxiety disorder by the fact that a sub- stance or medication (e.g., a drug of abuse, exposure to a toxin) is judged to be etiologically related to the anxiety. For example, severe anxiety that occurs only in the context of heavy coffee consumption would be diagnosed as caffeine-induced anxiety disorder.

1	Social anxiety disorder. Individuals with social anxiety disorder often have anticipa- tory anxiety that is focused on upcoming social situations in which they must perform or be evaluated by others, whereas individuals with generalized anxiety disorder worry, whether or not they are being evaluated. Obsessive-compulsive disorder. Several features distinguish the excessive worry of generalized anxiety disorder from the obsessional thoughts of obsessive-compulsive dis- order. In generalized anxiety disorder the focus of the worry is about forthcoming prob- lems, and it is the excessiveness of the worry about future events that is abnormal. In obsessive-compulsive disorder, the obsessions are inappropriate ideas that take the form of intrusive and unwanted thoughts, urges, or images.

1	Posttraumatic stress disorder and adjustment disorders. Anxiety is invariably pres- ent in posttraumatic stress disorder. Generalized anxiety disorder is not diagnosed if the anxiety and worry are better explained by symptoms of posttraumatic stress disorder. Anxiety may also be present in adjustment disorder, but this residual category should be used only when the criteria are not met for any other disorder (including generalized anx- iety disorder). Moreover, in adjustment disorders, the anxiety occurs in response to an identifiable stressor within 3 months of the onset of the stressor and does not persist for more than 6 months after the termination of the stressor or its consequences. Depressive, bipolar, and psychotic disorders. Generalized anxiety/worry is a common associated feature of depressive, bipolar, and psychotic disorders and should not be di- agnosed separately if the excessive worry has occurred only during the course of these conditions.

1	Individuals whose presentation meets criteria for generalized anxiety disorder are likely to have met, or currently meet, criteria for other anxiety and unipolar depressive disor- ders. The neuroticism or emotional liability that underpins this pattern of comorbidity is associated with temperamental antecedents and genetic and environmental risk factors shared between these disorders, although independent pathways are also possible. Co- morbidity with substance use, conduct, psychotic, neurodevelopmental, and neurocogni- tive disorders is less common. A. Panic attacks or anxiety is predominant in the clinical picture. B. There is evidence from the history. physical examination, or laboratory findings of both (1) and (2): 1. The symptoms in Criterion A developed during or soon after substance intoxication or withdrawal or after exposure to a medication. 2. The involved substance/medication is capable of producing the symptoms in Crite- rion A.

1	2. The involved substance/medication is capable of producing the symptoms in Crite- rion A. C. The disturbance is not better explained by an anxiety disorder that is not substance/ medication-induced. Such evidence of an independent anxiety disorder could include the following: The symptoms precede the onset of the substance/medication use; the symptoms persist for a substantial period of time (e.g., about 1 month) after the cessation of acute withdrawal or severe intoxication; or there is other evidence suggesting the existence of an independent non-substance/medication-induced anxiety disorder (e.g., a history of recurrent non-substance/medication-related episodes). D. The disturbance does not occur exclusively during the course of a delirium. E. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning.

1	E. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. Note: This diagnosis should be made instead of a diagnosis of substance intoxication or substance withdrawal only when the symptoms in Criterion A predominate in the clinical picture and they are sufficiently severe to warrant clinical attention.

1	Coding note: The |CD-9-CM and |CD-10-CM codes for the [specific substance/medica- tion]-induced anxiety disorders are indicated in the table below. Note that the |CD-10-CM code depends on whether or not there is a comorbid substance use disorder present for the same class of substance. If a mild substance use disorder is comorbid with the sub- stance-induced anxiety disorder, the 4th position character is “1 and the clinician should record “mild [substance] use disorder" before the substance-induced anxiety disorder (e.g., “mild cocaine use disorder with cocaine-induced anxiety disorder'). It a moderate or severe substance use disorder is comorbid with the substance-induced anxiety disorder, the 4th position character is “,"2 and the clinician should record “moderate [substance] use disorder” or “severe [substance] use disorder," depending on the severity of the comorbid substance use disorder. If there is no comorbid substance use disorder (e.g., after a one- time heavy use of the

1	or “severe [substance] use disorder," depending on the severity of the comorbid substance use disorder. If there is no comorbid substance use disorder (e.g., after a one- time heavy use of the substance), then the 4th position character is “9," and the clinician should record only the substance-induced anxiety disorder.

1	With use disorder, Without disorder, moderate use Alcohol 291.89 F10.180 F10.280 F10.980 Caffeine 292.89 F15.180 F15.280 F15.980 Cannabis 292.89 F12.180 F12.280 F12.980 Phencyclidine 292.89 F16.180 F16.280 F16.980 Other hallucinogen 292.89 F16.180 F16.280 F16.980 Inhalant 292.89 F18.180 F18.280 F18.980 Opioid 292.89 F11.188 F11.288 F11.988 Sedative, hypnotic, or anxiolytic 292.89 F13.180 F13.280 F13.980 Amphetamine (or other 292.89 F15.180 F15.280 F15.980 Cocaine 292.89 F14.180 F14.280 F14.980 Other (or unknown) substance 292.89 F19.180 F19.280 F19.980 Specify it (see Table 1 in the chapter “Substance-Fielated and Addictive Disorders" tor di- agnoses associated with substance class): With onset during intoxication: This specifier applies if criteria are met for intoxica- tion with the substance and the symptoms develop during intoxication.

1	With onset during intoxication: This specifier applies if criteria are met for intoxica- tion with the substance and the symptoms develop during intoxication. With onset during withdrawai: This specifier applies if criteria are met for withdrawal from the substance and the symptoms develop during, or shortly after, withdrawal. With onset after medication use: Symptoms may appear either at initiation of medi- cation or after a modification or change in use.

1	With onset after medication use: Symptoms may appear either at initiation of medi- cation or after a modification or change in use. |CD-9-CM. The name of the substance/medication-induced anxiety disorder begins with the specific substance (e.g., cocaine, salbutamol) that is presumed to be causing the anxiety symptoms. The diagnostic code is selected from the table included in the criteria set, which is based on the drug class. For substances that do not fit into any of the classes (e.g., salbutamol), the code for "other substance" should be used; and in cases in which a substance is judged to be an etiological factor but the specific class of substance is un- known, the category ”unknown substance” should be used.

1	The name of the disorder is followed by the specification of onset (i.e., onset during in- toxication, onset during withdrawal, with onset during medication use). Unlike the record- ing procedures for ICD-lO-CM, which combine the substance-induced disorder and substance use disorder into a single code, for ICD—9-CM a separate diagnostic code is given for the substance use disorder. For example, in the case of anxiety symptoms occurring dur- ing withdrawal in a man with a severe lorazepam use disorder, the diagnosis is 292.89 loraz- epam-induced anxiety disorder, with onset during withdrawal. An additional diagnosis of 304.10 severe lorazepam use disorder is also given. When more than one substance is judged to play a significant role in the development of anxiety symptoms, each should be listed sep- arately (e.g., 292.89 methylphenidate-induced anxiety disorder, with onset during intoxica- tion; 292.89 salbutamol-induced anxiety disorder, with onset after medication use).

1	|CD-10-CM. The name of the substance/medication-induced anxiety disorder begins with the specific substance (e.g., cocaine, salbutamol) that is presumed to be causing the anxiety symptoms. The diagnostic code is selected from the table included in the criteria set, Which is based on the drug class and presence or absence of a comorbid substance use disorder. For substances that do not fit into any of the classes (e.g., salbutamol), the code for ”other substance” should be used; and in cases in which a substance is judged to be an etiological factor but the specific class of substance is unknown, the category “unknown substance” should be used.

1	When recording the name of the disorder, the comorbid substance use disorder (if any) is listed first, followed by the word ”with,” followed by the name of the substance-induced anxiety disorder, followed by the specification of onset (i.e., onset during intoxication, onset during withdrawal, with onset during medication use). For example, in the case of anxiety symptoms occurring during withdrawal in a man with a severe lorazepam use dis- order, the diagnosis is F13.280 severe lorazepam use disorder with lorazepam-induced anxiety disorder, with onset during withdrawal. A separate diagnosis of the comorbid se- vere lorazepam use disorder is not given. If the substance-induced anxiety disorder occurs without a comorbid substance use disorder (e.g., after a one-time heavy use of the substance), no accompanying substance use disorder is noted (e.g., F16.980 psilocybin—induced anxi- ety disorder, with onset during intoxication). When more than one substance is judged to play a significant

1	no accompanying substance use disorder is noted (e.g., F16.980 psilocybin—induced anxi- ety disorder, with onset during intoxication). When more than one substance is judged to play a significant role in the development of anxiety symptoms, each should be listed sep- arately (e.g., F15.280 severe methylphenidate use disorder with methylphenidate—induced anxiety disorder, with onset during intoxication; F19.980 salbutamol-induced anxiety dis- order, with onset after medication use).

1	The essential features of substance/medication-induced anxiety disorder are prominent symptoms of panic or anxiety (Criterion A) that are judged to be due to the effects of a sub- stance (e.g., a drug of abuse, a medication, or a toxin exposure). The panic or anxiety symp- after exposure to a medication, and the substances or medications must be capable of pro— ducing the symptoms (Criterion B2). Substance/medication-induced anxiety disorder due to a prescribed treatment for a mental disorder or another medical condition must have its onset while the individual is receiving the medication (or during withdrawal, if a withdrawal is associated with the medication). Once the treatment is discontinued, the panic or anxiety symptoms will usually improve or remit within days to several weeks to a month (depending on the half-life of the substance/ medication and the presence of with- drawal). The diagnosis of substance/medication-induced anxiety disorder should not be given if the onset of

1	a month (depending on the half-life of the substance/ medication and the presence of with- drawal). The diagnosis of substance/medication-induced anxiety disorder should not be given if the onset of the panic or anxiety symptoms precedes the substance/ medication in- toxication or withdrawal, or if the symptoms persist for a substantial period of time (i.e., usually longer than 1 month) from the time of severe intoxication or withdrawal. If the panic or anxiety symptoms persist for substantial periods of time, other causes for the symptoms should be considered.

1	The substance/medication-induced anxiety disorder diagnosis should be made in- stead of a diagnosis of substance intoxication or substance withdrawal only when the symptoms in Criterion A are predominant in the clinical picture and are sufficiently severe to warrant independent clinical attention.

1	Panic or anxiety can occur in association with intoxication with the following classes of sub- stances: alcohol, caffeine, cannabis, phencyclidine, other hallucinogens, inhalants, stimu— lants (including cocaine), and other (or unknown) substances. Panic or anxiety can occur in association with withdrawal from the following classes of substances: alcohol; opioids; sed- atives, hypnotics, and anxiolytics; stimulants (including cocaine); and other (or unknown) substances. Some medications that evoke anxiety symptoms include anesthetics and anal- gesics, sympathomimetics or other bronchodilators, anticholinergics, insulin, thyroid prep- arations, oral contraceptives, antihistamines, antiparkinsonian medications, corticosteroids, antihypertensive and cardiovascular medications, anticonvulsants, lithium carbonate, an- tipsychotic medications, and antidepressant medications. Heavy metals and toxins (e.g., organophosphate insecticide, nerve gases, carbon monoxide, carbon dioxide, volatile

1	lithium carbonate, an- tipsychotic medications, and antidepressant medications. Heavy metals and toxins (e.g., organophosphate insecticide, nerve gases, carbon monoxide, carbon dioxide, volatile sub- stances such as gasoline and paint) may also cause panic or anxiety symptoms.

1	The prevalence of substance/medication-induced anxiety disorder is not clear. General population data suggest that it may be rare, with a 12-month prevalence of approximately 0.002%. However, in clinical populations, the prevalence is likely to be higher. Laboratory assessments (e.g., urine toxicology) may be useful to measure substance intox- ication as part of an assessment for substance/medication-induced anxiety disorder.

1	Laboratory assessments (e.g., urine toxicology) may be useful to measure substance intox- ication as part of an assessment for substance/medication-induced anxiety disorder. Substance intoxication and substance withdrawal. Anxiety symptoms commonly oc- cur in substance intoxication and substance withdrawal. The diagnosis of the substance— specific intoxication or substance-specific withdrawal will usually suffice to categorize the symptom presentation. A diagnosis of substance/medication-induced anxiety disorder should be made in addition to substance intoxication or substance withdrawal when the panic or anxiety symptoms are predominant in the clinical picture and are sufficiently se- vere to warrant independent clinical attention. For example, panic or anxiety symptoms are characteristic of alcohol withdrawal.

1	Anxiety disorder (i.e., not induced by a substance/medication). Substance/medication- induced anxiety disorder is judged to be etiologically related to the substance/medication.

1	Substance/medication-induced anxiety disorder is distinguished from a primary anxiety disorder based on the onset, course, and other factors with respect to substances/medica- tions. For drugs of abuse, there must be evidence from the history, physical examination, or laboratory findings for use, intoxication, or withdrawal. Substance/medication-induced anxiety disorders arise only in association with intoxication or withdrawal states, whereas primary anxiety disorders may precede the onset of substance/medication use. The pres- ence of features that are atypical of a primary anxiety disorder, such as atypical age at onset (e.g., onset of panic disorder after age 45 years) or symptoms (e.g., atypical panic attack symptoms such as true vertigo, loss of balance, loss of consciousness, loss of bladder con- trol, headaches, slurred speech) may suggest a substance/medication-induced etiology. A primary anxiety disorder diagnosis is warranted if the panic or anxiety symptoms persist for a

1	of bladder con- trol, headaches, slurred speech) may suggest a substance/medication-induced etiology. A primary anxiety disorder diagnosis is warranted if the panic or anxiety symptoms persist for a substantial period of time (about 1 month or longer) after the end of the substance in- toxication or acute withdrawal or there is a history of an anxiety disorder.

1	Delirium. If panic or anxiety symptoms occur exclusively during the course of delirium, they are considered to be an associated feature of the delirium and are not diagnosed sep- arately.

1	Anxiety disorder due to another medical condition. If the panic or anxiety symptoms are attributed to the physiological consequences of another medical condition (i.e., rather than to the medication taken for the medical condition), anxiety disorder due to another medical condition should be diagnosed. The history often provides the basis for such a judgment. At times, a change in the treatment for the other medical condition (e.g., med- ication substitution or discontinuation) may be needed to determine whether the medica- tion is the causative agent (in which case the symptoms may be better explained by substance/medication-induced anxiety disorder). If the disturbance is attributable to both another medical condition and substance use, both diagnoses (i.e., anxiety disorder due to given. When there is insufficient evidence to determine whether the panic or anxiety symp- toms are attributable to a substance/medication or to another medical condition or are pri- mary (i.e., not

1	given. When there is insufficient evidence to determine whether the panic or anxiety symp- toms are attributable to a substance/medication or to another medical condition or are pri- mary (i.e., not attributable to either a substance or another medical condition), a diagnosis of other specified or unspecified anxiety disorder would be indicated.

1	Diagnostic Criteria 293.84 (F06.4) A. Panic attacks or anxiety is predominant in the clinical picture. B. There is evidence from the history, physical examination, or laboratory findings that the dis- turbance is the direct pathophysiological consequence of another medical condition. C. The disturbance is not better explained by another mental disorder. D. The disturbance does not occur exclusively during the course of a delirium. E. The disturbance causes clinically significant distress or impairment in social. occupa- tional, or other important areas of functioning. Coding note: Include the name of the other medical condition within the name of the men- tal disorder (e.g.. 293.84 [F06.4] anxiety disorder due to pheochromocytoma). The other medical condition should be coded and listed separately immediately before the anxiety disorder due to the medical condition (e.g., 227.0 [D3500] pheochromocytoma; 293.84 [F06.4] anxiety disorder due to pheochromocytoma.

1	The essential feature of anxiety disorder due to another medical condition is clinically signifi- cant anxiety that is judged to be best explained as a physiological effect of another medical con- dition. Symptoms can include prominent anxiety symptoms or panic attacks (Criterion A). The judgment that the symptoms are best explained by the associated physical condition must be based on evidence from the history, physical examination, or laboratory findings (Criterion B). Additionally, it must be judged that the symptoms are not better accounted for by another mental disorder, in particular, adjustment disorder, with anxiety, in which the stressor is the medical condition (Criterion C). In this case, an individual with adjustment disorder is espe- cially distressed about the meaning or the consequences of the associated medical condition.

1	By contrast, there is often a prominent physical component to the anxiety (e.g., shortness of breath) when the anxiety is due to another medical condition. The diagnosis is not made if the anxiety symptoms occur only during the course of a delirium (Criterion D). The anxiety symp- toms must cause clinically significant distress or impairment in social, occupational, or other important areas of functioning (Criterion E). In determining whether the anxiety symptoms are attributable to another medical con- dition, the clinician must first establish the presence of the medical condition. Further- more, it must be established that anxiety symptoms can be etiologically related to the is the best explanation for the symptoms in a specific individual. A careful and compre-

1	Anxiety Disorder Due to Another Medical Condition 231 hensive assessment of multiple factors is necessary to make this judgment. Several aspects of the clinical presentation should be considered: 1) the presence of a clear temporal asso- ciation between the onset, exacerbation, or remission of the medical condition and the anx- iety symptoms; 2) the presence of features that are atypical of a primary anxiety disorder (e.g., atypical age at onset or course); and 3) evidence in the literature that a known phys- iological mechanism (e.g., hyperthyroidism) causes anxiety. In addition, the disturbance must not be better explained by a primary anxiety disorder, a substance/medication- induced anxiety disorder, or another primary mental disorder (e.g., adjustment disorder).

1	A number of medical conditions are known to include anxiety as a symptomatic manifes- tation. Examples include endocrine disease (e.g., hyperthyroidism, pheochromocytoma, hypoglycemia, hyperadrenocortisolism), cardiovascular disorders (e.g., congestive heart failure, pulmonary embolism, arrhythmia such as atrial fibrillation), respiratory illness (e.g., chronic obstructive pulmonary disease, asthma, pneumonia), metabolic distur— bances (e.g., vitamin B12 deficiency, porphyria), and neurological illness (e.g., neoplasms, vestibular dysfunction, encephalitis, seizure disorders). Anxiety due to another medical condition is diagnosed when the medical condition is known to induce anxiety and when the medical condition preceded the onset of the anxiety.

1	The prevalence of anxiety disorder due to another medical condition is unclear. There ap- pears to be an elevated prevalence of anxiety disorders among individuals with a variety of medical conditions, including asthma, hypertension, ulcers, and arthritis. However, this increased prevalence may be due to reasons other than the anxiety disorder directly caus- ing the medical condition. The development and course of anxiety disorder due to another medical condition gen- erally follows the course of the underlying illness. This diagnosis is not meant to include primary anxiety disorders that arise in the context of chronic medical illness. This is im- portant to consider with older adults, who may experience chronic medical illness and then develop independent anxiety disorders secondary to the chronic medical illness. Laboratory assessments and/ or medical examinations are necessary to confirm the diag- nosis of the associated medical condition.

1	Laboratory assessments and/ or medical examinations are necessary to confirm the diag- nosis of the associated medical condition. Delirium. A separate diagnosis of anxiety disorder due to another medical condition is not given if the anxiety disturbance occurs exclusively during the course of a delirium. However, a diagnosis of anxiety disorder due to another medical condition may be given in addition to a diagnosis of major neurocognitive disorder (dementia) if the etiology of anxiety is judged to be a physiological consequence of the pathological process causing the neurocognitive disorder and if anxiety is a prominent part of the clinical presentation. Mixed presentation of symptoms (e.g., mood and anxiety). If the presentation includes a mix of different types of symptoms, the specific mental disorder due to another medical condition depends on which symptoms predominate in the clinical picture.

1	Substance/medication-induced anxiety disorder. If there is evidence of recent or pro- longed substance use (including medications with psychoactive effects), withdrawal from a substance, or exposure to a toxin, a substance/medication-induced anxiety disorder should be considered. Certain medications are known to increase anxiety (e.g., corticoste- roids, estrogens, metoclopramide), and when this is the case, the medication may be the most likely etiology, although it may be difficult to distinguish whether the anxiety is at- tributable to the medications or to the medical illness itself. When a diagnosis of substance- induced anxiety is being made in relation to recreational or nonprescribed drugs, it may be useful to obtain a urine or blood drug screen or other appropriate laboratory evaluation.

1	Symptoms that occur during or shortly after (i.e., within 4 weeks of) substance intoxication or withdrawal or after medication use may be especially indicative of a substance/ medi- cation-induced anxiety disorder, depending on the type, duration, or amount of the sub- stance used. If the disturbance is associated with both another medical condition and substance use, both diagnoses (i.e., anxiety disorder due to another medical condition and substance/medication-induced anxiety disorder) can be given. Features such as onset af- ter age 45 years or the presence of atypical symptoms during a panic attack (e.g., vertigo, loss of consciousness, loss of bladder or bowel control, slurred speech, amnesia) suggest the possibility that another medical condition or a substance may be causing the panic at- tack symptoms.

1	Anxiety disorder (not due to a known medical condition). Anxiety disorder due to an- panic disorder and generalized anxiety disorder). In other anxiety disorders, no specific and direct causative physiological mechanisms associated with another medical condition can be demonstrated. Late age at onset, atypical symptoms, and the absence of a personal or family history of anxiety disorders suggest the need for a thorough assessment to rule out the diagnosis of anxiety disorder due to another medical condition. Anxiety disorders can exacerbate or pose increased risk for medical conditions such as cardiovascular events and myocardial infarction and should not be diagnosed as anxiety disorder due to another medical condition in these cases.

1	Illness anxiety disorder. Anxiety disorder due to another medical condition should be distinguished from illness anxiety disorder. Illness anxiety disorder is characterized by worry about illness, concern about pain, and bodily preoccupations. In the case of illness anxiety disorder, individuals may or may not have diagnosed medical conditions. Al- though an individual with illness anxiety disorder and a diagnosed medical condition is likely to experience anxiety about the medical condition, the medical condition is not physiologically related to the anxiety symptoms.

1	Adjustment disorders. Anxiety disorder due to another medical condition should be distinguished from adjustment disorders, with anxiety, or with anxiety and depressed mood. Adjustment disorder is warranted when individuals experience a maladaptive re- sponse to the stress of having another medical condition. The reaction to stress usually concerns the meaning or consequences of the stress, as compared with the experience of anxiety or mood symptoms that occur as a physiological consequence of the other medical condition. In adjustment disorder, the anxiety symptoms are typically related to coping with the stress of having a general medical condition, whereas in anxiety disorder due to another medical condition, individuals are more likely to have prominent physical symp- toms and to be focused on issues other than the stress of the illness itself.

1	Associated feature of another mental disorder. Anxiety symptoms may be an associ- ated feature of another mental disorder (e.g., schizophrenia, anorexia nervosa). Other specified or unspecified anxiety disorder. This diagnosis is given if it cannot be determined whether the anxiety symptoms are primary, substance-induced, or associated with another medical condition. 300.09 (F41.8) This category applies to presentations in which symptoms characteristic of an anxiety dis- order that cause clinically significant distress or impairment in social. occupational, or oth- er important areas of functioning predominate but do not meet the full criteria for any of the disorders in the anxiety disorders diagnostic class. The other specified anxiety disorder category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for any specific anxiety disorder.

1	This is done by recording “other specified anxiety disorder” followed by the specific reason (e.g., “generalized anxiety not occurring more days than not"). Examples of presentations that can be specified using the “other specified" designation include the following: 1. Limited-symptom attacks. 2. Generalized anxiety not occurring more days than not. 3. Khyél cap (wind attacks): See “Glossary of Cultural Concepts of Distress" in the Ap- pendix. 4. Ataque de nervios (attack of nerves): See “Glossary of Cultural Concepts of Distress" in the Appendix. 300.00 (F41.9)

1	4. Ataque de nervios (attack of nerves): See “Glossary of Cultural Concepts of Distress" in the Appendix. 300.00 (F41.9) This category applies to presentations in which symptoms characteristic of an anxiety dis- order that cause clinically significant distress or impairment in social, occupational, or oth- er important areas of functioning predominate but do not meet the full criteria for any of the disorders in the anxiety disorders diagnostic class. The unspecified anxiety disorder cate- gory is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for a specific anxiety disorder, and includes presentations in which there is insufficient information to make a more specific diagnosis (e.g., in emergency room settings).

1	.. r.,,__.... .0. W .._,. W disorder (OCD), body dysmorphic disorder, hoarding disorder, trichotillomania (hair- pulling disorder), excoriation (skin—picking) disorder, substance/medication-induced ob- sessive-compulsive and related disorder, obsessive-compulsive and related disorder due to another medical condition, and other specified obsessive—compulsive and related dis— order and unspecified obsessive-compulsive and related disorder (e.g., body-focused re- petitive behavior disorder, obsessional jealousy).

1	OCD is characterized by the presence of obsessions and / or compulsions. Obsessions are recurrent and persistent thoughts, urges, or images that are experienced as intrusive and unwanted, whereas compulsions are repetitive behaviors or mental acts that an indi- vidual feels driven to perform in response to an obsession or according to rules that must be applied rigidly. Some other obsessive-compulsive and related disorders are also char- acterized by preoccupations and by repetitive behaviors or mental acts in response to the preoccupations. Other obsessive-compulsive and related disorders are characterized pri- marily by recurrent body-focused repetitive behaviors (e.g., hair pulling, skin picking) and repeated attempts to decrease or stop the behaviors.

1	The inclusion of a chapter on obsessive-compulsive and related disorders in DSM-5 re- ﬂects the increasing evidence of these disorders’ relatedness to one another in terms of a range of diagnostic validators as well as the clinical utility of grouping these disorders in the same chapter. Clinicians are encouraged to screen for these conditions in individuals who present with one of them and be aware of overlaps between these conditions. At the same time, there are important differences in diagnostic validators and treatment ap- proaches across these disorders. Moreover, there are close relationships between the anx- iety disorders and some of the obsessive-compulsive and related disorders (e.g., 0CD), which is reﬂected in the sequence of DSM-5 chapters, with obsessive—compulsive and re- lated disorders following anxiety disorders.

1	The obsessive-compulsive and related disorders differ from developmentally norma- appropriate periods. The distinction between the presence of subclinical symptoms and a clinical disorder requires assessment of a number of factors, including the individual’s level of distress and impairment in functioning. The chapter begins with 0CD. It then covers body dysmorphic disorder and hoarding disorder, which are characterized by cognitive symptoms such as perceived defects or ﬂaws in physical appearance or the perceived need to save possessions, respectively. The disorder, which are characterized by recurrent body-focused repetitive behaviors. Finally, it covers substance/medication-induced obsessive-compulsive and related disorder, obsessive-compulsive and related disorder due to another medical condition, and other sive and related disorder.

1	While the specific content of obsessions and compulsions varies among individuals, certain symptom dimensions are common in 0CD, including those of cleaning (contami- ing, ordering, and counting compulsions); forbidden or taboo thoughts (e.g., aggressive, sexual, and religious obsessions and related compulsions); and harm (e.g., fears of harm to oneself or others and related checking compulsions). The tic-related specifier of 0CD is used when an individual has a current or past history of a tic disorder.

1	Body dysmorphic disorder is characterized by preoccupation with one or more per- ceived defects or ﬂaws in physical appearance that are not observable or appear only slight to others, and by repetitive behaviors (e.g., mirror checking, excessive grooming, skin picking, or reassurance seeking) or mental acts (e.g., comparing one’s appearance with that of other people) in response to the appearance concerns. The appearance preoccupations are not better explained by concerns with body fat or weight in an individual with an eat- ing disorder. Muscle dysmorphia is a form of body dysmorphic disorder that is character— ized by the belief that one’s body build is too small or is insufficiently muscular.

1	Hoarding disorder is characterized by persistent difficulty discarding or parting with possessions, regardless of their actual value, as a result of a strong perceived need to save the items and to distress associated with discarding them. Hoarding disorder differs from normal collecting. For example, symptoms of hoarding disorder result in the accumula- tion of a large number of possessions that congest and clutter active living areas to the ex- tent that their intended use is substantially compromised. The excessive acquisition form of hoarding disorder, which characterizes most but not all individuals with hoarding dis- order, consists of excessive collecting, buying, or stealing of items that are not needed or for which there is no available space. Trichotillomania (hair-pulling disorder) is characterized by recurrent pulling out of one's hair resulting in hair loss, and repeated attempts to decrease or stop hair pulling.

1	Excoriation (skin-picking) disorder is characterized by recurrent picking of one’s skin re- sulting in skin lesions and repeated attempts to decrease or stop skirt picking. The body- focused repetitive behaviors that characterize these two disorders are not triggered by ob- sessions or preoccupations; however, they may be preceded or accompanied by various emotional states, such as feelings of anxiety or boredom. They may also be preceded by an increasing sense of tension or may lead to gratification, pleasure, or a sense of relief when the hair is pulled out or the skin is picked. Individuals with these disorders may have vary- ing degrees of conscious awareness of the behavior while engaging in it, with some indi- viduals displaying more focused attention on the behavior (with preceding tension and subsequent relief) and other individuals displaying more automatic behavior (with the be- haviors seeming to occur without full awareness).

1	symptoms that are due to substance intoxication or withdrawal or to a medication. Obses- sive-compulsive and related disorder due to another medical condition involves symptoms characteristic of obsessive-compulsive and related disorders that are the direct pathophysio- logical consequence of a medical disorder. Other specified obsessive-compulsive and related disorder and unspecified obsessive-compulsive and related disorder consist of symptoms that do not meet criteria for a specific obsessive—compulsive and related disorder because of atypical presentation or uncertain etiology; these categories are also used for other specific syndromes that are not listed in Section [I and when insufficient information is available to di- agnose the presentation as another obsessive—compulsive and related disorder. Examples of specific syndromes not listed in Section II, and therefore diagnosed as other specified obses— sive-compulsive and related disorder or as unspecified obsessive—compulsive

1	disorder. Examples of specific syndromes not listed in Section II, and therefore diagnosed as other specified obses— sive-compulsive and related disorder or as unspecified obsessive—compulsive and related disorder include body—focused repetitive behavior disorder and obsessional jealousy.

1	sight as the basis for specifiers; in each of these disorders, insight ranges from ”good or fair insight” to ”poor insight" to "absent insight/delusional beliefs” with respect to disorder- related beliefs. For individuals whose obsessive-compulsive and related disorder symp- toms warrant the “with absent insight/delusional beliefs” specifier, these symptoms should not be diagnosed as a psychotic disorder. Diagnostic Criteria 300.3 (F42) A. Presence of obsessions, compulsions, or both: Obsessions are defined by (1) and (2): 1. Recurrent and persistent thoughts, urges, or images that are experienced, at some time during the disturbance. as intrusive and unwanted, and that in most individuals cause marked anxiety or distress. 2. The individual attempts to ignore or suppress such thoughts, urges, or images, or to neutralize them with some other thought or action (i.e., by performing a compulsion).

1	2. The individual attempts to ignore or suppress such thoughts, urges, or images, or to neutralize them with some other thought or action (i.e., by performing a compulsion). Compulsions are defined by (1) and (2): 1. Repetitive behaviors (e.g., hand washing, ordering, checking) or mental acts (e.g., praying, counting, repeating words silently) that the individual feels driven to per- form in response to an obsession or according to rules that must be applied rigidly. 2. The behaviors or mental acts are aimed at preventing or reducing anxiety or dis- tress, or preventing some dreaded event or situation; however, these behaviors or mental acts are not connected in a realistic way with what they are designed to neu- tralize or prevent, or are clearly excessive. Note: Young children may not be able to articulate the aims of these behaviors or mental acts.

1	Note: Young children may not be able to articulate the aims of these behaviors or mental acts. B. The obsessions or compulsions are time-consuming (e.g., take more than 1 hour per day) or cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. C. The obsessive-compulsive symptoms are not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication) or another medical condition.

1	D. The disturbance is not better explained by the symptoms of another mental disorder (e.g.. excessive worries. as in generalized anxiety disorder; preoccupation with ap- pearance, as in body dysmorphic disorder; difficulty discarding or parting with posses- sions, as in hoarding disorder; hair pulling, as in trichotillomania [hair-pulling disorder]; skin picking, as in excoriation [skin-picking] disorder; stereotypies, as in stereotypic movement disorder; ritualized eating behavior, as in eating disorders; preoccupation with substances or gambling, as in substance-related and addictive disorders; preoc- cupation with having an illness, as in illness anxiety disorder; sexual urges or fantasies, as in paraphilic disorders; impulses, as in disruptive, impulse-control, and conduct dis- orders; guilty ruminations, as in major depressive disorder; thought insertion or delu- sional preoccupations, as in schizophrenia spectrum and other psychotic disorders; or repetitive patterns of

1	orders; guilty ruminations, as in major depressive disorder; thought insertion or delu- sional preoccupations, as in schizophrenia spectrum and other psychotic disorders; or repetitive patterns of behavior, as in autism spectrum disorder).

1	Specify it: With good or fair insight: The individual recognizes that obsessive-compulsive dis- order beliefs are definitely or probably not true or that they may or may not be true. With poor insight: The individual thinks obsessive-compulsive disorder beliefs are probably true. With absent insightldeiuslonai beiiets: The individual is completely convinced that obsessive-compulsive disorder beliefs are true. Specify it: Tic-related: The individual has a current or past history of a tie disorder. Many individuals with obsessive-compulsive disorder (OCD) have dysfunctional beliefs. These beliefs can include an inﬂated sense of responsibility and the tendency to overesti- mate threat; perfectionism and intolerance of uncertainty; and over—importance of thoughts (e.g., believing that having a forbidden thought is as bad as acting on it) and the need to control thoughts.

1	Individuals with OCD vary in the degree of insight they have about the accuracy of the beliefs that underlie their obsessive-compulsive symptoms. Many individuals have good or fair insight (e.g., the individual believes that the house definitely will not, probably will not, or may or may not burn down if the stove is not checked 30 times). Some have poor insight (e.g., the individual believes that the house will probably burn down if the stove is not checked 30 times), and a few (4% or less) have absent insight/delusional beliefs (e.g., the in- dividual is convinced that the house will burn down if the stove is not checked 30 times). Insight can vary within an individual over the course of the illness. Poorer insight has been linked to worse long-term outcome.

1	Insight can vary within an individual over the course of the illness. Poorer insight has been linked to worse long-term outcome. Up to 30% of individuals with OCD have a lifetime tic disorder. This is most common in males with onset of 0CD in childhood. These individuals tend to differ from those with- out a history of tic disorders in the themes of their 0CD symptoms, comorbidity, course, and pattern of familial transmission.

1	The characteristic symptoms of 0CD are the presence of obsessions and compulsions (Cri- terion A). Obsessions are repetitive and persistent thoughts (e.g., of contamination), images (e.g., of violent or horrific scenes), or urges (e.g., to stab someone). Importantly, obsessions are not pleasurable or experienced as voluntary: they are intrusive and unwanted and cause marked distress or anxiety in most individuals. The individual attempts to ignore or suppress these obsessions (e.g., avoiding triggers or using thought suppression) or to neu- tralize them with another thought or action (e.g., performing a compulsion). Compulsions (or rituals) are repetitive behaviors (e.g., washing, checking) or mental acts (e.g., counting, repeating words silently) that the individual feels driven to perform in response to an obsession or according to rules that must be applied rigidly. Most individuals with OCD have both obsessions and compulsions. Compulsions are typically performed in response to an

1	in response to an obsession or according to rules that must be applied rigidly. Most individuals with OCD have both obsessions and compulsions. Compulsions are typically performed in response to an obsession (e.g., thoughts of contamination leading to washing rituals or that some- thing is incorrect leading to repeating rituals until it feels "just right”). The aim is to reduce the distress triggered by obsessions or to prevent a feared event (e.g., becoming ill). How- ever, these compulsions either are not connected in a realistic way to the feared event (e.g., arranging items symmetrically to prevent harm to a loved one) or are clearly excessive (e.g., showering for hours each day). Compulsions are not done for pleasure, although some individuals experience relief from anxiety or distress.

1	Criterion B emphasizes that obsessions and compulsions must be time-consuming (e.g., more than 1 hour per day) or cause clinically significant distress or impairment to warrant a diagnosis of OCD. This criterion helps to distinguish the disorder from the occasional in— trusive thoughts or repetitive behaviors that are common in the general population (e.g., double-checking that a door is locked). The frequency and severity of obsessions and com- pulsions vary across individuals with 0CD (e.g., some have mild to moderate symptoms, spending 1—3 hours per day obsessing or doing compulsions, whereas others have nearly constant intrusive thoughts or compulsions that can be incapacitating).

1	The specific content of obsessions and compulsions varies between individuals. However, certain themes, or dimensions, are common, including those of cleaning (contamination obsessions and cleaning compulsions); symmetry (symmetry obsessions and repeating, ordering, and counting compulsions); forbidden or taboo thoughts (e.g., aggressive, sexual, or religious obsessions and related compulsions); and harm (e.g., fears of harm to oneself or others and checking compulsions). Some individuals also have difficulties discarding and accumulate (hoard) objects as a consequence of typical obsessions and compulsions, such as fears of harming others. These themes occur across different cultures, are rela— tively consistent over time in adults with the disorder, and may be associated with differ- ent neural substrates. Importantly, individuals often have symptoms in more than one dimension.

1	Individuals with 0CD experience a range of affective responses when confronted with situations that trigger obsessions and compulsions. For example, many individuals expe- rience marked anxiety that can include recurrent panic attacks. Others report strong feel- ings of disgust. While performing compulsions, some individuals report a distressing sense of "incompleteness” or uneasiness until things look, feel, or sound ”just right.” It is common for individuals with the disorder to avoid people, places, and things that trigger obsessions and compulsions. For example, individuals with contamination con- cerns might avoid public situations (e.g., restaurants, public restrooms) to reduce ex- posure to feared contaminants; individuals with intrusive thoughts about causing harm might avoid social interactions.

1	The 12-month prevalence of 0CD in the United States is 1.2%, with a similar prevalence in— ternationally 0.1%—1.8%). Females are affected at a slightly higher rate than males in adulthood, although males are more commonly affected in childhood. In the United States, the mean age at onset of OCD is 19.5 years, and 25% of cases start by age 14 years. Onset after age 35 years is unusual but does occur. Males have an earlier age at onset than females: nearly 25% of males have onset before age 10 years. The onset of symptoms is typically gradual; however, acute onset has also been reported.

1	If 0CD is untreated, the course is usually chronic, often with waxing and waning symp- toms. Some individuals have an episodic course, and a minority have a deteriorating course. Without treatment, remission rates in adults are low (e.g., 20% for those reevalu- ated 40 years later). Onset in childhood or adolescence can lead to a lifetime of OCD. How- ever, 40% of individuals with onset of 0CD in childhood or adolescence may experience remission by early adulthood. The course of 0CD is often complicated by the co—occurrence of other disorders (see section ”Comorbidity" for this disorder).

1	Compulsions are more easily diagnosed in children than obsessions are because com- pulsions are observable. However, most children have both obsessions and compulsions (as do most adults). The pattern of symptoms in adults can be stable over time, but it is more variable in children. Some differences in the content of obsessions and compulsions adult samples. These differences likely reﬂect content appropriate to different develop- mental stages (e.g., higher rates of sexual and religious obsessions in adolescents than in children; higher rates of harm obsessions [e.g., fears of catastrophic events, such as death or illness to self or loved ones] in children and adolescents than in adults). Temperamental. Greater internalizing symptoms, higher negative emotionality, and behavioral inhibition in childhood are possible temperamental risk factors.

1	Temperamental. Greater internalizing symptoms, higher negative emotionality, and behavioral inhibition in childhood are possible temperamental risk factors. Environmental. Physical and sexual abuse in childhood and other stressful or traumatic events have been associated with an increased risk for developing OCD. Some children may develop the sudden onset of obsessive-compulsive symptoms, which has been asso- ciated with different environmental factors, including various infectious agents and a post-infectious autoimmune syndrome. Genetic and physiological. The rate of 0CD among first-degree relatives of adults with OCD is approximately two times that among first-degree relatives of those without the disorder; however, among first-degree relatives of individuals with onset of 0CD in child- hood or adolescence, the rate is increased 10-fold. Familial transmission is due in part to genetic factors (e.g., a concordance rate of 0.57 for monozygotic vs. 0.22 for dizygotic twins).

1	Dysfunction in the orbitofrontal cortex, anterior cingulate cortex, and striatum have been most strongly implicated. 0CD occurs across the world. There is substantial similarity across cultures in the gender distribution, age at onset, and comorbidity of OCD. Moreover, around the globe, there is a similar symptom structure involving cleaning, symmetry, hoarding, taboo thoughts, or fear of harm. However, regional variation in symptom expression exists, and cultural factors may shape the content of obsessions and compulsions.

1	Males have an earlier age at onset of OCD than females and are more likely to have co- morbid tic disorders. Gender differences in the pattern of symptom dimensions have been reported, with, for example, females more likely to have symptoms in the cleaning dimen- sion and males more likely to have symptoms in the forbidden thoughts and symmetry di- mensions. Onset or exacerbation of 0CD, as well as symptoms that can interfere with the mother-infant relationship (e.g., aggressive obsessions leading to avoidance of the infant), have been reported in the peripartum period. Suicidal thoughts occur at some point in as many as about half of individuals with OCD. Suicide attempts are also reported in up to one-quarter of individuals with 0CD; the pres- ence of comorbid major depressive disorder increases the risk.

1	0CD is associated with reduced quality of life as well as high levels of social and occupa- tional impairment. Impairment occurs across many different domains of life and is asso- ciated with symptom severity. Impairment can be caused by the time spent obsessing and doing compulsions. Avoidance of situations that can trigger obsessions or compulsions can also severely restrict functioning. In addition, specific symptoms can create specific obstacles. For example, obsessions about harm can make relationships with family and friends feel hazardous; the result can be avoidance of these relationships. Obsessions about symmetry can derail the timely completion of school or work projects because the project never feels “just right,” potentially resulting in school failure or job loss. Health consequences can also occur. For example, individuals with contamination concerns may avoid doctors’ offices and hospitals (e.g., because of fears of exposure to germs) or develop dermatological

1	consequences can also occur. For example, individuals with contamination concerns may avoid doctors’ offices and hospitals (e.g., because of fears of exposure to germs) or develop dermatological problems (e.g., skin lesions due to excessive washing). Sometimes the symptoms of the disorder interfere with its own treatment (e.g., when medications are con- sidered contaminated). When the disorder starts in childhood or adolescence, individuals may experience developmental difficulties. For example, adolescents may avoid socializ- ing with peers; young adults may struggle when they leave home to live independently.

1	The result can be few significant relationships outside the family and a lack of autonomy and financial independence from their family of origin. In addition, some individuals with OCD try to impose rules and prohibitions on family members because of their disorder (e.g., no one in the family can have visitors to the house for fear of contamination), and this can lead to family dysfunction.

1	Anxiety disorders. Recurrent thoughts, avoidant behaviors, and repetitive requests for reassurance can also occur in anxiety disorders. However, the recurrent thoughts that are present in generalized anxiety disorder (i.e., worries) are usually about real-life concerns, whereas the obsessions of OCD usually do not involve real-life concerns and can include content that is odd, irrational, or of a seemingly magical nature; moreover, compulsions are often present and usually linked to the obsessions. Like individuals with 0CD, indi— viduals with specific phobia can have a fear reaction to specific objects or situations; how- ever, in specific phobia the feared object is usually much more circumscribed, and rituals are not present. In social anxiety disorder (social phobia), the feared objects or situations are limited to social interactions, and avoidance or reassurance seeking is focused on re— ducing this social fear.

1	Major depressive disorder. OCD can be distinguished from the rumination of major depressive disorder, in which thoughts are usually mood-congruent and not necessarily experienced as intrusive or distressing; moreover, ruminations are not linked to compul— sions, as is typical in OCD.

1	Other obsessive-compulsive and related disorders. In body dysmorphic disorder, the obsessions and compulsions are limited to concerns about physical appearance; and in trichotillomania (hair-pulling disorder), the compulsive behavior is limited to hair pulling in the absence of obsessions. Hoarding disorder symptoms focus exclusively on the per- sistent difficulty discarding or parting with possessions, marked distress associated with discarding items, and excessive accumulation of objects. However, if an individual has ob— sessions that are typical of 0CD (e.g., concerns about incompleteness or harm), and these obsessions lead to compulsive hoarding behaviors (e.g., acquiring all objects in a set to at- tain a sense of completeness or not discarding old newspapers because they may contain information that could prevent harm), a diagnosis of OCD should be given instead.

1	Eating disorders. OCD can be distinguished from anorexia nervosa in that in OCD the obsessions and compulsions are not limited to concerns about weight and food. Tics (in tic disorder) and stereotyped movements. A tic is a sudden, rapid, recurrent, nonrhythmic motor movement or vocalization (e.g., eye blinking, throat clearing). A ste— reotyped movement is a repetitive, seemingly driven, nonfunctional motor behavior (e.g., head banging, body rocking, self—biting). Tics and stereotyped movements are typically less complex than compulsions and are not aimed at neutralizing obsessions. However, distinguishing between complex tics and compulsions can be difficult. Whereas compul- sions are usually preceded by obsessions, tics are often preceded by premonitory sensory urges. Some individuals have symptoms of both OCD and a tic disorder, in which case both diagnoses may be warranted.

1	Psychotic disorders. Some individuals with 0CD have poor insight or even delusional 0CD beliefs. However, they have obsessions and compulsions (distinguishing their condition from delusional disorder) and do not have other features of schizophrenia or schizoaffective disorder (e.g., hallucinations or formal thought disorder). Other compulsive-like behaviors. Certain behaviors are sometimes described as ”com- pulsive,” including sexual behavior (in the case of paraphilias), gambling (i.e., gambling disorder), and substance use (e.g., alcohol use disorder). However, these behaviors differ from the compulsions of OCD in that the person usually derives pleasure from the activity and may wish to resist it only because of its deleterious consequences.

1	Obsessive-compulsive personality disorder. Although obsessive-compulsive person- ality disorder and 0CD have similar names, the clinical manifestations of these disorders are quite different. Obsessive-compulsive personality disorder is not characterized by in- trusive thoughts, images, or urges or by repetitive behaviors that are performed in re- sponse to these intrusions; instead, it involves an enduring and pervasive maladaptive pattern of excessive perfectionism and rigid control. If an individual manifests symptoms of both OCD and obsessive-compulsive personality disorder, both diagnoses can be given.

1	Individuals with OCD often have other psychopathology. Many adults with the disorder have a lifetime diagnosis of an anxiety disorder (76%; e.g., panic disorder, social anxiety disorder, generalized anxiety disorder, specific phobia) or a depressive or bipolar disorder (63% for any depressive or bipolar disorder, with the most common being major depres- sive disorder [41%]). Onset of OCD is usually later than for most comorbid anxiety disor- ders (with the exception of separation anxiety disorder) and PTSD but often precedes that of depressive disorders. Comorbid obsessive-compulsive personality disorder is also common in individuals with OCD (e.g., ranging from 23% to 32%).

1	Up to 30% of individuals with OCD also have a lifetime tic disorder. A comorbid tic disorder is most common in males with onset of 0CD in childhood. These individuals tend to differ from those without a history of tic disorders in the themes of their OCD symptoms, comorbidity, course, and pattern of familial transmission. A triad of OCD, tic disorder, and attention-deficit/hyperactivity disorder can also be seen in children. Disorders that occur more frequently in individuals with OCD than in those without the disorder include several obsessive-compulsive and related disorders such as body dysmorphic disorder, trichotillomania (hair-pulling disorder), and excoriation (skin—pick- ing) disorder. Finally, an association between OCD and some disorders characterized by impulsivity, such as oppositional defiant disorder, has been reported.

1	OCD is also much more common in individuals with certain other disorders than would be expected based on its prevalence in the general population; when one of those other disorders is diagnosed, the individual should be assessed for 0CD as well. For ex- ample, in individuals with schizophrenia or schizoaffective disorder, the prevalence of 0CD is approximately 12%. Rates of 0CD are also elevated in bipolar disorder; eating dis- orders, such as anorexia nervosa and bulimia nervosa; and Tourette’s disorder. Diagnostic Criteria 300.7 (F45.22) A. Preoccupation with one or more perceived defects or ﬂaws in physical appearance that are not observable or appear slight to others. B. At some point during the course of the disorder, the individual has performed repetitive behaviors (e.g., mirror checking, excessive grooming, skin picking, reassurance seek- ing) or mental acts (e.g., comparing his or her appearance with that of others) in re- sponse to the appearance concerns.

1	C. The preoccupation causes clinically significant distress or impairment in social, occu- pational, or other important areas of functioning. D. The appearance preoccupation is not better explained by concerns with body fat or weight in an individual whose symptoms meet diagnostic criteria for an eating disorder. Specify it: With muscle dysmorphia: The individual is preoccupied with the idea that his or her body build is too small or insufficiently muscular. This specifier is used even if the indi- vidual is preoccupied with other body areas, which is often the case. Specify it: Indicate degree of insight regarding body dysmorphic disorder beliefs (e.g., “I look ugly" or “I look deformed"). With good or fair insight: The individual recognizes that the body dysmorphic disor- der beliefs are definitely or probably not true or that they may or may not be true. With poor insight: The individual thinks that the body dysmorphic disorder beliefs are probably true.

1	With poor insight: The individual thinks that the body dysmorphic disorder beliefs are probably true. With absent insight/delusionai beliefs: The individual is completely convinced that the body dysmorphic disorder beliefs are true.

1	Individuals with body dysmorphic disorder (formerly known as dysmorphophobia) are pre- occupied with one or more perceived defects or ﬂaws in their physical appearance, which they believe look ugly, unattractive, abnormal, or deformed (Criterion A). The perceived ﬂaws are not observable or appear only slight to other individuals. Concerns range from looking ”unattractive" or "not right” to looking ”hideous" or ”like a monster.” Preoccu- pations can focus on one or many body areas, most commonly the skin (e.g., perceived acne, scars, lines, wrinkles, paleness), hair (e.g., ”thinning" hair or ”excessive” body or fa— cial hair), or nose (e.g., size or shape). However, any body area can be the focus of concern (e.g., eyes, teeth, weight, stomach, breasts, legs, face size or shape, lips, chin, eyebrows, genitals). Some individuals are concerned about perceived asymmetry of body areas. The preoccupations are intrusive, unwanted, time-consuming (occurring, on average, 3—8 hours per day), and

1	eyebrows, genitals). Some individuals are concerned about perceived asymmetry of body areas. The preoccupations are intrusive, unwanted, time-consuming (occurring, on average, 3—8 hours per day), and usually difficult to resist or control.

1	Excessive repetitive behaviors or mental acts (e.g., comparing) are performed in re— sponse to the preoccupation (Criterion B). The individual feels driven to perform these be- haviors, which are not pleasurable and may increase anxiety and dysphoria. They are typically time-consuming and difficult to resist or control. Common behaviors are com- paring one's appearance with that of other individuals; repeatedly checking perceived grooming (e.g., combing, styling, shaving, plucking, or pulling hair); camouﬂaging (e.g., repeatedly applying makeup or covering disliked areas with such things as a hat, clothing, makeup, or hair); seeking reassurance about how the perceived ﬂaws look; touching dis- liked areas to check them; excessively exercising or weight lifting; and seeking cosmetic procedures. Some individuals excessively tan (e.g., to darken ”pale” skin or diminish per- ceived acne), repeatedly change their clothes (e.g., to camouﬂage perceived defects), or compulsively shop (e.g.,

1	Some individuals excessively tan (e.g., to darken ”pale” skin or diminish per- ceived acne), repeatedly change their clothes (e.g., to camouﬂage perceived defects), or compulsively shop (e.g., for beauty products). Compulsive skin picking intended to improve perceived skin defects is common and can cause skin damage, infections, or ruptured blood vessels. The preoccupation must cause clinically significant distress or im- pairment in social, occupational, or other important areas of functioning (Criterion C); usually both are present. Body dysmorphic disorder must be differentiated from an eating disorder.

1	Muscle dysmorphia, a form of body dysmorphic disorder occurring almost exclusively in males, consists of preoccupation with the idea that one’s body is too small or insuffi- ciently lean or muscular. Individuals with this form of the disorder actually have a nor- mal-looking body or are even very muscular. They may also be preoccupied with other body areas, such as skin or hair. A majority (but not all) diet, exercise, and/ or lift weights excessively, sometimes causing bodily damage. Some use potentially dangerous anabolic- androgenic steroids and other substances to try to make their body bigger and more mus- cular. Body dysmorphic disorder by proxy is a form of body dysmorphic disorder in which individuals are preoccupied with defects they perceive in another person’s appear- ance.

1	Insight regarding body dysmorphic disorder beliefs can range from good to absent/ delusional (i.e., delusional beliefs consisting of complete conviction that the individual’s view of their appearance is accurate and undistorted). On average, insight is poor; one- third or more of individuals currently have delusional body dysmorphic disorder beliefs. Individuals with delusional body dysmorphic disorder tend to have greater morbidity in some areas (e.g., suicidality), but this appears accounted for by their tendency to have more severe body dysmorphic disorder symptoms.

1	Many individuals with body dysmorphic disorder have ideas or delusions of reference, believing that other people take special notice of them or mock them because of how they look. Body dysmorphic disorder is associated with high levels of anxiety, social anxiety, social avoidance, depressed mood, neuroticism, and perfectionism as well as low extro- version and low self—esteem. Many individuals are ashamed of their appearance and their excessive focus on how they look, and are reluctant to reveal their concerns to others. A majority of individuals receive cosmetic treatment to try to improve their perceived de- fects. Dermatological treatment and surgery are most common, but any type (e.g., dental, electrolysis) may be received. Occasionally, individuals may perform surgery on them- selves. Body dysmorphic disorder appears to respond poorly to such treatments and sometimes becomes worse. Some individuals take legal action or are violent toward the clinician because they are

1	selves. Body dysmorphic disorder appears to respond poorly to such treatments and sometimes becomes worse. Some individuals take legal action or are violent toward the clinician because they are dissatisfied with the cosmetic outcome.

1	Body dysmorphic disorder has been associated with executive dysfunction and visual processing abnormalities, with a bias for analyzing and encoding details rather than ho— listic or configural aspects of visual stimuli. Individuals with this disorder tend to have a bias for negative and threatening interpretations of facial expressions and ambiguous sce- narios. The point prevalence among US. adults is 2.4% (2.5% in females and 2.2% in males). Out- side the United States (i.e., Germany), current prevalence is approximately 1.7%—1.8%, with a gender distribution similar to that in the United States. The current prevalence is 9%—15% among dermatology patients, 7%—8% among US. cosmetic surgery patients, 3%— 16% among international cosmetic surgery patients (most studies), 8% among adult orth- odontia patients, and 10% among patients presenting for oral or maxillofacial surgery.

1	The mean age at disorder onset is 16—17 years, the median age at onset is 15 years, and the most common age at onset is 12—13 years. Two-thirds of individuals have disorder onset before age 18. Subclinical body dysmorphic disorder symptoms begin, on average, at age 12 or 13 years. Subclinical concerns usually evolve gradually to the full disorder, although some individuals experience abrupt onset of body dysmorphic disorder. The disorder appears to usually be chronic, although improvement is likely when evidence-based treatment is received. The disorder’s clinical features appear largely similar in children/ adolescents and adults. Body dysmorphic disorder occurs in the elderly, but little is known about the disorder in this age group. Individuals with disorder onset before age 18 years are more likely to attempt suicide, have more comorbidity, and have gradual (rather than acute) disorder onset than those with adult-onset body dysmorphic disorder.

1	Environmental. \ Body dysmorphic disorder has been associated with high rates of child- hood neglect and abuse. Genetic and physiological. The prevalence of body dysmorphic disorder is elevated in first-degree relatives of individuals with obsessive-compulsive disorder (OCD). Body dysmorphic disorder has been reported internationally. It appears that the disorder values and preferences may inﬂuence symptom content to some degree. Taijin kyofusho, included in the traditional Japanese diagnostic system, has a subtype similar to body dys- morphic disorder: shubo—kyofu (”the phobia of a deformed body”).

1	Females and males appear to have more similarities than differences in terms of most clin- ical features— for example, disliked body areas, types of repetitive behaviors, symptom severity, suicidality, comorbidity, illness course, and receipt of cosmetic procedures for body dysmorphic disorder. However, males are more likely to have genital preoccupa- tions, and females are more likely to have a comorbid eating disorder. Muscle dysmorphia occurs almost exclusively in males.

1	Rates of suicidal ideation and suicide attempts are high in both adults and children/ado- lescents with body dysmorphic disorder. Furthermore, risk for suicide appears high in ad- olescents. A substantial proportion of individuals attribute suicidal ideation or suicide attempts primarily to their appearance concerns. Individuals with body dysmorphic dis- order have many risk factors for completed suicide, such as high rates of suicidal ideation and suicide attempts, demographic characteristics associated with suicide, and high rates of comorbid major depressive disorder.

1	Nearly all individuals with body dysmorphic disorder experience impaired psychosocial functioning because of their appearance concerns. Impairment can range from moderate (e.g., avoidance of some social situations) to extreme and incapacitating (e.g., being com- pletely housebound). On average, psychosocial functioning and quality of life are mark— edly poor. More severe body dysmorphic disorder symptoms are associated with poorer functioning and quality of life. Most individuals experience impairment in their job, aca- demic, or role functioning (e.g., as a parent or caregiver), which is often severe (e.g., per- forming poorly, missing school or work, not working). About 20% of youths with body dysmorphic disorder report dropping out of school primarily because of their body dys- morphic disorder symptoms. Impairment in social functioning (e.g., social activities, rela- tionships, intimacy), including avoidance, is common. Individuals may be housebound because of their body

1	morphic disorder symptoms. Impairment in social functioning (e.g., social activities, rela- tionships, intimacy), including avoidance, is common. Individuals may be housebound because of their body dysmorphic disorder symptoms, sometimes for years. A high pro- portion of adults and adolescents have been psychiatrically hospitalized.

1	Normal appearance concerns and cleady noticeable physical defects. Body dysmor- sive appearance-related preoccupations and repetitive behaviors that are time-consuming, are usually difficult to resist or control, and cause clinically significant distress or impair- ment in functioning. Physical defects that are clearly noticeable (i.e., not slight) are not diagnosed as body dysmorphic disorder. However, skin picking as a symptom of body dysmorphic disorder can cause noticeable skin lesions and scarring; in such cases, body dys- morphic disorder should be diagnosed. Eating disorders. In an individual with an eating disorder, concerns about being fat are considered a symptom of the eating disorder rather than body dysmorphic disorder. However, weight concerns may occur in body dysmorphic disorder. Eating disorders and body dysmorphic disorder can be comorbid, in which case both should be diagnosed.

1	However, weight concerns may occur in body dysmorphic disorder. Eating disorders and body dysmorphic disorder can be comorbid, in which case both should be diagnosed. Other obsessive-compulsive and related disorders. The preoccupations and repetitive behaviors of body dysmorphic disorder differ from obsessions and compulsions in 0CD in that the former focus only on appearance. These disorders have other differences, such as poorer insight in body dysmorphic disorder. When skin picking is intended to improve the appearance of perceived skin defects, body dysmorphic disorder, rather than excoria- tion (skin-picking) disorder, is diagnosed. When hair removal (plucking, pulling, or other types of removal) is intended to improve perceived defects in the appearance of facial or body hair, body dysmorphic disorder is diagnosed rather than trichotillomania (hair- pulling disorder).

1	Illness anxiety disorder. Individuals with body dysmorphic disorder are not preoccu- pied with having or acquiring a serious illness and do not have particularly elevated levels of somatization. Major depressive disorder. The prominent preoccupation with appearance and exces- pressive disorder. However, major depressive disorder and depressive symptoms are common in individuals with body dysmorphic disorder, often appearing to be secondary to the distress and impairment that body dysmorphic disorder causes. Body dysmorphic morphic disorder are met. ‘

1	Anxiety disorders. Social anxiety and avoidance are common in body dysmorphic dis- order. However, unlike social anxiety disorder (social phobia), agoraphobia, and avoidant personality disorder, body dysmorphic disorder includes prominent appearance-related preoccupation, which may be delusional, and repetitive behaviors, and the social anxiety and avoidance are due to concerns about perceived appearance defects and the belief or fear that other people will consider these individuals ugly, ridicule them, or reject them be- cause of their physical features. Unlike generalized anxiety disorder, anxiety and worry in body dysmorphic disorder focus on perceived appearance ﬂaws.

1	Psychotic disorders. Many individuals with body dysmorphic disorder have delu- sional appearance beliefs (i.e., complete conviction that their view of their perceived de- fects is accurate), which is diagnosed as body dysmorphic disorder, with absent insight/ delusional beliefs, not as delusional disorder. Appearance-related ideas or delusions of reference are common in body dysmorphic disorder; however, unlike schizophrenia or schizoaffective disorder, body dysmorphic disorder involves prominent appearance pre- occupations and related repetitive behaviors, and disorganized behavior and other psy— chotic symptoms are absent (except for appearance beliefs, which may be delusional).

1	Other disorders and symptoms. Body dysmorphic disorder should not be diagnosed if the preoccupation is limited to discomfort with or a desire to be rid of one's primary and / or secondary sex characteristics in an individual with gender dysphoria or if the preoccu- pation focuses on the belief that one emits a foul or offensive body odor as in olfactory reference syndrome (which is not a DSM-5 disorder). Body identity integrity disorder (apotemnophilia) (which is not a DSM-5 disorder) involves a desire to have a limb ampu- tated to correct an experience of mismatch between a person’s sense of body identity and his or her actual anatomy. However, the concern does not focus on the limb’s appearance, as it would in body dysmorphic disorder. Karo, a culturally related disorder that usually occurs in epidemics in Southeastern Asia, consists of a fear that the penis (labia, nipples, or breasts in females) is shrinking or retracting and will disappear into the abdomen, often accompanied by a

1	in epidemics in Southeastern Asia, consists of a fear that the penis (labia, nipples, or breasts in females) is shrinking or retracting and will disappear into the abdomen, often accompanied by a belief that death will result. Koro differs from body dysmorphic disor- der in several ways, including a focus on death rather than preoccupation with perceived ugliness. Dysmorphic concern (which is not a DSM-5 disorder) is a much broader construct than, and is not equivalent to, body dysmorphic disorder. It involves symptoms reﬂecting an overconcern with slight or imagined ﬂaws in appearance.

1	Major depressive disorder is the most common comorbid disorder, with onset usually af- ter that of body dysmorphic disorder. Comorbid social anxiety disorder (social phobia), OCD, and substance-related disorders are also common. Diagnostic Criteria 300.3 (F42) A. Persistent difficulty discarding or parting with possessions, regardless of their actual value. B. This difficulty is due to a perceived need to save the items and to distress associated with discarding them. C. The difficulty discarding possessions results in the accumulation of possessions that use. If living areas are uncluttered, it is only because of the interventions of third parties (e.g., family members, cleaners, authorities). D. The hoarding causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning (including maintaining a safe environ- ment for self and others).

1	E. The hoarding is not attributable to another medical condition (e.g., brain injury, cere- brovascular disease, Prader-Willi syndrome). F. The hoarding is not better explained by the symptoms of another mental disorder (e.g., obsessions in obsessive-compulsive disorder, decreased energy in major depressive disorder, delusions in schizophrenia or another psychotic disorder, cognitive deficits in major neurocognitive disorder, restricted interests in autism spectrum disorder). Specify it: With excessive acqulsition: If difficulty discarding possessions is accompanied by ex- cessive acquisition of items that are not needed or for which there is no available space. Specify it: With good or fair insight: The individual recognizes that hoarding-related beliefs and behaviors (pertaining to difficulty discarding items, clutter, or excessive acquisition) are problematic.

1	With good or fair insight: The individual recognizes that hoarding-related beliefs and behaviors (pertaining to difficulty discarding items, clutter, or excessive acquisition) are problematic. With poor insight: The individual is mostly convinced that hoarding-related beliefs and behaviors (pertaining to difficulty discarding items, clutter. or excessive acquisi- tion) are not problematic despite evidence to the contrary. With absent insight/delusional beliefs: The individual is completely convinced that hoarding-related beliefs and behaviors (pertaining to difficulty discarding items, clutter, or excessive acquisition) are not problematic despite evidence to the contrary. With excessive acquisition. Approximately 80%—90% of individuals with hoarding disorder display excessive acquisition. The most frequent form of acquisition is excessive buying, followed by acquisition of free items (e.g., leaﬂets, items discarded by others).

1	Stealing is less common. Some individuals may deny excessive acquisition when first as- sessed, yet it may appear later during the course of treatment. Individuals with hoarding disorder typically experience distress if they are unable to or are prevented from acquiring items. The essential feature of hoarding disorder is persistent difficulties discarding or parting with possessions, regardless of their actual value (Criterion A). The term persistent indi- to excessive clutter, such as inheriting property. The difficulty in discarding possessions noted in Criterion A refers to any form of discarding, including throwing away, selling, giving away, or recycling. The main reasons given for these difficulties are the perceived utility or aesthetic value of the items or strong sentimental attachment to the possessions.

1	Some individuals feel responsible for the fate of their possessions and often go to great lengths to avoid being wasteful. Fears of losing important information are also common. The most commonly saved items are newspapers, magazines, old clothing, bags, books, mail, and paperwork, but virtually any item can be saved. The nature of items is not lim- ited to possessions that most other people would define as useless or of limited value. Many individuals collect and save large numbers of valuable things as well, which are of- ten found in piles mixed with other less valuable items.

1	Many individuals collect and save large numbers of valuable things as well, which are of- ten found in piles mixed with other less valuable items. Individuals with hoarding disorder purposefully save possessions and experience dis- tress when facing the prospect of discarding them (Criterion B). This criterion emphasizes that the saving of possessions is intentional, which discriminates hoarding disorder from other forms of psychopathology that are characterized by the passive accumulation of items or the absence of distress when possessions are removed.

1	Individuals accumulate large numbers of items that fill up and clutter active living ar- eas to the extent that their intended use is no longer possible (Criterion C). For example, the individual may not be able to cook in the kitchen, sleep in his or her bed, or sit in a chair. If the space can be used, it is only with great difficulty. Clutter is defined as a large group of usually unrelated or marginally related objects piled together in a disorganized fashion in spaces designed for other purposes (e.g., tabletops, ﬂoor, hallway). Criterion C emphasizes the ”active" living areas of the home, rather than more peripheral areas, such as garages, attics, or basements, that are sometimes cluttered in homes of individuals with- out hoarding disorder. However, individuals with hoarding disorder often have posses— sions that spill beyond the active living areas and can occupy and impair the use of other spaces, such as vehicles, yards, the workplace, and friends’ and relatives’ houses. In

1	often have posses— sions that spill beyond the active living areas and can occupy and impair the use of other spaces, such as vehicles, yards, the workplace, and friends’ and relatives’ houses. In some cases, living areas may be uncluttered because of the intervention of third parties (e.g., family members, cleaners, local authorities). Individuals who have been forced to clear the lack of clutter is due to a third-party intervention. Hoarding disorder contrasts with normative collecting behavior, which is organized and systematic, even if in some cases the actual amount of possessions may be similar to the amount accumulated by an indi- vidual with hoarding disorder. Normative collecting does not produce the clutter, dis- tress, or impairment typical of hoarding disorder.

1	Symptoms (i.e., difficulties discarding and / or clutter) must cause clinically significant distress or impairment in social, occupational, or other important areas of functioning, in- cluding maintaining a safe environment for self and others (Criterion D). In some cases, particularly when there is poor insight, the individual may not report distress, and the im— pairment may be apparent only to those around the individual. However, any attempts to discard or clear the possessions by third parties result in high levels of distress. Other common features of hoarding disorder include indecisiveness, perfectionism, avoidance, procrastination, difficulty planning and organizing tasks, and distractibility.

1	Some individuals with hoarding disorder live in unsanitary conditions that may be a log- ical consequence of severely cluttered spaces and/ or that are related to planning and or- ganizing difficulties. Animal hoarding can be defined as the accumulation of a large number of animals and a failure to provide minimal standards of nutrition, sanitation, and veter- inary care and to act on the deteriorating condition of the animals (including disease, star- vation, or death) and the environment (e.g., severe overcrowding, extremely unsanitary conditions). Animal hoarding may be a special manifestation of hoarding disorder. Most individuals who hoard animals also hoard inanimate objects. The most prominent differ- ences between animal and object hoarding are the extent of unsanitary conditions and the poorer insight in animal hoarding.

1	Nationally representative prevalence studies of hoarding disorder are not available. Com— munity surveys estimate the point prevalence of clinically significant hoarding in the United States and Europe to be approximately 2%—6%. Hoarding disorder affects both males and females, but some epidemiological studies have reported a significantly greater prevalence among males. This contrasts with clinical samples, which are predominantly female. Hoarding symptoms appear to be almost three times more prevalent in older adults (ages 55—94 years) compared with younger adults (ages 34—44 years).

1	Hoarding appears to begin early in life and spans well into the late stages. Hoarding symp- toms may first emerge around ages 11—15 years, start interfering with the individual’s ev- eryday functioning by the mid—ZOs, and cause clinically significant impairment by the mid-30s. Participants in clinical research studies are usually in their 505. Thus, the severity of hoarding increases with each decade of life. Once symptoms begin, the course of hoard- ing is often chronic, with few individuals reporting a waxing and waning course. Pathological hoarding in children appears to be easily distinguished from develop- mentally adaptive saving and collecting behaviors. Because children and adolescents typically do not control their living environment and discarding behaviors, the possible intervention of third parties (e.g., parents keeping the spaces usable and thus reducing in- terference) should be considered when making the diagnosis.

1	Temperamental. Indecisiveness is a prominent feature of individuals with hoarding dis- order and their first-degree relatives. Environmental. Individuals with hoarding disorder often retrospectively report stressful and traumatic life events preceding the onset of the disorder or causing an exacerbation. Genetic and physiological. Hoarding behavior is familial, with about 50% of individu- als who hoard reporting having a relative who also hoards. Twin studies indicate that ap- proximately 50% of the variability in hoarding behavior is attributable to additive genetic factors. While most of the research has been done in Western, industrialized countries and urban communities, the available data from non-Westem and developing countries suggest that hoarding is a universal phenomenon with consistent clinical features.

1	The key features of hoarding disorder (i.e., difficulties discarding, excessive amount of clutter) are generally comparable in males and females, but females tend to display more excessive acquisition, particularly excessive buying, than do males. Functional Consequences of Hoarding Disorder

1	Functional Consequences of Hoarding Disorder Clutter impairs basic activities, such as moving through the house, cooking, cleaning, per- sonal hygiene, and even sleeping. Appliances may be broken, and utilities such as water and electricity may be disconnected, as access for repair work may be difficult. Quality of life is often considerably impaired. In severe cases, hoarding can put individuals at risk for fire, falling (especially elderly individuals), poor sanitation, and other health risks. Hoard- ing disorder is associated with occupational impairment, poor physical health, and high social service utilization. Family relationships are frequently under great strain. Conﬂict with neighbors and local authorities is common, and a substantial proportion of individ- uals with severe hoarding disorder have been involved in legal eviction proceedings, and some have a history of eviction.

1	Other medical conditions. Hoarding disorder is not diagnosed if the symptoms are judged to be a direct consequence of another medical condition (Criterion E), such as trau- matic brain injury, surgical resection for treatment of a tumor or seizure control, cerebro- vascular disease, infections of the central nervous system (e.g., herpes simplex encephalitis), or neurogenetic conditions such as Prader-Willi syndrome. Damage to the anterior ven- tromedial prefrontal and cingulate cortices has been particularly associated with the ex- cessive accumulation of objects. In these individuals, the hoarding behavior is not present prior to the onset of the brain damage and appears shortly after the brain damage occurs. Some of these individuals appear to have little interest in the accumulated items and are able to discard them easily or do not care if others discard them, whereas others appear to be very reluctant to discard anything.

1	Neurodevelopmental disorders. Hoarding disorder is not diagnosed if the accumula- tion of objects is judged to be a direct consequence of a neurodevelopmental disorder, such as autism spectrum disorder or intellectual disability (intellectual developmental disorder). Schizophrenia spectrum and other psychotic disorders. Hoarding disorder is not di— agnosed if the accumulation of objects is judged to be a direct consequence of delusions or negative symptoms in schizophrenia spectrum and other psychotic disorders. Major depressive episode. Hoarding disorder is not diagnosed if the accumulation of objects is judged to be a direct consequence of psychomotor retardation, fatigue, or loss of energy during a major depressive episode.

1	Obsessive-compulsive disorder. Hoarding disorder is not diagnosed if the symptoms are judged to be a direct consequence of typical obsessions or compulsions, such as fears of contamination, harm, or feelings of incompleteness in obsessive-compulsive disorder (OCD). Feelings of incompleteness (e.g., losing one’s identity, or having to document and preserve all life experiences) are the most frequent OCD symptoms associated with this form of hoarding. The accumulation of objects can also be the result of persistently avoid- ing onerous rituals (e.g., not discarding objects in order to avoid endless washing or check- ing rituals). \

1	In OCD, the behavior is generally unwanted and highly distressing, and the individual ex- periences no pleasure or reward from it. Excessive acquisition is usually not present; if exces- sive acquisition is present, items are acquired because of a specific obsession (e.g., the need to buy items that have been accidentally touched in order to avoid contaminating other people), not because of a genuine desire to possess the items. Individuals who hoard in the context of OCD are also more likely to accumulate bizarre items, such as trash, feces, urine, nails, hair, used diapers, or rotten food. Accumulation of such items is very unusual in hoarding disorder. When severe hoarding appears concurrently with other typical symptoms of 0CD but is judged to be independent from these symptoms, both hoarding disorder and 0CD may be diagnosed.

1	When severe hoarding appears concurrently with other typical symptoms of 0CD but is judged to be independent from these symptoms, both hoarding disorder and 0CD may be diagnosed. Neurocognitive disorders. Hoarding disorder is not diagnosed if the accumulation of objects is judged to be a direct consequence of a degenerative disorder, such as neurocog- nitive disorder associated with frontotemporal lobar degeneration or Alzheimer’s disease. Typically, onset of the accumulating behavior is gradual and follows onset of the neuro- cognitive disorder. The accumulating behavior may be accompanied by self—neglect and severe domestic squalor, alongside other neuropsychiatric symptoms, such as disinhibi- tion, gambling, rituals/stereotypies, tics, and self—injurious behaviors.

1	Approximately 75% of individuals with hoarding disorder have a comorbid mood or anx- iety disorder. The most common comorbid conditions are major depressive disorder (up to 50% of cases), social anxiety disorder (social phobia), and generalized anxiety disorder. Approximately 20% of individuals with hoarding disorder also have symptoms that meet diagnostic criteria for OCD. These comorbidities may often be the main reason for consul- tation, because individuals are unlikely to spontaneously report hoarding symptoms, and these symptoms are often not asked about in routine clinical interviews. Diagnostic Criteria 312.39 (F63.2) A. Recurrent pulling out of one’s hair, resulting in hair loss. B. Repeated attempts to decrease or stop hair pulling. C. The hair pulling causes clinically significant distress or impairment in social. occupa- tional, or other important areas of functioning.

1	B. Repeated attempts to decrease or stop hair pulling. C. The hair pulling causes clinically significant distress or impairment in social. occupa- tional, or other important areas of functioning. D. The hair pulling or hair loss is not attributable to another medical condition (e.g., a der- matological condition). E. The hair pulling is not better explained by the symptoms of another mental disorder (e.g., attempts to improve a perceived defect or flaw in appearance in body dysmorphic disorder).

1	The essential feature of trichotillomania (hair-pulling disorder) is the recurrent pulling out of one’s own hair (Criterion A). Hair pulling may occur from any region of the body in which hair grows; the most common sites are the scalp, eyebrows, and eyelids, while less common sites are axillary, facial, pubic, and peri-rectal regions. Hair-pulling sites may vary over time. Hair pulling may occur in brief episodes scattered throughout the day or during less frequent but more sustained periods that can continue for hours, and such hair pulling may endure for months or years. Criterion A requires that hair pulling lead to hair loss, although individuals with this disorder may pull hair in a widely distributed pattern (i.e., pulling single hairs from all over a site) such that hair loss may not be clearly visible.

1	Alternatively, individuals may attempt to conceal or camouﬂage hair loss (e.g., by using makeup, scarves, or wigs). Individuals with trichotillomania have made repeated at- tempts to decrease or stop hair pulling (Criterion B). Criterion C indicates that hair pulling causes clinically significant distress or impairment in social, occupational, or other impor- tant areas of functioning. The term distress includes negative affects that may be experi- enced by individuals with hair pulling, such as feeling a loss of control, embarrassment, and shame. Significant impairment may occur in several different areas of functioning (e.g., social, occupational, academic, and leisure), in part because of avoidance of work, school, or other public situations.

1	Hair pulling may be accompanied by a range of behaviors or rituals involving hair. Thus, individuals may search for a particular kind of hair to pull (e.g., hairs with a specific tex- ture or color), may try to pull out hair in a specific way (e.g., so that the root comes out in- tact), or may visually examine or tactilely or orally manipulate the hair after it has been pulled (e.g., rolling the hair between the fingers, pulling the strand between the teeth, bit- ing the hair into pieces, or swallowing the hair).

1	be triggered by feelings of anxiety or boredom, may be preceded by an increasing sense of tension (either immediately before pulling out the hair or when attempting to resist the urge to pull), or may lead to gratification, pleasure, or a sense of relief when the hair is pulled out. Hair-pulling behavior may involve varying degrees of conscious awareness, with some individuals displaying more focused attention on the hair pulling (with pre- ceding tension and subsequent relief), and other individuals displaying more automatic behavior (in which the hair pulling seems to occur without full awareness). Many individ- uals report a mix of both behavioral styles. Some individuals experience an "itch-like” or tingling sensation in the scalp that is alleviated by the act of pulling hair. Pain does not usually accompany hair pulling.

1	Patterns of hair loss are highly variable. Areas of complete alopecia, as well as areas of thinned hair density, are common. When the scalp is involved, there may be a predilection for pulling out hair in the crown or parietal regions. There may be a pattern of nearly com- plete baldness except for a narrow perimeter around the outer margins of the scalp, par- ticularly at the nape of the neck (”tonsure trichotillomania”). Eyebrows and eyelashes may be completely absent. Hair pulling does not usually occur in the presence of other individuals, except imme— diate family members. Some individuals have urges to pull hair from other individuals and may sometimes try to find opportunities to do so surreptitiously. Some individuals may pull hairs from pets, dolls, and other fibrous materials (e.g., sweaters or carpets).

1	Some individuals may deny their hair pulling to others. The majority of individuals with trichotillomania also have one or more other body-focused repetitive behaviors, including skin picking, nail biting, and lip chewing. In the general population, the 12—month prevalence estimate for trichotillomania in adults and adolescents is 1%—2%. Females are more frequently affected than males, at a ratio of approximately 10:1. This estimate likely reﬂects the true gender ratio of the condition, al- titudes regarding appearance (e.g., acceptance of normative hair 1055 among males). Among children with trichotillomania, males and females are more equally represented.

1	Among children with trichotillomania, males and females are more equally represented. Hair pulling may be seen in infants, and this behavior typically resolves during early devel- opment. Onset of hair pulling in trichotillomania most commonly coincides with, or follows the onset of, puberty. Sites of hair pulling may vary over time. The usual course of trichotillo- mania is chronic, with some waxing and waning if the disorder is untreated. Symptoms may possibly worsen in females accompanying hormonal changes (e.g., menstruation, perimeno— pause). For some individuals, the disorder may come and go for weeks, months, or years at a time. A minority of individuals remit without subsequent relapse within a few years of onset. Genetic and physiological. There is evidence for a genetic vulnerability to trichotillo— mania. The disorder is more common in individuals with obsessive-compulsive disorder (OCD) and their first-degree relatives than in the general population.

1	Trichotillomania appears to manifest similarly across cultures, although there is a paucity of data from non-Westem regions. Most individuals with trichotillomania admit to hair pulling; thus, dermatopathological diagnosis is rarely required. Skin biopsy and dermoscopy (or trichoscopy) of trichotillo- mania are able to differentiate the disorder from other causes of alopecia. In trichotil- lomania, dermoscopy shows a range of characteristic features, including decreased hair density, short vellus hair, and broken hairs with different shaft lengths.

1	Trichotillomania is associated with distress as well as with social and occupational impair- ment. There may be irreversible damage to hair growth and hair quality. Infrequent med- ical consequences of trichotillomania include digit purpura, musculoskeletal injury (e.g., carpal tunnel syndrome; back, shoulder and neck pain), blepharitis, and dental damage (e.g., worn or broken teeth due to hair biting). Swallowing of hair (trichophagia) may lead to trichobezoars, with subsequent anemia, abdominal pain, hematemesis, nausea and vomiting, bowel obstruction, and even perforation.

1	Normative hair removal/manipulation. Trichotillomania should not be diagnosed when hair removal is performed solely for cosmetic reasons (i.e., to improve one’s physical ap- pearance). Many individuals twist and play with their hair, but this behavior does not usu- ally qualify for a diagnosis of trichotillomania. Some individuals may bite rather than pull hair; again, this does not qualify for a diagnosis of trichotillomania. Other obsessive—compulsive and related disorders. Individuals with OCD and sym- metry concerns may pull out hairs as part of their symmetry rituals, and individuals with body dysmorphic disorder may remove body hair that they perceive as ugly, asymmetri- cal, or abnormal; in such cases a diagnosis of trichotillomania is not given. The description related disorder excludes individuals who meet diagnostic criteria for trichotillomania.

1	Neurodevelopmental disorders. In neurodevelopmental disorders, hair pulling may meet the definition of stereotypies (e.g., in stereotypic movement disorder). Tics (in tic dis- orders) rarely lead to hair pulling. Psychotic disorder. Individuals with a psychotic disorder may remove hair in response to a delusion or hallucination. Trichotillomania is not diagnosed in such cases.

1	Psychotic disorder. Individuals with a psychotic disorder may remove hair in response to a delusion or hallucination. Trichotillomania is not diagnosed in such cases. Another medical condition. Trichotillomania is not diagnosed if the hair pulling or hair loss is attributable to another medical condition (e.g., inﬂammation of the skin 01‘ other der- matological conditions). Other causes of scarring alopecia (e.g., alopecia areata, androgenic alopecia, telogen efﬂuvium) or nonscarring alopecia (e.g., chronic discoid lupus erythema- tosus, lichen planopilaris, central centrifugal cicatricial alopecia, pseudopelade, folliculitis decalvans, dissecting folliculitis, acne keloidalis nuchae) should be considered in individu- als with hair loss who deny hair pulling. Skirt biopsy or dermoscopy can be used to differ- entiate individuals with trichotillomania from those with dermatological disorders.

1	Substance-related disorders. Hair-pulling symptoms may be exacerbated by certain substances—for example, stimulants—but it is less likely that substances are the primary cause of persistent hair pulling. Trichotillomania is often accompanied by other mental disorders, most commonly major depressive disorder and excoriation (skin-picking) disorder. Repetitive body-focused symptoms other than hair pulling or skin picking (e.g. nail biting) occur in the majority of individuals with trichotillomania and may deserve an additional diagnosis of other spec- ified obsessive-compulsive and related disorder (i.e., body-focused repetitive behavior disorder). Diagnostic Criteria 698.4 (L98.1) A. Recurrent skin picking resulting in skin lesions. B. Repeated attempts to decrease or stop skin picking. C. The skin picking causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning.

1	B. Repeated attempts to decrease or stop skin picking. C. The skin picking causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. D. The skin picking is not attributable to the physiological effects of a substance (e.g., co- caine) or another medical condition (e.g., scabies). E. The skin picking is not better explained by symptoms of another mental disorder (e.g., delusions or tactile hallucinations in a psychotic disorder, attempts to improve a per- ceived detect or flaw in appearance in body dysmorphic disorder, stereotypies in ste- reotypic movement disorder, or intention to harm oneself in nonsuicidal self-injury).

1	The essential feature of excoriation (skin-picking) disorder is recurrent picking at one’s own skin (Criterion A). The most commonly picked sites are the face, arms, and hands, but many individuals pick from multiple body sites. Individuals may pick at healthy skin, at minor skin irregularities, at lesions such as pimples or calluses, or at scabs from previous picking. Most individuals pick with their fingernails, although many use tweezers, pins, or other objects. In addition to skin picking, there may be skin rubbing, squeezing, lancing, and biting. Individuals with excoriation disorder often spend significant amounts of time on their picking behavior, sometimes several hours per day, and such skin picking may endure for months or years. Criterion A requires that skin picking lead to skin lesions, al- though individuals with this disorder often attempt to conceal or camouﬂage such lesions (e.g., with makeup or clothing). Individuals with excoriation disorder have made repeated

1	to skin lesions, al- though individuals with this disorder often attempt to conceal or camouﬂage such lesions (e.g., with makeup or clothing). Individuals with excoriation disorder have made repeated attempts to decrease or stop skin picking (Criterion B).

1	ment in social, occupational, or other important areas of functioning. The term distress in- cludes negative affects that may be experienced by individuals with skin picking, such as feeling a loss of control, embarrassment, and shame. Significant impairment may occur in several different areas of functioning (e.g., social, occupational, academic, and leisure), in part because of avoidance of social situations. Skin picking may be accompanied by a range of behaviors or rituals involving skin or scabs.

1	Thus, individuals may search for a particular kind of scab to pull, and they may examine, play with, or mouth or swallow the skin after it has been pulled. Skin picking may also be pre- ceded or accompanied by various emotional states. Skin picking may be triggered by feelings of anxiety or boredom, may be preceded by an increasing sense of tension (either immedi- ately before picking the skin or when attempting to resist the urge to pick), and may lead to gratification, pleasure, or a sense of relief when the skin or scab has been picked. Some indi- viduals report picking in response to a minor skin irregularity or to relieve an uncomfortable bodily sensation. Pain is not routinely reported to accompany skin picking. Some individuals engage in skin picking that is more focused (i.e., with preceding tension and subsequent re- lief), whereas others engage in more automatic picking (i.e., when skin picking occurs with- out preceding tension and without full awareness), and many have a

1	preceding tension and subsequent re- lief), whereas others engage in more automatic picking (i.e., when skin picking occurs with- out preceding tension and without full awareness), and many have a mix of both behavioral styles. Skin picking does not usually occur in the presence of other individuals, except im- mediate family members. Some individuals report picking the skin of others.

1	In the general population, the lifetime prevalence for excoriation disorder in adults is 1.4% or somewhat higher. Three-quarters or more of individuals with the disorder are female. This likely reﬂects the true gender ratio of the condition, although it may also reﬂect dif- ferential treatment seeking based on gender or cultural attitudes regarding appearance. Although individuals with excoriation disorder may present at various ages, the skin pick- ing most often has onset during adolescence, commonly coinciding with or following the onset of puberty. The disorder frequently begins with a dermatological condition, such as acne. Sites of skin picking may vary over time. The usual course is chronic, with some waxing and waning if untreated. For some individuals, the disorder may come and go for weeks, months, or years at a time. Genetic and physiological. Excoriation disorder is more common in individuals with general population.

1	Genetic and physiological. Excoriation disorder is more common in individuals with general population. Most individuals with excoriation disorder admit to skin picking; therefore, dermato- pathological diagnosis is rarely required. However, the disorder may have characteristic features on histopathology.

1	Excoriation disorder is associated with distress as well as with social and occupational im- pairment. The majority of individuals with this condition spend at least 1 hour per day picking, thinking about picking, and resisting urges to pick. Many individuals report avoiding social or entertainment events as well as going out in public. A majority of indi- viduals with the disorder also report experiencing work interference from skin picking on at least a daily or weekly basis. A significant proportion of students with excoriation disor- der report having missed school, having experienced difficulties managing responsibilities at school, or having had difficulties studying because of skin picking. Medical complica- tions of skin picking include tissue damage, scarring, and infection and can be life-threaten- ing. Rarely, synovitis of the wrists due to chronic picking has been reported. Skin picking often results in significant tissue damage and scarring. It frequently requires

1	and can be life-threaten- ing. Rarely, synovitis of the wrists due to chronic picking has been reported. Skin picking often results in significant tissue damage and scarring. It frequently requires antibiotic treat- ment for infection, and on occasion it may require surgery.

1	Psychotic disorder. Skin picking may occur in response to a delusion (i.e., parasitosis) or tactile hallucination (i.e., formication) in a psychotic disorder. In such cases, excoriation disorder should not be diagnosed. Other obsessive-compulsive and related disorders. Excessive washing compulsions in response to contamination obsessions in individuals with 0CD may lead to skin lesions, and skin picking may occur in individuals with body dysmorphic disorder who pick their skin solely because of appearance concerns; in such cases, excoriation disorder should not be diagnosed. The description of body-focused repetitive behavior disorder in other spec- meet diagnostic criteria for excoriation disorder. Neurodevelopmental disorders. While stereotypic movement disorder may be charac- terized by repetitive self—injurious behavior, onset is in the early developmental period.

1	Neurodevelopmental disorders. While stereotypic movement disorder may be charac- terized by repetitive self—injurious behavior, onset is in the early developmental period. For example, individuals with the neurogenetic condition Prader-Willi syndrome may have early onset of skin picking, and their symptoms may meet criteria for stereotypic movement disorder. While tics in individuals with Tourette’s disorder may lead to self- injury, the behavior is not tic-like in excoriation disorder. Somatic symptom and related disorders. Excoriation disorder is not diagnosed if the skin lesion is primarily attributable to deceptive behaviors in factitious disorder. Other disorders. Excoriation disorder is not diagnosed if the skin picking is primarily attributable to the intention to harm oneself that is characteristic of nonsuicidal self-injury.

1	Other disorders. Excoriation disorder is not diagnosed if the skin picking is primarily attributable to the intention to harm oneself that is characteristic of nonsuicidal self-injury. Other medical conditions. Excoriation disorder is not diagnosed if the skin picking is primarily attributable to another medical condition. For example, scabies is a dermatolog- ical condition invariably associated with severe itching and scratching. However, excori- condition. For example, acne may lead to some scratching and picking, which may also be associated with comorbid excoriation disorder. The differentiation between these two clinical situations (acne with some scratching and picking vs. acne with comorbid excori- ation disorder) requires an assessment of the extent to which the individual’s skin picking has become independent of the underlying dermatological condition.

1	Substance/medication-induced disorders. Skin-picking symptoms may also be induced by certain substances (e.g., cocaine), in which case excoriation disorder should not be di- agnosed. If such skin picking is clinically significant, then a diagnosis of substance/med- ication-induced obsessive-compulsive and related disorder should be considered. Excoriation disorder is often accompanied by other mental disorders. Such disorders in- clude OCD and trichotillomania (hair-pulling disorder), as well as major depressive dis- order. Repetitive body-focused symptoms other than skin picking and hair pulling (e.g., nail biting) occur in many individuals with excoriation disorder and may deserve an ad- ditional diagnosis of other specified obsessive-compulsive and related disorder (i.e., body-focused repetitive behavior disorder).

1	A. Obsessions, compulsions. skin picking, hair pulling, other body-tocused repetitive be- haviors, or other symptoms characteristic of the obsessive-compulsive and related dis- orders predominate in the clinical picture. B. There is evidence from the history, physical examination, or laboratory findings of both (1) and (2): 1. The symptoms in Criterion A developed during or soon after substance intoxication or withdrawal or after exposure to a medication. 2. The involved substance/medication is capable of producing the symptoms in Crite- rion A. C. The disturbance is not better explained by an obsessive-compulsive and related disor- der that is not substance/medication-induced. Such evidence of an independent ob- sessive-compulsive and related disorder could include the following:

1	The symptoms precede the onset of the substance/medication use; the symptoms persist for a substantial period of time (e.g., about 1 month) after the cessation of acute withdrawal or severe intoxication; or there is other evidence suggesting the exis- tence of an independent non-substance/medication-induced obsessive-compul- sive and related disorder (e.g., a history of recurrent non-substance/medication- related episodes). D. The disturbance does not occur exclusively during the course of a delirium. E. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. Note: This diagnosis should be made in addition to a diagnosis of substance intoxication or substance withdrawal only when the symptoms in Criterion A predominate in the clinical picture and are sufficiently severe to warrant clinical attention.

1	Coding note: The lCD-Q-CM and ICD-10-CM codes for the [specific substance/medica- tion]-induced obsessive-compulsive and related disorders are indicated in the table below.

1	Note that the |CD-10-CM code depends on whether or not there is a comorbid substance use disorder present for the same class of substance. If a mild substance use disorder is comorbid with the substance-induced obsessive-compulsive and related disorder, the 4th position character is “1’ and the clinician should record “mild [substance] use disorder" before the substance-induced obsessive-compulsive and related disorder (e.g., “mild co- caine use disorder with cocaine-induced obsessive-compulsive and related disorder"). It a moderate or severe substance use disorder is comorbid with the substance-induced ob- sessive-compulsive and related disorder, the 4th position character is ‘,"2 and the clinician should record “moderate [substance] use disorder” or “severe [substance] Use disorder," depending on the severity of the comorbid substance use disorder. If there is no comorbid substance use disorder (e.g., after a one—time heavy use of the substance), then the 4th position character is

1	on the severity of the comorbid substance use disorder. If there is no comorbid substance use disorder (e.g., after a one—time heavy use of the substance), then the 4th position character is “9,” and the clinician should record only the substance-induced ob- sessive-oompulsive and related disorder.

1	With use disorder, Without disorder, moderate use Amphetamine (or other 292.89 F15.188 F15.288 F15.988 Cocaine 292.89 F14.188 Fl 4.288 F14.988 Other (or unknown) substance 292.89 F19.188 F19.288 F19.988 Specify it (see Table 1 in the chapter “Su bstance-Related and Addictive Disorders” for di- agnoses associated with substance class): With onset during intoxication: If the criteria are met for intoxication with the sub- stance and the symptoms develop during intoxication. Witii onset during withd rawai: If criteria are met for withdrawal from the substance and the symptoms develop during, or shorﬂy after, withdrawal. Witli onset after medication use: Symptoms may appear either at initiation of medi- cation or after a modification or change in use.

1	Witli onset after medication use: Symptoms may appear either at initiation of medi- cation or after a modification or change in use. ICD-9-CM. The name of the substance/ medication—induced obsessive—compulsive and related disorderbegins With the specific substance (e.g., cocaine) that is presumed to be causing the obsessive—compulsive and related symptoms. The diagnostic code is selected from the table included in the criteria set, Which is based on the drug class. For substances that do not fit into any ofthe classes, the code for "other sub stance" should be used; and in cases in Which a substance isjudged to be an etiological factor but the specific class ofsub stance is unknown, the category "unknown substance" should be used.

1	The name ofthe disord er is follow ed by the specification ofonset (i.e., onset during in toxication, onset during Withdraw a1, With onset after med ication use). Unlike the record ing procedures for ICD—IO—CM, Which combine the substance—induced disorder and substance use disorder into a single code, for ICD—9—CM a separate diagnostic code is given for the substance use disorder. For example, in the case ofrepetitive behaviors oc curring during intoxication in a man With a severe cocaine use disorder, the diagno sis is 292.89 cocaine—induced obsessive—compulsive and related disorder, With onset during in toxication. An additional diagnosis of 304.20 severe cocaine use disorder is also given. When more than one substance is jud ged to play a significant role in the development of the obsessive—compulsive and related disorder, each should be listed separately.

1	When more than one substance is jud ged to play a significant role in the development of the obsessive—compulsive and related disorder, each should be listed separately. |CD-10-CM. The name ofthe substance/ medication—induced obsessive—compulsive and re lated disorder begins With the specific substance (e.g., cocaine) that is presumed to be causing the obsessive—compulsive and related symptoms. The diagnostic code is selected from the ta ble included in the criteria set, Which is based on the drug class and presence or absence ofa comorbid substance use disorder. For substances that do not fit into any of the classes, the code for "other substance" With no comorbid substance use should be used; and in cases in Which a sub stance isjudged to be an etiological factorbut the specific class ofsub stance is un known, the category "unknown substance" With no comorbid substance use should be used.

1	When recording the name of the disorder, the comorbid substance use disorder (if any) is listed first, followed by the word "with," followed by the name of the substance-induced ob- sessive-compulsive and related disorder, followed by the specification of onset (i.e., onset dur- ing intoxication, onset during withdrawal, with onset after medication use). For example, in the case of repetitive behaviors occurring during intoxication in a man with a severe cocaine use disorder, the diagnosis is F14.288 severe cocaine use disorder with cocaine-induced obses- sive—compulsive and related disorder, with onset during intoxication. A separate diagnosis of the comorbid severe cocaine use disorder is not given. If the substance-induced obsessive- compulsive and related disorder occurs without a comorbid substance use disorder (e.g., after a one-time heavy use of the substance), no accompanying substance use disorder is noted (e.g.,

1	F15.988 amphetamine—induced obsessive—compulsive and related disorder, with onset during intoxication). When more than one substance is judged to play a significant role in the devel- opment of the obsessive—compulsive and related disorder, each should be listed separately. The essential features of substance/medication-induced obsessive-compulsive and related disorder are prominent symptoms of an obsessive—compulsive and related disorder (Criterion

1	A) that are judged to be attributable to the effects of a substance (e.g., drug of abuse, medica- tion). The obsessive-compulsive and related disorder symptoms must have developed during or soon after substance intoxication or withdrawal or after exposure to a medication or toxin, and the substance/medication must be capable of producing the symptoms (Criterion B). Sub- stance/medication—induced obsessive-compulsive and related disorder due to a prescribed treatment for a mental disorder or general medical condition must have its onset while the in- dividual is receiving the medication. Once the treatment is discontinued, the obsessive-com- pulsive and related disorder symptoms will usually improve or remit within days to several weeks to 1 month (depending on the half-life of the substance/medication). The diagnosis of given if onset of the obsessive-compulsive and related disorder symptoms precedes the sub- stance intoxication or medication use, or if the symptoms persist for a

1	The diagnosis of given if onset of the obsessive-compulsive and related disorder symptoms precedes the sub- stance intoxication or medication use, or if the symptoms persist for a substantial period of time, usually longer than 1 month, from the time of severe intoxication or withdrawal. If the obsessive—compulsive and related disorder symptoms persist for a substantial period of time, other causes for the symptoms should be considered. The substance/ medication-induced ob- sessive-compulsive and related disorder diagnosis should be made in addition to a diagnosis of substance intoxication only when the symptoms in Criterion A predominate in the clinical picture and are sufficiently severe to warrant independent clinical attention

1	Obsessions, compulsions, hair pulling, skin picking, or other body-focused repetitive be- haviors can occur in association with intoxication with the following classes of substances: stimulants (including cocaine) and other (or unknown) substances. Heavy metals and tox- ins may also cause obsessive-compulsive and related disorder symptoms. Laboratory as- sessments (e.g., urine toxicology) may be useful to measure substance intoxication as part of an assessment for obsessive-compulsive and related disorders. In the general population, the very limited data that are available indicate that substance- induced obsessive-compulsive and related disorder is very rare.

1	In the general population, the very limited data that are available indicate that substance- induced obsessive-compulsive and related disorder is very rare. Substance intoxication. Obsessive-compulsive and related disorder symptoms may oc~ cur in substance intoxication. The diagnosis of the substance-specific intoxication will usu- ally suffice to categorize the symptom presentation. A diagnosis of an obsessive-compulsive and related disorder should be made in addition to substance intoxication when the symp- toms are judged to be in excess of those usually associated with intoxication and are suf- ficiently severe to warrant independent clinical attention.

1	Obsessive-compulsive and related disorder (i.e., not induced by a substance). Sub- stance/ medication-induced obsessive-compulsive and related disorder is judged to be etiologically related to the substance/medication. Substance/medication-induced obses- sive-compulsive and related disorder is distinguished from a primary obsessive-compul- sive and related disorder by considering the onset, course, and other factors with respect to substances/ medications. For drugs of abuse, there must be evidence from the history, physical examination, or laboratory findings for use or intoxication. Substance/medica— tion-induced obsessive-compulsive and related disorder arises only in association with in- toxication, whereas a primary obsessive-compulsive and related disorder may precede the onset of substance/medication use. The presence of features that are atypical of a primary obsessive-compulsive and related disorder, such as atypical age at onset of symptoms, may suggest a substance-induced

1	substance/medication use. The presence of features that are atypical of a primary obsessive-compulsive and related disorder, such as atypical age at onset of symptoms, may suggest a substance-induced etiology. A primary obsessive-compulsive and related disorder diagnosis is warranted if the symptoms persist for a substantial period of time (about 1 month or longer) after the end of the substance intoxication or the individual has a history of an obsessive-compulsive and related disorder.

1	Obsessive-compulsive and related disorder due to another medical condition. If the obsessive-compulsive and related disorder symptoms are attributable to another medical condition (i.e., rather than to the medication taken for the other medical condition), obses- sive-compulsive and related disorder due to another medical condition should be diag- nosed. The history often provides the basis for judgment. At times, a change in the treatment for the other medical condition (e.g., medication substitution or discontinua- tion) may be needed to determine whether or not the medication is the causative agent (in which case the symptoms may be better explained by substance/medication-induced ob- sessive-compulsive and related disorder). If the disturbance is attributable to both another medical condition and substance use, both diagnoses (i.e., obsessive-compulsive and related disorder due to another medical condition and substance/medication-induced obsessive- compulsive and related

1	condition and substance use, both diagnoses (i.e., obsessive-compulsive and related disorder due to another medical condition and substance/medication-induced obsessive- compulsive and related disorder) may be given. When there is insufficient evidence to de- termine whether the symptoms are attributable to either a substance/medication or an- other medical condition or are primary (i.e., attributable to neither a substance/medication nor another medical condition), a diagnosis of other specified or unspecified obsessive- compulsive and related disorder would be indicated.

1	Delirium. If obsessive-compulsive and related disorder symptoms occur exclusively during the course of delirium, they are considered to be an associated feature of the delir- ium and are not diagnosed separately. Due to Another Medical Condition Diagnostic Criteria 294.8 (F06.8) A. Obsessions, compulsions. preoccupations with appearance, hoarding, skin picking, hair pulling, other body-focused repetitive behaviors, or other symptoms characteristic of obsessive-compulsive and related disorder predominate in the clinical picture. B. There is evidence from the history, physical examination, or laboratory findings that the disturbance is the direct pathophysiological consequence of another medical condition. C. The disturbance is not better explained by another mental disorder. Obsessive-Compulsive and Related Disorder Due to Another Medical Condition 261 D. The disturbance does not occur exclusively during the course of a delirium.

1	Obsessive-Compulsive and Related Disorder Due to Another Medical Condition 261 D. The disturbance does not occur exclusively during the course of a delirium. E. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. Specify it: With obsessive-compulsive disorder—like symptoms: It obsessive-compulsive dis- order—like symptoms predominate in the clinical presentation. With appearance preoccupations: If preoccupation with perceived appearance de- fects or flaws predominates in the clinical presentation. With hoarding symptoms: It hoarding predominates in the clinical presentation. With hair-pulling symptoms: If hair pulling predominates in the clinical presentation. With skin-picking symptoms: It skin picking predominates in the clinical presenta- tion.

1	With hair-pulling symptoms: If hair pulling predominates in the clinical presentation. With skin-picking symptoms: It skin picking predominates in the clinical presenta- tion. Coding note: Include the name of the other medical condition in the name of the mental disorder (e.g., 294.8 [F06.8] obsessive-compulsive and related disorder due to cerebral infarction). The other medical condition should be coded and listed separately immediately before the obsessive-compulsive and related disorder due to the medical condition (e.g., 438.89 [l69.398] cerebral infarction; 294.8 [F06.8] obsessive-compulsive and related dis- order due to cerebral infarction).

1	The essential feature of obsessive-compulsive and related disorder due to another medical condition is clinically significant obsessive-compulsive and related symptoms that are judged to be best explained as the direct pathophysiological consequence of another med— ical condition. Symptoms can include prominent obsessions, compulsions, preoccu- pations with appearance, hoarding, hair pulling, skin picking, or other body-focused repetitive behaviors (Criterion A). The judgment that the symptoms are best explained by the associated medical condition must be based on evidence from the history, physical ex- amination, or laboratory findings (Criterion B). Additionally, it must be judged that the symptoms are not better explained by another mental disorder (Criterion C). The diagno- sis is not made if the obsessive-compulsive and related symptoms occur only during the course of a delirium (Criterion D). The obsessive—compulsive and related symptoms must cause clinically significant

1	is not made if the obsessive-compulsive and related symptoms occur only during the course of a delirium (Criterion D). The obsessive—compulsive and related symptoms must cause clinically significant distress or impairment in social, occupational, or other impor- tant areas of functioning (Criterion E).

1	In determining whether the obsessive-compulsive and related symptoms are attribut— able to another medical condition, a relevant medical condition must be present. Further- more, it must be established that obsessive-compulsive and related symptoms can be etiologically related to the medical condition through a pathophysiological mechanism and that this best explains the symptoms in the individual. Although there are no infallible guidelines for determining whether the relationship between the obsessive-compulsive and related symptoms and the medical condition is etiological, considerations that may provide some guidance in making this diagnosis include the presence of a clear temporal association between the onset, exacerbation, or remission of the medical condition and the obsessive-compulsive and related symptoms; the presence of features that are atypical of a primary obsessive—compulsive and related disorder (e.g., atypical age at onset or course); and evidence in the literature

1	and related symptoms; the presence of features that are atypical of a primary obsessive—compulsive and related disorder (e.g., atypical age at onset or course); and evidence in the literature that a known physiological mechanism (e.g., striatal dam- age) causes obsessive-compulsive and related symptoms. In addition, the disturbance cannot be better explained by a primary obsessive-compulsive and related disorder, a sub- stance/medication-induced obsessive-compulsive and related disorder, or another men- tal disorder.

1	There is some controversy about whether obsessive-compulsive and related disorders can be attributed to Group A streptococcal infection. Sydenham’s chorea is the neurolog- ical manifestation of rheumatic fever, which is in turn due to Group A streptococcal in- fection. Sydenham’s chorea is characterized by a combination of motor and nonmotor features. Nonmotor features include obsessions, compulsions, attention deficit, and emo- tional lability. Although individuals with Sydenham’s chorea may present with non- neuropsychiatric features of acute rheumatic fever, such as carditis and arthritis, they may present with obsessive-compulsive disorder—like symptoms; such individuals should be diagnosed with obsessive-compulsive and related disorder due to another medical condition.

1	Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infec- tions (PANDAS) has been identified as another post-infectious autoimmune disorder characterized by the sudden onset of obsessions, compulsions, and/ or tics accompanied by a variety of acute neuropsychiatric symptoms in the absence of chorea, carditis, or ar- thritis, after Group A streptococcal infection. Although there is a body of evidence that supports the existence of PANDAS, it remains a controversial diagnosis. Given this ongo- ing controversy, the description of PANDAS has been modified to eliminate etiological factors and to designate an expanded clinical entity: pediatric acute-onset neuropsychiat- ric syndrome (PANS) or idiopathic childhood acute neuropsychiatric symptoms (CANS), which deserves further study.

1	A number of other medical disorders are known to include obsessive-compulsive and re- lated symptoms as a manifestation. Examples include disorders leading to striatal dam- age, such as cerebral infarction. The development and course of obsessive-compulsive and related disorder due to another medical condition generally follows the course of the underlying illness. Laboratory assessments and/ or medical examinations are necessary to confirm the diag- nosis of another medical condition.

1	Laboratory assessments and/ or medical examinations are necessary to confirm the diag- nosis of another medical condition. Delirium. A separate diagnosis of obsessive-compulsive and related disorder due to an- other medical condition is not given if the disturbance occurs exclusively during the course of a delirium. However, a diagnosis of obsessive—compulsive and related disorder due to another medical condition may be given in addition to a diagnosis of major neuro- cognitive disorder (dementia) if the etiology of the obsessive-compulsive symptoms is judged to be a physiological consequence of the pathological process causing the dementia and if obsessive-compulsive symptoms are a prominent part of the clinical presentation.

1	Mixed presentation of symptoms (e.g., mood and obsessive-compulsive and related disorder symptoms). If the presentation includes a mix of different types of symptoms, the specific mental disorder due to another medical condition depends on which symp- toms predominate in the clinical picture.

1	Substance/medication-induced obsessive-compulsive and related disorders. If there is evidence of recent or prolonged substance use (including medications with psychoac— tive effects), withdrawal from a substance, or exposure to a toxin, a substance/ medication— induced obsessive-compulsive and related disorder should be considered. When a sub- stance/medication-induced obsessive—compulsive and related disorder is being diag- nosed in relation to drugs of abuse, it may be useful to obtain a urine or blood drug screen or other appropriate laboratory evaluation. Symptoms that occur during or shortly after (i.e., within 4 weeks of) substance intoxication or withdrawal or after medication use may be especially indicative of a substance/medication—induced obsessive-compulsive and re— lated disorder, depending on the type, duration, or amount of the substance used.

1	Obsessive-compulsive and related disorders (primary). Obsessive-compulsive and re- lated disorder due to another medical condition should be distinguished from a primary obsessive-compulsive and related disorder. In primary mental disorders, no specific and direct causative physiological mechanisms associated with a medical condition can be demonstrated. Late age at onset or atypical symptoms suggest the need for a thorough as- sessment to rule out the diagnosis of obsessive-compulsive and related disorder due to an- other medical condition. Illness anxiety disorder. Illness anxiety disorder is characterized by a preoccupation with having or acquiring a serious illness. In the case of illness anxiety disorder, individuals may or may not have diagnosed medical conditions. Associated feature of another mental disorder. Obsessive-compulsive and related symp- toms may be an associated feature of another mental disorder (e.g., schizophrenia, an- orexia nervosa).

1	Associated feature of another mental disorder. Obsessive-compulsive and related symp- toms may be an associated feature of another mental disorder (e.g., schizophrenia, an- orexia nervosa). compulsive and related disorder. These diagnoses are given if it is unclear whether the obsessive-compulsive and related symptoms are primary, substance-induced, or due to another medical condition. 300.3 (F42)

1	300.3 (F42) This category applies to presentations in which symptoms characteristic of an obsessive- social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the obsessive-compulsive and related disorders diagnostic class. The other specified obsessive-compulsive and related disorder category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for any specific obsessive-compulsive and re- lated disorder. This is done by recording “other specified obsessive-compulsive and relat- ed disorder” followed by the specific reason (e.g.. “body-tocused repetitive behavior disorder”).

1	Examples of presentations that can be specified using the "other specified" designation include the following: 1. Body dysmorphic—like disorder with actual flaws: This is similar to body dysmor- phic disorder except that the defects or flaws in physical appearance are clearly ob- servable by others (i.e., they are more noticeable than “slight"). In such cases, the preoccupation with these flaws is clearly excessive and causes significant impairment or distress. 2. Body dysmorphic—like disorder without repetitive behaviors: Presentations that meet body dysmorphic disorder except that the individual has not performed repetitive behaviors or mental acts in response to the appearance concerns.

1	3. Body-focused repetitive behavior disorder: This is characterized by recurrent body- tocused repetitive behaviors (e.g., nail biting, lip biting, cheek chewing) and repeated attempts to decrease or stop the behaviors. These symptoms cause clinically significant distress or impairment in social, occupational, or other important areas of functioning and are not better explained by trichotillomania (hair-pulling disorder), excoriation (skin- picking) disorder. stereotypic movement disorder, or nonsuicidal self-injury.

1	4. Obsessional iealousy: This is characterized by nondelusional preoccupation with a partner’s perceived infidelity. The preoccupations may lead to repetitive behaviors or mental acts in response to the infidelity concerns; they cause clinically significant dis- tress or impairment in social, occupational, or other important areas of functioning; and they are not better explained by another mental disorder such as delusional disorder, jealous type, or paranoid personality disorder. 5. Shubo-kyofu: A variant of taijin kyofusho (see ”Glossary of Cultural Concepts of Dis- tress" in the Appendix) that is similar to body dysmorphic disorder and is characterized by excessive fear of having a bodily deformity. 6. Koro: Related to dhat syndrome (see “Glossary of Cultural Concepts of Distress” in the Appendix), an episode of sudden and intense anxiety that the penis (or the vulva and nipples in females) will recede into the body, possibly leading to death.

1	7. Jikoshu-kyofu: A variant of taijin kyofusho (see “Glossary of Cultural Concepts of Dis- tress” in the Appendix) characterized by fear of having an offensive body odor (also termed olfactory reference syndrome). 300.3 (F42) This category applies to presentations in which symptoms characteristic of an obsessive- social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the obsessive-compulsive and related disorders diagnostic class. The unspecified obsessive-compulsive and related disorder category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for a specific obsessive-compulsive and related disorder, and includes presen- tations in which there is insufficient information to make a more specific diagnosis (e.g., in emergency room settings).

1	a traumatic or stressful event is listed explicitly as a diagnostic criterion. These include reactive attachment disorder, disinhibited social engagement disorder, posttraumatic stress disor— der (PTSD), acute stress disorder, and adjustment disorders. Placement of this chapter reﬂects the close relationship between these diagnoses and disorders in the surrounding chapters on anxiety disorders, obsessive-compulsive and related disorders, and dissociative disorders.

1	Psychological distress following exposure to a traumatic or stressful event is quite vari- able. In some cases, symptoms can be well understood within an anxiety- or fear-based context. It is clear, however, that many individuals who have been exposed to a traumatic or stressful event exhibit a phenotype in which, rather than anxiety- or fear-based symp- toms, the most prominent clinical characteristics are anhedonic and dysphoric symptoms, externalizing angry and aggressive symptoms, or dissociative symptoms. Because of these variable expressions of clinical distress following exposure to catastrophic or aversive events, the aforementioned disorders have been grouped under a separate category: trauma- and stressor—related disorders. Furthermore, it is not uncommon for the clinical pic- ture to include some combination of the above symptoms (with or without anxiety- or fear-based symptoms). Such a heterogeneous picture has long been recognized in adjust- ment disorders, as well.

1	ture to include some combination of the above symptoms (with or without anxiety- or fear-based symptoms). Such a heterogeneous picture has long been recognized in adjust- ment disorders, as well. Social neglect—that is, the absence of adequate caregiving during childhood—is a diagnostic requirement of both reactive attachment disorder and disin- hibited social engagement disorder. Although the two disorders share a common etiology, the former is expressed as an internalizing disorder with depressive symptoms and with- drawn behavior, while the latter is marked by disinhibition and externalizing behavior.

1	Diagnostic Criteria 313.89 (F94.1) A. A consistent pattern of inhibited, emotionally withdrawn behavior toward adult caregiv- ers, manifested by both of the following: 1. The child rarely or minimally seeks comfort when distressed. 2. The child rarely or minimally responds to comfort when distressed. B. A persistent social and emotional disturbance characterized by at least two of the following: 1. Minimal social and emotional responsiveness to others. 2. Limited positive affect. 3. Episodes of unexplained irritability, sadness, or fearfulness that are evident even during nonthreatening interactions with adult caregivers. C. The child has experienced a pattern of extremes of insufficient care as evidenced by at least one of the following: 1. Social neglect or deprivation in the form of persistent lack of having basic emotional needs for comfort, stimulation, and affection met by caregiving adults.

1	2. Repeated changes of primary caregivers that limit opportunities to form stable at- tachments (e.g., frequent changes in foster care). 3. Rearing in unusual settings that severely limit opportunities to form selective at- tachments (e.g., institutions with high chiId-to-caregiver ratios). D. The care in Criterion C is presumed to be responsible for the disturbed behavior in Cri- terion A (e.g., the disturbances in Criterion A began following the lack of adequate care in Criterion C). E. The criteria are not met for autism spectrum disorder. F. The disturbance is evident before age 5 years. G. The child has a developmental age of at least 9 months. Specify it: Persistent: The disorder has been present for more than 12 months. Specify current severity: Reactive attachment disorder is specified as severe when a child exhibits all symp- toms of the disorder, with each symptom manifesting at relatively high levels.

1	Reactive attachment disorder of infancy or early childhood is characterized by a pattern of markedly disturbed and developmentally inappropriate attachment behaviors, in which a child rarely or minimally turns preferentially to an attachment figure for comfort, support, protection, and nurturance. The essential feature is absent or grossly underdeveloped at- tachment between the child and putative caregiving adults. Children with reactive attach- ment disorder are believed to have the capacity to form selective attachments. However, because of limited opportunities during early development, they fail to show the behavioral manifestations of selective attachments. That is, when distressed, they show no consistent effort to obtain comfort, support, nurturance, or protection from caregivers. Furthermore, when distressed, children with this disorder do not respond more than minimally to com- forting efforts of caregivers. Thus, the disorder is associated with the absence of expected

1	Furthermore, when distressed, children with this disorder do not respond more than minimally to com- forting efforts of caregivers. Thus, the disorder is associated with the absence of expected comfort seeking and response to comforting behaviors. As such, children with reactive attachment disorder show diminished or absent expression of positive emotions during routine interactions with caregivers. In addition, their emotion regulation capacity is com- promised, and they display episodes of negative emotions of fear, sadness, or irritability that are not readily explained. A diagnosis of reactive attachment disorder should not be made in children who are developmentally unable to form selective attachments. For this reason, the child must have a developmental age of at least 9 months.

1	Because of the shared etiological association with social neglect, reactive attachment dis- order often co-occurs with developmental delays, especially in delays in cognition and language. Other associated features include stereotypies and other signs of severe neglect (e.g., malnutrition or signs of poor care). The prevalence of reactive attachment disorder is unknown, but the disorder is seen rela- tively rarely in clinical settings. The disorder has been found in young children exposed to severe neglect before being placed in foster care or raised in institutions. However, even in populations of severely neglected children, the disorder is uncommon, occurring in less than 10% of such children.

1	Conditions of social neglect are often present in the first months of life in children diag- nosed with reactive attachment disorder, even before the disorder is diagnosed. The clin- ical features of the disorder manifest in a similar fashion between the ages of 9 months and 5 years. That is, signs of absent-to-minimal attachment behaviors and associated emotion- ally aberrant behaviors are evident in children throughout this age range, although differ- ing cognitive and motor abilities may affect how these behaviors are expressed. Without remediation and recovery through normative caregiving environments, it appears that signs of the disorder may persist, at least for several years. It is unclear whether reactive attachment disorder occurs in older children and, if so, how it differs from its presentation in young children. Because of this, the diagnosis should be made with caution in children older than 5 years.

1	Environmental. Serious social neglect is a diagnostic requirement for reactive attach- ment disorder and is also the only known risk factor for the disorder. However, the ma- jority of severely neglected children do not develop the disorder. Prognosis appears to depend on the quality of the caregiving environment following serious neglect. cultures around the world. However, caution should be exercised in making the diagnosis of reactive attachment disorder in cultures in which attachment has not been studied. Reactive attachment disorder significantly impairs young children’s abilities to relate in- terpersonally to adults or peers and is associated with functional impairment across many domains of early childhood. Autism spectrum disorder. Aberrant social behaviors manifest in young children with reactive attachment disorder, but they also are key features of autism spectrum disorder.

1	Autism spectrum disorder. Aberrant social behaviors manifest in young children with reactive attachment disorder, but they also are key features of autism spectrum disorder. Specifically, young children with either condition can manifest dampened expression of positive emotions, cognitive and language delays, and impairments in social reciprocity.

1	As a result, reactive attachment disorder must be differentiated from autism spectrum dis- order. These two disorders can be distinguished based on differential histories of neglect and on the presence of restricted interests or ritualized behaviors, specific deficit in social communication, and selective attachment behaviors. Children with reactive attachment disorder have experienced a history of severe social neglect, although it is not always pos— sible to obtain detailed histories about the precise nature of their experiences, especially in initial evaluations. Children with autistic spectrum disorder will only rarely have a history of social neglect. The restricted interests and repetitive behaviors characteristic of autism spectrum disorder are not a feature of reactive attachment disorder. These clinical features manifest as excessive adherence to rituals and routines; restricted, fixated interests; and unusual sensory reactions. However, it is important to note that children

1	disorder. These clinical features manifest as excessive adherence to rituals and routines; restricted, fixated interests; and unusual sensory reactions. However, it is important to note that children with either con- dition can exhibit stereotypic behaviors such as rocking or ﬂapping. Children with either disorder also may exhibit a range of intellectual functioning, but only children with autis- tic spectrum disorder exhibit selective impairments in social communicative behaviors, such as intentional communication (i.e., impairment in communication that is deliberate, goal-directed, and aimed at inﬂuencing the behavior of the recipient). Children with reac— tive attachment disorder show social communicative functioning comparable to their overall level of intellectual functioning. Finally, children with autistic spectrum disorder regularly show attachment behavior typical for their developmental level. In contrast, children with reactive attachment disorder do so only rarely or

1	children with autistic spectrum disorder regularly show attachment behavior typical for their developmental level. In contrast, children with reactive attachment disorder do so only rarely or inconsistently, if at all.

1	Intellectual disability (intellectual developmental disorder). Developmental delays of- ten accompany reactive attachment disorder, but they should not be confused with the disorder. Children with intellectual disability should exhibit social and emotional skills comparable to their cognitive skills and do not demonstrate the profound reduction in positive affect and emotion regulation difficulties evident in children with reactive attach- ment disorder. In addition, developmentally delayed children who have reached a cogni- tive age of 7—9 months should demonstrate selective attachments regardless of their chronological age. In contrast, children with reactive attachment disorder show lack of preferred attachment despite having attained a developmental age of at least 9 months.

1	Depressive disorders. Depression in young children is also associated with reductions in positive affect. There is limited evidence, however, to suggest that children with depres— sive disorders have impairments in attachment. That is, young children who have been di- agnosed with depressive disorders still should seek and respond to comforting efforts by caregivers. Conditions associated with neglect, including cognitive delays, language delays, and ste- reotypies, often co-occur with reactive attachment disorder. Medical conditions, such as severe malnutrition, may accompany signs of the disorder. Depressive symptoms also may co-occur with reactive attachment disorder. Diagnostic Criteria 313.89 (F94.2) A. A pattern of behavior in which a child actively approaches and interacts with unfamiliar adults and exhibits at least two of the following: 1. Reduced or absent reticence in approaching and interacting with untamiliar adults.

1	2. Overly familiar verbal or physical behavior (that is not consistent with culturally sanctioned and with age-appropriate social boundaries). 3. Diminished or absent checking back with adult caregiver after venturing away, even in unfamiliar settings. 4. Willingness to go off with an unfamiliar adult with minimal or no hesitation. B. The behaviors in Criterion A are not limited to impulsivity (as in attention-deficit/hyper- activity disorder) but include socially disinhiblted behavior. C. The child has experienced a pattern of extremes of insufficient care as evidenced by at least one of the following: 1. Social neglect or deprivation in the form of persistent lack of having basic emotional needs for comfort, stimulation, and affection met by caregiving adults. 2. Repeated changes of primary caregivers that limit opportunities to form stable at- tachments (e.g., frequent changes in foster care).

1	2. Repeated changes of primary caregivers that limit opportunities to form stable at- tachments (e.g., frequent changes in foster care). 3. Hearing in unusual settings that severely limit opportunities to form selective at- tachments (e.g., institutions with high chiId-to-caregiver ratios). D. The care in Criterion C is presumed to be responsible forthe disturbed behavior in Cri- terion A (e.g.} the disturbances in Criterion A began following the pathogenic care in Criterion C). E. The child has a developmental age of at least 9 months. Specify if: Persistent: The disorder has been present for more than 12 months. Specify current severity: Disinhibited social engagement disorder is specified as severe when the child exhibits all symptoms of the disorder. with each symptom manifesting at relatively high levels.

1	Disinhibited social engagement disorder is specified as severe when the child exhibits all symptoms of the disorder. with each symptom manifesting at relatively high levels. The essential feature of disinhibited social engagement disorder is a pattern of behavior that involves culturally inappropriate, overly familiar behavior with relative strangers (Criterion A). This overly familiar behavior violates the social boundaries of the culture. A diagnosis of disinhibited social engagement disorder should not be made before children are developmentally able to form selective attachments. For this reason, the child must have a developmental age of at least 9 months.

1	Because of the shared etiological association with social neglect, disinhibited social en- gagement disorder may co-occur with developmental delays, especially cognitive and lan- guage delays, stereotypies, and other signs of severe neglect, such as malnutrition or poor care. However, signs of the disorder often persist even after these other signs of neglect are no longer present. Therefore, it is not uncommon for children with the disorder to present with no current signs of neglect. Moreover, the condition can present in children who show no signs of disordered attachment. Thus, disinhibited social engagement disorder may be seen in children with a history of neglect who lack attachments or whose attach- ments to their caregivers range from disturbed to secure.

1	The prevalence of disinhibited social attachment disorder is unknown. Nevertheless, the disorder appears to be rare, occurring in a minority of children, even those who have been severely neglected and subsequently placed in foster care or raised in institutions. In such high-risk populations, the condition occurs in only about 20% of children. The condition is seen rarely in other clinical settings.

1	Conditions of social neglect are often present in the first months of life in children diag- nosed with disinhibited social engagement disorder, even before the disorder is diag- nosed. However, there is no evidence that neglect beginning after age 2 years is associated with manifestations of the disorder. If neglect occurs early and signs of the disorder appear, clinical features of the disorder are moderately stable over time, particularly if conditions of neglect persist. Indiscriminate social behavior and lack of reticence with un- familiar adults in toddlerhood are accompanied by attention-seeking behaviors in pre- schoolers. When the disorder persists into middle childhood, clinical features manifest as verbal and physical overfamiliarity as well as inauthentic expression of emotions. These signs appear particularly apparent when the child interacts with adults. Peer relationships are most affected in adolescence, with both indiscriminate behavior and conﬂicts appar- ent. The

1	These signs appear particularly apparent when the child interacts with adults. Peer relationships are most affected in adolescence, with both indiscriminate behavior and conﬂicts appar- ent. The disorder has not been described in adults.

1	Disinhibited social engagement disorder has been described from the second year of life through adolescence. There are some differences in manifestations of the disorder from early childhood through adolescence. At the youngest ages, across many cultures, children show reticence when interacting with strangers. Young children with the disorder fail to show reticence to approach, engage with, and even accompany adults. In preschool children, verbal and social intrusiveness appear most prominent, often accompanied by attention-seeking behavior. Verbal and physical overfamiliarity continue through middle childhood, accompanied by inauthentic expressions of emotion. In adolescence, indis- criminate behavior extends to peers. Relative to healthy adolescents, adolescents with the disorder have more "superficial” peer relationships and more peer conﬂicts. Adult man- ifestations of the disorder are unknown.

1	Environmental. Serious social neglect is a diagnostic requirement for disinhibited social engagement disorder and is also the only known risk factor for the disorder. However, the majority of severely neglected children do not develop the disorder. Neurobiological vul- nerability may differentiate neglected children who do and do not develop the disorder. However, no clear link with any specific neurobiological factors has been established. The 2 years. Prognosis is only modestly associated with quality of the caregiving environment following serious neglect. In many cases, the disorder persists, even in children whose caregiving environment becomes markedly improved. Course modifiers. Caregiving quality seems to moderate the course of disinhibited so- cial engagement disorder. Nevertheless, even after placement in normative caregiving environments, some children show persistent signs of the disorder, at least through ado- lescence. relate interpersonally to adults and peers.

1	relate interpersonally to adults and peers. Attention-deficit/hyperactivity disorder. Because of social impulsivity that sometimes accompanies attention-deficit/hyperactivity disorder (ADHD), it is necessary to differ- entiate the two disorders. Children with disinhibited social engagement disorder may be distinguished from those with ADHD because the former do not show difficulties with at- tention or hyperactivity. Limited research has examined the issue of disorders comorbid with disinhibited social engagement disorder. Conditions associated with neglect, including cognitive delays, language delays, and stereotypies, may co-occur with disinhibited social engagement dis- order. In addition, children may be diagnosed with ADHD and disinhibited social engage- ment disorder concurrently. Diagnostic Criteria 309.81 (F43.10) Note: The following criteria apply to adults, adolescents, and children older than 6 years. For children 6 years and younger, see corresponding criteria below.

1	Diagnostic Criteria 309.81 (F43.10) Note: The following criteria apply to adults, adolescents, and children older than 6 years. For children 6 years and younger, see corresponding criteria below. A. Exposure to actual or threatened death, serious injury, or sexual violence in one (or more) of the following ways: 1. 2. 3. Directly experiencing the traumatic event(s). Witnessing, in person, the event(s) as it occurred to others. Learning that the traumatic event(s) occurred to a close family member or close friend. In cases of actual or threatened death of a family member or friend, the event(s) must have been violent or accidental. Experiencing repeated or extreme exposure to aversive details of the traumatic event(s) (e.g., first responders collecting human remains; police officers repeatedly exposed to details of child abuse). Note: Criterion A4 does not apply to exposure through electronic media, television, movies, or pictures, unless this exposure is work related.

1	Note: Criterion A4 does not apply to exposure through electronic media, television, movies, or pictures, unless this exposure is work related. B. Presence of one (or more) of the following intrusion symptoms associated with the traumatic event(s), beginning after the traumatic event(s) occurred: 1. Recurrent, involuntary, and intrusive distressing memories of the traumatic event(s). Note: In children older than 6 years, repetitive play may occur in which themes or aspects of the traumatic event(s) are expressed. . Recurrent distressing dreams in which the content and/or affect of the dream are related to the traumatic event(s). Note: In children, there may be frightening dreams without recognizable content. Dissociative reactions (e.g., flashbacks) in which the individual feels or acts as if the traumatic event(s) were recurring. (Such reactions may occur on a continuum, with the most extreme expression being a complete loss of awareness of present surroundings.)

1	Note: In children, trauma-specitic reenactment may occur in play. Intense or prolonged psychological distress at exposure to internal or external cues that symbolize or resemble an aspect of the traumatic event(s). Marked physiological reactions to internal or external cues that symbolize or re- semble an aspect of the traumatic event(s). C. Persistent avoidance of stimuli associated with the traumatic event(s), beginning after the traumatic event(s) occurred, as evidenced by one or both of the following: 1. 2. Avoidance of or efforts to avoid distressing memories, thoughts, or feelings about or closely associated with the traumatic event(s). Avoidance of or efforts to avoid external reminders (people, places, conversations, activities, objects, situations) that arouse distressing memories, thoughts, or feel- ings about or closely associated with the traumatic event(s).

1	D. Negative alterations in cognitions and mood associated with the traumatic event(s), beginning or worsening after the traumatic event(s) occurred, as evidenced by two (or more) of the following: 1. Inability to remember an important aspect of the traumatic event(s) (typically due to dis- sociative amnesia and not to other factors such as head injury. alcohol, or drugs). G. H. 2. Persistent and exaggerated negative beliefs or expectations about oneself, others, or the world (e.g., “I am bad,” "No one can be trusted," “The world is completely dangerous," “My whole nervous system is permanently ruined"). Persistent, distorted cognitions about the cause or consequences of the traumatic event(s) that lead the individual to blame himself/herself or others. Persistent negative emotional state (e.g., fear, horror, anger, guilt, or shame). Markedly diminished interest or participation in significant activities. Feelings of detachment or estrangement from others.

1	Markedly diminished interest or participation in significant activities. Feelings of detachment or estrangement from others. Persistent inability to experience positive emotions (e.g., inability to experience happiness, satisfaction, or loving feelings). .0 N991? . Marked alterations in arousal and reactivity associated with the traumatic event(s). be- ginning or worsening after the traumatic event(s) occurred, as evidenced by two (or more) of the following: 1. Irritable behavior and angry outbursts (with little or no provocation) typically ex- pressed as verbal or physical aggression toward people or objects. Reckless or seIf-destructive behavior. Hypervigilance. Exaggerated startle response. Problems with concentration. Sleep disturbance (e.g., difficulty falling or staying asleep or restless sleep). Duration of the disturbance (Criteria B, C, D, and E) is more than 1 month.

1	Problems with concentration. Sleep disturbance (e.g., difficulty falling or staying asleep or restless sleep). Duration of the disturbance (Criteria B, C, D, and E) is more than 1 month. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. The disturbance is not attributable to the physiological effects of a substance (e.g., medication, alcohol) or another medical condition. Specify whether:

1	The disturbance is not attributable to the physiological effects of a substance (e.g., medication, alcohol) or another medical condition. Specify whether: With dissociative symptoms: The individual's symptoms meet the criteria for post- traumatic stress disorder, and in addition. in response to the stressor, the individual ex- periences persistent or recurrent symptoms of either of the following: 1. Depersonalization: Persistent or recurrent experiences of feeling detached from, and as if one were an outside observer of, one‘s mental processes or body (e.g., feeling as though one were in a dream; feeling a sense of unreality of self or body or of time moving slowly). 2. Derealization: Persistent or recurrent experiences of unreality of surroundings (e.g., the world around the individual is experienced as unreal, dreamlike. distant, or distorted).

1	2. Derealization: Persistent or recurrent experiences of unreality of surroundings (e.g., the world around the individual is experienced as unreal, dreamlike. distant, or distorted). Note: To use this subtype. the dissociative symptoms must not be attributable to the physiological effects of a substance (e.g., blackouts, behavior during alcohol intoxica- tion) or another medical condition (e.g., complex partial seizures). Specify it: With delayed expression: If the full diagnostic criteria are not met until at least 6 months after the event (although the onset and expression of some symptoms may be immediate). A. In children 6 years and younger, exposure to actual or threatened death, serious injury, or sexual violence in one (or more) of the following ways: 1. Directly experiencing the traumatic event(s). 2. Witnessing, in person. the event(s) as it occurred to others, especially primary care- givers.

1	2. Witnessing, in person. the event(s) as it occurred to others, especially primary care- givers. Note: Witnessing does not include events that are witnessed only in electronic me- dia, television, movies, or pictures. 3. Learning that the traumatic event(s) occurred to a parent or caregiving figure. B. Presence of one (or more) of the following intrusion symptoms associated with the traumatic event(s), beginning after the traumatic event(s) occurred: 1. Recurrent, involuntary, and intrusive distressing memories of the traumatic event(s). Note: Spontaneous and intrusive memories may not necessarily appear distress- ing and may be expressed as play reenactment. 2. Recurrent distressing dreams in which the content and/or affect of the dream are related to the traumatic event(s). Note: It may not be possible to ascertain that the frightening content is related to the traumatic event.

1	Note: It may not be possible to ascertain that the frightening content is related to the traumatic event. 3. Dissociative reactions (e.g., flashbacks) in which the child feels or acts as if the traumatic event(s) were recurring. (Such reactions may occur on a continuum, with the most extreme expression being a complete loss of awareness of present sur- roundings.) Such trauma-specitic reenactment may occur in play. 4. Intense or prolonged psychological distress at exposure to internal or external cues that symbolize or resemble an aspect of the traumatic event(s). 5. Marked physiological reactions to reminders of the traumatic event(s). C. One (or more) of the following symptoms, representing either persistent avoidance of stimuli associated with the traumatic event(s) or negative alterations in cognitions and mood associated with the traumatic event(s), must be present, beginning after the event(s) or worsening after the event(s):

1	Persistent Avoidance of Stimuli 1. Avoidance of or efforts to avoid activities, places, or physical reminders that arouse recollections of the traumatic event(s). 2. Avoidance of or efforts to avoid people, conversations, or interpersonal situations that arouse recollections of the traumatic event(s). 3. Substantially increased frequency of negative emotional states (e.g., fear, guilt, sadness, shame, contusion). 4. Markedly diminished interest or participation in significant activities, including con- striction of play. 5. Socially withdrawn behavior. 6. Persistent reduction in expression of positive emotions.

1	5. Socially withdrawn behavior. 6. Persistent reduction in expression of positive emotions. D. Alterations in arousal and reactivity associated with the traumatic event(s), beginning or worsening after the traumatic event(s) occurred. as evidenced by two (or more) of the following: 1. Irritable behavior and angry outbursts (with little or no provocation) typically ex- pressed as verbal or physical aggression toward people or objects (including ex- treme temper tantrums). Hypervigilance. Exaggerated startle response. Problems with concentration. 5. Sleep disturbance (e.g., difficulty falling or staying asleep or restless sleep). E. The duration of the disturbance is more than 1 month. :‘itFt’i‘J F. The disturbance causes clinically significant distress or impairment in relationships with parents, siblings, peers, or other caregivers or with school behavior.

1	:‘itFt’i‘J F. The disturbance causes clinically significant distress or impairment in relationships with parents, siblings, peers, or other caregivers or with school behavior. G. The disturbance is not attributable to the physiological effects of a substance (e.g., medication or alcohol) or another medical condition. Specify whether: With dissociative symptoms: The individual’s symptoms meet the criteria for post- traumatic stress disorder, and the individual experiences persistent or recurrent symp- toms of either of the following: 1. Depersonalization: Persistent or recurrent experiences of feeling detached from, and as if one were an outside observer of, one's mental processes or body (e.g., feeling as though one were in a dream; feeling a sense of unreality of self or body or of time moving slowly).

1	2. Derealization: Persistent or recurrent experiences of unreality of surroundings (e.g., the world around the individual is experienced as unreal, dreamlike, distant, or distorted). Note: To use this subtype, the dissociative symptoms must not be attributable to the physiological effects of a substance (e.g., blackouts) or another medical condition (e.g., complex partial seizures). Specify it: With delayed expression: It the full diagnostic criteria are not met until at least 6 months after the event (although the onset and expression of some symptoms may be immediate). The essential feature of posttraumatic stress disorder (PTSD) is the development of char- acteristic symptoms following exposure to one or more traumatic events. Emotional re- actions to the traumatic event (e.g., fear, helplessness, horror) are no longer a part of

1	Criterion A. The clinical presentation of PTSD varies. In some individuals, fear-based re- experiencing, emotional, and behavioral symptoms may predominate. In others, anhe- donic or dysphoric mood states and negative cognitions may be most distressing. In some other individuals, arousal and reactive-externalizing symptoms are prominent, while in others, dissociative symptoms predominate. Finally, some individuals exhibit combina- tions of these symptom patterns.

1	The directly experienced traumatic events in Criterion A include, but are not limited to, exposure to war as a combatant or civilian, threatened or actual physical assault (e.g., physical attack, robbery, mugging, childhood physical abuse), threatened or actual sexual violence (e.g., forced sexual penetration, alcohol/drug-facilitated sexual penetration, abu- sive sexual contact, noncontact sexual abuse, sexual trafficking), being kidnapped, being taken hostage, terrorist attack, torture, incarceration as a prisoner of war, natural or hu- man-made disasters, and severe motor vehicle accidents. For children, sexually violent violence or injury. A life-threatening illness or debilitating medical condition is not neces- sarily considered a traumatic event. Medical incidents that qualify as traumatic events in- volve sudden, catastrophic events (e.g., waking during surgery, anaphylactic shock).

1	Witnessed events include, but are not limited to, observing threatened or serious injury, unnatural death, physical or sexual abuse of another person due to violent assault, domes- tic violence, accident, war or disaster, or a medical catastrophe in one’s child (e.g., a life- threatening hemorrhage). Indirect exposure through learning about an event is limited to experiences affecting close relatives or friends and experiences that are Violent or acciden- tal (e.g., death due to natural causes does not qualify). Such events include violent per- sonal assault, suicide, serious accident, and serious injury. The disorder may be especially severe or long-Iasting when the stressor is interpersonal and intentional (e.g., torture, sex- ual violence).

1	The traumatic event can be reexperienced in various ways. Commonly, the individual has recurrent, involuntary, and intrusive recollections of the event (Criterion B1). Intrusive recollections in PTSD are distinguished from depressive rumination in that they apply only to involuntary and intrusive distressing memories. The emphasis is on recurrent memories of the event that usually include sensory, emotional, or physiological behavioral components. A common reexperiencing symptom is distressing dreams that replay the event itself or that are representative or thematically related to the major threats involved in the traumatic event (Criterion B2). The individual may experience dissociative states that last from a few seconds to several hours or even days, during which components of the event are relived and the individual behaves as if the event were occurring at that mo- ment (Criterion B3). Such events occur on a continuum from brief visual or other sensory intrusions about part of

1	are relived and the individual behaves as if the event were occurring at that mo- ment (Criterion B3). Such events occur on a continuum from brief visual or other sensory intrusions about part of the traumatic event without loss of reality orientation, to complete loss of awareness of present surroundings. These episodes, often referred to as ”ﬂash- backs,” are typically brief but can be associated with prolonged distress and heightened arousal. For young children, reenactment of events related to trauma may appear in play or in dissociative states. Intense psychological distress (Criterion B4) or physiological re- activity (Criterion B5) often occurs when the individual is exposed to triggering events that resemble or symbolize an aspect of the traumatic event (e.g., windy days after a hurricane; seeing someone who resembles one’s perpetrator). The triggering cue could be a physical sensation (e.g., dizziness for survivors of head trauma; rapid heartbeat for a previously traumatized

1	seeing someone who resembles one’s perpetrator). The triggering cue could be a physical sensation (e.g., dizziness for survivors of head trauma; rapid heartbeat for a previously traumatized child), particularly for individuals with highly somatic presentations.

1	Stimuli associated with the trauma are persistently (e.g., always or almost always) avoided. The individual commonly makes deliberate efforts to avoid thoughts, memories, feelings, or talking about the traumatic event (e.g., utilizing distraction techniques to avoid internal reminders) (Criterion C1) and to avoid activities, objects, situations, or people who arouse recollections of it (Criterion C2). Negative alterations in cognitions or mood associated with the event begin or worsen after exposure to the event. These negative alterations can take various forms, including an inability to remember an important aspect of the traumatic event; such amnesia is typically due to dissociative amnesia and is not due to head injury, alcohol, or drugs (Criterion D1).

1	Another form is persistent (i.e., always or almost always) and exaggerated negative ex- pectations regarding important aspects of life applied to oneself, others, or the future (e.g., ifest as a negative change in perceived identity since the trauma (e.g., ”I can’t trust anyone ever again”; Criterion D2). Individuals with PTSD may have persistent erroneous cogni- tions about the causes of the traumatic event that lead them to blame themselves or others (e.g., ”It’s all my fault that my uncle abused me”) (Criterion D3). A persistent negative mood state (e.g., fear, horror, anger, guilt, shame) either began or worsened after exposure to the event (Criterion D4). The individual may experience markedly diminished interest or participation in previously enjoyed activities (Criterion D5), feeling detached or es- tranged from other people (Criterion D6), or a persistent inability to feel positive emotions (especially happiness, joy, satisfaction, or emotions associated with intimacy,

1	feeling detached or es- tranged from other people (Criterion D6), or a persistent inability to feel positive emotions (especially happiness, joy, satisfaction, or emotions associated with intimacy, tenderness, and sexuality) (Criterion D7).

1	Individuals with PTSD may be quick tempered and may even engage in aggressive verbal and / or physical behavior with little or no provocation (e.g., yelling at people, get- ting into fights, destroying objects) (Criterion E1). They may also engage in reckless or self- destructive behavior such as dangerous driving, excessive alcohol or drug use, or self- injurious or suicidal behavior (Criterion E2). PTSD is often characterized by a heightened sensitivity to potential threats, including those that are related to the traumatic experience (e.g., following a motor vehicle accident, being especially sensitive to the threat potentially caused by cars or trucks) and those not related to the traumatic event (e.g., being fearful of suffering a heart attack) (Criterion E3). Individuals with PTSD may be very reactive to un- expected stimuli, displaying a heightened startle response, or jumpiness, to loud noises or unexpected movements (e.g., jumping markedly in response to a telephone ringing)

1	be very reactive to un- expected stimuli, displaying a heightened startle response, or jumpiness, to loud noises or unexpected movements (e.g., jumping markedly in response to a telephone ringing) (Cri- terion E4). Concentration difficulties, including difficulty remembering daily events (e.g., forgetting one’s telephone number) or attending to focused tasks (e.g., following a conver- sation for a sustained period of time), are commonly reported (Criterion E5). Problems with sleep onset and maintenance are common and may be associated with nightmares and safety concerns or with generalized elevated arousal that interferes with adequate sleep (Criterion E6). Some individuals also experience persistent dissociative symptoms of de- tachment from their bodies (depersonalization) or the world around them (derealization); this is reﬂected in the ”with dissociative symptoms” specifier.

1	Developmental regression, such as loss of language in young children, may occur. Audi— tory pseudo-hallucinations, such as having the sensory experience of hearing one’s thoughts spoken in one or more different voices, as well as paranoid ideation, can be pres- ent. Following prolonged, repeated, and severe traumatic events (e.g., childhood abuse, torture), the individual may additionally experience difficulties in regulating emotions or maintaining stable interpersonal relationships, or dissociative symptoms. When the trau- matic event produces violent death, symptoms of both problematic bereavement and PTSD may be present.

1	In the United States, projected lifetime risk for PTSD using DSM-IV criteria at age 75 years is 8.7%. Twelve—month prevalence among U.S. adults is about 3.5%. Lower estimates are seen in Europe and most Asian, African, and Latin American countries, clustering around 0.5"o—1.0%. Although different groups have different levels of exposure to traumatic events, the conditional probability of developing PTSD following a similar level of expo- sure may also vary across cultural groups. Rates of PTSD are higher among veterans and others whose vocation increases the risk of traumatic exposure (e.g., police, firefighters, emergency medical personnel). Highest rates (ranging from one-third to more than one— half of those exposed) are found among survivors of rape, military combat and captivity, and ethnically or politically motivated internment and genocide. The prevalence of PTSD may vary across development; children and adolescents, including preschool children, generally have displayed lower

1	ethnically or politically motivated internment and genocide. The prevalence of PTSD may vary across development; children and adolescents, including preschool children, generally have displayed lower prevalence following exposure to serious traumatic events; however, this may be because previous criteria were insufficiently developmen- tally informed. The prevalence of full-threshold PTSD also appears to be lower among older adults compared with the general population; there is evidence that subthreshold presentations are more common than full PTSD in later life and that these symptoms are associated with substantial clinical impairment. Compared with US. non-Latino whites, higher rates of PTSD have been reported among US. Latinos, African Americans, and

1	American Indians, and lower rates have been reported among Asian Americans, after ad- justment for traumatic exposure and demographic variables. PTSD can occur at any age, beginning after the first year of life. Symptoms usually begin within the first 3 months after the trauma, although there may be a delay of months, or even years, before criteria for the diagnosis are met. There is abundant evidence for what DSM-IV called ”delayed onset” but is now called "delayed expression," with the recogni- tion that some symptoms typically appear immediately and that the delay is in meeting full criteria.

1	DSM-IV called ”delayed onset” but is now called "delayed expression," with the recogni- tion that some symptoms typically appear immediately and that the delay is in meeting full criteria. Frequently, an individual’s reaction to a trauma initially meets criteria for acute stress disorder in the immediate aftermath of the trauma. The symptoms of PTSD and the rela- tive predominance of different symptoms may vary over time. Duration of the symptoms also varies, with complete recovery within 3 months occurring in approximately one—half of adults, while some individuals remain symptomatic for longer than 12 months and sometimes for more than 50 years. Symptom recurrence and intensification may occur in response to reminders of the original trauma, ongoing life stressors, or newly experienced traumatic events. For older individuals, declining health, worsening cognitive function- ing, and social isolation may exacerbate PTSD symptoms.

1	The clinical expression of reexperiencing can vary across development. Young children may report new onset of frightening dreams without content specific to the traumatic event.

1	Before age 6 years (see criteria for preschool subtype), young children are more likely to ex- press reexperiencing symptoms through play that refers directly or symbolically to the trauma. They may not manifest fearful reactions at the time of the exposure or during reex- periencing. Parents may report a wide range of emotional or behavioral changes in young children. Children may focus on imagined interventions in their play or storytelling. In ad- dition to avoidance, children may become preoccupied with reminders. Because of young children’s limitations in expressing thoughts or labeling emotions, negative alterations in mood or cognition tend to involve primarily mood changes. Children may experience co- occurring traumas (e.g., physical abuse, witnessing domestic violence) and in chronic cir- cumstances may not be able to identify onset of symptomatology. Avoidant behavior may be associated with restricted play or exploratory behavior in young children; reduced par- ticipation

1	cir- cumstances may not be able to identify onset of symptomatology. Avoidant behavior may be associated with restricted play or exploratory behavior in young children; reduced par- ticipation in new activities in school-age children; or reluctance to pursue developmental op- portunities in adolescents (e.g., dating, driving). Older children and adolescents may judge themselves as cowardly. Adolescents may harbor beliefs of being changed in ways that make them socially undesirable and estrange them from peers (e.g., ”Now I’ll never fit in”) and lose aspirations for the future. Irritable or aggressive behavior in children and adoles- cents can interfere with peer relationships and school behavior. Reckless behavior may lead to accidental injury to self or others, thrill-seeking, or high-risk behaviors. Individuals who continue to experience PTSD into older adulthood may express fewer symptoms of hy- perarousal, avoidance, and negative cognitions and mood compared with younger adults

1	behaviors. Individuals who continue to experience PTSD into older adulthood may express fewer symptoms of hy- perarousal, avoidance, and negative cognitions and mood compared with younger adults with PTSD, although adults exposed to traumatic events during later life may display more avoidance, hyperarousal, sleep problems, and crying spells than do younger adults exposed to the same traumatic events. In older individuals, the disorder is associated with negative health perceptions, primary care utilization, and suicidal ideation.

1	Risk (and protective) factors are generally divided into pretraumatic, peritraumatic, and posttraumatic factors. Temperamental. These include childhood emotional problems by age 6 years (e.g., prior traumatic exposure, externalizing or anxiety problems) and prior mental disorders (e.g., panic disorder, depressive disorder, PTSD, or obsessive-compulsive disorder [OCD]). Environmental. These include lower socioeconomic status; lower education; exposure to prior trauma (especially during childhood); childhood adversity (e.g., economic depriva- tion, family dysfunction, parental separation or death); cultural characteristics (e.g., fatal- and a family psychiatric history. Social support prior to event exposure is protective. Genetic and physiological. These include female gender and younger age at the time of trauma exposure (for adults). Certain genotypes may either be protective or increase risk of PTSD after exposure to traumatic events.

1	Environmental. These include severity (dose) of the trauma (the greater the magnitude of trauma, the greater the likelihood of PTSD), perceived life threat, personal injury, in- nessed threat to a caregiver in children), and, for military personnel, being a perpetrator, witnessing atrocities, or killing the enemy. Finally, dissociation that occurs during the trauma and persists afterward is a risk factor. Temperamental. These include negative appraisals, inappropriate coping strategies, and development of acute stress disorder. Environmental. These include subsequent exposure to repeated upsetting reminders, subse— quent adverse life events, and financial or other trauma-related losses. Social support (includ- ing family stability, for children) is a protective factor that moderates outcome after trauma.

1	The risk of onset and severity of PTSD may differ across cultural groups as a result of vari- ation in the type of traumatic exposure (e.g., genocide), the impact on disorder severity of the meaning attributed to the traumatic event (e.g., inability to perform funerary rites after a mass killing), the ongoing sociocultural context (e.g., residing among unpunished per- petrators in postconﬂict settings), and other cultural factors (e.g., acculturative stress in immigrants). The relative risk for PTSD of particular exposures (e.g., religious persecu- tion) may vary across cultural groups. The clinical expression of the symptoms or symp- tom clusters of PTSD may vary culturally, particularly with respect to avoidance and numbing symptoms, distressing dreams, and somatic symptoms (e.g., dizziness, short- ness of breath, heat sensations).

1	Cultural syndromes and idioms of distress inﬂuence the expression of PTSD and the range of comorbid disorders in different cultures by providing behavioral and cognitive templates that link traumatic exposures to specific symptoms. For example, panic attack symptoms may be salient in PTSD among Cambodians and Latin Americans because of the association of traumatic exposure with panic-like khytil attacks and atuque de nervios. Comprehensive evaluation of local expressions of PTSD should include assessment of cul- tural concepts of distress (see the chapter "Cultural Formulation" in Section III). PTSD is more prevalent among females than among males across the lifespan. Females in the general population experience PTSD for a longer duration than do males. At least some of the increased risk for PTSD in females appears to be attributable to a greater likelihood of exposure to traumatic events, such as rape, and other forms of interpersonal violence.

1	Within populations exposed specifically to such stressors, gender differences in risk for PTSD are attenuated or nonsignificant. Traumatic events such as childhood abuse increase a person’s suicide risk. PTSD is associated with suicidal ideation and suicide attempts, and presence of the disorder may indicate which individuals with ideation eventually make a suicide plan or actually attempt suicide. PTSD is associated with high levels of social, occupational, and physical disability, as well as considerable economic costs and high levels of medical utilization. Impaired function- ing is exhibited across social, interpersonal, developmental, educational, physical health, and occupational domains. In community and veteran samples, PTSD is associated with poor social and family relationships, absenteeism from work, lower income, and lower ed- ucational and occupational success.

1	Adjustment disorders. In adjustment disorders, the stressor can be of any severity or type rather than that required by PTSD Criterion A. The diagnosis of an adjustment dis- order is used when the response to a stressor that meets PTSD Criterion A does not meet all other PTSD criteria (or criteria for another mental disorder). An adjustment disorder is also diagnosed when the symptom pattern of PTSD occurs in response to a stressor that does not meet PTSD Criterion A (e.g., spouse leaving, being fired).

1	Other posttraumatic disorders and conditions. Not all psychopathology that occurs in individuals exposed to an extreme stressor should necessarily be attributed to PTSD. The diagnosis requires that trauma exposure precede the onset or exacerbation of pertinent symptoms. Moreover, if the symptom response pattern to the extreme stressor meets cri- teria for another mental disorder, these diagnoses should be given instead of, or in addi- tion to, PTSD. Other diagnoses and conditions are excluded if they are better explained by PTSD (e.g., symptoms of panic disorder that occur only after exposure to traumatic re- minders). If severe, symptom response patterns to the extreme stressor may warrant a sep- arate diagnosis (e.g., dissociative amnesia). Acute stress disorder. Acute stress disorder is distinguished from PTSD because the symptom pattern in acute stress disorder is restricted to a duration of 3 days to 1 month following exposure to the traumatic event.

1	Anxiety disorders and obsessive-compulsive disorder. In OCD, there are recurrent intrusive thoughts, but these meet the definition of an obsession. In addition, the intrusive thoughts are not related to an experienced traumatic event, compulsions are usually pres- ent, and other symptoms of PTSD or acute stress disorder are typically absent. Neither the arousal and dissociative symptoms of panic disorder nor the avoidance, irritability, and anxiety of generalized anxiety disorder are associated with a specific traumatic event. The symptoms of separation anxiety disorder are clearly related to separation from home or family, rather than to a traumatic event. Major depressive disorder. Major depression may or may not be preceded by a trau- matic event and should be diagnosed if other PTSD symptoms are absent. Specifically, ma- jor depressive disorder does not include any PTSD Criterion B or C symptoms. Nor does it include a number of symptoms from PTSD Criterion D or E.

1	Personality disorders. Interpersonal difficulties that had their onset, or were greatly ex- acerbated, after exposure to a traumatic event may be an indication of PTSD, rather than a personality disorder, in which such difficulties would be expected independently Of any traumatic exposure. Dissociative disorders. Dissociative amnesia, dissociative identity disorder, and de- personalization-derealization disorder may or may not be preceded by exposure to a trau- matic event or may or may not have co—occurring PTSD symptoms. When full PTSD criteria are also met, however, the I’ISD ”with dissociative symptoms” subtype should be considered. Conversion disorder (functional neurological symptom disorder). New onset of somatic symptoms within the context of posttraumatic distress might be an indication of PTSD rather than conversion disorder (functional neurological symptom disorder).

1	Psychotic disorders. Flashbacks in PTSD must be distinguished from illusions, halluci- nations, and other perceptual disturbances that may occur in schizophrenia, brief psy- chotic disorder, and other psychotic disorders; depressive and bipolar disorders with orders due to another medical condition.

1	Traumatic brain injury. When a brain injury occurs in the context of a traumatic event (e.g., traumatic accident, bomb blast, acceleration/ deceleration trauma), symptoms of PTSD may appear. An event causing head trauma may also constitute a psychological traumatic event, and tramautic brain injury (TBI)—related neurocognitive symptoms are not mutually exclusive and may occur concurrently. Symptoms previously termed postcancussive (e.g., headaches, dizziness, sensitivity to light or sound, irritability, concentration deficits) can occur in brain- injured and non-brain—injured populations, including individuals with PTSD. Because symp- toms of PTSD and TBI-related neurocognitive symptoms can overlap, a differential diagnosis between PTSD and neurocognitive disorder symptoms attributable to TBI may be possible based on the presence of symptoms that are distinctive to each presentation. Whereas reexpe- riencing and avoidance are characteristic of PTSD and not the effects of TBI,

1	to TBI may be possible based on the presence of symptoms that are distinctive to each presentation. Whereas reexpe- riencing and avoidance are characteristic of PTSD and not the effects of TBI, persistent disori- entation and confusion are more specific to TBI (neurocognitive effects) than to PTSD.

1	Individuals with PTSD are 80% more likely than those without PTSD to have symptoms that meet diagnostic criteria for at least one other mental disorder (e.g., depressive, bipo- lar, anxiety, or substance use disorders). Comorbid substance use disorder and conduct disorder are more common among males than among females. Among US. military per- sonnel and combat veterans who have been deployed to recent wars in Afghanistan and Iraq, co-occurrence of PTSD and mild TBI is 48%. Although most young children with PTSD also have at least one other diagnosis, the patterns of comorbidity are different than in adults, with oppositional defiant disorder and separation anxiety disorder predominat- ing. Finally, there is considerable comorbidity between PTSD and major neurocognitive disorder and some overlapping symptoms between these disorders. Diagnostic Criteria 308.3 (F43.0)

1	Diagnostic Criteria 308.3 (F43.0) A. Exposure to actual or threatened death, serious injury. or sexual violation in one (or more) of the following ways: 1. Directly experiencing the traumatic event(s). 2. Witnessing, in person, the event(s) as it occurred to others. 3. Learning that the event(s) occurred to a close family member or close friend. Note: In cases of actual or threatened death of a family member or friend, the event(s) must have been violent or accidental. 4. Experiencing repeated or extreme exposure to aversive details of the traumatic event(s) (e.g., first responders collecting human remains, police officers repeatedly exposed to details of child abuse). Note: This does not apply to exposure through electronic media, television, mov- ies, or pictures, unless this exposure is work related.

1	Note: This does not apply to exposure through electronic media, television, mov- ies, or pictures, unless this exposure is work related. B. Presence of nine (or more) of the following symptoms from any of the five categories of intrusion, negative mood, dissociation, avoidance, and arousal, beginning or wors- ening after the traumatic event(s) occurred: 1. Recurrent, involuntary, and intrusive distressing memories of the traumatic event(s). Note: In children, repetitive play may occur in which themes or aspects of the traumatic event(s) are expressed. 2. Recurrent distressing dreams in which the content and/or affect of the dream are related to, the event(s). Note: In children, there may be frightening dreams without recognizable content.

1	2. Recurrent distressing dreams in which the content and/or affect of the dream are related to, the event(s). Note: In children, there may be frightening dreams without recognizable content. 3. Dissociative reactions (e.g., flashbacks) in which the individual feels or acts as if the traumatic event(s) were recurring. (Such reactions may occur on a continuum, with the most extreme expression being a complete loss of awareness of present surroundings.) Note: In children, trauma-specitic reenactment may occur in play. 4. Intense or prolonged psychological distress or marked physiological reactions in re- sponse to internal or external cues that symbolize or resemble an aspect of the traumatic event(s). 5. Persistent inability to experience positive emotions (e.g., inability to experience happiness, satisfaction, or loving feelings). 6. An altered sense of the reality of one's surroundings or oneself (e.g., seeing oneself from another's perspective, being in a daze, time slowing).

1	6. An altered sense of the reality of one's surroundings or oneself (e.g., seeing oneself from another's perspective, being in a daze, time slowing). 7. Inability to remember an important aspect of the traumatic event(s) (typically due to dissociative amnesia and not to other factors such as head injury. alcohol, or drugs). 8. Efforts to avoid distressing memories, thoughts, or feelings about or closely asso- ciated with the traumatic event(s). 9. Efforts to avoid external reminders (people. places, conversations, activities, ob- jects, situations) that arouse distressing memories, thoughts, or feelings about or closely associated with the traumatic event(s). 10. Sleep disturbance (e.g., difficulty falling or staying asleep, restless sleep). 11. Irritable behavior and angry outbursts (with little or no provocation), typically ex- pressed as verbal or physical aggression toward people or objects. 12. Hypervigilance. 13. Problems with concentration.

1	12. Hypervigilance. 13. Problems with concentration. 14. Exaggerated startle response. C. Duration of the disturbance (symptoms in Criterion B) is 3 days to 1 month after trauma exposure. Note: Symptoms typically begin immediately after the trauma. but persistence for at least 3 days and up to a month is needed to meet disorder criteria. D. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. E. The disturbance is not attributable to the physiological effects of a substance (e.g., medication or alcohol) or another medical condition (e.g., mild traumatic brain injury) and is not better explained by brief psychotic disorder. The essential feature of acute stress disorder is the development of characteristic symp- toms lasting from 3 days to 1 month following exposure to one or more traumatic events.

1	The essential feature of acute stress disorder is the development of characteristic symp- toms lasting from 3 days to 1 month following exposure to one or more traumatic events. Traumatic events that are experienced directly include, but are not limited to, exposure to war as a combatant or civilian, threatened or actual violent personal assault (e.g., sexual violence, physical attack, active combat, mugging, childhood physical and / or sexual vio- lence, being kidnapped, being taken hostage, terrorist attack, torture), natural or human- made disasters (e.g., earthquake, hurricane, airplane crash), and severe accident (e.g., severe motor vehicle, industrial accident). For children, sexually traumatic events may include inappropriate sexual experiences without violence or injury. A life-threatening illness or debilitating medical condition is not necessarily considered a traumatic event.

1	Medical incidents that qualify as traumatic events involve sudden, catastrophic events (e.g., waking during surgery, anaphylactic shock). Stressful events that do not possess the severe and traumatic components of events encompassed by Criterion A may lead to an adjust- ment disorder but not to acute stress disorder.

1	The clinical presentation of acute stress disorder may vary by individual but typically involves an anxiety response that includes some form of reexperiencing of or reactivity to the traumatic event. In some individuals, a dissociative or detached presentation can pre- dominate, although these individuals typically will also display strong emotional or phys- iological reactivity in response to trauma reminders. In other individuals, there can be a strong anger response in which reactivity is characterized by irritable or possibly aggres- sive responses. The full symptom picture must be present for at least 3 days after the trau- matic event and can be diagnosed only up to 1 month after the event. Symptoms that occur immediately after the event but resolve in less than 3 days would not meet criteria for acute stress disorder.

1	Witnessed events include, but are not limited to, observing threatened or serious in- jury, unnatural death, physical or sexual violence inﬂicted on another individual as a re- sult of violent assault, severe domestic violence, severe accident, war, and disaster; it may also include witnessing a medical catastrophe (e.g., a life-threatening hemorrhage) involv- ing one’s child. Events experienced indirectly through learning about the event are limited to close relatives or close friends. Such events must have been violent or accidental—death due to natural causes does not qualify—and include violent personal assault, suicide, se- rious accident, or serious injury. The disorder may be especially severe when the stressor is interpersonal and intentional (e.g., torture, rape). The likelihood of developing this dis- order may increase as the intensity of and physical proximity to the stressor increase.

1	The traumatic event can be reexperienced in various ways. Commonly, the individual has recurrent and intrusive recollections of the event (Criterion Bl). The recollections are spontaneous or triggered recurrent memories of the event that usually occur in response to a stimulus that is reminiscent of the traumatic experience (e.g., the sound of a backfiring car triggering memories of gunshots). These intrusive memories often include sensory (e.g., sensing the intense heat that was perceived in a house fire), emotional (e.g., experi- encing the fear of believing that one was about to be stabbed), or physiological (e.g., expe- riencing the shortness of breath that one suffered during a near-drowning) components.

1	Distressing dreams may contain themes that are representative of or thematically re- lated to the major threats involved in the traumatic event. (For example, in the case of a motor vehicle accident survivor, the distressing dreams may involve crashing cars gener- ally; in the case of a combat soldier, the distressing dreams may involve being harmed in ways other than combat.)

1	Dissociative states may last from a few seconds to several hours, or even days, during which components of the event are relived and the individual behaves as though experi- encing the event at that moment. While dissociative responses are common during a trau- matic event, only dissociative responses that persist beyond 3 days after trauma exposure are considered for the diagnosis of acute stress disorder. For young children, reenactment of events related to trauma may appear in play and may include dissociative moments (e.g., a child who survives a motor vehicle accident may repeatedly crash toy cars during play in a focused and distressing manner). These episodes, often referred to as ﬂashbacks, are typically brief but involve a sense that the traumatic event is occurring in the present rather than being remembered in the past and are associated with significant distress.

1	Some individuals with the disorder do not have intrusive memories of the event itself, they are exposed to triggering events that resemble or symbolize an aspect of the traumatic event (e.g., windy days for children after a hurricane, entering an elevator for a male or fe- male who was raped in an elevator, seeing someone who resembles one’s perpetrator). The triggering cue could be a physical sensation (e.g., a sense of heat for a burn victim, diz- ziness for survivors of head trauma), particularly for individuals with highly somatic pre- sentations. The individual may have a persistent inability to feel positive emotions (e.g., happiness, joy, satisfaction, or emotions associated with intimacy, tenderness, or sexual- ity) but can experience negative emotions such as fear, sadness, anger, guilt, or shame.

1	Alterations in awareness can include depersonalization, a detached sense of oneself (e.g., seeing oneself from the other side of the room), or derealization, having a distorted view of one’s surroundings (e.g., perceiving that things are moving in slow motion, seeing things in a daze, not being aware of events that one would normally encode). Some individuals also report an inability to remember an important aspect of the traumatic event that was presumably encoded. This symptom is attributable to dissociative amnesia and is not at- tributable to head injury, alcohol, or drugs.

1	Stimuli associated with the trauma are persistently avoided. The individual may refuse to discuss the traumatic experience or may engage in avoidance strategies to minimize awareness of emotional reactions (e.g., excessive alcohol use when reminded of the ex- perience). This behavioral avoidance may include avoiding watching news coverage of the traumatic experience, refusing to return to a workplace where the trauma occurred, or avoiding interacting with others who shared the same traumatic experience.

1	It is very common for individuals with acute stress disorder to experience problems with sleep onset and maintenance, which may be associated with nightmares or with gen- eralized elevated arousal that prevents adequate sleep. Individuals with acute stress dis- behavior with little provocation. Acute stress disorder is often characterized by a height- ened sensitivity to potential threats, including those that are related to the traumatic ex- perience (e.g., a motor vehicle accident victim may be especially sensitive to the threat potentially caused by any cars or trucks) or those not related to the traumatic event (e.g., fear of having a heart attack). Concentration difficulties, including difficulty remembering daily events (e.g., forgetting one’s telephone number) or attending to focused tasks (e.g., following a conversation for a sustained period of time), are commonly reported. Individ- uals with acute stress disorder may be very reactive to unexpected stimuli, displaying a

1	focused tasks (e.g., following a conversation for a sustained period of time), are commonly reported. Individ- uals with acute stress disorder may be very reactive to unexpected stimuli, displaying a heightened startle response or jumpiness to loud noises or unexpected movements (e.g., the individual may jump markedly in the response to a telephone ringing).

1	Individuals with acute stress disorder commonly engage in catastrophic or extremely neg- ative thoughts about their role in the traumatic event, their response to the traumatic ex- perience, or the likelihood of future harm. For example, an individual with acute stress disorder may feel excessively guilty about not having prevented the traumatic event or about not adapting to the experience more successfully. Individuals with acute stress dis- order may also interpret their symptoms in a catastrophic manner, such that ﬂashback memories or emotional numbing may be interpreted as a sign of diminished mental ca- pacity. It is common for individuals with acute stress disorder to experience panic attacks in the initial month after trauma exposure that may be triggered by trauma reminders or may apparently occur spontaneously. Additionally, individuals with acute stress disorder may display chaotic or impulsive behavior. For example, individuals may drive reck- lessly, make irrational

1	or may apparently occur spontaneously. Additionally, individuals with acute stress disorder may display chaotic or impulsive behavior. For example, individuals may drive reck- lessly, make irrational decisions, or gamble excessively. In children, there may be sig- nificant separation anxiety, possibly manifested by excessive needs for attention from caregivers. In the case of bereavement following a death that occurred in traumatic cir- cumstances, the symptoms of acute stress disorder can involve acute grief reactions. In such cases, reexperiencing, dissociative, and arousal symptoms may involve reactions to the loss, such as intrusive memories of the circumstances of the individual’s death, disbe- lief that the individual has died, and anger about the death. Postconcussive symptoms (e.g., headaches, dizziness, sensitivity to light or sound, irritability, concentration deficits), which occur frequently following mild traumatic brain injury, are also frequently seen in individuals

1	headaches, dizziness, sensitivity to light or sound, irritability, concentration deficits), which occur frequently following mild traumatic brain injury, are also frequently seen in individuals with acute stress disorder. Postconcussive symptoms are equally common in brain-injured and non—brain-injured populations, and the frequent occurrence of postcon- cussive symptoms could be attributable to acute stress disorder symptoms.

1	The prevalence of acute stress disorder in recently trauma-exposed populations (i.e., within 1 month of trauma exposure) varies according to the nature of the event and the context in which it is assessed. In both US. and non-U.S. populations, acute stress disorder tends to be identified in less than 20% of cases following traumatic events that do not in- volve interpersonal assault; 13%—21% of motor vehicle accidents, 14% of mild traumatic brain injury, 19% of assault, 10% of severe burns, and 6%—12% of industrial accidents. Higher rates (i.e., 20%—50%) are reported following interpersonal traumatic events, in- cluding assault, rape, and witnessing a mass shooting.

1	Higher rates (i.e., 20%—50%) are reported following interpersonal traumatic events, in- cluding assault, rape, and witnessing a mass shooting. Acute stress disorder cannot be diagnosed unti13 days after a traumatic event. Although acute stress disorder may progress to posttraumatic stress disorder (PTSD) after 1 month, it may also be a transient stress response that remits within 1 month of trauma exposure and does not result in PTSD. Approximately half of individuals who eventually develop PTSD initially present with acute stress disorder. Symptom worsening during the initial month can occur, often as a result of ongoing life stressors or further traumatic events.

1	The forms of reexperiencing can vary across development. Unlike adults or adoles- cents, young children may report frightening dreams without content that clearly reﬂects aspects of the trauma (e.g., waking in fright in the aftermath of the trauma but being unable to relate the content of the dream to the traumatic event). Children age 6 years and younger are more likely than older children to express reexperiencing symptoms through play that refers directly or symbolically to the trauma. For example, a very young child who sur- vived a fire may draw pictures of ﬂames. Young children also do not necessarily manifest fearful reactions at the time of the exposure or even during reexperiencing. Parents typi- cally report a range of emotional expressions, such as anger, shame, or withdrawal, and even excessively bright positive affect, in young children who are traumatized. Although children may avoid reminders of the trauma, they sometimes become preoccupied with reminders (e.g., a young

1	even excessively bright positive affect, in young children who are traumatized. Although children may avoid reminders of the trauma, they sometimes become preoccupied with reminders (e.g., a young child bitten by a dog may talk about dogs constantly yet avoid g0- ing outside because of fear of coming into contact with a dog).

1	Temperamental. Risk factors include prior mental disorder, high levels of negative af- fectivity (neuroticism), greater perceived severity of the traumatic event, and an avoidant coping style. Catastrophic appraisals of the traumatic experience, often characterized by exaggerated appraisals of future harm, guilt, or hopelessness, are strongly predictive of acute stress disorder. Environmental. First and foremost, an individual must be exposed to a traumatic event to be at risk for acute stress disorder. Risk factors for the disorder include a history of prior trauma. Genetic and physiological. Females are at greater risk for developing acute stress dis- order. \ Elevated reactivity, as reﬂected by acoustic startle response, prior to trauma exposure increases the risk for developing acute stress disorder.

1	Elevated reactivity, as reﬂected by acoustic startle response, prior to trauma exposure increases the risk for developing acute stress disorder. The profile of symptoms of acute stress disorder may vary cross-culturally, particularly with respect to dissociative symptoms, nightmares, avoidance, and somatic symptoms (e.g., dizziness, shortness of breath, heat sensations). Cultural syndromes and idioms of distress shape the local symptom profiles of acute stress disorder. Some cultural groups may display variants of dissociative responses, such as possession or trancelike behaviors in the initial month after trauma exposure. Panic symptoms may be salient in acute stress disorder among Cambodians because of the association of traumatic exposure with panic- like khyfil attacks, and ataque de nervios among Latin Americans may also follow a traumatic exposure.

1	Acute stress disorder is more prevalent among females than among males. Sex-linked neu- robiological differences in stress response may contribute to females’ increased risk for acute stress disorder. The increased risk for the disorder in females may be attributable in part to a greater likelihood of exposure to the types of traumatic events with a high con- ditional risk for acute stress disorder, such as rape and other interpersonal violence. Functional Consequences of Acute Stress Disorder

1	Functional Consequences of Acute Stress Disorder Impaired functioning in social, interpersonal, or occupational domains has been shown across survivors of accidents, assault, and rape who develop acute stress disorder. The ex- treme levels of anxiety that may be associated with acute stress disorder may interfere with sleep, energy levels, and capacity to attend to tasks. Avoidance in acute stress dis- order can result in generalized withdrawal from many situations that are perceived as potentially threatening, which can lead to nonattendance of medical appointments, avoid- ance of driving to important appointments, and absenteeism from work.

1	Adjustment disorders. In acute stress disorder, the stressor can be of any severity rather than of the severity and type required by Criterion A of acute stress disorder. The diagnosis of an adjustment disorder is used when the response to a Criterion A event does not meet the cri- teria for acute stress disorder (or another specific mental disorder) and when the symptom pat- tern of acute stress disorder occurs in response to a stressor that does not meet Criterion A for exposure to actual or threatened death, serious injury, or sexual violence (e.g., spouse leaving, being fired). For example, severe stress reactions to life—threatening illnesses that may include some acute stress disorder symptoms may be more appropriately described as an adjustment disorder. Some forms of acute stress response do not include acute stress disorder symptoms and may be characterized by anger, depression, or guilt. These responses are more appro- priately described as primarily an adjustment disorder.

1	response do not include acute stress disorder symptoms and may be characterized by anger, depression, or guilt. These responses are more appro- priately described as primarily an adjustment disorder. Depressive or anger responses in an adjustment disorder may involve rumination about the traumatic event, as opposed to invol- untary and intrusive distressing memories in acute stress disorder.

1	Panic disorder. Spontaneous panic attacks are very common in acute stress disorder. However, panic disorder is diagnosed only if panic attacks are unexpected and there is anxiety about future attacks or maladaptive changes in behavior associated with fear of dire consequences of the attacks. Dissociative disorders. Severe dissociative responses (in the absence of characteristic acute stress disorder symptoms) may be diagnosed as derealization/depersonalization disorder. If severe amnesia of the trauma persists in the absence of characteristic acute stress disorder symptoms, the diagnosis of dissociative amnesia may be indicated. Posttraumatic stress disorder. Acute stress disorder is distinguished from PTSD because the symptom pattern in acute stress disorder must occur within 1 month of the traumatic event and resolve within that 1-month period. If the symptoms persist for more than 1 month and meet criteria for PTSD, the diagnosis is changed from acute stress disorder to PTSD.

1	Obsessive-compulsive disorder. In obsessive-compulsive disorder, there are recurrent intrusive thoughts, but these meet the definition of an obsession. In addition, the intrusive thoughts are not related to an experienced traumatic event, compulsions are usually pres- ent, and other symptoms of acute stress disorder are typically absent. Psychotic disorders. Flashbacks in acute stress disorder must be distinguished from il- lusions, hallucinations, and other perceptual disturbances that may occur in schizophre- nia, other psychotic disorders, depressive or bipolar disorder with psychotic features, a delirium, substance/medication—induced disorders, and psychotic disorders due to an- other medical condition. Acute stress disorder ﬂashbacks are distinguished from these other perceptual disturbances by being directly related to the traumatic experience and by occurring in the absence of other psychotic or substance—induced features.

1	Traumatic brain injury. When a brain injury occurs in the context of a traumatic event (e.g., traumatic accident, bomb blast, acceleration/deceleration trauma), symptoms of acute stress disorder may appear. An event causing head trauma may also constitute a psychological traumatic event, and tramautic brain injury (TBI)—related neurocognitive symptoms are not mutually exclusive and may occur concurrently. Symptoms previously termed postconcussive (e.g., headaches, dizziness, sensitivity to light or sound, irritability, concentration deficits) can occur in brain-injured and non—brain injured populations, in- cluding individuals with acute stress disorder. Because symptoms of acute stress disorder and TBI-related neurocognitive symptoms can overlap, a differential diagnosis between acute stress disorder. and neurocognitive disorder symptoms attributable to TBI may be possible based on the presence of symptoms that are distinctive to each presenta- tion. Whereas reexperiencing and

1	stress disorder. and neurocognitive disorder symptoms attributable to TBI may be possible based on the presence of symptoms that are distinctive to each presenta- tion. Whereas reexperiencing and avoidance are characteristic of acute stress disorder and not the effects of TBI, persistent disorientation and confusion are more specific to TBI (neu- rocognitive effects) than to acute stress disorder. Furthermore, differential is aided by the fact that symptoms of acute stress disorder persist for up to only 1 month following trauma exposure.

1	A. The development of emotional or behavioral symptoms in response to an identifiable stressor(s) occurring within 3 months of the onset of the stressor(s). B. These symptoms or behaviors are clinically significant, as evidenced by one or both of the following: 1. Marked distress that is out of proportion to the severity or intensity of the stressor, taking into account the external context and the cultural factors that might influence symptom severity and presentation. 2. Significant impairment in social, occupational, or other important areas of functioning. C. The stress-related disturbance does not meet the criteria for another mental disorder and is not merely an exacerbation of a preexisting mental disorder. D. The symptoms do not represent normal bereavement. E. Once the stressor or its consequences have terminated, the symptoms do not persist for more than an additional 6 months.

1	D. The symptoms do not represent normal bereavement. E. Once the stressor or its consequences have terminated, the symptoms do not persist for more than an additional 6 months. Specify whether: 309.0 (F43.21) With depressed mood: Low mood, tearfulness, or feelings of hope- lessness are predominant. 309.24 (F43.22) With anxiety: Nervousness, worry, jitteriness, or separation anxiety is predominant. 309.28 (F43.23) With mixed anxiety and depressed mood: A combination of de- pression and anxiety is predominant. 309.3 (F43.24) With disturbance of conduct: Disturbance of conduct is predominant. 309.4 (F4325) With mixed disturbance of emotions and conduct: Both emotional symptoms (e.g., depression, anxiety) and a disturbance of conduct are predominant. 309.9 (F43.20) Unspecified: For maladaptive reactions that are not classifiable as one of the specific subtypes of adjustment disorder.

1	The presence of emotional or behavioral symptoms in response to an identifiable stressor is the essential feature of adjustment disorders (Criterion A). The stressor may be a single event (e.g., a termination of a romantic relationship), or there may be multiple stressors (e.g., marked business difficulties and marital problems). Stressors may be recurrent (e.g., associated with seasonal business crises, unfulfilling sexual relationships) or continuous (e.g., a persistent painful illness with increasing disability, living in a crime-ridden neigh- borhood). Stressors may affect a single individual, an entire family, or a larger group or community (e.g., a natural disaster). Some stressors may accompany specific developmen- tal events (e.g., going to school, leaving a parental home, reentering a parental home, get- ting married, becoming a parent, failing to attain occupational goals, retirement).

1	Adjustment disorders may be diagnosed following the death of a loved one when the intensity, quality, or persistence of grief reactions exceeds what normally might be ex- pected, when cultural, religious, or age-appropriate norms are taken into account. A more specific set of bereavement-related symptoms has been designated persistent complex be— reavement disorder. Adjustment disorders are associated with an increased risk of suicide attempts and completed suicide. Adjustment disorders are common, although prevalence may vary widely as a function of the population studied and the assessment methods used. The percentage of individuals in outpatient mental health treatment with a principal diagnosis of an adjustment disorder ranges from approximately 5% to 20%. In a hospital psychiatric consultation setting, it is often the most common diagnosis, frequently reaching 50%.

1	By definition, the disturbance in adjustment disorders begins within 3 months of onset of a stressor and lasts no longer than 6 months after the stressor or its consequences have ceased. If the stressor is an acute event (e.g., being fired from a job), the onset of the dis- turbance is usually immediate (i.e., within a few days) and the duration is relatively brief (i.e., no more than a few months). If the stressor or its consequences persist, the adjustment disorder may also continue to be present and become the persistent form. Environmental. Individuals from disadvantaged life circumstances experience a high rate of stressors and may be at increased risk for adjustment disorders.

1	Environmental. Individuals from disadvantaged life circumstances experience a high rate of stressors and may be at increased risk for adjustment disorders. The context of the individual’s cultural setting should be taken into account in making the clinical judgment of whether the individual’s response to the stressor is maladaptive or whether the associated distress is in excess of what would be expected. The nature, mean- ing, and experience of the stressors and the evaluation of the response to the stressors may vary across cultures. Functional Consequences of Adjustment Disorders

1	Functional Consequences of Adjustment Disorders The subjective distress or impairment in functioning associated with adjustment disorders is frequently manifested as decreased performance at work or school and temporary changes in social relationships. An adjustment disorder may complicate the course of ill- ness in individuals who have a general medical condition (e.g., decreased compliance with the recommended medical regimen; increased length of hospital stay). Major depressive disorder. If an individual has symptoms that meet criteria for a major depressive disorder in response to a stressor, the diagnosis of an adjustment disorder is not applicable. The symptom profile of major depressive disorder differentiates it from ad- justment disorders.

1	Posttraumatic stress disorder and acute stress disorder. In adjustment disorders, the stressor can be of any severity rather than of the severity and type required by Criterion A of acute stress disorder and posttraumatic stress disorder (PTSD). In distinguishing ad- justment disorders from these two posttraumatic diagnoses, there are both timing and symptom profile considerations. Adjustment disorders can be diagnosed immediately and persist up to 6 months after exposure to the traumatic event, whereas acute stress dis- order can only occur between 3 days and 1 month of exposure to the stressor, and PTSD cannot be diagnosed until at least 1 month has passed since the occurrence of the traumatic stressor. The required symptom profile for PTSD and acute stress disorder differentiates them from the adjustment disorders. With regard to symptom profiles, an adjustment dis- of either acute stress disorder or PTSD that do not meet or exceed the diagnostic threshold for either disorder. An

1	from the adjustment disorders. With regard to symptom profiles, an adjustment dis- of either acute stress disorder or PTSD that do not meet or exceed the diagnostic threshold for either disorder. An adjustment disorder should also be diagnosed for individuals who have not been exposed to a traumatic event but who otherwise exhibit the full symptom pro- file of either acute stress disorder or PTSD.

1	Personality disorders. With regard to personality disorders, some personality features may be associated with a vulnerability to situational distress that may resemble an adjust- ment disorder. The lifetime history of personality functioning will help inform the in- terpretation of distressed behaviors to aid in distinguishing a long-standing personality disorder from an adjustment disorder. In addition to some personality disorders incurring vulnerability to distress, stressors may also exacerbate personality disorder symptoms. In the presence of a personality disorder, if the symptom criteria for an adjustment disorder are met, and the stress-related disturbance exceeds what may be attributable to maladap- tive personality disorder symptoms (i.e., Criterion C is met), then the diagnosis of an ad- justment disorder should be made.

1	Psychological factors affecting other medical conditions. In psychological factors af- fecting other medical conditions, specific psychological entities (e.g., psychological symp- toms, behaviors, other factors) exacerbate a medical condition. These psychological factors can precipitate, exacerbate, or put an individual at risk for medical illness, or they can worsen an existing condition. In contrast, an adjustment disorder is a reaction to the stressor (e.g., having a medical illness). Normative stress reactions. When bad things happen, most people get upset. This is not an adjustment disorder. The diagnosis should only be made when the magnitude of the distress (e.g., alterations in mood, anxiety, or conduct) exceeds what would normally be expected (which may vary in different cultures) or when the adverse event precipitates functional impairment. Adjustment disorders can accompany most mental disorders and any medical disorder.

1	Adjustment disorders can accompany most mental disorders and any medical disorder. Adjustment disorders can be diagnosed in addition to another mental disorder only if the latter does not explain the particular symptoms that occur in reaction to the stressor. For example, an individual may develop an adjustment disorder, with depressed mood, after losing a job and at the same time have a diagnosis of obsessive-compulsive disorder. Or, long as the criteria for both are met. Adjustment disorders are common accompaniments of medical illness and may be the major psychological response to a medical disorder. 309.89 (F43.8)

1	309.89 (F43.8) This category applies to presentations in which symptoms characteristic of a trauma- and stressor-related disorder that cause clinically significant distress or impairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the trauma- and stressor-related disorders diagnostic class. The other specified trauma- and stressor-related disorder category is used in situa- tions in which the clinician chooses to communicate the specific reason that the presenta- tion does not meet the criteria for any specific trauma- and stressor-related disorder. This the specific reason (e.g., “persistent complex bereavement disorder”). Examples of presentations that can be specified using the “other specified" designation include the following: 1 . Adjustment-Iike disorders with delayed onset of symptoms that occur more than 3 months after the stressor.

1	2. Adiustment-like disorders with prolonged duration of more than 6 months with- out prolonged duration of stressor. 3. Ataque de nervios: See “Glossary of Cultural Concepts of Distress" in the Appendix. 4. Other cultural syndromes: See “Glossary of Cultural Concepts of Distress" in the Ap- pendix. 5. Persistent complex bereavement disorder: This disorder is characterized by severe and persistent grief and mourning reactions (see the chapter "Conditions for Further Study”). 309.9 (F43.9) This category applies to presentations in which symptoms characteristic of a trauma- and stressor-related disorder that cause clinically significant distress or impairment in social, oc- cupational, or other important areas of functioning predominate but do not meet the full cri- teria for any of the disorders in the trauma- and stressor-related disorders diagnostic class.

1	The unspecified trauma- or stressor-related disorder category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for a specific trauma- and stressor-related disorder, and includes presentations in which there is insuffi- cient information to make a more specific diagnosis (e.g., in emergency room settings). «.5 M» fissociative Dissociative d isorde rs are characterized by a disruption of and / or discontinuity in the normal integration of consciousness, memory, identity, emotion, perception, body representation, motor control, and behavior. Dissociative symptoms can potentially dis- rupt every area of psychological functioning. This chapter includes dissociative identity disorder, dissociative amnesia, depersonalization/derealization disorder, other specified dissociative disorder, and unspecified dissociative disorder.

1	Dissociative symptoms are experienced as a) unbidden intrusions into awareness and behavior, with accompanying losses of continuity in subjective experience (i.e., “positive” dissociative symptoms such as fragmentation of identity, depersonalization, and dereal- ization) and / or b) inability to access information or to control mental functions that nor- mally are readily amenable to access or control (i.e., "negative” dissociative symptoms such as amnesia).

1	The dissociative disorders are frequently found in the aftermath of trauma, and many of the symptoms, including embarrassment and confusion about the symptoms or a desire to hide them, are inﬂuenced by the proximity to trauma. In DSM-5, the dissociative disor- ders are placed next to, but are not part of, the trauma- and stressor—related disorders, re- ﬂecting the close relationship between these diagnostic classes. Both acute stress disorder and posttraumatic stress disorder contain dissociative symptoms, such as amnesia, ﬂash- backs, numbing, and depersonalization/derealization.

1	Depersonalization/derealization disorder is characterized by clinically significant persis- tent or recurrent depersonalization (i.e., experiences of unreality or detachment from one’s mind, self, or body) and/ or derealization (i.e., experiences of unreality or detachment from one's surroundings). These alterations of experience are accompanied by intact reality testing. There is no evidence of any distinction between individuals with predominantly depersonalization versus derealization symptoms. Therefore, individuals with this disor- der can have depersonalization, derealization, or both.

1	Dissociative amnesia is characterized by an inability to recall autobiographical informa- tion. This amnesia may be localized (i.e., an event or period of time), selective (i.e., a specific aspect of an event), or generalized (i.e., identity and life history). Dissociative amnesia is fun- damentally an inability to recall autobiographical information that is inconsistent with nor- mal forgetting. It may or may not involve purposeful travel or bewildered wandering (i.e., fugue). Although some individuals with amnesia promptly notice that they have ”lost time” or that they have a gap in their memory, most individuals with dissociative disorders are ini- tially unaware of their amnesias. For them, awareness of amnesia occurs only when personal identity is lost or when circumstances make these individuals aware that autobiographical information is missing (e.g., when they discover evidence of events they cannot recall or when others tell them or ask them about events they cannot recall).

1	individuals aware that autobiographical information is missing (e.g., when they discover evidence of events they cannot recall or when others tell them or ask them about events they cannot recall). Until and unless this hap- pens, these individuals have ”amnesia for their amnesia.” Amnesia is experienced as an es- sential feature of dissociative amnesia; individuals may experience localized or selective amnesia most commonly, or generalized amnesia rarely. Dissociative fugue is rare in per— sons with dissociative amnesia but common in dissociative identity disorder.

1	Dissociative identity disorder is characterized by a) the presence of two or more distinct personality states or an experience of possession and b) recurrent episodes of amnesia. The fragmentation of identity may vary with culture (e.g., possession-form presentations) and cir- cumstance. Thus, individuals may experience discontinuities in identity and memory that may not be immediately evident to others or are obscured by attempts to hide dysfunction. In- dividuals with dissociative identity disorder experience a) recurrent, inexplicable intrusions into their conscious functioning and sense of self (e.g., voices; dissociated actions and speech; intrusive thoughts, emotions, and impulses), b) alterations of sense of self (e.g., attitudes, pref- erences, and feeling like one’s body or actions are not one’s own), c) odd changes of perception (e.g., depersonalization or derealization, such as feeling detached from one’s body while cut- ting), and d) intermittent functional neurological

1	are not one’s own), c) odd changes of perception (e.g., depersonalization or derealization, such as feeling detached from one’s body while cut- ting), and d) intermittent functional neurological symptoms. Stress often produces transient exacerbation of dissociative symptoms that makes them more evident.

1	The residual category of other specified dissociative disorder has seven examples: chronic or recurrent mixed dissociative symptoms that approach, but fall short of, the diagnostic cri- teria for dissociative identity disorder; dissociative states secondary to brainwashing or thought reform; two acute presentations, of less than 1 month’s duration, of mixed dissociative symptoms, one of which is also marked by the presence of psychotic symptoms; and three sin- gle-symptom dissociative presentations—dissociative trance, dissociative stupor or coma, and Ganser’s syndrome (the giving of approximate and vague answers). Diagnostic Criteria 300.14 (F44.81)

1	Ganser’s syndrome (the giving of approximate and vague answers). Diagnostic Criteria 300.14 (F44.81) A. Disruption of identity characterized by two or more distinct personality states, which may be described in some cultures as an experience of possession. The disruption in identity involves marked discontinuity in sense of self and sense of agency, accompa- nied by related alterations in attect, behavior, consciousness, memory, perception, cognition, and/or sensory-motor functioning. These signs and symptoms may be ob- served by others or reported by the individual. B. Recurrent gaps in the recall of everyday events, important personal information, and/ or traumatic events that are inconsistent with ordinary forgetting. C. The symptoms cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. D. The disturbance is not a normal part of a broadly accepted cultural or religious practice.

1	D. The disturbance is not a normal part of a broadly accepted cultural or religious practice. Note: In children, the symptoms are not better explained by imaginary playmates or other fantasy play. E. The symptoms are not attributable to the physiological el'Iects of a substance (e.g., (e.g., complex partial seizures).

1	The defining feature of dissociative identity disorder is the presence of two or more dis- tinct personality states or an experience of possession (Criterion A). The overtness or covertness of these personality states, however, varies as a function of psychological motivation, current level of stress, culture, internal conﬂicts and dynamics, and emotional resilience. Sustained periods of identity disruption may occur when psychosocial pres- sures are severe and / or prolonged. In many possession-form cases of dissociative identity disorder, and in a small proportion of non—possession-form cases, manifestations of alter- nate identities are highly overt. Most individuals with non-possession—form dissociative identity disorder do not overtly display their discontinuity of identity for long periods of time; only a small minority present to clinical attention with observable alternation of identities. When alternate personality states are not directly observed, the disorder can be

1	long periods of time; only a small minority present to clinical attention with observable alternation of identities. When alternate personality states are not directly observed, the disorder can be identified by two clusters of symptoms: 1) sudden alterations or discontinuities in sense of self and sense ofagency (Criterion A), and 2) recurrent dissociative amnesias (Criterion B).

1	Criterion A symptoms are related to discontinuities of experience that can affect any aspect of an individual’s functioning. Individuals with dissociative identity disorder may report the feeling that they have suddenly become depersonalized observers of their ”own" speech and actions, which they may feel powerless to stop (sense of self). Such in- dividuals may also report perceptions of voices (e.g., a child’s voice; crying; the voice of a spiritual being). In some cases, voices are experienced as multiple, perplexing, indepen- dent thought streams over which the individual experiences no control. Strong emotions, impulses, and even speech or other actions may suddenly emerge, without a sense of per— sonal ownership or control (sense of agency). These emotions and impulses are frequently reported as ego-dystonic and puzzling. Attitudes, outlooks, and personal preferences (e.g., about food, activities, dress) may suddenly shift and then shift back. Individuals may report that their

1	reported as ego-dystonic and puzzling. Attitudes, outlooks, and personal preferences (e.g., about food, activities, dress) may suddenly shift and then shift back. Individuals may report that their bodies feel different (e.g., like a small child, like the opposite gender, huge and muscular). Alterations in sense of self and loss of personal agency may be accompa- nied by a feeling that these attitudes, emotions, and behaviors—even one’s body—are ”not mine" and / or are "not under my control." Although most Criterion A symptoms are subjective, many of these sudden discontinuities in speech, affect, and behavior can be wit- nessed by family, friends, or the clinician. Non—epileptic seizures and other conversion symptoms are prominent in some presentations of dissociative identity disorder, espe- cially in some non—Western settings.

1	The dissociative amnesia of individuals with dissociative identity disorder manifests in three primary ways: as 1) gaps in remote memory of personal life events (e.g., periods of childhood or adolescence; some important life events, such as the death of a grandparent, getting married, giving birth); 2) lapses in dependable memory (e.g., of what happened today, of well-leamed skills such as how to do their job, use a computer, read, drive); and 3) discovery of evidence of their everyday actions and tasks that they do not recollect do- ing (e.g., finding unexplained objects in their shopping bags or among their possessions; ”coming to” in the midst of doing something). Dissociative fugues, wherein the person discovers dissociated travel, are common. Thus, individuals with dissociative identity dis- order may report that they have suddenly found themselves at the beach, at work, in a night- club, or somewhere at home (e.g., in the closet, on a bed or sofa, in the corner) with no memory

1	dis- order may report that they have suddenly found themselves at the beach, at work, in a night- club, or somewhere at home (e.g., in the closet, on a bed or sofa, in the corner) with no memory of how they came to be there. Amnesia in individuals with dissociative identity dis- order is not limited to stressful or traumatic events; these individuals often cannot recall everyday events as well.

1	Individuals with dissociative identity disorder vary in their awareness and attitude to- ward their amnesias. It is common for these individuals to minimize their amnestic symp- toms. Some of their amnestic behaviors may be apparent to others—as when these persons do not recall something they were witnessed to have done or said, when they cannot remember their own name, or when they do not recognize their spouse, children, or close friends.

1	Possession-form identities in dissociative identity disorder typically manifest as be- haviors that appear as if a "spirit,” supernatural being, or outside person has taken control, such that the individual begins speaking or acting in a distinctly different manner. For ex- ample, an individual’s behavior may give the appearance that her identity has been replaced by the "ghost” of a girl who committed suicide in the same community years before, speaking and acting as though she were still alive. Or an individual may be ”taken over" by a demon or deity, resulting in profound impairment, and demanding that the in- dividual or a relative be punished for a past act, followed by more subtle periods of iden- tity alteration. However, the majority of possession states around the world are normal, usually part of spiritual practice, and do not meet criteria for dissociative identity disor- der. The identities that arise during possession-form dissociative identity disorder present

1	are normal, usually part of spiritual practice, and do not meet criteria for dissociative identity disor- der. The identities that arise during possession-form dissociative identity disorder present recurrently, are unwanted and involuntary, cause clinically significant distress or impair- ment (Criterion C), and are not a normal part of a broadly accepted cultural or religious practice (Criterion D).

1	Individuals with dissociative identity disorder typically present with comorbid depression, anxiety, substance abuse, self—injury, non-epileptic seizures, or another common symp- tom. They often conceal, or are not fully aware of, disruptions in consciousness, amnesia, or other dissociative symptoms. Many individuals with dissociative identity disorder re- port dissociative ﬂashbacks during which they undergo a sensory reliving of a previous event as though it were occurring in the present, often with a change of identity, a partial or complete loss of contact with or disorientation to current reality during the ﬂashback, and a subsequent amnesia for the content of the ﬂashback. Individuals with the disorder typically report multiple types of interpersonal maltreatment during childhood and adult- hood. Nonmaltreatment forms of overwhelming early life events, such as multiple long, painful, early-life medical procedures, also may be reported. Self-mutilation and suicidal behavior are

1	adult- hood. Nonmaltreatment forms of overwhelming early life events, such as multiple long, painful, early-life medical procedures, also may be reported. Self-mutilation and suicidal behavior are frequent. On standardized measures, these individuals report higher levels of hypnotizability and dissociativity compared with other clinical groups and healthy con- trol subjects. Some individuals experience transient psychotic phenomena or episodes.

1	Several brain regions have been implicated in the pathophysiology of dissociative identity disorder, including the orbitofrontal cortex, hippocampus, parahippocampal gyrus, and amygdala. The 12-month prevalence of dissociative identity disorder among adults in a small U.S. community study was 1.5%. The prevalence across genders in that study was 1.6% for males and 1.4% for females.

1	Dissociative identity disorder is associated with overwhelming experiences, traumatic events, and / or abuse occurring in childhood. The full disorder may first manifest at al- most any age (from earliest childhood to late life). Dissociation in children may generate problems with memory, concentration, attachment, and traumatic play. Nevertheless, chil- dren usually do not present with identity changes; instead they present primarily with over- lap and interference among mental states (Criten'on A phenomena), with symptoms related to discontinuities of experience. Sudden changes in identity during adolescence may ap- pear to be just adolescent turmoil or the early stages of another mental disorder. Older individuals may present to treatment with what appear to be late-life mood disorders, ob- sessive-compulsive disorder, paranoia, psychotic mood disorders, or even cognitive dis- orders due to dissociative amnesia. In some cases, disruptive affects and memories may increasingly

1	ob- sessive-compulsive disorder, paranoia, psychotic mood disorders, or even cognitive dis- orders due to dissociative amnesia. In some cases, disruptive affects and memories may increasingly intrude into awareness with advancing age.

1	moval from the traumatizing situation (e.g., through leaving home); 2) the individual's children reaching the same age at which the individual was originally abused or trauma- tized; 3) later traumatic experiences, even seemingly inconsequential ones, like a minor motor vehicle accident; or 4) the death of, or the onset of a fatal illness in, their abuser(s). Environmental. Interpersonal physical and sexual abuse is associated with an increased risk of dissociative identity disorder. Prevalence of childhood abuse and neglect in the United States, Canada, and Europe among those with the disorder is about 90%. Other forms of traumatizing experiences, including childhood medical and surgical procedures, war, childhood prostitution, and terrorism, have been reported. Course modifiers. Ongoing abuse, later-life retraumatization, comorbidity with mental disorders, severe medical illness, and delay in appropriate treatment are associated with poorer prognosis.

1	Many features of dissociative identity disorder can be inﬂuenced by the individual’s cul- tural background. Individuals with this disorder may present with prominent medically unexplained neurological symptoms, such as non—epileptic seizures, paralyses, or sensory loss, in cultural settings where such symptoms are common. Similarly, in settings where normative possession is common (e.g., rural areas in the developing world, among certain religious groups in the United States and Europe), the fragmented identities may take the form of possessing spirits, deities, demons, animals, or mythical figures. Acculturation or prolonged intercultural contact may shape the characteristics of the other identities (e.g., identities in India may speak English exclusively and wear Western clothes). Possession- sion states in that the former is involuntary, distressing, uncontrollable, and often recur- rent or persistent; involves conﬂict between the individual and his or her surrounding family,

1	Possession- sion states in that the former is involuntary, distressing, uncontrollable, and often recur- rent or persistent; involves conﬂict between the individual and his or her surrounding family, social, or work milieu; and is manifested at times and in places that violate the norms of the culture or religion.

1	Females with dissociative identity disorder predominate in adult clinical settings but not in child clinical settings. Adult males with dissociative identity disorder may deny their symptoms and trauma histories, and this can lead to elevated rates of false negative di- agnosis. Females with dissociative identity disorder present more frequently with acute dissociative states (e.g., ﬂashbacks, amnesia, fugue, functional neurological [conversion] symptoms, hallucinations, self—mutilation). Males commonly exhibit more criminal or vi— olent behavior than females; among males, common triggers of acute dissociative states in- clude combat, prison conditions, and physical or sexual assaults.

1	Over 70% of outpatients with dissociative identity disorder have attempted suicide; mul- tiple attempts are common, and other self-injurious behavior is frequent. Assessment of suicide risk may be complicated when there is amnesia for past suicidal behavior or when the presenting identity does not feel suicidal and is unaware that other dissociated iden- tities do.

1	Impairment varies widely, from apparently minimal (e.g., in high-functioning profession- als) to profound. Regardless of level of disability, individuals with dissociative identity disorder commonly minimize the impact of their dissociative and posttraumatic symp- toms. The symptoms of higher-functioning individuals may impair their relational, mar- ital, family, and parenting functions more than their occupational and professional life (although the latter also may be affected). With appropriate treatment, many impaired in- dividuals show marked improvement in occupational and personal functioning. How- ever, some remain highly impaired in most activities of living. These individuals may only respond to treatment very slowly, with gradual reduction in or improved tolerance of their dissociative and posttraumatic symptoms. Long-term supportive treatment may slowly increase these individuals’ ability to manage their symptoms and decrease use of more restrictive levels of care.

1	Other specified dissociative disorder. The core of dissociative identity disorder is the division of identity, with recurrent disruption of conscious functioning and sense of self. This central feature is shared with one form of other specified dissociative disorder, which may be distinguished from dissociative identity disorder by the presence of chronic or re- disorder or are not accompanied by recurrent amnesia. Major depressive disorder. Individuals with dissociative identity disorder are often de- pressed, and their symptoms may appear to meet the criteria for a major depressive episode. major depressive disorder. Other specified depressive disorder in individuals with dissocia- tive identity disorder often has an important feature: the depressed mood and cognitionsﬂuc- tuate because they are experienced in some identity states but not others.

1	Bipolar disorders. Individuals with dissociative identity disorder are often misdiag- nosed with a bipolar disorder, most often bipolar II disorder. The relatively rapid shifts in mood in individuals with this disorder—typically within minutes or hours, in contrast to the slower mood changes typically seen in individuals with bipolar disorders—are due to the rapid, subjective shifts in mood commonly reported across dissociative states, some- times accompanied by ﬂuctuation in levels of activation. Furthermore, in dissociative identity disorder, elevated or depressed mood may be displayed in conjunction with overt identities, so one or the other mood may predominate for a relatively long period of time (often for days) or may shift within minutes.

1	Posttraumatic stress disorder. Some traumatized individuals have both posttraumatic stress disorder (PTSD) and dissociative identity disorder. Accordingly, it is crucial to dis- tinguish between individuals with PTSD only and individuals who have both PTSD and dissociative identity disorder. This differential diagnosis requires that the clinician estab- lish the presence or absence of dissociative symptoms that are not characteristic of acute stress disorder or PTSD. Some individuals with PTSD manifest dissociative symptoms that also occur in dissociative identity disorder: 1) amnesia for some aspects of trauma, 2) dis- sociative ﬂashbacks (i.e., reliving of the trauma, with reduced awareness of one’s current orientation), and 3) symptoms of intrusion and avoidance, negative alterations in cogni- tion and mood, and hyperarousal that are focused around the traumatic event. On the other hand, individuals with dissociative identity disorder manifest dissociative symptoms that are not a

1	in cogni- tion and mood, and hyperarousal that are focused around the traumatic event. On the other hand, individuals with dissociative identity disorder manifest dissociative symptoms that are not a manifestation of PTSD: 1) amnesias for many everyday (i.e., nontraumatic) events, 2) dissociative ﬂashbacks that may be followed by amnesia for the content of the ﬂashback, 3) disruptive intrusions (unrelated to traumatic material) by dissociated identity states into the individual’s sense of self and agency, and 4) infrequent, full-blown changes among different identity states.

1	Psychotic disorders. Dissociative identity disorder may be confused with schizophre- nia or other psychotic disorders. The personified, internally communicative inner voices of dissociative identity disorder, especially of a child (e.g., ”I hear a little girl crying in a closet and an angry man yelling at her"), may be mistaken for psychotic hallucinations.

1	Dissociative experiences of identity fragmentation or possession, and of perceived loss of control over thoughts, feelings, impulses, and acts, may be confused with signs of formal thought disorder, such as thought insertion or withdrawal. Individuals with dissociative identity disorder may also report visual, tactile, olfactory, gustatory, and somatic halluci- nations, which are usually related to posttraumatic and dissociative factors, such as partial ﬂashbacks. Individuals with dissociative identity disorder experience these symptoms as caused by alternate identities, do not have delusional explanations for the phenomena, and often describe the symptoms in a personified way (e.g., ”I feel like someone else wants to cry with my eyes”). Persecutory and derogatory internal voices in dissociative identity disorder associated with depressive symptoms may be misdiagnosed as major depression with psychotic features. Chaotic identity change and acute intrusions that disrupt thought

1	dissociative identity disorder associated with depressive symptoms may be misdiagnosed as major depression with psychotic features. Chaotic identity change and acute intrusions that disrupt thought processes may be distinguished from brief psychotic disorder by the predominance of dis- sociative symptoms and amnesia for the episode, and diagnostic evaluation after cessation of the crisis can help confirm the diagnosis.

1	Substance/medication-induced disorders. Symptoms associated with the physiological effects of a substance can be distinguished from dissociative identity disorder if the sub- stance in question is judged to be etiologically related to the disturbance. Personality disorders. Individuals with dissociative identity disorder often present identi- ties that appear to encapsulate a variety of severe personality disorder feahires, suggesting a differential diagnosis of personality disorder, especially of the borderline type. Importantly, however, the individual’s longitudinal variability in personality style (due to inconsistency among identities) differs from the pervasive and persistent dysfimction in affect management and interpersonal relationships typical of those with personality disorders.

1	Conversion disorder (functional neurological symptom disorder). This disorder may be distinguished from dissociative identity disorder by the absence of an identity disruption characterized by two or more distinct personality states or an experience of possession. Dissociative amnesia in conversion disorder is more limited and circumscribed (e.g., am- nesia for a non-epileptic seizure).

1	Dissociative amnesia in conversion disorder is more limited and circumscribed (e.g., am- nesia for a non-epileptic seizure). Seizure disorders. Individuals with dissociative identity disorder may present with sei- zurelike symptoms and behaviors that resemble complex partial seizures with temporal lobe foci. These include déja vu, jamais vu, depersonalization, derealization, out—of—body experiences, amnesia, disruptions of consciousness, hallucinations, and other intrusion phenomena of sensation, affect, and thought. Normal electroencephalographic findings, including telemetry, differentiate non—epileptic seizures from the seizurelike symptoms of dissociative identity disorder. Also, individuals with dissociative identity disorder obtain very high dissociation scores, whereas individuals with complex partial seizures do not.

1	Factitious disorder and malingering. Individuals who feign dissociative identity disor- der do not report the subtle symptoms of intrusion characteristic of the disorder; instead they tend to overreport well-publicized symptoms of the disorder, such as dissociative amnesia, while underreporting less-publicized comorbid symptoms, such as depression. Individuals who feign dissociative identity disorder tend to be relatively undisturbed by or may even seem to enjoy ”having” the disorder. In contrast, individuals with genuine dissociative identity disorder tend to be ashamed of and overwhelmed by their symptoms and to underreport their symptoms or deny their condition. Sequential observation, cor- roborating history, and intensive psychometric and psychological assessment may be helpful in assessment.

1	Individuals who malinger dissociative identity disorder usually create limited, stereo- typed alternate identities, with feigned amnesia, related to the events for which gain is sought. For example, they may present an ”all-good” identity and an "all-bad” identity in hopes of gaining exculpation for a crime. Many individuals with dissociative identity disorder present with a comorbid disorder. If not assessed and treated specifically for the dissociative disorder, these individuals often receive prolonged treatment for the comorbid diagnosis only, with limited overall treat- ment response and resultant demoralization, and disability.

1	Individuals with dissociative identity disorder usually exhibit a large number of co- morbid disorders. In particular, most develop PTSD. Other disorders that are highly co- morbid with dissociative identity disorder include depressive disorders, trauma- and stressor-related disorders, personality disorders (especially avoidant and borderline per- sonality disorders), conversion disorder (functional neurological symptom disorder), somatic symptom disorder, eating disorders, substance-related disorders, obsessive- compulsive disorder, and sleep disorders. Dissociative alterations in identity, memory, and consciousness may affect the symptom presentation of comorbid disorders. Diagnostic Criteria 300.12 (F44.0) A. An inability to recall important autobiographical information, usually of a traumatic or stressful nature, that is inconsistent with ordinary forgetting.

1	Diagnostic Criteria 300.12 (F44.0) A. An inability to recall important autobiographical information, usually of a traumatic or stressful nature, that is inconsistent with ordinary forgetting. Note: Dissociative amnesia most often consists of localized or selective amnesia tor a specific event or events; or generalized amnesia for identity and life history. B. The symptoms cause clinically significant distress or impairment in social, occupa— tional, or other important areas of functioning. C. The disturbance is not attributable to the physiological effects of a substance (e.g., al- cohol or other drug of abuse, a medication) or a neurological or other medical condition (e.g., partial complex seizures, transient global amnesia, sequelae of a closed head in- jury/traumatic brain injury, other neurological condition).

1	D. The disturbance is not better explained by dissociative identity disorder, posttraumatic stress disorder, acute stress disorder, somatic symptom disorder, or major or mild neu- rocognitive disorder. Coding note: The code for dissociative amnesia without dissociative fugue is 300.12 (F44.0). The code for dissociative amnesia with dissociative fugue is 300.13 (F44.1). Specify it: 300.13 (F44.1) With dissociative fugue: Apparently purposeful travel or bewildered wandering that is associated with amnesia for identity or for other important autobio- graphical information. The defining characteristic of dissociative amnesia is an inability to recall important auto- ily would be readily remembered (Criterion A). Dissociative amnesia differs from the permanent amnesias due to neurobiological damage or toxicity that prevent memory stor- age or retrieval in that it is always potentially reversible because the memory has been suc- cessfully stored.

1	Localized amnesia, a failure to recall events during a circumscribed period of time, is the most common form of dissociative amnesia. Localized amnesia may be broader than am- nesia for a single traumatic event (e.g., months or years associated with child abuse or in— tense combat). In selective amnesia, the individual can recall some, but not all, of the events during a circumscribed period of time. Thus, the individual may remember part of a trau- matic event but not other parts. Some individuals report both localized and selective am- nesias.

1	Generalized amnesia, a complete loss of memory for one’s life history, is rare. Individuals with generalized amnesia may forget personal identity. Some lose previous knowledge about the world (i.e., semantic knowledge) and can no longer access well-learned skills (i.e., procedural knowledge). Generalized amnesia has an acute onset; the perplexity, dis- orientation, and purposeless wandering of individuals with generalized amnesia usually bring them to the attention of the police or psychiatric emergency services. Generalized amnesia may be more common among combat veterans, sexual assault victims, and indi- viduals experiencing extreme emotional stress or conﬂict.

1	Individuals with dissociative amnesia are frequently unaware (or only partially aware) of their memory problems. Many, especially those with localized amnesia, minimize the importance of their memory loss and may become uncomfortable when prompted to ad- dress it. In systematized amnesia, the individual loses memory for a specific category of in- formation (e.g., all memories relating to one’s family, a particular person, or childhood sexual abuse). In continuous amnesia, an individual forgets each new event as it occurs.

1	Many individuals with dissociative amnesia are chronically impaired in their ability to form and sustain satisfactory relationships. Histories of trauma, child abuse, and victim- ization are common. Some individuals with dissociative amnesia report dissociative ﬂash- backs (i.e., behavioral reexperiencing of traumatic events). Many have a history of self- mutilation, suicide attempts, and other high-risk behaviors. Depressive and functional neurological symptoms are common, as are depersonalization, auto-hypnotic symptoms, and high hypnotizability. Sexual dysfunctions are common. Mild traumatic brain injury may precede dissociative amnesia. The 12-month prevalence for dissociative amnesia among adults in a small U.S. commu- nity study was 1.8% (1.0% for males; 2.6% for females). Onset of generalized amnesia is usually sudden. Less is known about the onset of localized and selective amnesias because these amnesias are seldom evident, even to the individual.

1	Onset of generalized amnesia is usually sudden. Less is known about the onset of localized and selective amnesias because these amnesias are seldom evident, even to the individual. Although overwhelming or intolerable events typically precede localized amnesia, its on- set may be delayed for hours, days, or longer.

1	Although overwhelming or intolerable events typically precede localized amnesia, its on- set may be delayed for hours, days, or longer. Individuals may report multiple episodes of dissociative amnesia. A single episode may predispose to future episodes. In between episodes of amnesia, the individual may or may not appear to be acutely symptomatic. The duration of the forgotten events can range from minutes to decades. Some episodes of dissociative amnesia resolve rapidly (e.g., when the person is removed from combat or some other stressful situation), whereas other episodes persist for long periods of time. Some individuals may gradually recall the dis- sociated memories years later. Dissociative capacities may decline with age, but not al- ways. As the amnesia remits, there may be considerable distress, suicidal behavior, and symptoms of posttraumatic stress disorder (PTSD). Dissociative amnesia has been observed in young children, adolescents, and adults.

1	Dissociative amnesia has been observed in young children, adolescents, and adults. Children may be the most difficult to evaluate because they often have difficulty under- standing questions about amnesia, and interviewers may find it difficult to formulate child- friendly questions about memory and amnesia. Observations of apparent dissociative am- nesia are often difficult to differentiate from inattention, absorption, anxiety, oppositional behavior, and learning disorders. Reports from several different sources (e.g., teacher, therapist, case worker) may be needed to diagnose amnesia in children.

1	Environmental. Single or repeated traumatic experiences (e.g., war, childhood maltreat- ment, natural disaster, internment in concentration camps, genocide) are common ante- cedents. Dissociative amnesia is more likely to occur with 1) a greater number of adverse childhood experiences, particularly physical and/ or sexual abuse, 2) interpersonal vio- lence; and 3) increased severity, frequency, and violence of the trauma. Genetic and physiological. There are no genetic studies of dissociative amnesia. Stud- ies of dissociation report significant genetic and environmental factors in both clinical and nonclinical samples.

1	Course modifiers. Removal from the traumatic circumstances underlying the dissociative amnesia (e.g., combat) may bring about a rapid return of memory. The memory loss of indi- viduals with dissociative fugue may be particularly refractory. Onset of PTSD symptoms may decrease localized, selective, or systematized amnesia. The returning memory, however, may be experienced as ﬂashbacks that alternate with amnesia for the content of the ﬂashbacks. In Asia, the Middle East, and Latin America, non-epileptic seizures and other functional neurological symptoms may accompany dissociative amnesia. In cultures with highly re- strictive social traditions, the precipitants of dissociative amnesia often do not involve frank trauma. Instead, the amnesia is preceded by severe psychological stresses or con- ﬂicts (e.g., marital conﬂict, other family disturbances, attachment problems, conﬂicts due to restriction or oppression).

1	Suicidal and other self-destructive behaviors are common in individuals with dissociative amnesia. Suicidal behavior may be a particular risk when the amnesia remits suddenly and overwhelms the individual with intolerable memories. Functional Consequences of Dissociative Amnesia The impairment of individuals with localized, selective, or systematized dissociative am- nesia ranges from limited to severe. Individuals with chronic generalized dissociative am- nesia usually have impairment in all aspects of functioning. Even when these individuals "re-learn" aspects of their life history, autobiographical memory remains very impaired. Most become vocationally and interpersonally disabled.

1	Most become vocationally and interpersonally disabled. Dissociative identity disorder. Individuals with dissociative amnesia may report de- personalization and auto-hypnotic symptoms. Individuals with dissociative identity dis- order report pervasive discontinuities in sense of self and agency, accompanied by many other dissociative symptoms. The amnesias of individuals with localized, selective, and/ or systematized dissociative amnesias are relatively stable. Amnesias in dissociative iden- tity disorder include amnesia for everyday events, finding of unexplained possessions, sudden ﬂuctuations in skills and knowledge, major gaps in recall of life history, and brief amnesic gaps in interpersonal interactions.

1	Posttraumatic stress disorder. Some individuals with PTSD cannot recall part or all of a specific traumatic event (e.g., a rape Victim with depersonalization and/ or derealization symptoms who cannot recall most events for the entire day of the rape). When that amne- sia extends beyond the immediate time of the trauma, a comorbid diagnosis of dissociative amnesia is warranted. Neurocognitive disorders. In neurocognitive disorders, memory loss for personal infor- mation is usually embedded in cognitive, linguistic, affective, attentional, and behavioral disturbances. In dissociative amnesia, memory deficits are primarily for autobiographical information; intellectual and cognitive abilities are preserved.

1	Substance-related disorders. In the context of repeated intoxication with alcohol or other substances/medications, there may be episodes of ”black outs” or periods for which the individual has no memory. To aid in distinguishing these episodes from dissociative am- nesia, a longitudinal history noting that the amnestic episodes occur only in the context of intoxication and do not occur in other situations would help identify the source as sub- stance-induced; however the distinction may be difficult when the individual with dis- sociative amnesia may also misuse alcohol or other substances in the context of stressful situations that may also exacerbate dissociative symptoms. Some individuals with comor- lems solely to the substance use. Prolonged use of alcohol or other substances may result in a substance-induced neurocognitive disorder that may be associated with impaired cog- nitive function, but in this context the protracted history of substance use and the persis- tent deficits

1	in a substance-induced neurocognitive disorder that may be associated with impaired cog- nitive function, but in this context the protracted history of substance use and the persis- tent deficits associated with the neurocognitive disorder would serve to distinguish it from dissociative amnesia, where there is typically no evidence of persistent impairment in intellectual functioning.

1	Posttraumatic amnesia due to brain injury. Amnesia may occur in the context of a trau- matic brain injury (TBI) when there has been an impact to the head or other mechanisms of rapid movement or displacement of the brain within the skull TBI. Other characteristics of TBI include loss of consciousness, disorientation and confusion, or, in more severe cases, neurological signs (e.g., abnormalities on neuroimaging, a new onset of seizures or a marked worsening of a preexisting seizure disorder, visual field cuts, anosmia). A neurocognitive disorder attributable to TBI must present either immediately after brain injury occurs or im- mediately after the individual recovers consciousness after the injury, and persist past the acute post-injury period. The cognitive presentation of a neurocognitive disorder following

1	TBI is variable and includes difficulties in the domains of complex attention, executive func- tion, learning and memory as well as slowed speed of information processing and distur- bances in social cognition. These additional features help distinguish it from dissociative amnes1a.

1	Seizure disorders. Individuals with seizure disorders may exhibit complex behavior dur- ing seizures or post—ictally with subsequent amnesia. Some individuals with a seizure disorder engage in nonpurposive wandering that is limited to the period of seizure activity. Con- versely, behavior during a dissociative fugue is usually purposeful, complex, and goal- directed and may last for days, weeks, or longer. Occasionally, individuals with a seizure dis- order will report that earlier autobiographical memories have been ”wiped out” as the seizure disorder progresses. Such memory loss is not associated with traumatic circumstances and ap— pears to occur randomly. Serial electroencephalogram usually show abnormalities. Telemet- ric electroencephalographic monitoring usually shows an association between the episodes of amnesia and seizure activity. Dissociative and epileptic amnesias may coexist.

1	Catatonic stupor. Mutism in catatonic stupor may suggest dissociative amnesia, but fail- ure of recall is absent. Other catatonic symptoms (e.g., rigidity, posturing, negativism) are usually present. Factitious disorder and malingering. There is no test, battery of tests, or set of procedures that invariably distinguishes dissociative amnesia from feigned amnesia. Individuals with factitious disorder or malingering have been noted to continue their deception even during hypnotic or barbiturate-facilitated interviews. Feigned amnesia is more common in individ- uals with 1) acute, ﬂorid dissociative amnesia; 2) financial, sexual, or legal problems; or 3) a wish to escape stressful circumstances. True amnesia can be associated with those same cir- cumstances. Many individuals who malinger confess spontaneously or when confronted.

1	Normal and age-related changes in memory. Memory decrements in major and mild neurocognitive disorders differ from those of dissociative amnesia, which are usually as— sociated with stressful events and are more specific, extensive, and / or complex. As dissociative amnesia begins to remit, a wide variety of affective phenomena may sur- face: dysphoria, grief, rage, shame, guilt, psychological conﬂict and turmoil, and suicidal and homicidal ideation, impulses, and acts. These individuals may have symptoms that der, with depressed mood; or adjustment disorder, with mixed disturbance of emotions and conduct. Many individuals with dissociative amnesia develop PTSD at some point during their life, especially when the traumatic antecedents of their amnesia are brought into conscious awareness.

1	Many individuals with dissociative amnesia have symptoms that meet diagnostic cri- teria for a comorbid somatic symptom or related disorder (and Vice versa), including so- disorder). Many individuals with dissociative amnesia have symptoms that meet diagnos- tic criteria for a personality disorder, especially dependent, avoidant, and borderline. Diagnostic Criteria 300.6 (F48.1) A. The presence of persistent or recurrent experiences of depersonalization, derealiza- tion, or both: 1. Depersonalization: Experiences of unreality, detachment, or being an outside ob- server with respect to one's thoughts. teelings, sensations, body, or actions (e.g., perceptual alterations, distorted sense of time, unreal or absent self, emotional and/ or physical numbing). 2. Derealization: Experiences of unreality or detachment with respect to surround- ings (e.g., individuals or objects are experienced as unreal, dreamlike, foggy, life- less, or visually distorted).

1	2. Derealization: Experiences of unreality or detachment with respect to surround- ings (e.g., individuals or objects are experienced as unreal, dreamlike, foggy, life- less, or visually distorted). B. During the depersonalization or derealization experiences, reality testing remains intact. C. The symptoms cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. D. The disturbance is not attributable to the physiological effects of a substance (e.g., a drug of abuse, medication) or another medical condition (e.g., seizures). E. The disturbance is not better explained by another mental disorder, such as schizo- phrenia, panic disorder, major depressive disorder. acute stress disorder, posttrau- matic stress disorder, or another dissociative disorder.

1	The essential features of depersonalization/derealization disorder are persistent or recur- rent episodes of depersonalization, derealization, or both. Episodes of depersonalization are characterized by a feeling of unreality or detachment from, or unfamiliarity with, one’s whole self or from aspects of the self (Criterion A1). The individual may feel detached from his or her entire being (e.g., ”I am no one,” ”I have no self”). He or she may also feel subjectively detached from aspects of the self, including feelings (e.g., hypoemotionality: ”I know I have feelings but I don’t feel them"), thoughts (e.g., ”My thoughts don’t feel like my own,” ”head\filled with cotton”), whole body or body parts, or sensations (e.g., touch, proprioception, hunger, thirst, libido). There may also be a diminished sense of agency (e.g., feeling robotic, like an automaton; lacking control of one’s speech or movements).

1	The depersonalization experience can sometimes be one of a split self, with one part ob- serving and one participating, known as an ”out-of—body experience" in its most extreme form. The unitary symptom of ”depersonalization” consists of several symptom factors: anomalous body experiences (i.e., unreality of the self and perceptual alterations); emo- tional or physical numbing; and temporal distortions with anomalous subjective recall.

1	Episodes of derealization are characterized by a feeling of unreality or detachment from, or unfamiliarity with, the world, be it individuals, inanimate objects, or all surround- ings (Criterion A2). The individual may feel as if he or she were in a fog, dream, or bubble, or as if there were a veil or a glass wall between the individual and world around. Surround- ings may be experienced as artificial, colorless, or lifeless. Derealization is commonly ac- companied by subjective visual distortions, such as blurriness, heightened acuity, widened or narrowed visual field, two-dimensionality or ﬂatness, exaggerated three-dimensional- ity, or altered distance or size of objects (i.e., macropsia or rnicropsia). Auditory distortions can also occur, whereby voices or sounds are muted or heightened. In addition, Criterion

1	C requires the presence of clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning, and Criteria D and E describe exclusionary diagnoses. Individuals with depersonalization/derealization disorder may have difficulty describ- ing their symptoms and may think they are ”crazy” or “going crazy”. Another common experience is the fear of irreversible brain damage. A commonly associated symptom is a subjectively altered sense of time (i.e., too fast or too slow), as well as a subjective difficulty in vividly recalling past memories and owning them as personal and emotional. Vague so— matic symptoms, such as head fullness, tingling, or lightheadedness, are not uncommon.

1	Individuals may suffer extreme rumination or obsessional preoccupation (e.g., constantly obsessing about whether they really exist, or checking their perceptions to determine whether they appear real). Varying degrees of anxiety and depression are also common as- sociated features. Individuals with the disorder have been found to have physiological hyporeactivity to emotional stimuli. Neural substrates of interest include the hypotha- lamic-pituitary-adrenocortical axis, inferior parietal lobule, and prefrontal cortical-lirnbic circuits.

1	Transient depersonalization/ derealization symptoms lasting hours to days are common in the general population. The 12-month prevalence of depersonalization/derealization disorder is thought to be markedly less than for transient symptoms, although precise es- timates for the disorder are unavailable. In general, approximately one-half of all adults have experienced at least one lifetime episode of depersonalization/ derealization. How- ever, symptomatology that meets full criteria for depersonalization/derealization disor- der is markedly less common than transient symptoms. Lifetime prevalence in US. and non-U.S. countries is approximately 2% (range of 0.8% to 2.8%). The gender ratio for the disorder is 1:1.

1	The mean age at onset of depersonalization/ derealization disorder is 16 years, although the the symptoms. Less than 20% of individuals experience onset after age 20 years and only 5% after age 25 years. Onset in the fourth decade of life or later is highly unusual. Onset can range from extremely sudden to gradual. Duration of depersonalization/derealization disorder episodes can vary greatly, from brief (hours or days) to prolonged (weeks, months, or years). Given the rarity of disorder onset after age 40 years, in such cases the in- dividual should be examined more closely for underlying medical conditions (e.g., brain lesions, seizure disorders, sleep apnea). The course of the disorder is often persistent. About one-third of cases involve discrete episodes; another third, continuous symptoms from the start; and still another third, an initially episodic course that eventually becomes continuous.

1	About one-third of cases involve discrete episodes; another third, continuous symptoms from the start; and still another third, an initially episodic course that eventually becomes continuous. While in some individuals the intensity of symptoms can wax and wane considerably, others report an unwavering level of intensity that in extreme cases can be constantly pres- ent for years or decades. Internal and external factors that affect symptom intensity vary between individuals, yet some typical patterns are reported. Exacerbations can be trig- gered by stress, worsening mood or anxiety symptoms, novel or overstimulating settings, and physical factors such as lighting or lack of sleep.

1	Temperamental. Individuals with depersonalization/derealization disorder are charac- terized by harm—avoidant temperament, immature defenses, and both disconnection and overconnection schemata. Immature defenses such as idealization/devaluation, projec- tion and acting out result in denial of reality and poor adaptation. Cognitive disconnection schemata reﬂect defectiveness and emotional inhibition and subsume themes of abuse, ne- glect, and deprivation. Overconnection schemata involve impaired autonomy with themes of dependency, vulnerability, and incompetence.

1	Environmental. There is a clear association between the disorder and childhood interper- sonal traumas in a substantial portion of individuals, although this association is not as prev- alent or as extreme in the nature of the traumas as in other dissociative disorders, such as dissociative identity disorder. In particular, emotional abuse and emotional neglect have been most strongly and consistently associated with the disorder. Other stressors can include phys- ical abuse; witnessing domestic violence; growing up with a seriously impaired, mentally ill parent; or unexpected death or suicide of a family member or close friend. Sexual abuse is a much less common antecedent but can be encountered. The most common proximal precipi- tants of the disorder are severe stress (interpersonal, financial, occupational), depression, anx- iety (particularly panic attacks), and illicit drug use. Symptoms may be specifically induced by substances such as tetrahydrocannabinol, hallucinogens,

1	financial, occupational), depression, anx- iety (particularly panic attacks), and illicit drug use. Symptoms may be specifically induced by substances such as tetrahydrocannabinol, hallucinogens, ketamine, MDMA (3,4-methylene— dioxymethamphetamine; ”ecstasy”) and salvia. Marijuana use may precipitate new-onset panic attacks and depersonalization/derealization symptoms simultaneously.

1	Volitionally induced experiences of depersonalization/ derealization can be a part of med- itative practices that are prevalent in many religions and cultures and should not be diag- nosed as a disorder. However, there are individuals who initially induce these states related practices. Symptoms of depersonalization/ derealization disorder are highly distressing and are as- sociated with major morbidity. The affectively ﬂattened and robotic demeanor that these individuals often demonstrate may appear incongruent with the extreme emotional pain reported by those with the disorder. Impairment is often experienced in both interpersonal and occupational spheres, largely due to the hypoemotionality with others, subjective diffi- culty in focusing and retaining information, and a general sense of disconnectedness from life.

1	Illness anxiety disorder. Although individuals with depersonalization/derealization dis- order can present with vague somatic complaints as well as fears of permanent brain dam- age, the diagnosis of depersonalization/derealization disorder is characterized by the presence of a constellation of typical depersonalization/derealization symptoms and the ab- sence of other manifestations of illness anxiety disorder. Major depressive disorder. Feelings of numbness, deadness, apathy, and being in a dream are not uncommon in major depressive episodes. However, in depersonalization/ derealization disorder, such symptoms are associated with further symptoms of the dis- order. If the depersonalization/ derealization clearly precedes the onset of a major depres- sive episode or clearly continues after its resolution, the diagnosis of depersonalization/ derealization disorder applies.

1	Obsessive-compulsive disorder. Some individuals with depersonalization/ dereal- ization disorder can become obsessively preoccupied with their subjective experience or develop rituals checking on the status of their symptoms. However, other symptoms of obsessive-compulsive disorder unrelated to depersonalization/derealization are not present. Other dissociative disorders. In order to diagnose depersonalization/ derealization disorder, the symptoms should not occur in the context of another dissociative disorder, such as dissociative identity disorder. Differentiation from dissociative amnesia and con- version disorder (functional neurological symptom disorder) is simpler, as the symptoms of these disorders do not overlap with those of depersonalization/ derealization disorder.

1	Anxiety disorders. Depersonalization/derealization is one of the symptoms of panic at- tacks, increasingly common as panic attack severity increases. Therefore, depersonal— ization/derealization disorder should not be diagnosed when the symptoms occur only during panic attacks that are part of panic disorder, social anxiety disorder, or specific phobia. In addition, it is not uncommon for depersonalization/derealization symptoms to first begin in the context of new-onset panic attacks or as panic disorder progresses and worsens. In such presentations, the diagnosis of depersonalization/ derealization disorder can be made if 1) the depersonalization/ derealization component of the presentation is very prominent from the start, clearly exceeding in duration and intensity the occurrence of actual panic attacks; or 2) the depersonalization/ derealization continues after panic dis- order has remitted or has been successfully treated.

1	Psychotic disorders. The presence of intact reality testing specifically regarding the depersonalization/derealization symptoms is essential to differentiating depersonal- ization/derealization disorder from psychotic disorders. Rarely, positive-symptom schizophrenia can pose a diagnostic challenge when nihilistic delusions are present. For example, an individual may complain that he or she is dead or the world is not real; this could be either a subjective experience that the individual knows is not true or a delusional conviction.

1	Substance/medication-induced disorders. Depersonalization/derealization associated with the physiological effects of substances during acute intoxication or withdrawal is not diagnosed as depersonalization/derealization disorder. The most common precipitating substances are the illicit drugs marijuana, hallucinogens, ketamine, ecstasy, and salvia. In about 15% of all cases of depersonalization/derealization disorder, the symptoms are pre- cipitated by ingestion of such substances. If the symptoms persist for some time in the ab- sence of any further substance or medication use, the diagnosis of depersonalization/ derealization disorder applies. This diagnosis is usually easy to establish since the vast ma- jority of individuals with this presentation become highly phobic and aversive to the trig- gering substance and do not use it again.

1	Mental disorders due to another medical condition. Features such as onset after age 40 years or the presence of atypical symptoms and course in any individual suggest the possibility of an underlying medical condition. In such cases, it is essential to conduct a thorough medical and neurological evaluation, which may include standard laboratory studies, Viral titers, an electroencephalogram, vestibular testing, visual testing, sleep stud- ies, and/ or brain imaging. When the suspicion of an underlying seizure disorder proves difficult to confirm, an ambulatory electroencephalogram may be indicated; although temporal lobe epilepsy is most commonly implicated, parietal and frontal lobe epilepsy may also be associated.

1	In a convenience sample of adults recruited for a number of depersonalization research studies, lifetime comorbidities were high for unipolar depressive disorder and for any anxiety disorder, with a significant proportion of the sample having both disorders. Comor- bidity with posttraumatic stress disorder was low. The three most commonly co-occurring personality disorders were avoidant, borderline, and obsessive-compulsive. 300.15 (F44.89)

1	300.15 (F44.89) This category applies to presentations in which symptoms characteristic of a dissociative disorder that cause clinically significant distress or impairment in social. occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the dissociative disorders diagnostic class. The other specified dissocia- tive disorder category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for any specific disso- ciative disorder. This is done by recording “other specified dissociative disorder” followed by the specific reason (e.g., “dissociative trance").

1	Examples of presentations that can be specified using the “other specified" designation include the following: 1. Chronic and recurrent syndromes of mixed dissociative symptoms: This cate- gory includes identity disturbance associated with less-than-marked discontinuities in sense of self and agency, or alterations of identity or episodes of possession in an in- dividual who reports no dissociative amnesia. 2. Identity disturbance due to proionged and intense coerclve persuasion: Individ- uals who have been subjected to intense coercive persuasion (e.g., brainwashing, thought reform, indoctrination while captive, torture, long-term political imprisonment, recruitment by sects/cults or by terror organizations) may present with prolonged changes in. or conscious questioning of, their identity.

1	3. Acute dissociative reactions to stressfui events: This category is for acute, tran- sient conditions that typically last less than 1 month, and sometimes only a few hours or days. These conditions are characterized by constriction of consciousness; deper- sonalization; derealization; perceptual disturbances (e.g., time slowing. macropsia); micro-amnesias; transient stupor; and/or alterations in sensory-motor functioning (e.g., analgesia, paralysis).

1	4. Dissociative trance: This condition is characterized by an acute narrowing or com— plete loss of awareness of immediate surroundings that manifests as profound unre- sponsiveness or insensitivity to environmental stimuli. The unresponsiveness may be accompanied by minor stereotyped behaviors (e.g., finger movements) of which the in- dividual is unaware and/or that he or she cannot control, as well as transient paralysis or loss of consciousness. The dissociative trance is not a normal part of a broadly ac- cepted collective cultural or religious practice. 300.15 (F44.9)

1	300.15 (F44.9) This category applies to presentations in which symptoms characteristic of a dissociative disorder that cause clinically significant distress or impairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any 01 the disorders in the dissociative disorders diagnostic class. The unspecified dissociative disorder category is used in situations in which the clinician chooses not to specify the rea- son that the criteria are not met for a specific dissociative disorder, and includes presen- tations for which there is insufficient information to make a more specific diagnosis (e.g., in emergency room settings). Somatic, T‘_

1	Somatic sym ptom d isorder and other disorders with prominent somatic symp- toms constitute a new category in DSM-S called somatic symptom and related disorders. This chapter includes the diagnoses of somatic symptom disorder, illness anxiety disorder, con- version disorder (functional neurological symptom disorder), psychological factors affect- ing other medical conditions, factitious disorder, other specified somatic symptom and related disorder, and unspecified somatic symptom and related disorder. All of the disor- ders in this chapter share a common feature: the prominence of somatic symptoms associ— ated with significant distress and impairment. Individuals with disorders with prominent somatic symptoms are commonly encountered in primary care and other medical settings but are less commonly encountered in psychiatric and other mental health settings. These reconceptualized diagnoses, based on a reorganization of DSM-IV somatoform disorder di- agnoses, are more useful for

1	less commonly encountered in psychiatric and other mental health settings. These reconceptualized diagnoses, based on a reorganization of DSM-IV somatoform disorder di- agnoses, are more useful for primary care and other medical (nonpsychiatric) clinicians.

1	The major diagnosis in this diagnostic class, somatic symptom disorder, emphasizes diagnosis made on the basis of positive symptoms and signs (distressing somatic symp- toms plus abnormal thoughts, feelings, and behaviors in response to these symptoms) rather than the absence of a medical explanation for somatic symptoms. A distinctive char- acteristic of many individuals with somatic symptom disorder is not the somatic symp- toms per se, but instead the way they present and interpret them. Incorporating affective, cognitive, and behavioral components into the criteria for somatic symptom disorder pro— Vides a more comprehensive and accurate reﬂection of the true clinical picture than can be achieved by assessing the somatic complaints alone. The principles behind the changes in the somatic symptom and related diagnoses from

1	The principles behind the changes in the somatic symptom and related diagnoses from DSM-IV are crucial in understanding the DSM-S diagnoses. The DSM-IV term somatoform disorders was confusing and is replaced by somatic symptom and related disorders. In DSM-IV there was a great deal of overlap across the somatoform disorders and a lack of clarity about the boundaries of diagnoses. Although individuals with these disorders primarily present in medical rather than mental health settings, nonpsychiatric physicians found the DSM-IV somatoform diagnoses difficult to understand and use. The current DSM-S clas- sification recognizes this overlap by reducing the total number of disorders as well as their subcategories. The previous criteria overemphasized the centrality of medically unexplained symptoms.

1	Such symptoms are present to various degrees, particularly in conversion disorder, but so- matic symptom disorders can also accompany diagnosed medical disorders. The reli- ability of determining that a somatic symptom is medically unexplained is limited, and grounding a diagnosis on the absence of an explanation is problematic and reinforces mind-body dualism. It is not appropriate to give an individual a mental disorder diagnosis solely because a medical cause cannot be demonstrated. Furthermore, the presence of a medical diagnosis does not exclude the possibility of a comorbid mental disorder, includ- ing a somatic symptom and related disorder. Perhaps because of the predominant focus on lack of medical explanation, individuals regarded these diagnoses as pejorative and de- meaning, implying that their physical symptoms were not ”real." The new classification defines the major diagnosis, somatic symptom disorder, on the basis of positive symptoms (distressing somatic symptoms plus

1	that their physical symptoms were not ”real." The new classification defines the major diagnosis, somatic symptom disorder, on the basis of positive symptoms (distressing somatic symptoms plus abnormal thoughts, feelings, and behaviors in response to these symptoms). However, medically unexplained symptoms remain a key feature in order) because it is possible to demonstrate definitively in such disorders that the symp- toms are not consistent with medical pathophysiology.

1	It is important to note that some other mental disorders may initially manifest with pri- marily somatic symptoms (e.g., major depressive disorder, panic disorder). Such diagno- ses may account for the somatic symptoms, or they may occur alongside one of the somatic symptom and related disorders in this chapter. There is also considerable medical comor- bidity among somatizing individuals. Although somatic symptoms are frequently associ— ated with psychological distress and psychopathology, some somatic symptom and related disorders can arise spontaneously, and their causes can remain obscure. Anxiety ders. The somatic component adds severity and complexity to depressive and anxiety dis- orders and results in higher severity, functional impairment, and even refractoriness to traditional treatments. In rare instances, the degree of preoccupation may be so severe as to warrant consideration of a delusional disorder diagnosis.

1	A number of factors may contribute to somatic symptom and related disorders. These include genetic and biological vulnerability (e.g., increased sensitivity to pain), early trau- matic experiences (e.g., violence, abuse, deprivation), and learning (e.g., attention ob- tained from illness, lack of reinforcement of nonsomatic expressions of distress), as well as cultural/social norms that devalue and stigmatize psychological suffering as compared with physical suffering. Differences in medical care across cultures affect the presentation, recognition, and management of these somatic presentations. Variations in symptom pre- sentation are likely the result of the interaction of multiple factors within cultural con- texts that affect how individuals identify and classify bodily sensations, perceive illness, and seek medical attention for them. Thus, somatic presentations can be viewed as expres- sions of personal suffering inserted in a cultural and social context.

1	All of these disorders are characterized by the prominent focus on somatic concerns and their initial presentation mainly in medical rather than mental health care settings. 50- matic symptom disorder offers a more clinically useful method of characterizing individ- uals who may have been considered in the past for a diagnosis of somatization disorder.

1	Furthermore, approximately 75% of individuals previously diagnosed with hypochon- driasis are subsumed under the diagnosis of somatic symptom disorder. However, about 25% of individuals with hypochondriasis have high health anxiety in the absence of so- matic symptoms, and many such individuals’ symptoms would not qualify for an anxiety disorder diagnosis. The DSM-5 diagnosis of ilhiess anxiety disorder is for this latter group of individuals. Illness anxiety disorder can be considered either in this diagnostic section or as an anxiety disorder. Because of the strong focus on somatic concerns, and because ill- ness anxiety disorder is most often encountered in medical settings, for utility it is listed with the somatic symptom and related disorders. In conversion disorder, the essential fea— ture is neurological symptoms that are found, after appropriate neurological assessment, to be incompatible with neurological pathophysiology. Psychological factors affecting other medical

1	fea— ture is neurological symptoms that are found, after appropriate neurological assessment, to be incompatible with neurological pathophysiology. Psychological factors affecting other medical conditions is also included in this chapter. Its essential feature is the pres- ence of one or more clinically significant psychological or behavioral factors that adversely affect a medical condition by increasing the risk for suffering, death, or disability. Like the other somatic symptom and related disorders, factitious disorder embodies persistent problems related to illness perception and identity. In the great majority of reported cases of factitious disorder, both imposed on self and imposed on another, individuals present with somatic symptoms and medical disease conviction. Consequently, DSM-5 factitious disorder is included among the somatic symptom and related disorders. Other specified order include conditions for which some, but not all, of the criteria for somatic symptom

1	DSM-5 factitious disorder is included among the somatic symptom and related disorders. Other specified order include conditions for which some, but not all, of the criteria for somatic symptom disorder or illness anxiety disorder are met, as well as pseudocyesis.

1	. Somatic Symptom Disorder Diagnostic Criteria 300.82 (F45.1) A. One or more somatic symptoms that are distressing or result in significant disruption of daily life. B. Excessive thoughts. feelings. or behaviors related to the somatic symptoms or associ- ated health concerns as manifested by at least one of the following: 1. Disproportionate and persistent thoughts about the seriousness of one’s symptoms. 2. Persistently high level of anxiety about health or symptoms. 3. Excessive time and energy devoted to these symptoms or health concerns. C. Although any one somatic symptom may not be continuously present. the state of be- ing symptomatic is persistent (typically more than 6 months). Specify it: With predominant pain (previously pain disorder): This specifier is for individuals whose somatic symptoms predominantly involve pain. Specify if: Persistent: A persistent course is characterized by severe symptoms, marked impair- ment, and long duration (more than 6 months).

1	Specify if: Persistent: A persistent course is characterized by severe symptoms, marked impair- ment, and long duration (more than 6 months). Specify current severity: Mild: Only one of the symptoms specified in Criterion B is fulfilled. Moderate: Two or more of the symptoms specified in Criterion B are fulfilled. Severe: Two or more of the symptoms specified in Criterion B are fulfilled, plus there are multiple somatic complaints (or one very severe somatic symptom).

1	Severe: Two or more of the symptoms specified in Criterion B are fulfilled, plus there are multiple somatic complaints (or one very severe somatic symptom). Individuals with somatic symptom disorder typically have multiple, current, somatic symp- toms that are distressing or result in significant disruption of daily life (Criterion A), al- though sometimes only one severe symptom, most commonly pain, is present. Symptoms may be specific (e.g., localized pain) or relatively nonspecific (e.g., fatigue). The symptoms nify serious disease. Somatic symptoms without an evident medical explanation are not sufficient to make this diagnosis. The individual’s suffering is authentic, whether or not it is medically explained.

1	The symptoms may or may not be associated with another medical condition. The di- agnoses of somatic symptom disorder and a concurrent medical illness are not mutually exclusive, and these frequently occur together. For example, an individual may become se- riously disabled by symptoms of somatic symptom disorder after an uncomplicated myo- cardial infarction even if the myocardial infarction itself did not result in any disability. If another medical condition or high risk for developing one is present (e.g., strong family history), the thoughts, feelings, and behaviors associated with this condition are excessive (Criterion B).

1	Individuals with somatic symptom disorder tend to have very high levels of worry about illness (Criterion B). They appraise their bodily symptoms as unduly threatening, harmful, or troublesome and often think the worst about their health. Even when there is evidence to the contrary, some patients still fear the medical seriousness of their symp- toms. In severe somatic symptom disorder, health concerns may assume a central role in the individual’s life, becoming a feature of his or her identity and dominating interper- sonal relationships.

1	Individuals typically experience distress that is principally focused on somatic symp- toms and their significance. When asked directly about their distress, some individuals de- scribe it in relation to other aspects of their lives, while others deny any source of distress other than the somatic symptoms. Health—related quality of life is often impaired, both physically and mentally. In severe somatic symptom disorder, the impairment is marked, and when persistent, the disorder can lead to invalidism.

1	There is often a high level of medical care utilization, which rarely alleviates the individ- ual’s concerns. Consequently, the patient may seek care from multiple doctors for the same symptoms. These individuals often seem unresponsive to medical interventions, and new interventions may only exacerbate the presenting symptoms. Some individuals with the dis- order seem unusually sensitive to medication side effects. Some feel that their medical as- sessment and treatment have been inadequate.

1	Cognitive features include attention focused on somatic symptoms, attribution of normal bodily sensations to physical illness (possibly with catastrophic interpretations), worry about illness, and fear that any physical activity may damage the body. The relevant as- sociated behavioral features may include repeated bodily checking for abnormalities, re— peated seeking of medical help and reassurance, and avoidance of physical activity. These behavioral features are most pronounced in severe, persistent somatic symptom disorder.

1	These features are usually associated with frequent requests for medical help for different somatic symptoms. This may lead to medical consultations in which individuals are so fo- cused on their concerns about somatic symptom(s) that they cannot be redirected to other matters. Any reassurance by the doctor that the symptoms are not indicative of serious physical illness tends to be short-lived and/ or is experienced by the individuals as the doctor not taking their symptoms with due seriousness. As the focus on somatic symp- toms is a primary feature of the disorder, individuals with somatic symptom disorder typ- ically present to general medical health services rather than mental health services. The suggestion of referral to a mental health specialist may be met with surprise or even frank refusal by individuals with somatic symptom disorder.

1	Since somatic symptom disorder is associated with depressive disorders, there is an in- creased suicide risk. It is not known whether somatic symptom disorder is associated with suicide risk independent of its association with depressive disorders. The prevalence of somatic symptom disorder is not known. However, the prevalence of somatic symptom disorder is expected to be higher than that of the more restrictive DSM- IV somatization disorder (<1%) but lower than that of undifferentiated somatoform dis- order (approximately 19%). The prevalence of somatic symptom disorder in the general adult population may be around 5%—7%. Females tend to report more somatic symptoms than do males, and the prevalence of somatic symptom disorder is consequently likely to be higher in females.

1	In older individuals, somatic symptoms and concurrent medical illnesses are common, and a focus on Criterion B is crucial for making the diagnosis. Somatic symptom disorder may be underdiagnosed in older adults either because certain somatic symptoms (e.g., pain, fatigue) are considered part of normal aging or because illness worry is considered tions than do younger people. Concurrent depressive disorder is common in older people who present with numerous somatic symptoms.

1	In children, the most common symptoms are recurrent abdominal pain, headache, fa- tigue, and nausea. A single prominent symptom is more common in children than in adults. While young children may have somatic complaints, they rarely worry about ”ill- ness” per se prior to adolescence. The parents’ response to the symptom is important, as this may determine the level of associated distress. It is the parent who may determine the interpretation of symptoms and the associated time off school and medical help seeking. Temperamental. The personality trait of negative affectivity (neuroticism) has been identi- fied as an independent correlate/ risk factor of a high number of somatic symptoms. Comorbid anxiety or depression is common and may exacerbate symptoms and impairment. Environmental. Somatic symptom disorder is more frequent in individuals with few years of education and low socioeconomic status, and in those who have recently experienced stressful life events.

1	Environmental. Somatic symptom disorder is more frequent in individuals with few years of education and low socioeconomic status, and in those who have recently experienced stressful life events. Course modifiers. Persistent somatic symptoms are associated with demographic fea- tures (female sex, older age, fewer years of education, lower socioeconomic status, un- employment), a reported history of sexual abuse or other childhood adversity, concurrent chronic physical illness or psychiatric disorder (depression, anxiety, persistent depressive disorder [dysthymia], panic), social stress, and reinforcing social factors such as illness benefits. Cognitive factors that affect clinical course include sensitization to pain, height- ened attention to bodily sensations, and attribution of bodily symptoms to a possible med- ical illness rather than recognizing them as a normal phenomenon or psychological stress.

1	Somatic symptoms are prominent in various ”culture—bound syndromes.” High numbers of somatic symptoms are found in population-based and primary care studies around the world, with a similar pattern of the most commonly reported somatic symptoms, impair- ment, and treatment seeking. The relationship between number of somatic symptoms and illness worry is similar in different cultures, and marked illness worry is associated with impairment and greater treatment seeking across cultures. The relationship between nu- merous somatic symptoms and depression appears to be very similar around the world and between different cultures within one country.

1	Despite these similarities, there are differences in somatic symptoms among cultures and ethnic groups. The description of somatic symptoms varies with linguistic and other local cultural factors. These somatic presentations have been described as ”idioms of dis- tress” because somatic symptoms may have special meanings and shape patient-clinician interactions in the particular cultural contexts. ”Burnout,” the sensation of heaviness or the complaints of ”gas”; too much heat in the body; or burning in the head are examples of symptoms that are common in some cultures or ethnic groups but rare in others. Explan- atory models also vary, and somatic symptoms may be attributed variously to particular family, work, or environmental stresses; general medical illness; the suppression of feel- ings of anger and resentment; or certain culture-specific phenomena, such as semen 1055.

1	There may also be differences in medical treatment seeking among cultural groups, in ad- dition to differences due to variable access to medical care services. Seeking treatment for multiple somatic symptoms in general medical clinics is a worldwide phenomenon and occurs at similar rates among ethnic groups in the same country. Functional Consequences of Somatic Symptom Disorder The disorder is associated with marked impairment of health status. Many individuals with severe somatic symptom disorder are likely to have impaired health status scores more than 2 standard deviations below population norms.

1	If the somatic symptoms are consistent with another mental disorder (e.g., panic disorder), and the diagnostic criteria for that disorder are fulfilled, then that mental disorder should be considered as an alternative or additional diagnosis. A separate diagnosis of somatic symptom disorder is not made if the somatic symptoms and related thoughts, feelings, or behaviors occur only during major depressive episodes. If, as commonly occurs, the crite- ria for both somatic symptom disorder and another mental disorder diagnosis are ful- filled, then both should be coded, as both may require treatment.

1	Other medical conditions. The presence of somatic symptoms of unclear etiology is not in itself sufficient to make the diagnosis of somatic symptom disorder. The symptoms of many individuals with disorders like irritable bowel syndrome or fibromyalgia would not satisfy the criterion necessary to diagnose somatic symptom disorder (Criterion B). Con- versely, the presence of somatic symptoms of an established medical disorder (e.g., diabe- tes or heart disease) does not exclude the diagnosis of somatic symptom disorder if the criteria are otherwise met. Panic disorder. In panic disorder, somatic symptoms and anxiety about health tend to occur in acute episodes, whereas in somatic symptom disorder, anxiety and somatic symp- toms are more persistent.

1	Panic disorder. In panic disorder, somatic symptoms and anxiety about health tend to occur in acute episodes, whereas in somatic symptom disorder, anxiety and somatic symp- toms are more persistent. Generalized anxiety disorder. Individuals with generalized anxiety disorder worry about multiple events, situations, or activities, only one of which may involve their health. The main focus is not usually somatic symptoms or fear of illness as it is in somatic symptom disorder. Depressive disorders. Depressive disorders are commonly accompanied by somatic symptoms. However, depressive disorders are differentiated from somatic symptom dis- order by the core depressive symptoms of low (dysphoric) mood and anhedonia. Illness anxiety disorder. If the individual has extensive worries about health but no or minimal somatic symptoms, it may be more appropriate to consider illness anxiety disorder.

1	Illness anxiety disorder. If the individual has extensive worries about health but no or minimal somatic symptoms, it may be more appropriate to consider illness anxiety disorder. Conversion disorder (functional neurological symptom disorder). In conversion disor- der, the presenting symptom is loss of function (e.g., of a limb), whereas in somatic symp— tom disorder, the focus is on the distress that particular symptoms cause. The features listed under Criterion B of somatic symptom disorder may be helpful in differentiating the two disorders. Delusional disorder. In somatic symptom disorder, the individual’s beliefs that somatic symptoms might reﬂect serious underlying physical illness are not held with delusional intensity. Nonetheless, the individual’s beliefs concerning the somatic symptoms can be firmly held. In contrast, in delusional disorder, somatic subtype, the somatic symptom be- liefs and behavior are stronger than those found in somatic symptom disorder.

1	Body dysmorphic disorder. In body dysmorphic disorder, the individual is excessively concerned about, and preoccupied by, a perceived defect in his or her physical features. In contrast, in somatic symptom disorder, the concern about somatic symptoms reﬂects fear of underlying illness, not of a defect in appearance. Obsessive—compulsive disorder. In somatic symptom disorder, the recurrent ideas about somatic symptoms or illness are less intrusive, and individuals with this disorder do not exhibit the associated repetitive behaviors aimed at reducing anxiety that occur in obses- sive—compulsive disorder.

1	Somatic symptom disorder is associated with high rates of comorbidity with medical dis- orders as well as anxiety and depressive disorders. When a concurrent medical illness is present, the degree of impairment is more marked than would be expected from the phys- symptom disorder, the disorder should be diagnosed; however, in view of the frequent co- morbidity, especially with anxiety and depressive disorders, evidence for these concur- rent diagnoses should be sought. Diagnostic Criteria 300.7 (F45.21) A. Preoccupation with having or acquiring a serious illness. B. Somatic symptoms are not present or, if present, are only mild in intensity. If another medical condition is present or there is a high risk for developing a medical condition (e.g., strong family history is present), the preoccupation is clearly excessive or dispro- portionate. C. There is a high level of anxiety about health, and the individual is easily alarmed about personal health status.

1	C. There is a high level of anxiety about health, and the individual is easily alarmed about personal health status. D. The individual performs excessive health-related behaviors (e.g., repeatedly checks his or her body for signs of illness) or exhibits maladaptive avoidance (e.g., avoids doc- tor appointments and hospitals). E. Illness preoccupation has been present for at least 6 months, but the specific illness that is feared may change over that period of time. F. The illness-related preoccupation is not better explained by another mental disorder, such as somatic symptom disorder, panic disorder, generalized anxiety disorder, body dysmor- phic disorder, obsessive-compulsive disorder, or delusional disorder, somatic type. Specify whether: Care-seeking type: Medical care, including physician visits or undergoing tests and procedures, is frequently used. Care—avoidant type: Medical care is rarely used.

1	Most individuals with hypochondriasis are now classified as having somatic symptom disorder; however, in a minority of cases, the diagnosis of illness anxiety disorder applies instead. Illness anxiety disorder entails a preoccupation with having or acquiring a seri- ous, undiagnosed medical illness (Criterion A). Somatic symptoms are not present or, if present, are only mild in intensity (Criterion B). A thorough evaluation fails to identify a serious medical condition that accounts for the individual’s concerns. While the concern may be derived from a nonpathological physical sign or sensation, the individual’s dis- tress emanates not primarily from the physical complaint itself but rather from his or her anxiety about the meaning, significance, or cause of the complaint (i.e., the suspected med- ical diagnosis). If a physical sign or symptom is present, it is often a normal physiological sensation (e.g., orthostatic dizziness), a benign and self—limited dysfunction (e.g., transient

1	med- ical diagnosis). If a physical sign or symptom is present, it is often a normal physiological sensation (e.g., orthostatic dizziness), a benign and self—limited dysfunction (e.g., transient tinnitus), or a bodily discomfort not generally considered indicative of disease (e.g., belch- ing). If a diagnosable medical condition is present, the individual’s anxiety and preoccu- pation are clearly excessive and disproportionate to the severity of the condition (Criterion

1	B). Empirical evidence and existing literature pertain to previously defined DSM hypo- chondriasis, and it is unclear to what extent and how precisely they apply to the descrip- tion of this new diagnosis.

1	The preoccupation with the idea that one is sick is accompanied by substantial anxiety about health and disease (Criterion C). Individuals with illness anxiety disorder are easily alarmed about illness, such as by hearing about someone else falling ill or reading a health- related news story. Their concerns about undiagnosed disease do not respond to appro— priate medical reassurance, negative diagnostic tests, or benign course. The physician’s at- tempts at reassurance and symptom palliation generally do not alleviate the individual’s concerns and may heighten them. Illness concerns assume a prominent place in the indi- vidual’s life, affecting daily activities, and may even result in invalidism. Illness becomes a central feature of the individual’s identity and self—image, a frequent topic of social dis- course, and a characteristic response to stressful life events. Individuals with the disorder often examine themselves repeatedly (e.g., examining one’s throat in the mirror)

1	topic of social dis- course, and a characteristic response to stressful life events. Individuals with the disorder often examine themselves repeatedly (e.g., examining one’s throat in the mirror) (Crite- rion D). They research their suspected disease excessively (e.g., on the Internet) and re- peatedly seek reassurance from family, friends, or physicians. This incessant worrying often becomes frustrating for others and may result in considerable strain within the family. In some cases, the anxiety leads to maladaptive avoidance of situations (e.g., visiting sick family members) or activities (e.g., exercise) that these individuals fear might jeopardize their health.

1	Because they believe they are medically ill, individuals with illness anxiety disorder are encountered far more frequently in medical than in mental health settings. The majority of individuals with illness anxiety disorder have extensive yet unsatisfactory medical care, though some may be too anxious to seek medical attention. They generally have elevated rates of medical utilization but do not utilize mental health services more than the general population. They often consult multiple physicians for the same problem and obtain re- peatedly negative diagnostic test results. At times, medical attention leads to a paradoxical exacerbation of anxiety or to iatrogenic complications from diagnostic tests and proce- dures. Individuals with the disorder are generally dissatisfied with their medical care and find it unhelpful, often feeling they are not being taken seriously by physicians. At times, these concerns may be justified, since physicians sometimes are dismissive or respond with

1	care and find it unhelpful, often feeling they are not being taken seriously by physicians. At times, these concerns may be justified, since physicians sometimes are dismissive or respond with frustration or hostility. This response can occasionally result in a failure to diagnose a medical condition that is present.

1	Prevalence estimates of illness anxiety disorder are based on estimates of the DSM-III and DSM-IV diagnosis hypochondriasis. The 1- to 2-year prevalence of health anxiety and/ or disease conviction in community surveys and population—based samples ranges from 1.3% to 10%. In ambulatory medical populations, the 6-month/1-year prevalence rates are be- tween 3% and 8%. The prevalence of the disorder is similar in males and females. The development and course of illness anxiety disorder are unclear. Illness anxiety disor- der is generally thought to be a chronic and relapsing condition with an age at onset in early and middle adulthood. In population-based samples, health-related anxiety in- creases with age, but the ages of individuals with high health anxiety in medical settings do not appear to differ from those of other patients in those settings. In older individuals, health—related anxiety often focuses on memory loss; the disorder is thought to be rare in children.

1	Environmental. Illness anxiety disorder may sometimes be precipitated by a major life stress or a serious but ultimately benign threat to the individual's health. A history of child- hood abuse or of a serious childhood illness may predispose to development of the disor- der in adulthood. Course modifiers. Approximately one-third to one-half of individuals with illness anx- iety disorder have a transient form, which is associated with less psychiatric comorbidity, more medical comorbidity, and less severe illness anxiety disorder. The diagnosis should be made with caution in individuals whose ideas about disease are congruent with widely held, culturally sanctioned beliefs. Little is known about the phe- nomenology of the disorder across cultures, although the prevalence appears to be similar across different countries with diverse cultures.

1	Functional Consequences of Illness Anxiety Disorder function and health-related quality of life. Health concerns often interfere with interper- sonal relationships, disrupt family life, and damage occupational performance. Other medical conditions. The first differential diagnostic consideration is an underly- ing medical condition, including neurological or endocrine conditions, occult malignan- cies, and other diseases that affect multiple body systems. The presence of a medical condition does not rule out the possibility of coexisting illness anxiety disorder. If a med- ical condition is present, the health-related anxiety and disease concerns are clearly dis- proportionate to its seriousness. Transient preoccupations related to a medical condition do not constitute illness anxiety disorder.

1	Adjustment disorders. Health-related anxiety is a normal response to serious illness and is not a mental disorder. Such nonpathological health anxiety is clearly related to the medical condition and is typically time-limited. If the health anxiety is severe enough, an adjustment disorder may be diagnosed. However, only when the health anxiety is of suf- ficient duration, severity, and distress can illness anxiety disorder be diagnosed. Thus, the diagnosis requires the continuous persistence of disproportionate health—related anxiety for at least 6 months. Somatic symptom disorder. Somatic symptom disorder is diagnosed when significant somatic symptoms are present. In contrast, individuals with illness anxiety disorder have minimal somatic symptoms and are primarily concerned with the idea they are ill.

1	Anxiety disorders. In generalized anxiety disorder, individuals worry about multiple events, situations, or activities, only one of which may involve health. In panic disorder, the individual may be concerned that the panic attacks reﬂect the presence of a medical ill- ness; however, although these individuals may have health anxiety, their anxiety is typi- cally very acute and episodic. In illness anxiety disorder, the health anxiety and fears are more persistent and enduring. Individuals with illness anxiety disorder may experience panic attacks that are triggered by their illness concerns.

1	Obsessive-compulsive and related disorders. Individuals with illness anxiety disor- compulsive behaviors (e.g., seeking reassurance). However, in illness anxiety disorder, the preoccupations are usually focused on having a disease, whereas in obsessive-compulsive disorder (OCD), the thoughts are intrusive and are usually focused on fears of getting a disease in the future. Most individuals with 0CD have obsessions or compulsions involv- ing other concerns in addition to fears about contracting disease. In body dysmorphic dis- order, concerns are limited to the individual’s physical appearance, which is viewed as defective or ﬂawed.

1	Major depressive disorder. Some individuals with a major depressive episode rumi- nate about their health and worry excessively about illness. A separate diagnosis of illness anxiety disorder is not made if these concerns occur only during major depressive epi- sodes. However, if excessive illness worry persists after remission of an episode of major depressive disorder, the diagnosis of illness anxiety disorder should be considered.

1	Psychotic disorders. Individuals with illness anxiety disorder are not delusional and can acknowledge the possibility that the feared disease is not present. Their ideas do not attain the rigidity and intensity seen in the somatic delusions occurring in psychotic dis- orders (e.g., schizophrenia; delusional disorder, somatic type; major depressive disorder, with psychotic features). True somatic delusions are generally more bizarre (e.g., that an organ is rotting or dead) than the concerns seen in illness anxiety disorder. The concerns seen in illness anxiety disorder, though not founded in reality, are plausible.

1	Because ilhiess anxiety disorder is a new disorder, exact comorbidities are unknown. Hy- pochondriasis co—occurs with anxiety disorders (in particular, generalized anxiety disor- der, panic disorder, and OCD) and depressive disorders. Approximately two-thirds of individuals with illness anxiety disorder are likely to have at least one other comorbid ma- jor mental disorder. Individuals with illness anxiety disorder may have an elevated risk for somatic symptom disorder and personality disorders. A. One or more symptoms of altered voluntary motor or sensory function. B. Clinical findings provide evidence of incompatibility between the symptom and recog- nized neurological or medical conditions. C. The symptom or deficit is not better explained by another medical or mental disorder. D. The symptom or deticit causes clinically significant distress or impairment in social, oc- cupational, or other important areas of functioning or warrants medical evaluation.

1	D. The symptom or deticit causes clinically significant distress or impairment in social, oc- cupational, or other important areas of functioning or warrants medical evaluation. Coding note: The |CD-9-CM code for conversion disorder is 300.11, which is assigned regardless of the symptom type. The |CD-10-CM code depends on the symptom type (see below). Specify symptom type: (F44.4) Wlth weakness or paralysis (F44.4) With abnormal movement (e.g., tremor, dystonic movement, myoclonus, gait (F44.4) With swallowing symptoms (F44.4) With speech symptom (e.g., dysphonia, slurred speech) (F44.5) With attacks or seizures (F44.6) With anesthesia or sensory loss (F44.6) With special sensory symptom (e.g., visual, olfactory, or hearing distur- (F44.7) With mixed symptoms Specify if: Acute episode: Symptoms present for less than 6 months. Persistent: Symptoms occurring for 6 months or more. Specify it:

1	Many clinicians use the alternative names of "functional" (referring to abnormal central nervous system functioning) or ”psychogenic” (referring to an assumed etiology) to de- scribe the symptoms of conversion disorder (functional neurological symptom disor- der). In conversion disorder, there may be one or more symptoms of various types. Motor symptoms include weakness or paralysis; abnormal movements, such as tremor or dys- tonic movements; gait abnormalities; and abnormal limb posturing. Sensory symptoms include altered, reduced, or absent skin sensation, vision, or hearing. Episodes of abnor- mal generalized limb shaking with apparent impaired or loss of consciousness may resem- ble epileptic seizures (also called psychogenic or non-epileptic seizures). There may be episodes of unresponsiveness resembling syncope or coma. Other symptoms include re- duced or absent speech volume (dysphonia/aphonia), altered articulation (dysarthria), a sensation of a lump in the throat (globus),

1	resembling syncope or coma. Other symptoms include re- duced or absent speech volume (dysphonia/aphonia), altered articulation (dysarthria), a sensation of a lump in the throat (globus), and diplopia.

1	Although the diagnosis requires that the symptom is not explained by neurological disease, it should not be made simply because results from investigations are normal or because the symptom is ”bizarre.” There must be clinical findings that show clear evidence of incompatibility with neurological disease. Internal inconsistency at examination is one way to demonstrate incompatibility (i.e., demonstrating that physical signs elicited through one examination method are no longer positive when tested a different way). Ex- amples of such examination findings include ' Hoover’s sign, in which weakness of hip extension returns to normal strength with con— tralateral hip ﬂexion against resistance.

1	0 Marked weakness of ankle plantar-ﬂexion when tested on the bed in an individual who is able to walk on tiptoes; 0 Positive findings on the tremor entrainment test. On this test, a unilateral tremor may be identified as functional if the tremor changes when the individual is distracted away from it. This may be observed if the individual is asked to copy the examiner in making a rhythmical movement with their unaffected hand and this causes the functional tremor to change such that it copies or “entrains” to the rhythm of the unaffected hand or the functional tremor is suppressed, or no longer makes a simple rhythmical move- ment. 0 In attacks resembling epilepsy or syncope ("psychogenic” non-epileptic attacks), the occurrence of closed eyes with resistance to opening or a normal simultaneous electro- encephalogram (although this alone does not exclude all forms of epilepsy or syncope). 0 For visual symptoms, a tubular visual field (i.e., tunnel vision).

1	0 For visual symptoms, a tubular visual field (i.e., tunnel vision). It is important to note that the diagnosis of conversion disorder should be based on the overall clinical picture and not on a single clinical finding. A number of associated features can support the diagnosis of conversion disorder. There may be a history of multiple similar somatic symptoms. Onset may be associated with stress or trauma, either psychological or physical in nature. The potential etiological rele- vance of this stress or trauma may be suggested by a close temporal relationship. However, while assessment for stress and trauma is important, the diagnosis should not be withheld if none is found. Conversion disorder is often associated with dissociative symptoms, such as deperson- alization, derealization, and dissociative amnesia, particularly at symptom onset or during attacks.

1	Conversion disorder is often associated with dissociative symptoms, such as deperson- alization, derealization, and dissociative amnesia, particularly at symptom onset or during attacks. The diagnosis of conversion disorder does not require the judgment that the symptoms are not intentionally produced (i.e., not feigned), as the definite absence of feigning may not be reliably discerned. The phenomenon of la belle indifférence (i.e., lack of concern about the nature or implications of the symptom) has been associated with conversion disorder but it is not specific for conversion disorder and should not be used to make the diagnosis.

1	Similarly the concept of secondary gain (i.e., when individuals derive external benefits such as money or release from responsibilities) is also not specific to conversion disorder and particularly in the context of definite evidence for feigning, the diagnoses that should be considered instead would include factitious disorder or malingering (see the section ”Dif- ferential Diagnosis” for this disorder). Transient conversion symptoms are common, but the precise prevalence of the disorder is unknown. This is partly because the diagnosis usually requires assessment in secondary care, where it is found in approximately 5% of referrals to neurology clinics. The incidence of individual persistent conversion symptoms is estimated to be 2—5/ 100,000 per year. Onset has been reported throughout the life course. The onset of non-epileptic attacks peaks in the third decade, and motor symptoms have their peak onset in the fourth decade.

1	Onset has been reported throughout the life course. The onset of non-epileptic attacks peaks in the third decade, and motor symptoms have their peak onset in the fourth decade. The symptoms can be transient or persistent. The prognosis may be better in younger chil— dren than in adolescents and adults. Temperamental. Maladaptive personality traits are commonly associated with conver- sion disorder. Environmental. There may be a history of childhood abuse and neglect. Stressful life events are often, but not always, present. Genetic and physiological. The presence of neurological disease that causes similar symp- toms is a risk factor (e.g., non-epileptic seizures are more common in patients who also have epilepsy). Course modifiers. Short duration of symptoms and acceptance of the diagnosis are pos— itive prognostic factors. Maladaptive personality traits, the presence of comorbid physical disease, and the receipt of disability benefits may be negative prognostic factors.

1	Changes resembling conversion (and dissociative) symptoms are common in certain culturally sanctioned rituals. If the symptoms are fully explained within the particular cultural context and do not result in clinically significant distress or disability, then the di- agnosis of conversion disorder is not made. Conversion disorder is two to three times more common in females. Functional Consequences of Conversion Disorder Individuals withconversion symptoms may have substantial disability. The severity of dis- ability can be similar to that experienced by individuals with comparable medical diseases. If another mental disorder better explains the symptoms, that diagnosis should be made. However the diagnosis of conversion disorder may be made in the presence of another mental disorder.

1	If another mental disorder better explains the symptoms, that diagnosis should be made. However the diagnosis of conversion disorder may be made in the presence of another mental disorder. Neurological disease. The main differential diagnosis is neurological disease that might better explain the symptoms. After a thorough neurological assessment, an unexpected neurological disease cause for the symptoms is rarely found at follow up. However, reas- sessment may be required if the symptoms appear to be progressive. Conversion disorder may coexist with neurological disease.

1	Somatic symptom disorder. Conversion disorder may be diagnosed in addition to so- matic symptom disorder. Most of the somatic symptoms encountered in somatic symptom disorder cannot be demonstrated to be clearly incompatible with pathophysiology (e.g., pain, fatigue), whereas in conversion disorder, such incompatibility is required for the di- agnosis. The excessive thoughts, feelings, and behaviors characterizing somatic symptom disorder are often absent in conversion disorder.

1	Factitious disorder and malingering. The diagnosis of conversion disorder does not re- quire the judgment that the symptoms are not intentionally produced (i.e., not feigned), because assessment of conscious intention is unreliable. However definite evidence of feigning (e.g., clear evidence that loss of function is present during the examination but not at home) would suggest a diagnosis of factitious disorder if the individual’s apparent aim is to assume the sick role or malingering if the aim is to obtain an incentive such as money. Dissociative disorders. Dissociative symptoms are common in individuals with con- version disorder. If both conversion disorder and a dissociative disorder are present, both diagnoses should be made. Body dysmorphic disorder. Individuals with body dysmorphic disorder are exces- symptoms of sensory or motor functioning in the affected body part.

1	Body dysmorphic disorder. Individuals with body dysmorphic disorder are exces- symptoms of sensory or motor functioning in the affected body part. Depressive disorders. In depressive disorders, individuals may report general heavi- ness of their limbs, whereas the weakness of conversion disorder is more focal and prom- inent. Depressive disorders are also differentiated by the presence of core depressive symptoms. Panic disorder. Episodic neurological symptoms (e.g., tremors and paresthesias) can occur in both conversion disorder and panic attacks. In panic attacks, the neurological symptoms are typically transient and acutely episodic with characteristic cardiorespira- tory symptoms. Loss of awareness with amnesia for the attack and violent limb move- ments occur in non-epileptic attacks, but not in panic attacks.

1	Anxiety disorders, especially panic disorder, and depressive disorders commonly co—occur with conversion disorder. Somatic symptom disorder may co-occur as well. Psychosis, sub- stance use disorder, and alcohol misuse are uncommon. Personality disorders are more common in individuals with conversion disorder than in the general population. Neuro- logical or other medical conditions commonly coexist with conversion disorder as well. A. A medical symptom or condition (other than a mental disorder) is present. B. Psychological or behavioral factors adversely affect the medical condition in one of the following ways: 1. The factors have inﬂuenced the course of the medical condition as shown by a close temporal association between the psychological factors and the development or exacerbation of, or delayed recovery from, the medical condition. 2. The factors interfere with the treatment of the medical condition (e.g., poor adher- ence).

1	2. The factors interfere with the treatment of the medical condition (e.g., poor adher- ence). 3. The factors constitute additional weII-established health risks for the individual. 4. The factors influence the underlying pathophysiology, precipitating or exacerbating symptoms or necessitating medical attention. C. The psychological and behavioral factors in Criterion B are not better explained by an- other mental disorder (e.g., panic disorder, major depressive disorder, posttraumatic stress disorder). Specify current severity: Mild: Increases medical risk (e.g.. inconsistent adherence with antihypertension treat- ment). Moderate: Aggravates underlying medical condition (e.g., anxiety aggravating asthma). Severe: Results in medical hospitalization or emergency room visit. Extreme: Results in severe, life-threatening risk (e.g., ignoring heart attack symp- toms).

1	Severe: Results in medical hospitalization or emergency room visit. Extreme: Results in severe, life-threatening risk (e.g., ignoring heart attack symp- toms). The essential feature of psychological factors affecting other medical conditions is the presence of one or more clinically significant psychological or behavioral factors that ad- versely affect a medical condition by increasing the risk for suffering, death, or disability (Criterion B). These factors can adversely affect the medical condition by inﬂuencing its course or treatment, by constituting an additional well—established health risk factor, or by inﬂuencing the underlying pathophysiology to precipitate or exacerbate symptoms or to necessitate medical attention.

1	Psychological or behavioral factors include psychological distress, patterns of interper- sonal interaction, coping styles, and maladaptive health behaviors, such as denial of symp- toms or poor adherence to medical recommendations. Common clinical examples are anxiety-exacerbating asthma, denial of need for treatment for acute chest pain, and manip- ulation of insulin by an individual with diabetes wishing to lose weight. Many different psychological factors have been demonstrated to adversely inﬂuence medical conditions— for example, symptoms of depression or anxiety, stressful life events, relationship style, personality traits, and coping styles. The adverse effects can range from acute, with imme- diate medical consequences (e.g., Takotsubo cardiomyopathy) to chronic, occurring over a long period of time (e.g., chronic occupational stress increasing risk for hypertension). Af- fected medical conditions can be those with clear pathophysiology (e.g., diabetes, cancer, coronary

1	a long period of time (e.g., chronic occupational stress increasing risk for hypertension). Af- fected medical conditions can be those with clear pathophysiology (e.g., diabetes, cancer, coronary disease), functional syndromes (e.g., migraine, irritable bowel syndrome, fibro- myalgia), or idiopathic medical symptoms (e.g., pain, fatigue, dizziness).

1	This diagnosis should be reserved for situations in which the effect of the psychological factor on the medical condition is evident and the psychological factor has clinically sig- nificant effects ori the course or outcome of the medical condition. Abnormal psychologi- cal or behavioral symptoms that develop in response to a medical condition are more properly coded as an adjustment disorder (a clinically significant psychological response to an identifiable stressor). There must be reasonable evidence to suggest an association between the psychological factors and the medical condition, although it may often not be possible to demonstrate direct causality or the mechanisms underlying the relationship. The prevalence of psychological factors affecting other medical conditions is unclear. In US. private insurance billing data, it is a more common diagnosis than somatic symptom disorders.

1	The prevalence of psychological factors affecting other medical conditions is unclear. In US. private insurance billing data, it is a more common diagnosis than somatic symptom disorders. Psychological factors affecting other medical conditions can occur across the lifespan. Par- ticularly with young children, corroborative history from parents or school can assist the di- agnostic evaluation. Some conditions are characteristic of particular life stages (e.g., in older individuals, the stress associated with acting as a caregiver for an ill spouse or partner).

1	on medical conditions, such as those in language and communication style, explanatory models of illness, patterns of seeking health care, service availability and organization, doctor-patient relationships and other healing practices, family and gender roles, and at- titudes toward pain and death. Psychological factors affecting other medical conditions must be differentiated from culturally specific behaviors such as using faith or spiritual healers or other variations in illness management that are acceptable within a culture and represent an attempt to help the medical condition rather than interfere with it. These local practices may complement rather than obstruct evidence-based interventions. If they do not adversely affect outcomes, they should not be pathologized as psychological factors affecting other medical conditions. Functional Consequences of Psychological Factors

1	Functional Consequences of Psychological Factors Psychological and behavioral factors have been demonstrated to affect the course of many medical diseases. Mental disorder due to another medical condition. A temporal association between symptoms of a mental disorder and those of a medical condition is also characteristic of a mental disorder due to another medical condition, but the presumed causality is in the op- posite direction. In a mental disorder due to another medical condition, the medical condition is judged to be causing the mental disorder through a direct physiological mech- anism. In psychological factors affecting other medical conditions, the psychological or be- havioral factors are judged to affect the course of the medical condition.

1	Adjustment disorders. Abnormal psychological or behavioral symptoms that develop in response to a medical condition are more properly coded as an adjustment disorder (a clin- ically significant psychological response to an identifiable stressor). For example, an indi- vidual with angina that is precipitated whenever he becomes enraged would be diagnosed as having psychological factors affecting other medical conditions, whereas an individual with angina who developed maladaptive anticipatory anxiety would be diagnosed as hav- ing an adjustment disorder with anxiety. In clinical practice, however, psychological fac- tors and a medical condition are often mutually exacerbating (e.g., anxiety as both a precipitant and a consequence of angina), in which case the distinction is arbitrary. Other mental disorders frequently result in medical complications, most notably substance use disorders (e.g., alcohol use disorder, tobacco use disorder). If an individual has a coexisting major mental

1	mental disorders frequently result in medical complications, most notably substance use disorders (e.g., alcohol use disorder, tobacco use disorder). If an individual has a coexisting major mental disorder that adversely affects or causes another medical condition, diagno- ses of the mental disorder and the medical condition are usually sufficient. Psychological factors affecting other medical conditions is diagnosed when the psychological traits or behaviors do not meet criteria for a mental diagnosis.

1	Somatic symptom disorder. Somatic symptom disorder is characterized by a combina- tion of distressing somatic symptoms and excessive or maladaptive thoughts, feelings, and behavior in response to these symptoms or associated health concerns. The individual may or may not have a diagnosable medical condition. In contrast, in psychological factors affecting other medical conditions, the psychological factors adversely affect a medical condition; the individual’s thoughts, feelings, and behavior are not necessarily excessive.

1	The difference is one of emphasis, rather than a clear-cut distinction. In psychological fac- tors affecting other medical conditions, the emphasis is on the exacerbation of the medical condition (e.g., an individual with angina that is precipitated whenever he becomes anx- ious). In somatic symptom disorder, the emphasis is on maladaptive thoughts, feelings, and behavior (e.g., an individual with angina who worries constantly that she will have a heart attack, takes her blood pressure multiple times per day, and restricts her activities).

1	Illness anxiety disorder. Illness anxiety disorder is characterized by high illness anxiety that is distressing and / or disruptive to daily life with minimal somatic symptoms. The fo- cus of clinical concern is the individual's worry about having a disease; in most cases, no serious disease is present. In psychological factors affecting other medical conditions, anx- iety may be a relevant psychological factor affecting a medical condition, but the clinical concern is the adverse effects on the medical condition. By definition, the diagnosis of psychological factors affecting other medical conditions entails a relevant psychological or behavioral syndrome or trait and a comorbid medical condition. Diagnostic Criteria 300.19 (F68.10) A. Falsification of physical or psychological signs or symptoms, or induction of injury or disease, associated with identified deception. B. The individual presents himself or herself to others as ill, impaired, or injured.

1	B. The individual presents himself or herself to others as ill, impaired, or injured. C. The deceptive behavior is evident even in the absence of obvious external rewards. D. The behavior is not better explained by another mental disorder. such as delusional disorder or another psychotic disorder. Specify: Recurrent episodes (two or more events of falsification of illness and/or induction of A. Falsification of physical or psychological signs or symptoms, or induction of injury or disease, in another, associated with identified deception. B. The individual presents another individual (victim) to others as ill, impaired, or injured. C. The deceptive behavior is evident even in the absence of obvious external rewards. D. The behavior is not better explained by another mental disorder, such as delusional disorder or another psychotic disorder. Note: The perpetrator, not the victim, receives this diagnosis. Specify:

1	Note: The perpetrator, not the victim, receives this diagnosis. Specify: Recurrent episodes (two or more events of falsification of illness and/or induction of When an individual falsifies illness in another (e.g., children, adults, pets), the diagnosis is factitious disorder imposed on another. The perpetrator, not the victim, is given the diag- nosis. The victim may be given an abuse diagnosis (e.g., 995.54 [T74.12X]; see the chapter ”Other Conditions That May Be a Focus of Clinical Attention”).

1	The essential feature of factitious disorder is the falsification of medical or psychological signs and symptoms in oneself or others that are associated with the identified deception. Indi- viduals with factitious disorder can also seek treatment for themselves or another following induction of injury or disease. The diagnosis requires demonstrating that the individual is taking surreptitious actions to misrepresent, simulate, or cause signs or symptoms of ill- ness or injury in the absence of obvious external rewards. Methods of illness falsification can include exaggeration, fabrication, simulation, and induction. While a preexisting med— ical condition may be present, the deceptive behavior or induction of injury associated with deception causes others to view such individuals (or another) as more ill or impaired, and this can lead to excessive clinical intervention. Individuals with factitious disorder might, for example, report feelings of depression and suicidality following

1	as more ill or impaired, and this can lead to excessive clinical intervention. Individuals with factitious disorder might, for example, report feelings of depression and suicidality following the death of a spouse despite the death not being true or the individual’s not having a spouse; decep- tively report episodes of neurological symptoms (e.g., seizures, dizziness, or blacking out); manipulate a laboratory test (e.g., by adding blood to urine) to falsely indicate an abnor- mality; falsify medical records to indicate an illness; ingest a substance (e.g., insulin or warfarin) to induce an abnormal laboratory result or illness; or physically injure them- selves or induce illness in themselves or another (e.g., by injecting fecal material to produce an abscess or to induce sepsis).

1	Individuals with factitious disorder imposed on self or factitious disorder imposed on an- other are at risk for experiencing great psychological distress or functional impairment by causing harm to themselves and others. Family, friends, and health care professionals are also often adversely affected by their behavior. Factitious disorders have similarities to substance use disorders, eating disorders, impulse-control disorders, pedophilic disorder, and some other established disorders related to both the persistence of the behavior and the intentional efforts to conceal the disordered behavior through deception. Whereas some aspects of factitious disorders might represent criminal behavior (e.g., factitious dis- order imposed on another, in which the parent’s actions represent abuse and maltreat- ment of a child), such criminal behavior and mental illness are not mutually exclusive. The diagnosis of factitious disorder emphasizes the objective identification of falsification of

1	and maltreat- ment of a child), such criminal behavior and mental illness are not mutually exclusive. The diagnosis of factitious disorder emphasizes the objective identification of falsification of signs and symptoms of illness, rather than an inference about intent or possible underly- ing motivation. Moreover, such behaviors, including the induction of injury or disease, are associated with deception.

1	The prevalence of factitious disorder is unknown, likely because of the role of deception in this population. Among patients in hospital settings, it is estimated that about 1% of indi- viduals have presentations that meet the criteria for factitious disorder. The course of factitious disorder is usually one of intermittent episodes. Single episodes and episodes that are characterized as persistent and unremitting are both less common. Onset is usually in early adulthood, often after hospitalization for a medical condition or a mental disorder. When imposed on another, the disorder may begin after hospitalization of the individual’s child or other dependent. In individuals with recurrent episodes of fal- sification of signs and symptoms of illness and / or induction of injury, this pattern of suc- cessive deceptive contact with medical personnel, including hospitalizations, may become lifelong.

1	Caregivers who lie about abuse injuries in dependents solely to protect themselves from lia- bility are not diagnosed with factitious disorder imposed on another because protection from liability is an external reward (Criterion C, the deceptive behavior is evident even in the ab- sence of obvious external rewards). Such caregivers who, upon observation, analysis of med- ical records, and / or interviews with others, are found to lie more extensively than needed for immediate self—protection are diagnosed with factitious disorder imposed on another. Somatic symptom disorder. In somatic symptom disorder, there may be excessive at- tention and treatment seeking for perceived medical concerns, but there is no evidence that the individual is providing false information or behaving deceptively.

1	Malingerlng. Malingering is differentiated from factitious disorder by the intentional re- porting of symptoms for personal gain (e.g., money, time off work). In contrast, the diag- nosis of factitious disorder requires the absence of obvious rewards. Conversion disorder (functional neurological symptom disorder). Conversion disorder is characterized by neurological symptoms that are inconsistent with neurological patho- physiology. Factitious disorder with neurological symptoms is distinguished from con- version disorder by evidence of deceptive falsification of symptoms. Borderiine personality disorder. Deliberate physical self-harm in the absence of suicidal intent can also occur in association with other mental disorders such as borderline person- ality disorder. Factitious disorder requires that the induction of injury occur in association with deception.

1	Medical condition or mental disorder not associated with intentional symptom falsifi- cation. Presentation of signs and symptoms of illness that do not conform to an identi- fiable medical condition or mental disorder increases the likelihood of the presence of a factitious disorder. However, the diagnosis of factitious disorder does not exclude the presence of true medical condition or mental disorder, as comorbid illness often occurs in the individual along with factitious disorder. For example, individuals who might manip- ulate blood sugar levels to produce symptoms may also have diabetes. 300.89 (F45.8) This category applies to presentations in which symptoms characteristic of a somatic cial, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the somatic symptom and related disorders diagnos- tic class.

1	Examples of presentations that can be specified using the “other specified" designation include the following: 1. Brief somatic symptom disorder: Duration of symptoms is less than 6 months. 2. Brief illness anxiety disorder: Duration of symptoms is less than 6 months. 3. Illness anxiety disorder without excessive health-related behaviors: Criterion D for illness anxiety disorder is not met. 4. Pseudocyesis: A false belief of being pregnant that is associated with objective signs and reported symptoms of pregnancy. 300.82 (F45.9)

1	4. Pseudocyesis: A false belief of being pregnant that is associated with objective signs and reported symptoms of pregnancy. 300.82 (F45.9) This category applies to presentations in which symptoms characteristic of a somatic cial, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the somatic symptom and related disorders diagnos- tic class. The unspecified somatic symptom and related disorder category should not be used unless there are decidedly unusual situations where there is insufficient information to make a more specific diagnosis. , Feedinga Eating Disorde?

1	, Feedinga Eating Disorde? Feed | rig an d eati ng disorders are characterized by a persistent disturbance of eat- ing or eating-related behavior that results in the altered consumption or absorption of food and that significantly impairs physical health or psychosocial functioning. Diagnos- tic criteria are provided for pica, rumination disorder, avoidant/restrictive food intake disorder, anorexia nervosa, bulimia nervosa, and binge-eating disorder.

1	The diagnostic criteria for rumination disorder, avoidant/restrictive food intake dis- order, anorexia nervosa, bulimia nervosa, and binge-eating disorder result in a classifica- tion scheme that is mutually exclusive, so that during a single episode, only one of these diagnoses can be assigned. The rationale for this approach is that, despite a number of common psychological and behavioral features, the disorders differ substantially in clin- ical course, outcome, and treatment needs. A diagnosis of pica, however, may be assigned in the presence of any other feeding and eating disorder.

1	Some individuals with disorders described in this chapter report eating-related symp- toms resembling those typically endorsed by individuals with substance use disorders, such as craving and patterns of compulsive use. This resemblance may reﬂect the involve- ment of the same neural systems, including those implicated in regulatory self-control and reward, in both groups of disorders. However, the relative contributions of shared and distinct factors in the development and perpetuation of eating and substance use disor- ders remain insufficiently understood.

1	Finally, obesity is not included in DSM-5 as a mental disorder. Obesity (excess body fat) results from the long—term excess of energy intake relative to energy expenditure. A range of genetic, physiological, behavioral, and environmental factors that vary across individ- uals contributes to the development of obesity; thus, obesity is not considered a mental disorder. However, there are robust associations between obesity and a number of mental disorders (e.g., binge-eating disorder, depressive and bipolar disorders, schizophrenia). The side effects of some psychotropic medications contribute importantly to the develop- ment of obesity, and obesity may be a risk factor for the development of some mental dis- orders (e.g., depressive disorders). A. Persistent eating of nonnutritive, nonfood substances over a period of at least 1 month. B. The eating of nonnutritive, nontood substances is inappropriate to the developmental level of the individual.

1	B. The eating of nonnutritive, nontood substances is inappropriate to the developmental level of the individual. . The eating behavior is not part of a culturally supported or socially normative practice. . If the eating behavior occurs in the context of another mental disorder (e.g., intellectual disability [intellectual developmental disorder], autism spectrum disorder, schizophre- nia) or medical condition (including pregnancy), it is sufficiently severe to warrant ad- ditional clinical attention. Coding note: The |CD-9-CM code for pica is 307.52 and is used for children or adults. The ICD-10-CM codes for pica are (F98.3) in children and (F50.8) in adults. Specify it: In remission: After full criteria for pica were previously met. the criteria have not been met for a sustained period of time.

1	The essential feature of pica is the eating of one or more nonnutritive, nonfood substances on a persistent basis over a period of at least 1 month (Criterion A) that is severe enough to warrant clinical attention. Typical substances ingested tend to vary with age and availability and might include paper, soap, cloth, hair, string, wool, soil, chalk, talcum powder, paint, gum, metal, pebbles, charcoal or coal, ash, clay, starch, or ice. The term nonfood is included because the di- agnosis of pica does not apply to ingestion of diet products that have minimal nutritional con- tent. There is typically no aversion to food in general. The eating of nonnutritive, nonfood substances must be developmentally inappropriate (Criterion B) and not part of a culturally supported or socially normative practice (Criterion C). A minimum age of 2 years is suggested for a pica diagnosis to exclude developmentally normal mouthing of objects by infants that re- sults in ingestion. The eating of

1	practice (Criterion C). A minimum age of 2 years is suggested for a pica diagnosis to exclude developmentally normal mouthing of objects by infants that re- sults in ingestion. The eating of nonnutritive, nonfood substances can be an associated feature of other mental disorders (e.g., intellectual disability [intellectual developmental disorder], autism spectrum disorder, schizophrenia). If the eating behavior occurs exclusively in the con- text of another mental disorder, a separate diagnosis of pica should be made only if the eating behavior is sufficiently severe to warrant additional clinical attention (Criterion D).

1	Although deficiencies in vitamins or minerals (e.g., zinc, iron) have been reported in some instances, often no specific biological abnormalities are found. In some cases, pica comes to clinical attention only following general medical complications (e.g., mechanical bowel problems; intestinal obstruction, such as that resulting from a bezoar; intestinal perfora- tion; infections such as toxoplasmosis and toxocariasis as a result of ingesting feces or dirt; poisoning, such as by ingestion of lead-based paint). The prevalence of pica is unclear. Among individuals with intellectual disability, the prev- alence of pica appears to increase with the severity of the condition.

1	Onset of pica can occur in childhood, adolescence, or adulthood, although childhood onset is most commonly reported. Pica can occur in otherwise normally developing children, whereas in adults, it appears more likely to occur in the context of intellectual disability or other mental disorders. The eating of nonnutritive, nonfood substances may also manifest in pregnancy, when specific cravings (e.g., chalk or ice) might occur. The diagnosis of pica during pregnancy is only appropriate if such cravings lead to the ingestion of nonnutri- tive, nonfood substances to the extent that the eating of these substances poses potential medical risks. The course of the disorder can be protracted and can result in medical emer- gencies (e.g., intestinal obstruction, acute weight loss, poisoning). The disorder can poten- tially be fatal depending on substances ingested. Environmental. Neglect, lack of supervision, and developmental delay can increase the risk for this condition.

1	Environmental. Neglect, lack of supervision, and developmental delay can increase the risk for this condition. In some populations, the eating of earth or other seemingly nonnutritive substances is believed to be of spiritual, medicinal, or other social value, or may be a culturally supported or socially normative practice. Such behavior does not warrant a diagnosis of pica (Criterion C). Pica occurs in both males and females. It can occur in females during pregnancy; however, little is known about the course of pica in the postpartum period. Abdominal ﬂat plate radiography, ultrasound, and other scanning methods may reveal obstructions related to pica. Blood tests and other laboratory tests can be used to ascertain levels of poisoning or the nature of infection. Functional Consequences of Pica

1	Functional Consequences of Pica Pica can significantly impair physical functioning, but it is rarely the sole cause of impair- ment in social functioning. Pica often occurs with other disorders associated with im- paired social functioning. Eating of nonnutritive, nonfood substances may occur during the course of other mental disorders (e.g., autism spectrum disorder, schizophrenia) and in Kleine-Levin syndrome. In any such instance, an additional diagnosis of pica should be given only if the eating be- havior is sufficiently persistent and severe to warrant additional clinical attention.

1	In any such instance, an additional diagnosis of pica should be given only if the eating be- havior is sufficiently persistent and severe to warrant additional clinical attention. Anorexia nervosa. Pica can usually be distinguished from the other feeding and eating disorders by the consumption of nonnutritive, nonfood substances. It is important to note, however, that some presentations of anorexia nervosa include ingestion of nonnutritive, nonfood substances, such as paper tissues, as a means of attempting to control appetite. In such cases, when the eating of nonnutritive, nonfood substances is primarily used as a means of weight control, anorexia nervosa should be the primary diagnosis. Factitious disorder. Some individuals with factitious disorder may intentionally ingest foreign objects as part of the pattern of falsification of physical symptoms. In such in— stances, there is an element of deception that is consistent with deliberate induction of in- jury or disease.

1	Nonsuicidal seIf-injury and nonsuicidal seIf-injury behaviors in personality disorders. Some individuals may swallow potentially harmful items (e.g., pins, needles, knives) in the context of maladaptive behavior patterns associated with personality disorders or nonsuicidal self-injury.

1	Some individuals may swallow potentially harmful items (e.g., pins, needles, knives) in the context of maladaptive behavior patterns associated with personality disorders or nonsuicidal self-injury. Disorders most commonly comorbid with pica are autism spectrum disorder and intellec- tual disability (intellectual developmental disorder), and, to a lesser degree, schizophrenia and obsessive-compulsive disorder. Pica can be associated with trichotillomania (hair— pulling disorder) and excoriation (skin-picking) disorder. In comorbid presentations, the hair or skin is typically ingested. Pica can also be associated with avoidant/ restrictive food intake disorder, particularly in individuals with a strong sensory component to their pre- sentation. When an individual is known to have pica, assessment should include con- sideration of the possibility of gastrointestinal complications, poisoning, infection, and nutritional deficiency. Diagnostic Criteria 307.53 (F9821)

1	Diagnostic Criteria 307.53 (F9821) A. Repeated regurgitation of food over a period of at least 1 month. Regurgitated food may be re-chewed, re-swallowed, or spit out. B. The repeated regurgitation is not attributable to an associated gastrointestinal or other medical condition (e.g.. gastroesophageal reflux, pyloric stenosis). C. The eating disturbance does not occur exclusively during the course of anorexia nervosa, bulimia nervosa, binge-eating disorder, or avoidant/restrictive food intake disorder. D. It the symptoms occur in the context of another mental disorder (e.g., intellectual dis- ability [intellectual developmental disorder] or another neurodevelopmental disorder), they are sufficiently severe to warrant additional clinical attention. Specify it: In remission: After full criteria for rumination disorder were previously met, the criteria have not been met for a sustained period of time.

1	The essential feature of rumination disorder is the repeated regurgitation of food occur- ring after feeding or eating over a period of at least 1 month (Criterion A). Previously swal- lowed food that may be partially digested is brought up into the mouth without apparent nausea, involuntary retching, or disgust. The food may be re-chewed and then ejected from the mouth or re—swallowed. Regurgitation in rumination disorder should be fre- quent, occurring at least several times per week, typically daily. The behavior is not better explained by an associated gastrointestinal or other medical condition (e.g., gastroesoph- ageal reflux, pyloric stenosis) (Criterion B) and does not occur exclusively during the course of anorexia nervosa, bulimia nervosa, binge-eating disorder, or avoidant/restric- tive food intake disorder (Criterion C). If the symptoms occur in the context of another mental disorder (e.g., intellectual disability [intellectual developmental disorder], neuro- developmental

1	tive food intake disorder (Criterion C). If the symptoms occur in the context of another mental disorder (e.g., intellectual disability [intellectual developmental disorder], neuro- developmental disorder), they must be sufficiently severe to warrant additional clinical attention (Criterion D) and should represent a primary aspect of the individual’s presen- tation requiring intervention. The disorder may be diagnosed across the life span, par- ticularly in individuals who also have intellectual disability. Many individuals with rumination disorder can be directly observed engaging in the behavior by the clinician. In other instances diagnosis can be made on the basis of self—report or corroborative informa- tion from parents or caregivers. Individuals may describe the behavior as habitual or out- side of their control.

1	Infants with rumination disorder display a characteristic position of straining and arching the back with the head held back, making sucking movements with their tongue. They may give the impression of gaining satisfaction from the activity. They may be irritable and hungry between episodes of regurgitation. Weight loss and failure to make expected weight gains are common features in infants with rumination disorder. Malnutrition may occur despite the infant’s apparent hunger and the ingestion of relatively large amounts of food, particularly in severe cases, when regurgitation immediately follows each feeding episode and regurgitated food is expelled. Malnutrition might also occur in older children and adults, particularly when the regurgitation is accompanied by restriction of intake.

1	Adolescents and adults may attempt to disguise the regurgitation behavior by placing a hand over the mouth or coughing. Some will avoid eating with others because of the ac- knowledged social undesirability of the behavior. This may extend to an avoidance of eat- ing prior to social situations, such as work or school (e.g., avoiding breakfast because it may be followed by regurgitation). Prevalence data for rumination disorder are inconclusive, but the disorder is commonly reported to be higher in certain groups, such as individuals with intellectual disability. Onset of rumination disorder can occur in infancy, childhood, adolescence, or adulthood. The age at onset in infants is usually between ages 3 and 12 months. In infants, the disorder frequently remits spontaneously, but its course can be protracted and can result in medical emergencies (e.g., severe malnutrition). It can potentially be fatal, particularly in infancy.

1	Rumination disorder can have an episodic course or occur continuously until treated. In infants, as well as in older individuals with intellectual disability (intellectual developmen- tal disorder) or other neurodevelopmental disorders, the regurgitation and rumination be- havior appears to have a self—soothing or self—stimulating function, similar to that of other repetitive motor behaviors such as head banging. Environmental. Psychosocial problems such as lack of stimulation, neglect, stressful life situations, and problems in the parent—child relationship may be predisposing factors in infants and young children. Functional Consequences of Rumination Disorder

1	Functional Consequences of Rumination Disorder Malnutrition secondary to repeated regurgitation may be associated with growth delay and have a negative effect on development and learning potential. Some older individuals with rumination disorder deliberately restrict their food intake because of the social un- desirability of regurgitation. They may therefore present with weight loss or low weight. In older children, adolescents, and adults, social functioning is more likely to be adversely affected. Gastrointestinal conditions. It is important to differentiate regurgitation in rumination disorder from other conditions characterized by gastroesophageal reﬂux or vomiting. Con- ditions such as gastroparesis, pyloric stenosis, hiatal hernia, and Sandifer syndrome in in- fants should be ruled out by appropriate physical examinations and laboratory tests.

1	Anorexia nervosa and bulimia nervosa. Individuals with anorexia nervosa and bulimia nervosa may also engage in regurgitation with subsequent spitting out of food as a means of disposing of ingested calories because of concerns about weight gain. Regurgitation with associated rumination can occur in the context of a concurrent medical condition or another mental disorder (e.g., generalized anxiety disorder). When the regur- gitation occurs in this context, a diagnosis of rumination disorder is appropriate only when the severity of the disturbance exceeds that routinely associated with such conditions or disorders and warrants additional clinical attention. Diagnostic Criteria 307.59 (F50.8)

1	Diagnostic Criteria 307.59 (F50.8) A. An eating orfeeding disturbance (e.g., apparent lack of interest in eating or food; avoid- ance based on the sensory characteristics of food; concern about aversive conse- quences of eating) as manifested by persistent failure to meet appropriate nutritional and/or energy needs associated with one (or more) of the following: 1. Significant weight loss (or failure to achieve expected weight gain or faltering growth in children). 2. Significant nutritional deficiency. 3. Dependence on enteral feeding or oral nutritional supplements. 4. Marked interference with psychosocial functioning. B. The disturbance is not better explained by lack of available food or by an associated culturally sanctioned practice. C. The eating disturbance does not occur exclusively during the course of anorexia ner- vosa or bulimia nervosa, and there is no evidence of a disturbance in the way in which one’s body weight or shape is experienced.

1	D. The eating disturbance is not attributable to a concurrent medical condition or not bet- ter explained by another mental disorder. When the eating disturbance occurs in the context of another condition or disorder, the severity of the eating disturbance exceeds that routinely associated with the condition or disorder and warrants additional clinical attention. Specify it: In remission: After full criteria for avoidant/restrictive food intake disorder were previ- ously met, the criteria have not been met for a sustained period of time.

1	Avoidant/restrictive food intake disorder replaces and extends the DSM-IV diagnosis of feeding disorder of infancy or early childhood. The main diagnostic feature of avoidant/ restrictive food intake disorder is avoidance or restriction of food intake (Criterion A) manifested by clinically significant failure to meet requirements for nutrition or insuffi- cient energy intake through oral intake of food. One or more of the following key features must be present: significant weight loss, significant nutritional deficiency (or related health impact), dependence on enteral feeding or oral nutritional supplements, or marked interference with psychosocial functioning. The determination of whether weight loss is significant (Criterion A1) is a clinical judgment; instead of losing weight, children and ad- along their developmental trajectory. '

1	Determination of significant nutritional deficiency (Criterion A2) is also based on clin- ical assessment (e.g., assessment of dietary intake, physical examination, and laboratory testing), and related impact on physical health can be of a similar severity to that seen in anorexia nervosa (e.g., hypothermia, bradycardia, anemia). In severe cases, particularly in infants, malnutrition can be life threatening. "Dependence” on enteral feeding or oral nu- tritional supplements (Criterion A3) means that supplementary feeding is required to sus- tain adequate intake. Examples of individuals requiring supplementary feeding include infants with failure to thrive who require nasogastric tube feeding, children with neuro- developmental disorders who are dependent on nutritionally complete supplements, and in the absence of an underlying medical condition. Inability to participate in normal social activities, such as eating with others, or to sustain relationships as a result of the distur-

1	and in the absence of an underlying medical condition. Inability to participate in normal social activities, such as eating with others, or to sustain relationships as a result of the distur- bance would indicate marked interference with psychosocial functioning (Criterion A4).

1	food intake related to lack of availability of food or to cultural practices (e.g., religious fast- ing or normal dieting) (Criterion B), nor does it include developmentally normal behaviors (e.g., picky eating in toddlers, reduced intake in older adults). The disturbance is not better medical factors or mental disorders (Criterion D). In some individuals, food avoidance or restriction may be based on the sensory char- acteristics of qualities of food, such as extreme sensitivity to appearance, color, smell, texture, temperature, or taste. Such behavior has been described as ”restrictive eating,” "selective eating," ”choosy eating,” ”perseverant eating, chronic food refusal,” and “food neophobia” and may manifest as refusal to eat particular brands of foods or to tol- erate the smell of food being eaten by others. Individuals with heightened sensory sensi- tivities associated with autism may show similar behaviors.

1	sociated with food intake following, or in anticipation of, an aversive experience, such as choking; a traumatic investigation, usually involving the gastrointestinal tract (e.g., esoph— agoscopy); or repeated vomiting. The termsfunctional dysphagia and globus hystericus have also been used for such conditions. Several features may be associated with food avoidance or reduced food intake, including a lack of interest in eating or food, leading to weight loss or faltering growth. Very young infants may present as being too sleepy, distressed, or agitated to feed. Infants and young children may not engage with the primary caregiver during feeding or communicate hun- ger in favor of other activities. In older children and adolescents, food avoidance or restric- tion may be associated with more generalized emotional difficulties that do not meet diagnostic criteria for an anxiety, depressive, or bipolar disorder, sometimes called ”food avoidance emotional disorder.”

1	Food avoidance or restriction associated with insufficient intake or lack of interest in eat- ing most commonly develops in infancy or early childhood and may persist in adulthood. Likewise, avoidance based on sensory characteristics of food tends to arise in the first de- cade of life but may persist into adulthood. Avoidance related to aversive consequences can arise at any age. The scant literature regarding long—term outcomes suggests that food avoidance or restriction based on sensory aspects is relatively stable and long-standing, but when persisting into adulthood, such avoidance / restriction can be associated with rel- atively normal functioning. There is currently insufficient evidence directly linking avoid- ant/restrictive food intake disorder and subsequent onset of an eating disorder.

1	Infants with avoidant/ restrictive food intake disorder may be irritable and difficult to console during feeding, or may appear apathetic and withdrawn. In some instances, par- ent-child interaction may contribute to the infant’s feeding problem (e.g., presenting food inappropriately, or interpreting the infant’s behavior as an act of aggression or rejection).

1	Inadequate nutritional intake may exacerbate the associated features (e.g., irritability, de- velopmental lags) and further contribute to feeding difficulties. Associated factors include to feeding. Coexisting parental psychopathology, or child abuse or neglect, is suggested if feeding and weight improve in response to changing caregivers. In infants, children, and prepubertal adolescents, avoidant/ restrictive food intake disorder may be associated with growth delay, and the resulting malnutrition negatively affects development and learning potential. In older children, adolescents, and adults, social functioning tends to be ad- versely affected. Regardless of the age, family function may be affected, with heightened stress at mealtimes and in other feeding or eating contexts involving friends and relatives.

1	in adults, and there may be a long delay between onset and clinical presentation. Triggers for presentation vary considerably and include physical, social, and emotional difficulties. Temperamental. Anxiety disorders, autism spectrum disorder, obsessive-compulsive disorder, and attention-deficit/hyperactivity disorder may increase risk for avoidant or restrictive feeding or eating behavior characteristic of the disorder. Environmental. Environmental risk factors for avoidant/ restrictive food intake disor- der include familial anxiety. Higher rates of feeding disturbances may occur in children of mothers with eating disorders. Genetic and physiological. History of gastrointestinal conditions, gastroesophageal re- ﬂux disease, vomiting, and a range of other medical problems has been associated with feeding and eating behaviors characteristic of avoidant/restrictive food intake disorder.

1	Presentations similar to avoidant/restrictive food intake disorder occur in various popu- lations, including in the United States, Canada, Australia, and Europe. Avoidant/restrictive food intake disorder should not be diagnosed when avoidance of food intake is solely re- lated to specific religious or cultural practices. Avoidant/restrictive food intake disorder is equally common in males and females in in- fancy and early childhood, but avoidant/restrictive food intake disorder comorbid with autism spectrum disorder has a male predominance. Food avoidance or restriction related to altered sensory sensitivities can occur in some physiological conditions, most notably pregnancy, but is not usually extreme and does not meet full criteria for the disorder. Diagnostic markers include malnutrition, low weight, growth delay, and the need for ar- tificial nutrition in the absence of any clear medical condition other than poor intake. Functionai Consequences of Avoidanthestrictive

1	Functionai Consequences of Avoidanthestrictive Associated developmental and functional limitations include impairment of physical de- function. Appetite loss preceding restricted intake is a nonspecific symptom that can accompany a number of mental diagnoses. Avoidant/restrictive food intake disorder can be diagnosed concurrently with the disorders below if all criteria are met, and the eating disturbance re- quires specific clinical attention.

1	Other medical conditions (e.g., gastrointestinal disease, food allergies and intoler- ances, occult malignancies). Restriction of food intake may occur in other medical condi- tions, especially those with ongoing symptoms such as vomiting, loss of appetite, nausea, ab- dominal pain, or diarrhea. A diagnosis of avoidant/ restrictive food intake disorder requires that the disturbance of intake is beyond that directly accounted for by physical symptoms con- sistent with a medical condition; the eating disturbance may also persist after being triggered by a medical condition and following resolution of the medical condition. Underlying medical or comorbid mental conditions may complicate feeding and eating. Because older individuals, postsurgical patients, and individuals receiving chemotherapy often lose their appetite, an additional diagnosis of avoidant/restrictive food intake dis— order requires that the eating disturbance is a primary focus for intervention.

1	Specific neurological/neuromuscular, structural, or congenital disorders and condi- tions associated with feeding difficulties. Feeding difficulties are common in a number of congenital and neurological conditions often related to problems with oral/esophageal/ pharyngeal structure and function, such as hypotonia of musculature, tongue protrusion, and unsafe swallowing. Avoidant/ restrictive food intake disorder can be diagnosed in in- dividuals with such presentations as long as all diagnostic criteria are met. Reactive attachment disorder. Some degree of withdrawal is characteristic of reactive attachment disorder and can lead to a disturbance in the caregiver-child relationship that can affect feeding and the child’s intake. Avoidant/restrictive food intake disorder should be diagnosed concurrently only if all criteria are met for both disorders and the feeding disturbance is a primary focus for intervention.

1	Autism spectrum disorder. Individuals with autism spectrum disorder often present with rigid eating behaviors and heightened sensory sensitivities. However, these features do not always result in the level of impairment that would be required for a diagnosis of avoidant/ restrictive food intake disorder. Avoidant/restrictive food intake disorder should be diagnosed concurrently only if all criteria are met for both disorders and when the eat- ing disturbance requires specific treatment. Specific phobia, social anxiety disorder (social phobia), and other anxiety disorders. Specific phobia, other type, specifies ”situations that may lead to choking or vomiting” and can represent the primary trigger for the fear, anxiety, or avoidance required for diagnosis.

1	Specific phobia, other type, specifies ”situations that may lead to choking or vomiting” and can represent the primary trigger for the fear, anxiety, or avoidance required for diagnosis. ficult when a fear of choking or vomiting has resulted in food avoidance. Although avoid- ance or restriction of food intake secondary to a pronounced fear of choking or vomiting can be conceptualized as specific phobia, in situations when the eating problem becomes the primary focus of clinical attention, avoidant/ restrictive food intake disorder becomes the appropriate diagnosis. In social anxiety disorder, the individual may present with a fear of being observed by others while eating, Which can also occur in avoidant/ restrictive food intake disorder.

1	Anorexia nervosa. Restriction of energy intake relative to requirements leading to sig- nificantly low body weight is a core feature of anorexia nervosa. However, individuals with anorexia nervosa also display a fear of gaining weight or of becoming fat, or persis- tent behavior that interferes with weight gain, as well as specific disturbances in relation to perception and experience of their own body weight and shape. These features are not present in avoidant/ restrictive food intake disorder, and the two disorders should not be diagnosed concurrently. Differential diagnosis between avoidant/restrictive food intake disorder and anorexia nervosa may be difficult, especially in late childhood and early ad- olescence, because these disorders may share a number of common symptoms (e.g., food avoidance, low weight). Differential diagnosis is also potentially difficult in individuals with anorexia nervosa who deny any fear of fatness but nonetheless engage in persistent behaviors that

1	food avoidance, low weight). Differential diagnosis is also potentially difficult in individuals with anorexia nervosa who deny any fear of fatness but nonetheless engage in persistent behaviors that prevent weight gain and who do not recognize the medical seriousness of their low weight—a presentation sometimes termed ”non-fat phobic anorexia nervosa."

1	Full consideration of symptoms, course, and family history is advised, and diagnosis may be best made in the context of a clinical relationship over time. In some individuals, avoid- ant/restrictive food intake disorder might precede the onset of anorexia nervosa. Obsessive-compulsive disorder. Individuals with obsessive-compulsive disorder may present with avoidance or restriction of intake in relation to preoccupations with food or ritualized eating behavior. Avoidant/restrictive food intake disorder should be diagnosed concurrently only if all criteria are met for both disorders and when the aberrant eating is a major aspect of the clinical presentation requiring specific intervention.

1	Major depressive disorder. In major depressive disorder, appetite might be affected to such an extent that individuals present with significantly restricted food intake, usually in relation to overall energy intake and often associated with weight loss. Usually appetite loss and related reduction of intake abate with resolution of mood problems. Avoidant/ restrictive food intake disorder should only be used concurrently if full criteria are met for both disorders and when the eating disturbance requires specific treatment.

1	Schizophrenia spectrum disorders. Individuals with schizophrenia, delusional disor- der, or other psychotic disorders may exhibit odd eating behaviors, avoidance of specific foods because of delusional beliefs, or other manifestations of avoidant or restrictive in- take. In some cases, delusional beliefs may contribute to a concern about negative conse- quences of ingesting certain foods. Avoidant/ restrictive food intake disorder should be used concurrently only if all criteria are met for both disorders and when the eating dis- turbance requires specific treatment.

1	Factitious disorder or factitious disorder imposed on another. Avoidant/restrictive der imposed on another. In order to assume the sick role, some individuals with factitious disorder may intentionally describe diets that are much more restrictive than those they are actually able to consume, as well as complications of such behavior, such as a need for enteral feedings or nutritional supplements, an inability to tolerate a normal range of foods, and/ or an inability to participate normally in age-appropriate situations involving food. The presentation may be impressively dramatic and engaging, and the symptoms re- ported inconsistently. In factitious disorder imposed on another, the caregiver describes symptoms consistent with avoidant/restrictive food intake disorder and may induce physical symptoms such as failure to gain weight. As with any diagnosis of factitious dis- order imposed on another, the caregiver receives the diagnosis rather than the affected in- dividual, and

1	physical symptoms such as failure to gain weight. As with any diagnosis of factitious dis- order imposed on another, the caregiver receives the diagnosis rather than the affected in- dividual, and diagnosis should be made only on the basis of a careful, comprehensive assessment of the affected individual, the caregiver, and their interaction.

1	The most commonly observed disorders comorbid with avoidant/restrictive food intake disorder are anxiety disorders, obsessive-compulsive disorder, and neurodevelopmental disorders (specifically autism spectrum disorder, attention-deficit/hyperactivity disor- der, and intellectual disability [intellectual developmental disorder]). A. Restriction of energy intake relative to requirements, leading to a significantly low body weight in the context of age, sex, developmental trajectory, and physical health. Sig- nificantly low weight is defined as a weight that is less than minimally normal or, for children and adolescents, less than that minimally expected. B. Intense fear of gaining weight or of becoming fat, or persistent behavior that interferes with weight gain, even though at a significantly low weight.

1	B. Intense fear of gaining weight or of becoming fat, or persistent behavior that interferes with weight gain, even though at a significantly low weight. C. Disturbance in the way in which one’s body weight or shape is experienced, undue in- fluence of body weight or shape on seIf-evaluation, or persistent lack of recognition of the seriousness of the current low body weight. Coding note: The |CD-9-CM code for anorexia nervosa is 307.1, which is assigned re- gardless of the subtype. The lCD-10-CM code depends on the subtype (see below). Specify whether: (F50.01) Restricting type: During the last 3 months, the individual has not engaged in re- current episodes of binge eating or purging behavior (i.e., self—induced vomiting or the mis- use of laxatives, diuretics, or enemas). This subtype describes presentations in which weight loss is accomplished primarily through dieting, fasting, and/or excessive exercise.

1	(F50.02) Binge-eating/purging type: During the last 3 months, the individual has en- gaged in recurrent episodes of binge eating or purging behavior (i.e., self-induced vomiting or the misuse of laxatives, diuretics, or enemas). Specify if: In partial remission: Al'ter full criteria for anorexia nervosa were previously met, Cri- terion A (low body weight) has not been met for a sustained period, but either Criterion B (intense fear of gaining weight or becoming fat or behavior that interferes with weight gain) or Criterion C (disturbances in seIf-perception of weight and shape) is still met. In full remission: After full criteria for anorexia nervosa were previously met, none of the criteria have been met for a sustained period of time. Specify current severity:

1	In full remission: After full criteria for anorexia nervosa were previously met, none of the criteria have been met for a sustained period of time. Specify current severity: The minimum level of severity is based, for adults, on current body mass index (BMI) (see below) or, for children and adolescents, on BMI percentile. The ranges below are derived cents, corresponding BMI percentiles should be used. The level of severity may be in— creased to reflect clinical symptoms, the degree of functional disability, and the need for supervision. Mild: BM|217 kg/m2 Moderate: BMI 16—16.99 kg/m2 Severe: BMI 15—15.99 kg/m2 Extreme: BMI < 15 kg/m2 Most individuals with the binge-eating/purging type of anorexia nervosa who binge eat also purge through self—induced vomiting or the misuse of laxatives, diuretics, or enemas. Some individuals with this subtype of anorexia nervosa do not binge eat but do regularly purge after the consumption of small amounts of food.

1	Some individuals with this subtype of anorexia nervosa do not binge eat but do regularly purge after the consumption of small amounts of food. Crossover between the subtypes over the course of the disorder is not uncommon; therefore, subtype description should be used to describe current symptoms rather than longitudinal course.

1	Crossover between the subtypes over the course of the disorder is not uncommon; therefore, subtype description should be used to describe current symptoms rather than longitudinal course. There are three essential features of anorexia nervosa: persistent energy intake restriction; intense fear of gaining weight or of becoming fat, or persistent behavior that interferes with weight gain; and a disturbance in self—perceived weight or shape. The individual main- tains a body weight that is below a minimally normal level for age, sex, developmental tra- jectory, and physical health (Criterion A). Individuals’ body weights frequently meet this criterion following a significant weight loss, but among children and adolescents, there may alternatively be failure to make expected weight gain or to maintain a normal devel- opmental trajectory (i.e., while growing in height) instead of weight loss.

1	Criterion A requires that the individual’s weight be significantly low (i.e., less than minimally normal or, for children and adolescents, less than that minimally expected). Weight assessment can be challenging because normal weight range differs among indi- viduals, and different thresholds have been published defining thinness or underwzeight status. Body mass index (BMI; calculated as weight 1n kilograms /he1ght1n mezters 2') 1s a useful measure to assess body weight for height. For adults, a BMI of 18.5 kg/m 2has been employed by the Centers for Disease Control and Prevention (CDC) and the World Health

1	Organization (WHO) as the lower limit of2 normal body weight. Therefore, most adults with a BMI greater than or equal to 18.5 kg/m2 would not be considered tzo have a significantly low body weight. On the other hand, a BMI of lower than 17.0 kg/m 2has been considered by the WHO to indiczate moderate or severe thinness; therefore, an individual with a BMI less than 17. 0 kg/m 2would likely be considzered to have a significantly low weight An adult with a BMI between 17. 0 and 18.5 kg/mz, or even above 18.5 kg/m ,might be consid- ered to have a significantly low weight if clinical history or other physiological informa- tion supports this judgment.

1	For children and adolescents, determining a BMI-for-age percentile is useful (see, e.g., the CDC BMI percentile calculator for children and teenagers. As for adults, it is not pos- sible to provide definitive standards for judging whether a child’s or an adolescent’s weight is significantly low, and variations in developmental trajectories among youth limit the utility of simple numerical guidelines. The CDC has used a BMI-for-age below the 5th per- centile as suggesting underweight; however, children and adolescents with a BMI above this benchmark may be judged to be significantly underweight in light of failure to main- tain their expected growth trajectory. In summary, in determining whether Criterion A is met, the clinician should consider available numerical guidelines, as well as the individual’s body build, weight history, and any physiological disturbances.

1	Individuals with this disorder typically display an intense fear of gaining weight or of becoming fat (Criterion B). This intense fear of becoming fat is usually not alleviated by weight loss. In fact, concern about weight gain may increase even as weight falls. Younger individuals with anorexia nervosa, as well as some adults, may not recognize or acknowl- edge a fear of weight gain. In the absence of another explanation for the significantly low weight, clinician inference drawn from collateral history, observational data, physical and laboratory findings, or longitudinal course either indicating a fear of weight gain or sup- porting persistent behaviors that prevent it may be used to establish Criterion B.

1	The experience and significance of body weight and shape are distorted in these indi- viduals (Criterion C). Some individuals feel globally overweight. Others realize that they are thin but are still concerned that certain body parts, particularly the abdomen, buttocks, and thighs, are ”too fat." They may employ a variety of techniques to evaluate their body size or weight, including frequent weighing, obsessive measuring of body parts, and per- sistent use of a mirror to check for perceived areas of ”fat.” The self-esteem of individuals with anorexia nervosa is highly dependent on their perceptions of body shape and weight. Weight loss is often viewed as an impressive achievement and a sign of extraordinary self- discipline, whereas weight gain is perceived as an unacceptable failure of self—control. Al- though some individuals with this disorder may acknowledge being thin, they often do not recognize the serious medical implications of their malnourished state.

1	Often, the individual is brought to professional attention by family members after marked weight loss (or failure to make expected weight gains) has occurred. If individuals seek help on their own, it is usually because of distress over the somatic and psychological sequelae of starvation. It is rare for an individual with anorexia nervosa to complain of weight loss per se. In fact, individuals with anorexia nervosa frequently either lack insight into or deny the problem. It is therefore often important to obtain information from family members or other sources to evaluate the history of weight loss and other features of the illness.

1	The semi-starvation of anorexia nervosa, and the purging behaviors sometimes associated with it, can result in significant and potentially life-threatening medical conditions. The nutritional compromise associated with this disorder affects most major organ systems and can produce a variety of disturbances. Physiological disturbances, including amenor- rhea and vital sign abnormalities, are common. While most of the physiological distur- bances associated with malnutrition are reversible with nutritional rehabilitation, some, including loss of bone mineral density, are often not completely reversible. Behaviors such as self—induced vomiting and misuse of laxatives, diuretics, and enemas may cause a num- ber of disturbances that lead to abnormal laboratory findings; however, some individuals with anorexia nervosa exhibit no laboratory abnormalities.

1	When seriously underweight, many individuals with anorexia nervosa have depressive signs and symptoms such as depressed mood, social withdrawal, irritability, insomnia, and diminished interest in sex. Because these features are also observed in individuals without anorexia nervosa who are significantly undernourished, many of the depressive features may be secondary to the physiological sequelae of semi-starvation, although they may also be sufficiently severe to warrant an additional diagnosis of major depressive disorder. Obsessive-compulsive features, both related and unrelated to food, are often prominent.

1	Obsessive-compulsive features, both related and unrelated to food, are often prominent. Most individuals with anorexia nervosa are preoccupied with thoughts of food. Some col- lect recipes or hoard food. Observations of behaviors associated with other forms of star- vation suggest that obsessions ancl compulsions related to food may be exacerbated by undemutrition. When individuals with anorexia nervosa exhibit obsessions and compul- sions that are not related to food, body shape, or weight, an additional diagnosis of obses- sive-compulsive disorder (OCD) may be warranted.

1	Other features sometimes associated with anorexia nervosa include concerns about eating in public, feelings of ineffectiveness, a strong desire to control one’s environment, inflexible thinking, limited social spontaneity, and overly restrained emotional ex- pression. Compared with individuals with anorexia nervosa, restricting type, those with binge-eating/purging type have higher rates of impulsivity and are more likely to abuse alcohol and other drugs. A subgroup of individuals with anorexia nervosa show excessive levels of physical ac- tivity. Increases in physical activity often precede onset of the disorder, and over the course of the disorder increased activity accelerates weight loss. During treatment, exces- sive activity may be difficult to control, thereby jeopardizing weight recovery.

1	Individuals with anorexia nervosa may misuse medications, such as by manipulating dosage, in order to achieve weight loss or avoid weight gain. Individuals with diabetes mellitus may omit or reduce insulin doses in order to minimize carbohydrate metabolism. The 12-month prevalence of anorexia nervosa among young females is approximately 0.4%. Less is known about prevalence among males, but anorexia nervosa is far less com— mon in males than in females, with clinical populations generally reﬂecting approximately a 10:1 female-to-male ratio. Anorexia nervosa commonly begins during adolescence or young adulthood. It rarely be- gins before puberty or after age 40, but cases of both early and late onset have been de- scribed. The onset of this disorder is often associated with a stressful life event, such as leaving home for college. The course and outcome of anorexia nervosa are highly variable.

1	Younger individuals may manifest atypical features, including denying "fear of fat.” Older individuals more likely have a longer duration of illness, and their clinical presentation may include more signs and symptoms of long-standing disorder. Clinicians should not exclude anorexia nervosa from the differential diagnosis solely on the basis of older age.

1	Many individuals have a period of changed eating behavior prior to full criteria for the disorder being met. Some individuals with anorexia nervosa recover fully after a single episode, with some exhibiting a ﬂuctuating pattern of weight gain followed by relapse, and others experiencing a chronic course over many years. Hospitalization may be re— quired to restore weight and to address medical complications. Most individuals with an- orexia nervosa experience remission within 5 years of presentation. Among individuals admitted to hospitals, overall remission rates may be lower. The crude mortality rate (CMR) for anorexia nervosa is approximately 5% per decade. Death most commonly results from medical complications associated with the disorder itself or from suicide. Temperamental. Individuals who develop anxiety disorders or display obsessional traits in childhood are at increased risk of developing anorexia nervosa.

1	Temperamental. Individuals who develop anxiety disorders or display obsessional traits in childhood are at increased risk of developing anorexia nervosa. Environmental. Historical and cross-cultural variability in the prevalence of anorexia nervosa supports its association with cultures and settings in which thinness is valued. Oc- cupations and avocations that encourage thinness, such as modeling and elite athletics, are also associated with increased risk.

1	Genetic and physiological. There is an increased risk of anorexia nervosa and bulimia nervosa among first-degree biological relatives of individuals with the disorder. An in— creased risk of bipolar and depressive disorders has also been found among first-degree relatives of individuals with anorexia nervosa, particularly relatives of individuals with the binge—eating/purging type. Concordance rates for anorexia nervosa in monozygotic twins are significantly higher than those for dizygotic twins. A range of brain abnormali- tional magnetic resonance imaging, positron emission tomography). The degree to which these findings reﬂect changes associated with malnutrition versus primary abnormalities associated with the disorder is unclear.

1	Anorexia nervosa occurs across culturally and socially diverse populations, although available evidence suggests cross-cultural variation in its occurrence and presentation. Anorexia ner- vosa is probably most prevalent in post-industrialized, high-income countries such as in the United States, many European countries, Australia, New Zealand, and Japan, but its incidence in most low- and middle-income countries is uncertain. Whereas the prevalence of anorexia nervosa appears comparatively low among Latinos, African Americans, and Asians in the

1	United States, clinicians should be aware that mental health service utilization among individ- uals with an eating disorder is significantly lower in these ethnic groups and that the low rates may reﬂect an ascertainment bias. The presentation of weight concerns among individuals with eating and feeding disorders varies substantially across cultural contexts. The absence of an expressed intense fear of weight gain, sometimes referred to as ”fat phobia,” appears to be relatively more common in populations in Asia, where the rationale for dietary restriction is commonly related to a more culturally sanctioned complaint such as gastrointestinal discom- fort. Within the United States, presentations without a stated intense fear of weight gain may be comparatively more common among Latino groups. The following laboratory abnormalities may be observed in anorexia nervosa; their pres- ence may serve to increase diagnostic confidence.

1	The following laboratory abnormalities may be observed in anorexia nervosa; their pres- ence may serve to increase diagnostic confidence. Hematology. Leukopenia is common, with the loss of all cell types but usually with ap- parent lymphocytosis. Mild anemia can occur, as well as thrombocytopenia and, rarely, bleeding problems. Serum chemistry. Dehydration may be reﬂected by an elevated blood urea nitrogen level. Hypercholesterolemia is common. Hepatic enzyme levels may be elevated. Hypo- magnesemia, hypozincemia, hypophosphatemia, and hyperamylasemia are occasionally observed. Self-induced vomiting may lead to metabolic alkalosis (elevated serum bicarbon- ate), hypochloremia, and hypokalernia; laxative abuse may cause a mild metabolic acidosis.

1	Endocrine. Serum thyroxine (T4) levels are usually in the low-normal range; triiodothy- ronine (T3) levels are decreased, while reverse T3 levels are elevated. Females have low se- rum estrogen levels, whereas males have low levels of serum testosterone. Electrocardiography. Sinus bradycardia is common, and, rarely, arrhythmias are noted. Significant prolongation of the QTc interval is observed in some individuals. Bone mass. Low bone mineral density, with specific areas of osteopenia or osteoporo- sis, is often seen. The risk of fracture is significantly elevated. Electroencephalography. Diffuse abnormalities, reﬂecting a metabolic encephalopa- thy, may result from significant ﬂuid and electrolyte disturbances. Resting energy expenditure. There is often a significant reduction in resting energy ex— penditure.

1	Resting energy expenditure. There is often a significant reduction in resting energy ex— penditure. Physical signs and symptoms. Many of the physical signs and symptoms of anorexia nervosa are attributable to starvation. Amenorrhea is commonly present and appears to be an indicator of physiological dysfunction. If present, amenorrhea is usually a conse- quence of the weight loss, but in a minority of individuals it may actually precede the weight loss. In prepubertal females, menarche may be delayed. In addition to amenorrhea, there may be complaints of constipation, abdominal pain, cold intolerance, lethargy, and excess energy.

1	The most remarkable finding on physical examination is emaciation. Commonly, there is also significant hypotension, hypothermia, and bradycardia. Some individuals develop lanugo, a fine downy body hair. Some develop peripheral edema, especially during weight restoration or upon cessation of laxative and diuretic abuse. Rarely, petechiae or ecchymoses, usually on the extremities, may indicate a bleeding diathesis. Some individ- uals evidence a yellowing of the skin associated with hypercarotenemia. As may be seen in individuals with bulimia nervosa, individuals with anorexia nervosa who self-induce vomiting may have hypertrophy of the salivary glands, particularly the parotid glands, as well as dental enamel erosion. Some individuals may have scars or calluses on the dorsal surface of the hand from repeated contact with the teeth while inducing vomiting. Suicide risk is elevated in anorexia nervosa, with rates reported as 12 per 100,000 per year.

1	Suicide risk is elevated in anorexia nervosa, with rates reported as 12 per 100,000 per year. Comprehensive evaluation of individuals with anorexia nervosa should include assess- ment of suicide—related ideation and behaviors as well as other risk factors for suicide, in— cluding a history of suicide attempt(s). Functional Consequences of Anorexia Nervosa Individuals with anorexia nervosa may exhibit a range of functional limitations associated with the disorder. While some individuals remain active in social and professional func- tioning, others demonstrate significant social isolation and / or failure to fulfill academic or career potential. Other possible causes of either significantly low body weight or significant weight loss should be considered in the differential diagnosis of anorexia nervosa, especially when the presenting features are atypical (e.g., onset after age 40 years).

1	Medical conditions (e.g., gastrointestinal disease, hyperthyroidism, occult malignan- cies, and acquired immunodeficiency syndrome [AIDS]). Serious weight loss may oc- cur in medical conditions, but individuals with these disorders usually do not also mani- fest a disturbance in the way their body weight or shape is experienced or an intense fear of weight gain or persist in behaviors that interfere with appropriate weight gain. Acute weight loss associated with a medical condition can occasionally be followed by the onset or recurrence of anorexia nervosa, which can initially be masked by the comorbid medical condition. Rarely, anorexia nervosa develops after bariatric surgery for obesity. Major depressive disorder. In major depressive disorder, severe weight loss may occur, but most individuals with major depressive disorder do not have either a desire for exces- sive weight loss or an intense fear of gaining weight.

1	Schizophrenia. Individuals with schizophrenia may exhibit odd eating behavior and oc- casionally experience significant weight loss, but they rarely show the fear of gaining weight and the body image disturbance required for a diagnosis of anorexia nervosa. Substance use disorders. Individuals with substance use disorders may experience low weight due to poor nutritional intake but generally do not fear gaining weight and do not manifest body image disturbance. Individuals who abuse substances that reduce appetite (e.g., cocaine, stimulants) and who also endorse fear of weight gain should be carefully evaluated for the possibility of comorbid anorexia nervosa, given that the substance use may represent a persistent behavior that interferes with weight gain (Criterion B).

1	Social anxiety disorder (social phobia), obsessive-compulsive disorder, and body dys- morphic disorder. Some of the features of anorexia nervosa overlap with the criteria for social phobia, OCD, and body dysmorphic disorder. Specifically, individuals may feel hu- miliated or embarrassed to be seen eating in public, as in social phobia; may exhibit obses- sions and compulsions related to food, as in 0CD; or may be preoccupied with an imagined defect in bodily appearance, as in body dysmorphic disorder. If the individual with anorexia nervosa has social fears that are limited to eating behavior alone, the diagnosis of social pho- bia should not be made, but social fears unrelated to eating behavior (e.g., excessive fear of speaking in public) may warrant an additional diagnosis of social phobia. Similarly, an ad— ditional diagnosis of 0CD should be considered only if the individual exhibits obsessions and compulsions unrelated to food (e.g., an excessive fear of contamination), and an

1	Similarly, an ad— ditional diagnosis of 0CD should be considered only if the individual exhibits obsessions and compulsions unrelated to food (e.g., an excessive fear of contamination), and an addi- tional diagnosis of body dysmorphic disorder should be considered only if the distortion is unrelated to body shape and size (e.g., preoccupation that one’s nose is too big).

1	Bulimia nervosa. Individuals with bulimia nervosa exhibit recurrent episodes of binge eating, engage in inappropriate behavior to avoid weight gain (e.g., self—induced vomit- ing), and are overly concerned with body shape and weight. However, unlike individuals with anorexia nervosa, binge-eating/purging type, individuals with bulimia nervosa main— tain body weight at or above a minimally normal level. Avoidant/restrictive food intake disorder. Individuals with this disorder may exhibit significant weight loss or significant nutritional deficiency, but they do not have a fear of gaining weight or of becoming fat, nor do they have a disturbance in the way they expe- rience their body shape and weight. Bipolar, depressive, and anxiety disorders commonly co-occur with anorexia nervosa.

1	Bipolar, depressive, and anxiety disorders commonly co-occur with anorexia nervosa. Many individuals with anorexia nervosa report the presence of either an anxiety disorder or symptoms prior to onset of their eating disorder. OCD is described in some individuals with anorexia nervosa, especially those with the restricting type. Alcohol use disorder and other substance use disorders may also be comorbid with anorexia nervosa, especially among those with the binge-eating/purging type. Diagnostic Criteria 307.51 (F50.2) A. Recurrent episodes of binge eating. An episode of binge eating is characterized by both of the following: 1. Eating, in a discrete period of time (e.g., within any 2-hour period), an amount of food that is definitely larger than what most individuals would eat in a similar period of time under similar circumstances. 2. A sense of lack of control over eating during the episode (e.g., a feeling that one cannot stop eating or control what or how much one is eating).

1	2. A sense of lack of control over eating during the episode (e.g., a feeling that one cannot stop eating or control what or how much one is eating). B. Recurrent inappropriate compensatory behaviors in order to prevent weight gain, such as selt-induced vomiting; misuse of laxatives, diuretics, or other medications; fasting; or excessive exercise. C. The binge eating and inappropriate compensatory behaviors both occur, on average, at least once a week for 3 months. D. Self-evaluation is unduly influenced by body shape and weight. E. The disturbance does not occur exclusively during episodes of anorexia nervosa. Specify it: In partial remission: After full criteria for bulimia nervosa were previously met, some, but not all, of the criteria have been met for a sustained period of time. In full remission: After full criteria for bulimia nervosa were previously met. none of the criteria have been met for a sustained period of time. Specify current severity:

1	In full remission: After full criteria for bulimia nervosa were previously met. none of the criteria have been met for a sustained period of time. Specify current severity: The minimum level of severity is based on the frequency of inappropriate compensatory behaviors (see below). The level of severity may be increased to reflect other symptoms and the degree of functional disability. Mild: An average of 1—3 episodes of inappropriate compensatory behaviors per week. Moderate: An average 014—7 episodes of inappropriate compensatory behaviors per week. Severe: An average of 8—13 episodes of inappropriate compensatory behaviors per week. Extreme: An average of 14 or more episodes of inappropriate compensatory behav- iors per week.

1	Severe: An average of 8—13 episodes of inappropriate compensatory behaviors per week. Extreme: An average of 14 or more episodes of inappropriate compensatory behav- iors per week. There are three essential features of bulimia nervosa: recurrent episodes of binge eating (Criterion A), recurrent inappropriate compensatory behaviors to prevent weight gain (Criterion B), and self—evaluation that is unduly inﬂuenced by body shape and weight (Criterion D). To qualify for the diagnosis, the binge eating and inappropriate compensa- tory behaviors must occur, on average, at least once per week for 3 months (Criterion C).

1	An ”episode of binge eating” is defined as eating, in a discrete period of time, an amount of food that is definitely larger than most individuals would eat in a similar period of time under similar circumstances (Criterion A1). The context in which the eating occurs may affect the clinician’s estimation of whether the intake is excessive. For example, a quantity of food that might be regarded as excessive for a typical meal might be consid- ered normal during a celebration or holiday meal. A ”discrete period of time” refers to a limited period, usually less than 2 hours. A single episode of binge eating need not be re- stricted to one setting. For example, an individual may begin a binge in a restaurant and then continue to eat on returning home. Continual snacking on small amounts of food throughout the day would not be considered an eating binge.

1	An occurrence of excessive food consumption must be accompanied by a sense of lack of control (Criterion A2) to be considered an episode of binge eating. An indicator of loss of control is the inability to refrain from eating or to stop eating once started. Some indi- viduals describe a dissociative quality during, or following, the binge-eating episodes. The impairment in control associated with binge eating may not be absolute; for example, an individual may continue binge eating while the telephone is ringing but will cease if a roommate or spouse unexpectedly enters the room. Some individuals report that their binge-eating episodes are no longer characterized by an acute feeling of loss of control but rather by a more generalized pattern of uncontrolled eating. If individuals report that they have abandoned efforts to control their eating, loss of control should be considered as present. Binge eating can also be planned in some instances.

1	The type of food consumed during binges varies both across individuals and for a given individual. Binge eating appears to be characterized more by an abnormality in the amount of food consumed than by a craving for a specific nutrient. However, during binges, individuals tend to eat foods they would otherwise avoid. Individuals with bulimia nervosa are typically ashamed of their eating problems and attempt to conceal their symptoms. Binge eating usually occurs in secrecy or as inconspic- uously as possible. The binge eating often continues until the individual is uncomfortably, or even painfully, full. The most common antecedent of binge eating is negative affect. to body weight, body shape, and food; and boredom. Binge eating may minimize or mit- igate factors that precipitated the episode in the short-term, but negative self-evaluation and dysphoria often are the delayed consequences.

1	Another essential feature of bulimia nervosa is the recurrent use of inappropriate com— pensatory behaviors to prevent weight gain, collectively referred to as purge behaviors or purging (Criterion B). Many individuals with bulimia nervosa employ several methods to compensate for binge eating. Vomiting is the most common inappropriate compensatory behavior. The immediate effects of vomiting include relief from physical discomfort and re- duction of fear of gaining weight. In some cases, vomiting becomes a goal in itself, and the individual will binge eat in order to vomit or will vomit after eating a small amount of food.

1	Individuals with bulimia nervosa may use a variety of methods to induce vomiting, includ- ing the use of fingers or instruments to stimulate the gag reﬂex. Individuals generally become adept at inducing vomiting and are eventually able to vomit at will. Rarely, indi- viduals consume syrup of ipecac to induce vomiting. Other purging behaviors include the misuse of laxatives and diuretics. A number of other compensatory methods may also be used in rare cases. Individuals with bulimia nervosa may misuse enemas following epi- sodes of binge eating, but this is seldom the sole compensatory method employed. Individ- uals with this disorder may take thyroid hormone in an attempt to avoid weight gain.

1	Individuals with diabetes mellitus and bulimia nervosa may omit or reduce insulin doses in order to reduce the metabolism of food consumed during eating binges. Individuals with bulimia nervosa may fast for a day or more or exercise excessively in an attempt to prevent weight gain. Exercise may be considered excessive when it significantly interferes with im- portant activities, when it occurs at inappropriate times or in inappropriate settings, or when the individual continues to exercise despite injury or other medical complications.

1	Individuals with bulimia nervosa place an excessive emphasis on body shape or weight in their self-evaluation, and these factors are typically extremely important in determining self—esteem (Criterion D). Individuals with this disorder may closely resemble those with anorexia nervosa in their fear of gaining weight, in their desire to lose weight, and in the level of dissatisfaction with their bodies. However, a diagnosis of bulimia nervosa should not be given when the disturbance occurs only during episodes of anorexia nervosa (Cri- terion E). Individuals with bulimia nervosa typically are within the normal weight or overweight range (body mass index [BMI] 2 18.5 and < 30 in adults). The disorder occurs but is un- common among obese individuals. Between eating binges, individuals with bulimia ner- (”diet") foods while avoiding foods that they perceive to be fattening or likely to trigger a binge.

1	Menstrual irregularity or amenorrhea often occurs among females with bulimia ner- vosa; it is uncertain whether such disturbances are related to weight ﬂuctuations, to nu- tritional deficiencies, or to emotional distress. The fluid and electrolyte disturbances resulting from the purging behavior are sometimes sufficiently severe to constitute med- ically serious problems. Rare but potentially fatal complications include esophageal tears, gastric rupture, and cardiac arrhythmias. Serious cardiac and skeletal myopathies have been reported among individuals following repeated use of syrup of ipecac to induce vom- iting. Individuals who chronically abuse laxatives may become dependent on their use to stimulate bowel movements. Gastrointestinal symptoms are commonly associated with bulimia nervosa, and rectal prolapse has also been reported among individuals with this disorder.

1	Twelve-month prevalence of bulimia nervosa among young females is 1%—1.5%. Point prevalence is highest among young adults since the disorder peaks in older adolescence and young adulthood. Less is known about the point prevalence of bulimia nervosa in males, but bulimia nervosa is far less common in males than it is in females, with an ap- proximately 10:1 female-to-male ratio. Bulimia nervosa commonly begins in adolescence or young adulthood. Onset before pu- berty or after age 40 is uncommon. The binge eating frequently begins during or after an episode of dieting to lose weight. Experiencing multiple stressful life events also can pre- cipitate onset of bulimia nervosa.

1	clinic samples. The course may be chronic or intermittent, with periods of remission alternating with recurrences of binge eating. However, over longer-term follow-up, the symptoms of many individuals appear to diminish with or without treatment, although treatment clearly impacts outcome. Periods of remission longer than 1 year are associated with better long-term outcome. individuals with bulimia nervosa. The CMR (crude mortality rate) for bulimia nervosa is nearly 2% per decade.

1	individuals with bulimia nervosa. The CMR (crude mortality rate) for bulimia nervosa is nearly 2% per decade. Diagnostic cross-over from initial bulimia nervosa to anorexia nervosa occurs in a mi- nority of cases (10%—15%). Individuals who do experience cross-over to anorexia nervosa commonly will revert back to bulimia nervosa or have multiple occurrences of cross-overs between these disorders. A subset of individuals with bulimia nervosa continue to binge eat but no longer engage in inappropriate compensatory behaviors, and therefore their symptoms meet criteria for binge-eating disorder or other specified eating disorder. Diag- nosis should be based on the current (i.e., past 3 months) clinical presentation. Temperamental. Weight concerns, low self—esteem, depressive symptoms, social anxi- ety disorder, and overanxious disorder of childhood are associated with increased risk for the development of bulimia nervosa.

1	Environmental. Internalization of a thin body ideal has been found to increase risk for developing weight concerns, which in turn increase risk for the development of bulimia nervosa. Individuals who experienced childhood sexual or physical abuse are at increased risk for developing bulimia nervosa. Genetic and physiological. Childhood obesity and early pubertal maturation increase risk for bulimia nervosa. Familial transmission of bulimia nervosa may be present, as well as genetic vulnerabilities for the disorder. Course modifiers. Severity of psychiatric comorbidity predicts worse long—term outcome of bulimia nervosa. Bulimia nervosa has been reported to occur with roughly similar frequencies in most in- dustrialized countries, including the United States, Canada, many European countries,

1	Bulimia nervosa has been reported to occur with roughly similar frequencies in most in- dustrialized countries, including the United States, Canada, many European countries, Australia, Japan, New Zealand, and South Africa. In clinical studies of bulimia nervosa in the United States, individuals presenting with this disorder are primarily white. However, the disorder also occurs in other ethnic groups and with prevalence comparable to esti- mated prevalences observed in white samples. Bulimia nervosa is far more common in females than in males. Males are especially under- represented in treatment-seeking samples, for reasons that have not yet been systemati- cally examined.

1	Bulimia nervosa is far more common in females than in males. Males are especially under- represented in treatment-seeking samples, for reasons that have not yet been systemati- cally examined. No specific diagnostic test for bulimia nervosa currently exists. However, several labora- tory abnormalities may occur as a consequence of purging and may increase diagnostic certainty. These include ﬂuid and electrolyte abnormalities, such as hypokalemia (which can provoke cardiac arrhythmias), hypochloremia, and hyponatremia. The loss of gastric acid through vomiting may produce a metabolic alkalosis (elevated serum bicarbonate), and the frequent induction of diarrhea or dehydration through laxative and diuretic abuse can cause metabolic acidosis. Some individuals with bulimia nervosa exhibit mildly ele— vated levels of serum amylase, probably reﬂecting an increase in the salivary isoenzyme.

1	Physical examination usually yields no physical findings. However, inspection of the mouth may reveal significant and permanent loss of dental enamel, especially from lin- gual surfaces of the front teeth due to recurrent vomiting. These teeth may become chipped and appear ragged and ”moth—eaten.” There may also be an increased frequency of dental caries. In some individuals, the salivary glands, particularly the parotid glands, may become notably enlarged. Individuals who induce vomiting by manually stimulating the gag reﬂex may develop calluses or scars on the dorsal surface of the hand from re- peated contact with the teeth. Serious cardiac and skeletal myopathies have been reported among individuals following repeated use of syrup of ipecac to induce vomiting.

1	Suicide risk is elevated in bulimia nervosa. Comprehensive evaluation of individuals with this disorder should include assessment of suicide-related ideation and behaviors as well as other risk factors for suicide, including a history of suicide attempts. Functional Consequences of Bulimia Nervosa Individuals with bulimia nervosa may exhibit a range of functional limitations associated with the disorder. A minority of individuals report severe role impairment, with the so— cial-life domain most likely to be adversely affected by bulimia nervosa.

1	Anorexia nervosa, binge-eating/purging type. Individuals whose binge-eating behav- ior occurs only during episodes of anorexia nervosa are given the diagnosis anorexia ner- vosa, binge-eating/purging type, and should not be given the additional diagnosis of bulimia nervosa. For individuals with an initial diagnosis of anorexia nervosa who binge and purge but whose presentation no longer meets the full criteria for anorexia nervosa, binge-eating/purging type (e.g., when weight is normal), a diagnosis of bulimia ner- 3 months. Binge-eating disorder. Some individuals binge eat but do not engage in regular inap- propriate compensatory behaviors. In these cases, the diagnosis of binge-eating disorder should be considered. Kleine-Levin syndrome. In certain neurological or other medical conditions, such as

1	Kleine-Levin syndrome. In certain neurological or other medical conditions, such as Kleine-Levin syndrome, there is disturbed eating behavior, but the characteristic psycho- logical features of bulimia nervosa, such as overconcern with body shape and weight, are not present. Major depressive disorder, with atypical features. Overeating is common in major de- pressive disorder, with atypical features, but individuals with this disorder do not engage in inappropriate compensatory behaviors and do not exhibit the excessive concern with body shape and weight characteristic of bulimia nervosa. If criteria for both disorders are met, both diagnoses should be given. Borderline personality disorder. Binge-eating behavior is included in the impulsive be- havior criterion that is part of the definition of borderline personality disorder. If the cri- teria for both borderline personality disorder and bulimia nervosa are met, both diagnoses should be given.

1	Comorbidity with mental disorders is common in individuals with bulimia nervosa, with morbidities. Comorbidity is not limited to any particular subset but rather occurs across a wide range of mental disorders. There is an increased frequency of depressive symptoms (e.g., low self-esteem) and bipolar and depressive disorders (particularly depressive dis- orders) in individuals with bulimia nervosa. In many individuals, the mood disturbance begins at the same time as or following the development of bulimia nervosa, and individ- uals often ascribe their mood disturbances to the bulimia nervosa. However, in some in- dividuals, the mood disturbance clearly precedes the development of bulimia nervosa.

1	There may also be an increased frequency of anxiety symptoms (e.g., fear of social situa- tions) or anxiety disorders. These mood and anxiety disturbances frequently remit follow- ing effective treatment of the bulimia nervosa. The lifetime prevalence of substance use, particularly alcohol or stimulant use, is at least 30% among individuals with bulimia ner- vosa. Stimulant use often begins in an attempt to control appetite and weight. A substan- tial percentage of individuals with bulimia nervosa also have personality features that meet criteria for one or more personality disorders, most frequently borderline personality disorder. Diagnostic Criteria 307.51 (F50.8) A. Recurrent episodes of binge eating. An episode of binge eating is characterized by both of the following: 1. Eating, in a discrete period of time (e.g., within any 2-hour period), an amount of food that is definitely larger than what most people would eat in a similar period of time under similar circumstances.

1	2. A sense of lack of control over eating during the episode (e.g., a feeling that one cannot stop eating or control what or how much one is eating). B. The binge-eating episodes are associated with three (or more) of the following: Eating much more rapidly than normal. Eating until feeling uncomfortably full. Eating large amounts of food when not feeling physically hungry. Eating alone because of feeling embarrassed by how much one is eating. Feeling disgusted with oneself, depressed, or very guilty aftenNard. @PWP.‘ . Marked distress regarding binge eating is present. . The binge eating occurs, on average, at least once a week for 3 months. . The binge eating is not associated with the recurrent use of inappropriate compensa- tory behavior as in bulimia nervosa and does not occur exclusively during the course of bulimia nervosa or anorexia nervosa. Specify it:

1	Specify it: In partial remission: After full criteria for binge-eating disorder were previously met, binge eating occurs at an average frequency of less than one episode per week for a sustained period of time. In full remission: After full criteria for binge-eating disorder were previously met, none of the criteria have been met for a sustained period of time. Specify current severity: The minimum level of severity is based on the frequency of episodes of binge eating (see below). The level of severity may be increased to reflect other symptoms and the degree of functional disability. Mild: 1—3 binge-eating episodes per week. Moderate: 4—7 binge-eating episodes per week. Severe: 8—13 binge-eating episodes per week. Extreme: 14 or more binge-eating episodes per week.

1	The essential feature of binge-eating disorder is recurrent episodes binge eating that must occur, on average, at least once per week for 3 months (Criterion D). An ”episode of binge eating" is defined as eating, in a discrete period of time, an amount of food that is defi- nitely larger than most people would eat in a similar period of time under similar circum- stances (Criterion A1). The context in which the eating occurs may affect the clinician’s estimation of whether the intake is excessive. For example, a quantity of food that might be regarded as excessive for a typical meal might be considered normal during a celebration or holiday meal. A ”discrete period of time” refers to a limited period, usually less than 2 hours. A single episode of binge eating need not be restricted to one setting. For example, an individual may begin a binge in a restaurant and then continue to eat on returning home. Continual snacking on small amounts of food throughout the day would not be con-

1	one setting. For example, an individual may begin a binge in a restaurant and then continue to eat on returning home. Continual snacking on small amounts of food throughout the day would not be con- sidered an eating binge.

1	An occurrence of excessive food consumption must be accompanied by a sense of lack of control (Criterion A2) to be considered an episode of binge eating. An indicator of loss of control is the inability to refrain from eating or to stop eating once started. Some indi- viduals describe a dissociative quality during, or following, the binge-eating episodes. The impairment in control associated with binge eating may not be absolute; for example, an individual may continue binge eating while the telephone is ringing but will cease if a roommate or spouse unexpectedly enters the room. Some individuals report that their binge-eating episodes are no longer characterized by an acute feeling of loss of control but rather by a more generalized pattern of uncontrolled eating. If individuals report that they have abandoned efforts to control their eating, loss of control may still be considered as present. Binge eating can also be planned in some instances.

1	The type of food consumed during binges varies both across individuals and for a given individual. Binge eating appears to be characterized more by an abnormality in the amount of food consumed than by a craving for a specific nutrient. of the following features: eating much more rapidly than normal; eating until feeling un- comfortably full; eating large amounts of food when not feeling physically hungry; eating alone because of feeling embarrassed by how much one is eating; and feeling disgusted with oneself, depressed, or very guilty afterward (Criterion B). Individuals with binge—eating disorder are typically ashamed of their eating problems and attempt to conceal their symptoms. Binge eating usually occurs in secrecy or as incon- spicuously as possible. The most common antecedent of binge eating is negative affect.

1	to body weight, body shape, and food; and boredom. Binge eating may minimize or mit- igate factors that precipitated the episode in the short-term, but negative self-evaluation and dysphoria often are the delayed consequences. Binge-eating disorder occurs in normal—weight/overweight and obese individuals. It is re- liably associated with overweight and obesity in treatment-seeking individuals. Never- theless, binge-eating disorder is distinct from obesity. Most obese individuals do not engage in recurrent binge eating. In addition, compared with weight-matched obese indi- viduals without binge-eating disorder, those with the disorder consume more calories in laboratory studies of eating behavior and have greater functional impairment, lower qual- ity of life, more subjective distress, and greater psychiatric comorbidity.

1	Twelve-month prevalence of binge-eating disorder among U.S. adult (age 18 or older) fe- males and males is 1.6% and 0.8%, respectively. The gender ratio is far less skewed in binge- eating disorder than in bulimia nervosa. Binge-eating disorder is as prevalent among fe- males from racial or ethnic minority groups as has been reported for white females. The disorder is more prevalent among individuals seeking weight—loss treatment than in the general population. Little is known about the development of binge-eating disorder. Both binge eating and associated with increased body fat, weight gain, and increases in psychological symptoms. Binge eating is common in adolescent and college-age samples. Loss-of-control eating or episodic binge eating may represent a prodromal phase of eating disorders for some indi- viduals.

1	Binge eating is common in adolescent and college-age samples. Loss-of-control eating or episodic binge eating may represent a prodromal phase of eating disorders for some indi- viduals. Dieting follows the development of binge eating in many individuals with binge- eating disorder. (This is in contrast to bulimia nervosa, in which dysfunctional dieting usually precedes the onset of binge eating.) Binge-eating disorder typically begins in ad- olescence or young adulthood but can begin in later adulthood. Individuals with binge- eating disorder who seek treatment usually are older than individuals with either bulimia nervosa or anorexia nervosa who seek treatment.

1	Remission rates in both natural course and treatment outcome studies are higher for binge-eating disorder than for bulimia nervosa or anorexia nervosa. Binge-eating disorder appears to be relatively persistent, and the course is comparable to that of bulimia nervosa in terms of severity and duration. Crossover from binge-eating disorder to other eating disorders is uncommon. Genetic and physiological. Binge—eating disorder appears to run in families, which may reﬂect additive genetic inﬂuences. Binge-eating disorder occurs with roughly similar frequencies in most industrialized countries, including the United States, Canada, many European countries, Australia, and New Zealand. In the United States, the prevalence of binge-eating disorder appears com- parable among non-Latino whites, Latinos, Asians, and African Americans. Functionai Consequences of Binge-Eating Disorder

1	Functionai Consequences of Binge-Eating Disorder Binge-eating disorder is associated with a range of functional consequences, including so- cial role adjustment problems, impaired health-related quality of life and life satisfaction, increased medical morbidity and mortality, and associated increased health care utiliza- tion compared with body mass index (BMI)—matched control subjects. It may also be as- sociated with an increased risk for weight gain and the development of obesity.

1	Bulimia nervosa. Binge-eating disorder has recurrent binge eating in common with bu- limia nervosa but differs from the latter disorder in some fundamental respects. In terms of clinical presentation, the recurrent inappropriate compensatory behavior (e.g., purging, driven exercise) seen in bulimia nervosa is absent in binge-eating disorder. Unlike in- dividuals with bulimia nervosa, individuals with binge-eating disorder typically do not show marked or sustained dietary restriction designed to inﬂuence body weight and shape between binge-eating episodes. They may, however, report frequent attempts at dieting. Binge-eating disorder also differs from bulimia nervosa in terms of response to treat- ment. Rates of improvement are consistently higher among individuals with binge-eating disorder than among those with bulimia nervosa.

1	Obesity. Binge—eating disorder is associated with overweight and obesity but has several key features that are distinct from obesity. First, levels of overvaluation of body weight and shape are higher in obese individuals with the disorder than in those without the disorder. Second, rates of psychiatric comorbidity are significantly higher among obese individuals with the disorder compared with those without the disorder. Third, the long-term successful outcome of evidence-based psychological treatments for binge- eating disorder can be contrasted with the absence of effective long-term treatments for obesity.

1	Bipolar and depressive disorders. Increases in appetite and weight gain are included in the criteria for major depressive episode and in the atypical features specifiers for de- pressive and bipolar disorders. Increased eating in the context of a major depressive epi- sode may or may not be associated with loss of control. If the full criteria for both disorders are met, both diagnoses can be given. Binge eating and other symptoms of disordered eat- ing are seen in association with bipolar disorder. If the full criteria for both disorders are met, both diagnoses should be given. Borderline personality disorder. Binge eating is included in the impulsive behavior cri- terion that is part of the definition of borderline personality disorder. If the full criteria for both disorders are met, both diagnoses should be given.

1	Binge-eating disorder is associated with significant psychiatric comorbidity that is com- parable to that of bulimia nervosa and anorexia nervosa. The most common comorbid dis- orders are bipolar disorders, depressive disorders, anxiety disorders, and, to a lesser degree, substance use disorders. The psychiatric comorbidity is linked to the severity of binge eating and not to the degree of obesity. 307.59 (F50.8)

1	307.59 (F50.8) This category applies to presentations in which symptoms characteristic of a feeding and eating disorder that cause clinically significant distress or impairment in social, occupation- al, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the feeding and eating disorders diagnostic class. The other spec- ified feeding or eating disorder category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for any specific feeding and eating disorder. This is done by recording “other specified feeding or eating disorder” followed by the specific reason (e.g., “bulimia nervosa of low frequency”).

1	Examples of presentations that can be specified using the “other specified" designation include the following: 1. Atypical anorexia nervosa: All of the criteria for anorexia nervosa are met, except that despite significant weight loss, the individual’s weight is within or above the normal range. 2. Bulimia nervosa (of low frequency andlor limited duration): All of the criteria for bulimia nervosa are met, except that the binge eating and inappropriate compensatory behaviors occur, on average, less than once a week and/or for less than 3 months. 3. BInge—eating disorder (of low frequency andlor limited duration): All of the criteria for binge-eating disorder are met, except that the binge eating occurs, on average, less than once a week and/or for less than 3 months. 4. Purging disorder: Recurrent purging behavior to influence weight or shape (e.g., selt- induced vomiting; misuse of laxatives, diuretics, or other medications) in the absence of binge eating.

1	4. Purging disorder: Recurrent purging behavior to influence weight or shape (e.g., selt- induced vomiting; misuse of laxatives, diuretics, or other medications) in the absence of binge eating. 5. Night eating syndrome: Recurrent episodes of night eating, as manifested by eating after awakening from sleep or by excessive food consumption after the evening meal. There is awareness and recall of the eating. The night eating is not better explained by external influences such as changes in the individual’s sleep-wake cycle or by local so- cial norms. The night eating causes significant distress andlor impairment in function- ing. The disordered pattern of eating is not better explained by binge-eating disorder or another mental disorder, including substance use, and is not attributable to another medical disorder or to an effect of medication. 307.50 (F50.9)

1	307.50 (F50.9) This category applies to presentations in which symptoms characteristic of a feeding and eating disorder that cause clinically significant distress or impairment in social, occupation- al, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the feeding and eating disorders diagnostic class. The unspecified feeding and eating disorder category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for a specific feeding and eating dis- order, and includes presentations in which there is insufficient information to make a more specific diagnosis (e.g., in emergency room settings).

1	Elimination disorders all involve the inappropriate elimination of urine or feces and are usually first diagnosed in childhood or adolescence. This group of disorders in- cludes enuresis, the repeated voiding of urine into inappropriate places, and encopresis, the repeated passage of feces into inappropriate places. Subtypes are provided to differentiate nocturnal from diurnal (i.e., during waking hours) voiding for enuresis and the presence or absence of constipation and overﬂow incontinence for encopresis. Although there are min- imum age requirements for diagnosis of both disorders, these are based on developmental age and not solely on chronological age. Both disorders may be voluntary or involuntary. Although these disorders typically occur separately, co-occurrence may also be observed. Diagnostic Criteria 307.6 (F98.0) A. Repeated voiding of urine into bed or clothes, whether involuntary or intentional.

1	Diagnostic Criteria 307.6 (F98.0) A. Repeated voiding of urine into bed or clothes, whether involuntary or intentional. B. The behavior is clinically significant as manifested by either a frequency of at least twice a week for at least 3 consecutive months or the presence of clinically significant distress or impairment in social, academic (occupational), or other important areas of functioning. C. Chronological age is at least 5 years (or equivalent developmental level). D. The behavior is not attributable to the physiological effects of a substance (e.g., a di- uretic, an antipsychotic medication) or another medical condition (e.g., diabetes, spina bifida, a seizure disorder). Specify whether: Nocturnal only: Passage of urine only during nighttime sleep. Diurnal only: Passage of urine during waking hours. Nocturnal and diurnal: A combination of the two subtypes above.

1	Specify whether: Nocturnal only: Passage of urine only during nighttime sleep. Diurnal only: Passage of urine during waking hours. Nocturnal and diurnal: A combination of the two subtypes above. The nocturnal—only subtype of enuresis, sometimes referred to as monosymptomutic enu re- sis, is the most common subtype and involves incontinence only during nighttime sleep, typically during the first one—third of the night. The diurnal-only subtype occurs in the absence of noctumal enuresis and may be referred to simply as urinary incontinence. Indi- viduals with this subtype can be divided into two groups. Individuals with ”urge incon- tinence” have sudden urge symptoms and detrusor instability, whereas individuals with ”voiding postponement” consciously defer micturition urges until incontinence results. The nocturnal-and-diumal subtype is also known as nonmonosymptomutz'c enuresis.

1	The nocturnal-and-diumal subtype is also known as nonmonosymptomutz'c enuresis. The essential feature of enuresis is repeated voiding of urine during the day or at night into bed or clothes (Criterion A). Most often the voiding is involuntary, but occasionally it may be intentional. To qualify for a diagnosis of enuresis, the voiding of urine must occur at tress or impairment in social, academic (occupational), or other important areas of func- tioning (Criterion B). The individual must have reached an age at which continence is expected (i.e., a chronological age of at least 5 years or, for children with developmental delays, a mental age of at least 5 years) (Criterion C). The urinary incontinence is not at- tributable to the physiological effects of a substance (e.g., a diuretic, an antipsychotic med- ication) or another medical condition (e.g., diabetes, spina bifida, a seizure disorder) (Criterion D).

1	During nocturnal enuresis, occasionally the voiding takes place during rapid eye movement (REM) sleep, and the child may recall a dream that involved the act of urinating. During day- time (diurnal) enuresis, the child defers voiding until incontinence occurs, sometimes because of a reluctance to use the toilet as a result of social anxiety or a preoccupation with school or play activity. The enuretic event most commonly occurs in the early afternoon on school days and may be associated with symptoms of disruptive behavior. The enuresis commonly per- sists after appropriate treatment of an associated infection. The prevalence of enuresis is 5%—10% among 5-year-olds, 3%—5% among 10-year-olds, and around 1% among individuals 15 years or older.

1	The prevalence of enuresis is 5%—10% among 5-year-olds, 3%—5% among 10-year-olds, and around 1% among individuals 15 years or older. Two types of course of enuresis have been described: a ”primary" type, in which the indi— vidual has never established urinary continence, and a I“secondary” type, in which the dis- turbance develops after a period of established urinary continence. There are no differences in prevalence of comorbid mental disorders between the two types. By definition, primary enuresis begins at age 5 years. The most common time for the onset of secondary enuresis is between ages 5 and 8 years, but it may occur at any time. After age 5 years, the rate of spon- taneous remission is 5%—10% per year. Most children with the disorder become continent by adolescence, but in approximately 1% of cases the disorder continues into adulthood.

1	Diurnal enuresis is uncommon after age 9 years. While occasional diurnal incontinence is not uncommon in middle childhood, it is substantially more common in those who also have persistent nocturnal enuresis. When enuresis persists into late childhood or adoles— cence, the frequency of incontinence may increase, whereas continence in early childhood is usually associated with a declining frequency of wet nights. Environmental. A number of predisposing factors for enuresis have been suggested, in- cluding delayed or lax toilet training and psychosocial stress.

1	Environmental. A number of predisposing factors for enuresis have been suggested, in- cluding delayed or lax toilet training and psychosocial stress. Genetic and physiological. Enuresis has been associated with delays in the develop- ment of normal circadian rhythms of urine production, with resulting nocturnal polyuria or abnormalities of central vasopressin receptor sensitivity, and reduced functional blad- der capacities with bladder hyperreactivity (unstable bladder syndrome). Nocturnal en- uresis is a genetically heterogeneous disorder. Heritability has been shown in family, twin, and segregation analyses. Risk for childhood nocturnal enuresis is approximately 3.6 times higher in offspring of enuretic mothers and 10.1 times higher in the presence of paternal urinary incontinence. The risk magnitudes for nocturnal enuresis and diurnal incontinence are similar.

1	Enuresis has beeh reported in a variety of European, African, and Asian countries as well as in the United States. At a national level, prevalence rates are remarkably similar, and there is great similarity in the developmental trajectories found in different countries. There are very high rates of enuresis in orphanages and other residential institutions, likely related to the mode and environment in which toilet training occurs. Noctumal enuresis is more common in males. Diurnal incontinence is more common in fe- males. The relative risk of having a child who develops enuresis is greater for previously enuretic fathers than for previously enuretic mothers. Functional Consequences of Enuresis

1	Functional Consequences of Enuresis The amount of impairment associated with enuresis is a function of the limitation on the child’s social activities (e.g., ineligibility for sleep-away camp) or its effect on the child’s self—esteem, the degree of social ostracism by peers, and the anger, punishment, and rejec- tion on the part of caregivers. Neurogenic bladder or another medical condition. The diagnosis of enuresis is not made in the presence of a neurogenic bladder or another medical condition that causes polyuria or urgency (e.g., untreated diabetes mellitus or diabetes insipidus) or during an acute urinary tract infection. However, a diagnosis is compatible with such conditions if urinary inconti- nence was regularly present prior to the development of another medical condition or if it per- sists after the institution of appropriate treatment of the medical condition.

1	Medication side effects. Enuresis may occur during treatment with antipsychotic med- ications, diuretics, or other medications that may induce incontinence. In this case, the di- agnosis should not be made in isolation but may be noted as a medication side effect. However, a diagnosis of enuresis may be made if urinary incontinence was regularly pres- ent prior to treatment with the medication. Although most children with enuresis do not have a comorbid mental disorder, the prevalence of comorbid behavioral symptoms is higher in children with enuresis than in children without enuresis. Developmental delays, including speech, language, learning, and motor skills delays, are also present in a portion of children with enuresis. Encopresis, sleepwalking, and sleep terror disorder may be present. Urinary tract infections are more common in children with enuresis, especially the diurnal subtype, than in those who are continent. Diagnostic Criteria 307.7 (F98.1)

1	Diagnostic Criteria 307.7 (F98.1) A. Repeated passage of feces into inappropriate places (e.g., clothing, floor), whether in- voluntary or intentional. B. At least one such event occurs each month for at least 3 months. 0. Chronological age is at least 4 years (or equivalent developmental level). D. The behavior is not attributable to the physiological effects of a substance (e.g., laxa- tives) or another medical condition except through a mechanism involving constipation. Specify whether: With constipation and overflow incontinence: There is evidence of constipation on physical examination or by history. Without constipation and overflow incontinence: There is no evidence of constipa- tion on physical examination or by history.

1	Without constipation and overflow incontinence: There is no evidence of constipa- tion on physical examination or by history. Feces in the with constipation and overﬂow incontinence subtype are characteristically (but not invariably) poorly formed, and leakage can be infrequent to continuous, occur- ring mostly during the day and rarely during sleep. Only part of the feces is passed during toileting, and the incontinence resolves after treatment of the constipation. In the without constipation and overﬂow incontinence subtype, feces are likely to be of normal form and consistency, and soiling is intermittent. Feces may be deposited in a prominent location. This is usually associated with the presence of oppositional defiant disorder or conduct disorder or may be the consequence of anal masturbation. Soiling without constipation appears to be less common than soiling with constipation.

1	The essential feature of encopresis is repeated passage of feces into inappropriate places (e.g., clothing or ﬂoor) (Criterion A). Most often the passage is involuntary but occasionally may be intentional. The event must occur at least once a month for at least 3 months (Criterion B), and the chronological age of the child must be at least 4 years (or for children with developmental delays, the mental age must be at least 4 years) (Criterion C). The fecal incontinence must not be exclusively attributable to the physiological effects of a substance (e.g., laxatives) or another medical condition except through a mechanism involving constipation (Criterion D).

1	When the passage of feces is involuntary rather than intentional, it is often related to constipation, impaction, and retention with subsequent overﬂow. The constipation may develop for psychological reasons (e.g., anxiety about defecating in a particular place, a more general pattern of anxious or oppositional behavior), leading to avoidance of defeca- tion. Physiological predispositions to constipation include ineffectual straining or paradox- ical defecation dynamics, with contraction rather than relaxation of the external sphincter or pelvic ﬂoor during straining for defecation. Dehydration associated with a febrile ill- ness, hypothyroidism, or a medication side effect can also induce constipation. Once con- stipation has developed, it may be complicated by an anal fissure, painful defecation, and further fecal retention. The consistency of the stool may vary. In some individuals the stool may be of normal or near-normal consistency. In other individuals—such as those with

1	defecation, and further fecal retention. The consistency of the stool may vary. In some individuals the stool may be of normal or near-normal consistency. In other individuals—such as those with overﬂow incontinence secondary to fecal retention—it may be liquid.

1	The child with encopresis often feels ashamed and may wish to avoid situations (e.g., camp, school) that might lead to embarrassment. The amount of impairment is a function of the effect on the child‘s self-esteem, the degree of social ostracism by peers, and the an- ger, punishment, and rejection on the part of caregivers. Smearing feces may be deliberate or accidental, resulting from the child’s attempt to clean or hide feces that were passed in— voluntarily. When the incontinence is clearly deliberate, features of oppositional defiant disorder or conduct disorder may also be present. Many children with encopresis and in the bladder or ureters that may lead to chronic urinary infections, the symptoms of which may remit with treatment of the constipation. It is estimated that approximately 1% of 5-year-olds have encopresis, and the disorder is more common in males than in females.

1	It is estimated that approximately 1% of 5-year-olds have encopresis, and the disorder is more common in males than in females. Encopresis is not diagnosed until a child has reached a chronological age of at least 4 years (or for children with developmental delays, a mental age of at least 4 years). Inadequate, inconsistent toilet training and psychosocial stress (e.g., entering school, the birth of a sib- ling) may be predisposing factors. Two types of course have been described: a "primary” type, in which the individual has never established fecal continence, and a ”secondary" type, in which the disturbance develops after a period of established fecal continence. En- copresis can persist, with intermittent exacerbations, for years.

1	Genetic and physiological. Painful defecation can lead to constipation and a cycle of with- holding behaviors that make encopresis more likely. Use of some medications (e.g., anti- convulsants, cough suppressants) may increase constipation and make encopresis more likely. In addition to physical examination, gastrointestinal imaging (e.g., abdominal radiograph) may be informative to assess retained stool and gas in the colon. Additional tests, such as barium enema and anorectal manography, may be used to help exclude other medical conditions, such as Hirschsprung’s disease. A diagnosis of encopresis in the presence of another medical condition is appropriate only if the mechanism involves constipation that cannot be explained by other medical condi- tions. Fecal incontinence related to other medical conditions (e.g., chronic diarrhea, spina bifida, anal stenosis) would not warrant a DSM-S diagnosis of encopresis.

1	Urinary tract infections can be comorbid with encopresis and are more common in females. This category applies to presentations in which symptoms characteristic of an elimination disorder that cause clinically significant distress or impairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the elimination disorders diagnostic class. The other specified elimination disorder category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for any specific elimination disorder. This is done by recording “other specified elimination disorder" followed by the specific reason (e.g., “Iow-frequency enuresis"). Coding note: Code 788.39 (N39.498) for other specified elimination disorder with urinary symptoms; 787.60 (R15.9) for other specified elimination disorder with fecal symptoms.

1	Coding note: Code 788.39 (N39.498) for other specified elimination disorder with urinary symptoms; 787.60 (R15.9) for other specified elimination disorder with fecal symptoms. This category applies to presentations in which symptoms characteristic of an elimination disorder that cause clinically significant distress or impairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the elimination disorders diagnostic class. The unspecified elimination dis- order category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for a specific elimination disorder, and includes presentations in which there is insufficient information to make a more specific diagnosis (e.g., in emer- gency room settings).

1	Coding note: Code 788.30 (R32) for unspecified elimination disorder with urinary symp— toms; 787.60 (R15.9) for unspecified elimination disorder with fecal symptoms.

1	The DSM-5 C I ass Ifl cation of sleep-wake disorders is intended for use by general mental health and medical clinicians (those caring for adult, geriatric, and pediatric pa- tients). Sleep-wake disorders encompass 10 disorders or disorder groups: insomnia disor- der, hypersomnolence disorder, narcolepsy, breathing-related sleep disorders, circadian rhythm sleep-wake disorders, non—rapid eye movement (N REM) sleep arousal disorders, nightmare disorder, rapid eye movement (REM) sleep behavior disorder, restless legs syn- drome, and substance/medication-induced sleep disorder. Individuals with these disor- ders typically present with sleep-wake complaints of dissatisfaction regarding the quality, timing, and amount of sleep. Resulting daytime distress and impairment are core features shared by all of these sleep-wake disorders.

1	The organization of this chapter is designed to facilitate differential diagnosis of sleep- wake complaints and to clarify when referral to a sleep specialist is appropriate for further assessment and treatment planning. The DSM-S sleep disorders nosology uses a simple, clinically useful approach, while also reﬂecting scientific advances in epidemiology, ge- netics, pathophysiology, assessment, and interventions research since DSM-IV. In some cases (e.g., insomnia disorder), a ”lumping” approach has been adopted, whereas in oth- ers (e.g., narcolepsy), a ”splitting” approach has been taken, reﬂecting the availability of validators derived from epidemiological, neurobiological, and interventions research.

1	Sleep disorders are often accompanied by depression, anxiety, and cognitive changes that must be addressed in treatment planning and management. Furthermore, persistent sleep disturbances (both insomnia and excessive sleepiness) are established risk factors for the subsequent development of mental illnesses and substance use disorders. They may also represent a prodromal expression of an episode of mental illness, allowing the possi- bility of early intervention to preempt or to attenuate a full-blown episode. The differential diagnosis of sleep-wake complaints necessitates a multidimensional approach, with consideration of possibly coexisting medical and neurological conditions.

1	The differential diagnosis of sleep-wake complaints necessitates a multidimensional approach, with consideration of possibly coexisting medical and neurological conditions. Coexisting clinical conditions are the rule, not the exception. Sleep disturbances furnish a clinically useful indicator of medical and neurological conditions that often coexist with depression and other common mental disorders. Prominent among these comorbidities are breathing—related sleep disorders, disorders of the heart and lungs (e.g., congestive heart failure, chronic obstructive pulmonary disease), neurodegenerative disorders (e.g., Alzheimer’s disease), and disorders of the musculoskeletal system (e.g., osteoarthritis).

1	Alzheimer’s disease), and disorders of the musculoskeletal system (e.g., osteoarthritis). sleep (e.g., prolonged apneas or electrocardiographic arrhythmias during REM sleep; con- fusional arousals in patients with dementing illness; seizures in persons with complex partial seizures). REM sleep behavior disorder is often an early indicator of neurodegen- erative disorders (alpha synucleinopathies) like Parkinson’s disease. For all of these rea- sons—related to differential diagnosis, clinical comorbidity, and facilitation of treatment planning—sleep disorders are included in DSM-S.

1	The approach taken to the classification of sleep-wake disorders in DSM-S can be under- stood within the context of ”lumping versus splitting.” DSM-IV represented an effort to simplify sleep-wake disorders classification and thus aggregated diagnoses under broader, less differentiated labels. At the other pole, the International Classification of Sleep Disorders, 2nd Edition (ICSD—Z) elaborated numerous diagnostic subtypes. DSM-IV was prepared for use by mental health and general medical clinicians who are not experts in sleep medicine. ICSD-2 reﬂected the science and opinions of the sleep specialist community and was pre- pared for use by specialists.

1	ICSD-2 reﬂected the science and opinions of the sleep specialist community and was pre- pared for use by specialists. The weight of available evidence supports the superior performance characteristics (interrater reliability, as well as convergent, discriminant, and face validity) of simpler, less- differentiated approaches to diagnosis of sleep-wake disorders. The text accompanying each set of diagnostic criteria provides linkages to the corresponding disorders included in ICSD-Z. The DSM-5 sleep-Wake disorders classification also specifies corresponding non- psychiatric listings (e.g., neurology codes) from the International Classification of Diseases (ICD).

1	ICSD-Z. The DSM-5 sleep-Wake disorders classification also specifies corresponding non- psychiatric listings (e.g., neurology codes) from the International Classification of Diseases (ICD). The field of sleep disorders medicine has progressed in this direction since the publi- cation of DSM-IV. The use of biological validators is now embodied in the DSM—5 classi- fication of sleep-wake disorders, particularly for disorders of excessive sleepiness, such as narcolepsy; for breathing-related sleep disorders, for which formal sleep studies (i.e., polysomnography) are indicated; and for restless legs syndrome, which can often coexist with periodic limb movements during sleep, detectable via polysomnography. Diagnostic Criteria 780.52 (647.00)

1	Diagnostic Criteria 780.52 (647.00) A. A predominant complaint of dissatisfaction with sleep quantity or quality, associated with one (or more) of the following symptoms: 1. Difficulty initiating sleep. (In children, this may manifest as difficulty initiating sleep without caregiver intervention.) 2. Difficulty maintaining sleep, characterized by frequent awakenings or problems re- turning to sleep after awakenings. (In children, this may manifest as difficulty return- ing to sleep without caregiver intervention.) 3. EarIy-morning awakening with inability to return to sleep. . The sleep disturbance causes clinically significant distress or impairment in social, oc- cupational, educational, academic, behavioral, or other important areas of functioning. . The sleep difficulty occurs at least 3 nights per week. . The sleep difficulty is present for at least 3 months. The sleep difficulty occurs despite adequate opportunity for sleep.

1	. The sleep difficulty occurs at least 3 nights per week. . The sleep difficulty is present for at least 3 months. The sleep difficulty occurs despite adequate opportunity for sleep. The insomnia is not better explained by and does not occur exclusively during the course of another sleep-wake disorder (e.g., narcolepsy, a breathing-related sleep dis- order, a circadian rhythm sleep-wake disorder, a parasomnia). G. The insomnia is not attributable to the physiological eftects of a substance (e.g., a drug of abuse, a medication). H. Coexisting mental disorders and medical conditions do not adequately explain the pre- dominant complaint of insomnia. Specify it: With non-sleep disorder mental comorbidity, including substance use disorders

1	Specify it: With non-sleep disorder mental comorbidity, including substance use disorders Coding note: The code 780.52 (647.00) applies to all three specifiers. Code also the relevant associated mental disorder, medical condition, or other sleep disorder imme- diately after the code for insomnia disorder in order to indicate the association. .TIWUO Specify if: Episodic: Symptoms last at least 1 month but less than 3 months. Persistent: Symptoms last 3 months or longer. Recurrent: Two (or more) episodes within the space of 1 year. Note: Acute and short-term insomnia (i.e., symptoms lasting less than 3 months but oth- erwise meeting all criteria with regard to frequency, intensity, distress, and/or impairment) should be coded as an other specified insomnia disorder.

1	Note. The diagnosis of insomnia disorder is given whether it occurs as an independent condition or is comorbid with another mental disorder (e.g., major depressive disorder), medical condition (e.g., pain), or another sleep disorder (e.g., a breathing-related sleep dis- order). For instance, insomnia may develop its own course with some anxiety and depres- sive features but in the absence of criteria being met for any one mental disorder. Insomnia may also manifest as a clinical feature of a more predominant mental disorder. Persistent insomnia may even be a risk factor for depression and is a common residual symptom af- ter treatment for this condition. With comorbid insomnia and a mental disorder, treatment may also need to target both conditions. Given these different courses, it is often impossi- ble to establish the precise nature of the relationship between these clinical entities, and this relationship may change over time. Therefore, in the presence of insomnia and a co—

1	is often impossi- ble to establish the precise nature of the relationship between these clinical entities, and this relationship may change over time. Therefore, in the presence of insomnia and a co— morbid disorder, it is not necessary to make a causal attribution between the two condi- tions. Rather, the diagnosis of insomnia disorder is made with concurrent specification of the clinically comorbid conditions. A concurrent insomnia diagnosis should only be con- sidered when the insomnia is sufficiently severe to warrant independent clinical attention; otherwise, no separate diagnosis is necessary.

1	The essential feature of insomnia disorder is dissatisfaction with sleep quantity or quality with complaints of difficulty initiating or maintaining sleep. The sleep complaints are ac- companied by clinically significant distress or impairment in social, occupational, or other important areas of functioning. The sleep disturbance may occur during the course of an- other mental disorder or medical condition, or it may occur independently. Different manifestations of insomnia can occur at different times of the sleep period. Sleep— onset insomnia (or initial insomnia) involves difficulty initiating sleep at bedtime. Sleep mainte- the night. late insomnia involves early-morning awakening with an inability to return to sleep.

1	Difficulty maintaining sleep is the most common single symptom of insomnia, followed by difficulty falling asleep, while a combination of these symptoms is the most common presen— tation overall. The specific type of sleep complaint often varies over time. Individuals who complain of difficulty falling asleep at one time may later complain of difficulty maintaining sleep, and vice versa. Symptoms of difficulty falling asleep and difficulty maintaining sleep can be quantified by the individual’s retrospective seIf—report, sleep diaries, or other methods, such as actigraphy or polysomnography, but the diagnosis of insomnia disorder is based on the individual’s subjective perception of sleep or a caretaker’s report.

1	Nonrestoratz've sleep, a complaint of poor sleep quality that does not leave the individual rested upon awakening despite adequate duration, is a common sleep complaint usually occurring in association with difficulty initiating or maintaining sleep, or less frequently in isolation. This complaint can also be reported in association with other sleep disorders (e.g., breathing-related sleep disorder). When a complaint of nonrestorative sleep occurs in isolation (i.e., in the absence of difficulty initiating and/ or maintaining sleep) but all di- agnostic criteria with regard to frequency, duration, and daytime distress and impairments are otherwise met, a diagnosis of other specified insomnia disorder or unspecified insom- nia disorder is made.

1	Aside from the frequency and duration criteria required to make the diagnosis, addi- tional criteria are useful to quantify insomnia severity. These quantitative criteria, while arbitrary, are provided for illustrative purpose only. For instance, difficulty initiating sleep is defined by a subjective sleep latency greater than 20—30 minutes, and difficulty maintain~ ing sleep is defined by a subjective time awake after sleep onset greater than 20—30 min- utes. Although there is no standard definition of early-morning awakening, this symptom involves awakening at least 30 minutes before the scheduled time and before total sleep time reaches 61/2 hours. It is essential to take into account not only the final awakening time but also the bedtime on the previous evening. Awakening at 4:00 A.M. does not have the same clinical significance in those who go to bed at 9:00 PM. as in those who go to bed at 1 1:00 PM. Such a symptom may also reﬂect an age-dependent decrease in the ability to sus-

1	does not have the same clinical significance in those who go to bed at 9:00 PM. as in those who go to bed at 1 1:00 PM. Such a symptom may also reﬂect an age-dependent decrease in the ability to sus- tain sleep or an age-dependent shift in the timing of the main sleep period.

1	Insomnia disorder involves daytime impairments as well as nighttime sleep difficulties.

1	These include fatigue or, less commonly, daytime sleepiness; the latter is more common among older individuals and when insomnia is comorbid with another medical condition (e.g., chronic pain) or sleep disorder (e.g., sleep apnea). Impairment in cognitive performance may include difficulties with attention, concentration and memory, and even with performing simple manual skills. Associated mood disturbances are typically described as irritability or mood lability and less commonly as depressive or anxiety symptoms. Not all individuals with nighttime sleep disturbances are distressed or have functional impairment. For example, sleep continuity is often interrupted in healthy older adults who nevertheless identify themselves as good sleepers. A diagnosis of insomnia disorder should be reserved for those individuals with significant daytime distress or impairment related to their nighttime sleep difficulties.

1	Insomnia is often associated with physiological and cognitive arousal and conditioning factors that interfere with sleep. A preoccupation with sleep and distress due to the inabil— ity to sleep may lead to a vicious cycle: the more the individual strives to sleep, the more frustration builds and further impairs sleep. Thus, excessive attention and efforts to sleep, which override normal sleep-onset mechanisms, may contribute to the development of in- somnia. Individuals with persistent insomnia may also acquire maladaptive sleep habits (e.g., spending excessive time in bed; following an erratic sleep schedule; napping) and cognitions (e.g., fear of sleeplessness; apprehensions of daytime impairments; clock mon- itoring) during the course of the disorder. Engaging in such activities in an environment in which the individual has frequently spent sleepless nights may further compound the con- ditioned arousal and perpetuate sleep difficulties. Conversely, the individual may fall asleep

1	environment in which the individual has frequently spent sleepless nights may further compound the con- ditioned arousal and perpetuate sleep difficulties. Conversely, the individual may fall asleep more easily when not trying to do so. Some individuals also report better sleep when away from their own bedrooms and their usual routines.

1	Insomnia may be accompanied by a variety of daytime complaints and symptoms, in- cluding fatigue, decreased energy, and mood disturbances. Symptoms of anxiety or de— pression that do not meet criteria for a specific mental disorder may be present, as well as an excessive focus on the perceived effects of sleep loss on daytime functioning. Individuals with insomnia may have elevated scores on self—report psychological or personality inventories with profiles indicating mild depression and anxiety, a worrisome cognitive style, an emotion-focused and intemalizing style of conﬂict resolution, and a so- matic focus. Patterns of neurocognitive impairment among individuals with insomnia dis- order are inconsistent, although there may be impairments in performing tasks of higher complexity and those requiring frequent changes in performance strategy. Individuals with insomnia often require more effort to maintain cognitive performance.

1	Population-based estimates indicate that about one-third of adults report insomnia symp- toms, 10%—15% experience associated daytime impairments, and 6%—10% have symptoms that meet criteria for insomnia disorder. Insomnia disorder is the most prevalent of all sleep disorders. In primary care settings, approximately 10%—20% of individuals complain of significant insomnia symptoms. Insomnia is a more prevalent complaint among fe- males than among males, with a gender ratio of about 1.44:1. Although insomnia can be a symptom or an independent disorder, it is most frequently observed as a comorbid con- dition with another medical condition or mental disorder. For instance, 40%—50% of indi- viduals with insomnia also present with a comorbid mental disorder.

1	The onset of insomnia symptoms can occur at any time during life, but the first episode is more common in young adulthood. Less frequently, insomnia begins in childhood or ad- olescence. In women, new-onset insomnia may occur during menopause and persist even after other symptoms (e.g., hot ﬂashes) have resolved. Insomnia may have a late-life onset, which is often associated with the onset of other health-related conditions.

1	Insomnia can be situational, persistent, or recurrent. Situational or acute insomnia usu- ally lasts a few days or a few weeks and is often associated with life events or rapid changes in sleep schedules or environment. It usually resolves once the initial precipitating event subsides. For some individuals, perhaps those more vulnerable to sleep disturbances, in- somnia may persist long after the initial triggering event, possibly because of conditioning factors and heightened arousal. The factors that precipitate insomnia may differ from those that perpetuate it. For example, an individual who is bedridden with a painful injury and has difficulty sleeping may then develop negative associations for sleep. Conditioned arousal may then persist and lead to persistent insomnia. A similar course may develop in the context of an acute psychological stress or a mental disorder. For instance, insomnia that occurs during an episode of major depressive disorder can become a focus of attention,

1	may develop in the context of an acute psychological stress or a mental disorder. For instance, insomnia that occurs during an episode of major depressive disorder can become a focus of attention, with consequent negative conditioning, and persist even after resolution of the depressive episode. In some cases, insomnia may also have an insidious onset without any identifi- able precipitating factor.

1	The course of insomnia may also be episodic, with recurrent episodes of sleep difficul- ties associated with the occurrence of stressful events. Chronicity rates range from 45% to 75% for follow-ups of 1—7 years. Even when the course of the insomnia has become chronic, there is night—to—night variability in sleep patterns, with an occasional restful night’s sleep interspersed with several nights of poor sleep. The characteristics of insomnia may also change over time. Many individuals with insomnia have a history of ”light” or easily disturbed sleep prior to onset of more persistent sleep problems. Insomnia complaints are more prevalent among middle-age and older adults. The type of insomnia symptom changes as a function of age, with difficulties initiating sleep being quently among middle-age and older individuals.

1	Difficulties initiating and maintaining sleep can also occur in children and adolescents, but there are more limited data on prevalence, risk factors, and comorbidity during these developmental phases of the lifespan. Sleep difficulties in childhood can result from con- ditioning factors (e.g., a child who does not learn to fall asleep or return to sleep without the presence of a parent) or from the absence of consistent sleep schedules and bedtime routines. Insomnia in adolescence is often triggered or exacerbated by irregular sleep sched- ules (e.g., phase delay). In both children and adolescents, psychological and medical fac- tors can contribute to insomnia.

1	The increased prevalence of insomnia in older adults is partly explained by the higher incidence of physical health problems with aging. Changes in sleep patterns associated with the normal developmental process must be differentiated from those exceeding age-related changes. Although polysomnography is of limited value in the routine evaluation of in- somnia, it may be more useful in the differential diagnosis among older adults because the etiologies of insomnia (e.g., sleep apnea) are more often identifiable in older individuals.

1	While the risk and prognostic factors discussed in this section increase vulnerability to in- somnia, sleep disturbances are more likely to occur when predisposed individuals are ex- posed to precipitating events, such as major life events (e.g., illness, separation) or less severe but more chronic daily stress. Most individuals resume normal sleep patterns after the initial triggering event has disappeared, but others—perhaps those more vulnerable to insomnla—continue experiencing persistent sleep difficulties. Perpetuating factors such as poor sleep habits, irregular sleep scheduling, and the fear of not sleeping feed into the in- somnia problem and may contribute to a Vicious cycle that may induce persistent insomnia. Temperamental. Anxiety or worry-prone personality or cognitive styles, increased arousal predisposition, and tendency to repress emotions can increase vulnerability to insomnia.

1	Temperamental. Anxiety or worry-prone personality or cognitive styles, increased arousal predisposition, and tendency to repress emotions can increase vulnerability to insomnia. Environmental. Noise, light, uncomfortably high or low temperature, and high altitude may also increase vulnerability to insomnia. Genetic and physiological. Female gender and advancing age are associated with in- creased vulnerability to insomnia. Disrupted sleep and insomnia display a familial dispo- sition. The prevalence of insomnia is higher among monozygotic twins relative to dizygotic twins; it is also higher in first-degree family members compared with the general population. The extent to which this link is inherited through a genetic predisposition, learned by observations of parental models, or established as a by-product of another psy- chopathology remains undetermined.

1	Course modifiers. Deleterious course modifiers include poor sleep hygiene practices (e.g., excessive caffeine use, irregular sleep schedules). Insomnia is a more prevalent complaint among females than among males, with first onset often associated with the birth of a new child or with menopause. Despite higher preva- lence among older females, polysomnographic studies suggest better preservation of sleep continuity and slow-wave sleep in older females than in older males. Polysomnography usually shows impairments of sleep continuity (e.g., increased sleep la- tency and time awake after sleep onset and decreased sleep efficiency [percentage of time in bed asleep] and may show increased stage 1 sleep and decreased stages 3 and 4 sleep.

1	The severity of these sleep impairments does not always match the individual’s clinical presentation or subjective complaint of poor sleep, as individuals with insomnia often un- derestimate sleep duration and overestimate wakefulness relative to polysomnography. Quantitative electroencephalographic analyses may indicate that individuals with insom- nia have greater high—frequency electroencephalography power relative to good sleepers both around the sleep onset period and during non—rapid eye movement sleep, a feature suggestive of increased cortical arousal. Individuals with insomnia disorder may have a tive sleep laboratory measures compared with individuals without sleep disorders.

1	Other laboratory measures show evidence, although not consistently, of increased arousal and a generalized activation of the hypothalamic-pituitary-adrenal axis (e.g., in- creased cortisol levels, heart rate variability, reactivity to stress, metabolic rate). In general, findings are consistent with the hypothesis that increased physiological and cognitive arousal plays a significant role in insomnia disorder. Individuals with insomnia disorder may appear either fatigued or haggard or, con- versely, overaroused and ”wired.” However, there are no consistent or characteristic abnormalities on physical examination. There may be an increased incidence of stress- related psychophysiological symptoms (e.g., tension headache, muscle tension or pain, gastrointestinal symptoms). Functional Consequences of Insomnia Disorder

1	Functional Consequences of Insomnia Disorder Interpersonal, social, and occupational problems may develop as a result of insomnia or excessive concern with sleep, increased daytime irritability, and poor concentration. De- creased attention and concentration are common and may be related to higher rates of ac- cidents observed in insomnia. Persistent insomnia is also associated with long-term consequences, including increased risks of major depressive disorder, hypertension, and quality of life; and increased economic burden. Normal sleep variations. Normal sleep duration varies considerably across individuals.

1	Normal sleep variations. Normal sleep duration varies considerably across individuals. sleep duration. Short sleepers differ from individuals with insomnia disorder by the lack of difficulty falling or staying asleep and by the absence of characteristic daytime symptoms (e.g., fatigue, concentration problems, irritability). However, some short sleepers may desire or attempt to sleep for a longer period of time and, by prolonging time in bed, may create an insomnia-like sleep pattern. Clinical insomnia also should be distinguished from normal, age—related sleep changes. Insomnia must also be distinguished from sleep deprivation due to inadequate opportunity or circumstance for sleep resulting, for example, from an emer- gency or from professional or family obligations forcing the individual to stay awake.

1	Situational/acute insomnia. Situational/acute insomnia is a condition lasting a few days to a few weeks, often associated with life events or with changes in sleep schedules. These fere with social, personal, and occupational functioning. When such symptoms are fre- quent enough and meet all other criteria except for the 3-month duration, a diagnosis of other specified insomnia disorder or unspecified insomnia disorder is made. Delayed sleep phase and shift work types of circadian rhythm sleep-wake disorder. Individuals with the delayed sleep phase type of circadian rhythm sleep-wake disorder re- port sleep-onset insomnia only when they try to sleep at socially normal times, but they do delayed and coincide with their endogenous circadian rhythm. Shift work type differs from insomnia disorder by the history of recent shift work.

1	Restless legs syndrome. Restless legs syndrome often produces difficulties initiating and maintaining sleep. However, an urge to move the legs and any accompanying unpleas- ant leg sensations are features that differentiate this disorder from insomnia disorder. Breathing-related sleep disorders. Most individuals with a breathing—related sleep dis- order have a history of loud snoring, breathing pauses during sleep, and excessive daytime sleepiness. Nonetheless, as many as 50% of individuals with sleep apnea may also report insomnia symptoms, a feature that is more common among females and older adults. Narcolepsy. Narcolepsy may cause insomnia complaints but is distinguished from in- somnia disorder by the predominance of symptoms of excessive daytime sleepiness, cat- aplexy, sleep paralysis, and sleep—related hallucinations.

1	Parasomnias. Parasomnias are characterized by a complaint of unusual behavior or events during sleep that may lead to intermittent awakenings and difficulty resuming sleep. However, it is these behavioral events, rather than the insomnia per se, that dominate the clinical picture. Substance/medication-induced sleep disorder, insomnia type. Substance/medication— induced sleep disorder, insomnia type, is distinguished from insomnia disorder by the fact that a substance (i.e., a drug of abuse, a medication, or exposure to a toxin) is judged to be etiologically related to the insomnia (see ”Substance/Medication-Induced Sleep Disor- der” later in this chapter). For example, insomnia occurring only in the context of heavy coffee consumption would be diagnosed as caffeine-induced sleep disorder, insomnia type, with onset during intoxication.

1	Insomnia is a common comorbidity of many medical conditions, including diabetes, cor- onary heart disease, chronic obstructive pulmonary disease, arthritis, fibromyalgia, and other chronic pain conditions. The risk relationship appears to be bidirectional: insomnia increases the risk of medical conditions, and medical problems increase the risk of insom- nia. The direction of the relationship is not always clear and may change over time; for this reason, comorbid insomnia is the preferred terminology in the presence of coexisting in- somnia with another medical condition (or mental disorder).

1	Individuals with insomnia disorder frequently have a comorbid mental disorder, par- ticularly bipolar, depressive, and anxiety disorders. Persistent insomnia represents a risk factor or an early symptom of subsequent bipolar, depressive, anxiety, and substance use disorders. Individuals with insomnia may misuse medications or alcohol to help with nighttime sleep, anxiolytics to combat tension or anxiety, and caffeine or other stimulants to combat excessive fatigue. In addition to worsening the insomnia, this type of substance use may in some cases progress to a substance use disorder. Relationship to International Classification of

1	Relationship to International Classification of There are several distinct insomnia phenotypes relating to the perceived source of the in- somnia that are recognized by the International Classification of Sleep Disorders, 2nd Edition (ICSD—Z). These include psychophysiologicul insomnia, idiopathic insomnia, sleep—stute mispercep- tion, and inadequate sleep hygiene. Despite their clinical appeal and heuristic value, there is limited evidence to support these distinct phenotypes. Diagnostic Criteria 780.54 (647.10) A. Self—reported excessive sleepiness (hypersomnolence) despite a main sleep period lasting at least 7 hours, with at least one of the following symptoms: 1. Recurrent periods of sleep or lapses into sleep within the same day. 2. A prolonged main sleep episode of more than 9 hours per day that is nonrestorative (i.e., unretreshing). 3. Difficulty being fully awake after abrupt awakening.

1	2. A prolonged main sleep episode of more than 9 hours per day that is nonrestorative (i.e., unretreshing). 3. Difficulty being fully awake after abrupt awakening. B. The hypersomnolence occurs at least three times per week, for at least 3 months. C. The hypersomnolence is accompanied by significant distress or impairment in cogni- tive, social, occupational, or other important areas of functioning. D. The hypersomnolence is not better explained by and does not occur exclusively during the course of another sleep disorder (e.g., narcolepsy, breathing-related sleep disor- der, circadian rhythm sleep-wake disorder, or a parasomnia). E. The hypersomnolence is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication). F. Coexisting mental and medical disorders do not adequately explain the predominant complaint of hypersomnolence. Specify it: With mental disorder, including substance use disorders

1	F. Coexisting mental and medical disorders do not adequately explain the predominant complaint of hypersomnolence. Specify it: With mental disorder, including substance use disorders Coding note: The code 780.54 (647.10) applies to all three specifiers. Code also the relevant associated mental disorder, medical condition, or other sleep disorder im- mediately after the code for hypersomnolence disorder in order to indicate the associ- ation. Specify if: Acute: Duration of less than 1 month. Subacute: Duration of 1—3 months. Persistent: Duration of more than 3 months. Specify current severity: Specify severity based on degree of difficulty maintaining daytime alertness as manifested by the occurrence of multiple attacks of irresistible sleepiness within any given day occur- ring, for example, while sedentary, driving, visiting with friends, or working. Mild: Difficulty maintaining daytime alertness 1—2 days/week.

1	Mild: Difficulty maintaining daytime alertness 1—2 days/week. Moderate: Difficulty maintaining daytime alertness 3—4 days/week. Severe: Difficulty maintaining daytime alertness 5—7 days/week.

1	Moderate: Difficulty maintaining daytime alertness 3—4 days/week. Severe: Difficulty maintaining daytime alertness 5—7 days/week. Hypersomnolence is a broad diagnostic term and includes symptoms of excessive quantity of sleep (e.g., extended nocturnal sleep or involuntary daytime sleep), deteriorated quality of wakefulness (i.e., sleep propensity during wakefulness as shown by difficulty awaken- ing or inability to remain awake when required), and sleep inertia (i.e., a period of im- paired performance and reduced vigilance following awakening from the regular sleep episode or from a nap) (Criterion A). Individuals with this disorder fall asleep quickly and have a good sleep efficiency (>90%). They may have difficulty waking up in the morning, sometimes appearing confused, combative, or ataxic. This prolonged impairment of alert- ness at the sleep—wake transition is often referred to as sleep inertia (i.e., sleep drunkenness).

1	It can also occur upon awakening from a daytime nap. During that period, the individual appears awake, but there is a decline in motor dexterity, behavior may be very inappro- priate, and memory deficits, disorientation in time and space, and feelings of grogginess may occur. This period may last some minutes to hours. The persistent need for sleep can lead to automatic behavior (usually of a very routine, low-complexity type) that the individual carries out with little or no subsequent recall. For example, individuals may find themselves having driven several miles from where they thought they were, unaware of the ”automatic” driving they did in the preceding minutes.

1	For some individuals with hypersomnolence disorder, the major sleep episode (for most individuals, nocturnal sleep) has a duration of 9 hours or more. However, the sleep is often nonrestorative and is followed by difficulty awakening in the morning. For other individ- uals with hypersomnolence disorder, the major sleep episode is of normal nocturnal sleep duration (6—9 hours). In these cases, the excessive sleepiness is characterized by several un- intentional daytime naps. These daytime naps tend to be relatively long (often lasting 1 hour or more), are experienced as nonrestorative (i.e., unrefreshing), and do not lead to improved alertness. Individuals with hypersomnolence have daytime naps nearly everyday regard- less of the nocturnal sleep duration. Subjective sleep quality may or may not be reported as good. Individuals typically feel sleepiness developing over a period of time, rather than experiencing a sudden sleep ”attack.” Unintentional sleep episodes typically occur in

1	may not be reported as good. Individuals typically feel sleepiness developing over a period of time, rather than experiencing a sudden sleep ”attack.” Unintentional sleep episodes typically occur in low- stimulation and low-activity situations (e.g., while attending lectures, reading, watching television, or driving long distances), but in more severe cases they can manifest in high- attention situations such as at work, in meetings, or at social gatherings.

1	Nonrestorative sleep, automatic behavior, difficulties awakening in the morning, and sleep inertia, although common in hypersomnolence disorder, may also be seen in a variety of conditions, including narcolepsy. Approximately 80% of individuals with hyper- somnolence report that their sleep is nonrestorative, and as many have difficulties awak- ening in the morning. Sleep inertia, though less common (i.e., observed in 36%—50% of individuals with hypersomnolence disorder), is highly specific to hypersomnolence. Short naps (i.e., duration of less than 30 minutes) are often unrefreshing. Individuals with hy- persomnolence often appear sleepy and may even fall asleep in the clinician’s waiting area. A subset of individuals with hypersomnolence disorder have a family history of hy- persomnolence and also have symptoms of autonomic nervous system dysfunction, in— cluding recurrent vascular-type headaches, reactivity of the peripheral vascular system (Raynaud’s phenomenon), and fainting.

1	Approximately 5%—10% of individuals who consult in sleep disorders clinics with com- plaints of daytime sleepiness are diagnosed as having hypersomnolence disorder. It is es- timated that about 1% of the European and US. general population has episodes of sleep inertia. Hypersomnolence occurs with relatively equal frequency in males and females.

1	Hypersomnolence disorder has a persistent course, with a progressive evolution in the se— verity of symptoms. In most extreme cases, sleep episodes can last up to 20 hours. How- ever, the average nighttime sleep duration is around 9% hours. While many individuals with hypersomnolence are able to reduce their sleep time during working days, weekend and holiday sleep is greatly increased (by up to 3 hours). Awakenings are very difficult and accompanied by sleep inertia episodes in nearly 40% of cases. Hypersomnolence fully manifests in most cases in late adolescence or early adulthood, with a mean age at onset of 17—24 years. Individuals with hypersomnolence disorder are diagnosed, on average, 10—15 years after the appearance of the first symptoms. Pediatric cases are rare.

1	Hypersomnolence has a progressive onset, with symptoms beginning between ages 15 and 25 years, with a gradual progression over weeks to months. For most individuals, the course is then persistent and stable, unless treatment is initiated. The development of other sleep disorders (e.g., breathing-related sleep disorder) may worsen the degree of sleepi- ness. Although hyperactivity may be one of the presenting signs of daytime sleepiness in children, voluntary napping increases with age. This normal phenomenon is distinct from hypersomnolence.

1	Environmental. Hypersomnolence can be increased temporarily by psychological stress and alcohol use, but they have not been documented as environmental precipitating factors. Viral infections have been reported to have preceded or accompanied hyper- somnolence in about 10% of cases. Viral infections, such as HIV pneumonia, infectious mononucleosis, and Guillain-Barré syndrome, can also evolve into hypersomnolence within months after the infection. Hypersomnolence can also appear within 6—18 months follow- ing a head trauma. Genetic and physiological. Hypersomnolence may be familial, with an autosomal- dominant mode of inheritance. Nocturnal polysomnography demonstrates a normal to prolonged sleep duration, short sleep latency, and normal to increased sleep continuity. The distribution of rapid eye movement (REM) sleep is also normal. Sleep efficiency is mostly greater than 90%. Some individuals with hypersomnolence disorder have increased amounts of slow-wave sleep.

1	The multiple sleep latency test documents sleep tendency, typically indicated by mean sleep latency values of less than 8 minutes. In hypersomnolence disorder, the mean sleep latency is typically less than 10 minutes and frequently 8 minutes or less. Sleep—onset REM periods (SOREMPs; i.e., the occurrence of REM sleep within 20 minutes of sleep onset) may be present but occur less than two times in four to five nap opportunities. Functional Consequences of Hypersomnoience Disorder

1	Functional Consequences of Hypersomnoience Disorder The low level of alertness that occurs while an individual fights the need for sleep can lead to reduced efficiency, diminished concentration, and poor memory during daytime activ- ities. Hypersomnolence can lead to significant distress and dysfunction in work and social relationships. Prolonged nocturnal sleep and difficulty awakening can result in difficulty in meeting morning obligations, such as arriving at work on time. Unintentional daytime sleep episodes can be embarrassing and even dangerous, if, for instance, the individual is driving or operating machinery when the episode occurs.

1	Normative variation in sleep. ”Normal” sleep duration varies considerably in the general population. ”Long sleepers" (i.e., individuals who require a greater than average amount of sleep) do not have excessive sleepiness, sleep inertia, or automatic behavior when they obtain their required amount of nocturnal sleep. Sleep is reported to be refreshing. If social or occupational demands lead to shorter nocturnal sleep, daytime symptoms may appear.

1	In hypersomnolence disorder, by contrast, symptoms of excessive sleepiness occur regard- less of nocturnal sleep duration. An inadequate amount of nocturnal sleep, or behaviorally induced insuﬂicient sleep syndrome, can produce symptoms of daytime sleepiness very similar to those of hypersomnolence. An average sleep duration of fewer than 7 hours per night strongly suggests inadequate nocturnal sleep, and an average of more than 9—10 hours of sleep per 24—hour period suggests hypersomnolence. Individuals with inadequate noctur- nal sleep typically ”catch up” with longer sleep durations on days when they are free from social or occupational demands or on vacations. Unlike hypersomnolence, insufficient nocturnal sleep is unlikely to persist unabated for decades. A diagnosis of hypersomno- lence disorder should not be made if there is a question regarding the adequacy of noctur- nal sleep duration. A diagnostic and therapeutic trial of sleep extension for 10—14 days can often clarify the

1	disorder should not be made if there is a question regarding the adequacy of noctur- nal sleep duration. A diagnostic and therapeutic trial of sleep extension for 10—14 days can often clarify the diagnosis.

1	Poor sleep quality and fatigue. Hypersomnolence disorder should be distinguished from excessive sleepiness related to insufficient sleep quantity or quality and fatigue (i.e., tiredness not necessarily relieved by increased sleep and unrelated to sleep quantity or quality). Excessive sleepiness and fatigue are difficult to differentiate and may overlap considerably. Breathing-related sleep disorders. Individuals with hypersomnolence and breathing- related sleep disorders may have similar patterns of excessive sleepiness. Breathing- related sleep disorders are suggested by a history of loud snoring, pauses in breathing during sleep, brain injury, or cardiovascular disease and by the presence of obesity, oro- pharyngeal anatomical abnormalities, hypertension, or heart failure on physical examina— tion. Polysomnographic studies can confirm the presence of apneic events in breathing— related sleep disorder (and their absence in hypersomnolence disorder).

1	Circadian rhythm sleep-wake disorders. Circadian rhythm sleep-wake disorders are often characterized by daytime sleepiness. A history of an abnormal sleep-wake schedule (with shifted or irregular hours) is present in individuals with a circadian rhythm sleep- wake disorder. Parasomnias. Parasomnias rarely produce the prolonged, undisturbed nocturnal sleep or daytime sleepiness characteristic of hypersomnolence disorder. Other mental disorders. Hypersomnolence disorder must be distinguished from mental disorders that include hypersomnolence as an essential or associated feature. In particular, complaints of daytime sleepiness may occur in a major depressive episode, with atypical fea- tures, and in the depressed phase of bipolar disorder. Assessment for other mental disorders is essential before a diagnosis of hypersomnolence disorder is considered. A diagnosis of hyper- somnolence disorder can be made in the presence of another current or past mental disorder.

1	Hypersomnolence can be associated with depressive disorders, bipolar disorders (during a depressive episode), and major depressive disorder, with seasonal pattern. Many individu- als with hypersomnolence disorder have symptoms of depression that may meet criteria for a depressive disorder. This presentation may be related to the psychosocial consequences of persistent increased sleep need. Individuals with hypersomnolence disorder are also at risk for substance-related disorders, particularly related to self—medication with stimulants. This general lack of specificity may contribute to very heterogeneous profiles among indi- viduals whose symptoms meet the same diagnostic criteria for hypersomnolence disorder. Neurodegenerative conditions, such as Alzheimer’s disease, Parkinson’s disease, and mul- tiple system atrophy, may also be associated with hypersomnolence. Relationship to Internatlonai Classification of

1	Relationship to Internatlonai Classification of The International Classification of Sleep Disorders, 2nd Edition (ICSD-2), differentiates nine subtypes of ”hypersomnias of central origin,” including recurrent hypersomnia (Kleine- Levin syndrome). A. Recurrent periods of an irrepressible need to sleep. Iapsing into sleep, or napping oc- curring within the same day. These must have been occurring at least three times per week over the past 3 months. B. The presence of at least one of the following: 1. Episodes of cataplexy, defined as either (a) or (b), occurring at least a few times per month: a. In individuals with Iong-standing disease, brief (seconds to minutes) episodes of sudden bilateral loss of muscle tone with maintained consciousness that are precipitated by laughter or joking. b. In children or in individuals within 6 months of onset, spontaneous grimaces or jaw-opening episodes with tongue thrusting or a global hypotonia, without any obviods emotional triggers.

1	b. In children or in individuals within 6 months of onset, spontaneous grimaces or jaw-opening episodes with tongue thrusting or a global hypotonia, without any obviods emotional triggers. 2. Hypocretin deficiency, as measured using cerebrospinal fluid (CSF) hypocretin-1 immunoreactivity values (less than or equal to one-third of values obtained in healthy subjects tested using the same assay, or less than or equal to 110 pg/mL). Low CSF levels of hypocretin-1 must not be obsen/ed in the context of acute brain injury, inflammation. or infection. 3. Nocturnal sleep polysomnography showing rapid eye movement (REM) sleep Ia- tency less than or equal to 15 minutes, or a multiple sleep latency test showing a mean sleep latency less than or equal to 8 minutes and two or more sleep-onset REM periods.

1	REM periods. Specify whether: 347.00 (647.419) Narcolepsy without cataplexy but with hypocretin deficiency: Cri- terion B requirements of low CSF hypocretin-1 levels and positive polysomnography/ multiple sleep latency test are met. but no cataplexy is present (Criterion B1 not met). 347.01 (647.411) Narcolepsy with cataplexy but without hypocretin deficiency: In this rare subtype (less than 5% of narcolepsy cases), Criterion B requirements of cataplexy and positive polysomnography/rnultiple sleep latency test are met, but CSF hypocretin-1 levels are normal (Criterion B2 not met). 347.00 (647.419) Autosomal dominant cerebellar ataxia, deafness, and narco- lepsy: This subtype is caused by exon 21 DNA (cytosine-5)-methyltransferase-1 mu- tations and is characterized by Iate-onset (age 30—40 years) narcolepsy (with low or intermediate CSF hypocretin-1 levels), deafness, cerebellar ataxia, and eventually de- mentia.

1	347.00 (647.419) Autosomal dominant narcolepsy, obesity, and type 2 diabetes: Narcolepsy, obesity, and type 2 diabetes and low CSF hypocretin-1 levels have been described in rare cases and are associated with a mutation in the myelin oligodendro- cyte glycoprotein gene. 347.10 (647.429) Narcolepsy secondary to another medical condition: This sub- type is for narcolepsy that develops secondary to medical conditions that cause infec- tious (e.g., Whipple‘s disease. sarcoidosis), traumatic, or tumoral destruction of hypocretin neurons. Coding note (for |CD-9-CM code 347.10 only): Code first the underlying medical con- dition (e.g., 040.2 Whipple’s disease; 347.10 narcolepsy secondary to Whipple’s dis- ease). Specify current severity: Mild: Infrequent cataplexy (less than once per week), need for naps only once or twice per day, and less disturbed nocturnal sleep. Moderate: Cataplexy once daily or every few days, disturbed nocturnal sleep, and need for multiple naps daily.

1	Moderate: Cataplexy once daily or every few days, disturbed nocturnal sleep, and need for multiple naps daily. Severe: Drug-resistant cataplexy with multiple attacks daily, nearly constant sleepi- ness, and disturbed nocturnal sleep (i.e., movements, insomnia, and vivid dreaming).

1	In narcolepsy without cataplexy but with hypocretin deficiency, unclear ”cataplexy-like” symptoms may be reported (e.g., the symptoms are not triggered by emotions and are un- usually long lasting). In extremely rare cases, cerebrospinal ﬂuid (CSF) levels of hypocre— tin-1 are low, and polysomnographic/multiple sleep latency test (MSLT) results are negative: repeating the test is advised before establishing the subtype diagnosis. In narco- lepsy with cataplexy but without hypocretin deficiency, test results for human leukocyte antigen (HLA) DQBl‘06202 may be negative. Seizures, falls of other origin, and conversion disorder (functional neurological symptom disorder) should be excluded. In narcolepsy secondary to infectious (e.g., Whipple’s disease, sarcoidosis), traumatic, or tumoral de- struction of hypocretin neurons, test results for HLA DQB1‘06202 may be positive and may result from the insult triggering the autoimmune process. In other cases, the destruction of hypocretin neurons

1	of hypocretin neurons, test results for HLA DQB1‘06202 may be positive and may result from the insult triggering the autoimmune process. In other cases, the destruction of hypocretin neurons may be secondary to trauma or hypothalamic surgery. Head trauma or infections of the central nervous system can, however, produce transitory decreases in

1	CSF hypocretin-l levels without hypocretin cell loss, complicating the diagnosis. The essential features of sleepiness in narcolepsy are recurrent daytime naps or lapses into sleep. Sleepiness typically occurs daily but must occur at a minimum three times a week for at least 3 months (Criterion A). Narcolepsy generally produces cataplexy, which most commonly presents as brief episodes (seconds to minutes) of sudden, bilateral loss of mus- cle tone precipitated by emotions, typically laughing and joking. Muscles affected may include those of the neck, jaw, arms, legs, or whole body, resulting in head bobbing, jaw dropping, or complete falls. Individuals are awake and aware during cataplexy. To meet Criterion B1(a), cataplexy must be triggered by laughter or joking and must occur at least a few times per month when the condition is untreated or in the past.

1	Criterion B1(a), cataplexy must be triggered by laughter or joking and must occur at least a few times per month when the condition is untreated or in the past. Cataplexy should not be confused with ”weakness” occurring in the context of athletic activities (physiological) or exclusively after unusual emotional triggers such as stress or anxiety (suggesting possible psychopathology). Episodes lasting hours or days, or those not triggered by emotions, are unlikely to be cataplexy, nor is rolling on the ﬂoor while laugh- ing hysterically. In children close to onset, genuine cataplexy can be atypical, affecting primarily the face, causing grimaces or jaw opening with tongue thrusting (”cataplectic faces”). Alter- natively, cataplexy may present as low-grade continuous hypotonia, yielding a wobbling walk. In these cases, Criterion Bl (b) can be met in children or in individuals within 6 months of a rapid onset.

1	Narcolepsy-cataplexy nearly always results from the loss of hypothalamic hypocretin (orexin)—producing cells, causing hypocretin deficiency (less than or equal to one-third of control values, or 110 pg/mL in most laboratories). Cell loss is likely autoimmune, and ap- proximately 99% of affected individuals carry HLA-DQB1*06:02 (vs. 12%—38% of control subjects). Thus, checking for the presence of DQB1*06:02 prior to a lumbar puncture for eval- uation of CSF hypocretin-l immunoreactivity may be useful. Rarely, low CSF levels of hypo- cretin-I occur without cataplexy, notably in youths who may develop cataplexy later. CSF hypocretin-l measurement represents the gold standard, excepting associated severe con- ditions (neurological, inﬂammatory, infectious, trauma) that can interfere with the assay.

1	confirm the diagnosis (Criterion B3). These tests must be performed after the individual has stopped all psychotropic medications, following 2 weeks of adequate sleep time (as documented with sleep diaries, actigraphy). Short rapid eye movement (REM) latency (sleep-onset REM period, REM latency less than or equal to 15 minutes) during polysom- nography is sufficient to confirm the diagnosis and meets Criterion B3. Alternatively, the MSLT result must be positive, showing a mean sleep latency of less than or equal to 8 min- utes and two or more sleep-onset REM periods in four to five naps.

1	When sleepiness is severe, automatic behaviors may occur, with the individual continuing his or her activities in a semi-automatic, hazelike fashion without memory or conscious- ness. Approximately 20%—60% of individuals experience vivid hypnagogic hallucinations before or upon falling asleep or hypnopompic hallucinations just after awakening. These hallucinations are distinct from the less Vivid, nonhallucinatory dreamlike mentation at sleep onset that 6ccurs in normal sleepers. Nightmares and vivid dreaming are also fre- quent in narcolepsy, as is REM sleep behavior disorder. Approximately 20%—60% of indi- viduals experience sleep paralysis upon falling asleep or awakening, leaving them awake but unable to move or speak. However, many normal sleepers also report sleep paralysis, especially with stress or sleep deprivation. Nocturnal eating may occur. Obesity is com- mon. Nocturnal sleep disruption with frequent long or short awakenings is common and can be disabling.

1	Individuals may appear sleepy or fall asleep in the waiting area or during clinical ex- amination. During cataplexy, individuals may slump in a chair and have slurred speech or drooping eyelids. If the clinician has time to check reﬂexes during cataplexy (most attacks are less than 10 seconds), reﬂexes are abolished—an important finding distinguishing gen— uine cataplexy from conversion disorder. Narcolepsy-cataplexy affects 0.02%—0.04% of the general population in most countries. Narcolepsy affects both genders, with possibly a slight male preponderance. Onset is typically in children and adolescents/young adults but rarely in older adults.

1	Narcolepsy affects both genders, with possibly a slight male preponderance. Onset is typically in children and adolescents/young adults but rarely in older adults. Two peaks of onset are suggested, at ages 15—25 years and ages 30—35 years. Onset can be abrupt or progressive (over years). Severity is highest when onset is abrupt in children, and then decreases with age or with treatment, so that symptoms such as cataplexy can oc- casionally disappear. Abrupt onset in young, prepubescent children can be associated with obesity and premature puberty, a phenotype more frequently observed since 2009. In adolescents, onset is more difficult to pinpoint. Onset in adults is often unclear, with some individuals reporting having had excessive sleepiness since birth. Once the disorder has manifested, the course is persistent and lifelong.

1	In 90% of cases, the first symptom to manifest is sleepiness or increased sleep, followed by cataplexy (within 1 year in 50% of cases, within 3 years in 85%). Sleepiness, hypnagogic hallucinations, vivid dreaming, and REM sleep behavior disorder (excessive movements during REM sleep) are early symptoms. Excessive sleep rapidly progresses to an inability to stay awake during the day, and to maintain good sleep at night, without a clear increase in total 24—hour sleep needs. In the first months, cataplexy may be atypical, especially in children. Sleep paralysis usually develops around puberty in children with prepubertal onset. Exacerbations of symptoms suggest lack of compliance with medications or devel- opment of a concurrent sleep disorder, notably sleep apnea.

1	Young children and adolescents with narcolepsy often develop aggression or behav- ioral problems secondary to sleepiness and / or nighttime sleep disruption. Workload and social pressure increase through high school and college, reducing available sleep time at night. Pregnancy does not seem to modify symptoms consistently. After retirement, indi- viduals typically have more opportunity for napping, reducing the need for stimulants. Maintaining a regular schedule benefits individuals at all ages. Temperamental. Parasomnias, such as sleepwalking, bruxism, REM sleep behavior dis- order, and enuresis, may be more common in individuals who develop narcolepsy. Indi- viduals commonly report that they need more sleep than other family members. Environmental. Group A streptococcal throat infection, inﬂuenza (notably pandemic

1	Environmental. Group A streptococcal throat infection, inﬂuenza (notably pandemic HINI 2009), or other winter infections are likely triggers of the autoimmune process, pro- ducing narcolepsy a few months later. Head trauma and abrupt changes in sleep-wake patterns (e.g., job changes, stress) may be additional triggers. Genetic and physiological. Monozygotic twins are 25%—32% concordant for narcolepsy. The prevalence of narcolepsy is 1%—2% in first-degree relatives (a 10- to 40-fold increase overall). Narcolepsy is strongly associated with DQBI‘06202 (99% vs. 12%—38% in control subjects of various ethnic groups; 25% in the general US. population). DQB1*03:01 in- creases, while DQB1‘O5:01, DQBI‘06:01, and DQB1*06:03 reduce risk in the presence of DQBI‘06202, but the effect is small. Polymorphisms within the T-cell receptor alpha gene and other immune modulating genes also modulate risk slightly. Narcolepsy has been described in all ethnic groups and in many cultures. Among African

1	Narcolepsy has been described in all ethnic groups and in many cultures. Among African Americans, more cases present without cataplexy or with atypical cataplexy, complicating diagnosis, especially in the presence of obesity and obstructive sleep apnea. Functional imaging suggests impaired hypothalamic responses to humorous stimuli. Nocturnal polysomnography followed by an MSLT is used to confirm the diagnosis of narcolepsy, especially when the disorder is first being diagnosed and before treatment has begun, and if hypocretin deficiency has not been documented biochemically. The poly- somnography/MSLT should be performed after the individual is no longer taking any psychotropic drugs and after regular sleep-wake patterns, without shift work or sleep de- privation, have been documented.

1	A sleep-onset REM period during the polysomnography (REM sleep latency less than or equal to 15 minutes) is highly specific (approximately 1% positive in control subjects) but moderately sensitive (approximately 50%). A positive MSLT result displays an aver- age sleep latency of less than or equal to 8 minutes, and sleep-onset REM periods in two or more naps on a four- or five-nap test. The MSLT result is positive in 90%—95% of individ- uals with narcolepsy versus 2%—4% of control subjects or individuals with other sleep dis- orders. Additional polysomnographic findings often include frequent arousals, decreased sleep efficiency, and increased stage 1 sleep. Periodic limb movements (found in about 40% of individuals with narcolepsy) and sleep apnea are often noted.

1	Hypocretin deficiency is demonstrated by measuring CSF hypocretin-l immunoreac- tivity. The test is particularly useful in individuals with suspected conversion disorder and those without typical cataplexy, or in treatment-refractory cases. The diagnostic value of the test is not affected by medications, sleep deprivation, or circadian time, but the find- ings are uninterpretable when the individual is severely ill with a concurrent infection or head trauma or is comatose. CSF cytology, protein, and glucose are within normal range even when sampled within weeks of rapid onset. CSF hypocretin-l in these incipient cases is typically already very diminished or undetectable. Functional Consequences of Narcolepsy

1	Functional Consequences of Narcolepsy Driving and working are impaired, and individuals with narcolepsy should avoid jobs that place themselves (e.g., working with machinery) or others (e.g., bus driver, pilot) in danger. Once the narcolepsy is controlled with therapy, patients can usually drive, al- though rarely long distances alone. Untreated individuals are also at risk for social isola- tion and accidental injury to themselves or others. Social relations may suffer as these individuals strive to avert cataplexy by exerting control over emotions.

1	Other hypersomnias. Hypersomnolence and narcolepsy are similar with respect to the degree of daytime sleepiness, age at onset, and stable course over time but can be distin- guished based on distinctive clinical and laboratory features. Individuals with hypersom- nolence typically have longer and less disrupted nocturnal sleep, greater difficulty awakening, more persistent daytime sleepiness (as opposed to more discrete ”sleep at- tacks" in narcolepsy), longer and less refreshing daytime sleep episodes, and little or no dreaming during daytime naps. By contrast, individuals with narcolepsy have cataplexy and recurrent intrusions of elements of REM sleep into the transition between sleep and wakefulness (e.g., sleep-related hallucinations and sleep paralysis). The MSLT typically demonstrates shorter sleep latencies (i.e., greater physiological sleepiness) as well as the presence of multiple sleep-onset REM periods in individuals with narcolepsy.

1	Sleep deprivation and insufficient nocturnal sleep. Sleep deprivation and insufficient nocturnal sleep are common in adolescents and shift workers. In adolescents, difficulties falling asleep at night are common, causing sleep deprivation. The MSLT result may be positive if conducted while the individual is sleep deprived or while his or her sleep is phase delayed. Sleep apnea syndromes. Sleep apneas are especially likely in the presence of obesity. Because obstructive sleep apnea is more frequent than narcolepsy, cataplexy may be over- looked (or absent), and the individual is assumed to have obstructive sleep apnea unre- sponsive to usual therapies. Major depressive disorder. Narcolepsy or hypersomnia may be associated or confused with depression. Cataplexy is not present in depression. The MSLT results are most often normal, and there is dissociation between subjective and objective sleepiness, as measured by the mean sleep latency during the MSLT.

1	Conversion disorder (functional neurological symptom disorder). Atypical features, such as long-lasting cataplexy or unusual triggers, may be present in conversion disorder (functional neurological symptom disorder). Individuals may report sleeping and dream- ing, yet the MSLT does not show the characteristic sleep-onset REM period. Full-blown, long-lasting pseudocataplexy may occur during consultation, allowing the examining physician enough time to verify reﬂexes, which remain intact. Attention-deficit/hyperactivity disorder or other behavioral problems. In children and adolescents, sleepiness can cause behavioral problems, including aggressiveness and in- attention, leading to a misdiagnosis of attention-deficit/hyperactivity disorder.

1	Seizures. In young children, cataplexy can be misdiagnosed as seizures. Seizures are not commonly triggered by emotions, and when they are, the trigger is not usually laughing or joking. During a seizure, individuals are more likely to hurt themselves when falling. Sei- zures characterized by isolated atonia are rarely seen in isolation of other seizures, and they also have signatures on the electroencephalogram. Chorea and movement disorders. In young children, cataplexy can be misdiagnosed as chorea or pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections, especially in the context of a strep throat infection and high antistreptolysin 0 antibody levels. Some children may have an overlapping movement disorder close to on— set of the cataplexy.

1	Schizophrenia. In the presence of ﬂorid and vivid hypnagogic hallucinations, individuals may think these experiences are real—a feature that suggests schizophrenia. Similarly, with stimulant treatment, persecutory delusions may develop. If cataplexy is present, the clinician should first assume that these symptoms are secondary to narcolepsy before con- sidering a co-occurring diagnosis of schizophrenia. Narcolepsy can co—occur with bipolar, depressive, and anxiety disorders, and in rare cases with schizophrenia. Narcolepsy is also associated with increased body mass index or obe- sity, especially when the narcolepsy is untreated. Rapid weight gain is common in young children with a sudden disease onset. Comorbid sleep apnea should be considered if there is a sudden aggravation of preexisting narcolepsy. Relationship to International Classification of The International Classification of Sleep Disorders, 2nd Edition (ICSD-Z), differentiates five subtypes of narcolepsy.

1	Relationship to International Classification of The International Classification of Sleep Disorders, 2nd Edition (ICSD-Z), differentiates five subtypes of narcolepsy. The breathing-related sleep disorders category encompasses three relatively distinct dis- orders: obstructive sleep apnea hypopnea, central sleep apnea, and sleep-related hypo- ventilation. Diagnostic Criteria 327.23 (647.33) A. Either (1) or (2): 1. Evidence by polysomnography of at least five obstructive apneas or hypopneas per hour of sleep and either of the following sleep symptoms: a. Nocturnal breathing disturbances: snoring, snorting/gasping, or breathing pauses during sleep. b. Daytime sleepiness, fatigue, or unrefreshing sleep despite sufficient opportuni- ties to sleep that is not better explained by another mental disorder (including a sleep disorder) and is not attributable to another medical condition.

1	2. Evidence by polysomnography 0t 15 or more obstructive apneas andlor hypopneas per hour of sleep regardless of accompanying symptoms. Specify current severity: Mild: Apnea hypopnea index is less than 15. Moderate: Apnea hypopnea index is 15-30. Severe: Apnea hypopnea index is greater than 30. Disease severity is measured by a count of the number of apneas plus hypopneas per hour of sleep (apnea hypopnea index) using polysomnography or other overnight monitoring.

1	Overall severity is also informed by levels of nocturnal desaturation and sleep fragmen- tation (measured by brain cortical arousal frequency and sleep stages) and degree of as- sociated symptoms and daytime impairment. However, the exact number and thresholds may vary according to the specific measurement techniques used, and these numbers may change over time. Regardless of the apnea hypopnea index (count) per se, the disorder is considered to be more severe when apneas and hypopneas are accompanied by significant oxygen hemoglobin desaturation (e.g., when more than 10% of the sleep time is spent at desaturation levels of less than 90%) or when sleep is severely fragmented as shown by an elevated arousal index (arousal index greater than 30) or reduced stages in deep sleep (e.g., percentage stage N3 [slow-wave sleep] less than 5%). Obstructive sleep apnea hypopnea is the most common breathing-related sleep disorder.

1	Obstructive sleep apnea hypopnea is the most common breathing-related sleep disorder. It is characterized by repeated episodes of upper (pharyngeal) airway obstruction (apneas and hypopneas) during sleep. Apnea refers to the total absence of airﬂow, and hypopnea re- fers to a reduction in airﬂow. Each apnea or hypopnea represents a reduction in breathing of at least 10 seconds in duration in adults or two missed breaths in children and is typi- cally associated with drops in oxygen saturation of 3% or greater and / or an electroenceph— alographic arousal. Both sleep-related (nocturnal) and wake-time symptoms are common. The cardinal symptoms of obstructive sleep apnea hypopnea are snoring and daytime sleepiness.

1	The cardinal symptoms of obstructive sleep apnea hypopnea are snoring and daytime sleepiness. Obstructive sleep apnea hypopnea in adults is diagnosed on the basis of polysom- nographic findings and symptoms. The diagnosis is based on symptoms of 1) nocturnal breathing disturbances (i.e., snoring, snorting/gasping, breathing pauses during sleep), or 2) daytime sleepiness, fatigue, or unrefreshing sleep despite sufficient opportunities to sleep that are not better explained by another mental disorder and not attributable to an- other medical condition, along with 3) evidence by polysomnography of five or more ob- structive apneas or hypopneas per hour of sleep (Criterion A1). Diagnosis can be made in the absence of these symptoms if there is evidence by polysomnography of 15 or more ob- structive apneas and / or hypopneas per hour of sleep (Criterion A2).

1	Specific attention to disturbed sleep occurring in association with snoring or breathing pauses and physical findings that increase risk of obstructive sleep apnea hypopnea (e.g., central obesity, crowded pharyngeal airway, elevated blood pressure) is needed to reduce the chance of misdiagnosing this treatable condition. Because of the frequency of nocturnal awakenings that occur with obstructive sleep apnea hypopnea, individuals may report symptoms of insomnia. Other common, though non— specific, symptoms of obstructive sleep apnea hypopnea are heartburn, nocturia, morning headaches, dry mouth, erectile dysfunction, and reduced libido. Rarely, individuals may complain of difficulty breathing while lying supine or sleeping. Hypertension may occur in more than 60% of individuals with obstructive sleep apnea hypopnea.

1	Obstructive sleep apnea hypopnea is a very common disorder, affecting at least 1%—2% of children, 2%—15% of middle-age adults, and more than 20% of older individuals. In the general community, prevalence rates of undiagnosed obstructive sleep apnea hypopnea may be very high in elderly individuals. Since the disorder is strongly associated with obe- sity, increases in obesity rates are likely to be accompanied by an increased prevalence of this disorder. Prevalence may be particularly high among males, older adults, and certain racial/ethnic groups. In adults, the male-to—female ratio of obstructive sleep apnea hypop- nea ranges from 2:1 to 4:1. Gender differences decline in older age, possibly because of an increased prevalence in females after menopause. There is no gender difference among prepubertal children.

1	The age distribution of obstructive sleep apnea hypopnea likely follows a J-shaped distri- bution. There is a peak in children ages 3—8 years when the nasopharynx may be compro- mised by a relatively large mass of tonsillar tissue compared with the size of the upper airway. With growth of the airway and regression of lymphoid tissue during later child- hood, there is reduction in prevalence. Then, as obesity prevalence increases in midlife and females enter menopause, obstructive sleep apnea hypopnea again increases. The course in older age is unclear; the disorder may level off after age 65 years, but in other individ- uals, prevalence may increase with aging. Because there is some age dependency of the oc- currence of apneas and hypopneas, polysomnographic results must be interpreted in light of other clinical data. In particular, significant clinical symptoms of insomnia or hyper- somnia should be investigated regardless of the individual’s age.

1	Obstructive sleep apnea hypopnea usually has an insidious onset, gradual progression, and persistent course. Typically the loud snoring has been present for many years, often since childhood, but an increase in its severity may lead the individual to seek evaluation. Weight gain may precipitate an increase in symptoms. Although obstructive sleep apnea hypopnea can occur at any age, it most commonly manifests among individuals ages 40—60 years. Over 4—5 years, the average apnea hypopnea index increases in adults and older individuals by ap— proximately two apneas/hypopneas per hour. The apnea hypopnea index is increased and in- cident obstructive sleep apnea hypopnea is greater among individuals who are older, who are male, or who have a higher baseline body mass index (BMI) or increase their BMI over time.

1	Spontaneous resolution of obstructive sleep apnea hypopnea has been reported with weight loss, particularly after bariatric surgery. In children, seasonal variation in obstructive sleep ap- nea hypopnea has been observed, as has improvement with overall growth.

1	In young children, the signs and symptoms of obstructive sleep apnea hypopnea may be more subtle than in adults, making diagnosis more difficult to establish. Polysomnography is useful in confirming diagnosis. Evidence of fragmentation of sleep on the polysomnogram may not be as apparent as in studies of older individuals, possibly because of the high homeo- static drive in young individuals. Symptoms such as snoring are usually parent—reported and thus have reduced sensitivity. Agitated arousals and unusual sleep postures, such as sleeping on the hands and knees, may occur. Nocturnal enuresis also may occur and should raise the suspicion of obstructive sleep apnea hypopnea if it recurs in a child who was previously dry at night. Children may also manifest excessive daytime sleepiness, although this is not as com- mon or pronounced as in adults. Daytime mouth breathing, difficulty in swallowing, and poor speech articulation are also common features in children. Children younger than 5

1	this is not as com- mon or pronounced as in adults. Daytime mouth breathing, difficulty in swallowing, and poor speech articulation are also common features in children. Children younger than 5 years more often present with nighttime symptoms, such as observed apneas or labored breathing, than with behavioral symptoms (i.e., the nighttime symptoms are more noticeable and more often bring the child to clinical attention). In children older than 5 years, daytime symptoms such as sleepiness and behavioral problems (e.g., impulsivity and hyperactivity), attention-deficit/ hyperactivity disorder, learning difficulties, and morning headaches are more often the focus of concern. Children with obstructive sleep apnea hypopnea also may present with failure to thrive and developmental delays. In young children, obesity is a less common risk factor, while delayed growth and "failure to thrive" may be present.

1	Genetic and physiological. The major risk factors for obstructive sleep apnea hypopnea are obesity and male gender. Others include maxillary-mandibular retrognathia or micro— gnathia, positive family history of sleep apnea, genetic syndromes that reduce upper airway patency (e.g., Down’s syndrome, Treacher Collin’s syndrome), adenotonsillar hy- pertrophy (especially in young children), menopause (in females), and various endocrine syndromes (e.g., acromegaly). Compared with premenopausal females, males are at in- creased risk for obstructive sleep apnea hypopnea, possibly reﬂecting the inﬂuences of sex hormones on ventilatory control and body fat distribution, as well as because of gender differences in airway structure. Medications for mental disorders and medical conditions that tend to induce somnolence may worsen the course of apnea symptoms if these med- ications are not managed carefully.

1	Obstructive sleep apnea hypopnea has a strong genetic basis, as evidenced by the sig- nificant familial aggregation of the apnea hypopnea index. The prevalence of obstructive sleep apnea hypbpnea is approximately twice as high among the first—degree relatives of probands with obstructive sleep apnea hypopnea as compared with members of control families. One-third of the variance in the apnea hypopnea index is explained by shared fa- milial factors. Although genetic markers with diagnostic or prognostic value are not yet available for use, eliciting a family history of obstructive sleep apnea hypopnea should in- crease the clinical suspicion for the disorder.

1	There is a potential for sleepiness and fatigue to be reported differently across cultures. In some groups, snoring may be considered a sign of health and thus may not trigger con- cerns. Individuals of Asian ancestry may be at increased risk for obstructive sleep apnea hypopnea despite relatively low BMI, possibly reﬂecting the influence of craniofacial risk factors that narrow the nasopharynx. snoring. Polysomnography provides quantitative data on frequency of sleep-related respiratory disturbances and associated changes in oxygen saturation and sleep continuity. Polysom— labored breathing, partial obstructive hypoventilation with cyclical desaturations, hyper- capnia and paradoxical movements. Apnea hypopnea index levels as low as 2 are used to define thresholds of abnormality in children.

1	Arterial blood gas measurements while the individual is awake are usually normal, but some individuals can have waking hypoxemia or hypercapnia. This pattern should alert the clinician to the possibility of coexisting lung disease or hypoventilation. Imaging procedures may reveal narrowing of the upper airway. Cardiac testing may show evidence of impaired ventricular function. Individuals with severe noctumal oxygen desaturation may also have el- evated hemoglobin or hematocrit values. Validated sleep measures (e.g., multiple sleep la— tency test [MSLT], maintenance of wakefulness test) may identify sleepiness.

1	More than 50% of individuals with moderate to severe obstructive sleep apnea hypopnea report symptoms of daytime sleepiness. A twofold increased risk of occupational accidents has been reported in association with symptoms of snoring and sleepiness. Motor vehicle crashes also have been reported to be as much as sevenfold higher among individuals with elevated apnea hypopnea index values. Clinicians should be cognizant of state govern- ment requirements for reporting this disorder, especially in relationship to commercial drivers. Reduced scores on measures of health-related quality of life are common in individ- uals with obstructive sleep apnea hypopnea, with the largest decrements observed in the physical and vitality subscales.

1	Primary snoring and other sleep disorders. Individuals with obstructive sleep apnea hypopnea must be differentiated from individuals with primary snoring (i.e., otherwise nography). Individuals with obstructive sleep apnea hypopnea may additionally report nocturnal gasping and choking. The presence of sleepiness or other daytime symptoms not explained by other etiologies suggests the diagnosis of obstructive sleep apnea hypop— nea, but this differentiation requires polysomnography. Definitive differential diagnosis between hypersomnia, central sleep apnea, sleep-related hypoventilation, and obstructive sleep apnea hypopnea also requires polysomnographic studies.

1	Obstructive sleep apnea hypopnea must be differentiated from other causes of sleepi- ness, such as narcolepsy, hypersomnia, and circadian rhythm sleep disorders. Obstructive sleep apnea hypopnea can be differentiated from narcolepsy by the absence of cataplexy, sleep-related hallucinations, and sleep paralysis and by the presence of loud snoring, gasping during sleep, or observed apneas in sleep. Daytime sleep episodes in narcolepsy are characteristically shorter, more refreshing, and more often associated with dreaming. gen desaturation during nocturnal polysomnographic studies. Narcolepsy results in mul- tiple sleep-onset rapid eye movement (REM) periods during the MSLT. Narcolepsy, like obstructive sleep apnea hypopnea, may be associated with obesity, and some individuals have concurrent narcolepsy and obstructive sleep apnea hypopnea. A diagnosis of narco- lepsy does not exclude the diagnosis of obstructive sleep apnea hypopnea, as the two con- ditions may co—occur.

1	Insomnia disorder. For individuals complaining of difficulty initiating or maintaining sleep or early-moming awakenings, insomnia disorder can be differentiated from obstruc- tive sleep apnea hypopnea by the absence of snoring and the absence of the history, signs, and symptoms characteristic of the latter disorder. However, insomnia and obstructive sleep apnea hypopnea may coexist, and if so, both disorders may need to be addressed concurrently to improve sleep.

1	Panic attacks. Nocturnal panic attacks may include symptoms of gasping or choking during sleep that may be difficult to distinguish clinically from obstructive sleep apnea hy- popnea. However, the lower frequency of episodes, intense autonomic arousal, and lack of popnea. Polysomnography in individuals with nocturnal panic attacks does not reveal the typical pattern of apneas or oxygen desaturation characteristic of obstructive sleep apnea hypopnea. Individuals with obstructive sleep apnea hypopnea do not provide a history of daytime panic attacks.

1	Attention-deficit/hyperactivity disorder. Attention—deficit/hyperactivity disorder in chil— dren may include symptoms of inattention, academic impairment, hyperactivity, and in- ternalizing behaviors, all of which may also be symptoms of childhood obstructive sleep apnea hypopnea. The presence of other symptoms and signs of childhood obstructive sleep apnea hypopnea (e.g., labored breathing or snoring during sleep and adenotonsillar hypertrophy) would suggest the presence of obstructive sleep apnea hypopnea. Obstruc- co-occur, and there may be causal links between them; therefore, risk factors such as en- larged tonsils, obesity, or a family history of sleep apnea may help alert the clinician to their co-occurrence.

1	Substance/medication-induced insomnia or hypersomnia. Substance use and substance withdrawal (including medications) can produce insomnia or hypersomnia. A careful his- tory is usually sufficient to identify the relevant substance/medication, and follow-up shows improvement of the sleep disturbance after discontinuation of the substance/med- ication. In other cases, the use of a substance/ medication (e.g., alcohol, barbiturates, ben- zodiazepines, tobacco) has been shown to exacerbate obstructive sleep apnea hypopnea. An individual with symptoms and signs consistent with obstructive sleep apnea hypop- nea should receive that diagnosis, even in the presence of concurrent substance use that is exacerbating the condition.

1	Systemic hypertension, coronary artery disease, heart failure, stroke, diabetes, and increased mortality are consistently associated with obstructive sleep apnea hypopnea. Risk esti- mates vary from 30% to as much as 300% for moderate to severe obstructive sleep apnea hypopnea. Evidence of pulmonary hypertension and right heart failure (e.g., cor pulmo- nale, ankle edema, hepatic congestion) are rare in obstructive sleep apnea hypopnea and pulmonary comorbidities. Obstructive sleep apnea hypopnea also may occur with in- creased frequency in association with a number of medical or neurological conditions (e.g., cerebrovascular disease, Parkinson’s disease). Physical findings reﬂect the co-occurrence of these conditions.

1	As many as one-third of individuals referred for evaluation of obstructive sleep apnea hypopnea report symptoms of depression, with as many of 10% having depression scores consistent with moderate to severe depression. Severity of obstructive sleep apnea hypop- nea, as measured by the apnea hypopnea index, has been found to be correlated with se- verity of symptoms of depression. This association may be stronger in males than in females. Reiationship to International Classification of The International Classification of Sleep Disorders, 2nd Edition (ICSD-Z), differentiates 11 sub- types of ”sleep-related breathing disorders,” including primary central sleep apnea, ob- structive sleep apnea, and sleep-related hypoventilation. A. Evidence by polysomnography of five or more central apneas per hour of sleep. B. The disorder is not better explained by another current sleep disorder.

1	A. Evidence by polysomnography of five or more central apneas per hour of sleep. B. The disorder is not better explained by another current sleep disorder. Specify whether: 327.21 (647.31) Idiopathic central sleep apnea: Characterized by repeated epi- sodes of apneas and hypopneas during sleep caused by variability in respiratory effort but without evidence of airway obstruction. 786.04 (R063) Cheyne-Stokes breathing: A pattern of periodic crescendo- a frequency of at least five events per hour, accompanied by frequent arousal. 780.57 (647.37) Central sleep apnea comorbid with opioid use: The pathogenesis of this subtype is attributed to the effects of opioids on the respiratory rhythm genera- tors in the medulla as well as the differential effects on hypoxic versus hypercapnic re- spiratory drive.

1	Coding note (for 780.57 [647.37] code only): When an opioid use disorder is present, first code the opioid use disorder: 305.50 (F1 1.10) mild opioid use disorder or 304.00 (F1 1.20) moderate or severe opioid use disorder; then code 780.57 (647.37) central sleep apnea comorbid with opioid use. When an opioid use disorder is not present (e.g., after a one- time heavy use of the substance), code only 780.57 (647.37) central sleep apnea comor- bid with opioid use. Note: See the section “Diagnostic Features” in text. Specify current severity: Severity of central sleep apnea is graded according to the frequency of the breathing disturbances as well as the extent of associated oxygen desaturation and sleep frag- mentation that occur as a consequence of repetitive respiratory disturbances.

1	Idiopathic central sleep apnea and Cheyne-Stokes breathing are characterized by increased gain of the ventilatory control system, also referred to as high loop gain, which leads to in- stability in ventilation and PaCOz levels. This instability is termed periodic breathing and can be recognized by hyperventilation alternating with hypoventilation. Individuals with these disorders typically have pC02 levels while awake that are slightly hypocapneic or normocapneic. Central sleep apnea may also manifest during initiation of treatment of ob- structive sleep apnea hypopnea or may occur in association with obstructive sleep apnea hypopnea syndrome (termed complex sleep apnea). The occurrence of central sleep apnea in association with obstructive sleep apnea is also considered to be due to high loop gain. In contrast, the pathogenesis of central sleep apnea comorbid with opioid use has been at- tributed to the effects of opioids on the respiratory rhythm generators in the medulla as well as

1	loop gain. In contrast, the pathogenesis of central sleep apnea comorbid with opioid use has been at- tributed to the effects of opioids on the respiratory rhythm generators in the medulla as well as to its differential effects on hypoxic versus hypercapneic respiratory drive. These individuals may have elevated pC02 levels while awake. Individuals receiving chronic methadone maintenance therapy have been noted to have increased somnolence and de- pression, although the role of breathing disorders associated with opioid medication in caus- ing these problems has not been studied.

1	An increase in the central apnea index (i.e., number of central apneas per hour of sleep) re- ﬂects an increase in severity of central sleep apnea. Sleep continuity and quality may be markedly impaired with reductions in restorative stages of non—rapid eye movement (REM) sleep (i.e., decreased slow-wave sleep [stage N3]). In individuals with severe Cheyne- Stokes breathing, the pattern can also be observed during resting wakefulness, a finding that is thought to be a poor prognostic marker for mortality. Central sleep apnea disorders are characterized by repeated episodes of apneas and hy- popneas during sleep caused by variability in respiratory effort. These are disorders of ventilatory control in which respiratory events occur in a periodic or intermittent pattern.

1	Idiopathic central sleep apnea is characterized by sleepiness, insomnia, and awakenings due to dyspnea in association with five or more central apneas per hour of sleep. Central sleep apnea occurring in individuals with heart failure, stroke, or renal failure typically have a breathing pattern called Cheyne-Stokes breathing, which is characterized by a pattern of and hypopneas occurring at a frequency of at least five events per hour that are accompa- nied by frequent arousals. Central and obstructive sleep apneas may coexist; the ratio of central to obstructive apneas/hypopneas may be used to identify which condition is pre- dominant.

1	Alterations in neuromuscular control of breathing can occur in association with med- ications or substances used in individuals with mental health conditions, which can cause or exacerbate impairments of respiratory rhythm and ventilation. Individuals taking these medications have a sleep-related breathing disorder that could contribute to sleep distur- bances and symptoms such as sleepiness, confusion, and depression. Specifically, chronic use of long—acting opioid medications is often associated with impairment of respiratory con- trol leading to central sleep apnea. Individuals with central sleep apnea hypopneas can manifest with sleepiness or insomnia. There can be complaints of sleep fragmentation, including awakening with dyspnea. Some individuals are asymptomatic. Obstructive sleep apnea hypopnea can coexist with Cheyne—Stokes breathing, and thus snoring and abruptly terminating apneas may be ob- served during sleep.

1	Some individuals are asymptomatic. Obstructive sleep apnea hypopnea can coexist with Cheyne—Stokes breathing, and thus snoring and abruptly terminating apneas may be ob- served during sleep. The prevalence of idiopathic central sleep apnea is unknown but thought to be rare. The prevalence of Cheyne-Stokes breathing is high in individuals with depressed cardiac ven- tricular ejection fraction. In individuals with an ejection fraction of less than 45%, the prev- alence has been reported to be 20% or higher. The male-to-female ratio for prevalence is even more highly skewed toward males than for obstructive sleep apnea hypopnea. Prev- alence increases with age, and most patients are older than 60 years. Cheyne-Stokes breath- ing occurs in approximately 20% of individuals with acute stroke. Central sleep apnea comorbid with opioid use occurs in approximately 30% of individuals taking chronic opi- nance therapy.

1	The onset of Cheyne-Stokes breathing appears tied to the development of heart failure. The Cheyne-Stokes breathing pattern is associated with oscillations in heart rate, blood pres- sure and oxygen desaturation, and elevated sympathetic nervous system activity that can promote progression of heart failure. The clinical significance of Cheyne-Stokes breathing in the setting of stroke is not known, but Cheyne-Stokes breathing may be a transient find- ing that resolves with time after acute stroke. Central sleep apnea comorbid with opioid use has been documented with chronic use (i.e., several months).

1	Genetic and physiological. Cheyne-Stokes breathing is frequently present in individu- als with heart failure. The coexistence of atrial fibrillation further increases risk, as do older age and male gender. Cheyne—Stokes breathing is also seen in association with acute stroke and possibly renal failure. The underlying ventilatory instability in the setting of heart fail- ure has been attributed to increased ventilatory chemosensitivity and hyperventilation due to pulmonary vascular congestion and circulatory delay. Central sleep apnea is seen in individuals taking long-acting opioids.

1	Physical findings seen in individuals with a Cheyne-Stokes breathing pattern relate to its risk factors. Findings consistent with heart failure, such as jugular venous distension, S3 heart sound, lung crackles, and lower extremity edema, may be present. Polysomnogra- phy is used to characterize the breathing characteristics of each breathing-related sleep disorder subtype. Central sleep apneas are recorded when periods of breathing cessation for longer than 10 seconds occur. Cheyne-Stokes breathing is characterized by a pattern of and hypopneas occurring at a frequency of at least five events per hour that are accompa- nied by frequent arousals. The cycle length of Cheyne-Stokes breathing (or time from end of one central apnea to the end of the next apnea) is about 60 seconds. Functional Consequences of Central Sleep Apnea

1	Functional Consequences of Central Sleep Apnea Idiopathic central sleep apnea has been reported to cause symptoms of disrupted sleep, in- cluding insomnia and sleepiness. Cheyne-Stokes breathing with comorbid heart failure has been associated with excessive sleepiness, fatigue, and insomnia, although many in— dividuals may be asymptomatic. Coexistence of heart failure and Cheyne-Stokes breath- ing may be associated with increased cardiac arrhythmias and increased mortality or cardiac transplantation. Individuals with central sleep apnea comorbid with opioid use may present with symptoms of sleepiness or insomnia. disorders, other sleep disorders, and medical conditions and mental disorders that cause sleep fragmentation, sleepiness, and fatigue. This is achieved using polysomnography.

1	disorders, other sleep disorders, and medical conditions and mental disorders that cause sleep fragmentation, sleepiness, and fatigue. This is achieved using polysomnography. Other breathing-related sleep disorders and sleep disorders. Central sleep apnea can be distinguished from obstructive sleep apnea hypopnea by the presence of at least five central apneas per hour of sleep. These conditions may co-occur, but central sleep apnea is considered to predominate when the ratio of central to obstructive respiratory events ex- ceeds 50%. Cheyne-Stokes breathing can be distinguished from other mental disorders, including other sleep disorders, and other medical conditions that cause sleep fragmentation, sleep- iness, and fatigue based on the presence of a predisposing condition (e.g., heart failure or stroke) and signs and polysomnographic evidence of the characteristic breathing pattern.

1	from insomnia due to other medical conditions. High-altitude periodic breathing has a pattern that resembles Cheyne-Stokes breathing but has a shorter cycle time, occurs only at high altitude, and is not associated with heart failure. Central sleep apnea comorbid with opioid use can be differentiated from other types of breathing-related sleep disorders based on the use of long-acting opioid medications in conjunction with polysomnographic evidence of central apneas and periodic or ataxic breathing. It can be distinguished from insomnia due to drug or substance use based on polysomnographic evidence of central sleep apnea.

1	Central sleep apnea disorders are frequently present in users of long-acting opioids, such as methadone. Individuals taking these medications have a sleep-related breathing disor- der that could contribute to sleep disturbances and symptoms such as sleepiness, confu- sion, and depression. While the individual is asleep, breathing patterns such as central apneas, periodic apneas, and ataxic breathing may be observed. Obstructive sleep apnea hypopnea may coexist with central sleep apnea, and features consistent with this condi- tion can also be present (see ”Obstructive Sleep Apnea Hypopnea" earlier in this chapter). Cheyne-Stokes breathing is more commonly observed in association with conditions that include heart failure, stroke, and renal failure and is seen more frequently in individuals with atrial fibrillation. Individuals with Cheyne-Stokes breathing are more likely to be older, to be male, and to have lower weight than individuals with obstructive sleep apnea hypopnea.

1	A. Polysomnograpy demonstrates episodes of decreased respiration associated with eI- evated 002 levels. (Note: In the absence of objective measurement of 002, persistent low levels of hemoglobin oxygen saturation unassociated with apneic/hypopneic events may indicate hypoventilation.) B. The disturbance is not better explained by another current sleep disorder. Specify whether: 327.24 (647.34) Idiopathic hypoventilation: This subtype is not attributable to any readily identified condition. 327.25 (647.35) Congenital central alveolar hypoventilation: This subtype is a rare congenital disorder in which the individual typically presents in the perinatal period with shallow breathing, or cyanosis and apnea during sleep.

1	327.26 (647.36) Comorbid sleep-related hypoventilation: This subtype occurs as a consequence of a medical condition, such as a pulmonary disorder (e.g., interstitial lung disease, chronic obstructive pulmonary disease) or a neuromuscular or chest wall disorder (e.g., muscular dystrophies, postpolio syndrome, cervical spinal cord injury, kyphoscoliosis), or medications (e.g., benzodiazepines. opiates). It also occurs with obesity (obesity hypoventilation disorder). where it reflects a combination of increased work of breathing due to reduced chest wall compliance and ventilation-perfusion mis- match and variably reduced ventilatory drive. Such individuals usually are character- ized by body mass index of greater than 30 and hypercapnia during wakefulness (with a p002 of greater than 45), without other evidence of hypoventilation. Specify current severity:

1	Specify current severity: Severity is graded according to the degree of hypoxemia and hypercarbia present dur- ing sleep and evidence of end organ impairment due to these abnormalities (e.g., right- sided heart failure). The presence of blood gas abnormalities during wakefulness is an indicator of greater severity. Regarding obesity hypoventilation disorder, the prevalence of obesity hypoventilation in the general population is not known but is thought to be increasing in association with the increased prevalence of obesity and extreme obesity. Sleep-related hypoventilation can occur independently or, more frequently, comorbid with medical or neurological disorders, medication use, or substance use disorder. Al- though symptoms are not mandatory to make this diagnosis, individuals often report excessive daytime sleepiness, frequent arousals and awakenings during sleep, morning headaches, and insomnia complaints.

1	Individuals with sleep—related hypoventilation can present with sleep-related complaints of insomnia or sleepiness. Episodes of orthopnea can occur in individuals with diaphragm weakness. Headaches upon awakening may be present. During sleep, episodes of shallow breathing may be observed, and obstructive sleep apnea hypopnea or central sleep apnea may coexist. Consequences of ventilatory insufficiency, including pulmonary hyperten- sion, cor pulmonale (right heart failure), polycythemia, and neurocognitive dysfunction, can be present. With progression of ventilatory insufficiency, blood gas abnormalities ex- tend into wakefulness. Features of the medical condition causing sleep-related hypoven- tilation can also be present. Episodes of hypoventilation may be associated with frequent arousals or bradytachycardia. Individuals may complain of excessive sleepiness and in- somnia or morning headaches or may present with findings of neurocognitive dysfunction or depression. Hypoventilation

1	or bradytachycardia. Individuals may complain of excessive sleepiness and in- somnia or morning headaches or may present with findings of neurocognitive dysfunction or depression. Hypoventilation may not be present during wakefulness.

1	Idiopathic sleep-related hypoventilation in adults is very uncommon. The prevalence of congenital central alveolar hypoventilation is unknown, but the disorder is rare. Comor- bid sleep-related hypoventilation (i.e., hypoventilation comorbid with other conditions, such as chronic obstructive pulmonary disease [COPD], neuromuscular disorders, or obe- sity) is more common. Idiopathic sleep-related hypoventilation is thought to be a slowly progressive disorder of respiratory impairment. When this disorder occurs comorbidly with other disorders (e.g., COPD, neuromuscular disorders, obesity), disease severity reﬂects the severity of the un- derlying condition, and the disorder progresses as the condition worsens. Complications such as pulmonary hypertension, cor pulmonale, cardiac dysrhythmias, polycythemia, neurocognitive dysfunction, and worsening respiratory failure can develop with increas- ing severity of blood gas abnormalities.

1	Congenital central alveolar hypoventilation usually manifests at birth with shallow, erratic, or absent breathing. This disorder can also manifest during infancy, childhood, and adulthood because of variable penetrance of the PHOXZB mutation. Children with congenital central alveolar hypoventilation are more likely to have disorders of the auto- nomic nervous system, Hirschsprung’s disease, neural crest tumors, and characteristic box- shaped face (i.e., the face is short relative to its width). Environmental. Ventilatory drive can be reduced in individuals using central nervous system depressants, including benzodiazepines, opiates, and alcohol.

1	Environmental. Ventilatory drive can be reduced in individuals using central nervous system depressants, including benzodiazepines, opiates, and alcohol. Genetic and physiological. Idiopathic sleep-related hypoventilation is associated with reduced ventilatory drive due to a blunted chemoresponsiveness to C02 (reduced respi- ratory drive; i.e., “won’t breathe”), reﬂecting underlying neurological deficits in centers governing the control of ventilation. More commonly, sleep-related hypoventilation is co- morbid with another medical condition, such as a pulmonary disorder, a neuromuscular or chest wall disorder, or hypothyroidism, or with use of medications (e.g., benzodiaze- pines, opiates). In these conditions, the hypoventilation may be a consequence of in- creased work of breathing and / or impairment of respiratory muscle function (i.e., ”can’t breathe") or reduced respiratory drive (i.e., ”won’t breathe").

1	Neuromuscular disorders inﬂuence breathing through impairment of respiratory mo- tor innervation or respiratory muscle function. They include conditions such as amyo- trophic lateral sclerosis, spinal cord injury, diaphragmatic paralysis, myasthenia gravis, Lambert-Eaton syndrome, toxic or metabolic myopathies, postpolio syndrome, and Char- cot-Marie-Tooth syndrome. Congenital central alveolar hypoventilation is a genetic disorder attributable to muta- tions of PHOXZB, a gene that is crucial for the development of the embryonic autonomic nervous system and neural crest derivatives. Children with congenital central alveolar hy- poventilation show blunted ventilatory responses to hypercapnia, especially in non—rapid eye movement sleep. Gender distributions for sleep-related hypoventilation occurring in association with co- morbid conditions reﬂect the gender distributions of the comorbid conditions. For exam- ple, COPD is more frequently present in males and with increasing age.

1	Sleep-related hypoventilation is diagnosed using polysomnography showing sleep-related hypoxemia and hypercapnia that is not better explained by another breathing-related sleep disorder. The documentation of increased arterial pCOz levels to greater than 55 mmHg 50 mmHg) during sleep in comparison to awake supine values, for 10 minutes or longer, is the gold standard for diagnosis. However, obtaining arterial blood gas determinations dur- ing sleep is impractical, and non-invasive measures of pC02 have not been adequately val- idated during sleep and are not widely used during polysomnography in adults. Prolonged and sustained decreases in oxygen saturation (oxygen saturation of less than 90% for more than 5 minutes with a nadir of at least 85%, or oxygen saturation of less than 90% for at least 30% of sleep time) in the absence of evidence of upper airway obstruction are often used as an indication of sleep-related hypoventilation; however, this finding is not specific, as there are

1	least 30% of sleep time) in the absence of evidence of upper airway obstruction are often used as an indication of sleep-related hypoventilation; however, this finding is not specific, as there are other potential causes of hypoxemia, such as that due to lung disease.

1	The consequences of sleep-related hypoventilation are related to the effects of chronic ex- posure to hypercapnia and hypoxemia. These blood gas derangements cause vasocon- striction of the pulmonary vasculature leading to pulmonary hypertension, which, if severe, can result in right-sided heart failure (cor pulmonale). Hypoxemia can lead to dys- function of organs such as the brain, blood, and heart, leading to outcomes such as cog- nitive dysfunction, polycythemia, and cardiac arrhythmias. Hypercapnia can depress ventilatory drive, leading to progressive respiratory failure.

1	Other medical conditions affecting ventilation. In adults, the idiopathic variety of sleep- related hypoventilation is very uncommon and is determined by excluding the presence of lung diseases, skeletal malformations, neuromuscular disorders, and other medical and neurological disorders or medications that affect ventilation. Sleep—related hypoventila- tion must be distinguished from other causes of sleep-related hypoxemia, such as that due to lung disease. Other breathing-related sleep disorders. Sleep-related hypoventilation can be distin- features and findings on polysomnography. Sleep-related hypoventilation typically shows more sustained periods of oxygen desaturation rather that the periodic episodes seen in obstructive sleep apnea hypopnea and central sleep apnea. Obstructive sleep apnea hy- popnea and central sleep apnea also show a pattern of discrete episodes of repeated air- ﬂow decreases that can be absent in sleep-related hypoventilation.

1	Sleep-related hypoventilation often occurs in association with a pulmonary disorder (e.g., in- terstitial lung disease, COPD), with a neuromuscular or chest wall disorder (e.g., muscular dystrophies, post—polio syndrome, cervical spinal cord injury, obesity, kyphoscoliosis), or, most relevant to the mental health provider, with medication use (e.g., benzodiazepines, opi- ates). Congenital central alveolar hypoventilation often occurs in association with autonomic dysfunction and may occur in association with Hirschsprung’s disease. COPD, a disorder of lower airway obstruction usually associated with cigarette smoking, can result in sleep- related hypoventilation and hypoxemia. The presence of coexisting obstructive sleep apnea hypopnea is thought to exacerbate hypoxemia and hypercapnia during sleep and wakeful- ness. The relationship between congenital central alveolar hypoventilation and idiopathic sleep-related hypoventilation is unclear; in some individuals, idiopathic

1	during sleep and wakeful- ness. The relationship between congenital central alveolar hypoventilation and idiopathic sleep-related hypoventilation is unclear; in some individuals, idiopathic sleep-related hy— poventilation may represent cases of late-onset congenital central alveolar hypoventilation.

1	Relationship to Internationai Ciassification of The International Classification of Sleep Disorders, 2nd Edition (ICSD-Z), combines sleep- related hypoventilation and sleep-related hypoxemia under the category of sleep-related hypoventilation/hypoxemic syndromes. This approach to classification reﬂects the fre- quent co-occurrence of disorders that lead to hypoventilation and hypoxemia. In contrast, the classification used in DSM-5 reflects evidence that there are distinct sleep-related pathogenetic processes leading to hypoventilation. A. A persistent or recurrent pattern of sleep disruption that is primarily due to an alteration of the circadian system or to a misalignment between the endogenous circadian rhythm and the sleep—wake schedule required by an individual’s physical environment or social or professional schedule. B. The sleep disruption leads to excessive sleepiness or insomnia, or both.

1	B. The sleep disruption leads to excessive sleepiness or insomnia, or both. C. The sleep disturbance causes clinically significant distress or impairment in social, oc- cupational, and other important areas of functioning. Coding note: For |CD-9-CM. code 307.45 for all subtypes. For |CD-10-CM. code is based on subtype. Specify whether: 307.45 (647.21) Delayed sleep phase type: A pattern of delayed sleep onset and awakening times, with an inability to fall asleep and awaken at a desired or convention- ally acceptable earlier time. Specify if: Familial: A family history of delayed sleep phase is present. Specify if: Overlapping with non-24-hour sleep-wake type: Delayed sleep phase type may overlap with another circadian rhythm sleep-wake disorder, non-24-hour sleep-wake type.

1	Specify if: Overlapping with non-24-hour sleep-wake type: Delayed sleep phase type may overlap with another circadian rhythm sleep-wake disorder, non-24-hour sleep-wake type. 307.45 (647.22) Advanced sleep phase type: A pattern of advanced sleep onset and awakening times, with an inability to remain awake or asleep until the desired or con- ventionally acceptable later sleep or wake times. Specify if: Familial: A family history of advanced sleep phase is present. 307.45 (647.23) Irregular sleep—wake type: A temporally disorganized sleep-wake pattern, such that the timing of sleep and wake periods is variable throughout the 24- hour period. 307.45 (647.24) Non-24-hour sleep—wake type: A pattern of sleep-wake cycles that is not synchronized to the 24-hour environment, with a consistent daily drift (usually to later and later times) of sleep onset and wake times.

1	307.45 (647.26) Shift work type: Insomnia during the major sleep period and/or ex- cessive sleepiness (including inadvertent sleep) during the major awake period asso- ciated with a shift work schedule (i.e., requiring unconventional work hours). 307.45 (647.20) Unspecified type Specify it: Episodic: Symptoms last at least 1 month but less than 3 months. Persistent: Symptoms last 3 months or longer. Recurrent: Two or more episodes occur within the space of 1 year. The delayed sleep phase type is based primarily on a history of a delay in the timing of the major sleep period (usually more than 2 hours) in relation to the desired sleep and wake— up time, resulting in symptoms of insomnia and excessive sleepiness. When allowed to set their own schedule, individuals with delayed sleep phase type exhibit normal sleep qual- ity and duration for age. Symptoms of sleep—onset insomnia, difficulty waking in the morning, and excessive early day sleepiness are prominent.

1	Common associated features of delayed sleep phase type include a history of mental dis- orders or a concurrent mental disorder. Extreme and prolonged difficulty awakening with morning confusion is also common. Psychophysiological insomnia may develop as a re- sult of maladaptive behaviors that impair sleep and increase arousal because of repeated attempts to fall asleep at an earlier time. Prevalence of delayed sleep phase type in the general population is approximately 0.17% but appears to be greater than 7% in adolescents. Although the prevalence of familial de- layed sleep phase type has not been established, a family history of delayed sleep phase is present in individuals with delayed sleep phase type.

1	Course is persistent, lasting longer than 3 months, with intermittent exacerbations through- out adulthood. Although age at onset is variable, symptoms begin typically in adolescence and early adulthood and persist for several months to years before diagnosis is estab- lished. Severity may decrease with age. Relapse of symptoms is common. Clinical expression may vary across the lifespan depending on social, school, and work obligations. Exacerbation is usually triggered by a change in work or school schedule that requires an early rise time. Individuals who can alter their work schedules to accommo- date the delayed circadian sleep and wake timing can experience remission of symptoms.

1	Increased prevalence in adolescence may be a consequence of both physiological and be- havioral factors. Hormonal changes may be involved specifically, as delayed sleep phase is as- sociated with the onset of puberty. Thus, delayed sleep phase type in adolescents should be differentiated from the common delay in the tinting of circadian rhythms in this age group. In the familial form, the course is persistent and may not improve significantly with age.

1	Genetic and physiological. Predisposing factors may include a longer than average cir- cadian period, changes in light sensitivity, and impaired homeostatic sleep drive. Some in- dividuals with delayed sleep phase type may be hypersensitive to evening light, which can serve as a delay signal to the circadian clock, or they may be hyposensitive to morning light such that its phase-advancing effects are reduced. Genetic factors may play a role in the pathogenesis of familial and sporadic forms of delayed sleep phase type, including mutations in circadian genes (e.g., PER3, CKIe).

1	Confirmation of the diagnosis includes a complete history and use of a sleep diary or actigra— phy (i.e., a wrist-worn motion detector that monitors motor activity for prolonged periods and can be used as a proxy for sleep-wake patterns for at least 7 days). The period covered should include weekends, when social and occupational obligations are less strict, to ensure that the individual exhibits a consistently delayed sleep-wake pattern. Biomarkers such as salivary dim light melatonin onset should be obtained only when the diagnosis is unclear. Functional Consequences of Delayed Sleep Phase Type Excessive early day sleepiness is prominent. Extreme and prolonged difficulty awakening with morning confusion (i.e., sleep inertia) is also common. The severity of insomnia and pends on the occupational and social demands on the individual.

1	Normative variations in sleep. Delayed sleep phase type must be distinguished from personal, social, or occupational distress (most commonly seen in adolescents and young adults). Other sleep disorders. Insomnia disorder and other circadian rhythm sleep-wake dis- orders should be included in the differential. Excessive sleepiness may also be caused by other sleep disturbances, such as breathing-related sleep disorders, insomnias, sleep- related movement disorders, and medical, neurological, and mental disorders. Overnight polysomnography may help in evaluating for other comorbid sleep disorders, such as sleep apnea. The circadian nature of delayed sleep phase type, however, should differen- tiate it from other disorders with similar complaints.

1	Delayed sleep phase type is strongly associated with depression, personality disorder, and somatic symptom disorder or illness anxiety disorder. In addition, comorbid sleep disor- ders, such as insomnia disorder, restless legs syndrome, and sleep apnea, as well as depres- sive and bipolar disorders and anxiety disorders, can exacerbate symptoms of insomnia and excessive sleepiness. Delayed sleep phase type may overlap with another circadian rhythm sleep-wake disorder, non-24—hour sleep-wake type. Sighted individuals with non- 24-hour sleep-wake type disorder commonly also have a history of delayed circadian sleep phase. Advanced sleep phase type may be documented with the specified ”familial." Although the prevalence of familial advanced sleep phase type has not been established, a family history of advanced sleep phase is present in individuals with advanced sleep phase type. In this type, specific mutations demonstrate an autosomal dominant mode of inheritance.

1	In this type, specific mutations demonstrate an autosomal dominant mode of inheritance. In the familial form, onset of symptoms may occur earlier (during childhood and early adulthood), the course is persistent, and the severity of symptoms may increase with age. Advanced sleep phase type is characterized by sleep-wake times that are several hours earlier than desired or conventional times. Diagnosis is based primarily on a history of an advance in the timing of the major sleep period (usually more than 2 hours) in relation to the desired sleep and wake—up time, with symptoms of early morning insomnia and ex- cessive daytime sleepiness. When allowed to set their schedule, individuals with ad- vanced sleep phase type will exhibit normal sleep quality and duration for age.

1	Individuals with advanced sleep phase type are ”morning types,” having earlier sleep— wake times, with the timing of circadian biomarkers such as melatonin and core body tem- perature rhythms occurring 2—4 hours earlier than normal. When required to keep a con- ventional schedule requiring a delay of bedtime, these individuals will continue to have an early rise time, leading to persistent sleep deprivation and daytime sleepiness. Use of hyp- notics or alcohol to combat sleep-maintenance insomnia and stimulants to reduce daytime sleepiness may lead to substance abuse in these individuals. The estimated prevalence of advanced sleep phase type is approximately 1% in middle— age adults. Sleep-wake times and circadian phase advance in older individuals, probably accounting for increased prevalence in this population.

1	Onset is usually in late adulthood. In the familial form, onset can be earlier. The course is typ- ically persistent, lasting more than 3 months, but the severity may increase depending on work and social schedules. The advanced sleep phase type is more common in older adults.

1	Clinical expression may vary across the lifespan depending on social, school, and work obligations. Individuals who can alter their work schedules to accommodate the advanced circadian sleep and wake timing can experience remission of symptoms. Increasing age tends to advance the sleep phase, however, it is unclear whether the common age-associ- ated advanced sleep phase type is due solely to a change in circadian timing (as seen in the familial form) or also to age-related changes in the homeostatic regulation of sleep, result- ing in earlier awakening. Severity, remission, and relapse of symptoms suggest lack of ad- herence to behavioral and environmental treatments designed to control sleep and wake structure and light exposure.

1	Environmental. Decreased late afternoon/early evening exposure to light and / or expo- sure to early morning light due to early morning awakening can increase the risk of ad- vanced sleep phase type by advancing circadian rhythms. By going to bed early, these individuals are not exposed to light in the phase delay region of the curve, resulting in per- petuation of advanced phase. In familial advanced sleep phase type, a shortening of the endogenous circadian period can result in an advanced sleep phase, although circadian pe- riod does not appear to systematically decrease with age. Genetic and physiological. Advanced sleep phase type has demonstrated an autoso- mal dominant mode of inheritance, including a PERZ gene mutation causing hypophos- phorylation of the PERZ protein and a missense mutation in CKI. vances to light than do Caucasians, possibly increasing the risk for development of ad- vanced sleep phase type in this population.

1	vances to light than do Caucasians, possibly increasing the risk for development of ad- vanced sleep phase type in this population. A sleep diary and actigraphy may be used as diagnostic markers, as described earlier for delayed sleep phase type. Functional Consequences of Advanced Sieep Phase Type Excessive sleepiness associated with advanced sleep phase can have a negative effect on cognitive performance, social interaction, and safety. Use of wake-promoting agents to substance abuse. Other sleep disorders. Behavioral factors such as irregular sleep schedules, voluntary early awakening, and exposure to light in the early morning should be considered, partic- ularly in older adults. Careful attention should be paid to rule out other sleep-wake dis- orders, such as insomnia disorder, and other mental disorders and medical conditions that can cause early morning awakening.

1	Depressive and bipolar disorders. Because early morning awakening, fatigue, and sleep- iness are prominent features of major depressive disorder, depressive and bipolar disor- ders must also be considered. Medical conditions and mental disorders with the symptom of early morning awakening, such as insomnia, can co-occur with the advance sleep phase type. The diagnosis of irregular sleep-wake type is based primarily on a history of symptoms of insomnia at night (during the usual sleep period) and excessive sleepiness (napping) dur- ing the day. Irregular sleep-wake type is characterized by a lack of discernable sleep-wake circadian rhythm. There is no major sleep period, and sleep is fragmented into at least three periods during the 24—hour day.

1	Individuals with irregular sleep-wake type typically present with insomnia or excessive sleepiness, depending on the time of day. Sleep and wake periods across 24 hours are frag- mented, although the longest sleep period tends to occur between 2:00 AM. and 6:00 AM. and is usually less than 4 hours. A history of isolation or reclusion may occur in association with the disorder and contribute to the symptoms via a lack of external stimuli to help en— train a normal pattern. Individuals or their caregivers report frequent naps throughout the day. Irregular sleep-wake type is most commonly associated with neurodegenerative dis- orders, such as major neurocognitive disorder, and many neurodevelopmental disorders in children. Prevalence of irregular sleep-wake type in the general population is unknown. The course of irregular sleep-wake type is persistent. Age at onset is variable, but the dis- order is more common in older adults.

1	The course of irregular sleep-wake type is persistent. Age at onset is variable, but the dis- order is more common in older adults. Temperamental. Neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, and neurodevelopmental disorders in children in- crease the risk for irregular sleep-wake type. Environmental. Decreased exposure to environmental light and structured daytime ac- tivity can be associated with a low-amplitude circadian rhythm. Hospitalized individuals are especially prone to such weak external entraining stimuli, and even outside the hospi— tal setting, individuals with major neurocognitive disorder (i.e., dementia) are exposed to significantly less bright light. irregular sleep-wake pattern.

1	irregular sleep-wake pattern. Lack of a clearly discernible major sleep and wake period in irregular sleep-wake type re- sults in insomnia or excessive sleepiness, depending on the time of day. Disruption of the caregiver’s sleep also often occurs and is an important consideration. Normative variations in sleep. Irregular sleep-wake type should be distinguished from a voluntary irregular sleep-wake schedule and poor sleep hygiene, which can result in in- somnia and excessive sleepiness. Other medical conditions and mental disorders. Other causes of insomnia and daytime sleepiness, including comorbid medical conditions and mental disorders or medication, should be considered.

1	Other medical conditions and mental disorders. Other causes of insomnia and daytime sleepiness, including comorbid medical conditions and mental disorders or medication, should be considered. Irregular sleep-wake type is often comorbid with neurodegenerative and neurodevelop- mental disorders, such as major neurocognitive disorder, intellectual disability (intellec- tual developmental disorder), and traumatic brain injury. It is also comorbid with other medical conditions and mental disorders in which there is social isolation and/ or lack of light and structured activities.

1	The diagnosis of non-24-hour sleep-wake type is based primarily on a history of symp- toms of insomnia or excessive sleepiness related to abnormal synchronization between the 24-hour light—dark cycle and the endogenous circadian rhythm. Individuals typically pre- sent with periods of insomnia, excessive sleepiness, or both, which alternate with short asymptomatic periods. Starting with the asymptomatic period, when the individual's sleep phase is aligned to the external environment, sleep latency will gradually increase and the individual will complain of sleep-onset insomnia. As the sleep phase continues to drift so that sleep time is now in the daytime, the individual will have trouble staying awake during the day and will complain of sleepiness. Because the circadian period is not aligned to the external 24—hour environment, symptoms will depend on when an individ- ual tries to sleep in relation to the circadian rhythm of sleep propensity.

1	Non-24-hour sleep-wake type is most common among blind or visually impaired individ- uals who have decreased light perception. In sighted individuals, there is often a history of delayed sleep phase and of decreased exposure to light and structured social and physical activity. Sighted individuals with non-24-hour sleep-wake type also demonstrate in- creased sleep duration. Prevalence of non-24—hour sleep-wake type in the general population is unclear, but the disorder appears rare in sighted individuals. The prevalence in blind individuals is esti- mated to be 50%.

1	Prevalence of non-24—hour sleep-wake type in the general population is unclear, but the disorder appears rare in sighted individuals. The prevalence in blind individuals is esti- mated to be 50%. Course of non-24-hour sleep—wake type is persistent, with intermittent remission and ex- acerbations due to changes in work and social schedules throughout the lifespan. Age at onset is variable, depending on the onset of visual impairment. In sighted individuals, be- cause of the overlap with delayed sleep phase type, non-24—hour sleep-wake type may de- velop in adolescence or early adulthood. Remission and relapse of symptoms in blind and sighted individuals largely depend on adherence to treatments designed to control sleep and wake structure and light exposure.

1	Clinical expression may vary across the lifespan depending on social, school, and work obligations. In adolescents and adults, irregular sleep-wake schedules and exposure to light or lack of light at critical times of the day can exacerbate the effects of sleep loss and disrupt circadian entrainment. Consequently, symptoms of insomnia, daytime sleepiness, and school, professional, and interpersonal functioning may worsen.

1	Environmental. In sighted individuals, decreased exposure or sensitivity to light and so- cial and physical activity cues may contribute to a free-running circadian rhythm. With the high frequency of mental disorders involving social isolation and cases of non-24—hour sleep-wake typexdeveloping after a change in sleep habits (e.g., night shift work, job loss), behavioral factors in combination with physiological tendency may precipitate and per- petuate this disorder in sighted individuals. Hospitalized individuals with neurological and psychiatric disorders can become insensitive to social cues, predisposing them to the de- velopment of non—24-hour sleep-wake type. Genetic and physiological. Blindness is a risk factor for non-24-hour sleep-wake type. Non-24—hour sleep-wake type has been associated with traumatic brain injury. Diagnosis is confirmed by history and sleep diary or actigraphy for an extended period.

1	Non-24—hour sleep-wake type has been associated with traumatic brain injury. Diagnosis is confirmed by history and sleep diary or actigraphy for an extended period. Sequential measurement of phase markers (e.g., melatonin) can help determine circadian phase in both sighted and blind individuals. Complaints of insomnia (sleep onset and sleep maintenance), excessive sleepiness, or both are prominent. The unpredictability of sleep and wake times (typically a daily delay drift) results in an inability to attend school or maintain a steady job and may increase potential for social isolation. Circadian rhythm sleep-wake disorders. In sighted individuals, non-24—hour sleep-wake type should be differentiated from delayed sleep phase type, as individuals with delayed sleep phase type may display a similar progressive delay in sleep period for several days. Depressive disorders. Depressive symptoms and depressive disorders may result in similar circadian dysregulation and symptoms.

1	Depressive disorders. Depressive symptoms and depressive disorders may result in similar circadian dysregulation and symptoms. Blindness is often comorbid with non-24—hour sleep-wake type, as are depressive and bi- polar disorders with social isolation. Diagnosis is primarily based on a history of the individual working outside of the normal 8:00 AM. to 6:00 PM. daytime window (particularly at night) on a regularly scheduled (i.e., non-overtime) basis. Symptoms of excessive sleepiness at work, and impaired sleep at home, on a persistent basis are prominent. Presence of both sets of symptoms are usually required for a diagnosis of shift work type. Typically, when the individual reverts to a day- work routine, symptoms resolve. Although the etiology is slightly different, individuals to those experienced by individuals with shift work type who work rotating shifts.

1	The prevalence of shift work type is unclear, but the disorder is estimated to affect 5%—10% of the night worker population (16%—20% of the workforce). Prevalence rises with advance- ment into middle-age and beyond (Drake et a1. 2004). Shift work type can appear in individuals of any age but is more prevalent in individuals older than 50 years and typically worsens with the passage of time if the disruptive work hours persist. Although older adults may show similar rates of circadian phase adjust- ment to a change in routine as do younger adults, they appear to experience significantly more sleep disruption as a consequence of the circadian phase shift.

1	Temperamental. Predisposing factors include a morning-type disposition, a need for long (i.e., more than 8 hours) sleep durations in order to feel well rested, and strong com- peting social and domestic needs (e.g., parents of young children). Individuals who are able to commit to a nocturnal lifestyle, with few competing day-oriented demands, appear at lower risk for shift work type. Genetic and physiological. Because shift workers are more likely than day workers to be obese, obstructive sleep apnea may be present and may exacerbate the symptoms. A history and sleep diary or actigraphy may be useful in diagnosis, as discussed earlier for delayed sleep phase type. Functional Consequences of Shift Work Type

1	A history and sleep diary or actigraphy may be useful in diagnosis, as discussed earlier for delayed sleep phase type. Functional Consequences of Shift Work Type Individuals with shift work type not only may perform poorly at work but also appear to be at risk for accidents both at work and on the drive home. They may also be at risk for poor mental health (e.g., alcohol use disorder, substance use disorder, depression) and physical health (e.g., gastrointestinal disorders, cardiovascular disease, diabetes, cancer). Individuals with a history of bipolar disorder are particularly Vulnerable to shift work typebrelated episodes of mania resulting from missed nights of sleep. Shift work type of- ten results in interpersonal problems.

1	Normative variations in sleep with shift work. The diagnosis of shift work type, as op- posed to the ”normal” difficulties of shift work, must depend to some extent on the sever- ity of symptoms and / or level of distress experienced by the individual. Presence of shift work type symptoms even when the individual is able to live on a day-oriented routine for several weeks at a time may suggest the presence of other sleep disorders, such as sleep ap- nea, insomnia, and narcolepsy, which should be ruled out. Shift work type has been associated with increased alcohol use disorder, other substance use disorders, and depression. A variety of physical health disorders (e.g., gastrointestinal disorders, cardiovascular disease, diabetes, cancer) have been found to be associated with prolonged exposure to shift work. Relationship to International Classification of

1	Relationship to International Classification of The International Classification of Sleep Disorders, 2nd Edition (ICSD-Z), differentiates nine circadian rhythm sleep disorders, including jet lag type. . Parasomnias Parasomnias are disorders characterized by abnormal behavioral, experiential, or physio- logical events occurring in association with sleep, specific sleep stages, or sleep-wake tran— sitions. The most common parasomnias—non—rapid eye movement (NREM) sleep mixtures of wakefulness and NREM sleep and wakefulness and REM sleep, respectively. These conditions serve as a reminder that sleep and wakefulness are not mutually exclu- sive and that sleep is not necessarily a global, whole-brain phenomenon.

1	These conditions serve as a reminder that sleep and wakefulness are not mutually exclu- sive and that sleep is not necessarily a global, whole-brain phenomenon. A. Recurrent episodes of incomplete awakening from sleep, usually occurring during the first third of the major sleep episode, accompanied by either one of the following: 1. Sleepwalking: Repeated episodes of rising from bed during sleep and walking about. While sleepwalking, the individual has a blank, staring face; is relatively un- responsive to the efforts of others to communicate with him or her; and can be awakened only with great difficulty. 2. Sleep terrors: Recurrent episodes of abrupt terror arousals from sleep, usually be- ginning with a panicky scream. There is intense fear and signs of autonomic arousal, such as mydriasis, tachycardia, rapid breathing, and sweating, during each episode. There is relative unresponsiveness to efforts of others to comfort the individual during the episodes.

1	. No or little (e.g., only a single visual scene) dream imagery is recalled. . Amnesia for the episodes is present. . The episodes cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. E. The disturbance is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication). F. Coexisting mental and medical disorders do not explain the episodes of sleepwatking or sleep terrors. Coding note: For ICD-9-CM, code 307.46 for all subtypes. For ICD-10-CM, code is based on subtype. Specify whether: 307.46 (F51.3) Sleepwalking type Specify if: 307.46 (F51.4) Sleep terror type

1	Coding note: For ICD-9-CM, code 307.46 for all subtypes. For ICD-10-CM, code is based on subtype. Specify whether: 307.46 (F51.3) Sleepwalking type Specify if: 307.46 (F51.4) Sleep terror type The essential feature of non—rapid eye movement (NREM) sleep arousal disorders is the repeated occurrence of incomplete arousals, usually beginning during the first third of the major Sleep episode (Criterion A), that typically are brief, lasting 1—10 minutes, but may be protracted, lasting up to 1 hour. The maximum duration of an event is unknown. The eyes are typically open during these events. Many individuals exhibit both subtypes of arousals on different occasions, which underscores the unitary underlying pathophysiology. The subtypes reﬂect varying degrees of simultaneous occurrence of wakefulness and NREM sleep, resulting in complex behaviors arising from sleep with varying degrees of conscious awareness, motor activity, and autonomic activation.

1	The essential feature of Sleepwalking is repeated episodes of complex motor behavior initiated during sleep, including rising from bed and walking about (Criterion A1). Sleep- walking episodes begin during any stage of NREM sleep, most commonly during slow- wave sleep and therefore most often occurring during the first third of the night. During episodes, the individual has reduced alertness and responsiveness, a blank stare, and rel- ative unresponsiveness to communication with others or efforts by others to awaken the individual. If awakened during the episode (or on awakening the following morning), the individual has limited recall for the episode. After the episode, there may initially be a brief period of confusion or difficulty orienting, followed by full recovery of cognitive function and appropriate behavior.

1	The essential feature of sleep terrors is the repeated occurrence of precipitous awaken- ings from sleep, usually beginning with a panicky scream or cry (Criterion A2). Sleep ter- rors usually begin during the first third of the major sleep episode and last 1—10 minutes, but they may last considerably longer, particularly in children. The episodes are accom- panied by impressive autonomic arousal and behavioral manifestations of intense fear.

1	During an episode, the individual is difficult to awaken or comfort. If the individual awak- ens after the sleep terror, little or none of the dream, or only fragmentary, single images, are recalled. During a typical episode of sleep terrors, the individual abruptly sits up in bed screaming or crying, with a frightened expression and autonomic signs of intense anx- iety (e.g., tachycardia, rapid breathing, sweating, dilation of the pupils). The individual may be inconsolable and is usually unresponsive to the efforts of others to awaken or com- fort him or her. Sleep terrors are also called “night terrors” or “pavor nocturnus.”

1	Sleepwalking episodes can include a wide variety of behaviors. Episodes may begin with confusion: the individual may simply sit up in bed, look about, or pick at the blanket or sheet. This behavior then becomes progressively complex. The individual may actually leave the bed and walk into closets, out of the room, and even out of buildings. Individuals may use the bathroom, eat, talk, or engage in more complex behaviors. Running and fran- tic attempts to escape some apparent threat can also occur. Most behaviors during sleep- walking episodes are routine and of low complexity. However, cases of unlocking doors and even operating machinery (driving an automobile) have been reported. Sleepwalking can also include inappropriate behavior (e.g., commonly, urinating in a closet or waste- basket). Most episodes last for several minutes to a half hour but may be more protracted.

1	Inasmuch as sleep is a state of relative analgesia, painful injuries sustained during sleep- walking may not be appreciated until awakening after the fact. There are two ”specialized” forms of Sleepwalking: sleep-related eating behavior and sleep-related sexual behavior (sexsomnia or sleep sex). Individuals with sleep-reluted eating experience unwanted recurrent episodes of eating with varying degrees of amnesia, rang- ing from no awareness to full awareness without the ability to not eat. During these epi- sodes, inappropriate foods may be ingested. Individuals with sleep-related eating disorder may find evidence of their eating only the next morning. In sexsomnia, varying degrees of sexual activity (e.g., masturbation, fondling, groping, sexual intercourse) occur as complex behaviors arisin from sleep without conscious awareness. This condition is more common consequences.

1	During a typical episode of sleep terrors, there is often a sense of overwhelming dread, with a compulsion to escape. Although fragmentary vivid dream images may occur, a story- like dream sequence (as in nightmares) is not reported. Most commonly, the individual does not awaken fully, but returns to sleep and has amnesia for the episode on awakening the next morning. Usually only one episode will occur on any one night. Occasionally several episodes may occur at intervals throughout the night. These events rarely arise during daytime naps.

1	Isolated or infrequent NREM sleep arousal disorders are very common in the general pop- ulation. From 10% to 30% of children have had at least one episode of sleepwalking, and 2%—3% sleepwalk often. The prevalence of sleepwalking disorder, marked by repeated ep- isodes and impairment or distress, is much lower, probably in the range of 1%—5%. The prevalence of sleepwalking episodes (not sleepwalking disorder) is 10%—7.0% among adults, with weekly to monthly episodes occurring in 0.5%—0.7%. The lifetime prevalence of sleepwalking in adults is 29.2%, with a past-year prevalence of sleepwalking of 3.6%. The prevalence of sleep terrors in the general population is unknown. The prevalence of sleep terror episodes (as opposed to sleep terror disorder, in which there is recurrence and distress or impairment) is approximately 36.9% at 18 months of age, 19.7% at 30 months of age, and 2.2% in adults.

1	quency with increasing age. The onset of sleepwalking in adults with no prior history of sleepwalking as children should prompt a search for specific etiologies, such as obstruc- tive sleep apnea, nocturnal seizures, or effect of medication. Environmental. Sedative use, sleep deprivation, sleep-wake schedule disruptions, fa- tigue, and physical or emotional stress increase the likelihood of episodes. Fever and sleep deprivation can produce an increased frequency of NREM sleep arousal disorders. Genetic and physiological. A family history for sleepwalking or sleep terrors may oc- cur in up to 80% of individuals who sleepwalk. The risk for sleepwalking is further in- creased (to as much as 60% of offspring) when both parents have a history of the disorder.

1	Individuals with sleep terrors frequently have a positive family history of either sleep terrors or sleepwalking, with as high as a 10-fold increase in the prevalence of the disorder among first-degree biological relatives. Sleep terrors are much more common in monozy- gotic twins as compared with dizygotic twins. The exact mode of inheritance is unknown. Violent or sexual activity during sleepwalking episodes is more likely to occur in adults. Eating during sleepwalking episodes is more commonly seen in females. Sleepwalking oc- curs more often in females during childhood but more often in males during adulthood. Older children and adults provide a more detailed recollection of fearful images asso- ciated with sleep terrors than do younger children, who are more likely to have complete amnesia or report only a vague sense of fear. Among children, sleep terrors are more com- mon in males than in females. Among adults, the sex ratio is even.

1	NREM sleep arousal disorders arise from any stage of NREM sleep but most commonly from deep NREM sleep (slow-wave sleep). They are most likely to appear in the first third of the night and do not commonly occur during daytime naps. During the episode, the polysomnogram may be obscured with movement artifact. In the absence of such artifact, the electroencephalogram typically shows theta or alpha frequency activity during the ep- isode, indicating partial or incomplete arousal.

1	Polysomnography in conjunction with audiovisual monitoring can be used to document episodes of sleepwalking. In the absence of actually capturing an event during a polysomno- graphic recording, there are no polysomnographic features that can serve as a marker for sleepwalking. Sleep deprivation may increase the likelihood of capturing an event. As a group, individuals who sleepwalk show instability of deep NREM sleep, but the overlap in findings with individuals who do not sleepwalk is great enough to preclude use of this indicator in es- tablishing a diagnosis. Unlike arousals from REM sleep associated with nightmares, in which there is an increase in heart rate and respiration prior to the arousal, the NREM sleep arousals of sleep terrors begin precipitously from sleep, without anticipatory autonomic changes. The arousals are associated with impressive autonomic activity, with doubling or tripling of the heart rate. The pathophysiology is poorly understood, but there appears to be

1	autonomic changes. The arousals are associated with impressive autonomic activity, with doubling or tripling of the heart rate. The pathophysiology is poorly understood, but there appears to be instability in the deeper stages of NREM sleep. Absent capturing an event during a formal sleep study, there are no reliable polysomnographic indicators of the tendency to experience sleep terrors.

1	For the diagnosis of a NREM sleep arousal disorder to be made, the individual or house- hold members must experience clinically significant distress or impairment, although pa- subthreshold for the diagnosis. Embarrassment concerning the episodes can impair social relationships. Social isolation or occupational difficulties can result. The determination of a ”disorder” depends on a number of factors, which may vary on an individual basis and will depend on the frequency of events, potential for violence or injurious behaviors, em- barrassment, or disruption/ distress of other household members. Severity determination is best made based on the nature or consequence of the behaviors rather than simply on fre- quency. Uncommonly, NREM sleep arousal disorders may result in serious injury to the individual or to someone trying to console the individual. Injuries to others are confined to those in close proximity; individuals are not ”sought out." Typically, sleepwalking in both children

1	the individual or to someone trying to console the individual. Injuries to others are confined to those in close proximity; individuals are not ”sought out." Typically, sleepwalking in both children and adults is not associated with significant mental disorders. For individuals with sleep-related eating behaviors, unknowingly preparing or eating food during the sleep period may create problems such as poor diabetes control, weight gain, injury (cuts and burns), or consequences of eating dangerous or toxic inedibles. NREM sleep arousal disorders may rarely result in violent or injurious behaviors with forensic implications.

1	Nightmare disorder. In contrast to individuals with NREM sleep arousal disorders, in- dividuals with nightmare disorder typically awaken easily and completely, report vivid storylike dreams accompanying the episodes, and tend to have episodes later in the night. NREM sleep arousal disorders occur during NREM sleep, whereas nightmares usually oc- cur during REM sleep. Parents of children with NREM sleep arousal disorders may mis- interpret reports of fragmentary imagery as nightmares. Breathing-related sleep disorders. Breathing disorders during sleep can also produce confusional arousals with subsequent amnesia. However, breathing-related sleep disor- ders are also characterized by characteristic symptoms of snoring, breathing pauses, and daytime sleepiness. In some individuals, a breathing-related sleep disorder may precipi- tate episodes of sleepwalking.

1	REM sleep behavior disorder. REM sleep behavior disorder may be difficult to distin- guish from NREM sleep arousal disorders. REM sleep behavior disorder is characterized by episodes of prominent, complex movements, often involving personal injury arising from sleep. In contrast to NREM sleep arousal disorders, REM sleep behavior disorder oc- curs during REM sleep. Individuals with REM sleep behavior disorder awaken easily and report more detailed and vivid dream content than do individuals with NREM sleep arousal disorders. They often report that they “act out dreams.” Parasomnia overlap syndrome. Parasomnia overlap syndrome consists of clinical and polysomnographic features of both sleepwalking and REM sleep behavior disorder.

1	Parasomnia overlap syndrome. Parasomnia overlap syndrome consists of clinical and polysomnographic features of both sleepwalking and REM sleep behavior disorder. SIeep-related seizures. Some types of seizures can produce episodes of very unusual behaviors that occur predominantly or exclusively during sleep. Nocturnal seizures may closely mimic NREM sleep arousal disorders but tend to be more stereotypic in nature, oc- cur multiple times nightly, and be more likely to occur from daytime naps. The presence of sleep-related seizures does not preclude the presence of NREM sleep arousal disorders. Sleep-related seizures should be classified as a form of epilepsy.

1	Sleep-related seizures should be classified as a form of epilepsy. Alcohol-induced blackouts. Alcohol-induced blackouts may be associated with extremely complex behaviors in the absence of other suggestions of intoxication. They do not involve the loss of consciousness but rather reﬂect an isolated disruption of memory for events during a drinking episode. By history, these behaviors may be indistinguishable from those seen in NREM sleep arousal disorders. Dissociative amnesia, with dissociative fugue. Dissociative fugue may be extremely difficult to distinguish from sleepwalking. Unlike all other parasornnias, nocturnal disso- ciative fugue arises from a period of wakefulness during sleep, rather than precipitously from sleep without intervening wakefulness. A history of recurrent childhood physical or sexual abuse is usually present (but may be difficult to obtain).

1	Malingering or other voluntary behavior occurring during wakefulness. As with disso- ciative fugue, malingering or other voluntary behavior occurring during wakefulness arises from wakefulness. Panic disorder. Panic attacks may also cause abrupt awakenings from deep NREM sleep accompanied by fearfulness, but these episodes produce rapid and complete awakening with- out the confusion, amnesia, or motor activity typical of NREM sleep arousal disorders.

1	Medication-induced complex behaviors. Behaviors similar to those in NREM sleep arousal disorders can be induced by use of, or withdrawal from, substances or medica- tions (e.g., benzodiazepines, nonbenzodiazepine sedative-hypnotics, opiates, cocaine, nic- otine, antipsychotics, tricyclic antidepressants, chloral hydrate). Such behaviors may arise from the sleep period and may be extremely complex. The underlying pathophysiology appears to be a relatively isolated amnesia. In such cases, substance/medication-induced sleep disorder, parasomnia type, should be diagnosed (see “Substance/Medication- Induced Sleep Disorder” later in this chapter). Night eating syndrome. The sleep-related eating disorder form of sleepwalking is to be differentiated from night eating syndrome, in which there is a delay in the circadian rhythm of food ingestion and an association with insomnia and / or depression.

1	In adults, there is an association between sleepwalking and major depressive episodes and obsessive-compulsive disorder. Children or adults with sleep terrors may have elevated scores for depression and anxiety on personality inventories. Relationship to International Classification of The International Classification of Sleep Disorders, 2nd Edition, includes ”confusional arousal” as a NREM sleep arousal disorder. Diagnostic Criteria 307.47 (F51.5) A. Repeated occurrences of extended, extremely dysphoric, and weII-remembered dreams that usually involve efforts to avoid threats to survival, security, or physical in- tegrity and that generally occur during the second half of the major sleep episode. B. On awakening from the dysphoric dreams, the individual rapidly becomes oriented and alert. C. The sleep disturbance causes clinically significant distress or impairment in social, oc— cupational, or other important areas of functioning.

1	C. The sleep disturbance causes clinically significant distress or impairment in social, oc— cupational, or other important areas of functioning. D. The nightmare symptoms are not attributable to the physiological effects of a sub- stance (e.g., a drug of abuse, a medication). E. Coexisting mental and medical disorders do not adequately explain the predominant complaint of dysphoric dreams. Specify if: Specify if: With associated non—sleep disorder, including substance use disorders Coding note: The code 307.47 (F51.5) applies to all three specifiers. Code also the relevant associated mental disorder, medical condition, or other sleep disorder imme- diately after the code for nightmare disorder in order to indicate the association. Specify if: Acute: Duration of period of nightmares is 1 month or less. Subacute: Duration of period of nightmares is greater than 1 month but less than 6 months. Persistent: Duration of period of nightmares is 6 months or greater.

1	Subacute: Duration of period of nightmares is greater than 1 month but less than 6 months. Persistent: Duration of period of nightmares is 6 months or greater. Specify current severity: Severity can be rated by the frequency with which the nightmares occur: Mild: Less than one episode per week on average. Moderate: One or more episodes per week but less than nightly. Severe: Episodes nightly. N ightmures are typically lengthy, elaborate, storylike sequences of dream imagery that seem real and that incite anxiety, fear, or other dysphoric emotions. Nightmare content typically focuses on attempts to avoid or cope with imminent danger but may involve themes that evoke other negative emotions. Nightmares occurring after traumatic experi- ences may replicate the threatening situation ("replicative nightmares”), but most do not.

1	On awakening, nightmares are well remembered and can be described in detail. They arise out sleep but are more likely in the second half of the major sleep episode when dreaming is longer and mdre intense. Factors that increase early-night REM intensity, such as sleep fragmentation or deprivation, jet lag, and REM-sensitive medications, might facilitate nightmares earlier in the night, including at sleep onset. Nightmares usually terminate with awakening and rapid return of full alertness. How- ever, the dysphoric emotions may persist into wakefulness and contribute to difficulty re- turning to sleep and lasting daytime distress. Some nightmares, known as ”bad dreams,” may not induce awakening and are recalled only later. If nightmares occur during sleep- onset REM periods (hypnagogic), the dysphoric emotion is frequently accompanied by a sense of being both awake and unable to move voluntarily (isolated sleep paralysis).

1	Mild autonomic arousal, including sweating, tachycardia, and tachypnea, may character- ize nightmares. Body movements and vocalizations are not characteristic because of REM sleep—related loss of skeletal muscle tone, but such behaviors may occur under situations of emotional stress or sleep fragmentation and in posttraumatic stress disorder (PTSD). When talking or emoting occurs, it is typically a brief event terminating the nightmare. Individuals with frequent nightmares are at substantially greater risk for suicidal ide- ation and suicide attempts, even when gender and mental illness are taken into account. Prevalence of nightmares increases through childhood into adolescence. From 1.3% to 3.9% of parents report that their preschool children have nightmares ”often” or "always”.

1	Prevalence of nightmares increases through childhood into adolescence. From 1.3% to 3.9% of parents report that their preschool children have nightmares ”often” or "always”. Prevalence increases from ages 10 to 13 for both males and females but continues to in- crease to ages 20—29 for females (while decreasing for males), when it can be twice as high for females as for males. Prevalence decreases steadily with age for both sexes, but the gen- der difference remains. Among adults, prevalence of nightmares at least monthly is 6%, whereas prevalence for frequent nightmares is 1%—2%. Estimates often combine idio- pathic and posttraumatic nightmares indiscriminately.

1	verity in late adolescence or early adulthood. Nightmares most likely appear in children exposed to acute or chronic psychosocial stressors and thus may not resolve spontane- ously. In a minority, frequent nightmares persist into adulthood, becoming virtually a life- long disturbance. Although specific nightmare content may reﬂect the individual’s age, the essential features of the disorder are the same across age groups. Temperamental. Individuals who experience nightmares report more frequent past ad- verse events, but not necessarily trauma, and often display personality disturbances or psychiatric diagnosis. Environmental. Sleep deprivation or fragmentation, and irregular sleep-wake schedules that alter the timing, intensity, or quantity of REM sleep, can put individuals at risk for nightmares. Genetic and physiological. Twin studies have identified genetic effects on the disposi- tion to nightmares and their co-occurrence with other parasomnias (e.g., sleeptalking).

1	Genetic and physiological. Twin studies have identified genetic effects on the disposi- tion to nightmares and their co-occurrence with other parasomnias (e.g., sleeptalking). Course modifiers. Adaptive parental bedside behaviors, such as soothing the child fol- lowing nightmares, may protect against developing chronic nightmares. The significance attributed to nightmares may vary by culture, and sensitivity to such be- liefs may facilitate disclosure. Adult females report having nightmares more frequently than do adult males. Nightmare content differs by sex, with adult females tending to report themes of sexual harassment or of loved ones disappearing/dying, and adult males tending to report themes of physical aggression or war/terror.

1	Polysomnographic studies demonstrate abrupt awakenings from REM sleep, usually during the second half of the night, prior to report of a nightmare. Heart, respiratory, and eye move- ment rates may quicken or increase in variability before awakening. N ightmares following traumatic events may also arise during non-REM (NREM), particularly stage 2, sleep. The typ- ical sleep of individuals with nightmares is mildly impaired (e.g., reduced efficiency, less slow- wave sleep, more awakenings), with more frequent periodic leg movements in sleep and rel- ative sympathetic nervous system activation after REM sleep deprivation. Functional Consequences of Nightmare Disorder pational impairment. However, if awakenings are frequent or result in sleep avoidance, individuals may experience excessive daytime sleepiness, poor concentration, depression, anxiety, or irritability. Frequent childhood nightmares (e.g., several per week), may cause significant distress to parents and child.

1	Sleep terror disorder. Both nightmare disorder and sleep terror disorder include awak- enings or partial awakenings with fearfulness and autonomic activation, but the two dis- orders are differentiable. Nightmares typically occur later in the night, during REM sleep, and produce vivid, storylike, and clearly recalled dreams; mild autonomic arousal; and complete awakenings. Sleep terrors typically arise in the first third of the night during rate storylike quality. The terrors lead to partial awakenings that leave the individual con— fused, disoriented, and only partially responsive and with substantial autonomic arousal. There is usually amnesia for the event in the morning.

1	There is usually amnesia for the event in the morning. REM sleep behavior disorder. The presence of complex motor activity during fright- ening dreams should prompt further evaluation for REM sleep behavior disorder, which occurs more typically among late middle-age males and, unlike nightmare disorder, is as- sociated with often violent dream enactments and a history of nocturnal injuries. The dream disturbance of REM sleep behavior disorder is described by patients as nightmares but is controlled by appropriate medication. Bereavement. Dysphoric dreams may occur during bereavement but typically involve loss and sadness and are followed by self—reﬂection and insight, rather than distress, on awakening. Narcolepsy. Nightmares are a frequent complaint in narcolepsy, but the presence of ex- cessive sleepiness and cataplexy differentiates this condition from nightmare disorder.

1	Narcolepsy. Nightmares are a frequent complaint in narcolepsy, but the presence of ex- cessive sleepiness and cataplexy differentiates this condition from nightmare disorder. Nocturnal seizures. Seizures may rarely manifest as nightmares and should be evalu- ated with polysomnography and continuous video electroencephalography. Nocturnal seizures usually involve stereotypical motor activity. Associated nightmares, if recalled, are often repetitive in nature or reﬂect epileptogenic features such as the content of diurnal auras (e.g., unmotivated dread), phosphenes, or ictal imagery. Disorders of arousal, espe- cially confusional arousals, may also be present. Breathing-related sleep disorders. Breathing-related sleep disorders can lead to awaken- ings with autonomic arousal, but these are not usually accompanied by recall of nightmares.

1	Breathing-related sleep disorders. Breathing-related sleep disorders can lead to awaken- ings with autonomic arousal, but these are not usually accompanied by recall of nightmares. Panic disorder. Attacks arising during sleep can produce abrupt awakenings with au- tonomic arousal and fearfulness, but nightmares are typically not reported and symptoms are similar to panic attacks arising during wakefulness. Sleep-related dissociative disorders. Individuals may recall actual physical or emo- tional trauma as a ”dream" during electroencephalography-documented awakenings.

1	Sleep-related dissociative disorders. Individuals may recall actual physical or emo- tional trauma as a ”dream" during electroencephalography-documented awakenings. Medication or substance use. Numerous substances/medications can precipitate night- mares, including dopaminergics; beta—adrenergic antagonists and other antihypertensives; amphetamine, cocaine, and other stimulants; antidepressants; smoking cessation aids; and melatonin. Withdrawal of REM sleep—suppressant medications (e.g., antidepressants) and alcohol can produce REM sleep rebound accompanied by nightmares. If nightmares are sufficiently severe to warrant independent clinical attention, a diagnosis of substance/ medication—induced sleep disorder should be considered.

1	Nightmares may be comorbid with several medical conditions, including coronary heart disease, cancer, parkinsonism, and pain, and can accompany medical treatments, such as he- modialysis, or withdrawal from medications or substances of abuse. Nightmares frequently are comorbid with other mental disorders, including PTSD; insomnia disorder; schizophrenia; psychosis; mood, anxiety, adjustment, and personality disorders; and grief during be- reavement. A concurrent nightmare disorder diagnosis should only be considered when in- dependent clinical attention is warranted (i.e., Criteria A—C are met). Otherwise, no separate diagnosis is necessary. These conditions should be listed under the appropriate comorbid category specifier. However, nightmare disorder may be diagnosed as a separate disorder in individuals with PTSD if the nightmares are temporally unrelated to PTSD (i.e., preceding other PTSD symptoms or persisting after other PTSD symptoms have resolved).

1	Nightmares are normally characteristic of REM sleep behavior disorder, PTSD, and acute stress disorder, but nightmare disorder may be independently coded if nightmares preceded the condition and their frequency or severity necessitates independent clinical attention. The latter may be determined by asking whether nightmares were a problem before onset of the other disorder and whether they continued after other symptoms had remitted. Relationshlp to International Ciassification of The International Classification of Sleep Disorders, 2nd Edition (ICSD-Z), presents similar di- agnostic criteria for nightmare disorder. Diagnostic Criteria 327.42 (647.52) A. Repeated episodes of arousal during sleep associated with vocalization and/or com- plex motor behaviors.

1	Diagnostic Criteria 327.42 (647.52) A. Repeated episodes of arousal during sleep associated with vocalization and/or com- plex motor behaviors. B. These behaviors arise during rapid eye movement (REM) sleep and therefore usually occur more than 90 minutes after sleep onset, are more frequent during the later por- tions of the sleep period, and uncommonly occur during daytime naps. C. Upon awakening from these episodes. the individual is completely awake, alert, and not confused or disoriented. D. Either of the following: 1. REM sleep without atonia on polysomnographic recording. 2. A history suggestive of REM sleep behavior disorder and an established synuclein- opathy diagnosis (e.g., Parkinson’s disease, multiple system atrophy). E. The behaviors cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning (which may include injury to self or the bed partner).

1	E. The behaviors cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning (which may include injury to self or the bed partner). F. The disturbance is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication) or another medical condition. 6. Coexisting mental and medical disorders do not explain the episodes.

1	The essential feature of rapid eye movement (REM) sleep behavior disorder is repeated episodes of arousal, often associated with vocalizations and / or complex motor behaviors arising from REM sleep (Criterion A). These behaviors often reﬂect motor responses to the content of action-filled or violent dreams of being attacked or trying to escape from a threatening situation, which may be termed dream enacting behaviors. The vocalizations are often loud, emotion-filled, and profane. These behaviors may be very bothersome to the individual and the bed partner and may result in significant injury (e.g., falling, jumping, or ﬂying out of bed; running, punching, thrusting, hitting, or kicking). Upon awakening, the individual is immediately awake, alert, and oriented (Criterion C) and is often able to recall dream mentation, which closely correlates with the observed behavior. The eyes typically remain closed during these events. The diagnosis of REM sleep behavior disor- will depend on a

1	able to recall dream mentation, which closely correlates with the observed behavior. The eyes typically remain closed during these events. The diagnosis of REM sleep behavior disor- will depend on a number of factors, including the frequency of events, the potential for vi- olence or injurious behaviors, embarrassment, and distress in other household members.

1	Severity determination is best made based on the nature or consequence of the behavior rather than simply on frequency. Although the behaviors are typically prominent and vi- olent, lesser behaviors may also occur. The prevalence of REM sleep behavior disorder is approximately O.38%—0.5% in the gen- eral population. Prevalence in patients with psychiatric disorders may be greater, possibly related to medications prescribed for the psychiatric disorder.

1	The onset of REM sleep behavior disorder may be gradual or rapid, and the course is usu- ally progressive. REM sleep behavior disorder associated with neurodegenerative disor- ders may improve as the underlying neurodegenerative disorder progresses. Because of the very high association with the later appearance of an underlying neurodegenerative disorder, most notably one of the synucleinopathies (Parkinson’s disease, multiple system atrophy, or major or mild neurocognitive disorder with Lewy bodies), the neurological status of individuals with REM sleep behavior disorder should be closely monitored. REM sleep behavior disorder overwhelmingly affects males older than 50 years, but in- creasingly this disorder is being identified in females and in younger individuals. Symp- toms in young individuals, particularly young females, should raise the possibility of narcolepsy or medication-induced REM sleep behavior disorder.

1	Genetic and physiological. Many widely prescribed medications, including tricyclic antidepressants, selective serotonin reuptake inhibitors, serotonin-norepinephrine reup- take inhibitors, and beta-blockers, may result in polysomnographic evidence of REM sleep without atonia and in frank REM sleep behavior disorder. It is not known whether the predisposition.

1	phasic electromyographic activity during REM sleep that is normally associated with mus- cle atonia. The increased muscle activity variably affects different muscle groups, mandating more extensive electromyographic monitoring than is employed in conventional sleep stud- ies. For this reason, it is suggested that electromyographic monitoring include the submen- talis, bilateral extensor digitorum, and bilateral anterior tibialis muscle groups. Continuous video monitoring is mandatory. Other polysomnographic findings may include very fre- (NREM) sleep. This polysomnography observation, termed REM sleep without atonia, is pres- ent in virtually all cases of REM sleep behavior disorder but may also be an asymptomatic polysomnographic finding. Clinical dream—enacting behaviors coupled with the polysom- nographic finding of REM without atonia is necessary for the diagnosis of REM sleep behav- ior disorder. REM sleep without atonia without a clinical history of dream-enacting behaviors is

1	polysom- nographic finding of REM without atonia is necessary for the diagnosis of REM sleep behav- ior disorder. REM sleep without atonia without a clinical history of dream-enacting behaviors is simply an asymptomatic polysomnographic observation. It is not known whether isolated REM sleep without atonia is a precursor to REM sleep behavior disorder.

1	dividuals. Embarrassment concerning the episodes can impair social relationships. Indi- viduals may avoid situations in which others might become aware of the disturbance, visiting friends overnight, or sleeping with bed partners. Social isolation or occupational difficulties can result. Uncommonly, REM sleep behavior disorder may result in serious injury to the victim or to the bed partner. Other parasomnias. Confusional arousals, sleepwalking, and sleep terrors can easily be confused with REM sleep behavior disorder. In general, these disorders occur in younger individuals. Unlike REM sleep behavior disorder, they arise from deep NREM sleep and therefore tend to occur in the early portion of the sleep period. Awakening from a confu- sional arousal is associated with confusion, disorientation, and incomplete recall of dream mentation accompanying the behavior. Polysomnographic monitoring in the disorders of arousal reveals normal REM atonia.

1	Nocturnal seizures. Nocturnal seizures may perfectly mimic REM sleep behavior disor- der, but the behaviors are generally more stereotyped. Polysomnographic monitoring em- ploying a full electroencephalographic seizure montage may differentiate the two. REM sleep without atonia is not present on polysomnographic monitoring. Obstructive sleep apnea. Obstructive sleep apnea may result in behaviors indistin- guishable from REM sleep behavior disorder. Polysomnographic monitoring is necessary to differentiate between the two. In this case, the symptoms resolve following effective treatment of the obstructive sleep apnea, and REM sleep without atonia is not present on polysomnography monitoring. Other specified dissociative disorder (sleep-related psychogenic dissociative disorder).

1	Other specified dissociative disorder (sleep-related psychogenic dissociative disorder). Unlike virtually all other parasomnias, which arise precipitously from NREM or REM sleep, psychogenic dissociative behaviors arise from a period of well-defined wakefulness during the sleep period. Unlike REM sleep behavior disorder, this condition is more prev- alent in young females. Malingering. Many cases of malingering in which the individual reports problematic sleep movements perfectly mimic the clinical features of REM sleep behavior disorder, and polysomnographic documentation is mandatory.

1	REM sleep behavior disorder is present concurrently in approximately 30% of patients with narcolepsy. When it occurs in narcolepsy, the demographics reﬂect the younger age range of narcolepsy, with equal frequency in males and females. Based on findings from individuals presenting to sleep clinics, most individuals (>50%) with initially ”idiopathic” notably, one of the synucleinopathies (Parkinson's disease, multiple system atrophy, or major or mild neurocognitive disorder with Lewy bodies). REM sleep behavior disorder often predates any other sign of these disorders by many years (often more than a decade). Relationship to International Classification of REM sleep behavior disorder is virtually identical to REM sleep behavior disorder in the International Classification of Sleep Disorders, 2nd Edition (ICSD-2). Diagnostic Criteria 333.94 (625.81)

1	REM sleep behavior disorder is virtually identical to REM sleep behavior disorder in the International Classification of Sleep Disorders, 2nd Edition (ICSD-2). Diagnostic Criteria 333.94 (625.81) A. An urge to move the legs, usually accompanied by or in response to uncomfortable and unpleasant sensations in the legs, characterized by all of the following: 1. The urge to move the legs begins or worsens during periods of rest or inactivity. 2. The urge to move the legs is partially or totally relieved by movement. 3. The urge to move the legs is worse in the evening or at night than during the day, or occurs only in the evening or at night. B. The symptoms in Criterion A occur at least three times per week and have persisted for at least 3 months. C. The symptoms in Criterion A are accompanied by significant distress or impairment in social. occupational, educational, academic, behavioral, or other important areas of functioning.

1	C. The symptoms in Criterion A are accompanied by significant distress or impairment in social. occupational, educational, academic, behavioral, or other important areas of functioning. D. The symptoms in Criterion A are not attributable to another mental disorder or medical condition (e.g., arthritis, leg edema, peripheral ischemia. leg cramps) and are not better explained by a behavioral condition (e.g., positional discomfort, habitual foot tapping). E. The symptoms are not attributable to the physiological effects of a drug of abuse or medication (e.g., akathisia).

1	E. The symptoms are not attributable to the physiological effects of a drug of abuse or medication (e.g., akathisia). Restless legs syndrome (RLS) is a sensorimotor, neurological sleep disorder characterized by a desire to move the legs or arms, usually associated with uncomfortable sensations typically described as creeping, crawling, tingling, burning, or itching (Criterion A). The diagnosis of RLS is based primarily on patient self—report and history. Symptoms are worse when the individual is at rest, and frequent movements of the legs occur in an effort to relieve the uncomfortable sensations. Symptoms are worse in the evening or night, and in some individuals they occur only in the evening or night. Evening worsening occurs in— dependently of any differences in activity. It is important to differentiate RLS from other conditions such as positional discomfort and leg cramps (Criterion D).

1	The symptoms of RLS can delay sleep onset and awaken the individual from sleep and are associated with significant sleep fragmentation. The relief obtained from moving the legs may no longer be apparent in severe cases. RLS is associated with daytime sleepiness and is frequently accompanied by significant clinical distress or functional impairment. Periodic leg movements in sleep (PLMS) can serve as corroborating evidence for RLS, with up to 90% of individuals diagnosed with RLS demonstrating PLMS when recordings are taken over multiple nights. Periodic leg movements during wakefulness are supportive of an RLS diagnosis. Reports of difficulty initiating and maintaining sleep and of excessive daytime sleepiness may also support the diagnosis of RLS. Additional supportive features include a family history of RLS among first-degree relatives and a reduction in symptoms, at least initially, with dopaminergic treatment.

1	Prevalence rates of RLS vary widely when broad criteria are utilized but range from 2% to 7.2% when more defined criteria are employed. When frequency of symptoms is at least three times per week with moderate or severe distress, the prevalence rate is 1.6%; when frequency of symptoms is a minimum of one time per week, the prevalence rate is 4.5%. Females are 1.5—2 times more likely than males to have RLS. RLS also increases with age. The prevalence of RLS may be lower in Asian populations. The onset of RLS typically occurs in the second or third decade. Approximately 40% of in- dividuals diagnosed with RLS during adulthood report having experienced symptoms before age 20 years, and 20% report having experienced symptoms before age 10 years.

1	Prevalence rates of RLS increase steadily with age until about age 60 years, with symptoms remaining stable or decreasing slightly in older age groups. Compared with nonfamilial cases, familial RLS usually has a younger age at onset and a slower progressive course. The clinical course of RLS differs by age at onset. When onset occurs before age 45, there is of— ten a slow progression of symptoms. In late-onset RLS, rapid progression is typical, and aggravating factors are common. Symptoms of RLS appear similar across the lifespan, re- maining stable or decreasing slightly in older age groups. Diagnosis of RLS in children can be difficult because of the self—report component.

1	Diagnosis of RLS in children can be difficult because of the self—report component. While Criterion A for adults assumes that the description of ”urge to move" is by the pa- tient, pediatric diagnosis requires a description in the child’s own words rather than by a parent or caretaker. Typically children age 6 years or older are able to provide detailed, ad— equate descriptors of RLS. However, children rarely use or understand the word "urge," reporting instead that their legs ”have to” or ”got to" move. Also, potentially related to prolonged periods of sitting during class, two-thirds of children and adolescents report daytime leg sensations. Thus, for diagnostic Criterion A3, it is important to compare equal duration of sitting or lying down in the day to sitting or lying down in the evening or night.

1	Nocturnal worsening tends to persist even in the context of pediatric RLS. As with RLS in adults, there is a significant negative impact on sleep, mood, cognition, and function. Im- pairment in children and adolescents is manifested more often in behavioral and educa- tional domains. Genetic and physiological. Predisposing factors include female gender, advancing age, genetic risk variants, and family history of RLS. Precipitating factors are often time- limited, such as iron deficiency, with most individuals resuming normal sleep patterns after the initial triggering event has disappeared. Genetic risk variants also play a role in RLS secondary to such disorders as uremia, suggesting that individuals with a genetic sus- ceptibility develop RLS in the presence of further risk factors. RLS has a strong familial component.

1	RLS secondary to such disorders as uremia, suggesting that individuals with a genetic sus- ceptibility develop RLS in the presence of further risk factors. RLS has a strong familial component. There are defined pathophysiological pathways subserving RLS. Genome-wide asso- ciation studies have found that RLS is significantly associated with common genetic vari- ants in intronic or intergenic regions in MEISI, BTBD9, and MAP2K5 on chromosomes 2p, 6p, and 15q, respectively. The association of these three variants with RLS has been inde- pendently replicated. B TBD9 confers a very large (80%) excessive risk when even a single allele is present. Because of the high frequency of this variant in individuals of European descent, the population attributable risk (PAR) approximates 50%. At-risk alleles associ- ated with MEISI and BTBD9 are less common in individuals of African or Asian descent, perhaps suggesting lower risk for RLS in these populations.

1	Pathophysiological mechanisms in RLS also include disturbances in the central dopa- minergic system and disturbances in iron metabolism. The endogenous opiate system may also be involved. Treatment effects of dopaminergic drugs (primarily D2 and D3 non- ergot agonists) provide further support that RLS is grounded in dysfunctional central dopaminergic pathways. While the effective treatment of RLS has also been shown to sig- nificantly reduce depressive symptoms, serotonergic antidepressants can induce or aggra- vate RLS in some individuals.

1	Although RLS is more prevalent in females than in males, there are no diagnostic differ- ences according to gender. However, the prevalence of RLS during pregnancy is two to three times greater than in the general population. RLS associated with pregnancy peaks during the third trimester and improves or resolves in most cases soon after delivery. The gender difference in prevalence of RLS is explained at least in part by parity, with nullipa- rous females being at the same risk of RLS as age—matched males. Polysomnography demonstrates significant abnormalities in RLS, commonly increased latency to sleep, and higher arousal index. Polysomnography with a preceding immobili- zation test may provide an indicator of the motor sign of RLS, periodic limb movements, under standard conditions of sleep and during quiet resting, both of which can provoke RLS symptoms. Functionai Consequences of Restless Legs Syndrome

1	Functionai Consequences of Restless Legs Syndrome Forms of RLS severe enough to significantly impair functioning or associated with mental dis- orders, including depression and anxiety, occur in approximately 2%—3% of the population. Although the impact of milder symptoms is less well characterized, individuals with RLS complain of disruption in at least one activity of daily living, with up to 50% reporting a negative impact on mood, and 476% reporting a lack of energy. The most common conse- quences of RLS are sleep disturbance, including reduced sleep time, sleep fragmentation, and overall distuibance; depression, generalized anxiety disorder, panic disorder, and post- traumatic stress disorder; and quality-of-life impairments. RLS can result in daytime sleep- iness or fatigue and is frequently accompanied by significant distress or impairment in affective, social, occupational, educational, academic, behavioral, or cognitive functioning.

1	The most important conditions in the differential diagnosis of RLS are leg cramps, posi- tional discomfort, arthralgias/arthritis, myalgias, positional ischemia (numbness), leg edema, peripheral neuropathy, radiculopathy, and habitual foot tapping. ”I(notting” of the muscle (cramps), relief with a single postural shift, limitation to joints, soreness to pal- pation (myalgias), and other abnormalities on physical examination are not characteristic of RLS. Unlike RLS, nocturnal leg cramps do not typically present with the desire to move the limbs nor are there frequent limb movements. Less common conditions to be differen- tiated from RLS include neuroleptic-induced akathisia, myelopathy, symptomatic venous insufficiency, peripheral artery disease, eczema, other orthopedic problems, and anxiety- induced restlessness. Worsening at night and periodic limb movements are more common in RLS than in medication-induced akathisia or peripheral neuropathy.

1	While is it important that RLS symptoms not be solely accounted for by another medical or behavioral condition, it should also be appreciated that any of these similar conditions can occur in an individual with RLS. This necessitates a separate focus on each possible condi- tion in the diagnostic process and when assessing impact. For cases in which the diagnosis of RLS is not certain, evaluation for the supportive features of RLS, particularly PLMS or a fam- ily history of RLS, may be helpful. Clinical features, such as response to a dopaminergic agent and positive family history for RLS, can help with the differential diagnosis. Depressive disorders, anxiety disorders, and attentional disorders are commonly comor- bid with RLS and are discussed in the section ”Functional Consequences of Restless Legs Syndrome." The main medical disorder comorbid with RLS is cardiovascular disease.

1	Syndrome." The main medical disorder comorbid with RLS is cardiovascular disease. There may be an association with numerous other medical disorders, including hyperten- sion, narcolepsy, migraine, Parkinson’s disease, multiple sclerosis, peripheral neuropathy, obstructive sleep apnea, diabetes mellitus, fibromyalgia, osteoporosis, obesity, thyroid disease, and cancer. Iron deficiency, pregnancy, and chronic renal failure are also comor- bid with RLS. Reiationship to Internationai Ciassification of The International Classification of Sleep Disorders, 2nd Edition (ICSD-2), presents similar diag- symptoms. A. A prominent and severe disturbance in sleep. B. There is evidence from the history, physical examination, or laboratory findings of both (1) and (2): 1. The symptoms in Criterion A developed during or soon after substance intoxication or after withdrawal from or exposure to a medication.

1	2. The involved substance/medication is capable of producing the symptoms in Crite- rion A. C. The disturbance is not better explained by a sleep disorder that is not substance/ medication-induced. Such evidence of an independent sleep disorder could include the following: The symptoms precede the onset of the substance/medication use; the symptoms persist for a substantial period of time (e.g., about 1 month) after the cessation of acute withdrawal or severe intoxication; or there is other evidence suggesting the existence of an independent non-substance/medication-induced sleep disorder (e.g., a history of recurrent non-substance/medication-related episodes). D. The disturbance does not occur exclusively during the course of a delirium. E. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning.

1	E. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. Note: This diagnosis should be made instead of a diagnosis of substance intoxication or substance withdrawal only when the symptoms in Criterion A predominate in the clinical picture and when they are sufficiently severe to warrant clinical attention.

1	Coding note: The ICD-9-CM and ICD-10-CM codes for the [specific substance/medica- tion]-induced sleep disorders are indicated in the table below. Note that the ICD—10-CM code depends on whether or not there is a comorbid substance use disorder present for the same class of substance. If a mild substance use disorder is comorbid with the sub- stance-induced sleep disorder, the 4th position character is “,"1 and the clinician should record “mild [substance] use disorder” before the substance-induced sleep disorder (e.g., “mild cocaine use disorder with cocaine-induced sleep disorder”). If a moderate or severe substance use disorder is comorbid with the substance-induced sleep disorder, the 4th po- sition character is “2," and the clinician should record “moderate [substance] use disorder” or “severe [substance] use disorder,” depending on the severity of the comorbid substance use disorder. If there is no comorbid substance use disorder (e.g., after a one-time heavy use of the

1	or “severe [substance] use disorder,” depending on the severity of the comorbid substance use disorder. If there is no comorbid substance use disorder (e.g., after a one-time heavy use of the substance). then the 4th position character is “9" and the clinician should record only the substance-induced sleep disorder. A moderate or severe tobacco use disorder is required in order to code a tobacco-induced sleep disorder; it is not permissible to code a comorbid mild tobacco use disorder or no tobacco use disorder with a tobacco—induced sleep disorder.

1	Specify whether: Insomnia type: Characterized by difficulty falling asleep or maintaining sleep, frequent nocturnal awakenings, or nonrestorative sleep. Daytime sleepiness type: Characterized by predominant complaint of excessive sleepiness/fatigue during waking hours or. less commonly, a long sleep period. Parasomnia type: Characterized by abnormal behavioral events during sleep. Mixed type: Characterized by a substance/medication-induced sleep problem charac- terized by multiple types of sleep symptoms, but no symptom clearly predominates. Specify it (see Table 1 in the chapter “Substance—Related and Addictive Disorders" for di- agnoses associated with substance class): With onset during intoxication: This specifier should be used if criteria are met for intoxication with the substance/medication and symptoms developed during the intox- ication period.

1	With onset during intoxication: This specifier should be used if criteria are met for intoxication with the substance/medication and symptoms developed during the intox- ication period. With onset during discontinuation/withdrawal: This specifier should be used it cri- teria are met for discontinuation/withdrawal from the substance/medication and symp- toms developed during. or shortly after, discontinuation of the substance/medication. With use disorder, Without disorder, moderate use Alcohol 291.82 F10.182 F10.282 F10.982 Caffeine 292.85 F15.182 F15.282 F15.982 Cannabis 292.85 F12.188 F12.288 F12.988 Opioid 292.85 F11.182 F11.282 F11.982 Sedative, hypnotic, or anxiolytic 292.85 F13.182 F13.282 F13.982 Amphetamine (or other 292.85 F15.182 F15.282 F15.982 Cocaine 292.85 F14.182 F14.282 F14.982 Tobacco 292.85 NA F17.208 NA

1	Sedative, hypnotic, or anxiolytic 292.85 F13.182 F13.282 F13.982 Amphetamine (or other 292.85 F15.182 F15.282 F15.982 Cocaine 292.85 F14.182 F14.282 F14.982 Tobacco 292.85 NA F17.208 NA Other (or unknown) substance 292.85 F19.182 F19.282 F19.982 |CD-9-CM. The name of the substance/ medication-induced sleep disorder begins with the specific substance (e.g., cocaine, bupropion) that is presumed to be causing the sleep disturbance. The diagnostic code is selected from the table included in the criteria set, which is based on the drug class. For substances that do not fit into any of the classes (e.g., bupropion), the code for ”other substance” should be used; and in cases in which a sub- stance is judged to be an etiological factor but the specific class of substance is unknown, the category ”unknown substance” should be used.

1	The name of the disorder is followed by the specification of onset (i.e., onset during in- toxication, onset during discontinuation/withdrawal), followed by the subtype designa- tion (i.e., insomnia type, daytime sleepiness type, parasomnia type, mixed type). Unlike the recording procedures for ICD-IO-CM, which combine the substance-induced disorder and substance use disorder into a single code, for ICD-9-CM a separate diagnostic code is given for the substance use disorder. For example, in the case of insomnia occurring during withdrawal in a man with a severe lorazepam use disorder, the diagnosis is 292.85 lorazepam-induced sleep disorder, with onset during withdrawal, insomnia type. An ad- ditional diagnosis of 304.10 severe lorazepam use disorder is also given. When more than one substance is judged to play a significant role in the development of the sleep distur- bance, each should be listed separately (e.g., 292.85 alcohol-induced sleep disorder, With onset during intoxication,

1	is judged to play a significant role in the development of the sleep distur- bance, each should be listed separately (e.g., 292.85 alcohol-induced sleep disorder, With onset during intoxication, insomnia type; 292.85 cocaine—induced sleep disorder, with on- set during intoxication, insomnia type).

1	|CD-1 O-CM. The name of the substance/medication-induced sleep disorder begins with the specific substance (e.g., cocaine, bupropion) that is presumed to be causing the sleep distur- bance. The diagnostic code is selected from the table included in the criteria set, which is based on the drug class and presence or absence of a comorbid substance use disorder. For sub- stances that do not fit into any of the classes (e.g., bupropion), the code for ”other substance” should be used; and in cases in which a substance is judged to be an etiological factor but the specific class of substance is unknown, the category "unknown substance" should be used.

1	When recording the name of the disorder, the comorbid substance use disorder (if any) is listed first, followed by the word “with,” followed by the name of the substance-induced sleep disorder, followed by the specification of onset (i.e., onset during intoxication, onset during discontinuation/withdrawal), followed by the subtype designation (i.e., insomnia type, daytime sleepiness type, parasomnia type, mixed type). For example, in the case of insomnia occurring during withdrawal in a man with a severe lorazepam use disorder, the diagnosis is F13.282 severe lorazepam use disorder with lorazepam-induced sleep disor— der, with onset during withdrawal, insomnia type. A separate diagnosis of the comorbid severe lorazepam use disorder is not given. If the substance-induced sleep disorder occurs without a comorbid substance use disorder (e.g., with medication use), no accompanying substance use disorder is noted (e.g., F19.982 bupropion-induced sleep disorder, with on- set during

1	occurs without a comorbid substance use disorder (e.g., with medication use), no accompanying substance use disorder is noted (e.g., F19.982 bupropion-induced sleep disorder, with on- set during medication use, insomnia type). When more than one substance is judged to play a significant role in the development of the sleep disturbance, each should be listed separately (e.g., F10.282 severe alcohol use disorder with alcohol-induced sleep disorder, with onset during intoxication, insomnia type; F14.282 severe cocaine use disorder with cocaine-induced sleep disorder, with onset during intoxication, insomnia type).

1	The essential feature of substance/medication-induced sleep disorder is a prominent sleep disturbance that is sufficiently severe to warrant independent clinical attention (Criterion A) and that is judged to be primarily associated with the pharmacological effects of a substance (i.e., a drug of abuse, a medication, toxin exposure) (Criterion B). Depending on the sub- stance involved, one of four types of sleep disturbances is reported. Insomnia type and day- time sleepiness type are most common, while parasomnia type is seen less often. The mixed type is noted when more than one type of sleep disturbance—related symptom is present and none predominates. The disturbance must not be better explained by another sleep disorder (Criterion C). A substance / medication-induced sleep disorder is distinguished from insom- nia disorder or a disorder associated with excessive daytime sleepiness by considering onset and course. For drugs of abuse, there must be evidence of intoxication or

1	is distinguished from insom- nia disorder or a disorder associated with excessive daytime sleepiness by considering onset and course. For drugs of abuse, there must be evidence of intoxication or withdrawal from the history, physical examination, or laboratory findings. Substance/ medication-induced sleep disorder arises only in association with intoxication or discontinuation/withdrawal states, whereas other sleep disorders may precede the onset of substance use or occur during times of sustained abstinence. As discontinuation/ withdrawal states for some substances can be protracted, onset of the sleep disturbance can occur 4 weeks after cessation of sub- stance use, and the disturbance may have features atypical of other sleep disorders (e.g., atypical age at onset or course). The diagnosis is not made if the sleep disturbance occurs only during a delirium (Criterion D). The symptoms must cause clinically significant dis- tress or impairment in social, occupational, or other

1	diagnosis is not made if the sleep disturbance occurs only during a delirium (Criterion D). The symptoms must cause clinically significant dis- tress or impairment in social, occupational, or other important areas of functioning (Crite— rion E). This diagnosis should be made instead of a diagnosis of substance intoxication or substance withdrawal only when the symptoms in Criterion A predominate in the clinical picture and when the symptoms warrant independent clinical attention.

1	During periods of substance/ medication use, intoxication, or withdrawal, individuals fre- quently complain of dysphoric mood, including depression and anxiety, irritability, cog- nitive impairment, inability to concentrate, and fatigue.

1	Prominent and severe sleep disturbances can occur in association with intoxication with the following classes of substances: alcohol; caffeine; cannabis; opioids; sedatives, hypnotics, 0r anxiolytics; stimulants (including cocaine); and other (or unknown) sub- stances. Prominent and severe sleep disturbances can occur in association with withdrawal from the following classes of substances: alcohol; caffeine; cannabis; opioids; sedatives, hypnotics, or anxiolytics; stimulant (including cocaine); tobacco; and other (or unknown) substances. Some medications that invoke sleep disturbances include adrenergic agonists and antagonists, dopamine agonists and antagonists, cholinergic agonists and antagonists, serotonergic agonists and antagonists, antihistamines, and corticosteroids.

1	Alcohol. Alcohol-induced sleep disorder typically occurs as insomnia type. During acute intoxication, alcohol produces an immediate sedative effect depending on close, ac- duced rapid eye movement (REM) sleep. Following these initial effects, there may be increased wakefulness, restless sleep, and vivid and anxiety-laden dreams for the remain- ing sleep period. In parallel, stages 3 and 4 sleep are reduced, and wakefulness and REM sleep are increased. Alcohol can aggravate breathing-related sleep disorder. With habitual use, alcohol continues to show a short—lived sedative effect in the first half of the night, fol- lowed by sleep continuity disruption in the second half. During alcohol withdrawal, there is extremely disrupted sleep continuity, and an increased amount and intensity of REM sleep, associated frequently with vivid dreaming, which in extreme form, constitutes part of alcohol withdrawal delirium. After acute withdrawal, chronic alcohol users may con- tinue to complain of

1	sleep, associated frequently with vivid dreaming, which in extreme form, constitutes part of alcohol withdrawal delirium. After acute withdrawal, chronic alcohol users may con- tinue to complain of light, fragmented sleep for weeks to years associated with a persistent deficit in slow-wave sleep.

1	Caffeine. Caffeine-induced sleep disorder produces insomnia in a dose-dependent man- ner, with some individuals presenting with daytime sleepiness related to withdrawal. Cannabis. Acute administration of cannabis may shorten sleep latency, though arous- ing effects with increments in sleep latency also occur. Cannabis enhances slow-wave sleep and suppresses REM sleep after acute administration. In chronic users, tolerance to the sleep-inducing and slow-wave sleep—enhancing effects develops. Upon withdrawal, sleep difficulties and unpleasant dreams have been reported lasting for several weeks. during this phase. Opioids. Opioids may produce an increase in sleepiness and in subjective depth of sleep, and reduced REM sleep, during acute short-term use. With continued administration, tol- erance to the sedative effects of opioids develops and there are complaints of insomnia. Consistent with their respiratory depressant effects, opioids exacerbate sleep apnea.

1	Consistent with their respiratory depressant effects, opioids exacerbate sleep apnea. Sedative, hypnotic, or anxiolytic substances. Sedatives, hypnotics, and anxiolytics (e.g., barbiturates, benzodiazepines receptor agonists, meprobamate, glutethimide, methypry- Ion) have similar effects as opioids on sleep. During acute intoxication, sedative-hypnotic drugs produce the expected increase in sleepiness and decrease in wakefulness. Chronic use (particularly of barbiturates and the older nonbarbiturate, nonbenzodiazepine drugs) may cause tolerance with subsequent return of insomnia. Daytime sleepiness may occur.

1	Sedative-hypnotic drugs can increase the frequency and severity of obstructive sleep ap- nea events. Parasomnias are associated with use of benzodiazepine receptor agonists, es- pecially when these medications are taken at higher doses and when they are combined with other sedative drugs. Abrupt discontinuation of chronic sedative, hypnotic, or anx- iolytic use can lead to withdrawal but more commonly rebound insomnia, a condition of an exacerbation of insomnia upon drug discontinuation for 1-2 days reported to occur even with short-term use. Sedative, hypnotic, or anxiolytic drugs with short durations of action are most likely to produce complaints of rebound insomnia, whereas those with longer durations of action are more often associated with daytime sleepiness. Any sedative, hypnotic, or anxiolytic drug can potentially cause daytime sedation, withdrawal, or re- bound insomnia.

1	Amphetamines and related substances and other stimulants. Sleep disorders induced by amphetamine and related substances and other stimulants are characterized by insomnia during intoxication and excessive sleepiness during withdrawal. During acute intoxication, stimulants reduce the total amount of sleep, increase sleep latency and sleep continuity distur- bances, and decrease REM sleep. Slow-wave sleep tends to be reduced. During withdrawal from chronic stimulant use, there is both prolonged nocturnal sleep duration and excessive daytime sleepiness. Multiple sleep latency tests may show increased daytime sleepiness dur- ing the withdrawal phase. Drugs like 3,4-methylenedioxymethamphetamine (MDMA; “ec- stasy”) and related substances lead to restless and disturbed sleep within 48 hours of intake; frequent use of these compounds is associated with persisting symptoms of anxiety, depres- sion, and sleep disturbances, even during longer-term abstinence.

1	Tobacco. Chronic tobacco consumption is associated primarily with symptoms of insom- nia, decreased slow-wave sleep with a reduction of sleep efficiency, and increased daytime sleepiness. Withdrawal from tobacco can lead to impaired sleep. Individuals who smoke heavily may experience regular nocturnal awakenings caused by tobacco craving. Other or unknown substances/medications. Other substances/ medications may pro- duce sleep disturbances, particularly medications that affect the central or autonomic nervous systems (e.g., adrenergic agonists and antagonists, dopamine agonists and antag- onists, cholinergic agonists and antagonists, serotonergic agonists and antagonists, anti- histamines, corticosteroids).

1	Insomnia in children can be identified by either a parent or the child. Often the child has a clear sleep disturbance associated with initiation of a medication but may not report symptoms, although parents observe the sleep disturbances. The use of some illicit sub- stances (e.g., cannabis, ecstasy) is prevalent in adolescence and early adulthood. Insomnia sideration of whether the sleep disturbance is due to consumption of these substances. Help-seeking behavior for the sleep disturbance in these age groups is limited, and thus corroborative report may be elicited from a parent, caregiver, or teacher. Older individuals take more medications and are at increased risk for developing a substance/medication- induced sleep disorder. They may interpret sleep disturbance as part of normal aging and fail to report symptoms. Individuals with major neurocognitive disorder (e.g., dementia) toms, making corroborative report from caregiver(s) particularly important.

1	are normative for certain age groups. They are relevant for, and likely applicable to, the type of sleep disturbance encountered (see the chapter "Substance-Related and Addictive Disorders” for descriptions of respective substance use disorders). Temperamental. Substance use generally precipitates or accompanies insomnia in vul- nerable individuals. Thus, presence of insomnia in response to stress or change in sleep en- sleep disorder. A similar risk may be present for individuals with other sleep disorders (e.g., individuals with hypersomnia who use stimulants). The consumption of substances, including prescribed medications, may depend in part on cultural background and specific local drug regulations.

1	The consumption of substances, including prescribed medications, may depend in part on cultural background and specific local drug regulations. Gender-specific prevalences (i.e., females affected more than males at a ratio of about 2:1) exist for patterns of consumption of some substances (e.g., alcohol). The same amount and duration of consumption of a given substance may lead to highly different sleep-related outcomes in males and females based on, for example, gender-specific differences in hepatic functioning.

1	Each of the substance/medication-induced sleep disorders produces electroencephalo- graphic sleep patterns that are associated with, but cannot be considered diagnostic of, other disorders. The electroencephalographic sleep profile for each substance is related to the stage of use, whether intake/ intoxication, chronic use, or withdrawal following discontinu- ation of the substance. All—night polysomnography can help define the severity of insomnia complaints, while the multiple sleep latency test provides information about the severity of daytime sleepiness. Monitoring of nocturnal respiration and periodic limb movements with havior. Sleep diaries for 2 weeks and actigraphy are considered helpful in confirming the presence of substance/medication-induced sleep disorder. Drug screening can be of use when the individual is not aware or unwilling to relate information about substance intake.

1	While there are many functional consequences associated with sleep disorders, the only unique consequence for substance/medication-induced sleep disorder is increased risk for relapse. The degree of sleep disturbance during alcohol withdrawal (e.g., REM sleep rebound predicts risk of relapse of drinking). Monitoring of sleep quality and daytime whether an individual is at increased risk for relapse. Substance intoxication or substance withdrawal. Sleep disturbances are commonly en- countered in the context of substance intoxication or substance discontinuation/with- drawal. A diagnosis of substance/ medication-induced sleep disorder should be made instead of a diagnosis of substance intoxication or substance withdrawal only when the sleep disturbance is predominant in the clinical picture and is sufficiently severe to war- rant independent clinical attention.

1	Delirium. If the substance/ medication-induced sleep disturbance occurs exclusively dur- ing the course of a delirium, it is not diagnosed separately.

1	Other sleep disorders. A substance/ medication—induced sleep disorder is distinguished from another sleep disorder if a substance/medication is judged to be etiologically related to the symptoms. A substance/medication-induced sleep disorder attributed to a prescribed medication for a mental disorder or medical condition must have its onset while the individual is receiving the medication or during discontinuation, if there is a discontinuation/with- drawal syndrome associated with the medication. Once treatment is discontinued, the sleep disturbance will usually remit within days to several weeks. If symptoms persist beyond 4 weeks, other causes for the sleep disturbancehrelated symptoms should be considered. Not infrequently, individuals with another sleep disorder use medications or drugs of abuse to self—medicate their symptoms (e.g., alcohol for management of insomnia). If the substance/ medication is judged to play a significant role in the exacerbation of the sleep disturbance,

1	of abuse to self—medicate their symptoms (e.g., alcohol for management of insomnia). If the substance/ medication is judged to play a significant role in the exacerbation of the sleep disturbance, an additional diagnosis of a substance/medication-induced sleep disorder may be warranted.

1	Sleep disorder due to another medical condition. Substance/medication-induced sleep disorder and sleep disorder associated with another medical condition may produce sim- ilar symptoms of insomnia, daytime sleepiness, or a parasomnia. Many individuals with other medical conditions that cause sleep disturbance are treated with medications that may also cause sleep disturbances. The chronology of symptoms is the most important fac- tor in distinguishing between these two sources of sleep symptoms. Difficulties with sleep that clearly preceded the use of any medication for treatment of a medical condition would suggest a diagnosis of sleep disorder associated with another medical condition. Con— versely, sleep symptoms that appear only after the initiation of a particular medication/ substance suggest a substance/medication-induced sleep disorder. If the disturbance is comorbid with another medical condition and is also exacerbated by substance use, both diagnoses (i.e., sleep disorder

1	suggest a substance/medication-induced sleep disorder. If the disturbance is comorbid with another medical condition and is also exacerbated by substance use, both diagnoses (i.e., sleep disorder associated with another medical condition and substance/ medication-induced sleep disorder) are given. When there is insufficient evidence to de- termine whether the sleep disturbance is attributable to a substance/ medication or to an— other medical condition or is primary (i.e., not due to either a substance/medication or another medical condition), a diagnosis of other specified sleep-wake disorder or unspec- ified sleep—wake disorder is indicated.

1	See the ”Comorbidity" sections for other sleep disorders in this chapter, including insom- nia, hypersomnolence, central sleep apnea, sleep-related hypoventilation, and circadian rhythm sleep-wake disorders, shift work type. Relationship to Internationai Ciassification of The International Classification of Sleep Disorders, 2nd Edition (ICSD-2), lists sleep disorders “due to drug or substance” under their respective phenotypes (e.g., insomnia, hypersomnia). 780.52 (647.09)

1	780.52 (647.09) This category applies to presentations in which symptoms characteristic of insomnia disorder that cause clinically significant distress or impairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for insomnia disorder or any of the disorders in the sleep-wake disorders diagnostic class. The other specified insomnia dis- order category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for insomnia disorder or any specific sleep—wake disorder. This is done by recording ”other specified insomnia disorder” followed by the specific reason (e.g., “brief insomnia disorder). Examples of presentations that can be specified using the “other specified" designation include the following: 1. Brief insomnia disorder: Duration is less than 3 months.

1	Examples of presentations that can be specified using the “other specified" designation include the following: 1. Brief insomnia disorder: Duration is less than 3 months. 2. Restricted to nonrestorative sleep: Predominant complaint is nonrestorative sleep unaccompanied by other sleep symptoms such as difficulty falling asleep or remaining asleep. 780.52 (647.00)

1	2. Restricted to nonrestorative sleep: Predominant complaint is nonrestorative sleep unaccompanied by other sleep symptoms such as difficulty falling asleep or remaining asleep. 780.52 (647.00) This category applies to presentations in which symptoms characteristic of insomnia disor- der that cause clinically significant distress or impairment in social, occupational. or other important areas of functioning predominate but do not meet the full criteria for insomnia dis- order or any of the disorders in the sleep-wake disorders diagnostic class. The unspecified insomnia disorder category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for insomnia disorder or a specific sleep-wake dis- order, and includes presentations in which there is insufficient information to make a more specific diagnosis. 780.54 (647.19)

1	780.54 (647.19) This category applies to presentations in which symptoms characteristic of hypersomno- lence disorder that cause clinically significant distress or impairment in social, occupation- al, or other important areas of functioning predominate but do not meet the full criteria for hypersomnolence disorder or any of the disorders in the sleep-wake disorders diagnostic class. The other specified hypersomnolence disorder category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for hypersomnolence disorder or any specific sleep-wake disorder. This is done by recording “other specified hypersomnolence disorder” followed by the spe- cific reason (e.g., “brief-duration hypersomnolence," as in Kieine-Levin syndrome). 780.54 (647.10)

1	This is done by recording “other specified hypersomnolence disorder” followed by the spe- cific reason (e.g., “brief-duration hypersomnolence," as in Kieine-Levin syndrome). 780.54 (647.10) This category applies to presentations in which symptoms characteristic of hypersomno- lence disorder that cause clinically significant distress or impairment in social, occupation- al, or other important areas of functioning predominate but do not meet the full criteria for hypersomnolence disorder or any of the disorders in the sleep-wake disorders diagnostic class. The unspecified hypersomnolence disorder category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for hypersom- nolence disorder or a specific sleep-wake disorder. and includes presentations in which there is insufficient information to make a more specific diagnosis. 780.59 (647.8)

1	780.59 (647.8) This category applies to presentations in which symptoms characteristic of a sleep-wake disorder that cause clinically significant distress or impairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the sleep-wake disorders diagnostic class and do not qualify for a diagno- sis of other specified insomnia disorder or other specified hypersomnolence disorder. The other specified sleep-wake disorder category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for any specific sleep-wake disorder. This is done by recording “other specified sleep-wake disorder" followed by the specific reason (e.g., "repeated arousals during rapid eye movement sleep without polysomnography or history of Parkinson's disease or other synucleinopathy"). 780.59 (0.47.9)

1	780.59 (0.47.9) This category applies to presentations in which symptoms characteristic of a sleep-wake disorder that cause clinically significant distress or impairment in social, occupational, or other important areas of functioning predominate but do not meet the tull criteria for any of the disorders in the sleep-wake disorders diagnostic class and do not qualify for a diagno- sis of unspecified insomnia disorder or unspecified hypersomnolence disorder. The un- specified sleep-wake disorder category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for a specific sleep-wake disorder, and includes presentations in which there is insufficient information to make a more spe- cific diagnosis.

1	Sexual dYSIU I'lCIIOITS include delayed ejaculation, erectile disorder, female orgasmic disorder, female sexual interest/arousal disorder, genito—pelvic pain/penetration disorder, male hypoactive sexual desire disorder, premature (early) ejaculation, substance/medication- induced sexual dysfunction, other specified sexual dysfunction, and unspecified sexual dys- function. Sexual dysfunctions are a heterogeneous group of disorders that are typically char- acterized by a clinically significant disturbance in a person’s ability to respond sexually or to experience sexual pleasure. An individual may have several sexual dysfunctions at the same time. In such cases, all of the dysfunctions should be diagnosed.

1	Clinical judgment should be used to determine if the sexual difficulties are the result of inadequate sexual stimulation; in these cases, there may still be a need for care, but a di- agnosis of a sexual dysfunction would not be made. These cases may include, but are not limited to, conditions in which lack of knowledge about effective stimulation prevents the experience of arousal or orgasm. Subtypes are used to designate the onset of the difficulty. In many individuals with sexual dysfunctions, the time of onset may indicate different etiologies and interventions.

1	Subtypes are used to designate the onset of the difficulty. In many individuals with sexual dysfunctions, the time of onset may indicate different etiologies and interventions. Lifelong refers to a sexual problem that has been present from first sexual experiences, and acquired applies to sexual disorders that develop after a period of relatively normal sexual function. Generalized refers to sexual difficulties that are not limited to certain types of stimulation, situations, or partners, and situational refers to sexual difficulties that only oc- cur with certain types of stimulation, situations, or partners.

1	In addition to the lifelong/ acquired and generalized/situational subtypes, a number of factors must be considered during the assessment of sexual dysfunction, given that they may be relevant to etiology and/ or treatment, and that may contribute, to varying degrees, across individuals: 1) partner factors (e.g., partner’s sexual problems; partner’s health sta— tus); 2) relationship factors (e.g., poor communication; discrepancies in desire for sexual activity); 3) individual vulnerability factors (e.g., poor body image; history of sexual or emo- tional abuse), psychiatric comorbidity (e.g., depression, anxiety), or stressors (e.g., job loss, bereavement); 4) cultural or religious factors (e.g., inhibitions related to prohibitions against prognosis, course, or treatment.

1	Clinical judgment about the diagnosis of sexual dysfunction should take into consideration cultural factors that may inﬂuence expectations or engender prohibitions about the experience of sexual pleasure. Aging may be associated with a normative decrease in sexual response. Sexual response has a requisite biological underpinning, yet is usually experienced in an intrapersonal, interpersonal, and cultural context. Thus, sexual function involves a com- plex interaction among biological, sociocultural, and psychological factors. In many clinical contexts, a precise understanding of the etiology of a sexual problem is unknown. Nonethe- less, a sexual dysfunction diagnosis requires ruling out problems that are better explained by a nonsexual mental disorder, by the effects of a substance (e.g., drug or medication), by a medical condition (e.g., due to pelvic nerve damage), or by severe relationship distress, partner violence, or other stressors.

1	If the sexual dysfunction is mostly explainable by another nonsexual mental disorder (e.g., depressive or bipolar disorder, anxiety disorder, posttraumatic stress disorder, psychotic dis- order), then only the other mental disorder diagnosis should be made. If the problem is thought to be better explained by the use / misuse or discontinuation of a drug or substance, it should be diagnosed accordingly as a substance/medication-induced sexual dysfunction. If the sexual dysfunction is attributable to another medical condition (e.g., peripheral neuropa- thy), the individual would not receive a psychiatric diagnosis. If severe relationship distress, partner violence, or significant stressors better explain the sexual difficulties, then a sexual dys- function diagnosis is not made, but an appropriate V or Z code for the relationship problem or stressor may be listed. In many cases, a precise etiological relationship between another con- dition (e.g., a medical condition) and a sexual

1	appropriate V or Z code for the relationship problem or stressor may be listed. In many cases, a precise etiological relationship between another con- dition (e.g., a medical condition) and a sexual dysfunction cannot be established.

1	Diagnostic Criteria 302.74 (F5232) A. Either of the following symptoms must be experienced on almost all or all occasions (approximately 75%—100%) of partnered sexual activity (in identified situational con- texts or, ii generalized, in all contexts), and without the individual desiring delay: 1. Marked delay in ejaculation. 2. Marked infrequency or absence of ejaculation. B. The symptoms in Criterion A have persisted for a minimum duration of approximately 6 months. C. The symptoms in Criterion A cause clinically significant distress in the individual. D. The sexual dysfunction is not better explained by a nonsexual mental disorder or as a consequence of severe relationship distress or other significant stressors and is not at- tributable to the effects of a substance/medication or another medical condition. Specify whether: Lifelong: The disturbance has been present since the individual became sexually active.

1	Specify whether: Lifelong: The disturbance has been present since the individual became sexually active. Acquired: The disturbance began after a period of relatively normal sexual function. Specify whether: Generalized: Not limited to certain types of stimulation, situations, or partners. Situational: Only occurs with certain types of stimulation, situations, or partners. Specify current severity: Mild: Evidence of mild distress over the symptoms in Criterion A. Moderate: Evidence of moderate distress over the symptoms in Criterion A. Severe: Evidence of severe or extreme distress over the symptoms in Criterion A.

1	Moderate: Evidence of moderate distress over the symptoms in Criterion A. Severe: Evidence of severe or extreme distress over the symptoms in Criterion A. The distinguishing feature of delayed ejaculation is a marked delay in or inability to achieve ejaculation (Criterion A). The man reports difficulty or inability to ejaculate de- spite the presence of adequate sexual stimulation and the desire to ejaculate. The present- ing complaint usually involves partnered sexual activity. In most cases, the diagnosis will be made by self—report of the individual. The definition of "delay” does not have precise boundaries, as there is noconsensus as to what constitutes a reasonable time to reach or- gasm or what is unacceptably long for most men and their sexual partners.

1	The man and his partner may report prolonged thrusting to achieve orgasm to the point of exhaustion or genital discomfort and then ceasing efforts. Some men may report avoiding sexual activity because of a repetitive pattern of difficulty ejaculating. Some sexual partners may report feeling less sexually attractive because their partner cannot ejaculate easily.

1	In addition to the subtypes ”lifelong/acquired” and ”generalized/ situational,” the fol— lowing five factors must be considered during assessment and diagnosis of delayed ejacu- lation, given that they may be relevant to etiology and/ or treatment: 1) partner factors (e.g., partner’s sexual problems, partner’s health status); 2) relationship factors (e.g., poor com- munication, discrepancies in desire for sexual activity); 3) individual vulnerability factors (e.g., poor body image; history of sexual or emotional abuse), psychiatric comorbidity (e.g., depression, anxiety), or stressors (e.g., job loss, bereavement); 4) cultural/religious factors (e.g., inhibitions related to prohibitions against sexual activity; attitudes toward sexuality); and 5) medical factors relevant to prognosis, course, or treatment. Each of these factors may contribute differently to the presenting symptoms of different men with this disorder.

1	Prevalence is unclear because of the lack of a precise definition of this syndrome. It is the least common male sexual complaint. Only 75% of men report always ejaculating during sexual activity, and less than 1% of men will complain of problems with reaching ejacula- tion that last more than 6 months. Lifelong delayed ejaculation begins with early sexual experiences and continues through- out life. By definition, acquired delayed ejaculation begins after a period of normal sexual function. There is minimal evidence concerning the course of acquired delayed ejacula- tion. The prevalence of delayed ejaculation appears to remain relatively constant until around age 50 years, when the incidence begins to increase significantly. Men in their 805 report twice as much difficulty ejaculating as men younger than 59 years.

1	Genetic and physiological. Age-related loss of the fast-conducting peripheral sensory nerves and age-related decreased sex steroid secretion may be associated with the increase in delayed ejaculation in men older than 50 years. Complaints of ejaculatory delay vary across countries and cultures. Such complaints are more common among men in Asian populations than in men living in Europe, Australia, or the United States. This variation may be attributable to cultural or genetic differences between cultures. Functional Consequences of Delayed Ejaculation Difficulty with ejaculation may contribute to difficulties in conception. Delayed ejacula- tion is often associated with considerable psychological distress in one or both partners.

1	Another medical condition. The major differential diagnosis is between delayed ejacu- lation fully explained by another medical illness or injury and delayed ejaculation with a psychogenic, idiopathic, or combined psychological and medical etiology. A situational aspect to the complaint is suggestive of a psychological basis for the problem (e.g., men who can ejaculate during sexual activity with one sex but not the other; men who can ejac— ulate with one partner but not another of the same sex; men with paraphilic arousal pat— terns; men who require highly ritualized activity to ejaculate during partnered sexual activity). Another medical illness or injury may produce delays in ejaculation independent of psychological issues. For example, inability to ejaculate can be caused by interruption of the nerve supply to the genitals, such as can occur after traumatic surgical injury to the lumbar sympathetic ganglia, abdominoperitoneal surgery, or lumbar sympathectomy.

1	Ejaculation is thought to be under autonomic nervous system control involving the hypo- gastric (sympathetic) and pudendal (parasympathetic) nerves. A number of neurodegen- erative diseases, such as multiple sclerosis and diabetic and alcoholic neuropathy, can cause inability to ejaculate. Delayed ejaculation should also be differentiated from retro— grade ejaculation (i.e., ejaculation into the bladder), which may follow transurethral pros- tatic resection. Substance/medication use. A number of pharmacological agents, such as antidepres- sants, antipsychotics, alpha sympathetic drugs, and opioid drugs, can cause ejaculatory problems. Dysfunction with orgasm. It is important in the history to ascertain whether the com- plaint concerns delayed ejaculation or the sensation of orgasm, or both. Ejaculation occurs in the genitals, whereas the experience of orgasm is believed to be primarily subjective.

1	Ejaculation and orgasm usually occur together but not always. For example, a man with a normal ejaculatory pattern may complain of decreased pleasure (i.e., anhedonic ejacula- tion). Such a complaint would not be coded as delayed ejaculation but could be coded as other specified sexual dysfunction or unspecified sexual dysfunction. There is some evidence to suggest that delayed ejaculation may be more common in severe forms of major depressive disorder. Diagnostic Criteria 302.72 (F52.21) A. At least one of the three following symptoms must be experienced on almost all or all (approximately 75%—100%) occasions of sexual activity (in identified situational con- texts or, if generalized, in all contexts): 1. Marked difficulty in obtaining an erection during sexual activity. 2. Marked difficulty in maintaining an erection until the completion of sexual activity. 3. Marked decrease in erectile rigidity.

1	2. Marked difficulty in maintaining an erection until the completion of sexual activity. 3. Marked decrease in erectile rigidity. B. The symptoms in Criterion A have persisted for a minimum duration of approximately 6 months. C. The symptoms in Criterion A cause clinically significant distress in the individual. D. The sexual dysfunction is not better explained by a nonsexual mental disorder or as a consequence of severe relationship distress or other significant stressors and is not at- tributable to the effects of a substance/medication or another medical condition. Specify whether: Lifelong: The disturbance has been present since the individual became sexually ac- tive. Acquired: The disturbance began after a period of relatively normal sexual function. Specify whether: Generalized: Not limited to certain types of stimulation, situations, or partners. Situational: Only occurs with certain types of stimulation, situations, or partners. Specify current severity:

1	Generalized: Not limited to certain types of stimulation, situations, or partners. Situational: Only occurs with certain types of stimulation, situations, or partners. Specify current severity: Mild: Evidence of mild distress over the symptoms in Criterion A. Moderate: Evidence of moderate distress over the symptoms in Criterion A. Severe: Evidence of severe or extreme distress over the symptoms in Criterion A.

1	Moderate: Evidence of moderate distress over the symptoms in Criterion A. Severe: Evidence of severe or extreme distress over the symptoms in Criterion A. The essential feature of erectile disorder is the repeated failure to obtain or maintain erec- tions during partnered sexual activities (Criterion A). A careful sexual history is necessary to ascertain that the problem has been present for a significant duration of time (i.e., at least approximately 6 months) and occurs on the majority of sexual occasions (i.e., at least 75% of the time). Symptoms may occur only in specific situations involving certain types of stimulation or partners, or they may occur in a generalized manner in all types of situa- tions, stimulation, or partners.

1	Many men with erectile disorder may have low self—esteem, low self—confidence, and a de- creased sense of masculinity, and may experience depressed affect. Fear and/ or avoid- ance of future sexual encounters may occur. Decreased sexual satisfaction and reduced sexual desire in the individual’s partner are common.

1	In addition to the subtypes ”lifelong/acquired” and “generalized/ situational,” the fol- lowing five factors must be considered during assessment and diagnosis of erectile disorder given that they may be relevant to etiology and/ or treatment: 1) partner factors (e.g., part- ner’s sexual problems, partner’s health status); 2) relationship factors (e.g., poor communi- cation, discrepancies in desire for sexual activity); 3) individual vulnerability factors (e.g., poor body image, history of sexual or emotional abuse), psychiatric comorbidity (e.g., de- pression, anxiety), or stressors (e.g., job loss, bereavement); 4) cultural/religious factors (e.g., inhibitions related to prohibitions against sexual activity; attitudes toward sexuality); and 5) medical factors relevant to prognosis, course, or treatment. Each of these factors may con- tribute differently to the presenting symptoms of different men with this disorder.

1	The prevalence of lifelong versus acquired erectile disorder is unknown. There is a strong age-related increase in both prevalence and incidence of problems with erection, particu- larly after age 50 years. Approximately 13%—21% of men ages 40—80 years complain of oc— casional problems with erections. Approximately 2% of men younger than age 40—50 years complain of frequent problems with erections, whereas 40%—50% of men older than 60—70 years may have significant problems with erections. About 20% of men fear erectile prob- lems on their first sexual experience, whereas approximately 8% experienced erectile prob- lems that hindered penetration during their first sexual experience.

1	Erectile failure on first sexual attempt has been found to be related to having sex with a previously unknown partner, concomitant use of drugs or alcohol, not wanting to have sex, and peer pressure. There is minimal evi—2t most of these problems spontaneously re- mit without professional intervention, but some men may continue to have episodic prob- lems. In contrast, acquired erectile disorder is often associated with biological factors such as diabetes and cardiovascular disease. Acquired erectile disorder is likely to be persistent in most men.

1	The natural history of lifelong erectile disorder is unknown. Clinical observation sup- ports the association of lifelong erectile disorder with psychological factors that are self- limiting or responsive to psychological interventions, whereas, as noted above, acquired erectile disorder is more likely to be related to biological factors and to be persistent. The incidence of erectile disorder increases with age. A minority of men diagnosed as having moderate erectile failure may experience spontaneous remission of symptoms without medical intervention. Distress associated with erectile disorder is lower in older men as compared with younger men.

1	Temperamental. Neurotic personality traits may be associated with erectile problems in col- lege students, and submissive personality traits may be associated with erectile problems in men age 40 years and older. Alexithymiu (i.e., deficits in cognitive processing of emotions) is common in men diagnosed with "psychogenic” erectile dysfunction. Erectile problems are common in men diagnosed with depression and posttraumatic stress disorder. Course modifiers. Risk factors for acquired erectile disorder include age, smoking to- bacco, lack of physical exercise, diabetes, and decreased desire. Complaints of erectile disorder have been found to vary across countries. It is unclear to what extent these differences represent differences in cultural expectations as opposed to genuine differences in the frequency of erectile failure.

1	employed to help differentiate organic from psychogenic erectile problems on the as- ical etiology to the problem. A number of other diagnostic procedures may be employed depending on the clinician’s assessment of their relevance given the individual’s age, co- morbid medical problems, and clinical presentation. Doppler ultrasonography and intra- vascular injection of vasoactive drugs, as well as invasive diagnostic procedures such as dynamic infusion cavernosography, can be used to assess vascular integrity. Pudendal nerve conduction studies, including somatosensory evoked potentials, can be employed when a peripheral neuropathy is suspected. In men also complaining of decreased sexual desire, serum bioavailable or free testosterone is frequently assessed to determine if the difficulty is secondary to endocrinological factors. Thyroid function may also be assessed.

1	Determination of fasting serum glucose is useful to screen for the presence of diabetes mel- litus. The assessment of serum lipids is important, as erectile disorder in men 40 years and older is predictive of the future risk of coronary artery disease. Functionai Consequences of Erectiie Disorder Erectile disorder can interfere with fertility and produce both individual and interpersonal distress. Fear and/ or avoidance of sexual encounters may interfere with the ability to de- velop intimate relationships. Nonsexual mental disorders. Major depressive disorder and erectile disorder are closely associated, and erectile disorder accompanying severe depressive disorder may occur. Normal erectile function. The differential should include consideration of normal erec- tile function in men with excessive expectations.

1	Normal erectile function. The differential should include consideration of normal erec- tile function in men with excessive expectations. Substance/medication use. Another major differential diagnosis is whether the erectile problem is secondary to substance/medication use. An onset that coincides with the be- ginning of substance/medication use and that dissipates with discontinuation of the sub- stance/medication or dose reduction is suggestive of a substance/ medication-induced sexual dysfunction.

1	Another medical condition. The most difficult aspect of the differential diagnosis of erec- tile disorder is ruling out erectile problems that are fully explained by medical factors. Such cases would not receive a diagnosis of a mental disorder. The distinction between erectile disorder as a mental disorder and erectile dysfunction as the result of another medical con- dition is usually unclear, and many cases will have complex, interactive biological and psy- chiatric etiologies. If the individual is older than 40—50 years and/ or has concomitant medical problems, the differential diagnosis should include medical etiologies, especially vascular disease. The presence of an organic disease known to cause erectile problems does not confirm a causal relationship. For example, a man with diabetes mellitus can develop erectile disorder in response to psychological stress. In general, erectile dysfunction due to organic factors is generalized and gradual in onset. An exception would be

1	diabetes mellitus can develop erectile disorder in response to psychological stress. In general, erectile dysfunction due to organic factors is generalized and gradual in onset. An exception would be erectile problems after traumatic injury to the nervous innervation of the genital organs (e.g., spinal cord injury).

1	Erectile problems that are situational and inconsistent and that have an acute onset after a stressful life event are most often due to psychological events. An age of less than 40 years is also suggestive of a psychological etiology to the difficulty. Other sexual dysfunctions. Erectile disorder may coexist with premature (early) ejacu- lation and male hypoactive sexual desire disorder. Erectile disorder can be comorbid with other sexual diagnoses, such as premature (early) ejaculation and male hypoactive sexual desire disorder, as well as with anxiety and de- pressive disorders. Erectile disorder is common in men with lower urinary tract symptoms related to prostatic hypertrophy. Erectile disorder may be comorbid with dyslipidemia, car- diovascular disease, hypogonadism, multiple sclerosis, diabetes mellitus, and other diseases that interfere with the vascular, neurological, or endocrine function necessary for normal erectile function.

1	Relationship to International Classification of Diseases Erectile response is coded as failure of genital response in ICD-lO (F22). Diagnostic Criteria 302.73 (F52.31) A. Presence of either of the following symptoms and experienced on almost all or all (ap- proximately 75%—100%) occasions of sexual activity (in identified situational contexts or, if generalized, in all contexts): 1. Marked delay in, marked infrequency of, or absence of orgasm. 2. Markedly reduced intensity of orgasmic sensations. B. The symptoms in Criterion A have persisted tor a minimum duration of approximately 6 months. C. The symptoms in Criterion A cause clinically significant distress in the individual. D. The sexual dysfunction is not better explained by a nonsexual mental disorder or as a consequence of severe relationship distress (e.g., partner violence) or other significant stressors and is not attributable to the effects of a substance/medication or another medical condition. Specify whether:

1	Specify whether: Lifelong: The disturbance has been present since the individual became sexually active. Acquired: The disturbance began after a period of relatively normal sexual function. Specify whether: Generalized: Not limited to certain types of stimulation, situations, or partners. Situational: Only occurs with certain types of stimulation, situations, or partners. Specify it: Never experienced an orgasm under any situation. Specify current severity: Mild: Evidence of mild distress over the symptoms in Criterion A. Moderate: Evidence of moderate distress over the symptoms in Criterion A. Severe: Evidence of severe or extreme distress over the symptoms in Criterion A.

1	Moderate: Evidence of moderate distress over the symptoms in Criterion A. Severe: Evidence of severe or extreme distress over the symptoms in Criterion A. Female orgasmic disorder is characterized by difficulty experiencing orgasm and / or markedly reduced intensity of orgasmic sensations (Criterion A). Women show wide vari- ability in the type or intensity of stimulation that elicits orgasm. Similarly, subjective descrip- tions of orgasm are extremely varied, suggesting that it is experienced in very different ways, both across women and on different occasions by the same woman. For a diagnosis of female orgasmic disorder, symptoms must be experienced on almost all or all (approx- imately 75%—100%) occasions of sexual activity (in identified situational contexts or, if generalized, in all contexts) and have a minimum duration of approximately 6 months.

1	The use of the minimum severity and duration criteria is intended to distinguish transient orgasm difficulties from more persistent orgasmic dysfunction. The inclusion of ”approx- does not meet the recommended 6-month threshold. For a woman to have a diagnosis of female orgasmic disorder, clinically significant dis- tress must accompany the symptoms (Criterion C). In many cases of orgasm problems, the causes are multifactorial or cannot be determined. If female orgasmic disorder is deemed to be better explained by another mental disorder, the effects of a substance/ medication, or a medical condition, then a diagnosis of female orgasmic disorder would not be made. Finally, if interpersonal or significant contextual factors, such as severe relationship dis- tress, intimate partner violence, or other significant stressors, are present, then a diagnosis of female orgasmic disorder would not be made.

1	Many women require clitoral stimulation to reach orgasm, and a relatively small pro- portion of women report that they always experience orgasm during penile-vaginal inter- course. Thus, a woman’s experiencing orgasm through clitoral stimulation but not during intercourse does not meet criteria for a clinical diagnosis of female orgasmic disorder. It is also important to consider whether orgasmic difficulties are the result of inadequate sex- ual stimulation; in these cases, there may still be a need for care, but a diagnosis of female orgasmic disorder would not be made.

1	Associations between specific patterns of personality traits or psychopathology and orgas- mic dysfunction have generally not been supported. Compared with women without the disorder, some women with female orgasmic disorder may have greater difficulty com- municating about sexual issues. Overall sexual satisfaction, however, is not strongly cor- related with orgasmic experience. Many women report high levels of sexual satisfaction despite rarely or never experiencing orgasm. Orgasmic difficulties in women often co- occur with problems related to sexual interest and arousal.

1	In addition to the subtypes "lifelong/ acquired” and ”generalized / situational,” the fol- lowing five factors must be considered during assessment and diagnosis of female orgas- mic disorder given that they may be relevant to etiology and/ or treatment: 1) partner factors (e.g., partner’s sexual problems, partner’s health status); 2) relationship factors (e.g., poor communication, discrepancies in desire for sexual activity); 3) individual vul- nerability factors (e.g., poor body image, history of sexual or emotional abuse), psychiatric comorbidity (e.g., depression, anxiety), or stressors (e.g., job loss, bereavement); (4) cul- tural/religious factors (e.g., inhibitions related to prohibitions against sexual activity; attitudes toward sexuality); and 5) medical factors relevant to prognosis, course, or treat- ment. Each of these factors may contribute differently to the presenting symptoms of dif- ferent women with this disorder.

1	Reported prevalence rates for female orgasmic problems in women vary widely, from 10% to 42%, depending on multiple factors (e.g., age, culture, duration, and severity of symp- toms); however, these estimates do not take into account the presence of distress. Only a proportion of women experiencing orgasm difficulties also report associated distress. Variation in how symptoms are assessed (e.g., the duration of symptoms and the recall pe- riod) also inﬂuence prevalence rates. Approximately 10% of women do not experience or- gasm throughout their lifetime. By definition, lifelong female orgasmic disorder indicates that the orgasmic difficulties have always been present, whereas the acquired subtype would be assigned if the woman’s or- gasmic difficulties developed after a period of normal orgasmic functioning.

1	A woman’s first experience of orgasm can occur any time from the prepubertal period to well into adulthood. Women show a more variable pattern in age at first orgasm than do men, and women’s reports of having experienced orgasm increase with age. Many women learn to experience orgasm as they experience a wide variety of stimulation and acquire more knowledge about their bodies. Women’s rates of orgasm consistency (defined as ”usually or always” experiencing orgasm) are higher during masturbation than during sexual activity with a partner. Temperamental. A wide range of psychological factors, such as anxiety and concerns about pregnancy, can potentially interfere with a woman’s ability to experience orgasm.

1	Temperamental. A wide range of psychological factors, such as anxiety and concerns about pregnancy, can potentially interfere with a woman’s ability to experience orgasm. Environmental. There is a strong association between relationship problems, physical health, and mental health and orgasm difficulties in women. Sociocultural factors (e.g., gender role expectations and religious norms) are also important inﬂuences on the expe- rience of orgasmic difficulties.

1	Genetic and physiological. Many physiological factors may inﬂuence a woman’s expe- rience of orgasm, including medical conditions and medications. Conditions such as mul- tiple sclerosis, pelvic nerve damage from radical hysterectomy, and spinal cord injury can all inﬂuence orgasmic functioning in women. Selective serotonin reuptake inhibitors are known to delay or inhibit orgasm in women. Women with vulvovaginal atrophy (charac- terized by symptoms such as vaginal dryness, itching, and pain) are significantly more likely to report orgasm difficulties than are women without this condition. Menopausal status is not consistently associated with the likelihood of orgasm difficulties. There may be a significant genetic contribution to variation in female orgasmic function. However, psychological, sociocultural, and physiological factors likely interact in complex ways to inﬂuence women’s experience of orgasm and of orgasm difficulties.

1	The degree to which lack of orgasm in women is regarded as a problem that requires treat- ment may vary depending on cultural context. In addition, women differ in how important orgasm is to their sexual satisfaction. There may be marked sociocultural and generational differences in women’s orgasmic ability. For example, the prevalence of inability to reach or- gasm has ranged from 17.7% (in Northern Europe) to 42.2% (in Southeast Asia). Although measurable physiological changes occur during female orgasm, including changes in hormones, pelvic ﬂoor musculature, and brain activation, there is significant variability in these indicators of orgasm across women. In clinical situations, the diagnosis of female orgasmic disorder is based on a woman’s self—report. Functional Consequences of Female Orgasmic Disorder

1	Functional Consequences of Female Orgasmic Disorder The functional consequences of female orgasmic disorder are unclear. Although there is a strong association between relationship problems and orgasmic difficulties in women, it is unclear whether relationship factors are risk factors for orgasmic difficulties or are conse- quences of those difficulties. Nonsexual mental disorders. Nonsexual mental disorders, such as major depressive disorder, which is characterized by markedly diminished interest or pleasure in all, or al- most all, activities, may explain female orgasmic disorder. If the orgasmic difficulties are better explained by another mental disorder, then a diagnosis of female orgasmic disorder would not be made. Substance/medication-induced sexual dysfunction. Substance/medication use may explain the orgasmic difficulties.

1	Substance/medication-induced sexual dysfunction. Substance/medication use may explain the orgasmic difficulties. Another medical condition. If the disorder is due to another medical condition (e.g., multiple sclerosis, spinal cord injury), then a diagnosis of female orgasmic disorder would not be made. Interpersonal factors. If interpersonal or significant contextual factors, such as severe relationship distress, intimate partner violence, or other significant stressors, are associ- ated with the orgasmic difficulties, then a diagnosis of female orgasmic disorder would not be made.

1	Other sexual dysfunctions. Female orgasmic disorder may occur in association with other sexual dysfunctions (e.g., female sexual interest/ arousal disorder). The presence of another sexual dysfunction does not rule out a diagnosis of female orgasmic disorder. Occasional or- gasmic difficulties that are short-term or infrequent and are not accompanied by clinically sig- nificant distress or impairment are not diagnosed as female orgasmic disorder. A diagnosis is also not appropriate if the problems are the result of inadequate sexual stimulation. Women with female orgasmic disorder may have co-occurring sexual interest/arousal difficulties. Women with diagnoses of other nonsexual mental disorders, such as major de- pressive disorder, may experience lower sexual interest/arousal, and this may indirectly increase the likelihood of orgasmic difficulties. Diagnostic Criteria 302.72 (F5222)

1	Diagnostic Criteria 302.72 (F5222) A. Lack of, or significantly reduced, sexual interest/arousal, as manifested by at least three of the following: 1. Absent/reduced interest in sexual activity. 2. Absent/reduced sexual/erotic thoughts or fantasies. 3. No/reduced initiation of sexual activity, and typically unreceptive to a partner’s at- tempts to initiate. 4. Absent/reduced sexual excitemenl/pleasure during sexual activity in almost all or or, if generalized, in all contexts). 5. Absent/reduced sexual interest/arousal in response to any internal or external sex- ual/erotic cues (e.g., written, verbal, visual). 6. Absent/reduced genital or nongenital sensations during sexual activity in almost all texts or, if generalized, in all contexts). B. The symptoms in Criterion A have persisted for a minimum duration of approximately 6 months. C. The symptoms in Criterion A cause clinically significant distress in the individual.

1	B. The symptoms in Criterion A have persisted for a minimum duration of approximately 6 months. C. The symptoms in Criterion A cause clinically significant distress in the individual. D. The sexual dysfunction is not better explained by a nonsexuai mental disorder or as a consequence of severe relationship distress (e.g., partner violence) or other significant stressors and is not attributable to the effects of a substance/medication or another medical condition. Specify whether: Lifelong: The disturbance has been present since the individual became sexually active. Acquired: The disturbance began after a period of relatively normal sexual function. Specify whether: Generalized: Not limited to certain types of stimulation, situations, or partners. Situational: Only occurs with certain types of stimulation, situations, or partners. Specify current severity: Mild: Evidence of mild distress over the symptoms in Criterion A.

1	Situational: Only occurs with certain types of stimulation, situations, or partners. Specify current severity: Mild: Evidence of mild distress over the symptoms in Criterion A. Moderate: Evidence of moderate distress over the symptoms in Criterion A. Severe: Evidence of severe or extreme distress over the symptoms in Criterion A. In assessing female sexual interest/arousal disorder, interpersonal context must be taken into account. A "desire discrepancy," in which a woman has lower desire for sexual activ- ity than her partner, is not sufficient to diagnose female sexual interest/arousal disorder.

1	In order for the criteria for the disorder to be met, there must be absence or reduced fre- quency or intensity of at least three of six indicators (Criterion A) for a minimum duration of approximately 6 months (Criterion B). There may be different symptom profiles across women, as well as variability in how sexual interest and arousal are expressed. For exam- ple, in one woman, sexual interest/arousal disorder may be expressed as a lack of interest in sexual activity, an absence of erotic or sexual thoughts, and reluctance to initiate sexual activity and respond to a partner’s sexual invitations. In another woman, an inability to be- come sexually excited, to respond to sexual stimuli with sexual desire, and a correspond- ing lack of signs of physical sexual arousal may be the primary features. Because sexual desire and arousal frequently coexist and are elicited in response to adequate sexual cues, the criteria for female sexual interest/arousal disorder take into account that

1	features. Because sexual desire and arousal frequently coexist and are elicited in response to adequate sexual cues, the criteria for female sexual interest/arousal disorder take into account that difficulties in desire and arousal often simultaneously characterize the complaints of women with this disorder. Short-term changes in sexual interest or arousal are common and may be adaptive responses to events in a woman’s life and do not represent a sexual dysfunction. Diagnosis of female sexual interest/arousal disorder requires a minimum duration of symptoms of approximately 6 months as a reﬂection that the symptoms must be a persistent problem.

1	The estimation of persistence may be determined by clinical judgment when a duration of 6 months cannot be ascertained precisely.

1	There may be absent or reduced frequency or intensity of interest in sexual activity (Crite- rion A1), which was previously termed hypoactive sexual desire disorder. The frequency or inten— sity of sexual and erotic thoughts or fantasies may be absent or reduced (Criterion A2). The expression of fantasies varies widely across women and may include memories of past sexual experiences. The normative decline in sexual thoughts with age should be taken into account when this criterion is being assessed. Absence or reduced frequency of initiating sexual activ- ity and of receptivity to a partner’s sexual invitations (Criterion A3) is a behaviorally focused criterion. A couple’s beliefs and preferences for sexual initiation patterns are highly relevant to the assessment of this criterion. There may be absent or reduced sexual excitement or pleasure terion A4). Lack of pleasure is a common presenting clinical complaint in women with low de- sire. Among women who report low sexual desire,

1	may be absent or reduced sexual excitement or pleasure terion A4). Lack of pleasure is a common presenting clinical complaint in women with low de- sire. Among women who report low sexual desire, there are fewer sexual or erotic cues that elicit sexual interest or arousal (i.e., there is a lack of ”responsive desire”). Assessment of the adequacy of sexual stimuli will assist in determining if there is a difficulty with responsive sex- ual desire (Criterion A5). Frequency or intensity of genital or nongenital sensations during sex- ual activity may be reduced or absent (Criterion A6). This may include reduced vaginal lubrication/vasocongestion, but because physiological measures of genital sexual response do not differentiate women who report sexual arousal concerns from those who do not, the self- report of reduced or absent genital or nongenital sensations is sufficient.

1	For a diagnosis of female sexual interest/arousal disorder to be made, clinically signif- icant distress must accompany the symptoms in Criterion A. Distress may be experienced as a result of the lack of sexual interest/arousal or as a result of significant interference in a woman's life and well-being. If a lifelong lack of sexual desire is better explained by one’s self—identification as ”asexual,” then a diagnosis of female sexual interest/arousal disor- der would not be made.

1	Female sexual interest/ arousal disorder is frequently associated with problems in experi- encing orgasm, pain experienced during sexual activity, infrequent sexual activity, and couple-level discrepancies in desire. Relationship difficulties and mood disorders are also frequently associated features of female sexual interest/ arousal disorder. Unrealistic ex- pectations and norms regarding the ”appropriate" level of sexual interest or arousal, along with poor sexual techniques and lack of information about sexuality, may also be evident in women diagnosed with female sexual interest/arousal disorder. The latter, as well as normative beliefs about gender roles, are important factors to consider.

1	In addition to the subtypes ”lifelong/acquired” and "generalized/situational,” the follow- ing five factors must be considered during assessment and diagnosis of female sexual interest/ arousal disorder given that they may be relevant to etiology and / or treatment: 1) partner fac- tors (e.g., partner’s sexual problems, partner’s health status); 2) relationship factors (e.g., poor communication, discrepancies in desire for sexual activity); 3) individual vulnerability factors (e.g., poor body image, history of sexual or emotional abuse), psychiatric comorbidity (e.g., de- pression, anxiety), or stressors (e.g., job loss, bereavement); 4) cultural/ religious factors (e.g., inhibitions related to prohibitions against sexual activity; attitudes toward sexuality); and 5) medical factors relevant to prognosis, course, or treatment. Note that each of these factors may contribute (fifferently to the presenting symptoms of different women with this disorder.

1	The prevalence of female sexual interest/arousal disorder, as defined in this manual, is unknown. The prevalence of low sexual desire and of problems with sexual arousal (with and without associated distress), as defined by DSM-IV or ICD-lO, may vary markedly in relation to age, cultural setting, duration of symptoms, and presence of distress. Regard- ing duration of symptoms, there are striking differences in prevalence estimates between short-term and persistent problems related to lack of sexual interest. When distress about sexual functioning is required, prevalence estimates are markedly lower. Some older women report less distress about low sexual desire than younger women, although sexual desire may decrease with age. By definition, lifelong female sexual interest/arousal disorder suggests that the lack of sexual interest or arousal has been present for the woman’s entire sexual life. For Criteria

1	By definition, lifelong female sexual interest/arousal disorder suggests that the lack of sexual interest or arousal has been present for the woman’s entire sexual life. For Criteria A3, A4, and A6, which assess functioning during sexual activity, a subtype of lifelong would mean presence of symptoms since the individual’s first sexual experiences. The ac- quired subtype would be assigned if the difficulties with sexual interest or arousal de- veloped after a period of nonproblematic sexual functioning. Adaptive and normative changes in sexual functioning may result from partner-related, interpersonal, or personal events and may be transient in nature. However, persistence of symptoms for approxi- mately 6 months or more would constitute a sexual dysfunction.

1	There are normative changes in sexual interest and arousal across the life span. Fur- thermore, women in relationships of longer duration are more likely to report engaging in sex despite no obvious feelings of sexual desire at the outset of a sexual encounter com- pared with women in shorter-duration relationships. Vaginal dryness in older women is related to age and menopausal status. Temperamental. Temperamental factors include negative cognitions and attitudes about sexuality and past history of mental disorders. Differences in propensity for sexual excitation and sexual inhibition may also predict the likelihood of developing sexual problems. Environmental. Environmental factors include relationship difficulties, partner sexual functioning, and developmental history, such as early relationships with caregivers and childhood stressors.

1	Environmental. Environmental factors include relationship difficulties, partner sexual functioning, and developmental history, such as early relationships with caregivers and childhood stressors. Genetic and physiological. Some medical conditions (e.g., diabetes mellitus, thyroid dysfunction) can be risk factors for female sexual interest/ arousal disorder. There appears to be a strong inﬂuence of genetic factors on vulnerability to sexual problems in women. ences between women with and without perceived lack of genital arousal. There is marked variability in prevalence rates of low desire across cultures. Lower rates of sexual desire may be more common among East Asian women compared with Euro-

1	There is marked variability in prevalence rates of low desire across cultures. Lower rates of sexual desire may be more common among East Asian women compared with Euro- Canadian women. Although the lower levels of sexual desire and arousal found in men and women from East Asian countries compared with Euro-American groups may reﬂect less interest in sex in those cultures, the possibility remains that such group differences are an artifact of the measures used to quantify desire. A judgment about whether low sexual sexual interest/arousal disorder must take into account the fact that different cultures may pathologize some behaviors and not others. By definition, the diagnosis of female sexual interest/arousal disorder is only given to women. Distressing difficulties with sexual desire in men would be considered under male hypoactive sexual desire disorder. Difficulties in sexual interest/arousal are often associated with decreased relationship sat- isfaction.

1	Difficulties in sexual interest/arousal are often associated with decreased relationship sat- isfaction. Nonsexual mental disorders. Nonsexual mental disorders, such as major depressive disorder, in which there is “markedly diminished interest or pleasure in all, or almost all, activities most of the day, nearly every day,” may explain the lack of sexual interest/ arousal. If the lack of interest or arousal is completely attributable to another mental dis- order, then a diagnosis of female sexual interest/arousal disorder would not be made. Substance/medication use. Substance or medication use may explain the lack of inter- est/arousal. Another medical condition. If the sexual symptoms are considered to be almost exclu- sively associated with the effects of another medical condition (e.g., diabetes mellitus, en- dothelial disease, thyroid dysfunction, central nervous system disease), then a diagnosis of female sexual interest/arousal disorder would not be made.

1	Interpersonal factors. If interpersonal or significant contextual factors, such as severe relationship distress, intimate partner violence, or other significant stressors, explain the sexual interest/arousal symptoms, then a diagnosis of female sexual interest/arousal dis- order would not be made. Other sexual dysfunctions. The presence of another sexual dysfunction does not rule out a diagnosis of female sexual interest/arousal disorder. It is common for women to ex- perience more than one sexual dysfunction. For example, the presence of chronic genital pain may lead to a lack of desire for the (painful) sexual activity. Lack of interest and arousal during sexual activity may impair orgasmic ability. For some women, all aspects of the sexual response may be unsatisfying and distressing.

1	Inadequate or absent sexual stimuli. When differentialdiagnoses are being considered, it is important to assess the adequacy of sexual stimuli within the woman’s sexual experi- ence. In cases where inadequate or absent sexual stimuli are contributing to the clinical pic- ture, there may be evidence for clinical care, but a sexual dysfunction diagnosis would not be made. Similarly, transient and adaptive alterations in sexual functioning that are second- ary to a significant life or personal event must be considered in the differential diagnosis.

1	extremely common. Sexual distress and dissatisfaction with sex life are also highly cor— related in women with low sexual desire. Distressing low desire is associated with depres- sion, thyroid problems, anxiety, urinary incontinence, and other medical factors. Arthritis and inﬂammatory or irritable bowel disease are also associated with sexual arousal prob- lems. Low desire appears to be comorbid with depression, sexual and physical abuse in adulthood, global mental functioning, and use of alcohol. Diagnostic Criteria 302.76 (F52.6) A. Persistent or recurrent difficulties with one (or more) of the following: 1. Vaginal penetration during intercourse. 2. Marked vulvovaginal or pelvic pain during vaginal intercourse or penetration attempts. 3. Marked fear or anxiety about vulvovaginal or pelvic pain in anticipation of, during, or as a result of vaginal penetration. 4. Marked tensing or tightening of the pelvic floor muscles during attempted vaginal penetration.

1	4. Marked tensing or tightening of the pelvic floor muscles during attempted vaginal penetration. B. The symptoms in Criterion A have persisted for a minimum duration of approximately 6 months. C. The symptoms in Criterion A cause clinically significant distress in the individual. D. The sexual dysfunction is not better explained by a nonsexual mental disorder or as a consequence of a severe relationship distress (e.g., partner violence) or other signifi- cant stressors and is not attributable to the effects of a substance/medication or an- other medical condition. Specify whether: Lifelong: The disturbance has been present since the individual became sexually active. Acquired: The disturbance began after a period of relatively normal sexual function. Specify current severity: Mild: Evidence of mild distress over the symptoms in Criterion A. Moderate: Evidence of moderate distress over the symptoms in Criterion A.

1	Specify current severity: Mild: Evidence of mild distress over the symptoms in Criterion A. Moderate: Evidence of moderate distress over the symptoms in Criterion A. Severe: Evidence of severe or extreme distress over the symptoms in Criterion A. Genito-pelvic pain/ penetration disorder refers to four commonly comorbid symptom di- mensions: 1) difficulty having intercourse, 2) genito-pelvic pain, 3) fear of pain or vaginal penetration, and 4) tension of the pelvic ﬂoor muscles (Criterion A). Because major diffi- culty in any one of these symptom dimensions is often sufficient to cause clinically sig- nificant distress, a diagnosis can be made on the basis of marked difficulty in only one symptom dimension. However, all four symptom dimensions should be assessed even if a diagnosis can be made on the basis of only one symptom dimension.

1	ability to experience vaginal penetration in any situation (e.g., intercourse, gynecological ex- aminations, tampon insertion) to the ability to easily experience penetration in one situation and but not in another. Although the most common clinical situation is when a woman is un- able to experience intercourse or penetration with a partner, difficulties in undergoing re- quired gynecological examinations may also be present. Marked vulvovaginal or pelvic pain during vaginal intercourse or penetration attempts (Criterion A2) refers to pain occurring in differ- ent locations in the genito-pelvic area. Location of pain as well as intensity should be assessed. Typically, pain can be characterized as superficial (vulvovaginal or occurring during penetra- tion) or deep (pelvic; i.e., not felt until deeper penetration). The intensity of the pain is often not linearly related to distress or interference with sexual intercourse or other sexual activities.

1	Some genito-pelvic pain only occurs when provoked (i.e., by intercourse or mechanical stim- ulation); other genito-pelvic pain may be spontaneous as well as provoked. Genito-pelvic pain can also be usefully characterized qualitatively (e.g., “burning," ”cutting,” ”shooting,” ”throb- bing”). The pain may persist for a period after intercourse is completed and may also occur during urination. Typically, the pain experienced during sexual intercourse can be reproduced during a gynecological examination.

1	Markedfear or anxiety about vulvovaginal or pelvic pain either in anticipation of, or during, or us a result of vaginal penetration (Criterion A3) is commonly reported by women who have regularly experienced pain during sexual intercourse. This "normal” reaction may lead to avoidance of sexual/intimate situations. In other cases, this marked fear does not appear to be closely related to the experience of pain but nonetheless leads to avoidance of inter- course and vaginal penetration situations. Some have described this as similar to a phobic reaction except that the phobic object may be vaginal penetration or the fear of pain.

1	Marked tensing or tightening of the pelvic ﬂoor muscles during attempted vaginal penetration (Criterion A4) can vary from reﬂexive-like spasm of the pelvic floor in response to at- tempted vaginal entry to ”normal/voluntary" muscle guarding in response to the antici- pated or the repeated experience of pain or to fear or anxiety. In the case of ”normal/ guarding” reactions, penetration may be possible under circumstances of relaxation. The characterization and assessment of pelvic ﬂoor dysfunction is often best undertaken by a specialist gynecologist or by a pelvic ﬂoor physical therapist.

1	Genito-pelvic pain/ penetration disorder is frequently associated with other sexual dysfunc- tions, particularly reduced sexual desire and interest (female sexual interest/arousal disor- der). Sometimes desire and interest are preserved in sexual situations that are not painful or do not require penetration. Even when individuals with genito—pelvic pain/penetration dis- order report sexual interest/motivation, there is often behavioral avoidance of sexual situ- ations and opportunities. Avoidance of gynecological examinations despite medical recommendations is also frequent. The pattern of avoidance is similar to that seen in phobic disorders. It is common for women who have not succeeded in having sexual intercourse to come for treatment only when they wish to conceive. Many women with genito-pelvic pain/ ten report that the symptoms significantly diminish their feelings of femininity.

1	In addition to the subtype “lifelong/acquired,” five factors should be considered dur- ing assessment and diagnosis of genito-pelvic pain/penetration disorder because they may be relevant to etiology and/ or treatment: 1) partner factors (e.g., partner’s sexual problems, partner’s health status); 2) relationship factors (e.g., poor communication, dis- crepancies in desire for sexual activity); 3) individual vulnerability factors (e.g., poor body image, history of sexual or emotional abuse), psychiatric comorbidity (e.g., depression, anxiety), or stressors (e.g., job loss, bereavement); 4) cultural/religious factors (e.g., inhi- bitions related to prohibitions against sexual activity; attitudes toward sexuality); and 5) medical factors relevant to prognosis, course, or treatment. Each of these factors may contribute differently to the presenting symptoms of different women with this disorder.

1	There are no valid physiological measures of any of the component symptom dimen- sions of genito-pelvic pain/penetration disorder. Validated psychometric inventories may be used to formally assess the pain and anxiety components related to genito-pelvic pain/ penetration disorder. The prevalence of genito-pelvic pain/ penetration disorder is unknown. However, approx- imately 15% of women in North America report recurrent pain during intercourse. Diffi- culties having intercourse appear to be a frequent referral to sexual dysfunction clinics and to specialist clinicians.

1	The development and course of genito-pelvic pain/ penetration disorder is unclear. Because women generally do not seek treatment until they experience problems in sexual functioning, it can, in general, be difficult to characterize genito—pelvic pain/penetration disorder as life- long (primary) or acquired (secondary). Although women typically come to clinical atten- tion after the initiation of sexual activity, there are often earlier clinical signs. For example, difficulty with or the avoidance of use of tampons is an important predictor of later problems.

1	Difficulties with vaginal penetration (inability or fear or pain) may not be obvious until sex- ual intercourse is attempted. Even once intercourse is attempted, the frequency of attempts may not be significant or regular. In cases where it is difficult to establish whether symptom- atology is lifelong or acquired, it is useful to determine the presence of any consistent period of successful pain-, fear-, and tension—free intercourse. If the experience of such a period can be established, then genito-pelvic pain/penetration disorder can be characterized as ac- quired. Once symptomatology is well established for a period of approximately 6 months, the probability of spontaneous and significant symptomatic remission appears to diminish.

1	Complaints related to genito-pelvic pain peak during early adulthood and in the peri- and postmenopausal period. Women with complaints about difficulty having intercourse appear to be primarily premenopausal. There may also be an increase in genito-pelvic pain—related symptoms in the postpartum period. Environmental. Sexual and / or physical abuse have often been cited as predictors of the DSM-IV-defined sexual pain disorders dyspareunia and vaginismus. This is a matter of con- troversy in the current literature. Genetic and physiological. Women experiencing superficial pain during sexual inter— course often report the onset of the pain after a history of vaginal infections. Even after the in- fections have resolved and there are no known residual physical findings, the pain persists. Pain during tampon insertion or the inability to insert tampons before any sexual contact has been attempted is an important risk factor for genit0<pelvic pain/penetration disorder.

1	Pain during tampon insertion or the inability to insert tampons before any sexual contact has been attempted is an important risk factor for genit0<pelvic pain/penetration disorder. In the past, inadequate sexual education and religious orthodoxy have often been consid- ered to be culturally related predisposing factors to the DSM-IV diagnosis of vaginismus. This perception appears to be confirmed by recent reports from Turkey, a primarily Mus- lim country, indicating a strikingly high prevalence for the disorder. However, most avail- able research, although limited in scope, does not support this notion (Lahaie et al. 2010).

1	By definition, the diagnosis of genito-pelvic pain/penetration disorder is only given to women. There is relatively new research concerning urological chronic pelvic pain syn- drome in men, suggesting that men may experience some similar problems. The research and clinical experience are not sufficiently developed yet to justify the application of this diagnosis to men. Other specified sexual dysfunction or unspecified sexual dysfunction may be diagnosed in men appearing to fit this pattern. Functional difficulties in genito-pelvic pain/penetration disorder are often associated with interference in relationship satisfaction and sometimes with the ability to conceive via penile / vaginal intercourse.

1	Another medical condition. In many instances, women with genito—pelvic pain/pene- tration disorder will also be diagnosed with another medical condition (e.g., lichen scle— rosus, endometriosis, pelvic inﬂammatory disease, vulvovaginal atrophy). In some cases, treating the medical condition may alleviate the genito-pelvic pain/penetration disorder. Much of the time, this is not the case. There are no reliable tools or diagnostic methods to allow clinicians to know whether the medical condition or genito—pelvic pain/penetration disorder is primary. Often, the associated medical conditions are difficult to diagnose and treat. For example, the increased incidence of postmenopausal pain during intercourse may sometimes be attributable to vaginal dryness or vulvovaginal atrophy associated with declining estrogen levels. The relationship, however, between vulvovaginal atrophy/ dry- ness, estrogen, and pain is not well understood.

1	Somatic symptom and related disorders. Some women with genito-pelvic pain/pene- tration disorder may also be diagnosable with somatic symptom disorder. Since both genito-pelvic pain/penetration disorder and the somatic symptom and related disorders are new diagnoses, it is not yet clear whether they can be reliably differentiated. Some women diagnosed with genito-pelvic pain/penetration disorder will also be diagnosed with a specific phobia.

1	Inadequate sexual stimuli. It is important that the clinician, in considering differential diag- noses, assess the adequacy of sexual stimuli within the woman’s sexual experience. Sexual sit- uations in which there is inadequate foreplay or arousal may lead to difficulties in penetration, pain, or avoidance. Erectile dysfunction or premature ejaculation in the male partner may result in difficulties with penetration. These conditions should be carefully assessed. In some situations, a diagnosis of genito-pelvic pain/penetration disorder may not be appropriate.

1	ties appears to be common. Comorbidity with relationship distress is also common. This is not surprising, since in Western cultures the inability to have (pain-free) intercourse with a desired partner and the avoidance of sexual opportunities may be either a contributing factor to or the result of other sexual or relationship problems. Because pelvic ﬂoor symp- toms are implicated in the diagnosis of genito-pelvic pain/penetration disorder, there is likely to be a higher prevalence of other disorders related to the pelvic ﬂoor or reproduc- tive organs (e.g., interstitial cystitis, constipation, vaginal infection, endometriosis, irrita- ble bowel syndrome). Diagnostic Criteria 302.71 (F52.0)

1	Diagnostic Criteria 302.71 (F52.0) A. Persistently or recurrently deficient (or absent) sexuaI/erotic thoughts or fantasies and desire for sexual activity. The judgment of deficiency is made by the clinician, taking into account factors that affect sexual functioning. such as age and general and socio- cultural contexts of the individual’s life. B. The symptoms in Criterion A have persisted for a minimum duration of approximately 6 months. C. The symptoms in Criterion A cause clinically significant distress in the individual. D. The sexual dysfunction is not better explained by a nonsexual mental disorder or as a consequence of severe relationship distress or other significant stressors and is not at- tributable to thes effects of a substance/medication or another medical condition. Specify whether: Lifelong: The disturbance has been present since the individual became sexually active. Acquired: The disturbance began after a period of relatively normal sexual function.

1	Specify whether: Lifelong: The disturbance has been present since the individual became sexually active. Acquired: The disturbance began after a period of relatively normal sexual function. Specify whether: Generaiized: Not limited to certain types of stimulation, situations, or partners. Situational: Only occurs with certain types of stimulation, situations, or partners. Specify current severity: Mild: Evidence of mild distress over the symptoms in Criterion A. Moderate: Evidence of moderate distress over the symptoms in Criterion A. Severe: Evidence of severe or extreme distress over the symptoms in Criterion A.

1	Moderate: Evidence of moderate distress over the symptoms in Criterion A. Severe: Evidence of severe or extreme distress over the symptoms in Criterion A. When an assessment for male hypoactive sexual desire disorder is being made, inter— personal context must be taken into account. A “desire discrepancy,” in which a man has lower desire for sexual activity than his partner, is not sufficient to diagnose male hypo- active sexual desire disorder. Both low/absent desire for sex and deficient/absent sexual thoughts or fantasies are required for a diagnosis of the disorder. There may be variation across men in how sexual desire is expressed. The lack of desire for sex and deficient/absent erotic thoughts or fantasies must be per- sistent or recurrent and must occur for a minimum duration of approximately 6 months.

1	The lack of desire for sex and deficient/absent erotic thoughts or fantasies must be per- sistent or recurrent and must occur for a minimum duration of approximately 6 months. The inclusion of this duration criterion is meant to safeguard against making a diagnosis in cases in which a man’s low sexual desire may represent an adaptive response to adverse life conditions (e.g., concern about a partner’s pregnancy when the man is considering ter- minating the relationship). The introduction of "approximately" in Criterion B allows for clinician judgment in cases in which symptom duration does not meet the recommended 6-month threshold.

1	Male hypoactive sexual desire disorder is sometimes associated with erectile and/ or ejac- ulatory concerns. For example, persistent difficulties obtaining an erection may lead a man to lose interest in sexual activity. Men with hypoactive sexual desire disorder often report that they no longer initiate sexual activity and that they are minimally receptive to a part- ner’s attempt to initiate. Sexual activities (e.g., masturbation or partnered sexual activity) may sometimes occur even in the presence of low sexual desire. Relationship-specific pref- erences regarding patterns of sexual initiation must be taken into account when making a diagnosis of male hypoactive sexual desire disorder. Although men are more likely to ini- tiate sexual activity, and thus low desire may be characterized by a pattern of non—initiation, many men may prefer to have their partner initiate sexual activity. In such situations, the man’s lack of receptivity to a partner’s initiation should be considered

1	by a pattern of non—initiation, many men may prefer to have their partner initiate sexual activity. In such situations, the man’s lack of receptivity to a partner’s initiation should be considered when evaluating low desire.

1	In addition to the subtypes ”lifelong /acquired" and "generalized / situational,” the fol- lowing five factors must be considered during assessment and diagnosis of male hypo- active sexual desire disorder given that they may be relevant to etiology and / or treatment: 1) partner factors (e.g., partner’s sexual problems, partner’s health status); 2) relationship factors (e.g., poor communication, discrepancies in desire for sexual activity); 3) individ- ual vulnerability factors (e.g., poor body image, history of sexual or emotional abuse), psy- chiatric comorbidity (e.g., depression, anxiety), or stressors (e.g., job loss, bereavement); 4) cultural/ religious factors (e.g., inhibitions related to prohibitions against sexual activity; attitudes toward sexuality); and 5) medical factors relevant to prognosis, course, or treat- ment. Each of these factors may contribute differently to the presenting symptoms of dif- ferent men with this disorder.

1	The prevalence of male hypoactive sexual desire disorder varies depending on country of origin and method of assessment. Approximately 6% of younger men (ages 18—24 years) and 41% of older men (ages 66—74 years) have problems with sexual desire. However, a persistent lack of interest in sex, lasting 6 months or more, affects only a small proportion of men ages 16—44 (1.8%). By definition, lifelong male hypoactive sexual desire disorder indicates that low or no sex- ual desire has always been present, whereas the acquired subtype would be assigned if the man’s low desire developed after a period of normal sexual desire. There is a requirement that low desire persist for approximately 6 months or more; thus, short-term changes in sexual desire should not be diagnosed as male hypoactive sexual desire disorder.

1	There is a normative age-related decline in sexual desire. Like women, men identify a variety of triggers for their sexual desire, and they describe a wide range of reasons that they choose to engage in sexual activity. Although erotic visual cues may be more potent elicitors of desire in younger men, the potency of sexual cues may decrease with age and must be considered when evaluating men for hypoactive sexual desire disorder. Temperamental. Mood and anxiety symptoms appear to be strong predictors of low de- sire in men. Up to half of men with a past history of psychiatric symptoms may have mod- erate or severe loss of desire, compared with only 15% of those without such a history. A man's feelings about himself, his perception of his partner’s sexual desire toward him, feelings of being emotionally connected, and contextual variables may all negatively (as well as positively) affect sexual desire.

1	Environmental. Alcohol use may increase the occurrence of low desire. Among gay men, self—directed homophobia, interpersonal problems, attitudes, lack of adequate sex educa- tion, and trauma resulting from early life experiences must be taken into account in ex- plaining the low desire. Social and cultural contextual factors should also be considered. Genetic and physiological. Endocrine disorders such as hyperprolactinemia signifi- cantly affect sexual desire in men. Age is a significant risk factor for low desire in men. It is unclear whether or not men with low desire also have abnormally low levels of testoster- one; however, among hypogonadal men, low desire is common. There also may be a crit- there is little effect of testosterone on men’s desire.

1	There is marked variability in prevalence rates of low desire across cultures, ranging from 12.5% in Northern European men to 28% in Southeast Asian men ages 40—80 years. Just as there are higher rates of low desire among East Asian subgroups of women, men of East Asian ancestry also have higher rates of low desire. Guilt about sex may mediate this as- sociation between East Asian ethnicity and sexual desire in men. In contrast to the classification of sexual disorders in women, desire and arousal disorders have been retained as separate constructs in men. Despite some similarities in the experi- ence of desire across men and women, and the fact that desire ﬂuctuates over time and is dependent on contextual factors, men do report a significantly higher intensity and fre- quency of sexual desire compared with women.

1	Nonsexual mental disorders. Nonsexual mental disorders, such as major depressive disorder, which is characterized by "markedly diminished interest or pleasure in all, or al- most all, activities," may explain the lack of sexual desire. If the lack of desire is better explained by another mental disorder, then a diagnosis of male hyqaoactive sexual desire disorder would not be made. Substance/medication use. Substance/medication use may explain the lack of sexual desire. Another medical condition. If the low/absent desire and deficient/absent erotic thoughts or fantasies are better explained by the effects of another medical condition (e.g., hypogo- nadism, diabetes mellitus, thyroid dysfunction, central nervous system disease), then a di- agnosis of male hypoactive sexual desire disorder would not be made.

1	Interpersonal factors. If interpersonal or significant contextual factors, such as severe relationship distress or other significant stressors, are associated with the loss of desire in the man, then a diagnosis of male hypoactive sexual desire disorder would not be made. Other sexual dysfunctions. The presence of another sexual dysfunction does not rule out a diagnosis of male hypoactive sexual desire disorder; there is some evidence that up to one-half of men with low sexual desire also have erectile difficulties, and slightly fewer may also have early ejaculation difficulties. If the man’s low desire is explained by self—identification as an asexual, then a diagnosis of male hypoactive sexual desire disorder is not made. Depression and other mental disorders, as well as endocrinological factors, are often co- morbid with male hypoactive sexual desire disorder. Diagnostic Criteria 302.75 (F52.4)

1	Depression and other mental disorders, as well as endocrinological factors, are often co- morbid with male hypoactive sexual desire disorder. Diagnostic Criteria 302.75 (F52.4) A. A persistent or recurrent pattern of ejaculation occurring during partnered sexual activ- ity within approximately 1 minute following vaginal penetration and before the individ- ual wishes it. Note: Although the diagnosis of premature (early) ejaculation may be applied to indi- viduals engaged in nonvaginal sexual activities, specific duration criteria have not been established for these activities. B. The symptom in Criterion A must have been present for at least 6 months and must be experienced on almost all or all (approximately 75%—100%) occasions of sexual activ- ity (in identified situational contexts or, if generalized, in all contexts). C. The symptom in Criterion A causes clinically significant distress in the individual.

1	C. The symptom in Criterion A causes clinically significant distress in the individual. D. The sexual dysfunction is not better explained by a nonsexual mental disorder or as a consequence of severe relationship distress or other significant stressors and is not at- tributable to the effects of a substance/medication or another medical condition. Specify whether: Lifelong: The disturbance has been present since the individual became sexually active. Acquired: The disturbance began after a period of relatively normal sexual function. Specify whether: Generalized: Not limited to certain types of stimulation, situations, or partners. Situational: Only occurs with certain types of stimulation, situations, or partners. Specify current severity: Mild: Ejaculation occurring within approximately 30 seconds to 1 minute of vaginal penetration. Moderate: Ejaculation occurring within approximately 15—30 seconds of vaginal pen- etration.

1	Mild: Ejaculation occurring within approximately 30 seconds to 1 minute of vaginal penetration. Moderate: Ejaculation occurring within approximately 15—30 seconds of vaginal pen- etration. Severe: Ejaculation occurring prior to sexual activity, at the start of sexual activity, or within approximately 15 seconds of vaginal penetration. Premature (early) ejaculation is manifested by ejaculation that occurs prior to or shortly af- ter vaginal penetration, operationalized by an individual’s estimate of ejaculatory latency (i.e., elapsed time before ejaculation) after vaginal penetration. Estimated and measured intravaginal ejaculatory latencies are highly correlated as long as the ejaculatory latency is of short duration; therefore, self—reported estimates of ejaculatory latency are sufficient for diagnostic purposes. A 60-second intravaginal ejaculatory latency time is an appropriate cutoff for the diagnosis of lifelong premature (early) ejaculation in heterosexual men.

1	There are insufficient data to determine if this duration criterion can be applied to ac- quired premature (early) ejaculation. The durational definition may apply to males of varying sexual orientations, since ejaculatory latencies appear to be similar across men of different sexual orientations and across different sexual activities. Many males with premature (early) ejaculation complain of a sense of lack of control over ejaculation and report apprehension about their anticipated inability to delay ejaculation on future sexual encounters.

1	The following factors may be relevant in the evaluation of any sexual dysfunction: 1) partner factors (e.g., partner’s sexual problems, partner’s health status); 2) relationship fac- tors (e.g., poor communication, discrepancies in desire for sexual activity); 3) individual vulnerability factors (e.g., poor body image, history of sexual or emotional abuse), psychiatric comorbidity (e.g., depression, anxiety), and stressors (e.g., job loss, bereavement); 4) cultural/ religious factors (e.g., inhibitions related to prohibitions against sexual activity; attitudes toward sexuality); and 5) medical factors relevant to prognosis, course, or treatment.

1	Estimates of the prevalence of premature (early) ejaculation vary widely depending on the definition utilized. Internationally, more than 20%-30% of men ages 18—70 years report concern about how rapidly they ejaculate. With the new definition of premature (early) ejaculation (i.e., ejaculation occurring within approximately 1 minute of vaginal penetra- tion), only 1%—3% of men would be diagnosed with the disorder. Prevalence of premature (early) ejaculation may increase with age.

1	By definition, lifelong premature (early) ejaculation starts during a male’s initial sexual ex- periences and persists thereafter. Some men may experience premature (early) ejaculation during their initial sexual encounters but gain ejaculatory control over time. It is the persis- tence of ejaculatory problems for longer than 6 months that determines the diagnosis of pre— mature (early) ejaculation. In contrast, some men develop the disorder after a period of having a normal ejaculatory latency, known as acquired premature (early) ejaculation. There is (early) ejaculation. The acquired form likely has a later onset, usually appearing during or af— ter the fourth decade of life. Lifelong is relatively stable throughout life. Little is known about the course of acquired premature (early) ejaculation. Reversal of medical conditions such as hyperthyroidism and prostatitis appears to restore ejaculatory latencies to baseline values.

1	Lifelong premature (early) ejaculation begins with early sexual experiences and persists throughout an individual’s life. In approximately 20% of men with premature (early) ejacu- lation, ejaculatory latencies decrease further with age. Age and relationship length have been found to be negatively associated with prevalence of premature (early) ejaculation. Temperamental. Premature (early) ejaculation may be more common in men with anx- iety disorders, especially social anxiety disorder (social phobia).

1	Temperamental. Premature (early) ejaculation may be more common in men with anx- iety disorders, especially social anxiety disorder (social phobia). Genetic and physiological. There is a moderate genetic contribution to lifelong prema- ture (early) ejaculation. Premature (early) ejaculation may be associated with dopamine transporter gene polymorphism or serotonin transporter gene polymorphism. Thyroid disease, prostatitis, and drug withdrawal are associated with acquired premature (early) ejaculation. Positron emission tomography measures of regional cerebral blood ﬂow dur- ing ejaculation have shown primary activation in the mesocephalic transition zone, includ- ing the ventral tegmental area. Perception of what constitutes a normal ejaculatory latency is different in many cultures. Measured ejaculatory latencies may differ in some countries. Such differences may be ex— plained by cultural or religious factors as well as genetic differences between populations.

1	Measured ejaculatory latencies may differ in some countries. Such differences may be ex— plained by cultural or religious factors as well as genetic differences between populations. Premature (early) ejaculation is a sexual disorder in males. Males and their sexual partners may differ in their perception of what constitutes an acceptable ejaculatory latency. There may be increasing concerns in females about early ejaculation in their sexual partners, which may be a reﬂection of changing societal attitudes concerning female sexual activity. Ejaculatory latency is usually monitored in research settings by the sexual partner utilizing a timing device (e.g., stopwatch), though this is not ideal in real-life sexual situations. For vaginal intercourse, the time between intravaginal penetration and ejaculation is measured.

1	A pattern of premature (early) ejaculation may be associated with decreased self-esteem, a sense of lack of control, and adverse consequences for partner relationships. It may also cause personal distress in the sexual partner and decreased sexual satisfaction in the sexual partner. Ejaculation prior to penetration may be associated with difficulties in conception. Substance/medication-induced sexual dysfunction. When problems with premature ejaculation are due exclusively to substance use, intoxication, or withdrawal, substance/ medication-induced sexual dysfunction should be diagnosed.

1	Ejaculatory concerns that do not meet diagnostic criteria. It is necessary to identify males with normal ejaculatory latencies who desire longer ejaculatory latencies and males who have episodic premature (early) ejaculation (e.g., during the first sexual encounter with a new partner when a short ejaculatory latency may be common or normative). Neither of these situations would lead to a diagnosis of premature (early) ejaculation, even though these situations may be distressing to some males. Premature (early) ejaculation may be associated with erectile problems. In many cases, it may be difficult to determine which difficulty preceded the other. Lifelong premature (early) ejaculation may be associated with certain anxiety disorders. Acquired premature (early) ejaculation may be associated with prostatitis, thyroid disease, or drug withdrawal (e.g., during opioid withdrawal). A. A clinically significant disturbance in sexual function is predominant in the clinical picture.

1	A. A clinically significant disturbance in sexual function is predominant in the clinical picture. B. There is evidence from the history, physical examination, or laboratory findings of both (1) and (2): 1. The symptoms in Criterion A developed during or soon after substance intoxication or withdrawal or after exposure to a medication. 2. The involved substance/medication is capable of producing the symptoms in Crite- rion A. C. The disturbance is not better explained by a sexual dysfunction that is not substance/ medication-induced. Such evidence of an independent sexual dysfunction could in- clude the following:

1	C. The disturbance is not better explained by a sexual dysfunction that is not substance/ medication-induced. Such evidence of an independent sexual dysfunction could in- clude the following: The symptoms precede the onset of the substance/medication use; the symptoms persist for a substantial period of time (e.g., about 1 month) after the cessation of acute withdrawal or severe intoxication; or there is other evidence suggesting the existence of an independent non-substance/medication-induced sexual dysfunc- tion (e.g., a history of recurrent non-substance/medication-related episodes). D. The disturbance does not occur exclusively during the course of a delirium. E. The disturbance causes clinically significant distress in the individual. Note: This diagnosis should be made instead of a diagnosis of substance intoxication or substance withdrawal only when the symptoms in Criterion A predominate in the clinical picture and are sufficiently severe to warrant clinical attention.

1	Coding note: The |CD-9-CM and |CD-10-CM codes for the [specific substance/medica- tion]-induced sexual dysfunctions are indicated in the table below. Note that the |CD-10-CM code depends on whether or not there is a comorbid substance use disorder present for the same class of substance. If a mild substance use disorder is comorbid with the substance— induced sexual dysfunction, the 4th position character is “1," and the clinician should record “mild [substance] use disorder” before the substance-induced sexual dysfunction (e.g., “mild cocaine use disorder with cocaine-induced sexual dysfunction”). If a moderate or severe substance use disorder is comorbid with the substance-induced sexual dysfunction, the 4th position character is “2" and the clinician should record “moderate [substance] use disorder” or “severe [substance] use disorder," depending on the severity of the comorbid substance use disorder. If there is no comorbid substance use disorder (e.g., after a one-time heavy use

1	use disorder” or “severe [substance] use disorder," depending on the severity of the comorbid substance use disorder. If there is no comorbid substance use disorder (e.g., after a one-time heavy use of the substance), then the 4th position character is “9" and the clinician should record only the substance-induced sexual dysfunction.

1	With use disorder, Without disorder, moderate use Alcohol 291.89 F10.181 F10.281 F10.981 Opioid 292.89 F11.181 F11.281 F11.981 Sedative, hypnotic, or 292.89 F13.181 F13.281 F13.981 Amphetamine (or other 292.89 F15.181 F15.281 F15.981 Cocaine 292.89 F14.181 F14.281 F14.981 Other (or unknown) substance 292.89 F19.181 F19.281 F19.981 Specify it (see Table 1 in the chapter “Substance-Related and Addictive Disorders" for di- agnoses associated with substance class): With onset during intoxication: If the criteria are met for intoxication with the sub- stance and the symptoms develop during intoxication. With onset during withdrawal: If criteria are met for withdrawal from the substance and the symptoms develop during, or shortly after, withdrawal. With onset after medication use: Symptoms may appear either at initiation of medi- cation or after a modification or change in use. Specify current severity: Mild: Occurs on 25%—50% of occasions of sexual activity.

1	Specify current severity: Mild: Occurs on 25%—50% of occasions of sexual activity. Moderate: Occurs on 50%-75% of occasions of sexual activity. Severe: Occurs on 75% or more of occasions of sexual activity. |CD-9-CM. The name of the substance/medication-induced sexual dysfunction begins with the specific substance (e.g., alcohol, ﬂuoxetine) that is presumed to be causing the sexual dysfunction. The diagnostic code is selected from the table included in the criteria set, which is based on the drug class. For substances that do not fit into any of the classes (e.g., ﬂuoxetine), the code for ”other substance” should be used; and in cases in which a substance is judged to be an etiological factor but the specific class of substance is un- known, the category ”unknown substance” should be used.

1	The name of the disorder is followed by the specification of onset (i.e., onset during in- toxication, onset during withdrawal, with onset after medication use), followed by the se- verity specifier (e.g., mild, moderate, severe). Unlike the recording procedures for ICD-10- CM, which combine the substance-induced disorder and substance use disorder into a sin- gle code, for ICD-9-CM a separate diagnostic code is given for the substance use disorder.

1	CM, which combine the substance-induced disorder and substance use disorder into a sin- gle code, for ICD-9-CM a separate diagnostic code is given for the substance use disorder. For example, in the case of erectile dysfunction occurring during intoxication in a man with a severe alcohol use disorder, the diagnosis is 291.89 alcohol-induced sexual dysfunc- tion, with onset during intoxication, moderate. An additional diagnosis of 303.90 severe alcohol use disorder is also given. When more than one substance is judged to play a sig- nificant role in the development of the sexual dysfunction, each should be listed separately (e.g., 292.89 cocaine-induced sexual dysfunction with onset during intoxication, moderate; 292.89 ﬂuoxetine-induced sexual dysfunction, with onset after medication use).

1	|CD-10-CM. The name of the substance/medication-induced sexual dysfunction begins with the specific substance (e.g., alcohol, ﬂuoxetine) that is presumed to be causing the sexual dysfunction. The diagnostic code is selected from the table included in the criteria set, which is based on the drug class and presence or absence of a comorbid substance use disorder. For substances that do not fit into any of the classes (e.g., ﬂuoxetine), the code for ”other substance” should be used; and in cases in which a substance is judged to be an eti- ological factor but the specific class of substance is unknown, the category "unknown sub- stance” should be used.

1	When recording the name of the disorder, the comorbid substance use disorder (if any) is listed first, followed by the word “with,” followed by the name of the substance-induced sexual dysfunction, followed by the specification of onset (i.e., onset during intoxication, onset during withdrawal, with onset after medication use), followed by the severity spec- ifier (e.g., mild, moderate, severe). For example, in the case of erectile dysfunction occur- ring during intoxication in a man with a severe alcohol use disorder, the diagnosis is

1	F10.281 moderate alcohol use disorder with alcohol-induced sexual dysfunction, with on- set during intoxication, moderate. A separate diagnosis of the comorbid severe alcohol use disorder is not given. If the substance-induced sexual dysfunction occurs without a co- morbid substance use disorder (e.g., after a one-time heavy use of the substance), no ac- companying substance use disorder is noted (e.g., F15.981 amphetamine-induced sexual dysfunction, with onset during intoxication). When more than one substance is judged to play a significant role in the development of the sexual dysfunction, each should be listed separately (e.g., F14.181 mild cocaine use disorder with cocaine-induced sexual dysfunc- tion, with onset during intoxication, moderate; F19.981 ﬂuoxetine-induced sexual dys- function, with onset after medication use, moderate).

1	The major feature is a disturbance in sexual function that has a temporal relationship with substance/medication initiation, dose increase, or substance/ medication discontinuation. Sexual dysfunctions can occur in association with intoxication with the following classes of substances: alcohol; opioids; sedatives, hypnotics, or anxiolytics; stimulants (including co- caine); and other (or unknown) substances. Sexual dysfunctions can occur in association with withdrawal from the following classes of substances: alcohol; opioids; sedatives, hyp- notics, or anxiolytics; and other (or unknown) substances. Medications that can induce sex- ual dysfunctions include antidepressants, antipsychotics, and hormonal contraceptives.

1	The most commonly reported side effect of antidepressant drugs is difficulty with or- gasm or ejaculation. Problems with desire and erection are less frequent. Approximately 30% of sexual complaints are clinically significant. Certain agents, such as bupropion and mirtazapine, appear not to be associated with sexual side effects. The sexual problems associated with antipsychotic drugs, including problems with sexual desire, erection, lubrication, ejaculation, or orgasm, have occurred with typical as well as atypical agents. However, problems are less common with prolactin-sparing anti- psychotics than with agents that cause significant prolactin elevation.

1	Although the effects of mood stabilizers on sexual function are unclear, it is possible that lithium and anticonvulsants, with the possible exception of lamotrigine, have adverse effects on sexual desire. Problems with orgasm may occur with gabapentin. Similarly, there may be a higher prevalence of erectile and orgasmic problems associated with benzodiaz- epines. There have not been such reports with buspirone. Many nonpsychiatric medications, such as cardiovascular, cytotoxic, gastrointestinal, and hormonal agents, are associated with disturbances in sexual function. Illicit substance use is associated with decreased sexual desire, erectile dysfunction, and difficulty reach- ing orgasm. Sexual dysfunctions are also seen in individuals receiving methadone but are seldom reported by patients receiving buprenorphine. Chronic alcohol abuse and chronic nicotine abuse are associated with erectile problems.

1	The prevalence and the incidence of substance/medication-induced sexual dysfunction are unclear, likely because of underreporting of treatment-emergent sexual side effects. Data on substance / medication-induced sexual dysfunction typically concern the effects of antidepressant drugs. The prevalence of antidepressant-induced sexual dysfunction var- ies in part depending on the specific agent. Approximately 25%—80% of individuals taking monoamine oxidase inhibitors, tricyclic antidepressants, serotonergic antidepressants, and combined serotonergic-adrenergic antidepressants report sexual side effects. There are differences in the incidence of sexual side effects between some serotonergic and com- bined adrenergic-serotonergic antidepressants, although it is unclear if these differences are clinically significant.

1	Approximately 50% of individuals taking antipsychotic medications will experience adverse sexual side effects, including problems with sexual desire, erection, lubrication, ejaculation, or orgasm. The incidence of these side effects among different antipsychotic agents is unclear.

1	Exact prevalence and incidence of sexual dysfunctions among users of nonpsychiatric medications such as cardiovascular, cytotoxic, gastrointestinal, and hormonal agents are unknown. Elevated rates of sexual dysfunction have been reported with methadone or high-dose opioid drugs for pain. There are increased rates of decreased sexual desire, erec- tile dysfunction, and difficulty reaching orgasm associated with illicit substance use. The prevalence of sexual problems appears related to chronic drug abuse and appears higher amphetamines or 3,4—methylenedioxymethamphetamine (i.e., MDMA, ecstasy). Elevated rates of sexual dysfunction are also seen in individuals receiving methadone but are sel- dom reported by patients receiving buprenorphine. Chronic alcohol abuse and chronic nicotine abuse are related to higher rates of erectile problems.

1	The onset of antidepressant-induced sexual dysfunction may be as early as 8 days after the agent is first taken. Approximately 30% of individuals with mild to moderate orgasm de- lay will experience spontaneous remission of the dysfunction within 6 months. In some cases, serotonin reuptake inhibitor-induced sexual dysfunction may persist after the agent is discontinued. The time to onset of sexual dysfunction after initiation of antipsy- chotic drugs or drugs of abuse is unknown. It is probable that the adverse effects of nico- tine and alcohol may not appear until after years of use. Premature (early) ejaculation can sometimes occur after cessation of opioid use. There is some evidence that disturbances in sexual function related to substance/medication use increase with age. There may be an interaction among cultural factors, the inﬂuence of medications on sexual functioning, and the response of the individual to those changes.

1	There may be an interaction among cultural factors, the inﬂuence of medications on sexual functioning, and the response of the individual to those changes. Some gender differences in sexual side effects may exist. Medication-induced sexual dysfunction may result in medication noncompliance.

1	Non-substance/medication-induced sexual dysfunctions. Many mental conditions, such as depressive, bipolar, anxiety, and psychotic disorders, are associated with disturbances of sexual function. Thus, differentiating a substance/ medication-induced sexual dys— function from a manifestation of the underlying mental disorder can be quite difficult. The diagnosis is usually established if a close relationship between substance/medication ini- tiation or discontinuation is observed. A clear diagnosis can be established if the problem occurs after substance/medication initiation, dissipates with substance/medication dis- continuation, and recurs with introduction of the same agent. Most substance/medication-in- duced side effects occur shortly after initiation or discontinuation. Sexual side effects that only occur after chronic use of a substance/medication may be extremely difficult to di- agnose with certainty. 302.79 (F528)

1	302.79 (F528) This category applies to presentations in which symptoms characteristic of a sexual dys- function that cause clinically significant distress in the individual predominate but do not meet the full criteria for any of the disorders in the sexual dysfunctions diagnostic class. The other specified sexual dysfunction category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the cri- teria for any specific sexual dysfunction. This is done by recording “other specified sexual dysfunction" followed by the specific reason (e.g., “sexual aversion”). 302.70 (F52.9) This category applies to presentations in which symptoms characteristic of a sexual dys- function that cause clinically significant distress in the individual predominate but do not meet the full criteria for any of the disorders in the sexual dysfunctions diagnostic class.

1	The unspecified sexual dysfunction category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for a specific sexual dys- function, and includes presentations for which there is insufficient information to make a more specific diagnosis. "‘ . . (a‘uelidel‘I In this Chapte r, there is one overarching diagnosis of gender dysphoria, with sepa- rate developmentally appropriate criteria sets for children and for adolescents and adults.

1	The area of sex and gender is highly controversial and has led to a proliferation of terms whose meanings vary over time and within and between disciplines. An additional source of confusion is that in English ”sex" connotes both male/ female and sexuality. This chapter employs constructs and terms as they are widely used by clinicians from various disci- plines with specialization in this area. In this chapter, sex and sexual refer to the biological indicators of male and female (understood in the context of reproductive capacity), such as in sex chromosomes, gonads, sex hormones, and nonambiguous internal and external genitalia. Disorders of sex development denote conditions of inborn somatic deviations of the reproductive tract from the norm and / or discrepancies among the biological indica- tors of male and female. Cross-sex hormone treatment denotes the use of feminizing hor- the use of masculinizing hormones in an individual assigned female at birth.

1	The need to introduce the term gender arose with the realization that for individuals with conﬂicting or ambiguous biological indicators of sex (i.e., ”intersex"), the lived role in society and/ or the identification as male or female could not be uniformly associated with or predicted from the biological indicators and, later, that some individuals develop an identity as female or male at variance with their uniform set of classical biological indica- tors. Thus, gender is used to denote the public (and usually legally recognized) lived role as boy or girl, man or woman, but, in contrast to certain social constructionist theories, biolog- ical factors are seen as contributing, in interaction with social and psychological factors, to gender development. Gender assignment refers to the initial assignment as male or female.

1	This occurs usually at birth and, thereby, yields the "natal gender.” Gender—atypicul refers to somatic features or behaviors that are not typical (in a statistical sense) of individuals with the same assigned gender in a given society and historical era; for behavior, gender-noncon- forming is an alternative descriptive term. Gender reassignment denotes an official (and usu- ally legal) change of gender. Gender identity is a category of social identity and refers to an individual’s identification as male, female, or, occasionally, some category other than male or female. Gender dysphoria as a general descriptive term refers to an individual’s affective/ cognitive discontent with the assigned gender but is more specifically defined when used as a diagnostic category. Trunsgender refers to the broad spectrum of individuals who tran- siently or persistently identify with a gender different from their natal gender. Transsexual denotes an individual who seeks, or has undergone, a social

1	the broad spectrum of individuals who tran- siently or persistently identify with a gender different from their natal gender. Transsexual denotes an individual who seeks, or has undergone, a social transition from male to female or female to male, which in many, but not all, cases also involves a somatic transition by cross-sex hormone treatment and genital surgery (sex reassignment surgery).

1	Gender dysphoria refers to the distress that may accompany the incongruence between one’s experienced or expressed gender and one’s assigned gender. Although not all indi- viduals will experience distress as a result of such incongruence, many are distressed if the desired physical interventions by means of hormones and / or surgery are not available. The current term is more descriptive than the previous DSM-IV term gender identity disor- der and focuses on dysphoria as the clinical problem, not identity per se. Gender Dysphoria in Children 302.6 (F64.2) A. A marked incongruence between one’s experienced/expressed gender and assigned gender, of at least 6 months’ duration, as manifested by at least six of the following (one of which must be Criterion A1): 1. A strong desire to be of the other gender or an insistence that one is the other gen- der (or some alternative gender different from one's assigned gender).

1	2. In boys (assigned gender). a strong preference for cross-dressing or simulating fe- male attire; or in girls (assigned gender), a strong preference for wearing only typ- ical masculine clothing and a strong resistance to the wearing of typical feminine clothing. A strong preference for cross-gender roles in make-believe play or fantasy play. 4. A strong preference for the toys, games, or activities stereotypically used or en- gaged in by the other gender. 5. A strong preference for playmates of the other gender. 6. In boys (assigned gender), a strong rejection of typically masculine toys, games, and activities and a strong avoidance of rough-and-tumble play; or in girls (as- signed gender), a strong rejection of typically feminine toys, games, and activities. 7. A strong dislike of one's sexual anatomy. 8. A strong desire for the primary and/or secondary sex characteristics that match one’s experienced gender.

1	7. A strong dislike of one's sexual anatomy. 8. A strong desire for the primary and/or secondary sex characteristics that match one’s experienced gender. B. The condition is associated with clinically significant distress or impairment in social, school, or other important areas of functioning. Specify if: With a disorder of sex development (e.g., a congenital adrenogenital disorder such as 255.2 [E250] congenital adrenal hyperplasia or 259.50 [E3450] androgen insensi- tivity syndrome). Coding note: Code the disorder of sex development as well as gender dysphoria. Gender Dysphoria in Adolescents and Adults 302.85 (F64.1)

1	Coding note: Code the disorder of sex development as well as gender dysphoria. Gender Dysphoria in Adolescents and Adults 302.85 (F64.1) A. A marked incongruence between one’s experienced/expressed gender and assigned gender, of at least 6 months’ duration. as manifested by at least two of the following: 1. A marked incongruence between one’s experienced/expressed gender and pri- mary and/or secondary sex characteristics (or in young adolescents. the antici- pated secondary sex characteristics). 2. A strong desire to be rid of one’s primary and/or secondary sex characteristics be- cause of a marked incongruence with one’s experienced/expressed gender (or in young adolescents. a desire to prevent the development of the anticipated second- ary sex characteristics). 3. A strong desire for the primary and/or secondary sex characteristics of the other gender. 4. A strong desire to be of the other gender (or some alternative gender different from one's assigned gender).

1	4. A strong desire to be of the other gender (or some alternative gender different from one's assigned gender). 5. A strong desire to be treated as the other gender (or some alternative gender dif- ferent from one’s assigned gender). 6. A strong conviction that one has the typical feelings and reactions of the other gen- der (or some alternative gender different from one's assigned gender). B. The condition is associated with clinically significant distress or impairment in social, occupationalxor other important areas of functioning. Specify it: With a disorder 01 sex development (e.g., a congenital adrenogenital disorder such as 255.2 [E25.0] congenital adrenal hyperplasia or 259.50 [E3450] androgen insensi- tivity syndrome). Coding note: Code the disorder of sex development as well as gender dysphoria. Specify if:

1	Coding note: Code the disorder of sex development as well as gender dysphoria. Specify if: Posttransition: The individual has transitioned to full-time living in the desired gender (with or without legalization of gender change) and has undergone (or is preparing to have) at least one cross-sex medical procedure or treatment regimen—namely, regu- lar cross-sex hormone treatment or gender reassignment surgery confirming the desired gender (e.g., penectomy, vaginoplasty in a natal male; mastectomy or phalloplasty in a natal female). The posttransition specifier may be used in the context of continuing treatment procedures that serve to support the new gender assignment.

1	The posttransition specifier may be used in the context of continuing treatment procedures that serve to support the new gender assignment. Individuals with gender dysphoria have a marked incongruence between the gender they have been assigned to (usually at birth, referred to as natal gender) and their experienced/ expressed gender. This discrepancy is the core component of the diagnosis. There must also be evidence of distress about this incongruence. Experienced gender may include al- ternative gender identities beyond binary stereotypes. Consequently, the distress is not limited to a desire to simply be of the other gender, but may include a desire to be of an al- ternative gender, provided that it differs from the individual’s assigned gender.

1	Gender dysphoria manifests itself differently in different age groups. Prepubertal natal girls with gender dysphoria may express the wish to be a boy, assert they are a boy, or as- sert they will grow up to be a man. They prefer boys’ clothing and hairstyles, are often perceived by strangers as boys, and may ask to be called by a boy’s name. Usually, they dis- play intense negative reactions to parental attempts to have them wear dresses or other feminine attire. Some may refuse to attend school or social events where such clothes are required. These girls may demonstrate marked cross-gender identification in role-playing, dreams, and fantasies. Contact sports, rough-and-tumble play, traditional boyhood games, and boys as playmates are most often preferred. They show little interest in stereotypically feminine toys (e.g., dolls) or activities (e.g., feminine dress—up or role—play). Occasionally, they refuse to urinate in a sitting position. Some natal girls may express a desire to

1	stereotypically feminine toys (e.g., dolls) or activities (e.g., feminine dress—up or role—play). Occasionally, they refuse to urinate in a sitting position. Some natal girls may express a desire to have a penis or claim to have a penis or that they will grow one when older. They may also state that they do not want to develop breasts or menstruate.

1	Prepubertal natal boys with gender dysphoria may express the wish to be a girl or as- sert they are a girl or that they will grow up to be a woman. They have a preference for (e.g., using towels, aprons, and scarves for long hair or skirts). These children may role- play female figures (e.g., playing "mother”) and often are intensely interested in female fantasy figures. Traditional feminine activities, stereotypical games, and pastimes (e.g., ”playing house”; drawing feminine pictures; watching television or videos of favorite fe- male characters) are most often preferred. Stereotypical female-type dolls (e.g., Barbie) are often favorite toys, and girls are their preferred playmates. They avoid rough-and-tumble play and competitive sports and have little interest in stereotypically masculine toys (e.g., cars, trucks). Some may pretend not to have a penis and insist on sitting to urinate. More rarely, they may state that they find their penis or testes disgusting, that they wish them

1	toys (e.g., cars, trucks). Some may pretend not to have a penis and insist on sitting to urinate. More rarely, they may state that they find their penis or testes disgusting, that they wish them re- moved, or that they have, or wish to have, a vagina.

1	In young adolescents with gender dysphoria, clinical features may resemble those of children or adults with the condition, depending on developmental level. As secondary sex characteristics of young adolescents are not yet fully developed, these individuals may not state dislike of them, but they are concerned about imminent physical changes.

1	In adults with gender dysphoria, the discrepancy between experienced gender and physical sex characteristics is often, but not always, accompanied by a desire to be rid of primary and / or secondary sex characteristics and / or a strong desire to acquire some pri- mary and / or secondary sex characteristics of the other gender. To varying degrees, adults with gender dysphoria may adopt the behavior, clothing, and mannerisms of the experi- enced gender. They feel uncomfortable being regarded by others, or functioning in soci- ety, as members of their assigned gender. Some adults may have a strong desire to be of a different gender and treated as such, and they may have an inner certainty to feel and re- spond as the experienced gender without seeking medical treatment to alter body char- acteristics. They may find other ways to resolve the incongruence between experienced/ expressed and assigned gender by partially living in the desired role or by adopting a gen- der role neither

1	acteristics. They may find other ways to resolve the incongruence between experienced/ expressed and assigned gender by partially living in the desired role or by adopting a gen- der role neither conventionally male nor conventionally female.

1	When visible signs of puberty develop, natal boys may shave their legs at the first signs of hair growth. They sometimes bind their genitals to make erections less visible. Girls may bind their breasts, walk with a stoop, or use loose sweaters to make breasts less visible. In- creasingly, adolescents request, or may obtain without medical prescription and supervi- sion, hormonal suppressors (“blockers") of gonadal steroids (e.g., gonadotropin—releasing hormone [GnRH] analog, spironolactone). Clinically referred adolescents often want hor- mone treatment and many also wish for gender reassignment surgery. Adolescents living in an accepting environment may openly express the desire to be and be treated as the experi- enced gender and dress partly or completely as the experienced gender, have a hairstyle typ- ical of the experienced gender, preferentially seek friendships with peers of the other gender, and/ or adopt a new first name consistent with the experienced gender. Older

1	have a hairstyle typ- ical of the experienced gender, preferentially seek friendships with peers of the other gender, and/ or adopt a new first name consistent with the experienced gender. Older adolescents, when sexually active, usually do not show or allow partners to touch their sexual organs. For adults with an aversion toward their genitals, sexual activity is constrained by the preference that their genitals not be seen or touched by their partners. Some adults may seek hormone ment surgery. Others are satisfied with either hormone treatment or surgery alone.

1	Adolescents and adults with gender dysphoria before gender reassignment are at in- creased risk for suicidal ideation, suicide attempts, and suicides. After gender reassign- ment, adjustment may vary, and suicide risk may persist. For natal adult males, prevalence ranges from 0.005% to 0.014%, and for natal females, from 0.002% to 0.003%. Since not all adults seeking hormone treatment and surgical reas- signment attend specialty clinics, these rates are likely modest underestimates. Sex differ- ences in rate of referrals to specialty clinics vary by age group. In children, sex ratios of natal boys to girls range from 2:1 to 45:1. In adolescents, the sex ratio is close to parity; in adults, the sex ratio favors natal males, with ratios ranging from 1:1 to 61:1. In two coun- tries, the sex ratio appears to favor natal females (J apan: 2.2:1; Poland: 3.421).

1	Because expression of gender dysphoria varies with age, there are separate criteria sets for children versus adolescents and adults. Criteria for children are defined in a more con- crete, behavioral manner than those for adolescents and adults. Many of the core criteria boys and girls. Young children are less likely than older children, adolescents, and adults to express extreme and persistent anatomic dysphoria. In adolescents and adults, incon- gruence between experienced gender and somatic sex is a central feature of the diagnosis.

1	Factors related to distress and impairment also vary with age. A very young child may show signs of distress (e.g., intense crying) only when parents tell the child that he or she is “really" not a member of the other gender but only ”desires” to be. Distress may not be manifest in social environments supportive of the child’s desire to live in the role of the other gender and may emerge only if the desire is interfered with. In adolescents and adults, distress may manifest because of strong incongruence between experienced gender and somatic sex. Such distress may, however, be mitigated by supportive environments and knowledge that biomedical treatments exist to reduce incongruence. Impairment (e.g., school refusal, development of depression, anxiety, and substance abuse) may be a conse- quence of gender dysphoria.

1	Gender dysphoria without a disorder of sex development. For clinic-referred children, onset of cross-gender behaviors is usually between ages 2 and 4 years. This corresponds to the developmental time period in which most typically developing children begin ex- pressing gendered behaviors and interests. For some preschool-age children, both perva- sive cross-gender behaviors and the expressed desire to be the other gender may be present, or, more rarely, labeling oneself as a member of the other gender may occur. In some cases, the expressed desire to be the other gender appears later, usually at entry into elementary school. A small minority of children express discomfort with their sexual anat- omy or will state the desire to have a sexual anatomy corresponding to the experienced gender ("anatomic dysphoria”). Expressions of anatomic dysphoria become more com- mon as children with gender dysphoria approach and anticipate puberty.

1	Rates of persistence of gender dysphoria from childhood into adolescence or adulthood vary. In natal males, persistence has ranged from 2.2% to 30%. In natal females, persistence has ranged from 12% to 50%. Persistence of gender dysphoria is modestly correlated with dimensional measures of severity ascertained at the time of a childhood baseline assess- ment. In one sample of natal males, lower socioeconomic background was also modestly correlated with persistence. It is unclear if particular therapeutic approaches to gender dysphoria in children are related to rates of long-term persistence. Extant follow-up sam- ples consisted of children receiving no formal therapeutic intervention or receiving ther- apeutic interventions of various types, ranging from active efforts to reduce gender dysphoria to a more neutral, “watchful waiting” approach. It is unclear if children ”en- couraged" or supported to live socially in the desired gender will show higher rates of per- sistence, since

1	dysphoria to a more neutral, “watchful waiting” approach. It is unclear if children ”en- couraged" or supported to live socially in the desired gender will show higher rates of per- sistence, since such children have not yet been followed longitudinally in a systematic manner. For both natal male and female children showing persistence, almost all are sexually attracted to individuals of their natal sex. For natal male children whose gender dysphoria does not persist, the majority are androphilic (sexually attracted to males) and of- ten self—identify as gay or homosexual (ranging from 63% to 100%). In natal female chil- dren whose gender dysphoria does not persist, the percentage who are gynephilic (sexually attracted to females) and self—identify as lesbian is lower (ranging from 32% to 50%).

1	In both adolescent and adult natal males, there are two broad trajectories for develop- ment of gender dysphoria: early onset and late onset. EarIy-onset gender dysphoria starts in childhood and continues into adolescence and adulthood; or, there is an intermittent pe- riod in which the gender dysphoria desists and these individuals self-identify as gay or ho— mosexual, followed by recurrence of gender dysphoria. Lute—onset gender dysphoria occurs around puberty or much later in life. Some of these individuals report having had a desire to be of the other gender in childhood that was not expressed verbally to others. Others do not recall any signs of childhood gender dysphoria. For adolescent males with late-onset gender dysphoria, parents often report surprise because they did not see signs of gender dysphoria during childhood. Expressions of anatomic dysphoria are more common and salient in adolescents and adults once secondary sex characteristics have developed.

1	Adolescent and adult natal males with early-onset gender dysphoria are almost al- ways sexually attracted to men (androphilic). Adolescents and adults with late-onset gen- der dysphoria frequently engage in transvestic behavior with sexual excitement. The majority of these individuals are gynephilic or sexually attracted to other posttransition natal males with late-onset gender dysphoria. A substantial percentage of adult males with late-onset gender dysphoria cohabit with or are married to natal females. After gen- der transition, many self—identify as lesbian. Among adult natal males with gender dyspho- ria, the early-onset group seeks out clinical care for hormone treatment and reassignment surgery at an earlier age than does the late-onset group. The late-onset group may have more ﬂuctuations in the degree of gender dysphoria and be more ambivalent about and less likely satisfied after gender reassignment surgery.

1	In both adolescent and adult natal females, the most common course is the early-onset form of gender dysphoria. The late-onset form is much less common in natal females com- pared with natal males. As in natal males with gender dysphoria, there may have been a period in which the gender dysphoria desisted and these individuals self—identified as les- bian; however, with recurrence of gender dysphoria, clinical consultation is sought, often with the desire for hormone treatment and reassignment surgery. Parents of natal adoles- cent females with the late-onset form also report surprise, as no signs of childhood gender dysphoria were evident. Expressions of anatomic dysphoria are much more common and salient in adolescents and adults than in children.

1	Adolescent and adult natal females with early-onset gender dysphoria are almost always gynephilic. Adolescents and adults with the late-onset form of gender dysphoria are usually androphilic and after gender transition self-identify as gay men. Natal females with the late-onset form do not have co-occurring transvestic behavior with sexual ex- citement. Gender dysphoria in association with a disorder of sex development. Most individuals with a disorder of sex development who develop gender dysphoria have already come to medical attention at an early age. For many, starting at birth, issues of gender assignment were raised by physicians and parents. Moreover, as infertility is quite common for this group, physicians are more willing to perform cross-sex hormone treatments and genital surgery before adulthood.

1	Disorders of sex development in general are frequently associated with gender—atypi- cal behavior starting in early childhood. However, in the majority of cases, this does not lead to gender dysphoria. As individuals with a disorder of sex development become aware of their medical history and condition, many experience uncertainty about their gender, as opposed to developing a firm conviction that they are another gender. How— ever, most do not progress to gender transition. Gender dysphoria and gender transition may vary considerably as a function of a disorder of sex development, its severity, and as- signed gender. Temperamental. For individuals with gender dysphoria without a disorder of sex de- velopment, atypical gender behavior among individuals with early-onset gender dyspho- ria develops in early preschool age, and it is possible that a high degree of atypicality makes the development of gender dysphoria and its persistence into adolescence and adulthood more likely.

1	Environmental. Among individuals with gender dysphoria without a disorder of sex de- velopment, males with gender dysphoria (in both childhood and adolescence) more com- monly have older brothers than do males without the condition. Additional predisposing factors under consideration, especially in individuals with late-onset gender dysphoria (ad- olescence, adulthood), include habitual fetishistic transvestism developing into autogyne- philia (i.e., sexual arousal associated with the thought or image of oneself as a woman) and other forms of more general social, psychological, or developmental problems.

1	Genetic and physiological. For individuals with gender dysphoria without a disorder of sex development, some genetic contribution is suggested by evidence for (weak) familial- ity of transsexualism among nontwin siblings, increased concordance for transsexualism in monozygotic compared with dizygotic same-sex twins, and some degree of heritability of gender dysphoria. As to endocrine findings, no endogenous systemic abnormalities in sex-hormone levels have been found in 46,XY individuals, whereas there appear to be in- creased androgen levels (in the range found in hirsute women but far below normal male levels) in 46,XX individuals. Overall, current evidence is insufficient to label gender dys- phoria without a disorder of sex development as a form of intersexuality limited to the cen— tral nervous system.

1	In gender dysphoria associated with a disorder of sex development, the likelihood of later gender dysphoria is increased if prenatal production and utilization (via receptor sensitivity) of androgens are grossly atypical relative to what is usually seen in individuals with the same assigned gender. Examples include 46,XY individuals with a history of nor- bladder exst-rophy or penile agenesis) and who have been assigned to the female gender.

1	The likelihood of gender dysphoria is further enhanced by additional, prolonged, highly gender-atypical postnatal androgen exposure with somatic virilization as may occur in fe- male-raised and noncastrated 46,XY individuals with 5-alpha reductase-2 deficiency or 17-beta—hydroxysteroid dehydrogenase-3 deficiency or in female—raised 46,XX individuals with classical congenital adrenal hyperplasia with prolonged periods of non-adherence to glucocorticoid replacement therapy. However, the prenatal androgen milieu is more closely related to gendered behavior than to gender identity. Many individuals with dis- orders of sex development and markedly gender-atypical behavior do not develop gender dysphoria. Thus, gender-atypical behavior by itself should not be interpreted as an indi- cator of current or future gender dysphoria. There appears to be a higher rate of gender dysphoria and patient-initiated gender change from assigned female to male than from as- signed male to female in 46,XY

1	current or future gender dysphoria. There appears to be a higher rate of gender dysphoria and patient-initiated gender change from assigned female to male than from as- signed male to female in 46,XY individuals with a disorder of sex development.

1	Individuals with gender dysphoria have been reported across many countries and cul- tures. The equivalent of gender dysphoria has also been reported in individuals living in cultures with institutionalized gender categories other than male or female. It is unclear whether with these individuals the diagnostic criteria for gender dysphoria would be met. Individuals with a somatic disorder of sex development show some correlation of final gender identity outcome with the degree of prenatal androgen production and utilization. However, the correlation is not robust enough for the biological factor, where ascertain- able, to replace a detailed and comprehensive diagnostic interview evaluation for gender dysphoria. Functional Consequences of Gender Dysphoria

1	Functional Consequences of Gender Dysphoria Preoccupation with cross-gender wishes may develop at all ages after the first 2—3 years of childhood and often interfere with daily activities. In older children, failure to develop age-typical same-sex peer relationships and skills may lead to isolation from peer groups and to distress. Some children may refuse to attend school because of teasing and harass- ment or pressure to dress in attire associated with their assigned sex. Also in adolescents and adults, preoccupation with cross-gender wishes often interferes with daily activities.

1	Relationship difficulties, including sexual relationship problems, are common, and func- tioning at school or at work may be impaired. Gender dysphoria, along with atypical gender expression, is associated with high levels of stigmatization, discrimination, and victimization, leading to negative self—concept, increased rates of mental disorder comor- bidity, school dropout, and economic marginalization, including unemployment, with at- tendant social and mental health risks, especially in individuals from resource-poor family backgrounds. In addition, these individuals’ access to health services and mental health services may be impeded by structural barriers, such as institutional discomfort or inex- perience in working with this patient population.

1	Nonconformity to gender roles. Gender dysphoria should be distinguished from sim- ple nonconformity to stereotypical gender role behavior by the strong desire to be of an- other gender than the assigned one and by the extent and pervasiveness of gender-variant activities and interests. The diagnosis is not meant to merely describe nonconformity to stereotypical gender role behavior (e.g., "tomboyism” in girls, ”girly-boy” behavior in boys, occasional cross-dressing in adult men). Given the increased openness of atypical gender expressions by individuals across the entire range of the transgender spectrum, it is important that the clinical diagnosis be limited to those individuals whose distress and impairment meet the specified criteria.

1	Transvestic disorder. Transvestic disorder occurs in heterosexual (or bisexual) adoles- gender into question. It is occasionally accompanied by gender dysphoria. An individual both diagnoses. In many cases of late-onset gender dysphoria in gynephilic natal males, transvestic behavior with sexual excitement is a precursor. Body dysmorphic disorder. An individual with body dysmorphic disorder focuses on the alteration or removal of a specific body part because it is perceived as abnormally formed, not because it represents a repudiated assigned gender. When an individual’s presenta— tion meets criteria for both gender dysphoria and body dysmorphic disorder, both diag- noses can be given. Individuals wishing to have a healthy limb amputated (termed by some body integrity identity disorder) because it makes them feel more ”complete” usually do not wish to change gender, but rather desire to live as an amputee or a disabled person.

1	Schizophrenia and other psychotic disorders. In schizophrenia, there may rarely be delusions of belonging to some other gender. In the absence of psychotic symptoms, in- sistence by an individual with gender dysphoria that he or she is of some other gender is not considered a delusion. Schizophrenia (or other psychotic disorders) and gender dys- phoria may co—occur. Other clinical presentations. Some individuals with an emasculinization desire who develop an alternative, nonmale/nonfemale gender identity do have a presentation that meets criteria for gender dysphoria. However, some males seek castration and / or penec- tomy for aesthetic reasons or to remove psychological effects of androgens without chang- ing male identity; in these cases, the criteria for gender dysphoria are not met.

1	Clinically referred children with gender dysphoria show elevated levels of emotional and behavioral problems—most commonly, anxiety, disruptive and impulse—control, and de- pressive disorders. In prepubertal children, increasing age is associated with having more behavioral or emotional problems; this is related to the increasing non—acceptance of gen- der-variant behavior by others. In older children, gender-variant behavior often leads to peer ostracism, which may lead to more behavioral problems. The prevalence of mental health problems differs among cultures; these differences may also be related to differences in attitudes toward gender variance in children. However, also in some non—Western cul- tures, anxiety has been found to be relatively common in individuals with gender dysphoria, even in cultures with accepting attitudes toward gender-variant behavior. Autism spec- trum disorder is more prevalent in clinically referred children with gender dysphoria than in the general

1	even in cultures with accepting attitudes toward gender-variant behavior. Autism spec- trum disorder is more prevalent in clinically referred children with gender dysphoria than in the general population. Clinically referred adolescents with gender dysphoria appear to have comorbid mental disorders, with anxiety and depressive disorders being the most common. As in children, autism spectrum disorder is more prevalent in clinically referred adolescents with gender dysphoria than in the general population. Clinically referred adults with gender dysphoria may have coexisting mental health problems, most commonly anxiety and depressive disorders.

1	302.6 (F64.8) This category applies to presentations in which symptoms characteristic of gender dys- phoria that cause clinically significant distress or impairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for gender dys- phoria. The other specified gender dysphoria category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for gender dysphoria. This is done by recording “other specified gender dys- phoria" followed by the specific reason (e.g., “brief gender dysphoria"). An example of a presentation that can be specified using the "other specified" desig- nation is the following: The current disturbance meets symptom criteria for gender dysphoria, but the duration is less than 6 months. 302.6 (F64.9)

1	The current disturbance meets symptom criteria for gender dysphoria, but the duration is less than 6 months. 302.6 (F64.9) This category applies to presentations in which symptoms characteristic of gender dys- phoria that cause clinically significant distress or impairment in social, occupational, 0r oth- er important areas of functioning predominate but do not meet the full criteria for gender dysphoria. The unspecified gender dysphoria category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for gender dyspho- ria, and includes presentations in which there is insufficient information to make a more specific diagnosis. Disru'ptIVe, lmp'ﬂlse—Contﬂl,

1	Disru'ptIVe, lmp'ﬂlse—Contﬂl, D I S TU pIIVG, | m p U I SG-COIIIFOI, and conduct disorders include conditions involv- ing problems in the self—control of emotions and behaviors. While other disorders in DSM- 5 may also involve problems in emotional and/ or behavioral regulation, the disorders in this chapter are unique in that these problems are manifested in behaviors that violate the rights of others (e.g., aggression, destruction of property) and / or that bring the individual into significant conﬂict with societal norms or authority figures. The underlying causes of the problems in the self-control of emotions and behaviors can vary greatly across the dis- orders in this chapter and among individuals within a given diagnostic category. The chapter includes oppositional defiant disorder, intermittent explosive disorder, con- duct disorder, antisocial personality disorder (which is described in the chapter ”Personality

1	Disorders”), pyromania, kleptomania, and other specified and unspecified disruptive, im- pulse-control, and conduct disorders. Although all the disorders in the chapter involve problems in both emotional and behavioral regulation, the source of variation among the disorders is the relative emphasis on problems in the two types of self-control. For example, the criteria for conduct disorder focus largely on poorly controlled behaviors that violate the rights of others or that violate major societal norms. Many of the behavioral symptoms (e.g., aggression) can be a result of poorly controlled emotions such as anger. At the other extreme, the criteria for intermittent explosive disorder focus largely on such poorly controlled emo- tion, outbursts of anger that are disproportionate to the interpersonal or other provocation or to other psychosocial stressors. Intermediate in impact to these two disorders is opposi- tional defiant disorder, in which the criteria are more evenly distributed

1	or other provocation or to other psychosocial stressors. Intermediate in impact to these two disorders is opposi- tional defiant disorder, in which the criteria are more evenly distributed between emotions (anger and irritation) and behaviors (argumentativeness and defiance). I’yromania and kleptomania are less commonly used diagnoses characterized by poor impulse control re- lated to specific behaviors (fire setting or stealing) that relieve internal tension. Other speci- fied disruptive, impulse-control, and conduct disorder is a category for conditions in which there are symptoms of conduct disorder, oppositional defiant disorder, or other disruptive, impulse-control, and conduct disorders, but the number of symptoms does not meet the di— agnostic threshold for any of the disorders in this chapter, even though there is evidence of clinically significant impairment associated with the symptoms.

1	The disruptive, impulse-control, and conduct disorders all tend to be more common in males than in females, although the relative degree of male predominance may differ both across disorders and within a disorder at different ages. The disorders in this chapter tend to have first onset in childhood or adolescence. In fact, it is very rare for either conduct disorder or oppositional defiant disorder to first emerge in adulthood. There is a developmental relation- ship between oppositional defiant disorder and conduct disorder, in that most cases of con— duct disorder previously would have met criteria for oppositional defiant disorder, at least in those cases in which conduct disorder emerges prior to adolescence. However, most children with oppositional defiant disorder do not eventually develop conduct disorder. Furthermore, children with oppositional defiant disorder are at risk for eventually developing other prob- lems besides conduct disorder, including anxiety and depressive

1	develop conduct disorder. Furthermore, children with oppositional defiant disorder are at risk for eventually developing other prob- lems besides conduct disorder, including anxiety and depressive disorders.

1	Many of the symptoms that define the disruptive, impulse—control, and conduct disor- ders are behaviors that can occur to some degree in typically developing individuals. Thus, it is critical that the frequency, persistence, pervasiveness across situations, and im- 462 Disruptive, lmpulse-Control, and Conduct Disorders pairment associated with the behaviors indicative of the diagnosis be considered relative to what is normative for a person’s age, gender, and culture when determining if they are symptomatic of a disorder.

1	The disruptive, impulse—control, and conduct disorders have been linked to a common externalizing spectrum associated with the personality dimensions labeled as disinhibition and (inversely) constraint and, to a lesser extent, negative emotionality. These shared per- sonality dimensions could account for the high level of comorbidity among these disorders and their frequent comorbidity with substance use disorders and antisocial personality disorder. However, the specific nature of the shared diathesis that constitutes the exter- nalizing spectrum remains unknown. Diagnostic Criteria 313.81 (F91.3) A. A pattern of angryfirritable mood, argumentative/defiant behavior, or vindictiveness lasting at least 6 months as evidenced by at least four symptoms from any of the following cate— gories, and exhibited during interaction with at least one individual who is not a sibling. 1. Often loses temper. 2. Is often touchy or easily annoyed. 3. Is often angry and resentful.

1	1. Often loses temper. 2. Is often touchy or easily annoyed. 3. Is often angry and resentful. 4. Often argues with authority figures or, for children and adolescents, with adults. 5. Often actively defies or refuses to comply with requests from authority figures or with rules. 6. Often deliberately annoys others. 7. Often blames others for his or her mistakes or misbehavior. 8. Has been spiteful or vindictive at least twice within the past 6 months.

1	6. Often deliberately annoys others. 7. Often blames others for his or her mistakes or misbehavior. 8. Has been spiteful or vindictive at least twice within the past 6 months. Note: The persistence and frequency of these behaviors should be used to distinguish a behavior that is within normal limits from a behavior that is symptomatic. For children younger than 5 years, the behavior should occur on most days for a period of at least 6 months unless otherwise noted (Criterion A8). For individuals 5 years or older, the behavior should occur at least once per week for at least 6 months, unless otherwise noted (Criterion A8). While these frequency criteria provide guidance on a minimal lev- el of frequency to define symptoms, other factors should also be considered, such as whether the frequency and intensity of the behaviors are outside a range that is nor- mative for the individual’s developmental level, gender, and culture.

1	B. The disturbance in behavior is associated with distress in the individual or others in his or her immediate social context (e.g., family, peer group, work colleagues), or it impacts neg- atively on social, educational, occupational, or other important areas of functioning. C. The behaviors do not occur exclusively during the course of a psychotic, substance use. depressive, or bipolar disorder. Also, the criteria are not met for disruptive mood dysregulation disorder. Specify current severity: Miid: Symptoms are confined to only one setting (e.g., at home, at school, at work, with peers). Moderate: Some symptoms are present in at least two settings. Severe: Some symptoms are present in three or more settings. It is not uncommon for individuals with oppositional defiant disorder to show symptoms only at home and only with family members. However, the pervasiveness of the symp- toms is an indicator of the severity of the disorder.

1	The essential feature of oppositional defiant disorder is a frequent and persistent pattern of angry/irritable mood, argumentative/defiant behavior, or vindictiveness (Criterion A). It is not unusual for individuals with oppositional defiant disorder to show the behav- ioral features of the disorder without problems of negative mood. However, individuals with the disorder who show the angry/irritable mood symptoms typically show the be- havioral features as well.

1	The symptoms of oppositional defiant disorder may be confined to only one setting, and this is most frequently the home. Individuals who show enough symptoms to meet the diagnostic threshold, even if it is only at home, may be significantly impaired in their social functioning. However, in more severe cases, the symptoms of the disorder are pres- ent in multiple settings. Given that the pervasiveness of symptoms is an indicator of the severity of the disorder, it is critical that the individual’s behavior be assessed across mul- tiple settings and relationships. Because these behaviors are common among siblings, they must be observed during interactions with persons other than siblings. Also, because symptoms of the disorder are typically more evident in interactions with adults or peers whom the individual knows well, they may not be apparent during a clinical examination.

1	The symptoms of oppositional defiant disorder can occur to some degree in individu- als without this disorder. There are several key considerations for determining if the be- haviors are symptomatic of oppositional defiant disorder. First, the diagnostic threshold of four or more symptoms within the preceding 6 months must be met. Second, the per- sistence and frequency of the symptoms should exceed what is normative for an individ- ual’s age, gender, and culture. For example, it is not unusual for preschool children to show temper tantrums on a weekly basis. Temper outbursts for a preschool child would be considered a symptom of oppositional defiant disorder only if they occurred on most days for the preceding 6 months, if they occurred with at least three other symptoms of the dis- order, and if the temper outbursts contributed to the significant impairment associated with the disorder (e.g., led to destruction of property during outbursts, resulted in the child being asked to leave

1	and if the temper outbursts contributed to the significant impairment associated with the disorder (e.g., led to destruction of property during outbursts, resulted in the child being asked to leave a preschool).

1	The symptoms of the disorder often are part of a pattern of problematic interactions with others. Furthermore, individuals with this disorder typically do not regard themselves as angry, oppositional, or defiant. Instead, they often justify their behavior as a response to unreasonable demands or circumstances. Thus, it can be difficult to disentangle the rela- tive contribution of the individual with the disorder to the problematic interactions he or she experiences. For example, children with oppositional defiant disorder may have ex- perienced a history of hostile parenting, and it is often impossible to determine if the child’s behavior caused the parents to act in a more hostile manner toward the child, if the parents’ hostility led to the child’s problematic behavior, or if there was some combination of both. Whether or not the clinician can separate the relative contributions of potential causal factors should not inﬂuence whether or not the diagnosis is made. In the event that

1	some combination of both. Whether or not the clinician can separate the relative contributions of potential causal factors should not inﬂuence whether or not the diagnosis is made. In the event that the child may be living in particularly poor conditions where neglect or mistreatment may occur (e.g., in institutional settings), clinical attention to reducing the contribution of the environment may be helpful.

1	464 Disruptive, Impulse-Control, and Conduct Disorders In children and adolescents, oppositional defiant disorder is more prevalent in families in which child care is disrupted by a succession of different caregivers or in families in which harsh, inconsistent, or neglectful child-rearing practices are common. Two of the most common co-occurring conditions with oppositional defiant disorder are attention—deficit/ hyperactivity disorder (ADHD) and conduct disorder (see the section ”Comorbidity” for this disorder). Oppositional defiant disorder has been associated with increased risk for suicide attempts, even after comorbid disorders are controlled for.

1	The prevalence of oppositional defiant disorder ranges from 1% to 11%, with an average prevalence estimate of around 3.3%. The rate of oppositional defiant disorder may vary depending on the age and gender of the child. The disorder appears to be somewhat more prevalent in males than in females (1.4:1) prior to adolescence. This male predominance is not consistently found in samples of adolescents or adults.

1	The first symptoms of oppositional defiant disorder usually appear during the preschool years and rarely later than early adolescence. Oppositional defiant disorder often precedes the development of conduct disorder, especially for those with the childhood-onset type of conduct disorder. However, many children and adolescents with oppositional defiant disorder do not subsequently develop conduct disorder. Oppositional defiant disorder also conveys risk for the development of anxiety disorders and major depressive disorder, even in the absence of conduct disorder. The defiant, argumentative, and vindictive symp- toms carry most of the risk for conduct disorder, whereas the angry-irritable mood symp- toms carry most of the risk for emotional disorders.

1	Manifestations of the disorder across development appear consistent. Children and adolescents with oppositional defiant disorder are at increased risk for a number of prob— lems in adjustment as adults, including antisocial behavior, impulse-control problems, substance abuse, anxiety, and depression. Many of the behaviors associated with oppositional defiant disorder increase in fre- quency during the preschool period and in adolescence. Thus, it is especially critical dur- ing these development periods that the frequency and intensity of these behaviors be evaluated against normative levels before it is decided that they are symptoms of opposi- tional defiant disorder. Temperamental. Temperamental factors related to problems in emotional regulation (e.g., high levels of emotional reactivity, poor frustration tolerance) have been predictive of the disorder.

1	Temperamental. Temperamental factors related to problems in emotional regulation (e.g., high levels of emotional reactivity, poor frustration tolerance) have been predictive of the disorder. Environmental. Harsh, inconsistent, or neglectful child-rearing practices are common in families of children and adolescents with oppositional defiant disorder, and these parent- ing practices play an important role in many causal theories of the disorder. Genetic and physiological. A number of neurobiological markers (e.g., lower heart rate and skin conductance reactivity; reduced basal cortisol reactivity; abnormalities in the pre- frontal cortex and amygdala) have been associated with oppositional defiant disorder. However, the vast majority of studies have not separated children with oppositional de- fiant disorder from those with conduct disorder. Thus, it is unclear whether there are markers specific to oppositional defiant disorder.

1	The prevalence of the disorder in children and adolescents is relatively consistent across countries that differ in race and ethnicity. When oppositional defiant disorder is persistent throughout development, individuals with the disorder experience frequent conﬂicts with parents, teachers, supervisors, peers, and romantic partners. Such problems often result in significant impairments in the iridi- vidual’s emotional, social, academic, and occupational adjustment. Conduct disorder. Conduct disorder and oppositional defiant disorder are both related to conduct problems that bring the individual in conﬂict with adults and other authority figures (e.g., teachers, work supervisors). The behaviors of oppositional defiant disorder are typically of a less severe nature than those of conduct disorder and do not include ag- gression toward people or animals, destruction of property, or a pattern of theft or deceit.

1	Furthermore, oppositional defiant disorder includes problems of emotional dysregulation (i.e., angry and irritable mood) that are not included in the definition of conduct disorder. Attention-deficit/hyperactivity disorder. ADHD is often comorbid with oppositional de- fiant disorder. To make the additional diagnosis of oppositional defiant disorder, it is impor- tant to determine that the individual’s failure to conform to requests of others is not solely in sihiations that demand sustained effort and attention or demand that the individual sit still. Depressive and bipolar disorders. Depressive and bipolar disorders often involve neg- ative affect and irritability. As a result, a diagnosis of oppositional defiant disorder should not be made if the symptoms occur exclusively during the course of a mood disorder.

1	Disruptive mood dysregulation disorder. Oppositional defiant disorder shares with dis- ruptive mood dysregulation disorder the symptoms of chronic negative mood and temper outbursts. However, the severity, frequency, and chronicity of temper outbursts are more severe in individuals with disruptive mood dysregulation disorder than in those with oppositional defiant disorder. Thus, only a minority of children and adolescents whose symptoms meet criteria for oppositional defiant disorder would also be diagnosed with dis- ruptive mood dysregulation disorder. When the mood disturbance is severe enough to meet criteria for disruptive mood dysregulation disorder, a diagnosis of oppositional defiant dis- order is not given, even if all criteria for oppositional defiant disorder are met.

1	Intermittent explosive disorder. Intermittent explosive disorder also involves high rates of anger. However, individuals with this disorder show serious aggression toward others that is not part of the definition of oppositional defiant disorder. Intellectual disability (intellectual developmental disorder). In individuals with intel- lectual disability, a diagnosis of oppositional defiant disorder is given only if the opposi- tional behavior is markedly greater than is commonly observed among individuals of comparable mental age and with comparable severity of intellectual disability. Language disorder. Oppositional defiant disorder must also be distinguished from a failure to follow directions that is the result of impaired language comprehension (e.g., hearing loss). Social anxiety disorder (social phobia). Oppositional defiant disorder must also be dis- tinguished from defiance due to fear of negative evaluation associated with social anxiety disorder.

1	Social anxiety disorder (social phobia). Oppositional defiant disorder must also be dis- tinguished from defiance due to fear of negative evaluation associated with social anxiety disorder. 466 Disruptive, Impulse-Control, and Conduct Disorders

1	466 Disruptive, Impulse-Control, and Conduct Disorders Rates of oppositional defiant disorder are much higher in samples of children, adoles- cents, and adults with ADHD, and this may be the result of shared temperamental risk fac- tors. Also, oppositional defiant disorder often precedes conduct disorder, although this appears to be most common in children with the childhood-onset subtype. Individuals with oppositional defiant disorder are also at increased risk for anxiety disorders and ma- jor depressive disorder, and this seems largely attributable to the presence of the angry- irritable mood symptoms. Adolescents and adults with oppositional defiant disorder also show a higher rate of substance use disorders, although it is unclear if this association is in- dependent of the comorbidity with conduct disorder. Diagnostic Criteria 312.34 (F63.81)

1	Diagnostic Criteria 312.34 (F63.81) A. Recurrent behavioral outbursts representing a failure to control aggressive impulses as manifested by either of the following: 1. Verbal aggression (e.g., temper tantrums, tirades, verbal arguments or fights) or physical aggression toward property, animals. or other individuals. occurring twice weekly, on average, for a period of 3 months. The physical aggression does not re- sult in damage or destruction of property and does not result in physical injury to animals or other individuals. 2. Three behavioral outbursts involving damage or destruction of property and/or ring within a 12-month period. B. The magnitude of aggressiveness expressed during the recurrent outbursts is grossly out of proportion to the provocation or to any precipitating psychosocial stressors.

1	B. The magnitude of aggressiveness expressed during the recurrent outbursts is grossly out of proportion to the provocation or to any precipitating psychosocial stressors. C. The recurrent aggressive outbursts are not premeditated (i.e., they are impulsive and/ or anger—based) and are not committed to achieve some tangible objective (e.g., money, power, intimidation). D. The recurrent aggressive outbursts cause either marked distress in the individual or impairment in occupational or interpersonal functioning. or are associated with finan- cial or legal consequences. E. Chronological age is at least 6 years (or equivalent developmental level).

1	E. Chronological age is at least 6 years (or equivalent developmental level). F. The recurrent aggressive outbursts are not better explained by another mental disor- der (e.g., major depressive disorder, bipolar disorder, disruptive mood dysregulation disorder, a psychotic disorder, antisocial personality disorder, borderline personality disorder) and are not attributable to another medical condition (e.g., head trauma. Alz- heimer's disease) or to the physiological effects of a substance (e.g.. a drug of abuse, a medication). For children ages 6—18 years. aggressive behavior that occurs as part of an adjustment disorder should not be considered for this diagnosis.

1	Note: This diagnosis can be made in addition to the diagnosis of attention-deficit/hyper- activity disorder, conduct disorder, oppositional defiant disorder, or autism spectrum dis- order when recurrent impulsive aggressive outbursts are in excess of those usually seen in these disorders and warrant independent clinical attention. The impulsive (or anger-based) aggressive outbursts in intermittent explosive disorder have a rapid onset and, typically, little or no prodromal period. Outbursts typically last for less than 30 minutes and commonly occur in response to a minor provocation by a close intimate or associate. Individuals with intermittent explosive disorder often have less severe epi- sodes of verbal and / or nondamaging, nondestructive, or noninjurious physical assault (Cri- terion A1) in between more severe destructive /assault1ve episodes (Criterion A2). Criterion

1	A1 defines frequent (i.e., twice weekly, on average, for a period of 3 months) aggressive out- bursts characterized by temper tantrums, tirades, verbal arguments or fights, or assault without damage to objects or without injury to animals or other individuals. Criterion A2 defines infrequent (i.e., three in a 1-year period) impulsive aggressive outbursts character- ized by damaging or destroying an object, regardless of its tangible value, or by assaulting/ striking or otherwise causing physical injury to an animal or to another individual. Regard- less of the nature of the impulsive aggressive outburst, the core feature of intermittent explosive disorder is failure to control impulsive aggressive behavior in response to subjec- tively experienced provocation (i.e., psychosocial stressor) that would not typically result in an aggressive outburst (Criterion B). The aggressive outbursts are generally impulsive and/ or anger-based, rather than premeditated or instrumental (Criterion C)

1	that would not typically result in an aggressive outburst (Criterion B). The aggressive outbursts are generally impulsive and/ or anger-based, rather than premeditated or instrumental (Criterion C) and are associated with significant distress or impairment in psychosocial function (Criterion D). A diagnosis of intermittent explosive disorder should not be given to individuals younger than 6 years, or the equivalent developmental level (Criterion E), or to individuals whose aggressive out- bursts are better explained by another mental disorder (Criterion F). A diagnosis of intermit- tent explosive disorder should not be given to individuals with disruptive mood dysregulation disorder or to individuals whose impulsive aggressive outbursts are attribut- able to another medical condition or to the physiological effects of a substance (Criterion F).

1	In addition, children ages 6—18 years should not receive this diagnosis when impulsive ag- gressive outbursts occur in the context of an adjustment disorder (Criterion F). Mood disorders (unipolar), anxiety disorders, and substance use disorders are associated with intermittent explosive disorder, although onset of these disorders is typically later than that of intermittent explosive disorder. One-year prevalence data for intermittent explosive disorder in the United States is about 2.7% (narrow definition). Intermittent explosive disorder is more prevalent among younger individuals (e.g., younger than 35—40 years), compared with older individuals (older than 50 years), and in individuals with a high school education or less.

1	The onset of recurrent, problematic, impulsive aggressive behavior is most common in late childhood or adolescence and rarely begins for the first time after age 40 years. The core features of intermittent explosive disorder, typically, are persistent and continue for many years. The course of the disorder may be episodic, with recurrent periods of impulsive ag— gressive outbursts. Intermittent explosive disorder appears to follow a chronic and persis— tent course over many years. It also appears to be quite common regardless of the presence or absence of attention-deficit/hyperactivity disorder (ADHD) or disruptive, impulse- control, and conduct disorders (e.g., conduct disorder, oppositional defiant disorder). Environmental. Individuals with a history of physical and emotional trauma during the first two decades of life are at increased risk for intermittent explosive disorder. 468 Disruptive, Impulse-Control, and Conduct Disorders

1	468 Disruptive, Impulse-Control, and Conduct Disorders Genetic and physiological. First-degree relatives of individuals with intermittent ex- plosive disorder are at increased risk for intermittent explosive disorder, and twin studies have demonstrated a substantial genetic inﬂuence for impulsive aggression. Research provides neurobiological support for the presence of serotonergic abnormal- ities, globally and in the brain, specifically in areas of the limbic system (anterior cingulate) and orbitofrontal cortex in individuals with intermittent explosive disorder. Amygdala responses to anger stimuli, during functional magnetic resonance imaging scanning, are greater in individuals with intermittent explosive disorder compared with healthy indi- viduals. The lower prevalence of intermittent explosive disorder in some regions (Asia, Middle

1	The lower prevalence of intermittent explosive disorder in some regions (Asia, Middle East) or countries (Romania, Nigeria), compared with the United States, suggests that in- formation about recurrent, problematic, impulsive aggressive behaviors either is not elic- ited on questioning or is less likely to be present, because of cultural factors. In some studies the prevalence of intermittent explosive disorder is greater in males than in females (odds ratio = 1.4—2.3); other studies have found no gender difference. Social (e.g., loss of friends, relatives, marital instability), occupational (e.g., demotion, loss of employment), financial (e.g., due to value of objects destroyed), and legal (e.g., civil suits as a result of aggressive behavior against person or property; criminal charges for as- sault) problems often develop as a result of intermittent explosive disorder.

1	A diagnosis of intermittent explosive disorder should not be made when Criteria A1 and / or A2 are only met during an episode of another mental disorder (e.g., major depressive disorder, bipolar disorder, psychotic disorder), or when impulsive aggressive outbursts are attributable to another medical condition or to the physiological effects of a substance or medication. This diagnosis also should not be made, particularly in children and ado- lescents ages 6—18 years, when the impulsive aggressive outbursts occur in the context of an adjustment disorder. Other examples in which recurrent, problematic, impulsive ag- gressive outbursts may, or may not, be diagnosed as intermittent explosive disorder in- clude the following.

1	Disruptive mood dysregulation disorder. In contrast to intermittent explosive disorder, disruptive mood dysregulation disorder is characterized by a persistently negative mood state (i.e., irritability, anger) most of the day, nearly every day, between impulsive aggressive out- bursts. A diagnosis of disruptive mood dysregulation disorder can only be given when the on- set of recurrent, problematic, impulsive aggressive outbursts is before age 10 years. Finally, a diagnosis of disruptive mood dysregulation disorder should not be made for the first time after age 18 years. Otherwise, these diagnoses are mutually exclusive.

1	Antisocial personality disorder or borderline personality disorder. Individuals with an- tisocial personality disorder or borderline personality disorder often display recurrent, problematic impulsive aggressive outbursts. However, the level of impulsive aggression in individuals with antisocial personality disorder or borderline personality disorder is lower than that in individuals with intermittent explosive disorder. Delirium, major neurocognitive disorder, and personality change due to another med- ical condition, aggressive type. A diagnosis of intermittent explosive disorder should not be made when aggressive outbursts are judged to result from the physiological effects of an- other diagnosable medical condition (e.g., brain injury associated with a change in personality

1	Characterized by aggressive outbursts; complex partial epilepsy). Nonspecific abnormalities on neurological examination (e.g., ”soft signs") and nonspecific electroencephalographic changes are compatible with a diagnosis of intermittent explosive disorder unless there is a di- agnosable medical condition that better explains the impulsive aggressive outbursts. Substance intoxication or substance withdrawal. A diagnosis of intermittent explosive disorder should not be made when impulsive aggressive outbursts are nearly always as- sociated with intoxication with or withdrawal from substances (e.g., alcohol, phencyclidine, cocaine and other stimulants, barbiturates, inhalants). However, when a sufficient number of impulsive aggressive outbursts also occur in the absence of substance intoxication or withdrawal, and these warrant independent clinical attention, a diagnosis of intermittent explosive disorder may be given.

1	Attention-deficit/hyperactivity disorder, conduct disorder, oppositional defiant disor- der, or autism spectrum disorder. Individuals with any of these childhood-onset dis- orders may exhibit impulsive aggressive outbursts. Individuals with ADHD are typically impulsive and, as a result, may also exhibit impulsive aggressive outbursts. While indi- viduals with conduct disorder can exhibit impulsive aggressive outbursts, the form of ag- gression characterized by the diagnostic criteria is proactive and predatory. Aggression in oppositional defiant disorder is typically characterized by temper tantrums and verbal ar- guments with authority figures, whereas impulsive aggressive outbursts in intermittent explosive disorder are in response to a broader array of provocation and include physical assault. The level of impulsive aggression in individuals with a history of one or more of these disorders has been reported as lower than that in comparable individuals whose symptoms also meet

1	assault. The level of impulsive aggression in individuals with a history of one or more of these disorders has been reported as lower than that in comparable individuals whose symptoms also meet intermittent explosive disorder Criteria A through E. Accordingly, if

1	Criteria A through E are also met, and the impulsive aggressive outbursts warrant inde- pendent clinical attention, a diagnosis of intermittent explosive disorder may be given. Depressive disorders, anxiety disorders, and substance use disorders are most commonly comorbid with intermittent explosive disorder. In addition, individuals with antisocial personality disorder or borderline personality disorder, and individuals with a history of disorders with disruptive behaviors (e.g., ADHD, conduct disorder, oppositional defiant disorder), are at greater risk for comorbid intermittent explosive disorder. A. A repetitive and persistent pattern of behavior in which the basic rights of others or ma- jor age—appropriate societal norms or rules are violated, as manifested by the presence of at least three of the following 15 criteria in the past 12 months from any of the cate- gories below, with at least one criterion present in the past 6 months:

1	Aggression to People and Animals 1. Often bullies, threatens, or intimidates others. 2. Often initiates physical fights. 3. Has used a weapon that can cause serious physical harm to others (e.g., a bat, brick, broken bottle, knife, gun). 470 Disruptive, lmpulse-Control, and Conduct Disorders 4. Has been physically cruel to people. 5. Has been physically cruel to animals. 6. Has stolen while confronting a victim (e.g., mugging, purse snatching, extortion, armed robbery). 7. Has forced someone into sexual activity. Destruction of Property 8. Has deliberately engaged in fire setting with the intention of causing serious damage. 9. Has deliberately destroyed others‘ property (other than by fire setting). 10. Has broken into someone else's house, building, or car. 11. Often lies to obtain goods or favors or to avoid obligations (i.e., “cons” others).

1	10. Has broken into someone else's house, building, or car. 11. Often lies to obtain goods or favors or to avoid obligations (i.e., “cons” others). 12. Has stolen items of nontrivial value without confronting a victim (e.g., shoplifting, but without breaking and entering; forgery). Serious Violations of Rules 13. Often stays out at night despite parental prohibitions. beginning before age 13 years. 14. Has run away from home overnight at least twice while living in the parental or pa- rental surrogate home, or once without returning for a lengthy period. 15. Is often truant from school, beginning before age 13 years. B. The disturbance in behavior causes clinically significant impairment in social. aca- demic, or occupational functioning. C. If the individual is age 18 years or older, criteria are not met for antisocial personality disorder.

1	C. If the individual is age 18 years or older, criteria are not met for antisocial personality disorder. Specify whether: 312.81 (F91.1) Childhood-onset type: Individuals show at least one symptom char- acteristic of conduct disorder prior to age 10 years. 312.82 (F91.2) Adolescent—onset type: Individuals show no symptom characteristic of conduct disorder prior to age 10 years. 312.89 (F91.9) Unspecified onset: Criteria for a diagnosis of conduct disorder are met, but there is not enough information available to determine whether the onset of the first symptom was before or after age 10 years. Specify it:

1	Specify it: With limited prosocial emotions: To qualify for this specifier, an individual must have dis- played at least two of the following characteristics persistently over at least 12 months and in multiple relationships and settings. These characteristics reflect the individual’s typical pattern of interpersonal and emotional functioning over this period and not just occasional occurrences in some situations. Thus, to assess the criteria for the specifier, multiple infor- mation sources are necessary. In addition to the individual’s seIf-report, it is necessary to consider reports by others who have known the individual for extended periods of time (e.g., parents, teachers, co—workers, extended family members, peers).

1	Lack of remorse or guilt: Does not feel bad or guilty when he or she does some- punishment). The individual shows a general lack of concern about the negative consequences of his or her actions. For example, the individual is not remorseful after hurting someone or does not care about the consequences of breaking rules. Callous—lack of empathy: Disregards and is unconcerned about the feelings of others. The individual is described as cold and uncaring. The person appears more concerned about the effects of his or her actions on himself or herself, rather than their effects on others, even when they result in substantial harm to others. Unconcerned about performance: Does not show concern about poor/problem- atic performance at school, at work, or in other important activities. The individual does not put forth the effort necessary to perlorm well, even when expectations are clear, and typically blames others for his or her poor performance.

1	Shallow or deficient affect: Does not express feelings or show emotions to others, except in ways that seem shallow, insincere. or superficial (e.g., actions contradict the sions are used for gain (e.g., emotions displayed to manipulate or intimidate others). Specify current severity: Mild: Few if any conduct problems in excess of those required to make the diagnosis are present, and conduct problems cause relatively minor harm to others (e.g., lying, truancy, staying out after dark without permission, other rule breaking). Moderate: The number of conduct problems and the effect on others are intermediate between those specified in “mild” and those in “severe" (e.g., stealing without confront- ing a victim, vandalism). Severe: Many conduct problems in excess of those required to make the diagnosis are present, or conduct problems cause considerable harm to others (e.g., forced sex, physical cruelty, use of a weapon, stealing while confronting a victim, breaking and entering).

1	Three subtypes of conduct disorder are provided based on the age at onset of the disorder. Onset is most accurately estimated with information from both the youth and the care- giver; estimates are often 2 years later than actual onset. Both subtypes can occur in a mild, moderate, or severe form. An unspecified-onset subtype is designated when there is in- sufficient information to determine age at onset.

1	In childhood-onset conduct disorder, individuals are usually male, frequently display physical aggression toward others, have disturbed peer relationships, may have had op- positional defiant disorder during early childhood, and usually have symptoms that meet full criteria for conduct disorder prior to puberty. Many children with this subtype also opmental difficulties. Individuals with childhood-onset type are more likely to have per- sistent conduct disorder into adulthood than are those with adolescent-onset type. As compared with individuals with childhood-onset type, individuals with adolescent-onset conduct disorder are less likely to display aggressive behaviors and tend to have more normative peer relationships (although they often display conduct problems in the com- pany of others). These individuals are less likely to have conduct disorder that persists into adulthood. The ratio of males to females with conduct disorder is more balanced for the adolescent-onset type than

1	These individuals are less likely to have conduct disorder that persists into adulthood. The ratio of males to females with conduct disorder is more balanced for the adolescent-onset type than for the childhood-onset type.

1	A minority of individuals with conduct disorder exhibit characteristics that qualify for the ”with limited prosocial emotions” specifier. The indicators of this specifier are those that have often been labeled as callous and unemotional traits in research. Other personality features, such as thrill seeking, fearlessness, and insensitivity to punishment, may also dis- tinguish those with characteristics described in the specifier. Individuals with character- istics described in this specifier may be more likely than other individuals with conduct disorder to engage in aggression that is planned for instrumental gain. Individuals with conduct disorder of any subtype or any level of severity can have characteristics that qual- ify for the specifier "with limited prosocial emotions," although individuals with the spec- ifier are more likely to have childhood-onset type and a severity specifier rating of severe. 472 Disruptive, Impulse-Control, and Conduct Disorders

1	472 Disruptive, Impulse-Control, and Conduct Disorders Although the validity of self-report to assess the presence of the specifier has been sup- ported in some research contexts, individuals with conduct disorder with this specifier may not readily admit to the traits in a clinical interview. Thus, to assess the criteria for the specifier, multiple information sources are necessary. Also, because the indicators of the specifier are characteristics that reﬂect the individual’s typical pattern of interpersonal and emotional functioning, it is important to consider reports by others who have known the individual for extended periods of time and across relationships and settings (e.g., par- ents, teachers, co-workers, extended family members, peers).

1	The essential feature of conduct disorder is a repetitive and persistent pattern of behavior in which the basic rights of others or major age-appropriate societal norms or rules are vi- olated (Criterion A). These behaviors fall into four main groupings: aggressive conduct that causes or threatens physical harm to other people or animals (Criteria A1—A7); non- theft (Criteria A10—A12); and serious violations of rules (Criteria A13—A15). Three or more characteristic behaviors must have been present during the past 12 months, with at least one behavior present in the past 6 months. The disturbance in behavior causes clinically significant impairment in social, academic, or occupational functioning (Criterion B). The behavior pattern is usually present in a variety of settings, such as home, at school, or in the community. Because individuals with conduct disorder are likely to minimize their conduct problems, the clinician often must rely on additional informants. However, infor- mants’

1	at school, or in the community. Because individuals with conduct disorder are likely to minimize their conduct problems, the clinician often must rely on additional informants. However, infor- mants’ knowledge of the individual’s conduct problems may be limited if they have inad- equately supervised the individual or the individual has concealed symptom behaviors.

1	Individuals with conduct disorder often initiate aggressive behavior and react aggres- sively to others. They may display bullying, threatening, or intimidating behavior (includ- physical fights (Criterion A2); use a weapon that can cause serious physical harm (e.g., a bat, brick, broken bottle, knife, gun) (Criterion A3); be physically cruel to people (Criterion A4) or animals (Criterion A5); steal while confronting a victim (e.g., mugging, purse snatching, extortion, armed robbery) (Criterion A6); or force someone into sexual activity (Criterion A7).

1	Physical violence may take the form of rape, assault, or, in rare cases, homicide. Deliberate destruction of others’ property may include deliberate fire setting with the intention of caus- ing serious damage (Criterion A8) or deliberate destroying of other people’s property in other ways (e.g., smashing car windows, vandalizing school property) (Criterion A9). Acts of deceitfulness or theft may include breaking into someone else’s house, building, or car (Crite- rion A10); frequently lying or breaking promises to obtain goods or favors or to avoid debts or obligations (e.g., ”conning” other individuals) (Criterion All); or stealing items of non- trivial value without confronting the victim (e.g., shoplifting, forgery, fraud) (Criterion A12).

1	Individuals with conduct disorder may also frequently commit serious violations of rules (e.g., school, parental, workplace). Children with conduct disorder often have a pat- tern, beginning before age 13 years, of staying out late at night despite parental prohi- bitions (Criterion A13). Children may also show a pattern of running away from home overnight (Criterion A14). To be considered a symptom of conduct disorder, the running away must have occurred at least twice (or only once if the individual did not return for a lengthy period). Runaway episodes that occur as a direct consequence of physical or sex- ual abuse do not typically qualify for this criterion. Children with conduct disorder may often be truant from school, beginning prior to age 13 years (Criterion A15).

1	Especially in ambiguous situations, aggressive individuals with conduct disorder fre- quently misperceive the intentions of others as more hostile and threatening than is the case and respond with aggression that they then feel is reasonable and justified. Person— ality features of trait negative emotionality and poor self—control, including poor frustra- tion tolerance, irritability, temper outbursts, suspiciousness, insensitivity to punishment, thrill seeking, and recklessness, frequently co-occur with conduct disorder. Substance misuse is often an associated feature, particularly in adolescent females. Suicidal ideation, suicide attempts, and completed suicide occur at a higher-than-expected rate in individu- als with conduct disorder.

1	One-year population prevalence estimates range from 2% to more than 10%, with a median of 4%. The prevalence of conduct disorder appears to be fairly consistent across various countries that differ in race and ethnicity. Prevalence rates rise from childhood to adoles- cence and are higher among males than among females. Few children with impairing con- duct disorder receive treatment.

1	The onset of conduct disorder may occur as early as the preschool years, but the first sig- nificant symptoms usually emerge during the period from middle childhood through middle adolescence. Oppositional defiant disorder is a common precursor to the child- hood-onset type of conduct disorder. Conduct disorder may be diagnosed in adults, how- ever, symptoms of conduct disorder usually emerge in childhood or adolescence, and onset is rare after age 16 years. The course of conduct disorder after onset is variable. In a majority of individuals, the disorder remits by adulthood. Many individuals with conduct disorder—particularly those with adolescent-onset type and those with few and milder symptoms—achieve adequate social and occupational adjustment as adults. However, the early-onset type predicts a worse prognosis and an increased risk of criminal behavior, conduct disorder, and substance-related disorders in adulthood. Individuals with conduct disorder are at risk for later mood

1	predicts a worse prognosis and an increased risk of criminal behavior, conduct disorder, and substance-related disorders in adulthood. Individuals with conduct disorder are at risk for later mood disorders, anxiety disorders, posttraumatic stress dis- order, impulse—control disorders, psychotic disorders, somatic symptom disorders, and substance-related disorders as adults.

1	Symptoms of the disorder vary with age as the individual develops increased physical strength, cognitive abilities, and sexual maturity. Symptom behaviors that emerge first tend to be less serious (e.g., lying, shoplifting), whereas conduct problems that emerge last tend to be more severe (e.g., rape, theft while confronting a victim). However, there are wide differences among individuals, with some engaging in the more damaging behaviors at an early age (which is predictive of a worse prognosis). When individuals with conduct disorder reach adulthood, symptoms of aggression, property destruction, deceitfulness, and rule violation, including Violence against co-workers, partners, and children, may be ex- hibited in the workplace and the home, such that antisocial personality disorder may be considered. Temperamental. Temperamental risk factors include a difficult undercontrolled infant temperament and lower-than-average intelligence, particularly with regard to verbal IQ.

1	Temperamental. Temperamental risk factors include a difficult undercontrolled infant temperament and lower-than-average intelligence, particularly with regard to verbal IQ. Environmental. Family-level risk factors include parental rejection and neglect, inconsis— tent child-rearing practices, harsh discipline, physical or sexual abuse, lack of supervision, early institutional living, frequent changes of caregivers, large family size, parental criminal- ity, and certain kinds of familial psychopathology (e.g., substance-related disorders). Com— munity-level risk factors include peer rejection, association with a delinquent peer group, and neighborhood exposure to violence. Both types of risk factors tend to be more common and severe among individuals with the childhood-onset subtype of conduct disorder. 474 Disruptive, Impulse-Control, and Conduct Disorders

1	474 Disruptive, Impulse-Control, and Conduct Disorders Genetic and physiological. Conduct disorder is inﬂuenced by both genetic and envi- ronmental factors. The risk is increased in children with a biological or adoptive parent or a sibling with conduct disorder. The disorder also appears to be more common in children of biological parents with severe alcohol use disorder, depressive and bipolar disorders, or schizophrenia or biological parents who have a history of ADHD or conduct disorder.

1	Family history particularly characterizes individuals with the childhood-onset subtype of conduct disorder. Slower resting heart rate has been reliably noted in individuals with conduct disorder compared with those without the disorder, and this marker is not char- acteristic of any other mental disorder. Reduced autonomic fear conditioning, particularly low skin conductance, is also well documented. However, these psychophysiological find- ings are not diagnostic of the disorder. Structural and functional differences in brain areas associated with affect regulation and affect processing, particularly frontotemporal-limbic connections involving the brain’s ventral prefrontal cortex and amygdala, have been con- sistently noted in individuals with conduct disorder compared with those without the dis— order. However, neuroimaging findings are not diagnostic of the disorder.

1	Course modifiers. Persistence is more likely for individuals with behaviors that meet criteria for the childhood-onset subtype and qualify for the specifier ”with limited pro- social emotions”. The risk that conduct disorder will persist is also increased by co-occurring ADHD and by substance abuse. Conduct disorder diagnosis may at times be potentially misapplied to individuals in set- tings where patterns of disruptive behavior are viewed as near-normative (e.g., in very threatening, high-crime areas or war zones). Therefore, the context in which the undesir- able behaviors have occurred should be considered. Males with a diagnosis of conduct disorder frequently exhibit fighting, stealing, vandalism, and school discipline problems. Females with a diagnosis of conduct disorder are more likely to exhibit lying, truancy, running away, substance use, and prostitution. Whereas males tend lationships of others), females tend to exhibit relatively more relational aggression.

1	Functional Consequences of Conduct Disorder

1	Conduct disorder behaviors may lead to school suspension or expulsion, problems in work adjustment, legal difficulties, sexually transmitted diseases, unplanned pregnancy, and physical injury from accidents or fights. These problems may preclude attendance in ordinary schools or living in a parental or foster home. Conduct disorder is often associ- ated with an early onset of sexual behavior, alcohol use, tobacco smoking, use of illegal substances, and reckless and risk-taking acts. Accident rates appear to be higher among in— dividuals with conduct disorder compared with those without the disorder. These func- tional consequences of conduct disorder may predict health difficulties when individuals reach midlife. It is not uncommon for individuals with conduct disorder to come into con- tact with the criminal justice system for engaging in illegal behavior. Conduct disorder is a common reason for treatment referral and is frequently diagnosed in mental health fa- cilities for

1	con- tact with the criminal justice system for engaging in illegal behavior. Conduct disorder is a common reason for treatment referral and is frequently diagnosed in mental health fa- cilities for children, especially in forensic practice. It is associated with impairment that is more severe and chronic than that experienced by other clinic-referred children.

1	Oppositional defiant disorder. Conduct disorder and oppositional defiant disorder are both related to symptoms that bring the individual in conﬂict with adults and other au- thority figures (e.g., parents, teachers, work supervisors). The behaviors of oppositional defiant disorderare typically of a less severe nature than those of individuals with conduct disorder and do not include aggression toward individuals or animals, destruction of property, or a pattern of theft or deceit. Furthermore, oppositional defiant disorder in- cludes problems of emotional dysregulation (i.e., angry and irritable mood) that are not in- cluded in the definition of conduct disorder. When criteria are met for both oppositional defiant disorder and conduct disorder, both diagnoses can be given.

1	Attention-deficit/hyperactivity disorder. Although children with ADHD often exhibit hyperactive and impulsive behavior that may be disruptive, this behavior does not by it— self violate societal norms or the rights of others and therefore does not usually meet cri- teria for conduct disorder. When criteria are met for both ADHD and conduct disorder, both diagnoses should be given.

1	Depressive and bipolar disorders. Irritability, aggression, and conduct problems can occur in children or adolescents with a major depressive disorder, a bipolar disorder, or disruptive mood dysregulation disorder. The behaviorial problems associated with these mood disorders can usually be distinguished from the pattern of conduct problems seen in conduct disorder based on their course. Specifically, persons with conduct disorder will display substantial levels of aggressive or non-aggressive conduct problems during peri- ods in which there is no mood disturbance, either historically (i.e., a history of conduct problems predating the onset of the mood disturbance) or concurrently (i.e., display of some conduct problems that are premeditated and do not occur during periods of intense emotional arousal). In those cases in which criteria for conduct disorder and a mood dis- order are met, both diagnoses can be given.

1	Intermittent explosive disorder. Both conduct disorder and intermittent explosive dis- order involve high rates of aggression. However, the aggression in individuals with inter- mittent explosive disorder is limited to impulsive aggression and is not premeditated, and it is not committed in order to achieve some tangible objective (e.g., money, power, intim- idation). Also, the definition of intermittent explosive disorder does not include the non- aggressive symptoms of conduct disorder. If criteria for both disorders are met, the diag- nosis of intermittent explosive disorder should be given only when the recurrent impul- sive aggressive outbursts warrant independent clinical attention.

1	Adjustment disorders. The diagnosis of an adjustment disorder (with disturbance of con- duct or with mixed disturbance of emotions and conduct) should be considered if clinically significant conduct problems that do not meet the criteria for another specific disorder de- velop in clear association with the onset of a psychosocial stressor and do not resolve within 6 months of the termination of the stressor (or its consequences). Conduct disorder is diag- nosed only when the conduct problems represent a repetitive and persistent pattern that is associated with impairment in social, academic, or occupational functioning.

1	ADHD and oppositional defiant disorder are both common in individuals with conduct disorder, and this comorbid presentation predicts worse outcomes. Individuals who show the personality features associated with antisocial personality disorder often violate the basic rights of others or violate major age-appropriate societal norms, and as a result their pattern of behavior often meets criteria for conduct disorder. Conduct disorder may also co-occur with one or more of the following mental disorders: specific learning disorder, anxiety disorders, depressive or bipolar disorders, and substance-related disorders. Aca- demic achievement, particularly in reading and other verbal skills, is often below the level expected on the basis of age and intelligence and may justify the additional diagnosis of specific learning disorder or a communication disorder. 476 Disruptive, Impulse—Control, and Conduct Disorders

1	476 Disruptive, Impulse—Control, and Conduct Disorders Criteria and text for antisocial personality disorder can be found in the chapter ”Person- ality Disorders.” Because this disorder is closely connected to the spectrum of ”external- izing” conduct disorders in this chapter, as well as to the disorders in the adjoining chapter ”Substance—Related and Addictive Disorders,” it is dual coded here as well as in the chap- ter ”Personality Disorders.” Diagnostic Criteria 312.33 (F63.1) A. Deliberate and purposeful fire setting on more than one occasion. B. Tension or affective arousal before the act. C. Fascination with, interest in, curiosity about. or attraction to fire and its situational con- texts (e.g., paraphernalia, uses, consequences). D. Pleasure, gratification, or relief when setting fires or when witnessing or participating in their aftermath.

1	D. Pleasure, gratification, or relief when setting fires or when witnessing or participating in their aftermath. E. The fire setting is not done for monetary gain. as an expression of sociopolitical ideol- ogy, to conceal criminal activity, to express anger or vengeance, to improve one’s living circumstances, in response to a delusion or hallucination, or as a result of impaired judgment (e.g., in major neurocognitive disorder, intellectual disability [intellectual de- velopmental disorder], substance intoxication). F. The fire setting is not better explained by conduct disorder, a manic episode. or anti- social personality disorder.

1	The essential feature of pyromania is the presence of multiple episodes of deliberate and purposeful fire setting (Criterion A). Individuals with this disorder experience tension or at- fective arousal before setting a fire (Criterion B). There is a fascination with, interest in, cu- riosity about, or attraction to fire and its situational contexts (e.g., paraphernalia, uses, consequences) (Criterion C). Individuals with this disorder are often regular ”watchers” at fires in their neighborhoods, may set off false alarms, and derive pleasure from institutions, equipment, and personnel associated with fire. They may spend time at the local fire depart- ment, set fires to be affiliated with the fire department, or even become firefighters. Individ- uals with this disorder experience pleasure, gratification, or relief when setting the fire, witnessing its effects, or participating in its aftermath (Criterion D). The fire setting is not done for monetary gain, as an expression of

1	pleasure, gratification, or relief when setting the fire, witnessing its effects, or participating in its aftermath (Criterion D). The fire setting is not done for monetary gain, as an expression of sociopolitical ideology, to conceal criminal ac- tivity, to express anger or vengeance, to improve one’s living circumstances, or in response to a delusion or a hallucination (Criterion E). The fire setting does not result from impaired judgment (e.g., in major neurocognitive disorder or intellectual disability [intellectual devel- opmental disorder]). The diagnosis is not made if the fire setting is better explained by con- duct disorder, a manic episode, or antisocial personality disorder (Criterion F).

1	Individuals with pyromania may make considerable advance preparation for starting a fire. They may be indifferent to the consequences to life or property caused by the fire, or they may derive satisfaction from the resulting property destruction. The behaviors may lead to property damage, legal consequences, or injury or loss of life to the fire setter or to others. Individuals who impulsively set fires (who may or may not have pyromania) often have a current or past history of alcohol use disorder.

1	The population prevalence of pyromania is not known. The lifetime prevalence of fire set— ting, which is just one component of pyromania and not sufficient for a diagnosis by itself, was reported as 1.13% in a population sample, but the most common comorbidities were antisocial personality disorder, substance use disorder, bipolar disorder, and pathological gambling (gambling disorder). In contrast, pyromania as a primary diagnosis appears to be very rare. Among a sample of persons reaching the criminal system with repeated fire setting, only 3.3% had symptoms that met full criteria for pyromania. There are insufficient data to establish a typical age at onset of pyromania. The relation- mented. In individuals with pyromania, fire-setting incidents are episodic and may wax and wane in frequency. Longitudinal course is unknown. Although fire setting is a major problem in children and adolescents (over 40% of those arrested for arson offenses in the

1	United States are younger than 18 years), pyromania in childhood appears to be rare. Ju- venile fire setting is usually associated with conduct disorder, attention—deficit/hyperac- tivity disorder, or an adjustment disorder. Pyromania occurs much more often in males, especially those with poorer social skills and learning difficulties. Other causes of intentional fire setting. It is important to rule out other causes of fire setting before giving the diagnosis of pyromania. Intentional fire setting may occur for profit, sabotage, or revenge; to conceal a crime; to make a political statement (e.g., an act of terrorism or protest); or to attract attention or recognition (e.g., setting a fire in order to dis- cover it and save the day). Fire setting may also occur as part of developmental experi- mentation in childhood (e.g., playing with matches, lighters, or fire).

1	Other mental disorders. A separate diagnosis of pyromania is not given when fire set— ting occurs as part of conduct disorder, a manic episode, or antisocial personality disorder, or if it occurs in response to a delusion or a hallucination (e.g., in schizophrenia) or is at- tributable to the physiological effects of another medical condition (e.g., epilepsy). The di- agnosis of pyromania should also not be given when fire setting results from impaired judgment associated with major neurocognitive disorder, intellectual disability, or sub- stance intoxication. There appears to be a high co-occurrence of substance use disorders, gambling disorder, depressive and bipolar disorders, and other disruptive, impulse-control, and conduct dis- orders with pyromania. 478 Disruptive, Impulse-Control, and Conduct Disorders Diagnostic Criteria 312.32 (F63.3) A. Recurrent failure to resist impulses to steal objects that are not needed for personal use or for their monetary value.

1	Diagnostic Criteria 312.32 (F63.3) A. Recurrent failure to resist impulses to steal objects that are not needed for personal use or for their monetary value. B. Increasing sense of tension immediately before committing the theft. C. Pleasure, gratification, or relief at the time of committing the theft. D. The stealing is not committed to express anger or vengeance and is not in response to a delusion or a hallucination. E. The stealing is not better explained by conduct disorder, a manic episode, or antisocial personality disorder. The essential feature of kleptomania is the recurrent failure to resist impulses to steal items even though the items are not needed for personal use or for their monetary value (Criterion

1	A). The individual experiences a rising subjective sense of tension before the theft (Criterion B) and feels pleasure, gratification, or relief when committing the theft (Criterion C). The stealing is not committed to express anger or vengeance, is not done in response to a delusion or hal- lucination (Criterion D), and is not better explained by conduct disorder, a manic episode, or antisocial personality disorder (Criterion E). The objects are stolen despite the fact that they are typically of little value to the individual, who could have afforded to pay for them and often gives them away or discards them. Occasionally the individual may hoard the stolen objects or surreptitiously return them. Although individuals with this disorder will generally avoid stealing when immediate arrest is probable (e.g., in full view of a police officer), they usually do not preplan the thefts or fully take into account the chances of apprehension. The stealing is done without assistance from, or

1	is probable (e.g., in full view of a police officer), they usually do not preplan the thefts or fully take into account the chances of apprehension. The stealing is done without assistance from, or collaboration with, others.

1	Individuals with kleptomania typically attempt to resist the impulse to steal, and they are aware that the act is wrong and senseless. The individual frequently fears being appre- hended and often feels depressed or guilty about the thefts. Neurotransmitter pathways associated with behavioral addictions, including those associated with the serotonin, d0- pamine, and opioid systems, appear to play a role in kleptomania as well. Kleptomania occurs in about 4%—24% of individuals arrested for shoplifting. Its preva- lence in the general population is very rare, at approximately 0.3%—0.6%. Females outnum- ber males at a ratio of 3:].

1	Age at onset of kleptomania is variable, but the disorder often begins in adolescence. How- ever, the disorder may begin in childhood, adolescence, or adulthood, and in rare cases in late adulthood. There is little systematic information on the course of kleptomania, but three typical courses have been described: sporadic with brief episodes and long periods of remission; episodic with protracted periods of stealing and periods of remission; and chronic with some degree of ﬂuctuation. The disorder may continue for years, despite multiple convictions for shoplifting. Other Specified Disruptive, Impulse-Control, and Conduct Disorder 479

1	Other Specified Disruptive, Impulse-Control, and Conduct Disorder 479 Genetic and physiological. There are no controlled family history studies of kleptoma- nia. However, first-degree relatives of individuals with kleptomania may have higher rates of obsessive-compulsive disorder than the general population. There also appears to be a higher rate of substance use disorders, including alcohol use disorder, in relatives of individuals with kleptomania than in the general population. Functionai Consequences of Kleptomania The disorder may cause legal, family, career, and personal difficulties.

1	Functionai Consequences of Kleptomania The disorder may cause legal, family, career, and personal difficulties. Ordinary theft. Kleptomania should be distinguished from ordinary acts of theft or shoplifting. Ordinary theft (whether planned or impulsive) is deliberate and is motivated by the usefulness of the object or its monetary worth. Some individuals, especially adoles- cents, may also steal on a dare, as an act of rebellion, or as a rite of passage. The diagnosis is not made unless other characteristic features of kleptomania are also present. Klepto- mania is exceedingly rare, whereas shoplifting is relatively common. Malingering. In malingering, individuals may simulate the symptoms of kleptomania to avoid criminal prosecution. Antisocial personality disorder and conduct disorder. Antisocial personality disorder and conduct disorder are distinguished from kleptomania by a general pattern of antiso— cial behavior.

1	Antisocial personality disorder and conduct disorder. Antisocial personality disorder and conduct disorder are distinguished from kleptomania by a general pattern of antiso— cial behavior. Manic episodes, psychotic episodes, and major neurocognitive disorder. Kleptomania manic episode, in response to delusions or hallucinations (as in, e.g., schizophrenia), or as a result of a major neurocognitive disorder. Kleptomania may be associated with compulsive buying as well as with depressive and bipolar disorders (especially major depressive disorder), anxiety disorders, eating disor- ders (particularly bulimia nervosa), personality disorders, substance use disorders (espe- cially alcohol use disorder), and other disruptive, impulse-control, and conduct disorders. Other Specified Disruptive, Impulse-Control, 312.89 (F91.8)

1	Other Specified Disruptive, Impulse-Control, 312.89 (F91.8) This category applies to presentations in which symptoms characteristic of a disruptive, impulse-control, and conduct disorder that cause clinically significant distress or impair- ment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the disruptive, impulse-control, and con- duct disorders diagnostic class. The other specified disruptive. impulse-control, and con- duct disorder category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for any specific disrup- tive, impulse-control, and conduct disorder. This is done by recording “other specified dis- ruptive, impulse-control, and conduct disorder" followed by the specific reason (e.g., “recurrent behavioral outbursts of insufficient frequency").

1	480 Disruptive, Impulse-Control, and Conduct Disorders Unspecified Disruptive, Impulse-Control, 312.9 (F91.9) This category applies to presentations in which symptoms characteristic of a disruptive, impulse-control, and conduct disorder that cause clinically significant distress or impair- ment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the disruptive, impulse-control, and con- duct disorders diagnostic class. The unspecified disruptive, impulse-control, and conduct disorder category is used in situations in which the clinician chooses not to specify the rea- son that the criteria are not met for a specific disruptive. impulse-control, and conduct dis- order, and includes presentations in which there is insufficient information to make a more specific diagnosis (e.g., in emergency room settings).

1	The substanoe—related disorders encompass 10 separate classes of drugs: alco- sedatives, hypnotics, and anxiolytics; stimulants (amphetamine-type substances, cocaine, and other stimulants); tobacco; and other (or unknown) substances. These 10 classes are not fully distinct. All drugs that are taken in excess have in common direct activation of the brain reward system, which is involved in the reinforcement of behaviors and the pro- duction of memories. They produce such an intense activation of the reward system that normal activities may be neglected. Instead of achieving reward system activation through adaptive behaviors, drugs of abuse directly activate the reward pathways. The pharmacological mechanisms by which each class of drugs produces reward are different, but the drugs typically activate the system and produce feelings of pleasure, often re- ferred to as a ”high.” Furthermore, individuals with lower levels of self-control, which may reﬂect impairments of brain inhibitory

1	activate the system and produce feelings of pleasure, often re- ferred to as a ”high.” Furthermore, individuals with lower levels of self-control, which may reﬂect impairments of brain inhibitory mechanisms, may be particularly predisposed to develop substance use disorders, suggesting that the roots of substance use disorders for some persons can be seen in behaviors long before the onset of actual substance use itself.

1	In addition to the substance-related disorders, this chapter also includes gambling dis- order, reflecting evidence that gambling behaviors activate reward systems similar to those activated by drugs of abuse and produce some behavioral symptoms that appear comparable to those produced by the substance use disorders. Other excessive behavioral patterns, such as Internet gaming, have also been described, but the research on these and other behavioral syndromes is less clear. Thus, groups of repetitive behaviors, which some term behavioral addictions, with such subcategories as ”sex addiction,” “exercise addiction,” or "shopping addiction,” are not included because at this time there is insufficient peer-re- viewed evidence to establish the diagnostic criteria and course descriptions needed to identify these behaviors as mental disorders.

1	The substance-related disorders are divided into two groups: substance use disorders and substance-induced disorders. The following conditions may be classified as sub- stance-induced: intoxication, withdrawal, and other substance/ medication-induced men- tal disorders (psychotic disorders, bipolar and related disorders, depressive disorders, anxiety disorders, obsessive-compulsive and related disorders, sleep disorders, sexual dys- functions, delirium, and neurocognitive disorders).

1	The current section begins with a general discussion of criteria sets for a substance use disorder, substance intoxication and withdrawal, and other substance/medication- induced mental disorders, at least some of which are applicable across classes of sub- stances. Reﬂecting some unique aspects of the 10 substance classes relevant to this chapter, the remainder of the chapter is organized by the class of substance and describes their unique aspects. To facilitate differential diagnosis, the text and criteria for the remaining substance/medication-induced mental disorders are included with disorders with which they share phenomenology (e.g., substance/medication-induced depressive disorder is in the chapter "Depressive Disorders”). The broad diagnostic categories associated with each specific group of substances are shown in Table 1.

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1	x x x 5 5 3\ 5 3\ 5 3\ 5 3\ 5 3\ 5 5 23:85:65.6 .50 .monoErE x x x 3: 5s: 35 3 35 852.50 x x 5 5 5 5 .5 8:525:85 50550 m:omo:5u==m5.5 x x x 5 3\ 5 5 5 553:3 E5585 cozao 209.85“. @6985 E25560 wee: 22:86 22:85 969686 0585208555 20.23:. b.5936 -555 -_xo5:_ ow: o>Ecmoo 0:295... 6006 50290.. Ucm 5550555555 02.». 5m_oq_m 25.050566 00:05.8 oocﬂw oocﬂmoam -o._:oz _msxow 02$:an0 89500 .555 .556 038030 00.20 00:52:50 5555 5.285083 032585 w u4n<h The essential feature of a substance use disorder is a cluster of cognitive, behavioral, and physiological symptoms indicating that the individual continues using the substance de- spite significant substance-related problems. As seen in Table 1, the diagnosis of a sub- stance use disorder can be applied to all 10 classes included in this chapter except caffeine.

1	For certain classes some symptoms are less salient, and in a few instances not all symptoms apply (e.g., withdrawal symptoms are not specified for phencyclidine use disorder, other hallucinogen use disorder, or inhalant use disorder). An important characteristic of substance use disorders is an underlying change in brain cir- cuits that may persist beyond detoxification, particularly in individuals with severe disorders. The behavioral effects of these brain changes may be exhibited in the repeated relapses and in- tense drug craving when the individuals are exposed to drug-related stimuli. These persistent drug effects may benefit from long-term approaches to treatment.

1	Overall, the diagnosis of a substance use disorder is based on a pathological pattern of behaviors related to use of the substance. To assist with organization, Criterion A criteria can be considered to fit within overall groupings of impaired control, social impairment, risky use, and pharmacological criteria. Impaired control over substance use is the first criteria grouping (Criteria 1—4). The individual may take the substance in larger amounts or over a longer pe- riod than was originally intended (Criterion 1). The individual may express a persistent de- sire to cut down or regulate substance use and may report multiple unsuccessful efforts to decrease or discontinue use (Criterion 2). The individual may spend a great deal of time ob- taining the substance, using the substance, or recovering from its effects (Criterion 3). In some instances of more severe substance use disorders, virtually all of the individual’s daily activities revolve around the substance. Craving (Criterion

1	from its effects (Criterion 3). In some instances of more severe substance use disorders, virtually all of the individual’s daily activities revolve around the substance. Craving (Criterion 4) is manifested by an intense de- sire or urge for the drug that may occur at any time but is more likely when in an environ- ment where the drug previously was obtained or used. Craving has also been shown to involve classical conditioning and is associated with activation of specific reward structures in the brain. Craving is queried by asking if there has ever been a time when they had such strong urges to take the drug that they could not think of anything else. Current craving is of- ten used as a treatment outcome measure because it may be a signal of impending relapse.

1	Social impairment is the second grouping of criteria (Criteria 5—7). Recurrent substance use may result in a failure to fulfill major role obligations at work, school, or home (Crite- rion 5). The individual may continue substance use despite having persistent or recurrent social or interpersonal problems caused or exacerbated by the effects of the substance (Cri- terion 6). Important social, occupational, or recreational activities may be given up or re- duced because of substance use (Criterion 7). The individual may withdraw from family activities and hobbies in order to use the substance.

1	Risky use of the substance is the third grouping of criteria (Criteria 8—9). This may take the form of recurrent substance use in situations in which it is physically hazardous (Cri- terion 8). The individual may continue substance use despite knowledge of having a per- sistent or recurrent physical or psychological problem that is likely to have been caused or exacerbated by the substance (Criterion 9). The key issue in evaluating this criterion is not the existence of the problem, but rather the individual’s failure to abstain from using the substance despite the difficulty it is causing.

1	Pharmacological criteria are the final grouping (Criteria 10 and 11). Tolerance (Crite- rion 10) is signaled by requiring a markedly increased dose of the substance to achieve the desired effect or a markedly reduced effect when the usual dose is consumed. The degree to which tolerance develops varies greatly across different individuals as well as across substances and may involve a variety of central nervous system effects. For example, tol- erance to respiratory depression and tolerance to sedating and motor coordination may develop at different rates, depending on the substance. Tolerance may be difficult to de- termine by history alone, and laboratory tests may be helpful (e.g., high blood levels of the substance coupled with little evidence of intoxication suggest that tolerance is likely). Tol— erance must also be distinguished from individual variability in the initial sensitivity to the effects of particular substances. For example, some first-time alcohol drinkers show very

1	likely). Tol— erance must also be distinguished from individual variability in the initial sensitivity to the effects of particular substances. For example, some first-time alcohol drinkers show very little evidence of intoxication with three or four drinks, whereas others of similar weight and drinking histories have slurred speech and incoordination.

1	Withdrawal (Criterion 11) is a syndrome that occurs when blood or tissue concentra— tions of a substance decline in an individual who had maintained prolonged heavy use of the substance. After developing withdrawal symptoms, the individual is likely to con- sume the substance to relieve the symptoms. Withdrawal symptoms vary greatly across the classes of substances, and separate criteria sets for withdrawal are provided for the drug classes. Marked and generally easily measured physiological signs of withdrawal are common With alcohol, opioids, and sedatives, hypnotics, and anxiolytics. Withdrawal signs and symptoms with stimulants (amphetamines and cocaine), as well as tobacco and cannabis, are often present but may be less apparent. Significant withdrawal has not been documented in humans after repeated use of phencyclidine, other hallucinogens, and in- halants; therefore, this criterion is not included for these substances. Neither tolerance nor withdrawal is necessary for a

1	humans after repeated use of phencyclidine, other hallucinogens, and in- halants; therefore, this criterion is not included for these substances. Neither tolerance nor withdrawal is necessary for a diagnosis of a substance use disorder. However, for most classes of substances, a past history of withdrawal is associated with a more severe clinical course (i.e., an earlier onset of a substance use disorder, higher levels of substance intake, and a greater number of substance—related problems).

1	Symptoms of tolerance and withdrawal occurring during appropriate medical treat- ment with prescribed medications (e.g., opioid analgesics, sedatives, stimulants) are spe- cifically not counted when diagnosing a substance use disorder. The appearance of normal, expected pharmacological tolerance and withdrawal during the course of medical treat- ment has been known to lead to an erroneous diagnosis of ”addiction” even when these were the only symptoms present. Individuals whose only symptoms are those that occur as a result of medical treatment (i.e., tolerance and withdrawal as part of medical care when the medications are taken as prescribed) should not receive a diagnosis solely on the basis of these symptoms. However, prescription medications can be used inappropriately, and a substance use disorder can be correctly diagnosed when there are other symptoms of compulsive, drug-seeking behavior.

1	Substance use disorders occur in a broad range of severity, from mild to severe, with se- verity based on the number of symptom criteria endorsed. As a general estimate of sever- ity, a mild substance use disorder is suggested by the presence of two to three symptoms, moderate by four to five symptoms, and severe by six or more symptoms. Changing severity across time is also reﬂected by reductions or increases in the frequency and/ or close of substance use, as assessed by the individual’s own report, report of knowledgeable others, clinician’s observations, and biological testing. The following course specifiers and descrip- tive features specifiers are also available for substance use disorders: “in early remission,” ”in sustained remission," "on maintenance therapy,” and ”in a controlled environment.” Definitions of each are provided within respective criteria sets.

1	The clinician should use the code that applies to the class of substances but record the name of the specific substance. For example, the clinician should record 304.10 (F1320) moderate alprazolam use disorder (rather than moderate sedative, hypnotic, or anxiolytic use disorder) or 305.70 (F1510) mild methamphetamine use disorder (rather than mild stimulant use disorder). For substances that do not fit into any of the classes (e.g., anabolic steroids), the appropriate code for ”other substance use disorder” should be used and the specific substance indicated (e.g., 305.90 [F19.10] mild anabolic steroid use disorder). If the substance taken by the individual is unknown, the code for the class ”other (or unknown)” should be used (e.g., 304.90 [F19.20] severe unknown substance use disorder). If criteria are met for more than one substance use disorder, all should be diagnosed (e.g., 304.00 [F1120] severe heroin use disorder; 304.20 [F1420] moderate cocaine use disorder).

1	The appropriate ICD-lO-CM code for a substance use disorder depends on whether there is a comorbid substance-induced disorder (including intoxication and withdrawal). In the above example, the diagnostic code for moderate alprazolam use disorder, F13.20, re- ﬂects the absence of a comorbid alprazolam-induced mental disorder. Because ICD-lO—CM codes for substance-induced disorders indicate both the presence (or absence) and severity of the substance use disorder, ICD-lO—CM codes for substance use disorders can be used only in the absence of a substance-induced disorder. See the individual substance-specific sections for additional coding information.

1	Note that the word addiction is not applied as a diagnostic term in this classification, although it is in common usage in many countries to describe severe problems related to compulsive and habitual use of substances. The more neutral term substance use disorder is used to describe the wide range of the disorder, from a mild form to a severe state of chron- ically relapsing, compulsive drug taking. Some clinicians will choose to use the word ud- diction to describe more extreme presentations, but the word is omitted from the official DSM-S substance use disorder diagnostic terminology because of its uncertain definition and its potentially negative connotation. The overall category of substance-induced disorders includes intoxication, withdrawal, and other substance/medication-induced mental disorders (e.g., substance-induced psy- chotic disorder, substance-induced depressive disorder).

1	Criteria for substance intoxication are included within the substance—specific sections of this chapter. The essential feature is the development of a reversible substance-specific syndrome due to the recent ingestion of a substance (Criterion A). The clinically significant problematic behavioral or psychological changes associated with intoxication (e.g., bellig- erence, mood lability, impaired judgment) are attributable to the physiological effects of the substance on the central nervous system and develop during or shortly after use of the substance (Criterion B). The symptoms are not attributable to another medical condition and are not better explained by another mental disorder (Criterion D). Substance intoxi- cation is common among those with a substance use disorder but also occurs frequently in individuals without a substance use disorder. This category does not apply to tobacco.

1	The most common changes in intoxication involve disturbances of perception, wake- fulness, attention, thinking, judgment, psychomotor behavior, and interpersonal behav- ior. Short-term, or "acute,” intoxications may have different signs and symptoms than sustained, or "chronic,” intoxications. For example, moderate cocaine doses may initially produce gregariousness, but social withdrawal may develop if such doses are frequently repeated over days or weeks. When used in the physiological sense, the term intoxication is broader than substance intoxication as defined here. Many substances may produce physiological or psychologi- cal changes that are not necessarily problematic. For example, an individual with tachy- cardia from substance use has a physiological effect, but if this is the only symptom in the absence of problematic behavior, the diagnosis of intoxication would not apply. Intoxica- tion may sometimes persist beyond the time when the substance is detectable in the body.

1	This may be due to enduring central nervous system effects, the recovery of which takes longer than the time for elimination of the substance. These longer-term effects of intoxi- cation must be distinguished from withdrawal (i.e., symptoms initiated by a decline in blood or tissue concentrations of a substance).

1	Criteria for substance withdrawal are included within the substance-specific sections of this chapter. The essential feature is the development of a substance-specific problematic be- havioral change, with physiological and cognitive concomitants, that is due to the cessation of, or reduction in, heavy and prolonged substance use (Criterion A). The substance-specific syn- drome causes clinically significant distress or impairment in social, occupational, or other im- portant areas of functioning (Criterion C). The symptoms are not due to another medical condition and are not better explained by another mental disorder (Criterion D). Withdrawal is usually, but not always, associated with a substance use disorder. Most individuals with withdrawal have an urge to re—administer the substance to reduce the symptoms. Route of Administration and Speed of Substance Effects

1	Route of Administration and Speed of Substance Effects Routes of administration that produce more rapid and efficient absorption into the blood- stream (e.g., intravenous, smoking, intranasal ”snorting") tend to result in a more intense intoxication and an increased likelihood of an escalating pattern of substance use leading to withdrawal. Similarly, rapidly acting substances are more likely than slower-acting substances to produce immediate intoxication. Duration of Effects

1	Duration of Effects Within the same drug category, relatively short-acting substances tend to have a higher potential for the development of withdrawal than do those with a longer duration of ac- tion. However, longer-acting substances tend to have longer withdrawal duration. The half-life of the substance parallels aspects of withdrawal: the longer the duration of action, the longer the time between cessation and the onset of withdrawal symptoms and the lon- ger the withdrawal duration. In general, the longer the acute withdrawal period, the less intense the syndrome tends to be. Use of Multiple Substances ously or sequentially. In these cases, each diagnosis should be recorded separately.

1	Use of Multiple Substances ously or sequentially. In these cases, each diagnosis should be recorded separately. Laboratory analyses of blood and urine samples can help determine recent use and the specific substances involved. However, a positive laboratory test result does not by itself indicate that the individual has a pattern of substance use that meets criteria for a substance—induced or sub- stance use disorder, and a negative test result does not by itself rule out a diagnosis. Laboratory tests can be useful in identifying withdrawal. If the individual presents with withdrawal from an unknown substance, laboratory tests may help identify the sub- stance and may also be helpful in differentiating withdrawal from other mental disorders. In addition, normal functioning in the presence of high blood levels of a substance sug- gests considerable tolerance.

1	In addition, normal functioning in the presence of high blood levels of a substance sug- gests considerable tolerance. Individuals ages 18—24 years have relatively high prevalence rates for the use of virtually every substance. Intoxication is usually the initial substance-related disorder and often be- gins in the teens. Withdrawal can occur at any age as long as the relevant drug has been taken in sufficient doses over an extended period of time.

1	The clinician should use the code that applies to the class of substances but record the name of the specific substance. For example, the clinician should record 292.0 (F13.239) seco- barbital withdrawal (rather than sedative, hypnotic, or anxiolytic withdrawal) or 292.89 (F15.129) methamphetamine intoxication (rather than stimulant intoxication). Note that the appropriate ICD-lO-CM diagnostic code for intoxication depends on whether there is a comorbid substance use disorder. In this case, the F15.129 code for methamphetamine in- dicates the presence of a comorbid mild methamphetamine use disorder. If there had been no comorbid methamphetamine use disorder, the diagnostic code would have been

1	F15.929. ICD-lO-CM coding rules require that all withdrawal codes imply a comorbid moderate to severe substance use disorder for that substance. In the above case, the code for secobarbital withdrawal (F13.239) indicates the comorbid presence of a moderate to se- vere secobarbital use disorder. See the coding note for the substance-specific intoxication and withdrawal syndromes for the actual coding options.

1	For substances that do not fit into any of the classes (e.g., anabolic steroids), the appropriate code for "other substance intoxication" should be used and the specific substance indicated (e.g., 292.89 [F19.929] anabolic steroid intoxication). If the substance taken by the individual is unknown, the code for the class ”other (or unknown)” should be used (e.g., 292.89 [F19.929] unknown substance intoxication). If there are symptoms or problems associated with a partic- ular substance but criteria are not met for any of the substance-specific disorders, the unspec- ified category can be used (e.g., 292.9 [F1299] unspecified cannabis-related disorder).

1	As noted above, the substance—related codes in ICDlOCM combine the substance use dis- order aspect of the clinical picture and the substance-induced aspect into a single combined code. Thus, if both heroin withdrawal and moderate heroin use disorder are present, the single code F1123 is given to cover both presentations. In ICD-9-CM, separate diagnostic codes (292.0 and 304.00) are given to indicate withdrawal and a moderate heroin use disorder, re- spectively. See the individual substance—specific sections for additional coding information.

1	The substance/medication-induced mental disorders are potentially severe, usually tem- porary, but sometimes persisting central nervous system (CNS) syndromes that develop in the context of the effects of substances of abuse, medications, or several toxins. They are distinguished from the substance use disorders, in which a cluster of cognitive, behav- ioral, and physiological symptoms contribute to the continued use of a substance despite significant substance-related problems. The substance / medication-induced mental disor- ders may be induced by the 10 classes of substances that produce substance use disorders, or by a great variety of other medications used in medical treatment. Each substance- induced mental disorder is described in the relevant chapter (e.g., ”Depressive Disorders,” ”Neurocognitive Disorders”), and therefore, only a brief description is offered here. All substance/medication-induced disorders share common characteristics. It is important to recognize these

1	”Neurocognitive Disorders”), and therefore, only a brief description is offered here. All substance/medication-induced disorders share common characteristics. It is important to recognize these common features to aid in the detection of these disorders. These features are described as follows:

1	A. The disorder represents a clinically significant symptomatic presentation of a relevant mental disorder. B. There is evidence from the history, physical examination, or laboratory findings of both of the following: 1. The disorder developed during or within 1 month of a substance intoxication or 2. The involved substance/medication is capable of producing the mental disorder.

1	C. The disorder is not better explained by an independent mental disorder (i.e., one that is not substance- or medication-induced). Such evidence of an independent mental dis- order could include the following: 1. The disorder preceded the onset of severe intoxication or withdrawal or exposure to the medication; or 2. The full mental disorder persisted for a substantial period of time (e.g., at least 1 month) after the cessation of acute withdrawal or severe intoxication or taking the medica- tion. This criterion does not apply to substance-induced neurocognitive disorders or hallucinogen persisting perception disorder, which persist beyond the cessation of acute intoxication or withdrawal. D. The disorder does not occur exclusively during the course of a delirium. E. The disorder causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning.

1	E. The disorder causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. Some generalizations can be made regarding the categories of substances capable of produc- ing clinically relevant substance-induced mental disorders. In general, the more sedating drugs (sedative, hypnotics, or anxiolytics, and alcohol) can produce prominent and clini- cally significant depressive disorders during intoxication, while anxiety conditions are likely to be observed during withdrawal syndromes from these substances. Also, during intoxica- tion, the more stimulating substances (e.g., amphetamines and cocaine) are likely to be as- sociated with substance-induced psychotic disorders and substance-induced anxiety disorders, with substance-induced major depressive episodes observed during withdrawal.

1	Both the more sedating and more stimulating drugs are likely to produce significant but temporary sleep and sexual disturbances. An overview of the relationship between specific categories of substances and specific psychiatric syndromes is presented in Table 1.

1	The medication-induced conditions include what are often idiosyncratic CNS reac- tions or relatively extreme examples of side effects for a wide range of medications taken for a variety of medical concerns. These include neurocognitive complications of anesthet- ics, antihistamines, antihypertensives, and a variety of other medications and toxins (e.g., organophosphates, insecticides, carbon monoxide), as described in the chapter on neuro- cognitive disorders. Psychotic syndromes may be temporarily experienced in the context of anticholinergic, cardiovascular, and steroid drugs, as well as during use of stimulant- like and depressant-like prescription or over-the-counter drugs. Temporary but severe mood disturbances can be observed with a wide range of medications, including steroids, antihypertensives, disulfiram, and any prescription or over-the—counter depressant or stimulant—like substances. A similar range of medications can be associated with tempo- rary anxiety syndromes,

1	antihypertensives, disulfiram, and any prescription or over-the—counter depressant or stimulant—like substances. A similar range of medications can be associated with tempo- rary anxiety syndromes, sexual dysfunctions, and conditions of disturbed sleep.

1	In general, to be considered a substance/medication—induced mental disorder, there must be evidence that the disorder being observed is not likely to be better explained by an independent mental condition. The latter are most likely to be seen if the mental disorder was present before the severe intoxication or withdrawal or medication administration, or, with the exception of several substance—induced persisting disorders listed in Table 1, con- tinued more than 1 month after cessation of acute withdrawal, severe intoxication, or use of the medications. When symptoms are only observed during a delirium (e.g., alcohol withdrawal delirium), the mental disorder should be diagnosed as a delirium, and the psychiatric syndrome occurring during the delirium should not also be diagnosed sepa- rately, as many symptoms (including disturbances in mood, anxiety, and reality testing) are commonly seen during agitated, confused states. The features associated with each rel- evant major mental

1	rately, as many symptoms (including disturbances in mood, anxiety, and reality testing) are commonly seen during agitated, confused states. The features associated with each rel- evant major mental disorder are similar whether observed with independent or sub- stance/medication-induced mental disorders. However, individuals with substance/ medication-induced mental disorders are likely to also demonstrate the associated fea- tures seen with the specific category of substance or medication, as listed in other subsec- tions of this chapter.

1	Substance-induced mental disorders develop in the context of intoxication or withdrawal from substances of abuse, and medication-induced mental disorders are seen with pre- scribed or over-the-counter medications that are taken at the suggested doses. Both condi- tions are usually temporary and likely to disappear within 1 month or so of cessation of acute withdrawal, severe intoxication, or use of the medication. Exceptions to these generaliza- tions occur for certain long-duration substance-induced disorders: substance-associated neurocognitive disorders that relate to conditions such as alcohol-induced neurocognitive disorder, inhalant-induced neurocognitive disorder, and sedative-, hypnotic-, or anxiolytic- backs"; see the section "Hallucinogen-Related Disorders” later in this chapter). However, most other substance/ medication-induced mental disorders, regardless of the severity of the symptoms, are likely to improve relatively quickly with abstinence and unlikely to re- main

1	However, most other substance/ medication-induced mental disorders, regardless of the severity of the symptoms, are likely to improve relatively quickly with abstinence and unlikely to re- main clinically relevant for more than 1 month after complete cessation of use.

1	As is true of many consequences of heavy substance use, some individuals are more and others less prone toward specific substance-induced disorders. Similar types of pre— dispositions may make some individuals more likely to develop psychiatric side effects of some types of medications, but not others. However, it is unclear whether individuals with family histories or personal prior histories with independent psychiatric syndromes are more likely to develop the induced syndrome once the consideration is made as to whether the quantity and frequency of the substance was sufficient to lead to the devel- opment of a substance-induced syndrome.

1	There are indications that the intake of substances of abuse or some medications with psychiatric side effects in the context of a preexisting mental disorder is likely to result in an intensification of the preexisting independent syndrome. The risk for substance/med- ication—induced mental disorders is likely to increase with both the quantity and the fre- quency of consumption of the relevant substance. The symptom profiles for the substance/medication-induced mental disorders resem- ble independent mental disorders. While the symptoms of substance/medication-in- duced mental disorders can be identical to those of independent mental disorders (e.g., delusions, hallucinations, psychoses, major depressive episodes, anxiety syndromes), and although they can have the same severe consequences (e.g., suicide), most induced mental disorders are likely to improve in a matter of days to weeks of abstinence.

1	The substance/medication-induced mental disorders are an important part of the dif- ferential diagnoses for the independent psychiatric conditions. The importance of recog- nizing an induced mental disorder is similar to the relevance of identifying the possible role of some medical conditions and medication reactions before diagnosing an indepen- dent mental disorder. Symptoms of substance- and medication-induced mental disorders may be identical cross-sectionally to those of independent mental disorders but have dif- ferent treatments and prognoses from the independent condition. Functional Consequences of SubstancelMedication-

1	Functional Consequences of SubstancelMedication- The same consequences related to the relevant independent mental disorder (e.g., suicide attempts) are likely to apply to the substance/medication-induced mental disorders, but these are likely to disappear within 1 month after abstinence. Similarly, the same func- tional consequences associated with the relevant substance use disorder are likely to be seen for the substance-induced mental disorders. other specific substance/medication—induced mental disorders are provided in other chapters of the manual with disorders with which they share phenomenology (see the sub- stance/medicat'ion-induced mental disorders in these chapters: ”Schizophrenia Spectrum and Other Psychotic Disorders,” “Bipolar and Related Disorders," ”Depressive Disor- ders,” ”Anxiety Disorders,” ”Obsessive-Compulsive and Related Disorders,” "Sleep-

1	Wake Disorders,” ”Sexual Dysfunctions,” and ”Neurocognitive Disorders”). Generally, for ICD—9-CM, if a mental disorder is induced by a substance use disorder, a separate di- agnostic code is given for the specific substance use disorder, in addition to the code for the substance/medication-induced mental disorder. For ICD-IO-CM, a single code combines the substance-induced mental disorder with the substance use disorder. A separate diag- nosis of the comorbid substance use disorder is not given, although the name and severity of the specific substance use disorder (when present) are used when recording the sub- stance/medication-induced mental disorder. ICD-IO-CM codes are also provided for sit- uations in which the substance/medication-induced mental disorder is not induced by a substance use disorder (e.g., when a disorder is induced by one-time use of a substance or medication). Additional information needed to record the diagnostic name of the sub- stance/medication-induced mental

1	use disorder (e.g., when a disorder is induced by one-time use of a substance or medication). Additional information needed to record the diagnostic name of the sub- stance/medication-induced mental disorder is provided in the section ”Recording Proce- dures” for each substance/ medication—induced mental disorder in its respective chapter.

1	A. A problematic pattern of alcohol use leading to clinically significant impairment or dis- tress. as manifested by at least two of the following, occurring within a 12-month period: 1. Alcohol is often taken in larger amounts or over a longer period than was intended. 2. There is a persistent desire or unsuccessful efforts to cut down or control alcohol use. 3. A great deal of time is spent in activities necessary to obtain alcohol. use alcohol, or recover from its effects. 4. Craving, or a strong desire or urge to use alcohol. 5. Recurrent alcohol use resulting in a failure to fulfill major role obligations at work, school, or home. 6. Continued alcohol use despite having persistent or recurrent social or interpersonal problems caused or exacerbated by the effects of alcohol. 7. Important social, occupational, or recreational activities are given up or reduced be- cause of alcohol use. 8. Recurrent alcohol use in situations in which it is physically hazardous.

1	7. Important social, occupational, or recreational activities are given up or reduced be- cause of alcohol use. 8. Recurrent alcohol use in situations in which it is physically hazardous. 9. Alcohol use is continued despite knowledge of having a persistent or recurrent physical or psychological problem that is likely to have been caused or exacerbated by alcohol. 10. Tolerance, as defined by either of the following: a. A need for markedly increased amounts of alcohol to achieve intoxication or de- sired effect. b. A markedly diminished effect with continued use of the same amount of alcohol. 11. Withdrawal, as manifested by either of the following: a. The characteristic withdrawal syndrome for alcohol (refer to Criteria A and B of the criteria set for alcohol withdrawal, pp. 499—500). b. Alcohol (or a closely related substance, such as a benzodiazepine) is taken to relieve or avoid withdrawal symptoms. Specify if:

1	b. Alcohol (or a closely related substance, such as a benzodiazepine) is taken to relieve or avoid withdrawal symptoms. Specify if: In early remission: After full criteria for alcohol use disorder were previously met, none of the criteria for alcohol use disorder have been met for at least 3 months but for less than 12 months (with the exception that Criterion A4, “Craving, or a strong desire or urge to use alcohol,” may be met). In sustained remission: After full criteria for alcohol use disorder were previously met, none of the criteria for alcohol use disorder have been met at any time during a period of 12 months or longer (with the exception that Criterion A4, “Craving, or a strong desire or urge to use alcohol,” may be met). Specify it: In a controlled environment: This additional specifier is used it the individual is in an environment where access to alcohol is restricted.

1	Specify it: In a controlled environment: This additional specifier is used it the individual is in an environment where access to alcohol is restricted. Code based on current severity: Note for |CD-10—CM codes: If an alcohol intoxication, alcohol withdrawal, or another alcohoI-induced mental disorder is also present, do not use the codes below for alcohol use disorder. Instead, the comorbid alcohol use disorder is indicated in the 4th character of the alcohol-induced disorder code (see the coding note for alcohol intoxication, alcohol withdrawal, or a specific alcohol-induced mental disorder). For example, if there is comorbid alcohol intoxication and alcohol use disorder, only the alcohol intoxication code is given, with the 4th character indicating whether the comorbid alcohol use disorder is mild, moderate, or severe: F10.129 for mild alcohol use disorder with alcohol intoxication or F10.229 tor a moderate or severe alcohol use disorder with al- cohol intoxication.

1	Specify current severity: 305.00 (F10.10) Mild: Presence 012—3 symptoms. 303.90 (F10.20) Moderate: Presence of 4—5 symptoms. 303.90 (F10.20) Severe: Presence of 6 or more symptoms. ”In a controlled environment” applies as a further specifier of remission if the individual is both in remission and in a controlled environment (i.e., in early remission in a controlled environment or in sustained remission in a controlled environment). Examples of these environments are closely supervised and substance-free jails, therapeutic communities, and locked hospital units.

1	Severity of the disorder is based on the number of diagnostic criteria endorsed. For a given individual, changes in severity of alcohol use disorder across time are also reﬂected by reductions in the frequency (e.g., days of use per month) and / or dose (e.g., number of standard drinks consumed per day) of alcohol used, as assessed by the individual’s self- report, report of knowledgeable others, clinician observations, and, when practical, bio- logical testing (e.g., elevations in blood tests as described in the section “Diagnostic Mark- ers” for this disorder).

1	Alcohol use disorder is defined by a cluster of behavioral and physical symptoms, which can include withdrawal, tolerance, and craving. Alcohol withdrawal is characterized by withdrawal symptoms that develop approximately 4—12 hours after the reduction of in- take following prolonged, heavy alcohol ingestion. Because withdrawal from alcohol can be unpleasant and intense, individuals may continue to consume alcohol despite adverse consequences, often to avoid or to relieve withdrawal symptoms. Some withdrawal symp- toms (e.g., sleep problems) can persist at lower intensities for months and can contribute to relapse. Once a pattern of repetitive and intense use develops, individuals with alcohol use disorder may devote substantial periods of time to obtaining and consuming alcoholic beverages.

1	Craving for alcohol is indicated by a strong desire to drink that makes it difficult to think of anything else and that often results in the onset of drinking. School and job per- formance may also suffer either from the aftereffects of drinking or from actual intoxica- tion at school or on the job; child care or household responsibilities may be neglected; and alcohol-related absences may occur from school or work. The individual may use alcohol in physically hazardous circumstances (e.g., driving an automobile, swimming, operating machinery while intoxicated). Finally, individuals with an alcohol use disorder may con- tinue to consume alcohol despite the knowledge that continued consumption poses sig- nificant physical (e.g., blackouts, liver disease), psychological (e.g., depression), social, or interpersonal problems (e.g., violent arguments with spouse while intoxicated, child abuse).

1	Alcohol use disorder is often associated with problems similar to those associated with other substances (e.g., cannabis; cocaine; heroin; amphetamines; sedatives, hypnotics, or anxiolytics). Alcohol may be used to alleviate the unwanted effects of these other substances or to substitute for them when they are not available. Symptoms of conduct problems, depression, anxiety, and insomnia frequently accompany heavy drinking and sometimes precede it. Repeated intake of high doses of alcohol can affect nearly every organ system, espe- cially the gastrointestinal tract, cardiovascular system, and the central and peripheral ner— vous systems. Gastrointestinal effects include gastritis, stomach or duodenal ulcers, and, in about 15% of individuals who use alcohol heavily, liver cirrhosis and/ or pancreatitis.

1	There is also an increased rate of cancer of the esophagus, stomach, and other parts of the gastrointestinal tract. One of the most commonly associated conditions is low-grade hy- pertension. Cardiomyopathy and other myopathies are less common but occur at an in- creased rate among those who drink very heavily. These factors, along with marked increases in levels of triglycerides and low-density lipoprotein cholesterol, contribute to an elevated risk of heart disease. Peripheral neuropathy may be evidenced by muscular weakness, paresthesias, and decreased peripheral sensation. More persistent central ner- vous system effects include cognitive deficits, severe memory impairment, and degener- ative changes in the cerebellum. These effects are related to the direct effects of alcohol or of trauma and to vitamin deficiencies (particularly of the B vitamins, including thiamine).

1	One devastating central nervous system effect is the relatively rare alcohol-induced per- sisting amnestic disorder, or Wernicke-Korsakoff syndrome, in which the ability to encode new memory is severely impaired. This condition would now be described within the chap- cognitive disorder. Alcohol use disorder is an important contributor to suicide risk during severe intoxi- cation and in the context of a temporary alcohol-induced depressive and bipolar disorder. There is an increased rate of suicidal behavior as well as of completed suicide among in- dividuals with the disorder.

1	There is an increased rate of suicidal behavior as well as of completed suicide among in- dividuals with the disorder. Alcohol use disorder is a common disorder. In the United States, the 12-month prevalence of alcohol use disorder is estimated to be 4.6% among 12- to 17-year-olds and 8.5% among adults age 18 years and older in the United States. Rates of the disorder are greater among adult men (12.4%) than among adult women (4.9%). Twelve-month prevalence of alcohol use disorder among adults decreases in middle age, being greatest among individuals 18- to 29-years-old (16.2%) and lowest among individuals age 65 years and older (1.5%). Twelve-month prevalence varies markedly across race/ethnic subgroups of the US. population. For 12- to 17-year-olds, rates are greatest among Hispanics (6.0%) and Native

1	Twelve-month prevalence varies markedly across race/ethnic subgroups of the US. population. For 12- to 17-year-olds, rates are greatest among Hispanics (6.0%) and Native Americans and Alaska Natives (5.7%) relative to whites (5.0%), African Americans (1.8%), and Asian Americans and Pacific Islanders (1.6%). In contrast, among adults, the 12-month prevalence of alcohol use disorder is clearly greater among Native Americans and Alaska Natives (12.1%) than among whites (8.9%), Hispanics (7.9%), African Americans (6.9%), and Asian Americans and Pacific Islanders (4.5%).

1	Natives (12.1%) than among whites (8.9%), Hispanics (7.9%), African Americans (6.9%), and Asian Americans and Pacific Islanders (4.5%). The first episode of alcohol intoxication is likely to occur during the mid—teens. Alcohol- occur prior to age 20 years, but the age at onset of an alcohol use disorder with two or more of the criteria clustered together peaks in the late teens or early to mid 205. The large ma- jority of individuals who develop alcohol-related disorders do so by their late 305. The first evidence of withdrawal is not likely to appear until after many other aspects of an alcohol use disorder have developed. An earlier onset of alcohol use disorder is observed in ado- lescents with preexisting conduct problems and those with an earlier onset of intoxication.

1	Alcohol use disorder has a variable course that is characterized by periods of remission and relapse. A decision to stop drinking, often in response to a crisis, is likely to be followed by a period of weeks or more of abstinence, which is often followed by limited periods of controlled or nonproblematic drinking. However, once alcohol intake resumes, it is highly develop. Alcohol use disorder is often erroneously perceived as an intractable condition, per- haps based on the fact that individuals who present for treatment typically have a history of many years of severe alcohol-related problems. However, these most severe cases rep- resent only a small proportion of individuals with this disorder, and the typical individual with the disorder has a much more promising prognosis.

1	Among adolescents, conduct disorder and repeated antisocial behavior often co-occur with alcohol- and with other substance-related disorders. While most individuals with al- cohol use disorder develop the condition before age 40 years, perhaps 10% have later onset. Age-related physical changes in older individuals result in increased brain suscep- tibility to the depressant effects of alcohol; decreased rates of liver metabolism of a variety of substances, including alcohol; and decreased percentages of body water. These changes can cause older people to develop more severe intoxication and subsequent problems at lower levels of consumption. Alcohol-related problems in older people are also especially likely to be associated with other medical complications.

1	Environmental. Environmental risk and prognostic factors may include cultural atti- tudes toward drinking and intoxication, the availability of alcohol (including price), acquired personal experiences with alcohol, and stress levels. Additional potential medi- ators of how alcohol problems develop in predisposed individuals include heavier peer substance use, exaggerated positive expectations of the effects of alcohol, and suboptimal ways of coping with stress. Genetic and physiological. Alcohol use disorder runs in families, with 40%-60% of the variance of risk explained by genetic inﬂuences. The rate of this condition is three to four times higher in close relatives of individuals with alcohol use disorder, with values highest for individuals with a greater number of affected relatives, closer genetic relationships to the affected person, and higher severity of the alcohol-related problems in those relatives.

1	A significantly higher rate of alcohol use disorders exists in the monozygotic twin than in the dizygotic twin of an individual with the condition. A three- to fourfold increase in risk has been observed in children of individuals with alcohol use disorder, even when these have the disorder. Recent advances in our understanding of genes that operate through intermediate characteristics (or phenotypes) to affect the risk of alcohol use disorder can help to identify individuals who might be at particularly low or high risk for alcohol use disorder. Among the low-risk phenotypes are the acute alcohol-related skin ﬂush (seen most prominently in

1	Asians). High vulnerability is associated with preexisting schizophrenia or bipolar disor- der, as well as impulsivity (producing enhanced rates of all substance use disorders and gambling disorder), and a high risk specifically for alcohol use disorder is associated with a low level of response (low sensitivity) to alcohol. A number of gene variations may ac- count for low response to alcohol or modulate the dopamine reward systems; it is impor- tant to note, however, that any one gene variation is likely to explain only 1%—2% of the risk for these disorders. Course modifiers. In general, high levels of impulsivity are associated with an earlier onset and more severe alcohol use disorder.

1	Course modifiers. In general, high levels of impulsivity are associated with an earlier onset and more severe alcohol use disorder. In most cultures, alcohol is the most frequently used intoxicating substance and contrib- utes to considerable morbidity and mortality. An estimated 3.8% of all global deaths and 4.6% of global disability—adjusted life-years are attributable to alcohol. In the United States, 80% of adults (age 18 years and older) have consumed alcohol at some time in their lives, and 65% are current drinkers (last 12 months). An estimated 3.6% of the world population (15—64 years old) has a current (12-month) alcohol use disorder, with a lower prevalence (1.1%) found in the African region, a higher rate (5.2%) found in the American region (North, South, and Central America and the Caribbean), and the highest rate (10.9%) found in the Eastern Europe region.

1	South, and Central America and the Caribbean), and the highest rate (10.9%) found in the Eastern Europe region. Polymorphisms of genes for the alcohol-metabolizing enzymes alcohol dehydroge- nase and aldehyde dehydrogenase are most often seen in Asians and affect the response to alcohol. When consuming alcohol, individuals with these gene variations can experience a ﬂushed face and palpitations, reactions that can be so severe as to limit or preclude future alcohol consumption and diminish the risk for alcohol use disorder. These gene variations are seen in as many as 40% of Japanese, Chinese, Korean, and related groups worldwide and are related to lower risks for the disorder. Despite small variations regarding individual criterion items, the diagnostic criteria perform equally well across most race/ethnicity groups.

1	Despite small variations regarding individual criterion items, the diagnostic criteria perform equally well across most race/ethnicity groups. Males have higher rates of drinking and related disorders than females. However, because females generally weigh less than males, have more fat and less water in their bodies, and metabolize less alcohol in their esophagus and stomach, they are likely to develop higher blood alcohol levels per drink than males. Females who drink heavily may also be more vulnerable than males to some of the physical consequences associated with alcohol, in- cluding liver disease.

1	results likely to be seen with regular heavier drinking. These measures do not establish a diagnosis of an alcohol-related disorder but can be useful in highlighting individuals for whom more information should be gathered. The most direct test available to measure a1- cohol consumption cross-sectionally is blood alcohol concentration, which can also be used to judge tolerance to alcohol. For example, an individual with a concentration of 150 mg of ethanol per deciliter (dL) of blood who does not show signs of intoxication can be pre- sumed to have acquired at least some degree of tolerance to alcohol. At 200 mg/dL, most nontolerant individuals demonstrate severe intoxication. Regarding laboratory tests, one sensitive laboratory indicator of heavy drinking is a modest elevation or high-normal levels (>35 units) of gamma-glutamyltransferase (GGT).

1	Regarding laboratory tests, one sensitive laboratory indicator of heavy drinking is a modest elevation or high-normal levels (>35 units) of gamma-glutamyltransferase (GGT). This may be the only laboratory finding. At least 70% of individuals with a high GGT level are persistent heavy drinkers (i.e., consuming eight or more drinks daily on a regular basis).

1	A second test with comparable or even higher levels of sensitivity and specificity is carbo- hydrate—deficient transferrin (CDT), with levels of 20 units or higher useful in identifying in- dividuals who regularly consume eight or more drinks daily. Since both GGT and CDT levels return toward normal within days to weeks of stopping drinking, both state markers may be useful in monitoring abstinence, especially when the clinician observes increases, rather than decreases, in these values over time—a finding indicating that the person is likely to have returned to heavy drinking. The combination of tests for CDT and GGT may have even higher levels of sensitivity and specificity than either test used alone. Additional useful tests include the mean corpuscular volume (MCV), which may be elevated to high- normal values in individuals who drink heavily—a change that is due to the direct toxic ef— fects of alcohol on erythropoiesis. Although the MCV can be used to help identify those who

1	to high- normal values in individuals who drink heavily—a change that is due to the direct toxic ef— fects of alcohol on erythropoiesis. Although the MCV can be used to help identify those who drink heavily, it is a poor method of monitoring abstinence because of the long half-life of red blood cells. Liver function tests (e.g., alanine aminotransferase [ALT] and alkaline phos- phatase) can reveal liver injury that is a consequence of heavy drinking. Other potential markers of heavy drinking that are more nonspecific for alcohol but can help the clinician think of the possible effects of alcohol include elevations in blood levels or lipids (e.g., tri- glycerides and high-density lipoprotein cholesterol) and high-normal levels of uric acid.

1	Additional diagnostic markers relate to signs and symptoms that reﬂect the consequences often associated with persistent heavy drinking. For example, dyspepsia, nausea, and bloat— ing can accompany gastritis, and hepatomegaly, esophageal varices, and hemorrhoids may reﬂect alcohol-induced changes in the liver. Other physical signs of heavy drinking include tremor, unsteady gait, insomnia, and erectile dysfunction. Males with chronic alcohol use dis- order may exhibit decreased testicular size and feminizing effects associated with reduced testosterone levels. Repeated heavy drinking in females is associated with menstrual irregu- larities and, during pregnancy, spontaneous abortion and fetal alcohol syndrome. Individu- als with preexisting histories of epilepsy or severe head trauma are more likely to develop alcohol-related seizures. Alcohol withdrawal may be associated with nausea, vomiting, gas- tritis, hematemesis, dry mouth, puffy blotchy complexion, and mild peripheral edema.

1	Functional Consequences of Alcohol Use Disorder The diagnostic features of alcohol use disorder highlight major areas of life functioning likely to be impaired. These include driving and operating machinery, school and work, interpersonal relationships and communication, and health. Alcohol-related disorders contribute to absenteeism from work, job-related accidents, and low employee productiv- ity. Rates are elevated in homeless individuals, perhaps reﬂecting a downward spiral in social and occupational functioning, although most individuals with alcohol use disorder continue to live with their families and function within their jobs.

1	Alcohol use disorder is associated with a significant increase in the risk of accidents, vi— olence, and suicide. It is estimated that one in five intensive care unit admissions in some urban hospitals is related to alcohol and that 40% of individuals in the United States ex- perience an alcohol-related adverse event at some time in their lives, with alcohol account- ing for up to 55% of fatal driving events. Severe alcohol use disorder, especially in individuals with antisocial personality disorder, is associated with the commission of criminal acts, including homicide. Severe problematic alcohol use also contributes to dis- inhibition and feelings of sadness and irritability, which contribute to suicide attempts and completed suicides. alcohol use disorder has been overlooked can add to the risks and costs of hospitalization and to time spent in the hospital.

1	alcohol use disorder has been overlooked can add to the risks and costs of hospitalization and to time spent in the hospital. Nonpathological use of alcohol. The key element of alcohol use disorder is the use of heavy doses of alcohol with resulting repeated and significant distress or impaired func- tioning. While most drinkers sometimes consume enough alcohol to feel intoxicated, only a minority (less than 20%) ever develop alcohol use disorder. Therefore, drinking, even daily, in low doses and occasional intoxication do not by themselves make this diagnosis. Sedative, hypnotic, or anxiolytic use disorder. The signs and symptoms of alcohol use disorder are similar to those seen in sedative, hypnotic, or anxiolytic use disorder. The two must be distinguished, however, because the course may be different, especially in rela- tion to medical problems.

1	Conduct disorder in childhood and adult antisocial personality disorder. Alcohol use disorder, along with other substance use disorders, is seen in the majority of individuals with antisocial personality and preexisting conduct disorder. Because these diagnoses are associated with an early onset of alcohol use disorder as well as a worse prognosis, it is im- portant to establish both conditions.

1	Bipolar disorders, schizophrenia, and antisocial personality disorder are associated with a markedly increased rate of alcohol use disorder, and several anxiety and depressive disorders may relate to alcohol use disorder as well. At least a part of the reported association between depression and moderate to severe alcohol use disorder may be attributable to temporary, al- cohol-induced comorbid depressive symptoms resulting from the acute effects of intoxication or withdrawal. Severe, repeated alcohol intoxication may also suppress immune mechanisms and predispose individuals to infections and increase the risk for cancers. A. Recent ingestion of alcohol. B. Clinically significant problematic behavioral or psychological changes (e.g., inappropri- ate sexual or aggressive behavior, mood lability, impaired judgment) that developed during, or shortly after, alcohol ingestion. C. One (or more) of the following signs or symptoms developing during, or shortly after, alcohol use:

1	C. One (or more) of the following signs or symptoms developing during, or shortly after, alcohol use: Slurred speech. Incoordination. Unsteady gait. Nystagmus. Impairment in attention or memory. Stupor or coma. 9791:5939)? D. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication with another substance. Coding note: The |CD-9-CM code is 303.00. The |CD-10-CM code depends on whether there is a comorbid alcohol use disorder. It a mild alcohol use disorder is comorbid, the |CD-10-CM code is F10.129, and if a moderate or severe alcohol use disorder is comorbid, the |CD-10-CM code is F10.229. If there is no comorbid alcohol use disorder, then the |CD-10-CM code is F10.929.

1	The essential feature of alcohol intoxication is the presence of clinically significant problematic behavioral or psychological changes (e.g., inappropriate sexual or aggressive behavior, mood lability, impaired judgment, impaired social or occupational functioning) that develop during, or shortly after, alcohol ingestion (Criterion B). These changes are accompanied by evidence of impaired functioning and judgment and, if intoxication is intense, can result in a life-threaten— ing coma. The symptoms must not be attributable to another medical condition (e.g., diabetic ketoacidosis), are not a reﬂection of conditions such as delirium, and are not related to intoxi- cation with other depressant drugs (e.g., benzodiazepines) (Criterion D). The levels of incoor- dination can interfere with driving abilities and performance of usual activities to the point of causing accidents. Evidence of alcohol use can be obtained by smelling alcohol on the individ- ual’s breath, eliciting a history

1	driving abilities and performance of usual activities to the point of causing accidents. Evidence of alcohol use can be obtained by smelling alcohol on the individ- ual’s breath, eliciting a history from the individual or another observer, and, when needed, having the individual provide breath, blood, or urine samples for toxicology analyses.

1	Alcohol intoxication is sometimes associated with amnesia for the events that occurred during the course of the intoxication (”blackouts"). This phenomenon may be related to the presence of a high blood alcohol level and, perhaps, to the rapidity with which this level is reached. During even mild alcohol intoxication, different symptoms are likely to be observed at different time points. Evidence of mild intoxication with alcohol can be seen in most individuals after approximately two drinks (each standard drink is approximately 10—12 grams of ethanol and raises the blood alcohol concentration approximately 20 mg/ dL). Early in the drinking period, when blood alcohol levels are rising, symptoms often include talkativeness, a sensation of well-being, and a bright, expansive mood. Later, es- pecially when blood alcohol levels are falling, the individual is likely to become progres- sively more depressed, withdrawn, and cognitively impaired. At very high blood alcohol levels (e.g.,

1	es- pecially when blood alcohol levels are falling, the individual is likely to become progres- sively more depressed, withdrawn, and cognitively impaired. At very high blood alcohol levels (e.g., 200—300 mg/dL), an individual who has not developed tolerance for alcohol is likely to fall asleep and enter a first stage of anesthesia. Higher blood alcohol levels (e.g., in excess of 300—400 mg/dL) can cause inhibition of respiration and pulse and even death in nontolerant individuals. The duration of intoxication depends on how much alcohol was consumed over what period of time. In general, the body is able to metabolize approxi- mately one drink per hour, so that the blood alcohol level generally decreases at a rate of 15—20 mg/dL per hour. Signs and symptoms of intoxication are likely to be more intense when the blood alcohol level is rising than when it is falling.

1	Alcohol intoxication is an important contributor to suicidal behavior. There appears to be an increased rate of suicidal behavior, as well as of completed suicide, among persons intoxicated by alcohol. The large majority of alcohol consumers are likely to have been intoxicated to some degree at some point in their lives. For example, in 2010, 44% of 12th—grade students admitted to having been ”drunk in the past year,” with more than 70% of college students reporting the same. Intoxication usually occurs as an episode usually developing over minutes to hours and typi— cally lasting several hours. In the United States, the average age at first intoxication is approx— imately 15 years, with the highest prevalence at approximately 18—25 years. Frequency and intensity usually decrease with further advancing age. The earlier the onset of regular intoxi— cation, the greater the likelihood the individual will go on to develop alcohol use disorder.

1	Temperamental. Episodes of alcohol intoxication increase with personality characteris- tics of sensation seeking and impulsivity. Environmental. Episodes of alcohol intoxication increase with a heavy drinking envi- ronment. The major issues parallel the cultural differences regarding the use of alcohol overall. Thus, college fraternities and sororities may encourage alcohol intoxication. This condi- tion is also frequent on certain dates of cultural significance (e.g., New Year’s Eve) and, for some subgroups, during specific events (e.g., wakes following funerals). Other subgroups encourage drinking at religious celebrations (e.g., Jewish and Catholic holidays), while still others strongly discourage all drinking or intoxication (e.g., some religious groups, such as Mormons, fundamentalist Christians, and Muslims).

1	Historically, in many Western societies, acceptance of drinking and drunkenness is more tolerated for males, but such gender differences may be much less prominent in recent years, especially during adolescence and young adulthood. Intoxication is usually established by observing an individual’s behavior and smelling alcohol on the breath. The degree of intoxication increases with an individual’s blood or breath alcohol level and with the ingestion of other substances, especially those with sedating effects. Functional Consequences of Alcohol Intoxication Alcohol intoxication contributes to the more than 30,000 alcohol-related drinking deaths in the United States each year. In addition, intoxication with this drug contributes to huge costs associated with drunk driving, lost time from school or work, as well as interpersonal arguments and physical fights.

1	Other medical conditions. Several medical (e.g., diabetic acidosis) and neurological condi— tions (e.g., cerebellar ataxia, multiple sclerosis) can temporarily resemble alcohol intoxication.

1	Sedative, hypnotic, or anxiolytic intoxication. Intoxication with sedative, hypnotic, or anxiolytic drugs or with other sedating substances (e.g., antihistamines, anticholinergic drugs) can be mistaken for alcohol intoxication. The differential requires observing alco- hol on the breath, measuring blood or breath alcohol levels, ordering a medical workup, and gathering a good history. The signs and symptoms of sedative-hypnotic intoxication are very similar to those observed with alcohol and include similar problematic behavioral or psychological changes. These changes are accompanied by evidence of impaired func- tioning and judgment—Which, if intense, can result in a life-threatening coma—and levels of incoordination that can interfere with driving abilities and with performing usual activities. However, there is no smell as there is with alcohol, but there is likely to be evi- dence of misuse of the depressant drug in the blood or urine toxicology analyses.

1	Alcohol intoxication may occur comorbidly with other substance intoxication, especially in individuals with conduct disorder or antisocial personality disorder. A. Cessation of (or reduction in) alcohol use that has been heavy and prolonged. B. Two (or more) of the following, developing within several hours to a few days after the cessation of (or reduction in) alcohol use described in Criterion A: Autonomic hyperactivity (e.g.. sweating or pulse rate greater than 100 bpm). Increased hand tremor. Insomnia. Nausea or vomiting. Transient visual, tactile. or auditory hallucinations or illusions. Psychomotor agitation. Anxiety. Generalized tonic-clonic seizures. C. The signs or symptoms in Criterion B cause clinically significant distress or impairment in social, occupational, or other important areas of functioning.

1	Generalized tonic-clonic seizures. C. The signs or symptoms in Criterion B cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. D. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication or withdrawal from another substance. Specify it: With perceptual disturbances: This specifier applies in the rare instance when hal- lucinations (usually visual or tactile) occur with intact reality testing, or auditory, visual, or tactile illusions occur in the absence of a delirium.

1	Coding note: The |CD-9-CM code is 291.81. The |CD-10-CM code for alcohol withdrawal without perceptual disturbances is F10.239, and the |CD-10-CM code for alcohol withdrawal with perceptual disturbances is F10.232. Note that the |CD-10-CM code indicates the comor- bid presence of a moderate or severe alcohol use disorder, reflecting the fact that alcohol with- drawal can only occur in the presence of a moderate or severe alcohol use disorder. It is not permissible to code a comorbid mild alcohol use disorder with alcohol withdrawal. When hallucinations occur in the absence of delirium (i.e., in a clear sensorium), a diagno- sis of substance/medication-induced psychotic disorder should be considered.

1	When hallucinations occur in the absence of delirium (i.e., in a clear sensorium), a diagno- sis of substance/medication-induced psychotic disorder should be considered. The essential feature of alcohol withdrawal is the presence of a characteristic withdrawal syndrome that develops within several hours to a few days after the cessation of (or re- duction in) heavy and prolonged alcohol use (Criteria A and B). The withdrawal syn- drome includes two or more of the symptoms reflecting autonomic hyperactivity and anxiety listed in Criterion B, along with gastrointestinal symptoms.

1	Withdrawal symptoms cause clinically significant distress or impairment in social, oc- cupational, or other important areas of functioning (Criterion C). The symptoms must not be attributable to another medical condition and are not better explained by another men- tal disorder (e.g., generalized anxiety disorder), including intoxication or withdrawal from another substance (e.g., sedative, hypnotic, or anxiolytic withdrawal) (Criterion D). Symptoms can be relieved by administering alcohol or benzodiazepines (e.g., diazepam).

1	Symptoms can be relieved by administering alcohol or benzodiazepines (e.g., diazepam). The withdrawal symptoms typically begin when blood concentrations of alcohol decline sharply (i.e., within 4—12 hours) after alcohol use has been stopped or reduced. Reﬂecting the relatively fast metabolism of alcohol, symptoms of alcohol withdrawal usually peak in inten- sity during the second day of abstinence and are likely to improve markedly by the fourth or fifth day. Following acute withdrawal, however, symptoms of anxiety, insomnia, and auto- nomic dysfunction may persist for up to 3—6 months at lower levels of intensity. Fewer than 10% of individuals who develop alcohol withdrawal will ever develop dra- matic symptoms (e.g., severe autonomic hyperactivity, tremors, alcohol withdrawal delir- ium). Tonic-clonic seizures occur in fewer than 3% of individuals.

1	Although confusion and changes in consciousness are not core criteria for alcohol with- drawal, alcohol withdrawal delirium (see ”Delirium” in the chapter "Neurocognitive Dis- orders”) may occur in the context of withdrawal. As is true for any agitated, confused state, regardless of the cause, in addition to a disturbance of consciousness and cognition, with- drawal delirium can include visual, tactile, or (rarely) auditory hallucinations (delirium tre- mens). When alcohol withdrawal delirium develops, it is likely that a clinically relevant medical condition may be present (e.g., liver failure, pneumonia, gastrointestinal bleeding, sequelae of head trauma, hypoglycemia, an electrolyte imbalance, postoperative status).

1	It is estimated that approximately 50% of middle-class, highly functional individuals with alcohol use disorder have ever experienced a full alcohol withdrawal syndrome. Among individuals with alcohol use disorder who are hospitalized or homeless, the rate of al- cohol withdrawal may be greater than 80%. Less than 10% of individuals in withdrawal ever demonstrate alcohol withdrawal delirium or withdrawal seizures. Acute alcohol withdrawal occurs as an episode usually lasting 4—5 days and only after extended periods of heavy drinking. Withdrawal is relatively rare in individuals younger than 30 years, and the risk and severity increase with increasing age.

1	Environmental. The probability of developing alcohol withdrawal increases with the quantity and frequency of alcohol consumption. Most individuals with this condition are drinking daily, consuming large amounts (approximately more than eight drinks per day) for multiple days. However, there are large inter-individual differences, with enhanced risks for individuals with concurrent medical conditions, those with family histories of al- cohol withdrawal (i.e., a genetic component), those with prior withdrawals, and individ- uals who consume sedative, hypnotic, or anxiolytic drugs. Autonomic hyperactivity in the context of moderately high but falling blood alcohol levels and a history of prolonged heavy drinking indicate a likelihood of alcohol withdrawal. Functional Consequences of Alcohol Withdrawal

1	Functional Consequences of Alcohol Withdrawal Symptoms of withdrawal may serve to perpetuate drinking behaviors and contribute to relapse, resulting in persistently impaired social and occupational functioning. Symptoms work productivity. Overall, the presence of withdrawal is associated with greater func- tional impairment and poor prognosis. Other medical conditions. The symptoms of alcohol withdrawal can also be mimicked by some medical conditions (e.g., hypoglycemia and diabetic ketoacidosis). Essential tremor, a disorder that frequently runs in families, may erroneously suggest the tremu- lousness associated with alcohol withdrawal. Sedative, hypnotic, or anxiolytic withdrawal. Sedative, hypnotic, or anxiolytic with- drawal produces a syndrome very similar to that of alcohol withdrawal. Withdrawal is more likely to occur with heavier alcohol intake, and that might be most of— ten observed in individuals with conduct disorder and antisocial personality disorder.

1	Withdrawal is more likely to occur with heavier alcohol intake, and that might be most of— ten observed in individuals with conduct disorder and antisocial personality disorder. Withdrawal states are also more severe in older individuals, individuals who are also de- pendent on other depressant drugs (sedative-hypnotics), and individuals who have had more alcohol withdrawal experiences in the past.

1	The following alcohol-induced disorders are described in other chapters of the manual with disorders with which they share phenomenology (see the substance/medication-induced mental disorders in these chapters): alcohol—induced psychotic disorder (”Schizophrenia Spec- induced major or mild neurocognitive disorder (”Neurocognitive Disorders”). For alcohol intoxication delirium and alcohol withdrawal delirium, see the criteria and discussion of de- lirium in the chapter ”Neurocognitive Disorders.” These alcohol—induced disorders are diag- nosed instead of alcohol intoxication or alcohol withdrawal only when the symptoms are sufficiently severe to warrant independent clinical attention.

1	The symptom profiles for an alcohol-induced condition resemble independent mental disor- ders as described elsewhere in DSM-5. However, the alcohol—induced disorder is temporary and observed after severe intoxication with and / or withdrawal from alcohol. While the symp- toms can be identical to those of independent mental disorders (e.g., psychoses, major depres- sive disorder), and while they can have the same severe consequences (e.g., suicide attempts), alcohol-induced conditions are likely to improve without formal treatment in a matter of days to weeks after cessation of severe intoxication and / or withdrawal.

1	Each alcohol—induced mental disorder is listed in the relevant diagnostic section and there- fore only a brief description is offered here. Alcohol-induced disorders must have developed in the context of severe intoxication and / or withdrawal from the substance capable of produc- ing the mental disorder. In addition, there must be evidence that the disorder being observed is not likely to be better explained by another non-alcohol-induced mental disorder. The latter is likely to occur if the mental disorder was present before the severe intoxication or with- drawal, or continued more than 1 month after the cessation of severe intoxication and / or with- drawal. When symptoms are observed only during a delirium, they should be considered part of the delirium and not diagnosed separately, as many symptoms (including disturbances in mood, anxiety, and reality testing) are commonly seen during agitated, confused states. The a1- cohol-induced disorder must be clinically relevant, causing

1	as many symptoms (including disturbances in mood, anxiety, and reality testing) are commonly seen during agitated, confused states. The a1- cohol-induced disorder must be clinically relevant, causing significant levels of distress or sig- nificant functional impairment. Finally, there are indications that the intake of substances of abuse in the context of a preexisting mental disorder are likely to result in an intensification of the preexisting independent syndrome.

1	The features associated with each relevant major mental disorder (e.g., psychotic epi- sodes, major depressive disorder) are similar whether observed with an independent or an alcohol-induced condition. However, individuals with alcohol-induced disorders are likely to also demonstrate the associated features seen with an alcohol use disorder, as listed in the subsections of this chapter. Rates of alcohol-induced disorders vary somewhat by diagnostic category. For exam- ple, the lifetime risk for major depressive episodes in individuals with alcohol use disorder is approximately 40%, but only about one-third to one-half of these represent independent major depressive syndromes observed outside the context of intoxication. Similar rates of alcohol-induced sleep and anxiety conditions are likely, but alcohol-induced psychotic ep— isodes are fairly rare.

1	Once present, the symptoms of an alcohol-induced condition are likely to remain clinically relevant as long as the individual continues to experience severe intoxication and / or with- drawal. While the symptoms are identical to those of independent mental disorders (e.g., psychoses, major depressive disorder), and while they can have the same severe conse- quences (e.g., suicide attempts), all alcohol-induced syndromes other than alcohol- induced neurocognitive disorder, amnestic confabulatory type (alcohol-induced persist- ing amnestic disorder), regardless of the severity of the symptoms, are likely to improve relatively quickly and unlikely to remain clinically relevant for more than 1 month after cessation of severe intoxication and/ or withdrawal.

1	The alcohol-induced disorders are an important part of the differential diagnoses for the independent mental conditions. Independent schizophrenia, major depressive disor- der, bipolar disorder, and anxiety disorders, such as panic disorder, are likely to be asso- ciated with much longer-lasting periods of symptoms and often require longer-term medications to optimize the probability of improvement or recovery. The alcohol-induced conditions, on the other hand, are likely to be much shorter in duration and disappear within several days to 1 month after cessation of severe intoxication and / or withdrawal, even without psychotropic medications.

1	The importance of recognizing an alcohol-induced disorder is similar to the relevance of identifying the possible role of some endocrine conditions and medication reactions be- fore diagnosing an independent mental disorder. In light of the high prevalence of alcohol use disorders worldwide, it is important that these alcohol-induced diagnoses be consid- ered before independent mental disorders are diagnosed. 291.9 (F10.99) This category applies to presentations in which symptoms characteristic of an alcohol- related disorder that cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning predominate but do not meet the full criteria for any specific alcohoI-related disorder or any of the disorders in the substance-related and addictive disorders diagnostic class. Diagnostic Criteria 305.90 (F15.929) A. Flecent consumption of caffeine (typically a high dose well in excess of 250 mg).

1	Diagnostic Criteria 305.90 (F15.929) A. Flecent consumption of caffeine (typically a high dose well in excess of 250 mg). B. Five (or more) of the following signs or symptoms developing during, or shortly after, caffeine use: 1. Restlessness. 2. Nervousness. 3. Excitement. 4. Insomnia. 5. Flushed face. 6. Diuresis. 7. Gastrointestinaldisturbance. 8. Muscle twitching. 9. Rambling flow of thought and speech. 10. Tachycardia or cardiac arrhythmia. 11. Periods of inexhaustibility. 12. Psychomotor agitation. C. The signs or symptoms in Criterion B cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. D. The signs or symptoms are not attributable to another medical condition and are not bet- ter explained by another mental disorder, including intoxication with another substance.

1	D. The signs or symptoms are not attributable to another medical condition and are not bet- ter explained by another mental disorder, including intoxication with another substance. Caffeine can be consumed from a number of different sources, including coffee, tea, caf- feinated soda, "energy” drinks, over-the-counter analgesics and cold remedies, energy aids (e.g., drinks), weight-loss aids, and chocolate. Caffeine is also increasingly being used as an additive to vitamins and to food products. More than 85% of children and adults con- sume caffeine regularly. Some caffeine users display symptoms consistent with problem- atic use, including tolerance and withdrawal (see ”Caffeine Withdrawal” later in this chapter); the data are not available at this time to determine the clinical significance of a caffeine use disorder and its prevalence. In contrast, there is evidence that caffeine with- drawal and caffeine intoxication are clinically significant and sufficiently prevalent.

1	The essential feature of caffeine intoxication is recent consumption of caffeine and five and B). Symptoms include restlessness, nervousness, excitement, insomnia, ﬂushed face, diuresis, and gastrointestinal complaints, which can occur with low doses (e.g., 200 mg) in vulnerable individuals such as children, the elderly, or individuals who have not been ex- posed to caffeine previously. Symptoms that generally appear at levels of more than 1 g / day include muscle twitching, rambling flow of thought and speech, tachycardia or car- diac arrhythmia, periods of inexhaustibility, and psychomotor agitation. Caffeine intoxi- cation may not occur despite high caffeine intake because of the development of tolerance.

1	The signs or symptoms must cause clinically significant distress or impairment in social, occupational, or other important areas of functioning (Criterion C). The signs or symp- toms must not be attributable to another medical condition and are not better explained by another mental disorder (e.g., an anxiety disorder) or intoxication with another substance (Criterion D).

1	Mild sensory disturbances (e.g., ringing in the ears and ﬂashes of light) may occur with high doses of caffeine. Although large doses of caffeine can increase heart rate, smaller doses can slow heart rate. Whether excess caffeine intake can cause headaches is unclear. On physical examination, agitation, restlessness, sweating, tachycardia, flushed face, and increased bowel motility may be seen. Caffeine blood levels may provide important information for diagnosis, particularly when the individual is a poor historian, although these levels are not diagnostic by themselves in view of the individual variation in response to caffeine. The prevalence of caffeine intoxication in the general population is unclear. In the United States, approximately 7% of individuals in the population may experience five or more symp- toms along with functional impairment consistent with a diagnosis of caffeine intoxication.

1	States, approximately 7% of individuals in the population may experience five or more symp- toms along with functional impairment consistent with a diagnosis of caffeine intoxication. Consistent with a half-life of caffeine of approximately 4—6 hours, caffeine intoxication symptoms usually remit within the first day or so and do not have any known long-lasting consequences. However, individuals who consume very high doses of caffeine (i.e., 5—10 g) may require immediate medical attention, as such doses can be lethal.

1	With advancing age, individuals are likely to demonstrate increasingly intense reac- tions to caffeine, with greater complaints of interference with sleep or feelings of hyper- arousal. Caffeine intoxication among young individuals after consumption of highly caffeinated products, including energy drinks, has been observed. Children and adoles- cents may be at increased risk for caffeine intoxication because of low body weight, lack of tolerance, and lack of knowledge about the pharmacological effects of caffeine. Environmental. Caffeine intoxication is often seen among individuals who use caffeine tial amount. Furthermore, oral contraceptives significantly decrease the elimination of caf— feine and consequently may increase the risk of intoxication. Genetic and physiological. Genetic factors may affect risk of caffeine intoxication. Functional Consequences of Caffeine Intoxication

1	Genetic and physiological. Genetic factors may affect risk of caffeine intoxication. Functional Consequences of Caffeine Intoxication Impairment from caffeine intoxication may have serious consequences, including dys- function at work or school, social indiscretions, or failure to fulfill role obligations. More- over, extremely high doses of caffeine can be fatal. In some cases, caffeine intoxication may precipitate a caffeine-induced disorder.

1	Other mental disorders. Caffeine intoxication may be characterized by symptoms (e.g., panic attacks) that resemble primary mental disorders. To meet criteria for caffeine intoxica- tion, the symptoms must not be associated with another medical condition or another mental disorder, such as an anxiety disorder, that could better explain them. Manic episodes; panic disorder; generalized anxiety disorder; amphetamine intoxication; sedative, hypnotic, or anx- fects (e.g., akathisia) can cause a clinical picture that is similar to that of caffeine intoxication.

1	Other caffeine—induced disorders. The temporal relationship of the symptoms to increased caffeine use or to abstinence from caffeine helps to establish the diagnosis. Caffeine intoxica- tion is differentiated from caffeine-induced anxiety disorder, with onset during intoxication (see ”Substance/Medication-Induced Anxiety Disorder” in the chapter ”Anxiety Disorders”), and caffeine—induced sleep disorder, with onset during intoxication (see ”Substance/Medica- tion—Induced Sleep Disorder” in the chapter ”Sleep-Wake Disorders”), by the fact that the symptoms in these latter disorders are in excess of those usually associated with caffeine in- toxication and are severe enough to warrant independent clinical attention.

1	Typical dietary doses of caffeine have not been consistently associated with medical prob- lems. However, heavy use (e.g., >400 mg) can cause or exacerbate anxiety and somatic symptoms and gastrointestinal distress. With acute, extremely high doses of caffeine, grand mal seizures and respiratory failure may result in death. Excessive caffeine use is as- sociated with depressive disorders, bipolar disorders, eating disorders, psychotic disor- ders, sleep disorders, and substance-related disorders, whereas individuals with anxiety disorders are more likely to avoid caffeine. Diagnostic Criteria 292.0 (F15.93) A. Prolonged daily use of caffeine. B. Abrupt cessation of or reduction in caffeine use, followed within 24 hours by three (or more) of the following signs or symptoms: Headache. Marked fatigue or drowsiness. Dysphoric mood. depressed mood, or irritability. Difficulty concentrating. Flu— like symptoms (nausea vomiting, or muscle pain/stiffness).

1	Headache. Marked fatigue or drowsiness. Dysphoric mood. depressed mood, or irritability. Difficulty concentrating. Flu— like symptoms (nausea vomiting, or muscle pain/stiffness). C. The signs or symptoms' In Criterion B cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. D. The signs or symptoms are not associated with the physiological effects of another medical condition (e.g., migraine, viral illness) and are not better explained by another mental disorder, including intoxication or withdrawal from another substance.

1	The essential feature of caffeine withdrawal is the presence of a characteristic withdrawal syndrome that develops after the abrupt cessation of (or substantial reduction in) pro- longed daily caffeine ingestion (Criterion B). The caffeine withdrawal syndrome is indi- cated by three or more of the following (Criterion B): headache; marked fatigue or drowsiness; dysphoric mood, depressed mood, or irritability; difficulty concentrating; and ﬂu-like symptoms (nausea, vomiting, or muscle pain/stiffness). The withdrawal syn- drome causes clinical significant distress or impairment in social, occupational, or other important areas of functioning (Criterion C). The symptoms must not be associated with the physiological effects of another medical condition and are not better explained by an- other mental disorder (Criterion D).

1	Headache is the hallmark feature of caffeine withdrawal and may be diffuse, gradual in development, throbbing, severe, and sensitive to movement. However, other symptoms of caffeine withdrawal can occur in the absence of headache. Caffeine is the most widely used behaviorally active drug in the world and is present in many different types of bev- erages (e.g., coffee, tea, maté, soft drinks, energy drinks), foods, energy aids, medications, and dietary supplements. Because caffeine ingestion is often integrated into social customs and daily rituals (e.g., coffee break, tea time), some caffeine consumers may be unaware of their physical dependence on caffeine. Thus, caffeine withdrawal symptoms could be un- expected and misattributed to other causes (e.g., the ﬂu, migraine). Furthermore, caffeine withdrawal symptoms may occur when individuals are required to abstain from foods and beverages prior to medical procedures or when a usual caffeine dose is missed be- cause of a change in

1	caffeine withdrawal symptoms may occur when individuals are required to abstain from foods and beverages prior to medical procedures or when a usual caffeine dose is missed be- cause of a change in routine (e.g., during travel, weekends).

1	The probability and severity of caffeine withdrawal generally increase as a function of usual daily caffeine dose. However, there is large variability among individuals and within individuals across different episodes in the incidence, severity, and time course of withdrawal symptoms. Caffeine withdrawal symptoms may occur after abrupt cessation of relatively low chronic daily doses of caffeine (i.e., 100 mg). Caffeine abstinence has been shown to be associated with impaired behavioral and cogni- tive performance (e.g., sustained attention). Electroencephalographic studies have shown that caffeine withdrawal symptoms are significantly associated with increases in theta power and decreases in beta-2 power. Decreased motivation to work and decreased socia- bility have also been reported during caffeine withdrawal. Increased analgesic use during caffeine withdrawal has been documented.

1	More than 85% of adults and children in the United States regularly consume caffeine, with adult caffeine consumers ingesting about 280 mg/day on average. The incidence and prevalence of the caffeine withdrawal syndrome in the general population are unclear. In the United States, headache may occur in approximately 50% of cases of caffeine absti- nence. In attempts to permanently stop caffeine use, more than 7 0% of individuals may ex- perience at least one caffeine withdrawal symptom (47% may experience headache), and 24% may experience headache plus one or more other symptoms as well as functional impairment due to withdrawal. Among individuals who abstain from caffeine for at least 24 hours but are not trying to permanently stop caffeine use, 11% may experience head- ache plus one or more other symptoms as well as functional impairment. Caffeine con- sumers can decrease the incidence of caffeine withdrawal by using caffeine daily or only infrequently (e.g., no more than 2

1	one or more other symptoms as well as functional impairment. Caffeine con- sumers can decrease the incidence of caffeine withdrawal by using caffeine daily or only infrequently (e.g., no more than 2 consecutive days). Gradual reduction in caffeine over a period of days or weeks may decrease the incidence and severity of caffeine withdrawal.

1	Symptoms usually begin 12—24 hours after the last caffeine dose and peak after 1—2 days of abstinence. Caffeine withdrawal symptoms last for 2—9 days, with the possibility of withdrawal headaches occurring for up to 21 days. Symptoms usually remit rapidly (within 30—60 minutes) after re—ingestion of caffeine. Caffeine is unique in that it is a behaviorally active drug that is consumed by individ- uals of nearly all ages. Rates of caffeine consumption and overall level of caffeine con- sumption increase with age until the early to mid-305 and then level off. Although caffeine withdrawal among children and adolescents has been documented, relatively little is known about risk factors for caffeine withdrawal among this age group. The use of highly caffeinated energy drinks is increasing with in young individuals, which could increase the risk for caffeine withdrawal.

1	Temperamental. Heavy caffeine use has been observed among individuals with mental disorders, including eating disorders; smokers; prisoners; and drug and alcohol abusers. Thus, these individuals could be at higher risk for caffeine withdrawal upon acute caffeine abstinence. Environmental. The unavailability of caffeine is an environmental risk factor for incipi- ent withdrawal symptoms. While caffeine is legal and usually widely available, there are conditions in which caffeine use may be restricted, such as during medical procedures, pregnancy, hospitalizations, religious Observances, wartime, travel, and research partici- pation. These external environmental circumstances may precipitate a withdrawal syn- drome in vulnerable individuals. Genetic and physiological factors. Genetic factors appear to increase vulnerability to caffeine withdrawal, but no specific genes have been identified.

1	Genetic and physiological factors. Genetic factors appear to increase vulnerability to caffeine withdrawal, but no specific genes have been identified. Course modifiers. Caffeine withdrawal symptoms usually remit within 30—60 minutes of reexposure to caffeine. Doses of caffeine significantly less than one’s usual daily dose may be sufficient to prevent or attenuate caffeine withdrawal symptoms (e.g., consump- tion of 25 mg by an individual who typically consumes 300 mg). feine withdrawal.

1	feine withdrawal. Caffeine withdrawal symptoms can vary from mild to extreme, at times causing functional impairment in normal daily activities. Rates of functional impairment range from 10% to 55% (median 13%), with rates as high as 73% found among individuals who also show other problematic features of caffeine use. Examples of functional impairment include be- ing unable to work, exercise, or care for children; staying in bed all day; missing religious services; ending a vacation early; and cancelling a social gathering. Caffeine withdrawal headaches may be described by individuals as ”the worst headaches” ever experienced. Decrements in cognitive and motor performance have also been observed.

1	Decrements in cognitive and motor performance have also been observed. Other medical disorders and medical side effects. Several disorders should be consid- ered in the differential diagnosis of caffeine withdrawal. Caffeine withdrawal can mimic migraine and other headache disorders, viral illnesses, sinus conditions, tension, other drug withdrawal states (e.g., from amphetamines, cocaine), and medication side effects. The final determination of caffeine withdrawal should rest on a determination of the pat- tern and amount consumed, the time interval between caffeine abstinence and onset of symptoms, and the particular clinical features presented by the individual. A challenge dose of caffeine followed by symptom remission may be used to confirm the diagnosis.

1	Caffeine withdrawal may be associated with major depressive disorder, generalized anx- iety disorder, panic disorder, antisocial personality disorder in adults, moderate to severe alcohol use disorder, and cannabis and cocaine use. The following caffeine-induced disorders are described in other chapters of the manual with disorders with which they share phenomenology (see the substance/medication- induced mental disorders in these chapters): caffeine-induced anxiety disorder (”Anxiety Disorders”) and caffeine-induced sleep disorder (”Sleep-Wake Disorders"). These caf- feine-induced disorders are diagnosed instead of caffeine intoxication or caffeine with- drawal only when the symptoms are sufficiently severe to warrant independent clinical attention. 292.9 (F15.99)

1	292.9 (F15.99) This category applies to presentations in which symptoms characteristic of a caffeine- related disorder that cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning predominate but do not meet the full criteria for any specific caffeine-related disorder or any of the disorders in the substance-related and addictive disorders diagnostic class. A. A problematic pattern of cannabis use leading to clinically significant impairment or dis- tress, as manifested by at least two of the following, occurring within a 12-month period: 1. 2. 3. 10. 11. Cannabis is often taken in larger amounts or over a longer period than was intended. There is a persistent desire or unsuccessful efforts to cut down or control cannabis use. A great deal of time is spent in activities necessary to obtain cannabis, use canna- bis, or recover from its effects. Craving, or a strong desire or urge to use cannabis.

1	A great deal of time is spent in activities necessary to obtain cannabis, use canna- bis, or recover from its effects. Craving, or a strong desire or urge to use cannabis. Recurrent cannabis use resulting in a failure to fulfill major role obligations at work, school, or home. sonal problems caused or exacerbated by the effects of cannabis. Important social. occupational. or recreational activities are given up or reduced be- cause of cannabis use. Recurrent cannabis use in situations in which it is physically hazardous. Cannabis use is continued despite knowledge of having a persistent or recurrent physical or psychological problem that is likely to have been caused or exacerbated by cannabis. Tolerance, as defined by either of the following: a. A need for markedly increased amounts of cannabis to achieve intoxication or desired effect. b. Markedly diminished effect with continued use of the same amount of cannabis.

1	b. Markedly diminished effect with continued use of the same amount of cannabis. Withdrawal, as manifested by either of the following: a. The characteristic withdrawal syndrome for cannabis (refer to Criteria A and B of the criteria set for cannabis withdrawal. pp. 517—518). b. Cannabis (or a closely related substance) is taken to relieve or avoid withdrawal symptoms. Specify it: In early remission: After full criteria for cannabis use disorder were previously met, none of the criteria for cannabis use disorder have been met for at least 3 months but for less than 12 months (with the exception that Criterion A4, "Craving, or a strong de- sire or urge to use cannabis,” may be met).

1	In sustained remission: After full criteria for cannabis use disorder were previously met, none of the criteria for cannabis use disorder have been met at any time during a period of 12 months or longer (with the exception that Criterion A4, “Craving. or a strong desire or urge to use cannabis," may be present). Specify it: In a controlled environment: This additional specifier is used it the individual is in an environment where access to cannabis is restricted.

1	Specify it: In a controlled environment: This additional specifier is used it the individual is in an environment where access to cannabis is restricted. Code based on current severity: Note for |CD-10-CM codes: If a cannabis intoxication, cannabis withdrawal, or another cannabis-induced mental disorder is also present, do not use the codes below for cannabis use disorder. Instead, the comorbid cannabis use disorder is indicated in the 4th character of the cannabis-induced disorder code (see the coding note for cannabis intoxication. cannabis withdrawal. or a specific cannabis-induced mental disor- der). For example. if there is comorbid cannabis-induced anxiety disorder and cannabis use disorder, only the cannabis-induced anxiety disorder code is given. with the 4th character indicating whether the comorbid cannabis use disorder is mild, moderate, or severe:

1	F12.180 for mild cannabis use disorder with cannabis-induced anxiety disorder or F12.280 for a moderate or severe cannabis use disorder with cannabis-induced anxiety disorder. Specify current severity: 305.20 (F12.10) Mild: Presence of 2-3 symptoms. 304.30 (F12.20) Moderate: Presence of 4—5 symptoms. 304.30 (F12.20) Severe: Presence of 6 or more symptoms. ”In a controlled environment" applies as a further specifier of remission if the individual is both in remission and in a controlled environment (i.e., in early remission in a controlled environment or in sustained remission in a controlled environment). Examples of these environments are closely supervised and substance-free jails, therapeutic communities, and locked hospital units. frequency (e.g., days of use per month or times used per day) and / or dose (e.g., amount used per episode) of cannabis, as assessed by individual self—report, report of knowledge- able others, clinician’s observations, and biological testing.

1	Cannabis use disorder and the other cannabis-related disorders include problems that are associated with substances derived from the cannabis plant and chemically similar syn- thetic compounds. Over time, this plant material has accumulated many names (e.g., weed, pot, herb, grass, reefer, mary jane, dagga, dope, bhang, skunk, boom, gangster, kit, and ganja). A concentrated extraction of the cannabis plant that is also commonly used is hashish. Cannabis is the generic and perhaps the most appropriate scientific term for the psychoactive substance(s) derived from the plant, and as such it is used in this manual to refer to all forms of cannabis-like substances, including synthetic cannabinoid com- pounds. Synthetic oral formulations (pill/capsules) of delta-9-tetrahydrocannabinol (delta-9-

1	Synthetic oral formulations (pill/capsules) of delta-9-tetrahydrocannabinol (delta-9- THC) are available by prescription for a number of approved medical indications (e.g., for with AIDS). Other synthetic cannabinoid compounds have been manufactured and dis- tributed for nonmedical use in the form of plant material that has been sprayed with a can- nabinoid formulation (e.g., K2, Spice, IWH-018, IWH-073). The cannabinoids have diverse effects in the brain, prominent among which are actions on C31 and C32 cannabinoid receptors that are found throughout the central nervous sys- tem. Endogenous ligands for these receptors behave essentially like neurotransmitters. The potency of cannabis (delta-9-THC concentration) that is generally available varies greatly, ranging from 1% to approximately 15% in typical cannabis plant material and 10%—20% in hashish. During the past two decades, a steady increase in the potency of seized cannabis has been observed.

1	Cannabis is most commonly smoked via a variety of methods: pipes, water pipes (bongs or hookahs), cigarettes (joints or reefers), or, most recently, in the paper from hol- lowed out cigars (blunts). Cannabis is also sometimes ingested orally, typically by mixing it into food. More recently, devices have been developed in which cannabis is "vapor- ized.” Vaporization involves heating the plant material to release psychoactive cannabi- noids for inhalation. As with other psychoactive substances, smoking (and vaporization) typically produces more rapid onset and more intense experiences of the desired effects.

1	Individuals who regularly use cannabis can develop all the general diagnostic features of a substance use disorder. Cannabis use disorder is commonly observed as the only sub— stance use disorder experienced by the individual; however, it also frequently occurs con- currently with other types of substance use disorders (i.e., alcohol, cocaine, opioid). In cases for which multiple types of substances are used, many times the individual may minimize the symptoms related to cannabis, as the symptoms may be less severe or cause less harm than those directly related to the use of the other substances. Pharmacological and behavioral tolerance to most of the effects of cannabis has been reported in individuals who use cannabis persistently. Generally, tolerance is lost when cannabis use is discontin- ued for a significant period of time (i.e., for at least several months).

1	New to DSM—5 is the recognition that abrupt cessation of daily or near-daily cannabis use often results in the onset of a cannabis withdrawal syndrome. Common symptoms of withdrawal include irritability, anger or aggression, anxiety, depressed mood, restless- ness, sleep difficulty, and decreased appetite or weight loss. Although typically not as severe as alcohol or opiate withdrawal, the cannabis withdrawal syndrome can cause sig- nificant distress and contribute to difficulty quitting or relapse among those trying to abstain. Individuals with cannabis use disorder may use cannabis throughout the day over a period of months or years, and thus may spend many hours a day under the inﬂuence.

1	Others may use less frequently, but their use causes recurrent problems related to family, school, work, or other important activities (e.g., repeated absences at work; neglect of fam- ily obligations). Periodic cannabis use and intoxication can negatively affect behavioral and cognitive functioning and thus interfere with optimal performance at work or school, or place the individual at increased physical risk when performing activities that could be physically hazardous (e.g., driving a car; playing certain sports; performing manual work activities, including operating machinery). Arguments with spouses or parents over the use of cannabis in the home, or its use in the presence of children, can adversely impact family functioning and are common features of those with cannabis use disorder. Last, in- dividuals with cannabis use disorder may continue using despite knowledge of physical problems (e.g., chronic cough related to smoking) or psychological problems (e.g., exces- sive

1	Last, in- dividuals with cannabis use disorder may continue using despite knowledge of physical problems (e.g., chronic cough related to smoking) or psychological problems (e.g., exces- sive sedation or exacerbation of other mental health problems) associated with its use.

1	Whether or not cannabis is being used for legitimate medical reasons may also affect diagnosis. When a substance is taken as indicated for a medical condition, symptoms of tolerance and withdrawal will naturally occur and should not be used as the primary cri- teria for determining a diagnosis of a substance use disorder. Although medical uses of cannabis remain controversial and equivocal, use for medical circumstances should be considered when a diagnosis is being made.

1	Individuals who regularly use cannabis often report that it is being used to cope with mood, sleep, pain, or other physiological or psychological problems, and those diagnosed with cannabis use disorder frequently do have concurrent other mental disorders. Careful assessment typically reveals reports of cannabis use contributing to exacerbation of these same symptoms, as well as other reasons for frequent use (e.g., to experience euphoria, to forget about problems, in response to anger, as an enjoyable social activity). Related to this issue, some individuals who use cannabis multiple times per day for the aforementioned reasons do not perceive themselves as (and thus do not report) spending an excessive amount of time under the inﬂuence or recovering from the effects of cannabis, despite be- ing intoxicated on cannabis or coming down from it effects for the majority of most days.

1	An important marker of a substance use disorder diagnosis, particularly in milder cases, is continued use despite a clear risk of negative consequences to other valued activities or re- lationships (e.g., school, work, sport activity, partner or parent relationship). Because some cannabis users are motivated to minimize their amount or frequency of use, it is important to be aware of common signs and symptoms of cannabis use and intox- ication so as to better assess the extent of use. As with other substances, experienced users to detect when they are under the inﬂuence. Signs of acute and chronic use include red eyes (conjunctival injection), cannabis odor on clothing, yellowing of finger tips (from smoking joints), chronic cough, burning of incense (to hide the odor), and exaggerated craving and impulse for specific foods, sometimes at unusual times of the day or night.

1	Cannabinoids, especially cannabis, are the most widely used illicit psychoactive sub- stances in the United States. The 12-month prevalence of cannabis use disorder (DSM-IV abuse and dependence rates combined) is approximately 3.4% among 12- to 17—year-olds and 1.5% among adults age 18 years and older. Rates of cannabis use disorder are greater among adult males (2.2%) than among adult females (0.8%) and among 12- to 17-year-old males (3.8%) than among 12- to 17—year-old females (3.0%). Twelve-month prevalence rates of cannabis use disorder among adults decrease with age, with rates highest among 18- to 29-year—olds (4.4%) and lowest among individuals age 65 years and older (0.01%). The high prevalence of cannabis use disorder likely reﬂects the much more widespread use of cannabis relative to other illicit drugs rather than greater addictive potential.

1	The high prevalence of cannabis use disorder likely reﬂects the much more widespread use of cannabis relative to other illicit drugs rather than greater addictive potential. Ethnic and racial differences in prevalence are moderate. Twelve-month prevalences of cannabis use disorder vary markedly across racial-ethnic subgroups in the United

1	Ethnic and racial differences in prevalence are moderate. Twelve-month prevalences of cannabis use disorder vary markedly across racial-ethnic subgroups in the United States. For 12- to 17-year-olds, rates are highest among Native American and Alaska Na- tives (7.1%) compared with Hispanics (4.1%), whites (3.4%), African Americans (2.7%), and Asian Americans and Pacific Islanders (0.9%). Among adults, the prevalence of can- nabis use disorder is also highest among Native Americans and Alaska Natives (3.4%) rel- ative to rates among African Americans (1.8%), whites (1.4%), Hispanics (1.2%), and Asian and Pacific Islanders (1.2%). During the past decade the prevalence of cannabis use disor- der has increased among adults and adolescents. Gender differences in cannabis use dis- order generally are concordant with those in other substance use disorders. Cannabis use disorder is more commonly observed in males, although the magnitude of this difference is less among adolescents.

1	The onset of cannabis use disorder can occur at any time during or following adolescence, but onset is most commonly during adolescence or young adulthood. Although much less frequent, onset of cannabis use disorder in the preteen years or in the late 205 or older can occur. Recent acceptance by some of the use and availability of “medical marijuana” may increase the rate of onset of cannabis use disorder among older adults. Generally, cannabis use disorder develops over an extended period of time, although the progression appears to be more rapid in adolescents, particularly those with pervasive conduct problems. Most people who develop a cannabis use disorder typically establish a pattern of cannabis use that gradually increases in both frequency and amount. Cannabis, along with tobacco and alcohol, is traditionally the first substance that adolescents try.

1	Many perceive cannabis use as less harmful than alcohol or tobacco use, and this percep- tion likely contributes to increased use. Moreover, cannabis intoxication does not typically result in as severe behavioral and cognitive dysfunction as does significant alcohol intox— ication, which may increase the probability of more frequent use in more diverse situa- tions than with alcohol. These factors likely contribute to the potential rapid transition from cannabis use to a cannabis use disorder among some adolescents and the common pattern of using throughout the day that is commonly observed among those with more severe cannabis use disorder.

1	Cannabis use disorder among preteens, adolescents, and young adults is typically ex- pressed as excessive use with peers that is a component of a pattern of other delinquent behaviors usually associated with conduct problems. Milder cases primarily reﬂect con- tinued use despite clear problems related to disapproval of use by other peers, school ad- ministration, or family, which also places the youth at risk for physical or behavioral consequences. In more severe cases, there is a progression to using alone or using through— out the day such that use interferes with daily functioning and takes the place of previ- ously established, prosocial activities.

1	With adolescent users, changes in mood stability, energy level, and eating patterns are commonly observed. These signs and symptoms are likely due to the direct effects of can- nabis use (intoxication) and the subsequent effects following acute intoxication (coming down), as well as attempts to conceal use from others. School-related problems are com— monly associated with cannabis use disorder in adolescents, particularly a dramatic drop in grades, truancy, and reduced interest in general school activities and outcomes.

1	Cannabis use disorder among adults typically involves well-established patterns of daily cannabis use that continue despite clear psychosocial or medical problems. Many adults have experienced repeated desire to stop or have failed at repeated cessation attempts. Milder adult cases may resemble the more common adolescent cases in that cannabis use is not as frequent or heavy but continues despite potential significant consequences of sustained use. The rate of use among middle-age and older adults appears to be increasing, likely because of a cohort ef— fect resulting from high prevalence of use in the late 1960s and the 19705.

1	Early onset of cannabis use (e.g., prior to age 15 years) is a robust predictor of the de- velopment of cannabis use disorder and other types of substance use disorders and mental disorders during young adulthood. Such early onset is likely related to concurrent other externalizing problems, most notably conduct disorder symptoms. However, early onset is also a predictor of internalizing problems and as such probably reﬂects a general risk factor for the development of mental health disorders.

1	Temperamental. A history of conduct disorder in childhood or adolescence and antiso- cial personality disorder are risk factors for the development of many substance-related disorders, including cannabis-related disorders. Other risk factors include externalizing or internalizing disorders during childhood or adolescence. Youths with high behavioral disinhibition scores show early-onset substance use disorders, including cannabis use dis- order, multiple substance involvement, and early conduct problems. Environmental. Risk factors include academic failure, tobacco smoking, unstable or abu- sive family situation, use of cannabis among immediate family members, a family history of a substance use disorder, and low socioeconomic status. As with all substances of abuse, the ease of availability of the substance is a risk factor; cannabis is relatively easy to obtain in most cultures, which increases the risk of developing a cannabis use disorder.

1	Genetic and physiological. Genetic inﬂuences contribute to the development of canna- bis use disorders. Heritable factors contribute between 30% and 80% of the total variance in risk of cannabis use disorders. It should be noted that common genetic and shared en- vironmental inﬂuences between cannabis and other types of substance use disorders sug- gest a common genetic basis for adolescent substance use and conduct problems. Cannabis is probably the world’s most commonly used illicit substance. Occurrence of cannabis use disorder across countries is unknown, but the prevalence rates are likely sim- ilar among developed countries. It is frequently among the first drugs of experimentation (often in the teens) of all cultural groups in the United States. Acceptance of cannabis for medical purposes varies widely across and within cultures.

1	Acceptance of cannabis for medical purposes varies widely across and within cultures. Cultural factors (acceptability and legal status) that might impact diagnosis relate to dif- ferential consequences across cultures for detection of use (i.e., arrest, school suspensions, or employment suspension). The general change in substance use disorder diagnostic cri- teria from DSM-IV to DSM-5 (i.e., removal of the recurrent substance-related legal prob- lems criterion) mitigates this concern to some degree.

1	Biological tests for cannabinoid metabolites are useful for determining if an individual has recently used cannabis. Such testing is helpful in making a diagnosis, particularly in milder cases if an individual denies using while others (family, work, school) purport con- cern about a substance use problem. Because cannabinoids are fat soluble, they persist in bodily ﬂuids for extended periods of time and are excreted slowly. Expertise in urine test- ing methods is needed to reliably interpret results. Functional Consequences of Cannabis Use Disorder

1	Functional consequences of cannabis use disorder are part of the diagnostic criteria. Many areas of psychosocial, cognitive, and health functioning may be compromised in relation to cannabis use disorder. Cognitive function, particularly higher executive function, ap- pears to be compromised in cannabis users, and this relationship appears to be dose de- pendent (both acutely and chronically). This may contribute to increased difficulty at school or work. Cannabis use has been related to a reduction in prosocial goal-directed ac- tivity, which some have labeled an amotivational syndrome, that manifests itself in poor school performance and employment problems. These problems may be related to perva- sive intoxication or recovery from the effects of intoxication. Similarly, cannabis-associated problems with social relationships are commonly reported in those with cannabis use dis- order. Accidents due to engagement in potentially dangerous behaviors while under the inﬂuence (e.g.,

1	problems with social relationships are commonly reported in those with cannabis use dis- order. Accidents due to engagement in potentially dangerous behaviors while under the inﬂuence (e.g., driving, sport, recreational or employment activities) are also of concern.

1	Cannabis smoke contains high levels of carcinogenic compounds that place chronic users at risk for respiratory illnesses similar to those experienced by tobacco smokers. Chronic cannabis use may contribute to the onset or exacerbation of many other mental disorders. In particular, concern has been raised about cannabis use as a causal factor in schizophrenia and other psychotic disorders. Cannabis use can contribute to the onset of an acute psy- chotic episode, can exacerbate some symptoms, and can adversely affect treatment of a major psychotic illness.

1	Nonproblematic use of cannabis. The distinction between nonproblematic use of can- nabis and cannabis use disorder can be difficult to make because social, behavioral, or psy- chological problems may be difficult to attribute to the substance, especially in the context of use of other substances. Also, denial of heavy cannabis use and the attribution that can- nabis is related to or causing substantial problems are common among individuals who are referred to treatment by others (i.e., school, family, employer, criminal justice system).

1	Other mental disorders. Cannabis-induced disorder may be characterized by symp- toms (e.g., anxiety) that resemble primary mental disorders (e.g., generalized anxiety dis- order vs. cannabis-induced anxiety disorder, with generalized anxiety, with onset during intoxication). Chronic intake of cannabis can produce a lack of motivation that resembles persistent depressive disorder (dysthymia). Acute adverse reactions to cannabis should be differentiated from the symptoms of panic disorder, major depressive disorder, delusional disorder, bipolar disorder, or schizophrenia, paranoid type. Physical examination will usually show an increased pulse and conjunctival injection. Urine toxicological testing can be helpful in making a diagnosis.

1	Cannabis has been commonly thought of as a ”gateway” drug because individuals who frequently use cannabis have a much greater lifetime probability than nonusers of using what are commonly considered more dangerous substances, like opioids or cocaine. Can- nabis use and cannabis use disorder are highly comorbid with other substance use disor- ders. Co-occurring mental conditions are common in cannabis use disorder. Cannabis use has been associated with poorer life satisfaction; increased mental health treatment and hospitalization; and higher rates of depression, anxiety disorders, suicide attempts, and conduct disorder. Individuals with past-year or lifetime cannabis use disorder have high rates of alcohol use disorder (greater than 50%) and tobacco use disorder (53%). Rates of other substance use disorders are also likely to be high among individuals with cannabis use disorder. Among those seeking treatment for a cannabis use disorder, 74% report problematic use of a secondary or

1	use disorders are also likely to be high among individuals with cannabis use disorder. Among those seeking treatment for a cannabis use disorder, 74% report problematic use of a secondary or tertiary substance: alcohol (40%), cocaine (12%), meth- amphetamine (6%), and heroin or other opiates (2%). Among those younger than 18 years, 61% reported problematic use of a secondary substance: alcohol (48%), cocaine (4%), meth- amphetamine (2%), and heroin or other opiates (2%). Cannabis use disorder is also often observed as a secondary problem among those with a primary diagnosis of other substance use disorders, with approximately 25%—80% of those in treatment for another substance use disorder reporting use of cannabis.

1	Individuals with past-year or lifetime diagnoses of cannabis use disorder also have high rates of concurrent mental disorders other than substance use disorders. Major de- pressive disorder (11%), any anxiety disorder (24%), and bipolar I disorder (13%) are quite common among individuals with a past-year diagnosis of a cannabis use disorder, as are antisocial (30%), obsessive-compulsive, (19%), and paranoid (18%) personality disorders. Approximately 33% of adolescents with cannabis use disorder have internalizing disor- ders (e.g., anxiety, depression, posttraumatic stress disorder), and 60% have externalizing disorders (e.g., conduct disorder, attention-deficit/hyperactivity disorder).

1	Although cannabis use can impact multiple aspects of normal human functioning, in- cluding the cardiovascular, immune, neuromuscular, ocular, reproductive, and respira- tory systems, as well as appetite and cognition/perception, there are few clear medical conditions that commonly co-occur with cannabis use disorder. The most significant health effects of cannabis involve the respiratory system, and chronic cannabis smokers exhibit high rates of respiratory symptoms of bronchitis, sputum production, shortness of breath, and wheezing. A. Recent use of cannabis. B. Clinically significant problematic behavioral or psychological changes (e.g., impaired motor coordination, euphoria, anxiety, sensation of slowed time, impaired judgment, social withdrawal) that developed during, or shortly after. cannabis use. C. Two (or more) of the following signs or symptoms developing within 2 hours of canna- bis use: 1. Conjunctival injection. 2. Increased appetite. 3. Dry mouth. 4. Tachycardia.

1	C. Two (or more) of the following signs or symptoms developing within 2 hours of canna- bis use: 1. Conjunctival injection. 2. Increased appetite. 3. Dry mouth. 4. Tachycardia. D. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication with another substance. Specify if: With perceptual disturbances: Hallucinations with intact reality testing or auditory, vi- sual. or tactile illusions occur in the absence of a delirium. Coding note: The ICD-9-CM code is 292.89. The |CD-10-CM code depends on whether or not there is a comorbid cannabis use disorder and whether or not there are perceptual disturbances.

1	Coding note: The ICD-9-CM code is 292.89. The |CD-10-CM code depends on whether or not there is a comorbid cannabis use disorder and whether or not there are perceptual disturbances. For cannabis intoxication, without perceptual disturbances: If a mild cannabis use disorder is comorbid, the lCD-10-CM code is F12.129, and if a moderate or severe cannabis use disorder is comorbid, the |CD-10-CM code is F12.229. If there is no co- morbid cannabis use disorder, then the |CD-10-CM code is F12.929. For cannabis intoxication, with perceptual disturbances: If a mild cannabis use disorder is comorbid, the |CD-10-CM code is F12.122, and if a moderate or severe cannabis use disorder is comorbid, the |CD-10-CM code is F12.222. If there is no co- morbid cannabis use disorder, then the |CD-10-CM code is F12.922. When hallucinations occur in the absence of intact reality testing, a diagnosis of substance/ medication—induced psychotic disorder should be considered.

1	The essential feature of cannabis intoxication is the presence of clinically significant prob- lematic behavioral or psychological changes that develop during, or shortly after, canna- bis use (Criterion B). Intoxication typically begins with a ”high" feeling followed by symptoms that include euphoria with inappropriate laughter and grandiosity, sedation, lethargy, impairment in short-term memory, difficulty carrying out complex mental pro- cesses, impaired judgment, distorted sensory perceptions, impaired motor performance, and the sensation that time is passing slowly. Occasionally, anxiety (which can be severe), dysphoria, or social withdrawal occurs. These psychoactive effects are accompanied by two or more of the following signs, developing within 2 hours of cannabis use: conjuncti- val injection, increased appetite, dry mouth, and tachycardia (Criterion C).

1	Intoxication develops within minutes if the cannabis is smoked but may take a few hours to develop if the cannabis is ingested orally. The effects usually last 3—4 hours, with the duration being somewhat longer when the substance is ingested orally. The magnitude of the behavioral and physiological changes depends on the dose, the method of adminis- tration, and the characteristics of the individual using the substance, such as rate of absorp- tion, tolerance, and sensitivity to the effects of the substance. Because most cannabinoids, including delta-9—tetrahydrocannabinol (delta-9-THC), are fat soluble, the effects of canna- bis or hashish may occasionally persist or reoccur for 12—24 hours because of the slow re- lease of psychoactive substances from fatty tissue or to enterohepatic circulation.

1	The prevalence of actual episodes of cannabis intoxication in the general population is un- known. However, it is probable that most cannabis users would at some time meet criteria for cannabis intoxication. Given this, the prevalence of cannabis users and the prevalence of individuals experiencing cannabis intoxication are likely similar. Functional Consequences of Cannabis Intoxication Impairment from cannabis intoxication may have serious consequences, including dys- function at work or school, social indiscretions, failure to fulfill role obligations, traffic ac- cidents, and having unprotected sex. In rare cases, cannabis intoxication may precipitate a psychosis that may vary in duration. Note that if the clinical presentation includes hallucinations in the absence of intact reality testing, a diagnosis of substance/medication-induced psychotic disorder should be con- sidered.

1	Note that if the clinical presentation includes hallucinations in the absence of intact reality testing, a diagnosis of substance/medication-induced psychotic disorder should be con- sidered. Other substance intoxication. Cannabis intoxication may resemble intoxication with other types of substances. However, in contrast to cannabis intoxication, alcohol intoxica- tion and sedative, hypnotic, or anxiolytic intoxication frequently decrease appetite, in- crease aggressive behavior, and produce nystagmus or ataxia. Hallucinogens in low doses may cause a clinical picture that resembles cannabis intoxication. Phencyclidine, like can— nabis, can be smoked and also causes perceptual changes, but phencyclidine intoxication is much more likely to cause ataxia and aggressive behavior.

1	Other cannabis-induced disorders. Cannabis intoxication is distinguished from the other cannabis-induced disorders (e.g., cannabis-induced anxiety disorder, with onset during intoxication) because the symptoms in these latter disorders predominate the clinical pre- sentation and are severe enough to warrant independent clinical attention. Diagnostic Criteria 292.0 (F12.288) A. Cessation of cannabis use that has been heavy and prolonged (i.e., usually daily or almost daily use over a period of at least a few months). B. Three (or more) of the following signs and symptoms develop within approximately 1 week after Criterion A: Irritability, anger, or aggression. Nervousness or anxiety. Sleep difficulty (e.g., insomnia, disturbing dreams). Decreased appetite or weight loss. Restlessness. Depressed mood. At least one of the following physical symptoms causing significant discomfort: ab- dominal pain, shakiness/tremors, sweating, fever, chills, or headache. .‘IP’S’IPPNT‘

1	Depressed mood. At least one of the following physical symptoms causing significant discomfort: ab- dominal pain, shakiness/tremors, sweating, fever, chills, or headache. .‘IP’S’IPPNT‘ C. The signs or symptoms in Criterion B cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. D. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication or withdrawal from another substance. Coding note: The |CD-9-CM code is 292.0. The |CD-10-CM code for cannabis withdrawal is F12.288. Note that the |CD-10-CM code indicates the comorbid presence of a moderate or severe cannabis use disorder, reflecting the fact that cannabis withdrawal can only oc- cur in the presence of a moderate or severe cannabis use disorder. It is not permissible to code a comorbid mild cannabis use disorder with cannabis withdrawal.

1	The essential feature of cannabis withdrawal is the presence of a characteristic withdrawal syndrome that develops after the cessation of or substantial reduction in heavy and pro- longed cannabis use. In addition to the symptoms in Criterion B, the following may also be observed postabstinence: fatigue, yawning, difficulty concentrating, and rebound periods of increased appetite and hypersomnia that follow initial periods of loss of appetite and in- somnia. For the diagnosis, withdrawal symptoms must cause clinically significant distress or impairment in social, occupational, or other important areas of functioning (Criterion

1	C). Many cannabis users report smoking cannabis or taking other substances to help re- lieve withdrawal symptoms, and many report that withdrawal symptoms make quitting difficult or have contributed to relapse. The symptoms typically are not of sufficient se- verity to require medical attention, but medication or behavioral strategies may help alle— viate symptoms and improve prognosis in those trying to quit using cannabis. Cannabis withdrawal is commonly observed in individuals seeking treatment for can- nabis use as well as in heavy cannabis users who are not seeking treatment. Among indi- viduals who have used cannabis regularly during some period of their lifetime, up to one- third report having experienced cannabis withdrawal. Among adults and adolescents en— rolled in treatment or heavy cannabis users, 50%—95% report cannabis withdrawal. These findings indicate that cannabis withdrawal occurs among a substantial subset of regular cannabis users who try to quit.

1	The amount, duration, and frequency of cannabis smoking that is required to produce an associated withdrawal disorder during a quit attempt are unknown. Most symptoms have their onset within the first 24—72 hours of cessation, peak within the first week, and last approximately 1—2 weeks. Sleep difficulties may last more than 30 days. Cannabis with- drawal has been documented among adolescents and adults. Withdrawal tends to be more common and severe among adults, most likely related to the more persistent and greater frequency and quantity of use among adults. Environmental. \Most likely, the prevalence and severity of cannabis withdrawal are greater among heavier cannabis users, and particularly among those seeking treatment for cannabis use disorders. Withdrawal severity also appears to be positively related to the se- verity of comorbid symptoms of mental disorders. Functional Consequences of Cannabis Withdrawal

1	Functional Consequences of Cannabis Withdrawal Cannabis users report using cannabis to relieve withdrawal symptoms, suggesting that withdrawal might contribute to ongoing expression of cannabis use disorder. Worse out- comes may be associated with greater withdrawal. A substantial proportion of adults and adolescents in treatment for moderate to severe cannabis use disorder acknowledge mod- erate to severe withdrawal symptoms, and many complain that these symptoms make ces- sation more difficult. Cannabis users report having relapsed to cannabis use or initiating use of other drugs (e.g., tranquilizers) to provide relief from cannabis withdrawal symp— toms. Last, individuals living with cannabis users observe significant withdrawal effects, suggesting that such symptoms are disruptive to daily living.

1	Because many of the symptoms of cannabis withdrawal are also symptoms of other sub- stance withdrawal syndromes or of depressive or bipolar disorders, careful evaluation should focus on ensuring that the symptoms are not better explained by cessation from an- other substance (e.g., tobacco or alcohol withdrawal), another mental disorder (general— ized anxiety disorder, major depressive disorder), or another medical condition. The following cannabis—induced disorders are described in other chapters of the manual with disorders with which they share phenomenology (see the substance/medication-induced mental disorders in these chapters): cannabis-induced psychotic disorder ("Schizophrenia

1	Disorders”); and cannabis-induced sleep disorder (”Sleep-Wake Disorders"). For cannabis intoxication delirium, see the criteria and discussion of delirium in the chapter ”Neurocog- nitive Disorders.” These cannabis-induced disorders are diagnosed instead of cannabis in- toxication or cannabis withdrawal when the symptoms are sufficiently severe to warrant independent clinical attention. 292.9 (F12.99) This category applies to presentations in which symptoms characteristic of a cannabis- related disorder that cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning predominate but do not meet the full criteria for any specific cannabis-related disorder or any of the disorders in the substance-related and addictive disorders diagnostic class.

1	A. A pattern of phencyclidine (or a pharmacologically similar substance) use leading to clinically significant impairment or distress, as manifested by at least two of the follow- ing, occurring within a 12-month period: 1. 2. 10. Phencyclidine is often taken in larger amounts or over a longer period than was in- tended. There is a persistent desire or unsuccessful efforts to cut down or control phency— clidine use. A great deal of time is spent in activities necessary to obtain phencyclidine, use the phencyclidine, or recover from its effects. Craving, or a strong desire or urge to use phencyclidine. Recurrent phencyclidine use resulting in a failure to fulfill major role obligations at work, school, or home (e.g., repeated absences from work or poor work performance related to phencyclidine use: phencyclidine-related absences, suspensions, or ex- pulsions from school; neglect of children or household).

1	personal problems caused or exacerbated by the effects of the phencyclidine (e.g.. arguments with a spouse about consequences of intoxication; physical fights). Important social, occupational, or recreational activities are given up or reduced be- cause of phencyclidine use. Recurrent phencyclidine use in situations in which it is physically hazardous (e.g., driving an automobile or operating a machine when impaired by a phencyclidine). Phencyclidine use is continued despite knowledge of having a persistent or recur- rent physical or psychological problem that is likely to have been caused or exac- erbated by the phencyclidine. Tolerance, as defined by either of the following: a. A need for markedly increased amounts of the phencyclidine to achieve intoxi- cation or desired effect. b. A markedly diminished effect with continued use of the same amount of the phencyclidine.

1	b. A markedly diminished effect with continued use of the same amount of the phencyclidine. Note: Withdrawal symptoms and signs are not established for phencyclidines, and so this criterion does not apply. (Withdrawal from phencyclidines has been reported in animals but not documented in human users.) Specify if: In early remission: After full criteria for phencyclidine use disorder were previously met, none of the criteria for phencyclidine use disorder have been met for at least 3 months but for less than 12 months (with the exception that Criterion A4, “Craving, or a strong desire or urge to use the phencyclidine," may be met). In sustained remission: After full criteria for phencyclidine use disorder were previ- ously met, none of the criteria for phencyclidine use disorder have been met at any time during a period of 12 months or longer (with the exception that Criterion A4, “Craving, or a strong desire or urge to use the phencyclidine," may be met). Specify if:

1	Specify if: In a controlled environment: This additional specifier is used if the individual is in an environment where access to phencyclidines is restricted.

1	Coding based on current severity: Note for ICD-10-CM codes: If a phencyclidine intoxica- tion or another phencyclidine-induced mental disorder is also present, do not use the codes below for phencyclidine use disorder. Instead, the comorbid phencyclidine use disorder is in- dicated in the 4th character of the phencyclidine-induced disorder code (see the coding note for phencyclidine intoxication or a specific phencyclidine-induced mental disorder). For ex- ample, it there is comorbid phencyclidine-induced psychotic disorder, only the phencyclidine- induced psychotic disorder code is given, with the 4th character indicating whether the co- morbid phencyclidine use disorder is mild, moderate, or severe: F16.159 for mild phencycli- dine use disorder with phencyclidine-induced psychotic disorder or F16.259 for a moderate or severe phencyclidine use disorder with phencyclidine-induced psychotic disorder. Specify current severity: 305.90 (F16.10) Mild: Presence of 2—3 symptoms.

1	Specify current severity: 305.90 (F16.10) Mild: Presence of 2—3 symptoms. 304.60 (F16.20) Moderate: Presence of 4—5 symptoms. 304.60 (F16.20) Severe: Presence of 6 or more symptoms. "In a controlled environment" applies as a further specifier of remission if the individual is both in remission and in a controlled environment (i.e., in early remission in a controlled environment or in sustained remission in a controlled environment). Examples of these environments are closely supervised and substance-free jails, therapeutic communities, and locked hospital units.

1	The phencyclidines (or phencyclidine-like substances) include phencyclidine (e.g., PCP, ”angel dust”) and less potent but similarly acting compounds such as ketamine, cyclohex- amine, and dizocilpine. These substances were first developed as dissociative anesthetics in the 19505 and became street drugs in the 19605. They produce feelings of separation from mind and body (hence ”dissociative”) in low doses, and at high doses, stupor and coma can result. These substances are most commonly smoked or taken orally, but they may also be snorted or injected. Although the primary psychoactive effects of PCP last for a few hours, the total elimination rate of this drug from the body typically extends 8 days or longer. The hallucinogenic effects in vulnerable individuals may last for weeks and may precipitate a persistent psychotic episode resembling schizophrenia. Ketamine has been observed to have utility in the treatment of major depressive disorder. Withdrawal symp- toms have not been

1	may precipitate a persistent psychotic episode resembling schizophrenia. Ketamine has been observed to have utility in the treatment of major depressive disorder. Withdrawal symp- toms have not been clearly established in humans, and therefore the withdrawal criterion is not included in the diagnosis of phencyclidine use disorder.

1	Phencyclidine may be detected in urine for up to 8 days or even longer at very high doses. In addition to laboratory tests to detect its presence, characteristic symptoms resulting from intoxication with phencyclidine or related substances may aid in its diagnosis. Phencycli- dine is likely to produce dissociative symptoms, analgesia, nystagmus, and hypertension, with risk of hypotension and shock. Violent behavior can also occur with phencyclidine use, as intoxicated persons may believe that they are being attacked. Residual symptoms following use may resemble schizophrenia.

1	The prevalence of phencyclidine use disorder is unknown. Approximately 2.5% of the pop- ulation reports having ever used phencyclidine. The proportion of users increases with age, from 0.3% of 12- to 17-year-olds, to 1.3% of 18- to 25-year-olds, to 2.9% of those age 26 years and older reporting ever using phencyclidine. There appears to have been an in— crease among 12th graders in both ever used (to 2.3% from 1.8%) and past-year use (to 1.3% from 1.0%) of phencyclidine. Past-year use of ketamine appears relatively stable among 12th graders (1.6%—1.7% over the past 3 years). There is little information about risk factors for phencyclidine use disorder. Among indi- viduals admitted to substance abuse treatment, those for whom phencyclidine was the primary substance were younger than those admitted for other substance use, had lower educational levels, and were more likely to be located in the West and Northeast regions of the United States, compared with other admissions.

1	Ketamine use in youths ages 16—23 years has been reported to be more common among whites (0.5%) than among other ethnic groups (range 0%—0.3%). Among individuals ad— mitted to substance abuse treatment, those for whom phencyclidine was the primary sub- stance were predominantly black (49%) or Hispanic (29%). Males make up about three-quarters of those with phencyclidine-related emergency room visits. Laboratory testing may be useful, as phencyclidine is present in the urine in intoxicated in- dividuals up to 8 days after ingestion. The individual’s history, along with certain physical signs, such as nystagmus, analgesia and prominent hypertension, may aid in distinguish- ing the phencyclidine clinical picture from that of other hallucinogens. Functional Consequences of Phencyclidine Use Disorder

1	Functional Consequences of Phencyclidine Use Disorder In individuals with phencyclidine use disorder, there may be physical evidence of injuries from accidents, fights, and falls. Chronic use of phencyclidine may lead to deficits in mem- ory, speech, and cognition that may last for months. Cardiovascular and neurological tox- icities (e.g., seizures, dystonias, dyskinesias, catalepsy, hypothermia or hyperthermia) may result from intoxication with phencyclidine. Other consequences include intracranial hemorrhage, rhabdomyolysis, respiratory problems, and (occasionally) cardiac arrest. Other substance use disorders. Distinguishing the effects of phencyclidine from those of other substances is important, since it may be a common additive to other substances (e.g., cannabis, cocaine).

1	Schizophrenia and other mental disorders. Some of the effects of phencyclidine and related substance use may resemble symptoms of other psychiatric disorders, such as psy- chosis (schizophrenia), low mood (major depressive disorder), violent aggressive be- haviors (conduct disorder, antisocial personality disorder). Discerning whether these behaviors occurred before the intake of the drug is important in the differentiation of acute drug effects from preexisting mental disorder. Phencyclidine-induced psychotic disorder should be considered when there is impaired reality testing in individuals experiencing disturbances in perception resulting from ingestion of phencyclidine. A. A problematic pattern of hallucinogen (other than phencyclidine) use leading to clini- cally significant impairment or distress, as manifested by at least two of the following. occurring within a 12-month period: 1. The hallucinogen is often taken in larger amounts or over a longer period than was intended.

1	occurring within a 12-month period: 1. The hallucinogen is often taken in larger amounts or over a longer period than was intended. 2. There is a persistent desire or unsuccessful efforts to cut down or control halluci- nogen use. 3. A great deal of time is spent in activities necessary to obtain the hallucinogen, use the hallucinogen, or recover from its effects. 4. Craving, or a strong desire or urge to use the hallucinogen. 5. Recurrent hallucinogen use resulting in a failure to fulfill major role obligations at work, school, or home (e.g., repeated absences from work or poor work perfor- mance related to hallucinogen use; hallucinogen-related absences. suspensions, or expulsions from school; neglect of children or household). 6. Continued hallucinogen use despite having persistent or recurrent social or inter- personal problems caused or exacerbated by the effects of the hallucinogen (e.g., arguments with a spouse about consequences of intoxication; physical tights).

1	7. Important social, occupational, or recreational activities are given up or reduced be- cause of hallucinogen use. 8. Recurrent hallucinogen use in situations in which it is physically hazardous (e.g., driving an automobile or operating a machine when impaired by the hallucinogen). 9. Hallucinogen use is continued despite knowledge of having a persistent or recur- rent physical or psychological problem that is likely to have been caused or exac- erbated by the hallucinogen. 10. Tolerance, as defined by either of the following: a. A need for markedly increased amounts of the hallucinogen to achieve intoxi- cation or desired effect. b. A markedly diminished effect with continued use of the same amount of the hal- lucinogen. Note: Withdrawal symptoms and signs are not established for hallucinogens, and so this criterion does not apply. Specify the particular hallucinogen. Specify ii:

1	Note: Withdrawal symptoms and signs are not established for hallucinogens, and so this criterion does not apply. Specify the particular hallucinogen. Specify ii: In early remission: After full criteria for other hallucinogen use disorder were previ- ously met, none of the criteria for other hallucinogen use disorder have been met for at least 3 months but for less than 12 months (with the exception that Criterion A4, “Craving, or a strong desire or urge to use the hallucinogen,” may be met). In sustained remission: After full criteria for other hallucinogen use disorder were previously met, none of the criteria for other hallucinogen use disorder have been met at any time during a period of 12 months or longer (with the exception that Criterion A4, “Craving. or a strong desire or urge to use the hallucinogen," may be met). Specify if:

1	Specify if: In a controlled environment: This additional specifier is used if the individual is in an environment where access to hallucinogens is restricted.

1	Coding based on current severity: Note for ICD-10-CM codes: It a hallucinogen intoxication or another hallucinogen-induced mental disorder is also present, do not use the codes below for hallucinogen use disorder. Instead, the comorbid hallucinogen use disorder is indicated in the 4th character of the hallucinogen-induced disorder code (see the coding note for halluci- nogen intoxication or specific hallucinogen-induced mental disorder). For example, if there is comorbid hallucinogen-induced psychotic disorder and hallucinogen use disorder, only the hallucinogen-induced psychotic disorder code is given, with the 4th character indicating wheth- er the comorbid hallucinogen use disorder is mild, moderate, or severe: F16.159 for mild hal- lucinogen use disorder with hallucinogen-induced psychotic disorder or F16.259 for a moderate or severe hallucinogen use disorder with hallucinogen-induced psychotic disorder. Specify current severity: 305.30 (F16.10) Mild: Presence of 2—3 symptoms.

1	Specify current severity: 305.30 (F16.10) Mild: Presence of 2—3 symptoms. 304.50 (F16.20) Moderate: Presence 014—5 symptoms. 304.50 (F16.20) Severe: Presence of 6 or more symptoms. "In a controlled environment" applies as a further specifier of remission if the individual is both in remission and in a controlled environment (i.e., in early remission in a controlled environment or in sustained remission in a controlled environment). Examples of these environments are closely supervised and substance—free jails, therapeutic communities, and locked hospital units.

1	Hallucinogens comprise a diverse group of substances that, despite having different chem— ical structures and possibly involving different molecular mechanisms, produce similar alterations of perception, mood, and cognition in users. Hallucinogens included are phenyl- alkylamines (e.g., mescaline, DOM [2,5-dimethoxy—4-methylamphetamine], and MDMA [3,4—methylenedioxymethamphetamine; also called "ecstasy”]); the indoleamines, includ- ing psilocybin (i.e., psilocin) and dimethyltryptamine (DMT); and the ergolines, such as LSD (lysergic acid diethylamide) and morning glory seeds. In addition, miscellaneous other ethnobotanical compounds are classified as "hallucinogens," of which Salviu divinorum and jimsonweed are two examples. Excluded from the hallucinogen group are cannabis and its active compound, delta-9—tetrahydrocannabinol (THC) (see the section "Cannabis-Related

1	Disorders”). These substances can have hallucinogenic effects but are diagnosed separately because of significant differences in their psychological and behavioral effects. Hallucinogens are usually taken orally, although some forms are smoked (e.g., DMT, salvia) or (rarely) taken intranasally or by injection (e.g., ecstasy). Duration of effects varies across types of hallucinogens. Some of these substances (i.e., LSD, MDMA) have a long half-life and extended duration such that users may spend hours to days using and / or re- covering from the effects of these drugs. However, other hallucinogenic drugs (e.g., DMT, salvia) are short acting. Tolerance to hallucinogens develops with repeated use and has been reported to have both autonomic and psychological effects. Cross-tolerance exists be- tween LSD and other hallucinogens (e.g., psilocybin, mescaline) but does not extend to other drug categories such as amphetamines and cannabis.

1	MDMA /ecstasy as a hallucinogen may have distinctive effects attributable to both its hal— lucinogenic and its stimulant properties. Among heavy ecstasy users, continued use despite physical or psychological problems, tolerance, hazardous use, and spending a great deal of time obtaining the substance are the most commonly reported criteria—over 50% in adults and over 30% in a younger sample, while legal problems related to substance use and persis- tent desire/inability to quit are rarely reported. As found for other substances, diagnostic cri- teria for other hallucinogen use disorder are arrayed along a single continuum of severity.

1	One of the generic criteria for substance use disorders, a clinically significant with— drawal syndrome, has not been consistently documented in humans, and therefore the di- agnosis of hallucinogen withdrawal syndrome is not included in DSM-5. However, there is evidence of withdrawal from MDMA, with endorsement of two or more withdrawal symptoms observed in 59%—98% in selected samples of ecstasy users. Both psychological and physical problems have been commonly reported as withdrawal problems. The characteristic symptom features of some of the hallucinogens can aid in diagnosis if urine or blood toxicology results are not available. For example, individuals who use LSD tend to experience visual hallucinations that can be frightening. Individuals intoxicated with hallucinogens may exhibit a temporary increase in suicidality.

1	Of all substance use disorders, other hallucinogen use disorder is one of the rarest. The 12-month prevalence is estimated to be 0.5% among 12- to 17-year-olds and 0.1% among adults age 18 and older in the United States. Rates are higher in adult males (0.2%) compared with females (0.1%), but the opposite is observed in adolescent samples ages 12—17, in which the 12-month rate is slightly higher in females (0.6%) than in males (0.4%). Rates are highest in individuals younger than 30 years, with the peak occurring in individuals ages 18—29 years (0.6%) and decreasing to virtually 00% among individuals age 45 and older. There are marked ethnic differences in 12-month prevalence of other hallucinogen use disorder. Among youths ages 12—17 years, 12-month prevalence is higher among Native

1	There are marked ethnic differences in 12-month prevalence of other hallucinogen use disorder. Among youths ages 12—17 years, 12-month prevalence is higher among Native Americans and Alaska Natives (1.2%) than among Hispanics (0.6%), whites (0.6%), Afri- can Americans (0.2%), and Asian Americans and Pacific Islanders (0.2%). Among adults, 12-month prevalence of other hallucinogen use disorder is similar for Native Americans and Alaska Natives, whites, and Hispanics (all 0.2%) but somewhat lower for Asian Amer- icans and Pacific Islanders (0.07%) and African Americans (0.03%). Past-year prevalence is higher in clinical samples (e.g., 19% in adolescents in treatment). Among individuals cur- rently using hallucinogens in the general population, 7.8% (adult) to 17% (adolescent) had a problematic pattern of use that met criteria for past-year other hallucinogen use disorder.

1	Among select groups of individuals who use hallucinogens (e.g., recent heavy ecstasy use), 73.5% of adults and 77% of adolescents have a problematic pattern of use that may meet other hallucinogen use disorder criteria. Unlike most substances where an early age at onset is associated with elevations in risk for the corresponding use disorder, it is unclear whether there is an association of an early age at onset with elevations in risk for other hallucinogen use disorder. However, patterns of drug consumption have been found to differ by age at onset, with early-onset ecstasy users more likely to be polydrug users than their later-onset counterparts. There may be a dis- proportionate inﬂuence of use of specific hallucinogens on risk of developing other hallu- cinogen use disorder, with use of ecstasy/MDMA increasing the risk of the disorder relative to use of other hallucinogens.

1	Little is known regarding the course of other hallucinogen use disorder, but it is generally thought to have low incidence, low persistence, and high rates of recovery. Adolescents are es- pecially at risk for using these drugs, and it is estimated that 2.7% of youths ages 12—17 years have used one or more of these drugs in the past 12 months, with 44% having used ecstasy/ MDMA. Other hallucinogen use disorder is a disorder observed primarily in individuals younger than 30 years, with rates vanishingly rare among older adults.

1	Temperamental. In adolescents but not consistently in adults, MDMA use is associated with an elevated rate of other hallucinogen use disorder. Other substance use disorders, particu- larly alcohol, tobacco, and cannabis, and major depressive disorder are associated with ele- vated rates of other hallucinogen use disorder. Antisocial personality disorder may be elevated among individuals who use more than two other drugs in addition to hallucinogens, compared with their counterparts with less extensive use history. The inﬂuence of adult anti- lucinogen use disorder may be stronger in females than in males. Use of specific hallucinogens (e.g., salvia) is prominent among individuals ages 18—25 years with other risk-taking behaviors and illegal activities. Cannabis use has also been implicated as a precursor to initiation of use of hallucinogens (e.g., ecstasy), along with early use of alcohol and tobacco. Higher drug use by peers and high sensation seeking have also been associated with

1	as a precursor to initiation of use of hallucinogens (e.g., ecstasy), along with early use of alcohol and tobacco. Higher drug use by peers and high sensation seeking have also been associated with elevated rates of ecstasy use.

1	MDMA/ecstasy use appears to signify a more severe group of hallucinogen users. Genetic and physiological. Among male twins, total variance due to additive genetics has been estimated to range from 26% to 79%, with inconsistent evidence for shared envi- ronmental inﬂuences. Historically, hallucinogens have been used as part of established religious practices, such as the use of peyote in the Native American Church and in Mexico. Ritual use by indige- nous populations of psilocybin obtained from certain types of mushrooms has occurred in South America, Mexico, and some areas in the United States, or of ayahuasca in the Santo Daime and Uniéo de Vegetal sects. Regular use of peyote as part of religious rituals is not linked to neuropsychological or psychological deficits. For adults, no race or ethnicity dif- ferences for the full criteria or for any individual criterion are apparent at this time.

1	In adolescents, females may be less likely than males to endorse "hazardous use,” and fe— male gender may be associated with increased odds of other hallucinogen use disorder. Laboratory testing can be useful in distinguishing among the different hallucinogens. However, because some agents (e.g., LSD) are so potent that as little as 75 micrograms can produce severe reactions, typical toxicological examination will not always reveal which substance has been used. There is evidence for long-term neurotoxic effects of MDMA/ecstasy use, including im- pairments in memory, psychological function, and neuroendocrine function; serotonin system dysfunction; and sleep disturbance; as well as adverse effects on brain microvas— culature, white matter maturation, and damage to axons. Use of MDMA/ecstasy may di- minish functional connectivity among brain regions.

1	Other substance use disorders. The effects of hallucinogens must be distinguished from those of other substances (e.g., amphetamines), especially because contamination of the hallucinogens with other drugs is relatively common. Schizophrenia. Schizophrenia also must be ruled out, as some affected individuals (e.g., individuals with schizophrenia who exhibit paranoia) may falsely attribute their symp- toms to use of hallucinogens. Other mental disorders or medical conditions. Other potential disorders or conditions to consider include panic disorder, depressive and bipolar disorders, alcohol or sedative withdrawal, hypoglycemia and other metabolic conditions, seizure disorder, stroke, oph- thalmological disorder, and central nervous system tumors. Careful history of drug tak- ing, collateral reports from family and friends (if possible), age, clinical history, physical examination, and toxicology reports should be useful in arriving at the final diagnostic de- c1s1on.

1	Adolescents who use MDMA/ecstasy and other hallucinogens, as well as adults who have recently used ecstasy, have a higher prevalence of other substance use disorders compared with nonhallucinogen substance users. Individuals who use hallucinogens exhibit eleva- tions of nonsubstance mental disorders (especially anxiety, depressive, and bipolar disor- ders), particularly with use of ecstasy and salvia. Rates of antisocial personality disorder (but not conduct disorder) are significantly elevated among individuals with other hallucinogen use disorder, as are rates of adult antisocial behavior. However, it is unclear whether the mental illnesses may be precursors to rather than consequences of other hallucinogen use disorder (see the section ”Risk and Prognostic Factors” for this disorder). Both adults and adolescents who use ecstasy are more likely than other drug users to be polydrug users and to have other drug use disorders.

1	A. Recent use of phencyclidine (or a pharmacologically similar substance). B. Clinically significant problematic behavioral changes (e.g., belligerence, assaultive- ness, impulsiveness. unpredictability, psychomotor agitation, impaired judgment) that developed during. or shortly after, phencyclidine use. C. Within 1 hour, two (or more) of the following signs or symptoms: Note: When the drug is smoked, "snorted," or used intravenously, the onset may be particularly rapid. 1. Vertical or horizontal nystagmus. 2. Hypertension or tachycardia. Numbness or diminished responsiveness to pain. Ataxia. Dysarthria. Muscle rigidity. Seizures or coma. Hyperacusis. D. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication with another substance.

1	Hyperacusis. D. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication with another substance. Coding note: The |CD-9-CM code is 292.89. The |CD-10-CM code depends on whether there is a comorbid phencyclidine use disorder. If a mild phencyclidine use disorder is co- morbid, the |CD-10-CM code is F16.129, and if a moderate or severe phencyclidine use disorder is comorbid, the |CD-10-CM code is F16.229. If there is no comorbid phencycli- dine use disorder, then the |CD-10-CM code is F16.929. @NQWP?’ Note: In addition to the section ”Functional Consequences of Phencyclidine Intoxication,” see the corresponding section in phencyclidine use disorder.

1	@NQWP?’ Note: In addition to the section ”Functional Consequences of Phencyclidine Intoxication,” see the corresponding section in phencyclidine use disorder. Phencyclidine intoxication reﬂects the clinically significant behavioral changes that occur shortly after ingestion of this substance (or a pharmacologically similar substance). The most common clinical presentations of phencyclidine intoxication include disorientation, confusion without hallucinations, hallucinations or delusions, a catatonic-like syndrome, and coma of varying severity. The intoxication typically lasts for several hours but, de- pending on the type of clinical presentation and whether other drugs besides phencycli- dine were consumed, may last for several days or longer.

1	Use of phencyclidine or related substances may be taken as an estimate of the prevalence of intoxication. Approximately 2.5% of the population reports having ever used phency- clidine. Among high school students, 2.3% of 12th graders report ever using phencycli- dine, with 57% having used in the past 12 months. This represents an increase from prior to 2011. Past—year use of ketamine, which is assessed separately from other substances, has remained stable over time, with about 1.7% of 12th graders reporting use. Laboratory testing may be useful, as phencyclidine is detectable in urine for up to 8 days following use, although the levels are only weakly associated with an individual’s clinical presentation and may therefore not be useful for case management. Creatine phosphoki- nase and aspartate aminotransferase levels may be elevated. Functional Consequences of Phencyclidine Intoxication

1	Functional Consequences of Phencyclidine Intoxication Phencyclidine intoxication produces extensive cardiovascular and neurological (e.g., sei- zures, dystonias, dyskinesias, catalepsy, hypothermia or hyperthermia) toxicity. In particular, in the absence of intact reality testing (i.e., without insight into any percep- tual abnormalities), an additional diagnosis of phencyclidine-induced psychotic disorder should be considered. Other substance intoxication. Phencyclidine intoxication should be differentiated from intoxication due to other substances, including other hallucinogens; amphetamine, co- caine, or other stimulants; and anticholinergics, as well as withdrawal from benzodiaze- pines. Nystagmus and bizarre and violent behavior may distinguish intoxication due to phencyclidine from that due to other substances. Toxicological tests may be useful in mak- ing this distinction, since phencyclidine is detectable in urine for up to 8 days after use.

1	However, there is a weak correlation between quantitative toxicology levels of phencycli- dine and clinical presentation that diminishes the utility of the laboratory findings for pa- tient management. Other conditions. Other conditions to be considered include schizophrenia, depression, withdrawal from other drugs (e.g., sedatives, alcohol), certain metabolic disorders like hy- poglycemia and hyponatremia, central nervous system tumors, seizure disorders, sepsis, neuroleptic malignant syndrome, and vascular insults. A. Recent use of a hallucinogen (other than phencyclidine). B. Clinically significant problematic behavioral or psychological changes (e.g., marked anxiety or depression, ideas of reference, fear of “losing one's mind," paranoid ide- ation, impaired judgment) that developed during, or shortly after, hallucinogen use.

1	C. Perceptual changes occurring in a state of full wakefulness and alertness (e.g., sub- jective intensification of perceptions, depersonalization, derealization, illusions, hallu- cinations, synesthesias) that developed during, or shortly after, hallucinogen use. D. Two (or more) of the following signs developing during, or shortly after, hallucinogen use: Pupillary dilation. Tachycardia. Sweating. Palpitations. Blurring of vision. Tremors. 7. Incoordination. E. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication with another sub- stance.

1	E. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication with another sub- stance. Coding note: The |CD-9-CM code is 292.89. The lCD-10-CM code depends on whether there is a comorbid hallucinogen use disorder. It a mild hallucinogen use disorder is co- morbid, the |CD-10-CM code is F16.129, and if a moderate or severe hallucinogen use disorder is comorbid, the |CD-10-CM code is F16.229. If there is no comorbid hallucinogen use disorder, then the |CD-10-CM code is F16.929. Note: For information on Associated Features Supporting Diagnosis and Culture-Related Diagnostic Issues, see the corresponding sections in other hallucinogen use disorder.

1	Note: For information on Associated Features Supporting Diagnosis and Culture-Related Diagnostic Issues, see the corresponding sections in other hallucinogen use disorder. Other hallucinogen intoxication reﬂects the clinically significant behavioral or psycholog- ical changes that occur shortly after ingestion of a hallucinogen. Depending on the specific hallucinogen, the intoxication may last only minutes (e.g., for salvia) or several hours or longer (e.g., for LSD [lysergic acid diethylamide] or MDMA [3,4-methylenedioxymetham- phetamine]).

1	The prevalence of other hallucinogen intoxication may be estimated by use of those sub— stances. In the United States, 1.8% of individuals age 12 years or older report using hallu- cinogens in the past year. Use is more prevalent among younger individuals, with 3.1% of 12- to 17-year-olds and 7.1% of 18- to 25-year-olds using hallucinogens in the past year, compared with only 0.7% of individuals age 26 years or older. Twelve-month prevalence for hallucinogen use is more common in males (2.4%) than in females (1.2%), and even more so among 18- to 25-year-olds (9.2% for males vs. 5.0% for females). In contrast, among individuals ages 12—17 years, there are no gender differences (3.1% for both gen- ders). These figures may be used as proxy estimates for gender-related differences in the prevalence of other hallucinogen intoxication. Other hallucinogen intoxication may lead to increased suicidality, although suicide is rare among users of hallucinogens.

1	Other hallucinogen intoxication may lead to increased suicidality, although suicide is rare among users of hallucinogens. Other hallucinogen intoxication can have serious consequences. The perceptual distur- bances and impaired judgment associated with other hallucinogen intoxication can result in injuries or fatalities from automobile crashes, physical fights, or unintentional self- injury (e.g., attempts to ”ﬂy” from high places). Environmental factors and the personality and expectations of the individual using the hallucinogen may contribute to the nature of and severity of hallucinogen intoxication. Continued use of hallucinogens, particularly MDMA, has also been linked with neurotoxic effects.

1	MDMA, has also been linked with neurotoxic effects. Other substance intoxication. Other hallucinogen intoxication should be differentiated from intoxication with amphetamines, cocaine, or other stimulants; anticholinergics; in— halants; and phencyclidine. Toxicological tests are useful in making this distinction, and determining the route of administration may also be useful. Other conditions. Other disorders and conditions to be considered include schizophre- nia, depression, withdrawal from other drugs (e.g., sedatives, alcohol), certain metabolic disorders (e.g., hypoglycemia), seizure disorders, tumors of the central nervous system, and vascular insults. Hallucinogen persisting perception disorder. Other hallucinogen intoxication is dis- tinguished from hallucinogen persisting perception disorder because the symptoms in the latter continue episodically or continuously for weeks (or longer) after the most recent in- toxication.

1	Other hallucinogen-induced disorders. Other hallucinogen intoxication is distinguished from the other hallucinogen-induced disorders (e.g., hallucinogen—induced anxiety disor— der, with onset during intoxication) because the symptoms in these latter disorders pre- dominate the clinical presentation and are severe enough to warrant independent clinical attention. Diagnostic Criteria 292.89 (F16.983) A. Following cessation of use of a hallucinogen, the reexperiencing of one or more of the perceptual symptoms that were experienced while intoxicated with the hallucinogen (e.g., geometric hallucinations, false perceptions of movement in the peripheral visual fields, flashes of color, intensified colors, trails of images of moving objects, positive afterimages, halos around objects, macropsia and micropsia). B. The symptoms in Criterion A cause clinically significant distress or impairment in so- cial, occupational, or other important areas of functioning.

1	B. The symptoms in Criterion A cause clinically significant distress or impairment in so- cial, occupational, or other important areas of functioning. C. The symptoms are not attributable to another medical condition (e.g., anatomical le- sions and infections of the brain, visual epilepsies) and are not better explained by an- other mental disorder (e.g., delirium, major neurocognitive disorder, schizophrenia) or hypnopompic hallucinations.

1	The hallmark of hallucinogen persisting perception disorder is the reexperiencing, when the individual is sober, of the perceptual disturbances that were experienced while the individ- ual was intoxicated with the hallucinogen (Criterion A). The symptoms may include any perceptual perturbations, but visual disturbances tend to be predominant. Typical of the ab— normal visual perceptions are geometric hallucinations, false perceptions of movement in the peripheral visual fields, ﬂashes of color, intensified colors, trails of images of moving ob- jects (i.e., images left suspended in the path of a moving object as seen in stroboscopic pho- tography), perceptions of entire objects, positive afterimages (i.e., a same-colored or complementary-colored ”shadow” of an object remaining after removal of the object), halos around objects, or misperception of images as too large (macropsia) or too small (micropsia).

1	Duration of the visual disturbances may be episodic or nearly continuous and must cause clinically significant distress or impairment in social, occupational, or other important areas of functioning (Criterion B). The disturbances may last for weeks, months, or years. Other explanations for the disturbances (e.g., brain lesions, preexisting psychosis, seizure disor- ders, migraine aura without headaches) must be ruled out (Criterion C). diethylamide) use, but not exclusively. There does not appear to be a strong correlation be- tween hallucinogen persisting perception disorder and number of occasions of hallucino- gen use, with some instances of hallucinogen persisting perception disorder occurring in individuals with minimal exposure to hallucinogens. Some instances of hallucinogen per- sisting perception disorder may be triggered by use of other substances (e.g., cannabis or alcohol) or in adaptation to dark environments.

1	Reality testing remains intact in individuals with hallucinogen persisting perception dis- order (i.e., the individual is aware that the disturbance is linked to the effect of the drug). If this is not the case, another disorder might better explain the abnormal perceptions. Prevalence estimates of hallucinogen persisting perception disorder are unknown. Initial prevalence estimates of the disorder among individuals who use hallucinogens is approx- imately 4.2%. Little is known about the development of hallucinogen persisting perception disorder. Its course, as suggested by its name, is persistent, lasting for weeks, months, or even years in certain individuals. There is little evidence regarding risk factors for hallucinogen persisting perception dis- order, although genetic factors have been suggested as a possible explanation underlying the susceptibility to LSD effects in this condition.

1	some cases, many individuals with the disorder are able to suppress the disturbances and continue to function normally. Conditions to be ruled out include schizophrenia, other drug effects, neurodegenerative disorders, stroke, brain tumors, infections, and head trauma. Neuroimaging results in hal- lucinogen persisting perception disorder cases are typically negative. As noted earlier, re- ality testing remains intact (i.e., the individual is aware that the disturbance is linked to the effect of the drug); if this is not the case, another disorder (e.g., psychotic disorder, another medical condition) might better explain the abnormal perceptions. disorder are panic disorder, alcohol use disorder, and major depressive disorder.

1	disorder are panic disorder, alcohol use disorder, and major depressive disorder. Other phencyclidine-induced disorders are described in other chapters of the manual with disorders with which they share phenomenology (see the substance/medication-induced mental disorders in these chapters): phencyclidine-induced psychotic disorder (”Schizo- ders”). For phencyclidine-induced intoxication delirium, see the criteria and discussion of delirium in the chapter ”Neurocognitive Disorders.” These phencyclidine-induced disor- ders are diagnosed instead of phencyclidine intoxication only when the symptoms are suf- ficiently severe to warrant independent clinical attention.

1	The following other hallucinogen-induced disorders are described in other Chapters of the manual with disorders with which they share phenomenology (see the substance/medi- cation-induced mental disorders in these chapters): other hallucinogen—induced psychotic disorder ("Anxiety Disorders"). For other hallucinogen intoxication delirium, see the cri- teria and discussion of delirium in the chapter "Neurocognitive Disorders.” These hallu— cinogen-induced disorders are diagnosed instead of other hallucinogen intoxication only when the symptoms are sufficiently severe to warrant independent clinical attention. 292.9 (F16.99)

1	292.9 (F16.99) This category applies to presentations in which symptoms characteristic of a phencycli- dine-related disorder that cause clinically significant distress or impairment in social, oc- cupational, or other important areas of functioning predominate but do not meet the full criteria for any specific phencyclidine-related disorder or any of the disorders in the sub- stance-related and addictive disorders diagnostic class. 292.9 (F16.99) This category applies to presentations in which symptoms characteristic of a hallucinogen- related disorder that cause clinically significant distress or impairment in social, occupa- tional, or other imponant areas of functioning predominate but do not meet the full criteria for any specific hallucinogen-related disorder or any of the disorders in the substance- related and addictive disorders diagnostic class.

1	A. A problematic pattern of use of a hydrocarbon-based inhalant substance leading to clinically significant impairment or distress, as manifested by at least two of the follow- ing, occurring within a 12-month period: 1. The inhalant substance is often taken in larger amounts or over a longer period than was intended. 2. There is a persistent desire or unsuccessful efforts to cut down or control use of the inhalant substance. 3. A great deal of time is spent in activities necessary to obtain the inhalant substance, use it, or recover from its effects. 4. Craving, or a strong desire or urge to use the inhalant substance. 5. Recurrent use of the inhalant substance resulting in a failure to fulfill major role ob- ligations at work, school, or home. 6. Continued use of the inhalant substance despite having persistent or recurrent so- cial or interpersonal problems caused or exacerbated by the effects of its use.

1	6. Continued use of the inhalant substance despite having persistent or recurrent so- cial or interpersonal problems caused or exacerbated by the effects of its use. 7. Important social. occupational, or recreational activities are given up or reduced be- cause of use of the inhalant substance. 8. Recurrent use of the inhalant substance in situations in which it is physically haz- ardous. 9. Use of the inhalant substance is continued despite knowledge of having a persis- tent or recurrent physical or psychological problem that is likely to have been caused or exacerbated by the substance. 10. Tolerance, as defined by either of the following: a. A need for markedly increased amounts of the inhalant substance to achieve intoxication or desired effect. b. A markedly diminished effect with continued use of the same amount of the in- halant substance. Specify the particular inhalant: When possible, the particular substance involved should be named (e.g., “solvent use disorder”).

1	Specify the particular inhalant: When possible, the particular substance involved should be named (e.g., “solvent use disorder”). Specify it: In early remission: After full criteria for inhalant use disorder were previously met, none of the criteria for inhalant use disorder have been met for at least 3 months but for less than 12 months (with the exception that Criterion A4, “Craving, or a strong de- sire or urge to use the inhalant substance,” may be met). In sustained remission: After full criteria for inhalant use disorder were previously met, none of the criteria for inhalant use disorder have been met at any time during a period of 12 months or longer (with the exception that Criterion A4, “Craving, or a strong desire or urge to use the inhalant substance,” may be met). Specify if: In a controlled environment: This additional specifier is used if the individual is in an environment where access to inhalant substances is restricted.

1	Specify if: In a controlled environment: This additional specifier is used if the individual is in an environment where access to inhalant substances is restricted. Coding based on current severity: Note for |CD-10-CM codes: If an inhalant intoxication or another inhalant-induced mental disorder is also present, do not use the codes below for inhalant use disorder. Instead, the comorbid inhaIant use disorder is indicated in the 4th character of the inhalant—induced disorder code (see the coding note for inhalant intox- ication or a specific inhalant-induced mental disorder). For example, if there is comorbid inhalant—induced depressive disorder and inhalant use disorder, only the inhalant-induced depressive disorder code is given, with the 4th character indicating whether the comorbid inhalant use disorder is mild, moderate, or severe: F18.14 for mild inhalant use disorder disorder with inhalant-induced depressive disorder.

1	Specify current severity: 305.90 (F18.10) Mild: Presence of 2—3 symptoms. 304.60 (F18.20) Moderate: Presence of 4—5 symptoms. 304.60 (F1820) Severe: Presence of 6 or more symptoms. This manual reeognizes volatile hydrocarbon use meeting the above diagnostic criteria as inhalant use disorder. Volatile hydrocarbons are toxic gases from glues, fuels, paints/ and other volatile compounds. When possible, the particular substance involved should be named (e.g., ”toluene use disorder”). However, most compounds that are inhaled are a mixture of several substances that can produce psychoactive effects, and it is often difficult to ascertain the exact substance responsible for the disorder. Unless there is clear evidence that a single, unmixed substance has been used, the general term inhalant should be used in recording the diagnosis. Disorders arising from inhalation of nitrous oxide or of amyl-, buty1-, or isobutylnitrite are considered as other (or unknown) substance use disorder.

1	"In a controlled environment” applies as a further specifier of remission if the individ- ual is both in remission and in a controlled environment (i.e., in early remission in a con- trolled environment or in sustained remission in a controlled environment). Examples of these environments are closely supervised and substance—free jails, therapeutic communi- ties, and locked hospital units. The severity of individuals’ inhalant use disorder is assessed by the number of diag- nostic criteria endorsed. Changing severity of individuals’ inhalant use disorder across time is reﬂected by reductions in the frequency (e.g., days used per month) and / or dose (e.g., tubes of glue per day) used, as assessed by the individual’s self—report, report of oth- ers, clinician’s observations, and biological testing (when practical).

1	Features of inhalant use disorder include repeated use of an inhalant substance despite the individual’s knowing that the substance is causing serious problems for the individual (Criterion A9). Those problems are reﬂected in the diagnostic criteria. Missing work or school or inability to perform typical responsibilities at work or school (Criterion A5), and continued use of the inhalant substance even though it causes arguments with family or friends, fights, and other social or interpersonal problems (Criterion A6), may be seen in inhalant use disorder. Limiting family contact, work or school obligations, or rec- reational activities (e.g., sports, games, hobbies) may also occur (Criterion A7). Use of inhal- ants when driving or operating dangerous equipment (Criterion A8) is also seen. Tolerance (Criterion A10) and mild withdrawal are each reported by about 10% of in- dividuals who use inhalants, and a few individuals use inhalants to avoid withdrawal.

1	Tolerance (Criterion A10) and mild withdrawal are each reported by about 10% of in- dividuals who use inhalants, and a few individuals use inhalants to avoid withdrawal. However, because the withdrawal symptoms are mild, this manual neither recognizes a diagnosis of inhalant withdrawal nor counts withdrawal complaints as a diagnostic crite- rion for inhalant use disorder.

1	A diagnosis of inhalant use disorder is supported by recurring episodes of intoxication with negative results in standard drug screens (which do not detect inhalants); possession, or lingering odors, of inhalant substances; peri-oral or peri-nasal ”glue-sniffer's rash”; as- sociation with other individuals known to use inhalants; membership in groups with prev- alent inhalant use (e.g., some native or aboriginal communities, homeless children in street gangs); easy access to certain inhalant substances; paraphernalia possession; presence of the disorder’s characteristic medical complications (e.g., brain white matter pathology, rhabdomyolysis); and the presence of multiple substance use disorders. Inhalant use and inhalant use disorder are associated with past suicide attempts, especially among adults reporting previous episodes of low mood or anhedonia.

1	About 0.4% of Americans ages 12—17 years have a pattern of use that meets criteria for in- halant use disorder in the past 12 months. Among those youths, the prevalence is highest in Native Americans and lowest in African Americans. Prevalence falls to about 0.1% among Americans ages 18—29 years, and only 0.02% when all Americans 18 years or older are con- sidered, with almost no females and a preponderance of European Americans. Of course, in isolated subgroups, prevalence may differ considerably from these overall rates. About 10% of 13-year-old American children report having used inhalants at least once; that percentage remains stable through age 17 years. Among those 12- to 17-year-olds who use inhalants, the more-used substances include glue, shoe polish, or toluene; gasoline or lighter ﬂuid; or spray paints.

1	Only 0.4% of 12- to 17-year-olds progress to inhalant use disorder; those youths tend to exhibit multiple other problems. The declining prevalence of inhalant use disorder after adolescence indicates that this disorder usually remits in early adulthood. Volatile hydrocarbon use disorder is rare in prepubertal children, most common in ad- olescents and young adults, and uncommon in older persons. Calls to poison-control cen- ters for ”intentional abuse" of inhalants peak with calls involving individuals at age 14 years.

1	Of adolescents who use inhalants, perhaps one-fifth develop inhalant use disorder; a few die from inhalant-related accidents, or “sudden sniffing death”. But the disorder apparently remits in many individuals after adolescence. Prevalence declines dramatically among in- dividuals in their 205. Those with inhalant use disorder extending into adulthood often have severe problems: substance use disorders, antisocial personality disorder, and sui- cidal ideation with attempts. Temperamental. Predictors of progression from nonuse of inhalants, to use, to inhalant disorder or antisocial personality disorder. Other predictors are earlier onset of inhalant use and prior use of mental health services. Environmental. Inhalant gases are widely and legally available, increasing the risk of mis- use. Childhood maltreatment or trauma also is associated with youthful progression from inhalant non-use to inhalant use disorder.

1	Genetic and physiological. Behavioral disinhibition is a highly heritable general propensity to not constrain behavior in socially acceptable ways, to break social norms and rules, and to take dangerous risks, pursuing rewards excessively despite dangers of adverse consequences. Youths with strong behavioral disinhibition show risk factors for inhalant use disorder: early- onset substance use disorder, multiple substance involvement, and early conduct problems. Because behavioral disinhibition is under strong genetic inﬂuence, youths in families with substance and antisocial problems are at elevated risk for inhalant use disorder. Certain native or aboriginal communities have experienced a high prevalence of inhalant problems. Also, in some countries, groups of homeless children in street gangs have ex- tensive inhalant use problems. Although the prevalence of inhalant use disorder is almost identical in adolescent males and females, the disorder is very rare among adult females.

1	Although the prevalence of inhalant use disorder is almost identical in adolescent males and females, the disorder is very rare among adult females. Urine, breath, or saliva tests may be valuable for assessing concurrent use of non-inhalant substances by individuals with inhalant use disorder. However, technical problems and the considerable expense of analyses make frequent biological testing for inhalants them- selves impractical. Functional Consequences of Inhalant Use Disorder Because of inherent toxicity, use of butane or propane is not infrequently fatal. Moreover, rhythmia. Fatalities may occur even on the first inhalant exposure and are not thought to be dose-related. Volatile hydrocarbon use impairs neurobehavioral function and causes various neurological, gastrointestinal, cardiovascular, and pulmonary problems.

1	Long-term inhalant users are at increased risk for tuberculosis, HIV/AIDS, sexually transmitted diseases, depression, anxiety, bronchitis, asthma, and sinusitis. Deaths may occur from respiratory depression, arrhythmias, asphyxiation, aspiration of vomitus, or accident and injury. Inhalant exposure (unintentional) from industrial or other accidents. This designation is used when findings suggest repeated or continuous inhalant exposure but the involved individual and other informants deny any history of purposeful inhalant use. Inhalant use (intentional), without meeting criteria for inhalant use disorder. Inhalant use is common among adolescents, but for most of those individuals, the inhalant use does not meet the diagnostic standard of two or more Criterion A items for inhalant use disorder in the past year.

1	Inhalant intoxication, without meeting criteria for inhalant use disorder. Inhalant intox- uals whose use does not meet criteria for inhalant use disorder, which requires at least two of the 10 diagnostic criteria in the past year. Inhalant-induced disorders (i.e., inhalant-induced psychotic disorder, depressive dis- order, anxiety disorder, neurocognitive disorder, other inhalant-induced disorders) without meeting criteria for inhalant use disorder. Criteria are met for a psychotic, de- pressive, anxiety, or major neurocognitive disorder, and there is evidence from history, physical examination, or laboratory findings that the deficits are etiologically related to the effects of inhalant substances. Yet, criteria for inhalant use disorder may not be met (i.e., fewer than 2 of the 10 criteria were present).

1	Other substance use disorders, especially those involving sedating substances (e.g., alcohol, benzodiazepines. barbiturates). Inhalant use disorder commonly co-occurs with other substance use disorders, and the symptoms of the disorders may be similar and overlapping. To disentangle symptom patterns, it is helpful to inquire about which symp- toms persisted during periods when some of the substances were not being used.

1	Other toxic, metabolic, traumatic, neoplastic, or infectious disorders impairing central or peripheral nervous system function. Individuals with inhalant use disorder may pre- sent with symptoms of pernicious anemia, subacute combined degeneration of the spinal cord, psychosis, major or minor cognitive disorder, brain atrophy, leukoencephalopathy, and many other nervous system disorders. Of course, these disorders also may occur in the absence of inhalant use disorder. A history of little or no inhalant use helps to exclude inhalant use disorder as the source of these problems. Disorders of other organ systems. Individuals with inhalant use disorder may present with symptoms of hepatic or renal damage, rhabdomyolysis, methemoglobinemia, or symp- toms of other gastrointestinal, cardiovascular, or pulmonary diseases. A history of little or no inhalant use helps to exclude inhalant use disorder as the source of such medical problems.

1	Individuals with inhalant use disorder receiving clinical care often have numerous other substance use disorders. Inhalant use disorder commonly co-occurs with adolescent con- duct disorder and adult antisocial personality disorder. Adult inhalant use and inhalant use disorder also are strongly associated with suicidal ideation and suicide attempts. A. Recent intended or unintended short-term, high-dose exposure to inhalant sub- stances, including volatile hydrocarbons such as toluene or gasoline. B. Clinically significant problematic behavioral or psychological changes (e.g., belliger- ence, assaultiveness, apathy, impaired judgment) that developed during, or shortly af- ter, exposure to inhalants. C. Two (or more) of the following signs or symptoms developing during, or shortly after, inhalant use or exposure: Dizziness. Nystagmus. Incoordination. Slurred speech. Unsteady gait. Lethargy. Depressed reflexes. Psychomotor retardation. 9. Tremor.

1	Dizziness. Nystagmus. Incoordination. Slurred speech. Unsteady gait. Lethargy. Depressed reflexes. Psychomotor retardation. 9. Tremor. 10. Generalized muscle weakness. 11. Blurred vision or diplopia. 12. Stupor or coma. 13. Euphoria. D. The signs or symptoms are not attributable to another medical condition and are not bet- ter explained by another mental disorder, including intoxication with another substance. Coding note: The |CD-9-CM code is 292.89. The |CD-10-CM code depends on whether there is a comorbid inhalant use disorder. It a mild inhalant use disorder is comorbid, the |CD-10-CM code is F18.129, and if a moderate or severe inhalant use disorder is comor- bid, the |CD-10—CM code is F18.229. If there is no comorbid inhalant use disorder, then the ICD-10-CM code is F18.929. Note: For information on Development and Course, Risk and Prognostic Factors, Culture-

1	Note: For information on Development and Course, Risk and Prognostic Factors, Culture- Related Diagnostic Issues, and Diagnostic Markers, see the corresponding sections in in- halant use disorder. Inhalant intoxication is an inhalant-related, clinically significant mental disorder that de- velops during, or immediately after, intended or unintended inhalation of a volatile hy- drocarbon substance. Volatile hydrocarbons are toxic gases from glues, fuels, paints, and other volatile compounds. When it is possible to do so, the particular substance involved should be named (e.g., toluene intoxication). Among those who do, the intoxication clears within a few minutes to a few hours after the exposure ends. Thus, inhalant intoxication usually occurs in brief episodes that may recur.

1	Inhalant intoxication may be indicated by evidence of possession, or lingering odors, of in- halant substances (e.g., glue, paint thinner, gasoline, butane lighters); apparent intoxica- tion occurring in the age range with the highest prevalence of inhalant use (12—17 years); and apparent intoxication with negative results from the standard drug screens that usu- ally fail to identify inhalants. The prevalence of actual episodes of inhalant intoxication in the general population is un- known, but it is probable that most inhalant users would at some time exhibit use that would meet criteria for inhalant intoxication disorder. Therefore, the prevalence of inhal- ant use and the prevalence of inhalant intoxication disorder are likely similar. In 2009 and 2010, inhalant use in the past year was reported by 0.8% of all Americans older than 12 years; the prevalence was highest in younger age groups (3.6% for individuals 12 to 17 years old, and 1.7% for individuals 18 to 25 years old).

1	Gender differences in the prevalence of inhalant intoxication in the general population are unknown. However, if it is assumed that most inhalant users eventually experience inhal- ant intoxication, gender differences in the prevalence of inhalant users likely approximate those in the proportions of males and females experiencing inhalant intoxication. Regard- ing gender differences in the prevalence of inhalant users in the United States, 1% of males older than 12 years and 0.7% of females older than 12 years have used inhalants in the pre- vious year, but in the younger age groups more females than males have used inhalants (e.g., among 12- to 17-year-olds, 3.6% of males and 4.2% of females). Functional Consequences of Inhalant Intoxication

1	Functional Consequences of Inhalant Intoxication Use of inhaled substances in a Closed container, such as a plastic bag over the head, may lead to unconsciousness, anoxia, and death. Separately, ”sudden sniffing death,” likely from cardiac arrhythmia or arrest, may occur with various volatile inhalants. The en- hanced toxicity of certain volatile inhalants, such as butane or propane, also causes fatal- ities. Although inhalant intoxication itself is of short duration, it may produce persisting medical and neurological problems, especially if the intoxications are frequent. Inhalant exposure, without meeting the criteria for inhalant intoxication disorder. The individual intentionally or unintentionally inhaled substances, but the dose was in- sufficient for the diagnostic criteria for inhalant use disorder to be met.

1	stances, especially from sedating substances (e.g., alcohol, benzodiazepines, barbi- turates). These disorders may have similar signs and symptoms, but the intoxication is attributable to other intoxicants that may be identified via a toxicology screen. Differenti- ating the source of the intoxication may involve discerning evidence of inhalant exposure as described for inhalant use disorder. A diagnosis of inhalant intoxication may be sug- gested by possession, or lingering odors, of inhalant substances (e.g., glue, paint thinner, gasoline, butane lighters,); paraphernalia possession (e.g., rags or bags for concentrating the intoxicated individual possesses or uses inhalants; apparent intoxication despite neg- ative results on standard drug screens (which usually fail to identify inhalants); apparent intoxication occurring in that age range with the highest prevalence of inhalant use (12—17 years); association with others known to use inhalants; membership in certain small com-

1	apparent intoxication occurring in that age range with the highest prevalence of inhalant use (12—17 years); association with others known to use inhalants; membership in certain small com- munities with prevalent inhalant use (e.g., some native or aboriginal communities, home- less street children and adolescents); or unusual access to certain inhalant substances.

1	Other inhalant-related disorders. Episodes of inhalant intoxication do occur during, but are not identical with, other inhalant—related disorders. Those inhalant-related disorders are recognized by their respective diagnostic criteria: inhalant use disorder, inhalant- induced neurocognitive disorder, inhalant—induced psychotic disorder, inhalant-induced depressive disorder, inhalant-induced anxiety disorder, and other inhalant-induced dis- orders. Other toxic, metabolic, traumatic, neoplastic, or infectious disorders that impair brain function and cognition. Numerous neurological and other medical conditions may pro- duce the clinically significant behavioral or psychological changes (e.g., belligerence, as- saultiveness, apathy, impaired judgment) that also characterize inhalant intoxication.

1	The following inhalant-induced disorders are described in other chapters of the manual with disorders with which they share phenomenology (see the substance/medication- induced mental disorders in these chapters): inhalant-induced psychotic disorder (”Schizo- halant-induced major or mild neurocognitive disorder (”Neurocognitive Disorders"). For inhalant intoxication delirium, see the criteria and discussion of delirium in the chapter "Neurocognitive Disorders.” These inhalant-induced disorders are diagnosed instead of in- halant intoxication only when symptoms are sufficiently severe to warrant independent clinical attention. 292.9 (F18.99)

1	292.9 (F18.99) This category applies to presentations in which symptoms characteristic of an inhalant- related disorder that cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning predominate but do not meet the full criteria for any specific inhalant—related disorder or any of the disorders in the substance-related and addictive disorders diagnostic class. A. A problematic pattern of opioid use leading to clinically significant impairment or distress, as manifested by at least two of the following, occurring within a 12-month period: 1. 2. 3. 10. 11. Opioids are often taken in larger amounts or over a longer period than was in- tended. There is a persistent desire or unsuccessful efforts to cut down or control opioid use. A great deal of time is spent in activities necessary to obtain the opioid, use the opi- oid, or recover from its effects. Craving, or a strong desire or urge to use opioids.

1	A great deal of time is spent in activities necessary to obtain the opioid, use the opi- oid, or recover from its effects. Craving, or a strong desire or urge to use opioids. Recurrent opioid use resulting in a failure to fulfill major role obligations at work, school. or home. problems caused or exacerbated by the effects of opioids. Important social, occupational, or recreational activities are given up or reduced be- cause of opioid use. Recurrent opioid use in situations in which it is physically hazardous. Continued opioid use despite knowledge of having a persistent or recurrent physi- cal or psychological problem that is likely to have been caused or exacerbated by the substance. Tolerance, as defined by either of the following: a. A need for markedly increased amounts of opioids to achieve intoxication or de- sired effect. b. A markedly diminished effect with continued use of the same amount of an opioid.

1	b. A markedly diminished effect with continued use of the same amount of an opioid. Note: This criterion is not considered to be met for those taking opioids solely under appropriate medical supervision. Withdrawal, as manifested by either of the following: a. The characteristic opioid withdrawal syndrome (refer to Criteria A and B of the criteria set for opioid withdrawal, pp. 547—548). b. Opioids (or a closely related substance) are taken to relieve or avoid withdrawal symptoms. Note: This criterion is not considered to be met for those individuals taking opioids solely under appropriate medical supervision. Specify it: In early remission: After full criteria for opioid use disorder were previously met, none of the criteria for opioid use disorder have been met for at least 3 months but for less than 12 months (with the exception that Criterion A4, "Craving, or a strong desire or urge to use opioids," may be met).

1	In sustained remission: After full criteria for opioid use disorder were previously met, none of the criteria for opioid use disorder have been met at any time during a period of 12 months or longer (with the exception that Criterion A4, “Craving, or a strong de- sire or urge to use opioids," may be met). Specify it: On maintenance therapy: This additional specifier is used if the individual is taking a prescribed agonist medication such as methadone or buprenorphine and none of the criteria for opioid use disorder have been met for that class of medication (except tol- erance to, or withdrawal from, the agonist). This category also applies to those individ- uals being maintained on a partial agonist, an agonist/antagonist, or a full antagonist such as oral naltrexone or depot naltrexone. In a controlled environment: This additional specifier is used if the individual is in an environment where access to opioids is restricted.

1	Coding based on current severity: Note for |CD-10-CM codes: If an opioid intoxication, opioid withdrawal, or another opioid-induced mental disorder is also present, do not use the codes below for opioid use disorder. Instead, the comorbid opioid use disorder is indi- cated in the 4th character of the opioid-induced disorder code (see the coding note for opi- oid intoxication, opioid withdrawal, or a specific opioid-induced mental disorder). For example, if there is comorbid opioid-induced depressive disorder and opioid use disorder, only the opioid-induced depressive disorder code is given, with the 4th character indicating whether the comorbid opioid use disorder is mild, moderate, or severe: F1 1 .14 for mild opi- oid use disorder with opioid-induced depressive disorder or F11.24 for a moderate or se- vere opioid use disorder with opioid-induced depressive disorder. Specify current severity: 305.50 (F11.10) Mild: Presence of 2—3 symptoms.

1	Specify current severity: 305.50 (F11.10) Mild: Presence of 2—3 symptoms. 304.00 (F11.20) Moderate: Presence of 4—5 symptoms. 304.00 (F11.20) Severe: Presence of 6 or more symptoms. The ”on maintenance therapy” specifier applies as a further specifier of remission if the in- dividual is both in remission and receiving maintenance therapy. "In a controlled environ— ment” applies as a further specifier of remission if the individual is both in remission and in a controlled environment (i.e., in early remission in a controlled environment or in sustained remission in a controlled environment). Examples of these environments are closely super- vised and substance-free jails, therapeutic communities, and locked hospital units.

1	Changing severity across time in an individual is also reﬂected by reductions in the fre- quency (e.g., days of use per month) and / or dose (e.g., injections or number of pills) of an opioid, as assessed by the individual’s self—report, report of knowledgeable others, clini— cian’s observations, and biological testing.

1	Opioid use disorder includes signs and symptoms that reﬂect compulsive, prolonged self— administration of opioid substances that are used for no legitimate medical purpose or, if another medical condition is present that requires opioid treatment, that are used in doses greatly in excess of the amount needed for that medical condition. (For example, an indi- more than prescribed and not only because of persistent pain.) Individuals with opioid use disorder tend to develop such regular patterns of compulsive drug use that daily activities are planned around obtaining and administering opioids. Opioids are usually purchased on the illegal market but may also be obtained from physicians by falsifying or exagger- physicians. Health care professionals with opioid use disorder will often obtain opioids by patients or from pharmacy supplies. Most individuals with opioid use disorder have significant levels of tolerance and will experience withdrawal on abrupt discontinuation of opioid

1	obtain opioids by patients or from pharmacy supplies. Most individuals with opioid use disorder have significant levels of tolerance and will experience withdrawal on abrupt discontinuation of opioid substances. Individuals with opioid use disorder often develop conditioned responses to drug-related stimuli (e.g., craving on seeing any heroin powder—like sub- stance)—a phenomenon that occurs with most drugs that cause intense psychological changes. These responses probably contribute to relapse, are difficult to extinguish, and typ- ically persist long after detoxification is completed.

1	Opioid use disorder can be associated with a history of drug-related crimes (e.g., posses- sion or distribution of drugs, forgery, burglary, robbery, larceny, receiving stolen goods). Among health care professionals and individuals who have ready access to controlled substances, there is often a different pattern of illegal activities involving problems with state licensing boards, professional staffs of hospitals, or other administrative agencies. Marital difficulties (including divorce), unemployment, and irregular employment are of- ten associated with opioid use disorder at all socioeconomic levels.

1	The 12-month prevalence of opioid use disorder is approximately 0.37% among adults age 18 years and older in the community population. This may be an underestimate because of the large number of incarcerated individuals with opioid use disorders. Rates are higher in males than in females (0.49% vs. 0.26%), with the male-to-female ratio typically being 1.521 for opioids other than heroin (i.e., available by prescription) and 3:1 for heroin. Female ad- olescents may have a higher likelihood of developing opioid use disorders. The preva- lence decreases with age, with the prevalence highest (0.82%) among adults age 29 years or younger, and decreasing to 0.09% among adults age 65 years and older. Among adults, the prevalence of opioid use disorder is lower among African Americans at 0.18% and over- represented among Native Americans at 1.25%. It is close to average among whites (0.38%), Asian or Pacific Islanders (0.35%), and Hispanics (0.39%).

1	Asian or Pacific Islanders (0.35%), and Hispanics (0.39%). Among individuals in the United States ages 12—17 years, the overall 12-month prev- alence of opioid use disorder in the community population is approximately 1.0%, but the prevalence of heroin use disorder is less than 0.1%. By contrast, analgesic use disorder is prevalent in about 1.0% of those ages 12—17 years, speaking to the importance of opioid an- algesics as a group of substances with significant health consequences. The 12-month prevalence of problem opioid use in European countries in the commu- nity population ages 15—64 years is between 0.1% and 0.8%. The average prevalence of problem opioid use in the European Union and Norway is between 0.36% and 0.44%.

1	Opioid use disorder can begin at any age, but problems associated with opioid use are most commonly first observed in the late teens or early 205. Once opioid use disorder develops, it usually continues over a period of many years, even though brief periods of abstinence are frequent. In treated populations, relapse following abstinence is common. Even though relapses do occur, and while some long—term mortality rates may be as high as 2% per year, about 20%—30% of individuals with opioid use disorder achieve long-term abstinence. An exception concerns that of military service personnel who became depen- dent on opioids in Vietnam; over 90% of this population who had been dependent on opi- oids during deployment in Vietnam achieved abstinence after they returned, but they experienced increased rates of alcohol or amphetamine use disorder as well as increased suicidality.

1	Increasing age is associated with a decrease in prevalence as a result of early mortality and the remission of symptoms after age 40 years (i.e., ”maturing out”). However, many individuals continue have presentations that meet opioid use disorder criteria for decades. Genetic and physiological. The risk for opiate use disorder can be related to individual, family, peer, and social environmental factors, but within these domains, genetic factors play a particularly important role both directly and indirectly. For instance, impulsivity and novelty seeking are individual temperaments that relate to the propensity to develop a substance use disorder but may themselves be genetically determined. Peer factors may relate to genetic predisposition in terms of how an individual selects his or her environ- ment.

1	Despite small variations regarding individual criterion items, opioid use disorder diag- nostic criteria perform equally well across most race/ethnicity groups. Individuals from resented among individuals with opioid use disorder. However, over time, opioid use disorder is seen more often among white middle-class individuals, especially females, suggesting that differences in use reﬂect the availability of opioid drugs and that other so- cial factors may impact prevalence. Medical personnel who have ready access to opioids may be at increased risk for opioid use disorder.

1	Routine urine toxicology test results are often positive for opioid drugs in individuals with- opioid use disorder. Urine test results remain positive for most opioids (e.g., heroin, mor- phine, codeine, oxycodone, propoxyphene) for 12—36 hours after administration. Fentanyl dures for several days. Methadone, buprenorphine (or buprenorphine/naloxone combi- nation), and LAAM (L-alpha-acetylmethadol) have to be specifically tested for and will not cause a positive result on routine tests for opiates. They can be detected for several days up to more than 1 week. Laboratory evidence of the presence of other substances (e.g., co- caine, marijuana, alcohol, amphetamines, benzodiazepines) is common. Screening test re- sults for hepatitis A, B, and C virus are positive in as many as 80%—90% of injection opioid users, either for hepatitis antigen (signifying active infection) or for hepatitis antibody (sig- nifying past infection). HIV is prevalent in injection opioid users as well. Mildly

1	injection opioid users, either for hepatitis antigen (signifying active infection) or for hepatitis antibody (sig- nifying past infection). HIV is prevalent in injection opioid users as well. Mildly elevated liver function test results are common, either as a result of resolving hepatitis or from toxic injury to the liver due to contaminants that have been mixed with the injected opioid. Sub- served for up to 6 months following opioid detoxification.

1	Similar to the risk generally observed for all substance use disorders, opioid use disorder is associated with a heightened risk for suicide attempts and completed suicides. Particu- larly notable are both accidental and deliberate opioid overdoses. Some suicide risk factors overlap with risk factors for an opioid use disorder. In addition, repeated opioid intoxica- tion or withdrawal may be associated with severe depressions that, although temporary, can be intense enough to lead to suicide attempts and completed suicides. Available data suggest that nonfatal accidental opioid overdose (which is common) and attempted sui- cide are distinct clinically significant problems that should not be mistaken for each other. Functional Consequences of Opioid Use Disorder

1	Functional Consequences of Opioid Use Disorder Opioid use is associated with a lack of mucous membrane secretions, causing dry mouth and nose. Slowing of gastrointestinal activity and a decrease in gut motility can produce severe constipation. Visual acuity may be impaired as a result of pupillary constriction with acute administration. In individuals who inject opioids, sclerosed veins (”tracks”) and puncture marks on the lower portions of the upper extremities are common. Veins sometimes become so severely sclerosed that peripheral edema develops, and individuals switch to injecting in veins in the legs, neck, or groin. When these veins become unusable, individuals often inject directly into their subcutaneous tissue ("skin-popping"), resulting in cellulit‘is, abscesses, and circular-appearing scars from healed skin lesions. Tetanus and

1	Clostridium botulinum infections are relatively rare but extremely serious consequences of injecting opioids, especially with contaminated needles. Infections may also occur in other organs and include bacterial endocarditis, hepatitis, and HIV infection. Hepatitis C infec- tions, for example, may occur in up to 90% of persons who inject opioids. In addition, the prevalence of HIV infection can be high among individuals who inject drugs, a large pro- portion of whom are individuals with opioid use disorder. HIV infection rates have been reported to be as high as 60% among heroin users with opioid use disorder in some areas of the United States or the Russian Federation. However, the incidence may also be 10% or less in other areas, especially those where access to clean injection material and parapher- nalia is facilitated.

1	Tuberculosis is a particularly serious problem among individuals who use drugs in- travenously, especially those who are dependent on heroin; infection is usually asymptom- atic and evident only by the presence of a positive tuberculin skin test. However, many cases of active tuberculosis have been found, especially among those who are infected with HIV. These individuals often have a newly acquired infection but also are likely to experience reactivation of a prior infection because of impaired immune function. Individuals who sniff heroin or other opioids into the nose (“snorting") often develop irritation of the nasal mucosa, sometimes accompanied by perforation of the nasal septum. Difficulties in sexual functioning are common. Males often experience erectile dysfunction during intoxication or chronic use. Females commonly have disturbances of reproductive function and irregular menses.

1	In relation to infections such as cellulitis, hepatitis, HIV infection, tuberculosis, and en- docarditis, opioid use disorder is associated with a mortality rate as high as 1.5%—2% per year. Death most often results from overdose, accidents, injuries, AIDS, or other general medical complications. Accidents and injuries due to violence that is associated with buy- ing or selling drugs are common. In some areas, violence accounts for more opioid-related deaths than overdose or HIV infection. Physiological dependence on opioids may occur in about half of the infants born to females with opioid use disorder; this can produce a se- vere withdrawal syndrome requiring medical treatment. Although low birth weight is also seen in children of mothers with opioid use disorder, it is usually not marked and is generally not associated with serious adverse consequences.

1	Opioid-induced mental disorders. Opioid-induced disorders occur frequently in individ- uals with opioid use disorder. Opioid-induced disorders may be characterized by symptoms (e.g., depressed mood) that resemble primary mental disorders (e.g., persistent depressive dis- order [dysthymia] vs. opioid-induced depressive disorder, with depressive features, with on- set during intoxication). Opioids are less likely to produce symptoms of mental disturbance than are most other drugs of abuse. Opioid intoxication and opioid withdrawal are distin- guished from the other opioid—induced disorders (e.g., opioid-induced depressive disorder, with onset during intoxication) because the symptoms in these latter disorders predominate the clinical presentation and are severe enough to warrant independent Clinical attention.

1	Other substance intoxication. Alcohol intoxication and sedative, hypnotic, or anxiolytic intoxication can cause a clinical picture that resembles that for opioid intoxication. A diag- nosis of alcohol or sedative, hypnotic, or anxiolytic intoxication can usually be made based on the absence of pupillary constriction or the lack of a response to naloxone challenge. In some cases, intoxication may be due both to opioids and to alcohol or other sedatives. In these cases, the naloxone challenge will not reverse all of the sedative effects. Other withdrawal disorders. The anxiety and restlessness associated with opioid with- drawal resemble symptoms seen in sedative-hypnotic withdrawal. However, opioid

1	Other withdrawal disorders. The anxiety and restlessness associated with opioid with- drawal resemble symptoms seen in sedative-hypnotic withdrawal. However, opioid Withdrawal is also accompanied by rhinorrhea, lacrimation, and pupillary dilation, which are not seen in sedative-type withdrawal. Dilated pupils are also seen in hallucinogen intoxication and stimulant intoxication. However, other signs or symptoms of opioid withdrawal, such as nausea, vomiting, diarrhea, abdominal cramps, rhinorrhea, or lacri— mation, are not present. The most common medical conditions associated with opioid use disorder are viral (e.g., HIV, hepatitis C virus) and bacterial infections, particularly among users of opioids by in- jection. These infections are less common in opioid use disorder with prescription opioids.

1	HIV, hepatitis C virus) and bacterial infections, particularly among users of opioids by in- jection. These infections are less common in opioid use disorder with prescription opioids. Opioid use disorder is often associated with other substance use disorders, especially those involving tobacco, alcohol, cannabis, stimulants, and benzodiazepines, which are often taken to reduce symptoms of opioid withdrawal or craving for opioids, or to enhance the ef- fects of administered opioids. Individuals with opioid use disorder are at risk for the devel- opment of mild to moderate depression that meets symptomatic and duration criteria for persistent depressive disorder (dysthymia) or, in some cases, for major depressive disorder.

1	of a preexisting primary depressive disorder. Periods of depression are especially common during chronic intoxication or in association with physical or psychosocial stressors that are related to the opioid use disorder. Insomnia is common, especially during withdrawal. An- tisocial personality disorder is much more common in individuals with opioid use disorder than in the general population. Posttraumatic stress disorder is also seen with increased fre- quency. A history of conduct disorder in childhood or adolescence has been identified as a significant risk factor for substance-related disorders, especially opioid use disorder. A. Recent use of an opioid. B. Clinically significant problematic behavioral or psychological changes (e.g.. initial eu- phoria followed by apathy, dysphoria, psychomotor agitation or retardation, impaired judgment) that developed during. or shortly after, opioid use.

1	C. Pupillary constriction (or pupillary dilation due to anoxia from severe overdose) and one (or more) of the following signs or symptoms developing during, or shortly after, opioid use: 1. Drowsiness or coma. 2. Slurred speech. 3. Impairment in attention or memory. D. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication with another sub- stance. Specify if: With perceptual disturbances: This specifier may be noted in the rare instance in which hallucinations with intact reality testing or auditory, visual, or tactile illusions oc- cur in the absence of a delirium. Coding note: The ICD-9-CM code is 292.89. The |CD-10-CM code depends on whether or not there is a comorbid opioid use disorder and whether or not there are perceptual dis- turbances.

1	Coding note: The ICD-9-CM code is 292.89. The |CD-10-CM code depends on whether or not there is a comorbid opioid use disorder and whether or not there are perceptual dis- turbances. For oploid Intoxication without perceptual disturbances: If a mild opioid use dis- order is comorbid, the |CD-10-CM code is F11.129, and it a moderate or severe opioid use disorder is comorbid, the |CD-10-CM code is F11.229. If there is no comorbid opi- oid use disorder, then the |CD-10-CM code is F11.929. For opioid intoxication with perceptual disturbances: If a mild opioid use disorder is comorbid, the |CD-10-CM code is F11.122, and if a moderate or severe opioid use disorder is comorbid, the lCD-10-CM code is F11.222. If there is no comorbid opioid use disorder, then the lCD-10-CM code is F11.922.

1	The essential feature of opioid intoxication is the presence of clinically significant prob— lematic behavioral or psychological changes (e.g., initial euphoria followed by apathy, dysphoria, psychomotor agitation or retardation, impaired judgment) that develop dur- ing, or shortly after, opioid use (Criteria A and B). Intoxication is accompanied by pupil- lary constriction (unless there has been a severe overdose with consequent anoxia and pupillary dilation) and one or more of the following signs: drowsiness (described as be- ing ”on the nod”), slurred speech, and impairment in attention or memory (Criterion C); drowsiness may progress to coma. Individuals with opioid intoxication may demonstrate inattention to the environment, even to the point of ignoring potentially harmful events. The signs or symptoms must not be attributable to another medical condition and are not better explained by another mental disorder (Criterion D).

1	The signs or symptoms must not be attributable to another medical condition and are not better explained by another mental disorder (Criterion D). Other substance intoxication. Alcohol intoxication and sedative-hypnotic intoxication can cause a clinical picture that resembles opioid intoxication. A diagnosis of alcohol or sedative-hypnotic intoxication can usually be made based on the absence of pupillary con- striction or the lack of a response to a naloxone challenge. In some cases, intoxication may be due both to opioids and to alcohol or other sedatives. In these cases, the naloxone chal- lenge will not reverse all of the sedative effects. Other opioid-related disorders. Opioid intoxication is distinguished from the other opioid-induced disorders (e.g., opioid—induced depressive disorder, with onset during in- toxication) because the symptoms in the latter disorders predominate in the clinical pre- sentation and meet full criteria for the relevant disorder.

1	Diagnostic Criteria 292.0 (F11.23) A. Presence of either of the following: 1. Cessation of (or reduction in) opioid use that has been heavy and prolonged (i.e., several weeks or longer). 2. Administration of an opioid antagonist after a period of opioid use. B. Three (or more) of the following developing within minutes to several days after Criterion A: 1. Dysphoric mood. Nausea or vomiting. Muscle aches. Lacrimation or rhinorrhea. Pupillary dilation, piloerection, or sweating. 91:59)!" Diarrhea. Yawning. Fever. Insomnia. 509.“? C. The signs or symptoms in Criterion B cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. D. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication or withdrawal from another substance. Coding note: The lCD-9-CM code is 292.0. The |CD-10-CM code for opioid withdrawal is

1	Coding note: The lCD-9-CM code is 292.0. The |CD-10-CM code for opioid withdrawal is F11.23. Note that the |CD-10-CM code indicates the comorbid presence of a moderate or severe opioid use disorder, reflecting the fact that opioid withdrawal can only occur in the presence of a moderate or severe opioid use disorder. It is not permissible to code a co- morbid mild opioid use disorder with opioid withdrawal. The essential feature of opioid withdrawal is the presence of a characteristic withdrawal syndrome that develops after the cessation of (or reduction in) opioid use that has been heavy and prolonged (Criterion A1). The withdrawal syndrome can also be precipitated by administration of an opioid antagonist (e.g., naloxone or naltrexone) after a period of opioid use (Criterion A2). This may also occur after administration of an opioid partial ag- onist such as buprenorphine to a person Currently using a full opioid agonist.

1	Opioid withdrawal is characterized by a pattern of signs and symptoms that are oppo- site to the acute agonist effects. The first of these are subjective and consist of complaints of anxiety, restlessness, and an "achy feeling” that is often located in the back and legs, along with irritability and increased sensitivity to pain. Three or more of the following must be present to make a diagnosis of opioid withdrawal: dysphoric mood; nausea or vomiting; muscle aches; lacrimation or rhinorrhea; pupillary dilation, piloerection, or increased sweating; diarrhea; yawning; fever; and insomnia (Criterion B). Piloerection and fever are associated with more severe withdrawal and are not often seen in routine clinical practice because individuals with opioid use disorder usually obtain substances before with- drawal becomes that far advanced. These symptoms of opioid withdrawal must cause clinically significant distress or impairment in social, occupational, or other important ar- eas of

1	before with- drawal becomes that far advanced. These symptoms of opioid withdrawal must cause clinically significant distress or impairment in social, occupational, or other important ar- eas of functioning (Criterion C). The symptoms must not be attributable to another med- ical condition and are not better explained by another mental disorder (Criterion D).

1	Meeting diagnostic criteria for opioid withdrawal alone is not sufficient for a diagnosis of opioid use disorder, but concurrent symptoms of craving and drug-seeking behavior are suggestive of comorbid opioid use disorder. ICD—10-CM codes only allow a diagnosis of opioid withdrawal in the presence of comorbid moderate to severe opioid use disorder. The speed and severity of withdrawal associated with opioids depend on the half-life of the opioid used. Most individuals who are physiologically dependent on short-acting drugs such as heroin begin to have withdrawal symptoms within 6—12 hours after the last dose. Symptoms may take 2—4 days to emerge in the case of longer-acting drugs such as metha- done, LAAM (L—alpha-acetylmethadol), or buprenorphine. Acute withdrawal symptoms for a short—acting opioid such as heroin usually peak within 1—3 days and gradually subside over a period of 5—7 days. Less acute withdrawal symptoms can last for weeks to months.

1	These more chronic symptoms include anxiety, dysphoria, anhedonia, and insomnia. Males with opioid withdrawal may experience piloerection, sweating, and spontaneous ejaculations while awake. Opioid withdrawal is distinct from opioid use disorder and does not necessarily occur in the presence of the drug—seeking behavior associated with opioid use disorder. Opioid withdrawal may occur in any individual after cessation of re- peated use of an opioid, whether in the setting of medical management of pain, during opioid agonist therapy for opioid use disorder, in the context of private recreational use, or following attempts to self—treat symptoms of mental disorders with opioids. Among individuals from various clinical settings, opioid withdrawal occurred in 60% of individuals who had used heroin at least once in the prior 12 months.

1	Among individuals from various clinical settings, opioid withdrawal occurred in 60% of individuals who had used heroin at least once in the prior 12 months. Opioid withdrawal is typical in the course of an opioid use disorder. It can be part of an es- calating pattern in which an opioid is used to reduce withdrawal symptoms, in turn lead- ing to more withdrawal at a later time. For persons with an established opioid use disorder, withdrawal and attempts to relieve withdrawal are typical. Other withdrawal disorders. The anxiety and restlessness associated with opioid with- drawal resemble symptoms seen in sedative-hypnotic withdrawal. However, opioid with- drawal is also accompanied by rhinorrhea, lacrimation, and pupillary dilation, which are not seen in sedative-type withdrawal.

1	Other substance intoxication. Dilated pupils are also seen in hallucinogen intoxication and stimulant intoxication. However, other signs or symptoms of opioid withdrawal, such as nausea, vomiting, diarrhea, abdominal cramps, rhinorrhea, and lacrimation, are not present. Other opioid-induced disorders. Opioid withdrawal is distinguished from the other opioid-induced disorders (e.g., opioid-induced depressive disorder, with onset during withdrawal) because the symptoms in these latter disorders are in excess of those usually associated with opioid withdrawal and meet full criteria for the relevant disorder.

1	The following opioid-induced disorders are described in other chapters of the manual with disorders with which they share phenomenology (see the substance/ medication-induced mental disorders in these chapters): opioid-induced depressive disorder (”Depressive Dis- functions”). For opioid intoxication delirium and opioid withdrawal delirium, see the crite- ria and discussion of delirium in the chapter ”Neurocognitive Disorders.” These opioid- induced disorders are diagnosed instead of opioid intoxication or opioid withdrawal only when the symptoms are sufficiently severe to warrant independent clinical attention. 292.9 (F11.99)

1	292.9 (F11.99) This category applies to presentations in which symptoms characteristic of an opioid- related disorder that cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning predominate but do not meet the full criteria for any specific opioid-related disorder or any of the disorders in the substance-related and addictive disorders diagnostic class. Sedative-, Hypnotic-, Sedative, Hypnotic, or Anxiolytic Use Disorder Sedative, Hypnotic, or Anxiolytic Intoxication Sedative, Hypnotic, or Anxiolytic Withdrawal Other Sedative-, Hypnotic-, or Anxiolytic-lnduced Disorders Unspecified Sedative-, Hypnotic-, or Anxiolytic-Related Disorder Sedative, Hypnotic, or Anxiolytic Use Disorder A. A problematic pattern of sedative, hypnotic, or anxiolytic use leading to clinically signif- icant impairment or distress, as manifested by at least two of the following, occurring within a 12-month period: 1. 2.

1	2. Sedatives, hypnotics, or anxiolytics are often taken in larger amounts or over a lon- ger period than was intended. There is a persistent desire or unsuccessful efforts to cut down or control sedative, hypnotic, or anxiolytic use. A great deal of time is spent in activities necessary to obtain the sedative, hypnotic, or anxiolytic; use the sedative, hypnotic, or anxiolytic; or recover from its effects. Craving, or a strong desire or urge to use the sedative, hypnotic, or anxiolytic. Recurrent sedative, hypnotic, or anxiolytic use resulting in a failure to fulfill major role obligations at work. school, or home (e.g., repeated absences from work or poor work performance related to sedative, hypnotic, or anxiolytic use; sedative-, hypnotic-, or anxiolytic-related absences, suspensions, or expulsions from school; neglect of children or household).

1	Continued sedative, hypnotic, or anxiolytic use despite having persistent or re- current social or interpersonal problems caused or exacerbated by the effects of sedatives, hypnotics, or anxiolytics (e.g., arguments with a spouse about conse- quences of intoxication; physical tights). Important social, occupational, or recreational activities are given up or reduced be- cause of sedative. hypnotic, or anxiolytic use. Sedative, Hypnotic, or Anxiolytic Use Disorder 551 8. Recurrent sedative, hypnotic, or anxiolytic use in situations in which it is physically hazardous (e.g., driving an automobile or operating a machine when impaired by sedative, hypnotic, or anxiolytic use). 9. Sedative. hypnotic, or anxiolytic use is continued despite knowledge of having a persistent or recurrent physical or psychological problem that is likely to have been caused or exacerbated by the sedative, hypnotic, or anxiolytic.

1	10. Tolerance, as defined by either of the following: a. A need for markedly increased amounts of the sedative, hypnotic, or anxiolytic to achieve intoxication or desired effect. b. A markedly diminished effect with continued use of the same amount of the sed- ative, hypnotic, or anxiolytic. Note: This criterion is not considered to be met for individuals taking sedatives, hypnotics, or anxiolytics under medical supervision. 11. Withdrawal, as manifested by either of the following: a. The characteristic withdrawal syndrome for sedatives, hypnotics, or anxiolytics (refer to Criteria A and B of the criteria set for sedative. hypnotic, or anxiolytic withdrawal, pp. 557—558). D. Sedatives, hypnotics, or anxiolytics (or a closely related substance, such as al- cohol) are taken to relieve or avoid withdrawal symptoms. Note: This criterion is not considered to be met for individuals taking sedatives, hypnotics, or anxiolytics under medical supervision. Specify it:

1	Note: This criterion is not considered to be met for individuals taking sedatives, hypnotics, or anxiolytics under medical supervision. Specify it: In early remission: After full criteria for sedative, hypnotic, or anxiolytic use disorder were previously met, none of the criteria for sedative, hypnotic, or anxiolytic use disor- der have been met for at least 3 months but for less than 12 months (with the exception that Criterion A4, “Craving, or a strong desire or urge to use the sedative, hypnotic, or anxiolytic,” may be met). In sustained remission: After full criteria for sedative, hypnotic, or anxiolytic use dis— order were previously met, none of the criteria for sedative, hypnotic, or anxiolytic use disorder have been met at any time during a period of 12 months or longer (with the exception that Criterion A4. “Craving. or a strong desire or urge to use the sedative, hypnotic, or anxiolytic,” may be met). Specify if:

1	Specify if: In a controlled environment: This additional specifier is used it the individual is in an environment where access to sedatives, hypnotics, or anxiolytics is restricted.

1	Coding based on current severity: Note for |CD-10-CM codes: If a sedative, hypnotic. or anxiolytic intoxication; sedative, hypnotic, or anxiolytic withdrawal; or another sedative-, hypnotic-, or anxiolytic-induced mental disorder is also present, do not use the codes be- low for sedative, hypnotic, or anxiolytic use disorder. Instead the comorbid sedative, hyp- notic, or anxiolytic use disorder is indicated in the 4th character of the sedative, hypnotic-, or anxiolytic-induced disorder (see the coding note for sedative, hypnotic, or anxiolytic in- toxication; sedative, hypnotic, or anxiolytic withdrawal; or specific sedative-, hypnotic-, or anxiolytic-induced mental disorder). For example, if there is comorbid sedative-, hypnotic-, or anxiolytic-induced depressive disorder and sedative, hypnotic, or anxiolytic use disor- der, only the sedative-, hypnotic-, or anxiolytic-induced depressive disorder code is given with the 4th character indicating whether the comorbid sedative, hypnotic,

1	or anxiolytic use disor- der, only the sedative-, hypnotic-, or anxiolytic-induced depressive disorder code is given with the 4th character indicating whether the comorbid sedative, hypnotic, or anxiolytic use disorder is mild, moderate, or severe: F13.14 for mild sedative, hypnotic, or anxiolytic use disorder with sedative-, hypnotic-, or anxiolytic-induced depressive disorder or F13.24 for a moderate or severe sedative, hypnotic, or anxiolytic use disorder with sedative-, hypnotic-, or anxiolytic-induced depressive disorder.

1	Specify current severity: 305.40 (F13.10) Mild: Presence of 2-3 symptoms. 304.10 (F13.20) Moderate: Presence of 4—5 symptoms. 304.10 (F13.20) Severe: Presence of 6 or more symptoms. "In a controlled environment” applies as a further specifier of remission if the individual is both in remission and in a controlled environment (i.e., in early remission in a controlled environment or in sustained remission in a controlled environment). Examples of these environments are closely supervised and substance-free jails, therapeutic communities, and locked hospital units.

1	Sedative, hypnotic, or anxiolytic substances include benzodiazepines, benzodiazepine- like drugs (e.g., zolpidem, zaleplon), carbamates (e.g., glutethimide, meprobamate), barbiturates (e.g., secobarbital), and barbiturate-like hypnotics (e.g., glutethimide, meth- aqualone). This class of substances includes all prescription sleeping medications and almost all prescription antianxiety medications. Nonbenzodiazepine antianxiety agents (e.g., buspirone, gepirone) are not included in this class because they do not appear to be associated with significant misuse.

1	Like alcohol, these agents are brain depressants and can produce similar substance/ medication-induced and substance use disorders. Sedative, hypnotic, or anxiolytic sub- stances are available both by prescription and illegally. Some individuals who obtain these substances by prescription will develop a sedative, hypnotic, or anxiolytic use disorder, use disorder. In particular, sedatives, hypnotics, or anxiolytics with rapid onset and / or short to intermediate lengths of action may be taken for intoxication purposes, although longer acting substances in this class may be taken for intoxication as well.

1	Craving (Criterion A4), either while using or during a period of abstinence, is a typical feature of sedative, hypnotic, or anxiolytic use disorder. Misuse of substances from this class may occur on its own or in conjunction with use of other substances. For example, in- dividuals may use intoxicating doses of sedatives or benzodiazepines to "come down” from cocaine or amphetamines or use high doses of benzodiazepines in combination with methadone to ”boost” its effects.

1	Repeated absences or poor work performance, school absences, suspensions or expul- sions, and neglect of children or household (Criterion A5) may be related to sedative, hyp- notic, or anxiolytic use disorder, as may the continued use of the substances despite arguments with a spouse about consequences of intoxication or despite physical fights (Criterion A6). Limiting contact with family or friends, avoiding work or school, or stop- ping participation in hobbies, sports, or games (Criterion A7) and recurrent sedative, hypnotic, or anxiolytic use when driving an automobile or operating a machine when im- paired by sedative, hypnotic, or anxiolytic use (Criterion A8) are also seen in sedative, hypnotic, or anxiolytic use disorder.

1	Very significant levels of tolerance and withdrawal can develop to the sedative, hyp- notic, or anxiolytic. There may be evidence of tolerance and withdrawal in the absence of a diagnosis of a sedative, hypnotic, or anxiolytic use disorder in an individual who has abruptly discontinued use of benzodiazepines that were taken for long periods of time at prescribed and therapeutic doses. In these cases, an additional diagnosis of sedative, hyp— notic, or anxiolytic use disorder is made only if other criteria are met. That is, sedative, hypnotic, or anxiolytic medications may be prescribed for appropriate medical purposes, and depending on the dose regimen, these drugs may then produce tolerance and with-

1	Sedative, Hypnotic, or Anxiolytic Use Disorder 553 drawal. If these drugs are prescribed or recommended for appropriate medical purposes, and if they are used as prescribed, the resulting tolerance or withdrawal does not meet the criteria for diagnosing a substance use disorder. However, it is necessary to determine whether the drugs were appropriately prescribed and used (e.g., falsifying medical symp- toms to obtain the medication; using more medication than prescribed; obtaining the med— ication from several doctors without informing them of the others’ involvement). Given the unidimensional nature of the symptoms of sedative, hypnotic, or anxiolytic use disorder, severity is based on the number of criteria endorsed.

1	Given the unidimensional nature of the symptoms of sedative, hypnotic, or anxiolytic use disorder, severity is based on the number of criteria endorsed. Sedative, hypnotic, or anxiolytic use disorder is often associated with other substance use dis- orders (e.g., alcohol, cannabis, opioid, stimulant use disorders). Sedatives are often used to al- leviate the unwanted effects of these other substances. With repeated use of the substance, tolerance develops to the sedative effects, and a progressively higher dose is used. However, tolerance to brain stem depressant effects develops much more slowly, and as the individual takes more substance to achieve euphoria or other desired effects, there may be a sudden onset of respiratory depression and hypotension, which may result in death. Intense or repeated sedative, hypnotic, or anxiolytic intoxication may be associated with severe depression that, although temporary, can lead to suicide attempt and completed suicide.

1	The 12-month prevalences of DSM-IV sedative, hypnotic, or anxiolytic use disorder are es- timated to be 0.3% among 12- to 17-year-olds and 0.2% among adults age 18 years and older. Rates of DSM-IV sedative, hypnotic, or anxiolytic use disorder are slightly greater among adult males (0.3%) than among adult females, but for 12- to 17-year-olds, the rate for females (0.4%) exceeds that for males (0.2%). The 12-month prevalence of DSM-IV sedative, hypnotic, or anxiolytic use disorder decreases as a function of age and is great- est among 18- to 29-year-olds (0.5%) and lowest among individuals 65 years and older (0.04%).

1	Twelve-month prevalence of sedative, hypnotic, or anxiolytic use disorder varies across racial/ethnic subgroups of the US. population. For 12- to 17-year-olds, rates are greatest among whites (0.3%) relative to African Americans (0.2%), Hispanics (0.2%), Native Amer- icans (0.1%), and Asian Americans and Pacific Islanders (0.1%). Among adults, 12—month prevalence is greatest among Native Americans and Alaska Natives (0.8%), with rates of approximately 0.2% among African Americans, whites, and Hispanics and 0.1% among Asian Americans and Pacific Islanders.

1	Asian Americans and Pacific Islanders. The usual course of sedative, hypnotic, or anxiolytic use disorder involves individuals in their teens or 205 who escalate their occasional use of sedative, hypnotic, or anxiolytic agents to the point at which they develop problems that meet criteria for a diagnosis. This ders (e.g., alcohol, opioids, stimulants). An initial pattern of intermittent use socially (e.g., at parties) can lead to daily use and high levels of tolerance. Once this occurs, an increasing level of interpersonal difficulties, as well as increasingly severe episodes of cognitive dys- fimction and physiological withdrawal, can be expected.

1	The second and less frequently observed clinical course begins with an individual who originally obtained the medication by prescription from a physician, usually for the treat- ment of anxiety, insomnia, or somatic complaints. As either tolerance or a need for higher doses of the medication develops, there is a gradual increase in the dose and frequency of self—administration. The individual is likely to continue to justify use on the basis of his or her original symptoms of anxiety or insomnia, but substance—seeking behavior becomes more prominent, and the individual may seek out multiple physicians to obtain sufficient supplies of the medication. Tolerance can reach high levels, and withdrawal (including seizures and withdrawal delirium) may occur.

1	As with many substance use disorders, sedative, hypnotic, or anxiolytic use disorder gen- erally has an onset during adolescence or early adult life. There is an increased risk for misuse and problems from many psychoactive substances as individuals age. In particular, cognitive impairment increases as a side effect with age, and the metabolism of sedatives, hypnotics, or anxiolytics decreases with age among older individuals. Both acute and chronic toxic effects of these substances, especially effects on cognition, memory, and motor coordination, are likely to increase with age as a consequence of pharmacodynamic and pharmacokinetic age- related changes. Individuals with major neurocognitive disorder (dementia) are more likely to develop intoxication and impaired physiological functioning at lower doses.

1	Deliberate intoxication to achieve a ”high" is most likely to be observed in teenagers and individuals in their 205. Problems associated with sedatives, hypnotics, or anxiolytics are also seen in individuals in their 403 and older who escalate the dose of prescribed med- ications. In older individuals, intoxication can resemble a progressive dementia. Temperamental. Impulsivity and novelty seeking are individual temperaments that re- late to the propensity to develop a substance use disorder but may themselves be geneti- cally determined. Environmental. Since sedatives, hypnotics, or anxiolytics are all pharmaceuticals, a key risk factor relates to availability of the substances. In the United States, the historical pat- terns of sedative, hypnotic, or anxiolytic misuse relate to the broad prescribing patterns.

1	For instance, a marked decrease in prescription of barbiturates was associated with an in- crease in benzodiazepine prescribing. Peer factors may relate to genetic predisposition in terms of how individuals select their environment. Other individuals at heightened risk might include those with alcohol use disorder who may receive repeated prescriptions in response to their complaints of alcohol-related anxiety or insomnia. Genetic and physiological. As for other substance use disorders, the risk for sedative, hypnotic, or anxiolytic use disorder can be related to individual, family, peer, social, and environmental factors. Within these domains, genetic factors play a particularly important role both directly and indirectly. Overall, across development, genetic factors seem to play a larger role in the onset of sedative, hypnotic, or anxiolytic use disorder as individuals age through puberty into adult life.

1	Course modifiers. Early onset of use is associated with greater likelihood for develop- ing a sedative, hypnotic, or anxiolytic use disorder. There are marked variations in prescription patterns (and availability) of this class of sub- stances in different countries, which may lead to variations in prevalence of sedative, hyp- notic, or anxiolytic use disorders. Females may be at higher risk than males for prescription drug misuse of sedative, hyp- notic, or anxiolytic substances. Almost all sedative, hypnotic, or anxiolytic substances can be identified through labora- tory evaluations of urine or blood (the latter of which can quantify the amounts of these Sedative, Hypnotic, or Anxiolytic Use Disorder 555 agents in the body). Urine tests are likely to remain positive for up to approximately 1 week after the use of long-acting substances, such as diazepam or ﬂurazepam. Sedative, Hypnotic, or Anxioiytic Use Disorder

1	Sedative, Hypnotic, or Anxioiytic Use Disorder The social and interpersonal consequences of sedative, hypnotic, or anxiolytic use disorder mimic those of alcohol in terms of the potential for disinhibited behavior. Accidents, interper- sonal difficulties (such as arguments or fights), and interference with work or school perfor- mance are all common outcomes. Physical examination is likely to reveal evidence of a mild decrease in most aspects of autonomic nervous system functioning, including a slower pulse, a slightly decreased respiratory rate, and a slight drop in blood pressure (most likely to occur with postural changes). At high doses, sedative, hypnotic, or anxiolytic substances can be le- thal, particularly when mixed with alcohol, although the lethal dosage varies considerably among the specific substances. Overdoses may be associated with a deterioration in vital signs that signals an impending medical emergency (e.g., respiratory arrest from barbiturates).

1	There may be consequences of trauma (e.g., internal bleeding or a subdural hematoma) from accidents that occur while intoxicated. Intravenous use of these substances can result in med- ical complications related to the use of contaminated needles (e.g., hepatitis and HIV).

1	Acute intoxication can result in accidental injuries and automobile accidents. For elderly individuals, even short—term use of these sedating medications at prescribed doses can be as- sociated with an increased risk for cognitive problems and falls. The disinhibiting effects of these agents, like alcohol, may potentially contribute to overly aggressive behavior, with sub- sequent interpersonal and legal problems. Accidental or deliberate overdoses, similar to those observed for alcohol use disorder or repeated alcohol intoxication, can occur. In contrast to their wide margin of safety when used alone, benzodiazepines taken in combination with al- cohol can be particularly dangerous, and accidental overdoses are reported commonly. Acci- and other nonbenzodiazepine sedatives (e.g., methaqualone), but since these agents are much less available than the benzodiazepines, the frequency of overdosing is low in most settings.

1	Other mental disorders or medical conditions. Individuals with sedative-, hypnotic-, or anxiolytic-induced disorders may present with symptoms (e.g., anxiety) that resemble primary mental disorders (e.g., generalized anxiety disorder vs. sedative-, hypnotic-, or anxiolytic-induced anxiety disorder, with onset during withdrawal). The slurred speech, incoordination, and other associated features characteristic of sedative, hypnotic, or anx- iolytic intoxication could be the result of another medical condition (e.g., multiple sclero- sis) or of a prior head trauma (e.g., a subdural hematoma). Alcohol use disorder. Sedative, hypnotic, or anxiolytic use disorder must be differenti- ated from alcohol use disorder.

1	Alcohol use disorder. Sedative, hypnotic, or anxiolytic use disorder must be differenti- ated from alcohol use disorder. Clinically appropriate use of sedative, hypnotic, or anxiolytic medications. Individuals may continue to take benzodiazepine medication according to a physician’s direction for a legitimate medical indication over extended periods of time. Even if physiological signs of tolerance or withdrawal are manifested, many of these individuals do not develop symp- toms that meet the criteria for sedative, hypnotic, or anxiolytic use disorder because they are not preoccupied with obtaining the substance and its use does not interfere with their performance of usual social or occupational roles.

1	Nonmedical use of sedative, hypnotic, or anxiolytic agents is associated with alcohol use disorder, tobacco use disorder, and, generally, illicit drug use. There may also be an over- lap between sedative, hypnotic, or anxiolytic use disorder and antisocial personality dis- order; depressive, bipolar, and anxiety disorders; and other substance use disorders, such as alcohol use disorder and illicit drug use disorders. Antisocial behavior and antisocial personality disorder are especially associated with sedative, hypnotic, or anxiolytic use disorder when the substances are obtained illegally. Sedative, Hypnotic, or Anxiolytic Intoxication A. Recent use of a sedative, hypnotic, or anxiolytic. B. Clinically significant maladaptive behavioral or psychological changes (e.g., inappro- priate sexual or aggressive behavior, mood lability, impaired judgment) that developed during, or shortly after, sedative, hypnotic, or anxiolytic use.

1	C. One (or more) of the following signs or symptoms developing during. or shortly after, sedative, hypnotic. or anxiolytic use: Slurred speech. Incoordination. Unsteady gait. Nystagmus. Impairment in cognition (e.g., attention, memory). 6. Stupor or coma. W959i“? D. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication with another sub- stance. Coding note: The lCD-9-CM code is 292.89. The |CD-10-CM code depends on whether there is a comorbid sedative, hypnotic, or anxiolytic use disorder. It a mild sedative, hyp- notic, or anxiolytic use disorder is comorbid, the ICD-10-CM code is F13.129, and if a mod- erate or severe sedative, hypnotic, or anxiolytic use disorder is comorbid, the |CD-10-CM code is F13.229. If there is no comorbid sedative, hypnotic, or anxiolytic use disorder, then the |CD-10-CM code is F13.929.

1	Note: For information on Development and Course; Risk and Prognostic Factors; Culture- Related Diagnostic Issues; Diagnostic Markers; Functional Consequences of Sedative, Hypnotic, or Anxiolytic Intoxication; and Comorbidity, see the corresponding sections in sedative, hypnotic, or anxiolytic use disorder.

1	The essential feature of sedative, hypnotic, or anxiolytic intoxication is the presence of clini- cally significant maladaptive behavioral or psychological changes (e.g., inappropriate sexual or aggressive behavior, mood lability, impaired judgment, impaired social or occupational functioning) that develop during, or shortly after, use of a sedative, hypnotic, or anxiolytic (Criteria A and B). As with other brain depressants, such as alcohol, these behaviors may be ac- companied by slurred speech, incoordination (at levels that can interfere with driving abilities and with performing usual activities to the point of causing falls or automobile accidents), an unsteady gait, nystagmus, impairment in cognition (e.g., attentional or memory problems), and stupor or coma (Criterion C). Memory impairment is a prominent feature of sedative, hyp- notic, or anxiolytic intoxication and is most often characterized by an anterograde amnesia that resembles ”alcoholic blackouts,” which can be

1	impairment is a prominent feature of sedative, hyp- notic, or anxiolytic intoxication and is most often characterized by an anterograde amnesia that resembles ”alcoholic blackouts,” which can be disturbing to the individual. The symptoms must not be attributable to another medical condition and are not better explained by another

1	Sedative, Hypnotic, or Anxiolytic Withdrawal 557 mental disorder (Criterion D). Intoxication may occur in individuals who are receiving these substances by prescription, are borrowing the medication from friends or relatives, or are de- liberately taking the substance to achieve intoxication. Associated features include taking more medication than prescribed, taking multiple dif— ferent medications, or mixing sedative, hypnotic, or anxiolytic agents with alcohol, which can markedly increase the effects of these agents.

1	The prevalence of sedative, hypnotic, or anxiolytic intoxication in the general population is unclear. However, it is probable that most nonmedical users of sedatives, hypnotics, or anxiolytics would at some time have signs or symptoms that meet criteria for sedative, hypnotic, or anxiolytic intoxication; if so, then the prevalence of nonmedical sedative, hypnotic, or anxiolytic use in the general population may be similar to the prevalence of sedative, hypnotic, or anxiolytic intoxication. For example, tranquilizers are used non- medically by 2.2% of Americans older than 12 years.

1	Alcohol use disorders. Since the clinical presentations may be identical, distinguishing sed- ative, hypnotic, or anxiolytic intoxication from alcohol use disorders requires evidence for re- cent ingestion of sedative, hypnotic, or anxiolytic medications by self-report, informant report, or toxicological testing. Many individuals who misuse sedatives, hypnotics, or anxiolytics may also misuse alcohol and other substances, and so multiple intoxication diagnoses are possible. Alcohol intoxication. Alcohol intoxication may be distinguished from sedative, hypnotic, or anxiolytic intoxication by the smell of alcohol on the breath. Otherwise, the features of the two disorders may be similar.

1	_Other sedative-, hypnotic-, or anxiolytic-induced disorders. Sedative, hypnotic, or anx- iolytic intoxication is distinguished from the other sedative—, hypnotic-, or anxiolytic- induced disorders (e.g., sedative, hypnotic-, or anxiolytic-induced anxiety disorder, with onset during withdrawal) because the symptoms in the latter disorders predominate in the clinical presentation and are severe enough to warrant clinical attention. Neurocognitive disorders. In situations of cognitive impairment, traumatic brain in- jury, and delirium from other causes, sedatives, hypnotics, or anxiolytics may be intoxi- cating at quite low dosages. The differential diagnosis in these complex settings is based on the predominant syndrome. An additional diagnosis of sedative, hypnotic, or anxio- lytic intoxication may be appropriate even if the substance has been ingested at a low dos- age in the setting of these other (or similar) co-occurring conditions. Sedative, Hypnotic, or Anxiolytic Withdrawal

1	Sedative, Hypnotic, or Anxiolytic Withdrawal A. Cessation of (or reduction in) sedative, hypnotic, or anxiolytic use that has been pro- longed. B. Two (or more) of the following, developing within several hours to a few days after the ces- sation of (or reduction in) sedative, hypnotic. or anxiolytic use described in Criterion A: 1. Autonomic hyperactivity (e.g., sweating or pulse rate greater than 100 bpm). 2. Hand tremor. Insomnia. Nausea or vomiting. Transient visual, tactile, or auditory hallucinations or illusions. Psychomotor agitation. Anxiety. Grand mal seizures. @NQWP.“ C. The signs or symptoms in Criterion B cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. D. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication or withdrawal from another substance. Specify if:

1	Specify if: With perceptual disturbances: This specifier may be noted when hallucinations with in- tact reality testing or auditory, visual, or tactile illusions occur in the absence of a delirium.

1	Coding note: The |CD-9-CM code is 292.0. The |CD-10-CM code for sedative, hypnotic, or anxiolytic withdrawal depends on whether or not there is a comorbid moderate or se- vere sedative, hypnotic, or anxiolytic use disorder and whether or not there are perceptual disturbances. For sedative, hypnotic, or anxiolytic withdrawal without perceptual distur- bances, the |CD-10—CM code is F13.239. For sedative, hypnotic, or anxiolytic withdrawal with perceptual disturbances, the ICD-10-CM code is F13.232. Note that the lCD-10-CM codes indicate the comorbid presence of a moderate or severe sedative, hypnotic, or anx- iolytic use disorder, reflecting the fact that sedative, hypnotic, or anxiolytic withdrawal can only occur in the presence of a moderate or severe sedative, hypnotic, or anxiolytic use disorder. It is not permissible to code a comorbid mild sedative, hypnotic, or anxiolytic use disorder with sedative, hypnotic, or anxiolytic withdrawal.

1	Note: For information on Development and Course; Risk and Prognostic Factors; Culture- Related Diagnostic Issues; Functional Consequences of Sedative, Hypnotic, or Anxiolytic Withdrawal; and Comorbidity, see the corresponding sections in sedative, hypnotic, or anxiolytic use disorder.

1	The essential feature of sedative, hypnotic, or anxiolytic withdrawal is the presence of a char- acteristic syndrome that develops after a marked decrease in or cessation of intake after several weeks or more of regular use (Criteria A and B). This withdrawal syndrome is characterized by two or more symptoms (similar to alcohol withdrawal) that include autonomic hyperactivity (e.g., increases in heart rate, respiratory rate, blood pressure, or body temperature, along with sweating); a tremor of the hands; insomnia; nausea, sometimes accompanied by vomiting; anxiety; and psychomotor agitation. A grand mal seizure may occur in perhaps as many as 20%—30% of individuals undergoing untreated withdrawal from these substances. In severe withdrawal, visual, tactile, or auditory hallucinations or illusions can occur but are usually in the context of a delirium. If the individual’s reality testing is intact (i.e., he or she knows the substance is causing the hallucinations) and the illusions

1	illusions can occur but are usually in the context of a delirium. If the individual’s reality testing is intact (i.e., he or she knows the substance is causing the hallucinations) and the illusions occur in a clear sensorium, the spec- ifier “with perceptual disturbances” can be noted. When hallucinations occur in the absence of intact reality testing, a diagnosis of substance/medication-induced psychotic disorder should be considered. The symptoms cause clinically significant distress or impairment in social, oc- cupational, or other important areas of functioning (Criterion C). The symptoms must not be attributable to another medical condition and are not better explained by another mental dis- order (e.g., alcohol withdrawal or generalized anxiety disorder) (Criterion D). Relief of with- drawal symptoms with administration of any sedative-hypnotic agent would support a diagnosis of sedative, hypnotic, or anxiolytic withdrawal.

1	Sedative, Hypnotic, or Anxiolytic Withdrawal 559

1	The timing and Severity of the withdrawal syndrome will differ depending on the specific substance and its pharmacokinetics and pharmacodynamics. For example, withdrawal from shorter-acting substances that are rapidly absorbed and that have no active metabo— lites (e.g., triazolam) can begin within hours after the substance is stopped; withdrawal from substances with long—acting metabolites (e.g., diazepam) may not begin for 1—2 days or longer. The withdrawal syndrome produced by substances in this class may be charac- terized by the development of a delirium that can be life-threatening. There may be evi- dence of tolerance and withdrawal in the absence of a diagnosis of a substance use for long periods of time at prescribed and therapeutic doses. However, ICD-10-CM codes only allow a diagnosis of sedative, hypnotic, or anxiolytic withdrawal in the presence of comorbid moderate to severe sedative, hypnotic, or anxiolytic use disorder.

1	The time course of the withdrawal syndrome is generally predicted by the half—life of the substance. Medications whose actions typically last about 10 hours or less (e.g., loraz- epam, oxazepam, temazepam) produce withdrawal symptoms within 6—8 hours of de- creasing blood levels that peak in intensity on the second day and improve markedly by the fourth or fifth day. For substances with longer half—lives (e.g., diazepam), symptoms may not develop for more than 1 week, peak in intensity during the second week, and de- crease markedly during the third or fourth week. There may be additional longer-term symptoms at a much lower level of intensity that persist for several months,

1	The longer the substance has been taken and the higher the dosages used, the more likely it is that there will be severe withdrawal. However, withdrawal has been reported with as little as 15 mg of diazepam (or its equivalent in other benzodiazepines) when taken daily for several months. Doses of approximately 40 mg of diazepam (or its equivalent) daily are more likely to produce clinically relevant withdrawal symptoms, and even higher doses (e.g., 100 mg of di- azepam) are more likely to be followed by withdrawal seizures or delirium. Sedative, hyp- notic, or anxiolytic withdrawal delirium is characterized by disturbances in consciousness and cognition, with visual, tactile, or auditory hallucinations. When present, sedative, hypnotic, or anxiolytic withdrawal delirium should be diagnosed instead of withdrawal. The prevalence of sedative, hypnotic, or anxiolytic withdrawal is unclear.

1	The prevalence of sedative, hypnotic, or anxiolytic withdrawal is unclear. Seizures and autonomic instability in the setting of a history of prolonged exposure to sed- ative, hypnotic, or anxiolytic medications suggest a high likelihood of sedative, hypnotic, or anxiolytic withdrawal. Other medical disorders. The symptoms of sedative, hypnotic, or anxiolytic with- drawal may be mimicked by other medical conditions (e.g., hypoglycemia, diabetic keto- acidosis). If seizures are a feature of the sedative, hypnotic, or anxiolytic withdrawal, the differential diagnosis includes the various causes of seizures (e.g., infections, head injury, poisonings). Essential tremor. Essential tremor, a disorder that frequently runs in families, may erroneously suggest the tremulousness associated with sedative, hypnotic, or anxiolytic withdrawal. Alcohol withdrawal. Alcohol withdrawal produces a syndrome very similar to that of sedative, hypnotic, or anxiolytic withdrawal.

1	Alcohol withdrawal. Alcohol withdrawal produces a syndrome very similar to that of sedative, hypnotic, or anxiolytic withdrawal. Other sedative-, hypnotic-, or anxiolytic-induced disorders. Sedative, hypnotic, or anx- iolytic withdrawal is distinguished from the other sedative-, hypnotic-, or anxiolytic- induced disorders (e.g., sedative—, hypnotic-, or anxiolytic-induced anxiety disorder, with onset during withdrawal) because the symptoms in the latter disorders predominate in the clinical presentation and are severe enough to warrant clinical attention.

1	Anxiety disorders. Recurrence or worsening of an underlying anxiety disorder pro- duces a syndrome similar to sedative, hypnotic, or anxiolytic withdrawal. Withdrawal would be suspected with an abrupt reduction in the dosage of a sedative, hypnotic, or anx- iolytic medication. When a taper is under way, distinguishing the withdrawal syndrome from the underlying anxiety disorder can be difficult. As with alcohol, lingering with— drawal symptoms (e.g., anxiety, moodiness, and trouble sleeping) can be mistaken for non-substance/medication-induced anxiety or depressive disorders (e.g., generalized anxiety disorder). Other Sedative-, Hypnotic-, The following sedative-, hypnotic-, or anxiolytic-induced disorders are described in other chapters of the manual with disorders with which they share phenomenology (see the sub- stance/medication-induced mental disorders in these chapters): sedative-, hypnotic, or

1	Disorders”); sedative-, hypnotic-, or anxiolytic-induced bipolar disorder (”Bipolar and Re- lated Disorders”); sedative-, hypnotic-, or anxiolytic-induced depressive disorder (”De- pressive Disorders”); sedative-, hypnotic-, or anxiolytic-induced anxiety disorder (”Anxiety Disorders”); sedative-, hypnotic-, or anxiolytic-induced sleep disorder (”Sleep-

1	Wake Disorders"); sedative-, hypnotic-, or anxiolytic-induced sexual dysfunction (”Sex- ual Dysfunctions”); and sedative-, hypnotic-, or anxiolytic—induced major or mild neuro- cognitive disorder ("Neurocognitive Disorders"). For sedative, hypnotic, or anxiolytic intoxication delirium and sedative, hypnotic, or anxiolytic withdrawal delirium, see the criteria and discussion of delirium in the chapter ”Neurocognitive Disorders.” These sed- ative-, hypnotic-, or anxiolytic-induced disorders are diagnosed instead of sedative, hyp- notic, or anxiolytic intoxication or sedative, hypnotic, or anxiolytic withdrawal only when the symptoms are sufficiently severe to warrant independent clinical attention. Unspecified Sedative-, Hypnotic-, 292.9 (F13.99)

1	Unspecified Sedative-, Hypnotic-, 292.9 (F13.99) This category applies to presentations in which symptoms characteristic of a sedative-, hypnotic-, or anxiolytic-related disorder that cause clinically significant distress or impair- ment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any specific sedative-. hypnotic-, or anxiolytic-related disorder or any of the disorders in the substance-related and addictive disorders diagnostic class. A. A pattern of amphetamine-type substance, cocaine, or other stimulant use leading to clinically significant impairment or distress, as manifested by at least two of the follow- ing, occurring within a 12-month period: 1. 2. 3. 10. 11. The stimulant is often taken in larger amounts or over a longer period than was in- tended. There is a persistent desire or unsuccessful efforts to cut down or control stimulant use.

1	3. 10. 11. The stimulant is often taken in larger amounts or over a longer period than was in- tended. There is a persistent desire or unsuccessful efforts to cut down or control stimulant use. A great deal of time is spent in activities necessary to obtain the stimulant, use the stimulant, or recover from its effects. Craving, or a strong desire or urge to use the stimulant. Recurrent stimulant use resulting in a failure to fulfill major role obligations at work, school, or home. sonal problems caused or exacerbated by the effects of the stimulant. Important social, occupational. or recreational activities are given up or reduced be- cause of stimulant use. Recurrent stimulant use in situations in which it is physically hazardous. Stimulant use is continued despite knowledge of having a persistent or recurrent physical or psychological problem that is likely to have been caused or exacerbated by the stimulant.

1	Stimulant use is continued despite knowledge of having a persistent or recurrent physical or psychological problem that is likely to have been caused or exacerbated by the stimulant. Tolerance. as defined by either of the following: a. A need for markedly increased amounts of the stimulant to achieve intoxication or desired effect. b. A markedly diminished effect with continued use of the same amount of the stimulant. Note: This criterion is not considered to be met for those taking stimulant medica- tions solely under appropriate medical supervision, such as medications for atten- tion-deficit/hyperactivity disorder or narcolepsy. Withdrawal, as manifested by either of the following: a. The characteristic withdrawal syndrome tor the stimulant (refer to Criteria A and B of the criteria set for stimulant withdrawal, p. 569). b. The stimulant (or a closely related substance) is taken to relieve or avoid with- drawal symptoms.

1	B of the criteria set for stimulant withdrawal, p. 569). b. The stimulant (or a closely related substance) is taken to relieve or avoid with- drawal symptoms. Note: This criterion is not considered to be met for those taking stimulant medica- tions solely under appropriate medical supervision, such as medications for atten- tion-deficit/hyperactivity disorder or narcolepsy. Specify if: In early remission: After full criteria for stimulant use disorder were previously met, none of the criteria for stimulant use disorder have been met for at least 3 months but for less than 12 months (with the exception that Criterion A4, “Craving, or a strong de- sire or urge to use the stimulant," may be met).

1	In sustained remission: After full criteria for stimulant use disorder were previously met, none of the criteria for stimulant use disorder have been met at any time during a period of 12 months or longer (with the exception that Criterion A4, “Craving, or a strong desire or urge to use the stimulant," may be met). Specify it: In a controlled environment: This additional specifier is used it the individual is in an environment where access to stimulants is restricted.

1	Specify it: In a controlled environment: This additional specifier is used it the individual is in an environment where access to stimulants is restricted. Coding based on current severity: Note for |CD-10-CM codes: If an amphetamine in- toxication, amphetamine withdrawal, or another amphetamine-induced mental disorder is also present, do not use the codes below for amphetamine use disorder. Instead, the co- morbid amphetamine use disorder is indicated in the 4th character of the amphetamine- induced disorder code (see the coding note for amphetamine intoxication, amphetamine withdrawal, or a specific amphetamine-induced mental disorder). For example, if there is amine-type or other stimulant use disorder, only the amphetamine-type or other stimulant- induced depressive disorder code is given, with the 4th character indicating whether the comorbid amphetamine-type or other stimulant use disorder is mild, moderate, or severe:

1	F15.14 for mild amphetamine-type or other stimulant use disorder with amphetamine-type or other stimulant-induced depressive disorder or F15.24 for a moderate or severe am- phetamine-type or other stimulant use disorder with amphetamine-type or other stimulant- induced depressive disorder. Similarly. if there is comorbid cocaine-induced depressive disorder and cocaine use disorder, only the cocaine-induced depressive disorder code is given, with the 4th character indicating whether the comorbid cocaine use disorder is mild, moderate, or severe: F14.14 for mild cocaine use disorder with cocaine-induced depressive disorder or F14.24 for a moderate or severe cocaine use disorder with cocaine-induced depressive disorder. Specify current severity: Mild: Presence of 2—3 symptoms. 305.70 (F15.10) Amphetamine-type substance 305.60 (F14.10) Cocaine 305.70 (F15.10) Other or unspecified stimulant Moderate: Presence of 4—5 symptoms.

1	Mild: Presence of 2—3 symptoms. 305.70 (F15.10) Amphetamine-type substance 305.60 (F14.10) Cocaine 305.70 (F15.10) Other or unspecified stimulant Moderate: Presence of 4—5 symptoms. 304.40 (F15.20) Amphetamine-type substance 304.20 (F14.20) Cocaine 304.40 (F15.20) Other or unspecified stimulant Severe: Presence of 6 or more symptoms. 304.40 (F15.20) Amphetamine-type substance 304.20 (F14.20) Cocaine 304.40 (F15.20) Other or unspecified stimulant ”In a controlled environment” applies as a further specifier of remission if the individual is both in remission and in a controlled environment (i.e., in early remission in a controlled environment or in sustained remission in a controlled environment). Examples of these environments are closely supervised and substance-free jails, therapeutic communities, and locked hospital units.

1	The amphetamine and amphetamine—type stimulants include substances with a substi- tuted-phenylethylamine structure, such as amphetamine, dextroamphetamine, and meth- amphetamine. Also included are those substances that are structurally different but have similar effects, such as methylphenidate. These substances are usually taken orally or in- travenously, although methamphetamine is also taken by the nasal route. In addition to the synthetic amphetamine—type compounds, there are naturally occurring, plant-derived stimulants such as khfit. Amphetamines and other stimulants may be obtained by prescrip- tion for the treatment of obesity, attention-deficit/hyperactivity disorder, and narcolepsy.

1	Consequently, prescribed stimulants may be diverted into the illegal market. The effects of amphetamines and amphetamine-like drugs are similar to those of cocaine, such that the criteria for stimulant use disorder are presented here as a single disorder with the ability to specify the particular stimulant used by the individual. Cocaine may be consumed in sev- eral preparations (e.g., coca leaves, coca paste, cocaine hydrochloride, and cocaine alka- loids such as freebase and crack) that differ in potency because of varying levels of purity and speed of onset. However, in all forms of the substance, cocaine is the active ingredient. Cocaine hydrochloride powder is usually ”snorted” through the nostrils or dissolved in water and injected intravenously.

1	Cocaine hydrochloride powder is usually ”snorted” through the nostrils or dissolved in water and injected intravenously. Individuals exposed to amphetamine-type stimulants or cocaine can develop stimu- lant use disorder as rapidly as 1 week, although the onset is not always this rapid. Re- gardless of the route of administration, tolerance occurs with repeated use. Withdrawal symptoms, particularly hypersomnia, increased appetite, and dysphoria, can occur and can enhance craving. Most individuals with stimulant use disorder have experienced tol- erance or withdrawal.

1	Use patterns and course are similar for disorders involving amphetamine-type stimu- lants and cocaine, as both substances are potent central nervous system stimulants with similar psychoactive and sympathomimetic effects. Amphetamine-type stimulants are longer acting than cocaine and thus are used fewer times per day. Usage may be chronic or episodic, with binges punctuated by brief non-use periods. Aggressive or violent behavior is common when high doses are smoked, ingested, or administered intravenously. Intense temporary anxiety resembling panic disorder or generalized anxiety disorder, as well as paranoid ideation and psychotic episodes that resemble schizophrenia, is seen with high— dose use.

1	Withdrawal states are associated with temporary but intense depressive symptoms that can resemble a major depressive episode; the depressive symptoms usually resolve within 1 week. Tolerance to amphetamine-type stimulants develops and leads to escalation of the dose. Conversely, some users of amphetamine-type stimulants develop sensitization, characterized by enhanced effects. When injected or smoked, stimulants typically produce an instant feeling of well—being, confidence, and euphoria. Dramatic behavioral changes can rapidly develop with stimu— lant use disorder. Chaotic behavior, social isolation, aggressive behavior, and sexual dys- function can result from long-term stimulant use disorder.

1	Individuals with acute intoxication may present with rambling speech, headache, tran- sient ideas of reference, and tinnitus. There may be paranoid ideation, auditory halluci- nations in a clear sensorium, and tactile hallucinations, which the individual usually recognizes as drug effects. Threats or acting out of aggressive behavior may occur. Depres— sion, suicidal ideation, irritability, anhedonia, emotional lability, or disturbances in atten- tion and concentration commonly occur during withdrawal. Mental disturbances associated with cocaine use usually resolve hours to days after cessation of use but can persist for 1 month. Physiological changes during stimulant withdrawal are opposite to those of the intoxication phase, sometimes including bradycardia. Temporary depressive symptoms may meet symptomatic and duration criteria for major depressive episode. Histories con- sistent with repeated panic attacks, social anxiety disorder (social phobia)—like behavior, and generalized

1	may meet symptomatic and duration criteria for major depressive episode. Histories con- sistent with repeated panic attacks, social anxiety disorder (social phobia)—like behavior, and generalized anxiety—like syndromes are common, as are eating disorders. One ex- treme instance of stimulant toxicity is stimulant-induced psychotic disorder, a disorder that resembles schizophrenia, with delusions and hallucinations.

1	Individuals with stimulant use disorder often develop conditioned responses to drug- related stimuli (e.g., craving on seeing any white powderlike substance). These responses contribute to relapse, are difficult to extinguish, and persist after detoxification. Depressive symptoms with suicidal ideation or behavior can occur and are generally the most serious problems seen during stimulant withdrawal.

1	Stimulant use disorder: amphetamine—type stimulants. Estimated 12-month prevalence of amphetamine—type stimulant use disorder in the United States is 0.2% among 12- to 17- year-olds and 0.2% among individuals 18 years and older. Rates are similar among adult males and females (0.2%), but among 12- to 17-year-olds, the rate for females (0.3%) is greater than that for males (0.1%). Intravenous stimulant use has a male-to-female ratio of 3:1 or 4:1, but rates are more balanced among non-injecting users, with males representing 54% of primary treatment admissions. Twelve-month prevalence is greater among 18- to 29-year-olds (0.4%) compared with 45— to 64-year-olds (0.1%). For 12- to 17-year-olds, rates are highest among whites and African Americans (0.3%) compared with Hispanics (0.1%) and Asian Americans and Pacific Islanders (0.01%), with amphetamine-type stimulant use disorder virtually absent among Native Americans. Among adults, rates are highest among

1	Native Americans and Alaska Natives (0.6%) compared with whites (0.2%) and Hispanics (0.2%), with amphetamine-type stimulant use disorder virtually absent among African Americans and Asian Americans and Pacific Islanders. Past-year nonprescribed use of prescription stimulants occurred among 5%—9% of children through high school, with 5%—35% of college-age persons reporting past-year use.

1	Stimulant use disorder: cocaine. Estimated 12-month prevalence of cocaine use disorder in the United States is 0.2% among 12- to 17—year-olds and 0.3% among individuals 18 years and older. Rates are higher among males (0.4%) than among females (0.1%). Rates are highest among 18- to 29-year-olds (0.6%) and lowest among 45- to 64-year-olds (0.1%). Among adults, rates are greater among Native Americans (0.8%) compared with African Ameri- cans (0.4%), Hispanics (0.3%), whites (0.2%), and Asian Americans and Pacific Islanders (0.1%). In contrast, for 12- to 17-year-olds, rates are similar among Hispanics (0.2%), whites (0.2%), and Asian Americans and Pacific Islanders (0.2%); and lower among African Amer- icans (0.02%); with cocaine use disorder virtually absent among Native Americans and Alaska Natives.

1	Alaska Natives. Stimulant use disorders occur throughout all levels of society and are more common among individuals ages 12—25 years compared with individuals 26 years and older. First regular use among individuals in treatment occurs, on average, at approximately age 23 years. For pri- mary methamphetamine—primary treatment admissions, the average age is 31 years. Some individuals begin stimulant use to control weight or to improve performance in school, work, or athletics. This includes obtaining medications such as methylphenidate or amphetamine salts prescribed to others for the treatment of attention-deficit/hyperac- tivity disorder. Stimulant use disorder can develop rapidly with intravenous or smoked administration; among primary admissions for amphetamine-type stimulant use, 66% re- ported smoking, 18% reported injecting, and 10% reported snorting. Patterns of stimulant administration include episodic or daily (or almost daily) use.

1	Patterns of stimulant administration include episodic or daily (or almost daily) use. Episodic use tends to be separated by 2 or more days of non—use (e.g., intense use over a weekend or on one or more weekdays). "Binges" involve continuous high-dose use over hours or days and are often associated with physical dependence. Binges usually termi- nate only when stimulant supplies are depleted or exhaustion ensues. Chronic daily use may involve high or low doses, often with an increase in dose over time. Stimulant smoking and intravenous use are associated with rapid progression to se- vere-level stimulant use disorder, often occurring over weeks to months. Intranasal use of cocaine and oral use of amphetamine-type stimulants result in more gradual progression occurring over months to years. With continuing use, there is a diminution of pleasurable effects due to tolerance and an increase in dysphoric effects.

1	Temperamental. Comorbid bipolar disorder, schizophrenia, antisocial personality disor- der, and other substance use disorders are risk factors for developing stimulant use disorder and for relapse to cocaine use in treatment samples. Also, impulsivity and similar personality traits may affect treatment outcomes. Childhood conduct disorder and adult antisocial per- sonality disorder are associated with the later development of stimulant—related disorders. Environmental. Predictors of cocaine use among teenagers include prenatal cocaine ex- posure, postnatal cocaine use by parents, and exposure to community Violence during childhood. For youths, especially females, risk factors include living in an unstable home environment, having a psychiatric condition, and associating with dealers and users.

1	Stimulant use—attendant disorders affect all racial / ethnic, socioeconomic, age, and gender groups. Diagnostic issues may be related to societal consequences (e.g., arrest, school sus- pensions, employment suspension). Despite small variations, cocaine and other stimulant use disorder diagnostic criteria perform equally across gender and race/ethnicity groups. Chronic use of cocaine impairs cardiac left ventricular function in African Americans. Approximately 66% of individuals admitted for primary methamphetamine/amphet- amine-related disorders are non-Hispanic white, followed by 21% of Hispanic origin, 3% Asian and Pacific Islander, and 3% non-Hispanic black. Benzoylecgonine, a metabolite of cocaine, typically remains in the urine for 1—3 days after a single dose and may be present for 7—12 days in individuals using repeated high doses.

1	Benzoylecgonine, a metabolite of cocaine, typically remains in the urine for 1—3 days after a single dose and may be present for 7—12 days in individuals using repeated high doses. Mildly elevated liver function tests can be present in cocaine injectors or users with con- comitant alcohol use. There are no neurobiological markers of diagnostic utility. Discon- tinuation of chronic cocaine use may be associated with electroencephalographic changes, suggesting persistent abnormalities; alterations in secretion patterns of prolactin; and downregulation of dopamine receptors.

1	Short-half—life amphetamine-type stimulants (MDMA [3,4-methylenedioxy-N-methyl- amphetamine], methamphetamine) can be detected for 1—3 days, and possibly up to 4 days depending on dosage and metabolism. Hair samples can be used to detect presence of am- phetamine~type stimulants for up to 90 days. Other laboratory findings, as well as physical findings and other medical conditions (e.g., weight loss, malnutrition; poor hygiene), are similar for both cocaine and amphetamine-type stimulant use disorder. Functional Consequences of Stimulant Use Disorder

1	Functional Consequences of Stimulant Use Disorder Various medical conditions may occur depending on the route of administration. Intrana- sal users often develop sinusitis, irritation, bleeding of the nasal mucosa, and a perforated nasal septum. Individuals who smoke the drugs are at increased risk for respiratory prob- lems (e.g., coughing, bronchitis, and pneumonitis). Injectors have puncture marks and ”tracks," most commonly on their forearms. Risk of HIV infection increases with frequent intravenous injections and unsafe sexual activity. Other sexually transmitted diseases, hepatitis, and tuberculosis and other lung infections are also seen. Weight loss and mal- nutrition are common.

1	Chest pain may be a common symptom during stimulant intoxication. Myocardial in- farction, palpitations and arrhythmias, sudden death from respiratory or cardiac arrest, and stroke have been associated with stimulant use among young and otherwise healthy individuals. Seizures can occur with stimulant use. Pneumothorax can result from per- forming Valsalva-like maneuvers done to better absorb inhaled smoke. Traumatic injuries due to violent behavior are common among individuals trafficking drugs. Cocaine use is associated with irregularities in placental blood ﬂow, abruptio placentae, premature labor and delivery, and an increased prevalence of infants with very low birth weights. Individuals with stimulant use disorder may become involved in theft, prostitution, or drug dealing in order to acquire drugs or money for drugs.

1	Individuals with stimulant use disorder may become involved in theft, prostitution, or drug dealing in order to acquire drugs or money for drugs. Neurocognitive impairment is common among methamphetamine users. Oral health problems include "meth mouth" with gum disease, tooth decay, and mouth sores related to the toxic effects of smoking the drug and to bruxism while intoxicated. Adverse pulmo- nary effects appear to be less common for amphetamine-type stimulants because they are smoked fewer times per day. Emergency department visits are common for stimulant-re— lated mental disorder symptoms, injury, skin infections, and dental pathology.

1	Primary mental disorders. Stimulant-induced disorders may resemble primary mental disorders (e.g., major depressive disorder) (for discussion of this differential diagnosis, see ”Stimulant Withdrawal"). The mental disturbances resulting from the effects of stimulants should be distinguished from the symptoms of schizophrenia; depressive and bipolar dis- orders; generalized anxiety disorder; and panic disorder. Phencyclidine intoxication. Intoxication with phencyclidine ("PCP” or ”angel dust") or synthetic ”designer drugs” such as mephedrone (known by different names, including ulant intoxication by the presence of cocaine or amphetamine—type substance metabolites in a urine or plasma sample.

1	Stimulant intoxication and withdrawal. Stimulant intoxication and withdrawal are dis- tinguished from the other stimulant-induced disorders (e.g., anxiety disorder, with onset during intoxication) because the symptoms in the latter disorders predominate the clinical presentation and are severe enough to warrant independent clinical attention.

1	Stimulant-related disorders often co-occur with other substance use disorders, especially those involving substances with sedative properties, which are often taken to reduce in- somnia, nervousness, and other unpleasant side effects. Cocaine users often use alcohol, while amphetamine-type stimulant users often use cannabis. Stimulant use disorder may be associated with posttraumatic stress disorder, antisocial personality disorder, atten- tion-deficit/hyperactivity disorder, and gambling disorder. Cardiopulmonary problems are often present in individuals seeking treatment for cocaine-related problems, with chest pain being the most common. Medical problems occur in response to adulterants used as ”cutting” agents. Cocaine users who ingest cocaine cut with levamisole, an antimicrobial and veterinary medication, may experience agranulocytosis and febrile neutropenia. A. Recent use of an amphetamine-type substance. cocaine, or other stimulant.

1	A. Recent use of an amphetamine-type substance. cocaine, or other stimulant. B. Clinically significant problematic behavioral or psychological changes (e.g., euphoria anxiety, tension, or anger; stereotyped behaviors; impaired judgment) that developed during, or shortly after, use of a stimulant. C. Two (or more) of the following signs or symptoms, developing during. or shortly after, stimulant use: Tachycardia or bradycardia. Pupillary dilation. Elevated or lowered blood pressure. Perspiration or chills. Nausea or vomiting. Evidence of weight loss. Psychomotor agitation or retardation. Muscular weakness, respiratory depression, chest pain, or cardiac arrhythmias. Confusion. seizures, dyskinesias, dystonias, or come. D. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication with another sub- stance.

1	D. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication with another sub- stance. Specify the specific intoxicant (i.e., amphetamine-type substance, cocaine, or other stimulant). Specify it: With perceptual disturbances: This specifier may be noted when hallucinations with intact reality testing or auditory, visual, or tactile illusions occur in the absence of a de- lirium. Coding note: The |CD-9-CM code is 292.89. The |CD-10-CM code depends on whether the stimulant is an amphetamine, cocaine, or other stimulant; whether there is a comorbid amphetamine, cocaine, or other stimulant use disorder; and whether or not there are per- ceptual disturbances.

1	For amphetamine, cocaine, or other stimulant intoxication, without perceptual dis- turbances: If a mild amphetamine or other stimulant use disorder is comorbid, the ICD- 10-CM code is F15.129, and it a moderate or severe amphetamine or other stimulant use disorder is comorbid, the |CD-10-CM code is F15.229. If there is no comorbid amphet- amine or other stimulant use disorder, then the |CD-10—CM code is F15.929. Similarly, it a mild cocaine use disorder is comorbid. the lCD-10-CM code is F14.129, and it a mod- erate or severe cocaine use disorder is comorbid, the |CD-10-CM code is F14.229. If there is no comorbid cocaine use disorder, then the lCD-10-CM code is F14.929. For amphetamine, cocaine, or other stimulant intoxication, with perceptual distur- bances: If a mild amphetamine or other stimulant use disorder is comorbid, the |CD-10-

1	For amphetamine, cocaine, or other stimulant intoxication, with perceptual distur- bances: If a mild amphetamine or other stimulant use disorder is comorbid, the |CD-10- CM code is F15.122, and if a moderate or severe amphetamine or other stimulant use disorder is comorbid, the |CD-10-CM code is F15.222. If there is no comorbid amphet- amine or other stimulant use disorder, then the lCD-10—CM code is F15.922. Similarly, if a mild cocaine use disorder is comorbid, the |CD-10-CM code is F14.122, and it a mod- erate or severe cocaine use disorder is comorbid, the lCD-10-CM code is F14.222. If there is no comorbid cocaine use disorder, then the |CD-10-CM code is F14.922.

1	The essential feature of stimulant intoxication, related to amphetamine-type stimulants and cocaine, is the presence of clinically significant behavioral or psychological changes that develop during, or shortly after, use of stimulants (Criteria A and B). Auditory hallu— cinations may be prominent, as may paranoid ideation, and these symptoms must be dis- tinguished from an independent psychotic disorder such as schizophrenia. Stimulant intoxication usually begins with a ”high" feeling and includes one or more of the follow- ing: euphoria with enhanced vigor, gregariousness, hyperactivity, restlessness, hypervig- ilance, interpersonal sensitivity, talkativeness, anxiety, tension, alertness, grandiosity, stereotyped and repetitive behavior, anger, impaired judgment, and, in the case of chronic intoxication, affective blunting with fatigue or sadness and social withdrawal. These be- havioral and psychological changes are accompanied by two or more of the following signs and symptoms

1	chronic intoxication, affective blunting with fatigue or sadness and social withdrawal. These be- havioral and psychological changes are accompanied by two or more of the following signs and symptoms that develop during or shortly after stimulant use: tachycardia or bra- nausea or vomiting; evidence of weight loss; psychomotor agitation or retardation; mus- cular weakness, respiratory depression, chest pain, or cardiac arrhythmias; and confu- sion, seizures, dyskinesias, dystonias, or coma (Criterion C). Intoxication, either acute or chronic, is often associated with impaired social or occupational functioning. Severe in- toxication can lead to convulsions, cardiac arrhythmias, hyperpyrexia, and death. For the diagnosis of stimulant intoxication to be made, the symptoms must not be attributable rion D). While stimulant intoxication occurs in individuals with stimulant use disorders, in- toxication is not a criterion for stimulant use disorder, which is confirmed by the presence of two

1	rion D). While stimulant intoxication occurs in individuals with stimulant use disorders, in- toxication is not a criterion for stimulant use disorder, which is confirmed by the presence of two of the 11 diagnostic criteria for use disorder.

1	The magnitude and direction of the behavioral and physiological changes depend on many variables, including the dose used and the characteristics of the individual using the sub- stance or the context (e.g., tolerance, rate of absorption, chronicity of use, context in which it is taken). Stimulant effects such as euphoria, increased pulse and blood pressure, and psychomotor activity are most commonly seen. Depressant effects such as sadness, brady- cardia, decreased blood pressure, and decreased psychomotor activity are less common and generally emerge only with chronic high-dose use.

1	Stimulant—induced disorders. Stimulant intoxication is distinguished from the other stimulant-induced disorders (e.g., stimulant-induced depressive disorder, bipolar disor- der, psychotic disorder, anxiety disorder) because the severity of the intoxication symp- toms exceeds that associated with the stimulant-induced disorders, and the symptoms warrant independent clinical attention. Stimulant intoxication delirium would be distin- guished by a disturbance in level of awareness and change in cognition. Other mental disorders. Salient mental disturbances associated with stimulant intoxi- cation should be distinguished from the symptoms of schizophrenia, paranoid type; bi- described 1n DSM- 5. A. Cessation of (or reduction in) prolonged amphetamine-type substance, cocaine, or other stimulant use. B. Dysphoric mood and two (or more) of the following physiological changes, developing within a few hours to several days after Criterion A: Fatigue. Vivid, unpleasant dreams.

1	B. Dysphoric mood and two (or more) of the following physiological changes, developing within a few hours to several days after Criterion A: Fatigue. Vivid, unpleasant dreams. Insomnia or hypersomnia. Increased appetite. Psychomotor retardation or agitation. WPWN.‘ C. The signs or symptoms in Criterion B cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. D. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication or withdrawal from another substance. Specify the specific substance that causes the withdrawal syndrome (i.e., amphet- amine-type substance, cocaine, or other stimulant).

1	Specify the specific substance that causes the withdrawal syndrome (i.e., amphet- amine-type substance, cocaine, or other stimulant). Coding note: The |CD-9-CM code is 292.0. The |CD-10-CM code depends on whether the stimulant is an amphetamine, cocaine, or other stimulant. The |CD-10-CM code for amphetamine or an other stimulant withdrawal is F15.23, and the |CD-10-CM for cocaine withdrawal is F14.23. Note that the |CD-10-CM code indicates the comorbid presence of a moderate or severe amphetamine, cocaine, or other stimulant use disorder, reflecting the fact that amphetamine, cocaine, or other stimulant withdrawal can only occur in the presence of a moderate or severe amphetamine, cocaine, or other stimulant use disorder. It is not permissible to code a comorbid mild amphetamine, cocaine, or other stimulant use disorder with amphetamine, cocaine, or other stimulant withdrawal.

1	It is not permissible to code a comorbid mild amphetamine, cocaine, or other stimulant use disorder with amphetamine, cocaine, or other stimulant withdrawal. The essential feature of stimulant withdrawal is the presence of a characteristic With- drawal syndrome that develops within a few hours to several days after the cessation of terion A). The withdrawal syndrome is characterized by the development of dysphoric mood accompanied by two or more of the following physiological changes: fatigue, vivid and unpleasant dreams, insomnia or hypersomnia, increased appetite, and psychomotor retardation or agitation (Criterion B). Bradycardia is often present and is a reliable mea- sure of stimulant withdrawal.

1	Anhedonia and drug craving can often be present but are not part of the diagnostic cri- teria. These symptoms cause clinically significant distress or impairment in social, occu- pational, or other important areas of functioning (Criterion C). The symptoms must not be attributable to another medical condition and are not better explained by another mental disorder (Criterion D).

1	Acute withdrawal symptoms (”a crash”) are often seen after periods of repetitive high-dose use (”runs” or ”binges”). These periods are characterized by intense and unpleasant feelings of lassitude and depression and increased appetite, generally requiring several days of rest and recuperation. Depressive symptoms with suicidal ideation or behavior can occur and are gen- erally the most serious problems seen during ”crashing” or other forms of stimulant with- drawal. The majority of individuals with stimulant use disorder experience a withdrawal syndrome at some point, and virtually all individuals with the disorder report tolerance.

1	Stimulant use disorder and other stimulant-induced disorders. Stimulant withdrawal is distinguished from stimulant use disorder and from the other stimulant-induced disor- ders (e.g., stimulant-induced intoxication delirium, depressive disorder, bipolar disorder, psychotic disorder, anxiety disorder, sexual dysfunction, sleep disorder) because the symptoms of withdrawal predominate the clinical presentation and are severe enough to warrant independent clinical attention.

1	The following stimulant-induced disorders (which include amphetamine-, cocaine-, and other stimulant—induced disorders) are described in other chapters of the manual with dis— orders with which they share phenomenology (see the substance/medication-induced mental disorders in these chapters): stimulant—induced psychotic disorder (”Schizophrenia ual Dysfunctions”). For stimulant intoxication delirium, see the criteria and discussion of delirium in the chapter "Neurocognitive Disorders.” These stimulant-induced disorders are diagnosed instead of stimulant intoxication or stimulant withdrawal only when the symptoms are sufficiently severe to warrant independent clinical attention.

1	This category applies to presentations in which symptoms characteristic of a stimulant- related disorder that cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning predominate but do not meet the full criteria for any specific stimulant-related disorder or any of the disorders in the substance-related and addictive disorders diagnostic class. Coding note: The |CD-9-CM code is 292.9. The |CD-10-CM code depends on whether the stimulant is an amphetamine, cocaine, or another stimulant. The |CD-10-CM code for an unspecified amphetamine- or other stimulant-related disorder is F15.99. The |CD-10- CM code for an unspecified cocaine-related disorder is F14.99. A. A problematic pattern of tobacco use leading to clinically significant impairment or dis- tress, as manifested by at least two of the following, occurring within a 12-month period: 10. 11.

1	A. A problematic pattern of tobacco use leading to clinically significant impairment or dis- tress, as manifested by at least two of the following, occurring within a 12-month period: 10. 11. Tobacco is often taken in larger amounts or over a longer period than was intended. There is a persistent desire or unsuccessful efforts to cut down or control tobacco use. A great deal of time is spent in activities necessary to obtain or use tobacco. Craving, or a strong desire or urge to use tobacco. Recurrent tobacco use resulting in a failure to fulfill major role obligations at work, school, or home (e.g., interference with work). sonal problems caused or exacerbated by the effects of tobacco (e.g., arguments with others about tobacco use). Important social, occupational, or recreational activities are given up or reduced be- cause of tobacco use. Recurrent tobacco use in situations in which it is physically hazardous (e.g., smok- ing in bed).

1	Recurrent tobacco use in situations in which it is physically hazardous (e.g., smok- ing in bed). Tobacco use is continued despite knowledge of having a persistent or recurrent physical or psychological problem that is likely to have been caused or exacerbated by tobacco. Tolerance, as defined by either of the following: a. A need for markedly increased amounts of tobacco to achieve the desired effect. b. A markedly diminished effect with continued use of the same amount of tobacco. Withdrawal, as manifested by either of the following: a. The characteristic withdrawal syndrome for tobacco (refer to Criteria A and B of the criteria set for tobacco withdrawal). b. Tobacco (or a closely related substance, such as nicotine) is taken to relieve or avoid withdrawal symptoms. Specify it:

1	b. Tobacco (or a closely related substance, such as nicotine) is taken to relieve or avoid withdrawal symptoms. Specify it: In early remission: After lull criteria for tobacco use disorder were previously met, none of the criteria for tobacco use disorder have been met for at least 3 months but for less than 12 months (with the exception that Criterion A4, "Craving, or a strong de- sire or urge to use tobacco,” may be met). In sustained remission: After full criteria for tobacco use disorder were previously met, none of the criteria for tobacco use disorder have been met at any time during a period of 12 months or longer (with the exception that Criterion A4, “Craving, or a strong desire or urge to use tobacco," may be met). Specify if:

1	Specify if: On maintenance therapy: The individual is taking a long-term maintenance medica- tion, such as nicotine replacement medication, and no criteria for tobacco use disorder have been met for that class of medication (except tolerance to, or withdrawal from, the nicotine replacement medication). In a controlled environment: This additional specifier is used if the individual is in an environment where access to tobacco is restricted.

1	In a controlled environment: This additional specifier is used if the individual is in an environment where access to tobacco is restricted. Coding based on current severity: Note for lCD-10-CM codes: If a tobacco withdrawal or tobacco-induced sleep disorder is also present, do not use the codes below for tobacco use disorder. Instead, the comorbid tobacco use disorder is indicated in the 4th character of the tobacco-induced disorder code (see the coding note for tobacco withdrawal or tobacco- induced sleep disorder). For example, if there is comorbid tobacco-induced sleep disorder and tobacco use disorder, only the tobacco—induced sleep disorder code is given, with the 4th char- acter indicating whether the comorbid tobacco use disorder is moderate or severe: F17.208 for moderate or severe tobacco use disorder with tobacco-induced sleep disorder. It is not per— missible to code a comorbid mild tobacco use disorder with a tobacco-induced sleep disorder.

1	Specify current severity: 305.1 (272.0) Mild: Presence of 2—3 symptoms. 305.1 (F17.200) Moderate: Presence of 4—5 symptoms. 305.1 (F17.200) Severe: Presence of 6 or more symptoms. "On maintenance therapy" applies as a further specifier to individuals being maintained on other tobacco cessation medication (e.g., bupropion, varenicline) and as a further specifier of remission if the individual is both in remission and on maintenance therapy. ”In a controlled environment” applies as a further specifier of remission if the individual is both in remission and in a controlled environment (i.e., in early remission in a controlled environment or in sus- tained remission in a controlled environment). Examples of these environments are closely su- pervised and substance-free jails, therapeutic communities, and locked hospital units.

1	Tobacco use disorder is common among individuals who use cigarettes and smokeless to- bacco daily and is uncommon among individuals who do not use tobacco daily or who use nicotine medications. Tolerance to tobacco is exemplified by the disappearance of nausea and dizziness after repeated intake and with a more intense effect of tobacco the first time it is used during the day. Cessation of tobacco use can produce a well-defined withdrawal syndrome. Many individuals with tobacco use disorder use tobacco to relieve or to avoid withdrawal symptoms (e.g., after being in a situation where use is restricted). Many indi- tinue to smoke. The large majority report craving when they do not smoke for several hours.

1	Spending excessive time using tobacco can be exemplified by chain-smoking (i.e., smok- ing one cigarette after another with no time between cigarettes). Because tobacco sources are readily and legally available, and because nicotine intoxication is very rare, spending a great deal of time attempting to procure tobacco or recovering from its effects is uncom- mon. Giving up important social, occupational, or recreational activities can occur when an individual forgoes an activity because it occurs in tobacco use—restricted areas. Use of tobacco rarely results in failure to fulfill major role obligations (e.g., interference with work, interference with home obligations), but persistent social or interpersonal problems (e.g., having arguments with others about tobacco use, avoiding social situations because of others’ disapproval of tobacco use) or use that is physically hazardous (e.g., smoking in bed, smoking around ﬂammable chemicals) occur at an intermediate prevalence. Although

1	situations because of others’ disapproval of tobacco use) or use that is physically hazardous (e.g., smoking in bed, smoking around ﬂammable chemicals) occur at an intermediate prevalence. Although these criteria are less often endorsed by tobacco users, if endorsed, they can indicate a more severe disorder.

1	Smoking within 30 minutes of waking, smoking daily, smoking more cigarettes per day, and waking at night to smoke are associated with tobacco use disorder. Environmental cues can evoke craving and withdrawal. Serious medical conditions, such as lung and other cancers, cardiac and pulmonary disease, perinatal problems, cough, shortness of breath, and accelerated skin aging, often occur. Cigarettes are the most commonly used tobacco product, representing over 90% of to- bacco/nicotine use. In the United States, 57% of adults have never been smokers, 22% are former smokers, and 21% are current smokers. Approximately 20% of current US. smok- ers are nondaily smokers. The prevalence of smokeless tobacco use is less than 5%, and the prevalence of tobacco use in pipes and cigars is less than 1%.

1	DSM-IV nicotine dependence criteria can be used to estimate the prevalence of tobacco use disorder, but since they are a subset of tobacco use disorder criteria, the prevalence of tobacco use disorder will be somewhat greater. The 12-month prevalence of DSM-IV nic- otine dependence in the United States is 13% among adults age 18 years and older. Rates among 18— to 29-year-olds to 4% among individuals age 65 years and older. The prevalence of current nicotine dependence is greater among Native American and Alaska Natives (23%) than among whites (14%) but is less among African Americans (10%), Asian Amer- icans and Pacific Islanders (6%), and Hispanics (6%). The prevalence among current daily smokers is approximately 50%. In many developing nations, the prevalence of smoking is much greater in males than in females, but this is not the case in developed nations. However, there often is a lag in the demographic transition such that smoking increases in females at a later time.

1	The majority of US. adolescents experiment with tobacco use, and by age 18 years, about 20% smoke at least monthly. Most of these individuals become daily tobacco users. Initi- ation of smoking after age 21 years is rare. In general, some of the tobacco use disorder cri- teria symptoms occur soon after beginning tobacco use, and many individuals’ pattern of use meets current tobacco use disorder criteria by late adolescence. More than 80% of in- dividuals who use tobacco attempt to quit at some time, but 60% relapse within 1 week and less than 5% remain abstinent for life. However, most individuals who use tobacco make multiple attempts such that one-half of tobacco users eventually abstain. Individuals who use tobacco who do quit usually do not do so until after age 30 years. Although non- daily smoking in the United States was previously rare, it has become more prevalent in the last decade, especially among younger individuals who use tobacco.

1	Temperamental. Individuals with externalizing personality traits are more likely to initiate tobacco use. Children with attention-deficit/hyperactivity disorder or conduct disorder, and adults with depressive, bipolar, anxiety, personality, psychotic, or other substance use disorders, are at higher risk of starting and continuing tobacco use and of to- bacco use disorder. Environmental. Individuals with low incomes and low educational levels are more likely to initiate tobacco use and are less likely to stop. Genetic and physiological. Genetic factors contribute to the onset of tobacco use, the continuation of tobacco use, and the development of tobacco use disorder, with a degree of heritability equivalent to that observed with other substance use disorders (i.e., about 50%). Some of this risk is specific to tobacco, and some is common with the vulnerability to developing any substance use disorder.

1	Cultures and subcultures vary widely in their acceptance of the use of tobacco. The prev- alence of tobacco use declined in the United States from the 19605 through the 19905, but this decrease has been less evident in African American and Hispanic populations. Also, smoking in developing countries is more prevalent than in developed nations. The degree to which these cultural differences are due to income, education, and tobacco control ac— tivities in a country is unclear. Non-Hispanic white smokers appear to be more likely to develop tobacco use disorder than are smokers. Some ethnic differences may be biologi- cally based. African American males tend to have higher nicotine blood levels for a given number of cigarettes, and this might contribute to greater difficulty in quitting. Also, the speed of nicotine metabolism is significantly different for whites compared with African Americans and can vary by genotypes associated with ethnicities.

1	Americans and can vary by genotypes associated with ethnicities. Carbon monoxide in the breath, and nicotine and its metabolite cotinine in blood, saliva, or urine, can be used to measure the extent of current tobacco or nicotine use; however, these are only weakly related to tobacco use disorder. Functional Consequences of Tobacco Use Disorder Medical consequences of tobacco use often begin when tobacco users are in their 405 and usually become progressively more debilitating over time. One-half of smokers who do not stop using tobacco will die early from a tobacco-related illness, and smoking-related morbidity occurs in more than one-half of tobacco users. Most medical conditions result from exposure to carbon monoxide, tars, and other non-nicotine components of tobacco. The major predictor of reversibility is duration of smoking. Secondhand smoke increases the risk of heart disease and cancer by 30%. Long-term use of nicotine medications does not appear to cause medical harm.

1	The most common medical diseases from smoking are cardiovascular illnesses, chronic obstructive pulmonary disease, and cancers. Smoking also increases perinatal problems, such as low birth weight and miscarriage. The most common psychiatric comorbidities are alcohol/substance, depressive, bipolar, anxiety, personality, and attention-deficit/hyper- activity disorders. In individuals with current tobacco use disorder, the prevalence of cur- rent alcohol, drug, anxiety, depressive, bipolar, and personality disorders ranges from 22% to 32%. Nicotine-dependent smokers are 2.7—8.1 times more likely to have these dis- orders than nondependent smokers, never-smokers, or ex-smokers. . Tobacco Withdrawal Diagnostic Criteria 292.0 (F17.203) A. Daily use of tobacco for at least several weeks. B. Abrupt cessation of tobacco use, or reduction in the amount of tobacco used, followed within 24 hours by four (or more) of the following signs or symptoms: Irritability, frustration, or anger.

1	B. Abrupt cessation of tobacco use, or reduction in the amount of tobacco used, followed within 24 hours by four (or more) of the following signs or symptoms: Irritability, frustration, or anger. Anxiety. Difficulty concentrating. Increased appetite. Restlessness. Depressed mood. Insomnia. C. The signs or symptoms in Criterion B cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. D. The signs or symptoms are not attributed to another medical condition and are not bet- ter explained by another mental disorder, including intoxication or withdrawal from an- other substance.

1	D. The signs or symptoms are not attributed to another medical condition and are not bet- ter explained by another mental disorder, including intoxication or withdrawal from an- other substance. Coding note: The ICD-9-CM code is 292.0. The lCD-10-CM code for tobacco withdrawal is F17.203. Note that the |CD-10-CM code indicates the comorbid presence of a moderate or severe tobacco use disorder, reflecting the fact that tobacco withdrawal can only occur in the presence of a moderate or severe tobacco use disorder. It is not permissible to code a comorbid mild tobacco use disorder with tobacco withdrawal.

1	Withdrawal symptoms impair the ability to stop tobacco use. The symptoms after absti- nence from tobacco are in large part due to nicotine deprivation. Symptoms are much among those who use nicotine medications. This difference in symptom intensity is likely due to the more rapid onset and higher levels of nicotine with cigarette smoking. Tobacco withdrawal is common among daily tobacco users who stop or reduce but can also occur among nondaily users. Typically, heart rate decreases by 5—12 beats per minute in the first few days after stopping smoking, and weight increases an average of 4—7 1b (2—3 kg) over the first year after stopping smoking. Tobacco withdrawal can produce clinically signifi- cant mood changes and functional impairment.

1	are associated with tobacco withdrawal. Abstinence can increase constipation, coughing, dizziness, dreaming/nightmares, nausea, and sore throat. Smoking increases the metab— olism of many medications used to treat mental disorders; thus, cessation of smoking can increase the blood levels of these medications, and this can produce clinically significant outcomes. This effect appears to be due not to nicotine but rather to other compounds in tobacco. Approximately 50% of tobacco users who quit for 2 or more days will have symptoms that meet criteria for tobacco withdrawal. The most commonly endorsed signs and symptoms are anxiety, irritability, and difficulty concentrating. The least commonly endorsed symp- toms are depression and insomnia.

1	Tobacco withdrawal usually begins within 24 hours of stopping or cutting down on to- bacco use, peaks at 2—3 days after abstinence, and lasts 2—3 weeks. Tobacco withdrawal symptoms can occur among adolescent tobacco users, even prior to daily tobacco use. Pro- longed symptoms beyond 1 month are uncommon. Temperamental. Smokers with depressive disorders, bipolar disorders, anxiety disor- ders, attention-deficit/hyperactivity disorder, and other substance use disorders have more severe withdrawal. Genetic and physiological. Genotype can inﬂuence the probability of withdrawal upon abstinence. Carbon monoxide in the breath, and nicotine and its metabolite cotinine in blood, saliva, or urine, can be used to measure the extent of tobacco or nicotine use but are only weakly re- lated to tobacco withdrawal. Functional Consequences of Tobacco Withdrawal

1	Functional Consequences of Tobacco Withdrawal Abstinence from cigarettes can cause clinically significant distress. Withdrawal impairs the ability to stop or control tobacco use. Whether tobacco withdrawal can prompt a new mental disorder or recurrence of a mental disorder is debatable, but if this occurs, it would be in a small minority of tobacco users.

1	The symptoms of tobacco withdrawal overlap with those of other substance withdrawal syndromes (e.g., alcohol withdrawal; sedative, hypnotic, or anxiolytic withdrawal; stim- ulant withdrawal; caffeine withdrawal; opioid withdrawal); caffeine intoxication; anxiety, depressive, bipolar, and sleep disorders; and medication-induced akathisia. Admission to toms that mimic, intensify, or disguise other disorders or adverse effects of medications used to treat mental disorders (e.g., irritability thought to be due to alcohol withdrawal could be due to tobacco withdrawal). Reduction in symptoms with the use of nicotine medications confirms the diagnosis. Tobacco-induced sleep disorder is discussed in the chapter ”Sleep-Wake Disorders” (see “Substance/ Medication-Induced Sleep Disorder”). 292.9 (F17.209)

1	Tobacco-induced sleep disorder is discussed in the chapter ”Sleep-Wake Disorders” (see “Substance/ Medication-Induced Sleep Disorder”). 292.9 (F17.209) This category applies to presentations in which symptoms characteristic of a tobacco- related disorder that cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning predominate but do not meet the full criteria for any specific tobacco-related disorder or any of the disorders in the substance-related and addictive disorders diagnostic class. A. A problematic pattern of use of an intoxicating substance not able to be classified within the alcohol; caffeine; cannabis; hallucinogen (phencyclidine and others); inhal- ant; opioid; sedative, hypnotic, or anxiolytic; stimulant; ortobacco categories and lead- ing to clinically significant impairment or distress, as manifested by at least two of the following, occurring within a 12-month period: 1. 2.

1	2. The substance is often taken in larger amounts or over a longer period than was intended. There is a persistent desire or unsuccessful efforts to cut down or control use of the substance. A great deal of time is spent in activities necessary to obtain the substance, use the substance, or recover from its effects. Craving, or a strong desire or urge to use the substance. Recurrent use of the substance resulting in a failure to fulfill major role obligations at work, school, or home. Continued use of the substance despite having persistent or recurrent social or in- terpersonal problems caused or exacerbated by the effects of its use. Important social, occupational, or recreational activities are given up or reduced be- cause of use of the substance. Recurrent use of the substance in situations in which it is physically hazardous.

1	Recurrent use of the substance in situations in which it is physically hazardous. Use of the substance is continued despite knowledge of having a persistent or re- current physical or psychological problem that is likely to have been caused or ex- acerbated by the substance. 10. Tolerance, as defined by either of the following: a. A need for markedly increased amounts of the substance to achieve intoxication or desired effect. b. A markedly diminished el'lect with continued use of the same amount of the sub- stance. 11. Withdrawal, as manifested by either of the following: a. The characteristic withdrawal syndrome for other (or unknown) substance (refer to Criteria A and B of the criteria sets for other [or unknown] substance withdrawal, p. 583). b. The substance (or a closely related substance) is taken to relieve or avoid with- drawal symptoms. Specify if:

1	b. The substance (or a closely related substance) is taken to relieve or avoid with- drawal symptoms. Specify if: In early remission: Afterfull criteria for other (or unknown) substance use disorder were previously met, none of the criteria for other (or unknown) substance use disorder have been met for at least 3 months but for less than 12 months (with the exception that Cri- terion A4, “Craving, or a strong desire or urge to use the substance," may be met). In sustained remission: After full criteria for other (or unknown) substance use disor- der were previously met, none of the criteria for other (or unknown) substance use dis- order have been met at any time during a period of 12 months or longer (with the exception that Criterion A4, “Craving, or a strong desire or urge to use the substance," may be met). Specify if: In a controlled environment: This additional specifier is used if the individual is in an environment where access to the substance is restricted.

1	Specify if: In a controlled environment: This additional specifier is used if the individual is in an environment where access to the substance is restricted. Coding based on current severity: Note for |CD-10-CM codes: If an other (or unknown) sub- stance intoxication, other (or unknown) substance withdrawal, or another other (or unknown) substance—induced mental disorder is present, do not use the codes below for other (or un- known) substance use disorder. Instead, the comorbid other (or unknown) substance use dis- order is indicated in the 4th character of the other (or unknown) substance—induced disorder code (see the coding note for other (or unknown) substance intoxication, other (or unknown) substance withdrawal, or specific other (or unknown) substance—induced mental disorder).

1	For example, it there is comorbid other (or unknown) substance—induced depressive disorder and other (or unknown) substance use disorder, only the other (or unknown) substance— induced depressive disorder code is given, with the 4th character indicating whether the co- morbid other (or unknown) substance use disorder is mild, moderate, or severe: F19.14 for other (or unknown) substance use disorder with other (or unknown) substance—induced de- order with other (or unknown) substance—induced depressive disorder. Specify current severity: 305.90 (F19.10) Mild: Presence of 2—3 symptoms. 304.90 (F19.20) Moderate: Presence of 4—5 symptoms. 304.90 (F19.20) Severe: Presence of 6 or more symptoms.

1	Specify current severity: 305.90 (F19.10) Mild: Presence of 2—3 symptoms. 304.90 (F19.20) Moderate: Presence of 4—5 symptoms. 304.90 (F19.20) Severe: Presence of 6 or more symptoms. “In a controlled environment” applies as a further specifier of remission if the individual is both in remission and in a controlled environment (i.e., in early remission in a controlled environment or in sustained remission in a controlled environment). Examples of these environments are closely supervised and substance-free jails, therapeutic communities, and locked hospital units.

1	The diagnostic class other (or unknown) substance use and related disorders comprises substance-related disorders unrelated to alcohol; caffeine; cannabis; hallucinogens (phen- cyclidine and others); inhalants; opioids; sedative, hypnotics, or anxiolytics; stimulants (including amphetamine and cocaine); or tobacco. Such substances include anabolic ste- tihistamines; nitrous oxide; amyl-, butyl-, or isobutyl-nitrites; betel nut, which is chewed in many cultures to produce mild euphoria and a ﬂoating sensation; kava (from a South

1	Pacific pepper plant), which produces sedation, incoordination, weight loss, mild hepati- tis, and lung abnormalities; or cathinones (including khfit plant agents and synthetic chem- ical derivatives) that produce stimulant effects. Unknown substance-related disorders are associated with unidentified substances, such as intoxications in which the individual can- not identify the ingested drug, or substance use disorders involving either new, black mar- ket drugs not yet identified or familiar drugs illegally sold under false names. Other (or unknown) substance use disorder is a mental disorder in which repeated use of an other or unknown substance typically continues, despite the individual’s knowing that the substance is causing serious problems for the individual. Those problems are re- ﬂected in the diagnostic criteria. When the substance is known, it should be reﬂected in the name of the disorder upon coding (e.g., nitrous oxide use disorder).

1	A diagnosis of other (or unknown) substance use disorder is supported by the individual’s statement that the substance involved is not among the nine classes listed in this chapter; by re- curring episodes of intoxication with negative results in standard drug screens (which may not detect new or rarely used substances); or by the presence of symptoms characteristic of an un- identified substance that has newly appeared in the individual’s community.

1	Because of increased access to nitrous oxide (”laughing gas"), membership in certain populations is associated with diagnosis of nitrous oxide use disorder. The role of this gas as an anesthetic agent leads to misuse by some medical and dental professionals. Its use as a propellant for commercial products (e.g., whipped cream dispensers) contributes to misuse by food service workers. With recent widespread availability of the substance in ”Whippet” cartridges for use in home whipped cream dispensers, nitrous oxide misuse by adolescents and young adults is significant, especially among those who also inhale vola- tile hydrocarbons. Some continuously using individuals, inhaling from as many as 240 whippets per day, may present with serious medical complications and mental conditions, including myeloneuropathy, spinal cord subacute combined degeneration, peripheral neuropathy, and psychosis. These conditions are also associated with a diagnosis of ni- trous oxide use disorder.

1	Use of amyl-, butyl-, and isobutyl nitrite gases has been observed among homosexual men and some adolescents, especially those with conduct disorder. Membership in these populations may be associated with a diagnosis of amy1—, butyl-, or isobutyl—nitrite use dis- order. However, it has not been determined that these substances produce a substance use disorder. Despite tolerance, these gases may not alter behavior through central effects, and they may be used only for their peripheral effects. Substance use disorders generally are associated with elevated risks of suicide, but there is no evidence of unique risk factors for suicide with other (or unknown) substance use disorder. Based on extremely limited data, the prevalence of other (or unknown) substance use disorder is likely lower than that of use disorders involving the nine substance classes in this chapter.

1	Based on extremely limited data, the prevalence of other (or unknown) substance use disorder is likely lower than that of use disorders involving the nine substance classes in this chapter. No single pattern of development or course characterizes the pharmacologically varied other (or unknown) substance use disorders. Often unknown substance use disorders will be reclassified when the unknown substance eventually is identified. Risk and prognostic factors for other (or unknown) substance use disorders are thought to be similar to those for most substance use disorders and include the presence of any other substance use disorders, conduct disorder, or antisocial personality disorder in the indi- vidual or the individual’s family; early onset of substance problems; easy availability of the substance in the individual’s environment; childhood maltreatment or trauma; and ev- idence of limited early self—control and behavioral disinhibition.

1	Certain cultures may be associated with other (or unknown) substance use disorders in- volving specific indigenous substances within the cultural region, such as betel nut. Urine, breath, or saliva tests may correctly identify a commonly used substance falsely sold as a novel product. However, routine clinical tests usually cannot identify truly un- usual or new substances, which may require testing in specialized laboratories. Use of other or unknown substances without meeting criteria for other (or unknown) substance use disorder. Use of unknown substances is not rare among adolescents, but most use does not meet the diagnostic standard of two or more criteria for other (or un- known) substance use disorder in the past year.

1	Substance use disorders. Other (or unknown) substance use disorder may co—occur with various substance use disorders, and the symptoms of the disorders may be similar and overlapping. To disentangle symptom patterns, it is helpful to inquire about which symptoms persisted during periods when some of the substances were not being used. Other (or unknown) substance/medication-induced disorder. This diagnosis should be differentiated from instances when the individual’s symptoms meet full criteria for one of the following disorders, and that disorder is caused by an other or unknown substance: delirium, major or mild neurocognitive disorder, psychotic disorder, depressive disorder, anxiety disorder, sexual dysfunction, or sleep disorder.

1	Other medical conditions. Individuals with substance use disorders, including other (or unknown) substance use disorder, may present with symptoms of many medical dis- orders. These disorders also may occur in the absence of other (or unknown) substance use disorder. A history of little or no use of other or unknown substances helps to exclude other (or unknown) substance use disorder as the source of these problems. Substance use disorders, including other (or unknown) substance use disorder, are com- monly comorbid with one another, with adolescent conduct disorder and adult antisocial personality disorder, and with suicidal ideation and suicide attempts. A. The development of a reversible substance-specific syndrome attributable to recent in- gestion of (or exposure to) a substance that is not listed elsewhere or is unknown.

1	A. The development of a reversible substance-specific syndrome attributable to recent in- gestion of (or exposure to) a substance that is not listed elsewhere or is unknown. B. Clinically significant problematic behavioral or psychological changes that are attribut- able to the effect of the substance on the central nervous system (e.g., impaired motor coordination, psychomotor agitation or retardation, euphoria. anxiety, belligerence, mood lability, cognitive impairment, impaired judgment, social withdrawal) and develop during, or shortly after, use of the substance. C. The signs or symptoms are not attributable to another medical condition and are not bet- ter explained by another mental disorder, including intoxication with another substance.

1	C. The signs or symptoms are not attributable to another medical condition and are not bet- ter explained by another mental disorder, including intoxication with another substance. Coding note: The lCD-9-CM code is 292.89. The lCD-10-CM code depends on whether there is a comorbid other (or unknown) substance use disorder involving the same sub- stance. If a mild other (or unknown) substance use disorder is comorbid, the |CD-10-CM code is F19.129, and it a moderate or severe other (or unknown) substance use disorder is comorbid, the |CD-10-CM code is F19.229. If there is no comorbid other (or unknown) sub- stance use disorder involving the same substance, then the ICD-10-CM code is F19.929. Note: For information on Risk and Prognostic Factors, Culture-Related Diagnostic Issues, and Diagnostic Markers, see the corresponding sections in other (or unknown) substance use disorder.

1	Note: For information on Risk and Prognostic Factors, Culture-Related Diagnostic Issues, and Diagnostic Markers, see the corresponding sections in other (or unknown) substance use disorder. Other (or unknown) substance intoxication is a clinically significant mental disorder that develops during, or immediately after, use of either a) a substance not elsewhere ad- dressed in this chapter (i.e., alcohol; caffeine; cannabis; phencyclidine and other halluci- nogens; inhalants; opioids; sedatives, hypnotics, or anxiolytics; stimulants; or tobacco) or b) an unknown substance. If the substance is known, it should be reﬂected in the name of the disorder upon coding.

1	Application of the diagnostic criteria for other (or unknown) substance intoxication is very challenging. Criterion A requires development of a reversible “substance-specific syndrome," but if the substance is unknown, that syndrome usually will be unknown. To resolve this conflict, clinicians may ask the individual or obtain collateral history as to whether the individual has experienced a similar episode after using substances with the same ”street” name or from the same source. Similarly, hospital emergency departments sometimes recognize over a few days numerous presentations of a severe, unfamiliar in- toxication syndrome from a newly available, previously unknown substance. Because of the great variety of intoxicating substances, Criterion B can provide only broad examples of signs and symptoms from some intoxications, with no threshold for the number of symptoms required for a diagnosis; clinical judgment guides those decisions. Criterion C requires ruling out other medical

1	and symptoms from some intoxications, with no threshold for the number of symptoms required for a diagnosis; clinical judgment guides those decisions. Criterion C requires ruling out other medical conditions, mental disorders, 01' intoxications.

1	The prevalence of other (or unknown) substance intoxication is unknown.

1	Intoxications usually appear and then peak minutes to hours after use of the substance, but the onset and course vary with the substance and the route of administration. Generally, substances used by pulmonary inhalation and intravenous injection have the most rapid onset of action, while those ingested by mouth and requiring metabolism to an active product are much slower. (For example, after ingestion of certain mushrooms, the first signs of an eventually fatal intoxication may not appear for a few days.) Intoxication ef- fects usually resolve within hours to a very few days. However, the body may completely eliminate an anesthetic gas such as nitrous oxide just minutes after use ends. At the other extreme, some ”hit-and-run" intoxicating substances poison systems, leaving permanent impairments. For example, MPTP (1-methy1—4-phenyl-1,2,3,6-tetrahydropyridine), a con- taminating by-product in the synthesis of a certain opioid, kills dopaminergic cells and in- duces permanent

1	impairments. For example, MPTP (1-methy1—4-phenyl-1,2,3,6-tetrahydropyridine), a con- taminating by-product in the synthesis of a certain opioid, kills dopaminergic cells and in- duces permanent parkinsonism in users who sought opioid intoxication.

1	Impairment from intoxication with any substance may have serious consequences, includ- ing dysfunction at work, social indiscretions, problems in interpersonal relationships, fail- ure to fulfill role obligations, traffic accidents, fighting, high-risk behaviors (i.e., having unprotected sex), and substance or medication overdose. The pattern of consequences will vary with the particular substance. Use of other or unknown substance, without meeting criteria for other (or unknown) substance intoxication. The individual used an other or unknown substance(s), but the dose was insufficient to produce symptoms that meet the diagnostic criteria required for the diagnosis. Substance intoxication or other substance/medication-induced disorders. Familiar sub- stances may be sold in the black market as novel products, and individuals may experience intoxication from those substances. History, toxicology screens, or chemical testing of the substance itself may help to identify it.

1	Different types of other (or unknown) substance—related disorders. Episodes of other (or unknown) substance intoxication may occur during, but are distinct from, other (or un- known) substance use disorder, unspecified other (or unknown) substance—related disor- der, and other (or unknown) substance—induced disorders. Other toxic, metabolic, traumatic, neoplastic, vascular, or infectious disorders that impair brain function and cognition. Numerous neurological and other medical conditions may produce rapid onset of signs and symptoms mimicking those of intoxications, including the examples in Criterion B. Paradoxically, drug withdrawals also must be ruled out, because, for example, lethargy may indicate withdrawal from one drug or intoxication with another drug. As with all substance-related disorders, adolescent conduct disorder, adult antisocial per- sonality disorder, and other substance use disorders tend to co-occur with other (or un- known) substance intoxication.

1	Diagnostic Criteria 292.0 (F19.239) A. Cessation of (or reduction in) use of a substance that has been heavy and prolonged. B. The development of a substance-specific syndrome shortly after the cessation of (or reduction in) substance use. C. The substance-specific syndrome causes clinically significant distress or impairment in social, occupational, or other important areas of functioning. D. The symptoms are not attributable to another medical condition and are not better ex- plained by another mental disorder, including withdrawal from another substance. E. The substance involved cannot be classified under any of the other substance catego- ries (alcohol; caffeine; cannabis; opioids; sedatives, hypnotics, or anxiolytics; stimu- lants; or tobacco) or is unknown.

1	Coding note: The ICD-9-CM code is 292.0. The |CD-10-CM code for other (or unknown) sub- stance withdrawal is F19.239. Note that the |CD-10-CM code indicates the comorbid presence of a moderate or severe other (or unknown) substance use disorder. It is not permissible to code a comorbid mild other (or unknown) substance use disorder with other (or unknown) sub- stance withdrawal. Note: For information on Risk and Prognostic Factors and Diagnostic Markers, see the cor- responding sections in other (or unknown) substance use disorder.

1	Other (or unknown) substance withdrawal is a clinically significant mental disorder that develops during, or within a few hours to days after, reducing or terminating dosing with a substance (Criteria A and B). Although recent dose reduction or termination usually is clear in the history, other diagnostic procedures are very challenging if the drug is un- known. Criterion B requires development of a "substance-specific syndrome” (i.e., the in- dividual’s signs and symptoms must correspond with the known withdrawal syndrome for the recently stopped drug)—a requirement that rarely can be met with an unknown substance. Consequently, clinical judgment must guide such decisions when information is this limited. Criterion D requires ruling out other medical conditions, mental disorders, or withdrawals from familiar substances. When the substance is known, it should be re- ﬂected in the name of the disorder upon coding (e.g., betel nut withdrawal).

1	The prevalence of other (or unknown) substance withdrawal is unknown. Withdrawal signs commonly appear some hours after use of the substance is terminated, but the onset and course vary greatly, depending on the dose typically used by the person and the rate of elimination of the specific substance from the body. At peak severity, with- drawal symptoms from some substances involve only moderate levels of discomfort, whereas withdrawal from other substances may be fatal. Withdrawal-associated dyspho- ria often motivates relapse to substance use. Withdrawal symptoms slowly abate over days, weeks, or months, depending on the particular drug and doses to which the indi- vidual became tolerant. Culture-related issues in diagnosis will vary with the particular substance.

1	Culture-related issues in diagnosis will vary with the particular substance. Withdrawal from any substance may have serious consequences, including physical signs and symptoms (e.g., malaise, vital sign changes, abdominal distress, headache), intense drug craving, anxiety, depression, agitation, psychotic symptoms, or cognitive impairments. These consequences may lead to problems such as dysfunction at work, problems in in- terpersonal relationships, failure to fulfill role obligations, traffic accidents, fighting, high- risk behavior (e.g., having unprotected sex), suicide attempts, and substance or medica- tion overdose. The pattern of consequences will vary with the particular substance. Dose reduction after extended dosing, but not meeting the criteria for other (or un- known) substance withdrawal. The individual used other (or unknown) substances, but the dose that was used was insufficient to produce symptoms that meet the criteria re- quired for the diagnosis.

1	Substance withdrawal or other substance/medication-induced disorders. Familiar substances may be sold in the black market as novel products, and individuals may expe- rience withdrawal when discontinuing those substances. History, toxicology screens, or chemical testing of the substance itself may help to identify it. Different types of other (or unknown) substance—related disorders. Episodes of other (or unknown) substance withdrawal may occur during, but are distinct from, other (or un- known) substance use disorder, unspecified other (or unknown) substance—related disor- der, and unspecified other (or unknown) substance—induced disorders. Other toxic, metabolic, traumatic, neoplastic, vascular, or infectious disorders that im- pair brain function and cognition. Numerous neurological and other medical condi- tions may produce rapid onset of signs and symptoms mimicking those of withdrawals.

1	Paradoxically, drug intoxications also must be ruled out, because, for example, lethargy may indicate withdrawal from one drug or intoxication with another drug. As with all substance-related disorders, adolescent conduct disorder, adult antisocial per- sonality disorder, and other substance use disorders likely co-occur with other (or un- known) substance withdrawal.

1	Because the category of other or unknown substances is inherently ill-defined, the extent and range of induced disorders are uncertain. Nevertheless, other (or unknown) sub- stance—induced disorders are possible and are described in other chapters of the manual with disorders with which they share phenomenology (see the substance/medication- induced mental disorders in these chapters): other (or unknown) substance—induced psy- rocognitive disorder (”Neurocognitive Disorders”). For other (or unknown) substance— delirium, see the criteria and discussion of delirium in the chapter "Neurocognitive Dis- orders.” These other (or unknown) substance—induced disorders are diagnosed instead of only when the symptoms are sufficiently severe to warrant independent clinical attention. 292.9 (F19.99)

1	292.9 (F19.99) This category applies to presentations in which symptoms characteristic of an other (or un- in social, occupational, or other important areas at functioning predominate but do not meet the full criteria for any specific other (or unknown) substance—related disorder or any of the disorders in the substance—related disorders diagnostic class. Diagnostic Criteria 312.31 (F63.0) A. Persistent and recurrent problematic gambling behavior leading to clinically significant impairment or distress, as indicated by the individual exhibiting tour (or more) of the fol- lowing in a 12-month period: 1. Needs to gamble with increasing amounts of money in order to achieve the desired excitement. 2. Is restless or irritable when attempting to cut down or stop gambling. 3. Has made repeated unsuccessful efforts to control, cut back, or stop gambling.

1	2. Is restless or irritable when attempting to cut down or stop gambling. 3. Has made repeated unsuccessful efforts to control, cut back, or stop gambling. 4. Is often preoccupied with gambling (e.g., having persistent thoughts of reliving past gambling experiences, handicapping or planning the next venture, thinking of ways to get money with which to gamble). 5. Often gambles when feeling distressed (e.g., helpless, guilty, anxious, depressed). 6. After losing money gambling, often returns another day to get even ("chasing" one‘s losses). 7. Lies to conceal the extent of involvement with gambling. 8. Has jeopardized or lost a significant relationship, job. or educational or career op- portunity because of gambling. 9. Relies on others to provide money to relieve desperate financial situations caused by gambling. B. The gambling behavior is not better explained by a manic episode. Specify if:

1	9. Relies on others to provide money to relieve desperate financial situations caused by gambling. B. The gambling behavior is not better explained by a manic episode. Specify if: Episodic: Meeting diagnostic criteria at more than one time point, with symptoms sub- siding between periods of gambling disorder for at least several months. Perslstent: Experiencing continuous symptoms, to meet diagnostic criteria for multiple years. Specify if: In early remission: After tull criteria for gambling disorder were previously met, none of the criteria for gambling disorder have been met for at least 3 months but for less than 12 months. In sustained remission: After full criteria for gambling disorder were previously met, none of the criteria for gambling disorder have been met during a period of 12 months or longer. Specify current severity: Mlld: 4—5 criteria met. Moderate: 6—7 criteria met. Severe: 8—9 criteria met.

1	Specify current severity: Mlld: 4—5 criteria met. Moderate: 6—7 criteria met. Severe: 8—9 criteria met. Note: Although some behavioral conditions that do not involve ingestion of substances have similarities to substance-related disorders, only one disorder—gambling disorder— has sufficient data to be included in this section.

1	Severity is based on the number of criteria endorsed. Individuals with mild gambling dis- order may exhibit only 4—5 of the criteria, with the most frequently endorsed criteria usu- ally related to preoccupation with gambling and "chasing" losses. Individuals with moderately severe gambling disorder exhibit more of the criteria (i.e., 6—7). Individuals with the most severe form will exhibit all or most of the nine criteria (i.e., 8—9). Ieopardiz- ing relationships or career opportunities due to gambling and relying on others to provide money for gambling losses are typically the least often endorsed criteria and most often oc- cur among those with more severe gambling disorder. Furthermore, individuals present- ing for treatment of gambling disorder typically have moderate to severe forms of the disorder.

1	Gambling involves risking something of value in the hopes of obtaining something of greater value. In many cultures, individuals gamble on games and events, and most do so without experiencing problems. However, some individuals develop substantial impair- ment related to their gambling behaviors. The essential feature of gambling disorder is persistent and recurrent maladaptive gambling behavior that disrupts personal, family, and / or vocational pursuits (Criterion A). Gambling disorder is defined as a cluster of four or more of the symptoms listed in Criterion A occurring at any time in the same 12-month period.

1	A pattern of ”chasing one’s losses” may develop, with an urgent need to keep gam- bling (often with the placing of larger bets or the taking of greater risks) to undo a loss or series of losses. The individual may abandon his or her gambling strategy and try to win back losses all at once. Although many gamblers may ”chase” for short periods of time, it is the frequent, and often long-term, ”chase” that is characteristic of gambling disorder (Criterion A6). Individuals may lie to family members, therapists, or others to conceal the extent of involvement with gambling; these instances of deceit may also include, but are not limited to, covering up illegal behaviors such as forgery, fraud, theft, or embez- zlement to obtain money with which to gamble (Criterion A7). Individuals may also en- gage in “bailout” behavior, turning to family or others for help with a desperate financial situation that was caused by gambling (Criterion A9).

1	Distortions in thinking (e.g., denial, superstitions, a sense of power and control over the outcome of chance events, overconfidence) may be present in individuals with gambling disorder. Many individuals with gambling disorder believe that money is both the cause of and the solution to their problems. Some individuals with gambling disorder are im- pulsive, competitive, energetic, restless, and easily bored; they may be overly concerned with the approval of others and may be generous to the point of extravagance when win- ning. Other individuals with gambling disorder are depressed and lonely, and they may gamble when feeling helpless, guilty, or depressed. Up to half of individuals in treatment for gambling disorder have suicidal ideation, and about 17% have attempted suicide.

1	The past-year prevalence rate of gambling disorder is about 0.2%—0.3% in the general pop- ulation. In the general population, the lifetime prevalence rate is about 0.4%—1.0%. For fe- males, the lifetime prevalence rate of gambling disorder is about 0.2%, and for males it is about 0.6%. The lifetime prevalence of pathological gambling among African Americans is about 0.9%, among whites about 0.4%, and among Hispanics about 0.3%.

1	The onset of gambling disorder can occur during adolescence or young adulthood, but in other individuals it manifests during middle or even older adulthood. Generally, gam- bling disorder develops over the course of years, although the progression appears to be more rapid in females than in males. Most individuals who develop a gambling disorder evidence a pattern of gambling that gradually increases in both frequency and amount of wagering. Certainly, milder forms can develop into more severe cases. Most individuals with gambling disorder report that one or two types of gambling are most problematic for them, although some individuals participate in many forms of gambling. Individuals are likely to engage in certain types of gambling (e.g., buying scratch tickets daily) more fre- quently than others (e.g., playing slot machines or blackjack at the casino weekly). Fre- quency of gambling can be related more to the type of gambling than to the severity of the overall gambling disorder.

1	than others (e.g., playing slot machines or blackjack at the casino weekly). Fre- quency of gambling can be related more to the type of gambling than to the severity of the overall gambling disorder. For example, purchasing a single scratch ticket each day may not be problematic, while less frequent casino, sports, or card gambling may be part of a gambling disorder. Similarly, amounts of money spent wagering are not in themselves in- dicative of gambling disorder. Some individuals can wager thousands of dollars per month and not have a problem with gambling, while others may wager much smaller amounts but experience substantial gambling-related difficulties.

1	Gambling patterns may be regular or episodic, and gambling disorder can be persis- tent or in remission. Gambling can increase during periods of stress or depression and during periods of substance use or abstinence. There may be periods of heavy gambling and severe problems, times of total abstinence, and periods of nonproblematic gambling. Gambling disorder is sometimes associated with spontaneous, long-term remissions. Nevertheless, some individuals underestimate their vulnerability to develop gambling disorder or to return to gambling disorder following remission. When in a period of re- mission, they may incorrectly assume that they will have no problem regulating gambling and that they may gamble on some forms nonproblematically, only to experience a retum to gambling disorder.

1	Early expression of gambling disorder is more common among males than among fe- males. Individuals who begin gambling in youth often do so with family members or friends. Development of early-life gambling disorder appears to be associated with impul- sivity and substance abuse. Many high school and college students who develop gambling disorder grow out of the disorder over time, although it remains a lifelong problem for some. Mid- and later-life onset of gambling disorder is more common among females than among males.

1	There are age and gender variations in the type of gambling activities and the preva- lence rates of gambling disorder. Gambling disorder is more common among younger and middle-age persons than among older adults, Among adolescents and young adults, the disorder is more prevalent in males than in females. Younger individuals prefer different forms of gambling (e.g., sports betting), while older adults are more likely to develop problems with slot machine and bingo gambling. Although the proportions of individuals who seek treatment for gambling disorder are low across all age groups, younger individ- uals are especially unlikely to present for treatment.

1	Males are more likely to begin gambling earlier in life and to have a younger age at on- set of gambling disorder than females, who are more likely to begin gambling later in life and to develop gambling disorder in a shorter time frame. Females with gambling disor- der are more likely than males with gambling disorder to have depressive, bipolar, and anxiety disorders. Females also have a later age at onset of the disorder and seek treatment sooner, although rates of treatment seeking are low (<10%) among individuals with gam- bling disorder regardless of gender. Temperamental. Gambling that begins in childhood or early adolescence is associated with increased rates of gambling disorder. Gambling disorder also appears to aggregate with antisocial personality disorder, depressive and bipolar disorders, and other sub- stance use disorders, particularly with alcohol disorders.

1	Genetic and physiological. Gambling disorder can aggregate in families, and this effect appears to relate to both environmental and genetic factors. Gambling problems are more frequent in monozygotic than in dizygotic twins. Gambling disorder is also more preva— lent among first-degree relatives of individuals with moderate to severe alcohol use dis- order than among the general population. Course modifiers. Many individuals, including adolescents and young adults, are likely to resolve their problems with gambling disorder over time, although a strong predictor of future gambling problems is prior gambling problems.

1	Individuals from specific cultures and races/ethnicities are more likely to participate in some types of gambling activities than others (e.g., pai gow, cockfights, blackjack, horse rac— ing). Prevalence rates of gambling disorder are higher among African Americans than among European Americans, with rates for Hispanic Americans similar to those of Euro— pean Americans. Indigenous populations have high prevalence rates of gambling disorder. Males develop gambling disorder at higher rates than females, although this gender gap may be narrowing. Males tend to wager on different forms of gambling than females, with cards, sports, and horse race gambling more prevalent among males, and slot machine and bingo gambling more common among females. Functional Consequences of Gambling Disorder

1	Functional Consequences of Gambling Disorder Areas of psychoSocial, health, and mental health functioning may be adversely affected by gambling disorder. Specifically, individuals with gambling disorder may, because of their involvement with gambling, jeopardize or lose important relationships with family mem— bers or friends. Such problems may occur from repeatedly lying to others to cover up the extent of gambling or from requesting money that is used for gambling or to pay off gam- bling debts. Employment or educational activities may likewise be adversely impacted by gambling disorder; absenteeism or poor work or school performance can occur with gam- bling disorder, as individuals may gamble during work or school hours or be preoccupied with gambling or its adverse consequence when they should be working or studying. In- dividuals with gambling disorder have poor general health and utilize medical services at high rates.

1	Nondisordered gambling. Gambling disorder must be distinguished from professional and social gambling. In professional gambling, risks are limited and discipline is central. Social gambling typically occurs with friends or colleagues and lasts for a limited period of time, with acceptable losses. Some individuals can experience problems associated with gambling (e.g., short-term chasing behavior and loss of control) that do not meet the full criteria for gambling disorder.

1	Manic episode. Loss of judgment and excessive gambling may occur during a manic ep- isode. An additional diagnosis of gambling disorder should be given only if the gambling behavior is not better explained by manic episodes (e.g., a history of maladaptive gam- bling behavior at times other than during a manic episode). Alternatively, an individual with gambling disorder may, during a period of gambling, exhibit behavior that resembles a manic episode, but once the individual is away from the gambling, these manic-like fea- tures dissipate. Personality disorders. Problems with gambling may occur in individuals with antisocial personality disorder and other personality disorders. If the criteria are met for both disor- ders, both can be diagnosed. Other medical conditions. Some patients taking dopaminergic medications (e.g., for

1	Other medical conditions. Some patients taking dopaminergic medications (e.g., for Parkinson’s disease) may experience urges to gamble. If such symptoms dissipate when dopaminergic medications are reduced in dosage or ceased, then a diagnosis of gambling disorder would not be indicated.

1	Gambling disorder is associated with poor general health. In addition, some specific med— ical diagnoses, such as tachycardia and angina, are more common among individuals with gambling disorder than in the general population, even when other substance use disor- ders, including tobacco use disorder, are controlled for. Individuals with gambling disor- der have high rates of comorbidity with other mental disorders, such as substance use disorders, depressive disorders, anxiety disorders, and personality disorders. In some in- dividuals, other mental disorders may precede gambling disorder and be either absent or present during the manifestation of gambling disorder. Gambling disorder may also occur prior to the onset of other mental disorders, especially anxiety disorders and substance use disorders. The neurocog nitive d isorders (NCDs) (referred to in DSM-IV as ”Dementia,

1	Delirium, Amnestic, and Other Cognitive Disorders”) begin with delirium, followed by the syndromes of major NCD, mild NCD, and their etiological subtypes. The major or mild NCD subtypes are NCD due to Alzheimer’s disease; vascular NCD; NCD with Lewy bodies; NCD due to Parkinson’s disease; frontotemporal NCD; NCD due to traumatic brain injury; NCD due to HIV infection; substance/medication—induced NCD; NCD due to Huntington’s disease; NCD due to prion disease; NCD due to another medical condi- tion; NCD due to multiple etiologies; and unspecified NCD. The NCD category encom- passes the group of disorders in which the primary clinical deficit is in cognitive function, and that are acquired rather than developmental. Although cognitive deficits are present in many if not all mental disorders (e.g., schizophrenia, bipolar disorders), only disorders whose core features are cognitive are included in the NCD category. The NCDs are those in which impaired cognition has not been present since

1	(e.g., schizophrenia, bipolar disorders), only disorders whose core features are cognitive are included in the NCD category. The NCDs are those in which impaired cognition has not been present since birth or very early life, and thus represents a decline from a previously attained level of functioning.

1	The NCDs are unique among DSM—S categories in that these are syndromes for which the underlying pathology, and frequently the etiology as well, can potentially be deter- mined. The various underlying disease entities have all been the subject of extensive re- search, clinical experience, and expert consensus on diagnostic criteria. The DSM-S criteria for these disorders have been developed in close consultation with the expert groups for each of the disease entities and align as closely as possible with the current consensus cri- teria for each of them. The potential utility of biomarkers is also discussed in relation to diagnosis. Dementia is subsumed under the newly named entity major neurocognitive dis- order, although the term dementia is not precluded from use in the etiological subtypes in which that term is standard. Furthermore, DSM—5 recognizes a less severe level of cogni- tive impairment, mild neurocognitive disorder, which can also be a focus of care, and which in DSM-IV

1	in which that term is standard. Furthermore, DSM—5 recognizes a less severe level of cogni- tive impairment, mild neurocognitive disorder, which can also be a focus of care, and which in DSM-IV was subsumed under ”Cognitive Disorder Not Otherwise Specified.” Diagnos- tic criteria are provided for both these syndromic entities, followed by diagnostic criteria for the different etiological subtypes. Several of the NCDs frequently coexist with one an- other, and their relationships may be multiply characterized under different chapter sub- headings, including ”Differential Diagnosis” (e.g., NCD due to Alzheimer’s disease vs.

1	vascular NCD), ”Risk and Prognostic Factors” (e.g., vascular pathology increasing the clinical expression of Alzheimer’s disease), and / or ”Comorbidity” (e.g., mixed Alzhei- mer’s disease—vascular pathology).

1	The term dementia is retained in DSM-5 for continuity and may be used in settings where physicians and patients are accustomed to this term. Although dementia is the cus- tomary term for disorders like the degenerative dementias that usually affect older adults, the term neurocognitive disorder is widely used and often preferred for conditions affect- ing younger individuals, such as impairment secondary to traumatic brain injury or HIV infection. Furthermore, the major NCD definition is somewhat broader than the term dementia, in that individuals with substantial decline in a single domain can receive this di- agnosis, most notably the DSM-IV category of ”Amnestic Disorder," which would now be diagnosed as major NCD due to another medical condition and for which the term demen- tia would not be used.

1	The criteria for the various NCDs are all based on defined cognitive domains. Table 1 pro- vides for each of the key domains a working definition, examples of symptoms or obser- vations regarding impairments in everyday activities, and examples of assessments. The domains thus defined, along with guidelines for clinical thresholds, form the basis on which the NCDs, their levels, and their subtypes may be diagnosed.

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1	3mg 6.355 2 5mm: 55 :8 @593 :25me 2.5..— ..555855 5:53 @5555— 5 @5533 $350me $555.58 mm 553 @593 555 .mmm 58 5555 55me 55“...on 9 5me .wfixumn E 35qu m5. m5 .555 _.5 wacmbumufib 5: 553 555.3 555.55 5 555 5% 5:5 >32 .8395 355 m 2 5w 9 @555 m305£ 555 35: mme £55855 .55 9555 .5 59: .5 5.55 55.. 2 5mm: 52 55.5% .5258me ww5m5u 5m: 50 .5535 9.5.532 55m m3o5m5m 553 5555 E 505555 5.55 5550 m5 $Em~5505>5w 55555 55 wczmwgmc 52559, .555 @5355 £502 mini 95: $525 55553 595555 553 9555555 5:555an mam “8.55% 5329:953an .355 £33855 5.5.5 BEE 555 .5553 555555 85.5“ $355 55:55 025500 «55:55:08 wEmEou 035589.52 w ”45:. A. B. A disturbance in attention (i.e., reduced ability to direct, focus, sustain, and shift atten- tion) and awareness (reduced orientation to the environment). The disturbance develops over a short period of time (usually hours to a few days), rep- resents a change from baseline attention and awareness, and tends to fluctuate in se- verity during the course of a day.

1	. An additional disturbance in cognition (e.g., memory deficit, disorientation, language, visuospatial ability, or perception). . The disturbances in Criteria A and C are not better explained by another preexisting, established, or evolving neurocognitive disorder and do not occur in the context of a severely reduced level of arousal, such as coma. There is evidence from the history, physical examination, or laboratory findings that the disturbance is a direct physiological consequence of another medical condition, sub- stance intoxication or withdrawal (i.e., due to a drug of abuse or to a medication), or exposure to a toxin, or is due to multiple etiologies. Specify whether: Substance intoxication delirium: This diagnosis should be made instead of sub- stance intoxication when the symptoms in Criteria A and C predominate in the clinical picture and when they are sufficiently severe to warrant clinical attention.

1	Coding note: The |CD-9-CM and ICD-10-CM codes for the [specific substance] in- toxication delirium are indicated in the table below. Note that the lCD-10-CM code depends on whether or not there is a comorbid substance use disorder present for the same class of substance. If a mild substance use disorder is comorbid with the substance intoxication delirium, the 4th position character is “1," and the clinician should record “mild [substance] use disorder‘ before the substance intoxication de- lirium (e.g., “mild cocaine use disorder with cocaine intoxication delirium"). If a mod- erate or severe substance use disorder is comorbid with the substance intoxication delirium, the 4th position character is “2,” and the clinician should record “moderate [substance] use disordei” or “severe [substance] use disorder,” depending on the severity of the comorbid substance use disorder. If there is no comorbid substance use disorder (e.g., after a one-time heavy use of the substance), then the 4th

1	use disorder,” depending on the severity of the comorbid substance use disorder. If there is no comorbid substance use disorder (e.g., after a one-time heavy use of the substance), then the 4th posi- tion character is “9," and the clinician should record only the substance intoxication delirium.

1	With use disorder, disorder, moderate or Without use Alcohol 291.0 F10.121 F10.221 F10.921 Cannabis 292.81 F12.121 F12.221 F12.921 Phencyclidine 292.81 F16.121 F16.221 F16.921 Other hallucinogen 292.81 F16.121 F16.221 F16.921 Inhalant 292.81 F18.121 F18.221 F18.921 Opioid 292.81 F11.121 F11.221 F11.921 With use disorder, disorder, moderate or Without use Sedative, hypnotic, or anxiolytic 292.81 F13.121 F13.221 F13.921 Amphetamine (or other 292.81 F15.121 F15.221 F15.921 Cocaine 292.81 F14.121 F14.221 F14.921 Other (or unknown) substance 292.81 F19.121 F19.221 F19.921 Substance withdrawal delirium: This diagnosis should be made instead of sub- stance withdrawal when the symptoms in Criteria A and C predominate in the clinical picture and when they are sufficiently severe to warrant clinical attention.

1	Code [specific substance] withdrawal delirium: 291.0 (F10.231) alcohol; 292.0 (F11.23) opioid; 292.0 (F13.231) sedative, hypnotic, or anxiolytic; 292.0 (F19.231) other (or unknown) substance/medication. Medication-induced delirium: This diagnosis applies when the symptoms in Criteria A and C arise as a side effect of a medication taken as prescribed. Coding note: The |CD-9-CM code for [specific medication]-induced delirium is 292.81. The |CD-10-CM code depends on the type of medication. If the medication is an opioid taken as prescribed, the code is F11.921. It the medication is a seda- tive, hypnotic, or anxiolytic taken as prescribed, the code is F13.921. If the medica- tion is an amphetamine-type or other stimulant taken as prescribed, the code is F15.921. For medications that do not fit into any of the classes (e.g., dexametha- sone) and in cases in which a substance is judged to be an etiological factor but the specific class of substance is unknown. the code is F19.921.

1	293.0 (F05) Delirium due to another medical condition: There is evidence from the history, physical examination, or laboratory findings that the disturbance is attributable to the physiological consequences of another medical condition. Coding note: Include the name of the other medical condition in the name of the delirium (e.g., 293.0 [F05] delirium due to hepatic encephalopathy). The other med- delirium due to another medical condition (e.g.. 572.2 [K7290] hepatic encepha- lopathy; 293.0 [F05] delirium due to hepatic encephalopathy). 293.0 (F05) Delirium due to multiple etiologies: There is evidence from the history, physical examination, or laboratory findings that the delirium has more than one etiol- ogy (e.g., more than one etiological medical condition; another medical condition plus substance intoxication or medication side effect).

1	Coding note: Use multiple separate codes reﬂecting specific delirium etiologies (e.g.. 572.2 [K7290] hepatic encephalopathy, 293.0 [F05] delirium due to hepatic failure; 291.0 [F10.231] alcohol withdrawal delirium). Note that the etiological med- ical condition both appears as a separate code that precedes the delirium code and is substituted into the delirium due to another medical condition rubric. Specify if: Acute: Lasting a few hours or days. Persistent: Lasting weeks or months. Specify if: Hyperactive: The individual has a hyperactive level of psychomotor activity that may be accompanied by mood lability, agitation, and/or refusal to cooperate with medical care. Hypoactive: The individual has a hypoactive level of psychomotor activity that may be accompanied by sluggishness and lethargy that approaches stupor.

1	Hypoactive: The individual has a hypoactive level of psychomotor activity that may be accompanied by sluggishness and lethargy that approaches stupor. Mixed level of activity: The individual has a normal level of psychomotor activity even though attention and awareness are disturbed. Also includes individuals whose activity level rapidly ﬂuctuates. ICD-9-CM. The name of the substance/medication intoxication delirium begins with the specific substance (e.g., cocaine, dexamethasone) that is presumed to be causing the delirium. The diagnostic code is selected from the table included in the criteria set, which is based on the drug class. For substances that do not fit into any of the classes (e.g., dexa- methasone), the code for "other substance" should be used; and in cases in which a sub- stance is judged to be an etiological factor but the specific class of substance is unknown, the category "unknown substance” should be used.

1	The name of the disorder is followed by the course (i.e., acute, persistent), followed by the specifier indicating level of psychomotor activity (i.e., hyperactive, hypoactive, mixed level of activity). Unlike the recording procedures for ICD-lO-CM, which combine the sub- stance/medication intoxication delirium and substance use disorder into a single code, for ICD-9-CM a separate diagnostic code is given for the substance use disorder. For example, in the case of acute hyperactive intoxication delirium occurring in a man with a severe co- caine use disorder, the diagnosis is 292.81 cocaine intoxication delirium, acute, hyperac- tive. An additional diagnosis of 304.20 severe cocaine use disorder is also given. If the intoxication delirium occurs without a comorbid substance use disorder (e.g., after a one- time heavy use of the substance), no accompanying substance use disorder is noted (e.g., 292.81 phencyclidine intoxication delirium, acute, hypoactive).

1	I CD-10-CM . The name of the substance/ medication intoxication delirium begins with the specific substance (e.g., cocaine, dexamethasone) that is presumed to be causing the delirium. The diagnostic code is selected from the table included in the criteria set, which is based on the drug class and presence or absence of a comorbid substance use disorder. For substances that do not fit into any of the classes (e.g., dexamethasone), the code for ”other substance” should be used; and in cases in which a substance is judged to be an etiological factor but the specific class of substance is unknown, the category ”unknown substance” should be used.

1	When recording the name of the disorder, the comorbid substance use disorder (if any) is listed first, followed by the word ”with,” followed by the name of the substance intoxication delirium, followed by the course (i.e., acute, persistent), followed by the specifier indicating level of psychomotor activity (i.e., hyperactive, hypoactive, mixed level of activity). For exam- ple, in the case of acute hyperactive intoxication delirium occurring in a man with a severe c0- caine use disorder, the diagnosis is F14.221 severe cocaine use disorder with cocaine intoxication delirium, acute, hyperactive. A separate diagnosis of the comorbid severe cocaine use disorder is not given. If the intoxication delirium occurs without a comorbid substance use disorder (e.g., after a one-time heavy use of the substance), no accompanying substance use disorder is noted (e.g., F16.921 phencyclidine intoxication delirium, acute, hypoactive).

1	ICD-9-CM. The name of the substance/medication withdrawal delirium begins with the specific substance (e.g., alcohol) that is presumed to be causing the withdrawal delirium. The diagnostic code is selected from substance—specific codes included in the coding note included in the criteria set. The name of the disorder is followed by the course (i.e., acute, persistent), fol- lowed by the specifier indicating level of psychomotor activity (i.e., hyperactive, hypoactive, mixed level of activity). Unlike the recording procedures for ICD-IOCM, which combine the substance/medication withdrawal delirium and substance use disorder into a single code, for

1	ICD—9—CM a separate diagnostic code is given for the substance use disorder. For example, in the case of acute hyperactive withdrawal delirium occurring in a man with a severe alcohol use disorder, the diagnosis is 291.0 alcohol withdrawal delirium, acute, hyperactive. An additional diagnosis of 303.90 severe alcohol use disorder is also given.

1	I CD-10-CM. The name of the substance/medication withdrawal delirium begins with the specific substance (e.g., alcohol) that is presumed to be causing the withdrawal delir- ium. The diagnostic code is selected from substance-specific codes included in the coding note included in the criteria set. When recording the name of the disorder, the comorbid moderate or severe substance use disorder (if any) is listed first, followed by the word ”with,” followed by the substance withdrawal delirium, followed by the course (i.e., acute, persistent), followed by the specifier indicating level of psychomotor activity (i.e., hyper- active, hypoactive, mixed level of activity). For example, in the case of acute hyperactive withdrawal delirium occurring in a man with a severe alcohol use disorder, the diagnosis is F10.231 severe alcohol use disorder with alcohol withdrawal delirium, acute, hyperac- tive. A separate diagnosis of the comorbid severe alcohol use disorder is not given.

1	Medication-induced delirium. The name of the medication—induced delirium begins with the specific substance (e.g., dexamethasone) that is presumed to be causing the de- lirium. The name of the disorder is followed by the course (i.e., acute, persistent), followed by the specifier indicating level of psychomotor activity (i.e., hyperactive, hypoactive, mixed level of activity). For example, in the case of acute hyperactive medication-induced delirium occurring in a man using dexamethasone as prescribed, the diagnosis is 292.81 (F19.921) dexamethasone-induced delirium, acute, hyperactive. Regarding course, in hospital settings, delirium usually lasts about 1 week, but some symptoms often persist even after individuals are discharged from the hospital.

1	Regarding course, in hospital settings, delirium usually lasts about 1 week, but some symptoms often persist even after individuals are discharged from the hospital. Individuals with delirium may rapidly switch between hyperactive and hypoactive states. The hyperactive state may be more common or more frequently recognized and often is associated with medication side effects and drug withdrawal. The hypoactive state may be more frequent in older adults.

1	The essential feature of delirium is a disturbance of attention or awareness that is accom- or evolving neurocognitive disorder (NCD). The disturbance in attention (Criterion A) is manifested by reduced ability to direct, focus, sustain, and shift attention. Questions must be repeated because the individual’s attention wanders, or the individual may perseverate with an answer to a previous question rather than appropriately shift attention. The indi- vidual is easily distracted by irrelevant stimuli. The disturbance in awareness is mani- fested by a reduced orientation to the environment or at times even to oneself.

1	The disturbance develops over a short period of time, usually hours to a few days, and tends to ﬂuctuate during the course of the day, often with worsening in the evening and night when external orienting stimuli decrease (Criterion B). There is evidence from the history, physical examination, or laboratory findings that the disturbance is a physiologi- cal consequence of an underlying medical condition, substance intoxication or with- drawal, use of a medication, or a toxin exposure, or a combination of these factors (Criterion E). The etiology should be coded according to the etiologically appropriate sub- type (i.e., substance or medication intoxication, substance withdrawal, another medical condition, or multiple etiologies). Delirium often occurs in the context of an underlying NCD. The impaired brain function of individuals with mild and major NCD renders them more vulnerable to delirium.

1	There is an accompanying change in at least one other area that may include memory and learning (particularly recent memory), disorientation (particularly to time and place), alteration in language, or perceptual distortion or a perceptual-motor disturbance (Crite- rion C). The perceptual disturbances accompanying delirium include misinterpretations, illusions, or hallucinations; these disturbances are typically visual, but may occur in other modalities as well, and range from simple and uniform to highly complex. Normal atten- tion/arousal, delirium, and coma lie on a continuum, with coma defined as the lack of any response to verbal stimuli. The ability to evaluate cognition to diagnose delirium depends on there being a level of arousal sufficient for response to verbal stimulation; hence, delir- ium should not be diagnosed in the context of coma (Criterion D). Many noncomatose pa- tients have a reduced level of arousal. Those patients who show only minimal responses to verbal

1	hence, delir- ium should not be diagnosed in the context of coma (Criterion D). Many noncomatose pa- tients have a reduced level of arousal. Those patients who show only minimal responses to verbal stimulation are incapable of engaging with attempts at standardized testing or even interview. This inability to engage should be classified as severe inattention. Low-arousal states (of acute onset) should be recognized as indicating severe inattention and cognitive change, and hence delirium. They are clinically indistinguishable from delirium diag- nosed on the basis of inattention or cognitive change elicited through cognitive testing and interview.

1	Delirium is often associated with a disturbance in the sleep-wake cycle. This disturbance can include daytime sleepiness, nighttime agitation, difficulty falling asleep, excessive sleepiness throughout the day, or wakefulness throughout the night. In some cases, com- plete reversal of the night-day sleep-wake cycle can occur. Sleep—wake cycle disturbances are very common in delirium and have been proposed as a core criterion for the diagnosis. The individual with delirium may exhibit emotional disturbances, such as anxiety, fear, depression, irritability, anger, euphoria, and apathy. There may be rapid and unpre- dictable shifts from one emotional state to another. The disturbed emotional state may also be evident in calling out, screaming, cursing, muttering, moaning, or making other sounds. These behaviors are especially prevalent at night and under conditions in which stimulation and environmental cues are lacking.

1	The prevalence of delirium is highest among hospitalized older individuals and varies depending on the individuals’ characteristics, setting of care, and sensitivity of the detec- tion method. The prevalence of delirium in the community overall is low (1%—2%) but in- creases with age, rising to 14% among individuals older than 85 years. The prevalence is 10%—30% in older individuals presenting to emergency departments, where the delirium often indicates a medical illness. The prevalence of delirium when individuals are admitted to the hospital ranges from 14% to 24%, and estimates of the incidence of delirium arising during hospitalization range from 6% to 56% in general hospital populations. Delirium occurs in 15%—53% of older individuals postoperatively and in 70%—87% of those in intensive care. Delirium oc- curs in up to 60% of individuals in nursing homes or post—acute care settings and in up to 83% of all individuals at the end of life.

1	While the majority of individuals with delirium have a full recovery with or without treatment, early recognition and intervention usually shortens the duration of the delir- ium. Delirium may progress to stupor, coma, seizures, or death, particularly if the under- lying cause remains untreated. Mortality among hospitalized individuals with delirium is high, and as many as 40% of individuals with delirium, particularly those with malignan- cies and other significant underlying medical illness, die within a year after diagnosis. Environmental. Delirium may be increased in the context of functional impairment, im- mobility, a history of falls, low levels of activity, and use of drugs and medications with psychoactive properties (particularly alcohol and anticholinergics).

1	Genetic and physiological. Both major and mild NCDs can increase the risk for delir- ium and complicate the course. Older individuals are especially susceptible to delirium compared With younger adults. Susceptibility to delirium in infancy and through child- hood may be greater than in early and middle adulthood. In childhood, delirium may be related to febrile illnesses and certain medications (e.g., anticholinergics). In addition to laboratory findings characteristic of underlying medical conditions (or in- toxication or withdrawal states), there is often generalized slowing on electroencephalog- raphy, and fast activity is occasionally found (e.g., in some cases of alcohol withdrawal delirium). However, electroencephalography is insufficiently sensitive and specific for di- agnostic use. Functional Consequences of Deiirium

1	Functional Consequences of Deiirium Delirium itself is associated with increased functional decline and risk of institutional placement. Hospitalized individuals 65 years or older with delirium have three times the risk of nursing home placement and about three times the functional decline as hospital- ized patients without delirium at both discharge and 3 months postdischarge. Psychotic disorders and bipolar and depressive disorders with psychotic features. Delirium that is characterized by vivid hallucinations, delusions, language disturbances, and agitation must be distinguished from brief psychotic disorder, schizophrenia, schizo- phreniform disorder, and other psychotic disorders, as well as from bipolar and depres- sive disorders with psychotic features.

1	Acute stress disorder. Delirium associated with fear, anxiety, and dissociative symptoms, such as depersonalization, must be distinguished from acute stress disorder, which is pre- cipitated by exposure to a severely traumatic event. Malingering and factitious disorder. Delirium can be distinguished from these disor- ders on the basis of the often atypical presentation in malingering and factitious disorder and the absence of another medical condition or substance that is etiologically related to the apparent cognitive disturbance. Other neurocognitive disorders. The most common differential diagnostic issue when evaluating confusion in older adults is disentangling symptoms of delirium and dementia.

1	Other neurocognitive disorders. The most common differential diagnostic issue when evaluating confusion in older adults is disentangling symptoms of delirium and dementia. The clinician must determine whether the individual has delirium; a delirium superim- posed on a preexisting NCD, such as that due to Alzheimer’s disease; or an NCD without delirium. The traditional distinction between delirium and dementia according to acute- ness of onset and temporal course is particularly difficult in those elderly individuals who had a prior NCD that may not have been recognized, or who develop persistent cognitive impairment following an episode of delirium. 780.09 (R41.0)

1	780.09 (R41.0) This category applies to presentations in which symptoms characteristic of delirium that cause clinically significant distress or impairment in social, occupational, or other impor- tant areas of functioning predominate but do not meet the full criteria for delirium or any of the disorders in the neurocognitive disorders diagnostic class. The other specified delirium category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for delirium or any specific neuro- cognitive disorder. This is done by recording “other specified delirium" followed by the spe- cific reason (e.g., “attenuated delirium syndrome"). An example of a presentation that can be specified using the “other specified” desig- nation is the following:

1	An example of a presentation that can be specified using the “other specified” desig- nation is the following: Attenuated delirium syndrome: This syndrome applies in cases of delirium in which the severity of cognitive impairment falls short of that required for the diagnosis, or in which some, but not all, diagnostic criteria for delirium are met. 780.09 (R41.0)

1	780.09 (R41.0) This category applies to presentations in which symptoms characteristic of delirium that cause clinically significant distress or impairment in social, occupational, or other impor- tant areas of functioning predominate but do not meet the full criteria for delirium or any of the disorders in the neurocognitive disorders diagnostic class. The unspecified delirium category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for delirium, and includes presentations for which there is insuffi- cient information to make a more specific diagnosis (e.g., in emergency room settings).

1	A. Evidence of significant cognitive decline from a previous level of performance in one or more cognitive domains (complex attention. executive function, learning and mem- ory, language, perceptuaI—motor, or social cognition) based on: 1. Concern ot the individual, a knowledgeable informant, or the clinician that there has 2. A substantial impairment in cognitive performance. preferably documented by stan- dardized neuropsychological testing or, in its absence, another quantified clinical assessment. B. The cognitive deficits interfere with independence in everyday activities (i.e., at a min- imum, requiring assistance with complex instrumental activities of daily living such as paying bills or managing medications). C. The cognitive deficits do not occur exclusively in the context of a delirium. D. The cognitive deficits are not better explained by another mental disorder (e.g., major depressive disorder, schizophrenia). Specify whether due to:

1	D. The cognitive deficits are not better explained by another mental disorder (e.g., major depressive disorder, schizophrenia). Specify whether due to: Alzheimer’s disease (pp. 611—614) Frontotemporal lobar degeneration (pp. 614—618) Lewy body disease (pp. 618—621) Vascular disease (pp. 621—624) Traumatic brain injury (pp. 624—627) Substance/medication use (pp. 627-632) HIV infection (pp. 632—634) Prion disease (pp. 634—636) Parkinson’s disease (pp. 636—638) Huntington's disease (pp. 638—641) Another medical condition (pp. 641—642) Multiple etiologies (pp. 642—643) Unspecified (p. 643) Coding note: Code based on medical or substance etiology. In some cases, there is need for an additional code for the etiological medical condition, which must immediately pre- cede the diagnostic code for major neurocognitive disorder, as follows: Probable: 331.0 (G309) Probable: 294.1x 331.83 (G31.84)

1	Probable: 331.0 (G309) Probable: 294.1x 331.83 (G31.84) Possible: no additional (F02.8x) (Do not use addi- medical code Possible: 331.9 tional code for (631.9)C Alzheimer‘s disease.) Probable: 331.19 Probable: 294.1x 331.83 (G31.84) (G31.09) (F02.8x) (Do not use addi- Possible: no additional Possible: 331.9 tional code for medical code (631.9)c frontotemporal disease.) Probable: 331.82 Probable: 294.1x 331.83 (G31.84) (631.83) (F02.8x) (Do not use addi- Possible: no additional Possible: 331.9 tional code for medical code (631.9)C Lewy body disease.) No additional medical Probable: 290.40 331.83 (G31.84) code (F01.5x) (Do not use addi- Possible: 331.9 tional code for the (631.9)c vascular disease.) 907.0 ($06.2X93) 294.1x (F02.8x) 331.83 (631.84) code for the trau- matic brain injury.) No additional medical Code based on the Code based on the code type of substance causing the major type of substance causing the mild Due to another Due to multiple 046.79 (A819)

1	No additional medical Code based on the Code based on the code type of substance causing the major type of substance causing the mild Due to another Due to multiple 046.79 (A819) Probable: 332.0 (620) Possible: No additional 333.4 (G10) Code the other medical (e.g., 340 [635] Code all of the etiological (with the exception of 294.1x (F02.8x) 294.1 x (F02.8x) Probable: 294.1x (F02.8x) Possible: 331.9 (631 .9)0 294.1x (F02.8x) 294.1x (F02.8x) 294.1x (F02.8x) (Plus the code for the relevant sub- etiology.) 799.59 (R413) 331.83 (G31.84) infection.) 331.83 (631.84) prion disease.) 331.83 (G31.a4) disease.) 331.83 (G31.84) disease.) 331.83 (G31.84) tions.) 331.83 (G31.84) (Plus the code for the relevant sub- a role in the etiol- ogy. Do not use ad- the presumed conditions.) 799.59 (R41.9) aCode first, before code for major neurocognitive disorder.

1	bCode fifth character based on symptom specifier: .xO without behavioral disturbance; .x1 with be- havioral disturbance (e.g., psychotic symptoms, mood disturbance, agitation, apathy, or other be- havioral symptoms). CNote: Behavioral disturbance specifier cannot be coded but should still be indicated in writing. dSee “Substance/Medication-Induced Major or Mild Neurocognitive Disorder.” Specify: Without behavioral disturbance: If the cognitive disturbance is not accompanied by any clinically significant behavioral disturbance. With behavioral disturbance (specify disturbance): If the cognitive disturbance is ac- companied by a clinically significant behavioral disturbance (e.g., psychotic symptoms, mood disturbance, agitation, apathy, or other behavioral symptoms). Specify current severity: Mild: Difficulties with instrumental activities of daily living (e.g., housework, managing money). Moderate: Difficulties with basic activities of daily living (e.g., feeding, dressing).

1	Mild: Difficulties with instrumental activities of daily living (e.g., housework, managing money). Moderate: Difficulties with basic activities of daily living (e.g., feeding, dressing). Severe: Fully dependent. A. Evidence of modest cognitive decline from a previous level of performance in one or more cognitive domains (complex attention, executive function, learning and memory, language, perceptual motor, or social cognition) based on: 1. Concern oi the individual, a knowledgeable informant, or the clinician that there has 2. A modest impairment in cognitive performance, preferably documented by stan- dardized neuropsychological testing or, in its absence, another quantified clinical assessment.

1	B. The cognitive deficits do not interfere with capacity for independence in everyday activities (i.e., complex instrumental activities of daily living such as paying bills or managing medications are preserved, but greater effort, compensatory strategies, or accommodation may be required). C. The cognitive deficits do not occur exclusively in the context of a delirium. D. The cognitive deficits are not better explained by another mental disorder (e.g., major depressive disorder, schizophrenia). Specify whether due to: Alzheimer's disease (pp. 611—614) Frontotemporal lobar degeneration (pp. 614—618) Lewy body disease (pp. 618—621) Vascular disease (pp. 621—624) Traumatic brain injury (pp. 624—627) Substance/medication use (pp. 627—632) HIV infection (pp. 632—634) Prion disease (pp. 634—636) Parkinson’s disease (pp. 636—638) Huntington’s disease (pp. 638-641) Another medical condition (pp. 641—642) Multiple etiologies (pp. 642—643) Unspecified (p. 643)

1	Prion disease (pp. 634—636) Parkinson’s disease (pp. 636—638) Huntington’s disease (pp. 638-641) Another medical condition (pp. 641—642) Multiple etiologies (pp. 642—643) Unspecified (p. 643) Coding note: For mild neurocognitive disorder due to any of the medical etiologies listed above, code 331.83 (631.84). Do not use additional codes for the presumed etiological medical conditions. For substance/medication-induced mild neurocognitive disorder, code based on type of substance; see “Substance/Medication-Induced Major or Mild Neurocog- nitive Disorder." For unspecified mild neurocognitive disorder, code 799.59 (R41.9). Specify: Without behavioral disturbance: If the cognitive disturbance is not accompanied by any clinically significant behavioral disturbance.

1	Specify: Without behavioral disturbance: If the cognitive disturbance is not accompanied by any clinically significant behavioral disturbance. With behavioral disturbance (specify disturbance): If the cognitive disturbance is ac- companied by a clinically significant behavioral disturbance (e.g., psychotic symptoms, mood disturbance, agitation, apathy, or other behavioral symptoms).

1	Major and mild neurocognitive disorders (NCDs) are primarily subtyped according to the known or presumed etiological/ pathological entity or entities underlying the cognitive de- cline. These subtypes are distinguished on the basis of a combination of time course, charac- teristic domains affected, and associated symptoms. For certain etiological subtypes, the diagnosis depends substantially on the presence of a potentially causative entity, such as Par- kinson’s or Huntington’s disease, or a traumatic brain injury or stroke in the appropriate time period. For other etiological subtypes (generally the neurodegenerative diseases like Alzhei- mer’s disease, frontotemporal lobar degeneration, and Lewy body disease), the diagnosis is based primarily on the cognitive, behavioral, and functional symptoms. Typically, the differ- clearer at the level of major NCD than at the level of mild NCD, but sometimes characteristic symptoms and associated features are present at the mild level as well.

1	NCDs are frequently managed by clinicians in multiple disciplines. For many sub- types, multidisciplinary international expert groups have developed specialized consen- sus criteria based on clinicopathological correlation with underlying brain pathology. The subtype criteria here have been harmonized with those expert criteria. Evidence for distinct behavioral features in NCDs has been recognized, particularly in the areas of psychotic symptoms and depression. Psychotic features are common in many

1	Evidence for distinct behavioral features in NCDs has been recognized, particularly in the areas of psychotic symptoms and depression. Psychotic features are common in many NCDs, particularly at the mild-to-moderate stage of major NCDs due to Alzheimer’s dis- ease, Lewy body disease, and frontotemporal lobar degeneration. Paranoia and other delusions are common features, and often a persecutory theme may be a prominent aspect of delusional ideation. In contrast to psychotic disorders with onset in earlier life (e.g., schizophrenia), disorganized speech and disorganized behavior are not characteristic of psychosis in NCDs. Hallucinations may occur in any modality, although visual hallucina- tions are more common in NCDs than in depressive, bipolar, or psychotic disorders.

1	Mood disturbances, including depression, anxiety, and elation, may occur. Depression is common early in the course (including at the mild NCD level) of NCD due to Alzhei- mer’s disease and Parkinson’s disease, while elation may occur more commonly in fron- totemporal lobar degeneration. When a full affective syndrome meeting diagnostic criteria for a depressive or bipolar disorder is present, that diagnosis should be coded as well. Mood symptoms are increasingly recognized to be a significant feature in the earliest stages of mild NCDs such that clinical recognition and intervention may be important.

1	Mood symptoms are increasingly recognized to be a significant feature in the earliest stages of mild NCDs such that clinical recognition and intervention may be important. Agitation is common in a wide variety of NCDs, particularly in major NCD of moder- ate to severe severity, and often occurs in the setting of confusion or frustration. It may arise as combative behaviors, particularly in the context of resisting caregiving duties such as bathing and dressing. Agitation is characterized as disruptive motor or vocal activity and tends to occur with advanced stages of cognitive impairment across all of the NCDs. Individuals with NCD can present with a wide variety of behavioral symptoms that are the focus of treatment. Sleep disturbance is a common symptom that can create a need for clinical attention and may include symptoms of insomnia, hypersomnia, and circadian rhythm disturbances.

1	Apathy is common in mild and mild major NCD. It is observed particularly in NCD due to Alzheimer’s disease and may be a prominent feature of NCD due to frontotemporal lobar degeneration. Apathy is typically characterized by diminished motivation and re- duced goal—directed behavior accompanied by decreased emotional responsiveness. Symptoms of apathy may manifest early in the course of NCDs when a loss of motivation to pursue daily activities or hobbies may be observed. Other important behavioral symptoms include wandering, disinhibition, hyperpha- gia, and hoarding. Some of these symptoms are characteristic of specific disorders, as dis- cussed in the relevant sections. When more than one behavioral disturbance is observed, each type should be noted in writing with the specifier ”with behavioral symptoms.” Major and mild NCDs exist on a spectrum of cognitive and functional impairment. Major

1	NCD corresponds to the condition referred to in DSM-IV as dementia, retained as an alter- native in this volume. The core feature of NCDs is acquired cognitive decline in one or part of the individual, a knowledgeable informant, or the clinician, and 2) performance on an objective assessment that falls below the expected level or that has been observed to de- cline over time. Both a concern and objective evidence are required because they are com- plementary. When there is an exclusive focus on objective testing, a disorder may go tually represents a substantial decline in abilities, or an illness may be incorrectly diag- from their own baseline or is a result of extraneous factors like test conditions or a passing illness. Alternatively, excessive focus on subjective symptoms may fail to diagnose illness in individuals with poor insight, or whose informants deny or fail to notice their symptoms, or it may be overly sensitive in the so-called worried well.

1	taneously. Rather, it may need to be elicited by careful questioning about specific symp- toms that commonly occur in individuals with cognitive deficits (see Table 1 in the introduction to this chapter). For example, memory concerns include difficulty remember— ing a short grocery list or keeping track of the plot of a television program; executive con- cerns include difficulty resuming a task when interrupted, organizing tax records, or planning a holiday meal. At the mild NCD level, the individual is likely to describe these tasks as being more difficult or as requiring extra time or effort or compensatory strategies.

1	At the major NCD level, such tasks may only be completed with assistance or may be abandoned altogether. At the mild NCD level, individuals and their families may not no- tice such symptoms or may view them as normal, particularly in the elderly; thus, careful history taking is of paramount importance. The difficulties must represent changes rather than lifelong patterns: the individual or informant may clarify this issue, or the clinician can infer change from prior experience with the patient or from occupational or other clues. It is also critical to determine that the difficulties are related to cognitive loss rather than to motor or sensory limitations. Neuropsychological testing, with performance compared with norms appropriate to the patient’s age, educational attainment, and cultural background, is part of the standard evaluation of NCDs and is particularly critical in the evaluation of mild NCD. For major

1	NCD, performance is typically 2 or more standard deviations below appropriate norms (3rd percentile or below). For mild NCD, performance typically lies in the 1—2 standard de- viation range (between the 3rd and 16th percentiles). However, neuropsychological test- ing is not available in all settings, and neuropsychological thresholds are sensitive to the specific test(s) and norms employed, as well as to test conditions, sensory limitations, and intercurrent illness. A variety of brief office-based or ”bedside” assessments, as described in Table 1, can also supply objective data in settings where such testing is unavailable or infeasible. In any case, as with cognitive concerns, objective performance must be inter- preted in light of the individual’s prior performance. Optimally, this information would be available from a prior administration of the same test, but often it must be inferred based on appropriate norms, along with the individual’s educational history, occupation, and

1	information would be available from a prior administration of the same test, but often it must be inferred based on appropriate norms, along with the individual’s educational history, occupation, and other factors. Norms are more challenging to interpret in individuals with very high or very low levels of education and in individuals being tested outside their own language or cultural background.

1	Criterion B relates to the individual's level of independence in everyday functioning. Individuals with major NCD will have impairment of sufficient severity so as to interfere with independence, such that others will have to take over tasks that the individuals were previously able to complete on their own. Individuals with mild NCD will have preserved independence, although there may be subtle interference with function or a report that tasks require more effort or take more time than previously. The distinction between major and mild NCD is inherently arbitrary, and the disorders exist along a continuum. Precise thresholds are therefore difficult to determine. Careful history taking, observation, and integration with other findings are required, and the im- plications of diagnosis should be considered when an individual’s clinical manifestations lie at a boundary.

1	Typically the associated features that support a diagnosis of major or mild NCD will be specific to the etiological subtype (e.g., neuroleptic sensitivity and visual hallucinations in NCD due to Lewy body disease). Diagnostic features specific to each of the subtypes are found in the relevant sections.

1	NCD due to Lewy body disease). Diagnostic features specific to each of the subtypes are found in the relevant sections. The prevalence of NCD varies widely by age and by etiological subtype. Overall preva- lence estimates are generally only available for older populations. Among individuals older than 60 years, prevalence increases steeply with age, so prevalence estimates are more accurate for narrow age bands than for broad categories such as "over 65" (where the mean age can vary greatly with the life expectancy of the given population). For those eti- ological subtypes occurring across the lifespan, prevalence estimates for NCD are likely to be available, if at all, only as the fraction of individuals who develop NCD among those with the relevant condition (e.g., traumatic brain injury, HIV infection). Overall prevalence estimates for dementia (which is largely congruent with major

1	Overall prevalence estimates for dementia (which is largely congruent with major NCD) are approximately 1%—2% at age 65 years and as high as 30% by age 85 years. The prevalence of mild NCD is very sensitive to the definition of the disorder, particularly in community settings, where evaluations are less detailed. In addition, in contrast with clin- ical settings, where cognitive concern must be high to seek and locate care, there may be a less clear decline from baseline functioning. Estimates of the prevalence of mild cognitive impairment (which is substantially congruent with mild NCD) among older individuals are fairly variable, ranging from 2% to 10% at age 65 and 5% to 25% by age 85.

1	The course of NCD varies across etiological subtypes, and this variation can be useful in differential diagnosis. Some subtypes (e.g., those related to traumatic brain injury or stroke) typically begin at a specific time and (at least after initial symptoms related to in- ﬂammation or swelling subside) remain static. Others may fluctuate over time (although if this occurs, the possibility of delirium superimposed on NCD should be considered). NCDs due to neurodegenerative diseases like Alzheimer's disease or frontotemporal lobar degeneration typically are marked by insidious onset and gradual progression, and the pattern of onset of cognitive deficits and associated features helps to distinguish among them.

1	NCDs with ohset in childhood and adolescence may have broad repercussions for so- cial and intellectual development, and in this setting intellectual disability (intellectual nosed to capture the full diagnostic picture and ensure the provision of a broad range of services. In older individuals, NCDs often occur in the setting of medical illnesses, frailty, and sensory loss, which complicate the clinical picture for diagnosis and treatment.

1	When cognitive loss occurs in youth to midlife, individuals and families are likely to seek care. NCDs are typically easiest to identify at younger ages, although in some settings malingering or other factitious disorders may be a concern. Very late in life, cognitive symptoms may not cause concern or may go unnoticed. In late life, mild NCD must also be distinguished from the more modest deficits associated with “normal aging,” although a substantial fraction of what has been ascribed to normal aging likely represents prodromal phases of various NCDs. In addition, it becomes harder to recognize mild NCD with age because of the increasing prevalence of medical illness and sensory deficits. It becomes harder to differentiate among subtypes with age because there are multiple potential sources of neurocognitive decline.

1	subtypes. Some subtypes are distributed throughout the lifespan, whereas others occur exclusively or primarily in late life. Even within the NCDs of aging, the relative prevalence varies with age: Alzheimer's disease is uncommon before age 60 years, and the prevalence increases steeply thereafter, while the overall less common frontotemporal lobar degener- ation has earlier onset and represents a progressively smaller fraction of NCDs with age. Genetic and physiological. The strongest risk factor for major and mild NCDs is age, primarily because age increases the risk of neurodegenerative and cerebrovascular dis- ease. Female gender is associated with higher prevalence of dementia overall, and especially Alzheimer’s disease, but this difference is largely, if not wholly, attributable to greater lon- gevity in females.

1	Alzheimer’s disease, but this difference is largely, if not wholly, attributable to greater lon- gevity in females. Individuals’ and families’ level of awareness and concern about neurocognitive symp- toms may vary across ethnic and occupational groups. Neurocognitive symptoms are more likely to be noticed, particularly at the mild level, in individuals who engage in com- plex occupational, domestic, or recreational activities. In addition, norms for neuropsy- chological testing tend to be available only for broad populations, and thus they may not be easily applicable to individuals with less than high school education or those being evaluated outside their primary language or culture.

1	Like age, culture, and occupation, gender issues may affect the level of concern and aware- ness of cognitive symptoms. In addition, for late-life NCDs, females are likely to be older, to have more medical comorbidity, and to live alone, which can complicate evaluation and treatment. In addition, there are gender differences in the frequency of some of the etio- logical subtypes.

1	In addition to a careful history, neuropsychological assessments are the key measures for diagnosis of NCDs, particularly at the mild level, where functional changes are minimal and symptoms more subtle. Ideally, individuals will be referred for formal neuropsycho— logical testing, which will provide a quantitative assessment of all relevant domains and thus help with diagnosis; provide guidance to the family on areas where the individual may require more support; and serve as a benchmark for further decline or response to therapies. When such testing is unavailable or not feasible, the brief assessments in Table 1 can provide insight into each domain. More global brief mental status tests may be helpful but may be insensitive, particularly to modest changes in a single domain or in those with high premorbid abilities, and may be overly sensitive in those with low premorbid abilities.

1	In distinguishing among etiological subtypes, additional diagnostic markers may come into play, particularly neuroimaging studies such as magnetic resonance imaging scans and positron emission tomography scans. In addition, specific markers may be in- volved in the assessment of specific subtypes and may become more important as addi- tional research findings accumulate over time, as discussed in the relevant sections. By definition, major and mild NCDs affect functioning, given the central role of cognition in human life. Thus, the criteria for the disorders, and the threshold for differentiating mild from major NCD, are based in part on functional assessment. Within major NCD there is a broad range of functional impairment, as implemented in the severity specifiers. In addition, the specific functions that are compromised can help identify the cognitive domains affected, particularly when neuropsychological testing is not available or is difficult to interpret.

1	Normal cognition. The differential diagnosis between normal cognition and mild NCD, as between mild and major NCD, is challenging because the boundaries are inherently ar- bitrary. Careful history taking and objective assessment are critical to these distinctions. A longitudinal evaluation using quantified assessments may be key in detecting mild NCD. Delirium. Both mild and major NCD may be difficult to distinguish from a persistent de- lirium, which can co-occur. Careful assessment of attention and arousal will help to make the distinction.

1	Delirium. Both mild and major NCD may be difficult to distinguish from a persistent de- lirium, which can co-occur. Careful assessment of attention and arousal will help to make the distinction. Major depressive disorder. The distinction between mild NCD and major depressive disorder, which may co-occur with NCD, can also be challenging. Specific patterns of cog- nitive deficits may be helpful. For example, consistent memory and executive function deficits are typical of Alzheimer’s disease, whereas nonspecific or more variable perfor- mance is seen in major depression. Alternatively, treatment of the depressive disorder with repeated observation over time may be required to make the diagnosis.

1	Specific learning disorder and other neurodevelopmental disorders. A careful clari- fication of the individual’s baseline status will help distinguish an NCD from a specific learning disorder or other neurodevelopmental disorders. Additional issues may enter the differential for specific etiological subtypes, as described in the relevant sections. NCDs are common in older individuals and thus often co-occur with a wide variety of age- related diseases that may complicate diagnosis or treatment. Most notable of these is delirium, for which NCD increases the risk. In older individuals, a delirium during hos- pitalization is, in many cases, the first time that an NCD is noticed, although a careful his- tory will often reveal evidence of earlier decline. Mixed NCDs are also common in older individuals, as many etiological entities increase in prevalence with age. In younger indi- viduals, NCD often co-occurs with neurodevelopmental disorders; for example, a head in-

1	Major or Mild Neurocognitive Disorder Due to Alzheimer's Disease 611 jury in a preschool child may also lead to significant developmental and learning issues. Additional comorbidity of NCD is often related to the etiological subtype, as discussed in the relevant sections. Due to Alzheimer’s Disease A. The criteria are met for major or mild neurocognitive disorder. B. There is insidious onset and gradual progression of impairment in one or more cognitive domains (for major neurocognitive disorder, at least two domains must be impaired). C. Criteria are met for either probable or possible Alzheimer’s disease as follows: For major neurocognitive disorder: Probable Alzheimer’s disease is diagnosed if either of the following is present; oth- erwise, possible Alzheimer’s disease should be diagnosed. 1. Evidence of a causative Alzheimer’s disease genetic mutation from family history or genetic testing.

1	1. Evidence of a causative Alzheimer’s disease genetic mutation from family history or genetic testing. 2. All three of the following are present: a. Clear evidence of decline in memory and learning and at least one other cogni- tive domain (based on detailed history or serial neuropsychological testing). b. Steadily progressive, gradual decline in cognition, without extended plateaus. c. No evidence of mixed etiology (i.e., absence of other neurodegenerative or cerebrovascular disease, or another neurological, mental, or systemic disease or condition likely contributing to cognitive decline). For mild neurocognitive disorder: Probable Alzheimer's disease is diagnosed if there is evidence of a causative Alz- heimer’s disease genetic mutation from either genetic testing or family history.

1	For mild neurocognitive disorder: Probable Alzheimer's disease is diagnosed if there is evidence of a causative Alz- heimer’s disease genetic mutation from either genetic testing or family history. Possible Alzheimer's disease is diagnosed if there is no evidence of a causative Alz- heimer’s disease genetic mutation from either genetic testing or family history, and all three of the following are present: 1. Clear evidence of decline in memory and learning. 2. Steadily progressive, gradual decline in cognition, without extended plateaus. 3. No evidence of mixed etiology (i.e., absence of other neurodegenerative or cere- brovascular disease, or another neurological or systemic disease or condition likely contributing to cognitive decline). D. The disturbance is not better explained by cerebrovascular disease. another neurode- generative disease, the effects of a substance, or another mental, neurological, or sys- temic disorder.

1	D. The disturbance is not better explained by cerebrovascular disease. another neurode- generative disease, the effects of a substance, or another mental, neurological, or sys- temic disorder. Coding note: For probable major neurocognitive disorder due to Alzheimer‘s disease, with behavioral disturbance, code first 331.0 (G303) Alzheimer‘s disease, followed by 294.11 (F02.81) major neurocognitive disorder due to Alzheimer’s disease. For probable neurocognitive disorder due to Alzheimer's disease, without behavioral disturbance, code first 331.0 (G309) Alzheimer's disease, followed by 294.10 (F02.80) major neurocognitive disorder due to Alzheimer's disease. without behavioral disturbance.

1	For possible major neurocognitive disorder due to Alzheimer’s disease, code 331.9 (G31.9) possible major neurocognitive disorder due to Alzheimer‘s disease. (Note: Do not use the additional code for Alzheimer’s disease. Behavioral disturbance cannot be coded but should still be indicated in writing.) For mild neurocognitive disorder due to Alzheimer’s disease, code 331.83 (G31.84). (Note: Do not use the additional code for Alzheimer’s disease. Behavioral disturbance cannot be coded but should still be indicated in writing.) Beyond the neurocognitive disorder (NCD) syndrome (Criterion A), the core features of ma- jor or mild NCD due to Alzheimer’s disease include an insidious onset and gradual pro- gression of cognitive and behavioral symptoms (Criterion B). The typical presentation is amnestic (i.e., with impairment in memory and learning). Unusual nonamnestic presen— tations, particularly visuospatial and logopenic aphasic variants, also exist. At the mild

1	NCD phase, Alzheimer’s disease manifests typically with impairment in memory and leam- ing, sometimes accompanied by deficits in executive function. At the major NCD phase, visuoconstructional/perceptual motor ability and language will also be impaired, partic- ularly when the NCD is moderate to severe. Social cognition tends to be preserved until late in the course of the disease.

1	A level of diagnostic certainty must be specified denoting Alzheimer’s disease as the ”probable” or ”possible” etiology (Criterion C). Probable Alzheimer’s disease is diagnosed in both major and mild NCD if there is evidence of a causative Alzheimer’s disease gene, ei- ther from genetic testing or from an autosomal dominant family history coupled with au- topsy confirmation or a genetic test in an affected family member. For major NCD, a typical clinical picture, without extended plateaus or evidence of mixed etiology, can also be diagnosed as due to probable Alzheimer’s disease. For mild NCD, given the lesser de- gree of certainty that the deficits will progress, these features are only sufficient for a possible Alzheimer’s etiology. If the etiology appears mixed, mild NCD due to multiple eti- ologies should be diagnosed. In any case, for both mild and major NCD due to Alzhei- mer’s disease, the clinical features must not suggest another primary etiology for the NCD (Criterion D).

1	In specialty clinical settings, approximately 80% of individuals with major NCD due to also frequent at the mild NCD stage of impairment. These symptoms are as or more dis- tressing than cognitive manifestations and are frequently the reason that health care is sought. At the mild NCD stage or the mildest level of major NCD, depression and / or ap- athy are often seen. With moderately severe major NCD, psychotic features, irritability, agitation, combativeness, and wandering are common. Late in the illness, gait distur- bance, dysphagia, incontinence, myoclonus, and seizures are observed. The prevalence of overall dementia (major NCD) rises steeply with age. In high—income countries, it ranges from 5% to 10% in the seventh decade to at least 25% thereafter. US.

1	The prevalence of overall dementia (major NCD) rises steeply with age. In high—income countries, it ranges from 5% to 10% in the seventh decade to at least 25% thereafter. US. census data estimates suggest that approximately 7% of individuals diagnosed with Alz- heimer’s disease are between ages 65 and 74 years, 53% are between ages 75 and 84 years, and 40% are 85 years and older. The percentage of dementias attributable to Alzheimer’s disease ranges from about 60% to over 90%, depending on the setting and diagnostic cri- teria. Mild NCD due to Alzheimer’s disease is likely to represent a substantial fraction of mild cognitive impairment (MCI) as well. Major or mild NCD due to Alzheimer’s disease progresses gradually, sometimes with brief plateaus, through severe dementia to death. The mean duration of survival after di-

1	Major or mild NCD due to Alzheimer’s disease progresses gradually, sometimes with brief plateaus, through severe dementia to death. The mean duration of survival after di- Major or Mild Neurocognitive Disorder Due to Alzheimer’s Disease 613 agnosis is approximately 10 years, reﬂecting the advanced age of the majority of individ- uals rather than the course of the disease; some individuals can live with the disease for as long as 20 years. Iate-stage individuals are eventually mute and bedbound. Death most commonly results from aspiration in those who survive through the full course. In mild NCD due to Alzheimer’s disease, impairments increase over time, and functional status gradually declines until symptoms reach the threshold for the diagnosis of major NCD.

1	NCD due to Alzheimer’s disease, impairments increase over time, and functional status gradually declines until symptoms reach the threshold for the diagnosis of major NCD. The onset of symptoms is usually in the eighth and ninth decades; early-onset forms seen in the fifth and sixth decades are often related to known causative mutations. Symp- toms and pathology do not differ markedly at different onset ages. However, younger in- dividuals are more likely to survive the full course of the disease, while older individuals are more likely to have numerous medical comorbidities that affect the course and man- agement of the illness. Diagnostic complexity is higher in older adults because of the in- creased likelihood of comorbid medical illness and mixed pathology. Environmental. Traumatic brain injury increases risk for major or mild NCD due to Alz- heimer’s disease.

1	Environmental. Traumatic brain injury increases risk for major or mild NCD due to Alz- heimer’s disease. Genetic and physiological. Age is the strongest risk factor for Alzheimer’s disease. The at onset, particularly in homozygous individuals. There are also extremely rare causative Alzheimer’s disease genes. Individuals with Down’s syndrome (trisomy 21) develop Alz- heimer’s disease if they survive to midlife. Multiple vascular risk factors influence risk for direct effects on Alzheimer pathology. Detection of an NCD may be more difficult in cultural and socioeconomic settings where memory loss is considered normal in old age, where older adults face fewer cognitive de- mands in everyday life, or where very low educational levels pose greater challenges to objective cognitive assessment.

1	Cortical atrophy, amyloid-predominant neuritic plaques, and tau-predominant neurofibril— lary tangles are hallmarks of the pathological diagnosis of Alzheimer’s disease and may be continued via postmortem histopathological examination. For early-onset cases with auto- somal dominant inheritance, a mutation in one of the known causative Alzheimer’s disease genes—amyloid precursor protein (APP), presenilin 1 (PSENl), or presenilin 2 (PSEN2)— may be involved, and genetic testing for such mutations is commercially available, at least for PSEN1. Apolipoprotein E4 cannot serve as a diagnostic marker because it is only a risk factor and neither necessary nor sufficient for disease occurrence.

1	Since amyloid beta-42 deposition in the brain occurs early in the pathophysiological cascade, amyloid-based diagnostic tests such as amyloid imaging on brain positron emis- sion tomography (PET) scans and reduced levels of amyloid beta—42 in the cerebrospinal ﬂuid (CSF) may have diagnostic value. Signs of neuronal injury, such as hippocampal and temporoparietal cortical atrophy on a magnetic resonance image scan, temporoparietal hypometabolism on a ﬂuorodeoxyglucose PET scan, and evidence for elevated total tau and phospho—tau levels in CSF, provide evidence of neuronal damage but are less specific for Alzheimer’s disease. At present, these biomarkers are not fully validated, and many are available only in tertiary care settings. However, some of them, along with novel bio- markers, will likely move into wider clinical practice in the coming years. Functional Consequences of Major or Mild Neurocognitive Disorder Due to Alzheimer’s Disease

1	Functional Consequences of Major or Mild Neurocognitive Disorder Due to Alzheimer’s Disease The prominence of memory loss can cause significant difficulties relatively early in the course. Social cognition (and thus social functioning) and procedural memory (e.g., danc- ing, playing musical instruments) may be relatively preserved for extended periods. Other neurocognitive disorders. Major and mild NCDs due to other neurodegenera- tive processes (e.g., Lewy body disease, frontotemporal lobar degeneration) share the in- features of their own. In major or mild vascular NCD, there is typically history of stroke temporally related to the onset of cognitive impairment, and infarcts or white matter hy- perintensities are judged sufficient to account for the clinical picture. However, particu- larly when there is no clear history of stepwise decline, major or mild vascular NCD can share many clinical features with Alzheimer’s disease.

1	Other concurrent, active neurological or systemic illness. Other neurological or sys- temic illness should be considered if there is an appropriate temporal relationship and severity to account for the clinical picture. At the mild NCD level, it may be difficult to dis- tinguish an Alzheimer’s disease etiology from that of another medical condition (e.g., thy- roid disorders, vitamin B12 deficiency). Major depressive disorder. Particularly at the mild NCD level, the differential diagnosis also includes major depression. The presence of depression may be associated with re- duced daily functioning and poor concentration that may resemble an NCD, but improve- ment with treatment of depression may be useful in making the distinction. Most individuals with Alzheimer’s disease are elderly and have multiple medical conditions that can complicate diagnosis and inﬂuence the clinical course. Major or mild NCD due to

1	Most individuals with Alzheimer’s disease are elderly and have multiple medical conditions that can complicate diagnosis and inﬂuence the clinical course. Major or mild NCD due to Alzheimer’s disease commonly co-occurs with cerebrovascular disease, which contributes to the clinical picture. When a comorbid condition contributes to the NCD in an individual with Alzheimer’s disease, then NCD due to multiple etiologies should be diagnosed. A. The criteria are met for major or mild neurocognitive disorder. B. The disturbance has insidious onset and gradual progression. C. Either (1) or (2): 1. Behavioral variant: a. Three or more of the following behavioral symptoms: i. Behavioraldisinhibition. ii. Apathy or inertia. iii. Loss of sympathy or empathy. v. Perseverative, stereotyped or compulsive/ritualistic behavior. v. Hyperorality and dietary changes. b. Prominent decline in social cognition and/or executive abilities.

1	v. Perseverative, stereotyped or compulsive/ritualistic behavior. v. Hyperorality and dietary changes. b. Prominent decline in social cognition and/or executive abilities. 2. Language variant: a. Promihent decline in language ability. in the form of speech production, word finding, object naming, grammar, or word comprehension. D. Relative sparing of learning and memory and perceptual-motor function. E. The disturbance is not better explained by cerebrovascular disease, another neurode- generative disease, the effects of a substance, or another mental, neurological. or sys- temic disorder. Probable frontotemporal neurocognitive disorder is diagnosed if either of the following is present; otherwise, possible frontotemporal neurocognitive disorder should be di- agnosed: 1 . Evidence of a causative frontotemporal neurocognitive disorder genetic mutation. from either family history or genetic testing.

1	2. Evidence of disproportionate frontal and/or temporal lobe involvement from neuroim- aging. Possible frontotemporal neurocognitive disorder is diagnosed it there is no evidence of a genetic mutation, and neuroimaging has not been performed. Coding note: For probable major neurocognitive disorder due to frontotemporal lobar de- generation, with behavioral disturbance, code first 331.19 (631.09) frontotemporal dis- ease, followed by 294.11 (F02.81) probable major neurocognitive disorder due to frontotemporal lobar degeneration, with behavioral disturbance. For probable major neu- rocognitive disorder due to frontotemporal lobar degeneration, without behavioral distur- bance, code first 331.19 (631.09) frontotemporal disease, followed by 294.10 (F02.80) probable major neurocognitive disorder due to frontotemporal lobar degeneration, without behavioral disturbance.

1	For possible major neurocognitive disorder due to frontotemporal lobar degeneration, code 331.9 (631.9) possible major neurocognitive disorder due to frontotemporal lobar degen— eration. (Note: Do not use the additional code for frontotemporal disease. Behavioral distur- bance cannot be coded but should still be indicated in writing.) For mild neurocognitive disorder due to frontotemporal lobar degeneration, code 331.83 (631.84). (Note: Do not use the additional code for frontotemporal disease. Behavioral disturbance cannot be coded but should still be indicated in writing.)

1	Major or mild frontotemporal neurocognitive disorder (NCD) comprises a number of syn- dromic variants characterized by the progressive development of behavioral and personality change and/ or language impairment. The behavioral variant and three language variants (se- mantic, agrammatic/nonﬂuent, and logopenic) exhibit distinct patterns of brain atrophy and some distinctive neuropathology. The criteria must be met for either the behavioral or the lan- guage variant to make the diagnosis, but many individuals present with features of both.

1	Individuals with behavioral—variant major or mild frontotemporal NCD present with varying degrees of apathy or disinhibition. They may lose interest in socialization, self- care, and personal responsibilities, or display socially inappropriate behaviors. Insight is usually impaired, and this often delays medical consultation. The first referral is often to a psychiatrist. Individuals may develop changes in social style, and in religious and political beliefs, with repetitive movements, hoarding, changes in eating behavior, and hyperoral- ity. In later stages, loss of sphincter control may occur. Cognitive decline is less prominent, and formal testing may show relatively few deficits in the early stages. Common neuro- cognitive symptoms are lack of planning and organization, distractibility, and poor judg- ment. Deficits in executive function, such as poor performance on tests of mental ﬂexibility, abstract reasoning, and response inhibition, are present, but learning and mem- ory are

1	and poor judg- ment. Deficits in executive function, such as poor performance on tests of mental ﬂexibility, abstract reasoning, and response inhibition, are present, but learning and mem- ory are relatively spared, and perceptual motor abilities are almost always preserved in the early stages.

1	Individuals with language-variant major or mild frontotemporal NCD present with pri- mary progressive aphasia with gradual onset, with three subtypes commonly described: semantic variant, agrammatic/nonﬂuent variant, and logopenic variant, and each variant has distinctive features and corresponding neuropathology. ”Probable” is distinguished from ”possible” frontotemporal NCD by the presence of causative genetic factors (e.g., mutations in the gene coding for microtubule—associated pro- tein tau) or by the presence of distinctive atrophy or reduced activity in frontotemporal re- gions on structural or functional imaging. Extrapyramidal features may be prominent in some cases, with an overlap with syn- dromes such as progressive supranuclear palsy and corticobasal degeneration. Features of motor neuron disease may be present in some cases (e.g., muscle atrophy, weakness). A subset of individuals develop visual hallucinations.

1	Major or mild frontotemporal NCD is a common cause of early-onset NCD in individuals younger than 65 years. Population prevalence estimates are in the range of 2—10 per 100,000. Approximately 20%—25% of cases of frontotemporal NCD occur in individuals older than 65 years. Frontotemporal NCD accounts for about 5% of all cases of dementia in unselected autopsy series. Prevalence estimates of behavioral variant and semantic lan- guage variant are higher among males, and prevalence estimates of nonﬂuent language variant are higher among females. Individuals with major or mild frontotemporal NCD commonly present in the sixth de- cade of life, although the age at onset varies from the third to the ninth decades. The dis- ease is gradually progressive, with median survival being 6-11 years after symptom onset and 3—4 years after diagnosis. Survival is shorter and decline is faster in major or mild fron- totemporal NCD than in typical Alzheimer’s disease.

1	Genetic and physiological. Approximately 40% of individuals with major or mild fron- totemporal NCD have a family history of early-onset NCD, and approximately 10% show an autosomal dominant inheritance pattern. A number of genetic factors have been identified, such as mutations in the gene encoding the microtubule associated protein tau (MAPT), the granulin gene (GRN), and the C9ORF72 gene. A number of families with causative muta- tions have been identified (see the section ”Diagnostic Markers” for this disorder), but many individuals with known familial transmission do not have a known mutation. The presence of motor neuron disease is associated with a more rapid deterioration.

1	distinct patterns of atrophy. In behavioral-variant major or mild frontotemporal NCD, both frontal lobes (especially the medial frontal lobes) and the anterior temporal lobes are atrophic. In semantic language—variant major or mild frontotemporal NCD, the middle, inferior, and anterior temporal lobes are atrophic bilaterally but asymmetrically, with the left side usually being more affected. Nonﬂuent language—variant major or mild fronto- temporal NCD is associated with predominantly left posterior frontal-insular atrophy.

1	The logopenic variant of major or mild frontotemporal NCD is associated with predomi- nantly left posterior perisylvian or parietal atrophy. Functional imaging demonstrates hy- poperfusion and/ or cortical hypometabolism in the corresponding brain regions, which may be present in the early stages in the absence of structural abnormality. Emerging bio— markers for Alzheimer’s disease (e.g., cerebrospinal ﬂuid amyloid-beta and tau levels, and amyloid imaging) may help in the differential diagnosis, but the distinction from Alzhei- mer’s disease can remain difficult (the logopenic variant is in fact often a manifestation of Alzheimer’s disease). In familial cases of frontotemporal NCD, the identification of genetic mutations may help confirm the diagnosis. Mutations associated with frontotemporal NCD include the genes encoding microtubule-associated protein tau (MAPT) and granulin (GRN),

1	C9ORF72, transactive response DNA-binding protein of 43 kDa (TDP—43, or TARDBP), valosin-containing protein (VCP), chromatin modifying protein ZB (CHMPZB), and fused in sarcoma protein (FUS). Functional Consequences of Major or Mild

1	Functional Consequences of Major or Mild Because of the relative early age at onset of the disorder, the disorder oftens affects work- place and family life. Because of the involvement of language and / or behavior, function is often more severely impaired relatively early in the course. For individuals with the be- havioral variant, prior to diagnostic clarification there may be significant family disrup- tion, legal involvement, and problems in the workplace because of socially inappropriate behaviors. The functional impairment due to behavioral change and language dysfunc- tion, which can include hyperorality, impulsive wandering, and other dishinhibited be- haviors, may far exceed that due to the cognitive disturbance and may lead to nursing home placement or institutionalization. These behaviors can be severely disruptive, even in structured care settings, particularly when the individuals are otherwise healthy, non- frail, and free of other medical comorbidities.

1	Other neurocognitive disorders. Other neurodegenerative diseases may be distinguished from major or mild frontotemporal NCD by their characteristic features. In major or mild NCD due to Alzheimer’s disease, decline in learning and memory is an early feature. However, 10%—30% of patients presenting with a syndrome suggestive of major or mild frontotemporal NCD are found at autopsy to have Alzheimer’s disease pathology. This oc- curs more frequently in individuals who present with progressive dysexecutive syn- dromes in the absence of behavioral changes or movement disorder or in those with the logopenic variant.

1	In major or mild NCD with Lewy bodies, core and suggestive features of Lewy bodies must be present. In major or mild NCD due to Parkinson’s disease, spontaneous parkin- sonism emerges well before the cognitive decline. In major or mild vascular NCD, depend- ing on affected brain regions, there may also be loss of executive ability and behavioral changes such as apathy, and this disorder should be considered in the differential diagno- sis. However, history of a cerebrovascular event is temporally related to the onset of cog- nitive impairment in major or mild vascular NCD, and neuroimaging reveals infarctions or white matter lesions sufficient to account for the clinical picture.

1	Other neurological conditions. Major or mild frontotemporal NCD overlaps with pro- gressive supranuclear palsy, corticobasal degeneration, and motor neuron disease clinically as well as pathologically. Progressive supranuclear palsy is characterized by supranuclear gaze palsies and axial—predominant parkinsonism. Pseudobulbar signs may be present, and retropulsion is often prominent. Neurocognitive assessment shows psy- chomotor slowing, poor working memory, and executive dysfunction. Corticobasal degen- eration presents with asymmetric rigidity, limb apraxia, postural instability, myoclonus, alien limb phenomenon, and cortical sensory loss. Many individuals with behavioral-variant major or mild frontotemporal NCD show features of motor neuron disease, which tend to be mixed upper and predominantly lower motor neuron disease.

1	Other mental disorders and medical conditions. Behavioral—variant major or mild fron— totemporal NCD may be mistaken for a primary mental disorder, such as major depression, bipolar disorders, or schizophrenia, and individuals with this variant often present initially to psychiatry. Over time, the development of progressive neurocognitive difficulties will help to make the distinction. A careful medical evaluation will help to exclude treatable causes of NCDs, such as metabolic disturbances, nutritional deficiencies, and infections. A. The criteria are met for major or mild neurocognitive disorder. B. The disorder has an insidious onset and gradual progression. C. The disorder meets a combination of core diagnostic features and suggestive diagnos- tic features for either probable or possible neurocognitive disorder with Lewy bodies.

1	C. The disorder meets a combination of core diagnostic features and suggestive diagnos- tic features for either probable or possible neurocognitive disorder with Lewy bodies. For probable major or mild neurocognitive disorder with Lewy bodies, the indi- vidual has two core features, or one suggestive feature with one or more core features. For possible major or mild neurocognitive disorder with Lewy bodies, the individ- ual has only one core feature, or one or more suggestive features. 1. Core diagnostic features: a. Fluctuating cognition with pronounced variations in attention and alertness. b. Recurrent visual hallucinations that are well formed and detailed. c. Spontaneous features of parkinsonism, with onset subsequent to the develop- ment of cognitive decline. 2. Suggestive diagnostic features: a. Meets criteria for rapid eye movement sleep behavior disorder. b. Severe neuroleptic sensitivity.

1	2. Suggestive diagnostic features: a. Meets criteria for rapid eye movement sleep behavior disorder. b. Severe neuroleptic sensitivity. D. The disturbance is not better explained by cerebrovascular disease, another neurode- generative disease, the effects of a substance, or another mental, neurological. or sys- temic disorder. Coding note: For probable major neurocognitive disorder with Lewy bodies, with behav- ioral disturbance, code first 331.82 (631.83) Lewy body disease, followed by 294.11 (F02.81) probable major neurocognitive disorder with Lewy bodies, with behavioral distur- bance. For probable major neurocognitive disorder with Lewy bodies, without behavioral disturbance, code first 331.82 (631.83) Lewy body disease, followed by 294.10 (F02.80) probable major neurocognitive disorder with Lewy bodies, without behavioral disturbance.

1	For possible major neurocognitive disorder with Lewy bodies, code 331.9(631.9) possible major neurocognitive disorder with Lewy bodies. (Note: Do not use the additional code for Lewy body disease. Behavioral disturbance cannot be coded but should still be indicated in writing.) For mild neurocognitive disorder with Lewy bodies, code 331.83 (631.84). (Note: Do not use the additional code for Lewy body disease. Behavioral disturbance cannot be coded but should still be indicated in writing.) Major or mild neurocognitive disorder with Lewy bodies (NCDLB), in the case of major neurocognitive disorder (NCD), corresponds to the condition known as dementia with Lewy bodies (DLB). The disorder includes not only progressive cognitive impairment tation); as well as hallucinations in other sensory modalities, depression, and delusions.

1	The symptoms ﬂuctuate in a pattern that can resemble a delirium, but no adequate under- lying cause can be found. The variable presentation of NCDLB symptoms reduces the like- lihood of all symptoms being observed in a brief clinic visit and necessitates a thorough assessment of caregiver observations. The use of assessment scales specifically designed to assess ﬂuctuation may aid in diagnosis. Another core feature is spontaneous parkinson- ism, which must begin after the onset of cognitive decline; by convention, major cognitive deficits are observed at least 1 year before the motor symptoms. The parkinsonism must also be distinguished from neuroleptic-induced extrapyramidal signs. Accurate diagnosis is essential to safe treatment planning, as up to 50% of individuals with NCDLB have se— vere sensitivity to neuroleptic drugs, and these medications should be used with extreme caution in managing the psychotic manifestations.

1	The diagnosis of mild NCDLB is appropriate for individuals who present with the core or suggestive features at a stage when cognitive or functional impairments are not of suf- ficient severity to fulfill criteria for major NCD. However, as for all mild NCDs, there will often be insufficient evidence to justify any single etiology, and use of the unspecified di- agnosis is most appropriate. Individuals with NCDLB frequently experience repeated falls and syncope and transient episodes of unexplained loss of consciousness. Autonomic dysfunction, such as ortho- static hypotension and urinary incontinence, may be observed. Auditory and other nonvisual hallucinations are common, as are systematized delusions, delusional misiden- tification, and depression.

1	The few population—based prevalence estimates for NCDLB available range from 0.1% to 5% of the general elderly population, and from 1.7% to 30.5% of all dementia cases. In brain bank (autopsy) series, the pathological lesions known as Lewy bodies are present in 20%—35% of cases of dementia. The male-to-female ratio is approximately 1.521.

1	NCDLB is a gradually progressive disorder with insidious onset. However, there is often a prodromal history of confusional episodes (delirium) of acute onset, often precipitated by illness or surgery. The distinction between NCDLB, in which Lewy bodies are primar- ily cortical in location, and major or mild NCD due to Parkinson’s disease, in which the pa- thology is primarily in the basal ganglia, is the order in which the cognitive and motor symptoms emerge. In NCDLB, the cognitive decline is manifested early in the course of ill- ness, at least a year before the onset of motor symptoms (see the section ”Differential Di- agnosis” for this disorder). Disease course may be characterized by occasional plateaus but eventually progresses through severe dementia to death. Average duration of survival is 5—7 years in clinical series. Onset of symptoms is typically observed from the sixth through the ninth decades of life, with most cases having their onset when affected indi- viduals are in

1	is 5—7 years in clinical series. Onset of symptoms is typically observed from the sixth through the ninth decades of life, with most cases having their onset when affected indi- viduals are in their mid-7OS.

1	Genetic and physiological. Familial aggregation may occur, and several risk genes have been identified, but in most cases of NCDLB, there is no family history. The underlying neurodegenerative disease is primarily a synucleinopathy due to alpha- synuclein misfolding and aggregation. Cognitive testing beyond the use of a brief screen- ing instrument may be necessary to define deficits clearly. Assessment scales developed to measure ﬂuctuation can be useful. The associated condition REM sleep behavior disorder may be diagnosed through a formal sleep study or identified by questioning the patient or informant about relevant symptoms. Neuroleptic sensitivity (challenge) is not recom— mended as a diagnostic marker but raises suspicion of NCDLB if it occurs. A diagnosti- cally suggestive feature is low striatal dopamine transporter uptake on single photon emission computed tomography (SPECT) or positron emission tomography (PET) scan.

1	Other clinically useful markers potentially include relative preservation of medial tempo- uptake on SPECT/PET perfusion scan with reduced occipital activity; abnormal (low up- slow-wave activity on the electroencephalogram with temporal lobe transient waves. Functional Consequences of Major or Miid Individuals with NCDLB are more functionally impaired than would be expected for their cognitive deficits when contrasted to individuals with other neurodegenerative diseases, such as Alzheimer’s disease. This is largely a result of motor and autonomic impairments, which cause problems with toileting, transferring, and eating. Sleep disorders and prom- inent psychiatric symptoms may also add to functional difficulties. Consequently, the qual- ity of life of individuals with NCDLB is often significantly worse than that of individuals with Alzheimer’s disease.

1	Major or mild neurocognitive disorder due to Parkinson’s disease. A key differenti- ating feature in clinical diagnosis is the temporal sequence in which the parkinsonism and the NCD appear. For NCD due to Parkinson’s disease, the individual must develop cog- nitive decline in the context of established Parkinson’s disease; by convention, the decline should not reach the stage of major NCD until at least 1 year after Parkinson’s is diagnosed. If less than a year has passed since the onset of motor symptoms, the diagnosis is NCDLB. This distinction is clearer at the major NCD level than at the mild NCD level. The timing and sequence of parkinsonism and mild NCD may be more difficult to de- termine because the onset and clinical presentation can be ambiguous, and unspecified mild NCD should be diagnosed if the other core and suggestive features are absent.

1	Lewy body pathology frequently coexists with Alzheimer’s disease and cerebrovascular disease pathology, particularly among the oldest age groups. In Alzheimer’s disease, there is concomitant synuclein pathology in 60% of cases (if amygdala-restricted cases are in- cluded). In general, there is a higher rate of Lewy body pathology in individuals with de- mentia than in older individuals without dementia. A. The criteria are met for major or mild neurocognitive disorder. B. The clinical features are consistent with a vascular etiology, as suggested by either of the following: 1. Onset of the cognitive deficits is temporally related to one or more cerebrovascular events. 2. Evidence for decline is prominent in complex attention (including processing speed) and frontal-executive function. C. There is evidence of the presence of cerebrovascular disease from history, physical examination, andlor neuroimaging considered sufficient to account for the neurocog- nitive deficits.

1	C. There is evidence of the presence of cerebrovascular disease from history, physical examination, andlor neuroimaging considered sufficient to account for the neurocog- nitive deficits. D. The symptoms are not better explained by another brain disease or systemic disorder. Probable vascular neurocognitive disorder is diagnosed if one of the following is pres- ent; otherwise possible vascular neurocognitive disorder should be diagnosed: 1. Clinical criteria are supported by neuroimaging evidence of significant parenchymal in- jury attributed to cerebrovascular disease (neuroimaging-supported). 2. The neurocognitive syndrome is temporally related to one or more documented cere- brovascular events. 3. Both clinical and genetic (e.g., cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) evidence of cerebrovascular disease is present.

1	3. Both clinical and genetic (e.g., cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) evidence of cerebrovascular disease is present. Possible vascular neurocognitive disorder is diagnosed if the clinical criteria are met but neuroimaging is not available and the temporal relationship of the neurocognitive syn- drome with one or more cerebrovascular events is not established. Coding note: For probable major vascular neurocognitive disorder, with behavioral dis— turbance, code 290.40 (F01.51). For probable major vascular neurocognitive disorder, without behavioral disturbance, code 290.40 (F01.50). For possible major vascular neuro- cognitive disorder, with or without behavioral disturbance, code 331.9 (631.9). An addi- tional medical code for the cerebrovascular disease is not needed.

1	For mild vascular neurocognitive disorder, code 331.83 (631.84). (Note: Do not use an additional code for the vascular disease. Behavioral disturbance cannot be coded but should still be indicated in writing.) The diagnosis of major or mild vascular neurocognitive disorder (NCD) requires the es- tablishment of an NCD (Criterion A) and the determination that cerebrovascular disease is the dominant if not exclusive pathology that accounts for the cognitive deficits (Criteria B and C). Vascular etiology may range from large vessel stroke to microvascular disease; the presentation is therefore very heterogeneous, stemming from the types of vascular lesions and their extent and location. The lesions may be focal, multifocal, or diffuse and occur in various combinations.

1	Many individuals with major or mild vascular NCD present with multiple infarctions, with an acute stepwise or ﬂuctuating decline in cognition, and intervening periods of stability and even some improvement. Others may have gradual onset with slow pro- gression, a rapid development of deficits followed by relative stability, or another complex presentation. Major or mild vascular NCD with a gradual onset and slow progression is generally due to small vessel disease leading to lesions in the white matter, basal ganglia, and/ or thalamus. The gradual progression in these cases is often punctuated by acute events that leave subtle neurological deficits. The cognitive deficits in these cases can be at- tributed to disruption of cortical-subcortical circuits, and complex attention, particularly speed of information processing, and executive ability are likely to be affected.

1	Assessing for the presence of sufficient cerebrovascular disease relies on history, phys- ical examination, and neuroimaging (Criterion C). Etiological certainty requires the dem- onstration of abnormalities on neuroimaging. The lack of neuroimaging can result in ter lesions. However, if the neurocognitive impairment is temporally associated with one or more well-documented strokes, a probable diagnosis can be made in the absence of neu- roimaging. Clinical evidence of cerebrovascular disease includes documented history of stroke, with cognitive decline temporally associated with the event, or physical signs con- sistent with stroke (e.g., hemiparesis; pseudobulbar syndrome, visual field defect). Neuro- cerebrovascular disease comprises one or more of the following: one or more large vessel infarcts or hemorrhages, a strategically placed single infarct or hemorrhage (e.g., in angu- lar gyms, thalamus, basal forebrain), two or more lacunar infarcts outside the brain stem, or extensive

1	infarcts or hemorrhages, a strategically placed single infarct or hemorrhage (e.g., in angu- lar gyms, thalamus, basal forebrain), two or more lacunar infarcts outside the brain stem, or extensive and conﬂuent white matter lesions. The latter is often termed small vessel dis- ease or subcortical ischemic changes on clinical neuroimaging evaluations.

1	For mild vascular NCD, history of a single stroke or extensive white matter disease is gen- erally sufficient. For major vascular NCD, two or more strokes, a strategically placed stroke, or a combination of white matter disease and one or more lacunes is generally necessary. The disorder must not be better explained by another disorder. For example, promi- nent memory deficit early in the course might suggest Alzheimer's disease, early and prominent parkinsonian features would suggest Parkinson's disease, and a close associa- tion between onset and depression would suggest depression. A neurological assessment often reveals history of stroke and/ or transient ischemic epi- sodes, and signs indicative of brain infarctions. Also commonly associated are personality and mood changes, abulia, depression, and emotional lability. The development of late- function is a common presentation among older adults with progressive small vessel isch- emic disease (”vascular depression”).

1	Major or mild vascular NCD is the second most common cause of NCD after Alzheimer’s disease. In the United States, population prevalence estimates for vascular dementia range from 0.2% in the 65—70 years age group to 16% in individuals 80 years and older. Within 3 months following stroke, 20%—30% of individuals are diagnosed with dementia. In neu- ropathology series, the prevalence of vascular dementia increases from 13% at age 70 years to 44.6% at age 90 years or older, in comparison with Alzheimer’s disease (23.6%—51%) and combined vascular dementia and Alzheimer’s disease (2%—46.4%). Higher prevalence has been reported in African Americans compared with Caucasians, and in East Asian countries (e.g., Japan, China). Prevalence is higher in males than in females.

1	Major or mild vascular NCD can occur at any age, although the prevalence increases ex- ponentially after age 65 years. In older individuals, additional pathologies may partly ac- count for the neurocognitive deficits. The course may vary from acute onset with partial improvement to stepwise decline to progressive decline, with ﬂuctuations and plateaus of varying durations. Pure subcortical major or mild vascular NCD can have a slowly pro- gressive course that simulates major or mild NCD due to Alzheimer’s disease. Environmental. The neurocognitive outcomes of vascular brain injury are inﬂuenced by neuroplasticity factors such as education, physical exercise, and mental activity.

1	Environmental. The neurocognitive outcomes of vascular brain injury are inﬂuenced by neuroplasticity factors such as education, physical exercise, and mental activity. Genetic and physiological. The major risk factors for major or mild vascular NCD are the same as those for cerebrovascular disease, including hypertension, diabetes, smoking, obesity, high cholesterol levels, high homocysteine levels, other risk factors for atherosclerosis and ar- teriolosclerosis, atrial fibrillation, and other conditions increasing the risk of cerebral emboli. Cerebral amyloid angiopathy is an important risk factor in which amyloid deposits occur within arterial vessels. Another key risk factor is the hereditary condition cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL. Structural neuroimaging, using MRI or CT, has an important role in the diagnostic pro- cess. There are no other established biomarkers of major or mild vascular NCD.

1	Structural neuroimaging, using MRI or CT, has an important role in the diagnostic pro- cess. There are no other established biomarkers of major or mild vascular NCD. Major or mild vascular NCD is commonly associated with physical deficits that cause ad- ditional disability. Other neurocognitive disorders. Since incidental brain infarctions and white matter le- sions are common in older individuals, it is important to consider other possible etiologies when an NCD is present. A history of memory deficit early in the course, and progressive worsening of memory, language, executive function, and perceptual-motor abilities in the absence of corresponding focal lesions on brain imaging, are suggestive of Alzheimer’s disease as the primary diagnosis. Potential biomarkers currently being validated for Alz- heimer’s disease, such as cerebrospinal ﬂuid levels of beta-amyloid and phosphorylated tau, and amyloid imaging, may prove to be helpful in the differential diagnosis. NCD with

1	Lewy bodies is distinguished from major or mild vascular NCD by its core features of ﬂuc- tuating cognition, visual hallucinations, and spontaneous parkinsonism. While deficits in executive function and language occur in major or mild vascular NCD, the insidious onset and gradual progression of behavioral features or language impairment are characteristic of frontotemporal NCD and are not typical of vascular etiology. Other medical conditions. A diagnosis of major or mild vascular NCD is not made if other diseases (e.g., brain tumor, multiple sclerosis, encephalitis, toxic or metabolic disor- ders) are present and are of sufficient severity to account for the cognitive impairment.

1	Other mental disorders. A diagnosis of major or mild vascular NCD is inappropriate if the symptoms can be entirely attributed to delirium, although delirium may sometimes be superimposed on a preexisting major or mild vascular NCD, in which case both diagnoses can be made. If the criteria for major depressive disorder are met and the cognitive impair- ment is temporally related to the likely onset of the depression, major or mild vascular NCD should not be diagnosed. However, if the NCD preceded the development of the de- pression, or the severity of the cognitive impairment is out of proportion to the severity of the depression, both should be diagnosed. Major or mild NCD due to Alzheimer’s disease commonly co—occurs with major or mild vascular NCD, in which case both diagnoses should be made. Major or mild vascular NCD and depression frequently co-occur. Due to Traumatic Brain Injury A. The criteria are met for major or mild neurocognitive disorder.

1	Due to Traumatic Brain Injury A. The criteria are met for major or mild neurocognitive disorder. B. There is evidence of a traumatic brain injury—that is, an impact to the head or other mechanisms of rapid movement or displacement of the brain within the skull, with one or more of the following: 1. Loss of consciousness. 2. Posttraumatic amnesia. 3. Disorientation and confusion. 4. Neurological signs (e.g., neuroimaging demonstrating injury; a new onset of sei- zures; a marked worsening of a preexisting seizure disorder; visual field cuts; an- osmia; hemiparesis). C. The neurocognitive disorder presents immediately after the occurrence of the trau- matic brain injury or immediately after recovery of consciousness and persists past the acute post-injury period.

1	Coding note: For major neurocognitive disorder due to traumatic brain injury, with behavioral disturbance: For |CD-9-CM, first code 907.0 late effect of intracranial injury without skull frac- ture, followed by 294.11 major neurocognitive disorder due to traumatic brain injury, with be- havioral disturbance. For |CD-10-CM, first code $06.2X98 diffuse traumatic brain injury with loss of consciousness of unspecified duration, sequela; followed by F02.81 major neurocog- nitive disorder due to traumatic brain injury, with behavioral disturbance.

1	For major neurocognitive disorder due to traumatic brain injury, without behavioral distur- bance: For |CD-9-CM, first code 907.0 late effect of intracranial injury without skull fracture, followed by 294.10 major neurocognitive disorder due to traumatic brain injury, without be- havioral disturbance. For |CD-10-CM, first code $06.2X9S diffuse traumatic brain injury with loss of consciousness of unspecified duration, sequela; followed by. F02.80 major neurocog- nitive disorder due to traumatic brain injury, without behavioral disturbance. For mild neurocognitive disorder due to traumatic brain injury, code 331.83 (631.84). (Note: Do not use the additional code for traumatic brain injury. Behavioral disturbance cannot be coded but should still be indicated in writing.) Major or Mild Neurocognitive Disorder Due to Traumatic Brain Injury 625

1	Major or Mild Neurocognitive Disorder Due to Traumatic Brain Injury 625 Rate the severity of the neurocognitive disorder (NCD), not the underlying traumatic brain injury (see the section ”Development and Course” for this disorder).

1	Major or mild NCD due to traumatic brain injury (TBI) is caused by an impact to the head, or other mechanisms of rapid movement or displacement of the brain within the skull, as can happen with blast injuries. Traumatic brain injury is defined as brain trauma with spe- cific characteristics that include at least one of the following: loss of consciousness, post— traumatic amnesia, disorientation and confusion, or, in more severe cases, neurological signs (e.g., positive neuroimaging, a new onset of seizures or a marked worsening of a pre- existing seizure disorder, visual field cuts, anosmia, hemiparesis) (Criterion B). To be at- tributable to TBI, the NCD must present either immediately after the brain injury occurs or immediately after the individual recovers consciousness after the injury and persist past the acute post-injury period (Criterion C).

1	The cognitive presentation is variable. Difficulties in the domains of complex attention, executive ability, learning, and memory are common as well as slowing in speed of infor- mation processing and disturbances in social cognition. In more severe TBI in which there is brain contusion, intracranial hemorrhage, or penetrating injury, there may be additional neurocognitive deficits, such as aphasia, neglect, and constructional dyspraxia.

1	Major or mild NCD due to TBI may be accompanied by disturbances in emotional function (e.g., irritability, easy frustration, tension and anxiety, affective lability); personality changes (e.g., disinhibition, apathy, suspiciousness, aggression); physical disturbances (e.g., headache, fatigue, sleep disorders, vertigo or dizziness, tinnitus or hyperacusis, pho- tosensitivity, anosmia, reduced tolerance to psychotropic medications); and, particularly in more severe TBI, neurological symptoms and signs (e.g., seizures, hemiparesis, visual disturbances, cranial nerve deficits) and evidence of orthopedic injuries. In the United States, 1.7 million TBIs occur annually, resulting in 1.4 million emergency de- partment visits, 275,000 hospitalizations, and 52,000 deaths. About 2% of the population lives with TBI-associated disability. Males account for 59% of TBIs in the United States.

1	The most common etiologies of TBI in the United States are falls, vehicular accidents, and being struck on the head. Collisions and blows to the head that occur in the course of con- tact sports are increasingly recognized as sources of mild TBI, with a concern that repeated mild TBI may have cumulatively persisting sequelae. The severity of a TBI is rated at the time of injury/initial assessment as mild, moderate, or severe according to the thresholds in Table 2. The severity rating of the TBI itself does not necessarily correspond to the severity of the resulting NCD. The course of recovery from TBI is variable, depending not only on the specifics of the injury but also on cofactors, such as age, prior history of brain damage, or substance abuse, that may favor or impede recovery. Loss of consciousness <30 min 30 minutes—24 hours >24 hours Neurobehavioral symptoms tend to be most severe in the immediate aftermath of the

1	Loss of consciousness <30 min 30 minutes—24 hours >24 hours Neurobehavioral symptoms tend to be most severe in the immediate aftermath of the TBI. Except in the case of severe TBI, the typical course is that of complete or substantial improvement in associated neurocognitive, neurological, and psychiatric symptoms and signs. Neurocognitive symptoms associated with mild TBI tend to resolve within days to weeks after the injury with complete resolution typical by 3 months. Other symptoms that may potentially co-occur with the neurological symptoms (e.g., depression, irritability, fatigue, headache, photosensitivity, sleep disturbance) also tend to resolve in the weeks following mild TBI. Substantial subsequent deterioration in these areas should trigger con- sideration of additional diagnoses. However, repeated mild TBI may be associated with persisting neurocognitive disturbance.

1	With moderate and severe TBI, in addition to persistence of neurocognitive deficits, there may be associated neurophysiological, emotional, and behavioral complications. These include seizures (particularly in the first year), photosensitivity, hyperacusis, irritabil- ity, aggression, depression, sleep disturbance, fatigue, apathy, inability to resume occu- pational and social functioning at pre-injury level, and deterioration in interpersonal relationships. Moderate and severe TBI have been associated with increased risk of depres- sion, aggression, and possibly neurodegenerative diseases such as Alzheimer’s disease.

1	The features of persisting major or mild NCD due to TBI will vary by age, specifics of the injury, and cofactors. Persisting TBI-related impairment in an infant or child may be re- ﬂected in delays in reaching developmental milestones (e.g., language acquisition), worse academic performance, and possibly impaired social development. Among older teenag- ers and adults, persisting symptoms may include various neurocognitive deficits, irrita- bility, hypersensitivity to light and sound, easy fatigability, and mood changes, including depression, anxiety, hostility, or apathy. In older individuals with depleted cognitive re- serve, mild TBI is more likely to result in incomplete recoveries.

1	Risk factors for traumatic brain injury. Traumatic brain injury rates vary by age, with the highest prevalence among individuals younger than 4 years, older adolescents, and in- dividuals older than 65 years. Falls are the most common cause of TBI, with motor vehicle accidents being second. Sports concussions are frequent causes of TBI in older children, teenagers, and young adults. Risk factors for neurocognitive disorder after traumatic brain injury. Repeated con- cussions can lead to persistent NCD and neuropathological evidence of traumatic enceph- alopathy. Co-occurring intoxication with a substance may increase the severity of a TBI from a motor vehicle accident, but whether intoxication at the time of injury worsens neu- rocognitive outcome is unknown.

1	Course modifiers. Mild TBI generally resolves within a few weeks to months, although res- olution may be delayed or incomplete in the context of repeated TBI. Worse outcome from moderate to severe TBI is associated with older age (older than 40 years) and initial clinical pa- rameters, such as low Glasgow Coma Scale score; worse motor function; pupillary nonreac- tivity; and computed tomography (CT) evidence of brain injury (e.g., petechial hemorrhages, subarachnoid hemorrhage, midline shift, obliteration of third ventricle). Beyond neuropsychological testing, CT scanning may reveal petechial hemorrhages, subarachnoid hemorrhage, or evidence of contusion. Magnetic resonance image scanning may also reveal hyperintensities suggestive of microhemorrhages. Functionai Consequences of Major or Miid Neurocognitive Disorder Due to Traumatic Brain Injury

1	Functionai Consequences of Major or Miid Neurocognitive Disorder Due to Traumatic Brain Injury With mild NCD due to TBI, individuals may report reduced cognitive efficiency, difficulty concentrating, and lessened ability to perform usual activities. With major NCD due to TBI, an individual may have difficulty in independent living and self—care. Prominent neuromotor features, such as severe incoordination, ataxia, and motor slowing, may be present in major NCD due to TBI and may add to functional difficulties. Individuals with TBI histories report more depressive symptoms, and these can amplify cognitive complaints and worsen func- tional outcome. Additionally, loss of emotional control, including aggressive or inappropriate affect and apathy, may be present after more severe TBI with greater neurocognitive impair- ment. These features may compound difficulties with independent living and self-care.

1	In some instances, severity of neurocognitive symptoms may appear to be inconsistent with the severity of the TBI. After previously undetected neurological complications (e.g., chronic hematoma) are excluded, the possibility of diagnoses such as somatic symptom disorder or factitious disorder need to be considered. Posttraumatic stress disorder (PTSD) can co-occur with the NCD and have overlapping symptoms (e.g., difficulty concentrat- ing, depressed mood, aggressive behavioral disinhibition). Among individuals with substance use disorders, the neurocognitive effects of the sub- stance contribute to or compound the TBI-associated neurocognitive change. Some symp- toms associated with TBI may overlap with symptoms found in cases of PTSD, and the two disorders may co-occur, especially in military populations. A. The criteria are met for major or mild neurocognitive disorder.

1	A. The criteria are met for major or mild neurocognitive disorder. B. The neurocognitive impairments do not occur exclusively during the course of a delir- ium and persist beyond the usual duration of intoxication and acute withdrawal. C. The involved substance or medication and duration and extent of use are capable of producing the neurocognitive impairment. D. The temporal course of the neurocognitive deficits is consistent with the timing of sub- stance or medication use and abstinence (e.g., the deficits remain stable or improve after a period of abstinence). E. The neurocognitive disorder is not attributable to another medical condition or is not better explained by another mental disorder.

1	Coding note: The lCD-9-CM and lCD-10-CM codes for the [specific substance/medica- tion]-induced neurocognitive disorders are indicated in the table below. Note that the ICD- 10-CM code depends on whether or not there is a comorbid substance use disorder present for the same class of substance. If a mild substance use disorder is comorbid with the sub- stance-induced neurocognitive disorder, the 4th position character is "1," and the clinician should record “mild [substance] use disorder” before the substance-induced neurocognitive disorder (e.g., “mild inhalant use disorder with inhalant-induced major neurocognitive disor- der"). If a moderate or severe substance use disorder is comorbid with the substance- induced neurocognitive disorder. the 4th position character is “2" and the clinician should record “moderate [substance] use disordel” or “severe [substance] use disorder,” depending on the severity of the comorbid substance use disorder. If there is no comorbid substance use

1	should record “moderate [substance] use disordel” or “severe [substance] use disorder,” depending on the severity of the comorbid substance use disorder. If there is no comorbid substance use disorder, then the 4th position character is “9" and the clinician should record only the substance-induced neurocognitive disorder. For some classes of substances (i.e., alcohol; sedatives, hypnotics, anxiolytics), it is not permissible to code a comorbid mild substance use disorder with a substance-induced neurocognitive disorder; only a comorbid moderate or severe substance use disorder, or no substance use disorder, can be diagnosed. Behav- ioral disturbance cannot be coded but should still be indicated in writing.

1	With use disorder, disorder, moderateor Without use Alcohol (major neurocognitive 291.2 NA F1027 F1097 disorder), nonamnestic- Alcohol (major neurocognitive 291.1 NA F1026 F1096 disorder), amnestic- Alcohol (mild neurocognitive 291.89 NA F10.288 F10.988 Inhalant (major neurocognitive 292.82 F1817 F1827 F1897 Inhalant (mild neurocognitive 292.89 F18.1BB F18.288 F18.988 Sedative, hypnotic, or anxiolytic 292.82 NA F1327 F1397 Sedative, hypnotic, or anxiolytic 292.89 NA F13.288 F13.988 Other (or unknown) substance 292.82 F1917 F1927 F1997 Other (or unknown) substance 292.89 F19.188 F19.288 F19.988 Specify if: PersistentaNeurocognitive impairment continues to be significant after an extended period of abstinence.

1	Other (or unknown) substance 292.89 F19.188 F19.288 F19.988 Specify if: PersistentaNeurocognitive impairment continues to be significant after an extended period of abstinence. |CD-9-CM. The name of the substance/medication-induced neurocognitive disorder be- gins with the specific substance/medication (e.g., alcohol) that is presumed to be causing the neurocognitive symptoms. The diagnostic code is selected from the table included in the criteria set, which is based on the drug class. For substances that do not fit into any of the classes, the code for “other substance” should be used; and in cases in which a substance is judged to be an etiological factor but the specific class of substance is unknown, the cat— egory ”unknown substance” should be used.

1	The name of the disorder (i.e., [specific substance]-induced major neurocognitive dis- order or [specific substance]-induced mild neurocognitive disorder) is followed by the type in the case of alcohol (i.e., nonamnestic-confabulatory type, amnestic-confabulatory type), followed by specification of duration (i.e., persistent). Unlike the recording procedures for ICD-IO-CM, which combine the substance/medication-induced disorder and sub- stance use disorder into a single code, for ICD-9-CM a separate diagnostic code is given for the substance use disorder. For example, in the case of persistent amnestic-confabulatory symptoms in a man with a severe alcohol use disorder, the diagnosis is 291.1 alcohol- induced major neurocognitive disorder, amnestic-confabulatory type, persistent. An addi- tional diagnosis of 303.90 severe alcohol use disorder is also given. If the substance/medi- (e.g., after a sporadic heavy use of inhalants), no accompanying substance use disorder is noted (e.g.,

1	tional diagnosis of 303.90 severe alcohol use disorder is also given. If the substance/medi- (e.g., after a sporadic heavy use of inhalants), no accompanying substance use disorder is noted (e.g., 292.82 inhalant-induced mild neurocognitive disorder).

1	lCD-10-CM. The name of the substance/medication-induced neurocognitive disorder begins with the specific substance (e.g., alcohol) that is presumed to be causing the neuro- cognitive symptoms. The diagnostic code is selected from the table included in the criteria set, which is based on the drug class and presence or absence of a comorbid substance use disorder. For substances that do not fit into any of the classes, the code for ”other sub- stance” should be used; and in cases in which a substance is judged to be an etiological fac- tor but the specific class of substance is unknown, the category "unknown substance" should be used.

1	When recording the name of the disorder, the comorbid substance use disorder (if any) is listed first, followed by the word "with,” followed by the name of the disorder (i.e., [specific neurocognitive disorder), followed by the type in the case of alcohol (i.e., nonamnestic-con- fabulatory type, amnestic-confabulatory type), followed by specification of duration (i.e., persistent). For example, in the case of persistent amnestic-confabulatory symptoms in a man with a severe alcohol use disorder, the diagnosis is F1026 severe alcohol use disorder with alcohol-induced major neurocognitive disorder, amnestic-confabulatory type, persis- tent. A separate diagnosis of the comorbid severe alcohol use disorder is not given. If the order (e.g., after a sporadic heavy use of inhalants), no accompanying substance use disor- der is noted (e.g., F18.988 inhalant-induced mild neurocognitive disorder).

1	Substance/ medication-induced major or mild NCD is characterized by neurocognitive impairments that persist beyond the usual duration of intoxication and acute withdrawal (Criterion B). Initially, these manifestations can reﬂect slow recovery of brain functions from a period of prolonged substance use, and improvements in neurocognitive as well as brain imaging indicators may be seen over many months. If the disorder continues for an extended period, persistent should be specified. The given substance and its use must be known to be capable of causing the observed impairments (Criterion C). While nonspecific decrements in a range of cognitive abilities can occur with nearly any substance of abuse and a variety of medications, some patterns occur more frequently with selected drug classes. For example, NCD due to sedative, hypnotic, or anxiolytic drugs (e.g., benzodiaz- epines, barbiturates) may show greater disturbances in memory than in other cognitive functions. NCD induced by

1	For example, NCD due to sedative, hypnotic, or anxiolytic drugs (e.g., benzodiaz- epines, barbiturates) may show greater disturbances in memory than in other cognitive functions. NCD induced by alcohol frequently manifests with a combination of impair- ments in executive-function and memory and learning domains. The temporal course of the substance-induced NCD must be consistent with that of use of the given substance (Criterion D). In alcohol-induced amnestic confabulatory (Korsakoff’s) NCD, the features include prominent amnesia (severe difficulty learning new information with rapid forget- ting) and a tendency to confabulate. These manifestations may co—occur with signs of thi- amine encephalopathy (Wernicke’s encephalopathy) with associated features such as nystagmus and ataxia. Ophthalmoplegia of Wernicke’s encephalopathy is typically charac- terized by a lateral gaze paralysis.

1	In addition to or independent of the more common neurocognitive symptoms related to methamphetamine use (e.g., difficulties with learning and memory; executive func- tion), methamphetamine use can also be associated with evidence of vascular injury (e.g., focal weakness, unilateral incoordination, asymmetrical reﬂexes). The most common neu- rocognitive profile approximates that seen in vascular NCD.

1	Intermediate-duration NCD induced by drugs with central nervous system depressant effects may manifest with added symptoms of increased irritability, anxiety, sleep disturbance, and dysphoria. Intermediate-duration NCD induced by stimulant drugs may manifest with re- bound depression, hypersomnia, and apathy. In severe forms of substance/medication— induced major NCD (e.g., associated with long-term alcohol use), there may be prominent neuromotor features, such as incoordination, ataxia, and motor slowing. There may also be loss of emotional control, including aggressive or inappropriate affect, or apathy. The prevalence of these conditions is not known. Prevalence figures for substance abuse are available, and substance/medication—induced major or mild NCDs are more likely in those who are older, have longer use, and have other risk factors such as nutritional deficits.

1	For alcohol abuse, the rate of mild NCD of intermediate duration is approximately 30%— 40% in the first 2 months of abstinence. Mild NCD may persist, particularly in those who do not achieve stable abstinence until after age 50 years. Major NCD is rare and may result from concomitant nutritional deficits, as in alcohol—induced amnestic confabulatory NCD.

1	For individuals quitting cocaine, methamphetamine, opioids, phencyclidine, and sed- ative, hypnotics, or anxiolytics, substance / medication-induced mild NCD of intermediate duration may occur in one-third or more, and there is some evidence that these substances may also be associated with persistent mild NCD. Major NCD associated with these sub- stances is rare, if it occurs at all. In the case of methamphetamine, cerebrovascular disease can also occur, resulting in diffuse or focal brain injury that can be of mild or major neu- rocognitive levels. Solvent exposure has been linked to both major and mild NCD of both intermediate and persistent duration. The presence of NCD induced by cannabis and various hallucinogens is controversial. With cannabis, intoxication is accompanied by various neurocognitive disturbances, but these tend to clear with abstinence.

1	Substance use disorders tend to commence during adolescence and peak in the 205 and 30s. Although longer history of severe substance use disorder is associated with greater likelihood of NCD, the relationships are not straightforward, with substantial and even complete recovery of neurocognitive functions being common among individuals who achieve stable abstinence prior to age 50 years. Substance/medication—induced major or mild NCD is most likely to become persistent in individuals who continue abuse of sub- stances past age 50 years, presumably because of a combination of lessened neural plas- ticity and beginnings of other age-related brain changes. Earlier commencement of abuse, particularly of alcohol, may lead to defects in later neural development (e.g., later stages of maturation of frontal circuitries), which may have effects on social cognition as well as other neurocognitive abilities. For alcohol-induced NCD, there may be an additive effect of aging and alcohol-induced

1	of frontal circuitries), which may have effects on social cognition as well as other neurocognitive abilities. For alcohol-induced NCD, there may be an additive effect of aging and alcohol-induced brain injury.

1	Risk factors for substance/medication—induced NCDs include older age, longer use, and persistent use past age 50 years. In addition, for alcohol—induced NCD, long-term nutri— tional deficiencies, liver disease, vascular risk factors, and cardiovascular and cerebrovas- cular disease may contribute to risk.

1	Magnetic resonance imaging (MRI) of individuals with chronic alcohol abuse frequently reveals cortical thinning, white matter loss, and enlargement of sulci and ventricles. While neuroimaging abnormalities are more common in those with NCDs, it is possible to ob- serve NCDs without neuroimaging abnormalities, and vice versa. Specialized techniques (e.g., diffusion tensor imaging) may reveal damage to specific white matter tracts. Mag- netic resonance spectroscopy may reveal reduction in N-acetylaspartate, and increase in markers of inﬂammation (e.g., myoinositol) or white matter injury (e.g., choline). Many of cessful abstinence. In individuals with methamphetamine use disorder, MRI may also re— veal hyperintensities suggestive of microhemorrhages or larger areas of infarction. Functional Consequences of SubstancelMedication-

1	Functional Consequences of SubstancelMedication- The functional consequences of substance/medication—induced mild NCD are sometimes in many other NCDs. In addition, at both major and mild levels, substance/medication- induced NCDs may have associated motor syndromes that increase the level of functional impairment. Individuals with substance use disorders, substance intoxication, and substance withdrawal are at increased risk for other conditions that may independently, or through a compounding effect, result in neurocognitive disturbance. These include history of traumatic brain injury and infections that can accompany substance use disorder (e.g., HIV, hepatitis C virus, syph- ilis). Therefore, presence of substance/medication—induced major or mild NCD should be differentiated from NCDs arising outside the context of substance use, intoxication, and with- drawal, including these accompanying conditions (e.g., traumatic brain injury).

1	Substance use disorders, substance intoxication, and substance withdrawal are highly co- morbid with other mental disorders. Comorbid posttraumatic stress disorder, psychotic disorders, depressive and bipolar disorders, and neurodevelopmental disorders can con- tribute to neurocognitive impairment in substance users. Traumatic brain injury occurs more frequently with substance use, complicating efforts to determine the etiology of NCD in such cases. Severe, long-term alcohol use disorder can be associated with major organ system disease, including cerebrovascular disease and cirrhosis. Amphetamine-induced NCD may be accompanied by major or mild vascular NCD, also secondary to amphet- amine use. Due to HIV Infection A. The criteria are met for major or mild neurocognitive disorder. B. There is documented infection with human immunodeficiency virus (HIV).

1	Due to HIV Infection A. The criteria are met for major or mild neurocognitive disorder. B. There is documented infection with human immunodeficiency virus (HIV). C. The neurocognitive disorder is not better explained by non-HIV conditions, including secondary brain diseases such as progressive multifocal leukoencephalopathy or cryptococcal meningitis. D. The neurocognitive disorder is not attributable to another medical condition and is not better explained by a mental disorder. Coding note: For major neurocognitive disorder due to HIV infection, with behavioral dis- turbance, code first 042 (320) HIV infection, followed by 294.11 (F02.81) major neurocog- nitive disorder due to HIV infection, with behavioral disturbance. For major neurocognitive disorder due to HIV infection, without behavioral disturbance, code first 042 (320) HIV in- fection, followed by 294.10 (F02.80) major neurocognitive disorder due to HIV infection, without behavioral disturbance.

1	For mild neurocognitive disorder due to HIV infection, code 331.83 (631.84). (Note: Do not use the additional code for HIV infection. Behavioral disturbance cannot be coded but should still be indicated in writing.)

1	HIV disease is caused by infection with human immunodeficiency Virus type-l (HIV—l), which is acquired through exposure to bodily ﬂuids of an infected person through injection drug use, unprotected sexual contact, or accidental or iatrogenic exposure (e.g., contami- nated blood supply, needle puncture injury to medical personnel). HIV infects several types of cells, most particularly immune cells. Over time, the infection can cause severe depletion of ”T-helper” (CD4) lymphocytes, resulting in severe immunocompromise, often leading to opportunistic infections and neoplasms. This advanced form of HIV infection is termed acquired immune deficiency syndrome (AIDS). Diagnosis of HIV is confirmed by established laboratory methods such as enzyme-linked immunosorbent assay for HIV antibody with Western blot confirmation and/ or polymerase chain reaction—based assays for HIV.

1	Western blot confirmation and/ or polymerase chain reaction—based assays for HIV. Some individuals with HIV infection develop an NCD, which generally shows a ”sub- cortical pattern” with prominently impaired executive function, slowing of processing speed, problems with more demanding attentional tasks, and difficulty in learning new information, but fewer problems with recall of learned information. In major NCD, slow- ing may be prominent. Language difficulties, such as aphasia, are uncommon, although reductions in ﬂuency may be observed. HIV pathogenic processes can affect any part of the brain; therefore, other patterns are possible. Major or Mild Neurocognitive Disorder Due to HIV Infection 633

1	Major or Mild Neurocognitive Disorder Due to HIV Infection 633 Major or mild NCD due to HIV infection is usually more prevalent in individuals with prior episodes of severe immunosuppression, high viral loads in the cerebrospinal ﬂuid, and indicators of advanced HIV disease such as anemia and hypoalbuminemia. Individ- uals with advanced NCD may experience prominent neuromotor features such as severe incoordination, ataxia, and motor slowing. There may be loss of emotional control, includ- ing aggressive or inappropriate affect or apathy. Depending on stage of HIV disease, approximately one-third to over one-half of HIV- infected individuals have at least mild neurocognitive disturbance, but some of these dis- turbances may not meet the full criteria for mild NCD. An estimated 25% of individuals with HIV will have signs and symptoms that meet criteria for mild NCD, and in fewer than 5% would criteria for major NCD be met.

1	An NCD due to HIV infection can resolve, improve, slowly worsen, or have a ﬂuctuating course. Rapid progression to profound neurocognitive impairment is uncommon in the context of currently available combination antiviral treatment; consequently, an abrupt change in mental status in an individual with HIV may prompt an evaluation of other medical sources for the cognitive change, including secondary infections. Because HIV in- fection preferentially affects subcortical regions over the course of illness, including deep white matter, the progression of the disorder follows a ”subcortical” pattern. Since HIV can affect a variety of brain regions, and the illness can take on many different trajectories depending on associated comorbidities and consequences of HIV, the overall course of an

1	NCD due to HIV infection has considerable heterogeneity. A subcortical neurocognitive profile may interact with age over the life course, when psychomotor slowing and motor impairments such as slowed gait may occur as a consequence of other age-related condi- tions so that the overall progression may appear more pronounced in later life. In developed countries, HIV disease is primarily a condition of adults, with acquisition via risky behaviors (e.g., unprotected sex, injection drug use) beginning in late adolescence and peaking during young and middle adulthood. In developing countries, particularly sub-Saharan Africa, where HIV testing and antiretroviral treatments for pregnant women are not readily available, perinatal transmission is common. The NCD in such infants and children may present primarily as neurodevelopmental delay. As individuals treated for

1	HIV survive into older age, additive and interactive neurocognitive effects of HIV and aging, including other NCDs (e.g., due to Alzheimer’s disease, due to Parkinson’s dis- ease), are possible. Risk and prognostic factors for HIV infection. Risk factors for HIV infection include injec- tion drug use, unprotected sex, and unprotected blood supply and other iatrogenic factors.

1	Risk and prognostic factors for major or mild neurocognitive disorder due to HIV in- fection. Paradoxically, NCD due to HIV infection has not declined significantly with the advent of combined antiretroviral therapy, although the most severe presentations (con- sistent with the diagnosis of major NCD) have decreased sharply. Contributory factors may include inadequate control of HIV in the central nervous System (CNS), the evolution of drug-resistant viral strains, the effects of chronic long-term systemic and brain inﬂam- mation, and the effects of comorbid factors such as aging, drug abuse, past history of CNS trauma, and co-infections, such as with the hepatitis C virus. Chronic exposure to antiret- roviral drugs also raises the possibility of neurotoxicity, although this has not been defin- itively established.

1	Serum HIV testing is required for the diagnosis. In addition, HIV characterization of the cere- ﬂuid versus in the plasma. Neuroimaging (i.e., magnetic resonance imaging [MRI]) may reveal reduction in total brain volume, cortical thinning, reduction in white matter volume, and patchy areas of abnormal white matter (hyperintensities). MRI or lumbar puncture may be helpful to exclude a specific medical condition such as cryptococcus infection or herpes en- cephalitis that may contribute to CNS changes in the context of AIDS. Specialized techniques such as diffusion tensor imaging may reveal damage to specific white matter tracts. Functional Consequences of Major or Mild Neurocognitive Disorder Due to HIV Infection

1	Functional Consequences of Major or Mild Neurocognitive Disorder Due to HIV Infection Functional consequences of major or mild NCD due to HIV infection are variable across individuals. Thus, impaired executive abilities and slowed information processing may substantially interfere with the complex disease management decisions required for ad- herence to the combined antiretroviral therapy regimen. The likelihood of comorbid dis- ease may further create functional challenges.

1	In the presence of comorbidities, such as other infections (e.g., hepatitis C virus, syphilis), drug abuse (e.g., methamphetamine abuse), or prior head injury or neurodevelopmental conditions, major or mild NCD due to HIV infection can be diagnosed provided there is ev- idence that infection with HIV has worsened any N CDs due to such preexisting or comorbid conditions. Among older adults, onset of neurocognitive decline related to cerebrovascular disease or neurodegeneration (e.g., major or mild NCD due to Alzheimer’s disease) may need to be differentiated. In general, stable, ﬂuctuating (without progression) or improving neurocognitive status would favor an HIV etiology, whereas steady or stepwise deter- ioration would suggest neurodegenerative or vascular etiology. Because more severe im- munodeficiency can result in opportunistic infections of the brain (e.g., toxoplasmosis; cryptococcosis) and neoplasia (e.g., CNS lymphoma), sudden onset of an NCD or sudden worsening of that

1	im- munodeficiency can result in opportunistic infections of the brain (e.g., toxoplasmosis; cryptococcosis) and neoplasia (e.g., CNS lymphoma), sudden onset of an NCD or sudden worsening of that disorder demands active investigation of non-HIV etiologies.

1	HIV disease is accompanied by chronic systemic and neuro-inﬂammation that can be as- sociated with cerebrovascular disease and metabolic syndrome. These complications can be part of the pathogenesis of major or mild NCD due to HIV infection. HIV frequently co- occurs with conditions such as substance use disorders when the substance has been in- jected and other sexually transmitted disorders. Due to Prion Disease A. The criteria are met for major or mild neurocognitive disorder. B. There is insidious onset, and rapid progression of impairment is common. C. There are motor features of prion disease, such as myoclonus or ataxia, or biomarker evidence. Major or Mild Neurocognitive Disorder Due to Prion Disease 635 D. The neurocognitive disorder is not attributable to another medical condition and is not better explained by another mental disorder.

1	D. The neurocognitive disorder is not attributable to another medical condition and is not better explained by another mental disorder. Coding note: For major neurocognitive disorder due to prion disease, with behavioral dis- turbance, code first 046.79 (A81.9) prion disease, followed by 294.11 (F02.81) major neurocognitive disorder due to prion disease, with behavioral disturbance. For major neu- rocognitive disorder due to prion disease, without behavioral disturbance, code first 046.79 (A81.9) prion disease. followed by 294.10 (F02.80) major neurocognitive disorder due to prion disease, without behavioral disturbance. For mild neurocognitive disorder due to prion disease, code 331.83 (631.84). (Note: Do not use the additional code for prion disease. Behavioral disturbance cannot be coded but should still be indicated in writing.)

1	The classification of major or mild neurocognitive disorder (NCD) due to prion disease in- cludes NCDs due to a group of subacute spongiform encephalopathies (including Creutz- feldt-Jakob disease, variant Creutzfeldt-Jakob disease, kuru, Gerstmann-Straussler- Scheinker syndrome, and fatal insomnia) caused by transmissible agents known as prions. The most common type is sporadic Creutzfeldt-Iakob disease, typically referred to as

1	Creutzfeldt-Jakob disease (CID). Variant CID is much rarer and is associated with trans- mission of bovine spongiform encephalopathy, also called "mad cow disease." Typically, individuals with CID present with neurocognitive deficits, ataxia, and abnormal move- ments such as myoclonus, chorea, or dystonia; a startle reﬂex is also common. Typically, the history reveals rapid progression to major NCD over as little as 6 months, and thus the disorder is typically seen only at the major level. However, many individuals with the dis- order may have atypical presentations, and the disease can be confirmed only by biopsy or at autopsy. Individuals with variant CID may present with a greater preponderance of psychiatric symptoms, characterized a by low mood, withdrawal, and anxiety. Prion dis- ease is typically not diagnosed without at least one of the characteristic biomarker fea- tures: recognized lesions on magnetic resonance imaging with DWI (diffusion-weighted imaging) or FLAIR

1	dis- ease is typically not diagnosed without at least one of the characteristic biomarker fea- tures: recognized lesions on magnetic resonance imaging with DWI (diffusion-weighted imaging) or FLAIR (ﬂuid-attenuated inversion recovery), tau or 14-3-3 protein in cerebro- spinal ﬂuid, characteristic triphasic waves on electroencephalogram, or, for rare familial forms, family history or genetic testing.

1	The annual incidence of sporadic CID is approximately one or two cases per million peo- ple. Prevalence is unknown but very low given the short survival. Prion disease may develop at any age in adults—the peak age for the sporadic CID is ap- proximately 67 years—although it has been reported to occur in individuals spanning the teenage years to late life. Prodromal symptoms of prion disease may include fatigue, anx- iety, problems with appetite or sleeping, or difficulties with concentration. After several weeks, these symptoms may be followed by incoordination, altered vision, or abnormal gait or other movements that may be myoclonic, choreoathetoid, or ballistic, along with a rapidly progressive dementia. The disease typically progresses very rapidly to the major level of impairment over several months. More rarely, it can progress over 2 years and ap- pear similar in its course to other NCDs.

1	Environmental. Cross-species transmission of prion infections, with agents that are closely related to the human form, has been demonstrated (e.g., the outbreak of bovine spongiform encephalopathy inducing variant CID in the United Kingdom during the mid- 19905). Transmission by corneal transplantation and by human growth factor injection has been documented, and anecdotal cases of transmission to health care workers have been reported. Genetic and physiological. There is a genetic component in up to 15% of cases, associ- ated with an autosomal dominant mutation.

1	Genetic and physiological. There is a genetic component in up to 15% of cases, associ- ated with an autosomal dominant mutation. Prion disease can be definitively confirmed only by biopsy or at autopsy. Although there are no distinctive findings on cerebrospinal ﬂuid analysis across the prion diseases, reliable bio- markers are being developed and include 14-3-3 protein (particularly for sporadic CID) as well as tau protein. Magnetic resonance brain imaging is currently considered the most sen- sitive diagnostic test when DWI is performed, with the most common finding being multi~ focal gray matter hyperintensities in subcortical and cortical regions. In some individuals, the electroencephalogram reveals periodic sharp, often triphasic and synchronous dis- charges at a rate of 0.5—2 Hz at some point during the course of the disorder.

1	Other major neurocognitive disorders. Major NCD clue to prion disease may appear similar in its course to other NCDs, but prion diseases are typically distinguished by their rapid progression and prominent cerebellar and motor symptoms. Due to Parkinson’s Disease The criteria are met for major or mild neurocognitive disorder. The disturbance occurs in the setting of established Parkinson's disease. . There is insidious onset and gradual progression of impairment. . The neurocognitive disorder is not attributable to another medical condition and is not better explained by another mental disorder.

1	. The neurocognitive disorder is not attributable to another medical condition and is not better explained by another mental disorder. Major or mild neurocognitive disorder probably due to Parkinson’s disease should be diagnosed if 1 and 2 are both met. Major or miid neurocognitive disorder possibly due to Parkinson’s disease should be diagnosed if 1 or 2 is met: 1. There is no evidence of mixed etiology (i.e., absence of other neurodegenerative or cerebrovascular disease or another neurological, mental, or systemic disease or con- dition likely contributing to cognitive decline). 2. The Parkinson’s disease clearly precedes the onset of the neurocognitive disorder. com?

1	2. The Parkinson’s disease clearly precedes the onset of the neurocognitive disorder. com? Coding note: For major neurocognitive disorder probably due to Parkinson’s disease, with behavioral disturbance, code first 332.0 (620) Parkinson’s disease, followed by 294.11 (F02.81) major neurocognitive disorder probably due to Parkinson’s disease, with behavioral disturbance. For major neurocognitive disorder probably due to Parkinson’s disease, without behavioral disturbance, code first 332.0 (620) Parkinson’s disease, fol- Major or Mild Neurocognitive Disorder Due to Parkinson's Disease 637 lowed by 294.10 (F02.80) major neurocognitive disorder probably due to Parkinson’s dis- ease, without behavioral disturbance.

1	Major or Mild Neurocognitive Disorder Due to Parkinson's Disease 637 lowed by 294.10 (F02.80) major neurocognitive disorder probably due to Parkinson’s dis- ease, without behavioral disturbance. For major neurocognitive disorder possibly due to Parkinson’s disease, code 331.9 (631.9) major neurocognitive disorder possibly due to Parkinson’s disease. (Note: Do not use the additional code for Parkinson's disease. Behavioral disturbance cannot be coded but should still be indicated in writing.) For mild neurocognitive disorder due to Parkinson's disease, code 331.83 (631.84). (Note: Do not use the additional code for Parkinson’s disease. Behavioral disturbance cannot be coded but should still be indicated in writing.)

1	(Note: Do not use the additional code for Parkinson’s disease. Behavioral disturbance cannot be coded but should still be indicated in writing.) The essential feature of major or mild neurocognitive disorder (NCD) due to Parkinson’s disease is cognitive decline following the onset of Parkinson’s disease. The disturbance must occur in the setting of established Parkinson’s disease (Criterion B), and deficits must have developed gradually (Criterion C). The NCD is viewed as probably due to Parkinson’s disease when there is no evidence of another disorder that might be contributing to the cognitive decline and when the Parkinson’s disease clearly precedes onset of the NCD. The NCD is considered possibly due to Parkinson’s disease either when there is no evidence of another disorder that might be contributing to the cognitive decline or when the Parkin- son’s disease precedes onset of the NCD, but not both.

1	Frequently present features include apathy, depressed mood, anxious mood, hallucina- tions, delusions, personality changes, rapid eye movement sleep behavior disorder, and excessive daytime sleepiness. The prevalence of Parkinson’s disease in the United States steadily increases with age from approximately 0.5% between ages 65 and 69 to 3% at age 85 years and older. Parkinson’s disease is more common in males than in females. Among individuals with Parkinson’s disease, as many as 75% will develop a major NCD sometime in the course of their disease. The prevalence of mild NCD in Parkinson’s disease has been estimated at 27%. Onset of Parkinson’s disease is typically between the sixth and ninth decades of life, with most expression in the early 605. Mild NCD often develops relatively early in the course of Parkinson’s disease, whereas major impairment typically does not occur until late.

1	Parkinson’s disease, whereas major impairment typically does not occur until late. Environmental. Risk factors for Parkinson’s disease include exposure to herbicides and pesticides. Genetic and physiological. Potential risk factors for NCD among individuals with Par— kinson’s disease include older age at disease onset and increasing duration of disease. Neuropsychological testing, with a focus on tests that do not rely on motor function, is crit- ical in detecting the core cognitive deficits, particularly at the mild NCD phase. Structural neuroimaging and dopamine transporter scans, such as DaT scans, may differentiate Lewy body—related dementias (Parkinson’s and dementia with Lewy bodies) from non— Lewy body—related dementias (e.g., Alzheimer’s disease) and can sometimes be helpful in the evaluation of major or mild NCD due to Parkinson’s disease.

1	Major or mild neurocognitive disorder with Lewy bodies. This distinction is based sub- stantially on the timing and sequence of motor and cognitive symptoms. For NCD to be at- tributed to Parkinson’s disease, the motor and other symptoms of Parkinson’s disease must be present well before (by convention, at least 1 year prior) cognitive decline has reached the level of major NCD, whereas in major or mild NCD with Lewy bodies, cognitive symp- toms begin shortly before, or concurrent with, motor symptoms. For mild NCD, the timing is harder to establish because the diagnosis itself is less clear and the two disorders exist on a continuum. Unless Parkinson’s disease has been established for some time prior to the onset of cognitive decline, or typical features of major or mild NCD with Lewy bodies are present, it is preferable to diagnose unspecified mild NCD.

1	Major or mild neurocognitive disorder due to Alzheimer’s disease. The motor features are the key to distinguishing major or mild NCD due to Parkinson’s disease from major or mild NCD due to Alzheimer’s disease. However, the two disorders can co-occur. Major or mild vascular neurocognitive disorder. Major or mild vascular NCD may pre- sent with parkinsonian features such as psychomotor slowing that may occur as a conse- quence of subcortical small vessel disease. However, the parkinsonian features typically are not sufficient for a diagnosis of Parkinson’s disease, and the course of the NCD usually has a clear association with cerebrovascular changes.

1	Neurocognitive disorder due to another medical condition (e.g., neurodegenerative disorders). When a diagnosis of major or mild NCD due to Parkinson’s disease is being considered, the distinction must also be made from other brain disorders, such as progres- sive supranuclear palsy, corticobasal degeneration, multiple system atrophy, tumors, and hydrocephalus. Neuroleptic-induced parkinsonism. Neuroleptic-induced parkinsonism can occur in individuals with other NCDs, particularly when dopamine-blocking drugs are prescribed for the behavioral manifestations of such disorders Other medical conditions. Delirium and NCDs due to side effects of dopamine-blocking drugs and other medical conditions (e.g., sedation or impaired cognition, severe hypothy- roidism, B12 deficiency) must also be ruled out.

1	Parkinson’s disease may coexist with Alzheimer’s disease and cerebrovascular disease, espe- cially in older individuals. The compounding of multiple pathological features may diminish the functional abilities of individuals with Parkinson’s disease. Motor symptoms and frequent co—occurrence of depression or apathy can make functional impairment worse. Due to Huntington’s Disease A. The criteria are met for major or mild neurocognitive disorder. B. There is insidious onset and gradual progression. C. There is clinically established Huntington‘s disease, or risk for Huntington’s disease based on family history or genetic testing. Major or Mild Neurocognitive Disorder Due to Huntington’s Disease 639 D. The neurocognitive disorder is not attributable to another medical condition and is not better explained by another mental disorder.

1	D. The neurocognitive disorder is not attributable to another medical condition and is not better explained by another mental disorder. Coding note: For major neurocognitive disorder due to Huntington’s disease, with behav- ioral disturbance, code first 333.4 (610) Huntington’s disease, followed by 294.11 (F02.81) major neurocognitive disorder due to Huntington’s disease, with behavioral dis- turbance. For major neurocognitive disorder due to Huntington’s disease, without behav- ioral disturbance, code first 333.4 (610) Huntington's disease, followed by 294.10 (F02.80) major neurocognitive disorder due to Huntington’s disease, without behavioral disturbance. For mild neurocognitive disorder due to Huntington’s disease, code 331.83 (631.84). (Note: Do not use the additional code for Huntington's disease. Behavioral disturbance cannot be coded but should still be indicated in writing.)

1	(Note: Do not use the additional code for Huntington's disease. Behavioral disturbance cannot be coded but should still be indicated in writing.) Progressive cognitive impairment is a core feature of Huntington’s disease, with early changes in executive function (i.e., processing speed, organization, and planning) rather than leam- ing and memory. Cognitive and associated behavioral changes often precede the emergence of the typical motor abnormalities of bradykinesia (i.e., slowing of voluntary movement) and chorea (i.e., involuntary jerking movements). A diagnosis of definite Huntington’s dis- ease is given in the presence of unequivocal, extrapyramidal motor abnormalities in an in- dividual with either a family history of Huntington’s disease or genetic testing showing a CAG trinucleotide repeat expansion in the HTT gene, located on chromosome 4.

1	CAG trinucleotide repeat expansion in the HTT gene, located on chromosome 4. Depression, irritability, anxiety, obsessive-compulsive symptoms, and apathy are fre- quently, and psychosis more rarely, associated with Huntington’s disease and often pre- cede the onset of motor symptoms. Neurocognitive deficits are an eventual outcome of Huntington’s disease; the worldwide prevalence is estimated to be 2.7 per 100,000. The prevalence of Huntington’s disease in North America, Europe, and Australia is 5.7 per 100,000, with a much lower prevalence of 0.40 per 100,000 in Asia.

1	North America, Europe, and Australia is 5.7 per 100,000, with a much lower prevalence of 0.40 per 100,000 in Asia. The average age at diagnosis of Huntington’s disease is approximately 40 years, although this varies widely. Age at onset is inversely correlated with CAG expansion length. Juve- nile Huntington’s disease (onset before age 20) may present more commonly with brady- kinesia, dystonia, and rigidity than with the choreic movements characteristic of the adult- onset disorder. The disease is gradually progressive, with median survival approximately 15 years after motor symptom diagnosis.

1	Phenotypic expression of Huntington’s disease varies by presence of motor, cognitive, and psychiatric symptoms. Psychiatric and cognitive abnormalities can predate the motor abnormality by at least 15 years. Initial symptoms requiring care often include irritabity, anxiety, or depressed mood. Other behavioral disturbances may include pronounced ap- athy, disinhibition, impulsivity, and impaired insight, with apathy often becoming more progressive over time. Early movement symptoms may involve the appearance of fidget- iness of the extremities as well as mild apmxia (i.e., difficulty with purposeful movements), particularly with fine motor tasks. As the disorder progresses, other motor problems in- clude impaired gait (ataxia) and postural instability. Motor impairment eventually affects speech production (dysarthria) such that the speech becomes very difficult to understand, which may result in significant distress resulting from the communication barrier in the context of

1	affects speech production (dysarthria) such that the speech becomes very difficult to understand, which may result in significant distress resulting from the communication barrier in the context of comparatively intact cognition. Advanced motor disease severely affects gait with progressive ataxia. Eventually individuals become nonambulatory. End-stage motor disease impairs motor control of eating and swallowing, typically a major contributor to the death of the individual from aspiration pneumonia.

1	Genetic and physiological. The genetic basis of Huntington’s disease is a fully penetrant autosomal dominant expansion of the CAG trincleotide, often called a CAG repeat in the huntingtin gene. A repeat length of 36 or more is invariably associated with Huntington’s disease, with longer repeat lengths associated with early age at onset. A CAG repeat length of 36 or more is invariably associated with Huntington’s disease.

1	Genetic testing is the primary laboratory test for the determination of Huntington’s dis- ease, which is an autosomal dominant disorder with complete penetrance. The trinucleo- tide CAG is observed to have a repeat expansion in the gene that encodes huntingtin protein on chromosome 4. A diagnosis of Huntington’s disease is not made in the presence of the gene expansion alone, but the diagnosis is made only after symptoms become man- ifest. Some individuals with a positive family history request genetic testing in a presymp- tomatic stage. Associated features may also include neuroimaging changes; volume loss in the basal ganglia, particularly the caudate nucleus and putamen, is well known to occur and progresses over the course of illness. Other structural and functional changes have been observed in brain imaging but remain research measures. Functionai Consequences of Major or Mild Neurocognitlve Disorder Due to Huntington’s Disease

1	Functionai Consequences of Major or Mild Neurocognitlve Disorder Due to Huntington’s Disease In the prodromal phase of illness and at early diagnosis, occupational decline is most com— mon, with most individuals reporting some loss of ability to engage in their typical work.

1	The emotional, behavioral, and cognitive aspects of Huntington’s disease, such as disin- hibition and personality changes, are highly associated with functional decline. Cognitive deficits that contribute most to functional decline may include speed of processing, initi- ation, and attention rather than memory impairment. Given that Huntington’s disease on- set occurs in productive years of life, it may have a very disruptive effect on performance in the work setting as well as social and family life. As the disease progresses, disability from problems such as impaired gait, dysarthria, and impulsive or irritable behaviors may substantially add to the level of impairment and daily care needs, over and above the care needs attributable to the cognitive decline. Severe choreic movements may substantially interfere with provision of care such as bathing, dressing, and toileting.

1	Other mental disorders. Early symptoms of Huntington’s disease may include instabil- ity of mood, irritability, or compulsive behaviors that may suggest another mental disor- der. However, genetic testing or the development of motor symptoms will distinguish the presence of Huntington’s disease. Other neurocognitive disorders. The early symptoms of Huntington’s disease, particu- larly symptoms of executive dysfunction and impaired psychomotor speed, may resemble other neurocognitive disorders (NCDs), such as major or mild vascular NCD. Major or Mild Neurocognitive Disorder Due to Another Medical Condition 641 Other movement disorders. Huntington’s disease must also be differentiated from other disorders or conditions associated with chorea, such as Wilson’s disease, drug-induced tardive dyskinesia, Sydenham’s chorea, systemic lupus erythematosus, or senile chorea.

1	Rarely, individuals may present with a course similar to that of Huntington’s disease but without positive genetic testing; this is considered to be a Huntington’s disease pheno- copy that results from a variety of potential genetic factors. Due to Another Medical Condition A. The criteria are met for major or mild neurocognitive disorder. B. There is evidence from the history, physical examination. or laboratory findings that the neurocognitive disorder is the pathophysiological consequence of another medical condition. C. The cognitive deficits are not better explained by another mental disorder or another specific neurocognitive disorder (e.g., Alzheimer’s disease, HIV infection).

1	C. The cognitive deficits are not better explained by another mental disorder or another specific neurocognitive disorder (e.g., Alzheimer’s disease, HIV infection). Coding note: For major neurocognitive disorder due to another medical condition, with behavioral disturbance, code first the other medical condition, followed by the major neu- rocognitive disorder due to another medical condition, with behavioral disturbance (e.g., 340 [635] multiple sclerosis, 294.11 [F02.81] major neurocognitive disorder due to multi- ple sclerosis, with behavioral disturbance). For major neurocognitive disorder due to an- other medical condition, without behavioral disturbance, code first the other medical condition, followed by the major neurocognitive disorder due to another medical condition, without behavioral disturbance (e.g., 340 [635] multiple sclerosis, 294.10 [F02.80] major neurocognitive disorder due to multiple sclerosis, without behavioral disturbance).

1	For mild neUrocognitive disorder due to another medical condition, code 331.83 (631.84). (Note: Do not use the additional code for the other medical condition. Behavioral distur- bance cannot be coded but should still be indicated in writing.)

1	A number of other medical conditions can cause neurocognitive disorders (NCDs). These conditions include structural lesions (e.g., primary or secondary brain tumors, subdural hematoma, slowly progressive or normal-pressure hydrocephalus), hypoxia related to hy- poperfusion from heart failure, endocrine conditions (e.g., hypothyroidism, hypercalce- mia, hypoglycemia), nutritional conditions (e.g., deficiencies of thiamine or niacin), other infectious conditions (e.g., neurosyphilis, cryptococcosis), immune disorders (e.g., tempo- ral arteritis, systemic lupus erythematosus), hepatic or renal failure, metabolic conditions (e.g., Kufs’ disease, adrenoleukodystrophy, metachromatic leukodystrophy, other storage diseases of adulthood and childhood), and other neurological conditions (e.g., epilepsy, multiple sclerosis). Unusual causes of central nervous system injury, such as electrical shock or intracranial radiation, are generally evident from the history. The temporal asso- ciation

1	epilepsy, multiple sclerosis). Unusual causes of central nervous system injury, such as electrical shock or intracranial radiation, are generally evident from the history. The temporal asso- ciation between the onset or exacerbation of the medical condition and the development of the cognitive deficit offers the greatest support that the NCD is induced by the medical condition. Diagnostic certainty regarding this relationship may be increased if the neuro- cognitive deficits ameliorate partially or stabilize in the context of treatment of the medical condition.

1	Typically the course of the NCD progresses in a manner that is commensurate with progres- sion of the underlying medical disorder. In circumstances where the medical disorder is treat- able (e.g., hypothyroidism), the neurocognitive deficit may improve or at least not progress. When the medical condition has a deteriorative course (e.g., secondary progressive multiple sclerosis), the neurocognitive deficits will progress along with the temporal course of illness. pend on the nature and severity of the medical condition. Other major or mild neurocognitive disorder. The presence of an attributable medical condition does not entirely exclude the possibility of another major or mild NCD. If cog- nitive deficits persist following successful treatment of an associated medical condition, then another etiology may be responsible for the cognitive decline. Due to Multiple Etiologies A. The criteria are met for major or mild neurocognitive disorder.

1	Due to Multiple Etiologies A. The criteria are met for major or mild neurocognitive disorder. B. There is evidence from the history. physical examination, or laboratory findings that the neurocognitive disorder is the pathophysiological consequence of more than one etio- logical process, excluding substances (e.g., neurocognitive disorder due to Alzhei- mer’s disease with subsequent development of vascular neurocognitive disorder). Note: Please refer to the diagnostic criteria for the various neurocognitive disorders due to specific medical conditions for guidance on establishing the particular etiologies. C. The cognitive deficits are not better explained by another mental disorder and do not occur exclusively during the course of a delirium.

1	C. The cognitive deficits are not better explained by another mental disorder and do not occur exclusively during the course of a delirium. Coding note: For major neurocognitive disorder due to multiple etiologies, with behavioral disturbance, code 294.11 (F02.81); for major neurocognitive disorder due to multiple etiolo- gies, without behavioral disturbance, code 294.10 (F02.80). All of the etiological medical conditions (with the exception of vascular disease) should be coded and listed separately immediately before major neurocognitive disorder due to multiple etiologies (e.g., 331.0 [630.9] Alzheimer’s disease; 331.82 [631.83] Lewy body disease; 294.11 [F02.81] major neurocognitive disorder due to multiple etiologies, with behavioral disturbance).

1	When a cerebrovascular etiology is contributing to the neurocognitive disorder, the diagno- sis of vascular neurocognitive disorder should be listed in addition to major neurocognitive disorder due to multiple etiologies. For example, for a presentation of major neurocognitive disorder due to both Alzheimer's disease and vascular disease, with behavioral disturbance, code the following: 331.0 (630.9) Alzheimer’s disease; 294.11 (F02.81) major neurocogni- tive disorder due to multiple etiologies, with behavioral disturbance; 290.40 (F01.51) major vascular neurocognitive disorder, with behavioral disturbance. For mild neurocognitive disorder due to multiple etiologies, code 331.83 (631.84). (Note: Do not use the additional codes for the etiologies. Behavioral disturbance cannot be coded but should still be indicated in writing.)

1	Do not use the additional codes for the etiologies. Behavioral disturbance cannot be coded but should still be indicated in writing.) This category is included to cover the clinical presentation of a neurocognitive disorder (NCD) development of the NCD. In addition to evidence indicative of the presence of multiple med- ical conditions that are known to cause NCD (i.e., findings from the history and physical ex- amination, and laboratory findings), it may be helpful to refer to the diagnostic criteria and text for the various medical etiologies (e.g., NCD due to Parkinson’s disease) for more information on establishing the etiological connection for that particular medical condition. 799.59 (R41.9)

1	799.59 (R41.9) This category applies to presentations in which symptoms characteristic of a neurocogni- tive disorder that cause clinically significant distress or impairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the neurocognitive disorders diagnostic class. The unspecified neuro- cognitive disorder category is used in situations in which the precise etiology cannot be determined with sufficient certainty to make an etiological attribution. Coding note: For unspecified major or mild neurocognitive disorder, code 799.59 (R419). (Note: Do not use additional codes for any presumed etiological medical conditions. Be- havioral disturbance cannot be coded but may be indicated in writing.)

1	(Note: Do not use additional codes for any presumed etiological medical conditions. Be- havioral disturbance cannot be coded but may be indicated in writing.) Th lS Chapter beg l US with a general definition of personality disorder that applies to each of the 10 specific personality disorders. A personality disorder is an enduring pattern of inner experience and behavior that deviates markedly from the expectations of the in- dividual’s culture, is pervasive and inﬂexible, has an onset in adolescence or early adult- hood, is stable over time, and leads to distress or impairment.

1	With any ongoing review process, especially one of this complexity, different view- points emerge, and an effort was made to accommodate them. Thus, personality disorders are included in both Sections II and III. The material in Section II represents an update of text associated with the same criteria found in DSM-IV-TR, whereas Section III includes the proposed research model for personality disorder diagnosis and conceptualization de- veloped by the DSM-S Personality and Personality Disorders Work Group. As this field evolves, it is hoped that both versions will serve clinical practice and research initiatives, respectively. The following personality disorders are included in this chapter. 0 Paranoid personality disorder is a pattern of distrust and suspiciousness such that oth- ers’ motives are interpreted as malevolent. 0 Schizoid personality disorder is a pattern of detachment from social relationships and a restricted range of emotional expression.

1	0 Schizoid personality disorder is a pattern of detachment from social relationships and a restricted range of emotional expression. 0 Schizotypal personality disorder is a pattern of acute discomfort in close relationships, cognitive or perceptual distortions, and eccentricities of behavior. 0 Antisocial personality disorder is a pattern of disregard for, and violation of, the rights of others. 0 Borderline personality disorder is a pattern of instability in interpersonal relation- ships, self—image, and affects, and marked impulsivity. 0 Histrionic personality disorder is a pattern of excessive emotionality and attention seeking. - Narcissistic personality disorder is a pattern of grandiosity, need for admiration, and lack of empathy. - Avoidant personality disorder is a pattern of social inhibition, feelings of inadequacy, and hypersensitivity to negative evaluation.

1	- Avoidant personality disorder is a pattern of social inhibition, feelings of inadequacy, and hypersensitivity to negative evaluation. 0 Dependent personality disorder is a pattern of submissive and clinging behavior re- lated to an excessive need to be taken care of. 0 Obsessive-compulsive personality disorder is a pattern of preoccupation with order- liness, perfectionism, and control. 0 Personality change due to another medical condition is a persistent personality dis- turbance that is judged to be due to the direct physiological effects of a medical condi- tion (e.g., frontal lobe lesion).

1	. Other specified personality disorder and unspecified personality disorder is a cate- gory provided for two situations: 1) the individual’s personality pattern meets the gen- eral criteria for a personality disorder, and traits of several different personality disorders are present, but the criteria for any specific personality disorder are not met; or 2) the individual’s personality pattern meets the general criteria for a personality dis- order, but the individual is considered to have a personality disorder that is not in- cluded in the DSM-5 classification (e.g., passive-aggressive personality disorder). The personality disorders are grouped into three clusters based on descriptive similarities.

1	The personality disorders are grouped into three clusters based on descriptive similarities. Cluster A includes paranoid, schizoid, and schizotypal personality disorders. Individuals with these disorders often appear odd or eccentric. Cluster B includes antisocial, borderline, histri- onic, and narcissistic personality disorders. Individuals with these disorders often appear dra- matic, emotional, or erratic. Cluster C includes avoidant, dependent, and obsessive— compulsive personality disorders. Individuals with these disorders often appear anxious or fearful. It should be noted that this clustering system, although useful in some research and ed- ucational situations, has serious limitations and has not been consistently validated.

1	Moreover, individuals frequently present with co-occurring personality disorders from different clusters. Prevalence estimates for the different clusters suggest 5.7% for dis- orders in Cluster A, 1.5% for disorders in Cluster B, 6.0% for disorders in Cluster C, and 9.1% for any personality disorder, indicating frequent co-occurrence of disorders from dif- ferent clusters. Data from the 2001—2002 National Epidemiologic Survey on Alcohol and Related Conditions suggest that approximately 15% of US. adults have at least one per- sonality disorder.

1	The diagnostic approach used in this manual represents the categorical perspective that personality disorders are qualitatively distinct clinical syndromes. An alternative to the categorical approach is the dimensional perspective that personality disorders represent maladaptive variants of personality traits that merge imperceptibly into normality and into one another. See Section III for a full description of a dimensional model for person- ality disorders. The DSM—IV personality disorder clusters (i.e., odd-eccentric, dramatic- emotional, and anxious-fearful) may also be viewed as dimensions representing spectra of personality dysfunction on a continuum with other mental disorders. The alternative di- mensional models have much in common and together appear to cover the important ar- eas of personality dysfunction. Their integration, clinical utility, and relationship with the personality disorder diagnostic categories and various aspects of personality dysfunction are under active

1	of personality dysfunction. Their integration, clinical utility, and relationship with the personality disorder diagnostic categories and various aspects of personality dysfunction are under active investigation.

1	A. An enduring pattern of inner experience and behavior that deviates markedly from the expectations of the individual’s culture. This pattern is manifested in two (or more) of the following areas: 1. Cognition (i.e., ways of perceiving and interpreting self, other people, and events). 2. Affectivity (i.e., the range, intensity, lability, and appropriateness of emotional re- sponse). 3. Interpersonalfunctioning. 4. Impulse control. B. The enduring pattern is inflexible and pervasive across a broad range of personal and social situations. C. The enduring pattern leads to clinically significant distress or impairment in social, oc- cupational, or other important areas of functioning. D. The pattern is stable and of long duration, and its onset can be traced back at least to adolescence-pr early adulthood. E. The enduring pattern is not better explained as a manifestation or consequence of an- other mental disorder.

1	E. The enduring pattern is not better explained as a manifestation or consequence of an- other mental disorder. F. The enduring pattern is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication) or another medical condition (e.g., head trauma). Personality traits are enduring patterns of perceiving, relating to, and thinking about the en- vironment and oneself that are exhibited in a wide range of social and personal contexts.

1	Only when personality traits are inﬂexible and maladaptive and cause significant func- tional impairment or subjective distress do they constitute personality disorders. The essen- tial feature of a personality disorder is an enduring pattern of inner experience and behavior that deviates markedly from the expectations of the individual’s culture and is manifested in at least two of the following areas: cognition, affectivity, interpersonal functioning, or im- pulse control (Criterion A). This enduring pattern is inﬂexible and pervasive across a broad range of personal and social situations (Criterion B) and leads to clinically significant dis- tress or impairment in social, occupational, or other important areas of functioning (Crite- rion C). The pattern is stable and of long duration, and its onset can be traced back at least to adolescence or early adulthood (Criterion D). The pattern is not better explained as a manifestation or consequence of another mental disorder (Criterion

1	and its onset can be traced back at least to adolescence or early adulthood (Criterion D). The pattern is not better explained as a manifestation or consequence of another mental disorder (Criterion E) and is not attribut- able to the physiological effects of a substance (e.g., a drug of abuse, a medication, exposure to a toxin) or another medical condition (e.g., head trauma) (Criterion F). Specific diagnostic criteria are also provided for each of the personality disorders included in this chapter.

1	The diagnosis of personality disorders requires an evaluation of the individual’s long- term patterns of functioning, and the particular personality features must be evident by early adulthood. The personality traits that define these disorders must also be distin- guished from characteristics that emerge in response to specific situational stressors or more transient mental states (e.g., bipolar, depressive, or anxiety disorders; substance in- toxication). The clinician should assess the stability of personality traits over time and across different situations. Although a single interview with the individual is sometimes sufficient for making the diagnosis, it is often necessary to conduct more than one inter- view and to space these over time. Assessment can also be complicated by the fact that the the individual (i.e., the traits are often ego-syntonic). To help overcome this difficulty, sup- plementary information from other informants may be helpful.

1	The features of a personality disorder usually become recognizable during adolescence or early adult life. By definition, a personality disorder is an enduring pattern of thinking, feeling, and behaving that is relatively stable over time. Some types of personality disorder (notably, antisocial and borderline personality disorders) tend to become less evident or to remit with age, whereas this appears to be less true for some other types (e.g., obsessive- compulsive and schizotypal personality disorders). Personality disorder categories may be applied with children or adolescents in those relatively unusual instances in which the individual’s particular maladaptive personality traits appear to be pervasive, persistent, and unlikely to be limited to a particular devel- opmental stage or another mental disorder. It should be recognized that the traits of a per- sonality disorder that appear in childhood will often not persist unchanged into adult life.

1	For a personality disorder to be diagnosed in an individual younger than 18 years, the fea- tures must have been present for at least 1 year. The one exception to this is antisocial per— sonality disorder, which cannot be diagnosed in individuals younger than 18 years. Al- though, by definition, a personality disorder requires an onset no later than early adulthood, individuals may not come to clinical attention until relatively late in life. A per- sonality disorder may be exacerbated following the loss of significant supporting persons (e.g., a spouse) or previously stabilizing social situations (e.g., a job). However, the devel- opment of a change in personality in middle adulthood or later life warrants a thorough evaluation to determine the possible presence of a personality change due to another med- ical condition or an unrecognized substance use disorder.

1	Judgments about personality functioning must take into account the individual’s ethnic, cul- tural, and social background. Personality disorders should not be confused with problems as- sociated with acculturation following immigration or with the expression of habits, customs, or religious and political values professed by the individual’s culture of origin. It is useful for the clinician, especially when evaluating someone from a different background, to obtain ad- ditional information from informants who are familiar with the person’s cultural background.

1	Certain personality disorders (e.g., antisocial personality disorder) are diagnosed more frequently in males. Others (e.g., borderline, histrionic, and dependent personality disor- ders) are diagnosed more frequently in females. Although these differences in prevalence probably reﬂect real gender differences in the presence of such patterns, clinicians must be cautious not to overdiagnose or underdiagnose certain personality disorders in females or in males because of social stereotypes about typical gender roles and behaviors.

1	Other mental disorders and personality traits. Many of the specific criteria for the per- sonality disorders describe features (e.g., suspiciousness, dependency, insensitivity) that are also characteristic of episodes of other mental disorders. A personality disorder should be diagnosed only when the defining characteristics appeared before early adulthood, are typical of the individual’s long-term functioning, and do not occur exclusively during an episode of another mental disorder. It may be particularly difficult (and not particularly useful) to distinguish personality disorders from persistent mental disorders such as per- sistent depressive disorder that have an early onset and an enduring, relatively stable course. Some personality disorders may have a ”spectrum” relationship to other mental disorders (e.g., schizotypal personality disorder with schizophrenia; avoidant personality disorder with social anxiety disorder [social phobia]) based on phenomenological or bio- logical

1	mental disorders (e.g., schizotypal personality disorder with schizophrenia; avoidant personality disorder with social anxiety disorder [social phobia]) based on phenomenological or bio- logical similarities or familial aggregation.

1	the threshold for a personality disorder. Personality traits are diagnosed as a personality disorder only when they are inﬂexible, maladaptive, and persisting and cause significant functional impairment or subjective distress. Psychotic disorders. For the three personality disorders that may be related to the psy- chotic disorders (i.e., paranoid, schizoid, and schizotypal), there is an exclusion criterion stating that the pattern of behavior must not have occurred exclusively during the course of schizophrenia, a bipolar or depressive disorder with psychotic features, or another psy- chotic disorder. When an individual has a persistent mental disorder (e.g., schizophrenia) that was preceded by a preexisting personality disorder, the personality disorder should also be recorded, followed by ”premorbid" in parentheses.

1	Anxiety and depressive disorders. The clinician must be cautious in diagnosing per- sonality disorders during an episode of a depressive disorder or an anxiety disorder, be- traits and may make it more difficult to evaluate retrospectively the individual’s long-term patterns of functioning. Posttraumatic stress disorder. When personality changes emerge and persist after an individual has been exposed to extreme stress, a diagnosis of posttraumatic stress disorder should be considered. Substance use disorders. When an individual has a substance use disorder, it is impor- tant not to make a personality disorder diagnosis based solely on behaviors that are con- sequences of substance intoxication or withdrawal or that are associated with activities in the service of sustaining substance use (e.g., antisocial behavior).

1	Personality change due to another medical condition. When enduring changes in per- sonality arise as a result of the physiological effects of another medical condition (e.g., brain tumor), a diagnosis of personality change due to another medical condition should be considered. Diagnostic Criteria 301.0 (F60.0) A. A pervasive distrust and suspiciousness of others such that their motives are inter- preted as malevolent, beginning by early adulthood and present in a variety of con- texts, as indicated by four (or more) of the following: 1. Suspects, without sufficient basis, that others are exploiting, harming, or deceiving him or her. 2. Is preoccupied with unjustified doubts about the loyalty or trustworthiness of friends or associates. 3. Is reluctant to confide in others because of unwarranted fear that the information will be used maliciously against him or her. 4. Heads hidden demeaning or threatening meanings into benign remarks or events.

1	4. Heads hidden demeaning or threatening meanings into benign remarks or events. 5. Persistently bears grudges (i.e., is unforgiving of insults, injuries, or slights). 6. Perceives attacks on his or her character or reputation that are not apparent to oth- ers and is quick to react angrily or to counterattack. 7. Has recurrent suspicions, withoutjustification, regarding fidelity of spouse or sexual partner. B. Does not occur exclusively during the course of schizophrenia, a bipolar disorder or depressive disorder with psychotic features, or another psychotic disorder and is not attributable to the physiological effects of another medical condition. Note: If criteria are met prior to the onset of schizophrenia, add “premorbid,” i.e., “paranoid personality disorder (premorbid).”

1	Note: If criteria are met prior to the onset of schizophrenia, add “premorbid,” i.e., “paranoid personality disorder (premorbid).” The essential feature of paranoid personality disorder is a pattern of pervasive distrust and suspiciousness of others such that their motives are interpreted as malevolent. This pattern begins by early adulthood and is present in a variety of contexts.

1	Individuals with this disorder assume that other people will exploit, harm, or deceive them, even if no evidence exists to support this expectation (Criterion A1). They suspect on the basis of little or no evidence that others are plotting against them and may attack them suddenly, at any time and without reason. They often feel that they have been deeply and irreversibly injured by another person or persons even when there is no objective evidence for this. They are preoccupied with unjustified doubts about the loyalty or trustworthiness of their friends and associates, whose actions are minutely scrutinized for evidence of hos- tile intentions (Criterion A2). Any perceived deviation from trustworthiness or loyalty serves to support their underlying assumptions. They are so amazed when a friend or as- sociate shows loyalty that they cannot trust or believe it. If they get into trouble, they ex- pect that friends and associates will either attack or ignore them.

1	Individuals with paranoid personality disorder are reluctant to confide in or become close to others because they fear that the information they share will be used against them (Criterion A3). They may refuse to answer personal questions, saying that the information is ”nobody’s business.” They read hidden meanings that are demeaning and threatening into benign remarks or events (Criterion A4). For example, an individual with this disor- der may misinterpret an honest mistake by a store clerk as a deliberate attempt to short- change, or view a casual humorous remark by a co-worker as a serious character attack. Compliments are often misinterpreted (e.g., a compliment on a new acquisition is mis- interpreted as a criticism for selfishness; a compliment on an accomplishment is misinter- preted as an attempt to coerce more and better performance). They may view an offer of help as a criticism that they are not doing well enough on their own.

1	Individuals with this disorder persistently bear grudges and are unwilling to forgive the insults, injuries, or slights that they think they have received (Criterion A5). Minor slights arouse major hostility, and the hostile feelings persist for a long time. Because they are constantly vigilant to the harmful intentions of others, they very often feel that their way. They are quick to counterattack and react with anger to perceived insults (Criterion A6). Individuals with this disorder may be pathologically jealous, often suspecting that their spouse or sexual partner is unfaithful without any adequate justification (Criterion A7). They may gather trivial and circumstantial "evidence” to support their jealous beliefs. They want to maintain complete control of intimate relationships to avoid being betrayed and may constantly question and challenge the whereabouts, actions, intentions, and fi- delity of their spouse or partner.

1	Paranoid personality disorder should not be diagnosed if the pattern of behavior oc- curs exclusively during the course of schizophrenia, a bipolar disorder or depressive dis- order with psychotic features, or another psychotic disorder, or if it is attributable to the physiological effects of a neurological (e.g., temporal lobe epilepsy) or another medical condition (Criterion B).

1	Individuals with paranoid personality disorder are generally difficult to get along with and often have problems with close relationships. Their excessive suspiciousness and hos- tility may be expressed in overt argumentativeness, in recurrent complaining, or by quiet, apparently hostile aloofness. Because they are hypervigilant for potential threats, they may act in a guarded, secretive, or devious manner and appear to be “cold” and lacking in tender feelings. Although they may appear to be objective, rational, and unemotional, they more often display a labile range of affect, with hostile, stubborn, and sarcastic expressions predominating. Their combative and suspicious nature may elicit a hostile response in others, which then serves to confirm their original expectations.

1	Because individuals with paranoid personality disorder lack trust in others, they have an excessive need to be self-sufficient and a strong sense of autonomy. They also need to have a high degree of control over those around them. They are often rigid, critical of oth- ers, and unable to collaborate, although they have great difficulty accepting criticism them- selves. They may blame others for their own shortcomings. Because of their quickness to counterattack in response to the threats they perceive around them, they may be litigious and frequently become involved in legal disputes. Individuals with this disorder seek to confirm their preconceived negative notions regarding people or situations they encounter, attributing malevolent motivations to others that are projections of their own fears. They may exhibit thinly hidden, unrealistic grandiose fantasies, are often attuned to issues of power and rank, and tend to develop negative stereotypes of others, particularly those from

1	own fears. They may exhibit thinly hidden, unrealistic grandiose fantasies, are often attuned to issues of power and rank, and tend to develop negative stereotypes of others, particularly those from population groups distinct from their own. Attracted by simplistic formulations of the world, they are often wary of ambiguous situations. They may be perceived as ”fanatics" and form tightly knit "cults” or groups with others who share their paranoid belief systems.

1	Particularly in response to stress, individuals with this disorder may experience very brief psychotic episodes (lasting minutes to hours). In some instances, paranoid personal- ity disorder may appear as the premorbid antecedent of delusional disorder or schizo- phrenia. Individuals with paranoid personality disorder may develop major depressive order. Alcohol and other substance use disorders frequently occur. The most common co- occurring personality disorders appear to be schizotypal, schizoid, narcissistic, avoidant, and borderline. Part II of the National Comorbidity Survey Replication suggests a prevalence of 2.3%, while the National Epidemiologic Survey on Alcohol and Related Conditions data suggest a prevalence of paranoid personality disorder of 4.4%.

1	solitariness, poor peer relationships, social anxiety, underachievement in school, hyper- sensitivity, peculiar thoughts and language, and idiosyncratic fantasies. These children may appear to be ”odd" or ”eccentric” and attract teasing. In clinical samples, this disorder appears to be more commonly diagnosed in males. Genetic and physiological. There is some evidence for an increased prevalence of par- anoid personality disorder in relatives of probands with schizophrenia and for a more spe- cific familial relationship with delusional disorder, persecutory type.

1	Some behaviors that are inﬂuenced by sociocultural contexts or specific life circumstances may be erroneously labeled paranoid and may even be reinforced by the process of clinical evaluation. Members of minority groups, immigrants, political and economic refugees, or individuals of different ethnic backgrounds may display guarded or defensive behaviors because of unfamiliarity (e.g., language barriers or lack of knowledge of rules and regula- tions) or in response to the perceived neglect or indifference of the majority society. These behaviors can, in turn, generate anger and frustration in those who deal with these indi- viduals, thus setting up a vicious cycle of mutual mistrust, which should not be confused with paranoid personality disorder. Some ethnic groups also display culturally related be- haviors that can be misinterpreted as paranoid.

1	Other mental disorders with psychotic symptoms. Paranoid personality disorder can be distinguished from delusional disorder, persecutory type; schizophrenia; and a bipolar or depressive disorder with psychotic features because these disorders are all characterized by a period of persistent psychotic symptoms (e.g., delusions and hallucinations). For an additional diagnosis of paranoid personality disorder to be given, the personality disorder must have been present before the onset of psychotic symptoms and must persist when the psychotic symptoms are in remission. When an individual has another persistent mental disorder (e.g., schizophrenia) that was preceded by paranoid personality disorder, paranoid personality dis- order should also be recorded, followed by ”premorbid” in parentheses.

1	Personality change due to another medical condition. Paranoid personality disorder must be distinguished from personality change due to another medical condition, in which the traits that emerge are attributable to the direct effects of another medical condi- tion on the central nervous system. Substance use disorders. Paranoid personality disorder must be distinguished from symptoms that may develop in association with persistent substance use. Paranoid traits associated with physical handicaps. The disorder must also be distin- guished from paranoid traits associated with the development of physical handicaps (e.g., a hearing impairment). Other personality disorders and personality traits. Other personality disorders may be confused with paranoid personality disorder because they have certain features in common.

1	It is therefore important to distinguish among these disorders based on differences in their characteristic features. However, if an individual has personality features that meet criteria for one or more personality disorders in addition to paranoid personality disorder, all can be diagnosed. Paranoid personality disorder and schizotypal personality disorder share the traits of suspiciousness, interpersonal aloofness, and paranoid ideation, but schizotypal per- sonality disorder also includes symptoms such as magical thinking, unusual perceptual ex- periences, and odd thinking and speech. Individuals with behaviors that meet criteria for schizoid personality disorder are often perceived as strange, eccentric, cold, and aloof, but they do not usually have prominent paranoid ideation. The tendency of individuals with paranoid personality disorder to react to minor stimuli with anger is also seen in borderline and histrionic personality disorders. However, these disorders are not

1	The tendency of individuals with paranoid personality disorder to react to minor stimuli with anger is also seen in borderline and histrionic personality disorders. However, these disorders are not necessarily associ- ated with pervasive suspiciousness. People with avoidant personality disorder may also be reluctant to confide in others, but more from fear of being embarrassed or found inadequate than from fear of others’ malicious intent. Although antisocial behavior may be present in some individuals with paranoid personality disorder, it is not usually motivated by a desire for personal gain or to exploit others as in antisocial personality disorder, but rather is more often attributable to a desire for revenge. Individuals with narcissistic personality disorder may occasionally display suspiciousness, social withdrawal, or alienation, but this derives primarily from fears of having their imperfections or ﬂaws revealed.

1	Paranoid traits may be adaptive, particularly in threatening environments. Paranoid personality disorder should be diagnosed only when these traits are inﬂexible, maladap- tive, and persisting and cause significant functional impairment or subjective distress. Diagnostic Criteria 301.20 (F60.1) A. A pervasive pattern of detachment from social relationships and a restricted range of expression of emotions in interpersonal settings, beginning by early adulthood and present in a variety of contexts. as indicated by four (or more) of the following: Neither desires nor enjoys close relationships, including being part of a family. Almost always chooses solitary activities. Has little, if any. interest in having sexual experiences with another person. Takes pleasure in few, if any, activities. Lacks close friends or confidants other than first-degree relatives. Appears indifferent to the praise or criticism of others.

1	Takes pleasure in few, if any, activities. Lacks close friends or confidants other than first-degree relatives. Appears indifferent to the praise or criticism of others. 7. Shows emotional coldness, detachment, or flattened affectivity. ?QPWN.‘ B. Does not occur exclusively during the course of schizophrenia, a bipolar disorder or depressive disorder with psychotic features, another psychotic disorder, or autism spectrum disorder and is not attributable to the physiological effects of another medical condition. Note: If criteria are met prior to the onset of schizophrenia, add “premorbid,” i.e., “schiz- oid personality disorder (premorbid).” The essential feature of schizoid personality disorder is a pervasive pattern of detachment from social relationships and a restricted range of expression of emotions in interpersonal settings. This pattern begins by early adulthood and is present in a variety of contexts.

1	Individuals with schizoid personality disorder appear to lack a desire for intimacy, seem indifferent to opportunities to develop close relationships, and do not seem to derive much satisfaction from being part of a family or other social group (Criterion A1). They prefer spending time by themselves, rather than being with other people. They often ap- pear to be socially isolated or ”loners" and almost always choose solitary activities or hob- bies that do not include interaction with others (Criterion A2). They prefer mechanical or abstract tasks, such as computer or mathematical games. They may have very little interest in having sexual experiences with another person (Criterion A3) and take pleasure in few, if any, activities (Criterion A4). There is usually a reduced experience of pleasure from sen- sory, bodily, or interpersonal experiences, such as walking on a beach at sunset or having sex. These individuals have no close friends or confidants, except possibly a first-degree

1	from sen- sory, bodily, or interpersonal experiences, such as walking on a beach at sunset or having sex. These individuals have no close friends or confidants, except possibly a first-degree relative (Criterion A5).

1	Individuals with schizoid personality disorder often seem indifferent to the approval or criticism of others and do not appear to be bothered by what others may think of them (Criterion A6). They may be oblivious to the normal subtleties of social interaction and of- ten do not respond appropriately to social cues so that they seem socially inept or super- ficial and self—absorbed. They usually display a ”bland” exterior without visible emotional reactivity and rarely reciprocate gestures or facial expressions, such as smiles or nods (Cri- terion A7). They claim that they rarely experience strong emotions such as anger and joy. They often display a constricted affect and appear cold and aloof. However, in those very able in revealing themselves, they may acknowledge having painful feelings, particularly related to social interactions.

1	Schizoid personality disorder should not be diagnosed if the pattern of behavior occurs exclusively during the course of schizophrenia, a bipolar or depressive disorder with psy- chotic features, another psychotic disorder, or autism spectrum disorder, or if it is attrib- utable to the physiological effects of a neurological (e.g., temporal lobe epilepsy) or another medical condition (Criterion B).

1	Individuals with schizoid personality disorder may have particular difficulty expressing anger, even in response to direct provocation, which contributes to the impression that they lack emotion. Their lives sometimes seem directionless, and they may appear to ”drift" in their goals. Such individuals often react passively to adverse circumstances and have difficulty responding appropriately to important life events. Because of their lack of social skills and lack of desire for sexual experiences, individuals with this disorder have few friendships, date infrequently, and often do not marry. Occupational functioning may be impaired, particularly if interpersonal involvement is required, but individuals with this disorder may do well when they work under conditions of social isolation. Particu- larly in response to stress, individuals with this disorder may experience very brief psy- chotic episodes (lasting minutes to hours). In some instances, schizoid personality disorder may appear

1	larly in response to stress, individuals with this disorder may experience very brief psy- chotic episodes (lasting minutes to hours). In some instances, schizoid personality disorder may appear as the premorbid antecedent of delusional disorder or schizophre- nia. Individuals with this disorder may sometimes develop major depressive disorder.

1	Schizoid personality disorder most often co-occurs with schizotypal, paranoid, and avoid- ant personality disorders. Schizoid personality disorder is uncommon in clinical settings. A prevalence estimate for schizoid personality based on a probability subsample from Part II of the National Co- morbidity Survey Replication suggests a prevalence of 4.9%. Data from the 2001—2002 3.1%. solitariness, poor peer relationships, and underachievement in school, which mark these children or adolescents as different and make them subject to teasing. Genetic and physiological. Schizoid personality disorder may have increased preva- lence in the relatives of individuals with schizophrenia or schizotypal personality disorder.

1	Genetic and physiological. Schizoid personality disorder may have increased preva- lence in the relatives of individuals with schizophrenia or schizotypal personality disorder. Individuals from a variety of cultural backgrounds sometimes exhibit defensive behaviors and interpersonal styles that may be erroneously labeled as ”schizoid.” For example, those who have moved from rural to metropolitan environments may react with ”emotional freezing” that may last for several months and manifest as solitary activities, constricted affect, and other deficits in communication. Immigrants from other countries are some- times mistakenly perceived as cold, hostile, or indifferent. Schizoid personality disorder is diagnosed slightly more often in males and may cause more impairment in them.

1	Schizoid personality disorder is diagnosed slightly more often in males and may cause more impairment in them. Other mental disorders with psychotic Symptoms. Schizoid personality disorder can be distinguished from delusional disorder, schizophrenia, and a bipolar or depressive dis- order with psychotic features because these disorders are all characterized by a period of persistent psychotic symptoms (e.g., delusions and hallucinations). To give an additional diagnosis of schizoid personality disorder, the personality disorder must have been present before the onset of psychotic symptoms and must persist when the psychotic symptoms are in remission. When an individual has a persistent psychotic disorder (e.g., schizophre- nia) that was preceded by schizoid personality disorder, schizoid personality disorder should also be recorded, followed by ”premorbid" in parentheses.

1	Autism spectrum disorder. There may be great difficulty differentiating individuals with schizoid personality disorder from those with milder forms of autism spectrum disorder, behaviors and interests. Personality change due to another medical condition. Schizoid personality disorder must be distinguished from personality change due to another medical condition, in which the traits that emerge are attributable to the effects of another medical condition on the central nervous system. Substance use disorders. Schizoid personality disorder must also be distinguished from symptoms that may develop in association with persistent substance use.

1	Other personality disorders and personality traits. Other personality disorders may be confused with schizoid personality disorder because they have certain features in com- mon. It is, therefore, important to distinguish among these disorders based on differences in their characteristic features. However, if an individual has personality features that meet criteria for one or more personality disorders in addition to schizoid personality dis- order, all can be diagnosed. Although characteristics of social isolation and restricted af- fectivity are common to schizoid, schizotypal, and paranoid personality disorders, by the lack of cognitive and perceptual distortions and from paranoid personality disorder by the lack of suspiciousness and paranoid ideation. The social isolation of schizoid per- sonality disorder can be distinguished from that of avoidant personality disorder, which is attributable to fear of being embarrassed or found inadequate and excessive anticipation of

1	schizoid per- sonality disorder can be distinguished from that of avoidant personality disorder, which is attributable to fear of being embarrassed or found inadequate and excessive anticipation of rejection. In contrast, people with schizoid personality disorder have a more pervasive detachment and limited desire for social intimacy. Individuals with obsessive-compulsive tion to work and discomfort with emotions, but they do have an underlying capacity for intimacy.

1	Individuals who are "loners" may display personality traits that might be considered schizoid. Only when these traits are inﬂexible and maladaptive and cause significant func- tional impairment or subjective distress do they constitute schizoid personality disorder. Diagnostic Criteria 301.22 (F21) A. A pen/asive pattern of social and interpersonal deficits marked by acute discomfort with, and reduced capacity for, close relationships as well as by cognitive or perceptual distortions and eccentricities of behavior, beginning by early adulthood and present in a variety of contexts, as indicated by five (or more) of the following: 1. Ideas of reference (excluding delusions of reference). 2. Odd beliefs or magical thinking that influences behavior and is inconsistent with subcultural norms (e.g., superstitiousness. belief in clairvoyance, telepathy, or “sixth sense"; in children and adolescents, bizarre fantasies or preoccupations).

1	3. Unusual perceptual experiences, including bodily illusions. 4. Odd thinking and speech (e.g., vague, circumstantial, metaphorical, overelaborate, or stereotyped). 5. Suspiciousness or paranoid ideation. Inappropriate or constricted affect. Behavior or appearance that is odd, eccentric. or peculiar. Lack of close friends or contidants other than first—degree relatives. Excessive social anxiety that does not diminish with familiarity and tends to be as- sociated with paranoid fears rather than negative judgments about self. B. Does not occur exclusively during the course of schizophrenia, a bipolar disorder or depressive disorder with psychotic features, another psychotic disorder, or autism spectrum disorder. $099.95» Note: If criteria are met prior to the onset of schizophrenia, add “premorbid," e.g., “schizo- typal personality disorder (premorbid)."

1	$099.95» Note: If criteria are met prior to the onset of schizophrenia, add “premorbid," e.g., “schizo- typal personality disorder (premorbid)." The essential feature of schizotypal personality disorder is a pervasive pattern of social and interpersonal deficits marked by acute discomfort with, and reduced capacity for, close relationships as well as by cognitive or perceptual distortions and eccentricities of be- havior. This pattern begins by early adulthood and is present in a variety of contexts.

1	Individuals with schizotypal personality disorder often have ideas of reference (i.e., in- correct interpretations of casual incidents and external events as having a particular and unusual meaning specifically for the person) (Criterion A1). These should be distin- guished from delusions of reference, in which the beliefs are held with delusional convic- tion. These individuals may be superstitious or preoccupied with paranormal phenomena that are outside the norms of their subculture (Criterion A2). They may feel that they have special powers to sense events before they happen or to read others’ thoughts. They may believe that they have magical control over others, which can be implemented directly (e.g., believing that their spouse’s taking the dog out for a walk is the direct result of think— ing an hour earlier it should be done) or indirectly through compliance with magical rit- uals (e.g., walking past a specific object three times to avoid a certain harmful outcome).

1	Perceptual alterations may be present (e.g., sensing that another person is present or hear— ing a voice murmuring his or her name) (Criterion A3). Their speech may include unusual or idiosyncratic phrasing and construction. It is often loose, digressive, or vague, but with- out actual derailment or incoherence (Criterion A4). Responses can be either overly con— crete or overly abstract, and words or concepts are sometimes applied in unusual ways (e.g., the individual may state that he or she was not "talkable" at work).

1	Individuals with this disorder are often suspicious and may have paranoid ideation (e.g., believing their colleagues at work are intent on undermining their reputation with the boss) (Criterion A5). They are usually not able to negotiate the full range of affects and interpersonal cuing required for successful relationships and thus often appear to interact with others in an inappropriate, stiff, or constricted fashion (Criterion A6). These individ- uals are often considered to be odd or eccentric because of unusual mannerisms, an often unkempt manner of dress that does not quite ”fit together,” and inattention to the usual social conventions (e.g., the individual may avoid eye contact, wear clothes that are ink stained and ill-fitting, and be unable to join in the give-and-take banter of co-workers) (Criterion A7).

1	Individuals with schizotypal personality disorder experience interpersonal related- ness as problematic and are uncomfortable relating to other people. Although they may express unhappiness about their lack of relationships, their behavior suggests a decreased desire for intimate contacts. As a result, they usually have no or few close friends or con- fidants other than a first-degree relative (Criterion A8). They are anxious in social situa- tions, particularly those involving unfamiliar people (Criterion A9). They will interact with other individuals when they have to but prefer to keep to themselves because they feel that they are different and just do not ”fit in.” Their social anxiety does not easily abate, even when they spend more time in the setting or become more familiar with the other people, because their anxiety tends to be associated with suspiciousness regarding others’ motivations. For example, when attending a dinner party, the individual with schizotypal personality

1	other people, because their anxiety tends to be associated with suspiciousness regarding others’ motivations. For example, when attending a dinner party, the individual with schizotypal personality disorder will not become more relaxed as time goes on, but rather may become increasingly tense and suspicious.

1	Schizotypal personality disorder should not be diagnosed if the pattem of behavior oc- curs exclusively during the course of schizophrenia, a bipolar or depressive disorder with psychotic features, another psychotic disorder, or autism spectrum disorder (Criterion B). Individuals with schizotypal personality disorder often seek treatment for the associated symptoms of anxiety or depression rather than for the personality disorder features per se.

1	Individuals with schizotypal personality disorder often seek treatment for the associated symptoms of anxiety or depression rather than for the personality disorder features per se. Particularly in response to stress, individuals with this disorder may experience transient psychotic episodes (lasting minutes to hours), although they usually are insufficient in du- ration to warrant an additional diagnosis such as brief psychotic disorder or schizophreni- form disorder. In some cases, clinically significant psychotic symptoms may develop that meet criteria for brief psychotic disorder, schizophreniform disorder, delusional disorder, or schizophrenia. Over half may have a history of at least one major depressive episode. From 30% to 50% of individuals diagnosed with this disorder have a concurrent diagnosis of major depressive disorder when admitted to a clinical setting. There is considerable co- occurrence with schizoid, paranoid, avoidant, and borderline personality disorders.

1	In community studies of schizotypal personality disorder, reported rates range from 0.6% in Norwegian samples to 4.6% in a U5. community sample. The prevalence of schizotypal personality disorder in clinical populations seems to be infrequent (0%—1.9%), with a higher estimated prevalence in the general population (3.9%) found in the National Epi- demiologic Survey on Alcohol and Related Conditions. Schizotypal personality disorder has a relatively stable course, with only a small propor- tion of individuals going on to develop schizophrenia or another psychotic disorder. solitariness, poor peer relationships, social anxiety, underachievement in school, hyper- sensitivity, peculiar thoughts and language, and bizarre fantasies. These children may ap- pear ”odd” or ”eccentric” and attract teasing.

1	Genetic and physiological. Schizotypal personality disorder appears to aggregate fa- milially and is more prevalent among the first-degree biological relatives of individuals with schizophrenia than among the general population. There may also be a modest in- crease in schizophrenia and other psychotic disorders in the relatives of probands with schizotypal personality disorder. Cognitive and perceptual distortions must be evaluated in the context of the individual’s cultural milieu. Pervasive culturally determined characteristics, particularly those regard- ing religious beliefs and rituals, can appear to be schizotypal to the uninformed outsider (e.g., voodoo, speaking in tongues, life beyond death, shamanism, mind reading, sixth sense, evil eye, magical beliefs related to health and illness). Schizotypal personality disorder may be slightly more common in males.

1	Schizotypal personality disorder may be slightly more common in males. Other mental disorders with psychotic symptoms. Schizotypal personality disorder can be distinguished from delusional disorder, schizophrenia, and a bipolar or depressive disorder with psychotic features because these disorders are all characterized by a period of persistent psychotic symptoms (e.g., delusions and hallucinations). To give an addi- tional diagnosis of schizotypal personality disorder, the personality disorder must have been present before the onset of psychotic symptoms and persist when the psychotic symptoms are in remission. When an individual has a persistent psychotic disorder (e.g., schizophrenia) that was preceded by schizotypal personality disorder, schizotypal per- sonality disorder should also be recorded, followed by ”premorbid" in parentheses.

1	Neurodevelopmental disorders. There may be great difficulty differentiating children with schizotypal personality disorder from the heterogeneous group of solitary, odd chil- dren whose behavior is characterized by marked social isolation, eccentricity, or peculiar- ities of language and whose diagnoses would probably include milder forms of autism spectrum disorder or language communication disorders. Communication disorders may be differentiated by the primacy and severity of the disorder in language and by the char- acteristic features of impaired language found in a specialized language assessment. Milder forms of autism spectrum disorder are differentiated by the even greater lack of so- cial awareness and emotional reciprocity and stereotyped behaviors and interests.

1	Milder forms of autism spectrum disorder are differentiated by the even greater lack of so- cial awareness and emotional reciprocity and stereotyped behaviors and interests. Personality change due to another medical condition. Schizotypal personality disor- der must be distinguished from personality change due to another medical condition, in which the traits that emerge are attributable to the effects of another medical condition on the central nervous system. Substance use disorders. Schizotypal personality disorder must also be distinguished from symptoms that may develop in association with persistent substance use.

1	Other personality disorders and personality traits. Other personality disorders may be confused with schizotypal personality disorder because they have certain features in common. It is, therefore, important to distinguish among these disorders based on differ- ences in their characteristic features. However, if an individual has personality features that meet criteria for one or more personality disorders in addition to schizotypal person- ality disorder, all can be diagnosed. Although paranoid and schizoid personality disor- ders may also be characterized by social detachment and restricted affect, schizotypal personality disorder can be distinguished from these two diagnoses by the presence of cognitive or perceptual distortions and marked eccentricity or oddness. Close relation- ships are limited in both schizotypal personality disorder and avoidant personality dis- order; however, in avoidant personality disorder an active desire for relationships is constrained by a fear of

1	are limited in both schizotypal personality disorder and avoidant personality dis- order; however, in avoidant personality disorder an active desire for relationships is constrained by a fear of rejection, whereas in schizotypal personality disorder there is a lack of desire for relationships and persistent detachment. Individuals with narcissistic personality disorder may also display suspiciousness, social withdrawal, or alienation, but in narcissistic personality disorder these qualities derive primarily from fears of hav- ing imperfections or ﬂaws revealed. Individuals with borderline personality disorder may also have transient, psychotic-like symptoms, but these are usually more closely related to affective shifts in response to stress (e.g., intense anger, anxiety, disappointment) and are usually more dissociative (e.g., derealization, depersonalization). In contrast, individuals with schizotypal personality disorder are more likely to have enduring psychotic-like symp- toms

1	and are usually more dissociative (e.g., derealization, depersonalization). In contrast, individuals with schizotypal personality disorder are more likely to have enduring psychotic-like symp- toms that may worsen under stress but are less likely to be invariably associated with pro- nounced affective symptoms. Although social isolation may occur in borderline personality disorder, it is usually secondary to repeated interpersonal failures due to angry outbursts and frequent mood shifts, rather than a result of a persistent lack of social contacts and de- sire for intimacy. Furthermore, individuals with schizotypal personality disorder do not usually demonstrate the impulsive or manipulative behaviors of the individual with bor- derline personality disorder. However, there is a high rate of co-occurrence between the two disorders, so that making such distinctions is not always feasible. Schizotypal features during adolescence may be reﬂective of transient emotional turmoil, rather

1	of co-occurrence between the two disorders, so that making such distinctions is not always feasible. Schizotypal features during adolescence may be reﬂective of transient emotional turmoil, rather than an endur- ing personality disorder.

1	Diagnostic Criteria 301.7 (F60.2) A. A pervasive pattern of disregard for and violation of the rights of others, occurring since age 15 years, as indicated by three (or more) of the following: 1. Failure to conform to social norms with respect to lawful behaviors, as indicated by repeatedly performing acts that are grounds for arrest. 2. Deceitfulness, as indicated by repeated lying, use of aliases, or conning others for personal profit or pleasure. Impulsivity or failure to plan ahead. lrritability and aggressiveness, as indicated by repeated physical fights or assaults. Reckless disregard for safety of self or others. Consistent irresponsibility. as indicated by repeated failure to sustain consistent work behavior or honor financial obligations. 7. Lack of remorse, as indicated by being indifferent to or rationalizing having hurt, mistreated, or stolen from another. 93.01:“? . The individual is at least age 18 years.

1	7. Lack of remorse, as indicated by being indifferent to or rationalizing having hurt, mistreated, or stolen from another. 93.01:“? . The individual is at least age 18 years. . There is evidence of conduct disorder with onset before age 15 years. . The occurrence of antisocial behavior is not exclusively during the course of schizo- phrenia or bipolar disorder. The essential feature of antisocial personality disorder is a pervasive pattern of disregard for, and violation of, the rights of others that begins in childhood or early adolescence and continues into adulthood. This pattern has also been referred to as psychopathy, sociopathy, or dyssocial personality disorder. Because deceit and manipulation are central features of an- tisocial personality disorder, it may be especially helpful to integrate information acquired from systematic clinical assessment with information collected from collateral sources.

1	For this diagnosis to be given, the individual must be at least age 18 years (Criterion B) and must have had a history of some symptoms of conduct disorder before age 15 years (Criterion C). Conduct disorder involves a repetitive and persistent pattern of behavior in which the basic rights of others or major age-appropriate societal norms or rules are vio- lated. The specific behaviors characteristic of conduct disorder fall into one of four cate- gories: aggression to people and animals, destruction of property, deceitfulness or theft, or serious violation of rules.

1	The pattern of antisocial behavior continues into adulthood. Individuals with antiso- cial personality disorder fail to conform to social norms with respect to lawful behavior (Criterion A1). They may repeatedly perform acts that are grounds for arrest (whether they are arrested or not), such as destroying property, harassing others, stealing, or pur- suing illegal occupations. Persons with this disorder disregard the wishes, rights, or feel- ings of others. They are frequently deceitful and manipulative in order to gain personal profit or pleasure (e.g., to obtain money, sex, or power) (Criterion A2). They may repeat- edly lie, use an alias, con others, or malinger. A pattern of impulsivity may be manifested by a failure to plan ahead (Criterion A3). Decisions are made on the spur of the moment, without forethought and without consideration for the consequences to self or others; this may lead to sudden changes of jobs, residences, or relationships. Individuals with antiso- cial

1	the moment, without forethought and without consideration for the consequences to self or others; this may lead to sudden changes of jobs, residences, or relationships. Individuals with antiso- cial personality disorder tend to be irritable and aggressive and may repeatedly get into physical fights or commit acts of physical assault (including spouse beating or child beat- ing) (Criterion A4). (Aggressive acts that are required to defend oneself or someone else are not considered to be evidence for this item.) These individuals also display a reckless disregard for the safety of themselves or others (Criterion A5). This may be evidenced in their driving behavior (i.e., recurrent speeding, driving while intoxicated, multiple acci- dents). They may engage in sexual behavior or substance use that has a high risk for harm- ful consequences. They may neglect or fail to care for a child in a way that puts the child in danger.

1	Individuals with antisocial personality disorder also tend to be consistently and ex- tremely irresponsible (Criterion A6). Irresponsible work behavior may be indicated by sig- nificant periods of unemployment despite available job opportunities, or by abandonment of several jobs without a realistic plan for getting another job. There may also be a pattern of repeated absences from work that are not explained by illness either in themselves or in their family. Financial irresponsibility is indicated by acts such as defaulting on debts, fail- ing to provide child support, or failing to support other dependents on a regular basis. In- dividuals with antisocial personality disorder show little remorse for the consequences of their acts (Criterion A7). They may be indifferent to, or provide a superficial rationaliza- tion for, having hurt, mistreated, or stolen from someone (e.g., ”life’s unfair,” "losers de- serve to lose”). These individuals may blame the victims for being foolish,

1	a superficial rationaliza- tion for, having hurt, mistreated, or stolen from someone (e.g., ”life’s unfair,” "losers de- serve to lose”). These individuals may blame the victims for being foolish, helpless, or deserving their fate (e.g., "he had it coming anyway”); they may minimize the harmful consequences of their actions; or they may simply indicate complete indifference. They generally fail to compensate or make amends for their behavior. They may believe that everyone is out to "help number one” and that one should stop at nothing to avoid being pushed around.

1	The antisocial behavior must not occur exclusively during the course of schizophrenia or bipolar disorder (Criterion D).

1	Individuals with antisocial personality disorder frequently lack empathy and tend to be callous, cynical, and contemptuous of the feelings, rights, and sufferings of others. They may have an inﬂated and arrogant self—appraisal (e.g., feel that ordinary work is beneath excessively opinionated, self—assured, or cocky. They may display a glib, superficial charm and can be quite voluble and verbally facile (e.g., using technical terms or jargon that might impress someone who is unfamiliar with the topic). Lack of empathy, inﬂated self- appraisal, and superficial charm are features that have been commonly included in tradi- tional conceptions of psychopathy that may be particularly distinguishing of the disorder and more predictive of recidivism in prison or forensic settings, where criminal, delin— quent, or aggressive acts are likely to be nonspecific. These individuals may also be irre- sponsible and exploitative in their sexual relationships. They may have a history of many sexual

1	delin— quent, or aggressive acts are likely to be nonspecific. These individuals may also be irre- sponsible and exploitative in their sexual relationships. They may have a history of many sexual partners and may never have sustained a monogamous relationship. They may be irresponsible as parents, as evidenced by malnutrition of a child, an illness in the child re- sulting from a lack of minimal hygiene, a child’s dependence on neighbors or nonresident relatives for food or shelter, a failure to arrange for a caretaker for a young child when the individual is away from home, or repeated squandering of money required for household necessities. These individuals may receive dishonorable discharges from the armed ser- vices, may fail to be self-supporting, may become impoverished or even homeless, or may spend many years in penal institutions. Individuals with antisocial personality disorder are more likely than people in the general population to die prematurely by violent means (e.g.,

1	or may spend many years in penal institutions. Individuals with antisocial personality disorder are more likely than people in the general population to die prematurely by violent means (e.g., suicide, accidents, homicides).

1	Individuals with antisocial personality disorder may also experience dysphoria, in- cluding complaints of tension, inability to tolerate boredom, and depressed mood. They may have associated anxiety disorders, depressive disorders, substance use disorders, 50- matic symptom disorder, gambling disorder, and other disorders of impulse control. In- dividuals with antisocial personality disorder also often have personality features that meet criteria for other personality disorders, particularly borderline, histrionic, and nar- cissistic personality disorders. The likelihood of developing antisocial personality disor- der in adult life is increased if the individual experienced childhood onset of conduct disorder (before age 10 years) and accompanying attention-deficit/hyperactivity disorder. Child abuse or neglect, unstable or erratic parenting, or inconsistent parental discipline may increase the likelihood that conduct disorder will evolve into antisocial personality disorder.

1	Child abuse or neglect, unstable or erratic parenting, or inconsistent parental discipline may increase the likelihood that conduct disorder will evolve into antisocial personality disorder. Twelve-month prevalence rates of antisocial personality disorder, using criteria from pre— vious DSMs, are between 0.2% and 3.3%. The highest prevalence of antisocial personality disorder (greater than 70%) is among most severe samples of males with alcohol use dis- order and from substance abuse clinics, prisons, or other forensic settings. Prevalence is higher in samples affected by adverse socioeconomic (i.e., poverty) or sociocultural (i.e., migration) factors.

1	as the individual grows older, particularly by the fourth decade of life. Although this re- mission tends to be particularly evident with respect to engaging in criminal behavior, there is likely to be a decrease in the full spectrum of antisocial behaviors and substance use. By definition, antisocial personality cannot be diagnosed before age 18 years. Genetic and physiological. Antisocial personality disorder is more common among the first-degree biological relatives of those with the disorder than in the general population.

1	The risk to biological relatives of females with the disorder tends to be higher than the risk to biological relatives of males with the disorder. Biological relatives of individuals with this disorder are also at increased risk for somatic symptom disorder and substance use disorders. Within a family that has a member with antisocial personality disorder, males more often have antisocial personality disorder and substance use disorders, whereas fe- males more often have somatic symptom disorder. However, in such families, there is an increase in prevalence of all of these disorders in both males and females compared with the general population. Adoption studies indicate that both genetic and environmental factors contribute to the risk of developing antisocial personality disorder. Both adopted and biological children of parents with antisocial personality disorder have an increased risk of developing antisocial personality disorder, somatic symptom disorder, and sub- stance use

1	adopted and biological children of parents with antisocial personality disorder have an increased risk of developing antisocial personality disorder, somatic symptom disorder, and sub- stance use disorders. Adopted-away children resemble their biological parents more than their adoptive parents, but the adoptive family environment inﬂuences the risk of devel- oping a personality disorder and related psychopathology.

1	Antisocial personality disorder appears to be associated with low socioeconomic status and urban settings. Concerns have been raised that the diagnosis may at times be misap— plied to individuals in settings in which seemingly antisocial behavior may be part of a protective survival strategy. In assessing antisocial traits, it is helpful for the clinician to consider the social and economic context in which the behaviors occur. Antisocial personality disorder is much more common in males than in females. There has males, particularly because of the emphasis on aggressive items in the definition of con- duct disorder. The diagnosis of antisocial personality disorder is not given to individuals younger than 18 years and is given only if there is a history of some symptoms of conduct disorder be- fore age 15 years. For individuals older than 18 years, a diagnosis of conduct disorder is given only if the criteria for antisocial personality disorder are not met.

1	Substance use disorders. When antisocial behavior in an adult is associated with a substance use disorder, the diagnosis of antisocial personality disorder is not made unless the signs of antisocial personality disorder were also present in childhood and have con- tinued into adulthood. When substance use and antisocial behavior both began in childhood and continued into adulthood, both a substance use disorder and antisocial personality disorder should be diagnosed if the criteria for both are met, even though some antisocial acts may be a consequence of the substance use disorder (e.g., illegal selling of drugs, thefts to obtain money for drugs). Schizophrenia and bipolar disorders. Antisocial behavior that occurs exclusively dur- ing the course of schizophrenia or a bipolar disorder should not be diagnosed as antisocial personality disorder.

1	Other personality disorders. Other personality disorders may be confused with antiso- cial personality disorder because they have certain features in common. It is therefore im- portant to distinguish among these disorders based on differences in their characteristic features. However, if an individual has personality features that meet criteria for one or more personality disorders in addition to antisocial personality disorder, all can be diag- nosed. Individuals with antisocial personality disorder and narcissistic personality disor- der share a tendency to be tough-minded, glib, superficial, exploitative, and lack empathy.

1	However, narcissistic personality disorder does not include characteristics of impulsivity, aggression, and deceit. In addition, individuals with antisocial personality disorder may not be as needy of the admiration and envy of others, and persons with narcissistic per- sonality disorder usually lack the history of conduct disorder in childhood or criminal behavior in adulthood. Individuals with antisocial personality disorder and histrionic personality disorder share a tendency to be impulsive, superficial, excitement seeking, reckless, seductive, and manipulative, but persons with histrionic personality disorder tend to be more exaggerated in their emotions and do not characteristically engage in an- tisocial behaviors. Individuals with histrionic and borderline personality disorders are manipulative to gain nurturance, whereas those with antisocial personality disorder are manipulative to gain profit, power, or some other material gratification. Individuals with antisocial

1	are manipulative to gain nurturance, whereas those with antisocial personality disorder are manipulative to gain profit, power, or some other material gratification. Individuals with antisocial personality disorder tend to be less emotionally unstable and more aggressive than those with borderline personality disorder. Although antisocial behavior may be present in some individuals with paranoid personality disorder, it is not usually moti- vated by a desire for personal gain or to exploit others as in antisocial personality disorder, but rather is more often attributable to a desire for revenge.

1	Criminal behavior not associated with a personality disorder. Antisocial personality disorder must be distinguished from criminal behavior undertaken for gain that is not ac- companied by the personality features characteristic of this disorder. Only when antisocial personality traits are inﬂexible, maladaptive, and persistent and cause significant func- tional impairment or subjective distress do they constitute antisocial personality disorder. Diagnostic Criteria 301.83 (F60.3)

1	Diagnostic Criteria 301.83 (F60.3) A pervasive pattern of instability of interpersonal relationships, seIf-image, and affects, and marked impulsivity, beginning by early adulthood and present in a variety of contexts, as indicated by five (or more) of the following: 1. Frantic efforts to avoid real or imagined abandonment. (Note: Do not include suicidal or self-mutilating behavior covered in Criterion 5.) 2. A pattern of unstable and intense interpersonal relationships characterized by alternat- ing between extremes of idealization and devaluation. Identity disturbance: markedly and persistently unstable seIf-image or sense of self. 4. impulsivity in at least two areas that are potentially self—damaging (e.g., spending, sex, substance abuse, reckless driving, binge eating). (Note: Do not include suicidal or self- mutilating behavior covered in Criterion 5.) 5. Recurrent suicidal behavior, gestures. or threats. or seIf-mutilating behavior.

1	6. Affective instability due to a maiked reactivity of mood (e.g., intense episodic dysphoria, irritability, or anxiety usually lasting a few hours and only rarely more than a few days). 7. Chronic feelings of emptiness. 8. Inappropriate, intense anger or difficulty controlling anger (e.g., frequent displays of temper, constant anger, recurrent physical fights). 9. Transient, stress-related paranoid ideation or severe dissociative symptoms. .09 The essential feature of borderline personality disorder is a pervasive pattern of instability of interpersonal relationships, self—image, and affects, and marked impulsivity that begins by early adulthood and is present in a variety of contexts.

1	Individuals with borderline personality disorder make frantic efforts to avoid real or imagined abandonment (Criterion 1). The perception of impending separation or rejection, or the loss of external structure, can lead to profound changes in self—image, affect, cognition, and behavior. These individuals are very sensitive to environmental circumstances. They ex- perience intense abandonment fears and inappropriate anger even when faced with a real- istic time-limited separation or when there are unavoidable changes in plans (e.g., sudden despair in reaction to a clinician’s announcing the end of the hour; panic or fury when some- one important to them is just a few minutes late or must cancel an appointment). They may believe that this “abandonment” implies they are ”bad.” These abandonment fears are re- lated to an intolerance of being alone and a need to have other people with them. Their frantic efforts to avoid abandonment may include impulsive actions such as self—mutilating or

1	fears are re- lated to an intolerance of being alone and a need to have other people with them. Their frantic efforts to avoid abandonment may include impulsive actions such as self—mutilating or sui- cidal behaviors, which are described separately in Criterion 5.

1	Individuals with borderline personality disorder have a pattern of unstable and intense relationships (Criterion 2). They may idealize potential caregivers or lovers at the first or second meeting, demand to spend a lot of time together, and share the most intimate details early in a relationship. However, they may switch quickly from idealizing other people to devaluing them, feeling that the other person does not care enough, does not give enough, or is not ”there” enough. These individuals can empathize with and nurture other people, but only with the expectation that the other person will "be there” in return to meet their own needs on demand. These individuals are prone to sudden and dramatic shifts in their view of others, who may alternatively be seen as beneficent supports or as cruelly punitive. Such shifts often reﬂect disillusionment with a caregiver whose nurturing qualities had been idealized or whose rejection or abandonment is expected.

1	Such shifts often reﬂect disillusionment with a caregiver whose nurturing qualities had been idealized or whose rejection or abandonment is expected. stable self—image or sense of self (Criterion 3). There are sudden and dramatic shifts in self- image, characterized by shifting goals, values, and vocational aspirations. There may be sudden changes in opinions and plans about career, sexual identity, values, and types of friends. These individuals may suddenly change from the role of a needy supplicant for help to that of a righteous avenger of past mistreatment. Although they usually have a self- image that is based on being bad or evil, individuals with this disorder may at times have feelings that they do not exist at all. Such experiences usually occur in situations in which the individual feels a lack of a meaningful relationship, nurturing, and support. These in- dividuals may show worse performance in unstructured work or school situations.

1	Individuals with borderline personality disorder display impulsivity in at least two areas that are potentially self—damaging (Criterion 4). They may gamble, spend money irrespon- sibly, binge eat, abuse substances, engage in unsafe sex, or drive recklessly. Individuals with this disorder display recurrent suicidal behavior, gestures, or threats, or self—mutilat- ing behavior (Criterion 5). Completed suicide occurs in 8%—10% of such individuals, and self-mutilative acts (e.g., cutting or burning) and suicide threats and attempts are very common. Recurrent suicidality is often the reason that these individuals present for help. These self—destructive acts are usually precipitated by threats of separation or rejection or by expectations that the individual assumes increased responsibility. Self—mutilation may occur during dissociative experiences and often brings relief by reaffirming the ability to feel or by expiating the individual’s sense of being evil.

1	Individuals with borderline personality disorder may display affective instability that is due to a marked reactivity of mood (e.g., intense episodic dysphoria, irritability, or anx- iety usually lasting a few hours and only rarely more than a few days) (Criterion 6). The basic dysphoric mood of those with borderline personality disorder is often disrupted by periods of anger, panic, or despair and is rarely relieved by periods of well-being or satis- faction. These episodes may reflect the individual’s extreme reactivity to interpersonal stresses. Individuals with borderline personality disorder may be troubled by chronic feel- ings of emptiness (Criterion 7). Easily bored, they may constantly seek something to do.

1	Individuals with this disorder frequently express inappropriate, intense anger or have dif- ficulty controlling their anger (Criterion 8). They may display extreme sarcasm, enduring bitterness, or verbal outbursts. The anger is often elicited when a caregiver or lover is seen as neglectful, withholding, uncaring, or abandoning. Such expressions of anger are often followed by shame and guilt and contribute to the feeling they have of being evil. During periods of extreme stress, transient paranoid ideation or dissociative symptoms (e.g., de- personalization) may occur (Criterion 9), but these are generally of insufficient severity or duration to warrant an additional diagnosis. These episodes occur most frequently in re- sponse to a real or imagined abandonment. Symptoms tend to be transient, lasting min- utes or hours. The real or perceived return of the caregiver’s nurturance may result in a remission of symptoms.

1	Individuals with borderline personality disorder may have a pattern of undermining themselves at the moment a goal is about to be realized (e.g., dropping out of school just before graduation; regressing severely after a discussion of how well therapy is going; de- stroying a good relationship just when it is clear that the relationship could last). Some in- dividuals develop psychotic—like symptoms (e.g., hallucinations, body-image distortions, ideas of reference, hypnagogic phenomena) during times of stress. Individuals with this disorder may feel more secure with transitional objects (i.e., a pet or inanimate possession) than in interpersonal relationships. Premature death from suicide may occur in individu- als with this disorder, especially in those with co-occurring depressive disorders or sub- stance use disorders. Physical handicaps may result from self—inﬂicted abuse behaviors or failed suicide attempts. Recurrent job losses, interrupted education, and separation or di- vorce

1	or sub- stance use disorders. Physical handicaps may result from self—inﬂicted abuse behaviors or failed suicide attempts. Recurrent job losses, interrupted education, and separation or di- vorce are common. Physical and sexual abuse, neglect, hostile conflict, and early parental loss are more common in the childhood histories of those with borderline personality dis- order. Common co-occurring disorders include depressive and bipolar disorders, sub- stance use disorders, eating disorders (notably bulimia nervosa), posttraumatic stress disorder, and attention—deficit/hyperactivity disorder. Borderline personality disorder also frequently co-occurs with the other personality disorders.

1	The median population prevalence of borderline personality disorder is estimated to be 1.6% but may be as high as 5.9%. The prevalence of borderline personality disorder is about 6% in primary care settings, about 10% among individuals seen in outpatient mental health clinics, and about 20% among psychiatric inpatients. The prevalence of borderline personality disorder may decrease in older age groups.

1	There is considerable variability in the course of borderline personality disorder. The most common pattern is one of chronic instability in early adulthood, with episodes of serious affective and impulsive dyscontrol and high levels of use of health and mental health re— sources. The impairment from the disorder and the risk of suicide are greatest in the young-adult years and gradually wane with advancing age. Although the tendency to- ward intense emotions, impulsivity, and intensity in relationships is often lifelong, indi- sometime during the first year. During their 305 and 40s, the majority of individuals with this disorder attain greater stability in their relationships and vocational functioning. Fol- low-up studies of individuals identified through outpatient mental health clinics indicate that after about 10 years, as many as half of the individuals no longer have a pattern of be- havior that meets full criteria for borderline personality disorder.

1	Genetic and physiological. Borderline personality disorder is about five times more common among first-degree biological relatives of those with the disorder than in the gen- eral population. There is also an increased familial risk for substance use disorders, anti- social personality disorder, and depressive or bipolar disorders. The pattern of behavior seen in borderline personality disorder has been identified in many settings around the world. Adolescents and young adults with identity problems (especially give the impression of borderline personality disorder. Such situations are characterized by emotional instability, ”existential” dilemmas, uncertainty, anxiety-provoking choices, con- ﬂicts about sexual orientation, and competing social pressures to decide on careers. Borderline personality disorder is diagnosed predominantly (about 75%) in females.

1	Borderline personality disorder is diagnosed predominantly (about 75%) in females. Depressive and bipolar disorders. Borderline personality disorder often co-occurs with depressive or bipolar disorders, and when criteria for both are met, both may be diagnosed. Because the cross-sectional presentation of borderline personality disorder can be mimicked by an episode of depressive or bipolar disorder, the clinician should avoid giving an addi- tional diagnosis of borderline personality disorder based only on cross-sectional presenta- tion without having documented that the pattern of behavior had an early onset and a long- standing course.

1	Other personality disorders. Other personality disorders may be confused with border- line personality disorder because they have certain features in common. It is therefore im- portant to distinguish among these disorders based on differences in their characteristic features. However, if an individual has personality features that meet criteria for one or more personality disorders in addition to borderline personality disorder, all can be diag— nosed. Although histrionic personality disorder can also be characterized by attention seek- ing, manipulative behavior, and rapidly shifting emotions, borderline personality disorder is distinguished by self-destructiveness, angry disruptions in close relationships, and chronic feelings of deep emptiness and loneliness. Paranoid ideas or illusions may be pres- ent in both borderline personality disorder and schizotypal personality disorder, but these symptoms are more transient, interpersonally reactive, and responsive to external structur-

1	may be pres- ent in both borderline personality disorder and schizotypal personality disorder, but these symptoms are more transient, interpersonally reactive, and responsive to external structur- ing in borderline personality disorder. Although paranoid personality disorder and narcis- sistic personality disorder may also be characterized by an angry reaction to minor stimuli, the relative stability of self-image, as well as the relative lack of self-destmctiveness, impul- sivity, and abandonment concerns, distinguishes these disorders from borderline person- ality disorder. Although antisocial personality disorder and borderline personality disorder are both characterized by manipulative behavior, individuals with antisocial personality disorder are manipulative to gain profit, power, or some other material gratification, whereas the goal in borderline personality disorder is directed more toward gaining the con— cern of caretakers. Both dependent personality disorder and borderline

1	some other material gratification, whereas the goal in borderline personality disorder is directed more toward gaining the con— cern of caretakers. Both dependent personality disorder and borderline personality disorder are characterized by fear of abandonment; however, the individual with borderline person- ality disorder reacts to abandonment with feelings of emotional emptiness, rage, and de- mands, whereas the individual with dependent personality disorder reacts with increasing appeasement and submissiveness and urgently seeks a replacement relationship to provide caregiving and support. Borderline personality disorder can further be distinguished from dependent personality disorder by the typical pattern of Lmstable and intense relationships.

1	Personality change due to another medical condition. Borderline personality disor- der must be distinguished from personality change due to another medical condition, in which the traits that emerge are attributable to the effects of another medical condition on the central nervous system. Substance use disorders. Borderline personality disorder must also be distinguished from symptoms that may develop in association with persistent substance use. Identity problems. Borderline personality disorder should be distinguished from an identity problem, which is reserved for identity concerns related to a developmental phase (e.g., adolescence) and does not qualify as a mental disorder. .. Histrionic Personality Disorder Diagnostic Criteria 301.50 (F60.4)

1	.. Histrionic Personality Disorder Diagnostic Criteria 301.50 (F60.4) A pervasive pattern of excessive emotionality and attention seeking, beginning by early adult- hood and present in a variety of contexts, as indicated by five (or more) of the following: 1. Is uncomfortable in situations in which he or she is not the center of attention. 2. Interaction with others is often characterized by inappropriate sexually seductive or provocative behavior. Displays rapidly shifting and shallow expression of emotions. Consistently uses physical appearance to draw attention to self. Has a style of speech that is excessively impressionistic and lacking in detail. Shows self-dramatization, theatricality, and exaggerated expression of emotion. Is suggestible (i.e., easily influenced by others or circumstances). Considers relationships to be more intimate than they actually are.

1	Is suggestible (i.e., easily influenced by others or circumstances). Considers relationships to be more intimate than they actually are. The essential feature of histrionic personality disorder is pervasive and excessive emotion- ality and attention-seeking behavior. This pattern begins by early adulthood and is pres- ent in a variety of contexts. Individuals with histrionic personality disorder are uncomfortable or feel unappreci- ated when they are not the center of attention (Criterion 1). Often lively and dramatic, they tend to draw attention to themselves and may initially charm new acquaintances by their enthusiasm, apparent openness, or ﬂirtatiousness. These qualities wear thin, however, as these individuals continually demand to be the center of attention. They commandeer the role of ”the life of the party." If they are not the center of attention, they may do something dramatic (e.g., make up stories, create a scene) to draw the focus of attention to themselves.

1	This need is often apparent in their behavior with a clinician (e.g., being ﬂattering, bring- ing gifts, providing dramatic descriptions of physical and psychological symptoms that are replaced by new symptoms each visit).

1	The appearance and behavior of individuals with this disorder are often inappropri- ately sexually provocative or seductive (Criterion 2). This behavior not only is directed to- ward persons in whom the individual has a sexual or romantic interest but also occurs in a wide variety of social, occupational, and professional relationships beyond what is ap- propriate for the social context. Emotional expression may be shallow and rapidly shifting (Criterion 3). Individuals with this disorder consistently use physical appearance to draw attention to themselves (Criterion 4). They are overly concerned with impressing others by their appearance and expend an excessive amount of time, energy, and money on clothes and grooming. They may “fish for compliments” regarding appearance and may be easily they regard as unﬂattering.

1	These individuals have a style of speech that is excessively impressionistic and lacking in detail (Criterion 5). Strong opinions are expressed with dramatic ﬂair, but underlying reasons are usually vague and diffuse, without supporting facts and details. For example, an individual with histrionic personality disorder may comment that a certain individual is a wonderful human being, yet be unable to provide any specific examples of good qual- ities to support this opinion. Individuals with this disorder are characterized by self- dramatization, theatricality, and an exaggerated expression of emotion (Criterion 6). They may embarrass friends and acquaintances by an excessive public display of emotions (e.g., embracing casual acquaintances with excessive ardor, sobbing uncontrollably on minor sentimental occasions, having temper tantrums). However, their emotions often seem to be turned on and off too quickly to be deeply felt, which may lead others to accuse the in- dividual of faking

1	sentimental occasions, having temper tantrums). However, their emotions often seem to be turned on and off too quickly to be deeply felt, which may lead others to accuse the in- dividual of faking these feelings.

1	Individuals with histrionic personality disorder have a high degree of suggestibility (Cri- terion 7). Their opinions and feelings are easily inﬂuenced by others and by current fads. They may be overly trusting, especially of strong authority figures whom they see as mag- ically solving their problems. They have a tendency to play hunches and to adopt convic- tions quickly. Individuals with this disorder often consider relationships more intimate than they actually are, describing almost every acquaintance as ”my dear, dear friend” or referring to physicians met only once or twice under professional circumstances by their first names (Criterion 8).

1	Individuals with histrionic personality disorder may have difficulty achieving emotional in- timacy in romantic or sexual relationships. Without being aware of it, they often act out a role (e.g., ”victim” or ”princess”) in their relationships to others. They may seek to control their partner through emotional manipulation or seductiveness on one level, while display- ing a marked dependency on them at another level. Individuals with this disorder often have impaired relationships with same—sex friends because their sexually provocative inter- personal style may seem a threat to their friends’ relationships. These individuals may also alienate friends with demands for constant attention. They often become depressed and up- set when they are not the center of attention. They may crave novelty, stimulation, and ex- citement and have a tendency to become bored with their usual routine. These individuals are often intolerant of, or frustrated by, situations that involve delayed

1	crave novelty, stimulation, and ex- citement and have a tendency to become bored with their usual routine. These individuals are often intolerant of, or frustrated by, situations that involve delayed gratification, and their actions are often directed at obtaining immediate satisfaction. Although they often ini- tiate a job or project with great enthusiasm, their interest may lag quickly. Longer-term re- lationships may be neglected to make way for the excitement of new relationships.

1	The actual risk of suicide is not known, but clinical experience suggests that individu- als with this disorder are at increased risk for suicidal gestures and threats to get attention and coerce better caregiving. Histrionic personality disorder has been associated with higher rates of somatic symptom disorder, conversion disorder (functional neurological symptom disorder), and major depressive disorder. Borderline, narcissistic, antisocial, and dependent personality disorders often co—occur. Data from the 2001—2002 National Epidemiologic Survey on Alcohol and Related Condi- tions suggest a prevalence of histrionic personality of 1.84%.

1	Data from the 2001—2002 National Epidemiologic Survey on Alcohol and Related Condi- tions suggest a prevalence of histrionic personality of 1.84%. Norms for interpersonal behavior, personal appearance, and emotional expressiveness vary widely across cultures, genders, and age groups. Before considering the various traits (e.g., emotionality, seductiveness, dramatic interpersonal style, novelty seeking, sociabil- ity, charm, impressionability, a tendency to somatization) to be evidence of histrionic per- sonality disorder, it is important to evaluate whether they cause clinically significant impairment or distress. In clinical settings, this disorder has been diagnosed more frequently in females; however, the sex ratio is not significantly different from the sex ratio of females within the respective clinical setting. In contrast, some studies using structured assessments report similar prev- alence rates among males and females.

1	Other personality disorders and personality traits. Other personality disorders may be confused with histrionic personality disorder because they have certain features in common. It is therefore important to distinguish among these disorders based on differ- ences in their characteristic features. However, if an individual has personality features that meet criteria for one or more personality disorders in addition to histrionic personal- ity disorder, all can be diagnosed. Although borderline personality disorder can also be characterized by attention seeking, manipulative behavior, and rapidly shifting emotions, it is distinguished by self—destructiveness, angry disruptions in close relationships, and chronic feelings of deep emptiness and identity disturbance. Individuals with antisocial personality disorder and histrionic personality disorder share a tendency to be impulsive, superficial, excitement seeking, reckless, seductive, and manipulative, but persons with histrionic

1	personality disorder and histrionic personality disorder share a tendency to be impulsive, superficial, excitement seeking, reckless, seductive, and manipulative, but persons with histrionic personality disorder tend to be more exaggerated in their emotions and do not characteristically engage in antisocial behaviors. Individuals with histrionic personality disorder are manipulative to gain nurturance, whereas those with antisocial personality disorder are manipulative to gain profit, power, or some other material gratification. Al- though individuals with narcissistic personality disorder also crave attention from others, they usually want praise for their ”superiority,” whereas individuals with histrionic per- sonality disorder are willing to be viewed as fragile or dependent if this is instrumental in getting attention. Individuals with narcissistic personality disorder may exaggerate the intimacy of their relationships with other people, but they are more apt to emphasize the

1	is instrumental in getting attention. Individuals with narcissistic personality disorder may exaggerate the intimacy of their relationships with other people, but they are more apt to emphasize the ”VIP” status or wealth of their friends. In dependent personality disorder, the individual is excessively dependent on others for praise and guidance, but is without the ﬂamboyant, exaggerated, emotional features of individuals with histrionic personality disorder.

1	Many individuals may display histrionic personality traits. Only when these traits are inﬂexible, maladaptive, and persisting and cause significant functional impairment or subjective distress do they constitute histrionic personality disorder. Personality change due to another medical condition. Histrionic personality disorder must be distinguished from personality change due to another medical condition, in which the traits that emerge are attributable to the effects of another medical condition on the central nervous system. Substance use disorders. The disorder must also be distinguished from symptoms that may develop in association with persistent substance use. Diagnostic Criteria 301.81 (F60.81)

1	Substance use disorders. The disorder must also be distinguished from symptoms that may develop in association with persistent substance use. Diagnostic Criteria 301.81 (F60.81) A pervasive pattern of grandiosity (in fantasy or behavior), need for admiration. and lack of empathy, beginning by early adulthood and present in a variety of contexts, as indicated by five (or more) of the following: 1. Has a grandiose sense of seIf-importance (e.g., exaggerates achievements and talents, expects to be recognized as superior without commensurate achievements). 2. Is preoccupied with fantasies of unlimited success, power, brilliance, beauty. or ideal love. 3. Believes that he or she is “special” and unique and can only be understood by, or should associate with, other special or high-status people (or institutions). 4. Requires excessive admiration.

1	3. Believes that he or she is “special” and unique and can only be understood by, or should associate with, other special or high-status people (or institutions). 4. Requires excessive admiration. 5. Has a sense of entitlement (i.e., unreasonable expectations of especially favorable treatment or automatic compliance with his or her expectations). 6. Is interpersonally exploitative (i.e., takes advantage of others to achieve his or her own ends). 7. Lacks empathy: is unwilling to recognize or identify with the feelings and needs of others. Is often envious of others or believes that others are envious of him or her. 9. Shows arrogant, haughty behaviors or attitudes. The essential feature of narcissistic personality disorder is a pervasive pattern of grandi- osity, need for admiration, and lack of empathy that begins by early adulthood and is pres- ent in a variety of contexts. Individuals with this disorder have a grandiose sense of self-importance (Criterion 1).

1	Individuals with this disorder have a grandiose sense of self-importance (Criterion 1). They routinely overestimate their abilities and inﬂate their accomplishments, often appealing boastful and pretentious. They may blithely assume that others attribute the same value to their efforts and may be surprised when the praise they expect and feel they deserve is not forthcoming. Often implicit in the inﬂated judgments of their own accomplishments is an un- derestimation (devaluation) of the contributions of others. Individuals with narcissistic per- sonality disorder are often preoccupied with fantasies of unlimited success, power, brilliance, beauty, or ideal love (Criterion 2). They may ruminate about “long overdue" admiration and privilege and compare themselves favorably with famous or privileged people.

1	Individuals with narcissistic personality disorder believe that they are superior, spe- cial, or unique and expect others to recognize them as such (Criterion 3). They may feel that they can only be understood by, and should only associate with, other people who are special or of high status and may attribute “unique,” “perfect,” or "gifted” qualities to those with whom they associate. Individuals with this disorder believe that their needs are spe- cial and beyond the ken of ordinary people. Their own self—esteem is enhanced (i.e., ”mir- rored”) by the idealized value that they assign to those with whom they associate. They are likely to insist on having only the ”top" person (doctor, lawyer, hairdresser, instructor) or being affiliated with the ”best" institutions but may devalue the credentials of those who dis- appoint them.

1	Individuals with this disorder generally require excessive admiration (Criterion 4). Their self—esteem is almost invariably very fragile. They may be preoccupied with how well they are doing and how favorably they are regarded by others. This often takes the form of a need for constant attention and admiration. They may expect their arrival to be greeted with great fanfare and are astonished if others do not covet their possessions. They may constantly fish for compliments, often with great charm. A sense of entitlement is evident in these individ- uals’ unreasonable expectation of especially favorable treatment (Criterion 5). They expect to be catered to and are puzzled or furious when this does not happen. For example, they may assume that they do not have to wait in line and that their priorities are so important that others should defer to them, and then get irritated when others fail to assist ”in their very important work.” This sense of entitlement, combined with a lack of

1	priorities are so important that others should defer to them, and then get irritated when others fail to assist ”in their very important work.” This sense of entitlement, combined with a lack of sensitivity to the wants and needs of others, may result in the conscious or unwitting exploitation of others (Criterion 6). They expect to be given whatever they want or feel they need, no matter what it might mean to others. For example, these individuals may expect great dedication from others and may overwork them without regard for the impact on their lives. They tend to form friendships or romantic relationships only if the other person seems likely to advance their purposes or otherwise enhance their self—esteem. They often usurp special privileges and extra resources that they believe they deserve because they are so special.

1	Individuals with narcissistic personality disorder generally have a lack of empathy and have difficulty recognizing the desires, subjective experiences, and feelings of others (Crite- rion 7). They may assume that others are totally concerned about their welfare. They tend to discuss their own concerns in inappropriate and lengthy detail, while failing to recognize that others also have feelings and needs. They are often contemptuous and impatient with others who talk about their own problems and concerns. These individuals may be oblivious to the hurt their remarks may inﬂict (e.g., exuberantly telling a former lover that ”I am now in the relationshii) of a lifetimel”; boasting of health in front of someone who is sick). When recognized, the needs, desires, or feelings of others are likely to be viewed disparagingly as signs of weakness or vulnerability. Those who relate to individuals with narcissistic person- ality disorder typically find an emotional coldness and lack of

1	are likely to be viewed disparagingly as signs of weakness or vulnerability. Those who relate to individuals with narcissistic person- ality disorder typically find an emotional coldness and lack of reciprocal interest.

1	These individuals are often envious of others or believe that others are envious of them (Criterion 8). They may begrudge others their successes or possessions, feeling that they better deserve those achievements, admiration, or priw'leges. They may harshly devalue the contri- butions of others, particularly when those individuals have received acknowledgment or praise for their accomplishments. Arrogant, haughty behaviors characterize these individuals; they often display snobbish, disdainful, or patronizing attitudes (Criterion 9). For example, an individual with this disorder may complain about a clumsy waiter’s ”rudeness” or ”stupidity” or conclude a medical evaluation with a condescending evaluation of the physician.

1	Vulnerability in self-esteem makes individuals with narcissistic personality disorder very sensitive to “injury” from criticism or defeat. Although they may not show it outwardly, criticism may haunt these individuals and may leave them feeling humiliated, degraded, hollow, and empty. They may react with disdain, rage, or defiant counterattack. Such ex- periences often lead to social withdrawal or an appearance of humility that may mask and protect the grandiosity. Interpersonal relations are typically impaired because of problems derived from entitlement, the need for admiration, and the relative disregard for the sen— sitivities of others. Though overweening ambition and confidence may lead to high achievement, performance may be disrupted because of intolerance of criticism or defeat.

1	Sometimes vocational functioning can be very low, reﬂecting an unwillingness to take a risk in competitive or other situations in which defeat is possible. Sustained feelings of shame or humiliation and the attendant self—criticism may be associated with social with- drawal, depressed mood, and persistent depressive disorder (dysthymia) or major de- pressive disorder. In contrast, sustained periods of grandiosity may be associated with a hypomanic mood. Narcissistic personality disorder is also associated with anorexia ner- vosa and substance use disorders (especially related to cocaine). Histrionic, borderline, antisocial, and paranoid personality disorders may be associated with narcissistic person- ality disorder. Prevalence estimates for narcissistic personality disorder, based on DSM-IV definitions, range from 0% to 6.2% in community samples.

1	Prevalence estimates for narcissistic personality disorder, based on DSM-IV definitions, range from 0% to 6.2% in community samples. dicate that the individual will go on to have narcissistic personality disorder. Individuals with narcissistic personality disorder may have special difficulties adjusting to the onset of physical and occupational limitations that are inherent in the aging process. Of those diagnosed with narcissistic personality disorder, 50%—75% are male.

1	Of those diagnosed with narcissistic personality disorder, 50%—75% are male. Other personality disorders and personality traits. Other personality disorders may be confused with narcissistic personality disorder because they have certain features in common. It is, therefore, important to distinguish among these disorders based on differ- ences in their characteristic features. However, if an individual has personality features that meet criteria for one or more personality disorders in addition to narcissistic person- ality disorder, all can be diagnosed. The most useful feature in discriminating narcissistic personality disorder from histrionic, antisocial, and borderline personality disorders, in

1	Which the interactive styles are coquettish, callous, and needy, respectively, is the grandi- osity characteristic of narcissistic personality disorder. The relative stability of self-image as well as the relative lack of self—destructiveness, impulsivity, and abandonment concerns der. Excessive pride in achievements, a relative lack of emotional display, and disdain for personality disorder. Although individuals with borderline, histrionic, and narcissistic personality disorders may require much attention, those with narcissistic personality dis- order specifically need that attention to be admiring. Individuals with antisocial and nar- cissistic personality disorders share a tendency to be tough-minded, glib, superficial, exploitative, and unempathic. However, narcissistic personality disorder does not neces- sarily include characteristics of impulsivity, aggression, and deceit. In addition, individu- als with antisocial personality disorder may not be as needy of the admiration and

1	does not neces- sarily include characteristics of impulsivity, aggression, and deceit. In addition, individu- als with antisocial personality disorder may not be as needy of the admiration and envy of others, and persons with narcissistic personality disorder usually lack the history of con- duct disorder in childhood or criminal behavior in adulthood. In both narcissistic person- ality disorder and obsessive-compulsive personality disorder, the individual may profess a commitment to perfectionism and believe that others cannot do things as well. In con- trast to the accompanying self—criticism of those with obsessive-compulsive personality disorder, individuals with narcissistic personality disorder are more likely to believe that they have achieved perfection. Suspiciousness and social withdrawal usually distinguish those with schizotypal or paranoid personality disorder from those with narcissistic per- sonality disorder. When these qualities are present in individuals with

1	social withdrawal usually distinguish those with schizotypal or paranoid personality disorder from those with narcissistic per- sonality disorder. When these qualities are present in individuals with narcissistic person- ality disorder, they derive primarily from fears of having imperfections or ﬂaws revealed.

1	narcissistic. Only when these traits are inﬂexible, maladaptive, and persisting and cause sonality disorder. Mania or hypomania. Grandiosity may emerge as part of manic or hypomanic episodes, but the association with mood change or functional impairments helps distinguish these episodes from narcissistic personality disorder. Substance use disorders. Narcissistic personality disorder must also be distinguished from symptoms that may develop in association with persistent substance use. Diagnostic Criteria 301.82 (F60.6) A pervasive pattern of social inhibition, feelings of inadequacy, and hypersensitivity to neg- ative evaluation, beginning by early adulthood and present in a variety of contexts, as in- dicated by four (or more) of the following: 1. Avoids occupational activities that involve significant interpersonal contact because of fears of criticism, disapproval, or rejection. 2. Is unwilling to get involved with people unless certain of being liked.

1	2. Is unwilling to get involved with people unless certain of being liked. Shows restraint within intimate relationships because of the fear of being shamed or ridiculed. ‘ ls preoccupied with being criticized or rejected in social situations. ls inhibited in new interpersonal situations because of feelings of inadequacy. Views self as socially inept, personally unappealing, or inferior to others. ls unusually reluctant to take personal risks or to engage in any new activities because they may prove embarrassing. 39591:" The essential feature of avoidant personality disorder is a pervasive pattern of social inhi- bition, feelings of inadequacy, and hypersensitivity to negative evaluation that begins by early adulthood and is present in a variety of contexts.

1	Individuals with avoidant personality disorder avoid work activities that involve sig- nificant interpersonal contact because of fears of criticism, disapproval, or rejection (Cri- terion 1). Offers of job promotions may be declined because the new responsibilities might result in criticism from co-workers. These individuals avoid making new friends unless they are certain they will be liked and accepted without criticism (Criterion 2). Until they pass stringent tests proving the contrary, other people are assumed to be critical and dis- approving. Individuals with this disorder will not join in group activities unless there are repeated and generous offers of support and nurturance. Interpersonal intimacy is often difficult for these individuals, although they are able to establish intimate relationships when there is assurance of uncritical acceptance. They may act with restraint, have diffi- culty talking about themselves, and withhold intimate feelings for fear of being exposed,

1	relationships when there is assurance of uncritical acceptance. They may act with restraint, have diffi- culty talking about themselves, and withhold intimate feelings for fear of being exposed, ridiculed, or shamed (Criterion 3).

1	Because individuals With this disorder are preoccupied with being criticized or re- jected in social situations, they may have a markedly low threshold for detecting such re- actions (Criterion 4). If someone is even slightly disapproving or critical, they may feel extremely hurt. They tend to be shy, quiet, inhibited, and “invisible" because of the fear that any attention would be degrading or rejecting. They expect that no matter what they say, others will see it as "wrong,” and so they may say nothing at all. They react strongly to subtle cues that are suggestive of mockery or derision. Despite their longing to be active participants in social life, they fear placing their welfare in the hands of others. Individuals with avoidant personality disorder are inhibited in new interpersonal situations because they feel inadequate and have low self—esteem (Criterion 5). Doubts concerning social actions with strangers. These individuals believe themselves to be socially inept, person- ally

1	because they feel inadequate and have low self—esteem (Criterion 5). Doubts concerning social actions with strangers. These individuals believe themselves to be socially inept, person- ally unappealing, or inferior to others (Criterion 6). They are unusually reluctant to take personal risks or to engage in any new activities because these may prove embarrassing (Criterion 7). They are prone to exaggerate the potential dangers of ordinary situations, and a restricted lifestyle may result from their need for certainty and security. Someone with this disorder may cancel a job interview for fear of being embarrassed by not dressing appropriately. Marginal somatic symptoms or other problems may become the reason for avoiding new activities.

1	Individuals with avoidant personality disorder often vigilantly appraise the movements and expressions of those with whom they come into contact. Their fearful and tense de- meanor may elicit ridicule and derision from others, which in turn confirms their self- doubts. These individuals are very anxious about the possibility that they will react to crit- icism with blushing or crying. They are described by others as being ”shy,” ”timid,” ”lonely,” and ”isolated." The major problems associated with this disorder occur in social and occupational functioning. The low self—esteem and hypersensitivity to rejection are associated with restricted interpersonal contacts. These individuals may become relatively crises. They desire affection and acceptance and may fantasize about idealized relation- ships with others. The avoidant behaviors can also adversely affect occupational function- ing because these individuals try to avoid the types of social situations that may be important for meeting

1	ships with others. The avoidant behaviors can also adversely affect occupational function- ing because these individuals try to avoid the types of social situations that may be important for meeting the basic demands of the job or for advancement.

1	Other disorders that are commonly diagnosed with avoidant personality disorder in- clude depressive, bipolar, and anxiety disorders, especially social anxiety disorder (social phobia). Avoidant personality disorder is often diagnosed with dependent personality disorder, because individuals with avoidant personality disorder become very attached to and dependent on those few other people with whom they are friends. Avoidant per- sonality disorder also tends to be diagnosed with borderline personality disorder and with the Cluster A personality disorders (i.e., paranoid, schizoid, or schizotypal personality disorders). Data from the 2001—2002 National Epidemiologic Survey on Alcohol and Related Condi- tions suggest a prevalence of about 2.4% for avoidant personality disorder.

1	Data from the 2001—2002 National Epidemiologic Survey on Alcohol and Related Condi- tions suggest a prevalence of about 2.4% for avoidant personality disorder. The avoidant behavior often starts in infancy or childhood with shyness, isolation, and fear of strangers and new situations. Although shyness in childhood is a common precur- sor of avoidant personality disorder, in most individuals it tends to gradually dissipate as they get older. In contrast, individuals who go on to develop avoidant personality disor— der may become increasingly shy and avoidant during adolescence and early adulthood, when social relationships with new people become especially important. There is some evidence that in adults, avoidant personality disorder tends to become less evident or to remit with age. This diagnosis should be used with great caution in children and adoles- cents, for whom shy and avoidant behavior may be developmentally appropriate.

1	There may be variation in the degree to which different cultural and ethnic groups regard diffidence and avoidance as appropriate. Moreover, avoidant behavior may be the result of problems in acculturation following immigration. Avoidant personality disorder appears to be equally frequent in males and females. Anxiety disorders. There appears to be a great deal of overlap between avoidant person- ality disorder and social anxiety disorder (social phobia), so much so that they may be alternative conceptualizations of the same or similar conditions. Avoidance also character- izes both avoidant personality disorder and agoraphobia, and they often co-occur.

1	Other personality disorders and personality traits. Other personality disorders may be confused with avoidant personality disorder because they have certain features in com- mon. It is, therefore, important to distinguish among these disorders based on differences in their characteristic features. However, if an individual has personality features that meet criteria for one or more personality disorders in addition to avoidant personality dis- order, all can be diagnosed. Both avoidant personality disorder and dependent personal- ity disorder are characterized by feelings of inadequacy, hypersensitivity to criticism, and a need for reassurance. Although the primary focus of concern in avoidant personality disorder is avoidance of humiliation and rejection, in dependent personality disorder the focus is on being taken care of. However, avoidant personality disorder and dependent personality disorder are particularly likely to co—occur. Like avoidant personality disor- der, schizoid

1	the focus is on being taken care of. However, avoidant personality disorder and dependent personality disorder are particularly likely to co—occur. Like avoidant personality disor- der, schizoid personality disorder and schizotypal personality disorder are characterized by social isolation. However, individuals with avoidant personality disorder want to have relationships with others and feel their loneliness deeply, whereas those with schizoid or schizotypal personality disorder may be content with and even prefer their social isola- tion. Paranoid personality disorder and avoidant personality disorder are both character- ized by a reluctance to confide in others. However, in avoidant personality disorder, this reluctance is attributable more to a fear of being embarrassed or being found inadequate than to a fear of others’ malicious intent.

1	Many individuals display avoidant personality traits. Only when these traits are in- ﬂexible, maladaptive, and persisting and cause significant functional impairment or sub- jective distress do they constitute avoidant personality disorder. Personality change due to another medical condition. Avoidant personality disorder must be distinguished from personality change due to another medical condition, in which the traits that emerge are attributable to the effects of another medical condition on the central nervous system. Substance use disorders. Avoidant personality disorder must also be distinguished from symptoms that may develop in association with persistent substance use. Diagnostic Criteria 301.6 (F60.7)

1	Substance use disorders. Avoidant personality disorder must also be distinguished from symptoms that may develop in association with persistent substance use. Diagnostic Criteria 301.6 (F60.7) A pervasive and excessive need to be taken care of that leads to submissive and clinging behavior and fears of separation, beginning by early adulthood and present in a variety of contexts, as indicated by five (or more) of the following: 1. Has difficulty making everyday decisions without an excessive amount of advice and reassurance from others. 2. Needs others to assume responsibility for most major areas of his or her life. 3. Has difficulty expressing disagreement with others because of fear of loss of support or approval. (Note: Do not include realistic fears of retribution.) 4. Has difficulty initiating projects or doing things on his or her own (because of a lack of self-confidence in judgment or abilities rather than a lack of motivation or energy).

1	5. Goes to excessive lengths to obtain nurturance and support from others, to the point of volunteering to do things that are unpleasant. 6. Feels uncomfortable or helpless when alone because of exaggerated fears of being unable to care for himself or herself. 7. Urgently seeks another relationship as a source of care and support when a close re- lationship ends. 8. Is unrealistically preoccupied with fears of being left to take care of himself or herself. The essential feature of dependent personality disorder is a pervasive and excessive need to be taken care of that leads to submissive and clinging behavior and fears of separation. This pattern begins by early adulthood and is present in a variety of contexts. The dependent and submissive behaviors are designed to elicit caregiving and arise from a self—perception of being unable to function adequately without the help of others.

1	Individuals with dependent personality disorder have great difficulty making every- day decisions (e.g., what color shirt to wear to work or whether to carry an umbrella) without an excessive amount of advice and reassurance from others (Criterion 1). These individu- als tend to be passive and to allow other people (often a single other person) to take the ini- tiative and assume responsibility for most major areas of their lives (Criterion 2). Adults with this disorder typically depend on a parent or spouse to decide where they should live, what kind of job they should have, and which neighbors to befriend. Adolescents with this disorder may allow their parent(s) to decide what they should wear, with whom they should associate, how they should spend their free time, and what school or college they should attend. This need for others to assume responsibility goes beyond age-appro- priate and situation-appropriate requests for assistance from others (e.g., the specific needs of

1	or college they should attend. This need for others to assume responsibility goes beyond age-appro- priate and situation-appropriate requests for assistance from others (e.g., the specific needs of children, elderly persons, and handicapped persons). Dependent personality dis- order may occur in an individual who has a serious medical condition or disability, but in such cases the difficulty in taking responsibility must go beyond what would normally be associated with that condition or disability.

1	Because they fear losing support or approval, individuals with dependent personality disorder often have difficulty expressing disagreement with other individuals, especially those on whom they are dependent (Criterion 3). These individuals feel so unable to func- tion alone that they will agree with things that they feel are wrong rather than risk losing the help of those to whom they look for guidance. They do not get appropriately angry at others whose support and nurturance they need for fear of alienating them. If the individ- ual’s concerns regarding the consequences of expressing disagreement are realistic (e.g., realistic fears of retribution from an abusive spouse), the behavior should not be consid- ered to be evidence of dependent personality disorder.

1	Individuals with this disorder have difficulty initiating projects or doing things inde- pendently (Criterion 4). They lack self—confidence and believe that they need help to begin and carry through tasks. They will wait for others to start things because they believe that as a rule others can do them better. These individuals are convinced that they are incapable of functioning independently and present themselves as inept and requiring constant as- sistance. They are, however, likely to function adequately if given the assurance that some- one else is supervising and approving. There may be a fear of becoming or appearing to be more competent, because they may believe that this will lead to abandonment. Because they rely on others to handle their problems, they often do not learn the skills of indepen- dent living, thus perpetuating dependency.

1	Individuals with dependent personality disorder may go to excessive lengths to obtain nurturance and support from others, even to the point of volunteering for unpleasant tasks if such behavior will bring the care they need (Criterion 5). They are willing to submit to what others want, even if the demands are unreasonable. Their need to maintain an im- portant bond will often result in imbalanced or distorted relationships. They may make ex- traordinary self—sacrifices or tolerate verbal, physical, or sexual abuse. (It should be noted that this behavior should be considered evidence of dependent personality disorder only when it can clearly be established that other options are available to the individual.) Indi- viduals with this disorder feel uncomfortable or helpless when alone, because of their ex— aggerated fears of being unable to care for themselves (Criterion 6). They will "tag along" with important others just to avoid being alone, even if they are not interested or involved

1	of their ex— aggerated fears of being unable to care for themselves (Criterion 6). They will "tag along" with important others just to avoid being alone, even if they are not interested or involved in what is happening.

1	When a close relationship ends (e.g., a breakup with a lover; the death of a caregiver), in- dividuals with dependent personality disorder may urgently seek another relationship to provide the care and support they need (Criterion 7). Their belief that they are unable to function in the absence of a close relationship motivates these individuals to become quickly and indiscriminately attached to another individual. Individuals with this disorder are often preoccupied with fears of being left to care for themselves (Criterion 8). They see themselves as so totally dependent on the advice and help of an important other person that they worry about being abandoned by that person when there are no grounds to justify such fears. To be considered as evidence of this criterion, the fears must be excessive and unrealistic. For ex- ample, an elderly man with cancer who moves into his son’s household for care is exhibiting dependent behavior that is appropriate given this person’s life

1	must be excessive and unrealistic. For ex- ample, an elderly man with cancer who moves into his son’s household for care is exhibiting dependent behavior that is appropriate given this person’s life circumstances.

1	Individuals with dependent personality disorder are often characterized by pessimism and self—doubt, tend to belittle their abilities and assets, and may constantly refer to them- selves as “stupid.” They take criticism and disapproval as proof of their worthlessness and lose faith in themselves. They may seek overprotection and dominance from others. Oc— cupational functioning may be impaired if independent initiative is required. They may avoid positions of responsibility and become anxious when faced with decisions. Social re- lations tend to be limited to those few people on whom the individual is dependent. There may be an increased risk of depressive disorders, anxiety disorders, and adjustment dis- orders. Dependent personality disorder often co-occurs with other personality disorders, especially borderline, avoidant, and histrionic personality disorders. Chronic physical ill- ness or separation anxiety disorder in childhood or adolescence may predispose the indi- vidual to the

1	especially borderline, avoidant, and histrionic personality disorders. Chronic physical ill- ness or separation anxiety disorder in childhood or adolescence may predispose the indi- vidual to the development of this disorder.

1	Data from the 2001—2002 National Epidemiologic Survey on Alcohol and Related Condi- tions yielded an estimated prevalence of dependent personality disorder of 0.49%, and de- pendent personality was estimated, based on a probability subsample from Part II of the National Comorbidity Survey Replication, to be 0.6%. This diagnosis should be used with great caution, if at all, in children and adolescents, for whom dependent behavior may be developmentally appropriate.

1	This diagnosis should be used with great caution, if at all, in children and adolescents, for whom dependent behavior may be developmentally appropriate. The degree to which dependent behaviors are considered to be appropriate varies sub- stantially across different age and sociocultural groups. Age and cultural factors need to be considered in evaluating the diagnostic threshold of each criterion. Dependent behav— ior should be considered characteristic of the disorder only when it is clearly in excess of the individual’s cultural norms or reﬂects unrealistic concerns. An emphasis on passivity, politeness, and deferential treatment is characteristic of some societies and may be mis- interpreted as traits of dependent personality disorder. Similarly, societies may differen— tially foster and discourage dependent behavior in males and females.

1	In clinical settings, dependent personality disorder has been diagnosed more frequently in females, although some studies report similar prevalence rates among males and females. Other mental disorders and medical conditions. Dependent personality disorder must be distinguished from dependency arising as a consequence of other mental disorders (e.g., depressive disorders, panic disorder, agoraphobia) and as a result of other medical conditions.

1	Other personality disorders and personality traits. Other personality disorders may be confused with dependent personality disorder because they have certain features in com- mon. It is therefore important to distinguish among these disorders based on differences in their characteristic features. However, if an individual has personality features that meet cri- teria for one or more personality disorders in addition to dependent personality disorder, all can be diagnosed. Although many personality disorders are characterized by dependent features, dependent personality disorder can be distinguished by its predominantly submis- sive, reactive, and clinging behavior. Both dependent personality disorder and borderline personality disorder are characterized by fear of abandonment; however, the individual with borderline personality disorder reacts to abandonment with feelings of emotional emp- tiness, rage, and demands, whereas the individual with dependent personality disorder re- acts

1	individual with borderline personality disorder reacts to abandonment with feelings of emotional emp- tiness, rage, and demands, whereas the individual with dependent personality disorder re- acts with increasing appeasement and submissiveness and urgently seeks a replacement relationship to provide caregiving and support. Borderline personality disorder can further be distinguished from dependent personality disorder by a typical pattern of unstable and intense relationships. Individuals with histrionic personality disorder, like those with de- pendent personality disorder, have a strong need for reassurance and approval and may ap- pear childlike and clinging. However, unlike dependent personality disorder, which is characterized by self-effacing and docile behavior, histrionic personality disorder is charac- terized by gregarious ﬂamboyance with active demands for attention. Both dependent personality disorder and avoidant personality disorder are characterized by feelings of in-

1	disorder is charac- terized by gregarious ﬂamboyance with active demands for attention. Both dependent personality disorder and avoidant personality disorder are characterized by feelings of in- adequacy, hypersensitivity to criticism, and a need for reassurance; however, individuals with avoidant personality disorder have such a strong fear of humiliation and rejection that they withdraw until they are certain they will be accepted. In contrast, individuals with de- pendent personality disorder have a pattern of seeking and maintaining connections to im- portant others, rather than avoiding and withdrawing from relationships.

1	Many individuals display dependent personality traits. Only when these traits are in- ﬂexible, maladaptive, and persisting and cause significant functional impairment or sub— jective distress do they constitute dependent personality disorder. Personality change due to another medical condition. Dependent personality disor- der must be distinguished from personality change due to another medical condition, in which the traits that emerge are attributable to the effects of another medical condition on the central nervous system. Substance use disorders. Dependent personality disorder must also be distinguished from symptoms that may develop in association with persistent substance use. Diagnostic Criteria 301.4 (F60.5)

1	Substance use disorders. Dependent personality disorder must also be distinguished from symptoms that may develop in association with persistent substance use. Diagnostic Criteria 301.4 (F60.5) A pervasive pattern of preoccupation with orderliness, perfectionism, and mental and in- terpersonal control. at the expense of flexibility, openness, and efficiency, beginning by early adulthood and present in a variety of contexts. as indicated by four (or more) of the following: 1. Is preoccupied with details. rules. lists, order, organization, or schedules to the extent that the major point of the activity is lost. 2. Shows perfectionism that interferes with task completion (e.g., is unable to complete a project because his or her own overly strict standards are not met). 3. Is excessively devoted to work and productivity to the exclusion of leisure activities and friendships (not accounted for by obvious economic necessity).

1	3. Is excessively devoted to work and productivity to the exclusion of leisure activities and friendships (not accounted for by obvious economic necessity). 4. Is overconscientious, scrupulous, and inflexible about matters of morality, ethics, or values (not accounted for by cultural or religious identification). 5. Is unable to discard worn-out or worthless objects even when they have no sentimental value. 6. Is reluctant tb delegate tasks or to work with others unless they submit to exactly his or her way of doing things. 7. Adopts a miserly spending style toward both self and others; money is viewed as something to be hoarded for future catastrophes. 8. Shows rigidity and stubbornness. The essential feature of obsessive-compulsive personality disorder is a preoccupation with orderliness, perfectionism, and mental and interpersonal control, at the expense of ﬂexibility, openness, and efficiency. This pattern begins by early adulthood and is present in a variety of contexts.

1	Individuals with obsessive-compulsive personality disorder attempt to maintain a sense of control through painstaking attention to rules, trivial details, procedures, lists, schedules, or form to the extent that the major point of the activity is lost (Criterion 1). They are excessively careful and prone to repetition, paying extraordinary attention to detail and repeatedly checking for possible mistakes. They are oblivious to the fact that other people tend to become very annoyed at the delays and inconveniences that result from this behavior. For example, when such individuals misplace a list of things to be done, they will spend an inordinate amount of time looking for the list rather than spending a few moments re-creating it from memory and proceeding to accomplish the tasks. Time is poorly allocated, and the most important tasks are left to the last moment. The perfection- ism and self—imposed high standards of performance cause significant dysfunction and distress in these

1	is poorly allocated, and the most important tasks are left to the last moment. The perfection- ism and self—imposed high standards of performance cause significant dysfunction and distress in these individuals. They may become so involved in making every detail of a project absolutely perfect that the project is never finished (Criterion 2). For example, the completion of a written report is delayed by numerous time-consuming rewrites that all come up short of ”perfection." Deadlines are missed, and aspects of the individual’s life that are not the current focus of activity may fall into disarray.

1	Individuals with obsessive—compulsive personality disorder display excessive devotion to work and productivity to the exclusion of leisure activities and friendships (Criterion 3). This behavior is not accounted for by economic necessity. They often feel that they do not have time to take an evening or a weekend day off to go on an outing or to just relax. They may keep postponing a pleasurable activity, such as a vacation, so that it may never occur.

1	When they do take time for leisure activities or vacations, they are very uncomfortable un- less they have taken along something to work on so they do not "waste time." There may be a great concentration on household chores (e.g., repeated excessive cleaning so that ”one could eat off the ﬂoor”). If they spend time with friends, it is likely to be in some kind of for- mally organized activity (e.g., sports). Hobbies or recreational activities are approached as serious tasks requiring careful organization and hard work to master. The emphasis is on perfect performance. These individuals turn play into a structured task (e.g., correcting an infant for not putting rings on the post in the right order; telling a toddler to ride his or her tri- cycle in a straight line; turning a baseball game into a harsh "lesson”).

1	Individuals with obsessive-compulsive personality disorder may be excessively con- scientious, scrupulous, and inﬂexible about matters of morality, ethics, or values (Crite- rion 4). They may force themselves and others to follow rigid moral principles and very strict standards of performance. They may also be mercilessly self-critical about their own mistakes. Individuals with this disorder are rigidly deferential to authority and rules and insist on quite literal compliance, with no rule bending for extenuating circumstances. For example, the individual will not lend a quarter to a friend who needs one to make a tele- phone call because ”neither a borrower nor a lender be” or because it would be ”bad” for the person’s character. These qualities should not be accounted for by the individual’s culv tural or religious identification.

1	Individuals with this disorder may be unable to discard worn-out or worthless objects, even when they have no sentimental value (Criterion 5). Often these individuals will ad- mit to being ”pack rats.” They regard discarding objects as wasteful because ”you never know when you might need something” and will become upset if someone tries to get rid of the things they have saved. Their spouses or roommates may complain about the amount of space taken up by old parts, magazines, broken appliances, and so on.

1	Individuals with obsessive-compulsive personality disorder are reluctant to delegate tasks or to work with others (Criterion 6). They stubbornly and unreasonably insist that everything be done their way and that people conform to their way of doing things. They often give very detailed instructions about how things should be done (e.g., there is one and only one way to mow the lawn, wash the dishes, build a doghouse) and are surprised and irritated if others suggest creative alternatives. At other times they may reject offers of help even when behind schedule because they believe no one else can do it right.

1	Individuals with this disorder may be miserly and stingy and maintain a standard of living far below what they can afford, believing that spending must be tightly controlled to provide for future catastrophes (Criterion 7). Obsessive-compulsive personality disorder is characterized by rigidity and stubbornness (Criterion 8). Individuals with this disorder are so concerned about having things done the one "correct” way that they have trouble going along with anyone else's ideas. These individuals plan ahead in meticulous detail and are unwilling to consider changes. Totally wrapped up in their own perspective, they have difficulty acknowledging the viewpoints of others. Friends and colleagues may be- come frustrated by this constant rigidity. Even when individuals with obsessive—compul- sive personality disorder recognize that it may be in their interest to compromise, they may stubbomly refuse to do so, arguing that it is ”the principle of the thing.”

1	When rules and established procedures do not dictate the correct answer, decision making may become a time-consuming, often painful process. Individuals with obsessive- ority or what is the best way of doing some particular task that they may never get started on anything. They are prone to become upset or angry in situations in which they are not able to maintain control of their physical or interpersonal environment, although the an- ger is typically not expressed directly. For example, an individual may be angry when ser- vice in a restaurant is poor, but instead of complaining to the management, the individual ruminates about how much to leave as a tip. On other occasions, anger may be expressed with righteous indignation over a seemingly minor matter. Individuals with this disorder may be especially attentive to their relative status in dominance-submission relationships and may display excessive deference to an authority they respect and excessive resistance to authority they do

1	be especially attentive to their relative status in dominance-submission relationships and may display excessive deference to an authority they respect and excessive resistance to authority they do not respect.

1	Individuals with this disorder usually express affection in a highly controlled or stilted fashion and may be very uncomfortable in the presence of others who are emotionally ex- pressive. Their everyday relationships have a formal and serious quality, and they may be stiff in situations in which others would smile and be happy (e.g., greeting a lover at the airport). They carefully hold themselves back until they are sure that whatever they say will be perfect. They may be preoccupied with logic and intellect, and intolerant of affec- tive behavior in others. They often have difficulty expressing tender feelings, rarely pay- ing compliments. Individuals with this disorder may experience occupational difficulties and distress, particularly when confronted with new situations that demand ﬂexibility and compromise.

1	Individuals with anxiety disorders, including generalized anxiety disorder, social anx- iety disorder (social phobia), and specific phobias, and obsessive-compulsive disorder (OCD) have an increased likelihood of having a personality disturbance that meets criteria for ob- sessive-compulsive personality disorder. Even so, it appears that the majority of individ- uals with OCD do not have a pattern of behavior that meets criteria for this personality disorder. Many of the features of obsessive-compulsive personality disorder overlap with "type A" personality characteristics (e.g., preoccupation with work, competitiveness, time urgency), and these features may be present in people at risk for myocardial infarction. pressive and bipolar disorders and eating disorders. Obsessive-compulsive personality disorder is one of the most prevalent personality dis- orders in the general population, with estimated prevalence ranging from 2.1% to 7.9%.

1	Obsessive-compulsive personality disorder is one of the most prevalent personality dis- orders in the general population, with estimated prevalence ranging from 2.1% to 7.9%. In assessing an individual for obsessive-compulsive personality disorder, the clinician should not include those behaviors that reﬂect habits, customs, or interpersonal styles that are culturally sanctioned by the individual’s reference group. Certain cultures place sub- stantial emphasis on work and productivity; the resulting behaviors in members of those societies need not be considered indications of obsessive-compulsive personality disorder. In systematic studies, obsessive—compulsive personality disorder appears to be diagnosed about twice as often among males.

1	In systematic studies, obsessive—compulsive personality disorder appears to be diagnosed about twice as often among males. Obsessive-compulsive disorder. Despite the similarity in names, 0CD is usually easily distinguished from obsessive-compulsive personality disorder by the presence of true ob- sessions and compulsions in OCD. When criteria for both obsessive-compulsive person- ality disorder and 0CD are met, both diagnoses should be recorded. Hoarding disorder. A diagnosis of hoarding disorder should be considered especially when hoarding is extreme (e.g., accumulated stacks of worthless objects present a fire haz- ard and make it difficult for others to walk through the house). When criteria for both ob- sessive-compulsive personality disorder and hoarding disorder are met, both diagnoses should be recorded.

1	Other personality disorders and personality traits. Other personality disorders may be confused with obsessive-compulsive personality disorder because they have certain features in common. It is, therefore, important to distinguish among these disorders based on differences in their characteristic features. However, if an individual has personality features that meet criteria for one or more personality disorders in addition to obsessive- compulsive personality disorder, all can be diagnosed. Individuals with narcissistic per- sonality disorder may also profess a commitment to perfectionism and believe that others cannot do things as well, but these individuals are more likely to believe that they have achieved perfection, whereas those with obsessive-compulsive personality disorder are usually self-critical. Individuals with narcissistic or antisocial personality disorder lack generosity but will indulge themselves, whereas those with obsessive-compulsive person- ality disorder adopt

1	self-critical. Individuals with narcissistic or antisocial personality disorder lack generosity but will indulge themselves, whereas those with obsessive-compulsive person- ality disorder adopt a miserly spending style toward both self and others. Both schizoid by an apparent formality and social detachment. In obsessive—compulsive personality dis— order, this stems from discomfort with emotions and excessive devotion to work, whereas in schizoid personality disorder there is a fundamental lack of capacity for intimacy.

1	Obsessive—compulsive personality traits in moderation may be especially adaptive, par- ticularly in situations that reward high performance. Only when these traits are inﬂexible, maladaptive, and persisting and cause significant functional impairment or subjective dis- tress do they constitute obsessive-compulsive personality disorder. Personality change due to another medical condition. Obsessive-compulsive person- ality disorder must be distinguished from personality change due to another medical con- dition, in which the traits emerge attributable to the effects of another medical condition on the central nervous system. Substance use disorders. Obsessive-compulsive personality disorder must also be dis- tinguished from symptoms that may develop in association with persistent substance use. Due to Another Medical Condition Diagnostic Criteria 310.1 (F07.0)

1	Due to Another Medical Condition Diagnostic Criteria 310.1 (F07.0) A. A persistent personality disturbance that represents a change from the individual’s pre- vious characteristic personality pattern. Note: In children. the disturbance involves a marked deviation from normal devel- opment or a significant change in the child’s usual behavior patterns, lasting at least 1 year. B. There is evidence from the history, physical examination, or laboratory findings that the disturbance is the direct pathophysiological consequence of another medical condition. C. The disturbance is not better explained by another mental disorder (including another mental disorder due to another medical condition). D. The disturbance does not occur exclusively during the course of a delirium. E. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. Specify whether:

1	E. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. Specify whether: Labile type: If the predominant feature is affective lability. Disinhibited type: If the predominant feature is poor impulse control as evidenced by sexual indiscretions, etc. Aggressive type: If the predominant feature is aggressive behavior. Apathetic type: If the predominant feature is marked apathy and indifference. Paranoid type: It the predominant feature is suspiciousness or paranoid ideation. Other type: If the presentation is not characterized by any of the above subtypes. Combined type: If more than one feature predominates in the clinical picture.

1	Other type: If the presentation is not characterized by any of the above subtypes. Combined type: If more than one feature predominates in the clinical picture. Coding note: Include the name of the other medical condition (e.g., 310.1 [FO7.0] person- ality change due to temporal lobe epilepsy). The other medical condition should be coded and listed separately immediately before the personality disorder due to another medical condition (e.g., 345.40 [640.209] temporal lobe epilepsy; 310.1 [FO7.0] personality change due to temporal lobe epilepsy). Personality Change Due to Another Medical Condition 683 The particular personality change can be specified by indicating the symptom presenta— tion that predominates in the clinical presentation.

1	The essential feature of a personality change due to another medical condition is a persis- tent personality disturbance that is judged to be due to the direct pathophysiological ef- fects of a medical condition. The personality disturbance represents a change from the individual’s previous characteristic personality pattern. In children, this condition may be manifested as a marked deviation from normal development rather than as a change in a stable personality pattern (Criterion A). There must be evidence from the history, physical examination, or laboratory findings that the personality change is the direct physiological consequence of another medical condition (Criterion B). The diagnosis is not given if the disturbance is better explained by another mental disorder (Criterion C). The diagnosis is not given if the disturbance occurs exclusively during the course of a delirium (Criterion

1	D). The disturbance must also cause clinically significant distress or impairment in social, occupational, or other important areas of functioning (Criterion E). Common manifestations of the personality change include affective instability, poor impulse control, outbursts of aggression or rage grossly out of proportion to any precipi- tating psychosocial stressor, marked apathy, suspiciousness, or paranoid ideation. The phenomenology of the change is indicated using the subtypes listed in the criteria set. An individual with the disorder is often characterized by others as ”not himself [or herself].” Although it shares the term “personality” with the other personality disorders, this diag— nosis is distinct by virtue of its specific etiology, different phenomenology, and more vari- able onset and course.

1	The clinical presentation in a given individual may depend on the nature and localiza- tion of the pathological process. For example, injury to the frontal lobes may yield symp- toms such as lack of judgment or foresight, facetiousness, disinhibition, and euphoria. Right hemisphere strokes have often been shown to evoke personality changes in asso- ciation with unilateral spatial neglect, anosognosia (i.e., inability of the individual to recognize a bodily or functional deficit, such as the existence of hemiparesis), motor im- persistence, and other neurological deficits. A variety of neurological and other medical conditions may cause personality changes, including central nervous system neoplasms, head trauma, cerebrovascular disease,

1	A variety of neurological and other medical conditions may cause personality changes, including central nervous system neoplasms, head trauma, cerebrovascular disease, Huntington’s disease, epilepsy, infectious conditions with central nervous system in- volvement (e.g., HIV), endocrine conditions (e.g., hypothyroidism, hypo- and hyperadre- nocorticism), and autoimmune conditions with central nervous system involvement (e.g., systemic lupus erythematosus). The associated physical examination findings, laboratory findings, and patterns of prevalence and onset reﬂect those of the neurological or other medical condition involved.

1	Chronic medical conditions associated with pain and disability. Chronic medical con- ditions associated with pain and disability can also be associated with changes in person- ality. The diagnosis of personality change due to another medical condition is given only if a direct pathophysiological mechanism can be established. This diagnosis is not given if the change is due to a behavioral or psychological adjustment or response to another med- ical condition (e.g., dependent behaviors that result from a need for the assistance of others following a severe head trauma, cardiovascular disease, or dementia).

1	Delirium or major neurocognitive disorder. Personality change is a frequently associated feature of a delirium or major neurocognitive disorder. A separate diagnosis of personal— ity change due to another medical condition is not given if the change occurs exclusively during the course of a delirium. However, the diagnosis of personality change due to an- other medical condition may be given in addition to the diagnosis of major neurocognitive disorder if the personality change is a prominent part of the clinical presentation. Another mental disorder due to another medical condition. The diagnosis of person- ality change due to another medical condition is not given if the disturbance is better ex- plained by another mental disorder due to another medical condition (e.g., depressive disorder due to brain tumor).

1	Substance use disorders. Personality changes may also occur in the context of substance use disorders, especially if the disorder is longstanding. The clinician should inquire carefully about the nature and extent of substance use. If the clinician wishes to indicate an etiological re- lationship between the personality change and substance use, the unspecified category for the specific substance (e.g., unspecified stimulant-related disorder) can be used. Other mental disorders. Marked personality changes may also be an associated feature of other mental disorders (e.g., schizophrenia; delusional disorder; depressive and bipolar disorders; other specified and unspecified disruptive behavior, impulse-control, and con- duct disorders; panic disorder). However, in these disorders, no specific physiological fac- tor is judged to be etiologically related to the personality change.

1	Other personality disorders. Personality change due to another medical condition can be distinguished from a personality disorder by the requirement for a clinically significant change from baseline personality functioning and the presence of a specific etiological medical condition. 301.89 (F60.89)

1	301.89 (F60.89) This category applies to presentations in which symptoms characteristic of a personality disorder that cause clinically significant distress or impairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the personality disorders diagnostic class. The other specified personality disorder category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for any specific personality disorder. This is done by recording “other specified personality disorder” followed by the specific reason (e.g., “mixed personality features”). 301.9 (F60.9)

1	301.9 (F60.9) This category applies to presentations in which symptoms characteristic of a personality disorder that cause clinically significant distress or impairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the personality disorders diagnostic class. The unspecified personality disorder category is used in situations in which the clinician chooses not to specify the rea- son that the criteria are not met for a specific personality disorder, and includes presenta- tions in which there is insufficient information to make a more specific diagnosis.

1	Paraphilic disorders included in this manual are voyeuristic disorder (spying on others in private activities), exhibitionistic disorder (exposing the genitals), frotteuristic disorder (touching or rubbing against a nonconsenting individual), sexual masochism disorder (undergoing humiliation, bondage, or suffering), sexual sadism disorder (inﬂict- ing humiliation, bondage, or suffering), pedophilic disorder (sexual focus on children), fe- body parts), and transvestic disorder (engaging in sexually arousing cross-dressing). These disorders have traditionally been selected for specific listing and assignment of ex- plicit diagnostic criteria in DSM for two main reasons: they are relatively common, in re— lation to other paraphilic disorders, and some of them entail actions for their satisfaction that, because of their noxiousness or potential harm to others, are classed as criminal of- fenses. The eight listed disorders do not exhaust the list of possible paraphilic disorders.

1	Many dozens of distinct paraphilias have been identified and named, and almost any of them could, by virtue of its negative consequences for the individual or for others, rise to the level of a paraphilic disorder. The diagnoses of the other specified and unspecified paraphilic disorders are therefore indispensable and will be required in many cases.

1	In this chapter, the order of presentation of the listed paraphilic disorders generally corresponds to common classification schemes for these conditions. The first group of disorders is based on anomalous activity preferences. These disorders are subdivided into courtship disorders, which resemble distorted components of human courtship behavior (voyeuristic disorder, exhibitionistic disorder, and frotteuristic disorder), and algolagnic disorders, which involve pain and suffering (sexual masochism disorder and sexual sadism disorder). The second group of disorders is based on anomalous target preferences. These elsewhere (fetishistic disorder and transvestic disorder).

1	The term paraphilia denotes any intense and persistent sexual interest other than sexual interest in genital stimulation or preparatory fondling with phenotypically normal, phys- ically mature, consenting human partners. In some circumstances, the criteria “intense and persistent” may be difficult to apply, such as in the assessment of persons who are very old or medically ill and who may not have ”intense” sexual interests of any kind. In such circumstances, the term paraphilia may be defined as any sexual interest greater than or equal to normophilic sexual interests. There are also specific paraphilias that are gen- erally better described as preferential sexual interests than as intense sexual interests.

1	Some paraphilias primarily concern the individual’s erotic activities, and others pri- marily concern the individual’s erotic targets. Examples of the former would include in- tense and persistent interests in spanking, whipping, cutting, binding, or strangulating another person, or an interest in these activities that equals or exceeds the individual’s in— terest in copulation or equivalent interaction with another person. Examples of the latter would include intense or preferential sexual interest in children, corpses, or amputees (as a class), as well as intense or preferential interest in nonhuman animals, such as horses or dogs, or in inanimate objects, such as shoes or articles made of rubber.

1	A paraphilic disorder is a paraphilia that is currently causing distress or impairment to the individual or a paraphilia whose satisfaction has entailed personal harm, or risk of harm, to others. A paraphilia is a necessary but not a sufficient condition for having a paraphilic dis- order, and a paraphilia by itself does not necessarily justify or require clinical intervention. In the diagnostic criteria set for each of the listed paraphilic disorders, Criterion A specifies the qualitative nature of the paraphilia (e.g., an erotic focus on children or on exposing the gen- itals to strangers), and Criterion B specifies the negative consequences of the paraphilia (i.e., distress, impairment, or harm to others). In keeping with the distinction between paraphilias and paraphilic disorders, the term diagnosis should be reserved for individuals who meet both

1	Criteria A and B (i.e., individuals who have a paraphilic disorder). If an individual meets Cri- a benign paraphilia is discovered during the clinical investigation of some other condition— then the individual may be said to have that paraphilia but not a paraphilic disorder. It is not rare for an individual to manifest two or more paraphilias. In some cases, the para- philic foci are closely related and the connection between the paraphilias is intuitively com— prehensible (e.g., foot fetishism and shoe fetishism). In other cases, the connection between the paraphilias is not obvious, and the presence of multiple paraphilias may be coincidental or else related to some generalized vulnerability to anomalies of psychosexual development. In any event, comorbid diagnoses of separate paraphilic disorders may be warranted if more than one paraphilia is causing suffering to the individual or harm to others.

1	Because of the two—pronged nature of diagnosing paraphilic disorders, clinician-rated or self-rated measures and severity assessments could address either the strength of the paraphilia itself or the seriousness of its consequences. Although the distress and impair- ment stipulated in the Criterion B are special in being the immediate or ultimate result of the paraphilia and not primarily the result of some other factor, the phenomena of reactive depression, anxiety, guilt, poor work history, impaired social relations, and so on are not unique in themselves and may be quantified with multipurpose measures of psychosocial functioning or quality of life.

1	The most widely applicable framework for assessing the strength of a paraphilia itself is one in which examinees’ paraphilic sexual fantasies, urges, or behaviors are evaluated in relation to their normophilic sexual interests and behaviors. In a clinical interview or on self—administered questionnaires, examinees can be asked whether their paraphilic sexual fantasies, urges, or behaviors are weaker than, approximately equal to, or stronger than their normophilic sexual interests and behaviors. This same type of comparison can be, and usually is, employed in psychophysiological measures of sexual interest, such as pe- nile plethysmography in males or viewing time in males and females. Diagnostic Criteria 302.82 (F65.3) A. Over a period of at least 6 months, recurrent and intense sexual arousal from observ- ing an unsuspecting person who is naked, in the process of disrobing, or engaging in sexual activity. as manifested by fantasies, urges, or behaviors.

1	B. The individual has acted on these sexual urges with a nonconsenting person, or the sexual urges or fantasies cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. C. The individual experiencing the arousal andlor acting on the urges is at least 18 years of age. Specify if: In a controlled environment: This specifier is primarily applicable to individuals living in institutional or other settings where opportunities to engage in voyeuristic behavior are restricted. In full remission: The individual has not acted on the urges with a nonconsenting per- son, and there has been no distress or impairment in social, occupational, or other ar- eas of functioning, for at least 5 years while In an uncontrolled envrronment. The ”in full remission” specifier does not address the continued presence or absence of voyeurism per se, which may still be present after behaviors and distress have remitted.

1	The diagnostic criteria for voyeuristic disorder can apply both to individuals who more or less freely disclose this paraphilic interest and to those who categorically deny any sexual arousal from observing an unsuspecting person who is naked, disrobing, or engaged in sexual activity despite substantial objective evidence to the contrary. If disclosing individuals also report dis- tress or psychosocial problems because of their voyeuristic sexual preferences, they could be diagnosed with voyeuristic disorder. On the other hand, if they declare no distress, demon- strated by lack of anxiety, obsessions, guilt, or shame, about these paraphilic impulses and are not impaired in other important areas of functioning because of this sexual interest, and their psychiatric or legal histories indicate that they do not act on it, they could be ascertained as having voyeuristic sexual interest but should not be diagnosed with voyeuristic disorder.

1	Nondisclosing individuals include, for example, individuals known to have been spy- ing repeatedly on unsuspecting persons who are naked or engaging in sexual activity on separate occasions but who deny any urges or fantasies concerning such sexual behavior, and who may report that known episodes of watching unsuspecting naked or sexually ac- tive persons were all accidental and nonsexual. Others may disclose past episodes of ob— sustained sexual interest in this behavior. Since these individuals deny having fantasies or impulses about watching others nude or involved in sexual activity, it follows that they would also reject feeling subjectively distressed or socially impaired by such impulses. De- spite their nondisclosing stance, such individuals may be diagnosed with voyeuristic dis— order. Recurrent voyeuristic behavior constitutes sufficient support for voyeurism (by behavior is causing harm to others (by fulfilling Criterion B).

1	”Recurrent” spying on unsuspecting persons who are naked or engaging in sexual ac- tivity (i.e., multiple victims, each on a separate occasion) may, as a general rule, be inter- preted as three or more victims on separate occasions. Fewer victims can be interpreted as satisfying this criterion if there were multiple occasions of watching the same victim or if there is corroborating evidence of a distinct or preferential interest in secret watching of naked or sexually active unsuspecting persons. Note that multiple victims, as suggested earlier, are a sufficient but not a necessary condition for diagnosis; the criteria may also be met if the individual acknowledges intense voyeuristic sexual interest.

1	The Criterion A time frame, indicating that signs or symptoms of voyeurism must have persisted for at least 6 months, should also be understood as a general guideline, not a strict threshold, to ensure that the sexual interest in secretly watching unsuspecting naked or sexually active others is not merely transient. Adolescence and puberty generally increase sexual curiosity and activity. To alleviate the risk of pathologizing normative sexual interest and behavior during pubertal adoles- cence, the minimum age for the diagnosis of voyeuristic disorder is 18 years (Criterion C). Voyeuristic acts are the most common of potentially law-breaking sexual behaviors. The population prevalence of voyeuristic disorder is unknown. However, based on voyeuris- tic sexual acts in nonclinical samples, the highest possible lifetime prevalence for voyeuris— tic disorder is approximately 12% in males and 4% in females.

1	Adult males with voyeuristic disorder often first become aware of their sexual interest in secretly watching unsuspecting persons during adolescence. However, the minimum age for a diagnosis of voyeuristic disorder is 18 years because there is substantial difficulty in differentiating it from age-appropriate puberty-related sexual curiosity and activity. The persistence of voyeurism over time is unclear. Voyeuristic disorder, however, per defini- tion requires one or more contributing factors that may change over time with or without treatment: subjective distress (e.g., guilt, shame, intense sexual frustration, loneliness), psychiatric morbidity, hypersexuality, and sexual impulsivity; psychosocial impairment; and / or the propensity to act out sexually by spying on unsuspecting naked or sexually ac- tive persons. Therefore, the course of voyeuristic disorder is likely to vary with age.

1	Temperamental. Voyeurism is a necessary precondition for voyeuristic disorder; hence, risk factors for voyeurism should also increase the rate of voyeuristic disorder. Environmental. Childhood sexual abuse, substance misuse, and sexual preoccupation/ hypersexuality have been suggested as risk factors, although the causal relationship to voyeurism is uncertain and the specificity unclear. Voyeuristic disorder is very uncommon among females in clinical settings, while the male- to-female ratio for single sexually arousing voyeuristic acts might be 3:1. Conduct disorder and antisocial personality disorder. Conduct disorderinadolescents and antisocial behaviors, and the specific sexual interest in secretly watching unsuspect- ing others who are naked or engaging in sexual activity should be lacking.

1	Substance use disorders. Substance use disorders might involve single voyeuristic ep- isodes by intoxicated individuals but should not involve the typical sexual interest in se- cretly watching unsuspecting persons being naked or engaging in sexual activity. Hence, recurrent voyeuristic sexual fantasies, urges, or behaviors that occur also when the indi— vidual is not intoxicated suggest that voyeuristic disorder might be present.

1	Known comorbidities in voyeuristic disorder are largely based on research with males suspected of or convicted for acts involving the secret watching of unsuspecting nude or sexually active persons. Hence, these comorbidities might not apply to all individuals with voyeuristic disorder. Conditions that occur comorbidly with voyeuristic disorder include hypersexuality and other paraphilic disorders, particularly exhibitionistic disorder. De- pressive, bipolar, anxiety, and substance use disorders; attention-deficit/hyperactivity disorder; and conduct disorder and antisocial personality disorder are also frequent co- morbid conditions. Diagnostic Criteria 302.4 (F65.2) A. Over a period of at least 6 months, recurrent and intense sexual arousal from the ex- posure of one's genitals to an unsuspecting person, as manifested by fantasies, urges, or behaviors.

1	A. Over a period of at least 6 months, recurrent and intense sexual arousal from the ex- posure of one's genitals to an unsuspecting person, as manifested by fantasies, urges, or behaviors. B. The individual has acted on these sexual urges with a nonconsenting person, or the sexual urges or fantasies cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. Specify whether: Sexually aroused by exposing genitals to prepubertal children Sexually aroused by exposing genitals to physically mature individuals Sexually aroused by exposing genitals to prepubertal children and to physically Specify if: In a controlled environment: This specifier is primarily applicable to individuals living in institutional or other settings where opportunities to expose one’s genitals are restricted.

1	Specify if: In a controlled environment: This specifier is primarily applicable to individuals living in institutional or other settings where opportunities to expose one’s genitals are restricted. In full remission: The individual has not acted on the urges with a nonconsenting per- son, and there has been no distress or impairment in social, occupational, or other ar- eas of functioning, for at least 5 years while in an uncontrolled environment.

1	The subtypes for exhibitionistic disorder are based on the age or physical maturity of the non- consenting individuals to whom the individual prefers to expose his or her genitals. The non- consenting individuals could be prepubescent children, adults, or both. This specifier should help draw adequate attention to characteristics of victims of individuals with exhibitionistic disorder to prevent co-occuiring pedophilic disorder from being overlooked. However, indi- cations that the individual with exhibitionistic disorder is sexually attracted to exposing his or her genitals to children should not preclude a diagnosis of pedophilic disorder. The ”in full remission” specifier does not address the continued presence or absence of ex- hibitionism per se, which may still be present after behaviors and distress have remitted.

1	The diagnostic criteria for exhibitionistic disorder can apply both to individuals who more or less freely disclose this paraphilia and to those who categorically deny any sexual attraction to exposing their genitals to unsuspecting persons despite substantial objective evidence to the contrary. If disclosing individuals also report psychosocial difficulties because of their sexual attractions or preferences for exposing, they may be diagnosed with exhibitionistic disorder. In contrast, if they declare no distress (exemplified by absence of anxiety, obsessions, and guilt or shame about these paraphilic impulses) and are not impaired by this sexual interest in other important areas of functioning, and their self—reported, psychiatric, or legal histories indicate that they do not act on them, they could be ascertained as having exhibitionistic sexual interest but not be diagnosed with exhibitionistic disorder.

1	Examples of nondisclosing individuals include those who have exposed themselves repeatedly to unsuspecting persons on separate occasions but who deny any urges or fan- tasies about such sexual behavior and who report that known episodes of exposure were all accidental and nonsexual. Others may disclose past episodes of sexual behavior involv- ior. Since these individuals deny having urges or fantasies involving genital exposure, it such impulses. Such individuals may be diagnosed with exhibitionistic disorder despite their negative self—report. Recurrent exhibitionistic behavior constitutes sufficient support motivated behavior is causing harm to others (Criterion B).

1	”Recurrent” genital exposure to unsuspecting others (i.e., multiple victims, each on a separate occasion) may, as a general rule, be interpreted as three or more victims on sep- arate occasions. Fewer victims can be interpreted as satisfying this criterion if there were multiple occasions of exposure to the same victim, or if there is corroborating evidence of a strong or preferential interest in genital exposure to unsuspecting persons. Note that multiple victims, as suggested earlier, are a sufficient but not a necessary condition for di- agnosis, as criteria may be met by an individual’s acknowledging intense exhibitionistic sexual interest with distress and / or impairment.

1	The Criterion A time frame, indicating that signs or symptoms of exhibitionism must have persisted for at least 6 months, should also be understood as a general guideline, not a strict threshold, to ensure that the sexual interest in exposing one’s genitals to unsuspect- ing others is not merely transient. This might be expressed in clear evidence of repeated behaviors or distress over a nontransient period shorter than 6 months. The prevalence of exhibitionistic disorder is unknown. However, based on exhibitionistic sexual acts in nonclinical or general populations, the highest possible prevalence for exhi- bitionistic disorder in the male population is 2%—4%. The prevalence of exhibitionistic dis- order in females is even more uncertain but is generally believed to be much lower than in males.

1	Adult males with exhibitionistic disorder often report that they first became aware of sex— ual interest in exposing their genitals to unsuspecting persons during adolescence, at a somewhat later time than the typical development of normative sexual interest in women or men. Although there is no minimum age requirement for the diagnosis of exhibitionis— tic disorder, it may be difficult to differentiate exhibitionistic behaviors from age-appro- priate sexual curiosity in adolescents. Whereas exhibitionistic impulses appear to emerge in adolescence or early adulthood, very little is known about persistence over time. By def- inition, exhibitionistic disorder requires one or more contributing factors, which may change over time with or without treatment; subjective distress (e.g., guilt, shame, intense sexual frustration, loneliness), mental disorder comorbidity, hypersexuality, and sexual impulsivity; psychosocial impairment; and / or the propensity to act out sexually by expos- ing

1	shame, intense sexual frustration, loneliness), mental disorder comorbidity, hypersexuality, and sexual impulsivity; psychosocial impairment; and / or the propensity to act out sexually by expos- ing the genitals to unsuspecting persons. Therefore, the course of exhibitionistic disorder is likely to vary with age. As with other sexual preferences, advancing age may be associ- ated with decreasing exhibitionistic sexual preferences and behavior.

1	Temperamental. Since exhibitionism is a necessary precondition for exhibitionistic dis- order, risk factors for exhibitionism should also increase the rate of exhibitionistic disor- der. Antisocial history, antisocial personality disorder, alcohol misuse, and pedophilic sexual preference might increase risk of sexual recidivism in exhibitionistic offenders. Hence, antisocial personality disorder, alcohol use disorder, and pedophilic interest may be considered risk factors for exhibitionistic disorder in males with exhibitionistic sexual preferences. Environmental. Childhood sexual and emotional abuse and sexual preoccupation/hyper— sexuality have been suggested as risk factors for exhibitionism, although the causal rela- tionship to exhibitionism is uncertain and the specificity unclear. Exhibitionistic disorder is highly unusual in females, whereas single sexually arousing ex- hibitionistic acts might occur up to half as often among women compared with men.

1	Exhibitionistic disorder is highly unusual in females, whereas single sexually arousing ex- hibitionistic acts might occur up to half as often among women compared with men. Functionai Consequences of Exhibitionistic Disorder The functional consequences of exhibitionistic disorder have not been addressed in re- tress over these preferences. Potential differential diagnoses for exhibitionistic disorder sometimes occur also as co- morbid disorders. Therefore, it is generally necessary to evaluate the evidence for exhibi- tionistic disorder and other possible conditions as separate questions. Conduct disorder and antisocial personality disorder. Conduct disorder in adolescents antisocial behaviors, and the specific sexual interest in exposing the genitals should be lacking.

1	Conduct disorder and antisocial personality disorder. Conduct disorder in adolescents antisocial behaviors, and the specific sexual interest in exposing the genitals should be lacking. Substance use disorders. Alcohol and substance use disorders might involve single exhibitionistic episodes by intoxicated individuals but should not involve the typical sex- ual interest in exposing the genitals to unsuspecting persons. Hence, recurrent exhibition- istic sexual fantasies, urges, or behaviors that occur also when the individual is not intoxicated suggest that exhibitionistic disorder might be present.

1	Known comorbidities in exhibitionistic disorder are largely based on research with indi- viduals (almost all males) convicted for criminal acts involving genital exposure to non- consenting individuals. Hence, these comorbidities might not apply to all individuals who qualify for a diagnosis of exhibitionistic disorder. Conditions that occur comorbidly with exhibitionistic disorder at high rates include depressive, bipolar, anxiety, and substance disorders; and antisocial personality disorder. Diagnostic Criteria 302.89 (F65.81) A. Over a period of at least 6 months, recurrent and intense sexual arousal from touching or rubbing against a nonconsenting person, as manifested by fantasies, urges, or be- haviors. B. The individual has acted on these sexual urges with a nonconsenting person, or the sexual urges or fantasies cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. Specify it:

1	Specify it: In a controlled environment: This specifier is primarily applicable to individuals living in institutional or other settings where opportunities to touch or rub against a noncon- senting person are restricted. In full remission: The individual has not acted on the urges with a nonconsenting per- son, and there has been no distress or impairment in social, occupational, or other ar- eas of functioning, for at least 5 years while in an uncontrolled environment. The ”in remission” specifier does not address the continued presence or absence of frot- teurism per se, which may still be present after behaviors and distress have remitted.

1	The diagnostic criteria for frotteuristic disorder can apply both to individuals who relatively freely disclose this paraphilia and to those who firmly deny any sexual attraction from touch- ing or rubbing against a nonconsenting individual regardless of considerable objective evi- dence to the contrary. If disclosing individuals also report psychosocial impairment due to their sexual preferences for touching or rubbing against a nonconsenting individual, they could be diagnosed with frotteuristic disorder. In contrast, if they declare no distress (demon- strated by lack of anxiety, obsessions, guilt, or shame) about these paraphilic impulses and are not impaired in other important areas of functioning because of this sexual interest, and their psychiatric or legal histories indicate that they do not act on it, they could be ascertained as having frotteuristic sexual interest but should not be diagnosed with frotteuristic disorder.

1	Nondisclosing individuals include, for instance, individuals known to have been contest any urges or fantasies concerning such sexual behavior. Such individuals may re- port that identified episodes of touching or rubbing against an unwilling individual were all unintentional and nonsexual. Others may disclose past episodes of touching or rubbing this. Since these individuals deny having fantasies or impulses about touching or rubbing, impulses. Despite their nondisclosing position, such individuals may be diagnosed with frotteuristic disorder. Recurrent frotteuristic behavior constitutes satisfactory support for cally motivated behavior is causing harm to others (by fulfilling Criterion B).

1	”Recurrent” touching or rubbing against a nonconsenting individual (i.e., multiple vic- tims, each on a separate occasion) may, as a general rule, be interpreted as three or more vic- tims on separate occasions. Fewer victims can be interpreted as satisfying this criterion if there were multiple occasions of touching or rubbing against the same unwilling individ- ual, or corroborating evidence of a strong or preferential interest in touching or rubbing against nonconsenting individuals. Note that multiple victims are a sufficient but not a nec- essary condition for diagnosis; criteria may also be met if the individual acknowledges in- tense frotteuristic sexual interest with clinically significant distress and/ or impairment.

1	The Criterion A time frame, indicating that signs or symptoms of frotteurism must persist for at least 6 months, should also be interpreted as a general guideline, not a strict threshold, to ensure that the sexual interest in touching or rubbing against a nonconsenting individual is not transient. Hence, the duration part of Criterion A may also be met if there is clear evidence of recurrent behaviors or distress over a shorter but nontransient time period. Frotteuristic acts, including the uninvited sexual touching of or rubbing against another individual, may occur in up to 30% of adult males in the general population. Approximately 10%—14% of adult males seen in outpatient settings for paraphilic disorders and hypersex- uality have a presentation that meets diagnostic criteria for frotteuristic disorder. Hence, whereas the population prevalence of frotteuristic disorder is unknown, it is not likely that it exceeds the rate found in selected clinical settings.

1	Adult males with frotteuristic disorder often report first becoming aware of their sexual in- adulthood. However, children and adolescents may also touch or rub against unwilling oth- ers in the absence of a diagnosis of frotteuristic disorder. Although there is no minimum age for the diagnosis, frotteuristic disorder can be difficult to differentiate from conduct-disor- dered behavior without sexual motivation in individuals at younger ages. The persistence of frotteurism over time is unclear. Frotteuristic disorder, however, by definition requires one or more contributing factors that may change over time with or without treatment: subjec- tive distress (e.g., guilt, shame, intense sexual frustration, loneliness); psychiatric morbidity; hypersexuality and sexual impulsivity; psychosocial impairment; and/ or the propensity to act out sexually by touching or rubbing against unconsenting persons. Therefore, the course of frotteuristic disorder is likely to vary with age. As with other

1	impairment; and/ or the propensity to act out sexually by touching or rubbing against unconsenting persons. Therefore, the course of frotteuristic disorder is likely to vary with age. As with other sexual preferences, advanc- ing age may be associated with decreasing frotteuristic sexual preferences and behavior.

1	Temperamental. Nonsexual antisocial behavior and sexual preoccupation/hypersexuality might be nonspecific risk factors, although the causal relationship to frotteurism is uncertain and the specificity unclear. However, frotteurism is a necessary precondition for frotteuristic disorder, so risk factors for frotteurism should also increase the rate of frotteuristic disorder. There appear to be substantially fewer females with frotteuristic sexual preferences than males. Conduct disorder and antisocial personality disorder. Conduct disorder in adolescents and antisocial behaviors, and the specific sexual interest in touching or rubbing against a nonconsenting individual should be lacking.

1	Substance use disorders. Substance use disorders, particularly those involving stimu- lants such as cocaine and amphetamines, might involve single frotteuristic episodes by in— toxicated individuals but should not involve the typical sustained sexual interest in touching or rubbing against unsuspecting persons. Hence, recurrent frotteuristic sexual fantasies, urges, or behaviors that occur also when the individual is not intoxicated sug- gest that frotteuristic disorder might be present.

1	Known comorbidities in frotteuristic disorder are largely based on research with males suspected of or convicted for criminal acts involving sexually motivated touching of or rubbing against a nonconsenting individual. Hence, these comorbidities might not apply to other individuals with a diagnosis of frotteuristic disorder based on subjective distress over their sexual interest. Conditions that occur comorbidly with frotteuristic disorder in- clude hypersexuality and other paraphilic disorders, particularly exhibitionistic disorder and voyeuristic disorder. Conduct disorder, antisocial personality disorder, depressive disorders, bipolar disorders, anxiety disorders, and substance use disorders also co-occur. Potential differential diagnoses for frotteuristic disorder sometimes occur also as comor- bid disorders. Therefore, it is generally necessary to evaluate the evidence for frotteuristic disorder and possible comorbid conditions as separate questions.

1	Diagnostic Criteria 302.83 (F65.51) A. Over a period of at least 6 months, recurrent and intense sexual arousal from the act of being humiliated, beaten, bound, or otherwise made to suffer. as manifested by fan- tasies, urges, or behaviors. B. The fantasies, sexual urges, or behaviors cause clinically significant distress or impair- ment in social, occupational, or other important areas of functioning. Specify if: With asphyxiophilia: If the individual engages in the practice of achieving sexual arousal related to restriction of breathing. Specify if: In a controlled environment: This specifier is primarily applicable to individuals living in institutional or other settings where opportunities to engage in masochistic sexual behaviors are restricted. In full remission: There has been no distress or impairment in social, occupational, or other areas of functioning for at last 5 years while in an uncontrolled environment.

1	The diagnostic criteria for sexual masochism disorder are intended to apply to individuals who freely admit to having such paraphilic interests. Such individuals openly acknowl- edge intense sexual arousal from the act of being humiliated, beaten, bound, or otherwise made to suffer, as manifested by fantasies, urges, or behaviors. If these individuals also re- port psychosocial difficulties because of their sexual attractions or preferences for being humiliated, beaten, bound, or otherwise made to suffer, they may be diagnosed with sex- ual masochism disorder. In contrast, if they declare no distress, exemplified by anxiety, ob- sessions, guilt, or shame, about these paraphilic impulses, and are not hampered by them in pursuing other personal goals, they could be ascertained as having masochistic sexual interest but should not be diagnosed with sexual masochism disorder.

1	The Criterion A time frame, indicating that the signs or symptoms of sexual masoch- ism must have persisted for at least 6 months, should be understood as a general guideline, not a strict threshold, to ensure that the sexual interest in being humiliated, beaten, bound, or otherwise made to suffer is not merely transient. However, the disorder can be diag- nosed in the context of a clearly sustained but shorter time period. The extensive use of pornography involving the act of being humiliated, beaten, bound, or oth- erwise made to suffer is sometimes an associated feature of sexual masochism disorder. The population prevalence of sexual masochism disorder is unknown. In Australia, it has been estimated that 2.2% of males and 1.3% of females had been involved in bondage and discipline, sadomasochism, or dominance and submission in the past 12 months.

1	Community individuals with paraphilias have reported a mean age at onset for masoch- ism of 19.3 years, although earlier ages, including puberty and childhood, have also been reported for the onset of masochistic fantasies. Very little is known about persistence over time. Sexual masochism disorder per definition requires one or more contributing factors, which may change over time with or without treatment. These include subjective distress (e.g., guilt, shame, intense sexual frustration, loneliness), psychiatric morbidity, hypersex- uality and sexual impulsivity, and psychosocial impairment. Therefore, the course of sex- ual masochism disorder is likely to vary with age. Advancing age is likely to have the same reducing effect on sexual preference involving sexual masochism as it has on other para- philic or normophilic sexual behavior. Functional Consequences of Sexual Masochism Disorder

1	Functional Consequences of Sexual Masochism Disorder The functional consequences of sexual masochism disorder are unknown. However, mas- ochists are at risk of accidental death while practicing asphyxiophilia or other autoerotic procedures. Many of the conditions that could be differential diagnoses for sexual masochism disorder (e.g., transvestic fetishism, sexual sadism disorder, hypersexuality, alcohol and substance use disorders) sometimes occur also as comorbid diagnoses. Therefore, it is necessary to carefully evaluate the evidence for sexual masochism disorder, keeping the possibility of other paraphilias or other mental disorders as part of the differential diagnosis. Sexual masochism in the absence of distress (i.e., no disorder) is also included in the differential, as individuals who conduct the behaviors may be satisfied with their masochistic orienta- tion.

1	Known comorbidities with sexual masochism disorder are largely based on individuals in treatment. Disorders that occur comorbidly with sexual masochism disorder typically in- clude other paraphilic disorders, such as transvestic fetishism. Diagnostic Criteria 302.84 (F6552) A. Over a period of at least 6 months, recurrent and intense sexual arousal from the phys- ical or psychological suffering of another person, as manifested by fantasies, urges, or behaviors. B. The individual has acted on these sexual urges with a nonconsenting person, or the sexual urges or fantasies cause clinically significant distress or impairment in social, occupational. or other important areas of functioning. Specify it: In a controlled environment: This specifier is primarily applicable to individuals living in institutional or other settings where opportunities to engage in sadistic sexual behav- iors are restricted.

1	In full remission: The individual has not acted on the urges with a nonconsenting per- son, and there has been no distress or impairment in social, occupational, or other ar- eas of functioning, for at least 5 years while in an uncontrolled environment.

1	The diagnostic criteria for sexual sadism disorder are intended to apply both to individuals who freely admit to having such paraphilic interests and to those who deny any sexual interest in the physical or psychological suffering of another individual despite substantial objective evidence to the contrary. Individuals who openly acknowledge intense sexual interest in the physical or psychological suffering of others are referred to as ”admitting individuals.” If these individuals also report psychosocial difficulties because of their sexual attractions or prefer— ences for the physical or psychological suffering of another individual, they may be diagnosed with sexual sadism disorder. In contrast, if admitting individuals declare no distress, exempli- fied by anxiety, obsessions, guilt, or shame, about these paraphilic impulses, and are not ham- pered by them in pursuing other goals, and their self-reported, psychiatric, or legal histories indicate that they do not act on them, then

1	or shame, about these paraphilic impulses, and are not ham- pered by them in pursuing other goals, and their self-reported, psychiatric, or legal histories indicate that they do not act on them, then they could be ascertained as having sadistic sexual interest but they would not meet criteria for sexual sadism disorder.

1	Examples of individuals who deny any interest in the physical or psychological suffering of another individual include individuals known to have inﬂicted pain or suffering on mul— behavior and who may further claim that known episodes of sexual assault were either un- intentional or nonsexual. Others may admit past episodes of sexual behavior involving the inﬂiction of pain or suffering on a nonconsenting individual but do not report any significant or sustained sexual interest in the physical or psychological suffering of another individual.

1	Since these individuals deny having urges or fantasies involving sexual arousal to pain and suffering, it follows that they would also deny feeling subjectively distressed or socially im- paired by such impulses. Such individuals may be diagnosed with sexual sadism disorder despite their negative self—report. Their recurrent behavior constitutes clinical support for the presence of the paraphilia of sexual sadism (by satisfying Criterion A) and simultane— ously demonstrates that their paraphilically motivated behavior is causing clinically signif- icant distress, harm, or risk of harm to others (satisfying Criterion B).

1	"Recurrent” sexual sadism involving nonconsenting others (i.e., multiple victims, each on a separate occasion) may, as general rule, be interpreted as three or more victims on separate occasions. Fewer victims can be interpreted as satisfying this criterion, if there are multiple instances of inﬂiction of pain and suffering to the same victim, or if there is cor— roborating evidence of a strong or preferential interest in pain and suffering involving multiple victims. Note that multiple victims, as suggested earlier, are a sufficient but not a necessary condition for diagnosis, as the criteria may be met if the individual acknowl- edges intense sadistic sexual interest.

1	The Criterion A time frame, indicating that the signs or symptoms of sexual sadism must have persisted for at least 6 months, should also be understood as a general guide- line, not a strict threshold, to ensure that the sexual interest in inﬂicting pain and suffering on nonconsenting victims is not merely transient. However, the diagnosis may be met if there is a clearly sustained but shorter period of sadistic behaviors. The extensive use of pornography involving the inﬂiction of pain and suffering is some- times an associated feature of sexual sadism disorder. The population prevalence of sexual sadism disorder is unknown and is largely based on individuals in forensic settings. Depending on the criteria for sexual sadism, prevalence varies widely, from 2% to 30%. Among civilly committed sexual offenders in the United

1	States, less than 10% have sexual sadism. Among individuals who have committed sexu- ally motivated homicides, rates of sexual sadism disorder range from 37% to 75%. Individuals with sexual sadism in forensic samples are almost exclusively male, but a rep- resentative sample of the population in Australia reported that 2.2% of men and 1.3% of women said they had been involved in bondage and discipline, "sadomasochism," or dom- inance and submission in the previous year. Information on the development and course of sexual sadism disorder is extremely limited. One study reported that females became aware of their sadomasochistic orientation as young adults, and another reported that the mean age at onset of sadism in a group of males was 19.4 years. Whereas sexual sadism per se is probably a lifelong characteristic, sexual sadism disorder may ﬂuctuate according to the individual’s subjective distress or his or her propensity to harm nonconsenting others.

1	Advancing age is likely to have the same reducing effect on this disorder as it has on other paraphilic or normophilic sexual behavior. Many of the conditions that could be differential diagnoses for sexual sadism disorder (e.g., antisocial personality disorder, sexual masochism disorder, hypersexuality, sub— stance use disorders) sometimes occur also as comorbid diagnoses. Therefore, it is neces- sary to carefully evaluate the evidence for sexual sadism disorder, keeping the possibility of other paraphilias or mental disorders as part of the differential diagnosis. The majority of individuals who are active in community networks that practice sadistic and masoch- istic behaviors do not express any dissatisfaction with their sexual interests, and their be- havior would not meet DSM-5 criteria for sexual sadism disorder. Sadistic interest, but not the disorder, may be considered in the differential diagnosis.

1	Known comorbidities with sexual sadism disorder are largely based on individuals (al- Victims. Hence, these comorbidities might not apply to all individuals who never engaged in sadistic activity with a nonconsenting victim but who qualify for a diagnosis of sexual sadism disorder based on subjective distress over their sexual interest. Disorders that are commonly comorbid with sexual sadism disorder include other paraphilic disorders. Diagnostic Criteria 302.2 (F65.4) A. Over a period of at least 6 months, recurrent, intense sexually arousing fantasies, sex- ual urges, or behaviors involving sexual activity with a prepubescent child or children (generally age 13 years or younger). B. The individual has acted on these sexual urges, or the sexual urges or fantasies cause marked distress or interpersonal difficulty. C. The individual is at least age 16 years and at least 5 years older than the child or chil- dren in Criterion A.

1	C. The individual is at least age 16 years and at least 5 years older than the child or chil- dren in Criterion A. Note: Do not include an individual in late adolescence involved in an ongoing sexual relationship with a 12- or 13-year-old. Specify whether: Exclusive type (attracted only to children) Specify if: Sexually attracted to males Sexually attracted to females Sexually attracted to both Specify if: Limited to incest

1	The diagnostic criteria for pedophilic disorder are intended to apply both to individuals who freely disclose this paraphilia and to individuals who deny any sexual attraction to prepuber- tal children (generally age 13 years or younger), despite substantial objective evidence to the contrary. Examples of disclosing this paraphilia include candidly acknowledging an intense sexual interest in children and indicating that sexual interest in children is greater than or equal to sexual interest in physically mature individuals. If individuals also complain that their sex- ual attractions or preferences for children are causing psychosocial difficulties, they may be di- agnosed with pedophilic disorder. However, if they report an absence of feelings of guilt, shame, or anxiety about these impulses and are not functionally limited by their paraphilic im- pulses (according to self-report, objective assessment, or both), and their self-reported and le- gally recorded histories indicate that

1	and are not functionally limited by their paraphilic im- pulses (according to self-report, objective assessment, or both), and their self-reported and le- gally recorded histories indicate that they have never acted on their impulses, then these individuals have a pedophilic sexual orientation but not pedophilic disorder.

1	Examples of individuals who deny attraction to children include individuals who are known to have sexually approached multiple children on separate occasions but who deny any urges or fantasies about sexual behavior involving children, and who may further claim that the known episodes of physical contact were all unintentional and nonsexual. Other indi- viduals may acknowledge past episodes of sexual behavior involving children but deny any significant or sustained sexual interest in children. Since these individuals may deny experi- ences impulses or fantasies involving children, they may also deny feeling subjectively dis— tressed. Such individuals may still be diagnosed with pedophilic disorder despite the absence of self-reported distress, provided that there is evidence of recurrent behaviors persisting for 6 months (Criterion A) and evidence that the individual has acted on sexual urges or experi- enced interpersonal difficulties as a consequence of the disorder (Criterion B).

1	Presence of multiple Victims, as discussed above, is sufficient but not necessary for di- agnosis; that is, the individual can still meet Criterion A by merely acknowledging intense or preferential sexual interest in children. The Criterion A clause, indicating that the signs or symptoms of pedophilia have per- sisted for 6 months or longer, is intended to ensure that the sexual attraction to children is not merely transient. However, the diagnosis may be made if there is clinical evidence of sustained persistence of the sexual attraction to children even if the 6-month duration can- not be precisely determined. The extensive use of pornography depicting prepubescent children is a useful diagnostic indicator of pedophilic disorder. This is a specific instance of the general case that individ- uals are likely to choose the kind of pornography that corresponds to their sexual interests.

1	The population prevalence of pedophilic disorder is unknown. The highest possible prev- alence for pedophilic disorder in the male population is approximately 3%—5%. The pop- ulation prevalence of pedophilic disorder in females is even more uncertain, but it is likely a small fraction of the prevalence in males. Adult males with pedophilic disorder may indicate that they become aware of strong or preferential sexual interest in children around the time of puberty—the same time frame in which males who later prefer physically mature partners became aware of their sexual interest in women or men. Attempting to diagnose pedophilic disorder at the age at which it first manifests is problematic because of the difficulty during adolescent development in differentiating it from age-appropriate sexual interest in peers or from sexual curiosity. Hence, Criterion C requires for diagnosis a minimum age of 16 years and at least 5 years older than the child or children in Criterion A.

1	Hence, Criterion C requires for diagnosis a minimum age of 16 years and at least 5 years older than the child or children in Criterion A. Pedophilia per se appears to be a lifelong condition. Pedophilic disorder, however, necessarily includes other elements that may change over time with or without treatment: subjective distress (e.g., guilt, shame, intense sexual frustration, or feelings of isolation) or psychosocial impairment, or the propensity to act out sexually with children, or both. Therefore, the course of pedophilic disorder may ﬂuctuate, increase, or decrease with age. Adults with pedophilic disorder may report an awareness of sexual interest in children that preceded engaging in sexual behavior involving children or self-identification as a pedo- phile. Advanced age is as likely to similarly diminish the frequency of sexual behavior involv— ing children as it does other paraphilically motivated and normophilic sexual behavior.

1	Temperamental. There appears to be an interaction between pedophilia and antisocial- ity, such that males with both traits are more likely to act out sexually with children. Thus, males with pedophilia. Environmental. Adult males with pedophilia often report that they were sexually abused as children. It is unclear, however, whether this correlation reﬂects a causal inﬂuence of childhood sexual abuse on adult pedophilia. Genetic and physiological. Since pedophilia is a necessary condition for pedophilic dis- order, any factor that increases the probability of pedophilia also increases the risk of pe- dophilic disorder. There is some evidence that neurodevelopmental perturbation in utero increases the probability of development of a pedophilic orientation.

1	Psychophysiological laboratory measures of sexual interest, which are sometimes useful in di- agnosing pedophilic disorder in males, are not necessarily useful in diagnosing this disorder in females, even when an identical procedure (e.g., viewing time) or analogous procedures (e.g., penile plethysmography and vaginal photoplethysmography) are available.

1	Psychophysiological measures of sexual interest may sometimes be useful when an indi- vidual’s history suggests the possible presence of pedophilic disorder but the individual denies strong or preferential attraction to children. The most thoroughly researched and longest used of such measures is penile plethysmography, although the sensitivity and spec- ificity of diagnosis may vary from one site to another. Viewing time, using photographs of nude or minimally clothed persons as visual stimuli, is also used to diagnose pedophilic disorder, especially in combination with self—report measures. Mental health professionals in the United States, however, should be aware that possession of such visual stimuli, even for diagnostic purposes, may violate American law regarding possession of child pornog- raphy and leave the mental health professional susceptible to criminal prosecution.

1	Many of the conditions that could be differential diagnoses for pedophilic disorder also sometimes occur as comorbid diagnoses. It is therefore generally necessary to evaluate the evidence for pedophilic disorder and other possible conditions as separate questions. Antisocial personality disorder. This disorder increases the likelihood that a person who is primarily attracted to the mature physique will approach a child, on one or a few occa- sions, on the basis of relative availability. The individual often shows other signs of this personality disorder, such as recurrent law-breaking. Alcohol and substance use disorders. The disinhibiting effects of intoxication may also increase the likelihood that a person who is primarily attracted to the mature physique will sexually approach a child.

1	Obsessive-compulsive disorder. There are occasional individuals who complain about ego-dystonic thoughts and worries about possible attraction to children. Clinical inter- viewing usually reveals an absence of sexual thoughts about children during high states of sexual arousal (e.g., approaching orgasm during masturbation) and sometimes additional ego-dystonic, intrusive sexual ideas (e.g., concerns about homosexuality). Psychiatric comorbidity of pedophilic disorder includes substance use disorders; depres- sive, bipolar, and anxiety disorders; antisocial personality disorder; and other paraphilic disorders. However, findings on comorbid disorders are largely among individuals con- izable to other individuals with pedophilic disorder (e.g., individuals who have never approached a child sexually but who qualify for the diagnosis of pedophilic disorder on the basis of subjective distress). Diagnostic Criteria 302.81 (F65.0)

1	Diagnostic Criteria 302.81 (F65.0) A. Over a period of at least 6 months, recurrent and intense sexual arousal from either the use of nonliving objects or a highly specific focus on nongenital body pad(s), as manifested by fantasies. urges, or behaviors. B. The fantasies, sexual urges, or behaviors cause clinically significant distress or impair- ment in social, occupational, or other important areas of functioning. C. The fetish objects are not limited to articles of clothing used in cross-dressing (as in transvestic disorder) or devices specifically designed for the purpose of tactile genital stimulation (e.g.. vibrator). Specify: Specify if: in a controlled environment: This specifier is primarily applicable to individuals living in institutional or other settings where opportunities to engage in fetishistic behaviors are restricted.

1	In full remIssIon: There has been no distress or impairment in social, occupational, or other areas of functioning for at least 5 years while in an uncontrolled environment. Although individuals with fetishistic disorder may report intense and recurrent sexual arousal to inanimate objects or a specific body part, it is not unusual for non—mutually ex- clusive combinations of fetishes to occur. Thus, an individual may have fetishistic disorder associated with an inanimate object (e.g., female undergarments) or an exclusive focus on an intensely eroticized body part (e.g., feet, hair), or their fetishistic interest may meet cri- teria for various combinations of these specifiers (e.g., socks, shoes and feet).

1	The paraphilic focus of fetishistic disorder involves the persistent and repetitive use of or de as primary elements associated with sexual arousal (Criterion A). A diagnosis of fetishistic dis- (Criterion B). Common fetish objects include female undergarments, male or female footwear, rubber articles, leather clothing, or other wearing apparel. Highly eroticized body parts asso- ciated with fetishistic disorder include feet, toes, and hair. It is not uncommon for sexualized fetishes to include both inanimate objects and body parts (e.g., dirty socks and feet), and for this reason the definition of fetishistic disorder now re-incorporates partiulism (i.e., an exclusive focus on a body part) into its boundaries. Partialism, previously considered a paraphilia not otherwise specified disorder, had historically been subsumed in fetishism prior to DSM-Il].

1	Many individuals who self—identify as fetishist practitioners do not necessarily report clinical impairment in association with their fetish-associated behaviors. Such individuals could be considered as having a fetish but not fetishistic disorder. A diagnosis of fetishistic disorder requires concurrent fulfillment of both the behaviors in Criterion A and the clin- ically significant distress or impairment in ftmctioning noted in Criterion B. Fetishistic disorder can be a multisensory experience, including holding, tasting, rubbing, inserting, or smelling the fetish object while masturbating, or preferring that a sexual part- ner wear or utilize a fetish object during sexual encounters. Some individuals may acquire extensive collections of highly desired fetish objects.

1	Usually paraphilias have an onset during puberty, but fetishes can develop prior to ado- lescence. Once established, fetishistic disorder tends to have a continuous course that ﬂuc- tuates in intensity and frequency of urges or behavior. Knowledge of and appropriate consideration for normative aspects of sexual behavior are important factors to explore to establish a clinical diagnosis of fetishistic disorder and to distinguish a clinical diagnosis from a socially acceptable sexual behavior. Fetishistic disorder has not been systematically reported to occur in females. In clinical samples, fetishistic disorder is nearly exclusively reported in males. Functional Consequences of Fetishistic Disorder

1	Functional Consequences of Fetishistic Disorder Typical impairments associated with fetishistic disorder include sexual dysfunction during romantic reciprocal relationships when the preferred fetish object or body part is unavailable during foreplay or coitus. Some individuals with fetishistic disorder may pre- fer solitary sexual activity associated with their fetishistic preference(s) even while in- volved in a meaningful reciprocal and affectionate relationship. Although fetishistic disorder is relatively uncommon among arrested sexual offenders with paraphilias, males with fetishistic disorder may steal and collect their particular fe- tishistic objects of desire. Such individuals have been arrested and charged for nonsexual antisocial behaviors (e.g., breaking and entering, theft, burglary) that are primarily moti- vated by the fetishistic disorder.

1	Transvestic disorder. The nearest diagnostic neighbor of fetishistic disorder is transves- tic disorder. As noted in the diagnostic criteria, fetishistic disorder is not diagnosed when fetish objects are limited to articles of clothing exclusively worn during cross-dressing (as in transvestic disorder), or when the object is genitally stimulating because it has been de~ signed for that purpose (e.g., a vibrator). Sexual masochism disorder or other paraphilic disorders. Fetishes can co-occur with other paraphilic disorders, especially ”sadomasochism” and transvestic disorder. When an individual fantasizes about or engages in ”forced cross-dressing” and is primarily sex- ually aroused by the domination or humiliation associated with such fantasy or repetitive activity, the diagnosis of sexual masochism disorder should be made.

1	Fetishistic behavior without fetishistic disorder. Use of a fetish object for sexual arousal quence would not meet criteria for fetishistic disorder, as the threshold required by Crite- rion B would not be met. For example, an individual whose sexual partner either shares or can successfully incorporate his interest in caressing, smelling, or licking feet or toes as an important element of foreplay would not be diagnosed with fetishistic disorder; nor would an individual who prefers, and is not distressed or impaired by, solitary sexual be- havior associated with wearing rubber garments or leather boots. Fetishistic disorder may co-occur with other paraphilic disorders as well as hypersexual- ity. Rarely, fetishistic disorder may be associated with neurological conditions. Diagnostic Criteria 302.3 (F65.1) A. Over a period of at least 6 months. recurrent and intense sexual arousal from cross- dressing, as manifested by fantasies, urges, or behaviors.

1	Diagnostic Criteria 302.3 (F65.1) A. Over a period of at least 6 months. recurrent and intense sexual arousal from cross- dressing, as manifested by fantasies, urges, or behaviors. B. The fantasies. sexual urges. or behaviors cause clinically significant distress or impair- ment in social. occupational, or other important areas of functioning. Specify if: Wlth fetishism: If sexually aroused by fabrics, materials, or garments. With autogynephllia: If sexually aroused by thoughts or images of self as female. Specify it: In a controiled environment: This specifier is primarily applicable to individuals living in institutional or other settings where opportunities to cross-dress are restricted. In full remisslon: There has been no distress or impairment in social, occupational, or other areas of functioning for at least 5 years while in an uncontrolled environment.

1	In full remisslon: There has been no distress or impairment in social, occupational, or other areas of functioning for at least 5 years while in an uncontrolled environment. The presence of fetishism decreases the likelihood of gender dysphoria in men with trans- vestic disorder. The presence of autogynephilia increases the likelihood of gender dyspho- ria in men with transvestic disorder.

1	The diagnosis of transvestic disorder does not apply to all individuals who dress as the op- posite sex, even those who do so habitually. It applies to individuals whose cross-dressing or thoughts of cross-dressing are always or often accompanied by sexual excitement (Cri- terion A) and who are emotionally distressed by this pattern or feel it impairs social or in- terpersonal functioning (Criterion B). The cross-dressing may involve only one or two articles of clothing (e.g., for men, it may pertain only to women’s undergarments), or it may involve dressing completely in the inner and outer garments of the other sex and (in men) may include the use of women’s wigs and make-up. Transvestic disorder is nearly exclusively reported in males. Sexual arousal, in its most obvious form of penile erection, may co-occur with cross-dressing in various ways. In younger males, cross-dressing often leads to masturbation, following which any female clothing is removed. Older males often learn to

1	erection, may co-occur with cross-dressing in various ways. In younger males, cross-dressing often leads to masturbation, following which any female clothing is removed. Older males often learn to avoid masturbating or doing anything to stimulate the penis so that the avoidance of ejaculation allows them to prolong their cross-dressing session. Males with female part- ners sometimes complete a cross-dressing session by having intercourse with their part- ners, and some have difficulty maintaining a sufficient erection for intercourse without cross-dressing (or private fantasies of cross-dressing).

1	Clinical assessment of distress or impairment, like clinical assessment of transvestic sexual arousal, is usually dependent on the individual’s self—report. The pattern of behav- ior "purging and acquisition” often signifies the presence of distress in individuals with transvestic disorder. During this behavioral pattern, an individual (usually a man) who has spent a great deal of money on women’s clothes and other apparel (e.g., shoes, wigs) discards the items (i.e., purges them) in an effort to overcome urges to cross-dress, and then begins acquiring a woman’s wardrobe all over again. Transvestic disorder in men is often accompanied by autogynephilia (i.e., a male’s para- philic tendency to be sexually aroused by the thought or image of himself as a woman).

1	Transvestic disorder in men is often accompanied by autogynephilia (i.e., a male’s para- philic tendency to be sexually aroused by the thought or image of himself as a woman). Autogynephilic fantasies and behaviors may focus on the idea of exhibiting female phys- iological functions (e.g., lactation, menstruation), engaging in stereotypically feminine be- havior (e.g., knitting), or possessing female anatomy (e.g., breasts). The prevalence of transvestic disorder is unknown. Transvestic disorder is rare in males and extremely rare in females. Fewer than 3% of males report having ever been sexually aroused by dressing in women’s attire. The percentage of individuals who have cross- dressed with sexual arousal more than once or a few times in their lifetimes would be even lower. The majority of males with transvestic disorder identify as heterosexual, although some individuals have occasional sexual interaction with other males, especially when they are cross-dressed.

1	In males, the first signs of transvestic disorder may begin in childhood, in the form of strong fascination with a particular item of women’s attire. Prior to puberty, cross-dress- ing produces generalized feelings of pleasurable excitement. With the arrival of puberty, dressing in women’s clothes begins to elicit penile erection and, in some cases, leads di- rectly to first ejaculation. In many cases, cross-dressing elicits less and less sexual ex- citement as the individual grows older; eventually it may produce no discernible penile response at all. The desire to cross-dress, at the same time, remains the same or grows even stronger. Individuals who report such a diminution of sexual response typically report that the sexual excitement of cross-dressing has been replaced by feelings of comfort or well-being.

1	In some cases, the course of transvestic disorder is continuous, and in others it is epi- sodic. It is not rare for men with transvestic disorder to lose interest in cross-dressing when they first fall in love with a woman and begin a relationship, but such abatement usually proves temporary. When the desire to cross-dress returns, so does the associated distress. Some cases of transvestic disorder progress to gender dysphoria. The males in these cases, who may be indistinguishable from others with transvestic disorder in adolescence or early childhood, gradually develop desires to remain in the female role for longer pe- riods and to feminize their anatomy. The development of gender dysphoria is usually ac- companied by a (self-reported) reduction or elimination of sexual arousal in association with cross-dressing.

1	The manifestation of transvestism in penile erection and stimulation, like the manifesta- tion of other paraphilic as well as normophilic sexual interests, is most intense in adolescence and early adulthood. The severity of transvestic disorder is highest in adulthood, when the transvestic drives are most likely to conﬂict with performance in heterosexual intercourse and desires to marry and start a family. Middle—age and older men with a history of trans- vestism are less likely to present with transvestic disorder than with gender dysphoria. Functional Consequences of Transvestic Disorder Engaging in transvestic behaviors can interfere with, or detract from, heterosexual rela- tionships. This can be a source of distress to men who wish to maintain conventional mar— riages or romantic partnerships with women. Fetishistic disorder. This disorder may resemble transvestic disorder, in particular, in men with fetishism who put on women’s undergarments while masturbating with them.

1	Fetishistic disorder. This disorder may resemble transvestic disorder, in particular, in men with fetishism who put on women’s undergarments while masturbating with them. Distinguishing transvestic disorder depends on the individual’s specific thoughts during such activity (e.g., are there any ideas of being a woman, being like a woman, or being dressed as a woman?) and on the presence of other fetishes (e.g., soft, silky fabrics, whether these are used for garments or for something else). Gender dysphoria. Individuals with transvestic disorder do not report an incongruence be- tween their experienced gender and assigned gender nor a desire to be of the other gender; and they typically do not have a history of childhood cross-gender behaviors, which would be present in individuals with gender dysphoria. Individuals with a presentation that meets full criteria for transvestic disorder as well as gender dysphoria should be given both diagnoses.

1	Transvestism (and thus transvestic disorder) is often found in association with other para- philias. The most frequently co-occurring paraphilias are fetishism and masochism. One particularly dangerous form of masochism, au toerotic asphyxia, is associated with transves- tism in a substantial proportion of fatal cases. 302.89 (F65.89)

1	302.89 (F65.89) This category applies to presentations in which symptoms characteristic of a paraphilic disor- der that cause clinically significant distress or impairment in social, occupational, or other im- portant areas of functioning predominate but do not meet the full criteria for any of the disorders in the paraphilic disorders diagnostic class. The other specified paraphilic disorder category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for any specific paraphilic disorder. This is done by re- cording “other specified paraphilic disorder’ followed by the specific reason (e.g., “zoophilia”).

1	Examples of presentations that can be specified using the "other specified" designation include, but are not limited to, recurrent and intense sexual arousal involving telephone scatologia (obscene phone calls), necrophilia (corpses), zoophilia (animals), coprophilia (feces), klismaphilia (enemas), or urophilia (urine) that has been present for at least 6 months and causes marked distress or impairment in social, occupational, or other important ar- eas of functioning. Other specified paraphilic disorder can be specified as in remission and/or as occurring in a controlled environment. 302.9 (F65.9)

1	302.9 (F65.9) This category applies to presentations in which symptoms characteristic of a paraphilic disorder that cause clinically significant distress or impairment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any of the disorders in the paraphilic disorders diagnostic class. The unspecified paraphilic dis- order category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for a specific paraphilic disorder, and includes presentations in which there is insufficient information to make a more specific diagnosis.

1	FOUF disorders are included in this chapter: other specified mental disorder due to another medical condition; unspecified mental disorder due to another medical condition; other specified mental disorder; and unspecified mental disorder. This residual category applies to presentations in which symptoms characteristic of a mental disorder that cause clinically significant distress or impairment in social, occupational, or other important ar- eas of functioning predominate but do not meet the full criteria for any other mental dis- order in DSM—5. For other specified and unspecified mental disorders due to another medical condition, it must be established that the disturbance is caused by the physiolog- ical effects of another medical condition. If other specified and unspecified mental disor- ders are due to another medical condition, it is necessary to code and list the medical condition first (e.g., 042 [820] HIV disease), followed by the other specified or unspecified mental

1	disor- ders are due to another medical condition, it is necessary to code and list the medical condition first (e.g., 042 [820] HIV disease), followed by the other specified or unspecified mental disorder (use appropriate code).

1	Due to Another Medical Condition 294.8 (F06.8)

1	This category applies to presentations in which symptoms characteristic of a mental dis- order due to another medical condition that cause clinically significant distress or impair— ment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any specific mental disorder attributable to another medical condition. The other specified mental disorder due to another medical condition category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for any specific mental disorder attributable to another medical condition. This is done by recording the name of the disorder, with the specific etiological medical condition inserted in place of “another medical condition," fol- lowed by the specific symptomatic manifestation that does not meet the criteria for any specific mental disorder due to another medical condition. Furthermore, the diagnostic code

1	condition," fol- lowed by the specific symptomatic manifestation that does not meet the criteria for any specific mental disorder due to another medical condition. Furthermore, the diagnostic code for the specific medical condition must be listed immediately before the code for the other specified mental disorder due to another medical condition. For example, dissocia- tive symptoms due to complex partial seizures would be coded and recorded as 345.40 (640.209), complex partial seizures 294.8 (F06.8) other specified mental disorder due to complex partial seizures, dissociative symptoms.

1	An example of a presentation that can be specified using the “other specified" desig- nation is the following: Dissociative symptoms: This includes symptoms occurring, for example, in the con- text of complex partial seizures. Due to Another Medical Condition 294.9 (F09) This category applies to presentations in which symptoms characteristic of a mental dis- order due to another medical condition that cause clinically significant distress or impair- ment in social, occupational, or other important areas of functioning predominate but do not meet the full criteria for any specific mental disorder due to another medical condition.

1	The unspecified mental disorder due to another medical condition category is used in sit- uations in which the clinician chooses not to specify the reason that the criteria are not met for a specific mental disorder due to another medical condition, and includes presentations for which there is insufficient information to make a more specific diagnosis (e.g., in emer- gency room settings). This is done by recording the name of the disorder, with the specific etiological medical condition inserted in place of “another medical condition." Furthermore, the diagnostic code for the specific medical condition must be listed immediately before the code for the unspecified mental disorder due to another medical condition. For exam- ple, dissociative symptoms due to complex partial seizures would be coded and recorded as 345.40 (640.209) complex partial seizures, 294.9 (F069) unspecified mental disorder due to complex partial seizures. 300.9 (F99)

1	300.9 (F99) This category applies to presentations in which symptoms characteristic of a mental dis- order that cause clinically significant distress or impairment in social, occupational. or oth- er important areas of functioning predominate but do not meet the full criteria for any specific mental disorder. The other specified mental disorder category is used in situations in which the clinician chooses to communicate the specific reason that the presentation does not meet the criteria for any specific mental disorder. This is done by recording "other specified mental disorder” followed by the specific reason. 300.9 (F99)

1	300.9 (F99) This category applies to presentations in which symptoms characteristic of a mental dis- order that cause clinically significant distress or impairment in social, occupational, or oth- er important areas of functioning predominate but do not meet the full criteria for any mental disorder. The unspecified mental disorder category is used in situations in which the clinician chooses not to specify the reason that the criteria are not met for a specific mental disorder, and includes presentations for which there is insufficient information to make a more specific diagnosis (e.g., in emergency room settings). MedlCaftiOn-lnduced.MOvem‘e' Disorders and ,Other‘ Advers

1	MedlCaftiOn-lnduced.MOvem‘e' Disorders and ,Other‘ Advers Medication—induced movement disorders are included in Section II because of their frequent importance in 1) the management by medication of mental disorders or oth- er medical conditions and 2) the differential diagnosis of mental disorders (e.g., anxiety malignant syndrome). Although these movement disorders are labeled “medication in- duced,” it is often difficult to establish the causal relationship between medication expo- sure and the development of the movement disorder, especially because some of these movement disorders also occur in the absence of medication exposure. The conditions and problems listed in this chapter are not mental disorders.

1	The term neuroleptic is becoming outdated because it highlights the propensity of an- tipsychotic medications to cause abnormal movements, and it is being replaced with the term antipsychotic in many contexts. Nevertheless, the term neuroleptic remains appropri- ate in this context. Although newer antipsychotic medications may be less likely to cause some medication-induced movement disorders, those disorders still occur. Neuroleptic medications include so-called conventional, ”typical,” or first-generation antipsychotic agents (e.g., chlorpromazine, haloperidol, ﬂuphenazine); ”atypical” or second-generation antipsychotic agents (e.g., clozapine, risperidone, olanzapine, quetiapine); certain dopa- mine receptor—blocking drugs used in the treatment of symptoms such as nausea and gas- troparesis (e.g., prochlorperazine, promethazine, trimethobenzamide, thiethylperazine, metoclopramide); and amoxapine, which is marketed as an antidepressant.

1	332.1 (G21.11) Neuroleptic-Induced Parkinsonism 332.1 (621.19) Other Medication-Induced Parkinsonism Parkinsonian tremor, muscular rigidity, akinesia (i.e., loss of movement or difficulty ini- tiating movement), or bradykinesia (i.e., slowing movement) developing within a few weeks of starting or raising the dosage of a medication (e.g., a neuroleptic) or after reduc- ing the dosage of a medication used to treat extrapyramidal symptoms. 333.92 (G21.0) Neuroleptic Malignant Syndrome Although neuroleptic malignant syndrome is easily recognized in its classic full-blown form, it is often heterogeneous in onset, presentation, progression, and outcome. The clin- ical features described below are those considered most important in making the diagno- sis of neuroleptic malignant syndrome based on consensus recommendations.

1	Patients have generally been exposed to a dopamine antagonist within 72 hours prior to symptom development. Hyperthermia (>100.4°F or >38.0°C on at least two occasions, measured orally), associated with profuse diaphoresis, is a distinguishing feature of neu- roleptic malignant syndrome, setting it apart from other neurological side effects of anti- psychotic medications. Extreme elevations in temperature, reflecting a breakdown in central thermoregulation, are more likely to support the diagnosis of neuroleptic malig- nant syndrome. Generalized rigidity, described as ”lead pipe” in its most severe form and usually unresponsive to antiparkinsonian agents, is a cardinal feature of the disorder and may be associated with other neurological symptoms (e.g., tremor, sialorrhea, akinesia, dystonia, trismus, myoclonus, dysarthria, dysphagia, rhabdomyolysis). Creatine kinase elevation of at least four times the upper limit of normal is commonly seen. Changes in mental status, characterized by

1	trismus, myoclonus, dysarthria, dysphagia, rhabdomyolysis). Creatine kinase elevation of at least four times the upper limit of normal is commonly seen. Changes in mental status, characterized by delirium or altered consciousness ranging from stupor to coma, are often an early sign. Affected individuals may appear alert but dazed and unre- sponsive, consistent with catatonic stupor. Autonomic activation and instability—mani- fested by tachycardia (rate>25% above baseline), diaphoresis, blood pressure elevation 225 mmHg systolic change within 24 hours), urinary incontinence, and pallor—may be seen at any time but provide an early clue to the diagnosis. Tachypnea (rate >50°/o above baseline) is common, and respiratory distress—resulting from metabolic acidosis, hyper- metabolism, chest wall restriction, aspiration pneumonia, or pulmonary emboli—can oc- cur and lead to sudden respiratory arrest.

1	A workup, including laboratory investigation, to exclude other infectious, toxic, met- abolic, and neuropsychiatric etiologies or complications is essential (see the section ”Dif- ferential Diagnosis” later in this discussion). Although several laboratory abnormalities are associated with neuroleptic malignant syndrome, no single abnormality is specific to the diagnosis. Individuals with neuroleptic malignant syndrome may have leukocytosis, metabolic acidosis, hypoxia, decreased serum iron concentrations, and elevations in se- rum muscle enzymes and catecholamines. Findings from cerebrospinal ﬂuid analysis and neuroimaging studies are generally normal, whereas electroencephalography shows gen- eralized slowing. Autopsy findings in fatal cases have been nonspecific and variable, de- pending on complications.

1	drome of 0.01%—0.02% among individuals treated with antipsychotics. The temporal pro- gression of signs and symptoms provides important clues to the diagnosis and prognosis of neuroleptic malignant syndrome. Alteration in mental status and other neurological signs typically precede systemic signs. The onset of symptoms varies from hours to days after drug initiation. Some cases develop within 24 hours after drug initiation, most within the first week, and virtually all cases within 30 days. Once the syndrome is diagnosed and oral antipsychotic drugs are discontinued, neuroleptic malignant syndrome is self—limited in most cases. The mean recovery time after drug discontinuation is 7—10 days, with most individuals recovering within 1 week and nearly all within 30 days. The duration may be prolonged when long-acting antipsychotics are implicated. There have been reports of in- dividuals in whom residual neurological signs persisted for weeks after the acute hyper- metabolic symptoms

1	be prolonged when long-acting antipsychotics are implicated. There have been reports of in- dividuals in whom residual neurological signs persisted for weeks after the acute hyper- metabolic symptoms resolved. Total resolution of symptoms can be obtained in most cases of neuroleptic malignant syndrome; however, fatality rates of 10%—20% have been reported when the disorder is not recognized. Although many individuals do not experi- ence a recurrence of neuroleptic malignant syndrome when rechallenged with antipsy- chotic medication, some do, especially when antipsychotics are reinstituted soon after an episode.

1	Neuroleptic malignant syndrome is a potential risk in any individual after antipsychotic drug administration. It is not specific to any neuropsychiatric diagnosis and may occur in individuals without a diagnosable mental disorder who receive dopamine antagonists. Clinical, systemic, and metabolic factors associated with a heightened risk of neuroleptic malignant syndrome include agitation, exhaustion, dehydration, and iron deficiency. A prior episode associated with antipsychotics has been described in 15%—20% of index cases, suggesting underlying vulnerability in some patients; however, genetic findings based on neurotransmitter receptor polymorphisms have not been replicated consistently.

1	Nearly all dopamine antagonists have been associated with neuroleptic malignant syndrome, although high-potency antipsychotics pose a greater risk compared with low- potency agents and newer atypical antipsychotics. Partial or milder forms may be associ- ated with newer antipsychotics, but neuroleptic malignant syndrome varies in severity even with older drugs. Dopamine antagonists used in medical settings (e.g., metoclopra- mide, prochlorperazine) have also been implicated. Parenteral administration routes, rapid titration rates, and higher total drug dosages have been associated with increased risk; however, neuroleptic malignant syndrome usually occurs within the therapeutic dos- age range of antipsychotics. or medical conditions, including central nervous system infections, inﬂammatory or au- toimmune conditions, status epilepticus, subcortical structural lesions, and systemic con- ditions (e.g., pheochromocytoma, thyrotoxicosis, tetanus, heat stroke).

1	resulting from the use of other substances or medications, such as serotonin syndrome; parkinsonian hyperthermia syndrome following abrupt discontinuation of dopamine ag- thesia; hyperthermia associated with abuse of stimulants and hallucinogens; and atropine poisoning from anticholinergics. In rare instances, individuals with schizophrenia or a mood disorder may present with malignant catatonia, which may be indistinguishable from neuroleptic malignant syn- drome. Some investigators consider neuroleptic malignant syndrome to be a drug- induced form of malignant catatonia. 333.72 (624.02) Medication-Induced Acute Dystonia Abnormal and prolonged contraction of the muscles of the eyes (oculogyric crisis), head, neck (torticollis or retrocollis), limbs, or trunk developing within a few days of starting or raising the dosage of a medication (such as a neuroleptic) or after reducing the dosage of a medication used to treat extrapyramidal symptoms.

1	333.99 (G25.71) Medication-Induced Acute Akathisia Subjective complaints of restlessness, often accompanied by observed excessive move- ments (e.g., fidgety movements of the legs, rocking from foot to foot, pacing, inability to sit or stand still), developing within a few weeks of starting or raising the dosage of a medi- cation (such as a neuroleptic) or after reducing the dosage of a medication used to treat ex- trapyramidal symptoms. 333.85 (624.01) Tardive Dyskinesia the tongue, lower face and jaw, and extremities (but sometimes involving the pharyngeal, diaphragmatic, or trunk muscles) developing in association with the use of a neuroleptic medication for at least a few months.

1	Symptoms may develop after a shorter period of medication use in older persons. In some patients, movements of this type may appear after discontinuation, or after change or reduction in dosage, of neuroleptic medications, in which case the condition is called neuroleptic withdrawal—emergent dyskinesia. Because withdrawal-emergent dyskinesia is usually time-limited, lasting less than 4—8 weeks, dyskinesia that persists beyond this win- dow is considered to be tardive dyskinesia. 333.72 (624.09) Tardive Dystonia 333.99 (G25.71) Tardive Akathisia Tardive syndrome involving other types of movement problems, such as dystonia or akathisia, which are distinguished by their late emergence in the course of treatment and their potential persistence for months to years, even in the face of neuroleptic discontinu- ation or dosage reduction. 333.1 (625.1) Medication-Induced Postural Tremor

1	333.1 (625.1) Medication-Induced Postural Tremor Fine tremor (usually in the range of 8—12 Hz) occurring during attempts to maintain a pos- ture and developing in association with the use of medication (e.g., lithium, antidepres- sants, valproate). This tremor is very similar to the tremor seen with anxiety, caffeine, and other stimulants. 333.99 (625.79) Other Medication-Induced Movement Disorder This category is for medication-induced movement disorders not captured by any of the specific disorders listed above. Examples include 1) presentations resembling neuroleptic malignant syndrome that are associated with medications other than neuroleptics and 2) other medication-induced tardive conditions. 995.29 (T43.205A) Initial encounter 995.29 (T 43.205D) Subsequent encounter 995.29 (T 43.2053) Sequelae

1	Antidepressant discontinuation syndrome is a set of symptoms that can occur after an abrupt cessation (or marked reduction in dose) of an antidepressant medication that was taken continuously for at least 1 month. Symptoms generally begin within 2—4 days and typically include specific sensory, somatic, and cognitive-emotional manifestations. Fre- quently reported sensory and somatic symptoms include ﬂashes of lights, ”electric shock” sensations, nausea, and hyperresponsivity to noises or lights. Nonspecific anxiety and feelings of dread may also be reported. Symptoms are alleviated by restarting the same medication or starting a different medication that has a similar mechanism of action— for example, discontinuation symptoms after withdrawal from a serotonin-norepineph- rine reuptake inhibitor may be alleviated by starting a tricyclic antidepressant. To qualify as antidepressant discontinuation syndrome, the symptoms should not have been present before the antidepressant dosage was

1	may be alleviated by starting a tricyclic antidepressant. To qualify as antidepressant discontinuation syndrome, the symptoms should not have been present before the antidepressant dosage was reduced and are not better explained by another mental disorder (e.g., manic or hypomanic episode, substance intoxication, substance withdrawal, somatic symptom disorder).

1	Discontinuation symptoms may occur following treatment with tricyclic antidepressants (e.g., imipramine, amitriptyline, desipramine), serotonin reuptake inhibitors (e.g., ﬂuox- etine, paroxetine, sertraline), and monoamine oxidase inhibitors (e.g., phenelzine, selegi- line, pargyline). The incidence of this syndrome depends on the dosage and half-life of the medication being taken, as well as the rate at which the medication is tapered. Short-acting medications that are stopped abruptly rather than tapered gradually may pose the great- est risk. The short-acting selective serotonin reuptake inhibitor (SSRI) paroxetine is the agent most commonly associated with discontinuation symptoms, but such symptoms oc- cur for all types of antidepressants.

1	Unlike withdrawal syndromes associated with opioids, alcohol, and other substances of abuse, antidepressant discontinuation syndrome has no pathognomonic symptoms. In- stead, the symptoms tend to be vague and variable and typically begin 2—4 days after the last dose of the antidepressant. For SSRIs (e.g., paroxetine), symptoms such as dizziness, ringing in the ears, "electric shocks in the head," an inability to sleep, and acute anxiety are described. The antidepressant use prior to discontinuation must not have incurred hypo- mania or euphoria (i.e., there should be confidence that the discontinuation syndrome is not the result of ﬂuctuations in mood stability associated with the previous treatment).

1	The antidepressant discontinuation syndrome is based solely on pharmacological factors and is not related to the reinforcing effects of an antidepressant. Also, in the case of stim- ulant augmentation of an antidepressant, abrupt cessation may result in stimulant with- drawal symptoms (see ”Stimulant Withdrawal" in the chapter ”Substance-Related and Addictive Disorders”) rather than the antidepressant discontinuation syndrome described here. The prevalence of antidepressant discontinuation syndrome is unknown but is thought to vary according to the dosage prior to discontinuation, the half-life and receptor-binding affinity of the medication, and possibly the individual’s genetically inﬂuenced rate of me- tabolism for this medication.

1	Because longitudinal studies are lacking, little is known about the clinical course of anti- depressant discontinuation syndrome. Symptoms appear to abate over time with very gradual dosage reductions. After an episode, some individuals may prefer to resume med- ication indefinitely if tolerated. The differential diagnosis of antidepressant discontinuation syndrome includes anxiety and depressive disorders, substance use disorders, and tolerance to medications. Anxiety and depressive disorders. Discontinuation symptoms often resemble symptoms of a persistent anxiety disorder or a return of somatic symptoms of depression for which the medication was initially given.

1	Substance use disorders. Antidepressant discontinuation syndrome differs from sub- fects. The medication dosage has usually not been increased without the clinician’s permission, and the individual generally does not engage in drug-seeking behavior to ob- tain additional medication. Criteria for a substance use disorder are not met. Tolerance to medications. Tolerance and discontinuation symptoms can occur as a normal physiological response to stopping medication after a substantial duration of exposure. Most cases of medication tolerance can be managed through carefully con- trolled tapering. Typically, the individual was initially started on the medication for a major depressive dis- order; the original symptoms may return during the discontinuation syndrome. Other Adverse Effect of Medication 995.20 (T 50.905A) Initial encounter 995.20 (T 50.905D) Subsequent encounter 995.20 (T 50.9058) Sequelae

1	Other Adverse Effect of Medication 995.20 (T 50.905A) Initial encounter 995.20 (T 50.905D) Subsequent encounter 995.20 (T 50.9058) Sequelae This category is available for optional use by clinicians to code side effects of medication (other than movement symptoms) when these adverse effects become a main focus of clin- ical attention. Examples include severe hypotension, cardiac arrhythmias, and priapism. a Focus of Cllnical Attention

1	a Focus of Cllnical Attention This d iscussion covers other conditions and problems that may be a focus of clini- cal attention or that may otherwise affect the diagnosis, course, prognosis, or treatment of a patient’s mental disorder. These conditions are presented with their corresponding codes from ICD-9-CM (usually V codes) and ICD-10-CM (usually Z codes). A condition or problem in this chapter may be coded if it is a reason for the current visit or helps to explain the need for a test, procedure, or treatment. Conditions and problems in this chap- ter may also be included in the medical record as useful information on circumstances that may affect the patient’s care, regardless of their relevance to the current visit.

1	The conditions and problems listed in this chapter are not mental disorders. Their in- clusion in DSM-5 is meant to draw attention to the scope of additional issues that may be encountered in routine clinical practice and to provide a systematic listing that may be useful to clinicians in documenting these issues.

1	Key relationships, especially intimate adult partner relationships and parent/caregiver- child relationships, have a significant impact on the health of the individuals in these re- lationships. These relationships can be health promoting and protective, neutral, or detri- mental to health outcomes. In the extreme, these close relationships can be associated with maltreatment or neglect, which has significant medical and psychological consequences for the affected individual. A relational problem may come to clinical attention either as the reason that the individual seeks health care or as a problem that affects the course, prognosis, or treatment of the individual’s mental or other medical disorder. Problems Related to Family Upbringing V61.20 (Z62.820) Parent-Child Relational Problem

1	For this category, the term parent is used to refer to one of the child’s primary caregivers, who may be a biological, adoptive, or foster parent or may be another relative (such as a grandparent) who fulfills a parental role for the child. This category should be used when the main focus of clinical attention is to address the quality of the parent-child relationship or when the quality of the parent—child relationship is affecting the course, prognosis, or treatment of a mental or other medical disorder. Typically, the parent-child relational problem is associated with impaired functioning in behavioral, cognitive, 0r affective do- mains. Examples of behavioral problems include inadequate parental control, supervision, and involvement with the child; parental overprotection; excessive parental pressure; ar- guments that escalate to threats of physical violence; and avoidance without resolution of problems. Cognitive problems may include negative attributions of the other’s

1	parental pressure; ar- guments that escalate to threats of physical violence; and avoidance without resolution of problems. Cognitive problems may include negative attributions of the other’s intentions, hostility toward or scapegoating of the other, and unwarranted feelings of estrangement.

1	Affective problems may include feelings of sadness, apathy, or anger about the other in- dividual in the relationship. Clinicians should take into account the developmental needs of the child and the cultural context. 716 Other Conditions That May Be a Focus of Clinical Attention V61.8 (262.891) Sibling Relational Problem This category should be used when the focus of clinical attention is a pattern of interaction among siblings that is associated with significant impairment in individual or family function- ing or with development of symptoms in one or more of the siblings, or when a sibling relational problem is affecting the course, prognosis, or treatment of a sibling’s mental or other medical disorder. This category can be used for either children or adults if the focus is on the sibling re- lationship. Siblings in this context include full, half-, step-, foster, and adopted siblings. V61.8 (262.29) Upbringing Away From Parents

1	V61.8 (262.29) Upbringing Away From Parents This category should be used when the main focus of clinical attention pertains to issues regarding a child being raised away from the parents or when this separate upbringing af- fects the course, prognosis, or treatment of a mental or other medical disorder. The child could be one who is under state custody and placed in kin care or foster care. The child could also be one who is living in a nonparental relative’s home, or with friends, but whose out—of—home placement is not mandated or sanctioned by the courts. Problems related to a child living in a group home or orphanage are also included. This category excludes issues related to V60.6 (Z593) children in boarding schools. V61.29 (262.898) Child Affected by Parental Relationship Distress

1	V61.29 (262.898) Child Affected by Parental Relationship Distress This category should be used when the focus of clinical attention is the negative effects of parental relationship discord (e.g., high levels of conﬂict, distress, or disparagement) on a child in the family, including effects on the child’s mental or other medical disorders. Other Problems Related to Primary Support Group V61.10 (263.0) Relationship Distress With Spouse or Intimate Partner

1	Other Problems Related to Primary Support Group V61.10 (263.0) Relationship Distress With Spouse or Intimate Partner This category should be used when the major focus of the clinical contact is to address the quality of the intimate (spouse or partner) relationship or when the quality of that rela- tionship is affecting the course, prognosis, or treatment of a mental or other medical dis- order. Partners can be of the same or different genders. Typically, the relationship distress is associated with impaired functioning in behavioral, cognitive, or affective domains. Ex- amples of behavioral problems include conﬂict resolution difficulty, withdrawal, and overinvolvement. Cognitive problems can manifest as chronic negative attributions of the other’s intentions or dismissals of the partner’s positive behaviors. Affective problems would include chronic sadness, apathy, and / or anger about the other partner.

1	Note: This category excludes clinical encounters for V61.1x (Z69.1x) mental health ser- vices for spousal or partner abuse problems and V65.49 (270.9) sex counseling. V61.03 (263.5) Disruption of Family by Separation or Divorce This category should be used when partners in an intimate adult couple are living apart due to relationship problems or are in the process of divorce. V61.8 (263.8) High Expressed Emotion Level Within Family Expressed emotion is a construct used as a qualitative measure of the ”amount" of emo- tion—in particular, hostility, emotional overinvolvement, and criticism directed toward a family member who is an identified patient—displayed in the family environment. This category should be used when a family’s high level of expressed emotion is the focus of clinical attention or is affecting the course, prognosis, or treatment of a family member’s mental or other medical disorder. V62.82 (263.4) Uncomplicated Bereavement

1	V62.82 (263.4) Uncomplicated Bereavement This category can be used when the focus of clinical attention is a normal reaction to the death of a loved one. As part of their reaction to such a loss, some grieving individuals present with symptoms characteristic of a major depressive episodchfor example, feel- Other Conditions That May Be a Focus of Clinical Attention 717 ings of sadness and associated symptoms such as insonmia, poor appetite, and weight loss. The bereaxed individual typically regards the depressed mood as ”normal,” al- though the individual may seek professional help for relief of associated symptoms such as insomnia or anorexia. The duration and expression of "normal” bereavement vary con- siderably among different cultural groups. Further guidance in distinguishing grief from a major depressive episode is provided in the criteria for major depressive episode.

1	Maltreatment by a family member (e.g., caregiver, intimate adult partner) or by a nonrel- ative can be the area of current clinical focus, or such maltreatment can be an important factor in the assessment and treatment of patients with mental or other medical disorders. Because of the legal implications of abuse and neglect, care should be used in assessing these conditions and assigning these codes. Having a past history of abuse or neglect can inﬂuence diagnosis and treatment response in a number of mental disorders, and may also be noted along with the diagnosis. For the following categories, in addition to listings of the confirmed or suspected event of abuse or neglect, other codes are provided for use if the current clirtical encounter is to provide mental health services to either the victim or the perpetrator of the abuse or ne- glect. A separate code is also provided for designating a past history of abuse or neglect.

1	For T codes only, the 7th character should be coded as follows: A (initial encounter)—Use while the patient is receiving active treatment for the condition (e.g., surgical treatment, emergency department encounter, eval- D (subsequent encounter)—Use for encounters after the patient has received active treatment for the condition and when he or she is receiving routine care for the condition during the healing or recovery phase (e.g., cast change or re- moval, removal of external or internal fixation device, medication adjustment, other aftercare and follow-up Visits).

1	Child physical abuse is nonaccidental physical injury to a child—ranging from minor bruises to severe fractures or death—occurring as a result of punching, beating, kicking, biting, shaking, throwing, stabbing, choking, hitting (with a hand, stick, strap, or other object), burning, or any other method that is inﬂicted by a parent, caregiver, or other individual who has responsibility for the child. Such injury is considered abuse regardless of whether the caregiver intended to hurt the child. Physical discipline, such as spanking or paddling, is not considered abuse as long as it is reasonable and causes no bodily injury to the child. Child Physical Abuse, Confirmed 995.54 (T74.12XA) Initial encounter 995.54 (T74.12XD) Subsequent encounter Child Physical Abuse, Suspected 995.54 (T76.12XA) Initial encounter 995.54 (T76.12XD) Subsequent encounter 718 Other Conditions That May Be a Focus of Clinical Attention Other Circumstances Related to Child Physical Abuse

1	Other Circumstances Related to Child Physical Abuse V61 .21 (269.010) Encounter for mental health services for victim of child abuse by parent V61.21 (269.020) Encounter for mental health services for victim of nonparental child V15.41 (262.810) Personal history (past history) of physical abuse in childhood V61 .22 (269.011) Encounter for mental health services for perpetrator of parental child V62.83 (269.021) Encounter for mental health services for perpetrator of nonparental

1	V61 .22 (269.011) Encounter for mental health services for perpetrator of parental child V62.83 (269.021) Encounter for mental health services for perpetrator of nonparental Child sexual abuse encompasses any sexual act involving a child that is intended to pro- vide sexual gratification to a parent, caregiver, or other individual who has responsibility for the child. Sexual abuse includes activities such as fondling a child’s genitals, penetra- tion, incest, rape, sodomy, and indecent exposure. Sexual abuse also includes noncontact exploitation of a child by a parent or caregiver—for example, forcing, tricking, enticing, threatening, or pressuring a child to participate in acts for the sexual gratification of others, without direct physical contact between child and abuser. Child Sexual Abuse, Confirmed 995.53 (T 74.22XA) Initial encounter 995.53 (T 74.22XD) Subsequent encounter

1	Child Sexual Abuse, Confirmed 995.53 (T 74.22XA) Initial encounter 995.53 (T 74.22XD) Subsequent encounter Child Sexual Abuse, Suspected 995.53 (T76.22XA) Initial encounter 995.53 (T76.22XD) Subsequent encounter Other Circumstances Related to Child Sexual Abuse V61.21 (269.010) Encounter for mental health services for victim of child sexual abuse V61.21 (269.020) Encounter for mental health services for victim of nonparental child V15.41 (262.810) Personal history (past history) of sexual abuse in childhood V61.22 (269.011) Encounter for mental health services for perpetrator of parental child V62.83 (269.021) Encounter for mental health services for perpetrator of nonparental

1	V61.22 (269.011) Encounter for mental health services for perpetrator of parental child V62.83 (269.021) Encounter for mental health services for perpetrator of nonparental Child neglect is defined as any confirmed or suspected egregious act or omission by a child’s parent or other caregiver that deprives the child of basic age-appropriate needs and thereby results, or has reasonable potential to result, in physical or psychological harm to the child. Child neglect encompasses abandonment; lack of appropriate supervision; fail- ure to attend to necessary emotional or psychological needs; and failure to provide neces- sary education, medical care, nourishment, shelter, and/ or clothing. Child Neglect, Confirmed 995.52 (T74.02XA) Initial encounter 995.52 (T74.02XD) Subsequent encounter Other Conditions That May Be a Focus of Clinical Attention 719 Child Neglect, Suspected 995.52 (T 76.02“) Initial encounter 995.52 (T76.02XD) Subsequent encounter

1	Other Conditions That May Be a Focus of Clinical Attention 719 Child Neglect, Suspected 995.52 (T 76.02“) Initial encounter 995.52 (T76.02XD) Subsequent encounter Other Circumstances Related to Child Neglect V61.21 (269.010) Encounter for mental health services for victim of child neglect by V61.21 (269.020) Encounter for mental health services for victim of nonparental child V1 5.42 (262.81 2) Personal history (past history) of neglect in childhood V61.22 (269.011) Encounter for mental health services for perpetrator of parental child V62.83 (269.021) Encounter for mental health services for perpetrator of nonparental

1	Child psychological abuse is nonaccidental verbal or symbolic acts by a child’s parent or caregiver that result, or have reasonable potential to result, in significant psychological harm to the child. (Physical and sexual abusive acts are not included in this category.) Ex- amples of psychological abuse of a child include berating, disparaging, or humiliating the child; threatening the child; harming/abandoning—or indicating that the alleged offender will harm/abandon—people or things that the child cares about; confining the child (as by tying a child’s arms or legs together or binding a child to furniture or another object, or confining a child to a small enclosed area [e.g., a closet]); egregious scapegoating of the child; coercing the child to inﬂict pain on himself or herself; and disciplining the child excessively (i.e., at an extremely high frequency or duration, even if not at a level of physical abuse) through physical or nonphysical means.

1	Child Psychological Abuse, Confirmed 995.51 (T74.32XA) Initial encounter 995.51 (T74.32XD) Subsequent encounter Child Psychological Abuse, Suspected 995.51 (T76.32XA) Initial encounter 995.51 (T76.32XD) Subsequent encounter Other Circumstances Related to Child Psychological Abuse V61 .21 (269.010) Encounter for mental health services for victim of child psychological V61.21 (269.020) Encounter for mental health services for victim of nonparental child V15.42 (262.811) Personal history (past history) of psychological abuse in childhood V61.22 (269.011) Encounter for mental health services for perpetrator of parental child V62.83 (269.021) Encounter for mental health services for perpetrator of nonparental 720 Other Conditions That May Be a Focus of Clinical Attention Spouse or Partner Violence, Physical

1	V62.83 (269.021) Encounter for mental health services for perpetrator of nonparental 720 Other Conditions That May Be a Focus of Clinical Attention Spouse or Partner Violence, Physical This category should be used when nonaccidental acts of physical force that result, or have reasonable potential to result, in physical harm to an intimate partner or that evoke signif- icant fear in the partner have occurred during the past year. Nonaccidental acts of physical force include shoving, slapping, hair pulling, pinching, restraining, shaking, throwing, biting, kicking, hitting with the fist or an object, burning, poisoning, applying force to the throat, cutting off the air supply, holding the head under water, and using a weapon. Acts for the purpose of physically protecting oneself or one’s partner are excluded. Spouse or Partner Violence, Physical, Confirmed 995.81 (T74.11XA) Initial encounter 995.81 (T 74.1 1 XD) Subsequent encounter

1	Spouse or Partner Violence, Physical, Confirmed 995.81 (T74.11XA) Initial encounter 995.81 (T 74.1 1 XD) Subsequent encounter Spouse or Partner Violence, Physical, Suspected 995.81 (T 76.1 1 XA) Initial encounter 995.81 (T 76.1 1 XD) Subsequent encounter Other Circumstances Related to Spouse or Partner Violence, Physical V61.11 (269.11) Encounter for mental health services for Victim of spouse or partner violence, physical V15.41 (291 .410) Personal history (past history) of spouse or partner violence, physical V61.12 (269.12) Encounter for mental health services for perpetrator of spouse or partner violence, physical Spouse or Partner Violence, Sexual

1	V61.12 (269.12) Encounter for mental health services for perpetrator of spouse or partner violence, physical Spouse or Partner Violence, Sexual This category should be used when forced or coerced sexual acts with an intimate partner have occurred during the past year. Sexual violence may involve the use of physical force or psychological coercion to compel the partner to engage in a sexual act against his or her will, whether or not the act is completed. Also included in this category are sexual acts with an intimate partner who is unable to consent. Spouse or Partner Violence, Sexual, Confirmed 995.83 (T7 4.21 XA) Initial encounter 995.83 (T7 4.21 XD) Subsequent encounter Spouse or Partner Violence, Sexual, Suspected 995.83 (17 6.21 XA) Initial encounter 995.83 (T7 6.21 XD) Subsequent encounter Other Circumstances Related to Spouse or Partner Violence, Sexual V61.11 (269.81) Encounter for mental health services for victim of spouse or partner violence, sexual

1	Other Circumstances Related to Spouse or Partner Violence, Sexual V61.11 (269.81) Encounter for mental health services for victim of spouse or partner violence, sexual V15.41 (291.410) Personal history (past history) of spouse or partner violence, sexual V61.12 (269.12) Encounter for mental health services for perpetrator of spouse or partner violence, sexual Other Conditions That May Be a Focus of Clinical Attention 721

1	V61.12 (269.12) Encounter for mental health services for perpetrator of spouse or partner violence, sexual Other Conditions That May Be a Focus of Clinical Attention 721 Partner neglect is any egregious act or omission in the past year by one partner that de- prives a dependent partner of basic needs and thereby results, or has reasonable potential to result, in physical or psychological harm to the dependent partner. This category is used in the context of relationships in which one partner is extremely dependent on the other partner for care or for assistance in navigating ordinary daily activities—for example, a partner who is incapable of self—care owing to substantial physical, psychological/intel- lectual, or cultural limitations (e.g., inability to communicate with others and manage ev- eryday activities due to living in a foreign culture). Spouse or Partner Neglect, Confirmed 995.85 (T 74.01 XA) Initial encounter 995.85 (T 74.01 XD) Subsequent encounter

1	Spouse or Partner Neglect, Confirmed 995.85 (T 74.01 XA) Initial encounter 995.85 (T 74.01 XD) Subsequent encounter Spouse or Partner Neglect, Suspected 995.85 (T7 6.01 XA) Initial encounter 995.85 (T 76.01 XD) Subsequent encounter Other Circumstances Related to Spouse or Partner Neglect V61.11 (269.11) Encounter for mental health services for victim of spouse or partner V15.42 (291 .412) Personal history (past history) of spouse or partner neglect V61.12 (269.12) Encounter for mental health services for perpetrator of spouse or

1	V61.12 (269.12) Encounter for mental health services for perpetrator of spouse or Spouse or Partner Abuse, Psychological partner that result, or have reasonable potential to result, in significant harm to the other partner. This category should be used when such psychological abuse has occurred during the past year. Acts of psychological abuse include berating or humiliating the victim; inter- rogating the victim; restricting the victim’s ability to come and go freely; obstructing the vic— tim’s access to assistance (e.g., law enforcement; legal, protective, or medical resources); threatening the victim with physical harm or sexual assault; harming, or threatening to harm, people or things that the victim cares about; unwarranted restriction of the victim’s ac— cess to or use of economic resources; isolating the victim from family, friends, or social sup- port resources; stalking the victim; and trying to make the victim think that he or she is crazy.

1	Spouse or Partner Abuse, Psychological, Confirmed 995.82 (T74.31XA) Initial encounter 995.82 (T74.31XD) Subsequent encounter Spouse or Partner Abuse, Psychological, Suspected 995.82 (T7 6.31 XA) Initial encounter 995.82 (T7 6.31 XD) Subsequent encounter Other Circumstances Related to Spouse or Partner Abuse, Psychological V61.11 (269.11) Encounter for mental health services for victim of spouse or partner 722 Other Conditions That May Be a Focus of Clinical Attention V15.42 (291 .41 1) Personal history (past history) of spouse or partner psychological abuse

1	V61 .12 (269.12) Encounter for mental health services for perpetrator of spouse or part- not an intimate partner. Such maltreatment may involve acts of physical, sexual, or emo- tional abuse. Examples of adult abuse include nonaccidental acts of physical force (e.g., pushing/shoving, scratching, slapping, throwing something that could hurt, punching, biting) that have resulted—or have reasonable potential to result—in physical harm or with the potential to cause psychological harm (e.g., berating or humiliating the person; interrogating the person; restricting the person’s ability to come and go freely; obstructing the person’s access to assistance; threatening the person; harming or threatening to harm people or things that the person cares about; restricting the person’s access to or use of eco- nomic resources; isolating the person from family, friends, or social support resources; stalking the person; trying to make the person think that he or she is crazy). Acts for the purpose

1	of eco- nomic resources; isolating the person from family, friends, or social support resources; stalking the person; trying to make the person think that he or she is crazy). Acts for the purpose of physically protecting oneself or the other person are excluded.

1	Adult Physical Abuse by Nonspouse or N onpartner, Confirmed 995.81 (T 74.1 1 XA) Initial encounter 995.81 (T 74.1 1 XD) Subsequent encounter Adult Physical Abuse by Nonspouse or Nonpartner, Suspected 995.81 (T 76.1 1XA) Initial encounter 995.81 (T 76.1 1 XD) Subsequent encounter Adult Sexual Abuse by Nonspouse or Nonpartner, Confirmed 995.83 (T 74.21 XA) Initial encounter 995.83 (T74.21XD) Subsequent encounter Adult Sexual Abuse by Nonspouse or Nonpartner, Suspected 995.83 (T76.21XA) Initial encounter 995.83 (T 76.21 XD) Subsequent encounter Adult Psychological Abuse by Nonspouse or Nonpartner, Confirmed 995.82 (T 74.31 XA) Initial encounter 995.82 (T74.31XD) Subsequent encounter Adult Psychological Abuse by Nonspouse or Nonpartner, Suspected 995.82 (T76.31XA) Initial encounter 995.82 (T 76.31 XD) Subsequent encounter Other Circumstances Related to Adult Abuse by Nonspouse or Nonpartner V65.49 (269.81) Encounter for mental health services for victim of nonspousal or non—

1	Other Circumstances Related to Adult Abuse by Nonspouse or Nonpartner V65.49 (269.81) Encounter for mental health services for victim of nonspousal or non— V62.83 (269.82) Encounter for mental health services for perpetrator of nonspousal or Other Conditions That May Be a Focus of Clinical Attention 723 V62.3 (255.9) Academic or Educational Problem This category should be used when an academic or educational problem is the focus of clinical attention or has an impact on the individual’s diagnosis, treatment, or prognosis.

1	This category should be used when an academic or educational problem is the focus of clinical attention or has an impact on the individual’s diagnosis, treatment, or prognosis. Problems to be considered include illiteracy or low-level literacy; lack of access to school- ing owing to unavailability or unattainability; problems with academic performance (e.g., failing school examinations, receiving failing marks or grades) or underachievement (be- low what would be expected given the individual’s intellectual capacity); discord with teachers, school staff, or other students; and any other problems related to education and / or literacy. V62.21 (256.82) Problem Related to Current Military Deployment Status

1	V62.21 (256.82) Problem Related to Current Military Deployment Status This category should be used when an occupational problem directly related to an indi- vidual’s military deployment status is the focus of clinical attention or has an impact on the individual’s diagnosis, treatment, or prognosis. Psychological reactions to deployment are not included in this category; such reactions would be better captured as an adjustment disorder or another mental disorder. V62.29 (256.9) Other Problem Related to Employment

1	V62.29 (256.9) Other Problem Related to Employment This category should be used when an occupational problem is the focus of clinical atten- tion or has an impact on the individual’s treatment or prognosis. Areas to be considered include problems with employment or in the work environment, including unemploy- ment; recent change of job; threat of job loss; job dissatisfaction; stressful work schedule; uncertainty about career choices; sexual harassment on the job; other discord with boss, supervisor, co-workers, or others in the work environment; uncongenial or hostile work environments; other psychosocial stressors related to work; and any other problems re- lated to employment and / or occupation. V60.0(259.0) Homelessness

1	V60.0(259.0) Homelessness This category should be used when lack of a regular dwelling or living quarters has an im- pact on an individual’s treatment or prognosis. An individual is considered to be homeless if his or her primary nighttime residence is a homeless shelter, a warming shelter, a do— mestic violence shelter, a public space (e.g., tunnel, transportation station, mall), a build- ing not intended for residential use (e.g., abandoned structure, unused factory), a cardboard box or cave, or some other ad hoc housing situation. V60.1 (259.1) Inadequate Housing

1	V60.1 (259.1) Inadequate Housing This category should be used when lack of adequate housing has an impact on an individ- ual’s treatment or prognosis. Examples of inadequate housing conditions include lack of heat (in cold temperatures) or electricity, infestation by insects or rodents, inadequate plumbing and toilet facilities, overcrowding, lack of adequate sleeping space, and exces- sive noise. It is important to consider cultural norms before assigning this category. V60.89 (259.2) Discord With Neighbor, Lodger, or Landlord This category should be used when discord with neighbors, lodgers, or a landlord is a fo- cus of clinical attention or has an impact on the individual's treatment or prognosis. 724 Other Conditions That May Be a Focus of Clinical Attention V60.6 (259.3) Problem Related to Living in a Residential Institution

1	724 Other Conditions That May Be a Focus of Clinical Attention V60.6 (259.3) Problem Related to Living in a Residential Institution This category should be used when a problem (or problems) related to living in a residen- tial institution is a focus of clinical attention or has an impact on the individual’s treatment or prognosis. Psychological reactions to a change in living situation are not included in this category; such reactions would be better captured as an adjustment disorder. V60.2 (259.4) Lack of Adequate Food or Safe Drinking Water V60.2 (259.5) Extreme Poverty V60.2 (259.6) Low Income

1	V60.2 (259.4) Lack of Adequate Food or Safe Drinking Water V60.2 (259.5) Extreme Poverty V60.2 (259.6) Low Income V60.2 (259.7) Insufficient Social Insurance or Welfare Support fare support but are not receiving such support, who receive support that is insufficient to address their needs, or who otherwise lack access to needed insurance or support pro- grams. Examples include inability to qualify for welfare support owing to lack of proper documentation or evidence of address, inability to obtain adequate health insurance be- cause of age or a preexisting condition, and denial of support owing to excessively strin- gent income or other requirements. V60.9 (259.9) Unspecified Housing or Economic Problem This category should be used when there is a problem related to housing or economic cir- cumstances other than as specified above. Other Problems Related to the Social Environment V62.89 (260.0) Phase of Life Problem

1	Other Problems Related to the Social Environment V62.89 (260.0) Phase of Life Problem This category should be used when a problem adjusting to a life-cycle transition (a partic- ular developmental phase) is the focus of clinical attention or has an impact on the indi- vidual’s treatment or prognosis. Examples of such transitions include entering or completing school, leaving parental control, getting married, starting a new career, be- coming a parent, adjusting to an ”empty nest” after children leave home, and retiring. V60.3 (260.2) Problem Related to Living Alone This category should be used when a problem associated with living alone is the focus of clinical attention or has an impact on the individual’s treatment or prognosis. Examples of such problems include chronic feelings of loneliness, isolation, and lack of structure in car— rying out activities of daily living (e.g., irregular meal and sleep schedules, inconsistent performance of home maintenance chores).

1	V62.4 (260.3) Acculturation Difficulty This category should be used when difficulty in adjusting to a new culture (e.g., following migration) is the focus of clinical attention or has an impact on the individual’s treatment or prognosis. V62.4 (260.4) Social Exclusion or Rejection This category should be used when there is an imbalance of social power such that there is recurrent social exclusion or rejection by others. Examples of social rejection include bul- lying, teasing, and intimidation by others; being targeted by others for verbal abuse and humiliation; and being purposefully excluded from the activities of peers, workmates, or others in one’s social environment. V62.4 (260.5) Target of (Perceived) Adverse Discrimination or Persecution This category should be used when there is perceived or experienced discrimination against or persecution of the individual based on his or her membership (or perceived

1	This category should be used when there is perceived or experienced discrimination against or persecution of the individual based on his or her membership (or perceived Other Conditions That May Be a Focus of Clinical Attention 725 membership) in a specific category. Typically, such categories include gender or gender identity, race, ethnicity, religion, sexual orientation, country of origin, political beliefs, dis- ability status, caste, social status, weight, and physical appearance. V62.9 (260.9) Unspecified Problem Related to Social Environment This category should be used when there is a problem related to the individual's social en- vironment other than as specified above. Problems Related to Crime or Interaction With the Legal System V62.89 (265.4) Victim of Crime V62.5 (265.0) Conviction in Civil or Criminal Proceedings Without Imprisonment V62.5 (265.1) Imprisonment or Other Incarceration V62.5 (265.2) Problems Related to Release From Prison

1	V62.5 (265.0) Conviction in Civil or Criminal Proceedings Without Imprisonment V62.5 (265.1) Imprisonment or Other Incarceration V62.5 (265.2) Problems Related to Release From Prison V62.5 (265.3) Problems Related to Other Legal Circumstances V65.49 (270.9) Sex Counseling This category should be used when the individual seeks counseling related to sex educa- tion, sexual behavior, sexual orientation, sexual attitudes (embarrassment, timidity), oth- ers’ sexual behavior or orientation (e.g., spouse, partner, child), sexual enjoyment, or any other sex-related issue. V65.40 (271.9) Other Counseling or Consultation This category should be used when counseling is provided or advice/consultation is sought for a problem that is not specified above or elsewhere in this chapter. Examples in- clude spiritual or religious counseling, dietary counseling, and counseling on nicotine use. Problems Related to Other Psychosocial, Personal, V62.89 (265.8) Religious or Spiritual Problem

1	Problems Related to Other Psychosocial, Personal, V62.89 (265.8) Religious or Spiritual Problem This category can be used when the focus of clinical attention is a religious or spiritual problem. Examples include distressing experiences that involve loss or questioning of faith, problems associated with conversion to a new faith, or questioning of spiritual val- ues that may not necessarily be related to an organized church or religious institution. V61.7 (264.0) Problems Related to Unwanted Pregnancy V61.5 (264.1) Problems Related to Multiparity V62.89 (264.4) Discord With Social Service Provider, Including Probation Officer, Case Manager, or Social Services Worker V62.89 (265.4) Victim of Terrorism or Torture V62.22 (265.5) Exposure to Disaster, War, or Other Hostilities V62.89 (265.8) Other Problem Related to Psychosocial Circumstances

1	V62.89 (265.4) Victim of Terrorism or Torture V62.22 (265.5) Exposure to Disaster, War, or Other Hostilities V62.89 (265.8) Other Problem Related to Psychosocial Circumstances V62.9 (265.9) Unspecified Problem Related to Unspecified Psychosocial Circum- 726 Other Conditions That May Be a Focus of Clinical Attention Other Circumstances of Personal History V15.49 (291.49) Other Personal History of Psychological Trauma V15.59 (291.5) Personal History of Self-Harm V62.22 (291.82) Personal History of Military Deployment V15.89 (291.89) Other Personal Risk Factors V69.9 (272.9) Problem Related to Lifestyle

1	V15.59 (291.5) Personal History of Self-Harm V62.22 (291.82) Personal History of Military Deployment V15.89 (291.89) Other Personal Risk Factors V69.9 (272.9) Problem Related to Lifestyle This category should be used when a lifestyle problem is a specific focus of treatment or di- rectly affects the course, prognosis, or treatment of a mental or other medical disorder. Ex- amples of lifestyle problems include lack of physical exercise, inappropriate diet, high-risk sexual behavior, and poor sleep hygiene. A problem that is attributable to a symptom of a mental disorder should not be coded unless that problem is a specific focus of treatment or directly affects the course, prognosis, or treatment of the individual. In such cases, both the mental disorder and the lifestyle problem should be coded. V71.01 (272.811) Adult Antisocial Behavior

1	V71.01 (272.811) Adult Antisocial Behavior This category can be used when the focus of clinical attention is adult antisocial behavior that is not due to a mental disorder (e.g., conduct disorder, antisocial personality disor- der). Examples include the behavior of some professional thieves, racketeers, or dealers in illegal substances. V71.02 (272.810) Child or Adolescent Antisocial Behavior This category can be used when the focus of clinical attention is antisocial behavior in a child or adolescent that is not due to a mental disorder (e.g., intermittent explosive disor- der, conduct disorder). Examples include isolated antisocial acts by children or adoles- cents (not a pattern of antisocial behavior). Problems Related to Access to Medical V63.9 (275.3) Unavailability or Inaccessibility of Health Care Facilities V63.8 (275.4) Unavailability or Inaccessibility of Other Helping Agencies Nonadherence to Medical Treatment V15.81 (291.19) Nonadherence to Medical Treatment

1	V63.8 (275.4) Unavailability or Inaccessibility of Other Helping Agencies Nonadherence to Medical Treatment V15.81 (291.19) Nonadherence to Medical Treatment This category can be used when the focus of clinical attention is nonadherence to an im- portant aspect of treatment for a mental disorder or another medical condition. Reasons for such nonadherence may include discomfort resulting from treatment (e.g., medication side effects), expense of treatment, personal value judgments or religious or cultural be- liefs about the proposed treatment, age-related debility, and the presence of a mental dis- order (e.g., schizophrenia, personality disorder). This category should be used only when the problem is sufficiently severe to warrant independent clinical attention and does not meet diagnostic criteria for psychological factors affecting other medical conditions. 278.00 (E66.9) Overweight or Obesity

1	278.00 (E66.9) Overweight or Obesity This category may be used when overweight or obesity is a focus of clinical attention. V65.2 (276.5) Malingering The essential feature of malingering is the intentional production of false or grossly exag- gerated physical or psychological symptoms, motivated by external incentives such as avoiding military duty, avoiding work, obtaining financial compensation, evading crimi- nal prosecution, or obtaining drugs. Under some circumstances, malingering may repre- Other Conditions That May Be a Focus of Clinical Attention 727 sent adaptive behavior—for example, feigning illness while a captive of the enemy during wartime. Malingering should be strongly suspected if any combination of the following is noted: 1. Medicolegal context of presentation (e.g., the individual is referred by an attorney to the clinician for examination, or the individual self—refers while litigation or criminal charges are pending).

1	2. Marked discrepancy between the individual’s claimed stress or disability and the ob- jective findings and observations. 3. Lack of cooperation during the diagnostic evaluation and in complying with the pre- scribed treatment regimen. 4. The presence of antisocial personality disorder. Malingering differs from factitious disorder in that the motivation for the symptom production in malingering is an external incentive, whereas in factitious disorder external incentives are absent. Malingering is differentiated from conversion disorder and somatic symptom—related mental disorders by the intentional production of symptoms and by the obvious external incentives associated with it. Definite evidence of feigning (such as clear evidence that loss of function is present during the examination but not at home) would suggest a diagnosis of factitious disorder if the individual’s apparent aim is to assume the sick role, or malingering if it is to obtain an incentive, such as money.

1	V40.31 (291.83) Wandering Associated With a Mental Disorder This category is used for individuals with a mental disorder whose desire to walk about leads to significant clinical management or safety concerns. For example, individuals with wander that places them at risk for falls and causes them to leave supervised settings with- out needed accompaniment. This category excludes individuals whose intent is to escape an unwanted housing situation (e.g., children who are running away from home, patients who no longer wish to remain in the hospital) or those who walk or pace as a result of med- ication-induced akathisia. Coding note: First code associated mental disorder (e.g., major neurocognitive disor- der, autism spectrum disorder), then code V40.31 (291.83) wandering associated with [specific mental disorder]. V62.89 (R41.83) Borderline Intellectual Functioning

1	V62.89 (R41.83) Borderline Intellectual Functioning This category can be used when an individual’s borderline intellectual functioning is the fo- cus of clinical attention or has an impact on the individual’s treatment or prognosis. Differ- developmental disorder) requires careful assessment of intellectual and adaptive functions and their discrepancies, particularly in the presence of co-occurring mental disorders that may affect patient compliance with standardized testing procedures (e.g., schizophrenia or attention—deficit/hyperactivity disorder with severe impulsivity). Assessment Measures ....................................... 733 Cross-Cutting Symptom Measures ........................... 734 Symptom Measure—Adult ............................. 738 Symptom Measure—Child Age 6—17 .................... 740 CIinician-Rated Dimensions of Psychosis Symptom Severity ...... 742

1	Symptom Measure—Adult ............................. 738 Symptom Measure—Child Age 6—17 .................... 740 CIinician-Rated Dimensions of Psychosis Symptom Severity ...... 742 World Health Organization Disability Assessment Schedule 2.0 (WHODAS 2.0) .......................................... 745 Cultural Formulation ......................................... 749 Cultural Formulation Interview (CFI) ........................... 750 Cultural Formulation Interview (CFI)—|nformant Version .......... 755 Alternative DSM-5 Model for Personality Disorders ................ 761 Conditions for Further Study .................................. 783 Attenuated Psychosis Syndrome ............................. 783 Depressive Episodes With Short-Duration Hypomania ........... 786 Persistent Complex Bereavement Disorder .................... 789 Caffeine Use Disorder ...................................... 792 Internet Gaming Disorder ................................... 795

1	Persistent Complex Bereavement Disorder .................... 789 Caffeine Use Disorder ...................................... 792 Internet Gaming Disorder ................................... 795 Prenatal Alcohol Exposure ................................ 798 Suicidal Behavior Disorder .................................. 801 Nonsuicidal SeIf-Injury ..................................... 803 This section contains tools and techniques to enhance the clinical deci- sion-making process, understand the cultural context of mental disorders, and recognize emerging diagnoses for further study. It provides strategies to en- hance clinical practice and new criteria to stimulate future research, represent- ing a dynamic DSM-5 that will evolve with advances in the field.

1	Among the tools in Section III is a Level 1 cross—cutting seIf/informant-rated measure that serves as a review of systems across mental disorders. A clini- is provided, as well as the World Health Organization Disability Assessment Schedule, Version 2 (WHODAS 2.0). Level 2 severity measures are available online (www.psychiatry.org/dsm5) and may be used to explore significant re- sponses to the Level 1 screen. A comprehensive review of the cultural context of mental disorders, and the Cultural Formulation Interview (CFI) for clinical use, are provided. Proposed disorders for future study are provided, which include a new model for the diagnosis of personality disorders as an alternative to the estab- lished diagnostic criteria; the proposed model incorporates impairments in per- sonality functioning as well as pathological personality traits. Also included are new conditions that are the focus of active research, such as attenuated psy- chosis syndrome and nonsuicidal seIf-injury.

1	A growing body of scientific evidence favors dimensional concepts in the diagnosis of mental disorders. The limitations of a categorical approach to diagnosis include the fail- ure to find zones of rarity between diagnoses (i.e., delineation of mental disorders from one another by natural boundaries), the need for intermediate categories like schizoaffective dis- order, high rates of comorbidity, frequent not—otherwise—specified (NOS) diagnoses, relative lack of utility in furthering the identification of unique antecedent validators for most men- tal disorders, and lack of treatment specificity for the various diagnostic categories.

1	From both clinical and research perspectives, there is a need for a more dimensional approach that can be combined with DSM’s set of categorical diagnoses. Such an approach incorporates variations of features within an individual (e.g., differential severity of indi— vidual symptoms both within and outside of a disorder’s diagnostic criteria as measured by intensity, duration, or number of symptoms, along with other features such as type and severity of disabilities) rather than relying on a simple yes-or-no approach. For diagnoses for which all symptoms are needed for a diagnosis (a monothetic criteria set), different se- verity levels of the constituent symptoms may be noted. If a threshold endorsement of multiple symptoms is needed, such as at least five of nine symptoms for major depressive disorder (a polythetic criteria set), both severity levels and different combinations of the criteria may identify more homogeneous diagnostic groups.

1	symptom experiences along with the clinician’s interpretation is consistent with current diagnostic practice. It is expected that as our understanding of basic disease mechanisms based on pathophysiology, neurocircuitry, gene-environment interactions, and laboratory tests increases, approaches that integrate both objective and subjective patient data will be developed to supplement and enhance the accuracy of the diagnostic process.

1	Cross—cutting symptom measures modeled on general medicine’s review of systems can serve as an approach for reviewing critical psychopathological domains. The general med- ical review of systems is crucial to detecting subtle changes in different organ systems that can facilitate diagnosis and treatment. A similar review of various mental functions can aid in a more comprehensive mental status assessment by drawing attention to symptoms that may not fit neatly into the diagnostic criteria suggested by the individual’s presenting symptoms, but may nonetheless be important to the individual’s care. The cross-cutting measures have two levels: Level 1 questions are a brief survey of 13 symptom domains for adult patients and 12 domains for child and adolescent patients. Level 2 questions provide a more in-depth assessment of certain domains. These measures were developed to be administered both at initial interview and over time to track the patient’s symptom status and response to

1	provide a more in-depth assessment of certain domains. These measures were developed to be administered both at initial interview and over time to track the patient’s symptom status and response to treatment.

1	Severity measures are disorder-specific, corresponding closely to the criteria that consti- tute the disorder definition. They may be administered to individuals who have received a diagnosis or who have a clinically significant syndrome that falls short of meeting full criteria for a diagnosis. Some of the assessments are self—completed by the individual, while others require a clinician to complete. As with the cross-cutting symptom measures, these measures were developed to be administered both at initial interview and over time to track the severity of the individual’s disorder and response to treatment.

1	The World Health Organization Disability Assessment Schedule, Version 2.0 (WHODAS 2.0) was developed to assess a patient's ability to perform activities in six areas: understanding and communicating; getting around; self—care; getting along with people; life activities (e.g., household, work/school); and participation in society. The scale is self—administered and was developed to be used in patients with any medical disorder. It corresponds to concepts contained in the WHO International Classification of Functioning, Disability and Health. This assessment can also be used over time to track changes in a patient’s dis- abilities. This chapter focuses on the DSM-5 Level 1 Cross—Cutting Symptom Measure (adult self-rated and parent/guardian versions); the Clinician-Rated Dimensions of Psychosis

1	This chapter focuses on the DSM-5 Level 1 Cross—Cutting Symptom Measure (adult self-rated and parent/guardian versions); the Clinician-Rated Dimensions of Psychosis Symptom Severity; and the WHODAS 2.0. Clinician instructions, scoring information, and interpretation guidelines are included for each. These measures and additional dimensional assessments, including those for diagnostic severity, can be found online at www.psychiatryorg/dsmS. The DSM-5 Level 1 Cross-Cutting Symptom Measure is a patient— or informant-rated mea- sure that assesses mental health domains that are important across psychiatric diagnoses. It is intended to help clinicians identify additional areas of inquiry that may have signifi- cant impact on the individual’s treatment and prognosis. In addition, the measure may be used to track changes in the individual’s symptom presentation over time.

1	The adult version of the measure consists of 23 questions that assess 13 psychiatric do- mains, including depression, anger, mania, anxiety, somatic symptoms, suicidal ideation, psychosis, sleep problems, memory, repetitive thoughts and behaviors, dissociation, per— sonality functioning, and substance use (Table 1). Each domain consists of one to three questions. Each item inquires about how much (or how often) the individual has been bothered by the specific symptom during the past 2 weeks. If the individual is of impaired capacity and unable to complete the form (e.g., an individual with dementia), a know]- edgeable adult informant may complete this measure. The measure was found to be clin- ically useful and to have good reliability in the DSM-5 field trials that were conducted in adult clinical samples across the United States and in Canada.

1	The parent/guardian-rated version of the measure (for children ages 6—17) consists of 25 questions that assess 12 psychiatric domains, including depression, anger, irritability, mania, anxiety, somatic symptoms, inattention, suicidal ideation/attempt, psychosis, sleep disturbance, repetitive thoughts and behaviors, and substance use (Table 2). Each item asks the parent or guardian to rate how much (or how often) his or her child has been bothered by the specific psychiatric symptom during the past 2 weeks. The measure was also found to be clinically useful and to have good reliability in the DSM-5 field trials that were conducted in pediatric clinical samples across the United States. For children ages 11—17, along with the parent/guardian rating of the child’s symptoms, the clinician may consider having the child complete the child-rated version of the measure. The child—rated version of the measure can be found online at www.psychiatry.org/dsm5.

1	Scoring and interpretation. On the adult self—rated version of the measure, each item is rated on a 5-point scale (0=none or not at all; 1=slight or rare, less than a day or two; 2=mild or several days; 3=moderate or more than half the days; and 4=severe or nearly every day). The score on each item within a domain should be reviewed. However, a rating of mild (i.e., 2) or greater on any item within a domain, except for substance use, suicidal ideation, and psychosis, may serve as a guide for additional inquiry and follow-up to determine if a more detailed assessment is necessary, which may include the Level 2 cross-cutting symptom as- sessment for the domain (see Table 2). For substance use, suicidal ideation, and psychosis, a TABLE 1 Adult DSM-5 Self-Rated Level 1 Cross-Cutting Symptom Measure: 13 domains, thresholds for further inquiry, and associated DSM-5 Threshold to guide DSM-5 Level 2 Cross-Cutting Symptom I. Depression Mild or greater Level 2—Depression—Adult (PROMIS

1	Threshold to guide DSM-5 Level 2 Cross-Cutting Symptom I. Depression Mild or greater Level 2—Depression—Adult (PROMIS II. Anger Mild or greater Level 2—Anger—Adult (PROMIS Emo— 111. Mania Mild or greater Level 2—Mania—Adult(A1tman Self-Rating IV. Anxiety Mild or greater Level 2—Anxiety—Adult (PROMIS V. Somatic symptoms Mild or greater Level 2—Somatic Symptom—Adult (Patient VI. Suicidal ideation Slight or greater None VII. Psychosis Slight or greater None VIII. Sleep problems Mild or greater Level 2—Sleep Disturbance—Adult IX. Memory Mild or greater None X. Repetitive thoughts Mild or greater Level 2—Repetitive Thoughts and XI. Dissociation Mild or greater None XII. Personality Mild or greater None XIII. Substance use Slight or greater Level 2—Substance Use—Adult (adapted from the NIDA-Modified ASSIST) Note. NIDA=National Institute on Drug Abuse. aAvailable at www.psychiatry.org/dsm5.

1	XIII. Substance use Slight or greater Level 2—Substance Use—Adult (adapted from the NIDA-Modified ASSIST) Note. NIDA=National Institute on Drug Abuse. aAvailable at www.psychiatry.org/dsm5. rating of slight (i.e., 1) or greater on any item within the domain may serve as a guide for ad- ditional inquiry and follow-up to determine if a more detailed assessment is needed. As such, indicate the highest score within a domain in the ”Highest domain score” column. Table 1 outlines threshold scores that may guide further inquiry for the remaining domains.

1	Table 1 outlines threshold scores that may guide further inquiry for the remaining domains. On the parent/guardian—rated version of the measure (for children ages 6—17), 19 of the 25 items are each rated on a 5—point scale (0=none or not at all; 1=slight or rare, less than a day or two; =mild or several days; 3=moderate or more than half the days; and 4=severe or nearly every day). The suicide ideation, suicide attempt, and substance abuse items are each rated on a ”Yes, No, or Don’t Know” scale. The score on each item within a domain should be re- viewed. However, with the exception of inattention and psychosis, a rating of mild (i.e., 2) or greater on any item within a domain that is scored on the 5-point scale may serve as a guide for additional inquiry and follow-up to determine if a more detailed assessment is necessary, which may include the Level 2 cross-cutting symptom assessment for the domain (see

1	Table 2). For inattention or psychosis, a rating of slight or greater (i.e., 1 or greater) may be for child age 6—17: 12 domains, thresholds for further inquiry, and Threshold to guide DSM-5 Level 2 Cross-Cutting Symptom I. Somatic symptoms Mild or greater Level 2—Somatic Symptoms—Parent/Guard- ian of Child Age 6—17 (Patient Health 11. Sleep problems Mild or greater Level 2—Sleep Disturbance—Parent/Guard- ian of Child Age 6—17 (PROMIS Sleep III. Inattention Slight or greater Level 2—Inattention—Parent/Guardian of Child Age 6—17(Swanson, Nolan, and Pel- ham, Version IV [SNAP-IV]) IV. Depression Mild or greater bevel 2—Depression—Parent/Guardian of V. Anger Mild or greater Level 2—Anger—Parent/Guardian of Child VI. Irritability Mild or greater Level 2—Irritability—Parent/Guardian of VII. Mania Mild or greater Level 2—Mania—Parent/Guardic'1 of Child VIII. Anxiety Mild or greater Level 2—Anxiety—Parent/Guardian of Child IX. Psychosis Slight or greater None

1	VII. Mania Mild or greater Level 2—Mania—Parent/Guardic'1 of Child VIII. Anxiety Mild or greater Level 2—Anxiety—Parent/Guardian of Child IX. Psychosis Slight or greater None X. Repetitive thoughts Mild or greater None XI. Substance use Yes Level 2—Substance Use—Parent/Guardian of Child (adapted from the NIDA-modified XII. Suicidal ideation/ Yes None Note. NIDA=National Institute on Drug Abuse. aAvailable at www.psychiatry.org/dsm5.

1	Child (adapted from the NIDA-modified XII. Suicidal ideation/ Yes None Note. NIDA=National Institute on Drug Abuse. aAvailable at www.psychiatry.org/dsm5. used as an indicator for additional inquiry. A parent or guardian’s rating of "Don’t Know” on the suicidal ideation, suicide attempt, and any of the substance use items, especially for chil- dren ages 11—17 years, may result in additional probing of the issues with the child, including using the child-rated Level 2 Cross-Cutting Symptom Measure for the relevant domain. Be- cause additional inquiry is made on the basis of the highest score on any item within a do- main, clinicians should indicate that score in the ”Highest Domain Score” column. Table 2 outlines threshold scores that may guide further inquiry for the remaining domains.

1	Any threshold scores on the Level 1 Cross-Cutting Symptom Measure (as noted in Tables clinical inquiry. Level 2 Cross-Cutting Symptom Measures provide one method of obtain- ing more in-depth information on potentially significant symptoms to inform diagnosis, treatment planning, and follow-up. They are available online at www.psychiatry.org/ dsm5. Tables 1 and 2 outline each Level 1 domain and identify the domains for which DSM-S Leve12 Cross-Cutting Symptom Measures are available for more detailed assess- ments. Adult and pediatric (parent and child) versions are available online for most Level 1 symptom domains at www.psychiatry.org/dsm5. Frequency of Use of the Cross-CUuing

1	Frequency of Use of the Cross-CUuing To track change in the individual’s symptom presentation over time, the Level 1 and rel- clinically indicated, depending on the stability of the individual’s symptoms and treat- ment status. For individuals with impaired capacity and for children ages 6—17 years, it is preferable for the measures to be completed at follow-up appointments by the same knowledgeable informant and by the same parent or guardian. Consistently high scores on a particular domain may indicate significant and problematic symptoms for the indi- vidual that might warrant further assessment, treatment, and follow-up. Clinical judg— ment should guide decision making. gmmusox wits: 33306 We meaofimswsoco bmsotom 5x3 mien so: 2a 889:: 59» .65 wifiom . , ﬂowed defied“? tmufiom £63.68: rwov gem bum mones voiﬂmxwab «Umsexfim :0» 912: «9.: 956.55% wEEo>< 28:85.de wEon— no umcmn— wczoom

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1	As described in the chapter "Schizophrenia Spectrum and Other Psychotic Disorders,” psychotic disorders are heterogeneous, and symptom severity can predict important as- pects of the illness, such as the degree of cognitive and / or neurobiological deficits. Dimen- sional assessments capture meaningful variation in the severity of symptoms, which may help with treatment planning, prognostic decision-making, and research on pathophysi- ological mechanisms. The Clinician-Rated Dimensions of Psychosis Symptom Severity provides scales for the dimensional assessment of the primary symptoms of psychosis, in- cluding hallucinations, delusions, disorganized speech, abnormal psychomotor behavior, and negative symptoms. A scale for the dimensional assessment of cognitive impairment is also included. Many individuals with psychotic disorders have impairments in a range of cognitive domains, which predict functional abilities. In addition, scales for dimensional assessment of depression and mania

1	Many individuals with psychotic disorders have impairments in a range of cognitive domains, which predict functional abilities. In addition, scales for dimensional assessment of depression and mania are provided, which may alert clinicians to mood pa- thology. The severity of mood symptoms in psychosis has prognostic value and guides treatment.

1	The Clinician—Rated Dimensions of Psychosis Symptom Severity is an 8-item measure that may be completed by the clinician at the time of the clinical assessment. Each item asks the clinician to rate the severity of each symptom as experienced by the individual during the past 7 days. Each item on the measure is rated on a 5-point scale (0=none; 1=equivoca1; 2=present, but mild; 3=present and moderate; and 4=present and severe) with a symptom-specific defi- nition of each rating level. The clinician may review all of the individual’s available infor- mation and, based on clinical judgment, select (by circling) the level that most accurately describes the severity of the individual’s condition. The clinician then indicates the score for each item in the ”Score” column provided. Frequency of Use

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1	The adult self-admim‘stered version of the World Health Organization Disability Assessment Schedule 2.0 (WHODAS 2.0) is a 36-item measure that assesses disability in adults age 18 years and older. It assesses disability across six domains, including understanding and communicating, getting around, self—Care, getting along with people, life activities (i.e., household, work, and / or school activities), and participation in society. If the adult indi- vidual is of impaired capacity and unable to complete the form (e.g., a patient with demen- tia), a knowledgeable informant may complete the proxy—administered version of the measure, which is available at www.psychiatry.org/dsm5. Each item on the self-administered version of the WHODAS 2.0 asks the individual to rate how much difficulty he or she has had in specific areas of functioning during the past 30 days. WHODAS 2.0 Scoring Instructions Provided by WHO

1	WHODAS 2.0 Scoring Instructions Provided by WHO WHODAS 2.0 summary scores. There are two basic options for computing the summary scores for the WHODAS 2.0 316-item full version. Simple: The scores assigned to each of the items—"none" (1), "mild” (2), ”moderate” (3), ”severe” (4), and ”extreme” (5)—are summed. This method is referred to as simple scoring because the scores from each of the items are simply added up without recoding or collaps- ing of response categories; thus, there is no weighting of individual items. This approach is practical to use as a hand-scoring approach, and may be the method of choice in busy clin- ical settings or in paper-and-pencil interview situations. As a result, the simple sum of the scores of the items across all domains constitutes a statistic that is sufficient to describe the degree of functional limitations.

1	Complex: The more complex method of scoring is called ”item-response-theory” (IRT)—based scoring. It takes into account multiple levels of difficulty for each WHODAS 2.0 item. It takes the coding for each item response as "none,” ”mild,” ”moderate,” ”se- vere," and ”extreme” separately, and then uses a computer to determine the summary score by differentially weighting the items and the levels of severity. The computer pro- gram is available from the WHO Web site. The scoring has three steps: 0 Step l—Summing of recoded item scores within each domain. 0 Step 2—Summing of all six domain scores. ' Step 3—Converting the summary score into a metric ranging from 0 to 100 (where 0=no disability; 100=full disability). WHODAS 2.0 domain scores. WHODAS 2.0 produces domain-specific scores for six different functioning domains: cognition, mobility, self—care, getting along, life activities (household and work/school), and participation.

1	WHODAS 2.0 population norms. For the population norms for IRT-based scoring of the WHODAS 2.0 and for the population distribution of IRT-based scores for WHODAS 2.0, please see www.Who.int/classifications / icf/ P0p_norms_distrib_IRT_scores.pdf.

1	The clinician is asked to review the individual’s response on each item on the measure during the clinical interview and to indicate the self—reported score for each item in the sec- tion provided for "Clinician Use Only." However, if the clinician determines that the score on an item should be different based on the clinical interview and other information avail- able, he or she may indicate a corrected score in the raw item score box. Based on findings from the DSM-S Field Trials in adult patient samples across six sites in the United States and one in Canada, DSM-5 recommends calculation and use of average scores for each domain and for general disability. The average scores are comparable to the WHODAS 5-point scale, which allows the Clinician to think of the individual’s disability in terms of none (1), mild (2), moderate (3), severe (4), or extreme (5). The average domain and general disability scores were found to be reliable, easy to use, and clinically useful to the

1	in terms of none (1), mild (2), moderate (3), severe (4), or extreme (5). The average domain and general disability scores were found to be reliable, easy to use, and clinically useful to the clinicians in the

1	DSM-S Field Trials. The average domain score is calculated by dividing the raw domain score by the number of items in the domain (e.g., if all the items within the "understanding and communicating” domain are rated as being moderate then the average domain score would be 18 / 6:3, indicating moderate disability). The average general disability score is cal- culated by dividing the raw overall score by number of items in the measure (i.e., 36). The individual should be encouraged to complete all of the items on the WHODAS 2.0. If no re- sponse is given on 10 or more items of the measure (i.e., more than 25% of the 36 total items), calculation of the simple and average general disability scores may not be helpful. If 10 or more of the total items on the measure are missing but the items for some of the do- mains are 75%—100% complete, the simple or average domain scores may be used for those domains.

1	If 10 or more of the total items on the measure are missing but the items for some of the do- mains are 75%—100% complete, the simple or average domain scores may be used for those domains. Frequency of use. To track change in the individual’s level of disability over time, the measure may be completed at regular intervals as clinically indicated, depending on the stability of the individual's symptoms and treatment status. Consistently high scores on a particular domain may indicate significant and problematic areas for the individual that might warrant further assessment and intervention. \ WHODAS 2.0 World Health Organization Disability Assessment Schedule 2.0 36-item version, self—administered Patient Name: Age: Sex: El Male CI Female Date:

1	\ WHODAS 2.0 World Health Organization Disability Assessment Schedule 2.0 36-item version, self—administered Patient Name: Age: Sex: El Male CI Female Date: This questionnaire asks about difficulties due to healthimental health conditions. Health conditions include diseases or illnesses, other health problems that may be short or long lasting, injuries, mental or emotional problems, and problems with alcohol or drugs. Think back over the past 30 days and answer these questions thinking about how much difficulty you had doing the following activities, For each question, please circle only m response. : Only Numeric scores assigned to each of the items: 4' ; ' 1 I 1 l 3 i 4 l1. 5 g c m z . _ u, ._ In the last 30 days, how much difficulty did you have in: g E i E § g g E . . . ‘ . a . Extreme or 01.1 Concegtgtmg on doung something for ten minutes? None Mlld Moderate Severe cannot do — 01.2 Remembering to do imgortant things? None Mild Moderate Severe :33“;

1	D13 . m all“ 0 ‘ems j‘ av None Mild Moderate Severe Extreme or e ay life? a cannot do i Learnin a new task for xam le, le ' how t 30 5 01.4 e p ammg 0 None Mild Moderate Severe Extreme or get to a new place? cannot do D13 neral und n in what people say? None Mild Moderate Severe 2mm” °' 01.6 Starting and maintaining a conversation? None Mild Moderate Severe :33; . . . - . Extreme or 02.1 Standing for long periods, §UCh as 30 minutes?‘ None Mlld Moderate Severe mm do . t . _ Extreme or 02.2 Standing ug from Slttlng down? None Mlld Moderate Severe cannot do D23 Moving around inside your home? None Mild Moderate Severe Emma °r _._. .— .. cannot do 25 5 D2.4 Getting out Of your home? None Mild Moderate Severe :3?er W “ti 3 lo ce such as a kilom r r D2.5 . ate (0 None Mild Moderate Severe Extreme or Fquwalem)? cannot do s . 7‘ ‘ Extreme Of 03.1 nghlng your whole body? None Mild Moderate Severe cannot do 03.2 Getting dressed? None Mild Moderate Severe “'3'“ °' . Extreme or 20 5

1	Fquwalem)? cannot do s . 7‘ ‘ Extreme Of 03.1 nghlng your whole body? None Mild Moderate Severe cannot do 03.2 Getting dressed? None Mild Moderate Severe “'3'“ °' . Extreme or 20 5 D33 Eggs? None Mild Moderate Severe cannot do . . Extreme or 03.4 Staying by yourself for a few days? None Mlld Moderate Severe cannot do Getting along with people . t Extreme or 04.1 9:31!th with people ou do not know? None Mild Moderate Seven cannot do . . . l . Extreme Of D4.2 Maintaining a friendshlg. None Mlld Moderate Severe cannot do . . . Extreme or D43 Gefimg along wuth people who are close to you? None Mlld Moderate Severe “mm do —-25 —5 D4i4 Making new friends. None Mlld Moderate Severe cannot do . . . Extreme or 04.5 Sexual actrvltles. None Mlld Moderate Severe cannot do Only . Numeric scores assigned to each of the items: I 1 I 2 l 3 l 4 l 5 E c w c In the last 30 days how much difficulty did you have in: g g E fig g E g

1	Only . Numeric scores assigned to each of the items: I 1 I 2 l 3 l 4 l 5 E c w c In the last 30 days how much difficulty did you have in: g g E fig g E g DS.1 Taking care of your hgusghglg responsibilities? ., None M Mlld Moderate Severe mm“ °' ; . « cannot do .u , DSTZ Doing most important household tasks well? None Mild Moderate Severe 2:39er ” _ Getting all of the household work done that you " 5‘ i ‘ Extreme or I ‘ ~ J . , -—-- ~- - 20 5 053‘ NEW to do? None Mild" kModerate Severe cannot do _" 054 Getting your household work done as guuckly as None Mild Moderate Severe Extreme ar ' ’1 ; a needed? cannot do . . Life activities—School/Work E 7 '- 7‘ . If you work (paid, non»paid, self~employedl or go to school, complete questions 05.5—05.8, below. “‘ ‘ ’“ Otherwise, skip to D6.1. ‘ ., Because of your health condition, in the past 30 days how much difficulg did you have in: , 7 ’ , 05.5 Your day-to«day wogfichool? 3 : None Mild Moderate“ same“ :32: ,

1	Otherwise, skip to D6.1. ‘ ., Because of your health condition, in the past 30 days how much difficulg did you have in: , 7 ’ , 05.5 Your day-to«day wogfichool? 3 : None Mild Moderate“ same“ :32: , D5.6 Doing your most important work/school tasks well? None Mild Moderate Severe E25212: " I "~ ' ’: 55.7 us all of the work done that you rfeed to do? 5 None Mlld Moderate Severe :33: —‘ 2° 5 D5.8 Getting your work done as Quickly as needed? None Mild Moderate Severe :33?er In the past 30 days: " How much of a problem did you have in 'olning in : “.- V : _ (for example festivities 3‘ 7 f : Extreme or ' .1 , , . . ' ' " N M” ' Mod at St? ' 2 , D6 lous, or other actwntles) m the same way as s ; me ' ~ ' er 5 vere cannot do - . nyone else can? - “ =.

1	D6 lous, or other actwntles) m the same way as s ; me ' ~ ' er 5 vere cannot do - . nyone else can? - “ =. D6 2 How much of a problem did you have because of Extreme or :, ' barriers or hindrances around you? None Mild Moderate Severe cannot do l much of a problem did you have Iivlng with E 96.3 ‘ ‘ because of the attitudes and actions of None Mﬂd Moderate Severe $32? 06.4 Howmuch tltne dId M spend on your health None Some Mademe A Lot Extreme or 40 5 condition or Its consequences? cannot do 06.5 health condition? , , None Mild Moderate Severe cannot do _ D6.6 l-tow much has your health been a drain on the None Mild Modems Severe Extreme or ‘ fmanual resources of you or your family? cannot do 05.7 How much of a problem dld your family have None Mud M Meme Severe Extreme or so of your health problems? cannot do D6. 8 How much of a problem dld you have In domg None M" d Moderate Severe Extreme or thlngs by yourself for relaxation or pleasure? cannot do

1	D6. 8 How much of a problem dld you have In domg None M" d Moderate Severe Extreme or thlngs by yourself for relaxation or pleasure? cannot do General Dlsablllty Score (Total): E T © World Health Organiution, 2012. All rights reserved. Measuring health and disability: manual for WHO Disability Assessment Schedule (WHODAS 2.0), World Health Organization, 2010, Geneva. The World Health Organization has granted the Publisher permission for the reproduction of this instrument. This material can be reproduced without permission by clinicians for use with their own patients, Any other use, including electronic use, requires written permission from WHO. _ Formul : tion E ass: .— U nd erstand in g the CU llU ral context of illness experience is essential for effec- tive diagnostic assessment and clinical management. Culture refers to systems of knowl- edge, concepts, rules, and practices that are learned and transmitted across generations.

1	Culture includes language, religion and spirituality, family structures, life-cycle stages, ceremonial rituals, and customs, as well as moral and legal systems. Cultures are open, dynamic systems that undergo continuous change over time; in the contemporary world, most individuals and groups are exposed to multiple cultures, which they use to fashion their own identities and make sense of experience. These features of culture make it cru- cial not to overgeneralize cultural information or stereotype groups in terms of fixed cul- tural traits.

1	Race is a culturally constructed category of identity that divides humanity into groups based on a variety of superficial physical traits attributed to some hypothetical intrinsic, biological characteristics. Racial categories and constructs have varied widely over history and across societies. The construct of race has no consistent biological definition, but it is socially important because it supports racial ideologies, racism, discrimination, and social exclusion, which can have strong negative effects on mental health. There is evidence that racism can exacerbate many psychiatric disorders, contributing to poor outcome, and that racial biases can affect diagnostic assessment.

1	Ethnicity is a culturally constructed group identity used to define peoples and communi- ties. It may be rooted in a common history, geography, language, religion, or other shared characteristics of a group, which distinguish that group from others. Ethnicity may be self- assigned or attributed by outsiders. Increasing mobility, intermarriage, and intermixing of cultures has defined new mixed, multiple, or hybrid ethnic identities. Culture, race, and ethnicity are related to economic inequities, racism, and discrimina- tion that result in health disparities. Cultural, ethnic, and racial identities can be sources of strength and group support that enhance resilience, but they may also lead to psycholog- ical, interpersonal, and intergenerational conﬂict or difficulties in adaptation that require diagnostic assessment.

1	The Outline for Cultural Formulation introduced in DSM-IV provided a framework for as— sessing information about cultural features of an individual’s mental health problem and how it relates to a social and cultural context and history. DSM-S not only includes an up- dated version of the Outline but also presents an approach to assessment, using the Cul- tural Formulation Interview (CFI), which has been field-tested for diagnostic usefulness among clinicians and for acceptability among patients. The revised Outline for Cultural Formulation calls for systematic assessment of the fol- lowing categories:

1	The revised Outline for Cultural Formulation calls for systematic assessment of the fol- lowing categories: I Cultural identity of the individual: Describe the individual’s racial, ethnic, or cultural reference groups that may inﬂuence his or her relationships with others, access to re- sources, and developmental and current challenges, conﬂicts, or predicaments. For im- migrants and racial or ethnic minorities, the degree and kinds of involvement with both the culture of origin and the host culture or majority culture should be noted separately. Language abilities, preferences, and patterns of use are relevant for identifying difficul- ties with access to care, social integration, and the need for an interpreter. Other clini— cally relevant aspects of identity may include religious affiliation, socioeconomic background, personal and family places of birth and growing up, migrant status, and sexual orientation.

1	0 Cultural conceptualizations of distress: Describe the cultural constructs that inﬂuence how the individual experiences, understands, and communicates his or her symptoms or problems to others. These constructs may include cultural syndromes, idioms of dis- tress, and explanatory models or perceived causes. The level of severity and meaning of the distressing experiences should be assessed in relation to the norms of the individ- ual’s cultural reference groups. Assessment of coping and help-seeking patterns should consider the use of professional as well as traditional, alternative, or complementary sources of care.

1	. Psychosocial stressors and cultural features of vulnerability and resilience: Identify key stressors and supports in the individual’s social environment (which may include both local and distant events) and the role of religion, family, and other social networks (e.g., friends, neighbors, coworkers) in providing emotional, instrumental, and infor- mational support. Social stressors and social supports vary with cultural interpreta- tions of events, family structure, developmental tasks, and social context. Levels of functioning, disability, and resilience should be assessed in light of the individual’s cul- tural reference groups.

1	0 Cultural features of the relationship between the individual and the clinician: Iden- tify differences in culture, language, and social status between an individual and clini- treatment. Experiences of racism and discrimination in the larger society may impede establishing trust and safety in the clinical diagnostic encounter. Effects may include problems eliciting symptoms, misunderstanding of the cultural and clinical signifi- cance of symptoms and behaviors, and difficulty establishing or maintaining the rap- port needed for an effective clinical alliance. 0 Overall cultural assessment: Summarize the implications of the components of the cul- tural formulation identified in earlier sections of the Outline for diagnosis and other clinically relevant issues or problems as well as appropriate management and treat- ment intervention.

1	The Cultural Formulation Interview (CFI) is a set of 16 questions that clinicians may use to obtain information during a mental health assessment about the impact of culture on key aspects of an individual’s clinical presentation and care. In the CPI, culture refers to 0 The values, orientations, knowledge, and practices that individuals derive from mem- bership in diverse social groups (e.g., ethnic groups, faith communities, occupational groups, veterans groups). 0 Aspects of an individual’s background, developmental experiences, and current social contexts that may affect his or her perspective, such as geographical origin, migration, language, religion, sexual orientation, or race/ethnicity. 0 The inﬂuence of family, friends, and other community members (the individual’s social network) on the individual’s illness experience.

1	0 The inﬂuence of family, friends, and other community members (the individual’s social network) on the individual’s illness experience. The CFI is a brief semistructured interview for systematically assessing cultural factors in the clinical eneounter that may be used with any individual. The CFI focuses on the in- dividual’s experience and the social contexts of the clinical problem. The CFI follows a per- son-centered approach to cultural assessment by eliciting information from the individual about his or her own views and those of others in his or her social network. This approach is designed to avoid stereotyping, in that each individual’s cultural knowledge affects how he or she interprets illness experience and guides how he or she seeks help. Because the CFI concerns the individual’s personal views, there are no right or wrong answers to these questions. The interview follows and is available online at www.psychiatry.org/dsm5.

1	CFI concerns the individual’s personal views, there are no right or wrong answers to these questions. The interview follows and is available online at www.psychiatry.org/dsm5. The CFI is formatted as two text columns. The left-hand column contains the instruc- tions for administering the CFI and describes the goals for each interview domain. The questions in the right-hand column illustrate how to explore these domains, but they are not meant to be exhaustive. Follow-up questions may be needed to clarify individuals’ an- swers. Questions may be rephrased as needed. The CFI is intended as a guide to cultural as- sessment and should be used ﬂexibly to maintain a natural ﬂow of the interview and rapport with the individual.

1	The CFI is best used in conjunction with demographic information obtained prior to the interview in order to tailor the CPI questions to address the individual’s background and current situation. Specific demographic domains to be explored with the CFI will vary across individuals and settings. A comprehensive assessment may include place of birth, age, gender, racial/ethnic origin, marital status, family composition, education, language ﬂuencies, sexual orientation, religious or spiritual affiliation, occupation, employment, in- come, and migration history.

1	The CFI can be used in the initial assessment of individuals in all clinical settings, regard- less of the cultural background of the individual or of the clinician. Individuals and clini- cians who appear to share the same cultural background may nevertheless differ in ways that are relevant to care. The CFI may be used in its entirety, or components may be incor- porated into a clinical evaluation as needed. The CFI may be especially helpful when there is 0 Difficulty in diagnostic assessment owing to significant differences in the cultural, re- ligious, or socioeconomic backgrounds of clinician and the individual. Uncertainty about the fit between culturally distinctive symptoms and diagnostic criteria. Difficulty in judging illness severity or impairment. Disagreement between the individual and clinician on the course of care. Limited engagement in and adherence to treatment by the individual.

1	Difficulty in judging illness severity or impairment. Disagreement between the individual and clinician on the course of care. Limited engagement in and adherence to treatment by the individual. The CFI emphasizes four domains of assessment: Cultural Definition of the Problem (questions 1-3); Cultural Perceptions of Cause, Context, and Support (questions 4—10); Cul- Factors Affecting Current Help Seeking (questions 14—16). Both the person-centered process of conducting the CFI and the information it elicits are intended to enhance the cultural va- lidity of diagnostic assessment, facilitate treatment planning, and promote the individual’s engagement and satisfaction. To achieve these goals, the information obtained from the CFI should be integrated with all other available clinical material into a comprehensive clinical and contextual evaluation. An Informant version of the CFI can be used to collect collateral information on the CFI domains from family members or caregivers.

1	Supplementary modules have been developed that expand on each domain of the CFI and guide clinicians who wish to explore these domains in greater depth. Supplementary modules have also been developed for specific populations, such as children and adoles- cents, elderly individuals, and immigrants and refugees. These supplementary modules are referenced in the CFI under the pertinent subheadings and are available online at www.psychiatry.org / dsm5. Supplementary modules used to expand each CFI subtopic are noted in parentheses. GUIDE TO INTERVIEWER ITALICIZED.

1	Supplementary modules used to expand each CFI subtopic are noted in parentheses. GUIDE TO INTERVIEWER ITALICIZED. Thefollowing questions aim to clarify key aspects of INTRODUCTION FOR THE INDIVIDUAL: the presenting Clinical problem from the point of I would like to understand the problems that view of the individual and other members of the bring you here so that 1 can help you more individual's social network (i.e.,family,friends, or effectively. I want to know about your experi- athers involved in current problem). This includes ence and ideas. 1 will ask some questions the problem’s meaning, potential 50“"55 ofhelp, about what is going on and how you are deal- and expectations for services ing with it. Please remember there are no right or wrong answers. (Explanatory Model, Level of Functioning) Elicit the individual’s view of core problems and key 1. What brings you here today? concerns. IF INDIVIDUAL GIVES FEW DETAILS OR

1	(Explanatory Model, Level of Functioning) Elicit the individual’s view of core problems and key 1. What brings you here today? concerns. IF INDIVIDUAL GIVES FEW DETAILS OR Focus on the individual’s own way ofunderstand- ONLY MENTIONS SYMPTOMS OR A ing the problem. MEDICAL DIAGNOSIS, PROBE: Use the term, expression, or briefdescription elicited People often understand their problems in in question 1 to identify the problem in subsequent their own way, which may be similar to or questions (e.g., “your conﬂict with your son"). different from how doctors describe the problem. How would you describe your problem? Ask how individualframes the problem for members 2. Sometimes people have different ways of of the social network. describing their problem to their family, friends, or others in their community. How would you describe your problem to them? Focus on the aspects of the problem that matter most 3. What troubles you most about your prob- to the individual. lem?

1	Focus on the aspects of the problem that matter most 3. What troubles you most about your prob- to the individual. lem? CULTURAL PERCEPTIONS 0F CAUSE, CONTEXT, AND SUPPORT (Explanatory Model, Social Network, Older Adults) This question indicates the meaning of the condition 4. Why do you think this is happening to for the individual, which may be relevant for clin— you? What do you think are the causes of ical care. your [PROBLEM]? Note that individuals may identify multiple causes, PROMPT FURTHER IF REQUIRED: depending 0" thefacet 0f the problem they are con- Some people may explain their problem as sidering. the result of bad things that happen in their life, problems with others, a physical ill- ness, a spiritual reason, or many other causes. Focus on the views of members of the individual’s 5. What do others in your family, your social network. These may be diverse and vary from friends, or others in your community think the individual’s. is causing your [PROBLEM]?

1	Supplementary modules used to expand each CFI subtopic are noted in parentheses. GUIDE TO INTERVIEWER ITALICIZED. (Social Network, Caregivers, Psychosocial Stressors, Religion and Spirituality, Immigrants and Refugees, Cultural Identity, Older Adults, Coping and Help Seeking) Elicit information on the individual’s life context, 6. Are there any kinds of support that make focusing on resources, social supports, and resil— your [PROBLEM] better, such as support ience. May also probe other supports (e.g., from co- from family, friends, or others? workers, from participation in religion or spiritu- ality). Focus on stressful aspects of the individual’s envi- 7. Are there any kinds of stresses that make ronment. Can also probe, e.g., relationship prob» your [PROBLEM] worse, such as difficul- lems, difi‘iculties at work or school, or ties with money, or family problems? discrimination. (Cultural Identity, Psychosocial Stressors, Religion and Spirituality, Immigrants and Refugees,

1	discrimination. (Cultural Identity, Psychosocial Stressors, Religion and Spirituality, Immigrants and Refugees, Older Adults, Children and Adolescents) Sometimes, aspects of people’s back- LEM] better or worse. By background or identity, I mean, for example, the commu- nities you belong to, the languages you speak, where you or your family are from, your race or ethnic background, your gen- der or sexual orientation, or your faith or religion. Ask the individual to reﬂect on the most salient ele— 8. For you, what are the most important ments of his or her cultural identity. Use this aspects of your background or identity? information to tailor questions 9—10 as needed. Elicit aspects of identity that make the problem bet- 9. Are there any aspects of your background ter or worse. or identity that make a difference to your Probe as needed (e.g., clinical worsening as a result [PROBLEM]? of discrimination due to migration status, race/ ethnicity, or sexual orientation).

1	Probe as needed (e.g., clinical worsening as a result [PROBLEM]? of discrimination due to migration status, race/ ethnicity, or sexual orientation). Probe as needed (e.g., migration-related problems; 10. Are there any aspects of your background conﬂict across generations or due to gender roles). or identity that are causing other concerns or difficulties for you? (Coping and Help Seeking, Religion and Spirituality, Older Adults, Caregivers, Clarify self-coping for the problem. 11. Sometimes people have various ways of dealing with problems like [PROBLEM]. What have you done on your own to cope with your [PROBLEM]? Supplementary modules used to expand each CFI subtopic are noted in parentheses. GUIDE TO INTERVIEWER ITALICIZED. (Coping and Help Seeking, Religion and Spirituality, Older Adults, Caregivers, Psychosocial Stressors, Immigrants and Refugees, Social Network, ClinicianvPatient Relationship)

1	(Coping and Help Seeking, Religion and Spirituality, Older Adults, Caregivers, Psychosocial Stressors, Immigrants and Refugees, Social Network, ClinicianvPatient Relationship) Elicit various sources ofhelp (e.g., medical care, 12. Often, people look for help from many dif- mental health treatment, support groups, work- ferent sources, including different kinds of based counseling,folk healing, religious or spiri- doctors, helpers, or healers. In the past, tual counseling, other forms of traditional or alter— what kinds of treatment, help, advice, or native healing). healing have you sought for your [PROB- Probe as needed (e.g., "What other sources of help LEM]? have you used?"). PROBE IF DOES NOT DESCRIBE USE— Clarify the individual’s experience and regard for FULNESS OF HELP RECEIVED: previous help. What types of help or treatment were most useful? Not useful?

1	Clarify the individual’s experience and regard for FULNESS OF HELP RECEIVED: previous help. What types of help or treatment were most useful? Not useful? (Coping and Help Seeking, Religion and Spirituality, Older Adults, Psychosocial Stressors, Immi- grants and Refugees, Social Network, Clinician-Patient Relationship) Clarify the role of social barriers to help seeking, 13. Has anything prevented you from getting access to care, and problems engaging in previous the help you need? treatment. PROBE AS NEEDED: Probe details as needed (e.g., ”What got in the 1:01- example, money, work or family com- way? ”)~ mitments, stigma or discrimination, or lack or background? (Social Network, Caregivers, Religion and Spirituality, Older Adults, Coping and Help Seeking) Clarify individual’s current perceived needs and Now let’s talk some more about the help expectations of help, broadly defined. you need.

1	Clarify individual’s current perceived needs and Now let’s talk some more about the help expectations of help, broadly defined. you need. Probe 1f individual lists only one source ofhelp (eg., 14‘ What kinds of help do you think would be “What other kinds of help would be useful to you most useful to you at this time for your at this time?”). [PROBLEM]? Focus on the views of the social network regarding 15. Are there other kinds of help that your fam— help seeking. ily, friends, or other people have suggested would be helpful for you now? (Clinician—Patient Relationship, Older Adults) Elicit possible concerns about the clinic or the clini— Sometimes doctors and patients misunder- cian-patient relationship, including perceived rac- stand each other because they come from ism, language barriers, or cultural differences that different backgrounds or have different may undermine goodwill, communication, or care expectations. delivery. 16. Have you been concerned about this and is

1	delivery. 16. Have you been concerned about this and is Probe details as needed (e.g., ”In what way?"). there anything that we can do to provide Address possible barriers to care or concerns about You With the care You need? the clinic and the clinician-patient relationship raised previously. The CFI—Inforniant Version collects collateral information from an informant who is knowledgeable about the clinical problems and life circumstances of the identified indi- vidual. This version can be used to supplement information obtained from the core CFI or can be used instead of the core CFI when the individual is unable to provide information— as might occur, for example, with children or adolescents, ﬂoridly psychotic individuals, or persons with cognitive impairment. GUIDE TO INTERVIEWER ITALICIZED.

1	GUIDE TO INTERVIEWER ITALICIZED. The following questions aim to clarify key aspects of INTRODUCTION FOR THE INFORMANT: the presenting clinical problemfrom the infor- I would like to understand the problems that mant’s point ofview. This includes the problem’s bring your family member/friend here so meaning, potential sources ofhelp, “"d exaecta- that I can help you and him/her more effec- tions for services. tively. I want to know about your experience and ideas. I will ask some questions about what is going on and how you and your fam- ily member/friend are dealing with it. There are no right or wrong answers. Clarify the informant’s relationship with the indi- 1. How would you describe your relationship vidual and/or the individual'sfamily. to [INDIVIDUAL OR TO FAMILY]? PROBE IF NOT CLEAR: How often do you see [INDIVIDUAL]? Elicit the informant’s view of core problems and key 2. What brings your family member/friend concerns. here today?

1	PROBE IF NOT CLEAR: How often do you see [INDIVIDUAL]? Elicit the informant’s view of core problems and key 2. What brings your family member/friend concerns. here today? Focus on the informant’s way of understanding the IF INFORMANT GIVES FEW DETAILS OR individual's problem. ONLY MENTIONS SYMPTOMS OR A Use the term, expression, or briefdescription elicited MEDICAL DIAGNOSIS, PROBE: in question 1 to identify the problem in subsequent People often understand problems in their questions (e.g., ”her conﬂict with her son”). own way, which may be similar or differ- ent from how doctors describe the prob- lem. How would you describe [INDIVIDUAL’S] problem? Ask how informantframes the problem for members 3. Sometimes people have different ways of of the social network. describing the problem to family, friends, or others in their community. How would them? Focus on the aspects of the problem that matter most 4. What troubles you most about [INDIVID- to the informant. UAL’S] problem?

1	Focus on the aspects of the problem that matter most 4. What troubles you most about [INDIVID- to the informant. UAL’S] problem? GUIDE TO INTERVIEWER ITALICIZED. CULTURAL PERCEPTIONS OF CAUSE, CONTEXT, AND SUPPORT This question indicates the meaning of the condition 5. Why do you think this is happening to for the informant, which may be relevant for clini— [INDIVIDUAL]? What do you think are the cal care. causes of his / her [PROBLEM]? Note that informants may identify multiple causes PROMPT FURTHER IF REQUIRED: depending on the facet of the problem they are con- Some people may explajn the problem as the sidering. result of bad things that happen in their life, problems with others, a physical illness, a spiritual reason, or many other causes.

1	Focus on the views of members of the individual's 6. What do others in [INDIVIDUAL’S] fam- social network. These may be diverse and vary ily, his/her friends, or others in the com- from the informant's. munity think is causing [INDIVIDUAL’S] [PROBLEM]? Elicit information on the individual’s life context, 7. Are there any kinds of supports that make focusing on resources, social supports, and resil— his/her [PROBLEM] better, such as from ience. May also probe other supports ( e.g., from co- family, friends, or others? workers, from participation in religion or spiritu- ality). Focus on stressful aspects of the individual's environ— 8. Are there any kinds of stresses that make ment. Can also probe, e.g., relationship problems, dlf- his/her [PROBLEM] worse, such as diffi- ficulties at work or school, or discrimination. culties with money, or family problems?

1	Sometimes, aspects of people’s background or identity can make the [PROBLEM] better or worse. By background or identity, I mean, for example, the communities you belong to, the languages you speak, where you or your family are from, your race or ethnic back- ground, your gender or sexual orientation, and your faith or religion. Ask the informant to reflect on the most salient ele— 9. For you, what are the most important ments of the individual’s cultural identity. Use this aspects of [INDIVIDUAL’S] background or information to tailor questions 10—11 as needed. identity? Elicit aspects of identity that make the problem bet- 104 Are there any aspects of [INDIVIDUAL’S] ter or worse. background or identity that make a differ— Probe as needed (e.g., clinical worsening as a result ence to his/her [PROBLEM]? of discrimination due to migration status, race/ ethnicity, or sexual orientation).

1	Probe as needed (e.g., clinical worsening as a result ence to his/her [PROBLEM]? of discrimination due to migration status, race/ ethnicity, or sexual orientation). Probe as needed (erg., migration-related problems; 11. Are there any aspects of [INDIVIDUAL’S] conﬂict across generations or due to gender roles). background or identity that are causing other concerns or difficulties for him/her? GUIDE TO INTERVIEWER ITALICIZED. Clarify individual's self-coping for the problem. 12. Sometimes people have various ways of dealing with problems like [PROBLEM]. own to cope with his/her [PROBLEM]? Elicit various sources of help (e.g., medical care, mental health treatment, support groups, work— based counseling,folk healing, religious or spiri- tual counseling, other alternative healing). Probe as needed (e.g., ”What other sources of help has he/she used .7 ")t Clarify the individual’s experience and regard for previous help.

1	Probe as needed (e.g., ”What other sources of help has he/she used .7 ")t Clarify the individual’s experience and regard for previous help. 13. Often, people also look for help from many different sources, including different kinds of doctors, helpers, or healers. In the past, what kinds of treatment, help, advice, or her [PROBLEM]? FULNESS OF HELP RECEIVED: What types of help or treatment were most useful? Not useful? Clarify the role ofsocial barriers to help—seeking, access to care, and problems engaging in previous treatment. Probe details as needed (e.g., ”What got in the way?"). 14. Has anything prevented [INDIVIDUAL] from getting the help he/she needs? PROBE AS NEEDED: For example, money, work or family com— mitments, stigma or discrimination, or lack guage or background? expectations ofhelp, broadly defined, from the point of view of the informant.

1	For example, money, work or family com— mitments, stigma or discrimination, or lack guage or background? expectations ofhelp, broadly defined, from the point of view of the informant. Probe if informant lists only one source of help (e.g., ”What other kinds of help would be useful to [INDIVIDUAL] at this time?"). Focus on the views of the social network regarding help seeking. Now let’s talk about the help [INDIVID- UAL] needs. 15. What kinds of help would be most useful to LEM]? 16. Are there other kinds of help that [INDI- VIDUAL’S] family, friends, or other people her now? Elicit possible concerns about the clinic or the clini— cian—patient relationship, including perceived rac— ism, language barriers, or cultural differences that may undermine goodwill, communication, or care delivery. Probe details as needed (e.g., "In what way?”). Address possible barriers to care or concerns about the clinic and the clinician-patient relationship raised previously.

1	Probe details as needed (e.g., "In what way?”). Address possible barriers to care or concerns about the clinic and the clinician-patient relationship raised previously. stand each other because they come from expectations. 17. Have you been concerned about this, and is there anything that we can do to provide [INDIVIDUAL] with the care he/she needs? Cultural Concepts of Distress

1	Cultural concepts of distress refers to ways that cultural groups experience, understand, and communicate suffering, behavioral problems, or troubling thoughts and emotions. Three main types of cultural concepts may be distinguished. Cultural syndromes are clusters of symptoms and attributions that tend to co-occur among individuals in specific cultural groups, communities, or contexts and that are recognized locally as coherent patterns of experience. Cultural idioms of distress are ways of expressing distress that may not involve specific symptoms or syndromes, but that provide collective, shared ways of experiencing and talking about personal or social concerns. For example, everyday talk about ”nerves” or ”depression” may refer to widely varying forms of suffering without mapping onto a discrete set of symptoms, syndrome, or disorder. Cultural explanations or perceived causes are labels, attributions, or features of an explanatory model that indicate culturally recog- nized

1	onto a discrete set of symptoms, syndrome, or disorder. Cultural explanations or perceived causes are labels, attributions, or features of an explanatory model that indicate culturally recog- nized meaning or etiology for symptoms, illness, or distress.

1	These three concepts—syndromes, idioms, and explanations—are more relevant to clinical practice than the older formulation culture-bound syndrome. Specifically, the term culture-bound syndrome ignores the fact that clinically important cultural differences often involve explanations or experience of distress rather than culturally distinctive configura- tions of symptoms. Furthermore, the term culture-bound overemphasizes the local partic- ularity and limited distribution of cultural concepts of distress. The current formulation acknowledges that all forms of distress are locally shaped, including the DSM disorders.

1	From this perspective, many DSM diagnoses can be understood as operationalized proto- types that started out as cultural syndromes, and became widely accepted as a result of their clinical and research utility. Across groups there remain culturally patterned differ- ences in symptoms, ways of talking about distress, and locally perceived causes, which are in turn associated with coping strategies and patterns of help seeking. and emotional distress, and they may also reﬂect the inﬂuence of biomedical concepts. Cultural concepts have four key features in relation to the DSM-S nosology: - There is seldom a one-to—one correspondence of any cultural concept with a DSM diag- nostic entity; the correspondence is more likely to be one-to-many in either direction, included in a single folk concept, and diverse presentations that might be classified by DSM-5 as variants of a single disorder may be sorted into several distinct concepts by an indigenous diagnostic system.

1	DSM-5 as variants of a single disorder may be sorted into several distinct concepts by an indigenous diagnostic system. 0 Cultural concepts may apply to a wide range of severity, including presentations that do not meet DSM criteria for any mental disorder. For example, an individual with acute grief or a social predicament may use the same idiom of distress or display the same cultural syndrome as another individual with more severe psychopathology. 0 In common usage, the same cultural term frequently denotes more than one type of cultural concept. A familiar example may be the concept of ”depression,” which may be used to describe a syndrome (e.g., major depressive disorder), an idiom of distress (e.g., as in the common expression “I feel depressed"), or a perceived cause (similar to ”stress"). 0 Like culture and DSM itself, cultural concepts may change over time in response to both local and global inﬂuences.

1	0 Like culture and DSM itself, cultural concepts may change over time in response to both local and global inﬂuences. Cultural concepts are important to psychiatric diagnosis for several reasons: ' To avoid misdiagnosis: Cultural variation in symptoms and in explanatory models as- sociated with these cultural concepts may lead clinicians to misjudge the severity of a problem or assign the wrong diagnosis (e.g., unfamiliar spiritual explanations may be misunderstood as psychosis). 0 To obtain useful clinical information: Cultural variations in symptoms and attribu- tions may be associated with particular features of risk, resilience, and outcome. I To improve clinical rapport and engagement: ”Speaking the language of the patient,” both linguistically and in terms of his or her dominant concepts and metaphors, can re- sult in greater communication and satisfaction, facilitate treatment negotiation, and lead to higher retention and adherence.

1	0 To improve therapeutic efficacy: Culture inﬂuences the psychological mechanisms of disorder, which need to be understood and addressed to improve clinical efficacy. For example, culturally specific catastrophic cognitions can contribute to smptom escala— tion into panic attacks. 0 To guide clinical research: Locally perceived connections between cultural concepts may help identify patterns of comorbidity and underlying biological substrates. . To clarify the cultural epidemiology: Cultural concepts of distress are not endorsed uniformly by everyone in a given culture. Distinguishing syndromes, idioms, and ex- planations provides an approach for studying the distribution of cultural features of ill- ness across settings and regions, and over time. It also suggests questions about cultural determinants of risk, course, and outcome in clinical and community settings to en- hance the evidence base of cultural research.

1	DSM-S includes information on cultural concepts in order to improve the accuracy of diagnosis and the comprehensiveness of clinical assessment. Clinical assessment of indi- viduals presenting with these cultural concepts should determine whether they meet

1	DSM-5 criteria for a specified disorder or an other specified or unspecified diagnosis. Once the disorder is diagnosed, the cultural terms and explanations should be included in case for- wise be confusing. Individuals whose symptoms do not meet DSM criteria for a specific by—case basis. In addition to the CFI and its supplementary modules, DSM-S contains the in clinical practice: 0 Data in DSM-S criteria and text for specific disorders: The text includes information on cultural variations in prevalence, symptomatology, associated cultural concepts, and other clinical aspects. It is important to emphasize that there is no one-to-one cor- respondence at the categorical level between DSM disorders and cultural concepts. Dif- variation with information elicited by the CFI.

1	0 Other Conditions That May Be a Focus of Clinical Attention: Some of the clinical con- cerns identified by the CFI may correspond to V codes or Z codes—for example, accul- turation problems, parent—child relational problems, or religious or spiritual problems. . Glossary of Cultural Concepts of Distress: Located in the Appendix, this glossary pro- vides examples of well-studied cultural concepts of distress that illustrate the relevance of cultural information for clinical diagnosis and some of the interrelationships among cultural syndromes, idioms of distress, and causal explanations.

1	The current approach to personality disorders appears in Section II of DSM-S, and an alternative model developed for DSM-S is presented here in Section III. The inclu- sion of both models in DSM-S reﬂects the decision of the APA Board of Trustees to pre- serve continuity with current clinical practice, while also introducing a new approach that aims to address numerous shortcomings of the current approach to personality disorders. For example, the typical patient meeting criteria for a specific personality disorder fre- quently also meets criteria for other personality disorders. Similarly, other specified or un- specified personality disorder is often the correct (but mostly uninformative) diagnosis, in the sense that patients do not tend to present with patterns of symptoms that correspond with one and only one personality disorder.

1	In the following alternative DSM~5 model, personality disorders are characterized by impairments in personalityfunctioning and pathological personality traits. The specific personality disorder diagnoses that may be derived from this model include antisocial, avoidant, borderline, narcissistic, obsessive-compulsive, and schizotypal personality dis- orders. This approach also includes a diagnosis of personality disorder—trait specified (PD-TS) that can be made when a personality disorder is considered present but the crite- ria for a specific disorder are not met. The essential features of a personality disorder are A. Moderate or greater impairment in personality (self/interpersonal) functioning. B. One or more pathological personality traits. C. The impairments in personality functioning and the individual’s personality trait expres- sion are relatively inflexible and pervasive across a broad range of personal and social situations.

1	C. The impairments in personality functioning and the individual’s personality trait expres- sion are relatively inflexible and pervasive across a broad range of personal and social situations. D. The impairments in personality functioning and the individual's personality trait expres- sion are relatively stable across _time, with onsets that can be traced back to at least adolescence or early adulthood. E. The impairments in personality functioning and the individual’s personality trait expres- sion are not better explained by another mental disorder. F. The impairments in personality functioning and the individual’s personality trait expres- sion are not solely attributable to the physiological effects of a substance or another medical condition (e.g., severe head trauma). G. The impairments in personality functioning and the individual’s personality trait expres- sion are not better understood as normal for an individual’s developmental stage or so- ciocultural environment.

1	A diagnosis of a personality disorder requires two determinations: 1) an assessment of the level of impairment in personality functioning, which is needed for Criterion A, and 2) an evaluation of pathological personality traits, which is required for Criterion B. The im- pairments in personality functioning and personality trait expression are relatively inﬂex- ible and pervasive across a broad range of personal and social situations (Criterion C); relatively stable across time, with onsets that can be traced back to at least adolescence or E); not attributable to the effects of a substance or another medical condition (Criterion F); and not better understood as normal for an individual’s developmental stage or sociocul- tural environment (Criterion G). All Section III personality disorders described by criteria sets, as well as PD-TS, meet these general criteria, by definition. Crlterion A: Level of Personality Functioning

1	Crlterion A: Level of Personality Functioning Disturbances in self and interpersonal functioning constitute the core of personality psy- chopathology and in this alternative diagnostic model they are evaluated on a continuum. empathy and intimacy (see Table 1). The Level of Personality Functioning Scale (LPFS; see Table 2, pp. 775—778) uses each of these elements to differentiate five levels of impairment, ranging from little or no impairment (i.e., healthy, adaptive functioning; Level 0) to some (Level 1), moderate (Level 2), severe (Level 3), and extreme (Level 4) impairment. TABLE 1 Elements of personality tunctioning Self: 1. Identity: Experience of oneself as unique, with clear boundaries between self and others; sta— bility of self—esteem and accuracy of self—appraisal; capacity for, and ability to regulate, a range of emotional experience.

1	2. Self-direction:1’ursuit of coherent and meaningful short-term and life goals; utilization of constructive and prosocial internal standards of behavior; ability to self-reﬂect productively. Interpersonal: 1. Empathy: Comprehension and appreciation of others’ experiences and motivations; tolerance of differing perspectives; understanding the effects of one’s own behavior on others. 2. Intimacy: Depth and duration of connection with others; desire and capacity for closeness; mutuality of regard reﬂected in interpersonal behavior.

1	2. Intimacy: Depth and duration of connection with others; desire and capacity for closeness; mutuality of regard reﬂected in interpersonal behavior. Impairment in personality functioning predicts the presence of a personality disorder, and the severity of impairment predicts whether an individual has more than one person- ality disorder or one of the more typically severe personality disorders. A moderate level of impairment in personality functioning is required for the diagnosis of a personality dis- order; this threshold is based on empirical evidence that the moderate level of impairment maximizes the ability of clinicians to accurately and efficiently identify personality disor- der pathology. Crlterion B: Pathological Personallty Traits

1	Crlterion B: Pathological Personallty Traits Pathological personality traits are organized into five broad domains: Negative Affectiv- ity, Detachment, Antagonism, Disinhibition, and Psychoticism. Within the five broad trait domains are 25 specific trait facets that were developed initially from a review of existing trait models and subsequently through iterative research with samples of persons who sought mental health services. The full trait taxonomy is presented in Table 3 (see pp. 779— 781). The B criteria for the specific personality disorders comprise subsets of the 25 trait facets, based on meta-analytic reviews and empirical data on the relationships of the traits to DSM-IV personality disorder diagnoses. Criteria C and D: Pervasiveness and Stability

1	Impairments in personality functioning and pathological personality traits are relatively per- vasive across a range of personal and social contexts, as personality is defined as a pattern of perceiving, relating to, and thinking about the environment and oneself. The term relatively reﬂects the fact that all except the most extremely pathological personalities show some de- gree of adaptability. The pattern in personality disorders is maladaptive and relatively inﬂex- ible, which leads to disabilities in social, occupational, or other important pursuits, as individuals are unable to modify their thinking or behavior, even in the face of evidence that their approach is not working. The impairments in functioning and personality traits are also relatively stable. Personality traits—the dispositions to behave or feel in certain ways—are more stable than the symptomatic expressions of these dispositions, but personality traits can also change. Impairments in personality functioning are

1	to behave or feel in certain ways—are more stable than the symptomatic expressions of these dispositions, but personality traits can also change. Impairments in personality functioning are more stable than symptoms.

1	Criteria E, F, and G: Alternative Explanations for

1	On some occasions, what appears to be a personality disorder may be better explained by another mental disorder, the effects of a substance or another medical condition, or a nor— mal developmental stage (e.g., adolescence, late life) or the individual’s sociocultural en- vironment. When another mental disorder is present, the diagnosis of a personality disorder is not made, if the manifestations of the personality disorder clearly are an ex- pression of the other mental disorder (e.g., it features of schizotypal personality disorder are present only in the context of schizophrenia). On the other hand, personality disorders can be accurately diagnosed in the presence of another mental disorder, such as major de- pressive disorder, and patients with other mental disorders should be assessed for comor- bid personality disorders because personality disorders often impact the course of other mental disorders. Therefore, it is always appropriate to assess personality functioning and

1	for comor- bid personality disorders because personality disorders often impact the course of other mental disorders. Therefore, it is always appropriate to assess personality functioning and pathological personality traits to provide a context for other psychopathology.

1	Section 111 includes diagnostic criteria for antisocial, avoidant, borderline, narcissistic, ob— sessive-compulsive, and schizotypal personality disorders. Each personality disorder is pathological personality traits (Criterion B): 0 Typical features of antisocial personality disorder are a failure to conform to lawful and ethical behavior, and an egocentric, callous lack of concern for others, accompanied by deceitfulness, irresponsibility, manipulativeness, and / or risk taking. 0 Typical features of avoidant personality disorder are avoidance of social situations and inhibition in interpersonal relationships related to feelings of ineptitude and inade- quacy, anxious preoccupation with negative evaluation and rejection, and fears of rid- icule or embarrassment. 0 Typical features of borderline personality disorder are instability of self—image, per- sonal goals, interpersonal relationships, and affects, accompanied by impulsivity, risk taking, and / 0r hostility.

1	0 Typical features of narcissistic personality disorder are variable and vulnerable self- esteem, with attempts at regulation through attention and approval seeking, and either overt or covert grandiosity. 0 Typical features of obsessive-compulsive personality disorder are difficulties in estab- lishing and sustaining close relationships, associated with rigid perfectionism, inﬂexi- bility, and restricted emotional expression. ' Typical features of schizotypal personality disorder are impairments in the capacity for social and close relationships, and eccentricities in cognition, perception, and behav- ior that are associated with distorted self—image and incoherent personal goals and ac- companied by suspiciousness and restricted emotional expression. The A and B criteria for the six specific personality disorders and for PD-TS follow. All personality disorders also meet criteria C through G of the General Criteria for Personality Disorder.

1	The A and B criteria for the six specific personality disorders and for PD-TS follow. All personality disorders also meet criteria C through G of the General Criteria for Personality Disorder. Typical features of antisocial personality disorder are a failure to conform to lawful and ethical behavior, and an egocentric, callous lack of concern for others, accompanied by de- ceitfulness, irresponsibility, manipulativeness, and/ or risk taking. Characteristic difficul- ties are apparent in identity, self-direction, empathy, and / or intimacy, as described below, along with specific maladaptive traits in the domains of Antagonism and Disinhibition. A. Moderate or greater impairment in personality functioning, manifested by characteristic difficulties in two or more of the following four areas: 1. Identity: Egocentrism; seIf-esteem derived trom personal gain, power, or pleasure.

1	2. SeIt-direction: Goal setting based on personal gratification; absence of prosocial internal standards, associated with failure to conform to lawful or culturally norma- tive ethical behavior. 3. Empathy: Lack of concern for feelings, needs, or suffering of others; lack of re- morse after hurting or mistreating another. 4. Intimacy: Incapacity for mutually intimate relationships, as exploitation is a primary means of relating to others, including by deceit and coercion; use of dominance or intimidation to control others. B. Six or more of the following seven pathological personality traits: 1. Manipulativeness (an aspect of Antagonism): Frequent use of subterfuge to in- fluence or control others; use at seduction, charm, glibness, or ingratiation to achieve one’s ends. 2. Callousness (an aspect of Antagonism): Lack of concern for feelings or problems of others; lack of guilt or remorse about the negative or harmful effects of one's ac- tions on others; aggression; sadism.

1	3. Deceitfulness (an aspect of Antagonism): Dishonesty and fraudulence; misrepre- sentation of self; embellishment or fabrication when relating events. 4. Hostility (an aspect of Antagonism): Persistent or frequent angry feelings; anger or irritability in response to minor slights and insults; mean, nasty, or vengeful behavior. 5. Risk taking (an aspect of Disinhibition): Engagement in dangerous, risky. and poten- tially self-damaging activities, unnecessarily and without regard for consequences; boredom proneness and thoughtless initiation of activities to counter boredom; lack of concern for one’s limitations and denial of the reality of personal danger. 6. Impulsivity (an aspect of Disinhibition): Acting on the spur of the moment in re- sponse to immediate stimuli; acting on a momentary basis without a plan or consid- eration of outcomes; difficulty establishing and following plans.

1	7. irresponsibility (an aspect of Disinhibition): Disregard for—and failure to honor— tinancial and other obligations or commitments; lack of respect for—and lack of toi- Iow-through on—agreements and promises. Note. The individual is at least 18 years of age. Specify it: With psychopathic features. Specifiers. A distinct variant often termed psychopathy (or ”primary” psychopathy) is marked by a lack of anxiety or fear and by a bold interpersonal style that may mask mal- adaptive behaviors (e.g., fraudulence). This psychopathic variant is characterized by low levels of anxiousness (Negative Affectivity domain) and withdrawal (Detachment do- main) and high levels of attention seeking (Antagonism domain). High attention seeking and low withdrawal capture the social potency (assertive/dominant) component of psy- chopathy, whereas low anxiousness captures the stress immunity (emotional stability/re- silience) component.

1	In addition to psychopathic features, trait and personality functioning specifiers may be used to record other personality features that may be present in antisocial personality dis- order but are not required for the diagnosis. For example, traits of Negative Affectivity (e.g., anxiousness), are not diagnostic criteria for antisocial personality disorder (see Criterion B) but can be specified when appropriate. Furthermore, although moderate or greater impair- ment in personality functioning is required for the diagnosis of antisocial personality disor- der (Criterion A), the level of personality functioning can also be specified.

1	Typical features of avoidant personality disorder are avoidance of social situations and in— hibition in interpersonal relationships related to feelings of ineptitude and inadequacy, anxious preoccupation with negative evaluation and rejection, and fears of ridicule or em- barrassment. Characteristic difficulties are apparent in identity, self—direction, empathy, and/ or intimacy, as described below, along with specific maladaptive traits in the do- mains of Negative Affectivity and Detachment. A. Moderate or greater impairment in personality functioning, manifest by characteristic difficulties in two or more of the following tour areas: 1. Identity: Low self—esteem associated with seIf-appraisal as socially inept. person- ally unappealing, or interior; excessive feelings of shame. 2. Selt-direction: Unrealistic standards for behavior associated with reluctance to pursue goals, take personal risks, or engage in new activities involving interper- sonal contact.

1	2. Selt-direction: Unrealistic standards for behavior associated with reluctance to pursue goals, take personal risks, or engage in new activities involving interper- sonal contact. 3. Empathy: Preoccupation with, and sensitivity to, criticism or rejection, associated with distorted inference of others' perspectives as negative. 4. Intimacy: Reluctance to get involved with people unless being certain of being liked; diminished mutuality within intimate relationships because of fear of being shamed or ridiculed. B. Three or more of the following four pathological personality traits. one of which must be (1) Anxiousness: 1. Anxiousness (an aspect of Negative Attectivlty): Intense feelings of nervous- ness, tenseness, or panic. often in reaction to social situations; worry about the negative effects of past unpleasant experiences and future negative possibilities; feeling tearful, apprehensive, or threatened by uncertainty; tears of embarrass- ment.

1	2. Withdrawal (an aspect of Detachment): Reticence in social situations; avoidance of social contacts and activity; lack of initiation of social contact. 3. Anhedonia (an aspect of Detachment): Lack of enjoyment from, engagement in, or energy for life's experiences; deficits in the capacity to feel pleasure or take in- terest in things. 4. Intimacy avoidance (an aspect of Detachment): Avoidance of close or romantic relationships, interpersonal attachments, and intimate sexual relationships.

1	4. Intimacy avoidance (an aspect of Detachment): Avoidance of close or romantic relationships, interpersonal attachments, and intimate sexual relationships. Specifiers. Considerable heterogeneity in the form of additional personality traits is found among individuals diagnosed with avoidant personality disorder. Trait and level of personality functioning specifiers can be used to record additional personality features that may be present in avoidant personality disorder. For example, other Negative Affec- tivity traits (e.g., depressivity, separation insecurity, submissiveness, suspiciousness, hos- tility) are not diagnostic criteria for avoidant personality disorder (see Criterion B) but can be specified when appropriate. Furthermore, although moderate or greater impairment in personality functioning is required for the diagnosis of avoidant personality disorder (Cri- terion A), the level of personality functioning also can be specified.

1	Typical features of borderline personality disorder are instability of self-image, personal goals, interpersonal relationships, and affects, accompanied by impulsivity, risk taking, and/ or hostility. Characteristic difficulties are apparent in identity, self—direction, empa- thy, and / or intimacy, as described below, along with specific maladaptive traits in the do- main of Negative Affectivity, and also Antagonism and / or Disinhibition. A. Moderate or greater impairment in personality functioning, manifested by characteristic difficulties in two or more of the following tour areas: 1. Identity: Markedly impoverished, poorly developed, or unstable seIf-image, often associated with excessive self—criticism; chronic feelings of emptiness; dissociative states under stress. 2. SeIt-direction: Instability in goals, aspirations, values, or career plans.

1	2. SeIt-direction: Instability in goals, aspirations, values, or career plans. 3. Empathy: Compromised ability to recognize the feelings and needs of others asso- ciated with interpersonal hypersensitivity (i.e., prone to feel slighted or insulted); per- ceptions of others selectively biased toward negative attributes or vulnerabilities. 4. Intimacy: Intense, unstable, and conflicted close relationships, marked by mistrust, neediness, and anxious preoccupation with real or imagined abandonment; close relationships often viewed in extremes of idealization and devaluation and alternat- ing between overinvolvement and withdrawal.

1	B. Four or more of the following seven pathological personality traits, at least one of which must be (5) Impulsivity, (6) Risk taking, or (7) Hostility: 1. Emotional lability (an aspect of Negative Affectivity): Unstable emotional expe- riences and frequent mood changes; emotions that are easily aroused, intense, and/or out of proportion to events and circumstances. 2. Anxiousness (an aspect of Negative Affectivity): Intense feelings of nervous- ness, tenseness, or panic, often in reaction to interpersonal stresses; worry about the negative effects of past unpleasant experiences and future negative possibili- ties; feeling fearful, apprehensive, or threatened by uncertainty; fears of falling apart or losing control. 3. Separation insecurity (an aspect of Negative Affectivity): Fears of rejection by— and/or separation from—significant others, associated with tears of excessive de- pendency and complete loss of autonomy.

1	4. Depressivity (an aspect of Negative Affectivity): Frequent feelings of being down, miserable, and/or hopeless; difficulty recovering from such moods; pessimism about the future; pervasive shame; feelings of inferior seIt-worth; thoughts of sui- cide and suicidal behavior. 5. Impulsivity (an aspect of Disinhibition): Acting on the spur of the moment in re- sponse to immediate stimuli; acting on a momentary basis without a plan or consid- eration of outcomes; difficulty establishing or following plans; a sense of urgency and seIt-harming behavior under emotional distress. 6. Risk taking (an aspect of Disinhibition): Engagement in dangerous, risky, and po- tentially selt-damaging activities, unnecessarily and without regard to conse- quences; lack of concern for one's limitations and denial of the reality of personal danger. 7. Hostility (an aspect of Antagonism): Persistent or frequent angry feelings; anger or irritability in response to minor slights and insults.

1	7. Hostility (an aspect of Antagonism): Persistent or frequent angry feelings; anger or irritability in response to minor slights and insults. Specifiers. Trait and level of personality functioning specifiers may be used to record ad- not required for the diagnosis. For example, traits of Psychoticism (e.g., cognitive and per- ceptual dysregulation) are not diagnostic criteria for borderline personality disorder (see Criterion B) but can be specified when appropriate. Furthermore, although moderate or greater impairment in personality functioning is required for the diagnosis of borderline personality disorder (Criterion A), the level of personality functioning can also be specified.

1	Typical features of narcissistic personality disorder are variable and vulnerable self-esteem, with attempts at regulation through attention and approval seeking, and either overt 0r covert grandiosity. Characteristic difficulties are apparent in identity, self—direction, em- pathy, and/ or intimacy, as described below, along with specific maladaptive traits in the domain of Antagonism. A. Moderate or greater impairment in personality functioning, manifested by characteristic difficulties in two or more of the following tour areas: 1. Identity: Excessive reference to others for seIt-detinition and seif-esteem regula- tion; exaggerated seli—appraisal inflated or deflated, or vaciilating between extremes; emotional regulation mirrors fluctuations in self-esteem.

1	2. SeIt-direction: Goat setting based on gaining approval from others; personal stan- dards unreasonably high in order to see oneselt as exceptional, or too low based on a sense of entitlement; often unaware of own motivations. 3. Empathy: Impaired ability to recognize or identify with the feelings and needs of others; excessively attuned to reactions of others, but only if perceived as relevant to self; over- or underestimate of own effect on others. 4. Intimacy: Relationships largely superficial and exist to serve seli-esteem regula- dominance of a need for personal gain. B. Both of the following pathological personality traits: 1 . Grandiosity (an aspect of Antagonism): Feelings of entitlement, either overt or co- vert; selt-centeredness; firmly holding to the belief that one is better than others; condescension toward others. ' 2. Attention seeking (an aspect of Antagonism): Excessive attempts to attract and be the focus of the attention of others; admiration seeking.

1	Specifiers. Trait and personality functioning specifiers may be used to record additional personality features that may be present in narcissistic personality disorder but are not re- quired for the diagnosis. For example, other traits of Antagonism (e.g., manipulativeness, de- ceitfulness, callousness) are not diagnostic criteria for narcissistic personality disorder (see Criterion B) but can be specified when more pervasive antagonistic features (e.g., ”malignant narcissism”) are present. Other traits of Negative Affectivity (e.g., depressivity, anxiousness) can be specified to record more "vulnerable” presentations. Furthermore, although moderate or greater impairment in personality functioning is required for the diagnosis of narcissistic personality disorder (Criterion A), the level of personality functioning can also be specified.

1	Typical features of obsessive-compulsive personality disorder are difficulties in establish- ing and sustaining close relationships, associated with rigid perfectionism, inﬂexibility, and restricted emotional expression. Characteristic difficulties are apparent in identity, self—direction, empathy, and / or intimacy, as described below, along with specific mal- adaptive traits in the domains of Negative Affectivity and/ or Detachment. A. Moderate or greater impairment in personality functioning, manifested by characteristic difficulties in two or more of the following four areas: 1. Identity: Sense of self derived predominantly from work or productivity; constricted experience and expression of strong emotions. 2. SeIf-direction: Difficulty completing tasks and realizing goals, associated with rigid and unreasonably high and inflexible internal standards of behavior; overly consci- entious and moralistic attitudes.

1	3. Empathy: Difficulty understanding and appreciating the ideas, feelings, or behav- iors of others. 4. Intimacy: Relationships seen as secondary to work and productivity; rigidity and stubbornness negatively affect relationships with others. B. Three or more of the following four pathological personality traits, one of which must be (1) Rigid perfectionism: 1. Rigid perfectionism (an aspect of extreme Conscientiousness [the opposite pole of DetachmentD: Rigid insistence on everything being flawless, perfect, and without errors or faults, including one’s own and others’ performance; sacrificing of timeli- ness to ensure correctness in every detail; believing that there is only one right way to do things; difficulty changing ideas and/or viewpoint; preoccupation with details, organization, and order.

1	2. Perseveration (an aspect of Negative Affectivity): Persistence at tasks long after the behavior has ceased to be functional or effective; continuance of the same be- havior despite repeated failures. 3. Intimacy avoidance (an aspect of Detachment): Avoidance of close or romantic relationships, interpersonal attachments, and intimate sexual relationships. 4. Restricted affectivity (an aspect of Detachment): Little reaction to emotionally or coldness.

1	4. Restricted affectivity (an aspect of Detachment): Little reaction to emotionally or coldness. Specifiers. Trait and personality functioning specifiers may be used to record additional not required for the diagnosis. For example, other traits of Negative Affectivity (e.g., anxious- ness) are not diagnostic criteria for obsessive-compulsive personality disorder (see Criterion B) but can be specified when appropriate. Furthermore, although moderate or greater impair- ment in personality functioning is required for the diagnosis of obsessive-compulsive person- ality disorder (Criterion A), the level of personality functioning can also be specified.

1	Typical features of schizotypal personality disorder are impairments in the capacity for so- cial and close relationships and eccentricities in cognition, perception, and behavior that are associated with distorted self—image and incoherent personal goals and accompanied by suspiciousness and restricted emotional expression. Characteristic difficulties are ap- parent in identity, self—direction, empathy, and / or intimacy, along with specific maladap- tive traits in the domains of Psychoticism and Detachment. A. Moderate or greater impairment in personality functioning, manifested by characteristic difficulties in two or more of the following four areas: 1. Identity: Confused boundaries between self and others; distorted seIf-concept; emotional expression often not congruent with context or internal experience. 2. Self-direction: Unrealistic or incoherent goals; no clear set of internal standards.

1	2. Self-direction: Unrealistic or incoherent goals; no clear set of internal standards. 3. Empathy: Pronounced difficulty understanding impact of own behaviors on others; frequent misinterpretations of others’ motivations and behaviors. 4. Intimacy: Marked impairments in developing close relationships, associated with mistrust and anxiety. B. Four or more of the following six pathological personality traits: 1. Cognitive and perceptual dysregulation (an aspect of Psychoticism): Odd or unusual thought processes; vague, circumstantial, metaphorical, overelaborate, or stereotyped thought or speech; odd sensations in various sensory modalities. 2. Unusual beliefs and experiences (an aspect of Psychoticism): Thought content and views of reality that are viewed by others as bizarre or idiosyncratic; unusual experiences of reality. 3. Eccentricity (an aspect of Psychoticism): Odd, unusual, or bizarre behavior or appearance; saying unusual or inappropriate things.

1	3. Eccentricity (an aspect of Psychoticism): Odd, unusual, or bizarre behavior or appearance; saying unusual or inappropriate things. 4. Restricted affectivity (an aspect of Detachment): Little reaction to emotionally or coldness. 5. Withdrawal (an aspect of Detachment): Preference for being alone to being with others; reticence in social situations; avoidance of social contacts and activity; lack of initiation of social contact. 6. Suspiciousness (an aspect of Detachment): Expectations ot—and heightened sensitivity to—signs of interpersonal iII-intent or harm; doubts about loyalty and fi- delity of others; feelings of persecution.

1	Specifiers. Trait and personality functioning specifiers may be used to record additional personality features that may be present in schizotypal personality disorder but are not re- quired for the diagnosis. For example, traits of Negative Affectivity (e.g., depressivity, anxiousness) are not diagnostic criteria for schizotypal personality disorder (see Criterion B) but can be specified when appropriate. Furthermore, although moderate or greater im- pairment in personality functioning is required for the diagnosis of schizotypal personal- ity disorder (Criterion A), the level of personality functioning can also be specified. A. Moderate or greater impairment in personality functioning, manifested by difficulties in two or more of the following four areas: 1. Identity 2. SeIf-direction 3. Empathy 4. Intimacy

1	A. Moderate or greater impairment in personality functioning, manifested by difficulties in two or more of the following four areas: 1. Identity 2. SeIf-direction 3. Empathy 4. Intimacy B. One or more pathological personality trait domains OR specific trait facets within do- mains, considering ALL of the following domains: 1. Negative Affectivity (vs. Emotional Stability): Frequent and intense experiences of high levels of a wide range of negative emotions (e.g., anxiety, depression, guilt/ shame, worry, anger), and their behavioral (e.g., self—harm) and interpersonal (e.g., dependency) manifestations. 2. Detachment'(vs. Extraversion): Avoidance of socioemotional experience, includ- ing both withdrawal from interpersonal interactions, ranging from casual, daily in- teractions to friendships to intimate relationships, as well as restricted affective experience and expression, particularly limited hedonic capacity.

1	3. Antagonism (vs. Agreeableness): Behaviors that put the individual at odds with other people, including an exaggerated sense of selt-importance and a concomi- tant expectation of special treatment, as well as a callous antipathy toward others, encompassing both unawareness of others' needs and feelings, and a readiness to use others in the service of selt-enhancement. 4. Disinhibition (vs. Conscientiousness): Orientation toward immediate gratification, leading to impulsive behavior driven by current thoughts, feelings, and external stimuli, without regard for past learning or consideration of future consequences. 5. Psychoticism (vs. Lucidity): Exhibiting a wide range of culturally incongruent odd, eccentric, or unusual behaviors and cognitions, including both process (e.g., per- ception, dissociation) and content (e.g., beliefs).

1	Subtypes. Because personality features vary continuously along multiple trait dimen- sions, a comprehensive set of potential expressions of PD-TS can be represented by DSM- 5’s dimensional model of maladaptive personality trait variants (see Table 3, pp. 779—781). Thus, subtypes are unnecessary for PD-TS, and instead, the descriptive elements that con- stitute personality are provided, arranged in an empirically based model. This arrange- ment allows clinicians to tailor the description of each individual’s personality disorder profile, considering all five broad domains of personality trait variation and drawing on the descriptive features of these domains as needed to characterize the individual.

1	Specifiers. The specific personality features of individuals are always recorded in eval- uating Criterion'B, so the combination of personality features characterizing an individual directly constitutes the specifiers in each case. For example, two individuals who are both characterized by emotional lability, hostility, and depressivity may differ such that the first individual is characterized additionally by callousness, whereas the second is not.

1	The requirement for any two of the tour A criteria for each of the six personality disorders was based on maximizing the relationship of these criteria to their corresponding person- ality disorder. Diagnostic thresholds for the B criteria were also set empirically to minimize change in prevalence of the disorders from DSM-IV and overlap with other personality disorders, and to maximize relationships with functional impairment. The resulting diag- nostic criteria sets represent clinically useful personality disorders with high fidelity, in terms of core impairments in personality functioning of varying degrees of severity and constellations of pathological personality traits.

1	Individuals who have a pattern of impairment in personality functioning and maladaptive traits that matches one of the six defined personality disorders should be diagnosed with that personality disorder. If an individual also has one or even several prominent traits that may have clinical relevance in addition to those required for the diagnosis (e.g., see narcis- sistic personality disorder), the option exists for these to be noted as specifiers. Individuals whose personality functioning or trait pattern is substantially different from that of any of the six specific personality disorders should be diagnosed with PD—TS. The individual may not meet the required number of A or B criteria and, thus, have a subthreshold presentation of a personality disorder. The individual may have a mix of features of personality disorder types or some features that are less characteristic of a type and more accurately considered a mixed or atypical presentation. The specific level of impairment in

1	of features of personality disorder types or some features that are less characteristic of a type and more accurately considered a mixed or atypical presentation. The specific level of impairment in personality function- ing and the pathological personality traits that characterize the individual’s personality can be specified for PD-TS, using the Level of Personality Functioning Scale (Table 2) and the pathological trait taxonomy (Table 3). The current diagnoses of paranoid, schizoid, histri- onic, and dependent personality disorders are represented also by the diagnosis of PD—TS; these are defined by moderate or greater impairment in personality functioning and can be specified by the relevant pathological personality trait combinations.

1	Level of Personality Functioning

1	Like most human tendencies, personality functioning is distributed on a continuum. Cen- tral to functioning and adaptation are individuals’ characteristic ways of thinking about and understanding themselves and their interactions with others. An optimally function- ing individual has a complex, fully elaborated, and well—integrated psychological world that includes a mostly positive, volitional, and adaptive self—concept; a rich, broad, and ap- propriately regulated emotional life; and the capacity to behave as a productive member of society with reciprocal and fulfilling interpersonal relationships. At the opposite end of the continuum, an individual with severe personality pathology has an impoverished, dis- organized, and/ or conﬂicted psychological world that includes a weak, unclear, and mal- adaptive self—concept; a propensity to negative, dysregulated emotions; and a deficient capacity for adaptive interpersonal functioning and social behavior.

1	Generalized severity may be the most important single predictor of concurrent and pro- spective dysfunction in assessing personality psychopathology. Personality disorders are optimally characterized by a generalized personality severity continuum with additional specification of stylistic elements, derived from personality disorder symptom constella- tions and personality traits. At the same time, the core of personality psychopathology is tion is consistent with multiple theories of personality disorder and their research bases. The components of the Level of Personality Functioning Scale—identity, self—direction, empa- thy, and intimacy (see Table 1)—are particularly central in describing a personality func- tioning continuum.

1	Mental representations of the self and interpersonal relationships are reciprocally in- ﬂuential and inextricably tied, affect the nature of interaction with mental health pro- fessionals, and can have a significant impact on both treatment efficacy and outcome, underscoring the importance of assessing an individual’s characteristic self—concept as well as views of other people and relationships. Although the degree of disturbance in the self and interpersonal functioning is continuously distributed, it is useful to consider the level of impairment in functioning for clinical characterization and for treatment planning and prognosis. Rating Levei of Personaiity Functioning

1	Rating Levei of Personaiity Functioning To use the Level of Personality Functioning Scale (LPFS), the clinician selects the level that most closely captures the individual’s current overall level of impairment in personality func- tioning. The rating is necessary for the diagnosis of a personality disorder (moderate or greater impairment) and can be used to specify the severity of impairment present for an individual with any personality disorder at a given point in time. The LPFS may also be used as a global indicator of personality functioning without specification of a personality disorder diagnosis, or in the event that personality impairment is subthreshold for a disorder diagnosis.

1	Criterion B in the alternative model involves assessments of personality traits that are grouped into five domains. A personality trait is a tendency to feel, perceive, behave, and think in relatively consistent ways across time and across situations in which the trait may manifest. For example, individuals with a high level of the personality trait of anxiousness would tend to feel anxious readily, including in circumstances in which most people would be calm and relaxed. Individuals high in trait anxiousness also would perceive sit- uations to be anxiety-provoking more frequently than would individuals with lower lev- els of this trait, and those high in the trait would tend to behave so as to avoid situations that they think would make them anxious. They would thereby tend to think about the world as more anxiety provoking than other people.

1	Importantly, individuals high in trait anxiousness would not necessarily be anxious at all times and in all situations. Individuals' trait levels also can and do change throughout life. Some changes are very general and reﬂect maturation (e.g., teenagers generally are higher on trait impulsivity than are older adults), whereas other changes reﬂect individ- uals’ life experiences.

1	Dimensionality of personality traits. All individuals can be located on the spectrum of trait dimensions; that is, personality traits apply to everyone in different degrees rather than being present versus absent. Moreover, personality traits, including those identified specifically in the Section 111 model, exist on a spectrum with two opposing poles. For ex- ample, the opposite of the trait of callousness is the tendency to be empathic and kind- hearted, even in circumstances in which most persons would not feel that way. Hence, al- though in Section 111 this trait is labeled callousness, because that pole of the dimension is the primary focus, it could be described in full as callousness versus kind-heartedness. More- over, its opposite pole can be recognized and may not be adaptive in all circumstances (e.g., individuals who, due to extreme kind-heartedness, repeatedly allow themselves to be taken advantage of by unscrupulous others).

1	Hierarchical structure of personality. Some trait terms are quite specific (e.g., ”talkative") and describe a narrow range of behaviors, whereas others are quite broad (e.g., Detach- ment) and Characterize a wide range of behavioral propensities. Broad trait dimensions are called domains, and specific trait dimensions are calledfacets. Personality trait domains comprise a spectrum of more specific personalityfacets that tend to occur together. For ex- ample, withdrawal and anhedonia are specific traitfacets in the trait domain of Detachment. Despite some cross-cultural variation in personality trait facets, the broad domains they collectively comprise are relatively consistent across cultures. The Personality Trait Model The Section III personality trait system includes five broad domains of personality trait variation—Negative Affectivity (vs. Emotional Stability), Detachment (vs. Extraversion),

1	The Section III personality trait system includes five broad domains of personality trait variation—Negative Affectivity (vs. Emotional Stability), Detachment (vs. Extraversion), Antagonism (vs. Agreeableness), Disinhibition (vs. Conscientiousness), and Psychoticism (vs. Lucidity)—comprising 25 specific personality trait facets. Table 3 provides definitions of all personality domains and facets. These five broad domains are maladaptive variants of the five domains of the extensively validated and replicated personality model known as the ”Big Five", or Five Factor Model of personality (FFM), and are also similar to the do- mains of the Personality Psychopathology Five (PSY-5). The specific 25 facets represent a list of personality facets chosen for their clinical relevance.

1	Although the Trait Model focuses on personality traits associated with psychopathol- ogy, there are healthy, adaptive, and resilient personality traits identified as the polar opposites of these traits, as noted in the parentheses above (i.e., Emotional Stability, Ex- traversion, Agreeableness, Conscientiousness, and Lucidity). Their presence can greatly mitigate the effects of mental disorders and facilitate coping and recovery from traumatic injuries and other medical illness. Distinguishing Traits, Symptoms, and Specific Behaviors

1	Distinguishing Traits, Symptoms, and Specific Behaviors Although traits are by no means immutable and do change throughout the life span, they show relative consistency compared with symptoms and specific behaviors. For example, a person may behave impulsively at a specific time for a specific reason (e.g., a person who is rarely impulsive suddenly decides to spend a great deal of money on a particular item because of an unusual opportunity to purchase something of unique value), but it is only when behaviors aggregate across time and circumstance, such that a pattern of behavior distinguishes between individuals, that they reﬂect traits. Nevertheless, it is important to recognize, for example, that even people who are impulsive are not acting impulsively all of the time. A trait is a tendency or disposition toward specific behaviors; a specific behav- ior is an instance or manifestation of a trait.

1	Similarly, traits are distinguished from most symptoms because symptoms tend to wax and wane, whereas traits are relatively more stable. For example, individuals with higher levels of depressivity have a greater likelihood of experiencing discrete episodes of a depressive disorder and of showing the symptoms of these disorders, such difficulty con- centrating. However, even patients who have a trait propensity to depressivity typically cy— cle through distinguishable episodes of mood disturbance, and specific symptoms such as difficulty concentrating tend to wax and wane in concert with specific episodes, so they do not form part of the trait definition. Importantly, however, symptoms and traits are both amenable to intervention, and many interventions targeted at symptoms can affect the longer term patterns of personality functioning that are captured by personality traits. Assessment of the DSM-5 Section III

1	Assessment of the DSM-5 Section III The clinical utility of the Section IH multidimensional personality trait model lies in its ability to focus attention on multiple relevant areas of personality variation in each individual patient. Rather than focusing attention on the identification of one and only one optimal diagnostic label, clinical application of the Section III personality trait model involves reviewing all five broad personality domains portrayed in Table 3. The clinical approach to personality is similar to the well-known review of systems in clinical medicine. For example, an individual’s pre- senting complaint may focus on a specific neurological symptom, yet during an initial evaluation clinicians still systematically review functioning in all relevant systems (e.g., car- diovascular, respiratory, gastrointestinal), lest an important area of diminished functioning and corresponding opportunity for effective intervention be missed.

1	Clinical use of the Section III personality trait model proceeds similarly. An initial in- quiry reviews all five broad domains of personality. This systematic review is facilitated by the use of formal psychometric instruments designed to measure specific facets and do- mains of personality. For example, the personality trait model is operationalized in the

1	Personality Inventory for DSM-S (PID-S), which can be completed in its self—report form by patients and in its informant-report form by those who know the patient well (e.g., a spouse). A detailed clinical assessment would involve collection of both patient- and in- formant—report data on all 25 facets of the personality trait model. However, if this is not possible, due to time or other constraints, assessment focused at the five-domain level is an acceptable clinical option when only a general (vs. detailed) portrait of a patient’s person- ality is needed (see Criterion B of PD—TS). However, if personality-based problems are the focus of treatment, then it will be important to assess individuals’ trait facets as well as do- mains.

1	Because personality traits are continuously distributed in the population, an approach to making the judgment that a specific trait is elevated (and therefore is present for diag- nostic purposes) could involve comparing individuals’ personality trait levels with pop— ulation norms and / or clinical judgment. If a trait is elevated—that is, formal psychometric testing and/ or interview data support the clinical judgment of elevation—then it is con- sidered as contributing to meeting Criterion B of Section III personality disorders. Clinical Utility of the Multidimensional Personality

1	Clinical Utility of the Multidimensional Personality Disorder and trait constructs each add value to the other in predicting important anteced- ent (e.g., family history, history of child abuse), concurrent (e.g., functional impairment, medication use), and predictive (e.g., hospitalization, suicide attempts) variables. DSM-S ute independently to clinical decisions about degree of disability; risks for self—harm, vio- lence, and criminality; recommended treatment type and intensity; and prognosis—all important aspects of the utility of psychiatric diagnoses. Notably, knowing the level of an the clinician with a rich base of information and is valuable in treatment planning and in predicting the course and outcome of many mental disorders in addition to personality disorders. Therefore, assessment of personality functioning and pathological personality traits may be relevant whether an individual has a personality disorder or not.

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1	Instability of emotional experiences and mood; emotions that are easily aroused, intense, and/ or out of proportion to events and cir- Feelings of nervousness, tenseness, or panic in reaction to diverse situa- tions; frequent worry about the negative effects of past unpleasant apprehensive about uncertainty; expecting the worst to happen. Fears of being alone due to rejection by—and/ or separation from— significant others, based in a lack of confidence in one’s ability to care for oneself, both physically and emotionally. Adaptation of one’s behavior to the actual or perceived interests and desires of others even when doing so is antithetical to one’s own interests, needs, or desires. to minor slights and insults; mean, nasty, or vengeful behavior. See also Antagonism.

1	to minor slights and insults; mean, nasty, or vengeful behavior. See also Antagonism. Persistence at tasks or in a particular way of doing things long after the behavior has ceased to be functional or effective; continuance of the same behavior despite repeated failures or clear reasons for stopping. See Detachment. See Detachment. The lack of this facet characterizes low levels of Negative Affectivity. See Detachment for definition of this facet. Avoidance of socioemotiona] experience, including both withdrawal from interpersonal interactions (ranging from casual, daily interac- tions to friendships to intimate relationships) and restricted affective experience and expression, particularly limited hedonic capacity. Preference for being alone to being with others; reticence in social sit- uations; avoidance of social contacts and activity; lack of initiation of social contact.

1	Preference for being alone to being with others; reticence in social sit- uations; avoidance of social contacts and activity; lack of initiation of social contact. Avoidance of close or romantic relationships, interpersonal attach- ments, and intimate sexual relationships. Lack of enjoyment from, engagement in, or energy for life’s experiences; deficits in the capacity to feel pleasure and take interest in things Feelings of being down, miserable, and / or hopeless; difficulty recov- ering from such moods; pessimism about the future; pervasive shame and/ or guilt; feelings of inferior self—worth; thoughts of sui- cide and suicidal behavior. Little reaction to emotionally arousing situations; constricted emo- matively engaging situations. Expectations of—and sensitivity to—signs of interpersonal ill- intent or harm; doubts about loyalty and fidelity of others; feelings of being mistreated, used, and / or persecuted by others.

1	Expectations of—and sensitivity to—signs of interpersonal ill- intent or harm; doubts about loyalty and fidelity of others; feelings of being mistreated, used, and / or persecuted by others. TABLE 3 Definitions of DSM-5 personality disorder trait domains ANTAGONISM (vs. (vs. Conscientiousness) Behaviors that put the individual at odds with other people, includ- ing an exaggerated sense of self-importance and a concomitant expectation of special treatment, as well as a callous antipathy toward others, encompassing both an unawareness of others’ needs and feelings and a readiness to use others in the service of self—enhancement. Use of subterfuge to inﬂuence or control others; use of seduction, charm, glibness, or ingratiation to achieve one’s ends. Dishonesty and fraudulence; misrepresentation of self; embellish- ment or fabrication when relating events.

1	Dishonesty and fraudulence; misrepresentation of self; embellish- ment or fabrication when relating events. Believing that one is superior to others and deserves special treat- ment; self—centeredness; feelings of entitlement; condescension toward others. Engaging in behavior designed to attract notice and to make oneself the focus of others’ attention and admiration. Lack of concern for the feelings or problems of others; lack of guilt or remorse about the negative or harmful effects of one’s actions on others. See Negative Affectivity. Orientation toward immediate gratification, leading to impulsive behavior driven by current thoughts, feelings, and external stim- uli, without regard for past learning or consideration of future consequences. Disregard for—and failure to honor—financial and other obliga- tions or commitments; lack of respect for—and lack of follow- through on—agreements and promises; carelessness with others’ property.

1	Disregard for—and failure to honor—financial and other obliga- tions or commitments; lack of respect for—and lack of follow- through on—agreements and promises; carelessness with others’ property. Acting on the spur of the moment in response to immediate stimuli; urgency and self—harming behavior under emotional distress. Difficulty concentrating and focusing on tasks; attention is easily focused behavior, including both planning and completing tasks. Engagement in dangerous, risky, and potentially self—damaging activities, unnecessarily and without regard to consequences; lack of concern for one’s limitations and denial of the reality of per- sonal danger; reckless pursuit of goals regardless of the level of risk involved.

1	Rigid insistence on everything being ﬂawless, perfect, and without errors or faults, including one’s own and others’ performance; sac- rificing of timeliness to ensure correctness in every detail; believ- ing that there is only one right way to do things; difficulty changing ideas and / or viewpoint; preoccupation with details, organization, and order. The lack of this facet characterizes low levels of Disinhibition. TABLE 3 Definitions of DSM-S personality disorder trait domains PSYCHOTICISM Exhibiting a wide range of culturally incongruent odd, eccentric, 01' (VS. Lucidity) unusual behaviors and cognitions, including both process (e.g., perception, dissociation) and content (e.g., beliefs). Unusual beliefs and Belief that one has unusual abilities, such as mind reading, telekine- experiences sis, thought-action fusion, unusual experiences of reality, includ- ing hallucination-like experiences.

1	Eccentricity Odd, unusual, or bizarre behavior, appearance, and/ or speech; inappropriate things. Cognitive and perceptual Odd or unusual thought processes and experiences, including depersonalization, derealization, and dissociative experiences; mixed sleep-wake state experiences; thought-control experiences. Proposed criterla sets are presented for conditions on which future research is en- couraged. The specific items, thresholds, and durations contained in these research crite- ria sets were set by expert consensus—informed by literature review, data reanalysis, and field trial results, where available—and are intended to provide a common language for researchers and clinicians who are interested in studying these disorders. It is hoped that such research will allow the field to better understand these conditions and will inform decisions about possible placement in forthcoming editions of DSM. The DSM-S Task

1	Force and Work Groups subjected each of these proposed criteria sets to a careful empir- ical review and invited wide commentary from the field as well as from the general public. The Task Force determined that there was insufficient evidence to warrant inclusion of these proposals as official mental disorder diagnoses in Section 11. These proposed criteria sets are not intended for clinical use; only the criteria sets and disorders in Section II of DSM—5 are officially recognized and can be used for clinical purposes. A. At least one of the following symptoms is present in attenuated form, with relatively in- tact reality testing, and is of sufficient severity or frequency to warrant clinical attention: 1. Delusions. 2. Hallucinations. 3. Disorganized speech. B. Symptom(s) must have been present at least once per week for the past month. C. Symptom(s) must have begun or worsened in the past year.

1	2. Hallucinations. 3. Disorganized speech. B. Symptom(s) must have been present at least once per week for the past month. C. Symptom(s) must have begun or worsened in the past year. D. Symptom(s) is sufficiently distressing and disabling to the individual to warrant clinical attention. E. Symptom(s) is not better explained by another mental disorder, including a depressive or bipolar disorder with psychotic features, and is not attributable to the physiological effects of a substance or another medical condition. F. Criteria for any psychotic disorder have never been met.

1	F. Criteria for any psychotic disorder have never been met. Attenuated psychotic symptoms, as defined in Criterion A, are psychosis—like but below the threshold for a full psychotic disorder. Compared with psychotic disorders, the symptoms are less severe and more transient, and insight is relatively maintained. A diagnosis of atten- uated psychosis syndrome requires state psychopathology associated with functional impairment rather than long-standing trait pathology. The psychopathology has not pro- gressed to full psychotic severity. Attenuated psychosis syndrome is a disorder based on the manifest pathology and impaired function and distress. Changes in experiences and behav- iors are noted by the individual and / or others, suggesting a change in mental state (i.e., the symptoms are of sufficient severity or frequency to warrant clinical attention) (Criterion A).

1	tent, including persecutory ideas of reference. The individual may have a guarded, distrust- ful attitude. When the delusions are moderate in severity, the individual views others as untrustworthy and may be hypervigilant or sense ill will in others. When the delusions are severe but still within the attenuated range, the individual entertains loosely organized be- liefs about danger or hostile intention, but the delusions do not have the fixed nature that is necessary for the diagnosis of a psychotic disorder. Guarded behavior in the interview can interfere with the ability to gather information. Reality testing and perspective can be elic- ited with nonconfirming evidence, but the propensity for viewing the world as hostile and dangerous remains strong. Attenuated delusions may have grandiose content presenting as an unrealistic sense of superior capacity. When the delusions are moderate, the individual harbors notions of being gifted, inﬂuential, or special. When the delusions are

1	content presenting as an unrealistic sense of superior capacity. When the delusions are moderate, the individual harbors notions of being gifted, inﬂuential, or special. When the delusions are severe, the in- dividual has beliefs of superiority that often alienate friends and worry relatives. Thoughts of being special may lead to unrealistic plans and investments, yet skepticism about these at- titudes can be elicited with persistent questioning and confrontation.

1	Attenuated hallucinations (Criterion A2) include alterations in sensory perceptions, usually auditory and/ or visual. When the hallucinations are moderate, the sounds and images are often unformed (e.g., shadows, trails, halos, murmurs, rumbling), and they are experienced as unusual or puzzling. When the hallucinations are severe, these experiences become more vivid and frequent (i.e., recurring illusions or hallucinations that capture at- tention and affect thinking and concentration). These perceptual abnormalities may dis— rupt behavior, but skepticism about their reality can still be induced.

1	Disorganized communication (Criterion A3) may manifest as odd speech (vague, meta- phorical, overelaborate, stereotyped), unfocused speech (confused, muddled, too fast or too slow, wrong words, irrelevant context, off track), or meandering speech (circumstantial, tan- gential). When the disorganization is moderately severe, the individual frequently gets into irrelevant topics but responds easily to clarifying questions. Speech may be odd but under- standable. At the moderately severe level, speech becomes meandering and circumstantial, and when the disorganization is severe, the individual fails to get to the point without external guidance (tangential). At the severe level, some thought blocking and / or loose as- sociations may occur infrequently, especially when the individual is under pressure, but re- orienting questions quickly return structure and organization to the conversation.

1	The individual realizes that changes in mental state and / or in relationships are taking place. He or she maintains reasonable insight into the psychotic-like experiences and gen— erally appreciates that altered perceptions are not real and magical ideation is not compel- ling. The individual must experience distress and/ or impaired performance in social or role functioning (Criterion D), and the individual or responsible others must note the changes and express concern, such that clinical care is sought (Criterion A).

1	The individual may experience magical thinking, perceptual aberrations, difficulty in con- centration, some disorganization in thought or behavior, excessive suspiciousness, anxi- ety, social withdrawal, and disruption in sleep-wake cycle. Impaired cognitive function and negative symptoms are often observed. Neuroimaging variables distinguish cohorts with attenuated psychosis syndrome from normal control cohorts with patterns similar to, but less severe than, that observed in schizophrenia. However, neuroimaging data is not diagnostic at the individual level. The prevalence of attenuated psychosis syndrome is unknown. Symptoms in Criterion A are not uncommon in the non-help-seeking population, ranging from 23%—13% for hallu- cinatory experiences and delusional thinking. There appears to be a slight male prepon- derance for attenuated psychosis syndrome.

1	Onset of attenuated psychosis syndrome is usually in mid-to-late adolescence or early adulthood. It may be preceded by normal development or evidence for impaired cogni- tion, negative symptoms, and/ or impaired social development. In help-seeking cohorts, criteria for a psychotic disorder. In some cases, the syndrome may transition to a depres- sive or bipolar disorder with psychotic features, but development to a schizophrenia spec- trum disorder is more frequent. It appears that the diagnosis is best applied to individuals ages 15—35 years. Long—term course is not yet described beyond 7—12 years. Temperamental. Factors predicting prognosis of attenuated psychosis syndrome have not been definitively characterized, but the presence of negative symptoms, cognitive im- pairment, and poor functioning are associated with poor outcome and increase risk of transition to psychosis.

1	Genetic and physiological. A family history of psychosis places the individual with at- tenuated psychosis syndrome at increased risk for developing a full psychotic disorder. Structural, functional, and neurochemical imaging data are associated with increased risk of transition to psychosis. Many individuals may experience functional impairments. Modest-to-moderate impair— ment in social and role functioning may persist even with abatement of symptoms. A sub- stantial portion of individuals with the diagnosis will improve over time; many continue to have mild symptoms and impairment, and many others will have a full recovery. Brief psychotic disorder. When symptoms of attenuated psychosis syndrome initially manifest, they may resemble symptoms of brief psychotic disorder. However, in attenu- ated psychosis syndrome, the symptoms do not cross the psychosis threshold and reality testing/insight remains intact.

1	Schizotypal personality disorder. Schizotypal personality disorder, although having symptomatic features that are similar to those of attenuated psychosis syndrome, is a rel- atively stable trait disorder not meeting the state-dependent aspects (Criterion C) of atten- uated psychosis syndrome. In addition, a broader array of symptoms is required for schizotypal personality disorder, although in the early stages of presentation it may re- semble attenuated psychosis syndrome. Depressive or bipolar disorders. Reality distortions that are temporally limited to an episode of a major depressive disorder or bipolar disorder and are descriptively more characteristic of those disorders do not meet Criterion E for attenuated psychosis syn- drome. For example, feelings of low self—esteem or attributions of low regard from others in the context of major depressive disorder would not qualify for comorbid attenuated psychosis syndrome.

1	Anxiety disorders. Reality distortions that are temporally limited to an episode of an anxiety disorder and are descriptively more characteristic of an anxiety disorder do not meet Criterion E for attenuated psychosis syndrome. For example, a feeling of being the focus of undesired attention in the context of social anXiety disorder would not qualify for comorbid attenuated psychosis syndrome. Bipolar || disorder. Reality distortions that are temporally limited to an episode of ma- nia or hypomania and are descriptively more characteristic of bipolar disorder do not meet Criterion E for attenuated psychosis syndrome. For example, inflated self—esteem in the context of pressured speech and reduced need for sleep would not qualify for comorbid at- tenuated psychosis syndrome.

1	Borderline personality disorder. Reality distortions that are concomitant with border- line personality disorder and are descriptively more characteristic of it do not meet Crite- rion E for attenuated psychosis syndrome. For example, a sense of being unable to experience feelings in the context of an intense fear of real or imagined abandonment and recurrent self—mutilation would not qualify for comorbid attenuated psychosis syndrome. Adjustment reaction of adolescence. Mild, transient symptoms typical of normal de- velopment and consistent with the degree of stress experienced do not qualify for attenu- ated psychosis syndrome. Extreme end of perceptual aberration and magical thinking in the non-ill population. This diagnostic possibility should be strongly entertained when reality distortions are not associated with distress and functional impairment and need for care.

1	This diagnostic possibility should be strongly entertained when reality distortions are not associated with distress and functional impairment and need for care. Substance/medication-induced psychotic disorder. Substance use is common among individuals whose symptoms meet attenuated psychosis syndrome criteria. When other- wise qualifying characteristic symptoms are strongly temporally related to substance use episodes, Criterion E for attenuated psychosis syndrome may not be met, and a diagnosis Attention-deficit/hyperactivity disorder. A history of attentional impairment does not exclude a Current attenuated psychosis syndrome diagnosis. Earlier attentional impair- ment may be a prodromal condition or comorbid attention-deficit/hyperactivity disorder.

1	Individuals with attenuated psychosis syndrome often experience anxiety and/ or depres— sion. Some individuals with an attenuated psychosis syndrome diagnosis will progress to another diagnosis, including anxiety, depressive, bipolar, and personality disorders. In such cases, the psychopathology associated with the attenuated psychosis syndrome diagnosis is reconceptualized as the prodromal phase of another disorder, not a comorbid condition. Lifetime experience of at least one major depressive episode meetlng the following criteria:

1	Lifetime experience of at least one major depressive episode meetlng the following criteria: A. Five (or more) of the following criteria have been present during the same 2-week pe- riod and represent a change from previous functioning; at least one of the symptoms is either (1) depressed mood or (2) loss of interest or pleasure. (Note: Do not include symptoms that are clearly attributable to a medical condition.) 1. Depressed mood most of the day, nearly every day. as indicated by either subjec- tive report (e.g., feels sad, empty, or hopeless) or observation made by others (e.g., appears tearful). (Note: In children and adolescents, can be irritable mood.) 2. Markedly diminished interest or pleasure in all, or almost all, activities most of the day, nearly every day (as indicated by either subjective account or observation).

1	3. Significant weight loss when not dieting or weight gain (e.g., a change of more than 5% of body weight in a month), or decrease or increase in appetite nearly every day. (Note: In children, consider failure to make expected weight gain.) 4. Insomnia or hypersomnia nearly every day. 5. Psychomotor agitation or retardation nearly every day (observable by others, not merely subjective feelings of restlessness or being slowed down). 6. Fatigue or loss of energy nearly every day. 7. Feelings of worthlessness or excessive or inappropriate guilt (which may be delu- sional) nearly every day (not merely self-reproach or guilt about being sick). 8. Diminished ability to think or concentrate, or indecisiveness, nearly every day (ei- ther by subjective account or as observed by others). 9. Recurrent thoughts of death (not just fear of dying), recurrent suicidal ideation with- out a specific plan, or a suicide attempt or a specific plan for committing suicide.

1	9. Recurrent thoughts of death (not just fear of dying), recurrent suicidal ideation with- out a specific plan, or a suicide attempt or a specific plan for committing suicide. . The symptoms cause clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. . The disturbance is not attributable to the physiological effects of a substance or an- other medical condition. . The disturbance is not better explained by schizoaffective disorder and is not superim- posed on schizophrenia, schizophreniform disorder, delusional disorder, or other spec- ified or unspecified schizophrenia spectrum and other psychotic disorder. At least two lifetime episodes of hypomanic periods that involve the required crite- rion symptoms below but are of insufficient duration (at least 2 days but less than 4 consecutive days) to meet criteria for a hypomanic episode. The criterion symp- toms are as follows: A. B.

1	A. B. A distinct period of abnormally and persistently elevated, expansive, or irritable mood and abnormally and persistently increased goaI-directed activity or energy. During the period of mood disturbance and increased energy and activity, three (or more) of the following symptoms have persisted (four if the mood is only irritable), represent a no- ticeable change from usual behavior, and have been present to a significant degree: Inﬂated seIt-esteem or grandiosity. Decreased need for sleep (e.g., feels rested after only 3 hours of sleep). More talkative than usual or pressured to keep talking. Flight of ideas or subjective experience that thoughts are racing. Distractibility (i.e., attention too easily drawn to unimportant or irrelevant external stimuli), as reported or observed. Increase in goal-directed activity (either socially, at work or school, or sexually) or psychomotor agitation.

1	Increase in goal-directed activity (either socially, at work or school, or sexually) or psychomotor agitation. 7. Excessive involvement in activities that have a high potential for painful conse- quences (e.g., the individual engages in unrestrained buying sprees, sexual indis- cretions, or foolish business investments). . The episode is associated with an unequivocal change in functioning that is uncharac- teristic of the individual when not symptomatic. . The disturbance in mood and the change in functioning are observable by others. The episode is not severe enough to cause marked impairment in social or occupa- tional functioning or to necessitate hospitalization. If there are psychotic features, the episode is, by definition, manic. The episode is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication or other treatment).

1	The episode is not attributable to the physiological effects of a substance (e.g., a drug of abuse, a medication or other treatment). Individuals with short-duration hypomania have experienced at least one major depres- sive episode as well as at least two episodes of 2—3 days’ duration in which criteria for a hy- pomanic episode were met (except for symptom duration). These episodes are of sufficient intensity to be categorized as a hypomanic episode but do not meet the 4-day duration re- quirement. Symptoms are present to a significant degree, such that they represent a no— ticeable change from the individual’s normal behavior. An individual with a history of a syndromal hypomanic episode and a major depres- sive episode by definition has bipolar II disorder, regardless of current duration of hypo- manic symptoms.

1	An individual with a history of a syndromal hypomanic episode and a major depres- sive episode by definition has bipolar II disorder, regardless of current duration of hypo- manic symptoms. sive episode, with their increased comorbidity with substance use disorders and a greater family history of bipolar disorder, more closely resemble individuals with bipolar disor- der than those with major depressive disorder. Differences have also been found between individuals with short-duration hypomania and those with syndromal bipolar disorder. Work impairment was greater for individuals with syndromal bipolar disorder, as was the estimated average number of episodes. Indi- viduals with short-duration hypomania may exhibit less severity than individuals with syndromal hypomanic episodes, including less mood lability.

1	The prevalence of short-duration hypomania is unclear, since the criteria are new as of this edition of the manual. Using somewhat different criteria, however, it has been estimated that short-duration hypomania occurs in 2.8% of the population (compared with hypoma- nia or mania in 5.5% of the population). Short-duration hypomania may be more common in females, who may present with more features of atypical depression. Genetic and physiological. A family history of mania is two to three times more common in individuals with short-duration hypomania compared with the general population, but less than half as common as in individuals with a history of syndromal mania'or hypomania. Individuals with short-duration hypomania have higher rates of suicide attempts than healthy individuals, although not as high as the rates in individuals with syndromal bipo- lar disorder. Functional Consequences of Short-Duration Hypomania

1	Functional Consequences of Short-Duration Hypomania Functional impairments associated specifically with short-duration hypomania are as yet not fully determined. However, research suggests that individuals with this disorder have less work impairment than individuals with syndromal bipolar disorder but more comor- bid substance use disorders, particularly alcohol use disorder, than individuals with major depressive disorder. Bipolar II disorder. Bipolar II disorder is characterized by a period of at least 4 days of hypomanic symptoms, whereas short—duration hypomania is characterized by periods of 2—3 days of hypomanic symptoms. Once an individual has experienced a hypomanic ep- isode (4 days or; more), the diagnosis becomes and remains bipolar II disorder regardless of future duration of hypomanic symptom periods.

1	Major depressive disorder. Major depressive disorder is also Characterized by at least one lifetime major depressive episode. However, the additional presence of at least two life— time periods of 2—3 days of hypomanic symptoms leads to a diagnosis of short-duration hy- pomania rather than to major depressive disorder. Major depressive disorder with mixed features. Both major depressive disorder with mixed features and short-duration hypomania are characterized by the presence of some hypomanic symptoms and a major depressive episode. However, major depressive disor- der with mixed features is characterized by hypomanic features present concurrently with a major depressive episode, while individuals with short—duration hwomania experience subsyndromal hypomania and fully syndromal major depression at different times.

1	Bipolar I disorder. Bipolar I disorder is differentiated from short-duration hypomania by at least one lifetime manic episode, which is longer (at least 1 week) and more severe (causes more impaired social functioning) than a hypomanic episode. An episode (of any duration) that involves psychotic symptoms or necessitates hospitalization is by definition a manic episode rather than a hypomanic one. Cyclothymic disorder. While cyclothymic disorder is characterized by periods of de- pressive symptoms and periods of hypomanic symptoms, the lifetime presence of a major depressive episode precludes the diagnosis of cyclothymic disorder. Short-duration hypomania, similar to full hypomanic episodes, has been associated with higher rates of comorbid anxiety disorders and substance use disorders than are found in the general population. A. The individual experienced the death of someone with whom he or she had a close re- lationship.

1	A. The individual experienced the death of someone with whom he or she had a close re- lationship. B. Since the death, at least one of the following symptoms is experienced on more days than not and to a clinically significant degree and has persisted for at least 12 months after the death in the case of bereaved adults and 6 months for bereaved children: 1. Persistent yearning/Ionging for the deceased. In young children, yearning may be expressed in play and behavior, including behaviors that reflect being separated from, and also reuniting with, a caregiver or other attachment figure. 2. Intense sorrow and emotional pain in response to the death. Preoccupation with the deceased. 4. Preoccupation with the circumstances of the death. In children, this preoccupation with the deceased may be expressed through the themes of play and behavior and may extend to preoccupation with possible death of others close to them. .03

1	.03 C. Since the death, at least six of the following symptoms are experienced on more days than not and to a clinically significant degree, and have persisted for at least 12 months after the death in the case of bereaved adults and 6 months for bereaved children: Reactive distress to the death 1. Marked difficulty accepting the death. In children, this is dependent on the child’s capacity to comprehend the meaning and permanence of death. Experiencing disbelief or emotional numbness over the loss. Difficulty with positive reminiscing about the deceased. Bitterness or anger related to the loss. Maladaptive appraisals about oneself in relation to the deceased or the death (e.g., self-blame). Excessive avoidance of reminders of the loss (e.g., avoidance of individuals, places, or situations associated with the deceased; in children, this may include avoidance of thoughts and feelings regarding the deceased).

1	."":“S’--3!\J .07 7. A desire to die in order to be with the deceased. 8. Difficulty trusting other individuals since the death. 9. Feeling alone or detached from other individuals since the death. 10. Feeling that life is meaningless or empty without the deceased, or the belief that one cannot function without the deceased. 11. Confusion about one’s role in life, or a diminished sense of one’s identity (e.g., ieel- ing that a part of oneself died with the deceased). 12. Difficulty or reluctance to pursue interests since the loss or to plan for the future (e.g., friendships, activities). D. The disturbance causes clinically significant distress or impairment in social, occupa- tional, or other important areas of functioning. E. The bereavement reaction is out of proportion to or inconsistent with cultural, religious, or age-appropriate norms. Specify it:

1	E. The bereavement reaction is out of proportion to or inconsistent with cultural, religious, or age-appropriate norms. Specify it: With traumatic bereavement: Bereavement due to homicide or suicide with persis- tent distressing preoccupations regarding the traumatic nature of the death (often in re- sponse to loss reminders), including the deceased’s last moments, degree of suffering and mutilating injury, or the malicious or intentional nature of the death.

1	Persistent complex bereavement disorder is diagnosed only if at least 12 months (6 months in children) have elapsed since the death of someone with whom the bereaved had a close relationship (Criterion A). This time frame discriminates normal grief from persistent grief. The condition typically involves a persistent yearning/longing for the deceased (Criterion Bl), which may be associated with intense sorrow and frequent crying (Crite- rion 82) or preoccupation with the deceased (Criterion B3). The individual may also be preoccupied with the manner in which the person died (Criterion B4).

1	Six additional symptoms are required, including marked difficulty accepting that the in- dividual has died (Criterion C1) (e.g. preparing meals for them), disbelief that the individual is dead (Criterion C2), distressing memories of the deceased (Criterion C3), anger over the loss (Criterion C4), maladaptive appraisals about oneself in relation to the deceased or the death (Criterion C5), and excessive avoidance of reminders of the loss (Criterion C6). Individuals may also report a desire to die because they wish to be with the deceased (Criterion C7); be dis- trustful of others (Criterion C8); feel isolated (Criterion C9); believe that life has no meaning or purpose without the deceased (Criterion C10); experience a diminished sense of identity in which they feel a part of themselves has died or been lost (Criterion C11); or have difficulty en- gaging in activities, pursuing relationships, or planning for the future (Criterion C12).

1	pairment in psychosocial functioning (Criterion D). The nature and severity of grief must be beyond expected norms for the relevant cultural setting, religious group, or develop- mental stage (Criterion E). Although there are variations in how grief can manifest, the symptoms of persistent complex bereavement disorder occur in both genders and in di- verse social and cultural groups. Some individuals with persistent complex bereavement disorder experience hallucina- tions of the deceased (auditory or visual) in which they temporarily perceive the deceased’s presence (e.g., seeing the deceased sitting in his or her favorite chair). They may also ex- perience diverse somatic complaints (e.g., digestive complaints, pain, fatigue), including symptoms experienced by the deceased. The prevalence of persistent complex bereavement disorder is approximately 2.4%—4.8%. The disorder is more prevalent in females than in males.

1	The prevalence of persistent complex bereavement disorder is approximately 2.4%—4.8%. The disorder is more prevalent in females than in males. Persistent complex bereavement disorder can occur at any age, beginning after the age of 1 year. Symptoms usually begin within the initial months after the death, although there may be a delay of months, or even years, before the full syndrome appears. Although grief responses commonly appear immediately following bereavement, these reactions are not diagnosed as persistent complex bereavement disorder unless the symptoms persist be- yond 12 months (6 months for children).

1	Young children may experience the loss of a primary caregiver as traumatic, given the disorganizing effects the caregiver’s absence can have on a child’s coping response. In Chil- dren, the distress may be expressed in play and behavior, developmental regressions, and anxious or protest behavior at times of separation and reunion. Separation distress may be predominant in younger children, and social/identity distress and risk for comorbid de— pression can increasingly manifest in older children and adolescents. Environmental. Risk for persistent complex bereavement disorder is heightened by in- creased dependency on the deceased person prior to the death and by the death of a child. Disturbances in caregiver support increase the risk for bereaved children. Genetic and physiological. Risk for the disorder is heightened by the bereaved individ- ual being female.

1	Disturbances in caregiver support increase the risk for bereaved children. Genetic and physiological. Risk for the disorder is heightened by the bereaved individ- ual being female. The symptoms of persistent complex bereavement disorder are observed across cultural settings, but grief responses may manifest in culturally specific ways. Diagnosis of the dis- order requires that the persistent and severe responses go beyond cultural norms of grief responses and not be better explained by culturally specific mourning rituals. Individuals with persistent complex bereavement disorder frequently report suicidal ideation. Persistent complex bereavement disorder is associated with deficits in work and social func- tioning and with harmful health behaviors, such as increased tobacco and alcohol use. It is also associated with marked increases in risks for serious medical conditions, including cardiac dis- ease, hypertension, cancer, immunological deficiency, and reduced quality of life.

1	Normal grief. Persistent complex bereavement disorder is distinguished from normal grief by the presence of severe grief reactions that persist at least 12 months (or 6 months in children) after the death of the bereaved. It is only when severe levels of grief response per- sist at least 12 months following the death and interfere with the individual’s capacity to function that persistent complex bereavement disorder is diagnosed. Depressive disorders. Persistent complex bereavement disorder, major depressive dis- order, and persistent depressive disorder (dysthymia) share sadness, crying, and suicidal thinking. Whereas major depressive disorder and persistent depressive disorder can share depressed mood with persistent complex bereavement disorder, the latter is characterized by a focus on the loss. Posttraumatic stress disorder. Individuals who experience bereavement as a result of trau- bereavement disorder. Both conditions can involve intrusive thoughts and avoidance.

1	Posttraumatic stress disorder. Individuals who experience bereavement as a result of trau- bereavement disorder. Both conditions can involve intrusive thoughts and avoidance. Whereas intrusions in PTSD revolve around the traumatic event, intrusive memories in per- sistent complex bereavement disorder focus on thoughts about many aspects of the relation- ship with the deceased, including positive aspects of the relationship and distress over the separation. In individuals with the traumatic bereavement specifier of persistent complex be- reavement disorder, the distressing thoughts or feelings may be more overtly related to the manner of death, with distressing fantasies of what happened. Both persistent complex be- reavement disorder and PTSD can involve avoidance of reminders of distressing events.

1	Whereas avoidance in PTSD is characterized by consistent avoidance of internal and external reminders of the traumatic experience, in persistent complex bereavement disorder, there is also a preoccupation with the loss and yeaming for the deceased, which is absent in PTSD. Separation anxiety disorder. Separation anxiety disorder is characterized by anxiety about separation from current attachment figures, whereas persistent complex bereavement disorder involves distress about separation from a deceased individual. The most common comorbid disorders with persistent complex bereavement disorder are major depressive disorder, PTSD, and substance use disorders. PTSD is more frequently comorbid with persistent complex bereavement disorder when the death occurred in trau- matic or violent circumstances.

1	A problematic pattern of caffeine use leading to clinically significant impairment or distress, as manifested by at least the first three of the following criteria occurring within a 12-month period: 1. A persistent desire or unsuccessful efforts to cut down or control caffeine use. 2. Continued caffeine use despite knowledge of having a persistent or recurrent physical or psychological problem that is likely to have been caused or exacerbated by caffeine. 3. Withdrawal, as manifested by either of the following: a. The characteristic withdrawal syndrome ior caffeine. b. Caffeine (or a closely related) substance is taken to relieve or avoid withdrawal symptoms. 4. Caffeine is often taken in larger amounts or over a longer period than was intended. 5. Recurrent caffeine use resulting in a failure to fulfill major role obligations at work, school, or home (e.g., repeated tardiness or absences from work or school related to caffeine use or withdrawal).

1	6. Continued caffeine use despite having persistent or recurrent social or interpersonal problems caused or exacerbated by the effects of caffeine (e.g., arguments with spouse about consequences of use, medical problems, cost). 7. Tolerance, as defined by either of the following: a. A need for markedly increased amounts of caffeine to achieve desired effect. b. Markedly diminished effect with continued use of the same amount of caffeine. 8. A great deal of time is spent in activities necessary to obtain caffeine, use caffeine, or recover from its effects. 9. Craving or a strong desire or urge to use caffeine. A diagnosis of substance dependence due to caffeine is recognized by the World Health

1	Organization in ICD-10. Since the publication of DSM-IV in 1994, considerable research on caffeine dependence has been published, and several recent reviews provide a current analysis of this literature. There is now sufficient evidence to warrant inclusion of caffeine use disorder as a research diagnosis in DSM-S to encourage additional research. The work- ing diagnostic algorithm proposed for the study of caffeine use disorder differs from that of the other substance use disorders, reﬂecting the need to identify only cases that have sufficient clinical importance to warrant the labeling of a mental disorder. A key goal of in- cluding caffeine use disorder in this section of DSM-S is to stimulate research that will determine the reliability, validity, and prevalence of caffeine use disorder based on the proposed diagnostic schema, with particular attention to the association of the diagnosis with functional impairments as part of validity testing.

1	The proposed criteria for caffeine use disorder reﬂect the need for a diagnostic thresh- old higher than that used for the other substance use disorders. Such a threshold is in- tended to prevent overdiagnosis of caffeine use disorder due to the high rate of habitual nonproblematic daily caffeine use in the general population.

1	Caffeine use disorder is characterized by the continued use of caffeine and failure to con- trol use despite negative physical and / or psychological consequences. In a survey of the general population, 14% of caffeine users met the criterion of use despite harm, with most reporting that a physician or counselor had advised them to stop or reduce caffeine use within the last year. Medical and psychological problems attributed to caffeine included heart, stomach, and urinary problems, and complaints of anxiety, depression, insomnia, irritability, and difficulty thinking. In the same survey, 45% of caffeine users reported de- sire or unsuccessful efforts to control caffeine use, 18% reported withdrawal, 8% reported tolerance, 28% used more than intended, and 50% reported spending a great deal of time using caffeine. In addition, 19% reported a strong desire for caffeine that they could not re- sist, and less than 1% reported that caffeine had interfered with social activities.

1	Among those seeking treatment for quitting problematic caffeine use, 88% reported having made prior serious attempts to modify caffeine use, and 43% reported having been advised by a medical professional to reduce or eliminate caffeine. Ninety—three percent endorsed signs and symptoms meeting DSM-IV criteria for caffeine dependence, with the most commonly endorsed criteria being withdrawal (96%), persistent desire or unsuccess- ful efforts to control use (89%), and use despite knowledge of physical or psychological problems caused by caffeine (87%). The most common reasons for wanting to modify caf— feine use Were health-related (59%) and a desire to not be dependent on caffeine (35%). The DSM-S discussion of caffeine withdrawal in the Section 11 chapter ”Substance-

1	The DSM-S discussion of caffeine withdrawal in the Section 11 chapter ”Substance- Related and Addictive Disorders” provides information on the features of the withdrawal criterion. It is well documented that habitual caffeine users can experience a well-defined withdrawal syndrome upon acute abstinence from caffeine, and many caffeine-dependent individuals report continued use of caffeine to avoid experiencing withdrawal symptoms.

1	The prevalence of caffeine use disorder in the general population is unclear. Based on all seven generic DSM-IV-TR criteria for dependence, 30% of current caffeine users may have met DSM-IV criteria for a diagnosis of caffeine dependence, with endorsement of three or more dependence criteria, during the past year. When only four of the seven criteria (the three primary criteria proposed above plus tolerance) are used, the prevalence appears to drop to 9%. Thus, the expected prevalence of caffeine use disorder among regular caffeine users is likely less than 9%. Given that approximately 75%—80% of the general population uses caffeine regularly, the estimated prevalence would be less than 7%. Among regular caffeine drinkers at higher risk for caffeine use problems (e.g., high school and college stu- dents, individuals in drug treatment, and individuals at pain clinics who have recent his- tories of alcohol or illicit drug misuse), approximately 20% may have a pattern of use that meets

1	stu- dents, individuals in drug treatment, and individuals at pain clinics who have recent his- tories of alcohol or illicit drug misuse), approximately 20% may have a pattern of use that meets all three of the proposed criteria in Criterion A.

1	Individuals whose pattern of use meets criteria for a caffeine use disorder have shown a wide range of daily caffeine intake and have been consumers of various types of caffein- ated products (e.g., coffee, soft drinks, tea) and medications. A diagnosis of caffeine use disorder has been shown to prospectively predict a greater incidence of caffeine reinforce- ment and more severe withdrawal. order. Caffeine use disorder has been identified in both adolescents and adults. Rates of caffeine consumption and overall level of caffeine consumption tend to increase with age until the early to mid—30s and then level off. Age—related factors for caffeine use disorder are unknown, although concern is growing related to excessive caffeine consumption among adolescents and young adults through use of caffeinated energy drinks.

1	Genetic and physiological. Heritabilities of heavy caffeine use, caffeine tolerance, and caffeine withdrawal range from 35% to 77%. For caffeine use, alcohol use, and cigarette smoking, a common genetic factor (polysubstance use) underlies the use of these three substances, with 28%—41% of the heritable effects of caffeine use (or heavy use) shared with alcohol and smoking. Caffeine and tobacco use disorders are associated and substan— tially inﬂuenced by genetic factors unique to these licit drugs. The magnitude of heritabil- ity for caffeine use disorder markers appears to be similar to that for alcohol and tobacco use disorder markers. Functional Consequences of Caffeine Use Disorder

1	Functional Consequences of Caffeine Use Disorder Caffeine use disorder may predict greater use of caffeine during pregnancy. Caffeine with- drawal, a key feature of caffeine use disorder, has been shown to produce functional im- pairment in normal daily activities. Caffeine intoxication may include symptoms of nausea and vomiting, as well as impairment of normal activities. Significant disruptions in normal daily activities may occur during caffeine abstinence. Nonproblematic use of caffeine. The distinction between nonproblematic use of caf— feine and caffeine use disorder can be difficult to make because social, behavioral, or psy- chological problems may be difficult to attribute to the substance, especially in the context of use of other substances. Regular, heavy caffeine use that can result in tolerance and with- drawal is relatively common, which by itself should not be sufficient for making a diagnosis.

1	Other stimulant use disorder. Problems related to use of other stimulant medications or substances may approximate the features of caffeine use disorder. Anxiety disorders. Chronic heavy caffeine use may mimic generalized anxiety disorder, and acute caffeine consumption may produce and mimic panic attacks. There may be comorbidity between caffeine use disorder and daily cigarette smoking, a family or personal history of alcohol use disorder. Features of caffeine use disorder (e.g., tolerance, caffeine withdrawal) may be positively associated with several diagnoses: ma— jor depression, generalized anxiety disorder, panic disorder, adult antisocial personality disorder, and alcohol, cannabis, and cocaine use disorders.

1	Persistent and recurrent use of the Internet to engage in games, often with other players, leading to clinically significant impairment or distress as indicated by five (or more) of the following in a 12-month period: 1. Preoccupation with Internet games. (The individual thinks about previous gaming activity or anticipates playing the next game; Internet gaming becomes the dominant activity in daily life). Note: This disorder is distinct from Internet gambling, which is included under gam- bling disorder. 2. Withdrawal symptoms when Internet gaming is taken away. (These symptoms are typ- ically described as irritability, anxiety, or sadness, but there are no physical signs of pharmacological withdrawal.) 3. Tolerance—the need to spend increasing amounts of time engaged in Internet games. Unsuccessful attempts to control the participation in Internet games. 5. Loss of interests in previous hobbies and entertainment as a result of, and with the ex- ception of. Internet games.

1	Unsuccessful attempts to control the participation in Internet games. 5. Loss of interests in previous hobbies and entertainment as a result of, and with the ex- ception of. Internet games. Continued excessive use of Internet games despite knowledge of psychosocial problems. 7. Has deceived family members, therapists, or others regarding the amount of Internet gaming. 8. Use of Internet games to escape or relieve a negative mood (e.g., feelings of helpless- ness, guilt, anxiety). 9. Has jeopardized or lost a significant relationship, job. or educational or career oppor- tunity because of participation in Internet games. .0’ Note: Only nongambling Internet games are included in this disorder. Use of the Internet for required activities in a business or profession is not included; nor is the disorder intend- ed to include other recreational or social Internet use. Similarly, sexual Internet sites are excluded. Specify current severity:

1	Specify current severity: Internet gaming disorder can be mild, moderate, or severe depending on the degree of disruption of normal activities. Individuals with less severe Internet gaming disorder may exhibit fewer symptoms and less disruption of their lives. Those with severe Inter- net gaming disorder will have more hours spent on the computer and more severe loss of relationships or career or school opportunities. There are no well-researched subtypes for Internet gaming disorder to date. Internet gam- ing disorder most often involves specific Internet games, but it could involve non-Internet computerized games as well, although these have been less researched. It is likely that pre- ferred games will vary over time as new games are developed and popularized, and it is unclear if behaviors and consequence associated with Internet gaming disorder vary by game type.

1	Gambling disorder is currently the only non-substance-related disorder proposed for in- clusion with DSM-S substance—related and addictive disorders. However, there are other behavioral disorders that show some similarities to substance use disorders and gambling disorder for which the word addiction is commonly used in nonmedical settings, and the one condition with a considerable literature is the compulsive playing of Intemet games. Internet gaming has been reportedly defined as an ”addiction” by the Chinese govern- ment, and a treatment system has been set up. Reports of treatment of this condition have appeared in medical journals, mostly from Asian countries and some in the United States.

1	The DSM-5 work group reviewed more than 240 articles and found some behavioral similarities of Internet gaming to gambling disorder and to substance use disorders. The literature suffers, however, from lack of a standard definition from which to derive prev- alence data. An understanding of the natural histories of cases, with or without treatment, is also missing. The literature does describe many underlying similarities to substance ad- dictions, including aspects of tolerance, withdrawal, repeated unsuccessful attempts to cut back or quit, and impairment in normal functioning. Further, the seemingly high preva- lence rates, both in Asian countries and, to a lesser extent, in the West, justified inclusion of this disorder in Section III of DSM-S.

1	Internet gaming disorder has significant public health importance, and additional re- search may eventually lead to evidence that Internet gaming disorder (also commonly re- ferred to as Internet use disorder, Internet addiction, or gaming addiction) has merit as an independent disorder. As with gambling disorder, there should be epidemiological stud- ies to determine prevalence, clinical course, possible genetic inﬂuence, and potential bio- logical factors based on, for example, brain imaging data.

1	Internet gaming disorder is a pattern of excessive and prolonged Internet gaming that re- sults in a cluster of cognitive and behavioral symptoms, including progressive loss of control over gaming, tolerance, and withdrawal symptoms, analogous to the symptoms of sub- stance use disorders. As with substance-related disorders, individuals with Intemet gaming disorder continue to sit at a computer and engage in gaming activities despite neglect of other activities. They typically devote 8—10 hours or more per day to this activity and at least 30 hours per week. If they are prevented from using a computer and returning to the game, they become agitated and angry. They often go for long periods without food or sleep. Nor- mal obligations, such as school or work, or family obligations are neglected. This condition is separate from gambling disorder involving the Internet because money is not at risk.

1	The essential feature of Internet gaming disorder is persistent and recurrent participa- tion in computer gaming, typically group games, for many hours. These games involve competition between groups of players (often in different global regions, so that duration of play is encouraged by the time-zone independence) participating in complex structured activities that include a significant aspect of social interactions during play. Team aspects appear to be a key motivation. Attempts to direct the individual toward schoolwork or in- terpersonal activities are strongly resisted. Thus personal, family, or vocational pursuits are neglected. When individuals are asked, the major reasons given for using the com- puter are more likely to be ”avoiding boredom” rather than communicating or searching for information.

1	The description of criteria related to this condition is adapted from a study in China. Un- til the optimal criteria and threshold for diagnosis are determined empirically, conserva- tive definitions ought to be used, such that diagnoses are considered for endorsement of five or more of nine criteria. No consistent personality types associated with Internet gaming disorder have been iden- tified. Some authors describe associated diagnoses, such as depressive disorders, atten- tion-deficit/hyperactivity disorder (ADHD), or obsessive-compulsive disorder (OCD). Individuals with compulsive Internet gaming have demonstrated brain activation in spe- cific regions triggered by exposure to the Internet game but not limited to reward system

1	Individuals with compulsive Internet gaming have demonstrated brain activation in spe- cific regions triggered by exposure to the Internet game but not limited to reward system The prevalence of Internet gaming disorder is unclear because of the varying question- naires, criteria and thresholds employed, but it seems to be highest in Asian countries and in male adolescents 12—20 years of age. There is an abundance of reports from Asian coun- tries, especially China and South Korea, but fewer from Europe and North America, from which prevalence estimates are highly variable. The point prevalence in adolescents (ages 15—19 years) in one Asian study using a threshold of five criteria was 8.4% for males and 4.5% for females. Environmental. Computer availability with Internet connection allows access to the types of games with which Internet gaming disorder is most often associated. Genetic and physiological. Adolescent males seem to be at greatest risk of developing

1	Genetic and physiological. Adolescent males seem to be at greatest risk of developing Internet gaming disorder, and it has been speculated that Asian environmental and / or ge— netic background is another risk factor, but this remains unclear. Functional Consequences of Internet Gaming Disorder Internet gaming disorder may lead to school failure, job loss, or marriage failure. The com- pulsive gaming behavior tends to crowd out normal social, scholastic, and family activities. Students may show declining grades and eventually failure in school. Family responsibil- ities may be neglected.

1	Students may show declining grades and eventually failure in school. Family responsibil- ities may be neglected. Excessive use of the Internet not involving playing of online games (e.g., excessive use of social media, such as Facebook; viewing pornography online) is not considered analogous to Internet gaming disorder, and future research on other excessive uses of the Internet would need to follow similar guidelines as suggested herein. Excessive gambling online may qualify for a separate diagnosis of gambling disorder. Health may be neglected due to compulsive gaming. Other diagnoses that may be associ- ated with Internet gaming disorder include major depressive disorder, ADHD, and OCD. A. More than minimal exposure to alcohol during gestation. including prior to pregnancy recognition. Confirmation of gestational exposure to alcohol may be obtained from ma- ternal seIf-report of alcohol use in pregnancy, medical or other records, or clinical ob- servation.

1	B. Impaired neurocognitive functioning as manifested by one or more of the following: 1. Impairment in global intellectual performance (i.e., IQ of 70 or below, or a standard score of 70 or below on a comprehensive developmental assessment). 2. Impairment in executive functioning (e.g., poor planning and organization; inflexi- bility; difficulty with behavioral inhibition). 3. Impairment in learning (e.g., lower academic achievement than expected for intel- lectual level; specific learning disability). 4. Memory impairment (e.g., problems remembering information learned recently; repeatedly making the same mistakes; difficulty remembering lengthy verbal in- structions). 5. Impairment in visuaI-spatial reasoning (e.g., disorganized or poorly planned draw- ings or constructions; problems differentiating left from right).

1	5. Impairment in visuaI-spatial reasoning (e.g., disorganized or poorly planned draw- ings or constructions; problems differentiating left from right). C. Impaired seIf-regulation as manifested by one or more of the following: 1. Impairment in mood or behavioral regulation (e.g.. mood lability; negative affect or irritability; frequent behavioral outbursts). 2. Attention deficit (e.g., difficulty shifting attention; difficulty sustaining mental effort). 3. Impairment in impulse control (e.g., difficulty waiting turn; difficulty complying with rules). D. Impairment in adaptive functioning as manifested by two or more of the following, one of which must be (1) or (2): 1. Communication deficit (e.g., delayed acquisition of language; difficulty understand- ing spoken language). 2. Impairment in social communication and interaction (e.g., overly friendly with strang- ers; difficulty reading social cues; difficulty understanding social consequences).

1	2. Impairment in social communication and interaction (e.g., overly friendly with strang- ers; difficulty reading social cues; difficulty understanding social consequences). 3. Impairment in daily living skills (e.g., delayed toileting, feeding, or bathing; difficulty managing daily schedule). 4. Impairment in motor skills (e.g., poor fine motor development; delayed attainment ordination and balance). E. Onset of the disorder (symptoms in Criteria B, C, and D) occurs in childhood. F. The disturbance causes clinically significant distress or impairment in social, aca- demic, occupational, or other important areas of functioning. G. The disorder is not better explained by the direct physiological effects associated with postnatal use of a substance (e.g., a medication, alcohol or other drugs), a general medical condition (e.g., traumatic brain injury, delirium, dementia), another known te- ratogen (e.g., fetal hydantoin syndrome), a genetic condition (e.g., Williams syndrome,

1	Down syndrome, Cornelia de Lange syndrome), or environmental neglect. Alcohol is a neurobehavioral teratogen, and prenatal alcohol exposure has teratogenic effects on central nervous system (CNS) development and subsequent function. Neurobe- havioral disorder associated with prenatal alcohol exposure (ND—PAE) is a new clarifying term, intended to encompass the full range of developmental disabilities associated with expo- sure to alcohol in utero. The current diagnostic guidelines allow ND—PAE to be diagnosed both in the absence and in the presence of the physical effects of prenatal alcohol exposure (e.g., facial dysmorphology required for a diagnosis of fetal alcohol syndrome).

1	The essential features of ND-PAE are the manifestation of impairment in neurocognitive, behavioral, and adaptive functioning associated with prenatal alcohol exposure. Impair- ment can be documented based on past diagnostic evaluations (e.g., psychological or ed- ucational assessments) or medical records, reports by the individual or informants, and/ or observation by a clinician.

1	A clinical diagnosis of fetal alcohol syndrome, including specific prenatal alcohol- related facial dysmorphology and growth retardation, can be used as evidence of signifi- cant levels of prenatal alcohol exposure. Although both animal and human studies have documented adverse effects of lower levels of drinking, identifying how much prenatal exposure is needed to significantly impact neurodevelopmental outcome remains chal- lenging. Data suggest that a history of more than minimal gestational exposure (e.g., more than light drinking) prior to pregnancy recognition and / or following pregnancy recogni- tion may be required. Light drinking is defined as 1—13 drinks per month during preg- nancy with no more than 2 of these drinks consumed on any 1 drinking occasion. Identifying a minimal threshold of drinking during pregnancy will require consideration of a variety of factors known to affect exposure and / or interact to influence developmental outcomes, including stage of prenatal

1	threshold of drinking during pregnancy will require consideration of a variety of factors known to affect exposure and / or interact to influence developmental outcomes, including stage of prenatal development, gestational smoking, maternal and fetal genet- ics, and maternal physical status (i.e., age, health, and certain obstetric problems).

1	Symptoms of ND-PAE include marked impairment in global intellectual performance (IQ) 0r neurocognitive impairments in any of the following areas: executive functioning, learning, memory, and / or visual-spatial reasoning. Impairments in self-regulation are pres- ent and may include impairment in mood or behavioral regulation, attention deficit, or impairment in impulse control. Finally, impairments in adaptive functioning include com- munication deficits and impairment in social communication and interaction. Impairment in daily living (self-help) skills and impairment in motor skills may be present. As it may be difficult to obtain an accurate assessment of the neurocognitive abilities of very young chil- dren, it is appropriate to defer a diagnosis for children 3 years of age and younger.

1	Associated features vary depending on age, degree of alcohol exposure, and the individ- ual’s environment. An individual can be diagnosed with this disorder regardless of socio- economic or cultural background. However, ongoing parental alcohol/substance misuse, parental mental illness, exposure to domestic or community violence, neglect or abuse, disrupted caregiving relationships, multiple out-of-home placements, and lack of conti- nuity in medical or mental health care are often present. The prevalence rates of ND-PAE are unknown. However, estimated prevalence rates of clini- cal conditions associated with prenatal alcohol exposure are 2%—5% in the United States.

1	Among individuals with prenatal alcohol exposure, evidence of CNS dysfunction varies according to developmental stage. Although about one-half of young children prenatally exposed to alcohol show marked developmental delay in the first 3 years of life, other chil- dren affected by prenatal alcohol exposure may not exhibit signs of CNS dysfunction until they are preschool- or school-age. Additionally, impairments in higher order cognitive processes (i.e., executive functioning), which are often associated with prenatal alcohol ex- posure, may be more easily assessed in older children. When children reach school age, learning difficulties, impairment in executive function, and problems with integrative lan- guage functions usually emerge more clearly, and both social skills deficits and challeng- ing behavior may become more evident. In particular, as school and other requirements become more complex, greater deficits are noted. Because of this, the school years repre- sent the ages at

1	ing behavior may become more evident. In particular, as school and other requirements become more complex, greater deficits are noted. Because of this, the school years repre- sent the ages at which a diagnosis of ND-PAE would be most likely.

1	Suicide is a high-risk outcome, with rates increasing significantly in late adolescence and early adulthood. Functional Consequences of Neurobehavioral Disorder The CNS dysfunction seen in individuals with ND—PAE often leads to decrements in adap- tive behavior and to maladaptive behavior with lifelong consequences. Individuals affected by prenatal alcohol exposure have a higher prevalence of disrupted school expe- riences, poor employment records, trouble with the law, confinement (legal or psychiat- ric), and dependent living conditions.

1	Disorders that are attributable to the physiological effects associated with postnatal use of a substance, another medical condition, or environmental neglect. Other consid- erations include the physiological effects of postnatal substance use, such as a medication, alcohol, or other substances; disorders due to another medical condition, such as traumatic brain injury or other neurocognitive disorders (e.g., delirium, major neurocognitive dis- order [dementia]); or environmental neglect. Genetic and teratogenic conditions. Genetic conditions such as Williams syndrome, Down syndrome, or Cornelia de Lange syndrome and other teratogenic conditions such as behavioral characteristics. A careful review of prenatal exposure history is needed to clar- ify the teratogenic agent, and an evaluation by a clinical geneticist may be needed to dis- tinguish physical characteristics associated with these and other genetic conditions.

1	Mental health problems have been identified in more than 90% of individuals with histo- ries of significant prenatal alcohol exposure. The most common co-occurring diagnosis is attention-deficit/hyperactivity disorder, but research has shown that individuals with ND-PAE differ in neuropsychological characteristics and in their responsiveness to phar- macological interventions. Other high- probability co-occurring disorders include oppo- sitional defiant disorder and conduct disorder, but the appropriateness of these diagnoses should be weighed in the context of the significant impairments in general intellectual and executive functioning that are often associated with prenatal alcohol exposure. Mood symptoms, including symptoms of bipolar disorder and depressive disorders, have been described. History of prenatal alcohol exposure is associated with an increased risk for later tobacco, alcohol, and other substance use disorders.

1	A. Within the last 24 months, the individual has made a suicide attempt. Note: A suicide attempt is a seIf-initiated sequence of behaviors by an individual who, at the time of initiation, expected that the set of actions would lead to his or her own death. The “time of initiation" is the time when a behavior took place that involved ap- plying the method.) B. The act does not meet criteria for nonsuicidal seIf-injury—that is, it does not involve self-injury directed to the surface of the body undertaken to induce relief from a nega- tive feeling/cognitive state or to achieve a positive mood state. C. The diagnosis is not applied to suicidal ideation or to preparatory acts. D. The act was not initiated during a state of delirium or confusion. E. The act was not undertaken solely for a political or religious objective. Specify if: Current: Not more than 12 months since the last attempt. In early remission: 12—24 months since the last attempt.

1	Specify if: Current: Not more than 12 months since the last attempt. In early remission: 12—24 months since the last attempt. Suicidal behavior is often categorized in terms of violence of the method. Generally, over— doses with legal or illegal substances are considered nonviolent in method, whereas jump- ing, gunshot wounds, and other methods are considered violent. Another dimension for classification is medical consequences of the behavior, with high—lethality attempts being defined as those requiring medical hospitalization beyond a visit to an emergency depart- ment. An additional dimension considered includes the degree of planning versus impul— siveness of the attempt, a characteristic that might have consequences for the medical outcome of a suicide attempt.

1	If the suicidal behavior occurred 12—24 months prior to evaluation, the condition is considered to be in early remission. Individuals remain at higher risk for further suicide at- tempts and death in the 24 months after a suicide attempt, and the period 12—24 months af— ter the behavior took place is specified as "early remission." The essential manifestation of suicidal behavior disorder is a suicide attempt. A suicide at— tempt is a behavior that the individual has undertaken with at least some intent to die. The behavior might or might not lead to injury or serious medical consequences. Several fac- tors can inﬂuence the medical consequences of the suicide attempt, including poor plan- ning, lack of knowledge about the lethality of the method chosen, low intentionality or ambivalence, or chance intervention by others after the behavior has been initiated. These should not be considered in assigning the diagnosis.

1	Determining the degree of intent can be challenging. Individuals might not acknowl— edge intent, especially in situations where doing so could result in hospitalization or cause distress to loved ones. Markers of risk include degree of planning, including selection of a time and place to minimize rescue or interruption; the individual’s mental state at the time of the behavior, with acute agitation being especially concerning; recent discharge from inpatient care; or recent discontinuation of a mood stabilizer such as lithium or an anti- psychotic such as clozapine in the case of schizophrenia. Examples of environmental “trig- gers” include recently learning of a potentially fatal medical diagnosis such as cancer, experiencing the sudden and unexpected loss of a close relative or partner, loss of employ— ment, or displacement from housing. Conversely, features such as talking to others about future events or preparedness to sign a contract for safety are less reliable indicators.

1	In order for the criteria to be met, the individual must have made at least one suicide at- tempt. Suicide attempts can include behaviors in which, after initiating the suicide attempt, the individual changed his or her mind or someone intervened. For example, an individual might intend to ingest a given amount of medication or poison, but either stop or be stopped by another before ingesting the full amount. If the individual is dissuaded by another or changes his or her mind before initiating the behavior, the diagnosis should not be made.

1	The act must not meet criteria for nonsuicidal self—injury—that is, it should not involve re- peated (at least five times within the past 12 months) self-injurious episodes undertaken to induce relief from a negative feeling/ cognitive state or to achieve a positive mood state. The act should not have been initiated during a state of delirium or confusion. If the individual deliberately became intoxicated before initiating the behavior, to reduce anticipatory anxi— ety and to minimize interference with the intended behavior, the diagnosis should be made.

1	Suicidal behavior can occur at any time in the lifespan but is rarely seen in children under the age of 5. In prepubertal children, the behavior will often consist of a behavior (e.g., sit- ting on a ledge) that a parent has forbidden because of the risk of accident. Approximately 25%—30% of persons who attempt suicide will go on to make more attempts.There is sig- nificant variability in terms of frequency, method, and lethality of attempts. However, this is not different from what is observed in other illnesses, such as major depressive disorder, in which frequency of episode, subtype of episode, and impairment for a given episode can vary significantly.

1	Suicidal behavior varies in frequency and form across cultures. Cultural differences might be due to method availability (e.g., poisoning with pesticides in developing countries; gunshot wounds in the southwestern United States) or the presence of culturally specific syndromes (e.g., ataques de nervios, which in some Latino groups might lead to behaviors that closely resemble suicide attempts or might facilitate suicide attempts). Laboratory abnormalities consequent to the suicidal attempt are often evident. Suicidal behavior that leads to blood loss can be accompanied by anemia, hypotension, or shock. Overdoses might lead to coma or obtundation and associated laboratory abnormalities such as electrolyte imbalances. Functional Consequences of Suicidal Behavior Disorder

1	Overdoses might lead to coma or obtundation and associated laboratory abnormalities such as electrolyte imbalances. Functional Consequences of Suicidal Behavior Disorder Medical conditions (e.g., lacerations or skeletal trauma, cardiopulmonary instability, in- halation of vomit and suffocation, hepatic failure consequent to use of paracetamol) can occur as a consequence of suicidal behavior.

1	Suicidal behavior is seen in the context of a variety of mental disorders, most commonly bipo— lar disorder, major depressive disorder, schizophrenia, schizoaffective disorder, anxiety dis- orders (in particular, panic disorders associated with catastrophic content and PTSD ﬂashbacks), substance use disorders (especially alcohol use disorders), borderline personality disorder, antisocial personality disorder, eating disorders, and adjustment disorders. It is rarely manifested by individuals with no discernible pathology, unless it is undertaken be- cause of a painful medical condition with the intention of drawing attention to martyrdom for political or religious reasons, or in partners in a suicide pact, both of which are excluded from this diagnosis, or when thiId-party informants wish to conceal the nature of the behavior.

1	A. In the last year, the individual has, on 5 or more days, engaged in intentional seIf-inflicted damage to the surface of his or her body of a sort likely to induce bleeding, bruising, or pain (e.g., cutting, burning, stabbing, hitting, excessive rubbing), with the expectation that the injury will lead to only minor or moderate physical harm (i.e., there is no suicidal intent). Note: The absence of suicidal intent has either been stated by the individual or can be inferred by the individual’s repeated engagement in a behavior that the individual knows, or has learned, is not likely to result in death. B. The individual engages in the seIf-injurious behavior with one or more of the following expectations: 1. To obtain relief from a negative feeling or cognitive state. 2. To resolve an interpersonal difficulty. 3. To induce a positive feeling state.

1	2. To resolve an interpersonal difficulty. 3. To induce a positive feeling state. Note: The desired relief or response is experienced during or shortly after the self- injury, and the individual may display patterns of behavior suggesting a dependence on repeatedly engaging in it. C. The intentional self-injury is associated with at least one of the following: 1. Interpersonal difficulties or negative feelings or thoughts, such as depression, anx- iety, tension, anger, generalized distress, or seIf—criticism, occurring in the period immediately prior to the seIf-injurious act. 2. Prior to engaging in the act, a period of preoccupation with the intended behavior that is difficult to control. 3. Thinking about seIf-injury that occurs frequently, even when it is not acted upon. D. The behavior is not socially sanctioned (e.g., body piercing, tattooing, part of a religious or cultural ritual) and is not restricted to picking a scab or nail biting.

1	D. The behavior is not socially sanctioned (e.g., body piercing, tattooing, part of a religious or cultural ritual) and is not restricted to picking a scab or nail biting. E. The behavior or its consequences cause clinically significant distress or interference in interpersonal, academic, or other important areas of functioning. F. The behavior does not occur exclusively during psychotic episodes, delirium, sub- stance intoxication, or substance withdrawal. In individuals with a neurodevelopmental disorder, the behavior is not part of a pattern of repetitive stereotypies. The behavior is not better explained by another mental disorder or medical condition (e.g., psychotic disorder, autism spectrum disorder. intellectual disability, Lesch-Nyhan syndrome, ste- reotypic movement disorder with seIf-injury, trichotillomania [hair-pulling disorder]. ex- coriation [skin-picking] disorder).

1	The essential feature of nonsuicidal self-injury is that the individual repeatedly inﬂicts shallow, yet painful injuries to the surface of his or her body. Most commonly, the purpose is to reduce negative emotions, such as tension, anxiety, and self—reproach, and / or to re- solve an interpersonal difficulty. In some cases, the injury is conceived of as a deserved self—punishment. The individual will often report an immediate sensation of relief that oc- curs during the process. When the behavior occurs frequently, it might be associated with a sense of urgency and craving, the resultant behavioral pattern resembling an addiction. The inﬂicted wounds can become deeper and more numerous. The injury is most often inﬂicted with a knife, needle, razor, or other sharp object. Com- mon areas for injury include the frontal area of the thighs and the dorsal side of the forearm.

1	The injury is most often inﬂicted with a knife, needle, razor, or other sharp object. Com- mon areas for injury include the frontal area of the thighs and the dorsal side of the forearm. A single session of injury might involve a series of superficial, parallel cuts—separated by 1 or 2 centimeters—on a visible or accessible location. The resulting cuts will often bleed and will eventually leave a characteristic pattern of scars. Other methods used include stabbing an area, most often the upper arm, with a needle or sharp, pointed knife; inﬂicting a superficial burn with a lit cigarette end; or burning the skin by repeated rubbing with an eraser. Engagement in nonsuicidal self—injury with mul- tiple methods is associated with more severe psychopathology, including engagement in suicide attempts.

1	The great majority of individuals Who engage in nonsuicidal self—injury do not seek clinical attention. It is not known if this reﬂects frequency of engagement in the disorder, because accurate reporting is seen as stigmatizing, or because the behaviors are experi- enced positively by the individual who engages in them, who is unmotivated to receive treatment. Young Children might experiment with these behaviors but not experience re- lief. In such cases, youths often report that the procedure is painful or distressing and might then discontinue the practice.

1	Nonsuicidal self—injury most often starts in the early teen years and can continue for many years. Admission to hospital for nonsuicidal self—injury reaches a peak at 20—29 years of age and then declines. However, research that has examined age at hospitalization did not provide information on age at onset of the behavior, and prospective research is needed to outline the natural history of nonsuicidal self—injury and the factors that promote or in- hibit its course. Individuals often learn of the behavior on the recommendation or observa- tion of another. Research has shown that when an individual who engages in nonsuicidal self—injury is admitted to an inpatient unit, other individuals may begin to engage in the behavior. Male and female prevalence rates of nonsuicidal self—injury are closer to each other than in suicidal behavior disorder, in which the female-to-male ratio is about 3:1 or 4:1.

1	Male and female prevalence rates of nonsuicidal self—injury are closer to each other than in suicidal behavior disorder, in which the female-to-male ratio is about 3:1 or 4:1. Two theories of psychopathology—based on functional behavioral analyses—have been proposed: In the first, based on learning theory, either positive or negative reinforcement sustains the behavior. Positive reinforcement might result from punishing oneself in a way that the individual feels is deserved, with the behavior inducing a pleasant and relaxed state or generating attention and help from a significant other, or as an expression of anger. Neg- ative reinforcement results from affect regulation and the reduction of unpleasant emotions or avoiding distressing thoughts, including thinking about suicide. In the second theory, nonsuicidal self-injury is thought to be a form of self—punishment, in which self—punitive ac- tions are engaged in to make up for acts that caused distress or harm to others.

1	Functional Consequences of Nonsuicidal SeIf-lniury The act of cuttingkmight be performed with shared implements, raising the possibility of blood—bome disease transmission.

1	Borderline personality disorder. As indicated, nonsuicidal self-injury has long been re- garded as a ”symptom" of borderline personality disorder, even though comprehensive clinical evaluations have found that most individuals with nonsuicidal self-injury have symptoms that also meet criteria for other diagnoses, with eating disorders and substance use disorders being especially common. Historically, nonsuicidal self-injury was regarded as pathognomonic of borderline personality disorder. Both conditions are associated with several other diagnoses. Although frequently associated, borderline personality disorder is not invariably found in individuals with nonsuicidal self—injury. The two conditions dif- fer in several ways. Individuals with borderline personality disorder often manifest dis- turbed aggressive and hostile behaviors, whereas nonsuicidal self—injury is more often associated with phases of closeness, collaborative behaviors, and positive relationships. At a more

1	dis- turbed aggressive and hostile behaviors, whereas nonsuicidal self—injury is more often associated with phases of closeness, collaborative behaviors, and positive relationships. At a more fundamental level, there are differences in the involvement of different neurotrans— mitter systems, but these will not be apparent on clinical examination.

1	Suicidal behavior disorder. The differentiation between nonsuicidal self-injury and sui- cidal behavior disorder is based either on the stated goal of the behavior being a wish to die (suicidal behavior disorder) or, in nonsuicidal self—injury, to experience relief as de- scribed in the criteria. Depending on the circumstances, individuals may provide reports of convenience, and several studies report high rates of false intent declaration. Individu- als with a history of frequent nonsuicidal self—injury episodes have learned that a session of cutting, while painful, is, in the short-term, largely benign. Because individuals with nonsuicidal self—injury can and do attempt and commit suicide, it is important to check past history of suicidal behavior and to obtain information from a third party concerning any recent change in stress exposure and mood. Likelihood of suicide intent has been as- sociated with the use of multiple previous methods of self—harm.

1	In a follow-up study of cases of ”self—harm" in males treated at one of several multiple emergency centers in the United Kingdom, individuals with nonsuicidal self-injury were significantly more likely to commit suicide than other teenage individuals drawn from the same cohort. Studies that have examined the relationship between nonsuicidal self-injury and suicidal behavior disorder are limited by being retrospective and failing to obtain ver- ified accounts of the method used during previous "attempts.” A significant proportion of have ever engaged in self-cutting (or their preferred means of self—injury) with an intention to die. It is reasonable to conclude that nonsuicidal self-injury, while not presenting a high risk for suicide when first manifested, is an especially dangerous form of self—injurious behavior.

1	This conclusion is also supported by a multisite study of depressed adolescents who had previously failed to respond to antidepressant medication, which noted that those with pre- vious nonsuicidal self—injury did not respond to cognitive-behavioral therapy, and by a study that found that nonsuicidal self-injury is a predictor of substance use/misuse. Trichotillomania (hair-pulling disorder). Trichotillomania is an injurious behavior con- fined to pulling out one’s own hair, most commonly from the scalp, eyebrows, or eyelashes. The behavior occurs in ”sessions" that can last for hours. It is most likely to occur during a period of relaxation or distraction. Stereotypic seIf-injury. Stereotypic self—injury, which can include head banging, self- biting, or self—hitting, is usually associated with intense concentration or under conditions of low external stimulation and might be associated with developmental delay.

1	Excoriation (skin-picking) disorder. Excoriation disorder occurs mainly in females and is usually directed to picking at an area of the skin that the individual feels is unsightly or a blemish, usually on the face or the scalp. As in nonsuicidal self-injury, the picking is often preceded by an urge and is experienced as pleasurable, even though the individual real- izes that he or she is harming himself or herself. It is not associated with the use of any im- plement. Highlights of Changes From DSM-IV to DSM-5 ................... 809 Glossary of Technical Terms .................................. 817 Glossary of Cultural Concepts of Distress ....................... 833 Alphabetical Listing of DSM-5 Diagnoses and Codes (lCD-9-CM and lCD—10-CM) ................................. 839 Numerical Listing of DSM—5 Diagnoses and Codes (ICD-9-CM) ...... 863 Numerical Listing of DSM-5 Diagnoses and Codes (|CD-10-CM) ..... 877

1	Numerical Listing of DSM—5 Diagnoses and Codes (ICD-9-CM) ...... 863 Numerical Listing of DSM-5 Diagnoses and Codes (|CD-10-CM) ..... 877 DSM-5 Advisors and Other Contributors ......................... 897 ‘ Highlights °f Changes Fro . DSM-IV to DSM C ham 9% mad 9 IO DSM-5 diagnostic criteria and texts are outlined in this chapter in the same order in which they appear in the DSM-S classification. This abbreviated descrip- tion is intended to orient readers to only the most significant changes in each disorder cate- gory. An expanded description of nearly all changes (e.g., except minor text or wording changes needed for clarity) is available online (www.psychiatry.org/ dsmS). It should also be noted that Section I contains a description of changes pertaining to the chapter organization in DSM-S, the multiaxial system, and the introduction of dimensional assessments.

1	The term mental retardation was used in DSM-IV. However, intellectual disability (intel- lectual developmental disorder) is the term that has come into common use over the past two decades among medical, educational, and other professionals, and by the lay public and advocacy groups. Diagnostic criteria emphasize the need for an assessment of both cognitive capacity (IQ) and adaptive functioning. Severity is determined by adaptive func- tioning rather than IQ score. The communication disorders, which are newly named from DSM-IV phonological dis- order and stuttering, respectively, include language disorder (which combines the previous expressive and mixed receptive-expressive language disorders), speech sound disorder (pre- viously phonological disorder), and childhood-onset ﬂuency disorder (previously stutter- ing). Also included is social (pragmatic) communication disorder, a new condition involving persistent difficulties in the social uses of verbal and nonverbal communication.

1	Autism spectrum disorder is a new DSM-S disorder encompassing the previous DSM- IV autistic disorder (autism), Asperger’s disorder, Childhood disintegrative disorder, Rett’s disorder, and pervasive developmental disorder not otherwise specified. It is char- acterized by deficits in two core domains: 1) deficits in social communication and social in- teraction and 2) restricted repetitive patterns of behavior, interests, and activities.

1	Several changes have been made to the diagnostic criteria for attention-deficit/hyperactiv- ity disorder (ADHD). Examples have been added to the criterion items to facilitate application across the life span; the age at onset description has been changed (from “some hyperactive- to ”Several inattentive or hyperactive-impulsive symptoms were present prior to age 12”); subtypes have been replaced with presentation specifiers that map directly to the prior sub- types; a comorbid diagnosis with autism spectrum disorder is now allowed; and a symptom threshold change has been made for adults, to reﬂect the substantial evidence of Clinically sig- nificant ADHD impairment, with the cutoff for ADHD of five symptoms, instead of six re- quired for younger persons, both for inattention and for hyperactivity and impulsivity.

1	Specific learning disorder combines the DSM-IV diagnoses of reading disorder, math- ematics disorder, disorder of written expression, and learning disorder not otherwise specified. Learning deficits in the areas of reading, written expression, and mathematics are coded as separate specifiers. Acknowledgment is made in the text that specific types of reading deficits are described internationally in various ways as dyslexia and specific types of mathematics deficits as dyscalculia. 810 Highlights of Changes From DSM-IV to DSM-5 The following motor disorders are included in DSM-S: developmental coordination disor- der, stereotypic movement disorder, Tourette’s disorder, persistent (chronic) motor or vocal tic disorder, provisional tic disorder, other specified tic disorder, and unspecified tic disorder. The tic criteria have been standardized across all of these disorders in this chapter.

1	The tic criteria have been standardized across all of these disorders in this chapter. Two changes were made to Criterion A for schizophrenia: 1) the elimination of the special at- tribution of bizarre delusions and Schneiderian first-rank auditory hallucinations (e.g., two or more voices conversing), leading to the requirement of at least two Criterion A symptoms for any diagnosis of schizophrenia, and 2) the addition of the requirement that at least one of the Criterion A symptoms must be delusions, hallucinations, or disorganized speech. The DSM-IV subtypes of schizophrenia were eliminated due to their limited diagnostic stability, low reli- ability, and poor validity. Instead, a dimensional approach to rating severity for the core symp- toms of schizophrenia is included in DSM-S Section III to capture the important heterogeneity in symptom type and severity expressed across individuals with psychotic disorders.

1	Schizoaffective disorder is reconceptualized as a longitudinal instead of a cross-sectional di- agnosis—more comparable to schizophrenia, bipolar disorder, and major depressive disorder, which are bridged by this condition—and requires that a major mood episode be present for a majority of the total disorder’s duration after Criterion A has been met. Criterion A for delu- sional disorder no longer has the requirement that the delusions must be nonbizarre; a spec- ifier is now included for bizarre type delusions to provide continuity with DSM-IV. Criteria for catatonia are described uniformly across DSM-5. Furthermore, catatonia may be diagnosed with a specifier (for depressive, bipolar, and psychotic disorders, including schizophrenia), in the context of a known medical condition, or as an other specified diagnosis.

1	activity or energy. The DSM-IV diagnosis of bipolar I disorder, mixed episodes—requiring that the individual simultaneously meet full criteria for both mania and major depressive ep- isode—is replaced with a new specifier "with mixed features.“ Particular conditions can now be diagnosed under other specified bipolar and related disorder, including categori- zation for individuals with a past history of a major depressive disorder whose symptoms meet all criteria for hypomania except the duration criterion is not met (i.e., the episode lasts only 2 or 3 days instead of the required 4 consecutive days or more). A second condition con- stituting an other specified bipolar and related disorder variant is that too few symptoms of hypomania are present to meet criteria for the full bipolar II syndrome, although the dura- tion, at least 4 consecutive days, is sufficient. Finally, in both this chapter and in the chapter ”Depressive Disorders,” an anxious distress specifier is delineated.

1	To address concerns about potential overdiagnosis and overtreatment of bipolar disorder in children, a new diagnosis, disruptive mood dysregulation disorder, is included for children up to age 18 years who exhibit persistent irritability and frequent episodes of extreme behav- ioral dyscontrol. Premenstrual dysphoric disorder is now promoted from Appendix B, ”Cri- teria Sets and Axes Provided for Further Study,” in DSM-IV to the main body of DSM-S. What was referred to as dysthyrnia in DSM-IV now falls under the category of persistent depressive disorder, which includes both chronic major depressive disorder and the previous dysthymic disorder. The coexistence within a major depressive episode of at least three manic symp- toms (insufficient to satisfy criteria for a manic episode) is now acknowledged by the specifier

1	Highlights of Changes From DSM-IV to DSM-5 811 ”with mixed features." In DSM-IV, there was an exclusion criterion for a major depressive ep- isode that was applied to depressive symptoms lasting less than 2 months following the death of a loved one (i.e., the bereavement exclusion). This exclusion is omitted in DSM—S for several reasons, including the recognition that bereavement is a severe psychosocial stressor that can precipitate a major depressive episode in a vulnerable individual, generally beginning soon after the loss, and can add an additional risk for suffering, feelings of worthlessness, suicidal ideation, poorer medical health, and worse interpersonal and work functioning. It was critical to remove the implication that bereavement typically lasts only 2 months, when both physi- cians and grief counselors recognize that the duration is more commonly 1—2 years. A detailed footnote has replaced the more simplistic DSM-IV exclusion to aid clinicians in making the critical

1	cians and grief counselors recognize that the duration is more commonly 1—2 years. A detailed footnote has replaced the more simplistic DSM-IV exclusion to aid clinicians in making the critical distinction between the symptoms characteristic of bereavement and those of a major depressive disorder. Finally, a new specifier to indicate the presence of mixed symptoms has been added across both the bipolar and the depressive disorders.

1	The chapter on anxiety disorders no longer includes obsessive-compulsive disorder (which is in the new chapter ”Obsessive-Compulsive and Related Disorders”) or posttraumatic stress disorder (PTSD) and acute stress disorder (which are in the new chapter ”Trauma- and Stressor-Related Disorders"). Changes in criteria for specific phobia and social anxiety disorder (social phobia) include deletion of the requirement that individuals over age 18 years recognize that their anxiety is excessive or unreasonable. Instead, the anxiety must be out of proportion to the actual danger or threat in the situation, after cultural contextual fac- tors are taken into account. In addition, the 6-month duration is now extended to all ages.

1	Panic attacks can now be listed as a specifier that is applicable to all DSM-5 disorders. Panic disorder and agoraphobia are unlinked in DSM-S. Thus, the former DSM-IV diagnoses of panic disorder with agoraphobia, panic disorder without agoraphobia, and agoraphobia without history of panic disorder are now replaced by two diagnoses, panic disorder and ag- oraphobia, each with separate criteria. The ”generalized" specifier for social anxiety disor- der has been deleted and replaced with a ”performance only” specifier. Separation anxiety disorder and selective mutism are now classified as anxiety disorders. The wording of the criteria is modified to more adequately represent the expression of separation anxiety symp- toms in adulthood. Also, in contrast to DSM-IV, the diagnostic criteria no longer specify that onset must be before age 18 years, and a duration statement—"typically lasting for 6 months or more”—has been added for adults to minimize overdiagnosis of transient fears.

1	The chapter ”Obsessive-Compulsive and Related Disorders" is new in DSM-S. New disor- ders include hoarding disorder, excoriation (skin-picking) disorder, substance/medica- tion-induced obsessive-compulsive and related disorder, and obsessive-compulsive and related disorder due to another medical condition. The DSM-IV diagnosis of trichotillo- mania is now termed trichotillomania (hair-pulling disorder) and has been moved from a

1	DSM—IV classification of impulse—control disorders not elsewhere classified to obsessive- compulsive and related disorders in DSM-5. The DSM-IV ”with poor insight” specifier for obsessive-compulsive disorder has been refined to allow a distinction between individuals with good or fair insight, poor insight, and "absent insight/delusional” obsessive-compul- sive disorder beliefs (i.e., complete conviction that obsessive-compulsive disorder beliefs are true). Analogous "insight” specifiers have been included for body dysmorphic disorder and hoarding disorder. A "tic-related" specifier for obsessive-compulsive disorder has also been added, because presence of a comorbid tic disorder may have important clinical im- plications. A ”muscle dysmorphia” specifier for body dysmorphic disorder is added to re- flect a growing literature on the diagnostic validity and clinical utility of making this 812 Highlights of Changes From DSM-IV to DSM-5 distinction in individuals with body dysmorphic

1	added to re- flect a growing literature on the diagnostic validity and clinical utility of making this 812 Highlights of Changes From DSM-IV to DSM-5 distinction in individuals with body dysmorphic disorder. The delusional variant of body dysmorphic disorder (which identifies individuals who are completely convinced that their perceived defects or ﬂaws are truly abnormal appearing) is no longer coded as both delu- sional disorder, somatic type, and body dysmorphic disorder; in DSM-S, this presentation is designated only as body dysmorphic disorder with the absent insight/ delusional specifier.

1	Individuals can also be diagnosed with other specified obsessive-compulsive and related disorder, which can include conditions such as body-focused repetitive behavior disorder and obsessional jealousy, or unspecified obsessive-compulsive and related disorder. For a diagnosis of acute stress disorder, qualifying traumatic events are now explicit as to whether they were experienced directly, witnessed, or experienced indirectly. Also, the DSM-IV Criterion A2 regarding the subjective reaction to the traumatic event (e.g., expe- riencing ”fear, helplessness, or horror”) has been eliminated. Adjustment disorders are reconceptualized as a heterogeneous array of stress-response syndromes that occur after exposure to a distressing (traumatic or nontraumatic) event, rather than as a residual cat- not meet criteria for a more discrete disorder (as in DSM-IV).

1	DSM-S criteria for PTSD differ significantly from the DSM-IV criteria. The stressor cri- terion (Criterion A) is more explicit with regard to events that qualify as ”traumatic” ex- periences. Also, DSM-IV Criterion A2 (subjective reaction) has been eliminated. Whereas there were three major symptom clusters in DSM-IV—reexperiencing, avoidance/numb- ing, and arousal—there are now four symptom clusters in DSM-5, because the avoidance/ numbing cluster is divided into two distinct clusters: avoidance and persistent negative al- terations in cognitions and mood. This latter category, which retains most of the DSM-IV numbing symptoms, also includes new or reconceptualized symptoms, such as persistent negative emotional states. The final cluster—alterations in arousal and reactivity—retains most of the DSM-IV arousal symptoms. It also includes irritable behavior or angry out- bursts and reckless or self—destructive behavior. PTSD is now developmentally sensitive in that diagnostic thresholds

1	of the DSM-IV arousal symptoms. It also includes irritable behavior or angry out- bursts and reckless or self—destructive behavior. PTSD is now developmentally sensitive in that diagnostic thresholds have been lowered for children and adolescents. Furthermore, separate criteria have been added for children age 6 years or younger with this disorder.

1	The DSM-IV childhood diagnosis reactive attachment disorder had two subtypes: emotionally withdrawn/ inhibited and indiscriminately social/disinhibited. In DSM-S, these subtypes are defined as distinct disorders: reactive attachment disorder and disin- hibited social engagement disorder.

1	Major changes in dissociative disorders in DSM-S include the following: 1) derealization is included in the name and symptom structure of what previously was called depersonali- zation disorder (depersonalization/derealization disorder); 2) dissociative fugue is now a specifier of dissociative amnesia rather than a separate diagnosis, and 3) the criteria for dissociative identity disorder have been changed to indicate that symptoms of disruption of identity may be reported as well as observed, and that gaps in the recall of events may occur for everyday and not just traumatic events. Also, experiences of pathological pos- session in some cultures are included in the description of identity disruption.

1	In DSM-5, somatoform disorders are now referred to as somatic symptom and related dis- orders. The DSM-S classification reduces the number of these disorders and subcategories to avoid problematic overlap. Diagnoses of somatization disorder, hypochondriasis, pain dis- order, and undifferentiated somatoform disorder have been removed. Individuals previ-

1	Highlights of Changes From DSM-IV to DSM—5 813 ously diagnosed with somatization disorder will usually have symptoms that meet DSM-5 criteria for somatic symptom disorder, but only if they have the maladaptive thoughts, feel- ings, and behaviors that define the disorder, in addition to their somatic symptoms. Because the distinction between somatization disorder and undifferentiated somatoform disorder was arbitrary, they are merged in DSM—S under somatic symptom disorder. Individuals pre- viously diagnosed with hypochondriasis who have high health anxiety but no somatic symp- toms would receive a DSM-S diagnosis of illness anxiety disorder (unless their health anxiety was better explained by a primary anxiety disorder, such as generalized anxiety dis- order). Some individuals with chronic pain would be appropriately diagnosed as having so- matic symptom disorder, with predominant pain. For others, psychological factors affecting other medical conditions or an adjustment disorder

1	pain would be appropriately diagnosed as having so- matic symptom disorder, with predominant pain. For others, psychological factors affecting other medical conditions or an adjustment disorder would be more appropriate.

1	Psychological factors affecting other medical conditions is a new mental disorder in DSM-5, having formerly been listed in the DSM-IV chapter ”Other Conditions That May

1	DSM-5, having formerly been listed in the DSM-IV chapter ”Other Conditions That May Be a Focus of Clinical Attention.” This disorder and factitious disorder are placed among the somatic symptom and related disorders because somatic symptoms are predominant in both disorders, and both are most often encountered in medical settings. The variants of psychological factors affecting other medical conditions are removed in favor of the stern diagnosis. Criteria for conversion disorder (functional neurological symptom disorder) have been modified to emphasize the essential importance of the neurological examina- tion, and in recognition that relevant psychological factors may not be demonstrable at the time of diagnosis. Other specified somatic symptom disorder, other specified illness anx- iety disorder, and pseudocyesis are now the only exemplars of the other specified somatic symptom and related disorder classification.

1	Because of the elimination of the DSM-IV-TR chapter ”Disorders Usually First Diagnosed

1	During Infancy, Childhood, or Adolescence,” this chapter describes several disorders found in the DSM-IV section ”Feeding and Eating Disorders of Infancy or Early Childhood,” such as pica and rumination disorder. The DSM-IV category feeding disorder of infancy or early childhood has been renamed avoidant/restrictive food intake disorder, and the criteria are significantly expanded. The core diagnostic criteria for anorexia nervosa are conceptually un- changed from DSM-IV with one exception: the requirement for amenorrhea is eliminated. As in DSM—IV, individuals with this disorder are required by Criterion A to be at a significantly low body weight for their developmental stage. The wording of the criterion is changed for clarification, and guidance regarding how to judge whether an individual is at or below a sig- nificantly low weight is provided in the text. In DSM-S, Criterion B is expanded to include not only overtly expressed fear of weight gain but also persistent behavior that

1	is at or below a sig- nificantly low weight is provided in the text. In DSM-S, Criterion B is expanded to include not only overtly expressed fear of weight gain but also persistent behavior that interferes with weight gain. The only change in the DSM-IV criteria for bulimia nervosa is a reduction in the required minimum average frequency of binge eating and inappropriate compensatory be- havior frequency from twice to once weekly. The extensive research that followed the prom- ulgation of preliminary criteria for binge-eating disorder in Appendix B of DSM-IV documented the clinical utility and validity of binge—eating disorder. The only significant dif- ference from the preliminary criteria is that the minimum average frequency of binge eating re- quired for diagnosis is once weekly over the last 3 months, identical to the frequency criterion for bulimia nervosa (rather than at least 2 days a week for 6 months in DSM-IV).

1	There have been no significant changes in this diagnostic class from DSM-IV to DSM-S. The disorders in this chapter were previously classified under disorders usually first di- agnosed in infancy, childhood, or adolescence in DSM-IV and exist now as an independent classification in DSM-S. 814 Highlights of Changes From DSM-IV to DSM-5

1	In DSM-5, the DSM-IV diagnoses named sleep disorder related to another mental disorder and sleep disorder related to another medical condition have been removed, and instead greater specification of coexisting conditions is provided for each sleep-wake disorder. The diagnosis of primary insomnia has been renamed insomnia disorder to avoid the differen- tiation between primary and secondary insomnia. DSM-S also distinguishes narcolepsy— now known to be associated with hypocretin deficiency—from other forms of hypersomno- lence (hypersomnolence disorder). Finally, throughout the DSM-S classification of sleep- wake disorders, pediatric and developmental criteria and text are integrated where existing science and considerations of clinical utility support such integration. Breathing-related sleep disorders are divided into three relatively distinct disorders: obstructive sleep apnea hypopnea, central sleep apnea, and sleep-related hypoventilation. The subtypes of circadian rhythm sleep

1	sleep disorders are divided into three relatively distinct disorders: obstructive sleep apnea hypopnea, central sleep apnea, and sleep-related hypoventilation. The subtypes of circadian rhythm sleep disorders are expanded to include advanced sleep phase type and irregular sleep-wake type, whereas the jet lag type has been removed. The use of the former “not oth- ment sleep behavior disorder and restless legs syndrome to independent disorders.

1	In DSM-5, some gender-specific sexual dysfunctions have been added, and, for females, sexual desire and arousal disorders have been combined into one disorder: female sexual interestlarousal disorder. All of the sexual dysfunctions (except substance/medication-in- duced sexual dysfunction) now require a minimum duration of approximately 6 months and more precise severity criteria. Genito-pelvic pain/penetration disorder has been added to DSM-S and represents a merging of vaginismus and dyspareunia, which were highly co- morbid and difficult to distinguish. The diagnosis of sexual aversion disorder has been re- moved due to rare use and lack of supporting research.

1	There are now only two subtypes for sexual dysfunctions: lifelong versus acquired and generalized versus situational. T0 indicate the presence and degree of medical and other nonmedical correlates, the following associated features have been added to the text: partner factors, relationship factors, individual vulnerability factors, cultural or religious factors, and medical factors.

1	Gender dysphoria is a new diagnostic class in DSM-S and reﬂects a change in conceptual- ization of the disorder’s defining features by emphasizing the phenomenon of ”gender in- congruence” rather than cross-gender identification per se, as was the case in DSM-IV gender identity disorder. Gender dysphoria includes separate sets of criteria: for children and for adults and adolescents. For the adolescents and adults criteria, the previous Criterion A (cross-gender identification) and Criterion B (aversion toward one’s gender) are merged. In the wording of the criteria, ”the other sex” is replaced by ”the other gender" (or ”some alter- native gender").” Gender instead of sex is used systematically because the concept ”sex” is in- adequate when referring to individuals with a disorder of sex development. In the child criteria, ”strong desire to be of the other gender” replaces the previous ”repeatedly stated de- sire to be...the other sex” to capture the situation of some children who, in

1	In the child criteria, ”strong desire to be of the other gender” replaces the previous ”repeatedly stated de- sire to be...the other sex” to capture the situation of some children who, in a coercive envi- ronment, may not verbalize the desire to be of another gender. For children, Criterion A1 (”a strong desire to be of the other gender or an insistence that he or she is the other gender.. .)” is now necessary (but not sufficient), which makes the diagnosis more restrictive and conser- vative. The subtyping on the basis of sexual orientation is removed because the distinction is no longer considered clinically useful. A posttransition specifier has been added to identify

1	Highlights of Changes From DSM-IV to DSM-S 815 individuals who have undergone at least one medical procedure or treatment to support the new gender assignment (e.g., cross-sex hormone treatment). Although the concept of post- transition is modeled on the concept of full or partial remission, the term remission has impli- cations in terms of symptom reduction that do not apply directly to gender dysphoria. Disruptive, Impulse-Control, and Conduct Disorders

1	The chapter “Disruptive, Impulse—Control, and Conduct Disorders” is new to DSM-S and combines disorders that were previously included in the chapter “Disorders Usually First Di- agnosed in Infancy, Childhood, or Adolescence” (i.e., oppositional defiant disorder; conduct disorder; and disruptive behavior disorder not otherwise specified, now categorized as other specified and unspecified disruptive, impulse—control, and conduct disorders) and the chap- ter ”Impulse—Control Disorders Not Elsewhere Classified” (i.ei, intermittent explosive disor- der, pyromania, and kleptomania). These disorders are all characterized by problems in emotional and behavioral self—control. Notably, ADHD is frequently comorbid with the dis- orders in this chapter but is listed with the neurodevelopmental disorders. Because of its close association with conduct disorder, antisocial personality disorder is listed both in this chapter and in the chapter ”Personality Disorders,” where it is described in detail.

1	The criteria for oppositional defiant disorder are now grouped into three types: an- gry/irritable mood, argumentative/defiant behavior, and vindictiveness. Additionally, the exclusionary criterion for conduct disorder has been removed. The criteria for conduct the disorder but also present with limited prosocial emotions. The primary change in in- termittent explosive disorder is in the type of aggressive outbursts that should be consid- ered: DSM-IV required physical aggression, whereas in DSM-5 verbal aggression and nondestructive/noninjurious physical aggression also meet criteria. DSM-S also provides more specific criteria defining frequency needed to meet the criteria and specifies that the aggressive outbursts are impulsive and / or anger based in nature, and must cause marked distress, cause impairment in occupational or interpersonal functioning, or be associated with negative financial or legal consequences. Furthermore, a minimum age of 6 years (or equivalent developmental

1	cause impairment in occupational or interpersonal functioning, or be associated with negative financial or legal consequences. Furthermore, a minimum age of 6 years (or equivalent developmental level) is now required.

1	An important departure from past diagnostic manuals is that the chapter on substance—related disorders has been expanded to include gambling disorder. Another key change is that

1	DSM-S does not separate the diagnoses of substance abuse and dependence as in DSM-IV. Rather criteria are provided for substance use disorder, accompanied by criteria for intoxication, withdrawal, substance-induced disorders, and unspecified substance-related disorders, where relevant. Within substance use disorders, the DSM-IV recurrent substance-related legal problems criterion has been deleted from DSM-S, and a new criterion—craving, or a strong de- sire or urge to use a substance—has been added. In addition, the threshold for substance use disorder diagnosis in DSM—S is set at two or more criteria, in contrast to a threshold of one or more criteria for a diagnosis of DSM—IV substance abuse and three or more for DSM-IV depen- dence. Cannabis withdrawal and caffeine withdrawal are new disorders (the latter was in DSM-IV Appendix B, ”Criteria Sets and Axes Provided for Further Study").

1	DSM-IV Appendix B, ”Criteria Sets and Axes Provided for Further Study"). Severity of the DSM-S substance use disorders is based on the number of criteria en- dorsed. The DSM-IV specifier for a physiological subtype is eliminated in DSM-S, as is the DSM-IV diagnosis of polysubstance dependence. Early remission from a DSM-S substance use disorder is defined as at least 3 but less than 12 months without meeting substance use disorder criteria (except craving), and sustained remission is defined as at least 12 months without meeting criteria (except craving). Additional new DSM-S specifiers include ”in a controlled environment” and ”on maintenance therapy” as the situation warrants. 816 Highlights of Changes From DSM-IV to DSM-5

1	816 Highlights of Changes From DSM-IV to DSM-5 The DSM-IV diagnoses of dementia and amnestic disorder are subsumed under the newly named entity maj or neurocognitive disorder (NCD). The term dementia is not precluded from use in the etiological subtypes where that term is standard. Furthermore, DSM-S now recog- nizes a less severe level of cognitive impairment, mild NCD, which is a new disorder that per- mits the diagnosis of less disabling syndromes that may nonetheless be the focus of concern and treatment. Diagnostic criteria are provided for both of these disorders, followed by diag- nostic criteria for different etiological subtypes. In DSM-IV, individual diagnoses were desig- nated for dementia of the Alzheimer’s type, vascular dementia, and substance-induced dementia, whereas the other neurodegenerative disorders were classified as dementia due to another medical condition, with HIV, head trauma, Parkinson’s disease, Huntington’s disease,

1	Pick’s disease, Creutzfeldt-Jakob disease, and other medical conditions specified. In DSM-S, major or mild NCD due to Alzheimer's disease and major or mild vascular NCD have been re— tained, while new separate criteria are now presented for major or mild frontotemporal NCD, NCD with Lewy bodies, and NCDs due to traumatic brain injury, a substance/ medication, HIV infection, prion disease, Parkinson’s disease, Huntington’s disease, another medical con- dition, and multiple etiologies, respectively. Unspecified NCD is also included as a diagnosis. The criteria for personality disorders in Section II of DSM-5 have not changed from those in DSM-IV. An alternative approach to the diagnosis of personality disorders was developed for Personality Disorders”). For the general criteria for personality disorder, presented in

1	Section III, a revised personality functioning criterion (Criterion A) has been developed based on a literature review of reliable clinical measures of core impairments central to per- sonality pathology. A diagnosis of personality disorder—trait specified, based on moderate or greater impairment in personality functioning and the presence of pathological personal- ity traits, replaces personality disorder not otherwise specified and provides a much more in- formative diagnosis for individuals who are not optimally described as having a specific personality disorder. A greater emphasis on personality functioning and trait—based criteria increases the stability and empirical bases of the disorders. Personality functioning and per- sonality traits also can be assessed whether or not the individual has a personality disor- der—a feature that provides clinically useful information about all individuals.

1	An overarching change from DSM-IV is the addition of the course specifiers ”in a controlled environment” and ”in remission” to the diagnostic criteria sets for all the paraphilic disor- ders. These specifiers are added to indicate important changes in an individual’s status. In

1	DSM-S, paraphilias are not ipsofucto mental disorders. There is a distinction between paraphil- ias and paraphilic disorders. A paraphilic disorder is a paraphilia that is currently causing dis- tress or impairment to the individual or a paraphilia whose satisfaction has entailed personal harm, or risk of harm, to others. A paraphilia is a necessary but not a sufficient condition for having a paraphilic disorder, and a paraphilia by itself does not automatically justify or require clinical intervention. The distinction between paraphilias and paraphilic disorders was im- plemented without making any changes to the basic structure of the diagnostic criteria as they had existed since DSM-III-R. The change proposed for DSM-S is that individuals who meet both Criterion A and Criterion B would now be diagnosed as having a paraphilic disorder. A diagnosis would not be given to individuals whose symptoms meet Criterion A but not Cri- terion B—that is, to individuals who have a paraphilia but

1	now be diagnosed as having a paraphilic disorder. A diagnosis would not be given to individuals whose symptoms meet Criterion A but not Cri- terion B—that is, to individuals who have a paraphilia but not a paraphilic disorder.

1	*:._ , Glossary of affect A pattern of observable behaviors that is the expression of a subjectively experi- enced feeling state (emotion). Examples of affect include sadness, elation, and anger. In contrast to mood, which refers to a pervasive and sustained emotional ”climate,” affect refers to more ﬂuctuating changes in emotional "weather." What is considered the nor- mal range of the expression of affect varies considerably, both within and among dif- ferent cultures. Disturbances in affect include blunted Significant reduction in the intensity of emotional expression. flat Absence or near absence of any sign of affective expression. inappropriate Discordance between affective expression and the content of speech or ideation. labile Abnormal variability in affect with repeated, rapid, and abrupt shifts in af- fective expression. restricted or constricted Mild reduction in the range and intensity of emotional ex- pression. affective blunting See AFFECT.

1	restricted or constricted Mild reduction in the range and intensity of emotional ex- pression. affective blunting See AFFECT. agitation (psychomotor) See PSYCHOMOTOR AGITATION. agnosia Loss of ability to recognize objects, persons, sounds, shapes, or smells that occurs in the absence of either impairment of the specific sense or significant memory loss. alogia An impoverishment in thinking that is inferred from observing speech and lan- guage behavior. There may be brief and concrete replies to questions and restriction in the amount of spontaneous speech (termed poverty of speech). Sometimes the speech is adequate in amount but conveys little information because it is overconcrete, overab— stract, repetitive, or stereotyped (termed poverty of content). amnesia An inability to recall important autobiographical information that is inconsis- tent with ordinary forgetting.

1	amnesia An inability to recall important autobiographical information that is inconsis- tent with ordinary forgetting. anhedonia Lack of enjoyment from, engagement in, or energy for life’s experiences; def- icits in the capacity to feel pleasure and take interest in things. Anhedonia is a facet of the broad personality trait domain DETACHMENT. anosognosia A condition in which a person with an illness seems unaware of the exis- tence of his or her illness. antagonism Behaviors that put an individual at odds with other people, such as an ex- aggerated sense of self—importance with a concomitant expectation of Special treat— ment, as well as a callous antipathy toward others, encompassing both unawareness of others’ needs and feelings, and a readiness to use others in the service of self-enhance- ment. Antagonism is one of the five broad PERSONALITY TRAIT DOMAINS defined in Sec- tion 111 ”Alternative DSM-S Model for Personality Disorders.”

1	SMALL CAPS indicate term found elsewhere in this glossary. Glossary definitions were informed by DSM-5 Work Groups, publicly available Internet sources, and previously published glossaries for mental disorders (World Health Organization and American Psychiatric Association). 818 Glossary of Technical Terms antidepressant discontinuation syndrome A set of symptoms that can occur after abrupt cessation, or marked reduction in dose, of an antidepressant medication that had been taken continuously for at least 1 month. anxiety The apprehensive anticipation of future danger or misfortune accompanied by a feeling of worry, distress, and / or somatic symptoms of tension. The focus of antici- pated danger may be internal or external.

1	anxiousness Feelings of nervousness or tenseness in reaction to diverse situations; frequent worry about the negative effects of past unpleasant experiences and future negative possi- bilities; feeling fearful and apprehensive about uncertainty; expecting the worst to happen. Anxiousness is a facet of the broad personality trait domain N EGATIVE AFFECI'IVITY. arousal The physiological and psychological state of being awake or reactive to stimuli. asociality A reduced initiative for interacting with other people. attention The ability to focus in a sustained manner on a particular stimulus or activity. finishing tasks or in concentrating on work. attention seeking Engaging in behavior designed to attract notice and to make oneself the focus of others’ attention and admiration. Attention seeking is a facet of the broad personality trait domain ANTAGONISM. autogynephilia Sexual arousal of a natal male associated with the idea or image of being a woman.

1	autogynephilia Sexual arousal of a natal male associated with the idea or image of being a woman. avoidance The act of keeping away from stress-related circumstances; a tendency to cir- cumvent cues, activities, and situations that remind the individual of a stressful event experienced. avolition An inability to initiate and persist in goal-directed activities. When severe enough to be considered pathological, avolition is pervasive and prevents the person from com- pleting many different types of activities (e.g., work, intellectual pursuits, self-care). bereavement The state of having lost through death someone with whom one has had a close relationship. This state includes a range of grief and mourning responses. biological rhythms See CIRCADIAN RHYTHMS.

1	biological rhythms See CIRCADIAN RHYTHMS. callousness Lack of concern for the feelings or problems of others; lack of guilt or re- morse about the negative or harmful effects of one’s actions on others. Callousness is a facet of the broad personality trait domain ANTAGONISM. catalepsy Passive induction of a posture held against gravity. Compare with WAXY FLEX- IBILITY. cataplexy Episodes of sudden bilateral loss of muscle tone resulting in the individual collapsing, often occurring in association with intense emotions such as laughter, an— ger, fear, or surprise. circadian rhythms Cyclical variations in physiological and biochemical function, level of sleep-wake activity, and emotional state. Circadian rhythms have a cycle of about 24 hours, ultradian rhythms have a cycle that is shorter than 1 day, and infmdian rhythms have a cycle that may last weeks or months.

1	ences, including DEPERSONALIZATION, DEREALIZATION, and DISSOCIATION; mixed sleep- wake state experiences; and thought-control experiences. Cognitive and perceptual dysregulation is a facet of the broad personality trait domain PSYCHOTICISM. coma State of complete loss of consciousness. Glossary of Technical Terms 819 compulsion Repetitive behaviors (e.g., hand washing, ordering, checking) or mental acts (e.g., praying, counting, repeating words silently) that the individual feels driven to perform in response to an obsession, or according to rules that must be applied rig- idly. The behaviors or mental acts are aimed at preventing or reducing anxiety or dis- tress, or preventing some dreaded event or situation; however, these behaviors or mental acts are not connected in a realistic way with what they are designed to neutral- ize or prevent or are clearly excessive.

1	conversion symptom A loss of, or alteration in, voluntary motor or sensory functioning, with or without apparent impairment of consciousness. The symptom is not fully ex- plained by a neurological or another medical condition or the direct effects of a sub- stance and is not intentionally produced or feigned. deceitfulness Dishonesty and fraudulence; misrepresentation of self; embellishment or fabrication when relating events. Deceitfulness is a facet of the broad personality trait domain ANTAGONISM. defense mechanism Mechanisms that mediate the individual’s reaction to emotional conﬂicts and to external stressors. Some defense mechanisms (e.g., projection, splitting, acting out) are almost invariably maladaptive. Others (e.g., suppression, denial) may be either maladaptive or adaptive, depending on their severity, their inﬂexibility, and the context in which they occur.

1	delusion A false belief based on incorrect inference about external reality that is firmly vertible and obvious proof or evidence to the contrary. The belief is not ordinarily ac- cepted by other members of the person’s culture or subculture (i.e., it is not an article of religious faith). When a false belief involves a value judgment, it is regarded as a delusion only when the judgment is so extreme as to defy credibility. Delusional conviction can sometimes be inferred from an overvalued idea (in which case the individual has an un- reasonable belief or idea but does not hold it as firmly as is the case with a delusion). De- lusions are subdivided according to their content. Common types are listed below: bizarre A delusion that involves a phenomenon that the person’s culture would re- gard as physically impossible. delusional jealousy A delusion that one's sexual partner is unfaithful.

1	delusional jealousy A delusion that one's sexual partner is unfaithful. erotomanic A delusion that another person, usually of higher status, is in love with the individual. grandiose A delusion of inﬂated worth, power, knowledge, identity, or special re- lationship to a deity or famous person. mixed type Delusions of more than one type (e.g., EROTOMANIC, GRANDIOSE, PERSE- CUTORY, SOMATIC) in which no one theme predominates. mood-congruent See MOOD-CONGRUENT PSYCHOTIC FEATURES. mood-incongruent See MOOD—INCONGRUENT PSYCHOTIC FEATURES. of being controlled A delusion in which feelings, impulses, thoughts, or actions are experienced as being under the control of some external force rather than be- ing under one’s own control.

1	of being controlled A delusion in which feelings, impulses, thoughts, or actions are experienced as being under the control of some external force rather than be- ing under one’s own control. of reference A delusion in which events, objects, or other persons in one’s immedi- ate environment are seen as having a particular and unusual significance. These delusions are usually of a negative or pejorative nature but also may be grandiose in content. A delusion of reference differs from an idea of reference, in which the false belief is not as firmly held nor as fully organized into a true belief. persecutory A delusion in which the central theme is that one (or someone to whom one is close) is being attacked, harassed, cheated, persecuted, or conspired against. 820 Glossary of Technical Terms somatic A delusion whose main content pertains to the appearance or functioning of one’s body.

1	820 Glossary of Technical Terms somatic A delusion whose main content pertains to the appearance or functioning of one’s body. thought broadcasting A delusion that one’s thoughts are being broadcast out loud so that they can be perceived by others. thought insertion A delusion that certain of one’s thoughts are not one’s own, but rather are inserted into one’s mind. depersonalization The experience of feeling detached from, and as if one is an outside observer of, one’s mental processes, body, or actions (e.g., feeling like one is in a dream; a sense of unreality of self, perceptual alterations; emotional and / or physical numbing; temporal distortions; sense of unreality).

1	depressivity Feelings of being intensely sad, miserable, and / or hopeless. Some patients describe an absence of feelings and/ or dysphoria; difficulty recovering from such moods; pessimism about the future; pervasive shame and/ or guilt; feelings of inferior self—worth; and thoughts of suicide and suicidal behavior. Depressivity is a facet of the broad personality trait domain DETACHMENT. derealization The experience of feeling detached from, and as if one is an outside ob- server of, one’s surroundings (e.g., individuals or objects are experienced as unreal, dreamlike, foggy, lifeless, or visually distorted). detachment Avoidance of socioemotional experience, including both WTTHDRAWAL from interpersonal interactions (ranging from casual, daily interactions to friendships and inti- mate relationships [i.e., H\1TIMACY AVOIDANCED and RESTRICTED AFFECTIVITY, particularly limited hedonic capacity. Detachment is one of the five pathological PERSONALITY TRAIT

1	DOMAINS defined in Section III "Altemative DSM-5 Model for Personality Disorders.” disinhibition Orientation toward immediate gratification, leading to impulsive behav- ior driven by current thoughts, feelings, and external stimuli, without regard for past learning or consideration of future consequences. RIGID PERFECTIONISM, the opposite pole of this domain, reﬂects excessive constraint of impulses, risk avoidance, hyper- responsibility, hyperperfectionism, and rigid, rule—governed behavior. Disinhibition is one of the five pathological PERSONALITY TRAIT DOMAINS defined in Section 111 ”Al- ternative DSM-S Model for Personality Disorders." disorder of sex development Condition of significant inborn somatic deviations of the reproductive tract from the norm and / or of discrepancies among the biological indica- tors of male and female. disorientation Confusion about the time of day, date, or season (time); where one is (place); or who one is (person).

1	disorientation Confusion about the time of day, date, or season (time); where one is (place); or who one is (person). dissociation The splitting off of clusters of mental contents from conscious awareness. Dissociation is a mechanism central to dissociative disorders. The term is also used to describe the separation of an idea from its emotional significance and affect, as seen in the inappropriate affect in schizophrenia. Often a result of psychic trauma, dissociation may allow the individual to maintain allegiance to two contradictory truths while re- maining unconscious of the contradiction. An extreme manifestation of dissociation is dissociative identity disorder, in which a person may exhibit several independent per- sonalities, each unaware of the others.

1	distractibility Difficulty concentrating and focusing on tasks; attention is easily divert- ed by extraneous stimuli; difficulty maintaining goal-focused behavior, including both planning and completing tasks. Distractibility is a facet of the broad personality trait domain DISINHIBITION. dysarthria A disorder of speech sound production due to structural or motor impair- ment affecting the articulatory apparatus. Such disorders include cleft palate, muscle Glossary of Technical Terms 821 disorders, cranial nerve disorders, and cerebral palsy affecting bulbar structures (i.e., lower and upper motor neuron disorders). dyskinesia Distortion of voluntary movements with involuntary muscle activity. of depression, discontent, and in some cases indifference to the world around them. dyssomnias Primary disorders of sleep or wakefulness characterized by INSOMNIA or

1	of depression, discontent, and in some cases indifference to the world around them. dyssomnias Primary disorders of sleep or wakefulness characterized by INSOMNIA or HYPERSOMNIA as the major presenting symptom. Dyssomnias are disorders of the amount, quality, or timing of sleep. Compare with PARASOMNIAS. dysthymia Presence, while depressed, of two or more of the following: 1) poor appetite or overeating, 2) insomnia or hypersomnia, 3) low energy or fatigue, 4) low self—esteem, 5) poor concentration or difficulty making decisions, or 6) feelings of hopelessness. dystonia Disordered tonicity of muscles. eccentricity Odd, unusual, or bizarre behavior, appearance, and / or speech having strange and unpredictable thoughts; saying unusual or inappropriate things. Eccentric- ity is a facet of the broad personality trait domain PSYCHOTICISM. echolalia The pathological, parrotlike, and apparently senseless repetition (echoing) of a word or phrase just spoken by another person.

1	echolalia The pathological, parrotlike, and apparently senseless repetition (echoing) of a word or phrase just spoken by another person. echopraxia Mimicking the movements of another. emotional lability Instability of emotional experiences and mood; emotions that are easily aroused, intense, and/ or out of proportion to events and circumstances. Emo- tional lability is a facet of the broad personality trait domain NEGATIVE AFFECTIVITY. empathy Comprehension and appreciation of others’ experiences and motivations; tol- erance of differing perspectives; understanding the effects of own behavior on others.

1	empathy Comprehension and appreciation of others’ experiences and motivations; tol- erance of differing perspectives; understanding the effects of own behavior on others. episode (episodic) A specified duration of time during which the patient has developed or experienced symptoms that meet the diagnostic criteria for a given mental disorder. De- pending on the type of mental disorder, episode may denote a certain number of symptoms or a specified severity or frequency of symptoms. Episodes may be further differentiated as a single (first) episode or a recurrence or relapse of multiple episodes if appropriate. ings of well-being, elation, happiness, excitement, and joy. fatigability Tendency to become easily fatigued. See also FATIGUE.

1	ings of well-being, elation, happiness, excitement, and joy. fatigability Tendency to become easily fatigued. See also FATIGUE. fatigue A state (also called exhaustion, tiredness, lethargy, languidness, languor, lassi- tude, and listlessness) usually associated with a weakening or depletion of one’s phys- ical and / or mental resources, ranging from a general state of lethargy to a specific, work-induced burning sensation within one’s muscles. Physical fatigue leads to an in- ability to continue functioning at one’s normal level of activity. Although widespread in everyday life, this state usually becomes particularly noticeable during heavy exer- cise. Mental fatigue, by contrast, most often manifests as SOMNOLENCE (sleepiness). fear An emotional response to perceived imminent threat or danger associated with urges to flee or fight. ﬂashback A dissociative state during which aspects of a traumatic event are reexperi- enced as though they were occurring at that moment.

1	ﬂashback A dissociative state during which aspects of a traumatic event are reexperi- enced as though they were occurring at that moment. ﬂight of ideas A nearly continuous flow of accelerated speech with abrupt changes from topic to topic that are usually based on understandable associations, distracting stimuli, or plays on words. When the condition is severe, speech may be disorganized and incoherent. 822 Glossary of Technical Terms gender The public (and usually legally recognized) lived role as boy or girl, man or woman. Biological factors are seen as contributing in interaction with social and psy- chological factors to gender development. gender assignment The initial assignment as male or female, which usually occurs at birth and is subsequently referred to as the ”natal gender.” gender dysphoria Distress that accompanies the incongruence between one’s experi- enced and expressed gender and one’s assigned or natal gender.

1	gender experience The unique and personal ways in which individuals experience their gender in the context of the gender roles provided by their societies. gender expression The specific ways in which individuals enact gender roles provided in their societies. gender identity A category of social identity that refers to an individual’s identification as male, female or, occasionally, some category other than male or female. gender reassignment A change of gender that can be either medical (hormones, sur- gery) or legal (government recognition), or both. In case of medical interventions, often referred to as sex reassignment. geometric hallucination See HALLUCINATION. grandiosity Believing that one is superior to others and deserves special treatment; self- centeredness; feelings of entitlement; condescension toward others. Grandiosity is a facet of the broad personality trait domain ANTAGONISM.

1	grimace (grimacing) Odd and inappropriate facial expressions unrelated to situation (as seen in individuals with CATATONIA). hallucination A perception-like experience with the clarity and impact of a true percep- tion but without the external stimulation of the relevant sensory organ. Hallucinations should be distinguished from ILLUSIONS, in which an actual external stimulus is misperceived or misinterpreted. The person may or may not have insight into the non— veridical nature of the hallucination. One hallucinating person may recognize the false sensory experience, whereas another may be convinced that the experience is grounded in reality. The term hallucination is not ordinarily applied to the false perceptions that occur during dreaming, while falling asleep (hypnagogic), or upon awakening (hypno— pompic). Transient hallucinatory experiences may occur without a mental disorder. auditory A hallucination involving the perception of sound, most commonly of voice.

1	auditory A hallucination involving the perception of sound, most commonly of voice. geometric Visual hallucinations involving geometric shapes such as tunnels and funnels, spirals, lattices, or cobwebs. gustatory A hallucination involving the perception of taste (usually unpleasant). mood-congruent See MOOD-CONGRUENT PSYCHOTIC FEATURES. mood-incongruent See MOOD-INCONGRUENT PSYCHOTIC FEATURES. olfactory A hallucination involving the perception of odor, such as of burning rub- ber or decaying fish. somatic A hallucination involving the perception of physical experience localized within the body (e.g., a feeling of electricity). A somatic hallucination is to be dis- medical condition, from hypochondriacal preoccupation with normal physical sensations, or from a tactile hallucination. tactile A hallucination involving the perception of being touched or of something being under one’s skin. The most common tactile hallucinations are the sensation

1	tactile A hallucination involving the perception of being touched or of something being under one’s skin. The most common tactile hallucinations are the sensation Glossary of Technical Terms 823 of electric shocks and formication (the sensation of something creeping or crawl- ing on or under the skin). visual A hallucination involving sight, which may consist of formed images, such as of people, or of unformed images, such as ﬂashes of light. Visual hallucinations should be distinguished from ILLUSIONS, which are misperceptions of real external stimuli. hostility Persistent or frequent angry feelings; anger or irritability in response to minor slights and insults; mean, nasty, or vengeful behavior. Hostility is a facet of the broad personality trait domain ANTAGONISM. hyperacusis Increased auditory perception. hyperorality A condition in which inappropriate objects are placed in the mouth. hypersexuality A stronger than usual urge to have sexual activity.

1	hyperacusis Increased auditory perception. hyperorality A condition in which inappropriate objects are placed in the mouth. hypersexuality A stronger than usual urge to have sexual activity. hypersomnia Excessive sleepiness, as evidenced by prolonged nocturnal sleep, difficul- ty maintaining an alert awake state during the day, or undesired daytime sleep epi- sodes. See also SOMNOLENCE.

1	hypervigilance An enhanced state of sensory sensitivity accompanied by an exaggerated intensity of behaviors whose purpose is to detect threats. Hypervigilance is also accompa- nied by a state of increased anxiety which can cause exhaustion. Other symptoms include abnormally increased arousal, a high responsiveness to stimuli, and a continual scanning of the environment for threats. In hypervigilance, there is a perpetual scanning of the envi- ronment to search for sights, sounds, people, behaviors, smells, or anything else that is rem- iniscent of threat or trauma. The individual is placed on high alert in order to be certain danger is not near. Hypervigilance can lead to a variety of obsessive behavior patterns, as well as producing difficulties with social interaction and relationships. hypomania An abnormality of mood resembling mania but of lesser intensity. See also MANIA. hypopnea Episodes of overly shallow breathing or an abnormally low respiratory rate.

1	hypomania An abnormality of mood resembling mania but of lesser intensity. See also MANIA. hypopnea Episodes of overly shallow breathing or an abnormally low respiratory rate. ideas of reference The feeling that causal incidents and external events have a particu- lar and unusual meaning that is specific to the person. An idea of reference is to be dis- tinguished from a DELUSION OF REFERENCE, in which there is a belief that is held with delusional conviction. identity Experience of oneself as unique, with clear boundaries between self and others; stability of self—esteem and accuracy of self—appraisal; capacity for, and ability to regu- late, a range of emotional experience. illusion A misperception or misinterpretation of a real external stimulus, such as hear- ing the rustling of leaves as the sound of voices. See also HALLUCINATION.

1	illusion A misperception or misinterpretation of a real external stimulus, such as hear- ing the rustling of leaves as the sound of voices. See also HALLUCINATION. impulsivity Acting on the spur of the moment in response to immediate stimuli; acting on a momentary basis without a plan or consideration of outcomes; difficulty establish- ing and following plans; a sense of urgency and self—harming behavior under emotion- al distress. Impulsivity is a facet of the broad personality trait domain DISINHIBITION.

1	incoherence Speech or thinking that is essentially incomprehensible to others because word or phrases are joined together without a logical or meaningful connection. This disturbance occurs within clauses, in contrast to derailment, in which the disturbance is between clauses. This has sometimes been referred to a ”word salad” to convey the degree of linguistic disorganization. Mildly ungrammatical constructions or idiomatic usages characteristic of a particular regional or cultural backgrounds, lack of educa- tion, or low intelligence should not be considered incoherence. The term is generally not applied when there is evidence that the disturbance in speech is due to an aphasia. insomnia A subjective complaint of difficulty falling or staying asleep or poor sleep quality. 824 Glossary of Technical Terms ological indicators of sex. intimacy Depth and duration of connection with others; desire and capacity for close- ness; mutuality of regard reflected in interpersonal behavior.

1	intimacy Depth and duration of connection with others; desire and capacity for close- ness; mutuality of regard reflected in interpersonal behavior. intimacy avoidance Avoidance of close or romantic relationships, interpersonal attach- ments, and intimate sexual relationships. Intimacy avoidance is a facet of the broad personality trait domain DETACHMENT. irresponsibility Disregard for—and failure to honor—financial and other obligations or commitments; lack of respect for—and lack of follow-through on—agreements and promises; carelessness with others’ property. Irresponsibility is a facet of the broad per- sonality trait domain DISINHIBITION.

1	language pragmatics The understanding and use of language in a given context. For example, the warning ”Watch your hands” when issued to a child who is dirty is in- tended not only to prompt the child to look at his or her hands but also to communicate the admonition ”Don’t get anything dirty." lethargy A state of decreased mental activity, characterized by sluggishness, drowsi- ness, inactivity, and reduced alertness. macropsia The visual perception that objects are larger than they actually are. Compare with MICROPSIA. magical thinking The erroneous belief that one’s thoughts, words, or actions will cause cause and effect. Magical thinking may be a part of normal child development. mania A mental state of elevated, expansive, or irritable mood and persistently in- creased level of activity or energy. See also HYPOMANLA.

1	mania A mental state of elevated, expansive, or irritable mood and persistently in- creased level of activity or energy. See also HYPOMANLA. manipulativeness Use of subterfuge to inﬂuence or control others; use of seduction, charm, glibness, or ingratiation to achieve one’s ends. Manipulativeness is a facet of the broad personality trait domain ANTAGONISM. mannerism A peculiar and characteristic individual style of movement, action, thought, or speech. melancholia (melancholic) A mental state characterized by very severe depression. micropsia The Visual perception that objects are smaller than they actually are. Com- pare with MACROPSIA.

1	melancholia (melancholic) A mental state characterized by very severe depression. micropsia The Visual perception that objects are smaller than they actually are. Com- pare with MACROPSIA. mixed symptoms The specifier ”with mixed features” is applied to mood episodes during which subthreshold symptoms from the opposing pole are present. Whereas these con- current ”mixed” symptoms are relatively simultaneous, they may also occur closely juxtaposed in time as a waxing and waning of individual symptoms of the opposite pole (i.e., depressive symptoms during hypomanic or manic episodes, and vice versa).

1	mood A pervasive and sustained emotion that colors the perception of the world. Com- mon examples of mood include depression, elation, anger, and anxiety. In contrast to aﬂect, which refers to more ﬂuctuating changes in emotional ”weather," mood refers to a pervasive and sustained emotional ”climate." Types of mood include dysphoric An unpleasant mood, such as sadness, anxiety, or irritability. elevated An exaggerated feeling of well—being, or euphoria or elation. A person with elevated mood may describe feeling ”high,” ”ecstatic,” ”on top of the world," or "up in the clouds.” euthymic Mood in the ”normal” range, which implies the absence of depressed or elevated mood. Glossary of Technical Terms 825 expansive Lack of restraint in expressing one’s feelings, frequently with an over- valuation of one’s significance or importance. irritable Easily annoyed and provoked to anger.

1	irritable Easily annoyed and provoked to anger. mood-congruent psychotic features Delusions or hallucinations whose content is en- tirely consistent with the typical themes of a depressed or manic mood. If the mood is depressed, the content of the delusions or hallucinations would involve themes of per- sonal inadequacy, guilt, disease, death, nihilism, or deserved punishment. The content of the delusion may include themes of persecution if these are based on self—derogatory concepts such as deserved punishment. If the mood is manic, the content of the delusions or hallucinations would involve themes of inﬂated worth, power, knowledge, or iden- tity, or a special relationship to a deity or a famous person. The content of the delusion may include themes of persecution if these are based on concepts such as inflated worth or deserved punishment.

1	mood-incongruent psychotic features Delusions or hallucinations whose content is not consistent with the typical themes of a depressed or manic mood. In the case of depres- sion, the delusions or hallucinations would not involve themes of personal inadequacy, guilt, disease, death, nihilism, or deserved punishment. In the case of mania, the delu- sions or hallucinations would not involve themes of inﬂated worth, power, knowledge, or identity, or a special relationship to a deity or a famous person. multiple sleep latency test Polysomnographic assessment of the sleep-onset period, with several short sleep-wake cycles assessed during a single session. The test repeat- edly measures the time to daytime sleep onset (”sleep latency”) and occurrence of and time to onset of the rapid eye movement sleep phase. mutism No, or very little, verbal response (in the absence of known aphasia).

1	mutism No, or very little, verbal response (in the absence of known aphasia). narcolepsy Sleep disorder characterized by periods of extreme drowsiness and frequent daytime lapses into sleep (sleep attacks). These must have been occurring at least three times per week over the last 3 months (in the absence of treatment). negative affectivity Frequent and intense experiences of high levels of a wide range of negative emotions (e.g., anxiety, depression, guilt/shame, worry, anger), and their be- havioral (e.g., self—harm) and interpersonal (e.g., dependency) manifestations. Nega- tive Affectivity is one of the five pathological PERSONALITY TRAIT DOMAINS defined in Section III ”Altemative DSM-5 Model for Personality Disorders.” negativism Opposition to suggestion or advice; behavior opposite to that appropriate to a specific situation or against the wishes of others, including direct resistance to efforts to be moved.

1	night eating syndrome Recurrent episodes of night eating, as manifested by eating after awakening from sleep or excessive food consumption after the evening meal. There is awareness and recall of the eating. The night eating is not better accounted for by ex- ternal inﬂuences such as changes in the individual’s sleep-wake cycle or by local social norms. nightmare disorder Repeated occurrences of extended, extremely dysphoric, and well- remembered dreams that usually involve efforts to avoid threats to survival, security or physical integrity and that generally occur during the second half of the major sleep episode. On awakening from the dysphoric dreams, the individual rapidly becomes oriented and alert. lated to any substance of abuse that shares some features with substance-induced addiction.

1	lated to any substance of abuse that shares some features with substance-induced addiction. 826 Glossary of Technical Terms obsession Recurrent and persistent thoughts, urges, or images that are experienced, at some time during the disturbance, as intrusive and unwanted and that in most individ- uals cause marked anxiety or distress. The individual attempts to ignore or suppress such thoughts, urges, or images, or to neutralize them with some other thought or ac- tion (i.e., by performing a compulsion). overeating Eating too much food too quickly. overvalued idea An unreasonable and sustained belief that is maintained with less than delusional intensity (i.e., the person is able to acknowledge the possibility that the be- lief may not be true). The belief is not one that is ordinarily accepted by other members of the person’s culture or subculture.

1	panic attacks Discrete periods of sudden onset of intense fear or terror, often associated with feelings of impending doom. During these attacks there are symptoms such as shortness of breath or smothering sensations; palpitations, pounding heart, or acceler- ated heart rate; chest pain or discomfort; choking; and fear of going crazy or losing con- trol. Panic attacks may be unexpected, in which the onset of the attack is not associated with an obvious trigger and instead occurs ”out of the blue,” or expected, in which the panic attack is associated with an obvious trigger, either internal or external. paranoid ideation Ideation, of less than delusional proportions, involving suspicious- ness or the belief that one is being harassed, persecuted, or unfairly treated. parasomnias Disorders of sleep involving abnormal behaviors or physiological events occurring during sleep or sleep-wake transitions. Compare with DYSSOMNIAS.

1	parasomnias Disorders of sleep involving abnormal behaviors or physiological events occurring during sleep or sleep-wake transitions. Compare with DYSSOMNIAS. perseveration Persistence at tasks or in particular way of doing things long after the be- havior has ceased to be functional or effective; continuance of the same behavior de- spite repeated failures or clear reasons for stopping. Perseveration is a facet of the broad personality trait domain NEGATIVE AFFECTIVITY. personality Enduring patterns of perceiving, relating to, and thinking about the envi- ronment and oneself. PERSONALITY TRAITS are prominent aspects of personality that are exhibited in relatively consistent ways across time and across situations. Personality traits influence self and interpersonal functioning. Depending on their severity, im- presence of a personality disorder.

1	sonality Disorders,” a proposed diagnostic category for use when a personality disor- der is considered present but the criteria for a specific disorder are not met. Personality disorder—trait specified (PD-TS) is defined by significant impairment in personality functioning, as measured by the Level of Personality Functioning Scale and one or more pathological PERSONALITY TRAN" DOMAINS or PERSONALITY TRAIT FACETS. PD—TS is proposed in DSM-5 Section III for further study as a possible future replacement for other specified personality disorder and unspecified personality disorder. personality functioning Cognitive models of self and others that shape patterns of emo- tional and affiliative engagement. personality trait A tendency to behave, feel, perceive, and think in relatively consistent ways across time and across situations in which the trait may be manifest.

1	personality trait A tendency to behave, feel, perceive, and think in relatively consistent ways across time and across situations in which the trait may be manifest. personality trait facets Specific personality components that make up the five broad per- sonality trait domains in the dimensional taxonomy of Section III ”Altemative DSM-S Model for Personality Disorders." For example, the broad domain antagonism has the following component facets: MANIPULA'I'IVENESS, DECEITFULNESS, GRANDIOSITY, ATTEN- TION SEEKING, CALLOUSNESS, and HOSTILITY. Glossary of Technical Terms 827 personality trait domains In the dimensional taxonomy of Section III ”Alternative DSM- 5 Model for Rersonality Disorders,” personality traits are organized into five broad do- mains: NEGATIVE AFFECTIVITY, DETACHMENT, ANTAGONISM, DISINHIBITION, and PSY- CHOTICISM. Within these five broad trait domains are 25 specific personality trait facets (e.g., IMPULSIVITY, RIGID PERFECTIONISM).

1	CHOTICISM. Within these five broad trait domains are 25 specific personality trait facets (e.g., IMPULSIVITY, RIGID PERFECTIONISM). phobia A persistent fear of a specific object, activity, or situation (i.e., the phobic stimu- lus) out of proportion to the actual danger posed by the specific object or situation that results in a compelling desire to avoid it. If it cannot be avoided, the phobic stimulus is endured with marked distress. pica Persistent eating of nonnutritive nonfood substances over a period of at least 1 month. The eating of nonnutritive nonfood substances is inappropriate to the developmental level of the individual (a minimum age of 2 years is suggested for diagnosis). The eat- ing behavior is not part of a culturally supported or socially normative practice.

1	polysomnography Polysomnography (PSG), also known as a sleep study, is a multipa- rametric test used in the study of sleep and as a diagnostic tool in sleep medicine. The test result is called a polysomnogram, also abbreviated PSG. PSG monitors many body functions, including brain (electroencephalography), eye movements (electro-oculog- raphy), muscle activity or skeletal muscle activation (electromyography), and heart rhythm (electrocardiography). posturing Spontaneous and active maintenance of a posture against gravity (as seen in CATATONIA). Abnormal posturing may also be a sign of certain injuries to the brain or spinal cord, including the following: decerebrate posture The arms and legs are out straight and rigid, the toes point downward, and the head is arched backward. decorticate posture The body is rigid, the arms are stiff and bent, the fists are tight, and the legs are straight out. opisthotonus The back is rigid and arching, and the head is thrown backward.

1	decorticate posture The body is rigid, the arms are stiff and bent, the fists are tight, and the legs are straight out. opisthotonus The back is rigid and arching, and the head is thrown backward. An affected person may alternate between different postures as the condition changes. pressured speech Speech that is increased in amount, accelerated, and difficult or impossi- ble to interrupt. Usually it is also loud and emphatic. Frequently the person talks without any social stimulation and may continue to talk even though no one is listening. prodrome An early or premonitory sign or symptom of a disorder. pseudocyesis A false belief of being pregnant that is associated with objective signs and reported symptoms of pregnancy. psychological distress A range of symptoms and experiences of a person’s internal life that are commonly held to be troubling, confusing, or out of the ordinary.

1	psychological distress A range of symptoms and experiences of a person’s internal life that are commonly held to be troubling, confusing, or out of the ordinary. psychometric measures Standardized instruments such as scales, questionnaires, tests, and assessments that are designed to measure human knowledge, abilities, attitudes, or personality traits. psychomotor agitation Excessive motor activity associated with a feeling of inner tension. The activity is usually nonproductive and repetitious and consists of behaviors such as pac- ing, fidgeting, wringing of the hands, pulling of clothes, and inability to sit still. psychomotor retardation Visible generalized slowing of movements and speech. psychotic features Features characterized by delusions, hallucinations, and formal thought disorder.

1	psychomotor retardation Visible generalized slowing of movements and speech. psychotic features Features characterized by delusions, hallucinations, and formal thought disorder. psychoticism Exhibiting a wide range of culturally incongruent odd, eccentric, or un- usual behaviors and cognitions, including both process (e.g., perception, dissociation) 828 Glossary of Technical Terms and content (e.g., beliefs). Psychoticism is one of the five broad PERSONALITY TRAIT DO- MAINS defined in Section III ”Alternative DSM—S Model for Personality Disorders.” purging disorder Eating disorder characterized by recurrent purging behavior to inﬂu- ence weight or shape, such as self—induced vomiting, misuse of laxatives, diuretics, or other medications, in the absence of binge eating.

1	racing thoughts A state in which the mind uncontrollably brings up random thoughts and memories and switches between them very quickly. Sometimes the thoughts are related, with one thought leading to another; other times they are completely random. A person experiencing an episode of racing thoughts has no control over them and is unable to focus on a single topic or to sleep. rapid cycling Term referring to bipolar disorder characterized by the presence of at least four mood episodes in the previous 12 months that meet the criteria for a manic, hypo- manic, or major depressive episode. Episodes are demarcated either by partial or full remissions of at least 2 months or by a switch to an episode of the opposite polarity (e.g., major depressive episode to manic episode). The rapid cycling specifier can be ap- plied to bipolar I or bipolar II disorder.

1	rapid eye movement (REM) A behavioral sign of the phase of sleep during which the sleeper is likely to be experiencing dreamlike mental activity. repetitive speech Morphologically heterogeneous iterations of speech. residual phase Period after an episode of schizophrenia that has partly or completed re- mitted but in which some symptoms may remain, and symptoms such as listlessness, problems with concentrating, and withdrawal from social activities may predominate. restless legs syndrome An urge to move the legs, usually accompanied or caused by uncomfortable and unpleasant sensations in the legs (for pediatric restless legs syn- drome, the description of these symptoms should be in the child’s own words). The symptoms begin or worsen during periods of rest or inactivity. Symptoms are partially or totally relieved by movement. Symptoms are worse in the evening or at night than during the day or occur only in the night/evening.

1	restricted affectivity Little reaction to emotionally arousing situations; constricted situations. Restricted affectivity is a facet of the broad personality trait domain DETACH- MENT. rigid perfectionism Rigid insistence on everything being ﬂawless, perfect, and without errors or faults, including one’s own and others’ performance; sacrificing of timeliness to ensure correctness in every detail; believing that there is only one right way to do things; difficulty changing ideas and / or viewpoint; preoccupation with details, orga- nization, and order. Lack of rigid perfectionism is a facet of the broad personality trait domain DISINH'IBITION.

1	risk taking Engagement in dangerous, risky, and potentially self-damaging activities, un- necessarily and without regard to consequences; lack of concern for one’s limitations and denial of the reality of personal danger; reckless pursuit of goals regardless of the level of risk involved. Risk taking is a facet of the broad personality trait domain DISINHIBrI'ION. rumination (rumination disorders) Repeated regurgitation of food over a period of at least 1 month. Regurgitated food may be re—chewed, re-swallowed, or spit out. In rumination disorders, there is no evidence that an associated gastrointestinal or an- other medical condition (e.g., gastroesophageal reﬂux) is sufficient to account for the repeated regurgitation. Glossary of Technical Terms 829 seasonal pattern A pattern of the occurrence of a specific mental disorder in selected seasons of the year.

1	Glossary of Technical Terms 829 seasonal pattern A pattern of the occurrence of a specific mental disorder in selected seasons of the year. self-directedness, self—direction Pursuit of coherent and meaningful short—term and life goals; utilization of constructive and prosocial internal standards of behavior; ability to self—reﬂect productively. separation insecurity Fears of being alone due to rejection by and / or separation from significant others, based in a lack of confidence in one’s ability to care for oneself, both physically and emotionally. Separation insecurity is a facet of the broad personality trait domain NEGATIVE AFFECTIVITY. sex Biological indication of male and female (understood in the context of reproductive capacity), such as sex chromosomes, gonads, sex hormones, and nonambiguous inter- nal and external genitalia.

1	sex Biological indication of male and female (understood in the context of reproductive capacity), such as sex chromosomes, gonads, sex hormones, and nonambiguous inter- nal and external genitalia. sign An objective manifestation of a pathological condition. Signs are observed by the examiner rather than reported by the affected individual. Compare with SYMPTOM. sleep-onset REM Occurrence of the rapid eye movement (REM) phase of sleep within minutes after falling asleep. Usually assessed by a polysomnographic MULTIPLE SLEEP LATENCY TEST. sleep terrors Recurrent episodes of abrupt terror arousals from sleep, usually occurring during the first third of the major sleep episode and beginning with a panicky scream. There is intense fear and signs of autonomic arousal, such as mydriasis, tachycardia, rapid breathing, and sweating, during each episode.

1	There is intense fear and signs of autonomic arousal, such as mydriasis, tachycardia, rapid breathing, and sweating, during each episode. sleepwalking Repeated episodes of rising from bed during sleep and walking about, usually occurring during the first third of the major sleep episode. While sleepwalking, the person has a blank, staring face, is relatively unresponsive to the efforts of others to communicate with him or her, and can be awakened only with great difficulty. somnolence (or ”drowsiness”) A state of near—sleep, a strong desire for sleep, or sleep- ing for unusually long periods. It has two distinct meanings, referring both to the usual state preceding falling asleep and to the chronic condition that involves being in that state independent of a circadian rhythm. Compare with HYPERSOMNIA. specific food cravings Irresistible desire for special types of food.

1	specific food cravings Irresistible desire for special types of food. startle response (or "startle reaction”) An involuntary (reﬂexive) reaction to a sudden unexpected stimulus, such as a loud noise or sharp movement. stereotypies, stereotyped behaviors/movements Repetitive, abnormally frequent, non- goal-directed movements, seemingly driven, and nonfunctional motor behavior (e.g., hand shaking or waving, body rocking, head banging, self—biting). stress The pattern of specific and nonspecific responses a person makes to stimulus events that disturb his or her equilibrium and tax or exceed his or her ability to cope. stressor Any emotional, physical, social, economic, or other factor that disrupts the nor- mal physiological, cognitive, emotional, or behavioral balance of an individual. stressor, psychological Any life event or life change that may be associated temporally (and perhaps causally) with the onset, occurrence, or exacerbation of a mental disorder.

1	stressor, psychological Any life event or life change that may be associated temporally (and perhaps causally) with the onset, occurrence, or exacerbation of a mental disorder. stupor Lack of psychomotor activity, which may range from not actively relating to the environment to complete immobility. submissiveness Adaptation of one’s behavior to the actual or perceived interests and desires of others even when doing so is antithetical to one's own interests, needs, or desires. Submissiveness is a facet of the broad personality trait domain NEGATIVE AF- FECTIVITY. 830 Glossary of Technical Terms subsyndromal Below a specified level or threshold required to qualify for a particular condition. Subsyndromal conditions (formesfrustes) are medical conditions that do not meet full criteria for a diagnosis—for example, because the symptoms are fewer or less the ”full-blown” syndrome.

1	suicidal ideas (suicidal ideation) Thoughts about self—harm, with deliberate consider- ation or planning of possible techniques of causing one’s own death. suicide The act of intentionally causing one’s own death. suicide attempt An attempt to end one’s own life, which may lead to one’s death. suspiciousness Expectations of—and sensitivity to—signs of interpersonal ill intent or harm; doubts about loyalty and fidelity of others; feelings of being mistreated, used, and / or persecuted by others. Suspiciousness is a facet of the broad personality trait do- main DETACHMENT. symptom A subjective manifestation of a pathological condition. Symptoms are reported by the affected individual rather than observed by the examiner. Compare with SIGN. syndrome A grouping of signs and symptoms, based on their frequent co-occurrence that may suggest a common underlying pathogenesis, course, familial pattern, or treat- ment selection.

1	syndrome A grouping of signs and symptoms, based on their frequent co-occurrence that may suggest a common underlying pathogenesis, course, familial pattern, or treat- ment selection. synesthesias A condition in which stimulation of one sensory or cognitive pathway leads to automatic, involuntary experiences in a second sensory or cognitive pathway. temper outburst An emotional outburst (also called a "tantrum”), usually associated with children or those in emotional distress, and typically characterized by stubbom- ness, crying, screaming, defiance, angry ranting, a resistance to attempts at pacifica- tion, and in some cases hitting. Physical control may be lost, the person may be unable to remain still, and even if the ”goal” of the person is met, he or she may not be calmed. thought-action fusion The tendency to treat thoughts and actions as equivalent. tic An involuntary, sudden, rapid, recurrent, nonrhythmic motor movement or vocal- ization.

1	thought-action fusion The tendency to treat thoughts and actions as equivalent. tic An involuntary, sudden, rapid, recurrent, nonrhythmic motor movement or vocal- ization. tolerance A situation that occurs with continued use of a drug in which an individual requires greater dosages to achieve the same effect. transgender The broad spectrum of individuals who transiently or permanently identify with a gender different from their natal gender. transsexual An individual who seeks, or has undergone, a social transition from male to female or female to male, which in many, but not all cases may also involve a somatic surgery"). traumatic stressor Any event (or events) that may cause or threaten death, serious injury, or sexual violence to an individual, a close family member, or a close friend.

1	traumatic stressor Any event (or events) that may cause or threaten death, serious injury, or sexual violence to an individual, a close family member, or a close friend. unusual beliefs and experiences Belief that one has unusual abilities, such as mind reading, telekinesis, or THOUGHT-ACTION FUSION; unusual experiences of reality, in- cluding hallucinatory experiences. In general, the unusual beliefs are not held at the same level of conviction as DELUSIONS. Unusual beliefs and experiences are a facet of the personality trait domain PSYCHOTICISM. waxy ﬂexibility Slight, even resistance to positioning by examiner. Compare with CAT- ALEPSY. Glossary of Technical Terms 831 withdrawal, social Preference for being alone to being with others; reticence in social situations; AVOIDANCE of social contacts and activity; lack of initiation of social contact. Social withdrawal is a facet of the broad personality trait domain DETACHMENT.

1	Social withdrawal is a facet of the broad personality trait domain DETACHMENT. trying to turn the attention to other subjects. The worrying is often persistent, repeti- tive, and out of proportion to the topic worried about (it can even be about a triviality). Glossary of Cult Concepts of Dis“?

1	Ataque de nervios ("attack of nerves”) is a syndrome among individuals of Latino descent, characterized by symptoms of intense emotional upset, including acute anxiety, anger, or grief; screaming and shouting uncontrollably; attacks of crying; trembling; heat in the chest rising into the head; and becoming verbally and physically aggressive. Dissociative experi- ences (e.g., depersonalization, derealization, amnesia), seizure—like or fainting episodes, and suicidal gestures are prominent in some ataques but absent in others. A general feature of an ataque de nervios is a sense of being out of control. Attacks frequently occur as a direct result of a stressful event relating to the family, such as news of the death of a close relative, con- ﬂicts with a spouse or children, or witnessing an accident involving a family member. For a minority of individuals, no particular social event triggers their atuques; instead, their vul- nerability to losing control comes from the accumulated

1	an accident involving a family member. For a minority of individuals, no particular social event triggers their atuques; instead, their vul- nerability to losing control comes from the accumulated experience of suffering.

1	order, although several disorders, including panic disorder, other specified or unspecified dis- sociative disorder, and conversion disorder, have symptomatic overlap with atuque. In community samples, ataque is associated with suicidal ideation, disability, and out- patient psychiatric utilization, after adjustment for psychiatric diagnoses, traumatic expo- sure, and other covariates. However, some atuques represent normative expressions of acute distress (e.g., at a funeral) without clinical sequelae. The term utaque de nervios may also refer to an idiom of distress that includes any ”fit”-like paroxysm of emotionality (e.g., hysterical laughing) and may be used to indicate an episode of loss of control in response to an intense stressor. Related conditions in other cultural contexts: Indisposition in Haiti, blacking out in the Southern United States, and falling out in the West Indies.

1	Related conditions in other cultural contexts: Indisposition in Haiti, blacking out in the Southern United States, and falling out in the West Indies. Related conditions in DSM-S: Panic attack, panic disorder, other specified or unspec- ified dissociative disorder, conversion (functional neurologic symptom) disorder, inter- mittent explosive disorder, other specified or unspecified anxiety disorder, other specified or unspecified trauma and stressor-related disorder.

1	Dhat syndrome is a term that was coined in South Asia little more than half a century ago to account for common clinical presentations of young male patients who attributed their various symptoms to semen loss. Despite the name, it is not a discrete syndrome but rather a cultural explanation of distress for patients who refer to diverse symptoms, such as anx- iety, fatigue, weakness, weight loss, impotence, other multiple somatic complaints, and depressive mood. The cardinal feature is anxiety and distress about the loss of dhat in the absence of any identifiable physiological dysfunction. Dhat was identified by patients as a white discharge that was noted on defecation or urination. Ideas about this substance are related to the concept of dhatu (semen) described in the Hindu system of medicine, Ayurveda, as one of seven essential bodily ﬂuids whose balance is necessary to maintain health. 834 Glossary of Cultural Concepts of Distress

1	Ayurveda, as one of seven essential bodily ﬂuids whose balance is necessary to maintain health. 834 Glossary of Cultural Concepts of Distress Although dhat syndrome was formulated as a cultural guide to local clinical practice, related ideas about the harmful effects of semen loss have been shown to be widespread in the general population, suggesting a cultural disposition for explaining health problems and symptoms with reference to dhat syndrome. Research in health care settings has yielded diverse estimates of the syndrome’s prevalence (e.g., 64% of men attending psychiatric clinics in India for sexual complaints; 30% of men attending general medical clinics in Pakistan). Although dhat syndrome is most commonly identified with young men from lower socioeconomic backgrounds, mid- dle-aged men may also be affected. Comparable concerns about white vaginal discharge (leu— korrhea) have been associated with a variant of the concept for women.

1	Related conditions in other cultural contexts: koro in Southeast Asia, particularly Sin- gapore and shen-k'uei (”kidney deficiency”) in China. Related conditions in DSM-S: Major depressive disorder, persistent depressive disor- der (dysthymia), generalized anxiety disorder, somatic symptom disorder, illness anxiety disorder, erectile disorder, early (premature) ejaculation, other specified or unspecified sexual dysfunction, academic problem.

1	"Khyfil attacks” (khyzil cap), or "wind attacks,” is a syndrome found among Cambodians in the United States and Cambodia. Common symptoms include those of panic attacks, such as dizziness, palpitations, shortness of breath, and cold extremities, as well as other symp- toms of anxiety and autonomic arousal (e.g., tinnitus and neck soreness). Khyfil attacks in- clude catastrophic cognitions centered on the concern that khyril (a windlike substance) may rise in the body—along with blood—and cause a range of serious effects (e.g., com- pressing the lungs to cause shortness of breath and asphyxia; entering the cranium to cause tinnitus, dizziness, blurry vision, and a fatal syncope). Khyzil attacks may occur with- out warning, but are frequently brought about by triggers such as worrisome thoughts, standing up (i.e., orthostasis), specific odors with negative associations, and agoraphobic- type cues like going to crowded spaces or riding in a car. Khyfil attacks usually meet panic attack

1	standing up (i.e., orthostasis), specific odors with negative associations, and agoraphobic- type cues like going to crowded spaces or riding in a car. Khyfil attacks usually meet panic attack criteria and may shape the experience of other anxiety and trauma- and stressor- related disorders. Khyfil attacks may be associated with considerable disability.

1	Related conditions in other cultural contexts: Laos (pen lom), Tibet (srog rlung gz' nud), Sri Lanka (vate), and Korea (hwu byung). Related conditions in DSM-S: Panic attack, panic disorder, generalized anxiety disor- der, agoraphobia, posttraumatic stress disorder, illness anxiety disorder. Kufungisisu ("thinking too much” in Shona) is an idiom of distress and a cultural explana- tion among the Shona of Zimbabwe. As an explanation, it is considered to be causative of anxiety, depression, and somatic problems (e.g., ”my heart is painful because I think too much"). As an idiom of psychosocial distress, it is indicative of interpersonal and social difficulties (e.g., marital problems, having no money to take care of children). Kufungisisu involves ruminating on upsetting thoughts, particularly worries.

1	Kufungisisa is associated with a range of psychopathology, including anxiety symp- toms, excessive worry, panic attacks, depressive symptoms, and irritability. In a study of a random community sample, two—thirds of the cases identified by a general psychopathol- ogy measure were of this complaint. In many cultures, "thinking too much” is considered to be damaging to the mind and body and to cause specific symptoms like headache and dizziness. ”Thinking too much” may also be a key component of cultural syndromes such as “brain fag” in Nigeria. In the case of brain fag, “thinking too much” is primarily attributed to excessive study, which is considered to damage the brain in particular, with symptoms including feelings of heat or crawling sensations in the head. Glossary of Cultural Concepts of Distress 835

1	Glossary of Cultural Concepts of Distress 835 Related conditions in other cultural contexts: ”Thinking too much" is a common id- iom of distress and cultural explanation across many countries and ethnic groups. It has been described in Africa, the Caribbean and Latin America, and among East Asian and Native American groups. Related conditions in DSM-5: Major depressive disorder, persistent depressive disorder (dysthymia), generalized anxiety disorder, posttraumatic stress disorder, obsessive—compul— sive disorder, persistent complex bereavement disorder (see ”Conditions for Further Study").

1	Maladi moun (literally "humanly caused illness," also referred to as "sent sickness”) is a ders. In this explanatory model, interpersonal envy and malice cause people to harm their enemies by sending illnesses such as psychosis, depression, social or academic failure, and inability to perform activities of daily living. The etiological model assumes that illness may be caused by others' envy and hatred, provoked by the victim’s economic success as evidenced by a new job or expensive purchase. One person’s gain is assumed to produce another person’s loss, so visible success makes one vulnerable to attack. Assigning the la- bel of sent sickness depends on mode of onset and social status more than presenting symptoms. The acute onset of new symptoms or an abrupt behavioral change raises sus- picions of a spiritual attack. Someone who is attractive, intelligent, or wealthy is perceived as especially vuhierable, and even young healthy children are at risk.

1	Related conditions in other cultural contexts: Concerns about illness (typically, phys- ical illness) caused by envy or social conﬂict are common across cultures and often ex- pressed in the form of ”evil eye” (e.g. in Spanish, mal de ojo, in Italian, mul’occhiu). Related conditions in DSM-S: Delusional disorder, persecutory type; schizophrenia with paranoid features. Nervios (“nerves”) is a common idiom of distress among Latinos in the United States and

1	Latin America. Nervios refers to a general state of vulnerability to stressful life experiences and to difficult life circumstances. The term nervios includes a wide range of symptoms of emotional distress, somatic disturbance, and inability to function. The most common symptoms attributed to nervios include headaches and “brain aches” (occipital neck ten- sion), irritability, stomach disturbances, sleep difficulties, nervousness, easy tearfulness, inability to concentrate, trembling, tingling sensations, and mareos (dizziness with occa- sional vertigo-like exacerbations). Nervios is a broad idiom of distress that spans the range of severity from cases with no mental disorder to presentations resembling adjustment, anxiety, depressive, dissociative, somatic symptom, or psychotic disorders. “Being ner- vous since childhood” appears to be more of a trait and may precede social anxiety disor- der, while ”being ill with nerves” is more related than other forms of nervios to psychiatric

1	“Being ner- vous since childhood” appears to be more of a trait and may precede social anxiety disor- der, while ”being ill with nerves” is more related than other forms of nervios to psychiatric problems, especially dissociation and depression.

1	Related conditions in other cultural contexts: Nevra among Greeks in North America, nierbi among Sicilians in North America, and nerves among whites in Appalachia and Newfoundland. Related conditions in DSM-5: Major depressive disorder, peristent depressive disor- der (dysthymia), generalized anxiety disorder, social anxiety disorder, other specified or unspecified dissociative disorder, somatic symptom disorder, schizophrenia. Shenjz'ng shuairuo (”weakness of the nervous system” in Mandarin Chinese) is a cultural syndrome that integrates conceptual categories of traditional Chinese medicine with the 836 Glossary of Cultural Concepts of Distress

1	Western diagnosis of neurasthenia. In the second, revised edition of the Chinese Classifica- tion of Mental Disorders (CCMD-Z-R), shenjing shuairuo is defined as a syndrome composed of three out of five nonhierarchical symptom clusters: weakness (e.g., mental fatigue), emotions (e.g., feeling vexed), excitement (e.g., increased recollections), nervous pain (e.g., headache), and sleep (e.g., insomnia). Fan nuo (feeling vexed) is a form of irritability mixed with worry and distress over conﬂicting thoughts and unfulfilled desires. The third edi- tion of the CCMD retains shenjing shuairuo as a somatoform diagnosis of exclusion. Salient precipitants of shenjing shuairuo include work- or family-related stressors, loss of face (mianzi, liunzz'), and an acute sense of failure (e.g., in academic performance). Shenjing sh— uairuo is related to traditional concepts of weakness (xu) and health imbalances related to deficiencies of a vital essence (e.g., the depletion of qi [vital energy]

1	performance). Shenjing sh— uairuo is related to traditional concepts of weakness (xu) and health imbalances related to deficiencies of a vital essence (e.g., the depletion of qi [vital energy] following overstrain- ing or stagnation of 111' due to excessive worry). In the traditional interpretation, shenjing ulated as a result of various social and interpersonal stressors, such as the inability to change a chronically frustrating and distressing situation. Various psychiatric disorders are associated with shenjing shuairuo, notably mood, anxiety, and somatic symptom disor- ders. In medical clinics in China, however, up to 45% of patients with shenjing shuairuo do not meet criteria for any DSM-IV disorder.

1	Related conditions in other cultural contexts: Neurasthenia-spectrum idioms and syndromes are present in India (ashaktapanna) and Japan (shinkei-suijaku), among other set- tings. Other conditions, such as brain fag syndrome, burnout syndrome, and chronic fa- tigue syndrome, are also closely related. Related conditions in DSM-S: Major depressive disorder, persistent depressive disor- der (dysthymia), generalized anxiety disorder, somatic symptom disorder, social anxiety disorder, specific phobia, posttraumatic stress disorder. Susto (”fright”) is a cultural explanation for distress and misfortune prevalent among some Latinos in the United States and among people in Mexico, Central America, and

1	South America. It is not recognized as an illness category among Latinos from the Carib- bean. Susto is an illness attributed to a frightening event that causes the soul to leave the body and results in unhappiness and sickness, as well as difficulties functioning in key social roles. Symptoms may appear any time from days to years after the fright is experi- enced. In extreme cases, susto may result in death. There are no specific defining symp- toms for susto; however, symptoms that are often reported by people with susto include appetite disturbances, inadequate or excessive sleep, troubled sleep or dreams, feelings of sadness, low self—worth or dirtiness, interpersonal sensitivity, and lack of motivation to do anything. Somatic symptoms accompanying susto may include muscle aches and pains, cold in the extremities, pallor, headache, stomachache, and diarrhea. Precipitating events are diverse, and include natural phenomena, animals, interpersonal situations, and super- natural

1	and pains, cold in the extremities, pallor, headache, stomachache, and diarrhea. Precipitating events are diverse, and include natural phenomena, animals, interpersonal situations, and super- natural agents, among others.

1	Three syndromic types of susto (referred to as cibih in the local Zapotec language) have been identified, each having different relationships with psychiatric diagnoses. An interper- sonal susto characterized by feelings of loss, abandonment, and not being loved by family, with accompanying symptoms of sadness, poor self-image, and suicidal ideation, seemed to be closely related to major depressive disorder. When susto resulted from a traumatic event that played a major role in shaping symptoms and in emotional processing of the experience, the diagnosis of posttraumatic stress disorder appeared more appropriate. Susto character- ized by various recurrent somatic symptoms—for which the person sought health care from several practitioners—was thought to resemble a somatic symptom disorder. Related conditions in other cultural contexts: Similar etiological concepts and symp- tom configurations are found globally. In the Andean region, susto is referred to as espunto.

1	Related conditions in other cultural contexts: Similar etiological concepts and symp- tom configurations are found globally. In the Andean region, susto is referred to as espunto. Glossary of Cultural Concepts of Distress 837 Related conditions in DSM-S: Major depressive disorder, posttraumatic stress disor- der, other specified or unspecified trauma and stressor-related disorder, somatic symp- tom disorders.

1	Taijin kyofusho (”interpersonal fear disorder” in Japanese) is a cultural syndrome charac- terized by anxiety about and avoidance of interpersonal situations due to the thought, feel- ing, or conviction that one’s appearance and actions in social interactions are inadequate or offensive to others. In the United States, the variant involves having an offensive body odor and is termed olfactory reference syndrome. Individuals with tazjin kyofusho tend to focus on the impact of their symptoms and behaviors on others. Variants include major concerns about facial blushing (erythrophobia), having an offensive body odor (olfactory reference syndrome), inappropriate gaze (too much or too little eye contact), stiff or awkward facial expression or bodily movements (e.g., stiffening, trembling), or body deformity.

1	Taijin kyofusho is a broader construct than social anxiety disorder in DSM-S. In addition to performance anxiety, tuijin kyofusho includes two culture-related forms: a ”sensitive type,” with extreme social sensitivity and anxiety about interpersonal interactions, and an ”of- fensive type,” in which the major concern is offending others. As a category, tuijin kyofusho thus includes syndromes with features of body dysmorphic disorder as well as delusional disorder. Concerns may have a delusional quality, responding poorly to simple reassurance or counterexample.

1	The distinctive symptoms of tuijin kyofusho occur in specific cultural contexts and, to some extent, with more severe social anxiety across cultures. Similar syndromes are found in Korea and other societies that place a strong emphasis on the self-conscious mainte- nance of appropriate social behavior in hierarchical interpersonal relationships. Taijin kyo— fusho—like symptoms have also been described in other cultural contexts, including the United States, Australia, and New Zealand. Related conditions in other cultural contexts: Tuein kong pa in Korea.

1	United States, Australia, and New Zealand. Related conditions in other cultural contexts: Tuein kong pa in Korea. Related conditions in DSM-S: Social anxiety disorder, body dysmorphic disorder, de- lusional disorder, obsessive-compulsive disorder, olfactory reference syndrome (a type of other specified obsessive-compulsive and related disorder). Olfactory reference syndrome is related specifically to the jikoshu-kyofu variant of taijin kyofusho, whose core symptom is the concern that the person emits an offensive body odor. This presentation is seen in var— ious cultures outside Japan. ICD-9-CM codes are to be used for coding purposes in the United States through September 30, 2014. ICD—lO-CM codes are to be used starting October 1, 2014. lCD-9-CM |CD-10-CM Disorder, condition, or problem V62.3 V62.4 308.3 309.24 309.0 309.3 309.28 309.4 309.9 V71.01 307.0 995.81 995.81 995.81 995.81 995.82 995.82 995.82 995.82 995.83 995.83 995.83 995.83 260.3 F43.0 272.811 T74.11XA

1	V62.3 V62.4 308.3 309.24 309.0 309.3 309.28 309.4 309.9 V71.01 307.0 995.81 995.81 995.81 995.81 995.82 995.82 995.82 995.82 995.83 995.83 995.83 995.83 260.3 F43.0 272.811 T74.11XA T74.11XD T76.11XA T76.11XD T74.31XA T74.31XD T76.31XA T76.31XD T74.21XA T74.21XD T76.21XA T76.21XD With disturbance of conduct With mixed disturbance of emotions and conduct Adult physical abuse by nonspouse or nonpartner, Confirmed Adult physical abuse by nonspouse or nonpartner, Suspected Adult psychological abuse by nonspouse or nonpartner, Adult psychological abuse by nonspouse or nonpartner, Suspected Adult sexual abuse by nonspouse or nonpartner, Confirmed Adult sexual abuse by nonspouse or nonpartner, Suspected Alphabetical Listing of DSM-5 Diagnoses and Codes (lCD-Q-CM and |CD-10-CM) |CD-9-CM |CD-10-CM Disorder, condition, or problem 300.22 291.89 291.89 291.89 291.1 291.2 291.89 291.9 291.89 291.82 303.00 291.0 F40.00 F10.180 F10.280 F10.980 F10.14 F10.24 F10.94

1	F40.00 F10.180 F10.280 F10.980 F10.14 F10.24 F10.94 F10.14 F10.24 F10.94 F10.27 F10.288 F10.988 F10.159 F10.259 F10.959 F10.181 F10.281 F10.981 F10.182 F10.282 F10.982 F10.129 F10.229 F10.929 F10.121 F10.221 F10.921 Alcohol-induced major neurocognitive disorder, Amnestic Alcohol-induced major neurocognitive disorder, Nonamnestic Alphabetical Listing of DSM-5 Diagnoses and Codes (|CD-9-CM and ICD-10-CM) 841 ICD-9-CM ICDe10-CM Disorder, condition, or problem 305.00 303.90 303.90 291.81 291.0 292.89 292.84 292.84 292.89 292.9 292.89 292.85 292.89 F10.10 F10.232 F10.239 F10.231 F15.180 F15.280 F15.980 F15.14 F15.24 F15.94 F15.921 F15.14 F15.94 F15.188 F15.288 F15.988 F15.159 F15.259 F15.959 F15.181 F15.281 F15.981 F15.182 F15.282 F15.982 F15.122 F15.222 F15.922 Amphetamine or other stimulant intoxication, With perceptual

1	F15.188 F15.288 F15.988 F15.159 F15.259 F15.959 F15.181 F15.281 F15.981 F15.182 F15.282 F15.982 F15.122 F15.222 F15.922 Amphetamine or other stimulant intoxication, With perceptual Alphabetical Listing of DSM-5 Diagnoses and Codes (|CD-9-CM and |CD-10-CM) lCD-9-CM |CD-10-CM Disorder, condition, or problem 292.81 292.0 305.70 304.40 304.40 307.1 995.29 995.29 995.29 301.7 293.84 314.01 314.01 314.00 299.00 301.82 307.59 307.51 296.56 296.55 296.51 296.52 296.53 296.54 296.50 296.40 296.46 296.45 296.40 F15.129 F15.229 F15.929 F15.121 F15.221 F15.921 T43.205A T43.205D F90.1 Amphetamine or other stimulant intoxication, Without perceptual Anxiety disorder due to another medical condition Bipolar I disorder, Current or most recent episode depressed Bipolar I disorder, Current or most recent episode hypomanic

1	Anxiety disorder due to another medical condition Bipolar I disorder, Current or most recent episode depressed Bipolar I disorder, Current or most recent episode hypomanic Alphabetical Listing of DSM-5 Diagnoses and Codes (ICD-9-CM and |CD-10-CM) 843 |CD—9-CM lCD-10-CM Disorder, condition, or problem 296.46 296.45 296.41 296.42 296.43 296.44 296.40 296.7 296.89 293.83 300.7 V62.89 301.83 298.8 307.51 292.89 292.85 305.90 292.0 292.89 292.9 292.85 292.89 F31.74 F31.73 F31.11 F31.12 F31.13 F31.81 F15.180 F15.280 F15.980 F15.182 F 15.282 F15.982 F15.929 F15.93 F12.180 F12.280 F12.980 F12.159 F12.259 F12.959 F12.188 F12.288 F12.988 Bipolar I disorder, Current or most recent episode manic Bipolar I disorder, Current or most recent episode unspecified

1	F12.280 F12.980 F12.159 F12.259 F12.959 F12.188 F12.288 F12.988 Bipolar I disorder, Current or most recent episode manic Bipolar I disorder, Current or most recent episode unspecified Bipolar and related disorder due to another medical condition 844 Alphabetical Listing of DSM-5 Diagnoses and Codes (|CD-9-CM and |CD-10-CM) lCD-9-CM |CD-10-CM Disorder, condition, or problem 292.81 305.20 304.30 304.30 292.0 293.89 293.89 780.57 786.04 327.21 V61.29 995.52 995.52 995.52 995.52 V71.02 995.54 995.54 995.54 995.54 995.51 995.51 995.51 995.51 F12.122 F12.222 F12.922 F12.129 F12.229 F12.929 F12.121 F12.221 F12.921 F12.10 F12.20 F12.20 F12.288 G47.37 647.31 262.898 T74.02XA T74.02XD T76.02XA T76.02XD Z72.810 T74.12XA T74.12XD T76.12XA T76.12XD T74.32XA T74.32XD T76.32XA T76.32XD Cannabis intoxication, With perceptual disturbances Cannabis intoxication, Without perceptual disturbances Catatonia associated with another mental disorder (catatonia

1	T76.32XA T76.32XD Cannabis intoxication, With perceptual disturbances Cannabis intoxication, Without perceptual disturbances Catatonia associated with another mental disorder (catatonia Catatonic disorder due to another medical condition Central sleep apnea comorbid with opioid use Child neglect, Confirmed Child neglect, Suspected Child physical abuse, Confirmed Child physical abuse, Suspected Child psychological abuse, Confirmed Child psychological abuse, Suspected Alphabetical Listing of DSM-5 Diagnoses and Codes (ICD-9-CM and ICD-10-CM) 845 lCD-9-CM lCD-10-CM Disorder, condition, or problem 995.53 995.53 995.53 995.53 315.35 307.45 307.45 307.45 307.45 307.45 307.45 292.89 292.84 292.84 292.89 292.9 292.89 292.85 T74.22XA T74.22XD T76.22XA T76.22XD G47.22 G47.21 G47.23 G47.24 G47.26 G47.20 F14.180 F14.280 F14.980 F14.14 F14.188 F14.288 F14.988 F14.159 F14.259 F14.959 F14.181 F14.281 F14.981 F14.182 F14.282 F14.982

1	G47.22 G47.21 G47.23 G47.24 G47.26 G47.20 F14.180 F14.280 F14.980 F14.14 F14.188 F14.288 F14.988 F14.159 F14.259 F14.959 F14.181 F14.281 F14.981 F14.182 F14.282 F14.982 Child sexual abuse, Confirmed Child sexual abuse, Suspected 846 Alphabetical Listing of DSM-5 Diagnoses and Codes (|CD-9-CM and |CD-10-CM) |CD-9-CM |CD-10-CM Disorder, condition, or problem 292.89 292.81 305.60 304.20 304.20 292.0 312.32 312.81 312.89 300.1 1 V62.5 301.13 302.74 293.0 293.0 292.81 297.1 301.6 F14.122 F14.222 F14.922 F14.129 F14.229 F14.929 F14.121 F14.221 F14.921 F14.10 F44.4 F44.7 F44.4 F44.4 F44.4 265.0 Cocaine intoxication, With perceptual disturbances Cocaine intoxication, Without perceptual disturbances Delirium due to another medical condition Delirium due to multiple etiologies Medication-induced delirium (far ICD-10-CM codes, see specific

1	Cocaine intoxication, Without perceptual disturbances Delirium due to another medical condition Delirium due to multiple etiologies Medication-induced delirium (far ICD-10-CM codes, see specific Alphabetical Listing of DSM-5 Diagnoses and Codes (|CD-9-CM and lCD-10-CM) 847 |CD-9-CM ICD-10-CM Disorder, condition, or problem 300.6 293.83 315.4 V60.89 V62.89 313.89 V61.03 296.99 300.12 300.13 300.14 307.7 307.6 302.72 698.4 302.4 V62.22 V60.2 300.19 302.73 302.81 302.89 312.31 302.85 302.6 300.02 302.76 315.8 292.89 V61.8 301.50 300.3 V60.0 780.54 300.7 V62.5 V60.1 259.2 264.4 263.5 F44.1 265.5 259.5 F41.1 F52.6 F16.983 263.8 F60.4 259.0 647.10 265.1 259.1 Depressive disorder due to another medical condition Discord with neighbor, lodger, or landlord Discord with social service provider, including probation officer, case manager, or social services worker Disruption of family by separation or divorce Dissociative amnesia, with dissociative fugue

1	Disruption of family by separation or divorce Dissociative amnesia, with dissociative fugue Exposure to disaster, war, or other hostilities Alphabetical Listing of DSM-S Diagnoses and Codes (|CD-9-CM and |CD-10-CM) ICD-9-CM ICD-10-CM Disorder, condition, or problem 292.89 292.84 292.82 292.89 292.9 292.89 292.81 305.90 304.60 304.60 780.52 V60.2 312.34 312.32 V60.2 315.39 V60.2 F18.180 F18.280 F18.980 F18.188 F18.288 F18.988 F18.159 F18.259 F18.959 F18.129 F18.229 F18.929 F18.121 F18.221 F18.921 G47.00 259.7 259.4 259.6 Lack of adequate food or safe drinking water Alphabetical Listing of DSM-5 Diagnoses and Codes (lCD-9-CM and lCD-10-CM) 849 lCD-9-CM ICD-10-CM Disorder, condition, or problem Major depressive disorder, Recurrent episode 296.36 F3342 In full remission 296.35 F3341 In partial remission 296.31 F330 Mild 296.32 F331 Moderate 296.33 F332 Severe 296.34 F333 With psychotic features 296.30 F339 Unspecified

1	Major depressive disorder, Single episode 296.26 F325 In full remission 296.25 F324 In partial remission 296.21 F320 Mild 296.22 F321 Moderate 296.23 F322 Severe 296.24 F323 With psychotic features 296.20 F329 Unspecifed 331.9 631.9 Major frontotemporal neurocognitive disorder, Possible Major frontotemporal neurocognitive disorder, Probable (code first 331.19 [631.09] frontotemporal disease) 294.1 1 F02.81 With behavioral disturbance 294.10 F02.80 Without behavioral disturbance 331.9 631.9 Major neurocognitive disorder due to Alzheimer’s disease, Possible Major neurocognitive disorder due to Alzheimer’s disease, Probable (code first 331.0 [630.9] Alzheimer’s disease) 294.11 F02.81 With behavioral disturbance 294.10 F0280 Without behavioral disturbance Major neurocognitive disorder due to another medical condition 294.11 F02.81 With behavioral disturbance 294.10 F0280 Without behavioral disturbance

1	Major neurocognitive disorder due to another medical condition 294.11 F02.81 With behavioral disturbance 294.10 F0280 Without behavioral disturbance Major neurocognitive disorder due to HIV infection (codefirst O42 294.11 F02.81 With behavioral disturbance 294.10 F02.80 Without behavioral disturbance Major neurocognitive disorder due to Huntington’s disease (code first 333.4 [610] Huntington’s disease) 294.11 F02.81 With behavioral disturbance 294.10 F0280 Without behavioral disturbance 331.9 6319 Major neurocognitive disorder with Lewy bodies, Possible Major neurocognitive disorder with Lewy bodies, Probable (code first 331.82 [631.83] Lewy body disease) 294.11 F02.81 With behavioral disturbance 294.10 F02.80 Without behavioral disturbance Major neurocognitive disorder due to multiple etiologies 294.11 F02.81 With behavioral disturbance 294.10 F0280 Without behavioral disturbance

1	Major neurocognitive disorder due to multiple etiologies 294.11 F02.81 With behavioral disturbance 294.10 F0280 Without behavioral disturbance Alphabetical Listing of DSM-5 Diagnoses and Codes (ICD-9-CM and lCD—10-CM) lCD-9-CM |CD—10-CM Disorder, condition, or problem 331.9 294.11 294.10 294.11 294.10 294.11 294.10 331.9 290.40 290.40 302.71 V65.2 333.99 333.72 292.81 333.1 331.83 331.83 331.83 331.83 331.83 331.83 331.83 331.83 331.83 331.83 331.83 301.81 347.00 347.00 347.10 347.01 347.00 332.1 631.9 F02.81 F02.80 F02.81 631.9 F52.0 276.5 625.71 624.02 625.1 631.84 631.84 631.84 631.84 631.84 631.84 631.84 631.84 631.84 631.84 631.84 647.419 647.419 647.429 647.411 647.419 621.11 Major neurocognitive disorder clue to Parkinson’s disease, Possible Major neurocognitive disorder due to Parkinson’s disease, Probable (code first 332.0 [620] Parkinson’s disease) Major neurocognitive disorder due to prion disease (code first 046.79 [A819] prion disease)

1	Probable (code first 332.0 [620] Parkinson’s disease) Major neurocognitive disorder due to prion disease (code first 046.79 [A819] prion disease) Major neurocognitive disorder due to traumatic brain injury (code first 907.0 late effect of intracranial injury without skull fracture [506.2X9S diffuse traumatic brain injury with loss of conscious- ness of unspecified duration, sequela]) Major vascular neurocognitive disorder, Possible Major vascular neurocognitive disorder, Probable Medication-induced delirium (for ICD-IO-CM codes, see specific Mild neurocognitive disorder due to Alzheimer’s disease Mild neurocognitive disorder due to another medical condition Mild neurocognitive disorder due to HIV infection Mild neurocognitive disorder due to Huntington’s disease Mild neurocognitive disorder due to multiple etiologies Mild neurocognitive disorder due to Parkinson’s disease Mild neurocognitive disorder due to prion disease

1	Mild neurocognitive disorder due to multiple etiologies Mild neurocognitive disorder due to Parkinson’s disease Mild neurocognitive disorder due to prion disease Mild neurocognitive disorder due to traumatic brain injury Mild neurocognitive disorder with Lewy bodies Autosomal dominant cerebellar ataxia, deafness, and Autosomal dominant narcolepsy, obesity, and type 2 diabetes Narcolepsy secondary to another medical condition Narcolepsy with cataplexy but without hypocretin deficiency Narcolepsy without cataplexy but with hypocretin deficiency Alphabetical Listing of DSM-5 Diagnoses and Codes (ICD-Q-CM and |CD-10-CM) 851 |CD-9-CM lCD_-10-CM Disorder, condition. or problem 333.92 307.47 V15.81 307.46 300.3 301.4 294.8 327.23 292.89 292.84 292.89 292.85 292.89 292.81 305.50 304.00 304.00 621.0 F51.5 291.19 F51.4 647.33 F11.188 F11.288 F11.988 F11.921 F1 1.14 F11.24 F1 1.94 F11.181 F11.281 F11.981 F1 1.182 F11.282 F11.982 F11.122 F11.222 F11.922 F11.129

1	F51.5 291.19 F51.4 647.33 F11.188 F11.288 F11.988 F11.921 F1 1.14 F11.24 F1 1.94 F11.181 F11.281 F11.981 F1 1.182 F11.282 F11.982 F11.122 F11.222 F11.922 F11.129 F1 1.229 F11.929 F11.121 F11.221 F11.921 F11.10 Nonadherence to medical treatment Obsessive-compulsive and related disorder due to another Opioid intoxication, With perceptual disturbances Opioid intoxication, Without perceptual disturbances Alphabetical Listing of DSM-5 Diagnoses and Codes (|CD-9-CM and ICD-10-CM) ICD-9-CM ICD-10-CM Disorder, condition, or problem 292.0 292.0 313.81 995.20 995.20 995.20 V62.83 V65.49 V62.83 V61.21 V61.21 V15.42 V62.83 V61.22 V61.21 V61.21 V15.41 V62.83 V61.22 V61.21 V61.21 V15.42 V62.83 V61.22 T50.905A T50.905D 269.82 269.81 269.021 269.011 269.010 269.020 262.812 269.021 269.011 269.010 269.020 262.810 269.021 269.011 269.010 269.020 262.811 269.021 269.011 Other adverse effect of medication

1	T50.905D 269.82 269.81 269.021 269.011 269.010 269.020 262.812 269.021 269.011 269.010 269.020 262.810 269.021 269.011 269.010 269.020 262.811 269.021 269.011 Other adverse effect of medication Other circumstances related to adult abuse by nonspouse or nonpartner Encounter for mental health services for victim of nonspousal Other circumstances related to child neglect Encounter for mental health services for perpetrator of parental Encounter for mental health services for victim of child neglect by Encounter for mental health services for victim of nonparental Personal history (past history) of neglect in childhood Other circumstances related to child physical abuse Encounter for mental health services for perpetrator of parental Encounter for mental health services for victim of child abuse by Encounter for mental health services for victim of nonparental Personal history (past history) of physical abuse in childhood

1	Encounter for mental health services for victim of child abuse by Encounter for mental health services for victim of nonparental Personal history (past history) of physical abuse in childhood Other circumstances related to child psychological abuse Encounter for mental health services for perpetrator of parental Encounter for mental health services for victim of child Encounter for mental health services for Victim of nonparental Personal history (past history) of psychological abuse in childhood Other circumstances related to child sexual abuse Encounter for mental health services for perpetrator of parental Alphabetical Listing of DSM-5 Diagnoses and Codes (|CD-9-CM and |CD-10-CM) 853 ICD-9-CM |CD-10-CM Disorder, condition, or problem V61.21 V61.21 V15.41 V61.12 V61.11 V15.42 V61.12 V61.11 V15.42 V61.12 V61.11 V15.41 V61.12 V61.11

1	ICD-9-CM |CD-10-CM Disorder, condition, or problem V61.21 V61.21 V15.41 V61.12 V61.11 V15.42 V61.12 V61.11 V15.42 V61.12 V61.11 V15.41 V61.12 V61.11 V15.41 292.89 292.84 292.84 269.010 269.020 269.12 269.11 291.411 269.12 269.11 291.412 269.12 269.11 291.410 269.12 269.81 291.410 271.9 F16.180 F16.280 F16.980 Encounter for mental health services for victim of child sexual Encounter for mental health services for victim of nonparental Personal history (past history) of sexual abuse in childhood Other circumstances related to spouse or partner abuse, Psychological Encounter for mental health services for perpetrator of spouse or Encounter for mental health services for victim of spouse or Personal history (past history) of spouse or partner Other circumstances related to spouse or partner neglect Encounter for mental health services for perpetrator of spouse or Encounter for mental health services for victim of spouse or

1	Other circumstances related to spouse or partner neglect Encounter for mental health services for perpetrator of spouse or Encounter for mental health services for victim of spouse or Personal history (past history) of spouse or partner neglect Other circumstances related to spouse or partner violence, Physical Encounter for mental health services for perpetrator of spouse or partner violence, Physical Encounter for mental health services for victim of spouse or partner violence, Physical Personal history (past history) of spouse or partner violence, Other circumstances related to spouse or partner violence, Sexual Encounter for mental health services for perpetrator of spouse or partner violence, Sexual Encounter for mental health services for victim of spouse or partner violence, Sexual Personal history (past history) of spouse or partner violence, 854 Alphabetical Listing of DSM-5 Diagnoses and Codes (|CD-9-CM and |CD-10-CM)

1	Personal history (past history) of spouse or partner violence, 854 Alphabetical Listing of DSM-5 Diagnoses and Codes (|CD-9-CM and |CD-10-CM) ICD-9-CM lCD-10-CM Disorder, condition, or problem 292.9 292.89 292.81 305.30 304.50 304.50 333.99 332.1 V15.49 V15.89 V62.89 300.09 314.01 296.89 780.09 312.89 300.15 787.60 788.39 307.59 302.6 780.54 780.52 300.9 294.8 315.8 300.3 302.89 301.89 298.8 302.79 F16.159 F16.259 F16.959 F16.129 F16.229 F16.929 F16.121 F16.221 F16.921 625.79 621.19 291.49 291.89 256.9 265.8 F91.8 N39.498 647.19 647.09 Other personal history of psychological trauma Other problem related to employment Other problem related to psychosocial circumstances Other specified disruptive, impulse-control, and conduct disorder Other specified mental disorder due to another medical condition

1	Other problem related to psychosocial circumstances Other specified disruptive, impulse-control, and conduct disorder Other specified mental disorder due to another medical condition Alphabetical Listing of DSM—5 Diagnoses and Codes (lCD-9-CM and |CD-10-CM) 855 |CD-9-CM ICD—10-CM Disorder, condition, or problem 780.59 300.89 307.20 309.89 292.89 292.84 292.84 292.82 292.89 292.89 292.9 292.89 292.85 647.8 F19.180 F19.280 F19.980 F19.14 F19.921 F19.17 F19.188 F19.288 F19.988 F19.188 F19.288 F19.988 F19.159 F19.259 F19.959 F19.181 F19.281 F19.981 F19.182 F19.282 F19.982 856 Alphabetical Listing of DSM-5 Diagnoses and Codes (|CD-9—CM and |CD-10-CM) ICD-9-CM ICD-10-CM Disorder, condition, or problem 292.89 Other (or unknown) substance intoxication F19.129 With mild use disorder F19.229 With moderate or severe use disorder F19.929 Without use disorder 292.81 Other (or unknown) substance intoxication delirium F19.121 With mild use disorder

1	F19.129 With mild use disorder F19.229 With moderate or severe use disorder F19.929 Without use disorder 292.81 Other (or unknown) substance intoxication delirium F19.121 With mild use disorder F19.221 With moderate or severe use disorder F19.921 Without use disorder 305.90 F19.10 Mild 304.90 F1920 Moderate 304.90 F1920 Severe 292.0 F19.239 Other (or unknown) substance withdrawal 292.0 F19.231 Other (or unknown) substance withdrawal delirium 305.70 F15.10 Mild 304.40 F15.20 Moderate 304.40 F15.20 Severe 278.00 E669 Overweight or obesity 300.01 F41.0 Panic disorder 301.0 F60.0 Paranoid personality disorder V61.20 262.820 Parent-child relational problem 302.2 F65.4 Pedophilic disorder 307.22 F95.1 Persistent (chronic) motor or vocal tic disorder 300.4 F341 Persistent depressive disorder (dysthymia) V6222 291.82 Personal history of military deployment V1559 291.5 Personal history of self-harm 310.1 F07.0 Personality change due to another medical condition

1	V6222 291.82 Personal history of military deployment V1559 291.5 Personal history of self-harm 310.1 F07.0 Personality change due to another medical condition V62.89 260.0 Phase of life problem 292.89 Phencyclidine-induced anxiety disorder F16.180 With mild use disorder F16.280 With moderate or severe use disorder F16.980 Without use disorder 292.84 Phencyclidine-induced bipolar and related disorder F16.14 With mild use disorder F16.24 With moderate or severe use disorder 292.84 Phencyclidine-induced depressive disorder F16.14 With mild use disorder Alphabetical Listing of DSM-5 Diagnoses and Codes (ICD-9-CM and |CD-10-CM) 857 lCD-9-CM |CD-10-CM Disorder, condition, or problem 292.9 Phencyclidine-induced psychotic disorder F16.159 With mild use disorder F16.259 With moderate or severe use disorder F16.959 Without use disorder 292.89 Phencyclidine intoxication F16.129 With mild use disorder F16.229 With moderate or severe use disorder

1	F16.259 With moderate or severe use disorder F16.959 Without use disorder 292.89 Phencyclidine intoxication F16.129 With mild use disorder F16.229 With moderate or severe use disorder F16.929 Without use disorder 292.81 Phencyclidine intoxication delirium F16.121 With mild use disorder F16.221 With moderate or severe use disorder F16.921 Without use disorder 305.90 F1610 Mild 304.60 F1620 Moderate 304.60 F1620 Severe 307.52 Pica 309.81 F43.10 Posttraumatic stress disorder 302.75 F52.4 Premature (early) ejaculation 625.4 N943 Premenstrual dysphoric disorder V62.21 256.82 Problem related to current military deployment status V69.9 272.9 Problem related to lifestyle V60.3 260.2 Problem related to living alone V60.6 259.3 Problem related to living in a residential institution V61.5 264.1 Problems related to multiparity V62.5 265.3 Problems related to other legal circumstances V62.5 265.2 Problems related to release from prison

1	V61.5 264.1 Problems related to multiparity V62.5 265.3 Problems related to other legal circumstances V62.5 265.2 Problems related to release from prison V61.7 264.0 Problems related to unwanted pregnancy 307.21 F95.0 Provisional tic disorder Psychotic disorder due to another medical condition 293.82 F060 With hallucinations 312.33 F63.1 Pyromania 327.42 647.52 Rapid eye movement sleep behavior disorder 313.89 F94.1 Reactive attachment disorder V61.10 263.0 Relationship distress with spouse or intimate partner V62.89 265.8 Religious or spiritual problem 333.94 625.81 Restless legs syndrome 307.53 F9821 Rumination disorder 858 Alphabetical Listing of DSM-5 Diagnoses and Codes (lCD-9-CM and lCD-10—CM) |CD-9-CM |CD-10-CM Disorder, condition, or problem 295.70 295.70 301.20 295.90 295.40 301.22 292.89 292.84 292.84 292.82 292.89 292.9 292.89 292.85 292.89 F13.180 F13.280 F13.980 F13.14 F13.921 F13.14 F13.288 F13.988 F13.159 F13.259 F13.959 F13.181 F13.281 F13.981

1	F13.180 F13.280 F13.980 F13.14 F13.921 F13.14 F13.288 F13.988 F13.159 F13.259 F13.959 F13.181 F13.281 F13.981 F13.182 F13.282 F13.982 F13.129 F13.229 F13.929 Sedative-, hypnotic-, or anxiolytic-induced anxiety disorder Sedative-, hypnotic-, or anxiolytic-induced bipolar and related Sedative-, hypnotic-, or anxiolytic-induced delirium Sedative-, hypnotic-, or anxiolytic-induced depressive disorder Sedative-, hypnotic-, or anxiolytic-induced major neurocognitive Sedative-, hypnotic-, or anxiolytic-induced mild neurocognitive Sedative-, hypnotic-, or anxiolytic-induced psychotic disorder Sedative-, hypnotic-, or anxiolytic-induced sexual dysfunction Sedative-, hypnotic-, or anxiolytic-induced sleep disorder Sedative, hypnotic, or anxiolytic intoxication Alphabetical Listing of DSM—5 Diagnoses and Codes (|CD-9—CM and |CD-10-CM) 859 lCD-9—CM ICD-10-CM Disorder, condition, or problem 292.81 Sedative, hypnotic, or anxiolytic intoxication delirium

1	Alphabetical Listing of DSM—5 Diagnoses and Codes (|CD-9—CM and |CD-10-CM) 859 lCD-9—CM ICD-10-CM Disorder, condition, or problem 292.81 Sedative, hypnotic, or anxiolytic intoxication delirium F13.121 With mild use disorder F13.221 With moderate or severe use disorder F13.921 Without use disorder Sedative, hypnotic, or anxiolytic use disorder 305.40 F13.10 Mild 304.10 F1320 Moderate 304.10 F13.20 Severe 292.0 Sedative, hypnotic, or anxiolytic withdrawal F13.232 With perceptual disturbances F13.239 Without perceptual disturbances 292.0 F13.231 Sedative, hypnotic, or anxiolytic withdrawal delirium 312.23 F940 Selective mutism 309.21 F930 Separation anxiety disorder V65.49 270.9 Sex counseling 302.83 F6551 Sexual masochism disorder 302.84 F6552 Sexual sadism disorder

1	V65.49 270.9 Sex counseling 302.83 F6551 Sexual masochism disorder 302.84 F6552 Sexual sadism disorder V61.8 262.891 Sibling relational problem 327.26 647.36 Comorbid sleep-related hypoventilation 327.25 647.35 Congenital central alveolar hypoventilation 327.24 647.34 Idiopathic hypoventilation 300.23 F4010 Social anxiety disorder (social phobia) V62.4 260.4 Social exclusion or rejection 315.39 F8089 Social (pragmatic) communication disorder 300.82 F451 Somatic symptom disorder 315.1 F81.2 With impairment in mathematics 315.00 F81.0 With impairment in reading 315.2 F8181 With impairment in written expression 300.29 F40.218 Animal 300.29 Blood-injection-injury F40.230 Fear of blood F40.231 Fear of injections and transfusions F40.233 Fear of injury F40.232 Fear of other medical care 300.29 F40.228 Natural environment 300.29 F40.298 Other 300.29 F40.248 Situational 315.39 F800 Speech sound disorder

1	F40.233 Fear of injury F40.232 Fear of other medical care 300.29 F40.228 Natural environment 300.29 F40.298 Other 300.29 F40.248 Situational 315.39 F800 Speech sound disorder Spouse or partner abuse, Psychological, Confirmed 995.82 T74.31XA Initial encounter 995.82 T74.31XD Subsequent encounter Alphabetical Listing of DSM-S Diagnoses and Codes (|CD-9-CM and |CD-10-CM) lCD-9-CM |CD-10-CM Disorder, condition, or problem 995.82 995.82 995.85 995.85 995.85 995.85 995.81 995.81 995.81 995.81 995.83 995.83 995.83 995.83 307.3 T76.31XA T76.31XD T74.01XA T74.01XD T76.01XA T76.01XD T74.11XA T74.11XD T76.11XA T76.11XD T74.21XA T74.21XD T76.21XA T76.21XD Spouse or partner abuse, Psychological, Suspected Spouse or partner neglect, Confirmed Spouse or partner neglect, Suspected Spouse or partner violence, Physical, Confirmed Spouse or partner violence, Physical, Suspected Spouse or partner violence, Sexual, Confirmed Spouse or partner violence, Sexual, Suspected

1	Spouse or partner violence, Physical, Confirmed Spouse or partner violence, Physical, Suspected Spouse or partner violence, Sexual, Confirmed Spouse or partner violence, Sexual, Suspected Alphabetical Listing of DSM-5 Diagnoses and Codes (lCD-9-CM and |CD-10-CM) 861 |CD-9-CM ICD-10-CM Disorder, condition, or problem 333.99 625.71 Tardive akathisia 333.85 624.01 Tardive dyskinesia 333.72 624.09 Tardive dystonia V62.4 2605 Target of (perceived) adverse discrimination or persecution 292.85 Tobacco—induced sleep disorder F17.208 With moderate or severe use disorder 305.1 272.0 Mild 305.1 F17.200 Moderate 305.1 F17.200 Severe 292.0 F17.203 Tobacco withdrawal 307.23 F952 Tourette’s disorder 312.39 F632 Trichotillomania (hair-pulling disorder) V639 275.3 Unavailability or inaccessibility of health care facilities V63.8 275.4 Unavailability or inaccessibility of other helping agencies

1	V639 275.3 Unavailability or inaccessibility of health care facilities V63.8 275.4 Unavailability or inaccessibility of other helping agencies V62.82 263.4 Uncomplicated bereavement 291.9 F10.99 Unspecified alcohol-related disorder 300.00 F41.9 Unspecified anxiety disorder 314.01 F90.9 Unspecified attention-deficit/hyperactivity disorder 296.80 F319 Unspecified bipolar and related disorder 292.9 F1599 Unspecified caffeine-related disorder 292.9 F12.99 Unspecified cannabis-related disorder 293.89 F061 Unspecified catatonia (codefirst 781.99 [R29.818] other symptoms 307.9 F809 Unspecified communication disorder 780.09 R41.0 Unspecified delirium 312.9 F919 Unspecified disruptive, impulse-control, and conduct disorder 300.15 F449 Unspecified dissociative disorder 787.60 R159 With fecal symptoms 788.30 R32 With urinary symptoms 307.50 F50.9 Unspecified feeding or eating disorder 302.6 F649 Unspecified gender dysphoria 292.9 F1699 Unspecified hallucinogen-related disorder

1	V60.9 259.9 Unspecified housing or economic problem 780.54 647.10 Unspecified hypersomnolence disorder 292.9 F1899 Unspecified inhalant—related disorder 780.52 647.00 Unspecified insomnia disorder 300.9 F99 Unspecified mental disorder 294.9 F09 Unspecified mental disorder due to another medical condition 799.59 R419 Unspecified neurocognitive disorder Alphabetical Listing of DSM-5 Diagnoses and Codes (ICD-9-CM and ICD-10-CM) |CD-9-CM ICD-10-CM Disorder, condition, or problem 315.9 300.3 292.9 292.9 302.9 301.9 292.9 V62.9 V62.9 298.9 292.9 302.70 780.59 300.82 292.9 307.20 292.9 309.9 V61.8 V62.89 V62.89 302.82 V40.31 F1 1.99 F19.99 F16.99 260.9 265.9 647.9 F17.209 262.29 265.4 265.4 291.83 Unspecified problem related to social environment Unspecified problem related to unspecified psychosocial Unspecified sedative-, hypnotic-, or anxiolytic-related disorder Victim of crime Victim of terrorism or torture Wandering associated with a mental disorder

1	Unspecified sedative-, hypnotic-, or anxiolytic-related disorder Victim of crime Victim of terrorism or torture Wandering associated with a mental disorder ICD-9-CM codes are to be used for coding purposes in the United States through September 30, 2014. |CD-9-CM Disorder, condition, or problem 278.00 290.40 290.40 291.0 291.0 291.1 291.2 291.81 291.82 291.89 291.89 291.89 291.89 291.89 291.9 291.9 292.0 292.0 292.0 292.0 292.0 292.0 292.0 292.0 292.0 292.0 292.0 292.81 292.81 292.81 Probable major vascular neurocognitive disorder, With behavioral disturbance Probable major vascular neurocognitive disorder, Without behavioral Alcohol-induced major neurocognitive disorder, Amnestic confabulatory type Alcohol-induced major neurocognitive disorder, Nonarrmestic confabulatory type Sedative, hypnotic, or anxiolytic withdrawal Sedative, hypnotic, or anxiolytic withdrawal delirium

1	Alcohol-induced major neurocognitive disorder, Nonarrmestic confabulatory type Sedative, hypnotic, or anxiolytic withdrawal Sedative, hypnotic, or anxiolytic withdrawal delirium Numerical Listing of DSM-S Diagnoses and Codes (|CD-9-CM) lCD-9-CM Disorder, condition, or problem 292.81 292.81 292.81 292.81 292.81 292.81 292.81 292.82 292.82 292.82 292.84 292.84 292.84 292.84 292.84 292.84 292.84 292.84 292.84 292.84 292.84 292.84 292.84 292.84 292.85 292.85 292.85 292.85 292.85 292.85 292.85 292.85 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 Sedative, hypnotic, or anxiolytic intoxication delirium Sedative-, hypnotic-, or anxiolytic-induced major neurocognitive disorder Sedative-, hypnotic-, or anxiolytic-induced bipolar and related disorder Sedative-, hypnotic-, or anxiolytic-induced depressive disorder Sedative—, hypnotic-, or anxiolytic-induced sleep disorder

1	Sedative-, hypnotic-, or anxiolytic-induced bipolar and related disorder Sedative-, hypnotic-, or anxiolytic-induced depressive disorder Sedative—, hypnotic-, or anxiolytic-induced sleep disorder Numerical Listing of DSM-5 Diagnoses and Codes (ICD-9-CM) 865 |CD-9-CM Disorder, condition, or problem 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.89 292.9 292.9 292.9 292.9 292.9 292.9 292.9 292.9 292.9 292.9 292.9 292.9 292.9 292.9 292.9 292.9 292.9 292.9 293.0 293.0 293.81 293.82 293.83 293.83 293.84 293.89 Sedative-, hypnotic-, or anxiolytiC-induced anxiety disorder Sedative-, hypnotic—, or anxiolytic-induced mild neurocognitive disorder Sedative-, hypnotic-, or anxiolytic-induced sexual dysfunction Sedative, hypnotic, or anxiolytic intoxication Sedative-, hypnotic-, or anxiolytic—induced psychotic disorder Unspecified sedative-, hypnotic-, or anxiolytic-related disorder

1	Sedative, hypnotic, or anxiolytic intoxication Sedative-, hypnotic-, or anxiolytic—induced psychotic disorder Unspecified sedative-, hypnotic-, or anxiolytic-related disorder Delirium due to another medical condition Delirium due to multiple etiologies Psychotic disorder due to another medical condition, With delusions Psychotic disorder due to another medical condition, With hallucinations Bipolar and related disorder due to another medical condition Depressive disorder due to another medical condition Anxiety disorder due to another medical condition Catatonia associated with another mental disorder (catatonia specifier) Numerical Listing of DSM-S Diagnoses and Codes (|CD-9-CM) lCD-9-CM Disorder, condition, or problem 293.89 293.89 294.10 294.10 294.10 294.10 294.10 294.10 294.10 294.10 294.10 294.10 294.1 1 294.11 294.11 294.11 294.11 294.11 294.11 294.11 294.1 1 294.1 1 294.8 294.8 294.9 Catatonic disorder due to another medical condition

1	Catatonic disorder due to another medical condition Unspecified catatonia (codefirst 781.99 other symptoms involving nervous and Major neurocognitive disorder due to another medical condition, Without Major neurocognitive disorder due to HIV infection, Without behavioral Major neurocognitive disorder due to Huntington’s disease, Without behavioral disturbance (codefirst 333.4 Huntington’s disease) Major neurocognitive disorder due to multiple etiologies, Without behavioral Major neurocognitive disorder probably due to Parkinson’s disease, Without behavioral disturbance (codefirst 332.0 Parkinson’s disease) Major neurocognitive disorder due to prion disease, Without behavioral disturbance (code first 046.79 prion disease) Major neurocognitive disorder due to traumatic brain injury, Without behavioral disturbance(codefirst907.01ate effect of intracranial injury without

1	Major neurocognitive disorder due to traumatic brain injury, Without behavioral disturbance(codefirst907.01ate effect of intracranial injury without Probable major frontotemporal neurocognitive disorder, Without behavioral disturbance (codefirst 331.19 frontotemporal disease) Probable major neurocognitive disorder due to Alzheimer’s disease, Without behavioral disturbance (codeﬂrst 331.0 Alzheimer’s disease) Probable major neurocognitive disorder with Lewy bodies, Without behavioral disturbance (codefirst 331.82 Lewy body disease) Major neurocognitive disorder due to another medical condition, With Major neurocognitive disorder due to HIV infection, With behavioral Major neurocognitive disorder due to Huntington’s disease, With behavioral disturbance (codefirst 333.4 Huntington’s disease) Major neurocognitive disorder due to multiple etiologies, With behavioral

1	Major neurocognitive disorder due to multiple etiologies, With behavioral Major neurocognitive disorder probably due to Parkinson’s disease, With behavioral disturbance (code first 332.0 Parkinson’s disease) Major neurocognitive disorder due to prion disease, With behavioral disturbance (code first 046.79 prion disease) Major neurocognitive disorder due to traumatic brain injury, With behavioral disturbance (codefirst 907.0 late effect of intracranial injury without skull Probable major frontotemporal neurocognitive disorder, With behavioral disturbance (code first 331.19 frontotemporal disease) Probable major neurocognitive disorder due to Alzheimer’s disease, With behavioral disturbance (codefirst 331.0 Alzheimer’s disease) Probable major neurocognitive disorder with Lewy bodies, With behavioral disturbance (codefirst 331.82 Lewy body disease) Obsessive-compulsive and related disorder due to another medical condition

1	Probable major neurocognitive disorder with Lewy bodies, With behavioral disturbance (codefirst 331.82 Lewy body disease) Obsessive-compulsive and related disorder due to another medical condition Other specified mental disorder due to another medical condition Unspecified mental disorder due to another medical condition Numerical Listing of DSM-5 Diagnoses and Codes (|CD-9-CM) 867 ICD-9-CM Disorder. condition, or problem 295.40 295.70 295.70 295.90 296.20 296.21 296.22 296.23 296.24 296.25 296.26 296.30 296.31 296.32 296.33 296.34 296.35 296.36 296.40 296.40 296.40 296.41 296.42 296.43 296.44 296.45 296.45 296.46 296.46 296.50 296.51 296.52 296.53 296.54 296.55 296.56 296.7 296.80 296.89 296.89 296.99 297.1 298.8 298.8 Schizoaffective disorder, Bipolar type Schizoaffective disorder, Depressive type Major depressive disorder, Single episode, Unspecifed Major depressive disorder, Single episode, Mild Major depressive disorder, Single episode, Moderate

1	Schizoaffective disorder, Depressive type Major depressive disorder, Single episode, Unspecifed Major depressive disorder, Single episode, Mild Major depressive disorder, Single episode, Moderate Major depressive disorder, Single episode, Severe Major depressive disorder, Single episode, With psychotic features Major depressive disorder, Single episode, In partial remission Major depressive disorder, Single episode, In full remission Major depressive disorder, Recurrent episode, Unspecified Major depressive disorder, Recurrent episode, Mild Major depressive disorder, Recurrent episode, Moderate Major depressive disorder, Recurrent episode, Severe Major depressive disorder, Recurrent episode, With psychotic features Major depressive disorder, Recurrent episode, In partial remission Major depressive disorder, Recurrent episode, In full remission Bipolar I disorder, Current or most recent episode hypomanic

1	Major depressive disorder, Recurrent episode, In partial remission Major depressive disorder, Recurrent episode, In full remission Bipolar I disorder, Current or most recent episode hypomanic Bipolar I disorder, Current or most recent episode hypomanic, Unspecified Bipolar I disorder, Current or most recent episode manic, Unspecified Bipolar I disorder, Current or most recent episode manic, Mild Bipolar I disorder, Current or most recent episode manic, Moderate Bipolar I disorder, Current or most recent episode manic, Severe Bipolar I disorder, Current or most recent episode manic, With psychotic features Bipolar I disorder, Current or most recent episode hypomanic, In partial remission Bipolar I disorder, Current or most recent episode manic, In partial remission Bipolar I disorder, Current or most recent episode hypomanic, In full remission Bipolar I disorder, Current or most recent episode manic, In full remission

1	Bipolar I disorder, Current or most recent episode hypomanic, In full remission Bipolar I disorder, Current or most recent episode manic, In full remission Bipolar I disorder, Current or most recent episode depressed, Unspecified Bipolar I disorder, Current or most recent episode depressed, Mild Bipolar I disorder, Current or most recent episode depressed, Moderate Bipolar I disorder, Current or most recent episode depressed, Severe Bipolar I disorder, Current or most recent episode depressed, With psychotic Bipolar I disorder, Current or most recent episode depressed, In partial remission Bipolar I disorder, Current or most recent episode depressed, In full remission Bipolar I disorder, Current or most recent episode unspecified Numerical Listing of DSM-5 Diagnoses and Codes (lCD-9-CM)

1	Bipolar I disorder, Current or most recent episode unspecified Numerical Listing of DSM-5 Diagnoses and Codes (lCD-9-CM) ICD-9-CM Disorder, condition, or problem 298.9 299.00 300.00 300.01 300.02 300.09 300.11 300.12 300.13 300.14 300.15 300.15 300.19 300.22 300.23 300.29 300.29 300.29 300.29 300.29 300.3 300.3 300.3 300.3 300.4 300.6 300.7 300.7 300.82 300.82 300.89 300.9 300.9 301.0 301.13 301.20 301.22 301.4 301.50 301.6 301.7 301.81 301.82 301.83 301.89 Dissociative amnesia, With dissociative fugue Specific phobia, Animal Specific phobia, Blood-injection-injury Specific phobia, Natural environment Specific phobia, Other Specific phobia, Situational

1	Numerical Listing of DSM-5 Diagnoses and Codes (|CD-9-CM) lCD-9-CM Disorder, condition, or problem 301.9 Unspecified personality disorder 302.2 Pedophilic disorder 302.3 Transvestic disorder 302.4 Exhibitionistic disorder 302.6 Gender dysphoria in children 302.6 Other specified gender dysphoria 302.6 Unspecified gender dysphoria 302.70 Unspecified sexual dysfunction 302.71 Male hypoactive sexual desire disorder 302.72 Erectile disorder 302.72 Female sexual interest/arousal disorder 302.73 Female orgasmic disorder 302.74 Delayed ejaculation 302,75 Premature (early) ejaculation 302.76 Genito—pelvic pain/penetration disorder 302.79 Other specified sexual dysfunction 302.81 Fetishistic disorder 302.82 Voyeuristic disorder 302.83 Sexual masochism disorder 302.84 Sexual sadism disorder 302.85 Gender dysphoria in adolescents and adults 302.89 Frotteuristic disorder 302.89 Other specified paraphilic disorder 302.9 Unspecified paraphilic disorder 303.00 Alcohol intoxication 303.90 Alcohol use

1	dysphoria in adolescents and adults 302.89 Frotteuristic disorder 302.89 Other specified paraphilic disorder 302.9 Unspecified paraphilic disorder 303.00 Alcohol intoxication 303.90 Alcohol use disorder, Moderate 303.90 Alcohol use disorder, Severe 304.00 Opioid use disorder, Moderate 304.00 Opioid use disorder, Severe 304.10 Sedative, hypnotic, or anxiolytic use disorder, Moderate 304.10 Sedative, hypnotic, or anxiolytic use disorder, Severe 304.20 Cocaine use disorder, Moderate 304.20 Cocaine use disorder, Severe 304.30 Cannabis use disorder, Moderate 304.30 Cannabis use disorder, Severe 304.40 Amphetamine-type substance use disorder, Moderate 304.40 Amphetamine—type substance use disorder, Severe 304.40 Other or unspecified stimulant use disorder, Moderate 304.40 Other or unspecified stimulant use disorder, Severe 304.50 Other hallucinogen use disorder, Moderate 304.50 Other hallucinogen use disorder, Severe 304.60 Inhalant use disorder, Moderate 304.60 Inhalant use disorder,

1	stimulant use disorder, Severe 304.50 Other hallucinogen use disorder, Moderate 304.50 Other hallucinogen use disorder, Severe 304.60 Inhalant use disorder, Moderate 304.60 Inhalant use disorder, Severe 304.60 Phencyclidine use disorder, Moderate 304.60 Phencyclidine use disorder, Severe

1	Numerical Listing of DSM—5 Diagnoses and Codes (|CD-9-CM) |CD-9-CM Disorder, condition, or problem 304.90 304.90 305.00 305.1 305.1 305.1 305.20 305.30 305.40 305.50 305.60 305.70 305.70 305.90 305.90 305.90 305.90 307.0 307.1 307.20 307.20 307.21 307.22 307.23 307.3 307.45 307.45 307.45 307.45 307.45 307.45 307.46 307.46 307.47 307.50 307.51 307.51 307.52 307.53 307.59 307.59 307.6 307.7 307.9 308.3 Other (or unknown) substance use disorder, Moderate Other (or unknown) substance use disorder, Severe Alcohol use disorder, Mild Tobacco use disorder, Mild Tobacco use disorder, Moderate Tobacco use disorder, Severe Cannabis use disorder, Mild Other hallucinogen use disorder, Mild Sedative, hypnotic, or anxiolytic use disorder, Mild Opioid use disorder, Mild Cocaine use disorder, Mild Amphetamine-type substance use disorder, Mild Other or unspecified stimulant use disorder, Mild Inhalant use disorder, Mild Other (or unknown) substance use disorder, Mild

1	Amphetamine-type substance use disorder, Mild Other or unspecified stimulant use disorder, Mild Inhalant use disorder, Mild Other (or unknown) substance use disorder, Mild Phencyclidine use disorder, Mild Circadian rhythm sleep-wake disorders, Advanced sleep phase type Circadian rhythm sleep-wake disorders, Delayed sleep phase type Circadian rhythm sleep-wake disorders, Irregular sleep-wake type Circadian rhythm sleep-wake disorders, Non-24-hour sleep-wake type Circadian rhythm sleep-wake disorders, Shift work type Circadian rhythm sleep-wake disorders, Unspecified type Non—rapid eye movement sleep arousal disorders, Sleep terror type Non—rapid eye movement sleep arousal disorders, Sleepwalking type

1	Circadian rhythm sleep-wake disorders, Unspecified type Non—rapid eye movement sleep arousal disorders, Sleep terror type Non—rapid eye movement sleep arousal disorders, Sleepwalking type Numerical Listing of DSM-5 Diagnoses and Codes (|CD-9-CM) 871 lCD-9-CM Disorder, condition, or problem 309.0 309.21 309.24 309.28 309.3 309.4 309.81 309.89 309.9 309.9 310.1 312.23 312.31 312.32 312.32 312.33 312.34 312.39 312.81 312.89 312.89 312.9 313.81 313.89 313.89 314.00 314.01 314.01 314.01 314.01 315.00 315.1 315.2 315.35 315.39 315.39 315.39 315.4 315.8 315.8 315.9 Adjustment disorders, With depressed mood Adjustment disorders, With anxiety Adjustment disorders, With mixed anxiety and depressed mood Adjustment disorders, With disturbance of conduct Adjustment disorders, With mixed disturbance of emotions and conduct Adjustment disorders, Unspecified Personality change due to another medical condition Conduct disorder, Adolescent-onset type Conduct disorder, Childhood-onset type

1	Adjustment disorders, Unspecified Personality change due to another medical condition Conduct disorder, Adolescent-onset type Conduct disorder, Childhood-onset type Conduct disorder, Unspecified onset Other specified disruptive, impulse-control, and conduct disorder Unspecified disruptive, impulse—control, and conduct disorder Attention-deficit/hyperactivity disorder, Predominantly inattentive presentation Attention-deficit/ hyperactivity disorder, Combined presentation Attention-deficit/hyperactivity disorder, Predominantly hyperactive/ Specific learning disorder, With impairment in reading Specific learning disorder, With impairment in mathematics Specific learning disorder, With impairment in written expression

1	Specific learning disorder, With impairment in reading Specific learning disorder, With impairment in mathematics Specific learning disorder, With impairment in written expression Numerical Listing of DSM-5 Diagnoses and Codes (|CD-9-CM) |CD-9-CM Disorder, condition, or problem 327.21 327.23 327.24 327.25 327.26 327.42 331.83 331.83 331.83 331.83 331.83 331.83 331.83 331.83 331.83 331.83 331.83 331.9 331.9 331.9 331.9 333.1 332.1 332.1 333.72 333.72 333.85 333.92 333.94 333.99 333.99 333.99 347.00 347.00 347.00 347.01 347.10 625.4 698.4 780.09 780.09 780.52 Central sleep apnea, Idiopathic central sleep apnea Sleep-related hypoventilation, Idiopathic hypoventilation Sleep-related hypoventilation, Congenital central alveolar hypoventilation Sleep-related hypoventilation, Comorbid sleep—related hypoventilation Mild neurocognitive disorder due to Alzheimer’s disease Mild neurocognitive disorder due to another medical condition Mild neurocognitive disorder due to HIV infection

1	Mild neurocognitive disorder due to Alzheimer’s disease Mild neurocognitive disorder due to another medical condition Mild neurocognitive disorder due to HIV infection Mild neurocognitive disorder due to Huntington’s disease Mild neurocognitive disorder with Lewy bodies Mild neurocognitive disorder due to multiple etiologies Mild neurocognitive disorder due to Parkinson’s disease Mild neurocognitive disorder due to prion disease Mild neurocognitive disorder due to traumatic brain injury Major neurocognitive disorder possibly due to Parkinson’s disease Possible major neurocognitive disorder due to Alzheimer’s disease Possible major neurocognitive disorder with Lewy bodies Autosomal dominant cerebellar ataxia, deafness, and narcolepsy Autosomal dominant narcolepsy, obesity, and type 2 diabetes Narcolepsy without cataplexy but with hypocretin deficiency Narcolepsy with cataplexy but without hypocretin deficiency Narcolepsy secondary to another medical condition

1	Narcolepsy without cataplexy but with hypocretin deficiency Narcolepsy with cataplexy but without hypocretin deficiency Narcolepsy secondary to another medical condition Numerical Listing of DSM-5 Diagnoses and Codes (|CD-9-CM) 873 |CD-9-CM Disorder, condition, or problem 780.52 780.52 780.54 780.54 780.54 780.57 780.59 780.59 786.04 787.60 787.60 788.30 788.39 799.59 995.20 995.20 995.20 995.29 995.29 995.29 995.51 995.51 995.51 995.51 995.52 995.52 995.52 995.52 995.53 995.53 995.53 995.53 995.54 995.54 995.54 995.54 995.81 995.81 995.81 995.81 995.81 995.81 995.81 Central sleep apnea, Central sleep apnea comorbid with opioid use Central sleep apnea, Cheyne—Stokes breathing Other specified elimination disorder, With fecal symptoms Unspecified elimination disorder, With fecal symptoms Unspecified elimination disorder, With urinary symptoms Other specified elimination disorder, With urinary symptoms Other adverse effect of medication, Initial encounter

1	Unspecified elimination disorder, With urinary symptoms Other specified elimination disorder, With urinary symptoms Other adverse effect of medication, Initial encounter Other adverse effect of medication, Sequelae Other adverse effect of medication, Subsequent encounter Antidepressant discontinuation syndrome, Initial encounter Antidepressant discontinuation syndrome, Sequelae Antidepressant discontinuation syndrome, Subsequent encounter Child psychological abuse, Confirmed, Initial encounter Child psychological abuse, Confirmed, Subsequent encounter Child psychological abuse, Suspected, Initial encounter Child psychological abuse, Suspected, Subsequent encounter Child neglect, Confirmed, Initial encounter Child neglect, Confirmed, Subsequent encounter Child neglect, Suspected, Initial encounter Child neglect, Suspected, Subsequent encounter Child sexual abuse, Confirmed, Initial encounter Child sexual abuse, Confirmed, Subsequent encounter

1	Child neglect, Suspected, Initial encounter Child neglect, Suspected, Subsequent encounter Child sexual abuse, Confirmed, Initial encounter Child sexual abuse, Confirmed, Subsequent encounter Child sexual abuse, Suspected, Initial encounter Child sexual abuse, Suspected, Subsequent encounter Child physical abuse, Confirmed, Initial encounter Child physical abuse, Confirmed, Subsequent encounter Child physical abuse, Suspected, Initial encounter Child physical abuse, Suspected, Subsequent encounter Adult physical abuse by nonspouse or nonpartner, Confirmed, Initial encounter Adult physical abuse by nonspouse or nonpartner, Confirmed, Subsequent Adult physical abuse by nonspouse or nonpartner, Suspected, Initial encounter Adult physical abuse by nonspouse or nonpartner, Suspected, Subsequent Spouse or partner violence, Physical, Confirmed, Initial encounter Spouse or partner violence, Physical, Confirmed, Subsequent encounter

1	Spouse or partner violence, Physical, Confirmed, Initial encounter Spouse or partner violence, Physical, Confirmed, Subsequent encounter Spouse or partner violence, Physical, Suspected, Initial encounter Numerical Listing of DSM-5 Diagnoses and Codes (|CD-9-CM) |CD-9-CM Disorder, condition, or problem 995.81 995.82 995.82 995.82 995.82 995.82 995.82 995.82 995.82 995.83 995.83 995.83 995.83 995.83 995.83 995.83 995.83 995.85 995.85 995.85 995.85 V15.41 V15.41 V15.41 V15.41 V15.42 V15.42 V15.42 V15.42 V15.49 V15.81 V15.89 V40.31 V60.1 V60.2 V60.2 V60.2 V60.2 Spouse or partner violence, Physical, Suspected, Subsequent encounter Adult psychological abuse by nonspouse or nonpartner, Confirmed, Initial Adult psychological abuse by nonspouse or nonpartner, Confirmed, Adult psychological abuse by nonspouse or nonpartner, Suspected, Initial Adult psychological abuse by nonspouse or nonpartner, Suspected,

1	Adult psychological abuse by nonspouse or nonpartner, Suspected, Initial Adult psychological abuse by nonspouse or nonpartner, Suspected, Spouse or partner abuse, Psychological, Confirmed, Initial encounter Spouse or partner abuse, Psychological, Confirmed, Subsequent encounter Spouse or partner abuse, Psychological, Suspected, Initial encounter Spouse or partner abuse, Psychological, Suspected, Subsequent encounter Adult sexual abuse by nonspouse or nonpartner, Confirmed, Initial encounter Adult sexual abuse by nonspouse or nonpartner, Confirmed, Subsequent Adult sexual abuse by nonspouse or nonpartner, Suspected, Initial encounter Adult sexual abuse by nonspouse or nonpartner, Suspected, Subsequent Spouse or partner violence, Sexual, Confirmed, Initial encounter Spouse or partner violence, Sexual, Confirmed, Subsequent encounter Spouse or partner violence, Sexual, Suspected, Initial encounter Spouse or partner violence, Sexual, Suspected, Subsequent encounter

1	Spouse or partner violence, Sexual, Suspected, Initial encounter Spouse or partner violence, Sexual, Suspected, Subsequent encounter Spouse or partner neglect, Confirmed, Initial encounter Spouse or partner neglect, Confirmed, Subsequent encounter Spouse or partner neglect, Suspected, Initial encounter Spouse or partner neglect, Suspected, Subsequent encounter Personal history (past history) of physical abuse in childhood Personal history (past history) of sexual abuse in childhood Personal history (past history) of spouse or partner violence, Physical Personal history (past history) of spouse or partner violence, Sexual Personal history (past history) of neglect in childhood Personal history (past history) of psychological abuse in childhood Personal history (past history) of spouse or partner neglect Personal history (past history) of spouse or partner psychological abuse Other personal history of psychological trauma Personal history of self—harm

1	Personal history (past history) of spouse or partner psychological abuse Other personal history of psychological trauma Personal history of self—harm Nonadherence to medical treatment Wandering associated with a mental disorder Lack of adequate food or safe drinking water Numerical Listing of DSM—S Diagnoses and Codes (ICD—Q-CM) 875 |CD-9-CM Disorder, condition, or problem V60.3 V60.6 V60.89 V60.9 V61.10 V61.11 V61.11 V61.11 V61.11 V61.12 V61.12 V61.12 V61.12 V61.20 V61.21 V61.21 V61.21 V61.21 V61.21 V61.21 V61.21 V61.21 V61.22 V61.22 V61.22 V61.22 V61.29 V61.5 V61.7 V61.8 V62.21 Problem related to living alone Problem related to living in a residential institution Discord with neighbor, lodger, or landlord Disruption of family by separation or divorce Relationship distress with spouse or intimate partner Encounter for mental health services for victim of spouse or partner neglect

1	Disruption of family by separation or divorce Relationship distress with spouse or intimate partner Encounter for mental health services for victim of spouse or partner neglect Encounter for mental health services for victim of spouse or partner Encounter for mental health services for victim of spouse or partner violence, Encounter for mental health services for victim of spouse or partner violence, Encounter for mental health services for perpetrator of spouse or partner Encounter for mental health services for perpetrator of spouse or partner Encounter for mental health services for perpetrator of spouse or partner violence, Physical Encounter for mental health services for perpetrator of spouse or partner violence, Sexual Encounter for mental health services for victim of child abuse by parent Encounter for mental health services for victim of child neglect by parent Encounter for mental health services for victim of child psychological abuse by

1	Encounter for mental health services for victim of child neglect by parent Encounter for mental health services for victim of child psychological abuse by Encounter for mental health services for victim of child sexual abuse by parent Encounter for mental health services for victim of nonparental child abuse Encounter for mental health services for victim of nonparental child neglect Encounter for mental health services for victim of nonparental child EnCOunter for mental health services for victim of nonparental child sexual Encounter for mental health services for perpetrator of parental child abuse Encounter for mental health services for perpetrator of parental child neglect Encounter for mental health services for perpetrator of parental child Encounter for mental health services for perpetrator of parental child sexual Problems related to multiparity Problems related to unwanted pregnancy Problem related to current military deployment status

1	Problems related to multiparity Problems related to unwanted pregnancy Problem related to current military deployment status Exposure to disaster, war, or other hostilities Numerical Listing of DSM-5 Diagnoses and Codes (ICD-Q-CM) lCD-9-CM Disorder, condition, or problem V62.22 V62.3 V62.4 V62.4 V62.4 V62.5 V62.5 V62.5 V62.5 V62.82 V62.83 V62.83 V62.83 V62.83 V62.83 V62.89 V62.89 V62.89 V62.89 V62.89 V62.89 V62.89 V62.9 V62.9 V63.8 V65.40 V65.49 V65.49 V69.9 V71.01 V71.02 Personal history of military deployment Other problem related to employment Target of (perceived) adverse discrimination or persecution Problems related to other legal circumstances Problems related to release from prison Encounter for mental health services for perpetrator of nonparental child abuse Encounter for mental health services for perpetrator of nonparental child Encounter for mental health services for perpetrator of nonparental child

1	Encounter for mental health services for perpetrator of nonparental child Encounter for mental health services for perpetrator of nonparental child Encounter for mental health services for perpetrator of nonparental child Encounter for mental health services for perpetrator of nonspousal adult abuse Discord with social service provider, including probation officer, case manager, Other problem related to psychosocial circumstances Phase of life problem Victim of crime Victim of terrorism or torture Unspecified problem related to social environment Unspecified problem related to unspecified psychosocial circumstances Unavailability or inaccessibility of other helping agencies Unavailability or inaccessibility of health care facilities Encounter for mental health services for victim of nonspousal adult abuse Problem related to lifestyle ICD-lO-CM codes are to be used for coding purposes in the United States starting October 1, 2014.

1	Problem related to lifestyle ICD-lO-CM codes are to be used for coding purposes in the United States starting October 1, 2014. ICD-10-CM Disorder, condition, or problem F0150 Probable major vascular neurocognitive disorder, Without behavioral disturbance F0151 Probable major vascular neurocognitive disorder, With behavioral disturbance F0280 Major neurocognitive disorder due to another medical condition, Without F02.80 Major neurocognitive disorder due to HIV infection, Without behavioral F02.80 Major neurocognitive disorder due to Huntington’s disease, Without F0280 Major neurocognitive disorder due to multiple etiologies, Without behavioral F0280 Major neurocognitive disorder probably due to Parkinson’s disease, Without F0280 Major neurocognitive disorder due to prion disease, Without behavioral

1	F0280 Major neurocognitive disorder probably due to Parkinson’s disease, Without F0280 Major neurocognitive disorder due to prion disease, Without behavioral F0280 Major neurocognitive disorder due to traumatic brain injury, Without behavioral disturbance (codefirst SO6.2X9S diffuse traumatic brain injury with loss of consciousness of unspecified duration, sequela) F0280 Probable major frontotemporal neurocognitive disorder, Without behavioral disturbance (codefirst 631.09 frontotemporal disease) F0280 Probable major neurocognitive disorder due to Alzheimer’s disease, Without behavioral disturbance (codefirst 630.9 Alzheimer’s disease) F0280 Probable major neurocognitive disorder with Lewy bodies, Without behavioral disturbance (codefirst 631.83 Lewy body disease) F02.81 Major neurocognitive disorder due to another medical condition, With F02.81 Major neurocognitive disorder due to HIV infection, With behavioral

1	F02.81 Major neurocognitive disorder due to another medical condition, With F02.81 Major neurocognitive disorder due to HIV infection, With behavioral F02.81 Major neurocognitive disorder due to Huntington’s disease, With behavioral F02.81 Major neurocognitive disorder due to multiple etiologies, With behavioral Numerical Listing of DSM-5 Diagnoses and Codes (ICD-10-CM) ICD—10—CM Disorder, condition, or problem F02.81 F02.81 F02.81 F02.81 F02.81 F02.81 F10.10 F10.121 F10.129 F10.159 F10.180 F10.181 F10.182 F10.221 Major neurocognitive disorder probably due to Parkinson’s disease, With Major neurocognitive disorder due to prion disease, With behavioral Major neurocognitive disorder due to traumatic brain injury, With behavioral disturbance (codefirst $06.2X9S diffuse traumatic brain injury with loss of consciousness of unspecified duration, sequela)

1	Probable major frontotemporal neurocognitive disorder, With behavioral disturbance (code first 631.09 frontotemporal disease) Probable major neurocognitive disorder due to Alzheimer’s disease, With behavioral disturbance (codefirst 630.9 Alzheimer’s disease) Probable major neurocognitive disorder with Lewy bodies, With behavioral disturbance (code first 631.83 Lewy body disease) Delirium due to another medical condition Delirium due to multiple etiologies Psychotic disorder due to another medical condition, With hallucinations Catatonia associated with another mental disorder (catatonia specifier) Catatonic disorder due to another medical condition Unspecified catatonia (codefirst R29.818 other symptoms involving nervous Psychotic disorder due to another medical condition, With delusions Depressive disorder due to another medical condition, With depressive features Depressive disorder due to another medical condition, With major

1	Depressive disorder due to another medical condition, With depressive features Depressive disorder due to another medical condition, With major Bipolar and related disorder due to another medical condition, With manic features Bipolar and related disorder due to another medical condition, With manic- or Bipolar and related disorder due to another medical condition, With mixed Depressive disorder due to another medical condition, With mixed features Anxiety disorder due to another medical condition Obsessive—compulsive and related disorder due to another medical condition Other specified mental disorder due to another medical condition Personality change due to another medical condition Unspecified mental disorder due to another medical condition Alcohol use disorder, Mild Alcohol intoxication delirium, With mild use disorder Alcohol intoxication, With mild use disorder Alcohol-induced bipolar and related disorder, With mild use disorder

1	Alcohol use disorder, Mild Alcohol intoxication delirium, With mild use disorder Alcohol intoxication, With mild use disorder Alcohol-induced bipolar and related disorder, With mild use disorder Alcohol-induced depressive disorder, With mild use disorder Alcohol-induced psychotic disorder, With mild use disorder Alcohol-induced anxiety disorder, With mild use disorder Alcohol-induced sexual dysfunction, With mild use disorder Alcohol-induced sleep disorder, With mild use disorder Alcohol use disorder, Moderate Alcohol use disorder, Severe Alcohol intoxication delirium, With moderate or severe use disorder Numerical Listing of DSM-5 Diagnoses and Codes (|CD-10—CM) 879 |CD—10-CM Disorder, condition, or problem F10.229 F10.231 F10.232 F10.239 F10.259 F10.27 F10.280 F10.281 F10.282 F10.288 F10.921 F10.929 F10.94 F10.94 F10.959 F10.980 F10.981 F10.982 F10.988 F11.10 F11.121 F11.122 F11.129 F11.14 F11.181 F11.182 F11.188 F11.222 F11.229 F11.23

1	F10.282 F10.288 F10.921 F10.929 F10.94 F10.94 F10.959 F10.980 F10.981 F10.982 F10.988 F11.10 F11.121 F11.122 F11.129 F11.14 F11.181 F11.182 F11.188 F11.222 F11.229 F11.23 Alcohol intoxication, With moderate or severe use disorder Alcohol withdrawal, With perceptual disturbances Alcohol withdrawal, Without perceptual disturbances Alcohol-induced bipolar and related disorder, With moderate or severe use Alcohol-induced depressive disorder, With moderate or severe use disorder Alcohol-induced psychotic disorder, With moderate or severe use disorder Alcohol—induced major neurocognitive disorder, Amnestic confabulatory type, With moderate or severe use disorder Alcohol-induced major neurocognitive disorder, Nonamnestic confabulatory type, With moderate or severe use disorder Alcohol-induced anxiety disorder, With moderate or severe use disorder Alcohol-induced sexual dysfunction, With moderate or severe use disorder

1	Alcohol-induced anxiety disorder, With moderate or severe use disorder Alcohol-induced sexual dysfunction, With moderate or severe use disorder Alcohol-induced sleep disorder, With moderate or severe use disorder Alcohol-induced mild neurocognitive disorder, With moderate or severe use Alcohol intoxication delirium, Without use disorder Alcohol intoxication, Without use disorder Alcohol-induced bipolar and related disorder, Without use disorder Alcohol-induced depressive disorder, Without use disorder Alcohol-induced psychotic disorder, Without use disorder Alcohol-induced major neurocognitive disorder, Amnestic confabulatory type, Without use disorder Alcohol-induced major neurocognitive disorder, Nonamnestic confabulatory type, Without use disorder Alcohol-induced anxiety disorder, Without use disorder Alcohol-induced sexual dysfunction, Without use disorder Alcohol-induced sleep disorder, Without use disorder

1	Alcohol-induced anxiety disorder, Without use disorder Alcohol-induced sexual dysfunction, Without use disorder Alcohol-induced sleep disorder, Without use disorder Alcohol-induced mild neurocognitive disorder, Without use disorder Opioid use disorder, Mild Opioid intoxication delirium, With mild use disorder Opioid intoxication, With perceptual disturbances, With mild use disorder Opioid intoxication, Without perceptual disturbances, With mild use disorder Opioid-induced depressive disorder, With mild use disorder Opioid-induced sexual dysfunction, With mild use disorder Opioid-induced sleep disorder, With mild use disorder Opioid-induced anxiety disorder, With mild use disorder Opioid use disorder, Moderate Opioid use disorder, Severe Opioid intoxication delirium, With moderate or severe use disorder Opioid intoxication, With perceptual disturbances, With moderate or severe Opioid intoxication, Without perceptual disturbances, With moderate or

1	Opioid intoxication, With perceptual disturbances, With moderate or severe Opioid intoxication, Without perceptual disturbances, With moderate or Numerical Listing of DSM-5 Diagnoses and Codes (|CD-10-CM) lCD-10-CM Disorder, condition, or problem F11.281 F11.282 F11.288 F11.921 F11.921 F11.922 F11.929 F11.94 F11.981 F11.982 F11.988 F12.121 F12.122 F12.129 F12.159 F12.180 F12.188 F12.221 F12.222 F12.229 F12.259 F12.280 F12.288 F12.288 F12.921 F12.922 F12.929 F12.959 F12.980 F12.988 F13,121 F13.129 F13.14 F13.14 Opioid-induced depressive disorder, With moderate or severe use disorder Opioid-induced sexual dysfunction, With moderate or severe use disorder Opioid-induced sleep disorder, With moderate or severe use disorder Opioid-induced anxiety disorder, With moderate or severe use disorder Opioid intoxication delirium, Without use disorder Opioid intoxication, With perceptual disturbances, Without use disorder

1	Opioid-induced anxiety disorder, With moderate or severe use disorder Opioid intoxication delirium, Without use disorder Opioid intoxication, With perceptual disturbances, Without use disorder Opioid intoxication, Without perceptual disturbances, Without use disorder Opioid-induced depressive disorder, Without use disorder Opioid-induced sexual dysfunction, Without use disorder Opioid-induced sleep disorder, Without use disorder Opioid-induced anxiety disorder, Without use disorder Cannabis use disorder, Mild Cannabis intoxication delirium, With mild use disorder Cannabis intoxication, With perceptual disturbances, With mild use disorder Cannabis intoxication, Without perceptual disturbances, With mild use disorder Cannabis-induced psychotic disorder, With mild use disorder Cannabis-induced anxiety disorder, With mild use disorder Cannabis-induced sleep disorder, With mild use disorder Cannabis use disorder, Moderate Cannabis use disorder, Severe

1	Cannabis-induced anxiety disorder, With mild use disorder Cannabis-induced sleep disorder, With mild use disorder Cannabis use disorder, Moderate Cannabis use disorder, Severe Cannabis intoxication delirium, With moderate or severe use disorder Cannabis intoxication, With perceptual disturbances, With moderate or severe Cannabis intoxication, Without perceptual disturbances, With moderate or Cannabis—induced psychotic disorder, With moderate or severe use disorder Cannabis-induced anxiety disorder, With moderate or severe use disorder Cannabis-induced sleep disorder, With moderate or severe use disorder Cannabis intoxication delirium, Without use disorder Cannabis intoxication, With perceptual disturbances, Without use disorder Cannabis intoxication, Without perceptual disturbances, Without use disorder Cannabis-induced psychotic disorder, Without use disorder Cannabis-induced anxiety disorder, Without use disorder Cannabis-induced sleep disorder, Without use disorder

1	Cannabis-induced psychotic disorder, Without use disorder Cannabis-induced anxiety disorder, Without use disorder Cannabis-induced sleep disorder, Without use disorder Sedative, hypnotic, or anxiolytic use disorder, Mild Sedative, hypnotic, or anxiolytic intoxication delirium, With mild use disorder Sedative, hypnotic, or anxiolytic intoxication, With mild use disorder Sedative, hypnotic-, or anxiolytic-induced bipolar and related disorder, With Sedative—, hypnotic-, or anxiolytic-induced depressive disorder, With mild use Numerical Listing of DSM-5 Diagnoses and Codes (|CD-10-CM) 881 lCD-10-CM Disorder, condition, or problem F13.159 F13.180 F13.181 F13.182 F13.221 F13.229 F13.231 F13.232 F13.239 F13.259 F13.27 F13.280 F13.281 F13.282 F13.288 F13.921 F13.921 F13.929 F13.959 F13.97 F13.980 F13.981 F13.982 F13.988 Sedative-, hypnotic-, or anxiolytic-induced psychotic disorder, With mild use

1	F13.280 F13.281 F13.282 F13.288 F13.921 F13.921 F13.929 F13.959 F13.97 F13.980 F13.981 F13.982 F13.988 Sedative-, hypnotic-, or anxiolytic-induced psychotic disorder, With mild use Sedative, hypnotic-, or anxiolytic-induced anxiety disorder, With mild use Sedative-, hypnotic-, or anxiolytic-induced sexual dysfunction, With mild use Sedative, hypnotic-, or anxiolytic-induced sleep disorder, With mild use disorder Sedative, hypnotic, or anxiolytic use disorder, Moderate Sedative, hypnotic, or anxiolytic use disorder, Severe Sedative, hypnotic, or anxiolytic intoxication delirium, With moderate or Sedative, hypnotic, or anxiolytic intoxication, With moderate or severe use disorder Sedative, hypnotic, or anxiolytic withdrawal delirium Sedative, hypnotic, or anxiolytic withdrawal, With perceptual disturbances Sedative, hypnotic, or anxiolytic withdrawal, Without perceptual disturbances Sedative-, hypnotic-, or anxiolytic-induced bipolar and related disorder, With

1	Sedative, hypnotic, or anxiolytic withdrawal, Without perceptual disturbances Sedative-, hypnotic-, or anxiolytic-induced bipolar and related disorder, With Sedative-, hypnotic-, or anxiolytic-induced depressive disorder, With Sedative-, hypnotic-, or anxiolytic-induced psychotic disorder, With moderate Sedative-, hypnotic-, or anxiolytic-induced major neurocognitive disorder, Sedative-, hypnotic—, or anxiolytic-induced anxiety disorder, With moderate or Sedative-, hypnotic-, or anxiolytic-induced sexual dysfunction, With moderate Sedative-, hypnotic-, or anxiolytic-induced sleep disorder, With moderate or Sedative-, hypnotic-, or anxiolytic-induced mild neurocognitive disorder, Sedative-, hypnotic-, or anxiolytic-induced delirium Sedative, hypnotic, or anxiolytic intoxication delirium, Without use disorder Sedative, hypnotic, or anxiolytic intoxication, Without use disorder Sedative, hypnotic—, or anxiolytic-induced bipolar and related disorder,

1	Sedative, hypnotic, or anxiolytic intoxication, Without use disorder Sedative, hypnotic—, or anxiolytic-induced bipolar and related disorder, Sedative-, hypnotic-, or anxiolytic-induced depressive disorder, Without use Sedative-, hypnotic-, or anxiolytic-induced psychotic disorder, Without use Sedative, hypnotic-, or anxiolytic-induced major neurocognitive disorder, Sedative—, hypnotic-, or anxiolytic-induced anxiety disorder, Without use disorder Sedative-, hypnotic-, or anxiolytic-induced sexual dysfunction, Without use Sedative, hypnotic-, or anxiolytic-induced sleep disorder, Without use disorder Sedative, hypnotic-, or anxiolytic-induced mild neurocognitive disorder, Numerical Listing of DSM-5 Diagnoses and Codes (ICD-10-CM) ICD-10-CM Disorder, condition, or problem F14.10 F14.121 F14.122 F14.129 F14.14 F14.14 F14.159 F14.180 F14.181 F14.182 F14.188 F14.221 F14.222 F14.229 F14.259 F14.280 F14.281 F14.282 F14.288 F14.921 F14.922 F14.929 F14.959

1	F14.121 F14.122 F14.129 F14.14 F14.14 F14.159 F14.180 F14.181 F14.182 F14.188 F14.221 F14.222 F14.229 F14.259 F14.280 F14.281 F14.282 F14.288 F14.921 F14.922 F14.929 F14.959 F14.980 F14.981 F14.982 F14.988 F14.99 F15.10 F15.10 F15.121 F15.122 Unspecified sedative-, hypnotic-, or anxiolytic-related disorder Cocaine use disorder, Mild Cocaine intoxication delirium, With mild use disorder Cocaine intoxication, With perceptual disturbances, With mild use disorder Cocaine intoxication, Without perceptual disturbances, With mild use disorder Cocaine-induced bipolar and related disorder, With mild use disorder Cocaine-induced depressive disorder, With mild use disorder Cocaine-induced psychotic disorder, With mild use disorder Cocaine-induced anxiety disorder, With mild use disorder Cocaine-induced sexual dysfunction, With mild use disorder Cocaine-induced sleep disorder, With mild use disorder

1	Cocaine-induced anxiety disorder, With mild use disorder Cocaine-induced sexual dysfunction, With mild use disorder Cocaine-induced sleep disorder, With mild use disorder Cocaine-induced obsessive-compulsive and related disorder, With mild use Cocaine use disorder, Moderate Cocaine use disorder, Severe Cocaine intoxication delirium, With moderate or severe use disorder Cocaine intoxication, With perceptual disturbances, With moderate or severe Cocaine intoxication, Without perceptual disturbances, With moderate or Cocaine-induced bipolar and related disorder, With moderate or severe use Cocaine-induced depressive disorder, With moderate or severe use disorder Cocaine-induced psychotic disorder, With moderate or severe use disorder Cocaine-induced anxiety disorder, With moderate or severe use disorder Cocaine-induced sexual dysfunction, With moderate or severe use disorder Cocaine-induced sleep disorder, With moderate or severe use disorder

1	Cocaine-induced sexual dysfunction, With moderate or severe use disorder Cocaine-induced sleep disorder, With moderate or severe use disorder Cocaine-induced obsessive—compulsive and related disorder, With moderate Cocaine intoxication delirium, Without use disorder Cocaine intoxication, With perceptual disturbances, Without use disorder Cocaine intoxication, Without perceptual disturbances, Without use disorder Cocaine-induced bipolar and related disorder, Without use disorder Cocaine-induced depressive disorder, Without use disorder Cocaine-induced psychotic disorder, Without use disorder Cocaine-induced anxiety disorder, Without use disorder Cocaine-induced sexual dysfunction, Without use disorder Cocaine-induced sleep disorder, Without use disorder Cocaine-induced obsessive—compulsive and related disorder, Without use disorder Unspecified stimulant-related disorder, Unspecified Cocaine-related disorder Amphetamine-type substance use disorder, Mild

1	Unspecified stimulant-related disorder, Unspecified Cocaine-related disorder Amphetamine-type substance use disorder, Mild Other or unspecified stimulant use disorder, Mild Amphetamine (or other stimulant) intoxication delirium, With mild use disorder Amphetamine or other stimulant intoxication, With perceptual disturbances, Numerical Listing of DSM-5 Diagnoses and Codes (ICD-10-CM) 883 ICD-10—CM Disorder, condition, or problem F15.129 F15.14 F15.14 F15.159 F15.180 F15.180 F15.181 F15.182 F15.182 F15.188 F15.221 F15.222 F15.229 F15.259 F15.280 F15.280 F15.281 F15.282 F15.282 F15.288 F15.921 F15.921 F15.922 Amphetamine or other stimulant intoxication, Without perceptual disturbances, With mild use disorder Amphetamine (or other stimulant)—induced bipolar and related disorder, Amphetamine (or other stimulant)—induced depressive disorder, With mild Amphetamine (or other stimulant)—induced psychotic disorder, With mild use

1	Amphetamine (or other stimulant)—induced depressive disorder, With mild Amphetamine (or other stimulant)—induced psychotic disorder, With mild use Amphetamine (or other stimulant)—induced anxiety disorder, With mild use Caffeine-induced anxiety disorder, With mild use disorder Amphetamine (or other stimulant)—induced sexual dysfunction, With mild Amphetamine (or other sfimulant)—induced sleep disorder, With mild use disorder Caffeine-induced sleep disorder, With mild use disorder disorder, With mild use disorder Amphetamine-type substance use disorder, Moderate Amphetamine-type substance use disorder, Severe Other or unspecified stimulant use disorder, Moderate Other or unspecified stimulant use disorder, Severe Amphetamine (or other stimulant) intoxication delirium, With moderate or Amphetamine or other stimulant intoxication, With perceptual disturbances, Amphetamine or other stimulant intoxication, Without perceptual disturbances, With moderate or severe use disorder

1	Amphetamine or other stimulant intoxication, With perceptual disturbances, Amphetamine or other stimulant intoxication, Without perceptual disturbances, With moderate or severe use disorder Amphetamine (or other stimulant)—induced bipolar and related disorder, Amphetamine (or other stimulant)—induced depressive disorder, With Amphetamine (or other stimulant)—induced psychotic disorder, With Amphetamine (or other stimulant)—induced anxiety disorder, With moderate Caffeine-induced anxiety disorder, With moderate or severe use disorder Amphetamine (or other stimulant)—induced sexual dysfunction, With Amphetamine (or other stimulant)—induced sleep disorder, With moderate or Caffeine-induced sleep disorder, With moderate or severe use disorder disorder, With moderate or severe use disorder Amphetamine (or other stimulant) intoxication delirium, Without use disorder Amphetamine or other stimulant intoxication, With perceptual disturbances,

1	Amphetamine (or other stimulant) intoxication delirium, Without use disorder Amphetamine or other stimulant intoxication, With perceptual disturbances, Numerical Listing of DSM-5 Diagnoses and Codes (ICD-10-CM) lCD-10-CM Disorder, condition, or problem F15.929 F15.929 F15.94 F15.94 F15.959 F15.980 F15.980 F15.981 F15.982 F15.982 F15.988 F16.10 F16.10 F16.121 F16.121 F16.129 F16.129 F16.14 F16.14 F16.14 F16.14 F16.159 F16.159 F16.180 F16.180 F16.221 F16.221 F16.229 F16.229 Amphetamine or other stimulant intoxication, Without perceptual disturbances, Without use disorder Amphetamine (or other stimulant)—induced bipolar and related disorder, Amphetamine (or other stimulant)—induced depressive disorder, Without use Amphetamine (or other stimulant)—induced psychotic disorder, Without use Amphetamine (or other stimulant)—induced anxiety disorder, Without use Caffeine-induced anxiety disorder, Without use disorder

1	Amphetamine (or other stimulant)—induced anxiety disorder, Without use Caffeine-induced anxiety disorder, Without use disorder Amphetamine (or other stimulant)—induced sexual dysfunction, Without use Amphetamine (or other stimulant)—induced sleep disorder, Without use disorder Caffeine-induced sleep disorder, Without use disorder disorder, Without use disorder Other hallucinogen use disorder, Mild Phencyclidine use disorder, Mild Other hallucinogen intoxication delirium, With mild use disorder Phencyclidine intoxication delirium, With mild use disorder Other hallucinogen intoxication, With mild use disorder Phencyclidine intoxication, With mild use disorder Other hallucinogen—induced bipolar and related disorder, With mild use disorder Other hallucinogen—induced depressive disorder, With mild use disorder Phencyclidine-induced bipolar and related disorder, With mild use disorder Phencyclidine-induced depressive disorder, With mild use disorder

1	Phencyclidine-induced bipolar and related disorder, With mild use disorder Phencyclidine-induced depressive disorder, With mild use disorder Other hallucinogen—induced psychotic disorder, With mild use disorder Phencyclidine-induced psychotic disorder, With mild use disorder Other hallucinogen—induced anxiety disorder, With mild use disorder Phencyclidine-induced anxiety disorder, With mild use disorder Other hallucinogen use disorder, Moderate Other hallucinogen use disorder, Severe Phencyclidine use disorder, Moderate Phencyclidine use disorder, Severe Other hallucinogen intoxication delirium, With moderate or severe use disorder Phencyclidine intoxication delirium, With moderate or severe use disorder Other hallucinogen intoxication, With moderate or severe use disorder Phencyclidine intoxication, With moderate or severe use disorder Other hallucinogen—induced bipolar and related disorder, With moderate or

1	Phencyclidine intoxication, With moderate or severe use disorder Other hallucinogen—induced bipolar and related disorder, With moderate or Numerical Listing of DSM-5 Diagnoses and Codes (|CD-10-CM) 885 |CD-10-CM Disorder, condition, or problem F16.259 F16.259 F16.280 F16.280 F16.921 F16.921 F16.929 F16.929 F16.959 F16.959 F16.980 F16.980 F16.983 F17.200 F17.200 F17.203 F17.208 F17.209 F18.10 F18.121 F18.129 F18.14 F18.159 F18.180 F18.188 F18.221 F18.229 F18.24 F18.259 Other hallucinogen—induced depressive disorder, With moderate or severe Phencyclidine-induced bipolar and related disorder, With moderate or severe Phencyclidirie-induced depressive disorder, With moderate or severe use disorder Other hallucinogen—induced psychotic disorder, With moderate or severe use Phencyclidine-induced psychotic disorder, With moderate or severe use disorder Other hallucinogen—induced anxiety disorder, With moderate or severe use

1	Phencyclidine-induced psychotic disorder, With moderate or severe use disorder Other hallucinogen—induced anxiety disorder, With moderate or severe use Phencyclidine-induced anxiety disorder, With moderate or severe use disorder Other hallucinogen intoxication delirium, Without use disorder Phencyclidine intoxication delirium, Without use disorder Other hallucinogen intoxication, Without use disorder Phencyclidine intoxication, Without use disorder Other hallucinogen—induced bipolar and related disorder, Without use disorder Other hallucinogen—induced depressive disorder, Without use disorder Phencyclidine-induced bipolar and related disorder, Without use disorder Phencyclidine-induced depressive disorder, Without use disorder Other hallucinogen—induced psychotic disorder, Without use disorder Phencyclidine-induced psychotic disorder, Without use disorder Other hallucinogen—induced anxiety disorder, Without use disorder

1	Other hallucinogen—induced psychotic disorder, Without use disorder Phencyclidine-induced psychotic disorder, Without use disorder Other hallucinogen—induced anxiety disorder, Without use disorder Phencyclidine-induced anxiety disorder, Without use disorder Tobacco use disorder, Moderate Tobacco use disorder, Severe Tobacco-induced sleep disorder, With moderate or severe use disorder Inhalant use disorder, Mild Inhalant intoxication delirium, With mild use disorder Inhalant intoxication, With mild use disorder Inhalant—induced depressive disorder, With mild use disorder Inhalant-induced psychotic disorder, With mild use disorder Inhalant-induced major neurocognitive disorder, With mild use disorder Inhalant-induced anxiety disorder, With mild use disorder Inhalant-induced mild neurocognitive disorder, With mild use disorder Inhalant use disorder, Moderate Inhalant use disorder, Severe Inhalant intoxication delirium, With moderate or severe use disorder

1	Inhalant use disorder, Moderate Inhalant use disorder, Severe Inhalant intoxication delirium, With moderate or severe use disorder Inhalant intoxication, With moderate or severe use disorder Inhalant-induced depressive disorder, With moderate or severe use disorder Inhalant-induced psychotic disorder, With moderate or severe use disorder Numerical Listing of DSM-5 Diagnoses and Codes (|CD-10-CM) ICD-10-CM Disorder, condition, or problem F18.27 F18.280 F18.288 F18.921 F18.929 F18.959 F18.97 F18.980 F18.988 F19.121 F19.129 F19.159 F19.17 F19.180 F19.181 F19.182 F19.188 F19.188 F19.229 F19.231 F19.239 F19.259 F19.27 Inhalant-induced major neurocognitive disorder, With moderate or severe use Inhalant-induced anxiety disorder, With moderate or severe use disorder Inhalant-induced mild neurocognitive disorder, With moderate or severe use Inhalant intoxication delirium, Without use disorder Inhalant intoxication, Without use disorder

1	Inhalant-induced mild neurocognitive disorder, With moderate or severe use Inhalant intoxication delirium, Without use disorder Inhalant intoxication, Without use disorder Inhalant-induced depressive disorder, Without use disorder Inhalant-induced psychotic disorder, Without use disorder Inhalant-induced major neurocognitive disorder, Without use disorder Inhalant—induced anxiety disorder, Without use disorder Inhalant-induced mild neurocognitive disorder, Without use disorder Other (or unknown) substance use disorder, Mild Other (or unknown) substance intoxication delirium, With mild use disorder Other (or unknown) substance intoxication, With mild use disorder Other (or unknown) substanc&induced bipolar and related disorder, With Other (or unknown) substance—induced depressive disorder, With mild use Other (or unknown) substance—induced psychotic disorder, With mild use Other (or unknown) substance—induced major neurocognitive disorder, With

1	Other (or unknown) substance—induced psychotic disorder, With mild use Other (or unknown) substance—induced major neurocognitive disorder, With Other (or unknown) substance—induced anxiety disorder, With mild use disorder Other (or unknown) substance—induced sexual dysfunction, With mild use Other (or unknown) substance—induced sleep disorder, With mild use disorder Other (or unknown) substance—induced mild neurocognitive disorder, With disorder, With mild use disorder Other (or unknown) substance use disorder, Moderate Other (or unknown) substance use disorder, Severe Other (or unknown) substance intoxication delirium, With moderate or severe Other (or unknown) substance intoxication, With moderate or severe use disorder Other (or unknown) substance—induced bipolar and related disorder, With Other (or unknown) substance—induced depressive disorder, With moderate Other (or unknown) substance—induced psychotic disorder, With moderate or

1	Other (or unknown) substance—induced depressive disorder, With moderate Other (or unknown) substance—induced psychotic disorder, With moderate or Other (or unknown) substance—induced major neurocognitive disorder, With Numerical Listing of DSM-5 Diagnoses and Codes (ICD-10-CM) 887 |CD-10-CM Disorder, condition, or problem F19.280 F19.281 F19.282 F19.288 F19.288 F19.921 F19.921 F19.929 F19.959 F19.97 F19.980 F19.981 F19.982 F19.988 F19.988 F25.1 F31.0 F31.11 F31.12 F31.13 F31.4 Othet (or unknown) substance—induced anxiety disorder, With moderate or Other (or unknown) substance—induced sexual dysfunction, With moderate or Other (or unknown) substancehinduced sleep disorder, With moderate or Other (or unknown) substance—induced mild neurocognitive disorder, With disorder, With moderate or severe use disorder Other (or unknown) substance intoxication delirium, Without use disorder Other (or unknown) substance intoxication, Without use disorder

1	Other (or unknown) substance intoxication delirium, Without use disorder Other (or unknown) substance intoxication, Without use disorder Other (or unknown) substance—induced bipolar and related disorder, Without Other (or unknown) substance—induced depressive disorder, Without use disorder Other (or unknown) substance—induced psychotic disorder, Without use disorder Other (or unknown) substance—induced major neurocognitive disorder, Other (or unknown) substance—induced anxiety disorder, Without use disorder Other (or unknown) substance—induced sexual dysfunction, Without use disorder Other (or unknown) substance—induced sleep disorder, Without use disorder Other (or unknown) substance—induced mild neurocognitive disorder, disorder, Without use disorder Schizoaffective disorder, Bipolar type Schizoaffective disorder, Depressive type Bipolar I disorder, Current or most recent episode hypomanic Bipolar I disorder, Current or most recent episode manic, Mild

1	Schizoaffective disorder, Depressive type Bipolar I disorder, Current or most recent episode hypomanic Bipolar I disorder, Current or most recent episode manic, Mild Bipolar I disorder, Current or most recent episode manic, Moderate Bipolar I disorder, Current or most recent episode manic, Severe Bipolar I disorder, Current or most recent episode manic, With psychotic features Bipolar I disorder, Current or most recent episode depressed, Mild Bipolar I disorder, Current or most recent episode depressed, Moderate Bipolar I disorder, Current or most recent episode depressed, Severe Bipolar I disorder, Current or most recent episode depressed, With psychotic Numerical Listing of DSM-5 Diagnoses and Codes (|CD-10-CM) ICD-10-CM Disorder, condition, or problem F31.74 F31.74 F40.218 F40.228 F40.230 F40.231 F40.232 F40.233 F40.248 F40.298 F41.0 F41.1 Bipolar I disorder, Current or most recent episode hypomanic, In partial remission

1	F31.74 F31.74 F40.218 F40.228 F40.230 F40.231 F40.232 F40.233 F40.248 F40.298 F41.0 F41.1 Bipolar I disorder, Current or most recent episode hypomanic, In partial remission Bipolar I disorder, Current or most recent episode manic, In partial remission Bipolar I disorder, Current or most recent episode hypomanic, In full remission Bipolar I disorder, Current or most recent episode manic, In full remission Bipolar I disorder, Current or most recent episode depressed, In partial remission Bipolar I disorder, Current or most recent episode depressed, In full remission Bipolar I disorder, Current or most recent episode depressed, Unspecified Bipolar I disorder, Current or most recent episode hypomanic, Unspecified Bipolar I disorder, Current or most recent episode manic, Unspecified Bipolar I disorder, Current or most recent episode unspecified Major depressive disorder, Single episode, Mild Major depressive disorder, Single episode, Moderate

1	Bipolar I disorder, Current or most recent episode unspecified Major depressive disorder, Single episode, Mild Major depressive disorder, Single episode, Moderate Major depressive disorder, Single episode, Severe Major depressive disorder, Single episode, With psychotic features Major depressive disorder, Single episode, In partial remission Major depressive disorder, Single episode, In full remission Major depressive disorder, Single episode, Unspecifed Major depressive disorder, Recurrent episode, Mild Major depressive disorder, Recurrent episode, Moderate Major depressive disorder, Recurrent episode, Severe Major depressive disorder, Recurrent episode, With psychotic features Major depressive disorder, Recurrent episode, In partial remission Major depressive disorder, Recurrent episode, In full remission Major depressive disorder, Recurrent episode, Unspecified Specific phobia, Animal Specific phobia, Natural environment Specific phobia, Fear of blood

1	Major depressive disorder, Recurrent episode, Unspecified Specific phobia, Animal Specific phobia, Natural environment Specific phobia, Fear of blood Specific phobia, Fear of injections and transfusions Specific phobia, Fear of other medical care Specific phobia, Fear of injury Specific phobia, Situational Specific phobia, Other Numerical Listing of DSM-5 Diagnoses and Codes (ICD-10-CM) 889 ICD-10-CM Disorder, condition, or problem F41 .9 F44.1 F44.4 F44.4 F44.4 F44.4 F44.7 Adjustment disorders, Unspecified Adjustment disorders, With depressed mood Adjustment disorders, With anxiety Adjustment disorders, With mixed anxiety and depressed mood Adjustment disorders, With disturbance of conduct Adjustment disorders, With mixed disturbance of emotions and conduct Dissociative amnesia, With dissociative fugue Conversion disorder (functional neurological symptom disorder), With Conversion disorder (functional neurological symptom disorder), With

1	Dissociative amnesia, With dissociative fugue Conversion disorder (functional neurological symptom disorder), With Conversion disorder (functional neurological symptom disorder), With Conversion disorder (functional neurological symptom disorder), With Conversion disorder (functional neurological symptom disorder), With Conversion disorder (functional neurological symptom disorder), With Conversion disorder (functional neurological symptom disorder), With Conversion disorder (functional neurological symptom disorder), With Conversion disorder (functional neurological symptom disorder), With mixed Anorexia nervosa, Restricting type Anorexia nervosa, Binge-eating/purging type Numerical Listing of DSM-5 Diagnoses and Codes (|CD-10-CM) ICD-10-CM Disorder, condition, or problem F52.4 F63.1 F64.1 F65.1 Pica, in adults Non—rapid eye movement sleep arousal disorders, Sleepwalking type Non—rapid eye movement sleep arousal disorders, Sleep terror type

1	F52.4 F63.1 F64.1 F65.1 Pica, in adults Non—rapid eye movement sleep arousal disorders, Sleepwalking type Non—rapid eye movement sleep arousal disorders, Sleep terror type Numerical Listing of DSM-5 Diagnoses and Codes (ICD-10-CM) 891 |CD-10-CM Disorder, condition, or problem F90.1 F91.1 F91.3 F94.1 Intellectual disability (intellectual developmental disorder), Mild Intellectual disability (intellectual developmental disorder), Moderate Intellectual disability (intellectual developmental disorder), Severe Intellectual disability (intellectual developmental disorder), Profound Specific learning disorder, With impairment in reading Specific learning disorder, With impairment in mathematics Specific learning disorder, With impairment in written expression Attention-deficit/hyperactivity disorder, Predominantly inattentive presentation Attention-deficit/hyperactivity disorder, Predominantly hyperactive/ Attention-deficit/hyperactivity disorder, Combined presentation

1	Attention-deficit/hyperactivity disorder, Predominantly hyperactive/ Attention-deficit/hyperactivity disorder, Combined presentation Conduct disorder, Childhood-onset type Conduct disorder, Adolescent-onset type Other specified disruptive, impulse-control, and conduct disorder Conduct disorder, Unspecified onset Unspecified disruptive, impulse-control, and conduct disorder Numerical Listing of DSM-S Diagnoses and Codes (|CD-10-CM) ICD-10-CM Disorder, condition, or problem 621.0 621.11 621.19 624.01 624.02 624.09 625.1 625.71 625.71 625.79 625.81 631.84 631.84 631.84 631.84 631.84 631.84 631.84 631.84 631.84 631.84 631.84 631.9 631.9 631.9 631.9 631.9 647.00 647.00 647.09 647.10 647.10 647.19 647.20 647.21 647.22 647.23 647.24 647.26 647.31 Pica, in children Mild neurocognitive disorder due to Alzheimer’s disease Mild neurocognitive disorder due to another medical condition Mild neurocognitive disorder due to HIV infection

1	Pica, in children Mild neurocognitive disorder due to Alzheimer’s disease Mild neurocognitive disorder due to another medical condition Mild neurocognitive disorder due to HIV infection Mild neurocognitive disorder due to Huntington’s disease Mild neurocognitive disorder with Lewy bodies Mild neurocognitive disorder due to multiple etiologies Mild neurocognitive disorder due to Parkinson’s disease Mild neurocognitive disorder due to prion disease Mild neurocognitive disorder due to traumatic brain injury Major neurocognitive disorder possibly due to Parkinson’s disease Possible major neurocognitive disorder due to Alzheimer’s disease Possible major neurocognitive disorder with Lewy bodies Circadian rhythm sleep—wake disorders, Unspecified type Circadian rhythm sleep-wake disorders, Delayed sleep phase type Circadian rhythm sleep-wake disorders, Advanced sleep phase type Circadian rhythm sleep-wake disorders, Irregular sleep-wake type

1	Circadian rhythm sleep-wake disorders, Delayed sleep phase type Circadian rhythm sleep-wake disorders, Advanced sleep phase type Circadian rhythm sleep-wake disorders, Irregular sleep-wake type Circadian rhythm sleep-wake disorders, Non-24-hour sleep-wake type Circadian rhythm sleep-wake disorders, Shift work type Central sleep apnea, Idiopathic central sleep apnea Numerical Listing of DSM-5 Diagnoses and Codes (|CD-10-CM) 893 lCD-10-CM Disorder. condition, or problem 647.33 647.34 647.35 647.36 647.37 647.411 647.419 647.419 647.419 647.429 647.52 647.8 647.9 N39.498 T43.205A T43.205D T50.905A T50.905D T74.01XA T74.01XD T74.02XA T74.02XD T74.11XA T74.11XA T74.11XD T74.11XD T74.12XA T74.12XD T74.21XA T74.21XA T74.21XD Sleep-related hypoventilation, Idiopathic hypoventilation Sleep-related hypoventilation, Congenital central alveolar hypoventilation Sleep-related hypoventilation, Comorbid sleep-related hypoventilation

1	Sleep-related hypoventilation, Congenital central alveolar hypoventilation Sleep-related hypoventilation, Comorbid sleep-related hypoventilation Central sleep apnea comorbid with opioid use Narcolepsy with cataplexy but without hypocretin deficiency Autosomal dominant cerebellar ataxia, deafness, and narcolepsy Autosomal dominant narcolepsy, obesity, and type 2 diabetes Narcolepsy without cataplexy but with hypocretin deficiency Narcolepsy secondary to another medical condition Other specified elimination disorder, With urinary symptoms Central sleep apnea, Cheyne-Stokes breathing Other specified elimination disorder, With fecal symptoms Unspecified elimination disorder, With fecal symptoms Unspecified elimination disorder, With urinary symptoms Antidepressant discontinuation syndrome, Initial encounter Antidepressant discontinuation syndrome, Subsequent encounter Antidepressant discontinuation syndrome, Sequelae Other adverse effect of medication, Initial encounter

1	Antidepressant discontinuation syndrome, Subsequent encounter Antidepressant discontinuation syndrome, Sequelae Other adverse effect of medication, Initial encounter Other adverse effect of medication, Subsequent encounter Other adverse effect of medication, Sequelae Spouse or partner neglect, Confirmed, Initial encounter Spouse or partner neglect, Confirmed, Subsequent encounter Child neglect, Confirmed, Initial encounter Child neglect, Confirmed, Subsequent encounter Adult physical abuse by nonspouse or nonpartner, Confirmed, Initial encounter Spouse or partner violence, Physical, Confirmed, Initial encounter Adult physical abuse by nonspouse or nonpartner, Confirmed, Subsequent Spouse or partner violence, Physical, Confirmed, Subsequent encounter Child physical abuse, Confirmed, Initial encounter Child physical abuse, Confirmed, Subsequent encounter Adult sexual abuse by nonspouse or nonpartner, Confirmed, Initial encounter

1	Child physical abuse, Confirmed, Initial encounter Child physical abuse, Confirmed, Subsequent encounter Adult sexual abuse by nonspouse or nonpartner, Confirmed, Initial encounter Spouse or partner violence, Sexual, Confirmed, Initial encounter Adult sexual abuse by nonspouse or nonpartner, Confirmed, Subsequent Numerical Listing of DSM-5 Diagnoses and Codes (|CD-10-CM) Disorder, condition, or problem T74.21XD T74.22XA T74.22XD T74.31XA T74.31XA T74.31XD T74.31XD T74.32XA T74.32XD T76.01XA T76.01XD T76.02XA T76.02XD T76.11XA T76.11XA T76.11XD T76.11XD T76.12XA T76.12XD T76.21XA T76.21XA T76.21XD T76.21XD T76.22XA T76.22XD T76.31XA T76.31XA T76.31XD T76.31XD T76.32XA T76.32XD 255.9 256.82 256.9 259.0 259.1 259.2 259.3 259.4 259.5 Spouse or partner violence, Sexual, Confirmed, Subsequent encounter Child sexual abuse, Confirmed, Initial encounter Child sexual abuse, Confirmed, Subsequent encounter

1	Spouse or partner violence, Sexual, Confirmed, Subsequent encounter Child sexual abuse, Confirmed, Initial encounter Child sexual abuse, Confirmed, Subsequent encounter Adult psychological abuse by nonspouse or nonpartner, Confirmed, Initial Spouse or partner abuse, Psychological, Confirmed, Initial encounter Adult psychological abuse by nonspouse or nonpartner, Confirmed, Spouse or partner abuse, Psychological, Confirmed, Subsequent encounter Child psychological abuse, Confirmed, Initial encounter Child psychological abuse, Confirmed, Subsequent encounter Spouse or partner neglect, Suspected, Initial encounter Spouse or partner neglect, Suspected, Subsequent encounter Child neglect, Suspected, Initial encounter Child neglect, Suspected, Subsequent encounter Adult physical abuse by nonspouse or nonpartner, Suspected, Initial encounter Spouse or partner violence, Physical, Suspected, Initial encounter

1	Child neglect, Suspected, Subsequent encounter Adult physical abuse by nonspouse or nonpartner, Suspected, Initial encounter Spouse or partner violence, Physical, Suspected, Initial encounter Adult physical abuse by nonspouse or nonpartner, Suspected, Subsequent Spouse or partner violence, Physical, Suspected, Subsequent encounter Child physical abuse, Suspected, Initial encounter Child physical abuse, Suspected, Subsequent encounter Adult sexual abuse by nonspouse or nonpartner, Suspected, Initial encounter Spouse or partner violence, Sexual, Suspected, Initial encounter Adult sexual abuse by nonspouse or nonpartner, Suspected, Subsequent Spouse or partner Violence, Sexual, Suspected, Subsequent encounter Child sexual abuse, Suspected, Initial encounter Child sexual abuse, Suspected, Subsequent encounter Adult psychological abuse by nonspouse or nonpartner, Suspected, Initial Spouse or partner abuse, Psychological, Suspected, Initial encounter

1	Child sexual abuse, Suspected, Subsequent encounter Adult psychological abuse by nonspouse or nonpartner, Suspected, Initial Spouse or partner abuse, Psychological, Suspected, Initial encounter Adult psychological abuse by nonspouse or nonpartner, Suspected, Spouse or partner abuse, Psychological, Suspected, Subsequent encounter Child psychological abuse, Suspected, Initial encounter Child psychological abuse, Suspected, Subsequent encounter Problem related to current military deployment status Other problem related to employment Discord with neighbor, lodger, or landlord Problem related to living in a residential institution Lack of adequate food or safe drinking water Numerical Listing of DSM-5 Diagnoses and Codes (|CD-10-CM) 895

1	Problem related to living in a residential institution Lack of adequate food or safe drinking water Numerical Listing of DSM-5 Diagnoses and Codes (|CD-10-CM) 895 ICD-10-CM Disorder, condition, or problem 259.6 259.7 259 .9 260.0 260.2 260.3 260.4 260.5 260.9 262.29 262.810 262.810 262.811 262.812 262.820 262.891 262.898 263.0 263.4 263.5 263.8 264.0 264.1 264.4 265.0 265.1 265.2 265.3 265.4 265.4 265.5 265.8 265.8 265.9 269.010 269.010 269.010 269.010 269.011 269.011 269.011 269.011 269.020 Phase of life problem Problem related to living alone Target of (perceived) adverse discrimination or persecution Unspecified problem related to social environment Personal history (past history) of physical abuse in childhood Personal history (past history) of sexual abuse in childhood Personal history (past history) of psychological abuse in childhood Personal history (past history) of neglect in childhood Relationship distress with spouse or intimate partner

1	Personal history (past history) of psychological abuse in childhood Personal history (past history) of neglect in childhood Relationship distress with spouse or intimate partner Disruption of family by separation or divorce Problems related to unwanted pregnancy Problems related to multiparity Discord with social service provider, including probation officer, case manager, or social services worker Problems related to release from prison Problems related to other legal circumstances Victim of crime Victim of terrorism or torture Exposure to disaster, war, or other hostilities Other problem related to psychosocial circumstances Unspecified problem related to unspecified psychosocial circumstances Encounter for mental health services for victim of child abuse by parent Encounter for mental health services for victim of child neglect by parent Encounter for mental health services for victim of child psychological abuse by

1	Encounter for mental health services for victim of child neglect by parent Encounter for mental health services for victim of child psychological abuse by Encounter for mental health services for victim of child sexual abuse by parent Encounter for mental health services for perpetrator of parental child abuse Encounter for mental health services for perpetrator of parental child neglect Encounter for mental health services for perpetrator of parental child Encounter for mental health services for perpetrator of parental child sexual abuse Encounter for mental health services for victim of nonparental child abuse Numerical Listing of DSM-S Diagnoses and Codes (|CD-10-CM) lCD-10-CM Disorder, condition, or problem 269.020 269.020 269.020 269.021 269.021 269.021 269.021 269.11 269.11 269.11 269.12 269.12 269.12 269.12 269.81 269.81 269.82 270.9 271.9 272.0 272.810 272.811 272.9 275.3 275.4 276.5 291.19 291.410 291.410 291.411 291.412 291.49 291.5 291.82 291.83 291.89

1	Encounter for mental health services for victim of nonparental child neglect Encounter for mental health services for victim of nonparental child psycho- Encounter for mental health services for victim of nonparental child sexual abuse Encounter for mental health services for perpetrator of nonparental child abuse Encounter for mental health services for perpetrator of nonparental child neglect Encounter for mental health services for perpetrator of nonparental child Encounter for mental health services for perpetrator of nonparental child Encounter for mental health services for victim of spouse or partner neglect Encounter for mental health services for victim of spouse or partner Encounter for mental health services for victim of spouse or partner violence, Encounter for mental health services for perpetrator of spouse or partner neglect Encounter for mental health services for perpetrator of spouse or partner

1	Encounter for mental health services for perpetrator of spouse or partner neglect Encounter for mental health services for perpetrator of spouse or partner Encounter for mental health services for perpetrator of spouse or partner violence, Physical Encounter for mental health services for perpetrator of spouse or partner violence, Sexual Encounter for mental health services for victim of nonspousal adult abuse Encounter for mental health services for victim of spouse or partner violence, Encounter for mental health services for perpetrator of nonspousal adult abuse Tobacco use disorder, mild Problem related to lifestyle Unavailability or inaccessibility of health care facilities Unavailability or inaccessibility of other helping agencies Nonadherence to medical treatment Personal history (past history) of spouse or partner violence, Physical Personal history (past history) of spouse or partner violence, Sexual

1	Nonadherence to medical treatment Personal history (past history) of spouse or partner violence, Physical Personal history (past history) of spouse or partner violence, Sexual Personal history (past history) of spouse or partner psychological abuse Personal history (past history) of spouse or partner neglect Other personal history of psychological trauma Personal history of self-harm Personal history of military deployment Wandering associated with a mental disorder APA Board of Trustees DSM-5 Review Committees Kenneth S. Kendler, M.D. (Chair) Robert Freedman, M.D. (Co—chair) Dan 6. Blazer, M.D., Ph.D., M.P.H. David Brent, M.D. (2011—) Ellen Leibenluft, M.D. Sir Michael Rutter, M.D. (-2011) Paul S. Summergrad, M.D. Robert J. Ursano, M.D. (—2011) Myrna Weissman, PhD. (2011—) Joel Yager, M.D. Jill L. Opalesky M.S. (Administrative Support) John S. McIntyre, M.D. (Chair) Joel Yager, M.D. (Co-chair) Anita Everett M.D. Cathryn A. Galanter, M.D.

1	Joel Yager, M.D. Jill L. Opalesky M.S. (Administrative Support) John S. McIntyre, M.D. (Chair) Joel Yager, M.D. (Co-chair) Anita Everett M.D. Cathryn A. Galanter, M.D. Jeffrey M. Lyness, MD. James E. Nininger, M.D. Victor I. Reus, M.D. Michael I. Vergare, M.D. Carolyn Robinowitz, M.D. (Chair) Mary Badaracco, M.D. Ronald Burd, M.D. Robert Freedman, M.D. Jeffrey A. Lieberman, M.D. Kyla Pope, M.D. Victor I. Reus, M.D. Daniel K. Winstead, M.D. Joel Yager, M.D. Glenn A. Martin, MD. (Chair) R. Scott Benson, M.D. (Speaker of the William Cardasis, M.D. John M. de Figueiredo, M.D. Lawrence S. Gross, M.D. Brian 5. Hart, M.D. Stephen A. McLeod Bryant, M.D. Gregory A. Miller, M.D. Roger Peele, M.D. Charles S. Price, M.D. Deepika Sastry, M.D. John PD. Shemo, M.D. Eliot Sorel, M.D. Dilip V. Jeste, M.D. (Chair) R. Scott Benson, M.D. Kenneth S. Kendler, M.D. Helena C. Kraemer, PhD. David I. Kupfer, M.D. Jeffrey A. Lieberman, M.D. Glenn A. Martin, M.D.

1	Eliot Sorel, M.D. Dilip V. Jeste, M.D. (Chair) R. Scott Benson, M.D. Kenneth S. Kendler, M.D. Helena C. Kraemer, PhD. David I. Kupfer, M.D. Jeffrey A. Lieberman, M.D. Glenn A. Martin, M.D. John S. McIntyre, M.D. John M. Oldham, M.D. Roger Peele, M.D. Darrel A. Regier, M.D., M.P.H. James H. Scully ]r., M.D. Joel Yager, M.D. Paul S. Appelbaum, M.D. (Consultant) Michael B. First, M.D. (Consultant) Robert D. Gibbons, PhD. Craig Nelson, M.D. Paul S. Appelbaum, M.D. Lama Bazzi, M.D. Alec W. Buchanan, M.D., PhD. Carissa Caban Aleman, M.D. Michael Champion, M.D. Jeffrey C. Eisen, M.D. Elizabeth Ford, M.D. Daniel T. Hackman, M.D. Mark Hauser, M.D. Steven K. Hoge, M.D., M.B.A. Debra A. Pinals, M.D. Guillermo Portillo, M.D. Patricia Recupero, M.D., ].D. Robert Weinstock, M.D. Cheryl Wills, M.D. Howard V. Zonana, M.D. Erin I. Dalder-Alpher Leah I. Engel Elizabeth C. Martin Rocio I. Salvador Emil F. Coccaro, M.D. Deborah Dabrick, Ph.D.

1	Robert Weinstock, M.D. Cheryl Wills, M.D. Howard V. Zonana, M.D. Erin I. Dalder-Alpher Leah I. Engel Elizabeth C. Martin Rocio I. Salvador Emil F. Coccaro, M.D. Deborah Dabrick, Ph.D. Prudence W. Fisher, Ph.D. Benjamin B. Lahey, Ph.D. Salvatore Mannuzza, Ph.D. Mary Solanto, Ph.D. I. Blake Turner, Ph.D. Eric Youngstrom, Ph.D. Anxiety, Obsessive-Compulsive Spectrum, Posttraumatic, and Lynn E. Alden, Ph.D. David B. Arciniegas, M.D. David H. Barlow, Ph.D. Katja Beesdo-Baum, Ph.D. Chris R. Brewin, Ph.D. Richard I. Brown, Ph.D. Timothy A. Brown, Ph.D. Richard A. Bryant, Ph.D. Joan M. Cook, Ph.D. loop de Jong, M.D., Ph.D. Paul F. Dell, Ph.D. Damiaan Denys, M.D. Bruce P. Dohrenwend, Ph.D. Brian A. Fallon, M.D., M.P.H. Edna B. Foa, Ph.D. Martin E. Franklin, Ph.D. Wayne K. Goodman, M.D. Jon E. Grant, ].D., M.D. Bonnie L. Green, Ph.D. Richard 6. Heimberg, Ph.D. Judith L. Herman, M.D. Devon E. Hinton, M.D., Ph.D. Stefan 6. Hofmann, Ph.D.

1	Wayne K. Goodman, M.D. Jon E. Grant, ].D., M.D. Bonnie L. Green, Ph.D. Richard 6. Heimberg, Ph.D. Judith L. Herman, M.D. Devon E. Hinton, M.D., Ph.D. Stefan 6. Hofmann, Ph.D. Charles W. Hoge, M.D. Terence M. Keane, Ph.D. Nancy J. Keuthen, Ph.D. Dean 6. Kilpatrick, Ph.D. Katharina Kircanski, Ph.D. Laurence ]. Kirmayer, M.D. Donald F. Klein, M.D., D.Sc. Amaro J. Laria, Ph.D. Richard T. LeBeau, M.A. Richard J. Loewenstein, M.D. David Mataix-Cols, Ph.D. Thomas W. McAllister, M.D. Harrison 6. Pope, M.D., M.P.H. Ronald M. Rapee, Ph.D. Steven A. Rasmussen, M.D. Patricia A. Resick, Ph.D. Vedat Sar, M.D. Sanjaya Saxena, M.D. Paula P. Schnurr, PhD. M. Katherine Shear, M.D. Daphne Simeon, M.D. Harvey S. Singer, M.D. Melinda A. Stanley, Ph.D. James I. Strain, M.D. Kate Wolitzky Taylor, Ph.D. Onno van der Hart, Ph.D. Eric Vermetten, M.D., Ph.D. John T. Walkup, M.D. Sabine Wilhelm, Ph.D. Douglas W. Woods, Ph.D. Richard E. 2inbarg, Ph.D. Joseph Zohar, M.D.

1	Onno van der Hart, Ph.D. Eric Vermetten, M.D., Ph.D. John T. Walkup, M.D. Sabine Wilhelm, Ph.D. Douglas W. Woods, Ph.D. Richard E. 2inbarg, Ph.D. Joseph Zohar, M.D. Adrian Angold, Ph.D. Deborah Beidel, Ph.D. David Brent, M.D. John Campo, M.D. Gabrielle Carlson, M.D. Prudence W. Fisher, Ph.D. David Klonsky, Ph.D. Matthew Nock, Ph.D. ]. Blake Turner, Ph.D. Michael J. Devlin, M.D. Denise E. Wilﬂey, Ph.D. Susan 2. Yanovski, M.D. Boris Birmaher, M.D. Yeates Conwell, M.D. Ellen B. Dennehy, Ph.D. S. Ann Hartlage, Ph.D. Jack M. Hettema, M.D., Ph.D. Michael C. Neale, Ph.D. Gordon B. Parker, M.D., Ph.D., D.Sc. Roy H. Perlis, M.D. M.Sc. Holly 6. Prigerson, Ph.D. Norman E. Rosenthal, M.D. Peter ]. Schmidt, M.D. Mort M. Silverman, M.D. Meir Steiner, M.D., Ph.D. Mauricio Tohen, M.D., Dr.P.H., M.B.A. Sidney Zisook, M.D. Jiska Cohen-Mansfield, Ph.D. Vladimir Hachinski, M.D., C.M., D.Sc. Sharon Inouye, M.D., M.P.H. Grant Iverson, Ph.D. Laura Marsh, M.D.

1	Mauricio Tohen, M.D., Dr.P.H., M.B.A. Sidney Zisook, M.D. Jiska Cohen-Mansfield, Ph.D. Vladimir Hachinski, M.D., C.M., D.Sc. Sharon Inouye, M.D., M.P.H. Grant Iverson, Ph.D. Laura Marsh, M.D. Bruce Miller, M.D. Jacobo Mintzer, M.D., M.B.A. Bruce Pollock, M.D., Ph.D. George Prigatano, Ph.D. Ron Ruff, Ph.D. Ingmar Skoog, M.D., Ph.D. Robert Sweet, M.D. Paula Trzepacz, M.D. Nickola Nelson, Ph.D. Diane Paul, Ph.D. Eva Petrova, Ph.D. Andrew Pickles, Ph.D. Ian Piek, Ph.D. Helene Polatajko, Ph.D. Alya Reeve, M.D. Mabel Rice, Ph.D. Joseph Sergeant, Ph.D. Bennett Shaywitz, M.D. Sally Shaywitz, M.D. Audrey Thurm, Ph.D. Keith Widaman, Ph.D. Warren Zigman, Ph.D. Eran Chemerinski, M.D. Thomas N. Crawford, Ph.D. Harold W. Koenigsberg, M.D. Kristian E. Markon, Ph.D. Rebecca L. Shiner, Ph.D. Kenneth R. Silk, M.D. Jennifer L. Tackett, Ph.D. David Watson, Ph.D. Kamaldeep Bhui, M.D. Manuel J. Cuesta, M.D., Ph.D. Richard Douyon, M.D. Paolo Fusar—Poli, Ph.D.

1	Rebecca L. Shiner, Ph.D. Kenneth R. Silk, M.D. Jennifer L. Tackett, Ph.D. David Watson, Ph.D. Kamaldeep Bhui, M.D. Manuel J. Cuesta, M.D., Ph.D. Richard Douyon, M.D. Paolo Fusar—Poli, Ph.D. John H. Krystal, M.D. Thomas H. McGlashan, M.D. Victor Peralta, M.D., Ph.D. Anita Riecher-Rt'issler, M.D. Mary V. Seeman, M.D. Stan E. Althof, Ph.D. Richard Balon, MD. John H.]. Bancroft, M.D., M.A., D.P.M. Howard E. Barbaree, Ph.D., M.A. Rosemary J. Basson, M.D. Sophie Bergeron, Ph.D. Anita L. Clayton, M.D. David L. Delmonico, Ph.D. Domenico Di Ceglie, M.D. Esther Gomez-Gil, M.D. Jamison Green, Ph.D. Richard Green, MD, JD. R. Karl Hanson, Ph.D. Lawrence Hartmann, M.D. Stephen J. Hucker, M.B. Eric S. Janus, JD. Patrick M. Iem, Ph.D. Megan S. Kaplan, Ph.D. Raymond A. Knight, Ph.D. Ellen T.M. Laan, Ph.D. Stephen B. Levine, M.D. Christopher 6. McMahon, M.B. Marta Meana, Ph.D. Michael H. Miner, Ph.D., M.A. William T. O’Donohue, Ph.D. Michael A. Perelman, Ph.D.

1	Ellen T.M. Laan, Ph.D. Stephen B. Levine, M.D. Christopher 6. McMahon, M.B. Marta Meana, Ph.D. Michael H. Miner, Ph.D., M.A. William T. O’Donohue, Ph.D. Michael A. Perelman, Ph.D. Caroline F. Pukall, Ph.D. Robert E. Pyke, M.D., Ph.D. Vernon L. Quinsey, Ph.D. M.Sc. David L. Rowland, Ph.D., M.A. Michael Sand, Ph.D., M.P.H. Leslie R. Schover, Ph.D., M.A. Paul Stern, BS, ].D. David Thornton, Ph.D. Leonore Tiefer, Ph.D. Douglas E. Tucker, M.D. Jacques van Lankveld, Ph.D. Marcel D. Waldinger, M.D., Ph.D. Donald L. Bliwise, Ph.D. Daniel I. Buysse, M.D. Vishesh K. Kapur, M.D., M.P.H. Sanjeeve V. Kothare, M.D. Kenneth L. Lichstein, Ph.D. Mark W. Mahowald, M.D. Rachel Manber, Ph.D. Emmanuel Mignot, M.D., Ph.D. Timothy H. Monk, Ph.D., D.Sc. Thomas C. Neylan, M.D. Maurice M. Ohayon, M.D., D.Sc., Ph.D. Judith Owens, M.D., M.P.H. Daniel L. Picchietti, M.D. Stuart F. Quan, M.D. Thomas Roth, Ph.D. Daniel Weintraub, M.D. Theresa 8. Young, Ph.D.

1	Maurice M. Ohayon, M.D., D.Sc., Ph.D. Judith Owens, M.D., M.P.H. Daniel L. Picchietti, M.D. Stuart F. Quan, M.D. Thomas Roth, Ph.D. Daniel Weintraub, M.D. Theresa 8. Young, Ph.D. Phyllis C. Zee, M.D., Ph.D. Brenda Bursch, Ph.D. Kurt Kroenke, M.D. W. Curt LaFrance, Ir., M.D., M.P.H. Ion Stone, M.B., Ch.B., Ph.D. Lynn M. Wegner, M.D. Raymond F. Anton, Jr., M.D. Deborah A. Dawson, Ph.D. Roland R. Griffiths, Ph.D. Dorothy K. Hatsukami, Ph.D. John E. Helzer, M.D. Marilyn A. Huestis, Ph.D. John R. Hughes, M.D. Thomas R. Kosten, M.D. Nora D. Volkow, M.D. Christina Bryant, Ph.D. Amber Gum, Ph.D. Thomas Meeks, M.D. Jan Mohlman, Ph.D. Steven Thorp, Ph.D. Julie Wetherell, Ph.D. Neil K. Aggarwal, M.D., M.B.A., M.A. Sofie Béarnhielm, M.D., Ph.D. Iosé I. Bauermeister, Ph.D. James Boehnlein, M.D., M.Sc. Iaswant Guzder, M.D. Alejandro Interian, Ph.D. Sushrut S. Iadhav, M.B.B.S., M.D., Ph.D. Laurence J. Kirmayer, M.D. Alex J. Kopelowicz, M.D. Amaro J. Laria, Ph.D.

1	James Boehnlein, M.D., M.Sc. Iaswant Guzder, M.D. Alejandro Interian, Ph.D. Sushrut S. Iadhav, M.B.B.S., M.D., Ph.D. Laurence J. Kirmayer, M.D. Alex J. Kopelowicz, M.D. Amaro J. Laria, Ph.D. Steven R. Lopez, Ph.D. Kwame J. McKenzie, M.D. John R. Peteet, M.D. Hans (I.6.B.M.) Rohlof, M.D. Cecile Rousseau, M.D. Mitchell 6. Weiss, M.D., Ph.D. Daniel L. Coury, M.D. Bernard P. Dreyer, M.D. Danielle Laraque, M.D. Lynn M. Wegner, M.D. Prudence W. Fisher, Ph.D. Martin Prince, M.D., M.Sc. Michael R. Von Korff, Sc.D. Prudence W. Fisher, Ph.D. Robert D. Gibbons, Ph.D. Ruben Gur, Ph.D. John E. Helzer, M.D. John Houston, M.D., Ph.D. Kurt Kroenke, M.D. Patrick E. Shrout, Ph.D. Erik Willcutt, Ph.D. Anxiety, Obsessive-Compulsive Spectrum, Posttraumatic, and Eric Hollander, M.D. Charlie Marmar, M.D. Mark w. Miller, Ph.D. Mark H. Pollack, M.D. Heidi S. Resnick, Ph.D. Grace T. Baranek, Ph.D. Colleen Jacobson, Ph.D. Maria Oquendo, M.D. Sir Michael Rutter, M.D.

1	Charlie Marmar, M.D. Mark w. Miller, Ph.D. Mark H. Pollack, M.D. Heidi S. Resnick, Ph.D. Grace T. Baranek, Ph.D. Colleen Jacobson, Ph.D. Maria Oquendo, M.D. Sir Michael Rutter, M.D. Nancy L. Zucker, Ph.D. Keith Hawton, M.Sc. David A. Jobes, Ph.D. Maria A. Oquendo, M.D. J. Eric Ahlskog, M.D., Ph.D. Allen J. Aksamit, M.D. Marilyn Albert, Ph.D. Guy Mckhann, M.D. Bradley Boeve, M.D. Helena Chui, M.D. Sureyya Dikmen, Ph.D. Douglas 6alasko, M.D. Harvey Levin, Ph.D. Mark Lovell, Ph.D. Jeffery Max, M.B.B.Ch. Ian McKeith, M.D. Cynthia Munro, Ph.D. Marlene Oscar—Berman, Ph.D. Alexander Troster, Ph.D. Anna Barnett, Ph.D. Martha Denckla, M.D. Jack M. Fletcher, Ph.D. Dido Green, Ph.D. Stephen Greenspan, Ph.D. Bruce Pennington, Ph.D. Ruth Shalev, M.D. Larry B. Silver, M.D. Lauren Swineford, Ph.D. Michael Von Aster, M.D. Patricia R. Cohen, Ph.D. Jaime L. Derringer, Ph.D. Lauren Helm, M.D. Christopher J. Patrick, Ph.D. Anthony Pinto, Ph.D.

1	Larry B. Silver, M.D. Lauren Swineford, Ph.D. Michael Von Aster, M.D. Patricia R. Cohen, Ph.D. Jaime L. Derringer, Ph.D. Lauren Helm, M.D. Christopher J. Patrick, Ph.D. Anthony Pinto, Ph.D. Scott W. Woods, M.D. Alan I. Riley, M.Sc. Ray C. Rosen, Ph.D. Jack D. Edinger, Ph.D. David Gozal, M.D. Hochang B. Lee, M.D. Tore A. Nielsen, Ph.D. Michael ]. Sateia, M.D. Jamie M. Zeitzer, Ph.D. Chuck V. Ford, M.D. Patricia 1. Rosebush, M.Sc.N., M.D. Sally M. Anderson, Ph.D. Julie A. Kable, Ph.D. Christopher Martin, Ph.D. Sarah N. Mattson, Ph.D. Edward V. Nunes, I11, M.D. Mary J. O’Connor, Ph.D. Heather Carmichael Olson, Ph.D. Blair Paley, Ph.D. Edward P. Riley, Ph.D. Tulshi D. Saha, Ph.D. Wim van den Brink, M.D., Ph.D. George E. Woody, M.D. Bruce Cuthbert, Ph.D. Aartjan Beekman Ph.D. Alistair Flint, M.B. David Sultzer, M.D. Ellen Whyte, M.D. Sergio Aguilar—Gaxiola, M.D., Ph.D. Kavoos 6. Bassiri, M.S. Venkataramana Bhat, M.D. Marit Boiler, M.P.H.

1	Aartjan Beekman Ph.D. Alistair Flint, M.B. David Sultzer, M.D. Ellen Whyte, M.D. Sergio Aguilar—Gaxiola, M.D., Ph.D. Kavoos 6. Bassiri, M.S. Venkataramana Bhat, M.D. Marit Boiler, M.P.H. Denise Canso, M.Sc. Smita N. Deshpande, M.D., D.P.M. Ravi DeSilva, M.D. Esperanza Diaz, M.D. Byron I. 600d, Ph.D. Simon 6roen, M.A. Ladson Hinton, M.D. Lincoln I. Khasakhala, Ph.D. Francis 6. Lu, M.D. Athena Madan, M.A. Anne W. Mbwayo, Ph.D. Oanh Meyer, Ph.D. Victoria N. Mutiso, Ph.D., D.Sc. David M. Ndetei, M.D. Andel V. Nicasio, M.S.Ed. Vasudeo Paralikar, M.D., Ph.D. Kanak Patil, M.A. Filipa I. Santos, H.B.Sc. Sanjeev B. Sarmukaddam, Ph.D., M.Sc. Monica 2. Scalco, M.D., Ph.D. Katie Thompson, M.A. Hendry Ton, M.D., M.Sc. Rob CJ. van Dijk, M.Sc. Johann M. Vega-Dienstmaier, M.D. Joseph Westermeyer, M.D., Ph.D. Daniel I. Balog, M.D. Charles C. Engel, M.D., M.P.H. Charles D. Motsinger, M.D. Cille Kennedy, Ph.D. Paul I. Pikonis, Ph.D. a Focus of Clinical Attention

1	Joseph Westermeyer, M.D., Ph.D. Daniel I. Balog, M.D. Charles C. Engel, M.D., M.P.H. Charles D. Motsinger, M.D. Cille Kennedy, Ph.D. Paul I. Pikonis, Ph.D. a Focus of Clinical Attention William E. Narrow, M.D., M.P.H., Chair Roger Peele, M.D. Lawson R. Wulsin, M.D. Charles H. Zeanah, M.D. Prudence W. Fisher, Ph.D., Advisor Stanley N. Caroff, M.D., Contributor/Consultant James B. Lohr, M.D., Contributor/Consultant Marianne Wambolt, Ph.D., Contributor/Consultant Allan Donner, Ph.D. Kenneth Altshuler, M.D. Pedro 6. Alvarenga, M.D. Diana 1. Antonacci, M.D. Richard Balon, M.D. David H. Barlow, Ph.D. L. Jarrett Bamhill, M.D. Katja Beesdo-Baum, Ph.D. Marty Boman, Ed.D. Iames Bourgeois, M.D. David Braff, M.D. Harry Brandt, M.D. Kirk Brower, M.D. Rachel Bryant—Waugh, Ph.D. Jack D. Burke Jr., M.D., M.P.H. Brenda Bursch, Ph.D. Joseph Camilleri, M.D. Patricia Casey, M.D. F. Xavier Castellanos, M.D. Eran Chemerinski, M.D. Wai Chen, M.D.

1	Rachel Bryant—Waugh, Ph.D. Jack D. Burke Jr., M.D., M.P.H. Brenda Bursch, Ph.D. Joseph Camilleri, M.D. Patricia Casey, M.D. F. Xavier Castellanos, M.D. Eran Chemerinski, M.D. Wai Chen, M.D. Elie Cheniaux, M.D., D.Sc. Cheryl Chessick, MD, J. Richard Ciccone, M.D. Anita H. Clayton, M.D. Tihalia I. Coleman, Ph.D. John Csemansky, M.D. Manuel 1. Cuesta M.D., Ph.D. Joanne L. Davis, M.D. David L. Delmonico, Ph.D. Ray J. DePaulo, M.D. Dimitris Dikeos, M.D. Ina E. Djonlagic, M.D. C. Neill Epperson, M.D. Javier I. Escobar, M.D., M.Sc. Spencer Eth, M.D. David Fassler, M.D. Giovanni A. Fava, M.D. Robert Feinstein, M.D. Molly Finnerty, M.D. Mark H. Fleisher, M.D. Alessio Florentini, M.D. Laura Fochtmann, M.D. Marshal Forstein, M.D. William French, M.D. Maximillian Gahr, M.D. Cynthia Geppert, M.D. Ann Germaine, Ph.D. Marcia 60in, M.D. David A. Gorelick, M.D., Ph.D. David Graeber, M.D. Cynthia A. Graham, Ph.D. Andreas Hartmann, M.D. Victoria Hendrick, M.D.

1	Cynthia Geppert, M.D. Ann Germaine, Ph.D. Marcia 60in, M.D. David A. Gorelick, M.D., Ph.D. David Graeber, M.D. Cynthia A. Graham, Ph.D. Andreas Hartmann, M.D. Victoria Hendrick, M.D. Merrill Herman, M.D. David Herzog, M.D. Mardi Horowitz, M.D. Ya-fen Huang, M.D. Anthony Kales, MD Niranjan S. Karnik, M.D., Ph.D. Jeffrey Katzman, M.D. Bryan King, M.D. Cecilia Kjellgren, M.D. Harold W. Koenigsberg, M.D. Richard B. Krueger, M.D. Steven Lamberti, M.D. Ruth A. Lanius, M.D. John Lauriello, M.D. Anthony Lehman, M.D. Michael Linden, M.D. Mark W. Mahowald, M.D. Marsha D. Marcus, Ph.D. Stephen Marder, M.D. Wendy Marsh, M.D. Michael S. McCloskey, Ph.D. Jeffrey Metzner, M.D. Robert Michels, M.D. Laura Miller, MD. Michael C. Miller, M.D. Frederick Moeller, M.D. Peter T. Morgan, M.D., Ph.D. Madhav Muppa, M.D. Philip Muskin, M.D. Joachim Nitschke, M.D. Abraham Nussbaurn, M.D. Ann Olincy, M.D. \ Mark Onslow, Ph.D. Sally Ozonoff, Ph.D. John R. Peteet, M.D.

1	Madhav Muppa, M.D. Philip Muskin, M.D. Joachim Nitschke, M.D. Abraham Nussbaurn, M.D. Ann Olincy, M.D. \ Mark Onslow, Ph.D. Sally Ozonoff, Ph.D. John R. Peteet, M.D. Ismene L. Petrakis, M.D. Christophe M. Pfeiffer, M.D. Karen Pierce, M.D. Belinda Plattner, M.D. Franklin Putnam, M.D. Stuart F. Quan, M.D. John Racy, M.D. Phillip Resnick, M.D. Michele Riba, M.D. Ierold Rosenbaum, M.D. Stephen Ross, M.D. Lawrence Scahill, M.S.N., Ph.D. Daniel Schechter, M.D. Mary V. Seeman, M.D. Alessandro Serretti, M.D. Iianhua Shen, M.D. Ravi Kumar R. Singareddy, M.D. Ingmar Skoog, M.D., Ph.D. Gary Small, M.D. Paul Soloff, M.D. Christina Stadler, M.D., Ph.D. Nada Stotland, M.D. Neil Swerdlow, M.D. Kim Tillery, Ph.D. David Tolin, Ph.D. Jayne Trachman, M.D. Luke Tsai, M.D. Ming T. Tsuang, M.D., Ph.D. Richard Tuch, M.D. Johan Verhulst, M.D. B. Timothy Walsh, M.D. Michael Weissberg, M.D. Codehard Weniger, M.D. Keith Widaman, Ph.D. Thomas Wise, M.D.

1	Ming T. Tsuang, M.D., Ph.D. Richard Tuch, M.D. Johan Verhulst, M.D. B. Timothy Walsh, M.D. Michael Weissberg, M.D. Codehard Weniger, M.D. Keith Widaman, Ph.D. Thomas Wise, M.D. George E. Woods, M.D. Kimberly A. Yonkers, M.D. Alexander Young, M.D. David Geffen School of Medicine, University of California, Los Angeles Helen Lavretsky, M.D., Principal Investigator Jessica Brommelhoff, Ph.D. Xavier Cagigas, Ph.D. Paul Cemin, Ph.D. Linda Ercoli, Ph.D. Randall Espinoza, M.D. Helen Lavretsky, M.D. Jeanne Kim, Ph.D. David Merrill, M.D. Karen Miller, Ph.D. Christopher Nunez, Ph.D. Natalie St. Cyr, M.A., Lead Research Nora Nazarian, B.A. Colin Shinn, M.A. Centre for Addiction and Mental Health, Toronto, Ontario, Canada Bruce 6. Pollock, M.D., Ph.D., Lead Principal R. Michael Bagby, Ph.D., Principal Investigator Kwame I. McKenzie, M.D., Principal Tony P. George, M.D., Co-investigator Lena C. Quilty, Ph.D., Co—investigator Peter Voore, M.D., Co-investigator

1	R. Michael Bagby, Ph.D., Principal Investigator Kwame I. McKenzie, M.D., Principal Tony P. George, M.D., Co-investigator Lena C. Quilty, Ph.D., Co—investigator Peter Voore, M.D., Co-investigator Donna E. Al(man, Ph.D. R. Michael Bagby, Ph.D. Wayne C. V. Baici, M.D. Crystal Baluyut, M.D. Eva W. C. Chow, M.D., ].D., M.P.H. 2. I. Daskalakis, M.D., Ph.D. Pablo Diaz-Hermosillo, M.D. George Foussias, M.Sc., M.D. Paul A. Frewen, Ph.D. Ariel 6raff-6uerrero, M.D., M.Sc., Ph.D. Margaret K. Hahn, M.D. Lorena Hsu, Ph.D. Justine Joseph, Ph.D. Sean Kidd, Ph.D. Kwame I. McKenzie, M.D. Mahesh Menon, Ph.D. Romina Mizrahi, M.D., Ph.D. Daniel J. Mueller, M.D., Ph.D. Lena C. Quilty, Ph.D. Anthony C. Ruocco, Ph.D. Jorge Soni, M.D. Aristotle N. Voineskos, M.D., Ph.D. George Voineskos, M.D. Peter Voore, Ph.D. Chris Watson, Ph.D. Ofer Agid, M.D. Ash Bender, M.D. Patricia Cavanagh, M.D. Sarah Colman, M.D. Vincenzo Deluca, M.D. Justin Geagea, MD. David S. Goldbloom, M.D.

1	Peter Voore, Ph.D. Chris Watson, Ph.D. Ofer Agid, M.D. Ash Bender, M.D. Patricia Cavanagh, M.D. Sarah Colman, M.D. Vincenzo Deluca, M.D. Justin Geagea, MD. David S. Goldbloom, M.D. Daniel Greben, M.D. Malati Gupta, M.D. Ken Harrison, M.D. Imraan Ieeva, M.D. Joel Jeffries, M.B. Judith Laposa, Ph.D. Jan Malat, M.D. Shelley McMain, Ph.D. Bruce Pollock, M.D., Ph.D. Andriy V. Samokhvalov, M.D., Ph.D. Martin Strassnig, M.D. Albert H. C. Wong, M.D., Ph.D. Gloria I. Leo, M.A., Lead Research Coordinator Anissa D. Bachan, B.A. Bahar Haji—Khamneh, M.A. Olga Likhodi, M.Sc. Eleanor J. Liu, Ph.D. Sarah A. McGee Ng, B.B.A. Susan E. Dickens, M.A., Clinical Research Sandy Richards, B.Sc.N., Schizophrenia Dallas VA Medical Center, Dallas, Texas Carol 5. North, M.D., M.P.E., Principal Alina Suris, Ph.D., A.B.P.P., Principal Barry Ardolf, Psy.D. Abila Awan, M.D. Joel Baskin, M.D. John Black, Ph.D. Jeffrey Dodds, Ph.D. Gloria Emmett, Ph.D. Karma Hudson, M.D.

1	Alina Suris, Ph.D., A.B.P.P., Principal Barry Ardolf, Psy.D. Abila Awan, M.D. Joel Baskin, M.D. John Black, Ph.D. Jeffrey Dodds, Ph.D. Gloria Emmett, Ph.D. Karma Hudson, M.D. Iamylah Jackson, Ph.D., A.B.P.P. Lynda Kirkland-Culp, Ph.D., A.B.P.P. Heidi Koehler, Ph.D., A.B.P.P. Elizabeth Lewis, Psy.D. Aashish Parikh, M.D. Reed Robinson, Ph.D. Iheel Shah, M.D. Geetha Shivakumar, M.D. Sarah Spain, Ph.D., A.B.P.P. Lisa Thoman, Ph.D. Lia Thomas, M.D. Jamie Zabukovec, Psy.D. Mustafa Zaidi, M.D. Andrea 2artman, Ph.D. Robert Blake, L.M.S.W. Evelyn Gibbs, L.M.S.W. Michelle King-Thompson, L.M.S.W. Jeannie B. Whitman, Ph.D., Lead Research Sunday Adewuyi, M.D. Elizabeth Anderson, B.A. Solaleh Azimipour, BS. Carissa Barney, B.S. Kristie Cavazos, B.A. Robert Devereaux, BS. Dana Downs, M.S., M.S.W. Sharjeel Farooqui, M.D. Julia Smith, Psy.D. Kun—Ying H. Sung, BS. School of Medicine, The University of Texas San Antonio, San Antonio. Texas

1	Robert Devereaux, BS. Dana Downs, M.S., M.S.W. Sharjeel Farooqui, M.D. Julia Smith, Psy.D. Kun—Ying H. Sung, BS. School of Medicine, The University of Texas San Antonio, San Antonio. Texas Mauricio Tohen, M.D., Dr.P.H., M.B.A., Suman Baddam, Psy.D. Charles L. Bowden, M.D. Nancy Diazgranados, M.D., MS. Craig A. Dike, Psy.D. Dianne E. Dunn, Psy.D., M.P.H. Elena 6herman, M.D. Jodi M. Gonzalez, Ph.D. Pablo Gonzalez, M.D. Phillip Lai, Psy.D. Natalie Maples—Aguilar, M.A., L.P.A. Marlon P. Quinones, M.D. Ieslina J. Raj, Psy.D. David L. Roberts, Ph.D. Nancy Sandusky, R.N., F.P.M.H.N.P.-B.C., D.N.P.-C. Donna S. Stutes, M.S., L.P.C. Mauricio Tohen, M.D., Dr.PH, M.B.A. Dawn I. Velligan, Ph.D. Weiran Wu, M.D., Ph.D. Albana Dassori, M.D. Megan Frederick, M.A. Robert Gonzalez, M.D. Uma Kasinath, M.D. Camis Milam, M.D. Vivek Singh, M.D. Peter Thompson, M.D. Melissa Hernandez, B.A., Lead Research Fermin Alejandro Carrizales, B.A. Martha Dahl, R.N., B.S.N.

1	Uma Kasinath, M.D. Camis Milam, M.D. Vivek Singh, M.D. Peter Thompson, M.D. Melissa Hernandez, B.A., Lead Research Fermin Alejandro Carrizales, B.A. Martha Dahl, R.N., B.S.N. Patrick M. Smith, B.A. Nicole B. Watson, M.A. Michael E. DeBakey VA Medical Center and the Menninger Clinic, Houston, Texas (Joint Study Site) Michael E. DeBakey VA Medical Center Laura Marsh, M.D., Principal Investigator Shalini Aggarwal, M.D. Su Bailey, Ph.D. Minnete (Helen) Beckner, Ph.D. Crystal Clark, M.D. Charles Delohn, M.D. Robert Garza, M.D. Aruna Gottumakkla, M.D. Janet Hickey, M.D. James Ireland, M.D. Mary Lois Lacey, A.P.R.N. Wendy Leopoulos, M.D. Laura Marsh, M.D. Deleene Menefee, Ph.D. Brian I. Miller, Ph.D. Candy Smith, Ph.D. Avila Steele, Ph.D. Jill Wanner, Ph.D. Rachel Wells, Ph.D. Kaki York-Ward, Ph.D. Sara Allison, M.D. Leonard Denney, L.C.S.W. Catherine Flores, L.C.S.W. Nathalie Marie, M.D. Christopher Martin, M.D. Sanjay Mathew, M.D.

1	Rachel Wells, Ph.D. Kaki York-Ward, Ph.D. Sara Allison, M.D. Leonard Denney, L.C.S.W. Catherine Flores, L.C.S.W. Nathalie Marie, M.D. Christopher Martin, M.D. Sanjay Mathew, M.D. Erica Montgomery, M.D. Gregory Scholl, P.A. Iocelyn Ulanday, M.D., M.P.H. Sarah Neely Torres, 8.5., Lead Research Kathleen Grout, M.A. Lea Kiefer, M.P.H. Iana Tran, M.A. Efrain Bleiberg, M.D., Principal Investigator Jennifer Baumgardner, Ph.D. Elizabeth Dodd Conaway, L.C.S.W., B.C.D. Warren Christianson, D.O. Wesley Clayton, L.M.S.W. I. Christopher Fowler, Ph.D. Michael Croat, Ph.D. Edythe Harvey, M.D. Denise Kagan, Ph.D. Hans Meyer, L.C.S.W. Segundo Robert-Ibarra, M.D. Sandhya Trivedi, M.D. Rebecca Wagner, Ph.D. Harrell Woodson, Ph.D. Amanda Yoder, L.C.S.W. Iames Flack, MD. David Ness, M.D. Steve Herrera, B.S., M.T., Lead Research Allison Kalpakci, B.A. Mayo Clinic, Rochester, Minnesota Mark A. Frye, M.D., Principal Investigator

1	Iames Flack, MD. David Ness, M.D. Steve Herrera, B.S., M.T., Lead Research Allison Kalpakci, B.A. Mayo Clinic, Rochester, Minnesota Mark A. Frye, M.D., Principal Investigator Glenn E. Smith, Ph.D., Principal Investigator Ieffrey P. Staab M.D., M.S., Principal Osama Abulseoud, M.D. Jane Cerhan, Ph.D. Julie Fields, Ph.D. Mark A. Frye, M.D. Manuel Fuentes, M.D. Yonas Geda, M.D. Maria Harmandayan, M.D. Reba King, M.D. Simon Kung, M.D. Mary Machuda, Ph.D. Donald McAlpine, M.D. Alastair McKean, M.D. Juliana Moraes, M.D. Teresa Rummans, M.D. James R. Rundell, M.D. Richard Seime, Ph.D. Glenn E. Smith, Ph.D. Christopher Sola, D.O. Jeffrey P. Staab M.D., MS. Marin Veldic, M.D. Mark D. Williams, M.D. Maya Yustis, Ph.D. Lisa Seymour, 85., Lead Research Coordinator Scott Feeder, M.S. Lee Gunderson, B.S. Sherrie Hanna, M.A., L.P. Kelly Harper, B.A. Katie Mingo, B.A. Cynthia Stoppel, A.S. Perelman School of Medicine, University of Pennsylvania,

1	Scott Feeder, M.S. Lee Gunderson, B.S. Sherrie Hanna, M.A., L.P. Kelly Harper, B.A. Katie Mingo, B.A. Cynthia Stoppel, A.S. Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania Mahendra T. Bhati, M.D., Principal Investigator Mama S. Barrett, Ph.D., Co-investigator Michael E. Thase, M.D., Co-investigator Peter B. Bloom, M.D. Nicole K Chalmers L.C.S.W. Torrey A. Creed, Ph.D. Mario Cristancho, M.D. Amy Cunningham, Psy.D. John P. Dennis, Ph.D. Josephine Elia, M.D. Peter 6ariti, Ph.D., L.C.S.W. Philip Gehrman, Ph.D. Laurie Gray, M.D. Emily A.P. Haigh, Ph.D. Nora J. Johnson, M.B.A., M.S., Psy.D. Paulo Knapp, M.D. Yong-Tong Li, M.D. Bill Mace, Ph.D. Kevin S. McCarthy, Ph.D. Dimitri Perivoliot'is, Ph.D. Luke Schultz, Ph.D. Tracy Steen, Ph.D. Chris Tjoa, M.D. Nancy A. Wintering, L.C.S.W. Eleanor Ainslie, M.D. Kelly C. Allison, Ph.D. Rebecca Aspden, M.D. Claudia F. Baldassano, M.D. Vijayta Bansal, M.D. Rachel A. Bennett, M.D.

1	Chris Tjoa, M.D. Nancy A. Wintering, L.C.S.W. Eleanor Ainslie, M.D. Kelly C. Allison, Ph.D. Rebecca Aspden, M.D. Claudia F. Baldassano, M.D. Vijayta Bansal, M.D. Rachel A. Bennett, M.D. Richard Bollinger, Ph.D. Andrea Bowen, M.D. Karla Campanella, M.D. Anthony Carlino, M.D. Noah Carroll, M.S.S. Alysia Cirona, M.D. Samuel Collier, M.D. Andreea Crauciuc, L.C.S.W. Pilar Cristancho, M.D. Traci D'Almeida, M.D. Kathleen Diller, M.D. Benoit Dubé, M.D. Ion Dukes, M.S.W. Lauren Elliott, M.D. Mira Elwell, B.A. Mia Everett, M.D. Lucy F. Faulconbridge, Ph.D. Patricia Furlan, Ph.D. Joanna Goldstein, L.C.S.W. Paul Grant, Ph.D. Jillian Graves, L.C.S.W. Tamar 6ur, M.D., Ph.D. Alisa Gutman, M.D., Ph.D. Nora Hymowitz, M.D. Sofia Jensen, M.D. Tiffany King, M.S.W. Katherine Levine, M.D. Alice Li, M.D. Janet Light, L.C.S.W. Iohn Listerud, M.DV Ph.D. Emily Malcoun, Ph.D. Donovan Maust, M.D. Adam Meadows, M.D. Michelle Moyer, M.D. Rebecca Naugle, L.C.S.W.

1	Alice Li, M.D. Janet Light, L.C.S.W. Iohn Listerud, M.DV Ph.D. Emily Malcoun, Ph.D. Donovan Maust, M.D. Adam Meadows, M.D. Michelle Moyer, M.D. Rebecca Naugle, L.C.S.W. Cory Newman, Ph.D. John Northrop, M.D., Ph.D. Elizabeth A. Ellis 0hr, Psy.D. John O'Reardon, M.D. Abraham Pachikara, M.D. Andrea Perelman, M.S.W. Diana Perez, M.S.W. Bianca Previdi, M.D. ]. Russell Ramsay, Ph.D. Jorge Rivera-Colon, M.D. Ian Smedley, L.C.S.W. Katie Struble, M.S.W. Aita Susi, M.D. Yekaterina Tatarchuk, M.D. Ellen Tarves, M.A. Allison Tweedie, M.D. Holly Valerio, M.D. Thomas A. Wadden, Ph.D. Joseph Wright, Ph.D. Yan Xuan, M.D. David Yusko, Psy.D. Jordan A. Coello, B.A., Lead Research Eric Wang, B.S.E. Jeannine Barker, M.A., A.T.R. Martekuor Dodoo, B.A. Laura Heller, B.A. Leah Hull-Rawson, B.A. Jacquelyn Klehm, B.A. Dante Proetto, B.S. Stanford University School of Medicine, Stanford, California Carl Feinstein, M.D., Principal Investigator

1	Laura Heller, B.A. Leah Hull-Rawson, B.A. Jacquelyn Klehm, B.A. Dante Proetto, B.S. Stanford University School of Medicine, Stanford, California Carl Feinstein, M.D., Principal Investigator Debra Safer, M.D., Principal Investigator Kari Berquist, Ph.D. Eric Clausell, Ph.D. Danielle Colbom, Ph.D. Whitney Daniels, M.D. Alison Darcy, Ph.D. Krista Fielding, M.D. Mina Fisher, M.D. Kara Fitzpatrick, Ph.D. Wendy Froehlich, M.D. Grace Gengoux, Ph.D. Anna Cassandra Golding, Ph.D. Lisa Groesz, Ph.D. Kyle Hinman, M.D. Rob Holaway, Ph.D. Matthew Holve, M.D. Rex Huang, M.D. Nina Kirz, M.D. Megan Klabunde, Ph.D. John Leckie, Ph.D. Naomi Leslie, M.D. Adrianne Lona, M.D. Ranvinder Rai, M.D. Rebecca Rialon, Ph.D. Beverly Rodriguez, M.D., Ph.D. Debra Safer, M.D. Mary Sanders, Ph.D. Jamie Scaletta, Ph.D. Norah Simpson, Ph.D. Manpreet Singh, M.D. Maria-Christina Stewart, Ph.D. Melissa Vallas, M.D. Patrick Whalen, Ph.D. Sanno Zack, Ph.D. Robin Apple, Ph.D.

1	Jamie Scaletta, Ph.D. Norah Simpson, Ph.D. Manpreet Singh, M.D. Maria-Christina Stewart, Ph.D. Melissa Vallas, M.D. Patrick Whalen, Ph.D. Sanno Zack, Ph.D. Robin Apple, Ph.D. Victor Carrion, M.D. Carl Feinstein, M.D. Qhristine Gray, Ph.D. Antonio Hardan, M.D. Megan Jones, Psy.D. Linda Lotspeich, M.D. Lauren Mikula, Psy.D. Brandyn Street, Ph.D. Violeta Tan, M.D. Heather Taylor, Ph.D. Jacob Towery, M.D. Sharon Williams, Ph.D. Kate Amow, B.A., Lead Research Coordinator Nandini Datta, BS. Stephanie Manasse, B.A. Arianna Martin, M.S. Adriana Nevado, B.A. Children’s Hospital Colorado, Aurora, Colorado Marianne Wamboldt, M.D., Principal Galia Abadi, M.D. Steven Behling, Ph.D. Jamie Blume, Ph.D. Adam Burstein, M.D. Debbie Carter, M.D. Kelly Caywood, Ph.D. Meredith Chapman, M.D. Paulette Christian, A.P.P.M.H.N. Mary Cook, M.D. Anthony Cordaro, M.D. Audrey Dumas, M.D. Guido Frank, M.D. Karen Frankel, Ph.D. Darryl Graham, Ph.D. Yael Granader, Ph.D.

1	Paulette Christian, A.P.P.M.H.N. Mary Cook, M.D. Anthony Cordaro, M.D. Audrey Dumas, M.D. Guido Frank, M.D. Karen Frankel, Ph.D. Darryl Graham, Ph.D. Yael Granader, Ph.D. Isabelle Guillemet, M.D. Patrece Hairston, Ph.D. Charles Harrison, Ph.D. Tammy Herckner, L.C.S.W. Cassie Karlsson, M.D. Kimberly Kelsay, M.D. David Kieval, Ph.D. Megan Klabunde, Ph.D. Jaimelyn Kost, L.C.S.W. Harrison Levine, M.D. Raven Lipmanson, M.D. Susan Lurie, M.D. Asa Marokus, M.D. Idalia Massa, Ph.D. Christine McDunn, Ph.D. Scot McKay, M.D. Marissa Murgolo, L.C.S.W. Alyssa Oland, Ph.D. Lina Patel, Ph.D. Rheena I’ineda, Ph.D. 6autam Rajendran, M.D. Diane Reichmuth, PhD Michael Rollin, M.D. Marlena Romero, L.C.S.W. Michelle Roy, Ph.D. Celeste St. John-Larkin, M.D. Elise Sannar, Ph.D. Daniel Savin, M.D. Claire Dean Sinclair, Ph.D. Ashley Smith, L.C.S.W. Mindy Solomon, Ph.D. Sally Tarbell, Ph.D. Helen Thilly, L.C.S.W. Sara Tlustos-Carter, Ph.D. l-Iolly Vause, A.P.P.M.I-I.N

1	Daniel Savin, M.D. Claire Dean Sinclair, Ph.D. Ashley Smith, L.C.S.W. Mindy Solomon, Ph.D. Sally Tarbell, Ph.D. Helen Thilly, L.C.S.W. Sara Tlustos-Carter, Ph.D. l-Iolly Vause, A.P.P.M.I-I.N Marianne Wamboldt, M.D. Angela Ward, L.C.S.W. Jason Williams, Ph.D. Jason Willoughby, Ph.D. Brennan Young, Ph.D. Kelly Bhatnagar, Ph.D. Jeffery Dolgan, Ph.D. Jennifer Eichberg, L.C.S.W. Jennifer Hagman, M.D. James Masterson, L.C.S.W. Hy 6ia Park, M.D. Tami Roblek, Ph.D. Wendy Smith, Ph.D. David Williams, M.D. Laurie Burnside, M.S.M., C.C.R.C., Lead Darci Anderson, B.A., C.C.R.C. Heather Kennedy, M.P.H. Amanda Millar, B.A. Vanessa Waruinge, B.S. Elizabeth Wallace, B.A. Baystate Medical Center, Springfield, Massachusetts Bruce Waslick, M.D., Principal Investigator Cheryl Bonica, Ph.D., Co-investigator John Fanton, M.D., Co-investigator Barry Sarvet, M.D., Co-investigator Iulie Bermant, R.N., M.S.N., N.P. Cheryl Bonica, Ph.D. Jodi Devine, L.I.C.S.W.

1	Cheryl Bonica, Ph.D., Co-investigator John Fanton, M.D., Co-investigator Barry Sarvet, M.D., Co-investigator Iulie Bermant, R.N., M.S.N., N.P. Cheryl Bonica, Ph.D. Jodi Devine, L.I.C.S.W. William Fahey, Ph.D. John Fanton, M.D. Stephane Jacobus, Ph.D. Barry Sarvet, M.D. Peter Thunfors, Ph.D. Bruce Waslick, M.D. Vicki Weld, L.I.C.S.W. Sara Wiener, L.I.C.S.W. Shadi Zaghloul, M.D. Sarah Detenber, L.I.C.S.W. Gordon Garrison, L.I.C.S.W. Jacqueline Humpreys, L.I.C.S.W. Noreen Mc6irr, L.I.C.S.W. Sarah Marcotte, L.C.S.W. Patricia Rogowski, R.N., C.N.S. Julie Kingsbury, C.C.R.P., Lead Research Brenda Martin, B.A. New York State Psychiatric Institute, New York, N.Y., Weill Cornell Medical College, Payne Whitney and Westchester Divisions, New York and White Plains, N.Y., and North Shore Child and Family Guidance Center, Roslyn Heights. NY. (Joint Study Site) Prudence W. Fisher, Ph.D., Principal Julia K. Carmody, B.A., Lead Research 2vi R. Shapiro, B.A., Lead Research

1	Center, Roslyn Heights. NY. (Joint Study Site) Prudence W. Fisher, Ph.D., Principal Julia K. Carmody, B.A., Lead Research 2vi R. Shapiro, B.A., Lead Research Jeremy Litfin, M.A. Sarah L. Pearlstein, B.A. Michele Cohen, L.C.S.W. Eduvigis Cruz-Arrieta, Ph.D. Miriam Ehrensaft, Ph.D. Laurence Greenhill, M.D. Schuyler Henderson, M.D., M.P.H. Sharlene Jackson, Ph.D. Lindsay Moskowitz, M.D. Sweene C. Oscar, Ph.D. Xenia Protopopescu, M.D. James Rodriguez, Ph.D. Gregory Tau, M.D. Melissa Tebbs, L.C.S.W. Carolina Velez-Grau, L.C.S.W. Khadijah Booth Watkins, M.D. George Alvarado, M.D. Alison Baker, M.D. Elena Baron, Psy.D. Lincoln Bickford, M.D., Ph.D. Zachary Blumkin, Psy.D. Colleen Cullen, L.C.S.W. Chyristianne DeAlmeida, Ph.D. Matthew Ehrlich, M.D. Eve Friedl, M.D. Clare Gaskins, Ph.D. Alice Greenfield, L.C.S.W. Liora Hoffman, M.D. Kathleen lung, M.D. Karimi Mailutha, M.D., M.P.H. Valentina Nikulina, Ph.D. Tal Reis, Ph.D. Moira Rynn, M.D.

1	Clare Gaskins, Ph.D. Alice Greenfield, L.C.S.W. Liora Hoffman, M.D. Kathleen lung, M.D. Karimi Mailutha, M.D., M.P.H. Valentina Nikulina, Ph.D. Tal Reis, Ph.D. Moira Rynn, M.D. Jasmine Sawhney, M.D. Sarajbit Singh, M.D. Katherine Stratigos, M.D. Oliver Stroeh, M.D. Russell Tobe, M.D. Meghan Tomb, Ph.D. Michelle Tricamo, M.D. Angel A. Caraballo, M.D. Erica M. Chin, Ph.D. Daniel T. Chrzanowski, M.D. Tess Daugherty, B.A. Stephanie Hundt, M.A. Moira A. Rynn, M.D. Deborah Stedge, RN. Weill Cornell Medical College, Payne Whitney and Westchester Divisions Archana Basu, Ph.D. Shannon M. Bennett, M.D. Maria De Pena-Nowak, M.D. Jill Feldman, L.M.S.W. Dennis Gee, M.D. Io R. Hariton, Ph.D. Lakshmi P. Reddy, M.D. Margaret Yoon, M.D. Margo Benjamin, M.D. Vanessa Bobb, M.D. Elizabeth Bochtler, M.D. Katie Cave, L.C.S.W. Maalobeeka Gangopadhyay, M.D. Jodi Gold, M.D. Tejal Kaur, M.D. Aaron Krasner, M.D. Amy Miranda, L.C.S.W. Cynthia Pfeffer, M.D.

1	Elizabeth Bochtler, M.D. Katie Cave, L.C.S.W. Maalobeeka Gangopadhyay, M.D. Jodi Gold, M.D. Tejal Kaur, M.D. Aaron Krasner, M.D. Amy Miranda, L.C.S.W. Cynthia Pfeffer, M.D. James Rebeta, Ph.D. Sharon Skariah, M.D. Jeremy Stone, Ph.D. Dirk Winter, M.D. Alex Eve Keller, B.S., Lead Research Coordinator Barbara L. Flye, Ph.D. Jamie S. Neiman (volunteer) Rebecca L. Rendleman, M.D. Casye Brachfeld-Launer, L.C.S.W. Susan Klein Cohen, Ph.D. Amy Gelb, L.C.S.W.-R. Jodi 61asser, L.C.S.W. Elizabeth Goulding-Tag, L.C.S.W. Deborah B. Kassimir, L.C.S.W. Margo Posillico Messina, L.C.S.W. Andréa Moullin—Heddle, L.M.S.W. Lisa Pineda, L.C.S.W. Elissa Smilowitz, L.C.S.W. Regina Barros-Rivera, L.C.S.W.—R. Assistant Maria Christiansen, B.S. Amy Davies—I—Iollander, L.M.S.W. Eartha Hackett, M.S.Ed., M.Sc., B.Sc. Bruce Kaufstein, L.C.S.W.-R, Director of Kathy Knaust, L.C.S.W. John Levinson, L.C.S.W.-R, B.C.D. Andrew Maleckoff, L.C.S.W., Executive

1	Eartha Hackett, M.S.Ed., M.Sc., B.Sc. Bruce Kaufstein, L.C.S.W.-R, Director of Kathy Knaust, L.C.S.W. John Levinson, L.C.S.W.-R, B.C.D. Andrew Maleckoff, L.C.S.W., Executive Sarah Rosen, L.C.S.W.-R, A.C.S.W. Abigail Rothenberg, L.M.S.W. Christine Scotten, A.C.S.W. Michelle Spatano, L.C.S.W.—R. Diane Straneri, M.S., R.N., CS. Rosara Torrisi, L.M.S.W. Rob Vichnis, L.C.S.W. Toni Kolb-Papetti, L.C.S.W. Sheena M. Dauro (volunteer) DSM-5 Field Trials Pilot Study, Johns Hopkins Medical Institution, Baltimore, Maryland Community Psychiatry Outpatient Program. Department of Psychiatry Bernadette Cullen, M.B., B.Ch., B.A.O., Holly C. Wilcox, Ph.D., Principal Investigator Bernadette Cullen, M.B., B.Ch., B.A.O. Shane Grant, L.C.S.W.-C. Charee Green, L.C.P.C. Emily Lorensen, L.C.S.W.—C. Kathleen Malloy, L.C.P.C. Gary Pilarchik, L.C.S.W.-C Holly Slater, L.C.P.C. Stanislav Spivak, M.D. Tarcia Spencer Turner, L.C.P.C. Nicholas Seldes Windt, L.C.S.W.-C.

1	Kathleen Malloy, L.C.P.C. Gary Pilarchik, L.C.S.W.-C Holly Slater, L.C.P.C. Stanislav Spivak, M.D. Tarcia Spencer Turner, L.C.P.C. Nicholas Seldes Windt, L.C.S.W.-C. Mellisha McKitty, B.A. Alison Newcomer, M.H.S. Child and Adolescent Outpatient Program. Department of Psychiatry and Ioan P. Gerring, M.D., Principal Investigator Leslie Miller, M.D., Principal Investigator Holly C. Wilcox, Ph.D., Co—investigator Shannon Barnett, M.D. Gwen Condon, L.C.P.C. Brijan Fellows, L.C.S.W.-C. Heather Gamer, L.C.S.W.-C. Joan P. Gerring, M.D. Anna Gonzaga, M.D. Debra Jenkins, L.C.S.W.-C. Paige N. Johnston, L.C.P.C. Brenda Memel, D.N.P., R.N. Leslie Miller, M.D. Ryan Moore, L.C.S.W.-C. Shauna Reinblatt, M.D. Monique Vardi, L.C.P.C. Mellisha McKitty, B.A. Alison Newcomer, M.H.S. DSM-5 Field Trials in Routine Clinical Practice Settings: Archil Abashidze, M.D. Francis R. Abueg, Ph.D. Jennifer Louise Accuardi, M.S. Balkozar 5. Adam, M.D.

1	Alison Newcomer, M.H.S. DSM-5 Field Trials in Routine Clinical Practice Settings: Archil Abashidze, M.D. Francis R. Abueg, Ph.D. Jennifer Louise Accuardi, M.S. Balkozar 5. Adam, M.D. Miriam E. Adams, Sc.D., M.S.W., L.I.C.S.W. Suzanna C. Adams, M.A. Lawrence Adler, M.D. Rownak Afroz, M.D. Khalid I. Afzal, M.D. Joseph Alimasuya, M.D. Emily Allen, M.S. Katherine A. Allen, L.M.F.T., M.A. William D. Allen, MS. Jafar AlMashat, M.D. Anthony T. Alonzo, D.M.F.T. Guillermo Alvarez, B.A., M.A. Angela Amoia-Lutz, L.M.F.T. Krista A. Anderson, M.A., L.M.F.T. Lisa R. Anderson, M.Ed., L.C.P.C. Pamela M. Anderson, L.M.F.T. Shannon N . Anderson, M.A., L.P.C., N.C.C. Eric S. Andrews, M.A. Vicki Arbuckle, M.S., Nursing(N.P.) Namita K. Arora, M.D. Darryl Arrington, M.A. Bearlyn Y. Ash, M.S. Wylie I. Bagley, Ph.D. Kumar D. Bahl, M.D. Deborah C. Bailey, M.A., M.S., Ph.D. Carolyn Baird, D.N.P., M.B.A., R.N.-B.C., C.A.R.N.-A.P., I.C.C.D.P.D. Joelle Bangsund M.S.W.

1	Wylie I. Bagley, Ph.D. Kumar D. Bahl, M.D. Deborah C. Bailey, M.A., M.S., Ph.D. Carolyn Baird, D.N.P., M.B.A., R.N.-B.C., C.A.R.N.-A.P., I.C.C.D.P.D. Joelle Bangsund M.S.W. Maria Baratta, M.S.W., Ph.D. Stan Barnard, M.S.W. Deborah Barnes, MS. Margaret L. Barnes, Ph.D. David Barnum, Ph.D. Raymond M. Baum, M.D. Edward Wescott Beal, M.D. Michelle Beaudoin, M.A. Ernest E. Beckham, Ph.D. Lori L. Beckwith, M.Ed Emmet Bellville, M.A. Randall E. Bennett, M.A. Lynn Benson, Ph.D. Robert Scott Benson, M.D. Linda Benton, M.S.W. Ditza D. Berger, Ph.D. Louise I. Bertman, Ph.D. Robin Bieber, M.S., L.M.F.T. Diana M. Bigham, M.A. David R. Blackburn, Ph.D. Kelley Blackwell, L.M.F.T. Lancia Blatchley, B.A., L.M.F.T. Stacey L. Block, L.M.S.W., A.C.S.W. Karen J. Bloodworth, M.S., N.C.C., L.P.C. Lester Bloomenstiel, M.S. Christine M. Blue, DO. Marina Bluvshtein, Ph.D. Callie Gray Bobbitt, M.S.W., L.C.S.W. Moses L. Boone, Ir., L.M.S.W., B.C.D.

1	Karen J. Bloodworth, M.S., N.C.C., L.P.C. Lester Bloomenstiel, M.S. Christine M. Blue, DO. Marina Bluvshtein, Ph.D. Callie Gray Bobbitt, M.S.W., L.C.S.W. Moses L. Boone, Ir., L.M.S.W., B.C.D. Steffanie Boudreau—Thomas, M.A.-L.P.C. Iay L. Boulter, M.A. Aaron Daniel Boume, M.A. Helen F. Bowden, Ph.D. Aryn Bowley-Safranek, 8.5., Ms. Elizabeth Boyajian, Ph.D. Beth K. Boyarsky, M.D. Gail M. Boyd, Ph.D. Jeffrey M. Brandler, Ed.S., C.A.S., S.A.P. Sandra L. Branton, Ed.D. Karen J. Brocco-Kish, M.D. Kristin Brooks, P.M.H.N.P. Ann Marie Brown, M.S.W. Philip Brown, M.S.W. Kellie Buckner, Ed.S. Richard Bunt, M.D. Neil F. Buono, D.Min. Janice Bureau, M.S.W., L.C.S.W. Kimlee Butterfield, M.S.W. Claudia Byrne, Ph.D. Quinn Callicott, M.S.W., L.C.S.W. Alvaro Camacho, M.D., M.P.H. Sandra Cambra, Ph.D. Heather Campbell, M.A. Nancy Campbell, Ph.D., M.S.W. Karen Ranee Canada, L.M.F.T. Joseph P. Cannavo, M.D. Catherine F. Caporale, Ph.D. Frederick Capps, Ph.D., M.S.

1	Sandra Cambra, Ph.D. Heather Campbell, M.A. Nancy Campbell, Ph.D., M.S.W. Karen Ranee Canada, L.M.F.T. Joseph P. Cannavo, M.D. Catherine F. Caporale, Ph.D. Frederick Capps, Ph.D., M.S. Rebecca J. Carney, M.B.A., M.A., L.M.H.C. Kelly J. Carroll, M.S.W. Richard W. Carroll, Ph.D., L.P.C., A.C.S. Sherry Casper, Ph.D. Joseph A. Catania, L.I.S.W.S., L.C.D.C. III Manisha P. Cavendish, Ph.D. Kenneth M. Certa, M.D. Shambhavi Chandraiah, M.D. Calvin Chatlos, M.D. Daniel C. Chen, M.D. Darlene Cheryl, M.S.W. Matthew R. Chirman, M5. Carole A. Chisholm, M.S.W. Shobha A. Chottera, M.D. Joseph Logue Christenson, M.D. Pamela Christy, Psy.D. Sharon M. Freeman Clevenger, Ph.D., P.M.H.C.N.S.-B.C. Mary Ann Cohen, M.D. Mitchell J. Cohen, M.D. Diego L. Coira, M.D. Melinda A. Lawless Coker, Psy.D. Carol Cole, M.S.W., L.C.S.W. Caron Collins, M.A., L.M.F.T. Wanda Collins, M.S.N. Linda Cook Cason, M.A. Ayanna Cooke-Chen, M.D., Ph.D. Heidi B. Cooperstein, D.O.

1	Carol Cole, M.S.W., L.C.S.W. Caron Collins, M.A., L.M.F.T. Wanda Collins, M.S.N. Linda Cook Cason, M.A. Ayanna Cooke-Chen, M.D., Ph.D. Heidi B. Cooperstein, D.O. Ileana Corbelle, M.S.W. Kimberly Corbett, Ph.D. Angelina Cordova, M.A.Ed. Jennifer Carol Cox, L.P.C. Sheree Cox, M.A., R.N., N.C.C., D.C.C., L.M.H.C. William Frederick Cox, M.D. Sally M. Cox, M.S.Ed. Debbie Herman Crane, M.S.W. Arthur Ray Crawford, III, Ph.D. Roula Creighton, M.D. John R. Crossfield, L.M.H.C. Sue Cutbirth, R.N., M.S.N, C.S., P.M.H.N.P. Marco Antonio Cuyar, M.S. Rebecca Susan Daily, M.D. Lori S. Danenberg, Ph.D. Chan Dang—Vu, M.D. Mary Hynes Danielak, Psy.D. Cynthia A. Darby, M.Ed., Ed.S. Douglas Darnall, Ph.D. Christopher Davidson, M.D. Doreen Davis, Ph.D., L.C.S.W. Sandra Davis, Ph.D., L.M.I-I.C., N.C.C. Walter Pitts Davis, M.Th. Christian I. Dean, Ph.D. Kent Dean, Ph.D. Elizabeth Dear, M.A. Shelby DeBause, M.A. Rebecca B. DeLaney, M.S.S.W., L.C.S.W., B.C.D.

1	Walter Pitts Davis, M.Th. Christian I. Dean, Ph.D. Kent Dean, Ph.D. Elizabeth Dear, M.A. Shelby DeBause, M.A. Rebecca B. DeLaney, M.S.S.W., L.C.S.W., B.C.D. John R. Delatorre, M.A. Frank DeLaurentis, M.D. Eric Denner, M.A., M.B.A. Mary Dennihan, L.M.F.T. Kenny Dennis, M.A. Pamela L. Detrick, Ph.D., M.S., F.N.P.—B.C., P.M.H.N.P.-B.C., R.N.-B.C., C.A.P., 6.C.A.C. Robert Detrinis, M.D. Daniel A. Deutschman, M.D. Tania Diaz, Psy.D. Sharon Dobbs, M.S.W., L.C.S.W. David Doreau, M.Ed. Gayle L. Dosher, M.A. D'Ann Downey, Ph.D., M.S.W. Beth Doyle, M.A. Amy J. Driskill, M.S., L.C.M.F.T. James Drury, M.D. Brenda-Lee Duarte, M.Ed. Shane E. Dulemba, M.S.N. Nancy R. G. Dunbar, M.D. Cathy Duncan, M.A. Rebecca S. Dunn, M.S.N., A.R.N.P. Debbie Earnshaw, M.A. Shawna Eddy-Kissell, M.A. Momen El Nesr, M.D. Jeffrey Bruce Elliott, Psy.D. Leslie Ellis, Ph.D. Donna M. Emfield, L.C.P.C. Gretchen S. Enright, M.D. John C. Espy, Ph.D. Renuka Evani, M.B.B.S., M.D.

1	Momen El Nesr, M.D. Jeffrey Bruce Elliott, Psy.D. Leslie Ellis, Ph.D. Donna M. Emfield, L.C.P.C. Gretchen S. Enright, M.D. John C. Espy, Ph.D. Renuka Evani, M.B.B.S., M.D. Heather Evans, M.S.Ed, L.P.C.N.C.C. Cesar A. Fabiani, M.D. Fahim Fahim, M.D. Samuel Fam, M.D. Edward H. Fankhanel, Ph.D., Ed.D. Tamara Farmer, M.S.N, A.R.N.P. Farida Farzana, M.D. Philip Fast, M.S. Patricia Feltrup-Exum, M.A.M.F.T. Hector J. Femandez—Barillas, Ph.D. Julie Ferry, M.S.W., L.I.C.S.W. Jane Fink, Ph.D., M.S.S.A. Kathy Finkle, L.P.C.M.H. Steven Finlay, Ph.D. Rik Fire, M.S.W., L.C.S.W. Ann Flood, Ph.D. Jeanine Lee Foreman, M.S. Thyra Fossum, Ph.D. Karen 5. Franklin, L.I.C.S.W. Sherre K. Franklin, M.A. Helen R. Frey, M.A., E.D. Michael L. Freytag, B.S., M.A. Beth Gagnon, M.S.W. Patrice L.R. Gallagher, Ph.D. Angela I. Gallien, M.A. Robert Gallo, M.S.W. Mario Galvarino, M.D. Vladimir I. Gasca, M.D. Joshua Gates, Ph.D. Anthony Gaudioso, Ph.D.

1	Beth Gagnon, M.S.W. Patrice L.R. Gallagher, Ph.D. Angela I. Gallien, M.A. Robert Gallo, M.S.W. Mario Galvarino, M.D. Vladimir I. Gasca, M.D. Joshua Gates, Ph.D. Anthony Gaudioso, Ph.D. Michelle S. Gauthier, A.P.R.N., M.S.N, P.M.H.N.P.-B.C. Rachel E. Gearhart, L.C.S.W. Stephen D. Gelfond, M.D. Nancy S. Gerow, M.S. Michael J. 6erson, Ph.D. Susan M. A. Geyer, L.M.S.W. Lorrie Gfeller—Strouts, Ph.D. Shubu 6hosh, M.D. Richard Dorsey Gillespie, M.Div. Stuart A. 6itlin, M.S.S.A. Jeannette E. Given, Ph.D. Frances 6izzi, L.C.S.W. Stephen 1. Glicksman, Ph.D. Martha Glisky, Ph.D. Sonia Godbole, M.D. Howard M. Goldfischer, Psy.D. Mary Jane Gonzalez-Huss, Ph.D. Michael 1. Good, M.D. Dawn Goodman-Martin, M.A.-L.M.H.C. Robert Gorkin, Ph.D., M.D. Jeff Gorski, M.S.W. Linda 0. Graf, M.Ed., L.C.P.C. Ona Graham, Psy.D. Aubrie M. Graves, L.M.S.W., C.A.S.A.C. Howard S. Green, M.D. Karen Torry Green, M.S.W. Gary Greenberg, Ph.D. Marjorie Greenhut, M.A.

1	Linda 0. Graf, M.Ed., L.C.P.C. Ona Graham, Psy.D. Aubrie M. Graves, L.M.S.W., C.A.S.A.C. Howard S. Green, M.D. Karen Torry Green, M.S.W. Gary Greenberg, Ph.D. Marjorie Greenhut, M.A. James L. Greenstone, Ed.D., ].D. Raymond A. Griffin, Ph.D. Joseph 6rillo, Ph.D. Janeane M. Grisez, A.A., B.A. Lawrence 5. Gross, M.D. Robert ]. Gross, M.D. Sally I. Grosscup, Ph.D. Philip A. Grossi, M.D. Gabrielle Guedet, Ph.D. Nicholas Guenzel, B.A., B.S., M.S.N. Mary 6. Hales, M.A. Tara C. Haley, M.S., L.M.F.T. John D. Hall, M.D. Amy Hammer, M.S.W. Michael S. Hanau, M.D. Linda K.W. Hansen, M.A., L.P. Genevieve R. Hansler, M.S.W. Mary T. Harrington, L.C.S.W. Lois Hartman, Ph.D. Steven Lee Hartsock, Ph.D., M.S.W. Victoria Ann Harwood, M.S.W., L.C.S.W. Rossi A. Hassad, Ph.D., M.P.H. Erin V. Hatchet, M.S.N. Richard L. Hauger, M.D. Kimberly M. Haverly, M.A. Gale Eisner Heater, M.S., M.F.T. Katlin Hecox, M.A. Brenda Heideman, M.S.W. Melinda Heinen, M.Sc.

1	Erin V. Hatchet, M.S.N. Richard L. Hauger, M.D. Kimberly M. Haverly, M.A. Gale Eisner Heater, M.S., M.F.T. Katlin Hecox, M.A. Brenda Heideman, M.S.W. Melinda Heinen, M.Sc. Marie-Therese Heitkamp, M.S. Melissa B. Held, M.A. Jessica Hellings, M.D. Bonnie Helmick-O'Brien, M.A., L.M.F.T. MaLinda T. Henderson, M.S.N, F.P.M.I-I.N.P. Gwenn Herman, M.S.W. Martha W. Hernandez, M.S.N, A.P.R.N., P.M.H.C.N.S. Robin L. Hewitt, M.S. Kenneth Hoffman, Ph.D. Patricia E. Hogan, D.O. Peggy Holcomb, Ph.D. Garland H. Holloman, Jr., M.D. Kimberly Huegel, M.S.W., L.C.S.W. Jason Hughes, L.P.C.-S., N.C.C. Jennifer C. Hughes, Ph.D., M.S.W., L.I.S.W.-S. Michelle K. Humke, M.A. Judith 6. Hunt, L.M.F.T. Tasneem Hussainee, M.D. Sharlene I. Hutchinson, M.S.N. Muhammad Ikram, M.D. Sunday Ilechukwu, M.D., D.Psy. Cli. Douglas H. Ingram, M.D. Marilynn Irvine, Ph.D. Marjorie Isaacs, Psy.D. Raymond Isackila, Ed.S., P.C.C.-S., L.I.C.D.C. Mohammed A. Issa, M.D. John L. Jankord, M.A.

1	Douglas H. Ingram, M.D. Marilynn Irvine, Ph.D. Marjorie Isaacs, Psy.D. Raymond Isackila, Ed.S., P.C.C.-S., L.I.C.D.C. Mohammed A. Issa, M.D. John L. Jankord, M.A. Barbara P. Iannah, L.C.S.W. C. Stuart Johnson, M.S. Dawn M. Johnson, M.A. Deanna V. Johnson, M.S., A.P.R.N., B.C. Eric C. Johnson, M.F.T. ]oy Johnson, Ph.D., L.C.S.W. Willard Johnson, Ph.D. Xenia Iohnson-Bhembe, M.D. Vann S. Joines, Ph.D. Margaret Jones, Psy.D. Patricia Jorgenson, M.S.W. Steven M. Joseph, M.D. Taylere Joseph, M.A. Jeanette M. Joyner—Craddock, M.S.S.W. Melissa Kachapis, M.A. Charles T. Kaelber, M.D. Aimee C. Kaempf, M.D. Peter Andrew Kahn, M.D. Robert P. Kahn-Rose, M.D. Maher Karam-I—Iage, M.D. Todd H. Kasdan, Ph.D. Karen Kaufman, M.S., L.M.F.T. Rhesa Kaulia, M.A., M.F.T. Debbie Lynn Kelly, M.S.N, P.M.H.N.P.-B.C. W. Stephen Kelly, Ph.D. Selena Kennedy, M.A. Judith A. Kenney, M.S., L.P.C. Mark Patrick Kerekes, M.D. Alyse Kerr, M.S., N.C.C., N.A.D.D.-C.C., L.P.C.

1	Debbie Lynn Kelly, M.S.N, P.M.H.N.P.-B.C. W. Stephen Kelly, Ph.D. Selena Kennedy, M.A. Judith A. Kenney, M.S., L.P.C. Mark Patrick Kerekes, M.D. Alyse Kerr, M.S., N.C.C., N.A.D.D.-C.C., L.P.C. Karen L. Kerschmann, L.C.S.W. Marcia Kesner, M.S. Ashan Khan, Ph.D. Shaukat Khan, M.D. Audrey Khatchikian, Ph.D. Laurie B. Kimmel, M.S.W. Jason H. King, Ph.D. Nancy Leigh King, M.S.W., L.C.S.W., L.C.A.S. Kyle Kinne, M.S.C Cassandra M. Klyman, M.D. David R. Knapp, L.C.S.W. Margaret Knerr, MS. Michael R. Knox, Ph.D. Carolyn Koblin, MS. Valerie Kolbert, M.S., A.R.N.P.-B.C. Heather Koontz, M.S.W. Faye Koop, Ph.D., L.C.M.F.T. Fern M. Kopakin, M.S.W., L.C.S.W. Joel Kotin, M.D. Sharlene K. Kraemer, M.S.E. Marjorie Vego Krausz, M.A., Ed.D. Nancy J. Krell, M.S.W. Mindy E. Kronenberg, Ph.D. Dwayne Kruse, M.S., M.F.T. Ajay S. Kuchibhatla, M.D. Shubha N. Kumar, M.D. Helen H. Kyomen, M.D., M.S. Rebecca M. Lachut, M.Ed., Ed.S. Alexis Lake, M.S.S. Ramaswamy Lakshmanan, M.D.

1	Dwayne Kruse, M.S., M.F.T. Ajay S. Kuchibhatla, M.D. Shubha N. Kumar, M.D. Helen H. Kyomen, M.D., M.S. Rebecca M. Lachut, M.Ed., Ed.S. Alexis Lake, M.S.S. Ramaswamy Lakshmanan, M.D. Brigitta Lalone, L.C.S.W.-R John W. Lancaster, Ph.D. Patience R. Land, L.I.C.S.W., M.S.W., M.P.A. Amber Lange, M.A., Ph.D. Jeff K. Larsen, M.A. Nathan E. Lavid, M.D. Michelle Leader, Ph.D. Stephen E. Lee, M.D. Cathryn L. Leff, Ph.D., L.M.F.T. Rachael Kollar Leombruno, L.M.F.T. Arlene I. Lev, M.S.W., L.C.S.W.-R Gregory K. Lewis, M.A.-L.M.F.T. Jane Hart Lewis, M.S. Melissa S. Lewis, M.S.W., L.I.C.S.W. Norman Gerald Lewis, F.R.A.N.2.C.P. Robin Joy Lewis, Ph.D. Ryan Michael Ley, M.D. Tammy R. Lias, M.A. Russell F. Lim, M.D. Jana Lincoln, M.D. Ted Lindberg, L.M.S.W., L.M.F.T., M.S.W. Peggy Solow Liss, M.S.W. Andrea Loeb, Psy.D. William David Lohr, M.D. Mary L. Ludy, M.A., L.M.H.C., L.M.F.T. Nathan Lundin, M.A., L.P.C. Veena Luthra, M.D. Patti Lyerly, L.C.S.W.

1	Peggy Solow Liss, M.S.W. Andrea Loeb, Psy.D. William David Lohr, M.D. Mary L. Ludy, M.A., L.M.H.C., L.M.F.T. Nathan Lundin, M.A., L.P.C. Veena Luthra, M.D. Patti Lyerly, L.C.S.W. Denise E. Maas, M.A. Silvia MacAllister, L.M.F.T. Nicola MacCallum, M.S., M.F.C. Therapy Colin N. MacKenzie, M.D. Cynthia Mack—Emsdorff, Ph.D. John R. Madsen—Bibeau, M.S., M.Div Christopher I. Maglio, Ph.D. Deepak Mahajan, M.D. Debra Majewski, M.A. Harish Kumar Malhotra, M.D. Pamela Marcus, R.N., M.S. Mary P. Marshall, Ph.D. Flora Lyme Martin, M.A., L.P.C., A.D.C. Robert S. Martin, M.D. Jennifer L. Martinez, MS. Ninfa Martinez-Aguilar, M.A., M.F.T. Emily Martinsen, M.S.W. Farhan A. Matin, M.D. Janus Maybee, P.M.H.N.P. Karen Mazarin-Stanek, M.A. Eben L. McClenahan, M.D., M.B. Ierlyn C. McCleod, M.D. Susan E. McCue, M.S.W., L.C.S.W. Kent D. McDonald, M.S. Daniel McDonnell, M.S.N, P.M.H.-N.P. Robert McElhose, Ph.D. Lisa D. Mc6rath, Ph.D. Mark McGrosky, M.S.W.

1	Ierlyn C. McCleod, M.D. Susan E. McCue, M.S.W., L.C.S.W. Kent D. McDonald, M.S. Daniel McDonnell, M.S.N, P.M.H.-N.P. Robert McElhose, Ph.D. Lisa D. Mc6rath, Ph.D. Mark McGrosky, M.S.W. Katherine M. McKay, Ph.D. Darren D. McKinnis, M.S.W. Mona McNelis-Broadley, M.S.W., L.C.S.W. Rick McQuistion, Ph.D. Susan Joy Mendelsohn, Psy.D. Barbara S. Menninga, M.Ed. Hindi Mermelstein, M.D., F.A.P.M. Rachel B. Michaelsen, M.S.W. Thomas F. Micka, M.D. Tonya Miles, Psy.D. Matthew Miller, MS. Michael E. Miller, M.D. Noel Miller, L.M.S.W., M.B.A., M.P.S. Kalpana Miriyala, M.D. Sandra Moenssens, MS. Erin Mokhtar, M.A. Robert E. Montgomery, M.Ed. Susan Moon, M.A. Theresa K. Moon, M.D. David B. Moore, B.A., M.Div., M.S.S.W., Ph.D. Joanne M. Moore, M.S. Peter I. M. Moran, M.B.B.Ch. Anna Moriarty, M.P.S., L.P.C., L.M.H.C. Richard Dean Morris, M.A. Michael M. Morrison, M.A. Carlton E. Munson, Ph.D. Timothy A. Murphy, M.D. Beth L. Murphy, Psy.D. Melissa A. Myers, M.D.

1	Anna Moriarty, M.P.S., L.P.C., L.M.H.C. Richard Dean Morris, M.A. Michael M. Morrison, M.A. Carlton E. Munson, Ph.D. Timothy A. Murphy, M.D. Beth L. Murphy, Psy.D. Melissa A. Myers, M.D. Stefan Nawab, M.D. Allyson Matney Neal, D.N.P. Steven Nicholas, M.A. Aurelian N. Niculescu, M.D. Earl S. Nielsen, Ph.D. Terry Oleson, Ph.D. Julianne R. Oliver, B.S., M.S., Ph.D. Robert 0. Olsen, M.D. Amy O'Neill, M.D. Oscar H. 00, Psy.D., A.B.P.P. Laurie Orlando, I.D., M.A. Jill Osborne, M.S., Ed.S. Kimberly Overlie, M.S. L. Kola Oyewumi, Ph.D. Zachary I. Pacha, M.S.W. Suzette R. Papadakis, M.S. Amanda C. Parsons, M.A., L.P.C.C. Lee R. Pate, B.A., M.A. Eric L. Patterson, L.P.C. Sherri Paulson, M.Ed., L.S.C.W. Peter Dennis Pautz, B.A., M.S.W. Malinda I. Perkins, M.S.W., L.C.S.W. Eleanor F. Perlman, M.S.W. Deborah K. Perry, M.S.W. Amanda Peterman, L.M.F.T. Shawn Pﬂugardt, Psy.D. Robert I. Dean Phillips, M.S. Laura Pieper, M.S.W., L.C.S.W. Lori D. Pink, M.S.W., B.C.D

1	Eleanor F. Perlman, M.S.W. Deborah K. Perry, M.S.W. Amanda Peterman, L.M.F.T. Shawn Pﬂugardt, Psy.D. Robert I. Dean Phillips, M.S. Laura Pieper, M.S.W., L.C.S.W. Lori D. Pink, M.S.W., B.C.D Michae16. Pipich, M.S., L.M.F.T. Cynthia 6. Pizzulli, M.S.W., Ph.D. Kathy C. Points, M.A. Marya E. Pollack, M.D., M.P.H. Sanford E. Pomerantz, M.D. Eva Ponder, M.S.W., Psy.D. Ernest Poortinga, M.D. David Post, M.D. Laura L. Post, M.D., Ph.D., ].D. Patrick W. Powell, Ed.D. Beth M. Prewett, Psy.D. Robert Price, D.C.C., M.Ed. John Pruett, M.D. Aneita S. Radov, M.A. Dawn M. Raffa, Ph.D. Kavitha Raja, M.D. Ranjit Ram, M.D. Mohamed Ibrahim Ramadan, M.D., MS. Christopher S. Randolph, M.D. Nancy Rappaport, M.Ed. John Moir Rauenhorst, M.D. Laurel Jean Rebenstock, L.M.S.W. Edwin Renaud, Ph.D. Heather ]. Rhodes, M.A. Jennifer S. Ritchie-Goodline, Psy.D. Daniel G. Roberts, M.A. Brenda Rohren, M.A., M.P.S., L.I.M.H.P., L.A.D.C., M.A.C. Donna 6. Rolin-Kenny, Ph.D., A.P.R.N.,

1	Heather ]. Rhodes, M.A. Jennifer S. Ritchie-Goodline, Psy.D. Daniel G. Roberts, M.A. Brenda Rohren, M.A., M.P.S., L.I.M.H.P., L.A.D.C., M.A.C. Donna 6. Rolin-Kenny, Ph.D., A.P.R.N., P.M.H.C.N.S.-B.C. Sylvia E. Rosario, M.Ed. Mindy S. Rosenbloom, M.D. Harvey A. Rosenstock, M.D. Thalia Ross, M.S.S.W. Fernando Rosso, M.D. Barry H. Roth, M.D. Thomas S. Rue, M.A., L.M.H.C. Elizabeth Ruegg, L.C.S.W. Diane Rullo, Ph.D. Angie Rumaldo, Ph.D. Eric Rutberg, M.A., D.H.Ed. Joseph A. Sabella, L.M.H.C. Kemal Sagduyu, M.D. Adam H. Saltz, M.S.W. Jennifer A. Samardak, L.I.S.W.-S. George R. Samuels, M.A., M.S.W. Carmen Sanjurjo, M.A. John S. Saroyan, Ed.D. Brigid Kathleen Sboto, M.A., M.F.T. Lori Cluff Schade, M.S. Joan E. Schaper, M.S.N. Rae ]. Schilling, Ph.D. Larry Schor, Ph.D. Donna J. Schwartz, M.S.W., L.I.C.S.W. Amy J. Schwarzenbart, P.M.H.-C.N.S., B.C., A.P.N.P. John V. Scialli, M.D. Chad Scott, Ph.D., L.P.CC. Sabine Sell, M.F.T.

1	Larry Schor, Ph.D. Donna J. Schwartz, M.S.W., L.I.C.S.W. Amy J. Schwarzenbart, P.M.H.-C.N.S., B.C., A.P.N.P. John V. Scialli, M.D. Chad Scott, Ph.D., L.P.CC. Sabine Sell, M.F.T. Mina] Shah, N.S., N.C.C., L.P.C. Lynn Shell, M.S.N. Dharmesh Navin Sheth, M.D. S. Christopher Shim, M.D. Marta M. Shinn, Ph.D. Andreas Sidiropoulos, M.D., Ph.D. Michael Siegell, M.D. Michael G. Simonds, Psy.D. Gagandeep Singh, M.D. Melissa Rae Skrzypchak, M.S.S.W., L.C.S.W. Paula Slater, M.D. William Bill Slaughter, M.D., M.A. Aki Smith, Ph.D. Deborah L. Smith, Ed.M. Diane E. Smith, M.A., L.M.F.T. James S. Sommer, MS. J. Richard Spatafora, M.D. Judy Splittgerber, M.S.N., 6.5., N.P. Thiruneermalai T.G. Sriram, M.D. Martha W. St. John, M.D. Sybil Stafford, Ph.D. Timothy Stambaugh, M.A. Laura A. Stamboni, M.S.W. Carol L. R. Stark, M.D. Stephanie Steinman, M.S. Claudia M. Stevens, M.S.W. Jennifer Boyer Stevens, Psy.D. Dominique Stevens-Young, M.S.W., L.C.S.W.

1	Laura A. Stamboni, M.S.W. Carol L. R. Stark, M.D. Stephanie Steinman, M.S. Claudia M. Stevens, M.S.W. Jennifer Boyer Stevens, Psy.D. Dominique Stevens-Young, M.S.W., L.C.S.W. Kenneth Stewart, Ph.D. Daniel Storch, M.D. Suzanne Straebler, A.P.R.N. Dawn Stremel, M.A., L.M.F.T. Emel Stroup, Psy.D. John W. Stump, M.S., L.M.F.T. Thomas 6. Suk, M.A. Elizabeth Sunzeri, M.S. Linnea Swanson, M.A., Psy.D. Patricia Swanson, M.A. Fereidoon Taghizadeh, M.D. Bonnie L. Tardif, L.M.H.C., N.C.C., B.C.P.C.C. Joan Tavares, M.S.W. Ann Taylor, M.S.W. Dawn O'Dwyer Taylor, Ph.D. Chanel V. Tazza, L.M.H.C. Martha H. Teater, M.A. Clark D. Terrell, M.D. Mark R. Thelen, Psy.D. Norman E. Thibault, M.S., Ph.D. Tojuana L. Thomason, Ph.D. Paula Thomson, Psy.D. D. Chadwick Thompson, M.A. Susan Thome-Devin, A.M. Jean Eva Thumm, M.A.P.C., M.A.T., L.M.F.T., B.C.C. James E. Tille, Ph.D., D.Min. Iacalyn 6. Tippey, Ph.D. Saraswathi Tirumalasetty, M.D. Jacqueline A. Torrance, M.S.

1	Susan Thome-Devin, A.M. Jean Eva Thumm, M.A.P.C., M.A.T., L.M.F.T., B.C.C. James E. Tille, Ph.D., D.Min. Iacalyn 6. Tippey, Ph.D. Saraswathi Tirumalasetty, M.D. Jacqueline A. Torrance, M.S. Terrence Trobaugh, MS. Louisa V. Troemel, Psy.D., L.M.F.T. Susan Ullman, M.S.W. Jennifer M. Underwood, M.S.W., L.C.S.W. Rodney Dale Veldhuizen, M.A. Michelle Voegels, B.S.N., M.S.N., B.C. Wess Vogt, M.D. R. Christopher Votolato, Psy.D. John W. Waid, Ph.D. Christa A. Wallis, M.A. Dominique Walmsley, M.A. Bhupinder Singh Waraich, M.D. Joseph Ward, N.C.C., L.P.C. M.Ed. Robert Ward, M.S.W. Marilee L. M. Wasell, Ph.D. Gannon 1. Watts, L.P.C.-S., L.A.C., N.C.C., N.C.S.C., A.A.D.C., I.C.A.A.D.C. Sheila R. Webster, M.A., M.S.S.A. Burton Weiss, M.D. Dennis V. Weiss, M.D. Jonathan S. Weiss, MD. Richard Wendel, Ph.D. Paul L. West, Ed.D. Kris Sandra Wheatley, M.A., L.P.C., N.C.C. Leneigh White, M.A. Danny R. Whitehead, L.I.C.S.W. Jean Whitinger, M.A. Peter D. Wilk, M.D.

1	Richard Wendel, Ph.D. Paul L. West, Ed.D. Kris Sandra Wheatley, M.A., L.P.C., N.C.C. Leneigh White, M.A. Danny R. Whitehead, L.I.C.S.W. Jean Whitinger, M.A. Peter D. Wilk, M.D. Vanessa Wilkinson, L.P.C. Tim F. Willia, M.S., M.A.Ed., L.P.C. Cathy E. Willis, M.A., L.M.F.T., C.A.D.C. Jeffery John Wilson, M.D. Jacquie Wilson, M.Ed. David D. Wines, M.S.W. Barbara A. Wirebaugh, M.S.W. Daniel L. Wise, Ph.D. Christina Wong, M.S.W., L.C.S.W. Susanna Wood, M.S.W., L.C.S.W. Linda L. Woodall, M.D. Leoneen Woodard-Faust, M.D. Sheryl E. Woodhouse, L.M.F.T. Gregory I. Worthington, Psy.D. Tanya Wozniak, M.D. Kimberly Isaac Wright, M.A. Peter Yamamoto, M.D. Maria Ruiza Ang Yee, M.D. Michael B. Zafrani, M.D. Jafet E. Gonzalez Zakarchenco, M.D. John Zibert, Ph.D. Karen 2ilberstein, M.S.W. Cathi 2illmann, C.P.N.P., N.P.P. Gerald A. Zimmerman, Ph.D. Michele Zimmerman, M.A., P.M.H.C.N.S.-B.C. Judith A. link, M.A. Paul Harris, Ph.D. Sudah Kashyap, B.E. Ion Scherdin, M.A.

1	Cathi 2illmann, C.P.N.P., N.P.P. Gerald A. Zimmerman, Ph.D. Michele Zimmerman, M.A., P.M.H.C.N.S.-B.C. Judith A. link, M.A. Paul Harris, Ph.D. Sudah Kashyap, B.E. Ion Scherdin, M.A. Rob Taylor, M.A. Janey Wang, MS.

1	“Biochemistry is the study of carbon compounds that crawl.” “We think we have found the basic mechanism by which life comes from life.” —Francis H. C. Crick “The biochemistry and biophysics are the notes required for life; they conspire, collectively, to generate the real unit of life, the organism.” This high-yield material includes molecular biology, genetics, cell biology, and principles of metabolism (especially vitamins, cofactors, minerals, and single-enzyme-deficiency diseases). When studying metabolic pathways, emphasize important regulatory steps and enzyme deficiencies that result in disease, as well as reactions targeted by pharmacologic interventions. For example, understanding the defect in Lesch-Nyhan syndrome and its clinical consequences is higher yield than memorizing every intermediate in the purine salvage pathway.

1	Do not spend time learning details of organic chemistry, mechanisms, or physical chemistry. Detailed chemical structures are infrequently tested; however, many structures have been included here to help students learn reactions and the important enzymes involved. Familiarity with the biochemical techniques that have medical relevance—such as ELISA, immunoelectrophoresis, Southern blotting, and PCR—is useful. Review the related biochemistry when studying pharmacology or genetic diseases as a way to reinforce and integrate the material. H1 histone (linker) Supercoiled structure Heterochromatin Euchromatin Metaphase chromosome Nucleosome (H2A, H2B, H3, H4) 2 DNA DNA double-helix DNA exists in the condensed, chromatin form to fit into the nucleus. DNA loops twice around a histone octamer to form a nucleosome (“beads on a string”). H1 binds to the nucleosome and to “linker DNA,” thereby stabilizing the chromatin fiber.

1	Phosphate groups give DNA a ⊝ charge. Lysine and arginine give histones a ⊕ charge. In mitosis, DNA condenses to form chromosomes. DNA and histone synthesis occurs during S phase. Mitochondria have their own DNA, which is circular and does not utilize histones. Heterochromatin Condensed, appears darker on EM (labeled H HeteroChromatin = Highly Condensed. in A ; Nu, nucleolus). Sterically inaccessible, Barr bodies (inactive X chromosomes) may be thus transcriptionally inactive. • methylation, visible on the periphery of nucleus. • acetylation. Histone methylation Usually causes reversible transcriptional Histone Methylation Mostly Makes DNA Mute. suppression, but can also cause activation depending on location of methyl groups. Histone acetylation Removal of histone’s ⊕ charge  relaxed DNA Histone Acetylation makes DNA Active. coiling  transcription. Histone deacetylation Removal of acetyl groups  tightened DNA coiling • transcription.

1	Histone deacetylation Removal of acetyl groups  tightened DNA coiling • transcription. Nucleotides NucleoSide = base + (deoxy)ribose (Sugar). NucleoTide = base + (deoxy)ribose + phosphaTe; linked by 3′-5′ phosphodiester bond. PURines (A,G)—2 rings. PYrimidines (C,U,T)—1 ring. Deamination reactions: Cytosine  uracil Adenine  hypoxanthine Guanine  xanthine 5-methylcytosine  thymine Uracil found in RNA; thymine in DNA. Methylation of uracil makes thymine. Purine (A, G) Pyrimidine (C, U, T) 5′ end of incoming nucleotide bears the triphosphate (energy source for the bond). Triphosphate bond is target of 3′ hydroxyl attack. PURe As Gold. CUT the PY (pie). Thymine has a methyl. C-G bond (3 H bonds) stronger than A-T bond (2 H bonds).  C-G content  melting temperature of DNA. “C-G bonds are like Crazy Glue.” purr until they GAG): Glycine Aspartate Glutamine

1	De novo pyrimidine Various immunosuppressive, antineoplastic, and antibiotic drugs function by interfering with and purine synthesis nucleotide synthesis: (de novo requires aspartate, Glutamine + CO2 glycine, glutamine, and THF) Lefunomide 6-MP, Mycophenolate,(impaired in Pyrimidine synthesis: Leflunomide: inhibits dihydroorotate dehydrogenase 5-fluorouracil (5-FU) and its prodrug capecitabine: form 5-F-dUMP, which inhibits thymidylate synthase ( dTMP) Purine synthesis: 6-mercaptopurine (6-MP) and its prodrug azathioprine: inhibit de novo purine synthesis Mycophenolate and ribavirin: inhibit inosine monophosphate dehydrogenase Purine and pyrimidine synthesis: • Hydroxyurea: inhibits ribonucleotide reductase AMP GMP • Methotrexate (MTX), trimethoprim (TMP), and pyrimethamine: inhibit dihydrofolate reductase (• deoxythymidine monophosphate [dTMP]) in humans, bacteria, and protozoa, 5-FU, MTX, TMP, pyrimethamine

1	MTX, TMP, pyrimethamine ADA, adenosine deaminase; APRT, adenine phosphoribosyltransferase; HGPRT, hypoxanthine guanine phosphoribosyltransferase; XO, xanthine oxidase. Defective purine salvage due to absent HGPRT, which converts hypoxanthine to IMP and guanine to GMP. Results in excess uric acid production and de novo purine synthesis. X-linked recessive. Findings: intellectual disability, self-mutilation, aggression, hyperuricemia (orange “sand” [sodium urate crystals] in diaper), gout, dystonia, macrocytosis. Treatment: allopurinol or febuxostat (2nd line). HGPRT: Hyperuricemia Gout Pissed off (aggression, self-mutilation) Retardation (intellectual disability) DysTonia Unambiguous Each codon specifies only 1 amino acid. Commaless, Read from a fixed starting point as a continuous Exceptions: some viruses. nonoverlapping sequence of bases. Universal Genetic code is conserved throughout Exception in humans: mitochondria. evolution.

1	Universal Genetic code is conserved throughout Exception in humans: mitochondria. evolution. DNA replication Eukaryotic DNA replication is more complex than in prokaryotes but uses many enzymes analogous to those listed below. In both prokaryotes and eukaryotes, DNA replication is semiconservative, involves continuous and discontinuous (Okazaki fragment) synthesis, and occurs in the 5′  3′ direction. B Y-shaped region along DNA template where leading and lagging strands are synthesized. C Unwinds DNA template at replication fork. Helicase Halves DNA. Deficient in Bloom syndrome (BLM gene mutation). Single-stranded Prevent strands from reannealing. binding proteins F Makes an RNA primer on which DNA polymerase III can initiate replication. H Prokaryotes only. Degrades RNA primer; Same functions as DNA polymerase III, also replaces it with DNA. excises RNA primer with 5′  3′ exonuclease.

1	H Prokaryotes only. Degrades RNA primer; Same functions as DNA polymerase III, also replaces it with DNA. excises RNA primer with 5′  3′ exonuclease. I Catalyzes the formation of a phosphodiester Joins Okazaki fragments. bond within a strand of double-stranded DNA. Ligase Links DNA. Mutations in DNA Severity of damage: silent << missense < nonsense < frameshift. Types of single nucleotide (point) mutations: • Transition—purine to purine (eg, A to G) or pyrimidine to pyrimidine (eg, C to T). Transversion—purine to pyrimidine (eg, A to T) or pyrimidine to purine (eg, C to G). Single nucleotide substitutions Silent mutation Nucleotide substitution codes for same (synonymous) amino acid; often base change in 3rd position of codon (tRNA wobble). Missense mutation Nucleotide substitution results in changed amino acid (called conservative if new amino acid has similar chemical structure). Examples include sickle cell disease (substitution of glutamic acid with valine).

1	Nonsense mutation Nucleotide substitution results in early stop codon (UGA, UAA, UAG). Usually results in nonfunctional protein. Stop the nonsense! Other mutations Frameshift mutation Deletion or insertion of a number of nucleotides not divisible by 3 • misreading of all nucleotides downstream. Protein may be shorter or longer, and its function may be disrupted or altered. Examples include Duchenne muscular dystrophy, Tay-Sachs disease. Splice site mutation Retained intron in mRNA • protein with impaired or altered function. Examples include rare causes of cancers, dementia, epilepsy, some types of β-thalassemia, Gaucher disease, Marfan syndrome. Lac operon Classic example of a genetic response to an environmental change. Glucose is the preferred metabolic substrate in E coli, but when glucose is absent and lactose is available, the lac operon is activated to switch to lactose metabolism. Mechanism of shift:

1	Low glucose  adenylate cyclase activity  generation of cAMP from ATP  activation of catabolite activator protein (CAP) • transcription. High lactose  unbinds repressor protein from repressor/operator site • transcription. Binds CAP site, Lacl LacZ LacY LacACAP site Promoter Operator Repressor protein Lacoperon Bindsoperator, blockstranscription organization of a eukaryotic gene Exon Intron Exon Intron Exon Regulation of gene expression Silencer DNA locus where regulatory proteins (“repressors”) bind, decreasing expression of a gene on the same chromosome. Initial transcript is called heterogeneous nuclear RNA (hnRNA). hnRNA is then modified and becomes mRNA. The following processes occur in the nucleus: Capping of 5′ end (addition of 7-methylguanosine cap) Polyadenylation of 3′ end (≈ 200 As) Splicing out of introns Capped, tailed, and spliced transcript is called mRNA. mRNA is transported out of nucleus to be translated in cytosol.

1	Polyadenylation of 3′ end (≈ 200 As) Splicing out of introns Capped, tailed, and spliced transcript is called mRNA. mRNA is transported out of nucleus to be translated in cytosol. mRNA quality control occurs at cytoplasmic processing bodies (P-bodies), which contain exonucleases, decapping enzymes, and microRNAs; mRNAs may be degraded or stored in P-bodies for future translation. Poly-A polymerase does not require a template. AAUAAA = polyadenylation signal. Eukaryotes RNA polymerase I makes rRNA, the most common (rampant) type; present only in nucleolus. RNA polymerase II makes mRNA (massive), microRNA (miRNA), and small nuclear RNA (snRNA). RNA polymerase III makes 5S rRNA, tRNA (tiny). No proofreading function, but can initiate chains. RNA polymerase II opens DNA at promoter site. I, II, and III are numbered in the same order that their products are used in protein synthesis: rRNA, mRNA, then tRNA.

1	I, II, and III are numbered in the same order that their products are used in protein synthesis: rRNA, mRNA, then tRNA. α-amanitin, found in Amanita phalloides (death cap mushrooms), inhibits RNA polymerase II. Causes severe hepatotoxicity if ingested. Actinomycin D, also called dactinomycin, inhibits RNA polymerase in both prokaryotes and eukaryotes. Prokaryotes 1 RNA polymerase (multisubunit complex) Rifampin inhibits DNA-dependent RNA makes all 3 kinds of RNA. polymerase in prokaryotes. Splicing of pre-mRNA Part of process by which precursor mRNA (pre-mRNA) is transformed into mature mRNA. Alterations in snRNP assembly can cause clinical disease; eg, in spinal muscular atrophy, snRNP assembly is affected due to  SMN protein  congenital degeneration of anterior horns of spinal cord  symmetric weakness (hypotonia, or “floppy baby syndrome”). Primary transcript combines with small nuclear ribonucleoproteins (snRNPs) and other proteins to form spliceosome.

1	Primary transcript combines with small nuclear ribonucleoproteins (snRNPs) and other proteins to form spliceosome. RNA polymerases P P OOH 3˜OH 3˜Cleavage at 5˜splice site; lariat-shaped (loop) intermediate is generated. Cleavage at 3˜splice site; lariat is released to precisely remove intron and join 2 exons. Exon 1 Exon 2 Spliceosome+P UGAAG3˜Exon 1 Mature mRNA Exon 2 P UGAAG

1	Structure 75–90 nucleotides, 2º structure, cloverleaf form, anticodon end is opposite 3′ aminoacyl end. All tRNAs, both eukaryotic and prokaryotic, have CCA at 3′ end along with a high percentage of chemically modified bases. The amino acid is covalently bound to the 3′ end of the tRNA. CCA Can Carry Amino acids. T-arm: contains the TΨC (ribothymidine, pseudouridine, cytidine) sequence necessary for tRNAribosome binding. T-arm Tethers tRNA molecule to ribosome. D-arm: contains Dihydrouridine residues necessary for tRNA recognition by the correct aminoacyltRNA synthetase. D-arm allows Detection of the tRNA by aminoacyl-tRNA synthetase. Attachment site: the 5′-CCA-3′ is the amino acid acceptor site.

1	Charging Aminoacyl-tRNA synthetase (uses ATP; 1 unique enzyme per respective amino acid) and binding of charged tRNA to the codon are responsible for the accuracy of amino acid selection. Aminoacyl-tRNA synthetase matches an amino acid to the tRNA by scrutinizing the amino acid before and after it binds to tRNA. If an incorrect amino acid is attached, the bond is hydrolyzed. A mischarged tRNA reads the usual codon but inserts the wrong amino acid. 3. eIFs released when the mRNA and the ribosomal 60S subunit assemble with the complex. Requires GTP. Aminoacyl-tRNA binds to A site (except for initiator methionine, which binds the P site), requires an elongation factor and GTP. rRNA (“ribozyme”) catalyzes peptide bond formation, transfers growing polypeptide to amino acid in A site. Ribosome advances 3 nucleotides toward 3′ end of mRNA, moving peptidyl tRNA to P site (translocation). ATP—tRNA Activation (charging). GTP—tRNA Gripping and Going places (translocation).

1	Ribosome advances 3 nucleotides toward 3′ end of mRNA, moving peptidyl tRNA to P site (translocation). ATP—tRNA Activation (charging). GTP—tRNA Gripping and Going places (translocation). Think of “going APE”: A site = incoming Aminoacyl-tRNA. P site = accommodates growing Peptide. E site = holds Empty tRNA as it Exits. Termination Eukaryotic release factors (eRFs) recognize the stop codon and halt translation • completed polypeptide is released from ribosome. Requires GTP. Trimming Removal of Nor C-terminal propeptides from zymogen to generate mature protein (eg, trypsinogen to trypsin). Covalent alterations Phosphorylation, glycosylation, hydroxylation, methylation, acetylation, and ubiquitination. Chaperone protein Intracellular protein involved in facilitating and maintaining protein folding. In yeast, heat shock proteins (eg, HSP60) are expressed at high temperatures to prevent protein denaturing/misfolding.

1	Cell cycle phases Checkpoints control transitions between phases of cell cycle. This process is regulated by cyclins, cyclin-dependent kinases (CDKs), and tumor suppressors. M phase (shortest phase of cell cycle) includes mitosis (prophase, prometaphase, metaphase, anaphase, telophase) and cytokinesis (cytoplasm splits in two). G1 and G0 are of variable duration. GO G1Growth DNASynthesis INTERPHASERb, p53 modulate Grestriction point G2 M CytokinesisS Mitosis Site of synthesis of secretory (exported) proteins and of N-linked oligosaccharide addition to lysosomal and other proteins. Nissl bodies (RER in neurons)—synthesize peptide neurotransmitters for secretion. Free ribosomes—unattached to any membrane; site of synthesis of cytosolic, peroxisomal, and mitochondrial proteins. Mucus-secreting goblet cells of the small intestine and antibody-secreting plasma cells are rich in RER. Proteins within organelles (eg, ER, Golgi bodies, lysosomes) are formed in RER.

1	Mucus-secreting goblet cells of the small intestine and antibody-secreting plasma cells are rich in RER. Proteins within organelles (eg, ER, Golgi bodies, lysosomes) are formed in RER. Cell trafficking Golgi is distribution center for proteins and lipids from ER to vesicles and plasma membrane. Posttranslational events in Golgi include modifying N-oligosaccharides on asparagine, adding O-oligosaccharides on serine and threonine, and adding mannose-6-phosphate to proteins for lysosomal trafficking. Endosomes are sorting centers for material from outside the cell or from the Golgi, sending it to lysosomes for destruction or back to the membrane/Golgi for further use.

1	I-cell disease (inclusion cell disease/mucolipidosis type II)—inherited lysosomal storage disorder (autosomal recessive); defect in N-acetylglucosaminyl-1-phosphotransferase  failure of the Golgi to phosphorylate mannose residues ( mannose-6-phosphate) on glycoproteins  proteins are secreted extracellularly rather than delivered to lysosomes. Results in coarse facial features, gingival hyperplasia, clouded corneas, restricted joint movements, claw hand deformities, kyphoscoliosis, and high plasma levels of lysosomal enzymes. Often fatal in childhood. Key: Abundant, cytosolic ribonucleoprotein that traffics polypeptide-ribosome complex from the cytosol to the RER. Absent or COPI dysfunctional SRP  accumulation of protein in cytosol. trans COPI: Golgi  Golgi (retrograde); cis-Golgi Anterograde • ER. COPII: ER • cis-Golgi (anterograde).

1	trans COPI: Golgi  Golgi (retrograde); cis-Golgi Anterograde • ER. COPII: ER • cis-Golgi (anterograde). Golgi apparatus “Two (COPII) steps forward (anterograde); one (COPI) step back (retrograde).” Clathrin: trans-Golgi • lysosomes; plasma membrane • endosomes (receptormediated endocytosis [eg, LDL receptor Rough activity]). endoplasmic reticulum Peroxisome Membrane-enclosed organelle involved in: β-oxidation of very-long-chain fatty acids (VLCFA) (strictly peroxisomal process) α-oxidation of branched-chain fatty acids (strictly peroxisomal process) Catabolism of amino acids and ethanol Synthesis of cholesterol, bile acids, and plasmalogens (important membrane phospholipid, especially in white matter of brain) Zellweger syndrome—autosomal recessive disorder of peroxisome biogenesis due to mutated PEX genes. Hypotonia, seizures, hepatomegaly, early death.

1	Refsum disease—autosomal recessive disorder of α-oxidation  phytanic acid not metabolized to pristanic acid. Scaly skin, ataxia, cataracts/night blindness, shortening of 4th toe, epiphyseal dysplasia. Treatment: diet, plasmapheresis. Adrenoleukodystrophy—X-linked recessive disorder of β-oxidation due to mutation in ABCD1 gene  VLCFA buildup in adrenal glands, white (leuko) matter of brain, testes. Progressive disease that can lead to adrenal gland crisis, coma, and death. Proteasome Barrel-shaped protein complex that degrades damaged or ubiquitin-tagged proteins. Defects in the ubiquitin-proteasome system have been implicated in some cases of Parkinson disease. Cytoskeletal elements A network of protein fibers within the cytoplasm that supports cell structure, cell and organelle movement, and cell division.

1	Cytoskeletal elements A network of protein fibers within the cytoplasm that supports cell structure, cell and organelle movement, and cell division. Cylindrical outer structure composed of a helical array of polymerized heterodimers of αand β-tubulin. Each dimer has 2 GTP bound. Incorporated into flagella, cilia, mitotic spindles. Grows slowly, collapses quickly. Also involved in slow axoplasmic transport in neurons. Molecular motor proteins—transport cellular cargo toward opposite ends of microtubule. REtrograde to microtubule (+  −)—DYnein. Anterograde to microtubule (−  +)—Kinesin. Clostridium tetani, herpes simplex virus, poliovirus, and rabies virus use dynein for retrograde transport to the neuronal cell body. Drugs that act on microtubules (Microtubules Get Constructed Very Poorly): Paclitaxel (anticancer) Negative end Near Nucleus. Positive end Points to Periphery. REaDY? AttacK! Cilia structure 9 doublet + 2 singlet arrangement of microtubules

1	Paclitaxel (anticancer) Negative end Near Nucleus. Positive end Points to Periphery. REaDY? AttacK! Cilia structure 9 doublet + 2 singlet arrangement of microtubules A . Basal body (base of cilium below cell membrane) consists of 9 microtubule triplets B with no central microtubules. Axonemal dynein—ATPase that links peripheral 9 doublets and causes bending of cilium by differential sliding of doublets. Gap junctions enable coordinated ciliary movement. Kartagener syndrome (1° ciliary dyskinesia)— immotile cilia due to a dynein arm defect. Autosomal recessive. Results in  male and female fertility due to immotile sperm and dysfunctional fallopian tube cilia, respectively; of ectopic pregnancy. Can cause bronchiectasis, recurrent sinusitis, chronic ear infections, conductive hearing loss, and situs inversus (eg, dextrocardia on CXR C ). nasal nitric oxide (used as screening test). (Kartagener’s restaurant: take-out only; there’s no dynein “dine-in”.)

1	nasal nitric oxide (used as screening test). (Kartagener’s restaurant: take-out only; there’s no dynein “dine-in”.) Na+-K+ ATPase is located in the plasma membrane with ATP site on cytosolic side. For each ATP consumed, 3 Na+ leave the cell (pump phosphorylated) and 2 K+ enter the cell (pump dephosphorylated). Plasma membrane is an asymmetric lipid bilayer containing cholesterol, phospholipids, sphingolipids, glycolipids, and proteins. Pumpkin = pump K+ in. Ouabain (a cardiac glycoside) inhibits by binding to K+ site. Cardiac glycosides (digoxin and digitoxin) directly inhibit the Na+-K+ ATPase, which leads to indirect inhibition of Na+/Ca2+ exchange • [Ca2+]i  cardiac contractility. Cleavage of procollagen Cand N-terminals Formation of cross-links (stabilized by lysyl oxidase) Collagen fber

1	Synthesis—translation of collagen α chains (preprocollagen)—usually Gly-X-Y (X and Y are proline or lysine). Collagen is 1⁄3 glycine; glycine content of collagen is less variable than that of lysine and proline. Hydroxyproline is used for lab quantification of collagen. Hydroxylation—hydroxylation of specific proline and lysine residues. Requires vitamin C; deficiency • scurvy. Glycosylation—glycosylation of pro-α-chain hydroxylysine residues and formation of procollagen via hydrogen and disulfide bonds (triple helix of 3 collagen α chains). Problems forming triple helix • osteogenesis imperfecta. Exocytosis—exocytosis of procollagen into extracellular space. Proteolytic processing—cleavage of disulfide-rich terminal regions of procollagen insoluble tropocollagen. Cross-linking—reinforcement of many staggered tropocollagen molecules by covalent lysine-hydroxylysine cross-linkage (by copper-containing lysyl oxidase) to make collagen fibrils. Problems with cross-linking

1	Menkes disease. Genetic bone disorder (brittle bone disease) caused by a variety of gene defects (most commonly COL1A1 and COL1A2). Most common form is autosomal dominant with  production of otherwise normal type I collagen. Manifestations include: Multiple fractures and bone deformities after minimal trauma (eg, during birth) B due to the translucent connective tissue over choroidal veins Some forms have tooth abnormalities, including opalescent teeth that wear easily due to lack of dentin (dentinogenesis imperfecta) May be confused with child abuse. Treat with bisphosphonates to  fracture risk. Patients can’t BITE: Faulty collagen synthesis causing hyperextensible skin A , hypermobile joints B , and tendency to bleed (easy bruising). Multiple types. Inheritance and severity vary. Can be autosomal dominant or recessive. May be associated with joint dislocation, berry and aortic aneurysms, organ rupture. Hypermobility type (joint instability): most common type.

1	Hypermobility type (joint instability): most common type. Classical type (joint and skin symptoms): caused by a mutation in type V collagen (eg, COL5A1, COL5A2). Vascular type (fragile tissues including vessels [eg, aorta], muscles, and organs that are prone to rupture [eg, gravid uterus]): mutations in type III procollagen (eg, COL3A1). X-linked recessive connective tissue disease caused by impaired copper absorption and transport due to defective Menkes protein (ATP7A, vs ATP7B in Wilson disease). Low copper levels (vs high levels in Wilson disease). Leads to  activity of lysyl oxidase (copper is a necessary cofactor) • defective collagen. Results in brittle, “kinky” hair, growth retardation, hypotonia, • risk of cerebral aneurysms.

1	Elastin Stretchy protein within skin, lungs, large arteries, elastic ligaments, vocal cords, ligamenta flava (connect vertebrae  relaxed and stretched conformations). Rich in nonhydroxylated proline, glycine, and lysine residues, vs the hydroxylated residues of collagen. Tropoelastin with fibrillin scaffolding. Cross-linking takes place extracellularly and gives elastin its elastic properties. Broken down by elastase, which is normally inhibited by α1-antitrypsin. α1-Antitrypsin deficiency results in unopposed elastase activity, which can cause COPD. Changes with aging:  dermal collagen and elastin,  synthesis of collagen fibrils; cross-linking remains normal. Marfan syndrome—autosomal dominant (with variable expression) connective tissue disorder affecting skeleton, heart, and eyes. FBN1 gene mutation on chromosome 15 (fifteen) results in defective fibrillin, a glycoprotein that forms a sheath around elastin. Findings: tall with long extremities; pectus carinatum (more specific)

1	FBN1 gene mutation on chromosome 15 (fifteen) results in defective fibrillin, a glycoprotein that forms a sheath around elastin. Findings: tall with long extremities; pectus carinatum (more specific) or pectus excavatum A ; hypermobile joints; long, tapering fingers and toes (arachnodactyly); cystic medial necrosis of aorta; aortic root aneurysm rupture or dissection (most common cause of death); mitral valve prolapse. Subluxation of lenses, typically upward and temporally (vs downward and medially in homocystinuria).

1	Polymerase chain Molecular biology lab procedure used to amplify a desired fragment of DNA. Useful as a diagnostic reaction tool (eg, neonatal HIV, herpes encephalitis). Denaturation—DNA is heated to ~95°C to separate the strands. Annealing—Sample is cooled to ~55°C. DNA primers, a heat-stable DNA polymerase (Taq), and deoxynucleotide triphosphates (dNTPs) are added. DNA primers anneal to the specific sequence to be amplified on each strand. Elongation—Temperature is increased to ~72°C. DNA polymerase attaches dNTPs to the strand to replicate the sequence after each primer. Heating and cooling cycles continue until the DNA sample size is sufficient.

1	Heating and cooling cycles continue until the DNA sample size is sufficient. CRISPR/Cas9 A genome editing tool derived from bacteria. Consists of a guide RNA (gRNA) , which is complementary to a target DNA sequence, and an endonuclease (Cas9), which makes a single-or double-strand break at the target site . Break imperfectly repaired by nonhomologous end joining (NHEJ)  accidental frameshift mutations (“knock-out”) , or a donor DNA sequence can be added to fill in the gap using homology-directed repair (HDR) . Not used clinically. Potential applications include removing virulence factors from pathogens, replacing disease-causing alleles of genes with healthy variants, and specifically targeting tumor cells. Southern blot 1. DNA sample is enzymatically cleaved into smaller pieces, which are separated on a gel by electrophoresis, and then transferred to a filter. 2. Filter is exposed to radiolabeled DNA probe that recognizes and anneals to its complementary strand.

1	2. Filter is exposed to radiolabeled DNA probe that recognizes and anneals to its complementary strand. 3. Resulting double-stranded, labeled piece of DNA is visualized when filter is exposed to film. Northern blot Similar to Southern blot, except that an RNA sample is electrophoresed. Useful for studying DRoP: mRNA levels, which are reflective of gene Southern = DNAexpression. Northern = RNAWestern blot Sample protein is separated via gel electrophoresis Western = Protein and transferred to a membrane. Labeled antibody is used to bind to relevant protein. Southwestern blot Identifies DNA-binding proteins (eg, c-Jun, c-Fos [leucine zipper motif]) using labeled double-stranded DNA probes.

1	Southwestern blot Identifies DNA-binding proteins (eg, c-Jun, c-Fos [leucine zipper motif]) using labeled double-stranded DNA probes. Flow cytometry Laboratory technique to assess size, granularity, and protein expression (immunophenotype) of individual cells in a sample. Cells are tagged with antibodies specific to Commonly used in workup of hematologic abnormalities (eg, leukemia, paroxysmal nocturnal hemoglobinuria, fetal RBCs in mother’s blood) and immunodeficiencies (eg, CD4+ cell count in HIV). Fluorescent label Antibody Anti-CD3 Ab Laser makes label fuoresce Laser Detector 104 103 Anti-CD8 Ab Fluorescence is detected; labeled cells are counted Cell surface or intracellular proteins. Antibodies are then tagged with a unique fluorescent dye. Sample is analyzed one cell at a time by focusing a laser on the cell and measuring light scatter and intensity of fluorescence.

1	Data are plotted either as histogram (one measure) or scatter plot (any two measures, as shown). In illustration: Cells in left lower quadrant ⊝ for both CD8 and CD3. CD8-expressing cells also express CD3. Cells in left upper quadrant ⊕ for CD3 and 100 ⊝ for CD8. 100 101 102 103 104 CD8 CD8 and CD3. and ⊝ for CD3. In this example, right lower quadrant is empty because all Microarrays Thousands of nucleic acid sequences are arranged in grids on glass or silicon. DNA or RNA probes are hybridized to the chip, and a scanner detects the relative amounts of complementary binding. Used to profile gene expression levels of thousands of genes simultaneously to study certain diseases and treatments. Able to detect single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) for a variety of applications including genotyping, clinical genetic testing, forensic analysis, cancer mutations, and genetic linkage analysis.

1	Karyotyping Colchicine is added to cultured cells to halt chromosomes in metaphase. Chromosomes are stained, ordered, and numbered according to morphology, size, arm-length ratio, and banding pattern (arrows in A point to extensive abnormalities in a cancer cell). Can be performed on a sample of blood, bone marrow, amniotic fluid, or placental tissue. Used to diagnose chromosomal imbalances (eg, autosomal trisomies, sex chromosome disorders). Fluorescent DNA or RNA probe binds to specific gene site of interest on chromosomes (arrows in A point to abnormalities in a cancer cell, whose karyotype is seen above; each fluorescent color represents a chromosome-specific probe). Used for specific localization of genes and direct visualization of chromosomal anomalies at the molecular level. Microdeletion—no fluorescence on a chromosome compared to fluorescence at the same locus on the second copy of that chromosome.

1	Microdeletion—no fluorescence on a chromosome compared to fluorescence at the same locus on the second copy of that chromosome. Translocation—fluorescence signal that corresponds to one chromosome is found in a different chromosome (two white arrows in A show fragments of chromosome 17 that have translocated to chromosome 19). Duplication—a second copy of a chromosome, resulting in a trisomy or tetrasomy (two blue arrows show duplicated chromosomes 8, resulting in a tetrasomy). Molecular cloning Production of a recombinant DNA molecule in a bacterial host. Steps: 1. Isolate eukaryotic mRNA (post-RNA processing) of interest. 2. Add reverse transcriptase (an RNA-dependent DNA polymerase) to produce complementary DNA (cDNA, lacks introns). 3. Insert cDNA fragments into bacterial plasmids containing antibiotic resistance genes. 4. Transform (insert) recombinant plasmid into bacteria. 5. Surviving bacteria on antibiotic medium produce cloned DNA (copies of cDNA).

1	4. Transform (insert) recombinant plasmid into bacteria. 5. Surviving bacteria on antibiotic medium produce cloned DNA (copies of cDNA). Cre-lox system Can inducibly manipulate genes at specific developmental points (eg, to study a gene whose deletion causes embryonic death). RNA interference Process whereby small non-coding RNA molecules target mRNAs to inhibit gene expression. Codominance Both alleles contribute to the phenotype of the Blood groups A, B, AB; α1-antitrypsin heterozygote. deficiency; HLA groups. Variable expressivity Patients with the same genotype have varying 2 patients with neurofibromatosis type 1 (NF1) phenotypes. may have varying disease severity. Anticipation Increased severity or earlier onset of disease in Trinucleotide repeat diseases (eg, Huntington succeeding generations. disease). Presence of genetically distinct cell lines in the same individual.

1	Presence of genetically distinct cell lines in the same individual. Somatic mosaicism—mutation arises from mitotic errors after fertilization and propagates through multiple tissues or organs. Gonadal mosaicism—mutation only in egg or sperm cells. If parents and relatives do not have the disease, suspect gonadal (or germline) mosaicism. McCune-Albright syndrome—due to Gs-protein activating mutation. Presents with unilateral café-au-lait spots A with ragged edges, polyostotic fibrous dysplasia (bone is replaced by collagen and fibroblasts), and at least one endocrinopathy (eg, precocious puberty). Lethal if mutation occurs before fertilization (affecting all cells), but survivable in patients with mosaicism. Locus heterogeneity Mutations at different loci can produce a similar Albinism. phenotype. Allelic heterogeneity Different mutations in the same locus β-thalassemia. produce the same phenotype.

1	Locus heterogeneity Mutations at different loci can produce a similar Albinism. phenotype. Allelic heterogeneity Different mutations in the same locus β-thalassemia. produce the same phenotype. Heteroplasmy Presence of both normal and mutated mtDNA passed from mother to all children. mtDNA, resulting in variable expression in mitochondrially inherited disease. Uniparental disomy Offspring receives 2 copies of a chromosome from 1 parent and no copies from the other parent. HeterodIsomy (heterozygous) indicates a meiosis I error. IsodIsomy (homozygous) indicates a meiosis II error or postzygotic chromosomal duplication of one of a pair of chromosomes, and loss of the other of the original pair. Uniparental is euploid (correct number of chromosomes). Most occurrences of uniparental disomy (UPD)  normal phenotype. Consider isodisomy in an individual manifesting a recessive disorder when only one parent is a carrier. Examples: Prader-Willi and Angelman syndromes.

1	If p and q represent the frequencies of alleles A and a, respectively, in a population, then p + q = 1: p2 = frequency of homozygosity for allele A q2 = frequency of homozygosity for allele a 2pq = frequency of heterozygosity (carrier frequency, if an autosomal recessive disease) Therefore, the sum of the frequencies of these genotypes is p2 + 2pq + q2 = 1. The frequency of an X-linked recessive disease in males = q and in females = q2. Hardy-Weinberg law assumptions include: No mutation occurring at the locus Natural selection is not occurring If a population is in Hardy-Weinberg equilibrium, then the values of p and q remain constant from generation to generation. Disorders of imprinting Imprinting—one gene copy is silenced by methylation, and only the other copy is expressed  parent-of-origin effects. Modes of inheritance Autosomal dominant Often due to defects in structural genes. Many generations, both males and females are affected. AaaaAaAaaaaa

1	Modes of inheritance Autosomal dominant Often due to defects in structural genes. Many generations, both males and females are affected. AaaaAaAaaaaa Often pleiotropic (multiple apparently unrelated effects) and variably expressive (different between individuals). Family history crucial to diagnosis. With one affected (heterozygous) parent, on average, 1/2 of children affected. Autosomal recessive With 2 carrier (heterozygous) parents, on average: Often due to enzyme deficiencies. Usually seen ¼ of children will be affected (homozygous), in only 1 generation. Commonly more severe 1/2 of children will be carriers, and ¼ of than dominant disorders; patients often present children will be neither affected nor carriers. in childhood. Aa  risk in consanguineous families. Unaffected individual with affected sibling has 2/3 probability of being a carrier.

1	Aa  risk in consanguineous families. Unaffected individual with affected sibling has 2/3 probability of being a carrier. X-linked recessive Sons of heterozygous mothers have a 50% Commonly more severe in males. Females chance of being affected. No male-to-male usually must be homozygous to be affected. transmission. Skips generations. transmit to all daughters but no sons. XXXXXXXYXYXYXXXXXXXYXYXY X-linked dominant Transmitted through both parents. Mothers Examples: fragile X syndrome, Alport syndrome, transmit to 50% of daughters and sons; fathers hypophosphatemic rickets (also called X-linked hypophosphatemia)—phosphate wasting at proximal tubule • rickets-like presentation. Mitochondrial Transmitted only through the mother. All inheritance offspring of affected females may show signs of disease. Variable expression in a population or even within a family due to heteroplasmy.

1	Mitochondrial myopathies—rare disorders; often present with myopathy, lactic acidosis, and CNS disease, eg, MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). 2° to failure in oxidative phosphorylation. Muscle biopsy often shows “ragged red fibers” (due to accumulation of diseased mitochondria in the subsarcolemma of the muscle fiber). Leber hereditary optic neuropathy—cell death in optic nerve neurons  subacute bilateral vision loss in teens/young adults, 90% males. Usually permanent. = a˜ected female.

1	Leber hereditary optic neuropathy—cell death in optic nerve neurons  subacute bilateral vision loss in teens/young adults, 90% males. Usually permanent. = a˜ected female. Autosomal dominant Achondroplasia, autosomal dominant polycystic kidney disease, familial adenomatous polyposis, diseases familial hypercholesterolemia, hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu syndrome), hereditary spherocytosis, Huntington disease, Li-Fraumeni syndrome, Marfan syndrome, multiple endocrine neoplasias, myotonic muscular dystrophy, neurofibromatosis type 1 (von Recklinghausen disease), neurofibromatosis type 2, tuberous sclerosis, von Hippel-Lindau disease.

1	Autosomal recessive Oculocutaneous albinism, autosomal recessive polycystic kidney disease (ARPKD), cystic diseases fibrosis, Friedreich ataxia, glycogen storage diseases, hemochromatosis, Kartagener syndrome, mucopolysaccharidoses (except Hunter syndrome), phenylketonuria, sickle cell anemia, sphingolipidoses (except Fabry disease), thalassemias, Wilson disease. GENETICS Autosomal recessive; defect in CFTR gene on chromosome 7; commonly a deletion of Phe508. Most common lethal genetic disease in Caucasian population.

1	GENETICS Autosomal recessive; defect in CFTR gene on chromosome 7; commonly a deletion of Phe508. Most common lethal genetic disease in Caucasian population. PATHOPHYSIOlOGY CFTR encodes an ATP-gated Cl− channel that secretes Cl− in lungs and GI tract, and reabsorbs Cl− in sweat glands. Most common mutation  misfolded protein  protein retained in RER and not transported to cell membrane, causing  Cl− (and H2O) secretion;  intracellular Cl− results in compensatory • Na+ reabsorption via epithelial Na+ channels (ENaC)  H2O reabsorption  abnormally thick mucus secreted into lungs and GI tract.  Na+ reabsorption also causes more negative transepithelial potential difference.

1	DIAGNOSIS  Cl− concentration in pilocarpine-induced sweat test is diagnostic. Can present with contraction alkalosis and hypokalemia (ECF effects analogous to a patient taking a loop diuretic) because of ECF H2O/Na+ losses via sweating and concomitant renal K+/H+ wasting. • immunoreactive trypsinogen (newborn screening). COMPlICATIONS Recurrent pulmonary infections (eg, S aureus [infancy and early childhood], P aeruginosa [adulthood], allergic bronchopulmonary aspergillosis [ABPA]), chronic bronchitis and bronchiectasis  reticulonodular pattern on CXR, opacification of sinuses. Pancreatic insufficiency, malabsorption with steatorrhea, fat-soluble vitamin deficiencies (A, D, E, K), biliary cirrhosis, liver disease. Meconium ileus in newborns. Infertility in men (absence of vas deferens, spermatogenesis may be unaffected) and subfertility in women (amenorrhea, abnormally thick cervical mucus). Nasal polyps, clubbing of nails.

1	TREATMENT Multifactorial: chest physiotherapy, albuterol, aerosolized dornase alfa (DNase), and hypertonic saline facilitate mucus clearance. Azithromycin used as anti-inflammatory agent. Ibuprofen slows disease progression. Pancreatic enzyme replacement therapy for pancreatic insufficiency. In patients with Phe508 deletion: combination of lumacaftor (corrects misfolded proteins and improves their transport to cell surface) and ivacaftor (opens Cl– channels • improved chloride transport). Ornithine transcarbamylase deficiency, Fabry disease, Wiskott-Aldrich syndrome, Ocular albinism, G6PD deficiency, Hunter syndrome, Bruton agammaglobulinemia, Hemophilia A and B, Lesch-Nyhan syndrome, Duchenne (and Becker) muscular dystrophy. X-inactivation (lyonization)—one copy of female X chromosome forms a transcriptionally inactive Barr body. Female carriers variably affected depending on the pattern of inactivation of the X chromosome carrying the mutant vs normal gene.

1	X-linked disorder typically due to frameshift deletions or nonsense mutations  truncated or absent dystrophin protein • progressive myofiber damage. Weakness begins in pelvic girdle muscles and progresses superiorly. Pseudohypertrophy of calf muscles due to fibrofatty replacement of muscle A . Waddling gait. Onset before 5 years of age. Dilated cardiomyopathy is common cause of death. Gowers sign—patient uses upper extremities to help stand up. Classically seen in Duchenne muscular dystrophy, but also seen in other muscular dystrophies and inflammatory myopathies (eg, polymyositis). Females with Turner syndrome (45,XO) are more likely to have an X-linked recessive disorder. Duchenne = deleted dystrophin.

1	Females with Turner syndrome (45,XO) are more likely to have an X-linked recessive disorder. Duchenne = deleted dystrophin. Dystrophin gene (DMD) is the largest protein-coding human gene  chance of spontaneous mutation. Dystrophin helps anchor muscle fibers, primarily in skeletal and cardiac muscle. It connects the intracellular cytoskeleton (actin) to the transmembrane proteins αand β-dystroglycan, which are connected to the extracellular matrix (ECM). Loss of dystrophin • myonecrosis.  CK and aldolase; genetic testing confirms diagnosis. Rett syndrome Sporadic disorder seen almost exclusively in girls (affected males die in utero or shortly after birth). Most cases are caused by de novo mutation of MECP2 on X chromosome. Symptoms of Rett syndrome usually appear between ages 1–4 and are characterized by regression (Retturn) in motor, verbal, and cognitive abilities; ataxia; seizures; growth failure; and stereotyped hand-wringing.

1	Myotonic dystrophy (CTG)n AD Cataracts, Toupee (early balding in men), Gonadal atrophy in men, reduced fertility in women Fragile X syndrome (CGG)n XD Chin (protruding), Giant Gonads Findings: intellectual disability, flat facies, prominent epicanthal folds, single palmar crease, incurved 5th finger, gap between 1st 2 toes, duodenal atresia, Hirschsprung disease, congenital heart disease (eg, ASD), Brushfield spots. Associated with early-onset Alzheimer disease (chromosome 21 codes for amyloid precursor protein),  risk of AML/ALL. 95% of cases due to meiotic nondisjunction ( with advanced maternal age; from 1:1500 in women < 20 to 1:25 in women > 45 years old). 4% of cases due to unbalanced Robertsonian translocation, most typically between chromosomes 14 and 21. Only 1% of cases are due to postfertilization mitotic error. Incidence 1:700. Drinking age (21). Most common viable chromosomal disorder and most common cause of genetic intellectual disability.

1	Incidence 1:700. Drinking age (21). Most common viable chromosomal disorder and most common cause of genetic intellectual disability. translucency and hypoplastic nasal bone. Markers for Down syndrome are HI up: hCG, • inhibin. The 5 A’s of Down syndrome: Findings: severe intellectual disability, rocker-bottom feet, microphthalmia, microcephaly, cleft liP/Palate, holoProsencephaly, Polydactyly, cutis aPlasia, congenital heart (Pump) disease, Polycystic kidney disease, omphalocele. Death usually occurs by age 1. Incidence 1:15,000. Puberty (13). Defect in fusion of prechordal mesoderm  midline defects.

1	Incidence 1:15,000. Puberty (13). Defect in fusion of prechordal mesoderm  midline defects. Genetic disorders by chromosome 3 von Hippel-Lindau disease, renal cell carcinoma 4 ADPKD (PKD2), achondroplasia, Huntington disease 5 Cri-du-chat syndrome, familial adenomatous polyposis 7 Williams syndrome, cystic fibrosis 9 Friedreich ataxia, tuberous sclerosis (TSC1) 11 Wilms tumor, β-globin gene defects (eg, sickle cell disease, β-thalassemia), MEN1 13 Patau syndrome, Wilson disease, retinoblastoma (RB1), BRCA2 15 Prader-Willi syndrome, Angelman syndrome, Marfan syndrome 16 ADPKD (PKD1), α-globin gene defects (eg, α-thalassemia), tuberous sclerosis (TSC2) 17 Neurofibromatosis type 1, BRCA1, TP53 22 Neurofibromatosis type 2, DiGeorge syndrome (22q11) X Fragile X syndrome, X-linked agammaglobulinemia, Klinefelter syndrome (XXY)

1	X Fragile X syndrome, X-linked agammaglobulinemia, Klinefelter syndrome (XXY) Chromosomal translocation that commonly involves chromosome pairs 21, 22, 13, 14, and 15. One of the most common types of translocation. Occurs when the long arms of 2 acrocentric chromosomes (chromosomes with centromeres near their ends) fuse at the centromere and the 2 short arms are lost. Balanced translocations normally do not cause any abnormal phenotype. Unbalanced translocations can result in miscarriage, stillbirth, and chromosomal imbalance (eg, Down syndrome, Patau syndrome). Cri-du-chat syndrome Cri du chat = cry of the cat. Congenital deletion on short arm of chromosome 5 (46,XX or XY, 5p−). Findings: microcephaly, moderate to severe intellectual disability, high-pitched crying/meowing, epicanthal folds, cardiac abnormalities (VSD).

1	Williams syndrome Congenital microdeletion of long arm of chromosome 7 (deleted region includes elastin gene). Findings: distinctive “elfin” facies A , intellectual disability, hypercalcemia, well-developed verbal skills, extreme friendliness with strangers, cardiovascular problems (eg, supravalvular aortic stenosis, renal artery stenosis). Think Will Ferrell in Elf. Vitamins: water soluble B1 (thiamine: TPP) B2 (riboflavin: FAD, FMN) B3 (niacin: NAD+) B5 (pantothenic acid: CoA) B6 (pyridoxine: PLP) B7 (biotin) B9 (folate) B12 (cobalamin) C (ascorbic acid) All wash out easily from body except B12 and B9 (folate). B12 stored in liver for ~ 3–4 years. B9 stored in liver for ~ 3–4 months. B-complex deficiencies often result in dermatitis, glossitis, and diarrhea. Can be coenzymes (eg, ascorbic acid) or precursors to coenzymes (eg, FAD, NAD+). Vitamin A Includes retinal, retinol, retinoic acid.

1	Can be coenzymes (eg, ascorbic acid) or precursors to coenzymes (eg, FAD, NAD+). Vitamin A Includes retinal, retinol, retinoic acid. DEFICIENCY Night blindness (nyctalopia); dry, scaly skin (xerosis cutis); corneal squamous metaplasia • Bitot spots (keratin debris; foamy appearance on conjunctiva A ); corneal degeneration (keratomalacia); immunosuppression. EXCESS Acute toxicity—nausea, vomiting, vertigo, and Isotretinoin is teratogenic. blurred vision. Chronic toxicity—alopecia, dry skin (eg, scaliness), hepatic toxicity and enlargement, arthralgias, and idiopathic intracranial hypertension. Teratogenic (cleft palate, cardiac abnormalities), therefore a ⊝ pregnancy test and two forms of contraception are required before isotretinoin (vitamin A derivative) is prescribed. Vitamin B1 Also called thiamine.

1	Vitamin B1 Also called thiamine. DEFICIENCY Impaired glucose breakdown  ATP depletion worsened by glucose infusion; highly aerobic tissues (eg, brain, heart) are affected first. In alcoholic or malnourished patients, give thiamine before dextrose to  risk of precipitating Wernicke encephalopathy. Diagnosis made by  in RBC transketolase activity following vitamin B1 administration. chronic alcohol consumption; presents with confabulation, personality changes, memory loss (permanent). Wernicke-Korsakoff syndrome—damage to medial dorsal nucleus of thalamus, mammillary bodies. Presentation is combination of Wernicke encephalopathy and Korsakoff syndrome. Dry beriberi—polyneuropathy, symmetric muscle wasting. Wet beriberi—high-output cardiac failure (dilated cardiomyopathy), edema. DEFICIENCY Cheilosis (inflammation of lips, scaling and The 2 C’s of B2. fissures at the corners of the mouth), Corneal vascularization. Vitamin B3 Also called niacin, nicotinic acid.

1	DEFICIENCY Cheilosis (inflammation of lips, scaling and The 2 C’s of B2. fissures at the corners of the mouth), Corneal vascularization. Vitamin B3 Also called niacin, nicotinic acid. FUNCTION Constituent of NAD+, NADP+ (used in redox NAD derived from Niacin (B3 ≈ 3 ATP). reactions). Derived from tryptophan. Synthesis requires vitamins B2 and B6. Used to treat dyslipidemia; lowers levels of VLDL and raises levels of HDL. DEFICIENCY Glossitis. Severe deficiency leads to pellagra, which can also be caused by Hartnup disease, malignant carcinoid syndrome (• tryptophan metabolism), and isoniazid ( vitamin B6). Symptoms of pellagra: Diarrhea, Dementia (also hallucinations), Dermatitis (C3/C4 dermatome circumferential “broad collar” rash [Casal necklace], hyperpigmentation of sun-exposed limbs A ). The 3 D’s of B3.

1	A ). The 3 D’s of B3. Hartnup disease—autosomal recessive. Deficiency of neutral amino acid (eg, tryptophan) transporters in proximal renal tubular cells and on enterocytes • neutral aminoaciduria and  absorption from the gut  tryptophan for conversion to niacin  pellagra-like symptoms. Treat with high- protein diet and nicotinic acid. Deficiency of vitamin B3 • pellagra. EXCESS Facial flushing (induced by prostaglandin, not Excess of vitamin B3 • podagra. histamine; can avoid by taking aspirin with niacin), hyperglycemia, hyperuricemia. Vitamin B5 Also called pantothenic acid. FUNCTION Essential component of coenzyme A (CoA, B5 is “pento”thenic acid. a cofactor for acyl transfers) and fatty acid synthase. DEFICIENCY Dermatitis, enteritis, alopecia, adrenal insufficiency. Vitamin B6 Also called pyridoxine.

1	DEFICIENCY Dermatitis, enteritis, alopecia, adrenal insufficiency. Vitamin B6 Also called pyridoxine. FUNCTION Converted to pyridoxal phosphate (PLP), a cofactor used in transamination (eg, ALT and AST), decarboxylation reactions, glycogen phosphorylase. Synthesis of glutathione, cystathionine, heme, niacin, histamine, and neurotransmitters including serotonin, epinephrine, norepinephrine (NE), dopamine, and GABA. DEFICIENCY Convulsions, hyperirritability, peripheral neuropathy (deficiency inducible by isoniazid and oral contraceptives), sideroblastic anemia (due to impaired hemoglobin synthesis and iron excess). Vitamin B7 Also called biotin. a 1-carbon group): Pyruvate carboxylase: pyruvate (3C) • oxaloacetate (4C) Acetyl-CoA carboxylase: acetyl-CoA (2C) • malonyl-CoA (3C) Propionyl-CoA carboxylase: propionyl-CoA (3C)  methylmalonyl-CoA (4C)

1	Pyruvate carboxylase: pyruvate (3C) • oxaloacetate (4C) Acetyl-CoA carboxylase: acetyl-CoA (2C) • malonyl-CoA (3C) Propionyl-CoA carboxylase: propionyl-CoA (3C)  methylmalonyl-CoA (4C) DEFICIENCY Relatively rare. Dermatitis, enteritis, alopecia. “Avidin in egg whites avidly binds biotin.” Caused by long-term antibiotic use or excessive ingestion of raw egg whites. DEFICIENCY Macrocytic, megaloblastic anemia; hypersegmented PMNs; paresthesias and subacute combined degeneration (degeneration of dorsal columns, lateral corticospinal tracts, and spinocerebellar tracts) due to abnormal myelin. Associated with  serum homocysteine and methylmalonic acid levels, along with 2° folate deficiency. Prolonged deficiency  irreversible nerve damage. Vitamin C Also called ascorbic acid.

1	Vitamin C Also called ascorbic acid. (eg, sprue, enteritis, Diphyllobothrium latum, achlorhydria, bacterial overgrowth, alcohol excess), lack of intrinsic factor (eg, pernicious anemia, gastric bypass surgery), absence of terminal ileum (surgical resection, eg, for Crohn disease), certain drugs (eg, metformin), or insufficient intake (eg, veganism). Anti-intrinsic factor antibodies diagnostic for pernicious anemia. Folate supplementation can mask the hematologic symptoms of B12 deficiency, but not the neurologic symptoms. Fatty acids with odd number of carbons, branched-chain amino acids CH3 to anabolic pathways Methylmalonyl-CoA EXCESS Nausea, vomiting, diarrhea, fatigue, calcium oxalate nephrolithiasis. Can  iron toxicity in predisposed individuals by increasing dietary iron absorption (ie, can worsen hereditary hemochromatosis or transfusion-related iron overload).

1	REGUlATION  PTH, • Ca2+,  PO43– • 1,25-(OH)2D3 production. 1,25-(OH)2D3 feedback inhibits its own production.  PTH • Ca2+ reabsorption and  PO43– reabsorption in the kidney. DEFICIENCY Rickets in children (deformity, such as genu varum “bowlegs” A ), osteomalacia in adults (bone pain and muscle weakness), hypocalcemic tetany. Caused by malabsorption,  sun exposure, poor diet, chronic kidney disease (CKD), advanced liver disease. Give oral vitamin D to breastfed infants. Deficiency is exacerbated by pigmented skin, premature birth. EXCESS Hypercalcemia, hypercalciuria, loss of appetite, stupor. Seen in granulomatous diseases ( activation of vitamin D by epithelioid macrophages). Vitamin E Includes tocopherol, tocotrienol. FUNCTION Antioxidant (protects RBCs and membranes from free radical damage). EXCESS Risk of enterocolitis in infants. High-dose supplementation may alter metabolism of vitamin K  enhanced anticoagulant effects of warfarin.

1	EXCESS Risk of enterocolitis in infants. High-dose supplementation may alter metabolism of vitamin K  enhanced anticoagulant effects of warfarin. Vitamin K Includes phytomenadione, phylloquinone, phytonadione, menaquinone. FUNCTION Mineral essential for the activity of 100+ enzymes. Important in the formation of zinc fingers (transcription factor motif). DEFICIENCY Delayed wound healing, suppressed immunity, male hypogonadism,  adult hair (axillary, facial, pubic), dysgeusia, anosmia. Associated with acrodermatitis enteropathica ( A , defect in intestinal zinc absorption). May predispose to alcoholic cirrhosis. Kwashiorkor Protein malnutrition resulting in skin lesions, edema due to  plasma oncotic pressure, liver malfunction (fatty change due to  apolipoprotein synthesis). Clinical picture is small child with swollen abdomen A . Kwashiorkor results from protein- deficient MEALS: Malnutrition Edema Anemia Liver (fatty) Skin lesions (eg, hyperkeratosis,

1	A . Kwashiorkor results from protein- deficient MEALS: Malnutrition Edema Anemia Liver (fatty) Skin lesions (eg, hyperkeratosis, Marasmus Malnutrition not causing edema. Diet is deficient in calories but no nutrients are entirely absent. Marasmus results in Muscle wasting B . Fomepizole—blocks alcohol DH; antidote For Overdoses of Methanol or Ethylene glycol. • discouraging drinking. NAD+ is the limiting reagent. Alcohol dehydrogenase operates via zero-order kinetics. Ethanol metabolism  NADH/ NAD+ ratio in liver, causing: Lactic acidosis— pyruvate conversion to lactate Fasting hypoglycemia— gluconeogenesis due to of OAA to malate Ketoacidosis—diversion of acetyl-CoA into ketogenesis rather than TCA cycle Hepatosteatosis— • conversion of DHAP to glycerol-3-P 4B , which combines with glycerol-3-P to synthesize triglycerides  NADH/NAD+ ratio inhibits TCA cycle  acetyl-CoA used in ketogenesis (• ketoacidosis), lipogenesis (• hepatosteatosis).

1	Mitochondria Fatty acid oxidation (β-oxidation), acetyl-CoA production, TCA cycle, oxidative phosphorylation, ketogenesis. Cytoplasm Glycolysis, HMP shunt, and synthesis of cholesterol (SER), proteins (ribosomes, RER), fatty acids, and nucleotides. Both Heme synthesis, Urea cycle, Gluconeogenesis. HUGs take two (both). Enzyme terminology An enzyme’s name often describes its function. For example, glucokinase is an enzyme that catalyzes the phosphorylation of glucose using a molecule of ATP. The following are commonly used enzyme descriptors. Kinase Catalyzes transfer of a phosphate group from a high-energy molecule (usually ATP) to a substrate (eg, phosphofructokinase). Phosphorylase Adds inorganic phosphate onto substrate without using ATP (eg, glycogen phosphorylase). Phosphatase Removes phosphate group from substrate (eg, fructose-1,6-bisphosphatase). Dehydrogenase Catalyzes oxidation-reduction reactions (eg, pyruvate dehydrogenase).

1	Phosphatase Removes phosphate group from substrate (eg, fructose-1,6-bisphosphatase). Dehydrogenase Catalyzes oxidation-reduction reactions (eg, pyruvate dehydrogenase). Hydroxylase Adds hydroxyl group (−OH) onto substrate (eg, tyrosine hydroxylase). Carboxylase Transfers CO2 groups with the help of biotin (eg, pyruvate carboxylase). Mutase Relocates a functional group within a molecule (eg, vitamin B12–dependent methylmalonyl-CoA mutase). Synthase/synthetase Joins two molecules together using a source of energy (eg, ATP, acetyl-CoA, nucleotide sugar). Rate-determining enzymes of metabolic processes Summary of pathways

1	T B T T B B Glycogen UDP-glucose Glucose-1-phosphate Glucose Glucose-6-phosphate 6-phosphogluconolactone Fructose-6-phosphate Fructose-1,6-bisphosphate Glyceraldehyde-3-P DHAP 1,3-bisphosphoglycerate 3-phosphoglycerate 2-phosphoglycerate Phosphoenolpyruvate (PEP) Pyruvate Alanine Acetyl-CoA Glyceraldehyde Ribulose-5-phosphate Fructose-1-phosphate Fructose NH3 + CO2 Carbamoyl phosphate Citrulline Aspartate Argininosuccinate Urea cycle Ornithine Urea H2O Arginine Fumarate Oxaloacetate Malate TCA cycle Succinate Citrate Isocitrate ˜-ketoglutarate Succinyl-CoA Methylmalonyl-CoA Propionyl-CoA Odd-chain fatty acids, isoleucine, valine, methionine, threonine Acetoacetate ˜-hydroxybutyrate Mevalonate Galactose Galactose-1-phosphate HMP shunt Fructose metabolism Lipid metabolism Galactose metabolism Gluconeogenesis Ketogenesis Glycolysis Protein metabolism Glycogenesis / glycogenolysis Lactate Acetoacetyl-CoA HMG-CoA Malonyl-CoA Triglycerides Glycerol Cholesterol B12 Irreversible, important

1	Gluconeogenesis Ketogenesis Glycolysis Protein metabolism Glycogenesis / glycogenolysis Lactate Acetoacetyl-CoA HMG-CoA Malonyl-CoA Triglycerides Glycerol Cholesterol B12 Irreversible, important point of regulation Requires thiamine cofactor (TPP)Requires biotin cofactor B T Fatty acids #Hexokinase/glucokinase Glucose-6-phosphatase (von Gierke disease) Glucose-6-phosphate dehydrogenase Transketolase Pyruvate kinase Pyruvate dehydrogenase HMG-CoA reductase Pyruvate carboxylase PEP carboxykinase Citrate synthase Triose phosphate isomerase Phosphofructokinase-1 Fructose-1,6-bisphosphatase Fructokinase (essential fructosuria) Aldolase B (fructose intolerance) Aldolase B (liver), A (muscle) Isocitrate dehydrogenase ˜-ketoglutarate dehydrogenase Ornithine transcarbamylase Propionyl-CoA carboxylase Carbamoyl phosphate synthetase I Galactokinase (mild galactosemia) Galactose-1-phosphate uridyltransferase (severe galactosemia)

1	NADH, NADPH, FADH2 Electrons CoA, lipoamide Acyl groups Phosphorylation of glucose to yield glucose-6-phosphate is catalyzed by glucokinase in the liver and hexokinase in other tissues. Hexokinase sequesters glucose in tissues, where it is used even when glucose concentrations are low. At high glucose concentrations, glucokinase helps to store glucose in liver. Glucose-6-P Glucose-6-P ⊝ hexokinase. Fructose-6-P ⊝ glucokinase. Fructose-6-P Fructose-1,6-BP AMP ⊕, fructose-2,6-bisphosphate ⊕. ATP ⊝, citrate ⊝. Regulation by fructose-2,6bisphosphate Fructose bisphosphatase-2 (FBPase-2) and phosphofructokinase-2 (PFK-2) are the same bifunctional enzyme whose function is reversed by phosphorylation by protein kinase A. Fructose-1,6-BP Fructose-2,6-BP Fasting state:  glucagon  cAMP  protein FaBian the Peasant (FBP) has to work hard kinase A  FBPase-2,  PFK-2, less when starving. glycolysis, more gluconeogenesis.

1	Fructose-2,6-BP Fasting state:  glucagon  cAMP  protein FaBian the Peasant (FBP) has to work hard kinase A  FBPase-2,  PFK-2, less when starving. glycolysis, more gluconeogenesis. Fed state:  insulin  cAMP  protein Prince FredericK (PFK) works only when fed. kinase A  FBPase-2,  PFK-2, more glycolysis, less gluconeogenesis. Mitochondrial enzyme complex linking glycolysis and TCA cycle. Differentially regulated in fed (active)/fasting (inactive) states. Reaction: pyruvate + NAD+ + CoA  acetyl-CoA + CO2 + NADH. Contains 3 enzymes requiring 5 cofactors: 1. 2. 3. CoA (B5, pantothenic acid) 4. FAD (B2, riboflavin) 5. NAD+ (B3, niacin) Activated by: • NAD+/NADH ratio,  ADP • Ca2+ The complex is similar to the α-ketoglutarate dehydrogenase complex (same cofactors, similar substrate and action), which converts α-ketoglutarate  succinyl-CoA (TCA cycle). The Lovely Coenzymes For Nerds.

1	The Lovely Coenzymes For Nerds. Arsenic inhibits lipoic acid. Arsenic poisoning clinical findings: imagine a vampire (pigmentary skin changes, skin cancer), vomiting and having diarrhea, running away from a cutie (QT prolongation) with garlic breath. FINDINGS Neurologic defects, lactic acidosis,  serum alanine starting in infancy. TREATMENT  intake of ketogenic nutrients (eg, high fat content or • lysine and leucine). Functions of different pyruvate metabolic pathways (and their associated cofactors): Alanine aminotransferase (B6): alanine carries amino groups to the liver from muscle Pyruvate carboxylase (biotin): oxaloacetate can replenish TCA cycle or be used in gluconeogenesis Pyruvate dehydrogenase (B1, B2, B3, B5, lipoic acid): transition from glycolysis to the TCA cycle Lactic acid dehydrogenase (B3): end of anaerobic glycolysis (major pathway in RBCs, WBCs, kidney medulla, lens, testes, and cornea) Also called Krebs cycle. Pyruvate  acetyl-CoA produces 1 NADH, 1 CO2.

1	Also called Krebs cycle. Pyruvate  acetyl-CoA produces 1 NADH, 1 CO2. The TCA cycle produces 3 NADH, 1 FADH2, 2 CO2, 1 GTP per acetyl-CoA = 10 ATP/ acetyl-CoA (2× everything per glucose). TCA cycle reactions occur in the mitochondria. α-ketoglutarate dehydrogenase complex requires the same cofactors as the pyruvate dehydrogenase complex (vitamins B1, B2, B3, B5, lipoic acid). Citrate Is Krebs’ Starting Substrate For Making Oxaloacetate. Electron transport NADH electrons from glycolysis enter mitochondria via the malate-aspartate or glycerol-3chain and oxidative phosphate shuttle. FADH2 electrons are transferred to complex II (at a lower energy level than phosphorylation NADH). The passage of electrons results in the formation of a proton gradient that, coupled to oxidative phosphorylation, drives the production of ATP.

1	Complex I Complex II Complex III Complex IV Complex V Rotenone Antimycin A Azide, Oligomycin (succinate dehydrogenase) Cyanide, CO2 NADH NAD+ FADH2 FAD 1/2O2 + 2H+ H2O H+H+ H+ H+ CoQ Cyto-chrome c space2,4-Dinitrophenol Aspirin overdose 1 NADH  2.5 ATP; 1 FADH2  1.5 ATP. Gluconeogenesis, irreversible enzymes Pathway Produces Fresh Glucose. Pyruvate carboxylase In mitochondria. Pyruvate  oxaloacetate. Requires biotin, ATP. Activated by acetyl-CoA. Phosphoenolpyruvate In cytosol. Oxaloacetate Requires GTP. carboxykinase • phosphoenolpyruvate. Fructose-1,6-In cytosol. Fructose-1,6-bisphosphate Citrate ⊕, AMP ⊝, fructose 2,6-bisphosphate ⊝. bisphosphatase  fructose-6-phosphate. Glucose-6-In ER. Glucose-6-phosphate  glucose. phosphatase

1	Fructose-1,6-In cytosol. Fructose-1,6-bisphosphate Citrate ⊕, AMP ⊝, fructose 2,6-bisphosphate ⊝. bisphosphatase  fructose-6-phosphate. Glucose-6-In ER. Glucose-6-phosphate  glucose. phosphatase Occurs primarily in liver; serves to maintain euglycemia during fasting. Enzymes also found in kidney, intestinal epithelium. Deficiency of the key gluconeogenic enzymes causes hypoglycemia. (Muscle cannot participate in gluconeogenesis because it lacks glucose-6-phosphatase). Odd-chain fatty acids yield 1 propionyl-CoA during metabolism, which can enter the TCA cycle (as succinyl-CoA), undergo gluconeogenesis, and serve as a glucose source. Even-chain fatty acids cannot produce new glucose, since they yield only acetyl-CoA equivalents.

1	Also called HMP shunt. Provides a source of NADPH from abundantly available glucose-6-P (NADPH is required for reductive reactions, eg, glutathione reduction inside RBCs, fatty acid and cholesterol biosynthesis). Additionally, this pathway yields ribose for nucleotide synthesis. Two distinct phases (oxidative and nonoxidative), both of which occur in the cytoplasm. No ATP is used or produced. Sites: lactating mammary glands, liver, adrenal cortex (sites of fatty acid or steroid synthesis), RBCs. Transketolase, B˜ Fructose Nucleotide 1,6-bisphosphate synthesis

1	Sites: lactating mammary glands, liver, adrenal cortex (sites of fatty acid or steroid synthesis), RBCs. Transketolase, B˜ Fructose Nucleotide 1,6-bisphosphate synthesis NADPH is necessary to keep glutathione reduced, which in turn detoxifies free radicals and peroxides.  NADPH in RBCs leads to hemolytic anemia due to poor RBC defense against oxidizing agents (eg, fava beans, sulfonamides, nitrofurantoin, primaquine/ chloroquine, antituberculosis drugs). Infection (most common cause) can also precipitate hemolysis; inflammatory response produces free radicals that diffuse into RBCs, causing oxidative damage. X-linked recessive disorder; most common human enzyme deficiency; more prevalent among African Americans.  malarial resistance. Heinz bodies—denatured globin chains precipitate within RBCs due to oxidative stress. Bite cells—result from the phagocytic removal of Heinz bodies by splenic macrophages. Think, “Bite into some Heinz ketchup.” Disorders of fructose metabolism

1	Bite cells—result from the phagocytic removal of Heinz bodies by splenic macrophages. Think, “Bite into some Heinz ketchup.” Disorders of fructose metabolism Essential fructosuria Involves a defect in fructokinase. Autosomal recessive. A benign, asymptomatic condition (fructokinase deficiency is kinder), since fructose is not trapped in cells. Hexokinase becomes 1° pathway for converting fructose to fructose-6-phosphate. Symptoms: fructose appears in blood and urine. Disorders of fructose metabolism cause milder symptoms than analogous disorders of galactose metabolism.

1	Hereditary deficiency of aldolase B. Autosomal recessive. Fructose-1-phosphate accumulates, causing a  in available phosphate, which results in inhibition of glycogenolysis and gluconeogenesis. Symptoms present following consumption of fruit, juice, or honey. Urine dipstick will be ⊝ (tests for glucose only); reducing sugar can be detected in the urine (nonspecific test for inborn errors of carbohydrate metabolism). Symptoms: hypoglycemia, jaundice, cirrhosis, vomiting. Treatment:  intake of fructose, sucrose (glucose + fructose), and sorbitol (metabolized to fructose). Disorders of galactose metabolism Hereditary deficiency of galactokinase. Galactitol accumulates if galactose is present in diet. Fructose is to Aldolase B as Galactose is to UridylTransferase (FAB GUT). The more serious defects lead to PO43− depletion.

1	Fructose is to Aldolase B as Galactose is to UridylTransferase (FAB GUT). The more serious defects lead to PO43− depletion. Sorbitol An alternative method of trapping glucose in the cell is to convert it to its alcohol counterpart, sorbitol, via aldose reductase. Some tissues then convert sorbitol to fructose using sorbitol dehydrogenase; tissues with an insufficient amount/activity of this enzyme are at risk of intracellular sorbitol accumulation, causing osmotic damage (eg, cataracts, retinopathy, and peripheral neuropathy seen with chronic hyperglycemia in diabetes). High blood levels of galactose also result in conversion to the osmotically active galactitol via aldose reductase. Liver, Ovaries, and Seminal vesicles have both enzymes (they LOSe sorbitol). Lens has primarily aldose reductase. Retina, Kidneys, and Schwann cells have only aldose reductase (LuRKS).

1	Lens has primarily aldose reductase. Retina, Kidneys, and Schwann cells have only aldose reductase (LuRKS). Lactase deficiency Insufficient lactase enzyme  dietary lactose intolerance. Lactase functions on the intestinal brush border to digest lactose (in milk and milk products) into glucose and galactose. Primary: age-dependent decline after childhood (absence of lactase-persistent allele), common in people of Asian, African, or Native American descent. Secondary: loss of intestinal brush border due to gastroenteritis (eg, rotavirus), autoimmune disease. Congenital lactase deficiency: rare, due to defective gene. Stool demonstrates  pH and breath shows  hydrogen content with lactose hydrogen breath test. Intestinal biopsy reveals normal mucosa in patients with hereditary lactose intolerance. FINDINGS Bloating, cramps, flatulence, osmotic diarrhea. TREATMENT Avoid dairy products or add lactase pills to diet; lactose-free milk.

1	FINDINGS Bloating, cramps, flatulence, osmotic diarrhea. TREATMENT Avoid dairy products or add lactase pills to diet; lactose-free milk. Amino acids Only l-amino acids are found in proteins. Essential PVT TIM HaLL: Phenylalanine, Valine, Tryptophan, Threonine, Isoleucine, Methionine, Histidine, Leucine, Lysine. Glucogenic: Methionine, histidine, valine. We met his valentine, she is so sweet (glucogenic). Glucogenic/ketogenic: Isoleucine, phenylalanine, threonine, tryptophan. Ketogenic: Leucine, Lysine. The onLy pureLy ketogenic amino acids. Acidic Aspartic acid, glutamic acid. Negatively charged at body pH. Basic Arginine, histidine, lysine. Arginine is most basic. Histidine has no charge at body pH. Arginine and histidine are required during periods of growth. Arginine and lysine are  in histones which bind negatively charged DNA. His lys (lies) are basic. Transport of ammonia by alanine

1	Transport of ammonia by alanine Hyperammonemia Can be acquired (eg, liver disease) or hereditary Treatment: limit protein in diet. (eg, urea cycle enzyme deficiencies). May be given to  ammonia levels: Presents with flapping tremor (eg, asterixis), • Lactulose to acidify GI tract and trap NH4+ slurring of speech, somnolence, vomiting, for excretion. cerebral edema, blurring of vision. • Antibiotics (eg, rifaximin, neomycin) to  NH3 changes relative amounts of • ammoniagenic bacteria. α-ketoglutarate, glutamate, GABA, and • Benzoate, phenylacetate, or phenylbutyrate glutamine to favor  glutamine. CNS toxicity react with glycine or glutamine, forming may involve  GABA, • α-ketoglutarate, TCA products that are excreted renally. cycle inhibition, and cerebral edema due to glutamine-induced osmotic shifts. NH NH

1	Most common urea cycle disorder. X-linked recessive (vs other urea cycle enzyme deficiencies, which are autosomal recessive). Interferes with the body’s ability to eliminate ammonia. Often evident in the first few days of life, but may present later. Excess carbamoyl phosphate is converted to orotic acid (part of the pyrimidine synthesis pathway). Findings:  orotic acid in blood and urine,  BUN, symptoms of hyperammonemia. No megaloblastic anemia (vs orotic aciduria). , B6 Tryptophan BH4, B6 Serotonin Due to • phenylalanine hydroxylase or tetrahydrobiopterin (BH4) cofactor (malignant PKU). Tyrosine becomes essential. phenylalanine  phenyl ketones in urine. Findings: intellectual disability, growth retardation, seizures, fair complexion, eczema, musty body odor. Treatment: • phenylalanine and  tyrosine in diet, tetrahydrobiopterin supplementation.

1	Findings: intellectual disability, growth retardation, seizures, fair complexion, eczema, musty body odor. Treatment: • phenylalanine and  tyrosine in diet, tetrahydrobiopterin supplementation. Maternal PKU—lack of proper dietary therapy during pregnancy. Findings in infant: microcephaly, intellectual disability, growth retardation, congenital heart defects. Autosomal recessive. Incidence ≈ 1:10,000. Screening occurs 2–3 days after birth (normal at birth because of maternal enzyme during fetal life). Phenyl ketones—phenylacetate, phenyllactate, and phenylpyruvate. Disorder of aromatic amino acid metabolism  musty body odor. PKU patients must avoid the artificial sweetener aspartame, which contains phenylalanine. Autosomal recessive. Presentation: vomiting, poor feeding, urine smells like maple syrup/burnt sugar. Causes severe CNS defects, intellectual disability, death. I Love Vermont maple syrup from maple trees (with B1ranches).

1	I Love Vermont maple syrup from maple trees (with B1ranches). Blocked degradation of branched amino acids (Isoleucine, Leucine, Valine) due to • branched-chain α-ketoacid dehydrogenase (B1). Causes • α-ketoacids in the blood, especially those of leucine. Treatment: restriction of isoleucine, leucine, valine in diet, and thiamine supplementation. Congenital deficiency of homogentisate oxidase in the degradative pathway of tyrosine to fumarate • pigment-forming homogentisic acid builds up in tissue A . Autosomal recessive. Usually benign. Findings: bluish-black connective tissue, ear cartilage, and sclerae (ochronosis); urine turns black on prolonged exposure to air. May have debilitating arthralgias (homogentisic acid toxic to cartilage). Causes (all autosomal recessive): • Cystathionine synthase deficiency (treatment:  methionine, • cysteine,  B6, B12, and folate in diet) •  affinity of cystathionine synthase for pyridoxal phosphate (treatment:  B6 and

1	Methionine synthase (homocysteine methyltransferase) deficiency (treatment: Methylenetetrahydrofolate reductase (MTHFR) deficiency (treatment:  folate in diet) All forms result in excess homocysteine. HOMOCYstinuria: • Homocysteine in urine, Osteoporosis, Marfanoid habitus, Ocular changes (downward and inward lens subluxation), Cardiovascular effects (thrombosis and atherosclerosis • stroke and MI), kYphosis, intellectual disability, fair complexion. In homocystinuria, lens subluxes “down and in” (vs Marfan, “up and fans out”). Hereditary defect of renal PCT and intestinal amino acid transporter that prevents reabsorption of Cystine, Ornithine, Lysine, and Arginine (COLA). Excess cystine in the urine can lead to recurrent precipitation of hexagonal cystine stones A . Treatment: urinary alkalinization (eg, potassium citrate, acetazolamide) and chelating agents (eg, penicillamine)  solubility of cystine stones; good hydration.

1	Treatment: urinary alkalinization (eg, potassium citrate, acetazolamide) and chelating agents (eg, penicillamine)  solubility of cystine stones; good hydration. Autosomal recessive. Common (1:7000). Urinary cyanide-nitroprusside test is diagnostic. Cystine is made of 2 cysteines connected by a disulfide bond. Organic acidemias Most commonly present in infancy with poor feeding, vomiting, hypotonia, high anion gap metabolic acidosis, hepatomegaly, seizures. Organic acid accumulation: Inhibits gluconeogenesis  fasting blood glucose levels, • ketoacidosis  high anion gap metabolic acidosis Glycogen Branches have α-(1,6) bonds; linkages have α-(1,4) bonds. Skeletal muscle Glycogen undergoes glycogenolysis  glucose-1-phosphate  glucose-6-phosphate, which is rapidly metabolized during exercise.

1	Skeletal muscle Glycogen undergoes glycogenolysis  glucose-1-phosphate  glucose-6-phosphate, which is rapidly metabolized during exercise. Hepatocytes Glycogen is stored and undergoes glycogenolysis to maintain blood sugar at appropriate levels. Glycogen phosphorylase liberates glucose-1-phosphate residues off branched glycogen until 4 glucose units remain on a branch. Then 4-α-d-glucanotransferase (debranching enzyme ) moves 3 of the 4 glucose units from the branch to the linkage. Then α-1,6-glucosidase (debranching enzyme ) cleaves off the last residue, liberating glucose. “Limit dextrin” refers to the two to four residues remaining on a branch after glycogen phosphorylase (˜-1,6-glucosidase) ˜-1,4-glucosidase Note: A small amount of glycogen is degraded in lysosomes by α-1,4-glucosidase (acid maltase). has already shortened it. Gaucher disease Most common.

1	Note: A small amount of glycogen is degraded in lysosomes by α-1,4-glucosidase (acid maltase). has already shortened it. Gaucher disease Most common. Hepatosplenomegaly, pancytopenia, osteoporosis, avascular necrosis of femur, bone crises, Gaucher cells C (lipid-laden macrophages resembling crumpled tissue paper). (β-glucosidase); treat with recombinant glucocerebrosidase C hepatosplenomegaly, foam cells (lipid-laden macrophages) D , Niemann-Pick disease Progressive neurodegeneration, “cherry-red” spot on macula A . Sphingomyelinase Sphingomyelin AR D Mucopolysaccharidoses Hurler syndrome Developmental delay, gargoylism, α-l-iduronidase Heparan sulfate, AR airway obstruction, corneal clouding, dermatan sulfate hepatosplenomegaly. Hunter syndrome Mild Hurler + aggressive behavior, no Iduronate-2-sulfatase Heparan sulfate, XR corneal clouding. dermatan sulfate No man picks (Niemann-Pick) his nose with his sphinger (sphingomyelinase).

1	No man picks (Niemann-Pick) his nose with his sphinger (sphingomyelinase). aggressively aim for the X (X-linked recessive).  incidence of Tay-Sachs, Niemann-Pick, some forms of Gaucher disease in Ashkenazi Jews. Sphingolipidoses Tay-Sachs disease A Progressive neurodegeneration, developmental delay, hyperreflexia, hyperacusis, “cherry-red” spot on macula A , lysosomes with onion skin, no hepatosplenomegaly (vs Niemann-Pick). HeXosaminidase A (“TAy-SaX”) GM2 ganglioside AR Fabry disease B Early: triad of episodic peripheral neuropathy, angiokeratomas B , hypohidrosis. Late: progressive renal failure, cardiovascular disease. α-galactosidase A Ceramide trihexoside (globotriaosylce-ramide) XR Metachromatic leukodystrophy Central and peripheral demyelination with ataxia, dementia. Arylsulfatase A Cerebroside sulfate AR (palmitate, a 16C FA)

1	Fatty acid synthesis requires transport of citrate from mitochondria to cytosol. Predominantly occurs in liver, lactating mammary glands, and adipose tissue. Long-chain fatty acid (LCFA) degradation requires carnitine-dependent transport into the mitochondrial matrix. “SYtrate” = SYnthesis. CARnitine = CARnage of fatty acids. Systemic 1° carnitine deficiency—no cellular uptake of carnitine  no transport of LCFAs into mitochondria  toxic accumulation of LCFAs in the cytosol. Causes weakness, hypotonia, hypoketotic hypoglycemia, dilated cardiomyopathy. Medium-chain acyl-CoA dehydrogenase deficiency— ability to break down fatty acids into acetyl-CoA • accumulation of fatty acyl carnitines in the blood with hypoketotic hypoglycemia. Causes vomiting, lethargy, seizures, coma, liver dysfunction, hyperammonemia. Can lead to sudden death in infants or children. Treat by avoiding fasting.

1	Ketone bodies In the liver, fatty acids and amino acids are metabolized to acetoacetate and β-hydroxybutyrate (to be used in muscle and brain). In prolonged starvation and diabetic ketoacidosis, oxaloacetate is depleted for gluconeogenesis. In alcoholism, excess NADH shunts oxaloacetate to malate. All of these processes lead to a buildup of acetyl-CoA, which is shunted into ketone body synthesis. Ketone bodies: acetone, acetoacetate, β-hydroxybutyrate. Breath smells like acetone (fruity odor). Urine test for ketones can detect acetoacetate, but not β-hydroxybutyrate. RBCs cannot utilize ketones; they strictly use glucose. HMG-CoA lyase for ketone production. HMG-CoA reductase for cholesterol synthesis. (liver) Fatty acids, amino acids Acetoacetate AcetoacetateAcetoneATP˜-hydroxybutyrate Expired by lungs TCA cycle (eg, skeletal muscle) ˜-hydroxybutyrate Acetoacetyl-CoA Acetoacetate ˜-hydroxybutyrateAcetyl-CoA HMG-CoA 2 Acetyl-CoA Duration of exercise

1	Duration of exercise Fasting and starvation Priorities are to supply sufficient glucose to the brain and RBCs and to preserve protein. Fed state (after a Glycolysis and aerobic respiration. meal) Fasting (between Hepatic glycogenolysis (major); hepatic meals) gluconeogenesis, adipose release of FFA (minor). Starvation days 1–3 Blood glucose levels maintained by: Adipose release of FFA Muscle and liver, which shift fuel use from glucose to FFA Insulin stimulates storage of lipids, proteins, and glycogen. Glucagon and epinephrine stimulate use of fuel reserves. Glycogen reserves depleted after day 1. RBCs lack mitochondria and therefore cannot use ketones. Weeks of starvation Carbohydrate Protein Fat 1 2 3 4 5 6 7 80 2 10 12 0 Starvation after day 3 Adipose stores (ketone bodies become the main source of energy for the brain). After these are depleted, vital protein degradation accelerates, leading to organ failure and death. Amount of excess stores determines survival time.

1	tissue lactate and alanine, and from CoA (from odd-chain FFA—the only to the liver via Apo E 7 Endocytosis of LDL PCSK9 Degrades LDL receptor  serum LDL. Inhibition  LDL receptor recycling  serum LDL. Transfer of cholesteryl CETP esters to VLDL, IDL, LDL HDL Mediates reverse cholesterol transport from peripheral tissues to liver. Acts as a repository for apolipoproteins C and E (which are needed for chylomicron and VLDL metabolism). Secreted from both liver and intestine. Alcohol • synthesis. HDL is Healthy.

1	Abetalipoproteinemia Autosomal recessive. Mutation in gene that encodes microsomal transfer protein (MTP). Chylomicrons, VLDL, LDL absent. Deficiency in ApoB-48, ApoB-100. Affected infants present with severe fat malabsorption, steatorrhea, failure to thrive. Later manifestations include retinitis pigmentosa, spinocerebellar degeneration due to vitamin E deficiency, progressive ataxia, acanthocytosis. Intestinal biopsy shows lipid-laden enterocytes. Treatment: restriction of long-chain fatty acids, large doses of oral vitamin E. “I hate to disappoint you, but my rubber lips are immune to your charms.” —Batman & Robin “The fully engaged heart is the antibody for the infection of violence.”

1	“I hate to disappoint you, but my rubber lips are immune to your charms.” —Batman & Robin “The fully engaged heart is the antibody for the infection of violence.” Learning the components of the immune system and their roles in host defense at the cellular level is essential for both the understanding of disease pathophysiology and clinical practice. Know the immune mechanisms of responses to vaccines. Both congenital and acquired immunodeficiencies are very testable. Cell surface markers are high yield for understanding immune cell interactions and for laboratory diagnosis. Know the roles and functions of major cytokines and chemokines. Lymph node A 2° lymphoid organ that has many afferents, 1 or more efferents. Encapsulated, with trabeculae B . Functions are nonspecific filtration by macrophages, circulation of B and T cells, and immune response activation.

1	Lymph node cluster Cervical, supraclavicular Head and neck Upper respiratory tract infection Infectious mononucleosis Kawasaki disease Mediastinal Trachea and esophagus Pulmonary TB Sarcoidosis (bilateral) 1° lung cancer Granulomatous disease Axillary Upper limb, breast, skin above umbilicus Mastitis Metastasis (especially breast cancer) Hilar Lungs Celiac Liver, stomach, spleen, pancreas, upper duodenum Superior mesenteric Lower duodenum, jejunum, ileum, colon to splenic fexure Inferior mesenteric Colon from splenic fexure to upper rectum Para-aortic Testes, ovaries, kidneys, uterus Metastasis Internal iliac External iliac Cervix, superior bladder, and body of uterus Lower rectum to anal canal (above pectinate line), bladder, vagina (middle third), cervix, prostate Superfcial inguinal Palpable lymph node Non-palpable lymph node Popliteal Right lymphatic duct drains right side of body above diaphragm into junction of the right Dorsolateral foot, posterior calf Lateral foot/leg

1	Palpable lymph node Non-palpable lymph node Popliteal Right lymphatic duct drains right side of body above diaphragm into junction of the right Dorsolateral foot, posterior calf Lateral foot/leg cellulitis Anal canal (below pectinate line), skin below umbilicus (except popliteal area), scrotum, vulva Sexually transmitted infections Medial foot/leg cellulitis (superfcial inguinal) Mesenteric lymphadenitis Typhoid fever Ulcerative colitis Celiac disease Area of body drained Associated pathology

1	Thoracic duct drains below the diaphragm and left thorax and upper limb into junction of left subclavian and internal jugular veins (rupture of thoracic duct can cause chylothorax) Spleen Located in LUQ of abdomen, anterolateral to left kidney, protected by 9th-11th ribs. Sinusoids are long, vascular channels in red pulp (red arrows in A ) with fenestrated “barrel hoop” basement membrane. T cells are found in the periarteriolar lymphatic sheath (PALS) within the white pulp (white arrows in A ). B cells are found in follicles within the white pulp. The marginal zone, in between the red pulp and white pulp, contains macrophages and specialized B cells, and is where antigen-presenting cells (APCs) capture blood-borne antigens for recognition by lymphocytes. Splenic macrophages remove encapsulated bacteria.

1	Splenic macrophages remove encapsulated bacteria. Splenic dysfunction (eg, postsplenectomy state, sickle cell disease autosplenectomy):  IgM  complement activation  C3b opsonization  susceptibility to encapsulated organisms. Postsplenectomy blood findings: Thrombocytosis (loss of sequestration and removal) Lymphocytosis (loss of sequestration) Vaccinate patients undergoing splenectomy or with splenic dysfunction against encapsulated organisms (pneumococci, Hib, meningococci). Located in the anterosuperior mediastinum. Site of T-cell differentiation and maturation. Encapsulated. Thymus epithelium is derived from Third pharyngeal pouch (endoderm), whereas thymic lymphocytes are of mesodermal origin. Cortex is dense with immature T cells; Medulla is pale with Mature T cells and Hassall corpuscles A containing epithelial reticular cells. B , involutes by age 3 years. seen in some immunodeficiencies (eg, SCID, DiGeorge syndrome).

1	B , involutes by age 3 years. seen in some immunodeficiencies (eg, SCID, DiGeorge syndrome). Thymoma—neoplasm of thymus. Associated with myasthenia gravis, superior vena cava syndrome, pure red cell aplasia, Good syndrome. Innate vs adaptive immunity HLA subtypes associated with diseases Functions of natural Lymphocyte member of innate immune system. killer cells Use perforin and granzymes to induce apoptosis of virally infected cells and tumor cells. Activity enhanced by IL-2, IL-12, IFN-α, and IFN-β. Induced to kill when exposed to a nonspecific activation signal on target cell and/or to an absence of MHC I on target cell surface. Also kills via antibody-dependent cell-mediated cytotoxicity (CD16 binds Fc region of bound IgG, activating the NK cell). Major functions of B and T cells

1	Major functions of B and T cells B cells Humoral immunity. Recognize and present antigen—undergo somatic hypermutation to optimize antigen specificity. Produce antibody—differentiate into plasma cells to secrete specific immunoglobulins. Maintain immunologic memory—memory B cells persist and accelerate future response to antigen. T cells Cell-mediated immunity. CD4+ T cells help B cells make antibodies and produce cytokines to recruit phagocytes and activate other leukocytes. CD8+ T cells directly kill virus-infected and tumor cells via perforin and granzymes (similar to NK cells). Delayed cell-mediated hypersensitivity (type IV). Acute and chronic cellular organ rejection. Rule of 8: MHC II × CD4 = 8; MHC I × CD8 = 8. Differentiation of T cells Cytotoxic T cell Helper T cell IL-6 TGF-˜, IL-1, IL-6 IFN-° IFN-°, IL-4 TGF-˜, IL-2 IFN-°, IL-12 IL-4, IL-10 IL-2, IL-4 IFN-°, IL-2 Activate macrophages and cytotoxic T cells IL-4, IL-5, IL-6 Activate eosinophils, IL-10, IL-13 ˙ IgE

1	IFN-°, IL-2 Activate macrophages and cytotoxic T cells IL-4, IL-5, IL-6 Activate eosinophils, IL-10, IL-13 ˙ IgE IL-17, IL-21, IL-22 Induce neutrophilic infammation TGF-˜, IL-10, IL-35 Prevent autoimmunity (maintain tolerance) CD4 Tand B-cell activation APCs: B cells, dendritic cells, Langerhans cells, macrophages. Two signals are required for T-cell activation, B-cell activation, and class switching. Th-cell activation as above. R B-cell receptor–mediated endocytosis. Exogenous antigen is presented on MHC II and recognized by TCR on Th cell. CD40 receptor on B cell binds CD40 ligand (CD40L) on Th cell. Th cells secrete cytokines that determine Ig class switching of B cells. B cells are activated, undergo class switching and affinity maturation, and begin producing antibodies.

1	Antibody structure and function Fab (containing the variable/hypervariable regions) consisting of light (L) and heavy (H) chains recognizes antigens. Fc region of IgM and IgG fixes complement. Heavy chain contributes to Fc and Fab regions. Light chain contributes only to Fab region. VHJHJLDVLHinge Complement binding Macrophage binding Fc region C = Constant V = Variable SS = Disulfde bond Fab regionHypervariable regionsCLCLCH1CH1CH2CH3CH2CH3SS SS SS SS Light chain Heavy chain Epitope Fab: Fragment, antigen binding Complement binding • Determines idiotype: unique antigen-binding pocket; only 1 antigenic specificity expressed per B cell Fc (5C’s): • Confers (determines) isotype (IgM, IgD, etc) Generation of antibody diversity (antigen independent) 1. Random recombination of VJ (light-chain) or V(D)J (heavy-chain) genes 2. Random addition of nucleotides to DNA during recombination by terminal deoxynucleotidyl transferase (TdT) 3.

1	Random recombination of VJ (light-chain) or V(D)J (heavy-chain) genes 2. Random addition of nucleotides to DNA during recombination by terminal deoxynucleotidyl transferase (TdT) 3. Random combination of heavy chains with light chains Generation of antibody specificity (antigen dependent) 4. 5. ComplementactivationMembrane attack complex (MAC) Antibody activates complement, enhancing opsonization and lysis C3b OpsonizationNeutralizationAntibody prevents bacterial adherence Antibody promotes phagocytosis IgG Main antibody in 2° response to an antigen. Most abundant isotype in serum. Fixes complement, opsonizes bacteria, neutralizes bacterial toxins and viruses. Only isotype that crosses the placenta (provides infants with passive immunity that starts to wane after birth). “IgGGreets the Growing fetus.”

1	Prevents attachment of bacteria and viruses to mucous membranes; does not fix complement. Monomer (in circulation) or dimer (with J chain when secreted). Crosses epithelial cells by transcytosis. Produced in GI tract (eg, by Peyer patches) and protects against gut infections (eg, Giardia). Most produced antibody overall, but has lower serum concentrations. Released into secretions (tears, saliva, mucus) and breast milk. Picks up secretory component from epithelial cells, which protects the Fc portion from luminal proteases. IgM Produced in the 1° (immediate) response to an antigen. Fixes complement. Antigen receptor on the surface of B cells. Monomer on B cell, pentamer with J chain when secreted. Pentamer enables avid binding to antigen while humoral response evolves. IgD Unclear function. Found on surface of many B cells and in serum.

1	IgD Unclear function. Found on surface of many B cells and in serum. IgE Binds mast cells and basophils; cross-links when exposed to allergen, mediating immediate (type I) hypersensitivity through release of inflammatory mediators such as histamine. Contributes to immunity to parasites by activating eosinophils. Opsonins—C3b and IgG are the two 1° Opsonin (Greek) = to prepare for eating. opsonins in bacterial defense; enhance phagocytosis. C3b also helps clear immune complexes. Inhibitors—decay-accelerating factor (DAF, aka CD55) and C1 esterase inhibitor help prevent complement activation on self cells (eg, RBCs). Spontaneous and microbial surfaces Amplifes generation of C3b called C2a but is now referred to as C2b. C3 C3b B D Bb C3 C3aC3aC3bBb (C3 convertase) C3C2b C4b C2 C1 C4 C4a(C3 convertase) C1 C5 C5aC5b C6-C9 C3bBb3b (C5 convertase) C4b2b3b (C5 convertase) C4b2b C3bcomplex C1-like * * *Historically, the larger fragment of C2 was Lysis, (C5b-9) (eg, mannose)

1	Lysis, (C5b-9) (eg, mannose) C1 esterase inhibitor Causes hereditary angioedema due to unregulated activation of kallikrein  bradykinin. deficiency Characterized by  C4 levels. ACE inhibitors are contraindicated (also  bradykinin). Paroxysmal nocturnal A defect in the PIGA gene preventing the formation of glycosylphosphatidylinositol (GPI) anchors for hemoglobinuria complement inhibitors, such as decay-accelerating factor (DAF/CD55) and membrane inhibitor of reactive lysis (MIRL/CD59). Causes complement-mediated intravascular hemolysis  haptoglobin, dark urine A . Important cytokines Acute (IL-1, IL-6, TNF-α), then recruit (IL-8, IL-12).

1	A . Important cytokines Acute (IL-1, IL-6, TNF-α), then recruit (IL-8, IL-12). Respiratory burst Also called oxidative burst. Involves the activation of the phagocyte NADPH oxidase complex (eg, in neutrophils, monocytes), which utilizes O2 as a substrate. Plays an important role in the immune response  rapid release of reactive oxygen species (ROS). NADPH plays a role in both the creation and neutralization of ROS. Myeloperoxidase contains a blue-green, heme-containing pigment that gives sputum its color. Phagocytes of patients with CGD can utilize H2O2 generated by invading organisms and convert it to ROS. Patients are at  risk for infection by catalase ⊕ species (eg, S aureus, Aspergillus) capable of neutralizing their own H2O2, leaving phagocytes without ROS for fighting infections.

1	Pyocyanin of P aeruginosa generates ROS to kill competing pathogens. Oxidative burst also leads to K+ influx, which releases lysosomal enzymes. Lactoferrin is a protein found in secretory fluids and neutrophils that inhibits microbial growth via iron chelation. Interferons IFN-α, IFN-β, IFN-γ mechanISm A part of innate host defense, interferons interfere with both RNA and DNA viruses. Cells infected with a virus synthesize these glycoproteins, which act on local cells, priming them for viral defense by downregulating protein synthesis to resist potential viral replication and by upregulating MHC expression to facilitate recognition of infected cells. Also play a major role in activating antitumor immunity. clInIcal uSe Chronic HBV, Kaposi sarcoma, hairy cell leukemia, condyloma acuminatum, renal cell carcinoma, malignant melanoma, multiple sclerosis, chronic granulomatous disease.

1	clInIcal uSe Chronic HBV, Kaposi sarcoma, hairy cell leukemia, condyloma acuminatum, renal cell carcinoma, malignant melanoma, multiple sclerosis, chronic granulomatous disease. adVerSe eFFectS Flu-like symptoms, depression, neutropenia, myopathy, interferon-induced autoimmunity. T cells TCR (binds antigen-MHC complex) CD3 (associated with TCR for signal transduction) CD28 (binds B7 on APC) Helper T cells CD4, CD40L, CXCR4/CCR5 (co-receptors for HIV) Anergy State during which a cell cannot become activated by exposure to its antigen. T and B cells become anergic when exposed to their antigen without costimulatory signal (signal 2). Another mechanism of self-tolerance. Vaccination Induces an active immune response (humoral and/or cellular) to specific pathogens. Hypersensitivity types Four types (ABCD): Anaphylactic and Atopic (type I), AntiBody-mediated (type II), Immune Complex (type III), Delayed (cell-mediated, type IV). Types I, II, and III are all antibody-mediated.

1	Antibodies bind to cell-surface antigens  cellular destruction, inflammation, and cellular dysfunction. Cellular destruction—cell is opsonized (coated) by antibodies, leading to either: Phagocytosis and/or activation of complement system. NK cell killing (antibody-dependent cellular cytotoxicity). Inflammation—binding of antibodies to cell surfaces  activation of complement system and Fc receptor-mediated inflammation. Cellular dysfunction—antibodies bind to cell surface receptors  abnormal blockade or activation of downstream process. Direct Coombs test—detects antibodies attached directly to the RBC surface. Indirect Coombs test—detects presence of unbound antibodies in the serum Examples: Hemolytic disease of the newborn Examples: Hyperacute transplant rejection Examples: Immune complex—antigen-antibody (mostly IgG) complexes activate complement, which attracts neutrophils; neutrophils release lysosomal enzymes.

1	Examples: Hyperacute transplant rejection Examples: Immune complex—antigen-antibody (mostly IgG) complexes activate complement, which attracts neutrophils; neutrophils release lysosomal enzymes. Can be associated with vasculitis and systemic manifestations. Serum sickness—the prototypic immune complex disease. Antibodies to foreign proteins are produced and 1–2 weeks later, antibody-antigen complexes form and deposit in tissues  complement activation • inflammation and tissue damage. Arthus reaction—a local subacute immune complex-mediated hypersensitivity reaction. Intradermal injection of antigen into a presensitized (has circulating IgG) individual leads to immune complex formation in the skin (eg, enhanced local reaction to a booster vaccination). Characterized by edema, necrosis, and activation of complement. In type III reaction, imagine an immune complex as 3 things stuck together: antigenantibody-complement. Examples:

1	In type III reaction, imagine an immune complex as 3 things stuck together: antigenantibody-complement. Examples: Poststreptococcal glomerulonephritis Fever, urticaria, arthralgia, proteinuria, lymphadenopathy occur 1–2 weeks after antigen exposure. Serum sickness-like reactions are associated with some drugs (may act as haptens, eg, penicillin) and infections (eg, hepatitis B). Two mechanisms, each involving T cells: 1. Direct cell cytotoxicity: CD8+ cytotoxic T cells kill targeted cells. 2. Inflammatory reaction: effector CD4+ T cells recognize antigen and release inflammation-inducing cytokines (shown in illustration). Response does not involve antibodies (vs types I, II, and III). Examples: contact dermatitis (eg, poison ivy, nickel allergy) and graft-versus-host disease. Tests: PPD for TB infection; patch test for contact dermatitis; Candida skin test for T cell immune function. 4T’s: T cells, Transplant rejections, TB skin tests, Touching (contact dermatitis).

1	Tests: PPD for TB infection; patch test for contact dermatitis; Candida skin test for T cell immune function. 4T’s: T cells, Transplant rejections, TB skin tests, Touching (contact dermatitis). Fourth (type) and last (delayed). Allergic/ Type I hypersensitivity Within minutes Allergies: urticaria, anaphylactic reaction against plasma to 2-3 hr (due to pruritus reaction proteins in transfused release of preformed Anaphylaxis: blood inflammatory wheezing, IgA-deficient individuals mediators in hypotension, should receive blood degranulating mast respiratory arrest, Donor plasma proteins, Host mast cell Acute Type II hypersensitivity During transfusion Fever, hypotension, hemolytic reaction or within 24 hr tachypnea, transfusion Typically causes (due to preformed tachycardia, reaction intravascular hemolysis antibodies) flank pain, Donor RBC with A and/ Host anti-A, anti-B IgG, incompatibility) (intravascular), jaundice (extravascular)

1	Donor RBC with A and/ Host anti-A, anti-B IgG, incompatibility) (intravascular), jaundice (extravascular) Febrile Cytokines created by Within 1-6 hr (due Fever, headaches, nonhemolytic donor WBCs accumulate to preformed chills, flushing transfusion during storage of blood cytokines) More common in reaction products children Donor WBC releases Reactions prevented by leukoreduction of blood products Respiratory distress, noncardiogenic pulmonary edema Generally self limited and clinically silent Mild fever, hyperbilirubinemia Donor RBC with Host IgG foreign antigens Antinuclear (ANA) Nonspecific screening antibody, often associated with SLE Anticardiolipin, lupus anticoagulant SLE, antiphospholipid syndrome Anti-dsDNA, anti-Smith SLE Antisynthetase (eg, anti-Jo-1), anti-SRP, anti-Polymyositis, dermatomyositis helicase (anti-Mi-2) IgA anti-endomysial, IgA anti-tissue Celiac disease transglutaminase, IgA and IgG deamidated gliadin peptide

1	IgA anti-endomysial, IgA anti-tissue Celiac disease transglutaminase, IgA and IgG deamidated gliadin peptide Anti-glutamic acid decarboxylase, islet cell Type 1 diabetes mellitus cytoplasmic antibodies Antiparietal cell, anti-intrinsic factor Pernicious anemia Ataxia-telangiectasia A Defects in ATM gene  failure to detect DNA damage  failure to halt progression of cell cycle mutations accumulate; autosomal recessive Triad: cerebellar defects (Ataxia), spider Angiomas (telangiectasia A ), IgA deficiency  sensitivity to radiation (limit x-ray exposure) AFP  IgA, IgG, and IgE Lymphopenia, cerebellar atrophy  risk of lymphoma and leukemia Hyper-IgM syndrome Most commonly due to defective CD40L on Th cells  class switching defect; X-linked recessive Severe pyogenic infections early in life; opportunistic infection with Pneumocystis, Cryptosporidium, CMV Normal or  IgM  IgG, IgA, IgE Failure to make germinal centers

1	Defect in lysosomal trafficking PLAIN: Progressive Giant granules ( B , arrows) in regulator gene (LYST) neurodegeneration, granulocytes and platelets. Microtubule dysfunction in Lymphohistiocytosis, Pancytopenia phagosome-lysosome fusion; Albinism (partial), recurrent Mild coagulation defects autosomal recessive pyogenic Infections, Bacteria Sepsis Encapsulated (Please SHINE my SKiS): Pseudomonas aeruginosa, Streptococcus pneumoniae, Haemophilus Influenzae type b, Neisseria meningitidis, Escherichia coli, Salmonella, Klebsiella pneumoniae, Group B Streptococcus Some Bacteria Encapsulated Produce No species with early Serious granules: complement Staphylococcus, deficiencies Burkholderia cepacia, Neisseria with late Pseudomonas complement (C5– aeruginosa, Nocardia, C9) deficiencies Serratia Viruses CMV, EBV, JC Enteroviral N/A N/A virus, VZV, chronic encephalitis, infection with poliovirus respiratory/GI viruses (live vaccine

1	Viruses CMV, EBV, JC Enteroviral N/A N/A virus, VZV, chronic encephalitis, infection with poliovirus respiratory/GI viruses (live vaccine Fungi/parasites Candida (local), PCP, GI giardiasis (no IgA) Candida (systemic), N/A Cryptococcus Aspergillus, Mucor Note: B-cell deficiencies tend to produce recurrent bacterial infections, whereas T-cell deficiencies produce more fungal and viral infections.

1	Hyperacute A Within minutes Pre-existing recipient antibodies react to donor antigen (type II hypersensitivity reaction), activate complement Widespread thrombosis of graft vessels (arrows within glomerulus A ) ischemia/necrosis Graft must be removed Acute B Weeks to months Cellular: CD8+ T cells and/ or CD4+ T cells activated against donor MHCs (type IV hypersensitivity reaction) Humoral: similar to hyperacute, except antibodies develop after transplant Vasculitis of graft vessels with dense interstitial lymphocytic infiltrate B Prevent/reverse with immunosuppressants Chronic C Months to years CD4+ T cells respond to recipient APCs presenting donor peptides, including allogeneic MHC Both cellular and humoral components (type II and IV hypersensitivity reactions) Recipient T cells react and secrete cytokines proliferation of vascular smooth muscle, parenchymal atrophy, interstitial fibrosis Dominated by arteriosclerosis C Organ-specific examples: Chronic allograft nephropathy

1	and secrete cytokines proliferation of vascular smooth muscle, parenchymal atrophy, interstitial fibrosis Dominated by arteriosclerosis C Organ-specific examples: Chronic allograft nephropathy Bronchiolitis obliterans Accelerated atherosclerosis (heart) Vanishing bile duct syndrome Graft-versus-host disease Varies Grafted immunocompetent T cells proliferate in the Maculopapular rash, jaundice, diarrhea, hepatosplenomegaly reject host cells with “foreign”

1	For immunocompromised patients, irradiate blood products prior to transfusion to prevent GVHD Immunosuppressants Agents that block lymphocyte activation and proliferation. Reduce acute transplant rejection by suppressing cellular immunity (used as prophylaxis). Frequently combined to achieve greater efficacy with  toxicity. Chronic suppression  risk of infection and malignancy. Cyclosporine Calcineurin inhibitor; Psoriasis, rheumatoid Nephrotoxicity, binds cyclophilin arthritis hypertension, Blocks T-cell activation hyperlipidemia, by preventing IL-2 neurotoxicity, gingival transcription hyperplasia, hirsutism Tacrolimus (FK506) Calcineurin inhibitor; Similar to cyclosporine, binds FK506 binding  risk of diabetes protein (FKBP) and neurotoxicity; Blocks T-cell activation no gingival by preventing IL-2 hyperplasia or transcription hirsutism Both calcineurin inhibitors are highly nephrotoxic, especially in higher doses or in patients with decreased renal function

1	Both calcineurin inhibitors are highly nephrotoxic, especially in higher doses or in patients with decreased renal function Sirolimus (Rapamycin) mTOR inhibitor; binds “PanSirtopenia” Kidney “sir-vives.” FKBP (pancytopenia), Synergistic with Blocks T-cell insulin resistance, cyclosporine Basiliximab Monoclonal antibody; Edema, hypertension, blocks IL-2R tremor Azathioprine Antimetabolite Rheumatoid arthritis, Pancytopenia 6-MP degraded by precursor of Crohn disease, xanthine oxidase; 6-mercaptopurine glomerulonephritis, toxicity  by Mycophenolate Reversibly inhibits Lupus nephritis GI upset, Associated with Mofetil IMP dehydrogenase, pancytopenia, invasive CMV preventing purine hypertension, infection synthesis of B and T hyperglycemia cells Less nephrotoxic and  transcription of

1	Mofetil IMP dehydrogenase, pancytopenia, invasive CMV preventing purine hypertension, infection synthesis of B and T hyperglycemia cells Less nephrotoxic and  transcription of Many autoimmune and inflammatory disorders, adrenal insufficiency, asthma, CLL, non-Hodgkin lymphoma Cushing syndrome, osteoporosis, hyperglycemia, diabetes, amenorrhea, adrenocortical atrophy, peptic ulcers, psychosis, cataracts, avascular necrosis (femoral head) Demargination of WBCs causes artificial leukocytosis Adrenal insufficiency may develop if drug is stopped abruptly after chronic use “Support bacteria. They’re the only culture some people have.” “What lies behind us and what lies ahead of us are tiny matters compared to what lies within us.” —Henry S. Haskins “Infectious disease is merely a disagreeable instance of a widely prevalent tendency of all living creatures to save themselves the bother of building, by their own efforts, the things they require.”

1	Microbiology questions on the Step 1 exam often require two (or more) steps: Given a certain clinical presentation, you will first need to identify the most likely causative organism, and you will then need to provide an answer regarding some features of that organism or relevant antimicrobial agents. For example, a description of a child with fever and a petechial rash will be followed by a question that reads, “From what site does the responsible organism usually enter the blood?” This section therefore presents organisms in two major ways: in individual microbial “profiles” and in the context of the systems they infect and the clinical presentations they produce. You should become familiar with both formats. When reviewing the systems approach, remind yourself of the features of each microbe by returning to the individual profiles. Also be sure to memorize the laboratory characteristics that allow you to identify microbes.

1	Flagellum Proteins Motility Pilus/fimbria Glycoprotein Mediate adherence of bacteria to cell surface; sex pilus forms during conjugation Spore Keratin-like coat; dipicolinic acid; Gram ⊕ only peptidoglycan, DNA Survival: resist dehydration, heat, chemicals Capsule Discrete layer usually made of polysaccharides Protects against phagocytosis (and rarely proteins) Slime (S) layer Loose network of polysaccharides Mediates adherence to surfaces, especially foreign surfaces (eg, indwelling catheters) Outer membrane Outer leaflet: contains endotoxin (LPS/LOS) Gram ⊝ only Embedded proteins: porins and other outer Endotoxin: lipid A induces TNF and IL-1; membrane proteins (OMPs) antigenic O polysaccharide component Inner leaflet: phospholipids Most OMPs are antigenic Porins: transport across outer membrane

1	Periplasm Space between cytoplasmic membrane Accumulates components exiting gram and outer membrane in gram ⊝ bacterial ⊝ cells, including hydrolytic enzymes (peptidoglycan in middle) (eg, β-lactamases) Cell wall Peptidoglycan is a sugar backbone with peptide Net-like structure gives rigid support, protects side chains cross-linked by transpeptidase against osmotic pressure damage Cytoplasmic Phospholipid bilayer sac with embedded Site of oxidative and transport enzymes; PBPs membrane proteins (eg, penicillin-binding proteins involved in cell wall synthesis [PBPs]) and other enzymes Lipoteichoic acids induce TNF-α and IL-1 Lipoteichoic acids (gram positive) only extend from membrane to exterior

1	Gram stain First-line lab test in bacterial identification. Bacteria with thick peptidoglycan layer retain crystal violet dye (gram ⊕); bacteria with thin peptidoglycan layer turn red or pink (gram ⊝) with counterstain. These bugs do not Gram stain well (These Little Microbes May Unfortunately Lack Real Color But Are Everywhere): Treponema, Leptospira Too thin to be visualized Mycoplasma, Ureaplasma No cell wall India ink stain Cryptococcus neoformans D ; mucicarmine can also be used to stain thick polysaccharide capsule red Silver stain Fungi (eg, Coccidioides E , Pneumocystis jirovecii), Legionella, Helicobacter pylori Properties of growth media The same type of media can possess both (or neither) of these properties. Selective media Favors the growth of particular organism while preventing growth of other organisms. Example: Thayer-Martin agar contains antibiotics that allow the selective growth of Neisseria by inhibiting the growth of other sensitive organisms.

1	Aerobes Use an O2-dependent system to generate ATP. Examples include Nocardia, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Bordetella pertussis. Reactivation of M tuberculosis (eg, after immunocompromise or TNF-α inhibitor use) has a predilection for the apices of the lung. Anaerobes Examples include Clostridium, Bacteroides, Fusobacterium, and Actinomyces israelii. They lack catalase and/or superoxide dismutase and are thus susceptible to oxidative damage. Generally foul smelling (short-chain fatty acids), are difficult to culture, and produce gas in tissue (CO2 and H2). Anaerobes Can’t Breathe Fresh Air. Anaerobes are normal flora in GI tract, typically pathogenic elsewhere. AminO2glycosides are ineffective against anaerobes because these antibiotics require O2 to enter into bacterial cell.

1	Anaerobes are normal flora in GI tract, typically pathogenic elsewhere. AminO2glycosides are ineffective against anaerobes because these antibiotics require O2 to enter into bacterial cell. Examples are Pseudomonas aeruginosa, Streptococcus pneumoniae A , Haemophilus influenzae type b, Neisseria meningitidis, Escherichia coli, Salmonella, Klebsiella pneumoniae, and group B Strep. Their capsules serve as an antiphagocytic virulence factor. Capsular polysaccharide + protein conjugate serves as an antigen in vaccines. Please SHiNE my SKiS. Are opsonized, and then cleared by spleen. Asplenics (No Spleen Here) have  opsonizing ability and thus  risk for severe infections; need vaccines to protect against: Some vaccines containing polysaccharide capsule antigens are conjugated to a carrier protein, enhancing immunogenicity by promoting T-cell activation and subsequent class switching. A polysaccharide antigen alone cannot be presented to T cells.

1	Pneumococcal vaccines: PCV13 (pneumococcal conjugate vaccine), PPSV23 (pneumococcal polysaccharide vaccine with no conjugated protein). H influenzae type b (conjugate vaccine). Meningococcal vaccine (conjugate vaccine). Catalase-positive Catalase degrades H2O2 into H2O and bubbles of O2 A before it can be converted to microbicidal organisms products by the enzyme myeloperoxidase. People with chronic granulomatous disease (NADPH oxidase deficiency) have recurrent infections with certain catalase ⊕ organisms. Examples: Nocardia, Staphylococci, Serratia, Candida, Listeria, E coli, Burkholderia cepacia, Pseudomonas, Aspergillus, Helicobacter pylori, Bordetella pertussis. Viridans streptococci (S mutans, S sanguinis) Dental plaques, infective endocarditis P aeruginosa Respiratory tree colonization in patients with cystic fibrosis, ventilator-associated pneumonia Contact lens–associated keratitis

1	P aeruginosa Respiratory tree colonization in patients with cystic fibrosis, ventilator-associated pneumonia Contact lens–associated keratitis Some gram ⊕ bacteria can form spores A when nutrients are limited. Spores lack metabolic activity and are highly resistant to heat and chemicals. Core contains dipicolinic acid. Must autoclave to kill spores (as is done to surgical equipment) by steaming at 121°C for 15 minutes. Examples: B anthracis (anthrax), B cereus (food poisoning), C botulinum (botulism), C difficile (pseudomembranous colitis), C perfringens (gas gangrene), C tetani (tetanus). Bacterial virulence factors These promote evasion of host immune response. pieces of environmental naked bacterial chromosomal DNA (from bacterial cell lysis). The transfer and expression of newly transferred genes is called transformation. A feature of many bacteria, especially S pneumoniae, Hinfluenzae type b, and Neisseria (SHiN).

1	Adding deoxyribonuclease degrades naked DNA, preventing transformation. into bacterial DNA of part of the chromosome bacterial chromosomal DNA, termed high- frequency recombination (Hfr) cell. Transfer of leading part of plasmid and a few flanking F+ cell F– cell Hfr cellF– cell Hfr cell F– cell Hfr cell Recombinant chromosomal genes. High-frequency recombination may integrate some of those bacterial genes. Recipient cell remains F– but now may have new bacterial genes. Generalized A packaging “error.” Lytic phage infects Cleavage of Bacterial DNA packaged bacterium, leading to cleavage of bacterial phage DNA. Parts of bacterial chromosomal DNA may become packaged in phage capsid. Phage infects another bacterium, transferring these genes. Release of new phage Infects other Genes transferred from lysed cell bacteria to new bacteria

1	Release of new phage Infects other Genes transferred from lysed cell bacteria to new bacteria Specialized An “excision” event. Lysogenic phage infects bacterium; viral DNA incorporates into bacterial chromosome. When phage DNA is excised, flanking bacterial genes may be excised with it. DNA is packaged into phage capsid and can infect another bacterium. Genes for the following 5 bacterial toxins are encoded in a lysogenic phage (ABCD’S): Group A strep erythrogenic toxin, Botulinum toxin, Cholera toxin, Diphtheria toxin, Shiga toxin. Release of new phage Infects other Genes di˜erent from from lysed cell bacteria donor and recipient (specialized segment of DNA), which can copy and excise itself and then insert into the same DNA molecule or an unrelated DNA (eg, Integration of genes Transposon plasmid or chromosome). Critical in creating plasmids with multiple drug resistance and DNA transfer across species lines (eg, Tn1546 with vanA from Enterococcus to S aureus).

1	Main features of exotoxins and endotoxins Bacteria with exotoxins Downstream cellular reaction Exotoxin TNF, IL-1, IL-6 Host cell Endotoxin Toxin prevents release of inhibitory (GABA and glycine) neurotransmitters from Renshaw cells in spinal cord  spastic paralysis, risus sardonicus, trismus (lockjaw) Toxin prevents release of stimulatory (ACh) signals at neuromuscular junction  flaccid paralysis (floppy baby) aAn AB toxin (aka, two-component toxin [or three for anthrax]) with B enabling binding and triggering uptake (endocytosis) of the active A component. The A components are usually ADP ribosyltransferases; others have enzymatic activities as listed in chart. Bacteria with exotoxins (continued) Clostridium Alpha toxin Phospholipase (lecithinase) Degradation of phospholipids  myonecrosis perfringens that degrades tissue and (“gas gangrene”) and hemolysis (“double zone” cell membranes of hemolysis on blood agar)

1	Streptococcus Streptolysin O Protein that degrades cell Lyses RBCs; contributes to β-hemolysis; pyogenes membrane host antibodies against toxin (ASO) used to diagnose rheumatic fever (do not confuse with immune complexes of poststreptococcal glomerulonephritis) Cross-links β region of TCR to MHC class II on APCs outside of the antigen binding site  overwhelming release of IL-1, IL-2, IFN-γ, and TNF-α • shock Toxic shock syndrome: fever, rash, shock; other toxins cause scalded skin syndrome (exfoliative toxin) and food poisoning (heat-stable enterotoxin) Toxic shock–like syndrome: fever, rash, shock; scarlet fever

1	Toxic shock–like syndrome: fever, rash, shock; scarlet fever Endotoxin LPS found in outer membrane of gram ⊝ bacteria (both cocci and rods). Composed of O antigen + core polysaccharide + lipid A (the toxic component). Released upon cell lysis or by living cells by blebs detaching from outer surface membrane (vs exotoxin, which is actively secreted). Three main effects: macrophage activation (TLR4/CD14), complement activation, and tissue factor activation. ENDOTOXINS: Edema Nitric oxide DIC/Death Outer membrane TNF-α O-antigen + core polysaccharide + lipid A eXtremely heat stable IL-1 and IL-6 Neutrophil chemotaxis Shock IL-1, IL-6 Fever Fever and hypotension HypotensionNitric oxide TNF-α hemolysis, hemolysis, Important tests are in bold. Important pathogens are in bold italics. Note: Enterococcus is either ˜or °-hemolytic. (No hemolysis, grows in bile) Novobiocin sensitivity

1	Important tests are in bold. Important pathogens are in bold italics. Note: Enterococcus is either ˜or °-hemolytic. (No hemolysis, grows in bile) Novobiocin sensitivity Growth in 6.5% NaCl and PYR Status -hemolytic bacteria Gram ⊕ cocci. Partial oxidation of hemoglobin causes greenish or brownish color without clearing around growth on blood agar A . Include the following organisms: -hemolytic bacteria Gram ⊕ cocci. Complete lysis of RBCs  pale/clear area surrounding colony on blood agar A . Include the following organisms: Gram ⊕, β-hemolytic, catalase ⊕, coagulase ⊕ cocci in clusters A . Protein A (virulence factor) binds Fc-IgG, inhibiting complement activation and phagocytosis. Commonly colonizes the nares, ears, axilla, and groin. Causes: Inflammatory disease—skin infections, organ abscesses, pneumonia (often after influenza virus infection), endocarditis, septic arthritis, and osteomyelitis.

1	Causes: Inflammatory disease—skin infections, organ abscesses, pneumonia (often after influenza virus infection), endocarditis, septic arthritis, and osteomyelitis. Toxin-mediated disease—toxic shock syndrome (TSST-1), scalded skin syndrome (exfoliative toxin), rapid-onset food poisoning (enterotoxins). MRSA (methicillin-resistant S aureus)— important cause of serious nosocomial and community-acquired infections; resistance due to altered penicillin-binding protein. mecA gene from staphylococcal chromosomal cassette involved in penicillin resistance. TSST-1 is a superantigen that binds to MHC II and T-cell receptor, resulting in polyclonal T-cell activation and cytokine release. Staphylococcal toxic shock syndrome (TSS)— fever, vomiting, rash, desquamation, shock, end-organ failure. TSS results in  AST, • ALT,  bilirubin. Associated with prolonged use of vaginal tampons or nasal packing.

1	Compare with Streptococcus pyogenes TSS (a toxic shock–like syndrome associated with painful skin infection). S aureus food poisoning due to ingestion of preformed toxin  short incubation period (2–6 hr) followed by nonbloody diarrhea and emesis. Enterotoxin is heat stable  not destroyed by cooking. S aureus makes coagulase and toxins. Forms fibrin clot around itself  abscess. Gram ⊕, catalase ⊕, coagulase ⊝, urease ⊕ cocci in clusters. Novobiocin sensitive. Does not ferment mannitol (vs S aureus). Normal flora of skin; contaminates blood cultures. Infects prosthetic devices (eg, hip implant, heart valve) and IV catheters by producing adherent biofilms. Staphylococcus Gram ⊕, catalase ⊕, coagulase ⊝, urease ⊕ cocci in clusters. Novobiocin resistant. saprophyticus Normal flora of female genital tract and perineum. Second most common cause of uncomplicated UTI in young women (most common is E coli). Gram ⊕, α-hemolytic, lancet-shaped diplococci

1	Second most common cause of uncomplicated UTI in young women (most common is E coli). Gram ⊕, α-hemolytic, lancet-shaped diplococci A . Encapsulated. IgA protease. Optochin sensitive and bile soluble. Most commonly causes: Pneumococcus is associated with “rusty” sputum, sepsis in patients with sickle cell disease, and asplenic patients. No virulence without capsule. Gram ⊕, α-hemolytic cocci. Optochin resistant and bile insoluble. Normal flora of the oropharynx. Streptococcus mutans and S mitis cause dental caries. S sanguinis makes dextrans that bind to fibrin-platelet aggregates on damaged heart valves, causing subacute bacterial endocarditis. Viridans group strep live in the mouth, because they are not afraid of-the-chin (op-to-chin resistant). Sanguinis = blood. Think, “there is lots of blood in the heart” (endocarditis). Gram ⊕ cocci in chains A . Group A strep cause: Pyogenic—pharyngitis, cellulitis, impetigo (“honey-crusted” lesions), erysipelas

1	Gram ⊕ cocci in chains A . Group A strep cause: Pyogenic—pharyngitis, cellulitis, impetigo (“honey-crusted” lesions), erysipelas Toxigenic—scarlet fever, toxic shock–like syndrome, necrotizing fasciitis Immunologic—rheumatic fever, glomerulonephritis Bacitracin sensitive, β-hemolytic, pyrrolidonyl arylamidase (PYR) ⊕. Hyaluronic acid capsule and M protein inhibit phagocytosis. Antibodies to M protein enhance host defenses against S pyogenes but can give rise to rheumatic fever. ASO titer or anti-DNase B antibodies indicate recent S pyogenes infection. “Ph”yogenes pharyngitis can result in rheumatic “phever” and glomerulonephritis. Strains causing impetigo can induce glomerulonephritis. Scarlet fever—blanching, sandpaper-like body rash, strawberry tongue, and circumoral pallor in the setting of group A streptococcal pharyngitis (erythrogenic toxin ⊕).

1	Scarlet fever—blanching, sandpaper-like body rash, strawberry tongue, and circumoral pallor in the setting of group A streptococcal pharyngitis (erythrogenic toxin ⊕). Bacillus anthracis Gram ⊕, spore-forming rod that produces anthrax toxin (an exotoxin consisting of protective antigen, lethal factor, and edema factor). Has a polypeptide capsule (poly d-glutamate). Colonies show a halo of projections, sometimes referred to as “medusa head” appearance. Cutaneous anthrax Painless papule surrounded by vesicles  ulcer with black eschar A (painless, necrotic)  uncommonly progresses to bacteremia and death. Pulmonary anthrax Inhalation of spores, most commonly from contaminated animals or animal products, although also a potential bioweapon  flu-like symptoms that rapidly progress to fever, pulmonary hemorrhage, mediastinitis (CXR may show widened mediastinum), and shock. Also called woolsorter’s disease.

1	Gram ⊕ cocci, bacitracin resistant, β-hemolytic, colonizes vagina; causes pneumonia, meningitis, and sepsis, mainly in babies. Produces CAMP factor, which enlarges the area of hemolysis formed by S aureus. (Note: CAMP stands for the authors of the test, not cyclic AMP.) Hippurate test ⊕. PYR ⊝. Screen pregnant women at 35–37 weeks of gestation with rectal and vaginal swabs. Patients with ⊕ culture receive intrapartum penicillin/ampicillin prophylaxis. Bacillus cereus Gram ⊕ rod. Causes food poisoning. Spores survive cooking rice (reheated rice syndrome). Keeping rice warm results in germination of spores and enterotoxin formation. Emetic type causes nausea and vomiting within 1–5 hours. Caused by cereulide, a preformed toxin. Diarrheal type causes watery, nonbloody diarrhea and GI pain within 8–18 hours. Management: supportive care (antibiotics are ineffective against toxins).

1	Clostridia Gram ⊕, spore-forming, obligate anaerobic rods. Tetanus toxin and botulinum toxin are proteases that cleave SNARE proteins involved in neurotransmission. Clostridium tetani Produces tetanospasmin, an exotoxin causing Tetanus is tetanic paralysis. tetanus. Tetanospasmin blocks release of GABA and glycine from Renshaw cells in spinal cord. Causes spastic paralysis, trismus (lockjaw), risus sardonicus (raised eyebrows and open grin), opisthotonos (spasms of spinal extensors). Prevent with tetanus vaccine. Treat with antitoxin +/− vaccine booster, antibiotics, diazepam (for muscle spasms), and wound debridement. Produces a heat-labile toxin that inhibits ACh release at the neuromuscular junction, causing botulism. In adults, disease is caused by ingestion of preformed toxin. In babies, ingestion of spores (eg, in honey) leads to disease (floppy baby syndrome). Treat with human botulinum immunoglobulin.

1	Symptoms of botulism (the 4 D’s): Diplopia, Dysarthria, Dysphagia, Dyspnea. Botulinum is from bad bottles of food, juice, and honey (causes a descending flaccid paralysis). Local botulinum toxin A (Botox) injections used to treat focal dystonia, hyperhidrosis, muscle spasms, and cosmetic reduction of facial wrinkles. Produces α-toxin (lecithinase, a phospholipase) that can cause myonecrosis (gas gangrene A ; presents as soft tissue crepitus) and hemolysis. If heavily spore-contaminated food is cooked but left standing too long at < 60°C, spores germinate • vegetative bacteria • heat-labile enterotoxin • food poisoning symptoms in 10-12 hours, resolution in 24 hours. Perfringens perforates a gangrenous leg. Produces toxins A and B, which damage enterocytes. Both toxins lead to watery diarrhea B . Often 2° to antibiotic use, especially clindamycin or ampicillin; associated with PPIs.

1	Produces toxins A and B, which damage enterocytes. Both toxins lead to watery diarrhea B . Often 2° to antibiotic use, especially clindamycin or ampicillin; associated with PPIs. Diagnosed by PCR or antigen detection of one or both toxins in stool. Complications: toxic megacolon. Difficile causes diarrhea. Treatment: oral vancomycin, metronidazole, or fidaxomicin. For recurrent cases, consider repeating prior regimen or fecal microbiota transplant. Gram ⊕ rods occurring in angular arrangements; transmitted via respiratory droplets. Causes diphtheria via exotoxin encoded by β-prophage. Potent exotoxin inhibits protein synthesis via ADP-ribosylation of EF-2, leading to possible necrosis in pharynx, cardiac, and CNS tissue. Symptoms include pseudomembranous pharyngitis (grayish-white membrane A ) with lymphadenopathy. Toxin dissemination may cause myocarditis, arrhythmias, neuropathies.

1	Symptoms include pseudomembranous pharyngitis (grayish-white membrane A ) with lymphadenopathy. Toxin dissemination may cause myocarditis, arrhythmias, neuropathies. Lab diagnosis based on gram ⊕ rods with metachromatic (blue and red) granules and ⊕ Elek test for toxin. Toxoid vaccine prevents diphtheria. on Löffler media). Black colonies on cystine-tellurite agar. ABCDEFG: Elongation Factor 2 Granules Treatment: antibiotic therapy +/– diphtheria antitoxin. Gram ⊕, facultative intracellular rod; acquired by ingestion of unpasteurized dairy products and cold deli meats, transplacental transmission, by vaginal transmission during birth. Grows well at refrigeration temperatures (“cold enrichment”). Forms “rocket tails” (red in A ) via actin polymerization that allow intracellular movement and cell-to-cell spread across cell membranes, thereby avoiding antibody. Characteristic tumbling motility in broth.

1	Can cause amnionitis, septicemia, and spontaneous abortion in pregnant women; granulomatosis infantiseptica; meningitis in immunocompromised patients, neonates, and older adults; mild, self-limited gastroenteritis in healthy individuals. Treatment: ampicillin. Nocardia vs Both are gram ⊕ and form long, branching filaments resembling fungi. Actinomyces Found in soil Normal oral, reproductive, and GI flora Causes pulmonary infections in Causes oral/facial abscesses that drain through immunocompromised (can mimic TB but sinus tracts; often associated with dental caries/ with ⊝ PPD); cutaneous infections after extraction and other maxillofacial trauma; trauma in immunocompetent; can spread to forms yellow “sulfur granules”; can also cause CNS PID with IUDs Treat with sulfonamides (TMP-SMX) Treat with penicillin Treatment is a SNAP: Sulfonamides—Nocardia; Actinomyces—Penicillin

1	Treat with sulfonamides (TMP-SMX) Treat with penicillin Treatment is a SNAP: Sulfonamides—Nocardia; Actinomyces—Penicillin Gram ⊕ acid fast rods (pink rods, arrows in A ). Mycobacterium tuberculosis (TB, often resistant to multiple drugs). M avium–intracellulare (causes disseminated, non-TB disease in AIDS; often resistant to multiple drugs). Prophylaxis with azithromycin when CD4+ count < 50 cells/ mm . M scrofulaceum (cervical lymphadenitis in children). M marinum (hand infection in aquarium handlers). TB symptoms include fever, night sweats, weight loss, cough (nonproductive or productive), hemoptysis. Cord factor creates a “serpentine cord” appearance in virulent M tuberculosis strains; activates macrophages (promoting granuloma formation) and induces release of TNF-α. Sulfatides (surface glycolipids) inhibit phagolysosomal fusion.

1	Healing by fbrosis Progressive primary tuberculosis Calcifcation (AIDS, malnutrition) (PPD false positives from BCG vaccination. PPD ⊕ if current infection or past exposure. PPD ⊝ if no infection and in sarcoidosis or HIV infection (especially with low CD4+ cell count). Caseating granulomas with central necrosis and Langhans giant cell (single example in A ) are characteristic of 2° tuberculosis. Leprosy Also called Hansen disease. Caused by Mycobacterium leprae, an acid-fast bacillus that likes cool temperatures (infects skin and superficial nerves—“glove and stocking” loss of sensation A ) and cannot be grown in vitro. Diagnosed via skin biopsy or tissue PCR. Reservoir in United States: armadillos. Leprosy has 2 forms (many cases fall temporarily between two extremes):

1	Lepromatous—presents diffusely over the skin, with Leonine (Lion-like) facies B , and is communicable (high bacterial load); characterized by low cell-mediated immunity with a largely Th2 response. Lepromatous form can be Lethal. Tuberculoid—limited to a few hypoesthetic, hairless skin plaques; characterized by high cell- mediated immunity with a largely Th1-type response and low bacterial load. Treatment: dapsone and rifampin for tuberculoid form; clofazimine is added for lepromatous form. Important tests are in bold. Important pathogens are in bold italics. aPleomorphic rod/coccobacillus Neisseria Gram ⊝ diplococci. Metabolize glucose Acid production: MeninGococci—Maltose and and produce IgA proteases. Contain Glucose; Gonococci—Glucose. lipooligosaccharides (LOS) with strong endotoxin activity. N gonorrhoeae is often intracellular (within neutrophils) A . No vaccine due to antigenic variation of pilus Vaccine (type B vaccine available for at-risk proteins individuals)

1	A . No vaccine due to antigenic variation of pilus Vaccine (type B vaccine available for at-risk proteins individuals) Causes gonorrhea, septic arthritis, neonatal Causes meningococcemia with petechial conjunctivitis (2–5 days after birth), pelvic hemorrhages and gangrene of toes B , inflammatory disease (PID), and Fitz-Hugh– meningitis, Waterhouse-Friderichsen Curtis syndrome syndrome (adrenal insufficiency, fever, DIC, Diagnosed with NAT Diagnosed via culture-based tests or PCR Condoms  sexual transmission, erythromycin Rifampin, ciprofloxacin, or ceftriaxone eye ointment prevents neonatal blindness prophylaxis in close contacts Treatment: ceftriaxone (+ azithromycin Treatment: ceftriaxone or penicillin G or doxycycline, for possible chlamydial coinfection)

1	Treatment: ceftriaxone (+ azithromycin Treatment: ceftriaxone or penicillin G or doxycycline, for possible chlamydial coinfection) Small gram ⊝ (coccobacillary) rod. Aerosol transmission. Nontypeable (unencapsulated) strains are the most common cause of mucosal infections (otitis media, conjunctivitis, bronchitis) as well as invasive infections since the vaccine for capsular type b was introduced. Produces IgA protease. Culture on chocolate agar, which contains factors V (NAD+) and X (hematin) for growth; can also be grown with S aureus, which provides factor V via RBC hemolysis. A , can be “cherry red” in children; “thumb sign” on lateral neck x-ray B ), Meningitis, Otitis media, and Pneumonia. Vaccine contains type b capsular polysaccharide (polyribosylribitol phosphate) conjugated to diphtheria toxoid or other protein. Given between 2 and 18 months of age. Does not cause the flu (influenza virus does).

1	Does not cause the flu (influenza virus does). Treatment: amoxicillin +/− clavulanate for mucosal infections; ceftriaxone for meningitis; rifampin prophylaxis for close contacts. Acinetobacter Gram ⊝, strictly aerobic, oxidase ⊝ coccobacillus. Commensal opportunist but increasingly baumannii associated with resistant hospital-acquired infections, especially in ICU. Can cause ventilator-associated pneumonia and septicemia in immunocompromised patients. Gram ⊝ rod. Gram stains poorly—use silver stain. Grow on charcoal yeast extract medium with iron and cysteine. Detected by presence of antigen in urine. Labs may show hyponatremia. Aerosol transmission from environmental water source habitat (eg, air conditioning systems, hot water tanks). No person-to-person transmission. Treatment: macrolide or quinolone. Gram ⊝, aerobic coccobacillus. Virulence factors include pertussis toxin (disables Gi), adenylate cyclase toxin ( cAMP), and tracheal cytotoxin. Three clinical stages:

1	Gram ⊝, aerobic coccobacillus. Virulence factors include pertussis toxin (disables Gi), adenylate cyclase toxin ( cAMP), and tracheal cytotoxin. Three clinical stages: Catarrhal—low-grade fevers, Coryza. Paroxysmal—paroxysms of intense cough followed by inspiratory “whooP” (“whooping cough”), posttussive vomiting. Convalescent—gradual recovery of chronic cough. Prevented by Tdap, DTaP vaccines. May be mistaken as viral infection due to lymphocytic infiltrate resulting from immune response. Treatment: macrolides; if allergic use TMP-SMX. Gram ⊝, aerobic coccobacillus. Transmitted via ingestion of contaminated animal products (eg, unpasteurized milk). Survives in macrophages in the reticuloendothelial system. Can form noncaseating granulomas. Typically presents with undulant fever, night sweats, and arthralgia. Treatment: doxycycline + rifampin or streptomycin.

1	Treatment: doxycycline + rifampin or streptomycin. Think of a French legionnaire (soldier) with his silver helmet, sitting around a campfire (charcoal) with his iron dagger—he is no sissy (cysteine). Legionnaires’ disease—severe pneumonia (often unilateral and lobar A ), fever, GI and CNS symptoms. Common in smokers and in chronic lung disease. Pontiac fever—mild flu-like syndrome. Aeruginosa—aerobic; motile, catalase ⊕, gram ⊝ rod. Non-lactose fermenting. Oxidase ⊕. Frequently found in water. Has a grape-like odor. PSEUDOMONAS is associated with: Pneumonia, Sepsis, Ecthyma gangrenosum, UTIs, Diabetes, Osteomyelitis, Mucoid polysaccharide capsule, Otitis externa (swimmer’s ear), Nosocomial infections (eg, catheters, equipment), Addicts (drug abusers), Skin infections (eg, hot tub folliculitis, wound infection in burn victims). Mucoid polysaccharide capsule may contribute to chronic pneumonia in cystic fibrosis patients due to biofilm formation.

1	Mucoid polysaccharide capsule may contribute to chronic pneumonia in cystic fibrosis patients due to biofilm formation. Produces PEEP: Phospholipase C (degrades cell membranes); Endotoxin (fever, shock); Exotoxin A (inactivates EF-2); Pigments: pyoverdine and pyocyanin (blue-green pigment A ; also generates reactive oxygen species). Corneal ulcers/keratitis in contact lens wearers/ minor eye trauma. Ecthyma gangrenosum—rapidly progressive, necrotic cutaneous lesion B caused by Pseudomonas bacteremia. Typically seen in immunocompromised patients. Treatments include “CAMPFIRE” drugs: Polymyxins (eg, polymyxin B, colistin) Fluoroquinolones (eg, ciprofloxacin, levofloxacin) ThIRdand fourth-generation cephalosporins (eg, ceftazidime, cefepime) Extended-spectrum penicillins (eg, piperacillin, ticarcillin)

1	Fluoroquinolones (eg, ciprofloxacin, levofloxacin) ThIRdand fourth-generation cephalosporins (eg, ceftazidime, cefepime) Extended-spectrum penicillins (eg, piperacillin, ticarcillin) Yersinia enterocolitica Gram ⊝ pleomorphic rod/coccobacillus. Usually transmitted from pet feces (eg, puppies), contaminated milk, or pork. Can cause acute bloody diarrhea, pseudoappendicitis (right lower abdominal pain due to mesenteric adenitis and/or terminal ileitis), reactive arthritis in adults. Gram ⊝ rod; intestinal flora that causes lobar pneumonia in alcoholics and diabetics when aspirated. Very mucoid colonies A caused by abundant polysaccharide capsules. Dark red “currant jelly” sputum (blood/mucus). Also cause of nosocomial UTIs. Associated with evolution of multidrug resistance (MDR). ABCDE’s of Klebsiella: Aspiration pneumonia aBscess in lungs and liver “Currant jelly” sputum Diabetes EtOH abuse

1	ABCDE’s of Klebsiella: Aspiration pneumonia aBscess in lungs and liver “Currant jelly” sputum Diabetes EtOH abuse Campylobacter jejuni Gram ⊝, comma or S shaped (with polar flagella) A , oxidase ⊕, grows at 42°C (“Campylobacter likes the hot campfire”). Major cause of bloody diarrhea, especially in children. Fecal-oral transmission through person-to-person contact or via ingestion of undercooked contaminated poultry or meat, unpasteurized milk. Contact with infected animals (dogs, cats, pigs) is also a risk factor. Common antecedent to Guillain-Barré syndrome and reactive arthritis.

1	Common antecedent to Guillain-Barré syndrome and reactive arthritis. Vibrio cholerae Gram ⊝, flagellated, comma shaped A , oxidase ⊕, grows in alkaline media. Endemic to developing countries. Produces profuse rice-water diarrhea via enterotoxin that permanently activates Gs,  cAMP. Sensitive to stomach acid (acid labile); requires large inoculum (high ID50) unless host has  gastric acidity. Transmitted via ingestion of contaminated water or uncooked food (eg, raw shellfish). Treat promptly with oral rehydration solution. Curved, flagellated (motile), gram ⊝ rod A that is triple ⊕: catalase ⊕, oxidase ⊕, and urease ⊕ (can use urea breath test or fecal antigen test for diagnosis). Urease produces ammonia, creating an alkaline environment, which helps H pylori survive in acidic mucosa. Colonizes mainly antrum of stomach; causes gastritis and peptic ulcers (especially duodenal). Risk factor for peptic ulcer disease, gastric adenocarcinoma, and MALT lymphoma.

1	Most common initial treatment is triple therapy: Amoxicillin (metronidazole if penicillin allergy) + Clarithromycin + Proton pump inhibitor; Antibiotics Cure Pylori. Bismuth-based quadruple therapy if concerned about macrolide resistance. Jarisch-Herxheimer Flu-like syndrome (fever, chills, headache, myalgia) after antibiotics are started; due to killed reaction bacteria (usually spirochetes) releasing toxins. Chlamydiae cannot make their own ATP. They are obligate intracellular organisms that cause mucosal infections. 2 forms: Elementary body (small, dense) is “Enfectious” and Enters cell via Endocytosis; transforms into reticulate body. Reticulate body Replicates in cell by fission; Reorganizes into elementary bodies. A , nongonococcal urethritis, PID, and reactive arthritis. Chlamydophila pneumoniae and Chlamydophila psittaci cause atypical pneumonia; transmitted by aerosol.

1	A , nongonococcal urethritis, PID, and reactive arthritis. Chlamydophila pneumoniae and Chlamydophila psittaci cause atypical pneumonia; transmitted by aerosol. Chlamydial cell wall lacks classic peptidoglycan (due to reduced muramic acid), rendering β-lactam antibiotics ineffective. Chlamys = cloak (intracellular). C psittaci—has an avian reservoir (parrots), causes atypical pneumonia. Lab diagnosis: PCR, nucleic acid amplification test. Cytoplasmic inclusions (reticulate bodies) seen on Giemsa or fluorescent antibody– stained smear. Treatment: azithromycin (favored because one-time treatment) or doxycycline. Add ceftriaxone for possible concomitant gonorrhea. Rickettsial diseases and vector-borne illnesses Treatment: doxycycline. Rickettsia rickettsii, vector is tick. Despite its name, disease occurs primarily in the South Atlantic states, especially North Carolina. Rash typically starts at wrists A and ankles and then spreads to trunk, palms, and soles.

1	Classic triad—headache, fever, rash (vasculitis). Palms and soles rash is seen in Coxsackievirus A infection (hand, foot, and mouth disease), Rocky Mountain spotted fever, and 2° Syphilis (you drive CARS using your palms and soles). Q fever is caused by a Quite Complicated Bug because it has no rash or vector and its causative organism can survive outside in its endospore form. Not in the Rickettsia genus, but closely related. Classic cause of atypical “walking pneumonia” (insidious onset, headache, nonproductive cough, patchy or diffuse interstitial infiltrate). Occurs frequently in those <30 years old; outbreaks in military recruits, prisons, colleges. X-ray looks worse than patient. High titer of cold agglutinins (IgM), which can agglutinate RBCs. Treatment: macrolides, doxycycline, or fluoroquinolone (penicillin ineffective since Mycoplasma has no cell wall). Not seen on Gram stain. Pleomorphic A . Bacterial membrane contains sterols for stability. Grown on Eaton agar.

1	Not seen on Gram stain. Pleomorphic A . Bacterial membrane contains sterols for stability. Grown on Eaton agar. Mycoplasma gets cold without a coat (no cell wall). Can cause atypical variant of Stevens-Johnson syndrome, typically in children and adolescents. Mississippi and Ohio Macrophage filled River Valleys with Histoplasma (smaller than RBC) Eastern and Central Broad-based budding US, Great Lakes of Blastomyces (same size as RBC) Southwestern US, Spherule (much larger California than RBC) filled with endospores of Coccidioides Latin America Budding yeast of Paracoccidioides with “captain’s wheel” formation (much larger than RBC) Palatal/tongue ulcers, splenomegaly, pancytopenia Disseminates to bone/ skin (may mimic SCC) Disseminates to skin/ bone Similar to blastomycosis, males > females Histo hides (within macrophages) Associated with bird or bat droppings (eg, spelunking) Associated with dust exposure in endemic areas (eg, archeological excavations, earthquakes)

1	Associated with bird or bat droppings (eg, spelunking) Associated with dust exposure in endemic areas (eg, archeological excavations, earthquakes) Paracoccidio parasails with the captain’s wheel all the way to Latin America alba = white. Dimorphic; forms pseudohyphae and budding yeasts at 20°C A , germ tubes at 37°C B . Systemic or superficial fungal infection. Causes oral C and esophageal thrush in immunocompromised (neonates, steroids, diabetes, AIDS), vulvovaginitis (diabetes, use of antibiotics), diaper rash, endocarditis (IV drug users), disseminated candidiasis (especially in neutropenic patients), chronic mucocutaneous candidiasis. Treatment: oral fluconazole/topical azoles for vaginal; nystatin, azoles, or, rarely, echinocandins for oral; fluconazole, echinocandins, or amphotericin B for esophageal or systemic disease. E . 5–10 μm with narrow budding. Heavily encapsulated yeast. Not dimorphic.

1	E . 5–10 μm with narrow budding. Heavily encapsulated yeast. Not dimorphic. Found in soil, pigeon droppings. Acquired through inhalation with hematogenous dissemination to meninges. Highlighted with India ink (clear halo G ) and mucicarmine (red inner capsule H ). Latex agglutination test detects polysaccharide capsular antigen and is more sensitive and specific. Causes cryptococcosis, cryptococcal meningitis, cryptococcal encephalitis (“soap bubble” lesions in brain), primarily in immunocompromised. Treatment: amphotericin B + flucytosine followed by fluconazole for cryptococcal meningitis. Irregular, broad, nonseptate hyphae branching at wide angles I .

1	Treatment: amphotericin B + flucytosine followed by fluconazole for cryptococcal meningitis. Irregular, broad, nonseptate hyphae branching at wide angles I . Causes mucormycosis, mostly in ketoacidotic diabetic and/or neutropenic patients (eg, leukemia). Inhalation of spores  fungi proliferate in blood vessel walls, penetrate cribriform plate, and enter brain. Rhinocerebral, frontal lobe abscess; cavernous sinus thrombosis. Headache, facial pain, black necrotic eschar on face J ; may have cranial nerve involvement. Treatment: surgical debridement, amphotericin B or isavuconazole.

1	Treatment: surgical debridement, amphotericin B or isavuconazole. Pneumocystis jirovecii Causes Pneumocystis pneumonia (PCP), a diffuse interstitial pneumonia A . Yeast-like fungus (originally classified as protozoan). Most infections are asymptomatic. Immunosuppression (eg, AIDS) predisposes to disease. Diffuse, bilateral ground-glass opacities on chest imaging, with pneumatoceles B . Diagnosed by bronchoalveolar lavage or lung biopsy. Disc-shaped yeast seen on methenamine silver stain of lung tissue C or with fluorescent antibody. Treatment/prophylaxis: TMP-SMX, pentamidine, dapsone (prophylaxis as single agent, or treatment in combination with TMP), atovaquone. Start prophylaxis when CD4+ count drops to < 200 cells/mm3 in HIV patients.

1	Causes sporotrichosis. Dimorphic fungus. Exists as a cigar-shaped yeast at 37 ºC in the human body and as hyphae with spores in soil (conidia). Lives on vegetation. When spores are traumatically introduced into the skin, typically by a thorn (“rose gardener’s disease”), causes local pustule or ulcer with nodules along draining lymphatics (ascending lymphangitis A ). Disseminated disease possible in immunocompromised host. Treatment: itraconazole or potassium iodide (only for cutaneous/lymphocutaneous). Think of a rose gardener who smokes a cigar and pot. Toxoplasma Immunocompetent: gondii mononucleosis-like symptoms, ⊝ heterophile antibody test Reactivation in AIDS  brain abscesses usually seen as multiple ring-enhancing lesions on MRI A Congenital toxoplasmosis: classic triad of chorioretinitis, hydrocephalus, and intracranial calcifications Cysts in meat (most Serology, biopsy Sulfadiazine + common); oocysts (tachyzoite)

1	Plasmodium P vivax/ovale P falciparum P malariae A B Malaria—fever, headache, anemia, splenomegaly P vivax/ovale—48-hr cycle (tertian; includes fever on first day and third day, thus fevers are actually 48 hr apart); dormant form (hypnozoite) in liver P falciparum—severe; irregular fever patterns; parasitized RBCs occlude capillaries in brain (cerebral malaria), kidneys, lungs P malariae—72-hr cycle (quartan) Anopheles mosquito Blood smear: trophozoite ring form within RBC A , schizont containing merozoites; red granules (Schüffner stippling) B throughout RBC cytoplasm seen with P vivax/ovale Chloroquine (for sensitive species); if resistant, use mefloquine or atovaquone/ proguanil If life-threatening, use intravenous quinidine or artesunate (test for G6PD deficiency) For P vivax/ovale, add primaquine for hypnozoite (test for G6PD deficiency) Babesia C Babesiosis—fever and hemolytic anemia; predominantly in northeastern United States; asplenia  risk of severe disease Ixodes tick

1	add primaquine for hypnozoite (test for G6PD deficiency) Babesia C Babesiosis—fever and hemolytic anemia; predominantly in northeastern United States; asplenia  risk of severe disease Ixodes tick (also vector for Borrelia burgdorferi and Anaplasma spp) Blood smear: ring form C1 , “Maltese cross” C2 ; PCR Atovaquone + azithromycin

1	Intestinal tapeworm Ingestion of larvae encysted in Praziquantel undercooked pork Cysticercosis, Ingestion of eggs in food Praziquantel; albendazole for neurocysticercosis (cystic contaminated with human neurocysticercosis CNS lesions, seizures) D (“eggshell Ingestion of eggs in food Albendazole calcification”) in liver E ; cyst contaminated with dog feces rupture can cause anaphylaxis Sheep are an intermediate host Liver and spleen enlargement (S mansoni, egg with lateral spine A ), fibrosis, inflammation, portal hypertension Chronic infection with S haematobium (egg with terminal spine B ) can lead to squamous cell carcinoma of the bladder (painless hematuria) and pulmonary hypertension Snails are intermediate host; Praziquantel cercariae penetrate skin of humans in contact with contaminated fresh water (eg, swimming or bathing) Associated with cholangiocarcinoma

1	Associated with cholangiocarcinoma Sarcoptes scabiei Mites burrow into stratum corneum and Common in children, crowded populations cause scabies—pruritus (worse at night) and (jails, nursing homes); transmission through serpiginous burrows (lines) often between skin-to-skin contact (most common) or via fingers and toes A . fomites. Treatment: permethrin cream, washing/drying all clothing/bedding, treat close contacts. Pediculus humanus/ Blood-sucking lice that cause intense pruritus Phthirus pubis with associated excoriations, commonly on scalp and neck (head lice), waistband and axilla (body lice), or pubic and perianal regions (pubic lice). Body lice can transmit Rickettsia prowazekii (epidemic typhus), Borrelia recurrentis (relapsing fever), Bartonella quintana (trench fever). Treatment: pyrethroids, malathion, or ivermectin lotion, and nit B combing. Children with head lice can be treated at home without interrupting school attendance.

1	Treatment: pyrethroids, malathion, or ivermectin lotion, and nit B combing. Children with head lice can be treated at home without interrupting school attendance. Biliary tract disease, cholangiocarcinoma Clonorchis sinensis Brain cysts, seizures Taenia solium (neurocysticercosis) Naked virus Enveloped virus Enveloped virus Bacteriophage with icosahedral capsid with icosahedral capsid with helical capsid Generally, enveloped viruses acquire their envelopes from plasma membrane when they exit from cell. Exceptions include herpesviruses, which acquire envelopes from nuclear membrane. Naked (nonenveloped) viruses include Papillomavirus, Adenovirus, Parvovirus, Polyomavirus, Calicivirus, Picornavirus, Reovirus, and Hepevirus. Purified nucleic acids of most dsDNA viruses (except poxviruses and HBV) and ⊕ strand ssRNA (≈ mRNA) viruses are infectious. Naked nucleic acids of ⊝ strand ssRNA and dsRNA viruses are not infectious. They require polymerases contained in the complete virion.

1	DNA = PAPP; RNA = CPR and hepevirus. Give PAPP smears and CPR to a naked hippie (hepevirus). Enveloped DNA viruses Have Helpful Protection (Herpesvirus, Hepadnavirus, Poxvirus). Some general rules—all DNA viruses: DNA viruses All replicate in the nucleus (except poxvirus). “Pox is out of the box (nucleus).” Papillomavirus No DS and circular HPV–warts (serotypes 1, 2, 6, 11), CIN, cervical cancer (most commonly 16, 18) (PML) in HIV BK virus—transplant patients, commonly targets kidney JC: Junky Cerebrum; BK: Bad Kidney Parvovirus No SS and linear B19 virus—aplastic crises in sickle cell disease, (smallest DNA virus) “slapped cheek” rash in children (erythema infectiosum, or fifth disease); infects RBC precursors and endothelial cells  RBC destruction • hydrops fetalis and death in fetus, pure RBC aplasia and rheumatoid arthritis–like symptoms in adults Herpesviruses Enveloped, DS, and linear viruses

1	Herpesviruses Enveloped, DS, and linear viruses Herpes Respiratory Gingivostomatitis, keratoconjunctivitis A , Most commonly latent in trigeminal simplex secretions, saliva herpes labialis (cold sores) B , herpetic ganglia virus-1 whitlow on finger, temporal lobe encephalitis, Most common cause of sporadic esophagitis, erythema multiforme encephalitis, can present as altered mental status, seizures, and/or aphasia Herpes Sexual contact, Herpes genitalis C , neonatal herpes Most commonly latent in sacral simplex perinatal ganglia virus-2 Viral meningitis more common with HSV-2 than with HSV-1 Varicella-Respiratory Varicella-zoster (chickenpox D , shingles E ), Latent in dorsal root or trigeminal Zoster virus secretions, encephalitis, pneumonia ganglia; CN V1 branch (HHV-3) contact with fluid Most common complication of shingles is post-involvement can cause herpes Epstein-Barr Respiratory virus (HHV-4) secretions, saliva; aka “kissing disease,” (common in teens, young adults)

1	Epstein-Barr Respiratory virus (HHV-4) secretions, saliva; aka “kissing disease,” (common in teens, young adults) Mononucleosis—fever, hepatosplenomegaly F , pharyngitis, and lymphadenopathy (especially posterior cervical nodes); avoid contact sports until resolution due to risk of splenic rupture Associated with lymphomas (eg, endemic Burkitt lymphoma), nasopharyngeal carcinoma (especially Asian adults), lymphoproliferative disease in transplant patients Infects B cells through CD21, “Must be 21 to drink Beer in a Barr” ⊕ Monospot test—heterophile antibodies detected by agglutination of sheep or horse RBCs Use of amoxicillin in mononucleosis can cause characteristic maculopapular rash HSV identification Viral culture for skin/genitalia. CSF PCR for herpes encephalitis. Tzanck test—a smear of an opened skin vesicle to detect multinucleated giant cells A commonly seen in HSV-1, HSV-2, and VZV infection. PCR of skin lesions is test of choice. Tzanck heavens I do not have herpes.

1	Tzanck heavens I do not have herpes. Intranuclear eosinophilic Cowdry A inclusions also seen with HSV-1, HSV-2, VZV. HIV CD4, CXCR4, CCR5 Rhinovirus ICAM-1 (I came to see the rhino) RNA viruses All replicate in the cytoplasm (except retrovirus and influenza virus). “Retro flu is outta cyt (sight).” SS, single-stranded; DS, double-stranded; ⊕, positive sense; ⊝, negative sense; a= arbovirus, arthropod borne (mosquitoes, ticks). Influenza viruses Orthomyxoviruses. Enveloped, ⊝ ssRNA viruses with 8-segment genome. Contain hemagglutinin (binds sialic acid and promotes viral entry) and neuraminidase (promotes progeny virion release) antigens. Patients at risk for fatal bacterial superinfection, most commonly S aureus, S pneumoniae, and H influenzae. Reformulated vaccine (“the flu shot”) contains viral strains most likely to appear during the flu season, due to the virus’ rapid genetic change. Killed viral vaccine is most frequently used.

1	Reformulated vaccine (“the flu shot”) contains viral strains most likely to appear during the flu season, due to the virus’ rapid genetic change. Killed viral vaccine is most frequently used. Live attenuated vaccine contains temperature-sensitive mutant that replicates in the nose but not in the lung; administered intranasally. Treatment: supportive +/– neuraminidase inhibitor (eg, oseltamivir, zanamivir). Rubella virus A togavirus. Causes rubella, once known as German (3-day) measles. Fever, postauricular and other lymphadenopathy, arthralgias, and fine, maculopapular rash that starts on face and spreads centrifugally to involve trunk and extremities A . Causes mild disease in children but serious congenital disease (a TORCH infection). Congenital rubella findings include “blueberry muffin” appearance due to dermal extramedullary hematopoiesis.

1	Paramyxoviruses Paramyxoviruses cause disease in children. They include those that cause parainfluenza (croup), mumps, measles, RSV, and human metapneumovirus, which causes respiratory tract infection (bronchiolitis, pneumonia) in infants. All contain surface F (fusion) protein, which causes respiratory epithelial cells to fuse and form multinucleated cells. Palivizumab (monoclonal antibody against F protein) prevents pneumonia caused by RSV infection in premature infants. Palivizumab for Paramyxovirus (RSV) Prophylaxis in Preemies.

1	Acute Also called croup. Caused by parainfluenza viruses. Virus membrane contains hemagglutinin laryngotracheobronchitis (binds sialic acid and promotes viral entry) and neuraminidase (promotes progeny virion release) antigens. Results in a “seal-like” barking cough and inspiratory stridor. Narrowing of upper trachea and subglottis leads to characteristic steeple sign on x-ray A . Severe croup can result in pulsus paradoxus 2° to upper airway obstruction. Mumps virus Uncommon due to effectiveness of MMR Mumps makes your parotid glands and testes as vaccine. big as POM-Poms. Symptoms: Parotitis A , Orchitis (inflammation of testes), aseptic Meningitis, and Pancreatitis. Can cause sterility (especially after puberty).

1	Symptoms: Parotitis A , Orchitis (inflammation of testes), aseptic Meningitis, and Pancreatitis. Can cause sterility (especially after puberty). A . Negri bodies (cytoplasmic inclusions B ) commonly found in Purkinje cells of cerebellum and in hippocampal neurons. Rabies has long incubation period (weeks to months) before symptom onset. Postexposure prophylaxis is wound cleaning plus immunization with killed vaccine and rabies immunoglobulin. Example of passive-active immunity. Travels to the CNS by migrating in a retrograde fashion (via dynein motors) up nerve axons after binding to ACh receptors. Progression of disease: fever, malaise • agitation, photophobia, hydrophobia, hypersalivation • paralysis, coma • death. Infection more commonly from bat, raccoon, and skunk bites than from dog bites in the United States; aerosol transmission (eg, bat caves) also possible.

1	Infection more commonly from bat, raccoon, and skunk bites than from dog bites in the United States; aerosol transmission (eg, bat caves) also possible. A filovirus A that targets endothelial cells, phagocytes, hepatocytes. Following an incubation period of up to 21 days, presents with abrupt onset of flu-like symptoms, diarrhea/vomiting, high fever, myalgia. Can progress to DIC, diffuse hemorrhage, shock. Diagnosed with RT-PCR within 48 hr of symptom onset. High mortality rate. Transmission requires direct contact with bodily fluids, fomites (including dead bodies), infected bats or primates (apes/monkeys); high incidence of nosocomial infection. Supportive care, no definitive treatment. Strict isolation of infected individuals and barrier practices for health care workers are key to preventing transmission. Extrahepatic manifestations of hepatitis B and C Anti-HBe Antibody to HBeAg; indicates low transmissibility. Relative concentration of reactants Level of detection

1	Extrahepatic manifestations of hepatitis B and C Anti-HBe Antibody to HBeAg; indicates low transmissibility. Relative concentration of reactants Level of detection Incubation Prodrome, period acute disease gp120: gp41: Diploid genome (2 molecules of RNA). The 3 structural genes (protein coded for): p17: Matrix protein • env (gp120 and gp41): • Formed from cleavage of gp160 to form Lipid envelope envelope glycoproteins. • gp120—attachment to host CD4+ T cell. p24: • gp41—fusion and entry. RNA proteins, respectively. • pol—Reverse transcriptase, Integrase, Protease; RIP “Pol” (Paul) Reverse transcriptase synthesizes dsDNA from genomic RNA; dsDNA integrates into host genome. Virus binds CD4 as well as a coreceptor, either CCR5 on macrophages (early infection) or CXCR4 on T cells (late infection). Homozygous CCR5 mutation = immunity. Heterozygous CCR5 mutation = slower course.

1	Homozygous CCR5 mutation = immunity. Heterozygous CCR5 mutation = slower course. Presumptive diagnosis made with HIV-1/2 Ag/ Western blot tests are no longer recommended Ab immunoassays. These immunoassays by the CDC for confirmatory testing. detect viral p24 Ag capsid protein and IgG Abs HIV-1/2 Ag/Ab testing is not recommended in to HIV-1/2. Very high sensitivity/specificity. babies with suspected HIV due to maternally Viral load tests determine the amount of viral transferred antibody. Use HIV viral load RNA in the plasma. High viral load associated instead. with poor prognosis. Also use viral load to monitor effect of drug therapy. Use HIV genotyping to determine appropriate therapy. AIDS diagnosis: ≤ 200 CD4+ cells/mm3 (normal: 500–1500 cells/mm3) or HIV ⊕ with AIDS-defining condition (eg, Pneumocystis pneumonia). HIV-1 NAT HIV-1 NAT NAT: nucleic acid test

1	AIDS diagnosis: ≤ 200 CD4+ cells/mm3 (normal: 500–1500 cells/mm3) or HIV ⊕ with AIDS-defining condition (eg, Pneumocystis pneumonia). HIV-1 NAT HIV-1 NAT NAT: nucleic acid test Time course of untreated HIV infection +/– Acute HIV infection Skin and mucous Systemic immuno-Viral dissemination membrane defciency/AIDS-Seeding of lymphoid organs Clinical latency infections defning illnesses Dashed lines on CD4+ count axis indicate moderate immunocompromise Four stages of untreated infection: (< 400 CD4+ cells/mm3) and when AIDS-defining illnesses emerge (< 200 1. Flu-like (acute) CD4+ cells/mm3). 2. Feeling fine (latent) Most patients who do not receive treatment eventually die of complications of 3. Falling count HIV infection. 4. Final crisis During clinical latency phase, virus replicates in lymph nodes

1	Most patients who do not receive treatment eventually die of complications of 3. Falling count HIV infection. 4. Final crisis During clinical latency phase, virus replicates in lymph nodes Common diseases of  CD4+ cell count  reactivation of past infections (eg, TB, HSV, shingles), dissemination of HIV-positive adults bacterial infections and fungal infections (eg, coccidioidomycosis), and non-Hodgkin lymphomas. Prions Prion diseases are caused by the conversion of a normal (predominantly α-helical) protein termed prion protein (PrPc) to a β-pleated form (PrPsc), which is transmissible via CNS-related tissue (iatrogenic CJD) or food contaminated by BSE-infected animal products (variant CJD). PrPsc resists protease degradation and facilitates the conversion of still more PrPc to PrPsc. Resistant to standard sterilizing procedures, including standard autoclaving. Accumulation of PrPsc results in spongiform encephalopathy and dementia, ataxia, and death.

1	Creutzfeldt-Jakob disease—rapidly progressive dementia, typically sporadic (some familial forms). Bovine spongiform encephalopathy—also called “mad cow disease.” Kuru—acquired prion disease noted in tribal populations practicing human cannibalism. Normal flora: Neonates delivered by C-section have no flora but are rapidly colonized after birth. dominant B cereus Reheated rice. “Food poisoning from reheated rice? Be serious!” (B cereus) C botulinum Improperly canned foods (toxins), raw honey (spores) Salmonella Poultry, meat, and eggs S aureus Meats, mayonnaise, custard; preformed toxin a V vulnificus can also cause wound infections from contact with contaminated water or shellfish. Common causes of pneumonia Common causes of meningitis Give ceftriaxone and vancomycin empirically (add ampicillin if Listeria is suspected). Viral causes of meningitis: enteroviruses (especially coxsackievirus), HSV-2 (HSV-1 = encephalitis), HIV, West Nile virus (also causes encephalitis), VZV.

1	Viral causes of meningitis: enteroviruses (especially coxsackievirus), HSV-2 (HSV-1 = encephalitis), HIV, West Nile virus (also causes encephalitis), VZV. In HIV: Cryptococcus spp. Note: Incidence of Group B streptococcal meningitis in neonates has  greatly due to screening and antibiotic prophylaxis in pregnancy. Incidence of H influenzae meningitis has  greatly due to conjugate H influenzae vaccinations. Today, cases are usually seen in unimmunized children. Assume if no other information is available S aureus (most common overall) Vertebral involvement S aureus, M tuberculosis (Pott disease) IV drug abuse S aureus; also Pseudomonas, Candida Elevated ESR and CRP sensitive but not specific. Radiographs are insensitive early but can be useful in chronic osteomyelitis ( A , left). MRI is best for detecting acute infection and detailing anatomic involvement ( A , right). sigNs aND symPtoms No inflammation Thin, white discharge

1	sigNs aND symPtoms No inflammation Thin, white discharge A with fishy odor lab FiNDiNgs Clue cells pH > 4.5 ⊕ KOH whiff test Inflammation (“strawberry Inflammation cervix”) Thick, white, “cottage cheese” Frothy, yellow-green, foul-discharge B pH normal (4.0–4.5) pH > 4.5 Metronidazole Azoles Treat sexual partner(s) TORCH infections Microbes that may pass from mother to fetus. Transmission is transplacental in most cases, or via delivery (especially HSV-2). Nonspecific signs common to many ToRCHHeS infections include hepatosplenomegaly, jaundice, thrombocytopenia, and growth retardation. Other important infectious agents include Streptococcus agalactiae (group B streptococci), E coli, and Listeria monocytogenes—all causes of meningitis in neonates. Parvovirus B19 causes hydrops fetalis. Red rashes of childhood Sexually transmitted infections Top bugs—Chlamydia trachomatis (subacute, often undiagnosed), Neisseria gonorrhoeae (acute).

1	Red rashes of childhood Sexually transmitted infections Top bugs—Chlamydia trachomatis (subacute, often undiagnosed), Neisseria gonorrhoeae (acute). C trachomatis—most common bacterial STI in the United States. Signs include cervical motion tenderness, adnexal tenderness, purulent cervical discharge A . PID may include salpingitis, endometritis, hydrosalpinx, and tubo-ovarian abscess. Salpingitis is a risk factor for ectopic pregnancy, infertility, chronic pelvic pain, and adhesions. Can lead to perihepatitis (Fitz-Hugh–Curtis syndrome)—infection and inflammation of liver capsule and “violin string” adhesions of peritoneum to liver B . Nosocomial infections E coli (UTI) and S aureus (wound infection) are the two most common causes. Rash Beginning at head and moving down with Rubella virus postauricular lymphadenopathy Beginning at head and moving down; preceded by Measles virus cough, coryza, conjunctivitis, and Koplik spots

1	Rash Beginning at head and moving down with Rubella virus postauricular lymphadenopathy Beginning at head and moving down; preceded by Measles virus cough, coryza, conjunctivitis, and Koplik spots Can also lead to myalgia and paralysis Poliovirus Tetanus Muscle spasms and spastic paralysis (eg, lockjaw, Clostridium tetani Branching rods in oral infection, sulfur granules Actinomyces israelii Chronic granulomatous disease Catalase ⊕ microbes, especially S aureus Human bite Human oral flora (eg, Eikenella, Fusobacterium) Neutropenic patients Candida albicans (systemic), Aspergillus Pneumonia in cystic fibrosis, burn infection Pseudomonas aeruginosa Puncture wound, lockjaw Clostridium tetani Pus, empyema, abscess S aureus

1	Fluoroquinolones Ciprofoxacin Levofoxacin, etc Quinolone Nalidixic acid Chloramphenicol Clindamycin Linezolid Macrolides Azithromycin Clarithromycin Erythromycin Streptogramins Quinupristin Dalfopristin Rifampin Aminoglycosides Gentamicin Glycylcycline Tigecycline Neomycin Amikacin Tobramycin Streptomycin Tetracyclines Tetracycline Doxycycline Minocycline PROTEIN SYNTHESIS Penicillinase-sensitive penicillins Penicillin G, V Ampicillin Amoxicillin Penicillinase-resistant penicillins Oxacillin Nafcillin Dicloxacillin Antipseudomonal Ticarcillin Piperacillin Cephalosporins (I-V) 1st—Cefazolin, etc 2nd—Cefoxitin, etc 3rd—Ceftriaxone, etc 4th—Cefepime 5th—Ceftaroline PEPTIDOGLYCAN SYNTHESIS CELL WALL SYNTHESIS MEMBRANE INTEGRITY GYRASEDNA INTEGRITY (via free radicals) mRNA SYNTHESIS (RNA polymerase) Carbapenems Imipenem Meropenem Ertapenem Doripenem Monobactams Aztreonam PEPTIDOGLYCAN CROSS-LINKING Glycopeptides Vancomycin Bacitracin 30S SUBUNIT 50S SUBUNIT 50S 30S30S 50S 30S 50S

1	polymerase) Carbapenems Imipenem Meropenem Ertapenem Doripenem Monobactams Aztreonam PEPTIDOGLYCAN CROSS-LINKING Glycopeptides Vancomycin Bacitracin 30S SUBUNIT 50S SUBUNIT 50S 30S30S 50S 30S 50S Sulfonamides Sulfamethoxazole Sulfsoxazole Sulfadiazine FOLIC ACID SYNTHESIS AND REDUCTION (DNA methylation) PABA DHF THF Trimethoprim Cellmembrane Cellwall BACTERIAL CELL DNA mRNA DNA gyrase Ribosomes RNA polymerase – – – – – – – – – Metronidazole – Daptomycin (gram ˜) Polymyxins (gram °)

1	Penicillin G, V Penicillin G (IV and IM form), penicillin V (oral). Prototype β-lactam antibiotics. mecHaNism D-Ala-D-Ala structural analog. Bind penicillin-binding proteins (transpeptidases). Block transpeptidase cross-linking of peptidoglycan in cell wall. Activate autolytic enzymes. cliNical Use Mostly used for gram ⊕ organisms (S pneumoniae, S pyogenes, Actinomyces). Also used for gram ⊝ cocci (mainly N meningitidis) and spirochetes (mainly T pallidum). Bactericidal for gram ⊕ cocci, gram ⊕ rods, gram ⊝ cocci, and spirochetes. β-lactamase sensitive. aDVerse eFFects Hypersensitivity reactions, direct Coombs ⊕ hemolytic anemia, drug-induced interstitial nephritis. resistaNce β-lactamase cleaves the β-lactam ring. Mutations in PBPs. aDVerse eFFects Hypersensitivity reactions, rash, pseudomembranous colitis. mecHaNism oF resistaNce Penicillinase (a type of β-lactamase) cleaves β-lactam ring. Penicillinase-resistant penicillins Dicloxacillin, nafcillin, oxacillin.

1	mecHaNism oF resistaNce Penicillinase (a type of β-lactamase) cleaves β-lactam ring. Penicillinase-resistant penicillins Dicloxacillin, nafcillin, oxacillin. mecHaNism Same as penicillin. Narrow spectrum; penicillinase resistant because bulky R group blocks access of β-lactamase to β-lactam ring. cliNical Use S aureus (except MRSA). “Use naf (nafcillin) for staph.” aDVerse eFFects Hypersensitivity reactions, interstitial nephritis. mecHaNism oF resistaNce MRSA has altered penicillin-binding protein target site. Antipseudomonal penicillins Piperacillin, ticarcillin. mecHaNism Same as penicillin. Extended spectrum. Penicillinase sensitive; use with β-lactamase inhibitors. cliNical Use Pseudomonas spp. and gram ⊝ rods. aDVerse eFFects Hypersensitivity reactions. 4th generation (cefepime)—gram ⊝ organisms, with  activity against Pseudomonas and gram ⊕ organisms.

1	cliNical Use Pseudomonas spp. and gram ⊝ rods. aDVerse eFFects Hypersensitivity reactions. 4th generation (cefepime)—gram ⊝ organisms, with  activity against Pseudomonas and gram ⊕ organisms. 5th generation (ceftaroline)—broad gram ⊕ and gram ⊝ organism coverage; unlike 1st–4th generation cephalosporins, ceftaroline covers MRSA, and Enterococcus faecalis—does not cover Pseudomonas. aDVerse eFFects Hypersensitivity reactions, autoimmune hemolytic anemia, disulfiram-like reaction, vitamin K deficiency. Low rate of cross-reactivity even in penicillin-allergic patients. • nephrotoxicity of aminoglycosides. mecHaNism oF resistaNce Inactivated by cephalosporinases (a type of β-lactamase). Structural change in penicillin-binding proteins (transpeptidases).

1	• nephrotoxicity of aminoglycosides. mecHaNism oF resistaNce Inactivated by cephalosporinases (a type of β-lactamase). Structural change in penicillin-binding proteins (transpeptidases). cliNical Use Gram ⊕ cocci, gram ⊝ rods, and anaerobes. Wide spectrum and significant side effects limit use to life-threatening infections or after other drugs have failed. Meropenem has a  risk of seizures and is stable to dehydropeptidase I. aDVerse eFFects GI distress, rash, and CNS toxicity (seizures) at high plasma levels. mecHaNism oF resistaNce Inactivated by carbapenemases produced by, eg, K pneumoniae, E coli, E aerogenes. mecHaNism Less susceptible to β-lactamases. Prevents peptidoglycan cross-linking by binding to penicillin-binding protein 3. Synergistic with aminoglycosides. No cross-allergenicity with penicillins.

1	cliNical Use Gram ⊝ rods only—no activity against gram ⊕ rods or anaerobes. For penicillin-allergic patients and those with renal insufficiency who cannot tolerate aminoglycosides. aDVerse eFFects Usually nontoxic; occasional GI upset. mecHaNism Inhibits cell wall peptidoglycan formation by binding D-Ala-D-Ala portion of cell wall precursors. Bactericidal against most bacteria (bacteriostatic against C difficile). Not susceptible to β-lactamases. cliNical Use Gram ⊕ bugs only—for serious, multidrug-resistant organisms, including MRSA, S epidermidis, sensitive Enterococcus species, and Clostridium difficile (oral dose for pseudomembranous colitis). aDVerse eFFects Well tolerated in general but NOT trouble Free. Nephrotoxicity, Ototoxicity, Thrombophlebitis, diffuse Flushing (red man syndrome A idiopathic reaction largely preventable by pretreatment with antihistamines), DRESS syndrome.

1	mecHaNism oF resistaNce Occurs in bacteria (eg, Enterococcus) via amino acid modification of D-Ala-D-Ala to D-Ala-D-Lac. “If you Lack a D-Ala (dollar), you can’t ride the van (vancomycin).” Specifically target smaller bacterial ribosome (70S, made of 30S and 50S subunits), leaving human ribosome (80S) unaffected. All are bacteriostatic, except aminoglycosides (bactericidal) and linezolid (variable). Chloramphenicol, Clindamycin Erythromycin (macrolides) Linezolid “Buy AT 30, CCEL (sell) at 50.” cliNical Use Severe gram ⊝ rod infections. Synergistic with β-lactam antibiotics. Neomycin for bowel surgery. aDVerse eFFects Nephrotoxicity, Neuromuscular blockade (absolute contraindication with myasthenia gravis), Ototoxicity (especially with loop diuretics), Teratogenicity. mecHaNism oF resistaNce Bacterial transferase enzymes inactivate the drug by acetylation, phosphorylation, or adenylation. Tetracyclines Tetracycline, doxycycline, minocycline.

1	mecHaNism oF resistaNce Bacterial transferase enzymes inactivate the drug by acetylation, phosphorylation, or adenylation. Tetracyclines Tetracycline, doxycycline, minocycline. mecHaNism Bacteriostatic; bind to 30S and prevent attachment of aminoacyl-tRNA. Limited CNS penetration. Doxycycline is fecally eliminated and can be used in patients with renal failure. Do not take tetracyclines with milk (Ca2+), antacids (eg, Ca2+ or Mg2+), or iron-containing preparations because divalent cations inhibit drugs’ absorption in the gut. cliNical Use Borrelia burgdorferi, M pneumoniae. Drugs’ ability to accumulate intracellularly makes them very effective against Rickettsia and Chlamydia. Also used to treat acne. Doxycycline effective against community-acquired MRSA. aDVerse eFFects GI distress, discoloration of teeth and inhibition of bone growth in children, photosensitivity. Contraindicated in pregnancy.

1	aDVerse eFFects GI distress, discoloration of teeth and inhibition of bone growth in children, photosensitivity. Contraindicated in pregnancy. mecHaNism oF resistaNce  uptake or  efflux out of bacterial cells by plasmid-encoded transport pumps. mecHaNism Tetracycline derivative. Binds to 30S, inhibiting protein synthesis. Generally bacteriostatic. cliNical Use Broad-spectrum anaerobic, gram ⊝, and gram ⊕ coverage. Multidrug-resistant organisms (MRSA, VRE) or infections requiring deep tissue penetration. aDVerse eFFects GI symptoms: nausea, vomiting. mecHaNism Blocks peptidyltransferase at 50S ribosomal subunit. Bacteriostatic. cliNical Use Meningitis (Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae) and rickettsial diseases (eg, Rocky Mountain spotted fever [Rickettsia rickettsii]). Limited use due to toxicity but often still used in developing countries because of low cost.

1	aDVerse eFFects Anemia (dose dependent), aplastic anemia (dose independent), gray baby syndrome (in premature infants because they lack liver UDP-glucuronosyltransferase). mecHaNism oF resistaNce Plasmid-encoded acetyltransferase inactivates the drug. mecHaNism Blocks peptide transfer (translocation) at 50S ribosomal subunit. Bacteriostatic. aDVerse eFFects Pseudomembranous colitis (C difficile overgrowth), fever, diarrhea. mecHaNism Inhibits protein synthesis by binding to 50S subunit and preventing formation of the initiation complex. cliNical Use Gram ⊕ species including MRSA and VRE. Macrolides Azithromycin, clarithromycin, erythromycin. mecHaNism Inhibit protein synthesis by blocking translocation (“macroslides”); bind to the 23S rRNA of the 50S ribosomal subunit. Bacteriostatic. cliNical Use Atypical pneumonias (Mycoplasma, Chlamydia, Legionella), STIs (Chlamydia), gram ⊕ cocci (streptococcal infections in patients allergic to penicillin), and B pertussis.

1	cliNical Use Atypical pneumonias (Mycoplasma, Chlamydia, Legionella), STIs (Chlamydia), gram ⊕ cocci (streptococcal infections in patients allergic to penicillin), and B pertussis. aDVerse eFFects MACRO: Gastrointestinal Motility issues, Arrhythmia caused by prolonged QT interval, acute Cholestatic hepatitis, Rash, eOsinophilia. Increases serum concentration of theophylline, oral anticoagulants. Clarithromycin and erythromycin inhibit cytochrome P-450. mecHaNism oF resistaNce Methylation of 23S rRNA-binding site prevents binding of drug. Polymyxins Colistin (polymyxin E), polymyxin B. mecHaNism Cation polypeptides that bind to phospholipids on cell membrane of gram ⊝ bacteria. Disrupt cell membrane integrity  leakage of cellular components  cell death. cliNical Use Salvage therapy for multidrug-resistant gram ⊝ bacteria (eg, P aeruginosa, E coli, K pneumoniae). Polymyxin B is a component of a triple antibiotic ointment used for superficial skin infections.

1	aDVerse eFFects Nephrotoxicity, neurotoxicity (eg, slurred speech, weakness, paresthesias), respiratory failure. Sulfonamides Sulfamethoxazole (SMX), sulfisoxazole, sulfadiazine. mecHaNism Inhibit dihydropteroate synthase, thus inhibiting folate synthesis. Bacteriostatic (bactericidal when combined with trimethoprim). Sulfonamides, dapsonecliNical Use Gram ⊕, gram ⊝, Nocardia. TMP-SMX for synthase simple UTI. aDVerse eFFects Hypersensitivity reactions, hemolysis if G6PD deficient, nephrotoxicity (tubulointerstitial nephritis), photosensitivity, Stevens-Johnson syndrome, kernicterus in infants, displace other drugs from albumin (eg, warfarin). mecHaNism oF resistaNce Altered enzyme (bacterial dihydropteroate synthase),  uptake, or  PABA synthesis. Trimethoprim, mecHaNism Similar to sulfonamides, but structurally distinct Purines Thymidine Methionine agent.

1	Trimethoprim, mecHaNism Similar to sulfonamides, but structurally distinct Purines Thymidine Methionine agent. cliNical Use Leprosy (lepromatous and tuberculoid), DNA, RNA DNA Protein Pneumocystis jirovecii prophylaxis, or treatment when used in combination with TMP. aDVerse eFFects Hemolysis if G6PD deficient, methemoglobinemia, agranulocytosis. mecHaNism Inhibits bacterial dihydrofolate reductase. Bacteriostatic. cliNical Use Used in combination with sulfonamides (trimethoprim-sulfamethoxazole [TMPSMX]), causing sequential block of folate synthesis. Combination used for UTIs, Shigella, Salmonella, Pneumocystis jirovecii pneumonia treatment and prophylaxis, toxoplasmosis prophylaxis. aDVerse eFFects Hyperkalemia (high doses), megaloblastic anemia, leukopenia, granulocytopenia, which may be avoided with coadministration of leucovorin (folinic acid). TMP Treats Marrow Poorly.

1	aDVerse eFFects Hyperkalemia (high doses), megaloblastic anemia, leukopenia, granulocytopenia, which may be avoided with coadministration of leucovorin (folinic acid). TMP Treats Marrow Poorly. cliNical Use Gram ⊝ rods of urinary and GI tracts (including Pseudomonas), some gram ⊕ organisms, otitis externa. mecHaNism oF resistaNce Chromosome-encoded mutation in DNA gyrase, plasmid-mediated resistance, efflux pumps. mecHaNism Lipopeptide that disrupts cell membranes of gram ⊕ cocci by creating transmembrane channels. aDVerse eFFects Myopathy, rhabdomyolysis. mecHaNism Forms toxic free radical metabolites in the bacterial cell that damage DNA. Bactericidal, antiprotozoal. aDVerse eFFects Disulfiram-like reaction (severe flushing, tachycardia, hypotension) with alcohol; headache, metallic taste. Plasma Cell wall membrane Interior of cell Rifamycins Rifampin, rifabutin.

1	Plasma Cell wall membrane Interior of cell Rifamycins Rifampin, rifabutin. mecHaNism Inhibit DNA-dependent RNA polymerase. Rifampin’s 4R’s: cliNical Use Mycobacterium tuberculosis; delay resistance to dapsone when used for leprosy. Used for meningococcal prophylaxis and chemoprophylaxis in contacts of children with H influenzae type b. aDVerse eFFects Minor hepatotoxicity and drug interactions ( cytochrome P-450); orange body fluids (nonhazardous side effect). Rifabutin favored over rifampin in patients with HIV infection due to less cytochrome P-450 stimulation. Rifampin ramps up cytochrome P-450, but rifabutin does not. mecHaNism oF resistaNce Mutations reduce drug binding to RNA polymerase. Monotherapy rapidly leads to resistance. mecHaNism  synthesis of mycolic acids. Bacterial catalaseperoxidase (encoded by KatG) needed to convert INH to active metabolite. mecHaNism oF resistaNce Mutations leading to underexpression of KatG.

1	mecHaNism oF resistaNce Mutations leading to underexpression of KatG. mecHaNism Mechanism uncertain. Pyrazinamide is a prodrug that is converted to the active compound pyrazinoic acid. Works best at acidic pH (eg, in host phagolysosomes). cliNical Use Mycobacterium tuberculosis. aDVerse eFFects Hyperuricemia, hepatotoxicity. mecHaNism  carbohydrate polymerization of mycobacterium cell wall by blocking arabinosyltransferase. cliNical Use Mycobacterium tuberculosis. aDVerse eFFects Optic neuropathy (red-green color blindness, usually reversible). Pronounce “eyethambutol.” mecHaNism Interferes with 30S component of ribosome. cliNical Use Mycobacterium tuberculosis (2nd line). aDVerse eFFects Tinnitus, vertigo, ataxia, nephrotoxicity. Antimicrobial cliNicalsceNario meDicatioN prophylaxis Exposure to meningococcal infection Ceftriaxone, ciprofloxacin, or rifampin History of recurrent UTIs TMP-SMX

1	Antimicrobial cliNicalsceNario meDicatioN prophylaxis Exposure to meningococcal infection Ceftriaxone, ciprofloxacin, or rifampin History of recurrent UTIs TMP-SMX Malaria prophylaxis for travelers Atovaquone-proguanil, mefloquine, doxycycline, primaquine, or chloroquine (for areas with sensitive species) Prevention of gonococcal conjunctivitis in Erythromycin ointment on eyes newborn Prevention of postsurgical infection due to Cefazolin Prophylaxis of strep pharyngitis in child with Benzathine penicillin G or oral penicillin V prior rheumatic fever Treatment of highly MRSA: vancomycin, daptomycin, linezolid, tigecycline, ceftaroline, doxycycline. resistant bacteria VRE: linezolid, tigecycline, and streptogramins (quinupristin, dalfopristin). Multidrug-resistantP aeruginosa, multidrug-resistantAcinetobacter baumannii: polymyxins B and E (colistin).

1	aDVerse eFFects Fever/chills (“shake and bake”), hypotension, nephrotoxicity, arrhythmias, anemia, IV phlebitis (“amphoterrible”). Hydration  nephrotoxicity. toxicity. mecHaNism Same as amphotericin B. Topical use only as too toxic for systemic use. cliNical Use “Swish and swallow” for oral candidiasis (thrush); topical for diaper rash or vaginal candidiasis. mecHaNism Inhibits DNA and RNA biosynthesis by conversion to 5-fluorouracil by cytosine deaminase. cliNical Use Systemic fungal infections (especially meningitis caused by Cryptococcus) in combination with amphotericin B. aDVerse eFFects Bone marrow suppression. Azoles Clotrimazole, fluconazole, isavuconazole, itraconazole, ketoconazole, miconazole, voriconazole. mecHaNism Inhibit fungal sterol (ergosterol) synthesis by inhibiting the cytochrome P-450 enzyme that converts lanosterol to ergosterol.

1	mecHaNism Inhibit fungal sterol (ergosterol) synthesis by inhibiting the cytochrome P-450 enzyme that converts lanosterol to ergosterol. cliNical Use Local and less serious systemic mycoses. Fluconazole for chronic suppression of cryptococcal meningitis in AIDS patients and candidal infections of all types. Itraconazole may be used for Blastomyces, Coccidioides, Histoplasma, Sporothrix schenckii. Clotrimazole and miconazole for topical fungal infections. Voriconazole for Aspergillus and some Candida. Isavuconazole for serious Aspergillus and Mucor infections. aDVerse eFFects Testosterone synthesis inhibition (gynecomastia, especially with ketoconazole), liver dysfunction (inhibits cytochrome P-450). mecHaNism Inhibits the fungal enzyme squalene epoxidase. cliNical Use Dermatophytoses (especially onychomycosis—fungal infection of finger or toe nails). aDVerse eFFects GI upset, headaches, hepatotoxicity, taste disturbance. Echinocandins Anidulafungin, caspofungin, micafungin.

1	aDVerse eFFects GI upset, headaches, hepatotoxicity, taste disturbance. Echinocandins Anidulafungin, caspofungin, micafungin. mecHaNism Inhibit cell wall synthesis by inhibiting synthesis of β-glucan. cliNical Use Invasive aspergillosis, Candida. aDVerse eFFects GI upset, flushing (by histamine release). mecHaNism Interferes with microtubule function; disrupts mitosis. Deposits in keratin-containing tissues (eg, nails). cliNical Use Oral treatment of superficial infections; inhibits growth of dermatophytes (tinea, ringworm). aDVerse eFFects Teratogenic, carcinogenic, confusion, headaches, disulfiram-like reaction,  cytochrome P-450 and warfarin metabolism. mecHaNism Blocks detoxification of heme into hemozoin. Heme accumulates and is toxic to plasmodia.

1	mecHaNism Blocks detoxification of heme into hemozoin. Heme accumulates and is toxic to plasmodia. cliNical Use Treatment of plasmodial species other than P falciparum (frequency of resistance in P falciparum is too high). Resistance due to membrane pump that  intracellular concentration of drug. Treat P falciparum with artemether/lumefantrine or atovaquone/proguanil. For life-threatening malaria, use quinidine in US (quinine elsewhere) or artesunate. aDVerse eFFects Retinopathy; pruritus (especially in dark-skinned individuals). Antihelminthic Pyrantel pamoate, Ivermectin, Mebendazole (microtubule inhibitor), Praziquantel ( Ca2+ therapy permeability,  vacuolization), Diethylcarbamazine. Helminths get PIMP’D. Zidovudine (ZDV, Acyclovir, etc (HSV, VZV) Oseltamivir, zanamivir mecHaNism Inhibit influenza neuraminidase  release of progeny virus.

1	Zidovudine (ZDV, Acyclovir, etc (HSV, VZV) Oseltamivir, zanamivir mecHaNism Inhibit influenza neuraminidase  release of progeny virus. Infuenza A, B cliNical Use Treatment and prevention of influenza A and B. Beginning therapy within 48 hours of symptom onset may shorten duration of illness. Acyclovir, famciclovir, valacyclovir Ganciclovir mecHaNism 5′-monophosphate formed by a CMV viral kinase. Guanosine analog. Triphosphate formed by cellular kinases. Preferentially inhibits viral DNA polymerase. cliNical Use CMV, especially in immunocompromised patients. Valganciclovir, a prodrug of ganciclovir, has better oral bioavailability. mecHaNism Viral DNA/RNA polymerase inhibitor and Foscarnet = pyrofosphate analog. HIV reverse transcriptase inhibitor. Binds to pyrophosphate-binding site of enzyme. Does not require any kinase activation. cliNical Use CMV retinitis in immunocompromised patients when ganciclovir fails; acyclovir-resistant HSV.

1	cliNical Use CMV retinitis in immunocompromised patients when ganciclovir fails; acyclovir-resistant HSV. aDVerse eFFects Nephrotoxicity, electrolyte abnormalities (hypoor hypercalcemia, hypoor hyperphosphatemia, hypokalemia, hypomagnesemia) can lead to seizures. mecHaNism oF resistaNce Mutated DNA polymerase. mecHaNism Preferentially inhibits viral DNA polymerase. Does not require phosphorylation by viral kinase. cliNical Use CMV retinitis in immunocompromised patients; acyclovir-resistant HSV. Long half-life. aDVerse eFFects Nephrotoxicity (coadminister with probenecid and IV saline to  toxicity).

1	cliNical Use CMV retinitis in immunocompromised patients; acyclovir-resistant HSV. Long half-life. aDVerse eFFects Nephrotoxicity (coadminister with probenecid and IV saline to  toxicity). HIV therapy Antiretroviral therapy (ART): often initiated at the time of HIV diagnosis. Strongest indication for use with patients presenting with AIDS-defining illness, low CD4+ cell counts (< 500 cells/mm3), or high viral load. Regimen consists of 3 drugs to prevent resistance: 2 NRTIs and preferably an integrase inhibitor. All ARTs are active against HIV-1 and HIV-2 with the exception of NNRTIs and enfuvirtide. Abacavir (ABC) Didanosine (ddI) Emtricitabine (FTC) Lamivudine (3TC) Stavudine (d4T) Tenofovir (TDF) Zidovudine (ZDV, Competitively inhibit nucleotide binding to reverse transcriptase and terminate the DNA chain (lack a 3′ OH group). Tenofovir is a nucleoTide; the others are nucleosides. All need to be phosphorylated to be active.

1	ZDV can be used for general prophylaxis and during pregnancy to  risk of fetal transmission. Have you dined (vudine) with my nuclear (nucleosides) family? Bone marrow suppression (can be reversed with granulocyte colony-stimulating factor [G-CSF] and erythropoietin), peripheral neuropathy, lactic acidosis (nucleosides), anemia (ZDV), pancreatitis (didanosine). Abacavir contraindicated if patient has HLA-B*5701 mutation due to  risk of hypersensitivity. Hepatitis C therapy Chronic HCV infection treated with multidrug therapy that targets specific steps within HCV replication cycle (HCV-encoded proteins). Examples of drugs are provided. Grazoprevir Inhibits NS3/4A, a viral protease, preventing Grazoprevir: headache, fatigue Simeprevir viral replication Simeprevir: photosensitivity reactions, rash

1	Grazoprevir Inhibits NS3/4A, a viral protease, preventing Grazoprevir: headache, fatigue Simeprevir viral replication Simeprevir: photosensitivity reactions, rash Ribavirin Inhibits synthesis of guanine nucleotides by Hemolytic anemia, severe teratogen competitively inhibiting IMP dehydrogenase Used as adjunct in cases refractory to newer medications Disinfection and Goals include the reduction of pathogenic organism counts to safe levels (disinfection) and the sterilization inactivation of all microbes including spores (sterilization). Autoclave Pressurized steam at > 120°C. Sporicidal. May not reliably inactivate prions. Alcohols Denature proteins and disrupt cell membranes. Not sporicidal. Chlorhexidine Denatures proteins and disrupts cell membranes. Not sporicidal. Chlorine Oxidizes and denatures proteins. Sporicidal. SAFe Children Take Really Good Care.

1	Chlorhexidine Denatures proteins and disrupts cell membranes. Not sporicidal. Chlorine Oxidizes and denatures proteins. Sporicidal. SAFe Children Take Really Good Care. “Digressions, objections, delight in mockery, carefree mistrust are signs of health; everything unconditional belongs in pathology.” “You cannot separate passion from pathology any more than you can separate a person’s spirit from his body.”

1	The fundamental principles of pathology are key to understanding diseases in all organ systems. Major topics such as inflammation and neoplasia appear frequently in questions across different organ systems, and such topics are definitely high yield. For example, the concepts of cell injury and inflammation are key to understanding the inflammatory response that follows myocardial infarction, a very common subject of board questions. Similarly, a familiarity with the early cellular changes that culminate in the development of neoplasias—for example, esophageal or colon cancer—is critical. Finally, make sure you recognize the major tumor-associated genes and are comfortable with key cancer concepts such as tumor staging and metastasis.

1	Apoptosis ATP-dependent programmed cell death. Intrinsic and extrinsic pathways; both pathways activate caspases (cytosolic proteases)  cellular breakdown including cell shrinkage, chromatin condensation, membrane blebbing, and formation of apoptotic bodies, which are then phagocytosed. Characterized by deeply eosinophilic cytoplasm and basophilic nucleus, pyknosis, and karyorrhexis. Cell membrane typically remains intact without significant inflammation (unlike necrosis). DNA laddering (fragments in multiples of 180 bp) is a sensitive indicator of apoptosis. Executioner caspases Cytoplasmic bleb Apoptotic body Ligands for macrophage cell receptors Macrophage Cytotoxic T cell Perforin Granzyme B FasL TNF-αTNFRFas Misfolded proteins Hypoxia p53 activation BAX/BAK Cytochrome C Bcl-2 Initiator caspases Initiator caspases Nuclear fragmentation (mitochondrial) pathway (death receptor) pathway DNA damage Radiation, ROS, toxins Cytoskeletal dispersion

1	Necrosis Exogenous injury  plasma membrane damage  cell undergoes enzymatic degradation and protein denaturation, intracellular components leak  local inflammatory reaction (unlike apoptosis). Ischemia Inadequate blood supply to meet demand. Mechanisms include  arterial perfusion (eg, atherosclerosis),  venous drainage (eg, testicular torsion, Budd-Chiari syndrome), shock. Regions most vulnerable to hypoxia/ischemia and subsequent infarction: Brain ACA/MCA/PCA boundary areasa,b Colon Splenic flexure (Griffith point),a rectosigmoid junction (Sudeck point)a aWatershed areas (border zones) receive blood supply from most distal branches of 2 arteries with limited collateral vascularity. These areas are susceptible to ischemia from hypoperfusion. bNeurons most vulnerable to hypoxic-ischemic insults include Purkinje cells of the cerebellum and pyramidal cells of the hippocampus and neocortex (zones 3, 5, 6). Types of infarcts

1	Types of infarcts Red infarct Occurs in venous occlusion and tissues with multiple blood supplies (eg, liver, lung A , intestine, testes), and with reperfusion (eg, after angioplasty). Reperfusion injury is due to damage by free radicals. Pale infarct Occurs in solid organs with a single (endarterial) blood supply (eg, heart, kidney B ). Free radical injury Free radicals damage cells via membrane lipid peroxidation, protein modification, DNA breakage. Initiated via radiation exposure (eg, cancer therapy), metabolism of drugs (phase I), redox reactions, nitric oxide (eg, inflammation), transition metals, WBC (eg, neutrophils, macrophages) oxidative burst. Free radicals can be eliminated by scavenging enzymes (eg, catalase, superoxide dismutase, glutathione peroxidase), spontaneous decay, antioxidants (eg, vitamins A, C, E), and certain metal carrier proteins (eg, transferrin, ceruloplasmin). Examples:

1	Oxygen toxicity: retinopathy of prematurity (abnormal vascularization), bronchopulmonary dysplasia, reperfusion injury after thrombolytic therapy Drug/chemical toxicity: acetaminophen overdose (hepatotoxicity), carbon tetrachloride (converted by cytochrome P-450 into CCl3 free radical  fatty liver [cell injury • apolipoprotein synthesis  fatty change], centrilobular necrosis) Metal storage diseases: hemochromatosis (iron) and Wilson disease (copper) Types of calcification Calcium deposits appear deeply basophilic (arrow in A ) on H&E stain. Lipofuscin A yellow-brown “wear and tear” pigment A associated with normal aging. Composed of polymers of lipids and phospholipids complexed with protein. May be derived through lipid peroxidation of polyunsaturated lipids of subcellular membranes. Autopsy of elderly person will reveal deposits in heart, colon, liver, kidney, eye, and other organs.

1	Primary amyloidosis AL (from Ig Light chains) Seen in Plasma cell disorders Manifestations include: (eg, multiple myeloma) • Cardiac (eg, restrictive GI (eg, macroglossia, hepatomegaly) Renal (eg, nephrotic syndrome) Hematologic (eg, easy bruising, splenomegaly) Neurologic (eg, neuropathy) Musculoskeletal (eg, carpal tunnel syndrome) Inflammation Response to eliminate initial cause of cell injury, to remove necrotic cells resulting from the original insult, and to initiate tissue repair. Divided into acute and chronic. The inflammatory response itself can be harmful to the host if the reaction is excessive (eg, septic shock), prolonged (eg, persistent infections such as TB), or inappropriate (eg, autoimmune diseases such as SLE).

1	Fever Pyrogens (eg, LPS) induce macrophages to release IL-1 and TNF  COX activity in perivascular cells of hypothalamus • PGE2  temperature set point Acute inflammation Transient and early response to injury or infection. Characterized by neutrophils in tissue A , often with associated edema. Rapid onset (seconds to minutes) and short duration (minutes to days). Represents a reaction of the innate immune system (ie, less specific response than chronic inflammation). STIMuLI Infections, trauma, necrosis, foreign bodies. Leukocyte Extravasation predominantly occurs at postcapillary venules. extravasation GlyCAM-1, CD34 L-selectin CD11/18 integrins (LFA-1, Mac-1) VLA-4 integrin Chronic inflammation Prolonged inflammation characterized by mononuclear infiltration (macrophages, lymphocytes, plasma cells), which leads to simultaneous tissue destruction and repair (including angiogenesis and fibrosis). May be preceded by acute inflammation.

1	STIMuLI Persistent infections (eg, TB, T pallidum, certain fungi and viruses)  type IV hypersensitivity, autoimmune diseases, prolonged exposure to toxic agents (eg, silica) and foreign material. MeDIATOrS Macrophages are the dominant cells. Interaction of macrophages and T lymphocytes  chronic inflammation. OuTCOMeS Scarring, amyloidosis, and neoplastic transformation (eg, chronic HCV infection  chronic inflammation  hepatocellular carcinoma; Helicobacter pylori infection  chronic gastritis • gastric adenocarcinoma). FGF Stimulates angiogenesis TGF-β Angiogenesis, fibrosis EGF Stimulates cell growth via tyrosine kinases (eg, EGFR/ErbB1)

1	FGF Stimulates angiogenesis TGF-β Angiogenesis, fibrosis EGF Stimulates cell growth via tyrosine kinases (eg, EGFR/ErbB1) HISTOLOGY Focus of epithelioid cells (activated macrophages with abundant pink cytoplasm) surrounded by lymphocytes and multinucleated giant cells (formed by fusion of several activated macrophages). Two types: Caseating: associated with Central necrosis. Seen with infectious etiologies (eg, TB, fungal). Noncaseating A : no central necrosis. Seen with autoimmune diseases (eg, sarcoidosis, Crohn disease). APCs present antigens to CD4+ Th cells and secrete IL-12 • CD4+ Th cells differentiate into Th1 cells Th1 secretes IFN-γ • macrophage activation Macrophages  cytokine secretion (eg, TNF)  formation of epithelioid macrophages and giant cells. Anti-TNF therapy can cause sequestering granulomas to break down • disseminated disease. Always test for latent TB before starting anti-TNF therapy.

1	Associated with hypercalcemia due to  1α-hydroxylase activity in activated macrophages, resulting in  vitamin D activity. Bacterial: Mycobacteria (tuberculosis, leprosy), Bartonella henselae (cat scratch disease; stellate necrotizing granulomas), Listeria monocytogenes (granulomatosis infantiseptica), Treponema pallidum (3° syphilis) Fungal: endemic mycoses (eg, histoplasmosis) Parasitic: schistosomiasis Immune-mediated: sarcoidosis, Crohn disease, 1° biliary cholangitis, subacute (de Quervain/ granulomatous) thyroiditis Vasculitis: granulomatosis with polyangiitis (Wegener), eosinophilic granulomatosis with polyangiitis (Churg-Strauss), giant cell (temporal) arteritis, Takayasu arteritis Foreign bodies: berylliosis, talcosis, hypersensitivity pneumonitis

1	Foreign bodies: berylliosis, talcosis, hypersensitivity pneumonitis Scar formation Occurs when repair cannot be accomplished by cell regeneration alone. Nonregenerated cells (2° to severe acute or chronic injury) are replaced by connective tissue. 70–80% of tensile strength regained at 3 months; little tensile strength regained thereafter. Associated with excess TGF-β. Normal cells Normal cells with basal  apical polarity. See cervical example A , which shows normal cells and spectrum of dysplasia, as discussed below. Loss of uniformity in cell size and shape (pleomorphism); loss of tissue orientation; nuclear changes (eg,  nuclear:cytoplasmic ratio) A . Carcinoma in situ/ Irreversible severe dysplasia that involves the entire thickness of epithelium but does not preinvasive penetrate the intact basement membrane A . Cells have invaded basement membrane using collagenases and hydrolases (metalloproteinases). Cell-cell contacts lost by inactivation of E-cadherin.

1	Cells have invaded basement membrane using collagenases and hydrolases (metalloproteinases). Cell-cell contacts lost by inactivation of E-cadherin. Metastasis Spread to distant organ(s) via lymphatics or blood. Tumor nomenclature Carcinoma implies epithelial origin, whereas sarcoma denotes mesenchymal origin. Both terms generally imply malignancy. Benign tumors are usually well-differentiated and well-demarcated, with low mitotic activity, no metastases, and no necrosis. Malignant tumors (cancers) may show poor differentiation, erratic growth, local invasion, metastasis, and • apoptosis. Terms for non-neoplastic malformations include hamartoma (disorganized overgrowth of tissues in their native location, eg, Peutz-Jeghers polyps) and choristoma (normal tissue in a foreign location, eg, gastric tissue located in distal ileum in Meckel diverticulum). Epithelium Adenoma, papilloma Adenocarcinoma, papillary carcinoma Blood cells Leukemia, lymphoma

1	Epithelium Adenoma, papilloma Adenocarcinoma, papillary carcinoma Blood cells Leukemia, lymphoma Tumor grade vs stage Differentiation—degree to which a tumor resembles its tissue of origin. Well-differentiated tumors (often less aggressive) closely resemble their tissue of origin, whereas poorly differentiated tumors (often more aggressive) do not. Anaplasia—complete lack of differentiation of cells in a malignant neoplasm. Grade Degree of cellular differentiation and mitotic activity on histology. Ranges from low grade (well-differentiated) to high grade (poorly differentiated, undifferentiated, or anaplastic). Low grade High grade Stage Degree of localization/spread based on site and size of 1° lesion, spread to regional lymph nodes, presence of metastases. Based on clinical (c) or pathologic (p) findings. Stage T generally has more prognostic value than Lymphgrade (eg, a high-stage yet low-grade tumor is node N tumor). Stage determines Survival. Blood or lymphatic vessel

1	Lymphgrade (eg, a high-stage yet low-grade tumor is node N tumor). Stage determines Survival. Blood or lymphatic vessel TNM staging system (Stage = Spread): Spread to otherT = Tumor size/invasiveness, N = Node organs and tissues involvement, M = Metastases, eg, cT3N1M0. Each TNM factor has independent prognostic value; N and M are often most important. Cancer epidemiology Skin cancer (basal > squamous >> melanoma) is the most common cancer (not included below). Common metastases Most sarcomas spread hematogenously; most carcinomas spread via lymphatics. However, Four Carcinomas Route Hematogenously: Follicular thyroid carcinoma, Choriocarcinoma, Renal cell carcinoma, and Hepatocellular carcinoma. Brain Lung > breast > melanoma, colon, kidney 50% of brain tumors are from metastases A Commonly seen as multiple well-circumscribed tumors at gray/white matter junction CD and lung are the most common sites Sometimes Penetrates liver) of metastasis after the regional lymph nodes

1	CD and lung are the most common sites Sometimes Penetrates liver) of metastasis after the regional lymph nodes Bone Prostate, Breast > Kidney, Thyroid, Lung (Painful Bones Kill The Lungs) Bone metastasis E F >> 1° bone tumors (eg, G Bone metastasis can be: Lytic (eg, thyroid, kidney, non-small cell lung cancer) Blastic (eg, prostate, small cell lung cancer) Mixed (eg, breast cancer) Tumor suppressor Loss of function  cancer risk; both (two) alleles of a tumor suppressor gene must be lost for genes expression of disease. EBV Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal carcinoma, 1° CNS lymphoma (in immunocompromised patients) HBV, HCV Hepatocellular carcinoma Serum tumor markers Tumor markers should not be used as the 1° tool for cancer diagnosis or screening. They may be used to monitor tumor recurrence and response to therapy, but definitive diagnosis is made via biopsy. Some can be associated with non-neoplastic conditions.

1	P-glycoprotein Also known as multidrug resistance protein 1 (MDR1). Classically seen in adrenocortical carcinoma but also expressed by other cancer cells (eg, colon, liver). Used to pump out toxins, including chemotherapeutic agents (one mechanism of  responsiveness or resistance to chemotherapy over time). Psammoma bodies Laminated, concentric spherules with dystrophic calcification A , PSaMMOMa bodies are seen in: Papillary carcinoma of thyroid Cachexia Weight loss, muscle atrophy, and fatigue that occur in chronic disease (eg, cancer, AIDS, heart failure, COPD). Mediated by TNF-α, IFN-γ, IL-1, and IL-6. effusions, periostosis of tubular bones Endocrine “One pill makes you larger, and one pill makes you small.” “I was under medication when I made the decision not to burn the tapes.” “I wondher why ye can always read a doctor’s bill an’ ye niver can read his purscription.” “One of the first duties of the physician is to educate the masses not to take medicine.”

1	Preparation for pharmacology questions is straightforward. Know all the mechanisms, clinical use, and important adverse effects of key drugs and their major variants. Obscure derivatives are low-yield. Learn their classic and distinguishing toxicities as well as major drug-drug interactions. Reviewing associated biochemistry, physiology, and microbiology concepts can be useful while studying pharmacology. The exam has a strong emphasis on ANS, CNS, antimicrobial, and cardiovascular agents as well as on NSAIDs, which are covered throughout the text. Specific drug dosages or trade names are generally not testable. The exam may use graphs to test various pharmacology content, so make sure you are comfortable interpreting them. Michaelis-Menten Km is inversely related to the affinity of the [S] = concentration of substrate; V = velocity. kinetics enzyme for its substrate. Vmax is directly proportional to the enzyme concentration.

1	Vmax is directly proportional to the enzyme concentration. curve (ie, Michaelis-Menten kinetics); however, enzymatic reactions that exhibit a Effects of enzyme inhibition kinetics (eg, hemoglobin). Lineweaver-Burk plot The closer to 0 on the Y-axis, the higher the 1 Vmax. V The closer to 0 on the X-axis, the higher the Km. −K The higher the Km, the lower the affinity. 1 Competitive inhibitors cross each other, [S] whereas noncompetitive inhibitors do not. Effects of enzyme inhibition Kompetitive inhibitors increase Km. Bioavailability (F) Fraction of administered drug reaching systemic circulation unchanged. For an IV dose, F = 100%. Orally: F typically < 100% due to incomplete absorption and first-pass metabolism. Can be calculated from the area under the curve in a plot of plasma concentration over time.

1	Volume of distribution Theoretical volume occupied by the total amount of drug in the body relative to its plasma (Vd) concentration. Apparent Vd of plasma protein–bound drugs can be altered by liver and kidney disease ( protein binding,  Vd). Drugs may distribute in more than one compartment. amount of drug in the body Low Intravascular Large/charged molecules; plasma protein bound Medium ECF Small hydrophilic molecules High All tissues including Small lipophilic molecules, especially if bound fat to tissue protein Clearance (CL) The volume of plasma cleared of drug per unit time. Clearance may be impaired with defects in cardiac, hepatic, or renal function. rate of elimination of drug Half-life (t1/2) The time required to change the amount of drug in the body by 1⁄2 during elimination. In first-order kinetics, a drug infused at a constant rate takes 4–5 half-lives to reach steady state. It takes 3.3 half-lives to reach 90% of the steady-state level.

1	t1/2 = 0.7 × Vd in first-order elimination CL Drug metabolism Geriatric patients lose phase I first. Patients who are slow acetylators have  side effects from certain drugs because of  rate of metabolism (eg, isoniazid). Elimination of drugs Weak bases Examples: TCAs, amphetamines. Trapped in acidic environments. Treat overdose with ammonium chloride to acidify urine. TCA toxicity is generally treated with sodium bicarbonate to overcome the sodium channel-blocking activity of TCAs, but not for accelerating drug elimination. pKa pH at which drugs (weak acid or base) are 50% ionized and 50% nonionized. The pKa represents the strength of the weak acid or base. Efficacy Maximal effect a drug can produce. Represented by the y-value (Vmax).  y-value =  Vmax =  efficacy. Unrelated to potency (ie, efficacious drugs can have high or low potency). Partial agonists have less efficacy than full agonists.

1	Potency Amount of drug needed for a given effect. Represented by the x-value (EC50). Left shifting = • EC50 =  potency =  drug needed. Unrelated to efficacy (ie, potent drugs can have high or low efficacy). 0.1 1.0 10 100 1000 0.1 1.0 10 100 1000 0.1 1.0 10 100 1000 Agonist dose Agonist dose Agonist dose Therapeutic index Measurement of drug safety. TD50 median toxic dose Therapeutic window—dosage range that can safely and effectively treat disease. % of patients responding TITE: Therapeutic Index = TD50 / ED50. Safer drugs have higher TI values. Drugs with lower TI values frequently require monitoring (eg, Warfarin, Theophylline, Digoxin, Antiepileptic drugs, Lithium; Warning! These Drugs Are Lethal!). LD50 (lethal median dose) often replaces TD50 in animal studies. Types of drug interactions Autonomic receptors

1	LD50 (lethal median dose) often replaces TD50 in animal studies. Types of drug interactions Autonomic receptors SOMATICSmooth muscle, gland cells, nerve terminals, cardiac muscle Sweat glands Smooth muscle, gland cells, nerve terminals, cardiac muscle Renal vasculature smooth muscle Cardiac muscle, vessels Skeletal muscle ParasympatheticBrainstem Spinal cord Post (long) SympatheticPost (short) ACh NN Pre (long) ACh NN AChM ACh D NE M D1 ACh NM ACh NN ACh NN ACh NN Adrenal medulla Voluntary motor nerve Neuromuscular junction Catecholamine transmission Blood ˜1˜2°1 Epi ˜1˜2°1°2 NE ˜1˜2°1 Pre (short) Pelvic splanchnic nerves and CNs III, VII, IX and X are part of the parasympathetic nervous system. Adrenal medulla is directly innervated by preganglionic sympathetic fibers. Sweat glands are part of the sympathetic pathway but are innervated by cholinergic fibers (sympathetic nervous system results in a “chold” sweat).

1	M˜-receptor bladder function via coordination of sympathetic and parasympathetic nervous Pelvic nerve β˜-receptor (parasympatheticsystems. • ⊕ parasympathetic  urine voiding. Some (sympathetic input) sphincter autonomic drugs act on smooth muscle External urethral receptors to treat bladder dysfunction. q • vascular smooth muscle contraction,  pupillary dilator muscle contraction (mydriasis),  intestinal and bladder sphincter muscle contraction i • sympathetic (adrenergic) outflow,  insulin release,  lipolysis,  platelet aggregation,  aqueous humor production s • heart rate,  contractility (one heart),  renin release,  lipolysis s Vasodilation, bronchodilation (two lungs),  lipolysis,  insulin release, glycogenolysis,  uterine tone (tocolysis),  aqueous humor production, β3 s lipolysis,  thermogenesis in skeletal muscle,  bladder relaxation Cholinergic (accommodation),  insulin release, endothelium-mediated vasodilation Dopamine

1	D1 s Relaxes renal vascular smooth muscle, activates direct pathway of striatum D2 i Modulates transmitter release, especially in brain, inhibits indirect “After qisses (kisses), you get a qiq (kick) out of siq (sick) sqs (super qinky sex).” H1, ˜1, V1, Protein HAVe 1M&M. M1, M3 kinase C Lipids ° 1, ° 2, ° 3, D1, Receptor Gs ATP H2, V2 M2, ˜2, D2 inhibit themselves. Autonomic drugs Release of norepinephrine from a sympathetic nerve ending is modulated by NE itself, acting on presynaptic α2-autoreceptors • negative feedback. Amphetamines use the NE transporter (NET) to enter the presynaptic terminal, where they utilize the vesicular monoamine transporter (VMAT) to enter neurosecretory vesicles. This displaces NE from the vesicles. Once NE reaches a concentration threshold within the presynaptic terminal, the action of NET is reversed, and NE is expelled into the synaptic cleft, contributing to the characteristics and effects of  NE observed in patients taking amphetamines.

1	ACh NE Choline Choline+ Acetyl-CoA Ca2+ Choline + acetate AChE ACh receptor DOPA Dopamine Reuptake Di˜usion, metabolism NET VMAT Adrenoreceptors ˜ or °Cocaine, TCAs, amphetamine Botulinum toxin -Ca2+ NE + --AChE inhibitors AXON Tyrosine ACh Tyrosine ˜2 AT II Reserpine -Release-modulating receptors Negativefeedback AXON ChAT -+ Amphetamine, ephedrine + + represents transporters. Atropine Muscarinic antagonist. Used to treat bradycardia and for ophthalmic applications. Eye  pupil dilation, cycloplegia Blocks muscarinic effects (DUMBBELSS) of anticholinesterases, but not the nicotinic Airway Bronchodilation,  secretions effects. AdVERSE EFFECTS  body temperature (due to  sweating); • HR; dry mouth; dry, flushed skin; cycloplegia; constipation; disorientation Can cause acute angle-closure glaucoma in elderly (due to mydriasis), urinary retention in men with prostatic hyperplasia, and hyperthermia in infants.

1	Side effects: Hot as a hare Fast as a fiddle Dry as a bone Red as a beet Blind as a bat Mad as a hatter Full as a flask Jimson weed (Datura)  gardener’s pupil (mydriasis due to plant alkaloids) priapism. Indirect sympathomimetics Ephedrine Indirect general agonist, releases stored catecholamines Narcolepsy, obesity, ADHD. Causes vasoconstriction and local anesthesia. Caution when giving β-blockers if cocaine intoxication is suspected (can lead to unopposed α1 activation • extreme hypertension, coronary vasospasm). Nasal decongestion (pseudoephedrine), urinary incontinence, hypotension. β1, refex tachycardia Epinephrine response exhibits reversal of mean arterial Phenylephrine response is suppressed but not reversed pressure from a net increase (the α response) to a net because it is a “pure” α-agonist (lacks β-agonist decrease (the β2 response). properties). In platelets:  cAMP  inhibition of platelet aggregation

1	In platelets:  cAMP  inhibition of platelet aggregation Cardiac stress testing (dipyridamole only, due to coronary vasodilation) Prevention of coronary stent restenosis Nausea, headache, facial flushing, hypotension, abdominal pain aCilostazol is a PDE-3 inhibitor, but due to its indications is categorized as a platelet inhibitor together with dipyridamole. bDipyridamole is a nonspecific PDE inhibitor, leading to inhibition of platelet aggregation. It also prevents adenosine reuptake by platelets • extracellular adenosine • vasodilation. Beers criteria Widely used criteria developed to reduce potentially inappropriate prescribing and harmful polypharmacy in the geriatric population. Includes > 50 medications that should be avoided in elderly patients due to  efficacy and/or  risk of adverse events. Examples: α-blockers ( risk of hypotension) Anticholinergics, antidepressants, antihistamines, opioids ( risk of delirium, sedation, falls, constipation, urinary retention)

1	Anticholinergics, antidepressants, antihistamines, opioids ( risk of delirium, sedation, falls, constipation, urinary retention) Benzodiazepines ( risk of delirium, sedation, falls) NSAIDs ( risk of GI bleeding, especially with concomitant anticoagulation) PPIs ( risk of C difficile infection) AChE inhibitors, organophosphates Atropine > pralidoxime Antimuscarinic, anticholinergic agents Physostigmine (crosses BBB), control hyperthermia Arsenic Dimercaprol, succimer β-blockers Atropine, glucagon, saline Iron (Fe) Deferoxamine, deferasirox, deferiprone Salicylates NaHCO3 (alkalinize urine), dialysis Warfarin Vitamin K (delayed effect), PCC (prothrombin complex concentrate)/FFP (immediate effect) Anticholinergics (eg, atropine, TCAs, Sympatholytics (eg, α2-agonists) tropicamide, scopolamine, antihistamines) Drugs of abuse (eg, amphetamines, cocaine, Drugs of abuse (eg, heroin/opioids) LSD), meperidine

1	Drugs of abuse (eg, amphetamines, cocaine, Drugs of abuse (eg, heroin/opioids) LSD), meperidine Sympathomimetics Parasympathomimetics (eg, pilocarpine), organophosphates Sulfa drugs Sulfonamide antibiotics, Sulfasalazine, Scary Sulfa Pharm FACTS Probenecid, Furosemide, Acetazolamide, Celecoxib, Thiazides, Sulfonylureas. Patients with sulfa allergies may develop fever, urinary tract infection, Stevens-Johnson syndrome, hemolytic anemia, thrombocytopenia, agranulocytosis, acute interstitial nephritis, and urticaria (hives). “Medicine is a science of uncertainty and an art of probability.” “There are two kinds of statistics: the kind you look up and the kind you make up.” “On a long enough timeline, the survival rate for everyone drops to zero.” “There are three kinds of lies: lies, damned lies, and statistics.”

1	A heterogenous mix of epidemiology, biostatistics, ethics, law, healthcare delivery, patient safety, quality improvement, and more falls under the heading of public health sciences. Biostatistics and epidemiology are the foundations of evidence-based medicine and are very high yield. Make sure you can quickly apply biostatistical equations such as sensitivity, specificity, and predictive values in a problem-solving format. Also, know how to set up your own 2 × 2 tables. Quality improvement and patient safety topics were introduced a few years ago on the exam and represent trends in health system science. Medical ethics questions often require application of principles. Typically, you are presented with a patient scenario and then asked how you would respond.

1	Clinical trial Experimental study involving humans. Compares therapeutic benefits of ≥2 treatments, or of treatment and placebo. Study quality improves when study is randomized, controlled, and double-blinded (ie, neither patient nor doctor knows whether the patient is in the treatment or control group). Triple-blind refers to the additional blinding of the researchers analyzing the data. Four phases (“Does the drug SWIM?”). Evaluation of Sensitivity and specificity are fixed properties Disease diagnostic tests of a test. PPV and NPV vary depending on disease prevalence in population being tested. SN-N-OUT = highly SeNsitive test, when Negative, rules OUT disease SP-P-IN = highly SPecific test, when Positive, rules IN disease PPV varies directly with pretest probability (baseline risk, such as prevalence of disease): NPV varies inversely with prevalence or pretest Quantifying risk Definitions and formulas are based on the classic 2 × 2 or contingency table.

1	NPV varies inversely with prevalence or pretest Quantifying risk Definitions and formulas are based on the classic 2 × 2 or contingency table. Odds ratio Typically used in case-control If in a case-control study, 20/30 lung a/c ad studies. Represents the odds of cancer patients and 5/25 healthy b/d bc exposure among cases (a/c) vs individuals report smoking, the OR odds of exposure among controls is 8; so the lung cancer patients are 8 (b/d). times more likely to have a history of smoking. Relative risk Typically used in cohort studies. Risk of developing disease in the exposed group divided by risk in the unexposed group. RR = 1  no association between exposure and disease. RR > 1  exposure associated with occurrence. RR < 1  exposure associated with disease occurrence.

1	If 5/10 people exposed to radiation are a/(a + b) diagnosed with cancer, and 1/10 people c/(c + d) not exposed to radiation are diagnosed with cancer, the RR is 5; so people exposed to radiation have a 5 times greater risk of developing cancer. For rare diseases (low prevalence), OR approximates RR. Relative risk The proportion of risk reduction If 2% of patients who receive a flu RRR = 1 − RR reduction attributable to the intervention as shot develop the flu, while 8% of compared to a control. unvaccinated patients develop the flu, then RR = 2/8 = 0.25, and RRR = 0.75. Attributable The difference in risk between If risk of lung cancer in smokers is 21% a c risk exposed and unexposed groups. and risk in nonsmokers is 1%, then the a + b c + d attributable risk is 20%. RR − 1

1	Absolute The difference in risk (not the If 8% of people who receive a placebo c a risk proportion) attributable to the vaccine develop the flu vs 2% of people c + d a + b reduction intervention as compared to a who receive a flu vaccine, then ARR = control. 8%–2% = 6% = 0.06. Number Number of patients who need to NNT = 1/ARR needed to be treated for 1 patient to benefit. treat Lower number = better treatment. Number Number of patients who need to NNH = 1/AR needed to be exposed to a risk factor for 1 harm patient to be harmed. Higher number = safer exposure. Case fatality Percentage of deaths occurring If 4 patients die among 10 cases of deaths rate among those with disease. meningitis, case fatality rate is 40%. cases Incidence vs # of new cases Incidence looks at new cases(incidents). Incidence = (per unit of time) prevalence # of people at risk Prevalence =# of existing cases (at a point in Prevalence looks at all current cases.

1	Incidence = (per unit of time) prevalence # of people at risk Prevalence =# of existing cases (at a point in Prevalence looks at all current cases. Total # of people time) 1 – prevalence of disease Prevalence ≈ incidence for short duration disease Prevalence ∼ pretest probability. (eg, common cold).  prevalence  PPV and  NPV. Prevalence > incidence for chronic diseases, due to large # of existing cases (eg, diabetes). Precision (reliability) The consistency and reproducibility of a test. Random error  precision in a test. The absence of random variation in a test.  precision  standard deviation.  precision  statistical power (1 −β). Accuracy (validity) The closeness of test results to the true values. Systematic error  accuracy in a test. The absence of systematic error or bias in a test.

1	Accuracy (validity) The closeness of test results to the true values. Systematic error  accuracy in a test. The absence of systematic error or bias in a test. ROC curve demonstrates how well a diagnostic 1 test can distinguish between 2 groups (eg, disease vs healthy). Plots the true-positive rate (sensitivity) against the false-positive rate (1 – specificity). The better performing test will have a higher area under the curve (AUC), with the curve closer to the upper left corner. No predictive value (AUC = 0.5)TP rate (sensitivity) FP rate (1 – specifcity) 1 A ctual test (0.5 < AU C < 1 ) Selection bias Nonrandom sampling or treatment allocation of subjects such that study population is not representative of target population. Most commonly a sampling bias. Berkson bias—cases and/ or controls selected from hospitals are less healthy and have different exposures than general population

1	Berkson bias—cases and/ or controls selected from hospitals are less healthy and have different exposures than general population Attrition bias—participants lost to follow up have a different prognosis than those who complete the study Randomization Ensure the choice of the right comparison/reference group Recall bias Awareness of disorder alters Patients with disease recall Decrease time from exposure recall by subjects; common in exposure after learning of to follow-up retrospective studies similar cases Measurement bias Information is gathered in a Using a faulty automatic Use objective, standardized, systemically distorted manner sphygmomanometer to and previously tested methods measure BP of data collection that are Hawthorne effect—participants planned ahead of time change behavior upon Use placebo group awareness of being observed

1	Procedure bias Subjects in different groups are Patients in treatment group not treated the same spend more time in highly specialized hospital units Blinding (masking) and use of placebo reduce influence of participants and researchers on procedures and interpretation of outcomes as neither are aware of group assignments Confounding bias Factor related to both exposure and outcome (but not on causal path) distorts effect of exposure on outcome (vs effect modification, in which the exposure leads to different outcomes in subgroups stratified by the factor) An uncontrolled study shows an association between drinking coffee and lung cancer. However, coffee drinkers also smoke more, which can account for the association Multiple/repeated studies Crossover studies (subjects act as their own controls) Matching (patients with similar characteristics in both treatment and control groups)

1	Crossover studies (subjects act as their own controls) Matching (patients with similar characteristics in both treatment and control groups) Lead-time bias Early detection is confused Early detection makes it seem Measure “back-end” survival with  survival like survival has increased, (adjust survival according to but the disease’s natural the severity of disease at the history has not changed time of diagnosis) Length-time bias Screening test detects diseases A slowly progressive cancer A randomized controlled trial with long latency period, is more likely detected by a assigning subjects to the while those with shorter screening test than a rapidly screening program or to no latency period become progressive cancer screening symptomatic earlier Mode = most common value. Least affected by outliers. Measures of Standard deviation = how much variability σ = SD; n = sample size. dispersion exists in a set of values, around the mean of Variance = (SD)2. these values. SE = σ/√n.

1	Measures of Standard deviation = how much variability σ = SD; n = sample size. dispersion exists in a set of values, around the mean of Variance = (SD)2. these values. SE = σ/√n. Standard error = an estimate of how much SE  as n . variability exists in a (theoretical) set of sample means around the true population mean. Normal distribution Gaussian, also called bell-shaped. Mean = median = mode. 68% 95% 99.7% Bimodal Suggests two different populations (eg, metabolic polymorphism such as fast vs slow acetylators; age at onset of Hodgkin lymphoma; suicide rate by age). Positive skew Typically, mean > median > mode. Asymmetry with longer tail on right. Negative skew Typically, mean < median < mode. Mode Asymmetry with longer tail on left. Statistical hypotheses Outcomes of statistical hypothesis testing

1	Asymmetry with longer tail on right. Negative skew Typically, mean < median < mode. Mode Asymmetry with longer tail on left. Statistical hypotheses Outcomes of statistical hypothesis testing Correct result Stating that there is an effect or difference when Reality one exists (null hypothesis rejected in favor of HH alternative hypothesis). Stating that there is no effect or difference when none exists (null hypothesis not rejected). Blue shading = correct result. Confidence interval Range of values within which the true mean of the population is expected to fall, with a specified probability. CI for sample mean = x¯ ± Z(SE) The 95% CI (corresponding to α = .05) is often used. As sample size increases, CI narrows. For the 95% CI, Z = 1.96. For the 99% CI, Z = 2.58. If the 95% CI for a mean difference between 2 variables includes 0, then there is no significant difference and H0 is not rejected. If the 95% CI for odds ratio or relative risk includes 1, H0 is not rejected.

1	If the 95% CI for odds ratio or relative risk includes 1, H0 is not rejected. If the CIs between 2 groups do not overlap  statistically significant difference exists. If the CIs between 2 groups overlap  usually no significant difference exists. Meta-analysis A method of statistical analysis that pools summary data (eg, means, RRs) from multiple studies for a more precise estimate of the size of an effect. Also estimates heterogeneity of effect sizes between studies. Improves power, strength of evidence, and generalizability of study findings. Limited by quality of individual studies and bias in study selection. Autonomy Obligation to respect patients as individuals (truth-telling, confidentiality), to create conditions necessary for autonomous choice (informed consent), and to honor their preference in accepting or not accepting medical care.

1	Beneficence Physicians have a special ethical (fiduciary) duty to act in the patient’s best interest. May conflict with autonomy (an informed patient has the right to decide) or what is best for society (eg, mandatory TB treatment). Traditionally, patient interest supersedes. Nonmaleficence “Do no harm.” Must be balanced against beneficence; if the benefits outweigh the risks, a patient may make an informed decision to proceed (most surgeries and medications fall into this category). Justice To treat persons fairly and equitably. This does not always imply equally (eg, triage). Informed consent A process (not just a document/signature) that requires: Disclosure: discussion of pertinent information (using medical interpreter, if needed) Understanding: ability to comprehend Capacity: ability to reason and make one’s own decisions (distinct from competence, a legal determination) Voluntariness: freedom from coercion and manipulation

1	Capacity: ability to reason and make one’s own decisions (distinct from competence, a legal determination) Voluntariness: freedom from coercion and manipulation Patients must have an intelligent understanding of their diagnosis and the risks/benefits of proposed treatment and alternative options, including no treatment. Patient must be informed that he or she can revoke written consent at any time, even orally. Exceptions to informed consent (WIPE it away): Waiver—patient explicitly waives the right of informed consent Therapeutic Privilege—withholding information when disclosure would severely harm the patient or undermine informed decision-making capacity

1	Therapeutic Privilege—withholding information when disclosure would severely harm the patient or undermine informed decision-making capacity Consent for minors A minor is generally any person < 18 years old. Parental consent laws in relation to healthcare vary by state. In general, parental consent should be obtained, but exceptions exist for emergency treatment (eg, blood transfusions) or if minor is legally emancipated (eg, married, self-supporting, or in the military). Situations in which parental consent is usually not required: Sex (contraception, STIs, pregnancy) Rock and roll (emergency/trauma) Physicians should always encourage healthy minor-guardian communication. Physician should seek a minor’s assent even if their consent is not required.

1	Rock and roll (emergency/trauma) Physicians should always encourage healthy minor-guardian communication. Physician should seek a minor’s assent even if their consent is not required. Physician must determine whether the patient is psychologically and legally capable of making a particular healthcare decision. Note that decisions made with capacity cannot be revoked simply if the patient later loses capacity. Intellectual disability alone (eg, Down syndrome, autism) is not an exclusion criterion for informed decision-making. Capacity is determined by a physician for a specific healthcare-related decision (eg, to refuse medical care). Competency is determined by a judge and usually refers to more global categories of decision making (eg, legally unable to make any healthcare-related decision). Components (think GIEMSA): Decision is consistent with patient’s values and Goals Patient is Informed (knows and understands)

1	Components (think GIEMSA): Decision is consistent with patient’s values and Goals Patient is Informed (knows and understands) Decision is not a result of altered Mental status (eg, delirium, psychosis, intoxication), Mood disorder Patient is ≥ 18 years of Age or otherwise legally emancipated Advance directives Instructions given by a patient in anticipation of the need for a medical decision. Details vary per state law. Oral advance directive Incapacitated patient’s prior oral statements commonly used as guide. Problems arise from variance in interpretation. If patient was informed, directive was specific, patient made a choice, and decision was repeated over time to multiple people, then the oral directive is more valid. Do not resuscitate DNR order prohibits cardiopulmonary resuscitation (CPR). Other resuscitative measures that may order follow (eg, feeding tube) are also typically avoided.

1	Do not resuscitate DNR order prohibits cardiopulmonary resuscitation (CPR). Other resuscitative measures that may order follow (eg, feeding tube) are also typically avoided. Confidentiality Confidentiality respects patient privacy and autonomy. If the patient is incapacitated or the situation is emergent, disclosing information to family and friends should be guided by professional judgment of patient’s best interest. The patient may voluntarily waive the right to confidentiality (eg, insurance company request). General principles for exceptions to confidentiality: Potential physical harm to others is serious and imminent Alternative means to warn or protect those at risk is not possible Self-harm is likely Steps can be taken to prevent harm Examples of exceptions to patient confidentiality (many are state specific) include the following (“The physician’s good judgment SAVED the day”): Suicidal/homicidal patients. Abuse (children, elderly, and/or prisoners).

1	Suicidal/homicidal patients. Abuse (children, elderly, and/or prisoners). Duty to protect—state-specific laws that sometimes allow physician to inform or somehow protect potential Victim from harm. Epileptic patients and other impaired automobile drivers. Reportable Diseases (eg, STIs, hepatitis, food poisoning); physicians may have a duty to warn public officials, who will then notify people at risk. Dangerous communicable diseases, such as TB or Ebola, may require involuntary treatment. Patient is not adherent. Attempt to identify the reason for nonadherence and determine his/her willingness to change; do not coerce the patient into adhering and do not refer him/her to another physician. A patient’s family member asks you not to disclose the results of a test if the prognosis is poor because the patient will be “unable to handle it.”

1	A patient’s family member asks you not to disclose the results of a test if the prognosis is poor because the patient will be “unable to handle it.” Attempt to identify why the family member believes such information would be detrimental to the patient’s condition. Explain that as long as the patient has decision-making capacity and does not indicate otherwise, communication of information concerning his/her care will not be withheld. However, if you believe the patient might seriously harm himself/herself or others if informed, then you may invoke therapeutic privilege and withhold the information. Patient is suicidal. Assess the seriousness of the threat. If it is serious, suggest that the patient remain in the hospital voluntarily; patient can be hospitalized involuntarily if he/she refuses. Discounted fee-for-Patient pays for each individual service at a discounted rate predetermined by providers and payers service (eg, PPOs).

1	Discounted fee-for-Patient pays for each individual service at a discounted rate predetermined by providers and payers service (eg, PPOs). Medicare and Medicaid—federal social healthcare programs that originated from amendments to the Social Security Act. Medicare is available to patients ≥ 65 years old, < 65 with certain disabilities, and those with end-stage renal disease. Medicaid is joint federal and state health assistance for people with limited income and/ or resources. MedicarE is for Elderly. MedicaiD is for Destitute. The 4 parts of Medicare: Part A: HospitAl insurance, home hospice care Part B: Basic medical bills (eg, doctor’s fees, diagnostic testing) Part C: (parts A + B = Combo) delivered by approved private companies Part D: Prescription Drugs

1	Part B: Basic medical bills (eg, doctor’s fees, diagnostic testing) Part C: (parts A + B = Combo) delivered by approved private companies Part D: Prescription Drugs Hospice care Medical care focused on providing comfort and palliation instead of definitive cure. Available to patients on Medicare or Medicaid and in most private insurance plans whose life expectancy is < 6 months. During end-of-life care, priority is given to improving the patient’s comfort and relieving pain (often includes opioid, sedative, or anxiolytic medications). Facilitating comfort is prioritized over potential side effects (eg, respiratory depression). This prioritization of positive effects over negative effects is called the principle of double effect. Common causes of death (US) by age

1	Common causes of death (US) by age Safety culture Organizational environment in which everyone Event reporting systems collect data on errors for can freely bring up safety concerns without internal and external monitoring. fear of censure. Facilitates error identification. Human factors design Forcing functions (those that prevent undesirable actions [eg, connecting feeding syringe to IV tubing]) are the most effective. Standardization improves process reliability (eg, clinical pathways, guidelines, checklists). Simplification reduces wasteful activities (eg, consolidating electronic medical records). Deficient designs hinder workflow and lead to staff workarounds that bypass safety features (eg, patient ID barcodes affixed to computers due to unreadable wristbands). PDSA cycle Process improvement model to test changes in real clinical setting. Impact on patients:

1	PDSA cycle Process improvement model to test changes in real clinical setting. Impact on patients: Swiss cheese model Focuses on systems and conditions rather than an individual’s error. The risk of a threat becoming a reality is mitigated by differing layers and types of defenses. Patient harm can occur despite multiple safeguards when “the holes in the cheese line up.” Types of medical May involve patient identification, diagnosis, monitoring, nosocomial infection, medications, errors procedures, devices, documentation, handoffs. Medical errors should be disclosed to patients, independent of immediate outcome (harmful or not). “Symptoms, then, are in reality nothing but the cry from suffering organs.” “Man is an intelligence in servitude to his organs.” “When every part of the machine is correctly adjusted and in perfect harmony, health will hold dominion over the human organism by laws as natural and immutable as the laws of gravity.” —Andrew T. Still

1	Skin, and Connective Tissue 445 In this section, we have divided the High-Yield Facts into the major Organ Systems. Within each Organ System are several subsections, including Embryology, Anatomy, Physiology, Pathology, and Pharmacology. As you progress through each Organ System, refer back to information in the previous subsections to organize these basic science subsections into a “vertically integrated” framework for learning. Below is some general advice for studying the organ systems by these subsections. Relevant embryology is included in each organ system subsection. Embryology tends to correspond well with the relevant anatomy, especially with regard to congenital malformations.

1	Relevant embryology is included in each organ system subsection. Embryology tends to correspond well with the relevant anatomy, especially with regard to congenital malformations. Several topics fall under this heading, including gross anatomy, histology, and neuroanatomy. Do not memorize all the small details; however, do not ignore anatomy altogether. Review what you have already learned and what you wish you had learned. Many questions require two or more steps. The first step is to identify a structure on anatomic cross section, electron micrograph, or photomicrograph. The second step may require an understanding of the clinical significance of the structure.

1	When studying, stress clinically important material. For example, be familiar with gross anatomy and radiologic anatomy related to specific diseases (eg, Pancoast tumor, Horner syndrome), traumatic injuries (eg, fractures, sensory and motor nerve deficits), procedures (eg, lumbar puncture), and common surgeries (eg, cholecystectomy). There are also many questions on the exam involving x-rays, CT scans, and neuro MRI scans. Many students suggest browsing through a general radiology atlas, pathology atlas, and histology atlas. Focus on learning basic anatomy at key levels in the body (eg, sagittal brain MRI; axial CT of the midthorax, abdomen, and pelvis). Basic neuroanatomy (especially pathways, blood supply, and functional anatomy), associated neuropathology, and neurophysiology have good yield. Please note that many of the photographic images in this book are for illustrative purposes and are not necessarily reflective of Step 1 emphasis.

1	The portion of the examination dealing with physiology is broad and concept oriented and thus does not lend itself as well to fact-based review. Diagrams are often the best study aids, especially given the increasing number of questions requiring the interpretation of diagrams. Learn to apply basic physiologic relationships in a variety of ways (eg, the Fick equation, clearance equations). You are seldom asked to perform complex calculations. Hormones are the focus of many questions, so learn their sites of production and action as well as their regulatory mechanisms. A large portion of the physiology tested on the USMLE Step 1 is clinically relevant and involves understanding physiologic changes associated with pathologic processes (eg, changes in pulmonary function with COPD). Thus, it is worthwhile to review the physiologic changes that are found with common pathologies of the major organ systems (eg, heart, lungs, kidneys, GI tract) and endocrine glands.

1	Questions dealing with this discipline are difficult to prepare for because of the sheer volume of material involved. Review the basic principles and hallmark characteristics of the key diseases. Given the clinical orientation of Step 1, it is no longer sufficient to know only the “buzzword” associations of certain diseases (eg, café-au-lait macules and neurofibromatosis); you must also know the clinical descriptions of these findings. Given the clinical slant of the USMLE Step 1, it is also important to review the classic presenting signs and symptoms of diseases as well as their associated laboratory findings. Delve into the signs, symptoms, and pathophysiology of major diseases that have a high prevalence in the United States (eg, alcoholism, diabetes, hypertension, heart failure, ischemic heart disease, infectious disease). Be prepared to think one step beyond the simple diagnosis to treatment or complications.

1	The examination includes a number of color photomicrographs and photographs of gross specimens that are presented in the setting of a brief clinical history. However, read the question and the choices carefully before looking at the illustration, because the history will help you identify the pathologic process. Flip through an illustrated pathology textbook, color atlases, and appropriate Web sites in order to look at the pictures in the days before the exam. Pay attention to potential clues such as age, sex, ethnicity, occupation, recent activities and exposures, and specialized lab tests.

1	Preparation for questions on pharmacology is straightforward. Learning all the key drugs and their characteristics (eg, mechanisms, clinical use, and important side effects) is high yield. Focus on understanding the prototype drugs in each class. Avoid memorizing obscure derivatives. Learn the “classic” and distinguishing toxicities of the major drugs. Do not bother with drug dosages or trade names. Reviewing associated biochemistry, physiology, and microbiology can be useful while studying pharmacology. There is a strong emphasis on ANS, CNS, antimicrobial, and cardiovascular agents as well as NSAIDs. Much of the material is clinically relevant. Newer drugs on the market are also fair game.

1	“As for me, except for an occasional heart attack, I feel as young as I ever did.” “Hearts will never be practical until they are made unbreakable.” —The Wizard of Oz “As the arteries grow hard, the heart grows soft.” —H. L. Mencken “Nobody has ever measured, not even poets, how much the heart can hold.” “Only from the heart can you touch the sky.” “It is not the size of the man but the size of his heart that matters.”

1	The cardiovascular system is one of the highest yield areas for the boards and, for some students, may be the most challenging. Focusing on understanding the mechanisms instead of memorizing the details can make a big difference, especially for this topic. Pathophysiology of atherosclerosis and heart failure, MOA of drugs (particular physiology interactions) and their adverse effects, ECGs of heart blocks, the cardiac cycle, and the Starling curve are some of the more high-yield topics. Differentiating between systolic and diastolic dysfunction is also very important. Heart murmurs and maneuvers that affect these murmurs have also been high yield and may be asked in a multimedia format. Septation of the chambers

1	Septation of the chambers Septum primum grows toward endocardial cushions, narrowing foramen primum. Foramen secundum forms in septum primum (foramen primum regresses). Septum secundum develops on the right side of septum primum, as foramen secundum maintains right-to-left shunt. Septum secundum expands and covers most of foramen secundum. The residual foramen is the foramen ovale. Remaining portion of septum primum forms the one-way valve of the foramen ovale. 6. Septum primum closes against septum secundum, sealing the foramen ovale soon after birth because of  LA pressure and  RA pressure. 7. Septum secundum and septum primum fuse during infancy/early childhood, forming the atrial septum. Patent foramen ovale—caused by failure of septum primum and septum secundum to fuse after birth; most are left untreated. Can lead to paradoxical emboli (venous thromboemboli entering the systemic arterial circulation) as can occur in ASD.

1	Muscular interventricular septum forms. Opening is called interventricular foramen. Aorticopulmonary septum rotates and fuses with muscular ventricular septum to form membranous interventricular septum, closing interventricular foramen. Growth of endocardial cushions separates atria from ventricles and contributes to both atrial septation and membranous portion of the interventricular septum. Ventricular septal defect—most common congenital cardiac anomaly, usually occurs in membranous septum. Primitive ventricle Trabeculated part of left and right ventricles Primitive atrium Trabeculated part of left and right atria Left horn of sinus venosus Coronary sinus Right horn of sinus venosus Smooth part of right atrium (sinus venarum) Endocardial cushion Atrial septum, membranous interventricular septum; AV and semilunar valves Posterior, subcardinal, and supracardinal veins Inferior vena cava (IVC) Primitive pulmonary vein Smooth part of left atrium

1	Posterior, subcardinal, and supracardinal veins Inferior vena cava (IVC) Primitive pulmonary vein Smooth part of left atrium Prostaglandins E1 and E2 kEEp PDA open. Ductus arteriosus Ligamentum arteriosum Near the left recurrent laryngeal nerve Allantois  urachus Median umbilical ligament Urachus is part of allantoic duct between bladder and umbilicus Blood in umbilical vein has a Po2 of ≈ 30 mm Hg and is ≈ 80% saturated with O2. Umbilical arteries have low O2 saturation. 3 important shunts: Blood entering fetus through the umbilical vein is conducted via the ductus venosus into the IVC, bypassing hepatic circulation. of the highly Oxygenated blood reaching the heart via the IVC is directed through the foramen Ovale into the left atrium. blood from the SVC passes through the RA  RV  main pulmonary artery • Ductus arteriosus • Descending aorta; shunt is due to high fetal pulmonary artery resistance (due partly to low O2 tension).

1	blood from the SVC passes through the RA  RV  main pulmonary artery • Ductus arteriosus • Descending aorta; shunt is due to high fetal pulmonary artery resistance (due partly to low O2 tension). At birth, infant takes a breath • resistance in pulmonary vasculature  left atrial pressure vs right atrial pressure foramen ovale closes (now called fossa ovalis);  in O2 (from respiration) and  in prostaglandins (from placental separation)  closure of ductus arteriosus. Indomethacin helps close the patent Ductus arteriosus  ligamentum arteriosum (remnant of ductus arteriosus). Come In and close the Door. Anatomy of the heart LA is the most posterior part of the heart A ; enlargement of the LA (eg, in mitral stenosis) can lead to compression of the esophagus (dysphagia) and/or the left recurrent laryngeal nerve, a branch of the vagus nerve, causing hoarseness (Ortner syndrome). RV is the most anterior part of the heart and most commonly injured in trauma. Key:

1	RV is the most anterior part of the heart and most commonly injured in trauma. Key: LAD and its branches supply anterior 2/3 of interventricular septum, anterolateral papillary muscle, and anterior surface of LV. Most commonly occluded. PDA supplies AV node (dependent on dominance), posterior 1/3 of interventricular septum, posterior 2/3 walls of ventricles, and posteromedial papillary muscle. RCA supplies SA node (blood supply independent of dominance). Infarct may cause nodal dysfunction (bradycardia or heart block). Right (acute) marginal artery supplies RV. Dominance: Right-dominant circulation (85%) = PDA arises from RCA. Left-dominant circulation (8%) = PDA arises from LCX. from both LCX and RCA. Coronary blood flow peaks in early diastole. Force of contraction is proportional to end-diastolic length of cardiac muscle fiber (preload). contractility with catecholamines, positive inotropes (eg, digoxin).

1	Force of contraction is proportional to end-diastolic length of cardiac muscle fiber (preload). contractility with catecholamines, positive inotropes (eg, digoxin). contractility with loss of functional myocardium (eg, MI), β-blockers (acutely), non-dihydropyridine Ca2+ channel blockers, dilated cardiomyopathy. Resistance, pressure, flow ΔP = Q × R Similar to Ohm’s law: ΔV = I × R Volumetric flow rate (Q) = flow velocity (v) × Q πr4 Total resistance of vessels in series: RT = R1 + R2 + R3 . . . Total resistance of vessels in parallel: 1 111 Capillaries have highest total cross-sectional area and lowest flow velocity. Pressure gradient drives flow from high pressure to low pressure. Arterioles account for most of TPR. Veins provide most of blood storage capacity. Viscosity depends mostly on hematocrit. Viscosity  in hyperproteinemic states (eg, multiple myeloma), polycythemia. Viscosity  in anemia.

1	volume, venous tone TPR TPRCardiac output/venous return Cardiac output/venous return Vascularfunction curve Cardiac function curve ↓↓↓↓↓↓volume, venous tone inotropy inotropy Mean systemic pressure Normal Intersection of curves = operating point of heart (ie, venous return and CO are equal, as circulatory system is a closed system). Changes often occur in tandem, and may be reinforcing (eg, exercise  inotropy and  TPR to maximize CO) or compensatory (eg, HF  inotropy  fluid retention to  preload to maintain CO). Pressure-volume loops and cardiac cycle The black loop represents normal cardiac physiology. Phases—left ventricle: valve opening; period of highest O2 consumption Heart sounds: S1—mitral and tricuspid valve closure. Loudest at mitral area. S2—aortic and pulmonary valve closure. Loudest at left upper sternal border. Considered abnormal if palpable. Jugular venous pulse (JVP): a wave—atrial contraction. Absent in atrial fibrillation (AF).

1	Considered abnormal if palpable. Jugular venous pulse (JVP): a wave—atrial contraction. Absent in atrial fibrillation (AF). filling phase. Best heard at apex with patient in left lateral decubitus position. Associated with  filling pressures (eg, MR, AR, HF, ventricles (but can be normal in children, young adults, athletes, and pregnancy). S4—in late diastole (“atrial kick”). Best heard at apex with patient in left lateral decubitus position. High atrial pressure. Associated with ventricular noncompliance (eg, hypertrophy). Left atrium must push against stiff LV wall. bulging into atrium). x descent—downward displacement of closed (aka J.V.P) ejection phase. Reduced or absent in tricuspid regurgitation and right HF because pressure gradients are reduced. v wave— right atrial pressure due to filling 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 (“villing”) against closed tricuspid valve.

1	Time (sec) y descent—RA emptying into RV. Prominent in constrictive pericarditis, absent in cardiac tamponade. 120  ESV due to  resistance and due to  LA volume/pressure from because of impaired ventricular filling Splitting of S2 Wide splitting Seen in conditions that delay RV emptying (eg, E pulmonic stenosis, right bundle branch block). S1 A2P2 Causes delayed pulmonic sound (especially on inspiration). An exaggeration of normal Abnormal delay splitting. Fixed splitting Heard in ASD. ASD  left-to-right shunt  RA and RV volumes  flow through pulmonic valve  delayed pulmonic valve S1 A2P2 closure (independent of respiration). I closure (eg, aortic stenosis, left bundle branch S1 P2A2 block). Normal order of semilunar valve closure is reversed so that P2 sound occurs before delayed A2 sound. On inspiration, P2 closes later and moves closer to A2, “paradoxically” eliminating the split. On expiration, the split can be heard (opposite to physiologic splitting).

1	Auscultation of the heart (eg, physiologic murmur) Aortic valve sclerosis Where to listen: APT M 111234567777777 Left sternal border: Aortic regurgitation Pulmonic regurgitation cardiomyopathy PA M T Aortic area: Systolic murmur Pulmonic area: Tricuspid area: Mitral area (apex): Crescendo-decrescendo systolic ejection murmur and soft S2 (ejection click may be present). LV >> aortic pressure during systole. Loudest at heart base; radiates to carotids. “Pulsus parvus et tardus”—pulses are weak with a delayed peak. Can lead to Syncope, Angina, and Dyspnea on exertion (SAD). Most commonly due to age-related calcification in older patients (> 60 years old) or in younger patients with early-onset calcification of bicuspid aortic valve.

1	Holosystolic, high-pitched “blowing murmur.” Mitral—loudest at apex and radiates toward axilla. MR is often due to ischemic heart disease (post-MI), MVP, LV dilatation. Tricuspid—loudest at tricuspid area. TR commonly caused by RV dilatation. Rheumatic fever and infective endocarditis can cause either MR or TR. Late systolic crescendo murmur with midsystolic click (MC) due to sudden tensing of chordae tendineae as mitral leaflets prolapse into the LA (Chordae cause Crescendo with Click). Most frequent valvular lesion. Best heard over apex. Loudest just before S2. Usually benign. Can predispose to infective endocarditis. Can be caused by myxomatous degeneration (1° or 2° to connective tissue disease such as Marfan or Ehlers-Danlos syndrome), rheumatic fever (particularly in developing countries), chordae rupture.

1	High-pitched “blowing” early diastolic decrescendo murmur. Best heard at base (aortic root dilation) or left sternal border (valvular disease). Long diastolic murmur, hyperdynamic pulse, and head bobbing when severe and chronic. Wide pulse pressure. Causes include Bicuspid aortic valve, Endocarditis, Aortic root dilation, Rheumatic fever (BEAR). Progresses to left HF. Follows opening snap (OS; due to abrupt halt in leaflet motion in diastole, after rapid opening due to fusion at leaflet tips). Delayed rumbling mid-to-late diastolic murmur ( interval between S2 and OS correlates with  severity). LA >> LV pressure during diastole. Often a late (and highly specific) sequela of rheumatic fever. Chronic MS can result in pulmonary congestion/hypertension and LA dilation  atrial fibrillation and Ortner syndrome. Patent ductus arteriosus Continuous machine-like murmur. Best heard at left infraclavicular area. Loudest at S2. Often due to congenital rubella or prematurity.

1	Patent ductus arteriosus Continuous machine-like murmur. Best heard at left infraclavicular area. Loudest at S2. Often due to congenital rubella or prematurity. “PDAs (Public Displays of Affection) are continuously annoying.” Phase 0 = rapid upstroke and depolarization— voltage-gated Na+ channels open. Phase 1 = initial repolarization—inactivation of voltage-gated Na+ channels. Voltage-gated K+ channels begin to open. Phase 2 = plateau—Ca2+ influx through voltage-gated Ca2+ channels balances K+ efflux. Ca2+ influx triggers Ca2+ release from sarcoplasmic reticulum and myocyte contraction. Phase 3 = rapid repolarization—massive K+ efflux due to opening of voltage-gated slow delayed-rectifier K+ channels and closure of voltage-gated Ca2+ channels. Phase 4 = resting potential—high K+ permeability through K+ channels. In contrast to skeletal muscle: Cardiac muscle action potential has a plateau due to Ca2+ influx and K+ efflux.

1	Phase 4 = resting potential—high K+ permeability through K+ channels. In contrast to skeletal muscle: Cardiac muscle action potential has a plateau due to Ca2+ influx and K+ efflux. Cardiac muscle contraction requires Ca2+ influx from ECF to induce Ca2+ release from sarcoplasmic reticulum (Ca2+-induced Ca2+ release). Cardiac myocytes are electrically coupled to each other by gap junctions. Occurs in all cardiac myocytes except for those in the SA and AV nodes. Occurs in the SA and AV nodes. Key differences from the ventricular action potential include: Phase 0 = upstroke—opening of voltage-gated Ca2+ channels. Fast voltage-gated Na+ channels are permanently inactivated because of the less negative resting potential of these cells. Results in a slow conduction velocity that is used by the AV node to prolong transmission from the atria to ventricles. Phases 1 and 2 are absent.

1	Phases 1 and 2 are absent. Phase 3 = repolarization—inactivation of the Ca2+ channels and  activation of K+ channels  K+ efflux. Phase 4 = slow spontaneous diastolic depolarization due to If (“funny current”). If channels responsible for a slow, mixed Na+/K+ inward current; different from INa in phase 0 of ventricular action potential. Accounts for automaticity of SA and AV nodes. The slope of phase 4 in the SA node determines HR. ACh/adenosine  the rate of diastolic depolarization and  HR, while catecholamines • depolarization and  HR. Sympathetic stimulation  the chance that If channels are open and thus  HR. Electrocardiogram Conduction pathway: SA node  atria  bundle of His  right and left bundle branches  Purkinje fibers left bundle branch divides into left anterior and posterior fascicles. SA node—located at junction of RA and SVC; “pacemaker” inherent dominance with slow phase of upstroke.

1	SA node—located at junction of RA and SVC; “pacemaker” inherent dominance with slow phase of upstroke. AV node—located in posteroinferior part of interatrial septum. Blood supply usually from RCA. 100-msec delay allows time for ventricular filling. Pacemaker rates: SA > AV > bundle of His/ Purkinje/ventricles. Speed of conduction: His-Purkinje > Atria > Ventricles > AV node. He Parks At Ventura Avenue. P wave—atrial depolarization. PR interval—time from start of atrial depolarization to start of ventricular depolarization (normally 120-200 msec). QRS complex—ventricular depolarization (normally < 100 msec). QT interval—ventricular depolarization, mechanical contraction of the ventricles, ventricular repolarization.

1	QRS complex—ventricular depolarization (normally < 100 msec). QT interval—ventricular depolarization, mechanical contraction of the ventricles, ventricular repolarization. T wave—ventricular repolarization. T-wave inversion may indicate ischemia or recent MI. J point—junction between end of QRS complex and start of ST segment. ST segment—isoelectric, ventricles depolarized. U wave—prominent in hypokalemia (think hyp“U”kalemia), bradycardia. SA node AV node Bundle of His Torsades de pointes Polymorphic ventricular tachycardia, characterized by shifting sinusoidal waveforms on ECG; can progress to ventricular fibrillation (VF). Long QT interval predisposes to torsades de pointes. Caused by drugs,  K+,  Mg2+, • Ca2+, congenital abnormalities. Treatment includes magnesium sulfate. Drug-induced long QT (ABCDE): AntiArrhythmics (class IA, III) AntiBiotics (eg, macrolides) Anti“C”ychotics (eg, haloperidol) AntiDepressants (eg, TCAs) AntiEmetics (eg, ondansetron)

1	Drug-induced long QT (ABCDE): AntiArrhythmics (class IA, III) AntiBiotics (eg, macrolides) Anti“C”ychotics (eg, haloperidol) AntiDepressants (eg, TCAs) AntiEmetics (eg, ondansetron) Torsades de pointes = twisting of the points Brugada syndrome Autosomal dominant disorder most common in Asian males. ECG pattern of pseudo-right bundle branch block and ST elevations in V1-V3.  risk of ventricular tachyarrhythmias and SCD. Prevent SCD with implantable cardioverter-defibrillator (ICD). Atrial fibrillation Chaotic and erratic baseline with no discrete P waves in between RR˜ RR ˜RR˜RR irregularly spaced QRS complexes. Irregularly irregular heartbeat. Most common risk factors include hypertension and coronary artery disease (CAD). Occasionally seen after binge drinking (“holiday heart syndrome”). Can lead to thromboembolic events, particularly stroke. Treatment: anticoagulation, rate and rhythm control and/or cardioversion.

1	Treatment: anticoagulation, rate and rhythm control and/or cardioversion. First-degree The PR interval is prolonged (> 200 msec). Benign and AV block asymptomatic. No treatment required. <<PR1PR2PR3P wave, absent QRS =PR1PR2P wave, absent QRS Baroreceptors and chemoreceptors Receptors: • Aortic arch transmits via vagus nerve to solitary nucleus of medulla (responds to changes in BP). • Carotid sinus (dilated region at carotid bifurcation) transmits via glossopharyngeal nerve to solitary nucleus of medulla (responds to changes in BP). Baroreceptors: parasympathetic stimulation  vasoconstriction,  HR,  contractility,  BP. Important in the response to severe hemorrhage. Carotid massage— pressure on carotid sinus  stretch  afferent baroreceptor firing  AV node refractory period  HR.

1	Carotid massage— pressure on carotid sinus  stretch  afferent baroreceptor firing  AV node refractory period  HR. Component of Cushing reflex (triad of hypertension, bradycardia, and respiratory depression)— intracranial pressure constricts arterioles  cerebral ischemia • pCO2 and  pH  central reflex sympathetic  in perfusion pressure (hypertension)  stretch  peripheral reflex baroreceptor– induced bradycardia. Chemoreceptors: Peripheral—carotid and aortic bodies are stimulated by  Pco2,  pH of blood, and  Po2 (< 60 mm Hg). Central—are stimulated by changes in pH and Pco2 of brain interstitial fluid, which in turn are influenced by arterial CO2 as H+ cannot cross the blood-brain barrier. Do not directly respond to Po2. Central chemoreceptors become less responsive with chronically  Pco2 (eg, COPD) • dependence on peripheral chemoreceptors to detect  O2 to drive respiration.

1	pressures in mm Hg) is a good approximation of left atrial pressure. In mitral stenosis, PCWP > LV end diastolic pressure. PCWP is measured with pulmonary artery catheter (Swan-Ganz catheter). Autoregulation How blood flow to an organ remains constant over a wide range of perfusion pressures.

1	Autoregulation How blood flow to an organ remains constant over a wide range of perfusion pressures. Capillary fluid Starling forces determine fluid movement through capillary membranes: exchange • Pc = capillary hydrostatic pressure—pushes fluid out of capillary πi = interstitial fluid colloid osmotic pressure—pulls fluid out of capillary Jv = net fluid flow = Kf [(Pc − Pi) −σ(πc −πi)] Kf = capillary permeability to fluid σ= reflection coefficient (measure of capillary permeability to protein) Edema—excess fluid outflow into interstitium commonly caused by: •  capillary pressure ( Pc; eg, HF) •  capillary permeability ( Kf; eg, toxins, infections, burns) •  interstitial fluid colloid osmotic pressure (• πi; eg, lymphatic blockage) •  plasma proteins (• πc; eg, nephrotic syndrome, liver failure, protein malnutrition) D-transposition of great vessels leaves LV (posterior)  separation of systemic

1	D-transposition of great vessels leaves LV (posterior)  separation of systemic Pulmonary and pulmonary circulations. Not compatible artery with life unless a shunt is present to allow mixing of blood (eg, VSD, PDA, or patent foramen ovale). Left Due to failure of the aorticopulmonary septum to ventricle spiral (“egg on a string” appearance on CXR). Without surgical intervention, most infants die Right ventricle within the first few months of life. Tricuspid atresia Absence of tricuspid valve and hypoplastic RV; requires both ASD and VSD for viability. Tetralogy of Fallot Caused by anterosuperior displacement of the infundibular septum. Most common cause of early childhood cyanosis. Pulmonary infundibular stenosis (most important determinant for prognosis) Right ventricular hypertrophy (RVH)— boot-shaped heart on CXR

1	Pulmonary stenosis forces right-to-left flow across VSD  RVH, “tet spells” (often caused by crying, fever, and exercise due to exacerbation of RV outflow obstruction). PROVe. Squatting:  SVR,  right-to-left shunt, improves cyanosis. Associated with 22q11 syndromes. lEFT-TO-RIGHT SHuNTS Acyanotic at presentation; cyanosis may occur Right-to-Left shunts: eaRLy cyanosis. years later. Frequency: VSD > ASD > PDA. Left-to-Right shunts: “LateR” cyanosis. Ventricular septal Asymptomatic at birth, may manifest weeks O2 saturation  in RV and pulmonary artery. defect later or remain asymptomatic throughout life. Most self resolve; larger lesions B may lead to LV overload and HF.

1	Defect in interatrial septum C ; wide, fixed split S2. Ostium secundum defects most common and usually an isolated finding; ostium primum defects rarer and usually occur with other cardiac anomalies. Symptoms range from none to HF. Distinct from patent foramen ovale in that septa are missing tissue rather than unfused. O2 saturation  in RA, RV, and pulmonary artery. May lead to paradoxical emboli (systemic venous emboli use ASD to bypass lungs and become systemic arterial emboli). Associated with Down syndrome. In fetal period, shunt is right to left (normal). In neonatal period,  pulmonary vascular resistance  shunt becomes left to right  progressive RVH and/or LVH and HF. Associated with a continuous, “machine-like” murmur. Patency is maintained by PGE synthesis and low O2 tension. Uncorrected PDA D can eventually result in late cyanosis in the lower extremities (differential cyanosis). PDA is normal in utero and normally closes only after birth.

1	PDA is normal in utero and normally closes only after birth. Uncorrected left-to-right shunt (VSD, ASD, PDA)  pulmonary blood flow • pathologic remodeling of vasculature  pulmonary arterial hypertension. RVH occurs to compensate  shunt becomes right to left. Causes late cyanosis, clubbing E , and polycythemia. Age of onset varies. Coarctation of the aorta Aortic narrowing F near insertion of ductus arteriosus (“juxtaductal”). Associated with bicuspid aortic valve, other heart defects, and Turner syndrome. Hypertension in upper extremities and weak, delayed pulse in lower extremities (brachial-femoral delay). With age, intercostal arteries enlarge due to collateral circulation; arteries erode ribs  notched appearance on CXR. Complications include HF,  risk of cerebral hemorrhage (berry aneurysms), aortic rupture, and possible endocarditis. defect associations Alcohol exposure in utero (fetal alcohol VSD, PDA, ASD, tetralogy of Fallot syndrome)

1	defect associations Alcohol exposure in utero (fetal alcohol VSD, PDA, ASD, tetralogy of Fallot syndrome) Infant of diabetic mother Transposition of great vessels, VSD Marfan syndrome MVP, thoracic aortic aneurysm and dissection, aortic regurgitation Turner syndrome Bicuspid aortic valve, coarctation of aorta 22q11 syndromes Truncus arteriosus, tetralogy of Fallot Hypertension Persistent systolic BP ≥ 130 mm Hg and/or diastolic BP ≥ 80 mm Hg. RISK FACTORS  age, obesity, diabetes, physical inactivity, excess salt intake, excess alcohol intake, cigarette smoking, family history; African American > Caucasian > Asian.

1	RISK FACTORS  age, obesity, diabetes, physical inactivity, excess salt intake, excess alcohol intake, cigarette smoking, family history; African American > Caucasian > Asian. FEATuRES 90% of hypertension is 1° (essential) and related to  CO or  TPR. Remaining 10% mostly 2° to renal/renovascular diseases such as fibromuscular dysplasia (characteristic “string of beads” appearance of renal artery A , usually seen in women of child-bearing age) and atherosclerotic renal artery stenosis or to 1° hyperaldosteronism. Hypertensive urgency—severe (≥ 180/≥ 120 mm Hg) hypertension without acute end-organ damage. Hypertensive emergency—severe hypertension with evidence of acute end-organ damage (eg, encephalopathy, stroke, retinal hemorrhages and exudates, papilledema, MI, HF, aortic dissection, kidney injury, microangiopathic hemolytic anemia, eclampsia).

1	PREdISPOSES TO CAD, LVH, HF, atrial fibrillation; aortic dissection, aortic aneurysm; stroke; CKD (hypertensive nephropathy); retinopathy. Xanthomas Plaques or nodules composed of lipid-laden histiocytes in skin A , especially the eyelids (xanthelasma B ). Tendinous xanthoma Lipid deposit in tendon C , especially Achilles. Corneal arcus Lipid deposit in cornea. Common in elderly (arcus senilis D ), but appears earlier in life with hypercholesterolemia. Arteriosclerosis Hardening of arteries, with arterial wall thickening and loss of elasticity. Arteriolosclerosis Common. Affects small arteries and arterioles. Two types: hyaline (thickening of vessel walls 2° to plasma protein leak into endothelium in essential hypertension or diabetes mellitus A ) and hyperplastic (“onion skinning” in severe hypertension B with proliferation of smooth muscle cells).

1	Atherosclerosis Very common. Disease of elastic arteries and largeand medium-sized muscular arteries; a form of arteriosclerosis caused by buildup of cholesterol plaques in intima. lOCATION Abdominal aorta > Coronary artery > Popliteal artery > Carotid artery > circle of Willis. A CoPy Cat named Willis. RISK FACTORS Modifiable: smoking, hypertension, dyslipidemia ( LDL,  HDL), diabetes. Non-modifiable: age, sex ( in men and postmenopausal women), family history. SYMPTOMS Angina, claudication, but can be asymptomatic. PROGRESSION Inflammation important in pathogenesis: endothelial cell dysfunction  macrophage and LDL accumulation  foam cell formation  fatty streaks  smooth muscle cell migration (involves PDGF and FGF), proliferation, and extracellular matrix deposition  fibrous plaque  complex atheromas A  calcification (calcium content correlates with risk of complications). COMPlICATIONS Aneurysms, ischemia, infarcts, peripheral vascular disease, thrombus, emboli.

1	COMPlICATIONS Aneurysms, ischemia, infarcts, peripheral vascular disease, thrombus, emboli. Aortic aneurysm Localized pathologic dilation of the aorta. May cause abdominal and/or back pain, which is a sign of leaking, dissection, or imminent rupture. Abdominal aortic Usually associated with atherosclerosis. Risk factors include history of tobacco use,  age, male aneurysm sex, family history. May present as palpable pulsatile abdominal mass (arrows in A point to outer dilated calcified aortic wall, with partial crescent-shaped non-opacification of aorta due to flap/ clot). Most often infrarenal (distal to origin of renal arteries). Traumatic aortic Due to trauma and/or deceleration injury, most commonly at aortic isthmus (proximal descending rupture aorta just distal to origin of left subclavian artery). X-ray may reveal widened mediastinum.

1	Longitudinal intimal tear forming a false lumen. Associated with hypertension, bicuspid aortic valve, inherited connective tissue disorders (eg, Marfan syndrome). Can present with tearing, sudden-onset chest pain radiating to the back +/− markedly unequal BP in arms. CXR can show mediastinal widening. Can result in organ ischemia, aortic rupture, death. Two types: Stanford type A (proximal): involves Ascending aorta (red arrow in A ). May extend to aortic arch or descending aorta (blue arrow in A ). May result in acute aortic regurgitation or cardiac tamponade. Treatment: surgery. Stanford type B (distal): involves only descending aorta (Below left subclavian artery). Treatment: β-blockers, then vasodilators. Angina Chest pain due to ischemic myocardium 2° to coronary artery narrowing or spasm; no myocyte necrosis.

1	Angina Chest pain due to ischemic myocardium 2° to coronary artery narrowing or spasm; no myocyte necrosis. Stable—usually 2° to atherosclerosis (≥ 70% occlusion); exertional chest pain in classic distribution (usually with ST depression on ECG), resolving with rest or nitroglycerin. Vasospastic (also called Prinzmetal or Variant)—occurs at rest 2° to coronary artery spasm; transient ST elevation on ECG. Smoking is a risk factor; hypertension and hypercholesterolemia are not. Triggers include cocaine, alcohol, and triptans. Treat with Ca2+ channel blockers, nitrates, and smoking cessation (if applicable). Unstable—thrombosis with incomplete coronary artery occlusion; +/− ST depression and/or T-wave inversion on ECG but no cardiac biomarker elevation (unlike NSTEMI);  in frequency or intensity of chest pain or any chest pain at rest.

1	Sudden cardiac death Death from cardiac causes within 1 hour of onset of symptoms, most commonly due to a lethal arrhythmia (eg, VF). Associated with CAD (up to 70% of cases), cardiomyopathy (hypertrophic, dilated), and hereditary ion channelopathies (eg, long QT syndrome, Brugada syndrome). Prevent with ICD. Evolution of Commonly occluded coronary arteries: LAD > RCA > circumflex. myocardial infarction Symptoms: diaphoresis, nausea, vomiting, severe retrosternal pain, pain in left arm and/or jaw, shortness of breath, fatigue. 0–24 hr Dark mottling Early coagulative necrosis Ventricular arrhythmia, HF,  cell content released into cardiogenic shock blood; edema, hemorrhage, wavy fibers  hypercontraction of Dark mottling; stripes) pale with tetrazolium stain Tissue surrounding infarct pericarditis shows acute inflammation with neutrophils 3–14 days Macrophages, then granulation Free wall rupture  tamponade; tissue at margins papillary muscle rupture due to macrophage-mediated

1	LV pseudoaneurysm (risk of rupture) 2 weeks to several Contracted scar complete Dressler syndrome, HF, months arrhythmias, true ventricular aneurysm (risk of mural thrombus) Multiples of upper limit of normal return to normal after 48 hours. Large MIs lead to greater elevations in troponin I and CK-MB. Exact curves vary with testing procedure. ECG changes can include ST elevation (STEMI, transmural infarct), ST depression (NSTEMI, subendocardial infarct), hyperacute (peaked) T waves, T-wave inversion, new left bundle branch block, and pathologic Q waves or poor R wave progression (evolving or old transmural infarct). Ventricular 3–14 days: free wall rupture contained by adherent pericardium or scar tissue B ;  CO, risk of pseudoaneurysm arrhythmia, embolus from mural thrombus. formation Ventricular free wall 5–14 days: free wall rupture C  cardiac tamponade. LV hypertrophy and previous MI protect rupture against free wall rupture. Acute form usually leads to sudden death.

1	Ventricular free wall 5–14 days: free wall rupture C  cardiac tamponade. LV hypertrophy and previous MI protect rupture against free wall rupture. Acute form usually leads to sudden death. 60–70% of cases are familial, autosomal dominant (most commonly due to mutations in genes encoding sarcomeric proteins, such as myosin binding protein C and β-myosin heavy chain). Causes syncope during exercise and may lead to sudden death (eg, in young athletes) due to ventricular arrhythmia. Findings: S4, systolic murmur. May see mitral regurgitation due to impaired mitral valve closure. Treatment: cessation of high-intensity athletics, use of β-blocker or non-dihydropyridine Ca2+ channel blockers (eg, verapamil). ICD if syncope occurs. Diastolic dysfunction ensues. Marked ventricular concentric hypertrophy (sarcomeres added in parallel) B , often septal predominance. Myofibrillar disarray and fibrosis.

1	Diastolic dysfunction ensues. Marked ventricular concentric hypertrophy (sarcomeres added in parallel) B , often septal predominance. Myofibrillar disarray and fibrosis. Physiology of HOCM—asymmetric septal hypertrophy and systolic anterior motion of mitral valve  outflow obstruction  dyspnea, possible syncope. Other causes of concentric LV hypertrophy: chronic HTN, Friedreich ataxia. Postradiation fibrosis, Löffler endocarditis, Endocardial fibroelastosis (thick fibroelastic tissue in endocardium of young children), Amyloidosis, Sarcoidosis, Hemochromatosis (although dilated cardiomyopathy is more common) (Puppy LEASH). Diastolic dysfunction ensues. Can have low-voltage ECG despite thick myocardium (especially in amyloidosis). Löffler endocarditis—associated with hypereosinophilic syndrome; histology shows eosinophilic infiltrates in myocardium.

1	Löffler endocarditis—associated with hypereosinophilic syndrome; histology shows eosinophilic infiltrates in myocardium. Heart failure Clinical syndrome of cardiac pump dysfunction  congestion and low perfusion. Symptoms include dyspnea, orthopnea, fatigue; signs include S3 heart sound, rales, jugular venous distention (JVD), pitting edema A . Systolic dysfunction—reduced EF,  EDV;  contractility often 2° to ischemia/MI or dilated cardiomyopathy. Diastolic dysfunction—preserved EF, normal EDV; • compliance ( EDP) often 2° to myocardial hypertrophy. Right HF most often results from left HF. Cor pulmonale refers to isolated right HF due to pulmonary cause. ACE inhibitors or angiotensin II receptor blockers, β-blockers (except in acute decompensated HF), and spironolactone  mortality. Loop and thiazide diuretics are used mainly for symptomatic relief. Hydralazine with nitrate therapy improves both symptoms and mortality in select patients.

1	of hemosiderin-laden macrophages (“HF” cells) in lungs. Right heart failure Shock Inadequate organ perfusion and delivery of nutrients necessary for normal tissue and cellular function. Initially may be reversible but life threatening if not treated promptly. Compression of the heart by fluid (eg, blood, effusions [arrows in A ] in pericardial space)  CO. Equilibration of diastolic pressures in all 4 chambers. Findings: Beck triad (hypotension, distended neck veins, distant heart sounds),  HR, pulsus paradoxus. ECG shows low-voltage QRS and electrical alternans B (due to “swinging” movement of heart in large effusion). Pulsus paradoxus— in amplitude of systolic BP by > 10 mm Hg during inspiration. Seen in constrictive Pericarditis, obstructive pulmonary disease (eg, Croup, OSA, Asthma, COPD), cardiac Tamponade (Pea COAT).

1	Bacterial endocarditis Acute—S aureus (high virulence). Large vegetations on previously normal valves A . Rapid onset. Subacute—viridans streptococci (low virulence). Smaller vegetations on congenitally abnormal or diseased valves. Sequela of dental procedures. Gradual onset. Symptoms: fever (most common), new murmur, Roth spots (round white spots on retina surrounded by hemorrhage B ), Osler nodes (Ouchy raised lesions on finger or toe pads C due to immune complex deposition), Janeway lesions (small, painless, erythematous lesions on palm or sole) D , splinter hemorrhages E on nail bed. Associated with glomerulonephritis, septic arterial or pulmonary emboli. May be nonbacterial (marantic/thrombotic) 2° to malignancy, hypercoagulable state, or lupus. FROM JANE with ♥: Fever Roth spots Osler nodes Murmur Janeway lesions Anemia Nail-bed hemorrhage Emboli Requires multiple blood cultures for diagnosis. If culture ⊝, most likely Coxiella burnetii,

1	FROM JANE with ♥: Fever Roth spots Osler nodes Murmur Janeway lesions Anemia Nail-bed hemorrhage Emboli Requires multiple blood cultures for diagnosis. If culture ⊝, most likely Coxiella burnetii, Bartonella spp. Mitral valve is most frequently involved. Tricuspid valve endocarditis is associated with IV drug abuse (don’t “tri” drugs). Associated with S aureus, Pseudomonas, and Candida. S bovis (gallolyticus) is present in colon cancer, S epidermidis on prosthetic valves. Native valve endocarditis may be due to HACEK organisms (Haemophilus, Aggregatibacter [formerly Actinobacillus], Cardiobacterium, Eikenella, Kingella). Inflammation of the pericardium [ A , red arrows]. Commonly presents with sharp pain, aggravated by inspiration, and relieved by sitting up and leaning forward. Often complicated by pericardial effusion [between yellow arrows in A ]. Presents with friction rub. ECG changes include widespread ST-segment elevation and/or PR depression.

1	Causes include idiopathic (most common; presumed viral), confirmed infection (eg, coxsackievirus B), neoplasia, autoimmune (eg, SLE, rheumatoid arthritis), uremia, cardiovascular (acute STEMI or Dressler syndrome), radiation therapy. Treatment: NSAIDs, colchicine, glucocorticoids, dialysis (uremia). Myocarditis Inflammation of myocardium  global enlargement of heart and dilation of all chambers. Major cause of SCD in adults < 40 years old. Presentation highly variable, can include dyspnea, chest pain, fever, arrhythmias (persistent tachycardia out of proportion to fever is characteristic). Multiple causes: Viral (eg, adenovirus, coxsackie B, parvovirus B19, HIV, HHV-6); lymphocytic infiltrate with focal necrosis highly indicative of viral myocarditis. Parasitic (eg, Trypanosoma cruzi, Toxoplasma gondii) Bacterial (eg, Borrelia burgdorferi, Mycoplasma pneumoniae, Corynebacterium diphtheriae) Toxins (eg, carbon monoxide, black widow venom) Drugs (eg, doxorubicin, cocaine)

1	Bacterial (eg, Borrelia burgdorferi, Mycoplasma pneumoniae, Corynebacterium diphtheriae) Toxins (eg, carbon monoxide, black widow venom) Drugs (eg, doxorubicin, cocaine) Autoimmune (eg, Kawasaki disease, sarcoidosis, SLE, polymyositis/dermatomyositis) Complications include sudden death, arrhythmias, heart block, dilated cardiomyopathy, HF, mural thrombus with systemic emboli. Buerger disease Heavy smokers, males < 40 years old. Segmental thrombosing vasculitis with vein and (thromboangiitis Intermittent claudication. May lead to nerve involvement. obliterans) gangrene C , autoamputation of digits, Treatment: smoking cessation. superficial nodular phlebitis. Raynaud phenomenon is often present.

1	C , autoamputation of digits, Treatment: smoking cessation. superficial nodular phlebitis. Raynaud phenomenon is often present. Kawasaki disease Asian children < 4 years old. CRASH and burn on a Kawasaki. (mucocutaneous Conjunctival injection, Rash (polymorphous May develop coronary artery aneurysms E ; lymph node  desquamating), Adenopathy (cervical), thrombosis or rupture can cause death. syndrome) Strawberry tongue (oral mucositis) D , Hand-Treatment: IV immunoglobulin and aspirin. foot changes (edema, erythema), fever. Polyarteritis nodosa Usually middle-aged men. Hepatitis B seropositivity in 30% of patients. Fever, weight loss, malaise, headache. GI: abdominal pain, melena. Hypertension, neurologic dysfunction, cutaneous eruptions, renal damage. Typically involves renal and visceral vessels, not pulmonary arteries. Different stages of transmural inflammation with fibrinoid necrosis.

1	Typically involves renal and visceral vessels, not pulmonary arteries. Different stages of transmural inflammation with fibrinoid necrosis. Innumerable renal microaneurysms F and spasms on arteriogram (string of pearls appearance). Treatment: corticosteroids, cyclophosphamide. Granulomatosis with polyangiitis (Wegener) Upper respiratory tract: perforation of nasal septum, chronic sinusitis, otitis media, mastoiditis. Lower respiratory tract: hemoptysis, cough, dyspnea. Renal: hematuria, red cell casts. Triad: Necrotizing glomerulonephritis PR3-ANCA/c-ANCA H (anti-proteinase 3). CXR: large nodular densities. Treatment: cyclophosphamide, corticosteroids. Cardiac tumors Most common heart tumor is a metastasis (eg, melanoma).

1	Cardiac tumors Most common heart tumor is a metastasis (eg, melanoma). Myxomas Most common 1° cardiac tumor in adults (arrows in A ). 90% occur in the atria (mostly left atrium). Myxomas are usually described as a “ball valve” obstruction in the left atrium (associated with multiple syncopal episodes). IL-6 production by tumor  constitutional symptoms (eg, fever, weight loss). May auscultate early diastolic “tumor plop” sound. Histology: gelatinous material, myxoma cells immersed in glycosaminoglycans. Adults make myxed drinks. Calcium channel Amlodipine, clevidipine, nicardipine, nifedipine, nimodipine (dihydropyridines, act on vascular blockers smooth muscle); diltiazem, verapamil (non-dihydropyridines, act on heart). MECHANISM Block voltage-dependent L-type calcium channels of cardiac and smooth muscle  muscle contractility. Vascular smooth muscle—amlodipine = nifedipine > diltiazem > verapamil. Heart—verapamil > diltiazem > amlodipine = nifedipine (verapamil = ventricle).

1	ClINICAl uSE Dihydropyridines (except nimodipine): hypertension, angina (including vasospastic type), Raynaud phenomenon. Nimodipine: subarachnoid hemorrhage (prevents cerebral vasospasm). Nicardipine, clevidipine: hypertensive urgency or emergency. Non-dihydropyridines: hypertension, angina, atrial fibrillation/flutter. AdvERSE EFFECTS Gingival hyperplasia. Dihydropyridine: peripheral edema, flushing, dizziness. Non-dihydropyridine: cardiac depression, AV block, hyperprolactinemia (verapamil), constipation. MECHANISM  cGMP  smooth muscle relaxation. Vasodilates arterioles > veins; afterload reduction. ClINICAl uSE Severe hypertension (particularly acute), HF (with organic nitrate). Safe to use during pregnancy. Frequently coadministered with a β-blocker to prevent reflex tachycardia. AdvERSE EFFECTS Compensatory tachycardia (contraindicated in angina/CAD), fluid retention, headache, angina, drug-induced lupus.

1	AdvERSE EFFECTS Compensatory tachycardia (contraindicated in angina/CAD), fluid retention, headache, angina, drug-induced lupus. Nitrates Nitroglycerin, isosorbide dinitrate, isosorbide mononitrate. MECHANISM Vasodilate by  NO in vascular smooth muscle  in cGMP and smooth muscle relaxation. Dilate veins >> arteries.  preload. ClINICAl uSE Angina, acute coronary syndrome, pulmonary edema. AdvERSE EFFECTS Reflex tachycardia (treat with β-blockers), hypotension, flushing, headache, “Monday disease” in industrial exposure: development of tolerance for the vasodilating action during the work week and loss of tolerance over the weekend • tachycardia, dizziness, headache upon reexposure. Contraindicated in right ventricular infarction, hypertrophic cardiomyopathy, and with concurrent PDE-5 inhibitor use. Verapamil is similar to β-blockers in effect. Pindolol and acebutolol are partial β-agonists that should be used with caution in angina.

1	Verapamil is similar to β-blockers in effect. Pindolol and acebutolol are partial β-agonists that should be used with caution in angina. MECHANISM Inhibits the late phase of inward sodium current thereby reducing diastolic wall tension and oxygen consumption. Does not affect heart rate or blood pressure. ClINICAl uSE Angina refractory to other medical therapies. AdvERSE EFFECTS Constipation, dizziness, headache, nausea. MECHANISM A neprilysin inhibitor; prevents degradation of natriuretic peptides, angiotensin II, and substance P  vasodilation,  ECF volume. ClINICAl uSE Used in combination with valsartan (an ARB) to treat HFrEF. AdvERSE EFFECTS Hypotension, hyperkalemia, cough, dizziness; contraindicated with ACE inhibitors due to angioedema. Cardiac glycosides Digoxin. MECHANISM Direct inhibition of Na+/K+ ATPase inhibition of Na+/Ca2+ exchanger. [Ca2+]i • positive inotropy. Stimulates vagus nerve  HR.

1	Cardiac glycosides Digoxin. MECHANISM Direct inhibition of Na+/K+ ATPase inhibition of Na+/Ca2+ exchanger. [Ca2+]i • positive inotropy. Stimulates vagus nerve  HR. ClINICAl uSE HF ( contractility); atrial fibrillation ( conduction at AV node and depression of SA node). AdvERSE EFFECTS Cholinergic effects (nausea, vomiting, diarrhea), blurry yellow vision (think van Glow), arrhythmias, AV block. Can lead to hyperkalemia, which indicates poor prognosis. Factors predisposing to toxicity: renal failure ( excretion), hypokalemia (permissive for digoxin binding at K+-binding site on Na+/K+ ATPase), drugs that displace digoxin from tissue-binding sites, and  clearance (eg, verapamil, amiodarone, quinidine). ANTIdOTE Slowly normalize K+, cardiac pacer, anti-digoxin Fab fragments, Mg2+.

1	ANTIdOTE Slowly normalize K+, cardiac pacer, anti-digoxin Fab fragments, Mg2+. Antiarrhythmics— Slow or block () conduction (especially in depolarized cells).  slope of phase 0 depolarization. Are sodium channel state dependent (selectively depress tissue that is frequently depolarized [eg, tachycardia]). blockers (class I) Class IA Quinidine, Procainamide, Disopyramide. “The Queen Proclaims Diso’s pyramid.” MECHANISM Moderate Na+ channel blockade.  AP duration,  effective refractory period (ERP) in ventricular action potential,  QT interval, some potassium channel blocking effects. ClINICAl uSE Both atrial and ventricular arrhythmias, especially re-entrant and ectopic SVT and VT. AdvERSE EFFECTS Cinchonism (headache, tinnitus with quinidine), reversible SLE-like syndrome (procainamide), HF (disopyramide), thrombocytopenia, torsades de pointes due to  QT interval. Class IB Lidocaine, MexileTine. “I’d Buy Liddy’s Mexican Tacos.” 0 mV

1	Class IB Lidocaine, MexileTine. “I’d Buy Liddy’s Mexican Tacos.” 0 mV MECHANISM Weak Na+ channel blockade.  AP duration. Preferentially affect ischemic or depolarized Purkinje and ventricular tissue. Phenytoin can also fall into the IB category. ClINICAl uSE Acute ventricular arrhythmias (especially post-MI), digitalis-induced arrhythmias. IB is Best post-MI. AdvERSE EFFECTS CNS stimulation/depression, cardiovascular depression. Class IC Flecainide, Propafenone. “Can I have Fries, Please.” 0 mV MECHANISM Strong Na+ channel blockade. Significantly prolongs ERP in AV node and accessory bypass tracts. No effect on ERP in Purkinje and ventricular tissue. Minimal effect on AP duration. ClINICAl uSE SVTs, including atrial fibrillation. Only as a last resort in refractory VT. AdvERSE EFFECTS Proarrhythmic, especially post-MI (contraindicated). IC is Contraindicated in structural and ischemic heart disease.

1	AdvERSE EFFECTS Proarrhythmic, especially post-MI (contraindicated). IC is Contraindicated in structural and ischemic heart disease. Antiarrhythmics— Metoprolol, propranolol, esmolol, atenolol, timolol, carvedilol. MECHANISM Decrease SA and AV nodal activity by  cAMP,  Ca2+ currents. Suppress abnormal pacemakers by  slope of phase 4. AV node particularly sensitive— PR interval. Esmolol very short acting. ClINICAl uSE SVT, ventricular rate control for atrial fibrillation and atrial flutter.

1	ClINICAl uSE SVT, ventricular rate control for atrial fibrillation and atrial flutter. AdvERSE EFFECTS Impotence, exacerbation of COPD and asthma, cardiovascular effects (bradycardia, AV block, HF), CNS effects (sedation, sleep alterations). May mask the signs of hypoglycemia. Metoprolol can cause dyslipidemia. Propranolol can exacerbate vasospasm in vasospastic angina. β-blockers (except the nonselective αand β-antagonists carvedilol and labetalol) cause unopposed α1-agonism if given alone for pheochromocytoma or for cocaine toxicity (unsubstantiated). Treat β-blocker overdose with saline, atropine, glucagon. Antiarrhythmics— Amiodarone, Ibutilide, Dofetilide, Sotalol. AIDS. potassium channel blockers (class III) MECHANISM  AP duration,  ERP,  QT interval. ClINICAl uSE Atrial fibrillation, atrial flutter; ventricular tachycardia (amiodarone, sotalol). Antiarrhythmics— Diltiazem, Verapamil calcium channel blockers (class IV)

1	ClINICAl uSE Atrial fibrillation, atrial flutter; ventricular tachycardia (amiodarone, sotalol). Antiarrhythmics— Diltiazem, Verapamil calcium channel blockers (class IV) MECHANISM Decrease conduction Velocity,  ERP, • PR interval. ClINICAl uSE Prevention of nodal arrhythmias (eg, SVT), rate control in atrial fibrillation. AdvERSE EFFECTS Constipation, flushing, edema, cardiovascular effects (HF, AV block, sinus node depression). Adenosine  K+ out of cells  hyperpolarizing the cell and  ICa, decreasing AV node conduction. Drug of choice in diagnosing/terminating certain forms of SVT. Very short acting (~ 15 sec). Effects blunted by theophylline and caffeine (both are adenosine receptor antagonists). Adverse effects include flushing, hypotension, chest pain, sense of impending doom, bronchospasm. Magnesium Effective in torsades de pointes and digoxin toxicity.

1	Magnesium Effective in torsades de pointes and digoxin toxicity. MECHANISM IVabradine prolongs slow depolarization (phase “IV”) by selectively inhibiting “funny” sodium channels (If). ClINICAl uSE Chronic stable angina in patients who cannot take β-blockers. Chronic HFrEF. AdvERSE EFFECTS Luminous phenomena/visual brightness, hypertension, bradycardia. “If you skew the endocrine system, you lose the pathways to self.” “We have learned that there is an endocrinology of elation and despair, a chemistry of mystical insight, and, in relation to the autonomic nervous system, a meteorology and even . . . an astro-physics of changing moods.” “Chocolate causes certain endocrine glands to secrete hormones that affect your feelings and behavior by making you happy.” —Elaine Sherman, Book of Divine Indulgences

1	The endocrine system comprises widely distributed organs that work in a highly integrated manner to orchestrate a state of hormonal equilibrium within the body. Generally speaking, endocrine diseases can be classified either as diseases of underproduction or overproduction, or as conditions involving the development of mass lesions—which themselves may be associated with underproduction or overproduction of hormones. Therefore, study the endocrine system first by learning the glands, their hormones, and their regulation, and then by integrating disease manifestations with diagnosis and management. Take time to learn the multisystem connections. Thyroid diverticulum arises from floor of primitive pharynx and descends into neck. Connected to tongue by thyroglossal duct, which normally disappears but may persist as cysts or the pyramidal lobe of thyroid. Foramen cecum is normal remnant of thyroglossal duct.

1	Most common ectopic thyroid tissue site is the tongue (lingual thyroid). Removal may result in hypothyroidism if it is the only thyroid tissue present. Thyroglossal duct cyst A presents as an anterior midline neck mass that moves with swallowing or protrusion of the tongue (vs persistent cervical sinus leading to pharyngeal cleft cyst in lateral neck). Thyroid follicular cells derived from endoderm. Superior surface of kidney Secretes FSH, LH, ACTH, TSH, prolactin, Proopiomelanocortin derivatives—β-endorphin, GH, and β-endorphin. Melanotropin (MSH) ACTH, and MSH. Go pro with a BAM! secreted from intermediate lobe of pituitary. FLAT PiG: FSH, LH, ACTH, TSH, PRL, GH. Derived from oral ectoderm (Rathke pouch). B-FLAT: Basophils—FSH, LH, ACTH, TSH. α subunit—hormone subunit common to Acid PiG: Acidophils — PRL, GH. TSH, LH, FSH, and hCG.

1	α subunit—hormone subunit common to Acid PiG: Acidophils — PRL, GH. TSH, LH, FSH, and hCG. β subunit—determines hormone specificity. Stores and releases vasopressin (antidiuretic hormone, or ADH) and oxytocin, both made in the hypothalamus (supraoptic and paraventricular nuclei) and transported to posterior pituitary via neurophysins (carrier proteins). Derived from neuroectoderm. Adrenal cortex (derived from mesoderm) and medulla (derived from neural crest). CORTEX Zona Fasciculata ACTH, CRH Glucocorticoids Cortisol Capsule ACTH, CRHDHEA Zona Reticularis Preganglionic Catecholamines Epi, NEChroma˜n cells sympathetic fbers MEDULLA GFR corresponds with Salt (mineralocorticoids), Sugar (glucocorticoids), and Sex (androgens). “The deeper you go, the sweeter it gets.”

1	GFR corresponds with Salt (mineralocorticoids), Sugar (glucocorticoids), and Sex (androgens). “The deeper you go, the sweeter it gets.” Sleep, hypoglycemia, stress Growth hormone IGF-1 Anterior pituitary Posterior pituitary Somatostatin Amino acid uptake Protein synthesis Amino acid uptake Protein synthesis Glucose uptake Lipolysis DNA and RNA synthesis Chondroitin sulfate Collagen Cell size and number Aging, obesity, glucose GHRH Antidiuretic hormone Also called vasopressin. soUrce Synthesized in hypothalamus (supraoptic and paraventricular nuclei), stored and secreted by posterior pituitary. FUnction Regulates b1ood pressure (V1-receptors) and serum osmolality (V2-receptors). Primary function is serum osmolality regulation (ADH  serum osmolality,  urine osmolality) via regulation of aquaporin channel insertion in principal cells of renal collecting duct. regUlAtion Plasma osmolality (1°); hypovolemia. Also called somatotropin. Secreted by anterior pituitary.

1	regUlAtion Plasma osmolality (1°); hypovolemia. Also called somatotropin. Secreted by anterior pituitary. Stimulates linear growth and muscle mass through IGF-1 (somatomedin C) secretion by liver.  insulin resistance (diabetogenic). Released in pulses in response to growth hormone–releasing hormone (GHRH). Secretion  during exercise, deep sleep, puberty, hypoglycemia, CKD. Secretion  by glucose, somatostatin, somatomedin (regulatory molecule secreted by liver in response to GH acting on target tissues). Excess secretion of GH (eg, pituitary adenoma) may cause acromegaly (adults) or gigantism (children). Treatment: somatostatin analogs (eg, octreotide) or surgery. ADH level is  in central diabetes insipidus (DI), normal or  in nephrogenic DI. Nephrogenic DI can be caused by mutation in V2-receptor. Desmopressin (ADH analog) is a treatment for central DI and nocturnal enuresis.

1	Nephrogenic DI can be caused by mutation in V2-receptor. Desmopressin (ADH analog) is a treatment for central DI and nocturnal enuresis. Thyroid hormones Thyroid produces triiodothyronine (T3) and thyroxine (T4), iodine-containing hormones that control the body’s metabolic rate.

1	Thyroid hormones Thyroid produces triiodothyronine (T3) and thyroxine (T4), iodine-containing hormones that control the body’s metabolic rate. soUrce Follicles of thyroid. 5′-deiodinase converts T4 (the major thyroid product) to T3 in peripheral tissue (5, 4, 3). Peripheral conversion is inhibited by glucocorticoids, β-blockers, and propylthiouracil (PTU). Reverse T3 (rT3) is a metabolically inactive byproduct of the peripheral conversion of T4 and its production is increased by growth hormone and glucocorticoids. Functions of thyroid peroxidase include oxidation, organification of iodine, and coupling of monoiodotyrosine (MIT) and diiodotyrosine (DIT). Inhibited by PTU and methimazole. DIT + DIT = T4. DIT + MIT = T3. Wolff-Chaikoff effect—excess iodine temporarily turns off thyroid peroxidase  T3/T4 production (protective autoregulatory effect). FUnction Only free hormone is active. T3 binds nuclear receptor with greater affinity than T4. T3 functions —7B’s:

1	FUnction Only free hormone is active. T3 binds nuclear receptor with greater affinity than T4. T3 functions —7B’s: Bone growth (synergism with GH) β-adrenergic effects. • β1 receptors in heart  CO, HR, SV, contractility; β-blockers alleviate adrenergic symptoms in thyrotoxicosis Basal metabolic rate  (via Na+/K+-ATPase activity  O2 consumption, RR, body temperature) Blood sugar (• glycogenolysis, gluconeogenesis) regUlAtion TRH ⊕ TSH release • ⊕ follicular cells. Thyroid-stimulating immunoglobulin (TSI) may ⊕ follicular cells in Graves disease. Negative feedback primarily by free T3/T4: Anterior pituitary  sensitivity to TRH Thyroxine-binding globulin (TBG) binds most T3/T4 in blood. Bound T3/T4 = inactive. •  TBG in pregnancy, OCP use (estrogen  TBG)  total T3/T4 •  TBG in steroid use, nephrotic syndrome TSI T3, T4

1	TSI T3, T4 Downstream thyroid function T3 T4 T4 > T3 (to circulation) I– I– I2 Na+ MIT, DIT Thyroglobulin Oxidation + Proteases5'-deiodinase Tyrosine DITMIT DITDIT Thyroid peroxidase Thyroid peroxidase TG TG TG TG TG Endocytosis Organifcation Coupling reaction Deiodinase PTU MIT MIT DIT DIT T3 T3 T4 T3 T4 MIT PTU, methimazole soUrce Chief cells of parathyroid FUnction  free Ca2+ in the blood (1° function) 1,25-(OH)2D3 (calcitriol) production by activating 1α-hydroxylase in PCT Tri to make D3 in the PCT PTH  serum Ca2+,  serum PO43–,  urine PO43– ,  urine cAMP

1	PTH  serum Ca2+,  serum PO43–,  urine PO43– ,  urine cAMP RANK-L (receptor activator of NF-κB ligand) secreted by osteoblasts and osteocytes; binds RANK (receptor) on osteoclasts and their precursors to stimulate osteoclasts and • Ca2+ like PTH and is commonly increased in malignancies (eg, squamous cell carcinoma of the lung, renal cell carcinoma) serum Mg2+  PTH secretion  serum Mg2+  PTH secretion Common causes of  Mg2+ include diarrhea, aminoglycosides, diuretics, alcohol abuse ˛ ionized Ca2+, ˜ PO 3– , or 1,25-(OH)2 D 1,25-(OH)2 D3 Four para-thyroid glandsFeedback inhibition of PTH synthesis Vitamin D activity ˜ Ca2+ and PO4 3–˜ PTH released into circulation 25-OH D3 Bone Intestines 1,25-(OH)2 D3 1˜-hydroxylase ˜ Ca2+ and ˜ PO4 3– ˜ Ca2+ and ˜ PO4 3– released from bone ˜ absorption of Ca2+ and PO4 3– Renal tubular cells ˜ 1,25-(OH)2 D synthesis3 Urine Ca2+ , ˜ PO4 3– Reabsorption: ˜ Ca2+ , PO4 3–˜ ˜ ˜ Calcium homeostasis Plasma Ca2+ exists in three forms:

1	Calcium homeostasis Plasma Ca2+ exists in three forms: Ionized/free (~ 45%, active form) Bound to albumin (∼ 40%) Bound to anions (∼ 15%) pH (less H+)  albumin binds more Ca2+ • ionized Ca2+ (eg, cramps, pain, paresthesias, carpopedal spasm) • PTH Ionized/free Ca2+ is 1° regulator of PTH; changes in pH alter PTH secretion, whereas changes in albumin concentration do not soUrce Parafollicular cells (C cells) of thyroid. Calcitonin opposes actions of PTH. Not important in normal Ca2+ homeostasis FUnction  bone resorption of Ca2+. Calcitonin tones down serum Ca2+ levels and regUlAtion  serum Ca2+ • calcitonin secretion. soUrce Made by α cells of pancreas. FUnction Promotes glycogenolysis, gluconeogenesis, lipolysis, ketogenesis. Elevates blood sugar levels to maintain homeostasis when bloodstream glucose levels fall too low (ie, fasting state). regUlAtion Secreted in response to hypoglycemia. Inhibited by insulin, hyperglycemia, somatostatin.

1	regUlAtion Secreted in response to hypoglycemia. Inhibited by insulin, hyperglycemia, somatostatin. synthesis Preproinsulin (synthesized in RER of pancreatic β cells)  cleavage of “presignal” • proinsulin (stored in secretory granules)  cleavage of proinsulin  exocytosis of insulin and C-peptide equally. Insulin and C-peptide are  in insulinoma and sulfonylurea use, whereas exogenous insulin lacks C-peptide. regUlAtion Glucose is the major regulator of insulin release.  insulin response with oral vs IV glucose due to incretins (eg, glucagon-like peptide 1 [GLP-1], glucose-dependent insulinotropic polypeptide [GIP]), which are released after meals and • β cell sensitivity to glucose. Release  by α2,  by β2 stimulation (2 = regulates insulin) closes K+ channels (target of sulfonylureas) . Voltage-gated Ca2+ channels open and stimulation of insulin exocytosis

1	Tyrosine phosphorylationPhosphoinositide-3 kinase pathway RAS/MAPkinasepathwayVesicles Cell growth, containing GLUT4 DNAGlycogen, lipid, protein synthesis synthesis ), inducing glucose uptake (carriermediated transport) into insulin-dependent tissue and gene transcription. Anabolic effects of insulin: •  glucose transport in skeletal muscle and adipose tissue •  glycogen synthesis and storage •  triglyceride synthesis •  Na+ retention (kidneys) •  protein synthesis (muscles) •  cellular uptake of K+ and amino acids •  glucagon release •  lipolysis in adipose tissue Unlike glucose, insulin does not cross placenta. Insulin-dependent glucose transporters: • GLUT4: adipose tissue, striated muscle (exercise can also • GLUT4 expression) Insulin-independent transporters: GLUT1: RBCs, brain, cornea, placenta GLUT2 (bidirectional): β islet cells, liver, kidney, GI tract (think 2-way street) GLUT3: brain, placenta GLUT5 (Fructose): spermatocytes, GI tract

1	GLUT1: RBCs, brain, cornea, placenta GLUT2 (bidirectional): β islet cells, liver, kidney, GI tract (think 2-way street) GLUT3: brain, placenta GLUT5 (Fructose): spermatocytes, GI tract SGLT1/SGLT2 (Na+-glucose cotransporters): kidney, small intestine Brain prefers glucose, but may use ketone bodies during starvation. RBCs utilize glucose, as they lack mitochondria for aerobic metabolism. BRICK LIPS (insulin-independent glucose uptake): Brain, RBCs, Intestine, Cornea, Kidney, Liver, Islet (β) cells, Placenta, Spermatocytes.

1	BRICK LIPS (insulin-independent glucose uptake): Brain, RBCs, Intestine, Cornea, Kidney, Liver, Islet (β) cells, Placenta, Spermatocytes. Cholesterol desmolase Aldosterone synthase 21-hydroxylation 11˜-hydroxylation ZONA GLOMERULOSA Mineralocorticoids ZONA FASCICULATA Glucocorticoids Adrenal cortex Peripheral tissue ZONA RETICULARIS Androgens Estrogens, DHT Cholesterol (via StARa) Pregnenolone Progesterone 11-deoxycorticosterone Corticosterone Aldosterone 17-hydroxyprogesterone 17-hydroxypregnenolone 11-deoxycortisol Cortisone Glycyrrhetinic acid Cortisol Dehydroepiandrosterone (DHEA) Anastrozole, letrozole, exemestane Finasteride Androstenedione Testosterone Dihydrotestosterone (DHT) Angiotensin II 3˜-hydroxysteroid dehydrogenase Estrone Estradiol Aromatase Aromatase 17°-hydroxylase 17,20-lyase 17,20-lyase17°-hydroxylase 5°-reductase AMetyrapone aRate-limiting step.

1	17α-hydroxylasea  androstenedione XY: ambiguous genitalia, undescended testes XX: lacks 2° sexual development 21-hydroxylasea  renin activity  17-hydroxy-progesterone Most common Presents in infancy (salt wasting) or childhood (precocious puberty) XX: virilization 11β-hydroxylasea  aldosterone  11-deoxycorti-costerone (results in BP)  renin activity Presents in infancy (severe hypertension) or childhood (precocious puberty) XX: virilization aAll congenital adrenal enzyme deficiencies are autosomal recessive disorders and most are characterized by skin hyperpigmentation (due to  MSH production, which is coproduced and secreted with ACTH) and bilateral adrenal gland enlargement (due to • ACTH stimulation). If deficient enzyme starts with 1, it causes hypertension; if deficient enzyme ends with 1, it causes virilization in females.

1	If deficient enzyme starts with 1, it causes hypertension; if deficient enzyme ends with 1, it causes virilization in females. Ghrelin Stimulates hunger (orexigenic effect) and GH release (via GH secretagog receptor). Produced by stomach. Sleep deprivation, fasting, or Prader-Willi syndrome  ghrelin production. Ghrelin makes you hunghre and ghrow. Acts on lateral area of hypothalamus (hunger center) to • appetite. Leptin Satiety hormone. Produced by adipose tissue. Mutation of leptin gene  central obesity. (Obese people have  leptin due to  adipose tissue but also appear resistant to leptin’s anorexigenic effect.) Sleep deprivation or starvation • leptin production. Leptin keeps you thin. Acts on ventromedial area of hypothalamus (satiety center) to  appetite. Endocannabinoids Act at cannabinoid receptors in hypothalamus and nucleus accumbens, two key brain areas for the homeostatic and hedonic control of food intake • appetite. Exogenous cannabinoids cause “the munchies.”

1	Signaling pathways of endocrine hormones Signaling pathways of steroid hormones Binding to receptor Steroid hormones are lipophilic and therefore must circulate bound to specific binding globulins, which  their solubility. In men,  sex hormone–binding globulin (SHBG) lowers free testosterone gynecomastia. In women,  SHBG raises free testosterone hirsutism. estrogen (eg, OCPs, pregnancy)  SHBG. Syndrome of inappropriate antidiuretic hormone secretion Characterized by: Euvolemic hyponatremia with continued urinary Na+ excretion Urine osmolality > serum osmolality Body responds to water retention with aldosterone and  ANP and BNP  urinary Na+ secretion • normalization of extracellular fluid volume  euvolemic hyponatremia. Very low serum Na+ levels can lead to cerebral edema, seizures. Correct slowly to prevent osmotic demyelination syndrome (formerly called central pontine myelinolysis). SIADH causes include: Ectopic ADH (eg, small cell lung cancer)

1	SIADH causes include: Ectopic ADH (eg, small cell lung cancer) Drugs (eg, SSRIs, carbamazepine, cyclophosphamide) Treatment: fluid restriction (first line), salt tablets, IV hypertonic saline, diuretics, ADH antagonists (eg, conivaptan, tolvaptan, demeclocycline). aNo water intake for 2–3 hr followed by hourly measurements of urine volume and osmolality as well as plasma Na+ concentration and osmolality. ADH analog (desmopressin) is administered if serum osmolality > 295–300 mOsm/kg, plasma Na+ ≥ 145 mEq/L, or urine osmolality does not rise despite a rising plasma osmolality. Hypopituitarism Undersecretion of pituitary hormones due to: Nonsecreting pituitary adenoma, craniopharyngioma Sheehan syndrome—ischemic infarct of pituitary following postpartum bleeding; pregnancy-induced pituitary growth  susceptibility to hypoperfusion. Usually presents with failure to lactate, absent menstruation, cold intolerance

1	Empty sella syndrome—atrophy or compression of pituitary (which lies in the sella turcica), often idiopathic, common in obese women; associated with idiopathic intracranial hypertension Pituitary apoplexy—sudden hemorrhage of pituitary gland, often in the presence of an existing pituitary adenoma. Usually presents with sudden onset severe headache, visual impairment (eg, bitemporal hemianopia, diplopia due to CN III palsy), and features of hypopituitarism Treatment: hormone replacement therapy (corticosteroids, thyroxine, sex steroids, human growth hormone) Acromegaly Excess GH in adults. Typically caused by pituitary adenoma. Findings Large tongue with deep furrows, deep voice,  GH in children  gigantism ( linear bone large hands and feet, coarsening of facial growth). HF most common cause of death. features with aging A , frontal bossing, diaphoresis (excessive sweating), impaired glucose tolerance (insulin resistance), hypertension.  risk of colorectal polyps and cancer.

1	A , frontal bossing, diaphoresis (excessive sweating), impaired glucose tolerance (insulin resistance), hypertension.  risk of colorectal polyps and cancer. diAgnosis  serum IGF-1; failure to suppress serum GH following oral glucose tolerance test; pituitary mass seen on brain MRI. treAtment Pituitary adenoma resection. If not cured, treat with octreotide (somatostatin analog), pegvisomant (GH receptor antagonist), or dopamine agonists (eg, cabergoline).

1	treAtment Pituitary adenoma resection. If not cured, treat with octreotide (somatostatin analog), pegvisomant (GH receptor antagonist), or dopamine agonists (eg, cabergoline). metAbolic Cold intolerance,  sweating, weight gain Heat intolerance,  sweating, weight loss ( basal metabolic rate • calorigenesis), ( synthesis of Na+-K+ ATPase  basal hyponatremia ( free water clearance) metabolic rate • calorigenesis) sKin/hAir Dry, cool skin (due to  blood flow); coarse, Warm, moist skin (due to vasodilation); fine hair; brittle hair; diffuse alopecia; brittle nails; onycholysis ( A ); pretibial myxedema in Graves puffy facies and generalized nonpitting edema disease (myxedema) due to  GAGs in interstitial spaces  osmotic pressure • water retention Other causes Iodine deficiency (with goiter E ), goitrogens (eg, amiodarone, lithium), Wolff-Chaikoff effect (thyroid gland downregulation in response to • iodide).

1	Other causes Iodine deficiency (with goiter E ), goitrogens (eg, amiodarone, lithium), Wolff-Chaikoff effect (thyroid gland downregulation in response to • iodide). Graves disease Most common cause of hyperthyroidism. Thyroid-stimulating immunoglobulin (IgG, can cause transient neonatal hyperthyroidism; type II hypersensitivity) stimulates TSH receptors on thyroid (hyperthyroidism, diffuse goiter), dermal fibroblasts (pretibial myxedema), and orbital fibroblasts (Graves orbitopathy). Activation of T-cells  lymphocytic infiltration of retroorbital space • cytokines (eg, TNF-α, IFN-γ)  fibroblast secretion of hydrophilic GAGs  osmotic muscle swelling, muscle inflammation, and adipocyte count  exophthalmos A . Often presents during stress (eg, pregnancy). Associated with HLA-DR3 and HLA-B8. Histology: tall, crowded follicular epithelial cells; scalloped colloid.

1	Histology: tall, crowded follicular epithelial cells; scalloped colloid. Toxic multinodular Focal patches of hyperfunctioning follicular cells distended with colloid working independently goiter of TSH (due to TSH receptor mutations in 60% of cases).  release of T3 and T4. Hot nodules are rarely malignant. Thyroid storm Uncommon but serious complication that occurs when hyperthyroidism is incompletely treated/ untreated and then significantly worsens in the setting of acute stress such as infection, trauma, surgery. Presents with agitation, delirium, fever, diarrhea, coma, and tachyarrhythmia (cause of death). May see  LFTs. Treat with the 4 P’s: β-blockers (eg, Propranolol), Propylthiouracil, corticosteroids (eg, Prednisolone), Potassium iodide (Lugol iodine). Iodide load  T4 synthesis  Wolff-Chaikoff effect.

1	Jod-Basedow Iodine-induced hyperthyroidism. Occurs when a patient with iodine deficiency and partially phenomenon autonomous thyroid tissue (eg, autonomous nodule) is made iodine replete. Can happen after iodine IV contrast or amiodarone use. Opposite to Wolff-Chaikoff effect. Causes of goiter Smooth/diffuse: Graves disease, Hashimoto thyroiditis, iodine deficiency, TSH-secreting pituitary adenoma. Nodular: toxic multinodular goiter, thyroid adenoma, thyroid cancer, thyroid cyst. Thyroid adenoma Benign solitary growth of the thyroid. Most are nonfunctional (“cold”), can rarely cause hyperthyroidism via autonomous thyroid hormone production (“hot” or “toxic”). Most common histology is follicular (arrows in A ); absence of capsular or vascular invasion (unlike follicular carcinoma).

1	Thyroid cancer Typically diagnosed with fine needle aspiration; treated with thyroidectomy. Complications of surgery include hypocalcemia (due to removal of parathyroid glands), transection of recurrent laryngeal nerve during ligation of inferior thyroid artery (leads to dysphagia and dysphonia [hoarseness]), and injury to the external branch of the superior laryngeal nerve during ligation of superior thyroid vascular pedicle (may lead to loss of tenor usually noticeable in professional voice users). Papillary carcinoma Most common, excellent prognosis. Empty-appearing nuclei with central clearing (“Orphan Annie” eyes) A , psamMoma bodies, nuclear grooves (Papi and Moma adopted OrphanAnnie).  risk with RET/PTC rearrangements and BRAF mutations, childhood irradiation. Papillary carcinoma: most Prevalent, Palpable lymph nodes. Good prognosis.

1	 risk with RET/PTC rearrangements and BRAF mutations, childhood irradiation. Papillary carcinoma: most Prevalent, Palpable lymph nodes. Good prognosis. Follicular carcinoma Good prognosis. Invades thyroid capsule and vasculature (unlike follicular adenoma), uniform follicles; hematogenous spread is common. Associated with RAS mutation and PAX8-PPAR-γ translocations. Medullary carcinoma From parafollicular “C cells”; produces calcitonin, sheets of polygonal cells in an amyloid stroma B (stains with Congo red). Associated with MEN 2A and 2B (RET mutations). Undifferentiated/ Older patients; presents with rapidly enlarging neck mass  compressive symptoms (eg, dyspnea, anaplastic carcinoma dysphagia, hoarseness); very poor prognosis. Associated with TP53 mutation. (vitamin D defciency, ˜ Ca2+ intake, (hyperplasia, adenoma, chronic kidney disease) carcinoma) 1° hypoparathyroidism PTH-independent (surgical resection, hypercalcemia autoimmune) (excess Ca2+ intake, cancer, ° vitamin D)

1	Due to injury to parathyroid glands or their blood supply (usually during surgery), autoimmune destruction, or DiGeorge syndrome. Findings: tetany, hypocalcemia, hyperphosphatemia. Chvostek sign—tapping of facial nerve (tap the Cheek)  contraction of facial muscles. Trousseau sign—occlusion of brachial artery with BP cuff (cuff the Triceps)  carpal spasm. Pseudohypoparathyroidism type 1A—autosomal dominant, maternally transmitted mutations (imprinted GNAS gene). GNAS1-inactivating mutation (coupled to PTH receptor) that encodes the Gs protein α subunit  inactivation of adenylate cyclase when PTH binds to its receptor  end-organ resistance (kidney and bone) to PTH. Physical findings: Albright hereditary osteodystrophy (shortened 4th/5th digits A , short stature, round face, subcutaneous calcifications, developmental delay). Labs:  PTH, • Ca2+,  PO43–.

1	Pseudopseudohypoparathyroidism—autosomal dominant, paternally transmitted mutations (imprinted GNAS gene) but without end-organ resistance to PTH due to normal maternal allele maintaining renal responsiveness to PTH. Physical findings: same as Albright hereditary osteodystrophy. Labs: normal PTH, Ca2+, PO43–. Usually due to parathyroid adenoma or hyperplasia. Hypercalcemia, hypercalciuria (renal stones), polyuria (thrones), hypophosphatemia,  PTH,  ALP,  urinary cAMP. Most often asymptomatic. May present with bone pain, weakness, constipation (“groans”), abdominal/flank pain (kidney stones, acute pancreatitis), neuropsychiatric disturbances (“psychiatric overtones”). Osteitis fibrosa cystica—cystic bone spaces filled with brown fibrous tissue A (“brown tumor” consisting of osteoclasts and deposited hemosiderin from hemorrhages; causes bone pain). Due to  PTH, classically associated with 1° (but also seen with 2°) hyperparathyroidism.

1	“Stones, thrones, bones, groans, and psychiatric overtones.” AcUte mAniFestAtions Polydipsia, polyuria, polyphagia, weight loss, DKA (type 1), hyperosmolar hyperglycemic state (type 2). Rarely, can be caused by unopposed secretion of GH and epinephrine. Also seen in patients on glucocorticoid therapy (steroid diabetes). chronic comPlicAtions Nonenzymatic glycation: Small vessel disease (diffuse thickening of basement membrane)  retinopathy (hemorrhage, exudates, microaneurysms, vessel proliferation), glaucoma, nephropathy. Nodular glomerulosclerosis • progressive proteinuria (initially microalbuminuria; ACE inhibitors and ARBs are renoprotective) and arteriolosclerosis (causing hypertension)  chronic kidney disease. Large vessel atherosclerosis, CAD, peripheral vascular occlusive disease, gangrene  limb loss, cerebrovascular disease. MI most common cause of death. Osmotic damage (sorbitol accumulation in organs with aldose reductase and  or absent sorbitol dehydrogenase):

1	Osmotic damage (sorbitol accumulation in organs with aldose reductase and  or absent sorbitol dehydrogenase): Neuropathy (motor, sensory [glove and stocking distribution], and autonomic degeneration). Cataracts. ˜ muscle mass, weight loss ˛ plasma osmolality Osmotic diuresis Loss of water, Na+, and K+˛ thirst Hypovolemia Circulation failure, ˜ tissue perfusion Coma/death tissue glucose uptake ˛˛ glycogenolysis ˛ gluconeogenesis ˛ proteolysis Hyperventilation, Kussmaul respiration ˛ serum lactate ˛ lipolysis Hyperglycemia, glycosuria ˛ plasma free fatty acids ˛ ketogenesis, ketonemia, ketonuria Anion gap metabolic acidosis Vomiting Diabetic ketoacidosis Insulin absent, ketones present (• complications). Insulin noncompliance or  requirements from  stress (eg, infection)  excess fat breakdown and • ketogenesis from  free fatty acids • ketone bodies (β-hydroxybutyrate > acetoacetate). comPlicAtions Life-threatening mucormycosis, cerebral edema, cardiac arrhythmias, HF.

1	comPlicAtions Life-threatening mucormycosis, cerebral edema, cardiac arrhythmias, HF. treAtment IV fluids, IV insulin, K+ (to replete intracellular stores) +/– glucose to prevent hypoglycemia. comPlicAtions Can progress to coma and death if untreated. treAtment IV fluids, IV insulin, and K+ (to replete intracellular stores). etiology  cortisol due to a variety of causes: Exogenous corticosteroids  ACTH  bilateral adrenal atrophy. Most common cause. Primary adrenal adenoma, hyperplasia, or carcinoma  ACTH  atrophy of uninvolved adrenal gland. ACTH-secreting pituitary adenoma (Cushing disease); paraneoplastic ACTH secretion (eg, small cell lung cancer, bronchial carcinoids) bilateral adrenal hyperplasia. Cushing disease is responsible for the majority of endogenous cases of Cushing syndrome.

1	Findings CUSHING Syndrome: • Cholesterol, • Urinary free cortisol, Skin changes (thinning, striae A ), Hypertension, Immunosuppression, Neoplasm (a cause, not a finding), Growth retardation (in children), • Sugar (hyperglycemia, insulin resistance). Also, amenorrhea, moon facies B , buffalo hump, osteoporosis,  weight (truncal obesity), hirsutism. diAgnosis Screening tests include:  free cortisol on 24-hr urinalysis,  late night salivary cortisol, and no suppression with overnight low-dose dexamethasone test. ˜ 24-hr urine free cortisol, ˜ late night salivary cortisol, and/or inadequate suppression on 1 mg overnight dexamethasone test (consider adrenal CT to confrm) CT of the chest/abdomen/pelvis MRI of the pituitary CT of the chest/abdomen/pelvis

1	CT of the chest/abdomen/pelvis MRI of the pituitary CT of the chest/abdomen/pelvis Nelson syndrome Enlargement of pre-existing ACTH–secreting pituitary adenoma after bilateral adrenalectomy for refractory Cushing disease  ACTH (hyperpigmentation), mass effect (headaches, bitemporal hemianopia). Treatment: transsphenoidal resection, postoperative pituitary irradiation for residual tumor. Adrenal insufficiency Inability of adrenal glands to generate enough glucocorticoids +/− mineralocorticoids for the body’s needs. Symptoms include weakness, fatigue, orthostatic hypotension, muscle aches, weight loss, GI disturbances, sugar and/or salt cravings. Treatment: glucocorticoid/mineralocorticoid replacement.

1	Metyrapone stimulation test ( conversion of 11-deoxycortisol to cortisol) Indeterminate cortisol results ↓AM or random cortisol, or ACTH stimulation test with ↓ peak cortisol Measure random serum ACTH Check AM or random cortisol, or ACTH stimulation test ↓/−/↑ACTH ↓↓11-deoxycortisol ↑↑ACTH ↑↑11-deoxycortisol ↑↑ACTH ↓11-deoxycortisol ↓ ACTH ↑ ACTH 2°/3° adrenal Normal response 1° adrenal 2°/3° adrenal 1° adrenal insu˜ciency to ↓ cortisol insu˜ciency insu˜ciency insu˜ciency Most common tumor of the adrenal medulla in children, usually < 4 years old. Originates from Neural crest cells. Occurs anywhere along the sympathetic chain. Most common presentation is abdominal distension and a firm, irregular mass that can cross the midline (vs Wilms tumor, which is smooth and unilateral). Less likely to develop hypertension than with pheochromocytoma (Neuroblastoma is Normotensive). Can also present with opsoclonus-myoclonus syndrome (“dancing eyes-dancing feet”).

1	• HVA and VMA (catecholamine metabolites) in urine. Homer-Wright rosettes (neuroblasts surrounding a central lumen A ) characteristic of neuroblastoma and medulloblastoma. Bombesin and NSE ⊕. Associated with amplification of N-myc oncogene. MEN 1 Pituitary tumors (prolactin or GH) Pancreatic endocrine tumors—Zollinger-Ellison syndrome, insulinomas, VIPomas, glucagonomas (rare) Parathyroid adenomas Associated with mutation of MEN1 (menin, a tumor suppressor, chromosome 11), angiofibromas, collagenomas, meningiomas MEN 2A Parathyroid hyperplasia Medullary thyroid carcinoma—neoplasm of parafollicular C cells; secretes calcitonin; prophylactic thyroidectomy required Pheochromocytoma (secretes catecholamines) Associated with mutation in RET (codes for receptor tyrosine kinase) Associated with marfanoid habitus; mutation in RET gene MEN 1 = 3 P’s: Pituitary, Parathyroid, and Pancreas MEN 2A = 2 P’s: Parathyroid and Pheochromocytoma MEN 2B = 1 P: Pheochromocytoma

1	Associated with marfanoid habitus; mutation in RET gene MEN 1 = 3 P’s: Pituitary, Parathyroid, and Pancreas MEN 2A = 2 P’s: Parathyroid and Pheochromocytoma MEN 2B = 1 P: Pheochromocytoma Insulinoma Tumor of pancreatic β cells  overproduction of insulin • hypoglycemia. May see Whipple triad: low blood glucose, symptoms of hypoglycemia (eg, lethargy, syncope, diplopia), and resolution of symptoms after normalization of plasma glucose levels. Symptomatic patients have  blood glucose and • C-peptide levels (vs exogenous insulin use). ∼ 10% of cases associated with MEN 1 syndrome. Treatment: surgical resection. Glucagonoma Tumor of pancreatic α cells  overproduction of glucagon. Presents with 6 D’s: Dermatitis (necrolytic migratory erythema), Diabetes (hyperglycemia), DVT, Declining weight, Depression, Diarrhea. Treatment: octreotide, surgical resection.

1	Somatostatinoma Tumor of pancreatic δ cells  overproduction of somatostatin  secretion of secretin, cholecystokinin, glucagon, insulin, gastrin, gastric inhibitory peptide (GIP). May present with diabetes/glucose intolerance, steatorrhea, gallstones, achlorhydria. Treatment: surgical resection; somatostatin analogs (eg, octreotide) for symptom control. Carcinoid tumors arise from neuroendocrine cells most commonly in the intestine or lung. Rare and does not occur if tumor is limited to the GI tract. Prominent rosettes (arrow in A ), chromogranin A ⊕ and synaptophysin ⊕). Neuroendocrine cells secrete 5-HT → recurrent diarrhea, wheezing, right-sided valvular heart disease (eg, tricuspid regurgitation, pulmonic stenosis), niacin deficiency (pellagra). 5-HT undergoes hepatic first-pass metabolism and enzymatic breakdown by MAO in the lung. Treatment: surgical resection, somatostatin analog (eg, octreotide, telotristat) for symptom control.

1	Rule of thirds: 1/3 metastasize 1/3 present with 2nd malignancy 1/3 are multiple Rapid acting (1-hr Bind insulin receptor (tyrosine kinase activity) Hypoglycemia, lipodystrophy, hypersensitivity peak): Lispro, Aspart, Liver:  glucose storage as glycogen reactions (rare), weight gain Glulisine (no LAG) Muscle:  glycogen, protein synthesis Short acting (2–3 hr Fat:  TG storage peak): regular Cell membrane:  K+ uptake Intermediate acting (4–10 hr peak): NPH Long acting (no real peak): detemir, glargine Lispro, aspart, glulisine Regular NPH Detemir Glargine Chlorpropamide, tolbutamide Glipizide, glyburide Meglitinides “-gliNs” Nateglinide, Close K+ channels in pancreatic B cell membrane  cell depolarizes  insulin release via • Ca2+ influx. DisulFIRam-like reaction (FIRst-generation only). Rarely used. Hypoglycemia ( risk in renal insufficiency), weight gain.

1	DisulFIRam-like reaction (FIRst-generation only). Rarely used. Hypoglycemia ( risk in renal insufficiency), weight gain. Amylin analogs • glucagon release,  gastric emptying. Hypoglycemia, nausea.  satiety (often desired). Pramlintide Thionamides Propylthiouracil, methimazole. mechAnism Block thyroid peroxidase, inhibiting the oxidation of iodide as well as the organification and coupling of iodine  inhibition of thyroid hormone synthesis. PTU also blocks 5′-deiodinase • Peripheral conversion of T4 to T3. clinicAl Use Hyperthyroidism. PTU used in first trimester of pregnancy (due to methimazole teratogenicity); methimazole used in second and third trimesters of pregnancy (due to risk of PTU-induced hepatotoxicity). Not used to treat Graves ophthalmopathy (treated with corticosteroids). AdVerse eFFects Skin rash, agranulocytosis (rare), aplastic anemia, hepatotoxicity. Methimazole is a possible teratogen (can cause aplasia cutis).

1	AdVerse eFFects Skin rash, agranulocytosis (rare), aplastic anemia, hepatotoxicity. Methimazole is a possible teratogen (can cause aplasia cutis). Levothyroxine, liothyronine mechAnism Hormone replacement for T4 (levothyroxine) or T3 (liothyronine). clinicAl Use Hypothyroidism, myxedema. May be abused for weight loss. Distinguish exogenous hyperthyroidism from endogenous hyperthyroidism by using a combination of TSH receptor antibodies, radioactive iodine uptake, and/or measurement of thyroid blood flow on ultrasound. AdVerse eFFects Tachycardia, heat intolerance, tremors, arrhythmias. mechAnism Synthetic analog of aldosterone with little glucocorticoid effects. clinicAl Use Mineralocorticoid replacement in 1° adrenal insufficiency. AdVerse eFFects Similar to glucocorticoids; also edema, exacerbation of heart failure, hyperpigmentation. mechAnism Sensitizes Ca2+-sensing receptor (CaSR) in parathyroid gland to circulating Ca2+ • PTH.

1	mechAnism Sensitizes Ca2+-sensing receptor (CaSR) in parathyroid gland to circulating Ca2+ • PTH. clinicAl Use 2° hyperparathyroidism in patients with CKD receiving hemodialysis, hypercalcemia in 1° hyperparathyroidism (if parathyroidectomy fails), or in parathyroid carcinoma. AdVerse eFFects Hypocalcemia. mechAnism Nonabsorbable phosphate binder that prevents phosphate absorption from the GI tract. clinicAl Use Hyperphosphatemia in CKD. AdVerse eFFects Hypophosphatemia, GI upset. “A good set of bowels is worth more to a man than any quantity of brains.” “Man should strive to have his intestines relaxed all the days of his life.” “All right, let’s not panic. I’ll make the money by selling one of my livers. I can get by with one.”

1	When studying the gastrointestinal system, be sure to understand the normal embryology, anatomy, and physiology and how it is affected in the various pathologic diseases. Study not only what a disease entails, but also its specific findings, so that you can differentiate between two similar diseases. For example, what specifically makes ulcerative colitis different than Crohn disease? Also, it is important to understand bile metabolism and which lab values increase or decrease depending on the disease process. Be comfortable with basic interpretation of abdominal x-rays, CT scans, and endoscopic images. Ventral wall defects Developmental defects due to failure of rostral fold closure (eg, sternal defects [ectopia cordis]), lateral fold closure (eg, omphalocele, gastroschisis), or caudal fold closure (eg, bladder exstrophy). Congenital umbilical Failure of umbilical ring to close after physiologic herniation of the midgut. Small defects usually hernia close spontaneously.

1	Congenital umbilical Failure of umbilical ring to close after physiologic herniation of the midgut. Small defects usually hernia close spontaneously. Tracheoesophageal Esophageal atresia (EA) with distal tracheoesophageal fistula (TEF) is the most common (85%) anomalies and often presents as polyhydramnios in utero (due to inability of fetus to swallow amniotic fluid). Neonates drool, choke, and vomit with first feeding. TEFs allow air to enter stomach (visible on CXR). Cyanosis is 2° to laryngospasm (to avoid reflux-related aspiration). Clinical test: failure to pass nasogastric tube into stomach. In H-type, the fistula resembles the letter H. In pure EA, CXR shows gasless abdomen.

1	Intestinal atresia Presents with bilious vomiting and abdominal distension within first 1–2 days of life. Duodenal atresia—failure to recanalize. Abdominal x-ray A shows “double bubble” (dilated stomach, proximal duodenum). Associated with Down syndrome. Jejunal and ileal atresia—disruption of mesenteric vessels (typically SMA)  ischemic necrosis of fetal intestine  segmental resorption: bowel becomes discontinuous. X-ray shows dilated loops of small bowel with air-fluid levels. Most common cause of gastric outlet obstruction in infants (1:600). Palpable olive-shaped mass in epigastric region, visible peristaltic waves, and nonbilious projectile vomiting at ∼ 2–6 weeks old. More common in firstborn males; associated with exposure to macrolides. Results in hypokalemic hypochloremic metabolic alkalosis (2° to vomiting of gastric acid and subsequent volume contraction). Ultrasound shows thickened and lengthened pylorus A .

1	Results in hypokalemic hypochloremic metabolic alkalosis (2° to vomiting of gastric acid and subsequent volume contraction). Ultrasound shows thickened and lengthened pylorus A . Treatment: surgical incision of pyloric muscles (pyloromyotomy). Retroperitoneal Retroperitoneal structures A are posterior to structures (and outside of) the peritoneal cavity. Injuries to retroperitoneal structures can cause blood or gas accumulation in retroperitoneal space. SAD PUCKER: Suprarenal (adrenal) glands [not shown] Aorta and IVC Duodenum (2nd through 4th parts) Pancreas (except tail) Ureters [not shown] Colon (descending and ascending) Kidneys Esophagus (thoracic portion) [not shown] Rectum (partially) [not shown] Omental foramen (epiploic foramen of Winslow) Falciform ligament Liver to anterior abdominal Ligamentum teres hepatis Derivative of ventral mesentery wall (derivative of fetal umbilical vein), patent paraumbilical veins

1	Falciform ligament Liver to anterior abdominal Ligamentum teres hepatis Derivative of ventral mesentery wall (derivative of fetal umbilical vein), patent paraumbilical veins Hepatoduodenal Liver to duodenum Portal triad: proper hepatic ligament artery, portal vein, common bile duct Derivative of ventral mesentery Pringle maneuver—ligament is compressed manually or with a vascular clamp in omental foramen to control bleeding from hepatic inflow source Borders the omental foramen, which connects the greater and lesser sacs Part of lesser omentum Esophagus Nonkeratinized stratified squamous epithelium. Upper 1/3, striated muscle; middle and lower 2/3 smooth muscle, with some overlap at the transition. Ileum Peyer patches (arrow in D ; lymphoid aggregates in lamina propria, submucosa), plicae circulares (proximal ileum), and crypts of Lieberkühn. Largest number of goblet cells in the small intestine. Colon Crypts of Lieberkühn with abundant goblet cells, but no villi E .

1	Colon Crypts of Lieberkühn with abundant goblet cells, but no villi E . Arteries supplying GI structures are single and branch anteriorly. Arteries supplying non-GI structures are paired and branch laterally and posteriorly. Two areas of the colon have dual blood supply from distal arterial branches (“watershed regions”)  susceptible in colonic ischemia: Rectosigmoid junction—the last sigmoid arterial branch from the IMA and superior rectal artery Nutcracker syndrome—compression of left renal vein between superior mesenteric artery and aorta. Characterized by abdominal (flank) pain and gross hematuria (from rupture of thin-walled renal varicosities). Superior mesenteric artery syndrome— characterized by intermittent intestinal obstruction symptoms (primarily postprandial pain) when SMA and aorta compress transverse (third) portion of duodenum. Typically occurs in conditions associated with diminished mesenteric fat (eg, low body weight/malnutrition).

1	Celiac trunk Branches of celiac trunk: common hepatic, splenic, and left gastric. These constitute the main blood supply of the foregut. Strong anastomoses exist between: Pathologic blood in portal HTN Flow through TIPS, re-establishing normal fow direction Varicesof gut, butt, and caput (medusae) are commonly seen with portal hypertension. Treatment with a transjugular intrahepatic portosystemic shunt (TIPS) between the portal vein and hepatic vein relieves portal hypertension by shunting blood to the systemic circulation, bypassing the liver. TIPS can precipitate hepatic encephalopathy due to  clearance of ammonia from shunting. Pectinate line Also called dentate line. Formed where endoderm (hindgut) meets ectoderm. Above pectinate line: internal hemorrhoids, adenocarcinoma. Internal hemorrhoids receive visceral innervation and are therefore not painful. Below pectinate line: external hemorrhoids, anal fissures, squamous cell carcinoma.

1	Internal hemorrhoids receive visceral innervation and are therefore not painful. Below pectinate line: external hemorrhoids, anal fissures, squamous cell carcinoma. External hemorrhoids receive somatic innervation (inferior rectal branch of pudendal nerve) and are therefore painful if thrombosed. Anal fissure—tear in anal mucosa below Pectinate line. Pain while Pooping; blood on toilet Paper. Located Posteriorly because this area is Poorly Perfused. Innervated by Pudendal nerve. Associated with low-fiber diets and constipation. The functional unit of the liver is made up of hexagonally arranged lobules surrounding the central vein with portal triads on the edges (consisting of a portal vein, hepatic artery, bile ducts, as well as lymphatics) A . Apical surface of hepatocytes faces bile canaliculi. Basolateral surface faces sinusoids.

1	Apical surface of hepatocytes faces bile canaliculi. Basolateral surface faces sinusoids. Kupffer cells (specialized macrophages) located in sinusoids (black arrows in B ; yellow arrows show hepatic venule) clear bacteria and damaged or senescent RBCs. Hepatic stellate (Ito) cells in space of Disse store vitamin A (when quiescent) and produce extracellular matrix (when activated). Responsible for hepatic fibrosis. Branch of hepatic artery Branch of portal vein Zone I—periportal zone: Best oxygenated, most resistant to circulatory compromise Ingested toxins (eg, cocaine) Zone II—intermediate zone: Yellow fever Zone III—pericentral vein (centrilobular) zone: High concentration of cytochrome P-450 Most sensitive to metabolic toxins (eg, ethanol, CCl4, halothane, rifampin, acetaminophen) Site of alcoholic hepatitis Stellate cell Space of Disse

1	High concentration of cytochrome P-450 Most sensitive to metabolic toxins (eg, ethanol, CCl4, halothane, rifampin, acetaminophen) Site of alcoholic hepatitis Stellate cell Space of Disse Gallstones that reach the confluence of the common bile and pancreatic ducts at the ampulla of Vater can block both the common bile and pancreatic ducts (double duct sign), causing both cholangitis and pancreatitis, respectively. Tumors that arise in head of pancreas (usually ductal adenocarcinoma) can cause obstruction of common bile duct  enlarged gallbladder with painless jaundice (Courvoisier sign). Cholangiography shows filling defects in gallbladder (blue arrow) and cystic duct (red arrow) A . Femoral sheath Fascial tube 3–4 cm below inguinal ligament. Contains femoral vein, artery, and canal (deep inguinal lymph nodes) but not femoral nerve. Aponeurosis of external oblique muscle

1	Femoral sheath Fascial tube 3–4 cm below inguinal ligament. Contains femoral vein, artery, and canal (deep inguinal lymph nodes) but not femoral nerve. Aponeurosis of external oblique muscle Evagination of transversalis fascia Internal (deep) inguinal ring Ductus (vas) deferens Genital branch of genitofemoral Hernias Protrusion of peritoneum through an opening, usually at a site of weakness. Contents may be at risk for incarceration (not reducible back into abdomen/pelvis) and strangulation (ischemia and necrosis). Complicated hernias can present with tenderness, erythema, fever.

1	Direct inguinal hernia Protrudes through inguinal (Hesselbach) triangle. Bulges directly through parietal peritoneum medial to the inferior epigastric vessels but lateral to the rectus abdominis. Goes through external (superficial) inguinal ring only. Covered by external spermatic fascia. Usually occurs in older men due to acquired weakness of transversalis fascia. MDs don’t LIe: Medial to inferior epigastric vessels = Direct hernia. Lateral to inferior epigastric vessels = Indirect hernia. Femoral hernia Protrudes below inguinal ligament through femoral canal below and lateral to pubic tubercle. More common in females, but overall inguinal hernias are the most common. More likely to present with incarceration or strangulation (vs inguinal hernia).

1	Abdominal structures enter the thorax A ; may occur due to congenital defect of pleuroperitoneal membrane or from trauma. Commonly occurs on left side due to relative protection of right hemidiaphragm by liver. Most commonly a hiatal hernia, in which stomach herniates upward through the esophageal hiatus of the diaphragm. Sliding hiatal hernia—gastroesophageal junction is displaced upward as gastric cardia slides into hiatus; “hourglass stomach.” Most gastric fundus common type. Associated with GERD. Paraesophageal hiatal hernia— gastroesophageal junction is usually normal but gastric fundus protrudes into the thorax. Goes through the internal (deep) inguinal ring, external (superficial) inguinal ring, and into the groin. Enters internal inguinal ring lateral to inferior epigastric vessels. Caused by failure of processus vaginalis to close (can form hydrocele). May be noticed in infants or discovered in adulthood. Much more common in males B .

1	B . Follows the pathway of testicular descent. Covered by all 3 layers of spermatic fascia. Gastrin G cells (antrum of stomach, duodenum) in chronic atrophic gastritis (eg, H pylori) Intrinsic factor Parietal cells Vitamin B12–binding Autoimmune destruction (stomach A ) protein (required for B12 of parietal cells  chronic uptake in terminal ileum) gastritis and pernicious anemia. Gastric acid Parietal cells  stomach pH  by histamine, (stomach) vagal stimulation (ACh), gastrin  by somatostatin, GIP, prostaglandin, secretin Pepsin Chief cells Protein digestion  Pepsinogen (inactive) is (stomach) stimulation converted to pepsin (active) in (ACh), local the presence of H+. acid by pancreatic (stomach, and biliary the gastric epithelium. duodenum, secretion with salivary glands, secretin pancreas) and Brunner glands (duodenum)

1	Gastrin  acid secretion primarily through its effects on enterochromaffin-like (ECL) cells (leading to histamine release) rather than through its direct effect on parietal cells. Pancreatic secretions Isotonic fluid; low flow  high Cl−, high flow  high HCO3 − . Only monosaccharides (glucose, galactose, fructose) are absorbed by enterocytes. Glucose and galactose are taken up by SGLT1 (Na+ dependent). Fructose is taken up via Facilitated diffusion by GLUT5. All are transported to blood by GLUT2. d-xylose absorption test: simple sugar that requires intact mucosa for absorption, but does not require digestive enzymes. Helps distinguish GI mucosal damage from other causes of malabsorption. Iron Absorbed as Fe2+ in duodenum Iron Fist, Bro Clinically relevant in patients with small bowel disease or after resection (eg, vitamin B12 Vitamin B Absorbed in terminal ileum along with bile salts, requires intrinsic factor Think of IgA, the Intra-gut Antibody

1	Vitamin B Absorbed in terminal ileum along with bile salts, requires intrinsic factor Think of IgA, the Intra-gut Antibody Unencapsulated lymphoid tissue A found in lamina propria and submucosa of ileum. Contain specialized M cells that sample and present antigens to immune cells. B cells stimulated in germinal centers of Peyer patches differentiate into IgA-secreting plasma cells, which ultimately reside in lamina propria. IgA receives protective secretory component and is then transported across the epithelium to the gut to deal with intraluminal antigen. Bilirubin Heme is metabolized by heme oxygenase to biliverdin, which is subsequently reduced to bilirubin. Unconjugated bilirubin is removed from blood by liver, conjugated with glucuronate, and excreted in bile. Direct bilirubin: conjugated with glucuronic acid; water soluble (dissolves in water). Indirect bilirubin: unconjugated; water insoluble. Excreted in urine as urobilin (˜ yellow color)

1	Excreted in urine as urobilin (˜ yellow color) Excreted in feces as stercobilin (˜ brown color of stool) Most are benign and commonly affect parotid gland (80-85%). Nearly half of all submandibular gland neoplasms and most sublingual and minor salivary gland tumors are malignant. Typically present as painless mass/swelling. Facial paralysis or pain suggests malignant involvement. Pleomorphic adenoma (benign mixed tumor)—most common salivary gland tumor A . Composed of chondromyxoid stroma and epithelium and recurs if incompletely excised or ruptured intraoperatively. May undergo malignant transformation. Mucoepidermoid carcinoma—most common malignant tumor, has mucinous and squamous components. Warthin tumor (papillary cystadenoma lymphomatosum)—benign cystic tumor with germinal centers. Typically found in smokers. Bilateral in 10%; multifocal in 10%. “Warriors from Germany love smoking.”

1	Warthin tumor (papillary cystadenoma lymphomatosum)—benign cystic tumor with germinal centers. Typically found in smokers. Bilateral in 10%; multifocal in 10%. “Warriors from Germany love smoking.” Barrett esophagus Specialized intestinal metaplasia A —replacement of nonkeratinized stratified squamous epithelium with intestinal epithelium (nonciliated columnar with goblet cells [stained blue in B ]) in distal esophagus. Due to chronic gastroesophageal reflux disease (GERD). Associated with  risk of esophageal adenocarcinoma. Most commonly gastric adenocarcinoma; lymphoma, GI stromal tumor, carcinoid (rare). Early aggressive local spread with node/liver metastases. Often presents late, with weight loss, abdominal pain, early satiety, and in some cases acanthosis nigricans or Leser-Trélat sign. Associated with blood type A.

1	Intestinal—associated with H pylori, dietary nitrosamines (smoked foods), tobacco smoking, achlorhydria, chronic gastritis. Commonly on lesser curvature; looks like ulcer with raised margins. Diffuse—not associated with H pylori; most cases due to E-cadherin mutation; signet ring cells (mucin-filled cells with peripheral nuclei) A ; stomach wall grossly thickened and leathery (linitis plastica). Erosions can be caused by: Especially common among alcoholics and NSAIDs—• PGE2  gastric mucosa patients taking daily NSAIDs (eg, patients with protection rheumatoid arthritis) Burns (Curling ulcer)—hypovolemia Burned by the Curling iron Brain injury (Cushing ulcer)—• vagal Always Cushion the brain stimulation  ACh  H+ production Mucosal inflammation, often leading to atrophy (hypochlorhydria • hypergastrinemia) and intestinal metaplasia ( risk of gastric cancers)

1	Mucosal inflammation, often leading to atrophy (hypochlorhydria • hypergastrinemia) and intestinal metaplasia ( risk of gastric cancers) Hyperplasia of gastric mucosa  hypertrophied rugae (look like brain gyri A ). Causes excess mucus production with resultant protein loss and parietal cell atrophy with  acid production. Precancerous. Presents with Weight loss, Anorexia, Vomiting, Epigastric pain, Edema (due to protein loss) (WAVEE). Virchow node—involvement of left supraclavicular node by metastasis from stomach. Krukenberg tumor—bilateral metastases to ovaries. Abundant mucin-secreting, signet ring cells. Sister Mary Joseph nodule—subcutaneous periumbilical metastasis. Blumer shelf—palpable mass on digital rectal exam suggesting metastasis to rectouterine pouch (pouch of Douglas).

1	Sister Mary Joseph nodule—subcutaneous periumbilical metastasis. Blumer shelf—palpable mass on digital rectal exam suggesting metastasis to rectouterine pouch (pouch of Douglas). Hemorrhage Gastric, duodenal (posterior > anterior). Most common complication. Ruptured gastric ulcer on the lesser curvature of stomach  bleeding from left gastric artery. An ulcer on the posterior wall of duodenum • bleeding from gastroduodenal artery. Obstruction Pyloric channel, duodenal. Perforation Duodenal (anterior > posterior). Anterior duodenal ulcers can perforate into the anterior abdominal cavity, potentially leading to pneumoperitoneum. May see free air under diaphragm (pneumoperitoneum) A with referred pain to the shoulder via irritation of phrenic nerve. ComPliCations Malabsorption/malnutrition, colorectal cancer ( risk with pancolitis).

1	ComPliCations Malabsorption/malnutrition, colorectal cancer ( risk with pancolitis). Fistulas (eg, enterovesical fistulae, which can Fulminant colitis, toxic megacolon, perforation. cause recurrent UTI and pneumaturia), phlegmon/abscess, strictures (causing obstruction), perianal disease. intestinal maniFestation Diarrhea that may or may not be bloody. Bloody diarrhea. eXtraintestinal maniFestations Rash (pyoderma gangrenosum, erythema nodosum), eye inflammation (episcleritis, uveitis), oral ulcerations (aphthous stomatitis), arthritis (peripheral, spondylitis).

1	Appendicitis Acute inflammation of the appendix (yellow arrows in A ), can be due to obstruction by fecalith (red arrow in A ) (in adults) or lymphoid hyperplasia (in children). Proximal obstruction of appendiceal lumen produces closed-loop obstruction  intraluminal pressure  stimulation of visceral afferent nerve fibers at T8-T10  initial diffuse periumbilical pain  inflammation extends to serosa and irritates parietal peritoneum. Pain localized to RLQ/ McBurney point (1/3 the distance from right anterior superior iliac spine to umbilicus). Nausea, fever; may perforate  peritonitis; may elicit psoas, obturator, and Rovsing signs, guarding and rebound tenderness on exam. Differential: diverticulitis (elderly), ectopic pregnancy (use hCG to rule out), pseudoappendicitis. Treatment: appendectomy. Diverticula of the GI tract Zenker diverticulum

1	Diverticula of the GI tract Zenker diverticulum Pharyngoesophageal false diverticulum A . Esophageal dysmotility causes herniation of mucosal tissue at Killian triangle between the thyropharyngeal and cricopharyngeal parts of the inferior pharyngeal constrictor. Presenting symptoms: dysphagia, obstruction, gurgling, aspiration, foul breath, neck mass. Most common in elderly males. Elder MIKE has bad breath: Elderly Males Inferior pharyngeal constrictor Killian triangle Esophageal dysmotility Halitosis

1	Elder MIKE has bad breath: Elderly Males Inferior pharyngeal constrictor Killian triangle Esophageal dysmotility Halitosis Meckel diverticulum True diverticulum. Persistence of the vitelline (omphalomesenteric) duct. May contain ectopic acid–secreting gastric mucosa and/or pancreatic tissue. Most common congenital anomaly of GI tract. Can cause hematochezia/ melena (less common), RLQ pain, intussusception, volvulus, or obstruction near terminal ileum. Contrast with omphalomesenteric cyst = cystic dilation of vitelline duct. Diagnosis: 99mTc-pertechnetate scan (aka Meckel scan) for uptake by heterotopic gastric mucosa. The rule of 2’s: 2 times as likely in males. 2 inches long. 2 feet from the ileocecal valve. 2% of population. Commonly presents in first 2 years of life. May have 2 types of epithelia (gastric/ pancreatic).

1	Congenital megacolon characterized by lack of ganglion cells/enteric nervous plexuses (Auerbach and Meissner plexuses) in distal segment of colon. Due to failure of neural crest cell migration. Associated with loss of function mutations in RET. Presents with bilious emesis, abdominal distention, and failure to pass meconium within 48 hours • chronic constipation. Normal portion of the colon proximal to the aganglionic segment is dilated, resulting in a “transition zone.” Risk  with Down syndrome. Explosive expulsion of feces (squirt sign)  empty rectum on digital exam. Diagnosed by absence of ganglionic cells on rectal suction biopsy. Treatment: resection. RET mutation in the REcTum. Malrotation Anomaly of midgut rotation during fetal development  improper positioning of bowel (small bowel clumped on the right side) A , formation of fibrous bands (Ladd bands). Can lead to volvulus, duodenal obstruction.

1	Volvulus Twisting of portion of bowel around its mesentery; can lead to obstruction and infarction. Can occur throughout the GI tract. Chronic mesenteric “Intestinal angina”: atherosclerosis of celiac artery, SMA, or IMA  intestinal hypoperfusion ischemia • postprandial epigastric pain  food aversion and weight loss. Colonic ischemia Reduction in intestinal blood flow causes ischemia. Crampy abdominal pain followed by hematochezia. Commonly occurs at watershed areas (splenic flexure, rectosigmoid junction). Typically affects elderly. Thumbprint sign on imaging due to mucosal edema/hemorrhage. Ileus Intestinal hypomotility without obstruction  constipation and  flatus; distended/tympanic abdomen with  bowel sounds. Associated with abdominal surgeries, opiates, hypokalemia, sepsis. Treatment: bowel rest, electrolyte correction, cholinergic drugs (stimulate intestinal motility).

1	Colonic polyps Growths of tissue within the colon A . Grossly characterized as flat, sessile, or pedunculated on the basis of protrusion into colonic lumen. Generally classified by histologic type. Submucosal polyps May include lipomas, leiomyomas, fibromas, and other lesions. Malignant potential tooth” pattern of crypts on biopsy. Up to 20% of cases of sporadic CRC. A B C Polyp Polyp Cancer SessilePedunculated Lynch syndrome Previously called hereditary nonpolyposis colorectal cancer (HNPCC). Autosomal dominant mutation of mismatch repair genes (eg, MLH1, MSH2) with subsequent microsatellite instability. ∼ 80% progress to CRC. Proximal colon is always involved. Associated with endometrial, ovarian, and skin cancers. Diagnosis Iron deficiency anemia in males (especially > 50 years old) and postmenopausal females raises suspicion. Screening:

1	Diagnosis Iron deficiency anemia in males (especially > 50 years old) and postmenopausal females raises suspicion. Screening: Low risk: screen at age 50 with colonoscopy (polyp seen in A ); alternatives include flexible sigmoidoscopy, fecal occult blood testing (FOBT), fecal immunochemical testing (FIT), FIT-fecal DNA, CT colonography Patients with a first-degree relative who has colon cancer: screen at age 40 with colonoscopy, or 10 years prior to the relative's presentation Patients with IBD: distinct screening protocol “Apple core” lesion seen on barium enema x-ray B . CEA tumor marker: good for monitoring recurrence, should not be used for screening. ePiDemiology Most patients are > 50 years old. ~ 25% have a family history. Presentation Rectosigmoid > ascending > descending. Right side (cecal, ascending) associated with occult bleeding; left side (rectosigmoid) associated with hematochezia and obstruction (narrower lumen).

1	Right side (cecal, ascending) associated with occult bleeding; left side (rectosigmoid) associated with hematochezia and obstruction (narrower lumen). Ascending—exophytic mass, iron deficiency anemia, weight loss. Descending—infiltrating mass, partial obstruction, colicky pain, hematochezia. Can present with S bovis (gallolyticus) bacteremia/endocarditis or as an episode of diverticulitis. risK FaCtors Adenomatous and serrated polyps, familial cancer syndromes, IBD, tobacco use, diet of processed meat with low fiber. Molecular Chromosomal instability pathway: mutations in APC cause FAP and most sporadic cases of CRC pathogenesis of via adenoma-carcinoma sequence; (firing order of events is “AK-53”). colorectal cancer Microsatellite instability pathway: mutations or methylation of mismatch repair genes (eg, MLH1) cause Lynch syndrome and some sporadic CRC (via serrated polyp pathway). Overexpression of COX-2 has been linked to colorectal cancer, NSAIDs may be chemopreventive.

1	Loss of tumor suppressor Loss of APC gene KRAS mutation gene(s) (TP53, DCC) Cirrhosis—diffuse bridging fibrosis (via stellate cells) and regenerative nodules (red arrows in A ; white arrows show splenomegaly) disrupt normal architecture of liver;  risk for hepatocellular carcinoma (white arrow in B ). Etiologies include alcohol, nonalcoholic steatohepatitis, chronic viral hepatitis, autoimmune hepatitis, biliary disease, genetic/metabolic disorders. Portal hypertension— pressure in portal venous system. Etiologies include cirrhosis (most common cause in Western countries), vascular obstruction (eg, portal vein thrombosis, Budd- Chiari syndrome), schistosomiasis. Eects of portal hypertension *Due to estrogen Anorexia Nausea, vomiting Dull abdominal pain Serum markers of liver pathology Reye syndrome Rare, often fatal childhood hepatic encephalopathy.

1	Eects of portal hypertension *Due to estrogen Anorexia Nausea, vomiting Dull abdominal pain Serum markers of liver pathology Reye syndrome Rare, often fatal childhood hepatic encephalopathy. Associated with viral infection (especially VZV and influenza) that has been treated with aspirin. Aspirin metabolites • β-oxidation by reversible inhibition of mitochondrial enzymes. Findings: mitochondrial abnormalities, fatty liver (microvesicular fatty changes), hypoglycemia, vomiting, hepatomegaly, coma. Avoid aspirin in children, except in those with Kawasaki disease. Salicylates aren’t a ray (Reye) of sunSHINE for kids: Steatosis of liver/hepatocytes Hypoglycemia/Hepatomegaly Infection (VZV, influenza) Not awake (coma) Encephalopathy

1	Salicylates aren’t a ray (Reye) of sunSHINE for kids: Steatosis of liver/hepatocytes Hypoglycemia/Hepatomegaly Infection (VZV, influenza) Not awake (coma) Encephalopathy Alcoholic cirrhosis Final and usually irreversible form. Sclerosis around central vein (arrows in C ) may be seen in early disease. Regenerative nodules surrounded by fibrous bands in response to chronic liver injury  portal hypertension and end-stage liver disease. Nonalcoholic fatty Metabolic syndrome (insulin resistance); ALT > AST (Lipids) liver disease obesity • fatty infiltration of hepatocytes A  cellular “ballooning” and eventual necrosis. May cause cirrhosis and HCC. Independent of alcohol use. Most common 1° malignant tumor of liver in adults

1	Most common 1° malignant tumor of liver in adults A . Associated with HBV (+/− cirrhosis) and all other causes of cirrhosis (including HCV, alcoholic and nonalcoholic fatty liver disease, autoimmune disease, hemochromatosis, Wilson disease, α1-antitrypsin deficiency) and specific carcinogens (eg, aflatoxin from Aspergillus). May lead to Budd-Chiari syndrome. Findings: jaundice, tender hepatomegaly, ascites, polycythemia, anorexia. Spreads hematogenously. Diagnosis: • α-fetoprotein; ultrasound or contrast CT/MRI B , biopsy. Hepatic adenoma Rare, benign liver tumor, often related to oral contraceptive or anabolic steroid use; may regress spontaneously or rupture (abdominal pain and shock). Metastases GI malignancies, breast and lung cancer. Most common overall; metastases are rarely solitary.

1	Metastases GI malignancies, breast and lung cancer. Most common overall; metastases are rarely solitary. Budd-Chiari syndrome Thrombosis or compression of hepatic veins with centrilobular congestion and necrosis  congestive liver disease (hepatomegaly, ascites, varices, abdominal pain, liver failure). Absence of JVD. Associated with hypercoagulable states, polycythemia vera, postpartum state, HCC. May cause nutmeg liver (mottled appearance). 1-antitrypsin Misfolded gene product protein aggregates in In lungs, • α1-antitrypsin  uninhibited elastase deficiency hepatocellular ER  cirrhosis with in alveoli  elastic tissue • panacinar PAS ⊕ globules A in liver. Codominant trait. emphysema. Often presents in young patients with liver damage and dyspnea without a history of smoking. Jaundice Abnormal yellowing of the skin HOT Liver—common causes of • bilirubin and/or sclera A due to bilirubin deposition. level:

1	Jaundice Abnormal yellowing of the skin HOT Liver—common causes of • bilirubin and/or sclera A due to bilirubin deposition. level: Hyperbilirubinemia 2° to  production Hemolysis or  clearance (impaired hepatic uptake, Obstruction conjugation, excretion). Tumor Hereditary All autosomal recessive. hyperbilirubinemias Gilbert syndrome Mildly  UDP-glucuronosyltransferase conjugation and impaired bilirubin uptake. Asymptomatic or mild jaundice usually with stress, illness, or fasting.  unconjugated bilirubin without overt hemolysis. Relatively common, benign condition. Dubin-Johnson Conjugated hyperbilirubinemia due to defective liver excretion. Grossly black (Dark) liver due to syndrome impaired excretion of epinephrine metabolites. Benign. Similar to Dubin-Johnson syndrome, but milder in presentation without black (Regular) liver. Due to impaired hepatic uptake and excretion.

1	Similar to Dubin-Johnson syndrome, but milder in presentation without black (Regular) liver. Due to impaired hepatic uptake and excretion. Endothelial cells Hemoglobin circulating bilirubin Kuper cell (macrophage) Obstructive jaundice (downstream) (albumin bound, unconjugated, water insoluble) Space of Disse HEPATIC SINUSOID RSTQQBILIRUBIN UPTAKE CONJUGATION INTRACELLULAR TRANSPORT UDP-glucuronosyl-transferase Unconjugated bilirubin Conjugated bilirubin (bilirubin diglucuronide, water soluble) Bile canalicular lumen Hepatocyte Bile fow Unknown cause of concentric “onion skin” bile duct fibrosis  alternating strictures and dilation with “beading” of intraand extrahepatic bile ducts on ERCP, magnetic resonance cholangiopancreatography (MRCP).

1	Also called hepatolenticular degeneration. Autosomal recessive mutations in hepatocyte copper-transporting ATPase (ATP7B gene; chromosome 13) • copper incorporation into apoceruloplasmin and excretion into bile  serum ceruloplasmin. Copper accumulates, especially in liver, brain, cornea, kidneys;  urine copper. Presents before age 40 with liver disease (eg, hepatitis, acute liver failure, cirrhosis), neurologic disease (eg, dysarthria, dystonia, tremor, parkinsonism), psychiatric disease, Kayser-Fleischer rings (deposits in Descemet membrane of cornea) A , hemolytic anemia, renal disease (eg, Fanconi syndrome). Treatment: chelation with penicillamine or trientine, oral zinc. Liver transplant in acute liver failure related to Wilson disease.

1	Treatment: chelation with penicillamine or trientine, oral zinc. Liver transplant in acute liver failure related to Wilson disease. Autosomal recessive. On HFE gene, located on chromosome 6; associated with HLA-A3. Leads to abnormal iron sensing and  intestinal absorption ( ferritin,  iron,  TIBC  transferrin saturation). Iron overload can also be 2° to chronic transfusion therapy (eg, β-thalassemia major). Iron accumulates, especially in liver, pancreas, skin, heart, pituitary, joints. Hemosiderin (iron) can be identified on liver MRI or biopsy with Prussian blue stain A .

1	Presents after age 40 when total body iron > 20 g; iron loss through menstruation slows progression in women. Classic triad of cirrhosis, diabetes mellitus, skin pigmentation (“bronze diabetes”). Also causes restrictive cardiomyopathy (classic) or dilated cardiomyopathy (reversible), hypogonadism, arthropathy (calcium pyrophosphate deposition; especially metacarpophalangeal joints). HCC is common cause of death. Treatment: repeated phlebotomy, iron (Fe) chelation with deferasirox, deferoxamine, deferiprone. May present with pruritus, jaundice, dark urine, light-colored stool, hepatosplenomegaly. Typically with cholestatic pattern of LFTs ( conjugated bilirubin, • cholesterol,  ALP, • GGT). Classically in middle-aged men Associated with ulcerative with ulcerative colitis. colitis. p-ANCA ⊕.  IgM. Can lead to 2° biliary cholangitis.  risk of cholangiocarcinoma and gallbladder cancer.

1	Classically in middle-aged men Associated with ulcerative with ulcerative colitis. colitis. p-ANCA ⊕.  IgM. Can lead to 2° biliary cholangitis.  risk of cholangiocarcinoma and gallbladder cancer. Autoimmune reaction Classically in middle-aged Anti-mitochondrial antibody ⊕, infiltrate women.  IgM. Associated with other +/– granulomas autoimmune conditions of lobular bile (eg, Hashimoto thyroiditis, ducts. rheumatoid arthritis, celiac disease). Treatment: ursodiol. Extrahepatic biliary obstruction Patients with known May be complicated by  pressure in intrahepatic obstructive lesions (gallstones, ascending cholangitis. ducts  injury/ fibrosis and biliary strictures, pancreatic bile stasis. carcinoma).  cholesterol and/or bilirubin,  bile salts, and Gender (female), Chronic hemolysis, gallbladder stasis all cause stones. age, obesity, genetics, biliary tract infection 2 types of stones:

1	 cholesterol and/or bilirubin,  bile salts, and Gender (female), Chronic hemolysis, gallbladder stasis all cause stones. age, obesity, genetics, biliary tract infection 2 types of stones: Cholesterol stones (radiolucent with 10–20% opaque due to calcifications)—80% of stones. Associated with obesity, Crohn disease, advanced age, estrogen therapy, multiparity, rapid weight loss, Native American origin. Pigment stones A (black = radiopaque, Ca2+ bilirubinate, hemolysis; brown = radiolucent, infection). Associated with Crohn disease, chronic hemolysis, alcoholic cirrhosis, advanced age, biliary infections, total parenteral nutrition (TPN). Risk factors (4 F’s): 1. 2. 3. 4. Most common complication is cholecystitis; can also cause acute pancreatitis, ascending cholangitis. Diagnose with ultrasound. Treat with elective cholecystectomy if symptomatic.

1	2. 3. 4. Most common complication is cholecystitis; can also cause acute pancreatitis, ascending cholangitis. Diagnose with ultrasound. Treat with elective cholecystectomy if symptomatic. ↓cholesterol 7αhydroxylase ↑Cholesterol, ↓bile salts, gallbladder stasis ↑Unconjugated bilirubin, gallbladder stasis Supersaturation of bile with calcium bilirubinate Pigment stones Supersaturation of bile with cholesterol Cholesterol stones Choledocholithiasis Presence of gallstone(s) in common bile duct, often leading to elevated ALP, GGT, direct bilirubin, and/or AST/ALT.

1	Cholecystitis Acute or chronic inflammation of gallbladder. Calculous cholecystitis—most common type; due to gallstone impaction in the cystic duct resulting in inflammation and gallbladder wall thickening (arrows in B ); can produce 2° infection. Acalculous cholecystitis—due to gallbladder stasis, hypoperfusion, or infection (CMV); seen in critically ill patients. Murphy sign: inspiratory arrest on RUQ palpation due to pain. Pain may radiate to right shoulder (due to irritation of phrenic nerve).  ALP if bile duct becomes involved (eg, ascending cholangitis). Diagnose with ultrasound or cholescintigraphy (HIDA scan). Failure to visualize gallbladder on HIDA scan suggests obstruction. Gallstone ileus—fistula between gallbladder and GI tract  stone enters GI lumen  obstructs at ileocecal valve (narrowest point); can see air in biliary tree (pneumobilia). Rigler triad: radiographic findings of pneumobilia, small bowel obstruction, gallstone (usually in iliac fossa).

1	Porcelain gallbladder Calcified gallbladder due to chronic cholecystitis; usually found incidentally on imaging C . Treatment: prophylactic cholecystectomy generally recommended due to  risk of gallbladder cancer (mostly adenocarcinoma). Ascending cholangitis Infection of biliary tree usually due to obstruction that leads to stasis/bacterial overgrowth. Charcot triad of cholangitis includes jaundice, fever, RUQ pain. Reynolds pentad is Charcot triad plus altered mental status and shock (hypotension). Autodigestion of pancreas by pancreatic enzymes ( A shows pancreas [yellow arrows] surrounded by edema [red arrows]). Causes: Idiopathic, Gallstones, Ethanol, Trauma, Steroids, Mumps, Autoimmune disease, Scorpion sting, Hypercalcemia/Hypertriglyceridemia (> 1000 mg/dL), ERCP, Drugs (eg, sulfa drugs, NRTIs, protease inhibitors). I GET SMASHED.

1	Diagnosis by 2 of 3 criteria: acute epigastric pain often radiating to the back,  serum amylase or lipase (more specific) to 3× upper limit of normal, or characteristic imaging findings. Complications: pseudocyst B (lined by granulation tissue, not epithelium), abscess, necrosis, hemorrhage, infection, organ failure (ALI/ARDS, shock, renal failure), hypocalcemia (precipitation of Ca2+ soaps). Chronic pancreatitis Chronic inflammation, atrophy, calcification of the pancreas A . Major causes include alcohol abuse and genetic predisposition (ie, cystic fibrosis); can be idiopathic. Complications include pancreatic insufficiency and pseudocysts. Pancreatic insufficiency (typically when <10% pancreatic function) may manifest with steatorrhea, fat-soluble vitamin deficiency, diabetes mellitus. Amylase and lipase may or may not be elevated (almost always elevated in acute pancreatitis).

1	Very aggressive tumor arising from pancreatic ducts (disorganized glandular structure with cellular infiltration A ); often metastatic at presentation, with average survival ~ 1 year after diagnosis. Tumors more common in pancreatic head B (lead to obstructive jaundice). Associated with CA 19-9 tumor marker (also CEA, less specific). Risk factors: Jewish and African-American males Often presents with: Abdominal pain radiating to back Weight loss (due to malabsorption and anorexia) Migratory thrombophlebitis—redness and tenderness on palpation of extremities (Trousseau syndrome) Obstructive jaundice with palpable, nontender gallbladder (Courvoisier sign) Treatment: Whipple procedure (pancreaticoduodenectomy), chemotherapy, radiation therapy. HCO3 ”alkaline tide”— ˜ blood pH after gastric acid secretion (eg, after meals, vomiting) Misoprostol H+ K+ Sucralfate, bismuth

1	HCO3 ”alkaline tide”— ˜ blood pH after gastric acid secretion (eg, after meals, vomiting) Misoprostol H+ K+ Sucralfate, bismuth Histamine-2 blockers Cimetidine, ranitidine, famotidine, nizatidine. Take H2 blockers before you dine. Think “table for 2” to remember H2. meCHanism Reversible block of histamine H2-receptors  H+ secretion by parietal cells. CliniCal Use Peptic ulcer, gastritis, mild esophageal reflux. aDVerse eFFeCts Cimetidine is a potent inhibitor of cytochrome P-450 (multiple drug interactions); it also has antiandrogenic effects (prolactin release, gynecomastia, impotence,  libido in males); can cross blood-brain barrier (confusion, dizziness, headaches) and placenta. Both cimetidine and ranitidine  renal excretion of creatinine. Other H2 blockers are relatively free of these effects. Proton pump inhibitors Omeprazole, lansoprazole, esomeprazole, pantoprazole, dexlansoprazole. meCHanism Irreversibly inhibit H+/K+ ATPase in stomach parietal cells.

1	Proton pump inhibitors Omeprazole, lansoprazole, esomeprazole, pantoprazole, dexlansoprazole. meCHanism Irreversibly inhibit H+/K+ ATPase in stomach parietal cells. CliniCal Use Peptic ulcer, gastritis, esophageal reflux, Zollinger-Ellison syndrome, component of therapy for H pylori, stress ulcer prophylaxis. aDVerse eFFeCts  risk of C difficile infection, pneumonia, acute interstitial nephritis. Vitamin B12 malabsorption;  serum Mg2+ and • Ca2+ absorption (potentially leading to increased fracture risk in elderly). Antacids Can affect absorption, bioavailability, or urinary excretion of other drugs by altering gastric and urinary pH or by delaying gastric emptying. All can cause hypokalemia. Overuse can also cause the following problems: Bismuth, sucralfate meCHanism Bind to ulcer base, providing physical protection and allowing HCO3 secretion to reestablish pH gradient in the mucous layer. Sucralfate requires acidic environment, not given with PPIs/H2 blockers.

1	CliniCal Use  ulcer healing, travelers’ diarrhea (bismuth). Bismuth also used in quadruple therapy for H pylori gastritis. aDVerse eFFeCts Diarrhea. Contraindicated in women of childbearing potential (abortifacient). meCHanism Long-acting somatostatin analog; inhibits secretion of various splanchnic vasodilatory hormones. CliniCal Use Acute variceal bleeds, acromegaly, VIPoma, carcinoid tumors. aDVerse eFFeCts Nausea, cramps, steatorrhea.  risk of cholelithiasis due to CCK inhibition. meCHanism A combination of sulfapyridine (antibacterial) and 5-aminosalicylic acid (anti-inflammatory). Activated by colonic bacteria. CliniCal Use Ulcerative colitis, Crohn disease (colitis component). aDVerse eFFeCts Malaise, nausea, sulfonamide toxicity, reversible oligospermia. meCHanism Agonist at μ-opioid receptors; slows gut motility. Poor CNS penetration (low addictive potential). CliniCal Use Diarrhea. aDVerse eFFeCts Constipation, nausea.

1	meCHanism Agonist at μ-opioid receptors; slows gut motility. Poor CNS penetration (low addictive potential). CliniCal Use Diarrhea. aDVerse eFFeCts Constipation, nausea. meCHanism 5-HT3 antagonist;  vagal stimulation. Powerful central-acting antiemetic. CliniCal Use Control vomiting postoperatively and in patients undergoing cancer chemotherapy. aDVerse eFFeCts Headache, constipation, QT interval prolongation, serotonin syndrome. meCHanism D2 receptor antagonist.  resting tone, contractility, LES tone, motility, promotes gastric emptying. Does not influence colon transport time. CliniCal Use Diabetic and postoperative gastroparesis, antiemetic, persistent GERD. aDVerse eFFeCts • parkinsonian effects, tardive dyskinesia. Restlessness, drowsiness, fatigue, depression, diarrhea. Drug interaction with digoxin and diabetic agents. Contraindicated in patients with small bowel obstruction, Parkinson disease (due to D2-receptor blockade), • seizure threshold.

1	meCHanism Inhibits gastric and pancreatic lipase  breakdown and absorption of dietary fats. Taken with fat-containing meals. CliniCal Use Weight loss. aDVerse eFFeCts Abdominal pain, flatulence, bowel urgency/frequent bowel movements, steatorrhea;  absorption of fat-soluble vitamins. Laxatives Indicated for constipation or patients on opiates requiring a bowel regimen. meCHanism Substance P antagonist. Blocks NK1 (neurokinin-1) receptors in brain. CliniCal Use Antiemetic for chemotherapy-induced nausea and vomiting. “You’re always somebody’s type! (blood type, that is)” “All the soarings of my mind begin in my blood.” “The best blood will at some time get into a fool or a mosquito.”

1	“You’re always somebody’s type! (blood type, that is)” “All the soarings of my mind begin in my blood.” “The best blood will at some time get into a fool or a mosquito.” When studying hematology, pay close attention to the many cross connections to immunology. Make sure you master the different types of anemias. Be comfortable interpreting blood smears. When reviewing oncologic drugs, focus on mechanisms and adverse effects rather than details of clinical uses, which may be lower yield. Please note that solid tumors are covered in their respective organ system chapters. Fetal erythropoiesis Fetal erythropoiesis occurs in: Young Liver Synthesizes Blood. Bone marrow (18 weeks to adult) Embryonic globins: ζ and ε. Fetal hemoglobin (HbF) =αγ From fetal to adult hemoglobin: 2 2. Adult hemoglobin (HbA ) =αβ Alpha Always; Gamma Goes, Becomes Beta. 1 22.

1	Embryonic globins: ζ and ε. Fetal hemoglobin (HbF) =αγ From fetal to adult hemoglobin: 2 2. Adult hemoglobin (HbA ) =αβ Alpha Always; Gamma Goes, Becomes Beta. 1 22. HbF has higher affinity for O2 due to less avid binding of 2,3-BPG, allowing HbF to extract O2 from maternal hemoglobin (HbA1 and HbA2) across the placenta. HbA2 (α2δ2) is a form of adult hemoglobin present in small amounts. Site of erythropoiesis 50 40 % of total 30 globin synthesis 20 Hemolytic disease of Also known as erythroblastosis fetalis. the newborn Neutrophils Acute inflammatory response cells. Numbers Hypersegmented neutrophils (nucleus has 6+  in bacterial infections. Phagocytic. lobes) are seen in vitamin B / folate deficiency.

1	Neutrophils Acute inflammatory response cells. Numbers Hypersegmented neutrophils (nucleus has 6+  in bacterial infections. Phagocytic. lobes) are seen in vitamin B / folate deficiency. A 12 Multilobed nucleus A . Specific granules A left shift with  band cells (immature contain leukocyte alkaline phosphatase neutrophils) reflects states of  myeloid (LAP), collagenase, lysozyme, and proliferation (eg, bacterial infections, CML). lactoferrin. Azurophilic granules (lysosomes) Important neutrophil chemotactic agents: C5a, contain proteinases, acid phosphatase, IL-8, LTB4, kallikrein, platelet-activating factor. myeloperoxidase, and β-glucuronidase.

1	Carry O2 to tissues and CO2 to lungs. Anucleate and lack organelles; biconcave A , with large surface area-to-volume ratio for rapid gas exchange. Life span of 120 days. Source of energy is glucose (90% used in glycolysis, 10% used in HMP shunt). Membranes contain Cl−/HCO3 − antiporter, which allow RBCs to export HCO3 − and transport CO2 from the periphery to the lungs for elimination. Eryth = red; cyte = cell. Erythrocytosis = polycythemia =  Hct. Anisocytosis = varying sizes. Poikilocytosis = varying shapes. Reticulocyte = immature RBC; reflects erythroid proliferation. Bluish color (polychromasia) on Wright-Giemsa stain of reticulocytes represents residual ribosomal RNA.

1	Reticulocyte = immature RBC; reflects erythroid proliferation. Bluish color (polychromasia) on Wright-Giemsa stain of reticulocytes represents residual ribosomal RNA. Involved in 1° hemostasis. Small cytoplasmic fragments A derived from megakaryocytes. Life span of 8–10 days. When activated by endothelial injury, aggregate with other platelets and interact with fibrinogen to form platelet plug. Contain dense granules (Ca2+ , ADP, Serotonin, Histamine; CASH) and α granules (vWF, fibrinogen, fibronectin, platelet factor 4). Approximately 1⁄3 of platelet pool is stored in the spleen. in petechiae. vWF receptor: GpIb. Fibrinogen receptor: GpIIb/IIIa. Thrombopoietin stimulates megakaryocyte proliferation. Alfa granules contain vWF, fibrinogen, fibronectin, platelet factor four. Monocytes Found in blood, differentiate into macrophages Mono = one (nucleus); cyte = cell. in tissues. Large, kidney-shaped nucleus A . Extensive “frosted glass” cytoplasm.

1	Monocytes Found in blood, differentiate into macrophages Mono = one (nucleus); cyte = cell. in tissues. Large, kidney-shaped nucleus A . Extensive “frosted glass” cytoplasm. Phagocytose bacteria, cellular debris, and senescent RBCs. Long life in tissues. Differentiate from circulating blood monocytes A . Activated by γ-interferon. Can function as antigen-presenting cell via MHC II. Important cellular component of granulomas (eg, TB, sarcoidosis). Macro = large; phage = eater. Macrophage naming varies by specific tissue type (eg, Kupffer cells in liver, histiocytes in connective tissue, Langerhans cells in skin, osteoclasts in bone, microglial cells in brain). Lipid A from bacterial LPS binds CD14 on macrophages to initiate septic shock.

1	Lipid A from bacterial LPS binds CD14 on macrophages to initiate septic shock. Basophils Mediate allergic reaction. Densely basophilic Basophilic—stains readily with basic stains. granules A contain heparin (anticoagulant) Basophilia is uncommon, but can be a sign of and histamine (vasodilator). Leukotrienes myeloproliferative disorders, particularly CML. synthesized and released on demand. Dendritic cells Highly phagocytic antigen-presenting cells (APCs) A . Function as link between innate and adaptive immune systems. Express MHC class II and Fc receptors on surface. Defend against helminthic infections (major basic protein). Bilobate nucleus. Packed with large eosinophilic granules of uniform size A . Highly phagocytic for antigen-antibody complexes. Produce histaminase, major basic protein (MBP, a helminthotoxin), eosinophil peroxidase, eosinophil cationic protein, and eosinophilderived neurotoxin. Eosin = pink dye; philic = loving.

1	Produce histaminase, major basic protein (MBP, a helminthotoxin), eosinophil peroxidase, eosinophil cationic protein, and eosinophilderived neurotoxin. Eosin = pink dye; philic = loving. Causes of eosinophilia = PACCMAN: Parasites Asthma Eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome) Chronic adrenal insufficiency Myeloproliferative disorders Allergic processes Neoplasia (eg, Hodgkin lymphoma) Lymphocytes Refer to B cells, T cells, and NK cells. B cells and T cells mediate adaptive immunity. NK cells are part of the innate immune response. Round, densely staining nucleus with small amount of pale cytoplasm A .

1	A . Natural killer cells Important in innate immunity, especially against intracellular pathogens. Larger than B and T cells, with distinctive cytoplasmic lytic granules (containing perforin and granzymes) that, when released, act on target cells to induce apoptosis. Distinguish between healthy and infected cells by identifying cell surface proteins (induced by stress, malignant transformation, or microbial infections). Mediate humoral immune response. Originate from stem cells in bone marrow and matures in marrow. Migrate to peripheral lymphoid tissue (follicles of lymph nodes, white pulp of spleen, unencapsulated lymphoid tissue). When antigen is encountered, B cells differentiate into plasma cells (which produce antibodies) and memory cells. Can function as an APC. B = Bone marrow.

1	B = Bone marrow. Mediate cellular immune response. Originate from stem cells in the bone marrow, but mature in the thymus. Differentiate into cytotoxic T cells (express CD8, recognize MHC I), helper T cells (express CD4, recognize MHC II), and regulatory T cells. CD28 (costimulatory signal) necessary for T-cell activation. Most circulating lymphocytes are T cells (80%). T = Thymus. CD4+ helper T cells are the primary target of HIV. Rule of 8: MHC II × CD4 = 8; MHC I × CD8 = 8. Plasma cells Produce large amounts of antibody specific to Multiple myeloma is a plasma cell dyscrasia. a particular antigen. “Clock-face” chromatin distribution and eccentric nucleus, abundant RER, and well-developed Golgi apparatus (arrows in A ). Found in bone marrow and normally do not circulate in peripheral blood.

1	RER, and well-developed Golgi apparatus (arrows in A ). Found in bone marrow and normally do not circulate in peripheral blood. ACTIVATIONADP binding to P2Y12 receptor induces GpIIb/IIIa expression at platelet surface Platelets release ADP and Ca2+ (necessary for coagulation cascade), TXA2 ADHESIONPlatelets bind vWF via GpIb receptor at the site of injury only (specifc) ˜ platelets undergo conformational change EXPOSUREvWF binds to exposed collagen vWF is from Weibel-Palade bodies of endothelial cells and °-granules of platelets INJURYEndothelial damage ˜ transient vasoconstriction via neural stimulation refex and endothelin (released from damaged cell) 4A Temporary plug stops bleeding; unstable, easily dislodged

1	Thrombogenesis Formation of insoluble fibrin mesh. Aspirin irreversibly inhibits cyclooxygenase, thereby inhibiting TXA2 synthesis. Clopidogrel, prasugrel, and ticlopidine inhibit ADP-induced expression of GpIIb/IIIa by irreversibly blocking P2Y12 receptor. Abciximab, eptifibatide, and tirofiban inhibit GpIIb/IIIa directly. Ristocetin activates vWF to bind GpIb. Failure of aggregation with ristocetin assay occurs in von Willebrand disease and Bernard-Soulier syndrome. vWF carries/protects factor VIII; volksWagen Factories make gr8 cars. Platelet ADP (P2Y12) receptor GpIIb/IIIa insertion Subendothelial collagen GpIb GpIIb/IIIa Fibrinogen vWF Thrombin-thrombomodulin complex Protein C Activated protein C Vascular endothelial cells Inside endothelial cells (vWF + factor VIII) Thromboplastin tPA, PGI2 Arachidonic acid Aspirin COX TXA2 vWF Fibrinogen Inside platelets Clopidogrel, prasugrel, ticlopidine Defciency: Bernard-Soulier syndrome Defciency: Glanzmann thrombasthenia Defciency: von

1	tPA, PGI2 Arachidonic acid Aspirin COX TXA2 vWF Fibrinogen Inside platelets Clopidogrel, prasugrel, ticlopidine Defciency: Bernard-Soulier syndrome Defciency: Glanzmann thrombasthenia Defciency: von Willebrand disease Abciximab, eptifbatide, tirofban 4B 4A

1	Hemophilia A: defciency of factor VIII (XR) Hemophilia B: defciency of factor IX (XR) Hemophilia C: defciency of factor XI (AR) Note: Kallikrein activates bradykinin * = require Ca2+ , phospholipid; Fibrin degradation = inhibited by vitamin K antagonist warfarin products (eg, D-dimer) Fibrin mesh stabilizes = activates but not part of coagulation cascade

1	Fibrin mesh stabilizes = activates but not part of coagulation cascade Collagen, basement membrane, activated platelets Tissue factor (extrinsic) pathway Combined pathway Fibrinolytic system ANTICOAGULANTS: LMWH (eg, dalteparin, enoxaparin) heparin direct Xa inhibitors (eg, apixaban) fondaparinux ANTICOAGULANTS: -heparin -LMWH -direct thrombin inhibitors (eg, argatroban, bivalirudin, dabigatran) Tissue factor VII VIIa XII Prothrombin Kallikrein Bradykinin ˜vasodilation ˜ permeability ˜ painKinin cascade HMWK Thrombin Plasminogen Plasmin THROMBOLYTICS: alteplase, reteplase, streptokinase, tenecteplase ANTIFIBRINOLYTICS:aminocaproic acid, tranexamic acid tPA XI XIa IX X II IXa VIIIa VIII with vWF XIIa Xa Va* * IIa I Fibrinogen Aggregation Fibrin monomers Ia Ca2+ XIIIa XIII V Contact activation (intrinsic) pathway –––** * * * # # REGULATORY ANTICOAGULANT PROTEINS: -proteins C and S –C1-esterase inhibitor –––

1	Procoagulation Vitamin K deficiency:  synthesis of factors II, VII, IX, X, protein C, protein S. Reduced vitamin K (active) Inactive II, VII, IX, X, C, S Clotting factors Anti-coagulants -glutamyl carboxylase (vitamin K-dependent) Epoxide reductase Mature, carboxylated II, VII, IX, X, C, S Fibrinogen Fibrin Liver Oxidized vitamin K (inactive) Warfarin inhibits vitamin K epoxide reductase. Vitamin K administration can potentially reverse inhibitory effect of warfarin on clotting factor synthesis (delayed). FFP or PCC administration reverses action of warfarin immediately and can be given with vitamin K in cases of severe bleeding. Neonates lack enteric bacteria, which produce vitamin K. Early administration of vitamin K overcomes neonatal deficiency/coagulopathy. Factor VII (seven)—shortest half-life. Warfarin, Factor II (two)—longest (tallest) half-life. factors VIIa, IXa, Xa, XIa, XIIa. Heparin enhances the activity of antithrombin.

1	Factor VII (seven)—shortest half-life. Warfarin, Factor II (two)—longest (tallest) half-life. factors VIIa, IXa, Xa, XIa, XIIa. Heparin enhances the activity of antithrombin. Principal targets of antithrombin: thrombin and factor Xa. Antithrombin ProteinCpathway pathway Heparin-like molecule (enhances ATIII activity) Thrombin-thrombomodulin complex (endothelial cells) Antithrombin III Protein C Requires protein S Activated protein C Inhibits thrombin (and VIIa, IXa, Xa, XIa, XIIa) Cleaves and inactivates Va, VIIIa resistant to inhibition by activated protein C. tPA is used clinically as a thrombolytic. Liver disease, abetalipoproteinemia Projections of varying size at (“spur cells”) irregular intervals. Liver disease, ESRD, pyruvate Smaller and more uniform (“burr cells”) kinase deficiency projections than acanthocytes Bone marrow infiltration (eg, RBC “sheds a tear” because it’s (“teardrop cells”) myelofibrosis) mechanically squeezed out of its home in the bone marrow

1	Bone marrow infiltration (eg, RBC “sheds a tear” because it’s (“teardrop cells”) myelofibrosis) mechanically squeezed out of its home in the bone marrow MAHAs (eg, DIC, TTP/HUS, Fragmented RBCs (eg, “helmet” cells) HELLP syndrome), mechanical hemolysis (eg, heart valve prosthesis) G6PD deficiency Due to removal of Heinz bodies by cells”) splenic macrophages Hereditary elliptocytosis Caused by mutation in genes encoding RBC membrane proteins (eg, spectrin) Hereditary spherocytosis, Small, spherical cells without autoimmune hemolytic anemia central pallor HbC disease, Asplenia, “HALT,” said the hunter to his Liver disease, Thalassemia target Sickle cell anemia Sickling occurs with low O2 conditions (eg, high altitude, acidosis) Sideroblastic anemias (eg, lead Perinuclear mitochondria with (eg, in ringed poisoning, myelodysplastic excess iron (forming ring in sideroblasts) syndromes, alcoholism) ringed sideroblasts) Require Prussian blue stain to be visualized

1	Functional hyposplenia (eg, sickle Basophilic nuclear remnants (do cell disease), asplenia not contain iron) Usually removed by splenic macrophages Sideroblastic anemias, thalassemias Basophilic ribosomal precipitates (do not contain iron) G6PD deficiency Denatured and precipitated hemoglobin (contain iron) Phagocytic removal of Heinz bodies  bite cells Requires supravital stain (eg, crystal violet) to be visualized Reticulocyte index Also called corrected reticulocyte count. Used to correct falsely elevated reticulocyte count in anemia. Measures appropriate bone marrow response to anemic conditions (effective erythropoiesis). High reticulocyte index (RI) indicates compensatory RBC production; low RI indicates inadequate response to correct anemia. Calculated as: Microcytic, hypochromic anemias MCV < 80 fL.

1	Microcytic, hypochromic anemias MCV < 80 fL. Iron deficiency  iron due to chronic bleeding (eg, GI loss, menorrhagia), malnutrition, absorption disorders, GI surgery (eg, gastrectomy), or • demand (eg, pregnancy)  final step in heme synthesis. Labs:  iron, • TIBC, • ferritin,  free erythrocyte protoporphyrin,  RDW,  RI. Microcytosis and hypochromasia ( central pallor) A . Symptoms: fatigue, conjunctival pallor B , pica (persistent craving and compulsive eating of nonfood substances), spoon nails (koilonychia). May manifest as glossitis, cheilosis, Plummer-Vinson syndrome (triad of iron deficiency anemia, esophageal webs, and dysphagia). -thalassemia α-globin gene deletions on chromosome 16 • α-globin synthesis. cis deletion (deletions occur on same chromosome) prevalent in Asian populations; trans deletion (deletions occur on separate chromosomes) prevalent in African populations. Normal is αα/αα.

1	2 (α –/α –; trans) or α-thalassemia minor Mild microcytic, hypochromic (αα/– –; cis) anemia; cis deletion may worsen outcome for the carrier’s offspring 3 (– –/– α) Hemoglobin H disease (HbH); Moderate to severe microcytic excess β-globin forms β4 hypochromic anemia 4 (– –/– –) Hemoglobin Barts disease; Hydrops fetalis; incompatible no α-globin, excess γ-globin with life forms γ4 -thalassemia Point mutations in splice sites and promoter sequences on chromosome 11 • β-globin synthesis. Prevalent in Mediterranean populations. -thalassemia minor (heterozygote): β chain is underproduced. Usually asymptomatic. Diagnosis confirmed by • HbA2 (> 3.5%) on electrophoresis.

1	-thalassemia minor (heterozygote): β chain is underproduced. Usually asymptomatic. Diagnosis confirmed by • HbA2 (> 3.5%) on electrophoresis. -thalassemia major (homozygote): β chain is absent  severe microcytic, hypochromic anemia with target cells and increased anisopoikilocytosis C requiring blood transfusion (2° hemochromatosis). Marrow expansion (“crew cut” on skull x-ray)  skeletal deformities (eg, “chipmunk” facies). Extramedullary hematopoiesis  hepatosplenomegaly.  risk of parvovirus B19–induced aplastic crisis.  HbF (αγ ), HbA (αδ ). HbF is protective in the infant and disease becomes symptomatic only after 6 months, when fetal hemoglobin declines. HbS/β -thalassemia heterozygote: mild to moderate sickle cell disease depending on amount of β-globin production. Microcytic, hypochromic anemias (continued)

1	Microcytic, hypochromic anemias (continued) Lead poisoning Lead inhibits ferrochelatase and ALA dehydratase  heme synthesis and  RBC protoporphyrin. Also inhibits rRNA degradation  RBCs retain aggregates of rRNA (basophilic stippling). Symptoms of LEAD poisoning: Lead Lines on gingivae (Burton lines) and on metaphyses of long bones D on x-ray. Encephalopathy and Erythrocyte basophilic stippling. Abdominal colic and sideroblastic Anemia. Drops—wrist and foot drop. Dimercaprol and EDTA are 1st line of treatment. Succimer used for chelation for kids (It “sucks” to be a kid who eats lead). Exposure risk  in old houses with chipped paint.

1	Sideroblastic anemia Causes: genetic (eg, X-linked defect in ALA synthase gene), acquired (myelodysplastic syndromes), and reversible (alcohol is most common; also lead poisoning, vitamin B6 deficiency, copper deficiency, drugs [eg, isoniazid, linezolid]). Lab findings: • iron, normal/ TIBC, • ferritin. Ringed sideroblasts (with iron-laden, Prussian blue–stained mitochondria) seen in bone marrow E . Peripheral blood smear: basophilic stippling of RBCs. Some acquired variants may be normocytic or macrocytic. Treatment: pyridoxine (B6, cofactor for ALA synthase). Interpretation of iron studies  = 1° disturbance. Transferrin—transports iron in blood. TIBC—indirectly measures transferrin. Ferritin—1° iron storage protein of body. aEvolutionary reasoning—pathogens use circulating iron to thrive. The body has adapted a system in which iron is stored within the cells of the body and prevents pathogens from acquiring circulating iron. Macrocytic anemias MCV > 100 fL.

1	Macrocytic anemias MCV > 100 fL. Megaloblastic anemia Impaired DNA synthesis  maturation of RBC macrocytosis, hypersegmented neutrophils nucleus of precursor cells in bone marrow (arrow in A ), glossitis. delayed relative to maturation of cytoplasm. Causes: vitamin B12 deficiency, folate deficiency, medications (eg, hydroxyurea, phenytoin, methotrexate, sulfa drugs). Vitamin B12 Causes: pernicious anemia, malabsorption (cobalamin) (eg, Crohn disease), pancreatic insufficiency, deficiency gastrectomy, insufficient intake (eg, veganism), Diphyllobothrium latum (fish tapeworm).  homocysteine, • methylmalonic acid. Neurologic symptoms: reversible dementia, subacute combined degeneration (due to involvement of B12 in fatty acid pathways and myelin synthesis): spinocerebellar tract, lateral corticospinal tract, dorsal column dysfunction. Folate supplementation in vitamin B12 deficiency can correct the anemia, but worsens neurologic symptoms.

1	Historically diagnosed with the Schilling test, a test that determines if the cause is dietary insufficiency vs malabsorption. Anemia 2° to insufficient intake may take several years to develop due to liver’s ability to store B12 (as opposed to folate deficiency). Nonhemolytic, normocytic anemias Anemia of chronic disease Inflammation (eg,  IL-6)  hepcidin (released by liver, binds ferroportin on intestinal mucosal cells and macrophages, thus inhibiting iron transport)  release of iron from macrophages and • iron absorption from gut. Associated with conditions such as chronic infections, neoplastic disorders, chronic kidney disease, and autoimmune diseases (eg, SLE, rheumatoid arthritis).  iron,  TIBC,  ferritin. Normocytic, but can become microcytic. Treatment: address underlying cause of inflammation, judicious use of blood transfusion, consider erythropoiesisstimulating agents such as EPO (eg, in chronic kidney disease).

1	Aplastic anemia Caused by failure or destruction of hematopoietic stem cells due to: • Radiation and drugs (eg, benzene, chloramphenicol, alkylating agents, antimetabolites) Viral agents (eg, EBV, HIV, hepatitis viruses) Idiopathic (immune mediated, 1° stem cell defect); may follow acute hepatitis  reticulocyte count, • EPO. Pancytopenia characterized by anemia, leukopenia, and thrombocytopenia (not to be confused with aplastic crisis, which causes anemia only). Normal cell morphology, but hypocellular bone marrow with fatty infiltration A (dry bone marrow tap). Symptoms: fatigue, malaise, pallor, purpura, mucosal bleeding, petechiae, infection. Treatment: withdrawal of offending agent, immunosuppressive regimens (eg, antithymocyte globulin, cyclosporine), bone marrow allograft, RBC/platelet transfusion, bone marrow stimulation (eg, GM-CSF).  valine). Mutant HbA is termed HbS. Causes extravascular and intravascular hemolysis.

1	 valine). Mutant HbA is termed HbS. Causes extravascular and intravascular hemolysis. Pathogenesis: low O2, high altitude, or acidosis precipitates sickling (deoxygenated HbS polymerizes)  anemia, vaso-occlusive disease. Newborns are initially asymptomatic because of  HbF and  HbS. Heterozygotes (sickle cell trait) have resistance to malaria. 8% of African Americans carry an HbS allele. Sickle cells are crescent-shaped RBCs A . “Crew cut” on skull x-ray due to marrow expansion from  erythropoiesis (also seen in thalassemias). Complications in sickle cell disease: Aplastic crisis (transient arrest of erythropoiesis due to parvovirus B19). Autosplenectomy (Howell-Jolly bodies)  risk of infection by encapsulated organisms (eg, S pneumoniae). Splenic infarct/sequestration crisis. Salmonella osteomyelitis.

1	Autosplenectomy (Howell-Jolly bodies)  risk of infection by encapsulated organisms (eg, S pneumoniae). Splenic infarct/sequestration crisis. Salmonella osteomyelitis. Painful vaso-occlusive crises: dactylitis (painful swelling of hands/feet), priapism, acute chest syndrome (respiratory distress, new pulmonary infiltrates on CXR, common cause of death), avascular necrosis, stroke. papillary necrosis • hematuria. Hb electrophoresis:  HbA,  HbF, • HbS. Treatment: hydroxyurea ( HbF), hydration. A normocytic anemia that is usually idiopathic and Coombs ⊕. Two types: Warm AIHA–chronic anemia in which IgG causes RBC agglutination. Seen in SLE and CLL and with certain drugs (eg, α-methyldopa). “Warm weather is Good.” + complement causes RBC agglutination upon exposure to cold • painful, blue fingers and toes. Seen in CLL, Mycoplasma pneumoniae infections, infectious Mononucleosis. Spherocytes and agglutinated RBCs A on peripheral blood smear.

1	Spherocytes and agglutinated RBCs A on peripheral blood smear. Warm AIHA treatment: steroids, rituximab, splenectomy (if refractory). Cold AIHA treatment: cold avoidance, rituximab. Direct Coombs test—anti-Ig antibody (Coombs reagent) added to patient’s RBCs. RBCs agglutinate if RBCs are coated with Ig. For comparison, Indirect Coombs test—normal RBCs added to patient’s serum. If serum has anti-RBC surface Ig, RBCs agglutinate when Coombs reagent added. aCorticosteroids cause neutrophilia, despite causing eosinopenia and lymphopenia. Corticosteroids  activation of neutrophil adhesion molecules, impairing migration out of the vasculature to sites of inflammation. In contrast, corticosteroids sequester eosinophils in lymph nodes and cause apoptosis of lymphocytes. Neutrophil left shift  neutrophil precursors, such as band cells A left shift is a shift to a more immature cell in and metamyelocytes, in peripheral blood. the maturation process.

1	Neutrophil left shift  neutrophil precursors, such as band cells A left shift is a shift to a more immature cell in and metamyelocytes, in peripheral blood. the maturation process. Usually seen with neutrophilia in the acute response to infection or inflammation. Called leukoerythroblastic reaction when left shift is seen with immature RBCs. Occurs with severe response (eg, fibrosis, tumor taking up space in marrow). Ferrochelatase and Protoporphyrin, ALA ALA dehydratase (blood) Microcytic anemia (basophilic stippling in peripheral smear A , ringed sideroblasts in bone marrow), GI and kidney disease. Children—exposure to lead paint  mental deterioration. Adults—environmental exposure (eg, batteries, ammunition) • headache, memory loss, demyelination (peripheral neuropathy). B . Most common porphyria. Exacerbated with alcohol consumption. Causes: familial, hepatitis C. Treatment: phlebotomy, sun avoidance, antimalarials (eg, hydroxychloroquine).

1	B . Most common porphyria. Exacerbated with alcohol consumption. Causes: familial, hepatitis C. Treatment: phlebotomy, sun avoidance, antimalarials (eg, hydroxychloroquine). Hemophilia A, B, or C A — • Intrinsic pathway coagulation defect ( PTT). A: deficiency of factor VIII; X-linked recessive. B: deficiency of factor IX; X-linked recessive. C: deficiency of factor XI; autosomal recessive. Hemorrhage in hemophilia—hemarthroses (bleeding into joints, eg, knee A ), easy bruising, bleeding after trauma or surgery (eg, dental procedures). Treatment: desmopressin + factor VIII concentrate (A); factor IX concentrate (B); factor XI concentrate (C). Vitamin K deficiency • General coagulation defect. Bleeding time normal.  activity of factors II, VII, IX, X, protein C, protein S. Hereditary thrombosis syndromes leading to hypercoagulability

1	Vitamin K deficiency • General coagulation defect. Bleeding time normal.  activity of factors II, VII, IX, X, protein C, protein S. Hereditary thrombosis syndromes leading to hypercoagulability Blood transfusion risks include infection transmission (low), transfusion reactions, iron overload (may lead to 2° hemochromatosis), hypocalcemia (citrate is a Ca2+ chelator), and hyperkalemia (RBCs may lyse in old blood units). Leukemia Lymphoid or myeloid neoplasm with widespread involvement of bone marrow. Tumor cells are usually found in peripheral blood. Lymphoma Discrete tumor mass arising from lymph nodes. Presentations often blur definitions. Both may present with constitutional (“B”) signs/symptoms: low-grade fever, night sweats, weight loss. Localized, single group of nodes with Multiple lymph nodes involved; extranodal contiguous spread (stage is strongest predictor involvement common; noncontiguous spread. of prognosis). Better prognosis. Worse prognosis.

1	Characterized by Reed-Sternberg cells. Majority involve B cells; a few are of T-cell lineage. Bimodal distribution: young adulthood and Can occur in children and adults. > 55 years; more common in men except for nodular sclerosing type. Associated with EBV. May be associated with autoimmune diseases and viral infections (eg, HIV, EBV, HTLV). Hodgkin lymphoma Contains Reed-Sternberg cells: distinctive tumor giant cells; binucleate or bilobed with the 2 halves as mirror images (“owl eyes” A ). RS cells are CD15+ and CD30+ B-cell origin. 2 owl eyes × 15 = 30.

1	Plasma cell dyscrasias Characterized by monoclonal immunoglobulin (Ig) overproduction due to plasma cell disorder. Labs: serum protein electrophoresis (SPEP) or free light chain (FLC) assay for initial tests (M spike on SPEP represents overproduction of a monoclonal Ig fragment). For urinalysis, use 24-hr urine protein electrophoresis (UPEP) to detect light chain, as routine urine dipstick detects only albumin. Confirm with bone marrow biopsy. Multiple myeloma Overproduction of IgG (55% of cases) > IgA. Clinical features: CRAB Bone lytic lesions (“punched out” on X-ray A ) • Back pain. Peripheral blood smear shows Rouleaux formation B (RBCs stacked like poker chips). Urinalysis shows Ig light chains (Bence Jones proteinuria) with ⊖ urine dipstick. Bone marrow analysis shows > 10% monoclonal plasma cells with clock-face chromatin C and intracytoplasmic inclusions containing IgG. Complications:  infection risk, 1° amyloidosis (AL).

1	Overproduction of IgM (macroglobulinemia because IgM is the largest Ig). Clinical features: Hyperviscosity syndrome: Retinal hemorrhages Bone marrow analysis shows >10% small lymphocytes with IgM-containing vacuoles (lymphoplasmacytic lymphoma). Complication: thrombosis. syndromes hematopoiesis  defects in cell maturation with bilobed (“duet”) nuclei A . Typically seen of nonlymphoid lineages. Caused by de novo after chemotherapy. mutations or environmental exposure (eg, radiation, benzene, chemotherapy). Risk of transformation to AML. Leukemias Unregulated growth and differentiation of WBCs in bone marrow  marrow failure  anemia ( RBCs), infections ( mature WBCs), and hemorrhage ( platelets). Usually presents with  circulating WBCs (malignant leukocytes in blood); rare cases present with normal/• WBCs. Leukemic cell infiltration of liver, spleen, lymph nodes, and skin (leukemia cutis) possible.

1	A B C D E Chronic myeloproliferative disorders Malignant hematopoietic neoplasms with varying impacts on WBCs and myeloid cell lines. tumor cell lysis, most often in lymphomas/ leukemias. Release of K+  hyperkalemia, Arrhythmias, release of PO 3– • hyperphosphatemia, hypocalcemia due to Ca2+ sequestration Seizures, by PO43–.  nucleic acid breakdown tetany  hyperuricemia  acute kidney injury. hydration, allopurinol, rasburicase. injury Hemophagocytic Systemic overactivation of macrophages and cytotoxic T cells  fever, pancytopenia, lymphohistiocytosis hepatosplenomegaly,  serum ferritin levels. Can be inherited or 2° to strong immunologic activation (eg, after EBV infection, malignancy). Bone marrow biopsy shows macrophages phagocytosing marrow elements A .

1	A . Direct thrombin Bivalirudin, Argatroban, Dabigatran (only oral agent in class). inhibitors mechanISm Directly inhibits activity of free and clot-associated thrombin. clInIcal USe Venous thromboembolism, atrial fibrillation. Can be used in HIT, when heparin is BAD for the patient. Does not require lab monitoring. adVeRSe eFFectS Bleeding; can reverse dabigatran with idarucizumab. Consider PCC and/or antifibrinolytics (eg, tranexamic acid) if no reversal agent available. mechanISm Activates antithrombin, which  action of IIa (thrombin) and factor Xa. Short half-life. clInIcal USe Immediate anticoagulation for pulmonary embolism (PE), acute coronary syndrome, MI, deep venous thrombosis (DVT). Used during pregnancy (does not cross placenta). Follow PTT. adVeRSe eFFectS Bleeding, thrombocytopenia (HIT), osteoporosis, drug-drug interactions. For rapid reversal (antidote), use protamine sulfate (positively charged molecule that binds negatively charged heparin).

1	noteS Low-molecular-weight heparins (eg, enoxaparin, dalteparin)—act predominantly on factor Xa. Fondaparinux acts only on factor Xa. Have better bioavailability and 2–4× longer half life than unfractionated heparin; can be administered subcutaneously and without laboratory monitoring. LMWHs undergo renal clearance (vs hepatic clearance of unfractionated heparin) and are contraindicated in renal insufficiency. Not easily reversible. Heparin-induced thrombocytopenia (HIT) type 2—development of IgG antibodies against heparin-bound platelet factor 4 (PF4). Antibody-heparin-PF4 complex activates platelets • thrombosis and thrombocytopenia. Highest risk with unfractionated heparin. HIT type 1 characterized by nonimmunologic milder drop in platelet count, usually asymptomatic. mechanISm Inhibits epoxide reductase, which interferes The EX-PresidenT went to war(farin). with γ-carboxylation of vitamin K–dependent clotting factors II, VII, IX, X, and proteins C,

1	mechanISm Inhibits epoxide reductase, which interferes The EX-PresidenT went to war(farin). with γ-carboxylation of vitamin K–dependent clotting factors II, VII, IX, X, and proteins C, S. Metabolism affected by polymorphisms in the gene for vitamin K epoxide reductase complex (VKORC1). In laboratory assay, has effect on EXtrinsic pathway and  PT. Long half-life. clInIcal USe Chronic anticoagulation (eg, venous thromboembolism prophylaxis, and prevention of stroke in atrial fibrillation). Not used in pregnant women (because warfarin, unlike heparin, crosses placenta). Follow PT/INR. Bleeding, teratogenic, skin/tissue necrosis A , drug-drug interactions.

1	Bleeding, teratogenic, skin/tissue necrosis A , drug-drug interactions. Initial risk of hypercoagulation: protein C has a shorter half-life than factors II and X. Existing protein C depletes before existing factors II and X deplete, and before warfarin can reduce factors II and X production  hypercoagulation. Skin/tissue necrosis within first few days of large doses believed to be due to small vessel microthrombosis. For reversal of warfarin, give vitamin K. For rapid reversal, give fresh frozen plasma (FFP) or PCC. Heparin “bridging”: heparin frequently used when starting warfarin. Heparin’s activation of antithrombin enables anticoagulation during initial, transient hypercoagulable state caused by warfarin. Initial heparin therapy reduces risk of recurrent venous thromboembolism and skin/tissue necrosis. Metabolized by cytochrome P-450. Direct factor Xa inhibitors ApiXaban, rivaroXaban. mechanISm Bind to and directly inhibit factor Xa.

1	Metabolized by cytochrome P-450. Direct factor Xa inhibitors ApiXaban, rivaroXaban. mechanISm Bind to and directly inhibit factor Xa. clInIcal USe Treatment and prophylaxis for DVT and PE; stroke prophylaxis in patients with atrial fibrillation. Oral agents do not usually require coagulation monitoring. adVeRSe eFFectS Bleeding. Reverse with andeXanet alfa. Thrombolytics Alteplase (tPA), reteplase (rPA), streptokinase, tenecteplase (TNK-tPA). mechanISm Directly or indirectly aid conversion of plasminogen to plasmin, which cleaves thrombin and fibrin clots.  PT,  PTT, no change in platelet count. clInIcal USe Early MI, early ischemic stroke, direct thrombolysis of severe PE.

1	clInIcal USe Early MI, early ischemic stroke, direct thrombolysis of severe PE. adVeRSe eFFectS Bleeding. Contraindicated in patients with active bleeding, history of intracranial bleeding, recent surgery, known bleeding diatheses, or severe hypertension. Nonspecific reversal with antifibrinolytics (eg, aminocaproic acid, tranexamic acid), platelet transfusions, and factor corrections (eg, cryoprecipitate, FFP, PCC). ADP receptor inhibitors Clopidogrel, prasugrel, ticagrelor (reversible), ticlopidine. mechanISm Irreversibly block ADP (P2Y12) receptor, which prevents subsequent platelet aggregation. Prevent expression of glycoproteins IIb/IIIa on platelet surface. clInIcal USe Acute coronary syndrome; coronary stenting.  incidence or recurrence of thrombotic stroke. adVeRSe eFFectS Neutropenia (ticlopidine). TTP may be seen. Glycoprotein IIb/IIIa inhibitors Abciximab, eptifibatide, tirofiban.

1	adVeRSe eFFectS Neutropenia (ticlopidine). TTP may be seen. Glycoprotein IIb/IIIa inhibitors Abciximab, eptifibatide, tirofiban. mechanISm Bind to the glycoprotein receptor IIb/IIIa (fibrinogen receptor) on activated platelets, preventing aggregation. Abciximab is made from monoclonal antibody Fab fragments. clInIcal USe Unstable angina, percutaneous coronary intervention. adVeRSe eFFectS Bleeding, thrombocytopenia. Platinum agents (eg, cisplatin) Alkylating agents:Azathioprine Ifosfamide 5-fuorouracil Rb, p53 modulate Nitrosoureas (eg, carmustine) Hydroxyurea G restriction point Methotrexate 1 6-mercaptopurine

1	Alkylating agents:Azathioprine Ifosfamide 5-fuorouracil Rb, p53 modulate Nitrosoureas (eg, carmustine) Hydroxyurea G restriction point Methotrexate 1 6-mercaptopurine Nucleotide synthesis DNA RNA Cellular division MTX, 5-FU: ˜thymidine synthesis 6-MP: ˜de novo purine synthesis Hydroxyurea: inhibits ribonucleotide reductase Alkylating agents, platinum agents: cross-link DNA Bleomycin: DNA strand breakage Dactinomycin, doxorubicin: DNA intercalators Etoposide/teniposide: inhibits topoisomerase II Irinotecan/topotecan: inhibits topoisomerase I Vinca alkaloids: inhibit microtubule formation Paclitaxel: inhibits microtubule disassembly Protein

1	Azathioprine, Purine (thiol) analogs Preventing organ rejection, Myelosuppression; GI, liver 6-mercaptopurine  de novo purine synthesis. rheumatoid arthritis, IBD, toxicity. Activated by HGPRT. SLE; used to wean patients Azathioprine and 6-MP are Azathioprine is metabolized off steroids in chronic disease metabolized by xanthine into 6-MP. and to treat steroid-refractory oxidase; thus both have  risk chronic disease. of toxicity with allopurinol or febuxostat. Cladribine Purine analog • multiple Hairy cell leukemia. Myelosuppression, mechanisms (eg, inhibition nephrotoxicity, and of DNA polymerase, DNA neurotoxicity. strand breaks). Cytarabine Pyrimidine analog  DNA Leukemias (AML), lymphomas. Myelosuppression with (arabinofuranosyl chain termination. At higher megaloblastic anemia. cytidine) concentrations, inhibits DNA CYTarabine causes polymerase. panCYTopenia.

1	5-fluorouracil Pyrimidine analog bioactivated to 5-FdUMP, which covalently complexes with thymidylate synthase and folic acid. Capecitabine is a prodrug. This complex inhibits thymidylate synthase  dTMP  DNA synthesis. Colon cancer, pancreatic cancer, actinic keratosis, basal cell carcinoma (topical). Effects enhanced with the addition of leucovorin. Myelosuppression, palmarplantar erythrodysesthesia (hand-foot syndrome). Methotrexate Folic acid analog that competitively inhibits dihydrofolate reductase  dTMP  DNA synthesis. Cancers: leukemias (ALL), lymphomas, choriocarcinoma, sarcomas. Non-neoplastic: ectopic pregnancy, medical abortion (with misoprostol), rheumatoid arthritis, psoriasis, IBD, vasculitis. Myelosuppression, which is reversible with leucovorin (folinic acid) “rescue.” Hepatotoxicity. Mucositis (eg, mouth ulcers). Pulmonary fibrosis. Folate deficiency, which may be teratogenic (neural tube defects) without supplementation. Nephrotoxicity.

1	Hepatotoxicity. Mucositis (eg, mouth ulcers). Pulmonary fibrosis. Folate deficiency, which may be teratogenic (neural tube defects) without supplementation. Nephrotoxicity. aAll are S-phase specific except cladribine, which is cell cycle nonspecific. Cyclophosphamide, ifosfamide Myelosuppression; SIADH; Fanconi syndrome (ifosfamide); hemorrhagic cystitis and bladder cancer, prevented with mesna (sulfhydryl group of mesna binds toxic metabolites) and adequate hydration. Microtubule inhibitors Cisplatin, carboplatin, oxaliplatin mechanISm Cross-link DNA. clInIcal USe Testicular, bladder, ovary, GI, and lung carcinomas. adVeRSe eFFectS Nephrotoxicity (including Fanconi syndrome), peripheral neuropathy, ototoxicity. Prevent nephrotoxicity with amifostine (free radical scavenger) and chloride (saline) diuresis. Etoposide, teniposide mechanISm Inhibit topoisomerase II  DNA degradation (cell cycle arrest in G2 and S phases).

1	Etoposide, teniposide mechanISm Inhibit topoisomerase II  DNA degradation (cell cycle arrest in G2 and S phases). clInIcal USe Solid tumors (particularly testicular and small cell lung cancer), leukemias, lymphomas. adVeRSe eFFectS Myelosuppression, alopecia. Irinotecan, topotecan mechanISm Inhibit topoisomerase I and prevent DNA unwinding and replication. clInIcal USe Colon cancer (irinotecan); ovarian and small cell lung cancers (topotecan). adVeRSe eFFectS Severe myelosuppression, diarrhea. mechanISm Inhibits ribonucleotide reductase  DNA Synthesis (S-phase specific). clInIcal USe Myeloproliferative disorders (eg, CML, polycythemia vera), sickle cell disease (• HbF). adVeRSe eFFectS Severe myelosuppression, megaloblastic anemia. mechanISm Monoclonal antibody against VEGF. Inhibits angiogenesis (BeVacizumab inhibits Blood Vessel formation). clInIcal USe Solid tumors (eg, colorectal cancer, renal cell carcinoma), wet age-related macular degeneration.

1	clInIcal USe Solid tumors (eg, colorectal cancer, renal cell carcinoma), wet age-related macular degeneration. adVeRSe eFFectS Hemorrhage, blood clots, and impaired wound healing. mechanISm EGFR tyrosine kinase inhibitor. clInIcal USe Non-small cell lung cancer. adVeRSe eFFectS Rash, diarrhea. Cetuximab, panitumumab mechanISm Monoclonal antibodies against EGFR. clInIcal USe Stage IV colorectal cancer (wild-type KRAS), head and neck cancer. adVeRSe eFFectS Rash, elevated LFTs, diarrhea. Imatinib, dasatinib, nilotinib mechanISm Tyrosine kinase inhibitors of bcr-abl (encoded by Philadelphia chromosome fusion gene in CML) and c-kit (common in GI stromal tumors). clInIcal USe CML, GI stromal tumors (GISTs). adVeRSe eFFectS Fluid retention. mechanISm Monoclonal antibody against CD20, which is found on most B-cell neoplasms. clInIcal USe Non-Hodgkin lymphoma, CLL, ITP, rheumatoid arthritis, TTP, AIHA. adVeRSe eFFectS  risk of progressive multifocal leukoencephalopathy.

1	clInIcal USe Non-Hodgkin lymphoma, CLL, ITP, rheumatoid arthritis, TTP, AIHA. adVeRSe eFFectS  risk of progressive multifocal leukoencephalopathy. Bortezomib, carfilzomib mechanISm Proteasome inhibitors, induce arrest at G2-M phase and apoptosis. clInIcal USe Multiple myeloma, mantle cell lymphoma. adVeRSe eFFectS Peripheral neuropathy, herpes zoster reactivation. Tamoxifen, raloxifene mechanISm Selective estrogen receptor modulators (SERMs)—receptor antagonists in breast and agonists in bone. Block the binding of estrogen to ER ⊕ cells. clInIcal USe Breast cancer treatment (tamoxifen only) and prevention. Raloxifene also useful to prevent osteoporosis.

1	clInIcal USe Breast cancer treatment (tamoxifen only) and prevention. Raloxifene also useful to prevent osteoporosis. adVeRSe eFFectS Tamoxifen—partial agonist in endometrium, which  the risk of endometrial cancer. Raloxifene—no  in endometrial carcinoma (so you can relax!), because it is an estrogen receptor antagonist in endometrial tissue. Both  risk of thromboembolic events (eg, DVT, PE) and “hot flashes.” mechanISm Monoclonal antibody against HER-2 (c-erbB2), a tyrosine kinase receptor. Helps kill cancer cells that overexpress HER-2 through inhibition of HER-2 initiated cellular signaling and antibody-dependent cytotoxicity. clInIcal USe HER-2 ⊕ breast cancer and gastric cancer (tras2zumab). adVeRSe eFFectS Dilated cardiomyopathy. “Heartceptin” damages the heart. Dabrafenib, vemurafenib mechanISm Recombinant uricase that catalyzes metabolism of uric acid to allantoin. clInIcal USe Prevention and treatment of tumor lysis syndrome. Bleomycin, Busulfan

1	Dabrafenib, vemurafenib mechanISm Recombinant uricase that catalyzes metabolism of uric acid to allantoin. clInIcal USe Prevention and treatment of tumor lysis syndrome. Bleomycin, Busulfan Nonspecific common toxicities of nearly all cytotoxic chemotherapies include myelosuppression (neutropenia, anemia, thrombocytopenia), GI toxicity (nausea, vomiting, mucositis), alopecia. Musculoskeletal, Skin, and Connective Tissue “Rigid, the skeleton of habit alone upholds the human frame.” “Beauty may be skin deep, but ugly goes clear to the bone.” “The function of muscle is to pull and not to push, except in the case of the genitals and the tongue.” “To thrive in life you need three bones. A wishbone. A backbone. And a funny bone.”

1	This chapter provides information you will need to understand certain anatomical dysfunctions, rheumatic diseases, and dermatologic conditions. Be able to interpret 3D anatomy in the context of radiologic imaging. For the rheumatic diseases, create instructional cases or personas that include the most likely presentation and symptoms: risk factors, gender, important markers (eg, autoantibodies), and other epidemiologic factors. Doing so will allow you to answer the higher order questions that are likely to be asked on the exam. `mUsCUlosKElEtal, sKin, and ConnECtiVE tissUE—anatomy and physiology Rotator cuff muscles Shoulder muscles that form the rotator cuff: SItS (small t is for teres minor). Supraspinatus (suprascapular nerve)— abducts arm initially (before the action of the deltoid); most common rotator cuff injury (trauma or degeneration and impingement • tendinopathy or tear [arrow in

1	A ]), assessed by “empty/full can” test nerves)—internally rotates and adducts arm Innervated primarily by C5-C6. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 447 Axillary (C5-C6) Fractured surgical neck of humerus Flattened deltoid Anterior dislocation of humerus Loss of arm abduction at shoulder (> 15°) Loss of sensation over deltoid and lateral arm Musculocutaneous Upper trunk compression  biceps (C5-6) reflex (C5-C7) Weakness of forearm flexion and supination Loss of sensation over lateral forearm Radial (C5-T1) Compression of axilla, eg, due to crutches or Wrist drop: loss of elbow, wrist, and finger sleeping with arm over chair (“Saturday night extension palsy”)  grip strength (wrist extension necessary for Midshaft fracture of humerus maximal action of flexors) Repetitive pronation/supination of forearm, eg, Loss of sensation over posterior arm/forearm and due to screwdriver use (“finger drop”) dorsal hand

1	Median (C5-T1) Supracondylar fracture of humerus  proximal “Ape hand” and “Pope’s blessing” lesion of the nerve Loss of wrist flexion, flexion of lateral fingers, Carpal tunnel syndrome and wrist laceration thumb opposition, lumbricals of index and  distal lesion of the nerve middle fingers Loss of sensation over thenar eminence and dorsal and palmar aspects of lateral 31⁄2 fingers with proximal lesion Ulnar (C8-T1) Fracture of medial epicondyle of humerus “funny bone” (proximal lesion) Fractured hook of hamate (distal lesion) from fall on outstretched hand Radial deviation of wrist upon flexion (proximal lesion) Loss of wrist flexion, flexion of medial fingers, abduction and adduction of fingers (interossei), actions of medial 2 lumbrical muscles Loss of sensation over medial 11/2 fingers including hypothenar eminence Humerus fractures, proximally to distally, follow the ARM (Axillary • Radial • Median)

1	Loss of sensation over medial 11/2 fingers including hypothenar eminence Humerus fractures, proximally to distally, follow the ARM (Axillary • Radial • Median) MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 448 Erb palsy (“waiter’s tip”) Klumpke palsy (claw hand) Wrist drop Winged scapula Deltoid paralysis “Saturday night palsy” (wrist drop) Di⁄culty fexing elbow, variable sensory loss Decreased thumb function, “Pope’s blessing” Intrinsic muscles of hand, claw hand Erb palsy (“waiter’s Traction or tear Infants—lateral Deltoid, Abduction (arm tip”) of upper trunk: traction on neck supraspinatus hangs by side) C5-C6 roots during delivery medially rotated) Biceps brachii Flexion, supination Herb gets DIBs (arm extended and on tips pronated) Klumpke palsy Traction or tear Infants—upward Intrinsic hand Total claw hand: of lower trunk: force on arm muscles: lumbricals normally C8-T1 roots during delivery lumbricals, flex MCP joints and

1	Klumpke palsy Traction or tear Infants—upward Intrinsic hand Total claw hand: of lower trunk: force on arm muscles: lumbricals normally C8-T1 roots during delivery lumbricals, flex MCP joints and Adults—trauma interossei, extend DIP and PIP (eg, grabbing a thenar, joints tree branch to hypothenar break a fall) Thoracic outlet Compression Cervical rib Same as Klumpke Atrophy of intrinsic syndrome of lower trunk (arrows in A , palsy hand muscles; and subclavian Pancoast tumor ischemia, pain, vessels, most and edema commonly due to vascular within the compression scalene triangle Winged scapula Lesion of long Axillary node Serratus anterior Inability to anchor thoracic nerve, dissection after scapula to thoracic roots C5-C7 mastectomy, cage  cannot (“wings of stab wounds abduct arm heaven”) above horizontal position MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 449

1	MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 449 Wrist region Scaphoid, Lunate, Triquetrum, Pisiform, A Hamate, Capitate, Trapezoid, Trapezium A. (So Long To Pinky, Here Comes The Thumb) 1st MC Hamate is the most commonly fractured carpal bone, Trapezium typically due to a fall on an outstretched hand. Complications of proximal scaphoid fractures include avascular necrosis and nonunion due to retrograde blood supply from a branch of the radial artery. Fracture not always seen on initial x-ray. Dislocation of lunate may cause acute carpal tunnel syndrome. Plane of section MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 450 MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 451

1	Plane of section MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 450 MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 451 Distortions of the hand At rest, a balance exists between the extrinsic flexors and extensors of the hand, as well as the intrinsic muscles of the hand—particularly the lumbrical muscles (flexion of MCP, extension of DIP and PIP joints). “Clawing”—seen best with distal lesions of median or ulnar nerves. Remaining extrinsic flexors of the digits exaggerate the loss of the lumbricals  fingers extend at MCP, flex at DIP and PIP joints. Deficits less pronounced in proximal lesions; deficits present during voluntary flexion of the digits. Note: Atrophy of the thenar eminence (unopposable thumb  “ape hand”) can be seen in median nerve lesions, while atrophy of the hypothenar eminence can be seen in ulnar nerve lesions. Actions of hip muscles

1	Actions of hip muscles MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 452 Obturator (L2-L4) Sensory—medial thigh Motor—obturator externus, adductor longus, adductor brevis, gracilis, pectineus, adductor magnus Pelvic surgery  thigh sensation (medial) and adduction Femoral (L2-L4) Sensory—anterior thigh, medial leg Motor—quadriceps, iliacus, pectineus, sartorius Pelvic fracture  leg extension ( patellar reflex) MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 453 Superficial peroneal nerve: Sensory—dorsum of foot (except webspace between hallux and 2nd digit) and brevis Deep peroneal nerve: Motor—tibialis anterior Sensory—sole of foot Motor—biceps femoris (long head), triceps surae, plantaris, popliteus, flexor muscles of foot Motor—gluteus medius, gluteus minimus, tensor fascia latae Trauma or compression of lateral aspect of leg, fibular neck fracture

1	Motor—gluteus medius, gluteus minimus, tensor fascia latae Trauma or compression of lateral aspect of leg, fibular neck fracture Knee trauma, Baker cyst (proximal lesion); tarsal tunnel syndrome (distal lesion) Iatrogenic injury during intramuscular injection to superomedial gluteal region (prevent by choosing superolateral quadrant, preferably anterolateral region) PED = Peroneal Everts and Dorsiflexes; if injured, foot dropPED Loss of sensation on dorsum of foot Foot drop—inverted and plantarflexed at rest, loss of eversion and dorsiflexion; “steppage gait” TIP = Tibial Inverts and Plantarflexes; if injured, can’t stand on TIPtoes Inability to curl toes and loss of sensation on sole; in proximal lesions, foot everted at rest with loss of inversion and plantar flexion Trendelenburg sign/gait— pelvis tilts because weight-bearing leg cannot maintain alignment of pelvis through hip abduction

1	Trendelenburg sign/gait— pelvis tilts because weight-bearing leg cannot maintain alignment of pelvis through hip abduction Lesion is contralateral to the side of the hip that drops, ipsilateral to extremity on which the patient stands MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 454 Knee exam Lateral femoral condyle to anterior tibia: ACL. Medial femoral condyle to posterior tibia: PCL. LAMP. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 455 Ankle sprains Anterior TaloFibular ligament—most common ankle sprain overall, classified as a low ankle sprain. Due to overinversion/supination of foot. Anterior inferior tibiofibular ligament—most common high ankle sprain. Always Tears First. Neurovascular pairing Nerves and arteries are frequently named together by the bones/regions with which they are associated. The following are exceptions to this naming convention.

1	Neurovascular pairing Nerves and arteries are frequently named together by the bones/regions with which they are associated. The following are exceptions to this naming convention. Surgical neck of humerus Axillary Posterior circumflex MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 456 Motoneuron action T-tubules are extensions of plasma membrane in contact with the sarcoplasmic reticulum, allowing potential to muscle for coordinated contraction of striated muscles. contraction

1	Action potential opens presynaptic voltage-gated Ca2+ channels, inducing acetylcholine (ACh) release. Postsynaptic ACh binding leads to muscle cell depolarization at the motor end plate. Depolarization travels over the entire muscle cell and deep into the muscle via the T-tubules. Membrane depolarization induces conformational changes in the voltage-sensitive dihydropyridine receptor (DHPR) and its mechanically coupled ryanodine receptor (RR) • Ca2+ release from the sarcoplasmic reticulum into the cytoplasm. Tropomyosin is blocking myosin-binding sites on the actin filament. Released Ca2+ binds to troponin C (TnC), shifting tropomyosin to expose the myosin-binding sites. The myosin head binds strongly to actin, forming a crossbridge. Pi is then released, initiating the power stroke. During the power stroke, force is produced as myosin pulls on the thin filament

1	A . Muscle shortening occurs, with shortening of H and I bands and between Z lines (HIZ shrinkage). The A band remains the same length (A band is Always the same length). ADP is released at the end of the power stroke. Binding of new ATP molecule causes detachment of myosin head from actin filament. Ca2+ is resequestered. ATP hydrolysis into ADP and Pi results in myosin head returning to high-energy position (cocked). The myosin head can bind to a new site on actin to form a crossbridge if Ca2+ remains available. Sarcomere (Z line to Z line) MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 457 Types of muscle fibers Acetylcholine, bradykinin, etc MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 458 Muscle proprioceptors Specialized sensory receptors that relay information about muscle dynamics.

1	MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` anatomy and physiology SECTION III 458 Muscle proprioceptors Specialized sensory receptors that relay information about muscle dynamics. Membranous Bones of calvarium, facial bones, and clavicle. Woven bone formed directly without cartilage. Later ossification remodeled to lamellar bone. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 459 Cell biology of bone Osteoblast Builds bone by secreting collagen and catalyzing mineralization in alkaline environment via ALP. Differentiates from mesenchymal stem cells in periosteum. Osteoblastic activity measured by bone ALP, osteocalcin, propeptides of type I procollagen.

1	Osteoclast Dissolves (“crushes”) bone by secreting H+ and collagenases. Differentiates from a fusion of monocyte/macrophage lineage precursors. RANK receptors on osteoclasts are stimulated by RANKL (RANK ligand, expressed on osteoblasts). OPG (osteoprotegerin, a RANKL decoy receptor) binds RANKL to prevent RANK-RANKL interaction  osteoclast activity. `mUsCUlosKElEtal, sKin, and ConnECtiVE tissUE—pathology Overuse injuries of the elbow Metacarpal neck Also called boxer’s fracture. Common fracture fracture caused by direct blow with a closed fist (eg, from punching a wall). Most commonly seen in 4th and 5th metacarpals A . Entrapment of median nerve in carpal tunnel (between transverse carpal ligament and carpal bones)  nerve compression  paresthesia, pain, and numbness in distribution of median nerve. Thenar eminence atrophies B but sensation spared, because palmar cutaneous branch enters hand external to carpal tunnel.

1	B but sensation spared, because palmar cutaneous branch enters hand external to carpal tunnel. Suggested by ⊕ Tinel sign (percussion of wrist causes tingling) and Phalen maneuver (90° flexion of wrist causes tingling). Associated with pregnancy (due to edema), rheumatoid arthritis, hypothyroidism, diabetes, acromegaly, dialysis-related amyloidosis; may be associated with repetitive use. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 460 Clavicle fractures Common in children and as birth trauma. Usually caused by a fall on outstretched hand or by direct trauma to shoulder. Weakest point at the junction of middle and lateral thirds; fractures at the middle third segment are most common. Presents as shoulder drop, shortened clavicle (lateral fragment is depressed due to arm weight and medially rotated by arm adductors [eg, pectoralis major]).

1	“Unhappy triad” Common injury in contact sports due to lateral force applied to a planted foot. Consists of damage to the ACL A , MCL, and medial meniscus (attached to MCL). However, lateral meniscus involvement is more common than with ACL and MCL injury. Presents with acute pain and signs of joint instability. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 461 Plantar fasciitis Inflammation of plantar aponeurosis characterized by heel pain (worse with first steps in the morning or after period of inactivity) and tenderness. Developmental Abnormal acetabulum development in newborns. Major risk factor includes breech presentation. dysplasia of the hip Results in hip instability/dislocation. Commonly tested with Ortolani and Barlow maneuvers (manipulation of newborn hip reveals a “clunk”). Confirmed via ultrasound (x-ray not used until ~4–6 months because cartilage is not ossified).

1	Legg-Calvé-Perthes Idiopathic avascular necrosis of femoral head. Commonly presents between 5–7 years with disease insidious onset of hip pain that may cause child to limp. More common in males (4:1 ratio). Initial x-ray often normal. Osgood-Schlatter Also called traction apophysitis. Overuse injury caused by repetitive strain and chronic avulsion of disease the secondary ossification center of proximal tibial tubercle. Occurs in adolescents after growth spurt. Common in running and jumping athletes. Presents with progressive anterior knee pain. 462 SECTION III MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology

1	462 SECTION III MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology Achondroplasia Failure of longitudinal bone growth (endochondral ossification)  short limbs. Membranous ossification is not affected  large head relative to limbs. Constitutive activation of fibroblast growth factor receptor (FGFR3) actually inhibits chondrocyte proliferation. > 85% of mutations occur sporadically; autosomal dominant with full penetrance (homozygosity is lethal). Associated with  paternal age. Most common cause of short-limbed dwarfism. Trabecular (spongy) and cortical bone lose mass despite normal bone mineralization and lab values (serum Ca2+ and PO43−). Most commonly due to  bone resorption related to  estrogen levels and old age. Can be 2° to drugs (eg, steroids, alcohol, anticonvulsants, anticoagulants, thyroid replacement therapy) or other conditions (eg, hyperparathyroidism, hyperthyroidism, multiple myeloma, malabsorption syndromes, anorexia).

1	Diagnosed by bone mineral density measurement by DEXA (dual-energy X-ray absorptiometry) at the lumbar spine, total hip, and femoral neck, with a T-score of ≤−2.5 or by a fragility fracture (eg, fall from standing height, minimal trauma) at hip or vertebra. One time screening recommended in women ≥ 65 years old. Prophylaxis: regular weight-bearing exercise and adequate Ca2+ and vitamin D intake throughout adulthood. Treatment: bisphosphonates, teriparatide, SERMs, rarely calcitonin; denosumab (monoclonal antibody against RANKL). Can lead to vertebral compression fractures A —acute back pain, loss of height, kyphosis. Also can present with fractures of femoral neck, distal radius (Colles fracture). Central expansion Restricted of intervertebral intervertebral disc foramen MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 463

1	Central expansion Restricted of intervertebral intervertebral disc foramen MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 463 Osteopetrosis Failure of normal bone resorption due to defective osteoclasts  thickened, dense bones that are prone to fracture. Mutations (eg, carbonic anhydrase II) impair ability of osteoclast to generate acidic environment necessary for bone resorption. Overgrowth of cortical bone fills marrow space  pancytopenia, extramedullary hematopoiesis. Can result in cranial nerve impingement and palsies due to narrowed foramina. X-rays show diffuse symmetric sclerosis (bone-in-bone, “stone bone” A ). Bone marrow transplant is potentially curative as osteoclasts are derived from monocytes. Defective mineralization of osteoid (osteomalacia) or cartilaginous growth plates (rickets, only in children). Most commonly due to vitamin D deficiency.

1	Defective mineralization of osteoid (osteomalacia) or cartilaginous growth plates (rickets, only in children). Most commonly due to vitamin D deficiency. X-rays show osteopenia and “Looser zones” (pseudofractures) in osteomalacia, epiphyseal widening and metaphyseal cupping/fraying in rickets. Children with rickets have pathologic bow legs (genu varum A ), bead-like costochondral junctions (rachitic rosary B ), craniotabes (soft skull).  serum PO43− . Hyperactivity of osteoblasts  ALP. Also called Paget disease of bone. Common, localized disorder of bone remodeling caused by  osteoclastic activity followed by  osteoblastic activity that forms poor-quality bone. Serum Ca2+ , phosphorus, and PTH levels are normal.  ALP. Mosaic pattern of woven and lamellar bone (osteocytes within lacunae in chaotic juxtapositions); long bone chalk-stick fractures.  blood flow from • arteriovenous shunts may cause high-output heart failure.  risk of osteosarcoma.

1	Hat size can be increased due to skull thickening A ; hearing loss is common due to auditory foramen narrowing. Stages of Paget disease: activity Treatment: bisphosphonates. Avascular necrosis of bone Infarction of bone and marrow, usually very painful. Most common site is femoral head (watershed zone) A (due to insufficiency of medial circumflex femoral artery). Causes include Corticosteroids, Alcoholism, Sickle cell disease, Trauma, SLE, “the Bends” (caisson/decompression disease), LEgg-Calvé-Perthes disease (idiopathic), Gaucher disease, Slipped capital femoral epiphysis—CASTS Bend LEGS. Branch of obturator artery MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 464 MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 465 Anaplastic small blue cells of neuroectodermal origin (resemble lymphocytes) F .

1	MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 465 Anaplastic small blue cells of neuroectodermal origin (resemble lymphocytes) F . Differentiate from conditions with similar morphology (eg, lymphoma, chronic osteomyelitis) by testing for t(11;22) (fusion protein EWS-FLI1). “Onion skin” periosteal reaction in bone. Aggressive with early metastases, but responsive to chemotherapy. 11 + 22 = 33 (Patrick Ewing’s jersey number). MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 466 Joint Findings Osteophytes (bone spurs), joint space narrowing, subchondral sclerosis and cysts. Synovial fluid noninflammatory (WBC < 2000/mm3). Development of Heberden nodes B (at DIP) and Bouchard nodes C (at PIP), and 1st CMC; not MCP. Erosions, juxta-articular osteopenia, soft tissue swelling, subchondral cysts, joint space narrowing. Deformities: cervical subluxation, ulnar finger deviation, swan neck

1	Erosions, juxta-articular osteopenia, soft tissue swelling, subchondral cysts, joint space narrowing. Deformities: cervical subluxation, ulnar finger deviation, swan neck D , boutonniere E . Involves MCP, PIP, wrist; not DIP or 1st CMC. *Extraarticular manifestations include rheumatoid nodules (fibrinoid necrosis with palisading histiocytes) in subcutaneous tissue and lung (+ pneumoconiosis • Caplan syndrome), interstitial lung disease, pleuritis, pericarditis, anemia of chronic disease, neutropenia + splenomegaly (Felty syndrome), AA amyloidosis, Sjögren syndrome, scleritis, carpal tunnel syndrome. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 467 Findings Acute inflammatory monoarthritis caused by precipitation of monosodium urate crystals in joints A . Risk factors: male sex, hypertension, obesity, diabetes, dyslipidemia, alcohol use. Strongest risk factor is hyperuricemia, which can be caused by:

1	Underexcretion of uric acid (90% of patients)—largely idiopathic, potentiated by renal failure; can be exacerbated by certain medications (eg, thiazide diuretics). Overproduction of uric acid (10% of patients)—Lesch-Nyhan syndrome, PRPP excess,  cell turnover (eg, tumor lysis syndrome), von Gierke disease. Crystals are needle shaped and ⊝ birefringent under polarized light (yellow under parallel light, blue under perpendicular light B ). Serum uric acid levels may be normal during an acute attack.

1	Crystals are needle shaped and ⊝ birefringent under polarized light (yellow under parallel light, blue under perpendicular light B ). Serum uric acid levels may be normal during an acute attack. symptoms Asymmetric joint distribution. Joint is swollen, red, and painful. Classic manifestation is painful MTP joint of big toe (podagra). Tophus formation C (often on external ear, olecranon bursa, or Achilles tendon). Acute attack tends to occur after a large meal with foods rich in purines (eg, red meat, seafood), trauma, surgery, dehydration, diuresis, or alcohol consumption (alcohol metabolites compete for same excretion sites in kidney as uric acid  uric acid secretion and subsequent buildup in blood). tREatmEnt Acute: NSAIDs (eg, indomethacin), glucocorticoids, colchicine. Chronic (preventive): xanthine oxidase inhibitors (eg, allopurinol, febuxostat).

1	tREatmEnt Acute: NSAIDs (eg, indomethacin), glucocorticoids, colchicine. Chronic (preventive): xanthine oxidase inhibitors (eg, allopurinol, febuxostat). Previously called pseudogout. Deposition of The blue P’s—blue (when Parallel), Positive calcium pyrophosphate crystals within the birefringence, calcium Pyrophosphate, joint space. Occurs in patients > 50 years old; Pseudogout both sexes affected equally. Usually idiopathic, sometimes associated with hemochromatosis, hyperparathyroidism, joint trauma. Pain and swelling with acute inflammation (pseudogout) and/or chronic degeneration (pseudo-osteoarthritis). Most commonly affected joint is the knee. Chondrocalcinosis (cartilage calcification) on x-ray. Crystals are rhomboid and weakly ⊕ birefringent under polarized light (blue when parallel to light) A . Acute treatment: NSAIDs, colchicine, glucocorticoids. Prophylaxis: colchicine. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 468

1	A . Acute treatment: NSAIDs, colchicine, glucocorticoids. Prophylaxis: colchicine. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 468 Septic arthritis S aureus, Streptococcus, and Neisseria gonorrhoeae are common causes. Affected joint is swollen A , red, and painful. Synovial fluid purulent (WBC > 50,000/mm3). Gonococcal arthritis—STI that presents as either purulent arthritis (eg, knee) or triad of polyarthralgia, tenosynovitis (eg, hand), dermatitis (eg, pustules). MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 469 MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 470 Libman-Sacks Endocarditis—nonbacterial, verrucous thrombi usually on mitral or aortic valve and can be present on either surface of the valve (but usually on undersurface). LSE in SLE.

1	Libman-Sacks Endocarditis—nonbacterial, verrucous thrombi usually on mitral or aortic valve and can be present on either surface of the valve (but usually on undersurface). LSE in SLE. Lupus nephritis (glomerular deposition of DNA-anti-DNA immune complexes) can be nephritic or nephrotic (causing hematuria or proteinuria). Most common and severe type is diffuse proliferative. Common causes of death in SLE: Renal disease (most common), Infections, Cardiovascular disease (accelerated CAD). In an anti-SSA ⊕ pregnant woman,  risk of newborn developing neonatal lupus  congenital heart block, periorbital/diffuse rash, transaminitis, and cytopenias at birth. RASH OR PAIN: Rash (malar

1	In an anti-SSA ⊕ pregnant woman,  risk of newborn developing neonatal lupus  congenital heart block, periorbital/diffuse rash, transaminitis, and cytopenias at birth. RASH OR PAIN: Rash (malar A or discoid B ) Arthritis (nonerosive) Serositis (eg, pleuritis, pericarditis) Hematologic disorders (eg, cytopenias) Oral/nasopharyngeal ulcers (usually painless) Renal disease Photosensitivity Antinuclear antibodies Immunologic disorder (anti-dsDNA, anti-Sm, antiphospholipid) Neurologic disorders (eg, seizures, psychosis) Lupus patients die with Redness In their Cheeks. Mixed connective Features of SLE, systemic sclerosis, and/or tissue disease polymyositis. Associated with anti-U1 RNP antibodies (speckled ANA). 1° or 2° autoimmune disorder (most commonly in SLE). Diagnosed based on clinical criteria including history of thrombosis (arterial or venous) or spontaneous abortion along with laboratory findings of lupus anticoagulant, anticardiolipin, anti-β2 glycoprotein I antibodies.

1	Treatment: systemic anticoagulation. Anticardiolipin antibodies can cause false-positive VDRL/RPR. Lupus anticoagulant can cause prolonged PTT that is not corrected by the addition of normal platelet-free plasma. symptoms Pain and stiffness in proximal muscles (eg, shoulders, hips), often with fever, malaise, weight loss. Does not cause muscular weakness. More common in women > 50 years old; associated with giant cell (temporal) arteritis. Findings  ESR,  CRP, normal CK. tREatmEnt Rapid response to low-dose corticosteroids. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 471 MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 472

1	MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 471 MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` pathology SECTION III 472 Raynaud phenomenon  blood flow to skin due to arteriolar (small vessel) vasospasm in response to cold or stress: color change from white (ischemia) to blue (hypoxia) to red (reperfusion). Most often in the fingers A and toes. Called Raynaud disease when 1° (idiopathic), Raynaud syndrome when 2° to a disease process such as mixed connective tissue disease, SLE, or CREST syndrome (limited form of systemic sclerosis). Digital ulceration (critical ischemia) seen in 2° Raynaud syndrome. Treat with calcium2+ channel blockers. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` dERmatology SECTION III 473

1	MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` dERmatology SECTION III 473 Scleroderma Systemic sclerosis. Triad of autoimmunity, noninflammatory vasculopathy, and collagen deposition with fibrosis. Commonly sclerosis of skin, manifesting as puffy, taut skin A without wrinkles, fingertip pitting B . Can involve other systems, eg, renal (scleroderma renal crisis; treat with ACE inhibitors), pulmonary (interstitial fibrosis, pulmonary HTN), GI (esophageal dysmotility and reflux), cardiovascular. 75% female. 2 major types: Diffuse scleroderma—widespread skin involvement, rapid progression, early visceral involvement. Associated with anti-Scl-70 antibody (anti-DNA topoisomerase-I antibody) and anti-RNA polymerase III. Limited scleroderma—limited skin involvement confined to fingers and face. Also with CREST syndrome: Calcinosis cutis C , anti-Centromere antibody, Raynaud phenomenon, Esophageal dysmotility, Sclerodactyly, and Telangiectasia. More benign clinical course.

1	` mUsCUlosKElEtal, sKin,and ConnECtiVEtissUE—dERmatology Skin layers Skin has 3 layers: epidermis, dermis, subcutaneous fat (hypodermis, subcutis). Epidermal layers: Come, Let’s Get Sun Burned. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` dERmatology SECTION III 474 Tight junctions (zonula occludens) A –prevents paracellular movement of solutes; composed of claudins and occludins. Adherens junction (belt desmosome, zonula adherens) B –forms “belt” connecting actin cytoskeletons of adjacent cells with CADherins (Ca2+-dependent adhesion proteins). Loss of E-cadherin promotes metastasis. Desmosome (spot desmosome, macula adherens) C –structural support via intermediate filament interactions. Autoantibodies to desmoglein 1 and/or 3 • pemphigus vulgaris. D –channel proteins called connexons permit electrical and chemical communication between cells.

1	D –channel proteins called connexons permit electrical and chemical communication between cells. E –connects keratin in basal cells to underlying basement membrane. Autoantibodies • bullous pemphigoid. (Hemidesmosomes are down “bullow.”) Integrins–membrane proteins that maintain integrity of basolateral membrane by binding to collagen, laminin, and fibronectin in basement membrane. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` dERmatology SECTION III 475 MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` dERmatology SECTION III 476 Seborrheic dermatitis Erythematous, well-demarcated plaques A with greasy yellow scales in areas rich in sebaceous glands, such as scalp, face, and periocular region. Common in both infants (cradle cap) and adults, associated with Parkinson disease. Sebaceous glands are not inflamed, but play a role in disease development. Possibly associated with Malassezia spp. Treatment: topical antifungals and corticosteroids.

1	MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` dERmatology SECTION III 477 Acne Multifactorial etiology—• sebum/androgen production, abnormal keratinocyte desquamation, Cutibacterium acnes colonization of the pilosebaceous unit (comedones), and inflammation (papules/pustules A , nodules, cysts). Treatment: retinoids, benzoyl peroxide, and antibiotics. Pseudofolliculitis Foreign body inflammatory facial skin disorder characterized by firm, hyperpigmented papules and barbae pustules that are painful and pruritic. Located on cheeks, jawline, and neck. Commonly occurs as a result of shaving (“razor bumps”), primarily affects African-American males.

1	Psoriasis Papules and plaques with silvery scaling H , especially on knees and elbows. Acanthosis with parakeratotic scaling (nuclei still in stratum corneum), Munro microabscesses.  stratum spinosum,  stratum granulosum. Auspitz sign ( I )—pinpoint bleeding spots from exposure of dermal papillae when scales are scraped off. Associated with nail pitting and psoriatic arthritis. Rosacea Inflammatory facial skin disorder characterized by erythematous papules and pustules J , but no comedones. May be associated with facial flushing in response to external stimuli (eg, alcohol, heat). Phymatous rosacea can cause rhinophyma (bulbous deformation of nose).

1	Seborrheic keratosis Flat, greasy, pigmented squamous epithelial proliferation of immature keratinocytes with keratin-filled cysts (horn cysts) K . Looks “stuck on.” Lesions occur on head, trunk, and extremities. Common benign neoplasm of older persons. Leser-Trélat sign L —rapid onset of multiple seborrheic keratoses, indicates possible malignancy (eg, GI adenocarcinoma). Verrucae Warts; caused by low-risk HPV strains. Soft, tan-colored, cauliflower-like papules M. Epidermal hyperplasia, hyperkeratosis, koilocytosis. Condyloma acuminatum on anus or genitals N . Urticaria Hives. Pruritic wheals that form after mast cell degranulation O . Characterized by superficial dermal edema and lymphatic channel dilation. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` dERmatology SECTION III 478 Vascular tumors of skin Glomus tumor Benign, painful, red-blue tumor, commonly under fingernails C . Arises from modified smooth muscle cells of the thermoregulatory glomus body.

1	Vascular tumors of skin Glomus tumor Benign, painful, red-blue tumor, commonly under fingernails C . Arises from modified smooth muscle cells of the thermoregulatory glomus body. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` dERmatology SECTION III 479 Cellulitis Acute, painful, spreading infection of deeper dermis and subcutaneous tissues. Usually from S pyogenes or S aureus. Often starts with a break in skin from trauma or another infection D . Varicella zoster virus Causes varicella (chickenpox) and zoster (shingles). Varicella presents with multiple crops of lesions in various stages from vesicles to crusts. Zoster is a reactivation of the virus in dermatomal distribution (unless it is disseminated). Hairy leukoplakia Irregular, white, painless plaques on lateral tongue that cannot be scraped off J . EBV mediated. Occurs in HIV-positive patients, organ transplant recipients. Contrast with thrush (scrapable) and leukoplakia (precancerous).

1	J . EBV mediated. Occurs in HIV-positive patients, organ transplant recipients. Contrast with thrush (scrapable) and leukoplakia (precancerous). MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` dERmatology SECTION III 480 pathophysiology Potentially fatal. Most commonly seen in older adults. Type II hypersensitivity reaction. IgG antibodies against desmoglein-1 and/or desmoglein-3 (component of desmosomes, which connect keratinocytes in the stratum spinosum). Less severe than pemphigus vulgaris. Most commonly seen in older adults. Type II hypersensitivity reaction. IgG antibodies against hemidesmosomes (epidermal basement membrane; antibodies are “bullow” the epidermis). gRoss moRphology Flaccid intraepidermal bullae A caused by Tense blisters C containing eosinophils; oral acantholysis (separation of keratinocytes, “row mucosa spared. Nikolsky sign ⊝. of tombstones” on H&E stain); oral mucosa is involved. Nikolsky sign ⊕.

1	immUnoFlUoREsCEnCE Reticular pattern around epidermal cells B . Linear pattern at epidermal-dermal junction D . MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` dERmatology SECTION III 481 D . adverse drug reaction. Toxic epidermal necrolysis (TEN) EF is more severe form of SJS involving > 30% body surface area. 10–30% involvement denotes SJS-TEN. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` dERmatology SECTION III 482 Acanthosis nigricans Epidermal hyperplasia causing symmetric, hyperpigmented thickening of skin, especially in axilla AB . Associated with insulin resistance (eg, diabetes, obesity, Cushing syndrome, PCOS), visceral malignancy (eg, gastric adenocarcinoma). Actinic keratosis Premalignant lesions caused by sun exposure. Small, rough, erythematous or brownish papules or CD . Risk of squamous cell carcinoma is proportional to degree of epithelial dysplasia. Rule of 9’s The extent of a burn injury can be estimated as a percentage of the body surface area. H .

1	CD . Risk of squamous cell carcinoma is proportional to degree of epithelial dysplasia. Rule of 9’s The extent of a burn injury can be estimated as a percentage of the body surface area. H . MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` dERmatology SECTION III 483 Entire head 9% Entire torso 18% Entire arm (L) 9% Entire arm (R) 9% Entire abdomen 18% Perineum 1% Total 100% Entire leg (L) 18% Entire leg (R) 18% 4.5 4.5 4.5 4.5 1 9 9 9 9 4.5 9 9 9 9 4.5 MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` dERmatology SECTION III 484 Skin cancer Basal cell carcinoma more common above upper lip Squamous cell carcinoma more common below lower lip Sun exposure strongly predisposes to skin cancer.

1	Skin cancer Basal cell carcinoma more common above upper lip Squamous cell carcinoma more common below lower lip Sun exposure strongly predisposes to skin cancer. Basal cell carcinoma Most common skin cancer. Found in sun-exposed areas of body (eg, face). Locally invasive, but rarely metastasizes. Waxy, pink, pearly nodules, commonly with telangiectasias, rolled borders A , central crusting or ulceration. BCCs also appear as nonhealing ulcers with infiltrating growth B or as a scaling plaque (superficial BCC) C . Basal cell tumors have “palisading” (aligned) nuclei D . Keratoacanthoma Seen in middle-aged and elderly individuals. Rapidly growing, resembles squamous cell carcinoma. Presents as dome-shaped nodule with keratin-filled center. Grows rapidly (4-6 weeks) and may spontaneously regress E .

1	E . Melanoma Common tumor with significant risk of metastasis. S-100 tumor marker. Associated with dysplastic nevi; fair-skinned persons are at  risk. Depth of tumor (Breslow thickness) correlates with risk of metastasis. Look for the ABCDEs: Asymmetry, Border irregularity, Color variation, Diameter > 6 mm, and Evolution over time. At least 4 different types of melanoma, including superficial spreading F , nodular G , lentigo maligna H , and acral lentiginous (highest prevalence in African-Americans and Asians) I . Often driven by activating mutation in BRAF kinase. Primary treatment is excision with appropriately wide margins. Metastatic or unresectable melanoma in patients with BRAF V600E mutation may benefit from vemurafenib, a BRAF kinase inhibitor. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` phaRmaCology SECTION III 485 `mUsCUlosKElEtal, sKin, and ConnECtiVE tissUE—phaRmaCology vasodilation.

1	MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` phaRmaCology SECTION III 485 `mUsCUlosKElEtal, sKin, and ConnECtiVE tissUE—phaRmaCology vasodilation. DinoprostoneEpoprostenol CarboprostAlprostadil COX-2 ONLY Celecoxib NF-˜B I˜B COX-1, COX-2 ENDOPEROXIDE SYNTHESIS (cyclooxygenase)KetorolacNaproxenGlucocorticoids (corticosteroids) COX-2 COX-1 Cyclic endoperoxides ThromboxaneProstaglandinsProstacyclin •platelet aggregation •vascular tone •uterine tone •uterine tone •platelet aggregation •vascular tone PGI2 PGE1 TXA2PGF2˜PGE2 •vascular tone Aspirin (irreversible) Other NSAIDs (reversible) Diclofenac Ketorolac Ibuprofen Naproxen Indomethacin is a neutrophil chemotactic agent. inhibits platelet aggregation and promotes Neutrophils arrive “B4” others. Platelet-Gathering Inhibitor. mEChanism Reversibly inhibits cyclooxygenase, mostly in CNS. Inactivated peripherally.

1	mEChanism Reversibly inhibits cyclooxygenase, mostly in CNS. Inactivated peripherally. CliniCal UsE Antipyretic, analgesic, but not anti-inflammatory. Used instead of aspirin to avoid Reye syndrome in children with viral infection. adVERsE EFFECts Overdose produces hepatic necrosis; acetaminophen metabolite (NAPQI) depletes glutathione and forms toxic tissue byproducts in liver. N-acetylcysteine is antidote—regenerates glutathione. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` phaRmaCology SECTION III 486 mEChanism NSAID that irreversibly inhibits cyclooxygenase (both COX-1 and COX-2) by covalent acetylation  synthesis of TXA2 and prostaglandins.  bleeding time. No effect on PT, PTT. Effect lasts until new platelets are produced. CliniCal UsE Low dose (< 300 mg/day):  platelet aggregation. Intermediate dose (300–2400 mg/day): antipyretic and analgesic. High dose (2400–4000 mg/day): anti-inflammatory.

1	CliniCal UsE Low dose (< 300 mg/day):  platelet aggregation. Intermediate dose (300–2400 mg/day): antipyretic and analgesic. High dose (2400–4000 mg/day): anti-inflammatory. adVERsE EFFECts Gastric ulceration, tinnitus (CN VIII), allergic reactions (especially in patients with asthma or nasal polyps). Chronic use can lead to acute kidney injury, interstitial nephritis, GI bleeding. Risk of Reye syndrome in children treated with aspirin for viral infection. Toxic doses cause respiratory alkalosis early, but transitions to mixed metabolic acidosis-respiratory alkalosis. Treatment of overdose: NaHCO3. mEChanism Reversibly and selectively inhibits the cyclooxygenase (COX) isoform 2 (“Selecoxib”), which is found in inflammatory cells and vascular endothelium and mediates inflammation and pain; spares COX-1, which helps maintain gastric mucosa. Thus, does not have the corrosive effects of other NSAIDs on the GI lining. Spares platelet function as TXA2 production is dependent on COX-1.

1	CliniCal UsE Rheumatoid arthritis, osteoarthritis. adVERsE EFFECts  risk of thrombosis, sulfa allergy. Nonsteroidal Ibuprofen, naproxen, indomethacin, ketorolac, diclofenac, meloxicam, piroxicam. anti-inflammatory drugs mEChanism Reversibly inhibit cyclooxygenase (both COX-1 and COX-2). Block prostaglandin synthesis. CliniCal UsE Antipyretic, analgesic, anti-inflammatory. Indomethacin is used to close a PDA. adVERsE EFFECts Interstitial nephritis, gastric ulcer (prostaglandins protect gastric mucosa), renal ischemia (prostaglandins vasodilate afferent arteriole), aplastic anemia. mEChanism Reversibly inhibits dihydroorotate dehydrogenase, preventing pyrimidine synthesis. Suppresses T-cell proliferation. CliniCal UsE Rheumatoid arthritis, psoriatic arthritis. adVERsE EFFECts Diarrhea, hypertension, hepatotoxicity, teratogenicity. Bisphosphonates Alendronate, ibandronate, risedronate, zoledronate.

1	CliniCal UsE Rheumatoid arthritis, psoriatic arthritis. adVERsE EFFECts Diarrhea, hypertension, hepatotoxicity, teratogenicity. Bisphosphonates Alendronate, ibandronate, risedronate, zoledronate. mEChanism Pyrophosphate analogs; bind hydroxyapatite in bone, inhibiting osteoclast activity. CliniCal UsE Osteoporosis, hypercalcemia, Paget disease of bone, metastatic bone disease, osteogenesis imperfecta. adVERsE EFFECts Esophagitis (if taken orally, patients are advised to take with water and remain upright for 30 minutes), osteonecrosis of jaw, atypical femoral stress fractures. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` phaRmaCology SECTION III 487 mEChanism Recombinant PTH analog.  osteoblastic activity when administered in pulsatile fashion. CliniCal UsE Osteoporosis. Causes  bone growth compared to antiresorptive therapies (eg, bisphosphonates).

1	CliniCal UsE Osteoporosis. Causes  bone growth compared to antiresorptive therapies (eg, bisphosphonates). adVERsE EFFECts  risk of osteosarcoma (avoid use in patients with Paget disease of the bone or unexplained elevation of alkaline phosphatase). Avoid in patients who have had prior cancers or radiation therapy. Transient hypercalcemia. Allopurinol Competitive inhibitor of xanthine oxidase  conversion of hypoxanthine and xanthine to urate. Also used in lymphoma and leukemia to prevent tumor lysis–associated urate nephropathy.  concentrations of xanthine oxidase active metabolites, azathioprine, and 6-MP. Pegloticase Recombinant uricase catalyzing uric acid to allantoin (a more water-soluble product). Probenecid Inhibits reabsorption of uric acid in proximal Prevent APainful Flare. convoluted tubule (also inhibits secretion of penicillin). Can precipitate uric acid calculi. Allopurinol, Febuxostat Febuxostat Inhibits xanthine oxidase.

1	Allopurinol, Febuxostat Febuxostat Inhibits xanthine oxidase. NSAIDs Any NSAID. Use salicylates with caution (may decrease uric acid excretion, particularly at Diuretics,low doses). low-dose salicylates Glucocorticoids Oral, intra-articular, or parenteral. Colchicine Binds and stabilizes tubulin to inhibit microtubule polymerization, impairing neutrophil chemotaxis and degranulation. Acute and prophylactic value. GI, neuromyopathic side effects. Urine Probenecid, high-dose salicylates Tubular reabsorption Etanercept Fusion protein (decoy receptor for TNF-α + IgG1 Fc), produced by recombinant DNA. Etanercept intercepts TNF. Infliximab, adalimumab, certolizumab, golimumab Anti-TNF-α monoclonal antibody. Rheumatoid arthritis, psoriasis, ankylosing spondylitis Inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, psoriasis Predisposition to infection, including reactivation of latent TB, since TNF is important in granuloma formation and stabilization.

1	Can also lead to drug-induced lupus. MUSCULOSKELETAL, SKIN, AND CONNECTIVE TISSUE ` phaRmaCology SECTION II 488 “We are all now connected by the Internet, like neurons in a giant brain.” “Anything’s possible if you’ve got enough nerve.” —J.K. Rowling, Harry Potter and the Order of the Phoenix “I like nonsense; it wakes up the brain cells.” —Dr. Seuss “I believe in an open mind, but not so open that your brains fall out.” “The chief function of the body is to carry the brain around.” “Exactly how [the brain] operates remains one of the biggest unsolved mysteries, and it seems the more we probe its secrets, the more surprises we find.”

1	Understand the difference between upper motor neuron (UMN) and lower motor neuron (LMN) findings and the underlying anatomy. Know the major motor, sensory, cerebellar and visual pathways and their respective locations in the CNS. Connect key neurological associations with certain pathologies (eg, cerebellar lesions, stroke manifestations, Brown-Séquard syndrome). Recognize common findings on MRI/ CT (eg, ischemic and hemorrhagic stroke) and on neuropathology (eg, neurofibrillary tangles and Lewy bodies). High-yield medications include those used to treat epilepsy, Parkinson disease, migraine, and pain (eg, opioids). Neural development Notochord induces overlying ectoderm to differentiate into neuroectoderm and form neural plate. Neural plate Neural plate gives rise to neural tube and neural crest cells. Day 18 Notochord becomes nucleus pulposus of intervertebral disc in adults. Notochord Neural fold

1	Neural plate Neural plate gives rise to neural tube and neural crest cells. Day 18 Notochord becomes nucleus pulposus of intervertebral disc in adults. Notochord Neural fold Alar plate (dorsal): sensory; regulated by TGF-β (including bone morphogenetic protein [BMP]) Same orientation as spinal cord Basal plate (ventral): motor; regulated by Regional specification Telencephalon is the 1st part. Diencephalon is the 2nd part. The rest are arranged alphabetically: of developing brain mesencephalon, metencephalon, myelencephalon. Neural tube defects Neuropores fail to fuse (4th week)  persistent connection between amniotic cavity and spinal canal. Associated with maternal diabetes and folate deficiency. α-fetoprotein (AFP) in amniotic fluid and maternal serum (except spina bifida occulta = normal AFP). acetylcholinesterase (AChE) in amniotic fluid is a helpful confirmatory test.

1	Spina bifida occulta Failure of caudal neuropore to close, but no herniation. Usually seen at lower vertebral levels. Dura is intact. Associated with tuft of hair or skin dimple at level of bony defect. Meningocele Meninges (but no neural tissue) herniate through bony defect. Myelomeningocele Meninges and neural tissue (eg, cauda equina) herniate through bony defect. Myeloschisis Also called rachischisis. Exposed, unfused neural tissue without skin/meningeal covering. Anencephaly Failure of rostral neuropore to close  no forebrain, open calvarium. Clinical findings: polyhydramnios (no swallowing center in brain). +/− Tuft of hair Skin defect/thinning Skin thin or absent Skin

1	+/− Tuft of hair Skin defect/thinning Skin thin or absent Skin Holoprosencephaly Failure of the embryonic forebrain (prosencephalon) to separate into 2 cerebral hemispheres; usually occurs during weeks 5–6. May be related to mutations in sonic hedgehog signaling pathway. Associated with other midline defects including cleft lip/palate (moderate form) and ★ cyclopia (severe form). risk for pituitary dysfunction (eg, diabetes insipidus). Can be seen with Patau syndrome (trisomy 13). Monoventricle MRI reveals monoventricle A and fusion of basal ganglia (star in A ). Lissencephaly Failure of neuronal migration resulting in a “smooth brain” that lacks sulci and gyri. May be associated with microcephaly, ventriculomegaly. Chiari I malformation Ectopia of cerebellar tonsils inferior to foramen magnum (1 structure) A . Congenital, usually asymptomatic in childhood, manifests in adulthood with headaches and cerebellar symptoms. Associated with spinal cavitations (eg, syringomyelia).

1	Chiari II malformation Herniation of cerebellar vermis and tonsils (2 structures) through foramen magnum with aqueductal stenosis  noncommunicating hydrocephalus. Usually associated with lumbosacral myelomeningocele (may present as paralysis/sensory loss at and below the level of the lesion). More severe than Chiari I, usually presents early in life. Dandy-Walker Agenesis of cerebellar vermis  cystic enlargement of 4th ventricle (arrow in B ) that fills the malformation enlarged posterior fossa. Associated with noncommunicating hydrocephalus, spina bifida.

1	Syringomyelia Cystic cavity (syrinx) within central canal of spinal cord (yellow arrows in A ). Fibers crossing in anterior white commissure (spinothalamic tract) are typically damaged first. Results in a “capelike,” bilateral, symmetrical loss of pain and temperature sensation in upper extremities (fine touch sensation is preserved). Associated with Chiari I malformation (red arrow in A shows low-lying cerebellar tonsils), scoliosis and other congenital malformations; acquired causes include trauma and tumors. Most common location cervical > thoracic >> lumbar. Syrinx = tube, as in “syringe.”

1	Posterior fossa malformations A˜erent Lateral spinothalamic tract (pain, temperature) Posterior fossa malformations Posterior fossa malformations Posterior fossa malformations Posterior fossa malformations Posterior fossa malformations Posterior fossa malformations Posterior fossa malformations Posterior fossa malformations Posterior fossa malformations Posterior fossa malformations Posterior fossa malformations Posterior fossa malformations Posterior fossa malformations Posterior fossa malformations Posterior fossa malformations Posterior fossa malformations Posterior fossa malformations Expanding syrinx (can a˜ect multiple dermatomes) Anterior white commissure compressed by syrinx Dorsal root Posterior fossa malformations Posterior fossa malformations ganglion Loss of pain and temperature sensation at a˜ected dermatomes (C5-T4 shown here) 1st and 2nd pharyngeal arches form anterior 2/3 (thus sensation via CN V3, taste via CN VII).

1	Loss of pain and temperature sensation at a˜ected dermatomes (C5-T4 shown here) 1st and 2nd pharyngeal arches form anterior 2/3 (thus sensation via CN V3, taste via CN VII). 3rd and 4th pharyngeal arches form posterior 1/3(thus sensation and taste mainly via CN IX, extreme posterior via CN X). Motor innervation is via CN XII to hyoglossus (retracts and depresses tongue), genioglossus (protrudes tongue), and styloglossus (draws sides of tongue upward to create a trough for swallowing). Motor innervation is via CN X to palatoglossus (elevates posterior tongue during swallowing). Taste—CN VII, IX, X (solitary nucleus). Pain—CN V3, IX, X. Motor—CN X, XII. The Genie comes out of the lamp in style.

1	Taste—CN VII, IX, X (solitary nucleus). Pain—CN V3, IX, X. Motor—CN X, XII. The Genie comes out of the lamp in style. Neurons Signal-transmitting cells of the nervous system. Permanent cells—do not divide in adulthood. Signal-relaying cells with dendrites (receive input), cell bodies, and axons (send output). Cell bodies and dendrites can be seen on Nissl staining (stains RER). RER is not present in the axon. Neuron markers: neurofilament protein, synaptophysin. Astrocytes Most common glial cell type in CNS. Physical Derived from neuroectoderm. support, repair, extracellular K+ buffer, removal Astrocyte marker: GFAP. of excess neurotransmitter, component of blood-brain barrier, glycogen fuel reserve buffer. Reactive gliosis in response to neural injury.

1	of excess neurotransmitter, component of blood-brain barrier, glycogen fuel reserve buffer. Reactive gliosis in response to neural injury. Ependymal cells Ciliated simple columnar glial cells line the ventricles and central canal of spinal cord. Apical surfaces are covered in cilia (which circulate CSF) and microvilli (which help with CSF absorption). Specialized ependymal cells (choroid plexus) produce CSF. Myelin  conduction velocity of signals transmitted down axons  saltatory conduction of action potential at the nodes of Ranvier, where there are high concentrations of Na+ channels. In CNS (including CN II), myelin is synthesized by oligodendrocytes; in PNS (including CN III-XII), myelin is synthesized by Schwann cells. Wraps and insulates axons (arrow in A ):  space constant and  conduction velocity. COPS: CNS = Oligodendrocytes, PNS = Schwann cells.

1	Wraps and insulates axons (arrow in A ):  space constant and  conduction velocity. COPS: CNS = Oligodendrocytes, PNS = Schwann cells. Schwann cells Promote axonal regeneration. Derived from Each “Schwone” cell myelinates only 1 PNS neural crest. axon. Injured in Guillain-Barré syndrome. Oligodendrocytes Myelinate axons of neurons in CNS. Each Derived from neuroectoderm. oligodendrocyte can myelinate many axons “Fried egg” appearance histologically. (∼ 30). Predominant type of glial cell in white Injured in multiple sclerosis, progressive matter. multifocal leukoencephalopathy (PML), leukodystrophies. Pacinian corpuscles Large, myelinated fibers; adapt Deep skin layers, ligaments, High-frequency vibration, quickly joints pressure Merkel discs Large, myelinated fibers; adapt Finger tips, superficial skin Pressure, deep static touch (eg, slowly shapes, edges), position sense

1	Merkel discs Large, myelinated fibers; adapt Finger tips, superficial skin Pressure, deep static touch (eg, slowly shapes, edges), position sense Endoneurium—thin, supportive connective Endo = inner tissue that ensheathes and supports individual Peri = around myelinated nerve fibers. Epi = outer Perineurium (blood-nerve Permeability barrier)—surrounds a fascicle of nerve fibers. Epineurium—dense connective tissue that surrounds entire nerve (fascicles and blood vessels). Reaction of neuronal cell body to axonal injury. Changes reflect  protein synthesis in effort to repair the damaged axon. Characterized by: Displacement of the nucleus to the periphery Dispersion of Nissl substance throughout cytoplasm Wallerian degeneration—disintegration of the axon and myelin sheath distal to site of axonal injury with macrophages removing debris.

1	Dispersion of Nissl substance throughout cytoplasm Wallerian degeneration—disintegration of the axon and myelin sheath distal to site of axonal injury with macrophages removing debris. Proximal to the injury, the axon retracts, and the cell body sprouts new protrusions that grow toward other neurons for potential reinnervation. Serves as a preparation for axonal regeneration and functional recovery. Neurotransmitter changes with disease Acetylcholine Basal nucleus of Meynert Three membranes that surround and protect the CSF flows in the subarachnoid space, located brain and spinal cord: between arachnoid and pia mater. Dura mater—thick outer layer closest to Epidural space—potential space between skull. Derived from mesoderm. the dura mater and skull/vertebral column Arachnoid mater—middle layer, contains containing fat and blood vessels. Site of blood web-like connections. Derived from neural collection associated with middle meningeal crest. artery injury.

1	Arachnoid mater—middle layer, contains containing fat and blood vessels. Site of blood web-like connections. Derived from neural collection associated with middle meningeal crest. artery injury. Pia mater—thin, fibrous inner layer that firmly adheres to brain and spinal cord. Derived from neural crest. Prevents circulating blood substances Circumventricular organs with fenestrated (eg, bacteria, drugs) from reaching the CSF/ capillaries and no blood-brain barrier CNS. Formed by 3 structures: allow molecules in blood to affect brain Tight junctions between nonfenestrated function (eg, area postrema—vomiting after capillary endothelial cells chemotherapy; OVLT [organum vasculosum

1	Tight junctions between nonfenestrated function (eg, area postrema—vomiting after capillary endothelial cells chemotherapy; OVLT [organum vasculosum Astrocyte foot processes neurosecretory products to enter circulation Glucose and amino acids cross slowly by carrier-(eg, neurohypophysis—ADH release). mediated transport mechanisms. Infarction and/or neoplasm destroys endothelial Nonpolar/lipid-soluble substances cross rapidly cell tight junctions • vasogenic edema. via diffusion. Hyperosmolar agents (eg, mannitol) can disrupt the BBB • permeability of medications. Coordinated by nucleus tractus solitarius (NTS) in the medulla, which receives information from the chemoreceptor trigger zone (CTZ, located within area postrema in 4th ventricle), GI tract (via vagus nerve), vestibular system, and CNS. CTZ and adjacent vomiting center nuclei receive input from 5 major receptors: muscarinic (M1), dopamine (D2), histamine (H1), serotonin (5-HT3), and neurokinin (NK-1) receptors.

1	CTZ and adjacent vomiting center nuclei receive input from 5 major receptors: muscarinic (M1), dopamine (D2), histamine (H1), serotonin (5-HT3), and neurokinin (NK-1) receptors. 5-HT3, D2, and NK-1 antagonists used to treat chemotherapy-induced vomiting. H1 and M1 antagonists treat motion sickness; H1 antagonists treat hyperemesis gravidarum. Sleep physiology Sleep cycle is regulated by the circadian rhythm, which is driven by suprachiasmatic nucleus (SCN) of the hypothalamus. Circadian rhythm controls nocturnal release of ACTH, prolactin, melatonin, norepinephrine: SCN • norepinephrine release  pineal gland • melatonin. SCN is regulated by environment (eg, light). Two stages: rapid-eye movement (REM) and non-REM. Alcohol, benzodiazepines, and barbiturates are associated with  REM sleep and N3 sleep; norepinephrine also  REM sleep. Benzodiazepines are useful for night terrors and sleepwalking by  N3 and REM sleep.

1	Thalamus Major relay for all ascending sensory information except olfaction. Limbic system Collection of neural structures involved in The famous 5 F’s. emotion, long-term memory, olfaction, behavior modulation, ANS function. Consists of hippocampus (red arrows in A ), amygdalae, mammillary bodies, anterior thalamic nuclei, cingulate gyrus (yellow arrows in A ), entorhinal cortex. Responsible for Feeding, Fleeing, Fighting, Feeling, and Sex. A . Input: inferior cerebellar peduncle from spinal cord Output: The only output of cerebellar cortex = Purkinje cells (always inhibitory)  deep nuclei of cerebellum  contralateral cortex via superior cerebellar peduncle Deep nuclei (lateral  medial)—Dentate, Emboliform, Globose, Fastigial Lateral lesions—affect voluntary movement of extremities (lateral structures); when injured, propensity to fall toward injured (ipsilateral) side.

1	Lateral lesions—affect voluntary movement of extremities (lateral structures); when injured, propensity to fall toward injured (ipsilateral) side. Medial lesions (eg, vermis, fastigial nuclei, flocculonodular lobe)—truncal ataxia (widebased cerebellar gait), nystagmus, head tilting. Generally result in bilateral motor deficits affecting axial and proximal limb musculature (medial structures). Important in voluntary movements and adjusting posture A . D1 Receptor = D1Rect Receives cortical input, provides negative feedback to cortex to pathway. modulate movement. Indirect (D2) = Inhibitory. Striatum = putamen (motor) + Caudate (cognitive). Lentiform = putamen + globus pallidus. Direct (excitatory) pathway—SNc input to the striatum via the nigrostriatal dopaminergic pathway releases GABA, which inhibits GABA release from the GPi, disinhibiting the Thalamus via the GPi (• motion).

1	Indirect (inhibitory) pathway—SNc input to the striatum via the nigrostriatal dopaminergic pathway releases GABA that disinhibits STN via GPe inhibition, and STN stimulates GPi to inhibit the thalamus (• motion). Dopamine binds to D1, stimulating the excitatory pathway, and to D2, inhibiting the inhibitory pathway  motion. Cerebral perfusion Relies on tight autoregulation. Primarily driven by Pco2 (Po2 also modulates perfusion in severe hypoxia). Also relies on a pressure gradient between mean arterial pressure (MAP) and intracranial pressure (ICP).  blood pressure or  ICP  cerebral perfusion pressure (CPP).  vasoconstriction  cerebral blood flow  ICP. May be used to treat acute cerebral edema (eg, 2° to stroke) unresponsive to other interventions. CPP = MAP – ICP. If CPP = 0, there is no cerebral perfusion • brain death. Hypoxemia increases CPP only if Po2 < 50 mm Hg. CPP is directly proportional to Pco2 until Pco2 > 90 mm Hg.

1	CPP = MAP – ICP. If CPP = 0, there is no cerebral perfusion • brain death. Hypoxemia increases CPP only if Po2 < 50 mm Hg. CPP is directly proportional to Pco2 until Pco2 > 90 mm Hg. HipLeg HipTrunk NeckHeadShoulderArmElbow RingLittle MiddleIndexThumb ForearmWrist Toes Ankle KneeShoulder FingersFingers ElbowWristHandLittleRingMiddleIndexThumbNeckBrow Lips TongueJaw Swallowing Eyelid & eyeball TrunkEye Nose Face Upper lip Lower lipTeeth, gums Foot Toes TongueIntra-abdominalPharynx Genitals Homunculus Topographic representation of motor and sensory areas in the cerebral cortex. Distorted appearance is due to certain body regions being more richly innervated and thus having  cortical representation. Watershed zones Cortical border zones occur between anterior and middle cerebral arteries and posterior and middle cerebral arteries (blue areas in A ). Internal border zones occur between the superficial and deep vascular territories of the middle cerebral artery (red areas in A ).

1	A ). Infarct due to severe hypoperfusion • proximal upper and lower extremity weakness (“manin-the-barrel syndrome”), higher order visual dysfunction (if posterior cerebral/middle cerebral cortical border zone stroke). Circle of Willis System of anastomoses between anterior and posterior blood supplies to brain. Dural venous sinuses Large venous channels A that run through the periosteal and meningeal layers of the dura mater. Drain blood from cerebral veins (arrow) and receive CSF from arachnoid granulations. Empty into internal jugular vein. Venous sinus thrombosis—presents with signs/symptoms of • ICP (eg, headache, seizures, papilledema, focal neurologic deficits). May lead to venous hemorrhage. Associated with hypercoagulable states (eg, pregnancy, OCP use, factor V Leiden). (main location of CSF return via arachnoid granulations) Superior ophthalmic vein Great cerebral vein of Galen Confuence of the sinuses Sigmoid sinus

1	(main location of CSF return via arachnoid granulations) Superior ophthalmic vein Great cerebral vein of Galen Confuence of the sinuses Sigmoid sinus Lateral ventricles  3rd ventricle via right and left interventricular foramina of Monro. 3rd ventricle  4th ventricle via cerebral aqueduct of Sylvius. 4th ventricle  subarachnoid space via: Foramina of Luschka = Lateral. Foramen of Magendie = Medial. CSF made by choroid plexuses located in the lateral and fourth ventricles. Travels to subarachnoid space via foramina of Luschka and Magendie, is reabsorbed by arachnoid granulations, and then drains into dural venous sinuses. Brain stem—ventral view 4 CN are above pons (I, II, III, IV). 4 CN exit the pons (V, VI, VII, VIII). (CN I) 4 CN are in medulla (IX, X, XI, XII). Olfactory tract 4 CN nuclei are medial (III, IV, VI, XII). CN II “Factors of 12, except 1 and 2.” Middle cerebellar Anterior wall of peduncle fourth ventricle

1	Middle cerebellar Anterior wall of peduncle fourth ventricle Pineal gland—melatonin secretion, circadian rhythms. Superior colliculi—direct eye movements to stimuli (noise/movements) or objects of interest. Inferior colliculi—auditory. Your eyes are above your ears, and the superior colliculus (visual) is above the inferior colliculus (auditory). In adults, spinal cord ends at lower border of L1–L2 vertebrae. Subarachnoid Space (which contains the CSF) extends to lower border of S2 vertebra. Lumbar puncture is usually performed between L3–L4 or L4–L5 (level of cauda equina). Goal of lumbar puncture is to obtain sample of CSF without damaging spinal cord. To keep the cord alive, keep the spinal needle between L3 and L5. Needle passes through: skin fascia and fat supraspinous ligament interspinous ligament ligamentum flavum epidural space (epidural anesthesia needle stops here) dura mater arachnoid mater subarachnoid space (CSF collection occurs here)

1	Spinal tract anatomy Ascending tracts synapse and then cross. and functions Dorsal column Pressure, vibration, fine touch, proprioception Spinothalamic tract Lateral: pain, temperature Anterior: crude touch, pressure Sensory nerve ending  bypasses pseudounipolar cell body in dorsal root ganglion  enters spinal cord  ascends ipsilaterally in dorsal columns Sensory nerve ending (Aδ and C fibers)  bypasses pseudounipolar cell body in dorsal root ganglion • enters spinal cord Nucleus gracilis, nucleus cuneatus (ipsilateral medulla)  ascends contralaterally as the medial lemniscus Decussates in spinal cord as the anterior white commissure Clinical reflexes Reflexes count up in order (main nerve root in Additional reflexes: bold): Cremasteric reflex = L1, L2 (“testicles move”) Achilles reflex = S1, S2 (“buckle my shoe”) Anal wink reflex = S3, S4 (“winks galore”) C5, 6

1	Patellar reflex = L2-L4 (“kick the door”) C6, 7, 8 Biceps and brachioradialis reflexes = C5, C6 (“pick up sticks”) L2, 3, 4 Triceps reflex = C6, C7, C8 (“lay them S1, 2 straight”) Primitive reflexes CNS reflexes that are present in a healthy infant, but are absent in a neurologically intact adult. Normally disappear within 1st year of life. These primitive reflexes are inhibited by a mature/ developing frontal lobe. They may reemerge in adults following frontal lobe lesions  loss of inhibition of these reflexes. Moro reflex “Hang on for life” reflex—abduct/extend arms when startled, and then draw together Rooting reflex Movement of head toward one side if cheek or mouth is stroked (nipple seeking) Sucking reflex Sucking response when roof of mouth is touched Palmar reflex Curling of fingers if palm is stroked

1	Sucking reflex Sucking response when roof of mouth is touched Palmar reflex Curling of fingers if palm is stroked Plantar reflex Dorsiflexion of large toe and fanning of other toes with plantar stimulation Babinski sign—presence of this reflex in an adult, which may signify a UMN lesion Galant reflex Stroking along one side of the spine while newborn is in ventral suspension (face down) causes lateral flexion of lower body toward stimulated side C2 Posterior half of skull C3 High turtleneck shirt Diaphragm and gallbladder pain referred to the right shoulder via phrenic nerve C3, 4, 5 keeps the diaphragm alive T4 At the nipple T4 at the teat pore T7 At the xiphoid process 7 letters in xiphoid T10 At the umbilicus (belly butten) Point of referred pain in early appendicitis L1 At the Inguinal Ligament L4 Includes the kneecaps Down on ALL 4’s S2, S3, S4 Sensation of penile and anal zones S2, 3, 4 keep the penis off the floor

1	L1 At the Inguinal Ligament L4 Includes the kneecaps Down on ALL 4’s S2, S3, S4 Sensation of penile and anal zones S2, 3, 4 keep the penis off the floor Irreversible neuronal injury begins after 5 minutes of hypoxia. Most vulnerable: hippocampus, neocortex, cerebellum (Purkinje cells), watershed areas (“vulnerable hippos need pure water”). Stroke imaging: noncontrast CT to exclude hemorrhage (before tPA can be given). CT detects ischemic changes in 6–24 hr. Diffusion-weighted MRI can detect ischemia within 3–30 min. Acute blockage of vessels  disruption of blood flow and subsequent ischemia  infarction necrosis. 3 types: Thrombotic—due to a clot forming directly at site of infarction (commonly the MCA A ), usually over a ruptured atherosclerotic plaque. Embolic—embolus from another part of the body obstructs vessel. Can affect multiple vascular territories. Examples: atrial fibrillation, carotid artery stenosis, DVT with patent foramen ovale, infective endocarditis.

1	Hypoxic—due to hypoperfusion or hypoxemia. Common during cardiovascular surgeries, tends to affect watershed areas. Treatment: tPA (if within 3–4.5 hr of onset and no hemorrhage/risk of hemorrhage) and/or thrombectomy (if large artery occlusion). Reduce risk with medical therapy (eg, aspirin, clopidogrel); optimum control of blood pressure, blood sugars, lipids; smoking cessation; and treat conditions that  risk (eg, atrial fibrillation, carotid artery stenosis). Transient ischemic Brief, reversible episode of focal neurologic dysfunction without acute infarction (⊝ MRI), with the attack majority resolving in < 15 minutes; ischemia (eg, embolus, small vessel stenosis).

1	Bleeding into ventricles (arrow in coronal transcranial ultrasound A shows blood in right intraventricular space, extending into periventricular white matter). Increased risk in premature and low-birth-weight infants. Originates in germinal matrix, a highly vascularized layer within the subventricular zone. Due to reduced glial fiber support and impaired autoregulation of BP in premature infants. Can present with altered level of consciousness, bulging fontanelle, hypotension, seizures, coma. Epidural hematoma Rupture of middle meningeal artery (branch of maxillary artery), often 2° to skull fracture (circle in A ) involving the pterion (thinnest area of the lateral skull). Might present with transient loss of consciousness  recovery (“lucid interval”)  rapid deterioration due to hematoma expansion. Scalp hematoma (arrows in

1	A ) and rapid intracranial expansion (arrows in B ) under systemic arterial pressure  transtentorial herniation, CN III palsy. CT shows biconvex (lentiform), hyperdense blood collection B not crossing suture lines. Subdural hematoma Rupture of bridging veins. Can be acute (traumatic, high-energy impact  hyperdense on CT) or chronic (associated with mild trauma, cerebral atrophy, elderly, alcoholism  hypodense on CT). Also seen in shaken babies. Predisposing factors: brain atrophy, trauma. Crescent-shaped hemorrhage (red arrows in C and D ) that crosses suture lines. Can cause midline shift (yellow arrow in C ), findings of “acute on chronic” hemorrhage (blue arrows in D ).

1	Crescent-shaped hemorrhage (red arrows in C and D ) that crosses suture lines. Can cause midline shift (yellow arrow in C ), findings of “acute on chronic” hemorrhage (blue arrows in D ). EF due to trauma, or rupture of an aneurysm (such as a saccular aneurysm E ) or arteriovenous malformation. Rapid time course. Patients complain of “worst headache of my life.” Bloody or yellow (xanthochromic) lumbar puncture. Vasospasm can occur due to blood breakdown  ischemic infarct; nimodipine used to prevent/reduce vasospasm.  risk of developing communicating and/or obstructive hydrocephalus. Most commonly caused by systemic hypertension. Also seen with amyloid angiopathy (recurrent lobar hemorrhagic stroke in elderly), vasculitis, neoplasm. May be 2º to reperfusion injury in ischemic stroke. Hypertensive hemorrhages (Charcot-Bouchard microaneurysm) most often occur in putamen of basal ganglia (lenticulostriate vessels G ), followed by thalamus, pons, and cerebellum H .

1	Hypertensive hemorrhages (Charcot-Bouchard microaneurysm) most often occur in putamen of basal ganglia (lenticulostriate vessels G ), followed by thalamus, pons, and cerebellum H . Effects of strokes Anterior circulation Middle cerebral artery Motor and sensory cortices A —upper limb and face. Temporal lobe (Wernicke area); frontal lobe (Broca area). Contralateral paralysis and sensory loss—face and upper limb. Aphasia if in dominant (usually left) hemisphere. Hemineglect if lesion affects nondominant (usually right) hemisphere. Wernicke aphasia is associated with right superior quadrant visual field defect due to temporal lobe involvement. Lenticulo-striate artery Striatum, internal capsule. Contralateral paralysis. Absence of cortical signs (eg, neglect, aphasia, visual field loss). Pure motor stroke. Common location of lacunar infarcts B , due to hyaline arteriosclerosis (lipohyalinosis) 2° to unmanaged hypertension. Posterior circulation

1	Lateral medulla: Nucleus ambiguus (CN IX, X, XI) Vestibular nuclei Lateral spinothalamic tract, spinal trigeminal nucleus Dysphagia, hoarseness, • gag reflex, hiccups. Vomiting, vertigo, nystagmus  pain and temperature sensation from contralateral body, ipsilateral face. Ipsilateral Horner syndrome. Ipsilateral ataxia, dysmetria. Lateral medullary (Wallenberg) syndrome. Nucleus ambiguus effects are specific to PICA lesions C . “Don’t pick a (PICA) horse (hoarseness) that can’t eat (dysphagia).” Also supplies inferior cerebellar peduncle (part of cerebellum). Lateral pons: Facial nucleus Vestibular nuclei Spinothalamic tract, spinal trigeminal nucleus Paralysis of face (LMN lesion vs UMN lesion in cortical stroke),  lacrimation,  salivation, • taste from anterior 2⁄3 of tongue. Vomiting, vertigo, nystagmus  pain and temperature sensation from contralateral body, ipsilateral face. Ipsilateral Horner syndrome. Ipsilateral ataxia, dysmetria.

1	Ipsilateral Horner syndrome. Ipsilateral ataxia, dysmetria. Ipsilateral sensorineural deafness, vertigo. Lateral pontine syndrome. Facial nucleus effects are specific to AICA lesions. “Facial droop means AICA’s pooped.” Also supplies middle and inferior cerebellar peduncles (part of cerebellum). Effects of strokes (continued) Basilar artery Pons, medulla, lower midbrain. Corticospinal and corticobulbar tracts. Ocular cranial nerve nuclei, paramedian pontine reticular formation. If RAS spared, consciousness is preserved. Quadriplegia; loss of voluntary facial, mouth, and tongue movements. Loss of horizontal, but not vertical, eye movements. Locked-in syndrome (locked in the basement). Posterior cerebral artery Occipital lobe D . Contralateral hemianopia with macular sparing; alexia without agraphia (dominant hemisphere).

1	Locked-in syndrome (locked in the basement). Posterior cerebral artery Occipital lobe D . Contralateral hemianopia with macular sparing; alexia without agraphia (dominant hemisphere). Diffuse axonal injury Caused by traumatic shearing forces during rapid acceleration and/or deceleration of the brain (eg, motor vehicle accident). Usually results in devastating neurologic injury, often causing coma or persistent vegetative state. MRI A shows multiple lesions (punctate hemorrhages) involving the white matter tracts. Aphasia Aphasia—higher-order language deficit Good comprehension Poor comprehension (inability to understand/produce/use language appropriately); caused by pathology in dominant cerebral hemisphere (usually left). speech Dysarthria—motor inability to produce speech (movement deficit). Broca (expressive) Broca area in inferior frontal gyrus of frontal lobe. Patient appears frustrated, insight intact. Broca = Broken Boca (boca = mouth in Spanish).

1	Broca (expressive) Broca area in inferior frontal gyrus of frontal lobe. Patient appears frustrated, insight intact. Broca = Broken Boca (boca = mouth in Spanish). Wernicke (receptive) Wernicke area in superior temporal gyrus of temporal lobe. Patients do not have insight. Wernicke is a Word salad and makes no sense. Conduction Can be caused by damage to arCuate fasciculus. Global Broca and Wernicke areas affected. Transcortical motor Affects frontal lobe around Broca area, but Broca area is spared. Transcortical sensory Affects temporal lobe around Wernicke area, but Wernicke area is spared. Transcortical mixed Broca and Wernicke areas and arcuate fasciculus remain intact; surrounding watershed areas affected. Aneurysms Abnormal dilation of an artery due to weakening of vessel wall.

1	Transcortical mixed Broca and Wernicke areas and arcuate fasciculus remain intact; surrounding watershed areas affected. Aneurysms Abnormal dilation of an artery due to weakening of vessel wall. Also called berry aneurysm A . Occurs at bifurcations in the circle of Willis. Most common site is junction of ACom and ACA. Associated with ADPKD, Ehlers-Danlos syndrome. Other risk factors: advanced age, hypertension, smoking, race ( risk in African-Americans). Usually clinically silent until rupture (most common complication) • subarachnoid hemorrhage (“worst headache of my life” or “thunderclap headache”)  focal neurologic deficits. Can also cause symptoms via direct compression of surrounding structures by growing aneurysm. ACom—compression  bitemporal hemianopia (compression of optic chiasm); visual acuity deficits; rupture  ischemia in ACA distribution • contralateral lower extremity hemiparesis, sensory deficits.

1	ACom—compression  bitemporal hemianopia (compression of optic chiasm); visual acuity deficits; rupture  ischemia in ACA distribution • contralateral lower extremity hemiparesis, sensory deficits. MCA—rupture  ischemia in MCA distribution  contralateral upper extremity and lower facial hemiparesis, sensory deficits. PCom—compression  ipsilateral CN III palsy • mydriasis (“blown pupil”); may also see ptosis, “down and out” eye. Seizures Characterized by synchronized, high-frequency neuronal firing. Variety of forms. Impaired consciousness? Headaches Pain due to irritation of structures such as the dura, cranial nerves, or extracranial structures. More common in females, except cluster headaches. Unilateral 15 min–3 hr; Excruciating periorbital pain repetitive (“suicide headache”) with lacrimation and rhinorrhea. May present with Horner syndrome. More common in males.

1	Unilateral 15 min–3 hr; Excruciating periorbital pain repetitive (“suicide headache”) with lacrimation and rhinorrhea. May present with Horner syndrome. More common in males. Unilateral 4–72 hr Pulsating pain with nausea, photophobia, or phonophobia. May have “aura.” Due to irritation of CN V, meninges, or blood vessels (release of vasoactive neuropeptides [eg, substance P, calcitonin gene-related peptide]). Bilateral > 30 min Steady, “band-like” pain. No (typically 4–6 photophobia or phonophobia. hr); constant No aura. Acute: sumatriptan, 100% O2. Prophylaxis: verapamil. Acute: NSAIDs, triptans, dihydroergotamine. Prophylaxis: lifestyle changes (eg, sleep, exercise, diet), β-blockers, amitriptyline, topiramate, valproate, botulinum toxin, anti-CGRP monoclonal antibodies. POUND–Pulsatile, One-day duration, Unilateral, Nausea, Disabling. Acute: analgesics, NSAIDs, acetaminophen. Prophylaxis: TCAs (eg, amitriptyline), behavioral therapy.

1	POUND–Pulsatile, One-day duration, Unilateral, Nausea, Disabling. Acute: analgesics, NSAIDs, acetaminophen. Prophylaxis: TCAs (eg, amitriptyline), behavioral therapy. Other causes of headache include subarachnoid hemorrhage (“worst headache of my life”), meningitis, hydrocephalus, neoplasia, giant cell (temporal) arteritis. aCompare with trigeminal neuralgia, which produces repetitive, unilateral, shooting/shock-like pain in the distribution of CN V. Triggered by chewing, talking, touching certain parts of the face. Lasts (typically) for seconds to minutes, but episodes often increase in intensity and frequency over time. First-line therapy: carbamazepine. Athetosis Slow, snake-like, writhing Basal ganglia. Seen in Huntington disease. movements; especially seen in the fingers. Intention tremor Slow, zigzag motion when Cerebellar dysfunction. pointing/extending toward a target.

1	Intention tremor Slow, zigzag motion when Cerebellar dysfunction. pointing/extending toward a target. Hemiballismus Sudden, wild flailing of one Contralateral subthalamic Pronounce “Half-of-body side of the body. nucleus (eg, lacunar stroke). ballistic.” Parkinson TRAPSS your body: Tremor (pill-rolling tremor at rest) Rigidity (cogwheel) Akinesia (or bradykinesia) Postural instability Shuffling gait Small handwriting (micrographia) MPTP, a contaminant in illegal drugs, is metabolized to MPP+, which is toxic to substantia nigra.  in cognitive ability, memory, or function with intact consciousness. Must rule out depression as cause of dementia (called pseudodementia). Other reversible causes of dementia: hypothyroidism, vitamin B12 deficiency, neurosyphilis, normal pressure hydrocephalus. Loss of dopaminergic neurons (ie, depigmentation) of substantia nigra pars compacta. Lewy bodies: composed of α-synuclein (intracellular eosinophilic inclusions A ).

1	Loss of dopaminergic neurons (ie, depigmentation) of substantia nigra pars compacta. Lewy bodies: composed of α-synuclein (intracellular eosinophilic inclusions A ). Autosomal dominant trinucleotide (CAG)n repeat expansion in the huntingtin (HTT) gene on chromosome 4 (4 letters). Symptoms manifest between ages 20 and 50: chorea, athetosis, aggression, depression, dementia (sometimes initially mistaken for substance abuse). Anticipation results from expansion of CAG repeats. Caudate loses ACh and GABA. Atrophy of caudate and putamen with ex vacuo ventriculomegaly. • dopamine,  GABA,  ACh in brain. Neuronal death via NMDA-R binding and glutamate excitotoxicity. Most common cause of dementia in elderly. Down syndrome patients have  risk of developing Alzheimer disease, as APP is located on chromosome 21. ACh. Associated with the following altered proteins: ApoE-2:  risk of sporadic form

1	ACh. Associated with the following altered proteins: ApoE-2:  risk of sporadic form ApoE-4:  risk of sporadic form • APP, presenilin-1, presenilin-2: familial forms (10%) with earlier onset Widespread cortical atrophy (normal cortex B ; cortex in Alzheimer disease C ), especially hippocampus (arrows in B and C ). Narrowing of gyri and widening of sulci. Senile plaques D in gray matter: extracellular β-amyloid core; may cause amyloid angiopathy  intracranial hemorrhage; Aβ (amyloid-β) synthesized by cleaving amyloid precursor protein (APP). E : intracellular, hyperphosphorylated tau protein = insoluble cytoskeletal elements; number of tangles correlates with degree of dementia. Hirano bodies—intracellular eosinophilic proteinaceous rods in hippocampus. Formerly called Pick disease. Early changes in Frontotemporal lobe degeneration

1	Hirano bodies—intracellular eosinophilic proteinaceous rods in hippocampus. Formerly called Pick disease. Early changes in Frontotemporal lobe degeneration F . personality and behavior (behavioral variant), Inclusions of hyperphosphorylated tau (round or aphasia (primary progressive aphasia). Pick bodies G ) or ubiquitinated TDP-43. May have associated movement disorders. Lewy body dementia Visual hallucinations (“haLewycinations”), Intracellular Lewy bodies A primarily in cortex. dementia with fluctuating cognition/ alertness, REM sleep behavior disorder, and parkinsonism. Called Lewy body dementia if apart, otherwise considered dementia 2° to Parkinson disease.

1	dementia with fluctuating cognition/ alertness, REM sleep behavior disorder, and parkinsonism. Called Lewy body dementia if apart, otherwise considered dementia 2° to Parkinson disease. Also called pseudotumor cerebri.  ICP with no obvious findings on imaging. Risk factors include female sex, Tetracyclines, Obesity, vitamin A excess, Danazol (female TOAD). Associated with cerebral venous sinus stenosis. Findings: headache, tinnitus, diplopia (usually from CN VI palsy), no change in mental status. Impaired optic nerve axoplasmic flow  papilledema. Visual field testing shows enlarged blind spot and peripheral constriction. Lumbar puncture reveals • opening pressure and provides temporary headache relief. Treatment: weight loss, acetazolamide, invasive procedures for refractory cases (eg, CSF shunt placement, optic nerve sheath fenestration surgery for visual loss). Hydrocephalus  CSF volume  ventricular dilation +/− • ICP.

1	Hydrocephalus  CSF volume  ventricular dilation +/− • ICP. Noncommunicating Caused by structural blockage of CSF circulation within ventricular system (eg, stenosis of hydrocephalus aqueduct of Sylvius, colloid cyst blocking foramen of Monro, tumor B ). Multiple sclerosis Autoimmune inflammation and demyelination of CNS (brain and spinal cord) with subsequent axonal damage. Can present with: Acute optic neuritis (painful unilateral visual loss associated with Marcus Gunn pupil) Brain stem/cerebellar syndromes (eg, diplopia, ataxia, scanning speech, intention tremor, nystagmus/INO [bilateral > unilateral]) Pyramidal tract demyelination (eg, weakness, spasticity) Spinal cord syndromes (eg, electric shock-like sensation along cervical spine on neck flexion, neurogenic bladder, paraparesis, sensory manifestations affecting the trunk or one or more extremity)

1	Spinal cord syndromes (eg, electric shock-like sensation along cervical spine on neck flexion, neurogenic bladder, paraparesis, sensory manifestations affecting the trunk or one or more extremity) Symptoms may exacerbate with increased body temperature (eg, hot bath, exercise). Relapsing and remitting is most common clinical course. Most often affects women in their 20s and 30s; more common in individuals living farther from equator and with low serum vitamin D levels. Findings  IgG level and myelin basic protein in CSF. Oligoclonal bands are diagnostic. MRI is gold standard. Periventricular plaques A (areas of oligodendrocyte loss and reactive gliosis). Multiple white matter lesions disseminated in space and time.

1	treAtment Stop relapses and halt/slow progression with disease-modifying therapies (eg, β-interferon, glatiramer, natalizumab). Treat acute flares with IV steroids. Symptomatic treatment for neurogenic bladder (catheterization, muscarinic antagonists), spasticity (baclofen, GABAB receptor agonists), pain (TCAs, anticonvulsants). Osmotic demyelination Also called central pontine myelinolysis. Massive axonal demyelination in pontine white matter syndrome A 2° to rapid osmotic changes, most commonly iatrogenic correction of hyponatremia but also rapid shifts of other osmolytes (eg, glucose). Acute paralysis, dysarthria, dysphagia, diplopia, loss of consciousness. Can cause “locked-in syndrome.” Correcting serum Na+ too fast: “From low to high, your pons will die” (osmotic demyelination syndrome) “From high to low, your brains will blow” (cerebral edema/herniation)

1	Charcot-Marie-Tooth Also called hereditary motor and sensory neuropathy. Group of progressive hereditary nerve disease disorders related to the defective production of proteins involved in the structure and function of peripheral nerves or the myelin sheath. Typically autosomal dominant and associated with foot deformities (eg, pes cavus, hammer toe), lower extremity weakness (eg, foot drop), and sensory deficits. Most common type, CMT1A, is caused by PMP22 gene duplication. Progressive multifocal Demyelination of CNS B due to destruction of oligodendrocytes (2° to reactivation of latent leukoencephalopathy JC virus infection). Seen in 2–4% of patients with AIDS. Rapidly progressive, usually fatal. Predominantly involves parietal and occipital areas; visual symptoms are common.  risk associated with natalizumab. Other disorders Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy. Most common subtype of Guillain-Barré syndrome.

1	Other disorders Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy. Most common subtype of Guillain-Barré syndrome. Autoimmune condition that destroys Schwann cells via inflammation and demyelination of motor fibers, sensory fibers, peripheral nerves (including CN III-XII). Likely facilitated by molecular mimicry and triggered by inoculations or stress. Despite association with infections (eg, Campylobacter jejuni, viruses [eg, Zika]), no definitive causal link to any pathogen. Results in symmetric ascending muscle weakness/paralysis and depressed/absent DTRs beginning in lower extremities. Facial paralysis (usually bilateral) and respiratory failure are common. May see autonomic dysregulation (eg, cardiac irregularities, hypertension, hypotension) or sensory abnormalities. Almost all patients survive; majority recover completely after weeks to months. • CSF protein with normal cell count (albuminocytologic dissociation).

1	• CSF protein with normal cell count (albuminocytologic dissociation). Respiratory support is critical until recovery. Disease-modifying treatment: plasmapheresis or IV immunoglobulins. No role for steroids. Congenital nonhereditary anomaly of neural crest derivatives. Somatic mosaicism of an activating mutation in one copy of the GNAQ gene. B  seizures/epilepsy; intellectual disability; episcleral hemangioma  IOP • early-onset glaucoma. Also called encephalotrigeminal angiomatosis. SSTURGGE-Weber: Sporadic, port-wine Stain, Tram track calcifications (opposing gyri), Unilateral, intellectual disability (Retardation), Glaucoma, GNAQ gene, Epilepsy. Tuberous sclerosis AD, variable expression. Mutation in tumor suppressor genes TSC1 on chromosome 9 (hamartin), TSC2 on chromosome 16 (tuberin). Hamartomas in CNS and skin, Angiofibromas

1	Tuberous sclerosis AD, variable expression. Mutation in tumor suppressor genes TSC1 on chromosome 9 (hamartin), TSC2 on chromosome 16 (tuberin). Hamartomas in CNS and skin, Angiofibromas C , Mitral regurgitation, Ash-leaf spots D , cardiac Rhabdomyoma, (Tuberous sclerosis), autosomal dOminant; Mental retardation (intellectual disability), renal Angiomyolipoma E , Seizures, Shagreen patches. HAMARTOMASS.  incidence of Subependymal giant cell astrocytomas and ungual fibromas. AD, 100% penetrance. Mutation in NF1 tumor suppressor gene on chromosome 17 (encodes neurofibromin, a negative RAS regulator). Café-au-lait spots F , Intellectual disability, Cutaneous neurofibromas G , Lisch nodules (pigmented iris hamartomas H ), Optic gliomas, Pheochromocytomas, Seizures/focal neurologic Signs (often from meningioma), bone lesions (eg, sphenoid dysplasia). Also called von Recklinghausen disease. 17 letters in “von Recklinghausen.” CICLOPSS. cells D . H .

1	Also called von Recklinghausen disease. 17 letters in “von Recklinghausen.” CICLOPSS. cells D . H . Cingulate (subfalcine) herniation under Can compress anterior cerebral artery. Falx cerebri falx cerebri Uncal transtentorial herniation Uncus = medial temporal lobe. Early herniation compression), contralateral hemiparesis. Late herniation  coma, Kernohan phenomenon (misleading contralateral blown pupil and ipsilateral hemiparesis due to contralateral compression against Kernohan notch). Central/downward transtentorial Caudal displacement of brain stem  rupture of mass hemorrhages. Usually fatal. Cerebellar tonsillar herniation into the Coma and death result when these herniations foramen magnum compress the brain stem. (less muscle mass,  muscle tone,  reflexes, Upper motor neuron = everything up (tone, Reflexes • DTRs, toes) Tone • Fasciculations = muscle twitching Babinski +− Positive Babinski is normal in infants

1	Upper motor neuron = everything up (tone, Reflexes • DTRs, toes) Tone • Fasciculations = muscle twitching Babinski +− Positive Babinski is normal in infants Poliomyelitis Caused by poliovirus (fecal-oral transmission). Replicates in oropharynx and small intestine before spreading via bloodstream to CNS. Infection causes destruction of cells in anterior horn of spinal cord (LMN death). Signs of LMN lesion: asymmetric weakness (vs symmetric weakness in spinal muscular atrophy), hypotonia, flaccid paralysis, fasciculations, hyporeflexia, muscle atrophy. Respiratory muscle involvement leads to respiratory failure. Signs of infection: malaise, headache, fever, nausea, etc. CSF shows  WBCs (lymphocytic pleocytosis) and slight  of protein (with no change in CSF glucose). Virus recovered from stool or throat.

1	Hemisection of spinal cord. Findings: Ipsilateral loss of all sensation at level of lesion Ipsilateral LMN signs (eg, flaccid paralysis) at level of lesion Ipsilateral UMN signs below level of lesion (due to corticospinal tract damage) Ipsilateral loss of proprioception, vibration, light (2-point discrimination) touch, and tactile sense below level of lesion (due to dorsal column damage) Contralateral loss of pain, temperature, and crude (non-discriminative) touch below level of lesion (due to spinothalamic tract damage) If lesion occurs above T1, patient may present with ipsilateral Horner syndrome due to damage of oculosympathetic pathway. Level of lesion Loss of sensation LMN signs Impaired proprioception, vibration, light touch, tactile sense Impaired pain, temperature, crude touch sensation

1	Level of lesion Loss of sensation LMN signs Impaired proprioception, vibration, light touch, tactile sense Impaired pain, temperature, crude touch sensation Autosomal recessive trinucleotide repeat disorder (GAA)n on chromosome 9 in gene that encodes frataxin (iron-binding protein). Leads to impairment in mitochondrial functioning. Degeneration of lateral corticospinal tract (spastic paralysis), spinocerebellar tract (ataxia), dorsal columns ( vibratory sense, proprioception), and dorsal root ganglia (loss of DTRs). Staggering gait, frequent falling, nystagmus, dysarthria, pes cavus, hammer toes, diabetes mellitus, hypertrophic cardiomyopathy (cause of death). Presents in Friedreich is Fratastic (frataxin): he’s your favorite frat brother, always staggering and falling but has a sweet, big heart. B . Ataxic GAAit.

1	B . Ataxic GAAit. CN V motor lesion Jaw deviates toward side of lesion due to unopposed force from the opposite pterygoid muscle. CN X lesion Uvula deviates away from side of lesion. Weak side collapses and uvula points away. CN XI lesion Weakness turning head to contralateral side of lesion (SCM). Shoulder droop on side of lesion (trapezius). The left SCM contracts to help turn the head to the right. CN XII lesion LMN lesion. Tongue deviates toward side of lesion (“lick your wounds”) due to weakened tongue muscles on affected side. Facial nerve lesions Bell palsy is the most common cause of peripheral facial palsy A . Usually develops after HSV reactivation. Treatment: corticosteroids +/– acyclovir. Most patients gradually recover function, but aberrant regeneration can occur. Other causes of peripheral facial palsy include Lyme disease, herpes zoster (Ramsay Hunt syndrome), sarcoidosis, tumors (eg, parotid gland), diabetes mellitus.

1	lesion loCAtion Motor cortex, connection from motor cortex to Facial nucleus, anywhere along CN VII facial nucleus in pons musCles inVolVed Lower muscles of facial expression Upper and lower muscles of facial expression ForeheAd inVolVed? Spared, due to bilateral UMN innervation Affected other symPtoms None Incomplete eye closure (dry eyes, corneal ulceration), hyperacusis, loss of taste sensation to anterior tongue Face area of motor cortex Outer ear Visible portion of ear (pinna), includes auditory canal and tympanic membrane. Transfers sound waves via vibration of tympanic membrane. Middle ear Air-filled space with three bones called the ossicles (malleus, incus, stapes). Ossicles conduct and amplify sound from tympanic membrane to inner ear.

1	Middle ear Air-filled space with three bones called the ossicles (malleus, incus, stapes). Ossicles conduct and amplify sound from tympanic membrane to inner ear. Inner ear Snail-shaped, fluid-filled cochlea. Contains basilar membrane that vibrates 2° to sound waves. Vibration transduced via specialized hair cells  auditory nerve signaling • brain stem. Each frequency leads to vibration at specific location on basilar membrane (tonotopy): Low frequency heard at apex near helicotrema (wide and flexible). High frequency heard best at base of cochlea (thin and rigid). Weber test Tuning fork on vertex of skull Rinne test Tuning fork in front of ear (air conduction, AC), Tuning fork on mastoid process (bone conduction, BC) No localization BC > AC Localizes to una˜ected ear ˜ transmission of all sound AC > BCAC > BC Localizes to a˜ected ear ˜ transmission of background noise Types of hearing loss

1	Types of hearing loss Cholesteatoma Overgrowth of desquamated keratin debris within the middle ear space ( A , arrows); may erode ossicles, mastoid air cells  conductive hearing loss. Often presents with painless otorrhea. Conjunctivitis Inflammation of the conjunctiva  red eye A . Allergic—itchy eyes, bilateral. Bacterial—pus; treat with antibiotics. Viral—most common, often adenovirus; sparse mucous discharge, swollen preauricular node, • lacrimation; self-resolving. Cataract Painless, often bilateral, opacification of lens A , often resulting in glare and  vision, especially at night. Acquired risk factors:  age, smoking, excessive alcohol use, excessive sunlight, prolonged corticosteroid use, diabetes mellitus, trauma, infection. Congenital risk factors: classic galactosemia, galactokinase deficiency, trisomies (13, 18, 21), TORCH infections (eg, rubella), Marfan syndrome, Alport syndrome, myotonic dystrophy, neurofibromatosis 2.

1	Iris Lens Suspended from ciliary body by zonule fbers. Muscular fbers and position. Iris Dilator muscle (α1) Sphincter muscle (M3) Ciliary body Trabecularmeshwork Cornea Sclera Canal of Schlemm Anterior chamber “Angle” of the eye Lens Trabecular outfow (90%) Drainage through trabecular meshwork canal of Schlemm episcleral vasculature agonist (eg, carbachol, pilocarpine) ↓↓↓Uveoscleral outfow (10%) Drainage into uvea and sclera with prostaglandin agonists (eg, latanoprost, bimatoprost) Aqueous humor Posterior chamber Produced by nonpigmented epithelium on ciliary body and carbonic anhydrase inhibitors (eg, acetazolamide) by β-blockers (eg, timolol), α2-agonists (eg, brimonidine), Glaucoma Optic disc atrophy with characteristic cupping (normal A versus thinning of outer rim of optic nerve head B ), usually with elevated intraocular pressure (IOP) and progressive peripheral visual field loss if untreated. Treatment is through pharmacologic or surgical lowering of IOP.

1	Open-angle glaucoma Associated with  age, African-American race, family history. Painless, more common in US. Primary—cause unclear. Secondary—blocked trabecular meshwork from WBCs (eg, uveitis), RBCs (eg, vitreous hemorrhage), retinal elements (eg, retinal detachment). Uveitis Inflammation of uvea; specific name based on location within affected eye. Anterior uveitis: iritis; posterior uveitis: choroiditis and/or retinitis. May have hypopyon (accumulation of pus in anterior chamber A ) or conjunctival redness. Associated with systemic inflammatory disorders (eg, sarcoidosis, rheumatoid arthritis, juvenile idiopathic arthritis, HLA-B27–associated conditions). Degeneration of macula (central area of retina). Causes distortion (metamorphopsia) and eventual loss of central vision (scotomas).

1	Degeneration of macula (central area of retina). Causes distortion (metamorphopsia) and eventual loss of central vision (scotomas). Dry (nonexudative, > 80%)—Deposition of yellowish extracellular material (“Drusen”) in between Bruch membrane and retinal pigment epithelium A with gradual  in vision. Prevent progression with multivitamin and antioxidant supplements. Wet (exudative, 10–15%)—rapid loss of vision due to bleeding 2° to choroidal neovascularization. Treat with anti-VEGF (vascular endothelial growth factor) injections (eg, bevacizumab, ranibizumab). Diabetic retinopathy Retinal damage due to chronic hyperglycemia. Two types: (arrows in A ) and macular edema. Treatment: blood sugar control. Proliferative—chronic hypoxia results in new blood vessel formation with resultant traction on retina • retinal detachment. Treatment: anti-VEGF injections, peripheral retinal photocoagulation, surgery. Hypertensive Retinal damage due to chronic uncontrolled HTN.

1	Hypertensive Retinal damage due to chronic uncontrolled HTN. retinopathy Flame-shaped retinal hemorrhages, arteriovenous nicking, microaneurysms, macular star (exudate, red arrow in A ), cotton-wool spots (blue arrow in A ). Presence of papilledema requires immediate lowering of BP. Associated with  risk of stroke, CAD, kidney disease. Retinal vein occlusion Blockage of central or branch retinal vein due to compression from nearby arterial atherosclerosis. Retinal hemorrhage and venous engorgement (“blood and thunder appearance”; arrows in A ), edema in affected area. Separation of neurosensory layer of retina (photoreceptor layer with rods and cones) from outermost pigmented epithelium (normally shields excess light, supports retina)  degeneration of photoreceptors  vision loss. May be 2° to retinal breaks, diabetic traction, inflammatory effusions. Visualized on fundoscopy as crinkling of retinal tissue A and changes in vessel direction.

1	Breaks more common in patients with high myopia and/or history of head trauma. Often preceded by posterior vitreous detachment (“flashes” and “floaters”) and eventual monocular loss of vision like a “curtain drawn down.” Surgical emergency. Central retinal artery Acute, painless monocular vision loss. Retina cloudy with attenuated vessels and “cherry-red” spot occlusion at fovea (center of macula) A . Evaluate for embolic source (eg, carotid artery atherosclerosis, cardiac vegetations, patent foramen ovale). Retinitis pigmentosa Inherited progressive retinal degeneration. Nyctalopia (night blindness)  peripheral vision loss. Bone spicule-shaped deposits A . Papilledema Optic disc swelling (usually bilateral) due to  ICP (eg, 2° to mass effect). Enlarged blind spot and elevated optic disc with blurred margins A . Leukocoria Loss (whitening) of the red reflex. Important causes in children include retinoblastoma A , congenital cataract, toxocariasis.

1	Leukocoria Loss (whitening) of the red reflex. Important causes in children include retinoblastoma A , congenital cataract, toxocariasis. Miosis Constriction, parasympathetic: 1st neuron: Edinger-Westphal nucleus to ciliary ganglion via CN III 2nd neuron: short ciliary nerves to sphincter pupillae muscles Short ciliary nerves shorten the pupil diameter. Pupillary light reflex Light in either retina sends a signal via CN II to pretectal nuclei (dashed lines in image) in midbrain that activates bilateral Edinger-Westphal nuclei; pupils constrict bilaterally (direct and consensual reflex). Result: illumination of 1 eye results in bilateral pupillary constriction.

1	Mydriasis Dilation, sympathetic: 1st neuron: hypothalamus to ciliospinal center of Budge (C8–T2) 2nd neuron: exit at T1 to superior cervical ganglion (travels along cervical sympathetic chain near lung apex, subclavian vessels) 3rd neuron: plexus along internal carotid, through cavernous sinus; enters orbit as long ciliary nerve to pupillary dilator muscles. Sympathetic fibers also innervate smooth muscle of eyelids (minor retractors) and sweat glands of forehead and face. Long ciliary nerves make the pupil diameter longer. Marcus Gunn pupil Also called relative afferent pupillary defect (RAPD). When the light shines into a normal eye, constriction of the ipsilateral (direct reflex) and contralateral eye (consensual reflex) is observed. When the light is then swung to the affected eye, both pupils dilate instead of constrict due to impaired conduction of light signal along the injured optic nerve. Associated with optic neuritis, early multiple sclerosis.

1	Sympathetic denervation of face : PAM is horny (Horner).  Ptosis (slight drooping of eyelid: superior Hypothalamus Ophthalmic divisiontarsal muscle) of trigeminal nerve Anhidrosis (absence of sweating) and Long ciliary nerve flushing of affected side of face To sweat glands of forehead To smooth muscle of eyelid Associated with lesions along the sympathetic chain: Internal carotid To sweat glands of face  1st neuron: pontine hemorrhage, lateral artery medullary syndrome, spinal cord lesion Third neuron above T1 (eg, Brown-Séquard syndrome, First neuron 2nd neuron: stellate ganglion compression lateral horn 3rd neuron: carotid dissection (painful) Spinal cord Superior Superior Superior CN VI innervates the Lateral Rectus. oblique rectus oblique CN IV innervates the Superior Oblique. CN III innervates the Rest. Medial rectus The “chemical formula” LR6SO4R3. muscle Obliques go Opposite (left SO and IO tested with patient looking right). IOU: IO tested looking Up.

1	CN III innervates the Rest. Medial rectus The “chemical formula” LR6SO4R3. muscle Obliques go Opposite (left SO and IO tested with patient looking right). IOU: IO tested looking Up. CN III, IV, VI palsies CN III damage CN III has both motor (central) and parasympathetic (peripheral) components. Common causes include: Cavernous sinus thrombosis • proptosis, involvement of CNs IV, V1/V2, VI Motor output to extraocular muscles—affected primarily by vascular disease (eg, diabetes mellitus: glucose  sorbitol) due to  diffusion of oxygen and nutrients to the interior fibers from compromised vasculature that resides on outside of nerve. Signs: ptosis, “down-and-out” gaze. Parasympathetic output—fibers on the periphery are first affected by compression (eg, PCom aneurysm, uncal herniation). Signs: diminished or absent pupillary light reflex, “blown pupil” often with “down-and-out” gaze A .

1	A . Visual field defects 1. Right anopia (monocular vision loss) 4Opticchiasm7 Macula Opticnerve Optic tractVisual 13 52Dorsal optic radiation(parietallobe)Lt. Rt.Lateralgeniculatebody Defect in visual feld of L eye R eye 2. Bitemporal hemianopia (pituitary lesion, chiasm) 3. 4. Left upper quadrantanopia (right temporal lesion, MCA) 5. loop(right parietal lesion, MCA) (temporal 6. Left hemianopia with macular sparing (right occipital lesion, PCA) 7. Central scotoma (eg, macular degeneration) PCA infarct) 6 Calcarine cortex sulcus Meyer Loop—Lower retina; Loops around inferior horn of Lateral ventricle. Note: When an image hits 1° visual cortex, it is upsideDorsal optic radiation—superior retina; takes down and left-right reversed. shortest path via internal capsule.

1	Note: When an image hits 1° visual cortex, it is upsideDorsal optic radiation—superior retina; takes down and left-right reversed. shortest path via internal capsule. Cavernous sinus Collection of venous sinuses on either side of pituitary. Blood from eye and superficial cortex  cavernous sinus  internal jugular vein. CNs III, IV, V1, V2, and VI plus postganglionic sympathetic pupillary fibers en route to orbit all pass through cavernous sinus. Cavernous portion of internal carotid artery is also here. Cavernous sinus syndrome—presents with variable ophthalmoplegia,  corneal sensation, Horner syndrome and occasional decreased maxillary sensation. 2° to pituitary tumor mass effect, carotid-cavernous fistula, or cavernous sinus thrombosis related to infection.

1	3rd ventricle Oculomotor n. (CN III) Trochlear n. (CN IV) Ophthalmic n. (CN V1) Optic chiasma (CN II) Maxillary n. (CN V2) Pituitary Pia Dura Arachnoid Sphenoid sinus Subarachnoid space Anterior cerebral a. Internal carotid a. Cavernous sinus Abducens n. (CN VI) Medial longitudinal fasciculus (MLF): pair of tracts that allows for crosstalk between CN VI and CN III nuclei. Coordinates both eyes to move in same horizontal direction. Highly myelinated (must communicate quickly so eyes move at same time). Lesions may be unilateral or bilateral (latter classically seen in multiple sclerosis, stroke). Lesion in MLF = internuclear ophthalmoplegia (INO), a conjugate horizontal gaze palsy. Lack of communication such that when CN VI nucleus activates ipsilateral lateral rectus, contralateral CN III nucleus does not stimulate medial rectus to contract. Abducting eye displays nystagmus (CN VI overfires to stimulate CN III). Convergence normal. MLF in MS.

1	MLF in MS. When looking left, the left nucleus of CN VI fires, which contracts the left lateral rectus and stimulates the contralateral (right) nucleus of CN III via the right MLF to contract the right medial rectus. Directional term (eg, right INO, left INO) refers to the eye that is unable to adduct. INO = Ipsilateral adduction failure, Nystagmus Opposite. Sedation, tolerance, Also for eclampsia seizures (1st dependence, respiratory line is MgSO4) depression Diplopia, ataxia, blood 1st line for trigeminal neuralgia dyscrasias (agranulocytosis, aplastic anemia), liver toxicity, teratogenesis (cleft lip/palate, spina bifida), induction of cytochrome P-450, SIADH, SJS Sucks to have Silent causes Fatigue, GI distress, (absence) Seizures Headache, Itching (and urticaria), SJS

1	Sucks to have Silent causes Fatigue, GI distress, (absence) Seizures Headache, Itching (and urticaria), SJS PHENYTOIN: cytochrome P-450 induction, Hirsutism, Enlarged gums, Nystagmus, Yellow-brown skin, Teratogenicity (fetal hydantoin syndrome), Osteopenia, Inhibited folate absorption, Neuropathy. Rare: SJS, DRESS syndrome, SLE-like syndrome. Toxicity leads to diplopia, ataxia, sedation. Sedation, slow cognition, Also used for migraine kidney stones, skinny (weight prophylaxis loss), sight threatened (glaucoma), speech (wordfinding) difficulties GI distress, rare but fatal Also used for myoclonic seizures, bipolar disorder, hepatotoxicity (measure migraine prophylaxis LFTs), pancreatitis, neural tube defects, tremor, weight gain, contraindicated in Vision gone all bad with  GABA. Irreversible GABA * = Common use, ** = 1st line for acute, *** = 1st line for recurrent seizure prophylaxis. Barbiturates Phenobarbital, pentobarbital, thiopental, secobarbital.

1	Barbiturates Phenobarbital, pentobarbital, thiopental, secobarbital. meChAnism Facilitate GABAA action by • duration of Cl− channel opening, thus  neuron firing (barbidurates • duration). CliniCAl use Sedative for anxiety, seizures, insomnia, induction of anesthesia (thiopental). AdVerse eFFeCts Respiratory and cardiovascular depression (can be fatal); CNS depression (can be exacerbated by alcohol use); dependence; drug interactions (induces cytochrome P-450). Overdose treatment is supportive (assist respiration and maintain BP). Contraindicated in porphyria. Benzodiazepines Diazepam, lorazepam, triazolam, temazepam, oxazepam, midazolam, chlordiazepoxide, alprazolam. meChAnism Facilitate GABAA action by • frequency of Cl– channel opening (“frenzodiazepines” • frequency).  REM sleep. Most have long half-lives and active metabolites (exceptions [ATOM]: Alprazolam, Triazolam, Oxazepam, and Midazolam are short acting  higher addictive potential).

1	 REM sleep. Most have long half-lives and active metabolites (exceptions [ATOM]: Alprazolam, Triazolam, Oxazepam, and Midazolam are short acting  higher addictive potential). CliniCAl use Anxiety, panic disorder, spasticity, status epilepticus (lorazepam, diazepam, midazolam), eclampsia, detoxification (especially alcohol withdrawal– DTs), night terrors, sleepwalking, general anesthetic (amnesia, muscle relaxation), hypnotic (insomnia). Lorazepam, Oxazepam, and Temazepam can be used for those with liver disease who drink a LOT due to minimal first-pass metabolism. AdVerse eFFeCts Dependence, additive CNS depression effects with alcohol and barbiturates (all bind the GABAA receptor). Less risk of respiratory depression and coma than with barbiturates. Treat overdose with flumazenil (competitive antagonist at GABA benzodiazepine receptor). Can precipitate seizures by causing acute benzodiazepine withdrawal.

1	Nonbenzodiazepine Zolpidem, Zaleplon, esZopiclone. “These ZZZs put you to sleep.” hypnotics meChAnism Act via the BZ1 subtype of the GABA receptor. Effects reversed by flumazenil. Sleep cycle less affected as compared with benzodiazepine hypnotics. CliniCAl use Insomnia. AdVerse eFFeCts Ataxia, headaches, confusion. Short duration because of rapid metabolism by liver enzymes. Unlike older sedative-hypnotics, cause only modest day-after psychomotor depression and few amnestic effects.  dependence risk than benzodiazepines. meChAnism Orexin (hypocretin) receptor antagonist. Suvorexant is an orexin antagonist. CliniCAl use Insomnia. AdVerse eFFeCts CNS depression (somnolence), headache, abnormal sleep-related activities. Contraindications: narcolepsy, combination with strong CYP3A4 inhibitors. Not recommended in patients with liver disease. Limited physical dependence or abuse potential.

1	AdVerse eFFeCts Dizziness, nausea, fatigue, headache. No dependence (not a controlled substance). meChAnism 5-HT agonists. Inhibit trigeminal nerve A sumo wrestler trips and falls on his head. activation, prevent vasoactive peptide release, induce vasoconstriction. CliniCAl use Acute migraine, cluster headache attacks. AdVerse eFFeCts Coronary vasospasm (contraindicated in patients with CAD or vasospastic angina), mild paresthesia, serotonin syndrome (in combination with other 5-HT agonists). meChAnism • dopamine in brain. Unlike dopamine, l-DOPA can cross blood-brain barrier and is converted by dopa decarboxylase in the CNS to dopamine. Carbidopa, a peripheral DOPA decarboxylase inhibitor, is given with l-DOPA to  bioavailability of l-DOPA in the brain and to limit peripheral side effects. CliniCAl use Parkinson disease.

1	CliniCAl use Parkinson disease. AdVerse eFFeCts Nausea, hallucinations, postural hypotension. With progressive disease, l-DOPA can lead to “onoff” phenomenon with improved mobility during “on” periods, then impaired motor function during “off” periods when patient responds poorly to l-DOPA or medication wears off. Selegiline, rasagiline meChAnism Selectively inhibit MAO-B (metabolize dopamine) • dopamine availability. Selegiline selectively inhibits MAO-B and is more commonly found in the Brain than in the periphery. CliniCAl use Adjunctive agent to l-DOPA in treatment of Parkinson disease. AdVerse eFFeCts May enhance adverse effects of l-DOPA. Inhaled anesthetics Desflurane, halothane, enflurane, isoflurane, sevoflurane, methoxyflurane, N2O. meChAnism Mechanism unknown. eFFeCts Myocardial depression, respiratory depression, postoperative nausea/vomiting,  cerebral blood flow,  cerebral metabolic demand.

1	meChAnism Mechanism unknown. eFFeCts Myocardial depression, respiratory depression, postoperative nausea/vomiting,  cerebral blood flow,  cerebral metabolic demand. AdVerse eFFeCts Hepatotoxicity (halothane), nephrotoxicity (methoxyflurane), proconvulsant (enflurane, epileptogenic), expansion of trapped gas in a body cavity (N2O). Malignant hyperthermia—rare, life-threatening condition in which inhaled anesthetics or succinylcholine induce severe muscle contractions and hyperthermia. Susceptibility is often inherited as autosomal dominant with variable penetrance. Mutations in voltage-sensitive ryanodine receptor (RYR1 gene) cause • Ca2+ release from sarcoplasmic reticulum. Treatment: dantrolene (a ryanodine receptor antagonist). Local anesthetics Esters—procaine, tetracaine, benzocaine, chloroprocaine. Amides—lIdocaIne, mepIvacaIne, bupIvacaIne, ropIvacaIne (amIdes have 2 I’s in name).

1	Local anesthetics Esters—procaine, tetracaine, benzocaine, chloroprocaine. Amides—lIdocaIne, mepIvacaIne, bupIvacaIne, ropIvacaIne (amIdes have 2 I’s in name). meChAnism Block Na+ channels by binding to specific receptors on inner portion of channel. Most effective in rapidly firing neurons. 3° amine local anesthetics penetrate membrane in uncharged form, then bind to ion channels as charged form. Can be given with vasoconstrictors (usually epinephrine) to enhance local action— bleeding,  anesthesia by  systemic concentration. In infected (acidic) tissue, alkaline anesthetics are charged and cannot penetrate membrane effectively  need more anesthetic. Order of nerve blockade: small-diameter fibers > large diameter. Myelinated fibers > unmyelinated fibers. Overall, size factor predominates over myelination such that small myelinated fibers > small unmyelinated fibers > large myelinated fibers > large unmyelinated fibers.

1	Order of loss: (1) pain, (2) temperature, (3) touch, (4) pressure. CliniCAl use Minor surgical procedures, spinal anesthesia. If allergic to esters, give amides. AdVerse eFFeCts CNS excitation, severe cardiovascular toxicity (bupivacaine), hypertension, hypotension, arrhythmias (cocaine), methemoglobinemia (benzocaine). Spasmolytics, antispasmodics meChAnism Act as agonists at opioid receptors (μ = β-endorphin, δ = enkephalin, κ = dynorphin) to modulate synaptic transmission—close presynaptic Ca2+ channels, open postsynaptic K+ channels  synaptic transmission. Inhibit release of ACh, norepinephrine, 5-HT, glutamate, substance P. eFFiCACy Full agonist: morphine, heroin, meperidine, methadone, codeine, fentanyl. Partial agonist: buprenorphine. Mixed agonist/antagonist: nalbuphine, pentazocine, butorphanol. Antagonist: naloxone, naltrexone, methylnaltrexone.

1	CliniCAl use Moderate to severe or refractory pain, diarrhea (loperamide, diphenoxylate), acute pulmonary edema, maintenance programs for heroin addicts (methadone, buprenorphine + naloxone). AdVerse eFFeCts Nausea, vomiting, pruritus, addiction, respiratory depression, constipation, sphincter of Oddi spasm, miosis (except meperidine  mydriasis), additive CNS depression with other drugs. Tolerance does not develop to miosis and constipation. Treat toxicity with naloxone (competitive opioid receptor antagonist) and prevent relapse with naltrexone once detoxified. meChAnism Very weak opioid agonist; also inhibits the Tramadol is a Slight opioid agonist, and a reuptake of norepinephrine and serotonin. Serotonin and norepinephrine reuptake inhibitor. It is used for Stubborn pain, but CliniCAl use Chronic pain. can lower Seizure threshold, and may cause AdVerse eFFeCts Similar to opioids; decreases seizure threshold; Serotonin Syndrome. serotonin syndrome.

1	CliniCAl use Chronic pain. can lower Seizure threshold, and may cause AdVerse eFFeCts Similar to opioids; decreases seizure threshold; Serotonin Syndrome. serotonin syndrome. Glaucoma therapy  IOP via  amount of aqueous humor (inhibit synthesis/secretion or • drainage). BAD humor may not be Politically Correct. β-blockers Timolol, betaxolol, carteolol  aqueous humor synthesis No pupillary or vision changes α-agonists Epinephrine (α1),  aqueous humor synthesis via Mydriasis (α1); do not use in apraclonidine, vasoconstriction (epinephrine) closed-angle glaucoma brimonidine (α2)  aqueous humor synthesis Blurry vision, ocular (apraclonidine, brimonidine) hyperemia, foreign body sensation, ocular allergic reactions, ocular pruritus Diuretics Acetazolamide  aqueous humor synthesis No pupillary or vision changes via inhibition of carbonic anhydrase

1	Diuretics Acetazolamide  aqueous humor synthesis No pupillary or vision changes via inhibition of carbonic anhydrase Prostaglandins Bimatoprost, latanoprost  outflow of aqueous humor via Darkens color of iris (PGF2α)  resistance of flow through (browning), eyelash growth uveoscleral pathway

1	Prostaglandins Bimatoprost, latanoprost  outflow of aqueous humor via Darkens color of iris (PGF2α)  resistance of flow through (browning), eyelash growth uveoscleral pathway Cholinomimetics (M3) Direct: pilocarpine, carbachol  outflow of aqueous humor via Miosis (contraction of pupillary Indirect: physostigmine, contraction of ciliary muscle sphincter muscles) and echothiophate and opening of trabecular cyclospasm (contraction of meshwork ciliary muscle) Use pilocarpine in acute angle closure glaucoma—very effective at opening meshwork into canal of Schlemm “Words of comfort, skillfully administered, are the oldest therapy known to man.” “All men should strive to learn before they die what they are running from, and to, and why.” “The sorrow which has no vent in tears may make other organs weep.” “It’s no use going back to yesterday, because I was a different person then.” —Lewis Carroll, Alice in Wonderland

1	This chapter encompasses overlapping areas in psychiatry, psychology, sociology, and psychopharmacology. High-yield topics include schizophrenia, mood disorders, eating disorders, personality disorders, somatic symptom disorders, substance abuse, and antipsychotic agents. Know the DSM-5 criteria for diagnosing common psychiatric disorders. Operant conditioning Learning in which a particular action is elicited because it produces a punishment or reward. Usually elicits voluntary responses. Reinforcement Target behavior (response) is followed by desired Skinner operant conditioning quadrants: reward (positive reinforcement) or removal of aversive stimulus (negative reinforcement). Punishment Repeated application of aversive stimulus (positive punishment) or removal of desired reward (negative punishment) to extinguish unwanted behavior. Extinction Discontinuation of reinforcement (positive or negative) eventually eliminates behavior. Can occur in operant or classical conditioning.

1	Extinction Discontinuation of reinforcement (positive or negative) eventually eliminates behavior. Can occur in operant or classical conditioning. Transference Patient projects feelings about formative or other important persons onto physician (eg, psychiatrist is seen as parent). Countertransference Doctor projects feelings about formative or other important persons onto patient (eg, patient reminds physician of younger sibling). Ego defenses Thoughts and behaviors (voluntary or involuntary) used to resolve conflict and prevent undesirable feelings (eg, anxiety, depression). Denial Avoiding the awareness of some painful reality. A patient with cancer plans a full-time work schedule despite being warned of significant fatigue during chemotherapy. Mature adults wear a SASH. Lack of basic trust Reactive attachment disorder (infant withdrawn/unresponsive to comfort) Disinhibited social engagement (child indiscriminately attaches to strangers)

1	Lack of basic trust Reactive attachment disorder (infant withdrawn/unresponsive to comfort) Disinhibited social engagement (child indiscriminately attaches to strangers) Child neglect Failure to provide a child with adequate food, shelter, supervision, education, and/or affection. Most common form of child maltreatment. Signs: poor hygiene, malnutrition, withdrawal, impaired social/emotional development, failure to thrive. As with child abuse, suspected child neglect must be reported to local child protective services.

1	Onset before age 12. ≥ 6 months of limited attention span and/or poor impulse control. Characterized by hyperactivity, impulsivity, and/or inattention in ≥ 2 settings (eg, school, home, places of worship). Normal intelligence, but commonly coexists with difficulties in school. Often persists into adulthood. Commonly coexists with oppositional defiant disorder. Treatment: stimulants (eg, methylphenidate) +/– behavioral therapy; alternatives include atomoxetine, guanfacine, clonidine. Conduct disorder Repetitive, pervasive behavior violating societal norms or the basic rights of others (eg, aggression toward people and animals, destruction of property, theft). After age 18, often reclassified as antisocial personality disorder. Treatment: psychotherapy (eg, cognitive behavioral therapy [CBT]).

1	Intellectual disability Global cognitive deficits (vs specific learning disorder) that affect reasoning, memory, abstract thinking, judgment, language, learning. Adaptive functioning is impaired, leading to major difficulties with education, employment, communication, socialization, independence. Treatment: psychotherapy, occupational therapy, special education. Oppositional defiant Enduring pattern of anger and irritability with argumentative, vindictive, and defiant behavior disorder toward authority figures. Treatment: psychotherapy (eg, CBT). Selective mutism Onset before age 5. Anxiety disorder lasting ≥ 1 month involving refraining from speech in certain situations despite speaking in other, usually more comfortable situations. Development (eg, speech and language) not typically impaired. Interferes with social, academic, and occupational tasks. Commonly coexists with social anxiety disorder. Treatment: behavioral, family, and play therapy; SSRIs.

1	Tourette syndrome Onset before age 18. Sudden, Sudden, recurrent, nonrhythmic, stereotyped motor and vocal tics that persist for > 1 year. Coprolalia (involuntary obscene speech) found in some patients. Associated with OCD and ADHD. Treatment: psychoeducation, behavioral therapy. For intractable and distressing tics, high-potency antipsychotics (eg, haloperidol, fluphenazine), tetrabenazine, α2-agonists (eg, guanfacine, clonidine), or atypical antipsychotics. Orientation Patients’ ability to know the date and time, where they are, and who they are (order of loss: time  place  person). Common causes of loss of orientation: alcohol, drugs, fluid/electrolyte imbalance, head trauma, hypoglycemia, infection, nutritional deficiencies, hypoxia. Depersonalization/ Persistent feelings of detachment or estrangement from one’s own body, thoughts, perceptions, derealization and actions (depersonalization) or one’s environment (derealization). Intact reality testing (vs disorder psychosis).

1	Delirium “Waxing and waning” level of consciousness with acute onset,  attention span,  level of arousal. Characterized by disorganized thinking, hallucinations (often visual), misperceptions (eg, illusions), disturbance in sleep-wake cycle, cognitive dysfunction, agitation. Reversible. Usually 2° to other identifiable illness (eg, CNS disease, infection, trauma, substance abuse/ withdrawal, metabolic/electrolyte disturbances, hemorrhage, urinary/fecal retention), or medications (eg, anticholinergics), especially in the elderly. Most common presentation of altered mental status in inpatient setting, especially in the ICU or during prolonged hospital stays. EEG may show diffuse background rhythm slowing. Delirium = changes in sensorium.

1	Delirium = changes in sensorium. Treatment: identification and management of underlying condition. Orientation protocols (eg, keeping a clock or calendar nearby),  sleep disturbances, and  cognitive stimulation to manage symptoms. Antipsychotics as needed. Avoid unnecessary restraints and drugs that may worsen delirium (eg, anticholinergics, benzodiazepines, opioids). Psychosis Distorted perception of reality characterized by delusions, hallucinations, and/or disorganized thought/speech. Can occur in patients with medical illness, psychiatric illness, or both. Delusions False, fixed, idiosyncratic beliefs that persist despite evidence to the contrary and are not typical of a patient’s culture or religion (eg, a patient who believes that others are reading his thoughts). Types include erotomanic, grandiose, jealous, persecutory, somatic, mixed, and unspecified. Disorganized thought Speech may be incoherent (“word salad”), tangential, or derailed (“loose associations”).

1	Disorganized thought Speech may be incoherent (“word salad”), tangential, or derailed (“loose associations”). Hallucinations Perceptions in the absence of external stimuli (eg, seeing a light that is not actually present). Contrast with misperceptions (eg, illusions) of real external stimuli. Types include: Auditory—more commonly due to psychiatric illness (eg, schizophrenia) than medical illness. Visual—more commonly due to medical illness (eg, drug intoxication, delirium) than psychiatric illness. Tactile—common in alcohol withdrawal and stimulant use (eg, “cocaine crawlies,” a type of delusional parasitosis). Olfactory—often occur as an aura of temporal lobe epilepsy (eg, burning rubber) and in brain tumors. Gustatory—rare, but seen in epilepsy. Hypnagogic—occurs while going to sleep. Sometimes seen in narcolepsy. Hypnopompic—occurs while waking from sleep (“get pomped up in the morning”). Sometimes seen in narcolepsy.

1	Hypnagogic—occurs while going to sleep. Sometimes seen in narcolepsy. Hypnopompic—occurs while waking from sleep (“get pomped up in the morning”). Sometimes seen in narcolepsy. Schizophrenia Chronic illness causing profound functional impairment. Symptom categories include: Positive—hallucinations, delusions, unusual thought processes, disorganized speech, bizarre behavior Negative—flat or blunted affect, apathy, anhedonia, alogia, social withdrawal Cognitive—reduced ability to understand or make plans, diminished working memory, inattention Diagnosis requires ≥ 2 of the following active symptoms, including ≥ 1 from symptoms #1–3: 1. 2. Hallucinations, often auditory 3. 4. 5. Requires ≥ 1 month of active symptoms over the past 6 months; onset ≥ 6 months prior to diagnosis.

1	2. Hallucinations, often auditory 3. 4. 5. Requires ≥ 1 month of active symptoms over the past 6 months; onset ≥ 6 months prior to diagnosis. Associated with altered dopaminergic activity,  serotonergic activity, and  dendritic branching. Ventriculomegaly on brain imaging. Lifetime prevalence—1.5% (males > females). Presents earlier in men (late teens to early 20s) than in women (late 20s to early 30s).  suicide risk. Heavy cannabis use in adolescence is associated with  incidence and worsened course of psychotic, mood, and anxiety disorders. Treatment: atypical antipsychotics (eg, risperidone) are first line. Negative symptoms often persist after treatment, despite resolution of positive symptoms. Brief psychotic disorder—≥ 1 positive symptom(s) lasting < 1 month, usually stress-related. Schizophreniform disorder—≥ 2 symptoms lasting 1–6 months.

1	Brief psychotic disorder—≥ 1 positive symptom(s) lasting < 1 month, usually stress-related. Schizophreniform disorder—≥ 2 symptoms lasting 1–6 months. Schizoaffective Shares symptoms with both schizophrenia and mood disorders (major depressive or bipolar disorder disorder). To differentiate from a mood disorder with psychotic features, patient must have > 2 weeks of psychotic symptoms without a manic or depressive episode. Delusional disorder ≥ 1 delusion(s) lasting > 1 month, but without a mood disorder or other psychotic symptoms. Daily functioning, including socialization, may be impacted by the pathological, fixed belief but is otherwise unaffected. Can be shared by individuals in close relationships (folie à deux). Schizotypal personality Cluster A personality disorder that also falls on the schizophrenia spectrum. May include brief disorder psychotic episodes (eg, delusions) that are less frequent and severe than in schizophrenia.

1	Mood disorder Characterized by an abnormal range of moods or internal emotional states and loss of control over them. Severity of moods causes distress and impairment in social and occupational functioning. Includes major depressive, bipolar, dysthymic, and cyclothymic disorders. Episodic superimposed psychotic features (delusions, hallucinations, disorganized speech/behavior) may be present. Manic episode Distinct period of abnormally and persistently elevated, expansive, or irritable mood and  activity or energy lasting ≥ 1 week. Diagnosis requires hospitalization or marked functional impairment with ≥ 3 of the following (manics DIG FAST): Distractibility • Flight of ideas—racing thoughts Impulsivity/Indiscretion—seeks pleasure •  goal-directed Activity/psychomotor without regard to consequences (hedonistic) Agitation

1	Distractibility • Flight of ideas—racing thoughts Impulsivity/Indiscretion—seeks pleasure •  goal-directed Activity/psychomotor without regard to consequences (hedonistic) Agitation Hypomanic episode Similar to a manic episode except mood disturbance is not severe enough to cause marked impairment in social and/or occupational functioning or to necessitate hospitalization. Abnormally  activity or energy usually present. No psychotic features. Lasts ≥ 4 consecutive days. length of time). Bipolar II—a hypomanic and a depressive episode (no history of manic episodes). Patient’s mood and functioning usually normalize between episodes. Use of antidepressants can destabilize mood. High suicide risk. Treatment: mood stabilizers (eg, lithium, valproic acid, carbamazepine, lamotrigine), atypical antipsychotics.

1	Cyclothymic disorder—milder form of bipolar disorder fluctuating between mild depressive and hypomanic symptoms. Must last ≥ 2 years with symptoms present at least half of the time, with any remission lasting ≤ 2 months. Recurrent episodes lasting ≥ 2 weeks characterized by ≥ 5 of 9 diagnostic symptoms (must include depressed mood or anhedonia) (DIGS SPACE): Guilt or feelings of worthlessness Screen for previous manic or hypomanic episodes to rule out bipolar disorder. Treatment: CBT and SSRIs are first line. Also SNRIs, mirtazapine, bupropion, electroconvulsive therapy (ECT). Persistent depressive Often milder than MDD; ≥ 2 depressive symptoms lasting ≥ 2 years (≥ 1 year in children), with any disorder (dysthymia) remission lasting ≤ 2 months.

1	Persistent depressive Often milder than MDD; ≥ 2 depressive symptoms lasting ≥ 2 years (≥ 1 year in children), with any disorder (dysthymia) remission lasting ≤ 2 months. MDD with seasonal Formerly called seasonal affective disorder. Major depressive episodes occurring only during a pattern particular season (usually winter) in ≥ 2 consecutive years and in most years across a lifetime. Atypical symptoms common. Depression with atypical features Characterized by mood reactivity (transient improvement in response to a positive event), hypersomnia, hyperphagia, leaden paralysis (heavy feeling in arms and legs), long-standing interpersonal rejection sensitivity. Most common subtype of depression. Treatment: CBT and SSRIs are first line. MAO inhibitors (MAOIs) are effective but not first line because of their risk profile.

1	MDD with peripartum 10–15% incidence rate. Formerly called postpartum depression. Meets MDD criteria with onset no onset later than 1 year after delivery. Treatment: CBT and SSRIs are first line. Postpartum psychosis 0.1–0.2% incidence rate. Characterized by mood-congruent delusions, hallucinations, and thoughts of harming the baby or self. Risk factors include first pregnancy, family history, bipolar disorder, psychotic disorder, recent medication change. Treatment: hospitalization and initiation of atypical antipsychotic; if insufficient, ECT may be used.

1	Grief The five stages of grief per the Kübler-Ross model are denial, anger, bargaining, depression, and acceptance (may occur in any order). Other normal grief symptoms include shock, guilt, sadness, anxiety, yearning, and somatic symptoms that usually occur in waves. Simple hallucinations of the deceased person are common (eg, hearing the deceased speaking). Any thoughts of dying are limited to joining the deceased (vs complicated grief). Duration varies widely; usually resolves within 6–12 months. Persistent complex bereavement disorder involves obsessive preoccupation with the deceased and causes functional impairment, lasting at least 12 months (6 months in children). Can also meet criteria for major depressive episode. SAD PERSONS are more likely to complete suicide. Most common method in US is firearms; access to guns  risk of suicide completion. Women try more often; men complete more often.

1	SAD PERSONS are more likely to complete suicide. Most common method in US is firearms; access to guns  risk of suicide completion. Women try more often; men complete more often. Other risk factors include recent psychiatric hospitalization and family history of completed suicide. Anxiety disorders Inappropriate experiences of fear/worry and their physical manifestations incongruent with the magnitude of the stressors. Symptoms are not attributable to another psychiatric disorder, medical condition (eg, hyperthyroidism), or substance abuse. Includes panic disorder, phobias, generalized anxiety disorder, and selective mutism. Phobias Severe, persistent (≥ 6 months) fear or anxiety due to presence or anticipation of a specific object or situation. Person often recognizes fear is excessive. Treatment: CBT with exposure therapy.

1	Social anxiety disorder—exaggerated fear of embarrassment in social situations (eg, public speaking, using public restrooms). Treatment: CBT, SSRIs, venlafaxine. For performance type (eg, anxiety restricted to public speaking), use β-blockers or benzodiazepines as needed. Agoraphobia—irrational fear/anxiety while facing or anticipating ≥ 2 specific situations (eg, open/ closed spaces, lines, crowds, public transport). If severe, patients may refuse to leave their homes. Associated with panic disorder. Treatment: CBT, SSRIs. Trichotillomania Compulsively pulling out one’s hair. Causes significant distress and persists despite attempts to stop. Presents with areas of thinning hair or baldness on any area of the body, most commonly the scalp A . Incidence highest in childhood but spans all ages. Treatment: psychotherapy.

1	A . Incidence highest in childhood but spans all ages. Treatment: psychotherapy. Adjustment disorder Emotional or behavioral symptoms (eg, anxiety, outbursts) that occur within 3 months of an identifiable psychosocial stressor (eg, divorce, illness) lasting < 6 months once the stressor has ended. If symptoms persist > 6 months after stressor ends, it is GAD. Symptoms do not meet criteria for MDD. Treatment: CBT is first line; antidepressants and anxiolytics may be considered. Schizoid Voluntary social withdrawal (Aloof), limited emotional expression, content with social isolation (vs avoidant). Factitious disorders Symptoms are intentional, motivation is unconscious. Patient consciously creates physical and/or psychological symptoms in order to assume “sick role” and to get medical attention and sympathy (1° [internal] gain).

1	Conversion disorder Also called functional neurologic symptom disorder. Loss of sensory or motor function (eg, paralysis, blindness, mutism), often following an acute stressor; patient may be aware of but indifferent toward symptoms (la belle indifférence); more common in females, adolescents, and young adults. Illness anxiety Preoccupation with acquiring or having a serious illness, often despite medical evaluation and disorder reassurance; minimal to no somatic symptoms.

1	Illness anxiety Preoccupation with acquiring or having a serious illness, often despite medical evaluation and disorder reassurance; minimal to no somatic symptoms. Bulimia nervosa Recurring episodes of binge eating with compensatory purging behaviors at least weekly over the last 3 months. BMI often normal or slightly overweight (vs anorexia). Associated with parotid gland hypertrophy (may see • serum amylase), enamel erosion, Mallory-Weiss syndrome, electrolyte disturbances (eg,  K+ , • Cl−), metabolic alkalosis, dorsal hand calluses from induced vomiting (Russell sign). Treatment: psychotherapy, nutritional rehabilitation, antidepressants (eg, SSRIs). Bupropion is contraindicated due to seizure risk.

1	Sleep terror disorder Periods of inconsolable terror with screaming in the middle of the night. Most common in children. Occurs during slow-wave/deep (stage N3) non-REM sleep with no memory of the arousal episode, as opposed to nightmares that occur during REM sleep (remembering a scary dream). Triggers include emotional stress, fever, and lack of sleep. Usually self limited. Enuresis Nighttime urinary incontinence ≥ 2 times/week for ≥ 3 months in person > 5 years old. First-line treatment: behavioral modification (eg, scheduled voids, nighttime fluid restriction) and positive reinforcement. For refractory cases: bedwetting alarm, oral desmopressin (ADH analog; preferred over imipramine due to fewer side effects).

1	Narcolepsy Excessive daytime sleepiness (despite awakening well-rested) with recurrent episodes of rapid-onset, overwhelming sleepiness ≥ 3 times/week for the last 3 months. Due to • orexin (hypocretin) production in lateral hypothalamus and dysregulated sleep-wake cycles. Associated with: Hypnagogic (just before going to sleep) or hypnopompic (just before awakening; get pomped up in the morning) hallucinations. Nocturnal and narcoleptic sleep episodes that start with REM sleep (sleep paralysis). Cataplexy (loss of all muscle tone following strong emotional stimulus, such as laughter). Treatment: good sleep hygiene (scheduled naps, regular sleep schedule), daytime stimulants (eg, amphetamines, modafinil) and/or nighttime sodium oxybate (GHB). Stages of change in overcoming addiction Maladaptive pattern of substance use involving ≥ 2 of the following in the past year: Intense, distracting cravings Using more, or longer, than intended

1	Stages of change in overcoming addiction Maladaptive pattern of substance use involving ≥ 2 of the following in the past year: Intense, distracting cravings Using more, or longer, than intended Persistent desire but inability to cut down Time-consuming substance acquisition, use, or recovery Impaired functioning at work, school, or home > 1 episode of use involving danger (eg, unsafe sex, driving while impaired) Continued use despite awareness of harm 1. 2. Contemplation—acknowledging problem, but unwilling to change 3. 4. 5. 6. Relapse—(if applicable) returning to old behaviors and abandoning changes Nonspecific: mood elevation,  anxiety, sedation, Nonspecific: anxiety, tremor, seizures, behavioral disinhibition, respiratory depression. insomnia. Alcohol Emotional lability, slurred speech, ataxia, coma, blackouts. Serum γ-glutamyltransferase (GGT)—sensitive indicator of alcohol use. AST value is 2× ALT value (“ToAST 2 ALcohol”). Treatment: benzodiazepines.

1	Nonspecific: mood elevation, • appetite, Nonspecific: post-use “crash,” including psychomotor agitation, insomnia, cardiac depression, lethargy,  appetite, sleep arrhythmias, tachycardia, anxiety. disturbance, vivid nightmares. Alcohol use disorder Physiologic tolerance and dependence on alcohol with symptoms of withdrawal when intake is interrupted. Complications: vitamin B1 (thiamine) deficiency, alcoholic cirrhosis, hepatitis, pancreatitis, peripheral neuropathy, testicular atrophy. Treatment: naltrexone (reduces cravings), acamprosate, disulfiram (to condition the patient to abstain from alcohol use). Support groups such as Alcoholics Anonymous are helpful in sustaining abstinence and supporting patient and family.

1	Wernicke-Korsakoff Results from vitamin B1 deficiency. Symptoms can be precipitated by administering dextrose syndrome before vitamin B1. Triad of confusion, ophthalmoplegia, ataxia (Wernicke encephalopathy). May progress to irreversible memory loss, confabulation, personality change (Korsakoff syndrome). Treatment: IV vitamin B1 (before dextrose). Behavioral therapy Teaches patients how to identify and change maladaptive behaviors or reactions to stimuli. Examples include systematic desensitization for treatment of phobia. Cognitive behavioral Teaches patients to recognize distortions in their thought processes, develop constructive coping therapy skills, and • maladaptive coping behaviors • greater emotional control and tolerance of distress. Examples include recognizing triggers for alcohol consumption. Dialectical behavioral Designed for use in borderline personality disorder, but can be used in other psychiatric conditions therapy as well (eg, depression).

1	Dialectical behavioral Designed for use in borderline personality disorder, but can be used in other psychiatric conditions therapy as well (eg, depression). Interpersonal therapy Focused on improving interpersonal relationships and communication skills. Supportive therapy Utilizes empathy to help individuals during a time of hardship to maintain optimism or hope. for selected ADHD Stimulants (methylphenidate, amphetamines) psychiatric conditions Alcohol withdrawal Benzodiazepines (eg, chlordiazepoxide, lorazepam, diazepam) Generalized anxiety disorder SSRIs, SNRIs Obsessive-compulsive disorder SSRIs, venlafaxine, clomipramine Panic disorder SSRIs, venlafaxine, benzodiazepines PTSD SSRIs, venlafaxine MEchaNisM  catecholamines in the synaptic cleft, especially norepinephrine and dopamine. cliNical UsE ADHD, narcolepsy, binge-eating disorder. aDVErsE EFFEcts Nervousness, agitation, anxiety, insomnia, anorexia, tachycardia, hypertension, weight loss, tics, bruxism.

1	cliNical UsE ADHD, narcolepsy, binge-eating disorder. aDVErsE EFFEcts Nervousness, agitation, anxiety, insomnia, anorexia, tachycardia, hypertension, weight loss, tics, bruxism. Typical antipsychotics Haloperidol, pimozide, trifluoperazine, fluphenazine, thioridazine, chlorpromazine. MEchaNisM Block dopamine D2 receptor (• cAMP). cliNical UsE Schizophrenia (1° positive symptoms), psychosis, bipolar disorder, delirium, Tourette syndrome, Huntington disease, OCD. Use with caution in dementia. PotENcy High potency: Haloperidol, Trifluoperazine, Fluphenazine (Hal Tries to Fly High)—more neurologic side effects (eg, extrapyramidal symptoms [EPS]). Low potency: Chlorpromazine, Thioridazine (Cheating Thieves are low)—more anticholinergic, antihistamine, α1-blockade effects.

1	aDVErsE EFFEcts Lipid soluble  stored in body fat  slow to be removed from body. Endocrine: dopamine receptor antagonism • hyperprolactinemia  galactorrhea, oligomenorrhea, gynecomastia. Metabolic: dyslipidemia, weight gain, hyperglycemia. Antimuscarinic: dry mouth, constipation. Antihistamine: sedation. α1-blockade: orthostatic hypotension. Cardiac: QT prolongation. Ophthalmologic: Chlorpromazine—Corneal deposits; Thioridazine—reTinal deposits. Neuroleptic malignant syndrome. Extrapyramidal symptoms— ADAPT: Hours to days: Acute Dystonia (muscle spasm, stiffness, oculogyric crisis). Treatment: benztropine, diphenhydramine. Days to months: Akathisia (restlessness). Treatment: β-blockers, benztropine, benzodiazepines. Parkinsonism (bradykinesia). Treatment: benztropine, amantadine. Months to years: Tardive dyskinesia (chorea, especially orofacial). Treatment: atypical antipsychotics (eg, clozapine), valbenazine, deutetrabenazine.

1	Months to years: Tardive dyskinesia (chorea, especially orofacial). Treatment: atypical antipsychotics (eg, clozapine), valbenazine, deutetrabenazine. MAOIs AXON POSTSYNAPTIC NEURON Mirtazapine TCAs, SSRIs, SNRIs, trazodone MAO Metabolites ----NE NE reuptake ˜2 (autoreceptor) adrenergic receptor Metabolites 5-HT 5-HT reuptake 5-HT receptorNE receptor -MAO AXON -TCAs, SNRIs, bupropion Selective serotonin reuptake inhibitors Fluoxetine, fluvoxamine, paroxetine, sertraline, escitalopram, citalopram. MEchaNisM Inhibit 5-HT reuptake. It normally takes 4–8 weeks for antidepressants to show appreciable effect. cliNical UsE Depression, generalized anxiety disorder, panic disorder, OCD, bulimia, binge-eating disorder, social anxiety disorder, PTSD, premature ejaculation, premenstrual dysphoric disorder. aDVErsE EFFEcts Fewer than TCAs. Serotonin syndrome, GI distress, SIADH, sexual dysfunction (anorgasmia,  libido).

1	aDVErsE EFFEcts Fewer than TCAs. Serotonin syndrome, GI distress, SIADH, sexual dysfunction (anorgasmia,  libido). Serotoninnorepinephrine reuptake inhibitors Venlafaxine, desvenlafaxine, duloxetine, levomilnacipran, milnacipran. MEchaNisM Inhibit 5-HT and NE reuptake. cliNical UsE Depression, generalized anxiety disorder, diabetic neuropathy. Venlafaxine is also indicated for social anxiety disorder, panic disorder, PTSD, OCD. Duloxetine and milnacipran are also indicated for fibromyalgia. aDVErsE EFFEcts  BP, stimulant effects, sedation, nausea. Tricyclic Amitriptyline, nortriptyline, imipramine, desipramine, clomipramine, doxepin, amoxapine. antidepressants MEchaNisM TCAs inhibit 5-HT and NE reuptake. cliNical UsE MDD, peripheral neuropathy, chronic neuropathic pain, migraine prophylaxis, OCD (clomipramine), nocturnal enuresis (imipramine, although adverse effects may limit use).

1	cliNical UsE MDD, peripheral neuropathy, chronic neuropathic pain, migraine prophylaxis, OCD (clomipramine), nocturnal enuresis (imipramine, although adverse effects may limit use). aDVErsE EFFEcts Sedation, α1-blocking effects including postural hypotension, and atropine-like (anticholinergic) side effects (tachycardia, urinary retention, dry mouth). 3° TCAs (amitriptyline) have more anticholinergic effects than 2° TCAs (nortriptyline). Can prolong QT interval. Tri-CyCliC’s: Convulsions, Coma, Cardiotoxicity (arrhythmia due to Na+ channel inhibition); also respiratory depression, hyperpyrexia. Confusion and hallucinations are more common in the elderly due to anticholinergic side effects (2° amines [eg, nortriptyline] better tolerated). Treatment: NaHCO3 to prevent arrhythmia. Monoamine oxidase Tranylcypromine, Phenelzine, Isocarboxazid, Selegiline (selective MAO-B inhibitor). inhibitors (MAO Takes Pride In Shanghai).

1	Monoamine oxidase Tranylcypromine, Phenelzine, Isocarboxazid, Selegiline (selective MAO-B inhibitor). inhibitors (MAO Takes Pride In Shanghai). MEchaNisM Nonselective MAO inhibition  levels of amine neurotransmitters (norepinephrine, 5-HT, dopamine). cliNical UsE Atypical depression, anxiety. Parkinson disease (selegiline). aDVErsE EFFEcts CNS stimulation; hypertensive crisis, most notably with ingestion of tyramine. Contraindicated with SSRIs, TCAs, St. John’s wort, meperidine, dextromethorphan, linezolid (to avoid precipitating serotonin syndrome). Wait 2 weeks after stopping MAOIs before starting serotonergic drugs or stopping dietary restrictions. Bupropion Inhibits NE and DA reuptake. Also used for smoking cessation. Toxicity: stimulant effects (tachycardia, insomnia), headache, seizures in patients with bulimia and anorexia nervosa. Favorable sexual side effect profile.

1	Mirtazapine α2-antagonist ( release of NE and 5-HT), potent 5-HT2 and 5-HT3 receptor antagonist, and H1 antagonist. Toxicity: sedation (which may be desirable in depressed patients with insomnia), • appetite, weight gain (which may be desirable in underweight patients), dry mouth. Trazodone Primarily blocks 5-HT2, α1-adrenergic, and H1 receptors; also weakly inhibits 5-HT reuptake. Used primarily for insomnia, as high doses are needed for antidepressant effects. Toxicity: sedation, nausea, priapism, postural hypotension. Think traZZZobone due to sedative and male-specific side effects. Varenicline Nicotinic ACh receptor partial agonist. Used for smoking cessation. Toxicity: sleep disturbance, depressed mood, suicidal ideation. Varenicline helps nicotine cravings decline. Methadone Long-acting oral opiate used for heroin detoxification or long-term maintenance therapy. Buprenorphine Sublingual form (partial agonist) used to prevent relapse.

1	Methadone Long-acting oral opiate used for heroin detoxification or long-term maintenance therapy. Buprenorphine Sublingual form (partial agonist) used to prevent relapse. Naloxone Short-acting opioid antagonist given IM, IV, or as a nasal spray to treat acute opioid overdose, particularly to reverse respiratory and CNS depression. Naltrexone Long-acting oral opioid antagonist used after detoxification to prevent relapse. Use naltrexone for the long trex back to sobriety. “But I know all about love already. I know precious little still about kidneys.” —Aldous Huxley, Antic Hay “This too shall pass. Just like a kidney stone.” “I drink too much. The last time I gave a urine sample it had an olive in it.”

1	Being able to understand and apply renal physiology will be critical for the exam. Important topics include electrolyte disorders, acid-base derangements, glomerular disorders (including histopathology), acute and chronic kidney disease, urine casts, diuretics, ACE inhibitors, and AT-II receptor blockers. Renal anomalies associated with various congenital defects are also high-yield associations to think about when evaluating pediatric vignettes. Kidney embryology Pronephros—week 4; then degenerates. Mesonephros—functions as interim kidney for 1st trimester; later contributes to male genital system. Metanephros—permanent; first appears in 5th week of gestation; nephrogenesis continues through weeks 32–36 of gestation. Ureteric bud (metanephric diverticulum)— derived from caudal end of mesonephric duct; gives rise to ureter, pelvises, calyces, collecting ducts; fully canalized by 10th week

1	Ureteric bud (metanephric diverticulum)— derived from caudal end of mesonephric duct; gives rise to ureter, pelvises, calyces, collecting ducts; fully canalized by 10th week Metanephric mesenchyme (ie, metanephric blastema)—ureteric bud interacts with this tissue; interaction induces differentiation and formation of glomerulus through to distal convoluted tubule (DCT) Aberrant interaction between these 2 tissues may result in several congenital malformations of the kidney (eg, renal agenesis, multicystic dysplastic kidney) Ureteropelvic junction—last to canalize  congenital obstruction. Most common cause of prenatal hydronephrosis. Detected by prenatal ultrasound. Oligohydramnios  compression of developing fetus  limb deformities, facial anomalies (eg, low-set ears and retrognathia A , flattened nose), compression of chest and lack of amniotic fluid aspiration into fetal lungs  pulmonary hypoplasia (cause of death).

1	Causes include ARPKD, obstructive uropathy (eg, posterior urethral valves), bilateral renal agenesis, chronic placental insufficiency. Babies who can’t “Pee” in utero develop Potter sequence. POTTER sequence associated with: Pulmonary hypoplasia Oligohydramnios (trigger) Twisted face Twisted skin Extremity defects Renal failure (in utero) Inferior poles of both kidneys fuse abnormally A . As they ascend from pelvis during fetal development, horseshoe kidneys get trapped under inferior mesenteric artery and remain low in the abdomen. Kidneys function normally. Associated with hydronephrosis (eg, ureteropelvic junction obstruction), renal stones, infection,  risk of renal cancer. Higher incidence in chromosomal aneuploidy (eg, Turner syndrome, trisomies 13, 18, 21). *Components of glomerular fltration barrier. Cross-section of glomerulus Left kidney is taken during living donor transplantation because it has a longer renal vein. Afferent = Arriving. Efferent = Exiting.

1	Cross-section of glomerulus Left kidney is taken during living donor transplantation because it has a longer renal vein. Afferent = Arriving. Efferent = Exiting. Renal blood flow: renal artery  segmental artery • interlobar artery  arcuate artery A • efferent arteriole  vasa recta/peritubular capillaries • venous outflow. Left renal vein receives two additional veins: left suprarenal and left gonadal veins. Despite high overall renal blood flow, renal medulla receives significantly less blood flow than renal cortex  very sensitive to hypoxia  vulnerable to ischemic damage. Course of ureters Course of ureter A : arises from renal pelvis, Water (ureters) flows over the iliacs and under travels under gonadal arteries • over common the bridge (uterine artery or vas deferens). (retroperitoneal). Gynecologic procedures (eg, ligation of  ureteral obstruction or leak. (in female) Bladder contraction compresses the intravesical ureter, preventing urine reflux.

1	(retroperitoneal). Gynecologic procedures (eg, ligation of  ureteral obstruction or leak. (in female) Bladder contraction compresses the intravesical ureter, preventing urine reflux. Blood supply to ureter: Middle—gonadal artery, aorta, common and internal iliac arteries 3 common points of ureteral obstruction: ureteropelvic junction, pelvic inlet, ureterovesical junction. Body mass: 70 kg HIKIN’: HIgh K+ INtracellularly. 60–40–20 rule (% of body weight for average 60% of body mass = 42 kg 42 L 40% of body mass = 28 kg person): 40% ICF, mainly composed of K+, Mg2+, organic phosphates (eg, ATP) 20% ECF, mainly composed of Na+, Cl–, Extracellular fuid (ECF)~ 14 kg (20% of 70 kg) Intracellular fuid (ICF)~ 28 kg (40% of 70 kg) Interstitial fuid = 75% ECF 10.5 L 10.5 kg Blood volume 6 L Plasma = 25% ECF 3.5 L 3.5 kg RBC volume 2.8 L HCO3–, albumin Normal Hct = 45% Plasma volume can be measured by Hct (%) 3 [Hb] in g/dL radiolabeling albumin.

1	HCO3–, albumin Normal Hct = 45% Plasma volume can be measured by Hct (%) 3 [Hb] in g/dL radiolabeling albumin. or mannitol. Serum osmolality = 285–295 mOsm/kg H2O. Plasma volume = TBV × (1 – Hct). Glomerular filtration Responsible for filtration of plasma according to Charge barrier—all 3 layers contain ⊝ charged barrier size and charge selectivity. glycoproteins that prevent entry of ⊝ charged Composed of: Basement membrane with type IV collagen chains and heparan sulfate Visceral epithelial layer consisting of podocyte foot processes (FPs) molecules (eg, albumin). Size barrier—fenestrated capillary endothelium (prevents entry of > 100 nm molecules/blood cells); podocyte foot processes interpose with glomerular basement membrane (GBM); slit diaphragm (prevents entry of molecules > 50–60 nm). Glomerular filtration Inulin clearance can be used to calculate GFR 14 rate because it is freely filtered and is neither reabsorbed nor secreted. 12

1	Glomerular filtration Inulin clearance can be used to calculate GFR 14 rate because it is freely filtered and is neither reabsorbed nor secreted. 12 Creatinine clearance is an approximate measure of GFR. Slightly overestimates GFR because 2 creatinine is moderately secreted by renal tubules. coefficient). Normal GFR ≈ 100 mL/min. Filtration Filtration fraction (FF) = GFR/RPF. GFR can be estimated with creatinine Normal FF = 20%. clearance. Filtered load (mg/min) = GFR (mL/min) RPF is best estimated with PAH clearance. × plasma concentration (mg/mL). Prostaglandins Dilate Afferent arteriole (PDA). Angiotensin II Constricts Efferent arteriole (ACE). Prostaglandins preferentially dilate aƒerent arteriole Bowman capsule GFR, so no ˜ FF) (parietal layer) RPF, RPF, GFR, so Calculation of Filtered load = GFR × P. reabsorption and Excretion rate = V × Ux.

1	RPF, RPF, GFR, so Calculation of Filtered load = GFR × P. reabsorption and Excretion rate = V × Ux. secretion rate Reabsorption rate = filtered – excreted. Secretion rate = excreted – filtered. FeNa = fractional excretion of sodium. Glucose clearance Glucose at a normal plasma level (range 60–120 mg/dL) is completely reabsorbed in proximal convoluted tubule (PCT) by Na+/glucose cotransport. In adults, at plasma glucose of ∼ 200 mg/dL, glucosuria begins (threshold). At rate of ∼ 375 mg/min, all transporters are fully saturated (Tm). Normal pregnancy is associated with  GFR. With  filtration of all substances, including glucose, the glucose threshold occurs at lower plasma glucose concentrations  glucosuria at normal plasma glucose levels. Sodium-glucose cotransporter 2 (SGLT2) inhibitors (eg, -flozin drugs) result in glucosuria at plasma concentrations < 200 mg/dL. Glucosuria is an important clinical clue to diabetes mellitus.

1	Glucosuria is an important clinical clue to diabetes mellitus. Splay phenomenon—Tm for glucose is reached gradually rather than sharply due to the heterogeneity of nephrons (ie, different Tm points); represented by the portion of the titration curve between threshold and T . Early PCT—contains brush border. Reabsorbs all glucose and amino acids and most HCO3–, Na+, Cl–, PO43–, K+, H2O, and uric acid. Isotonic absorption. Generates and secretes NH3, which enables the kidney to secrete more H+. PTH—inhibits Na+/PO43– cotransport •• PO43– excretion. AT II—stimulates Na+/H+ exchange •• Na+, H2O, and HCO3− reabsorption (permitting contraction alkalosis). 65–80% Na+ and H2O reabsorbed. Thin descending loop of Henle—passively reabsorbs H2O via medullary hypertonicity (impermeable to Na+). Concentrating segment. Makes urine hypertonic. Mg2+, Ca2+ Interstitium blood Diusion down the electrochemical gradient

1	Mg2+, Ca2+ Interstitium blood Diusion down the electrochemical gradient Thick ascending loop of Henle—reabsorbs Na+, K+, and Cl−. Indirectly induces paracellular reabsorption of Mg2+ and Ca2+ through ˜ lumen potential generated by K+ backleak. Impermeable to H2O. Makes urine less concentrated as it ascends. 10–20% Na+ reabsorbed. Early DCT—reabsorbs Na+, Cl−. Impermeable to H2O. Makes urine fully dilute (hypotonic). PTH—• Ca2+/Na+ exchange •• Ca2+ reabsorption. 5–10% Na+ reabsorbed. Collecting tubule—reabsorbs Na+ in exchange for secreting K+ and H+ (regulated by aldosterone). Aldosterone—acts on mineralocorticoid receptor • mRNA • protein synthesis. In principal cells: • apical K+ conductance, • Na+/K+ pump, • epithelial Na+ channel (ENaC) activity • lumen negativity • K+ secretion. In ˜-intercalated cells: lumen negativity •• H+ ATPase activity •• H+ secretion •• HCO3−/Cl− exchanger activity.

1	ADH—acts at V2 receptor • insertion of aquaporin H2O channels on apical side. 3–5% Na+ reabsorbed. Renal tubular defects Order: Fanconi’s BaGeLS Gitelman syndrome Bartter syndrome Liddle syndrome, SAME Syndrome of Apparent Mineralocorticoid Excess Treatment: K+-sparing diuretics (• mineralocorticoid effects) or corticosteroids (exogenous corticosteroid • endogenous cortisol production • mineralocorticoid receptor activation) 0% 25% 50% 75% 100% % Distance along PCT length PAH Creatinine Inulin clearance = GFR Urea Cl−Glucose Amino acids HCO3 – K+ Osmolarity, Na+ 1.90 1.85 when solute is reabsorbed less quickly 1.80 Tubular inulin  in concentration (but not amount) along the PCT as a result of water reabsorption. Cl− reabsorption occurs at a slower rate than Na+ in early PCT and then matches the rate of Na+ reabsorption more distally. Thus, its relative concentration  before it plateaus.

1	Renal cells ↑BP ↑activity Na+ , HCOƒ , and H⁄O H+ secretion ↑receptor type I Vasoconstriction ↑FF arteriole Na+ reabsorption H⁄O reabsorption secretion pituitary) Preserves GFR (when RBF ) PCT cell α-intercalated cell Principal cell Na+/K+ ATPase, and ENaC activity) Renin Secreted by JG cells in response to  renal perfusion pressure (detected by renal baroreceptors in afferent arteriole),  renal sympathetic discharge (β1 effect), and • NaCl delivery to macula densa cells. AT II Helps maintain blood volume and blood pressure. Affects baroreceptor function; limits reflex bradycardia, which would normally accompany its pressor effects. ANP, BNP Released from atria (ANP) and ventricles (BNP) in response to  volume; inhibits renin-angiotensinaldosterone system; relaxes vascular smooth muscle via cGMP  GFR, • renin. Dilates afferent arteriole, promotes natriuresis.

1	ADH Primarily regulates serum osmolality; also responds to low blood volume states. Stimulates reabsorption of water in collecting ducts. Also stimulates reabsorption of urea in collecting ducts to maximizes corticopapillary osmotic gradient. Aldosterone Primarily regulates ECF volume and Na+ content;  release in  blood volume states. Responds to hyperkalemia by  K+ excretion. contraction alkalosis) activity) ( K+ conductance, JGA maintains GFR via renin-angiotensinaldosterone system. In addition to vasodilatory properties, β-blockers can decrease BP by inhibiting β1-receptors of the JGA  renin release. Calciferol (vitamin D) PCT cells convert 25-OH vitamin D to 1,25-25-OH D (OH)2 vitamin D3 (calcitriol, active form). (calcidiol) 1˜-hydroxylase (calcitriol) 1,25-(OH) D Secreted in response to ° atrial pressure. Causes ° GFR and ° Na+ fltration with no compensatory Na+ reabsorption + loss and volume loss.

1	Synthesized in response to reabsorption in proximal and distal nephron. Net e˜ect: BP. Causes e˜erent arteriole˜ ° GFR and ° FF but with compensatory Na+ preservation of renal function (° FF) in low-volume state with simultaneous Na+ reabsorption (both proximal and distal) to maintain circulating volume. Glomerulus Proximal convoluted tubule Loop of Henle Ascending limb, loop of Henle (permeable to salts) Collecting duct convoluted tubule Cortex Medulla Sugars Amino acids Na+ Na+ K+ 2CI– Ca2+ Mg2+ Mg2+ Na+ CI– H Ca2+ Parathyroid hormone Secreted in response to ˜ plasma [Ca2+], ° plasma [PO43–], or ˜ plasma 1,25-(OH)2 D3. Causes ° [Ca2+] reabsorption (DCT), ˜ [PO43–] reabsorption (PCT), and ° 1,25-(OH)2 D3 production (° Ca2+ and PO43– absorption from gut via vitamin D). Aldosterone Secreted in response to ˜ blood volume (via AT II) and ° plasma [K+ ]; causes ° Na+ reabsorption, ° K+ secretion, ° H+ secretion. ADH (vasopressin) Secreted in response to ° plasma osmolarity and ˜ blood

1	in response to ˜ blood volume (via AT II) and ° plasma [K+ ]; causes ° Na+ reabsorption, ° K+ secretion, ° H+ secretion. ADH (vasopressin) Secreted in response to ° plasma osmolarity and ˜ blood volume. Binds to receptors on principal cells, causing ° number of aquaporins and ° H2O reabsorption. ° reabsorption of urea in collecting ducts to maximize corticopapillary osmotic gradient. K+ H+ Na+

1	Features of renal disorders Key: • = compensatory response. [HCO3 −]Henderson-Hasselbalch equation: pH = 6.1 + log 0.03 P2 Predicted respiratory compensation for a simple metabolic acidosis can be calculated using the Winters formula. If measured Pco2 > predicted Pco2  concomitant respiratory acidosis; if measured Pco2 < predicted Pco2 • concomitant respiratory alkalosis: Pco2 = 1.5 [HCO3–] + 8 ± 2 Acidosis and alkalosis Check arterial pH pH < 7.35 Pco2 > 44 mm Hg HCO3 – < 20 mEq/L Acidemia Respiratory acidosis Metabolic acidosis Hypoventilation Airway obstruction Acute lung disease Chronic lung disease Opioids, sedatives Weakening of respiratory muscles •Anion gap MUDPILES: Normal anion gap Check anion gap Methanol (formic acid) Uremia Diabetic ketoacidosis Propylene glycol Iron tablets or INH Lactic acidosis Ethylene glycol (oxalic acid) Salicylates (late) HARDASS:= Na+ – (CI + HCO3 )– _

1	Anxiety/panic attack Hypoxemia (eg, high altitude) Salicylates (early) Tumor Pulmonary embolism pH > 7.45 6.97.0 7.17.2 7.37.4 7.57.6 7.77.8 7.9 Renal tubular Disorder of the renal tubules that causes normal anion gap (hyperchloremic) metabolic acidosis. acidosis Distal renal Inability of > 5.5 • Amphotericin B toxicity,  risk for calcium tubular acidosis α-intercalated cells to analgesic nephropathy, phosphate kidney (type 1) secrete H+  no new congenital anomalies stones (due to  urine HCO3 is generated (obstruction) of urinary pH and  bone • metabolic acidosis tract, autoimmune turnover related to diseases (eg, SLE) buffering) Defect in PCT > 5.5 when HCO3 reabsorption resorptive  excretion of threshold

1	Defect in PCT > 5.5 when HCO3 reabsorption resorptive  excretion of threshold Urine can be acidified by exceeded; α-intercalated cells in < 5.5 when collecting duct, but not HCO3 enough to overcome depleted • Fanconi syndrome,  risk for multiple myeloma, hypophosphatemic carbonic anhydrase rickets (in Fanconi inhibitors syndrome) (eg, diabetic hyporeninism, ACE inhibitors, ARBs, NSAIDs, heparin, cyclosporine, adrenal insufficiency) or aldosterone resistance (eg, K+-sparing diuretics, nephropathy due to obstruction, TMP-SMX) Casts in urine Presence of casts indicates that hematuria/pyuria is of glomerular or renal tubular origin. Bladder cancer, kidney stones  hematuria, no casts. Acute cystitis  pyuria, no casts. A Glomerulonephritis, hypertensive emergency. B Tubulointerstitial inflammation, acute pyelonephritis, transplant rejection.

1	A Glomerulonephritis, hypertensive emergency. B Tubulointerstitial inflammation, acute pyelonephritis, transplant rejection. Nomenclature of glomerular disorders Glomerular diseases  GBM damage  loss of RBCs into urine • hematuria Hematuria, RBC casts in urine  oliguria, azotemia, release, HTN Proteinuria often in the subnephrotic range (< 3.5 g/ day) but in severe cases may be in nephrotic range Massive proteinuria (> 3.5 g/ day) with hypoalbuminemia, edema Frothy urine with fatty casts Associated with hypercoagulable state due to antithrombin III loss in urine and  risk of infection (loss of IgGs in urine and soft tissue compromise by edema) May be 1° (eg, direct podocyte damage) or 2° (podocyte damage from systemic process):

1	Kidney Can lead to severe complications such as hydronephrosis, pyelonephritis, and acute kidney injury. Obstructed stones stone presents with unilateral flank tenderness, colicky pain radiating to groin, hematuria. Treat and prevent by encouraging fluid intake. Calcium Calcium Radiopaque Radiopaque Shaped like oxalate: envelope Calcium Radiopaque Radiopaque Wedge-phosphate: shaped • pH prism Calcium stones most common (80%); calcium oxalate more common than calcium phosphate stones. Can result from ethylene glycol (antifreeze) ingestion, vitamin C abuse, hypocitraturia (associated with  urine pH), malabsorption (eg, Crohn disease). Treatment: thiazides, citrate, low-sodium diet. Treatment: low-sodium diet, thiazides. B Account for 15% of stones. Caused by infection magnesium with urease ⊕ bugs (eg, Proteus mirabilis, phosphate Staphylococcus saprophyticus, Klebsiella) (struvite) that hydrolyze urea to ammonia  urine alkalinization. Commonly form staghorn calculi

1	C . Treatment: eradication of underlying infection, surgical removal of stone. D About 5% of all stones. Risk factors:  urine visible or rosettes volume, arid climates, acidic pH. Strong association with hyperuricemia (eg, gout). Often seen in diseases with  cell turnover (eg, leukemia). Treatment: alkalinization of urine, allopurinol. opaque radiopaque which Cystine-reabsorbing PCT transporter loses function, causing cystinuria. Transporter defect also results in poor reabsorption of Ornithine, Lysine, Arginine (COLA). Cystine is poorly soluble, thus stones form in urine. Usually begins in childhood. Can form staghorn calculi. Sodium cyanide nitroprusside test ⊕. “SIXtine” stones have SIX sides. Treatment: low sodium diet, alkalinization of urine, chelating agents (eg, penicillamine) if refractory.

1	“SIXtine” stones have SIX sides. Treatment: low sodium diet, alkalinization of urine, chelating agents (eg, penicillamine) if refractory. Hydronephrosis Distention/dilation of renal pelvis and calyces A . Usually caused by urinary tract obstruction (eg, renal stones, severe BPH, congenital obstructions, cervical cancer, injury to ureter); other causes include retroperitoneal fibrosis, vesicoureteral reflux. Dilation occurs proximal to site of pathology. Serum creatinine becomes elevated if obstruction is bilateral or if patient has an obstructed solitary kidney. Leads to compression and possible atrophy of renal cortex and medulla. Urinary incontinence Mixed incontinence has features of both stress and urgency incontinence.

1	Urinary incontinence Mixed incontinence has features of both stress and urgency incontinence. Acute cystitis Inflammation of urinary bladder. Presents as suprapubic pain, dysuria, urinary frequency, urgency. Systemic signs (eg, high fever, chills) are usually absent. Risk factors include female sex (short urethra), sexual intercourse, indwelling catheter, diabetes mellitus, impaired bladder emptying. Causes: Staphylococcus saprophyticus—seen in sexually active young women (E coli is still more common in this group) Proteus mirabilis—urine has ammonia scent Labs: ⊕ leukocyte esterase. ⊕ nitrites (indicate gram ⊝ organisms). Sterile pyuria (pyuria with ⊝ urine cultures) could suggest urethritis by Neisseria gonorrhoeae or Chlamydia trachomatis. Treatment: antibiotics (eg, TMP-SMX, nitrofurantoin).

1	Acute pyelonephritis Neutrophils infiltrate renal interstitium A . Affects cortex with relative sparing of glomeruli/vessels. Presents with fevers, flank pain (costovertebral angle tenderness), nausea/vomiting, chills. Causes include ascending UTI (E coli is most common), hematogenous spread to kidney. Presents with WBCs in urine +/− WBC casts. CT would show striated parenchymal enhancement B . Risk factors include indwelling urinary catheter, urinary tract obstruction, vesicoureteral reflux, diabetes mellitus, pregnancy. Complications include chronic pyelonephritis, renal papillary necrosis, perinephric abscess, urosepsis. Treatment: antibiotics.

1	Also called tubulointerstitial nephritis. Acute interstitial renal inflammation. Pyuria (classically eosinophils) and azotemia occurring after administration of drugs that act as haptens, inducing hypersensitivity (eg, diuretics, NSAIDs, penicillin derivatives, proton pump inhibitors, rifampin, quinolones, sulfonamides). Less commonly may be 2° to other processes such as systemic infections (eg, Mycoplasma) or autoimmune diseases (eg, Sjögren syndrome, SLE, sarcoidosis). Associated with fever, rash, hematuria, pyuria, and costovertebral angle tenderness, but can be asymptomatic. Remember these 5P’S: Acute tubular necrosis Most common cause of acute kidney injury in hospitalized patients. Spontaneously resolves in many cases. Can be fatal, especially during initial oliguric phase.  FENa. Key finding: granular casts (often muddy brown in appearance) A . 3 stages: 1. 2. Maintenance phase—oliguric; lasts 1–3 weeks; risk of hyperkalemia, metabolic acidosis, uremia 3.

1	2. Maintenance phase—oliguric; lasts 1–3 weeks; risk of hyperkalemia, metabolic acidosis, uremia 3. Recovery phase—polyuric; BUN and serum creatinine fall; risk of hypokalemia and renal wasting of other electrolytes and minerals Can be caused by ischemic or nephrotoxic injury: Ischemic—2° to  renal blood flow (eg, hypotension, shock, sepsis, hemorrhage, HF). Results in death of tubular cells that may slough into tubular lumen B (PCT and thick ascending limb are highly susceptible to injury). Nephrotoxic—2° to injury resulting from toxic substances (eg, aminoglycosides, radiocontrast agents, lead, cisplatin, ethylene glycol), crush injury (myoglobinuria), hemoglobinuria. Proximal tubules are particularly susceptible to injury. Diffuse cortical Acute generalized cortical infarction of both Associated with obstetric catastrophes (eg, necrosis kidneys. Likely due to a combination of abruptio placentae), septic shock. vasospasm and DIC.

1	Renal papillary Sloughing of necrotic renal papillae A • gross Associated with: Sickle cell disease or trait, necrosis hematuria and proteinuria. May be triggered Acute pyelonephritis, Analgesics (NSAIDs), by recent infection or immune stimulus. Diabetes mellitus (SAAD papa with papillary necrosis). Consequences of renal failure Decline in renal filtration can lead to excess retained nitrogenous waste products and electrolyte disturbances. Consequences (MAD HUNGER): Uremia—clinical syndrome marked by: Na+/H2O retention (HF, pulmonary edema, hypertension) 2 forms of renal failure: acute (eg, ATN) and chronic (eg, hypertension, diabetes mellitus, congenital anomalies). Incremental reductions in GFR define the stages of chronic kidney disease.

1	Incremental reductions in GFR define the stages of chronic kidney disease. Renal osteodystrophy Hypocalcemia, hyperphosphatemia, and failure of vitamin D hydroxylation associated with chronic kidney disease  2° hyperparathyroidism  3° hyperparathyroidism (if 2° poorly managed). High serum phosphate can bind with Ca2+  tissue deposits  serum Ca2+. • 1,25-(OH)2D3  intestinal Ca2+ absorption. Causes subperiosteal thinning of bones. Autosomal recessive Cystic dilation of collecting ducts B . Often presents in infancy. Associated with congenital polycystic kidney hepatic fibrosis. Significant oliguric renal failure in utero can lead to Potter sequence. Concerns disease beyond neonatal period include systemic hypertension, progressive renal insufficiency, and portal hypertension from congenital hepatic fibrosis.

1	Autosomal dominant Also called medullary cystic kidney disease. Causes tubulointerstitial fibrosis and progressive renal tubulointerstitial insufficiency with inability to concentrate urine. Medullary cysts usually not visualized; smaller kidney disease kidneys on ultrasound. Poor prognosis. Renal cell carcinoma Polygonal clear cells A filled with accumulated lipids and carbohydrate. Often golden-yellow B due to • lipid content. Originates from PCT  invades renal vein (may develop varicocele if left sided)  IVC • hematogenous spread  metastasis to lung and bone. Manifests with hematuria, palpable masses, 2° polycythemia, flank pain, fever, weight loss. Treatment: surgery/ablation for localized disease. Immunotherapy (eg, aldesleukin) or targeted therapy for metastatic disease, rarely curative. Resistant to chemotherapy and radiation therapy. C . Most common in men 50–70 years old, • incidence with smoking and obesity.

1	C . Most common in men 50–70 years old, • incidence with smoking and obesity. Associated with paraneoplastic syndromes, eg, PTHrP, Ectopic EPO, ACTH, Renin (“PEAR”-aneoplastic). Clear cell (most common subtype) associated with gene deletion on chromosome 3 (sporadic, or inherited as von Hippel-Lindau syndrome). RCC = 3 letters = chromosome 3. Benign epithelial cell tumor arising from collecting ducts (arrows in A point to well-circumscribed mass with central scar). Large eosinophilic cells with abundant mitochondria without perinuclear clearing B (vs chromophobe renal cell carcinoma). Presents with painless hematuria, flank pain, abdominal mass. Often resected to exclude malignancy (eg, renal cell carcinoma). Also called Wilms tumor. Most common renal malignancy of early childhood (ages 2–4). Contains embryonic glomerular structures. Presents with large, palpable, unilateral flank mass A and/or hematuria and possible HTN.

1	“Loss of function” mutations of tumor suppressor genes WT1 or WT2 on chromosome 11. May be a part of several syndromes: WAGR complex—Wilms tumor, Aniridia (absence of iris), Genitourinary malformations, mental Retardation/intellectual disability (WT1 deletion) Denys-Drash syndrome—Wilms tumor, Diffuse mesangial sclerosis (early-onset nephrotic syndrome), Dysgenesis of gonads (male pseudohermaphroditism), WT1 mutation Beckwith-Wiedemann syndrome—Wilms tumor, macroglossia, organomegaly, hemihyperplasia (WT2 mutation), omphalocele Urothelial carcinoma Also called transitional cell carcinoma. Most of the bladder common tumor of urinary tract system (can occur in renal calyces, renal pelvis, ureters, and bladder) A B . Can be suggested by painless hematuria (no casts). Associated with problems in your Pee SAC: Phenacetin, Smoking, Aniline dyes, and Cyclophosphamide.

1	Associated with problems in your Pee SAC: Phenacetin, Smoking, Aniline dyes, and Cyclophosphamide. Diuretics site of action 5Glomerulus A°erent Mannitol Acetazolamide Loop diuretics Thiazide diuretics K+ sparing diuretics Proximal convoluted tubule Loop of Henle Descending limb, loop of Henle (permeable to water) Ascending limb, loop of Henle (permeable to salts) Collecting duct Distal convoluted tubule Cortex Medulla 1234Sugars Amino acids Na+ Na+ K+ 2CI– Ca2+ Mg2+ Na+ CI– Na+ Na+ HCO3 – Ca2+ 34K+ H+ 5521E°erent H2O mEChANism Osmotic diuretic.  tubular fluid osmolarity  urine flow,  intracranial/intraocular pressure. CLiNiCAL UsE Drug overdose, elevated intracranial/intraocular pressure. ADVERsE EFFECts Pulmonary edema, dehydration, hypoor hypernatremia. Contraindicated in anuria, HF. mEChANism Carbonic anhydrase inhibitor. Causes self-limited NaHCO3 diuresis and  total body HCO3 − stores. Alkalinizes urine.

1	mEChANism Carbonic anhydrase inhibitor. Causes self-limited NaHCO3 diuresis and  total body HCO3 − stores. Alkalinizes urine. CLiNiCAL UsE Glaucoma, metabolic alkalosis, altitude sickness, idiopathic intracranial hypertension. ADVERsE EFFECts Proximal renal tubular acidosis, paresthesias, “Acid”azolamide causes Acidosis. NH3 toxicity, sulfa allergy, hypokalemia. Promotes calcium phosphate stone formation (insoluble at high pH). Furosemide, bumetanide, torsemide mEChANism Sulfonamide loop diuretics. Inhibit cotransport system (Na+/K+/2Cl−) of thick ascending limb of loop of Henle. Abolish hypertonicity of medulla, preventing concentration of urine. Associated with  PGE (vasodilatory effect on afferent arteriole); inhibited by NSAIDs.  Ca2+ excretion. Loops Lose Ca2+. CLiNiCAL UsE Edematous states (HF, cirrhosis, nephrotic syndrome, pulmonary edema), hypertension, hypercalcemia. CLiNiCAL UsE Diuresis in patients allergic to sulfa drugs.

1	CLiNiCAL UsE Edematous states (HF, cirrhosis, nephrotic syndrome, pulmonary edema), hypertension, hypercalcemia. CLiNiCAL UsE Diuresis in patients allergic to sulfa drugs. ADVERsE EFFECts Similar to furosemide, but more ototoxic. Loop earrings hurt your ears. Thiazide diuretics Hydrochlorothiazide, chlorthalidone, metolazone. mEChANism Inhibit NaCl reabsorption in early DCT  diluting capacity of nephron. • Ca2+ excretion. CLiNiCAL UsE Hypertension, HF, idiopathic hypercalciuria, nephrogenic diabetes insipidus, osteoporosis. ADVERsE EFFECts Hypokalemic metabolic alkalosis, hyponatremia, hyperGlycemia, hyperLipidemia, hyperUricemia, hyperCalcemia. Sulfa allergy. HyperGLUC. Potassium-sparing Spironolactone, Eplerenone, Amiloride, Keep your SEAT diuretics Triamterene. mEChANism Spironolactone and eplerenone are competitive aldosterone receptor antagonists in cortical collecting tubule. Triamterene and amiloride block Na+ channels at the same part of the tubule.

1	mEChANism Spironolactone and eplerenone are competitive aldosterone receptor antagonists in cortical collecting tubule. Triamterene and amiloride block Na+ channels at the same part of the tubule. CLiNiCAL UsE Hyperaldosteronism, K+ depletion, HF, hepatic ascites (spironolactone), nephrogenic DI (amiloride), antiandrogen. ADVERsE EFFECts Hyperkalemia (can lead to arrhythmias), endocrine effects with spironolactone (eg, gynecomastia, antiandrogen effects). Diuretics: electrolyte changes Urine NaCl  with all diuretics (strength varies based on potency of diuretic effect). Serum NaCl may decrease as a result. Urine Ca2+  with loop diuretics:  paracellular Ca2+ reabsorption  hypocalcemia.  with thiazides: enhanced Ca2+ reabsorption.

1	Urine Ca2+  with loop diuretics:  paracellular Ca2+ reabsorption  hypocalcemia.  with thiazides: enhanced Ca2+ reabsorption. Angiotensin-Captopril, enalapril, lisinopril, ramipril. converting enzyme inhibitors mEChANism Inhibit ACE  AT II  GFR by preventing constriction of efferent arterioles.  renin due to loss of negative feedback. Inhibition of ACE also prevents inactivation of bradykinin, a potent vasodilator. CLiNiCAL UsE Hypertension, HF, proteinuria, or chronic kidney disease (eg, diabetic nephropathy) with intolerance to ACE inhibitors (eg, cough, angioedema). ADVERsE EFFECts Hyperkalemia, • GFR, hypotension; teratogen. mEChANism Direct renin inhibitor, blocks conversion of angiotensinogen to angiotensin I. Aliskiren Kills Renin. CLiNiCAL UsE Hypertension. ADVERsE EFFECts Hyperkalemia, • GFR, hypotension, angioedema. Relatively contraindicated in patients already taking ACE inhibitors or ARBs and contraindicated in pregnancy.

1	ADVERsE EFFECts Hyperkalemia, • GFR, hypotension, angioedema. Relatively contraindicated in patients already taking ACE inhibitors or ARBs and contraindicated in pregnancy. “Artificial insemination is when the farmer does it to the cow instead of the bull.” Make no mistake about why these babies are here they are here to replace us. “Whoever called it necking was a poor judge of anatomy.” “See, the problem is that God gives men a brain and a penis, and only enough blood to run one at a time.” The reproductive system can be intimidating at first but is manageable once you organize the concepts into the pregnancy, endocrinologic, embryologic, and oncologic aspects of reproduction. Study the endocrine and reproductive chapters together, because mastery of the hypothalamic-pituitary-gonadal axis is key to answering questions on ovulation, menstruation, disorders of sexual development, contraception, and many pathologies.

1	Embryology is a nuanced subject that covers multiple organ systems. Approaching it from a clinical perspective will allow for better understanding. For instance, make the connection between the presentation of DiGeorge syndrome and the 3rd/4th pharyngeal pouch, and between the Müllerian/Wolffian systems and disorders of sexual development. As for oncology, don’t worry about remembering screening or treatment guidelines. It is more important to know how these cancers present (eg, signs and symptoms) and their associated labs, histopathology, and risk factors. In addition, some of the testicular and ovarian cancers have distinct patterns of hCG, AFP, LH, or FSH derangements that serve as helpful clues in exam questions. Important genes of embryogenesis Types of errors in morphogenesis Deformation Extrinsic disruption (eg, multiple gestations  crowding  foot deformities); occurs after embryonic period. Malformation Intrinsic disruption; occurs during embryonic period (weeks 3–8).

1	Malformation Intrinsic disruption; occurs during embryonic period (weeks 3–8). Sequence Abnormalities result from a single 1° embryologic event (eg, oligohydramnios  Potter sequence). Teratogens Most susceptible in 3rd–8th weeks (embryonic period—organogenesis) of pregnancy. Before week 3, “all-or-none” effects. After week 8, growth and function affected. ACE inhibitors Renal failure, oligohydramnios, hypocalvaria. Lithium Ebstein anomaly. Methimazole Aplasia cutis congenita (congenital absence of skin, particularly on scalp). Alcohol Fetal alcohol syndrome. Iodine (lack or excess) Congenital goiter or hypothyroidism (cretinism). Maternal diabetes Caudal regression syndrome, cardiac defects (eg, VSD), neural tube defects, macrosomia, neonatal hypoglycemia (due to islet cell hyperplasia), polycythemia. Methylmercury Neurotoxicity. Highest in swordfish, shark, tilefish, king mackerel.

1	Methylmercury Neurotoxicity. Highest in swordfish, shark, tilefish, king mackerel. Vitamin A excess Extremely high risk for spontaneous abortions and birth defects (cleft palate, cardiac). X-rays Microcephaly, intellectual disability. Minimized by lead shielding. One of the leading preventable causes of intellectual disability in the US. Newborns of mothers who consumed alcohol during any stage of pregnancy have  incidence of congenital abnormalities, including preand postnatal developmental retardation, microcephaly, facial abnormalities A (eg, smooth philtrum, thin vermillion border, small palpebral fissures), limb dislocation, heart defects. Heart-lung fistulas and holoprosencephaly in most severe form. One mechanism is due to impaired migration of neuronal and glial cells. Twinning Dizygotic (“fraternal”) twins arise from 2 eggs that are separately fertilized by 2 different sperm (always 2 zygotes) and will have 2 separate amniotic sacs and 2 separate placentas (chorions).

1	Monozygotic (“identical”) twins arise from 1 fertilized egg (1 egg + 1 sperm) that splits in early pregnancy. The timing of cleavage determines chorionicity (number of chorions) and amnionicity (number of amnions) (SCAB): Cleavage 0–4 days: Separate chorion and amnion Cleavage 4–8 days: shared Chorion Cleavage 8–12 days: shared Amnion Cleavage 13+ days: shared Body (conjoined) 2 eggs, 1 egg, 1 sperm 2 sperm Placenta 1º site of nutrient and gas exchange between mother and fetus. Decidua basalis Derived from endometrium. Maternal blood in lacunae. Branch villus Umbilical vein (O2 rich) Umbilical arteries (O2 poor) Endometrial vein Maternal circulation Chorionic plate Maternal blood Amnion Maternal circulation Fetal circulation CO2 H2O Urea, waste products Hormones Endometrial artery Decidua basalis Endothelial cell Syncytiotrophoblast Cytotrophoblast O2 H2O, electrolytes Nutrients Hormones IgG Drugs Viruses

1	Urachus Allantois forms from hindgut and extends into urogenital sinus. Allantois becomes the urachus, a duct between fetal bladder and umbilicus. Failure of urachus to involute can lead to anomalies that may increase risk of infection and/or malignancy (eg, adenocarcinoma) if not treated. Obliterated urachus is represented by the median umbilical ligament after birth, which is covered by median umbilical fold of the peritoneum. Vesicourachal Slight failure of urachus to obliterate  outpouching of bladder. diverticulum Vitelline duct 7th week—obliteration of vitelline duct (omphalomesenteric duct), which connects yolk sac to midgut lumen. Vitelline fistula Vitelline duct fails to close  meconium discharge from umbilicus. Meckel diverticulum Partial closure of vitelline duct, with patent portion attached to ileum (true diverticulum, white arrow in B ). May be asymptomatic. May have heterotopic gastric and/or pancreatic tissue  melena, hematochezia, abdominal pain.

1	Aortic arch derivatives Develop into arterial system. 1st Part of maxillary artery (branch of external 1st arch is maximal carotid) 3rd Common Carotid artery and proximal part of C is 3rd letter of alphabet internal Carotid artery 4th On left, aortic arch; on right, proximal part of 4th arch (4 limbs) = systemic right subclavian artery 6th Proximal part of pulmonary arteries and (on left only) ductus arteriosus 6th arch = pulmonary and the pulmonary-tosystemic shunt (ductus arteriosus) Pharyngeal apparatus Composed of pharyngeal clefts, arches, CAP covers outside to inside: pouches. Clefts = ectoderm Pharyngeal clefts—derived from ectoderm. Also Arches = mesoderm + neural crest called pharyngeal grooves. Pouches = endoderm Pharyngeal arches—derived from mesoderm (muscles, arteries) and neural crest (bones, cartilage). Pharyngeal pouches—derived from endoderm. • Maxilla, zygoMatic  Mandible, Malleus and incus, sphenoMandibular ligament

1	• Maxilla, zygoMatic  Mandible, Malleus and incus, sphenoMandibular ligament Reichert cartilage: Stapes, Styloid process, leSSer horn of hyoid, Stylohyoid ligament Muscles of Mastication (temporalis, Masseter, lateral and Medial pterygoids), Mylohyoid, anterior belly of digastric, tensor tympani, anterior 2/3 of tongue, tensor veli palatini Muscles of facial expression, Stapedius, Stylohyoid, platySma, posterior belly of digastric CN V3 chew Pierre Robin sequence— micrognathia, glossoptosis, cleft palate, airway obstruction

1	CN VII (facial • craniofacial expression) abnormalities (eg, smile zygomatic bone and mandibular hypoplasia), hearing loss, airway compromise 3rd pharyngeal Greater horn of hyoid Stylopharyngeus (think CN IX (styloarch of stylopharyngeus pharyngeus) innervated by swallow stylishly glossopharyngeal nerve) 4th and 6th Arytenoids, Cricoid, pharyngeal Corniculate, arches Cuneiform, Thyroid (used to sing and ACCCT) 4th arch: most pharyngeal 4th arch: CN Arches 3 and 4 form constrictors; cricothyroid, X (superior posterior 1/3 of tongue levator veli palatini laryngeal branch) Arch 5 makes no 6th arch: all intrinsic simply swallow major developmental muscles of larynx except 6th arch: CN contributions cricothyroid X (recurrent/ inferior laryngeal branch) speak aSensory and motor nerves are not pharyngeal arch derivatives. They grow into the arches and are derived from neural crest (sensory) and neuroectoderm (motor).

1	When at the restaurant of the golden arches, children tend to first chew (1), then smile (2), then swallow stylishly (3) or simply swallow (4), and then speak (6). 1st pharyngeal pouch Middle ear cavity, eustachian 1st pouch contributes to Ear, tonsils, bottom-to-top: tube, mastoid air cells endoderm-lined structures 1 (ear) of ear 2 (tonsils) 3 dorsal (bottom for inferior 2nd pharyngeal pouch Epithelial lining of palatine 3rd pharyngeal pouch Dorsal wings • inferior 3rd pouch contributes to 3 parathyroids structures (thymus, left and (C) cells of thyroid Cleft lip and cleft Distinct, multifactorial etiologies, but often occur together. palate Cleft lip Due to failure of fusion of the maxillary and merged medial nasal processes (formation of 1° palate). Cleft palate Due to failure of fusion of the two lateral palatine shelves or failure of fusion of lateral palatine shelf with the nasal septum and/or 1° palate (formation of 2° palate).

1	Cleft palate Due to failure of fusion of the two lateral palatine shelves or failure of fusion of lateral palatine shelf with the nasal septum and/or 1° palate (formation of 2° palate). Intermaxillary segment Roof of mouth (1° palate) Nasal septum Maxillary prominence Palatine shelves (2° palate) Female Default development. Mesonephric duct degenerates and paramesonephric duct develops.

1	Female Default development. Mesonephric duct degenerates and paramesonephric duct develops. Metanephric kidney Ureter Uterus Vagina Vas deferens Degenerated paramesonephric duct Degenerated mesonephric duct Oviduct Urinary bladder Testis Ovary Epididymis Testis-determining factor Androgens MIF Gubernaculum Mesonephros Paramesonephric duct Mesonephric duct Urogenital sinus No androgens (Müllerian) duct fallopian tubes, uterus, upper portion of vagina (lower portion from urogenital sinus). Male remnant is appendix testis. Müllerian agenesis (Mayer-Rokitansky-Küster-Hauser syndrome)—may present 2° sexual characteristics (functional ovaries). (Wolffian) duct prostate)—Seminal vesicles, Epididymis, Ejaculatory duct, Ductus deferens (SEED). Female remnant is Gartner duct. determining factor • testes development. Sertoli cells secrete Müllerian inhibitory factor (MIF, also called antimullerian hormone) that suppresses development of paramesonephric ducts.

1	determining factor • testes development. Sertoli cells secrete Müllerian inhibitory factor (MIF, also called antimullerian hormone) that suppresses development of paramesonephric ducts. Leydig cells secrete androgens that stimulate development of mesonephric ducts. Paramesonephric Develops into female internal structures— as 1° amenorrhea (due to a lack of uterine development) in females with fully developed Absence of Sertoli cells or lack of Müllerian inhibitory factor  develop both male and sexual differentiation. Sertoli Shuts down female (internal) sexual differentiation. genitalia, ambiguous external genitalia until In the testes: Leydig Leads to male (internal and external) Septate uterus Common anomaly vs normal uterus A . Incomplete resorption of septum B .  fertility and early miscarriage/pregnancy loss. Treat with septoplasty. Opening of sinus Scrotum Opening of Anus

1	Opening of sinus Scrotum Opening of Anus Hypospadias Abnormal opening of penile urethra on ventral Hypospadias is more common than surface of penis due to failure of urethral folds epispadias. Associated with inguinal hernia, to fuse. cryptorchidism, chordee (downward or upward bending of penis). Hypo is below. Can be seen in 5α-reductase deficiency. Epispadias Abnormal opening of penile urethra on dorsal Exstrophy of the bladder is associated with surface of penis due to faulty positioning of Epispadias. genital tubercle. When you have Epispadias, you hit your Eye when you pEE. Descent of testes and ovaries Adnexal torsion Twisting of ovary and fallopian tube around infundibulopelvic ligament and ovarian ligament  compression of ovarian vessels in infundibulopelvic ligament  blockage of lymphatic and venous outflow. Continued arterial perfusion  ovarian edema  complete blockage of arterial inflow  necrosis, local hemorrhage.

1	Associated with ovarian masses. Presents with acute pelvic pain, adnexal mass, nausea/vomiting. Vagina Stratified squamous epithelium, nonkeratinized Ectocervix Stratified squamous epithelium, nonkeratinized Uterus Simple columnar epithelium with long tubular glands in proliferative phase; coiled glands in secretory phase Pathway of sperm during ejaculation— SEVEN UP: Seminiferous tubules Epididymis Vas deferens Ejaculatory ducts (Nothing) Urethra Penis Autonomic innervation of male sexual response Occurs almost exclusively in men. Suspect if blood seen at urethral meatus. Urethral catheterization is relatively contraindicated. If Buck fascia is torn, urine escapes into perineal space Blood at urethral meatus and scrotal hematoma Blood at urethral meatus and high-riding prostate Erection—Parasympathetic nervous system (pelvic splanchnic nerves, S2-S4): NO  cGMP  smooth muscle relaxation  vasodilation  proerectile.

1	Erection—Parasympathetic nervous system (pelvic splanchnic nerves, S2-S4): NO  cGMP  smooth muscle relaxation  vasodilation  proerectile. antierectile. Emission—Sympathetic nervous system (hypogastric nerve, T11-L2). Expulsion—visceral and Somatic nerves (pudendal nerve). Point, Squeeze, and Shoot. S2, 3, 4 keep the penis off the floor. PDE-5 inhibitors (eg, sildenafil)  cGMP breakdown. local levels of testosterone Produce MIF Tight junctions between adjacent Sertoli cells  inhibin B with  temperature Line seminiferous tubules Non-germ cells Convert testosterone and androstenedione to estrogens via aromatase Sertoli cells are inSide Seminiferous tubules, Support Sperm Synthesis, and inhibit FSH Homolog of female granulosa cells  temperature seen in varicocele, cryptorchidism Leydig cells Secrete testosterone in the presence of LH; Interstitium temperature Homolog of female theca interna cells

1	Leydig cells Secrete testosterone in the presence of LH; Interstitium temperature Homolog of female theca interna cells Spermatogenesis Begins at puberty with spermatogonia. Full “Gonium” is going to be a sperm; “Zoon” is development takes 2 months. Occurs in “Zooming” to egg. seminiferous tubules. Produces spermatids Tail mobility impaired in ciliary dyskinesia/ that undergo spermiogenesis (loss of Kartagener syndrome  infertility. cytoplasmic contents, gain of acrosomal cap) Tail mobility normal in cystic fibrosis (in CF, to form mature spermatozoa. Spermatogonium 1° spermatocyte 2° spermatocyte Diploid Diploid Haploid (2N, 2C) (2N, 4C) (1N, 2C) absent vas deferens  infertility). Mature spermatozoon Haploid Haploid (1N, 1C) (1N, 1C) SOURCE Ovary (17β-estradiol), placenta (estriol), adipose Potency: estradiol > estrone > estriol. tissue (estrone via aromatization).

1	Mature spermatozoon Haploid Haploid (1N, 1C) (1N, 1C) SOURCE Ovary (17β-estradiol), placenta (estriol), adipose Potency: estradiol > estrone > estriol. tissue (estrone via aromatization). FUNCTION Development of genitalia and breast, female fat distribution. Growth of follicle, endometrial proliferation,  myometrial excitability. Upregulation of estrogen, LH, and progesterone receptors; feedback inhibition of FSH and LH, then LH surge; stimulation of prolactin secretion.  transport proteins, SHBG;  HDL;  LDL. Pregnancy: 1000-fold  in estriol (indicator of fetal wellbeing) Estrogen receptors expressed in cytoplasm; translocate to nucleus when bound by estrogen. SOURCE Corpus luteum, placenta, adrenal cortex, testes. Fall in progesterone after delivery disinhibits FUNCTION During luteal phase, prepares uterus for implantation of fertilized egg: Stimulation of endometrial glandular secretions and spiral artery development Production of thick cervical mucus

1	FUNCTION During luteal phase, prepares uterus for implantation of fertilized egg: Stimulation of endometrial glandular secretions and spiral artery development Production of thick cervical mucus Prevention of endometrial hyperplasia •  body temperature •  estrogen receptor expression •  gonadotropin (LH, FSH) secretion During pregnancy: • Maintenance of pregnancy •  myometrial excitability  contraction frequency and intensity •  prolactin action on breasts prolactin  lactation.  progesterone is indicative of ovulation. Progesterone is pro-gestation. Prolactin is pro-lactation. Oogenesis 1° oocytes begin meiosis I during fetal life and complete meiosis I just prior to ovulation. Meiosis I is arrested in prOphase I for years until Ovulation (1° oocytes). Meiosis II is arrested in metaphase II until fertilization (2° oocytes). “An egg met a sperm.” If fertilization does not occur within 1 day, the 2° oocyte degenerates.

1	Oogonium 1° oocyte 2° oocyte Ovum Diploid Diploid Haploid Haploid (2N, 2C) (2N, 4C) (1N, 2C) (1N, 1C) N = ploidy C = # of chromatids Menstrual cycle Follicular phase can vary in length. Luteal phase is 14 days. Ovulation day + 14 days = menstruation. Follicular growth is fastest during 2nd week of the follicular phase. Estrogen stimulates endometrial proliferation. Progesterone maintains endometrium to support implantation.  progesterone • fertility. Abnormal uterine Characterized as either heavy menstrual Terms such as dysfunctional uterine bleeding, bleeding bleeding (AUB/HMB) or intermenstrual menorrhagia, oligomenorrhea are no longer bleeding (AUB/IMB). recommended. These are further subcategorized by PALM COEIN: Structural causes (PALM): Polyp, Adenomyosis, Leiomyoma, or Malignancy/ hyperplasia Non-structural causes (COEIN): Coagulopathy, Ovulatory, Endometrial, Iatrogenic, Not yet classified

1	COEIN: Structural causes (PALM): Polyp, Adenomyosis, Leiomyoma, or Malignancy/ hyperplasia Non-structural causes (COEIN): Coagulopathy, Ovulatory, Endometrial, Iatrogenic, Not yet classified Pregnancy Fertilization most commonly occurs in upper end of fallopian tube (the ampulla). Occurs within 1 day of ovulation. Implantation within the wall of the uterus occurs 6 days after fertilization. Syncytiotrophoblasts secrete hCG, which is conception. Gestational age—calculated from date of last menstrual period. Physiologic adaptations in pregnancy: Weeks of pregnancy •  GFR  BUN and creatinine, Placental hormone secretion generally increases  glucosuria threshold over the course of pregnancy, but hCG peaks at • • cardiac output (• preload,  afterload, 8–10 weeks. Anemia ( plasma, • RBCs) •  lipolysis and fat utilization (due to maternal hypoglycemia and insulin resistance)  preserves glucose and amino acids for utilization by the fetus

1	Anemia ( plasma, • RBCs) •  lipolysis and fat utilization (due to maternal hypoglycemia and insulin resistance)  preserves glucose and amino acids for utilization by the fetus SOURCE Syncytiotrophoblast of placenta. FUNCTION Maintains corpus luteum (and thus progesterone) for first 8–10 weeks of pregnancy by acting like LH (otherwise no luteal cell stimulation  abortion). After 8–10 weeks, placenta synthesizes its own estriol and progesterone and corpus luteum degenerates. Used to detect pregnancy because it appears early in urine (see above). Has identical α subunit as LH, FSH, TSH (states of  hCG can cause hyperthyroidism). β subunit is unique (pregnancy tests detect β subunit). hCG is  in multiple gestations, hydatidiform moles, choriocarcinomas, and Down syndrome; hCG is  in ectopic/failing pregnancy, Edwards syndrome, and Patau syndrome. Human placental Also called chorionic somatomammotropin. lactogen SOURCE Syncytiotrophoblast of placenta.

1	Human placental Also called chorionic somatomammotropin. lactogen SOURCE Syncytiotrophoblast of placenta. FUNCTION Stimulates insulin production; overall  insulin resistance. Gestational diabetes can occur if maternal pancreatic function cannot overcome the insulin resistance. Assessment of newborn vital signs following delivery via a 10-point scale evaluated at 1 minute and 5 minutes. Apgar score is based on Appearance, Pulse, Grimace, Activity, and Respiration. Apgar scores < 7 may require further evaluation. If Apgar score remains low at later time points, there is  risk the child will develop long-term neurologic damage. Milestone dates are ranges that have been approximated and vary by source. Children not meeting milestones may need assessment for potential developmental delay. Cruises, takes first steps (by = age (yr) × 3 Cutlery—feeds self with fork and spoon (by 20 mo) Kicks ball (by 24 mo)

1	Cruises, takes first steps (by = age (yr) × 3 Cutlery—feeds self with fork and spoon (by 20 mo) Kicks ball (by 24 mo) Defined as < 2500 g. Caused by prematurity or intrauterine growth restriction (IUGR). Associated with  risk of sudden infant death syndrome (SIDS) and with  overall mortality.

1	Lactation After parturition and delivery of placenta, rapid  in progesterone disinhibits prolactin • initiation of lactation. Suckling is required to maintain milk production and ejection, since  nerve stimulation  oxytocin and prolactin. Prolactin—induces and maintains lactation and  reproductive function. Oxytocin—assists in milk letdown; also promotes uterine contractions. Breast milk is the ideal nutrition for infants < 6 months old. Contains maternal immunoglobulins (conferring passive immunity; mostly IgA), macrophages, lymphocytes. Breast milk reduces infant infections and is associated with  risk for child to develop asthma, allergies, diabetes mellitus, and obesity. Guidelines recommend exclusively breastfed infants get vitamin D and possibly iron supplementation. Breastfeeding  maternal risk of breast and ovarian cancer and facilitates mother-child bonding. Menopause Diagnosed by amenorrhea for 12 months.

1	Menopause Diagnosed by amenorrhea for 12 months. • estrogen production due to age-linked decline in number of ovarian follicles. Average age at onset is 51 years (earlier in smokers). Usually preceded by 4–5 years of abnormal menstrual cycles. Source of estrogen (estrone) after menopause becomes peripheral conversion of androgens,  androgens  hirsutism.  FSH is specific for menopause (loss of negative feedback on FSH due to  estrogen). Hormonal changes:  estrogen,  FSH,  LH (no surge),  GnRH. Causes HAVOCS: Hot flashes, Atrophy of the Vagina, Osteoporosis, Coronary artery disease, Sleep disturbances. Menopause before age 40 suggests 1° ovarian insufficiency (premature ovarian failure); may occur in women who have received chemotherapy and/or radiation therapy. Androgens Testosterone, dihydrotestosterone (DHT), androstenedione. SOURCE DHT and testosterone (testis), AnDrostenedione Potency: DHT > testosterone > (ADrenal) androstenedione. FUNCTION Testosterone:

1	SOURCE DHT and testosterone (testis), AnDrostenedione Potency: DHT > testosterone > (ADrenal) androstenedione. FUNCTION Testosterone: Differentiation of epididymis, vas deferens, seminal vesicles (internal genitalia, except prostate) Growth spurt: penis, seminal vesicles, sperm, muscle, RBCs Deepening of voice Closing of epiphyseal plates (via estrogen converted from testosterone) Libido DHT: Early—differentiation of penis, scrotum, prostate Late—prostate growth, balding, sebaceous gland activity Testosterone is converted to DHT by 5α-reductase, which is inhibited by finasteride. In the male, androgens are converted to estrogen by cytochrome P-450 aromatase (primarily in adipose tissue and testis). Aromatase is the key enzyme in conversion of androgens to estrogen.

1	In the male, androgens are converted to estrogen by cytochrome P-450 aromatase (primarily in adipose tissue and testis). Aromatase is the key enzyme in conversion of androgens to estrogen. Androgenic steroid abuse—abuse of anabolic steroids to  fat-free mass, muscle strength, and performance. Suspect in men who present with changes in behavior (eg, aggression), acne, gynecomastia,  Hb and Hct, small testes (exogenous testosterone • hypothalamic-pituitary-gonadal axis inhibition • intratesticular testosterone  testicular size,  sperm count, azoospermia). Women may present with virilization (eg, hirsutism, acne, breast atrophy, male pattern baldness). Tanner stages of sexual development Tanner stage is assigned independently to genitalia, pubic hair, and breast (eg, a person can have Tanner stage 2 genitalia, Tanner stage 3 pubic hair). Earliest detectable secondary sexual characteristic is breast bud development in girls, testicular enlargement in boys.

1	Coarsening of pubic Coarse hair across pubis, Coarse hair across pubis and Flat-appearing chest with (pubarche) hair Breast enlarges, raised adult size (thelarche), mound forms areola, mound on mound Adult breast contour, areola fattens Pre-pubertal ~ 811.5 years ~ 11.5–13 years ~ 13–15 years Usually > 15 years Precocious puberty Appearance of 2° sexual characteristics (eg, adrenarche, thelarche, menarche) before age 8 years in girls and 9 years in boys.  sex hormone exposure or production  linear growth, somatic and skeletal maturation (eg, premature closure of epiphyseal plates  short stature). Types include: Central precocious puberty ( GnRH secretion): idiopathic (most common; early activation of hypothalamic-pituitary gonadal axis), CNS tumors.

1	Central precocious puberty ( GnRH secretion): idiopathic (most common; early activation of hypothalamic-pituitary gonadal axis), CNS tumors. Peripheral precocious puberty (GnRH-independent;  sex hormone production or exposure to exogenous sex steroids): congenital adrenal hyperplasia, estrogen-secreting ovarian tumor (eg, granulosa cell tumor), Leydig cell tumor, McCune-Albright syndrome. Sex chromosome Aneuploidy most commonly due to meiotic nondisjunction. disorders Klinefelter syndrome Male, 47,XXY. Dysgenesis of seminiferous tubules Testicular atrophy, eunuchoid body shape,  inhibin B  FSH. tall, long extremities, gynecomastia, female Abnormal Leydig cell function  testosterone A . May present with  LH  estrogen. developmental delay. Presence of inactivated X chromosome (Barr body). Common cause of hypogonadism seen in infertility work-up.

1	A . May present with  LH  estrogen. developmental delay. Presence of inactivated X chromosome (Barr body). Common cause of hypogonadism seen in infertility work-up. Double Y males 47, XYY. Phenotypically normal (usually undiagnosed), very tall. Normal fertility. May be associated with severe acne, learning disability, autism spectrum disorders. Female, 45,XO. Short stature (associated with SHOX gene, preventable with growth hormone therapy), ovarian dysgenesis (streak ovary), shield chest B , bicuspid aortic valve, coarctation of the aorta (femoral < brachial pulse), lymphatic defects (result in webbed neck or cystic hygroma; lymphedema in feet, hands), horseshoe kidney, high-arched palate, shortened 4th metacarpals. Most common cause of 1° amenorrhea. No Barr body. Menopause before menarche.

1	Most common cause of 1° amenorrhea. No Barr body. Menopause before menarche.  estrogen leads to  LH, FSH. Sex chromosome (X, or rarely Y) loss often due to nondisjunction during meiosis or mitosis. Meiosis errors usually occur in paternal gametes  sperm missing the sex chromosome. Mitosis errors occur after zygote formation  loss of sex chromosome in some but not all cells  mosaic karyotype (eg. 45,X/46XX). (45,X/46,XY) mosaicism associated with increased risk for gonadoblastoma. Pregnancy is possible in some cases (IVF, exogenous estradiol-17β and progesterone). • Defective androgen receptor • Testosterone-secreting tumor, exogenous ⊕⊝ Hypergonadotropic hypogonadism (eg, Turner syndrome, genetic mosaicism, pure gonadal dysgenesis) Hypogonadotropic hypogonadism (eg, CNS lesions, Kallmann syndrome)

1	Kallmann syndrome Failure to complete puberty; a form of hypogonadotropic hypogonadism. Defective migration of neurons and subsequent failure of olfactory bulbs to develop  synthesis of GnRH in the hypothalamus; hyposmia/anosmia;  GnRH, FSH, LH, testosterone. Infertility (low sperm count in males; amenorrhea in females). Abruptio placentae Premature separation (partial or complete) of placenta from uterine wall before delivery of infant. Risk factors: trauma (eg, motor vehicle accident), smoking, hypertension, preeclampsia, cocaine abuse. Presentation: abrupt, painful bleeding (concealed or apparent) in third trimester; possible DIC (mediated by tissue factor activation), maternal shock, fetal distress. May be life threatening for mother and fetus. Complete abruption with Partial abruption (blue arrow) concealed hemorrhage with apparent hemorrhage (red arrow)

1	Complete abruption with Partial abruption (blue arrow) concealed hemorrhage with apparent hemorrhage (red arrow) Defective decidual layer  abnormal attachment and separation after delivery. Risk factors: prior C-section or uterine surgery involving myometrium, inflammation, placenta previa, advanced maternal age, multiparity. Three types distinguishable by the depth of penetration: Placenta accreta—placenta attaches to myometrium without penetrating it; most common type. Placenta increta—placenta penetrates into myometrium. Placenta percreta—placenta penetrates (“perforates”) through myometrium and into uterine serosa (invades entire uterine wall); can result in placental attachment to rectum or bladder (can result in hematuria). Presentation: often detected on ultrasound prior to delivery. No separation of placenta after delivery  postpartum bleeding (can cause Sheehan syndrome).

1	Presentation: often detected on ultrasound prior to delivery. No separation of placenta after delivery  postpartum bleeding (can cause Sheehan syndrome). Placenta previa Attachment of placenta over internal cervical os. Risk factors: multiparity, prior C-section. Associated with painless third-trimester bleeding. A “preview” of the placenta is visible through cervix. Low-lying placenta (< 2 cm from internal cervical os, but not over it) is managed differently from placenta previa. Implantation of fertilized ovum in a site other Isthmus (highest riskInterstitium than the uterus, most often in ampulla of Fallopian tube of tubal rupture) fallopian tube A . Suspect with history of amenorrhea, lower-than-expected rise in hCG based on dates, and sudden lower abdominal pain; confirm with ultrasound, which may show extraovarian adnexal mass. Often clinically mistaken for appendicitis. Pain +/− bleeding. Risk factors: History of infertility

1	Pain +/− bleeding. Risk factors: History of infertility Hydatidiform mole Cystic swelling of chorionic villi and proliferation of chorionic epithelium (only trophoblast). Presents with vaginal bleeding, emesis, uterine enlargement more than expected, pelvic pressure/ pain. Associated with hCG-mediated sequelae: early preeclampsia (before 20 weeks), theca-lutein cysts, hyperemesis gravidarum, hyperthyroidism. Treatment: dilation and curettage and methotrexate. Monitor hCG. Rare; can develop during or after pregnancy in mother or baby. Malignancy of trophoblastic tissue A (cytotrophoblasts, syncytiotrophoblasts); no chorionic villi present.  frequency of bilateral/ multiple theca-lutein cysts. Presents with abnormal • hCG, shortness of breath, hemoptysis. Hematogenous spread to lungs B . Treatment: methotrexate. Incidence (US)—endometrial > ovarian > cervical; cervical cancer is more common worldwide due to lack of screening or HPV vaccination.

1	B . Treatment: methotrexate. Incidence (US)—endometrial > ovarian > cervical; cervical cancer is more common worldwide due to lack of screening or HPV vaccination. Prognosis: Cervical (best prognosis, diagnosed < 45 years old) > Endometrial (middleaged, about 55 years old) > Ovarian (worst prognosis, > 65 years). CEOs often go from best to worst as they get older. Bartholin cyst and Due to blockage of Bartholin gland duct causing accumulation of gland fluid. May lead to abscess abscess 2° to obstruction and inflammation A . Usually in reproductive-age females. Lichen sclerosus Thinning of epidermis with fibrosis/sclerosis of dermis. Presents with porcelain-white plaques with a red or violet border. Skin fragility with erosions can be observed B . Most common in postmenopausal women. Benign, but slightly increased risk for SCC.

1	Lichen simplex Hyperplasia of vulvar squamous epithelium. Presents with leathery, thick vulvar skin with chronicus enhanced skin markings due to chronic rubbing or scratching. Benign, no risk of SCC. Neoplastic Vulvar carcinoma Carcinoma from squamous epithelial lining of vulva C . Rare. Presents with leukoplakia, biopsy often required to distinguish carcinoma from other causes. HPV-related vulvar carcinoma—associated with high-risk HPV types 16, 18. Risk factors: multiple partners, early coitarche. Usually in reproductive-age females. Non-HPV vulvar carcinoma—usually from long-standing lichen sclerosus. Females > 70 years old. Imperforate hymen Incomplete degeneration of the central portion of the hymen. Accumulation of vaginal mucus at birth  self-resolving bulge in introitus. If untreated, leads to 1° amenorrhea, cyclic abdominal pain, hematocolpos (accumulation of menstrual blood in vagina  bulging and bluish hymenal membrane).

1	Vaginal squamous cell Usually 2° to cervical SCC; 1° vaginal carcinoma rare. carcinoma Disordered epithelial growth; begins at basal layer of squamocolumnar junction (transformation zone) and extends outward. Classified as CIN 1, CIN 2, or CIN 3 (severe, irreversible dysplasia or carcinoma in situ), depending on extent of dysplasia. Associated with HPV-16 and HPV-18, which produce both the E6 gene product (inhibits TP53) and E7 gene product (inhibits pRb) (6 before 7; P before R). Koilocytes A are pathognomonic of HPV infection. May progress slowly to invasive carcinoma if left untreated. Typically asymptomatic (detected with Pap smear) or presents as abnormal vaginal bleeding (often postcoital). Risk factors: multiple sexual partners, HPV, smoking, early coitarche, DES exposure, immunocompromise (eg, HIV, transplant).

1	Risk factors: multiple sexual partners, HPV, smoking, early coitarche, DES exposure, immunocompromise (eg, HIV, transplant). Invasive carcinoma Often squamous cell carcinoma. Pap smear can detect cervical dysplasia before it progresses to invasive carcinoma. Diagnose via colposcopy and biopsy. Lateral invasion can block ureters • hydronephrosis • renal failure. Most common causes Pregnancy, polycystic ovarian syndrome, obesity, HPO axis abnormalities/immaturity, premature of anovulation ovarian failure, hyperprolactinemia, thyroid disorders, eating disorders, competitive athletics, Cushing syndrome, adrenal insufficiency, chromosomal abnormalities (eg, Turner syndrome). Also called exercise-induced amenorrhea. Severe caloric restriction,  energy expenditure, and/or stress  functional disruption of pulsatile GnRH secretion  LH, FSH, estrogen. Pathogenesis includes  leptin (due to  fat) and  cortisol (stress, excessive exercise).

1	Associated with eating disorders and “female athlete triad” ( calorie availability/excessive exercise,  bone mineral density, menstrual dysfunction). Hyperinsulinemia and/or insulin resistance hypothesized to alter hypothalamic hormonal feedback response • LH:FSH,  androgens (eg, testosterone) from theca interna cells,  rate of follicular maturation  unruptured follicles (cysts) + anovulation. Common cause of • fertility in women. Enlarged, bilateral cystic ovaries A ; presents with amenorrhea/oligomenorrhea, hirsutism, acne,  fertility. Associated with obesity, acanthosis nigricans.  risk of endometrial cancer 2° to unopposed estrogen from repeated anovulatory cycles. Treatment: cycle regulation via weight reduction ( peripheral estrone formation), OCPs (prevent endometrial hyperplasia due to unopposed estrogen); clomiphene (ovulation induction); spironolactone, finasteride, flutamide to treat hirsutism.

1	Primary dysmenorrhea Painful menses, caused by uterine contractions to  blood loss • ischemic pain. Mediated by prostaglandins. Treatment: NSAIDs. TyPE mAlIgNANT? CHARACTERISTICS arrow in E ). Tumor marker: AFP. Endometriosis Endometrium-like glands/stroma outside endometrial cavity, most commonly in the ovary (frequently bilateral), pelvis, peritoneum (yellow-brown “powder burn” lesions). In ovary, appears as endometrioma (blood-filled “chocolate cysts” [oval structures above and below asterisks in A ]). May be due to retrograde flow, metaplastic transformation of multipotent cells, transportation of endometrial tissue via lymphatic system. Characterized by cyclic pelvic pain, bleeding, dysmenorrhea, dyspareunia, dyschezia (pain with defecation), infertility; normal-sized uterus. Treatment: NSAIDs, OCPs, progestins, GnRH agonists, danazol, laparoscopic removal.

1	Endometritis Inflammation of endometrium B associated with retained products of conception following delivery, miscarriage, abortion, or with foreign body (eg, IUD). Retained material is nidus for bacteria from vagina or GI tract. Chronic endometritis shows plasma cells on histology. Treatment: gentamicin + clindamycin +/− ampicillin. Commonly postmenopausal. Often presents as a palpable hard mass A most often in the upper outer quadrant. Invasive cancer can become fixed to pectoral muscles, deep fascia, Cooper ligaments, and overlying skin  nipple retraction/skin dimpling. Usually arises from terminal duct lobular unit. Amplification/overexpression of estrogen/ progesterone receptors or c-erbB2 (HER2, an EGF receptor) is common; triple negative (ER ⊝, PR ⊝, and HER2/neu ⊝) form more aggressive.

1	Risk factors in women:  age; history of atypical hyperplasia; family history of breast cancer; race (Caucasians at highest risk, African Americans at  risk for triple ⊝ breast cancer); BRCA1/BRCA2 mutations;  estrogen exposure (eg, nulliparity); postmenopausal obesity (adipose tissue converts androstenedione to estrone);  total number of menstrual cycles; absence of breastfeeding; later age of first pregnancy; alcohol intake. In men: BRCA2 mutation, Klinefelter syndrome. Axillary lymph node metastasis most important prognostic factor in early-stage disease. Invasive ductal Firm, fibrous, “rock-hard” mass with sharp margins and small, glandular, duct-like cells in desmoplastic stroma. aAll types of invasive breast carcinoma can be either of tubular subtype (well-differentiated tubules that lack myoepithelium) or mucinous subtype (abundant extracellular mucin, seen in older women).

1	Peyronie disease Abnormal curvature of penis A due to fibrous plaque within tunica albuginea. Associated with erectile dysfunction. Can cause pain, anxiety. Consider surgical repair or treatment with collagenase injections once curvature stabilizes. Distinct from penile fracture (rupture of corpora cavernosa due to forced bending). Ischemic priapism Painful sustained erection lasting > 4 hours. Associated with sickle cell disease (sickled RBCs block venous drainage of corpus cavernosum vascular channels), medications (eg, sildenafil, trazodone). Treat immediately with corporal aspiration, intracavernosal phenylephrine, or surgical decompression to prevent ischemia. Squamous cell Seen in the US, but more common in Asia, Africa, South America. Precursor in situ lesions: carcinoma Bowen disease (in penile shaft, presents as leukoplakia “white plaque”), erythroplasia of Queyrat

1	B (carcinoma in situ of the glans B , presents as erythroplakia “red plaque”). Bowenoid papulosis (carcinoma in situ of unclear malignant potential, presenting as reddish papules). Associated with uncircumcised males and HPV. Cryptorchidism Descent failure of one A or both testes; impaired spermatogenesis (since sperm develop best at temperatures < 37°C); can have normal testosterone levels (Leydig cells are mostly unaffected by temperature); associated with  risk of germ cell tumors. Prematurity  risk of cryptorchidism.  inhibin B,  FSH,  LH; testosterone  in bilateral cryptorchidism, normal in unilateral. Most cases resolve spontaneously; otherwise, orchiopexy performed before 2 years of age.

1	 inhibin B,  FSH,  LH; testosterone  in bilateral cryptorchidism, normal in unilateral. Most cases resolve spontaneously; otherwise, orchiopexy performed before 2 years of age. Testicular torsion Rotation of testicle around spermatic cord and vascular pedicle. Commonly presents in males 12–18 years old. May occur after an inciting event (eg, trauma) or spontaneously. Characterized by acute, severe pain, high-riding testis, and absent cremasteric reflex. Treatment: surgical correction (orchiopexy) within 6 hours, manual detorsion if surgical option unavailable in timeframe. If testis is not viable, orchiectomy. Orchiopexy, when performed, should be bilateral because the contralateral testis is at risk for subsequent torsion.

1	Varicocele Dilated veins in pampiniform plexus due to  venous pressure; most common cause of scrotal enlargement in adult males; most often on left side because of  resistance to flow from left gonadal vein drainage into left renal vein; can cause infertility because of • temperature; diagnosed by standing clinical exam/Valsalva maneuver (distension on inspection and “bag of worms” on palpation; augmented by Valsalva) or ultrasound A ; does not transilluminate. Treatment: consider surgical ligation or embolization if associated with pain or infertility. Extragonadal germ cell Arise in midline locations. In adults, most commonly in retroperitoneum, mediastinum, pineal, and tumors suprasellar regions. In infants and young children, sacrococcygeal teratomas are most common. Scrotal masses Benign scrotal lesions present as testicular masses that can be transilluminated (vs solid testicular tumors).

1	Scrotal masses Benign scrotal lesions present as testicular masses that can be transilluminated (vs solid testicular tumors). Congenital hydrocele Common cause of scrotal swelling A in infants, Transilluminating swelling. due to incomplete obliteration of processus vaginalis. Most spontaneously resolve within 1 year. Germ cell tumors account for ∼ 95% of all testicular tumors. Arise from germ cells that produce sperm. Most often occur in young men. Risk factors: cryptorchidism, Klinefelter syndrome. Can present as a mixed germ cell tumor. Do not transilluminate. Usually not biopsied (risk of seeding scrotum), removed via radical orchiectomy. Sex cord stromal tumors develop from embryonic sex cord (develops into Sertoli and Leydig cells of seminiferous tubules, theca and granulosa cells of follicle) derivatives. 5% of all testicular tumors. Mostly benign.

1	Orchitis Inflammation of testis. Presents with testicular pain and swelling. Mumps orchitis  infertility risk. Rare in boys < 10 years old. Prostatitis Characterized by dysuria, frequency, urgency, low back pain. Warm, tender, enlarged prostate. Acute bacterial prostatitis—in older men most common bacterium is E coli; in young men consider C trachomatis, N gonorrhoeae. Chronic prostatitis—either bacterial or nonbacterial (eg, 2° to previous infection, nerve problems, chemical irritation). Common in men > 50 years old. Characterized by smooth, elastic, firm nodular enlargement (hyperplasia not hypertrophy) of periurethral (lateral and middle) lobes, which compress the urethra into a vertical slit. Not premalignant. Often presents with  frequency of urination, nocturia, difficulty starting and stopping urine stream, dysuria. May lead to distention and hypertrophy of bladder, hydronephrosis, UTIs.  free prostate-specific antigen (PSA).

1	 free prostate-specific antigen (PSA). Treatment: α1-antagonists (terazosin, tamsulosin), which cause relaxation of smooth muscle; 5α-reductase inhibitors (eg, finasteride); PDE-5 inhibitors (eg, tadalafil); surgical resection (eg, TURP, ablation). Control of reproductive hormones Goserelin, leuprolide mECHANISm GnRH analogs. When used in pulsatile Leuprolide can be used in lieu of GnRH. fashion act as GnRH agonists. When used in continuous fashion first transiently act as GnRH agonists (tumor flare), but subsequently act as GnRH antagonists (downregulate • LH). ClINICAl USE Uterine fibroids, endometriosis, precocious puberty, prostate cancer, infertility. ADVERSE EFFECTS Hypogonadism,  libido, erectile dysfunction, nausea, vomiting. mECHANISm GnRH antagonist. No start-up flare. ClINICAl USE Prostate cancer. ADVERSE EFFECTS Hot flashes, liver toxicity. Estrogens Ethinyl estradiol, DES, mestranol. mECHANISm Bind estrogen receptors.

1	ClINICAl USE Prostate cancer. ADVERSE EFFECTS Hot flashes, liver toxicity. Estrogens Ethinyl estradiol, DES, mestranol. mECHANISm Bind estrogen receptors. ClINICAl USE Hypogonadism or ovarian failure, menstrual abnormalities (combined OCPs), hormone replacement therapy in postmenopausal women. ADVERSE EFFECTS  risk of endometrial cancer (when given without progesterone), bleeding in postmenopausal women, clear cell adenocarcinoma of vagina in females exposed to DES in utero,  risk of thrombi. Contraindications—ER ⊕ breast cancer, history of DVTs, tobacco use in women > 35 years old. Raloxifene Antagonist at breast, uterus; agonist at bone;  risk of thromboembolic events (especially with smoking) but no increased risk of endometrial cancer (vs tamoxifen); used primarily to treat osteoporosis. Aromatase inhibitors Anastrozole, letrozole, exemestane. mECHANISm Inhibit peripheral conversion of androgens to estrogen. ClINICAl USE ER ⊕ breast cancer in postmenopausal women.

1	Aromatase inhibitors Anastrozole, letrozole, exemestane. mECHANISm Inhibit peripheral conversion of androgens to estrogen. ClINICAl USE ER ⊕ breast cancer in postmenopausal women. mECHANISm Bind progesterone receptors,  growth and  vascularization of endometrium, thicken cervical mucus. ClINICAl USE Contraception (forms include pill, intrauterine device, implant, depot injection), endometrial cancer, abnormal uterine bleeding. Progestin challenge: presence of withdrawal bleeding excludes anatomic defects (eg, Asherman syndrome) and chronic anovulation without estrogen. Antiprogestins Mifepristone, ulipristal. mECHANISm Competitive inhibitors of progestins at progesterone receptors. ClINICAl USE Termination of pregnancy (mifepristone with misoprostol); emergency contraception (ulipristal). mECHANISm Produces local inflammatory reaction toxic to sperm and ova, preventing fertilization and implantation; hormone free.

1	mECHANISm Produces local inflammatory reaction toxic to sperm and ova, preventing fertilization and implantation; hormone free. ClINICAl USE Long-acting reversible contraception. Most effective emergency contraception. ADVERSE EFFECTS Heavier or longer menses, dysmenorrhea. Risk of PID with insertion (contraindicated in active pelvic infection). Tocolytics Medications that relax the uterus; include terbutaline (β2-agonist action), nifedipine (Ca2+ channel blocker), indomethacin (NSAID). Used to  contraction frequency in preterm labor and allow time for administration of steroids (to promote fetal lung maturity) or transfer to appropriate medical center with obstetrical care. mECHANISm Synthetic androgen that acts as partial agonist at androgen receptors. ClINICAl USE Endometriosis, hereditary angioedema. ADVERSE EFFECTS Weight gain, edema, acne, hirsutism, masculinization,  HDL levels, hepatotoxicity, idiopathic intracranial hypertension.

1	ClINICAl USE Endometriosis, hereditary angioedema. ADVERSE EFFECTS Weight gain, edema, acne, hirsutism, masculinization,  HDL levels, hepatotoxicity, idiopathic intracranial hypertension. Testosterone, methyltestosterone mECHANISm Agonists at androgen receptors. ClINICAl USE Treat hypogonadism and promote development of 2° sex characteristics; stimulate anabolism to promote recovery after burn or injury. ADVERSE EFFECTS Masculinization in females;  intratesticular testosterone in males by inhibiting release of LH (via negative feedback)  gonadal atrophy. Premature closure of epiphyseal plates. • LDL, • HDL. Finasteride 5α-reductase inhibitor ( conversion of Testosterone DHT (more potent). testosterone to DHT). Used for BPH and male-pattern baldness. Adverse effects: gynecomastia and sexual dysfunction. Tamsulosin α -antagonist used to treat BPH by inhibiting smooth muscle contraction. Selective for α receptors (found on prostate) vs vascular α1B receptors.

1	Tamsulosin α -antagonist used to treat BPH by inhibiting smooth muscle contraction. Selective for α receptors (found on prostate) vs vascular α1B receptors. mECHANISm Direct arteriolar vasodilator. ClINICAl USE Androgenetic alopecia (pattern baldness), severe refractory hypertension. “There’s so much pollution in the air now that if it weren’t for our lungs, there’d be no place to put it all.” “Freedom is the oxygen of the soul.” “Whenever I feel blue, I start breathing again.” —L. Frank Baum “Life is not the amount of breaths you take; it’s the moments that take your breath away.” —Will Smith, Hitch Group key respiratory, cardiovascular, and renal concepts together for study whenever possible. Know obstructive vs restrictive lung disorders, V˙/Q˙ mismatch, lung volumes, mechanics of respiration, and hemoglobin physiology. Lung cancers and other causes of lung masses are high yield. Be comfortable reading basic chest x-rays, CT scans, and PFTs.

1	Club cells Nonciliated; low columnar/cuboidal with secretory granules. Located in bronchioles. Degrade toxins; secrete component of surfactant; act as reserve cells. Type I pneumocytes Squamous. 97% of alveolar surfaces. Thinly line the alveoli (two black arrows in A ) for optimal gas exchange. A . 2 (surface tension) 2 functions: radius A . (≥ 2 is healthy; < 1.5 predictive of NRDS), foam Risk factors: prematurity, maternal diabetes (due stability index, surfactant-albumin ratio. to  fetal insulin), C-section delivery ( release Persistently low O2 tension  risk of PDA. of fetal glucocorticoids; less stressful than vaginal delivery). Treatment: maternal steroids before birth; exogenous surfactant for infant. Retinopathy of prematurity, Intraventricular hemorrhage, Bronchopulmonary dysplasia (RIB).

1	Treatment: maternal steroids before birth; exogenous surfactant for infant. Retinopathy of prematurity, Intraventricular hemorrhage, Bronchopulmonary dysplasia (RIB). Conducting zone Large airways consist of nose, pharynx, larynx, trachea, and bronchi. Airway resistance highest in the largeto medium-sized bronchi. Small airways consist of bronchioles that further divide into terminal bronchioles (large numbers in parallel  least airway resistance). Warms, humidifies, and filters air but does not participate in gas exchange  “anatomic dead space.” Cartilage and goblet cells extend to the end of bronchi. Pseudostratified ciliated columnar cells primarily make up epithelium of bronchus and extend to beginning of terminal bronchioles, then transition to cuboidal cells. Clear mucus and debris from lungs (mucociliary escalator). Airway smooth muscle cells extend to end of terminal bronchioles (sparse beyond this point).

1	Respiratory zone Lung parenchyma; consists of respiratory bronchioles, alveolar ducts, and alveoli. Participates in gas exchange. Mostly cuboidal cells in respiratory bronchioles, then simple squamous cells up to alveoli. Cilia terminate in respiratory bronchioles. Alveolar macrophages clear debris and participate in immune response. Right lung has 3 lobes; Left has Less Lobes (2) and Lingula (homolog of right middle lobe). Instead of a middle lobe, left lung has a space occupied by the heart A . Relation of the pulmonary artery to the bronchus at each lung hilum is described by RALS—Right Anterior; Left Superior. Carina is posterior to ascending aorta and anteromedial to descending aorta B . Right lung is a more common site for inhaled foreign bodies because right main stem bronchus is wider, more vertical, and shorter than the left. If you aspirate a peanut: While supine—usually enters superior segment of right lower lobe.

1	While supine—usually enters superior segment of right lower lobe. While lying on right side—usually enters right upper lobe. While upright—usually enters right lower lobe. Structures perforating diaphragm: At T8: IVC, right phrenic nerve At T10: esophagus, vagus (CN 10; 2 trunks) At T12: aorta (red), thoracic duct (white), azygos vein (blue) (“At T-1-2 it’s the red, white, and blue”) Diaphragm is innervated by C3, 4, and 5 (phrenic nerve). Pain from diaphragm irritation (eg, air, blood, or pus in peritoneal cavity) can be referred to shoulder (C5) and trapezius ridge (C3, 4). Number of letters = T level: T8: vena cava (IVC) T10: (O)esophagus T12: aortic hiatus I ate (8) ten eggs at twelve. C3, 4, 5 keeps the diaphragm alive. Other bifurcations: The common carotid bifourcates at C4. The trachea bifourcates at T4. The abdominal aorta bifourcates at L4. Lung volumes Note: a capacity is a sum of ≥ 2 physiologic volumes. 6.0

1	The common carotid bifourcates at C4. The trachea bifourcates at T4. The abdominal aorta bifourcates at L4. Lung volumes Note: a capacity is a sum of ≥ 2 physiologic volumes. 6.0 Tidal volume Air that moves into lung with each quiet inspiration, typically 500 mL 2.7 2.2 1.2 FRC Determination of physiologic dead space VD = physiologic dead space = anatomic dead space of conducting airways plus alveolar dead space; apex of healthy lung is largest contributor of alveolar dead space. Volume of inspired air that does not take part in gas exchange. VT = tidal volume. Paco2 = arterial Pco2. Peco = expired air Pco 2 2. Taco, Paco, Peco, Paco (refers to order of variables in equation) Physiologic dead space—approximately equivalent to anatomic dead space in normal lungs. May be greater than anatomic dead space in lung diseases with V˙/Q˙ defects. Minute ventilation Total volume of gas entering lungs per minute Normal values: VE = VT × RR Respiratory rate (RR) = 12–20 breaths/min

1	Minute ventilation Total volume of gas entering lungs per minute Normal values: VE = VT × RR Respiratory rate (RR) = 12–20 breaths/min Alveolar ventilation Volume of gas that reaches alveoli each minute T V = 150 mL/breath Elastic recoil Tendency for lungs to collapse inward and chest wall to spring outward. At FRC, airway and alveolar pressures equal atmospheric pressure (called zero), and intrapleural pressure is negative (preventing atelectasis). The inward pull of the lung is balanced by the outward pull of the chest wall. System pressure is atmospheric. Pulmonary vascular resistance (PVR) is at a minimum. 0.5 0.0 +2 Compliance Change in lung volume for a change in pressure (ΔV/ΔP). Inversely proportional to wall stiffness and increased by surfactant. 6 •  compliance = lung easier to fill (eg, emphysema, aging) •  compliance = lung harder to fill (eg, 4 pulmonary fibrosis, pneumonia, ARDS, pulmonary edema)

1	Hysteresis Lung inflation follows a different pressure-2 volume curve than lung deflation due to need to overcome surface tension forces in inflation. Hemoglobin Oxygen content of blood O2 content = (1.34 × Hb × Sao2) + (0.003 × Pao2) Hb = hemoglobin concentration; Sao2 = arterial O2 saturation Pao2 = partial pressure of O2 in arterial blood Normally 1 g Hb can bind 1.34 mL O2; normal Hb amount in blood is 15 g/dL. O2 binding capacity ≈ 20 mL O2/dL of blood. With  Hb there is  O2 content of arterial blood, but no change in O2 saturation and Pao2. O2 delivery to tissues = cardiac output × O2 content of blood. ODC has sigmoidal shape due to positive cooperativity (ie, tetrameric Hb molecule can 100 bind 4 O2 molecules and has higher affinity for each subsequent O2 molecule bound). 80 Myoglobin is monomeric and thus does 70 not show positive cooperativity; curve lacks 60 sigmoidal appearance.

1	Shifting ODC to the right  Hb affinity for O2 (facilitates unloading of O2 to tissue)  P50 (higher Po2 required to maintain 50% saturation). Shifting ODC to the left  O2 unloading compensatory erythrocytosis. Fetal Hb (2 α and 2 γ subunits) has higher affinity for O2 than adult Hb (due to  affinity for 2,3-BPG)  dissociation curve is shifted left, driving diffusion of O2 across the placenta from mother to fetus. Cyanide vs carbon Both inhibit aerobic metabolism via inhibition of complex IV (cytochrome c oxidase)  hypoxia monoxide poisoning that does not fully correct with supplemental O2 and  anaerobic metabolism. Both can lead to pink or cherry red skin (usually postmortem finding), seizures, and coma. SOURCE Byproduct of synthetic product combustion, Odorless gas from fires, car exhaust, or gas ingestion of amygdalin (cyanogenic glucoside heaters. found in apricot seeds) or cyanide. TREATmENT Hydroxocobalamin (binds cyanide 100% O2, hyperbaric O2.

1	TREATmENT Hydroxocobalamin (binds cyanide 100% O2, hyperbaric O2. • cyanocobalamin • renal excretion). Nitrites (oxidize Hb • methemoglobin  binds cyanide • cyanomethemoglobin  less toxicity). Sodium thiosulfate (• cyanide conversion to thiocyanate • renal excretion). SIgNS/SYmPTOmS Breath has bitter almond odor; cardiovascular Headache, dizziness. collapse. Multiple individuals may be involved (eg, family with similar symptoms in winter). Classically associated with bilateral globus pallidus lesions on MRI A , although rarely seen with cyanide toxicity as well. normal initially. Left shift in curve  affinity for O2  O2 unloading in tissues. Binds competitively to Hb with 200× greater affinity than O2 to form carboxyhemoglobin • %O2 saturation of Hb. O2 bound to Hb (mL O2 /100 mL)

1	Binds competitively to Hb with 200× greater affinity than O2 to form carboxyhemoglobin • %O2 saturation of Hb. O2 bound to Hb (mL O2 /100 mL) Normally a low-resistance, high-compliance system. A  in Pao2 causes a hypoxic vasoconstriction that shifts blood away from poorly ventilated regions of lung to well-ventilated regions of lung. Perfusion limited—O2 (normal health), CO2, N2O. Gas equilibrates early along the length of the capillary. Exchange can be  only if blood flow . Diffusion limited—O2 (emphysema, fibrosis, exercise), CO. Gas does not equilibrate by the time blood reaches the end of the capillary. A consequence of pulmonary hypertension is cor pulmonale and subsequent right ventricular failure. P – P Diffusion: V˙ gas = A × Dk × 1 2 where Δ A = area, Δ x = alveolar wall thickness, Dk = diffusion coefficient of gas, P1 – P2 = difference in partial pressures. A  in emphysema. Δ x  in pulmonary fibrosis.

1	A = area, Δ x = alveolar wall thickness, Dk = diffusion coefficient of gas, P1 – P2 = difference in partial pressures. A  in emphysema. Δ x  in pulmonary fibrosis. DLCO is the extent to which CO passes from air sacs of lungs into blood. Ventilation/perfusion Ideally, ventilation is matched to perfusion (ie, mismatch V˙/Q˙ = 1) for adequate gas exchange. Lung zones: V˙/Q˙ at apex of lung = 3 (wasted ventilation) Q V˙/Q˙ at base of lung = 0.6 (wasted perfusion) Both ventilation and perfusion are greater at the base of the lung than at the apex of the lung. With exercise ( cardiac output), there is vasodilation of apical capillaries  V˙/Q˙ ratio approaches 1. Certain organisms that thrive in high O2 (eg, TB) flourish in the apex.

1	With exercise ( cardiac output), there is vasodilation of apical capillaries  V˙/Q˙ ratio approaches 1. Certain organisms that thrive in high O2 (eg, TB) flourish in the apex. V ˜˜ V˙/Q˙ = 0 = “oirway” obstruction (shunt). In Q shunt, 100% O2 does not improve Pao2 (eg, foreign body aspiration). V˙/Q˙ = ∞ = blood flow obstruction (physiologic dead space). Assuming < 100% dead space, 100% O2 improves Pao2 (eg, pulmonary embolus). CO2 is transported from tissues to lungs in 3 forms: HCO3 − (70%). Carbaminohemoglobin or HbCO2 (21–25%). CO2 bound to Hb at N-terminus of globin (not heme). CO2 favors deoxygenated form (O2 unloaded). Dissolved CO2 (5–9%). In lungs, oxygenation of Hb promotes dissociation of H+ from Hb. This shifts equilibrium toward CO2 formation; therefore, CO2 is released from RBCs (Haldane effect). In peripheral tissue,  H+ from tissue metabolism shifts curve to right, unloading O2 (Bohr effect). Majority of blood CO2 is carried as HCO3 − in the plasma.

1	In peripheral tissue,  H+ from tissue metabolism shifts curve to right, unloading O2 (Bohr effect). Majority of blood CO2 is carried as HCO3 − in the plasma. Response to high altitude sickness. Chronic  in ventilation. erythropoietin  Hct and Hb (due to chronic hypoxia). 2,3-BPG (binds to Hb causing rightward shift of the ODC so that Hb releases more O2). Cellular changes ( mitochondria). renal excretion of HCO3 − to compensate for respiratory alkalosis (can augment with acetazolamide). Chronic hypoxic pulmonary vasoconstriction results in pulmonary hypertension and RVH. Response to exercise  CO2 production.  O2 consumption. Right shift of ODC. ventilation rate to meet O2 demand. V˙/Q˙ ratio from apex to base becomes more uniform. pulmonary blood flow due to  cardiac output. pH during strenuous exercise (2° to lactic acidosis). No change in Pao2 and Paco2, but  in venous CO2 content and  in venous O2 content.

1	pulmonary blood flow due to  cardiac output. pH during strenuous exercise (2° to lactic acidosis). No change in Pao2 and Paco2, but  in venous CO2 content and  in venous O2 content. Obstruction of sinus drainage into nasal cavity  inflammation and pain over affected area. Typically affects maxillary sinuses, which drain against gravity due to ostia located superomedially (red arrow points to fluid-filled right maxillary sinus in A ). Superior meatus—drains sphenoid, posterior ethmoid; middle meatus—drains frontal, maxillary, and anterior ethmoid; inferior meatus—drains nasolacrimal duct. Most common acute cause is viral URI; may lead to superimposed bacterial infection, most commonly H influenzae, S pneumoniae, M catarrhalis. Paranasal sinus infections may extend to the orbits, cavernous sinus, and brain, causing complications (eg, orbital cellulitis, cavernous sinus syndrome, meningitis).

1	Paranasal sinus infections may extend to the orbits, cavernous sinus, and brain, causing complications (eg, orbital cellulitis, cavernous sinus syndrome, meningitis). Head and neck cancer Mostly squamous cell carcinoma. Risk factors include tobacco, alcohol, HPV-16 (oropharyngeal), EBV (nasopharyngeal). Field cancerization: carcinogen damages wide mucosal area • multiple tumors that develop independently after exposure. Blood clot within a deep vein  swelling, redness A , warmth, pain. Predisposed by Virchow triad (SHE): Stasis (eg, post-op, long drive/flight) Hypercoagulability (eg, defect in coagulation cascade proteins, such as factor V Leiden; oral contraceptive use; pregnancy) Most pulmonary emboli arise from proximal deep veins of lower extremity. d-dimer lab test used clinically to rule out DVT in low-to-moderate risk patients (high sensitivity, low specificity). Imaging test of choice is compression ultrasound with Doppler.

1	d-dimer lab test used clinically to rule out DVT in low-to-moderate risk patients (high sensitivity, low specificity). Imaging test of choice is compression ultrasound with Doppler. Use unfractionated heparin or low-molecular weight heparins (eg, enoxaparin) for prophylaxis and acute management. Use oral anticoagulants (eg, rivaroxaban, apixaban) for treatment and long-term prevention.

1	Pulmonary emboli V˙/Q˙ mismatch, hypoxemia, respiratory alkalosis. Sudden-onset dyspnea, pleuritic chest pain, tachypnea, tachycardia. Large emboli or saddle embolus A may cause sudden death due to electromechanical dissociation (pulseless electrical activity). CT pulmonary angiography is imaging test of choice for PE (look for filling defects) B . May have S1Q3T3 abnormality on ECG. Lines of Zahn C are interdigitating areas of pink (platelets, fibrin) and red (RBCs) found only in thrombi formed before death; help distinguish preand postmortem thrombi. Types: Fat, Air, Thrombus, Bacteria, Amniotic fluid, Tumor. An embolus moves like a FAT BAT. Fat emboli—associated with long bone fractures and liposuction; classic triad of hypoxemia, neurologic abnormalities, petechial rash. Air emboli—nitrogen bubbles precipitate in ascending divers (caisson disease/decompression sickness); treat with hyperbaric O2; or, can be iatrogenic 2° to invasive procedures (eg, central line placement).

1	bubbles precipitate in ascending divers (caisson disease/decompression sickness); treat with hyperbaric O2; or, can be iatrogenic 2° to invasive procedures (eg, central line placement). Amniotic fluid emboli—typically occurs during labor or postpartum, but can be due to uterine trauma. Can lead to DIC. Rare, but high mortality.

1	Mediastinal pathology Normal mediastinum contains heart, thymus, lymph nodes, esophagus, and aorta. Mediastinal masses Some pathologies (eg, lymphoma, lung cancer, abscess) can occur in any compartment, but there are common associations: Anterior—4T’s: Thyroid (substernal goiter), Thymic neoplasm, Teratoma, “Terrible” lymphoma. Middle—esophageal carcinoma, metastases, hiatal hernia, bronchogenic cysts. Posterior—neurogenic tumor (eg, neurofibroma), multiple myeloma. Mediastinitis Inflammation of mediastinal tissues. Commonly due to postoperative complications of cardiothoracic procedures (≤ 14 days), esophageal perforation, or contiguous spread of odontogenic/retropharyngeal infection. Chronic mediastinitis—also known as fibrosing mediastinitis; due to  proliferation of connective tissue in mediastinum. Histoplasma capsulatum is common cause. Clinical features: fever, tachycardia, leukocytosis, chest pain, and sternal wound drainage.

1	Pneumomediastinum Presence of gas (usually air) in the mediastinum (black arrows show air around the aorta, red arrow shows air dissecting into the neck A ). Can either be spontaneous (due to rupture of pulmonary bleb) or 2° (eg, trauma, iatrogenic, Boerhaave syndrome). Ruptured alveoli allow tracking of air into the mediastinum via peribronchial and perivascular sheaths. Clinical features: chest pain, dyspnea, voice change, subcutaneous emphysema, ⊕ Hamman sign (crepitus on cardiac auscultation). Emphysema (“pink puffer”) Normal Centriacinar emphysema Panacinar emphysema Findings: barrel-shaped chest D , exhalation through pursed lips (increases airway pressure and prevents airway collapse). Centriacinar—affects respiratory bronchioles while sparing distal alveoli, associated with B . Frequently in upper lobes (smoke rises up). Panacinar—affects respiratory bronchioles and alveoli, associated with α1-antitrypsin deficiency. Frequently in lower lobes.

1	B . Frequently in upper lobes (smoke rises up). Panacinar—affects respiratory bronchioles and alveoli, associated with α1-antitrypsin deficiency. Frequently in lower lobes. Enlargement of air spaces recoil, • compliance, from destruction of alveolar walls (arrow in C ) and  blood volume in pulmonary capillaries. Imbalance of proteases and antiproteases • elastase activity  loss of elastic fibers • lung compliance. CXR: • AP diameter, flattened diaphragm, • lung field lucency. Asthma Findings: cough, wheezing, tachypnea, dyspnea, hypoxemia, • inspiratory/ expiratory ratio, pulsus paradoxus, mucus plugging E . Triggers: viral URIs, allergens, stress. Hyperresponsive bronchi • reversible bronchoconstriction. Smooth muscle hypertrophy and hyperplasia, Curschmann spirals F (shed epithelium forms whorled mucous plugs), and Charcot-Leyden crystals G (eosinophilic, hexagonal, double-pointed crystals formed from breakdown of eosinophils in sputum). DLCO normal or .

1	F (shed epithelium forms whorled mucous plugs), and Charcot-Leyden crystals G (eosinophilic, hexagonal, double-pointed crystals formed from breakdown of eosinophils in sputum). DLCO normal or . Type I hypersensitivity reaction. Diagnosis supported by spirometry and methacholine challenge. NSAID-exacerbated respiratory disease is a combination of COX inhibition (leukotriene overproduction • airway constriction), chronic sinusitis with nasal polyps, and asthma symptoms. TYPE PRESENTATION PAThOlOgY OThER Bronchiectasis Findings: purulent sputum, recurrent infections (most often P aeruginosa), hemoptysis, digital clubbing. Chronic necrotizing infection of bronchi or obstruction permanently dilated airways. Associated with bronchial obstruction, poor ciliary motility (eg, smoking, Kartagener syndrome), cystic fibrosis H , allergic bronchopulmonary aspergillosis. A B C D E G HF

1	Restricted lung expansion causes  lung volumes ( FVC and TLC). PFTs:  FEV1/FVC ratio. Patient presents with short, shallow breaths. Types: Poor breathing mechanics (extrapulmonary, normal DLCO, normal A-a gradient): Poor muscular effort—polio, myasthenia gravis, Guillain-Barré syndrome Poor structural apparatus—scoliosis, morbid obesity Interstitial lung diseases (pulmonary,  DLCO,  A-a gradient): Pneumoconioses (eg, coal workers’ pneumoconiosis, silicosis, asbestosis) Sarcoidosis: bilateral hilar lymphadenopathy, noncaseating granulomas;  ACE and Ca2+ Idiopathic pulmonary fibrosis (repeated cycles of lung injury and wound healing with  collagen deposition, “honeycomb” lung appearance [red arrows in A ], traction bronchiectasis [blue arrow in A ] and digital clubbing). Granulomatosis with polyangiitis (Wegener) Drug toxicity (eg, bleomycin, busulfan, amiodarone, methotrexate)

1	Granulomatosis with polyangiitis (Wegener) Drug toxicity (eg, bleomycin, busulfan, amiodarone, methotrexate) Hypersensitivity pneumonitis—mixed type III/IV hypersensitivity reaction to environmental antigen. Causes dyspnea, cough, chest tightness, fever, headache. Often seen in farmers and those exposed to birds. Reversible in early stages if stimulus is avoided.

1	Sarcoidosis Characterized by immune-mediated, widespread noncaseating granulomas A , elevated serum ACE levels, and elevated CD4/CD8 ratio in bronchoalveolar lavage fluid. More common in African-American females. Often asymptomatic except for enlarged lymph nodes. CXR shows bilateral adenopathy and coarse reticular opacities B ; CT of the chest better demonstrates the extensive hilar and mediastinal adenopathy C . Associated with Bell palsy, Uveitis, Granulomas (noncaseating epithelioid, containing microscopic Schaumann and asteroid bodies), Lupus pernio (skin lesions on face resembling lupus), Interstitial fibrosis (restrictive lung disease), Erythema nodosum, Rheumatoid arthritis-like arthropathy, hypercalcemia (due to  1α-hydroxylase–mediated vitamin D activation in macrophages). A facial droop is UGLIER. Treatment: steroids (if symptomatic).

1	Complication of inhalation of noxious stimuli (eg, smoke). Caused by heat, particulates (< 1 µm diameter), or irritants (eg, NH3)  chemical tracheobronchitis, edema, pneumonia, ARDS. Many patients present 2° to burns, CO inhalation, cyanide poisoning, or arsenic poisoning. Singed nasal hairs or soot in oropharynx common on exam. Bronchoscopy shows severe edema, congestion of bronchus, and soot deposition ( A , 18 hours after inhalation injury; B , resolution at 11 days after injury). Pneumoconioses Asbestos is from the roof (was common in insulation), but affects the base (lower lobes). Silica and coal are from the base (earth), but affect the roof (upper lobes). Asbestosis Associated with shipbuilding, roofing, plumbing. “Ivory white,” calcified, supradiaphragmatic A and pleural B plaques are pathognomonic of asbestosis. Risk of bronchogenic carcinoma > risk of mesothelioma.  risk of Caplan syndrome (rheumatoid arthritis and pneumoconioses with intrapulmonary nodules).

1	Affects lower lobes. Asbestos (ferruginous) bodies are golden-brown fusiform rods resembling dumbbells C , found in alveolar sputum sample, visualized using Prussian blue stain, often obtained by bronchoalveolar lavage.  risk of pleural effusions. Berylliosis Associated with exposure to beryllium in Affects upper lobes. aerospace and manufacturing industries. Granulomatous (noncaseating) D on histology and therefore occasionally responsive to steroids.  risk of cancer and cor pulmonale. Mesothelioma Malignancy of the pleura associated with Psammoma bodies seen on histology. asbestosis. May result in hemorrhagic pleural Calretinin and cytokeratin 5/6 ⊕ in almost all effusion (exudative), pleural thickening A . mesotheliomas, ⊝ in most carcinomas. Smoking not a risk factor.

1	PAThOPhYSIOlOgY Alveolar insult  release of pro-inflammatory cytokines  neutrophil recruitment, activation, and release of toxic mediators (eg, reactive oxygen species, proteases, etc)  capillary endothelial damage and  vessel permeability  leakage of protein-rich fluid into alveoli • formation of intra-alveolar hyaline membranes (arrows in A ) and noncardiogenic pulmonary edema (normal PCWP). Loss of surfactant also contributes to alveolar collapse. CAUSES Sepsis (most common), aspiration, pneumonia, trauma, pancreatitis. DIAgNOSIS Diagnosis of exclusion with the following criteria (ARDS): Respiratory failure within 1 week of alveolar insult Decreased Pao2/Fio2 (ratio < 300, hypoxemia due to  intrapulmonary shunting and diffusion abnormalities) Symptoms of respiratory failure are not due to HF/fluid overload CONSEQUENCES Impaired gas exchange,  lung compliance; pulmonary hypertension.

1	Symptoms of respiratory failure are not due to HF/fluid overload CONSEQUENCES Impaired gas exchange,  lung compliance; pulmonary hypertension. mANAgEmENT Treat the underlying cause. Mechanical ventilation:  tidal volume, • PEEP. Sleep apnea Repeated cessation of breathing > 10 seconds during sleep  disrupted sleep  daytime somnolence. Diagnosis confirmed by sleep study. Nocturnal hypoxia • systemic/pulmonary hypertension, arrhythmias (atrial fibrillation/flutter), sudden death. Hypoxia  EPO release  erythropoiesis. Central sleep apnea Impaired respiratory effort due to CNS injury/toxicity, HF, opioids. May be associated with Cheyne-Stokes respirations (oscillations between apnea and hyperpnea). Think 3 C’s: Congestive HF, CNS toxicity, Cheyne-Stokes respirations. Treat with positive airway pressure.

1	Obesity Obesity (BMI ≥ 30 kg/m2)  hypoventilation • Paco2 during waking hours (retention); • Pao2 hypoventilation and • Paco2 during sleep. Also known as Pickwickian syndrome. syndrome Normal mean pulmonary artery pressure = 10–14 mm Hg; pulmonary hypertension ≥ 25 mm Hg at rest. Results in arteriosclerosis, medial hypertrophy, intimal fibrosis of pulmonary arteries, plexiform lesions. Course: severe respiratory distress • cyanosis and RVH • death from decompensated cor pulmonale. Pleural effusion • Dull • None if small Away from side of lesion if large Atelectasis • Dull • Toward side of lesion Tension • Hyperresonant • Away from side of lesion pneumothorax Atelectasis Alveolar collapse (right upper lobe collapse against mediastinum in A ). Multiple causes: Obstructive—airway obstruction prevents new air from reaching distal airways, old air is resorbed (eg, foreign body, mucous plug, tumor)

1	Obstructive—airway obstruction prevents new air from reaching distal airways, old air is resorbed (eg, foreign body, mucous plug, tumor) Compressive—external compression on lung decreases lung volumes (eg, space-occupying lesion, pleural effusion) Contraction (cicatrization)—scarring of lung parenchyma that distorts alveoli (eg, sarcoidosis) Adhesive—due to lack of surfactant (eg, NRDS in premature babies) Pleural e˜usion, Scarring air, tumor Pleural effusions Excess accumulation of fluid A between pleural layers  restricted lung expansion during inspiration. Can be treated with thoracentesis to remove/reduce fluid B . Lymphatic Also known as chylothorax. Due to thoracic duct injury from trauma or malignancy. Milkyappearing fluid;  triglycerides.

1	Lymphatic Also known as chylothorax. Due to thoracic duct injury from trauma or malignancy. Milkyappearing fluid;  triglycerides. Exudate  protein content (> 2.9 g/dL), cloudy (cellular). Due to malignancy, inflammation/infection (eg, pneumonia, collagen vascular disease), trauma (occurs in states of  vascular permeability). Must be drained due to risk of infection. Transudate  protein content (< 2.5 g/dL), clear (hypocellular). Due to  hydrostatic pressure (eg, HF, Na+ retention) or  oncotic pressure (eg, nephrotic syndrome, cirrhosis). Pneumothorax Accumulation of air in pleural space A . Dyspnea, uneven chest expansion. Chest pain,  tactile fremitus, hyperresonance, and diminished breath sounds, all on the affected side. Primary spontaneous Due to rupture of apical subpleural bleb or cysts. Occurs most frequently in tall, thin, young males pneumothorax and smokers.

1	Primary spontaneous Due to rupture of apical subpleural bleb or cysts. Occurs most frequently in tall, thin, young males pneumothorax and smokers. Traumatic Caused by blunt (eg, rib fracture), penetrating (eg, gunshot), or iatrogenic (eg, central line pneumothorax placement, lung biopsy, barotrauma due to mechanical ventilation) trauma. Can be from any of the above. Air enters pleural space but cannot exit. Increasing trapped air • tension pneumothorax. Trachea deviates away from affected lung B . May lead to increased intrathoracic pressure • mediastinal displacement  kinking of IVC  venous return  cardiac output. Needs immediate needle decompression and chest tube placement. Lobar pneumonia S pneumoniae most frequently, also Legionella, Intra-alveolar exudate • consolidation A ; may Klebsiella involve entire lobe B or the whole lung.

1	Lobar pneumonia S pneumoniae most frequently, also Legionella, Intra-alveolar exudate • consolidation A ; may Klebsiella involve entire lobe B or the whole lung. Etiology unknown. Secondary organizing pneumonia is caused by chronic inflammatory diseases (eg, rheumatoid arthritis) or medication side effects (eg, amiodarone). ⊝ sputum and blood cultures, often responds to steroids but not to antibiotics. Formerly known as bronchiolitis obliterans organizing pneumonia (BOOP). Noninfectious pneumonia characterized by inflammation of bronchioles and surrounding structure. Natural history of lobar pneumonia

1	Natural history of lobar pneumonia Lung cancer Leading cause of cancer death. Presentation: cough, hemoptysis, bronchial obstruction, wheezing, pneumonic “coin” lesion on CXR or noncalcified nodule on CT. Sites of metastases from lung cancer: Liver (jaundice, hepatomegaly), Adrenals, Bone (pathologic fracture), Brain; “Lung ‘mets’ Love Affective Boneheads and Brainiacs.” In the lung, metastases (usually multiple lesions) are more common than 1° neoplasms. Most often from breast, colon, prostate, and bladder cancer. SPHERE of complications: Superior vena cava/thoracic outlet syndromes Pancoast tumor Horner syndrome Endocrine (paraneoplastic) Recurrent laryngeal nerve compression Risk factors include smoking, secondhand smoke, radon, asbestos, family history. Squamous and Small cell carcinomas are Sentral (central) and often caused by Smoking.

1	Risk factors include smoking, secondhand smoke, radon, asbestos, family history. Squamous and Small cell carcinomas are Sentral (central) and often caused by Smoking. TYPE lOCATION ChARACTERISTICS hISTOlOgY Small cell Small cell (oat cell) carcinoma Central Undifferentiated  very aggressive. May produce ACTH (Cushing syndrome), ADH (SIADH), or Antibodies against presynaptic Ca2+ channels (Lambert-Eaton myasthenic syndrome) or neurons (paraneoplastic myelitis, encephalitis, subacute cerebellar degeneration). Amplification of myc oncogenes common. Managed with chemotherapy +/– radiation. Neoplasm of neuroendocrine Kulchitsky cells  small dark blue cells A . Chromogranin A ⊕, neuron-specific enolase ⊕, synaptophysin ⊕. Non-small cell

1	Adenocarcinoma Peripheral Most common 1° lung cancer. More common in women than men, most likely to arise in nonsmokers. Activating mutations include KRAS, EGFR, and ALK. Associated with hypertrophic osteoarthropathy (clubbing). Bronchioloalveolar subtype (adenocarcinoma in situ): CXR often shows hazy infiltrates similar to pneumonia; better prognosis. Glandular pattern on histology, often stains mucin ⊕ B . Bronchioloalveolar subtype: grows along alveolar septa  apparent “thickening” of alveolar walls. Tall, columnar cells containing mucus. Squamous cell carcinoma Central Hilar mass C arising from bronchus; Cavitation; Cigarettes; hyperCalcemia (produces PTHrP). Keratin pearls D and intercellular bridges. Large cell carcinoma Peripheral Highly anaplastic undifferentiated tumor; poor prognosis. Less responsive to chemotherapy; removed surgically. Strong association with smoking. Pleomorphic giant cells E . Localized collection of pus within parenchyma

1	Localized collection of pus within parenchyma A . Caused by aspiration of oropharyngeal contents (especially in patients predisposed to loss of consciousness [eg, alcoholics, epileptics]) or bronchial obstruction (eg, cancer). B often seen on CXR; presence suggests cavitation. Due to anaerobes (eg, Bacteroides, Fusobacterium, Peptostreptococcus) or S aureus. Treatment: antibiotics, drainage, or surgery. Lung abscess 2° to aspiration is most often found in right lung. Location depends on patient’s position during aspiration: RLL if upright, RUL or RML if recumbent. Pancoast tumor Also known as superior sulcus tumor. Carcinoma that occurs in the apex of lung A may cause Pancoast syndrome by invading/compressing local structures. Compression of locoregional structures may cause array of findings: Stellate ganglion • Horner syndrome (ipsilateral ptosis, miosis, anhidrosis)

1	Stellate ganglion • Horner syndrome (ipsilateral ptosis, miosis, anhidrosis) An obstruction of the SVC that impairs blood drainage from the head (“facial plethora”; note blanching after fingertip pressure in A ), neck (jugular venous distention), and upper extremities (edema). Commonly caused by malignancy (eg, mediastinal mass, Pancoast tumor) and thrombosis from indwelling catheters B . Medical emergency. Can raise intracranial pressure (if obstruction is severe) • headaches, dizziness,  risk of aneurysm/ rupture of intracranial arteries. Histamine-1 blockers Reversible inhibitors of H1 histamine receptors. First generation Diphenhydramine, dimenhydrinate, Names usually contain “-en/-ine” or “-en/-ate.” chlorpheniramine, doxylamine. ClINICAl USE Allergy, motion sickness, sleep aid. ADVERSE EFFECTS Sedation, antimuscarinic, anti-α-adrenergic. Second generation Loratadine, fexofenadine, desloratadine, Names usually end in “-adine.” cetirizine. ClINICAl USE Allergy.

1	ADVERSE EFFECTS Sedation, antimuscarinic, anti-α-adrenergic. Second generation Loratadine, fexofenadine, desloratadine, Names usually end in “-adine.” cetirizine. ClINICAl USE Allergy. ADVERSE EFFECTS Far less sedating than 1st generation because of  entry into CNS. Guaifenesin Expectorant—thins respiratory secretions; does not suppress cough reflex. N-acetylcysteine Mucolytic—liquifies mucus in chronic bronchopulmonary diseases (eg, COPD, CF) by disrupting disulfide bonds. Also used as an antidote for acetaminophen overdose. Dextromethorphan Antitussive (antagonizes NMDA glutamate receptors). Synthetic codeine analog. Has mild opioid effect when used in excess. Naloxone can be given for overdose. Mild abuse potential. May cause serotonin syndrome if combined with other serotonergic agents. Pseudoephedrine, phenylephrine mEChANISm α-adrenergic agonists. ClINICAl USE Reduce hyperemia, edema (used as nasal decongestants); open obstructed eustachian tubes.

1	Pseudoephedrine, phenylephrine mEChANISm α-adrenergic agonists. ClINICAl USE Reduce hyperemia, edema (used as nasal decongestants); open obstructed eustachian tubes. ADVERSE EFFECTS Hypertension. Rebound congestion if used more than 4–6 days. Can also cause CNS stimulation/ anxiety (pseudoephedrine). therapy IgE and blocks binding to FcεRI. Used in allergic asthma with  IgE levels resistant to inhaled steroids and long-acting β2-agonists. (leukotrienes, histamine, interleukins, etc) due to  cAMP hydrolysis. Limited use due to narrow therapeutic index (cardiotoxicity, neurotoxicity); metabolized by cytochrome ˜-agonists P-450. Blocks actions of adenosine. Muscarinic Chromones Cromolyn—prevents mast cell degranulation. antagonists Prevents acute asthma symptoms. Rarely used.

1	P-450. Blocks actions of adenosine. Muscarinic Chromones Cromolyn—prevents mast cell degranulation. antagonists Prevents acute asthma symptoms. Rarely used. Anti-IL-5 monoclonal Prevents eosinophil differentiation, maturation, therapy activation, and survival mediated by IL-5 Early response: Late response: stimulation. For maintenance therapy in severe eosinophilic asthma. Mepolizumab, reslizumab—against IL-5. Benralizumab—against IL-5 receptor α. “Study without thought is vain: thought without study is dangerous.” “It is better, of course, to know useless things than to know nothing.” “For every complex problem there is an answer that is clear, simple, and wrong.” —H. L. Mencken The following tables represent a collection of high-yield associations between diseases and their clinical findings, treatments, and key associations. They can be quickly reviewed in the days before the exam.

1	Gout, intellectual disability, self-mutilating behavior in a Lesch-Nyhan syndrome (HGPRT deficiency, X-linked 37 boy recessive) Arachnodactyly, lens dislocation (upward and temporal), Marfan syndrome (fibrillin defect) 52 aortic dissection, hyperflexible joints Calf pseudohypertrophy Muscular dystrophy (most commonly Duchenne, due to 61 X-linked recessive frameshift mutation of dystrophin gene) Child uses arms to stand up from squat Duchenne muscular dystrophy (Gowers sign) 61 Slow, progressive muscle weakness in boys Becker muscular dystrophy (X-linked non-frameshift 61 deletions in dystrophin; less severe than Duchenne) Infant with cleft lip/palate, microcephaly or Patau syndrome (trisomy 13) 63 holoprosencephaly, polydactyly, cutis aplasia Infant with microcephaly, rocker-bottom feet, clenched Edwards syndrome (trisomy 18) 63 hands, and structural heart defect Dilated cardiomyopathy, edema, alcoholism or Wet beriberi (thiamine [vitamin B1] deficiency) 66 malnutrition

1	Dilated cardiomyopathy, edema, alcoholism or Wet beriberi (thiamine [vitamin B1] deficiency) 66 malnutrition Dermatitis, dementia, diarrhea Pellagra (niacin [vitamin B3] deficiency) 67 Swollen gums, mucosal bleeding, poor wound healing, Scurvy (vitamin C deficiency: can’t hydroxylate proline/ 69 petechiae lysine for collagen synthesis) Chronic exercise intolerance with myalgia, fatigue, McArdle disease (skeletal muscle glycogen phosphorylase 87 painful cramps, myoglobinuria deficiency) Infant with hypoglycemia, hepatomegaly Cori disease (debranching enzyme deficiency) or Von 87 Gierke disease (glucose-6-phosphatase deficiency, more severe) Myopathy (infantile hypertrophic cardiomyopathy), Pompe disease (lysosomal α-1,4-glucosidase deficiency) 88 (sphingomyelin accumulation), central retinal artery occlusion Hepatosplenomegaly, pancytopenia, osteoporosis, Gaucher disease (glucocerebrosidase [β-glucosidase] avascular necrosis of femoral head, bone crises deficiency)

1	Hepatosplenomegaly, pancytopenia, osteoporosis, Gaucher disease (glucocerebrosidase [β-glucosidase] avascular necrosis of femoral head, bone crises deficiency) Male child, recurrent infections, no mature B cells Bruton disease (X-linked agammaglobulinemia) Recurrent cold (noninflamed) abscesses, eczema, high Hyper-IgE syndrome (Job syndrome: neutrophil 116 serum IgE,  eosinophils chemotaxis abnormality) “Strawberry tongue” Scarlet fever 136, Kawasaki disease 314 Abdominal pain, diarrhea, leukocytosis, recent antibiotic Clostridium difficile infection Back pain, fever, night sweats Pott disease (vertebral TB) Adrenal hemorrhage, hypotension, DIC Waterhouse-Friderichsen syndrome (meningococcemia) 142, 349 Large rash with bull’s-eye appearance Erythema migrans from Ixodes tick bite (Lyme disease: 146 Borrelia) Ulcerated genital lesion Nonpainful, indurated: chancre (1° syphilis, Treponema 147, pallidum) 184 Painful, with exudate: chancroid (Haemophilus ducreyi)

1	Ulcerated genital lesion Nonpainful, indurated: chancre (1° syphilis, Treponema 147, pallidum) 184 Painful, with exudate: chancroid (Haemophilus ducreyi) Smooth, moist, painless, wart-like white lesions on Condylomata lata (2° syphilis) 147 genitals Fever, chills, headache, myalgia following antibiotic Jarisch-Herxheimer reaction (rapid lysis of spirochetes 148 treatment for syphilis results in endotoxin-like release) Dog or cat bite resulting in infection Pasteurella multocida (cellulitis at inoculation site) 149 Rash on palms and soles Coxsackie A, 2° syphilis, Rocky Mountain spotted fever 150 Black eschar on face of patient with diabetic ketoacidosis Mucor or Rhizopus fungal infection 153 Chorioretinitis, hydrocephalus, intracranial calcifications Congenital toxoplasmosis 156 Child with fever later develops red rash on face that Erythema infectiosum/fifth disease (“slapped cheeks” 164 spreads to body appearance, caused by parvovirus B19)

1	Child with fever later develops red rash on face that Erythema infectiosum/fifth disease (“slapped cheeks” 164 spreads to body appearance, caused by parvovirus B19) Fever, cough, conjunctivitis, coryza, diffuse rash Measles 170 Small, irregular red spots on buccal/lingual mucosa with Koplik spots (measles [rubeola] virus) 170 blue-white centers Bounding pulses, wide pulse pressure, diastolic heart Aortic regurgitation 291 murmur, head bobbing Continuous “machine-like” heart murmur PDA (close with indomethacin; keep open with PGE Chest pain on exertion Angina (stable: with moderate exertion; unstable: with 304 Chest pain, pericardial effusion/friction rub, persistent Dressler syndrome (autoimmune-mediated post-MI Chest pain with ST depressions on ECG Angina (⊝ troponins) or NSTEMI (⊕ troponins) 307 fever following MI fibrinous pericarditis, 2 weeks to several months after acute episode)

1	Chest pain with ST depressions on ECG Angina (⊝ troponins) or NSTEMI (⊕ troponins) 307 fever following MI fibrinous pericarditis, 2 weeks to several months after acute episode) Painful, raised red lesions on pads of fingers/toes Osler nodes (infective endocarditis, immune complex Painless erythematous lesions on palms and soles Janeway lesions (infective endocarditis, septic emboli/ 311 microabscesses) Retinal hemorrhages with pale centers Roth spots (bacterial endocarditis) Distant heart sounds, distended neck veins, hypotension Beck triad of cardiac tamponade Cervical lymphadenopathy, desquamating rash, coronary Kawasaki disease (mucocutaneous lymph node syndrome, 314 aneurysms, red conjunctivae and tongue, hand-foot treat with IVIG and aspirin) changes Palpable purpura on buttocks/legs, joint pain, abdominal Immunoglobulin A vasculitis (Henoch-Schönlein 315 pain (child), hematuria purpura, affects skin and kidneys)

1	Palpable purpura on buttocks/legs, joint pain, abdominal Immunoglobulin A vasculitis (Henoch-Schönlein 315 pain (child), hematuria purpura, affects skin and kidneys) Telangiectasias, recurrent epistaxis, skin discoloration, Hereditary hemorrhagic telangiectasia (Osler-Weber-316 arteriovenous malformations, GI bleeding, hematuria Rendu syndrome) Skin hyperpigmentation, hypotension, fatigue 1° adrenocortical insufficiency  ACTH, • α-MSH (eg, 349 Addison disease) Cutaneous flushing, diarrhea, bronchospasm Carcinoid syndrome (right-sided cardiac valvular lesions, 352 • 5-HIAA) Cold intolerance, weight gain, brittle hair Hypothyroidism 341 Cutaneous/dermal edema due to deposition of Myxedema (caused by hypothyroidism, Graves disease 340 mucopolysaccharides in connective tissue [pretibial]) No lactation postpartum, absent menstruation, cold Sheehan syndrome (postpartum hemorrhage leading to 339 intolerance pituitary infarction)

1	No lactation postpartum, absent menstruation, cold Sheehan syndrome (postpartum hemorrhage leading to 339 intolerance pituitary infarction) Deep, labored breathing/hyperventilation Diabetic ketoacidosis (Kussmaul respirations) 347 Pancreatic, pituitary, parathyroid tumors MEN 1 (autosomal dominant) 351 Thyroid tumors, pheochromocytoma, MEN 2B (autosomal dominant RET mutation) 351 ganglioneuromatosis, Marfanoid habitus Thyroid and parathyroid tumors, pheochromocytoma MEN 2A (autosomal dominant RET mutation) 351 Jaundice, palpable distended non-tender gallbladder Courvoisier sign (distal malignant obstruction of biliary 398 tree) Vomiting blood following gastroesophageal lacerations Mallory-Weiss syndrome (alcoholic and bulimic patients) 377 Dysphagia (esophageal webs), glossitis, iron deficiency Plummer-Vinson syndrome (may progress to esophageal 377 anemia squamous cell carcinoma) Enlarged, hard left supraclavicular node Virchow node (abdominal metastasis) 379

1	Enlarged, hard left supraclavicular node Virchow node (abdominal metastasis) 379 Arthralgias, adenopathy, cardiac and neurological Whipple disease (Tropheryma whipplei) symptoms, diarrhea Severe RLQ pain with palpation of LLQ Rovsing sign (acute appendicitis) Severe RLQ pain with deep tenderness McBurney sign (acute appendicitis) Hamartomatous GI polyps, hyperpigmented macules on Peutz-Jeghers syndrome (inherited, benign polyposis can 387 mouth, feet, hands, genitalia cause bowel obstruction;  cancer risk, mainly GI) Multiple colon polyps, osteomas/soft tissue tumors, Gardner syndrome (subtype of FAP) Abdominal pain, ascites, hepatomegaly Budd-Chiari syndrome (posthepatic venous thrombosis) 392 Hyperphagia, hypersexuality, hyperorality Klüver-Bucy syndrome (bilateral amygdala lesion) Resting tremor, athetosis, chorea Basal ganglia lesion “Worst headache of my life” Subarachnoid hemorrhage

1	Hyperphagia, hypersexuality, hyperorality Klüver-Bucy syndrome (bilateral amygdala lesion) Resting tremor, athetosis, chorea Basal ganglia lesion “Worst headache of my life” Subarachnoid hemorrhage Resting tremor, rigidity, akinesia, postural instability, Parkinson disease (loss of dopaminergic neurons in Chorea, dementia, caudate degeneration Huntington disease (autosomal dominant CAG repeat 520 expansion) Nystagmus, intention tremor, scanning speech, bilateral Multiple sclerosis 523 internuclear ophthalmoplegia Café-au-lait spots, Lisch nodules (iris hamartoma), Neurofibromatosis type I 525 cutaneous neurofibromas, pheochromocytomas, optic gliomas Vascular birthmark (port-wine stain) of the face Nevus flammeus (benign, but associated with Sturge-525 Weber syndrome) Renal cell carcinoma (bilateral), hemangioblastomas, von Hippel-Lindau disease (dominant tumor suppressor 525 angiomatosis, pheochromocytoma gene mutation)

1	Renal cell carcinoma (bilateral), hemangioblastomas, von Hippel-Lindau disease (dominant tumor suppressor 525 angiomatosis, pheochromocytoma gene mutation) Hyperreflexia, hypertonia, Babinski sign present UMN damage 529 Hyporeflexia, hypotonia, atrophy, fasciculations LMN damage 529 Spastic weakness, sensory loss, bowel/bladder dysfunction Spinal cord lesion 530 Unilateral facial drooping involving forehead LMN facial nerve (CN VII) palsy; UMN lesions spare the 532 forehead Episodic vertigo, tinnitus, hearing loss Ménière disease 534 Ptosis, miosis, anhidrosis Horner syndrome (sympathetic chain lesion) 540 Conjugate horizontal gaze palsy, horizontal diplopia Internuclear ophthalmoplegia (damage to MLF; may be 543 unilateral or bilateral) Polyuria, renal tubular acidosis type II, growth failure, Fanconi syndrome (multiple combined dysfunction of the 586 electrolyte imbalances, hypophosphatemic rickets proximal convoluted tubule)

1	Polyuria, renal tubular acidosis type II, growth failure, Fanconi syndrome (multiple combined dysfunction of the 586 electrolyte imbalances, hypophosphatemic rickets proximal convoluted tubule) Athlete with polycythemia 2° to erythropoietin injection Periorbital and/or peripheral edema, proteinuria (> 3.5g/ Nephrotic syndrome day), hypoalbuminemia, hypercholesterolemia Hereditary nephritis, sensorineural hearing loss, Alport syndrome (mutation in collagen IV) retinopathy, lens dislocation Streak ovaries, congenital heart disease, horseshoe kidney, Turner syndrome (45,XO) 638 cystic hygroma at birth, short stature, webbed neck, lymphedema Red, itchy, swollen rash of nipple/areola Paget disease of the breast (sign of underlying neoplasm) 650 Fibrous plaques in tunica albuginea of penis with Peyronie disease (connective tissue disorder) 651 abnormal curvature Hypoxemia, polycythemia, hypercapnia Chronic bronchitis (hyperplasia of mucous cells, “blue

1	Hypoxemia, polycythemia, hypercapnia Chronic bronchitis (hyperplasia of mucous cells, “blue Pink complexion, dyspnea, hyperventilation Emphysema (“pink puffer,” centriacinar [smoking] or Bilateral hilar adenopathy, uveitis Sarcoidosis (noncaseating granulomas)  AFP in amniotic fluid/maternal serum Down syndrome, Edwards syndrome 63 Large granules in phagocytes, immunodeficiency Chédiak-Higashi disease (congenital failure of 117 phagolysosome formation) Recurrent infections, eczema, thrombocytopenia Wiskott-Aldrich syndrome 117 Optochin sensitivity Sensitive: S pneumoniae; resistant: viridans streptococci 134 (S mutans, S sanguis) Novobiocin response Sensitive: S epidermidis; resistant: S saprophyticus 134 Bacitracin response Sensitive: S pyogenes (group A); resistant: S agalactiae 134 (group B) Branching gram ⊕ rods with sulfur granules Actinomyces israelii 139

1	Branching gram ⊕ rods with sulfur granules Actinomyces israelii 139 Hilar lymphadenopathy, peripheral granulomatous lesion Ghon complex (1° TB: Mycobacterium bacilli) 140 in middle or lower lung lobes (can calcify) Cardiomegaly with apical atrophy Chagas disease (Trypanosoma cruzi) 158 Enlarged cells with intranuclear inclusion bodies “Owl eye” appearance of CMV 165 Eosinophilic globule in liver Councilman body (viral hepatitis, yellow fever), represents 168 hepatocyte undergoing apoptosis Eosinophilic inclusion bodies in cytoplasm of Negri bodies of rabies Ring-enhancing brain lesion on CT/MRI in AIDS Toxoplasma gondii, CNS lymphoma Psammoma bodies Meningiomas, papillary thyroid carcinoma, 211 mesothelioma, papillary serous carcinoma of the

1	Ring-enhancing brain lesion on CT/MRI in AIDS Toxoplasma gondii, CNS lymphoma Psammoma bodies Meningiomas, papillary thyroid carcinoma, 211 mesothelioma, papillary serous carcinoma of the Sheets of medium-sized lymphoid cells with scattered Burkitt lymphoma (t[8:14] c-myc activation, associated 430 pale, tingible body–laden macrophages (“starry sky” with EBV; “starry sky” made up of malignant cells) histology) • Monoclonal gammopathy of undetermined significance (MGUS consequence of aging) Stacks of RBCs Rouleaux formation (high ESR, multiple myeloma) 423 Azurophilic peroxidase ⊕ granular inclusions in Auer rods (AML, especially the promyelocytic [M3] type) 432 granulocytes and myeloblasts “Brown” tumor of bone Hyperparathyroidism or osteitis fibrosa cystica (deposited 464 hemosiderin from hemorrhage gives brown color) “Soap bubble” in femur or tibia on x-ray Giant cell tumor of bone (generally benign) 464

1	Raised periosteum (creating a “Codman triangle”) Aggressive bone lesion (eg, osteosarcoma, Ewing 465 sarcoma, osteomyelitis) “Onion skin” periosteal reaction Ewing sarcoma (malignant small blue cell tumor) 465 Anti-IgG antibodies Rheumatoid arthritis (systemic inflammation, joint 466 pannus, boutonniere and swan neck deformities) Rhomboid crystals, ⊕ birefringent Pseudogout (calcium pyrophosphate dihydrate crystals) 467 Needle-shaped, ⊝ birefringent crystals Gout (monosodium urate crystals) 467  uric acid levels Gout, Lesch-Nyhan syndrome, tumor lysis syndrome, 467 loop and thiazide diuretics “Bamboo spine” on x-ray Ankylosing spondylitis (chronic inflammatory arthritis: 469 HLA-B27) Antinuclear antibodies (ANAs: anti-Smith and anti-SLE (type III hypersensitivity) 470 dsDNA) Anti-histone antibodies Drug-induced SLE (eg, hydralazine, isoniazid, 250 phenytoin, procainamide)

1	Antinuclear antibodies (ANAs: anti-Smith and anti-SLE (type III hypersensitivity) 470 dsDNA) Anti-histone antibodies Drug-induced SLE (eg, hydralazine, isoniazid, 250 phenytoin, procainamide) Bloody or yellow tap on lumbar puncture Xanthochromia (due to subarachnoid hemorrhage) 513 Eosinophilic cytoplasmic inclusion in neuron Lewy body (Parkinson disease and Lewy body dementia) 520 Extracellular amyloid deposition in gray matter of brain Senile plaques (Alzheimer disease) Depigmentation of neurons in substantia nigra Parkinson disease (basal ganglia disorder: rigidity, resting 520 tremor, bradykinesia) Silver-staining spherical aggregation of tau proteins in Pick bodies (Pick disease: progressive dementia, changes 520 neurons in personality) Streptococcus pneumoniae Penicillin/cephalosporin (systemic infection, pneumonia), 187, vancomycin (meningitis) 190

1	Streptococcus pneumoniae Penicillin/cephalosporin (systemic infection, pneumonia), 187, vancomycin (meningitis) 190 Staphylococcus aureus MSSA: nafcillin, oxacillin, dicloxacillin 188, (antistaphylococcal penicillins); MRSA: vancomycin, 190, daptomycin, linezolid, ceftaroline 195 Enterococci Vancomycin, aminopenicillins/cephalosporins 189, 190 Rickettsia rickettsii Doxycycline, chloramphenicol 192 Clostridium difficile Oral metronidazole; if refractory, oral vancomycin 190, 195 Mycobacterium tuberculosis RIPE (rifampin, isoniazid, pyrazinamide, ethambutol) 196 Influenza Oseltamivir, zanamivir 201 CMV Ganciclovir, foscarnet, cidofovir 202 Patent ductus arteriosus Close with indomethacin; keep open with PGE analogs 282 Stable angina Sublingual nitroglycerin 304 Kawasaki disease IVIG, aspirin 314 Granulomatosis with polyangiitis (Wegener) Cyclophosphamide, corticosteroids 315

1	Kawasaki disease IVIG, aspirin 314 Granulomatosis with polyangiitis (Wegener) Cyclophosphamide, corticosteroids 315 Arrhythmia in damaged cardiac tissue Class IB antiarrhythmic (lidocaine, mexiletine) 322 Prolactinoma Cabergoline/bromocriptine (dopamine agonists) 330 Diabetes insipidus Desmopressin (central); hydrochlorothiazide, 338 indomethacin, amiloride (nephrogenic) SIADH Fluid restriction, IV hypertonic saline, conivaptan/ 338 tolvaptan, demeclocycline Diabetic ketoacidosis Fluids, insulin, K+ 347 Diabetes mellitus type 2 Dietary intervention, oral hypoglycemics, and insulin (if 347 refractory) Pheochromocytoma α-antagonists (eg, phenoxybenzamine) Crohn disease Corticosteroids, infliximab, azathioprine Ulcerative colitis 5-ASA preparations (eg, mesalamine), 6-mercaptopurine, 382 infliximab, colectomy Acute promyelocytic leukemia (M3) All-trans retinoic acid, arsenic trioxide Drug of choice for anticoagulation in pregnancy or renal Low-molecular-weight heparin

1	Acute promyelocytic leukemia (M3) All-trans retinoic acid, arsenic trioxide Drug of choice for anticoagulation in pregnancy or renal Low-molecular-weight heparin Long-term anticoagulation Warfarin, dabigatran, rivaroxaban and apixaban 436, 437 Warfarin reversal Fresh frozen plasma (acute), vitamin K (non-acute) 436 Osteoporosis Calcium/vitamin D supplementation (prophylaxis); 462 bisphosphonates, PTH analogs, SERMs, calcitonin, denosumab (treatment) Chronic gout Xanthine oxidase inhibitors (eg, allopurinol, febuxostat); 467 pegloticase; probenecid Acute gout attack NSAIDs, colchicine, glucocorticoids 467 Migraine Abortive therapies (eg, sumatriptan, NSAIDs); prophylaxis 518 (eg, propranolol, topiramate, CCBs, amitriptyline) Multiple sclerosis Disease-modifying therapies (eg, β-interferon, 523 natalizumab); for acute flares, use IV steroids Tonic-clonic seizures Levetiracetam, phenytoin, valproate, carbamazepine 544 Anorexia Nutrition, psychotherapy, SSRIs 567

1	Tonic-clonic seizures Levetiracetam, phenytoin, valproate, carbamazepine 544 Anorexia Nutrition, psychotherapy, SSRIs 567 Alcoholism Disulfiram, acamprosate, naltrexone, supportive care 571 ADHD Methylphenidate, amphetamines, CBT, atomoxetine, guanfacine, clonidine Bipolar disorder Mood stabilizers (eg, lithium, valproic acid, carbamazepine), atypical antipsychotics Generalized anxiety disorder SSRIs, SNRIs (first line); buspirone (second line) Benign prostatic hyperplasia α1-antagonists, 5α-reductase inhibitors, PDE-5 inhibitors 654 Infertility Leuprolide, GnRH (pulsatile), clomiphene Prostate adenocarcinoma/uterine fibroids Leuprolide, GnRH (continuous) Erectile dysfunction Sildenafil, tadalafil, vardenafil 686 Pulmonary arterial hypertension (idiopathic) Sildenafil, bosentan, epoprostenol 686 Mitochondrial inheritance Disease occurs in both males and females, inherited 59 through females only Intellectual disability Down syndrome, fragile X syndrome 62, 63

1	Mitochondrial inheritance Disease occurs in both males and females, inherited 59 through females only Intellectual disability Down syndrome, fragile X syndrome 62, 63 Vitamin deficiency (USA) Folate (pregnant women are at high risk; body stores only 68 3to 4-month supply; prevents neural tube defects) Bacterial meningitis (newborns and kids) Group B streptococcus/E coli/Listeria monocytogenes 180 (newborns), S pneumoniae/N meningitidis (kids/teens) HLA-DR3 Diabetes mellitus type 1, SLE, Graves disease, Hashimoto 100 thyroiditis (also associated with HLA-DR5), Addison disease HLA-DR4 Diabetes mellitus type 1, rheumatoid arthritis, Addison 100 disease Bacteria associated with gastritis, peptic ulcer disease, and H pylori 146 gastric malignancies (eg, adenocarcinoma, MALToma) Infection 2° to blood transfusion Hepatitis C Food poisoning (exotoxin mediated) S aureus, B cereus Osteomyelitis with IV drug use Pseudomonas, Candida, S aureus

1	Infection 2° to blood transfusion Hepatitis C Food poisoning (exotoxin mediated) S aureus, B cereus Osteomyelitis with IV drug use Pseudomonas, Candida, S aureus UTI E coli, Staphylococcus saprophyticus (young women) 181 Sexually transmitted disease C trachomatis (usually coinfected with N gonorrhoeae) Nosocomial pneumonia S aureus, Pseudomonas, other enteric gram ⊝ rods Pelvic inflammatory disease C trachomatis, N gonorrhoeae Infections in chronic granulomatous disease S aureus, E coli, Aspergillus (catalase ⊕) Metastases to bone Prostate, breast > kidney, thyroid, lung Metastases to brain Lung > breast > melanoma, colon, kidney Metastases to liver Colon >> stomach > pancreas 223 S3 heart sound  ventricular filling pressure (eg, mitral regurgitation, 287 HF), common in dilated ventricles S4 heart sound Stiff/hypertrophic ventricle (aortic stenosis, restrictive 287 cardiomyopathy)

1	S4 heart sound Stiff/hypertrophic ventricle (aortic stenosis, restrictive 287 cardiomyopathy) Constrictive pericarditis TB (developing world); idiopathic, viral illness (developed 287 world) Holosystolic murmur VSD, tricuspid regurgitation, mitral regurgitation 291 Heart murmur, congenital Mitral valve prolapse 291 Chronic arrhythmia Atrial fibrillation (associated with high risk of emboli) 295 Cyanosis (early; less common) Tetralogy of Fallot, transposition of great vessels, truncus 298 arteriosus, total anomalous pulmonary venous return, tricuspid atresia Late cyanotic shunt (uncorrected left to right becomes Eisenmenger syndrome (caused by ASD, VSD, PDA; 299 right to left) results in pulmonary hypertension/polycythemia) Hypertension, 2° Renal artery stenosis, chronic kidney disease (eg, 300 polycystic kidney disease, diabetic nephropathy),

1	Hypertension, 2° Renal artery stenosis, chronic kidney disease (eg, 300 polycystic kidney disease, diabetic nephropathy), Aortic aneurysm, thoracic Marfan syndrome (idiopathic cystic medial degeneration) 302 Aortic aneurysm, abdominal Atherosclerosis, smoking is major risk factor 302 Aortic aneurysm, ascending or arch 3° syphilis (syphilitic aortitis), vasa vasorum destruction 303 Sites of atherosclerosis Abdominal aorta > coronary artery > popliteal artery Right heart failure due to a pulmonary cause Cor pulmonale Heart valve in bacterial endocarditis Mitral > aortic (rheumatic fever), tricuspid (IV drug abuse) 310 Endocarditis presentation associated with bacterium S aureus (acute, IVDA, tricuspid valve), viridans 310 streptococci (subacute, dental procedure), S bovis (colon cancer), culture negative (Coxiella, Bartonella, HACEK) Temporal arteritis Risk of ipsilateral blindness due to occlusion of

1	Temporal arteritis Risk of ipsilateral blindness due to occlusion of Recurrent inflammation/thrombosis of small/medium Buerger disease (strongly associated with tobacco) 314 vessels in extremities Cardiac 1° tumor (kids) Rhabdomyoma, often seen in tuberous sclerosis Cardiac tumor (adults) Metastasis, myxoma (90% in left atrium; “ball valve”) Congenital adrenal hyperplasia, hypotension 21-hydroxylase deficiency 1° hyperparathyroidism Adenomas, hyperplasia, carcinoma 345 2° hyperparathyroidism Hypocalcemia of chronic kidney disease 345 • Paraneoplastic (due to ACTH secretion by tumors) Tumor of the adrenal medulla (kids) Neuroblastoma (malignant) 350 Tumor of the adrenal medulla (adults) Pheochromocytoma (usually benign) 350 Refractory peptic ulcers and high gastrin levels Zollinger-Ellison syndrome (gastrinoma of duodenum or 351, pancreas), associated with MEN1 352

1	Refractory peptic ulcers and high gastrin levels Zollinger-Ellison syndrome (gastrinoma of duodenum or 351, pancreas), associated with MEN1 352 Acute gastric ulcer associated with CNS injury Cushing ulcer ( intracranial pressure stimulates vagal 379 gastric H+ secretion) Acute gastric ulcer associated with severe burns Curling ulcer (greatly reduced plasma volume results in 379 sloughing of gastric mucosa) Bilateral ovarian metastases from gastric carcinoma Krukenberg tumor (mucin-secreting signet ring cells) 379 Chronic atrophic gastritis (autoimmune) Predisposition to gastric carcinoma (can also cause 379 pernicious anemia) Alternating areas of transmural inflammation and normal Skip lesions (Crohn disease) 382 colon Site of diverticula Sigmoid colon 383

1	Alternating areas of transmural inflammation and normal Skip lesions (Crohn disease) 382 colon Site of diverticula Sigmoid colon 383 Hepatocellular carcinoma Cirrhotic liver (associated with hepatitis B and C, alcoholism, and hemochromatosis) 1° liver cancer Hepatocellular carcinoma (chronic hepatitis, cirrhosis, 392 hemochromatosis, α1-antitrypsin deficiency, Wilson disease) Congenital conjugated hyperbilirubinemia (black liver) Dubin-Johnson syndrome (inability of hepatocytes to Hemochromatosis Multiple blood transfusions or hereditary HFE mutation 395 (can result in heart failure, “bronze diabetes,” and  risk of hepatocellular carcinoma) Pancreatitis (acute) Gallstones, alcohol Pancreatitis (chronic) Alcohol (adults), cystic fibrosis (kids) Bleeding disorder with GpIb deficiency Bernard-Soulier syndrome (defect in platelet adhesion to 427 von Willebrand factor) DIC Severe sepsis, obstetric complications, cancer, burns, 428 trauma, major surgery, acute pancreatitis, APL

1	DIC Severe sepsis, obstetric complications, cancer, burns, 428 trauma, major surgery, acute pancreatitis, APL Malignancy associated with noninfectious fever Hodgkin lymphoma 429 Type of Hodgkin lymphoma Nodular sclerosis (vs mixed cellularity, lymphocytic 429 predominance, lymphocytic depletion) t(14;18) Follicular lymphomas (BCL-2 activation, anti-apoptotic 430 oncogene) t(8;14) Burkitt lymphoma (c-myc fusion, transcription factor 430 oncogene) Type of non-Hodgkin lymphoma Diffuse large B-cell lymphoma 430 Age ranges for patient with ALL/CLL/AML/CML ALL: child, CLL: adult > 60, AML: adult ∼ 65, CML: 432, adult 45–85 433 Malignancy (kids) Leukemia, brain tumors 432, 526 t(9;22) Philadelphia chromosome, CML (BCR-ABL oncogene, 434 tyrosine kinase activation), more rarely associated with ALL Vertebral compression fracture Osteoporosis (type I: postmenopausal woman; type II: 462 elderly man or woman)

1	Vertebral compression fracture Osteoporosis (type I: postmenopausal woman; type II: 462 elderly man or woman) HLA-B27 Psoriatic arthritis, ankylosing spondylitis, IBD-associated 469 arthritis, reactive arthritis (formerly Reiter syndrome) Tumor of infancy Strawberry hemangioma (grows rapidly and regresses Actinic (solar) keratosis Precursor to squamous cell carcinoma Atrophy of the mammillary bodies Wernicke encephalopathy (thiamine deficiency causing ataxia, ophthalmoplegia, and confusion) “Some books are to be tasted, others to be swallowed, and some few to be chewed and digested.” “Always read something that will make you look good if you die in the middle of it.” —P.J. O’Rourke “So many books, so little time.” “If one cannot enjoy reading a book over and over again, there is no use in reading it at all.” to Use the Database 712 `Anatomy, Embryology, and Neuroscience 715

1	Embryology, and Neuroscience 715 This section is a database of top-rated basic science review books, sample examination books, software, websites, and apps that have been marketed to medical students studying for the USMLE Step 1. For each recommended resource, we list (where applicable) the Title, the First Author (or editor), the Current Publisher, the Copyright Year, the Number of Pages, the Approximate List Price, the Format of the resource, and the Number of Test Questions. Finally, each recommended resource receives a Rating. Within each section, resources are arranged first by Rating and then alphabetically by the first author within each Rating group. For a complete list of resources, including summaries that describe their overall style and utility, go to www.firstaidteam.com/bonus A letter rating scale with six different grades reflects the detailed student evaluations for Rated Resources. Each rated resource receives a rating as follows:

1	A letter rating scale with six different grades reflects the detailed student evaluations for Rated Resources. Each rated resource receives a rating as follows: The Rating is meant to reflect the overall usefulness of the resource in helping medical students prepare for the USMLE Step 1. This is based on a number of factors, including: The cost The readability of the text or usability of the app The appropriateness and accuracy of the material The quality and number of sample questions The quality of written answers to sample questions The quality and appropriateness of the illustrations (eg, graphs, diagrams, photographs) The length of the text (longer is not necessarily better) The quality and number of other resources available in the same discipline The importance of the discipline for the USMLE Step 1

1	The length of the text (longer is not necessarily better) The quality and number of other resources available in the same discipline The importance of the discipline for the USMLE Step 1 Please note that ratings do not reflect the quality of the resources for purposes other than reviewing for the USMLE Step 1. Many books with lower ratings are well written and informative but are not ideal for boards preparation. We have not listed or commented on general textbooks available in the basic sciences. Evaluations are based on the cumulative results of formal and informal surveys of thousands of medical students at many medical schools across the country. The ratings represent a consensus opinion, but there may have been a broad range of opinion or limited student feedback on any particular resource. Please note that the data listed are subject to change in that: Publishers’ prices change frequently. Bookstores often charge an additional markup.

1	Please note that the data listed are subject to change in that: Publishers’ prices change frequently. Bookstores often charge an additional markup. New editions come out frequently, and the quality of updating varies. The same book may be reissued through another publisher. We actively encourage medical students and faculty to submit their opinions and ratings of these basic science review materials so that we may update our database. (See p. xvii, How to Contribute.) In addition, we ask that publishers and authors submit for evaluation review copies of basic science review books, including new editions and books not included in our database. We also solicit reviews of new books or suggestions for alternate modes of study that may be useful in preparing for the examination, such as flash cards, computer software, commercial review courses, apps, and websites. Disclaimer/Conflict of Interest Statement

1	Disclaimer/Conflict of Interest Statement No material in this book, including the ratings, reflects the opinion or influence of the publisher. All errors and omissions will gladly be corrected if brought to the attention of the authors through our blog at www.firstaidteam.com. Please note that USMLE-Rx and the entire First Aid for the USMLE series are publications by certain authors of this book; the following ratings are based solely on recommendations from the student authors of this book as well as data from the student survey and feedback forms. UWorld Qbank UWorld www.uworld.com Test/2400 q $249–$749 A NBME Practice Exams National Board of Medical Examiners www.nbme.org/students/sas/ Comprehensive.html AMBOSS Amboss www.amboss.com Test/3500 USMLE-Rx Qmax USMLE-Rx www.usmle-rx.com Test/2300 Qbank Kaplan www.kaptest.com Test/2100 BoardVitals www.boardvitals.com Test/1750 q $59–$179 B Kaplan USMLE Step 1 Qbook Kaplan Kaplan, 2017, 468 pages Test/850 q $50 B

1	Qbank Kaplan www.kaptest.com Test/2100 BoardVitals www.boardvitals.com Test/1750 q $59–$179 B Kaplan USMLE Step 1 Qbook Kaplan Kaplan, 2017, 468 pages Test/850 q $50 B Pastest www.pastest.com Test/2100 Review www.truelearn.com Test/2200 Anki www.ankisrs.net Flash www.boardsbeyond.com Review/ Physeo www.physeo.com Review $30–$150 SketchyMedical www.sketchymedical.com Review $99–$369 Fighter www.cramfighter.com Study www.usmle-rx.com Review/Test $69–$299 Facts www.usmle-rx.com Flash Medbullets www.medbullets.com Review/ Pathology www.medicalschoolpathology.com Review Free www.onlinemeded.org Review Free www.osmosis.org Mastery builtbyhlt.com/medical/usmle-step- 1-mastery Test/1400 q $2–$10 B+ WebPath: The Internet Pathology Laboratory webpath.med.utah.edu Review/ Test/1300 q Free B Blue Histology www.lab.anhb.uwa.edu.au/mb140 Review/Test Free B Digital Anatomist Project: Interactive Atlases University of Washington da.si.washington.edu/da.html Review Free

1	B Digital Anatomist Project: Interactive Atlases University of Washington da.si.washington.edu/da.html Review Free Dr. Najeeb Lectures www.drnajeeblectures.com Review $99 B Firecracker Firecracker Inc. firecracker.lww.com Review/ Test/2800 q $39–$660 B KISSPrep www.kissprep.com Review $99–$135 www.lecturio.com Review/ Inc. www.memorangapp.com Flash Picmonic www.picmonic.com Review $25–$480 Radiopaedia.org www.radiopaedia.org Cases/Test Free B– The Pathology Guy Friedlander www.pathguy.com Review Free

1	A First Aid for the Basic Sciences: General Principles Le McGraw-Hill, 2017, 528 pages Review $55 A First Aid Cases for the USMLE Step 1 Le McGraw-Hill, 2018, 496 pages Cases $50 A– First Aid for the Basic Sciences: Organ Systems Le McGraw-Hill, 2017, 912 pages Review $72 A– Crush Step 1: The Ultimate USMLE Step 1 Review O’Connell Elsevier, 2017, 704 pages Review $45 A– Cracking the USMLE Step 1 Princeton Review Princeton Review, 2013, 832 pages Review $45 B+ USMLE Step 1 Secrets in Color Brown Elsevier, 2016, 800 pages, ISBN 9780323396790 Review $43 B+ Step-Up to USMLE Step 1 2015 Jenkins Lippincott Williams & Wilkins, 2014, 528 pages Review $50 B+ USMLE Step 1 Lecture Notes 2018 Kaplan Kaplan Medical, 2018, ~2700 pages Review $330 B+ USMLE Images for the Boards: A Comprehensive Image-Based Review Tully Elsevier, 2012, 296 pages Review $42 B USMLE Step 1 Made Ridiculously Simple Carl MedMaster, 2017, 416 pages, Review/Test 1000 q $30 B medEssentials for the USMLE Step 1 Manley

1	Review Tully Elsevier, 2012, 296 pages Review $42 B USMLE Step 1 Made Ridiculously Simple Carl MedMaster, 2017, 416 pages, Review/Test 1000 q $30 B medEssentials for the USMLE Step 1 Manley Kaplan, 2012, 588 pages Review $55

1	Anatomy, Embryology, and Neuroscience A– High-Yield Gross Anatomy Dudek Lippincott Williams & Wilkins, 2014, 320 pages Review $43 A– Clinical Anatomy Made Ridiculously Simple Goldberg MedMaster, 2016, 175 pages Review $30 B+ High-Yield Embryology Dudek Lippincott Williams & Wilkins, 2013, 176 pages Review $56 B+ High-Yield Neuroanatomy Fix Lippincott Williams & Wilkins, 2015, 208 pages Review/ Test/50 q $40 Anatomy, Embryology, and Neuroscience (continued)

1	Anatomy, Embryology, and Neuroscience (continued) B+ Anatomy—An Essential Textbook Gilroy Thieme, 2017, 528 pages Text/ Test/400 q $48 B+ Netter’s Anatomy Flash Cards Hansen Saunders, 2018, 688 flash cards Flash cards $40 B+ Crash Course: Anatomy Stenhouse Elsevier, 2015, 288 pages Review $45 B BRS Embryology Dudek Lippincott Williams & Wilkins, 2014, 336 pages Review/ Test/220 q $56 B Anatomy Flash Cards: Anatomy on the Go Gilroy Thieme, 2013, 752 flash cards Flash cards $60 B Clinical Neuroanatomy Made Ridiculously Simple Goldberg MedMaster, 2014, 90 pages + CD-ROM Review/Test/ Few q $26 B Netter’s Anatomy Coloring Book Hansen Elsevier, 2018, 392 pages Review $20 B Case Files: Anatomy Toy McGraw-Hill, 2014, 416 pages Cases $35 B– Case Files: Neuroscience Toy McGraw-Hill, 2014, 432 pages Cases $35 A– BRS Behavioral Science Fadem Lippincott Williams & Wilkins, 2016, 384 pages Review/ Test/700 q $52 B+

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1	pages Cases $62 B Pretest: Microbiology Kettering McGraw-Hill, 2013, 480 pages Test/500 q $38 B Case Files: Microbiology Toy McGraw-Hill, 2014, 416 pages Cases $36 B Lange Microbiology and Infectious Diseases Flash Cards, 3e Somers McGraw-Hill Education, 2017, 358 pages Flash cards $46 B– Lippincott Illustrated Reviews: Microbiology Cornelissen Lippincott Williams & Wilkins, 2019, 448 pages Review/Test/ Few q $73

1	A+ Pathoma: Fundamentals of Pathology Sattar Pathoma, 2019, 218 pages Review/ Lecture $85–$120 A– Rapid Review: Pathology Goljan Elsevier, 2018, 864 pages Review/ Test/500 q $65 A– Robbins and Cotran Review of Pathology Klatt Elsevier, 2014, 504 pages Test/1100 q $55 A– Crash Course: Pathology Xiu Elsevier, 2019, 438 pages Review $40 B High-Yield Histopathology Dudek Lippincott Williams & Wilkins, 2017, 320 pages Review $36 B Pathophysiology of Disease: Introduction to Clinical Medicine Hammer McGraw-Hill, 2018, 832 pages Text $90 B Haematology at a Glance Mehta Blackwell Science, 2014, 136 pages Review $49 B Pocket Companion to Robbins and Cotran Pathologic Basis of Disease Mitchell Elsevier, 2016, 896 pages Review $40 B BRS Pathology Schneider Lippincott Williams & Wilkins, 2013, 480 pages Review/ Test/450 q $54

1	B+ Crash Course: Pharmacology Battista Elsevier, 2019, 336 pages Review $40 B+ Master the Boards USMLE Step 1 Pharmacology Flashcards Fischer Kaplan, 2015, 200 flash cards Flash cards $55 B+ BRS Pharmacology Rosenfeld Lippincott Williams & Wilkins, 2019, 384 pages Review/ Test/200 q $55 B Lange Pharmacology Flash Cards Baron McGraw-Hill, 2017, 266 flash cards Flash cards $39 B Pharmacology Flash Cards Brenner Elsevier, 2017, 230 flash cards Flash cards $45 B Katzung & Trevor’s Pharmacology: Examination and Board Review Trevor McGraw-Hill, 2018, 592 pages Review/ Test/800 q $54 B Lippincott Illustrated Reviews: Pharmacology Whalen Lippincott Williams & Wilkins, 2018, 576 pages Review/ Test/380 q $75

1	A– BRS Physiology Costanzo Lippincott Williams & Wilkins, 2018, 304 pages Review/ Test/350 q $54 A– Pathophysiology of Heart Disease Lilly Lippincott Williams & Williams, 2015, 480 pages Review $57 A– PreTest Physiology Metting McGraw-Hill, 2013, 528 pages Test/500 q $38 A– Color Atlas of Physiology Silbernagl Thieme, 2015, 472 pages Review $50 B+ BRS Physiology Cases and Problems Costanzo Lippincott Williams & Wilkins, 2012, 368 pages Cases $58 B+ Physiology Costanzo Saunders, 2017, 528 pages Text $60 B+ Vander’s Renal Physiology Eaton McGraw-Hill, 2018, 224 pages Text $49 B+ Acid-Base, Fluids, and Electrolytes Made Ridiculously Simple Preston MedMaster, 2017, 166 pages Review $24 B+ Pulmonary Pathophysiology: The Essentials West Lippincott Williams & Wilkins, 2017, 264 pages Review/ Test/75 q $57 B Rapid Review: Physiology Brown Elsevier, 2011, 384 pages Test/350 q $39 B Endocrine Physiology Molina McGraw-Hill, 2018, 320 pages Review $59 B– Netter’s Physiology Flash Cards Mulroney

1	q $57 B Rapid Review: Physiology Brown Elsevier, 2011, 384 pages Test/350 q $39 B Endocrine Physiology Molina McGraw-Hill, 2018, 320 pages Review $59 B– Netter’s Physiology Flash Cards Mulroney Saunders, 2015, 450 flash cards Flash cards $40 calcinosis, Raynaud phenomenon, esophageal dysfunction, sclerosis, and telangiectasias [syndrome] left, liver

1	LA left atrial, left atrium LM lateral meniscus, left main coronary artery, light microscopy LT labile toxin, leukotriene LV left ventricle, left ventricular MAC membrane attack complex, minimum alveolar concentration MAP mean arterial pressure, mitogen-activated protein Med cond* medial condyle MELAS mitochondrial encephalopathy, lactic acidosis, and stroke-syndrome like episodes MGUS monoclonal gammopathy of undetermined significance MHC major histocompatibility complex MIRL membrane inhibitor of reactive lysis MMR measles, mumps, rubella [vaccine] MR medial rectus [muscle], mitral regurgitation NPH neutral protamine Hagedorn, normal pressure hydrocephalus 1,25-OH D3 calcitriol (active form of vitamin D) 25-OH D3 storage form of vitamin D OVLT organum vasculosum of the lamina terminalis P-450 cytochrome P-450 family of enzymes PA posteroanterior, pulmonary artery Pao2 partial pressure of oxygen in arterial blood Pao2 partial pressure of oxygen in alveolar blood

1	P-450 cytochrome P-450 family of enzymes PA posteroanterior, pulmonary artery Pao2 partial pressure of oxygen in arterial blood Pao2 partial pressure of oxygen in alveolar blood PAP Papanicolaou [smear], prostatic acid phosphatase PC platelet count, pyruvate carboxylase Pco2 partial pressure of carbon dioxide PCP phencyclidine hydrochloride, Pneumocystis jirovecii pneumonia PDA patent ductus arteriosus, posterior descending artery Pi plasma interstitial osmotic pressure, inorganic phosphate PICA posterior inferior cerebellar artery PIP2 phosphatidylinositol 4,5-bisphosphate PIP3 phosphatidylinositol 3,4,5-bisphosphate Po2 partial pressure of oxygen PV plasma volume, venous pressure R correlation coefficient, right, R variable [group] R3 Registration, Ranking, & Results [system] RANK-L receptor activator of nuclear factor-κ B ligand RR relative risk, respiratory rate RV residual volume, right ventricle, right ventricular SE standard error [of the mean]

1	RANK-L receptor activator of nuclear factor-κ B ligand RR relative risk, respiratory rate RV residual volume, right ventricle, right ventricular SE standard error [of the mean] SIADH syndrome of inappropriate [secretion of] antidiuretic hormone SV splenic vein, stroke volume TCA tricarboxylic acid [cycle], tricyclic antidepressant V1, V2 vasopressin receptors Vd volume of distribution V(D)J variable, (diversity), joining gene segments rearranged to form Ig genes VH variable region, heavy chain [antibody] VL variable region, light chain [antibody] VPL ventral posterior nucleus, lateral VPM ventral posterior nucleus, medial VPN vancomycin, polymyxin, nystatin [media] V˙ /Q˙ ventilation/perfusion [ratio] XR X-linked recessive XX/XY normal complement of sex chromosomes for female/male ZDV zidovudine [formerly AZT]

1	dendritic cells. 366 9-14 Cell-adhesion molecules mediate the initial interaction of naive T cells with antigen- presenting cells. 367 the clonal expansion and differentiation of naive T cells. 368 9-16 CD28-dependent co-stimulation of activated T cells induces expression of interleukin-2 and the high-affinity IL-2 receptor. 368 9-17 Additional co-stimulatory pathways are involved in T-cell activation. 369 9-18 Proliferating T cells differentiate into effector T cells that do not require co-stimulation to act. 370 9-19 CD8 T cells can be activated in different ways to become cytotoxic effector cells. 372 9-20 CD4 T cells differentiate into several subsets of functionally different effector cells. 372 9-21 Cytokines induce the differentiation of naive CD4

1	T cells down distinct effector pathways. 375 9-22 CD4 T-cell subsets can cross-regulate each other’s differentiation through the cytokines they produce. 377 9-23 Regulatory CD4 T cells are involved in controlling adaptive immune responses. 379 Summary. 380 General properties of effector T cells and their cytokines. 380 9-24 Effector T-cell interactions with target cells are molecules. 381 T cells and their targets to regulate signaling and to direct the release of effector molecules. 381 9-26 The effector functions of T cells are determined by the array of effector molecules that they produce. 383 9-27 Cytokines can act locally or at a distance. 383 9-28 T cells express several TNF-family cytokines as trimeric proteins that are usually associated with the cell surface. 386 Summary. 386

1	Summary. 386 T-cell-mediated cytotoxicity. 387 9-29 Cytotoxic T cells induce target cells to undergo pathways of apoptosis. 387 9-30 The intrinsic pathway of apoptosis is mediated by the release of cytochrome c from mitochondria. 389 9-31 Cytotoxic effector proteins that trigger apoptosis are contained in the granules of CD8 cytotoxic T cells. 390 9-32 Cytotoxic T cells are selective serial killers of targets expressing a specific antigen. 9-33 Cytotoxic T cells also act by releasing cytokines. 392 Summary. 392 Summary to Chapter 9. Questions. 393 References. 395 B-cell activation by antigen and helper T cells.

1	10-1 Activation of B cells by antigen involves signals from the B-cell receptor and either TFH cells or microbial antigens. 400 10-2 Linked recognition of antigen by T cells and B cells promotes robust antibody responses. 402 the boundaries between B-cell and T-cell areas in secondary lymphoid tissues. 403 activate B cells, which in turn promote TFH-cell development. 406 10-5 Activated B cells differentiate into antibody-secreting plasmablasts and plasma cells. 406 10-6 The second phase of a primary B-cell immune follicles and proliferate to form germinal centers. 408 hypermutation, and cells with mutations that improve affinity for antigen are selected. 410 10-8 Positive selection of germinal center B cells involves contact with TFH cells and CD40 signaling. 412 in B cells. 413 contribute to somatic hypermutation following initiation by AID. 414 10-11 AID initiates class switching to allow the same assembled VH exon to be associated with different

1	CH genes in the course of an immune response. 415 10-12 Cytokines made by TFH cells direct the choice of responses. 418 10-13 B cells that survive the germinal center reaction memory cells. 419 10-14 Some antigens do not require T-cell help to induce B-cell responses. Summary. 421 The distributions and functions of immunoglobulin classes. 422 10-15 Antibodies of different classes operate in distinct places and have distinct effector functions. 10-16 Polymeric immunoglobulin receptor binds to the Fc regions of IgA and IgM and transports them across epithelial barriers.

1	10-17 The neonatal Fc receptor carries IgG across the placenta and prevents IgG excretion from the body. 426 12-6 Priming of lymphocytes in one mucosal tissue may 13-4 SCID can also be due to defects in the purine induce protective immunity at other mucosal salvage pathway. 538 surfaces. 502 12-7 Distinct populations of dendritic cells control can result in SCID. 538 mucosal immune responses. 503 can cause severe immunodeficiency. 539 roles in mucosal immune responses. 505 12-9 Antigen-presenting cells in the intestinal mucosa development result in severe immunodeficiencies. 539 acquire antigen by a variety of routes. 505 12-10 Secretory IgA is the class of antibody associated in antibody production that cause an inability to with the mucosal immune system. 506 clear extracellular bacteria and some viruses. 541 12-11 T-independent processes can contribute to IgA 13-9 Immune deficiencies can be caused by defects in production in some species. 509

1	B-cell or T-cell activation and function that lead to abnormal antibody responses. 543 12-12 IgA deficiency is relatively common in humans but may be compensated for by secretory IgM. 509 13-10 Normal pathways for host defense against different infectious agents are pinpointed by genetic deficiencies 12-13 The intestinal lamina propria contains antigen of cytokine pathways central to type 1/TH1 and typeexperienced T cells and populations of unusual 3/TH17 responses. 546 innate lymphoid cells. 510 13-11 Inherited defects in the cytolytic pathway of 12-14 The intestinal epithelium is a unique compartment of the immune system. 511 proliferation and inflammatory responses to viral Summary. 514 infections. 548

1	The mucosal response to infection and regulation 13-12 X-linked lymphoproliferative syndrome is associated with fatal infection by Epstein–Barr virus and with the of mucosal immune responses. 514 development of lymphomas. 550 12-15 Enteric pathogens cause a local inflammatory response and the development of protective 13-13 Immunodeficiency is caused by inherited defects immunity. 515 in the development of dendritic cells. 551 regulatory proteins cause defective humoral immune function and tissue damage. 552 when innate defenses have been breached. 518 12-17 Effector T-cell responses in the intestine protect the function of the epithelium. 518 bacterial infections. 553 12-18 The mucosal immune system must maintain 13-16 Mutations in the molecular regulators of inflammation tolerance to harmless foreign antigens. 519 can cause uncontrolled inflammatory responses that 12-19 The normal intestine contains large quantities of result in ‘autoinflammatory disease.’ 556 bacteria that are

1	harmless foreign antigens. 519 can cause uncontrolled inflammatory responses that 12-19 The normal intestine contains large quantities of result in ‘autoinflammatory disease.’ 556 bacteria that are required for health. 520 therapy can be useful to correct genetic defects. 557 microbiota while preventing inflammation without 13-18 Noninherited, secondary immunodeficiencies are compromising the ability to react to invaders. 521 major predisposing causes of infection and death. 558 12-21 The intestinal microbiota plays a major role in

1	Summary. 559 shaping intestinal and systemic immune function. 522 12-22 Full immune responses to commensal bacteria Evasion and subversion of immune defenses. 560 provoke intestinal disease. 524 13-19 Extracellular bacterial pathogens have evolved different strategies to avoid detection by pattern Summary. 525 recognition receptors and destruction by antibody, Summary to Chapter 12. 525 complement, and antimicrobial peptides. 560 Questions. 526 13-20 Intracellular bacterial pathogens can evade the immune system by seeking shelter within phagocytes. 563 References. 527 13-21 Immune evasion is also practiced by protozoan parasites. 565 13-22 RNA viruses use different mechanisms of antigenic variation to keep a step ahead of the adaptive immune system. 566 Chapter 13 Failures of Host Defense Mechanisms 533 13-23 DNA viruses use multiple mechanisms to subvert NK-cell and CTL responses. 568

1	Chapter 13 Failures of Host Defense Mechanisms 533 13-23 DNA viruses use multiple mechanisms to subvert NK-cell and CTL responses. 568 Immunodeficiency diseases. 533 13-1 A history of repeated infections suggests a replicate until immunity wanes. 571 diagnosis of immunodeficiency. 534 Summary. 573 13-2 Primary immunodeficiency diseases are caused by inherited gene defects. 534 Acquired immune deficiency syndrome. 573 13-25 HIV is a retrovirus that establishes a chronic combined immunodeficiencies. 535 infection that slowly progresses to AIDS. 574 Fighting infectious diseases with vaccination. 729 A-14 Immunoblotting (Western blotting). A-15 Use of antibodies in the isolation and or material from killed organisms. 730 characterization of multiprotein complexes by mass spectrometry. 764 16-21 Most effective vaccines generate antibodies that prevent the damage caused by toxins or that

1	A-16 Isolation of peripheral blood lymphocytes by density- neutralize the pathogen and stop infection. 731 gradient fractionation. 766 A-17 Isolation of lymphocytes from tissues other than protection while being safe and inexpensive. 732 blood. 766 16-23 Live-attenuated viral vaccines are usually more A-18 Flow cytometry and FACS analysis. 767 by the use of recombinant DNA technology. 732 magnetic beads. 770 A-20 Isolation of homogeneous T-cell lines. 770 A-21 Limiting-dilution culture. 771 creating genetically attenuated parasites (GAPs). 734 A-22 ELISPOT assay. 773 16-25 The route of vaccination is an important A-23 Identification of functional subsets of T cells based determinant of success. 735 on cytokine production or transcription factor 16-26 Bordetella pertussis vaccination illustrates the expression. 773 importance of the perceived safety of a vaccine. 736

1	A-24 Identification of T-cell receptor specificity using peptide:MHC 16-27 Conjugate vaccines have been developed as a tetramers. 776 result of linked recognition between T and B cells. 737 A-25 Biosensor assays for measuring the rates of association and dissociation of antigen receptors immunity, but they require adjuvants and must for their ligands. 777 be targeted to the appropriate cells and cell A-26 Assays of lymphocyte proliferation. 778 compartment to be effective. 738 A-27 Measurements of apoptosis. 779 16-29 Adjuvants are important for enhancing the immunogenicity of vaccines, but few are approved A-28 Assays for cytotoxic T cells. 780 for use in humans. 739 A-29 Assays for CD4 T cells. 782 A-30 Transfer of protective immunity. 782 vaccination. 740 A-31 Adoptive transfer of lymphocytes. 783 A-32 Hematopoietic stem-cell transfers. 784 useful in controlling existing chronic infections. 741 A-33 In vivo administration of antibodies. 785 Summary. 742

1	A-32 Hematopoietic stem-cell transfers. 784 useful in controlling existing chronic infections. 741 A-33 In vivo administration of antibodies. 785 Summary. 742 A-34 Transgenic mice. 786 Summary to Chapter 16. 742 A-35 Gene knockout by targeted disruption. 786 Questions. 743 A-36 Knockdown of gene expression by References. 744 RNA interference (RNAi). 790 Appendix I The Immunologist's Toolbox 749 A-1. Immunization. 749 A-2 Antibody responses. 752 A-3 Affinity chromatography. 753 A-4 Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and competitive inhibition assay. 753 A-5 Hemagglutination and blood typing. 755 A-6 Coombs tests and the detection of rhesus incompatibility. 756 A-7 Monoclonal antibodies. 757 A-8 Phage display libraries for antibody V-region production. 758 A-9 Generation of human monoclonal antibodies from vaccinated individuals. 759 A-10 Microscopy and imaging using fluorescent dyes. 760 A-11 Immunoelectron microscopy. 761

1	A-9 Generation of human monoclonal antibodies from vaccinated individuals. 759 A-10 Microscopy and imaging using fluorescent dyes. 760 A-11 Immunoelectron microscopy. 761 A-12 Immunohistochemistry. 762 A-13 Immunoprecipitation and co-immunoprecipitation. 762 Immunology is the study of the body’s defense against infection. We are continually exposed to microorganisms, many of which cause disease, and yet become ill only rarely. How does the body defend itself? When infection does occur, how does the body eliminate the invader and cure itself? And why do we develop long-lasting immunity to many infectious diseases encountered once and overcome? These are the questions addressed by immunology, which we study to understand our body’s defenses against infection at the cellular and molecular levels.

1	The beginning of immunology as a science is usually attributed to Edward Jenner for his work in the late 18th century (Fig. 1.1). The notion of immunity— that surviving a disease confers greater protection against it later—was known since ancient Greece. Variolation—the inhalation or transfer into superficial skin wounds of material from smallpox pustules—had been practiced since at least the 1400s in the Middle East and China as a form of protection against that disease and was known to Jenner. Jenner had observed that the relatively mild disease of cowpox, or vaccinia, seemed to confer protection against the often fatal disease of smallpox, and in 1796, he demonstrated that inoculation with cowpox protected the recipient against smallpox. His scientific proof relied on the deliberate exposure of the inoculated individual to infectious smallpox material two months after inoculation. This scientific test was his original contribution.

1	Jenner called the procedure vaccination. This term is still used to describe the inoculation of healthy individuals with weakened or attenuated strains of disease-causing agents in order to provide protection from disease. Although Jenner’s bold experiment was successful, it took almost two centuries for smallpox vaccination to become universal. This advance enabled the World Health Organization to announce in 1979 that smallpox had been eradicated (Fig. 1.2), arguably the greatest triumph of modern medicine. Jenner’s strategy of vaccination was extended in the late 19th century by the discoveries of many great microbiologists. Robert Koch proved that infectious diseases are caused by specific microorganisms. In the 1880s, Louis Pasteur the origins of vertebrate immune cells. Principles of innate immunity. Principles of adaptive immunity. the effector mechanisms of immunity.

1	Principles of adaptive immunity. the effector mechanisms of immunity. Fig. 1.1 Edward Jenner. Portrait by John raphael Smith. reproduced courtesy of yale university, Harvey Cushing/John Hay Whitney medical library. Number of countries with one or more cases Fig. 1.2 The eradication of smallpox by vaccination. After a period of 3 years in which no cases of smallpox were recorded, the World Health organization was able to announce in 1979 that smallpox had been eradicated, and vaccination stopped (upper panel). A few laboratory stocks have been retained, however, and some fear that these are a source from which the virus might reemerge. Ali maow maalin (lower panel) contracted and survived the last case of smallpox in Somalia in 1977. Photograph courtesy of dr. Jason Weisfeld. devised a vaccine against cholera in chickens, and developed a rabies vaccine that proved to be a spectacular success upon its first trial in a boy bitten by a rabid dog.

1	devised a vaccine against cholera in chickens, and developed a rabies vaccine that proved to be a spectacular success upon its first trial in a boy bitten by a rabid dog. These practical triumphs led to a search for vaccination’s mechanism of protection and to the development of the science of immunology. In the early 1890s, Emil von Behring and Shibasaburo Kitasato discovered that the serum of animals immune to diphtheria or tetanus contained a specific ‘antitoxic activity’ that could confer short-lived protection against the effects of diphtheria or tetanus toxins in people. This activity was later determined to be due to the proteins we now call antibodies, which bind specifically to the toxins and neutralize their activity. That these antibodies might have a crucial role in immunity was reinforced by Jules Bordet’s discovery in 1899 of complement, a component of serum that acts in conjunction with antibodies to destroy pathogenic bacteria.

1	A specific response against infection by potential pathogens, such as the production of antibodies against a particular pathogen, is known as adaptive immunity, because it develops during the lifetime of an individual as an adaptation to infection with that pathogen. Adaptive immunity is distinguished from innate immunity, which was already known at the time von Behring was developing serum therapy for diphtheria chiefly through the work of the great Russian immunologist Elie Metchnikoff, who discovered that many microorganisms could be engulfed and digested by phagocytic cells, which thus provide defenses against infection that are nonspecific. Whereas these cells— which Metchnikoff called 'macrophages'—are always present and ready to act, adaptive immunity requires time to develop but is highly specific.

1	It was soon clear that specific antibodies could be induced against a vast range of substances, called antigens because they could stimulate antibody generation. Paul Ehrlich advanced the development of an antiserum as a treatment for diphtheria and developed methods to standardize therapeutic serums. Today the term antigen refers to any substance recognized by the adaptive immune system. Typically antigens are common proteins, glycoproteins, and polysaccharides of pathogens, but they can include a much wider range of chemical structures, for example, metals such as nickel, drugs such as penicillin, and organic chemicals such as the urushiol (a mix of pentadecylcatechols) in the leaves of poison ivy. Metchnikoff and Ehrlich shared the 1908 Nobel Prize for their respective work on immunity.

1	This chapter introduces the principles of innate and adaptive immunity, the cells of the immune system, the tissues in which they develop, and the tissues through which they circulate. We then outline the specialized functions of the different types of cells by which they eliminate infection. The origins of vertebrate immune cells.

1	The origins of vertebrate immune cells. The body is protected from infectious agents, their toxins, and the damage they cause by a variety of effector cells and molecules that together make up the immune system. Both innate and adaptive immune responses depend upon the activities of white blood cells or leukocytes. Most cells of the immune system arise from the bone marrow, where many of them develop and mature. But some, particularly certain tissue-resident macrophage populations (for example, the microglia of the central nervous system), originate from the yolk sack or fetal liver during embryonic development. They seed tissues before birth and are maintained throughout life as independent, self-renewing populations. Once mature, immune cells reside within peripheral tissues, circulate in the bloodstream, or circulate in a specialized system of vessels called Principles of innate immunity.

1	Principles of innate immunity. the lymphatic system. The lymphatic system drains extracellular fluid and immune cells from tissues and transports them as lymph that is eventually emptied back into the blood system. All the cellular elements of blood, including the red blood cells that transport oxygen, the platelets that trigger blood clotting in damaged tissues, and the white blood cells of the immune system, ultimately derive from the hematopoietic stem cells (HSCs) of the bone marrow. Because these can give rise to all the different types of blood cells, they are often known as pluripotent hematopoietic stem cells. The hematopoietic stem cells give rise to cells of more limited developmental potential, which are the immediate progenitors of red blood cells, platelets, and the two main categories of white blood cells, the lymphoid and myeloid lineages. The different types of blood cells and their lineage relationships are summarized in Fig. 1.3. Principles of innate immunity.

1	Principles of innate immunity. In this part of the chapter we will outline the principles of innate immunity and describe the molecules and cells that provide continuous defense against invasion by pathogens. Although the white blood cells known as lymphocytes possess the most powerful ability to recognize and target pathogenic microorganisms, they need the participation of the innate immune system to initiate and mount their offensive. Indeed, the adaptive immune response and innate immunity use many of the same destructive mechanisms to eliminate invading microorganisms. 1-1 Commensal organisms cause little host damage while pathogens damage host tissues by a variety of mechanisms.

1	We recognize four broad categories of disease-causing microorganisms, or pathogens: viruses, bacteria and archaea, fungi, and the unicellular and multicellular eukaryotic organisms collectively termed parasites (Fig. 1.4). These microorganisms vary tremendously in size and in how they damage host tissues. The smallest are viruses, which range from five to a few hundred nanometers in size and are obligate intracellular pathogens. Viruses can directly kills cells by inducing lysis during their replication. Somewhat larger are intracellular bacteria and mycobacteria. These can kill cells directly or damage cells by producing toxins. Many single-celled intracellular parasites, such as members of the Plasmodium genus that cause malaria, also directly kill infected cells. Pathogenic bacteria and fungi growing in extracellular spaces can induce shock and sepsis by releasing toxins into the blood or tissues. The largest pathogens—parasitic worms, or helminths—are too large to infect host

1	and fungi growing in extracellular spaces can induce shock and sepsis by releasing toxins into the blood or tissues. The largest pathogens—parasitic worms, or helminths—are too large to infect host cells but can injure tissues by forming cysts that induce damaging cellular responses in the tissues into which the worms migrate.

1	Not all microbes are pathogens. Many tissues, especially the skin, oral mucosa, conjunctiva, and gastrointestinal tract, are constantly colonized by microbial communities—called the microbiome—that consist of archaea, bacteria, and fungi but cause no damage to the host. These are also called commensal microorganisms, since they can have a symbiotic relationship with the host. Indeed, some commensal organisms perform important functions, as in the case of the bacteria that aid in cellulose digestion in the stomachs of ruminants. The difference between commensal organisms and pathogens lies in whether they induce damage. Even enormous numbers of microbes in the intestinal microbiome normally cause no damage and are confined within the intestinal lumen by a protective layer of mucus, whereas pathogenic bacteria can penetrate this barrier, injure intestinal epithelial cells, and spread into the underlying tissues.

1	Fig. 1.3 All the cellular elements of the blood, including the cells of the immune system, arise from pluripotent hematopoietic stem cells in the bone marrow. these pluripotent cells divide to produce two types of stem cells. A common lymphoid progenitor gives rise to the lymphoid lineage (blue background) of white blood cells or leukocytes—the innate lymphoid cells (IlCs) and natural killer (nK) cells and the t and B lymphocytes. A common myeloid progenitor gives rise to the myeloid lineage (pink and yellow backgrounds), which comprises the rest of the leukocytes, the erythrocytes (red blood cells), and the megakaryocytes that produce platelets important in blood clotting. t and B lymphocytes are distinguished from the other leukocytes by having antigen receptors and from each other by their sites of differentiation—the thymus and bone marrow, respectively. After encounter with antigen, B cells differentiate into antibody-secreting plasma cells, while t cells differentiate into

1	by their sites of differentiation—the thymus and bone marrow, respectively. After encounter with antigen, B cells differentiate into antibody-secreting plasma cells, while t cells differentiate into effector t cells with a variety of functions. unlike t and B cells, IlCs and nK cells lack antigen specificity. the remaining leukocytes are the monocytes, the dendritic cells, and the neutrophils, eosinophils, and basophils. the last three of these circulate in the blood and are termed granulocytes, because of the cytoplasmic granules whose staining gives these cells a distinctive appearance in blood smears, or polymorphonuclear leukocytes, because of their irregularly shaped nuclei. Immature dendritic cells (yellow background) are phagocytic cells that enter the tissues; they mature after they have encountered a potential pathogen. the majority of dendritic cells are derived from the common myeloid progenitor cells, but some may also arise from the common lymphoid progenitor. monocytes

1	have encountered a potential pathogen. the majority of dendritic cells are derived from the common myeloid progenitor cells, but some may also arise from the common lymphoid progenitor. monocytes enter tissues, where they differentiate into phagocytic macrophages or dendritic cells. mast cells also enter tissues and complete their maturation there.

1	Principles of innate immunity. Viruses Intracellular bacteria Extracellular bacteria, Archaea, Protozoa Fungi Parasites Log scale of size in meters 10–7 10–6 10–5 10–4 10–3 10–2 (1 cm) Fig. 1.4 Pathogens vary greatly in size and lifestyle. Intracellular pathogens include viruses, such as herpes simplex (first panel), and various bacteria, such as Listeria monocytogenes (second panel). many bacteria, such as Staphylococcus aureus (third panel), or fungi, such as Aspergillus fumigates (fourth panel), tissues, as do some archaea and protozoa (third panel). many parasites, such as the nematode Strongyloides stercoralis (fifth panel), are large multicellular organisms that can move throughout the body in a complex life cycle. Second panel courtesy of dan Portnoy. Fifth panel courtesy of James lok. can grow in the extracellular spaces and directly invade through 1-2 Anatomic and chemical barriers are the first defense against pathogens.

1	The host can adopt three strategies to deal with the threat posed by microbes: avoidance, resistance, and tolerance. Avoidance mechanisms prevent exposure to microbes, and include both anatomic barriers and behavior modifications. If an infection is established, resistance is aimed at reducing or eliminating pathogens. To defend against the great variety of microbes, the immune system has numerous molecular and cellular functions, collectively called mediators, or effector mechanisms, suited to resist different categories of pathogens. Their description is a major aspect of this book. Finally, tolerance involves responses that enhance a tissue’s capacity to resist damage induced by microbes. This meaning of the term ‘tolerance’ has been used extensively in the context of disease susceptibility in plants rather than animal immunity. For example, increasing growth by activating dormant meristems, the undifferentiated cells that generate new parts of the plant, is a common tolerance

1	in plants rather than animal immunity. For example, increasing growth by activating dormant meristems, the undifferentiated cells that generate new parts of the plant, is a common tolerance mechanism in response to damage. This should be distinguished from the term immunological tolerance, which refers to mechanisms that prevent an immune response from being mounted against the host’s own tissues.

1	Anatomic and chemical barriers are the initial defenses against infection (Fig. 1.5). The skin and mucosal surfaces represent a kind of avoidance strategy that prevents exposure of internal tissues to microbes. At most anatomic barriers, additional resistance mechanisms further strengthen host defenses. For example, mucosal surfaces produce a variety of antimicrobial proteins that act as natural antibiotics to prevent microbes from entering the body.

1	If these barriers are breached or evaded, other components of the innate immune system can immediately come into play. We mentioned earlier the discovery by Jules Bordet of complement, which acts with antibodies to lyse bacteria. Complement is a group of around 30 different plasma proteins that act together and are one of the most important effector mechanisms in serum and interstitial tissues. Complement not only acts in conjunction with antibodies, but can also target foreign organisms in the absence of a specific antibody; thus it contributes to both innate and adaptive responses. We will examine anatomic barriers, the antimicrobial proteins, and complement in greater detail in Chapter 2.

1	the first is the anatomic barrier provided by the body’s epithelial surfaces. Second, various chemical and enzymatic systems, including complement, act as an immediate antimicrobial barrier near these epithelia. If epithelia are breached, nearby various innate lymphoid cells can coordinate a rapid cell-mediated defense. If the pathogen overcomes these barriers, the slower-acting defenses of the adaptive immune system are brought to bear. Target tissues Production of antimicrobial proteins Induction of intracellular antiviral proteins Killing of infected cells Mediators Cytokines, cytotoxicity In˜ammatory inducers Bacterial lipopolysaccharides, ATP, urate crystals Sensor cells Macrophages, neutrophils, dendritic cells Fig. 1.6 Cell-mediated immunity proceeds in a series of steps.

1	Fig. 1.6 Cell-mediated immunity proceeds in a series of steps. Inflammatory inducers are chemical structures that indicate the presence of invading microbes or the cellular damage produced by them. Sensor cells detect these inducers by expressing various innate recognition receptors, and in response produce a variety of mediators that act directly in defense or that further propagate the immune response. mediators include many cytokines, and they act on various target tissues, such as epithelial cells, to induce antimicrobial proteins and resist intracellular viral growth; or on other immune cells, such as IlCs that produce other cytokines that amplify the immune response. 1-3 The immune system is activated by inflammatory inducers that indicate the presence of pathogens or tissue damage.

1	A pathogen that breaches the host’s anatomic and chemical barriers will encounter the cellular defenses of innate immunity. Cellular immune responses are initiated when sensor cells detect inflammatory inducers (Fig. 1.6). Sensor cells include many cell types that detect inflammatory mediators through expression of many innate recognition receptors, which are encoded by a relatively small number of genes that remain constant over an individual’s lifetime. Inflammatory inducers that trigger these receptors include molecular components unique to bacteria or viruses, such as bacterial lipopolysaccharides, or molecules such as ATP, which is not normally found in the extracellular space. Triggering these receptors can activate innate immune cells to produce various mediators that either act directly to destroy invading microbes, or act on other cells to propagate the immune response. For example, macrophages can ingest microbes and produce toxic chemical mediators, such as degradative

1	directly to destroy invading microbes, or act on other cells to propagate the immune response. For example, macrophages can ingest microbes and produce toxic chemical mediators, such as degradative enzymes or reactive oxygen intermediates, to kill them. Dendritic cells may produce cytokine mediators, including many cytokines that activate target tissues, such as epithelial or other immune cells, to resist or kill invading microbes more efficiently. We will discuss these receptors and mediators briefly below and in much greater detail in Chapter 3.

1	Innate immune responses occur rapidly on exposure to an infectious organism (Fig. 1.7). In contrast, responses by the adaptive immune system take days rather than hours to develop. However, the adaptive immune system is capable of eliminating infections more efficiently because of exquisite specificity Fig. 1.7 Phases of the immune response.

1	Innate immune response Phases of the immune response Response Duration of response Typical time after infection to start of response Infammation, complement activation, phagocytosis, and destruction of pathogen Emigration of effector lymphocytes from peripheral lymphoid organs Interaction of T cells with B cells, formation of germinal centers. Formation of effector B cells (plasma cells) and memory B cells. Production of antibody Interaction between antigen-presenting dendritic cells and antigen-specifc T cells: recognition of antigen, adhesion, co-stimulation, T-cell proliferation and differentiation Adaptive immune response Activation of antigen-specifc B cells Formation of effector and memory T cells Immunological memory Elimination of pathogen by effector cells and antibody Maintenance of memory B cells and T cells and high serum or mucosal antibody levels. Protection against reinfection Minutes Days Hours Days Hours Days Weeks Days Weeks Days A few days Weeks A few days Weeks

1	of memory B cells and T cells and high serum or mucosal antibody levels. Protection against reinfection Minutes Days Hours Days Hours Days Weeks Days Weeks Days A few days Weeks A few days Weeks Days to weeks Can be lifelong

1	Principles of innate immunity. of antigen recognition by its lymphocytes. In contrast to a limited repertoire of receptors expressed by innate immune cells, lymphocytes express highly specialized antigen receptors that collectively possess a vast repertoire of specificity. This enables the adaptive immune system to respond to virtually any pathogen and effectively focus resources to eliminate pathogens that have evaded or overwhelmed innate immunity. But the adaptive immune system interacts with, and relies on, cells of the innate immune system for many of its functions. The next several sections will introduce the major components of the innate immune system and prepare us to consider adaptive immunity later in the chapter. 1-4 The myeloid lineage comprises most of the cells of the innate immune system.

1	1-4 The myeloid lineage comprises most of the cells of the innate immune system. The common myeloid progenitor (CMP) is the precursor of the macrophages, granulocytes (the collective term for the white blood cells called neutrophils, eosinophils, and basophils), mast cells, and dendritic cells of the innate immune system. Macrophages, granulocytes, and dendritic cells make up the three types of phagocytes in the immune system. The CMP also generates megakaryocytes and red blood cells, which we will not be concerned with here. The cells of the myeloid lineage are shown in Fig. 1.8.

1	Macrophages are resident in almost all tissues. Many tissue-resident macrophages arise during embryonic development, but some macrophages that arise in the adult animal from the bone marrow are the mature form of monocytes, which circulate in the blood and continually migrate into tissues, where they differentiate. Macrophages are relatively long-lived cells and perform several different functions throughout the innate immune response and the subsequent adaptive immune response. One is to engulf and kill invading microorganisms. This phagocytic function provides a first defense in innate immunity. Macrophages also dispose of pathogens and infected cells targeted by an adaptive immune response. Both monocytes and macrophages are phagocytic, but most infections occur in the tissues, and so it is primarily macrophages that perform this important protective function. An additional and crucial role of macrophages is to orchestrate immune responses: they help induce inflammation, which, as

1	it is primarily macrophages that perform this important protective function. An additional and crucial role of macrophages is to orchestrate immune responses: they help induce inflammation, which, as we shall see, is a prerequisite to a successful immune response, and they produce many inflammatory mediators that activate other immune-system cells and recruit them into an immune response.

1	Local inflammation and the phagocytosis of invading bacteria can also be triggered by the activation of complement. Bacterial surfaces can activate the complement system, inducing a cascade of proteolytic reactions that coat the microbes with fragments of specific proteins of the complement system.

1	Fig. 1.8 Myeloid cells in innate and adaptive immunity. In the rest of the book, these cells will be represented in the schematic form shown on the left. A photomicrograph of each cell type is shown on the right. macrophages and neutrophils are primarily phagocytic cells that engulf pathogens and destroy them in intracellular vesicles, a function they perform in both innate and adaptive immune responses. dendritic cells are phagocytic when they are immature and can take up pathogens; after maturing, they function as specialized cells that present pathogen antigens to t lymphocytes in a form they can recognize, thus activating t lymphocytes and initiating adaptive immune responses. macrophages can also present antigens to t lymphocytes and can activate them. the other myeloid cells are primarily secretory cells that release the contents of their prominent granules upon activation via antibody during an adaptive immune response. eosinophils are thought to be involved in attacking large

1	secretory cells that release the contents of their prominent granules upon activation via antibody during an adaptive immune response. eosinophils are thought to be involved in attacking large antibody-coated parasites such as worms; basophils are also thought to be involved in anti-parasite immunity. mast cells are tissue cells that trigger a local inflammatory response to antigen by releasing substances that act on local blood vessels. mast cells, eosinophils, and basophils are also important in allergic responses. Photographs courtesy of n. rooney, r. Steinman, and d. Friend.

1	MOVIE 1.1 Microbes coated in this way are recognized by specific complement receptors on macrophages and neutrophils, taken up by phagocytosis, and destroyed. In addition to their specialized role in the immune system, macrophages act as general scavenger cells in the body, clearing it of dead cells and cell debris.

1	The granulocytes are named for the densely staining granules in their cytoplasm; they are also called polymorphonuclear leukocytes because of their oddly shaped nuclei. The three types of granulocytes—neutrophils, eosinophils, and basophils— are distinguished by the different staining properties of their granules, which serve distinct functions. Granulocytes are all relatively short-lived, surviving for only a few days. They mature in the bone marrow, and their production increases during immune responses, when they migrate to sites of infection or inflammation. The phagocytic neutrophils are the most numerous and important cells in innate immune responses: they take up a variety of microorganisms by phagocytosis and efficiently destroy them in intracellular vesicles by using degradative enzymes and other antimicrobial substances stored in their cytoplasmic granules. Hereditary deficiencies in neutrophil function open the way to overwhelming bacterial infection, which is fatal if

1	enzymes and other antimicrobial substances stored in their cytoplasmic granules. Hereditary deficiencies in neutrophil function open the way to overwhelming bacterial infection, which is fatal if untreated. Their role is discussed further in Chapter 3.

1	Eosinophils and basophils are less abundant than neutrophils, but like neutrophils, they have granules containing a variety of enzymes and toxic proteins, which are released when these cells are activated. Eosinophils and basophils are thought to be important chiefly in defense against parasites, which are too large to be ingested by macrophages or neutrophils. They can also contribute to allergic inflammatory reactions, in which their effects are damaging rather than protective. Mast cells begin development in the bone marrow, but migrate as immature precursors that mature in peripheral tissues, especially skin, intestines, and airway mucosa. Their granules contain many inflammatory mediators, such as histamine and various proteases, which play a role in protecting the internal surfaces from pathogens, including parasitic worms. We cover eosinophils, basophils, and mast cells and their role in allergic inflammation further in Chapters 10 and 14.

1	Dendritic cells were discovered in the 1970s by Ralph Steinman, for which he received half the 2011 Nobel Prize. These cells form the third class of phagocytic cells of the immune system and include several related lineages whose distinct functions are still being clarified. Most dendritic cells have elaborate membranous processes, like the dendrites of nerve cells. Immature dendritic cells migrate through the bloodstream from the bone marrow to enter tissues. They take up particulate matter by phagocytosis and also continually ingest large amounts of the extracellular fluid and its contents by a process known as macropinocytosis. They degrade the pathogens that they take up, but their main role in the immune system is not the clearance of microorganisms. Instead, dendritic cells are a major class of sensor cells whose encounter with pathogens triggers them to produce mediators that activate other immune cells. Dendritic cells were discovered because of their role in activating a

1	a major class of sensor cells whose encounter with pathogens triggers them to produce mediators that activate other immune cells. Dendritic cells were discovered because of their role in activating a particular class of lymphocytes—T lymphocytes—of the adaptive immune system, and we will return to this activity when we discuss T-cell activation in Section 1-15. But dendritic cells and the mediators they produce also play a critical role in controlling responses of cells of the innate immune system.

1	1-5 Sensor cells express pattern recognition receptors that provide an initial discrimination between self and nonself. Long before the mechanisms of innate recognition were discovered, it was recognized that purified antigens such as proteins often did not evoke an immune response in an experimental immunization—that is, they were not Principles of innate immunity. immunogenic. Rather, the induction of strong immune responses against purified proteins required the inclusion of microbial constituents, such as killed bacteria or bacterial extracts, famously called the immunologist’s ‘dirty little secret’ by Charles Janeway (see Appendix I, Sections A-1–A-4). This additional material was termed an adjuvant, because it helped intensify the response to the immunizing antigen (adjuvare is Latin for ‘to help’). We know now that adjuvants are needed, at least in part, to activate innate receptors on various types of sensor cells to help activate T cells in the absence of an infection.

1	Macrophages, neutrophils, and dendritic cells are important classes of sensor cells that detect infection and initiate immune responses by producing inflammatory mediators, although other cells, even cells of the adaptive immune system, can serve in this function. As mentioned in Section 1-3, these cells express a limited number of invariant innate recognition receptors as a means of detecting pathogens or the damage induced by them. Also called pattern recognition receptors (PRRs), they recognize simple molecules and regular patterns of molecular structure known as pathogen-associated molecular patterns (PAMPs) that are part of many microorganisms but not of the host body’s own cells. Such structures include mannose-rich oligosaccharides, peptidoglycans, and lipopolysaccharides of the bacterial cell wall, as well as unmethylated CpG DNA common to many pathogens. All of these microbial elements have been conserved during evolution, making them excellent targets for recognition because

1	cell wall, as well as unmethylated CpG DNA common to many pathogens. All of these microbial elements have been conserved during evolution, making them excellent targets for recognition because they do not change (Fig. 1.9). Some PRRs are transmembrane proteins, such as the Toll-like receptors (TLRs) that detect PAMPs derived from extracellular bacteria or bacteria taken into vesicular pathways by phagocytosis. The role of the Toll receptor in immunity was discovered first in Drosophila melanogaster by Jules Hoffman, and later extended to homologous TLRs in mice by Janeway and Bruce Beutler. Hoffman and Beutler shared the remaining half of the 2011 Nobel Prize (see Section 1-4) for their work in the activation of innate immunity. Other PRRs are cytoplasmic proteins, such as the NOD-like receptors (NLRs) that sense intracellular bacterial invasion. Yet other cytoplasmic receptors detect viral infection based on differences in the structures and locations of the host mRNA and virally

1	receptors (NLRs) that sense intracellular bacterial invasion. Yet other cytoplasmic receptors detect viral infection based on differences in the structures and locations of the host mRNA and virally derived RNA species, and between host and microbial DNA. Some receptors expressed by sensor cells detect cellular damage induced by pathogens, rather than the pathogens themselves. Much of our knowledge of innate recognition has emerged only within the past 15 years and is still an active area of discovery. We describe these innate recognition systems further in Chapter 3, and how adjuvants are used as a component of vaccines in Chapter 16.

1	1-6 Sensor cells induce an inflammatory response by producing mediators such as chemokines and cytokines. Activation of PRRs on sensor cells such as macrophages and neutrophils can directly induce effector functions in these cells, such as the phagocytosis and degradation of bacteria they encounter. But sensor cells serve to amplify the immune response by the production of inflammatory mediators. Two important categories of inflammatory mediators are the secreted proteins called cytokines and chemokines, which act in a manner similar to hormones to convey important signals to other immune cells.

1	‘Cytokine’ is a term for any protein secreted by immune cells that affects the behavior of nearby cells bearing appropriate receptors. There are more than 60 different cytokines; some are produced by many different cell types; others, by only a few specific cell types. Some cytokines influence many types of cells, while others influence only a few, through the expression pattern of each cytokine’s specific receptor. The response that a cytokine induces in a target cell is typically related to amplifying an effector mechanism of the target cell, as illustrated in the next section.

1	macrophages express a variety of receptors, each of which is able to recognize specific components of microbes. Some, like the mannose and glucan receptors and the scavenger receptor, bind cell-wall carbohydrates of bacteria, yeast, and fungi. the toll-like receptors (tlrs) are an important family of pattern recognition receptors present on macrophages, dendritic cells, and other immune cells. tlrs recognize different microbial components; for example, a heterodimer of tlr-1 and tlr-2 binds certain lipopeptides from pathogens such as gram-positive bacteria, while tlr-4 binds both lipopolysaccharides from gram-negative and lipoteichoic acids from gram-positive bacteria.

1	Fig. 1.10 Infection triggers an inflammatory response. macrophages encountering bacteria or other types of microorganisms in tissues are triggered to release cytokines (left panel) that increase the permeability of blood vessels, allowing fluid and proteins to pass into the tissues (center panel). macrophages also produce chemokines, which direct the migration of neutrophils to the site of infection. the stickiness of the endothelial cells of the blood vessel wall is also changed, so that circulating cells of the immune system adhere to the wall and are able to crawl through it; first neutrophils and then monocytes are shown entering the tissue from a blood vessel (right panel). the accumulation of fluid and cells at the site of infection causes the redness, swelling, heat, and pain known collectively as inflammation. neutrophils and macrophages are the principal inflammatory cells. later in an immune response, activated lymphocytes can also contribute to inflammation.

1	Vasodilation and increased vascular permeability cause redness, heat, and swelling fuids protein neutrophil Infammatory cells migrate into tissue, releasing infammatory mediators that cause pain monocyte Instead of presenting all the cytokines together all at once, we introduce each cytokine as it arises during our description of cellular and functional responses. We list the cytokines, their producer and target cells, and their general functions in Appendix III.

1	Chemokines are a specialized subgroup of secreted proteins that act as chemoattractants, attracting cells bearing chemokine receptors, such as neutrophils and monocytes, out of the bloodstream and into infected tissue (Fig. 1.10). Beyond this role, chemokines also help organize the various cells in lymphoid tissues into discrete regions where specialized responses can take place. There are on the order of 50 different chemokines, which are all related structurally but fall into two major classes. Appendix IV lists the chemokines, their target cells, and their general functions. We will discuss chemokines as the need arises during our descriptions of particular cellular immune processes.

1	The cytokines and chemokines released by activated macrophages act to recruit cells from the blood into infected tissues, a process, known as inflammation, that helps to destroy the pathogen. Inflammation increases the flow of lymph, which carries microbes or cells bearing their antigens from the infected tissue to nearby lymphoid tissues, where the adaptive immune response is initiated. Once adaptive immunity has been generated, inflammation also recruits these effector components to the site of infection.

1	Inflammation is described clinically by the Latin words calor, dolor, rubor, and tumor, meaning heat, pain, redness, and swelling. Each of these features reflects an effect of cytokines or other inflammatory mediators on the local blood vessels. Heat, redness, and swelling result from the dilation and increased permeability of blood vessels during inflammation, leading to increased local blood flow and leakage of fluid and blood proteins into the tissues. Cytokines and complement fragments have important effects on the endothelium that lines blood vessels; the endothelial cells themselves also produce cytokines in response to infection. These alter the adhesive properties of the endothelial cells and cause circulating leukocytes to stick to the endothelial cells and migrate between them into the site of infection, to which they are attracted by chemokines. The migration of cells into the tissue and their local actions account for the pain.

1	The main cell types seen in the initial phase of an inflammatory response are macrophages and neutrophils, the latter being recruited into the inflamed, infected tissue in large numbers. Macrophages and neutrophils are thus also known as inflammatory cells. The influx of neutrophils is followed a short time later by the increased entry of monocytes, which rapidly differentiate into macrophages, thus reinforcing and sustaining the innate immune response. Later, if the inflammation continues, eosinophils also migrate into inflamed tissues and contribute to the destruction of the invading microorganisms. Principles of adaptive immunity. 1-7 Innate lymphocytes and natural killer cells are effector cells that share similarities with lymphoid lineages of the adaptive immune system.

1	The common lymphoid progenitor (CLP) in the bone marrow gives rise both to antigen-specific lymphocytes of the adaptive immune system and to several innate lineages that lack antigen-specific receptors. Although the B and T lymphocytes of the adaptive immune system were recognized in the 1960s, the natural killer (NK) cells (Fig. 1.11) of the innate immune system were not discovered until the 1970s. NK cells are large lymphocyte-like cells with a distinctive granular cytoplasm that were identified because of their ability to recognize and kill certain tumor cells and cells infected with herpesviruses. Initially, the distinction between these cells and T lymphocytes was unclear, but we now recognize that NK cells are a distinct lineage of cells that arise from the CLP in the bone marrow. They lack the antigen-specific receptors of the adaptive immune system cells, but express members of various families of innate receptors that can respond to cellular stress and to infections by very

1	They lack the antigen-specific receptors of the adaptive immune system cells, but express members of various families of innate receptors that can respond to cellular stress and to infections by very specific viruses. NK cells play an important role in the early innate response to viral infections, before the adaptive immune response has developed.

1	More recently, additional lineages of cells related to NK cells have been identified. Collectively, these cells are called innate lymphoid cells (ILCs). Arising from the CLP, ILCs reside in peripheral tissues, such as the intestine, where they function as the sources of mediators of inflammatory responses. The functions of NK cells and ILC cells are discussed in Chapter 3. Summary.

1	Strategies of avoidance, resistance, and tolerance represent different ways to deal with pathogens. Anatomic barriers and various chemical barriers such as complement and antimicrobial proteins may be considered a primitive form of avoidance, and they are the first line of defense against entry of both commensal organisms and pathogens into host tissues. If these barriers are breached, the vertebrate immune response becomes largely focused on resistance. Inflammatory inducers, which may be either chemical structures unique to microbes (PAMPs) or the chemical signals of tissue damage, act on receptors expressed by sensor cells to inform the immune system of infection. Sensor cells are typically innate immune cells such as macrophages or dendritic cells. Sensor cells can either directly respond with effector activity or produce inflammatory mediators, typically cytokines and chemokines that act on other immune cells, such as the innate NK cells and ILCs. These cells then are recruited

1	with effector activity or produce inflammatory mediators, typically cytokines and chemokines that act on other immune cells, such as the innate NK cells and ILCs. These cells then are recruited into target tissues to provide specific types of immune-response effector activities, such as cell killing or production of cytokines that have direct antiviral activity, all aimed to reduce or eliminate infection by pathogens. Responses by mediators in target tissues can induce several types of inflammatory cells that are specially suited for eliminating viruses, intracellular bacteria, extracellular pathogens, or parasites.

1	Principles of adaptive immunity. these are large, granular, lymphoid-like cells with important functions in innate immunity, especially against intracellular infections, being able to kill other cells. unlike lymphocytes, they lack antigen-specific receptors. Photograph courtesy of B. Smith. We come now to the components of adaptive immunity, the antigen-specific lymphocytes. Unless indicated otherwise, we shall use the term lymphocyte to refer only to the antigen-specific lymphocytes. Lymphocytes allow responses against a vast array of antigens from various pathogens encountered during a person’s lifetime and confer the important feature of immunological memory. Lymphocytes make this possible through the highly variable antigen receptors

1	Fig. 1.12 Lymphocytes are mostly small and inactive cells. the left panel shows a light micrograph of a small lymphocyte in which the nucleus has been stained purple by hematoxylin and eosin dye, surrounded by red blood cells (which have no nuclei). note the darker purple patches of condensed chromatin of the lymphocyte nucleus, indicating little transcriptional activity and the relative absence of cytoplasm. the right panel shows a transmission electron micrograph of a small lymphocyte. Again, note the evidence of functional inactivity: the condensed chromatin, the scanty cytoplasm, and the absence of rough endoplasmic reticulum. Photographs courtesy of n. rooney.

1	on their surface, by which they recognize and bind antigens. Each lymphocyte matures bearing a unique variant of a prototype antigen receptor, so that the population of lymphocytes expresses a huge repertoire of receptors that are highly diverse in their antigen-binding sites. Among the billion or so lymphocytes circulating in the body at any one time there will always be some that can recognize a given foreign antigen. A unique feature of the adaptive immune system is that it is capable of generating immunological memory, so that having been exposed once to an infectious agent, a person will make an immediate and stronger response against any subsequent exposure to it; that is, the individual will have protective immunity against it. Finding ways of generating long-lasting immunity to pathogens that do not naturally provoke it is one of the greatest challenges facing immunologists today.

1	1-8 The interaction of antigens with antigen receptors induces lymphocytes to acquire effector and memory activity. There are two major types of lymphocytes in the vertebrate immune system, the B lymphocytes (B cells) and T lymphocytes (T cells). These express distinct types of antigen receptors and have quite different roles in the immune system, as was discovered in the 1960s. Most lymphocytes circulating in the body appear as rather unimpressive small cells with few cytoplasmic organelles and a condensed, inactive-appearing nuclear chromatin (Fig. 1.12). Lymphocytes manifest little functional activity until they encounter a specific antigen that interacts with an antigen receptor on their cell surface. Lymphocytes that have not yet been activated by antigen are known as naive lymphocytes; those that have met their antigen, become activated, and have differentiated further into fully functional lymphocytes are known as effector lymphocytes.

1	B cells and T cells are distinguished by the structure of the antigen receptor that they express. The B-cell antigen receptor, or B-cell receptor (BCR), is formed by the same genes that encode antibodies, a class of proteins also known as immunoglobulins (Ig) (Fig. 1.13). Thus, the antigen receptor of B lymphocytes is also known as membrane immunoglobulin (mIg) or surface immunoglobulin (sIg). The T-cell antigen receptor, or T-cell receptor (TCR), is related to the immunoglobulins but is quite distinct in its structure and recognition properties. After antigen binds to a B-cell antigen receptor, or B-cell receptor (BCR), the B cell will proliferate and differentiate into plasma cells. These are the effector form of B lymphocytes, and they secrete antibodies that have the same antigen specificity as the plasma cell’s B-cell receptor. Thus the antigen that activates a given B cell becomes the target of the antibodies produced by that B cell’s progeny. Principles of adaptive immunity.

1	Fig. 1.13 Schematic structure of antigen receptors. upper panel: an antibody molecule, which is secreted by activated B cells as an antigen-binding effector molecule. A membrane-bound version of this molecule acts as the B-cell antigen receptor (not shown). An antibody is composed of two identical heavy chains (green) and two identical light chains (yellow). each chain has a constant part (shaded blue) and a variable part (shaded red). each arm of the antibody molecule is formed by a light chain and a heavy chain, with the variable parts of the two chains coming together to create a variable region that contains the antigen-binding site. the stem is formed from the constant parts of the heavy chains and takes a limited number of forms. this constant region is involved in the elimination of the bound antigen. lower panel: a t-cell antigen receptor. this is also composed of two chains, an α chain (yellow) and a β chain (green), each of which has a variable and a constant part. As with

1	of the bound antigen. lower panel: a t-cell antigen receptor. this is also composed of two chains, an α chain (yellow) and a β chain (green), each of which has a variable and a constant part. As with the antibody molecule, the variable parts of the two chains create a variable region, which forms the antigen-binding site. the t-cell receptor is not produced in a secreted form.

1	When a T cell first encounters an antigen that its receptor can bind, it proliferates and differentiates into one of several different functional types of effector T lymphocytes. When effector T cells subsequently detect antigen, they can manifest three broad classes of activity. Cytotoxic T cells kill other cells that are infected with viruses or other intracellular pathogens bearing the antigen. Helper T cells provide signals, often in the form of specific cytokines that activate the functions of other cells, such as B-cell production of antibody and macrophage killing of engulfed pathogens. Regulatory T cells suppress the activity of other lymphocytes and help to limit the possible damage of immune responses. We discuss the detailed functions of cytotoxic, helper, and regulatory T cells in Chapters 9, 11, 12, and 15.

1	Some of the B cells and T cells activated by antigen will differentiate into memory cells, the lymphocytes that are responsible for the long-lasting immunity that can follow exposure to disease or vaccination. Memory cells will readily differentiate into effector cells on a second exposure to their specific antigen. Immunological memory is described in Chapter 11. 1-9 Antibodies and T-cell receptors are composed of constant and variable regions that provide distinct functions.

1	1-9 Antibodies and T-cell receptors are composed of constant and variable regions that provide distinct functions. Antibodies were studied by traditional biochemical techniques long before recombinant DNA technology allowed the study of the membrane-bound forms of the antigen receptors on B and T cells. These early studies found that antibody molecules are composed of two distinct regions. One is a constant region, also called the fragment crystallizable region, or Fc region, which takes one of only four or five biochemically distinguishable forms (see Fig. 1.13). The variable region, by contrast, can be composed of a vast number of different amino acid sequences that allow antibodies to recognize an equally vast variety of antigens. It was the uniformity of the Fc region relative to the variable region that allowed its early analysis by X-ray crystallography by Gerald Edelman and Rodney Porter, who shared the 1972 Nobel Prize for their work on the structure of antibodies.

1	The antibody molecule is composed of two identical heavy chains and two identical light chains. Heavy and light chains each have variable and constant regions. The variable regions of a heavy chain and a light chain combine to form an antigen-binding site that determines the antigen-binding specificity of the antibody. Thus, both heavy and light chains contribute to the antigen-binding specificity of the antibody molecule. Also, each antibody has two identical variable regions, and so has two identical antigen-binding sites. The constant region determines the effector function of the antibody, that is, how the antibody will interact with various immune cells to dispose of antigen once it is bound. Fig. 1.14 Antigens are the molecules recognized by the immune response, while epitopes are sites within antigens to which antigen receptors bind.

1	Fig. 1.14 Antigens are the molecules recognized by the immune response, while epitopes are sites within antigens to which antigen receptors bind. Antigens can be complex macromolecules such as proteins, as shown in yellow. most antigens are larger than the sites on the antibody or antigen receptor to which they bind, and the actual portion of the antigen that is bound is known as the antigenic determinant, or epitope, for that receptor. large antigens such as proteins can contain more than one epitope (indicated in red and blue) and thus may bind different antibodies (shown here in the same color as the epitopes they bind). Antibodies generally recognize epitopes on the surface of the antigen.

1	Fig. 1.15 T-cell receptors bind a complex of an antigen fragment and a self molecule. unlike most antibodies, t-cell receptors can recognize epitopes that are buried within antigens (first panel). these antigens must first be degraded by proteases (second panel) and the peptide epitope delivered to a self molecule, called an mHC molecule (third panel). It is in this form, as a complex of peptide and mHC molecule, that antigens are recognized by t-cell receptors (tCrs; fourth panel).

1	The T-cell receptor shows many similarities to the B-cell receptor and antibody (see Fig. 1.13). It is composed of two chains, the TCR αand β chains, that are roughly equal in size and which span the T-cell membrane. Like antibody, each TCR chain has a variable region and a constant region, and the combination of the αand β-chain variable regions creates a single site for binding antigen. The structures of both antibodies and T-cell receptors are described in detail in Chapter 4, and functional properties of antibody constant regions are discussed in Chapters 5 and 10. 1-10 Antibodies and T-cell receptors recognize antigens by fundamentally different mechanisms.

1	1-10 Antibodies and T-cell receptors recognize antigens by fundamentally different mechanisms. In principle, almost any chemical structure can be recognized as an antigen by the adaptive immune system, but the usual antigens encountered in an infection are the proteins, glycoproteins, and polysaccharides of pathogens. An individual antigen receptor or antibody recognizes a small portion of the antigen’s molecular structure, and the part recognized is known as an antigenic determinant or epitope (Fig. 1.14). Typically, proteins and glycoproteins have many different epitopes that can be recognized by different antigen receptors. Antibodies and B-cell receptors directly recognize the epitopes of native antigen in the serum or the extracellular spaces. It is possible for different antibodies to simultaneously recognize an antigen by its different epitopes; such simultaneous recognition increases the efficiency of clearing or neutralizing the antigen.

1	Whereas antibodies can recognize nearly any type of chemical structure, T-cell receptors usually recognize protein antigens and do so very differently from antibodies. The T-cell receptor recognizes a peptide epitope derived from a partially degraded protein, but only if the peptide is bound to specialized cell-surface glycoproteins called MHC molecules (Fig. 1.15). The members of this large family of cell-surface glycoproteins are encoded in a cluster of genes called the major histocompatibility complex (MHC). The antigens recognized by T cells can be derived from proteins arising from intracellular pathogens, such as a virus, or from extracellular pathogens. A further difference from the antibody molecule is that there is no secreted form of the T-cell receptor; the T-cell receptor functions solely to signal to the T cell that it has bound its antigen, and the subsequent immunological effects depend on the actions of the T cells themselves. We will further describe how epitopes from

1	solely to signal to the T cell that it has bound its antigen, and the subsequent immunological effects depend on the actions of the T cells themselves. We will further describe how epitopes from antigens are placed on MHC proteins in Chapter 6 and how T cells carry out their subsequent functions in Chapter 9.

1	TCRMHCmoleculeMHCmoleculeepitopepeptideThe epitopes recognized by T-cell receptors are often buried The antigen must ÿrst be broken down into peptide fragments The epitope peptide binds to a self molecule, an MHC molecule The T-cell receptor binds to a complex of MHC molecule and epitope peptide Principles of adaptive immunity. 1-11 Antigen-receptor genes are assembled by somatic gene rearrangements of incomplete receptor gene segments.

1	The innate immune system detects inflammatory stimuli by means of a relatively limited number of sensors, such as the TLR and NOD proteins, numbering fewer than 100 different types of proteins. Antigen-specific receptors of adaptive immunity provide a seemingly infinite range of specificities, and yet are encoded by a finite number of genes. The basis for this extraordinary range of specificity was discovered in 1976 by Susumu Tonegawa, for which he was awarded the 1987 Nobel Prize. Immunoglobulin variable regions are inherited as sets of gene segments, each encoding a part of the variable region of one of the immunoglobulin polypeptide chains. During B-cell development in the bone marrow, these gene segments are irreversibly joined by a process of DNA recombination to form a stretch of DNA encoding a complete variable region. A similar process of antigen-receptor gene rearrangement takes place for the T-cell receptor genes during development of T cells in the thymus.

1	Just a few hundred different gene segments can combine in different ways to generate thousands of different receptor chains. This combinatorial diversity allows a small amount of genetic material to encode a truly staggering diversity of receptors. During this recombination process, the random addition or subtraction of nucleotides at the junctions of the gene segments creates additional diversity known as junctional diversity. Diversity is amplified further by the fact that each antigen receptor has two different variable chains, each encoded by distinct sets of gene segments. We will describe the gene rearrangement process that assembles complete antigen receptors from gene segments in Chapter 5. 1-12 Lymphocytes activated by antigen give rise to clones of antigen-specific effector cells that mediate adaptive immunity.

1	There are two critical features of lymphocyte development that distinguish adaptive immunity from innate immunity. First, the process described above that assembles antigen receptors from incomplete gene segments is carried out in a manner that ensures that each developing lymphocyte expresses only one receptor specificity. Whereas the cells of the innate immune system express many different pattern recognition receptors and recognize features shared by many pathogens, the antigen-receptor expression of lymphocytes is ‘clonal,’ so that each mature lymphocyte differs from others in the specificity of its antigen receptor. Second, because the gene rearrangement process irreversibly changes the lymphocyte’s DNA, all its progeny inherit the same receptor specificity. Because this specificity is inherited by a cell’s progeny, the proliferation of an individual lymphocyte forms a clone of cells with identical antigen receptors.

1	There are lymphocytes of at least 108 different specificities in an individual human at any one time, comprising the lymphocyte receptor repertoire of the individual. These lymphocytes are continually undergoing a process similar to natural selection: only those lymphocytes that encounter an antigen to which their receptor binds will be activated to proliferate and differentiate into effector cells. This selective mechanism was first proposed in the 1950s by Macfarlane Burnet, who postulated the preexistence in the body of many different potential antibody-producing cells, each displaying on its surface a membrane-bound version of the antibody that served as a receptor for the antigen. On binding antigen, the cell is activated to divide and to produce many identical progeny, a process known as clonal expansion; this clone of identical cells can now secrete clonotypic antibodies with a specificity identical to that of the surface receptor that first triggered activation and clonal

1	known as clonal expansion; this clone of identical cells can now secrete clonotypic antibodies with a specificity identical to that of the surface receptor that first triggered activation and clonal expansion (Fig. 1.16). Burnet called this the clonal selection theory

1	A single progenitor cell gives rise to a large number of lymphocytes, each with a different speciÿcity Fig. 1.16 Clonal selection. each lymphoid progenitor gives rise to a large number of lymphocytes, each bearing a distinct antigen receptor. lymphocytes with receptors that bind ubiquitous self antigens are eliminated before they become fully mature, ensuring tolerance to such self antigens. When a foreign antigen (red dot) interacts with the receptor on a mature naive lymphocyte, that cell is activated and starts to divide. It gives rise to a clone of identical progeny, all of whose receptors bind the same antigen. Antigen specificity is thus maintained as the progeny proliferate and differentiate into effector cells. once antigen has been eliminated by these effector cells, the immune response ceases, although some lymphocytes are retained to mediate immunological memory. Each lymphocyte bears a single type of receptor with a unique speciÿcity

1	Each lymphocyte bears a single type of receptor with a unique speciÿcity Interaction between a foreign molecule and a lymphocyte receptor capable of binding that molecule with high afÿnity leads to lymphocyte activation The differentiated effector cells derived from an activated lymphocyte will bear receptors of identical speciÿcity to those of the parental cell from which that lymphocyte was derived Lymphocytes bearing receptors speciÿc for ubiquitous self molecules are deleted at an early stage in lymphoid cell development and are therefore absent from the repertoire of mature lymphocytes Fig. 1.17 The four basic principles of clonal selection. of antibody production; its four basic postulates are listed in Fig. 1.17. Clonal selection of lymphocytes is the single most important principle in adaptive immunity. 1-13 Lymphocytes with self-reactive receptors are normally eliminated during development or are functionally inactivated.

1	1-13 Lymphocytes with self-reactive receptors are normally eliminated during development or are functionally inactivated. When Burnet formulated his theory, nothing was known of the antigen receptors or indeed the function of lymphocytes themselves. In the early 1960s, James Gowans discovered that removal of the small lymphocytes from rats resulted in the loss of all known adaptive immune responses, which were restored when the small lymphocytes were replaced. This led to the realization that lymphocytes must be the units of clonal selection, and their biology became the focus of the new field of cellular immunology.

1	Clonal selection of lymphocytes with diverse receptors elegantly explained adaptive immunity, but it raised one significant conceptual problem. With so many different antigen receptors being generated randomly during the lifetime of an individual, there is a possibility that some receptors might react against an individual’s own self antigens. How are lymphocytes prevented from recognizing native antigens on the tissues of the body and attacking them? Ray Owen had shown in the late 1940s that genetically different twin calves with a common placenta, and thus a shared placental blood circulation, were immunologically unresponsive, or tolerant, to one another’s tissues. Peter Medawar then showed in 1953 that exposure to foreign tissues during embryonic development caused mice to become immunologically tolerant to these tissues. Burnet proposed that developing lymphocytes that are potentially self-reactive are removed before they can mature, a process known as clonal deletion. Medawar

1	tolerant to these tissues. Burnet proposed that developing lymphocytes that are potentially self-reactive are removed before they can mature, a process known as clonal deletion. Medawar and Burnet shared the 1960 Nobel Prize for their work on tolerance. This process was demonstrated to occur experimentally in the late 1980s. Some lymphocytes that receive either too much or too little signal through their antigen receptor during development are eliminated by a form of cell suicide called apoptosis—derived from a Greek word meaning the falling of leaves from trees— or programmed cell death. Other types of mechanisms of immunological tolerance have been identified since then that rely on the induction of an inactive state, called anergy, as well as mechanisms of active suppression of self-reactive lymphocytes. Chapter 8 will describe lymphocyte development and tolerance mechanisms that shape the lymphocyte receptor repertoire. Chapters 14 and 15 will discuss how immune tolerance

1	self-reactive lymphocytes. Chapter 8 will describe lymphocyte development and tolerance mechanisms that shape the lymphocyte receptor repertoire. Chapters 14 and 15 will discuss how immune tolerance mechanisms can sometimes fail.

1	Principles of adaptive immunity. 1-14 Lymphocytes mature in the bone marrow or the thymus and then congregate in lymphoid tissues throughout the body.

1	Lymphocytes circulate in the blood and the lymph and are also found in large numbers in lymphoid tissues or lymphoid organs, which are organized aggregates of lymphocytes in a framework of nonlymphoid cells. Lymphoid organs can be divided broadly into the central or primary lymphoid organs, where lymphocytes are generated, and the peripheral or secondary lymphoid organs, where mature naive lymphocytes are maintained and adaptive immune responses are initiated. The central lymphoid organs are the bone marrow and the thymus, an organ in the upper chest. The peripheral lymphoid organs comprise the lymph nodes, the spleen, and the mucosal lymphoid tissues of the gut, the nasal and respiratory tract, the urogenital tract, and other mucosa. The locations of the main lymphoid tissues are shown schematically in Fig. 1.18; we describe the individual peripheral lymphoid organs in more detail later in the chapter. Lymph nodes are interconnected by a system of lymphatic vessels, which drain

1	schematically in Fig. 1.18; we describe the individual peripheral lymphoid organs in more detail later in the chapter. Lymph nodes are interconnected by a system of lymphatic vessels, which drain extracellular fluid from tissues, carry it through the lymph nodes, and deposit it back into the blood.

1	The progenitors that give rise to B and T lymphocytes originate in the bone marrow. B cells complete their development within the bone marrow. Although the ‘B’ in B lymphocytes originally stood for the bursa of Fabricius, a lymphoid organ in young chicks in which lymphocytes mature, it is a useful mnemonic for bone marrow. The immature precursors of T lymphocytes migrate to the thymus, from which they get their name, and complete their development there. Once they have completed maturation, both types of lymphocytes enter the bloodstream as mature naive lymphocytes and continuously circulate through the peripheral lymphoid tissues.

1	Fig. 1.18 The distribution of lymphoid tissues in the body. lymphocytes arise from stem cells in bone marrow and differentiate in the central lymphoid organs (yellow)—B cells in the bone marrow and t cells in the thymus. they migrate from these tissues and are carried in the bloodstream to the peripheral lymphoid organs (blue). these include lymph nodes, spleen, and lymphoid tissues associated with mucosa, such as the gut-associated tonsils, Peyer’s patches, and appendix. the peripheral lymphoid organs are the sites of lymphocyte activation by antigen, and lymphocytes recirculate between the blood and these organs until they encounter their specific antigen. lymphatics drain extracellular fluid from the peripheral tissues, through the lymph nodes, and into the thoracic duct, which empties into the left subclavian vein. this fluid, known as lymph, carries antigen taken up by dendritic cells and macrophages to the lymph nodes, as well as recirculating lymphocytes from the lymph nodes

1	into the left subclavian vein. this fluid, known as lymph, carries antigen taken up by dendritic cells and macrophages to the lymph nodes, as well as recirculating lymphocytes from the lymph nodes back into the blood. lymphoid tissue is also associated with other mucosa such as the bronchial linings (not shown).

1	Fig. 1.19 Dendritic cells initiate adaptive immune responses. Immature dendritic cells residing in a tissue take up pathogens and their antigens by macropinocytosis and by receptor-mediated endocytosis. they are stimulated by recognition of the presence of pathogens to migrate through the lymphatics to regional lymph nodes, where they arrive as fully mature nonphagocytic dendritic cells that express both antigen and the co-stimulatory molecules necessary to activate a naive t cell that recognizes the antigen. thus the dendritic cells stimulate lymphocyte proliferation and differentiation. MOVIE 1.1

1	MOVIE 1.1 Fig. 1.20 Dendritic cells form a key link between the innate immune system and the adaptive immune system. like the other cells of innate immunity, dendritic cells recognize pathogens via invariant cell-surface receptors for pathogen molecules and are activated by these stimuli early in an infection. dendritic cells in tissues are phagocytic; they are specialized to ingest a wide range of pathogens and to display their antigens at the dendritic cell surface in a form that can be recognized by t cells. 1-15 Adaptive immune responses are initiated by antigen and antigen-presenting cells in secondary lymphoid tissues.

1	Adaptive immune responses are initiated when B or T lymphocytes encounter antigens for which their receptors have specific reactivity, provided that there are appropriate inflammatory signals to support activation. For T cells, this activation occurs via encounters with dendritic cells that have picked up antigens at sites of infection and migrated to secondary lymphoid organs. Activation of the dendritic cells’ PRRs by PAMPs at the site of infection stimulates the dendritic cells in the tissues to engulf the pathogen and degrade it intracellularly. They also take up extracellular material, including virus particles and bacteria, by receptor-independent macropinocytosis. These processes lead to the display of peptide antigens on the MHC molecules of the dendritic cells, a display that activates the antigen receptors of lymphocytes. Activation of PRRs also triggers the dendritic cells to express cell-surface proteins called co-stimulatory molecules, which support the ability of the T

1	the antigen receptors of lymphocytes. Activation of PRRs also triggers the dendritic cells to express cell-surface proteins called co-stimulatory molecules, which support the ability of the T lymphocyte to proliferate and differentiate into its final, fully functional form (Fig. 1.19). For these reasons dendritic cells are also called antigen-presenting cells (APCs), and as such, they form a crucial link between the innate immune response and the adaptive immune response (Fig. 1.20). In certain situations, macrophages and B cells can also act as antigen-presenting cells, but dendritic cells are the cells that are specialized in initiating the adaptive immune response. Free antigens can also stimulate the antigen receptors of B cells, but most B cells require ‘help’ from activated helper T cells for optimal antibody responses. The activation of naive T lymphocytes is therefore an essential first stage in virtually all adaptive immune responses. Chapter 6 returns to dendritic cells to

1	T cells for optimal antibody responses. The activation of naive T lymphocytes is therefore an essential first stage in virtually all adaptive immune responses. Chapter 6 returns to dendritic cells to discuss how antigens are processed for presentation to T cells. Chapters 7 and 9 discuss co-stimulation and lymphocyte activation. Chapter 10 describes how T cells help in activating B cells.

1	Principles of adaptive immunity. naive lymphocytes recirculate constantly through peripheral lymphoid tissue, here illustrated as a popliteal lymph node—a lymph node situated behind the knee. In the case of an infection in the foot, this will be the draining lymph node, where lymphocytes may encounter their specific antigens and become activated. Both activated and nonactivated lymphocytes are returned to the bloodstream via the lymphatic system. 1-16 Lymphocytes encounter and respond to antigen in the peripheral lymphoid organs.

1	1-16 Lymphocytes encounter and respond to antigen in the peripheral lymphoid organs. Antigen and lymphocytes eventually encounter each other in the peripheral lymphoid organs—the lymph nodes, spleen, and mucosal lymphoid tissues (see Fig. 1.18). Mature naive lymphocytes are continually recirculating through these tissues, to which pathogen antigens are carried from sites of infection, primarily by dendritic cells. The peripheral lymphoid organs are specialized to trap antigen-bearing dendritic cells and to facilitate the initiation of adaptive immune responses. Peripheral lymphoid tissues are composed of aggregations of lymphocytes in a framework of nonleukocyte stromal cells, which provide both the basic structural organization of the tissue and survival signals to help sustain the life of the lymphocytes. Besides lymphocytes, peripheral lymphoid organs also contain resident macrophages and dendritic cells.

1	When an infection occurs in a tissue such as the skin, free antigen and antigen-bearing dendritic cells travel from the site of infection through the afferent lymphatic vessels into the draining lymph nodes (Fig. 1.21)—peripheral lymphoid tissues where they activate antigen-specific lymphocytes. The activated lymphocytes then undergo a period of proliferation and differentiation, after which most leave the lymph nodes as effector cells via the efferent lymphatic vessel. This eventually returns them to the bloodstream (see Fig. 1.18), which then carries them to the tissues where they will act. This whole process takes about 4–6 days from the time that the antigen is recognized, which means that an adaptive immune response to an antigen that has not been encountered before does not become effective until about a week after infection (see Fig. 1.7). Naive lymphocytes that do not recognize their antigen also leave through the efferent lymphatic vessel and are returned to the blood, from

1	effective until about a week after infection (see Fig. 1.7). Naive lymphocytes that do not recognize their antigen also leave through the efferent lymphatic vessel and are returned to the blood, from which they continue to recirculate through lymphoid tissues until they recognize antigen or die.

1	The lymph nodes are highly organized lymphoid organs located at the points of convergence of vessels of the lymphatic system, which is the extensive system that collects extracellular fluid from the tissues and returns it to the blood (see Fig. 1.18). This extracellular fluid is produced continuously by filtration from the blood and is called lymph. Lymph flows away from the peripheral tissues under the pressure exerted by its continuous production, and is carried by lymphatic vessels, or lymphatics. One-way valves in the lymphatic vessels prevent a reverse flow, and the movements of one part of the body in relation to another are important in driving the lymph along.

1	As noted above, afferent lymphatic vessels drain fluid from the tissues and carry pathogens and antigen-bearing cells from infected tissues to the lymph nodes (Fig. 1.22). Free antigens simply diffuse through the extracellular fluid to the lymph node, while the dendritic cells actively migrate into the lymph node, attracted by chemokines. The same chemokines also attract lymphocytes from the blood, and these enter lymph nodes by squeezing through the walls of specialized blood vessels called high endothelial venules (HEV), named for their thicker, more rounded appearance relative to flatter endothelial cells in other locations. In the lymph nodes, B lymphocytes are localized in follicles, which make up the outer cortex of the lymph node, with T cells more Fig. 1.21 Circulating lymphocytes encounter antigen in peripheral lymphoid organs.

1	Fig. 1.21 Circulating lymphocytes encounter antigen in peripheral lymphoid organs. Lymphocytes and lymph return to blood via the thoracic duct Naive lymphocytes enter lymph nodes from blood infected peripheral tissue lymph node heart Antigens from sites of infection reach lymph nodes via lymphatics

1	Fig. 1.22 Organization of a lymph node. As shown at left in the diagram of a lymph node in longitudinal section, a lymph node consists of an outermost cortex and an inner medulla. the cortex is composed of an outer cortex of B cells organized into lymphoid follicles and of adjacent, or paracortical, areas made up mainly of t cells and dendritic cells. When an immune response is under way, some of the follicles—known as secondary lymphoid follicles— contain central areas of intense B-cell proliferation called germinal centers. these reactions are very dramatic, but eventually die out as germinal centers become senescent. lymph draining from the extracellular spaces of the body carries antigens in phagocytic dendritic cells and phagocytic macrophages from the tissues to the lymph node via the afferent lymphatics. these migrate directly from the sinuses into the cellular parts of the node. lymph leaves via the efferent lymphatics in the medulla. the medulla consists of strings of

1	via the afferent lymphatics. these migrate directly from the sinuses into the cellular parts of the node. lymph leaves via the efferent lymphatics in the medulla. the medulla consists of strings of macrophages and antibody-secreting plasma cells known as the medullary cords. naive lymphocytes enter the node from the bloodstream through specialized postcapillary venules (not shown) and leave with the lymph through the efferent lymphatic. the light micrograph (right) shows a transverse section through a lymph node, with prominent follicles containing germinal centers. magnification ×7. Photograph courtesy of n. rooney.

1	diffusely distributed in the surrounding paracortical areas, also referred to as the deep cortex or T-cell zones (see Fig. 1.22). Lymphocytes migrating from the blood into lymph nodes enter the paracortical areas first, and because they are attracted by the same chemokines, antigen-presenting dendritic cells and macrophages also become localized there. Free antigen diffusing through the lymph node can become trapped on these dendritic cells and macrophages. This juxtaposition of antigen, antigen-presenting cells, and naive T cells in the T-cell zone creates an ideal environment in which naive T cells can bind their specific antigen and thus become activated.

1	As noted earlier, activation of B cells usually requires not only antigen, which binds to the B-cell receptor, but also the cooperation of activated helper T cells, a type of effector T cell. The location of B cells and T cells within the lymph node is dynamically regulated by their state of activation. When they become activated, T cells and B cells both move to the border of the follicle and T-cell zone, where T cells can first provide their helper function to B cells. Some of the B-cell follicles include germinal centers, where activated B cells are undergoing intense proliferation and differentiation into plasma cells. These mechanisms are described in detail in Chapter 10.

1	In humans, the spleen is a fist-sized organ situated just behind the stomach (see Fig. 1.18). It has no direct connection with the lymphatic system; instead, it collects antigen from the blood and is involved in immune responses to blood-borne pathogens. Lymphocytes enter and leave the spleen via blood vessels. The spleen also collects and disposes of senescent red blood cells. Its organization is shown schematically in Fig. 1.23. The bulk of the spleen is composed of red pulp, which is the site of red blood cell disposal. The lymphocytes surround the arterioles running through the spleen, forming isolated Principles of adaptive immunity.

1	Fig. 1.23 Organization of the lymphoid tissues of the spleen. of lymphocytes. In each area of white pulp, blood carrying both the schematic at top left shows that the spleen consists of red pulp lymphocytes and antigen flows from a trabecular artery into a (pink areas), which is a site of red blood cell destruction, interspersed central arteriole. From this arteriole smaller blood vessels fan out, with the lymphoid white pulp. An enlargement of a small section eventually terminating in a specialized zone in the human spleen of a human spleen (top right) shows the arrangement of discrete called the perifollicular zone (PFZ), which surrounds each marginal areas of white pulp (yellow and blue) around central arterioles. zone. Cells and antigen then pass into the white pulp through open most of the white pulp is shown in transverse section, with two blood-filled spaces in the perifollicular zone. the light micrograph portions in longitudinal section. the two schematics below this at

1	most of the white pulp is shown in transverse section, with two blood-filled spaces in the perifollicular zone. the light micrograph portions in longitudinal section. the two schematics below this at bottom left shows a transverse section of white pulp of human diagram show enlargements of a transverse section (bottom center) spleen immunostained for mature B cells. Both follicle and PAlS are and longitudinal section (bottom right) of white pulp. Surrounding surrounded by the perifollicular zone. the follicular arteriole emerges the central arteriole is the periarteriolar lymphoid sheath (PAlS), in the PAlS (arrowhead at bottom), traverses the follicle, goes made up of t cells. lymphocytes and antigen-loaded dendritic through the marginal zone, and opens into the perifollicular zone cells come together here. the follicles consist mainly of B cells; (upper arrowheads). Co, follicular B-cell corona; gC, germinal center; in secondary follicles, a germinal center is surrounded by a B-cell

1	come together here. the follicles consist mainly of B cells; (upper arrowheads). Co, follicular B-cell corona; gC, germinal center; in secondary follicles, a germinal center is surrounded by a B-cell mZ, marginal zone; rP, red pulp; arrowheads, central arteriole. corona. the follicles are surrounded by a so-called marginal zone Photograph courtesy of n. m. milicevic.

1	areas of white pulp. The sheath of lymphocytes around an arteriole is called the periarteriolar lymphoid sheath (PALS) and contains mainly T cells. Lymphoid follicles occur at intervals along it, and these contain mainly B cells. An area called the marginal zone surrounds the follicle; it has few T cells, is rich in macrophages, and has a resident, noncirculating population of B cells known as marginal zone B cells. These B cells are poised to rapidly produce antibodies that have low affinity to bacterial capsular polysaccharides. These antibodies, which are discussed in Chapter 8, provide some degree of protection before the adaptive immune response is fully activated. Blood-borne microbes, soluble antigens, and antigen:antibody complexes are filtered from the blood by macrophages and immature dendritic cells within the marginal zone. Like the migration of immature dendritic cells from peripheral tissues to the T-cell areas of lymph nodes, dendritic cells in the marginal zones in the

1	dendritic cells within the marginal zone. Like the migration of immature dendritic cells from peripheral tissues to the T-cell areas of lymph nodes, dendritic cells in the marginal zones in the spleen migrate to the T-cell areas after taking up antigen and becoming activated; here they are able to present the antigens they carry to T cells.

1	villusgerminal center T cells follicle-associated epithelium subepithelial dome follicle efferent lymphatics Peyer’s patches are covered by an epithelial layer containing specialized cells called M cells, which have characteristic membrane ruf˜es M cell a dome TDA GC epithelium M cell b c

1	Fig. 1.24 Organization of a Peyer’s patch in the gut mucosa. As the diagram on the left shows, a Peyer’s patch contains numerous B-cell follicles with germinal centers. the areas between follicles are occupied by t cells and are therefore called the t-cell dependent areas. the layer between the surface epithelium and the follicles is known as the subepithelial dome, and is rich in dendritic cells, t cells, and B cells. Peyer’s patches have no afferent lymphatics, and the antigen enters directly from the gut across a specialized epithelium made up of so-called microfold (m) cells. Although this tissue looks very different from other lymphoid organs, the basic divisions are maintained. As in the lymph nodes, lymphocytes enter Peyer’s patches from the blood across the walls of high endothelial venules (not shown), and leave via the efferent lymphatic. the light micrograph in panel a shows a section through a Peyer’s patch in the gut wall of a mouse. the Peyer’s patch can be seen lying

1	venules (not shown), and leave via the efferent lymphatic. the light micrograph in panel a shows a section through a Peyer’s patch in the gut wall of a mouse. the Peyer’s patch can be seen lying beneath the epithelial tissues. gC, germinal center; tdA, t-cell dependent area. Panel b, a scanning electron micrograph of the follicle-associated epithelium boxed in panel a, shows the m cells, which lack the microvilli and the mucus layer present on normal epithelial cells. each m cell appears as a sunken area on the epithelial surface. Panel c, a higher-magnification view of the boxed area in panel b, shows the characteristic ruffled surface of an m cell. m cells are the portal of entry for many pathogens and other particles. Panel a, hematoxylin and eosin stain, magnification ×100; panel b, ×5000; panel c, ×23,000.

1	1-17 Mucosal surfaces have specialized immune structures that orchestrate responses to environmental microbial encounters.

1	Most pathogens enter the body through mucosal surfaces, and these are also exposed to a vast load of other potential antigens from the air, food, and the natural microbial flora of the body. Mucosal surfaces are protected by an extensive system of lymphoid tissues known generally as the mucosal immune system or mucosa-associated lymphoid tissues (MALT). Collectively, the mucosal immune system is estimated to contain as many lymphocytes as all the rest of the body, and they form a specialized set of cells obeying somewhat different rules of recirculation from those in the other peripheral lymphoid organs. The gut-associated lymphoid tissues (GALT) include the tonsils, adenoids, appendix, and specialized structures in the small intestine called Peyer’s patches, and they collect antigen from the epithelial surfaces of the gastrointestinal tract. In Peyer’s patches, which are the most important and highly organized of these tissues, the antigen is collected by specialized epithelial cells

1	the epithelial surfaces of the gastrointestinal tract. In Peyer’s patches, which are the most important and highly organized of these tissues, the antigen is collected by specialized epithelial cells called microfold or M cells (Fig. 1.24). The lymphocytes form a follicle consisting of a large central dome of B lymphocytes surrounded by smaller numbers of T lymphocytes. Dendritic cells resident within the Peyer’s patch present the antigen to T lymphocytes. Lymphocytes enter Peyer’s patches from the blood and leave through efferent lymphatics. Effector lymphocytes generated in Peyer’s patches travel through the lymphatic system and into the bloodstream, from where they are disseminated back into mucosal tissues to carry out their effector actions.

1	Similar but more diffuse aggregates of lymphocytes are present in the respiratory tract and other mucosa: nasal-associated lymphoid tissue (NALT) and bronchus-associated lymphoid tissue (BALT) are present in the respiratory tract. Like the Peyer’s patches, these mucosal lymphoid tissues are also overlaid by M cells, through which inhaled microbes and antigens that become trapped in the mucous covering of the respiratory tract can pass. The mucosal immune system is discussed in Chapter 12.

1	Although very different in appearance, the lymph nodes, spleen, and mucosa-associated lymphoid tissues all share the same basic architecture. They all operate on the same principle, trapping antigens and antigen-presenting cells from sites of infection in order to present antigen to migratory small lymphocytes, thus inducing adaptive immune responses. The peripheral lymphoid tissues also provide sustaining signals to lymphocytes that do not encounter their specific antigen immediately, so that they survive and continue to recirculate. Because they are involved in initiating adaptive immune responses, the peripheral lymphoid tissues are not static structures but vary quite markedly, Principles of adaptive immunity.

1	Because they are involved in initiating adaptive immune responses, the peripheral lymphoid tissues are not static structures but vary quite markedly, Principles of adaptive immunity. depending on whether or not infection is present. The diffuse mucosal lymphoid tissues may appear in response to infection and then disappear, whereas the architecture of the organized tissues changes in a more defined way during an infection. For example, the B-cell follicles of the lymph nodes expand as B lymphocytes proliferate to form germinal centers (see Fig. 1.22), and the entire lymph node enlarges, a phenomenon familiarly known as swollen glands.

1	Finally, specialized populations of lymphocytes and innate lymphoid cells can be found distributed throughout particular sites in the body rather than being found in organized lymphoid tissues. Such sites include the liver and the lamina propria of the gut, as well as the base of the epithelial lining of the gut, reproductive epithelia, and, in mice but not in humans, the epidermis. These lymphocyte populations seem to have an important role in protecting these tissues from infection, and are described further in Chapters 8 and 12. 1-18 Lymphocytes activated by antigen proliferate in the peripheral lymphoid organs, generating effector cells and immunological memory.

1	1-18 Lymphocytes activated by antigen proliferate in the peripheral lymphoid organs, generating effector cells and immunological memory. The great diversity of lymphocyte receptor repertoire means that there will usually be some lymphocytes bearing a receptor for any given foreign antigen. Recent experiments suggest this number to be perhaps a few hundred per mouse, certainly not enough to mount a response against a pathogen. To generate sufficient antigen-specific effector lymphocytes to fight an infection, a lymphocyte with an appropriate receptor specificity is activated first to proliferate. Only when a large clone of identical cells has been produced do these finally differentiate into effector cells, a process that requires 4 to 5 days. This means that the adaptive immune response to a pathogen occurs several days after the initial infection has occurred and been detected by the innate immune system.

1	On recognizing its specific antigen on an activated antigen-presenting cell, a naive lymphocyte stops migrating, the volume of the nucleus and cytoplasm increases, and new mRNAs and new proteins are synthesized. Within a few hours, the cell looks completely different and is known as a lymphoblast. Dividing lymphoblasts are able to duplicate themselves two to four times every 24 hours for 3–5 days, so that a single naive lymphocyte can produce a clone of around 1000 daughter cells of identical specificity. These then differentiate into effector cells. In the case of B cells, the differentiated effector cells are the plasma cells, which secrete antibody. In the case of T cells, the effector cells are either cytotoxic T cells, which are able to destroy infected cells, or helper T cells, which activate other cells of the immune system (see Section 1-8).

1	Effector lymphocytes do not recirculate like naive lymphocytes. Some effector T cells detect sites of infection and migrate into them from the blood; others stay in the lymphoid tissues to activate B cells. Some antibody-secreting plasma cells remain in the peripheral lymphoid organs, but most plasma cells generated in the lymph nodes and spleen will migrate to the bone marrow and take up residence there, secreting large amounts of antibodies into the blood system. Effector cells generated in the mucosal immune system generally stay within the mucosal tissues. Most lymphocytes generated by clonal expansion in an immune response will eventually die. However, a significant number of activated antigen-specific B cells and T cells persist after antigen has been eliminated. These cells are known as memory cells and form the basis of immunological memory. They can be reactivated much more quickly than naive lymphocytes, which ensures a more rapid and effective response on a second encounter

1	as memory cells and form the basis of immunological memory. They can be reactivated much more quickly than naive lymphocytes, which ensures a more rapid and effective response on a second encounter with a pathogen and thereby usually provides lasting protective immunity.

1	Fig. 1.25 The course of a typical antibody response. the first encounter with an antigen produces a primary response. Antigen A introduced at time zero encounters little specific antibody in the serum. After a lag phase (light blue), antibody against antigen A (dark blue) appears; its concentration rises to a plateau and then gradually declines, typical of a primary response. When the serum is tested for antibody against another antigen, B (yellow), there is little preset. When the animal is later challenged with a mixture of antigens A and B, a very rapid and intense antibody secondary response to A occurs, illustrating immunological memory. this is the main reason for giving booster injections after an initial vaccination. note that the response to B resembles the primary response to A, as this is the first encounter with antigen B.

1	The characteristics of immunological memory are readily observed by comparing an individual’s antibody response to a first or primary immunization with the response to a secondary or booster immunization with the same antigen. As shown in Fig. 1.25, the secondary antibody response occurs after a shorter lag phase and achieves a markedly higher level than in the primary response. During the secondary responses, antibodies can also acquire higher affinity, or strength of binding, for the antigen due to a process called affinity maturation, which takes place in the specialized germinal centers within B-cell follicles (see Section 1-16). Importantly, helper T cells are required for the process of affinity maturation, but T-cell receptors do not undergo affinity maturation. Compared with naive T cells, memory T cells show a lower threshold for activation, but as a result of changes in the responsiveness of the cell and not because of changes in the receptor. We describe the mechanisms of

1	T cells, memory T cells show a lower threshold for activation, but as a result of changes in the responsiveness of the cell and not because of changes in the receptor. We describe the mechanisms of affinity maturation in Chapters 5 and 10.

1	The cellular basis of immunological memory is the clonal expansion and clonal differentiation of cells that have a specific attraction for the eliciting antigen, and the memory is therefore entirely antigen-specific. It is immunological memory that enables successful vaccination and prevents reinfection with pathogens that have been repelled successfully by an adaptive immune response. In Chapter 11, we will return to immunological memory, which is perhaps the most important biological consequence of adaptive immunity. Summary.

1	Summary. While the innate immune system relies on invariant pattern recognition receptors to detect common microbial structures or the damage they cause, the adaptive immune system relies on a repertoire of antigen receptors to recognize structures that are specific to individual pathogens. This feature provides adaptive immunity with greater sensitivity and specificity. The clonal expansion of antigen-reactive lymphocytes also confers the property of immunological memory, which enhances protection against reinfection by the same pathogen. Adaptive immunity relies on two major types of lymphocytes. B cells mature in the bone marrow and are the source of circulating antibodies. T cells mature in the thymus and recognize peptides from pathogens presented by MHC the effector mechanisms of immunity.

1	molecules on infected cells or antigen-presenting cells. An adaptive response involves the selection and amplification of clones of lymphocytes bearing receptors that recognize the foreign antigen. This clonal selection provides the theoretical framework for understanding all the key features of an adaptive immune response. Each lymphocyte carries cell-surface receptors of a single antigen specificity. These receptors are generated by the random recombination of variable receptor gene segments and the pairing of distinct variable protein chains: heavy and light chains in immunoglobulins, or the two chains of T-cell receptors. The large antigen-receptor repertoire of lymphocytes can recognize virtually any antigen. Adaptive immunity is initiated when an innate immune response fails to eliminate a new infection and activated antigen-presenting cells—typically dendritic cells that bear antigens from pathogens and co-stimulatory receptors—migrate to the draining lymphoid tissues.

1	Immune responses are initiated in several peripheral lymphoid tissues. The spleen serves as a filter for blood-borne infections. Lymph nodes draining various tissues and the mucosal and gut-associated lymphoid tissues (MALT and GALT) are organized into specific zones where T and B cells can be activated efficiently by antigen-presenting cells or helper T cells. When a recirculating lymphocyte encounters its corresponding antigen in these peripheral lymphoid tissues, it proliferates, and its clonal progeny differentiate into effector T and B lymphocytes that can eliminate the infectious agent. A subset of these proliferating lymphocytes differentiates into memory cells, ready to respond rapidly to the same pathogen if it is encountered again. The details of these processes of recognition, development, and differentiation form the main material of the central three parts of this book. The effector mechanisms of immunity.

1	For activated innate and adaptive immune cells to destroy pathogens, they must employ an appropriate effector mechanism suited to each infecting agent. The different types of pathogens noted in Fig. 1.26 have different lifestyles and require different responses for both their recognition and their destruction. Perhaps it is not surprising, then, that defenses against different pathogen types are organized into effector modules suited for these different lifestyles. In this sense, an effector module is a collection of cell-mediated and humoral mechanisms, both innate and adaptive, that act together to achieve elimination of a particular category of pathogen. For example, defense against extracellular pathogens can involve both phagocytic cells and B cells, which recognize extracellular antigens and become plasma cells that secrete antibody into the extracellular environment. Defense against intracellular pathogens involves T cells that can detect peptides generated inside the infected

1	and become plasma cells that secrete antibody into the extracellular environment. Defense against intracellular pathogens involves T cells that can detect peptides generated inside the infected cell. Some effector T cells directly kill cells infected with intracellular pathogens such as viruses. Moreover, activated T cells differentiate into three major subsets of helper T cells, which produce different patterns of cytokines. These three subsets, discussed below, generally specialize in promoting defenses against pathogens having three major lifestyles: they can defend against intracellular infection, destroy extracellular bacteria and fungi, or provide barrier immunity directed at parasites. T cells also promote defense against extracellular pathogens by helping B cells make antibody.

1	Most of the other effector mechanisms used by an adaptive immune response to dispose of pathogens are the same as those of innate immunity and involve cells such as macrophages and neutrophils, and proteins such as complement. Indeed, it seems likely that the vertebrate adaptive immune response evolved by the addition of specific recognition properties to innate defense mechanisms already existing in invertebrates. This is supported by recent findings that the innate lymphoid cells—the ILCs—show similar patterns of differentiation into different cytokine-producing subsets to those of T cells. We begin this section by outlining the effector actions of antibodies, which depend almost entirely on recruiting cells and molecules of the innate immune system. 1-19 Innate immune responses can select from several effector modules to protect against different types of pathogens.

1	As we mentioned in Section 1-7, the innate immune system contains several types of cells—NK cells and ILCs—that have similarities to lymphocytes, particularly T cells. NK cells lack the antigen-specific receptors of T cells, but can exhibit the cytotoxic capacity of T cells and produce some of the cytokines that effector T cells produce. ILCs develop from the same progenitor cells in the bone marrow as NK cells, and they also lack antigen-specific receptors. Very recent discoveries indicate that ILCs actually comprise several closely related lineages that differ in the specific cytokines that they will produce when activated. Remarkably, there is a striking similarity between the patterns of cytokines produced by ILC subsets and helper T-cell subsets, as mentioned above. It appears that subsets of ILCs are the innate homologs of their helper T-cell counterparts, and NK cells are the innate homolog of cytotoxic T cells.

1	As mentioned in Section 1-6, there are a large number of cytokines with different functions (see Appendix III). A convenient way to organize the effects of cytokines is by the effector module that each cytokine promotes. Some cytokines tend to promote immunity to intracellular pathogens. One such cytokine is interferon-γ, which acts both by activating phagocytes to more efficiently kill intracellular pathogens and by inducing target tissues to resist intracellular pathogens. This is called type 1 immunity. IFN-γ is produced by some but not all subsets of innate and adaptive lymphocytes, and the subset of ILC making IFN-γ is called ILC1. Other ILC subsets produce cytokines favoring effector modules called type 2 and type 3, which coordinate defense against parasitic and extracellular pathogens, respectively. The modular nature of immune effector functions will be encountered frequently throughout this book. One principle seems to be that activated sensor cells from either the innate or

1	respectively. The modular nature of immune effector functions will be encountered frequently throughout this book. One principle seems to be that activated sensor cells from either the innate or the adaptive immune system can activate different subsets of innate or adaptive lymphocytes that are specialized for amplifying particular effector modules that are directed against different categories of pathogens (Fig. 1.27).

1	the effector mechanisms of immunity. Fig. 1.27 Innate and adaptive lymphocyte cells share a variety of functions. the different effector modules are served by both innate and adaptive immune mechanisms. For each of the four major types of innate lymphocytes, there is a corresponding type of t cell with generally similar functional characteristics. each set of innate lymphocyte and t cell exert an effector activity that is broadly directed at a distinct category of pathogen. 1-20 Antibodies protect against extracellular pathogens and their toxic products. Antibodies are found in plasma—the fluid component of blood—and in extracellular fluids. Because body fluids were once known as humors, immunity mediated by antibodies is known as humoral immunity.

1	Antibodies are found in plasma—the fluid component of blood—and in extracellular fluids. Because body fluids were once known as humors, immunity mediated by antibodies is known as humoral immunity. Antibodies are Y-shaped molecules with two identical antigen-binding sites and one constant, or Fc, region. As mentioned in Section 1-9, there are five forms of the constant region of an antibody, known as the antibody classes or isotypes. The constant region determines an antibody’s functional properties—how it will engage with the effector mechanisms that dispose of antigen once it is recognized. Each class carries out its particular function by engaging a distinct set of effector mechanisms. We describe the antibody classes and their actions in Chapters 5 and 10.

1	The first and most direct way in which antibodies can protect against pathogens or their products is by binding to them and thereby blocking their access to cells that they might infect or destroy (Fig. 1.28, left panels). This is known as neutralization and is important for protection against viruses, which become prevented from entering cells and replicating, and against bacterial toxin and is the form of immunity elicited by most vaccines.

1	For bacteria, however, binding by antibodies is not sufficient to stop their replication. In this case, the function of the antibody is to enable a phagocytic cell such as a macrophage or a neutrophil to ingest and destroy the bacterium. Many bacteria evade the innate immune system because they have an outer coat that is not recognized by the pattern recognition receptors of phagocytes. However, antigens in the coat can be recognized by antibodies, and phagocytes have receptors, called Fc receptors, that bind the constant region and facilitate phagocytosis of the bacterium (see Fig. 1.28, center panels). The coating of pathogens and foreign particles in this way is known as opsonization.

1	The third function of antibodies is complement activation. In Section 1-2 we briefly mentioned Bordet’s discovery of complement as a serum factor that ‘complements’ the activities of antibodies. Complement can be activated by microbial surfaces even without the help of antibodies, which leads to the covalent deposition of certain complement proteins onto the bacterial surface. But when an antibody binds first to the bacterial surface, its constant region provides a platform that is much more efficient in complement activation than the left panels show antibodies binding to and neutralizing a bacterial toxin, thus preventing it from interacting with host cells and causing pathology. unbound toxin can react with receptors on the host cell, whereas the toxin:antibody complex cannot. Antibodies also neutralize complete virus particles and bacterial cells by binding and inactivating them. the antigen:antibody complex is eventually scavenged and degraded by macrophages. Antibodies coating

1	also neutralize complete virus particles and bacterial cells by binding and inactivating them. the antigen:antibody complex is eventually scavenged and degraded by macrophages. Antibodies coating an antigen render it recognizable as foreign by phagocytes (macrophages and neutrophils), which then ingest and destroy it; this is called opsonization. the center panels show opsonization and phagocytosis of a bacterial cell. Antibody first binds to antigens (red) on the bacterial cell through the variable regions. then the antibody’s Fc region binds to Fc receptors (yellow) expressed by macrophages and other phagocytes, facilitating phagocytosis. the right panels show activation of the complement system by antibodies coating a bacterial cell. Bound antibodies form a platform that activates the first protein in the complement system, which deposits complement proteins (blue) on the surface of the bacterium. this can lead in some cases to formation of a pore that lyses the bacterium directly.

1	protein in the complement system, which deposits complement proteins (blue) on the surface of the bacterium. this can lead in some cases to formation of a pore that lyses the bacterium directly. more generally, complement proteins on the bacterium can be recognized by complement receptors on phagocytes; this stimulates the phagocytes to ingest and destroy the bacterium. thus, antibodies target pathogens and their toxic products for disposal by phagocytes.

1	microbial activation alone. Thus, once antibodies are produced, complement activation against a pathogen can be substantially increased.

1	Certain complement components that are deposited on the bacterial surface can directly lyse the membranes of some bacteria, and this is important in a few bacterial infections (see Fig. 1.28, right panels). The major function of complement, however, is to enable phagocytes to engulf and destroy bacteria that the phagocytes would not otherwise recognize. Most phagocytes express receptors that bind certain complement proteins; called complement receptors, these receptors bind to the complement proteins deposited onto the bacterial surface and thus facilitate bacterial phagocytosis. Certain other complement proteins also enhance the phagocytes’ bactericidal capacity. The end result is that all pathogens and free molecules bound by antibody are eventually delivered to phagocytes for ingestion, degradation, and removal from the body (see Fig. 1.28, bottom panels). The complement system and the phagocytes that antibodies recruit are not themselves antigen-specific; they depend upon antibody

1	degradation, and removal from the body (see Fig. 1.28, bottom panels). The complement system and the phagocytes that antibodies recruit are not themselves antigen-specific; they depend upon antibody molecules to mark the particles as foreign.

1	the effector mechanisms of immunity. 1-21 T cells orchestrate cell-mediated immunity and regulate B-cell responses to most antigens. Some bacteria and parasites, and all viruses, replicate inside cells, where they cannot be detected by antibodies, which access only the blood and extracellular space. The destruction of intracellular invaders is the function of the T lymphocytes, which are responsible for the cell-mediated immune responses of adaptive immunity. But T lymphocytes participate in responses to a wide variety of pathogens, including extracellular organisms, and so must exert a wide variety of effector activities.

1	T lymphocytes, of which there are several types, develop in the thymus. They are characterized by the type of T-cell receptors they express and by the expression of certain markers. The two main classes of T cells express either a cell-surface protein called CD8 or another called CD4. These are not just random markers, but are important for a T cell’s function, because they help to determine the interactions between the T cell and other cells. Recall from Section 1-10 that T cells detect peptides derived from foreign antigens that are displayed by MHC molecules on a cell’s surface. CD8 and CD4 function in antigen recognition by recognizing different regions of MHC molecules and by being involved in the signaling of the T-cell receptor that is engaged with its antigen. Thus, CD4 and CD8 are known as co-receptors and they provide a functional difference between CD8 and CD4 T cells,

1	Importantly, there are two main types of MHC molecules, called MHC class I and MHC class II. These have slightly different structures, but both have an elongated groove on the outer surface that can bind a peptide (Fig. 1.29). The peptide becomes trapped in this groove during the synthesis and assembly of the MHC molecule inside the cell, and the peptide:MHC complex is then transported to the cell surface and displayed to T cells (Fig. 1.30). Because CD8 recognizes a region of the MHC class I protein while CD4 recognizes a region of MHC class II protein, the two co-receptors functionally distinguish T cells. Therefore, CD8 T cells selectively recognize peptides that are bound to MHC class I molecules, while CD4 T cells recognize peptides presented by MHC class II.

1	The most direct action of T cells is cytotoxicity. Cytotoxic T cells are effector T cells that act against cells infected with viruses. Antigens derived from the virus multiplying inside the infected cell are displayed on the cell’s surface, where they are recognized by the antigen receptors of cytotoxic T cells. These T cells can then control the infection by directly killing the infected cell before viral replication is complete and new viruses are released (Fig. 1.31). Cytotoxic T cells carry CD8, and so recognize antigen presented by MHC class I molecules. Because MHC class I molecules are expressed on most cells of the body, they serve as an important mechanism to defend against viral infections.

1	Fig. 1.29 MHC molecules on the cell surface display peptide fragments of antigens. mHC molecules are membrane proteins whose outer extracellular domains form a cleft in which a peptide fragment is bound. these fragments are derived from proteins degraded inside the cell and include both self and foreign protein antigens. the peptides are bound by the newly synthesized mHC molecule before it reaches the cell surface. there are two kinds of mHC molecules, mHC class I and mHC class II; they have related but distinct structures and functions. Although not shown here for simplicity, both mHC class I and mHC class II molecules are trimers of two protein chains and the bound self or nonself peptide. Bound peptides transported by MHC class I to the cell surface Viral proteins synthesized in cytosol endoplasmic reticulumcytosol Peptide fragments of viral proteins bound by MHC class I in ER cytosol nucleus Virus infects cell

1	Fig. 1.30 MHC class I molecules present antigen derived from proteins in the cytosol. In cells infected with viruses, viral proteins are synthesized in the cytosol. Peptide fragments of viral proteins are transported into the endoplasmic reticulum (er), where they are bound by mHC class I molecules, which then deliver the peptides to the cell surface.

1	Fig. 1.31 Mechanism of host defense against intracellular infection by viruses. Cells infected by viruses are recognized by specialized t cells called cytotoxic t cells, which kill the infected cells directly. the killing mechanism involves the activation of enzymes known as caspases, which contain cysteine in their active site and cleave target proteins at aspartic acid. the caspases in turn activate a cytosolic nuclease that cleaves host and viral dnA in the infected cell. Panel a is a transmission electron micrograph showing the plasma membrane of a cultured CHo cell (the Chinese hamster ovary cell line) infected with influenza virus. many virus particles can be seen budding from the cell surface. Some of these have been labeled with a monoclonal antibody that is specific for a viral protein and is coupled to gold particles, which appear as the solid black dots in the micrograph. Panel b is a transmission electron micrograph of a virus-infected cell (V) surrounded by cytotoxic

1	protein and is coupled to gold particles, which appear as the solid black dots in the micrograph. Panel b is a transmission electron micrograph of a virus-infected cell (V) surrounded by cytotoxic t lymphocytes. note the close apposition of the membranes of the virus-infected cell and the t cell (t) in the upper left corner of the micrograph, and the clustering of the cytoplasmic organelles in the t cell between its nucleus and the point of contact with the infected cell. Panel a courtesy of m. Bui and

1	A. Helenius; panel b courtesy of n. rooney. MHC class I molecules bearing viral peptides are recognized by CD8-bearing cytotoxic T cells, which then kill the infected cell (Fig. 1.32).

1	CD4 T cells recognize antigen presented by MHC class II proteins, which are expressed by the predominant antigen-presenting cells of the immune system: dendritic cells, macrophages, and B cells (Fig. 1.33). Thus CD4 T cells tend to recognize antigens taken up by phagocytosis from the extracellular environment. CD4 T cells are the helper T cells mentioned earlier in the chapter. They develop into a variety of different effector subsets, called TH1 (for T helper type 1), TH2, TH17, and so on, and they produce cytokines in patterns similar to the subsets of ILCs mentioned earlier that activate effector modules protective against different pathogens. These subsets act primarily at sites of infection or injury in peripheral tissues. In the lymphoid tissues, a subset of CD4 T cells, called the T follicular helper (TFH) cell, interacts with B cells to regulate antibody production during the immune response. The various T helper subsets are described later, in Chapter 9.

1	For example, the TH1 subset of CD4 T cells helps to control certain bacteria that take up residence in membrane-enclosed vesicles inside macrophages. They produce the same cytokine as ILC1 cells, IFN-γ, which activates macrophages to increase their intracellular killing power and destroy these bacteria. Important infections that are controlled by this function are tuberculosis and leprosy, which are caused by the bacteria Mycobacterium tuberculosis and M. leprae, respectively. Mycobacteria survive intracellularly because they prevent the vesicles they occupy from fusing with lysosomes, which contain a variety of degradative enzymes and antimicrobial substances (Fig. 1.34). However, on its surface, the infected macrophage presents mycobacteriaderived antigens that can be recognized by activated antigen-specific TH1 cells, which in turn secrete particular cytokines that induce the macrophage to the effector mechanisms of immunity.

1	Fig. 1.33 CD4 T cells recognize antigen presented by MHC class II molecules. on recognition of their specific antigen on infected macrophages, tH1 cells activate the macrophage, leading to the destruction of the intracellular bacteria (top panel). When t follicular helper (tFH) cells recognize antigen on B cells (bottom panel), they activate these cells to proliferate and differentiate into antibody-producing plasma cells (not shown). overcome the block on vesicle fusion. TH2 and TH17 subsets produce cytokines that are specialized for promoting responses against parasites or extracellular bacteria and fungi, respectively. CD4 T cells, and their specialized subsets, play a pervasive role in adaptive immunity, and we will be returning to them many times in this book, including in Chapters 8, 9, 11 and 12. 1-22 Inherited and acquired defects in the immune system result in increased susceptibility to infection.

1	We tend to take for granted the ability of our immune systems to free our bodies of infection and prevent its recurrence. In some people, however, parts of the immune system fail. In the most severe of these immunodeficiency diseases, adaptive immunity is completely absent, and death occurs in infancy from overwhelming infection unless heroic measures are taken. Other less catastrophic failures lead to recurrent infections with particular types of pathogens, depending on the particular deficiency. Much has been learned about the functions of the different components of the human immune system through the study of these immunodeficiencies, many of which are caused by inherited genetic defects. Because understanding the features of immunodeficiencies requires a detailed knowledge of normal immune mechanisms, we have postponed discussion of most of these diseases until Chapter 13, where they can be considered together.

1	More than 30 years ago, a devastating form of immunodeficiency appeared, the acquired immune deficiency syndrome, or AIDS, which is caused by an infectious agent, the human immunodeficiency viruses HIV-1 and HIV-2. This disease destroys T cells, dendritic cells, and macrophages bearing CD4, leading to infections caused by intracellular bacteria and other pathogens

1	Fig. 1.34 Mechanism of host defense against intracellular infection by mycobacteria. mycobacteria are engulfed by macrophages but resist being destroyed by preventing the intracellular vesicles in which they reside from fusing with lysosomes containing bactericidal agents. thus the bacteria are protected from being killed. In resting macrophages, mycobacteria persist and replicate in these vesicles. When the phagocyte is recognized and activated by a tH1 cell, however, the phagocytic vesicles fuse with lysosomes, and the bacteria can be killed. macrophage activation is controlled by tH1 cells, both to avoid tissue damage and to save energy. the light micrographs (bottom row) show resting (left) and activated (right) macrophages infected with mycobacteria. the cells have been stained with an acid-fast red dye to reveal mycobacteria. these are prominent as red-staining rods in the resting macrophages but have been eliminated from the activated macrophages. Photographs courtesy of g.

1	an acid-fast red dye to reveal mycobacteria. these are prominent as red-staining rods in the resting macrophages but have been eliminated from the activated macrophages. Photographs courtesy of g. Kaplan.

1	Fig. 1.35 Immune responses can be beneficial or harmful, depending on the nature of the antigen. Beneficial responses are shown in white, harmful responses in red shaded boxes. Where the response is beneficial, its absence is harmful. normally controlled by such cells. These infections are the major cause of death from this increasingly prevalent immunodeficiency disease, which is discussed fully in Chapter 13 together with the inherited immunodeficiencies. 1-23 Understanding adaptive immune responses is important for the control of allergies, autoimmune disease, and the rejection of transplanted organs.

1	The main function of our immune system is to protect the human host from infectious agents. However, many medically important diseases are associated with a normal immune response directed against an inappropriate antigen, often in the absence of infectious disease. Immune responses directed at noninfectious antigens occur in allergy, in which the antigen is an innocuous foreign substance; in autoimmune disease, in which the response is to a self antigen; and in graft rejection, in which the antigen is borne by a transplanted foreign cell (discussed in Chapter 15). The major antigens provoking graft rejection are, in fact, the MHC molecules, as each of these is present in many different versions in the human population—that is, they are highly polymorphic—and most unrelated people differ in the set of MHC molecules they express, a property commonly known as their ‘tissue type.’ The MHC was originally recognized by the work of Peter Goren in the 1930s as a gene locus in mice, the H-2

1	in the set of MHC molecules they express, a property commonly known as their ‘tissue type.’ The MHC was originally recognized by the work of Peter Goren in the 1930s as a gene locus in mice, the H-2 locus, that controlled the acceptance or rejection of transplanted tumors, and later by George Snell, who examined their role in tissue transplantation by developing mouse strains differing only at these histocompatibility loci. The human MHC molecules were first discovered during the Second World War, when attempts were made to use skin grafts from donors to repair badly burned pilots and bomb victims. The patients rejected the grafts, which were recognized by their immune systems as being ‘foreign.’ What we call a successful immune response or a failure, and whether the response is considered harmful or beneficial to the host, depends not on the response itself but rather on the nature of the antigen and the circumstances in which the response occurs (Fig. 1.35). Snell was awarded the

1	harmful or beneficial to the host, depends not on the response itself but rather on the nature of the antigen and the circumstances in which the response occurs (Fig. 1.35). Snell was awarded the 1980 Nobel Prize for his work on MHC, together with Baruj Benacerraf and Jean Dausset.

1	Allergic diseases, which include asthma, are an increasingly common cause of disability in the developed world. Autoimmunity is also now recognized as the cause of many important diseases. An autoimmune response directed against pancreatic β cells is the leading cause of diabetes in the young. In allergies and autoimmune diseases, the powerful protective mechanisms of the adaptive immune response cause serious damage to the patient. Immune responses to harmless antigens, to body tissues, or to organ grafts are, like all other immune responses, highly specific. At present, the usual way to treat these responses is with immunosuppressive drugs, which inhibit all the effector mechanisms of immunity.

1	immune responses, desirable and undesirable alike. If it were possible to suppress only those lymphocyte clones responsible for the unwanted response, the disease could be cured or the grafted organ protected without impeding protective immune responses. At present, antigen-specific immunoregulation is outside the reach of clinical treatment. But as we shall see in Chapter 16, many new drugs have been developed recently that offer more selective immune suppression to control autoimmune and other unwanted immune responses. Among these, therapies using highly specific monoclonal antibodies were made possible by Georges Köhler and César Milstein, who shared the 1984 Nobel Prize for the discovery of their production. We shall discuss the present state of understanding of allergies, autoimmune disease, graft rejection, and immunosuppressive drugs and monoclonal antibodies in Chapters 14–16, and we shall see in Chapter 15 how the mechanisms of immune regulation are beginning to emerge from

1	disease, graft rejection, and immunosuppressive drugs and monoclonal antibodies in Chapters 14–16, and we shall see in Chapter 15 how the mechanisms of immune regulation are beginning to emerge from a better understanding of the functional subsets of lymphocytes and the cytokines that control them.

1	1-24 Vaccination is the most effective means of controlling infectious diseases. The deliberate stimulation of an immune response by immunization, or vaccination, has achieved many successes in the two centuries since Jenner’s pioneering experiment. Mass immunization programs have led to the virtual eradication of several diseases that used to be associated with significant morbidity (illness) and mortality (Fig. 1.36). Immunization is considered so safe 100 10 1.0 0.1 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 vaccine inactivated vaccine oral vaccine vaccine measles SSPE 40 30 20 10 0 1940 1950 1960 1970 1980 1990 600 500 400 300 200 100 0 1960 1965 1970 1975 1980 1985 1990 Reported cases per 100,000 population Reported cases per 100,000 population Reported cases per 100,000 population Reported SSPE cases in USA 60 50 40 30 20 10 0 DiphtheriaPolioMeasles

1	Fig. 1.36 Successful vaccination campaigns. diphtheria, polio, and measles and their consequences have been virtually eliminated in the united States, as shown in these three graphs. SSPe stands for subacute sclerosing panencephalitis, a brain disease that is a late consequence of measles infection in a few patients. When measles was prevented, SSPe disappeared 15–20 years later. However, because these diseases have not been eradicated worldwide, immunization must be maintained in a very high percentage of the population to prevent their reappearance.

1	and so important that most states in the United States require children to be immunized against up to seven common childhood diseases. Impressive as these accomplishments are, there are still many diseases for which we lack effective vaccines. And even where vaccines for diseases such as measles can be used effectively in developed countries, technical and economic problems can prevent their widespread use in developing countries, where mortality from these diseases is still high. The tools of modern immunology and molecular biology are being applied to develop new vaccines and improve old ones, and we discuss these advances in Chapter 16. The prospect of controlling these important diseases is tremendously exciting. The guarantee of good health is a critical step toward population control and economic development. At a cost of pennies per person, great hardship and suffering can be alleviated.

1	Many serious pathogens have resisted efforts to develop vaccines against them, often because they can evade or subvert the protective mechanisms of an adaptive immune response. We examine some of the evasive strategies used by successful pathogens in Chapter 13. The conquest of many of the world’s leading diseases, including malaria and diarrheal diseases (the leading killers of children) as well as the more recent threat from AIDS, depends on a better understanding of the pathogens that cause them and their interactions with the cells of the immune system. Summary.

1	The responses to infection can be organized into several effector modules that target the various types of pathogen lifestyles. Innate sensor cells that detect infection generate mediators that activate innate lymphoid cells (ILCs) and T cells, which amplify the immune response and also activate various effector modules. Innate lymphoid cells include subsets that produce different cytokines and activate distinct effector modules. T cells fall into two major classes that are based on the expression of the co-receptors CD8 and CD4; these T cells recognize antigen presented by MHC class I or MHC class II proteins, respectively. These subsets of T cells, like their ILC counterparts, also promote the actions of distinct effector modules. NK cells and CD8 T cells can exert cytotoxic activity to target intracellular infections such as viruses. Other subsets of innate lymphoid and helper T cells can secrete mediators that activate other effector functions, ones that target intracellular

1	to target intracellular infections such as viruses. Other subsets of innate lymphoid and helper T cells can secrete mediators that activate other effector functions, ones that target intracellular bacteria, extracellular bacteria and fungi, and parasites. T cells also provide signals that help regulate B cells and stimulate them to produce antibodies. Specific antibodies mediate the clearance and elimination of soluble toxins and extracellular pathogens. They interact not only with the toxins or the antigens on microbes, but also with the Fc region of specific receptors that are expressed by many types of phagocytes. Phagocytes also express receptors for complement proteins that are deposited on microbial surfaces, particularly in the presence of antibody.

1	Failures of immunity can be caused by genetic defects or by infections that target important components of the immune system. Misdirected immune responses can damage host tissues, as in autoimmune diseases or allergy, or lead to the failure of transplanted organs. While vaccination is still the greatest tool of immunology to fight diseases, modern approaches have added new tools, such as monoclonal antibodies, that have become progressively more important in the clinic over the past two decades. Summary to Chapter 1. The immune system defends the host against infection. Innate immunity serves as a first line of defense but lacks the ability to recognize certain pathogens and Questions.

1	to provide the specific protective immunity that prevents reinfection. Adaptive immunity is based on clonal selection from a repertoire of lymphocytes bearing highly diverse antigen-specific receptors that enable the immune system to recognize any foreign antigen. In the adaptive immune response, antigen-specific lymphocytes proliferate and differentiate into clones of effector lymphocytes that eliminate the pathogen. Figure1.7 summarizes the phases of the immune response and their approximate timings. Host defense requires different recognition systems and a wide variety of effector mechanisms to seek out and destroy the wide variety of pathogens in their various habitats within the body and at its external and internal surfaces. Not only can the adaptive immune response eliminate a pathogen, but, in the process, it also generates increased numbers of differentiated memory lymphocytes through clonal selection, and this allows a more rapid and effective response upon reinfection. The

1	but, in the process, it also generates increased numbers of differentiated memory lymphocytes through clonal selection, and this allows a more rapid and effective response upon reinfection. The regulation of immune responses, whether to suppress them when unwanted or to stimulate them in the prevention of infectious disease, is the major medical goal of research in immunology.

1	Questions. 1.1 Multiple Choice: Which of the following examples can be considered an illustration of vaccination? A. Inoculating an individual with cowpox in order to protect that individual against smallpox B. Administering the serum of animals immune to diphtheria to protect against the effect of diphtheria toxin in an exposed individual C. A bacterial infection that results in complement activation and destruction of the pathogen D. An individual that becomes ill with chickenpox, but does not develop it again due to the development of immunologic memory 1.2 Multiple Choice: Which of the following is an appropriate definition for immunological memory? A. the mechanism by which an organism prevents the development of an immune response against the host’s own tissues B. the mechanism by which an organism prevents exposure to microbes

1	A. the mechanism by which an organism prevents the development of an immune response against the host’s own tissues B. the mechanism by which an organism prevents exposure to microbes C. the persistence of pathogen-specific antibodies and lymphocytes after the original infection has been eliminated so that reinfection can be prevented D. the process of reducing or eliminating a pathogen 1.3 True or False: toll-like receptors (tlrs) recognize intracellular bacteria, while nod-like receptors (nlrs) recognize extracellular bacteria. 1.4 Matching: Classify the following as lymphoid or myeloid in origin: A. eosinophils B. B cells C. neutrophils D. nK cells E. mast cells F. macrophages

1	1.4 Matching: Classify the following as lymphoid or myeloid in origin: A. eosinophils B. B cells C. neutrophils D. nK cells E. mast cells F. macrophages G. red blood cells 1.5 Multiple Choice: the immunologist’s ‘dirty little secret’ involves the addition of microbial constituents in order to stimulate a strong immune response against the desired protein antigen of interest. Which of the following is not a receptor or receptor family that can recognize microbial products in order to achieve a potent immune response? A. toll-like receptors (tlrs) B. t-cell antigen receptor (tCr) C. nod-like receptors (nlrs) D. Pattern recognition receptors (Prrs) 1.6 True or False: Hematopoietic stem cells can develop into any cell type in the body. 1.7 Matching: match each of the following terms to the numbered phrase that describes it best: A. Allergy __ 1. Immunological response to an antigen present on a transplanted foreign cell

1	1.7 Matching: match each of the following terms to the numbered phrase that describes it best: A. Allergy __ 1. Immunological response to an antigen present on a transplanted foreign cell B. Immunological 2. Immunological response tolerance __ to an antigen that is an innocuous foreign substance C. Autoimmune 3. Immunological process disease __ that prevents an immune response to self antigens D. graft rejection __ 4. Immunological response to a self antigen 1.8 Multiple Choice: Which of the following processes is not a mechanism of maintaining immunologic tolerance? A. Clonal deletion B. Anergy C. Clonal expansion D. Suppression of self-reactive lymphocytes 1.9 Matching: Classify each of the following as a central/ primary or peripheral/secondary lymphoid organ: A. Bone marrow B. lymph node C. Spleen D. thymus E. Appendix 1.10 Matching: match the following region, structure, or subcompartments with the numbered organ they are present in: A. lymph node __ B. Spleen __

1	B. lymph node C. Spleen D. thymus E. Appendix 1.10 Matching: match the following region, structure, or subcompartments with the numbered organ they are present in: A. lymph node __ B. Spleen __ C. mucosa of the small intestine __ 1. 2. 3. 1.11 Multiple Choice: Which of the following events do not occur during inflammation? A. Cytokine secretion B. Chemokine secretion C. recruitment of innate immune cells D. Constriction of blood vessels 1.12 Fill-in-the-Blanks: ___________ t cells are able to kill infected cells, while ________ t cells activate other cells of the immune system. 1.13 True or False: Both t-cell and B-cell receptors undergo the process of affinity maturation in order to acquire progressively higher affinity for an antigen during an immune response. 1.14 True or False: each lymphocyte carries cell-surface receptors with multiple antigen specificity.

1	1.14 True or False: each lymphocyte carries cell-surface receptors with multiple antigen specificity. 1.15 Multiple Choice: Which cell type forms an important link between the innate immune response and the adaptive immune response? A. dendritic cell B. neutrophil C. B cell D. Innate lymphoid cell (IlC) 1.16 Multiple Choice: Which of the following options is not a mechanism by which an antibody can protect against a pathogen? A. neutralization B. Co-stimulation of t cells C. opsonization D. Complement activation/deposition 1.17 True or False: tH2 cells do not possess mHC class I molecules. General references. Burnet, F.M.: The Clonal Selection Theory of Acquired Immunity. London: Cambridge University Press, 1959. Gowans, J.L.: The lymphocyte—a disgraceful gap in medical knowledge. Immunol. Today 1996, 17:288–291. Landsteiner, K.: The Specificity of Serological Reactions, 3rd ed. Boston: Harvard University Press, 1964.

1	Landsteiner, K.: The Specificity of Serological Reactions, 3rd ed. Boston: Harvard University Press, 1964. Metchnikoff, É.: Immunity in the Infectious Diseases, 1st ed. New York: Macmillan Press, 1905. Silverstein, A.M.: History of Immunology, 1st ed. London: Academic Press, 1989. Alberts, B., Johnson, A., Lewis, J., Morgan, D., Raff, M., Roberts, K., and Walter, P.: Molecular Biology of the Cell, 6th ed. New York: Garland Science, 2015. Berg, J.M., Stryer, L., and Tymoczko, J.L.: Biochemistry, 5th ed. New York: W.H. Freeman, 2002. Geha, R.S., and Notarangelo, L.D.: Case Studies in Immunology: A Clinical Companion, 7th ed. New York: Garland Science, 2016. Harper, D.R.: Viruses: Biology, Applications, Control. New York: Garland Science, 2012. Kaufmann, S.E., Sher, A., and Ahmed, R. (eds): Immunology of Infectious Diseases. Washington, DC: ASM Press, 2001.

1	Kaufmann, S.E., Sher, A., and Ahmed, R. (eds): Immunology of Infectious Diseases. Washington, DC: ASM Press, 2001. Lodish, H., Berk, A., Kaiser, C.A., Krieger, M., Scott, M.P., Bretscher, A., Ploegh, H., and Matsudaira, P.: Molecular Cell Biology, 6th ed. New York: W.H. Freeman, 2008. Lydyard, P., Cole, M., Holton, J., Irving, W., Porakishvili, N., Venkatesan, P., and Ward, K.: Case Studies in Infectious Disease. New York: Garland Science, 2009. Mims, C., Nash, A., and Stephen, J.: Mims’ Pathogenesis of Infectious Disease, 5th ed. London: Academic Press, 2001. Ryan, K.J. (ed): Medical Microbiology, 3rd ed. East Norwalk, CT: Appleton-Lange, 1994. Advanced textbooks in immunology, compendia, etc. Lachmann, P.J., Peters, D.K., Rosen, F.S., and Walport, M.J. (eds): Clinical Aspects of Immunology, 5th ed. Oxford: Blackwell Scientific Publications, 1993. Mak, T.W., and Saunders, M.E.: The Immune Response: Basic and Clinical Principles. Burlington: Elsevier/Academic Press, 2006.

1	Mak, T.W., and Saunders, M.E.: The Immune Response: Basic and Clinical Principles. Burlington: Elsevier/Academic Press, 2006. Mak, T.W., and Simard, J.J.L.: Handbook of Immune Response Genes. New York: Plenum Press, 1998. Paul, W.E. (ed): Fundamental Immunology, 7th ed. New York: Lippincott Williams & Wilkins, 2012. Roitt, I.M., and Delves, P.J. (eds): Encyclopedia of Immunology, 2nd ed. (4 vols.). London and San Diego: Academic Press, 1998. Innate Immunity: The First Lines of Defense

1	Innate Immunity: The First Lines of Defense As introduced in Chapter 1, most microbial invaders can be detected and destroyed within minutes or hours by the body’s defense mechanisms of innate immunity, which do not rely on expansion of antigen-specific lymphocytes. The innate immune system uses a limited number of secreted proteins and cell-associated receptors to detect infection and to distinguish between pathogens and host tissues. These are called innate receptors because they are inborn; they are encoded by genes directly inherited from an individual’s parents, and do not need to be generated by the gene rearrangements used to assemble antigen receptors of lymphocytes described in Section 1-11. The importance of innate immunity is illustrated by several immunodeficiencies that result when it is impaired, discussed in Chapter 13, which increase susceptibility to infection even in the presence of an intact adaptive immune system.

1	As we saw in Fig. 1.5, an infection starts when a pathogen breaches one of the host’s anatomic barriers. Some innate immune mechanisms start acting immediately (Fig. 2.1). These immediate defenses include several classes of preformed soluble molecules that are present in extracellular fluid, blood, and epithelial secretions and that can either kill the pathogen or weaken its effect. Antimicrobial enzymes such as lysozyme begin to digest bacterial cell walls; antimicrobial peptides such as the defensins lyse bacterial cell membranes directly; and a system of plasma proteins known as the complement system targets pathogens both for lysis and for phagocytosis by cells of the innate immune system such as macrophages. If these fail, innate immune cells become activated by pattern recognition receptors (PRRs) that detect molecules called pathogen-associated molecular patterns (PAMPs) (see Section 1-5) that are typical of microbes. The activated innate cells can engage various effector

1	receptors (PRRs) that detect molecules called pathogen-associated molecular patterns (PAMPs) (see Section 1-5) that are typical of microbes. The activated innate cells can engage various effector mechanisms to eliminate the infection. By themselves, neither the soluble nor the cellular components of innate immunity generate long-term protective immunological memory. Only if an infectious organism breaches these first two lines of defense will mechanisms be engaged to induce an adaptive immune response—the third phase of the response to a pathogen. This leads to the expansion of antigen-specific lymphocytes that target the pathogen specifically and to the formation of memory cells that provide long-lasting specific immunity.

1	Fig. 2.1 The response to an initial infection occurs in three phases. These are the innate phase, the early induced innate response, and the adaptive immune response. The first two phases rely on the recognition of pathogens by germline-encoded receptors of the innate immune system, whereas adaptive immunity uses variable antigen-specific receptors that are produced as a result of gene segment rearrangements. Adaptive immunity occurs late, because the rare B cells and T cells specific for the invading pathogen must first undergo clonal expansion before they differentiate into effector cells that migrate to the site of infection and clear the infection. The effector mechanisms that remove the infectious agent are similar or identical in each phase. Anatomic barriers and initial chemical defenses. The complement system and innate immunity.

1	Anatomic barriers and initial chemical defenses. The complement system and innate immunity. Clonal expansion and differentiation to effector cells Removal of infectious agent Recognition by naive B and T cells Adaptive immune response (late: >96 hours) Transport of antigen to lymphoid organs Recognition of PAMPs. Activation of effector cells and infammation Removal of infectious agent Early induced innate response (early: 4–96 hours) Recruitment of effector cells FAIL Recognition by preformed nonspecifc and broadly specifc effectors Removal of infectious agent Infection FAIL Containment by an anatomic barrier Prevention of infection Pathogen FAIL

1	This chapter considers the first phase of the innate immune response. We first describe the anatomic barriers that protect the host against infection and examine the immediate innate defenses provided by various secreted soluble proteins. The anatomic barriers are fixed defenses against infection and consist of the epithelia that line the internal and external surfaces of the body along with the phagocytes residing beneath all epithelial surfaces. These phagocytes act directly by engulfing and digesting invading microorganisms. Epithelia are also protected by many kinds of chemical defenses, including antimicrobial enzymes and peptides. Next we describe the complement system, which directly kills some microorganisms and interacts with others to promote their removal by phagocytic cells. The complement system together with other soluble circulating defensive proteins is sometimes referred to as humoral innate immunity, from the old word ‘humor’ for body fluids. If these early defenses

1	complement system together with other soluble circulating defensive proteins is sometimes referred to as humoral innate immunity, from the old word ‘humor’ for body fluids. If these early defenses fail, the phagocytes at the site of infection help recruit new cells and circulating effector molecules, a process called inflammation, which we will discuss in Chapter 3.

1	Anatomic barriers and initial chemical defenses.

1	Microorganisms that cause disease in humans and animals enter the body at different sites and produce disease symptoms by a variety of mechanisms. Microorganisms that cause disease and produce damage, or pathology, to tissues are referred to as pathogenic microorganisms, or simply pathogens. As innate immunity eliminates most microorganisms that may occasionally cross an anatomic barrier, pathogens are microorganisms that have evolved ways of overcoming the body’s innate defenses more effectively than other microorganisms. Once infection is established, both innate and adaptive immune responses are typically required to eliminate pathogens from the body. Even in these cases, the innate immune system performs a valuable function by reducing pathogen numbers during the time needed for the adaptive immune system to gear up for action. In the first part of this chapter we briefly describe the different types of pathogens and their invasive strategies, and then examine the immediate innate

1	immune system to gear up for action. In the first part of this chapter we briefly describe the different types of pathogens and their invasive strategies, and then examine the immediate innate defenses that, in most cases, prevent microorganisms from establishing an infection.

1	2-1 Infectious diseases are caused by diverse living agents that replicate in their hosts. The agents that cause disease fall into five groups: viruses, bacteria, fungi, protozoa, and helminths (worms). Protozoa and worms are usually grouped together as parasites, and are the subject of the discipline of parasitology, whereas viruses, bacteria, and fungi are the subject of microbiology. Fig. 2.2 lists some examples of the different classes of microorganisms and parasites, and the diseases they cause. The characteristic features of each pathogen are its mode of transmission, its mechanism of replication, its mechanism of pathogenesis—the means by which it causes disease—and the response it elicits from the host. The distinct pathogen habitats and life cycles mean that a range of different innate and adaptive immune mechanisms have to be deployed for pathogen destruction.

1	Infectious agents can grow in all body compartments, as shown schematically in Fig. 2.3. We saw in Chapter 1 that two major compartments can be defined—extracellular and intracellular. Both innate and adaptive immune responses have different ways of dealing with pathogens found in these two Anatomic barriers and initial chemical defenses.

1	Physical contact Trichophyton Bacillus anthracis Clostridium tetani Francisella tularensis Athlete’s foot Mosquito bites (Aedes aegypti) Deer tick bites Mosquito bites (Anopheles) Flavivirus Borrelia burgdorferi Plasmodium spp. Yellow fever Lyme disease Malaria Minor skin abrasions Puncture wounds Handling infected animals Cutaneous anthrax Tetanus Tularemia Route of entry Mode of transmission Pathogen Disease External epithelia Mucosal surfaces Routes of infection for pathogens Mouth and respiratory tract Contaminated water or food Sexual transmission/ infected blood Sexual transmission Neisseria meningitidis Bacillus anthracis Meningococcal meningitis Inhalation anthrax Fungus Virus Bacterium (spirochete) Protozoan Gram-positive bacterium Gram-positive bacterium Gram-negative bacterium Type of pathogen Hepatitis B virus Hepatitis B Hepadnavirus Neisseria gonorrhoeae Gonorrhea Gram-negative bacterium Human immunodefciency virus (HIV) Acquired immunodefciency syndrome (AIDS)

1	Type of pathogen Hepatitis B virus Hepatitis B Hepadnavirus Neisseria gonorrhoeae Gonorrhea Gram-negative bacterium Human immunodefciency virus (HIV) Acquired immunodefciency syndrome (AIDS) Retrovirus Gram-negative bacterium Gram-positive bacterium Wounds and abrasions Insect bites Gastrointestinal tract Reproductive tract and other routes External surface Resident microbiota Candida albicans Candidiasis, thrush Opportunistic infections Fungus Resident lung microbiota Pneumocystis jirovecii Pneumonia Fungus Measles virus Measles Paramyxovirus Infuenza virus Infuenza Orthomyxovirus Varicella-zoster Chickenpox Herpesvirus Epstein–Barr virus Mononucleosis Herpesvirus Streptococcus pyogenes Tonsillitis Gram-positive bacterium Haemophilus infuenzae Pneumonia, meningitis Gram-negative bacterium Rotavirus Diarrhea Rotavirus Hepatitis A Jaundice Picornavirus Salmonella enteritidis, Food poisoning Gram-negative bacteriumS. typhimurium Vibrio cholerae Cholera Gram-negative bacterium Inhalation

1	Rotavirus Diarrhea Rotavirus Hepatitis A Jaundice Picornavirus Salmonella enteritidis, Food poisoning Gram-negative bacteriumS. typhimurium Vibrio cholerae Cholera Gram-negative bacterium Inhalation or ingestion of infective material (e.g., saliva droplets) Salmonella typhi Trichuris trichiura Typhoid fever Trichuriasis Gram-negative bacterium Helminth Treponema pallidum Syphilis Bacterium (spirochete) Spores

1	Fig. 2.2 A variety of microorganisms can cause disease. Pathogenic organisms are of five main types: viruses, bacteria, fungi, protozoa, and worms. Some well-known pathogens are listed. Virtually all pathogens have an extracellular phase in which they are vulnerable to the circulating molecules and cells of innate immunity and to the antibodies of the adaptive immune response. All these clear the microorganism mainly by promoting its uptake and destruction by the phagocytes of the immune system. Intracellular phases of pathogens such as viruses are not accessible to these mechanisms; instead, the infected cell is attacked by the NK cells of innate immunity or by the cytotoxic T cells of adaptive immunity. Activation of macrophages as a result of NK-cell or T-cell activity can induce the macrophage to kill pathogens that are living inside macrophage vesicles.

1	compartments. Many bacterial pathogens live and replicate in extracellular spaces, either within tissues or on the surface of the epithelia that line body cavities. Extracellular bacteria are usually susceptible to killing by phagocytes, an important arm of the innate immune system, but some pathogens, such as Staphylococcus and Streptococcus species, are protected by a polysaccharide capsule that resists engulfment. This can be overcome to some extent by the help of another component of innate immunity—complement—which renders the bacteria more susceptible to phagocytosis. In the adaptive immune response, bacteria are rendered more susceptible to phagocytosis by a combination of antibodies and complement.

1	Infectious diseases differ in their symptoms and outcome depending on where the causal pathogen replicates within the body—the intracellular or the extracellular compartment—and what damage it does to the tissues (Fig. 2.4). Pathogens that live intracellularly frequently cause disease by damaging or killing the cells they infect. Obligate intracellular pathogens, such as viruses, must invade host cells to replicate. Facultative intracellular pathogens, such as mycobacteria, can replicate either intracellularly or outside the cell. Two strategies of innate immunity defend against intracellular pathogens. One is to destroy pathogens before they infect cells. To this end, innate immunity includes soluble defenses such as antimicrobial peptides, as well as phagocytic cells that can engulf and destroy pathogens before they become intracellular. Alternatively, the innate immune system can recognize and kill cells infected by some pathogens. This is the role of the natural killer cells (NK

1	destroy pathogens before they become intracellular. Alternatively, the innate immune system can recognize and kill cells infected by some pathogens. This is the role of the natural killer cells (NK cells), which are instrumental in keeping certain viral infections in check before cytotoxic T cells of the adaptive immune system become functional. Intracellular pathogens can be subdivided further into those that replicate freely in the cell, such as viruses and certain bacteria (for example, Chlamydia, Rickettsia, and Listeria), and those that replicate inside intracellular vesicles, such as mycobacteria. Pathogens that live inside macrophage vesicles may become more susceptible to being killed after activation of the macrophage as a result of NK-cell or T-cell actions (see Fig. 2.3).

1	Many of the most dangerous extracellular bacterial pathogens cause disease by releasing protein toxins; these secreted toxins are called exotoxins (see Fig. 2.4). The innate immune system has little defense against such toxins, Anatomic barriers and initial chemical defenses.

1	Anatomic barriers and initial chemical defenses. Direct mechanisms of tissue damage by pathogens Direct cytopathic effect Variola Varicella-zoster Hepatitis B virus Polio virus Measles virus Infuenza virus Herpes simplex virus Human herpes virus 8 (HHV8) Smallpox Chickenpox, shingles Hepatitis Poliomyelitis Measles, subacute sclerosing panencephalitis Infuenza Cold Kaposi's sarcoma sores Exotoxin production Streptococcus pyogenes Staphylococcus aureus Corynebacterium diphtheriae Clostridium tetani Vibrio cholerae Tonsillitis, scarlet fever Boils, toxic shock syndrome, food poisoning Diphtheria Tetanus Cholera Endotoxin Escherichia coli Haemophilus infuenzae Salmonella typhi Shigella Pseudomonas aeruginosa Yersinia pestis Gram-negative sepsis Meningitis, pneumonia Typhoid fever Bacillarydysentery Wound infection Plague

1	Fig. 2.4 Pathogens can damage tissues in a variety of different ways. The mechanisms of damage, representative infectious agents, and the common names of the diseases associated with each are shown. Exotoxins are released by microorganisms and act at the surface of host cells, for example, by binding to receptors. Endotoxins, which are intrinsic components of microbial structure, trigger phagocytes to release cytokines that produce local or systemic symptoms. Many pathogens are cytopathic, directly damaging the cells they infect. Finally, an adaptive immune response to the pathogen can generate antigen:antibody complexes that activate neutrophils and macrophages, antibodies that can cross-react with host tissues, or T cells that kill infected cells. All of these have some potential to damage the host’s tissues. In addition, neutrophils, the most abundant cells early in infection, release many proteins and small-molecule inflammatory mediators that both control infection and cause

1	damage the host’s tissues. In addition, neutrophils, the most abundant cells early in infection, release many proteins and small-molecule inflammatory mediators that both control infection and cause tissue damage.

1	and highly specific antibodies produced by the adaptive immune system are required to neutralize their action (see Fig. 1.28). The damage caused by a particular infectious agent also depends on where it grows; Streptococcus pneumoniae in the lung causes pneumonia, for example, whereas in the blood it causes a potentially fatal systemic illness, pneumococcal sepsis. In contrast, nonsecreted constituents of bacterial structure that trigger phagocytes to release cytokines with local and systemic effects are called endotoxins. An endotoxin of major medical importance is the lipopolysaccharide (LPS) of the outer cell membrane of Gram-negative bacteria, such as Salmonella. Many of the clinical symptoms of infection by such bacteria—including fever, pain, rash, hemorrhage, septic shock—are due largely to LPS.

1	Most pathogenic microorganisms can overcome innate immune responses and continue to grow, making us ill. An adaptive immune response is required to eliminate them and to prevent subsequent reinfection. Certain pathogens are never entirely eliminated by the immune system, and persist in the body for years. But most pathogens are not universally lethal. Those that have lived for thousands of years in the human population are highly evolved to exploit their human hosts; they cannot alter their pathogenicity without upsetting the compromise they have achieved with the human immune system. Rapidly killing every host it infects is no better for the long-term survival of a pathogen than being wiped out by the immune response before the microbe has had time to infect someone else. In short, we have adapted to live with many microbes, and they with us. Nevertheless, the recent concern about highly pathogenic strains of avian influenza and the episode in 2002–2003 of SARS (severe acute

1	we have adapted to live with many microbes, and they with us. Nevertheless, the recent concern about highly pathogenic strains of avian influenza and the episode in 2002–2003 of SARS (severe acute respiratory syndrome), a severe pneumonia in humans that is caused by a coronavirus from bats, remind us that new and deadly infections can transfer from animal reservoirs to humans. Such transmission appears responsible for the Ebola virus epidemic in West Africa in 2014–2015. These are known as zoonotic infections—and we must be on the alert at all times for the emergence of new pathogens and new threats to health. The human immunodeficiency virus that causes AIDS (discussed in Chapter 13) serves as a warning that we remain constantly vulnerable.

1	2-2 Epithelial surfaces of the body provide the first barrier against infection. Our body surfaces are defended by epithelia, which impose a physical barrier between the internal milieu and the external world that contains pathogens. Epithelia comprise the skin and the linings of the body’s tubular structures— the respiratory, urogenital, and gastrointestinal tracts. Epithelia in these locations are specialized for their particular functions and possess unique innate defense strategies against the microbes they typically encounter (Fig. 2.5 and Fig. 2.6).

1	Epithelial cells are held together by tight junctions, which effectively form a seal against the external environment. The internal epithelia are known as mucosal epithelia because they secrete a viscous fluid called mucus, which contains many glycoproteins called mucins. Mucus has a number of protective functions. Microorganisms coated in mucus may be prevented from adhering to the epithelium, and in the respiratory tract, microorganisms can be expelled in the outward flow of mucus driven by the beating of cilia on the mucosal epithelium (Fig. 2.7). The importance of mucus flow in clearing infection is illustrated by people with the inherited disease cystic fibrosis, in which the mucus becomes abnormally thick and dehydrated due to defects in a gene, CFTR, encoding a chloride channel in the epithelium. Such individuals frequently develop lung infections caused by bacteria that colonize the epithelial surface but do not cross it (see Fig. 2.7). In the gut, peristalsis is an important

1	the epithelium. Such individuals frequently develop lung infections caused by bacteria that colonize the epithelial surface but do not cross it (see Fig. 2.7). In the gut, peristalsis is an important mechanism for keeping both food and infectious agents moving through the body. Failure of peristalsis is typically accompanied by the overgrowth of pathogenic bacteria within the lumen of the gut.

1	Fig. 2.5 Many barriers prevent pathogens from crossing epithelia and colonizing tissues. Surface epithelia provide mechanical, chemical, and microbiological barriers to infection. Skin Gut Lungs Eyes/nose/oral cavity Mechanical Epithelial cells joined by tight junctions Longitudinal fow of air or fuid Longitudinal fow of air or fuid Normal microbiota Movement of mucus by cilia Fatty acids Low pH Pulmonary surfactant Enzymes (pepsin) Enzymes in tears and saliva (lysozyme) Microbiological Chemical ˜-defensins Lamellar bodies Cathelicidin Histatins ˜-defensins °-defensins Cathelicidin Tears Nasal cilia °-defensins (cryptdins) RegIII (lecticidins) Cathelicidin Anatomic barriers and initial chemical defenses.

1	Fig. 2.6 Epithelia form specialized physical and chemical barriers that provide innate defenses in different locations. Top panel: the epidermis has multiple layers of keratinocytes in different stages of differentiation arising from the basal layer of stem cells. Differentiated keratinocytes in the stratum spinosum produce β-defensins and cathelicidins, which are incorporated into secretory organelles called lamellar bodies (yellow) and secreted into the intercellular space to form a waterproof lipid layer (the stratum corneum) containing antimicrobial activity. Center panel: in the lung, the airways are lined by ciliated epithelium. Beating of the cilia moves a continuous stream of mucus (green) secreted by goblet cells outward, trapping and ejecting potential pathogens. Type II pneumocytes in the lung alveoli (not shown) also produce and secrete antimicrobial defensins. Bottom panel: in the intestine, Paneth cells—specialized cells deep in the epithelial crypts— produce several

1	in the lung alveoli (not shown) also produce and secrete antimicrobial defensins. Bottom panel: in the intestine, Paneth cells—specialized cells deep in the epithelial crypts— produce several kinds of antimicrobial proteins: α-defensins (cryptdins) and the antimicrobial lectin RegIII.

1	Most healthy epithelial surfaces are also associated with a large population of normally nonpathogenic bacteria, known as commensal bacteria or the microbiota, that help keep pathogens at bay. The microbiota can also make antimicrobial substances, such as the lactic acid produced by vaginal lactobacilli, some strains of which also produce antimicrobial peptides (bacteriocins). Commensal microorganisms also induce responses that help to strengthen the barrier functions of epithelia by stimulating the epithelial cells to produce Lung of patient with cystic fbrosis Ciliated respiratory epithelium is covered by a layer of mucus Mucus Cilia Epithelium Fig. 2.7 Ciliated respiratory epithelium propels the overlying mucus layer for clearance of environmental microbes.

1	Fig. 2.7 Ciliated respiratory epithelium propels the overlying mucus layer for clearance of environmental microbes. Top panel: The ciliated respiratory epithelium in the airways of the lung is covered by a layer of mucus. The cilia propel the mucus outward and help prevent colonization of the airways by bacteria. Bottom panel: Section of a lung from a patient with cystic fibrosis. The dehydrated mucus layer impairs the ability of cilia to propel it, leading to frequent bacterial colonization and resulting inflammation of the airway. Courtesy of J. Ritter.

1	antimicrobial peptides. When commensal microorganisms are killed by antibiotic treatment, pathogens frequently replace them and cause disease (see Fig. 12.20). Under some circumstances commensal microbes themselves can cause disease if their growth is not kept in check or if the immune system is compromised. In Chapter 12, we will further discuss how commensal microorganisms play an important role in the setting of normal immunity, particularly in the intestine; and in Chapter 15, we will see how these normally nonpathogenic organisms can cause disease in the context of inherited immunodeficiencies. 2-3 Infectious agents must overcome innate host defenses to establish a focus of infection.

1	2-3 Infectious agents must overcome innate host defenses to establish a focus of infection. Our bodies are constantly exposed to microorganisms present in our environment, including infectious agents that have been shed by other individuals. Contact with these microorganisms may occur through external or internal epithelial surfaces. In order to establish an infection, a microorganism must first invade the body by binding to or crossing an epithelium (Fig. 2.8). With the epithelial damage that is common due to wounds, burns, or loss of the integrity of the body’s internal epithelia, infection is a major cause of mortality and morbidity. The body rapidly repairs damaged epithelial surfaces, but even without epithelial damage, pathogens may establish infection by specifically adhering to and colonizing epithelial surfaces, using the attachment to avoid being dislodged by the flow of air or fluid across the surface.

1	Disease occurs when a microorganism succeeds in evading or overwhelming innate host defenses to establish a local site of infection, and then replicates there to allow its further transmission within our bodies. The epithelium lining the respiratory tract provides a route of entry into tissues for airborne microorganisms, and the lining of the gastrointestinal tract does the same for microorganisms ingested in food and water. The intestinal pathogens Salmonella typhi, which causes typhoid fever, and Vibrio cholerae, which causes cholera, are spread through fecally contaminated food and water, respectively. Insect bites and wounds allow microorganisms to penetrate the skin, and direct contact between individuals offers opportunities for infection through the skin, the gut, and the reproductive tract (see Fig. 2.2).

1	In spite of this exposure, infectious disease is fortunately quite infrequent. Most of the microorganisms that succeed in crossing an epithelial surface are efficiently removed by innate immune mechanisms that function in the underlying tissues, preventing infection from becoming established. It is difficult to know how many infections are repelled in this way, because they cause no symptoms and pass undetected. In general, pathogenic microorganisms are distinguished from the mass of microorganisms in the environment by having special adaptations that evade the immune system. In some cases, such as the fungal disease athlete’s foot, the initial infection remains local and does not cause significant pathology. In other cases, such as tetanus, the bacterium (Clostridium tetani in this case) secretes a powerful neurotoxin, and the infection causes serious illness as it spreads through the lymphatics or the bloodstream, invades and destroys tissues, and disrupts the body’s workings.

1	The spread of a pathogen is often initially countered by an inflammatory response that recruits more effector cells and molecules of the innate immune system out of the blood and into the tissues, while inducing clotting in small blood vessels further downstream so that the microbe cannot spread through the circulation (see Fig. 2.8). The cellular responses of innate immunity act over several days. During this time, the adaptive immune response may also begin if antigens derived from the pathogen are delivered to local lymphoid tissues Anatomic barriers and initial chemical defenses.

1	Anatomic barriers and initial chemical defenses. Fig. 2.8 An infection and the response to it can be divided from becoming established. If not, it helps to contain the infection into a series of stages. These are illustrated here for an infectious and also delivers the infectious agent, carried in lymph and inside microorganism entering through a wound in the skin. The infectious dendritic cells, to local lymph nodes. This initiates the adaptive agent must first adhere to the epithelial cells and then cross the immune response and eventual clearance of the infection. epithelium. A local immune response may prevent the infection by dendritic cells (see Section 1-15). While an innate immune response may eliminate some infections, an adaptive immune response can target particular strains and variants of pathogens and protect the host against reinfection by using either effector T cells or antibodies to generate immunological memory.

1	2-4 Epithelial cells and phagocytes produce several kinds of antimicrobial proteins.

1	Our surface epithelia are more than mere physical barriers to infection; they also produce a wide variety of chemical substances that are microbicidal or that inhibit microbial growth. For example, the acid pH of the stomach and the digestive enzymes, bile salts, fatty acids, and lysolipids present in the upper gastrointestinal tract create a substantial chemical barrier to infection (see Fig. 2.5). One important group of antimicrobial proteins comprises enzymes that attack chemical features specific to bacterial cell walls. Such antibacterial enzymes include lysozyme and secretory phospholipase A2, which are secreted in tears and saliva and by phagocytes. Lysozyme is a glycosidase that breaks a specific chemical bond in the peptidoglycan component of the bacterial cell wall. Peptidoglycan is an alternating polymer of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), strengthened by cross-linking peptide bridges (Fig. 2.9). Lysozyme selectively cleaves the β-(1,4)

1	is an alternating polymer of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), strengthened by cross-linking peptide bridges (Fig. 2.9). Lysozyme selectively cleaves the β-(1,4) linkage between these two sugars and is more effective in acting against Gram-positive bacteria, in which the peptidoglycan cell wall is exposed, than against Gram-negative bacteria, which have an outer layer of LPS covering the peptidoglycan layer. Lysozyme is also produced by Paneth cells, specialized epithelial cells in the base of the crypts in the small intestine that secrete many antimicrobial proteins into the gut (see Fig. 2.6). Paneth cells also produce secretory phospholipase A2, a highly basic enzyme that can enter the bacterial cell wall to access and hydrolyze phospholipids in the cell membrane, killing the bacteria.

1	The second group of antimicrobial agents secreted by epithelial cells and phagocytes is the antimicrobial peptides. These represent one of the most ancient forms of defense against infection. Epithelial cells secrete these peptides into the fluids bathing the mucosal surface, whereas phagocytes secrete them in tissues. Three important classes of antimicrobial peptides in mammals are defensins, cathelicidins, and histatins.

1	Defensins are an ancient, evolutionarily conserved class of antimicrobial peptides made by many eukaryotic organisms, including mammals, insects, and plants (Fig. 2.10). They are short cationic peptides of around 30–40 amino acids that usually have three disulfide bonds stabilizing a common amphipathic structure—a positively charged region separated from a hydrophobic region. Defensins act within minutes to disrupt the cell membranes of bacteria and fungi, as well as the membrane envelopes of some viruses. The mechanism is thought to involve insertion of the hydrophobic region into the membrane bilayer and the formation of a pore that makes the membrane leaky (see Fig. 2.10). Most multicellular organisms make many different defensins

1	Fig. 2.9 Lysozyme digests the cell walls of Gram-positive and Gram-negative bacteria. Upper panels: the peptidoglycan of bacterial cell walls is a polymer of alternating residues of β-(1,4)-linked N-acetylglucosamine (GlcNAc) (large turquoise hexagons) and N-acetylmuramic acid (MurNAc) (purple circles) that are cross-linked by peptide bridges (red bars) into a dense three-dimensional network. In Gram-positive bacteria (upper left panel), peptidoglycan forms the outer layer in which other molecules are embedded such as teichoic acid and the lipoteichoic acids that link the peptidoglycan layer to the bacterial cell membrane itself. In Gram-negative bacteria (upper right panel), a thin inner wall of peptidoglycan is covered by an outer lipid membrane that contains proteins and lipopolysaccharide (LPS). Lipopolysaccharide is composed of a lipid, lipid A (turquoise circles), to which is attached a polysaccharide core (small turquoise hexagons). Lysozyme (lower panels) cleaves β-(1,4)

1	(LPS). Lipopolysaccharide is composed of a lipid, lipid A (turquoise circles), to which is attached a polysaccharide core (small turquoise hexagons). Lysozyme (lower panels) cleaves β-(1,4) linkages between GlcNAc and MurNAc, creating a defect in the peptidoglycan layer and exposing the underlying cell membrane to other antimicrobial agents. Lysozyme is more effective against Gram-positive bacteria because of the relatively greater accessibility of the peptidoglycan.

1	Anatomic barriers and initial chemical defenses. Fig. 2.10 Defensins are amphipathic peptides that disrupt the cell membranes of microbes. The structure of human β1-defensin is shown in the top panel. It is composed of a short segment of α helix (yellow) resting against three strands of antiparallel β sheet (green), generating an amphipathic peptide with charged and hydrophobic residues residing in separate regions. This general feature is shared by defensins from plants and insects and allows the defensins to interact with the charged surface of the cell membrane and become inserted in the lipid bilayer (center panel). Although the details are still unclear, a transition in the arrangement of the defensins in the membrane leads to the formation of pores and a loss of membrane integrity (bottom panel).

1	—the plant Arabidopsis thaliana produces 13 and the fruitfly Drosophila melanogaster at least 15. Human Paneth cells make as many as 21 different defensins, many of which are encoded by a cluster of genes on chromosome 8.

1	Three subfamilies of defensins—α-, β-, and θ-defensins—are distinguished on the basis of amino acid sequence, and each family has members with distinct activities, some being active against Gram-positive bacteria and some against Gram-negative bacteria, while others are specific for fungal pathogens. All the antimicrobial peptides, including the defensins, are generated by proteolytic processing from inactive propeptides (Fig. 2.11). In humans, developing neutrophils produce α-defensins by the processing of an initial propeptide of about 90 amino acids by cellular proteases to remove an anionic propiece, generating a mature cationic defensin that is stored in so-called primary granules. The primary granules of neutrophils are specialized membrane-enclosed vesicles, rather similar to lysosomes, that contain a number of other antimicrobial agents as well as defensins. We will explain in Chapter 3 how these primary granules within neutrophils are induced to fuse with phagocytic vesicles

1	that contain a number of other antimicrobial agents as well as defensins. We will explain in Chapter 3 how these primary granules within neutrophils are induced to fuse with phagocytic vesicles (phagosomes) after the cell has engulfed a pathogen, helping to kill the microbe. The Paneth cells of the gut constitutively produce α-defensins, called cryptdins, which are processed by proteases such as the metalloprotease matrilysin in mice, or trypsin in humans, before being secreted into the gut lumen. The β-defensins lack the long propiece of α-defensins and are generally produced specifically in response to the presence of microbial products. β-Defensins (and some α-defensins) are made by epithelia outside the gut, primarily in the respiratory and urogenital tracts, skin, and tongue. β-Defensins made by keratinocytes in the epidermis and by type II pneumocytes in the lungs are packaged into lamellar bodies (see Fig. 2.6), lipid-rich secretory organelles that release their contents into

1	made by keratinocytes in the epidermis and by type II pneumocytes in the lungs are packaged into lamellar bodies (see Fig. 2.6), lipid-rich secretory organelles that release their contents into the extracellular space to form a watertight lipid sheet in the epidermis and the pulmonary surfactant layer in the lung. The θ-defensins arose in the primates, but the single human θ-defensin gene has been inactivated by a mutation.

1	The antimicrobial peptides belonging to the cathelicidin family lack the disulfide bonds that stabilize the defensins. Humans and mice have one cathelicidin gene, but some other mammals, including cattle and sheep, have several. Cathelicidins are made constitutively by neutrophils and macrophages, and are made in response to infection by keratinocytes in the skin and epithelial cells in the lungs and intestine. They are made as inactive pro-peptides composed of two linked domains and are processed before secretion (see Fig. 2.11). In neutrophils, the inactive cathelicidin propeptides are stored in another type of specialized cytoplasmic granule called secondary granules. Cathelicidin is activated by proteolytic cleavage only when primary and secondary granules are induced to fuse with phagosomes, where it is cleaved by neutrophil elastase that has been stored in primary granules. Cleavage separates the two domains, and the cleavage products either remain in the phagosome or are

1	phagosomes, where it is cleaved by neutrophil elastase that has been stored in primary granules. Cleavage separates the two domains, and the cleavage products either remain in the phagosome or are released from the neutrophil by exocytosis. The carboxyterminal peptide is a cationic amphipathic peptide that disrupts membranes and is toxic to a wide range of microorganisms. The amino-terminal peptide is similar in structure to a protein called cathelin, an inhibitor of cathepsin L (a lysosomal enzyme involved in antigen processing and protein degradation), but its role in immune defense is unclear. In keratinocytes, cathelicidins, like β-defensins, are stored and processed in the lamellar bodies.

1	When αand β-defensins are first synthesized, they contain a signal peptide (not shown); a pro-region (blue), which is shorter in the β-defensins; and an amphipathic domain (AMPH, green–yellow); the pro-region represses the membrane-inserting properties of the amphipathic domain. After defensins are released from the cell, or into phagosomes, they undergo cleavage by proteases, which releases the amphipathic domain in active form. Newly synthesized cathelicidins contain a signal peptide, a cathelin domain, a short pro-region, and an amphipathic domain; they, too, are activated by proteolytic cleavage. RegIII contains a C-type lectin domain (CTLD), also known as a carbohydrate-recognition domain (CRD). After release of the signal peptide, further proteolytic cleavage of RegIII also regulates its antimicrobial activity.

1	A class of antimicrobial peptides called histatins are constitutively produced in the oral cavity by the parotid, sublingual, and submandibular glands. These short, histidine-rich, cationic peptides are active against pathogenic fungi such as Cryptococcus neoformans and Candida albicans. More recently histatins were found to promote the rapid wound healing that is typical in the oral cavity, but the mechanism of this effect is unclear.

1	Another type of bactericidal proteins made by epithelia is carbohydrate-binding proteins, or lectins. C-type lectins require calcium for the binding activity of their carbohydrate-recognition domain (CRD), which provides a variable interface for binding carbohydrate structures. C-type lectins of the RegIII family include several bactericidal proteins expressed by intestinal epithelium in humans and mice, comprising a family of ‘lecticidins.’ In mice, RegIIIγ is produced by Paneth cells and secreted into the gut, where it binds to peptidoglycans in bacterial cell walls and exerts direct bactericidal activity. Like other bactericidal peptides, RegIIIγ is produced in inactive form but is cleaved by the protease trypsin, which removes a short amino-terminal fragment to activate the bactericidal potential of RegIIIγ within the intestinal lumen (see Fig. 2.11). Human RegIIIα (also called HIP/PAP for hepatocarcinomaintestine-pancreas/pancreatitis-associated protein) kills bacteria directly

1	potential of RegIIIγ within the intestinal lumen (see Fig. 2.11). Human RegIIIα (also called HIP/PAP for hepatocarcinomaintestine-pancreas/pancreatitis-associated protein) kills bacteria directly by forming a hexameric pore in the bacterial membrane (Fig. 2.12). RegIII family proteins preferentially kill Gram-positive bacteria, in which the peptidoglycan is exposed on the outer surface (see Fig. 2.9). In fact, the LPS of Gram-negative bacteria inhibits the pore-forming ability of RegIIIα, further enforcing the selectivity of RegIII proteins for Gram-positive bacteria.

1	Summary. The mammalian immune response to invading organisms proceeds in three phases, beginning with immediate innate defenses, then the induced innate defenses, and finally adaptive immunity. The first phase of host defense consists of those mechanisms that are present and ready to resist an invader at any time. Epithelial surfaces provide a physical barrier against pathogen entry, but they also have other more specialized strategies. Mucosal surfaces have a protective barrier of mucus. Through particular cell-surface interactions, highly differentiated epithelia protect against both microbial colonization and invasion. Defense mechanisms of epithelia include the prevention of pathogen adherence, secretion of antimicrobial enzymes and bactericidal peptides, and the flow caused by the actions of cilia. Antimicrobial peptides and the bactericidal lectins of the RegIII family are made as inactive proproteins that require

1	Fig. 2.12 Pore formation by human RegIIIα. Top: a model of the RegIIIα pore was generated by docking the human pro-RegIIIα structure (PDB ID: 1UV0), shown as individual purple and turquoise ribbon diagrams, into the cryo-electron microscopic map of the RegIIIα filament. LPS blocks the pore-forming activity of RegIIIα, explaining its selective bactericidal activity against Gram-positive but not Gram-negative bacteria. Bottom: electron microscopic images of RegIIIα pores assembled in the presence of lipid bilayers. Top structure courtesy of L. Hooper. The complement system and innate immunity.

1	L. Hooper. The complement system and innate immunity. a proteolytic step to complete their activation, whereupon they become capable of killing microbes by forming pores in the microbial cell membranes. The actions of antimicrobial enzymes and peptides described in this section often involve binding to unique glycan/carbohydrate structures on the microbe. Thus, these soluble molecular defenses are both pattern recognition receptors and effector molecules at the same time, representing the simplest form of innate immunity. The complement system and innate immunity.

1	The complement system and innate immunity. When a pathogen breaches the host’s epithelial barriers and initial antimicrobial defenses, it next encounters a major component of innate immunity known as the complement system, or complement. Complement is a collection of soluble proteins present in blood and other body fluids. It was discovered in the 1890s by Jules Bordet as a heat-labile substance in normal plasma whose activity could ‘complement’ the bactericidal activity of immune sera. Part of the process is opsonization, which refers to coating a pathogen with antibodies and/or complement proteins so that it can be more readily taken up and destroyed by phagocytic cells. Although complement was first discovered as an effector arm of the antibody response, we now understand that it originally evolved as part of the innate immune system and that it still provides protection early in infection, in the absence of antibodies, through more ancient pathways of complement activation.

1	The complement system is composed of more than 30 different plasma proteins, which are produced mainly by the liver. In the absence of infection, these proteins circulate in an inactive form. In the presence of pathogens or of antibody bound to pathogens, the complement system becomes ‘activated.’ Particular complement proteins interact with each other to form several different pathways of complement activation, all of which have the final outcome of killing the pathogen, either directly or by facilitating its phagocytosis, and inducing inflammatory responses that help to fight infection. There are three pathways of complement activation. As the antibody-triggered pathway of complement activation was discovered first, this became known as the classical pathway of complement activation. The next to be discovered was called the alternative pathway, which can be activated by the presence of the pathogen alone; and the most recently discovered is the lectin pathway, which is activated by

1	next to be discovered was called the alternative pathway, which can be activated by the presence of the pathogen alone; and the most recently discovered is the lectin pathway, which is activated by lectin-type proteins that recognize and bind to carbohydrates on pathogen surfaces.

1	We learned in Section 2-4 that proteolysis can be used as a means of activating antimicrobial proteins. In the complement system, activation by proteolysis is inherent, with many of the complement proteins being proteases that successively cleave and activate one another. The proteases of the complement system are synthesized as inactive pro-enzymes, or zymogens, which become enzymatically active only after proteolytic cleavage, usually by another complement protein. The complement pathways are triggered by proteins that act as pattern recognition receptors to detect the presence of pathogens. This detection activates an initial zymogen, triggering a cascade of proteolysis in which complement zymogens are activated sequentially, each becoming an active protease that cleaves and activates many molecules of the next zymogen in the pathway, amplifying the signal as the cascade proceeds. This results in activation of three distinct effector pathways—inflammation, phagocytosis, and

1	many molecules of the next zymogen in the pathway, amplifying the signal as the cascade proceeds. This results in activation of three distinct effector pathways—inflammation, phagocytosis, and membrane attack—that help eliminate the pathogen. In this way, the detection of even a small number of pathogens produces a rapid response that is greatly amplified at each step. This overall scheme for complement is shown in Fig. 2.13.

1	Fig. 2.13 The complement system proceeds in distinct phases in the elimination of microbes. Proteins that can distinguish self from microbial surfaces (yellow box) activate a proteolytic amplification cascade that ends in the formation of the critical enzymatic activity (green box) of C3 convertase, a family of proteases. This activity is the gateway to three effector arms of complement that produce inflammation (purple), enhance phagocytosis of the microbe (blue), and lyse microbial membranes (pink). We will use this color scheme in the figures throughout this chapter to illustrate which activity each complement protein serves. MOVIE 2.1

1	Nomenclature for complement proteins can seem confusing, so we will start by explaining their names. The first proteins discovered belong to the classical pathway, and they are designated by the letter C followed by a number. The native complement proteins—such as the inactive zymogens—have a simple number designation, for example, C1 and C2. Unfortunately, they were named in the order of their discovery rather than the sequence of reactions. The reaction sequence in the classical pathway, for example, is C1, C4, C2, C3, C5, C6, C7, C8, and C9 (note that not all of these are proteases). Products of cleavage reactions are designated by adding a lowercase letter as a suffix. For example, cleavage of C3 produces a small protein fragment called C3a and the remaining larger fragment, C3b. By convention, the larger fragment for other factors is designated by the suffix b, with one exception. For C2, the larger fragment was named C2a by its discoverers, and this system has been maintained in

1	the larger fragment for other factors is designated by the suffix b, with one exception. For C2, the larger fragment was named C2a by its discoverers, and this system has been maintained in the literature, so we preserve it here. Another exception is the naming of C1q, C1r, and C1s: these are not cleavage products of C1 but are distinct proteins that together comprise C1. The proteins of the alternative pathway were discovered later and are designated by different capital letters, for example, factor B and factor D. Their cleavage products are also designated by the addition of lowercase a and b: thus, the large fragment of B is called Bb and the small fragment Ba. Activated complement components are sometimes designated by a horizontal line, for example, C2a; however, we will not use this convention. All the components of the complement system are listed in Fig. 2.14.

1	Besides acting in innate immunity, complement also influences adaptive immunity. Opsonization of pathogens by complement facilitates their uptake by phagocytic antigen-presenting cells that express complement receptors; this enhances the presentation of pathogen antigens to T cells, which we discuss in more detail in Chapter 6. B cells express receptors for complement proteins that enhance their responses to complement-coated antigens, as we describe later in Chapter 10. In addition, several of the complement fragments can act to influence cytokine production by antigen-presenting cells, thereby influencing the direction and extent of the subsequent adaptive immune response, as we describe in Chapter 11. 2-5 The complement system recognizes features of microbial with C3b.

1	Fig. 2.15 gives a highly simplified preview of the initiation mechanisms and outcomes of complement activation. The three pathways of complement activation are initiated in different ways. The lectin pathway is initiated by soluble carbohydrate-binding proteins—mannose-binding lectin (MBL) and the ficolins—that bind to particular carbohydrate structures on microbial surfaces. Specific proteases, called MBL-associated serine proteases (MASPs), that associate with these recognition proteins then trigger the cleavage of complement proteins and activation of the pathway. The classical pathway is initiated when the complement component C1, which comprises a recognition protein (C1q) associated with proteases (C1r and C1s), either recognizes a microbial surface directly or binds to antibodies already bound to a pathogen. Finally, the alternative pathway can be initiated by spontaneous hydrolysis and activation of the complement component C3, which can then bind directly to microbial

1	already bound to a pathogen. Finally, the alternative pathway can be initiated by spontaneous hydrolysis and activation of the complement component C3, which can then bind directly to microbial surfaces.

1	These three pathways converge at the central and most important step in complement activation. When any of the pathways interacts with a pathogen surface, the enzymatic activity of a C3 convertase is generated. There are various types of C3 convertase, depending on the complement pathway activated, but each is a multisubunit protein with protease activity that cleaves complement component 3 (C3). The C3 convertase is bound covalently to the pathogen surface, where it cleaves C3 to generate large amounts of C3b, the main effector molecule of the complement system; and C3a, a small peptide that binds to The complement system and innate immunity.

1	The complement system and innate immunity. C3a and C5a recruit phagocytic cells to the site of infection and promote infammation C3a infected tissue Phagocytes with receptors for C3b engulf and destroy the pathogen Completion of the complement cascade leads to formation of a membrane-attack complex (MAC), which disrupts cell membrane and causes cell lysis factor D factor B properdin C3 C1q, C1r, C1s C4 C2 MBL/fcolin, MASP-2 C4 C2 C3a C3 complement receptors All pathways generate a C3 convertase, which cleaves C3, leaving C3b bound to the microbial surface and releasing C3a C3b C6 C5b C8 C7 C9 MAC C3b Phagocyte

1	Fig. 2.15 Complement is a system of soluble pattern recognition receptors and effector molecules that detect and destroy microorganisms. The pathogen-recognition mechanisms of the three complement-activation pathways are shown in the top row, along with the complement components used in the proteolytic cascades leading to formation of a C3 convertase. This enzyme activity cleaves complement component C3 into the small soluble protein C3a and the larger component C3b, which becomes covalently bound to the pathogen surface (middle row). The components are listed by biochemical function in Fig, 2.14 and are described in detail in later figures. The lectin pathway of complement activation (top left) is triggered by the binding of mannose-binding lectin (MBL) or ficolins to carbohydrate residues in microbial cell walls and capsules. The classical pathway (top center) is triggered by binding of C1 either to the pathogen surface or to antibody bound to the pathogen. In the alternative

1	residues in microbial cell walls and capsules. The classical pathway (top center) is triggered by binding of C1 either to the pathogen surface or to antibody bound to the pathogen. In the alternative pathway (top right), soluble C3 undergoes spontaneous hydrolysis in the fluid phase, generating C3(H2O), which is augmented by the action of factors B, D, and P (properdin). All pathways thus converge on the formation of C3b bound to a pathogen and lead to all of the effector activities of complement, which are shown in the bottom row. C3b bound to a pathogen acts as an opsonin, enabling phagocytes that express receptors for C3b to ingest the complement-coated microbe more easily (bottom center). C3b can also bind to C3 convertases to produce another activity, a C5 convertase (detail not shown here), which cleaves C5 to C5a and C5b. C5b triggers the late events of the complement pathway in which the terminal components of complement—C6 to C9—assemble into a membrane-attack complex (MAC)

1	here), which cleaves C5 to C5a and C5b. C5b triggers the late events of the complement pathway in which the terminal components of complement—C6 to C9—assemble into a membrane-attack complex (MAC) that can damage the membrane of certain pathogens (bottom right). C3a and C5a act as chemoattractants that recruit immune-system cells to the site of infection and cause inflammation (bottom left).

1	specific receptors and helps induce inflammation. Cleavage of C3 is the critical step in complement activation and leads directly or indirectly to all the effector activities of the complement system (see Fig. 2.15). C3b binds covalently to the microbial surface and acts as an opsonin, enabling phagocytes that carry receptors for complement to take up and destroy the C3b-coated microbe. Later in the chapter, we will describe the different complement receptors that bind C3b that are involved in this function of complement and how C3b is degraded by a serum protease into inactive smaller fragments called C3f and C3dg. C3b can also bind to the C3 convertases produced by the classical and lectin pathways and form another multisubunit enzyme, the C5 convertase. This cleaves C5, liberating the highly inflammatory peptide C5a and generating C5b. C5b initiates the ‘late’ events of complement activation, in which additional complement proteins interact with C5b to form a membraneattack complex

1	inflammatory peptide C5a and generating C5b. C5b initiates the ‘late’ events of complement activation, in which additional complement proteins interact with C5b to form a membraneattack complex (MAC) on the pathogen surface, creating a pore in the cell membrane that leads to cell lysis (see Fig. 2.15, bottom right).

1	The key feature of C3b is its ability to form a covalent bond with microbial surfaces, which allows the innate recognition of microbes to be translated into effector responses. Covalent bond formation is due to a highly reactive thioester bond that is hidden inside the folded C3 protein and cannot react until C3 is cleaved. When C3 convertase cleaves C3 and releases the C3a fragment, large conformational changes occur in C3b that allow the thioester bond to react with a hydroxyl or amino group on the nearby microbial surface (Fig. 2.16). If no bond is made, the thioester is rapidly hydrolyzed, inactivating C3b, which is one way the alternative pathway is inhibited in healthy individuals. As we will see below, some of the individual components of C3 and C5 convertases differ between the various complement pathways; the components that are different are listed in Fig. 2.17.

1	C3b bound to pathogen surface C3b inactivated by hydrolysis ˜°Before cleavage by C3 convertase, the thioester bond within TED is protected from reacting Cleavage of C3 releases C3a, and a change in conformation of C3b allows the thioester bond to react with a chemical group on the pathogen surface C3b C3aTED TED 23 14 5 6 C3 convertase (C4b2a)pathogen surface thioester bond The reactive thioester group of C3b in TED Gly Glu GlnCys ˜°

1	Fig. 2.16 C3 convertase activates C3 for covalent bonding convertase C4b2a is shown here) and release of C3a from the amino to microbial surfaces by cleaving it into C3a and C3b and terminus of the α chain causes a conformational change in C3b exposing a highly reactive thioester bond in C3b. Top panel: that exposes the thioester bond. This can now react with hydroxyl C3 in blood plasma consists of an α chain and a β chain (formed by or amino groups on molecules on microbial surfaces, covalently proteolytic processing from the native C3 polypeptide) held together bonding C3b to the surface. Bottom right panels: schematic view by a disulfide bond. The thioester-containing domain (TED) of the α of the thioester reaction. If a bond is not made with a microbial chain contains a potentially highly reactive thioester bond (red spot). surface, the thioester is rapidly hydrolyzed (that is, cleaved by water), Bottom left panels: cleavage by C3 convertase (the lectin pathway rendering C3b

1	highly reactive thioester bond (red spot). surface, the thioester is rapidly hydrolyzed (that is, cleaved by water), Bottom left panels: cleavage by C3 convertase (the lectin pathway rendering C3b inactive.

1	The complement system and innate immunity.

1	Pathways leading to such potent inflammatory and destructive effects—and which have a series of built-in amplification steps—are potentially dangerous and must be tightly regulated. One important safeguard is that the key activated complement components are rapidly inactivated unless they bind to the pathogen surface on which their activation was initiated. There are also several points in the pathway at which regulatory proteins act to prevent the activation of complement on the surfaces of healthy host cells, thereby protecting them from accidental damage, as we shall see later in the chapter. Complement can, however, be activated by dying cells, such as those at sites of ischemic injury, and by cells undergoing apoptosis, or programmed cell death. In these cases, the complement coating helps phagocytes dispose of the dead and dying cells neatly, thus limiting the risk of cell contents being released and triggering an autoimmune response (discussed in Chapter 15).

1	Having introduced some of the main complement components, we are ready for a more detailed account of the three pathways. To help indicate the types of functions carried out by each of the complement components in the tables throughout the rest of the chapter, we will use the color code introduced in Fig. 2.13 and Fig. 2.14: yellow for recognition and activation, green for amplification, purple for inflammation, blue for phagocytosis, and pink for membrane attack. 2-6 The lectin pathway uses soluble receptors that recognize microbial surfaces to activate the complement cascade.

1	Microorganisms typically bear on their surface repeating patterns of molecular structures, known generally as pathogen-associated molecular patterns (PAMPs). The cell walls of Gram-positive and Gram-negative bacteria, for example, are composed of a matrix of proteins, carbohydrates, and lipids in a repetitive array (see Fig. 2.9). The lipoteichoic acids of Gram-positive bacterial cell walls and the lipopolysaccharide of the outer membrane of Gram-negative bacteria are not present on animal cells and are important in the recognition of bacteria by the innate immune system. Similarly, the glycans of yeast surface proteins commonly terminate in mannose residues rather than the sialic acid residues (N-acetylneuraminic acid) that terminate the glycans of vertebrate cells (Fig. 2.18). The lectin pathway uses these features of microbial surfaces to detect and respond to pathogens.

1	Fig. 2.18 The carbohydrate side chains on yeast and vertebrate glycoproteins are terminated with different patterns of sugars. N-linked glycosylation in fungi and animals is initiated by the addition of the same precursor oligosaccharide, Glc3Man9-GlcNAc2 (left panel), to an asparagine residue. In many yeasts this is processed to high-mannose glycans (middle panel). In contrast, in vertebrates, the initial glycan is trimmed and processed, and the N-linked glycoproteins of vertebrates have terminal sialic acid residues (right panel).

1	The lectin pathway can be triggered by any of four different pattern recognition receptors that circulate in blood and extracellular fluids and recognize carbohydrates on microbial surfaces. The first such receptor to be discovered was mannose-binding lectin (MBL), which is shown in Fig. 2.19, and which is synthesized in the liver. MBL is an oligomeric protein built up from a monomer that contains an amino-terminal collagen-like domain and a carboxy-terminal C-type lectin domain (see Section 2-4). Proteins of this type are called collectins. MBL monomers assemble into trimers through the formation of a triple helix by their collagen-like domains. Trimers then assemble into oligomers by disulfide bonding between the cysteine-rich collagen domains. The MBL present in the blood is composed of two to six trimers, with the major forms of human MBL being trimers and tetramers. A single carbohydrate-recognition domain of MBL has a low affinity for mannose, fucose, and N-acetylglucosamine

1	of two to six trimers, with the major forms of human MBL being trimers and tetramers. A single carbohydrate-recognition domain of MBL has a low affinity for mannose, fucose, and N-acetylglucosamine (GlcNAc) residues, which are common on microbial glycans, but does not bind sialic acid residues, which terminate vertebrate glycans. Thus, multimeric MBL has high total binding strength, or avidity, for repetitive carbohydrate structures on a wide variety of microbial surfaces, including Gram-positive and Gram-negative bacteria, mycobacteria, yeasts, and some viruses and parasites, while not interacting with host cells. MBL is present at low concentrations in the plasma of most individuals, but in the presence of infection, its production is increased during the acute-phase response. This is part of the induced phase of the innate immune response and is discussed in Chapter 3.

1	The other three pathogen-recognition molecules used by the lectin pathway are known as ficolins. Although related in overall shape and function to MBL, they have a fibrinogen-like domain, rather than a lectin domain, attached to

1	Mannose-binding lectin (MBL) (left panels) is an oligomeric protein in which two to six clusters of carbohydrate-binding heads arise from a central stalk formed from the collagen-like tails of the MBL monomers. An MBL monomer is composed of a collagen region (red), an α-helical neck region (blue), and a carbohydrate-recognition domain (yellow). Three MBL monomers associate to form a trimer, and between two and six trimers assemble to form a mature MBL molecule (bottom left panel). An MBL molecule associates with MBL-associated serine proteases (MASPs). MBL binds to bacterial surfaces that display a particular spatial arrangement of mannose or fucose residues. The ficolins (right panels) resemble MBL in their overall structure, are associated with MASP-1 and MASP-2, and can activate C4 and C2 after binding to carbohydrate molecules present on microbial surfaces. The carbohydrate-binding domain of ficolins is a fibrinogenlike domain, rather than the lectin domain present in MBL.

1	Fig. 2.19 Mannose-binding lectin and ficolins form complexes with serine proteases and recognize particular carbohydrates on microbial surfaces. collagen helices ˜-helical coiled coils carbohydrate-recognition domains MBL monomers form trimeric clusters of carbohydrate-recognition domains collagen helices ˜-helical coiled coils ÿbronectin domains Ficolins are similar in structure to MBL but have a different carbohydrate-binding domain The complement system and innate immunity. the collagen-like stalk (see Fig. 2.19). The fibrinogen-like domain gives ficolins a general specificity for oligosaccharides containing acetylated sugars, but it does not bind mannose-containing carbohydrates. Humans have three ficolins: L-ficolin (ficolin-2), M-ficolin (ficolin-1), and H-ficolin (ficolin-3). L- and H-ficolin are synthesized by the liver and circulate in the blood; M-ficolin is synthesized and secreted by lung and blood cells.

1	MBL in plasma forms complexes with the MBL-associated serine proteases MASP-1, MASP-2, and MASP-3, which bind MBL as inactive zymogens. When MBL binds to a pathogen surface, a conformational change occurs in MASP-1 that enables it to cleave and activate a MASP-2 molecule in the same MBL complex. Activated MASP-2 can then cleave complement components C4 and C2 (Fig. 2.20). Like MBL, ficolins form oligomers that make a complex with MASP-1 and MASP-2, which similarly activate complement upon recognition of a microbial surface by the ficolin. C4, like C3, contains a buried thioester bond. When MASP-2 cleaves C4, it releases C4a, allowing a conformational change in C4b that exposes the reactive thioester as described for C3b (see Fig. 2.16). C4b bonds covalently via this thioester to the microbial surface nearby, where it then binds one molecule of C2 (see Fig. 2.20). C2 is cleaved by MASP-2, producing C2a, an active serine protease that remains bound to C4b to form C4b2a, which is the C3

1	surface nearby, where it then binds one molecule of C2 (see Fig. 2.20). C2 is cleaved by MASP-2, producing C2a, an active serine protease that remains bound to C4b to form C4b2a, which is the C3 convertase of the lectin pathway. (Remember, C2a is the exception in complement nomenclature.) C4b2a now cleaves many molecules of C3 into C3a and C3b. The C3b fragments bond covalently to the nearby pathogen surface, and the released C3a initiates a local inflammatory response. The complement-activation pathway initiated by ficolins proceeds like the MBL lectin pathway (see Fig. 2.20).

1	Individuals deficient in MBL or MASP-2 experience substantially more respiratory infections by common extracellular bacteria during early childhood, indicating the importance of the lectin pathway for host defense. This susceptibility illustrates the particular importance of innate defense mechanisms in early childhood, when adaptive immune responses are not yet fully developed

1	Binding of mannose-binding lectin or ficolins to their carbohydrate cleaves many molecules of C3 to produce C3b, which binds to the ligands on microbial surfaces induces MASP-1 to cleave and activate pathogen surface, and C3a, an inflammatory mediator. The covalent the serine protease MASP-2. MASP-2 then cleaves C4, exposing attachment of C3b and C4b to the pathogen surface is important in the thioester bond in C4b that allows it to react covalently with the confining subsequent complement activity to pathogen surfaces. pathogen surface. C4b then binds C2, making C2 susceptible to Fig. 2.21 The first protein in the classical pathway of complement activation is C1, which is a complex of C1q, C1r, and C1s. As shown in the micrograph and drawing, C1q is composed of six identical subunits with globular heads (yellow) and long collagen-like tails (red); it has been described as looking like “a bunch of tulips.” The tails combine to bind to two molecules each of C1r and C1s, forming the C1

1	globular heads (yellow) and long collagen-like tails (red); it has been described as looking like “a bunch of tulips.” The tails combine to bind to two molecules each of C1r and C1s, forming the C1 complex C1q:C1r2:C1s2. The heads can bind to the constant regions of immunoglobulin molecules or directly to the pathogen surface, causing a conformational change in C1r, which then cleaves and activates the C1s zymogen (proenzyme). The C1 complex is similar in overall structure to the MBL–MASP complex, and it has an identical function, cleaving C4 and C2 to form the C3 convertase C4b2a (see Fig. 2.20). Photograph (×500,000) courtesy of K.B.M. Reid.

1	but the maternal antibodies transferred across the placenta and present in the mother’s milk are gone. Other members of the collectin family are the surfactant proteins A and D (SP-A and SP-D), which are present in the fluid that bathes the epithelial surfaces of the lung. There they coat the surfaces of pathogens, making them more susceptible to phagocytosis by macrophages that have left the subepithelial tissues to enter the alveoli. Because SP-A and SP-D do not associate with MASPs, they do not activate complement.

1	We have used MBL here as our prototype activator of the lectin pathway, but the ficolins are more abundant than MBL in plasma and so may be more important in practice. L-ficolin recognizes acetylated sugars such as GlcNAc and N-acetylgalactosamine (GalNAc), and particularly recognizes lipoteichoic acid, a component of the cell walls of Gram-positive bacteria that contains GalNAc. It can also activate complement after binding to a variety of capsulated bacteria. M-ficolin also recognizes acetylated sugar residues; H-ficolin shows a more restricted binding specificity, for d-fucose and galactose, and has only been linked to activity against the Gram-positive bacterium Aerococcus viridans, a cause of bacterial endocarditis. 2-7 The classical pathway is initiated by activation of the C1 complex and is homologous to the lectin pathway.

1	2-7 The classical pathway is initiated by activation of the C1 complex and is homologous to the lectin pathway. In its overall scheme, the classical pathway is similar to the lectin pathway, except that it uses a pathogen sensor known as the C1 complex, or C1. Because C1 interacts directly with some pathogens but can also interact with antibodies, C1 allows the classical pathway to function both in innate immunity, which we describe now, and in adaptive immunity, which we examine in more detail in Chapter 10.

1	Like the MBL–MASP complex, the C1 complex is composed of a large subunit (C1q), which acts as the pathogen sensor, and two serine proteases (C1r and C1s), which are initially in their inactive form (Fig. 2.21). C1q is a hexamer of trimers, composed of monomers that contain an amino-terminal globular domain and a carboxy-terminal collagen-like domain. The trimers assemble through interactions of the collagen-like domains, bringing the globular domains together to form a globular head. Six of these trimers assemble to form a complete C1q molecule, which has six globular heads held together by their collagen-like tails. C1r and C1s are closely related to MASP-2, and somewhat more distantly related to MASP-1 and MASP-3; all five enzymes are likely to have evolved from the duplication of a gene for a common precursor. C1r and C1s interact noncovalently and form tetramers that fold into the arms of C1q, with at least part of the C1r:C1s complex being external to C1q.

1	The recognition function of C1 resides in the six globular heads of C1q. When two or more of these heads interact with a ligand, this causes a conformational change in the C1r:C1s complex, which leads to the activation of an autocatalytic enzymatic activity in C1r; the active form of C1r then cleaves its associated C1s to generate an active serine protease. The activated C1s acts on the next two components of the classical pathway, C4 and C2. C1s cleaves C4 to produce C4b, which binds covalently to the pathogen surface as described earlier for the lectin pathway (see Fig. 2.20). C4b then also binds one molecule of C2, which is cleaved by C1s to produce the serine protease C2a. This produces the active C3 convertase C4b2a, which is the C3 convertase of both the lectin and the classical pathways. However, because it was first discovered as part of the classical pathway, C4b2a is often known as the classical C3 convertase (see Fig. 2.17). The proteins involved in the classical pathway,

1	However, because it was first discovered as part of the classical pathway, C4b2a is often known as the classical C3 convertase (see Fig. 2.17). The proteins involved in the classical pathway, and their active forms, are listed in Fig. 2.22.

1	C1q can attach itself to the surface of pathogens in several different ways. One is by binding directly to surface components on some bacteria, including The complement system and innate immunity.

1	certain proteins of bacterial cell walls and polyanionic structures such as the lipoteichoic acid on Gram-positive bacteria. A second is through binding to C-reactive protein, an acute-phase protein in human plasma that binds to phosphocholine residues in bacterial surface molecules such as pneumococcal C polysaccharide—hence the name C-reactive protein. We discuss the acute-phase proteins in detail in Chapter 3. However, a main function of C1q in an immune response is to bind to the constant, or Fc, regions of antibodies (see Section 1-9) that have bound pathogens via their antigen-binding sites. C1q thus links the effector functions of complement to recognition provided by adaptive immunity. This might seem to limit the usefulness of C1q in fighting the first stages of an infection, before the adaptive immune response has generated pathogen-specific antibodies. However, some antibodies, called natural antibodies, are produced by the immune system in the apparent absence of

1	before the adaptive immune response has generated pathogen-specific antibodies. However, some antibodies, called natural antibodies, are produced by the immune system in the apparent absence of infection. These antibodies have a low affinity for many microbial pathogens and are highly cross-reactive, recognizing common membrane constituents such as phosphocholine and even recognizing some antigens of the body’s own cells (that is, self antigens). Natural antibodies may be produced in response to commensal microbiota or to self antigens, but do not seem to be the consequence of an adaptive immune response to infection by pathogens. Most natural antibody is of the isotype, or class, known as IgM (see Sections 1-9 and 1-20) and represents a considerable amount of the total IgM circulating in humans. IgM is the class of antibody most efficient at binding C1q, making natural antibodies an effective means of activating complement on microbial surfaces immediately after infection and leading

1	humans. IgM is the class of antibody most efficient at binding C1q, making natural antibodies an effective means of activating complement on microbial surfaces immediately after infection and leading to the clearance of bacteria such as Streptococcus pneumoniae (the pneumococcus) before they become dangerous.

1	2-8 Complement activation is largely confined to the surface on which it is initiated. We have seen that both the lectin and the classical pathways of complement activation are initiated by proteins that bind to pathogen surfaces. During the triggered enzyme cascade that follows, it is important that activating events are confined to this same site, so that C3 activation also occurs on the surface of the Fig. 2.23 The alternative pathway of complement activation can amplify the classical or the lectin pathway by forming an alternative C3 convertase and depositing more C3b molecules on the pathogen. C3b deposited by the classical or lectin pathway can bind factor B, making it susceptible to cleavage by factor D. The C3bBb complex is the C3 convertase of the alternative pathway of complement activation, and its action, like that of C4b2a, results in the deposition of many molecules of C3b on the pathogen surface.

1	pathogen and not in the plasma or on host-cell surfaces. This is achieved principally by the covalent binding of C4b to the pathogen surface. In innate immunity, C4 cleavage is catalyzed by a ficolin or MBL complex that is bound to the pathogen surface, and so the C4b cleavage product can bind adjacent proteins or carbohydrates on the pathogen surface. If C4b does not rapidly form this bond, the thioester bond is cleaved by reaction with water and C4b is irreversibly inactivated. This helps to prevent C4b from diffusing from its site of activation on the microbial surface and becoming attached to healthy host cells.

1	C2 becomes susceptible to cleavage by C1s only when it is bound by C4b, and the active C2a serine protease is thereby also confined to the pathogen surface, where it remains associated with C4b, forming the C3 convertase C4b2a. Cleavage of C3 to C3a and C3b is thus also confined to the surface of the pathogen. Like C4b, C3b is inactivated by hydrolysis unless its exposed thioester rapidly makes a covalent bond (see Fig. 2.16), and it therefore opsonizes only the surface on which complement activation has taken place. Opsonization by C3b is more effective when antibodies are also bound to the pathogen surface, as phagocytes have receptors for both complement and Fc receptors that bind the Fc region of antibody (see Sections 1-20 and 10-20). Because the reactive forms of C3b and C4b are able to form a covalent bond with any adjacent protein or carbohydrate, when complement is activated by bound antibody, a proportion of the reactive C3b or C4b will become linked to the antibody

1	are able to form a covalent bond with any adjacent protein or carbohydrate, when complement is activated by bound antibody, a proportion of the reactive C3b or C4b will become linked to the antibody molecules themselves. Antibody that is chemically cross-linked to complement is likely the most efficient trigger for phagocytosis.

1	2-9 The alternative pathway is an amplification loop for C3b formation that is accelerated by properdin in the presence of pathogens.

1	Although probably the most ancient of the complement pathways, the alternative pathway is so named because it was discovered as a second, or ‘alternative,’ pathway for complement activation after the classical pathway had been defined. Its key feature is its ability to be spontaneously activated. It has a unique C3 convertase, the alternative pathway C3 convertase, that differs from the C4b2a convertase of the lectin or classical pathways (see Fig. 2.17). The alternative pathway C3 convertase is composed of C3b itself bound to Bb, which is a cleavage fragment of the plasma protein factor B. This C3 convertase, designated C3bBb, has a special place in complement activation because, by producing C3b, it can generate more of itself. This means that once some C3b has been formed, by whichever pathway, the alternative pathway can act as an amplification loop to increase C3b production rapidly.

1	The alternative pathway can be activated in two different ways. The first is by the action of the lectin or classical pathway. C3b generated by either of these pathways and covalently linked to a microbial surface can bind factor B (Fig. 2.23). This alters the conformation of factor B, enabling a plasma protease The complement system and innate immunity. The C3(H2O)Bb complex is a C3 convertase, cleaving more C3 into C3a and C3b. C3b is rapidly inactivated unless it binds to a cell surface

1	The complement system and innate immunity. The C3(H2O)Bb complex is a C3 convertase, cleaving more C3 into C3a and C3b. C3b is rapidly inactivated unless it binds to a cell surface Complement component C3 hydrolyzes spontaneously in plasma to give C3(H2O), which binds factor B and enables the bound factor B to be cleaved by factor D (first panel). The resulting ‘soluble C3 convertase’ cleaves C3 to give C3a and C3b, which can attach to host cells or pathogen surfaces (second panel). Covalently bound to the cell surface, C3b binds factor B; in turn, factor B is rapidly cleaved by factor D to Bb, which remains bound to C3b to form a C3 convertase (C3bBb), and Ba, which is released (third panel). This convertase functions in the alternative pathway as the C4b2a C3 convertase does in the lectin and classical pathways (see Fig. 2.17).

1	called factor D to cleave it into Ba and Bb. Bb remains stably associated with C3b, forming the C3bBb C3 convertase. The second way of activating the alternative pathway involves the spontaneous hydrolysis (known as ‘tickover’) of the thioester bond in C3 to form C3(H2O), as shown in Fig. 2.24. C3 is abundant in plasma, and tickover causes a steady, low-level production of C3(H2O). This C3(H2O) can bind factor B, which is then cleaved by factor D, producing a short-lived fluid-phase C3 convertase, C3(H2O)Bb. Although formed in only small amounts by C3 tickover, fluid-phase C3(H2O)Bb can cleave many molecules of C3 to C3a and C3b. Much of this C3b is inactivated by hydrolysis, but some attaches covalently via its thioester bond to the surfaces of any microbes present. C3b formed in this way is no different from C3b produced by the lectin or classical pathways and binds factor B, leading to the formation of C3 convertase and a stepping up of C3b production (see Fig. 2.23).

1	On their own, the alternative pathway C3 convertases C3bBb and C3(H2O) Bb are very short-lived. They are, however, stabilized by binding the plasma protein properdin (factor P) (Fig. 2.25). Properdin is made by neutrophils and stored in secondary granules. It is released when neutrophils are activated by the presence of pathogens. Properdin may have some properties of a pattern recognition receptor, since it can bind to some microbial surfaces. Properdindeficient patients are particularly susceptible to infections with Neisseria meningitidis, the main agent of bacterial meningitis. Properdin’s ability to bind to bacterial surfaces may direct the activity of the alternative complement pathway to these pathogens, thus aiding their removal by phagocytosis. Properdin can also bind to mammalian cells that are undergoing apoptosis or have been damaged or modified by ischemia, viral infection, or antibody binding, leading to the deposition of C3b on these cells and facilitating their removal

1	cells that are undergoing apoptosis or have been damaged or modified by ischemia, viral infection, or antibody binding, leading to the deposition of C3b on these cells and facilitating their removal by phagocytosis. The distinctive components of the alternative pathway are listed in Fig. 2.26.

1	Fig. 2.25 Properdin stabilizes the alternative pathway C3 convertase on pathogen surfaces. Bacterial surfaces do not express complement-regulatory proteins and favor the binding of properdin (factor P), which stabilizes the C3bBb convertase. This convertase activity is the equivalent of C4b2a of the classical pathway. C3bBb then cleaves many more molecules of C3, coating the pathogen surface with bound C3b. C3bBb complex is a C3 convertase and deposits many molecules of C3b on the pathogen surface Opsonization, activation of terminal complement components

1	C3bBb complex is a C3 convertase and deposits many molecules of C3b on the pathogen surface Opsonization, activation of terminal complement components Fig. 2.27 Complement activation spares host cells, which are protected by complement-regulatory proteins. If C3bBb forms on the surface of host cells, it is rapidly inactivated by complement-regulatory proteins expressed by the host cell: complement receptor 1 (CR1), decay-accelerating factor (DAF), and membrane cofactor of proteolysis (MCP). Host-cell surfaces also favor the binding of factor H from plasma. CR1, DAF, and factor H displace Bb from C3b, and CR1, MCP, and factor H catalyze the cleavage of bound C3b by the plasma protease factor I to produce inactive C3b (known as iC3b). 2-10 Membrane and plasma proteins that regulate the formation and stability of C3 convertases determine the extent of complement activation.

1	2-10 Membrane and plasma proteins that regulate the formation and stability of C3 convertases determine the extent of complement activation. Several mechanisms ensure that complement activation will proceed only on the surface of a pathogen or on damaged host cells, and not on normal host cells and tissues. After initial complement activation by any pathway, the extent of amplification via the alternative pathway is critically dependent on the stability of the C3 convertase C3bBb. This stability is controlled by both positive and negative regulatory proteins. We have already described how properdin acts as a positive regulatory protein on foreign surfaces, such as those of bacteria or damaged host cells, by stabilizing C3bBb.

1	Several negative regulatory proteins, present in plasma and in host-cell membranes, protect healthy host cells from the injurious effects of inappropriate complement activation on their surfaces. These complement-regulatory proteins interact with C3b and either prevent the convertase from forming or promote its rapid dissociation (Fig. 2.27). For example, a membrane-attached protein known as decay-accelerating factor (DAF or CD55) competes with factor B for binding to C3b on the cell surface and can displace Bb from a convertase that has already formed. Convertase formation can also be prevented by cleaving C3b to an inactive derivative, iC3b. This is achieved by a plasma protease, factor I, in conjunction with C3b-binding proteins that act as cofactors, such as membrane cofactor of proteolysis (MCP or CD46), another host-cell membrane protein (see Fig. 2.27). Cell-surface complement receptor type 1 (CR1, also known as CD35) behaves similarly to DAF and MCP in that it inhibits C3

1	(MCP or CD46), another host-cell membrane protein (see Fig. 2.27). Cell-surface complement receptor type 1 (CR1, also known as CD35) behaves similarly to DAF and MCP in that it inhibits C3 convertase formation and promotes the catabolism of C3b to inactive products, but it has a more limited tissue distribution. Factor H is another complement-regulatory protein in plasma that binds C3b, and like CR1, it is able to compete with factor B to displace Bb from the convertase; in addition, it acts as a cofactor for factor I. Factor H binds preferentially to C3b bound to vertebrate cells because it has an affinity for the sialic acid residues present on their cell surfaces (see Fig. 2.18). Thus, the amplification loop of the alternative pathway is allowed to proceed on the surface of a pathogen or on damaged host cells, but not on normal host cells or on tissues that express these negative regulatory proteins.

1	The C3 convertase of the classical and lectin pathways (C4b2a) is molecularly distinct from that of the alternative pathway (C3bBb). However, understanding of the complement system is simplified somewhat by recognition of the close evolutionary relationships between the different complement proteins (Fig. 2.28). Thus the complement zymogens, factor B and C2, are closely related proteins encoded by homologous genes located in tandem within the major The complement system and innate immunity. histocompatibility complex (MHC) on human chromosome 6. Furthermore, their respective binding partners, C3 and C4, both contain thioester bonds that provide the means of covalently attaching the C3 convertases to a pathogen surface.

1	Only one component of the alternative pathway seems entirely unrelated to its functional equivalents in the classical and lectin pathways: the initiating serine protease, factor D. Factor D can also be singled out as the only activating protease of the complement system to circulate as an active enzyme rather than a zymogen. This is both necessary for the initiation of the alternative pathway (through the cleavage of factor B bound to spontaneously activated C3) and safe for the host, because factor D has no other substrate than factor B bound to C3b. This means that factor D finds its substrate only at pathogen surfaces and at a very low level in plasma, where the alternative pathway of complement activation can be allowed to proceed. 2-11 Complement developed early in the evolution of multicellular organisms.

1	The complement system was originally known only from vertebrates, but homologs of C3 and factor B and a prototypical ‘alternative pathway’ have been discovered in nonchordate invertebrates. This is not altogether surprising since C3, which is cleaved and activated by serine proteases, is evolutionarily related to the serine protease inhibitor α2-macroglobulin, whose first appearance likely was in an ancestor to all modern vertebrates. The amplification loop of the alternative pathway also has an ancestral origin, as it is present in echinoderms (which include sea urchins and sea stars) and is based on a C3 convertase formed by the echinoderm homologs of C3 and factor B. These factors are expressed by phagocytic cells called amoeboid coelomocytes present in the coelomic fluid. Expression of C3 by these cells increases when bacteria are present. This simple system seems to function to opsonize bacterial cells and other foreign particles and facilitate their uptake by coelomocytes. C3

1	of C3 by these cells increases when bacteria are present. This simple system seems to function to opsonize bacterial cells and other foreign particles and facilitate their uptake by coelomocytes. C3 homologs in invertebrates are clearly related to each other. They all contain the distinctive thioester linkage and form a family of proteins, the thioester proteins, or TEPs. In the mosquito Anopheles, the production of protein TEP1 is induced in response to infection, and the protein may directly bind to bacterial surfaces to mediate phagocytosis of Gram-negative bacteria. Some form of C3 activity may even predate the evolution of the Bilateria—animals with bilateral symmetry, flatworms being

1	Fig. 2.28 There is a close evolutionary relationship among the factors of the alternative, lectin, and classical pathways of complement activation.

1	Most of the factors are either identical or the homologous products of genes that have duplicated and then diverged in sequence. The proteins C4 and C3 are homologous and contain the unstable thioester bond by which their large fragments, C4b and C3b, bind covalently to membranes. The genes encoding proteins C2 and factor B are adjacent in the MHC region of the genome and arose by gene duplication. The regulatory proteins factor H, CR1, and C4BP share a repeat sequence common to many complement-regulatory proteins. The greatest divergence between the pathways is in their initiation: in the classical pathway the C1 complex binds either to certain pathogens or to bound antibody, and in the latter circumstance it serves to convert antibody binding into enzyme activity on a specific surface; in the lectin pathway, mannose-binding lectin (MBL) associates with a serine protease, activating MBL-associated serine protease (MASP), to serve the same function as C1r:C1s; in the alternative

1	in the lectin pathway, mannose-binding lectin (MBL) associates with a serine protease, activating MBL-associated serine protease (MASP), to serve the same function as C1r:C1s; in the alternative pathway this enzyme activity is provided by factor D.

1	Fig. 2.29 Complement component C5 is cleaved when captured by a C3b molecule that is part of a C5 convertase complex. As shown in the top panel, C5 convertases are formed when C3b binds either the classical or lectin pathway C3 convertase C4b2a to form C4b2a3b, or the alternative pathway C3 convertase C3bBb to form C3b2Bb. C5 binds to C3b in these complexes (center panel). The bottom panel shows that C5 is cleaved by the active enzyme C2a or Bb to form C5b and the inflammatory mediator C5a. Unlike C3b and C4b, C5b is not covalently bound to the cell surface. The production of C5b initiates the assembly of the terminal complement components. the most primitive modern representatives—because genomic evidence of C3, factor B, and some later-acting complement components has been found in the Anthozoa (corals and sea anemones).

1	After its initial appearance, the complement system seems to have evolved by the acquisition of new activation pathways that allow specific targeting of microbial surfaces. The first to evolve was likely the ficolin pathway, which is present both in vertebrates and in some closely related invertebrates, such as the urochordates. Evolutionarily, the ficolins may predate the collectins, which are also first seen in the urochordates. Homologs of MBL and of the classical pathway complement component C1q, both collectins, have been identified in the genome of the ascidian urochordate Ciona (sea squirt). Two invertebrate homologs of mammalian MASPs also have been identified in Ciona, and it seems likely that they may be able to cleave and activate C3. Thus, the minimal complement system of the echinoderms appears to have been expanded in the urochordates by the recruitment of a specific activation system that may target C3 deposition onto microbial surfaces. This also suggests that when

1	echinoderms appears to have been expanded in the urochordates by the recruitment of a specific activation system that may target C3 deposition onto microbial surfaces. This also suggests that when adaptive immunity evolved, much later, the ancestral antibody molecule used an already diversified C1q-like collectin member to activate the complement pathway, and that the complement activation system evolved further by use of this collectin and its associated MASPs to become the initiating components of the classical complement pathway, namely, C1q, C1r, and C1s.

1	2-12 Surface-bound C3 convertase deposits large numbers of convertase activity.

1	We now return to the present-day complement system. The formation of C3 convertases is the point at which the three pathways of complement activation converge. The convertase of the lectin and classical pathways, C4b2a, and the convertase of the alternative pathway, C3bBb, initiate the same subsequent events—they cleave C3 to C3b and C3a. C3b binds covalently through its thioester bond to adjacent molecules on the pathogen surface; otherwise it is inactivated by hydrolysis. C3 is the most abundant complement protein in plasma, occurring at a concentration of 1.2 mg/ml, and up to 1000 molecules of C3b can bind in the vicinity of a single active C3 convertase (see Fig. 2.23). Thus, the main effect of complement activation is to deposit large quantities of C3b on the surface of the infecting pathogen, where the C3b forms a covalently bonded coat that can signal the ultimate destruction of the pathogen by phagocytes.

1	The next step in the complement cascade is the generation of the C5 convertases. C5 is a member of the same family of proteins as C3, C4, α2-macroglobulin, and the thioester-containing proteins (TEPs) of invertebrates. C5 does not form an active thioester bond during its synthesis but, like C3 and C4, it is cleaved by a specific protease into C5a and C5b fragments, each of which exerts specific downstream actions that are important in propagating the complement cascade. In the classical and the lectin pathways, a C5 convertase is formed by the binding of C3b to C4b2a to yield C4b2a3b. The C5 convertase of the alternative pathway is formed by the binding of C3b to the C3bBb convertase to form C3b2Bb. A C5 is captured by these C5 convertase complexes through binding to an acceptor site on C3b, and is thus rendered susceptible to cleavage by the serine protease activity of C2a or Bb. This reaction, which generates C5b and C5a, is much more limited than cleavage of C3, because C5 can be

1	C3b, and is thus rendered susceptible to cleavage by the serine protease activity of C2a or Bb. This reaction, which generates C5b and C5a, is much more limited than cleavage of C3, because C5 can be cleaved only when it binds to C3b that is in turn bound to C4b2a or C3bBb to form the active C5 convertase complex. Thus, complement activated by all three pathways leads to the binding of large numbers of C3b molecules on the surface of the pathogen, the generation of a more limited number of C5b molecules, and the release of C3a and a smaller amount of C5a (Fig. 2.29).

1	The complement system and innate immunity. 2-13 Ingestion of complement-tagged pathogens by phagocytes is mediated by receptors for the bound complement proteins. The most important action of complement is to facilitate the uptake and destruction of pathogens by phagocytic cells. This occurs by the specific recognition of bound complement components by complement receptors (CRs) on phagocytes. These complement receptors bind pathogens opsonized with complement components: opsonization of pathogens is a major function of C3b and its proteolytic derivatives. C4b also acts as an opsonin but has a relatively minor role, largely because so much more C3b than C4b is generated.

1	The known receptors for bound complement components C5a and C3a are listed, with their functions and distributions, in Fig. 2.30. The C3b receptor CR1, described in Section 2-10, is a negative regulator of complement activation (see Fig. 2.27). CR1 is expressed on many types of immune cells, including macrophages and neutrophils. Binding of C3b to CR1 cannot by itself stimulate phagocytosis, but it can lead to phagocytosis in the presence of other immune mediators that activate macrophages. For example, the small complement fragment C5a can activate macrophages to ingest bacteria bound to their CR1 receptors (Fig. 2.31). C5a binds to another receptor expressed by macrophages, the C5a receptor, which has seven membrane-spanning domains. Receptors of this type transduce their signals via intracellular guanine-nucleotide-binding proteins called G proteins and are known generally as G-protein-coupled receptors (GPCRs); they are discussed in Section 3-2. C5L2 (GPR77), expressed by

1	via intracellular guanine-nucleotide-binding proteins called G proteins and are known generally as G-protein-coupled receptors (GPCRs); they are discussed in Section 3-2. C5L2 (GPR77), expressed by neutrophils and macrophages, is a

1	Fig. 2.30 Distribution and function of cell-surface receptors for complement proteins. A variety of complement receptors are specific for bound C3b and its cleavage products (iC3b and C3dg). CR1 and CR3 are important in inducing the phagocytosis of bacteria with complement components bound to their surface. CR2 is found mainly on B cells, where it is part of the B-cell co-receptor complex CR1 and CR2 share structural features with the complement-regulatory proteins that bind C3b and C4b. CR3 and CR4 are integrins composed of integrin β2 paired with either integrin αM (CD11b) or integrin αX (CD11c), respectively (see Appendix II); CR3, also called Mac-1, is also important for leukocyte adhesion and migration, as we shall see in Chapter 3, whereas CR4 is only known to function in phagocytosis. The receptors for C5a and C3a are seven-span G-protein-coupled receptors. FDC, follicular dendritic cells; these are not involved in innate immunity and are discussed in later chapters.

1	Fig. 2.31 The anaphylatoxin C5a can enhance the phagocytosis of microorganisms opsonized in an innate immune response. Activation of complement leads to the deposition of C3b on the surface of microorganisms (left panel). C3b can be bound by the complement receptor CR1 on the surface of phagocytes, but this on its own is insufficient to induce phagocytosis (center panel). Phagocytes also express receptors for the anaphylatoxin C5a, and binding of C5a will now activate the cell to phagocytose microorganisms bound through CR1 (right panel).

1	Fig. 2.32 The cleavage products of C3b are recognized by different complement receptors. After C3b is deposited on the surface of pathogens, it can undergo several conformational changes that alter its interaction with complement receptors. Factor I and MCP can cleave the C3f fragment from C3b, producing iC3b, which is a ligand for the complement receptors CR2, CR3, and CR4, but not CR1. Factor I and CR1 cleave iC3b to release C3c, leaving C3dg bound. C3dg is then recognized by CR2. When only C3b binds to CR1, bacteria are not phagocytosed nonsignaling receptor that acts as a decoy receptor for C5a and may regulate activity of the C5a receptor. Proteins associated with the extracellular matrix, such as fibronectin, can also contribute to phagocyte activation; these are encountered when phagocytes are recruited to connective tissue and activated there.

1	Four other complement receptors—CR2 (also known as CD21), CR3 (CD11b:CD18), CR4 (CD11c:CD18), and CRIg (complement receptor of the immunoglobulin family)—bind to forms of C3b that have been cleaved by factor I but that remain attached to the pathogen surface. Like several other key components of complement, C3b is subject to regulatory mechanisms that cleave it into derivatives, such as iC3b, that cannot form an active convertase. C3b bound to the microbial surface can be cleaved by factor I and MCP to remove the small fragment C3f, leaving the inactive iC3b form bound to the surface (Fig. 2.32). iC3b is recognized by several complement receptors—CR2, CR3, CR4, and CRIg. Unlike the binding of iC3b to CR1, the binding of iC3b to the receptor CR3 is sufficient on its own to stimulate phagocytosis. Factor I and CR1 cleave iC3b to release C3c, leaving C3dg bound to the pathogen. C3dg is recognized only by CR2. CR2 is found on B cells as part of a co-receptor complex that can augment the

1	Factor I and CR1 cleave iC3b to release C3c, leaving C3dg bound to the pathogen. C3dg is recognized only by CR2. CR2 is found on B cells as part of a co-receptor complex that can augment the signal received through the antigen-specific immunoglobulin receptor. Thus, a B cell whose antigen receptor is specific for an antigen of a pathogen will receive a strong signal on binding this antigen if it or the pathogen is also coated with C3dg. The activation of complement can therefore contribute to producing a strong antibody response.

1	The importance of opsonization by C3b and its inactive fragments in destroying extracellular pathogens can be seen in the effects of various complement deficiencies. For example, individuals deficient in C3 or in molecules that catalyze C3b deposition show an increased susceptibility to infection by a wide range of extracellular bacteria, including Streptococcus pneumoniae. We describe the effects of various defects in complement and the diseases they cause in Chapter 13. The complement system and innate immunity. 2-14 The small fragments of some complement proteins initiate a local inflammatory response.

1	The small complement fragments C3a and C5a act on specific receptors on endothelial cells and mast cells (see Fig. 2.30) to produce local inflammatory responses. Like C5a, C3a also signals through a G-protein-coupled receptor, discussed in more detail in Chapter 3. C4a, although generated during C4 cleavage, is not potent at inducing inflammation, is inactive at C3a and C5a receptors, and seems to lack a receptor of its own. When produced in large amounts or injected systemically, C3a and C5a induce a generalized circulatory collapse, producing a shocklike syndrome similar to that seen in a systemic allergic reaction involving antibodies of the IgE class, discussed in Chapter 14. Such a reaction is termed anaphylactic shock, and these small fragments of complement are therefore often referred to as anaphylatoxins. C5a has the highest specific biological activity, but both C3a and C5a induce the contraction of smooth muscle and increase vascular permeability and act on the endothelial

1	to as anaphylatoxins. C5a has the highest specific biological activity, but both C3a and C5a induce the contraction of smooth muscle and increase vascular permeability and act on the endothelial cells lining blood vessels to induce the synthesis of adhesion molecules. In addition, C3a and C5a can activate the mast cells that populate submucosal tissues to release inflammatory molecules such as histamine and the cytokine tumor necrosis factor-α (TNF-α), which cause similar effects. The changes induced by C5a and C3a recruit antibody, complement, and phagocytic cells to the site of an infection (Fig. 2.33), and the increased fluid in the tissues hastens the movement of pathogen-bearing antigen-presenting cells to the local lymph nodes, contributing to the prompt initiation of the adaptive immune response.

1	Small complement-cleavage products act on blood vessels to increase vascular permeability and cell-adhesion molecules Migration of macrophages, polymorphonuclear leukocytes (PMNs), and lymphocytes is increased. Microbicidal activity of macrophages and PMNs is also increased

1	Fig. 2.33 Local inflammatory responses can be induced by small complement fragments, especially C5a. The small complement fragments are differentially active: C5a is more active than C3a; C4a is weak or inactive. C5a and C3a cause local inflammatory responses by acting directly on local blood vessels, stimulating an increase in blood flow, increased vascular permeability, and increased binding of phagocytes to endothelial cells. C3a and C5a also activate mast cells (not shown) to release mediators, such as histamine and TNF-α, that contribute to the inflammatory response. The increase in vessel diameter and permeability leads to the accumulation of fluid and protein in the surrounding tissue. Fluid accumulation increases lymphatic drainage, bringing pathogens and their antigenic components to nearby lymph nodes. The antibodies, complement, and cells thus recruited participate in pathogen clearance by enhancing phagocytosis. The small complement fragments can also directly increase the

1	to nearby lymph nodes. The antibodies, complement, and cells thus recruited participate in pathogen clearance by enhancing phagocytosis. The small complement fragments can also directly increase the activity of the phagocytes.

1	Deficiency of the C8 Complement Component C5a also acts directly on neutrophils and monocytes to increase their adherence to vessel walls, their migration toward sites of antigen deposition, and their ability to ingest particles; it also increases the expression of CR1 and CR3 on the surfaces of these cells. In this way, C5a, and to a smaller extent C3a and C4a, act in concert with other complement components to hasten the destruction of pathogens by phagocytes. 2-15 The terminal complement proteins polymerize to form pores in membranes that can kill certain pathogens.

1	2-15 The terminal complement proteins polymerize to form pores in membranes that can kill certain pathogens. One of the important effects of complement activation is the assembly of the terminal components of complement (Fig. 2.34) to form a membrane-attack complex. The reactions leading to the formation of this complex are shown schematically in Fig. 2.35. The end result is a pore in the lipid bilayer membrane that destroys membrane integrity. This is thought to kill the pathogen by destroying the proton gradient across the pathogen’s cell membrane.

1	The first step in the formation of the membrane-attack complex is the cleavage of C5 by a C5 convertase to release C5b (see Fig. 2.29). In the next stages (see Fig. 2.35), C5b initiates the assembly of the later complement components and their insertion into the cell membrane. The process begins when one molecule of C5b binds one molecule of C6, and the C5b6 complex then binds one molecule of C7. This reaction leads to a conformational change in the constituent molecules, with the exposure of a hydrophobic site on C7, which inserts into the lipid bilayer. Similar hydrophobic sites are exposed on the later components C8 and C9 when they are bound to the complex, allowing these proteins also to insert into the lipid bilayer. C8 is a complex of two proteins, C8β and C8α-γ. The C8β protein binds to C5b, and the binding of C8β to the membrane-associated C5b67 complex allows the hydrophobic domain of C8α-γ to insert into the lipid bilayer. Finally, C8α-γ induces the polymerization of 10–16

1	to C5b, and the binding of C8β to the membrane-associated C5b67 complex allows the hydrophobic domain of C8α-γ to insert into the lipid bilayer. Finally, C8α-γ induces the polymerization of 10–16 molecules of C9 into a pore-forming structure called the membrane-attack complex. The membrane-attack complex has a hydrophobic external face, allowing it to associate with the lipid bilayer, but a hydrophilic internal channel. The diameter of this channel is about 10 nm, allowing the free passage of solutes and water across the lipid bilayer. The pore damage to the lipid bilayer leads to the loss of cellular homeostasis, the disruption of the proton gradient across the membrane, the penetration of enzymes such as lysozyme into the cell, and the eventual destruction of the pathogen.

1	Although the effect of the membrane-attack complex is very dramatic, particularly in experimental demonstrations in which antibodies against red blood cell membranes are used to trigger the complement cascade, the significance The complement system and innate immunity.

1	Fig. 2.35 Assembly of the membrane-attack complex polymerization of C9, again with the exposure of a hydrophobic generates a pore in the lipid bilayer membrane. The sequence site. Up to 16 molecules of C9 are then added to the assembly of steps and their approximate appearance are shown here in to generate a channel 10 nm in diameter in the membrane. This schematic form. C5b triggers the assembly of a complex of channel disrupts the bacterial cell membrane, killing the bacterium. one molecule each of C6, C7, and C8, in that order. C7 and C8 The electron micrographs show erythrocyte membranes with undergo conformational changes, exposing hydrophobic domains membrane-attack complexes in two orientations, end on and side that insert into the membrane. This complex causes moderate on. Photographs courtesy of S. Bhakdi and J. Tranum-Jensen. membrane damage in its own right, and also serves to induce the of these components in host defense seems to be quite limited. So far, deficiencies in

1	courtesy of S. Bhakdi and J. Tranum-Jensen. membrane damage in its own right, and also serves to induce the of these components in host defense seems to be quite limited. So far, deficiencies in complement components C5–C9 have been associated with susceptibility only to Neisseria species, the bacteria that cause the sexually transmitted disease gonorrhea and a common form of bacterial meningitis. Thus, the opsonizing and inflammatory actions of the earlier components of the complement cascade are clearly more important for host defense against infection. Formation of the membrane-attack complex seems to be important only for the killing of a few pathogens, although, as we will see in Chapter 15, this complex might well have a major role in immunopathology.

1	of complement activation and protect the host from their destructive effects. Complement activation usually is initiated on a pathogen surface, and the activated complement fragments that are produced usually bind nearby on the pathogen surface or are rapidly inactivated by hydrolysis. Even so, all complement components are activated spontaneously at a low rate in plasma, and these activated complement components will sometimes bind proteins on host cells. Section 2-10 introduced the soluble host proteins factor I and factor H and the membrane-bound proteins MCP and DAF that regulate the alternative pathway of complement activation. In addition to these, several other soluble and membrane-bound complement-control proteins can regulate the complement cascade at various steps to protect normal host cells while allowing complement activation to proceed on pathogen surfaces (Fig. 2.36).

1	The activation of C1 is controlled by the C1 inhibitor (C1INH), which is a plasma serine protease inhibitor, or serpin. C1INH binds to the active enzymes C1r:C1s and causes them to dissociate from C1q, which remains bound to the pathogen (Fig. 2.37). In this way, C1INH limits the time during which active C1s is able to cleave C4 and C2. By the same means, C1INH limits the spontaneous activation of C1 in plasma. Its importance can be seen in the C1INH deficiency disease hereditary angioedema (HAE), in which chronic spontaneous complement activation leads to the production of excess cleaved fragments of C4 and C2. The large activated fragments from this cleavage, which normally combine to form the C3 convertase, do not damage host cells in such patients because C4b is rapidly inactivated by hydrolysis in plasma, The complement system and innate immunity.

1	The complement system and innate immunity. Fig. 2.37 Complement activation is regulated by a series of proteins that serve to protect host cells from accidental damage. These act on different stages of the complement cascade, dissociating complexes or catalyzing the enzymatic degradation of covalently bound complement proteins. Stages in the complement cascade are shown schematically down the left side of the figure, with the regulatory reactions on the right. The alternative-pathway C3 convertase is similarly regulated by DAF, CR1, MCP, and factor H.

1	and the convertase does not form. However, the small fragment of C2, C2b, is further cleaved into a peptide, the C2 kinin, that causes extensive swell-ing—the most dangerous being local swelling in the larynx that can lead to suffocation. Bradykinin, which has similar actions to those of C2 kinin, is also produced in an uncontrolled fashion in this disease, as a result of the lack of inhibition of kallikrein, another plasma protease and component of the kinin system (discussed in Section 3-3). Kallikrein is activated by tissue damage and is also regulated by C1INH. Hereditary angioedema is fully corrected by replacing C1INH. A similar, extremely rare human disease stems from a partial deficiency of carboxypeptidase N (CPN), a metalloproteinase that inactivates the anaphylatoxins C3a and C5a as well as bradykinin and kallikrein. Humans with partial CPN deficiency exhibit recurrent angioedema due to delayed inactivation of serum C3a and bradykinin.

1	Since the highly reactive thioester bond of activated C3 and C4 cannot distinguish between acceptor groups on a host cell from those on the surface of a pathogen, mechanisms have evolved that prevent the small amounts of C3 or C4 molecules deposited on host cells from fully triggering complement activation. We introduced these mechanisms in the context of control of the alternative pathway (see Fig. 2.27), but they are also important regulators of the classical pathway convertase (see Fig. 2.37, second and third rows). Section 2-10 described the proteins that inactivate any C3b or C4b that has bound to host cells. These are the plasma factor I and its cofactors MCP and CR1, which are membrane proteins. Circulating factor I is an active serine protease, but it can cleave C3b and C4b only when they are bound to MCP and CR1. In these circumstances, factor I cleaves C3b, first into iC3b and then further to C3dg, thus permanently inactivating it. C4b is similarly inactivated by cleavage

1	when they are bound to MCP and CR1. In these circumstances, factor I cleaves C3b, first into iC3b and then further to C3dg, thus permanently inactivating it. C4b is similarly inactivated by cleavage into C4c and C4d. Microbial cell walls lack MCP and CR1 and thus cannot promote the breakdown of C3b and C4b, which instead act as binding sites for factor B and C2, promoting complement activation. The importance of factor I can be seen in people with genetically determined factor I deficiency. Because of uncontrolled complement activation, complement proteins rapidly become depleted and such people suffer repeated bacterial infections, especially with ubiquitous pyogenic bacteria.

1	There are also plasma proteins with cofactor activity for factor I, most notably C4b-binding protein (C4BP) (see Fig. 2.36). It binds C4b and acts mainly as a regulator of the classical pathway in the fluid phase. Another is factor H, which binds C3b in the fluid phase as well as at cell membranes and helps to distinguish the C3b that is bound to host cells from that bound to microbial surfaces. The higher affinity of factor H for sialic acid residues on host membrane glycoproteins allows it to displace factor B in binding to C3b on host cells. Also, C3b at cell membranes is bound by cofactor proteins DAF and MCP. Factor H, DAF, and MCP effectively compete with factor B for binding to C3b bound to host cells, so that the bound C3b is catabolized by factor I into iC3b and C3dg and complement activation is inhibited. In contrast, factor B is favored for binding C3b on microbial membranes, which do not express DAF or MCP and which lack the sialic acid modifications that attract factor H.

1	activation is inhibited. In contrast, factor B is favored for binding C3b on microbial membranes, which do not express DAF or MCP and which lack the sialic acid modifications that attract factor H. The greater amount of factor B on a microbial surface stimulates formation of more C3bBb C3 convertase, and thus complement activation is amplified.

1	The critical balance between the inhibition and the activation of complement on cell surfaces is illustrated in individuals heterozygous for mutations in any of the regulatory proteins MCP, factor I, or factor H. In such individuals, the concentration of functional regulatory proteins is reduced, and the tipping of the balance toward complement activation leads to a predisposition to atypical hemolytic uremic syndrome, a condition characterized by damage to platelets and red blood cells and by kidney inflammation. Another serious health problem related to complement malfunction is a significantly increased The complement system and innate immunity.

1	The complement system and innate immunity. risk of age-related macular degeneration, the leading cause of blindness in the elderly in developed countries, which has been predominantly linked to single-nucleotide polymorphisms in the factor H gene. Polymorphisms in other complement genes have also been found to be either detrimental or protective for this disease. Thus, even small alterations in the efficiency of either the activation or the regulation of this powerful effector system can contribute to the progression of degenerative or inflammatory disorders.

1	The competition between DAF or MCP and factor B for binding to surface-bound C3b is an example of the second mechanism for inhibiting complement activation on host cells. By binding C3b and C4b on the cell surface, these proteins competitively inhibit the binding of C2 to cell-bound C4b and of factor B to cell-bound C3b, thereby inhibiting convertase formation. DAF and MCP also mediate protection against complement through a third mechanism, which is to augment the dissociation of C4b2a and C3bBb convertases that have already formed. Like DAF, CR1 is among the host-cell membrane molecules that regulate complement through both these mechanisms—that is, by promoting the dissociation of convertase and exhibiting cofactor activity. All the proteins that bind the homologous C4b and C3b molecules share one or more copies of a structural element called the short consensus repeat (SCR), the complement control protein (CCP) repeat, or (especially in Japan) the sushi domain.

1	In addition to the mechanisms for preventing C3 convertase formation and C4 and C3 deposition on cell membranes, there are also inhibitory mechanisms that prevent the inappropriate insertion of the membrane-attack complex (MAC) into membranes. We saw in Section 2-15 that the membrane-attack complex polymerizes onto C5b molecules created by the action of C5 convertase. The MAC complex mainly inserts into cell membranes adjacent to the site of the C5 convertase, that is, close to the site of complement activation on a pathogen. However, some newly formed membrane-attack complexes may diffuse from the site of complement activation and insert into adjacent host-cell membranes. Several plasma proteins, including, notably, vitronectin (also known as S-protein), bind to the C5b67, C5b678, and C5b6789 complexes and thereby inhibit their random insertion into cell membranes. Host-cell membranes also contain an intrinsic protein, CD59, or protectin, that inhibits the binding of C9 to the C5b678

1	complexes and thereby inhibit their random insertion into cell membranes. Host-cell membranes also contain an intrinsic protein, CD59, or protectin, that inhibits the binding of C9 to the C5b678 complex (see Fig. 2.37, bottom row). CD59 and DAF are both linked to the cell surface by a glycosylphosphatidylinositol (GPI) tail, like many other peripheral membrane proteins. One of the enzymes involved in the synthesis of GPI tails is encoded by a gene, PIGA, on the X chromosome. In people with a somatic mutation in this gene in a clone of hematopoietic cells, both CD59 and DAF fail to function. This causes the disease paroxysmal nocturnal hemoglobinuria, which is characterized by episodes of intravascular red blood cell lysis by complement. Red blood cells that lack only CD59 are also susceptible to destruction as a result of spontaneous activation of the complement cascade.

1	2-17 Pathogens produce several types of proteins that can inhibit complement activation. Bacterial pathogens have evolved various strategies to avoid activation of complement, and thereby to avoid elimination by this first line of innate defense (Fig. 2.38). One strategy that many pathogens employ is to mimic host surfaces by attracting host complement regulators to their own surfaces. A mechanism to achieve this is for the pathogen to express surface proteins that bind to soluble complement-regulatory proteins such as C4BP and factor H. For example, the Gram-negative pathogen Neisseria meningitidis produces factor H binding protein (fHbp), which recruits factor H (see Section 2-10), and the outer Fig. 2.38 Complement evasion proteins produced by various pathogens.

1	Fig. 2.38 Complement evasion proteins produced by various pathogens. membrane protein PorA, which binds to C4BP. By recruiting factor H and C4BP to the pathogen membrane, the pathogen is able to inactivate C3b that is deposited on its surface and thereby avoid the consequences of complement activation. Complement is important in defense against Neisseria species, and several complement deficiencies are associated with increased susceptibility to this pathogen.

1	Another strategy employed by pathogens is to secrete proteins that directly inhibit components of complement. The Gram-positive pathogen Staphylococcus aureus provides several examples of this type of strategy. Staphylococcal protein A (Spa) binds to the Fc regions of immunoglobulins and interferes with the recruitment and activation of C1. This binding specificity was used as an early biochemical technique in the purification of antibodies. The staphylococcal protein staphylokinase (SAK) acts by cleaving immunoglobulins bound to the pathogen membrane, preventing complement activation and avoiding phagocytosis. The staphylococcal complement inhibitor (SCIN) protein binds to the classical C3 convertase, C4b2a, and the alternative pathway C3 convertase, C3bBb, and inhibits their activity. Other stages of complement activation, including formation of the C5 convertase, are targets of inhibition by proteins produced by these and other pathogens. We will return to this topic of complement

1	stages of complement activation, including formation of the C5 convertase, are targets of inhibition by proteins produced by these and other pathogens. We will return to this topic of complement regulation in Chapter 13 when we discuss how the immune system sometimes fails or is evaded by pathogens.

1	Summary. The complement system is one of the major mechanisms by which pathogen recognition is converted into an effective host defense against initial infection. Complement is a system of plasma proteins that can be activated directly by pathogens or indirectly by pathogen-bound antibody, leading to a cascade of The complement system and innate immunity.

1	reactions that occurs on the surface of pathogens and generates active components with various effector functions. There are three pathways of complement activation: the lectin pathway, triggered by the pattern recognition receptors MBL and the ficolins; the classical pathway, triggered directly by antibody binding to the pathogen surface; and the alternative pathway, which utilizes spontaneous C3 deposition onto microbial surfaces, is augmented by proper-din, and provides an amplification loop for the other two pathways. The early events in all pathways consist of a sequence of cleavage reactions in which the larger cleavage product binds covalently to the pathogen surface and contributes to the activation of the next component. The pathways converge with the formation of a C3 convertase enzyme, which cleaves C3 to produce the active complement component C3b. The binding of large numbers of C3b molecules to the pathogen is the central event in complement activation. Bound complement

1	enzyme, which cleaves C3 to produce the active complement component C3b. The binding of large numbers of C3b molecules to the pathogen is the central event in complement activation. Bound complement components, especially bound C3b and its inactive fragments, are recognized by specific complement receptors on phagocytic cells, which engulf pathogens opsonized by C3b and its inactive fragments. The small cleavage fragments of C3 and C5 act on specific trimeric G-protein-coupled receptors to recruit phagocytes, such as neutrophils, to sites of infection. Together, these activities promote the uptake and destruction of pathogens by phagocytes. The molecules of C3b that bind the C3 convertase itself initiate the late events of complement, binding C5 to make it susceptible to cleavage by C2a or Bb. The larger C5b fragment triggers the assembly of a membrane-attack complex, which can result in the lysis of certain pathogens. A system of soluble and membrane-bound complement-regulatory

1	C2a or Bb. The larger C5b fragment triggers the assembly of a membrane-attack complex, which can result in the lysis of certain pathogens. A system of soluble and membrane-bound complement-regulatory proteins act to limit complement activation on host tissues, in order to prevent tissue damage from the inadvertent binding of activated complement components to host cells or from the spontaneous activation of complement components in plasma. Many pathogens produce a variety of soluble and membrane-associated proteins that can counteract complement activation and contribute to infection of the host by the microbe.

1	Summary to Chapter 2.

1	This chapter has described the preexisting, constitutive components of innate immunity. The body’s epithelial surfaces are a constant barrier to pathogen entry and have specialized adaptations, such as cilia, various antimicrobial molecules and mucus, that provide the simplest form of innate immunity. The complement system is a more specialized system that combines direct recognition of microbes with a complex effector system. Of the three pathways that can activate complement, two are devoted to innate immunity. The lectin pathway relies on pattern recognition receptors that detect microbial membranes, while the alternative pathway relies on spontaneous complement activation that is down-regulated by host molecules expressed on self membranes. The main event in complement activation is accumulation of C3b on microbial membranes, which is recognized by complement receptors on phagocytic cells to promote microbial clearance by cells recruited to sites of infection by C3a and C5a. In

1	accumulation of C3b on microbial membranes, which is recognized by complement receptors on phagocytic cells to promote microbial clearance by cells recruited to sites of infection by C3a and C5a. In addition, C5b initiates the membrane-attack complex that is able to lyse some microbes directly. The complement cascade is under regulation to prevent attack on host tissues, and genetic variation in regulatory pathways can result in autoimmune syndromes and age-related tissue damage.

1	Questions. 2.1 Multiple Choice: The widely used β-lactam antibiotics are mainly active against Gram-positive bacteria. These inhibit the transpeptidation step in synthesis of peptidoglycan, a major component of the bacterial cell wall that is critical for the survival of the microorganism. Which of the following is an antimicrobial enzyme that functions to disrupt the same bacterial structure that β-lactams ultimately target? A. Phospholipase A B. Lysozyme C. Defensins D. Histatins 2.2 Short Answer: Why is the capacity of mannose-binding lectin (MBL) trimers to oligomerize important for their function? 2.3 Multiple Choice: Choose the option that correctly describes ficolins. A. C-type lectin domain, affinity for carbohydrates such as fucose and N-acetylglucosamine (GlcNAc), synthesized in the liver B. Fibrinogen-like domain, affinity for oligosaccharides containing acetylated sugars, synthesized in the liver

1	B. Fibrinogen-like domain, affinity for oligosaccharides containing acetylated sugars, synthesized in the liver C. C-type lectin domain, affinity for oligosaccharides containing acetylated sugars, synthesized in the liver D. Fibrinogen-like domain, affinity for carbohydrates such as fucose and N-acetylglucosamine (GlcNAc), synthetized in the liver and lungs 2.4 Fill-in-the-Blanks: For each of the following sentences, fill in the blanks with the best word selected from the list below. Not all words will be used; each word should be used only once. Like MBLs, ficolins form oligomers with ________ and ________. Such interaction allows the oligomer to cleave the complement components ________ and ________. Once these are cleaved, they form ________, a C3 convertase, which cleaves ________ and permits the formation of the membrane-attack complex.

1	2.5 Short Answer: One way in which the alternative pathway activates is by spontaneous hydrolysis of the C3 thioester bond that is normally used to covalently attach to the pathogen’s surface. How can the alternative pathway proceed to form a membrane-attack complex if the C3 convertase that initiates this process is soluble? 2.6 Fill-in-the-Blanks: Paroxysmal nocturnal hemoglobinuria, a disease characterized by episodes of intravascular red blood cell lysis, is the result of red blood cells losing the expression of ________ and ________, which renders them susceptible to lysis by the ________ pathway of the complement system. 2.7 Matching: Match each of the following complement-regulatory proteins with the pathological manifestation that would develop if this factor were defective: A. C1INH 1. Atypical hemolytic uremic syndrome B. Factor H & factor I 2. Hereditary angioedema

1	A. C1INH 1. Atypical hemolytic uremic syndrome B. Factor H & factor I 2. Hereditary angioedema C. DAF 3. Paroxysmal nocturnal hemoglobinuria 2.8 Multiple Choice: Diseases such as cryoglobulinemia and systemic lupus erythematosus usually present with low C3 and C4 levels in blood due to the activation of the classical complement pathway. In contrast, diseases such as dense deposit disease or C3 glomerulonephritis generally have low C3 due to activation of the alternative complement pathway. What would be the expected levels of C2 and C4 in a patient suffering from dense deposit disease or C3 glomerulonephritis? A. Normal B. High C. Low D. High C4 and low C3 2.9 True or False: Mucins secreted at a mucosal surface exhibit direct microbicidal activities. 2.10 Short Answer: Neisseria meningitidis and Staphylococcus aureus each prevent complement activation in different ways. Explain how each does so.

1	2.10 Short Answer: Neisseria meningitidis and Staphylococcus aureus each prevent complement activation in different ways. Explain how each does so. 2.11 True or False: Both neutrophils and Paneth cells of the gut secrete antimicrobial peptides, such as defensins, only upon stimulation. 2.12 Short Answer: What are two products of the C3 convertase? Name three downstream events that can result from the formation of these products and lead to clearance of the microbe. 2.13 True or False: CD21 (CR1) is a complement receptor expressed on B cells that binds C3dg (a C3b breakdown product) and serves as a co-receptor to augment signaling and trigger a stronger antibody response. References. Section references. 2-1 Infectious diseases are caused by diverse living agents that replicate in their hosts. Kauffmann, S.H.E., Sher, A., and Ahmed, R.: Immunology of Infectious Diseases. Washington, DC: ASM Press, 2002.

1	Kauffmann, S.H.E., Sher, A., and Ahmed, R.: Immunology of Infectious Diseases. Washington, DC: ASM Press, 2002. Mandell, G.L., Bennett, J.E., and Dolin, R. (eds): Principles and Practice of Infectious Diseases, 4th ed. New York: Churchill Livingstone, 1995. 2-2 Epithelial surfaces of the body provide the first line of defense against infection. Gallo, R.L., and Hooper, L.V.: Epithelial antimicrobial defense of the skin and intestine. Nat. Rev. Immunol. 2012, 12:503–516. 2-3 Infectious agents must overcome innate host defenses to establish a focus of infection. Gorbach, S.L., Bartlett, J.G., and Blacklow, N.R. (eds): Infectious Diseases, 3rd ed. Philadelphia: Lippincott Williams & Wilkins, 2003. Hornef, M.W., Wick, M.J., Rhen, M., and Normark, S.: Bacterial strategies for overcoming host innate and adaptive immune responses. Nat. Immunol. 2002, 3:1033–1040. 2-4 Epithelial cells and phagocytes produce several kinds of antimicrobial proteins.

1	2-4 Epithelial cells and phagocytes produce several kinds of antimicrobial proteins. Cash, H.L., Whitham, C.V., Behrendt, C.L., and Hooper, L.H.: Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science 2006, 313:1126–1130. De Smet, K., and Contreras, R.: Human antimicrobial peptides: defensins, cathelicidins and histatins. Biotechnol. Lett. 2005, 27:1337–1347. Ganz, T.: Defensins: antimicrobial peptides of innate immunity. Nat. Rev. Immunol. 2003, 3:710–720. Mukherjee, S., Zheng, H., Derebe, M.G., Callenberg, K.M., Partch, C.L., Rollins, D., Propheter, D.C., Rizo, J., Grabe, M., Jiang, Q.X., and Hooper, L.V.: Antibacterial membrane attack by a pore-forming intestinal C-type lectin. Nature 2014, 505:103–107. Zanetti, M.: The role of cathelicidins in the innate host defense of mammals. Curr. Issues Mol. Biol. 2005, 7:179–196. 2-5 The complement system recognizes features of microbial surfaces and marks them for destruction by coating them with C3b.

1	2-5 The complement system recognizes features of microbial surfaces and marks them for destruction by coating them with C3b. Gros, P., Milder, F.J., and Janssen, B.J.: Complement driven by conformational changes. Nat. Rev. Immunol. 2008, 8:48–58. Janssen, B.J., Christodoulidou, A., McCarthy, A., Lambris, J.D., and Gros, P.: Structure of C3b reveals conformational changes that underlie complement activity. Nature 2006, 444:213–216. Janssen, B.J., Huizinga, E.G., Raaijmakers, H.C., Roos, A., Daha, M.R., Nilsson-Ekdahl, K., Nilsson, B., and Gros, P.: Structures of complement component C3 provide insights into the function and evolution of immunity. Nature 2005, 437:505–511. 2-6 The lectin pathway uses soluble receptors that recognize microbial surfaces to activate the complement cascade.

1	2-6 The lectin pathway uses soluble receptors that recognize microbial surfaces to activate the complement cascade. Bohlson, S.S., Fraser, D.A., and Tenner, A.J.: Complement proteins C1q and MBL are pattern recognition molecules that signal immediate and long-term protective immune functions. Mol. Immunol. 2007, 44:33–43. Fujita, T.: Evolution of the lectin-complement pathway and its role in innate immunity. Nat. Rev. Immunol. 2002, 2:346–353. Gál, P., Harmat, V., Kocsis, A., Bián, T., Barna, L., Ambrus, G., Végh, B., Balczer, J., Sim, R.B., Náray-Szabó, G., et al: A true autoactivating enzyme. Structural insight into mannose-binding lectin-associated serine protease-2 activations. J. Biol. Chem. 2005, 280:33435–33444.

1	Héja, D., Kocsis, A., Dobó, J., Szilágyi, K., Szász, R., Závodszky, P., Pál, G., Gál, P.: Revised mechanism of complement lectin-pathway activation revealing the role of serine protease MASP-1 as the exclusive activator of MASP-2. Proc. Natl Acad. Sci. USA 2012, 109:10498–10503. Wright, J.R.: Immunoregulatory functions of surfactant proteins. Nat. Rev. Immunol. 2005, 5:58–68. 2-7 The classical pathway is initiated by activation of the C1 complex and is homologous to the lectin pathway. McGrath, F.D., Brouwer, M.C., Arlaud, G.J., Daha, M.R., Hack, C.E., and Roos, A.: Evidence that complement protein C1q interacts with C-reactive protein through its globular head region. J. Immunol. 2006, 176:2950–2957. 2-8 Complement activation is largely confined to the surface on which it is initiated.

1	2-8 Complement activation is largely confined to the surface on which it is initiated. Cicardi, M., Bergamaschini, L., Cugno, M., Beretta, A., Zingale, L.C., Colombo, M., and Agostoni, A.: Pathogenetic and clinical aspects of C1 inhibitor deficiency. Immunobiology 1998, 199:366–376. 2-9 The alternative pathway is an amplification loop for C3b formation that is accelerated by properdin in the presence of pathogens. Fijen, C.A., van den Bogaard, R., Schipper, M., Mannens, M., Schlesinger, M., Nordin, F.G., Dankert, J., Daha, M.R., Sjoholm, A.G., Truedsson, L., et al.: Properdin deficiency: molecular basis and disease association. Mol. Immunol. 1999, 36:863–867. Kemper, C., and Hourcade, D.E.: Properdin: new roles in pattern recognition and target clearance. Mol. Immunol. 2008, 45:4048–4056.

1	Kemper, C., and Hourcade, D.E.: Properdin: new roles in pattern recognition and target clearance. Mol. Immunol. 2008, 45:4048–4056. Spitzer, D., Mitchell, L.M., Atkinson, J.P., and Hourcade, D.E.: Properdin can initiate complement activation by binding specific target surfaces and providing a platform for de novo convertase assembly. J. Immunol. 2007, 179:2600–2608. Xu, Y., Narayana, S.V., and Volanakis, J.E.: Structural biology of the alternative pathway convertase. Immunol. Rev. 2001, 180:123–135. 2-10 Membrane and plasma proteins that regulate the formation and stability of C3 convertases determine the extent of complement activation. Golay, J., Zaffaroni, L., Vaccari, T., Lazzari, M., Borleri, G.M., Bernasconi, S., Tedesco, F., Rambaldi, A., and Introna, M.: Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood 2000, 95:3900–3908.

1	Spiller, O.B., Criado-Garcia, O., Rodriguez De Cordoba, S., and Morgan, B.P.: Cytokine-mediated up-regulation of CD55 and CD59 protects human hepatoma cells from complement attack. Clin. Exp. Immunol. 2000, 121:234–241. Varsano, S., Frolkis, I., Rashkovsky, L., Ophir, D., and Fishelson, Z.: Protection of human nasal respiratory epithelium from complement-mediated lysis by cell-membrane regulators of complement activation. Am. J. Respir. Cell Mol. Biol. 1996, 15:731–737. 2-11 Complement developed early in the evolution of multicellular organisms. Fujita, T.: Evolution of the lectin-complement pathway and its role in innate immunity. Nat. Rev. Immunol. 2002, 2:346–353. Zhang, H., Song, L., Li, C., Zhao, J., Wang, H., Gao, Q., and Xu, W.: Molecular 2-16 Complement control proteins regulate all three pathways of cloning and characterization of a thioester-containing protein from Zhikong scallop Chlamys farreri. Mol. Immunol. 2007, 44:3492–3500.

1	2-12 Surface-bound C3 convertase deposits large numbers of C3b activity. Rawal, N., and Pangburn, M.K.: Structure/function of C5 convertases of complement. Int. Immunopharmacol. 2001, 1:415–422. 2-13 Ingestion of complement-tagged pathogens by phagocytes is mediated by receptors for the bound complement proteins. Gasque, P.: Complement: a unique innate immune sensor for danger signals. Mol. Immunol. 2004, 41:1089–1098. Helmy, K.Y., Katschke, K.J., Jr., Gorgani, N.N., Kljavin, N.M., Elliott, J.M., Diehl, L., Scales, S.J., Ghilardi, N., and van Lookeren Campagne, M.: CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens. Cell 2006, 124:915–927. 2-14 The small fragments of some complement proteins initiate a local inflammatory response. Barnum, S.R.: C4a: an anaphylatoxin in name only. J. Innate Immun. 2015, 7:333-9. Kohl, J.: Anaphylatoxins and infectious and noninfectious inflammatory diseases. Mol. Immunol. 2001, 38:175–187.

1	Barnum, S.R.: C4a: an anaphylatoxin in name only. J. Innate Immun. 2015, 7:333-9. Kohl, J.: Anaphylatoxins and infectious and noninfectious inflammatory diseases. Mol. Immunol. 2001, 38:175–187. Schraufstatter, I.U., Trieu, K., Sikora, L., Sriramarao, P., and DiScipio, R.: Complement C3a and C5a induce different signal transduction cascades in endothelial cells. J. Immunol. 2002, 169:2102–2110. 2-15 The terminal complement proteins polymerize to form pores in membranes that can kill certain pathogens. Hadders, M.A., Beringer, D.X., and Gros, P.: Structure of C8α-MACPF reveals mechanism of membrane attack in complement immune defense. Science 2007, 317:1552–1554. Parker, C.L., and Sodetz, J.M.: Role of the human C8 subunits in complement-mediated bacterial killing: evidence that C8γ is not essential. Mol. Immunol. 2002, 39:453–458.

1	Parker, C.L., and Sodetz, J.M.: Role of the human C8 subunits in complement-mediated bacterial killing: evidence that C8γ is not essential. Mol. Immunol. 2002, 39:453–458. Scibek, J.J., Plumb, M.E., and Sodetz, J.M.: Binding of human complement C8 to C9: role of the N-terminal modules in the C8α subunit. Biochemistry 2002, 41:14546–14551. complement activation and protect the host from their destructive effects. Ambati, J., Atkinson, J.P., and Gelfand, B.D.: Immunology of age-related macular degeneration. Nat. Rev. Immunol. 2013, 13:438–451. Atkinson, J.P., and Goodship, T.H.: Complement factor H and the hemolytic uremic syndrome. J. Exp. Med. 2007, 204:1245–1248. Jiang, H., Wagner, E., Zhang, H., and Frank, M.M.: Complement 1 inhibitor is a regulator of the alternative complement pathway. J. Exp. Med. 2001, 194:1609–1616.

1	Jiang, H., Wagner, E., Zhang, H., and Frank, M.M.: Complement 1 inhibitor is a regulator of the alternative complement pathway. J. Exp. Med. 2001, 194:1609–1616. Miwa, T., Zhou, L., Hilliard, B., Molina, H., and Song, W.C.: Crry, but not CD59 and DAF, is indispensable for murine erythrocyte protection in vivo from spontaneous complement attack. Blood 2002, 99:3707–3716. Singhrao, S.K., Neal, J.W., Rushmere, N.K., Morgan, B.P., and Gasque, P.: Spontaneous classical pathway activation and deficiency of membrane regulators render human neurons susceptible to complement lysis. Am. J. Pathol. 2000, 157:905–918. Smith, G.P., and Smith, R.A.: Membrane-targeted complement inhibitors. Mol. Immunol. 2001, 38:249–255.

1	Smith, G.P., and Smith, R.A.: Membrane-targeted complement inhibitors. Mol. Immunol. 2001, 38:249–255. Spencer, K.L., Hauser, M.A., Olson, L.M., Schmidt, S., Scott, W.K., Gallins, P., Agarwal, A., Postel, E.A., Pericak-Vance, M.A., and Haines, J.L.: Protective effect of complement factor B and complement component 2 variants in age-related macular degeneration. Hum. Mol. Genet. 2007, 16:1986–1992. Spencer, K.L., Olson, L.M., Anderson, B.M., Schnetz-Boutaud, N., Scott, W.K., Gallins, P., Agarwal, A., Postel, E.A., Pericak-Vance, M.A., and Haines, J.L.: C3 R102G polymorphism increases risk of age-related macular degeneration. Hum. Mol. Genet. 2008, 17:1821–1824. 2-17 Pathogens produce several types of proteins that can inhibit complement activation.

1	2-17 Pathogens produce several types of proteins that can inhibit complement activation. Blom, A.M., Rytkonen, A., Vasquez, P., Lindahl, G., Dahlback, B., and Jonsson, A.B.: A novel interaction between type IV pili of Neisseria gonorrhoeae and the human complement regulator C4B-binding protein. J. Immunol. 2001, 166:6764–6770. Serruto, D., Rappuoli, R., Scarselli, M., Gros, P., and van Strijp, J.A.: Molecular mechanisms of complement evasion: learning from staphylococci and meningococci. Nat. Rev. Microbiol. 2010, 8:393–399. The Induced Responses of Innate Immunity

1	In Chapter 2 we introduced the innate defenses—such as epithelial barriers, secreted antimicrobial proteins, and the complement system—that act immediately upon encounter with microbes to protect the body from infection. We also introduced the phagocytic cells that lie beneath the epithelial barriers and stand ready to engulf and digest invading microorganisms that have been flagged for destruction by complement. These phagocytes also initiate the next phase of the innate immune response, inducing an inflammatory response that recruits new phagocytic cells and circulating effector molecules to the site of infection. In this chapter we describe how phagocytic cells of the innate immune system detect microbes or the cellular damage they cause, how they destroy these pathogens, and how they orchestrate downstream inflammatory responses through production of cytokines and chemokines (chemoattractant cytokines). We also introduce other cells of the innate immune system—a diverse array of

1	orchestrate downstream inflammatory responses through production of cytokines and chemokines (chemoattractant cytokines). We also introduce other cells of the innate immune system—a diverse array of specialized innate lymphoid cells (ILCs) including the natural killer cells (NK cells)—that contribute to innate host defenses against viruses and other intracellular pathogens. Also in this stage of infection dendritic cells initiate adaptive immune responses, so that if the infection is not cleared by innate immunity, a full immune response will ensue.

1	Pattern recognition by cells of the innate immune system. The basis of the adaptive immune system’s enormous capacity for antigen recognition has long been appreciated. In contrast, the basis of recognition of microbial products by innate immune sensors was discovered only in the late 1990s. Initially, innate recognition was considered to be restricted to relatively few pathogen-associated molecular patterns, or PAMPs, and we have already seen examples of such recognition of microbial surfaces by complement (see Chapter 2). In the last several years, with the discovery of an increasing number of innate receptors that are capable of discriminating among a number of closely related molecules, we have come to realize that a much greater flexibility in innate recognition exists than had been previously thought.

1	The first part of this chapter examines the cellular receptors that recognize pathogens and signal for a cellular innate immune response. Regular patterns of molecular structure are present on many microorganisms but do not occur on the host body’s own cells. Receptors that recognize such features are expressed on macrophages, neutrophils, and dendritic cells, and they are similar to the secreted molecules, such as ficolins and histatins, described in Chapter 2. The general characteristics of these pattern recognition receptors (PRRs) are contrasted with those of the antigen-specific receptors of adaptive immunity in Fig. 3.1. A new insight is that self-derived host molecules can be induced that indicate cellular infection, damage, stress, or transformation, and that some innate receptors recognize such proteins to mediate responses by innate immune cells. Such indicator molecules have been termed ‘damageassociated molecular patterns,’ or DAMPs, and some of the molecules in this class

1	recognize such proteins to mediate responses by innate immune cells. Such indicator molecules have been termed ‘damageassociated molecular patterns,’ or DAMPs, and some of the molecules in this class can be recognized by receptors also involved in pathogen recognition, such as the Toll-like receptors (TLRs).

1	Pattern recognition by cells of the innate immune system. Induced innate responses to infection. Fig. 3.1 Comparison of the characteristics of recognition molecules of the innate and adaptive immune systems. The innate immune system uses germline-encoded receptors while the adaptive immune system uses antigen receptors of unique specificity assembled from incomplete gene segments during lymphocyte development. Antigen receptors of the adaptive immune system are clonally distributed on individual lymphocytes and their progeny. Typically, receptors of the innate immune system are expressed non-clonally, that is, they are expressed on all the cells of a given cell type. However, NK cells express various combinations of NK receptors from several families, making individual NK cells different from one another. A particular NK receptor may not be expressed on all NK cells.

1	Coordination of the innate immune response relies on the information provided by many types of receptors. Pattern recognition receptors can be classified into four main groups on the basis of their cellular localization and their function: free receptors in the serum, such as ficolins and histatins (discussed in Chapter 2); membrane-bound phagocytic receptors; membrane-bound signaling receptors; and cytoplasmic signaling receptors. Phagocytic receptors primarily signal for phagocytosis of the microbes they recognize. A diverse group of receptors, including chemotactic receptors, help to guide cells to sites of infection, and other receptors, including PRRs and cytokine receptors, can control the activity of effector molecules at those sites.

1	In this part of the chapter we first look at the recognition properties of phagocytic receptors and of signaling receptors that activate phagocytic microbial killing mechanisms. Next we describe an evolutionarily ancient pathogen system of recognition and signaling, the Toll-like receptors (TLRs), the first of the innate sensor systems to be discovered, and several recently discovered systems that detect intracellular infections by sensing cytoplasmic microbial cell-wall components, foreign RNA, or foreign DNA. 3-1 After entering tissues, many microbes are recognized, ingested, and killed by phagocytes.

1	If a microorganism crosses an epithelial barrier and begins to replicate in the tissues of the host, in most cases, it is immediately recognized by resident phagocytic cells. The main classes of phagocytic cells in the innate immune system are macrophages and monocytes, granulocytes, and dendritic cells. Macrophages are the major phagocyte population resident in most normal tissues at homeostasis. They can arise either from progenitor cells that enter the tissues during embryonic development, and then self-renew at steady state during life, or from circulating monocytes. Studies suggest that the embryonic progenitors arise from either the fetal liver, the yolk sac, or an embryonic region near the dorsal aorta called the aorta–gonad–mesonephros (AGM), although the relative contribution of these origins is still debated. Macrophages are found in especially large numbers in connective tissue: for example, in the submucosal layer of the gastrointestinal tract; in the submucosal layer of

1	these origins is still debated. Macrophages are found in especially large numbers in connective tissue: for example, in the submucosal layer of the gastrointestinal tract; in the submucosal layer of the bronchi, and in the lung interstitium—the tissue and intercellular spaces around the air sacs (alveoli)—and in the alveoli themselves; along some blood

1	Pattern recognition by cells of the innate immune system. vessels in the liver; and throughout the spleen, where they remove senescent blood cells. Macrophages in different tissues were historically given different names, for example, microglial cells in neural tissue and Kupffer cells in the liver. The self-renewal of these two types of cells is dependent on a cytokine called interleukin 34 (IL-34) that is produced in these tissues and acts on the same receptor as macrophage-colony stimulating factor (M-CSF).

1	During infection or inflammation, macrophages can also arise from monocytes that leave from the circulation to enter into tissues. Monocytes in both mouse and human develop in the bone marrow and circulate in the blood as two main populations. In humans, 90% of circulating monocytes are the ‘classical’ monocyte that expresses CD14, a co-receptor for a PRR described later, and function during infection by entering tissues and differentiating into activated inflammatory monocytes or macrophages. In mice, this monocyte population expresses high levels of the surface marker Ly6C. A smaller population are the ‘patrolling monocytes’ that roll along the endothelium rather than circulating freely in the blood. In humans, they express CD14 and CD16, a type of Fc receptor (FcγRIII; see Section 10-21), and are thought to survey for injury to the endothelium but do not differentiate into tissue macrophages. In mice, they express low levels of Ly6C.

1	The second major family of phagocytes comprises the granulocytes, which include neutrophils, eosinophils, and basophils. Of these, neutrophils have the greatest phagocytic activity and are the cells most immediately involved in innate immunity against infectious agents. Also called polymorphonuclear neutrophilic leukocytes (PMNs, or polys), they are short-lived cells that are abundant in the blood but are not present in healthy tissues. Macrophages and granulocytes have an important role in innate immunity because they can recognize, ingest, and destroy many pathogens without the aid of an adaptive immune response. Phagocytic cells that scavenge incoming pathogens represent an ancient mechanism of innate immunity, as they are found in both invertebrates and vertebrates.

1	The third class of phagocytes in the immune system is the immature dendritic cells that reside in lymphoid organs and in peripheral tissues. There are two main functional types of dendritic cells: conventional (or classical) dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). Both types of cells arise from progenitors within the bone marrow that primarily branch from cells of myeloid potential, and they migrate via the blood to tissues throughout the body and to peripheral lymphoid organs. Dendritic cells ingest and break down microbes, but, unlike macrophages and neutrophils, their primary role in immune defense is not the front-line, large-scale direct killing of microbes. A major role of cDCs is to process ingested microbes in order to generate peptide antigens that can activate T cells and induce an adaptive immune response. They also produce cytokines in response to microbial recognition that activate other types of cells against infection. cDCs are thus considered to

1	T cells and induce an adaptive immune response. They also produce cytokines in response to microbial recognition that activate other types of cells against infection. cDCs are thus considered to act as a bridge between innate and adaptive immune responses. pDCs are major producers of a class of cytokines known as type I interferons, or antiviral interferons, and are considered to be part of innate immunity; they are discussed in detail later in the chapter.

1	Because most microorganisms enter the body through the mucosa of the gut and respiratory system, skin, or urogenital tract, macrophages in the submucosal tissues are the first cells to encounter most pathogens, but they are soon reinforced by the recruitment of large numbers of neutrophils to sites of infection. Macrophages and neutrophils recognize pathogens by means of cell-surface receptors that can discriminate between the surface molecules of pathogens and those of the host. Although they are both phagocytic, macrophages and neutrophils have distinct properties and functions in innate immunity. MOVIE 3.1

1	MOVIE 3.1 MOVIE 3.2 lipid receptor (CD36) scavenger receptors (SR-A I/II, MARCO) mannose receptor CTLD Dectin-1 Bound material is internalized in phagosomes and broken down in phagolysosomes Macrophages have phagocytic receptors that bind microbes and their components mannose receptor Dectin-1 (˜-glucan receptor) scavenger receptor complement receptor Fc receptor complement receptor lipid receptor

1	The process of phagocytosis is initiated when certain receptors on the surface of the cell—typically a macrophage, neutrophil, or dendritic cell—interacts with the microbial surface. The bound pathogen is first surrounded by the phagocyte plasma membrane and then internalized in a large membrane-enclosed endocytic vesicle known as a phagosome. The phagosome fuses with one or more lysosomes to generate a phagolysosome, in which the lysosomal contents are released. The phagolysosome also becomes acidified, acquires antimicrobial peptides and enzymes, and undergoes enzymatic processes that produce highly reactive superoxide and nitric oxide radicals, which together kill the microbe (Fig. 3.2). Neutrophils are highly specialized for the intracellular killing of microbes, and contain different types of cytoplasmic granules—the primary granules and secondary granules described in Section 2-4. These granules fuse with phagosomes, releasing additional enzymes and antimicrobial peptides that

1	types of cytoplasmic granules—the primary granules and secondary granules described in Section 2-4. These granules fuse with phagosomes, releasing additional enzymes and antimicrobial peptides that attack the microbe. Another pathway by which extracellular material, including microbial material, can be taken up into the endosomal compartment of cells and degraded is receptor-mediated endocytosis, which is not restricted to phagocytes. Dendritic cells and other phagocytes can also take up pathogens by a nonspecific process called macropinocytosis, in which large amounts of extracellular fluid and its contents are ingested.

1	Macrophages and neutrophils constitutively express a number of cell-surface receptors that stimulate the phagocytosis and intracellular killing of microbes bound to them, although some also signal through other pathways to trigger responses such as cytokine production. These phagocytic receptors include several members of the C-type lectin-like family (see Fig. 3.2). For example, Dectin-1 is strongly expressed by macrophages and neutrophils and recognizes β-1,3-linked glucans (polymers of glucose), which are common components of fungal cell walls in particular. Dendritic cells also express Dectin-1, as well as several other C-type lectin-like phagocytic receptors, which will be discussed in relation to pathogen uptake for antigen processing and presentation in Chapter 9. Another C-type lectin, the mannose receptor (MR) expressed by macrophages and dendritic cells, recognizes various mannosylated ligands, including some present on fungi, bacteria, and viruses; it was once suspected to

1	the mannose receptor (MR) expressed by macrophages and dendritic cells, recognizes various mannosylated ligands, including some present on fungi, bacteria, and viruses; it was once suspected to have an important role in resistance to microbes. However, experiments with mice that lack this receptor do not support this idea. The macrophage mannose receptor is now thought to function mainly as a clearance receptor for host glycoproteins such as β-glucuronidase and lysosomal hydrolases, which have mannose-containing carbohydrate side chains and whose extracellular concentrations are raised during inflammation.

1	A second set of phagocytic receptors on macrophages, called scavenger receptors, recognize various anionic polymers and acetylated low-density lipoproteins. These receptors are structurally heterogeneous, consisting of at least six different molecular families. Class A scavenger receptors are membrane proteins composed of trimers of collagen domains (see Fig. 3.2).

1	Fig. 3.2 Macrophages express receptors that enable them to take up microbes by phagocytosis. First panel: macrophages residing in tissues throughout the body are among the first cells to encounter and respond to pathogens. They carry cell-surface receptors that bind to various molecules on microbes, in particular carbohydrates and lipids, and induce phagocytosis of the bound material. Second panel: Dectin-1 is a member of the C-type lectin-like family built around a single C-type lectin-like domain (CTLD). Lectins in general are based on a carbohydrate-recognition domain (CRD). The macrophage mannose receptor contains many CTLDs, with a fibronectin-like domain and a cysteine-rich region at its amino terminus. Class A scavenger receptors such as MARCO are built from collagen-like domains and form trimers. The receptor protein CD36 is a class B scavenger receptor that recognizes and internalizes lipids. Various complement receptors bind and internalize complement-coated bacteria. Third

1	form trimers. The receptor protein CD36 is a class B scavenger receptor that recognizes and internalizes lipids. Various complement receptors bind and internalize complement-coated bacteria. Third panel: phagocytosis of receptor-bound material is taken into intracellular phagosomes, which fuse with lysosomes to form an acidified phagolysosome in which the ingested material is broken down by lysosomal hydrolases.

1	Pattern recognition by cells of the innate immune system. They include SR-A I, SR-A II, and MARCO (macrophage receptor with a collagenous structure), which all bind various bacterial cell-wall components and help to internalize bacteria, although the basis of their specificity is poorly understood. Class B scavenger receptors bind high-density lipoproteins, and they internalize lipids. One of these receptors is CD36, which binds many ligands, including long-chain fatty acids. A third set of receptors of crucial importance in macrophage and neutrophil phagocytosis is the complement receptors and Fc receptors introduced in Chapters 1 and 2. These receptors bind to complement-coated microbes or to antibodies that have bound to the surface of microbes and facilitate the phagocytosis of a wide range of microorganisms. 3-2 G-protein-coupled receptors on phagocytes link microbe recognition with increased efficiency of intracellular killing.

1	3-2 G-protein-coupled receptors on phagocytes link microbe recognition with increased efficiency of intracellular killing. Phagocytosis of microbes by macrophages and neutrophils is generally followed by the death of the microbe inside the phagocyte. As well as the phagocytic receptors, macrophages and neutrophils have other receptors that signal to stimulate antimicrobial killing. These receptors belong to the evolutionarily ancient family of G-protein-coupled receptors (GPCRs), which are characterized by seven membrane-spanning segments. Members of this family are crucial to immune system function because they also direct responses to anaphylatoxins such as the complement fragment C5a (see Section 2-14) and to many chemokines, recruiting phagocytes to sites of infection and promoting inflammation.

1	The fMet-Leu-Phe (fMLF) receptor is a G-protein-coupled receptor that senses the presence of bacteria by recognizing a unique feature of bacterial polypeptides. Protein synthesis in bacteria is typically initiated with an N-formylmethionine (fMet) residue, an amino acid present in prokaryotes but not in eukaryotes. The fMLF receptor is named after a tripeptide, formylmethionyl-leucyl phenylalanine, for which it has a high affinity, although it also binds other peptide motifs. Bacterial polypeptides binding to this receptor activate intracellular signaling pathways that direct the cell to move toward the most concentrated source of the ligand. Signaling through the fMLF receptor also induces the production of microbicidal reactive oxygen species (ROS) in the phagolysosome. The C5a receptor recognizes the small fragment of C5 generated when the classical or lectin pathways of complement are activated, usually by the presence of microbes (see Section 2-14), and signals by a similar

1	recognizes the small fragment of C5 generated when the classical or lectin pathways of complement are activated, usually by the presence of microbes (see Section 2-14), and signals by a similar pathway as the fMLF receptor. Thus, stimulation of these receptors both guides monocytes and neutrophils toward a site of infection and leads to increased antimicrobial activity; these cell responses can be activated by directly sensing unique bacterial products or by messengers such as C5a that indicate previous recognition of a microbe.

1	The G-protein-coupled receptors are so named because ligand binding activates a member of a class of intracellular GTP-binding proteins called G proteins, sometimes referred to as heterotrimeric G proteins to distinguish them from the family of ‘small’ GTPases typified by Ras. Heterotrimeric G proteins are composed of three subunits: Gα, Gβ, and Gγ, of which the α subunit is similar to the small GTPases (Fig. 3.3). In the resting state, the G protein is inactive, not associated with the receptor, and a molecule of GDP is bound to the α subunit. Ligand binding induces conformational changes in the receptor that allow it to bind the G protein, which results in the displacement of the GDP from the G protein and its replacement with GTP. The active G protein dissociates into two components, the Gα subunit and a complex consisting of a Gβ and a Gγ subunit. Each of these components can interact with other intracellular signaling molecules to transmit and amplify the signal. G proteins can

1	the Gα subunit and a complex consisting of a Gβ and a Gγ subunit. Each of these components can interact with other intracellular signaling molecules to transmit and amplify the signal. G proteins can activate a wide variety of downstream enzymatic

1	MOVIE 3.3 GPCR chemokine or fMet-Leu-Phe Before ligand binding a GPCR is not associated with a G protein Ligand binding causes a conformational change in the receptor which enables it to associate with the G protein G protein dissociates into ˜ and °˛ subunits, both of which can activate other proteins The ˜ subunit cleaves GTP to GDP, allowing the ˜ and °˛ subunits to reassociate Inactive G protein has GDP bound G protein releases GDP and binds GTP Activation of the GTPases Rac, Rho, and Cdc42 stimulates chemotaxis or the respiratory burst Signaling terminates ˜°˛GTP GTP GDP GDP heterotrimeric G protein Rac/Rho Cdc42 Chemotaxis Respiratory burst

1	Fig. 3.3 G-protein-coupled receptors signal by coupling with intracellular heterotrimeric G proteins. First panel: G-protein-coupled receptors (GPCRs) such as the fMet-Leu-Phe (fMLF) and chemokine receptors signal through GTP-binding proteins known as heterotrimeric G proteins. In the inactive state, the α subunit of the G protein binds GDP and is associated with the β and γ subunits. Second panel: the binding of a ligand to the receptor induces a conformational change that allows the receptor to interact with the G protein, which results in the displacement of GDP and binding of GTP by the α subunit. Third panel: GTP binding triggers the dissociation of the G protein into the α subunit and the βγ subunit, each of which can activate other proteins at the inner face of the cell membrane. In the case of fMLF signaling in macrophages and neutrophils, the α subunit of the activated G protein indirectly activates the GTPases Rac and Rho, whereas the βγ subunit indirectly activates the

1	In the case of fMLF signaling in macrophages and neutrophils, the α subunit of the activated G protein indirectly activates the GTPases Rac and Rho, whereas the βγ subunit indirectly activates the GTPase Cdc42. The actions of these proteins result in the assembly of the NADPH oxidase, resulting in a respiratory burst. Chemokine signaling acts by a similar pathway and activates chemotaxis. Fourth panel: The activated response ceases when the intrinsic GTPase activity of the α subunit hydrolyzes GTP to GDP, and the α and βγ subunits reassociate. The intrinsic rate of GTP hydrolysis by α subunits is relatively slow, and signaling is regulated by additional GTPase-activating proteins (not shown), which accelerate the rate of GTP hydrolysis.

1	targets, such as adenylate cyclase, which produces the second messenger cyclic AMP; and phospholipase C, whose activation gives rise to the second messenger inositol 1,3,5-trisphosphate (IP3) and the release of free Ca2+.

1	Signaling by fMLF and C5a receptors influences cell motility, metabolism, gene expression, and cell division through activation of several Rho family small GTPase proteins. The α subunit of the activated G protein indirectly activates Rac and Rho, while the βγsubunit indirectly activates the small GTPase Cdc42 (see Fig. 3.3). Activation of these GTPases is controlled by guanine nucleotide exchange factors (GEFs) (see Fig. 7.4, which exchange GTP for GDP bound to the GTPase. The G proteins activated by fMLF activate the GEF protein PREX1 (phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein), which can directly activate Rac. Other GEFs, including members of the Vav family that are controlled by other types of receptors (see Section 7-19), can also activate Rac activity, and their activity synergizes with the actions of fMLF and C5a.

1	The activation of Rac and Rho helps to increase the microbicidal capacity of macrophages and neutrophils that have ingested pathogens. Upon phagocytosing microbes, macrophages and neutrophils produce a variety of toxic products that help to kill the engulfed microorganism (Fig. 3.4). The most important of these are the antimicrobial peptides described in Section 2-4, reactive nitrogen species such as nitric oxide (NO), and ROS, such as the super-oxide anion (O2–) and hydrogen peroxide (H2O2). Nitric oxide is produced by a high-output form of nitric oxide synthase, inducible NOS2 (iNOS2), whose expression is induced by a variety of stimuli, including fMLF.

1	Activation of the fMLF and C5a receptors is directly involved in generating ROS. Superoxide is generated by a multicomponent, membrane-associated NADPH oxidase, also called phagocyte oxidase. In unstimulated phagocytes, this enzyme is inactive because it is not fully assembled. One set of subunits, the cytochrome b558 complex (composed of p22 and gp91), is localized in the plasma membranes of resting macrophages and neutrophils, and it appears in lysosomes after the maturation of phagolysosomes. The other components, p40, p47, and p67, are in the cytosol. Activation of phagocytes induces the cytosolic subunits to join with the membrane-associated cytochrome b558 to form a complete, functional NADPH oxidase in the phagolysosome membrane (Fig. 3.5). The fMLF and C5a receptors participate in the process by activating Rac, which functions to promote the movement of the cytosolic components to the membrane to assemble the active NADPH oxidase.

1	Pattern recognition by cells of the innate immune system. ˜-Defensins (HNP1–4), °-defensin Cathelicidin, macrophage HBD4, cathelicidin, azurocidin, Antimicrobial peptides bacterial permeability inducing protein (BPI), lactoferricin Acidifcation Toxic nitrogen oxides Toxic oxygen-derived products pH°3.5–4.0, bacteriostatic or bactericidal Nitric oxide NO Superoxide O2 – , hydrogen peroxide H2O2, singlet oxygen 1O2 •, hydroxyl radical •OH, hypohalite OCl– Lysozyme: digests cell walls of some Gram-positive bacteria Acid hydrolases (e.g., elastase and other proteases): break down ingested microbes Lactoferrin (sequesters Fe2+), vitamin B12-binding protein

1	The NADPH oxidase reaction results in a transient increase in oxygen consumption by the cell, which is known as the respiratory burst. It generates superoxide anion within the lumen of the phagolysosome, and this is converted by the enzyme superoxide dismutase (SOD) into H2O2. Further chemical and enzymatic reactions produce a range of toxic ROS from H2O2, including the hydroxyl radical (•OH), hypochlorite (OCl–), and hypobromite (OBr–). In this way, the direct recognition of bacterially derived polypeptides or previous pathogen recognition by the complement system activates a potent killing mechanism within macrophages and neutrophils that have ingested microbes via their phagocytic receptors. However, phagocyte activation can also cause extensive tissue damage because hydrolytic enzymes, membrane-disrupting peptides, and reactive oxygen species can be released into the extracellular environment and are toxic to host cells.

1	Neutrophils use the respiratory burst described above in their role as an early responder to infection. Neutrophils are not tissue-resident cells, and they need to be recruited to a site of infection from the bloodstream. Their sole function is to ingest and kill microorganisms. Although neutrophils are eventually present in much larger numbers than macrophages in some types of acute infection, they are short-lived, dying soon after they have accomplished a round of phagocytosis and used up their primary and secondary granules. Dead and dying neutrophils are a major component of the pus that forms in abscesses and in wounds infected by certain extracellular capsulated bacteria such as streptococci and staphylococci, which are thus known as pus-forming, or pyogenic, bacteria. Macrophages, in contrast, are long-lived cells and continue to generate new lysosomes.

1	Patients with a disease called chronic granulomatous disease (CGD) have a genetic deficiency of the NADPH oxidase, which means that their phagocytes do not produce the toxic oxygen derivatives characteristic of the respiratory burst and so are less able to kill ingested microorganisms and clear an infection. The most common form of CGD is an X-linked heritable disease that arises from inactivating mutations in the gene encoding the gp91 subunit of cytochrome b558. People with this defect are unusually susceptible to bacterial and fungal infections, especially in infancy, though they remain susceptible for life. One autosomal recessive form of NADPH oxidase deficiency, p47phox deficiency, has very low but detectable activity and causes a milder form of CGD.

1	Fig. 3.4 Bactericidal agents produced or released by phagocytes after uptake of microorganisms. Most of the agents listed are directly toxic to microbes and can act directly in the phagolysosome. They can also be secreted into the extracellular environment, and many of these substances are toxic to host cells. Other phagocyte products sequester essential nutrients in the extracellular environment, rendering them inaccessible to microbes and hindering microbial growth. Besides being directly bacteriostatic or bactericidal, acidification of lysosomes also activates the many acid hydrolases that degrade the contents of the vacuole.

1	O2–OCl–H2O2phagosomeprimarygranuleNADPHoxidasesecondarygranulegp91p22Rac2fMLFlysosomeBacterial fMet-Leu-Phe peptides activate Rac2, and bacteria are taken up into phagosomes Phagosomes fuse with primary and secondary granules. Rac2 induces assembly of a functional NADPH oxidase in the phagolysosome membrane, leading to generation of O2 – . Acidifcation as a result of ion infux releases granule proteases from granule matrix p67p47p40Neutrophils engulf and kill the microbes to which they bind primarygranulesecondarygranulemicrobelysosomeK+K+K+SOD

1	Fig. 3.5 The microbicidal respiratory burst in phagocytes is initiated by activation-induced assembly of the phagocyte NADPH oxidase. First panel: neutrophils are highly specialized for the uptake and killing of pathogens, and contain several different kinds of cytoplasmic granules, such as the primary and secondary granules shown in the first panel. These granules contain antimicrobial peptides and enzymes. Second panel: in resting neutrophils, the cytochrome b558 subunits (gp91 and p22) of the NADPH oxidase are localized in the plasma membrane; the other oxidase components (p40, p47, and p67) are located in the cytosol. Signaling by phagocytic receptors and by fMLF or C5a receptors synergizes to activate Rac2 and induce the assembly of the complete, active NADPH oxidase in the membrane of the phagolysosome, which has formed by the fusion of the phagosome with lysosomes and primary and secondary granules. Third panel: active NADPH oxidase transfers an electron from its FAD cofactor

1	of the phagolysosome, which has formed by the fusion of the phagosome with lysosomes and primary and secondary granules. Third panel: active NADPH oxidase transfers an electron from its FAD cofactor to molecular oxygen, forming the superoxide ion O2– (blue) and other free oxygen radicals in the lumen of the phagolysosome. Potassium and hydrogen ions are then drawn into the phagolysosome to neutralize the charged superoxide ion, increasing acidification of the vesicle. Acidification dissociates granule enzymes such as cathepsin G and elastase (yellow) from their proteoglycan matrix, leading to their cleavage and activation by lysosomal proteases. O2– is converted by superoxide dismutase (SOD) to hydrogen peroxide (H2O2), which can kill microorganisms, and can be converted by myeloperoxidase, a heme-containing enzyme, to microbicidal hypochlorite (OCl–) and by chemical reaction with ferrous (Fe2+) ions to the hydroxyl (•OH) radical.

1	In addition to killing microbes engulfed by phagocytosis, neutrophils use another rather novel mechanism of destruction that is directed at extracellular pathogens. During infection, some activated neutrophils undergo a unique form of cell death in which the nuclear chromatin, rather than being degraded as occurs during apoptosis, is released into the extracellular space and forms a MOVIE 3.4 fibril matrix known as neutrophil extracellular traps, or NETs (Fig. 3.6). NETs act to capture microorganisms, which may then be more efficiently phagocytosed by other neutrophils or macrophages. NET formation requires the generation of ROS, and patients with CGD have reduced NET formation, which may contribute to their susceptibility to microorganisms.

1	Macrophages can phagocytose pathogens and produce the respiratory burst immediately upon encountering an infecting microorganism, and this can be sufficient to prevent an infection from becoming established. In the nineteenth century, the immunologist Élie Metchnikoff believed that the innate response Pattern recognition by cells of the innate immune system. of macrophages encompassed all host defenses; indeed, invertebrates such as the sea star that he was studying rely entirely on innate immunity to overcome infection. Although this is not the case in humans and other vertebrates, the innate response of macrophages still provides an important front line of defense that must be overcome if a microorganism is to establish an infection that can be passed on to a new host.

1	Pathogens have, however, developed a variety of strategies to avoid immediate destruction by macrophages and neutrophils. Many extracellular pathogenic bacteria coat themselves with a thick polysaccharide capsule that is not recognized by any phagocytic receptor. In such cases, however, the complement system can recognize microbial surfaces and coat them with C3b, thereby flagging them for phagocytosis via complement receptors, as described in Chapter 2. Other pathogens, for example, mycobacteria, have evolved ways to grow inside macrophage phagosomes by inhibiting their acidification and fusion with lysosomes. Without such devices, a microorganism must enter the body in sufficient numbers to overwhelm the immediate innate host defenses and to establish a focus of infection. 3-3 Microbial recognition and tissue damage initiate an inflammatory response.

1	3-3 Microbial recognition and tissue damage initiate an inflammatory response. An important effect of the interaction between microbes and tissue macrophages is the activation of macrophages and other immune cells to release small proteins called cytokines and chemokines, and other chemical mediators. Collectively, these proteins induce a state of inflammation in the tissue, attract monocytes and neutrophils to the infection, and allow plasma proteins to enter the tissue from the blood. An inflammatory response is usually initiated within hours of infection or wounding. Macrophages are stimulated to secrete pro-inflammatory cytokines, such as TNF-α, and chemokines by interactions between microbes and microbial products and specific receptors expressed by the macrophage. We will examine how the cytokines interact with pathogens later in the chapter, but first we describe some general aspects of inflammation and how it contributes to host defense.

1	Inflammation has three essential roles in combating infection. The first is to deliver additional effector molecules and cells from the blood into sites of infection, and so increase the destruction of invading microorganisms. The second is to induce local blood clotting, which provides a physical barrier to the spread of the infection in the bloodstream. The third is to promote the repair of injured tissue.

1	Inflammatory responses are characterized by pain, redness, heat, and swelling at the site of an infection, reflecting four types of change in the local blood vessels, as shown in Fig. 3.7. The first is an increase in vascular diameter, leading to increased local blood flow—hence the heat and redness—and a reduction in the velocity of blood flow, especially along the inner walls of small blood vessels. The second change is the activation of endothelial cells lining the blood vessel to express cell-adhesion molecules that promote the binding of circulating leukocytes. The combination of slowed blood flow and adhesion molecules allows leukocytes to attach to the endothelium and migrate into the tissues, a process known as extravasation. All these changes are initiated by the pro-inflammatory cytokines and chemokines produced by activated macrophages and parenchymal cells.

1	Once inflammation has begun, the first white blood cells attracted to the site are neutrophils. These are followed by monocytes (Fig. 3.8), which upon activation are called inflammatory monocytes and can produce various pro-inflammatory cytokines, but are distinguishable from macrophages by their lack of expression of the adhesion G-protein-coupled receptor E1, commonly Fig. 3.6 Neutrophil extracellular traps (NETs) can trap bacteria and fungi. This scanning electron micrograph of activated human neutrophils infected with a virulent strain of Shigella flexneri (pink rods) shows the stimulated neutrophils forming NETs (blue, indicated by arrows). Bacteria trapped within NETs are visible (lower arrow). Photo courtesy of Arturo Zychlinsky.

1	Fig. 3.7 Infection stimulates macrophages to release cytokines and chemokines that initiate an inflammatory response. Cytokines produced by tissue macrophages at the site of infection cause the dilation of local small blood vessels and changes in the endothelial cells of their walls. These changes lead to the movement of leukocytes, such as neutrophils and monocytes, out of the blood vessel (extravasation) and into the infected tissue; this movement is guided by chemokines produced by the activated macrophages. The blood vessels also become more permeable, allowing plasma proteins and fluid to leak into the tissues. Together, these changes cause the characteristic inflammatory signs of heat, pain, redness, and swelling at the site of infection.

1	Fig. 3.8 Monocytes circulating in the blood migrate into infected and inflamed tissues. Adhesion molecules on the endothelial cells of the blood vessel wall capture the monocyte and cause it to adhere to the vascular endothelium. Chemokines bound to the vascular endothelium then signal the monocyte to migrate across the endothelium into the underlying tissue. The monocyte, now differentiating into an inflammatory monocyte, continues to migrate, under the influence of chemokines released during inflammatory responses, toward the site of infection. Monocytes leaving the blood are also able to differentiate into dendritic cells (not shown), depending on the signals that they receive from their environment. called F4/80. Monocytes are also able to give rise to dendritic cells in the tissues, depending on signals that they receive from their environment. In the later stages of inflammation, other leukocytes such as eosinophils and lymphocytes also enter the infected site.

1	The third major change in local blood vessels is an increase in vascular permeability. Thus, instead of being tightly joined together, the endothelial cells lining the blood vessel walls become separated, leading to an exit of fluid and proteins from the blood and their local accumulation in the tissue. This accounts for the swelling, or edema, and pain—as well as the accumulation in tissues of plasma proteins such as complement and MBL that aid in host defense. The changes that occur in endothelium as a result of inflammation are known generally as endothelial activation. The fourth change, clotting in microvessels in the site of infection, prevents the spread of the pathogen via the blood.

1	These changes are induced by a variety of inflammatory mediators released as a consequence of the recognition of pathogens by macrophages, and later by neutrophils and other white blood cells. Macrophages and neutrophils secrete lipid mediators of inflammation—prostaglandins, leukotrienes, and platelet-activating factor (PAF)—which are rapidly produced by enzymatic pathways that degrade membrane phospholipids. Their actions are followed by those of the chemokines and cytokines that are synthesized and secreted Pattern recognition by cells of the innate immune system. by macrophages and inflammatory monocytes in response to pathogens. The cytokine tumor necrosis factor-α (TNF-α, also known simply as TNF), for example, is a potent activator of endothelial cells. We describe TNF-αand related cytokines in more detail in Section 3-15.

1	Besides stimulating the respiratory burst in phagocytes and acting as a chemoattractant for neutrophils and monocytes, C5a also promotes inflammation by increasing vascular permeability and inducing the expression of certain adhesion molecules on endothelium. C5a also activates local mast cells (see Section 1-4), which are stimulated to release their granules containing the small inflammatory molecule histamine as well as TNF-α and cathelicidins.

1	If wounding has occurred, the injury to blood vessels immediately triggers two protective enzyme cascades. One is the kinin system of plasma proteases that is triggered by tissue damage to generate several polypeptides that regulate blood pressure, coagulation, and pain. Although we will not fully describe its components here, one inflammatory mediator produced is the vasoactive peptide bradykinin, which increases vascular permeability to promote the influx of plasma proteins to the site of tissue injury. It also causes pain. Although unpleasant to the victim, pain draws attention to the problem and leads to immobilization of the affected part of the body, which helps to limit the spread of the infection.

1	The coagulation system is another protease cascade that is triggered in the blood after damage to blood vessels, although its full description is also outside our present scope. Its activation leads to the formation of a fibrin clot, whose normal role is to prevent blood loss. With regard to innate immunity, however, the clot physically encases the infectious microorganisms and prevents their entry into the bloodstream. The kinin and the coagulation cascades are also triggered by activated endothelial cells, and so they can have important roles in the inflammatory response to pathogens even if wounding or gross tissue injury has not occurred. Thus, within minutes of the penetration of tissues by a pathogen, the inflammatory response causes an influx of proteins and cells that may control the infection. Coagulation also forms a physical barrier in the form of blood clots to limit the spread of infection. Damage to tissues can occur in the absence of infection by microbes, such as

1	the infection. Coagulation also forms a physical barrier in the form of blood clots to limit the spread of infection. Damage to tissues can occur in the absence of infection by microbes, such as physical trauma, ischemia, and metabolic or autoimmune disorders. In such sterile injury, many of the changes associated with infection, such as neutrophil recruitment, can also occur, in addition to activation of the kinin system and clot formation.

1	3-4 Toll-like receptors represent an ancient pathogen-recognition system.

1	Section 1-5 introduced pattern recognition receptors (PRRs), which function as sensors for pathogen-associated molecular patterns (PAMPs). Cytokine and chemokine production by macrophages is the result of signaling by these PRRs that is induced by a wide variety of pathogen components. The existence of these receptors was predicted by Charles Janeway, Jr., before mechanisms of innate recognition were known, based on the requirement for adjuvants in driving immune responses to purified antigens. Jules Hoffmann discovered the first example of such a receptor, for which he was awarded part of the 2011 Nobel Prize in Physiology or Medicine. The receptor protein Toll was identified earlier as a gene controlling the correct dorso-ventral patterning embryo of the fruitfly Drosophila melanogaster. But in 1996, Hoffmann discovered that in the adult fly, Toll signaling induces the expression of several host-defense mechanisms, including antimicrobial peptides such as drosomycin, and is critical

1	But in 1996, Hoffmann discovered that in the adult fly, Toll signaling induces the expression of several host-defense mechanisms, including antimicrobial peptides such as drosomycin, and is critical for defense against Gram-positive bacteria and fungal pathogens.

1	Mutations in Drosophila Toll or in signaling proteins activated by Toll decreased the production of antimicrobial peptides and led to susceptibility of the adult fly Fig. 3.9 Toll is required for antifungal responses in Drosophila melanogaster. Flies that are deficient in the Toll receptor are dramatically more susceptible than wild-type flies to fungal infection. This is illustrated here by the uncontrolled hyphal growth (arrow) of the normally weak pathogen Aspergillus fumigatus in a Toll-deficient fly. Photo courtesy of J.A. Hoffmann.

1	MOVIE 3.5 to fungal infections (Fig. 3.9). Subsequently, homologs of Toll, called Toll-like receptors (TLRs), were found in other animals, including mammals, in which they are associated with resistance to viral, bacterial, and fungal infection. In plants, proteins with domains resembling the ligand-binding regions of TLR proteins are involved in the production of antimicrobial peptides, indicating the ancient association of these domains with this means of host defense. 3-5 Mammalian Toll-like receptors are activated by many different pathogen-associated molecular patterns.

1	There are 10 expressed TLR genes in humans and 12 in mice. Each TLR is devoted to recognizing a distinct set of molecular patterns that are essentially not found in healthy vertebrate cells. Initially called pathogen-associated molecular patterns (PAMPs), these molecules are general components of both pathogenic and nonpathogenic microorganisms, and so are sometimes called microbial-associated molecular patterns, or MAMPs. Between them, the mammalian TLRs recognize molecules characteristic of Gram-negative and Gram-positive bacteria, fungi, and viruses. Among these, the lipoteichoic acids of Gram-positive bacterial cell walls and the lipopolysaccharide (LPS) of the outer membrane of Gram-negative bacteria (see Fig. 2.9) are particularly important in the recognition of bacteria by the innate immune system, and are recognized by TLRs. Other microbial components also have a repetitive structure. Bacterial flagella are made of a repeated flagellin subunit, and bacterial DNA has abundant

1	immune system, and are recognized by TLRs. Other microbial components also have a repetitive structure. Bacterial flagella are made of a repeated flagellin subunit, and bacterial DNA has abundant repeats of unmethylated CpG dinucleotides (which are often methylated in mammalian DNA). In many viral infections, a double-stranded RNA intermediate is part of the viral life cycle, and frequently the viral RNA contains modifications that can be used to distinguish it from normal host RNA species.

1	The mammalian TLRs and their known microbial ligands are listed in Fig. 3.10. Because there are relatively few TLR genes, the TLRs have limited specificity compared with the antigen receptors of the adaptive immune system. However, they can recognize elements of most pathogenic microbes and are expressed by many types of cells, including macrophages, dendritic cells, B cells, stromal cells, and certain epithelial cells, enabling the initiation of antimicrobial responses in many tissues.

1	TLRs are sensors for microbes present in extracellular spaces. Some mammalian TLRs are cell-surface receptors similar to Drosophila Toll, but others are located intracellularly in the membranes of endosomes, where they detect pathogens or their components that have been taken into cells by phagocytosis, receptor-mediated endocytosis, or macropinocytosis (Fig. 3.11). TLRs are single-pass transmembrane proteins with an extracellular region composed of 18–25 copies of a leucine-rich repeat (LRR). Each LLR of a TLR protein is composed of around 20–25 amino acids, and multiple LRRs create a horseshoe-shaped protein scaffold that is adaptable for ligand binding and recognition on both the outer (convex) and inner (concave) surfaces. Signaling by mammalian TLRs is activated when binding of a ligand induces formation of a dimer, or induces conformational changes in a preformed TLR dimer. All mammalian TLR proteins have in their cytoplasmic tail a TIR (for Toll–IL-1 receptor) domain, which

1	ligand induces formation of a dimer, or induces conformational changes in a preformed TLR dimer. All mammalian TLR proteins have in their cytoplasmic tail a TIR (for Toll–IL-1 receptor) domain, which interacts with other TIR-type domains, usually in other signaling molecules, and is also found in the cytoplasmic tail of the receptor for the cytokine interleukin-1β(IL-1β). For years after the discovery of the mammalian TLRs it was not known whether they made direct contact with microbial products or whether they sensed the presence of microbes by some indirect means. Drosophila Toll, for example, does not recognize pathogen products directly, but instead it is activated when it binds a cleaved version of a self protein, Spätzle. Drosophila has other direct pathogen-recognition molecules, and these trigger the proteolytic cascade that ends in the cleavage

1	Pattern recognition by cells of the innate immune system. Fig. 3.10 Innate immune recognition by Toll-like receptors. Each of the human or mouse TLRs whose specificity is known recognizes one or more microbial molecular patterns, generally by direct interaction with molecules on the pathogen surface. Some Toll-like receptor proteins form heterodimers (e.g., TLR-1:TLR-2 and TLR-6:TLR-2). LPS, lipopolysaccharide. of Spätzle. In this sense Toll is not a classical pattern recognition receptor. However, X-ray crystal structures of several mammalian dimeric TLRs bound to their ligands show that at least some mammalian TLRs make direct contact with microbial ligands. Fig. 3.11 The cellular locations of the mammalian Toll-like receptors.

1	Fig. 3.11 The cellular locations of the mammalian Toll-like receptors. TLRs are transmembrane proteins whose extracellular region contains 18–25 copies of the leucine-rich repeat (LRR), but these cartoons depict only 9 LRRs for simplicity. Some TLRs are located on the cell surface of dendritic cells, macrophages, and other cells, where they are able to detect extracellular pathogen molecules. TLRs are thought to act as dimers. Only those that form heterodimers are shown in dimeric form here; the rest act as homodimers. TLRs located intracellularly, in the walls of endosomes, can recognize microbial components, such as DNA, that are accessible only after the microbe has been broken down. The diacyl and triacyl lipopeptides recognized by the heterodimeric receptors TLR-6:TLR-2 and TLR-1:TLR-2, respectively, are derived from the lipoteichoic acid of Gram-positive bacterial cell walls and the lipoproteins of Gram-negative bacterial surfaces.

1	Mammalian TLR-1, TLR-2, and TLR-6 are cell-surface receptors that are activated by various ligands, including lipoteichoic acid and the diacyl and triacyl lipoproteins of Gram-negative bacteria. They are found on macrophages, dendritic cells, eosinophils, basophils, and mast cells. Ligand binding induces the formation of heterodimers of TLR-2 and TLR-1, or of TLR-2 and TLR-6. The X-ray crystal structure of a synthetic triacyl lipopeptide ligand bound to TLR-1 and TLR-2 shows exactly how it induces dimerization (Fig. 3.12). Two of the three lipid chains bind to the convex surface of TLR-2, while the third binds to the convex surface of TLR-1. Dimerization brings the cytoplasmic TIR domains of the TLR chains into close proximity with each other to initiate signaling. Similar interactions are presumed to occur with the diacyl lipopeptide ligands that induce the dimerization of TLR-2 and TLR-6. The scavenger receptor CD36, which binds long-chain fatty acids, and Dectin-1, which binds

1	are presumed to occur with the diacyl lipopeptide ligands that induce the dimerization of TLR-2 and TLR-6. The scavenger receptor CD36, which binds long-chain fatty acids, and Dectin-1, which binds β-glucans (see Section 3-1), both cooperate with TLR-2 in ligand recognition.

1	TLR-5 is expressed on the cell surface of macrophages, dendritic cells, and intestinal epithelial cells; it recognizes flagellin, a protein subunit of bacterial flagella. TLR-5 recognizes a highly conserved site on flagellin that is buried and inaccessible in the assembled flagellar filament. This means that the receptor is activated only by monomeric flagellin, which is produced by the breakdown of flagellated bacteria in the extracellular space. Mice, but not humans, express TLR-11 and TLR-12, which share with TLR-5 the ability to recognize an intact protein. TLR-11 is expressed by macrophages and dendritic cells, and also by liver, kidney, and bladder epithelial cells.

1	TLR-12 is also expressed in macrophages and dendritic cells, and is more broadly expressed in hematopoietic cells than TLR-11, but is not expressed by the epithelial tissues where TLR-11 is expressed. TLR-11-deficient mice develop urinary infections caused by uropathogenic strains of Escherichia coli, although the bacterial ligand for TLR-11 has not yet been identified. TLR-11 and TLR-12 have an overlapping function in that both recognize protozoan parasites such as Toxoplasma gondii and Plasmodium falciparum. They bind to protein motifs that are present in the protozoan actin-binding protein profilin but absent in mammalian profilins. TLR-11 and TLR-12 are both required in macrophages and conventional dendritic cells for activation by T. gondii profilin, but TLR-12 plays a more dominant role. Mice lacking TLR-11 develop more severe tissue injury than normal mice on infection with

1	Fig. 3.12 Direct recognition of pathogen-associated molecular can activate TLR1:TLR2 dimers; it has three fatty-acid chains bound patterns by TLR-1 and TLR-2 induces dimerization of the TLRs to a polypeptide backbone. Two fatty-acid chains bind to a pocket and signaling. TLR-1 and TLR-2 are located on cell surfaces (left on the convex surface of the TLR-2 ectodomain, and the third chain panel), where they can directly recognize bacterial triacyl lipoproteins associates with a hydrophobic channel in the convex binding surface (middle panel). The convex surfaces of their extracellular domains of TLR-1, inducing dimerization of the two TLR subunits and bringing have binding sites for the lipid side chains of triacyl lipopeptides. In their cytoplasmic Toll–IL-1 receptor (TIR) domains together to initiate the crystal structure (right panel), the ligand is a synthetic lipid that signaling. Structure courtesy of Jie-Oh Lee. Pattern recognition by cells of the innate immune system.

1	Pattern recognition by cells of the innate immune system. Toxoplasma, whereas mice lacking TLR-12 die rapidly after infection. TLR-10 is expressed in humans, but TLR-10 is a pseudogene in mice. Its ligand and function are currently not known.

1	Not all mammalian TLRs are cell-surface receptors. The TLRs that recognize nucleic acids are located in the membranes of endosomes, to which they are transported via the endoplasmic reticulum. TLR-3 is expressed by macrophages, conventional dendritic cells, and intestinal epithelial cells; it recognizes double-stranded RNA (dsRNA), which is a replicative intermediate of many types of viruses, not only those with RNA genomes. dsRNA is internalized either by the direct endocytosis of viruses with double-stranded RNA genomes, such as rotavirus, or by the phagocytosis of dying cells in which viruses are replicating, and it encounters the TLRs when the incoming endocytic vesicle or phagosome fuses with the TLR-containing endosome. Crystallographic analysis shows that TLR-3 binds directly to dsRNA. The TLR-3 ectodomain (the ligand-binding domain) has two contact sites for dsRNA: one on the amino terminus and a second near the membrane-proximal carboxy terminus. The twofold symmetry of dsRNA

1	The TLR-3 ectodomain (the ligand-binding domain) has two contact sites for dsRNA: one on the amino terminus and a second near the membrane-proximal carboxy terminus. The twofold symmetry of dsRNA allows it to bind simultaneously to two TLR-3 ectodomains, inducing a dimerization that brings the TIR domains of TLR-3 together and activates intracellular signaling. This can be verified by using poly I:C to artificially induce signaling. A synthetic polymer composed of inosinic and cytidylic acid, poly I:C binds to TLR-3 and functions as an analog of dsRNA; poly I:C is often used experimentally to activate this pathway. Mutations in the ectodomain of human TLR-3, which produce a dominantly acting loss-of-function mutant receptor, have been associated with encephalitis that is caused by a failure to control the herpes simplex virus.

1	TLR-7, TLR-8, and TLR-9, like TLR-3, are endosomal nucleotide sensors involved in the recognition of viruses. TLR-7 and TLR-9 are expressed by plasmacytoid dendritic cells, B cells, and eosinophils; TLR-8 is expressed primarily by monocytes and macrophages. TLR-7 and TLR-8 are activated by single-stranded RNA (ssRNA), which is a component of healthy mammalian cells, but it is normally confined to the nucleus and cytoplasm and is not present in endosomes. Many virus genomes, for example, those of orthomyxoviruses (such as influenza) and flaviviruses (such as West Nile virus), consist of ssRNA. When extracellular particles of these viruses are endocytosed by macrophages or dendritic cells, they are uncoated in the acidic environment of endosomes and lysosomes, exposing the ssRNA genome for recognition by TLR-7. Mice lacking TLR-7 have impaired immune responses to viruses such as influenza. In abnormal settings, TLR-7 may be activated by self-derived ssRNA. Normally, extracellular RNases

1	by TLR-7. Mice lacking TLR-7 have impaired immune responses to viruses such as influenza. In abnormal settings, TLR-7 may be activated by self-derived ssRNA. Normally, extracellular RNases degrade the ssRNA released from apoptotic cells during tissue injury. But in a mouse model of lupus nephritis, an inflammatory condition of the kidney, TLR-7 recognition of self ssRNA was observed to contribute to disease. Several studies have identified polymorphisms in the human TLR-7 gene that are associated with increased risk of the autoimmune disease systemic lupus erythematosus, suggesting a potential role in this disease. The role for TLR-8 has not been established as clearly from mouse model systems as for TLR-7. TLR-9 recognizes unmethylated CpG dinucleotides. In mammalian genomes, CpG dinucleotides in genomic DNA are heavily methylated on the cytosine by DNA methyltransferases. But in the genomes of bacteria and many viruses, CpG dinucleotides remain unmethylated and represent another

1	in genomic DNA are heavily methylated on the cytosine by DNA methyltransferases. But in the genomes of bacteria and many viruses, CpG dinucleotides remain unmethylated and represent another pathogen-associated molecular pattern.

1	The delivery of TLR-3, TLR-7, and TLR-9 from the endoplasmic reticulum to the endosome relies on their interaction with a specific protein, UNC93B1, which is composed of 12 transmembrane domains. Mice lacking this protein have defects in signaling by these endosomal TLRs. Rare human mutations in UNC93B1 have been identified as causing susceptibility to herpes simplex encephalitis, similarly to TLR-3 deficiency, but do not impair immunity to many other viral pathogens, presumably because of the existence of other viral sensors, which are discussed later in this chapter. 3-6 TLR-4 recognizes bacterial lipopolysaccharide in association with the host accessory proteins MD-2 and CD14.

1	Not all mammalian TLRs bind their ligands so directly. TLR-4 is expressed by several types of immune-system cells, including dendritic cells and macrophages, and is important in sensing and responding to numerous bacterial infections. TLR-4 recognizes the LPS of Gram-negative bacteria by a mechanism that is partly direct and partly indirect. The systemic injection of LPS causes a collapse of the circulatory and respiratory systems, a condition known as shock. These dramatic effects of LPS are seen in humans as septic shock, which results from an uncontrolled systemic bacterial infection, or sepsis. In this case, LPS induces an overwhelming secretion of cytokines, particularly TNF-α (see Section 3-15), causing systemic vascular permeability, an undesirable effect of its normal role in containing local infections. Mutant mice lacking TLR-4 function are resistant to LPS-induced septic shock but are highly sensitive to LPS-bearing pathogens such as Salmonella typhimurium, a natural

1	containing local infections. Mutant mice lacking TLR-4 function are resistant to LPS-induced septic shock but are highly sensitive to LPS-bearing pathogens such as Salmonella typhimurium, a natural pathogen of mice. In fact, TLR-4 was identified as the receptor for LPS by positional cloning of its gene from the LPS-resistant C3H/HeJ mouse strain, which harbors a naturally occurring mutation in the cytoplasmic tail of TLR-4 that interferes with the receptor’s ability to signal. For this discovery, the 2011 Nobel Prize in Physiology or Medicine was partly awarded to Bruce Buetler.

1	LPS varies in composition among different bacteria but essentially consists of a polysaccharide core attached to an amphipathic lipid, lipid A, with a variable number of fatty-acid chains per molecule. To recognize LPS, the ectodomain of TLR-4 uses an accessory protein, MD-2. MD-2 initially binds to TLR-4 within the cell and is necessary both for the correct trafficking of TLR-4 to the cell surface and for the recognition of LPS. MD-2 associates with the central section of the curved ectodomain of TLR-4, binding off to one side as shown in Fig. 3.13. When the TLR4–MD-2 complex encounters LPS, five lipid chains of LPS bind to a deep hydrophobic pocket of MD-2, but not directly to TLR-4, while a sixth lipid chain remains exposed on the surface of MD-2. This last lipid chain and parts of the LPS polysaccharide backbone can then bind to the convex side of a second TLR-4 ectodomain, inducing TLR-4 dimerization that activates intracellular signaling pathways.

1	TLR-4 activation by LPS involves two other accessory proteins besides MD-2. While LPS is normally an integral component of the outer membrane of Gram-negative bacteria, during infections it can become detached from the membrane and be picked up by the host LPS-binding protein present in the blood and in extracellular fluid in tissues. LPS is transferred from LPS-binding protein to a second protein, CD14, which is present on the surface of macrophages, neutrophils, and dendritic cells. On its own, CD14 can act as a phagocytic receptor, but on macrophages and dendritic cells it also acts as an accessory protein for TLR-4. 3-7 TLRs activate NFκB, AP-1, and IRF transcription factors to induce the expression of inflammatory cytokines and type I interferons.

1	3-7 TLRs activate NFκB, AP-1, and IRF transcription factors to induce the expression of inflammatory cytokines and type I interferons. Signaling by mammalian TLRs in various cell types induces a diverse range of intracellular responses that together result in the production of inflammatory cytokines, chemotactic factors, antimicrobial peptides, and the antiviral cytokines interferon-α and -β (IFN-α and IFN-β), the type I interferons. TLR signaling achieves this by activating several different signaling pathways that each activate different transcription factors. As mentioned earlier, ligand-induced dimerization of two TLR ectodomains brings the cytoplasmic TIR domains together, allowing them to interact with the TIR domains of cytoplasmic adaptor molecules that initiate intracellular signaling. There are Pattern recognition by cells of the innate immune system.

1	Pattern recognition by cells of the innate immune system. four such adaptors used by mammalian TLRs: MyD88, MAL (also known as TIRAP), TRIF, and TRAM. It is significant that the TIR domains of the different TLRs interact with different combinations of these adaptors (Fig. 3.14). Most TLRs interact only with MyD88, which is required for their signaling.

1	Fig. 3.13 TLR-4 recognizes LPS in association with the accessory protein MD-2. Panel a: a side view of the symmetrical complex of TLR-4, MD-2, and LPS. TLR-4 polypeptide backbones are shown in green and dark blue. The structure shows the entire extracellular region of TLR-4, composed of the LRR region (shown in green and dark blue), but lacks the intracellular signaling domain. The MD-2 protein is shown in light blue. Five of the LPS acyl chains (shown in red) are inserted into a hydrophobic pocket within MD-2. The remainder of the LPS glycan and one lipid chain (orange) make contact with the convex surface of a TLR-4 monomer. Panel b: the top view of the structure shows that an LPS molecule makes contact with one TLR-4 subunit on its convex (outer) surface, while binding to an MD-2 molecule that is attached to the other TLR-4 subunit. The MD-2 protein binds off to one side of the TLR-4 LRR region. Panel c: schematic illustration of relative orientation of LPS binding to MD-2 and

1	that is attached to the other TLR-4 subunit. The MD-2 protein binds off to one side of the TLR-4 LRR region. Panel c: schematic illustration of relative orientation of LPS binding to MD-2 and TLR-4. Structures courtesy of Jie-Oh Lee.

1	Fig. 3.14 Mammalian TLRs interact with different TIR-domain adaptor molecules to activate downstream signaling pathways. The four signaling adaptor molecules used by mammalian TLRs are MyD88 (myeloid differentiation factor 88), MAL (MyD88 adaptor-like, also known as TIRAP, for TIR-containing adaptor protein), TRIF (TIR domain-containing adaptor-inducing IFN-β), and TRAM (TRIF-related adaptor molecule). All TLRs interact with MyD88, except TLR-3, which interacts only with TRIF. The table indicates the known pattern of adaptor interactions for the known TLRs. TLR-3 interacts only with TRIF. Other TLRs use either MyD88 paired with MAL, or TRIF paired with TRAM. Signaling by the TLR-2 heterodimers (TLR-2/1 and TLR-2/6) requires MyD88/MAL. TLR-4 signaling uses both of these adaptor pairs, MyD88/MAL and TRIF/TRAM, which is used during endosomal signaling by TLR-4. Importantly, the choice of adaptor influences which of the several downstream signals will be activated by the TLR.

1	Signaling by most TLRs activates the transcription factor NFκB (Fig. 3.15), which is related to DIF, the factor activated by Drosophila Toll. Mammalian TLRs also activate several members of the interferon regulatory factor (IRF) transcription factor family through a second pathway, and they activate members of the activator protein 1 (AP-1) family, such as c-Jun, through yet another signaling pathway involving mitogen-activated protein kinases (MAPKs). NFκB and AP-1 act primarily to induce the expression of pro-inflammatory cytokines and chemotactic factors. The IRF factors IRF3 and IRF7 are particularly important for inducing antiviral type I interferons, whereas a related factor, IRF5, is involved in the production of pro-inflammatory cytokines. Here we will describe how TLR signaling induces the transcription of various cytokine genes; later in the chapter, we will explain how those cytokines exert their various actions.

1	We consider first the signaling pathway triggered by TLRs that use MyD88. Two protein domains of MyD88 are responsible for its function as an adaptor. MyD88 has a TIR domain at its carboxy terminus that associates with the TIR domains in the TLR cytoplasmic tails. At its amino terminus, MyD88 has a death domain, so named because it was first identified in signaling proteins involved in apoptosis, a type of programmed cell death. The MyD88 death domain associates with a similar death domain present in other intracellular signaling proteins. Both MyD88 domains are required for signaling, since rare mutations in either domain are associated with immunodeficiency characterized by recurrent bacterial infections in humans. The MyD88 death domain recruits and activates two serine–threonine protein kinases—IRAK4 (IL-1receptor associated kinase 4) and IRAK1—via their death domains. This IRAK complex performs two functions: it recruits enzymes that produce a signaling scaffold, and uses this

1	kinases—IRAK4 (IL-1receptor associated kinase 4) and IRAK1—via their death domains. This IRAK complex performs two functions: it recruits enzymes that produce a signaling scaffold, and uses this scaffold to recruit other molecules that are then phosphorylated by the IRAKs.

1	To form a signaling scaffold, the IRAK complex recruits the enzyme TRAF6 (tumor necrosis factor receptor-associated factor 6), which is an E3 ubiquitin ligase that acts in cooperation with UBC13, an E2 ubiquitin ligase, and its cofactor Uve1A (together called TRIKA1) (see Fig. 3.15). The combined activity of TRAF-6 and UBC13 is to ligate (unite with a chemical bond) one ubiquitin molecule to another protein, which can be another ubiquitin molecule, and thereby generate protein polymers. The polyubiquitin involved in signaling contains linkages between the lysine 63 on one ubiquitin and the carboxy terminus of the next, forming K63 linkages. This polyubiquitin polymer can be initiated on other proteins, including TRAF-6 itself, or produced as free linear ubiquitin polymers, and can be extended to produce polyubiquitin chains that act as a platform—or scaffold—that bind to other signaling molecules. Next, the scaffold recruits a signaling complex consisting of the polyubiquitin-binding

1	to produce polyubiquitin chains that act as a platform—or scaffold—that bind to other signaling molecules. Next, the scaffold recruits a signaling complex consisting of the polyubiquitin-binding adaptor proteins TAB1, TAB2, and the serine–threonine kinase TAK1 (see Fig. 3.15). By being brought onto the scaffold, TAK1 is phosphorylated by the IRAK complex, and activated TAK1 propagates signaling by activating certain MAPKs, such as c-Jun terminal kinase (JNK) and MAPK14 (p38 MAPK). These then activate AP-1-family transcription factors that transcribe cytokine genes.

1	TAK1 also phosphorylates and activates the IκB kinase (IKK) complex, which is composed of three proteins: IKKα, IKKβ, and IKKγ (also known as NEMO, for NFκB essential modifier). NEMO functions by binding to polyubiquitin chains, which brings the IKK complex into proximity with TAK1. TAK1 Pattern recognition by cells of the innate immune system. NF˜B NF˜B p50 p65 I˜B I˜B I˜B degradation cytokine genes TRAF-6 UBC13, Uve1A MyD88 MAL nucleus TAK1 TAK1 TAB1/2 TAB1/2 IRAK4IRAK1 (E3 ligase) (E2 ligase) ubiquitin polyubiquitin chain IKK° IKK˛IKK˝ (NEMO) IKK˝ IKK°/˛ IKK I˜B is degraded, releasing NF˜B into the nucleus to induce expression of cytokine genes TAK1 associates with IKK and phosphorylates IKK°, which phosphorylates I˜B TRAF-6 is polyubiquitinated, creating a scaffold for activation of TAK1 Dimerized TLRs recruit IRAK1 and IRAK4, activating the E3 ubiquitin ligase TRAF-6

1	Fig. 3.15 TLR signaling can activate the transcription factor NFκB, which induces the expression of pro-inflammatory cytokines. First panel: TLRs signal via their cytoplasmic TIR domains, which are brought into proximity to each other by ligand-induced dimerization of their ectodomains. Some TLRs use the adaptor protein MyD88, and others use the MyD88/MAL pair to initiate signaling. The MyD88 death domain recruits the serine–threonine kinases IRAK1 and IRAK4, in association with the ubiquitin E3 ligase TRAF-6. IRAK undergoes autoactivation and phosphorylates TRAF-6, activating its E3 ligase activity. Second panel: TRAF-6 cooperates with an E2 ligase (UBC13) and a cofactor (Uve1A) to generate polyubiquitin scaffolds (yellow triangles) by attachment of ubiquitin through its lysine 63 (K63). This scaffold recruits a complex of proteins composed of the kinase TAK1 (transforming growth factor-β-activated kinase 1) and two adaptor proteins, TAB1 (TAK1-binding protein 1) and TAB2. TAB1 and

1	This scaffold recruits a complex of proteins composed of the kinase TAK1 (transforming growth factor-β-activated kinase 1) and two adaptor proteins, TAB1 (TAK1-binding protein 1) and TAB2. TAB1 and TAB2 function to bind to polyubiquitin, bringing TAK1 into proximity with IRAK to become phosphorylated (red dot). Third panel: activated TAK1 activates IKK, the IκB kinase complex. First, the IKKγ subunit (NEMO) binds to the polyubiquitin scaffold and brings the IKK complex into proximity to TAK1. TAK1 then phosphorylates and activates IKKβ. IKKβ then phosphorylates IκB, the cytoplasmic inhibitor of NFκB. Fourth panel: phosphorylated IκB is targeted by a process of ubiquitination (not shown) that leads to its degradation. This releases NFκB, which is composed of two subunits, p50 and p65, into the nucleus, driving the transcription of many genes including those encoding inflammatory cytokines. TAK1 also stimulates activation of the mitogen-activated protein kinases (MAPKs) JNK and p38,

1	the nucleus, driving the transcription of many genes including those encoding inflammatory cytokines. TAK1 also stimulates activation of the mitogen-activated protein kinases (MAPKs) JNK and p38, which phosphorylate and activate AP-1 transcription factors (not shown).

1	phosphorylates and activates IKKβ. IKKβ then phosphorylates IκB (inhibitor of κB), which is a distinct molecule whose name should not be confused with IKKβ. IκB is a cytoplasmic protein that constitutively binds to the transcription factor NFκB, which is composed of two subunits, p50 and p65. The binding of IκB traps the NFκB proteins in the cytoplasm. Phosphorylation by IKK induces the degradation of IκB, and this releases NFκB into the nucleus, where it can drive transcription of genes for pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. The actions of these cytokines in the innate immune response are described in the second half of this chapter. The outcome of TLR activation can also vary depending on the cell type in which it occurs. For example, activation of TLR-4 via MyD88 in specialized epithelial cells such as the Paneth cells of the intestine (see Section 2-4) results in the production of antimicrobial peptides, a mammalian example of the ancient function of

1	MyD88 in specialized epithelial cells such as the Paneth cells of the intestine (see Section 2-4) results in the production of antimicrobial peptides, a mammalian example of the ancient function of Toll-like proteins.

1	The ability of TLRs to activate NFκB is crucial to their role of alerting the immune system to the presence of bacterial pathogens. Rare instances of inactivating mutations in IRAK4 in humans cause an immunodeficiency, IRAK4 deficiency, which, like MyD88 deficiency, is characterized by recurrent bacterial infections. Mutations in human NEMO produce a syndrome known as X-linked hypohidrotic ectodermal dysplasia and immunodeficiency or NEMO deficiency, which is characterized by both immunodeficiency and developmental defects. The nucleic-acid-sensing TLRs—TLR-3, TLR-7, TLR-8, and TLR-9—activate members of the IRF family. IRF proteins reside in the cytoplasm and are inactive until they become phosphorylated on serine and threonine residues in

1	Left panel: TLR-3, expressed by dendritic cells and macrophages, senses double-stranded viral RNA (dsRNA). TLR-3 signaling uses the adaptor protein TRIF, which recruits the E3 ligase TRAF3 to generate K63-linked polyubiquitin chains. This scaffold recruits NEMO and TANK (TRAF family member-associated NFκB activator), which associate with the serine– threonine kinases IKKε (IκB kinase ε) and TBK1 (TANK-binding kinase 1). TBK1 phosphorylates (red dot) the transcription factor IRF3, and IRF3 then enters the nucleus and induces expression of type I interferon genes. Right panel: TLR-7, expressed by plasmacytoid dendritic cells, detects single-stranded RNA (ssRNA) and signals through MyD88. Here, IRAK1 directly recruits and phosphorylates IRF7, which is also highly expressed in plasmacytoid dendritic cells. IRF7 then enters the nucleus to induce expression of type I interferons.

1	their carboxy termini. They then move to the nucleus as active transcription factors. Of the nine IRF family members, IRF3 and IRF7 are particularly important for TLR signaling and expression of antiviral type I interferons. For TLR-3, expressed by macrophages and conventional dendritic cells, the cytoplasmic TIR domain interacts with the adaptor protein TRIF. TRIF interacts with the E3 ubiquitin ligase TRAF3, which, like TRAF6, generates a polyubiquitin scaffold. In TLR-3 signaling, this scaffold recruits a multiprotein complex containing the kinases IKKε and TBK1, which phosphorylate IRF3 (Fig. 3.16). TLR-4 also triggers this pathway by binding TRIF, but the IRF3 response induced by TLR-4 is relatively weak compared with that induced by TLR-3, and its functional role in vivo remains elusive. In contrast to TLR-3, TLR-7, TLR-8, and TLR-9 signal uniquely through MyD88. For TLR-7 and TLR-9 signaling in plasmacytoid dendritic cells, the MyD88 TIR domain recruits the IRAK1/IRAK4 complex

1	In contrast to TLR-3, TLR-7, TLR-8, and TLR-9 signal uniquely through MyD88. For TLR-7 and TLR-9 signaling in plasmacytoid dendritic cells, the MyD88 TIR domain recruits the IRAK1/IRAK4 complex as described above. Here, the IRAK complex carries out a distinct function beyond recruiting TRAFs that generate a signaling scaffold. In these cells, IRAK1 can also physically associate with IRF7, which is highly expressed by plasmacytoid dendritic cells. This allows IRF7 to become phosphorylated by IRAK1, leading to induction of type I interferons (see Fig. 3.16). Not all IRF factors regulate type I interferon genes; IRF5, for example, plays a role in the induction of pro-inflammatory cytokines.

1	The collective ability of TLRs to activate both IRFs and NFκB means that they can stimulate either antiviral or antibacterial responses as needed. In human IRAK4 deficiency, for example, no extra susceptibility to viral infections has been noted. This would suggest that IRF activation is not impaired and the production of antiviral interferons is not affected. TLRs are expressed by different types of cells involved in innate immunity and by some stromal and epithelial cells, and the responses generated will differ in some respects depending on what type of cell is being activated. 3-8 The NOD-like receptors are intracellular sensors of bacterial infection and cellular damage.

1	The TLRs, being expressed on the cell’s plasma membrane or endocytic vesicles, are primarily sensors of extracellular microbial products. Since the discovery of Toll and the mammalian TLRs, additional families of innate sensors have been identified that detect microbial products in the cytoplasm. One large group of cytoplasmic innate sensors has a centrally located nucleotidebinding oligomerization domain (NOD), and other variable domains that detect microbial products or cellular damage or that activate signaling pathways; collectively, these are the NOD-like receptors (NLRs). Some NLRs activate NFκB to initiate the same inflammatory responses as the TLRs, while other NLRs trigger a distinct pathway that induces cell death and the production of pro-inflammatory cytokines. The NLRs are considered a very ancient family of innate immunity receptors because the resistance (R) proteins that are part of plant defenses against pathogens are NLR homologs.

1	Subfamilies of NLRs can be distinguished on the basis of the other protein domains they contain. The NOD subfamily has an amino-terminal caspase recruitment domain (CARD) (Fig. 3.17). CARD was initially recognized in a family of proteases called caspases (for cysteine-aspartic acid proteases), which are important in many intracellular pathways, including those leading to cell death by apoptosis. CARD is structurally related to the TIR death domain in MyD88 and can dimerize with CARD domains on other proteins to induce signaling (Fig. 3.18). NOD proteins recognize fragments of bacterial cell-wall peptidoglycans, although it is not known whether this occurs through direct binding or via accessory proteins. NOD1 senses γ-glutamyl diaminopimelic acid (iE-DAP), a breakdown product of peptidoglycans of Gram-negative bacteria such as Salmonella and some Gram-positive bacteria such as Listeria, Pattern recognition by cells of the innate immune system.

1	Pattern recognition by cells of the innate immune system. LRR domain cytoplasm RIP2 RIP2 NF˜B I˜B Binding of bacterial ligands to NOD proteins induces recruitment of RIP2, which activates TAK1, leading to NF˜B activation NOD proteins reside in the cytoplasm in an inactive form CARD XIAP cIAP1/2 NOD CARD NOD intracellular bacteria, muramyl dipeptide or iE-DAP TAB1/2 NEMO polyubiquitin TAK1 IKK°IKK˛ Fig. 3.17 Intracellular NOD proteins sense the presence of bacteria by recognizing bacterial peptidoglycans and activate NFκB to induce the expression of pro-inflammatory genes. whereas NOD2 recognizes muramyl dipeptide (MDP), which is present in the peptidoglycans of most bacteria. NOD ligands may enter the cytoplasm as a result of intracellular infection, but may also be transported from materials captured by endocytosis, since mice lacking an oligopeptide transporter (SLC15A4) that is present in lysosomes have greatly reduced responses to NOD1 ligands.

1	When NOD1 or NOD2 recognizes its ligand, it recruits the CARD-containing serine–threonine kinase RIP2 (also known as RICK and RIPK2) (see Fig. 3.17). RIP2 associates with the E3 ligases cIAP1, cIAP2, and XIAP, whose activity generates a polyubiquitin scaffold as in TLR signaling. This scaffold recruits TAK1 and IKK and results in activation of NFκB as shown in Fig. 3.15. NFκB then induces the expression of genes for inflammatory cytokines and for enzymes involved in the production of nitric oxide (NO), which is toxic to bacteria and intracellular parasites. In keeping with their role as sensors of bacterial components, NOD proteins are expressed in cells that are routinely exposed to bacteria. These include epithelial cells forming the barrier that bacteria must cross to establish an infection in the body, and the macrophages and dendritic cells that ingest bacteria that have succeeded in entering the body. Macrophages and dendritic cells express TLRs as well as NOD1 and NOD2, and are

1	in the body, and the macrophages and dendritic cells that ingest bacteria that have succeeded in entering the body. Macrophages and dendritic cells express TLRs as well as NOD1 and NOD2, and are activated by both pathways. In epithelial cells, NOD1 is an important activator of responses against bacterial infections, and NOD1 may also function as a systemic activator of innate immunity. It seems that peptidoglycans from intestinal microbiota are transported via blood in amounts sufficient to

1	First panel: NOD proteins reside in an inactive state in the cytoplasm, where they serve as sensors for various bacterial components. Second panel: degradation of bacterial cell-wall peptidoglycans produces muramyl dipeptide, which is recognized by NOD2. NOD1 recognizes γ-glutamyl diaminopimelic acid (iE-DAP), a breakdown product of Gram-negative bacterial cell walls. The binding of these ligands to NOD1 or NOD2 induces aggregation, allowing CARD-dependent recruitment of the serine– threonine kinase RIP2, which associates with E3 ligases, including XIAP (X-linked inhibitor of apoptosis protein), cIAP1 (cellular inhibitor of apoptosis 1), and cIAP2. This recruited E3 ligase activity produces a polyubiquitin scaffold, as in TLR signaling, and the association of TAK1 and the IKK complex with this scaffold leads to the activation of NFκB, as shown in Fig. 3.15. In this pathway, RIP2 acts as a scaffold to recruit XIAP, and RIP2 kinase activity is not required for signaling.

1	Fig. 3.18 Protein-interaction domains contained in various immune signaling molecules. Signaling proteins contain protein-interaction domains that mediate the assembly of larger complexes. The table shows examples of proteins discussed in this chapter that contain the indicated domain. Proteins may have more than one domain, such as the adaptor protein MyD88, which can interact with TLRs via its TIR domain and with IRAK1/4 via its death domain (DD). MOVIE 3.6 Fig. 3.19 Cellular damage activates the NLRP3 inflammasome to produce pro-inflammatory cytokines.

1	The LRR domain of NLRP3 associates with chaperones (HSP90 and SGT1) that prevent NLRP3 activation. Damage to cells caused by bacterial pore-forming toxins or activation of the P2X7 receptor by extracellular ATP allows efflux of K+ ions from the cell; this may dissociate these chaperones from NLRP3 and induce multiple NLRP3 molecules to aggregate through interactions of their NOD domains (also called the NACHT domain). Reactive oxygen intermediates (ROS) and disruption of lysosomes also can activate NLRP3 (see text). The aggregated NLRP3 conformation brings multiple NLRP3 pyrin domains into close proximity, which then interact with the pyrin domains of the adaptor protein ASC (PYCARD). This conformation aggregates the ASC CARD domains, which in turn aggregate the CARD domains of pro-caspase 1. This aggregation of procaspase 1 induces proteolytic cleavage of itself to form the active caspase 1, which cleaves the immature forms of pro-inflammatory cytokines, releasing the mature

1	1. This aggregation of procaspase 1 induces proteolytic cleavage of itself to form the active caspase 1, which cleaves the immature forms of pro-inflammatory cytokines, releasing the mature cytokines that are then secreted.

1	increase basal activation of neutrophils. A reduction in neutrophils activated in this way may explain why mice lacking NOD1 show increased susceptibility even to pathogens that lack NOD ligands, such as the parasite Trypanosoma cruzi.

1	NOD2 seems to have a more specialized role, being strongly expressed in the Paneth cells of the gut, where it regulates the expression of potent antimicrobial peptides such as the αand β-defensins (see Chapter 2). Consistent with this, loss-of-function mutations in NOD2 in humans are associated with the inflammatory bowel condition known as Crohn’s disease (discussed in Chapter 15). Some patients with this condition carry mutations in the LRR domain of NOD2 that impair its ability to sense MDP and activate NFκB. This is thought to diminish the production of defensins and other antimicrobial peptides, thereby weakening the natural barrier function of the intestinal epithelium and leading to the inflammation characteristic of this disease. Gain-of-function mutations in human NOD2 are associated with the inflammatory disorders early-onset sarcoidosis and Blau syndrome, which are characterized by spontaneous inflammation in tissues such as the liver, or in the joints, eyes, and skin.

1	with the inflammatory disorders early-onset sarcoidosis and Blau syndrome, which are characterized by spontaneous inflammation in tissues such as the liver, or in the joints, eyes, and skin. Activating mutations in the NOD domain seem to promote the signaling cascade in the absence of ligand, leading to an inappropriate inflammatory response in the absence of pathogens. Besides NOD1 and NOD2, there are other members of the NOD family, such as the proteins NLRX1 and NLRC5, but their function is currently less well understood.

1	3-9 NLRP proteins react to infection or cellular damage through an inflammasome to induce cell death and inflammation. Another subfamily of NLR proteins has a pyrin domain in place of the CARD domain at their amino termini, and is known as the NLRP family. Pyrin domains are structurally related to the CARD and TIR domains, and interact with other pyrin domains (Fig. 3.19). Humans have 14 NLR proteins containing pyrin domains. The best characterized is NLRP3 (also known as NALP3 or cryopyrin), although the molecular details of its activation are still under active investigation. NLRP3 resides in an inactive form in the cytoplasm, Potassium ef˜ux induces NLRP3 forms oligomers with dissociation of chaperones ASC causing proteolytic that keep NLRP3 in an cleavage of pro-caspase 1 inactive conformation Pattern recognition by cells of the innate immune system.

1	where its LRR domains are thought to bind the heat-shock chaperone protein HSP90 and the co-chaperone SGT1, which may hold NLRP3 in an inactive state (see Fig. 3.19). Several events seem to induce NLRP3 signaling: reduced intracellular potassium, the generation of reactive oxygen species (ROS), or the disruption of lysosomes by particulate or crystalline matter. The loss of intracellular potassium through efflux can occur during infection with, for example, intracellular bacteria such as Staphylococcus aureus that produce pore-forming toxins. Also, death of nearby cells can release ATP into the extracellular space; this ATP would activate the purinergic receptor P2X7, which itself is a potassium channel, and allow K+ ion efflux. In one model, it is the reduction of intracellular K+ concentration that triggers NLRP3 signaling by causing the dissociation of HSP90 and SGT1. A model proposed for ROS-induced NLRP3 activation involves the intermediate oxidization of sensor proteins

1	concentration that triggers NLRP3 signaling by causing the dissociation of HSP90 and SGT1. A model proposed for ROS-induced NLRP3 activation involves the intermediate oxidization of sensor proteins collectively called thioredoxin (TRX). Normally TRX proteins are bound to thioredoxin-interacting protein (TXNIP), but oxidation of TRX by ROS causes the dissociation of TXNIP from TRX. The free TXNIP may then displace HSP90 and SGT1 from NLRP3, again causing its activation. In both of these cases, NLRP3 activation involves aggregation of multiple monomers via their LLR and NOD domains to induce signaling. Finally, phagocytosis of particulate matter, such as the adjuvant alum, a crystalline salt of aluminum potassium sulfate, may lead to the rupture of lysosomes and release of the active protease cathepsin B, which can activate NLRP3 by an unknown mechanism.

1	Rather than activating NFκB as in NOD1 and NOD2 signaling, NLRP3 signaling leads to the generation of pro-inflammatory cytokines and to cell death through formation of a multiprotein complex known as the inflammasome (see Fig. 3.19). Activation of the inflammasome proceeds in several stages. The first is the aggregation of LRR domains of several NLRP3 molecules, or other NLRP molecules, by a specific trigger or recognition event. This aggregation induces the pyrin domains of NLRP3 to interact with pyrin domains of another protein named ASC (also called PYCARD). ASC is an adaptor protein composed of an amino-terminal pyrin domain and a carboxy-terminal CARD domain. Pyrin and CARD domains are each able to form polymeric filamentous structures (Fig. 3.20). The interaction of NLRP3 with ASC further drives the formation of a polymeric ASC filament, with the pyrin domains in the center and CARD domains facing outward. These CARD domains then interact with CARD domains of the inactive

1	ASC further drives the formation of a polymeric ASC filament, with the pyrin domains in the center and CARD domains facing outward. These CARD domains then interact with CARD domains of the inactive protease pro-caspase 1, initiating

1	Fig. 3.20 The inflammasome is composed of several filamentous protein polymers created by aggregated CARD and pyrin domains. Top panel: an electron micrograph of structures formed by full-length ASC, the pyrin domain of AIM2, and the CARD domain of caspase 1. The central dark region represents anti-ASC staining with a gold-labeled (15 nm) antibody. The long outward filaments represent the polymer composed of the caspase 1 CARD domain. Bottom panel: Schematic interpretation of NLRP3 inflammasome assembly. In this model, CARD regions of ASC and caspase 1 aggregate into a filamentous structure. The adaptor ASC translates aggregation of NLRP3 into aggregation of pro-caspase 1. Electron micrograph courtesy of Hao Wu.

1	its CARD-dependent polymerization into discrete caspase 1 filaments. This aggregation seems to trigger the autocleavage of pro-caspase 1, which releases the active caspase 1 fragment from its autoinhibitory domains. Active caspase 1 then carries out the ATP-dependent proteolytic processing of pro-inflammatory cytokines, particularly IL-1β and IL-18, into their active forms (see Fig. 3.19). Caspase 1 activation also induces a form of cell death called pyroptosis (‘fiery death’) through an unknown mechanism that is associated with inflammation because of the release of these pro-inflammatory cytokines upon cell rupture.

1	For inflammasome activation to produce inflammatory cytokines, a priming step must first occur in which cells induce and translate the mRNAs that encode the pro-forms of IL-1β, IL-18, or other cytokines. This priming step can result from TLR signaling, which may help ensure that inflammasome activation proceeds primarily during infections. For example, the TLR-3 agonist poly I:C (see Section 3-5) can be used experimentally to prime cells for subsequent triggering of the inflammasome.

1	Several other NLR family members form inflammasomes with ASC and caspase 1 that activate these pro-inflammatory cytokines. NLRP1 is highly expressed in monocytes and dendritic cells and is activated directly by MDP, similar to NOD2, but can also be activated by other factors. For example, Bacillus anthracis expresses an endopeptidase, called anthrax lethal factor, which allows the pathogen to evade the immune system by killing macrophages. Lethal factor does this by cleaving NLRP1, activating an NLRP1 inflammasome and inducing pyroptosis in the infected macrophages. NLRC4 acts as an adaptor with two other NLR proteins, NAIP2 and NAIP5, that serve to detect various bacterial proteins that enter cells through specialized secretion systems used by pathogens to transport materials into or access nutrients from host cells. One such protein, PrgJ, from the pathogen Salmonella typhimurium, is a component of the type III secretion system (T3SS), a needle-like macromolecular complex. Upon

1	access nutrients from host cells. One such protein, PrgJ, from the pathogen Salmonella typhimurium, is a component of the type III secretion system (T3SS), a needle-like macromolecular complex. Upon infection of host cells by Salmonella, PrgJ enters the cytoplasm and is recognized by NLRC4 functioning together with NAIP2. Extracellular bacterial flagellin is recognized by TLR5, but flagellin may also enter host cells with PrgJ via the T3SS, and in this case can be recognized by NLRC4 in conjunction with NAIP5. Some NLR proteins may negatively regulate innate immunity, such as NLRP6, since mice lacking this protein exhibit increased resistance to certain pathogens. However, NLRP6 is highly expressed in intestinal epithelium, where it appears to play a positive role in promoting normal mucosal barrier function and is required for the normal secretion of mucus granules into the intestine by goblet cells. NLRP7, which is present in humans but not mice, recognizes microbial acylated

1	mucosal barrier function and is required for the normal secretion of mucus granules into the intestine by goblet cells. NLRP7, which is present in humans but not mice, recognizes microbial acylated lipopeptides and forms an inflammasome with ASC and caspase 1 to produce IL-1β and IL-18. Less is known about NLRP12, but like NLRP6, it initially was proposed to have an inhibitory function. Subsequent studies of mice lacking NLRP12 suggest it has a possible role in the detection of and response to certain bacterial species, including Yersinia pestis, the bacterium that causes bubonic plague, although the basis of this recognition is still unclear.

1	Inflammasome activation can also involve proteins of the PYHIN family, which contain an N-terminal pyrin domain but lack the LRR domains present in the NLR family. In place of an LRR domain, PYHIN proteins have a HIN (H inversion) domain, so named for the HIN DNA recombinase of Salmonella that mediates DNA inversion between flagellar H antigens. There are four PYHIN proteins in humans, and 13 in mice. In one of these, AIM2 (absent in melanoma 2), the HIN domain recognizes double-stranded DNA genomes and triggers caspase 1 activation through pyrin domain interactions with ASC. AIM2 is located in the cytoplasm and is important for responses in vitro to vaccinia virus, and its in vivo role has been demonstrated by the increased susceptibility of AIM2-deficient mice to infection by Francisella tularensis, the causative agent of tularemia. The related protein IFI16 (interferon inducible protein 16) contains two HIN domains; it is primarily located in the cell nucleus and recognizes viral

1	tularensis, the causative agent of tularemia. The related protein IFI16 (interferon inducible protein 16) contains two HIN domains; it is primarily located in the cell nucleus and recognizes viral double-stranded DNA, and will be described below in Section 3-11.

1	A ‘non-canonical’ inflammasome (caspase 1-independent) pathway uses the protease caspase 11 to detect intracellular LPS. The discovery of this pathway was initially confused as being dependent on caspase 1 because of a specific genetic difference between experimental mouse strains. Caspase 11 is encoded by the murine Casp4 gene and is homologous to human caspases 4 and 5. The mice in which the caspase 1 gene (Casp1) was initially disrupted and studied were originally found to be resistant to lethal shock (see Section 3-20) induced by administration of LPS. This led researchers to conclude that caspase 1 acted in the inflammatory response to LPS. But researchers later discovered that this mouse strain also carried a natural mutation that inactivated the related Casp4 gene. Because the Casp1 and Casp4 genes reside within 2 kilobases of each other on mouse chromosome 9, they failed to segregate independently during subsequent experimental genetic backcrosses to other mouse strains. Thus,

1	and Casp4 genes reside within 2 kilobases of each other on mouse chromosome 9, they failed to segregate independently during subsequent experimental genetic backcrosses to other mouse strains. Thus, mice initially thought to lack only caspase 1 protein in fact lacked both caspase 1 and caspase 11. Later, mice lacking only caspase 1 were generated by expressing functional Casp4 as a transgene; these mice became susceptible to LPS-induced shock. Mice were also generated that lacked only caspase 11, and these were found to be resistant to LPS-induced shock. These results indicated that caspase 11, and not caspase 1 as originally thought, is responsible for LPS-induced shock. Caspase 11 is responsible for inducing pyroptosis, but not for processing of IL-1β or IL-18. It was suspected that TLR-4 was not the sensor for LPS that activated the non-canonical imflammasome, since mice lacking TLR-4 remain susceptible to LPS-induced shock. Recent evidence has suggested that caspase 11 itself is

1	was not the sensor for LPS that activated the non-canonical imflammasome, since mice lacking TLR-4 remain susceptible to LPS-induced shock. Recent evidence has suggested that caspase 11 itself is the intracellular LPS sensor, making it an example of a protein that is both a sensor and an effector molecule.

1	Inappropriate inflammasome activation has been associated with various diseases. Gout has been known for many years to cause inflammation in the cartilaginous tissues by the deposition of monosodium urate crystals, but how urate crystals caused inflammation was a mystery. Although the precise mechanism is still unclear, urate crystals are known to activate the NLRP3 inflammasome, which induces the inflammatory cytokines associated with the symptoms of gout. Mutations in the NOD domain of NLRP2 and NLRP3 can activate inflammasomes inappropriately, and they are the cause of some inherited autoinflammatory diseases, in which inflammation occurs in the absence of infection. Mutations in NLRP3 in humans are associated with hereditary periodic fever syndromes, such as familial cold inflammatory syndrome and Muckle–Wells syndrome (discussed in more detail in Chapter 13). Macrophages from patients with these conditions show spontaneous production of inflammatory cytokines such as IL-1β. We

1	syndrome and Muckle–Wells syndrome (discussed in more detail in Chapter 13). Macrophages from patients with these conditions show spontaneous production of inflammatory cytokines such as IL-1β. We will also discuss how pathogens can interfere with formation of the inflammasome in Chapter 13.

1	3-10 The RIG-I-like receptors detect cytoplasmic viral RNAs and activate MAVS to induce type I interferon production and pro-inflammatory cytokines. TLR-3, TLR-7, and TLR-9 detect extracellular viral RNAs and DNAs that enter the cell from the endocytic pathway. By contrast, viral RNAs produced within a cell are sensed by a separate family of proteins called the RIG-I-like receptors (RLRs). These proteins serve as viral sensors by binding to viral RNAs using an RNA helicase-like domain in their carboxy terminal. The RLR helicase-like domain has a ‘DExH’ tetrapeptide amino acid motif and is a subgroup of DEAD-box family proteins. The RLR proteins also contain two amino-terminal CARD domains that interact with adaptor proteins and activate Fig. 3.21 RIG-I and other RLRs are cytoplasmic sensors of viral RNA.

1	First panel: before detecting viral RNA, RIG-I and MDA-5 are cytoplasmic and in inactive, auto-inhibited conformations. The adaptor protein MAVS is attached to the mitochondrial outer membrane. Second panel: detection of uncapped 5ʹ-triphosphate RNA by RIG-I, or viral dsRNA by MDA-5, changes the conformation of their CARD domains to become free to interact with the amino-terminal CARD domain of MAVS. This interaction involves the generation of K63-linked polyubiquitin from the E3 ligases TRIM25 or Riplet, although structural details are still unclear. Third panel: the aggregation induces a proline-rich region of MAVS to interact with TRAFs (see text) and leads to the generation of additional K63-linked polyubiquitin scaffold. As in TLR signaling, this scaffold recruits TBK1 and IKK complexes (see Figs. 3.15 and 3.16) to activate IRF and NFκB, producing type I interferons and pro-inflammatory cytokines, respectively.

1	signaling to produce type I interferons when viral RNAs are bound. The first of these sensors to be discovered was RIG-I (retinoic acid-inducible gene I). RIG-I is widely expressed across tissues and cell types and serves as an intracellular sensor for several kinds of infections. Mice deficient in RIG-I are highly susceptible to infection by several kinds of single-stranded RNA viruses, including paramyxoviruses, rhabdoviruses, orthomyxoviruses, and flaviviruses, but not picornaviruses.

1	RIG-I discriminates between host and viral RNA by sensing differences at the 5ʹ end of single-stranded RNA transcripts. Eukaryotic RNA is transcribed in the nucleus and contains a 5ʹ-triphosphate group on its initial nucleotide that undergoes subsequent enzymatic modification called capping by the addition of a 7-methylguanosine to the 5ʹ-triphosphate. Most RNA viruses, however, do not replicate in the nucleus, where capping normally occurs, and their RNA genomes do not undergo this modification. Biochemical studies have determined that RIG-I senses the unmodified 5ʹ-triphosphate end of the ssRNA viral genome. Flavivirus RNA transcripts have the unmodified 5ʹ-triphosphate, as do the transcripts of many other ssRNA viruses, and they are detected by RIG-I. In contrast, the picornaviruses, which include poliovirus and hepatitis A, replicate by a mechanism that involves the covalent attachment of a viral protein to the 5ʹ end of the viral RNA, so that the 5ʹ-triphosphate is absent, which

1	include poliovirus and hepatitis A, replicate by a mechanism that involves the covalent attachment of a viral protein to the 5ʹ end of the viral RNA, so that the 5ʹ-triphosphate is absent, which explains why RIG-I is not involved in sensing them.

1	MDA-5 (melanoma differentiation-associated 5), also called helicard, is similar in structure to RIG-I, but it senses dsRNA. In contrast to RIG-I-deficient mice, mice deficient in MDA-5 are susceptible to picornaviruses, indicating that these two sensors of viral RNAs have crucial but distinct roles in host defense. Inactivating mutations in alleles of human RIG-I or MDA-5 have been reported, but these mutations were not associated with immunodeficiency. The RLR family member LGP2 (encoded by DHX58) retains a helicase domain but lacks CARD domains. LGP2 appears to cooperate with RIG-I and MDA-5 in the recognition of viral RNA, since mice lacking LGP2 have impaired antiviral responses normally mediated by RIG-I or MDA-5. This cooperative viral recognition by LGP2 appears to depend on its helicase domain, since in mice, mutations that disrupt its ATPase activity result in impaired IFN-β production in response to various RNA viruses.

1	Sensing of viral RNAs activates signaling by RIG-I and MDA-5 that leads to type I interferon production appropriate for defense against viral infection (Fig. 3.21). Before infection by viruses, RIG-I and MDA-5 are in the cytoplasm in an autoinhibited configuration that is stabilized by interactions between the CARD and helicase domains. These interactions are disrupted upon infection when viral RNA associates with the helicase domains of RIG-I or MDA-5, freeing the two CARD domains for other interactions. The more amino-proximal portion of the two CARD domains can then recruit E3 ligases, including TRIM25 and Riplet (encoded by RNF153), which initiate K63-linked polyubiquitin scaffolds (see Section 3-7), either as free polyubiquitin chains or on linkages within the second CARD domain. Precise details are unclear, but this scaffold appears to help RIG-I and MDA-5 interact with a downstream adaptor protein called MAVS (mitochondrial antiviral signaling protein). MAVS is attached to the

1	details are unclear, but this scaffold appears to help RIG-I and MDA-5 interact with a downstream adaptor protein called MAVS (mitochondrial antiviral signaling protein). MAVS is attached to the outer mitochondrial membrane and contains a CARD domain that may bind RIG-I and MDA-5. This aggregation of CARD domains, as in the inflammasome, may initiate aggregation of MAVS. In this state, MAVS propagates signals by recruiting various TRAF family E3 ubiquitin ligases, including TRAF2, TRAF3, TRAF5, and TRAF6. The relative importance of each E3 ligase may differ between cell types, but their further production of K63linked polyubiquitin leads to activation of TBK1 and IRF3 and production of type I interferons, as described for TLR-3 signaling (see Fig. 3.16), and also to activation of NFκB. Some viruses have evolved countermeasures to thwart the protection conferred by RLRs. For example, even though the negative-sense RNA genome of influenza virus replicates in the nucleus, some viral RNA

1	viruses have evolved countermeasures to thwart the protection conferred by RLRs. For example, even though the negative-sense RNA genome of influenza virus replicates in the nucleus, some viral RNA transcripts produced during influenza infection are not capped but must be translated in the cytoplasm. The influenza A nonstructural protein 1(NS1) inhibits the activity of TRIM25, and thereby interrupts the antiviral actions that RIG-I might exert against infection.

1	3-11 Cytosolic DNA sensors signal through STING to induce production of type I interferons. Innate sensors that recognize cytoplasmic RNA use specific modifications, such as the 5ʹ cap, to discriminate between host and viral origin. Host DNA is generally restricted to the nucleus, but viral, microbial, or protozoan DNA may become located in the cytoplasm during various stages of infection. Several innate sensors of cytoplasmic DNA have been identified that can lead to the production of type I interferon in response to infections. One component of the DNA-sensing pathway, STING (stimulator of interferon genes), was identified in a functional screen for proteins that can induce expression of type I interferons. STING (encoded by TMEM173) is anchored to the endoplasmic reticulum membrane by an amino-terminal tetraspan transmembrane domain; its carboxy-terminal domain extends into the cytoplasm and interacts to form an inactive STING homodimer.

1	STING is known to serve as a sensor of intracellular infection, based on its recognition of bacterial cyclic dinucleotides (CDNs), including cyclic diguanylate monophosphate (c-di-GMP) and cyclic diadenylate monophosphate (c-di-AMP). These molecules are bacterial second messengers and are produced by enzymes present in most bacterial genomes. CDNs activate STING signaling by changing the conformation of the STING homodimer. This homodimer recruits and activates TBK1, which in turn phosphorylates and activates IRF3, leading to type I interferon production (Fig. 3.22), similar to signaling by TLR-3 and MAVS (see Figs. 3.16 and 3.21). TRIF (downstream of TLR3), MAVS, and STING each contain a similar amino acid sequence motif at their carboxy termini that becomes serine-phosphorylated when these molecules are activated. It appears that this motif, when phosphorylated, recruits both TBK1 and IRF3, allowing IRF3 to be efficiently phosphorylated and activated by TBK1.

1	STING also plays a role in viral infections, since mice lacking STING are susceptible to infection by herpesvirus. But until recently, it was unclear whether STING activates the kinase TBK1 to phosphorylate IRF3, which enters the nucleus and induces expression of type I interferon genes First panel: cGAS resides in the cytoplasm and serves as a sensor of double-stranded DNA (dsDNA) from viruses. When cGAS binds dsDNA, its enzymatic activity is stimulated, leading to production of cyclic-GMP-AMP (cGAMP). Bacteria that infect cells produce second messengers such as cyclic dinucleotides, including cyclic diguanylate monophosphate (c-di-GMP) and cyclic diadenylate monophosphate (c-di-AMP). Second panel: cGAMP and other bacterial dinucleotides can bind and activate the STING dimer present on the ER membrane. Third panel: in this state STING activates TBK1, although the details of this interaction are still unclear. Active TBK1 activates IRF3, as described in Fig. 3.16.

1	STING recognized viral DNA directly or acted only downstream of an unknown viral DNA sensor. It was found that the introduction of DNA into cells, even without live infection, generated another second messenger molecule that activated STING. This second messenger was identified as cyclic guano-sine monophosphate-adenosine monophosphate (cyclic GMP-AMP), or cGAMP. cGAMP, like bacterial CDNs, binds both subunits of the STING dimer and activates STING signaling. This result also suggested the presence of a DNA sensor acting upstream of STING. Purification of the enzyme that produces cGAMP in response to cytosolic DNA identified a previously unknown enzyme, which was named cGAS, for cyclic GAMP synthase. cGAS contains a protein motif present in the nucleotidyltransferase (NTase) family of enzymes, which includes adenylate cyclase (the enzyme that generates the second messenger molecule cyclic AMP) and various DNA polymerases. cGAS can bind directly to cytosolic DNA, and this stimulates

1	which includes adenylate cyclase (the enzyme that generates the second messenger molecule cyclic AMP) and various DNA polymerases. cGAS can bind directly to cytosolic DNA, and this stimulates its enzymatic activity to produce cGAMP from GTP and ATP in the cytoplasm, activating STING. Mice harboring an inactivated cGAS gene show increased susceptibility to herpesvirus infection, demonstrating its importance in immunity.

1	There are several other candidate DNA sensors, but less is known about the mechanism of their recognition and signaling, or their in vivo activity. IFI16 (IFN-γ-inducible protein 16) is a PYHIN family member related to AIM2, but appears to function in DNA sensing and acts through STING, TBK1, and IRF3 rather than activating an inflammasome pathway. DDX41 (DEAD box polypeptide 41) is an RLR related to RIG-I and is a member of the DEAD-box family, but appears to signal through STING rather than MAVS. MRE11A (meitotic recombination 11 homolog a) can sense cytosolic double-stranded DNA to activate the STING pathway, but its role in innate immunity is currently unknown. 3-12 Activation of innate sensors in macrophages and dendritic far-reaching effects on the immune response.

1	3-12 Activation of innate sensors in macrophages and dendritic far-reaching effects on the immune response. Besides activating effector functions and cytokine production, another outcome of the activation of innate sensing pathways is the induction of co-stimulatory molecules on tissue dendritic cells and macrophages (see Section 1-15). We will describe these in more detail later in the book, but mention them now because they provide an important link between innate and adaptive immune responses. Two important co-stimulatory molecules are the cell-surface proteins B7.1 (CD80) and B7.2 (CD86), which are induced on macrophages and tissue dendritic cells by innate sensors such as TLRs in response to pathogen recognition (Fig. 3.23). B7.1 and B7.2 are recognized by specific co-stimulatory receptors expressed by cells of the adaptive immune response, particularly CD4 T cells, and their activation by B7 is an important step in activating adaptive immune responses.

1	Substances such as LPS that induce co-stimulatory activity have been used for years in mixtures that are co-injected with protein antigens to enhance their immunogenicity. These substances are known as adjuvants (see Appendix I, Section A-1), and it was found empirically that the best adjuvants contain microbial components that induce macrophages and tissue dendritic cells to express co-stimulatory molecules and cytokines. As we shall see in Chapters 9 and 11, the cytokines produced in response to infections influence the functional character of the adaptive immune response that develops. In this way the ability of the innate immune system to discriminate among different types of pathogens is used by the organism to ensure an appropriate module of adaptive immune response. 3-13 Toll signaling in Drosophila is downstream of a distinct set of pathogen-recognition molecules.

1	Before leaving innate sensing, we shall look briefly at how Toll, TLRs, and NODs are used in invertebrate innate immunity. Although Toll is central to defense against both bacterial and fungal pathogens in Drosophila, Toll itself is not a pattern recognition receptor, but is downstream of other proteins that detect pathogens (Fig. 3.24). In Drosophila, there are 13 genes encoding peptidoglycan-recognition proteins (PGRPs) that bind the peptidoglycan components of bacterial cell walls. Another family, the Gram-negative binding proteins (GNBPs), recognizes LPS and β-1,3-linked glucans. GNBPs recognize Gram-negative bacteria and, unexpectedly, fungi, rather than Gram-positive bacteria. The family members GNBP1 and PGRP-SA cooperate in the recognition of peptidoglycan from Gram-positive bacteria. They interact with a serine protease called Grass, which initiates a proteolytic cascade that terminates in the cleavage of the protein Spätzle. One of the cleaved fragments forms a homodimer

1	They interact with a serine protease called Grass, which initiates a proteolytic cascade that terminates in the cleavage of the protein Spätzle. One of the cleaved fragments forms a homodimer that binds to Toll and induces its dimerization, which in turn stimulates the antimicrobial response. A fungus-specific recognition protein, GNBP3, also activates the proteolytic cascade, causing cleavage of Spätzle and activation of Toll.

1	In Drosophila, fat-body cells and hemocytes are phagocytic cells that act as part of the fly’s immune system. When the Spätzle dimer binds to Toll, hemocytes synthesize and secrete antimicrobial peptides. The Toll signaling pathway in Drosophila activates a transcription factor called DIF, which is related to mammalian NFκB. DIF enters the nucleus and induces the transcription of genes for antimicrobial peptides such as drosomycin. Another Drosophila factor in the NFκB family, Relish, induces the production of antimicrobial peptides in response to the Imd (immunodeficiency) signaling pathway, which is triggered in Drosophila by particular PGRPs that recognize Gram-negative bacteria. Relish induces expression of the antimicrobial peptides diptericin, attacin, and cecropin, which are distinct from the peptides induced by Toll signaling. Thus, the Toll and Imd pathways activate effector mechanisms to eliminate infection by different kinds of pathogens. Four mammalian PGRP homologs have

1	from the peptides induced by Toll signaling. Thus, the Toll and Imd pathways activate effector mechanisms to eliminate infection by different kinds of pathogens. Four mammalian PGRP homologs have been identified, but act differently than in Drosophila. One, PGLYRP-2, is secreted and functions as an amidase to hydrolyze bacterial peptidoglycans. The others are present in neutrophil granules and exert a bacteriostatic action through interactions with bacterial cell-wall peptidoglycan.

1	Fig. 3.23 Bacterial LPS induces changes in dendritic cells, stimulating them to migrate and to initiate adaptive immunity by activating T cells. Top panel: immature dendritic cells in the skin are highly phagocytic and macropinocytic, but lack the ability to activate T lymphocytes. Dendritic cells residing in the skin ingest microbes and their products and degrade them. During a bacterial infection, the dendritic cells are activated by various innate sensors and the activation induces two types of changes. Second panel: the dendritic cells migrate out of the tissues and enter the lymphatic system and begin to mature. They lose the ability to ingest antigen but gain the ability to stimulate T cells. Third panel: in the regional lymph nodes, they become mature dendritic cells. They change the character of their cell-surface molecules, increasing the number of MHC molecules on their surface and the expression of the co-stimulatory molecules CD80 (B7.1) and CD86 (B7.2).

1	Fig. 3.24 Drosophila Toll is activated as a result of a proteolytic cascade initiated by pathogen recognition. The peptidoglycan-recognition protein PGRP-SA and the Gram-negative binding protein GNBP1 cooperate in the recognition of bacterial pathogens and activate the first protease in a protease cascade that leads to cleavage of the Drosophila protein Spätzle (first panel). Cleavage alters the conformation of Spätzle, enabling it to bind Toll and induce Toll dimerization (second panel). Toll’s cytoplasmic TIR domains recruit the adaptor protein dMyD88 (third panel), which initiates a signaling pathway very similar to that leading to the release of NFκB from its cytoplasmic inhibitor in mammals. The Drosophila version of NFκB is the transcription factor DIF, which then enters the nucleus and activates the transcription of genes encoding antimicrobial peptides. Fungal recognition also leads to cleavage of Spätzle and the production of antimicrobial peptides by this pathway, although

1	and activates the transcription of genes encoding antimicrobial peptides. Fungal recognition also leads to cleavage of Spätzle and the production of antimicrobial peptides by this pathway, although the recognition proteins for fungi are as yet unidentified.

1	3-14 TLR and NOD genes have undergone extensive diversification in both invertebrates and some primitive chordates. There are only about a dozen mammalian TLR genes, but some organisms have diversified their repertoire of innate recognition receptors, especially those containing LRR domains, to a much greater degree. The sea urchin Strongylocentrotus purpuratus has an unprecedented 222 different TLR genes, more than 200 NOD-like receptor genes, and more than 200 scavenger receptor genes in its genome. The sea urchin also has an increased number of proteins that are likely to be involved in signaling from these receptors, there being, for example, four genes that are similar to the single mammalian MyD88 gene. However, there is no apparent increase in the number of downstream targets, such as the family of NFκB transcription factors, suggesting that the ultimate outcome of TLR signaling in the sea urchin may be very similar to that in other organisms.

1	Sea urchin TLR genes fall into two broad categories. One is a small set of 11 divergent genes. The other is a large family of 211 genes, which show a high degree of sequence variation within particular LRR regions; this, together with the large number of pseudogenes in this family, indicates rapid evolutionary turnover, suggesting rapidly changing receptor specificities, in contrast with the few stable mammalian TLRs. Although the pathogen specificity of sea urchin TLRs is unknown, the hypervariability in the LRR domains could be used to generate a highly diversified pathogen-recognition system based on Toll-like receptors. A similar expansion of innate receptors has occurred in some chordates, the phylum to which vertebrates belong. Amphioxus (the lancelet) is a nonvertebrate chordate lacking an adaptive immune system. The amphioxus genome contains 71 TLRs, more than 100 NOD-like receptors, and more than 200 scavenger receptors. As we will see in Chapter 5, a primitive vertebrate

1	lacking an adaptive immune system. The amphioxus genome contains 71 TLRs, more than 100 NOD-like receptors, and more than 200 scavenger receptors. As we will see in Chapter 5, a primitive vertebrate lineage—the jawless fish, which lack immunoglobulinand T-cell-based adaptive immunity—uses somatic gene rearrangement of LRR-containing proteins to provide a version of adaptive immunity (see Section 5-18).

1	Summary.

1	Innate immune cells express several receptor systems that recognize microbes and induce rapid defenses as well as delayed cellular responses. Several scavenger and lectin-like receptors on neutrophils, macrophages, and dendritic cells help rapidly eliminate microbes through phagocytosis. G-protein-coupled receptors for C5a (which can be produced by activation of the complement system’s innate pathogen-recognition ability) and for the bacterial peptide fMLF synergize with phagocytic receptors in activating the NADPH oxidase in phagosomes to generate antimicrobial reactive oxygen intermediates. Toll-like receptors (TLRs) on the cell surface and in the membranes of endosomes detect microbes outside the cell and activate several host defense signaling pathways. The NFκB and IRF pathways downstream of these receptors induce pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, and antiviral cytokines including type I interferons. Other receptor families detect microbial infection

1	of these receptors induce pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, and antiviral cytokines including type I interferons. Other receptor families detect microbial infection in the cytosol. NOD proteins detect bacterial products within the cytosol and activate NFκB and the production of pro-inflammatory cytokines. The related NLR family of proteins detects signs of cellular stress or damage, as well as certain microbial components. NLRs signal through the inflammasome, which generates pro-inflammatory cytokines and induces pyroptosis, a form of cell death. RIG-I and MDA-5 detect viral infection by sensing the presence of viral RNAs and activate the MAVS pathway, while sensors of cytosolic DNA, such as cGAS, activate the STING pathway; both of these pathways induce type I interferons. The signaling pathways activated by all of these primary sensors of pathogens induce a variety of genes, including those for cytokines, chemokines, and co-stimulatory molecules that

1	I interferons. The signaling pathways activated by all of these primary sensors of pathogens induce a variety of genes, including those for cytokines, chemokines, and co-stimulatory molecules that have essential roles in immediate defense and in directing the course of the adaptive immune response later in infection.

1	Induced innate responses to infection.

1	We will now examine the responses of innate immunity induced as an immediate consequence of pathogen recognition by the sensors described in the last section. We will focus on the major phagocytes—neutrophils, macrophages, and dendritic cells—and the cytokines they produce that induce and maintain inflammation. First, we will introduce the families of cytokines and chemokines that coordinate many cellular responses, such as the recruitment of neutrophils and other immune cells to sites of infection. We will discuss the various adhesion molecules that are induced on immune cells circulating in the blood and on endothelial cells of blood vessels to coordinate movement of cells out of the blood and into infected tissues. We will consider in some detail how macrophage-derived chemokines and cytokines promote the continued destruction of infecting microbes. This is achieved both by stimulating the production and recruitment of fresh phagocytes and by inducing another phase of the innate

1	cytokines promote the continued destruction of infecting microbes. This is achieved both by stimulating the production and recruitment of fresh phagocytes and by inducing another phase of the innate immune response—the acute-phase response—in which the liver produces proteins that act as opsonizing molecules, helping to augment the actions of complement. We will also look at the mechanism of action of antiviral interferons, the type I interferons, and finally examine the growing class of innate lymphoid cells, or ILCs, which include the NK cells long known to contribute to innate immune defense against viruses and other intracellular pathogens. ILCs exert a diverse array of effector function that contribute to a rapid innate immune response to infection. They respond to early cytokine signals provided by innate sensor cells, and amplify the response by producing various types of effector cytokines. If an infection is not cleared by the induced innate response, an adaptive response

1	signals provided by innate sensor cells, and amplify the response by producing various types of effector cytokines. If an infection is not cleared by the induced innate response, an adaptive response will ensue that uses many of the same effector mechanisms used by the innate immune system but targets them with much greater precision. The effector mechanisms described here therefore serve as a primer for the focus on adaptive immunity in the later parts of this book.

1	3-15 Cytokines and their receptors fall into distinct families of structurally related proteins. Cytokines are small proteins (about 25 kDa) that are released by various cells in the body, usually in response to an activating stimulus, and that induce responses through binding to specific receptors. Cytokines can act in an autocrine manner, affecting the behavior of the cell that releases the cytokine, or in a paracrine manner, affecting adjacent cells. Some cytokines are even stable enough to act in an endocrine manner, affecting distant cells, although this

1	Fig. 3.25 Cytokine receptors belong to families of receptor proteins, each with a distinctive structure. Many cytokines signal through receptors of the hematopoietin receptor superfamily, named after its first member, the erythropoietin receptor. The hematopoietin receptor superfamily includes homodimeric and heterodimeric receptors, which are subdivided into families on the basis of protein sequence and structure. Examples of these are given in the first three rows. Heterodimeric class I cytokine receptors have an α chain that often defines the ligand specificity of the receptor; they may share with other receptors a common β or γ chain that confers the intracellular signaling function. Heterodimeric class II cytokine receptors have no common chain and include receptors for interferons or interferon-like cytokines. All the cytokine receptors signal through the JAK-STAT pathway. The IL-1 receptor family have extracellular immunoglobulin domains and signal as dimers through TIR domains

1	interferon-like cytokines. All the cytokine receptors signal through the JAK-STAT pathway. The IL-1 receptor family have extracellular immunoglobulin domains and signal as dimers through TIR domains in their cytoplasmic tails and through MyD88. Other superfamilies of cytokine receptors are the tumor necrosis factor receptor (TNFR) family and the chemokine receptor family, the latter belonging to the very large family of G-protein-coupled receptors. The ligands of the TNFR family act as trimers and may be associated with the cell membrane rather than being secreted.

1	depends on their ability to enter the circulation and on their half-life in the blood. In an attempt to develop a standardized nomenclature for molecules secreted by, and acting on, leukocytes, many cytokines are called by the name interleukin (IL) followed by a number (for example, IL-1 or IL-2). However, not all cytokines are included in this system; thus students of immunology are still faced with a somewhat confusing and difficult task. The cytokines are listed alphabetically, together with their receptors, in Appendix III.

1	Cytokines can be grouped by structure into families—the IL-1 family, the hematopoietin superfamily, the interferons (described in Section 3-7), and the TNF family—and their receptors can likewise be grouped (Fig. 3.25). The IL-1 family contains 11 members, notably IL-1α, IL-1β, and IL-18. Most members of this family are produced as inactive proproteins that are cleaved (removing an amino-terminal peptide) to produce the mature cytokine. The exception to this rule is IL-1α, for which both the proprotein and its cleaved forms are biologically active. As discussed earlier, mature IL-1β and IL-18 are produced by macrophages through the action of caspase 1 in response to TLR signaling and inflammasome activation. The IL-1-family receptors have TIR domains in their cytoplasmic tails and signal by the NFκB pathway described earlier for TLRs. The IL-1 receptor functions in concert with a second transmembrane protein, the IL-1 receptor accessory protein (IL1RAP), that is required for IL-1

1	by the NFκB pathway described earlier for TLRs. The IL-1 receptor functions in concert with a second transmembrane protein, the IL-1 receptor accessory protein (IL1RAP), that is required for IL-1 signal transduction.

1	The hematopoietin superfamily of cytokines is quite large and includes non-immune-system growth and differentiation factors such as erythropoietin (which stimulates red blood cell development) and growth hormone, as well as interleukins with roles in innate and adaptive immunity. IL-6 is a member of this superfamily, as is the cytokine GM-CSF, which stimulates the production of new monocytes and granulocytes in the bone marrow. Many of the soluble cytokines made by activated T cells are members of the hematopoietin family. The receptors for the hematopoietin cytokines are tyrosine kinase-associated receptors that form dimers when their cytokine ligand binds. Dimerization initiates intracellular signaling from the tyrosine kinases associated with the cytoplasmic domains of the receptor. Some types of cytokine receptors are composed of two identical subunits, but others have two different subunits. An important feature of cytokine signaling is the large variety of different receptor

1	Some types of cytokine receptors are composed of two identical subunits, but others have two different subunits. An important feature of cytokine signaling is the large variety of different receptor subunit combinations that occur.

1	Tumor necrosis factor (TNF) receptors I and II, CD40, Fas (Apo1, CD95), CD30, CD27, nerve growth factor receptor CCR1–10, CXCR1–5, XCR1, CX3CR1 Homodimeric receptors Receptors for erythropoietin and growth hormone Heterodimeric receptors (no common chain) IL-1 family receptors Receptors for IL-13, IFN-˜, IFN-°, IFN-˛, IL-10 Heterodimeric receptors with a common chain Receptors for IL-3, IL-5, GM-CSF share a common chain, CD131 or °c (common °chain) °c Receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 share a common chain, CD132 or ˛c (common ˛chain). IL-2 receptor also has a third chain, a high-affnity subunit IL-2R˜(CD25) ˛c TNF receptor family Chemokine receptor family

1	These cytokines and their receptors can also be further divided into subfamilies characterized by functional similarities and genetic linkage. For instance, IL-3, IL-4, IL-5, IL-13, and GM-CSF are related structurally, their genes are closely linked in the genome, and they are often produced together by the same kinds of cells. In addition, they bind to closely related receptors, which belong to the family of class I cytokine receptors. The IL-3, IL-5, and GM-CSF receptors form a subgroup that shares a common β chain. Another subgroup of class I cytokine receptors is defined by the use of the common γ chain (γc) of the IL-2 receptor. This chain is shared by receptors for the cytokines IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, and is encoded by a gene located on the X chromosome. Mutations that inactivate γc cause an X-linked severe combined immunodeficiency (X-linked SCID) due to inactivation of the signaling pathways for several cytokines—IL-7, IL-15, and IL-2—that are required for

1	that inactivate γc cause an X-linked severe combined immunodeficiency (X-linked SCID) due to inactivation of the signaling pathways for several cytokines—IL-7, IL-15, and IL-2—that are required for normal lymphocyte development (see Section 13-3). More distantly related, the receptor for IFN-γ is a member of a small family of heterodimeric cytokine receptors with some similarities to the hematopoietin receptor family. These so-called class II cytokine receptors (also known as interferon receptors) include the receptors for IFN-α and IFN-β, and the IL-10 receptor. The hematopoietin and interferon receptors all signal through the JAK–STAT pathway described below and activate different combinations of STATs with different effects.

1	The TNF family, of which TNF-α is the prototype, contains more than 17 cytokines with important functions in adaptive and innate immunity. Unlike most of the other immunologically important cytokines, many members of the TNF family are transmembrane proteins, a characteristic that gives them distinct properties and limits their range of action. Some, however, can also be released from the membrane in some circumstances. They are usually found as homotrimers of a membrane-bound subunit, although some heterotrimers consisting of different subunits also occur. TNF-α (sometimes called simply TNF) is initially expressed as a trimeric membrane-bound cytokine but can be released from the membrane. The effects of TNF-α are mediated by either of two TNF receptors. TNF receptor I (TNFR-I) is expressed on a wide range of cells, including endothelial cells and macrophages, whereas TNFR-II is expressed largely by lymphocytes. The receptors for cytokines of the TNF family are structurally unrelated

1	on a wide range of cells, including endothelial cells and macrophages, whereas TNFR-II is expressed largely by lymphocytes. The receptors for cytokines of the TNF family are structurally unrelated to the receptors described above and also have to cluster to become activated. Since TNF-family cytokines are produced as trimers, the binding of these cytokines induces the clustering of three identical receptor subunits. The signaling pathway activated by these receptors is described in Chapter 7, where we see that signaling uses members of the TRAF family to activate the so-called non-canonical NFκB pathway.

1	Members of the chemokine receptor family are listed in Appendix IV, along with the chemokines they recognize. These receptors have a 7-transmembrane structure and signal by interacting with G-proteins as described in Section 3-2. 3-16 Cytokine receptors of the hematopoietin family are associated with the JAK family of tyrosine kinases, which activate STAT transcription factors. The signaling chains of the hematopoietin family of cytokine receptors are noncovalently associated with protein tyrosine kinases of the Janus kinase (JAK) family—so called because they have two tandem kinase-like domains and thus resemble the two-headed mythical Roman god Janus. There are four members of the JAK family: Jak1, Jak2, Jak3, and Tyk2. As mice deficient for individual JAK family members show different phenotypes, each kinase must Chapter 3: The Induced Responses of Innate Immunity MOVIE 3.7 Fig. 3.26 Many cytokine receptors signal using a rapid pathway called the JAK–STAT pathway.

1	First panel: many cytokines act via receptors that are associated with cytoplasmic Janus kinases (JAKs). The receptor consists of at least two chains, each associated with a specific JAK. Second panel: binding of ligand brings the two chains together, allowing the JAKs to phosphorylate and activate each other, and then to phosphorylate (red dots) specific tyrosines in the receptor tails. The STAT (signal transducer and activator of transcription) family of proteins have an N-terminal domain that homodimerizes STATs in the cytosol before activation, and an SH2 domain that binds to the tyrosinephosphorylated receptor tails. Third panel: upon binding, the STAT homodimers are phosphorylated by JAKs. Fourth panel: after phosphorylation, STAT proteins reconfigure into a dimer that is stabilized by SH2 domain binding to phosphotyrosine residues on the other STAT. They then translocate to the nucleus, where they bind to and activate the transcription of a variety of genes important for

1	by SH2 domain binding to phosphotyrosine residues on the other STAT. They then translocate to the nucleus, where they bind to and activate the transcription of a variety of genes important for adaptive immunity.

1	have a distinct function. For example, Jak3 is used by γc for signaling by several of the cytokines described above. Mutations that inactivate Jak3 cause a form of SCID that is not X-linked. The dimerization or clustering of receptor signaling chains brings the JAKs into close proximity, causing phosphorylation of each JAK on a tyrosine residue that stimulates its kinase activity. The activated JAKs then phosphorylate their associated receptors on specific tyrosine residues. This phosphotyrosine, and the specific amino acid sequence surrounding it, creates a binding site that is recognized by SH2 domains found in other proteins, in particular members of a family of transcription factors known as signal transducers and activators of transcription (STATs) (Fig. 3.26).

1	There are seven STATs (1–4, 5a, 5b, and 6), which reside in the cytoplasm in an inactive form until activated by cytokine receptors. Before activation, most STATs form homodimers, due to a specific homotypic interaction between domains present at the amino termini of the individual STAT proteins. The receptor specificity of each STAT is determined by the recognition of the distinctive phosphotyrosine sequence on each activated receptor by the different SH2 domains within the various STAT proteins. Recruitment of a STAT to the activated receptor brings the STAT close to an activated JAK, which can then phosphorylate a conserved tyrosine residue in the carboxy terminus of the particular STAT. This leads to a rearrangement, in which the phosphotyrosine of each STAT protein binds to the SH2 domain of the other STAT, forming a configuration that can bind DNA with high affinity. Activated STATs predominantly form homodimers, with a cytokine typically activating one type of STAT. For

1	SH2 domain of the other STAT, forming a configuration that can bind DNA with high affinity. Activated STATs predominantly form homodimers, with a cytokine typically activating one type of STAT. For example, IFN-γ activates STAT1 and generates STAT1 homodimers, whereas IL-4 activates STAT6, generating STAT6 homodimers. Other cytokine receptors can activate several STATs, and some STAT heterodimers can be formed. The phosphorylated STAT dimer enters the nucleus, where it acts as a transcription factor to initiate the expression of selected genes that can regulate growth and differentiation of particular subsets of lymphocytes.

1	Since signaling by these receptors depends on tyrosine phosphorylation, dephosphorylation of the receptor complex by tyrosine phosphatases is one way that cells can terminate signaling. A variety of tyrosine phosphatases have been implicated in the dephosphorylation of cytokine receptors, JAKs, and STATs. These include the nonreceptor tyrosine phosphatases SHP-1 and SHP-2 (encoded by PTPN6 and PTPN11), and the transmembrane receptor tyrosine phosphatase CD45, which is expressed as multiple isoforms on many hematopoietic cells. Cytokine signaling can also be terminated by negative feedback involving specific inhibitors that are induced by cytokine activation. The suppressor of cytokine signaling (SOCS) proteins are a class of inhibitors that terminate the signaling of many cytokine and hormone receptors. SOCS proteins contain an SH2 domain that can recruit them to the phosphorylated JAK kinase or receptor, and they can inhibit JAK kinases directly, compete for the receptor, and direct

1	receptors. SOCS proteins contain an SH2 domain that can recruit them to the phosphorylated JAK kinase or receptor, and they can inhibit JAK kinases directly, compete for the receptor, and direct the ubiquitination and subsequent degradation of JAKs and STATs. SOCS proteins are induced by STAT activation, and thus inhibit receptor signaling after the cytokine has had its effect. Their importance can be seen in SOCS1-deficient mice, which develop a multiorgan inflammatory infiltrate caused by increased signaling from interferon receptors, γc-containing receptors, and TLRs. Another class of inhibitory proteins consists of the protein inhibitors of activated STAT (PIAS) proteins, which also seem to be involved in promoting the degradation of receptors and pathway components.

1	Phosphorylated STATs form dimers that translocate into the nucleus to initiate gene transcription Transcription factors (STATs) bind to the phosphorylated receptors, and are in turn phosphorylated by the activated JAKs JAK kinase active STAT dimer 3-17 Chemokines released by macrophages and dendritic cells recruit effector cells to sites of infection.

1	All the cytokines produced by macrophages in innate immune responses have important local and systemic effects that contribute to both innate and adaptive immunity, and these are summarized in Fig. 3.27. The recognition of different classes of pathogens by phagocytes and dendritic cells may involve signaling through different receptors, such as the various TLRs, and can result in some variation in the cytokines expressed by stimulated macrophages and dendritic cells. This is one way in which appropriate immune responses can be selectively activated, as the released cytokines orchestrate the next phase of host defense. In response to activation by PRRs, macrophages and dendritic

1	Fig. 3.27 Important cytokines and chemokines secreted by dendritic cells and macrophages in response to bacterial products include IL-1β, IL-6, CXCL8, IL-12, and TNF-α. TNF-α is an inducer of a local inflammatory response that helps to contain infections. It also has systemic effects, many of which are harmful (discussed in Section 3-20). The

1	Local effects Systemic effects IL-12IL-1˜Fever Production of IL-6 Fever Mobilization of metabolites Shock Fever Induces acute-phase protein production Activated macrophages secrete a range of cytokines CXCL8TNF-°IL-6 Activates vascular endothelium Activates lymphocytes Local tissue destruction Increases access of effector cells Lymphocyte activation Increased antibody production Chemotactic factor recruits neutrophils, basophils, and T cells to site of infection Activates NK cells Induces the differentiation of CD4 T cells into TH1 cells Activates vascular endothelium and increases vascular permeability, which leads to increased entry of IgG, complement, and cells to tissues and increased ˜uid drainage to lymph nodes chemokine CXCL8 is also involved in the local inflammatory response, helping to attract neutrophils to the site of infection. IL-1β, IL-6, and TNF-α have a crucial role in inducing the acute-phase response in the liver and inducing fever, which favors effective host

1	helping to attract neutrophils to the site of infection. IL-1β, IL-6, and TNF-α have a crucial role in inducing the acute-phase response in the liver and inducing fever, which favors effective host defense in various ways. IL-12 activates natural killer (NK) cells and favors the differentiation of CD4 T cells into the TH1 subset in adaptive immunity.

1	MOVIE 3.8

1	Fig. 3.28 Properties of selected human chemokines. Chemokines fall mainly into two related but distinct groups: the CC chemokines, which have two adjacent cysteine residues near the amino terminus; and the CXC chemokines, in which the equivalent cysteine residues are separated by a single amino acid. In humans, the genes for CC chemokines are mostly clustered in one region of chromosome 4. Genes for CXC chemokine genes are found mainly in a cluster on chromosome 17. The two groups of chemokines act on different sets of receptors, all of which are G-proteincoupled receptors. CC chemokines bind to receptors designated CCR1–10. CXC chemokines bind to receptors designated CXCR1–7. Different receptors are expressed on different cell types, and so a particular chemokine can be used to attract a particular cell type. In general, CXC chemokines with a Glu-Leu-Arg tripeptide motif immediately before the first cysteine promote the migration of neutrophils. CXCL8 is an example of this type. Most

1	particular cell type. In general, CXC chemokines with a Glu-Leu-Arg tripeptide motif immediately before the first cysteine promote the migration of neutrophils. CXCL8 is an example of this type. Most of the other CXC chemokines, including those that interact with receptors CXCR3, 4, and 5, lack this motif. Fractalkine is unusual in several respects: it has three amino acid residues between the two cysteines, and it exists in two forms, one that is tethered to the membrane of the endothelial and epithelial cells that express it, where it serves as an adhesion protein, and a soluble form that is released from the cell surface and acts as a chemoattractant for a wide range of cell types. A more comprehensive list of chemokines and their receptors is given in Appendix IV.

1	cells secrete a diverse group of cytokines that includes IL-1β, IL-6, IL-12, TNF-α, and the chemokine CXCL8 (formerly known as IL-8).

1	Among the cytokines released by tissues in the earliest phases of infection are members of a family of chemoattractant cytokines known as chemokines. These small proteins induce directed chemotaxis in nearby responsive cells, resulting in the movement of the cells toward the source of the chemokine. Because chemokines were first detected in functional assays, they were initially given a variety of names, which are listed along with their standardized nomenclature in Appendix IV. All the chemokines are related in amino acid sequence, and their receptors are G-protein-coupled receptors (see Section 3-2). The signaling pathway stimulated by chemokines causes changes in cell adhesiveness and changes in the cell’s cytoskeleton that lead to directed migration. Chemokines can be produced and released by many different types of cells, not only those of the immune system. In the immune system they function mainly as chemoattractants for leukocytes, recruiting monocytes, neutrophils, and other

1	by many different types of cells, not only those of the immune system. In the immune system they function mainly as chemoattractants for leukocytes, recruiting monocytes, neutrophils, and other effector cells of innate immunity from the blood into sites of infection. They also guide lymphocytes in adaptive immunity, as we will learn in Chapters 9–11. Some chemokines also function in lymphocyte development and migration and in angiogenesis (the growth of new blood vessels). There are more than 50 known chemokines, and this striking multiplicity may reflect their importance in delivering cells to their correct locations, which seems to be their main function in the case of lymphocytes. Some of the chemokines that are produced by or that affect human innate immune cells are listed in Fig. 3.28 along with their properties.

1	Chemokines fall mainly into two related but distinct groups. CC chemokines have two adjacent cysteine residues near the amino terminus, whereas in CXC chemokines the corresponding two cysteine residues are separated by a single amino acid. The CC chemokines promote the migration of monocytes, lymphocytes, and other cell types. One example relevant to innate immunity is CCL2, which attracts monocytes through the receptor CCR2B, inducing their migration from the bloodstream to become tissue macrophages. In contrast, neutrophil migration is promoted by CXC chemokines. CXCL8, acting through CXCR2, mobilizes neutrophils from bone marrow and induces them to leave the blood and migrate into the surrounding tissues. CCL2 and CXCL8 therefore have similar but complementary functions in the innate immune response, attracting monocytes and neutrophils respectively.

1	The role of chemokines in cell recruitment is twofold. First, they act on the leukocyte as it rolls along endothelial cells at sites of inflammation, converting this rolling into stable binding by triggering a change of conformation in the adhesion molecules known as leukocyte integrins. These conformational changes enable integrins to bind strongly to their ligands on the endothelial cells, which allows the leukocyte to cross the blood vessel walls by squeezing between the endothelial cells. Second, the chemokine directs the migration of the leukocyte along a gradient of chemokine molecules bound to the extracellular matrix and the surfaces of endothelial cells. This gradient increases in concentration toward the site of infection.

1	Chemokines are produced by a wide variety of cell types in response to bacterial products, viruses, and agents that cause physical damage, such as silica, alum, or the urate crystals that occur in gout. Complement fragments such as C3a and C5a, and fMLF bacterial peptides, also act as chemoattractants for neutrophils. Thus, infection or physical damage to tissues induces the production of chemokine gradients that can direct phagocytes to the sites where they are needed. Neutrophils arrive rapidly in large numbers at a site of infection. The recruitment of monocytes occurs simultaneously, but they accumulate more slowly at the site of infection, perhaps because they are less abundant in the circulation. The complement fragment C5a and the chemokines CXCL8 and CCL2 activate their respective target cells, so that not only are neutrophils and monocytes brought to potential sites of infection but, in the process, they are armed to deal with the pathogens they encounter there. In

1	respective target cells, so that not only are neutrophils and monocytes brought to potential sites of infection but, in the process, they are armed to deal with the pathogens they encounter there. In particular, the signaling induced by C5a or CXCL8 in neutrophils serves to augment the respiratory burst that generates oxygen radicals and nitric oxide and to induce the neutrophils to release their stored antimicrobial granule contents (see Section 3-2).

1	Chemokines do not act alone in cell recruitment. They require the action of vasoactive mediators that bring leukocytes close to the blood vessel wall (see Section 3-3) and cytokines such as TNF-α to induce the necessary adhesion molecules on endothelial cells. We will return to the chemokines in later chapters, where they are discussed in the context of the adaptive immune response. Now, however, we turn to the molecules that enable leukocytes to adhere to the endothelium, and we shall then describe step by step the extravasation process by which monocytes and neutrophils enter infected sites. response. The recruitment of activated phagocytes to sites of infection is one of the most important functions of innate immunity. Recruitment occurs as part of the inflammatory response and is mediated by cell-adhesion molecules that are induced on the surface of the endothelial cells of local blood vessels. Here we MOVIE 3.9

1	MOVIE 3.9 Chapter 3: The Induced Responses of Innate Immunity consider those functions that participate in the recruitment of inflammatory MOVIE 3.10 cells in the hours to days after the establishment of an infection.

1	As with the complement components, a significant barrier to understanding the functions of cell-adhesion molecules is their nomenclature. Most adhesion molecules, especially those on leukocytes, which are relatively easy to analyze functionally, were originally named after the effects of specific monoclonal antibodies directed against them. Their names therefore bear no relation to their structural class. For instance, the leukocyte functional antigens LFA-1, LFA-2, and LFA-3 are actually members of two different protein families. In Fig. 3.29, the adhesion molecules relevant to innate immunity are grouped according to their molecular structure, which is shown in schematic form alongside their different names, sites of expression, and ligands. Three structural families of adhesion molecules are important for leukocyte recruitment. The selectins are membrane glycoproteins with a distal lectin-like domain that binds specific carbohydrate groups. Members of this family are induced on

1	are important for leukocyte recruitment. The selectins are membrane glycoproteins with a distal lectin-like domain that binds specific carbohydrate groups. Members of this family are induced on activated endothelium and initiate endothelium–leukocyte interactions by binding to fucosylated oligosaccharide ligands on passing leukocytes (see Fig. 3.29).

1	The next step in leukocyte recruitment depends on tighter adhesion, which is due to the binding of intercellular adhesion molecules (ICAMs) on the endothelium to heterodimeric proteins of the integrin family on leukocytes. ICAMs are single-pass membrane proteins that belong to the large superfamily of immunoglobulin-like proteins, which contain protein domains similar to those of immunoglobulins. The extracellular regions of ICAMs are composed of several immunoglobulin-like domains. An integrin molecule is composed

1	Fig. 3.29 Adhesion molecules involved in leukocyte interactions. Several structural families of adhesion molecules have a role in leukocyte migration, homing, and cell–cell interactions: the selectins, the integrins, and proteins of the immunoglobulin superfamily. The figure shows schematic representations of an example from each family, a list of other family members that participate in leukocyte interactions, their cellular distribution, and their ligand in adhesive interactions. The family members shown here are limited to those that participate in inflammation and other innate immune mechanisms. The same molecules and others participate in adaptive immunity and will be considered in Chapters 9 and 11. The nomenclature of the different molecules in these families is confusing because it often reflects the way in which the molecules were first identified rather than their related structural characteristics. Alternative names for each of the adhesion molecules are given in

1	because it often reflects the way in which the molecules were first identified rather than their related structural characteristics. Alternative names for each of the adhesion molecules are given in parentheses. Sulfated sialyl-LewisX, which is recognized by Pand E-selectin, is an oligosaccharide present on the cell-surface glycoproteins of circulating leukocytes.

1	˜°LFA-1 Integrins Bind to cell-adhesion molecules and extracellular matrix. Strong adhesion Monocytes, macrophages Fibronectin ˜5:°1 (VLA-5, CD49d:CD29) Monocytes, T cells, macrophages, neutrophils, dendritic cells, NK cells Neutrophils, monocytes, macrophages, NK cells Dendritic cells, macrophages, neutrophils, NK cells ICAM-1, ICAM-2 ICAM-1, iC3b, ÿbrinogen iC3b ˜L:°2 (LFA-1, CD11a:CD18) ˜M:°2 (CR3, Mac-1, CD11b:CD18) ˜X:°2 (CR4, p150.95, CD11c:CD18) of two transmembrane protein chains, α and β, of which there are numerous different types. Subsets of integrins have a common β chain partnered with different α chains. The leukocyte integrins important for extravasation are LFA-1 (αL:β2, also known as CD11a:CD18) and CR3 (αM:β2, complement receptor type 3, also known as CD11b:CD18 or Mac-1). We described CR3 in Section 2-13 as a receptor for iC3b, but it also binds other ligands. Both LFA-1 and CR3 bind to ICAM-1 and to ICAM-2 (Fig. 3.30). Even in the absence of infection, circulating

1	We described CR3 in Section 2-13 as a receptor for iC3b, but it also binds other ligands. Both LFA-1 and CR3 bind to ICAM-1 and to ICAM-2 (Fig. 3.30). Even in the absence of infection, circulating monocytes are continuously leaving the blood and entering certain tissues, such as the intestine, where they become resident macrophages. To navigate out of the blood vessel, they may adhere to ICAM-2, which is expressed at low levels by unactivated endothelium. CR3 also binds to fibrinogen and factor X, both substrates of the coagulation cascade.

1	Strong adhesion between leukocytes and endothelial cells is promoted by the induction of ICAM-1 on inflamed endothelium together with a conformational change in LFA-1 and CR3 that occurs on the leukocyte. Integrins can switch between an ‘active’ state, in which they bind strongly to their ligands, and an ‘inactive’ state, in which binding is easily broken. This enables cells to make and break integrin-mediated adhesions in response to signals received by the cell either through the integrin itself or through other receptors. In the activated state, an integrin molecule is linked via the intracellular protein talin to the actin cytoskeleton. In the case of migrating leukocytes, chemokines binding to their receptors on the leukocyte generate intracellular signals that cause talin to bind to the cytoplasmic tails of the β chains of LFA-1 and CD3, forcing the integrin extracellular regions to assume an active binding conformation. The importance of leukocyte integrin function in

1	bind to the cytoplasmic tails of the β chains of LFA-1 and CD3, forcing the integrin extracellular regions to assume an active binding conformation. The importance of leukocyte integrin function in inflammatory cell recruitment is illustrated by leukocyte adhesion deficiencies, which can be caused by defects in the integrins themselves or in the proteins required for modulating adhesion. People with these diseases suffer from recurrent bacterial infections and impaired healing of wounds.

1	Endothelial activation is driven by macrophage-produced cytokines, particularly TNF-α, which induce the rapid externalization of granules called Weibel–Palade bodies in the endothelial cells. These granules contain preformed P-selectin, which appears on the surfaces of local endothelial cells just minutes after macrophages have responded to the presence of microbes by producing TNF-α. Shortly after P-selectin gets to the cell surface, mRNA encoding E-selectin is synthesized, and within 2 hours the endothelial cells are expressing mainly E-selectin. Both P-selectin and E-selectin interact with sulfated sialyl-LewisX, a sulfated form of a carbohydrate structure that is also an important blood group antigen. Sulfated sialyl-LewisX is present on the surface of neutrophils, and its interactions with P-selectin and E-selectin are important for neutrophil rolling on the endothelium. Mutations in enzymes involved in its synthesis, such as fucosyltransferase, cause defective sialyl-LewisX

1	with P-selectin and E-selectin are important for neutrophil rolling on the endothelium. Mutations in enzymes involved in its synthesis, such as fucosyltransferase, cause defective sialyl-LewisX expression that results in an immunodeficiency, leukocyte adhesion deficiency type 2.

1	Integrins are also convenient cell-surface markers for distinguishing different cell types. Dendritic cells, macrophages, and monocytes express different integrin α chains and thus display distinct β2 integrins on their surface. The predominant leukocyte integrin on conventional dendritic cells is αX:β2, also known as CD11c:CD18 or complement receptor 4 (CR4) (see Fig. 3.29). This integrin is a receptor for the complement C3 cleavage product iC3b, fibrinogen, and ICAM-1. In contrast to conventional dendritic cells, most monocytes and macrophages express low levels of CD11c, and predominantly express the integrin αM:β2 (CD11b:CD18; CR3). However, patterns of integrin expression can vary, with some tissue macrophages, such as those in the lung, expressing high levels of CD11c:CD18. In the mouse, the two major branches of conventional dendritic cells can be distinguished by expression of CD11b:CD18: one branch characterized by high expression of CD11b:CD18,

1	Fig. 3.30 Phagocyte adhesion to vascular endothelium is mediated by integrins. When vascular endothelium is activated by inflammatory mediators it expresses two adhesion molecules, namely ICAM-1 and ICAM-2. These are ligands for integrins expressed by phagocytes— αM:β2 (also called CR3, Mac-1, or CD11b:CD18) and αL:β2 (also called LFA-1 or CD11a:CD18) MOVIE 3.11 MOVIE 3.12 MOVIE 3.13 and a second branch that lacks CD11b:CD18. Plasmacytoid dendritic cells (pDCs) express lower levels of CD11c, but can be distinguished from conventional dendritic cells using other markers; human pDCs express the C-type lectin BDCA-2 (blood dendritic cell antigen 2), and mouse pDCs express BST2 (bone marrow stromal antigen), neither of which is expressed by conventional dendritic cells. 3-19 Neutrophils make up the first wave of cells that cross the blood vessel wall to enter an inflamed tissue.

1	3-19 Neutrophils make up the first wave of cells that cross the blood vessel wall to enter an inflamed tissue. The migration of leukocytes out of blood vessels, the process known as extravasation, occurs in response to signals generated at sites of infection. Under normal conditions, leukocytes travel in the center of small blood vessels, where blood flow is fastest. Within sites of inflammation, the vessels are dilated and the consequent slower blood flow allows leukocytes to interact in large numbers with the vascular endothelium. During an inflammatory response, the induction of adhesion molecules on the endothelial cells of blood vessels within the infected tissue, as well as induced changes in the adhesion molecules expressed on leukocytes, recruits large numbers of circulating leukocytes to the site of infection. We will describe this process with regard to monocytes and neutrophils (Fig. 3.31).

1	Extravasation proceeds in four stages. In the first, induction of selectins induces leukocyte rolling along the endothelium. P-selectin appears on endothelial cell surfaces within a few minutes of exposure to leukotriene B4, C5a, or histamine, which is released from mast cells in response to C5a. P-selectin can also be induced by TNF-α or LPS, and both of these induce synthesis of E-selectin, which appears on the endothelial cell surface a few hours later. When the sulfated sialyl-LewisX on monocytes and neutrophils contacts these exposed P-and E-selectins, these cells adhere reversibly to the vessel wall and begin to ‘roll’ along endothelium (see Fig. 3.31, top panel), permitting stronger interactions of the next step in leukocyte migration. Neutrophils are particularly efficient at rolling along endothelium even under flow rates that prevent rolling by other cells. Such ‘shear-resistant rolling’ by neutrophils uses long extensions of plasma membrane, termed slings, that bind the

1	along endothelium even under flow rates that prevent rolling by other cells. Such ‘shear-resistant rolling’ by neutrophils uses long extensions of plasma membrane, termed slings, that bind the endothelium and wrap around the cell as it rolls, serving to tether the cell firmly to the endothelium and to promote rapid entry to sites of infection.

1	The second step depends on interactions between the leukocyte integrins LFA-1 and CR3 with adhesion molecules such as ICAM-1 (which can be induced on endothelial cells by TNF-α) and ICAM-2 on endothelium (see Fig. 3.31, bottom panel). LFA-1 and CR3 normally bind their ligands only weakly, but CXCL8 (or other chemokines), bound to proteoglycans on the surface of endothelial cells, binds to specific chemokine receptors on the leukocyte and signals the cell to trigger a conformational change in LFA-1 and CR3 on the rolling leukocyte; this greatly increases the adhesive properties of the leukocyte, as discussed in Section 3-18. The cell then attaches firmly to the endothelium, and its rolling is arrested.

1	In the third step the leukocyte extravasates, or crosses the endothelial wall. This step also involves LFA-1 and CR3, as well as a further adhesive interaction involving an immunoglobulin-related molecule called PECAM or CD31, which is expressed both on the leukocyte and at the intercellular junctions of endothelial cells. These interactions enable the phagocyte to squeeze between the endothelial cells. It then penetrates the basement membrane with the aid of enzymes that break down the extracellular matrix proteins of the basement membrane. The movement through the basement membrane is known as diapedesis, and it enables phagocytes to enter the subendothelial tissues. The fourth and final step in extravasation is the migration of leukocytes through the tissues under the influence of chemokines. Chemokines such as Selectin-mediated adhesion to leukocyte sialyl-Lewisx is weak, and allows leukocytes to roll along the vascular endothelial surface

1	CXCL8 and CCL2 (see Section 3-17) are produced at the site of infection and bind to proteoglycans in the extracellular matrix and on endothelial cell surfaces. In this way, a matrix-associated concentration gradient of chemokines is formed on a solid surface along which the leukocyte can migrate to the focus of infection (see Fig. 3.31). CXCL8 is released by the macrophages that first encounter pathogens; it recruits neutrophils, which enter the infected tissue in large numbers in the early part of the induced response. Their influx usually peaks within the first 6 hours of an inflammatory response. Monocytes are recruited through the action of CCL2, and accumulate more slowly than neutrophils. Once in the inflamed tissue, neutrophils are able to eliminate many pathogens by phagocytosis. In an innate immune response, neutrophils use their complement receptors and the direct pattern recognition receptors discussed earlier in this chapter (see Section 3-1) to recognize and phagocytose

1	In an innate immune response, neutrophils use their complement receptors and the direct pattern recognition receptors discussed earlier in this chapter (see Section 3-1) to recognize and phagocytose pathogens or pathogen components directly or after opsonization with complement (see Section 2-13). In addition, as we will see in Chapter 10, neutrophils act as phagocytic effectors in humoral adaptive immunity, taking up antibody-coated microbes by means of specific receptors.

1	The importance of neutrophils in immune defense is dramatically illustrated by diseases or medical treatments that severely reduce neutrophil numbers. Patients suffering this affliction are said to have neutropenia, and they are highly susceptible to deadly infection with a wide range of pathogens and commensal organisms. Restoring neutrophil levels in such patients by transfusion Induced innate responses to infection. 117

1	Fig. 3.31 Neutrophils leave the blood and migrate to sites of infection in a multi-step process involving adhesive interactions that are regulated by macrophage-derived cytokines and chemokines. Top panel: the first step involves the reversible binding of a neutrophil to vascular endothelium through interactions between selectins induced on the endothelium and their carbohydrate ligands on the neutrophil, shown here for E-selectin and its ligand, the sialyl-LewisX moiety (s-Lex). This interaction cannot anchor the cells against the shearing force of the flow of blood, and thus they roll along the endothelium, continually making and breaking contact. Bottom panel: the binding does, however, eventually trigger stronger interactions, which result only when binding of a chemokine such as CXCL8 to its specific receptor on the neutrophil triggers the activation of the integrins LFA-1 and CR3 (Mac-1; not shown). Inflammatory cytokines such as TNF-α are also necessary to induce the expression

1	to its specific receptor on the neutrophil triggers the activation of the integrins LFA-1 and CR3 (Mac-1; not shown). Inflammatory cytokines such as TNF-α are also necessary to induce the expression of adhesion molecules such as ICAM-1 and ICAM-2, the ligands for these integrins, on the vascular endothelium. Tight binding between ICAM-1 and the integrins arrests the rolling and allows the neutrophil to squeeze between the endothelial cells forming the wall of the blood vessel (i.e., to extravasate). The leukocyte integrins LFA-1 and CR3 are required for extravasation and for migration toward chemoattractants. Adhesion between molecules of CD31, expressed on both the neutrophil and the junction of the endothelial cells, is also thought to contribute to extravasation. The neutrophil also needs to traverse the basement membrane; it penetrates this with the aid of a matrix metalloproteinase enzyme, MMP-9, that it expresses at the cell surface. Finally, the neutrophil migrates along a

1	needs to traverse the basement membrane; it penetrates this with the aid of a matrix metalloproteinase enzyme, MMP-9, that it expresses at the cell surface. Finally, the neutrophil migrates along a concentration gradient of chemokines (shown here as CXCL8) secreted by cells at the site of infection. The electron micrograph shows a neutrophil extravasating between endothelial cells. The blue arrow indicates the pseudopod that the neutrophil is inserting between the endothelial cells. Photograph (×5500) courtesy of I. Bird and J. Spragg.

1	of neutrophil-rich blood fractions or by stimulating their production with specific growth factors largely corrects this susceptibility. 3-20 TNF-α is an important cytokine that triggers local containment of infection but induces shock when released systemically.

1	TNF-α acting on endothelial cells stimulates the expression of adhesion molecules and aids the extravasation of cells such as monocytes and neutrophils. Another important action of TNF-αis to stimulate endothelial cells to express proteins that trigger blood clotting in the local small vessels, occluding them and cutting off blood flow. This can be important in preventing the pathogen from entering the bloodstream and spreading through the blood to organs all over the body. The importance of TNF-α in the containment of local infection is illustrated by experiments in which rabbits were infected locally with a bacterium. Normally, the infection would be contained at the site of the inoculation; if, however, an injection of anti-TNF-α antibody was also given to block the action of TNF-α, the infection spread via the blood to other organs. In parallel, the fluid that has leaked into the tissue in the early phases of an infection carries the pathogen, usually enclosed in dendritic cells,

1	infection spread via the blood to other organs. In parallel, the fluid that has leaked into the tissue in the early phases of an infection carries the pathogen, usually enclosed in dendritic cells, via the lymph to the regional lymph nodes, where an adaptive immune response can be initiated.

1	Once an infection has spread to the bloodstream, however, the same mechanisms by which TNF-α so effectively contains local infection instead becomes catastrophic (Fig. 3.32). Although produced as a membrane-associated cytokine, TNF-α can be cleaved by a specific protease, TACE (TNF-α-converting enzyme, which is encoded by the ADAM17 gene), and released from the membrane as a soluble cytokine. The presence of infection in the bloodstream, or sepsis, is accompanied by a massive release of soluble TNF-α from macrophages in the liver, spleen, and other sites throughout the body. The systemic release of TNF-α into the bloodstream causes vasodilation, which leads to a loss of blood pressure and increased vascular permeability; this in turn leads to a loss of plasma volume and eventually to shock, known in this case as septic shock because the underlying cause is a bacterial infection. The TNF-α released in septic shock also triggers blood clotting in small vessels throughout the body—known

1	known in this case as septic shock because the underlying cause is a bacterial infection. The TNF-α released in septic shock also triggers blood clotting in small vessels throughout the body—known as disseminated intravascular coagulation—which leads to the massive consumption of clotting proteins, so that the patient’s blood cannot clot appropriately. Disseminated intravascular coagulation frequently leads to the failure of vital organs such as the kidneys, liver, heart, and lungs, which are quickly compromised by the failure of normal blood perfusion; consequently, septic shock has a very high mortality rate.

1	Mice with defective or no TNF-α receptors are resistant to septic shock but are also unable to control local infection. Mice in which the ADAM17 gene has been selectively inactivated in myeloid cells are also resistant to septic shock, confirming that the release of soluble TNF-αinto the circulation both depends on TACE and is the main factor responsible for septic shock. Blockade of TNF-α activity, either with specific antibodies or with soluble proteins that mimic the receptor, is a successful treatment for several inflammatory disorders, including rheumatoid arthritis. However, these treatments have been found to reactivate tuberculosis in some apparently well patients with evidence of previous infection (as demonstrated by skin test), which is a direct demonstration of the importance of TNF-α in keeping infection local and in check. 3-21 Cytokines made by macrophages and dendritic cells induce a systemic reaction known as the acute-phase response.

1	3-21 Cytokines made by macrophages and dendritic cells induce a systemic reaction known as the acute-phase response. As well as their important local effects, the cytokines produced by macrophages and dendritic cells have long-range effects that contribute to host defense. One of these is the elevation of body temperature, which is caused Local infection with Systemic infection with Gram-negative bacteria Gram-negative bacteria (sepsis) Macrophages activated to secrete Macrophages activated in the liver and TNF-˜ in the tissue spleen secrete TNF-˜ into the bloodstream Increased release of plasma proteins into Systemic edema causing decreased blood tissue. Increased phagocyte and volume, hypoproteinemia, and neutropenia, lymphocyte migration into tissue. Increased followed by neutrophilia. Decreased blood platelet adhesion to blood vessel wall volume causes collapse of vessels

1	Phagocytosis of bacteria. Local vessel Disseminated intravascular coagulation occlusion. Plasma and cells drain to leading to wasting and multiple local lymph node organ failure Removal of infection DeathAdaptive immunity mainly by TNF-α, IL-1β, and IL-6. These cytokines are termed endogenous pyrogens because they cause fever and derive from an endogenous source rather than from bacterial components such as LPS, which also induces fever and is an exogenous pyrogen. Endogenous pyrogens cause fever by inducing the synthesis of prostaglandin E2 by the enzyme cyclooxygenase-2, the expression of which is induced by these cytokines. Prostaglandin E2 then acts on the hypothalamus, resulting in an increase in both heat production from the catabolism of brown fat and heat retention from vasoconstriction, which decreases the loss of excess heat through the skin. Exogenous pyrogens are able to induce fever by promoting the production of the endogenous pyrogens

1	Induced innate responses to infection. 119

1	Fig. 3.32 The release of TNF-α by macrophages induces local protective effects, but TNF-α can be damaging when released systemically. The panels on the left show the causes and consequences of local release of TNF-α, and the panels on the right show the causes and consequences of systemic release. In both cases TNF-α acts on blood vessels, especially venules, to increase blood flow and vascular permeability to fluid, proteins, and cells, and to increase endothelial adhesiveness for leukocytes and platelets (center row). Local release thus allows an influx of fluid, cells, and proteins into the infected tissue, where they participate in host defense. Later, blood clots form in the small vessels (bottom left panel), preventing spread of infection via the blood, and the accumulated fluid and cells drain to regional lymph nodes, where an adaptive immune response is initiated. When there is a systemic infection, or sepsis, with bacteria that elicit TNF-α production, TNF-α is released into

1	cells drain to regional lymph nodes, where an adaptive immune response is initiated. When there is a systemic infection, or sepsis, with bacteria that elicit TNF-α production, TNF-α is released into the blood by macrophages in the liver and spleen and acts in a similar way on all small blood vessels in the body (bottom right panel). The result is shock, disseminated intravascular coagulation with depletion of clotting factors, and consequent bleeding, multiple organ failure, and frequently death.

1	Fig. 3.33 The cytokines TNF-α, IL-1β, and IL-6 have a wide spectrum of biological activities that help to coordinate the body’s responses to infection. IL-1β, IL-6, and TNF-α activate hepatocytes to synthesize acute-phase proteins, and bone marrow endothelium to release neutrophils. The acute-phase proteins act as opsonins, whereas the disposal of opsonized pathogens is augmented by the enhanced recruitment of neutrophils from the bone marrow. IL-1β, IL-6, and TNF-α are also endogenous pyrogens, raising body temperature, which is believed to help in eliminating infections. A major effect of these cytokines is to act on the hypothalamus, altering the body’s temperature regulation, and on muscle and fat cells, altering energy mobilization to increase the body temperature. At higher temperatures, bacterial and viral replication is less efficient, whereas the adaptive immune response operates more efficiently.

1	and also by directly inducing cyclooxygenase-2 as a consequence of signaling through TLR-4, leading to the production of prostaglandin E2. Fever is generally beneficial to host defense; most pathogens grow better at lower temperatures, whereas adaptive immune responses are more intense at elevated temperatures. Host cells are also protected from the deleterious effects of TNF-α at raised temperatures.

1	The effects of TNF-α, IL-1β, and IL-6 are summarized in Fig. 3.33. One of the most important of these occurs in the liver and is the initiation of a response known as the acute-phase response (Fig. 3.34). The cytokines act on hepatocytes, which respond by changing the profile of proteins that they synthesize and secrete into the blood. In the acute-phase response, blood levels of some proteins go down, whereas levels of others increase markedly. The proteins induced by TNF-α, IL-1β, and IL-6 are called the acute-phase proteins. Several of these are of particular interest because they mimic the action of antibodies, but unlike antibodies they have broad specificity for pathogen-associated molecular patterns and depend only on the presence of cytokines for their production.

1	One acute-phase protein, the C-reactive protein, is a member of the pentraxin protein family, so called because the proteins are formed from five identical subunits. C-reactive protein is yet another example of a multipronged pathogen-recognition molecule, and it binds to the phosphocholine portion of certain bacterial and fungal cell-wall lipopolysaccharides. Phosphocholine is also found in mammalian cell-membrane phospholipids, but it cannot be bound by C-reactive protein. When C-reactive protein binds to a bacterium, it not only is able to opsonize the bacterium but can also activate the complement cascade by binding to C1q, the first component of the classical pathway of complement activation (see Section 2-7). The interaction with C1q involves the collagen-like parts of C1q rather than the globular heads that make contact with pathogen surfaces, but the same cascade of reactions is initiated.

1	Mannose-binding lectin (MBL) is another acute-phase protein; it serves as an innate recognition molecule that can activate the lectin pathway of complement (see Section 2-6). MBL is present at low levels in the blood of healthy individuals, but it is produced in increased amounts during the acute-phase response. By recognizing mannose residues on microbial surfaces, MBL can act as an opsonin that is recognized by monocytes, which do not express the macrophage mannose receptor. Two other proteins with opsonizing properties that are also produced in increased amounts during an acute-phase response are the surfactant proteins, SP-A and SP-D. These are produced by the liver and a variety of epithelia. They are, for example, found along with macrophages in the alveolar fluid of the lung, where they are secreted by

1	Liver Activation of complement Opsonization Bone marrow endothelium Phagocytosis Hypothalamus Decreased viral and bacterial replication Increased antigen processing Increased speciÿc immune response Fat, muscle Dendritic cells Acute-phase proteins (C-reactive protein, mannose-binding lectin) Neutrophil mobilization Increased body temperature Protein and energy mobilization to allow increased body temperature TNF-˜stimulates migration to lymph nodes and maturation Initiation of adaptive immune response IL-1˜/IL-6/TNF-°

1	Fig. 3.34 The acute-phase response produces molecules that bind pathogens but not host cells. Acute-phase proteins are produced by liver cells in response to cytokines released by macrophages in the presence of bacteria (top panel). They include serum amyloid protein (SAP) (in mice but not humans), C-reactive protein (CRP), fibrinogen, and mannose-binding lectin (MBL). CRP binds phosphocholine on certain bacterial and fungal surfaces but does not recognize it in the form in which it is found in host cell membranes (middle panel). SAP and CRP are homologous in structure; both are pentraxins, forming five-membered discs, as shown for SAP (lower panel). SAP both acts as an opsonin in its own right and activates the classical complement pathway by binding C1q to augment opsonization. MBL is a member of the collectin family, which also includes the pulmonary surfactant proteins SP-A and SP-D. Like CRP, MBL can act as an opsonin in its own right, as can SP-A and SP-D. Model structure

1	is a member of the collectin family, which also includes the pulmonary surfactant proteins SP-A and SP-D. Like CRP, MBL can act as an opsonin in its own right, as can SP-A and SP-D. Model structure courtesy of J. Emsley.

1	pneumocytes, and are important in promoting the phagocytosis of opportunistic respiratory pathogens such as Pneumocystis jirovecii (formerly known as P. carinii), one of the main causes of pneumonia in patients with AIDS. Thus, within a day or two, the acute-phase response provides the host with several proteins with the functional properties of antibodies but able to bind a broad range of pathogens. However, unlike antibodies, which we describe in Chapters 4 and 10, acute-phase proteins have no structural diversity and are made in response to any stimulus that triggers the release of TNF-α, IL-1, and IL-6. Therefore, unlike antibodies, their synthesis is not specifically induced and targeted.

1	A final, distant effect of the cytokines produced by macrophages is to induce leukocytosis, an increase in the numbers of circulating neutrophils. The neutrophils come from two sources: the bone marrow, from which mature leukocytes are released in increased numbers; and sites in blood vessels, where they are attached loosely to endothelial cells. Thus the effects of these cytokines contribute to the control of infection while the adaptive immune response is being developed. As shown in Fig. 3.33, TNF-α also has a role in stimulating the migration of dendritic cells from their sites in peripheral tissues to the lymph nodes and in their maturation into nonphagocytic but highly co-stimulatory antigen-presenting cells. 3-22 Interferons induced by viral infection make several contributions to host defense.

1	3-22 Interferons induced by viral infection make several contributions to host defense. Viral infection induces the production of interferons, originally named because of their ability to interfere with viral replication in previously uninfected tissue culture cells. Interferons have a similar role in vivo, blocking the spread of viruses to uninfected cells. There are numerous genes encoding antiviral, or type I, interferons. Best understood are the IFN-α family of 12 closely related human genes and IFN-β, the product of a single gene; less well studied are IFN-κ, IFN-ε, and IFN-ω. IFN-γ is the sole type II interferon.

1	Type III interferons are a newly classified IFN family composed of the products of three IFN-λ genes, also known as IL-28A, IL-28B, and IL-29, which bind a heterodimeric IFN-λ receptor composed of a unique IL-28Rα subunit and the β subunit of the IL-10 receptor. While receptors for type I interferons and IFN-γ are widespread in their tissue distribution, type III receptors are more restricted, not expressed by fibroblasts or epithelial cells, but expressed on epithelial cells. Type I interferons are inducible and are synthesized by many cell types after infection by diverse viruses. Almost all types of cells can produce IFN-α and IFN-β in response to activation of several innate sensors. For example, type I interferons are induced by RIG-I and MDA-5 (the sensors of cytoplasmic viral RNA) downstream of MAVS, and by signaling from cGAS (the sensor of cytoplasmic DNA) downstream of STING (see Sections 3-10 and 3-11).

1	C-reactive protein binds phosphocholine on bacterial surfaces, acting as an opsonin, and also activating complement Serum amyloid protein IFN-˜, IFN-°Virus-infected host cells virus

1	IFN-˜, IFN-°Virus-infected host cells virus Fig. 3.35 Interferons are antiviral proteins produced by cells in response to viral infection. The interferons IFN-α and IFN-β have three major functions. First, they induce resistance to viral replication in uninfected cells by activating genes that cause the destruction of mRNA and inhibit the translation of viral proteins and some host proteins. These include the Mx proteins, oligoadenylate synthetase, PKR, and IFIT proteins. Second, they can induce MHC class I expression in most cell types in the body, thus enhancing their resistance to NK cells; they may also induce increased synthesis of MHC class I molecules in cells that are newly infected by virus, thus making them more susceptible to being killed by CD8 cytotoxic T cells (see Chapter 9). Third, they activate NK cells, which then selectively kill virus-infected cells.

1	However, some immune cells seem to be specialized for this task. In Section 3-1 we introduced the plasmacytoid dendritic cell (pDC). Also called interferon-producing cells (IPCs) or natural interferon-producing cells, human plasmacytoid dendritic cells were initially recognized as rare peripheral blood cells that accumulate in peripheral lymphoid tissues during a viral infection and make abundant type I interferons (IFN-α and IFN-β)—up to 1000 times more than other cell types. This abundant production of type I interferon may result from the efficient coupling of viral recognition by TLRs to the pathways of interferon production (see Section 3-7). Plasmacytoid dendritic cells express a subset of TLRs that includes TLR-7 and TLR-9, which are endosomal sensors of viral RNA and of the nonmethylated CpG residues present in the genomes of many DNA viruses (see Fig. 3.11). The requirement for TLR-9 in sensing infections caused by DNA viruses has been demonstrated, for example, by the

1	nonmethylated CpG residues present in the genomes of many DNA viruses (see Fig. 3.11). The requirement for TLR-9 in sensing infections caused by DNA viruses has been demonstrated, for example, by the inability of TLR-9-deficient plasmacytoid dendritic cells to generate type I interferons in response to herpes simplex virus. Plasmacytoid dendritic cells express CXCR3, a receptor for the chemokines CXCL9, CXCL10, and CXCL11, which are produced by T cells. This allows pDCs to migrate from the blood into lymph nodes in which there is an ongoing inflammatory response to a pathogen.

1	Interferons help defend against viral infection in several ways (Fig. 3.35). IFN-β is particularly important because it induces cells to make IFN-α, thus amplifying the interferon response. Interferons act to induce a state of resistance to viral replication in all cells. IFN-α and IFN-β bind to a common cell-surface receptor, known as the interferon-α receptor (IFNAR), which uses the JAK and STAT pathways described in Section 3-16. IFNAR uses the kinases Tyk2 and Jak1 to activate the factors STAT1 and STAT2, which can interact with IRF9 and form a complex called ISGF3 that binds to the promoters of many interferon stimulated genes (ISGs).

1	One ISG encodes the enzyme oligoadenylate synthetase, which polymerizes ATP into 2ʹ–5ʹ-linked oligomers (whereas nucleotides in nucleic acids are normally linked 3ʹ–5ʹ). These 2ʹ–5ʹ-linked oligomers activate an endoribonuclease that then degrades viral RNA. A second protein induced by IFN-α and IFN-βis a dsRNA-dependent protein kinase called PKR. This serine–threonine kinase phosphorylates the α subunit of eukaryotic initiation factor 2 (eIF2α), thus suppressing protein translation and contributing to the inhibition of viral replication. Mx (myxoma resistant) proteins are also induced by type I interferons. Humans and wild mice have two highly similar proteins, Mx1 and Mx2, which are GTPases belonging to the dynamin protein family, but how they interfere with viral replication is not understood. Oddly, most common laboratory strains of mice have inactivated both Mx genes, and in these mice, IFN-β cannot act to protect against influenza infection.

1	In the last few years, several novel ISGs have been identified and linked to antiviral functions. The IFIT (IFN-induced protein with tetratricoid repeats) family contains four human and three mouse proteins that function in restraining the translation of viral RNA into proteins. IFIT1 and IFIT2 can both suppress the translation of normal capped mRNAs by binding to subunits of the eukaryotic initiation factor 3 (eIF3) complex, which prevents eIF3 from interacting with eIF2 to form the 43S pre-initiation complex (Fig. 3.36). This action may be responsible in part for the reduction in cellular proliferation induced by type I interferons. Mice lacking IFIT1 or IFIT2 show increased susceptibility to infection by certain viruses, such as vesicular stomatitis virus.

1	Another function of IFIT1 is to suppress translation of viral RNA that lacks a normal host modification of the 5ʹ cap. Recall that the normal mammalian 5ʹ cap is initiated by linking a 7-methylguanosine nucleotide to the first ribose sugar of the mRNA by a 5ʹ–5ʹ triphosphate bridge, to produce a structure called cap-0. This structure is further modified by cytoplasmic methylation of the 2ʹ hydroxyl groups on the first and second ribose sugars of the RNA. Methylation of the first ribose sugar produces a structure called cap-1; methylation of the second generates cap-2. IFIT1 has a high affinity for cap-0, but much lower affinity for cap-1 and cap-2. Some viruses, such as Sindbis virus (family Togaviridae), lack 2ʹ-O-methylation, and therefore are restricted by this action of IFIT1. Many viruses, such as West Nile virus and SARS coronavirus, have acquired a 2ʹe-O-methyltransferase (MTase) that produce cap-1 or cap-2 on their viral transcripts. These viruses can thus evade restriction by

1	such as West Nile virus and SARS coronavirus, have acquired a 2ʹe-O-methyltransferase (MTase) that produce cap-1 or cap-2 on their viral transcripts. These viruses can thus evade restriction by IFIT1.

1	Members of the interferon-induced transmembrane protein (IFITM) family are expressed at a basal level on many types of tissues but are strongly induced by type I interferons. There are four functional IFITM genes in humans and in mice, and these encode proteins that have two transmembrane domains and are localized to various vesicular compartments of the cell. IFITM proteins act to inhibit, or restrict, viruses at early steps of infection. Although the molecular details are unclear, IFITM1 appears to interfere with the fusion of viral membranes with the membrane of the lysosome, which is required for introducing some viral genomes into the cytoplasm. Viruses that must undergo this fusion event in lysosomes, such as the Ebola virus, are restricted by IFITM1. Similarly, IFITM3 interferes with membrane fusion in late endosomes, and so restricts the influenza A virus, which undergoes fusion there. The importance of this mechanism is demonstrated by the increased viral load and higher

1	with membrane fusion in late endosomes, and so restricts the influenza A virus, which undergoes fusion there. The importance of this mechanism is demonstrated by the increased viral load and higher mortality in mice lacking IFITM3 that are infected with the influenza A virus.

1	Interferons also stimulate production of the chemokines CXCL9, CXCL10, and CXCL11, which recruit lymphocytes to sites of infection. They also increase the expression of MHC class I molecules on all types of cells, which facilitates recognition of virally infected cells by cytotoxic T lymphocytes via the display of viral peptides complexed to MHC class I molecules on the infected cell surface (see Fig. 1.30). Through these effects, interferons indirectly help promote the killing of virus-infected cells by CD8 cytotoxic T cells. Another way in which interferons act is to activate populations of innate immune cells, such as NK cells, that can kill virus-infected cells, as described below. 3-23 Several types of innate lymphoid cells provide protection in early infection.

1	A defining feature of adaptive immunity is the clonal expression of antigen receptors, produced by somatic gene rearrangements, that provide the extraordinarily diverse specificities of T and B lymphocytes (see Section 1-11). However, for several decades, immunologists have recognized cells that have lymphoid characteristics but which lack specific antigen receptors. Natural killer (NK) cells have been known the longest, but in the past several years other distinct groups of such cells have been identified. Collectively, these are now called innate lymphoid cells (ILCs) and include NK cells (Fig. 3.37). ILCs develop in the bone marrow from the same common lymphocyte progenitor (CLP) that gives rise to B and T cells. Expression of the transcription factor Id2 (inhibitor of DNA binding 2) in the CLP represses Band T-cell fates, and is required for the development of all ILCs. ILCs are identified by the absence of Tand B-cell antigen receptor and co-receptor complexes, but they express

1	in the CLP represses Band T-cell fates, and is required for the development of all ILCs. ILCs are identified by the absence of Tand B-cell antigen receptor and co-receptor complexes, but they express the receptor for IL-7. They migrate from the bone marrow and populate lymphoid tissues and peripheral organs, notably the dermis, liver, small intestine, and lung.

1	ILCs function in innate immunity as effector cells that amplify the signals delivered by innate recognition. They are stimulated by cytokines produced by other innate cells, such as macrophages or dendritic cells, that have been activated by innate sensors of microbial infection or cellular damage. Three major subgroups of ILCs are defined, largely on the basis of the types of cytokines that each produces. Group 1 ILCs (ILC1s) generate IFN-γ in response to activation by certain cytokines, in particular IL-12 and IL-18, made by dendritic cells and macrophages, and they function in protection against infection by viruses or intracellular pathogens. NK cells are now considered to be a type of ILC. ILC1s and NK cells are closely related, but have distinct functional properties and differ in the factors required for their development. NK cells are more similar to CD8 T cells in function, while ILC1s resemble more closely the TH1 subset of CD4 T cells (see Section 3-24). NK cells can be

1	the factors required for their development. NK cells are more similar to CD8 T cells in function, while ILC1s resemble more closely the TH1 subset of CD4 T cells (see Section 3-24). NK cells can be distinguished from recently identified ILC1 cells in several ways. NK cells can be found within tissues, but they also circulate through the blood, while ILC1 cells appear to be largely non-circulating tissue-resident cells. In the mouse, conventional NK cells express the integrin α2 (CD49b), while ILC1 cells, for example in the liver, lack CD49b but express the surface protein Ly49a. Both NK and ILC1 cells require the transcription factor Id2 for their development, but NK cells require the cytokine IL-15 and the transcription factors Nfil3 and eomesodermin, while liver ILC1 cells require the cytokine IL-7 and the transcription factor Tbet.

1	ILC2s produce the cytokines IL-4, IL-5, and IL-13, in response to various cytokines, particularly thymic stromal lymphopoietin (TSLP) and IL-33. ILC2 cytokines function in promoting mucosal and barrier immunity and aid in protection against parasites. ILC3s respond to the cytokines IL-1β and IL-23 and produce several cytokines, including IL-17 and IL-22, which increase defenses against extracellular bacteria and fungi. IL-17 functions by stimulating the production of chemokines that recruit neutrophils, while IL-22 acts directly on epithelial cells to stimulate the production of antimicrobial peptides such as RegIIIγ (see Section 2-4).

1	The classification of ILC subtypes and the analysis of their development and function is still an active area, and studies to define the relative importance of these cells in immune responses are ongoing. The ILC subgroups identified so far appear to be highly parallel in structure to the subsets of effector CD8 and CD4 T cells that were defined over the last three decades. The transcription factors that control the development of different ILC subsets seem, for now at least, to be the same as those that control the corresponding T-cell subsets. Because of these similarities, we will postpone a detailed description of ILC development until Chapter 9, where we will cover this topic along with the development of T-cell subsets. 3-24 NK cells are activated by type I interferon and macrophage-derived cytokines.

1	NK cells are larger than T and B cells, have distinctive cytoplasmic granules containing cytotoxic proteins, and are functionally identified by their ability to kill certain tumor cell lines in vitro without the need for specific immunization. NK cells kill cells by releasing their cytotoxic granules, which are similar to those of cytotoxic T cells and have the same effects (discussed in Chapter 9). In brief, the contents of cytotoxic granules, which contain granzymes and the pore-forming protein perforin, are released onto the surface of the target cell, and penetrate the cell membrane and induce programmed cell death. However, unlike T cells, killing by NK cells is triggered by germline-encoded receptors that recognize molecules on the surface of infected or malignantly transformed cells. A second pathway used by NK cells to kill target cells involves the TNF family member known as TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). NK cells express TRAIL on their cell

1	A second pathway used by NK cells to kill target cells involves the TNF family member known as TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). NK cells express TRAIL on their cell surface. TRAIL interacts with two TNFR superfamily ‘death’ receptors, DR4 and DR5 (encoded by TNFSF10A and B), that are expressed by many types of cells. When NK cells recognize a target cell, TRAIL stimulates DR4 and DR5 to activate the pro-enzyme caspase 8, which leads to apoptosis. In contrast to pyroptosis, induced by caspase 1 following inflammasome activation (see Section 3-9), apoptosis is not associated with production of inflammatory cytokines. We will return to discuss more details of the mechanisms of caspase-induced apoptosis when we discuss killing by cytotoxic T cells in Chapter 9. Finally, NK cells express Fc receptors (see Section 1-20); binding of antibodies to these receptors activates NK cells to release their cytotoxic granules, a process known as antibody-dependent

1	9. Finally, NK cells express Fc receptors (see Section 1-20); binding of antibodies to these receptors activates NK cells to release their cytotoxic granules, a process known as antibody-dependent cellular cytotoxicity, or ADCC, to which we will return in Chapter 10.

1	The ability of NK cells to kill target cells can be enhanced by interferons or certain cytokines. NK cells that can kill sensitive targets can be isolated from uninfected individuals, but this activity is increased 20to 100-fold when NK cells are exposed to IFN-αand IFN-β, or to IL-12, a cytokine produced by dendritic cells and macrophages during infection by many types of pathogens. Activated 1 2 3 4 5 6 7 8 9 10 time after viral infection (days) NK-cell-mediated killing of infected cells T-cell-mediated killing of infected cells virus titer production of IFN-˜, IFN-°, TNF-˜, and IL-12

1	Fig. 3.38 Natural killer cells (NK cells) are an early component of the host response to virus infection. Experiments in mice have shown that IFN-α, IFN-β, and the cytokines TNF-α and IL-12 are produced first, followed by a wave of NK cells, which together control virus replication but do not eliminate the virus. Virus elimination is accomplished when virus-specific CD8 T cells and neutralizing antibodies are produced. Without NK cells, the levels of some viruses are much higher in the early days of the infection, and the infection can be lethal unless treated vigorously with antiviral compounds.

1	NK cells serve to contain virus infections while the adaptive immune response is generating antigen-specific cytotoxic T cells and neutralizing antibodies that can clear the infection (Fig. 3.38). A clue to the physiological function of NK cells in humans comes from rare patients deficient in these cells, who are frequently susceptible to herpesvirus infection. For example, a selective NK-cell deficiency results from mutations in the human MCM4 (minichromosome maintenance-deficient 4) protein, which is associated with predisposition to viral infections.

1	IL-12, acting in synergy with the cytokine IL-18 produced by activated macrophages, can also stimulate NK cells to secrete large amounts of interferon (IFN)-γ, and this is crucial in controlling some infections before the IFN-γ produced by activated CD8 cytotoxic T cells becomes available. IFN-γ, whose receptor activates only the STAT1 transcription factor, is quite distinct functionally from the antiviral type I interferons IFN-α and IFN-β, and is not directly induced by viral infection. The production of IFN-γ by NK cells early in an immune response can directly activate macrophages to enhance their capacity to kill pathogens, augmenting innate immunity, but also influences adaptive immunity through actions on dendritic cells and in regulating the differentiation of CD4 T cells into the pro-inflammatory TH1 subset, which produces IFN-γ. NK cells also produce TNF-α, granulocyte-macrophage stimulating factor (GM-CSF), and the chemokines CCL3 (MIF 1-α), CCL4, and CCL5 (RANTES), which

1	pro-inflammatory TH1 subset, which produces IFN-γ. NK cells also produce TNF-α, granulocyte-macrophage stimulating factor (GM-CSF), and the chemokines CCL3 (MIF 1-α), CCL4, and CCL5 (RANTES), which act to recruit and activate macrophages.

1	3-25 NK cells express activating and inhibitory receptors to distinguish between healthy and infected cells.

1	For NK cells to defend against viruses or other pathogens, they should be able to distinguish infected cells from uninfected healthy cells. However, the mechanism used by NK cells is slightly more complicated than pathogen recognition by T or B cells. In general, it is thought that an individual NK cell expresses various combinations of germline-encoded activating receptors and inhibitory receptors. While the exact details are not clear in every case, it is thought that the overall balance of signaling by these receptors determines whether an NK cell engages and kills a target cell. The receptors on an NK cell are tuned to detect changes in expression of various surface proteins on a target cell, referred to as ‘dysregulated self.’ The activating receptors generally recognize cell-surface proteins that are induced on target cells by metabolic stress, such as malignant transformation or microbial infection. These changes are referred to as ‘stress-induced self.’ Specific cellular

1	proteins that are induced on target cells by metabolic stress, such as malignant transformation or microbial infection. These changes are referred to as ‘stress-induced self.’ Specific cellular events, including DNA damage, signals related to proliferation, heat-shock related stress, and signaling by innate sensors including TLRs can lead to expression by host cells of surface proteins that bind to the activating receptors on NK cells. Stimulation of activating receptors will add to the chance that the NK cells will release cytokines such as IFN-γ and activate the killing of the stimulating cell through the release of cytotoxic granules.

1	By contrast, inhibitory receptors on NK cells recognize surface molecules that are constitutively expressed at high levels by most cells, and the loss of these molecules is referred to as ‘missing self.’ Inhibitory receptors can recognize other molecules, but those recognizing MHC class I molecules have been studied the most so far. MHC molecules are glycoproteins expressed on nearly all cells of the body. We will discuss the role of MHC proteins in antigen presentation to T cells in Chapter 6, but for now we need only to introduce the two main classes of MHC molecules. MHC class I molecules are expressed on most of the cells of the body (except, notably, red blood cells), whereas the expression of MHC class II molecules is far more restricted, largely to immune cells.

1	Inhibitory receptors that recognize MHC class I molecules function to prevent NK cells from killing normal host cells. The greater the number of MHC class I molecules on a cell surface, the better protected that cell is against attack by NK cells. Interferons induce expression of MHC class I molecules, and protect uninfected host cells from being killed by NK cells, while also activating NK cells to kill virus-infected cells. Viruses and some other intracellular pathogens can cause downregulation of MHC class I molecules as a strategy to prevent the display of antigens as peptides to T cells, also discussed in Chapter 6. NK cells are able to sense this reduction in expression of MHC class I molecules through reduced signaling from their inhibitory receptors. Reduction in MHC class I expression is an example of ‘missing self,’ and increases the chance that an NK cell will kill the target cell. It is thought that the balance of signals from ‘stress-induced self’ and ‘missing self’

1	expression is an example of ‘missing self,’ and increases the chance that an NK cell will kill the target cell. It is thought that the balance of signals from ‘stress-induced self’ and ‘missing self’ determines whether an individual NK cell will be triggered to kill a particular target cell (Fig. 3.39). Thus receptors expressed on NK cells integrate the signals from two types of

1	Fig. 3.39 Killing by NK cells depends on the balance between activating and inhibitory signals. NK cells have several different activating receptors that signal the NK to kill the bound cell. However, NK cells are prevented from a wholesale attack by another set of inhibitory receptors that recognize MHC class I molecules (which are present on almost all cell types) and that inhibit killing by overruling the actions of the activating receptors. This inhibitory signal is lost when the target cells do not express MHC class I, such as in cells infected with viruses, many of which specifically inhibit MHC class I expression or alter its conformation so as to avoid recognition by CD8 T cells. NK cells may also kill target cells through their expression of the TNF family member TRAIL, which binds to TNFR members DR4 and DR5 expressed by some types of cells. DR4 and DR5 signal through FADD, an adaptor that activates pro-caspase 8, leading to induction of apoptosis of the target cell.

1	Fig. 3.40 The genes that encode NK receptors fall into two large families.

1	The first, the leukocyte receptor complex (LRC), comprises a large cluster of genes encoding a family of proteins composed of immunoglobulin-like domains. These include the killer cell immunoglobulin-like receptors (KIRs) expressed by NK cells, the ILT (immunoglobulin-like transcript) class, and the leukocyte-associated immunoglobulin-like receptor (LAIR) gene families. The sialic acid-binding Ig-like lectins (SIGLECs) and members of the CD66 family are located nearby. In humans, this complex is located on chromosome 19. The second gene cluster is called the NK receptor complex (NKC) and encodes killer cell lectin-like receptors (KLRs), a receptor family that includes the NKG2 proteins and CD94, with which some NKG2 molecules pair to form a functional receptor. This complex is located on human chromosome 12. Some NK receptor genes are found outside these two major gene clusters; for example, the genes for the natural cytotoxicity receptors NKp30 and NKp44 are located within the major

1	human chromosome 12. Some NK receptor genes are found outside these two major gene clusters; for example, the genes for the natural cytotoxicity receptors NKp30 and NKp44 are located within the major histocompatibility complex on chromosome 6. Figure based on data courtesy of J. Trowsdale University of Cambridge.

1	surface receptors, which together control the NK cell’s cytotoxic activity and cytokine production. 3-26 NK-cell receptors belong to several structural families, the KIRs, KLRs, and NCRs.

1	The receptors that regulate the activity of NK cells fall into two large families that contain a number of other cell-surface receptors in addition to NK receptors (Fig. 3.40). Members of the killer cell immunoglobulin-like receptor family, or KIRs, have differing numbers of immunoglobulin domains. Some, such as KIR-2D, have two immunoglobulin domains, whereas others, such as KIR-3D, have three. The KIR genes form part of a larger cluster of immunoglobulin-like receptor genes known as the leukocyte receptor complex (LRC). Another family, the killer cell lectin-like receptors, or KLRs, are C-type lectin-like proteins whose genes reside within a gene cluster called the NK receptor complex (NKC). Mice lack KIR genes, and instead predominantly express Ly49 receptors encoded in the NKC on mouse chromosome 6 to control their NK-cell activity. These receptors can be activating or inhibitory, and are highly polymorphic between different strains of mice. By contrast, humans lack functional

1	chromosome 6 to control their NK-cell activity. These receptors can be activating or inhibitory, and are highly polymorphic between different strains of mice. By contrast, humans lack functional Ly49 genes and rely on KIRs encoded in the LRC to control their NK-cell activity. An important feature of the NK-cell population is that any given NK cell expresses only a subset of the receptors in its potential repertoire, and so not all NK cells in the individual are identical.

1	Activating and inhibitory receptors are present within the same structural family. Whether a KIR protein is activating or inhibitory depends on the presence or absence of particular signaling motifs in its cytoplasmic domain. Inhibitory KIRs have long cytoplasmic tails that contain an immunoreceptor tyrosine-based inhibition motif (ITIM). The consensus sequence for the ITIM is V/I/LxYxxL/V, where x stands for any amino acid. For example, the cytoplasmic tails of the inhibitory receptors KIR-2DL and KIR-3DL each contain two ITIMs (Fig. 3.41). When ligands associate with an inhibitory KIR, the tyrosine in its ITIM becomes phosphorylated by the action of Src family protein tyrosine kinases. When phosphorylated, the ITIM can then bind the intracellular protein tyrosine phosphatases SHP-1 (src homology region 2-containing protein tyrosine phosphatase-1) and SHP-2, which become localized near the cell membrane. These phosphatases inhibit signaling induced by other receptors by removing

1	(src homology region 2-containing protein tyrosine phosphatase-1) and SHP-2, which become localized near the cell membrane. These phosphatases inhibit signaling induced by other receptors by removing phosphates from tyrosine residues on other intracellular signaling molecules.

1	Activating KIRs have short cytoplasmic tails, designated, for example, as KIR2DS and KIR-3DS (see Fig. 3.41). These receptors lack an ITIM and instead have a charged residue in their transmembrane regions that associates with an accessory signaling protein called DAP12. DAP12 is a transmembrane protein that contains an immunoreceptor tyrosine-based activation motif (ITAM, with consensus sequence YXX[L/I]X6–9YXX[L/I]) in its cytoplasmic tail and forms a disulfide-linked homodimer in the membrane. When a ligand binds to an activating KIR, the tyrosine residues in the ITAM of DAP12 become phosphorylated, turning on intracellular signaling pathways that activate the NK cell and lead to release of the cytotoxic granules. The phosphorylated ITAMs bind and activate intracellular tyrosine kinases such as Syk or ZAP-70, leading to further signaling events similar to those described for T cells in Chapter 7.

1	The KLR family also has both activating and inhibitory members. In mice, inhibitory Ly49 receptors have an ITIM in their cytoplasmic tail that recruits SHP-1. The latter’s importance is shown by the failure of Ly49 to inhibit NK activation upon binding to MHC class I in mice carrying the motheaten mutation, which inactivates SHP-1 protein. In humans and mice, NK cells express a heterodimer of two different C-type lectin-like receptors, CD94 and NKG2. This heterodimer interacts with nonpolymorphic MHC class I-like molecules, including HLA-E in humans and Qa1 in mice. HLA-E and Qa1 are unusual in that instead of binding peptides derived from pathogens, they bind fragments of the signal peptide derived from other MHC class I molecules during processing in the endoplasmic reticulum. This enables CD94:NKG2 to detect the presence of several different MHC class I variants, whose expression may be targeted by viruses, and kill cells in which overall MHC molecule expression is diminished. In

1	CD94:NKG2 to detect the presence of several different MHC class I variants, whose expression may be targeted by viruses, and kill cells in which overall MHC molecule expression is diminished. In humans there are four closely related NKG2 family proteins, NKG2A, C, E, and F (encoded by KLRC1–4), and a more distantly related protein, NKG2D (encoded by KLRK1). Of these, for example, NKG2A contains an ITIM and is inhibitory, whereas NKG2C has a charged transmembrane residue, associates with DAP12, and is activating (see Fig. 3.41). NKG2D is also activating but quite distinct from the other NKG2 receptors, and we will discuss it separately below.

1	The overall response of NK cells to differences in MHC expression is further complicated by the extensive polymorphism of KIR genes, with different numbers of activating and inhibitory KIR genes being found in different people. This may explain why NK cells are a barrier to bone marrow transplantation, since the NK cells of the recipient may react more strongly to donor MHC molecules than to the self MHC with which they developed. A similar phenomenon may occur during pregnancy, because of differences between fetal and maternal MHC molecules (see Section 15-38). The advantage of such extensive KIR polymorphism is not yet clear, and some genetic epidemiologic studies even suggest an association between certain alleles of KIR genes and earlier onset (although not absolute frequency) of rheumatoid arthritis. The KIR gene cluster is not present in mice, but some species, including some primates, contain genes of both the KIR and KLR families. This might suggest that both gene clusters are

1	arthritis. The KIR gene cluster is not present in mice, but some species, including some primates, contain genes of both the KIR and KLR families. This might suggest that both gene clusters are relatively ancient and that for some reason, one or the other gene cluster was lost by mice and humans.

1	Signaling by the inhibitory NK receptors suppresses the killing activity and cytokine production of NK cells. This means that NK cells will not kill healthy, genetically identical cells with normal expression of MHC class I molecules, such as the other cells of the body. Virus-infected cells, however, can become susceptible to being killed by NK cells by a variety of mechanisms. First, some viruses inhibit all protein synthesis in their host cells, so that synthesis of MHC class I proteins would be blocked in infected cells, even while their production in uninfected cells is being stimulated by the actions of type I interferons. The reduced level of MHC class I expression in infected cells would make them correspondingly less able to inhibit NK cells through their MHC-specific receptors, and they would become more susceptible to being killed. Second, many viruses can selectively prevent the export of MHC class I molecules to the cell surface, or induce their degradation once there.

1	and they would become more susceptible to being killed. Second, many viruses can selectively prevent the export of MHC class I molecules to the cell surface, or induce their degradation once there. This might allow the infected cell to evade recognition by cytotoxic T cells but would make it

1	Fig. 3.41 The structural families of NK receptors encode both activating and inhibitory receptors. The families of killer cell immunoglobulin-like receptors (KIRs) and killer cell lectin-like receptors (KLRs) have members that send activating signals to the NK cell (upper panel) and those that send inhibitory signals (lower panel). KIR family members are designated according to the number of immunoglobulin-like domains they possess and by the length of their cytoplasmic tails. Activating KIR receptors have short cytoplasmic tails and bear the designation ‘S.’ These associate with the signaling protein DAP12 via a charged amino acid residue in the transmembrane region. The cytoplasmic tails of DAP12 contain amino acid motifs called ITAMs, which are involved in signaling. NKG2 receptors belong to the KLR family, and, whether activating or inhibitory, form heterodimers with another C-type lectin-like family member, CD94. The inhibitory KIR receptors have longer cytoplasmic tails and are

1	to the KLR family, and, whether activating or inhibitory, form heterodimers with another C-type lectin-like family member, CD94. The inhibitory KIR receptors have longer cytoplasmic tails and are designated ‘L’; these do not associate constitutively with adaptor proteins but contain a signaling motif called an ITIM, which when phosphorylated is recognized by inhibitory phosphatases.

1	Fig. 3.42 Activating receptors of NK cells include the natural cytotoxicity receptors and NKG2D. The natural cytotoxicity receptors are immunoglobulinlike proteins. NKp30 and NKp44, for example, have an extracellular domain that resembles a single variable domain of an immunoglobulin molecule. NKp30 and NKp46 activate the NK cell through their association with homodimers of the CD3ζ chain, or the Fc receptor γ chain (not shown). These signaling proteins also associate with other types of receptors that are described in Chapter 7. NKp44 activates the NK cell through their association with homodimers of DAP12. NKp46 resembles the KIR-2D molecules in having two domains that resemble the constant domains of an immunoglobulin molecule. NKG2D is a member of the C-type lectin-like family and forms a homodimer, and it associates with DAP10. In mice, an alternatively spliced form of NKG2D also associates with DAP12 (not shown).

1	The ligands for NKG2D are MHC-like molecules, MIC-A, MIC-B, or RAET1 family members, whose expression is induced by cellular stress MIC-A or MIC-B RAET1 family (includes MULT1, ULBPs) susceptible to being killed by NK cells. Virally infected cells can still be killed by NK cells even if the cells do not downregulate MHC, provided that ligands for activating receptors are induced. However, some viruses target ligands for the activating receptors on NK cells, thwarting NK-cell recognition and killing of virus-infected cells 3-27 NK cells express activating receptors that recognize ligands induced on infected cells or tumor cells.

1	In addition to the KIRs and KLRs, which have a role in sensing the level of MHC class I proteins present on other cells, NK cells also express receptors that more directly sense the presence of infection or other perturbations in a cell. Activating receptors for the recognition of infected cells, tumor cells, and cells injured by physical or chemical damage include the natural cytotoxicity receptors (NCRs) NKp30, NKp44, and NKp46, which are immunoglobulin-like receptors, and the C-type lectin-like family members Ly49H and NKG2D (Fig. 3.42). Among NCRs, only NKp46 is conserved in humans and in mice, and it is the most selective marker of NK cells across mammalian species. The ligands recognized by the NCRs are still being defined, but some evidence suggests that they recognize viral proteins, including the hemagglutinin (HA) glycoprotein of the influenza virus. Ly49H is an activating receptor that recognizes the viral protein m157, an MHC class I-like structure encoded by the murine

1	including the hemagglutinin (HA) glycoprotein of the influenza virus. Ly49H is an activating receptor that recognizes the viral protein m157, an MHC class I-like structure encoded by the murine cytomegalovirus. The ligand for NKp30 is a protein named B7-H6, a member of the family of co-stimulatory proteins mentioned in Section 1-15, and is further described in Chapters 7 and 9.

1	NKG2D has a specialized role in activating NK cells. NKG2 family members form heterodimers with CD94 and bind the MHC class I molecule HLA-E. In contrast, two NKG2D molecules form a homodimer that binds to several MHC class I-like molecules that are induced by various types of cellular stress. These include the MIC molecules MIC-A and MIC-B, and the RAET1 family of proteins, which are similar to the α1 and α2 domains of MHC class I molecules (Fig. 3.43). The RAET1 family has 10 members, 3 of which were initially characterized as ligands for the cytomegalovirus UL16 protein and are also called UL16-binding proteins, or ULBPs. Mice do not have equivalents of the MIC molecules; the ligands for mouse NKG2D have a very similar structure to that of the RAET1 proteins, and are probably orthologs of them. In fact, these ligands were first identified in mice as the RAE1 (retinoic acid early inducible 1) protein family, and also include related proteins H60 and MULT1 (see Fig. 6.26). We will

1	of them. In fact, these ligands were first identified in mice as the RAE1 (retinoic acid early inducible 1) protein family, and also include related proteins H60 and MULT1 (see Fig. 6.26). We will return to these MHC-like molecules when we discuss the structure of the MHC molecule in Section 6-18.

1	The ligands for NKG2D are expressed in response to cellular or metabolic stress, and so are upregulated on cells infected with intracellular bacteria and most viruses, as well as on incipient tumor cells that have become malignantly transformed. Thus, recognition by NKG2D acts as a generalized ‘danger’ signal to the immune system. In addition to expression by a subset of NK cells, NKG2D is expressed by various T cells, including all human CD8 T cells, γ:δ T cells, activated murine CD8 T cells, and invariant NKT cells (described in Chapter 8). In these cells, recognition of NKG2D ligands provides a potent co-stimulatory signal that enhances their effector functions.

1	Fig. 3.43 The ligands for the activating NK receptor NKG2D are proteins that are expressed in conditions of cellular stress. The MIC proteins MIC-A and MIC-B are MHC-like molecules induced on epithelial and other cells by stress, such as DNA damage, cellular transformation, or infection. RAET1 family members, including the subset designated as UL16-binding proteins (ULBPs), also resemble a portion of an MHC class I molecule—the α1 and α2 domains—and most (but not all) are attached to the cell via a glycophosphatidylinositol linkage. Unlike MHC class I molecules, the NKG2D ligands do not bind processed peptides.

1	NKG2D also differs from other activating receptors on NK cells in the signaling pathway it engages within the cell. The other activating receptors are associated intracellularly with signaling proteins such as the CD3ζ chain, the Fc receptor γ chain, and DAP12, which all contain ITAMs. In contrast, NKG2D binds a different adaptor protein, DAP10, which does not contain an ITAM sequence and instead activates the intracellular lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), initiating a different series of intracellular signaling events in the NK cell (see Section 7-4). Generally, PI 3-kinase is considered to enhance the survival of cells in which it is activated, thereby augmenting the cell’s overall effector activity. In NK cells, activation of PI 3-kinase is directly linked to the induction of cytotoxic activity. In mice, the workings of NKG2D are even more complicated, because mouse NKG2D is produced in two alternatively spliced forms, one of which binds DAP12 and DAP10,

1	the induction of cytotoxic activity. In mice, the workings of NKG2D are even more complicated, because mouse NKG2D is produced in two alternatively spliced forms, one of which binds DAP12 and DAP10, whereas the other binds only DAP10. Mouse NKG2D can thus activate both signaling pathways, whereas human NKG2D seems to signal only through DAP10 to activate the PI 3-kinase pathway. Finally, NK cells express several receptors from the SLAM (signaling lymphocyte activation molecule) family, including 2B4, which recognizes the cell-surface molecule CD48 expressed by many cells including NK cells. Interactions between 2B4 and CD48 on nearby NK cells can release signals that promote survival and proliferation through SAP (SLAM-associated protein) and the Src kinase Fyn.

1	Summary.

1	Triggering of innate sensors on various cells—neutrophils, macrophages, and dendritic cells in particular—not only activates these cells’ individual effector functions, but also stimulates the release of pro-inflammatory chemokines and cytokines that act together to recruit more phagocytic cells to the site of infection. Especially prominent is the early recruitment of neutrophils and monocytes. Furthermore, cytokines released by tissue phagocytic cells can induce more systemic effects, including fever and the production of acute-phase response proteins, including mannose-binding lectin, C-reactive protein, fibrinogen, and pulmonary surfactant proteins, which add to a general state of augmented innate immunity. These cytokines also mobilize antigen-presenting cells that induce the adaptive immune response. The innate immune system has at its service several recently recognized subtypes of innate lymphoid cells which join the ranks of the long-recognized NK cells. ILCs exhibit

1	adaptive immune response. The innate immune system has at its service several recently recognized subtypes of innate lymphoid cells which join the ranks of the long-recognized NK cells. ILCs exhibit specialized effector activity in response to different signals, and act to amplify the strength of the innate response. The production of interferons in response to viral infections serves to inhibit viral replication and to activate NK cells. These in turn can distinguish healthy cells from those that are infected by virus or that are transformed or stressed in some way, based on the expression of class I MHC molecules and MHC-related molecules that are ligands for some NK receptors. As we will see later in the book, cytokines, chemokines, phagocytic cells, and NK cells are all effector mechanisms that are also employed in the adaptive immune response, which uses variable receptors to target specific pathogen antigens.

1	Summary to Chapter 3.

1	Innate immunity uses a variety of effector mechanisms to detect infection and eliminate pathogens, or hold them in check until an adaptive immune response develops. These effector mechanisms are all regulated by germline-encoded receptors on many types of cells that can detect molecules of microbial origin or that sense signs of host cellular damage. The induced responses of the innate immune system are based on several distinct components. After the initial barriers—the body’s epithelia and the soluble antimicrobial molecules described in Chapter 2—have been breached, the most important innate defenses rely on tissue macrophages and other tissue-resident sensor cells, such as dendritic cells. Macrophages provide a double service: they mediate rapid cellular defense at the borders of infection through phagocytosis and antimicrobial actions, and they also use their various innate sensors to activate the process of inflammation, which involves recruiting additional cells to sites of

1	through phagocytosis and antimicrobial actions, and they also use their various innate sensors to activate the process of inflammation, which involves recruiting additional cells to sites of infection. Innate sensors activate signaling pathways that lead to the production of pro-inflammatory and antiviral cytokines, which in turn stimulate innate effector responses while also helping to initiate an adaptive immune response. The uncovering of the pathogen-sensing mechanisms described in this chapter is still extremely active. It is providing new insights into human autoinflammatory conditions such as lupus, Crohn’s disease, and gout. Indeed, the induction of powerful effector mechanisms by innate immune recognition based on germline-encoded receptors clearly has some dangers. It is a double-edged sword, as is illustrated by the effects of the cytokine TNF-α—beneficial when released locally, but disastrous when produced systemically. This illustrates the evolutionary knife edge along

1	sword, as is illustrated by the effects of the cytokine TNF-α—beneficial when released locally, but disastrous when produced systemically. This illustrates the evolutionary knife edge along which all innate mechanisms of host defense travel. The innate immune system can be viewed as a defense system that mainly frustrates the establishment of a focus of infection; however, even when it proves inadequate in fulfilling this function, it has already set in motion—by recruiting and activating dendritic cells—the initiation of the adaptive immune response, which forms an essential part of humans’ defenses against infection.

1	Having introduced immunology with a consideration of innate immune function, we next turn our attention to the adaptive immune response, beginning with an explanation of the structure and function of the antigen receptors expressed by lymphocytes. Questions. 3.1 Matching: Match the Toll-like receptor (TLR) to its ligand: A. TLR-2:TLR-1 or i. ssRNA TLR-2:TLR-6 B. TLR-3 ii. Lipopolysaccharide C. TLR-4 iii. Lipoteichoic acid and di-/triacyl lipoproteins D. TLR-5 iv. dsRNA E. TLR-7 v. Flagellin F. TLR-9 vi. Unmethylated CpG DNA 3.2 Matching: Match the hereditary disorder to the gene affected: A. Chronic granulomatous i. NOD2 disease B. X-linked hypohidrotic ii. IKKγ (NEMO) ectodermal dysplasia and immunodeficiency C. Crohn’s disease iii. Jak3 D. X-linked SCID iv. NAPDH oxidase E. SCID (not X-linked) v. NLRP3 F. Familial cold vi. γc inflammatory syndrome 3.3 Multiple Choice: Which of the following does not occur during an inflammatory response? A. Local blood clotting

1	E. SCID (not X-linked) v. NLRP3 F. Familial cold vi. γc inflammatory syndrome 3.3 Multiple Choice: Which of the following does not occur during an inflammatory response? A. Local blood clotting B. Tissue injury repair C. Endothelial cell activation D. Decreased vascular permeability E. Extravasation of leukocytes into inflamed tissue 3.4 Short Answer: What is the difference between conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs)? 3.5 Multiple Choice: Which of the following is a G-proteincoupled receptor? A. fMLF receptor B. TLR-4 C. IL-1R D. CD14 E. STING F. B7.1 (CD80) 3.6 True or False: All forms of ubiquitination lead to proteasomal degradation. 3.7 Fill-in-the-Blanks: A. Toll-like receptors (TLRs) have a cytoplasmic signaling domain called TIR that is also shared with __________.

1	3.7 Fill-in-the-Blanks: A. Toll-like receptors (TLRs) have a cytoplasmic signaling domain called TIR that is also shared with __________. B. Cytokine receptors of the hematopoietin family activate tyrosine kinases of the _____ family, in order to signal these recruit SH2-domain-containing transcriptions factors called __________. C. Out of all the different TLRs, the only one that uses both MyD88/MAL and TRIF/TRAM adaptor pairs is __________. 3.8 True or False: Cytosolic DNA is sensed by cGAS, which signals through STING, while cytosolic ssRNA and dsRNA are sensed by RIG-I and MDA-5, respectively, which interact with the downstream adaptor protein MAVS. 3.9 Multiple Choice: Which of the following is not true? A. CCL2 attracts macrophages through CCR2. B. IL-3, IL-5, and GM-CSF are a subgroup of class I cytokine receptors that share a common β chain. C. IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 share a common γc.

1	B. IL-3, IL-5, and GM-CSF are a subgroup of class I cytokine receptors that share a common β chain. C. IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 share a common γc. D. The inflammasome is a large oligomer composed of the sensor NLRP3, the adaptor ASC, and caspase 8. E. CXCL8 attracts neutrophils through CXCR2. F. ILC1s secrete IFN-γ, ILC2s secrete IL-4, IL-5, and IL-13, and ILC3s secrete IL-17 and IL-22. Section references. 3‑1 After entering tissues, many microbes are recognized, ingested, and killed by phagocytes. Aderem, A., and Underhill, D.M.: Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 1999, 17:593–623. Auffray, C., Fogg, D., Garfa, M., Elain, G., Join-Lambert, O., Kayal, S., Sarnacki, S., Cumano, A., Lauvau, G., and Geissmann, F.: Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science 2007, 317:666–670.

1	Cervantes-Barragan, L., Lewis, K.L., Firner, S., Thiel, V., Hugues, S., Reith, W., Ludewig, B., and Reizis, B.: Plasmacytoid dendritic cells control T‑cell response to chronic viral infection. Proc. Natl Acad. Sci. USA 2012, 109:3012–3017. Goodridge, H.S., Wolf, A.J., and Underhill, D.M.: Beta‑glucan recognition by the innate immune system. Immunol. Rev. 2009, 230:38–50. Greaves, D.R., and Gordon, S.: The macrophage scavenger receptor at 30 years of age: current knowledge and future challenges. J. Lipid Res. 2009, 50:S282–S286. Greter, M., Lelios, I., Pelczar, P., Hoeffel, G., Price, J., Leboeuf, M., Kdig, T.M., Frei, K., Ginhoux, F., Merad, M., and Becher, B.: Stroma‑derived interleukin‑34 controls the development and maintenance of Langerhans cells and the maintenance of microglia. Immunity 2012, 37:1050–1060. Harrison, R.E., and Grinstein, S.: Phagocytosis and the microtubule cytoskeleton. Biochem. Cell Biol. 2002, 80:509–515.

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1	3.11 Matching: Match the step in neutrophil recruitment into inflamed tissues with the key effectors involved: A. Endothelial cell i. Neutrophil LFA-1 with activation endothelial ICAM-1 B. Rolling ii. Local secretion of TNF-α and other cytokines C. Neutrophil integrin iii.CXCL8 signaling through assuming ‘active’ CXCR2 leading to talin state activation D. Strong adhesion iv.Endothelial and neutrophil CD31 E. Diapedesis v. Interaction of endothelial P-and E-selectin with neutrophil sulfated sialyl-LewisX 3.12 Short Answer: What co-stimulatory molecules are induced on macrophages and dendritic cells upon pathogen recognition, and what is their function? McGreal, E.P., Miller, J.L., and Gordon, S.: Ligand recognition by antigen‑presenting cell C‑type lectin receptors. Curr. Opin. Immunol. 2005, 17:18–24.

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1	Pietras, E.M., Saha, S.K., and Cheng, G.: The interferon response to bacterial and viral infections. J. Endotoxin Res. 2006, 12:246–250. Pott, J., Mahlakv, T., Mordstein, M., Duerr, C.U., Michiels, T., Stockinger, S., Staeheli, P., and Hornef, M.W.: IFN‑lambda determines the intestinal epithelial antiviral host defense. Proc. Natl Acad. Sci. USA 2011, 108:7944–7949. 3‑23 Several types of innate lymphoid cells provide protection in early infection. Cortez, V.S., Robinette, M.L., and Colonna, M.: Innate lymphoid cells: new insights into function and development. Curr. Opin. Immunol. 2015, 32:71–77. Spits, H., Artis, D., Colonna, M., Diefenbach, A., Di Santo, J.P., Eberl, G., Koyasu, S., Locksley, R.M., McKenzie, A.N., Mebius, R.E., et al.: Innate lymphoid cells—a proposal for uniform nomenclature. Nat. Rev. Immunol. 2013, 13:145–149. 3‑24 NK cells are activated by type I interferon and macrophage‑derived cytokines.

1	3‑24 NK cells are activated by type I interferon and macrophage‑derived cytokines. Barral, D.C., and Brenner, M.B.: CD1 antigen presentation: how it works. Nat. Rev. Immunol. 2007, 7:929–941. Gineau, L., Cognet, C., Kara, N., Lach, F.P., Dunne, J., Veturi, U., Picard, C., Trouillet, C., Eidenschenk, C., Aoufouchi S., et al.: Partial MCM4 deficiency in patients with growth retardation, adrenal insufficiency, and natural killer cell deficiency. J. Clin. Invest. 2012, 122:821–832. Godshall, C.J., Scott, M.J., Burch, P.T., Peyton, J.C., and Cheadle, W.G.: Natural killer cells participate in bacterial clearance during septic peritonitis through interactions with macrophages. Shock 2003, 19:144–149. Lanier, L.L.: Evolutionary struggles between NK cells and viruses. Nat. Rev. Immunol. 2008, 8:259–268. Salazar-Mather, T.P., Hamilton, T.A., and Biron, C.A.: A chemokine‑to‑cytokine‑to‑chemokine cascade critical in antiviral defense. J. Clin. Invest. 2000, 105:985–993.

1	Salazar-Mather, T.P., Hamilton, T.A., and Biron, C.A.: A chemokine‑to‑cytokine‑to‑chemokine cascade critical in antiviral defense. J. Clin. Invest. 2000, 105:985–993. Seki, S., Habu, Y., Kawamura, T., Takeda, K., Dobashi, H., Ohkawa, T., and Hiraide, H.: The liver as a crucial organ in the first line of host defense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+ T cells in T helper 1 immune responses. Immunol. Rev. 2000, 174:35–46. Yokoyama, W.M., and Plougastel, B.F.: Immune functions encoded by the natural killer gene complex. Nat. Rev. Immunol. 2003, 3:304–316. 3‑25 NK cells express activating and inhibitory receptors to distinguish between healthy and infected cells. & 3‑26 NK‑cell receptors belong to several structural families, the KIRs, KLRs, and NCRs.

1	& 3‑26 NK‑cell receptors belong to several structural families, the KIRs, KLRs, and NCRs. Borrego, F., Kabat, J., Kim, D.K., Lieto, L., Maasho, K., Pena, J., Solana, R., and Coligan, J.E.: Structure and function of major histocompatibility complex (MHC) class I specific receptors expressed on human natural killer (NK) cells. Mol. Immunol. 2002, 38:637–660. Boyington, J.C., and Sun, P.D.: A structural perspective on MHC class I recognition by killer cell immunoglobulin‑like receptors. Mol. Immunol. 2002, 38:1007–1021. Brown, M.G., Dokun, A.O., Heusel, J.W., Smith, H.R., Beckman, D.L., Blattenberger, E.A., Dubbelde, C.E., Stone, L.R., Scalzo, A.A., and Yokoyama, W.M.: Vital involvement of a natural killer cell activation receptor in resistance to viral infection. Science 2001, 292:934–937. Long, E.O.: Negative signalling by inhibitory receptors: the NK cell paradigm. Immunol. Rev. 2008, 224:70–84.

1	Long, E.O.: Negative signalling by inhibitory receptors: the NK cell paradigm. Immunol. Rev. 2008, 224:70–84. Robbins, S.H., and Brossay, L.: NK cell receptors: emerging roles in host defense against infectious agents. Microbes Infect. 2002, 4:1523–1530. Trowsdale, J.: Genetic and functional relationships between MHC and NK receptor genes. Immunity 2001, 15:363–374. Vilches, C., and Parham, P.: KIR: diverse, rapidly evolving receptors of innate and adaptive immunity. Annu. Rev. Immunol. 2002, 20:217–251. Vivier, E., Raulet, D.H., Moretta, A., Caligiuri, M.A., Zitvogel, L., Lanier, L.L., Yokoyama, W.M., and Ugolini, S.: Innate or adaptive immunity? The example of natural killer cells. Science 2011, 331:44–49. Vivier, E., Tomasello, E., Baratin, M., Walzer, T., and Ugolini, S.: Functions of natural killer cells. Nat. Immunol. 2008, 9:503–510. 3‑27 NK cells express activating receptors that recognize ligands induced on infected cells or tumor cells.

1	3‑27 NK cells express activating receptors that recognize ligands induced on infected cells or tumor cells. Brandt, C.S., Baratin, M., Yi, E.C., Kennedy, J., Gao, Z., Fox, B., Haldeman, B., Ostrander, C.D., Kaifu, T., Chabannon, C., et al.: The B7 family member B7‑H6 is a tumor cell ligand for the activating natural killer cell receptor NKp30 in humans. J. Exp. Med. 2009, 206:1495–1503. Gasser, S., Orsulic, S., Brown, E.J., and Raulet, D.H.: The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor. Nature 2005, 436:1186–1190. Gonzalez, S., Groh, V., and Spies, T.: Immunobiology of human NKG2D and its ligands. Curr. Top. Microbiol. Immunol. 2006, 298:121–138. Lanier, L.L.: Up on the tightrope: natural killer cell activation and inhibition. Nat. Immunol. 2008, 9:495–502. Moretta, L., Bottino, C., Pende, D., Castriconi, R., Mingari, M.C., and Moretta, A.: Surface NK receptors and their ligands on tumor cells. Semin. Immunol. 2006, 18:151–158.

1	Moretta, L., Bottino, C., Pende, D., Castriconi, R., Mingari, M.C., and Moretta, A.: Surface NK receptors and their ligands on tumor cells. Semin. Immunol. 2006, 18:151–158. Parham, P.: MHC class I molecules and KIRs in human history, health and survival. Nat. Rev. Immunol. 2005, 5:201–214. Raulet, D.H., and Guerra, N.: Oncogenic stress sensed by the immune system: role of natural killer cell receptors. Nat. Rev. Immunol. 2009, 9:568–580. Upshaw, J.L., and Leibson, P.J.: NKG2D‑mediated activation of cytotoxic lymphocytes: unique signaling pathways and distinct functional outcomes. Semin. Immunol. 2006, 18:167–175. Vivier, E., Nunes, J.A., and Vely, F.: Natural killer cell signaling pathways. Science 2004, 306:1517–1519.

1	Innate immune responses are the body’s initial defense against infection, but these work only to control pathogens that have certain molecular patterns or that induce interferons and other nonspecific defenses. To effectively fight the wide range of pathogens an individual will encounter, the lymphocytes of the adaptive immune system have evolved to recognize a great variety of different antigens from bacteria, viruses, and other disease-causing organisms. An antigen is any molecule or part of a molecule that is specifically recognized by the highly specialized recognition proteins of lymphocytes. On B cells these proteins are the immunoglobulins (Igs), which these cells produce in a vast range of antigen specificities, each B cell producing immunoglobulins of a single specificity (see Section 1-12). A membrane-bound form of immunoglobulin on the B-cell surface serves as the cell’s receptor for antigen, and is known as the B-cell receptor (BCR). A secreted form of immunoglobulin of

1	Section 1-12). A membrane-bound form of immunoglobulin on the B-cell surface serves as the cell’s receptor for antigen, and is known as the B-cell receptor (BCR). A secreted form of immunoglobulin of the same antigen specificity is the antibody produced by terminally differentiated B cells—plasmablasts and plasma cells. The secretion of antibodies, which bind pathogens or their toxic products in the extracellular spaces of the body (see Fig. 1.25), is the main effector function of B cells in adaptive immunity.

1	Antibodies were the first proteins involved in specific immune recognition to be characterized, and are understood in great detail. The antibody molecule has two separate functions: one is to bind specifically to the pathogen or its products that have elicited the immune response; the other is to recruit other cells and molecules to destroy the pathogen once antibody has bound. For example, binding by antibodies can neutralize viruses and mark pathogens for destruction by phagocytes and complement, as described in Chapters 2 and 3. Recognition and effector functions are structurally separated in the antibody molecule, one part of which specifically binds to the antigen whereas the other engages the elimination mechanisms. The antigen-binding region varies extensively between antibody molecules and is known as the variable region or V region. The variability of antibody molecules allows each antibody to bind a different specific antigen, and the total repertoire of antibodies made by a

1	and is known as the variable region or V region. The variability of antibody molecules allows each antibody to bind a different specific antigen, and the total repertoire of antibodies made by a single individual is large enough to ensure that virtually any structure can be recognized. The region of the antibody molecule that engages the effector functions of the immune system does not vary in the same way and

1	The structure of a typical antibody molecule. The interaction of the antibody molecule with specific antigen. Antigen recognition by T cells. is known as the constant region or C region. It comes in five main forms, called isotypes, each of which is specialized for activating different effector mechanisms. The membrane-bound B-cell receptor does not have these effector functions, because the C region remains inserted in the membrane of the B cell. The function of the B-cell receptor is to recognize and bind antigen via the V regions exposed on the surface of the cell, thus transmitting a signal that activates the B cell, leading to clonal expansion and antibody production. To this end, the B-cell receptor is associated with a set of intracellular signaling proteins, which will be described in Chapter 7. Antibodies have become an important class of drug due to their highly specific activities, and we return to discuss their therapeutic uses in Chapter 16.

1	The antigen-recognition molecules of T cells are made solely as membrane-bound proteins, which are associated with an intracellular signaling complex and function only to signal T cells for activation. These T-cell receptors (TCRs) are related to immunoglobulins both in their protein structure—having both V and C regions—and in the genetic mechanism that produces their great variability, which is discussed in Chapter 5. The T-cell receptor differs from the B-cell receptor in an important way, however: it does not recognize and bind antigen by itself, but instead recognizes short peptide fragments of protein antigens that are presented to them by proteins known as MHC molecules on the surface of host cells.

1	The MHC molecules are transmembrane glycoproteins encoded in the large cluster of genes known as the major histocompatibility complex (MHC). The most striking structural feature of MHC molecules is a cleft in the extracellular face of the molecule in which peptides can be bound. MHC molecules are highly polymorphic—each type of MHC molecule occurs in many different versions—within the population. These are encoded by slightly different versions of individual genes called alleles. Most people are therefore heterozygous for the MHC molecules: that is, they express two different alleles for each type of MHC molecule, thus increasing the range of pathogen-derived peptides and self-peptides that can be bound. T-cell receptors recognize features of both the peptide antigen and the MHC molecule to which it is bound. This introduces an extra dimension to antigen recognition by T cells, known as MHC restriction because any given T-cell receptor is specific for a particular peptide bound to a

1	to which it is bound. This introduces an extra dimension to antigen recognition by T cells, known as MHC restriction because any given T-cell receptor is specific for a particular peptide bound to a particular MHC molecule.

1	In this chapter we focus on the structure and antigen-binding properties of immunoglobulins and T-cell receptors. Although B cells and T cells recognize foreign molecules in separate distinct fashions, the receptor molecules they use for this task are very similar in structure. We will see how this basic structure can accommodate great variability in antigen specificity, and how it enables immunoglobulins and T-cell receptors to perform their functions as the anti-gen-recognition molecules of the adaptive immune response. With this foundation, we will return to discuss the impact of MHC polymorphism on T-cell antigen recognition and T-cell development in Chapters 6 and 8, respectively. The structure of a typical antibody molecule.

1	The structure of a typical antibody molecule. Antibodies are the secreted form of the B-cell receptor. Because they are soluble and secreted into the blood in large quantities, antibodies are easily obtained and easily studied. For this reason, most of what we know about the B-cell receptor comes from the study of antibodies. Antibody molecules are roughly Y-shaped, as represented in Fig. 4.1 using three different schematic styles. This part of the chapter will explain how this structure is formed and allows the antibody molecule to perform its dual tasks of binding to a wide variety of antigens while also binding to effector molecules

1	Fig. 4.1 Structure of an antibody molecule. in panel a, the X-ray crystallographic structure of an igg antibody is illustrated as a ribbon diagram of the backbones of the polypeptide chains. The two heavy chains are shown in yellow and purple. The two light chains are both shown in red. Three globular regions form an approximate Y shape. The two antigen-binding sites are at the tips of the arms, which are tethered at their other end to the trunk of the Y by a flexible hinge region. The light-chain variable (VL) and constant region (cL) are indicated. The heavy-chain variable region (Vh) and VL together form the antigen-binding site of the antibody. in panel b, a schematic representation of the same structure denotes each immunoglobulin domain as a separate rectangle. The hinge that tethers each heavy chain’s first constant domain (ch1) to its second (ch2) is illustrated by a thin purple or yellow line, respectively. The antibody-binding sites are indicated by concave regions in VL and

1	each heavy chain’s first constant domain (ch1) to its second (ch2) is illustrated by a thin purple or yellow line, respectively. The antibody-binding sites are indicated by concave regions in VL and Vh. Positions of carbohydrate modifications and disulfide linkages are indicated. in panel c, a more simplified schematic is shown that will be used throughout this book with the variable region in red and the constant region in blue. c terminus, carboxy terminus; n terminus, amino terminus. Structure courtesy of R.L. Stanfield and i.A. Wilson.

1	and to cells that destroy the antigen. Each of these tasks is performed by different parts of the molecule. The ends of the two arms of the Y—the V regions—are involved in antigen binding, and they vary in their detailed structure between different antibody molecules. The stem of the Y—the C region—is far less variable and is the part that interacts with effector molecules and cells. There are five different classes of immunoglobulins, distinguished in their being constructed from C regions that have different structures and properties. These are known as immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE).

1	All antibodies are constructed in the same way from paired heavy and light polypeptide chains, and the generic term immunoglobulin is used for all such proteins. More subtle differences confined to the V region account for the specificity of antigen binding. We will use the IgG antibody molecule as an example to describe the general structural features of immunoglobulins. 4-1 IgG antibodies consist of four polypeptide chains.

1	4-1 IgG antibodies consist of four polypeptide chains. IgG antibodies are large molecules with a molecular weight of approximately 150 kDa and are composed of two different kinds of polypeptide chains. One, of approximately 50 kDa, is called the heavy or H chain, and the other, of 25 kDa, is the light or L chain (Fig. 4.2). Each IgG molecule consists of two heavy chains and two light chains. The two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. In any given immunoglobulin molecule, the two heavy chains and the two light chains are identical, giving an antibody molecule two identical antigen-binding sites. This gives the antibody the ability to bind simultaneously to two identical antigens on a surface, thereby increasing the total strength of the interaction, which is called its avidity. The strength of the interaction between a single antigen-binding site and its antigen is called its affinity.

1	Two types of light chains, lambda (λ) and kappa (κ), are found in antibodies. A given immunoglobulin has either κ chains or λ chains, never one of each. No functional difference has been found between antibodies having λ or κ light chains, and either type of light chain can be found in antibodies of any of the five major classes. The ratio of the two types of light chains varies from species light to species. In mice, the average κ to λ ratio is 20:1, whereas in humans it is 2:1 and in cattle it is 1:20. The reason for this variation is unknown. Distortions of this ratio can sometimes be used to detect the abnormal proliferation of a Fig. 4.2 Immunoglobulin molecules are composed of two types of protein chains: heavy chains and light chains. each immunoglobulin molecule is made up of two hinged heavy chains (green) and two light chains (yellow) joined by disulfide bonds so that each heavy chain is linked to a light chain and the two heavy chains are linked together.

1	B-cell clone, since all progeny of a particular B cell will express an identical light chain. For example, an abnormally high level of λ light chains in a person might indicate the presence of a B-cell tumor that is producing λ chains.

1	The class, and thus the effector function, of an antibody is defined by the structure of its heavy chain. There are five main heavy-chain classes, or isotypes, some of which have several subtypes, and these determine the functional activity of an antibody molecule. The five major classes of immunoglobulin are IgM, IgD, IgG, IgA, and IgE, and their heavy chains are denoted by the lowercase Greek letters μ, δ, γ, α, and ε, respectively. For example, the constant region of IgM is denoted by Cμ. IgG is by far the most abundant immunoglobulin in serum and has several subclasses (IgG1, 2, 3, and 4 in humans). The distinctive functional properties of the different classes and subclasses of antibodies are conferred by the carboxy-terminal part of the heavy chain, where this chain is not associated with the light chain. The general structural features of all the isotypes are similar, particularly with respect to antigen binding. Here we will consider IgG as a typical antibody molecule, and we

1	with the light chain. The general structural features of all the isotypes are similar, particularly with respect to antigen binding. Here we will consider IgG as a typical antibody molecule, and we will return to discuss the structural and functional properties of the different heavy-chain isotypes in Chapter 5.

1	The structure of a B-cell receptor is identical to that of its corresponding antibody except for a small portion of the carboxy terminus of the heavy-chain C region. In the B-cell receptor, the carboxy terminus is a hydrophobic amino acid sequence that anchors the molecule in the membrane, and in the antibody it is a hydrophilic sequence that allows secretion. 4-2 Immunoglobulin heavy and light chains are composed of constant and variable regions.

1	4-2 Immunoglobulin heavy and light chains are composed of constant and variable regions. The amino acid sequences of many immunoglobulin heavy and light chains have been determined and reveal two important features of antibody molecules. First, each chain consists of a series of similar, although not identical, sequences, each about 110 amino acids long. Each of these repeats corresponds to a discrete, compactly folded region of protein known as an immunoglobulin domain, or Ig domain. The light chain consists of two Ig domains, whereas the heavy chain of the IgG antibody contains four Ig domains (see Fig. 4.2). This suggests that the immunoglobulin chains have evolved by repeated duplication of ancestral gene segments corresponding to a single Ig domain.

1	The second important feature is that the amino-terminal amino acid sequences of the heavy and light chains vary greatly between different antibodies. The variability is limited to approximately the first 110 amino acids, corresponding to the first Ig domain, whereas the remaining domains are constant between immunoglobulin chains of the same isotype. The amino-terminal variable Ig domains (V domains) of the heavy and light chains (VH and VL, respectively) together make up the V region of the antibody and determine its antigen-binding specificity, whereas the constant Ig domains (C domains) of the heavy and light chains (CH and CL, respectively) make up the C region (see Fig. 4.1). The multiple heavy-chain C domains are numbered from the amino-terminal end to the carboxy terminus, for example, CH1, CH2, and so on. 4-3 The domains of an immunoglobulin molecule have similar structures.

1	4-3 The domains of an immunoglobulin molecule have similar structures. Immunoglobulin heavy and light chains are composed of a series of Ig domains that have a similar overall structure. Within this basic structure, there are distinct differences between V and C domains that are illustrated for the light chain in Fig. 4.3. Each V or C domain is constructed from two β sheets. A β sheet is

1	Fig. 4.3 The structure of immunoglobulin constant and variable domains. The upper panels show schematically the folding pattern of the constant (c) and variable (V) domains of an immunoglobulin light chain. each domain is a barrel-shaped structure in which strands of polypeptide chain (β strands) running in opposite directions (antiparallel) pack together to form two β sheets (shown in yellow and green for the c domain and red and blue for the V domain), which are held together by a disulfide bond. The way in which the polypeptide chain folds to give the final structure can be seen more clearly when the sheets are opened out, as shown in the lower panels. The β strands are lettered sequentially with respect to the order of their occurrence in the amino acid sequence of the domains; the order in each β sheet is characteristic of immunoglobulin domains. The β strands cʹ and cʹʹ that are found in the V domains but not in the c domains are indicated by a blue-shaded background. The

1	the order in each β sheet is characteristic of immunoglobulin domains. The β strands cʹ and cʹʹ that are found in the V domains but not in the c domains are indicated by a blue-shaded background. The characteristic four-strand plus three-strand (c-region type domain) or four-strand plus five-strand (V-region type domain) arrangements are typical immunoglobulin superfamily domain building blocks, found in a whole range of other proteins as well as antibodies and T-cell receptors.

1	built from several β strands, which are regions of protein where several consecutive polypeptides have their peptide backbone bonds arranged in an extended, or flat, conformation. β strands in proteins are sometimes shown as ‘ribbons with an arrow’ to indicate the direction of the polypeptide backbone (see Fig. 4.3). β strands can pack together in a side-by-side manner, being stabilized laterally by two or three backbone hydrogen bonds between adjacent strands. This arrangement is called a β sheet. The Ig domain has two β sheets that are folded onto each other, like two pieces of bread, into a structure called a β sandwich, and are covalently linked by a disulfide bond between cysteine residues from each β sheet. This distinctive structure is known as the immunoglobulin fold.

1	The similarities and differences between V and C domains can be seen in the bottom panels of Fig. 4.3. Here, the Ig domains have been opened out to show how their respective polypeptide chains fold to create each of the βsheets and how each polypeptide chain forms flexible loops between adjacent β strands as it turns to change direction. The main difference between the V and C domains is that the V domain is larger and contains extra β strands, called Cʹ and Cʹʹ. In the V domain, the flexible loops formed between some of the β strands contribute to the antigen-binding site of the immunoglobulin molecule.

1	Many of the amino acids that are common to the C and V domains are present in the core of the immunoglobulin fold and are essential for its stability. Other proteins with sequences similar to those of immunoglobulins have been found to have domains with a similar structure, called immunoglobulin-like domains (Ig-like domains). These domains are present in many proteins of the immune system, such as the KIRs expressed by NK cells described in Chapter 3. They are also frequently involved in cell–cell recognition and adhesion, and together with the immunoglobulins and the T-cell receptors, these proteins make up the extensive immunoglobulin superfamily. 4-4 The antibody molecule can readily be cleaved into functionally distinct fragments.

1	4-4 The antibody molecule can readily be cleaved into functionally distinct fragments. When fully assembled, an antibody molecule comprises three equal-sized globular portions, with its two arms joined to its trunk by a flexible stretch of polypeptide chain known as the hinge region (see Fig. 4.1b). Each arm of this Y-shaped structure is formed by the association of a light chain with the amino-terminal half of a heavy chain; the VH domain is paired with the VL domain, and the CH1 domain is paired with the CL domain. The two antigen-binding sites are formed by the paired VH and VL domains at the ends of the two arms of the Y (see Fig. 4.1b). The trunk of the Y is formed by the pairing of the carboxyterminal halves of the two heavy chains. The CH3 domains pair with each other but the CH2 domains do not interact. Carbohydrate side chains attached to the CH2 domains lie between the two heavy chains.

1	Proteolytic enzymes (proteases) were an important tool in early studies of antibody structure, and it is valuable to review the terminology they generated. Limited digestion with the protease papain cleaves antibody molecules into three fragments (Fig. 4.4). Papain cuts the antibody molecule on the amino-terminal side of the disulfide bonds that link the two heavy chains, releasing the two arms of the antibody molecule as two identical fragments that contain the antigen-binding activity. These are called the Fab fragments, for fragment antigen binding. The other fragment contains no antigen-binding activity, but because it crystallized readily, it was named the Fc fragment (fragment crystallizable). It corresponds to the paired CH2 and CH3 domains. The Fc fragment is the part of the antibody molecule that does not interact with antigen, but rather interacts with effector molecules and cells, and it differs between heavy-chain isotypes. Another protease, pepsin, cuts on the

1	the antibody molecule that does not interact with antigen, but rather interacts with effector molecules and cells, and it differs between heavy-chain isotypes. Another protease, pepsin, cuts on the carboxy-terminal side of the disulfide bonds (see Fig. 4.4). This produces a fragment, the F(abʹ)2 fragment, in which the two antigen-binding arms of the antibody molecule remain linked. Pepsin cuts the remaining part of the heavy chain into several small fragments. The F(abʹ)2 fragment has exactly the same antigen-binding characteristics as the original antibody but is unable to interact with any effector molecule, such as C1q or Fc receptors, and can be used experimentally to separate the antigen-binding functions from the antibody’s other effector functions.

1	Many antibody-related molecules can be constructed using genetic engineering techniques, and many antibodies and antibody-related molecules are being used therapeutically to treat a variety of diseases. We will return to this topic in Chapter 16, where we discuss the various therapeutic uses of antibodies that have been developed over the last two decades. Fig. 4.4 The Y-shaped immunoglobulin molecule can be dissected by partial digestion with proteases. Upper panels: papain cleaves the immunoglobulin molecule into three pieces, two fab fragments and one fc fragment. The fab fragment contains the V regions and binds antigen. The fc fragment is crystallizable and contains c regions. Lower panels: pepsin cleaves immunoglobulin to yield one f(abʹ)2 fragment and many small pieces of the fc fragment, the largest of which is called the pfcʹ fragment. f(abʹ)2 is written with a prime because it contains a few more amino acids than fab, including the cysteines that form the disulfide bonds.

1	Angle between arms is 90oAngle between arms is 60o(Micrograph ×300,000) 4-5 The hinge region of the immunoglobulin molecule allows flexibility in binding to multiple antigens.

1	The hinge region between the Fc and Fab portions of the IgG molecule allows for some degree of independent movement of the two Fab arms. For example, in the antibody molecule shown in Fig. 4.1a, not only are the two hinge regions clearly bent differently, but the angle between the V and C domains in each of the two Fab arms is also different. This range of motion has led to the junction between the V and C domains being referred to as a ‘molecular ball-and-socket joint.’ This flexibility can be revealed by studies of antibodies bound to small antigens known as haptens. These are molecules of various types that are typically about the size of a tyrosine side chain. Although hap-tens are specifically recognized by antibody, they can stimulate the production of anti-hapten antibodies only when linked to a protein (see Appendix I, Section A-1). Two identical hapten molecules joined by a short flexible region can link two or more anti-hapten antibodies, forming dimers, trimers, tetramers,

1	linked to a protein (see Appendix I, Section A-1). Two identical hapten molecules joined by a short flexible region can link two or more anti-hapten antibodies, forming dimers, trimers, tetramers, and so on, which can be seen by electron microscopy (Fig. 4.5). The shapes formed by these complexes show that antibody molecules are flexible at the hinge region. Some flexibility is also found at the junction between the V and C domains, allowing bending and rotation of the V domain relative to the C domain. Flexibility at both the hinge and the V–C junction enables the two arms of an antibody molecule to bind to sites some distance apart, such as the repeating sites on bacterial cell-wall polysaccharides. Flexibility at the hinge also enables antibodies to interact with the antibody-binding proteins that mediate immune effector mechanisms.

1	Summary.

1	The IgG antibody molecule is made up of four polypeptide chains, comprising two identical light chains and two identical heavy chains, and can be thought of as forming a flexible Y-shaped structure. Each of the four chains has a variable (V) region at its amino terminus, which contributes to the antigen-binding site, and a constant (C) region. The light chains are bound to the heavy chains by many noncovalent interactions and by disulfide bonds, and the V regions of the heavy and light chains pair in each arm of the Y to generate two identical antigen-binding sites, which lie at the tips of the arms of the Y. The possession of two antigen-binding sites allows antibody molecules to cross-link antigens and to bind them much more stably and with higher avidity. The trunk of the Y, also called the Fc fragment, is composed of the carboxy-terminal domains of the heavy chains, and it is these domains that determine the antibody’s isotype. Joining the arms of the Y to the trunk are the

1	called the Fc fragment, is composed of the carboxy-terminal domains of the heavy chains, and it is these domains that determine the antibody’s isotype. Joining the arms of the Y to the trunk are the flexible hinge regions. The Fc fragment and hinge regions differ in antibodies of different isotypes.

1	Fig. 4.5 Antibody arms are joined by a flexible hinge. An antigen consisting of two hapten molecules (red balls in diagrams) that can cross-link two antigen-binding sites is used to create antigen:antibody complexes, which can be seen in the electron micrograph. Linear, triangular, and square forms are seen, with short projections or spikes. Limited pepsin digestion removes these spikes (not shown in the figure), which therefore correspond to the fc portion of the antibody; the f(abʹ)2 pieces remain cross-linked by antigen. The interpretation of some of the complexes is shown in the diagrams. The angle between the arms of the antibody molecules varies. in the triangular forms, this angle is 60°, whereas it is 90° in the square forms, showing that the connections between the arms are flexible. Photograph courtesy of n.M. green.

1	Different isotypes have different properties and therefore differ in their interactions with effector molecules and cell types. However, the overall organization of the domains is similar in all isotypes. The interaction of the antibody molecule with specific antigen. In this part of the chapter we look at the antigen-binding site of an immunoglobulin molecule in more detail. We discuss the different ways in which antigens can bind to antibody, and address the question of how variation in the sequences of the antibody V domains determines the specificity for antigen. 4-6 Localized regions of hypervariable sequence form the antigen-binding site.

1	The V regions of any given antibody molecule differ from those of every other. Sequence variability is not, however, distributed evenly throughout the V region but is concentrated in certain segments, as is clearly seen in a variability plot (Fig. 4.6), in which the amino acid sequences of many different antibody V regions are compared. Three particularly variable segments can be identified in both the VH and VL domains. They are designated hypervariable regions and are denoted HV1, HV2, and HV3. In the heavy chains they are located at residues 30 to 36, 49 to 65, and 95 to 103, respectively, while in the light chains they are located at residues 28 to 35, 49 to 59, and 92 to 103, respectively. The most variable part of the domain is in the HV3 region. The regions between the hypervariable regions comprise the rest of the V domain; they show less variability and are termed the framework regions. There are four such regions in each V domain, designated FR1, FR2, FR3, and FR4.

1	The framework regions form the β sheets that provide the structural framework of the immunoglobulin domain. The hypervariable sequences correspond to three loops and are positioned near one another in the folded domain at the outer edge of the β sandwich (Fig. 4.7). Thus, not only is diversity concentrated in particular parts of the V domain sequence, but it is also localized to a particular Fig. 4.6 There are discrete regions of hypervariability in V domains.

1	Fig. 4.6 There are discrete regions of hypervariability in V domains. The hypervariability regions of both the heavy and the light chain contribute to antigen binding of an antibody molecule. A variability plot derived from comparison of the amino acid sequences of several dozen heavy-chain and light-chain V domains is shown. At each amino acid position, the degree of variability is the ratio of the number of different amino acids seen in all of the sequences together to the frequency of the most common amino acid. Three hypervariable regions (hV1, hV2, and hV3) are indicated in red. They are flanked by less variable framework regions (fR1, fR2, fR3, and fR4, shown in blue or yellow). Fig. 4.7 The hypervariable regions lie in discrete loops of the folded structure.

1	Fig. 4.7 The hypervariable regions lie in discrete loops of the folded structure. first panel: the hypervariable regions (red) are positioned on the structure of a map of the coding region of the V domain. Second panel: when shown as a flattened ribbon diagram, hypervariable regions are seen to occur in loops (red) that join particular β strands. Third panel: in the folded structure of the V domain, these loops (red) are brought together to form antigen-binding regions. fourth panel: in a complete antibody molecule, the pairing of a heavy chain and a light chain brings together the hypervariable loops from each chain to create a single hypervariable surface, which forms the antigen-binding site at the tip of each arm. Because they are complementary to the antigen surface, the hypervariable regions are also commonly known as the complementarity-determining regions (cDRs). c, carboxy terminus; n, amino terminus.

1	region on the surface of the molecule. When the VH and VL immunoglobulin domains are paired in the antibody molecule, the three hypervariable loops from each domain are brought together, creating a single hypervariable site at the tip of each arm of the molecule. This is the antigen-binding site, or antibody-combining site, which determines the antigen specificity of the antibody. These six hypervariable loops are more commonly termed the complementarity-determining regions, or CDRs, because the surface they form is complementary to that of the antigen they bind. There are three CDRs from each of the heavy and light chains, namely, CDR1, CDR2, and CDR3. In most cases, CDRs from both VH and VL domains contribute to the antigen-binding site; thus it is the combination of the heavy and the light chain that usually determines the final antigen specificity (see Fig. 4.6). However, there are some Fab crystal structures that show antigen interaction with just the heavy chain; for example, in

1	chain that usually determines the final antigen specificity (see Fig. 4.6). However, there are some Fab crystal structures that show antigen interaction with just the heavy chain; for example, in one influenza Fab, antigen interaction involves binding mostly to the VH CDR3, and only minor contacts with other CDRs. Thus, one way in which the immune system is able to generate antibodies of different specificities is by generating different combinations of heavy-chain and light-chain V regions. This is known as combinatorial diversity; we will encounter a second form of combinatorial diversity in Chapter 5 when we consider how the genes encoding the heavy-chain and light-chain V regions are created from smaller segments of DNA during the development of B cells in the bone marrow.

1	4-7 Antibodies bind antigens via contacts in CDRs that are complementary to the size and shape of the antigen.

1	In early investigations of antigen binding to antibodies, the only available sources of large quantities of a single type of antibody molecule were tumors of antibody-secreting cells. The antigen specificities of these antibodies were unknown, and therefore many compounds had to be screened to identify ligands that could be used to study antigen binding. In general, the substances found to bind to these antibodies were haptens (see Section 4-5) such as phosphocholine or vitamin K1. Structural analysis of complexes of antibodies with their hapten ligands provided the first direct evidence that the hypervariable regions form the antigen-binding site, and demonstrated the structural basis of specificity for the hapten. Subsequently, with the discovery of methods of generating monoclonal antibodies (see Appendix I, Section A-7), it became possible to make large amounts of pure antibody specific for a given antigen. This has provided a more general picture of how antibodies interact with

1	(see Appendix I, Section A-7), it became possible to make large amounts of pure antibody specific for a given antigen. This has provided a more general picture of how antibodies interact with their antigens, confirming and extending the view of antibody–antigen interactions derived from the study of haptens.

1	The surface of the antibody molecule formed by the juxtaposition of the CDRs of the heavy and light chains is the site to which an antigen binds. The amino acid sequences of the CDRs are different in different antibodies, and so too are the shapes and properties of the surfaces created by these CDRs. As a general principle, antibodies bind ligands whose surfaces are complementary to that of the antigen-binding site. A small antigen, such as a hapten or a short

1	The panels in the top row show schematic representations of the different types of binding sites in a fab fragment of an antibody: first panel, pocket; second panel, groove; third panel, extended surface; and fourth panel, protruding surface. Below are examples of each type. Panel a: the top image shows the molecular surface of the interaction of a small hapten with the complementarity-determining regions (cDRs) of a fab fragment as viewed looking into the antigen-binding site. The ferrocene hapten, shown in red, is bound into the antigen-binding pocket (yellow). in the bottom image (and in those of panels b, c, and d), the molecule has been rotated by about 90° to give a side-on view of the binding site. Panel b: in a complex of an antibody with a peptide from the human immunodeficiency virus (hiV), the peptide (red) binds along a groove (yellow) formed between the heavy-chain and light-chain V domains. Panel c: shown is a complex between hen egg-white lysozyme and the fab fragment

1	virus (hiV), the peptide (red) binds along a groove (yellow) formed between the heavy-chain and light-chain V domains. Panel c: shown is a complex between hen egg-white lysozyme and the fab fragment of its corresponding antibody (hyhel5). The surface on the antibody that comes into contact with the lysozyme is colored yellow. All six cDRs of the antigen-binding site are involved in the binding. Panel d: an antibody molecule against the hiV gp120 antigen has an elongated cDR3 loop (arrow) that protrudes into a recess on the side of the antigen. The structure of the complex between this antibody and gp120 has been solved, and in this case only the heavy chain interacts with gp120. Structures courtesy of R.L. Stanfield and i.A. Wilson.

1	Fig. 4.8 Antigens can bind in pockets, or grooves, or on extended surfaces in the binding sites of antibodies. peptide, generally binds in a pocket or groove lying between the heavy-chain and light-chain V domains (Fig. 4.8a and b). Some antigens, such as proteins, can be the same size as, or larger than, the antibody itself. In these cases, the interface between antigen and antibody is often an extended surface that involves all the CDRs and, in some cases, part of the framework region as well (see Fig. 4.8c). This surface need not be concave but can be flat, undulating, or even convex. In some cases, antibody molecules with elongated CDR3 loops can protrude a ‘finger’ into recesses in the surface of the antigen, as shown in Fig. 4.8d, where an antibody binding to the HIV gp120 antigen projects a long loop into its target. 4-8 Antibodies bind to conformational shapes on the surfaces of antigens using a variety of noncovalent forces.

1	The biological function of antibodies is to bind to pathogens and their products, and to facilitate their removal from the body. An antibody generally recognizes only a small region on the surface of a large molecule such as a polysaccharide or protein. The structure recognized by an antibody is called an antigenic determinant or epitope. Some of the most important pathogens have polysaccharide coats, and antibodies that recognize epitopes formed by the sugar subunits of these molecules are essential in providing immune protection against such pathogens. In many cases, however, the antigens that provoke an immune response are proteins. For example, many protective antibodies against viruses recognize viral coat proteins. In all such cases, the structures recognized by the antibody are located on the surface of the protein. Such sites are likely to be composed of amino acids from different parts of the polypeptide chain that have been brought together by protein folding. Antigenic

1	located on the surface of the protein. Such sites are likely to be composed of amino acids from different parts of the polypeptide chain that have been brought together by protein folding. Antigenic determinants of this kind are known as conformational or discontinuous epitopes because the structure recognized is composed of segments of the protein that are discontinuous in the amino acid sequence of the antigen but are brought together in the three-dimensional structure. In contrast, an epitope composed of a single segment of polypeptide chain is termed a continuous or linear epitope. Although most antibodies raised against intact, fully folded proteins recognize discontinuous epitopes, some will bind to peptide fragments of the protein. Conversely, antibodies raised against peptides of a protein or against synthetic peptides corresponding to part of its sequence are occasionally found to bind to the natural folded protein. This makes it possible, in some cases, to use synthetic

1	of a protein or against synthetic peptides corresponding to part of its sequence are occasionally found to bind to the natural folded protein. This makes it possible, in some cases, to use synthetic peptides in vaccines that aim to raise antibodies against a pathogen’s protein.

1	The interaction between an antibody and its antigen can be disrupted by high salt concentrations, by extremes of pH, by detergents, and sometimes by competition with high concentrations of the pure epitope itself. The binding is therefore a reversible noncovalent interaction. The forces, or bonds, involved in these noncovalent interactions are outlined in Fig. 4.9. Electrostatic interactions occur between charged amino acid side chains, as in salt bridges. Most antibody–antigen interactions involve at least one electrostatic interaction. Interactions also occur between electric dipoles, as in hydrogen bonds, or can involve short-range van der Waals forces. High salt concentrations and extremes of pH disrupt antigen–antibody binding by weakening electrostatic interactions and/or hydrogen bonds. This principle is employed in the purification of antigens by using affinity columns of immobilized antibodies (or in the purification of antibody by using antigens in a like manner)

1	hydrogen bonds. This principle is employed in the purification of antigens by using affinity columns of immobilized antibodies (or in the purification of antibody by using antigens in a like manner) (see Appendix I, Section A-3). Hydrophobic interactions occur when two hydrophobic surfaces come together to exclude water. The strength of a hydrophobic interaction is proportional to the surface area that is hidden from water, and for some antigens, hydrophobic interactions probably account for most of the binding energy. In some cases, water molecules are trapped in pockets in the interface between antigen and antibody. These trapped water molecules, especially those between polar amino acid residues, may also contribute to binding and hence to the specificity of the antibody.

1	The contribution of each of these forces to the overall interaction depends on the particular antibody and antigen involved. A striking difference between antibody interactions with protein antigens and most other natural protein– protein interactions is that antibodies often have many aromatic amino acids HHHHHH HH NH OOC3 N H O C ˜–˜–˜+˜–˜–˜+˜+˜–˜˜–˜–˜–˜–˜–˜––˜+˜+H H O H HO HH O Na+H H O Noncovalent forces Origin Electrostatic forces Hydrogen bonds Attraction between opposite charges Hydrogen shared between electronegative atoms (N, O) Van der Waals forces Hydrophobic forces Cation-pi interaction Fluctuations in electron clouds around molecules polarize neighboring atoms oppositely Hydrophobic groups interact unfavorably with water and tend to pack together to exclude water molecules. The attraction also involves van der Waals forces Non-covalent interaction between a cation and an electron cloud of a nearby aromatic group H H

1	Fig. 4.9 The noncovalent forces that hold together the antigen:antibody complex. Partial charges found in electric dipoles are shown as δ+ or δ–. electrostatic forces diminish as the inverse square of the distance separating the charges, whereas van der Waals forces, which are more numerous in most antigen–antibody contacts, fall off as the sixth power of the separation and therefore operate only over very short ranges. covalent bonds never occur between antigens and naturally produced antibodies.

1	VL VH D1.3 Gln121 in their antigen-binding sites. These amino acids participate mainly in van der Waals and hydrophobic interactions, and sometimes in hydrogen bonds and pi-cation interactions. Tyrosine, for example, can take part in both hydrogen bonding and hydrophobic interactions; it is therefore particularly suitable for providing diversity in antigen recognition and is overrepresented in antigen-binding sites. In general, the hydrophobic and van der Waals forces operate over very short ranges and serve to pull together two surfaces that are complementary in shape: hills on one surface must fit into valleys on the other for good binding to occur. In contrast, electrostatic interactions between charged side chains, and hydrogen bonds bridging oxygen and/or nitrogen atoms, accommodate more specific chemical interactions while strengthening the interaction overall. The side chains of aromatic amino acids such as tyrosine can interact noncovalently through their pi-electron system

1	more specific chemical interactions while strengthening the interaction overall. The side chains of aromatic amino acids such as tyrosine can interact noncovalently through their pi-electron system with nearby cations, including nitrogen-containing side chains that may be in a protonated cationic state.

1	4-9 Antibody interaction with intact antigens is influenced by steric constraints.

1	An example of an antibody–antigen interaction involving a specific amino acid in the antigen can be seen in the complex of hen egg-white lysozyme with the antibody D1.3 (Fig. 4.10). In this structure, strong hydrogen bonds are formed between the antibody and a particular glutamine in the lysozyme molecule that protrudes between the VH and VL domains. Lysozymes from partridge and turkey have another amino acid in place of the glutamine and do not bind to this antibody. In the high-affinity complex of hen egg-white lysozyme with another antibody, HyHel5 (see Fig. 4.8c), two salt bridges between two basic arginines on the surface of the lysozyme interact with two glutamic acids, one each from the VH CDR1 and CDR2 loops. Lysozymes that lack one of the two arginine residues show a 1000-fold decrease in affinity for HyHel5. Overall surface complementarity must have an important role in antigen–antibody interactions, but in most antibodies that have been studied at this level of detail, only

1	in affinity for HyHel5. Overall surface complementarity must have an important role in antigen–antibody interactions, but in most antibodies that have been studied at this level of detail, only a few residues make a major contribution to the binding energy and hence to the final specificity of the antibody. Although many antibodies naturally bind their ligands with high affinity, in the nanomolar range, genetic engineering by site-directed mutagenesis can tailor an antibody to bind even more strongly to its epitope.

1	Even when antibodies have high affinity for antigens on a larger structure, such as an intact viral particle, antibody binding may be prevented by their particular arrangement. For example, the intact West Nile virion is built from an icosahedral scaffold that has 90 homodimers of a membrane-anchored envelope glycoprotein, E, which has three domains, DI, DII, and DIII. The DIII domain has four polypeptide loops that protrude outward from the viral particle. A neutralizing antibody against West Nile virus, E16, recognizes these loops of DIII, as shown in Fig. 4.11. In theory, there should be 180 possible antigen-binding sites for the E16 antibody on the West Nile viral particle. However, a combination of crystallographic and electron micrographic studies show that even with an excess of the E16 Fab fragment, only about 120 of the total 180 DIII domains of E are able to bind E16 Fab fragment (see Fig. 4.11).

1	Fig. 4.10 The complex of lysozyme with the antibody D1.3. Top panel: The interaction of the fab fragment of D1.3 with hen egg-white lysozyme (heL) is shown. heL is depicted in yellow, the heavy chain (Vh) in turquoise, and the light chain (VL) in green. Bottom panel: A glutamine residue (gln121) that protrudes from heL (yellow) extends its side chain (shown in red) between the VL (green) and Vh (turquoise) domains of the antigen-binding site and makes hydrogen bonds with the hydroxyl group (red dots) of the indicated amino acids of both domains. These hydrogen bonds are important to the antigen–antibody binding. courtesy of R. Mariuzza and R.J. Poljak.

1	Fig. 4.11 Steric hindrance occludes the binding of antibody to native antigen in the intact West Nile viral particle. Top panel: the monoclonal antibody e16 recognizes Diii, one of the three structural domains in the West nile virus glycoprotein e. Shown is a crystal structure of the e16 fab bound to the Diii epitope. Bottom left panel: a computer model was used to dock e16 fab to the mature West nile virion. e16 fabs were able to bind 120 of the 180 Diii epitopes. Sixty of the five-fold clustered Diii epitopes are sterically hindered by the binding of fab to four nearby Diii epitopes. An example of an occluded epitope is the blue area indicated by the arrow. Bottom right panel: cryogenic electron microscopic reconstruction of saturating e16 fab bound to West nile virion confirmed the predicted steric hindrance. The vertices of the triangle shown in the figure indicate the icosahedral symmetry axes.

1	This appears to result from steric hindrance, with the presence of one Fab blocking the ability of another Fab to bind to some nearby E protein sites. Presumably, such steric hindrance would become more severe with intact antibody than is evident with the smaller Fab fragment. This study also showed that the Fab bound to the DIII region using only one of its antigen-binding arms, indicating that antibodies may not always bind to antigens with both antigen-binding sites, depending on the orientation of the antigens being recognized. These constraints will impact the ability of antibodies to neutralize their targets. 4-10 Some species generate antibodies with alternative structures.

1	Our focus in this chapter has been on the structure of antibodies produced by humans, which is generally similar in most mammalian species, including mice, an important model system for immunology research. However, some mammals have the ability to produce an alternative form of antibody that is based on the ability of a single VH domain to interact with antigen in the absence of a VL domain (Fig. 4.12). It has been known for some time that the serum of camels contained abundant immunoglobulin-like material composed of heavy-chain dimers that lack associated light chains but retain the capacity to bind antigens. These antibodies are called heavy-chain-only IgGs (hcIgGs). This property is shared by other camelids, including llamas and alpacas. These species have retained the genes for the immunoglobulin light chains, and some IgG-like material in their sera remains associated with light chains, and so it is unclear what led to this particular adaptation during their evolution. In

1	the immunoglobulin light chains, and some IgG-like material in their sera remains associated with light chains, and so it is unclear what led to this particular adaptation during their evolution. In camelids, the ability to produce hcIgGs arises from mutations that allow the alternative splicing of the heavy-chain mRNA, with loss of the CH1 exon and thus the joining of the VH directly to the CH2 domain in the protein. Other mutations stabilize this structure in the absence of VL domains.

1	Cartilaginous fish, in particular the shark, also have an antibody molecule that differs substantially from human or murine antibodies (see Fig. 4.12). Like camelids, the shark also has genes encoding both immunoglobulin heavy and light chains, and does produce immunoglobulins containing both Fig. 4.12 Camelid and shark antibody can consist of heavy chain only. in the camelid heavy-chain-only antibody, a splicing event of the mature heavy chain can delete the exon encoding the ch1 region and thereby create an in-frame hinge region linking the Vh1 to the ch2 region. in the shark, the heavy-chainonly ig molecule retains the ch1 region, suggesting that this form of antibody may predate the evolution of light chains. for both, the repertoire of antigen-binding sites involves extensive variations in long cDR3 regions of the Vh domain relative to other types of antibody.

1	heavy and light chains. But sharks also produce an immunoglobulin new antigen receptor (IgNAR), with heavy-chain-only antibody in which the VH is spliced to the CH1 exon, rather than the CH1 exon being spliced out as in camelids. These differences suggest that hcIgG production by camelids and sharks represents an event of convergent evolution. The ability of camelid VH domains to interact efficiently with antigens is the basis for producing so-called single-chain antibody. The simplification of using only a single domain for antigen recognition has prompted recent interest in single-chain monoclonal antibodies as an alternative to standard monoclonal antibodies, which we will discuss more in Chapter 16. Summary.

1	X-ray crystallographic analyses of antigen:antibody complexes have shown that the hypervariable loops (complementarity-determining regions, CDRs) of immunoglobulin V regions determine the binding specificity of an antibody. Contact between an antibody molecule and a protein antigen usually occurs over a broad area of the antibody surface that is complementary to the surface recognized on the antigen. Electrostatic interactions, hydrogen bonds, van der Waals forces, and hydrophobic and pi-cation interactions can all contribute to binding. Depending on the size of the antigen, amino acid side chains in most or all of the CDRs make contact with antigen and determine both the specificity and the affinity of the interaction. Other parts of the V region normally play little part in the direct contact with the antigen, but they provide a stable structural framework for the CDRs and help to determine their position and conformation. Antibodies raised against intact proteins usually bind to

1	contact with the antigen, but they provide a stable structural framework for the CDRs and help to determine their position and conformation. Antibodies raised against intact proteins usually bind to the surface of the protein and make contact with residues that are discontinuous in the primary structure of the molecule; they may, however, occasionally bind peptide fragments of the protein, and antibodies raised against peptides derived from a protein can sometimes be used to detect the native protein molecule. Peptides binding to antibodies usually bind in a cleft or pocket between the V regions of the heavy and light chains, where they make specific contact with some, but not necessarily all, of the CDRs. This is also the usual mode of binding for carbohydrate antigens and small molecules such as haptens.

1	Antigen recognition by T cells. In contrast to the immunoglobulins, which interact with pathogens and their toxic products in the extracellular spaces of the body, T cells recognize foreign antigens only when they are displayed on the surface of the body’s own cells. These antigens can derive from pathogens such as viruses or intracellular bacteria, which replicate within cells, or from pathogens or their products that have been internalized by endocytosis from the extracellular fluid.

1	T cells detect the presence of an intracellular pathogen because the infected cells display peptide fragments of the pathogen’s proteins on their surface. These foreign peptides are delivered to the cell surface by specialized host-cell glycoproteins—the MHC molecules. These are encoded in a large cluster of genes that were first identified by their powerful effects on the immune response to transplanted tissues. For that reason, the gene complex was called the major histocompatibility complex (MHC), and the peptide-binding glycoproteins are known as MHC molecules. The recognition of antigen as a small peptide fragment bound to an MHC molecule and displayed at the cell surface is one of the most distinctive features of T cells, and will be the focus of this part of the chapter. How the peptide fragments of antigen are generated and become associated with MHC molecules will be described in Chapter 6.

1	We describe here the structure and properties of the T-cell receptor (TCR). As might be expected from the T-cell receptors’ function as highly variable antigen-recognition structures, the genes for TCRs are closely related to those for immunoglobulins. There are, however, important differences between T-cell receptors and immunoglobulins, and these differences reflect the special features of antigen recognition by T cells. 4-11 The TCRα:β heterodimer is very similar to a Fab fragment of immunoglobulin.

1	T-cell receptors were first identified by using monoclonal antibodies that bound to a single cloned T-cell line: such antibodies either specifically inhibit antigen recognition by the clone or specifically activate it by mimicking the antigen (see Appendix I, Section A-20). These clonotypic antibodies were then used to show that each T cell bears about 30,000 identical antigen receptors on its surface, each receptor consisting of two different polypeptide chains, termed the T-cell receptor α (TCRα) and β (TCRβ) chains. Each chain of the α:β heterodimer is composed of two Ig domains, and the two chains are linked by a disulfide bond, similar to the structure of the Fab fragment of an immunoglobulin molecule (Fig. 4.13). α:β heterodimers account for antigen recognition by most T cells. A minority of T cells bear an alternative, but structurally similar, receptor made up of a different pair of polypeptide chains designated γ and δ. The γ:δ T-cell receptors seem to have different

1	A minority of T cells bear an alternative, but structurally similar, receptor made up of a different pair of polypeptide chains designated γ and δ. The γ:δ T-cell receptors seem to have different antigen-recognition properties from the α:β T-cell receptors, and the functions of γ:δ T cells in immune responses are still being clarified as the various ligands they recognize are identified (see Section 6-20). In the rest of this chapter and elsewhere in the book we use the term T-cell receptor to mean the α:β receptor, except where specified otherwise. Both types of T-cell receptors differ from the membrane-bound immunoglobulin that serves as the B-cell receptor in two main ways. A T-cell receptor has only one antigen-binding site, whereas a B-cell receptor has two, and T-cell receptors are never secreted, whereas immunoglobulins can be secreted as antibodies.

1	Further insights into the structure and function of the α:β T-cell receptor came from studies of cloned cDNA encoding the receptor chains. The amino acid sequences predicted from the cDNA showed that both chains of the T-cell receptor have an amino-terminal variable (V) region with sequence homology to an immunoglobulin V domain, a constant (C) region with homology to an immunoglobulin C domain, and a short stalk segment containing a cysteine residue that forms the interchain disulfide bond (Fig. 4.14). Each chain spans the lipid bilayer by a hydrophobic transmembrane domain, and ends in a short cytoplasmic tail. These close similarities of T-cell receptor chains to the heavy and light immunoglobulin chains first enabled prediction of the structural resemblance of the T-cell receptor heterodimer to a Fab fragment of immunoglobulin.

1	The three-dimensional structure of the T-cell receptor determined by X-ray crystallography in Fig. 4.15a shows that T-cell receptor chains fold in much the same way as the regions comprising the Fab fragment in Fig. 4.1a. Fig. 4.14 Structure of the T-cell receptor. The T-cell receptor heterodimer is composed of two transmembrane glycoprotein chains, α and β. The extracellular portion of each chain consists of two domains, resembling immunoglobulin V and c domains, respectively. Both chains have carbohydrate side chains attached to each domain. A short stalk segment, analogous to an immunoglobulin hinge region, connects the ig-like domains to the membrane and contains the cysteine residue that forms the interchain disulfide bond. The transmembrane helices of both chains are unusual in containing positively charged (basic) residues within the hydrophobic transmembrane segment. The α chain carries two such residues; the β chain has one. Antigen recognition by T cells. 153

1	Antigen recognition by T cells. 153 Fig. 4.13 The T-cell receptor resembles a membrane-bound Fab fragment. The fab fragment of an antibody molecule is a disulfide-linked heterodimer, each chain of which contains one immunoglobulin c domain and one V domain; the juxtaposition of the V domains forms the antigen-binding site (see Section 4-6). The T-cell receptor is also a disulfide-linked heterodimer, with each chain containing an immunoglobulin c-like domain and an immunoglobulin V-like domain. As in the fab fragment, the juxtaposition of the V domains forms the site for antigen recognition.

1	Fig. 4.15 The crystal structure of an α:β T-cell receptor resolved at 0.25 nm. in panels a and b, the α chain is shown in pink and the β chain in blue. Disulfide bonds are shown in green. in panel a, the T-cell receptor is viewed from the side as it would sit on a cell surface, with the cDR loops that form the antigen-binding site (labeled 1, 2, and 3) arrayed across its relatively flat top. in panel b, the cα and cβ domains are shown. The cα domain does not fold into a typical ig-like domain; the face of the domain away from the cβ domain is mainly composed of irregular strands of polypeptide rather than β sheet. The intramolecular disulfide bond (far left) joins a β strand to this segment of α helix. The interaction between the cα and cβ domains is assisted by carbohydrate (colored gray and labeled), with a sugar group from the cα domain making hydrogen bonds to the cβ domain. in panel c, the T-cell receptor is shown aligned with the antigen-binding sites from three different

1	gray and labeled), with a sugar group from the cα domain making hydrogen bonds to the cβ domain. in panel c, the T-cell receptor is shown aligned with the antigen-binding sites from three different antibodies. This view is looking down into the binding site. The Vα domain of the T-cell receptor is aligned with the VL domains of the antigen-binding sites of the antibodies, and the Vβ domain is aligned with the Vh domains. The cDRs of the T-cell receptor and immunoglobulin molecules are colored, with cDRs 1, 2, and 3 of the TcR shown in red and the hV4 loop in orange. for the immunoglobulin V domains, the cDR1 loops of the heavy chain (h1) and light chain (L1) are shown in light and dark blue, respectively, and the cDR2 loops (h2, L2) in light and dark purple, respectively. The heavy-chain cDR3 loops (h3) are in yellow; the light-chain cDR3s (L3) are in bright green. The hV4 loops of the TcR (orange) have no hypervariable counterparts in immunoglobulins. Model structures courtesy of

1	cDR3 loops (h3) are in yellow; the light-chain cDR3s (L3) are in bright green. The hV4 loops of the TcR (orange) have no hypervariable counterparts in immunoglobulins. Model structures courtesy of i.A. Wilson.

1	There are, however, some distinct structural differences between T-cell receptors and Fab fragments. The most striking is in the Cα domain, where the fold is unlike that of any other Ig-like domain. The half of the Cα domain that is juxtaposed with the Cβ domain forms a β sheet similar to that found in other Ig-like domains, but the other half of the domain is formed of loosely packed strands and a short segment of α helix (see Fig. 4.15b). In a Cα domain the intramolecular disulfide bond, which in Ig-like domains normally joins two β strands, joins a β strand to this segment of α helix.

1	There are also differences in the way in which the domains interact. The interface between the V and C domains of both T-cell receptor chains is more extensive than in most antibodies. The interaction between the Cα and Cβ domains is distinctive, as it might be assisted by carbohydrates, with a sugar group from the Cα domain making a number of hydrogen bonds to the Cβ domain (see Fig. 4.15b). Finally, a comparison of the variable binding sites shows that, although the CDR loops align fairly closely with those of antibody molecules, there is some relative displacement (see Fig. 4.15c). This is particularly marked in the Vα CDR2 loop, which is oriented at roughly right angles to the equivalent loop in antibody V domains, as a result of a shift in the β strand that anchors one end of the loop from one face of the domain to the other. A strand displacement also causes a change in the orientation of the Vβ CDR2 loop in some Vβ domains. These differences with antibodies influence the

1	loop from one face of the domain to the other. A strand displacement also causes a change in the orientation of the Vβ CDR2 loop in some Vβ domains. These differences with antibodies influence the ability of the T-cell receptor to recognize their specific ligands, as we will discuss in the next section. In addition to the three hypervariable regions shared with immunoglobulins, the T-cell receptor has a fourth hypervariability region, HV4, in both of its chains (see Fig. 4.15c). These regions occur away from the antigen-binding face of the receptor, and have been implicated in other functions of the TCR, such as superantigen binding, which we will describe in Section 6-14.

1	4-12 A T-cell receptor recognizes antigen in the form of a complex of a foreign peptide bound to an MHC molecule. Antigen recognition by T-cell receptors clearly differs from recognition by B-cell receptors and antibodies. The immunoglobulin on B cells binds directly to the intact antigen, and, as discussed in Section 4-8, antibodies typically bind to the surface of protein antigens, contacting amino acids that are discontinuous in the primary structure but are brought together in the folded protein. In contrast, αβ T cells respond to short, continuous amino acid sequences. As we described in Section 1-10, these peptide sequences are often buried within the native structure of the protein. Thus, antigens cannot be recognized directly by T-cell receptors unless the protein is unfolded and processed into peptide fragments (Fig. 4.16), and then presented by an MHC molecule (see Fig. 1.15). We will return to the issue of how this process occurs in Chapter 6.

1	The nature of the antigen recognized by T cells became clear with the realization that the peptides that stimulate T cells are recognized only when bound to an MHC molecule. The ligand recognized by the T cell is thus a complex of peptide and MHC molecule. The evidence for involvement of the MHC in T-cell recognition of antigen was at first indirect, but it has been proven conclusively by stimulating T cells with purified peptide:MHC complexes. The T-cell receptor interacts with this ligand by making contacts with both the MHC molecule and the antigen peptide. 4-13 There are two classes of MHC molecules with distinct subunit compositions but similar three-dimensional structures.

1	4-13 There are two classes of MHC molecules with distinct subunit compositions but similar three-dimensional structures. There are two classes of MHC molecules—MHC class I and MHC class II—and they differ in both their structure and their expression pattern in the tissues of the body. As shown in Figs. 4.17 and 4.18, MHC class I and MHC class II molecules are closely related in overall structure but differ in their subunit composition. In both classes, the two paired protein domains nearest to the membrane resemble immunoglobulin domains, whereas the two domains furthest away from the membrane fold together to create a long cleft, or groove, which is the site at which a peptide binds. Purified peptide:MHC class I and peptide:MHC class II complexes have been characterized structurally, allowing us to describe in detail both the MHC molecules themselves and the way in which they bind peptides.

1	MHC class I molecules (see Fig. 4.17) consist of two polypeptide chains. One chain, the α chain, is encoded in the MHC (on chromosome 6 in humans) and is noncovalently associated with a smaller chain, β2-microglobulin, which is encoded on a different chromosome—chromosome 15 in humans. Only the class I α chain spans the membrane. The complete MHC class I molecule has four domains, three formed from the MHC-encoded α chain, and one contributed by β2-microglobulin. The α3 domain and β2-microglobulin closely resemble Ig-like domains in their folded structure. The folded α1 and α2

1	Fig. 4.16 Differences in the recognition of hen egg-white lysozyme by immunoglobulins and T-cell receptors. Antibodies can be shown by X-ray crystallography to bind epitopes on the surface of proteins, as shown in panel a, where the epitopes for three antibodies are shown in different colors on the surface of hen egg-white lysozyme (see also fig. 4.10). in contrast, the epitopes recognized by T-cell receptors need not lie on the surface of the molecule, because the T-cell receptor recognizes not the antigenic protein itself but a peptide fragment of the protein. The peptides corresponding to two T-cell epitopes of lysozyme are shown in panel b. one epitope, shown in blue, lies on the surface of the protein, but a second, shown in red, lies mostly within the core and is inaccessible in the folded protein. This implies that T-cell receptors do not recognize their epitopes in the context of the native protein. Panel a courtesy of S. Sheriff.

1	domains form the walls of a cleft on the surface of the molecule; because this is where the peptide binds, this part of the MHC molecule is known as the peptide-binding cleft or peptide-binding groove. The MHC molecules are highly polymorphic, and the major differences between the different allelic

1	Fig. 4.17 The structure of an MHC class I molecule determined by X-ray crystallography. Panel a shows a computer graphic representation of a human Mhc class i molecule, hLA-A2, which has been cleaved from the cell surface by the enzyme papain. The surface of the molecule is shown, colored according to the domains shown in panels b–d and described below. Panels b and c show a ribbon diagram of that structure. Shown schematically in panel d, the Mhc class i molecule is a heterodimer of a membrane-spanning α chain (molecular weight 43 kDa) bound noncovalently to β2-microglobulin (12 kDa), which does not span the membrane. The α chain folds into three domains: α1, α2, and α3. The α3 domain and β2-microglobulin show similarities in amino acid sequence to immunoglobulin c domains and have similar folded structures, whereas the α1 and α2 domains are part of the same polypeptide and fold together into a single structure consisting of two separated α helices lying on a sheet of eight

1	similar folded structures, whereas the α1 and α2 domains are part of the same polypeptide and fold together into a single structure consisting of two separated α helices lying on a sheet of eight antiparallel β strands. The folding of the α1 and α2 domains creates a long cleft or groove, which is the site at which peptide antigens bind to the Mhc molecules. for class i molecules, this groove is open at only one end. The transmembrane region and the short stretch of peptide that connects the external domains to the cell surface are not seen in panels a and b because they have been removed by the digestion with papain. As can be seen in panel c, looking down on the molecule from above, the sides of the cleft are formed from the inner faces of the two α helices; the β pleated sheet formed by the pairing of the α1 and α2 domains creates the floor of the cleft.

1	forms are located in the peptide-binding cleft, influencing which peptides will bind and thus the specificity of the dual antigen presented to T cells. By contrast, β2-microglobulin, which does not contribute directly to peptide binding, is not polymorphic.

1	An MHC class II molecule consists of a noncovalent complex of two chains, α and β, both of which span the membrane (see Fig. 4.18). The MHC class II α chain is a different protein from the class I α chain. The MHC class II α and β chains are both encoded within the MHC. The crystallographic structure of the MHC class II molecule shows that it is folded very much like the MHC class I molecule, but the peptide-binding cleft is formed by two domains from different chains, the α1 and β1 domains. The major differences lie at the ends of the peptide-binding cleft, which are more open in MHC class II than in MHC class I molecules. Consequently, the ends of a peptide bound to an MHC class I molecule are substantially buried within the molecule, whereas the ends of peptides bound to MHC class II molecules are not. In both MHC class I and class II molecules, bound peptides are sandwiched between the two α-helical segments of the MHC molecule (Fig. 4.19). The T-cell receptor interacts with this

1	are not. In both MHC class I and class II molecules, bound peptides are sandwiched between the two α-helical segments of the MHC molecule (Fig. 4.19). The T-cell receptor interacts with this compound ligand, making contacts with both the MHC molecule and the peptide antigen. As in the case of MHC class I molecules, the sites of major polymorphism in MHC class II molecules are located in the peptide-binding cleft.

1	Fig. 4.18 MHC class II molecules resemble MHC class I molecules in overall structure. The Mhc class ii molecule is composed of two transmembrane glycoprotein chains, α (34 kDa) and β (29 kDa), as shown schematically in panel d. each chain has two domains, and the two chains together form a compact four-domain structure similar to that of the Mhc class i molecule (compare with panel d of fig. 4.17). Panel a shows a computer graphic representation of the surface of the Mhc class ii molecule, in this case the human protein hLA-DR1, and panel b shows the equivalent ribbon diagram. n, amino terminus; c, carboxy terminus. The α2 and β2 domains, like the α3 and β2-microglobulin domains of the Mhc class i molecule, have amino acid sequence and structural similarities to immunoglobulin c domains; in the Mhc class ii molecule the two domains forming the peptide-binding cleft are contributed by different chains and are therefore not joined by a covalent bond (see panels c and d). Another

1	in the Mhc class ii molecule the two domains forming the peptide-binding cleft are contributed by different chains and are therefore not joined by a covalent bond (see panels c and d). Another important difference, not apparent in this diagram, is that the peptide-binding groove of the Mhc class ii molecule is open at both ends.

1	Fig. 4.19 MHC molecules bind peptides tightly within the cleft. When Mhc molecules are crystallized with a single synthetic peptide antigen, the details of peptide binding are revealed. in Mhc class i molecules (panels a and c), the peptide is bound in an elongated conformation with both ends tightly bound at either end of the cleft. in Mhc class ii molecules (panels b and d), the peptide is also bound in an elongated conformation but the ends of the peptide are not tightly bound and the peptide extends beyond the cleft. The upper surface of the peptide:Mhc complex is recognized by T cells, and is composed of residues of the Mhc molecule and the peptide. The amino acid side chains of the peptide insert into pockets in the peptide-binding groove of the Mhc molecule; these pockets are lined with residues that are polymorphic within the Mhc. in representations c and d, the surfaces of the different pockets for the different amino acids are depicted as areas of different colors.

1	are lined with residues that are polymorphic within the Mhc. in representations c and d, the surfaces of the different pockets for the different amino acids are depicted as areas of different colors. Structures courtesy of

1	R.L. Stanfield and i.A. Wilson. 4-14 Peptides are stably bound to MHC molecules, and also serve to stabilize the MHC molecule on the cell surface. An individual can be infected by a wide variety of pathogens, whose proteins will not generally have peptide sequences in common. For T cells to be able to detect the widest possible array of infections, the MHC molecules (both class I and class II) of an individual should be able to bind stably to many different peptides. This behavior is quite distinct from that of other peptide-binding receptors, such as those for peptide hormones, which usually bind only a single type of peptide. The crystal structures of peptide:MHC complexes have helped to show how a single binding site can bind a peptide with high affinity while retaining the ability to bind a wide variety of different peptides.

1	An important feature of the binding of peptides to MHC molecules is that the peptide is bound as an integral part of the MHC molecule’s structure, and MHC molecules are unstable when peptides are not bound. This dependence on bound peptide applies to both MHC class I and MHC class II molecules. Stable peptide binding is important, because otherwise peptide exchanges occurring at the cell surface would prevent peptide:MHC complexes from being reliable indicators of infection or of uptake of a specific antigen. When MHC molecules are purified from cells, their stably bound peptides co-purify with them, and this fact has enabled the peptides bound by particular MHC molecules to be analyzed. Peptides are released from the MHC molecules by denaturing the complex in acid, and they are then purified and sequenced. Pure synthetic peptides can also be incorporated into empty MHC molecules and the structure of the complex determined, revealing details of the contacts between the MHC molecule

1	and sequenced. Pure synthetic peptides can also be incorporated into empty MHC molecules and the structure of the complex determined, revealing details of the contacts between the MHC molecule and the peptide. From such studies a detailed picture of the binding interactions has been built up. We first discuss the peptide-binding properties of MHC class I molecules.

1	4-15 MHC class I molecules bind short peptides of 8–10 amino acids by both ends.

1	Binding of a peptide to an MHC class I molecule is stabilized at both ends of the peptide-binding cleft by contacts between atoms in the free amino and carboxy termini of the peptide and invariant sites that are found at each end of the cleft in all MHC class I molecules (Fig. 4.20). These are thought to be the main stabilizing contacts for peptide:MHC class I complexes, because synthetic peptide analogs lacking terminal amino and carboxyl groups fail to bind stably to MHC class I molecules. Other residues in the peptide serve as additional anchors. Peptides that bind to MHC class I molecules are usually 8–10 amino acids long. Longer peptides are thought to bind, however, particularly if they can bind at their carboxy terminus, but they are subsequently shortened to 8–10 amino acids through cleavage by exopeptidases present in the endoplasmic reticulum, which is where MHC class I molecules bind peptides. The peptide lies in an elongated conformation along the cleft; variations in

1	cleavage by exopeptidases present in the endoplasmic reticulum, which is where MHC class I molecules bind peptides. The peptide lies in an elongated conformation along the cleft; variations in peptide length seem to be accommodated, in most cases, by a kinking in the peptide backbone. However, in some cases, length variation can also be accommodated in MHC class I molecules by allowing the peptide to extend out of the cleft at the carboxy terminus.

1	These interactions give MHC class I molecules a broad peptide-binding specificity. In addition, MHC molecules are highly polymorphic. As mentioned earlier, MHC genes are highly polymorphic and there are hundreds of different allelic variations of the MHC class I genes in the human population. Each individual carries only a small selection of these variants. The main differences between allelic MHC variants are found at certain sites in the peptide-binding cleft, resulting in different amino acids in key peptide-interaction sites. Because of this, different MHC variants preferentially bind different peptides. The peptides that can bind to a given MHC variant have the same or very similar amino acid residues at two or three particular positions along the peptide sequence. The amino acid side chains at these positions insert into pockets in the MHC molecule that are lined by the polymorphic amino acids. Because this binding anchors the peptide to the MHC molecule, the peptide residues

1	chains at these positions insert into pockets in the MHC molecule that are lined by the polymorphic amino acids. Because this binding anchors the peptide to the MHC molecule, the peptide residues involved are called the anchor residues, as illustrated in Fig. 4.21. Both the position and identity of these anchor residues can vary, depending on the particular MHC class I variant that is binding the peptide. However, most peptides that bind to MHC class I molecules have a hydrophobic (or sometimes basic) residue at the carboxy terminus that also serves to anchor the peptide in the groove. Whereas changing an anchor residue will in most cases prevent the peptide from binding, not every synthetic peptide of suitable length that

1	Antigen recognition by T cells. 159 Fig. 4.20 Peptides are bound to MHC class I molecules by their ends. Mhc class i molecules interact with the backbone of a bound peptide (shown in yellow) through a series of hydrogen bonds and ionic interactions (shown as dotted blue lines) at each end of the peptide. The amino terminus of the peptide is to the left, the carboxy terminus to the right. Black circles are carbon atoms; red are oxygen; blue are nitrogen. The amino acid residues in the Mhc molecule that form these bonds are common to all Mhc class i molecules, and their side chains are shown in full (in gray) on a ribbon diagram of the Mhc class i groove. A cluster of tyrosine residues common to all Mhc class i molecules forms hydrogen bonds to the amino terminus of the bound peptide, while a second cluster of residues forms hydrogen bonds and ionic interactions with the peptide backbone at the carboxy terminus and with the carboxy terminus itself.

1	Fig. 4.21 Peptides bind to MHC molecules through structurally related anchor residues. Peptides eluted from two different Mhc class i molecules are shown in the upper and lower panels, respectively. The anchor residues (green) differ for peptides that bind different allelic variants of Mhc class i molecules but are similar for all peptides that bind to the same Mhc molecule. The anchor residues that bind a particular Mhc molecule need not be identical, but are always related: for example, phenylalanine (f) and tyrosine (Y) are both aromatic amino acids, whereas valine (V), leucine (L), and isoleucine (i) are all large hydrophobic amino acids. Peptides also bind to Mhc class i molecules through their amino (blue) and carboxy (red) termini.

1	Fig. 4.22 Peptides bind to MHC class II molecules by interactions along the length of the binding groove. A peptide (yellow; shown as the peptide backbone only, with the amino terminus to the left and the carboxy terminus to the right) is bound by an Mhc class ii molecule through a series of hydrogen bonds (dotted blue lines) that are distributed along the length of the peptide. The hydrogen bonds toward the amino terminus of the peptide are made with the backbone of the Mhc class ii polypeptide chain, whereas throughout the peptide’s length bonds are made with residues that are highly conserved in Mhc class ii molecules. The side chains of these residues are shown in gray on the ribbon diagram of the Mhc class ii groove.

1	contains these anchor residues will bind the appropriate MHC class I molecule, and so the overall binding must also depend on the nature of the amino acids at other positions in the peptide. In some cases, particular amino acids are preferred in certain positions, whereas in others the presence of particular amino acids prevents binding. These additional amino acid positions are called ‘secondary anchors.’ These features of peptide binding enable an individual MHC class I molecule to bind a wide variety of different peptides, yet allow different MHC class I allelic variants to bind different sets of peptides. As we will see in Chapter 15, MHC polymorphisms also impact the binding of peptides derived from self-proteins and can influence the susceptibility of an individual to various autoimmune diseases. 4-16 The length of the peptides bound by MHC class II molecules is not constrained.

1	Like MHC class I molecules, MHC class II molecules that lack bound peptide are unstable. Peptide binding to MHC class II molecules has also been analyzed by elution of bound peptides and by X-ray crystallography, and differs in several ways from peptide binding to MHC class I molecules. Natural peptides that bind to MHC class II molecules are at least 13 amino acids long and can be much longer. The clusters of conserved residues that bind the two ends of a peptide in MHC class I molecules are not found in MHC class II molecules, and the ends of the peptide are not bound. Instead, the peptide lies in an extended conformation along the peptide-binding cleft. It is held there both by peptide side chains that protrude into shallow and deep pockets lined by polymorphic residues, and by interactions between the peptide backbone and the side chains of conserved amino acids that line the peptide-binding cleft in all MHC class II molecules (Fig. 4.22). Structural data show that amino acid side

1	between the peptide backbone and the side chains of conserved amino acids that line the peptide-binding cleft in all MHC class II molecules (Fig. 4.22). Structural data show that amino acid side chains at residues 1, 4, 6, and 9 of an MHC class II-bound peptide can be held in these binding pockets.

1	The binding pockets of MHC class II molecules accommodate a greater variety of side chains than those of MHC class I molecules, making it more difficult to define anchor residues and to predict which peptides will be able to bind

1	Fig. 4.23 Peptides that bind MHC class II molecules are peptides can vary, and so by convention the first anchor residue variable in length and their anchor residues lie at various is denoted as residue 1. note that all of the peptides share a distances from the ends of the peptide. The sequences of a hydrophobic residue in position 1, a negatively charged residue set of peptides that bind to the mouse Mhc class ii Ak allele are [aspartic acid (D) or glutamic acid (e)] in position 4, and a tendency shown in the upper panel. All contain the same core sequence to have a basic residue [lysine (K), arginine (R), histidine (h), (shaded) but differ in length. in the lower panel, different peptides glutamine (Q), or asparagine (n)] in position 6 and a hydrophobic binding to the human Mhc class ii allele hLA-DR3 are shown. residue [for example, tyrosine (Y), leucine (L), phenylalanine (f)] in Anchor residues are shown as green circles. The lengths of these position 9.

1	a particular MHC class II variant (Fig. 4.23). Nevertheless, by comparing the sequences of known binding peptides it is usually possible to detect patterns of amino acids that permit binding to different MHC class II variants, and to model how the amino acids of this peptide sequence motif will interact with the amino acids of the peptide-binding cleft. Because the peptide is bound by its backbone and allowed to emerge from both ends of the binding groove, there is, in principle, no upper limit to the length of peptides that could bind to MHC class II molecules. An example of this is the protein known as invariant chain, part of which lies entirely across the peptide-binding groove of nascent MHC class II molecules during their synthesis in the endoplasmic reticulum. We will return in Chapter 6 to the role of the invariant chain in the loading of peptides onto MHC class II molecules. In most cases, long peptides bound to MHC class II molecules are trimmed by peptidases to a length of

1	6 to the role of the invariant chain in the loading of peptides onto MHC class II molecules. In most cases, long peptides bound to MHC class II molecules are trimmed by peptidases to a length of around 13–17 amino acids.

1	4-17 The crystal structures of several peptide:MHC:T-cell receptor complexes show a similar orientation of the T-cell receptor over the peptide:MHC complex. At the time that the first X-ray crystallographic structure of a T-cell receptor was published, a structure of the same T-cell receptor bound to a peptide:MHC class I ligand was also produced. The orientation revealed by these structures showed that the T-cell receptor is aligned diagonally over the peptide and the peptide-binding cleft (Fig. 4.24). The TCRα chain lies over the α2 domain of the MHC molecule at the amino-terminal end of the bound peptide, as seen from the side view shown in Fig. 4.24a. The TCRβ chain lies over the MHC molecule’s α1 domain, closer to the carboxy-terminal end of the peptide. Figure 4.24b shows a view of this structure as if looking down through a transparent T-cell receptor to indicate where it contacts the MHC molecule. The CDR3 loops of both TCRα and TCRβ chains come together and lie over

1	Fig. 4.24 The T-cell receptor binds to the peptide:MHC complex. Panel a: the T-cell receptor binds to the top of a peptide:Mhc class i molecule, touching both the α1 and α2 domain helices. The cDRs of the T-cell receptor are shown in color: the cDR1 and cDR2 loops of the β chain are light and dark blue, respectively; and the cDR1 and cDR2 loops of the α chain are light and dark purple, respectively. The α-chain cDR3 loop is yellow, and the β-chain cDR3 loop is green. The β-chain hV4 loop is in red. The thick yellow line P1–P8 is the bound peptide. Panel b: the outline of the T-cell receptor’s antigen-binding site (thick black line) is superimposed over the top surface of the peptide:Mhc complex (the peptide is shaded dull yellow). The T-cell receptor lies at a somewhat diagonal angle across the peptide:Mhc complex, with the α and β cDR3 loops of the T-cell receptor (3α, 3β, yellow and green, respectively) contacting the center of the peptide. The α-chain cDR1 and cDR2 loops (1α, 2α,

1	the peptide:Mhc complex, with the α and β cDR3 loops of the T-cell receptor (3α, 3β, yellow and green, respectively) contacting the center of the peptide. The α-chain cDR1 and cDR2 loops (1α, 2α, light and dark purple, respectively) contact the Mhc helices at the amino terminus of the bound peptide, whereas the β-chain cDR1 and cDR2 loops (1β, 2β, light and dark blue, respectively) make contact with the helices at the carboxy terminus of the bound peptide. courtesy of i.A. Wilson.

1	central amino acids of the peptide. The T-cell receptor is threaded through a valley between the two high points on the two surrounding α helices that form the walls of the peptide-binding cleft. This can be seen in Fig. 4.25, which shows a view from the end of the peptide-binding groove of a peptide:MHC class II:T-cell receptor complex. Comparison of various peptide: MHC:T-cell receptor complexes shows that the axis of the TCR as it binds the surface of the MHC molecule is rotated somewhat relative to the peptide-binding groove of the MHC molecule (see Fig. 4.24b). In this orientation, the Vα domain makes contact primarily with the amino-terminal half of the bound peptide, whereas the Vβ domain contacts primarily the carboxy-terminal half. Both chains also interact with the α helices of the MHC class I molecule (see Fig. 4.24). The T-cell receptor contacts are not symmetrically distributed over the MHC molecule: whereas the Vα CDR1 and CDR2 loops are in close contact with the helices

1	MHC class I molecule (see Fig. 4.24). The T-cell receptor contacts are not symmetrically distributed over the MHC molecule: whereas the Vα CDR1 and CDR2 loops are in close contact with the helices of the peptide:MHC complex around the amino terminus of the bound peptide, the β-chain CDR1 and CDR2 loops, which interact with the complex at the carboxy terminus of the bound peptide, have variable contributions to the binding.

1	Comparison of the three-dimensional structure of an unliganded T-cell receptor and the same T-cell receptor complexed to its peptide:MHC ligand shows that the binding results in some degree of conformational change, or ‘induced fit,’ particularly within the Vα CDR3 loop. Subtle variations at amino acids that contact the T-cell receptor can have strikingly different effects on the recognition of an otherwise identical peptide:MHC ligand by the same T cell. The flexibility in the CDR3 loop demonstrated by these two structures helps to explain how the T-cell receptor can adopt conformations that recognize related, but different, peptide ligands.

1	The specificity of T-cell recognition involves both the peptide and its presenting MHC molecule. Kinetic analysis of T-cell receptor binding to peptide:MHC ligands suggests that the interactions with MHC molecules might predominate at the start of the contact, but that subsequent interactions with the peptide as well as the MHC molecule dictate the final outcome—binding or dissociation. As with antibody–antigen interactions, only a few amino acids at the interface might provide the essential contacts that determine the specificity and strength of binding. Simply changing a leucine to isoleucine in the peptide, for example, is sufficient to alter a T-cell response from strong killing to no response at all. Mutations of single residues in the presenting MHC molecules can have the same effect. This dual specificity for T-cell recognition of antigen underlies the MHC restriction of T-cell responses, a phenomenon that was observed long before the peptide-binding properties of MHC molecules

1	This dual specificity for T-cell recognition of antigen underlies the MHC restriction of T-cell responses, a phenomenon that was observed long before the peptide-binding properties of MHC molecules were known. Another consequence of this dual specificity is a need for T-cell receptors to exhibit some inherent specificity for MHC molecules in order to be able to interact appropriately with the antigen-presenting surface of MHC

1	Fig. 4.25 The T-cell receptor interacts with MHC class I and MHC class II molecules in a similar fashion. Shown is the structure of a T-cell receptor, specific for a peptide derived from chicken cytochrome c, bound to an Mhc class ii molecule. This T-cell receptor’s binding is at a site and orientation similar to that of the T-cell receptor bound to the Mhc class i molecule shown in fig. 4.24. The α and β chains of the T-cell receptor are colored in light and dark blue, respectively. The cytochrome c peptide is light orange. The T-cell receptor sits in a shallow saddle formed between the α-helical regions of the Mhc class ii α (brown) and β (yellow) chains at roughly 90° to the long axis of the Mhc class ii molecule and the bound peptide. Structure derived from PDB 3QiB. courtesy of K.c. garcia.

1	molecules. We will return to these issues in Chapter 6, where we recount the discovery of MHC restriction in the context of T-cell recognition and MHC polymorphisms, and in Chapter 8, where we discuss the impact of these phenomena on T-cell development in the thymus. 4-18 The CD4 and CD8 cell-surface proteins of T cells directly contact MHC molecules and are required to make an effective response to antigen.

1	As we introduced in Section 1-21, T cells fall into two major classes distinguished by the expression of the cell-surface proteins CD4 and CD8. CD8 is expressed by cytotoxic T cells, while CD4 is expressed by T cells whose function is to activate other cells. CD4 and CD8 were known as markers for these functional sets for some time before it became clear that the distinction was based on the ability of T cells to recognize different classes of MHC molecules. We now know that CD8 recognizes MHC class I molecules and CD4 recognizes MHC class II. During antigen recognition, CD4 or CD8 (depending on the type of T cell) associates with the T-cell receptor on the T-cell surface and binds to invariant sites on the MHC portion of the composite peptide:MHC ligand, away from the peptide-binding site. This binding contributes to the overall effectiveness of the T-cell response, and so CD4 and CD8 are called co-receptors.

1	CD4 is a single-chain protein composed of four Ig-like domains (Fig. 4.26). The first two domains (D1 and D2) are packed tightly together to form a rigid

1	Fig. 4.26 The structures of the CD4 and CD8 co-receptor molecules. The cD4 molecule contains four ig-like domains, shown in schematic form in panel a and as a ribbon diagram from the crystal structure in panel b. The amino-terminal domain, D1, is similar in structure to an immunoglobulin V domain. The second domain, D2, although related to an immunoglobulin domain, is different from both V and c domains and has been termed a c2 domain. The first two domains of cD4 form a rigid rodlike structure that is linked to the two carboxy-terminal domains by a flexible link. The binding site for Mhc class ii molecules involves mainly the D1 domain. The cD8 molecule is a heterodimer of an α and a β chain covalently linked by a disulfide bond. An alternative form of cD8 exists as a homodimer of α chains. The heterodimer is depicted in panel a, whereas the ribbon diagram in panel b is of the homodimer. cD8α and cD8β chains have very similar structures, each having a single domain resembling an

1	chains. The heterodimer is depicted in panel a, whereas the ribbon diagram in panel b is of the homodimer. cD8α and cD8β chains have very similar structures, each having a single domain resembling an immunoglobulin V domain and a stretch of polypeptide chain, believed to be in a relatively extended conformation, that anchors the V-like domain to the cell membrane.

1	rod about 6 nm long, which is joined by a flexible hinge to a similar rod formed by the third and fourth domains (D3 and D4). The MHC-binding region on CD4 is located mainly on a lateral face of the D1 domain, and CD4 binds to a hydrophobic crevice formed at the junction of the α2 and β2 domains of the MHC class II molecule (Fig. 4.27a). This site is well away from the site where the T-cell receptor binds, as shown by the complete crystal structure of a T-cell receptor bound to peptide:MHC class II with bound CD4 (Fig. 4.28). This structure demonstrates that the CD4 molecule and the T-cell receptor can bind simultaneously to the same peptide:MHC class II complex. CD4 enhances sensitivity to antigen, as the T cell is about 100-fold more sensitive to the antigen when CD4 is present. The enhancement process results from the ability of the intracellular portion of CD4 to bind to a cytoplasmic tyrosine kinase called Lck. As we will discuss in detail Chapter 7, bringing Lck into proximity

1	process results from the ability of the intracellular portion of CD4 to bind to a cytoplasmic tyrosine kinase called Lck. As we will discuss in detail Chapter 7, bringing Lck into proximity with the T-cell receptor complex helps activate the signaling cascade induced by antigen recognition.

1	The structure of CD8 is quite different. It is a disulfide-linked dimer of two different chains, called αand β, each containing a single Ig-like domain linked to the membrane by a segment of extended polypeptide (see Fig. 4.26). This segment

1	The binding sites for cD4 and cD8 on the Mhc class ii and class i molecules, respectively, lie in the ig-like domains nearest to the membrane and distant from the peptide-binding cleft. The binding of cD4 to an Mhc class ii molecule is shown as a ribbon structure in panel a and schematically in panel c. The α chain of the Mhc class ii molecule is shown in pink, and the β chain in white, while cD4 is in gold. only the D1 and D2 domains of the cD4 molecule are shown in panel a. The binding site for cD4 lies at the base of the β2 domain of an Mhc class ii molecule, in the hydrophobic crevice between the β2 and α2 domains. The binding of cD8αβ to an Mhc class i molecule is shown in panel b and schematically in panel d. The class i heavy chain and β2-microglobulin are shown in white and pink, respectively, and the two chains of the cD8 dimer are shown in light (cD8β) and dark (cD8α) purple. The binding site for cD8 on the Mhc class i molecule lies in a similar position to that of cD4 in

1	respectively, and the two chains of the cD8 dimer are shown in light (cD8β) and dark (cD8α) purple. The binding site for cD8 on the Mhc class i molecule lies in a similar position to that of cD4 in the Mhc class ii molecule, but cD8 binding also involves the base of the α1 and α2 domains, and thus the binding of cD8 to Mhc class i is not completely equivalent to the binding of cD4 to Mhc class ii. Structures derived from PDB 3S4S (cD4/ Mhc class ii) and PDB 3DMM (cD8αβ/ Mhc class i). courtesy of K.c. garcia.

1	Fig. 4.27 The binding sites for CD4 and CD8 on MHC class II and class I molecules lie in the Ig-like domains. is extensively glycosylated, which is thought to maintain it in an extended conformation and protect it from cleavage by proteases. CD8α chains can form homodimers, although these are usually not found when CD8β is expressed. Naive T cells express CD8αβ, but the CD8αα homodimer can be expressed by highly activated effector and memory T cells. CD8αα is also expressed by a population of intraepithelial lymphocytes known as mucosal associated invariant T cells (MAIT cells); these cells recognize metabolites of folic acid that are produced by bacteria in association with the nonclassical MHC class I molecule MR1, which we will describe in Chapter 6.

1	CD8αβ binds weakly to an invariant site in the α3 domain of an MHC class I molecule (see Fig. 4.27b). The CD8β chain interacts with residues in the base of the α2 domain of the MHC class I molecule, while the αchain is in a lower position interacting with the α3 domain of the MHC class I molecule. The strength of binding of CD8 to the MHC class I molecule is influenced by the glycosylation state of the CD8 molecule; increasing the number of sialic acid residues on CD8 carbohydrate structures decreases the strength of the interaction. The pattern of sialylation of CD8 changes during the maturation of T cells and also on activation, and this likely has a role in modulating antigen recognition.

1	Like the interactions with MHC class II molecules, the T-cell receptor and CD8 can interact simultaneously with one MHC class I molecule (Fig. 4.29). Like CD4, CD8 binds Lck through the cytoplasmic tail of the α chain, and CD8αβ increases the sensitivity of T cells to antigen presented by MHC class I molecules about 100-fold. Although the molecular details are unclear, the CD8αα homodimer appears to function less efficiently than CD8αβ as a co-receptor, and may negatively regulate activation. In contrast to CD8, CD4 is not thought to dimerize. Fig. 4.28 CD4 and the T-cell receptor bind to distinct regions of the MHC class II molecule. A ribbon diagram is shown from a crystal structure of a complete α:β TcR:peptide-Mhc:cD4 ternary complex. The α and β chains of the T-cell receptor (TcR) are blue and red, respectively. The Mhc class ii molecule is green, with the bound peptide shown in gray. cD4 is shown in orange. Structure derived from PDB 3T0e. courtesy of K.c. garcia.

1	Fig. 4.29 CD8 binds to a site on MHC class I molecules that is distant from where the T-cell receptor binds. The relative binding positions of both the T-cell receptor and cD8 molecules can be seen in this hypothetical reconstruction of their interaction with Mhc class i (α chain in dark green and β2-microglobulin in light green). The α and β chains of the T-cell receptor are shown in brown and purple, respectively. The cD8αβ heterodimer is shown bound to the Mhc class i α3 domain. The cD8 α chain is in blue, and the cD8β chain is in red. courtesy of chris nelson and David fremont.

1	Mhc class i molecules are expressed on all nucleated cells, although they are most highly expressed in hematopoietic cells. Mhc class ii molecules are normally expressed only by a subset of hematopoietic cells and by thymic stromal cells, although they may be expressed by other cell types on exposure to the inflammatory cytokine ifn-γ. *in humans, activated T cells express Mhc class ii molecules, whereas in mice all T cells are Mhc class ii-negative. †in the brain, most cell types are Mhc class ii-negative, but microglia, which are related to macrophages, are Mhc class ii-positive. 4-19 The two classes of MHC molecules are expressed differentially on cells.

1	MHC class I and MHC class II molecules have distinct distributions among cells, and these reflect the different effector functions of the T cells that recognize them (Fig. 4.30). MHC class I molecules present peptides from pathogens, commonly viruses, to CD8 cytotoxic T cells, which are specialized to kill any cell that they specifically recognize. Because viruses can infect any nucleated cell, almost all such cells express MHC class I molecules, although the level of constitutive expression varies from one cell type to the next. For example, cells of the immune system express abundant MHC class I on their surface, whereas liver cells (hepatocytes) express relatively low levels (see Fig. 4.30). Nonnucleated cells, such as mammalian red blood cells, express little or no MHC class I, and thus the interior of red blood cells is a site in which an infection can go undetected by cytotoxic T cells. Because red blood cells cannot support viral replication, this is of no great consequence for

1	the interior of red blood cells is a site in which an infection can go undetected by cytotoxic T cells. Because red blood cells cannot support viral replication, this is of no great consequence for viral infection, but it might be the absence of MHC class I that allows the Plasmodium parasites that cause malaria to live in this privileged site.

1	In contrast, a major function of the CD4 T cells that recognize MHC class II molecules is to activate other effector cells of the immune system. Thus, MHC class II molecules are normally found on dendritic cells, B lymphocytes, and macrophages—antigen-presenting cells that participate in immune responses—but not on other tissue cells (see Fig. 4.30). The peptides presented by MHC class II molecules expressed by dendritic cells can function to activate naive CD4 T cells. When previously activated CD4 T cells recognize peptides bound to MHC class II molecules on B cells, the T cells secrete cytokines that can influence the isotype of antibody that those B cells will choose to produce. Upon recognizing peptides bound to MHC class II molecules on macrophages, CD4 T cells activate these cells, again in part through cytokines, to destroy the pathogens in their vesicles.

1	The expression of both MHC class I and MHC class II molecules is regulated by cytokines, in particular, interferons, released in the course of an immune response. Interferon-α (IFN-α) and IFN-β increase the expression of MHC class I molecules on all types of cells, whereas IFN-γ increases the expression of both MHC class I and MHC class II molecules, and can induce the expression of MHC class II molecules on certain cell types that do not normally express them. Interferons also enhance the antigen-presenting function of MHC class I molecules by inducing the expression of key components of the intracellular machinery that enables peptides to be loaded onto the MHC molecules. 4-20 A distinct subset of T cells bears an alternative receptor made up of γ and δ chains.

1	4-20 A distinct subset of T cells bears an alternative receptor made up of γ and δ chains. During the search for the gene for the TCRα chain, another T-cell receptor-like gene was unexpectedly discovered. This gene was named TCRγ, and its discovery led to a search for further T-cell receptor genes. Another receptor chain was identified by using antibody against the predicted sequence of the γ chain and was called the δ chain. It was soon discovered that a minority population of T cells bore a distinct type of T-cell receptor made up of γ:δ heterodimers rather than α:β heterodimers. The development of these cells is described in Sections 8-11 and 8-12.

1	Like α:β T cells, γ:δ T cells can be found in the lymphoid tissues of all vertebrates, but they are also prominent as populations of intraepithelial lymphocytes, particularly in the skin and female reproductive tracts, where their receptors display very limited diversity. Unlike α:β T cells, γ:δ T cells do not generally recognize antigen as peptides presented by MHC molecules, and γ:δ T-cell receptors are not restricted by the ‘classical’ MHC class I and class II molecules that function in binding and presenting peptides to T cells. Instead, γ:δ T-cell receptors seem to recognize their target antigens directly and thus probably are able to recognize and respond rapidly to molecules expressed by many different cell types. Their ligands have been difficult to identify, but several have now been described and seem to indicate that γ:δ T cells play an intermediate, or transitional, role between wholly innate and fully adaptive immune responses.

1	Like NK-cell receptor ligands, such as the proteins MIC and RAET1 (see Section 3-27), many of the ligands seen by γ:δ T cells are induced by cellular stress or damage. γ:δ T cells may also bind antigens presented by ‘nonclassical’ MHC class Ib molecules, which we will discuss in Chapter 6. These proteins are related structurally to the MHC proteins we have already discussed, but have functions besides binding peptides for presentation to T cells. Additional ligands may include heat-shock proteins and nonpeptide ligands such as phosphorylated ligands or mycobacterial lipid antigens. γ:δ T cells can also respond to unorthodox nucleotides and phospholipids. Recognition of molecules expressed as a consequence of infection, rather than recognition of pathogen-specific antigens themselves, distinguishes intraepithelial γ:δT cells from other lymphocytes, and this would place them in the innate-like class. For these reasons, the term ‘transitional immunity’ has been proposed to clarify the

1	intraepithelial γ:δT cells from other lymphocytes, and this would place them in the innate-like class. For these reasons, the term ‘transitional immunity’ has been proposed to clarify the role of γ:δ T cells, since the function of these cells seems to be someplace between innate and adaptive responses.

1	The crystallographic structure of a γ:δ T-cell receptor reveals that, as expected, it is similar in shape to α:β T-cell receptors. Figure 4.31 shows a crystal structure of a γ:δ T-cell receptor complex bound to one of the nonclassical MHC class I molecules mentioned above, called T22. This structure shows that the overall orientation of the γ:δ T-cell receptor with the MHC molecule is strikingly different from that of an α:β T-cell receptor, in that it interacts primarily with one end of the T22 molecule. However, the CDR3 regions of the γ:δ T-cell receptor still play a critical role in recognition, similar to that of antibodies and of α:β T-cell receptors. Further, the CDR3 of the γ:δ T-cell receptor is longer than either of these other two antigen receptors, and this could have implications toward the type of ligand that the γ:δ T-cell receptor recognizes, as there is enormous combinatorial diversity from the CDR3 within the γ:δ T-cell receptor repertoire. We will return to discuss

1	toward the type of ligand that the γ:δ T-cell receptor recognizes, as there is enormous combinatorial diversity from the CDR3 within the γ:δ T-cell receptor repertoire. We will return to discuss further the ligands and the development of γ:δ T cells in Chapters 6 and 8.

1	Summary.

1	The receptor for antigen on most T cells, the α:β T-cell receptor, is composed of two protein chains, TCRα and TCRβ, and resembles in many respects a single Fab fragment of immunoglobulin. α:β T-cell receptors are always membrane-bound and recognize a composite ligand of a peptide antigen bound to an MHC molecule. Each MHC molecule binds a wide variety of different peptides, but the different variants of MHC each preferentially recognize sets of peptides with particular sequences and physical features. The peptide antigen is generated intracellularly, and is bound stably in a peptide-binding cleft on the surface of the MHC molecule. There are two classes of MHC molecules, and these are bound in their nonpolymorphic domains by CD8 and CD4 molecules that distinguish two different functional classes of α:β T cells. CD8 binds MHC class I molecules and can bind simultaneously to the same peptide:MHC class I complex being recognized by a T-cell receptor, thus acting as a co-receptor and

1	classes of α:β T cells. CD8 binds MHC class I molecules and can bind simultaneously to the same peptide:MHC class I complex being recognized by a T-cell receptor, thus acting as a co-receptor and enhancing the T-cell response; CD4 binds MHC class II molecules and acts as a co-receptor for T-cell receptors that recognize peptide:MHC class II ligands. A T-cell receptor interacts directly both with the antigenic peptide

1	Fig. 4.31 Structures of γ:δ T-cell receptor bound to the nonclassical MHC class I molecule T22. The γ:δ T-cell receptor has a similar overall structure to the α:β T-cell receptor and the fab fragment of an immunoglobulin. The cδ domain is more like an immunoglobulin domain than is the corresponding cα domain of the α:β T-cell receptor. in this structure, the overall orientation of the γ:δ T-cell receptor with respect to the nonclassical Mhc molecule T22 is very different from the orientation of an α:β T-cell receptor with either Mhc class i or class ii molecules. Rather than lying directly over the peptide-binding groove, the γ:δ T-cell receptor is engaged with one end much more than the other; this is consistent with a lack of peptide contact and absence of Mhc-restricted recognition.

1	and with polymorphic features of the MHC molecule that displays it, and this dual specificity underlies the MHC restriction of T-cell responses. A second type of T-cell receptor, composed of a γand a δ chain, is structurally similar to the α:β T-cell receptor, but it binds to different ligands, including nonpeptide ligands, nonpolymorphic nonclassical MHC molecules, and certain lipids. The receptor is thought not to be MHC-restricted and is found on a minority population of lymphoid and intraepithelial T cells, the γ:δ T cells. Summary to Chapter 4.

1	Summary to Chapter 4. B cells and T cells use different, but structurally similar, molecules to recognize antigen. The antigen-recognition molecules of B cells are immunoglobulins, and are made both as a membrane-bound receptor for antigen, the B-cell receptor, and as secreted antibodies that bind antigens and elicit humoral effector functions. The antigen-recognition molecules of T cells, in contrast, are made only as cell-surface receptors and so elicit only cellular effector functions. Immunoglobulins and T-cell receptors are highly variable molecules, with the variability concentrated in that part of the molecule—the variable (V) region—that binds to antigen. Immunoglobulins bind a wide variety of chemically different antigens, whereas the major type of T-cell receptor, the α:β T-cell receptor, predominantly recognizes peptide fragments of foreign proteins bound to MHC molecules, which are ubiquitous on cell surfaces.

1	Binding of antigen by immunoglobulins has chiefly been studied with antibodies. The binding of antibody to its antigen is highly specific, and is determined by the shape and physicochemical properties of the antigen-binding site. Located at the other end of the antibody from the antigen-binding site is the constant, or Fc, region, which influences the types of effector function the antibody can elicit. There are five main functional classes of antibodies, each encoded by a different type of constant region. As we will see in Chapter 10, these interact with different components of the immune system to incite an inflammatory response and eliminate the antigen.

1	T-cell receptors differ in several respects from the B-cell immunoglobulins. One is the absence of a secreted form of T-cell receptor, reflecting the functional differences between T cells and B cells. B cells deal with pathogens and their protein products circulating within the body; secretion of a soluble antigen-recognition molecule enables the B cell to act in the clearance of antigen effectively throughout the extracellular spaces of the body. T cells, in contrast, are specialized for active surveillance of pathogens, and T-cell recognition does not involve a soluble, secreted receptor. Some, such as CD8 T cells, are able to detect intracellular infections and are able to kill infected cells that bear foreign antigenic peptides on their surface. Others, such as CD4 T cells, interact with cells of the immune system that have taken up foreign antigen and are displaying it on the cell surface.

1	T-cell receptors also recognize a composite ligand made up of the foreign peptide bound to a self MHC molecule, and not intact antigen. This means that T cells can interact only with a body cell displaying the antigen, not with the intact pathogen or protein. Each T-cell receptor is specific for a particular combination of peptide and a self MHC molecule. MHC molecules are encoded by a family of highly polymorphic genes. Expression of multiple variant MHC molecules, each with a different peptide-binding repertoire, helps to ensure that T cells from an individual will be able to recognize at least some peptides generated from nearly every pathogen. Questions. 4.1 True or False: An antibody proteolytically cleaved by papain yields a fragment with higher avidity to the cognate antigen than an antibody cleaved by pepsin. 4.2 Short Answer: how is cD4 and cD8 co-receptor binding to Mhc important for T-cell receptor signaling?

1	4.2 Short Answer: how is cD4 and cD8 co-receptor binding to Mhc important for T-cell receptor signaling? 4.3 Short Answer: Why and how is it advantageous to have heterozygosity in the Mhc locus? 4.4 Matching: Match the term to the best description: A. Antigenic determinant i. The structure recognized by an antibody (that is, the epitope) B. conformational/ ii. Regions of the V region C. continuous/linear iii. An epitope composed epitope of a single segment of a polypeptide chain

1	B. conformational/ ii. Regions of the V region C. continuous/linear iii. An epitope composed epitope of a single segment of a polypeptide chain D. hypervariable region iv. An epitope composed of amino acids from different parts of a polypeptide chain brought together by protein folding 4.5 Fill-in-the-Blanks: Most vertebrates, including humans and mice, produce antibodies composed of ________ and ________ chains. These bear ____ regions that recognize the antigen and ____ regions that dictate the antibody class and isotype. camelids and cartilaginous fish, however, produce ________________ and _______________, respectively, which are forming the basis for single-chain antibody production for clinical applications. 4.6 Multiple Choice: Which of the following statements is not true? A. T-cell receptor α and β chains pair together, but the α chain can be switched out for a γ or a δ chain. B. electrostatic interactions (for example, a salt bridge) occur between charged amino acids.

1	B. electrostatic interactions (for example, a salt bridge) occur between charged amino acids. C. hydrophobic interactions occur between two hydrophobic surfaces and exclude water. D. Antibodies often have many aromatic amino acids such as tyrosine in their antigen-binding sites. E. Mhc restriction is the phenomenon where T cells will recognize a unique set of peptides bound to a particular Mhc molecule. 4.7 Multiple Choice: Which of the following is the most abundant immunoglobulin class in healthy adult humans and mice? A. igA B. igD C. ige D. igg E. igM 4.8 Multiple Choice: Which of the following describes the structure of an immunoglobulin fold? A. Two antiparallel β sheets with an α-helical linker and a disulfide bond link B. Two β strands linked by a disulfide bond C. four α helices linked by two disulfide bonds D. Seven antiparallel α helices in series

1	B. Two β strands linked by a disulfide bond C. four α helices linked by two disulfide bonds D. Seven antiparallel α helices in series E. one β sandwich of two β sheets folded together and linked by a disulfide bond 4.9 Multiple Choice: Antibodies have flexibility at various points in the molecule, particularly the hinge region between the fc and fab portion and, to some extent, the junction between the V and c regions. Which of the following properties of an antibody are not affected by its flexibility? A. Binding to small antigens (haptens) B. Avidity to antigen C. Affinity to antigen D. interaction with antibody-binding proteins E. Binding to distantly spaced antigens 4.10 Multiple Choice: Which region of the antigen receptor of B cells and T cells is most critical in antigen recognition and specificity? A. fR1 B. cDR1 C. fR2 D. cDR2 E. fR3 F. cDR3 G. fR4 General references.

1	A. fR1 B. cDR1 C. fR2 D. cDR2 E. fR3 F. cDR3 G. fR4 General references. Garcia, K.C., Degano, M., Speir, J.A., and Wilson, I.A.: Emerging principles for T cell receptor recognition of antigen in cellular immunity. Rev. Immunogenet. 1999, 1:75–90. Garcia, K.C., Teyton, L., and Wilson, I.A.: Structural basis of T cell recognition. Annu. Rev. Immunol. 1999, 17:369–397. Moller, G. (ed): Origin of major histocompatibility complex diversity. Immunol. Rev. 1995, 143:5–292. Poljak, R.J.: Structure of antibodies and their complexes with antigens. Mol. Immunol. 1991, 28:1341–1345. Rudolph, M.G., Stanfield, R.L., and Wilson, I.A: How TCRs bind MHCs, peptides, and coreceptors. Annu. Rev. Immunol. 2006, 24:419–466. Sundberg, E.J., and Mariuzza, R.A.: Luxury accommodations: the expanding role of structural plasticity in protein-protein interactions. Structure 2000, 8:R137–R142. Section references. 4-1 IgG antibodies consist of four polypeptide chains.

1	Section references. 4-1 IgG antibodies consist of four polypeptide chains. Edelman, G.M.: Antibody structure and molecular immunology. Scand. J. Immunol. 1991, 34:4–22. Faber, C., Shan, L., Fan, Z., Guddat, L.W., Furebring, C., Ohlin, M., Borrebaeck, C.A.K., and Edmundson, A.B.: Three-dimensional structure of a human Fab with high affinity for tetanus toxoid. Immunotechnology 1998, 3:253–270. Harris, L.J., Larson, S.B., Hasel, K.W., Day, J., Greenwood,A., and McPherson,A.: The three-dimensional structure of an intact monoclonal antibody for canine lymphoma. Nature 1992, 360:369–372. Barclay, A.N., Brown, M.H., Law, S.K., McKnight, A.J., Tomlinson, M.G., and van der Merwe, P.A. (eds): The Leukocyte Antigen Factsbook, 2nd ed. London: Academic Press, 1997. Brummendorf, T., and Lemmon, V.: Immunoglobulin superfamily receptors: cis-interactions, intracellular adapters and alternative splicing regulate adhesion. Curr. Opin. Cell Biol. 2001, 13:611–618.

1	Brummendorf, T., and Lemmon, V.: Immunoglobulin superfamily receptors: cis-interactions, intracellular adapters and alternative splicing regulate adhesion. Curr. Opin. Cell Biol. 2001, 13:611–618. Marchalonis, J.J., Jensen, I., and Schluter, S.F.: Structural, antigenic and evolutionary analyses of immunoglobulins and T cell receptors. J. Mol. Recog. 2002, 15:260–271. Ramsland, P.A., and Farrugia, W.: Crystal structures of human antibodies: a detailed and unfinished tapestry of immunoglobulin gene products. J. Mol. Recog. 2002, 15:248–259. 4-4 The antibody molecule can readily be cleaved into functionally distinct fragments. Porter, R.R.: Structural studies of immunoglobulins. Scand. J. Immunol. 1991, 34:382–389. Yamaguchi, Y., Kim, H., Kato, K., Masuda, K., Shimada, I., and Arata, Y.: Proteolytic fragmentation with high specificity of mouse IgG—mapping of proteolytic cleavage sites in the hinge region. J. Immunol. Methods. 1995, 181:259–267.

1	4-5 The hinge region of the immunoglobulin molecule allows flexibility in binding to multiple antigens. Gerstein, M., Lesk, A.M., and Chothia, C.: Structural mechanisms for domain movements in proteins. Biochemistry 1994, 33:6739–6749. Jimenez, R., Salazar, G., Baldridge, K.K., and Romesberg, F.E.: Flexibility and molecular recognition in the immune system. Proc. Natl Acad. Sci. USA 2003, 100:92–97. Saphire, E.O., Stanfield, R.L., Crispin, M.D., Parren, P.W., Rudd, P.M., Dwek, R.A., Burton, D.R., and Wilson, I.A.: Contrasting IgG structures reveal extreme asymmetry and flexibility. J. Mol. Biol. 2002, 319:9–18. 4-6 Localized regions of hypervariable sequence form the antigen-binding site. Chitarra, V., Alzari, P.M., Bentley, G.A., Bhat, T.N., Eiselé, J.-L., Houdusse, A., Lescar, J., Souchon, H., and Poljak, R.J.: Three-dimensional structure of a heteroclitic antigen-antibody cross-reaction complex. Proc. Natl Acad. Sci. USA 1993, 90:7711–7715.

1	Decanniere, K., Muyldermans, S., and Wyns, L.: Canonical antigen-binding loop structures in immunoglobulins: more structures, more canonical classes? J. Mol. Biol. 2000, 300:83–91. Gilliland, L.K., Norris, N.A., Marquardt, H., Tsu, T.T., Hayden, M.S., Neubauer, M.G., Yelton, D.E., Mittler, R.S., and Ledbetter, J.A.: Rapid and reliable cloning of antibody variable regions and generation of recombinant single-chain antibody fragments. Tissue Antigens 1996, 47:1–20. Johnson, G., and Wu, T.T.: Kabat Database and its applications: 30 years after the first variability plot. Nucleic Acids Res. 2000, 28:214–218. Wu, T.T., and Kabat, E.A.: An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J. Exp. Med. 1970, 132:211–250.

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1	A lymphocyte expresses many exact copies of a single antigen receptor that has a unique antigen-binding site (see Section 1-12). The clonal expression of antigen receptors means that each lymphocyte is unique among the billions of lymphocytes that each person possesses. Chapter 4 described the structural features of immunoglobulins and T-cell receptors, the antigen receptors on B cells and T cells, respectively. We saw that the vast repertoire of antigen receptors results from variations in the amino acid sequence at the antigen-binding site, which is composed of the two variable regions from the two chains of the receptor. In immunoglobulins, these are the heavy-chain variable region (VH) and the light-chain variable region (VL), and in T-cell receptors, the Vα and Vβ regions. The immunoglobulin domains of these regions contain three loops that comprise three hypervariable regions, or complementarity-determining regions (CDRs) (see Section 4-6) that determine the receptor’s antigen

1	domains of these regions contain three loops that comprise three hypervariable regions, or complementarity-determining regions (CDRs) (see Section 4-6) that determine the receptor’s antigen binding site and allow for seemingly limitless diversity in specificity.

1	In the 1960s and 1970s, immunologists recognized that the limited size of the genome (at roughly 3 billion nucleotides) meant that the genome could not directly encode a sufficient number of genes to account for the observed diversity of antigen receptors. For example, encoding each distinct antibody by its own gene could easily fill the genome with nothing but antibody genes. As we will see, variable regions of the receptor chains are not directly encoded as a complete immunoglobulin domain by a single DNA segment. Instead, the variable regions are initially specified by so-called gene segments that encode only a part of the immunoglobulin domain. During the development of each lymphocyte, these gene segments are rearranged by a process of somatic DNA recombination to form a complete and unique variable-region coding sequence. This process is known generally as gene rearrangement. A fully assembled variable region sequence is produced by combining two or three types of gene segments,

1	unique variable-region coding sequence. This process is known generally as gene rearrangement. A fully assembled variable region sequence is produced by combining two or three types of gene segments, each of which is present in multiple copies in the germline genome. The final diversity of the receptor repertoire is the result of assembling complete antigen receptors from the many different gene segments of each type during the development of each individual lymphocyte. This process gives each new lymphocyte only one of many possible combinations of antigen receptors, providing the repertoire of diverse antigen specificities of naive B cells and T cells.

1	The first and second parts of this chapter describe the gene rearrangements that generate the primary repertoire of immunoglobulins and T-cell receptors. The mechanism of gene rearrangement is common to both B cells and T cells, and its evolution was probably critical to the evolution of the vertebrate adaptive immune system. The third part of the chapter explains how the transition from production of transmembrane immunoglobulins by activated B cells results in the production of secreted antibodies by plasma cells. Immunoglobulins can be synthesized as either transmembrane receptors or secreted antibodies, unlike T-cell receptors, which exist only as trans-membrane receptors. Antibodies can also be produced with different types of constant regions, or isotypes (see Section 4-1). Here, we describe how the Primary immunoglobulin gene rearrangement. T-cell receptor gene rearrangement. Structural variation in immunoglobulin constant regions.

1	Primary immunoglobulin gene rearrangement. T-cell receptor gene rearrangement. Structural variation in immunoglobulin constant regions. Evolution of the adaptive immune response. Fig. 5.1 Three hypervariable regions are encoded within a single V-region exon. Panel a: the variable region is based on the immunoglobulin (Ig) fold that is supported by framework regions (yellow) composed of nine β sheets and contains three hypervariable (HV) regions (red) that determine its antigen specificity. Panel b: the three HV regions exist as loops of amino acids between the β sheets of B and C, between Cʹ and Cʹʹ, and between F and G. Panel c: a complete variable region in a lymphocyte is encoded within a single exon of the full antigen-receptor gene. The three HV regions are interspersed between four framework regions (FRs) made up of the β sheets of the Ig domain.

1	expression of the isotypes IgM and IgD is regulated, but we postpone describing how isotype switching occurs until Chapter 10, since that process and the affinity maturation of antibodies occurs normally in the context of an immune response. The last part of this chapter briefly examines alternative evolutionary forms of gene rearrangements that give rise to different forms of adaptive immunity in other species. Primary immunoglobulin gene rearrangement.

1	Primary immunoglobulin gene rearrangement. Virtually any substance can be the target of an antibody response, and the response to even a single epitope comprises many different antibody molecules, each with a subtly different specificity for the epitope and a unique affinity, or binding strength. The total number of antibody specificities available to an individual is known as the antibody repertoire or immunoglobulin repertoire, and in humans is at least 1011 and probably several orders of magnitude greater. The number of antibody specificities present at any one time is, however, limited by the total number of B cells in an individual, as well as by each individual’s previous encounters with antigens.

1	Before it was possible to examine the immunoglobulin genes directly, there were two main hypotheses for the origin of this diversity. The germline theory held that there is a separate gene for each different immunoglobulin chain and that the antibody repertoire is largely inherited. In contrast, somatic diversification theories proposed that the observed repertoire is generated from a limited number of inherited V-region sequences that undergo alteration within B cells during the individual’s lifetime. Cloning of the immunoglobulin genes revealed that elements of both theories were correct and that the DNA sequence encoding each variable region is generated by rearrangements of a relatively small group of inherited gene segments. Diversity is further enhanced by the process of somatic hypermutation in mature activated B cells. Thus, the somatic diversification theory was essentially correct, although the germline theory concept of the existence of multiple germline genes also proved

1	in mature activated B cells. Thus, the somatic diversification theory was essentially correct, although the germline theory concept of the existence of multiple germline genes also proved true.

1	5-1 Immunoglobulin genes are rearranged in the progenitors of antibody-producing cells.

1	Figure 5.1 shows the relationships between a light-chain variable region’s antigen-binding site, its domain structure, and the gene that encodes it. The variable regions of immunoglobulin heavy and light chains are based on the immunoglobulin fold, which is composed of nine β sheets. The anti-body-binding site is formed by three loops of amino acids known as hyper-variable regions HV1, HV2, and HV3, or also CDR1, CDR2, and CDR3 (see Fig. 5.1a). These loops are located between the pairs of β sheets B and C, Cʹ and Cʹʹ, and F and G (see Fig. 5.1b). In a mature B cell, the variable regions for heavy and light chains are encoded by a single exon, but are separated from one another within this coding sequence (see Fig. 5.1c). This exon is the gene’s second exon (exon 2). The first exon of the variable regions encodes the antibody’s leader sequence, which directs the antibody into the endoplasmic reticulum for surface expression or secretion.

1	Unlike most genes, the complete DNA sequence of the variable-region exon is not present in the germline of the individual, but is originally encoded by two separate DNA segments, as illustrated in Fig. 5.2. These two DNA segments are spliced together to form the complete exon 2 as the B cell develops in the bone marrow. The first 95–101 amino acids of the variable region, encoding βsheets A–F and the first two complete hypervariable regions, originate from a

1	Fig. 5.2 The CDR3 region originates from two or more individual gene segments that are joined during lymphocyte development. Panel a: a complete light-chain variable region encoding the CDR1, CDR2, and CDR3 loops resides in a single exon. Panel b: the complete variable region is derived from distinct germline DNA sequences. A V gene segment encodes the CDR1 and CDR2 loops, and the CDR3 loop is formed by sequences from the end of the V gene segment and the beginning of the J gene segment, and by nucleotides added or lost when these gene segments are joined during lymphocyte development. The exon for the CDR3 loop of the heavy chain is formed by the joining of sequences from V, D, and J gene segments (not shown).

1	variable or V gene segment (see Fig. 5.2). This segment also contributes part of the third hypervariable region. Other parts of the third hypervariable region, and the remainder of the variable region including β sheet G (up to 13 amino acids), originate from a joining or J gene segment. By convention, we will refer to the exon encoding the complete variable region formed by the splicing together of these gene segments as the V-region gene.

1	In nonlymphoid cells, the V-region gene segments remain in their original germline configuration, and are a considerable distance away from the sequence encoding the C region. In mature B lymphocytes, however, the assembled V-region sequence lies much closer to the C region, as a consequence of a splicing event of the gene’s DNA. Rearrangement within the immunoglobulin genes was originally discovered almost 40 years ago, when the techniques of restriction enzyme analysis first made it possible to study the organization of the immunoglobulin genes in both B cells and nonlymphoid cells. Such experiments showed that segments of genomic DNA within the immunoglobulin genes are rearranged in cells of the B-lymphocyte lineage, but not in other cells. This process of rearrangement is known as ‘somatic’ DNA recombination to distinguish it from the meiotic recombination that takes place during the production of gametes.

1	5-2 Complete genes that encode a variable region are generated by the somatic recombination of separate gene segments. The rearrangements that produce the complete immunoglobulin light-chain and heavy-chain genes are shown in Fig. 5.3. For the light chain, the joining of a VL and a JL gene segment creates an exon that encodes the whole light-chain VL region. In the unrearranged DNA, the VL gene segments are located relatively far away from the exons encoding the constant region of the light chain (CL region). The JL gene segments are located close to the CL region, however, and the joining of a VL gene segment to a JL gene segment also brings the VL gene segment close to a CL-region sequence. The JL gene segment of the rearranged VL region is separated from a CL-region sequence only by a short intron. To make a complete immunoglobulin light-chain messenger RNA, the V-region exon is joined to the C-region sequence by RNA splicing after transcription.

1	For the heavy-chain, there is one additional complication. The heavy-chain V region (VH) is encoded in three gene segments, rather than two. In addition to the V and J gene segments (denoted VH and JH to distinguish them from the light-chain VL and JL), the heavy chain uses a third gene segment called the diversity or DH gene segment, which lies between the VH and JH gene segments. The recombination process that generates a complete heavy-chain V region is shown in Fig. 5.3 (right panel), and occurs in two separate stages. First, a DH gene segment is joined to a JH gene segment; then a VH gene segment rearranges to DJH to make a complete VH-region exon. As with the light-chain genes, RNA splicing joins the assembled V-region sequence to the neighboring C-region gene.

1	Fig. 5.3 V-region genes are constructed from gene segments. Heavy-chain V regions are constructed from three gene segments Light-chain V-region genes are constructed from two segments (right panel). First, the diversity (D) and J gene segments join, and (center panel). A variable (V) and a joining (J) gene segment in the then the V gene segment joins to the combined DJ sequence, genomic DNA are joined to form a complete light-chain V-region forming a complete VH exon. A heavy-chain C-region gene is exon. Immunoglobulin chains are extracellular proteins, and the encoded by several exons. The C-region exons, together with the V gene segment is preceded by an exon encoding a leader peptide leader sequence, are spliced to the V-domain sequence during (L), which directs the protein into the cell’s secretory pathways processing of the heavy-chain RNA transcript. The leader sequence and is then cleaved. The light-chain C region is encoded in a is removed after translation, and the disulfide

1	secretory pathways processing of the heavy-chain RNA transcript. The leader sequence and is then cleaved. The light-chain C region is encoded in a is removed after translation, and the disulfide bonds that link the separate exon and is joined to the V-region exon by splicing of polypeptide chains are formed. The hinge region is shown in purple. the light-chain RNA to remove the L-to-V and the J-to-C introns.

1	5-3 Multiple contiguous V gene segments are present at each immunoglobulin locus.

1	For simplicity we have discussed the formation of a complete V-region sequence as though there were only a single copy of each gene segment. In fact, there are multiple copies of the V, D, and J gene segments in germline DNA. It is the random selection of just one gene segment of each type that produces the great diversity of V regions among immunoglobulins. The numbers of functional gene segments of each type in the human genome, as determined by gene cloning and sequencing, are shown in Fig. 5.4. Not all the gene segments discovered are functional, as some have accumulated mutations that prevent them from encoding a functional protein. Such genes are termed ‘pseudogenes.’ Because there are many V, D, and J gene segments in germline DNA, no single gene segment is essential, resulting in a relatively large number of pseudogenes. Since some of these can undergo rearrangement just like a functional gene segment, a significant proportion of rearrangements incorporate a pseudogene and

1	in a relatively large number of pseudogenes. Since some of these can undergo rearrangement just like a functional gene segment, a significant proportion of rearrangements incorporate a pseudogene and will thus be nonfunctional.

1	We saw in Section 4-1 that there are three sets of immunoglobulin chains— the heavy chain, and two equivalent types of light chains, the κ and λ chains. The immunoglobulin gene segments that encode these chains are organized into three clusters or genetic loci—the κ, λ, and heavy-chain loci—each of which can assemble a complete V-region sequence. Each locus is on a different chromosome and is organized slightly differently, as shown for the human loci in Fig. 5.5. At the λ light-chain locus, located on human chromosome 22, a cluster of Vλ gene segments is followed by four (or in some individuals five) sets of Jλ gene segments each linked to a single Cλ gene. In the κ light-chain locus, on chromosome 2, the cluster of Vκ gene segments is followed by a cluster of Jκ gene segments, and then by a single Cκ gene.

1	The organization of the heavy-chain locus, on chromosome 14, contains separate clusters of VH, DH, and JH gene segments and of CH genes. The heavy-chain locus differs in one important way: instead of a single C region, it contains a series of C regions arrayed one after the other, each of which corresponds to a different immunoglobulin isotype (see Fig. 5.19). While the Cλ locus contains several distinct C regions, these encode similar proteins, which function similarly, whereas the different heavy-chain isotypes are structurally quite distinct and have different functions.

1	B cells initially express the heavy-chain isotypes μ and δ (see Section 4-1), which is accomplished by alternative mRNA splicing and which leads to the expression of immunoglobulins IgM and IgD, as we shall see in Section 5-14. The expression of other isotypes, such as γ (giving IgG), occurs through DNA rearrangements referred to as class switching, and takes place at a later stage, after a B cell is activated by antigen in an immune response. We describe class switching in Chapter 10. The human V gene segments can be grouped into families in which each member shares at least 80% DNA sequence identity with all others in the

1	The human V gene segments can be grouped into families in which each member shares at least 80% DNA sequence identity with all others in the Fig. 5.4 The number of functional gene segments for the V regions of human heavy and light chains. The numbers shown are derived from exhaustive cloning and sequencing of DNA from one individual and exclude all pseudogenes (mutated and nonfunctional versions of a gene sequence). As a result of genetic polymorphism, the numbers will not be the same for all people.

1	Fig. 5.5 The germline organization of the immunoglobulin heavyand light-chain loci in the human genome. Depending on the individual, the genetic locus for the λ light chain (chromosome 22) has between 29 and 33 functional Vλ gene segments and four or five pairs of functional Jλ gene segments and Cλ genes. The κ locus (chromosome 2) is organized in a similar way, with about 38 functional Vκ gene segments accompanied by a cluster of five Jκ gene segments but with a single Cκ gene. In approximately 50% of individuals, the entire cluster of Vκ gene segments has undergone an increase by duplication (not shown, for simplicity). The heavy-chain locus (chromosome 14) has about 40 functional VH gene segments and a cluster of around 23 DH segments lying between these VH gene segments and 6 JH gene segments. The heavy-chain locus also contains a large cluster of CH genes (see Fig. 5.19). For simplicity, all V gene segments have been shown in the same chromosomal orientation; only the first

1	gene segments. The heavy-chain locus also contains a large cluster of CH genes (see Fig. 5.19). For simplicity, all V gene segments have been shown in the same chromosomal orientation; only the first CH gene (for Cμ) is shown, without illustrating its separate exons; and all pseudogenes have been omitted. This diagram is not to scale: the total length of the heavy-chain locus is more than 2 megabases (2 million bases), whereas some of the D gene segments are only 6 bases long.

1	Fig. 5.6 Recombination signal sequences are conserved heptamer and nonamer sequences that flank the gene segments encoding the V, D, and J regions of immunoglobulins.

1	Recombination signal sequences (RSSs) are composed of heptamer (CACAGTG) and nonamer (ACAAAAACC) sequences that are separated by either 12 bp or approximately 23 bp of nucleotides. The heptamer–12-bp spacer–nonamer motif is depicted here as an orange arrowhead; the motif that includes the 23-bp spacer is depicted as a purple arrowhead. Joining of gene segments almost always involves a 12-bp and a 23-bp RSS—the 12/23 rule. The arrangement of RSSs in the V (red), D (green), and J (yellow) gene segments of heavy (H) and light (λ and κ) chains of immunoglobulin is shown here. The RAG-1 recombinase (see Section 5-5) cuts the DNA precisely between the last nucleotide of the V gene segment and the first C of the heptamer; or between the last G of the heptamer and the first nucleotide of the D or J gene segment. Note that according to the 12/23 rule, the arrangement of RSSs in the immunoglobulin heavy-chain gene segments precludes direct V-to-J joining.

1	family. Both the heavy-chain and the κ-chain V gene segments can be subdivided into seven families, and there are eight families of Vλ gene segments. The families can be grouped into clans, made up of families that are more similar to each other than to families in other clans. Human VH gene segments fall into three clans. All the VH gene segments identified from amphibians, reptiles, and mammals also fall into the same three clans, suggesting that these clans existed in a common ancestor of these modern animal groups. Thus, the V gene segments that we see today have arisen by a series of gene duplications and diversification through evolutionary time. 5-4 Rearrangement of V, D, and J gene segments is guided by flanking DNA sequences.

1	For a complete immunoglobulin or T-cell receptor chain to be expressed, DNA rearrangements must take place at the correct locations relative to the V, D, or J gene segment coding regions. In addition, these DNA rearrangements must be regulated such that a V gene segment is joined to a D or a J and not joined to another V gene segment. DNA rearrangements are guided by conserved noncoding DNA sequences, called recombination signal sequences (RSSs), that are found adjacent to the points at which recombination takes place. The structure and arrangements of the RSSs are shown in Fig. 5.6 for the λ and κ light-chain loci and the heavy-chain loci. An RSS consists of a conserved block of seven nucleotides—the heptamer 5ʹCACAGTG3ʹ, which is always contiguous with the coding sequence; followed by a nonconserved region known as the spacer, which is either 12 or 23 base pairs (bp) long; followed by a second conserved block of nine nucleotides, the nonamer 5ʹACAAAAACC3ʹ.

1	The sequences given here are the consensus sequences, but they can vary substantially from one gene segment to another, even in the same individual, as there is some flexibility in the recognition of these sequences by the enzymes that carry out the recombination. The spacers vary in sequence, but their conserved lengths correspond to one turn (12 bp) or two turns (23 bp) of the DNA double helix. This is thought to bring the heptamer and nonamer sequences to the same side of the DNA helix to allow interactions with proteins that catalyze recombination, but this concept still lacks structural proof. The heptamer– spacer–nonamer sequence motif—the RSS—is always found directly adjacent to the coding sequence of V, D, or J gene segments. Recombination normally occurs between gene segments located on the same chromosome. A gene segment flanked by an RSS with a 12-bp spacer typically can be joined only to one flanked by a 23-bp spacer RSS. This is known as the 12/23 rule.

1	It is important to recognize that the pattern of 12and 23-bp spacers used by the various gene segments is different between the λ, κ, and heavy-chain loci (see Fig. 5.6). Thus, for the heavy chain, a DH gene segment can be joined to a JH gene segment and a VH gene segment to a DH gene segment, but VH gene segments cannot be joined to JH gene segments directly, as both VH and JH gene segments are flanked by 23-bp spacers. However, they can be joined with a DH gene segment between them, as DH segments have 12-bp spacers on both sides (see Fig. 5.6).

1	In the antigen-binding region of an immunoglobulin, CDR1 and CDR2 are encoded directly in the V gene segment (see Fig. 5.2). CDR3 is encoded by the additional DNA sequence that is created by the joining of the V and J gene segments for the light chain, and the V, D, and J gene segments for the heavy chain. Further diversity in the antibody repertoire can be supplied by CDR3 regions that result from the joining of one D gene segment to another D gene segment, before being joined by a J gene segment. Such D–D joining is infrequent and seems to violate the 12/23 rule, suggesting that such violations of the 12/23 rule can occur at low frequency. In humans, D–D joining is found in approximately 5% of antibodies and is the major mechanism accounting for the unusually long CDR3 loops found in some heavy chains.

1	The mechanism of DNA rearrangement is similar for the heavyand light-chain loci, although only one joining event is needed to generate a light-chain gene but two are required for a heavy-chain gene. When two gene segments are in the same transcriptional orientation in the germline DNA, their rearrangement involves the looping out and deletion of the DNA between them (Fig. 5.7, left panels). By contrast, when the gene segments have opposite transcriptional orientations, the rearrangement retains the intervening DNA in the chromosome but with an inverted orientation (see Fig. 5.7, right panels). This mode of recombination is less common, but it accounts for about half of all Vκ to Jκ joins in humans because the orientation of half the Vκ gene segments is opposite to that of the Jκ gene segments. 5-5 The reaction that recombines V, D, and J gene segments modifying enzymes.

1	The overall enzymatic mechanisms involved in V-region rearrangement, or V(D)J recombination, are illustrated in Fig. 5.8. Two RSSs are brought together by interactions between proteins that specifically recognize the length of the spacers and thus enforce the 12/23 rule for recombination. The DNA molecule is then precisely cleaved by endonuclease activity at two locations and is then rejoined in a different configuration. The ends of the heptamer sequences are joined in a head-to-head fashion to form a signal joint. In the majority of cases, no nucleotides are lost or added between the two heptamer sequences, creating a double-heptamer sequence 5ʹCACAGTGCACAGTG3ʹ within the DNA molecule. When the joining segments are in the same orientation, the signal joint is contained in a circular piece of extrachromosomal DNA (see Fig. 5.7, left panels), which is lost from the genome when the cell divides. The V and J gene segments, which remain on the chromosome, join to form what is called the

1	of extrachromosomal DNA (see Fig. 5.7, left panels), which is lost from the genome when the cell divides. The V and J gene segments, which remain on the chromosome, join to form what is called the coding joint. When the joining segments are in the opposite relative orientation to each other within the chromosome (see Fig. 5.7, right panels), the signal joint is also retained within the chromosome, and the region of DNA between the V gene segment and the RSS of the J gene segment is inverted to form the coding joint. This situation leads to rearrangement by inversion. As we shall see later, the coding joint junction is imprecise, meaning that nucleotides can be added or lost between joined segments during the rearrangement process. This imprecise nature of coding joint formation adds to the variability in the V-region sequence, called junctional diversity.

1	MOVIE 5.1

1	Fig. 5.7 V-region gene segments are joined by recombination. Top panel: in every V-region recombination event, the recombination signal sequences (RSSs) flanking the gene segments are brought together to allow recombination to take place. The 12-bp-spaced RSSs are shown in orange, the 23-bp-spaced RSSs in purple. For simplicity, the recombination of a light-chain gene is illustrated; for a heavy-chain gene, two separate recombination events are required to generate a functional V region. Left panels: in most cases, the two segments undergoing rearrangement (the V and J gene segments in this example) are arranged in the same transcriptional orientation in the chromosome, and juxtaposition of the RSSs results in the looping out of the intervening DNA. Recombination occurs at the ends of the heptamer sequences in the RSSs, creating the so-called signal joint and releasing the intervening DNA in the form of a closed circle. Subsequently, the joining of the V and J gene segments creates

1	heptamer sequences in the RSSs, creating the so-called signal joint and releasing the intervening DNA in the form of a closed circle. Subsequently, the joining of the V and J gene segments creates the coding joint in the chromosomal DNA. Right panels: in other cases, the V and J gene segments are initially oriented in opposite transcriptional directions. In this case, alignment of the RSSs requires the coiled configuration shown, rather than a simple loop, so that joining the ends of the two heptamer sequences now results in the inversion and integration of the intervening DNA into a new position on the chromosome. Again, the joining of the V and J segments creates a functional V-region exon.

1	direction of transcription L2 V2 Vn Ln L2 V2 L1 V1 JL1 V1 direction of transcription JLn Vn deleted inverted signal joint coding joint After recombination this loop is excised from the chromosome, taking the two RSS regions with it After recombination the coiled region is retained in the chromosome in an inverted orientation L1 V1 L2 L2 V2 V2 23 23 23 12 L2 V2 Vn Vn Ln Ln J J L1 V1 J Ln Vn L1 V1 V gene segments may be in either forward or reverse transcriptional orientation relative to downstream gene segments When a forward-oriented V gene segment recombines with a downstream gene segment, alignment of the two RSS regions loops out the intervening DNA When a reverse-oriented V gene segment recombines with a downstream gene segment, alignment of the RSS regions forms the intervening DNA into a coiled conÿguration

1	The complex of enzymes that act in concert to carry out somatic V(D)J recombination is termed the V(D)J recombinase. The lymphoid-specific components of the recombinase are called RAG-1 and RAG-2, and they are encoded by two recombination-activating genes, RAG1 and RAG2. This pair of genes is essential for V(D)J recombination, and they are expressed in developing lymphocytes only while the lymphocytes are engaged in assembling their antigen receptors, as described in more detail in Chapter 8. Indeed, the RAG genes expressed together can confer on nonlymphoid cells such as fibroblasts the capacity to rearrange exogenous segments of DNA containing the appropriate RSSs; this is how RAG-1 and RAG-2 were initially discovered.

1	DNA ligase IV:XRCC4 ligates DNA ends Ku70:Ku80 binds DNA ends Precise signal joint DNA ligase:XRCC4 Signal joints Ku80 Ku70 5´-phosphorylated blunt ends TdT processes DNA ends Ku70:Ku80 binds DNA ends DNA ligase IV:XRCC4 ligates DNA ends terminal deoxynucleotidyl transferase (TdT) DNA ligase:XRCC4 1223 JV RAG-1:2 binds RSS Synapsis of two RSSs Germline confguration Cleavage of RSSs Coding joints DNA-PK:Artemis opens hairpin RAG-1/2 Ku70 Ku80 Artemis DNA-PK covalently closed DNA hairpin ends Imprecise coding joint Fig. 5.8 Enzymatic steps in RAG-dependent V(D)J rearrangement.

1	Recombination of gene segments containing recombination signal sequences (RSSs, triangles) begins with the binding of a complex of RAG-1 (purple), RAG-2 (blue), and high-mobility-group (HMG) proteins (not shown) to one of the RSSs flanking the coding sequences to be joined (second row). The RAG complex then recruits the other RSS. In the cleavage step, the endonuclease activity of RAG makes single-stranded cuts in the DNA backbone precisely between each coding segment and its RSS. At each cutting point this creates a 3ʹ-OH group, which then reacts with a phosphodiester bond on the opposite DNA strand to generate a hairpin, leaving a blunt double-stranded break at the end of the RSS. These two types of DNA ends are resolved in different ways. At the coding ends (left panels), essential repair proteins such as Ku70:Ku80 (green) bind to the hairpin. Ku70:80 forms a ringlike structure as a heterodimer, but the monomers do not encircle the DNA. The DNA-PK:Artemis complex (purple) then

1	repair proteins such as Ku70:Ku80 (green) bind to the hairpin. Ku70:80 forms a ringlike structure as a heterodimer, but the monomers do not encircle the DNA. The DNA-PK:Artemis complex (purple) then joins the complex, and its endonuclease activity opens the DNA hairpin at a random site, yielding either two flush-ended DNA strands or a single-strand extension. The cut end is then modified by terminal deoxynucleotidyl transferase (TdT, pink) and exonuclease, which randomly add and remove nucleotides, respectively (this step is shown in more detail in Fig. 5.11). The two coding ends are finally ligated by DNA ligase IV in association with XRCC4 (turquoise). At the signal ends (right panels), Ku70:Ku80 binds to the RSS but the ends are not further modified. Instead, a complex of DNA ligase IV:XRCC4 joins the two ends precisely to form the signal joint.

1	The other proteins in the recombinase complex are members of the ubiquitously expressed nonhomologous end joining (NHEJ) pathway of DNA repair known as double-strand break repair (DSBR). In all cells, this process is responsible for rejoining the two ends at the site of a double-strand break in DNA. The DSBR joining process is imprecise, meaning that nucleotides are frequently gained or lost at the site of joining. This has evolutionary relevance as in most cells it would not be advantageous to gain or lose nucleotides when repairing DSBs. However, in lymphocytes, the imprecise nature of DSBR is critical for junctional diversity and adaptive immunity. Thus, this may be the driving pressure for NHEJ to mediate imprecise joining. One ubiquitous protein contributing to DSBR is Ku, which is a heterodimer (Ku70:Ku80); this forms a ring around the DNA and associates tightly with a protein kinase catalytic subunit, DNA-PKcs, to form the DNA-dependent protein kinase (DNA-PK). Another protein

1	heterodimer (Ku70:Ku80); this forms a ring around the DNA and associates tightly with a protein kinase catalytic subunit, DNA-PKcs, to form the DNA-dependent protein kinase (DNA-PK). Another protein that associates with DNA-PKcs is Artemis, which has nuclease activity. The DNA ends are finally joined together by the enzyme DNA ligase IV, which forms a complex with the DNA repair protein XRCC4. DNA polymerases μ and λ participate in DNA-end fill-in synthesis. In addition, polymerase μ can add nucleotides in a template-independent manner. In summary, lymphocytes have adapted several enzymes used in common DNA repair pathways to help complete the process of somatic V(D)J recombination that is initiated by the RAG-1 and RAG-2 V(D)J recombinases.

1	The first reaction is an endonucleolytic cleavage that requires the coordinated activity of both RAG proteins. Initially, a complex of RAG-1 and RAG-2 proteins, together with high-mobility group chromatin protein HMGB1 or HMGB2, recognizes and aligns the two RSSs that are the target of the cleavage reaction. RAG-1 operates as a dimer, with RAG-2 acting as a cofactor (Fig. 5.9). RAG-1 specifically recognizes and binds the heptamer and the nonamer of the RSS and contains the Zn2+-dependent endonuclease activity of the RAG protein complex. As a dimer, RAG-1 seems to align the two RSSs that will undergo rearrangement. Recent models suggest that the 12/23 rule may be established because an essential asymmetric orientation of the RAG-1:RAG-2 complex induces a preference for binding to RSS elements of different types (Fig. 5.10). The bound RAG complex makes a single-strand DNA break at the nucleotide just 5ʹ of the heptamer of the RSS, thus creating a free 3ʹ-OH group at the end of the

1	elements of different types (Fig. 5.10). The bound RAG complex makes a single-strand DNA break at the nucleotide just 5ʹ of the heptamer of the RSS, thus creating a free 3ʹ-OH group at the end of the coding segment. This nucleophilic 3ʹ-OH group immediately attacks the phosphodiester bond on the opposite DNA strand, making a double-strand break and creating a DNA ‘hairpin’ at the coding region and a flush double-strand break at the end of the heptamer sequence. This cutting process occurs twice, once for the each gene segment being joined, producing four ends: two hairpin ends at the coding regions and two flush ends at both heptamer sequences (see Fig. 5.8). These DNA ends do not float apart, however, but are held tightly in the complex until a joining step has been completed. The blunt ends of the heptamer sequence are precisely joined by a complex of DNA ligase IV and XRCC4 to form the signal joint.

1	Formation of the coding joint is more complex. The two coding hairpin ends are each bound by Ku, which recruits the DNA-PKcs subunit. Artemis is recruited into this complex and is phosphorylated by DNA-PK. Artemis then opens the DNA hairpins by making a single-strand nick in the DNA. This nicking can happen at various points along the hairpin, which leads to sequence variability in the final joint. The DNA repair enzymes in the complex modify the opened Shown as ribbon diagrams, the RAG-1:RAG-2 complex contains two RAG-1 (green and blue) and two RAG-2 proteins (purple). The first 383 amino acids of RAG-1 were truncated before crystallization. The N-terminal nonamer binding domain (NBD) of the two RAG-1 proteins undergoes domain swapping and mediates dimerization of the two proteins. The remainder of the RAG-1 protein contains the endonuclease activity that is dependent on the binding of a Zn2+ ion. Each RAG-1 protein binds a separate RAG-2 protein. Courtesy of Martin Gellert.

1	Zn2+ Zn2+ J region V region heptamer nonamer 23-bp RSS 12-bp RSS A 12-bp RSS bound to one RAG-1 favors binding of a 23-bp RSS to the other RAG-1 Fig. 5.10 The 12/23 base pair rule may result from asymmetric binding of RSSs to the RAG-1:RAG-2 dimer.

1	hairpins by removing nucleotides, while at the same time the lymphoidspecific enzyme terminal deoxynucleotidyl transferase (TdT), which is also part of the recombinase complex, adds nucleotides randomly to the single-strand ends. Addition and deletion of nucleotides can occur in any order, and one does not necessarily precede the other. Finally, DNA ligase IV joins the processed ends together, thus reconstituting a chromosome that includes the rearranged gene. This repair process creates diversity in the joint between gene segments while ensuring that the RSS ends are ligated without modification and that unintended genetic damage such as a chromosome break is avoided. Despite the use of some ubiquitous mechanisms of DNA repair, adaptive immunity based on the RAG-mediated generation of antigen receptors by somatic recombination seems to be unique to the jawed vertebrates, and its evolution is discussed in the last part of this chapter.

1	The in vivo roles of the enzymes involved in V(D)J recombination have been established through both natural and artificially induced mutations. Mice lacking TdT have about 10% of the normal level of non-templated nucleotides added to the joints between gene segments. This small remainder may result from the template-independent activity of DNA polymerase μ.

1	Mice in which either of the RAG genes has been inactivated, or which lack DNA-PKcs, Ku, or Artemis, suffer a complete block in lymphocyte development at the gene-rearrangement stage or make only trivial numbers of B and T cells. They are said to suffer from severe combined immune deficiency (SCID). The original scid mutation was discovered some time before the components of the recombination pathway were identified and was subsequently identified as a mutation in DNA-PKcs. In humans, mutations in RAG1 or RAG2 that result in partial V(D)J recombinase activity are responsible for an inherited disorder called Omenn syndrome, which is characterized by an absence of circulating B cells and an infiltration of skin by activated oligoclonal T lymphocytes. Mice deficient in components of ubiquitous DNA repair pathways, such as DNA-PKcs, Ku, or Artemis, are defective in double-strand break repair in general and are therefore also hypersensitive to ionizing radiation (which produces

1	of ubiquitous DNA repair pathways, such as DNA-PKcs, Ku, or Artemis, are defective in double-strand break repair in general and are therefore also hypersensitive to ionizing radiation (which produces double-strand breaks). Defects in Artemis in humans produce a combined immunodeficiency of B and T cells that is associated with increased radiosensitivity. SCID caused by mutations in DNA repair pathways is called irradiation-sensitive SCID (IR-SCID) to distinguish it from SCID due to lymphocyte-specific defects.

1	Another genetic condition in which radiosensitivity is associated with some degree of immunodeficiency is ataxia telangiectasia, which is due to mutations in the protein kinase ATM (ataxia telangiectasia mutated), which are also associated with cerebellar degeneration and increased radiation sensitivity

1	Left panel: This cartoon of the structure shown in Fig 5.9 illustrates the flexibility of the hinge connecting the NBD to the catalytic domain of RAG-1. Right panel: the NBD domain of RAG-1 interacts with the RSS nonamer sequence (blue), while the RSS heptamer sequence (red) is bound to the portion of RAG-1 that contains the Zn2+ endonuclease activity. In this cartoon model, the interaction of a 12-bp RSS with one of the RAG-1 subunits induces the NBD domain to rotate toward the catalytic domain of RAG-1, to accommodate the length of the RSS. Since the two NBD domains are coupled by domain swaps, this induced conformation pulls the other NBD away from its RAG-1 subunit, which then prefers binding of the 23-bp RSS. The endonucleolytic cleavage (arrows) of the DNA by RAG-1 occurs precisely at the junction between the heptamer and the respective V, D, or J gene segment.

1	and cancer risk. ATM is a serine/threonine kinase, like DNA-PKcs, and functions during V(D)J recombination by activating pathways that prevent the chromosomal translocations and large DNA deletions that can sometimes occur during resolution of DNA double-strand breaks. Some V(D)J recombination can occur in the absence of ATM, since the immune deficiencies seen in ataxia telangiectasia, which include low numbers of B and T cells and/or a deficiency in antibody class switching, are variable in their severity and are less severe than in SCID. Evidence that ATM and DNA-PKcs are partially redundant in their functions comes from the observation that B cells lacking both kinases show much more severely abnormal signal joining sequences compared with B cells lacking either enzyme alone. 5-6 The diversity of the immunoglobulin repertoire is generated by four main processes.

1	The gene rearrangements that combine gene segments to form a complete V-region exon generate diversity in two ways. First, there are multiple different copies of each type of gene segment, and different combinations of gene segments can be used in different rearrangement events. This combinatorial diversity is responsible for a substantial part of the diversity of V regions. Second, junctional diversity is introduced at the joints between the different gene segments as a result of the addition and subtraction of nucleotides by the recombination process. A third source of diversity is also combinatorial, arising from the many possible different combinations of heavyand light-chain V regions that pair to form the antigen-binding site in the immunoglobulin molecule. The two means of generating combinatorial diversity alone could give rise, in theory, to approximately 1.9 × 106 different antibody molecules, as we will see below. Coupled with junctional diversity, it is estimated that at

1	combinatorial diversity alone could give rise, in theory, to approximately 1.9 × 106 different antibody molecules, as we will see below. Coupled with junctional diversity, it is estimated that at least 1011 different receptors could make up the repertoire of receptors expressed by naive B cells, and diversity could even be several orders of magnitude greater, depending on how one calculates junctional diversity. Finally, somatic hypermutation, which we describe in Chapter 10, occurs only in B cells after the initiation of an immune response and introduces point mutations into the rearranged V-region genes. This process generates further diversity in the antibody repertoire that can be selected for enhanced binding to antigen.

1	5-7 The multiple inherited gene segments are used in different combinations.

1	There are multiple copies of the V, D, and J gene segments, each of which can contribute to an immunoglobulin V region. Many different V regions can therefore be made by selecting different combinations of these segments. For human κ light chains, there are approximately 40 functional Vκ gene segments and 5 Jκ gene segments, and thus potentially 200 different combinations of complete Vκ regions. For λ light chains there are approximately 30 functional Vλ gene segments and 4 to 5 Jλ gene segments, yielding at least 120 possible Vλ regions (see Fig. 5.4). So, in all, 320 different light chains can be made as a result of combining different light-chain gene segments. For the heavy chains of humans, there are 40 functional VH gene segments, approximately 25 DH gene segments, and 6 JH gene segments, and thus around 6000 different possible VH regions (40 ×25 ×6 = 6000). During B-cell development, rearrangement at the heavy-chain gene locus to produce a heavy chain is followed by several

1	segments, and thus around 6000 different possible VH regions (40 ×25 ×6 = 6000). During B-cell development, rearrangement at the heavy-chain gene locus to produce a heavy chain is followed by several rounds of cell division before light-chain gene rearrangement takes place, resulting in the same heavy chain being paired with different light chains in different cells. Because both the heavyand the light-chain V regions contribute to antibody specificity, each of the 320 different light chains could be combined with each of the approximately 6000 heavy chains to give around 1.9 × 106 different antibody specificities.

1	This theoretical estimate of combinatorial diversity is based on the number of germline V gene segments contributing to functional antibodies (see Fig. 5.4); the total number of V gene segments is larger, but the additional gene segments are pseudogenes and do not appear in expressed immunoglobulin molecules. In practice, combinatorial diversity is likely to be less than one might expect from the calculations above. One reason is that not all V gene segments are used at the same frequency; some are common in antibodies, while others are found only rarely. This bias for or against certain V gene segments relates to their proximity with intergenic control regions within the heavy-chain locus that activate V(D)J recombination in developing B cells. Also, not every heavy chain can pair with every light chain: certain combinations of VH and VL regions will not form a stable molecule. Cells in which heavy and light chains fail to pair may undergo further light-chain gene rearrangement until

1	light chain: certain combinations of VH and VL regions will not form a stable molecule. Cells in which heavy and light chains fail to pair may undergo further light-chain gene rearrangement until a suitable chain is produced or they will be eliminated. Nevertheless, it is thought that most heavy and light chains can pair with each other, and that this type of combinatorial diversity has a major role in forming an immunoglobulin repertoire with a wide range of specificities.

1	5-8 Variable addition and subtraction of nucleotides at the junctions between gene segments contributes to the diversity of the third hypervariable region. As noted earlier, of the three hypervariable loops in an immunoglobulin chain, CDR1 and CDR2 are encoded within the V gene segment. CDR3, however, falls at the joint between the V gene segment and the J gene segment, and in the heavy chain it is partly encoded by the D gene segment. In both heavy and light chains, the diversity of CDR3 is significantly increased by the addition and deletion of nucleotides at two steps in the formation of the junctions between gene segments. The added nucleotides are known as P-nucleotides and N-nucleotides, and their addition is illustrated in Fig. 5.11.

1	P-nucleotides are so called because they make up palindromic sequences added to the ends of the gene segments. As described in Section 5-5, the RAG proteins generate DNA hairpins at the coding ends of the V, D, or J segments, after which Artemis catalyzes a single-stranded cleavage at a random point within the coding sequence but near where the hairpin was first formed. When this cleavage occurs at a different point from the initial break induced by the RAG1/2 complex, a single-stranded tail is formed from a few nucleotides of the coding sequence plus the complementary nucleotides from the other DNA

1	Fig. 5.11 The introduction of Pand N-nucleotides diversifies the joints between gene segments during immunoglobulin gene rearrangement. The process is illustrated for a DH to JH rearrangement (first panel); however, the same steps occur in VH to DH and in VL to JL rearrangements. After formation of the DNA hairpins (second panel), the two heptamer sequences are ligated to form the signal joint (not shown here), while the Artemis:DNA-PK complex cleaves the DNA hairpin at a random site (indicated by the arrows) to yield a single-stranded DNA end (third panel). Depending on the site of cleavage, this single-stranded DNA may contain nucleotides that were originally complementary in the double-stranded DNA and which therefore form short DNA palindromes, such as TCGA and ATAT, as indicated by the light blue-shaded box. For example, the sequence GA at the end of the D segment shown is complementary to the preceding sequence TC. Such stretches of nucleotides that originate from the

1	by the light blue-shaded box. For example, the sequence GA at the end of the D segment shown is complementary to the preceding sequence TC. Such stretches of nucleotides that originate from the complementary strand are known as P-nucleotides. Where the enzyme terminal deoxynucleotidyl transferase (TdT) is present, nucleotides are added at random to the ends of the single-stranded segments (fourth panel); these nontemplated, or N, nucleotides are indicated by the shaded box. The two single-stranded ends then pair (fifth panel). Exonuclease trimming of unpaired nucleotides (sixth panel) and repair of the coding joint by DNA synthesis and ligation (bottom panel) leaves both Pand N-nucleotides (indicated by light blue shading) in the final coding joint. The randomness of insertion of Pand N-nucleotides makes an individual P–N region virtually unique and a valuable marker for following an individual B-cell clone as it develops, for instance in studies of somatic hypermutation.

1	TCGCCGGCAGATATATATGCGCATATPPNDJThe gaps are ÿlled by DNA synthesis and ligation to form coding joint TCCGGCAATGCTTAADJUnpaired nucleotides are removed by an exonuclease TCCGGCTAAATGCTTAAAATGCTCCGGCTATTAAATGCTTAADJPairing of strands DJN-nucleotide additions by TdT JDArtemis:DNA-PK complex opens DNA hairpins, generating palindromic P-nucleotides ATATATGCJDRAG complex generates DNA hairpin at coding ends RSSs brought together ATATATGCGCGCATATATGCGCGCGCATATATGCGCJD strand (see Fig. 5.11). In many light-chain gene rearrangements, DNA repair enzymes then fill in complementary nucleotides on the single-stranded tails, which would leave short palindromic sequences (the P-nucleotides) at the joint if the ends were rejoined without any further exonuclease activity.

1	In heavy-chain gene rearrangements and in a proportion of human light-chain gene rearrangements, however, N-nucleotides are added by a quite different mechanism before the ends are rejoined. N-nucleotides are so called because they are non-template-encoded. They are added by the enzyme TdT to the single-stranded ends of the coding DNA after hairpin cleavage. After the addition of up to 20 nucleotides, single-stranded stretches may have some complementary base pairs. Repair enzymes then trim off nonmatching nucleotides, synthesize complementary DNA to fill in the remaining single-stranded gaps, and ligate the new DNA to the palindromic region (see Fig. 5.11). TdT is maximally expressed during the period in B-cell development when the heavy-chain gene is being assembled, and so N-nucleotides are common in heavy-chain V–D and D–J junctions. N-nucleotides are less common in light-chain genes, which undergo rearrangement after heavy-chain genes, when TdT expression has been shut off, as we

1	are common in heavy-chain V–D and D–J junctions. N-nucleotides are less common in light-chain genes, which undergo rearrangement after heavy-chain genes, when TdT expression has been shut off, as we will explain further in Chapter 8 when discussing the specific developmental stages of B and T cells.

1	Nucleotides can also be deleted at gene segment junctions. This is accomplished by exonucleases, and although these have not yet been identified, Artemis has dual endonuclease and exonuclease activity and so could well be involved in this step. Thus, a heavy-chain CDR3 can be shorter than even the smallest D segment. In some instances it is difficult, if not impossible, to recognize the D segment that contributed to CDR3 formation because of the excision of most of its nucleotides. Deletions may also erase the traces of P-nucleotide palindromes introduced at the time of hairpin opening. For this reason, many completed VDJ joins do not show obvious evidence of P-nucleotides. As the total number of nucleotides added by these processes is random, the added nucleotides often disrupt the reading frame of the coding sequence beyond the joint. Such frameshifts will lead to a nonfunctional protein, and DNA rearrangements leading to such disruptions are known as nonproductive rearrangements.

1	frame of the coding sequence beyond the joint. Such frameshifts will lead to a nonfunctional protein, and DNA rearrangements leading to such disruptions are known as nonproductive rearrangements. As roughly two in every three rearrangements will be nonproductive, many B-cell progenitors never succeed in producing functional immunoglobulin and therefore never become mature B cells. Thus, junctional diversity is achieved only at the expense of considerable loss of cells during B-cell development. In Chapter 8, we return to this topic when we discuss the cellular stages of B-cell development and how they relate to the temporal sequence of rearrangement of the V, D, and J gene segments of the antigen receptor chains.

1	Summary.

1	The extraordinary diversity of the immunoglobulin repertoire is achieved in several ways. Perhaps the most important factor enabling this diversity is that V regions are encoded by separate gene segments (V, D, and J gene segments), which are brought together by a somatic recombination process— V(D)J recombination—to produce a complete V-region exon. Many different gene segments are present in the genome of an individual, thus providing a heritable source of diversity that this combinatorial mechanism can use. Unique lymphocyte-specific recombinases, the RAG proteins, are absolutely required to catalyze this rearrangement, and the evolution of RAG proteins coincided with the appearance of the modern vertebrate adaptive immune system. Another substantial fraction of the functional diversity of immunoglobulins comes from the imprecise nature of the joining process itself. Variability at the coding joints between gene segments is generated by the insertion of random numbers of Pand

1	of immunoglobulins comes from the imprecise nature of the joining process itself. Variability at the coding joints between gene segments is generated by the insertion of random numbers of Pand N-nucleotides and by the variable deletion of nucleotides at the ends of some segments. These are brought about by the random opening of the hairpin by Artemis and by the actions of TdT. The association of different lightand heavy-chain V regions to form the antigen-binding site of an immunoglobulin molecule contributes further diversity. The combination of all of these sources of diversity generates a vast primary repertoire of antibody specificities.

1	T-cell receptor gene rearrangement. The mechanism by which B-cell antigen receptors are generated is such a powerful means of creating diversity that it is not surprising that the antigen receptors of T cells bear structural resemblances to immunoglobulins and are generated by the same mechanism. In this part of the chapter we describe the organization of the T-cell receptor loci and the generation of the genes for the individual T-cell receptor chains. 5-9 The T-cell receptor gene segments are arranged in a similar pattern to immunoglobulin gene segments and are rearranged by the same enzymes.

1	Like immunoglobulin light and heavy chains, T-cell receptor (TCR) α and β chains each consist of a variable (V) amino-terminal region and a constant (C) region (see Section 4-10). The organization of the TCRα and TCRβ loci is shown in Fig. 5.12. The organization of the gene segments is broadly homologous to that of the immunoglobulin gene segments (see Sections 5-2 and 5-3). The TCRα locus, like the loci of the immunoglobulin light chains, contains V and J gene segments (Vα and Jα). The TCRβ locus, like the locus of the immunoglobulin heavy chain, contains D gene segments in addition to Vβ and Jβ gene segments. The T-cell receptor gene segments rearrange during T-cell development to form complete V-domain exons (Fig. 5.13). T-cell receptor gene rearrangement takes place in the thymus; the order and regulation of the rearrangements are dealt with in detail in Chapter 8. Essentially, however, the mechanics of gene rearrangement are similar for B and T cells. The T-cell receptor gene

1	the order and regulation of the rearrangements are dealt with in detail in Chapter 8. Essentially, however, the mechanics of gene rearrangement are similar for B and T cells. The T-cell receptor gene segments are flanked by 12-bp and 23-bp spacer recombination signal sequences (RSSs) that are homologous to those flanking immunoglobulin

1	Fig. 5.12 The germline organization of the human T-cell receptor α and β loci.

1	The arrangement of the gene segments for the T-cell receptor resembles that at the immunoglobulin loci, with separate variable (V), diversity (D), and joining (J) gene segments, and constant (C) genes. The TCRα locus (chromosome 14) consists of 70–80 Vα gene segments, each preceded by an exon encoding the leader sequence (L). How many of these Vα gene segments are functional is not known exactly. A cluster of 61 Jα gene segments is located a considerable distance from the Vα gene segments. The Jα gene segments are followed by a single C gene, which contains separate exons for the constant and hinge domains and a single exon encoding the transmembrane and cytoplasmic regions (not shown). The TCRβ locus (chromosome 7) has a different organization, with a cluster of 52 functional Vβ gene segments located distant from two separate clusters that each contain a single D gene segment together with six or seven J gene segments and a single C gene. Each TCRβ C gene has separate exons encoding

1	located distant from two separate clusters that each contain a single D gene segment together with six or seven J gene segments and a single C gene. Each TCRβ C gene has separate exons encoding the constant domain, the hinge, the transmembrane region, and the cytoplasmic region (not shown). The TCRα locus is interrupted between the J and V gene segments by another T-cell receptor locus—the TCRδ locus (not shown here; see Fig. 5.17).

1	The TCRαand β-chain genes are composed of discrete segments that are joined by somatic recombination during development of the T cell. Functional αand β-chain genes are generated in the same way that complete immunoglobulin genes are created. For the α chain (upper part of figure), a Vα gene segment rearranges to a Jα gene segment to create a functional V-region exon. Transcription and splicing of the VJα exon to Cα generates the mRNA that is translated to yield the T-cell receptor α-chain protein. For the β chain (lower part of figure), like the immunoglobulin heavy chain, the variable domain is encoded in three gene segments, Vβ, Dβ, and Jβ. Rearrangement of these gene segments generates a functional VDJβ V-region exon that is transcribed and spliced to join to Cβ; the resulting mRNA is translated to yield the T-cell receptor β chain. The α and β chains pair soon after their synthesis to yield the α:β T-cell receptor heterodimer. Not all J gene segments are shown, and the leader

1	is translated to yield the T-cell receptor β chain. The α and β chains pair soon after their synthesis to yield the α:β T-cell receptor heterodimer. Not all J gene segments are shown, and the leader sequences preceding each V gene segment are omitted for simplicity.

1	Fig. 5.14 Recombination signal sequences flank T-cell receptor gene segments. As in the immunoglobulin gene loci (see Fig. 5.6), the individual gene segments at the TCRα and TCRβ loci are flanked by heptamer–spacer–nonamer recombination signal sequences (RSSs). RSS motifs containing 12-bp spacers are depicted here as orange arrowheads, and those containing 23-bp spacers are shown in purple. Joining of gene segments almost always follows the 12/23 rule. Because of the disposition of heptamer and nonamer RSSs in the TCRβ and TCRδ loci, direct Vβ to Jβ joining is in principle allowed by the 12/23 rule (unlike in the immunoglobulin heavy-chain gene), although this occurs very rarely owing to other types of regulation. Fig. 5.13 T-cell receptor αand β-chain gene rearrangement and expression.

1	gene segments (Fig. 5.14; see Section 5-4) and are recognized by the same enzymes. The DNA circles resulting from gene rearrangement (see Fig. 5.7) are known as T-cell receptor excision circles (TRECs) and are used as markers for T cells that have recently emigrated from the thymus. All known defects in genes that control V(D)J recombination affect T cells and B cells equally, and animals with these genetic defects lack functional B and T lymphocytes altogether (see Section 5-5). A further shared feature of immunoglobulin and T-cell receptor gene rearrangement is the presence of Pand N-nucleotides in the junctions between the V, D, and J gene segments of the rearranged TCRβ gene. In T cells, Pand N-nucleotides are also added between the V and J gene segments of all rearranged TCRα genes, whereas only about half of the V–J joints in immunoglobulin light-chain genes are modified by N-nucleotide addition, and these are often left without any P-nucleotides as well (Fig. 5.15; see Section

1	whereas only about half of the V–J joints in immunoglobulin light-chain genes are modified by N-nucleotide addition, and these are often left without any P-nucleotides as well (Fig. 5.15; see Section 5-8).

1	The main differences between the immunoglobulin genes and those encoding T-cell receptors reflect the differences in how B cells and T cells function. All the effector functions of B cells depend upon secreted antibodies whose different heavy-chain C-region isotypes trigger distinct effector mechanisms. The effector functions of T cells, in contrast, depend upon cell–cell contact and are not mediated directly by the T-cell receptor, which serves only for antigen Fig. 5.15 The number of human T-cell receptor gene segments and the sources of T-cell receptor diversity compared with those of immunoglobulins. Note that only about half of human κ chains contain N-nucleotides. Somatic hypermutation as a source of diversity is not included in this figure because it does not occur in T cells.

1	recognition. Thus, the C regions of the TCRα and TCRβ loci are much simpler than those of the immunoglobulin heavy-chain locus. There is only one Cα gene, and although there are two Cβ genes, they are very closely homologous and there is no known functional distinction between their products. The T-cell receptor C-region genes encode only transmembrane polypeptides.

1	Another difference between the rearrangement of immunoglobulin genes and T-cell receptor genes is in the nature of the RSSs surrounding the D gene segments. For the immunoglobulin heavy chain, the D segment is surrounded by two RSSs, both with a 12-bp spacing (see Fig. 5.6), whereas the D segments in the TCRβ and TCRγ loci have a 5ʹ 12-bp RSS and a 3ʹ 23-bp RSS (see Fig. 5.14). The arrangement in the immunoglobulin locus naturally enforces the inclusion of D segments in the heavy-chain V region, since direct V to J joining would violate the 12/23 rule. However, in the T-cell receptor loci, direct V to J joining would not violate this rule, since the 23-bp RSS of the Vβ or Vγ segment is compatible with the 12-bp RSS of the J gene segment, and yet normally, little to no such direct joining is observed. Instead, regulation of gene rearrangements appears to be controlled by mechanisms beyond the 12/23 rule, and these mechanisms are still being investigated.

1	5-10 T-cell receptors concentrate diversity in the third hypervariable region.

1	The three-dimensional structure of the antigen-recognition site of a T-cell receptor looks much like that of the antigen-recognition site of an antibody molecule (see Sections 4-10 and 4-7, respectively). In an antibody, the center of the antigen-binding site is formed by the CDR3 loops of the heavy and light chains. The structurally equivalent third hypervariable loops of the T-cell receptor α and β chains, to which the D and J gene segments contribute, also form the center of the antigen-binding site of a T-cell receptor; the periphery of the site consists of the CDR1 and CDR2 loops, which are encoded within the germline V gene segments for the αand β chains. The extent and pattern of variability in T-cell receptors and immunoglobulins reflect the distinct nature of their ligands. Whereas the antigen-binding sites of immunoglobulins must conform to the surfaces of an almost infinite variety of different antigens, and thus come in a wide variety of shapes and chemical properties, the

1	the antigen-binding sites of immunoglobulins must conform to the surfaces of an almost infinite variety of different antigens, and thus come in a wide variety of shapes and chemical properties, the ligand for the major class of human T-cell receptors (α:β) is always a peptide bound to an MHC molecule. As a group, the antigen-recognition sites of T-cell receptors

1	Fig. 5.16 The most variable parts of the T-cell receptor interact with the peptide of a peptide:MHC complex. The CDR loops of a T-cell receptor are shown as colored tubes, which in this figure are superimposed on the peptide:MHC complex (MHC, gray; peptide, yellow-green with O atoms in red and N atoms in blue). The CDR loops of the α chain are in green, while those of the β chain are in magenta. The CDR3 loops lie in the center of the interface between the T-cell receptor and the peptide:MHC complex, and make direct contact with the antigenic peptide. would therefore be predicted to have a less variable shape, with most of the variability focused on the bound antigenic peptide occupying the center of the surface in contact with the receptor. Indeed, the less variable CDR1 and CDR2 loops of a T-cell receptor mainly contact the relatively less variable MHC component of the ligand, whereas the highly variable CDR3 regions mainly contact the unique peptide component (Fig. 5.16).

1	The structural diversity of T-cell receptors is attributable mainly to combinatorial and junctional diversity generated during the process of gene rearrangement. It can be seen from Fig. 5.15 that most of the variability in T-cell receptor chains is in the junctional regions, which are encoded by V, D, and J gene segments and modified by Pand N-nucleotides. The TCRα locus contains many more J gene segments than either of the immunoglobulin light-chain loci: in humans, 61 Jα gene segments are distributed over about 80 kb of DNA, whereas immunoglobulin light-chain loci have only 5 J gene segments at most (see Fig. 5.15). Because the TCRαlocus has so many J gene segments, the variability generated in this region is even greater for T-cell receptors than for immunoglobulins. Thus, most of the diversity resides in the CDR3 loops that contain the junctional region and form the center of the antigen-binding site. 5-11 γ:δ T-cell receptors are also generated by gene rearrangement.

1	5-11 γ:δ T-cell receptors are also generated by gene rearrangement. A minority of T cells bear T-cell receptors composed of γ and δ chains (see Section 4-20). The organization of the TCRγ and TCRδ loci (Fig. 5.17) resembles that of the TCRα and TCRβ loci, although there are important differences. The cluster of gene segments encoding the δ chain is found entirely within the TCRα locus, between the Vα and the Jα gene segments. Vδ genes are

1	Fig. 5.17 The organization of the T-cell receptor γand δ-chain loci in humans. The TCRγ and TCRδ loci, like the TCRα and TCRβ loci, have discrete V, D, and J gene segments, and C genes. Uniquely, the locus encoding the δ chain is located entirely within the α-chain locus. The three Dδ gene segments, four Jδ gene segments, and the single δ C gene lie between the cluster of Vα gene segments and the cluster of Jα gene segments. There are two Vδ gene segments (not shown) located near the δ C gene, one just upstream of the D regions and one in inverted orientation just downstream of the C gene. In addition, there are six Vδ gene segments interspersed among the Vα gene segments. Five are shared with Vα and can be used by either locus, and one is unique to the δ locus. The human TCRγ locus resembles the TCRβ locus in having two C genes, each with its own set of J gene segments. The mouse γ locus (not shown) has a more complex organization and there are three functional clusters of γ gene

1	the TCRβ locus in having two C genes, each with its own set of J gene segments. The mouse γ locus (not shown) has a more complex organization and there are three functional clusters of γ gene segments, each containing V and J gene segments and a C gene. Rearrangement at the γ and δ loci proceeds as for the other T-cell receptor loci, with the exception that during TCRδ rearrangement two D segments can be used in the same gene. The use of two D segments greatly increases the variability of the δ chain, mainly because extra N-region nucleotides can be added at the junction between the two D gene segments as well as at the V–D and D–J junctions.

1	interspersed with the Vα genes but are located primarily in the 3ʹ region of the locus. Because all Vα gene segments are oriented such that rearrangement will delete the intervening DNA, any rearrangement at the α locus results in the loss of the δ locus (Fig. 5.18). There are substantially fewer V gene segments at the TCRγ and TCRδ loci than at either the TCRα or TCRβ loci or any of the immunoglobulin loci. Increased junctional variability in the δ chains may compensate for the small number of V gene segments and has the effect of focusing almost all the variability in the γ:δreceptor in the junctional region. As we have seen for the α:β T-cell receptors, the amino acids encoded by the junctional regions lie at the center of the T-cell receptor binding site. T cells bearing γ:δ receptors are a distinct lineage of T cells, and as discussed in Chapter 4, some γ:δ T cells recognize nonclassical MHC class I molecules and other molecules whose expression may be an indication of cellular

1	are a distinct lineage of T cells, and as discussed in Chapter 4, some γ:δ T cells recognize nonclassical MHC class I molecules and other molecules whose expression may be an indication of cellular damage or infection. As we saw in Section 4-20, the CDR3 of a γ:δ T cell is frequently longer than the CDR3 in an α:β T-cell receptor; this permits the CDR of γ:δ T cell receptors to interact directly with ligand and also contributes to the great diversity of these receptors. We will discuss the regulation of the fate choice between the α:βand γ:δ T-cell lineages in Chapter 8.

1	Summary. T-cell receptors are structurally similar to immunoglobulins and are encoded by homologous genes. T-cell receptor genes are assembled by somatic recombination from sets of gene segments in the same way that the immunoglobulin genes are. Diversity is, however, distributed differently in immunoglobulins and T-cell receptors: the T-cell receptor loci have roughly the same number of V gene segments as the immunoglobulin loci but more J gene segments, and there is greater diversification of the junctions between gene segments during the process of gene rearrangement. Thus, the greatest diversity of the T-cell receptor is in the central part of the receptor, within the CDR3, which in the case of α:β T-cell receptors contacts the bound peptide fragment of the ligand. Most of the diversity among γ:δ T-cell receptors is also within the CDR3, which is frequently longer than the CDR3 of α:β T-cell receptors and can also directly interact with ligands recognized by the γ:δ T cells.

1	Structural variation in immunoglobulin constant regions.

1	This chapter so far has focused on the mechanisms of assembly of the V regions for immunoglobulins and T-cell receptors. We now turn to the C regions. The C regions of T-cell receptors act only to support the V regions and anchor the receptor into the membrane, and they do not vary after assembly of a complete receptor gene. Immunoglobulins, in contrast, can be made as both a trans-membrane receptor and a secreted antibody, and they can be made in several different classes, depending on the different C regions used by the heavy chain. The light-chain C regions (CL) provide only structural attachment for V regions, and there seem to be no functional differences between λand κ light chains. The heavy-chain locus encodes different C regions (CH) that are present as separate genes located downstream of the V-region segments. Initially, naive B cells use only the first two of these, the Cμ and Cδ genes, which are expressed along with the associated assembled V-region sequence to produce

1	of the V-region segments. Initially, naive B cells use only the first two of these, the Cμ and Cδ genes, which are expressed along with the associated assembled V-region sequence to produce transmembrane IgM and IgD on the surface of the naive B cell.

1	In this section, we introduce the different heavy-chain isotypes and discuss some of their special properties as well as the structural features that distinguish the CH regions of antibodies of the five major classes. We explain how Fig. 5.18 Deletion of the TCRδ locus is induced by rearrangement of a Vα to Jα gene segment. The TCRδ locus is entirely contained within the chromosomal region containing the TCRα locus. When any V region in the Vα/Vδ region rearranges to any one of the Jα segments, the intervening region, and the entire Vδ locus, is deleted. Thus, Vα rearrangement prevents any continued expression of a Vδ gene and precludes lineage development down the γ:δ pathway.

1	naive B cells express both Cμ and Cδ isotypes at the same time and how the same antibody gene can generate both membrane-bound immunoglobulin and secreted immunoglobulin through alternative mRNA splicing. During an antibody response, activated B cells can switch to the expression of CH genes other than Cμ and Cδ by a type of somatic recombination known as class switching (discussed in Chapter 10) that links different heavy-chain C regions (CH) to the rearranged VDJH gene segment. 5-12 Different classes of immunoglobulins are distinguished by the structure of their heavy-chain constant regions.

1	5-12 Different classes of immunoglobulins are distinguished by the structure of their heavy-chain constant regions. The five main classes of immunoglobulins are IgM, IgD, IgG, IgE, and IgA, all of which can occur as transmembrane antigen receptors or secreted antibodies (Fig. 5.19). In humans, IgG is found as four subclasses (IgG1, IgG2, IgG3, and IgG4), named by decreasing order of their abundance in serum, and IgA antibodies are found as two subclasses (IgA1 and IgA2). The different heavy chains that define these classes are known as isotypes and are designated by the lowercase Greek letters μ, δ, γ, ε, and α. The different heavy chains are encoded by different immunoglobulin CH genes located in a gene cluster that is 3ʹ of the JH segments as illustrated in Fig. 5.19. Figure 5.20 lists the major physical and functional properties of the different human antibody classes.

1	The functions of the immunoglobulin classes are discussed in detail in Chapter 10, in the context of the humoral immune response; here, we just touch on them briefly. IgM is the first class of immunoglobulin produced after activation of a B cell, and the IgM antibody is secreted as a pentamer (see Section 5-14 and Fig. 5.21). This accounts for the high molecular weight of IgM and the fact that it is normally present in the bloodstream but not in tissues. Being a pentamer

1	The general structure of the main immunoglobulin isotypes (above in upper panel) is indicated, with each rectangle denoting an immunoglobulin domain. These isotypes are encoded by separate heavy-chain C-region genes arranged in a cluster in both mouse and human (lower panel). The constant region of the heavy chain for each isotype is indicated by the same color as the C-region gene segment that encodes it. IgM and IgE lack a hinge region but each contains an extra heavy-chain domain. Note the differences in the number and location of the disulfide bonds (black lines) linking the chains. The isotypes also differ in the distribution of N-linked carbohydrate groups, shown as hexagons. In humans, the gene cluster shows evidence of evolutionary duplication of a unit consisting of two γ genes, an ε gene, and an α gene. One of the ε genes is a pseudogene (ψ); hence only one subtype of IgE is expressed. For simplicity, other pseudogenes are not illustrated, and the exon details within each C

1	an ε gene, and an α gene. One of the ε genes is a pseudogene (ψ); hence only one subtype of IgE is expressed. For simplicity, other pseudogenes are not illustrated, and the exon details within each C gene are not shown. The classes of immunoglobulins found in mice are called IgM, IgD, IgG1, IgG2a, IgG2b, IgG3, IgA, and IgE.

1	Fig. 5.20 The physical and functional properties of the human immunoglobulin isotypes. IgM is so called because of its size: although monomeric IgM is only 190 kDa, it normally forms pentamers, known as macroglobulin (hence the M), of very large molecular weight (see Fig. 5.23). IgA dimerizes to give an approximate molecular weight of around 390 kDa in secretions. IgE antibody is associated with immediate-type hypersensitivity. When fixed to tissue mast cells, IgE has a much longer half-life than its half-life in plasma shown here. The relative activities of the various isotypes are compared for several functions, ranging from inactive (–) to most active (++++). also increases the avidity of IgM for antigens before its affinity is increased through the process of affinity maturation.

1	also increases the avidity of IgM for antigens before its affinity is increased through the process of affinity maturation. IgG isotypes produced during an immune response are found in the bloodstream and in the extracellular spaces in tissues. IgM and most IgG isotypes can interact with the complement component C1 to activate the classical complement pathway (described in Section 2-7). IgA and IgE do not activate complement. IgA can be found in the bloodstream, but it also acts in the defense of mucosal surfaces; it is secreted into the gut and respiratory tract, and also into mother’s milk. IgE is particularly involved in defense against multicellular parasites (for example, schistosomes), but it is also the antibody involved in common allergic diseases such as allergic asthma. IgG and IgE are always monomers, but IgA can be secreted as either a monomer or a dimer.

1	Sequence differences in the constant regions of the immunoglobulin heavy chains produce the distinct characteristics of each antibody isotype. These characteristics include the number and location of interchain disulfide bonds, the number of attached carbohydrate groups, the number of C domains, and the length of the hinge region (see Fig. 5.19). IgM and IgE heavy chains contain an extra C domain that replaces the hinge region found in γ, δ, and α chains. The absence of the hinge region does not imply that IgM and IgE molecules lack flexibility; electron micrographs of IgM molecules binding to ligands show that the Fab arms can bend relative to the Fc portion. However, such a difference in structure may have functional consequences that are not yet characterized. Different isotypes and subtypes also differ in their ability to engage various effector functions, as described below. 5-13 The constant region confers functional specialization on the antibody.

1	5-13 The constant region confers functional specialization on the antibody. Antibodies can protect the body in a variety of ways. In some cases it is enough for the antibody simply to bind antigen. For instance, by binding tightly to a toxin or virus, an antibody can prevent it from recognizing its receptor on a host cell (see Fig. 1.25). The antibody V regions are sufficient for this activity. The C region is essential, however, for recruiting the help of other cells and molecules to destroy and dispose of pathogens to which the antibody has bound.

1	The Fc region contains all C regions of an antibody and has three main effector functions: Fc-receptor binding, complement activation, and regulation of secretion. First, the Fc region of certain isotypes binds to specialized Fc receptors expressed by immune effector cells. Fcγ receptors expressed on the surface of macrophages and neutrophils bind the Fc portions of IgG1 and IgG3 antibodies, facilitating the phagocytosis of pathogens coated with these antibodies. The Fc region of IgE binds to a high-affinity Fcε receptor on mast cells, basophils, and activated eosinophils, triggering the release of inflammatory mediators in response to antigens. We will return to this topic in Section 10-19.

1	Second, the Fc regions in antigen:antibody complexes can bind to the C1q complement protein (see Section 2-7) and initiate the classical complement cascade, which recruits and activates phagocytes to engulf and destroy pathogens. Third, the Fc portion can deliver antibodies to places they would not reach without active transport. These include transport of IgA into mucous secretions, tears, and milk, and the transfer of IgG from the pregnant mother into the fetal blood circulation. In both cases, the Fc portion of IgA or IgG engages a specific receptor, the neonatal Fc receptor (FcRn), that actively transports the immunoglobulin through cells to reach different body compartments. Podocytes in the kidney glomerulus express FcRn to help remove IgG that has been filtered from the blood and accumulated at the glomerular basement membrane.

1	The role of the Fc portion in these effector functions has been demonstrated by studying immunoglobulins that have had one or more Fc domains cleaved off enzymatically or modified genetically. Many microorganisms have responded to the destructive potential of the Fc portion by evolving proteins that either bind it or cleave it, and so prevent the Fc region from working; examples are Protein A and Protein G of Staphylococcus and Protein D of Haemophilus. Researchers have exploited these proteins to help map the Fc region and also as immunological reagents. Not all immunoglobulin classes have the same capacity to engage each of the effector functions (see Fig. 5.20). For example, IgG1 and IgG3 have a higher affinity than IgG2 for the most common type of Fc receptor. 5-14 IgM and IgD are derived from the same pre-mRNA transcript and are both expressed on the surface of mature B cells.

1	5-14 IgM and IgD are derived from the same pre-mRNA transcript and are both expressed on the surface of mature B cells. The immunoglobulin CH genes form a large cluster spanning about 200 kb to the 3ʹ side of the JH gene segments (see Fig. 5.19). Each CH gene is split into several exons (not shown in the figure), with each exon corresponding to an individual immunoglobulin domain in the folded C region. The gene encoding the μ C region lies closest to the JH gene segments, and therefore closest to the assembled VH-region exon (VDJ exon) after DNA rearrangement. Once rearrangement is completed, transcription from a promoter just 5ʹ to the rearranged VDJ exon produces a complete μ heavy-chain transcript. Any JH gene segments remaining between the assembled V gene and the Cμ gene are removed during RNA processing to generate the mature mRNA. The μ heavy chains are therefore the first to be expressed, and IgM is the first immunoglobulin to be produced during B-cell development.

1	Immediately 3ʹ to the μ gene lies the δ gene, which encodes the C region of the IgD heavy chain (see Fig. 5.19). IgD is coexpressed with IgM on the surface of almost all mature B cells, but is secreted in only small amounts by plasma cells. The unique function of IgD is still unclear and a matter of active research. Because IgD has hinge regions that are more flexible than those in IgM, IgD has been suggested to be an auxiliary receptor that may facilitate the binding of antigens by naive B cells. Mice lacking the Cδ exons show normal B-cell development and can generate largely normal antibody responses, but show a delay in the process of affinity maturation of antibody for antigens. We return to this topic in Chapter 10, when we discuss somatic hypermutation.

1	B cells expressing IgM and IgD have not undergone class switching, which requires irreversible changes to the DNA. Instead, these B cells produce a long primary mRNA transcript that is differentially spliced to yield either of two distinct mRNA molecules (Fig. 5.21). In one transcript, the VDJ exon is spliced to the Cμ exons and undergoes polyadenylation from a nearby site (pA1), to encode a complete IgM molecule. The second RNA transcript extends well beyond this site and includes the downstream Cδ exons. In this transcript, the VDJ exon is spliced to these Cδ exons and polyadenylation occurs at a separate site downstream (pA2). This transcript encodes an IgD molecule.

1	It has been known since the 1980s that the processing of the long mRNA transcript is developmentally regulated, with immature B cells making mostly the μ transcript and mature B cells making mostly the δ along with some μ, although until recently there was little to no molecular explanation. A recent forward genetic screen of N-ethyl-N-nitrosourea (ENU)-induced mutagenesis in mice identified a gene involved in IgD expression that regulates the alternative splicing process. The gene encodes ZFP318, a protein structurally related to the U1 small nuclear ribonucleoprotein of the spliceosome, the RNA– protein complex that is required for mRNA splicing. ZFP318 is not expressed in immature B cells, where the IgD transcript is not produced, but becomes expressed in mature and activated B cells that coexpress IgD with IgM. ZFP318 is required for alternative splicing of the long pre-mRNA from the VDJ exon to the Cδ exons, as mice with a fully inactivated ZFP318 gene fail to express IgD and

1	that coexpress IgD with IgM. ZFP318 is required for alternative splicing of the long pre-mRNA from the VDJ exon to the Cδ exons, as mice with a fully inactivated ZFP318 gene fail to express IgD and express increased levels of IgM. While the precise mechanism is unclear, it seems likely that ZFP318 may act directly on the pre-mRNA transcript during elongation, by suppressing splicing of the VDJ exon to the Cμ exons, allowing transcript elongation and promoting splicing to the Cδ exons. In short, expression of ZFP318 promotes IgD expression, although how ZFP318 expression itself is regulated in immature and mature B cells is still unknown.

1	5-15 Transmembrane and secreted forms of immunoglobulin are generated from alternative heavy-chain mRNA transcripts. Each of the immunoglobulin isotypes can be produced either as a membrane-bound receptor or as secreted antibodies. B cells initially express the transmembrane form of IgM; after stimulation by antigen, some of their In mature B cells, transcription initiated at the VH promoter extends through both Cμ and Cδ exons. This long primary transcript is then processed by cleavage and polyadenylation (AAA), and by splicing. Cleavage and polyadenylation at the μ site (pA1) and splicing between Cμ exons yields an mRNA encoding the μ heavy chain (left panel). Cleavage and polyadenylation at the δ site (pA2) and a different pattern of splicing that joins the V region exon to the Cδ exons and removes the Cμ exons yields mRNA encoding the complete δ heavy chain (right panel). For simplicity we have not shown all the individual C-region exons.

1	progeny differentiate into plasma cells producing IgM antibodies, whereas others undergo class switching to express transmembrane immunoglobulins of a different class, followed by the production of secreted antibody of the new class. The membrane-bound forms of all immunoglobulin classes are monomers comprising two heavy and two light chains. IgM and IgA polymerize only when they have been secreted. The membrane-bound form of immunoglobulin heavy chain has at the carboxy terminus a hydrophobic transmembrane domain of about 25 amino acid residues that anchors it to the surface of the B lymphocyte. The secreted form replaces this transmembrane domain with a carboxy terminus composed of a hydrophilic secretory tail. These two forms of carboxy termini are encoded by different exons found at the end of each CH gene as these exons undergo alternative RNA processing.

1	For example, the IgM heavy-chain gene contains four exons—Cμ1 to Cμ4— that encode its four heavy-chain Ig domains (Fig. 5.22). The end of the Cμ4 exon also encodes the carboxy terminus for the secreted form. Two additional downstream exons, M1 and M2, encode the transmembrane forms. If the primary transcript is cleaved at the polyadenylation site (pAs) located just downstream of the Cμ4 exon but before the last two exons, then only the secreted molecule can be produced. If the polymerase transcribes through this first polyadenylation site, then splicing can occur from a non-consensus splice-donor site within the Cμ4 exon to the M1 exon. In this case, polyadenylation occurs at a downstream site (pAm) and the cell-surface form of immunoglobulin can be produced. This alternative splicing is incompletely understood, but

1	AAA AAA L VDJ C˜1 C˜2 C˜3 C˜4SCpAs pAmM1 M2 carboxy terminusfor transmembrane IgM carboxy terminusfor secreted IgM AAA AAA Rearranged DNA mRNA Protein Transcription Different forms of RNA processing Translation, protein processing Primary transcript RNA Secreted IgMTransmembrane IgM L VDJ C˜1 C˜2 C˜3 C˜4SC pAs pAmM1 M2

1	Fig. 5.22 Transmembrane and secreted forms of immunoglobulins are derived from the same heavy-chain sequence by alternative RNA processing. At the end of the heavy-chain C gene, there are two exons (M1 and M2, yellow) that together encode the transmembrane region and cytoplasmic tail of the transmembrane form. Within the last C-domain exon, a secretion-coding (SC) sequence (orange) encodes the carboxy terminus of the secreted form. In the case of IgD, the SC sequence is in a separate exon (not shown), but for the other isotypes, including IgM as shown here, the SC sequence is contiguous with the last C-domain exon. The events that dictate whether a heavy-chain RNA will result in a secreted or a transmembrane immunoglobulin occur during processing of the pre-mRNA transcript. Each heavy-chain C gene has two potential polyadenylation sites (shown as pAs and pAm). Left panel: the transcript is cleaved and polyadenylated (AAA) at the second site (pAm). Splicing occurs from a site located

1	C gene has two potential polyadenylation sites (shown as pAs and pAm). Left panel: the transcript is cleaved and polyadenylated (AAA) at the second site (pAm). Splicing occurs from a site located within the last Cμ4 exon just upstream of the SC sequence (orange), to a second site at the 5ʹ end of the M1 exons (yellow). This results in removal of the SC sequence and joining of the Cμ4 exon to the exons M1 and M2 and generates the transmembrane form of the heavy chain. Right panel: polyadenylation occurs at the first poly(A) addition site (pAs), and transcription terminates before the exons M1 and M2, preventing the generation of the transmembrane form of the heavy chain, and producing the secreted form.

1	may involve the regulation of RNA polymerase activity as the polymerase transcribes through the IgM locus. One factor that regulates the polyadenylation of RNA transcripts is a cleavage stimulation factor subunit, CstF-64, which favors production of the transcript for secreted IgM. The transcription elongation factor ELL2, which is induced in plasma cells, also promotes polyadenylation at the pAs site and favors the secreted form. CstF-64 and ELL2 co-associate with RNA polymerase within the immunoglobulin locus. This differential RNA processing is illustrated for Cμ in Fig. 5.22, but it occurs in the same way for all isotypes. In activated B cells that differentiate to become antibody-secreting plasma cells, most of the transcripts are spliced to yield the secreted rather than the transmembrane form of whichever heavy-chain isotype the B cell is expressing. 5-16 IgM and IgA can form polymers by interacting with the J chain.

1	5-16 IgM and IgA can form polymers by interacting with the J chain. Although all immunoglobulin molecules are constructed from a basic unit of two heavy and two light chains, both IgM and IgA can form multimers of these basic units (Fig. 5.23). C regions of IgM and IgA can include a ‘tailpiece’ of 18 amino acids that contains a cysteine residue essential for polymerization. A separate 15-kDa polypeptide chain called the J chain promotes polymerization by linking to the cysteine of this tailpiece, which is found only in the secreted forms of the μand α chains. (This J chain should not be confused with the immunoglobulin J region encoded by a J gene segment; see Section 5-2.) In the case of IgA, dimerization is required for transport through epithelia, as we will discuss in Chapter 10. IgM molecules are found as pentamers, and occasionally hexamers (without J chain), in plasma, whereas IgA is found mainly as a dimer in mucous secretions but as a monomer in plasma.

1	Immunoglobulin polymerization is also thought to be important in the binding of antibody to repetitive epitopes. An antibody molecule has at least two identical antigen-binding sites, each of which has a given affinity, or binding strength, for antigen. If the antibody attaches to multiple identical epitopes on a target antigen, it will dissociate only when all binding sites dissociate. The dissociation rate of the whole antibody will therefore be much slower than the dissociation rate for a single binding site; multiple binding sites thus give the antibody a greater total binding strength, or avidity. This consideration is particularly relevant for pentameric IgM, which has 10 antigen-binding sites.

1	Fig. 5.23 The IgM and IgA molecules can form multimers. IgM and IgA are usually synthesized as multimers in association with an additional polypeptide chain, the J chain. In dimeric IgA (left panel), the monomers have disulfide bonds to the J chain as well as to each other. In pentameric IgM (right panel), the monomers are cross-linked by disulfide bonds to each other and to the J chain. IgM can also form hexamers that lack a J chain (not shown).

1	IgM antibodies frequently recognize repetitive epitopes such as those on bacterial cell-wall polysaccharides, but individual binding sites are often of low affinity because IgM is made early in immune responses, before somatic hypermutation and affinity maturation. Multisite binding makes up for this, markedly improving the overall functional binding strength. This implies that binding of a single IgM pentamer to a target could be sufficient to mediate biological effector activity, whereas in the case of IgGs, two independent target molecules may need to be located in close proximity. Summary.

1	The classes of immunoglobulins are defined by their heavy-chain C regions, with the different heavy-chain isotypes being encoded by different C-region genes. The heavy-chain C-region genes are present in a cluster 3ʹ to the V, D, and J gene segments. A productively rearranged V-region exon is initially expressed in association with μ and δ CH genes, which are coexpressed in naive B cells by alternative splicing of an mRNA transcript that contains both the μ and δ CH exons. In addition, B cells can express any class of immunoglobulin as a membrane-bound antigen receptor or as a secreted antibody. This is achieved by differential splicing of mRNA to include exons that encode either a hydrophobic membrane anchor or a secretable tailpiece. The antibody that a B cell secretes upon activation thus recognizes the antigen that initially activated the B cell via its antigen receptor. The same V-region exon can subsequently be associated with any one of the other isotypes to direct the

1	thus recognizes the antigen that initially activated the B cell via its antigen receptor. The same V-region exon can subsequently be associated with any one of the other isotypes to direct the production of antibodies of different classes by the process of class switching, which is described in Chapter 10.

1	Evolution of the adaptive immune response.

1	The form of adaptive immunity that we have discussed so far in this book depends on the action of the RAG-1/RAG-2 recombinase to generate an enormously diverse clonally distributed repertoire of immunoglobulins and T-cell receptors. This system is found only in the jawed vertebrates, the gnathostomes, which split off from the other vertebrates around 500 million years ago. Adaptive immunity seems to have arisen abruptly in evolution. Even the cartilaginous fishes, the earliest group of jawed fishes to survive to the present day, have organized lymphoid tissue, T-cell receptors and immunoglobulins, and the ability to mount adaptive immune responses. The diversity generated within the vertebrate adaptive immune system was once viewed as unique among animal immune systems. But we now know that organisms as different as insects, echinoderms, and mollusks use a variety of genetic mechanisms to increase their repertoires of pathogen-detecting molecules, although they do not achieve true

1	that organisms as different as insects, echinoderms, and mollusks use a variety of genetic mechanisms to increase their repertoires of pathogen-detecting molecules, although they do not achieve true adaptive immunity. Nearer to home, it has been found that the surviving species of jawless vertebrates, the agnathans—the lampreys and hagfish—have a form of adaptive or ‘anticipatory’ immunity that is based on non-immunoglobulin ‘antibody’-like proteins and involves a system of somatic gene rearrangement that is quite distinct from RAG-dependent V(D)J rearrangement. So we should now view our adaptive immune system as only one solution, albeit the most powerful, to the problem of generating highly diverse systems for pathogen recognition.

1	5-17 Some invertebrates generate extensive diversity in a repertoire of immunoglobulin-like genes. Until very recently, it was thought that invertebrate immunity was limited to an innate system that had a very restricted diversity in recognizing pathogens. This idea was based on the knowledge that innate immunity in vertebrates relied on around 10 distinct Toll-like receptors and a similar number of other receptors that also recognize PAMPs, and also on the assumption that the number of receptors in invertebrates was no greater. Recent studies have, however, uncovered at least two invertebrate examples of extensive diversification of an immunoglobulin superfamily member, which could potentially provide an extended range of recognition of pathogens.

1	In Drosophila, fat-body cells and hemocytes act as part of the immune system. Fat-body cells secrete proteins, such as the antimicrobial defensins (see Chapters 2 and 3), into the hemolymph. Another protein found in hemolymph is the Down syndrome cell adhesion molecule (Dscam), a member of the immunoglobulin superfamily. Dscam was originally discovered in the fly as a protein involved in specifying neuronal wiring. It is also made in fat-body cells and hemocytes, which can secrete it into the hemolymph, where it is thought to recognize invading bacteria and aid in their engulfment by phagocytes.

1	The Dscam protein contains multiple, usually 10, immunoglobulin-like domains. The gene that encodes Dscam has, however, evolved to contain a large number of alternative exons for several of these domains (Fig. 5.24). Exon 4 of the gene encoding the Dscam protein can be any 1 of 12 different exons, each specifying an immunoglobulin domain of differing sequence. Exon cluster 6 has 48 alternative exons, cluster 9 another 33, and cluster 17 a further 2: it is estimated that the Dscam gene could encode around 38,000 protein isoforms. A role for Dscam in immunity was proposed when it was found that in vitro phagocytosis of Escherichia coli by isolated hemocytes lacking Dscam was less efficient than by normal hemocytes. These observations suggest that at least some of this extensive repertoire of alternative exons may have evolved to diversify insects’ ability to recognize pathogens. This role for Dscam has been confirmed in the mosquito Anopheles gambiae, in which silencing of the Dscam

1	of alternative exons may have evolved to diversify insects’ ability to recognize pathogens. This role for Dscam has been confirmed in the mosquito Anopheles gambiae, in which silencing of the Dscam homolog AgDscam has been shown to weaken the mosquito’s normal resistance to bacteria and to the malaria parasite Plasmodium. There is also evidence from the mosquito that some Dscam exons have specificity for particular pathogens. It is not clear whether Dscam isoforms are expressed in a clonal manner.

1	Another invertebrate, this time a mollusk, uses a different strategy to diversify an immunoglobulin superfamily protein for use in immunity. The freshwater snail Biomphalaria glabrata expresses a small family of fibrinogen-related proteins (FREPs) thought to have a role in innate immunity. FREPs have one or two immunoglobulin domains at their amino-terminal end and a fibrinogen domain at their carboxy terminus. The immunoglobulin domains may interact with pathogens, while the fibrinogen domain may confer on the FREP lectin-like properties that help precipitate the complex. FREPs are produced by

1	Fig. 5.24 The Dscam protein of Drosophila innate immunity contains multiple immunoglobulin domains and is highly diversified through alternative splicing. The gene encoding Dscam in Drosophila contains several large clusters of alternative exons. The clusters encoding exon 4 (green), exon 6 (light blue), exon 9 (red), and exon 17 (orange) contain 12, 48, 33, and 2 alternative exons, respectively. For each of these clusters, only one alternative exon is used in the complete Dscam mRNA. There is some differential usage of exons in neurons, fat-body cells, and hemocytes. All three cell types use the entire range of alternative exons for exons 4 and 6. For exon 9, there is a restricted use of alternative exons in hemocytes and fat-body cells. The combinatorial use of alternative exons in the Dscam gene makes it possible to generate more than 38,000 protein isoforms. Adapted from Anastassiou, D.: Genome Biol. 2006, 7:R2.

1	hemocytes and secreted into the hemolymph. Their concentration increases when the snail is infected by parasites—it is, for example, the intermediate host for the parasitic schistosomes that cause human schistosomiasis.

1	The B. glabrata genome contains many copies of FREP genes that can be divided into approximately 13 subfamilies. A study of the sequences of expressed FREP3 subfamily members has revealed that the FREPs expressed in an individual organism are extensively diversified compared with the germline genes. There are fewer than five genes in the FREP3 subfamily, but an individual snail was found to generate more than 45 distinct FREP3 proteins, all with slightly different sequences. An analysis of the protein sequences suggested that this diversification was due to the accumulation of point mutations in one of the germline FREP3 genes. Although the precise mechanism of this diversification, and the cell type in which it occurs, are not yet known, it does suggest some similarity to somatic hypermutation that occurs in the immunoglobulins. Both the insect and Biomphalaria examples seem to represent a way of diversifying molecules involved in immune defense, but although they resemble in some

1	that occurs in the immunoglobulins. Both the insect and Biomphalaria examples seem to represent a way of diversifying molecules involved in immune defense, but although they resemble in some ways the strategy of an adaptive immune response, there is no evidence of clonal selection—the cornerstone of true adaptive immunity.

1	somatic gene rearrangement to diversify receptors built from LRR domains.

1	Since the early 1960s it has been known that certain jawless fishes, the hagfish and the lamprey, could mount a form of accelerated rejection of transplanted skin grafts and exhibit a kind of immunological delayed-type hypersensitivity. Their serum also seemed to contain an activity that behaved as a specific agglutinin, increasing in titer after secondary immunizations, in a similar way to an antibody response in higher vertebrates. Although these phenomena seemed reminiscent of adaptive immunity, there was no evidence of a thymus or of immunoglobulins, but these animals did have cells that could be considered to be genuine lymphocytes on the basis of morphological and molecular analysis. Analysis of the genes expressed by lymphocytes of the sea lamprey Petromyzon marinus revealed none related to T-cell receptor or immunoglobulin genes. However, these cells expressed large amounts of mRNAs from genes encoding proteins with multiple LRR domains, the same protein domain from which the

1	to T-cell receptor or immunoglobulin genes. However, these cells expressed large amounts of mRNAs from genes encoding proteins with multiple LRR domains, the same protein domain from which the pathogen-recognizing Toll-like receptors (TLRs) are built (see Section 3-5).

1	This might simply have meant that these cells are specialized for recognizing and reacting to pathogens, but the LRR proteins expressed had some surprises in store. Instead of being present in a relatively few forms (like the invariant TLRs), they were found to have highly variable amino acid sequences, with a large number of variable LRR units placed between less variable amino-terminal and carboxy-terminal LRR units. These LRR-containing proteins, called variable lymphocyte receptors (VLRs), have an invariant stalk region connecting them to the plasma membrane by a glycosylphosphatidylinositol linkage, and they can either be tethered to the cell or, at other times, like antibodies, be secreted into the blood.

1	Analysis of the expressed lamprey VLR genes indicates that they are assembled by a process of somatic gene rearrangement (Fig. 5.25). In the germline configuration, there are three incomplete VLR genes, VLRA, VLRB, and VLRC, each encoding a signal peptide, a partial amino-terminal LRR unit, and a partial carboxy-terminal LRR unit, but these three blocks of coding sequence are separated by noncoding DNA that contains neither typical signals for RNA splicing nor the RSSs present in immunoglobulin genes (see Section 5-4). Instead, the regions flanking the incomplete VLR genes include a large number of DNA ‘cassettes’ that contain LRR units—one, two, or three LRR domains at a time. Each mature lamprey lymphocyte expresses a complete and unique VLR gene, either VLRA, VLRB, or VLRC, which has undergone recombination of these flanking regions with the germline VLR gene.

1	The creation of a complete VLR gene is currently thought to occur during replication of lamprey lymphocyte DNA by a ‘copy-choice’ mechanism that is similar, but not identical, to gene conversion (described in Section 5-20). During DNA replication, LRR units flanking the VLR gene are copied into the VLR gene—presumably when a DNA strand being synthesized switches templates and copies sequences from one of these LRR units. Although final proof is still lacking, this template-switching mechanism may be triggered by enzymes of the AID-APOBEC family that are expressed by lamprey lymphocytes, and whose cytidine deaminase activity (CDA) could cause the single-strand DNA breaks that can start the copy-choice process. Lampreys possess two such enzymes: CDA1, which is expressed in VLRA-lineage lymphocytes, and CDA2, which is expressed in VLRB-lineage lymphocytes. It is not yet known if CDA1 or CDA2 is expressed in VLRC-expressing lymphocytes. The final VLR gene contains a complete

1	lymphocytes, and CDA2, which is expressed in VLRB-lineage lymphocytes. It is not yet known if CDA1 or CDA2 is expressed in VLRC-expressing lymphocytes. The final VLR gene contains a complete amino-terminal capping LRR subunit, followed by the addition of up to seven internal LRR domains, each 24 amino acids long, and the removal of the internal noncoding regions to complete the formation of the carboxy-terminal LRR domain (see Fig. 5.25).

1	It is estimated that this somatic rearrangement mechanism can generate as much diversity in the VLR proteins as is possible for immunoglobulins. Indeed, the crystal structure of a VLR protein shows that the concave surface formed by the series of LRR repeats interacts with a variable insert in the carboxy-terminal LRR to form a surface capable of interacting with a great diversity of antigens. Thus, the diversity of the anticipatory repertoire of agnathans may be limited not by the numbers of possible receptors they can generate but by the number of lymphocytes present in any individual, as in the adaptive immune system of their evolutionary cousins, the gnathostomes. As noted above, each lamprey lymphocyte rearranges only one of the two germline VLR genes, expressing either a complete VLRA or VLRB or VLRC protein. The first two cell populations seem to have some characteristics of mammalian T and B lymphocytes,

1	Top panel: an incomplete germline copy of a lamprey VLR gene contains a framework (right) for the complete gene: the portion encoding the signal peptide (SP), part of an amino-terminal LRR unit (NT, dark blue), and a carboxy-terminal LRR unit that is split into two parts (LRR, light red; and CT, red) by intervening noncoding DNA sequences. Nearby flanking regions (left) contain multiple copies of VLR gene-‘cassettes’ with single or double copies of variable LRR domains (green) and cassettes that encode part of the amino-terminal LRR domains (light blue, yellow). Middle panel: somatic recombination causes various LRR units to be copied into the original VLR gene. This creates a complete VLR gene that contains the assembled amino-terminal LRR cassette (LRR NT) and first LRR (yellow), followed by several variable LRR units (green) and the completed carboxy-terminal LRR unit, and ends with the portion that encodes the stalk region of the VLR receptor. The cytidine deaminases PmCDA1 and

1	by several variable LRR units (green) and the completed carboxy-terminal LRR unit, and ends with the portion that encodes the stalk region of the VLR receptor. The cytidine deaminases PmCDA1 and PmCDA2 from the lamprey

1	P. marinus are candidates for enzymes that may initiate this gene rearrangement. Expression of the rearranged gene results in a complete receptor that can be attached to the cell membrane by glycosylphosphatidylinositol (GPI) linkage of its stalk. Bottom panel: an individual lymphocyte undergoes somatic gene rearrangement to produce a unique VLR receptor. These receptors can be tethered to the surface of the lymphocyte via the GPI linkage or can be secreted into the blood. Unique somatic rearrangement events in each developing lymphocyte generate a repertoire of VLR receptors of differing specificities. Adapted from Pancer, Z., and Cooper, M.D.: Annu. Rev. Immunol. 2006, 24:497–518.

1	In vertebrates, evolution of the locus results in a multipart antigen-receptor locus that can be rearranged by RAG-mediated somatic recombination Recombination separates the RAG genes from the TR-tagged gene segments Insertion of the transposon into a V-type Ig receptor gene splits the gene into two TR TRRAG1 gene excision site RAG2 gene RAG1 gene RSS RSS RAG2 gene Transposon-derived RAG1/2-like gene cluster in a deuterostome ancestor Transposase activity can excise the transposon at terminal repeat sequences and reinsert it at a new location in the genome J JVV V-type Ig-like domain Fig. 5.26 Integration of a transposon into a V-type immunoglobulin receptor gene is thought to have given rise to the T-cell receptor and immunoglobulin genes.

1	Fig. 5.26 Integration of a transposon into a V-type immunoglobulin receptor gene is thought to have given rise to the T-cell receptor and immunoglobulin genes. respectively, and VLRC cells appear more closely related to the VLRA lineage. For example, VLRA-expressing lymphocytes also express genes similar to some mammalian T-cell cytokine genes, suggesting an even closer similarity to our own RAG-dependent adaptive immune system than was previously appreciated. repertoire of immunoglobulin-like genes appeared abruptly in the cartilaginous fishes.

1	Within the vertebrates, we can trace the development of immune functions from the agnathans through the cartilaginous fishes (sharks, skates, and rays) to the bony fishes, then to the amphibians, to reptiles and birds, and finally to mammals. RAG-dependent V(D)J recombination has not been found in agnathans, other chordates, or any invertebrate. The origins of RAG-dependent adaptive immunity are now becoming clearer as the genome sequences of many more animals become available. The first clue was that RAG-dependent recombination shares many features with the transposition mechanism of DNA transposons—mobile genetic elements that encode their own transposase, an enzymatic activity that allows them to excise from one site in the genome and reinsert themselves elsewhere. The mammalian RAG complex can act as a transposase in vitro, and even the structure of the RAG genes, which lie close together in the chromosome and lack the usual introns of mammalian genes, is reminiscent of a

1	RAG complex can act as a transposase in vitro, and even the structure of the RAG genes, which lie close together in the chromosome and lack the usual introns of mammalian genes, is reminiscent of a transposon.

1	All this provoked speculation that the origin of RAG-dependent adaptive immunity was the invasion of a DNA transposon into a gene similar to an immunoglobulin or a T-cell receptor V-region gene, an event that would have occurred in some ancestor of the jawed vertebrates (Fig. 5.26). DNA transposons carry inverted repeated sequences at either end, which are bound by the transposase for transposition to occur. These terminal repeats are considered to be the ancestors of the RSSs in present-day antigen-receptor genes (see Section 5-4), while the RAG-1 protein is believed to have evolved from a transposase. Subsequent duplication, reduplication, and recombination of the immune-receptor gene and its inserted RSSs eventually led to the separation of the RAG genes from the rest of the relic transposon and to the multisegmented immunoglobulin and T-cell receptor loci of present-day vertebrates.

1	The ultimate origins of the RSSs and the RAG-1 catalytic core are now thought to lie in the Transib superfamily of DNA transposons, and genome sequencing has led to the discovery of sequences related to RAG1 in animals as distantly related to vertebrates as the sea anemone Nematostella. The origin of RAG2 is

1	Top panel: a DNA transposon in an ancestor of the deuterostomes (the large group of phyla to which the chordates belong) is thought to have had genes related to RAG1 and RAG2—prototype RAG1 (purple) and RAG2 (blue), which acted as its transposase. DNA transposons are bounded by terminal inverted repeat (TR) sequences. Second panel: to excise a transposon from DNA, the transposase proteins (purple and blue) bind the TRs, bringing them together, and the transposase enzymatic activity cuts the transposon out of the DNA, leaving a footprint in the host DNA that resembles the TRs. After excision from one site, the transposon reinserts elsewhere in the genome, in this case into a V-type immunoglobulin receptor (green). The enzymatic activity of the transposase enables the transposon to insert into DNA in a reaction that is the reverse of the excision reaction. Third panel: the integration of the RAG1/2-like transposon into the middle of the gene for a V-type immunoglobulin receptor splits

1	into DNA in a reaction that is the reverse of the excision reaction. Third panel: the integration of the RAG1/2-like transposon into the middle of the gene for a V-type immunoglobulin receptor splits the V exon into two parts. Fourth and fifth panels: in the evolution of the immunoglobulin and T-cell receptor genes, the initial integration event has been followed by DNA rearrangements that separate the transposase genes (now known as the RAG1 and RAG2 genes) from the transposon TRs, which we now term the recombination signal sequences (RSSs). The purple sea urchin (an invertebrate deuterostome) has a RAG1/2-like gene cluster (not shown) and expresses proteins similar to RAG-1 and RAG-2 proteins, but does not have immunoglobulins, T-cell receptors, or adaptive immunity. The RAG-like proteins presumably retain some other cellular function (so far unknown) in this animal.

1	more obscure, but a RAG1–RAG2-related gene cluster was recently discovered in sea urchins, invertebrate relatives of the chordates. Sea urchins themselves show no evidence of immunoglobulins, T-cell receptors, or adaptive immunity, but the proteins expressed by the sea-urchin RAG genes form a complex with each other and with RAG proteins from the bull shark (Carcharhinus leucas), a primitive jawed vertebrate, but not with those from mammals. This suggests that these proteins could indeed be related to the vertebrate RAGs, and that RAG-1 and RAG-2 were already present in a common ancestor of chordates and echinoderms (the group to which sea urchins belong), presumably fulfilling some other cellular function.

1	The origin of somatic gene rearrangement in the excision of a transposable element makes sense of an apparent paradox in the rearrangement of immune-system genes. This is that the RSSs are joined precisely in the excised DNA (see Section 5-5), which has no further function and whose fate is irrelevant to the cell, whereas the cut ends in the genomic DNA, which form part of the immunoglobulin or T-cell receptor gene, are joined by an error-prone process, which could be viewed as a disadvantage. However, when looked at from the transposon’s point of view, this makes sense, because the transposon preserves its integrity by this excision mechanism, whereas the fate of the DNA it leaves behind is of no significance to it. As it turned out, the error-prone joining in the primitive immunoglobulin gene generated useful diversity in antigen-recognition molecules and was strongly selected for. The RAG-based rearrangement system also provided something else that mutations could not—a means of

1	gene generated useful diversity in antigen-recognition molecules and was strongly selected for. The RAG-based rearrangement system also provided something else that mutations could not—a means of rapidly modifying the size of the coding region, not just its diversity.

1	The next question is what sort of gene the transposon inserted into. Proteins containing Ig-like domains are ubiquitous throughout the plant, animal, and bacterial kingdoms, making this one of the most abundant protein super-families; in species whose genomes have been fully sequenced, the immunoglobulin superfamily is one of the largest families of protein domains in the genome. The functions of the members of this superfamily are very disparate, and they are a striking example of natural selection taking a useful structure— the basic Ig-domain fold—and adapting it to different purposes.

1	The immunoglobulin superfamily domains can be divided into four families on the basis of differences in structure and sequence of the immunoglobulin domain. These are V (resembling an immunoglobulin variable domain), C1 and C2 (resembling constant-region domains), and a type of immunoglobulin domain called an I domain (for intermediate). The target of the RSS-containing element is likely to have been a gene encoding a cell-surface receptor containing an Ig-like V domain, most probably a type similar to present-day VJ domains. These domains are found in some invariant receptor proteins and are so called because of the resemblance of one of the strands to a J segment. It is possible to imagine how transposon movement into such a gene could produce separate V and J gene segments (see Fig. 5.26). On the basis of phylogenetic analysis, agnathan paired receptors resembling Ag receptors, or APARs, which are encoded by a multigene family found in hagfish and lamprey, are currently the best

1	5.26). On the basis of phylogenetic analysis, agnathan paired receptors resembling Ag receptors, or APARs, which are encoded by a multigene family found in hagfish and lamprey, are currently the best candidates for being relatives of the ancestor of the antigen receptor. Their DNA sequences predict single-pass transmembrane proteins with a single extracellular VJ domain and a cytoplasmic region containing signaling modules. APARs are expressed in leukocytes.

1	5-20 Different species generate immunoglobulin diversity in different ways.

1	Most of the vertebrates we are familiar with generate a large part of their antigen receptor diversity in the same way as mice and humans, by putting together gene segments in different combinations. There are exceptions, however, even within the mammals. Some animals use gene rearrangement to always join together the same V and J gene segment initially, and then diversify this recombined V region. In birds, rabbits, cows, pigs, sheep, and horses, there is little or no germline diversity in the V, D, and J gene segments that are rearranged to form the genes for the initial B-cell receptors, and the rearranged V-region sequences are identical or similar in most immature B cells. These immature B cells migrate to specialized microenvironments—the bursa of Fabricius in the gut of chickens, and another intestinal lymphoid organ in rabbits. Here, B cells proliferate rapidly, and their rearranged immunoglobulin genes undergo further diversification.

1	In birds and rabbits this occurs mainly by gene conversion, a process by which short sequences in the expressed rearranged V-region gene are replaced with sequences from an upstream V gene segment pseudogene. The germline arrangement of the chicken heavy-chain locus is a single set of rearranging V, J, D, and C gene segments and multiple copies of V-segment pseudogenes. Diversity in this system is created by gene conversion in which sequences from the VH pseudogenes are copied into the single rearranged VH gene (Fig. 5.27). It seems that gene conversion is related to somatic hypermutation

1	Fig. 5.27 The diversification of chicken immunoglobulins occurs through gene conversion. In chickens, the immunoglobulin diversity that can be created by V(D)J recombination is extremely limited. Initially, there are only one active V, one J, and 15 D gene segments at the chicken heavy-chain locus and one active V and one J gene segment at the single light-chain locus (top left panel). Primary gene rearrangement can thus produce only a very limited number of receptor specificities (second panels). Immature B cells expressing this receptor migrate to the bursa of Fabricius, where the cross-linking of surface immunoglobulin (sIg) induces cell proliferation (second panels). The chicken genome contains numerous pseudogenes with a prerearranged VH–D structure. Gene conversion events introduce sequences from these adjacent V gene segment pseudogenes into the expressed gene, creating diversity in the receptors (third panels). Some of these gene conversions will inactivate the previously

1	sequences from these adjacent V gene segment pseudogenes into the expressed gene, creating diversity in the receptors (third panels). Some of these gene conversions will inactivate the previously expressed gene (not shown). If a B cell can no longer express sIg after such a gene conversion, it is eliminated. Repeated gene conversion events can continue to diversify the repertoire (bottom panels).

1	Sequences from V pseudogenes are introduced into rearranged V genes through gene conversion Multiple rounds of gene conversion can alter affnities of antibody for antigen C˜VDJ˜C°VJ°C˜VDJ˜C°VJ°C˜VDJ˜C°VJ°Immature chicken B cells. All have rearranged the same VH and V˜ genes V H D˜C˜J˜V°C°J°VH pseudogenes V° pseudogenes Germline chicken immunoglobulin genes in its mechanism, because gene conversion in a chicken B-cell line has been shown to require the enzyme activation-induced cytidine deaminase (AID). In Chapter 10, we will see that this same enzyme is involved in class switching and affinity maturation of the antibody response. For gene conversion, it is thought that single-strand cuts in DNA generated by the endonuclease apurinic/apyrimidinic endonuclease-1 (APE1) after the actions of AID are the signal that initiates a homology-directed repair process in which a homologous V pseudogene segment is used as the template for the DNA replication that repairs the V-region gene.

1	In sheep and cows, immunoglobulin diversification is the result of somatic hypermutation, which occurs in an organ known as the ileal Peyer’s patch. Somatic hypermutation, independent of T cells and a particular driving antigen, also contributes to immunoglobulin diversification in birds, sheep, and rabbits.

1	A more fundamentally different organization of immunoglobulin genes is found in the cartilaginous fish, the most primitive jawed vertebrates. Sharks have multiple copies of discrete VL–JL–CL and VH–DH–JH–CH cassettes, and activate rearrangement within individual cassettes (Fig. 5.28). Although this is somewhat different from the kind of combinatorial gene rearrangement of higher vertebrates, in most cases there is still a requirement for a RAG-mediated somatic rearrangement event. As well as rearranging genes, cartilaginous fish have multiple ‘rearranged’ VL regions (and sometimes rearranged VH regions) in the germline genome (see Fig. 5.28) and apparently generate diversity by activating the transcription of different copies. Even here, some diversity is also contributed by combinatorial means by the subsequent pairing of heavy and light chains.

1	This ‘germline-joined’ organization of the light-chain loci is unlikely to represent an intermediate evolutionary stage, because in that case the heavy-chain and light-chain genes would have had to independently acquire the capacity for rearrangement by convergent evolution. It is much more likely that, after the divergence of the cartilaginous fishes, some immunoglobulin loci became rearranged in the germline of various ancestors through activation of the RAG genes in germ cells, with the consequent inheritance of the rearranged loci by the offspring. In these species, the rearranged germline loci might confer some advantages, such as ensuring rapid responses to common pathogens by producing a preformed set of immunoglobulin chains. The IgM antibody isotype is thought to go back to the origins of adaptive immunity. It is the predominant form of immunoglobulin in cartilaginous

1	The IgM antibody isotype is thought to go back to the origins of adaptive immunity. It is the predominant form of immunoglobulin in cartilaginous Fig. 5.28 The organization of immunoglobulin genes is different in different species, but all can generate a diverse repertoire of receptors.

1	The organization of the immunoglobulin heavy-chain genes in mammals, in which there are separated clusters of repeated V, D, and J gene segments, is not the only solution to the problem of generating a diverse repertoire of receptors. Other vertebrates have found alternative solutions. In ‘primitive’ groups, such as the sharks, the locus consists of multiple repeats of a basic unit composed of a V gene segment, one or two D gene segments, a J gene segment, and a C gene segment. A more extreme version of this organization is found in the κ-like light-chain locus of some cartilaginous fishes such as the rays and the carcharhine sharks, in which the repeated unit consists of already rearranged VJ–C genes, from which a random choice is made for expression. In chickens, there is a single rearranging set of gene segments at the heavy-chain locus but there are multiple copies of pre-integrated VH–D segment pseudogenes. Diversity in this system is created by gene conversion, in which

1	rearranging set of gene segments at the heavy-chain locus but there are multiple copies of pre-integrated VH–D segment pseudogenes. Diversity in this system is created by gene conversion, in which sequences from the VH-–D pseudogenes are copied onto the single rearranged VH gene.

1	fishes and bony fishes. The cartilaginous fishes also have at least two other heavy-chain isotypes not found in more recently evolved species. One, IgW, has a constant region composed of six immunoglobulin domains, whereas the second, IgNAR, which we described in Section 4-10, seems to be related to IgW but has lost the first constant-region domain and does not pair with light chains. Instead, it forms a homodimer in which each heavy-chain V domain forms a separate antigen-binding site. IgW seems to be related to IgD (which is first found in bony fish) and, like IgM, seems to go back to the origin of adaptive immunity. 5-21 Both α:β and γ:δ T-cell receptors are present in cartilaginous fishes.

1	Neither the T-cell receptors nor the immunoglobulins have been found in any species evolutionarily earlier than the cartilaginous fishes, in which they have essentially the same form that we see in mammals. The identification of TCRβ-chain and δ-chain homologs from sharks, and of distinct TCRα, β, γ, and δ chains from a skate, show that even at the earliest time that these adaptive immune system receptors can be identified, they had already diversified into at least two recognition systems. Moreover, each lineage shows diversity resulting from combinatorial somatic rearrangement. The identification of many ligands recognized by γ:δ T cells has helped clarify their role in the immune response. Although a complete list is still lacking, the trend appears to be more similar to a kind of innate sensing rather than the fine peptide specificity of the α:β T cells. Ligands of γ:δ T cells include various lipids that may derive from microbes and nonclassical MHC class Ib molecules whose

1	innate sensing rather than the fine peptide specificity of the α:β T cells. Ligands of γ:δ T cells include various lipids that may derive from microbes and nonclassical MHC class Ib molecules whose expression may be an indication of infection or cellular stress (see Section 6-17). Even certain α:βT cells appear to participate in a form of innate recognition, such as the mucosa-associated invariant T cells described in Section 4-18. This could indicate that early in the evolution of RAG-dependent adaptive immunity, the receptors generated by excision of the primordial retrotransposon were useful in innate sensing of infections, and this role has persisted in certain minor T-cell populations to this day. In any case, the very early divergence of these two classes of T-cell receptors and their conservation through subsequent evolution suggests an important early separation of functions.

1	5-22 MHC class I and class II molecules are also first found in the cartilaginous fishes. One would expect to see the specific ligands of T-cell receptors, the MHC molecules, emerge at around the same time in evolution as the receptors. Indeed, MHC molecules are present in the cartilaginous fishes and in all higher vertebrates, but, like the T-cell receptors, they have not been found in agnathans or invertebrates. Both MHC class I and class II α-chain and β-chain genes are present in sharks, and their products seem to function in an identical way to mammalian MHC molecules. The key residues of the peptide-binding cleft that interact with the ends of the peptide in MHC class I molecules or with the central region of the peptide in MHC class II molecules are conserved in shark MHC molecules.

1	Moreover, the MHC genes are also polymorphic in sharks, with multiple alleles of class I and class II loci. In some species, more than 20 MHC class I alleles have been identified so far. For the shark MHC class II molecules, both the class II α and the class II β chains are polymorphic. Thus, not only has the function of the MHC molecules in selecting peptides for presentation evolved during the divergence of the agnathans and the cartilaginous fishes, but the continuous selection imposed by pathogens has also resulted in the polymorphism that is a characteristic feature of the MHC.

1	Section 4-20 introduced the division between classical MHC class I genes (sometimes called class Ia) and the nonclassical MHC class Ib genes, which will be discussed in Chapter 6. This division is also present in cartilaginous fishes, because the class I genes of sharks include some that resemble mammalian class Ib molecules. However, it is thought that the shark class Ib genes are not the direct ancestors of the mammalian class Ib genes. For the class I genes, it seems that within each of the five major vertebrate lineages studied (cartilaginous fishes, lobe-finned fishes, ray-finned fishes, amphibians, and mammals), these genes have independently separated into classical and nonclassical loci.

1	Thus, the characteristic features of the MHC molecules are all present when these molecules are first encountered, and there are no intermediate forms to guide our understanding of their evolution. Although we can trace the evolution of the components of the innate immune system, the mystery of the origin of the adaptive immune system still largely persists. But although we may not have a sure answer to the question of what selective forces led to RAG-dependent elaboration of adaptive immunity, it has never been clearer that, as Charles Darwin remarked about evolution in general, “from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved.” Summary.

1	Evolution of RAG-dependent adaptive immunity in jawed vertebrates was once considered a wholly unique and inexplicable ‘immunological Big Bang.’ However, we now understand that adaptive immunity has also evolved independently at least one other time during evolution. Our close vertebrate cousins, the jawless fishes, have evolved an adaptive immune system built on a completely different basis—the diversification of LRR domains rather than immunoglobulin domains—but which otherwise seems to have the essential features of clonal expression of receptors produced through a somatic rearrangement and with a form of immunological memory, all features of an adaptive immune system. We now appreciate that evolution of the RAG-dependent adaptive immune system is probably related to the insertion of a transposon into a member of a primordial immunoglobulin superfamily gene, which must have occurred in a germline cell in an ancestor of the vertebrates. By chance, the transposon terminal sequences,

1	transposon into a member of a primordial immunoglobulin superfamily gene, which must have occurred in a germline cell in an ancestor of the vertebrates. By chance, the transposon terminal sequences, the forerunners of the RSSs, were placed in an appropriate location within this primordial antigen-receptor gene to enable intramolecular somatic recombination, thus paving the way for the full-blown somatic gene rearrangement seen in present-day immunoglobulin and T-cell receptor genes. The MHC molecules that are the ligands for T-cell receptors first appear in the cartilaginous fishes, suggesting coevolution with RAG-dependent adaptive immunity. The transposase genes (the RAG genes) could have already been present and active in some other function in the genome of this ancestor. RAG1 seems to be of very ancient origin, as RAG1related sequences have been found in a wide variety of animal genomes.

1	Summary to Chapter 5.

1	The antigen receptors of lymphocytes are remarkably diverse, and developing B cells and T cells use the same basic mechanism to achieve this diversity. In each cell, functional genes for the immunoglobulin and T-cell receptor chains are assembled by somatic recombination from sets of separate gene segments that together encode the V region. The substrates for the joining process are arrays of V, D, and J gene segments, which are similar in all the antigen-receptor gene loci. The lymphoid-specific proteins RAG-1 and RAG-2 direct the site-specific cleavage of DNA at RSSs flanking the V, D, and J segments to form double-strand breaks that initiate the recombination process in both T and B cells. These proteins function in concert with ubiquitous DNA-modifying enzymes acting in the double-strand break repair pathway, and with at least one other lymphoid-specific enzyme, TdT, to complete the gene rearrangements. As each type of gene segment is present in multiple, slightly different,

1	break repair pathway, and with at least one other lymphoid-specific enzyme, TdT, to complete the gene rearrangements. As each type of gene segment is present in multiple, slightly different, versions, the random selection of one gene segment from each set is a source of substantial potential diversity. During assembly, the imprecise joining mechanisms at the coding junctions create a high degree of diversity concentrated in the CDR3 loops of the receptor, which lie at the center of the antigen-binding sites. The independent association of the two chains of immunoglobulins or T-cell receptors to form a complete antigen receptor multiplies the overall diversity available. An important difference between immunoglobulins and T-cell receptors is that immunoglobulins exist in both membrane-bound forms (B-cell receptors) and secreted forms (antibodies). The ability to express both a secreted and a membrane-bound form of the same molecule is due to alternative splicing of the heavy-chain mRNA

1	forms (B-cell receptors) and secreted forms (antibodies). The ability to express both a secreted and a membrane-bound form of the same molecule is due to alternative splicing of the heavy-chain mRNA to include exons that encode different forms of the carboxy terminus. Heavy-chain C regions of immunoglobulins contain three or four domains, whereas the T-cell receptor chains have only one. Other species have developed strategies to diversify receptors involved in immunity, and the agnathans use a system of VLRs that undergo somatic rearrangement that has some specific similarities to our own adaptive immune system. Adaptive immunity in jawed vertebrates—gnathostomes—appears to have arisen by the integration of a retrotransposon that encoded prototype RAG1/2 genes into a preexisting V-type immunoglobulin-like gene that subsequently diversified to generate Tand B-cell receptor genes.

1	Questions. 5.1 True or False: A developing T cell may by chance express both an αβ heterodimer and a γδ heterodimer if all the loci recombine successfully. 5.2 Multiple Choice: Which of the following factors involved in antigen-receptor recombination could be deleted without completing ablating antigen receptor formation? A. Artemis B. TdT C. RAG-2 D. Ku E. XRCC4 5.3 True or False: Both B and T cells can undergo somatic hypermutation of their antigen receptor in the context of an immune response in order to enhance antigen affinity. 5.4 Short Answer: What four processes contribute to the vast diversity of antibodies and B-cell receptors? 5.5 Matching: Match the protein(s) to its (their) function: A. RAG-1 and RAG-2 i. Nontemplate addition of N-nucleotides B. Artemis ii. Nuclease activity to open the DNA hairpin and generate P-nucleotides C. TdT iii. Recognize(s) RSS and create(s) single-stranded break D. DNA ligase IV and iv. Join(s) DNA ends XRCC4

1	B. Artemis ii. Nuclease activity to open the DNA hairpin and generate P-nucleotides C. TdT iii. Recognize(s) RSS and create(s) single-stranded break D. DNA ligase IV and iv. Join(s) DNA ends XRCC4 E. DNA-PKcs v. Form(s) a complex with Ku to hold DNA together and phosphorylate Artemis 5.6 Short Answer: What is the 12/23 rule and how does it ensure proper V(D)J segment joining? 5.7 Matching: Match the clinical disorder to the gene defects: A. Ataxia telangiectasia i. RAG-1 or RAG-2 mutations resulting in decreased recombinase activity B. Irradiation-sensitive ii. ATM mutations SCID (IR-SCID) C. Omenn syndrome iii. Artemis mutations 5.8 Matching: Match the immunoglobulin class to its main function: A. IgA i. Most abundant in serum and strongly induced during an immune response B. IgD ii. First one produced after B-cell activation C. IgE iii. Defense at mucosal sites D. IgG iv. Defense against parasites but also involved in allergic diseases

1	E. IgM v. Function not well known; may serve as auxiliary BCR 5.9 Fill-in-the-Blanks: Out of the five different antibody classes, two are secreted as multimers. _____ is secreted as a dimer and _______ is secreted as a pentamer, both of which have a(n) _______ as part of the multimeric complex. IgM and ______ are both expressed at the surface of mature B cells and are derived from the same pre-mRNA transcript. The balance of expression between these two is determined by alternative _______________ and is regulated by the snRNP __________. The process that regulates membrane-bound versus secreted forms of antibodies is determined by two factors: ___________ and ___________. Fcγ receptors on macrophages and neutrophils bind to the Fc portions of _____ and _____ isotype antibodies of the IgG class. Mast cells, basophils, and activated eosinophils, however, will bear Fcε receptors that bind to _______ class antibodies. IgA and IgG class antibodies are able to bind to ______, which

1	the IgG class. Mast cells, basophils, and activated eosinophils, however, will bear Fcε receptors that bind to _______ class antibodies. IgA and IgG class antibodies are able to bind to ______, which actively transports them to different body tissues and recycles them at the kidney glomerulus to prevent their loss and prolong their half-lives.

1	5.10 Multiple Choice: Which of the following is not true concerning the evolutionary history of the adaptive immune system? A. Adaptive immunity arose abruptly in evolution. B. Fruitflies and mosquitoes exhibit diversity in the secreted Dscam protein by alternative splicing of a vast array of different exons, while freshwater snails exhibit diversity in FREP genes by differential accumulation of genomic mutations in these genes. C. Jawless fish recombine VLR genes during DNA replication to engender diversity in these genes, which are expressed on lymphocytes and have GPI-anchored and secreted forms. D. RAG-1 arose from transposases while the RSSs it recognizes arose from terminal repeats from DNA transposons. E. MHC class I and class II genes arose before T cells and immunoglobulins in cartilaginous fish. General references.

1	E. MHC class I and class II genes arose before T cells and immunoglobulins in cartilaginous fish. General references. Fugmann, S.D., Lee, A.I., Shockett, P.E., Villey, I.J., and Schatz, D.G.: The RAG proteins and V(D)J recombination: complexes, ends, and transposition. Annu. Rev. Immunol. 2000, 18:495–527. Jung, D., Giallourakis, C., Mostoslavsky, R., and Alt, F.W.: Mechanism and control of V(D)J recombination at the immunoglobulin heavy chain locus. Annu. Rev. Immunol. 2006, 24:541–570. Schatz, D.G.: V(D)J recombination. Immunol. Rev. 2004, 200:5–11. Schatz, D.G., and Swanson, P.C.: V(D)J recombination: mechanisms of initiation. Annu. Rev. Genet. 2011, 45:167–202. Section references. 5-1 Immunoglobulin genes are rearranged in the progenitors of antibody-producing cells. Hozumi, N., and Tonegawa, S.: Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions. Proc. Natl Acad. Sci. USA 1976, 73:3628–3632.

1	Hozumi, N., and Tonegawa, S.: Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions. Proc. Natl Acad. Sci. USA 1976, 73:3628–3632. Seidman, J.G., and Leder, P.: The arrangement and rearrangement of antibody genes. Nature 1978, 276:790–795. Tonegawa, S., Brack, C., Hozumi, N., and Pirrotta, V.: Organization of immunoglobulin genes. Cold Spring Harbor Symp. Quant. Biol. 1978, 42:921–931. 5-2 Complete genes that encode a variable region are generated by the somatic recombination of separate gene segments. Early, P., Huang, H., Davis, M., Calame, K., and Hood, L.: An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH. Cell 1980, 19:981–992. Tonegawa, S., Maxam, A.M., Tizard, R., Bernard, O., and Gilbert, W.: Sequence of a mouse germ-line gene for a variable region of an immunoglobulin light chain. Proc. Natl Acad. Sci. USA 1978, 75:1485–1489.

1	5-3 Multiple contiguous V gene segments are present at each immunoglobulin locus. Maki, R., Traunecker, A., Sakano, H., Roeder, W., and Tonegawa, S.: Exon shuffling generates an immunoglobulin heavy chain gene. Proc. Natl Acad. Sci. USA 1980, 77:2138–2142. Matsuda, F., and Honjo, T.: Organization of the human immunoglobulin heavy-chain locus. Adv. Immunol. 1996, 62:1–29. Thiebe, R., Schable, K.F., Bensch,A., Brensing-Kuppers, J., Heim,V., Kirschbaum, T., Mitlohner, H., Ohnrich, M., Pourrajabi, S., Roschenthaler, F., et al.: The variable genes and gene families of the mouse immunoglobulin kappa locus. Eur. J. Immunol. 1999, 29:2072–2081. 5-4 Rearrangement of V, D, and J gene segments is guided by flanking DNA sequences. Grawunder, U., West, R.B., and Lieber, M.R.: Antigen receptor gene rearrangement. Curr. Opin. Immunol. 1998, 10:172–180. Lieber, M. R.: The mechanism of human nonhomologous DNA end joining. J. Biol. Chem. 2008, 283:1–5.

1	Lieber, M. R.: The mechanism of human nonhomologous DNA end joining. J. Biol. Chem. 2008, 283:1–5. Sakano, H., Huppi, K., Heinrich, G., and Tonegawa, S.: Sequences at the somatic recombination sites of immunoglobulin light-chain genes. Nature 1979, 280:288–294. 5-5 The reaction that recombines V, D, and J gene segments involves both lymphocyte-specific and ubiquitous DNA-modifying enzymes. Agrawal, A., and Schatz, D.G.: RAG1 and RAG2 form a stable postcleavage synaptic complex with DNA containing signal ends in V(D)J recombination. Cell 1997, 89:43–53. Ahnesorg, P., Smith, P., and Jackson, S.P.: XLF interacts with the XRCC4DNA ligase IV complex to promote nonhomologous end-joining. Cell 2006, 124:301–313.

1	Ahnesorg, P., Smith, P., and Jackson, S.P.: XLF interacts with the XRCC4DNA ligase IV complex to promote nonhomologous end-joining. Cell 2006, 124:301–313. Blunt, T., Finnie, N.J., Taccioli, G.E., Smith, G.C.M., Demengeot, J., Gottlieb, T.M., Ma, Y., Pannicke, U., Schwarz, K., and Lieber, M.R.: Hairpin opening and overhang processing by an Artemis:DNA-PKcs complex in V(D)J recombination and in nonhomologous end joining. Cell 2002, 108:781–794. Buck, D., Malivert, L., deChasseval, R., Barraud, A., Fondaneche, M.-C., Xanal, O., Plebani, A., Stephan, J.-L., Hufnagel, M., le Diest, F., et al.: Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly. Cell 2006, 124:287–299. Jung, D., Giallourakis, C., Mostoslavsky, R., and Alt, F.W.: Mechanism and control of V(D)J recombination at the immunoglobulin heavy chain locus. Annu. Rev. Immunol. 2006, 24:541–570.

1	Jung, D., Giallourakis, C., Mostoslavsky, R., and Alt, F.W.: Mechanism and control of V(D)J recombination at the immunoglobulin heavy chain locus. Annu. Rev. Immunol. 2006, 24:541–570. Kim, M.S., Lapkouski, M., Yang, W., and Gellert, M.: Crystal structure of the V(D)J recombinase RAG1-RAG2. Nature 2015, 518:507–511. Li, Z.Y., Otevrel, T., Gao, Y.J., Cheng, H.L., Seed, B., Stamato, T.D., Taccioli, G.E., and Alt, F.W.: The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination. Cell 1995, 83:1079–1089. Mizuta, R., Varghese, A.J., Alt, F.W., Jeggo, P.A., and Jackson, S.P.: Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA-repair defects associated with the murine scid mutation. Cell 1995, 80:813–823.

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1	5-6 The diversity of the immunoglobulin repertoire is generated by four main processes. Weigert, M., Perry, R., Kelley, D., Hunkapiller, T., Schilling, J., and Hood, L.: The joining of V and J gene segments creates antibody diversity. Nature 1980, 283:497–499. 5-7 The multiple inherited gene segments are used in different combinations. Lee, A., Desravines, S., and Hsu, E.: IgH diversity in an individual with only one million B lymphocytes. Dev. Immunol. 1993, 3:211–222. 5-8 Variable addition and subtraction of nucleotides at the junctions between gene segments contributes to the diversity of the third hypervariable region. Gauss, G.H., and Lieber, M.R.: Mechanistic constraints on diversity in human V(D)J recombination. Mol. Cell. Biol. 1996, 16:258–269. Gilfillan, S., Dierich, A., Lemeur, M., Benoist, C., and Mathis, D.: Mice lacking TdT: mature animals with an immature lymphocyte repertoire. Science 1993, 261:1755–1759.

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1	Bertocci, B., DeSmet, A., Weill, J.-C., and Reynaud, C.A. Non-overlapping functions of polX family DNA polymerases, pol μ, pol λ, and TdT, during immunoglobulin V(D)J recombination in vivo. Immunity 2006, 25:31–41. Lieber, M.R.: The polymerases for V(D)J recombination. Immunity 2006, 25:7–9. Rowen, L., Koop, B.F., and Hood, L.: The complete 685-kilobase DNA sequence of the human βT cell receptor locus. Science 1996, 272:1755–1762. Shinkai, Y., Rathbun, G., Lam, K.P., Oltz, E.M., Stewart, V., Mendelsohn, M., Charron, J., Datta, M., Young, F., Stall, A.M., et al.: RAG-2 deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 1992, 68:855–867. 5-10 T-cell receptors concentrate diversity in the third hypervariable region. Davis, M.M., and Bjorkman, P.J.: T-cell antigen receptor genes and T-cell recognition. Nature 1988, 334:395–402.

1	5-10 T-cell receptors concentrate diversity in the third hypervariable region. Davis, M.M., and Bjorkman, P.J.: T-cell antigen receptor genes and T-cell recognition. Nature 1988, 334:395–402. Garboczi, D.N., Ghosh, P., Utz, U., Fan, Q.R., Biddison, W.E., and Wiley, D.C.: Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature 1996, 384:134–141. Hennecke, J., Carfi, A., and Wiley, D.C.: Structure of a covalently stabilized complex of a human αβ T-cell receptor, influenza HA peptide and MHC class II molecule, HLA-DR1. EMBO J. 2000, 19:5611–5624. Hennecke, J., and Wiley, D.C.: T cell receptor–MHC interactions up close. Cell 2001, 104:1–4. Jorgensen, J.L., Esser, U., Fazekas de St. Groth, B., Reay, P.A., and Davis, M.M.: Mapping T-cell receptor–peptide contacts by variant peptide immunization of single-chain transgenics. Nature 1992, 355:224–230. 5-11 γ:δ T-cell receptors are also generated by gene rearrangement.

1	5-11 γ:δ T-cell receptors are also generated by gene rearrangement. Chien, Y.H., Iwashima, M., Kaplan, K.B., Elliott, J.F., and Davis, M.M.: A new T-cell receptor gene located within the alpha locus and expressed early in T-cell differentiation. Nature 1987, 327:677–682. Lafaille, J.J., DeCloux, A., Bonneville, M., Takagaki, Y., and Tonegawa, S.: Junctional sequences of T cell receptor gamma delta genes: implications for gamma delta T cell lineages and for a novel intermediate of V-(D)-J joining. Cell 1989, 59:859–870. Tonegawa, S., Berns, A., Bonneville, M., Farr, A.G., Ishida, I., Ito, K., Itohara, S., Janeway, C.A., Jr., Kanagawa, O., Kubo, R., et al.: Diversity, development, ligands, and probable functions of gamma delta T cells. Adv. Exp. Med. Biol. 1991, 292:53–61. 5-12 Different classes of immunoglobulins are distinguished by the structure of their heavy-chain constant regions.

1	5-12 Different classes of immunoglobulins are distinguished by the structure of their heavy-chain constant regions. Davies, D.R., and Metzger, H.: Structural basis of antibody function. Annu. Rev. Immunol. 1983, 1:87–117. 5-13 The constant region confers functional specialization on the antibody. Helm, B.A., Sayers, I., Higginbottom, A., Machado, D.C., Ling, Y., Ahmad, K., Padlan, E.A., and Wilson, A.P.M.: Identification of the high affinity receptor binding region in human IgE. J. Biol. Chem. 1996, 271:7494–7500. Nimmerjahn, F., and Ravetch, J.V.: Fc-receptors as regulators of immunity. Adv. Immunol. 2007, 96:179–204. Sensel, M.G., Kane, L.M., and Morrison, S.L.: Amino acid differences in the N-terminus of CH2 influence the relative abilities of IgG2 and IgG3 to activate complement. Mol. Immunol. 34:1019–1029. 5-14 IgM and IgD are derived from the same pre-mRNA transcript and are both expressed on the surface of mature B cells.

1	5-14 IgM and IgD are derived from the same pre-mRNA transcript and are both expressed on the surface of mature B cells. Abney, E.R., Cooper, M.D., Kearney, J.F., Lawton, A.R., and Parkhouse, R.M.: Sequential expression of immunoglobulin on developing mouse B lymphocytes: a systematic survey that suggests a model for the generation of immunoglobulin isotype diversity. J. Immunol. 1978, 120:2041–2049. Blattner, F.R., and Tucker, P.W.: The molecular biology of immunoglobulin D. Nature 1984, 307:417–422. Enders, A., Short, A., Miosge, L.A., Bergmann, H., Sontani, Y., Bertram, E.M., Whittle, B., Balakishnan, B., Yoshida, K., Sjollema, G., et al.: Zinc-finger protein ZFP318 is essential for expression of IgD, the alternatively spliced Igh product made by mature B lymphocytes. Proc. Natl Acad. Sci. USA 2014, 111:4513–4518. Goding, J.W., Scott, D.W., and Layton, J.E.: Genetics, cellular expression and function of IgD and IgM receptors. Immunol. Rev. 1977, 37:152–186.

1	Goding, J.W., Scott, D.W., and Layton, J.E.: Genetics, cellular expression and function of IgD and IgM receptors. Immunol. Rev. 1977, 37:152–186. 5-15 Transmembrane and secreted forms of immunoglobulin are generated from alternative heavy-chain mRNA transcripts. Early, P., Rogers, J., Davis, M., Calame, K., Bond, M., Wall, R., and Hood, L.: Two mRNAs can be produced from a single immunoglobulin μgene by alternative RNA processing pathways. Cell 1980, 20:313–319. Martincic, K., Alkan, S.A., Cheatle, A., Borghesi, L., and Milcarek, C.: Transcription elongation factor ELL2 directs immunoglobulin secretion in plasma cells by stimulating altered RNA processing. Nat. Immunol. 2009, 10:1102–1109. Peterson, M.L., Gimmi, E.R., and Perry, R.P.: The developmentally regulated shift from membrane to secreted μ mRNA production is accompanied by an increase in cleavage-polyadenylation efficiency but no measurable change in splicing efficiency. Mol. Cell. Biol. 1991, 11:2324–2327.

1	Rogers, J., Early, P., Carter, C., Calame, K., Bond, M., Hood, L., and Wall, R.: Two mRNAs with different 3ʹ ends encode membrane-bound and secreted forms of immunoglobulin μchain. Cell 1980, 20:303–312. Takagaki, Y., and Manley, J.L.: Levels of polyadenylation factor CstF-64 control IgM heavy chain mRNA accumulation and other events associated with B cell differentiation. Mol. Cell. 1998, 2:761–771. Takagaki, Y., Seipelt, R.L., Peterson, M.L., and Manley, J.L.: The polyadenylation factor CstF-64 regulates alternative processing of IgM heavy chain premRNA during B cell differentiation. Cell 1996, 87:941–952. 5-16 IgM and IgA can form polymers by interacting with the J chain. Hendrickson, B.A., Conner, D.A., Ladd, D.J., Kendall, D., Casanova, J.E., Corthesy, B., Max, E.E., Neutra, M.R., Seidman, C.E., and Seidman, J.G.: Altered hepatic transport of IgA in mice lacking the J chain. J. Exp. Med. 1995, 182:1905–1911.

1	Niles, M.J., Matsuuchi, L., and Koshland, M.E.: Polymer IgM assembly and secretion in lymphoid and nonlymphoid cell-lines—evidence that J chain is required for pentamer IgM synthesis. Proc. Natl Acad. Sci. USA 1995, 92:2884–2888. 5-17 Some invertebrates generate extensive diversity in a repertoire of immunoglobulin-like genes. Dong, Y., Taylor, H.E., and Dimopoulos, G.: AgDscam, a hypervariable immunoglobulin domain-containing receptor of the Anopheles gambiae innate immune system. PLoS Biol. 2006, 4:e229. Loker, E.S., Adema, C.M., Zhang, S.M., and Kepler, T.B.: Invertebrate immune systems—not homogeneous, not simple, not well understood. Immunol. Rev. 2004, 198:10–24. Watson, F.L., Puttmann-Holgado, R., Thomas, F., Lamar, D.L., Hughes, M., Kondo, M., Rebel, V.I., and Schmucker, D.: Extensive diversity of Ig-superfamily proteins in the immune system of insects. Science 2005, 309:1826–1827.

1	Zhang, S.M., Adema, C.M., Kepler, T.B., and Loker, E.S.: Diversification of Ig superfamily genes in an invertebrate. Science 2004, 305:251–254. 5-18 Agnathans possess an adaptive immune system that uses somatic gene rearrangement to diversify receptors built from LRR domains. Boehm, T., McCurley, N., Sutoh, Y., Schorpp, M., Kasahara, M., and Cooper, M.D.: VLR-based adaptive immunity. Annu. Rev. Immunol. 2012, 30:203–220. Finstad, J., and Good, R.A.: The evolution of the immune response. 3. Immunologic responses in the lamprey. J. Exp. Med. 1964, 120:1151–1168. Guo, P., Hirano, M., Herrin, B.R., Li, J., Yu, C., Sadlonova, A., and Cooper, M.D.: Dual nature of the adaptive immune system in lampreys. Nature 2009, 459:796–801. [Erratum: Nature 2009, 460:1044.] Han, B.W., Herrin, B.R., Cooper, M.D., and Wilson, I.A.: Antigen recognition by variable lymphocyte receptors. Science 2008, 321:1834–1837.

1	Han, B.W., Herrin, B.R., Cooper, M.D., and Wilson, I.A.: Antigen recognition by variable lymphocyte receptors. Science 2008, 321:1834–1837. Hirano, M., Guo, P., McCurley, N., Schorpp, M., Das, S., Boehm, T., and Cooper, M.D.: Evolutionary implications of a third lymphocyte lineage in lampreys. Nature 2013, 501:435–438. Litman, G.W., Finstad, F.J., Howell, J., Pollara, B.W., and Good, R.A.: The evolution of the immune response. 3. Structural studies of the lamprey immunoglobulin. J. Immunol. 1970, 105:1278–1285. of immunoglobulin-like genes appeared abruptly in the cartilaginous fishes. Fugmann, S.D., Messier, C., Novack, L.A., Cameron, R.A., and Rast, J.P.: An ancient evolutionary origin of the Rag1/2 gene locus. Proc. Natl Acad. Sci. USA 2006, 103:3728–3733. Kapitonov, V.V., and Jurka, J.: RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons. PLoS Biol. 2005, 3:e181.

1	Kapitonov, V.V., and Jurka, J.: RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons. PLoS Biol. 2005, 3:e181. Litman, G.W., Rast, J.P., and Fugmann, S.D.: The origins of vertebrate adaptive immunity. Nat. Rev. Immunol. 2010, 10:543–553. Suzuki, T., Shin-I, T., Fujiyama, A., Kohara, Y., and Kasahara, M.: Hagfish leukocytes express a paired receptor family with a variable domain resembling those of antigen receptors. J. Immunol. 2005, 174:2885–2891. 5-20 Different species generate immunoglobulin diversity in different ways. Becker, R.S., and Knight, K.L.: Somatic diversification of immunoglobulin heavy chain VDJ genes: evidence for somatic gene conversion in rabbits. Cell 1990, 63:987–997. Knight, K.L., and Crane, M.A.: Generating the antibody repertoire in rabbit. Adv. Immunol. 1994, 56:179–218. Kurosawa, K., and Ohta, K.: Genetic diversification by somatic gene conversion. Genes (Basel) 2011, 2:48–58.

1	Kurosawa, K., and Ohta, K.: Genetic diversification by somatic gene conversion. Genes (Basel) 2011, 2:48–58. Reynaud, C.A., Bertocci, B., Dahan, A., and Weill, J.C.: Formation of the chicken B-cell repertoire—ontogeny, regulation of Ig gene rearrangement, and diversification by gene conversion. Adv. Immunol. 1994, 57:353–378. Reynaud, C.A., Garcia, C., Hein, W.R., and Weill, J.C.: Hypermutation generating the sheep immunoglobulin repertoire is an antigen independent process. Cell 1995, 80:115–125. Vajdy, M., Sethupathi, P., and Knight, K.L.: Dependence of antibody somatic diversification on gut-associated lymphoid tissue in rabbits. J. Immunol. 1998, 160:2725–2729. Winstead, C.R., Zhai, S.K., Sethupathi, P., and Knight, K.L.: Antigen-induced somatic diversification of rabbit IgH genes: gene conversion and point mutation. J. Immunol. 1999, 162:6602–6612. 5-21 Both α:βand γ:δ T-cell receptors are present in cartilaginous fishes.

1	5-21 Both α:βand γ:δ T-cell receptors are present in cartilaginous fishes. Rast, J.P., Anderson, M.K., Strong, S.J., Luer, C., Litman, R.T., and Litman, G.W.: α, β, γ, and δ T-cell antigen receptor genes arose early in vertebrate phylo geny. Immunity 1997, 6:1–11. Rast, J.P., and Litman, G.W.: T-cell receptor gene homologs are present in the most primitive jawed vertebrates. Proc. Natl Acad. Sci. USA 1994, 91:9248–9252. 5-22 MHC class I and class II molecules are also first found in the cartilaginous fishes. Hashimoto, K., Okamura, K., Yamaguchi, H., Ototake, M., Nakanishi, T., and Kurosawa, Y.: Conservation and diversification of MHC class I and its related molecules in vertebrates. Immunol. Rev. 1999, 167:81–100. Kurosawa, Y., and Hashimoto, K.: How did the primordial T cell receptor and MHC molecules function initially? Immunol. Cell Biol. 1997, 75:193–196.

1	Kurosawa, Y., and Hashimoto, K.: How did the primordial T cell receptor and MHC molecules function initially? Immunol. Cell Biol. 1997, 75:193–196. Ohta, Y., Okamura, K., McKinney, E.C., Bartl, S., Hashimoto, K., and Flajnik, M.F.: Primitive synteny of vertebrate major histocompatibility complex class I and class II genes. Proc. Natl Acad. Sci. USA 2000, 97:4712–4717. Okamura, K., Ototake, M., Nakanishi, T., Kurosawa, Y., and Hashimoto, K.: The most primitive vertebrates with jaws possess highly polymorphic MHC class I genes comparable to those of humans. Immunity 1997, 7:777–790. Antigen Presentation to T Lymphocytes

1	Vertebrate adaptive immune cells possess two types of antigen receptors: the immunoglobulins that serve as antigen receptors on B cells, and the T-cell receptors. While immunoglobulins can recognize native antigens, T cells recognize only antigens that are displayed by MHC complexes on cell surfaces. The conventional α:β T cells recognize antigens as peptide:MHC complexes (see Section 4-13). The peptides recognized by α:β T cells can be derived from the normal turnover of self proteins, from intracellular pathogens, such as viruses, or from products of pathogens taken up from the extracellular fluid. Various tolerance mechanisms normally prevent self peptides from initiating an immune response; when these mechanisms fail, self peptides can become the target of autoimmune responses, as discussed in Chapter 15. Other classes of T cells, such as MAIT cells and γ:δ T cells (see Sections 4-18 and 4-20), recognize different types of surface molecules whose expression may indicate infection

1	in Chapter 15. Other classes of T cells, such as MAIT cells and γ:δ T cells (see Sections 4-18 and 4-20), recognize different types of surface molecules whose expression may indicate infection or cellular stress.

1	The first part of this chapter describes the cellular pathways used by various types of cells to generate peptide:MHC complexes recognized by α:β T cells. This process participates in adaptive immunity in at least two different ways. In somatic cells, peptide:MHC complexes can signal the presence of an intracellular pathogen for elimination by armed effector T cells. In dendritic cells, which may not themselves be infected, peptide:MHC complexes serve to activate antigen-specific effector T cells. We will also introduce mechanisms by which certain pathogens defeat adaptive immunity by blocking the production of peptide:MHC complexes.

1	The second part of this chapter focuses on the MHC class I and II genes and their tremendous variability. The MHC molecules are encoded within a large cluster of genes that were first identified by their powerful effects on the immune response to transplanted tissues and were therefore called the major histocompatibility complex (MHC). There are several different MHC molecules in each class, and each of their genes is highly polymorphic, with many variants present in the population. MHC polymorphism has a profound effect on antigen recognition by T cells, and the combination of multiple genes and polymorphism greatly extends the range of peptides that can be presented to T cells in each individual and in populations as a whole, thus enabling individuals to respond to the wide range of potential pathogens they will encounter. The MHC also contains genes other than those for the MHC molecules; some of these genes are involved in the processing of antigens to produce peptide:MHC

1	of potential pathogens they will encounter. The MHC also contains genes other than those for the MHC molecules; some of these genes are involved in the processing of antigens to produce peptide:MHC complexes.

1	The last part of the chapter discusses the ligands for unconventional classes of T cells. We will examine a group of proteins similar to MHC class I molecules that have limited polymorphism, some encoded within the MHC and others encoded outside the MHC. These so-called nonclassical MHC class I proteins serve various functions, some acting as ligands for γ:δ T-cell receptors and MAIT cells, or as ligands for NKG2D expressed by T cells and NK cells. In addition, we will introduce a special subset of α:β T cells known as invariant NKT cells that recognize microbial lipid antigens presented by these proteins. The generation of α:β T-cell receptor ligands. The major histocompatibility complex and its function. Generation of ligands for unconventional T-cell subsets. The generation of α:β T-cell receptor ligands.

1	The major histocompatibility complex and its function. Generation of ligands for unconventional T-cell subsets. The generation of α:β T-cell receptor ligands. The protective function of T cells depends on their recognition of cells harboring intracellular pathogens or that have internalized their products. As we saw in Chapter 4, the ligand recognized by an α:β T-cell receptor is a peptide bound to an MHC molecule and displayed on a cell surface. The generation of peptides from native proteins is commonly referred to as antigen processing, while peptide display at the cell surface by the MHC molecule is referred to as antigen presentation. We have already described the structure of MHC molecules and seen how they bind peptide antigens in a cleft, or groove, on their outer surface (see Sections 4-13 to 4-16). We will now look at how peptides are generated from the proteins derived from pathogens and how they are loaded onto MHC class I or MHC class II molecules.

1	and in triggering their effector functions to attack pathogen- infected cells.

1	The processing and presentation of pathogen-derived antigens has two distinct purposes: inducing the development of armed effector T cells, and triggering the effector functions of these armed cells at sites of infection. MHC class I molecules bind peptides that are recognized by CD8 T cells, and MHC class II molecules bind peptides that are recognized by CD4 T cells, a pattern of recognition determined by specific binding of the CD8 or CD4 molecules to the respective MHC molecules (see Section 4-18). The importance of this specificity of recognition lies in the different distributions of MHC class I and class II molecules on cells throughout the body. Nearly all somatic cells (except red blood cells) express MHC class I molecules. Consequently, the CD8 T cell is primarily responsible for pathogen surveillance and cytolysis of somatic cells. Also called cytotoxic T cells, their function is to kill the cells they recognize. CD8 T cells are therefore an important mechanism in

1	for pathogen surveillance and cytolysis of somatic cells. Also called cytotoxic T cells, their function is to kill the cells they recognize. CD8 T cells are therefore an important mechanism in eliminating sources of new viral particles and bacteria that live only in the cytosol, and thus freeing the host from infection.

1	By contrast, MHC class II molecules are expressed primarily only on cells of the immune system, and particularly by dendritic cells, macrophages, and B cells. Thymic cortical epithelial cells and activated, but not naive, T cells can express MHC class II molecules, which can also be induced on many cells in response to the cytokine IFN-γ. Thus, CD4 T cells can recognize their cognate antigens during their development in the thymus, on a limited set of ‘professional’ antigen-presenting cells, and on other somatic cells under specific inflammatory conditions. Effector CD4 T cells comprise several subsets with different activities that help eliminate the pathogens. Importantly, naive CD8 and CD4 T cells can become armed effector cells only after encountering their cognate antigen once it has been processed and presented by activated dendritic cells.

1	In considering antigen processing, it is important to distinguish between the various cellular compartments from which antigens can be derived (Fig. 6.1). These compartments, which are separated by membranes, include the cytosol and the various vesicular compartments involved in endocytosis and secretion. Peptides derived from the cytosol are transported into the endoplasmic reticulum and directly loaded onto newly synthesized MHC class I molecules on the same cell for recognition by T cells, as we will discuss below in greater detail. Because viruses and some bacteria replicate in the cytosol or in the contiguous nuclear compartment, peptides from their components can be loaded onto MHC class I molecules by this process (Fig. 6.2, first upper panel).

1	Fig. 6.1 There are two categories of major intracellular compartments, separated by membranes. One compartment is the cytosol, which communicates with the nucleus via pores in the nuclear membrane. The other is the vesicular system, which comprises the endoplasmic reticulum, Golgi apparatus, endosomes, lysosomes, and other intracellular vesicles. The vesicular system can be thought of as being continuous with the extracellular fluid. Secretory vesicles bud off from the endoplasmic reticulum and are transported via fusion with Golgi membranes to move vesicular contents out of the cell. Extracellular material is taken up by endocytosis or phagocytosis into endosomes or phagosomes, respectively. The fusion of incoming and outgoing vesicles is important both for pathogen destruction in cells such as neutrophils and for antigen presentation. Autophagosomes surround components in the cytosol and deliver them to lysosomes in a process known as autophagy.

1	This pathway of recognition is sometimes referred to as direct presentation, and can identify both somatic and immune cells that are infected by a pathogen. Certain pathogenic bacteria and protozoan parasites survive ingestion by macrophages and are able to replicate inside the intracellular vesicles of the endosomal–lysosomal system (Fig. 6.2, second panel). Other pathogenic bacteria proliferate outside cells, and can be internalized, along with their toxic products, by phagocytosis, receptor-mediated endocytosis, or macropinocytosis into endosomes and lysosomes, where they are broken down by digestive enzymes. For example, receptor-mediated endocytosis by B cells can efficiently internalize extracellular antigens through B-cell receptors (Fig. 6.2, third panel). Virus particles and parasite antigens in extracellular fluids can also be taken up by these routes and degraded, and their peptides presented to T cells.

1	either the cytosolic or the vesicular compartments. Top, first panel: viruses and some bacteria replicate in the cytosolic compartment. Their antigens are presented by MHC class I molecules to activate killing by cytotoxic CD8 T cells. Second panel: other bacteria and some parasites are taken up into endosomes, usually by specialized phagocytic cells such as macrophages. Here they are killed and degraded, or in some cases are able to survive and proliferate within the vesicle. Their antigens are presented by MHC class II molecules to activate cytokine production by CD4 T cells. Third panel: proteins derived from extracellular pathogens may bind to cell-surface receptors and enter the vesicular system by endocytosis, illustrated here for antigens bound by the surface immunoglobulin of B cells. These antigens are presented by MHC class II molecules to CD4 helper T cells, which can then stimulate the B cells to produce antibody.

1	Fig. 6.4 Autophagy pathways can deliver cytosolic antigens for presentation by MHC class II molecules. In the process of autophagy, portions of the cytoplasm are taken into autophagosomes, specialized vesicles that are fused with endocytic vesicles and eventually with lysosomes, where the contents are catabolized. Some of the resulting peptides of this process can be bound to MHC class II molecules and presented on the cell surface. In dendritic cells and macrophages, this can occur in the absence of activation, so that immature dendritic cells may express self peptides in a tolerogenic context, rather than inducing T-cell responses to self antigens.

1	Fig. 6.3 Cross-presentation of extracellular antigens on MHC class I molecules by dendritic cells. Certain subsets of dendritic cells are efficient in capturing exogenous proteins and loading peptides derived from them onto MHC class I molecules. There is evidence that several cellular pathways may be involved. One route may involve the translocation of ingested proteins from the phagolysosome into the cytosol for degradation by the proteasome, with the resultant peptides then passing through TAP (see Section 6-3) into the endoplasmic reticulum, where they load onto MHC class I molecules in the usual way. Another route may involve direct transport of antigens from the phagolysosome into a vesicular loading compartment—without passage through the cytosol—where peptides are allowed to be bound to mature MHC class I molecules.

1	For loading peptides onto MHC class II molecules, dendritic cells, macrophages, and B cells are able to capture exogenous proteins via endocytic vesicles and through specific cell-surface receptors. For B cells, this process of antigen capture can include the B-cell receptor. The peptides that are derived from these proteins are loaded onto MHC class II molecules in specially modified endocytic compartments in these antigen-presenting cells, which we will discuss in more detail later. In dendritic cells, this pathway operates to activate naive CD4 T cells to become effector T cells. Macrophages take up particulate material by phagocytosis and so mainly present pathogen-derived peptides on MHC class II molecules. In macrophages, such antigen presentation may be used to indicate the presence of a pathogen within its vesicular compartment. Effector CD4 T cells, on recognizing antigen, produce cytokines that can activate the macrophage to destroy the pathogen. Some intravesicular

1	presence of a pathogen within its vesicular compartment. Effector CD4 T cells, on recognizing antigen, produce cytokines that can activate the macrophage to destroy the pathogen. Some intravesicular pathogens have adapted to resist intracellular killing, and the macrophages in which they live require these cytokines to kill the pathogen: this is one of the roles of the TH1 subset of CD4 T cells. Other CD4 T cell subsets have roles in regulating other aspects of the immune response, and some CD4 T cells even have cytotoxic activity. In B cells, antigen presentation may serve to recruit help from CD4 T cells that recognize the same protein antigen as the B cell. By efficiently endocytosing a specific antigen via their surface immunoglobulin and presenting the antigen-derived peptides on MHC class II molecules, B cells can activate CD4 T cells that will in turn serve as helper T cells for the production of antibodies against that antigen.

1	Beyond the presentation of exogenous proteins, MHC class II molecules can also be loaded with peptides derived from cytosolic proteins by a ubiquitous pathway of autophagy, in which cytoplasmic proteins are delivered into the endocytic system for degradation in lysosomes (Fig. 6.4). This pathway can serve in the presentation of self-cytosolic proteins for the induction of tolerance to self antigens, and also as a means for presenting antigens from pathogens, such as herpes simplex virus, that have accessed the cell’s cytosol. 6-2 Peptides are generated from ubiquitinated proteins in the cytosol by the proteasome.

1	Proteins in cells are continually being degraded and replaced with newly synthesized proteins. Much cytosolic protein degradation is carried out by a large, multicatalytic protease complex called the proteasome (Fig. 6.5). A typical proteasome is composed of one 20S catalytic core and two 19S regulatory caps, one at each end; both the core and the caps are multisubunit complexes of proteins. The 20S core is a large cylindrical complex of some 28 subunits, arranged in four stacked rings of seven subunits each around a hollow core. The two outer rings are composed of seven distinct α subunits and are noncatalytic. The two inner rings of the 20S proteasome core are composed of seven distinct β subunits. The constitutively expressed proteolytic subunits are β1, β2, and β5, which form the catalytic chamber. The 19S regulator is composed of a base containing nine subunits that binds directly to the α ring of the 20S core particle and a lid that has up to 10 different subunits. The

1	the catalytic chamber. The 19S regulator is composed of a base containing nine subunits that binds directly to the α ring of the 20S core particle and a lid that has up to 10 different subunits. The association of the 20S core with a 19S cap requires ATP as well as the ATPase activity of many of the caps’ subunits. One of the 19S caps binds and delivers proteins into the proteasome, while the other keeps them from exiting prematurely.

1	Proteins in the cytosol are tagged for degradation via the ubiquitin–proteasome system (UPS). This begins with the attachment of a chain of several ubiquitin molecules to the target protein, a process called ubiquitination. First, a lysine residue on the targeted protein is chemically linked to the glycine at the carboxy terminus of one ubiquitin molecule. Ubiquitin chains are then formed by linking the lysine at residue 48 (K48) of the first ubiquitin to the carboxy-terminal glycine of a second ubiquitin, and so on until at least 4 ubiquitin molecules are bound. This K48-linked type of ubiquitin chain is recognized by the 19S cap of the proteasome, which then unfolds the tagged protein so that it can be introduced into the proteasome’s catalytic core. There the protein chain is degraded with a general lack of sequence specificity into short peptides, which are subsequently released into the cytosol. The general degradative functions of the proteasome have been co-opted for antigen

1	with a general lack of sequence specificity into short peptides, which are subsequently released into the cytosol. The general degradative functions of the proteasome have been co-opted for antigen presentation, so that MHC molecules have evolved to work with the peptides that the proteasome can produce.

1	Various lines of evidence implicate the proteasome in the production of peptide ligands for MHC class I molecules. Experimentally tagging proteins with ubiquitin results in more efficient presentation of their peptides by MHC class I molecules, and inhibitors of the proteolytic activity of the proteasome inhibit antigen presentation by MHC class I molecules. Whether the proteasome is the only cytosolic protease capable of generating peptides for transport into the endoplasmic reticulum is not known.

1	The constitutive β1, β2, and β5 subunits of the catalytic chamber are sometimes replaced by three alternative catalytic subunits that are induced by interferons. These induced subunits are called β1i (or LMP2), β2i (or MECL-1), and β5i (or LMP7). Both β1i and β5i are encoded by the PSMB9 and PSMB8 genes, which are located in the MHC locus, whereas β2i is encoded by PSMB10 outside the MHC locus. Thus, the proteasome can exist both as both a constitutive proteasome present in all cells and as the immunoproteasome, which is present in cells stimulated with interferons. MHC class I proteins are also induced by interferons. The replacement of the β subunits by their interferon-inducible counterparts alters the enzymatic specificity of the proteasome such that there is increased cleavage of polypeptides after hydrophobic residues, and decreased cleavage after acidic residues. This produces peptides with carboxy-terminal residues that are preferred anchor residues for binding to most MHC

1	polypeptides after hydrophobic residues, and decreased cleavage after acidic residues. This produces peptides with carboxy-terminal residues that are preferred anchor residues for binding to most MHC class I molecules (see Chapter 4) and are also the preferred structures for transport by TAP.

1	Another substitution for a β subunit in the catalytic chamber has been found to occur in cells in the thymus. Epithelial cells of the thymic cortex (cTECs) express a unique β subunit, called β5t, that is encoded by PSMB11. In cTECs, β5t becomes a component of the proteasome in association with β1i and β2i, and this specialized type of proteasome is called the thymoproteasome. Mice lacking expression of β5t have reduced numbers of CD8 T cells, indicating that the peptide:MHC complexes produced by the thymoproteasome are important in CD8 T-cell development in the thymus.

1	Interferon-γ (IFN-γ) can further increase the production of antigenic peptides by inducing expression of the PA28 proteasome-activator complex that binds to the proteasome. PA28 is a sixor seven-membered ring composed of two proteins, PA28α and PA28β, both of which are induced by IFN-γ. A PA28 ring, which can bind to either end of the 20S proteasome core in place of the 19S regulatory cap, acts to increase the rate at which peptides are released (Fig. 6.6). In addition to simply providing more peptides, the increased rate of Polyubiquitinated proteins are bound by the 19S cap and degraded within the catalytic core, releasing peptides into the cytosol

1	Polyubiquitinated proteins are bound by the 19S cap and degraded within the catalytic core, releasing peptides into the cytosol Fig. 6.5 Cytosolic proteins are degraded by the ubiquitin–proteasome system into short peptides. The proteasome is composed of a 20S catalytic core, which consists of four multisubunit rings (see text), and two 19S regulatory caps on either end. Proteins (orange) that are targeted become covalently tagged with K48-linked polyubiquitin chains (yellow) through the actions of various E3 ligases. The 19S regulatory cap recognizes polyubiquitin and draws the tagged protein inside the catalytic chamber; there, the protein is degraded, giving rise to small peptide fragments that are released back into the cytoplasm.

1	Panel a: in this side view cross-section, the heptamer rings of the PA28 proteasome activator (yellow) interact with the α subunits (pink) at either end of the core proteasome (the β subunits that make up the catalytic cavity of the core are in blue). Within this region is the α-annulus (green), a narrow ringlike opening that is normally blocked by other parts of the α subunits (shown in red). Panel b: a close-up view from the top, looking into the α-annulus without PA28 bound. Panel c: with the same perspective, the binding of PA28 to the proteasome changes the conformation of the α subunits, moving those parts of the molecule that block the α-annulus, and opening the end of the cylinder. For simplicity, PA28 is not shown. Structures courtesy of F. Whitby. Fig. 6.6 The PA28 proteasome activator binds to either end of the proteasome. flow allows potentially antigenic peptides to escape additional processing that might destroy their antigenicity.

1	Fig. 6.6 The PA28 proteasome activator binds to either end of the proteasome. flow allows potentially antigenic peptides to escape additional processing that might destroy their antigenicity. Translation of self or pathogen-derived mRNAs in the cytoplasm generates not only properly folded proteins but also a significant quantity—possibly up to 30%—of peptides and proteins that are known as defective ribosomal products (DRiPs). These include peptides translated from introns in improperly spliced mRNAs, translations of frameshifts, and improperly folded proteins, which are tagged by ubiquitin for rapid degradation by the proteasome. This seemingly wasteful process provides another source of peptides and ensures that both self proteins and proteins derived from pathogens generate abundant peptide substrates for eventual presentation by MHC class I proteins. 6-3 Peptides from the cytosol are transported by TAP into the to MHC class I molecules.

1	6-3 Peptides from the cytosol are transported by TAP into the to MHC class I molecules. The polypeptide chains of proteins destined for the cell surface, such as the two chains of MHC molecules, are translocated during synthesis into the lumen of the endoplasmic reticulum, where two chains fold correctly and assemble with each other. This means that the peptide-binding site of the MHC class I molecule is formed in the lumen of the endoplasmic reticulum and is never exposed to the cytosol. The antigen fragments that bind to MHC class I molecules, however, are typically derived from proteins made in the cytosol. This raises the question, How are these peptides able to bind to MHC class I molecules and be delivered to the cell surface?

1	The answer was aided by analysis of mutant cells that had a defect in antigen presentation by MHC class I molecules. These cells expressed far fewer MHC class I proteins than normal on their surface despite normal synthesis of these molecules in the cytoplasm. This defect could be corrected by adding synthetic peptides to the culture medium, suggesting that the supply of peptides to the MHC class I molecules in the endoplasmic reticulum might be the limiting factor. Analysis of the DNA of the mutant cells identified the problem responsible for this phenotype to be in genes for members of the ATP-binding cassette (ABC) family of proteins; the ABC proteins mediate the ATP-dependent transport of ions, sugars, amino acids, and peptides across membranes.

1	Missing from the mutant cells were two ABC proteins, called transporters associated with antigen processing-1 and -2 (TAP1 and TAP2), that are normally associated with the endoplasmic reticulum membrane. Transfection of the mutant cells with the missing genes restored the presentation of peptides by the cell’s MHC class I molecules. The two TAP proteins form a heterodimer in the membrane (Fig. 6.7), and mutations in either TAP gene can prevent antigen presentation by MHC class I molecules. The genes TAP1 and TAP2 are located in the MHC locus (see Section 6-10), near the PSMB9 and PSMB8 genes, and their basal level of expression is further enhanced by interferons produced in response to viral infection, similar to MHC class I and β1, β2, and β5 subunits of the proteasome. This induction results in increased delivery of cytosolic peptides into the endoplasmic reticulum.

1	Microsomal vesicles from non-mutant cells can mimic the endoplasmic reticulum in assays in vitro, by internalizing peptides that then bind to MHC class I molecules present in the microsome lumen. In contrast, vesicles from TAP1or TAP2-deficient cells do not take up peptides. Peptide transport into normal microsomes requires ATP hydrolysis, confirming that the TAP1:TAP2 complex is an ATP-dependent peptide transporter. The TAP complex has limited specificity for the peptides it will transport, transporting peptides of between 8 and 16 amino acids in length and preferring peptides that have hydrophobic or basic residues at the carboxy terminus—the precise features of peptides that bind MHC class I molecules (see Section 4-15). The TAP complex has a bias against proline in the first three amino-terminal residues, but lacks in any true peptide-sequence specificity. The discovery of TAP explained how viral peptides from proteins synthesized in the cytosol gain access to the lumen of the

1	amino-terminal residues, but lacks in any true peptide-sequence specificity. The discovery of TAP explained how viral peptides from proteins synthesized in the cytosol gain access to the lumen of the endoplasmic reticulum and are bound by MHC class I molecules.

1	Peptides produced in the cytosol are protected from complete degradation by cellular chaperones such as the TCP-1 ring complex (TRiC), but many of these peptides are longer than can be bound by MHC class I molecules. Evidence indicates that the carboxy terminus of peptide antigens is produced by cleavage in the proteasome. However, the amino terminus of peptides that are too long to bind MHC class I molecules can be trimmed by an enzyme called the endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP). Like other components of the antigen-processing pathway, expression of ERAAP is increased by IFN-γ stimulation. Mice lacking the enzyme ERAAP have an altered repertoire of peptides loaded onto MHC class I molecules. Although the loading of some peptides is not affected by the absence of ERAAP, other peptides fail to load normally, and many unstable and immunogenic peptides not normally present are found bound to MHC molecules on the cell surface. This causes

1	by the absence of ERAAP, other peptides fail to load normally, and many unstable and immunogenic peptides not normally present are found bound to MHC molecules on the cell surface. This causes cells from ERAAP-deficient mice to be immunogenic for T cells from wild-type mice, demonstrating that ERAAP is an important editor of the normal peptide:MHC repertoire.

1	6-4 Newly synthesized MHC class I molecules are retained in the endoplasmic reticulum until they bind a peptide. Binding a peptide is an important step in the assembly of a stable MHC class I molecule. When the supply of peptides into the endoplasmic reticulum is disrupted, as in TAP-mutant cells, newly synthesized MHC class I molecules

1	Fig. 6.7 TAP1 and TAP2 form a peptide transporter in the endoplasmic reticulum membrane. Upper panel: TAP1 and TAP2 are individual polypeptide chains, each with one hydrophobic and one ATP-binding domain. The two chains assemble into a heterodimer to form a four-domain transporter typical of the ATP-binding cassette (ABC) family. The hydrophobic transmembrane domains have multiple transmembrane regions (not shown here). The ATP-binding domains lie within the cytosol, whereas the hydrophobic domains project through the membrane into the lumen of the endoplasmic reticulum (ER) to form a channel through which peptides can pass. Lower panel: electron microscopic reconstruction of the structure of the TAP1:TAP2 heterodimer. Panel a shows the surface of the TAP transporter as seen from the lumen of the ER, looking down onto the top of the transmembrane domains, while panel b shows a lateral view of the TAP heterodimer in the plane of the membrane. The ATP-binding domains form two lobes

1	lumen of the ER, looking down onto the top of the transmembrane domains, while panel b shows a lateral view of the TAP heterodimer in the plane of the membrane. The ATP-binding domains form two lobes beneath the transmembrane domains; the bottom edges of these lobes are just visible at the back of the lateral view. TAP structures courtesy of G. Velarde.

1	are held in the endoplasmic reticulum in a partly folded state. This explains why the rare human patients who have been identified with immunodeficiency due to defects in TAP1 and TAP2 have few MHC class I molecules on their cell surfaces, a condition known as MHC class I deficiency. The folding and assembly of a complete MHC class I molecule (see Fig. 4.19) depends on the association of the MHC class I α chain first with β2-microglobulin and then with peptide, and this process involves a number of accessory proteins with chaperone-like functions. Only after peptide has bound is the MHC class I molecule released from the endoplasmic reticulum and transported to the cell surface.

1	Newly synthesized MHC class I αchains that enter the endoplasmic reticulum membranes bind to calnexin, a general-purpose chaperone protein that retains the MHC class I molecule in a partly folded state (Fig. 6.8). Calnexin also associates with partly folded T-cell receptors, immunoglobulins, and MHC class II molecules, and so has a central role in the assembly of many immunological as well as non-immunological proteins. When β2-microglobulin binds to the α chain, the partly folded MHC class I α:β2-microglobulin heterodimer dissociates from calnexin and binds to an assembly of proteins called the MHC class I peptide-loading complex (PLC). One component of ubiquitinated protein normal proteins (>70%) ribosome DRiPs (<30%) TAP tapasin ERp57calreticulin MHC class I ˜:˛2m complex is released from calnexin, binds a complex of chaperone proteins (calreticulin, ERp57) and binds to TAP via tapasin peptide fragments proteasome ERAAP˜2m MHC class I nucleus ER cytosol calnexin Partly folded MHC

1	from calnexin, binds a complex of chaperone proteins (calreticulin, ERp57) and binds to TAP via tapasin peptide fragments proteasome ERAAP˜2m MHC class I nucleus ER cytosol calnexin Partly folded MHC class I ˜ chains bind to calnexin until ˛2-microglobulin binds Cytosolic proteins and defective ribosomal products (DRiPs) are degraded to peptide fragments by the proteasome. TAP delivers peptides to the ER

1	Fig. 6.8 MHC class I molecules do not leave the endoplasmic reticulum unless they bind peptides. Newly synthesized MHC class I α chains assemble in the endoplasmic reticulum (ER) with the membrane-bound protein calnexin. When this complex binds β2-microglobulin (β2m), the MHC class I α:β2m dimer dissociates from calnexin, and the partly folded MHC class I molecule then binds to the TAP-associated protein tapasin. Two MHC:tapasin complexes may bind with the TAP dimer at the same time. The chaperone molecules ERp57, which forms a heterodimer with tapasin, and calreticulin also bind to form the MHC class I peptide-loading complex. The MHC class I molecule is retained within the ER until released by the binding of a peptide, which completes the folding of the MHC molecule. Even in the absence

1	A peptide binds the MHC class I molecule and completes its folding. The MHC class I molecule is released from the TAP complex and exported to the cell membrane of infection, there is a continual flow of peptides from the cytosol into the ER. Defective ribosomal products (DRiPs) and proteins marked for destruction by K48-linked polyubiquitin (yellow triangles) are degraded in the cytoplasm by the proteasome to generate peptides that are transported into the lumen of the endoplasmic reticulum by TAP. Some of these peptides will bind to MHC class I molecules. The aminopeptidase ERAAP trims the peptides at their amino termini, allowing peptides that are too long to bind to MHC class I molecules and thereby increasing the repertoire of potential peptides for presentation. Once a peptide has bound to the MHC molecule, the peptide:MHC complex leaves the endoplasmic reticulum and is transported through the Golgi apparatus and finally to the cell surface.

1	the PLC—calreticulin—is similar to calnexin and probably also has a general

1	MOVIE 6.1 chaperone function, like calnexin. A second component of the complex is the TAP-associated protein tapasin, encoded by a gene within the MHC. Tapasin forms a bridge between MHC class I molecules and TAP, allowing the partly folded α:β2-microglobulin heterodimer to await the transport of a suitable peptide from the cytosol. A third component of this complex is the chaperone ERp57, a thiol oxidoreductase that may have a role in breaking and re-forming the disulfide bond in the MHC class I α2 domain during peptide loading (Fig. 6.9). ERp57 forms a stable disulfide-linked heterodimer with tapasin. Tapasin seems to be a component of the PLC that is specific to antigen processing, while calnexin, ERp57, and calreticulin bind various other glycoproteins assembling in the endoplasmic reticulum and seem to be part of the cell’s general quality control machinery. TAP itself is the final component of the PLC, and it delivers peptides to the partially folded MHC class I molecule.

1	The PLC maintains the MHC class I molecule in a state that is receptive to peptide binding and mediates the exchange of low-affinity peptides bound to the MHC molecule for peptides of higher affinity, a process called peptide editing. The ERp57:tapasin heterodimer functions in editing peptides binding to MHC class I. Cells lacking calreticulin or tapasin show defects in the assembly of MHC class I molecules, and those molecules that reach the cell surface are bound to suboptimal, low-affinity peptides. The binding of a peptide to the partly folded MHC class I molecule releases it from the PLC, and the peptide:MHC complex leaves the endoplasmic reticulum and is transported to the cell surface. Most of the peptides transported by TAP will not bind to the MHC molecules and are rapidly cleared out of the endoplasmic reticulum; these appear to be transported back into the cytosol by Sec61, an ATP-dependent transport complex distinct from TAP.

1	As mentioned above, the MHC class I molecule must bind a peptide in order to be released from the PLC. In cells lacking functional TAP genes, the MHC class I molecules fail to exit the endoplasmic reticulum, and so must be degraded instead. Since the ubiquitin–proteasome system is located in the cytosol, these terminally misfolded MHC molecules must somehow be transported back into the cytoplasm for degradation. This is achieved by a system of quality control pathways called endoplasmic reticulum-associated protein degradation (ERAD). ERAD comprises several general cellular pathways that involve the recognition and delivery of misfolded proteins to a retrotranslocation complex that unfolds and translocates the proteins across the membrane of the endoplasmic reticulum and into the cytosol. The proteins are ubiquitinated during this process and so are targeted to the ubiquitin– proteasome system (UPS) for eventual degradation. We shall not delve deeply into the details of ERAD here,

1	The proteins are ubiquitinated during this process and so are targeted to the ubiquitin– proteasome system (UPS) for eventual degradation. We shall not delve deeply into the details of ERAD here, since these pathways are not unique to MHC

1	Fig. 6.9 The MHC class I peptide-loading complex includes the chaperones calreticulin, ERp57, and tapasin. This model shows a side (a) and top view (b) of the peptide-loading complex (PLC) oriented as it extends from the luminal surface of the endoplasmic reticulum. The newly synthesized MHC class I and β2-microglobulin are shown as yellow ribbons, with the α helices of the MHC peptide-binding groove clearly identifiable. The MHC and tapasin (cyan) would be tethered to the membrane of the endoplasmic reticulum by carboxy-terminal extensions not shown here. Tapasin and ERp57 (green) form a heterodimer linked by a disulfide bond, and tapasin makes contacts with the MHC molecule that stabilize the empty conformation of the peptide-binding groove; they function in editing peptides binding to the MHC class I molecule. Calreticulin (orange), like the calnexin it replaces (see Fig. 6.8), binds to the monoglucosylated N-linked glycan at asparagine 86 of the immature MHC molecule. The long,

1	the MHC class I molecule. Calreticulin (orange), like the calnexin it replaces (see Fig. 6.8), binds to the monoglucosylated N-linked glycan at asparagine 86 of the immature MHC molecule. The long, flexible P domain of calreticulin extends around the top of the peptide-binding groove of the MHC molecule to make contact with ERp57. The transmembrane region of tapasin (not shown) associates the PLC with TAP (see Fig. 6.8), bringing the empty MHC molecules into proximity with peptides arriving into the endoplasmic reticulum from the cytosol. Structure based on PDB file provided by Karin Reinisch and Peter Cresswell.

1	class I assembly or antigen processing. However, we will see in Chapter 13 how many viral pathogens co-opt the ERAD pathways to block assembly of MHC class I molecules as a way to evade recognition by CD8 T cells. In uninfected cells, peptides derived from self proteins fill the peptide-binding groove of the mature MHC class I molecules and are carried to the cell surface. In normal cells, MHC class I molecules are retained in the endoplasmic reticulum for some time, which suggests that they are present in excess of peptide. This is important for the immunological function of MHC class I molecules, which must be immediately available to transport viral peptides to the cell surface if the cell becomes infected. 6-5 Dendritic cells use cross-presentation to present exogenous proteins on MHC class I molecules to prime CD8 T cells.

1	The pathway described above explains how proteins synthesized in the cytosol can generate peptides that become displayed as complexes with MHC class I molecules on the cell surface. This pathway is sufficient to ensure detection and destruction of pathogen-infected cells by cytotoxic T cells. But how do these cytotoxic T cells first become activated? Our explanation so far would require that dendritic cells become infected as well, so that they express the peptide:MHC class I complex needed to activate naive CD8 T cells. But many viruses exhibit a restricted tropism for different cells types, and not all viruses will infect dendritic cells. This creates the chance that antigens from such pathogens might never be displayed by dendritic cells, and that cytotoxic T cells that recognize them might not be activated. As it turns out, certain dendritic cells are able to generate peptide:MHC class I complexes from peptides that were not generated within their own cytosol. Peptides from

1	them might not be activated. As it turns out, certain dendritic cells are able to generate peptide:MHC class I complexes from peptides that were not generated within their own cytosol. Peptides from extracellular sources—such as viruses, bacteria, and phagocytosed dying cells infected with cytosolic pathogens—can be presented on MHC class I molecules on the surface of these dendritic cells by the process of cross-presentation.

1	Long before its role in priming T-cell responses to viruses was appreciated, cross-presentation was observed in studies of minor histocompatibility antigens. These are non-MHC gene products that can elicit strong responses between mice of different genetic backgrounds. When spleen cells from B10 mice of MHC type H-2b were injected into BALB mice of MHC type H-2b×d (which express both b and d MHC types), BALB mice generated cytotoxic T cells reactive against minor antigens of the B10 background. Some of these cytotoxic T cells recognized minor antigens presented by the H-2b B10 cells used for immunization, as one might expect from direct priming of T cells by the B10 antigen-presenting cells. But other cytotoxic T cells recognized minor B10 antigens only when presented by cells of the H-2d MHC type. This meant that these CD8 T cells had been activated in vivo by recognizing the minor B10 antigens presented by the BALB host’s own H-2d molecules. In other words, the minor

1	of the H-2d MHC type. This meant that these CD8 T cells had been activated in vivo by recognizing the minor B10 antigens presented by the BALB host’s own H-2d molecules. In other words, the minor histocompatibility antigens must have become transferred from the original immunizing B10 cells to the BALB host’s dendritic cells and processed for MHC class I presentation. We now know that cross-presentation by MHC class I molecules occurs not only for antigens on tissue or cell grafts, as in the original experiment described above, but also for viral and bacterial antigens.

1	It appears that the capacity for cross-presentation is not equally distributed across all antigen-presenting cells. While still an area of active study, it seems that cross-presentation is most efficiently performed by certain subsets of dendritic cells that are present in both humans and mice. Dendritic cell subsets are not identified by the same markers in humans and mice, but in both species, one strongly cross-presenting dendritic cell subset requires the transcription factor BATF3 for its development, and these cells uniquely express the chemokine receptor XCR1. In lymphoid tissues such as the spleen, this lineage of dendritic cells expresses the CD8α molecule on the cell surface, and migratory dendritic cells in lymph nodes capable of cross-presentation are identified by their expression of the αE integrin (CD103). Mice lacking a functional BATF3 gene lack these types of dendritic cells and are also unable to generate normal CD8 T-cell responses to many viruses, including herpes

1	of the αE integrin (CD103). Mice lacking a functional BATF3 gene lack these types of dendritic cells and are also unable to generate normal CD8 T-cell responses to many viruses, including herpes simplex virus.

1	The biochemical mechanisms enabling cross-presentation are still unclear, and there may be several different pathways at work. It is not clear whether all proteins captured by phagocytic receptors and taken into endosomes need to be transported into the cytosol and degraded by the proteasome in order to be cross-presented. Some evidence supports a direct pathway in which the PLC is transported from the endoplasmic reticulum to the endosomal compartments, allowing exogenous antigens to be loaded onto newly synthesized MHC class I molecules in phagosomes (see Fig. 6.3). Another pathway of cross-presentation by dendritic cells may involve an interferon-γ-induced GTPase known as IRGM3 (short for immune-related GTPase family M protein 3). IRGM3 interacts with adipose differentiation related protein (ADRP) in the endoplasmic reticulum and regulates the generation of neutral lipid storage organelles called lipid bodies, which are thought to originate from ER membranes. Dendritic cells from

1	(ADRP) in the endoplasmic reticulum and regulates the generation of neutral lipid storage organelles called lipid bodies, which are thought to originate from ER membranes. Dendritic cells from mice lacking IRGM3 are selectively deficient in cross-presentation of antigens to CD8 T cells, but have a normal process for presenting antigens on MHC class II molecules. The relationship between this and other pathways remains an area of active research.

1	6-6 Peptide:MHC class II complexes are generated in acidified endocytosis, phagocytosis, and autophagy.

1	The immunological function of MHC class II molecules is to bind peptides generated in the intracellular vesicles of dendritic cells, macrophages, and B cells, and to present these peptides to CD4 T cells. The purpose for this pathway is different for each cell type. Dendritic cells primarily are concerned with activating CD4 T cells, while macrophages and B cells are concerned with receiving various forms of help from these CD4 T cells. For example, the intracellular vesicles of macrophages are the sites of replication for several types of pathogens, including the protozoan parasite Leishmania and the mycobacteria that cause leprosy and tuberculosis. Because these pathogens reside in membrane-enclosed vesicles, the proteins of these pathogens are not usually accessible to proteasomes in the cytosol. Instead, after activation of the macrophage, the pathogens are degraded by activated intravesicular proteases into peptide fragments that can bind to MHC class II molecules, which pass

1	the cytosol. Instead, after activation of the macrophage, the pathogens are degraded by activated intravesicular proteases into peptide fragments that can bind to MHC class II molecules, which pass through this compartment on their way from the endoplasmic reticulum to the cell surface. Like all membrane proteins, MHC class II molecules are first delivered into the endoplasmic reticulum membrane, and are then transported onward as part of membrane-enclosed vesicles that bud off the endoplasmic reticulum and are directed to intracellular vesicles containing internalized antigens. Complexes of peptides and MHC class II molecules are formed there and are then delivered to the cell surface, where they can be recognized by CD4 T cells.

1	Antigen processing for MHC class II molecules begins when extracellular pathogens and proteins are internalized into endocytic vesicles (Fig. 6.10). Proteins that bind to surface immunoglobulin on B cells and are internalized by receptor-mediated endocytosis are processed by this pathway. Larger particulate materials, such as fragments of dead cells, are internalized by phagocytosis, particularly by macrophages and dendritic cells. Soluble proteins, such as secreted toxins, are taken up by macropinocytosis. Proteins that enter cells through endocytosis are delivered to endosomes, which become Vesicles containing peptides fuse with vesicles containing MHC class II molecules Acidifcation of vesicles activates proteases to degrade antigen into peptide fragments Antigen is taken up from the extracellular space into intracellular vesicles extracellular space cytosol In early endosomes of neutral pH, endosomal proteases are inactive

1	Fig. 6.10 Peptides that bind to MHC class II molecules are generated in acidified endocytic vesicles. In the case illustrated here, extracellular foreign antigens, such as bacteria or bacterial antigens, have been taken up by an antigen-presenting cell such as a macrophage or an immature dendritic cell. In other cases, the source of the peptide antigen may be bacteria or parasites that have invaded the cell to replicate in intracellular vesicles. In both cases the antigen-processing pathway is the same. The pH of the endosomes containing the engulfed pathogens decreases progressively, activating proteases within the vesicles to degrade the engulfed material. At some point on their pathway to the cell surface, newly synthesized MHC class II molecules pass through such acidified vesicles and bind peptide fragments of the antigen, transporting the peptides to the cell surface.

1	increasingly acidic as they progress into the interior of the cell, eventually fusing with lysosomes. The endosomes and lysosomes contain proteases, known as acid proteases, that are activated at low pH and eventually degrade the protein antigens contained in the vesicles.

1	Drugs such as chloroquine that raise the pH of endosomes, making them less acidic, inhibit the presentation of intravesicular antigens, suggesting that acid proteases are responsible for processing internalized antigen. These proteases include the cysteine proteases—so called because they use a cysteine in their catalytic site—known as cathepsins B, D, S, and L, of which L is the most active. Antigen processing can be mimicked to some extent by the digestion of proteins with these enzymes in vitro at acid pH. Cathepsins S and L may be the predominant proteases in the processing of vesicular antigens; mice that lack cathepsin B or cathepsin D process antigens normally, whereas mice with no cathepsin S show some deficiencies, including in cross-presentation. Asparagine endopeptidase (AEP), a cysteine protease cleaving after asparagines, is important for processing some antigens, such as the tetanus toxin antigen for MHC class II presentation, but is not required in all cases where

1	a cysteine protease cleaving after asparagines, is important for processing some antigens, such as the tetanus toxin antigen for MHC class II presentation, but is not required in all cases where antigens contain asparagine residues near their relevant epitopes. It is likely that the overall repertoire of peptides produced within the vesicular pathway reflects the activities of the many proteases present in endosomes and lysosomes. Disulfide bonds, particularly intramolecular disulfide bonds, help in the denaturation process and facilitate proteolysis in endosomes. The enzyme IFN-γ-induced lysosomal thiol reductase (GILT) is present in endosomes and functions by breaking and re-forming disulfide bonds in the antigen-processing pathway. The various endosomal proteases act in a largely redundant and nonspecific manner to digest regions of the polypeptide that have become accessible to proteolysis by denaturation and previous steps of degradation. The peptides generated vary in sequence

1	and nonspecific manner to digest regions of the polypeptide that have become accessible to proteolysis by denaturation and previous steps of degradation. The peptides generated vary in sequence and abundance throughout the endocytic pathway, so that MHC class II molecules can bind and present many different peptides from these compartments.

1	A significant number of the self-peptides bound to MHC class II molecules arise from common proteins that are cytosolic in location, such as actin and ubiquitin. The most likely way in which cytosolic proteins are processed for MHC class II presentation is by the natural process of protein turnover known as autophagy, in which damaged organelles and cytosolic proteins are delivered to lysosomes for degradation. Here their peptides could encounter MHC class II molecules present in the lysosome membranes, and the resulting peptide:MHC class II complex could be transported to the cell surface via endolysosomal tubules (see Fig. 6.4). Autophagy is constitutive, but it is increased by cellular stresses such as starvation, when the cell catabolizes intracellular proteins to obtain energy. In microautophagy, cytosol is continuously internalized into the vesicular system by lysosomal invaginations, whereas in macroautophagy, which is induced by starvation, a double-membraned autophagosome

1	microautophagy, cytosol is continuously internalized into the vesicular system by lysosomal invaginations, whereas in macroautophagy, which is induced by starvation, a double-membraned autophagosome engulfs cytosol and fuses with lysosomes. A third autophagic pathway uses the heat-shock cognate protein 70 (Hsc70) and the lysosome-associated membrane protein-2 (LAMP-2) to transport cytosolic proteins to lysosomes. Autophagy has been shown to be involved in the processing of the Epstein–Barr virus nuclear antigen 1 (EBNA-1) for presentation on MHC class II molecules. Such presentation enables cytotoxic CD4 T cells to recognize and kill B cells infected with Epstein–Barr virus.

1	6-7 The invariant chain directs newly synthesized MHC class II molecules to acidified intracellular vesicles.

1	The biosynthetic pathway for MHC class II molecules begins with their translocation into the endoplasmic reticulum. Here, it is important to prevent them from prematurely binding to peptides transported into the endoplasmic reticulum lumen or to the cell’s own newly synthesized polypeptides. The endoplasmic reticulum is full of unfolded and partly folded polypeptide chains, and so a general mechanism is needed to prevent these from binding in the open-ended peptide-binding groove of the MHC class II molecule. Premature peptide binding is prevented by the assembly of newly synthesized MHC class II molecules with a membrane protein known as the MHC class II-associated invariant chain (Ii, CD74). Ii is a type II membrane glycoprotein; its amino terminus resides in the cytosol and its transmembrane region spans the membrane of the endoplasmic reticulum (Fig. 6.11). The remainder of Ii and its carboxy terminus reside within the endoplasmic reticulum. Ii has a unique cylindrical domain that

1	region spans the membrane of the endoplasmic reticulum (Fig. 6.11). The remainder of Ii and its carboxy terminus reside within the endoplasmic reticulum. Ii has a unique cylindrical domain that mediates formation of stable Ii trimers. Near this domain, Ii contains a peptide sequence, the class II-associated invariant chain peptide (CLIP), with which each Ii subunit of the trimer binds noncovalently to an MHC class II α:β heterodimer. Each Ii subunit binds to an MHC class II molecule with CLIP lying within the peptide-binding groove, thus blocking the groove and preventing the binding of either peptides or partly folded proteins. The binding site of an MHC class II molecule is open relative to the binding site of an MHC class I molecule.

1	Invariant chain (Ii) binds in the groove of MHC class II molecule Ii is cleaved initially to leave a fragment bound to the class II molecule and to the membrane Further cleavage leaves a short peptide fragment, CLIP, bound to the class II molecule Ii LIP10 cytosol ER C terminus trimerization domain transmembrane domainN terminus CLIP CLIP Fig. 6.11 The invariant chain is cleaved to leave a peptide fragment, CLIP, bound to the MHC class II molecule.

1	A model of the trimeric invariant chain bound to MHC class II α:β heterodimers is shown on the left. The CLIP portion is shown in purple, the rest of the invariant chain is shown in green, and the MHC class II molecules are shown in yellow (model, and leftmost of the three panels). In the endoplasmic reticulum, the invariant chain (Ii) binds to MHC class II molecules with the CLIP section of its polypeptide chain lying along the peptide-binding groove. After transport into an acidified vesicle, Ii is cleaved, initially just at one side of the MHC class II molecule (center panel), first by non-cysteine proteases to give a remaining portion of Ii known as the leupeptin-induced peptide LIP22 (not shown), and then by cysteine protease to the LIP10 fragment shown. LIP10 retains the transmembrane and cytoplasmic segments that contain the signals that target Ii:MHC class II complexes tothe endosomal pathway. Subsequent cleavage (right panel) of LIP10 leaves only a short peptide still bound

1	and cytoplasmic segments that contain the signals that target Ii:MHC class II complexes tothe endosomal pathway. Subsequent cleavage (right panel) of LIP10 leaves only a short peptide still bound by the class II molecule; this peptide is the CLIP fragment. Model structure courtesy of P. Cresswell.

1	Fig. 6.12 MHC class II molecules are loaded with peptide in a late endosomal compartment called the MIIC. MHC class II molecules are transported from the Golgi apparatus (labeled G in this electron micrograph of an ultrathin section of a B cell) to the cell surface via intracellular vesicles called the MHC class II compartment (MIIC). These have a complex morphology, showing internal vesicles and sheets of membrane. Antibodies labeled with different-sized gold particles identify the presence of both MHC class II molecules (visible as small dark spots) and the invariant chain (large dark spots) in the Golgi, whereas only MHC class II molecules are detectable in the MIIC. This compartment is thought to be a late endosome, an acidified compartment of the endocytic system (pH 4.5–5) in which the invariant chain is cleaved and peptide loading occurs. Photograph (×135,000) courtesy of H.J. Geuze.

1	This allows MHC class II molecules to more easily allow the CLIP region of Ii to pass through their binding sites. While this complex is being assembled in the endoplasmic reticulum, its component parts are associated with calnexin. Only when a nine-chain complex—three Ii chains, three α chains, and three β chains—has been assembled is the complex released from calnexin for transport out of the endoplasmic reticulum. As part of the nine-chain complex, the MHC class II molecules cannot bind peptides or unfolded proteins, so that peptides present in the endoplasmic reticulum are not usually presented by MHC class II molecules. There is evidence that in the absence of Ii many MHC class II molecules are retained in the endoplasmic reticulum as complexes with misfolded proteins.

1	Trafficking of membrane proteins is controlled by cytosolic sorting tags. In this regard, Ii has a second function, which is to target delivery of the MHC class II molecules to a low-pH endosomal compartment where peptide loading can occur. The complex of MHC class II α:β heterodimers with Ii trimers is retained for 2–4 hours in this compartment (see Fig. 6.11). During this time, the Ii molecule undergoes an initial cleavage by acid proteases to remove the trimerization domain, generating a truncated 22-kDa fragment of Ii called LIP22. This is further cleaved by cysteine proteases into a 10-kDa fragment called LIP10, which remains bound to the MHC class II molecule and retains it within the proteolytic compartment. A subsequent cleavage of LIP10 releases the MHC class II molecule from the membrane-associated Ii, leaving the CLIP fragment bound to the MHC molecule. This cleavage is carried out by cathepsin S in most MHC class II-positive cells but by cathepsin L in thymic epithelial

1	membrane-associated Ii, leaving the CLIP fragment bound to the MHC molecule. This cleavage is carried out by cathepsin S in most MHC class II-positive cells but by cathepsin L in thymic epithelial cells. Being associated with CLIP, the MHC class II molecules cannot yet bind other peptides. However, since CLIP does not carry the Ii-encoded signals that retain the complex in the endocytic compartment, the MHC–CLIP complex is now free to escape to the cell surface.

1	To allow another peptide to bind to the MHC class II molecule, CLIP must either dissociate or be displaced. Newly synthesized MHC class II molecules are brought toward the cell surface in vesicles, most of which at some point fuse with incoming endosomes. However, some MHC class II:Ii complexes may first be transported to the cell surface and reinternalized into endosomes. In either case, MHC class II:Ii complexes enter the endosomal pathway, where they encounter and bind peptides derived from either internalized pathogen proteins or self proteins. Initially, specialized endosomal compartments were thought to exist for antigen-presenting cells. One was an early endosomal compartment in dendritic cells that was called the CIIV (MHC class II vesicle). Another, a late endosomal compartment containing Ii and MHC class II molecules, was the MHC class II compartment, or MIIC (Fig. 6.12). The current view is that MHC class II molecules use many common endocytic compartments, including

1	containing Ii and MHC class II molecules, was the MHC class II compartment, or MIIC (Fig. 6.12). The current view is that MHC class II molecules use many common endocytic compartments, including lysosomes, to allow for the exchange of CLIP for as many peptides as possible. MHC class II molecules that do not bind peptide after dissociation from CLIP are unstable in the acidic pH after fusion with lysosomes, and they are rapidly degraded.

1	6-8 The MHC class II-like molecules HLA-DM and HLA-DO regulate exchange of CLIP for other peptides.

1	Because an MHC class II:CLIP complex cannot be released to the cell surface unless another peptide replaces it, antigen-presenting cells possess a mechanism that facilitates the efficient exchange of CLIP for other peptides. This process was uncovered by analysis of mutant human B-cell lines with a defect in antigen presentation. MHC class II molecules in these mutant cells assemble correctly with Ii and seem to follow the normal vesicular route, but fail to bind peptides derived from internalized proteins and often arrive at the cell surface with the CLIP peptide still bound. The defect in these cells lies in an MHC class II-like molecule called HLA-DM in humans (H-2DM in mice). The HLA-DM genes (see Section 6-10) are found near the TAP and PSMB8/9 genes in the MHC class II region (see Fig. 6.16); they encode an α chain and a β chain that closely resemble those of other MHC class II molecules. The HLA-DM molecule is not present at the cell surface, however, but is found predominantly

1	6.16); they encode an α chain and a β chain that closely resemble those of other MHC class II molecules. The HLA-DM molecule is not present at the cell surface, however, but is found predominantly in the endosomal compartment that contains Ii and MHC class II molecules. HLA-DM binds to and stabilizes empty MHC class II molecules and catalyzes the release of CLIP, thus allowing the binding of other peptides to the empty MHC class II molecule (Fig. 6.13). The HLA-DM molecule does not contain the open groove found in other MHC class II molecules, and it does not bind peptides. Instead, HLA-DM binds to the α chain of the MHC class II molecule near the region of the floor of the peptide-binding site (Fig. 6.14). This binding induces changes in the structure of the MHC class II molecule, and holds this part of the peptide binding groove in a partially ‘open’ configuration (see Fig. 6.14, right panel). In this way, HLA-DM catalyzes the release of CLIP and of other unstably bound peptides

1	holds this part of the peptide binding groove in a partially ‘open’ configuration (see Fig. 6.14, right panel). In this way, HLA-DM catalyzes the release of CLIP and of other unstably bound peptides from MHC class II molecules.

1	In the presence of a mixture of peptides capable of binding to MHC class II molecules, HLA-DM continuously binds and rebinds to newly formed peptide:MHC class II complexes, allowing for the dissociation of weakly bound peptides and for other peptides to replace them. Antigens presented by MHC class II molecules may have to persist on the surface of antigen-presenting cells for some days before encountering T cells able to recognize them. The ability of HLA-DM to remove unstably bound peptides, sometimes called peptide editing (see Section 6-4), ensures that the peptide:MHC class II complexes displayed on the surface of the antigen-presenting cell survive long enough to stimulate the appropriate CD4 T cells. During this process, it is likely that some peptides are captured first as longer polypeptides that undergo amino-terminal trimming by exopeptidases, further increasing the number of possible peptides that can be bound.

1	A second atypical MHC class II molecule, called HLA-DO in humans (H-2O in mice), is produced in thymic epithelial cells, B cells, and dendritic cells. MOVIE 6.2 Invariant chain (Ii) forms a complex with MHC class II molecule, blocking the binding of peptides and misfolded proteins Ii is cleaved in an acidifed endosome, leaving a short peptide fragment, CLIP, still bound to the MHC class II molecule Endocytosed antigens are degraded to peptides in endosomes, but the CLIP peptide blocks the binding of peptides to MHC class II molecules HLA-DM binds to the MHC class II molecule, releasing CLIP and allowing other peptides to bind. The MHC class II molecule then travels to the cell surface HLA-DM endoplasmic reticulum cytosol Ii LIP10 CLIP

1	Fig. 6.13 HLA-DM facilitates the loading of antigenic peptides leaving CLIP bound to the MHC class II molecules. Third panel: onto MHC class II molecules. First panel: the invariant chain pathogens and their proteins are broken down into peptides within (Ii) binds to newly synthesized MHC class II molecules and blocks acidified endosomes, but these peptides cannot bind to MHC peptides from binding class II molecules in the endoplasmic class II molecules that are occupied by CLIP. Fourth panel: the class reticulum and during their transport to acidified endosomes. Second II-like molecule HLA-DM binds to MHC class II:CLIP complexes, panel: in late endosomes, proteases cleave the invariant chain, catalyzing the release of CLIP and the binding of antigenic peptides.

1	Fig. 6.14 HLA-DM and HLA-DO regulate loading of peptides into MHC class II molecules. First panel: the HLA-DM dimer, composed of α (green) and β (turquoise) chains, binds to the HLA-DR MHC class II molecule (side view). HLA-DM contacts the MHC molecule near the peptide-binding groove where the peptide amino terminus would reside. Second panel: HLA-DO binds to HLA-DM in a similar configuration as HLA-DR, thus blocking DM’s peptide-editing activity. Third panel: top view of HLA-DR with bound peptide in the absence of HLA-DM. Fourth panel: top view of HLA-DR with HLA-DM bound. The amino-terminal end of the MHC peptide-binding groove is open and devoid of bound peptide, enabling peptide exchange.

1	This molecule is a heterodimer of the HLA-DOα chain and the HLA-DOβ chain. HLA-DO is not present at the cell surface, being found only in intracellular vesicles, and it does not seem to bind peptides. HLA-DO acts as a negative regulator of HLA-DM. HLA-DO binds to HLA-DM in the same manner as MHC class II molecules (see Fig. 6.14), and it must be bound to HLA-DM in order to leave the endoplasmic reticulum. When the DM–DO dimer reaches an acidified endocytic compartment, HLA-DO appears to dissociate slowly from HLA-DM, which is then free to catalyze peptide editing for MHC class II molecules. Moreover, IFN-γ increases the expression of HLA-DM, but not of the HLA-DOβ chain. Thus, during inflammatory responses, IFN-γ produced by T cells and NK cells can increase the expression of HLA-DM, and so overcome the inhibitory effects of HLA-DO. Why HLA-DO is expressed in this way remains obscure. The loss of HLA-DO in mice does not dramatically alter adaptive immunity, but does cause a

1	and so overcome the inhibitory effects of HLA-DO. Why HLA-DO is expressed in this way remains obscure. The loss of HLA-DO in mice does not dramatically alter adaptive immunity, but does cause a spontaneous production of autoantibodies with age. As thymic epithelial cells function in the selection of developing CD4 T cells, perhaps HLA-DO influences the repertoire of self peptides that these T cells encounter at different stages, as discussed further in Chapter 8.

1	The role of HLA-DM in peptide editing for MHC class II molecules parallels the role of tapasin in facilitating peptide binding to MHC class I molecules. HLA-DM carries out this function by mediating peptide exchange and driving the association of high-affinity peptides. Thus, it seems likely that specialized mechanisms of delivering peptides have coevolved with the MHC molecules themselves. It is also likely that pathogens have evolved strategies to inhibit the loading of peptides onto MHC class II molecules, much as viruses have found ways of subverting antigen processing and presentation through the MHC class I molecules. We will return to these topics in Chapter 13 when we discuss pathogen immunoevasion mechanisms.

1	The peptide editing conferred by DM and removing unstable MHC molecules provide important safeguards. To reveal the presence of an intracellular pathogen, the peptide:MHC complex must be stable at the cell surface. If peptides were to dissociate too readily, an infected cell could escape detection, and if peptides could too easily be acquired from other cells, then healthy cells might be mistakenly targeted for destruction. Tight binding of peptides to MHC molecules reduces the chance of these unwanted outcomes. MHC class I molecules display peptides derived largely from cytosolic proteins, so it is important that dissociation of a peptide from a cell-surface MHC molecule does not allow extracellular peptides to bind in the empty peptide-binding site. Fortunately, when an MHC class I molecule at the surface of a living cell loses its peptide, its conformation changes, the β2-microglobulin dissociates, and the α chain is internalized and rapidly degraded. Thus, most empty MHC class I

1	at the surface of a living cell loses its peptide, its conformation changes, the β2-microglobulin dissociates, and the α chain is internalized and rapidly degraded. Thus, most empty MHC class I molecules are quickly lost from the cell surface, largely preventing them from acquiring peptides directly from the surrounding extracellular fluid. This helps ensure that primed T cells target infected cells while sparing surrounding healthy cells.

1	Empty MHC class II molecules are also removed from the cell surface. Although at neutral pH, empty MHC class II molecules are more stable than empty MHC class I molecules, they aggregate readily, and internalization of such aggregates may account for their removal. Moreover, peptide loss from MHC class II molecules is most likely when the molecules are transiting through acidified endosomes as part of the normal process of cell-membrane recycling. At acidic pH, MHC class II molecules are able to bind peptides that are present in the vesicles, but those that fail to do so are rapidly degraded.

1	Some binding of extracellular peptides to MHC molecules at the cell surface can occur, however, as the addition of peptides to living or even chemically fixed cells in vitro can generate peptide:MHC complexes that are recognized by T cells specific for those peptides. This has been readily demonstrated for many peptides that bind MHC class II and class I molecules. Whether this phenomenon is due to the presence of empty MHC proteins on the cells or to peptide exchange is not clear. Nevertheless it can happen and is a widely used technique to load synthetic peptides for analyzing the specificity of T cells. 6-9 Cessation of antigen processing occurs in dendritic cells after their activation through reduced expression of the MARCH-1 E3 ligase.

1	Dendritic cells that have not yet been activated by infection carry out active surveillance of the antigens in their location, for example, through macropinocytosis of soluble proteins. Peptides derived from proteins are continuously processed and loaded onto MHC class II molecules for expression on the cell surface. In addition, peptide:MHC complexes are also continuously being recycled from the surface and degraded in cells by ubiquitination and proteasomal degradation. MHC class II molecules contain a conserved lysine residue in the cytoplasmic tails of the β chain; this lysine residue is a target of an E3 ligase (see Section 3-7) called membrane associated ring finger (C3HC4) 1, or MARCH-1, expressed in B cells, dendritic cells, and macrophages. MARCH-1 is expressed constitutively in B cells and induced by the cytokine IL-10 in other cells. It resides in the membrane of a recycling endosomal compartment, where it ubiquitinates the cytoplasmic tail of MHC class II molecules,

1	in B cells and induced by the cytokine IL-10 in other cells. It resides in the membrane of a recycling endosomal compartment, where it ubiquitinates the cytoplasmic tail of MHC class II molecules, leading to their eventual degradation in lysosomes, thereby regulating their steady-state level of expression (Fig. 6.15).

1	The MARCH-1 pathway is shut down during infection to increase the stability of peptide:MHC complexes. Dendritic cells that capture antigens at sites of infection must first migrate to local lymph nodes in order to activate naive T cells; this may take many hours. Since continuous recycling limits the lifetime of peptide:MHC complexes on the cell surface, pathogen-derived peptide:MHC complexes could be lost during this migration, preventing T-cell activation. To prevent this situation, when dendritic cells are activated by pathogens, expression of MARCH-1 is shut off. This may be mediated directly by innate pathogen sensors, since TLR signaling in dendritic cells rapidly reduces the level of mRNA for MARCH-1. The MARCH-1 protein half-life is only around 30 minutes, so that activated dendritic cells soon accumulate peptide:MHC complexes on their cell surface produced at the time of encounter with pathogen.

1	In addition to regulating MHC class II expression in dendritic cells, MARCH-1 similarly regulates expression in dendritic cells of the co-stimulatory molecule (see Section 1-15) CD86 (or B7-2), which like MHC class II molecules, is also MHC molecules accumulate on the cell surface, presenting peptides they acquired at the time of dendritic cell activation

1	Fig. 6.15 Activation of dendritic cells reduces MARCH-1 expression, thus increasing the lifetime of MHC molecules. Before activation by innate recognition of pathogens, dendritic cells express the membrane-associated E3 ligase MARCH-1, which resides in the recycling endosomes compartment, where it attaches K-48-linked ubiquitin chains to the β chain of MHC class II molecules. This causes MHC molecules to move from the recycling endosomes and eventually to be degraded, leading to a reduced overall half-life and level of MHC expression on the cell surface. Signals emanating from innate sensors, such as TLR-4, reduce the level of MARCH-1 mRNA, and with the half-life of MARCH-1 now being short, MHC molecules are free to accumulate on the cell surface. Because innate signaling also triggers acidification of endocytic compartments and activates caspases associated with antigen processing, the MHC molecules that accumulate on the cell surface will bear peptides from the pathogens captured

1	acidification of endocytic compartments and activates caspases associated with antigen processing, the MHC molecules that accumulate on the cell surface will bear peptides from the pathogens captured around the time of innate activation of the dendritic cells.

1	regulated by ubiquitination. This means that by the time dendritic cells arrive at lymph nodes, they express peptides derived from the pathogens that activated them and have higher CD86 levels that provide signals for greater CD4 T-cell activation. However, we will see in Chapter 13 that viral pathogens have taken advantage of this pathway by producing MARCH-1-like proteins to downregulate MHC class II molecules as a means of evading adaptive immunity. Summary.

1	The ligand recognized by the T-cell receptor is a peptide bound to an MHC molecule. MHC class I and MHC class II molecules acquire peptides at different intracellular sites and activate either CD8 or CD4 T cells, respectively. Infected cells presenting peptides derived from virus replication in the cytosol are thus recognized by CD8 cytotoxic T cells, which are specialized to kill any cells displaying foreign antigens. MHC class I molecules are synthesized in the endoplasmic reticulum and typically acquire their peptides at this location. The peptides loaded onto MHC class I molecules are derived from proteins degraded in the cytosol by the proteasome, transported into the endoplasmic reticulum by the heterodimeric ATP-binding protein TAP, and further processed by the aminopeptidase ERAAP before being loaded onto the MHC molecules. Peptide binding to MHC class I molecules is required for them to be released from chaperones in the endoplasmic reticulum and to travel to the cell

1	ERAAP before being loaded onto the MHC molecules. Peptide binding to MHC class I molecules is required for them to be released from chaperones in the endoplasmic reticulum and to travel to the cell surface. Certain subsets of dendritic cells are able to produce peptides from exogenous proteins and load them onto MHC class I molecules. Such cross-presentation of antigens ensures that CD8 T cells can be activated by pathogens that may not directly infect antigen-presenting cells.

1	MHC class II molecules do not acquire their peptide ligands in the endoplasmic reticulum, because the invariant chain (Ii) first inserts CLIP into their peptide-binding groove. Association with Ii targets these MHC molecules to an acidic endosomal compartment where active proteases cleave Ii, and HLA-DM helps to catalyze dissociation of CLIP. The MHC molecules can then associate with peptides derived from proteins that have entered the vesicular compartments of macrophages, dendritic cells, or B cells. The process of autophagy can deliver cytosolic proteins to the vesicular system for presentation by MHC class II molecules. The CD4 T cells that recognize peptide:MHC class II complexes have a variety of specialized effector activities. Subsets of CD4 T cells activate macrophages to kill the intravesicular pathogens they harbor, help B cells to secrete immunoglobulins against foreign molecules, and regulate immune responses. The major histocompatibility complex and its function.

1	The major histocompatibility complex and its function. The function of MHC molecules is to bind peptide fragments derived from pathogens and display them on the cell surface for recognition by the appropriate T cells. The consequences are almost always deleterious to the pathogen—virus-infected cells are killed, macrophages are activated to kill bacteria living in their intracellular vesicles, and B cells are activated to produce antibodies that eliminate or neutralize extracellular pathogens. Thus, there is strong selective pressure in favor of any pathogen that has mutated in such a way that it escapes presentation by an MHC molecule.

1	Two separate properties of the major histocompatibility complex (MHC) make it difficult for pathogens to evade immune responses in this way. First, the MHC is polygenic: it contains several different MHC class I and MHC class II genes, so that every individual possesses a set of MHC molecules with different ranges of peptide-binding specificities. Second, the MHC is highly polymorphic; that is, there are multiple variants, or alleles, of each gene within the population as a whole. The MHC genes are, in fact, the most polymorphic genes known. In this section we describe the organization of the genes in the MHC and discuss how the variation in MHC molecules arises. We also consider how the effect of polygeny and polymorphism on the range of peptides that can be bound contributes to the ability of the immune system to respond to the multitude of different and rapidly evolving pathogens.

1	6-10 Many proteins involved in antigen processing and presentation are encoded by genes within the MHC.

1	The MHC is located on chromosome 6 in humans and chromosome 17 in the mouse and extends over at least 4 million base pairs. In humans it contains more than 200 genes. As work continues to define the genes within and around the MHC, it becomes difficult to establish precise boundaries for this genetic region, which is now thought to span as many as 7 million base pairs. The genes encoding the α chains of MHC class I molecules and the αand βchains of MHC class II molecules are linked within the complex; the genes for β2-microglobulin and the invariant chain are on different chromosomes (chromosomes 15 and 5, respectively, in humans, and chromosomes 2 and 18 in the mouse). Figure 6.16 shows the general organization of the MHC class I and II genes in human and mouse. In humans these genes are called human leukocyte antigen or HLA genes, because they were first discovered through antigenic differences between white blood cells from different individuals; in the mouse they are known as the

1	called human leukocyte antigen or HLA genes, because they were first discovered through antigenic differences between white blood cells from different individuals; in the mouse they are known as the H-2 genes. The mouse MHC class II genes were in fact first identified as genes that controlled whether an immune response was made to a given antigen and were originally called Ir (immune response) genes. Because of this, the mouse MHC class II A and E genes were in the past referred to as I-A and I-E, but this terminology could be confused with MHC class I genes and it is no longer used.

1	Fig. 6.16 The genetic organization of the major histocompatibility complex (MHC) in humans and mice. The organization of the MHC genes is shown. In humans, the cluster is called HLA (short for human leukocyte antigen) and is on chromosome 6, and in mice, it is called H-2 (for histocompatibility) and is on chromosome 17. The organization is similar in both species, with separate clusters of MHC class I genes (red) and MHC class II genes (yellow). In mice, the MHC class I gene H-2K has been translocated relative to the human MHC, splitting the class I region in two. Both species have three main class I genes, which are called HLA-A, HLA-B, and HLA-C in humans, and H2-K, H2-D, and H2-L in the mouse. These encode the α chain of the respective MHC class I proteins, HLA-A, HLA-B, and so on. The other subunit of an MHC class I molecule, β2-microglobulin, is encoded by a gene located on a different chromosome—chromosome 15 in humans and chromosome 2 in the mouse. The class II region includes

1	other subunit of an MHC class I molecule, β2-microglobulin, is encoded by a gene located on a different chromosome—chromosome 15 in humans and chromosome 2 in the mouse. The class II region includes the genes for the α and β chains (designated A and B) of the MHC class II molecules HLA-DR, -DP, and -DQ (H-2A and -E in the mouse). Also in the MHC class II region are the genes for the TAP1:TAP2 peptide transporter, the PSMB (or LMP) genes that encode proteasome subunits, the genes encoding the DMα and DMβ chains (DMA and DMB), the genes encoding the α and β chains of the DO molecule (DOA and DOB, respectively), and the gene encoding tapasin (TAPBP). The so-called class III genes encode various other proteins with functions in immunity (see Fig. 6.17).

1	There are three class I α-chain genes in humans, called HLA-A, -B, and -C. There are also three pairs of MHC class II α-and β-chain genes, called HLA-DR, -DP, and -DQ. In many people, however, the HLA-DR cluster contains an extra β-chain gene whose product can pair with the DRα chain. This means that the three sets of genes can give rise to four types of MHC class II molecules. All the MHC class I and class II molecules can present peptides to T cells, but each protein binds a different range of peptides (see Sections 4-14 and 4-15). Thus, the presence of several different genes for each MHC class means that any one individual is equipped to present a much broader range of peptides than if only one MHC molecule of each class were expressed at the cell surface.

1	Figure 6.17 shows a more detailed map of the human MHC region. Many genes within this locus participate in antigen processing or antigen presentation, or have other functions related to the innate or adaptive immune response. The two TAP genes lie in the MHC class II region near the PSMB8 and PSMB9 genes, whereas the gene encoding tapasin (TAPBP) lies at the edge of the MHC nearest the centromere. The genetic linkage of the MHC class I genes (whose products deliver cytosolic peptides to the cell surface) with the TAP, tapasin, and proteasome (PSMB or LMP) genes (whose products deliver cytosolic peptides into the endoplasmic reticulum) suggests that the entire MHC has been selected during evolution for antigen processing and presentation.

1	When cells are treated with the interferons IFN-α, -β, or -γ, there is a marked increase in the transcription of MHC class I α-chain and β2-microglobulin genes and of the proteasome, tapasin, and TAP genes. Interferons are produced early in viral infections as part of the innate immune response, as described in Chapter 3. The increase in MHC expression they produce helps all cells to process viral proteins and present the resulting virus-derived peptides on their surface (except for red blood cells). On dendritic cells, this helps to activate the appropriate T cells and initiate the adaptive immune response to the virus. The coordinated regulation of the genes encoding these components may be facilitated by the linkage of many of them in the MHC.

1	The DMA and DMB genes encoding the subunits of the HLA-DM molecule that catalyzes peptide binding to MHC class II molecules are clearly related to the MHC class II genes, as are the DOA and DOB genes that encode the subunits of the regulatory HLA-DO molecule. Gene expression of the classical MHC class II proteins, along with the invariant-chain, DMα, DMβ, and DOα, but not DOβ, is coordinately increased by IFN-γ, which is produced by activated TH1 cells, CD8 T cells, and NK cells. This form of regulation allows dendritic cells and macrophages to upregulate molecules involved in processing of intravesicular antigens when presenting antigens to T cells and NK cells. Expression of all these molecules is induced by IFN-γ (but not by IFN-αor -β), via the production of a protein known as MHC class II transactivator (CIITA), which acts as a positive transcriptional co-activator of MHC class II genes. An absence of CIITA causes severe immunodeficiency due to the nonproduction of MHC class II

1	II transactivator (CIITA), which acts as a positive transcriptional co-activator of MHC class II genes. An absence of CIITA causes severe immunodeficiency due to the nonproduction of MHC class II molecules—MHC class II deficiency. Finally, the MHC contains many ‘non-classical’ MHC genes, so-called because while they resemble MHC genes in structure, their products do not function in presenting peptides to conventional α:β T cells. Many of these genes are now referred to as MHC class Ib genes, and their protein products have a variety of different functions, which we will describe in Section 6-16 , following our discussion of the conventional MHC genes.

1	Fig. 6.17 Detailed map of the human MHC. The organization of the class I, class II, and class III regions of the human MHC is shown, with approximate genetic distances given in thousands of base pairs. Most of the genes in the class I and class II regions are mentioned in the text. The additional genes indicated in the class I region (for example, E, F, and G) are class I-like genes encoding class Ib molecules; the additional class II genes are pseudogenes. The genes shown in the class III region encode the complement proteins C4 (two genes, shown as C4A and C4B), C2, and factor B (shown as Bf), as well as genes that encode the cytokines tumor necrosis factor-α (TNF) and lymphotoxin (LTA, LTB). Closely linked to the C4 genes is the gene encoding 21-hydroxylase (shown as CYP 21B), an enzyme involved in steroid biosynthesis. Immunologically important functional protein-coding genes mentioned in the text are color coded, with the MHC class I genes being shown in red, except for the MIC

1	involved in steroid biosynthesis. Immunologically important functional protein-coding genes mentioned in the text are color coded, with the MHC class I genes being shown in red, except for the MIC genes, which are shown in blue; these are distinct from the other class I-like genes and are under different transcriptional control. The immunologically important MHC class II genes are shown in yellow. Genes in the MHC region that have immune functions but are not related to the MHC class I and class II genes are shown in purple. Genes in dark gray are pseudogenes related to immune-function genes. Unnamed genes unrelated to immune function are shown in light gray.

1	Fig. 6.18 Human MHC genes are highly polymorphic. With the notable exception of the DRα locus, which is functionally monomorphic, each gene locus has many alleles. The number of functional proteins encoded is less than the total number of alleles. Shown in this figure as the heights of the bars are the number of different HLA proteins assigned by the WHO Nomenclature Committee for Factors of the HLA System as of January 2010. 6-11 The protein products of MHC class I and class II genes are highly polymorphic. Because of the polygeny of the MHC, every person expresses at least three different MHC class I molecules and three (or sometimes four) MHC class II molecules on his or her cells. In fact, the number of different MHC molecules expressed by most people is greater because of the extreme polymorphism of the MHC (Fig. 6.18).

1	The term polymorphism comes from the Greek poly, meaning many, and morphe, meaning shape or structure. As used here, it means within-species variation at a gene locus, and thus in the gene’s protein product; the variant genes that can occupy the locus are termed alleles. For several MHC class I and class II genes, there are more than 1000 alleles in the human population, far more than the number of alleles for other genes found within the MHC region. Each MHC class I and class II allele is relatively frequent in the population, so there is only a small chance that the corresponding gene loci on both homologous chromosomes of an individual will have the same allele; most individuals will be heterozygous for the genes encoding MHC class I and class II molecules. The particular combination of MHC alleles found on a single chromosome is known as an MHC haplotype. Expression of MHC alleles is codominant, meaning that the protein products of both of the alleles at a locus are expressed

1	of MHC alleles found on a single chromosome is known as an MHC haplotype. Expression of MHC alleles is codominant, meaning that the protein products of both of the alleles at a locus are expressed equally in the cell, and both gene products can present antigens to T cells. The number of MHC alleles discovered that do not code for a functional protein is remarkably small. The extensive polymorphism at each locus thus has the potential to double the number of different MHC molecules expressed in an individual and thereby increase the diversity already available through polygeny (Fig. 6.19).

1	Because most individuals are heterozygous, most matings will produce offspring that receive one of four possible combinations of the parental MHC haplotypes. Thus siblings are also likely to differ in the MHC alleles they express, there being one chance in four that an individual will share both haplotypes with a sibling. One consequence of this is the difficulty of finding suitable donors for tissue transplantation, even among siblings. All MHC class I and II proteins are polymorphic to a greater or lesser extent, with the exception of the DRα chain and its mouse homolog Eα. These chains do not vary in sequence between different individuals and are said to be monomorphic. This might indicate a functional constraint that prevents variation in the DRαand Eα proteins, but no such special function has been found. Fig. 6.19 Polymorphism and polygeny both contribute to the diversity of MHC molecules expressed by an individual.

1	Fig. 6.19 Polymorphism and polygeny both contribute to the diversity of MHC molecules expressed by an individual. Many mice, both domestic and wild, have a mutation in the Eα gene that prevents synthesis of the Eα protein. They thus lack cell-surface H-2E molecules, so if H2-E does have a special function it is unlikely to be essential.

1	MHC polymorphisms at individual MHC genes seem to have been strongly selected by evolutionary pressures. Several genetic mechanisms contribute to the generation of new alleles. Some new alleles arise by point mutations and others by gene conversion, a process in which a sequence in one gene is replaced, in part, by sequences from a different gene (Fig. 6.20). The effects of selective pressure in favor of polymorphism can be seen clearly in the pattern of point mutations in the MHC genes. Point mutations can be classified as replacement substitutions, which change an amino acid, or silent substitutions, which simply change the codon but leave the amino acid the same. Replacement substitutions occur within the MHC at a higher frequency relative to silent substitutions than would be expected, providing evidence that polymorphism has been actively selected for in the evolution of the MHC. influencing both peptide binding and the contacts between T-cell receptor and MHC molecule.

1	influencing both peptide binding and the contacts between T-cell receptor and MHC molecule. The next few sections describe how MHC polymorphisms benefit the immune response and how pathogen-driven selection can account for the large number of MHC alleles. The products of individual MHC alleles, often known as protein isoforms, can differ from one another by up to 20 amino acids, making each variant protein quite distinct. Most of the differences are localized to exposed surfaces of the extracellular domain furthest from the membrane, and to the peptide-binding groove in particular (Fig. 6.21). We have seen that peptides bind to MHC class I and class II molecules through the interaction of specific anchor residues with peptide-binding pockets in the peptide-binding groove (see Sections 4-15 and 4-16). Many of the polymorphisms in MHC molecules alter the amino acids that line these pockets and thus change

1	Fig. 6.20 Gene conversion can create new alleles by copying sequences from one MHC gene to another. Multiple MHC genes of generally similar structure were derived over evolutionary time by duplication of an unknown ancestral MHC gene (gray) followed by genetic divergence. Further interchange between these genes can occur by a process known as gene conversion, in which sequences can be transferred from part of one gene to a similar gene. For this to happen, the two genes must become apposed during meiosis. This can occur as a consequence of the misalignment of the two paired homologous chromosomes when there are many copies of similar genes arrayed in tandem—somewhat like buttoning in the wrong buttonhole. During the process of crossing-over and DNA recombination, a DNA sequence from one chromosome is sometimes copied to the other, replacing the original sequence. In this way, several nucleotide changes can be inserted all at once into a gene and can cause several simultaneous amino

1	chromosome is sometimes copied to the other, replacing the original sequence. In this way, several nucleotide changes can be inserted all at once into a gene and can cause several simultaneous amino acid changes in the gene product. Because of the similarity of the MHC genes to each other and their close linkage, gene conversion has occurred many times in the evolution of MHC alleles.

1	The high polymorphism of the classical MHC genes ensures diversity in MHC gene expression in the population as a whole. However, no matter how polymorphic a gene is, no individual can express more than two alleles at a single gene locus. Polygeny, the presence of several different related genes with similar functions, ensures that each individual produces a number of different MHC molecules. The combination of polymorphism and polygeny produces the diversity of MHC molecules seen both within an individual and in the population at large.

1	Fig. 6.21 Allelic variation in MHC molecules occurs Moreover, allelic variability is clustered in specific sites within the predominantly within the peptide-binding region. Variability amino-terminal domains, lying in positions that line the peptide-plots of the amino acid sequences of MHC molecules show that binding groove, either on the floor of the groove or inward from the the variation arising from genetic polymorphism is restricted to walls. For the MHC class II molecule, the variability of the HLA-DR the amino-terminal domains (α1 and α2 domains of MHC class I alleles is shown. For HLA-DR, and its homologs in other species, the molecules, and α1 and β1 domains of MHC class II molecules). α chain is essentially invariant and only the β chain shows significant These are the domains that form the peptide-binding groove. polymorphism.

1	the pockets’ binding specificities. This in turn changes the anchor residues of peptides that can bind to each MHC isoform. The set of anchor residues that allows binding to a given isoform of an MHC class I or class II molecule is called a sequence motif, and this can be used to predict peptides within a protein that might bind that variant (Fig. 6.22). Such predictions may be very important in designing peptide vaccines, as we will see in Chapter 16, where we discuss recent progress in cancer immunotherapy.

1	In rare cases, processing of a protein does not generate any peptides with a suitable sequence motif for binding to any of the MHC molecules expressed by an individual. This individual fails to respond to the antigen. Such failures in responsiveness to simple antigens were first reported in inbred animals and were called immune response (Ir) gene defects. These defects were mapped to genes within the MHC long before the structure or function of MHC molecules was understood, and they were the first clue to the antigen-presenting function of MHC molecules. We now understand that Ir gene defects are common in inbred strains of mice because the mice are homozygous at all their MHC gene loci, which limits the range of peptides they can present to T cells. Ordinarily, MHC polymorphism guarantees a sufficient number of different MHC molecules in a single individual to make this type of nonresponsiveness unlikely, even to relatively simple antigens such as small toxins.

1	Initially, the only evidence linking Ir gene defects to the MHC was genetic— mice of one MHC genotype could make antibody in response to a particular antigen, whereas mice of a different MHC genotype, but otherwise genetically identical, could not. The MHC genotype was somehow controlling the ability

1	Fig. 6.22 Different allelic variants of an MHC class I molecule bind different peptides. Shown are cutaway views of (a) ovalbumin peptide bound to the mouse H2-Kb MHC class I molecule and (b) influenza nucleoprotein (NP) peptide bound to the H2-Kd MHC class I molecule. The solvent-accessible surface of the MHC molecules is shown as a blue dotted surface. Class I MHC molecules typically have six pockets in the peptide-binding groove, which are conventionally called A–F. The bound peptides, shown as space-filling models, fit into the peptide-binding groove, with side chains from the anchor residues extending to fill the pockets. H2-Kb is binding SIINFEKL (single-letter amino acid code), a peptide of eight residues (P1–8) from ovalbumin, and H2-Kd is binding TYQRTRALV, a peptide of nine residues (P1–9) from the influenza nucleoprotein (NP). Anchor residues (shown in yellow) may be primary or secondary in their influence on peptide binding. For H2-Kb, the sequence motif is determined by

1	(P1–9) from the influenza nucleoprotein (NP). Anchor residues (shown in yellow) may be primary or secondary in their influence on peptide binding. For H2-Kb, the sequence motif is determined by two primary anchors, P5 and P8; the C pocket binds the P5 side chain of the peptide [a tyrosine (Y) or a phenylalanine (F)], and the F pocket binds the P8 residue [a non-aromatic hydrophobic side chain from leucine (L), isoleucine (I), methionine (M), or valine (V)]. The B pocket binds P2, a secondary anchor residue in H-2Kb. For H2-Kd, the sequence motif is primarily determined by the two primary anchors, P2 and P9. The B pocket accommodates a tyrosine side chain. The F pocket binds leucine, isoleucine, or valine. Beneath the structures are shown sequence motifs from peptides that are known to bind to the MHC molecule. CSP, circumsporozoite antigen; ERK4, extracellular signal-related kinase 4; HBV, hepatitis B virus; JAK1, Janus-associated kinase 1; LCMV, lymphocytic choriomeningitis virus;

1	to the MHC molecule. CSP, circumsporozoite antigen; ERK4, extracellular signal-related kinase 4; HBV, hepatitis B virus; JAK1, Janus-associated kinase 1; LCMV, lymphocytic choriomeningitis virus; NS2, NS2 protein; P198, modified tumor-cell antigen; P. berghei, Plasmodium berghei; P. yoelii, Plasmodium yoelii; VSV, vesicular stomatitis virus. An extensive collection of motifs can be found at http://www. syfpeithi.de. Structures courtesy of V.E. Mitaksov and D. Fremont.

1	of the immune system to detect or respond to specific antigens, but it was not clear at the time that direct recognition of MHC molecules was involved. Later experiments showed that the antigen specificity of T-cell recognition was controlled by MHC molecules. The immune responses affected by the Ir genes were known to depend on T cells, and this led to a series of experiments in mice to ascertain how MHC polymorphism might control T-cell responses. The earliest of these experiments showed that T cells could be activated only by macrophages or B cells that shared MHC alleles with the mouse in which the T cells originated. This was the first evidence that antigen recognition by T cells depends on the presence of specific MHC molecules in the antigen-presenting cell—the phenomenon we now know as MHC restriction.

1	Fig. 6.23 T-cell recognition of antigens is MHC-restricted. The antigen-specific T-cell receptor (TCR) recognizes a complex consisting of an antigenic peptide and a self MHC molecule. One consequence of this is that a T cell specific for peptide x and an MHC molecule that is the product of a particular MHC allele, MHCa (left panel), will usually not recognize the complex of peptide x bound to a different MHC allele product, MHCb (center panel), or the complex of a different peptide, peptide y, bound to MHCa (right panel). The co-recognition of a foreign peptide and an MHC molecule is known as MHC restriction because the particular MHC allele product is said to restrict the ability of the T cell to recognize antigen. This restriction may either result from direct contact between the MHC molecule and T-cell receptor or be an indirect effect of MHC polymorphism on the peptides that bind or on their bound conformation.

1	We first mentioned MHC restriction in Section 4-17 in the context of the crystal structure of the T-cell receptor bound to peptide:MHC complexes. But the phenomenon of MHC restriction was discovered much earlier and is illustrated by the studies of virus-specific cytotoxic T cells carried out by Peter Doherty and Rolf Zinkernagel, for which they received the Nobel Prize in 1996. When mice are infected with a virus, they generate cytotoxic T cells that kill the virus-infected cells while sparing both uninfected cells and cells infected with unrelated viruses. The cytotoxic T cells are thus virus-specific. The additional and striking outcome of these experiments was the demonstration that the ability of cytotoxic T cells to kill virus-infected cells was also affected by the polymorphism of MHC molecules. Cytotoxic T cells induced by viral infection in mice of MHC genotype a (MHCa) would kill any MHCa cell infected with that virus. But these same T cells would not kill cells of MHC

1	of MHC molecules. Cytotoxic T cells induced by viral infection in mice of MHC genotype a (MHCa) would kill any MHCa cell infected with that virus. But these same T cells would not kill cells of MHC genotype b, or c, and so on, even if they were infected with the same virus. In other words, cytotoxic T cells kill cells infected by virus only if those cells express the same MHC by which the T cells were primed. Because the MHC genotype ‘restricts’ the antigen specificity of the T cells, this effect was called MHC restriction. Together with the earlier studies on both B cells and macrophages, this work showed that MHC restriction is a critical feature of antigen recognition by all functional classes of T cells.

1	We now know that MHC restriction is due to the fact that the binding specificity of an individual T-cell receptor is not for its peptide antigen alone but for the complex of peptide and MHC molecule, as discussed in Section 4-17. MHC restriction is explained in part by the fact that different MHC molecules bind different peptides. In addition, some of the polymorphic amino acids in MHC molecules are located in the α helices that flank the peptide-binding groove but have side chains oriented toward the exposed surface of the peptide:MHC complex that can directly contact the T-cell receptor (see Figs. 6.21 and 4.24). In retrospect, it is therefore not surprising that T cells can distinguish between a peptide bound to MHCa and the same peptide bound to MHCb. This restricted recognition may sometimes be caused both by differences in the conformation of the bound peptide imposed by the different MHC molecules and by direct recognition of polymorphic amino acids in the MHC molecule itself.

1	be caused both by differences in the conformation of the bound peptide imposed by the different MHC molecules and by direct recognition of polymorphic amino acids in the MHC molecule itself. Thus, the specificity of a T-cell receptor is defined both by the peptide it recognizes and by the MHC molecule bound to it (Fig. 6.23).

1	6-13 Alloreactive T cells recognizing nonself MHC molecules are very abundant.

1	The discovery of MHC restriction also helped to explain the otherwise puzzling phenomenon of recognition of nonself MHC in the rejection of organs and tissues transplanted between members of the same species. Transplanted organs from donors bearing MHC molecules that differ from those of the recipient—even by as little as one amino acid—are rapidly rejected owing to the presence in any individual of large numbers of T cells that react to nonself, or allogeneic, MHC molecules. Early studies on T-cell responses to allogeneic MHC molecules used the mixed lymphocyte reaction, in which T cells from one individual are mixed with lymphocytes from a second individual. If the T cells of this individual recognize the other individual’s MHC molecules as ‘foreign,’ the T cells will divide and proliferate. The lymphocytes from the second individual are usually prevented from dividing by irradiation or treatment with the cytostatic drug mitomycin C. Such studies have shown that roughly 1–10% of all

1	The lymphocytes from the second individual are usually prevented from dividing by irradiation or treatment with the cytostatic drug mitomycin C. Such studies have shown that roughly 1–10% of all T cells in an individual will respond to stimulation by cells from another, unrelated, member of the same species. This type of T-cell response is called an alloreaction or alloreactivity, because it represents the recognition of allelic polymorphisms in MHC molecules.

1	Before the role of the MHC molecules in antigen presentation was understood, it was a mystery why so many T cells should recognize nonself MHC molecules, as there is no reason that the immune system should have evolved a defense against tissue transplants. Once it was realized that T-cell receptors have evolved to recognize foreign peptides in combination with polymorphic MHC molecules, alloreactivity became easier to explain. We now know of at least two processes that can contribute to the high frequency of alloreactive T cells. The first is the process of positive selection. When developing in the thymus, T cells whose T-cell receptors interact weakly with the self MHC molecules receive survival signals, and so are favored for representation in the peripheral repertoire. It is thought that T-cell receptors that interact with one type of MHC molecule are more likely to cross-react with other (nonself ) MHC variants. We discuss positive selection in greater detail in Chapter 8.

1	But positive selection is not the only basis for alloreactivity. This conclusion was implied by observations that T cells artificially driven to mature in animals lacking MHC class I and class II, in which positive selection in the thymus cannot occur, still display frequent alloreactivity. It appears that T-cell receptor genes encode the inherent ability to recognize MHC molecules. X-ray crystallographic studies of T-cell receptors bound to MHC molecules provide a structural basis for an inherent binding interaction (Fig. 6.24). Specific amino acid residues within the germline-encoded region of certain TCRβ genes interact with conserved features of the MHC molecule, implying a type of germlineencoded affinity. Given the large number of variable-region sequences in T-cell receptors, each T-cell receptor may bind MHC molecules in its own idiosyncratic way using both germline-encoded regions and variable regions.

1	In principle, alloreactive T cells might depend on recognizing either a foreign peptide antigen or the nonself MHC molecule to which it is bound for their reactivity against nonself MHC; these options have been called peptide-dependent and peptide-independent allorecognition. But as the number of individual alloreactive T-cell clones studied has increased, it seems that most alloreactive T cells actually recognize both; that is, most individual alloreactive T-cell clones respond to a foreign MHC molecule only when a particular peptide is bound to it. In this sense, the structural basis of allorecognition may be quite similar to normal MHC-restricted peptide recognition and be dependent on contacts with both peptide and MHC molecule (see Fig. 6.23, left panel), but in this case a foreign MHC molecule. In practice, alloreactive responses against a transplanted organ are likely to represent the combined

1	Fig. 6.24 Germline-encoded residues in CDR1 and CDR2 of V-region genes confer on T-cell receptors an inherent affinity for MHC molecules. Shown is the structure for several T-cell receptors bound to a class II MHC molecule. Conserved residues (Lys39, Gln57, and Gln61) within the α1 helix of the MHC (green) make an extended hydrogen-bonded network with germline-encoded and nonpolymorphic residues located in the CDR1 (Asn31) and CDR2 (Glu56, Tyr50) regions of the Vβ 8.2 gene, respectively. The configuration of these contacts is very similar between different structures, implying that the germline sequence of the CDR1 and CDR2 confers an inherent bias for T-cell receptor affinity for MHC. Courtesy of K.C. Garcia. activity of many alloreactive T cells, and it is not possible to determine what peptides from the donor might be involved in recognition by the alloreactive T cells. We will return to alloreactivity when we discuss organ transplantation in more detail in Chapter 15.

1	6-14 Many T cells respond to superantigens. Superantigens are a distinct class of antigens that stimulate a primary T-cell response similar in magnitude to a response to allogeneic MHC molecules. Such responses were first observed in mixed lymphocyte reactions using lymphocytes from strains of mice that were MHC-identical but otherwise genetically distinct. The antigens provoking this reaction were originally designated as minor lymphocyte stimulating (Mls) antigens, and it seemed reasonable to suppose that they might be functionally similar to the MHC molecules themselves. We now know that this is not true. The Mls antigens in these mouse strains are encoded by retroviruses, such as the mouse mammary tumor virus, that have become stably integrated at various sites in the mouse chromosomes. Superantigens are produced by many different pathogens, including bacteria, mycoplasmas, and viruses, and the responses they provoke are helpful to the pathogen rather than the host.

1	Mls proteins act as superantigens because they have a distinctive mode of binding to both MHC and T-cell receptor molecules that enables them to stimulate very large numbers of T cells. Superantigens are unlike other protein antigens, in that they are recognized by T cells without being processed into peptides that are captured by MHC molecules. Indeed, fragmentation of a superantigen destroys its biological activity, which depends on binding as an intact protein to the outside surface of an MHC class II molecule that has already bound peptide. In addition to binding MHC class II molecules, superantigens are able to bind the Vβ region of many T-cell receptors (Fig. 6.25). Bacterial superantigens bind mainly to the Vβ CDR2 loop, and, to a smaller extent, to the Vβ CDR1 loop and an additional loop called the hypervariable 4 or HV4 loop. The HV4 loop is the predominant binding site for viral superantigens, at least for the Mls antigens encoded by the endogenous mouse mammary tumor

1	loop called the hypervariable 4 or HV4 loop. The HV4 loop is the predominant binding site for viral superantigens, at least for the Mls antigens encoded by the endogenous mouse mammary tumor viruses.

1	Fig. 6.25 Superantigens bind directly to T-cell receptors and to MHC molecules. Superantigens can bind independently to MHC class II molecules and to T-cell receptors. As shown in the top panels, the superantigens (red bars) can bind to the Vβ domain of the T-cell receptor (TCR), away from the complementarity-determining regions, and to the outer faces of the MHC class II molecule, outside the peptide-binding site. In the bottom panel, a reconstruction of the interaction between a T-cell receptor, an MHC class II molecule, and a staphylococcal enterotoxin (SE) superantigen is shown by superimposing separate structures of an enterotoxin:MHC class II complex onto an enterotoxin:T-cell receptor complex. The two enterotoxin molecules (actually SEC3 and SEB) are shown in turquoise and blue, binding to the α chain of the MHC class II molecule (yellow) and to the β chain of the T-cell receptor (colored gray for the Vβ domain and pink for the Cβ domain). Molecular model courtesy of

1	H.M. Li, B.A. Fields, and R.A. Mariuzza. Thus, the α-chain V region and the CDR3 of the β chain of the T-cell receptor have little effect on superantigen recognition, which is determined largely by the germline-encoded V gene segments that encode the expressed Vβ chain. Each superantigen is specific for one or a few of the different Vβ gene products, of which there are 20–50 in mice and humans; a superantigen can thus stimulate 2–20% of all T cells.

1	This mode of stimulation does not prime an adaptive immune response specific for the pathogen. Instead, it causes a massive production of cytokines by CD4 T cells, the predominant responding population of T cells. These cytokines have two effects on the host: systemic toxicity and suppression of the adaptive immune response. Both these effects contribute to microbial pathogenicity. Among the bacterial superantigens are the staphylococcal enterotoxins (SEs), which cause food poisoning, and the toxic shock syndrome toxin-1 (TSST-1) of Staphylococcus aureus, the etiologic principle in toxic shock syndrome, which can be caused by a localized infection with toxin-producing strains of the bacterium. The role of viral superantigens in human disease is less clear. 6-15 MHC polymorphism extends the range of antigens to which the immune system can respond.

1	Most polymorphic genes encode proteins that vary by only one or a few amino acids, whereas the allelic variants of MHC proteins differ from each other by up to 20 amino acids. The extensive polymorphism of the MHC proteins has almost certainly evolved to outflank the evasive strategies of pathogens. The requirement that pathogen antigens must be presented by an MHC molecule provides two possible ways in which pathogens could evolve to evade detection. One is through mutations that eliminate from the pathogen’s proteins all peptides able to bind MHC molecules. The Epstein–Barr virus provides an example. There are small isolated populations in southeast China and Papua New Guinea in which about 60% of the people carry the HLA-A11 allele. Many isolates of the Epstein–Barr virus obtained from these populations have mutations in a dominant peptide epitope normally presented by HLA-A11; the mutant peptides no longer bind to HLA-A11 and cannot be recognized by HLA-A11-restricted T cells.

1	these populations have mutations in a dominant peptide epitope normally presented by HLA-A11; the mutant peptides no longer bind to HLA-A11 and cannot be recognized by HLA-A11-restricted T cells. This strategy is clearly much less successful if there are many different MHC molecules, and the polygeny of the MHC may have evolved in response.

1	In addition, in large outbred populations, polymorphism at each locus can potentially double the number of different MHC molecules expressed by an individual, as most individuals will be heterozygotes. Polymorphism has the additional advantage that individuals in a population will differ in the combinations of MHC molecules that they express and will therefore present different sets of peptides from each pathogen. This makes it unlikely that all individuals in a population will be equally susceptible to a given pathogen, and its spread will therefore be limited. The fact that exposure to pathogens over an evolutionary timescale can select for particular MHC alleles is indicated by the strong association of the HLA-B53 allele with recovery from a potentially lethal form of malaria. This allele is very common in people from West Africa, where malaria is endemic, and rare elsewhere, where lethal malaria is uncommon.

1	Similar arguments apply to a second strategy by which pathogens could evade recognition. Pathogens that can block the presentation of their peptides by MHC molecules can avoid the adaptive immune response. Adenoviruses encode a protein that binds to MHC class I molecules in the endoplasmic reticulum and prevents their transport to the cell surface, thus preventing the recognition of viral peptides by CD8 cytotoxic T cells. This viral MHC-binding protein interacts with a polymorphic region of the MHC class I molecule, as some allelic variants are retained in the endoplasmic reticulum by the adenoviral protein, whereas others are not. Increasing the variety of MHC molecules expressed reduces the likelihood that a pathogen will be able to block presentation by all of them and completely evade an immune response.

1	These arguments raise a question: if having three MHC class I loci is better than having one, why are there not far more? The probable explanation is that each time a distinct MHC molecule is added to the repertoire, all T cells that can respond to self peptides bound to that MHC molecule must be removed to maintain self tolerance. It seems that the number of MHC genes present in humans and mice is about optimal to balance the advantages of presenting an increased range of foreign peptides with the disadvantages of losing T cells from the repertoire. Summary.

1	The major histocompatibility complex (MHC) of genes consists of a linked set of genetic loci encoding many of the proteins involved in antigen presentation to T cells, most notably the MHC class I and class II glycoproteins (the MHC molecules) that present peptides to the T-cell receptor. The outstanding feature of the MHC molecules is their extensive polymorphism. This polymorphism is of critical importance in antigen recognition by T cells. A T cell recognizes antigen as a peptide bound by a particular allelic variant of an MHC molecule, and will not recognize the same peptide bound to other MHC molecules. This behavior of T cells is called MHC restriction. Most MHC alleles differ from one another by multiple amino acid substitutions, and these differences are focused on the peptide-binding site and the surface-exposed regions that make direct contact with the T-cell receptor. At least three properties of MHC molecules are affected by MHC polymorphism: the range of peptides bound;

1	site and the surface-exposed regions that make direct contact with the T-cell receptor. At least three properties of MHC molecules are affected by MHC polymorphism: the range of peptides bound; the conformation of the bound peptide; and the direct interaction of the MHC molecule with the T-cell receptor. Thus, the highly polymorphic nature of the MHC has functional consequences, and the evolutionary selection for this polymorphism suggests that it is critical to the role of the MHC molecules in the immune response. Powerful genetic mechanisms generate the variation that is seen among MHC alleles, and a compelling argument can be made that selective pressure to maintain a wide variety of MHC molecules in the population comes from infectious agents. As a consequence, the immune system is highly individualized—each individual responds differently to a given antigen.

1	Generation of ligands for unconventional T-cell subsets.

1	So far we have focused on how peptide:MHC complexes—the ligands for α:β T cells—are generated. We now turn to the question of how other types of T cells recognize their ligands and how these ligands are generated. Our current knowledge in this area is still incomplete, and is perhaps most apparent in the area of γ:δ T cells, where a growing list of ligands for individual γ:δT cells suggests an innate-like pattern of recognition. The recent discovery that the mucosal associated invariant T (MAIT) cells (see Section 4-18) recognize a microbial metabolite when it is presented by a nonpolymorphic MHC class I-like molecule solved a long-standing mystery regarding the function of this particular T-cell subset. Another invariant subset, the invariant NKT cells, provides a system for detecting and responding to lipid rather than peptide antigens. These findings suggest that these invariant and unconventional T cells operate somewhere between innate and adaptive immunity. In this part of the

1	responding to lipid rather than peptide antigens. These findings suggest that these invariant and unconventional T cells operate somewhere between innate and adaptive immunity. In this part of the chapter, we will discuss the ligands they recognize and what is known about how they are generated or expressed.

1	6-16 A variety of genes with specialized functions in immunity are also encoded in the MHC. In addition to the highly polymorphic ‘classical’ MHC class I and class II genes, there are many ‘nonclassical’ MHC genes, many encoded in the MHC but others encoded outside this region. The MHC class I-type molecules show comparatively little polymorphism; many of these have yet to be assigned a function. They are linked to the class I region of the MHC, and their exact number varies greatly among species and even among members of the same species. These genes have been termed MHC class Ib genes; like MHC class I genes, many, but not all, associate with β2-microglobulin when expressed on the cell surface. Their expression on cells is variable, both in the amount present at the cell surface and in tissue distribution. The characteristics of several MHC class Ib gene products are shown in Fig. 6.26.

1	One mouse MHC class Ib molecule, H2-M3, can present peptides with N-formylated amino termini, which is of interest because all bacteria initiate protein synthesis with N-formylmethionine. Cells infected with cytosolic bacteria can be killed by CD8 T cells that recognize N-formylated bacterial peptides bound to H2-M3. Whether an equivalent MHC class Ib molecule exists in humans is not known. Two other closely related mouse MHC class Ib genes, T22 and T10, are expressed by activated lymphocytes and are recognized by a subset of γ:δT cells. Although the precise purpose remains unclear, it has been proposed that this interaction allows γ:δ T cells to regulate these activated lymphocytes expressing T22 and T10 proteins.

1	The other genes that map within the MHC include some that encode complement components (for example, C2, C4, and factor B) and some that encode cytokines—for example, tumor necrosis factor-α (TNF-α) and lymphotoxin— all of which have important functions in immunity. These genes lie in the so-called ‘MHC class III’ region (see Fig. 6.17), a somewhat misleading name, since genes in this region are not MHC molecules at all.

1	Many studies have established associations between susceptibility to certain diseases and particular alleles of MHC genes (see Chapter 15), and we now have considerable insight into how polymorphism in the classical MHC class I and class II genes can affect disease resistance or susceptibility. Most MHC-influenced traits or diseases are known or suspected to have an immunological cause, but this is not so for all of them: some genes residing within the MHC have no known or suspected immunological function. For example, the class Ib gene M10 encodes a protein that functions in the vomeronasal organ as a chaperone to escort certain types of pheromone receptors to the cell surface. M10 could potentially influence mating preference, a trait that has been linked to the MHC region in rodents.

1	The HFE gene, encoding the hemochromatosis protein, lies some 4 million base pairs from HLA-A. Its protein product is expressed in cells of the intestinal cells (see the text and Section 3-24). HLA-C, which is a classical MHC molecule (class Ia), is included here because, in addition to presenting peptides to T-cell receptors, all HLA-C isoforms interact with the KIR class of NK-cell receptors to regulate NK-cell function in the innate immune response. CTL, cytotoxic T lymphocyte.

1	tract and acts in iron metabolism by regulating the uptake of dietary iron into the body. It seems to interact with the transferrin receptor and decrease the receptor’s affinity for iron-loaded transferrin. Individuals defective for this gene have an iron-storage disease, hereditary hemochromatosis, in which an abnormally high level of iron is retained in the liver and other organs. Mice lacking β2-microglobulin have defective expression of all class I molecules and thus also show a similar iron overload. Another MHC gene with a nonimmune function encodes the enzyme 21-hydroxylase, which, when deficient, causes congenital adrenal hyperplasia and, in severe cases, salt-wasting syndrome. Even where a disease-related gene is clearly homologous to immune-system genes, as is the case with HFE, the disease mechanism may not be immune-related. Disease associations mapping to the MHC must therefore be interpreted with caution, in the light of a detailed understanding of its genetic structure

1	the disease mechanism may not be immune-related. Disease associations mapping to the MHC must therefore be interpreted with caution, in the light of a detailed understanding of its genetic structure and the functions of its individual genes. Much remains to be learned about the significance of all the genetic variation localized within the MHC. For instance, the human complement component C4 comes in two versions, C4A and C4B, not to be confused with the C4 convertase cleavage products C4a and C4b, and different individuals have variable numbers of the gene for each type in their genomes, but the adaptive significance of this genetic variability is not well understood.

1	6-17 Specialized MHC class I molecules act as ligands for the activation and inhibition of NK cells and unconventional T-cell subsets.

1	In Sections 3-24 to 3-27, we introduced NK cells and briefly discussed their activation by members of the MIC gene family. These are MHC class Ib genes that are under a different regulatory control than the classical MHC class I genes and are induced in response to cellular stress (such as heat shock). There are seven MIC genes, but only two—MICA and MICB—are expressed and produce protein products (see Fig. 6.26). They are expressed in fibroblasts and epithelial cells, particularly in intestinal epithelial cells, and have a role in innate immunity or in the induction of immune responses in circumstances in which interferons are not produced. The MICA and MICB proteins are recognized by the NKG2D receptor expressed by NK cells. But in addition, NKG2D is also expressed by γ:δ T cells and some CD8 T cells, and it can activate these cells to kill MIC-expressing targets. NKG2D is an ‘activating’ member of the NKG2 family of NK-cell receptors (see Fig. 3.42); its cytoplasmic domain lacks

1	some CD8 T cells, and it can activate these cells to kill MIC-expressing targets. NKG2D is an ‘activating’ member of the NKG2 family of NK-cell receptors (see Fig. 3.42); its cytoplasmic domain lacks the inhibitory sequence motif found in other members of this family, which act as inhibitory receptors (see Section 3-26). NKG2D is coupled to the adaptor protein DAP10, which transmits the signal into the interior of the cell by interacting with and activating intracellular phosphatidylinositol 3-kinase.

1	Even more distantly related to MHC class I genes is a small family of proteins known in humans as the UL16-binding proteins (ULBPs) or the RAET1 proteins (see Fig. 6.26); the homologous proteins in mice are known as Rae1 (retinoic acid early inducible 1) and H60. These proteins also bind NKG2D (see Section 3-27). They seem to be expressed under conditions of cellular stress, such as when cells are infected with pathogens (UL16 is a human cytomegalovirus protein) or have undergone transformation to tumor cells. By expressing ULBPs, stressed or infected cells can bind and activate NKG2D molecules expressed on NK cells, γ:δ T cells, and CD8 cytotoxic α:β T cells, and so be recognized and eliminated.

1	The human MHC class Ib molecule HLA-E and its mouse counterpart Qa-1 (see Fig. 6.26) have an unusual and somewhat puzzling role in cell recognition by NK cells and CD8 T cells. HLA-E and Qa-1 bind a very restricted subset of nonpolymorphic peptides, called Qa-1 determinant modifiers (Qdm), that are derived from the leader peptides of other HLA class I molecules. These peptide:HLA-E complexes can bind to the inhibitory receptor NKG2A:CD94 expressed on NK cells, and so should inhibit the cytotoxic activity of NK cells. This function might seem redundant, since expression of other MHC class I molecules by cells should prevent NK-cell activation (see Section 3-25). Nonetheless, it has been shown that Qa-1 expression by activated CD4 T cells protects them from lysis by NK cells and so Qa-1 expression by other host cells may provide them with additional protection from being killed by NK cells. HLA-E and Qa-1 can also bind leader peptides from the heat shock protein Hsp60sp, and CD8 T cells

1	by other host cells may provide them with additional protection from being killed by NK cells. HLA-E and Qa-1 can also bind leader peptides from the heat shock protein Hsp60sp, and CD8 T cells that are specific for these complexes have been identified in mice and humans. Some recent evidence suggests that CD8 T cells restricted by HLA-E/Qa-1 may help maintain self-tolerance by killing or suppressing potentially autoreactive T cells.

1	In Section 3-26, we introduced the killer cell immunoglobulin-like receptors (KIRs) expressed by NK cells. Members of the KIRs recognize the classical class Ia MHC molecules HLA-A, -B, and -C, which present a diverse repertoire of peptides to CD8 T cells. Although KIRs interact with the same face of the MHC class I molecule as do T-cell receptors, the KIRs bind only at one end, and not over the whole area recognized by the T-cell receptor. Like MHC molecules, KIRs themselves are highly polymorphic, and they have undergone rapid evolution in humans. Only a few HLA-A and HLA-B alleles code for proteins that bind KIRs, but all HLA-C alleles express proteins that bind KIRs, indicating a specialization of HLA-C for regulating NK cells in humans.

1	Two other MHC class Ib molecules, HLA-F and HLA-G (see Fig. 6.26), can also inhibit cell killing by NK cells. HLA-G is expressed on fetus-derived placental cells that migrate into the uterine wall. These cells express no classical MHC class I molecules and cannot be recognized by CD8 T cells but, unlike other cells lacking such proteins, they are not killed by NK cells. This seems to be because HLA-G is recognized by another inhibitory receptor on NK cells, the leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), also called ILT-2 or LIR-1, which prevents the NK cell from killing the placental cells. HLA-F is expressed in a variety of tissues, although it is usually not detected at the cell surface except, for example, on some monocyte cell lines or on virus-transformed lymphoid cells. HLA-F is also thought to interact with LILRB1. 6-18 Members of the CD1 family of MHC class I-like molecules present microbial lipids to invariant NKT cells.

1	Some MHC class I-like genes map outside the MHC region. One small family of such genes is called CD1 and is expressed on dendritic cells, monocytes, and some thymocytes. Humans have five CD1 genes, CD1a through e, whereas mice express only two highly homologous versions of CD1d, namely, CD1d1 and CD1d2. CD1 proteins can present antigens to T cells, but they have two features that distinguish them from classical MHC class I molecules. The first is that CD1, although similar to an MHC class I molecule in its subunit organization and association with β2-microglobulin, behaves like an MHC class II molecule. It is not retained within the endoplasmic reticulum by association with the TAP complex but is targeted to vesicles, where it binds its ligand. The second unusual feature is that, unlike MHC class I, CD1 molecules have a hydrophobic channel that is specialized for binding hydrocarbon alkyl chains. This confers on CD1 molecules an ability to bind and present a variety of glycolipids.

1	CD1 molecules are classified into group 1, comprising CD1a, CD1b, and CD1c, and group 2, containing only CD1d; CD1e is considered intermediate. Group 1 molecules bind various microbial glycolipids, phospholipids, and lipopeptide antigens, such as the mycobacterial membrane components mycolic acid, glucose monomycolate, phosphoinositol mannosides, and lipoarabinomannan (Fig. 6.27). Group 2 CD1 molecules are thought to bind mainly self

1	Fig. 6.27 CD1c binds microbial lipids for presentation to iNKT cells. Top panel: the structure of mannosyl-β1-phosphomycoketides (MPMs) from the cell walls of Mycobacterium tuberculosis (Mtb) (R = C7H15) and M. avium (R = C5H11). Middle panel: MPM (stick figure) bound to CD1c (purple) as viewed from the top, facing the surface of the cell bearing CD1c. Bottom panel: side view of MPM bound to CD1c. The general resemblance with peptide:MHC complexes is apparent. Note: the long acyl chain of MPM extends deep into the binding groove of CD1c, to beneath the α1 helical domain. Courtesy of E. Adams.

1	lipid antigens such as sphingolipids and diacylglycerols. Structural studies show that the CD1 molecule has a deep binding groove into which the glycolipid antigens bind (Fig. 6.28). Unlike the binding of peptide to MHC, in which the peptide takes on a linear, extended conformation, CD1 molecules bind their antigens by anchoring the alkyl chains in the hydrophobic groove, which orients the variable carbohydrate headgroups (or other hydrophilic parts of these molecules) so that they protrude from the end of the binding groove, allowing recognition by the T-cell receptors on CD1-restricted T cells.

1	The T cells that recognize lipids presented by CD1 molecules are largely negative for CD4 and CD8 expression, although some express CD4. Most of the T cells recognizing lipids presented by group 1 CD1 molecules have a diverse repertoire of α:β receptors, and respond to these lipids presented by CD1a, CD1b, and CD1c. In contrast, CD1d-restricted T cells are less diverse, many using the same TCRα chain (Vα24–Jα18 in humans), but they also express NK-cell receptors. These CD1-restricted T cells are called invariant NKT (iNKT) cells.

1	One recognized ligand for CD1d molecules is α-galactoceramide (α-GalCer), which was isolated from an extract of marine sponge. Related glycosphingolipids are produced by various bacteria, including Bacteroides fragilis, which is present in the normal human microbiota. When α-galactoceramide is bound to CD1d, it forms a structure that is recognized by many iNKT cells. The ability of iNKT cells to recognize different glycolipid constituents from microorganisms presented by CD1d molecules places them in an ‘innate’ category, while their possession of a fully rearranged T-cell receptor, despite its relatively limited repertoire, makes them ‘adaptive.’

1	CD1 proteins have evolved as a separate lineage of antigen-presenting molecules able to present microbial lipids and glycolipids to T cells. Just as peptides are loaded onto classical MHC proteins at various cellular locations, the various CD1 proteins are transported differently through the endoplasmic reticulum and endocytic compartments; this provides access to different lipid antigens. Transport is regulated by an amino acid sequence motif at the terminus of the cytoplasmic domain of the CD1 protein through interaction with adaptor-protein (AP) complexes. CD1a lacks this binding motif and moves to the cell surface, where it is transported only through the early endocytic compartment. CD1c and CD1d have motifs that interact with the adaptor AP-2 and are transported through early and late endosomes; CD1d is also targeted to lysosomes. CD1b and mouse CD1d bind AP-2 and AP-3 and can be transported through late endosomes, lysosomes, and the MIIC. CD1 proteins can thus bind lipids

1	late endosomes; CD1d is also targeted to lysosomes. CD1b and mouse CD1d bind AP-2 and AP-3 and can be transported through late endosomes, lysosomes, and the MIIC. CD1 proteins can thus bind lipids delivered into and processed within the endocytic pathway, such as by the internalization of mycobacteria or the ingestion of mycobacterial lipoarabinomannans mediated by mannose receptors.

1	From an evolutionary perspective it is interesting that some class Ib genes seem to have evolved early, before the divergence of the cartilaginous fishes from the vertebrate line, and are likely to have homologs in all vertebrates. Other class I genes have independently evolved into classical and nonclassical loci within the vertebrate lineages that have been studied (for example, cartilaginous fishes, lobe-finned fishes, ray-finned fishes, amphibians, and mammals). Sequence data have also revealed homologs of the mammalian MHC-I and MHC-II gene families in virtually all jawed vertebrates including sharks, bony fishes, reptiles, and birds. In contrast, CD1 genes may not be as old as Chapter 6: Antigen Presentation to T Lymphocytes

1	Fig. 6.28 Structure of CD1 binding to a lipid antigen. Shown are top and side views of the structure of mouse CD1d bound to C8PhF, a synthetic lipid that is an analog of α-GalCer. The helical side chains of CD1d (blue) form a binding pocket that is generally similar in shape to the binding pockets of MHC class I and II molecules. However, the C8PhF (red) ligand binds to CD1 molecules in a distinctly different conformation from that of peptides. The two long alkyl side chains extend deep inside the binding groove (see side view), where they make contacts with hydrophobic residues. This orientation of the alkyl side chains places the carbohydrate component of α-GalCer to the outer surface of CD1, where it can be recognized by the T-cell receptor. In addition, the CD1 molecule contains an endogenous lipid molecule (yellow) derived from cellular sources that binds to a distinct region within the groove and prevents a large pocket adjacent to the α-GalCer-binding region from collapsing.

1	endogenous lipid molecule (yellow) derived from cellular sources that binds to a distinct region within the groove and prevents a large pocket adjacent to the α-GalCer-binding region from collapsing. The ability to incorporate additional ligands into the binding groove may provide flexibility to CD1d in accommodating a variety of exogenous glycosphingolipids from microorganisms. Courtesy of I.A. Wilson.

1	other MHC class Ib genes. They have been found only in a subset of these animal groups and appear to be missing in fish. This pattern of CD1 occurrence in the genomes of living species suggests the emergence of CD1 in an early terrestrial vertebrate. 6-19 The nonclassical MHC class I molecule MR1 presents microbial folate metabolites to MAIT cells.

1	Another nonclassical MHC class Ib molecule is MR1 (MHC-related protein 1). MR1 associates with β2-microglobulin and is encoded outside the MHC, but its function was originally known only in relation to a conserved population of α:β T cells known as mucosal associated invariant T cells (MAIT cells). In Section 4-18, we introduced MAIT cells as one population of T cells expressing the CD8α homodimer, but they are uniquely characterized by expressing an invariant α chain of the T-cell receptor, specifically human Vα7.2J2–Jα33 (or in mouse, Vα19) . This α chain pairs with a limited number of Vβ chains, typically Vβ2 or Vβ13. MAIT cells are very abundant in humans and can comprise up to 10% of the lymphocytes in the peripheral blood and tissues such as the liver. They are also present in mesenteric lymph nodes and the mucosa of the intestine. Studies of MAIT cells revealed that their development requires the expression of MR1, and further, that a wide spectrum of microbes, including

1	mesenteric lymph nodes and the mucosa of the intestine. Studies of MAIT cells revealed that their development requires the expression of MR1, and further, that a wide spectrum of microbes, including diverse bacteria and yeast, can activate MAIT cells. However, when they were originally identified around a decade ago, it was unclear what, if any, ligand is being recognized by these cells.

1	Structural studies of MR1 uncovered an important clue. The MR1 protein was unstable when produced in vitro by cell lines grown in typical tissue culture conditions. It was discovered that the protein was stabilized when it was refolded in media containing B vitamins or folic acid (vitamin B9). Chemical analysis revealed that a small molecule—identified as a folate derivative, 6-formyl pterin (6-FP)—was bound to the stabilized MR1. X-ray crystallographic studies showed that 6-FP was bound in the central groove of the MR1 molecule; this helped explain how folate derivatives might stabilize MR1. However, MAIT cells were not activated by cells expressing the 6-FP:MR1 complex, suggesting other molecules might be the physiological ligands to activate MAIT cells. Analysis of MR1 proteins that were refolded in the presence of supernatants from cultures of Salmonella typhimurium eventually led to the identification of several riboflavin metabolites that are formed by biosynthetic pathways in

1	refolded in the presence of supernatants from cultures of Salmonella typhimurium eventually led to the identification of several riboflavin metabolites that are formed by biosynthetic pathways in most bacteria and yeast. These metabolites not only bind to MR1, but also activate MAIT cells. Thus, MAIT cells are activated in response to infection by these organisms by detecting products specific to their folate metabolism. As such, MAIT cells appear to hold an intermediate place in the spectrum of innate and adaptive immunity, similar to iNKT cells, in that they use an antigen receptor assembled by somatic gene rearrangement, but recognize a molecular structure that falls within the definition of a PAMP.

1	6-20 γ:δ T cells can recognize a variety of diverse ligands. γ:δ T cells and α:β T cells have been known to be distinct developmental lineages almost since the T-cell receptor genes were identified. But unlike α:βT cells, the function of γ:δ T cells has remained somewhat obscure, due primarily to difficulty in identifying the ligands they recognize. Yet the abundance of γ:δ T cells across vertebrate species, their rapid expansion to form more than 50% of the blood lymphocytes during infections, and their abundant cytokine production all argue for an important role in immunity. Over time, many different ligands recognized by γ:δ T-cell clones have been identified (Fig. 6.29), and their diversity suggests that, like iNKT and MAIT cells, they hold an intermediate, or transitional, position in the spectrum of innate versus adaptive immunity.

1	In Section 4-20, we discussed how one γ:δ T-cell receptor binds to the non-classical MHC class I molecule T22. Instead of binding centrally over the MHC binding groove, similar to an α:β T-cell receptor, the γ:δ T-cell receptor interacts obliquely from one side of the T22 molecule. However, fewer than 1% of γ:δ T cells recognize this ligand. Other antigens recognized by murine γ:δT cells are the protein phycoerythrin (PE) from algae, the inner mitochondrial membrane lipid cardiolipin, glycoprotein I of herpes simplex virus, and a peptide derived from the hormone insulin. Among antigens that can activate human γ:δ T cells are the nonclassical MHC class I proteins MICA and ULBP4 and the endothelial protein C receptor (EPCR), which is expressed by endothelial cells. Like MICA and ULBPs, EPCR appears to be induced upon stress, such as during infection of cells by cytomegalovirus, suggesting that reactive γ:δ T cells could serve in an innate capacity similar to that of NK cells activated

1	appears to be induced upon stress, such as during infection of cells by cytomegalovirus, suggesting that reactive γ:δ T cells could serve in an innate capacity similar to that of NK cells activated by stress-induced nonclassical MHC class Ib molecules. Several other antigens can activate human γ:δ T cells (see Fig. 6.29), although there is still limited structural information about their interaction with the T-cell receptor, and even reservations about whether such an interaction is always

1	Chapter 6: Antigen Presentation to T Lymphocytes the basis for activation. Among these activating antigens is Skint-1 (selection and upkeep of intraepithelial T cells 1), an immunoglobulin superfamily member that is expressed by thymic epithelial cells and by keratinocytes. Skint-1 seems to be required for the generation of a subset of Vγ5:Vδ1 T cells that develop in the thymus and home to the skin to become ‘dendritic epidermal T cells’ (DETCs). Some evidence suggests a direct interaction between Skint-1 and the γ:δ T-cell receptor, although structural studies are not yet available. Conceivably, DETCs localize to the skin due to recognition by their T-cell receptor of Skint-1 expressed by keratinocytes. There, they might provide a ‘transitional’ mode of immune defense, becoming activated through innate receptors that are triggered locally during infections. Summary.

1	Antigen presentation to various nonconventional T-cell subsets and γ:δT cells generally does not involve the generation of peptide:MHC complexes. Instead, these cells recognize surface proteins, such as ULBPs and RAET-1 proteins, that may indicate cellular stress, transformation, or intracellular infection, or nonpeptide antigens, such as microbial glycolipids or folate metabolites presented by CD1 molecules. The MHC region contains many genes whose structure is closely related to the MHC class I molecules—the so-called non-classical, or class Ib, MHC. Some of these genes serve purposes that are unrelated to the immune system, but many are involved in recognition by activating and inhibitory receptors expressed by NK cells, γ:δ T cells, and α:β T cells. MHC class Ib proteins called CD1 molecules are encoded outside the MHC region. CD1c and CD1d can bind lipids and glycolipid antigens for presentation to iNKT cells expressing invariant T-cell receptors. The T-cell population called

1	molecules are encoded outside the MHC region. CD1c and CD1d can bind lipids and glycolipid antigens for presentation to iNKT cells expressing invariant T-cell receptors. The T-cell population called MAIT cells, which are abundant in humans, recognize vitamin B9 metabolites presented by the MR1 MHC class Ib molecule, suggesting that the MAIT cells have a ‘transitional’ role between innate and adaptive immunity. Likewise, many antigens that activate γ:δ T cells may be indicators of stress or infection, and these cells are able to generate cytokines that amplify immune defense pathways.

1	Summary to Chapter 6. T-cell receptors on conventional α:β T cells recognize peptides bound to MHC molecules. In the absence of infection, MHC molecules are occupied by self peptides, which do not normally provoke a T-cell response, because of various tolerance mechanisms. But during infections, pathogen-derived peptides become bound to MHC molecules and are displayed on the cell surface, where they can be recognized by T cells that have been previously activated and armed for the specific peptide:MHC complex. Naive T cells become activated when they encounter their specific antigen presented on activated dendritic cells. MHC class I molecules in most cells bind to peptides derived from proteins that have been synthesized and then degraded in the cytosol. Some dendritic cells can obtain and process exogenous antigens and present them on MHC class I molecules. This process of cross-presentation is important for priming CD8 T cells to many viral infections.

1	Through assembly with the invariant chain (Ii), MHC class II molecules bind peptides derived from proteins degraded in endocytic vesicles, but they can also acquire self antigens through autophagy. Stable peptides are bound after a process of peptide editing in the endocytic compartment involving HLA-DM and HLA-DO. CD8 T cells recognize peptide:MHC class I complexes and are activated to kill cells displaying foreign peptides derived from cytosolic pathogens, such as viruses. CD4 T cells recognize peptide:MHC class II complexes and are specialized to activate other immune effector cells, for example, B cells or macrophages, to act against the foreign antigens or pathogens that they have taken up.

1	For each class of MHC molecule, there are several genes arranged in clusters within a larger region known as the major histocompatibility complex (MHC). Within the MHC, the genes for the MHC molecules are closely linked to genes involved in the degradation of proteins into peptides, the formation of the complex of peptide and MHC molecule, and the transport of these complexes to the cell surface. Because the several different genes for the MHC class I and class II molecules are highly polymorphic and are expressed in a codominant fashion, each individual expresses a number of different MHC class I and class II molecules. Each different MHC molecule can bind stably to a range of different peptides, and thus the MHC repertoire of each individual can recognize and bind many different peptide antigens. Because the T-cell receptor binds a combined peptide:MHC ligand, T cells show MHC-restricted antigen recognition, such that a given T cell is specific for a particular peptide bound to a

1	antigens. Because the T-cell receptor binds a combined peptide:MHC ligand, T cells show MHC-restricted antigen recognition, such that a given T cell is specific for a particular peptide bound to a particular MHC molecule.

1	Unconventional T-cell subsets include iNKT cells, MAIT cells, and γ:δ T cells, which recognize nonpeptide ligands of various types. Some CD1 molecules bind self lipids and pathogen-derived lipid molecules and present them to iNKT cells. MAIT cells recognize vitamin metabolites that are specific to bacteria and yeast and that are presented by MR1. γ:δ T cells are activated by a diverse array of ligands, including MHC class Ib molecules and EPCR, that are induced by infection or cellular stress. These T-cell subsets function in the transitional area between innate and adaptive immunity, relying on a repertoire of receptors produced by somatic gene rearrangement but recognizing ligands in a manner somewhat similar to the way PAMPs are recognized by TLRs and other fully innate receptors. Questions.

1	Questions. 6.1 Short Answer: Dendritic cells are capable of efficiently acquiring antigens from exogenous sources and presenting these them to T cells on MHC class I molecules. How is this different from every other cell in the body and why is it important? 6.2 Matching: Match the following terms with the appropriate description: A. Proteasome i. Displace the constitutive β subunits of the catalytic chamber as a response to interferons B. 20S core ii. Composed of one catalytic core and two 19S regulatory caps C. LMP2, LMP7, iii. Large cylindrical complex of MECL-1 28 subunits arranged in four stacked rings D. PA28 iv. Targets protein for degradation E. Lysine 48 v. Binds the proteasome and ubiquitin increases the rate of protein release from the proteasome 6.3 True or False: MHC class I surface expression is not affected by the cell’s capacity to transport peptides into the endoplasmic reticulum.

1	6.4 Fill-in-the-Blanks: Cell membrane-destined polypeptides are translocated to the lumen of the endoplasmic reticulum, which is intriguing because the MHC class I presented peptides are found in the ________. Further research revealed that presentation of cytosolic peptides is possible due to a family of ABC transporters, ______, that mediate the ATP-dependent transport of peptides into the lumen of the _______. This transporter complex has limited specificities for the transported peptides; for example, peptides are generally ________ amino acids in length and transport is biased in favor of ________ residues in the 6.9 Multiple Choice: Defective function of which of the carboxy terminus and against _________ residues within the first _______ amino-terminal residues.

1	6.5 Multiple Choice: CD8 dendritic cells are uniquely capable of strongly cross-presenting antigens. Which of the following options correctly matches a transcription factor essential for CD8 dendritic cell development and a surface marker uniquely expressed by these cells? A. CIITA, CD74 B. BATF3, CD4 C. CIITA, CD94 D. BATF3, XCR1 6.6 Matching: Match the following terms with the appropriate description: A. TRiC i. Retains the MHC class I molecule α chain in a partly folded state B. ERAAP ii. Protects peptides produced in the cytosol from complete degradation C. Calnexin iii. Forms a bridge between the MHC class I molecule and the TAP complex D. ERp57 iv. Trims the amino terminus of peptides that are too long for MHC binding E. Tapasin v. Breaks and re-forms disulfide bonds in the MHC class I α domain during peptide loading 6.7 True or False: Cytosolic antigens are not presented through MHC class II molecules.

1	E. Tapasin v. Breaks and re-forms disulfide bonds in the MHC class I α domain during peptide loading 6.7 True or False: Cytosolic antigens are not presented through MHC class II molecules. 6.8 Matching: Order the following events in the sequence in which MHC class II processing happens in an antigen-presenting cell: ____The CD74 trimerization domain is cleaved. ____MHC class II is translocated into the endoplasmic reticulum. ____Cathepsin S cleaves LIP22 and leaves the CLIP fragment on the MHC molecule. ____CD74 trimers bind non-covalently to MHC class II α:β heterodimers. ____HLA-DM catalyzes the release of CLIP and promotes peptide editing. ____MHC class II heterodimers are released from calnexin for transport to a low-pH endosomal compartment. General references. Germain, R.N.: MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell 1994, 76:287–299.

1	General references. Germain, R.N.: MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell 1994, 76:287–299. Klein, J.: Natural History of the Major Histocompatibility Complex. New York: Wiley, 1986. Moller, G. (ed.): Origin of major histocompatibility complex diversity. Immunol. Rev. 1995, 143:5–292. Trombetta, E.S., and Mellman, I.: Cell biology of antigen processing in vitro and in vivo. Annu. Rev. Immunol. 2005, 23:975–1028. following proteins will result in failed CD8 T-cell priming? A. HLA-DM B. Cathepsin S C. TAP1/2 D. CD74 6.10 Multiple Choice: Defective function of which of the following proteins will result in decreased cytosolic peptide presentation on MHC class II? A. IRGM3 B. BATF3 C. MARCH-1 D. TAP1/2 6.11 True or False: Superantigens do not induce an adaptive immune response and are independent of peptide-specific MHC–TCR interactions. 6.12 Multiple Choice: Which of the following statements is false?

1	6.12 Multiple Choice: Which of the following statements is false? A. Polymorphisms at each locus can potentially double the number of different MHC molecules expressed by an individual. B. Pathogens can evade the immune system by mutating the immunodominant epitope, which results in loss of affinity for the specific MHC allele. C. Pathogens do not cause evolutionary pressure to select MHC alleles that confer protection against them. D. The DRα chain and its mouse homolog, Eα, are monomorphic. 6.13 True or False: Classical MHC class I molecules are highly polymorphic, as opposed to MHC class Ib, which are oligomorphic. 6.14 Matching: Match the following MHC class Ib genes with their appropriate description: Section references. 6-1 Antigen presentation functions both in arming effector T cells and in triggering their effector functions to attack pathogen-infected cells.

1	Section references. 6-1 Antigen presentation functions both in arming effector T cells and in triggering their effector functions to attack pathogen-infected cells. Guermonprez, P., Valladeau, J., Zitvogel, L., Théry, C., and Amigorena, S.: Antigen presentation and T cell stimulation by dendritic cells. Annu. Rev. Immunol. 2002, 20:621–667. Lee, H.K., Mattei, L.M., Steinberg, B.E., Alberts, P., Lee, Y.H., Chervonsky, A., Mizushima, N., Grinstein, S., and Iwasaki, A.: In vivo requirement for Atg5 in antigen presentation by dendritic cells. Immunity 2010, 32:227–239. Segura, E., and Villadangos, J.A.: Antigen presentation by dendritic cells in vivo. Curr. Opin. Immunol. 2009, 21:105–110. Vyas, J.M., Van der Veen, A.G., and Ploegh, H.L.: The known unknowns of antigen processing and presentation. Nat. Rev. Immunol. 2008, 8:607–618. 6-2 Peptides are generated from ubiquitinated proteins in the cytosol by the proteasome.

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1	Boyington, J.C., Riaz, A.N., Patamawenu, A., Coligan, J.E., Brooks, A.G., and Sun, P.D.: Structure of CD94 reveals a novel C-type lectin fold: implications for the NK cell-associated CD94/NKG2 receptors. Immunity 1999, 10:75–82. Braud, V.M., Allan, D.S., O’Callaghan, C.A., Söderström, K., D’Andrea, A., Ogg, G.S., Lazetic, S., Young, N.T., Bell, J.I., Phillips, J.H., et al.: HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 1998, 391:795–799. Braud, V.M., and McMichael, A.J.: Regulation of NK cell functions through interaction of the CD94/NKG2 receptors with the nonclassical class I molecule HLA-E. Curr. Top. Microbiol. Immunol. 1999, 244:85–95. Jiang, H., Canfield, S.M., Gallagher, M.P., Jiang, H.H., Jiang, Y., Zheng, Z., and Chess, L.: HLA-E-restricted regulatory CD8(+) T cells are involved in development and control of human autoimmune type 1 diabetes. J. Clin. Invest. 2010, 120:3641–3650.

1	Lanier, L.L.: NK cell recognition. Annu. Rev. Immunol. 2005, 23:225–274. Lopez-Botet, M., and Bellon, T.: Natural killer cell activation and inhibition by receptors for MHC class I. Curr. Opin. Immunol. 1999, 11:301–307. Lopez-Botet, M., Bellon, T., Llano, M., Navarro, F., Garcia, P., and de Miguel, M.: Paired inhibitory and triggering NK cell receptors for HLA class I molecules. Hum. Immunol. 2000, 61:7–17. Lopez-Botet, M., Llano, M., Navarro, F., and Bellon, T.: NK cell recognition of non-classical HLA class I molecules. Semin. Immunol. 2000, 12:109–119. Lu, L., Ikizawa, K., Hu, D., Werneck, M.B., Wucherpfennig, K.W., and Cantor, H.: Regulation of activated CD4+ T cells by NK cells via the Qa-1-NKG2A inhibitory pathway. Immunity 2007, 26:593–604. Pietra, G., Romagnani, C., Moretta, L., and Mingari, M.C.: HLA-E and HLA-Ebound peptides: recognition by subsets of NK and T cells. Curr. Pharm. Des. 2009, 15:3336–3344.

1	Pietra, G., Romagnani, C., Moretta, L., and Mingari, M.C.: HLA-E and HLA-Ebound peptides: recognition by subsets of NK and T cells. Curr. Pharm. Des. 2009, 15:3336–3344. Rodgers, J.R., and Cook, R.G.: MHC class Ib molecules bridge innate and acquired immunity. Nat. Rev. Immunol. 2005, 5:459–471. 6-18 Members of the CD1 family of MHC class I-like molecules present microbial lipids to invariant NKT cells. Gendzekhadze, K., Norman, P.J., Abi-Rached, L., Graef, T., Moesta, A.K., Layrisse, Z., and Parham, P.: Co-evolution of KIR2DL3 with HLA-C in a human population retaining minimal essential diversity of KIR and HLA class I ligands. Proc. Natl Acad. Sci. USA 2009, 106:18692–18697. Godfrey, D.I., Stankovic, S., and Baxter, A.G.: Raising the NKT cell family. Nat. Immunol. 2010, 11:197–206. Hava, D.L., Brigl, M., van den Elzen, P., Zajonc, D.M., Wilson, I.A., and Brenner, M.B.: CD1 assembly and the formation of CD1-antigen complexes. Curr. Opin. Immunol. 2005, 17:88–94.

1	Hava, D.L., Brigl, M., van den Elzen, P., Zajonc, D.M., Wilson, I.A., and Brenner, M.B.: CD1 assembly and the formation of CD1-antigen complexes. Curr. Opin. Immunol. 2005, 17:88–94. Moody, D.B., and Besra, G.S.: Glycolipid targets of CD1-mediated T-cell responses. Immunology 2001, 104:243–251. Moody, D.B., and Porcelli, S.A.: CD1 trafficking: invariant chain gives a new twist to the tale. Immunity 2001, 15:861–865. Moody, D.B., and Porcelli, S.A.: Intracellular pathways of CD1 antigen presentation. Nat. Rev. Immunol. 2003, 3:11–22. Scharf, L., Li, N.S., Hawk, A.J., Garzón, D., Zhang, T., Fox, L.M., Kazen, A.R., Shah, S., Haddadian, E.J., Gumperz, J.E., et al.: The 2.5Å structure of CD1c in complex with a mycobacterial lipid reveals an open groove ideally suited for diverse antigen presentation. Immunity 2010, 33:853–862.

1	Schiefner, A., Fujio, M., Wu, D., Wong, C.H., and Wilson, I.A.: Structural evaluation of potent NKT cell agonists: implications for design of novel stimulatory ligands. J. Mol. Biol. 2009, 394:71–82. 6-19 The nonclassical MHC class I molecule MR1 presents microbial folate metabolites to MAIT cells. Birkinshaw, R.W., Kjer-Nielsen, L., Eckle, S.B., McCluskey, J., and Rossjohn, J.: MAITs, MR1 and vitamin B metabolites. Curr. Opin. Immunol. 2014, 26:7–13. Kjer-Nielsen, L., Patel, O., Corbett, A.J., Le Nours, J., Meehan, B., Liu, L., Bhati, M., Chen, Z., Kostenko, L., Reantragoon, R., et al.: MR1 presents microbial vitamin B metabolites to MAIT cells. Nature 2012, 491:717–723. López-Sagaseta, J., Dulberger, C.L., Crooks, J.E., Parks, C.D., Luoma, A.M., McFedries, A., Van Rhijn, I., Saghatelian, A., and Adams, E.J.: The molecular basis for Mucosal-Associated Invariant T cell recognition of MR1 proteins. Proc. Natl Acad. Sci. USA 2013, 110:E1771–1778.

1	6-20 γ:δ T cells can recognize a variety of diverse ligands. Chien, Y.H., Meyer, C., and Bonneville, M.: γδ T cells: first line of defense and beyond. Annu. Rev. Immunol. 2014, 32:121–155. Turchinovich, G., and Hayday, A.C.: Skint-1 identifies a common molecular mechanism for the development of interferon-γ-secreting versus interleukin-17-secreting γδ T cells. Immunity 2011, 35:59–68. Uldrich, A.P., Le Nours, J., Pellicci, D.G., Gherardin, N.A., McPherson, K.G., Lim, R.T., Patel, O., Beddoe, T., Gras, S., Rossjohn, J., et al.: CD1d-lipid antigen recognition by the γδ TCR. Nat. Immunol. 2013, 14:1137–1145. Willcox, C.R., Pitard, V., Netzer, S., Couzi, L., Salim, M., Silberzahn, T., Moreau, J.F., Hayday, A.C., Willcox, B.E., and Déchanet-Merville, J.: Cytomegalovirus and tumor stress surveillance by binding of a human γδ T cell antigen receptor to endothelial protein C receptor. Nat. Immunol. 2012, 13:872–879.

1	T and B lymphocytes are cells of the adaptive immune system, each of which expresses a unique antigen receptor. These cells circulate between the blood, the lymph, and, most importantly, the secondary lymphoid organs, where they survey antigen-presenting cells for their specific antigen. Once that antigen is encountered, signals from the antigen receptor activate several downstream pathways that convert quiescent naive lymphocytes into metabolically active cells that are reorganizing their actin cytoskeleton, activating transcription factors, and synthesizing a wide range of new proteins. As a result of these events, naive T and B cells undergo rapid cell division and differentiate into armed effector cells, thus expanding lymphocyte populations during an immune response and equipping these cells with the machinery to combat infections.

1	We begin by discussing some general principles of intracellular signaling, and then outline the pathways activated when a naive lymphocyte encounters its specific antigen. Next, we briefly discuss the co-stimulatory signaling that is necessary to activate naive T cells and, in most cases, naive B cells. In the last part of the chapter we focus on inhibitory receptors, and their roles in down-regulating signaling pathways in T and B cells. General principles of signal transduction and propagation.

1	General principles of signal transduction and propagation. In this part of the chapter we review briefly some general principles of receptor action and signal transduction that are common to many of the pathways discussed here. All cell-surface receptors that have a signaling function either are transmembrane proteins themselves or form parts of protein complexes that link the exterior and interior of the cell. Different classes of receptors trans-duce extracellular signals in a variety of ways. A common theme among the receptors covered in this chapter is that ligand binding results in the activation of an intracellular enzymatic activity. General principles of signal transduction and propagation. antigen receptor signaling and lymphocyte activation. co-stimulatory and inhibitory receptors modulate antigen receptor signaling in T and B lymphocytes. 7-1 Transmembrane receptors convert extracellular signals into intracellular biochemical events.

1	co-stimulatory and inhibitory receptors modulate antigen receptor signaling in T and B lymphocytes. 7-1 Transmembrane receptors convert extracellular signals into intracellular biochemical events. The enzymes most commonly associated with receptor activation are the protein kinases. This large group of enzymes catalyzes the covalent attachment of a phosphate group to a protein, a reversible process called phosphorylation. For receptors that use protein kinases, the binding of ligand to the extracellular part of the receptor allows the receptor-associated protein kinase to become ‘active’—that is, to phosphorylate its intracellular substrate—and thus to propagate the signal. As we shall see, receptor-associated kinases can become activated in various ways, such as by undergoing modifications to the kinase itself that alter its intrinsic catalytic efficiency or by changes in subcellular localization that increase access to its biochemical substrates.

1	In animals, protein kinases phosphorylate proteins on three amino acids— tyrosine, serine, or threonine. Most of the enzyme-linked receptors we discuss in detail in this chapter activate tyrosine protein kinases. Tyrosine kinases are specific for tyrosine residues, whereas serine/threonine kinases phosphorylate serine and threonine residues; less common are dual-specificity kinases that phosphorylate both tyrosine and serine/threonine residues in their substrates. Protein tyrosine phosphorylation is much less common than serine/threonine phosphorylation in general, and is employed mainly in signaling pathways. One large group of receptors—the so-called receptor tyrosine kinases— carry a kinase activity within the cytoplasmic region of the receptor itself (Fig. 7.1, top panel). This group contains receptors for many growth factors; lymphocyte receptors of this type include Kit and FLT3, which are expressed on developing lymphocytes in addition to other hematopoietic progenitor cells

1	contains receptors for many growth factors; lymphocyte receptors of this type include Kit and FLT3, which are expressed on developing lymphocytes in addition to other hematopoietic progenitor cells and are discussed in Chapter 8. The receptor for transforming growth factor-β (TGF-β), an important regulatory cytokine produced by many cells, is a receptor serine/threonine kinase.

1	Even more important to the function of mature lymphocytes are receptors that have no intrinsic enzymatic activity themselves but associate with intracellular tyrosine kinases. The antigen receptors on B lymphocytes and T lymphocytes are of this type, as are the receptors for some types of cytokines. Ligand binding to the extracellular domain of such receptors causes particular amino acid residues in their cytoplasmic domains to become phosphorylated by specific cytoplasmic tyrosine kinases (see Fig. 7.1, bottom panel). These nonreceptor kinases either can be constitutively associated with the cytoplasmic domains of the receptors, as with many cytokine receptors, or may become associated with the receptors when they bind their ligands, as is the case for the antigen receptors.

1	For many cytokine receptors, ligand binding causes dimerization or clustering of individual receptor molecules, bringing the associated kinases together and enabling them to phosphorylate the cytoplasmic tail of adjacent receptors— thus initiating an intracellular signal. In the case of the lymphocyte antigen receptors, association with cytoplasmic tyrosine kinases occurs after ligand binding but is unlikely to be due to a simple clustering mechanism. Instead, the actions of co-receptors are required: these bring cytoplasmic tyrosine kinases into proximity with the cytoplasmic regions of the antigen receptor, a complex process that we will describe later.

1	Signaling is usually not a simple ‘on or off’ switch. Depending on the affinity of the receptor for the ligand, the abundance of the ligand, the concentrations of intracellular signaling components, and a complex network of positiveand negative-feedback pathways, receptor activation and downstream signaling occur when a minimum threshold determined by all of these factors is exceeded. These features are often merged into the simple term ‘signal

1	These receptors activate a protein kinase on the cytoplasmic side of the membrane to convey the information that a ligand has bound to their extracellular portion. Receptor tyrosine kinases (top panels) contain the kinase activity as part of the receptor itself. Ligand binding results in clustering of the receptor, activation of catalytic activity, and the consequent phosphorylation (denoted by red dots) of the receptor tails and other substrates, transmitting the signal onward. Receptors that lack intrinsic kinase activity associate with nonreceptor kinases (bottom panels). Receptor dimerization or clustering after ligand binding activates the associated enzyme. in all receptors of these types encountered in this chapter, the enzyme is a tyrosine kinase. strength.’ It is important to keep in mind that variations in signal strength will determine the magnitude of cellular responses—some will be all-or-nothing, whereas others will increase as the strength of signaling increases.

1	The role of protein kinases in cell signaling is not confined to receptor activation, as they act at many different stages in intracellular signaling pathways. Protein kinases figure largely in cell signaling because phosphorylation and dephosphorylation—the removal of a phosphate group—are the means of regulating the activity of many enzymes, transcription factors, and other proteins. Equally important to the workings of signaling pathways is the fact that phosphorylation generates sites on proteins to which other signaling proteins can bind.

1	Phosphate groups are removed from proteins by a large class of enzymes called protein phosphatases. Different classes of protein phosphatases remove phosphate groups from phosphotyrosine or from phosphoserine/ phosphothreonine, or both (as in dual-specificity phosphatases). Specific dephosphorylation by phosphatases is one important means of regulating signaling pathways by resetting a protein to its original state and thus switching signaling off. Dephosphorylation does not always inhibit a protein’s activity. In many instances the removal of a particular phosphate group by a specific phosphatase is needed to activate an enzyme. In other cases, the extent of phosphorylation of an enzyme determines its activity, and represents a balance between the activity of kinases and phosphatases.

1	Fig. 7.2 Signaling proteins interact with each other and with lipid signaling molecules via modular protein domains. a few of the most common protein domains used by immune-system signaling proteins are listed, together with some proteins that contain them and the general class of ligand bound by the interaction domain. The right-hand column lists specific examples of a protein motif (in single-letter amino acid code) or, for the phosphoinositide-binding domains, the particular phosphoinositide that they bind. all these domains are used in many other nonimmune signaling pathways as well. 7-2 Intracellular signal propagation is mediated by large multiprotein signaling complexes.

1	As we learned in Chapter 3, binding of a ligand to its receptor can initiate a cascade of events involving intracellular proteins that sequentially convey signaling information onward. The unique enzymes and other components assembled into a particular multiprotein receptor complex will determine the character of the signal it generates. These components may be shared by several receptor pathways, or they may be exclusive to one receptor pathway, thus allowing distinct signaling pathways to be built up from a relatively limited number of components. The assembly of multisubunit signaling complexes involves specific interactions of a number of distinct types of proteininteraction domains, or protein-interaction modules, carried by the signaling proteins. Figure 7.2 gives a few examples of such domains. Signaling proteins in general contain at least one such protein-interaction domain, but many contain multiple domains. These protein modules cooperate with each other, for example, to

1	such domains. Signaling proteins in general contain at least one such protein-interaction domain, but many contain multiple domains. These protein modules cooperate with each other, for example, to organize signaling proteins into the correct subcellular localizations, to enable specific binding between protein partners, and to modify enzymatic activity.

1	For the pathways considered in this chapter, the most important mechanism underlying the formation of signaling complexes is the phosphorylation of protein tyrosine residues. Phosphotyrosines are binding sites for a number of protein-interaction domains, including the SH2 (Src homology 2) domain (see Fig. 7.2). SH2 domains, built from approximately 100 amino acids, are present in many intracellular signaling proteins, where they are frequently linked to other types of enzymatic or other functional domains. SH2 domains recognize the phosphorylated tyrosine (pY) and, typically, the amino acid three positions away (pYXXZ, where X is any amino acid and Z is a specific amino acid); they bind in a sequence-specific fashion, with different SH2 domains preferring different combinations of amino acids. In this way, the unique SH2 domain of a signaling molecule can act as a ‘key’ that allows inducible and specific association with a protein containing the appropriate pY-containing amino acid

1	acids. In this way, the unique SH2 domain of a signaling molecule can act as a ‘key’ that allows inducible and specific association with a protein containing the appropriate pY-containing amino acid sequence.

1	Tyrosine kinase-associated receptors can assemble multiprotein signaling complexes by using proteins called scaffolds and adaptors. Scaffolds and adaptors lack enzymatic activity, and they function by recruiting other proteins into a signaling complex so that interactions among these proteins can take place.

1	Scaffolds are relatively large proteins that can, for example, become tyrosine phosphorylated on multiple sites in order to recruit many different proteins (Fig. 7.3, top panel). By specifying which proteins are recruited, scaffolds can define the character of a particular signaling response. This is accomplished by several mechanisms. For example, scaffolds can regulate the specificity of a recruited enzyme by recruiting one of the enzyme’s substrates. Binding to a scaffold can also change the conformation of a recruited protein, thereby revealing sites for protein modifications, such as phosphorylation or ubiquitination, or for protein–protein interactions. Finally, scaffolds can function to promote membrane localization of the signaling complex.

1	Adaptors are membrane-anchored or cytoplasmic proteins containing several signaling modules that serve to link two or more proteins together. The adaptor proteins Grb2 and Gads, for example, each contain an SH2 domain and two copies of another module called the SH3 domain (see Fig. 7.2). This arrangement of modules can be used to link tyrosine phosphorylation of a receptor to molecules acting in the next stage of signaling. For example, the SH2 domain of Grb2 binds to a phosphotyrosine residue on a receptor or a scaffold protein, while its two SH3 domains bind to proline-rich motifs on other signaling proteins (see Fig. 7.3, bottom panel), such as Sos, which we discuss in the next section.

1	Fig. 7.3 Assembly of signaling complexes is mediated by scaffold and adaptor proteins. assembly of signaling complexes is an important aspect of signal transduction. This is often achieved through scaffold and adaptor proteins. in general, scaffolds have numerous sites of phosphorylation that function to bring many different signaling proteins together (top panel). Scaffolds may also function to promote membrane localization, to bring enzymes into close proximity with their substrates, and to induce conformational changes in proteins that regulate their functions. an adaptor protein functions to bring two different proteins together (bottom panel). When erythropoietin (epo) binds to its receptor, associated tyrosine kinases phosphorylate (red dots) sites on the epo receptor cytoplasmic domain, generating binding sites for the Sh2 domain of an adaptor protein. The adaptor protein (green) shown here contains two Sh3 domains in addition to an Sh2 domain. With the Sh3 domains it can, for

1	generating binding sites for the Sh2 domain of an adaptor protein. The adaptor protein (green) shown here contains two Sh3 domains in addition to an Sh2 domain. With the Sh3 domains it can, for example, bind proline-rich sites on an intracellular signaling molecule (yellow).

1	Over time, the small G protein hydrolyzes the GTP to GDP and becomes inactive, a process that is accelerated by GAPs The GTP-bound small G protein is the active effector molecule Signaling activates guanine-nucleotide exchange factors (GEFs) such as Sos, which increase the rate of exchange of GDP for GTP In the resting state, small G proteins are bound to GDP and are inactive GTP GDP:Ras GDP:Ras GTP:Ras active Ras GEF

1	Fig. 7.4 Small G proteins are switched from inactive to active states by guanine-nucleotide exchange factors (GEFs) and the binding of GTP. Ras is a small GTp-binding protein with intrinsic GTpase activity. in its resting state, Ras is bound to Gdp. Receptor signaling activates guanine-nucleotide exchange factors (Gefs), such as Sos, which can bind to small G proteins such as Ras and increase the rate of exchange of Gdp for GTp (center panels). The GTp-bound form of Ras can then bind to a large number of effectors, recruiting them to the membrane. over time, the intrinsic GTpase activity of Ras will result in the hydrolysis of GTp to Gdp. GTpase-activating proteins (Gaps) can accelerate the hydrolysis of GTp to Gdp, thus shutting off the signal more rapidly. 7-3 Small G proteins act as molecular switches in many different signaling pathways.

1	7-3 Small G proteins act as molecular switches in many different signaling pathways. Monomeric GTP-binding proteins known as small G proteins or small GTPases are important in the signaling pathways leading from many tyrosine kinase-associated receptors. The small GTPases are distinct from the larger heterotrimeric G proteins associated with G-protein-coupled receptors such as the chemokine receptors discussed in Chapter 3. The superfamily of small GTPases comprises more than 100 different proteins, and many are important in lymphocyte signaling. One of these, Ras, is involved in many pathways leading to cell proliferation. Other small GTPases include Rac, Rho, and Cdc42, which control changes in the actin cytoskeleton caused by signals received through the T-cell receptor or B-cell receptor. We will describe their actions in more detail in Section 7-19.

1	Small GTPases exist in two states, depending on whether they are bound to GTP or to GDP. The GDP-bound form is inactive but is converted into the active form by exchange of the GDP for GTP. This reaction is mediated by proteins known as guanine-nucleotide exchange factors, or GEFs, which cause the GTPase to release GDP and to bind the more abundant GTP (Fig. 7.4). Sos, which is recruited to signaling pathways by the adaptor Grb2 (see Section 7-2), is one of the GEFs for Ras. The binding of GTP induces a conformational change in the small GTPase that enables it to bind to and induce effector activity in a variety of target proteins. Thus, GTP binding functions as an on/off switch for small GTPases.

1	This GTP-bound form does not remain permanently active but is eventually converted into the inactive GDP-bound form by the intrinsic GTPase activity in the G protein, which removes a phosphate group from the bound GTP. Regulatory cofactors known as GTPase-activating proteins (GAPs) accelerate the conversion of GTP to GDP, thus rapidly downregulating the activity of the small GTPase. Because of GAP activity, small GTPases are usually present in the inactive GDP-bound state and are activated only transiently in response to a signal from an activated receptor. RAS is frequently mutated in cancer cells, and the mutated Ras protein is thought to be a significant contributor to the cancerous state. The importance of GAPs in signaling regulation is indicated by the fact that some mutations in Ras found in cancer act by preventing GAP from enhancing the intrinsic GTPase activity of Ras, thus prolonging the duration for which Ras exists in the active GTP-bound state.

1	GEFs are the key to G-protein activation and are recruited to the site of receptor activation at the cell membrane by binding to adaptor proteins or to lipid metabolites produced by receptor activation. Once recruited, they are able to activate Ras or other small G proteins, which are localized to the inner surface of the plasma membrane via fatty acids that are attached to the G protein post-translationally. Thus, G proteins act as molecular switches, becoming switched on when a cell-surface receptor is activated and then being switched off. Each G protein has its own specific GEFs and GAPs, which help to confer specificity on the pathway. 7-4 Signaling proteins are recruited to the membrane by a variety of mechanisms.

1	7-4 Signaling proteins are recruited to the membrane by a variety of mechanisms. We have seen how receptors can recruit intracellular signaling proteins to the plasma membrane through tyrosine phosphorylation of the receptor itself or of an associated scaffold, followed by recruitment of SH2-domain-containing signaling proteins or adaptors, such as Grb2 (Fig. 7.5). A second mechanism for membrane recruitment of signaling proteins is via binding to small GTPases, such as Ras, following their activation. As described in Section 7-3, small GTPases are constitutively bound to the cytoplasmic surface of the plasma membrane due to their fatty acid modifications. Once activated by exchange of GDP for GTP, the activated GTPases bind to signaling proteins such as Sos, relocalizing the bound proteins to the plasma membrane (see Fig. 7.5).

1	Another way in which receptors can recruit signaling molecules to the plasma membrane is by the local production of modified membrane lipids. These lipids are produced by phosphorylation of the membrane phospholipid phosphatidylinositol by enzymes known as phosphatidylinositol kinases, which are activated as a result of receptor signaling. The inositol headgroup of phosphatidylinositol is a carbohydrate ring that can be phosphorylated at one or more positions to generate a wide variety of derivatives. The ones that we will focus on in this chapter are phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3), the latter of which is generated from PIP2 by the enzyme phosphatidylinositol 3-kinase (PI 3-kinase) (see Fig. 7.5). PI 3-kinase is often recruited by binding of the SH2 domain of its regulatory subunit to phosphotyrosines in a receptor tail, bringing its catalytic subunit into proximity with inositol phospholipid substrates in the membrane.

1	by binding of the SH2 domain of its regulatory subunit to phosphotyrosines in a receptor tail, bringing its catalytic subunit into proximity with inositol phospholipid substrates in the membrane. In this way, membrane phosphoinositides such as PIP3 are rapidly produced after receptor activation. This, combined with their short life span, makes them ideal signaling molecules. PIP3 is recognized specifically by proteins containing a pleckstrin homology (PH) domain or, less commonly, a PX domain (see Fig. 7.2), and one of its functions is to recruit such proteins to the membrane and in some cases contribute to enzyme activation.

1	7-5 Post-translational modifications of proteins can both activate and inhibit signaling responses. Protein phosphorylation is a common mechanism for transducing signals from cellular receptors to downstream pathways. These signals are terminated by the action of protein phosphatases, which dephosphorylate signaling intermediates (Fig. 7.6). The importance of protein phosphatases in terminating signaling is underscored by the existence of diseases, such as autoimmunity and cancer, which may result from absent or deficient protein phosphatase activity. However, protein dephosphorylation can also function as a mechanism of activation. Dephosphorylation can regulate protein–protein interactions, protein subcellular localization, or nucleic acid binding, thereby promoting downstream signaling events.

1	Another general mechanism of protein regulation by post-translational modification is the covalent attachment of one or more molecules of the small protein ubiquitin. Ubiquitination is a potent means of signal termination, as it often leads to protein degradation. Ubiquitin is attached by its carboxyterminal glycine to lysine residues of target proteins in a multi-step process.

1	Fig. 7.5 Signaling proteins can be recruited to the membrane in a variety of ways. Recruitment of signaling proteins to the plasma membrane is important in signal propagation because this is where receptors are usually located. Left panel: tyrosine phosphorylation of membrane-associated proteins, such as the scaffold LaT, recruits phosphotyrosine-binding proteins. This can also protect the scaffold from dephosphorylation by tyrosine phosphatases, which inhibit signaling. Second panel: small G proteins such as Ras can associate with the membrane by having lipid modifications (shown in red). When activated, they can bind a variety of signaling proteins. Right two panels: modifications to the membrane itself that result from receptor activation can recruit signaling proteins. in this example the membrane lipid pip3 has been produced in the inner leaflet of the plasma membrane by the phosphorylation of pip2 by pi 3-kinase. pip3 is recognized by the ph domains of signaling proteins such as

1	the membrane lipid pip3 has been produced in the inner leaflet of the plasma membrane by the phosphorylation of pip2 by pi 3-kinase. pip3 is recognized by the ph domains of signaling proteins such as the protein kinases akt and itk.

1	Fig. 7.6 Signaling must be turned off as well as turned on. The inability to terminate a signaling pathway can result in serious diseases such as autoimmunity or cancer. as a significant proportion of signaling events depend on protein phosphorylation, protein phosphatases, such as Shp, have an important part in shutting down signaling pathways (left panel). another common mechanism for terminating signaling is regulated protein degradation (center and right panels). phosphorylated proteins recruit ubiquitin ligases, such as cbl, that add the small protein ubiquitin to proteins, thus targeting them for degradation. cytoplasmic proteins are targeted for destruction in the proteasome by the addition of polyubiquitin chains, linked through lysine 48 (K48) of ubiquitin (center panel). membrane receptors that become ubiquitinated by individual ubiquitin molecules or di-ubiquitin are internalized and transported to the lysosome for destruction (right panel).

1	First, an E1 ubiquitin-activating enzyme promotes attachment of ubiquitin to an E2 ubiquitin-conjugating enzyme. The ubiquitin is then transferred to the protein substrate by an enzyme known as an E3 ubiquitin ligase. Ubiquitin ligases can continue to add ubiquitin molecules to form polyubiquitin. Importantly, different ubiquitin ligases add the carboxy terminus of one ubiquitin molecule to different lysine residues of the conjugated ubiquitin, typically either lysine 48 (K48) or lysine 63 (K63). These different forms of polyubiquitin produce divergent outcomes for signaling pathways.

1	When polyubiquitin chains are formed using K48 linkages, the outcome is to target the protein for degradation by the proteasome (see Fig 7.6). An important ubiquitin ligase of this kind in lymphocytes is Cbl, which selects its targets via its SH2 domain. Cbl can thus bind to specific tyrosine-phosphorylated targets, causing them to become ubiquitinated via K48 linkages. Proteins that recognize this form of polyubiquitin then target the ubiquitinated proteins to degradative pathways via the proteasome. Membrane proteins such as receptors can be tagged by single ubiquitin molecules or by di-ubiquitin. These are not recognized by the proteasome, but instead, are recognized by specific ubiquitin-binding proteins that target proteins for degradation in lysosomes (see Fig. 7.6). Thus, ubiquitination of proteins can inhibit signaling. Unlike phosphatases, where the mechanism of inhibition is reversible, inhibition by ubiquitin-mediated protein degradation is a more permanent means of

1	of proteins can inhibit signaling. Unlike phosphatases, where the mechanism of inhibition is reversible, inhibition by ubiquitin-mediated protein degradation is a more permanent means of terminating signaling.

1	Ubiquitination can also be used to activate signaling pathways. We have already discussed this aspect in Section 3-7 in connection with the NFκB signaling pathway from TLRs. There, the ubiquitin ligase TRAF-6 produces K63-linked polyubiquitin chains on TRAF-6 and NEMO. In lymphocytes, K63-linked polyubiquitination is a key step in signaling through tumor necrosis factor (TNF) receptor family members, as will be discussed in Section 7-23 (and Fig. 7.31). This form of polyubiquitin is recognized by specific domains in signaling proteins that recruit additional signaling molecules to the pathway (see Fig. 3.15). 7-6 The activation of some receptors generates small-molecule second messengers.

1	7-6 The activation of some receptors generates small-molecule second messengers. In many cases, intracellular signaling involves the activation of enzymes that produce small-molecule biochemical mediators known as second messengers (Fig. 7.7). These mediators can diffuse throughout the cell, enabling the signal to activate a variety of target proteins. The enzymatic production of second messengers also serves the dual purpose of achieving concentrations of them sufficient to activate the next stage of the pathway and of amplifying the signaling cascade. The second messengers generated by receptors that signal via tyrosine kinases include calcium ions (Ca2+) and a variety of membrane lipids and their soluble derivatives. Although some of these lipid messengers are confined to membranes, they can move within them. A second messenger binding to its target protein typically induces a conformational change that allows the protein to be activated. Summary.

1	Cell-surface receptors serve as the front line of a cell’s interaction with its environment; they sense extracellular events and convert them into biochemical signals for the cell. As most receptors sit in the plasma membrane, a critical step in the transduction of extracellular signals to the interior of the cell is recruitment of intracellular proteins to the membrane and changes in the composition of the membrane surrounding the receptor. Many immune receptors operate by activating tyrosine kinases to transmit their signals onward, often using scaffolds and adaptors to form large multiprotein signaling complexes. Both the qualitative and quantitative changes that take place in the composition of these signaling complexes determine the character of the response and biological outcomes. Formation of signaling complexes is mediated by a wide variety of protein-interaction domains, or modules, including the SH2, SH3, and PH domains found in proteins. In many cases, the increase in

1	Formation of signaling complexes is mediated by a wide variety of protein-interaction domains, or modules, including the SH2, SH3, and PH domains found in proteins. In many cases, the increase in enzymatically produced small-molecule signaling intermediates called second messengers regulates and amplifies the signaling cascade. Termination of signaling involves protein dephosphorylation as well as ubiquitin-mediated protein degradation.

1	Antigen receptor signaling and lymphocyte activation.

1	Fig. 7.7 Signaling pathways amplify the initial signal. amplification of the initial signal is an important element of most signal transduction pathways. one means of amplification is a kinase cascade (left panel), in which protein kinases successively phosphorylate and activate each other. in this example, taken from a commonly used kinase cascade (see fig. 7.19), activation of the kinase Raf results in the phosphorylation and activation of a second kinase, mek, that phosphorylates yet another kinase, erk. as each kinase can phosphorylate many different substrate molecules, the signal is amplified at each step, resulting in a huge amplification of the initial signal. another method of signal amplification is the generation of second messengers (center and right panels). in this example, signaling results in the release of the second messenger calcium (ca2+) from intracellular stores into the cytosol or its influx from the extracellular environment. ca2+ release from the endoplasmic

1	signaling results in the release of the second messenger calcium (ca2+) from intracellular stores into the cytosol or its influx from the extracellular environment. ca2+ release from the endoplasmic reticulum (eR) is shown here. The sharp increase in free ca2+ in the cytoplasm can potentially activate many downstream signaling molecules, such as the calcium-binding protein calmodulin. calcium binding induces a conformational change in calmodulin, which allows it to bind to and regulate a variety of effector proteins.

1	The ability of T cells and B cells to recognize and respond to their specific antigen is central to adaptive immunity. As described in Chapters 4 and 5, the B-cell antigen receptor (BCR) and the T-cell antigen receptor (TCR) are made up of antigen-binding chains—the heavy and light immunoglobulin chains

1	Fig. 7.8 The T-cell receptor complex is made up of variable antigen-recognition proteins and invariant signaling proteins. upper panel: the functional T-cell receptor (TcR) complex is composed of the antigen-binding TcRα:β heterodimer associated with six signaling chains: two ε, one δ, and one γ collectively called cd3, and a homodimer of ζ. cell-surface expression of the antigen-binding chains requires assembly of TcRα:β with the signaling subunits. each cd3 chain has one immunoreceptor tyrosine-based activation motif (iTam), shown as a yellow segment, whereas each ζ chain has three. The transmembrane regions of each chain have unusual acidic or basic residues as shown. Lower panel: the transmembrane regions of the various TcR subunits are represented in cross-section. it is thought that one of the positive charges, from a lysine (K) of the α chain, interacts with the two negative charges of aspartic acid (d) of the cd3δ:ε dimer, while the other positive charge, of arginine (R),

1	that one of the positive charges, from a lysine (K) of the α chain, interacts with the two negative charges of aspartic acid (d) of the cd3δ:ε dimer, while the other positive charge, of arginine (R), interacts with aspartic acids of the ζ homodimer. The positive arginine (K) charge of the β chain interacts with the negative charges of aspartic acid and glutamic acid (e) in the cd3γ:ε dimer.

1	in the B-cell receptor, and the TCRα and TCRβ chains in the T-cell receptor. These variable chains have exquisite specificity for antigen, allowing each lymphocyte to detect the presence of one type of pathogen. However, binding of antigen to the antigen receptor is not sufficient for a lymphocyte to respond— the information that antigen receptor engagement has occurred also needs to be transduced into the intracellular compartment of the lymphocyte. Thus, the fully functional antigen receptor complex must include proteins that can transduce a signal across the plasma membrane. For the B-cell antigen receptor and the T-cell antigen receptor, this function is mediated by invariant accessory proteins that initiate signaling when the receptors bind antigen. Assembly with these accessory proteins is also essential for transport of the receptor to the cell surface. In this part of the chapter we describe the structure of the antigen receptor complexes on T cells and B cells, and the

1	proteins is also essential for transport of the receptor to the cell surface. In this part of the chapter we describe the structure of the antigen receptor complexes on T cells and B cells, and the signaling pathways that lead from them.

1	7-7 Antigen receptors consist of variable antigen-binding chains associated with invariant chains that carry out the signaling function of the receptor.

1	In T cells, the highly variable TCRα:β heterodimer (see Chapter 5) is not sufficient on its own to make up a complete cell-surface antigen receptor. When cells were transfected with cDNAs encoding the TCRα and TCRβ chains, the heterodimers formed were degraded and did not appear on the cell surface. This implied that other molecules are required for the T-cell receptor to be expressed on the cell surface. In the T-cell receptor, the other required molecules are the CD3γ, CD3δ, and CD3ε chains, which together form the CD3 complex; and the ζ chain, which is present as a disulfide-linked homodimer (Fig. 7.8). The CD3 proteins have an extracellular immunoglobulin-like domain, whereas the ζ chain contains only a short extracellular domain. Throughout the remainder of the chapter, we will use the term T-cell receptor to refer to the entire T-cell receptor complex, including these associated signaling subunits.

1	Although the exact stoichiometry of the complete T-cell receptor is not definitively established, it is thought that the receptor α chain interacts with one CD3δ:CD3εdimer and one ζ dimer, while the receptor β chain interacts with one CD3γ:CD3ε dimer (see Fig. 7.8). These interactions are mediated by reciprocal charge interactions between basic and acidic intramembrane amino acids of the receptor subunits. There are two positive charges in the TCRα transmembrane region and one in the TCRβ transmembrane domain. Negative charges in the CD3 and ζ transmembrane domains interact with the positive charges in αand β. Assembly of CD3 and a ζ dimer with the α:β heterodimer stabilizes the αand β dimer during its production in the endoplasmic reticulum and allows the complex to be transported to the plasma membrane. These associations ensure that all T-cell receptors present on the plasma membrane are properly assembled.

1	Signaling from the T-cell receptor is initiated by tyrosine phosphorylation within cytoplasmic regions called immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3ε, γ, δ, and ζ chains. CD3γ, δ, and ε each contain one ITAM, and each ζ chain contains three, giving the T-cell receptor a total of 10 ITAMs. This motif is also present in the signaling chains of the B-cell receptor and in the NK-cell receptors described in Chapter 3, as well as in the receptors for the immunoglobulin constant region (Fc receptors) that are present on mast cells, macrophages, monocytes, neutrophils, and NK cells (see Section 7-11 below).

1	Each ITAM contains two tyrosine residues that become phosphorylated by specific protein tyrosine kinases when the receptor binds its ligand, providing sites for the recruitment of the SH2 domains of signaling proteins as described earlier in the chapter. Two YXXL/I motifs are separated by about six to nine amino acids within each ITAM, so the canonical ITAM sequence is …YXX[L/I] X6–9YXX[L/I]…, where Y is tyrosine, L is leucine, I is isoleucine, and X represents any amino acid. The two tyrosines of the ITAM make it particularly efficient in recruiting signaling proteins that contain two tandem SH2 domains (Fig. 7.9). When both tyrosines of the ITAM are phosphorylated, tandem SH2 domain-containing proteins such as Syk or ZAP-70 are recruited. This leads to the phosphorylation of Syk or ZAP-70, an essential step in the activation of these kinases, as will be discussed further below (see Section 7-10).

1	The antigen-binding immunoglobulin on the B-cell surface is also associated with invariant protein chains that mediate signaling. These two polypeptides, called Igαand Igβ, are required for transport of the receptor to the surface and for B-cell receptor signaling (Fig. 7.10). Igα and Igβ are single-chain proteins composed of an extracellular immunoglobulin-like domain connected by a transmembrane domain to a cytoplasmic tail. They form a disulfide-linked heterodimer that becomes associated with immunoglobulin heavy chains and enables their transport to the cell surface. The Igα:Igβdimer associates with the B-cell receptor through hydrophilic rather than charge interactions between their transmembrane regions. The complete B-cell receptor is thought to be a complex of six chains—two identical light chains, two identical heavy chains, and one associated heterodimer of Igα and Igβ. Like CD3 and the ζ chains of the T-cell receptor, Igα and Igβ have ITAMs, but just one each, and these are

1	light chains, two identical heavy chains, and one associated heterodimer of Igα and Igβ. Like CD3 and the ζ chains of the T-cell receptor, Igα and Igβ have ITAMs, but just one each, and these are essential for the ability of the B-cell receptor to signal.

1	7-8 Antigen recognition by the T-cell receptor and its co-receptors transduces a signal across the plasma membrane to initiate signaling. To make an effective immune response, T cells and B cells must be able to respond to their specific antigen even when it is present at extremely low levels. This is especially important for T cells, as an antigen-presenting cell will display on its surface many different peptides from both self and foreign proteins, and the number of peptide:MHC complexes specific for a particular T-cell receptor is likely to be very low. Some estimates suggest that a naive CD4 T

1	Fig. 7.10 The B-cell receptor complex is made up of cell-surface immunoglobulin with one each of the invariant signaling proteins Igα and Igβ. The immunoglobulin recognizes and binds antigen but cannot itself generate a signal. it is associated with antigen-nonspecific signaling molecules—igα and igβ. These each have a single iTam (yellow segment) in their cytosolic tails that enables them to signal when the B-cell receptor is ligated with antigen. igα and igβ form a disulfide-linked heterodimer that is non-covalently associated with the heavy chains.

1	Fig. 7.9 ITAMs recruit signaling proteins that have tandem SH2 domains. The iTams of the T-cell receptor (TcR) and B-cell receptor (BcR) contain tyrosine residues within the motif`…yXX[L/i] X6–9yXX[L/i]…. The spacing between the tyrosines is important in binding to tandem Sh2-containing proteins such as Syk and Zap-70. Left panel: prior to TcR or BcR stimulation, these kinases are in an inactive conformation, known as the autoinhibited conformation. The autoinhibited conformation is stabilized by interactions between the tandem Sh2 domain-kinase domain linker region and the kinase domain that hold the enzyme in a catalytically inactive state. Right panel: after phosphorylation of both tyrosines within one iTam (here depicted as two tyrosines separated by 9-12 amino acids), the tandem Sh2 domains of Syk or Zap-70 can dock cooperatively to both phosphotyrosines, as shown here for Zap-70. By being recruited into the active signaling complex, Zap-70 can itself be phosphorylated, so that

1	domains of Syk or Zap-70 can dock cooperatively to both phosphotyrosines, as shown here for Zap-70. By being recruited into the active signaling complex, Zap-70 can itself be phosphorylated, so that it becomes an active kinase that can then phosphorylate its substrates. This final step of Zap-70 activation requires phosphorylation on two tyrosines in the linker region between the tandem Sh2 domains and the kinase domain, together with phosphorylation of a tyrosine in the catalytic site of the kinase domain.

1	Fig. 7.11 Engagement of co-receptors with the T-cell receptor enhances phosphorylation of the ITAMs. upper panel: for simplicity, we show the cd4 co-receptor engaging the same mhc molecule as the T-cell receptor (TcR), although signaling within receptor microclusters may differ from this arrangement. When T-cell receptors and co-receptors are brought together by binding to peptide:mhc complexes on the surface of an antigen-presenting cell, recruitment of the co-receptor-associated kinase Lck leads to phosphorylation of iTams in cd3γ, δ, and ε, and in the ζ chains. Lower panel: the tyrosine kinase Zap-70 binds to phosphorylated iTams through its Sh2 domains, enabling Zap-70 to be phosphorylated and activated by Lck. Zap-70 then phosphorylates other intracellular signaling molecules.

1	cell can become activated by fewer than 50 antigenic peptide:MHC complexes displayed by an antigen-presenting cell, and effector CD8 cytotoxic T cells may be even more sensitive. B cells become activated when about 20 B-cell receptors are engaged. These estimates based on in vitro studies may not be precise for cells in vivo, but it is clear that the antigen receptors on T cells and B cells confer remarkable sensitivity to antigen.

1	For a peptide:MHC complex to activate T cells, it must bind directly to the T-cell receptor (Fig. 7.11, upper panel, and see Fig. 4.22). However, it remains unclear precisely how this extracellular recognition event is transmitted across the T-cell membrane to initiate signaling. Questions remain as to the stoichiometry and physical arrangement of T-cell receptors and peptide:MHC complexes required to initiate the signaling cascade. We will discuss this area of active research only briefly before moving on to explain well-understood intracellular events that occur after antigen recognition.

1	One suggestion is that signaling is initiated by T-cell receptor dimerization through formation of ‘pseudo-dimeric’ peptide:MHC complexes containing one antigen peptide:MHC molecule and one self peptide:MHC molecule on the surface of the antigen-presenting cell. This model relies on a weak interaction occurring between the T-cell receptor and self peptide:MHC complexes, stabilized by the CD4 or CD8 co-receptor interactions with the self peptide:MHC complexes, but it would explain signaling induced by low densities of antigenic peptides. An additional possibility is that the antigenic peptide:MHC complex induces a conformational change in the T-cell receptor, or its associated CD3 and ζ chains, that promotes receptor phosphorylation; however, direct structural evidence supporting this model is still lacking.

1	It has also been suggested that signaling might involve receptor oligomerization, or clustering, as antibodies that bind to and cross-link T-cell receptors can activate T cells. Since antigenic peptides are vastly outnumbered by other peptides displayed on the surface of the antigen-presenting cell, it is unlikely that physiologic amounts of antigen induce conventional oligomerization as observed with antibodies. However, assemblies of small numbers of T-cell receptors called microclusters have been observed in the zone of contact between the T cell and the antigen-presenting cell. These microclusters form rapidly following TCR stimulation, and quickly merge with microclusters containing downstream signaling components, such as scaffolds and adaptors. Current evidence indicates that signaling is initiated in these microclusters. One popular model proposes that signal initiation takes place when inhibitory signaling proteins are excluded from these complexes. A key component of this

1	is initiated in these microclusters. One popular model proposes that signal initiation takes place when inhibitory signaling proteins are excluded from these complexes. A key component of this model is that prior to TCR signaling, activating and inhibitory enzymes are in a balanced equilibrium; the initiation of signaling occurs when this equilibrium is perturbed in favor of the activating modifications.

1	7-9 Antigen recognition by the T-cell receptor and its co-receptors leads to phosphorylation of ITAMs by Src-family kinases, generating the first intracellular signal in a signaling cascade.

1	The first intracellular signal generated after the T cell has detected its specific antigen is the phosphorylation of both tyrosines in the ITAMs of the T-cell receptor. This signal is initiated with the help of the CD4 and CD8 co-receptors, which bind to MHC class II molecules and class I molecules, respectively, via their extracellular domains (see Section 4-18), and associate with nonreceptor kinases via their intracellular domains. The Src-family kinase Lck is constitutively associated with the cytoplasmic domains of CD4 and CD8 and is thought to be the kinase primarily responsible for phosphorylation of the ITAMs of the T-cell receptor (see Fig. 7.11). Evidence suggests that binding of the co-receptor to the peptide:MHC complex that binds the T-cell receptor enhances the recruitment of Lck to the engaged T-cell receptor, leading to more efficient phosphorylation of the T-cell receptor ITAMs. The importance of this event is demonstrated by the profound reduction in T-cell

1	of Lck to the engaged T-cell receptor, leading to more efficient phosphorylation of the T-cell receptor ITAMs. The importance of this event is demonstrated by the profound reduction in T-cell development in Lck-deficient mice. This indicates the essential role of Lck in T-cell receptor signaling during the selection of developing T cells in the thymus (discussed in Chapter 8). Lck is important for T-cell receptor signaling in naive T cells and effector T cells, but is less important for the activation or maintenance of memory CD8 T cells by their specific antigen. A related tyrosine kinase, Fyn, is weakly associated with the ITAMs of the T-cell receptor and may have some role in signaling. Whereas mice lacking Fyn develop normal CD4 and CD8 T cells that respond in essentially normal fashion to antigen, mice lacking both Lck and Fyn show a more complete loss of T-cell development than mice lacking Lck alone.

1	Another role of the co-receptors in T-cell receptor signaling may be to stabilize interactions between the receptor and the peptide:MHC complex. Affinities of individual receptors for their specific peptide:MHC complexes are in the micromolar range, which means that the T-cell receptor:peptide:MHC complexes have half-lives of less than 1 second and dissociate rapidly. The additional binding of a co-receptor to the MHC molecule is thought to stabilize the interaction by increasing its duration, thereby providing time for an intracellular signal to be generated.

1	The Lck bound to the cytoplasmic tails of CD4 or CD8 is brought near its substrate ITAMs in the T-cell receptor when the co-receptor binds the recep-tor:peptide:MHC complex (see Fig. 7.11). Lck’s activity is also regulated allosterically by phosphorylation of a tyrosine in its carboxy terminus by the C-terminal Src kinase (Csk). The resulting phosphotyrosine interacts with Lck’s SH2 domain and helps maintain Lck in a closed conformation, resulting in a catalytically inactive state (Fig. 7.12). The absence of Csk during T-cell development causes T cells to mature autonomously in the thymus without needing to bind peptide:MHC, presumably as a result of abnormal activation of TCR signaling by hyperactive Lck in Csk-deficient thymocytes. This suggests that Csk normally acts to reduce Lck activity and to attenuate TCR signaling. Dephosphorylation of the tyrosine or engagement of the SH2 or SH3 domains by their ligands releases Lck from its inactive conformation, resulting in a primed, but

1	and to attenuate TCR signaling. Dephosphorylation of the tyrosine or engagement of the SH2 or SH3 domains by their ligands releases Lck from its inactive conformation, resulting in a primed, but not fully active, Lck kinase (see Fig. 7.12). Full activation of catalytic activity also requires Lck autophosphorylation of a tyrosine in its kinase domain. In nonstimulated lymphocytes, phosphorylation of Lck is counteracted by the tyrosine phosphatase CD45, which can dephosphorylate both of the Lck tyrosine phosphorylation sites. Prior to TCR stimulation, multiple phosphorylated species of Lck are found in T cells, but antigen receptor stimulation is required to stabilize the activated form of Lck and lead to ITAM phosphorylation.

1	Fig. 7.12 Lck activity is regulated by tyrosine phosphorylation and dephosphorylation. Src kinases such as Lck contain Sh3 (blue) and Sh2 (orange) domains preceding the kinase domain (green). Lck also contains a unique amino-terminal motif (yellow) with two cysteine residues that bind a Zn ion that is also bound to a similar motif in the cytoplasmic domain of cd4 or cd8. upper panel: in inactive Lck, the two lobes of the kinase domain are constrained by interactions with both the Sh2 and Sh3 domains. The Sh2 domain interacts with a phosphorylated tyrosine (red) at the carboxy-terminal end of the kinase domain. The Sh3 domain interacts with a proline-rich sequence in the linker between the Sh2 domain and the kinase domain. middle panel: dephosphorylation of the carboxyterminal tyrosine by the phosphatase cd45 (not shown) releases the Sh2 domain. Binding of other ligands to the Sh3 region can facilitate release of the linker region (not shown). in this state, Lck is considered primed,

1	the phosphatase cd45 (not shown) releases the Sh2 domain. Binding of other ligands to the Sh3 region can facilitate release of the linker region (not shown). in this state, Lck is considered primed, but not fully activated. Lower panel: full activation of Lck catalytic activity requires autophosphorylation on the activation loop in the kinase domain. active Lck can then phosphorylate iTams in the signaling chains of the nearby T-cell receptor. Rephosphorylation of the carboxy-terminal tyrosine by the c-terminal Src kinase (csk) or loss of the Sh3 ligand returns Lck to the inactive state.

1	Fig. 7.13 Structure of the autoinhibited ZAP-70 kinase. The structure of the inactive autoinhibited conformation of Zap-70 is shown with the protein domains color-coded according to the domain map shown at the bottom; the dashed red line indicates a region of the protein that was not detected in the structural analysis. prior to T-cell receptor stimulation, the Zap-70 kinase is in this inactive conformation, based on interactions between the tandem Sh2 domain-kinase domain linker region (thick red line) and the kinase domain. This interaction stabilizes Zap-70 in a catalytically inactive state, referred to as the autoinhibited form of Zap-70, by locking the kinase domain in an inactive conformation. following T-cell receptor stimulation, Lck phosphorylates two tyrosine residues in this linker region, y315 and y319, shown in yellow. Lck also phosphorylates a tyrosine residue in the catalytic (kinase) domain. When y315 and y319 are phosphorylated, the linker region no longer binds to

1	linker region, y315 and y319, shown in yellow. Lck also phosphorylates a tyrosine residue in the catalytic (kinase) domain. When y315 and y319 are phosphorylated, the linker region no longer binds to the kinase domain, allowing the phosphorylated kinase domain to adopt an active conformation. courtesy of arthur Weiss.

1	Fc˜RIII (CD16) Fc˜RIV Fc°RI ˜˛˜˝˙NKG2C, D, E (CD94) DAP12or Receptors other than antigen receptors also associate with ITAM-containing chains that deliver activating signals NK cells Macrophages Neutrophils NK cells Mast cells Basophils 7-10 Phosphorylated ITAMs recruit and activate the tyrosine kinase ZAP-70.

1	The precise spacing of the two YXXL/I motifs in an ITAM suggests that the ITAM is a binding site for a signaling protein with two SH2 domains. In the case of the T-cell receptor, this protein is the tyrosine kinase ZAP-70 (ζ-chainassociated protein), which carries the activation signal onward. ZAP-70 has two tandem SH2 domains that can be simultaneously engaged by the two phosphorylated tyrosines in an ITAM (see Fig. 7.9). The affinity of the phosphorylated YXXL sequence for a single SH2 domain is low; binding of both SH2 domains to the ITAM is significantly stronger and confers specificity on ZAP-70 binding. Thus, when Lck has sufficiently phosphorylated an ITAM in the T-cell receptor, ZAP-70 binds to it. Once bound, ZAP-70 is phosphorylated by Lck at three tyrosine residues, two in the linker region between the tandem SH2 domains and the kinase domain, and a third residue in the catalytic domain. Together these phosphorylations activate ZAP-70 by disrupting the autoinhibited form of

1	region between the tandem SH2 domains and the kinase domain, and a third residue in the catalytic domain. Together these phosphorylations activate ZAP-70 by disrupting the autoinhibited form of inactive ZAP-70, allowing the ZAP-70 kinase domain to adopt the active conformation (Fig. 7.13). ZAP-70 can also be activated by autophosphorylation.

1	7-11 ITAMs are also found in other receptors on leukocytes that signal for cell activation.

1	The signaling subunits of the T-cell receptor and the B-cell receptor each contain ITAMs, which are essential for T-cell receptor and B-cell receptor signaling. Phosphorylation of both tyrosines in the ITAM functions to recruit a tyrosine kinase with tandem SH2 domains—ZAP-70 in the case of T cells, and a closely related kinase, Syk, in B cells. Other immune-system receptors also use ITAM-containing accessory chains to transduce activating signals (Fig. 7.14). One example is FcγRIII (CD16); this is a receptor for IgG that triggers anti-body-dependent cell-mediated cytotoxicity (ADCC) by NK cells, which we consider in Chapter 11; CD16 is also found on macrophages and neutrophils, where it facilitates the uptake and destruction of antibody-bound pathogens. To signal, FcγRIII must associate either with the ζchain found also in the T-cell receptor or with another member of the same protein family known as the Fcγ chain. The Fcγ chain is also the signaling component of another Fc

1	either with the ζchain found also in the T-cell receptor or with another member of the same protein family known as the Fcγ chain. The Fcγ chain is also the signaling component of another Fc receptor—the Fcε receptor I (FcεRI) on mast cells. As we discuss in Chapter 14, this receptor binds IgE antibodies, and on cross-linking by allergens, it triggers the degranulation of mast cells. Last, many activating receptors on NK cells are associated with DAP12, another ITAM-containing protein (see Section 3-26). Each of these additional ITAM-containing signaling chains becomes tyrosine phosphorylated following stimulation of its associated receptor, leading to the recruitment of a tyrosine kinase, either Syk or ZAP-70. With the exception of

1	Fig. 7.14 Other receptors that pair with ITAM-containing chains can deliver activating signals. cells other than B and T cells have receptors that pair with accessory chains containing iTams, which are phosphorylated when the receptor is cross-linked. These receptors deliver activating signals. The fcγ receptor iii (fcγRiii, or cd16) is found on nK cells, macrophages, and neutrophils. Binding of igG to this receptor activates the killing function of the nK cell, leading to the process known as antibody-dependent cell-mediated cytotoxicity (adcc). activating receptors on nK cells, such as nKG2c, nKG2d, and nKG2e, also associate with iTam-containing signaling chains. The fcε receptor (fcεRi) is found on mast cells and basophils. The α subunit binds to ige antibodies with very high affinity. The β subunit is a four-spanning transmembrane protein. When antigen subsequently binds to the ige, the mast cell is triggered to release granules containing inflammatory mediators. The γ chain

1	The β subunit is a four-spanning transmembrane protein. When antigen subsequently binds to the ige, the mast cell is triggered to release granules containing inflammatory mediators. The γ chain associated with the fc receptors, and the dap12 chain that associates with the nK killer-activating receptors, also contain one iTam per chain and are present as homodimers.

1	T cells, Syk is broadly expressed in all leukocyte subsets; in contrast, ZAP-70 has been found only in T cells and NK cells. Several viral pathogens seem to have acquired ITAM-containing receptors from their hosts. These include the Epstein–Barr virus (EBV), whose LMP2A gene encodes a membrane protein with a cytoplasmic tail containing an ITAM. This enables EBV to trigger B-cell proliferation by the signaling pathways discussed in Section 7-20, an important step in the development of EBV-induced malignancies. Another virus that expresses an ITAM-containing protein is the Kaposi sarcoma herpesvirus (KSHV or HHV8), which causes malignant transformation and proliferation of the cells it infects. 7-12 Activated ZAP-70 phosphorylates scaffold proteins and promotes PI 3-kinase activation.

1	7-12 Activated ZAP-70 phosphorylates scaffold proteins and promotes PI 3-kinase activation. As described in Section 7-10, phosphorylation of the tyrosines in the T-cell receptor ITAMs leads to the recruitment and activation of ZAP-70. This provides ZAP-70 proximity to the cell membrane, where it phosphorylates the scaffold protein LAT (linker for activated T cells), a transmembrane protein with a large cytoplasmic domain (Fig. 7.15). ZAP-70 also phosphorylates another adaptor protein, SLP-76. LAT and SLP-76 can be linked by the adaptor protein Gads; this three-protein complex, referred to as the LAT:Gads:SLP-76 complex, plays a central role in T-cell activation. This is illustrated by the profound TCR signaling and T-cell development defects seen in mice lacking any MOVIE 7.1

1	Fig. 7.15 ZAP-70 phosphorylates LAT and SLP-76, initiating four downstream signaling modules. activated Zap-70 phosphorylates the scaffold proteins LaT and SLp-76 and recruits them to the activated T-cell receptor (TcR) complex. an adaptor protein, Gads, holds the tyrosinephosphorylated LaT and SLp-76 together. The multiple binding sites on these scaffold proteins recruit several additional adaptors and enzymes that initiate four essential downstream signaling modules. one key component required for several of these modules is the activation of pi 3-kinase, which phosphorylates pip2 in the plasma membrane to generate pip3. These four modules include the activation of the serine/ threonine kinase akt, which promotes enhanced cellular metabolic activity; the activation of pLc-γ, which leads to transcription factor activation; the activation of vav, which induces actin polymerization and cytoskeletal reorganization; and the recruitment of adap, an adaptor protein that promotes enhanced

1	to transcription factor activation; the activation of vav, which induces actin polymerization and cytoskeletal reorganization; and the recruitment of adap, an adaptor protein that promotes enhanced integrin adhesiveness and clustering.

1	Akt activation leads to increased cellular metabolic activity PIP3AktPHdomainADAP recruitment leads to enhanced integrin adhesiveness and clustering ADAPVav activation leads to actin polymerization and cytoskeletal reorganization PLC-˜activation leads to transcription factor activation PLC-˜GadsVavZAP-70LATSLP-76Activated ZAP-70 phosphorylates LAT and SLP-76, initiating four signaling modules

1	Fig. 7.16 The recruitment of phospholipase C-γ by LAT and SLP-76, and its phosphorylation and activation by the protein kinase Itk, are crucial steps in T-cell activation. Zap-70 phosphorylates the scaffold proteins LaT and SLp-76, which are brought together to form a complex at the activated T-cell receptor by the adaptor protein Gads. This complex also promotes the activation of pi 3-kinase, leading to the production of pip3 (formed by the phosphorylation of pip2 by pi 3-kinase). phospholipase c-γ (pLc-γ) is recruited to the membrane by its ph domain binding to pip3 and then binds to phosphorylated sites in LaT and to the proline-rich domain of SLp-76. To be activated, pLc-γ must be phosphorylated by the Tec-family kinase itk. itk is recruited to the membrane by interaction of its ph domain with pip3, and by interactions with phosphorylated SLp-76. once phosphorylated by itk, phospholipase c-γ is active.

1	one of these components, and in humans lacking ZAP-70. A second essential event that occurs rapidly following ZAP-70 activation is the recruitment and activation of the enzyme PI 3-kinase (see Section 7.4); while the detailed mechanism linking PI 3-kinase activation to T-cell receptor stimulation is not well understood, current evidence suggests a role for the small GTPase Ras. In this case, Ras may be activated by recruitment of the Ras-GEF Sos to LAT via Sos binding to the small adaptor protein Grb2, forming a second three-protein complex containing LAT and Sos, bridged by Grb2.

1	Following formation of the LAT:Gads:SLP-76 complex and the activation of PI 3-kinase, the T-cell receptor signaling pathway branches into several downstream modules, each of which induces cellular changes that contribute to optimal T-cell activation (see Figure 7.15). Each module is initiated by the recruitment of a key intermediate to the active signaling complexes, either via binding to the LAT:Gads:SLP-76 complex, to the PIP3 generated by PI 3-kinase, or to both. In brief, these modules lead to the activation of phospholipase C-γ (PLC-γ), which affects transcription; the activation of the serine/threonine kinase Akt, which affects metabolism, among other things; the recruitment of the adaptor protein ADAP, which upregulates cell adhesion; and the activation of the protein Vav, which initiates actin polymerization. Each of these modules will be described in detail in the sections below.

1	7-13 Activated PLC-γ generates the second messengers diacylglycerol and inositol trisphosphate that lead to transcription factor activation. One important module of T-cell receptor signaling is the activation of the enzyme phospholipase C-γ (PLC-γ). First, PLC-γ is brought to the inner face of the plasma membrane by the binding of its PH domain to the PIP3 that has been formed by the phosphorylation of PIP2 by PI 3-kinase; it then binds to phosphorylated LAT and SLP-76. The actions of PLC-γ produce two second messengers that activate three distinct terminal branches of the T-cell receptor pathway leading to transcription factor activation.

1	Due to this crucial role in T-cell activation, PLC-γ activation is controlled at several different levels. Recruitment to the membrane, while necessary, is not sufficient to activate PLC-γ. PLC-γ activation requires phosphorylation by Itk, a member of the Tec family of cytoplasmic tyrosine kinases. Like PLC-γ, Tec kinases contain PH, SH2, and SH3 domains and are recruited to the plasma membrane by interactions via these domains; specifically, the PH domain interacts with PIP3 on the inner face of the membrane (Fig. 7.16), and the SH2 and SH3 domains interact with SLP-76. These interactions serve to localize Itk in close proximity to its substrate, PLC-γ.

1	Once PLC-γ has been recruited to the inner face of the plasma membrane and has been activated, it can catalyze the breakdown of the membrane lipid PIP2 (see Section 7-4 and Fig. 7.5) to generate two products, the membrane lipid diacylglycerol (DAG) and the diffusible second messenger inositol 1,4,5-trisphosphate (IP3) (not to be confused with the membrane lipid PIP3) (Fig. 7.17). DAG is confined to the membrane, but diffuses in the plane of the membrane and serves as a molecular target that recruits other signaling molecules to the membrane. IP3 diffuses into the cytosol and binds to IP3 receptors on the endoplasmic reticulum (ER) membrane. These receptors are Ca2+ channels, which open and release calcium stored in the ER into the cytosol. The consequent low levels of calcium in the ER then cause a conformational change in the transmembrane protein STIM1, promoting its clustering within the ER membrane. The STIM1 oligomers bind to the plasma membrane, where they interact directly with

1	cause a conformational change in the transmembrane protein STIM1, promoting its clustering within the ER membrane. The STIM1 oligomers bind to the plasma membrane, where they interact directly with ORAI1, the plasma membrane calcium channel (also known as the CRAC channel: calcium release-activated calcium channel). Binding of STIM1 to ORAI1 triggers calcium channel opening, allowing extracellular calcium to flow into the cell to activate further signaling pathways and to replenish ER calcium stores.

1	The activation of PLC-γ marks an important step in T-cell activation, because after this point the PLC-γ signaling module splits into three distinct branches— the stimulation of Ca2+ entry, the activation of Ras, and the activation of protein kinase C-θ (PKC-θ)—each of which ends in the activation of a different transcription factor. These signaling pathways are utilized by many cell types, in addition to lymphocytes. Their importance in T-cell activation is shown by the observation that treatment of T cells with phorbol myristate acetate (an analog of DAG) and ionomycin (a pore-forming drug that allows extracellular calcium to flow into the cell) can reconstitute many of the effects of T-cell receptor stimulation. Additionally, deficiencies in several of these signaling components, including Lck, Zap-70, Itk, CD45, Carma1, and ORAI1, have been found to be mutated in cases of severe combined immunodeficiency (SCID). 7-14 Ca2+ entry activates the transcription factor NFAT.

1	7-14 Ca2+ entry activates the transcription factor NFAT. One of the three signaling pathways leading from PLC-γ results in an influx of calcium ions into the cytosol. An important outcome of the increased cytosolic Ca2+ resulting from T-cell receptor signaling via PLC-γ is the activation of a family of transcription factors called NFAT (nuclear factor of activated T cells). NFAT is something of a misnomer, because the five members of this family are expressed in many different tissues. In the absence of activating

1	Fig. 7.17 Phospholipase C-γ cleaves inositol phospholipids to generate two important signaling molecules. Top panel: phosphatidylinositol bisphosphate (pip2) is a component of the inner leaflet of the plasma membrane. When pLc-γ is activated by phosphorylation, it cleaves pip2 into two parts: inositol trisphosphate (ip3), which diffuses away from the membrane into the cytosol, and diacylglycerol (daG), which stays in the membrane. Both of these molecules are important in signaling. middle panel: there are two phases of calcium release. ip3 binds to a receptor in the endoplasmic reticulum (eR) membrane, opening calcium channels (yellow) and allowing the early phase of calcium ions (ca2+) to enter the cytosol from the eR. The depletion of ca2+ stores in the eR causes the aggregation of an eR calcium sensor, STim1. Bottom panel: aggregated STim1 stimulates the second phase of calcium entry by binding to and opening calcium channels called oRai1 in the plasma membrane. This further

1	an eR calcium sensor, STim1. Bottom panel: aggregated STim1 stimulates the second phase of calcium entry by binding to and opening calcium channels called oRai1 in the plasma membrane. This further increases cytosolic calcium and restores eR ca2+ stores. daG binds and recruits signaling proteins to the membrane, most importantly the Ras-Gef called RasGRp and a serine/threonine kinase called protein kinase c-θ (pKc-θ). Recruitment of RasGRp to the plasma membrane activates Ras, and pKc-θ activation results in the activation of the transcription factor nfκB.

1	Left panel: nfaT is maintained in the cytoplasm by phosphorylation on serine and threonine residues. center panel: after antigen receptor stimulation, calcium enters the cytosol, first from the endoplasmic reticulum (not shown; see fig. 7.17) and later from the extracellular space (shown). after entering the cytosol, calcium binds to calmodulin, and the ca2+:calmodulin complex binds to the serine/threonine phosphatase calcineurin, activating it to dephosphorylate nfaT. Right panel: once dephosphorylated, nfaT moves into the nucleus, where it binds to promoter elements and activates the transcription of various genes.

1	MOVIE 7.2 signals, NFAT is kept in the cytosol of resting T cells by phosphorylation on serine/threonine residues. This phosphorylation is mediated by serine/threonine kinases, including glycogen synthase kinase 3 (GSK3) and casein kinase 2 (CK2). When phosphorylated, the nuclear localization sequence of NFAT is not recognized by nuclear transporters, and NFAT is unable to enter into the nucleus (Fig. 7.18).

1	The cytoplasmic Ca2+ resulting from T-cell receptor signaling binds to and induces a conformational change in a protein called calmodulin, which is then able to bind to and activate a wide range of different target enzymes. In T cells, an important target of calmodulin is calcineurin, a protein phosphatase that acts on NFAT. Dephosphorylation of NFAT by calcineurin allows the nuclear localization sequence to be recognized by nuclear transporters, and NFAT enters the nucleus (see Fig. 7.18). There it participates in turning on many of the genes crucial for T-cell activation, such as the gene for the cytokine interleukin-2 (IL-2).

1	The importance of NFAT in T-cell activation is illustrated by the effects of selective inhibitors of calcineurin called cyclosporin A (CsA) and tacrolimus (also known as FK506). CsA forms a complex with the protein cyclophilin A, and this complex inhibits calcineurin. Tacrolimus binds a different protein, FK-binding protein (FKBP), making a complex that similarly inhibits calcineurin. By inhibiting calcineurin, these drugs prevent the formation of active NFAT. T cells express low levels of calcineurin, so they are more sensitive to inhibition of this pathway than are many other cell types. Both cyclosporin A and tacrolimus thus act as effective immunosuppressants, and are widely used to prevent the rejection of organ transplants (see Chapter 16, Section 16-3). 7-15 Ras activation stimulates the mitogen-activated protein kinase (MAPK) relay and induces expression of the transcription factor AP-1.

1	A second branch of the PLC-γ signaling module is the activation of the small GTPase Ras. This can occur by various means. The most efficient mechanism for Ras activation in T cells is via the DAG generated by PLC-γ, which diffuses in the plasma membrane and activates a variety of proteins. One of these is the protein RasGRP, which is a guanine-nucleotide exchange factor that specifically activates Ras. RasGRP contains a protein-interaction module called a C1 domain that binds to DAG. This interaction recruits RasGRP to the membrane near active signaling complexes (Fig. 7.19), where it activates Ras by promoting the exchange of GDP for GTP. Ras is also activated in the T-cell receptor signaling pathway by the guanine-exchange factor Sos, which is recruited by the adaptor protein Grb2 (see Sections 7-2 and 7-3), which has itself been recruited by binding to phosphorylated LAT and SLP-76.

1	Ras is initially inactive. TCR signaling produces DAG, which recruits RasGRP to the membrane where it activates Ras Ras activates Raf, which phosphorylates Mek, which phosphorylates Erk Activated Erk enters the nucleus and activates transcription factors such as Elk-1 inactive Ras RasGRP active Ras DAG Raf transcription factor nucleus cytoplasm Mek Erk Activated Ras then triggers a three-kinase relay that ends in the activation of a serine/threonine kinase known as a mitogen-activated protein kinase or MAP kinase (MAPK) (see Fig. 7.19). In the case of antigen receptor signaling, the first member of the relay is a MAPK kinase kinase (MAP3K) called Raf. Raf is a serine/threonine kinase that phosphorylates the next member of the series, a MAPK kinase (MAP2K) called MEK1. MEK1 is a dual-specificity protein kinase that phosphorylates a tyrosine and a threonine residue on the last member of the relay, a MAPK which in T cells and B cells is Erk (extracellular signal-related kinase).

1	Signaling by MAPK cascades is facilitated by specialized scaffold proteins that bind to all three kinases in a particular MAPK relay, thereby accelerating their interactions. The scaffold protein kinase suppressor of Ras (KSR) functions in the Raf/MEK1/Erk pathway. During T-cell receptor signaling, KSR associates with Raf, MEK1, and Erk and localizes itself and its cargo to the membrane. In that location, activated Ras can engage with the Raf bound to KSR and trigger the kinase relay (see Fig. 7.19). An important function of MAPKs is to phosphorylate and activate transcription factors that can then induce new gene expression. Erk acts indirectly to generate the transcription factor AP-1, which is a heterodimer composed of one monomer each from the Fos and Jun families of transcription factors (Fig. 7.20). Active Erk phosphorylates the transcription factor Elk-1, which

1	Fig. 7.19 DAG activates MAPK cascades, leading to transcription factor activation. all mapK cascades are initiated by the activation of small G proteins, such as Ras in this example. Ras is switched from an inactive state (first panel) to an active state (second panel) by a guanine-nucleotide exchange factor (Gef), RasGRp, which is recruited to the membrane by daG. Ras activates the first enzyme of the cascade, a protein kinase called Raf, a mapK kinase kinase (map3K) (third panel). Raf phosphorylates mek, a map2K, which in turn phosphorylates and activates erk, a mapK. The scaffold protein KSR associates with Raf, mek, and erk to facilitate their efficient interactions (not shown). phosphorylation and activation of erk releases it from the complex so that it can diffuse within the cell and enter the nucleus (fourth panel). phosphorylation of transcription factors by erk results in new gene transcription.

1	Fig. 7.20 The transcription factor AP-1 is formed as a result of the Ras/ MAPK signaling pathway. Left panel: phosphorylation of the mapK erk activated as a result of the Ras–mapK cascade allows erk to enter the nucleus, where it phosphorylates the transcription factor elk-1. elk-1, along with serum response factor (SRf), binds to the serum response element (SRe) in the promoter of the gene (FOS) for the transcription factor c-fos, stimulating its transcription. Right panel: the protein kinase pKc-θ can induce the phosphorylation of another mapK called Jun kinase (JnK). This enables JnK to enter the nucleus and phosphorylate the transcription factor c-Jun, which forms a dimer with c-fos. The phosphorylated c-Jun/fos dimer is an active ap-1 transcription factor that binds to ap-1 sites and promotes transcription of many target genes.

1	cooperates with a transcription factor called serum response factor to initiate transcription of the FOS gene. Fos protein then associates with Jun to form the AP-1 heterodimer, but this remains transcriptionally inactive until another MAPK called Jun kinase (JNK) phosphorylates Jun. Similar to NFAT, AP-1 participates in turning on transcription of many genes important for T-cell activation, including the gene encoding the cytokine IL-2. 7-16 Protein kinase C activates the transcription factors NFκB and AP-1.

1	7-16 Protein kinase C activates the transcription factors NFκB and AP-1. The third signaling pathway leading from PLC-γ results in the activation of PKC-θ, an isoform of protein kinase C that is restricted to T cells and muscle. Mice lacking PKC-θ develop T cells in the thymus, but their mature T cells have a defect in the activation of two crucial transcription factors, NFκB and AP-1, in response to signaling by the T-cell receptor and CD28. These transcription factors also participate in turning on genes required for T-cell activation. For example, transcription of the gene for IL-2 requires NFκB in addition to NFAT and AP-1, making PKC-θ activation an important component of T-cell activation.

1	PKC-θ has a C1 domain and is recruited to the membrane when DAG is generated by activated PLC-γ (see Fig. 7.17). In this location, the kinase activity of PKC-θ initiates a series of steps that results in the activation of NFκB (Fig. 7.21). PKC-θ phosphorylates the large membrane-localized scaffold protein CARMA1, causing it to oligomerize and form a multisubunit complex with other proteins. This complex recruits and activates TRAF-6, the same protein that we encountered in Chapter 3 in its role in activating NFκB in the TLR signaling pathway (see Fig. 3.13). NFκB is the general name for a member of a family of homoand heterodimeric transcription factors made up of the Rel family of proteins. The most common NFκB activated in lymphocytes is a heterodimer of p50:p65Rel.

1	DAG recruits PKC-˜to the membrane where it phosphorylates CARMA1 Phosphorylated CARMA1 creates a scaffold with BCL10 and MALT1 TRAF-6 is recruited and makes a polyubiquitin scaffold on itself and on NEMO TAK1, recruited by TAB1/2, phosphorylates IKK-°, which phosphorylates I˛B, inducing I˛B ubiquitination and degradation Degradation of I˛B releases NF˛B, which activates transcription in the nucleus PKC-˜CARMA1I°BdegradedI°B˛˝ ˙(NEMO)IKKTAB1/2TAK1ubiquitinBCL10MALT1TRAF-6NF°B

1	Fig. 7.21 Activation of the transcription factor NFκB by antigen receptors is mediated by protein kinase C. diacylglycerol (daG), produced as a result of T-cell receptor signaling activating pLc-γ, recruits a protein kinase c (pKc-θ) to the membrane, where it phosphorylates a scaffold protein called caRma1. This forms a complex with BcL10 and maLT1 that recruits the e3 ubiquitin ligase TRaf-6. as described in fig. 3.13, the kinase TaK1 is recruited by the polyubiquitin scaffold produced by TRaf-6 and phosphorylates the iκB kinase (iKK) complex [iKKα:iKKβ:iKKγ (nemo)]. iKK phosphorylates iκB, stimulating its ubiquitination, which targets iκB for degradation by the proteasome. This releases nfκB to enter the nucleus and stimulate transcription of its target genes. a defect in nemo that prevents nfκB activation causes immunodeficiency that results in susceptibility to extracellular bacterial infections, along with a skin disease known as ectodermal dysplasia.

1	The dimer is held in an inactive state in the cytoplasm by binding to an inhibitory protein called inhibitor of κB (IκB). As described for TLR signaling (see Fig. 3.13), TRAF-6 stimulates the degradation of IκB by first activating the kinase TAK1, which activates a complex of serine kinases, IκB kinase (IKK). IKK phosphorylates IκB, causing its ubiquitination and subsequent degradation, and the consequent release and entry into the nucleus of active NFκB. Inherited deficiency of the IKKγ subunit (also called NEMO) leads to a syndrome known as X-linked hypohidrotic ectodermal dysplasia and immunodeficiency, which is characterized by developmental defects in ectodermal structures such as skin and teeth, as well as immunodeficiency.

1	PKC-θ can also activate JNK, and might be able to activate the transcription factor AP-1 by this route. However, T cells lacking PKC-θ have a defect in AP-1 activation in addition to their defect in NFκB activation, but no defect in JNK activation, indicating that our understanding of this pathway is still incomplete. 7-17 PI 3-kinase activation upregulates cellular metabolic pathways via the serine/threonine kinase Akt.

1	While the activation of transcription factors is an important outcome of antigen receptor signaling, a productive T-cell response also requires substantial changes in cellular metabolism, needed to accommodate the energetic and macromolecular demands of rapidly dividing cells. The PI 3-kinase pathway plays a central role in this response via the recruitment and activation of the second important signaling module, initiated by the serine/threonine kinase Akt (also known as protein kinase B). Akt, via its PH domain, binds to PIP3 in the membrane, which is generated by PI 3-kinase (Fig. 7.22; see also Fig. 7.5). In that location, Akt is phosphorylated by PDK1, and once activated, phosphorylates a variety of downstream proteins. One of its effects is to promote cell survival by inhibiting cell death via multiple mechanisms. A major mechanism is the phosphorylation of the pro-apoptotic protein Bad. When phosphorylated, Bad can no longer bind to and inhibit the antiapoptotic (pro-survival)

1	death via multiple mechanisms. A major mechanism is the phosphorylation of the pro-apoptotic protein Bad. When phosphorylated, Bad can no longer bind to and inhibit the antiapoptotic (pro-survival) protein Bcl-2 (see Fig. 7.22). Another effect of activated Akt is to regulate the expression of homing and adhesion receptors that orchestrate the migratory properties of activated T cells (discussed in detail in Chapters 9 and 11). Activated Akt also functions to stimulate the cell’s metabolism by increasing the utilization of glucose; this is mediated by increasing the activity of glycolytic enzymes and by inducing the upregulation of nutrient transporters on the T-cell membrane.

1	Fig. 7.22 The serine/threonine kinase Akt is activated by TCR signaling and promotes cell survival and enhanced metabolic activity via mTOR. panel one: T-cell receptor (TcR) signaling activates pi 3-kinase (not shown), generating pip3 in the plasma membrane; pip3 recruits and activates the kinase pdK1. akt, a second serine/threonine kinase, binds to pip3 via its ph domain, and is phosphorylated and activated by pdK1. panel two: active akt phosphorylates the pro-apoptotic protein Bad, which is binding to and inhibiting the anti-apoptotic protein Bcl-2 at the mitochondrial membrane. panel three: phosphorylated Bad binds to 14-3-3, releasing Bcl-2 to promote cell survival. panel four: a second function of active akt is to phosphorylate the TSc1/2 complex, a Gap for the small GTpase Rheb. panel five: When TSc1/2 is phosphorylated, it releases the inactive Rheb protein, leading to Rheb activation. active Rheb binds to and activates mToR (mammalian target of rapamycin). once activated, mToR

1	When TSc1/2 is phosphorylated, it releases the inactive Rheb protein, leading to Rheb activation. active Rheb binds to and activates mToR (mammalian target of rapamycin). once activated, mToR acts on multiple pathways that lead to increased lipid production, ribosome biosynthesis, mRna synthesis, and protein translation.

1	Akt Bcl-2 14-3-3 inactive Rheb active RhebTSC1/2 Bad outer mitochondrial membrane inner mitochondrial membrane matrix mTOR PDK1 lipid production ribosome biosynthesis mRNA synthesis protein translation PIP3 recruits PDK1 and Akt, allowing PDK1 to phosphorylate and activate Akt Active Akt phosphorylates Bad, releasing Bad from Bcl-2 Upon release, phosphorylated Bad binds 14-3-3, allowing Bcl-2 to promote cell survival Active Akt phosphorylates the TSC1/2 complex, a GAP for the small GTPase, Rheb Phosphorylated TSC1/2 releases Rheb, allowing active Rheb to bind mTOR Fig. 7.23 Recruitment of ADAP to the LAT:Gads:SLP-76 complex activates integrin adhesion and aggregation.

1	Fig. 7.23 Recruitment of ADAP to the LAT:Gads:SLP-76 complex activates integrin adhesion and aggregation. Left panel: prior to T-cell receptor (TcR) signaling, the integrin Lfa-1 is present on the T-cell membrane in a low-affinity conformation that binds weakly to icam-1 on antigen-presenting-cells. middle panel: following TcR signaling, the adaptor protein adap is recruited to the LaT:Gads:SLp-76 complex by an interaction between tyrosine-phosphorylated adap and the Sh2 domain of SLp-76. adap then recruits a complex of SKap and Riam (Rap1-GTpinteracting adaptor molecule), activating the small GTpase Rap1. Right panel: active Rap1 induces aggregation of Lfa-1 and a conformational change in Lfa-1 that leads to high-affinity binding to icam-1.

1	Yet another important function of activated Akt is to stimulate the mTOR (mammalian target of rapamycin) pathway, a key regulator of macromolecular biosynthesis (see Fig. 7.22). In this case, Akt phosphorylates and inactivates the TSC complex, a GAP for the small GTPase Rheb. This leads to Rheb activation, and in turn, to the activation of mTOR. The mTOR pathway has multiple effects on cellular metabolism; collectively, these changes are essential to provide the raw materials needed to carry out the increased gene expression, protein production, and cell division that accompany T-cell activation. Specifically, mTOR activation leads to increased lipid production, ribosome biosynthesis, mRNA synthesis, and protein translation. 7-18 T-cell receptor signaling leads to enhanced integrin-mediated cell adhesion.

1	The third signaling module induced by TCR stimulation leads to enhanced integrin adhesion. Together with cytoskeletal changes (discussed in the next section), this process promotes stability of the T cell-APC interaction and localizes active signaling complexes into a structure known as the ‘immune synapse’ described below in Section 7-19 (and see Fig. 7.25). The immune synapse, the region of the T-cell membrane that is in direct and stable contact with the APC or target cell, is formed within minutes of T-cell receptor recognition of MHC/peptide ligands. One important component of this is increased adhesiveness of the T-cell integrin LFA-1. On nonstimulated T cells, LFA-1 resides in a low-affinity state and is well dispersed on the T-cell membrane, resulting in weak binding to its ligand, ICAM-1. Following T-cell receptor stimulation, LFA-1 molecules aggregate at the synapse, and also undergo a conformational change that converts each LFA-1 molecule into a high-affinity binding

1	ICAM-1. Following T-cell receptor stimulation, LFA-1 molecules aggregate at the synapse, and also undergo a conformational change that converts each LFA-1 molecule into a high-affinity binding partner for ICAM-1. Together, these changes lead to enhanced adhesion between the T cell and the APC, and to stabilization of this cell–cell interaction. These effects on LFA-1 are induced by the recruitment of the adaptor protein ADAP to the LAT:Gads:SLP-76 scaffold complex (Fig. 7.23). In turn, ADAP recruits two additional proteins, SKAP55 and RIAM. The ADAP:SKAP55:RIAM complex binds to the small GTPase Rap1, activating Rap1 at the site of T-cell receptor signaling. GTP-bound Rap1 then promotes LFA-1 aggregation and the conformational change that converts LFA-1 into a high-affinity binding partner for ICAM-1. The importance of this pathway is underscored by the finding that ADAP-deficient T cells show impaired proliferation and cytokine production following T-cell receptor stimulation.

1	7-19 T-cell receptor signaling induces cytoskeletal reorganization by activating the small GTPase Cdc42.

1	The fourth TCR signaling module, also involved in the formation of a stable immune synapse, leads to reorganization of the actin cytoskeleton. Without this process, integrin aggregation would not occur, interactions between the T cell and the APC would not stabilize, and in fact, T-cell activation would completely fail. A major component of this T-cell receptor signal is transduced by Vav, a GEF that activates Rho-family GTPases, including Cdc42. Like PLC-γand Itk, Vav is recruited to the site of receptor activation by interactions of the Vav PH domain with PIP3 and of the Vav SH2 domain with the LAT:Gads:SLP-76 scaffold complex (Fig. 7.24). When Cdc42 is activated by Vav, the GTP-bound Cdc42 induces a conformational change in the protein WASp (Wiskott– Aldrich syndrome protein), which is also recruited to the LAT:Gads:SLP-76 scaffold complex by binding to the small adaptor protein Nck. This active form of WASp binds to WIP, and together, these proteins recruit Arp2/3, leading to

1	is also recruited to the LAT:Gads:SLP-76 scaffold complex by binding to the small adaptor protein Nck. This active form of WASp binds to WIP, and together, these proteins recruit Arp2/3, leading to actin polymerization. The importance of this pathway is underscored by the fact that defects in WASp are the basis for the immunodeficiency disease Wiskott–Aldrich syndrome. Due to the widespread expression of WASp, individuals suffering from this disease have impairments in multiple immune cell types, all of which depend on WASp-dependent actin polymerization for their functions. One major defect in this disease is in T cell-dependent antibody responses, due to the requirement for actin polymerization to ensure effective interactions between CD4 T cells and B cells. Thus, the failure of WASpdeficient T cells to provide adequate ‘help’ to B cells most likely results from a defect in the formation of the immune synapse, which is normally required to ensure directed secretion of cytokines

1	T cells to provide adequate ‘help’ to B cells most likely results from a defect in the formation of the immune synapse, which is normally required to ensure directed secretion of cytokines from the T cell onto the B-cell membrane (Fig. 7.25).

1	7-20 The logic of B-cell receptor signaling is similar to that of T-cell receptor signaling, but some of the signaling components are specific to B cells. There are many similarities between signaling from T-cell receptors and signaling from B-cell receptors. As with the T-cell receptor, the B-cell receptor is composed of antigen-specific chains associated with ITAM-containing signaling chains, in this case Igα and Igβ (see Fig. 7.10). In B cells, three protein tyrosine kinases of the Src family—Fyn, Blk, and Lyn—are thought to be responsible for phosphorylation of the ITAMs (Fig. 7.26). These kinases associate with

1	Fig. 7.24 Recruitment of Vav to the LAT:Gads:SLP-76 complex induces activation of Cdc42, leading to actin polymerization. Left panel: vav, a guanine-nucleotide exchange factor (Gef) for the small GTpase cdc42, is recruited to the activated T-cell receptor (TcR) complex by binding via its ph domain to pip3 in the membrane and by binding to phosphorylated SLp-76. The small adaptor protein nck binds to an adjacent phosphorylated tyrosine on SLp-76 and recruits the inactive form of the protein WaSp. middle panel: vav activates cdc42, which binds to and activates WaSp. Right panel: active WaSp binds to Wip, recruiting arp2/3 and inducing actin polymerization. The importance of this pathway is illustrated by the discovery of WaSp as the protein encoded by the gene responsible for the human immunodeficiency disease Wiskott– aldrich syndrome.

1	Fig. 7.25 The immune synapse provides a structure for directed secretion of T-cell cytokines. When the T-cell receptors (TcRs) on a T cell recognize peptide:mhc on an antigen-presenting cell, a process of receptor reorganization takes place on the plasma membranes of the two interacting cells. Left panel: when a cd4+ T cell recognizes its peptide:mhc ligand on a B cell, the immune synapse functions to direct T-cell-secreted cytokines onto the B-cell surface at the site of closest contact between the plasma membranes of the two cells. Right panel: confocal microscopy images of the TcR/peptide:mhc (red) and the Lfa-1/icam-1 (green) proteins 30 minutes after the initiation of signaling show a central accumulation of the TcR/peptide:mhc complexes and a peripheral ring of the Lfa-1/icam-1 complexes. These structures have been called the central supermolecular activation complex (cSmac, red) and the peripheral supermolecular activation complex (pSmac, green). The combined structure is known

1	These structures have been called the central supermolecular activation complex (cSmac, red) and the peripheral supermolecular activation complex (pSmac, green). The combined structure is known as the immune synapse. photograph courtesy of y. Kaizuka.

1	resting receptors via a low-affinity interaction with unphosphorylated ITAMs in Igα and Igβ. After the receptors have bound a multivalent antigen, which cross-links them, the receptor-associated kinases are activated and phosphorylate the tyrosine residues in the ITAMs. B cells do not express ZAP-70; instead, a closely related tyrosine kinase, Syk, containing two SH2 domains, is recruited to the phosphorylated ITAM. In contrast to ZAP-70, which requires additional Lck phosphorylation for activation, Syk is activated simply by its binding to the phosphorylated site.

1	For B cells, the co-receptor and co-stimulatory receptor functions are combined into one accessory receptor that is a complex of cell-surface proteins—CD19, CD21, and CD81—often referred to as the B-cell co-receptor (Fig. 7.27). As with T cells, antigen-dependent signaling from the B-cell receptor is enhanced if the B-cell co-receptor is simultaneously bound by its ligand and clusters with the antigen receptor. CD21 (also known as complement receptor 2, CR2) is a receptor for the C3dg fragment of complement. This means that antigens such as bacterial pathogens on which C3dg is bound (see Fig. 7.27) can cross-link the B-cell receptor with the CD21:CD19:CD81 complex. This induces phosphorylation of the cytoplasmic tail of CD19 by B-cell receptor-associated tyrosine kinases, which in turn leads to the binding of additional Src-family kinases, the augmentation of signaling through the B-cell receptor itself, and the recruitment of PI 3-kinase (see Section 7-4). PI 3-kinase

1	Fig. 7.26 Src-family kinases are associated with B-cell receptors and phosphorylate the tyrosines in ITAMs to create binding sites for Syk and Syk activation via transphosphorylation. The membrane-bound Src-family kinases fyn, Blk, and Lyn associate with the B-cell antigen receptor by binding to iTams, either (as shown in the figure) through their amino-terminal domains or by binding a single phosphorylated tyrosine through their Sh2 domains. after ligand binding and receptor clustering, the associated kinases phosphorylate tyrosines in the iTams on the cytoplasmic tails of igα and igβ. Subsequently, Syk binds to the phosphorylated iTams of the igβ chain. Because there are at least two receptor complexes in each cluster, Syk molecules become bound in close proximity and can activate each other by transphosphorylation, thus initiating further signaling.

1	initiates an additional signaling pathway leading from the B-cell receptor (see Fig. 7.27). Thus, the B-cell co-receptor serves to strengthen the signal resulting from antigen recognition. The role of the third component of the B-cell receptor complex, CD81 (TAPA-1), is as yet unknown.

1	Once activated, Syk phosphorylates the scaffold protein SLP-65 (also known as BLNK). Like LAT and SLP-76 in T cells, SLP-65 functions as a composite of these two proteins, providing multiple sites for tyrosine phosphorylation and recruiting a variety of SH2-containing proteins, including enzymes and adaptor proteins, to form several distinct multiprotein signaling complexes that can act in concert. As in T cells, a key signaling protein is the phospholipase PLC-γ, which is activated with the aid of the B cell-specific Tec kinase Bruton’s tyrosine kinase (Btk) and hydrolyzes PIP2 to form DAG and IP3 (see Fig. 7.27). As discussed for the T-cell receptor, signaling by calcium and DAG leads to the activation of downstream transcription factors. A deficiency in Btk (which is encoded by a gene on the X chromosome) prevents the development and functioning of B cells, resulting in the disease X-linked agammaglobulinemia, which is characterized by a lack of antibodies. Besides Btk, mutations

1	on the X chromosome) prevents the development and functioning of B cells, resulting in the disease X-linked agammaglobulinemia, which is characterized by a lack of antibodies. Besides Btk, mutations in other signaling molecules in B cells, including receptor chains and SLP-65, have been linked to B-cell immunodeficiencies (see Chapter 8).

1	Several other downstream pathways described for TCR signaling are also shared with BCR signaling, and are dependent on the adaptor protein SLP-65. These include the Vav-dependent induction of actin polymerization by Cdc42 and WASp, and the recruitment and activation of small GTPases that promote integrin adhesion (see Fig. 7.27). In the case of B-cell recognition of

1	B-cell receptor (BcR) signaling is greatly enhanced when the antigen is tagged by complement fragments, engaging the B-cell co-receptor together with the B-cell antigen receptor. cleavage of the antigen-bound complement component c3 to c3dg (see fig. 2.30) allows the tagged antigen to bind to the cell-surface protein cd21 (complement receptor 2, cR2), a component of the B-cell co-receptor complex, which also includes cd19 and cd81 (Tapa-1). cross-linking and clustering of the co-receptor with the antigen receptor result in the phosphorylation of tyrosine residues in the iTam sequences of the cytoplasmic domains of the BcR signaling subunits, igα and igβ. The Src-family kinase also phosphorylates tyrosine residues in the cytoplasmic domain of cd19. The phosphorylated iTams in igα and igβ recruit and activate the tyrosine kinase Syk, which functions similarly to Zap-70 in T cells. The phosphorylated tail of cd19 recruits pi 3-kinase, leading to pip3 generation in the plasma membrane.

1	recruit and activate the tyrosine kinase Syk, which functions similarly to Zap-70 in T cells. The phosphorylated tail of cd19 recruits pi 3-kinase, leading to pip3 generation in the plasma membrane. activated Syk phosphorylates the membrane-associated scaffold protein SLp-65, which associates with the plasma membrane by binding cin85. pip3 recruits pdK1 and akt, leading to akt activation. phosphorylated SLp-65 and pip3 recruit the Tec-family tyrosine kinase Btk and pLc-γ, leading to Btk phosphorylation and activation of pLc-γ. phosphorylated SLp-65 and pip3 also recruit vav, nck, and inactive WaSp. vav activates small GTpases that activate WaSp, leading to actin polymerization; the activated GTpases also induce integrin aggregation and conversion of Lfa-1 to the high-affinity binding state.

1	˜˜°˛˝˙˙ˆmIgMTCR Ig°Ig˛Tyrosine kinases Adaptors and scaffold proteins Phospholipases and lipid kinases GTPases, serine/threonine kinases, and phosphatases Transcription factors, cytoskeletal changes, adhesion, metabolism

1	Fig. 7.28 Summary of antigen receptor signaling pathways. as outlined in this section, the signal transduction pathways downstream of the T-cell and B-cell receptors occur in an orchestrated series of stages involving many categories of proteins that produce widespread changes in the cells. The first detectable events following antigen receptor stimulation are the activation of tyrosine kinases. following this, adaptor proteins and scaffolds are modified, recruiting phospholipases and lipid kinases to the activated receptor complexes. The next level of signaling amplifies these earlier stages by activating multiple small GTpases, serine/threonine kinases, and protein phosphatases. Together, these lead to transcription factor activation, cytoskeletal changes, and increases in cellular adhesion and metabolism, all of which contribute to T-and B-cell activation.

1	membrane-bound antigen, B-cell receptor signaling also produces an immune synapse that localizes signaling complexes to the cell–cell interface. One key function of the immune synapse in B cells is to promote antigen uptake by the B cell, a prerequisite to presenting that antigen in the form of peptide:MHC complexes to CD4 T cells. B-cell receptor signaling also induces metabolic changes in activated B cells. As is the case for T cells, this response is dependent on the action of PI 3-kinase, whose activation leads to formation of the membrane phosphoinositide PI(3,4,5)P3 at the site of the activated B-cell receptor. This response is augmented by combined signaling through the B-cell receptor plus the B-cell co-receptor complex of CD19/CD21/CD81. PI(3,4,5)P3 recruits Akt, which is then phosphorylated and activated, leading to downstream activation of mTOR, as well as additional Akt-dependent pathways promoting cell survival and proliferation (see Fig. 7.27). Summary.

1	The antigen receptors on the surface of lymphocytes are multiprotein complexes in which the antigen-binding chains interact with additional proteins that are responsible for signaling from the receptor. These protein chains carry tyrosine-containing signaling motifs known as ITAMs. Signaling chains containing ITAM motifs are widely used by activating receptors in many immune cell types in addition to lymphocytes. In lymphocytes, activation of the receptors by antigen results in a series of biochemical events that are broadly outlined in Fig. 7.28. This signaling cascade is initiated by the phosphorylation of the ITAMs by Src-family kinases. The phosphorylated ITAM then recruits another tyrosine kinase, ZAP-70 in T cells and Syk in B cells. Activation of ZAP-70 or Syk results in the phosphorylation of scaffolds called LAT and SLP-76 in T cells, and SLP-65 in B cells, and in the activation of PI 3-kinase. Multiple signaling proteins are recruited and activated by these phosphorylated

1	of scaffolds called LAT and SLP-76 in T cells, and SLP-65 in B cells, and in the activation of PI 3-kinase. Multiple signaling proteins are recruited and activated by these phosphorylated scaffolds, including phospholipase C-γ, ADAP, and Vav, whereas Akt is recruited and activated by the action of PI 3-kinase generating PIP3 at the plasma membrane. PLC-γ generates inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 has an important role in inducing changes in intracellular calcium concentrations, and DAG is involved in activating protein kinase C-θ and the small G protein Ras. In T cells, these pathways ultimately result in the activation of three transcription factors, namely, AP-1, NFAT, and NFκB; together, these transcription factors induce transcription of the cytokine IL-2, which is essential for the proliferation and further differentiation of the activated lymphocyte. In addition to transcription factor activation, antigen receptor signaling in both T cells and B cells

1	is essential for the proliferation and further differentiation of the activated lymphocyte. In addition to transcription factor activation, antigen receptor signaling in both T cells and B cells leads to enhanced cell survival, metabolic activity, adhesiveness, and cytoskeletal reorganization. Signaling by antigen receptors is facilitated by co-receptors that become engaged as a result of receptor–antigen binding. These co-receptors are the MHC-binding CD4 and CD8 transmembrane proteins in T cells and the complement-binding B-cell co-receptor complex containing CD19 in B cells.

1	Co-stimulatory and inhibitory receptors modulate antigen receptor signaling in T and B lymphocytes.

1	Signals initiated by the T-cell and B-cell antigen receptors are essential for lymphocyte activation, and determine the specificity of the adaptive immune response that is initiated. However, signaling from the antigen receptor is not on its own sufficient to activate a naive T cell or B cell. These naive lymphocytes require additional signals to achieve full activation. Receptors on T cells and B cells that can provide this necessary second signal are called costimulatory receptors, and are members of either the CD28 family of proteins or of the TNF receptor superfamily. While naive T cells primarily utilize CD28 as the co-stimulatory receptor, naive B cells use the TNF receptor family member CD40. The overall function of signaling through these co-stimulatory receptors is to enhance the antigen receptor signals that induce transcription factor activation and PI 3-kinase activation, thereby ensuring activation of the T cell or B cell. In contrast to these activating co-stimulatory

1	antigen receptor signals that induce transcription factor activation and PI 3-kinase activation, thereby ensuring activation of the T cell or B cell. In contrast to these activating co-stimulatory receptor signals, other cell-surface receptors on T cells and B cells function to downregulate activation signals. These inhibitory receptors are important in preventing excessive immune responses that can lead to destructive inflammatory or autoimmune conditions, particularly in the case of chronic infections that are inefficiently controlled by the immune system.

1	7-21 The cell-surface protein CD28 is a required co-stimulatory signaling receptor for naive T-cell activation. The signaling through the T-cell receptor complex described in the previous sections is not by itself sufficient to activate a naive T cell. As noted in Chapter 1, antigen-presenting cells that can activate naive T cells bear cell-surface proteins known as co-stimulatory molecules or co-stimulatory ligands. These interact with cell-surface receptors, known as co-stimulatory receptors, on the naive T cell to transmit a signal that is required, along with antigen stimulation, for T-cell activation—this signal is sometimes called ‘signal 2.’ We discuss the immunological consequences of this requirement for co-stimulation in detail in Chapter 9.

1	The best understood of these co-stimulatory receptors is the cell-surface protein CD28. CD28 is present on the surface of all naive T cells and binds the co-stimulatory ligands B7.1 (CD80) and B7.2 (CD86), which are expressed mainly on specialized antigen-presenting cells such as dendritic cells. To become activated, the naive lymphocyte must engage both antigen and a co-stimulatory ligand on the same antigen-presenting cell. CD28 signaling aids antigen-dependent T-cell activation mainly by promoting T-cell proliferation, cytokine production, and cell survival. All these effects are mediated by signaling motifs present in the cytoplasmic domain of CD28.

1	After engagement by B7 molecules, CD28 becomes tyrosine phosphorylated by Lck in its cytoplasmic domain on tyrosine residues in a YXN motif that can recruit the adaptor protein Grb2, and in a non-ITAM motif YMNM. The cytoplasmic tail of CD28 also carries a proline-rich motif (PXXP) that binds the SH3 domains of Lck and Itk. Although the details are uncertain, a major effect of CD28 phosphorylation is to activate PI 3-kinase to generate PIP3 (Fig. 7.29). By this mechanism, the co-stimulatory signal induced by CD28 cooperates with the T-cell receptor signal to ensure maximal activation of three of the four T-cell receptor signaling modules described above. Specifically, a high concentration of PIP3 recruits Itk to the membrane, where Lck can phosphorylate it, thereby enhancing PLC-γ activation. PIP3 also functions to recruit and activate Akt, which promotes cell survival and increased cellular metabolism (see Section 7.17). An additional function of Akt is to phosphorylate the

1	activation. PIP3 also functions to recruit and activate Akt, which promotes cell survival and increased cellular metabolism (see Section 7.17). An additional function of Akt is to phosphorylate the RNA-binding protein NF-90; when phosphorylated, NF-90 translocates from the nucleus to the cytoplasm and binds to and stabilizes the IL-2 mRNA, leading to increased IL-2 synthesis. Finally, PIP3 recruits Vav, leading to cytoskeletal reorganization (see Section 7.19). Thus, co-stimulatory signaling through CD28 functions to amplify most of the downstream responses to T-cell receptor stimulation (see Fig. 7.29).

1	MOVIE 7.3 CD28B7MHCPIP2PIP3PI3KPI3KPIP3 recruits PDK1 and Akt, allowing PDK1 to phosphorylate and activate Akt B7 binding induces CD28 phosphorylation, activating PI 3-kinase to produce PIP3 B7.1 and B7.2 are CD28 ligands expressed on specialized APCs PIP3 also recruits Itk, allowing it to phosphorylate PLC-˜PIP3 also recruits Vav, leading to Cdc24 activation SH2domainkinasedomainAktItkSLP-76LATPDK1PLC-˜activeCdc42inactiveCdc42Vav

1	Fig. 7.29 The T-cell co-stimulatory protein CD28 transduces signals that enhance antigen receptor signaling pathways. The ligands for cd28, namely B7.1 and B7.2, are expressed only on specialized antigen-presenting cells (apcs) such as dendritic cells (first panel). engagement of cd28 induces its tyrosine phosphorylation, which activates pi 3-kinase (pi3K), with subsequent production of pip3 that recruits several enzymes via their ph domains, thus bringing them together with their substrates in the membrane. The protein kinase akt, which becomes phosphorylated by phosphoinositide-dependent protein kinase-1 (pdK1), is activated and enhances cell survival and upregulates cell metabolism (see fig. 7.22). Recruitment of the kinase itk to the membrane is critical for the full activation of pLc-γ (see fig. 7.16). pip3 also recruits vav, leading to cdc42 activation and inducing actin polymerization (see fig. 7.24).

1	7-22 Maximal activation of PLC-γ, which is important for transcription factor activation, requires a co-stimulatory signal induced by CD28. One important function of co-stimulatory signaling through CD28 is to promote the maximal activation of PLC-γ via the local production of PIP3. This recruits Itk by its PH domain, enhancing Itk phosphorylation by Lck. Activated Itk is then recruited to the phosphorylated LAT:Gads:SLP-76 complex by its SH2 and SH3 domains binding to SLP-76, where it phosphorylates and activates PLC-γ (see Fig. 7.16). Activated PLC-γ cleaves PIP2 to produce the two second messengers DAG and IP3, ultimately leading to the activation of transcription factors NFAT, AP-1, and NFκB. Thus, the full activation of PLC-γ leading to transcription factor activation requires signals emanating from both the T-cell receptor and CD28.

1	In T cells, one of the major functions of NFAT, AP-1, and NFκB is to act together to stimulate expression of the gene for the cytokine IL-2, which is essential for promoting T-cell proliferation and differentiation into effector cells. The promoter for the IL-2 gene contains multiple regulatory elements that must be bound by transcription factors to initiate IL2 expression. Some control sites are already bound by transcription factors, such as Oct1, that are produced constitutively in lymphocytes, but this is not sufficient to switch on IL-2. Only when AP-1, NFAT, and NFκB are all activated and are bound to their control sites in the IL-2 promoter is the gene expressed. NFAT and AP-1 bind to the promoter cooperatively and with higher affinity by forming a heterotrimer of NFAT, Jun, and Fos. In addition, CD28 co-stimulation further enhances IL-2 transcription by increasing NFκB activation. Thus, the IL-2 promoter integrates signals from both the T-cell receptor and CD28 signaling

1	Fos. In addition, CD28 co-stimulation further enhances IL-2 transcription by increasing NFκB activation. Thus, the IL-2 promoter integrates signals from both the T-cell receptor and CD28 signaling pathways to ensure that IL-2 is produced only in appropriate circumstances (Fig. 7.30). Together with the CD28-induced phosphorylation of NF-90 leading to increased IL-2 mRNA stability, CD28 co-stimulation leads to substantially increased production of IL-2 by activated T cells.

1	7-23 TNF receptor superfamily members augment T-cell and B-cell activation. While naive Tand B-cell activation requires signaling through the antigen receptors on these cells, T-cell receptor or B-cell receptor signaling, Fig. 7.30 Simplified scheme depicting multiple signaling pathways that converge on the IL-2 promoter.

1	Fig. 7.30 Simplified scheme depicting multiple signaling pathways that converge on the IL-2 promoter. ap-1, nfaT, and nfκB binding to the promoter of the IL-2 gene integrate multiple signaling pathways emanating from the T-cell receptor (TcR) and cd28 into a single output, the production of the cytokine iL-2. The mapK pathway activates ap-1; calcium activates nfaT; and protein kinase c activates nfκB. all three pathways are required to stimulate IL-2 transcription. activation of the gene requires both the binding of nfaT and ap-1 to a specific promoter element, and the additional binding of ap-1 on its own to another site. oct1 is a transcription factor that is required for IL-2 transcription. unlike the other transcription factors, it is constitutively bound to the promoter and is therefore not regulated by T-cell receptor or cd28 signaling.

1	respectively, is not sufficient to activate the cells. For naive T cells, an additional co-stimulatory signal is required and, as discussed above (see Sections 7-21 and 7-22), is frequently provided by the CD28 receptor. For naive B cells, the additional activation signal can be contributed by direct interactions between the pathogen and an innate pattern recognition receptor (PRR), such as a TLR, on the B cell. However, for more effective B-cell activation leading to the production of all classes of antibodies and to the formation of memory B cells, additional B-cell activation signals are contributed by CD4 T cells. One component of this is the production of T-cell cytokines, which bind to and stimulate their receptors on the B-cell surface (see Chapter 10). The second and more essential component provided by the CD4 T cells is the stimulation of CD40 on the B cell by the CD40 ligand expressed on the T cell. The importance of the CD40–CD40-ligand interaction for B-cell responses to

1	component provided by the CD4 T cells is the stimulation of CD40 on the B cell by the CD40 ligand expressed on the T cell. The importance of the CD40–CD40-ligand interaction for B-cell responses to protein antigens is highlighted by the discovery that a severe immunodeficiency disease resulting from impaired antibody responses is caused by the absence of CD40 ligand expression on a patient’s CD4 T cells.

1	CD40 is a member of the large TNF receptor superfamily, which consists of more than 20 members. While some members of this family, such as Fas (see Chapter 11), are specialized to induce cell death, the majority of TNF receptor superfamily members, including CD40, activate both the NFκB and the PI 3-kinase pathways following receptor stimulation (Fig. 7.31). While NFκB activation leads to enhanced cell survival, the PI 3-kinase pathway has widespread and pleiotropic effects on B-cell physiology, and is a central feature of CD40 signaling. The major mediator of the PI 3-kinase signal is the serine/threonine kinase Akt, which is recruited and activated following the generation of PI(3,4,5)P3 at the B-cell membrane. Akt then stimulates multiple downstream pathways that induce cell survival, cell cycle progression, glucose uptake and metabolism, and mTOR activation, all of which are essential for the productive response of the activated B cell. In general terms, CD40 on the B cell

1	cell cycle progression, glucose uptake and metabolism, and mTOR activation, all of which are essential for the productive response of the activated B cell. In general terms, CD40 on the B cell functions in a manner analogous to CD28 on the T cell, as both receptors serve to enhance the levels of Akt activation induced by the B-cell receptor or T-cell receptor signaling pathways, respectively.

1	TNF receptor superfamily members, including CD40, signal by a mechanism distinct from that of antigen receptors, as it does not involve the activation of tyrosine kinases. Instead, stimulation of TNF receptors recruits adaptor proteins known as TRAFs (TNF receptor-associated factors). In addition to serving as simple adaptors that promote the assembly of multiprotein complexes, five

1	Fig. 7.31 The TNF receptor superfamily member CD40 is an important co-stimulatory molecule on B cells. Several members of the Tnf receptor superfamily are expressed on T cells and B cells. a key function of these receptors is the activation of nfκB, which occurs by a pathway distinct from the one initiated by antigen receptor stimulation and is often referred to as the non-canonical nfκB pathway. Tnf receptor superfamily members also activate pi 3-kinase signaling pathways. one important Tnf receptor superfamily member on B cells is cd40. prior to stimulation, TRaf molecules, which are ubiquitin ligases, are associated with ciap, another ubiquitin ligase, and the nfκB-inducing kinase niK. under these steady-state conditions, TRaf binding promotes ubiquitination and degradation of niK. When cd40 is stimulated by binding to cd40L, this complex is recruited to the intracellular domain of cd40. TRaf2 catalyzes K63-linked ubiquitination of ciap, leading to ciap-mediated K48-linked

1	niK. When cd40 is stimulated by binding to cd40L, this complex is recruited to the intracellular domain of cd40. TRaf2 catalyzes K63-linked ubiquitination of ciap, leading to ciap-mediated K48-linked ubiquitination of TRaf3. This leads to TRaf3 degradation, releasing niK, and allowing niK to phosphorylate and activate iκB kinase α (iKKα). iKKα phosphorylates the nfκB precursor protein p100, inducing its cleavage to form the active p52 subunit, which binds to relB to form the active nfκB transcription factor. cd40L stimulation of cd40 also activates pi 3-kinase, which leads to the activation of akt by pdK1.

1	of the six known TRAFs also function as E3 ubiquitin ligases. This activity contributes to the ability of most TNF receptor superfamily members to activate the NFκB pathway, using a pathway distinct from the one initiated by antigen receptor stimulation and often referred to as the non-canonical NFκB pathway (see Fig. 7.31). In contrast, the precise biochemical mechanism by which TNF receptors and TRAFs induce PI 3-kinase activation is not yet known.

1	CD40 is constitutively expressed on B cells and functions during B-cell activation in response to antigen recognition by the B-cell receptor. Additional TNF receptor superfamily members are also expressed on B cells, and each of them is important in B-cell survival at a particular stage of B-cell maturation, including B cells that have differentiated into antibody-secreting cells or memory cells. Similarly, TNF receptor superfamily members are expressed on T cells, many of which are upregulated following T-cell activation. These molecules, such as OX40, 4-1BB, CD30, and CD27, contribute important survival signals and function to enhance cellular metabolism at later stages of the T-cell response to infection (see Chapter 11). responses by interfering with co-stimulatory signaling pathways.

1	CD28 belongs to a family of structurally related receptors that are expressed by lymphocytes and bind B7-family ligands. Some, such as the receptor ICOS, which is discussed in Chapter 9, act as activating receptors, but others inhibit signaling by the antigen receptors, can stimulate apoptosis, and are important in regulating the immune response. Inhibitory receptors related to CD28 and expressed by T cells include CTLA-4 (CD152) and PD-1 (programmed death-1), while the B and T lymphocyte attenuator (BTLA) is expressed by both T cells and B cells. Of these, CTLA-4 seems to be the most important: mice lacking CTLA-4 die at a young age from an uncontrolled proliferation of T cells in multiple organs, whereas loss of PD-1 or BTLA causes less marked changes that alter the magnitude of responses following lymphocyte activation, rather than causing widespread spontaneous lymphoproliferation. Both CTLA-4 and PD-1 have been targeted for the development of protein-based therapeutics that

1	following lymphocyte activation, rather than causing widespread spontaneous lymphoproliferation. Both CTLA-4 and PD-1 have been targeted for the development of protein-based therapeutics that function to block the activities of these receptors. The goal of these therapeutics is to enhance T-cell responses by inhibiting these inhibitory receptors, a therapeutic strategy referred to as checkpoint blockade (see Chapter 16). Recent clinical trials demonstrate that both CTLA-4 and PD-1 blockade have remarkable efficacy in the treatment of cancer by enhancing the patient’s own antitumor T-cell responses.

1	CTLA-4 is induced on activated T cells and binds to the same co-stimulatory ligands (B7.1 and B7.2) as CD28, but CTLA-4 engagement is inhibitory for T-cell activation, rather than enhancing (Fig. 7.32). The function of CTLA-4 is controlled largely by regulation of its surface expression. Initially, CTLA-4 resides on intracellular membranes but moves to the cell surface after T-cell receptor signaling. The surface expression of CTLA-4 is controlled by phosphorylation of the tyrosine-based motif GVYVKM in its cytoplasmic tail. When this motif is not phosphorylated, it is able to bind to the clathrin adaptor molecule AP-2, which removes CTLA-4 from the surface. When it is phosphorylated, this motif cannot bind AP-2, and CTLA-4 remains in the membrane, where it can bind B7 molecules on antigen-presenting cells.

1	CTLA-4 has a higher affinity for its B7 ligands than does CD28, and, apparently of importance for its inhibitory function, it engages B7 molecules in a different orientation. CD28, CTLA-4, and B7.1 are all expressed as homodimers. A CD28 dimer engages one B7.1 dimer in a direct one-to-one correspondence, but a CTLA-4 dimer engages two different B7 dimers in a configuration that allows for extended cross-linkages that confer high avidity to the interaction (see Fig. 7.32). CTLA-4 was once presumed to act by recruiting inhibitory phosphatases, like some of the other inhibitory receptors described later, but this is no longer thought to be so. It is still not clear whether CTLA-4 directly activates inhibitory signaling pathways. Instead, its actions may result in part from blocking the binding of CD28 to B7, thereby reducing CD28-dependent co-stimulation.

1	CTLA-4-expressing T cells can also exert an inhibitory effect on the activation of other T cells. How they do this is not yet clear, but it might result from CTLA-4 binding to B7 molecules on antigen-presenting cells, in effect stealing the ligand for CD28 required by the other T cells. Direct actions of CTLA-4 on T cells have not been excluded, however. Notably, the regulatory T cells that are needed to suppress autoimmunity express high levels of CTLA-4 on their surface, and they require CTLA-4 to function normally. Regulatory cells are described in detail in Chapter 9. 7-25 Inhibitory receptors on lymphocytes downregulate immune responses by recruiting protein or lipid phosphatases.

1	Some other receptors that can inhibit lymphocyte activation possess motifs in their cytoplasmic regions that are known as the immunoreceptor tyrosine-based inhibitory motif (ITIM, consensus sequence [I/V]XYXX[L/I], where X is any amino acid) (Fig. 7.33) or the related immunoreceptor tyrosine-based switch motif (ITSM, consensus sequence TXYXX[V/I]). When the tyrosine in an ITIM or ITSM is phosphorylated, it can recruit either of two inhibitory phosphatases, called SHP (SH2-containing phosphatase) and SHIP (SH2containing inositol phosphatase), via their SH2 domains. SHP is a protein tyrosine phosphatase that removes phosphate groups added by tyrosine kinases to a variety of proteins. SHIP is an inositol phosphatase and removes the phosphate from PIP3 to generate PIP2, thus reversing the recruitment of proteins such as Tec kinases and Akt to the cell membrane and thereby inhibiting signaling.

1	CD28 B7.1 One dimer of CD28 engages just one dimer of B7 A distinct binding orientation allows one dimer of CTLA-4 to bind two different B7 dimers, providing for high-avidity clustering CTLA-4

1	Fig. 7.32 CTLA-4 has a higher affinity than CD28 for B7 and engages it in a multivalent orientation. cd28 and cTLa-4 are both expressed as dimers on the cell surface and both bind to two ligands of B7.1, which is a dimer, and B7.2, which is not. however, the orientations of the B7 binding of cd28 and of cTLa-4 differ in a way that contributes to the inhibitory action of cTLa-4. one dimer of cd28 engages just one dimer of B7.1. But one dimer of cTLa-4 binds in such a way that two different dimers of B7.1 are engaged at once, allowing these molecules to cluster into complexes of high avidity. This, and the higher affinity of cTLa-4 for B7 molecules, may give it an advantage in competing for available B7 molecules on an antigen-presenting cell, providing one mechanism by which it could block the co-stimulation of T cells. B cells, T cells, and NK cells express receptors that contain immunoreceptor tyrosine-based inhibitory motifs PIR-B Fc˜RIIB-1 CD22 BTLA, KIR2DL KIR3DL PD-1

1	B cells, T cells, and NK cells express receptors that contain immunoreceptor tyrosine-based inhibitory motifs PIR-B Fc˜RIIB-1 CD22 BTLA, KIR2DL KIR3DL PD-1 Fig. 7.33 Some lymphocyte cell-surface receptors contain motifs involved in downregulating activation. Several receptors that transduce signals that inhibit lymphocyte or nK-cell activation contain one or more iTims (immunoreceptor tyrosine-based inhibitory motifs) in their cytoplasmic tails (red rectangles). iTims bind to various phosphatases that, when activated, inhibit signals derived from iTamcontaining receptors.

1	One ITIM-containing receptor is PD-1 (see Fig. 7.33), which is induced transiently on activated T cells, B cells, and myeloid cells. It can bind to the B7-family ligands PD-L1 (programmed death ligand-1, B7-H1) and PD-L2 (programmed death ligand-2, B7-DC). Despite their names, we now understand that these proteins function as ligands for the inhibitory receptor PD-1, rather than acting directly in cell death. PD-L1 is constitutively expressed by a wide variety of cells, whereas PD-L2 expression is induced on antigen-presenting cells during inflammation. Because PD-L1 is expressed constitutively, regulation of PD-1 expression could have a critical role in controlling T-cell responses. For example, signaling by pro-inflammatory cytokines can repress PD-1, thus enhancing the T-cell response. Mice lacking PD-1 gradually develop autoimmunity, presumably because of an inability to regulate T-cell activation. In chronic infections, the widespread expression of PD-1 reduces the effector

1	Mice lacking PD-1 gradually develop autoimmunity, presumably because of an inability to regulate T-cell activation. In chronic infections, the widespread expression of PD-1 reduces the effector activity of T cells; this helps to limit potential damage to bystander cells, but at the expense of pathogen clearance.

1	BTLA contains an ITIM and an ITSM and is expressed on activated T cells and B cells, as well as on some cells of the innate immune system. Unlike other CD28-family members, however, BTLA does not interact with B7 ligands but binds a member of the TNF receptor family; called the herpesvirus entry molecule (HVEM), this receptor is highly expressed on resting T cells and immature dendritic cells. When BTLA and HVEM are co-expressed on the same cell, BTLA utilizes a second mechanism that further inhibits lymphocyte activation. In this configuration, BLTA binds to HVEM and prevents HVEM from binding to alternative partners that would stimulate NFκB-dependent pro-survival signaling pathways downstream of HVEM. Alternatively, when BTLA and HVEM are expressed on different cells, the interaction of these two receptors functions to stimulate the positive pro-survival signal in the HVEM-expressing cell.

1	Other receptors on B cells and T cells also contain ITIMs and can inhibit cell activation when ligated along with the antigen receptors. One example is the receptor FcγRIIB-1 on B cells, which binds the Fc region of IgG antibodies. As a result, antigens present as immune complexes containing IgG antibodies are poor at activating naive B cells, due to the co-engagement of the B-cell receptor with this inhibitory Fc receptor. The ITIM in FcγRIIB-1 recruits SHIP into a complex with the B-cell receptor to block the actions of PI 3-kinase (Fig. 7.34). Another inhibitory receptor on B cells is CD22, a transmembrane protein that recognizes sialic acid-modified glycoproteins commonly found on mammalian cells but rarely on microbial pathogen surfaces. CD22 contains an ITIM that interacts with SHP, a phosphatase that can dephosphorylate adaptors such as SLP-65 that associate with CD22, thereby inhibiting signaling from the B-cell receptor.

1	The ITIM motif is also an important motif in signaling by receptors on NK cells that inhibit the killer activity of these cells (see Section 3-26). These inhibitory receptors recognize MHC class I molecules and transmit signals that inhibit the release of the NK cell’s cytotoxic granules when the NK cell recognizes a healthy uninfected cell. In NK cells, ITIM-containing receptors play an important role in setting the threshold for NK cell activation by balancing positive signals from ITAM-containing receptors. Summary.

1	Summary. Signaling through the antigen receptors on T cells and B cells is essential for the activation of these cells. However, for naive T and B cells, the signal through the T-cell receptor or B-cell receptor, respectively, is not sufficient to initiate a response. In addition to the antigen receptor signals, these cells require signals through accessory receptors that serve to monitor the environment of the cell to ensure the presence of an infection. An important secondary signaling

1	Fig. 7.34 The ITIM-containing Fc receptor inhibits B-cell receptor signaling by recruiting the inositol phosphatase SHIP. When the B-cell receptor binds an antigen that is already present in immune complexes with igG, the iTim-containing fc receptor fcγRiiB is engaged at the same time as the B-cell receptor. The Src-family kinase present at the B-cell receptor (BcR) phosphorylates the iTim motif of fcγRiiB, which then recruits the Sh2 domain-containing inositol phosphatase Ship. Ship dephosphorylates pip3 in the plasma membrane, generating pip2. ph domain-containing enzymes, such as vav, Btk, and pLc-γ, depend on their ph domain binding to pip3 for their stable recruitment to the activated B-cell receptor complex. The loss of pip3 terminates the recruitment of these enzymes and inhibits B-cell receptor signaling.

1	system in naive T cells is provided by the CD28 family of co-stimulatory proteins, which bind members of the B7 family of proteins. Activating members of the CD28 family provide co-stimulatory signals that amplify the signal from the T-cell receptor and are important in ensuring the activation of naive T cells by the appropriate target cell. In B cells, these secondary signals are provided by members of the TNF receptor superfamily, such as CD40. Inhibitory members of the CD28 and other receptor families function to attenuate or completely block signaling by activating receptors. The regulated expression of activating and inhibitory receptors and their ligands generates a sophisticated level of control of immune responses that is only beginning to be understood. Summary to Chapter 7.

1	Signaling by cell-surface receptors of many different sorts is crucial to the ability of the immune system to respond appropriately to foreign pathogens. The importance of these signaling pathways is demonstrated by the many diseases that are due to aberrant signaling, which include both immunodeficiency diseases and autoimmune diseases. Common features of many signaling pathways are the generation of second messengers such as calcium and phosphoinositides and the activation of both serine/threonine and tyrosine kinases. An important concept in the initiation of signaling pathways by receptor proteins is the recruitment of signaling proteins to the plasma membrane and the assembly of multiprotein signaling complexes. In many cases, signal transduction leads to the activation of transcription factors that lead directly or indirectly to the proliferation, differentiation, and effector function of activated lymphocytes. Other roles of signal transduction are to mediate changes in the

1	factors that lead directly or indirectly to the proliferation, differentiation, and effector function of activated lymphocytes. Other roles of signal transduction are to mediate changes in the cytoskeleton that are important for cell functions such as migration and shape changes. These steps of the T-cell and B-cell receptor signaling pathways are summarized in Fig. 7.28.

1	While we are beginning to understand the basic circuitry of signal transduction pathways, it is important to keep in mind that we do not yet understand why these pathways are so complex. One reason might be that the signaling pathways have roles in properties such as amplification, robustness, diversity, and efficiency of signaling responses. An important goal for the future will be to understand how the design of each signaling pathway contributes to the particular quality and sensitivity needed for specific signaling responses. Questions. 7.1 True or False: antigen receptors bear intrinsic kinase activity that allows for phosphorylation of cytoplasmic proteins and subsequent downstream signaling events. 7.2 Matching: indicate whether the following receptors are receptor tyrosine kinases (RTKs), are receptor serine/ threonine kinases (RSTKs), or have no intrinsic enzymatic activity (null). A. ___ Kit B. ___ B-cell receptor C. ___ fLT3

1	A. ___ Kit B. ___ B-cell receptor C. ___ fLT3 D. ___ TGf-β receptor 7.3 Short Answer: how can scaffolds and adaptors modulate signaling responses if they have no intrinsic enzymatic activity? 7.4 Multiple Choice: Which of the following alterations would result in increased activity of Ras (one or more may apply): A. a mutation in Ras that enhances its GTpase activity B. overexpression of Gefs C. depletion of GTp in the cytoplasm D. overexpression of Gaps

1	A. a mutation in Ras that enhances its GTpase activity B. overexpression of Gefs C. depletion of GTp in the cytoplasm D. overexpression of Gaps E. a mutation in Ras that renders it unsusceptible to the activities of Gaps 7.5 Matching: order (by numbering 1–5) the downstream signaling events that occur immediately after T-cell receptor engagement: ____ LaT and SLp-76, scaffold proteins linked by Gads, are phosphorylated ____ Zap-70, a tandem Sh2 domain containing kinase, binds to iTams ____ Recruitment and activation of Sh2, ph, and pX domain-containing proteins ____ pi 3-kinase is activated and produces pip3 ____ phosphorylation of iTams by Lck, a Src-family kinase 7.6 Fill-in-the-Blanks: for each of the following sentences, fill in the blanks with the best word selected from the list below. each word should be used only once.

1	LaT:Gads:SLp-76 akt antigen receptor signaling leads to many downstream events that branch out into many signaling pathways or modules. These can be activated by the scaffold complex ___________, the generation of pip3 from pip2 by the enzyme ____________, or both. phosphorylated tyrosine residues on the scaffold recruit proteins containing ___________ domains, while pip3 recruits proteins containing ___________ domains. These four modules are the activation of (1) ___________, which cleaves pip2 to produce daG and ip3, (2) ___________, which binds to pip3 and activates the mToR pathway by phosphorylating and inactivating the TSc complex, (3) ____________, an adaptor that recruits SKap55 and Riam, and (4) __________, a Gef that leads to activation of WaSp. These pathways ultimately lead to increased transcription of key genes, increased cellular metabolism, increased cellular adhesion, and actin polymerization, respectively.

1	7.7 Matching: match the small G protein (GTpase) to its function. A. _____ Ras i. WaSp; actin polymerization B. _____ cdc42 ii. mToR; cellular metabolism (Rho family) C. _____ Rap1 iii. Lfa-1 aggregation; cellular adhesion D. _____ Rheb iv. mapK pathway; cellular proliferation 7.8 Multiple Choice: Which of the follow statements is false? A. K63 polyubiquitination leads to downstream cellular signaling. B. K48 polyubiquitination leads to degradation by the proteasome. C. out of the three families of enzymes involved in ubiquitination—e1 (ubiquitin-activating) enzymes, e2 (ubiquitin-conjugating) enzymes, and e3 enzymes (ubiquitin ligases)—cbl is an e3 enzyme that selects its target via its Sh2 domain. D. monoor di-ubiquitination of surface receptors leads to degradation by the proteasome. 7.9 Matching: match the human disease with the gene that is

1	D. monoor di-ubiquitination of surface receptors leads to degradation by the proteasome. 7.9 Matching: match the human disease with the gene that is D. ____ X-linked hypohidrotic iv. WaSp ectodermal dysplasia and immunodeficiency 7.10 Fill-in-the-Blanks: name the corresponding receptor or signaling component in its respective T/B cell counterpart: T cell B cell cd3ε:cd3δ:(cd3γ)2:(cd3ζ)2 A. __________ B. __________ cd21:cd19:cd81 cd28 C. __________ D. __________ fyn, Blk, Lyn E. __________ Syk LaT:Gads:SLp-76 F. __________ General references.

1	D. __________ fyn, Blk, Lyn E. __________ Syk LaT:Gads:SLp-76 F. __________ General references. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P.: Molecular Biology of the Cell, 6th ed. New York: Garland Science, 2015. Gomperts, B., Kramer, I., and Tatham, P.: Signal Transduction. San Diego: Elsevier, 2002. Marks, F., Klingmler, U., and Mler-Decker, K.: Cellular Signal Processing. New York: Garland Science, 2009. Samelson, L., and Shaw, A. (eds.): Immunoreceptor Signaling. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2010. Section references. 7-1 Transmembrane receptors convert extracellular signals into intracellular biochemical events. Lin, J., and Weiss, A.: T cell receptor signalling. J. Cell Sci. 2001, 114:243–244. Smith-Garvin, J.E., Koretzky, G.A., and Jordan, M.S.: T cell activation. Annu. Rev. Immunol. 2009, 27:591–619.

1	Lin, J., and Weiss, A.: T cell receptor signalling. J. Cell Sci. 2001, 114:243–244. Smith-Garvin, J.E., Koretzky, G.A., and Jordan, M.S.: T cell activation. Annu. Rev. Immunol. 2009, 27:591–619. 7.11 True or False: cTLa-4 and pd-1 are both iTim-containing inhibitory receptors that interfere with co-stimulatory signaling pathways by activating intracellular protein and/or lipid phosphatases. 7.12 Multiple Choice: intravenous administration of exogenous immunoglobulin is a widely used therapy for autoimmune disorders that involve the production of autoantibodies (antibodies against self antigens). Researchers have discovered that the presence of sialic acids on the infused immunoglobulins is critical for the inhibition of autoantibody production in the patient’s own B cells. Which of the following receptors could potentially be responsible for the inhibition of antibody production by B cells, given this finding? A. fcγRiiB B. cd22 C. pd-1 D. cd40

1	A. fcγRiiB B. cd22 C. pd-1 D. cd40 E. BTLa 7-2 Intracellular signal propagation is mediated by large multiprotein signaling complexes. Balagopalan, L., Coussens, N.P., Sherman, E., Samelson, L.E., and Sommers, C.L.: The LAT story: a tale of cooperativity, coordination, and choreography. Cold Spring Harbor Perspect. Biol. 2010, 2:a005512. Jordan, M.S., and Koretzky, G.A.: Coordination of receptor signaling in multiple hematopoietic cell lineages by the adaptor protein SLP-76. Cold Spring Harbor Perspect. Biol. 2010, 2:a002501. Lim, W.A., and Pawson, T.: Phosphotyrosine signaling: evolving a new cellular communication system. Cell 2010, 142:661–667. Scott, J.D., and Pawson, T.: Cell signaling in space and time: where proteins come together and when they’re apart. Science 2009, 326:1220–1224. 7-3 Small G proteins act as molecular switches in many different signaling pathways. Etienne-Manneville, S., and Hall, A.: Rho GTPases in cell biology. Nature 2002, 420:629–635.

1	7-3 Small G proteins act as molecular switches in many different signaling pathways. Etienne-Manneville, S., and Hall, A.: Rho GTPases in cell biology. Nature 2002, 420:629–635. Mitin, N., Rossman, K.L., and Der, C.J.: Signaling interplay in Ras superfamily function. Curr. Biol. 2005, 15:R563–R574. 7-4 Signaling proteins are recruited to the membrane by a variety of mechanisms. Buday, L.: Membrane-targeting of signalling molecules by SH2/ SH3 domain-containing adaptor proteins. Biochim. Biophys. Acta 1999, 1422:187–204. Kanai, F., Liu, H., Field, S.J., Akbary, H., Matsuo, T., Brown, G.E., Cantley, L.C., and Yaffe, M.B.: The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nat. Cell Biol. 2001, 3:675–678. Lemmon, M.A.: Membrane recognition by phospholipid-binding domains. Nat. Rev. Mol. Cell Biol. 2008, 9:99–111. 7-5 Post-translational modifications of proteins can both activate and inhibit signaling responses.

1	7-5 Post-translational modifications of proteins can both activate and inhibit signaling responses. Ciechanover, A.: Proteolysis: from the lysosome to ubiquitin and the proteasome. Nat. Rev. Mol. Cell Biol. 2005, 6:79–87. Hurley, J.H., Lee, S., and Prag, G.: Ubiquitin-binding domains. Biochem. J. 2006, 399:361–372. Liu, Y.C., Penninger, J., and Karin, M.: Immunity by ubiquitylation: a reversible process of modification. Nat. Rev. Immunol. 2005, 5:941–952. Wertz, I.E., and Dixit, V.M.: Signaling to NFκB: regulation by ubiquitination. Cold Spring Harbor Perspect. Biol. 2010, 2:a003350. 7-6 The activation of some receptors generates small-molecule second messengers. Kresge, N., Simoni, R.D., and Hill, R.L.: Earl W. Sutherland’s discovery of cyclic adenine monophosphate and the second messenger system. J. Biol. Chem. 2005, 280:39–40. Rall, T.W., and Sutherland, E.W.: Formation of a cyclic adenine ribonucleotide by tissue particles. J. Biol. Chem. 1958, 232:1065–1076.

1	Rall, T.W., and Sutherland, E.W.: Formation of a cyclic adenine ribonucleotide by tissue particles. J. Biol. Chem. 1958, 232:1065–1076. 7-7 Antigen receptors consist of variable antigen-binding chains associated with invariant chains that carry out the signaling function of the receptor. Brenner, M.B., Trowbridge, I.S., and Strominger, J.L.: Cross-linking of human T cell receptor proteins: association between the T cell idiotype beta subunit and the T3 glycoprotein heavy subunit. Cell 1985, 40:183–190. Call, M.E., Pyrdol, J., Wiedmann, M., and Wucherpfennig, K.W.: The organizing principle in the formation of the T cell receptor-CD3 complex. Cell 2002, 111:967–979. Kuhns, M.S., and Davis, M.M.: TCR signaling emerges from the sum of many parts. Front. Immunol. 2012, 3:159. Samelson, L.E., Harford, J.B., and Klausner, R.D.: Identification of the components of the murine T cell antigen receptor complex. Cell 1985, 43:223–231.

1	Samelson, L.E., Harford, J.B., and Klausner, R.D.: Identification of the components of the murine T cell antigen receptor complex. Cell 1985, 43:223–231. Tolar, P., Sohn, H.W., Liu, W., and Pierce, S.K.: The molecular assembly and organization of signaling active B-cell receptor oligomers. Immunol. Rev. 2009, 232:34–41. 7-8 Antigen recognition by the T-cell receptor and its co-receptors transduces a signal across the plasma membrane to initate signaling. Klausner, R.D., and Samelson, L.E.: T cell antigen receptor activation pathways: the tyrosine kinase connection. Cell 1991, 64:875–878. Weiss, A., and Littman, D.R.: Signal transduction by lymphocyte antigen receptors. Cell 1994, 76:263–274. 7-9 Antigen recognition by the T-cell receptor and its co-receptors leads to phosphorylation of ITAMs by Src-family kinases, generating the first intracellular signal in a signaling cascade.

1	7-9 Antigen recognition by the T-cell receptor and its co-receptors leads to phosphorylation of ITAMs by Src-family kinases, generating the first intracellular signal in a signaling cascade. Au-Yeung, B.B., Deindl, S., Hsu, L.-Y., Palacios, E.H., Levin, S.E., Kuriyan, J., and Weiss, A.: The structure, regulation, and function of ZAP-70. Immunol. Rev. 2009, 228:41–57. Bartelt, R.R., and Houtman, J.C.D.: The adaptor protein LAT serves as an integration node for signaling pathways that drive T cell activation. Wiley Interdiscip. Rev. Syst. Biol. Med. 2013, 5:101–110. Chan, A.C., Iwashima, M., Turck, C.W., and Weiss, A.: ZAP-70: a 70 Kd pro-tein-tyrosine kinase that associates with the TCR zeta chain. Cell 1992, 71:649–662. Iwashima, M., Irving, B.A., van Oers, N.S., Chan, A.C., and Weiss, A.: Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science 1994, 263:1136–1139.

1	Iwashima, M., Irving, B.A., van Oers, N.S., Chan, A.C., and Weiss, A.: Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science 1994, 263:1136–1139. Okkenhaug, K., and Vanhaesebroeck, B.: PI3K in lymphocyte development, differentiation and activation. Nat. Rev. Immunol. 2003, 3:317–330. Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R.P., and Samelson, L.E.: LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 1998, 92:83–92. 7-10 Phosphorylated ITAMs recruit and activate the tyrosine kinase ZAP-70. Yan, Q., Barros, T., Visperas, P.R., Deindl, S., Kadlecek, T.A., Weiss, A., and Kuriyan, J.: Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker. Mol. Cell. Biol. 2013, 33:2188–2201. 7-11 ITAMs are also found in other receptors on leukocytes that signal for cell activation.

1	7-11 ITAMs are also found in other receptors on leukocytes that signal for cell activation. Lanier, L.L.: Up on the tightrope: natural killer cell activation and inhibition. Nat. Immunol. 2008, 9:495–502. 7-12 Activated ZAP-70 phosphorylates scaffold proteins and promotes PI 3-kinase activation. Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R.P., and Samelson, L.E.: LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 1998, 92:83–92. 7-13 Activated PLC-γ generates the second messengers diacylglycerol and inositol trisphosphate that lead to transcription factor activation. Berg, L.J., Finkelstein, L.D., Lucas, J.A., and Schwartzberg, P.L.: Tec family kinases in T lymphocyte development and function. Annu. Rev. Immunol. 2005, 23:549–600. Yang, Y.R., Choi, J.H., Chang, J.-S., Kwon, H.M., Jang, H.-J., Ryu, S.H., and Suh, P.-G.: Diverse cellular and physiological roles of phospholipase C-γ1. Adv. Biol. Regul. 2012, 52:138–151.

1	Yang, Y.R., Choi, J.H., Chang, J.-S., Kwon, H.M., Jang, H.-J., Ryu, S.H., and Suh, P.-G.: Diverse cellular and physiological roles of phospholipase C-γ1. Adv. Biol. Regul. 2012, 52:138–151. 7-14 Ca2+ entry activates the transcription factor NFAT. Hogan, P.G., Chen, L., Nardone, J., and Rao, A.: Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev. 2003, 17:2205–2232. Hogan, P.G., Lewis, R.S., and Rao, A.: Molecular basis of calcium signaling in lymphocytes: STIM and ORAI. Annu. Rev. Immunol. 2010, 28:491–533. Picard, C., McCarl, C.A., Papolos, A., Khalil, S., Lhy, K., Hivroz, C., LeDeist, F., Rieux-Laucat, F., Rechavi, G., Rao, A., et al. STIM1 mutation associated with a syndrome of immunodeficiency and autoimmunity. N. Engl. J. Med. 2009, 360:1971–1980. Prakriya, M., Feske, S., Gwack, Y., Srikanth, S., Rao, A., and Hogan, P.G.: Orai1 is an essential pore subunit of the CRAC channel. Nature 2006, 443:230–233.

1	Prakriya, M., Feske, S., Gwack, Y., Srikanth, S., Rao, A., and Hogan, P.G.: Orai1 is an essential pore subunit of the CRAC channel. Nature 2006, 443:230–233. 7-15 Ras activation stimulates the mitogen-activated protein kinase (MAPK) relay and induces expression of the transcription factor AP-1. Downward, J., Graves, J.D., Warne, P.H., Rayter, S., and Cantrell, D.A.: Stimulation of P21ras upon T-cell activation. Nature 1990, 346:719–723. Leevers, S.J., and Marshall, C.J.: Activation of extracellular signal-regulated kinase, ERK2, by P21ras oncoprotein. EMBO J. 1992, 11:569–574. References. Roskoski, R.: ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol. Res. 2012, 66:105–143. Shaw, A.S., and Filbert, E.L.: Scaffold proteins and immune-cell signalling. Nat. Rev. Immunol. 2009, 9:47–56. 7-16 Protein kinase C activates the transcription factors NFκB and AP-1.

1	Shaw, A.S., and Filbert, E.L.: Scaffold proteins and immune-cell signalling. Nat. Rev. Immunol. 2009, 9:47–56. 7-16 Protein kinase C activates the transcription factors NFκB and AP-1. Blonska, M., and Lin, X.: CARMA1-mediated NFκB and JNK activation in lymphocytes. Immunol. Rev. 2009, 228:199–211. Matsumoto, R., Wang, D., Blonska, M., Li, H., Kobayashi, M., Pappu, B., Chen, Y., Wang, D., and Lin, X.: Phosphorylation of CARMA1 plays a critical role in T cell receptor-mediated NFκB activation. Immunity 2005, 23:575–585. Rueda, D., and Thome, M.: Phosphorylation of CARMA1: the link(Er) to NFκB activation. Immunity 2005, 23:551–553. Sommer, K., Guo, B., Pomerantz, J.L., Bandaranayake, A.D., Moreno-García, M.E., Ovechkina, Y.L., and Rawlings, D.J.: Phosphorylation of the CARMA1 linker controls NFκB activation. Immunity 2005, 23:561–574. Thome, M., and Weil, R.: Post-translational modifications regulate distinct functions of CARMA1 and BCL10. Trends Immunol. 2007, 28:281–288.

1	Thome, M., and Weil, R.: Post-translational modifications regulate distinct functions of CARMA1 and BCL10. Trends Immunol. 2007, 28:281–288. 7-17 PI 3-kinase activation upregulates cellular metabolic pathways via the serine/threonine kinase Akt. Gamper, C.J., and Powell, J.D.: All PI3Kinase signaling is not mTOR: dissecting mTOR-dependent and independent signaling pathways in T cells. Front. Immunol. 2012, 3:312. Kane, L.P., and Weiss, A.: The PI-3 kinase/Akt pathway and T cell activation: pleiotropic pathways downstream of PIP3. Immunol. Rev. 2003, 192:7–20. Pearce, E.L.: Metabolism in T cell activation and differentiation. Curr. Opin. Immunol. 2010, 22:314–320. 7-18 T-cell receptor signaling leads to enhanced integrin-mediated cell adhesion. Bezman, N., and Koretzky, G.A.: Compartmentalization of ITAM and integrin signaling by adapter molecules. Immunol. Rev. 2007, 218:9–28.

1	Bezman, N., and Koretzky, G.A.: Compartmentalization of ITAM and integrin signaling by adapter molecules. Immunol. Rev. 2007, 218:9–28. Mor, A., Dustin, M.L., and Philips, M.R.: Small GTPases and LFA-1 reciprocally modulate adhesion and signaling. Immunol. Rev. 2007, 218:114–125. 7-19 T-cell receptor signaling induces cytoskeletal reorganization by activating the small GTPase Cdc42. Burkhardt, J.K., Carrizosa, E., and Shaffer, M.H.: The actin cytoskeleton in T cell activation. Annu. Rev. Immunol. 2008, 26:233–259. Tybulewicz, V.L.J., and Henderson, R.B.: Rho family GTPases and their regulators in lymphocytes. Nat. Rev. Immunol. 2009, 9:630–644. 7-20 The logic of B-cell receptor signaling is similar to that of T-cell receptor signaling, but some of the signaling components are specific to B cells. Cambier, J.C., Pleiman, C.M., and Clark, M.R.: Signal transduction by the B cell antigen receptor and its coreceptors. Annu. Rev. Immunol. 1994, 12:457–486.

1	Cambier, J.C., Pleiman, C.M., and Clark, M.R.: Signal transduction by the B cell antigen receptor and its coreceptors. Annu. Rev. Immunol. 1994, 12:457–486. DeFranco, A.L., Richards, J.D., Blum, J.H., Stevens, T.L., Law, D.A., Chan, V.W., Datta, S.K., Foy, S.P., Hourihane, S.L., and Gold, M.R.: Signal transduction by the B-cell antigen receptor. Ann. N.Y. Acad. Sci. 1995, 766:195–201. Harwood, N.E., and Batista, F.D.: Early events in B cell activation. Annu. Rev. Immunol. 2010, 28:185–210. Koretzky, G.A., Abtahian, F., and Silverman, M.A.: SLP76 and SLP65: complex regulation of signalling in lymphocytes and beyond. Nat. Rev. Immunol. 2006, 6:67–78. Kurosaki, T., and Hikida, M.: Tyrosine kinases and their substrates in B lymphocytes. Immunol. Rev. 2009, 228:132–148. 7-21 The cell-surface protein CD28 is a required co-stimulatory signaling receptor for naive T cell activation.

1	7-21 The cell-surface protein CD28 is a required co-stimulatory signaling receptor for naive T cell activation. Acuto, O., and Michel, F.: CD28-mediated co-stimulation: a quantitative support for TCR signalling. Nat. Rev. Immunol. 2003, 3:939–951. Frauwirth, K.A., Riley, J.L., Harris, M.H., Parry, R.V., Rathmell, J.C., Plas, D.R., Elstrom, R.L., June, C.H., and Thompson, C.B.: The CD28 signaling pathway regulates glucose metabolism. Immunity 2002, 16: 769–777. Sharpe, A.H.: Mechanisms of costimulation. Immunol. Rev. 2009, 229:5–11. 7-22 Maximal activation of PLC-γ, which is important for transcription factor activation, requires a co-stimulatory signal induced by CD28. Chen, L., and Flies, D.B.: Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat. Rev. Immunol. 2013, 13:227–242. 7-23 TNF receptor superfamily members augment T-cell and B-cell activation.

1	7-23 TNF receptor superfamily members augment T-cell and B-cell activation. Chen, L., and Flies, D.B.: Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat. Rev. Immunol. 2013, 13:227–242. Rickert, R.C., Jellusova, J., and Miletic, A.V.: Signaling by the tumor necrosis factor receptor superfamily in B-cell biology and disease. Immunol. Rev. 2011, 244:115–133. 7-24 Inhibitory receptors on lymphocytes downregulate immune responses by interfering with co-stimulatory signaling pathways. Qureshi, O.S., Zheng, Y., Nakamura, K., Attridge, K., Manzotti, C., Schmidt, E.M., Baker, J., Jeffery, L.E., Kaur, S., Briggs, Z., et al.: Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsic function of CTLA-4. Science 2011, 332:600–603. Rudd, C.E., Taylor, A., and Schneider, H.: CD28 and CTLA-4 coreceptor expression and signal transduction. Immunol. Rev. 2009, 229:12–26.

1	Rudd, C.E., Taylor, A., and Schneider, H.: CD28 and CTLA-4 coreceptor expression and signal transduction. Immunol. Rev. 2009, 229:12–26. 7-25 Inhibitory receptors on lymphocytes downregulate immune responses by recruiting protein or lipid phosphatases. Acuto, O., Di Bartolo, V., and Michel, F.: Tailoring T-cell receptor signals by proximal negative feedback mechanisms. Nat. Rev. Immunol. 2008, 8:699–712. Chen, L., and Flies, D.B.: Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat. Rev. Immunol. 2013, 13:227–242. The Development of B and T Lymphocytes

1	The production of new lymphocytes, or lymphopoiesis, takes place in specialized lymphoid tissues—the central (or primary) lymphoid tissues— which are the bone marrow for most B cells and the thymus for most T cells. Precursors for both populations originate in the bone marrow, but whereas B cells complete most of their development there, the precursors of most T cells migrate to the thymus, where they develop into mature T cells. A major goal of lymphopoiesis is to generate a diverse repertoire of B-cell receptors and T-cell receptors on circulating B and T cells, respectively, thereby enabling an individual to make adaptive immune responses against the wide range of pathogens encountered during a lifetime. In the fetus and the juvenile, the central lymphoid tissues are the sources of large numbers of new lymphocytes, which migrate to populate the peripheral lymphoid tissues (also called secondary lymphoid tissues) such as lymph nodes, spleen, and mucosal lymphoid tissue. In mature

1	numbers of new lymphocytes, which migrate to populate the peripheral lymphoid tissues (also called secondary lymphoid tissues) such as lymph nodes, spleen, and mucosal lymphoid tissue. In mature individuals, the development of new T cells in the thymus slows down, and peripheral T-cell numbers are maintained by the division of mature T cells outside the central lymphoid organs. New B cells, in contrast, are continually produced from the bone marrow, even in adults. This chapter will focus on the development of T cells and B cells from their uncommitted progenitors, with an emphasis on the major populations of CD4+ and CD8+ T cells and B cells. The development of additional subsets of T cells and B cells, such as invariant NKT (iNKT) cells, Treg cells, γ:δ TCR+ T cells, B-1 B cells, and marginal zone B cells will be briefly discussed.

1	The structure of the antigen-receptor genes expressed by B cells and T cells, and the mechanisms by which a complete antigen receptor is assembled, were described in Chapters 4 and 5. Once an antigen receptor has been formed, rigorous testing is required to select lymphocytes that carry useful antigen receptors—that is, antigen receptors that can recognize a wide spectrum of pathogens and yet will not react against an individual’s own cells. Given the incredible diversity of receptors that the rearrangement process can generate, it is important that those lymphocytes that mature are likely to be useful in recognizing and responding to foreign antigens, especially as an individual can express only a small fraction of the total possible receptor repertoire in his or her lifetime. We describe how the specificity and affinity of the receptor for self ligands are tested to determine whether the immature lymphocyte will either survive and join the mature repertoire, or die. In general, it

1	how the specificity and affinity of the receptor for self ligands are tested to determine whether the immature lymphocyte will either survive and join the mature repertoire, or die. In general, it seems that developing lymphocytes whose receptors interact weakly with self antigens, or that bind self antigens in a particular way, receive a signal that enables them to survive. This process, known as positive selection, is particularly critical in the development of α:β T cells, which recognize composite antigens consisting of peptides bound to MHC molecules, because it ensures that an individual’s T cells will be able to respond to peptides bound to one’s own MHC molecules.

1	In contrast, lymphocytes with strongly self-reactive receptors must be eliminated to prevent autoimmune reactions; this process of negative selection is one of the ways in which the immune system is made self-tolerant. The default fate of developing lymphocytes, in the absence of any signal being received from the receptor, is death by apoptosis, and as we will see, the vast Development of B lymphocytes. Development of T lymphocytes. Positive and negative selection of T cells.

1	Fig. 8.1 B cells develop in the bone marrow and migrate to peripheral lymphoid organs, where they can be activated by antigens. In the first phase of development, progenitor B cells in the bone marrow rearrange their immunoglobulin genes. This phase is independent of antigen but is dependent on interactions with bone marrow stromal cells (first panels). It ends in an immature B cell that carries an antigen receptor in the form of cell-surface IgM (second panels), and in the second phase it can now interact with antigens in its environment. Immature B cells that are strongly stimulated by antigen at this stage either die or are inactivated in a process of negative selection, thus removing many self-reactive B cells from the repertoire. In the third phase of development, the surviving immature B cells emerge into the periphery and mature to express IgD as well as IgM. They can now be activated by encounter with their specific foreign antigen in a peripheral lymphoid organ (third

1	immature B cells emerge into the periphery and mature to express IgD as well as IgM. They can now be activated by encounter with their specific foreign antigen in a peripheral lymphoid organ (third panels). Activated B cells proliferate, and differentiate into antibody-secreting plasma cells and long-lived memory cells (fourth panels).

1	majority of developing lymphocytes die before emerging from the central lymphoid organs or before completing maturation in the peripheral lymphoid organs.

1	In this chapter we describe the different stages of the development of B cells and T cells in mice and humans from the uncommitted stem cell in the bone marrow up to the mature, functionally specialized lymphocyte with its unique antigen receptor ready to respond to a foreign antigen. The final stages in the life history of a mature lymphocyte, in which an encounter with its antigen activates it to become an effector or memory lymphocyte, are discussed in Chapters 9–11. We now know that the Band T-cell development that predominates during late fetal life and after birth is distinct from waves of lymphocyte development that take place earlier in fetal ontogeny. These earlier waves originate from stem cells found in the fetal liver and in even more primitive hematopoietic tissues in the developing embryo. Unlike the lymphocytes that develop from bone marrow stem cells, B and T cells that develop from these early fetal progenitors generally populate mucosal and epithelial tissues and

1	developing embryo. Unlike the lymphocytes that develop from bone marrow stem cells, B and T cells that develop from these early fetal progenitors generally populate mucosal and epithelial tissues and function in innate immune responses. In the adult, these subsets of lymphocytes are minority populations in secondary lymphoid tissues. This chapter will focus on B and T cells that develop from bone marrow stem cells and that comprise the cells of the adaptive immune response (see Figs 1.7 and 1.20). The chapter is divided into three parts. The first two describe B-cell and T-cell development, respectively. In the third section, we discuss the positive and negative selection of T cells in the thymus.

1	Development of B lymphocytes. The main phases of a B lymphocyte’s life history are shown in Fig. 8.1. The stages in both B-cell and T-cell development are defined mainly by the successive steps in the assembly and expression of functional antigen-receptor genes. Generation of B-cell receptors in the bone marrow Negative selection in the bone marrow Migration of B cells through the circulatory system to lymphoid organs and B-cell activation

1	Generation of B-cell receptors in the bone marrow Negative selection in the bone marrow Migration of B cells through the circulatory system to lymphoid organs and B-cell activation At each step of lymphocyte development, the progress of gene rearrangement is monitored; the major recurring theme is that successful gene rearrangement leads to the production of a protein chain that serves as a signal for the cell to progress to the next stage. We will see that a developing B cell is presented with opportunities for multiple rearrangements that increase the likelihood of expressing a functional antigen receptor, but that there are also checkpoints that reinforce the requirement that each B cell express just one receptor specificity. We will start by looking at how the earliest recognizable cells of the B-cell lineage develop from the multipotent hematopoietic stem cells in the bone marrow, and at what point the B-cell and T-cell lineages diverge.

1	8-1 Lymphocytes derive from hematopoietic stem cells in the bone marrow. The cells of the lymphoid lineage—B cells, T cells, and innate lymphoid cells (ILCs)—are all derived from common lymphoid progenitor cells, which themselves derive from the multipotent hematopoietic stem cells (HSCs) that give rise to all blood cells (see Fig. 1.3). Development from the precursor stem cell into cells that are committed to becoming B cells or T cells follows the basic principles of cell differentiation. Properties that are essential for the function of the mature cell are gradually acquired, along with the loss of properties that are more characteristic of the immature cell. In the case of lymphocyte development, cells become committed first to the lymphoid lineage, as opposed to the myeloid, and then to either the B-cell or the T-cell lineage (Fig. 8.2).

1	The specialized microenvironment of the bone marrow provides signals both for the development of lymphocyte progenitors from hematopoietic stem cells and for the subsequent differentiation of B cells. Such signals act on the developing lymphocytes to switch on key genes that direct the developmental program and are produced by the network of specialized nonlymphoid connective tissue stromal cells that are in intimate contact with the developing lymphocytes (Fig. 8.3). The contribution of the stromal cells is twofold. First, they form specific adhesive contacts with the developing lymphocytes by interactions between cell-adhesion molecules and their ligands. Second, they provide soluble and membrane-bound cytokines and chemokines that control lymphocyte differentiation and proliferation.

1	The hematopoietic stem cells first differentiate into multipotent progenitor cells (MPPs), which can produce both lymphoid and myeloid cells but are no longer self-renewing stem cells. Multipotent progenitors express a cell-surface receptor tyrosine kinase known as FLT3 that binds the membrane-bound FLT3 ligand on stromal cells. Additionally, MPPs express transcription factors and

1	Fig. 8.2 A multipotent hematopoietic stem cell generates all the cells of the immune system. In the bone marrow or other hematopoietic sites, the multipotent stem cell gives rise to cells with progressively more limited potential. A simplified progression is shown here. The multipotent progenitor (MPP), for example, has lost its stem-cell properties. The first branch leads to cells with myeloid and erythroid potential, on the one hand (CMPs and MEPs), and, on the other, to the common lymphoid progenitors (CLPs), with lymphoid potential. The former give rise to all nonlymphoid cellular blood elements, including circulating monocytes and granulocytes, as well as the macrophages and dendritic cells that reside in tissues and peripheral lymphoid organs (not shown). The CLP population is heterogeneous and single cells can give rise to NK cells, T cells, or B cells through successive stages of differentiation in either the bone marrow or thymus. There may be considerable plasticity in these

1	and single cells can give rise to NK cells, T cells, or B cells through successive stages of differentiation in either the bone marrow or thymus. There may be considerable plasticity in these pathways, in that in certain circumstances progenitor cells may switch their commitment. For example, a progenitor cell may give rise to either B cells or macrophages; however, for simplicity these alternative pathways are not shown. Some dendritic cells are also thought to be derived from the lymphoid progenitor.

1	Fig. 8.3 The early stages of B-cell development are dependent on bone marrow stromal cells. Interaction of B-cell progenitors with bone marrow stromal cells is required for development to the immature B-cell stage. The designations pro-B cell and pre-B cell refer to defined phases of B-cell development, as described in Fig. 8.4. Multipotent progenitor cells express the receptor tyrosine kinase FLT3, which binds to its ligand on stromal cells. Signaling through FLT3 is required for differentiation to the next stage, the common lymphoid progenitor. The chemokine CXCL12 (SDF-1) acts to retain stem cells and lymphoid progenitors at appropriate stromal cells in the bone marrow. The receptor for interleukin-7 (IL-7) is present at this stage, and IL-7 produced by stromal cells is required for the development of B-lineage cells. Progenitor cells bind to the adhesion molecule VCAM-1 on stromal cells through the integrin VLA-4 and also interact through other cell-adhesion molecules (CAMs). The

1	development of B-lineage cells. Progenitor cells bind to the adhesion molecule VCAM-1 on stromal cells through the integrin VLA-4 and also interact through other cell-adhesion molecules (CAMs). The adhesive interactions promote the binding of the receptor tyrosine kinase Kit (CD117) on the surface of the pro-B cell to stem-cell factor (SCF) on the stromal cell, which activates the kinase and induces the proliferation of B-cell progenitors. The actions of the listed transcription factors in B-cell development are discussed in the text. The pink horizontal bands denote the expression of particular proteins at the indicated stages of development.

1	receptors that are required for the development of multiple hematopoietic lineages, such as the transcription factor PU.1 and the receptor c-kit. In the next stage, MPPs produce two subsets of progenitor cells that give rise to all the lymphocyte lineages. One progenitor cell, as yet unnamed, produces the ILC subsets, ILC1, ILC2, and ILC3 cells. A second progenitor cell arising from the MPP is known as the common lymphoid progenitor (CLP). Differentiation of MPPs into CLPs requires signaling through the FLT3 receptor expressed on MPPs. Progenitor cell transfer and lineage repopulation experiments have shown that the CLP population is actually heterogeneous and represents a continuum of cells with decreasing multipotent potential. A subset of CLP cells with the broadest potential is able to generate B cells, T cells, and NK cells. A second subset of CLPs is able to generate only B cells and T cells, and a third subset of CLPs is committed exclusively to the B-cell lineage.

1	is able to generate B cells, T cells, and NK cells. A second subset of CLPs is able to generate only B cells and T cells, and a third subset of CLPs is committed exclusively to the B-cell lineage. B-cell-committed CLPs give rise to pro-B cells (see Fig. 8.3).

1	The production of lymphocyte progenitors from the multipotent progenitor cell is accompanied by expression of the receptor for interleukin-7 (IL-7), which is induced by FLT3 signaling together with the activity of PU.1. The cytokine IL-7, secreted by bone marrow stromal cells, is essential for the growth and survival of developing B cells in mice (but possibly not in humans). The IL-7 receptor is composed of two polypeptides, the IL-7 receptor α chain and the common cytokine receptor γ chain (γ-c), so called because it is also a subunit of five additional cytokine receptors. This family of cytokine receptors includes the receptors for IL-2, IL-4, IL-9, IL-15, and IL-21, in addition to IL-7. These receptors also share the tyrosine kinase Jak3, a signaling protein that binds exclusively to γ-c and is required for productive signaling by each of the receptors.

1	Due to the importance of IL-7 for murine B-cell development, mice with a genetic deficiency in IL-7, IL-7 receptor α, γ-c, or Jak3 all exhibit a severe block in B-cell development.

1	Another essential factor for B-cell development is stem-cell factor (SCF), a membrane-bound cytokine present on bone marrow stromal cells that stimulates the growth of hematopoietic stem cells and the earliest B-lineage progenitors. SCF interacts with the receptor tyrosine kinase Kit on the precursor cells (see Fig. 8.3). The chemokine CXCL12 (stromal cell-derived factor 1, SDF-1) is also essential for the early stages of B-cell development. It is produced constitutively by bone marrow stromal cells, and one of its roles may be to retain developing B-cell precursors in the marrow microenvironment. Thymic stroma-derived lymphopoietin (TSLP) resembles IL-7 and binds a receptor that includes the IL-7 receptor αchain, but not γ-c. Despite its name, TSLP may promote B-cell development in the embryonic liver and, in the perinatal period at least, in the mouse bone marrow.

1	A definitive B-cell stage, the pro-B cell, is specified by induction of the B-lineage-specific transcription factor E2A. It is not clear what initiates the expression of E2A in some progenitors, but it is known that the transcription factors PU.1 and Ikaros are required for E2A expression. E2A then induces the expression of the early B-cell factor (EBF). IL-7 signaling promotes the survival of these committed progenitors, while E2A and EBF act together to drive the expression of proteins that determine the pro-B-cell state.

1	As B-lineage cells mature, they migrate within the marrow, remaining in contact with the stromal cells. The earliest stem cells lie in a region called the endosteum, which lines the inner cavity of the long bones such as the femur and tibia. Developing B-lineage cells make contact with reticular stromal cells in the trabecular spaces, and as they mature they move toward the central sinus of the marrow cavity. The final stages of development of immature B cells into mature B cells occur in peripheral lymphoid organs such as the spleen, which we describe in Sections 8-7 and 8-8 of this chapter. 8-2 B-cell development begins by rearrangement of the heavy-chain locus.

1	The stages of B-cell development are, in the order they occur, early pro-B cell, late pro-B cell, large pre-B cell, small pre-B cell, immature B cell, and mature B cell (Fig. 8.4). Rearrangement of the heavy-chain locus is initiated in the pro-B cell when E2A and EBF induce the expression of several key proteins that enable gene rearrangement to occur, including the RAG-1 and RAG-2 components of the V(D)J recombinase (see Chapter 5). Only one gene locus is rearranged at a time, in a fixed sequence. The first rearrangement to take place is the joining of a D gene segment to a J segment at the immunoglobulin heavy-chain (IgH) locus. D to JH rearrangement takes place mostly in the early pro-B-cell stage, but can be seen as early as the common lymphoid progenitor. In the absence of E2A or EBF this initial rearrangement event fails to occur. Another key protein induced by E2A and EBF is the transcription factor Pax5, one isoform of which is known as the B-cell activator protein (BSAP) (see

1	this initial rearrangement event fails to occur. Another key protein induced by E2A and EBF is the transcription factor Pax5, one isoform of which is known as the B-cell activator protein (BSAP) (see Fig. 8.3). Among the targets of Pax5 are the gene for the B-cell co-receptor component CD19 and the gene for Igα, a signaling component of both the pre-B-cell receptor and the B-cell receptor (see Section 7-7). In the absence of Pax5, pro-B cells fail to develop further down the B-cell pathway but can be induced to give rise to T cells and myeloid cell types, indicating that Pax5 is required for commitment of the pro-B cell to the B-cell lineage. Pax5 also induces the expression of the B-cell linker protein (BLNK), an SH2-containing scaffold protein that is required for further development of the pro-B cell and for signaling from the mature B-cell antigen receptor lgD Stem cell Early pro-B cell Late pro-B cell Immature B cell Mature B cell Small pre-B cell Large pre-B cell L-chain genes

1	the pro-B cell and for signaling from the mature B-cell antigen receptor lgD Stem cell Early pro-B cell Late pro-B cell Immature B cell Mature B cell Small pre-B cell Large pre-B cell L-chain genes Surface Ig Absent Absent Absent Intracellular ˜ chain IgM expressed on cell surface Germline Germline GermlineGermline VJ rearranged VJ rearrangedV–J rearranging H-chain genes Germline D–J rearranging V–DJ rearranging VDJ rearranged VDJ rearranged VDJ rearranged VDJ rearranged lgM lgMpre-B receptor BP-1 Amino-peptidase CD19 Unknown CD43 CD24 Growth factor receptorIL-7R Kit TdT ˛5 Ig˝ VpreB Ig˙ CD45R Btk RAG-1 RAG-2 N-nucleotide addition Protein Function Surrogate light-chain components Lymphoid-speciÿc recombinase Signal transduction ˜ chain transiently at surface as part of pre-B-cell receptor. Mainly intracellular IgD and IgM made from alternatively spliced H-chain transcripts

1	Fig. 8.4 The development of a B-lineage cell proceeds through several stages marked by the rearrangement and expression of the immunoglobulin genes. The stem cell has not yet begun to rearrange its immunoglobulin (Ig) gene segments; they are in the germline configuration found in all nonlymphoid cells. The heavy-chain (H-chain) locus rearranges first. Rearrangement of a D gene segment to a JH gene segment starts in the common lymphoid progenitor and occurs mostly in early pro-B cells, generating late pro-B cells in which VH to DJH rearrangement occurs. A successful VDJH rearrangement leads to the expression of a complete immunoglobulin heavy chain as part of the pre-B-cell receptor, which signals via Igα, Igβ, and Btk (see Fig 7.27). Once this occurs, the cell is stimulated to become a large pre-B cell, which proliferates to become small resting pre-B cells; at this point the cells cease expression of the surrogate light chains (λ5 and VpreB) and express the μ heavy chain alone in the

1	pre-B cell, which proliferates to become small resting pre-B cells; at this point the cells cease expression of the surrogate light chains (λ5 and VpreB) and express the μ heavy chain alone in the cytoplasm. Small pre-B cells reexpress the RAG proteins and start to rearrange the light-chain (L-chain) genes. Upon successfully assembling a light-chain gene, a cell becomes an immature B cell that expresses a complete IgM molecule at the cell surface, which also signals via Igα and Igβ. Mature B cells produce a δ heavy chain as well as a μ heavy chain, by a mechanism of alternative mRNA splicing (see Fig. 5.17), and are marked by the additional appearance of IgD on the cell surface. All stages through the development of immature B cells takes place in the bone marrow; the final maturation to IgM+IgD+ mature B cells occurs in the spleen. The earliest B-lineage surface markers are CD19 and CD45R (B220 in the mouse), which are expressed throughout B-cell development. A pro-B cell is also

1	to IgM+IgD+ mature B cells occurs in the spleen. The earliest B-lineage surface markers are CD19 and CD45R (B220 in the mouse), which are expressed throughout B-cell development. A pro-B cell is also distinguished by the expression of CD43 (a marker of unknown function), Kit (CD117), and the IL-7 receptor. A late pro-B cell starts to express CD24 (a marker of unknown function). A pre-B cell is phenotypically distinguished by the expression of the enzyme BP-1, whereas Kit is no longer expressed.

1	(see Section 7-20). The temporal expressions of some of the transcription factors, surface proteins, and receptors required for B-cell development are listed in Fig. 8.3 and Fig. 8.4. Although the V(D)J recombinase system operates in both Band T-lineage cells and uses the same core enzymes, rearrangements of T-cell receptor genes do not occur in B-lineage cells, nor do complete rearrangements of immunoglobulin genes occur in T cells. The ordered rearrangement events that do occur are associated with lineage-specific low-level transcription of the gene segments about to be joined.

1	The initial D to JH rearrangements in the immunoglobulin heavy-chain locus (Fig. 8.5) typically occur on both alleles, at which point the cell becomes a late pro-B cell. Most D to JH joins in humans are potentially useful, because most human D gene segments can be translated in all three reading frames without encountering a stop codon. Thus, there is no need for a special mechanism to distinguish successful D to JH joints, and at this early stage there is also no need to ensure that only one allele undergoes rearrangement. Indeed, given the likely rate of failure at later stages, starting off with two successfully rearranged D–JH sequences may be an advantage.

1	Fig. 8.5 A productively rearranged immunoglobulin gene is immediately expressed as a protein by the developing B cell. In early pro-B cells, heavy-chain gene rearrangement is initiated with D to JH rearrangements. As shown in the top panels, no functional μ protein is expressed, although transcription occurs (red arrow). In late pro-B cells, VH to DJH rearrangement occurs on one chromosome first. If no functional H-chain is produced, VH to DJH rearrangement occurs on the second chromosome. As soon as a productive heavy-chain gene rearrangement takes place, μ chains are expressed by the cell in a complex with two other chains, λ5 and VpreB, which together make up a surrogate light chain. The whole immunoglobulin-like complex is known as the pre-B-cell receptor (center panels). It is associated with two other protein chains, Igα and Igβ, which signal the B cell to halt heavy-chain gene rearrangement; this drives the transition to the large pre-B-cell stage by inducing proliferation.

1	with two other protein chains, Igα and Igβ, which signal the B cell to halt heavy-chain gene rearrangement; this drives the transition to the large pre-B-cell stage by inducing proliferation. Failure to produce a functional H-chain leading to a pre-B-cell receptor signal leads to cell death. The progeny of large pre-B cells stop dividing and become small pre-B cells, in which light-chain gene rearrangements commence. Vκ–Jκ rearrangement (see Section 5-2) occurs first, and if unsuccessful, Vλ to Jλ rearrangement occurs next. Successful light-chain gene rearrangement results in the production of a light chain that binds the μ chain to form a complete IgM molecule, which is expressed together with Igα and Igβ at the cell surface, as shown in the bottom panels. Signaling via this surface receptor complex is thought to trigger the cessation of light-chain gene rearrangement. Failure to produce a functional L chain leads to cell death.

1	To produce a complete immunoglobulin heavy chain, the late pro-B cell now proceeds with a rearrangement of a VH gene segment to a DJH sequence. In contrast to D to JH rearrangement, VH to DJH rearrangement occurs first on only one chromosome. A successful rearrangement leads to the production of intact μheavy chains, after which VH to DJH rearrangement ceases and the cell becomes a pre-B cell. Pro-B cells that do not produce a μ chain are eliminated, as they fail to receive an important survival signal mediated by the pre-B-cell receptor (see Section 8-3). At least 45% of pro-B cells are lost at this stage. In at least two out of three cases, the first VH to DJH rearrangement is nonproductive as each amino acid is encoded by a triplet of nucleotides. When this initial rearrangement is out of frame, rearrangement then occurs on the other chromosome, again with a theoretical two in three chance of failure. A rough estimate of the chance of generating a pre-B cell is thus 55% [1/3 + (2/3

1	of frame, rearrangement then occurs on the other chromosome, again with a theoretical two in three chance of failure. A rough estimate of the chance of generating a pre-B cell is thus 55% [1/3 + (2/3 × 1/3) = 0.55]. The actual frequency is somewhat lower, because the V gene segment repertoire contains pseudogenes that can rearrange yet have major defects that prevent the expression of a functional protein. An initial nonproductive rearrangement does not automatically lead to pro-B cell elimination, as it is possible for most loci to undergo successive rearrangements on the same chromosome, and where that fails, the locus on the other chromosome will rearrange.

1	The diversity of the B-cell antigen-receptor repertoire is enhanced at this stage by the enzyme terminal deoxynucleotidyl transferase (TdT). TdT is expressed by the pro-B cell and adds nontemplated nucleotides (N-nucleotides) at the joints between rearranged gene segments (see Section 5-8). In adult humans, it is expressed in pro-B cells during heavy-chain gene rearrangement, but its expression declines at the pre-B-cell stage during light-chain gene rearrangement. This explains why N-nucleotides are found in the V–D and D–J joints of nearly all heavy-chain genes but only in about a quarter of human light-chain joints. N-nucleotides are rarely found in mouse light-chain V–J joints, showing that TdT is switched off slightly earlier in the development of mouse B cells. In fetal development, when the peripheral immune system is first being supplied with T and B lymphocytes, TdT is expressed only at low levels, if at all.

1	8-3 The pre-B-cell receptor tests for successful production of a complete heavy chain and signals for the transition from the pro-B cell to the pre-B cell stage.

1	The imprecise nature of V(D)J recombination is a double-edged sword. Although it produces increased diversity in the antibody repertoire, it also results in many unsuccessful rearrangements. Pro-B cells therefore need a way of testing whether a potentially functional heavy chain has been produced. They do this by incorporating a functional heavy chain into a receptor that can signal its successful production. This test takes place in the absence of light chains, whose loci have not yet rearranged. Instead, pro-B cells make two invariant ‘surrogate’ proteins that together have a structural resemblance to the light chain and can pair with the μ chain to form the pre-B-cell receptor (pre-BCR) (see Fig. 8.5). The assembly of a pre-B-cell receptor signals to the B cell that a productive rearrangement has been made, and the cell is then considered a pre-B cell.

1	The surrogate chains are encoded by nonrearranging genes separate from the antigen-receptor loci, and their expression is induced by E2A and EBF (see Fig. 8.4). One is called λ5 because of its close resemblance to the C domain of the λ light chain; the other, called VpreB, resembles a light-chain V domain but has an extra region at the amino-terminal end. Pro-B cells and pre-B cells also express the invariant proteins Igα and Igβ, introduced in Chapter 7 as the signaling components of the B-cell receptor complex on mature B cells. As components of the pre-B-cell receptor, Igα and Igβ transduce signals by interacting with intracellular tyrosine kinases through their cytoplasmic tails, just Amino-terminal tails on VpreB and ˜5 in adjacent pre-B-cell receptor molecules bind each other and cross-link the receptors, inducing clustering and signaling as they function to transduce signals through the antigen receptor on mature B cells (see Section 7-7).

1	Formation of the pre-B-cell receptor and signaling through this receptor provide an important checkpoint that mediates the transition between the pro-B cell and the pre-B cell. In mice that either lack λ5 or have mutant heavy-chain genes that cannot produce the transmembrane domain, the pre-B-cell receptor cannot be formed and B-cell development is blocked after heavy-chain gene rearrangement. In normal B-cell development, the pre-B-cell receptor complex is expressed transiently, perhaps because the production of λ5 mRNA stops as soon as pre-B-cell receptors begin to be formed. Although present at only low levels on the cell surface, the pre-B-cell receptor generates signals required for the transition from pro-B cell to pre-B cell. No antigen or other external ligand seems to be involved in signaling by the receptor. Instead, pre-B-cell receptors are thought to interact with each other, forming dimers or oligomers that generate signals as described in Section 7-16. Dimerization

1	in signaling by the receptor. Instead, pre-B-cell receptors are thought to interact with each other, forming dimers or oligomers that generate signals as described in Section 7-16. Dimerization involves ‘unique’ regions in the amino termini of λ5 and VpreB proteins that are not present in other immunoglobulin-like domains and which mediate the cross-linking of adjacent pre-B-cell receptors on the cell surface (Fig. 8.6). PreB-cell receptor signaling requires the scaffold protein BLNK and Bruton’s tyrosine kinase (Btk), an intracellular Tec-family tyrosine kinase (see Section 7-20). In humans and mice, deficiency of BLNK leads to a block in B-cell development at the pro-B-cell stage. In humans, mutations in the BTK gene cause a profound B-lineage-specific immune deficiency, Bruton’s X-linked agammaglobulinemia (XLA), in which no mature B cells are produced. The block in B-cell development caused by mutations in BTK is almost total, interrupting the transition from pre-B cell to

1	X-linked agammaglobulinemia (XLA), in which no mature B cells are produced. The block in B-cell development caused by mutations in BTK is almost total, interrupting the transition from pre-B cell to immature B cell. A similar, but less severe, defect called X-linked immunodeficiency, or xid, arises from mutations in the Btk gene in mice.

1	8-4 Pre-B-cell receptor signaling inhibits further heavy-chain locus rearrangement and enforces allelic exclusion. The signaling generated by pre-B-cell receptor clustering halts further rearrangement of the heavy-chain locus and allows the pro-B cell to become sensitive to IL-7. This induces cell proliferation, initiating the transition to the large

1	Fig. 8.6 The pre-B-cell receptor initiates signaling through spontaneous dimerization induced by the unique regions of VpreB and λ5. Two surrogate protein chains, VpreB (orange) and λ5 (green), substitute for a light chain and bind to a heavy chain, thus allowing its surface expression. VpreB substitutes for the light-chain V region in this surrogate interaction, while λ5 takes the part of the light-chain constant region. Both VpreB and λ5 contain ‘unique’ amino-terminal regions that are not present in other immunoglobulin-like domains, shown here as unstructured tails extending out from the globular domains. These amino-terminal regions associated with one pre-B-cell receptor can interact with the corresponding regions on the adjacent pre-B-cell receptor, promoting the spontaneous formation of pre-Bcell receptor dimers on the cell surface. Dimerization generates signaling from the pre-B-cell receptor that is dependent on the presence of the ITAM-containing signaling chains Igα and

1	of pre-Bcell receptor dimers on the cell surface. Dimerization generates signaling from the pre-B-cell receptor that is dependent on the presence of the ITAM-containing signaling chains Igα and Igβ. The signals cause the inhibition of RAG-1 and RAG-2 expression and the proliferation of the large pre-B cell. Courtesy of Chris Garcia.

1	Fig. 8.7 Allelic exclusion in individual B cells. Most species have genetic polymorphisms of the constant regions of their immunoglobulin heavy-chain and light-chain genes; these polymorphisms lead to amino acid differences between the encoded proteins. These variants of heavy-chain or light-chain proteins expressed by different individuals in a species are known as allotypes. In rabbits, for example, all of the B cells in an individual homozygous for the a allele of the immunoglobulin heavy-chain locus (Igha/a) will express immunoglobulin of allotype a, whereas in an individual homozygous for the b allele (Ighb/b) all the B cells make immunoglobulin of allotype b. In a heterozygous animal (Igha/b), which carries the a allele at one of the Igh loci and the b allele at the other, individual B cells can be shown to express surface immunoglobulin of either the a-allotype or the b-allotype, but not both (bottom panel). This allelic exclusion reflects the productive rearrangement of only

1	B cells can be shown to express surface immunoglobulin of either the a-allotype or the b-allotype, but not both (bottom panel). This allelic exclusion reflects the productive rearrangement of only one of the two Igh alleles in the B cell, because the production of a successfully rearranged immunoglobulin heavy chain forms a pre-Bcell receptor, which signals the cessation of further heavy-chain gene rearrangement.

1	pre-B cell. Successful rearrangements at both heavy-chain alleles could result in a B cell producing two receptors of different antigen specificities. To prevent this, signaling by the pre-B-cell receptor enforces allelic exclusion, the state in which only one of the two alleles of a gene is expressed in a diploid cell. Allelic exclusion, which occurs at both the heavy-chain locus and the light-chain loci, was discovered nearly 50 years ago and provided one of the original pieces of experimental support for the theory that one lymphocyte expresses one type of antigen receptor (Fig. 8.7).

1	Signaling from the pre-B-cell receptor promotes heavy-chain allelic exclusion in three ways. First, it reduces V(D)J recombinase activity by directly reducing the expression of the RAG-1 and RAG-2 genes. Second, it further reduces levels of RAG-2 by indirectly causing this protein to be targeted for degradation, which occurs when RAG-2 is phosphorylated in response to the entry of the pro-B cell into S phase (the DNA synthesis phase) of the cell cycle. Finally, preB-cell receptor signaling reduces access of the heavy-chain locus to the recombinase machinery, although the precise details of this are not clear. At a later stage of B-cell development, RAG proteins will again be expressed in order to carry out light-chain locus rearrangement, but at that point the heavy-chain locus does not undergo further rearrangement. In the absence of pre-B-cell receptor signaling, allelic exclusion of the heavy-chain locus does not occur. Since a second important role of pre-B-cell receptor signaling

1	further rearrangement. In the absence of pre-B-cell receptor signaling, allelic exclusion of the heavy-chain locus does not occur. Since a second important role of pre-B-cell receptor signaling is to stimulate proliferative expansion of B-cell precursors with a successful heavy-chain rearrangement, a deficiency in this signal causes a profound reduction in the numbers of pre-B cells and mature B cells that develop.

1	8-5 Pre-B cells rearrange the light-chain locus and express cell-surface immunoglobulin. The transition from the pro-B-cell to the large pre-B-cell stage is accompanied by several rounds of cell division, expanding the population of cells with successful in-frame joins by about 30to 60-fold before they become resting small pre-B cells. A large pre-B cell with a particular rearranged heavy-chain gene therefore gives rise to numerous small pre-B cells. RAG proteins are produced again in the small pre-B cells, and rearrangement of the light-chain locus begins. Each of these cells can make a different rearranged light-chain gene, and so cells with many different antigen specificities are generated from a single pre-B cell, which makes an important contribution to overall B-cell receptor diversity.

1	Light-chain rearrangement also exhibits allelic exclusion. Rearrangements at the light-chain locus generally take place at only one allele at a time, a process regulated by a mechanism not currently understood. The light-chain loci lack D segments, and rearrangement occurs by V to J joining; and if a particular VJ rearrangement fails to produce a functional light chain, repeated rearrangements of unused V and J gene segments at the same allele can occur (Fig. 8.8). Several attempts at productive rearrangement of a light-chain gene can therefore be made on one chromosome before initiating any rearrangements on the second chromosome. This greatly increases the chances of eventually generating an intact light chain, especially as there are two different light-chain loci. As a result, many cells that reach the preB-cell stage succeed in generating progeny that bear intact IgM molecules and can be classified as immature B cells. Figure 8.4 lists some of the proteins involved in V(D)J

1	cells that reach the preB-cell stage succeed in generating progeny that bear intact IgM molecules and can be classified as immature B cells. Figure 8.4 lists some of the proteins involved in V(D)J recombination and shows how their expression is regulated throughout B-cell development. Figure 8.5 summarizes the stages of B-cell development up to the point of assembly of a complete surface immunoglobulin. Developing B cells that fail to assemble a complete surface immunoglobulin undergo apoptosis in the bone marrow, and are eliminated from the B-cell pool.

1	Fig. 8.8 Nonproductive light-chain gene rearrangements can be rescued by further rearrangement. The organization of the light-chain loci in mice and humans offers many opportunities for the rescue of pre-B cells that initially make an out-offrame rearrangement. Light-chain rescue is illustrated here at the human κ locus. If the first rearrangement is nonproductive, a 5’ Vκ gene segment can recombine with a 3’ Jκ gene segment to remove the out-of-frame join located between them and to replace it with a new rearrangement. In principle, this can happen up to five times on each chromosome, because there are five functional Jκ gene segments in humans. If all rearrangements of κ-chain genes fail to yield a productive light-chain join, λ-chain gene rearrangement may succeed (not shown).

1	As well as allelic exclusion, light chains also display isotypic exclusion, that is, the expression of only one type of light chain—κ or λ—by an individual B cell. Again, the mechanism regulating this process is not known. In mice and humans, the κ light-chain locus tends to rearrange before the λ locus. This was first deduced from the observation that myeloma cells secreting λ light chains generally have both their κ and λ light-chain genes rearranged, whereas in myelomas secreting κ light chains, generally only the κ genes are rearranged. This order is occasionally reversed, however, and λ gene rearrangement does not absolutely require the previous rearrangement of the κ genes. The ratios of κ-expressing versus λ-expressing mature B cells vary from one extreme to the other in different species. In mice and rats it is 95% κ to 5% λ, in humans it is typically 65%:35%, and in cats it is 5%:95%, the opposite of that in mice. These ratios correlate most strongly with the number of

1	species. In mice and rats it is 95% κ to 5% λ, in humans it is typically 65%:35%, and in cats it is 5%:95%, the opposite of that in mice. These ratios correlate most strongly with the number of functional Vκ and Vλ gene segments in the genome of the species. They also reflect the kinetics and efficiency of gene segment rearrangements. The κ:λ ratio in the mature lymphocyte population is useful in clinical diagnostics, because an aberrant κ:λ ratio indicates the dominance of one clone and the presence of a lymphoproliferative disorder.

1	8-6 Immature B cells are tested for autoreactivity before they leave the bone marrow. Once a rearranged light chain has paired with a μ chain, IgM can be expressed on the cell surface (as a surface IgM, or sIgM) and the pre-B cell becomes an immature B cell. At this stage, the antigen receptor is first tested for reactivity to self antigens, or autoreactivity. The elimination or inactivation of autoreactive B cells ensures that the B-cell population as a whole will be tolerant of self antigens. The tolerance produced at this stage of B-cell development is known as central tolerance because it arises in a central lymphoid organ, the bone marrow. However, B cells leaving the bone marrow are not fully mature and require additional maturation steps that take place in peripheral lymphoid organs (see Section 8-8). As we shall see later in the chapter and in Chapter 15, self-reactive B cells that escape central tolerance may still be removed from

1	Fig. 8.9 Binding to self molecules in the bone marrow can lead to the death or inactivation of immature B cells.

1	First panels: immature B cells that do not encounter antigen mature normally; they migrate from the bone marrow to the peripheral lymphoid tissues, where they may become mature recirculating B cells bearing both IgM and IgD on their surface. Second panels: when developing B cells express receptors that recognize multivalent ligands, for example, ubiquitous cell-surface self molecules such as those of the MHC, these receptors are deleted from the repertoire. The B cells either undergo receptor editing (see Fig. 8.10), thereby eliminating the self-reactive receptor, or the cells themselves undergo programmed cell death (apoptosis), resulting in clonal deletion. Third panels: immature B cells that bind soluble self antigens able to cross-link the B-cell receptor are rendered unresponsive to the antigen (anergic) and bear little surface IgM. They migrate to the periphery, where they express IgD but remain anergic; if in competition with other B cells in the periphery, anergic B cells fail

1	antigen (anergic) and bear little surface IgM. They migrate to the periphery, where they express IgD but remain anergic; if in competition with other B cells in the periphery, anergic B cells fail to receive survival signals and die. Fourth panels: immature B cells whose antigen is inaccessible to them, or which bind monovalent or soluble self antigens with low affinity, do not receive any signal and mature normally. Such cells are potentially self-reactive, however, and are said to be clonally ignorant because their ligand is present but is unable to activate them.

1	the repertoire after they have left the bone marrow, a process that takes place during the final peripheral stages of B-cell maturation, and is referred to as peripheral tolerance, described in Section 8-7.

1	sIgM associates with Igα and Igβ to form a functional B-cell receptor complex, and the fate of an immature B cell in the bone marrow depends on signals delivered from this receptor complex when it interacts with ligands in the environment. Igα signaling is particularly important in dictating the emigration of B cells from the bone marrow and/or their survival in the periphery: mice that express Igα with a truncated cytoplasmic domain that cannot signal show a fourfold reduction in the number of immature B cells in the marrow, and a hundredfold reduction in the number of peripheral B cells. The release of immature B cells from the bone marrow into the circulation is also dependent on their expression of S1PR1, a G-protein-coupled receptor that binds to the lipid ligand S1P and promotes cell migration towards the high concentrations of S1P that exist in the blood (see Section 8-27).

1	Immature B cells that have no strong reactivity to self antigens continue to mature (Fig. 8.9, first panel). They leave the marrow via sinusoids that enter the central sinus, enter the circulation, and are carried by the venous blood supply to the spleen. If, however, the newly expressed receptor encounters a strongly cross-linking antigen in the bone marrow—that is, if the B cell is strongly self-reactive—development is arrested at this stage.

1	Experiments using genetically modified mice that enforce the expression of self-reactive B-cell receptors have shown that there are four possible fates for self-reactive immature B cells (see Fig. 8.9, last three panels). These fates are the production of a new receptor by a process known as receptor editing; cell death by apoptosis, resulting in clonal deletion; the induction of a permanent state of unresponsiveness to antigen, or anergy; and a state of immunological ignorance in which antigen concentrations are too low to stimulate B-cell receptor signaling. The outcome for each self-reactive B cell is dependent on the interaction of the B-cell receptor with the self antigen.

1	Fig. 8.10 Replacement of light chains by receptor editing can rescue some self-reactive B cells by changing their antigen specificity. When a developing B cell expresses antigen receptors that are strongly cross-linked by multivalent self antigens such as MHC molecules on cell surfaces (top panel), its development is arrested. The cell decreases surface expression of IgM and does not turn off the RAG genes (second panel). Continued synthesis of RAG proteins allows the cell to continue light-chain gene rearrangement. This usually leads to a new productive rearrangement and the expression of a new light chain, which combines with the previous heavy chain to form a new receptor; the process is called receptor editing (third panel). If this new receptor is not self-reactive, the cell is ‘rescued’ and continues normal development, much like a cell that had never reacted with self antigen (bottom right panel). If the cell remains self-reactive, it may be rescued by another cycle of

1	is ‘rescued’ and continues normal development, much like a cell that had never reacted with self antigen (bottom right panel). If the cell remains self-reactive, it may be rescued by another cycle of rearrangement; however, if it continues to react strongly with self antigen, it will undergo apoptosis, resulting in clonal deletion from the repertoire of B cells (bottom left panel).

1	Immature B cells that express an autoreactive receptor recognizing a multivalent self antigen can be rescued by further gene rearrangements that replace the autoreactive receptor with a new receptor that is not self-reactive. This mechanism is termed receptor editing (Fig. 8.10). When an immature B cell first produces sIgM, RAG proteins are still being made. If the receptor is not self-reactive, the absence of sIgM cross-linking allows gene rearrangement to cease and B-cell development continues, with RAG proteins eventually disappearing. For an autoreactive receptor, however, an encounter with the self antigen results in strong cross-linking of sIgM; RAG expression continues, and light-chain gene rearrangement can continue, as described in Fig. 8.8. These secondary rearrangements can rescue immature self-reactive B cells by deleting the self-reactive light-chain gene and replacing it with another sequence. If the new light chain is not autoreactive, the B cell continues normal

1	rescue immature self-reactive B cells by deleting the self-reactive light-chain gene and replacing it with another sequence. If the new light chain is not autoreactive, the B cell continues normal development. If the receptor remains autoreactive, rearrangement continues until a non-autoreactive receptor is produced or until no additional light-chain V and J gene segments are available for recombination. The importance of receptor editing as a mechanism of tolerance is well established, as defects in this process contribute to the human autoimmune diseases systemic lupus erythematosus and rheumatoid arthritis, two diseases characterized by high levels of autoreactive antibodies (see Chapter 15).

1	It was originally thought that the successful production of a heavy chain and a light chain caused the almost instantaneous shutdown of light-chain locus rearrangement and that this ensured both allelic and isotypic exclusion. The unexpected ability of self-reactive B cells to continue to rearrange their light-chain genes, even after having made a productive rearrangement, suggests an alternative mechanism of allelic exclusion, where the fall in the level of RAG proteins that follows a successful non-autoreactive rearrangement could be the principal means by which light-chain rearrangement is terminated. It is now apparent that allelic exclusion is not absolute, as there are rare B cells that express two different light chains.

1	Cells that remain autoreactive when receptor editing efforts fail to generate a non-autoreactive receptor undergo a process known as clonal deletion, in which they are subjected to cell death by apoptosis to eliminate their specific autoreactivity from the repertoire. Early experiments using transgenic mice expressing both chains of an immunoglobulin specific for H-2Kb MHC class I molecules, in which nearly all developing B cells expressed the anti-MHC immunoglobulin as sIgM, suggested that clonal deletion was a predominant mechanism of B-cell tolerance. These studies found that transgenic mice not expressing H-2Kb had normal numbers of B cells, all bearing the transgene-encoded anti-H-2Kb receptors. However, in mice expressing both H-2Kb and the immunoglobulin transgenes, B-cell development was blocked. Normal numbers of pre-B cells and immature B cells were found, but B cells expressing the anti-H-2Kb immunoglobulin as sIgM never matured to populate the spleen and lymph nodes;

1	was blocked. Normal numbers of pre-B cells and immature B cells were found, but B cells expressing the anti-H-2Kb immunoglobulin as sIgM never matured to populate the spleen and lymph nodes; instead, most of these immature B cells died in the bone marrow by apoptosis. However, more recent studies, using

1	Strong ligation of IgM by self antigen Arrest of B-cell development and continued light-chain rearrangement: low cell-surface IgM If the new receptor is still self-reactive, the B cell undergoes apoptosis If the new receptor is no longer self-reactive, the immature B cell migrates to the periphery and matures A new receptor speciÿcity is now expressed IgM mice bearing transgenes for autoantibody heavy and light chains that have been placed within the immunoglobulin loci by homologous recombination (see Appendix I, Section A-35, for details of this method), indicate that receptor editing, rather than clonal deletion, is the more likely outcome for immature autoreactive B cells.

1	We have so far discussed the fate of newly formed B cells that undergo multivalent cross-linking of their sIgM. Immature B cells that encounter more weakly cross-linking self antigens of low valence, such as small soluble proteins, respond differently. In this situation, some self-reactive B cells are inactivated and enter a state of permanent unresponsiveness, or anergy, but do not immediately die (see Fig. 8.9). Anergic B cells cannot be activated by their specific antigen even with help from antigen-specific T cells. Again, this phenomenon was elucidated using transgenic mice. Hen egg-white lysozyme (HEL) was expressed in soluble form from a transgene in mice that were also transgenic for high-affinity anti-HEL immunoglobulin. The HEL-specific B cells matured and emigrated from the bone marrow, but could not respond to antigen. Furthermore, the migration of anergic B cells is impaired, as the cells are detained in the T-cell areas of peripheral lymphoid tissues and are excluded

1	bone marrow, but could not respond to antigen. Furthermore, the migration of anergic B cells is impaired, as the cells are detained in the T-cell areas of peripheral lymphoid tissues and are excluded from lymphoid follicles, thereby reducing their life-span and their ability to compete with immunocompetent B cells (described further in Section 8-8). Under normal circumstances, where few self-reactive anergic B cells successfully mature, these cells die relatively quickly. This mechanism ensures that the long-lived pool of peripheral B cells is purged of potentially self-reactive cells.

1	The fourth potential fate of self-reactive immature B cells is that nothing happens to them; they remain in a state of immunological ignorance of their self antigen (see Fig. 8.9). Immunologically ignorant cells have affinity for a self antigen but for various reasons do not sense and respond to it. The antigen may not be accessible to developing B cells in the bone marrow or spleen, or may be in low concentration, or may bind so weakly to the B-cell receptor that it does not generate an activating signal. Because some ignorant cells can be (and in fact are) activated under certain conditions such as inflammation or when the self antigen becomes available or reaches an unusually high concentration, they should not be considered inert, and they are fundamentally different from cells with non-autoreactive receptors that could never be activated by self antigens.

1	The fact that central tolerance is not perfect and some self-reactive B cells are allowed to mature reflects the balance that the immune system strikes between purging all self-reactivity and maintaining the ability to respond to pathogens. If the elimination of self-reactive cells were too efficient, the receptor repertoire might become too limited and thus unable to recognize a wide variety of pathogens. Some autoimmune disease is the price of this balance: we shall see in Chapter 15 that ignorant self-reactive lymphocytes can be activated and cause disease under certain circumstances. Normally, however, ignorant B cells are held in check by a lack of T-cell help, the continued inaccessibility of the self antigen, or the tolerance that can be induced in mature B cells following their emigration from the bone marrow, which is described below. 8-7 Lymphocytes that encounter sufficient quantities of self antigens for the first time in the periphery are eliminated or inactivated.

1	While large numbers of autoreactive B cells are purged from the population of new lymphocytes in the bone marrow, only lymphocytes specific for autoantigens that are expressed in or can reach this organ are affected. Some antigens, like the thyroid product thyroglobulin, are highly tissue specific, or are compartmentalized so that little if any is available in the circulation. Therefore, newly emigrated self-reactive B cells that encounter their specific autoantigen for the first time in the periphery must be eliminated or inactivated also. This tolerance mechanism, which acts on newly emigrated B cells that are still immature, is known as peripheral tolerance. Like self-reactive lymphocytes in the central lymphoid organs, lymphocytes that encounter self antigens de novo in the periphery can have several fates: deletion, anergy, or survival (Fig. 8.11).

1	In the absence of an infection, newly emigrated B cells that encounter a strongly cross-linking antigen in the periphery will undergo clonal deletion. This was elegantly shown in studies of B cells expressing B-cell receptors specific for H-2Kb MHC class I molecules. These B cells are deleted even when, in transgenic animals, the expression of the H-2Kb molecule is restricted to the liver by the use of a liver-specific gene promoter. There is no receptor editing: B cells that encounter strongly cross-linking antigens in the periphery undergo apoptosis directly, unlike their counterparts in the bone marrow, which attempt further receptor rearrangements. This difference may be due to the fact that the B cells in the periphery are somewhat more mature and can no longer rearrange their light-chain loci.

1	As with immature B cells in the bone marrow, newly developed peripheral B cells that encounter and bind an abundant soluble antigen become unresponsive. This was demonstrated in mice by placing the HEL transgene under the control of an inducible promoter that can be regulated by changes in the diet. It is thus possible to induce the production of lysozyme at any time and thereby study its effects on HEL-specific B cells at different stages of maturation. These experiments have shown that both peripheral and immature bone marrow B cells are inactivated when they are chronically exposed to soluble antigen. 8-8 Immature B cells arriving in the spleen turn over rapidly and require cytokines and positive signals through the B-cell receptor for maturation and long-term survival.

1	Fig. 8.11 Transitional B cells that recognize self antigens undergo peripheral tolerance. After emigrating from the bone marrow and entering the circulation, immature B cells are known as transitional B cells. Not yet fully mature, these cells are still subject to tolerance in the spleen following engagement of their sIgM receptor by a self antigen. Transitional B cells that encounter a multivalent self antigen receive a strong B-cell receptor signal and undergo cell death. Transitional B cells with sIgM that binds to a soluble self molecule are rendered anergic, and ultimately die within a few days due to being excluded from the B-cell follicles in the spleen (see Fig. 8.12). Transitional B cells that bind with low affinity to a soluble self molecule remain clonally ignorant of the self antigen and continue their maturation. Transitional B cells with no self reaction also continue their maturation into mature B cells. The final stages of B-cell maturation lead to upregulation of

1	antigen and continue their maturation. Transitional B cells with no self reaction also continue their maturation into mature B cells. The final stages of B-cell maturation lead to upregulation of sIgD, and take place in the B-cell follicles in the spleen.

1	When B cells emerge from the bone marrow into the periphery, they are still functionally immature. As discussed above, their final maturation in the periphery Transitional B cells that enter the follicle receive maturation and survival signals and differentiate into follicular B cells or marginal zone B cells provides an opportunity for the immature B cells to encounter peripheral self antigens and to undergo tolerance. Immature B cells express high levels of sIgM but little sIgD, whereas mature B cells express low levels of IgM and high levels of IgD; while the changes in expression of sIgM and sIgD as B cells mature is well documented, the function of sIgD on mature B cells is not known.

1	Most immature B cells leaving the bone marrow will not survive to become fully mature B cells. Figure 8.12 shows the possible fates of newly produced B cells that enter the periphery. The daily output of new B cells from the bone marrow is roughly 5–10% of the total B-lymphocyte population in the steady-state peripheral pool. In unimmunized animals, the size of this pool seems to remain constant, due to homeostasis, which means that the stream of new B cells needs to be balanced by the removal of an equal number of peripheral B cells. However, the majority of peripheral mature B cells are long-lived, and only 1–2% of these die each day. Thus, most of the B cells that die are in the rapidly turning-over immature peripheral B-cell population, of which more than 50% die every 3 days. The failure of most newly formed B cells to survive for more than a few days in the periphery seems to be due to competition between peripheral B cells for access to the follicles in the spleen. If newly

1	failure of most newly formed B cells to survive for more than a few days in the periphery seems to be due to competition between peripheral B cells for access to the follicles in the spleen. If newly produced immature B cells do not enter a follicle, their passage through the periphery is halted and they eventually die. The limited number of lymphoid follicles cannot accommodate all of the B cells generated each day, and so there is continual competition for entry.

1	The follicle provides signals necessary for B-cell survival. In particular, the TNF-family member BAFF (for B-cell activating factor belonging to the TNF family) is made by several cell types, but is produced abundantly by the follicular dendritic cells (FDCs). FDCs are non-hematopoietic cells resident in the B-cell follicles that are specialized to capture antigens for recognition by B-cell antigen receptors (see Section 9-1). B cells express three different receptors for BAFF, namely BAFF-R, BCMA, and TACI. The BAFF-R is the most important for follicular B-cell survival, because mutant mice lacking BAFF-R have mainly immature B cells and few long-lived peripheral B cells. BCMA and TACI also bind the related TNF family cytokine APRIL, which is not required for the survival of immature B cells but is important for IgA antibody production, as we shall see in Chapter 10.

1	Fig. 8.12 Transitional B cells complete their maturation in B-cell follicles in the spleen. The micrograph in the top panel shows a cross-sectional view of a mouse spleen indicating the distribution of B cells (anti-B220, brown) and T cells (anti-CD3, blue), comprising the white pulp. Surrounding the B-cell-rich follicles (intense brown staining) are the marginal zones (also brown due to presence of B220+ B cells). The white pulp cords sit within the red pulp, which is rich in myeloid cells (mostly macrophages), plasma cells, and passing red blood cells. Transitional B cells that have left the bone marrow must complete their maturation in the B-cell follicles of the spleen, where they receive necessary maturation and survival signals (middle panel). One essential component is a low level of signaling through the B-cell receptor. A second essential factor is the expression of BAFF, a TNF-family member, on follicular dendritic cells (FDCs). BAFF stimulates the BAFF-R on transitional

1	of signaling through the B-cell receptor. A second essential factor is the expression of BAFF, a TNF-family member, on follicular dendritic cells (FDCs). BAFF stimulates the BAFF-R on transitional B cells, promoting B-cell survival. Newly emigrated transitional B cells (T1) exhibit high levels of surface IgM, little IgD, and the BAFF-R. In the B-cell follicles, these cells upregulate CD21 to become transitional stage 2 B cells (T2). Finally, the cells upregulate surface IgD, and become long-lived mature B cells. The majority of long-lived B cells are recirculating B cells, known as follicular B cells. A second, less numerous subset is the marginal zone B-cell population. Marginal zone B cells are thought to be weakly self-reactive and express very high levels of the complement receptor CD21. These cells migrate to the marginal zones of the splenic white pulp, an area at the white pulp/red pulp junctions. In this location, marginal zone B cells are poised to make rapid responses to

1	CD21. These cells migrate to the marginal zones of the splenic white pulp, an area at the white pulp/red pulp junctions. In this location, marginal zone B cells are poised to make rapid responses to blood-borne antigens or pathogens. Transitional T1 B cells that are excluded from the follicles fail to receive maturation and survival signals and will die within 2–3 days of leaving the bone marrow (bottom panel). Self-reactive anergic B cells are also excluded from the follicles and undergo cell death. Micrograph courtesy of Xiaoming Wang and Jason Cyster. Howard Hughes Medical Institute and Department of Microbiology and Immunology, UCSF.

1	Immature B cells in the spleen proceed through two distinct transitional stages, called T1 and T2, defined by the absence or presence of the B-cell co-receptor component CD21 (complement receptor 2) (see Section 2-13 and Section 7-20). In mice lacking BAFF, immature B cells progress to the T1 stage in the spleen but fail to express CD21, and the mice lack mature B cells. Signaling through the B-cell receptor is also required for immature B cells in the spleen to progress through the T1 and T2 stages and enter the long-lived peripheral B-cell pool. In this case, the B-cell receptor signals do not arise from high-affinity interactions between the sIgM of the B-cell receptor with an antigen, which would induce strong signals; instead, these B-cell receptor signals are thought to be weak, constitutive signals that are developmentally programmed into the maturing B cells, although the mechanism responsible for this constitutive signaling is not known. These weak B-cell receptor signals

1	constitutive signals that are developmentally programmed into the maturing B cells, although the mechanism responsible for this constitutive signaling is not known. These weak B-cell receptor signals together with the BAFF-R signals are essential to promote the final stages of B-cell maturation in the periphery. Disregulation of the appropriate balance between B-cell receptor and BAFF-R signaling occurs in individuals who overexpress BAFF, and has been linked to the development of autoimmune diseases, such as Sjögren’s syndrome, that result from a failure to purge auto-reactive B cells.

1	The majority of peripheral B cells that reside in the spleen and other secondary lymphoid organs are known as follicular B cells, often referred to as B-2 B cells. A second, minor population of B cells found in the spleen consists of marginal zone B cells, named for their predominance at the marginal zones that lie at the white pulp/red pulp junctions (see Fig. 8.12). Follicular and marginal zone B cells both derive from a common lineage that develops in the bone marrow and bifurcates during the final stage of B-cell maturation in the splenic follicles. Experiments reconstituting the signals promoting peripheral B-cell maturation in cell culture, starting from immature B-cell precursors, indicate that follicular versus marginal zone B-cell lineages diverge at the transitional T2 stage, as cells transition to the fully mature stage. Like follicular B cells, marginal zone B cells are dependent on BAFF signals, and are missing in mice lacking BAFF expression. Marginal zone B cells can be

1	cells transition to the fully mature stage. Like follicular B cells, marginal zone B cells are dependent on BAFF signals, and are missing in mice lacking BAFF expression. Marginal zone B cells can be identified by their expression of very high levels of the complement receptor CD21. Studies using rearranged immunoglobulin gene knock-in mice that express a single B-cell receptor specificity on all developing B cells have demonstrated that some B-cell receptors predominantly generate follicular B cells whereas others generate marginal zone B cells. These findings indicate that the specificity of the B-cell receptor is a major factor in determining the final commitment of transitional B cells to the follicular versus the marginal zone lineage; however, the details of this process are still not fully understood. Due to their location, marginal zone B cells are poised to make rapid responses to antigens or pathogens filtered from the blood. Therefore, it is thought that marginal zone B

1	not fully understood. Due to their location, marginal zone B cells are poised to make rapid responses to antigens or pathogens filtered from the blood. Therefore, it is thought that marginal zone B cells represent an early line of defense for blood-borne pathogens.

1	Peripheral B cells also include memory B cells, which are generated in addition to antibody-producing plasma cells from mature B cells after their first encounter with antigen; we will return to B-cell memory in Chapter 11. Competition for follicular entry favors mature B cells that are already established in the relatively long-lived and stable peripheral B-cell pool. Mature B cells have undergone phenotypic changes that might make their access to the follicles easier; for example, they express CXCR5, the receptor for CXCL13, which is expressed by FDCs (see Section 10-3). They also have increased expression of CD21 compared with immature newly developed B cells, which enhances the signaling capacity of the B cell.

1	The B-cell receptor plays a positive role in the maturation and continued recirculation of peripheral B cells. Mice that lack the tyrosine kinase Syk, which is involved in signaling from the B-cell receptor (see Section 7-20), have immature B cells but fail to develop mature B cells. Thus, a Syk-transduced signal may be required for final B-cell maturation or for the survival of mature B cells. Furthermore, continuous expression of the B-cell receptor is required for B-cell survival, as is evident from the loss of all B cells in mice whose BCR is conditionally deleted specifically in mature B cells. Although each B-cell receptor has a unique specificity, antigen-specific interactions may not induce the signals used for final B-cell maturation and survival; the receptor could, for example, be responsible for ‘tonic’ signaling, in which a weak but important signal is generated by the assembly of the receptor complex and infrequently triggers some or all of the downstream signaling

1	be responsible for ‘tonic’ signaling, in which a weak but important signal is generated by the assembly of the receptor complex and infrequently triggers some or all of the downstream signaling events.

1	8-9 B-1 B cells are an innate lymphocyte subset that arises early in development. Thus far, this chapter has focused on the development of the majority populations of B cells that reside in secondary lymphoid organs, such as follicular (B-2) B cells and marginal zone B cells. These two populations comprise the B-cell arm of the adaptive immune response. A third important subset of B cells, called B-1 B cells, is part of the innate immune system. These cells are present only in low numbers in secondary lymphoid organs, and are found in large numbers in the peritoneal and pleural cavities instead. B-1 B cells are the major source of ‘natural’ antibodies, which are constitutively produced circulating antibodies that are secreted by these B cells prior to any infections. Most antibodies made by B-1 B cells recognize capsular polysaccharide antigens, and B-1 B cells are important in controlling infections of pathogenic viruses and bacteria.

1	One important feature of B-1 B cells is that they can produce antibodies of the IgM class without ‘help’ from T cells. Although this response can be enhanced by T-cell cooperation, the antibodies first appear within 48 hours of exposure to antigen, when T cells cannot be involved. The lack of an antigen-specific interaction with helper T cells might explain why immunological memory is not generated as a result of B-1 cell responses: repeated exposures to the same antigen elicit similar, or decreased, responses with each exposure. While the precise functions of B-1 B cells are still not clear, mice deficient in B-1 cells are more susceptible to infection with Streptococcus pneumoniae because they fail to produce an anti-phosphocholine antibody that provides protection against this bacterium. Since a significant fraction of the B-1 cells can make antibodies of this specificity, and because no antigen-specific T-cell help is required, a potent response can be produced early in infection

1	Since a significant fraction of the B-1 cells can make antibodies of this specificity, and because no antigen-specific T-cell help is required, a potent response can be produced early in infection with this pathogen. Whether human B-1 cells have the same role is not certain.

1	Unlike follicular and marginal zone B cells that develop from bone marrow stem cells, the majority of B-1 B cells are generated from progenitor cells found in the fetal liver (Fig. 8.13). During late fetal and early neonatal stages in mice, B-1 B cells are produced in large numbers. After birth, the development of follicular and marginal zone B cells predominates, and few B-1 B cells are made. Current evidence indicates that the progenitor cells giving rise to B-1 B cells are committed to this lineage, and are distinct from those producing B-2 B cells. Whereas B-2 B cells are absent in mice lacking BAFF or the BAFF-R, these deficiencies have no effect on the development or survival of B-1 B cells. Additionally, the weak B-cell receptor signals that promote the final stages of B-2 B-cell maturation in the spleen require the non-canonical NF-κB activation pathway (see Section 7-23), a signaling pathway that is dispensable for B-1 B-cell development. Cytokine requirements also differ

1	maturation in the spleen require the non-canonical NF-κB activation pathway (see Section 7-23), a signaling pathway that is dispensable for B-1 B-cell development. Cytokine requirements also differ between these developmental pathways. B-1 B cells develop normally in mice lacking IL-7 or IL-7R signaling components, defects that prevent the development of B-2 B cells. B-2 B-cell development also requires the transcription factor PU.1, which is not needed for the development of B-1 B cells.

1	Summary.

1	In this section, we have followed B-cell development from the earliest progenitors in the bone marrow to the long-lived mature peripheral B-cell pool (Fig. 8.14). The heavy-chain locus is rearranged first and, if this is successful, a μ heavy chain is produced that combines with surrogate light chains to form the pre-B-cell receptor; this is the first checkpoint in B-cell development. Production of the pre-B-cell receptor signals successful heavy-chain gene rearrangement and causes cessation of this rearrangement, thus enforcing allelic exclusion. It also initiates pre-B-cell proliferation, generating numerous progeny in which subsequent light-chain rearrangement can be attempted. If the initial light-chain gene rearrangement is productive, a complete immunoglobulin B-cell receptor is formed, gene rearrangement again ceases, and the B cell continues its development. If the first light-chain gene rearrangement is unsuccessful, rearrangement continues until either a productive

1	is formed, gene rearrangement again ceases, and the B cell continues its development. If the first light-chain gene rearrangement is unsuccessful, rearrangement continues until either a productive rearrangement is made or all available J regions are used up. If no productive rearrangement is made, the developing B cell dies. Once a complete immunoglobulin receptor is expressed on the surface of the cell, immature B cells undergo tolerance to self antigens. This process begins in the bone marrow and continues for a short time after immature B cells emigrate to the periphery. For the majority population of B cells, the final stages of their maturation occur in the B-cell follicles of the spleen, and require the TNF family member BAFF as well as signals through the B-cell receptor.

1	Fig. 8.13 A comparison of the properties of B-1 cells, follicular B cells (B-2 cells), and marginal zone B cells. In addition to developing in the liver, B-1 cells can develop in unusual sites in the fetus, such as the omentum. B-1 cells predominate in the young animal, although they probably can be produced throughout life. Being produced mainly during fetal and neonatal life, their rearranged variable-region sequences contain few N-nucleotides. In contrast, marginal zone B cells accumulate after birth and do not reach peak levels in the mouse until 8 weeks of age. Follicular B-2 cells and marginal zone B cells share a common precursor population, the transitional T2 B cells in the spleen; as a consequence, both subsets are dependent on IL-7 and BAFF signals for their development. In contrast, B-1 cell development does not require IL-7 or BAFF. B-1 cells are best thought of as a partly activated self-renewing pool of lymphocytes that are selected by ubiquitous self and foreign

1	In contrast, B-1 cell development does not require IL-7 or BAFF. B-1 cells are best thought of as a partly activated self-renewing pool of lymphocytes that are selected by ubiquitous self and foreign antigens. Because of this selection, and possibly because the cells are produced early in life, the B-1 cells have a restricted repertoire of variable regions and antigen-binding specificities. Marginal zone B cells also have a restricted repertoire of V-region specificities that may be selected by a set of antigens similar to those that select B-1 cells. B-1 cells seem to be the major population of B cells in certain body cavities, most probably because of exposure at these sites to antigens that drive B-1 cell proliferation. Marginal zone B cells remain in the marginal zone of the spleen and are not thought to recirculate. Partial activation of B-1 cells leads to the secretion of mainly IgM antibody; B-1 cells contribute much of the IgM that circulates in the blood. The limited

1	and are not thought to recirculate. Partial activation of B-1 cells leads to the secretion of mainly IgM antibody; B-1 cells contribute much of the IgM that circulates in the blood. The limited diversity of both the B-1 and marginal zone B-cell repertoire and the propensity of these cells to react with common bacterial carbohydrate antigens suggest that they carry out a more primitive, less adaptive immune response than follicular B cells (B-2 cells). In this regard they are comparable to γ:δ T cells.

1	Fig. 8.14 A summary of the development of human conventional B-lineage cells. The state of the immunoglobulin genes, the expression of some essential intracellular proteins, and the expression of some cell-surface molecules are shown for successive stages of conventional B-2 B-cell development. During antigen-driven B-cell differentiation, the immunoglobulin genes undergo further changes, such as class switching and somatic hypermutation (see Chapter 5), which are evident in the immunoglobulins produced by memory cells and plasma cells. These antigen-dependent stages are described in more detail in Chapter 9.

1	lgGPlasmablast and plasma cell lgG Memory B cell CD135 Plasma cell antigen-1 CD38 Somatic hypermutation VJ rearranged Isotype switch to C˜, C°, or C˛. Somatic hypermutation Alternative splicing yields both membrane and secreted Ig lgM Lympho-blast lgMlgD Mature naive B cell CD45R MHC class II IgM, IgD CD19, CD20 CD21, CD40 CD45R MHC class II IgG, IgA CD19, CD20 CD21, CD40 CD45R MHC class II CD19, CD20 CD21, CD40 VDJ rearranged. ˝chain produced in membrane form. Alternative splicing yields ˝+ˆmRNA Alternative splicing yields secreted ˝chains Ig IgANTIGENDEPENDENTTERMINALDIFFERENTIATIONCD45R AA4.1, IL-7R MHC class II pre-B-R CD19, CD38 CD20, CD40 CD45R AA4.1 MHC class II CD19, CD38 CD20, CD40 Germline pre-B-cell receptor cytoplasmic ˝˝RAG-1 RAG-2 Large pre-B cell Small pre-B cell Late pro-B cell Germline TdT ˇ5, VpreB ˇ5, VpreB CD45R AA4.1, IL-7R MHC class II CD10, CD19 CD38, CD20 CD40 D–J rearranged V–DJ rearranged VDJ rearranged VDJ rearranged V–J rearrangement Early pro-B cell Stem

1	cell Germline TdT ˇ5, VpreB ˇ5, VpreB CD45R AA4.1, IL-7R MHC class II CD10, CD19 CD38, CD20 CD40 D–J rearranged V–DJ rearranged VDJ rearranged VDJ rearranged V–J rearrangement Early pro-B cell Stem cell Germline Germline Germline RAG-1 RAG-2 TdT ˇ5, VpreB CD34 CD45 AA4.1 CD34 CD45R AA4.1, IL-7R MHC class II CD10, CD19 CD38 Heavy-chain genesB cells Light-chain genes Intra-cellular proteins Surface marker proteinsANTIGENINDEPENDENT rearranged.

1	AA4.1 CD19, CD20 Development of T lymphocytes.

1	Like B cells, T lymphocytes derive from the multipotent hematopoietic stem cells in the bone marrow. However, their progenitor cells migrate from the bone marrow via the blood to the thymus, where they mature (Fig. 8.15); this is the reason for the name thymus-dependent (T) lymphocytes, or T cells. T-cell development parallels that of B cells in many ways, including the orderly and stepwise rearrangement of antigen-receptor genes, the sequential testing for successful gene rearrangement, and the eventual assembly of a heterodimeric antigen receptor. Nevertheless, T-cell development in the thymus has some features not seen for B cells, such as the generation of two distinct lineages of T cells expressing antigen receptors encoded by distinct genes, the γ:δ lineage and the α:β lineage. Developing T cells, which are known generally as thymocytes, also undergo rigorous selection that depends on interactions with thymic cells and that shapes the mature repertoire of T cells to ensure self

1	T cells, which are known generally as thymocytes, also undergo rigorous selection that depends on interactions with thymic cells and that shapes the mature repertoire of T cells to ensure self MHC restriction as well as self-tolerance. We begin with a general overview of the stages of thymocyte development and its relationship to thymic anatomy before considering gene rearrangement and the mechanisms of selection.

1	8-10 T-cell progenitors originate in the bone marrow, but all the important events in their development occur in the thymus. The thymus is situated in the upper anterior thorax, just above the heart. It consists of numerous lobules, each clearly differentiated into an outer cortical region—the thymic cortex—and an inner medulla (Fig. 8.16). In young individuals, the thymus contains large numbers of developing T-cell precursors embedded in a network of epithelia known as the thymic stroma. This provides a unique microenvironment for T-cell development analogous to that provided for B cells by the stromal cells of the bone marrow.

1	Fig. 8.15 T cells undergo development in the thymus and migrate to the peripheral lymphoid organs, where they are activated by foreign antigens. T-cell precursors migrate from the bone marrow to the thymus, where they commit to the T-cell lineage following Notch receptor signaling. In the thymus, T-cell receptor genes are rearranged (top first panel); α:β T-cell receptors that are compatible with self MHC molecules transmit a survival signal on interacting with thymic epithelium, leading to positive selection of the cells that bear them. Self-reactive receptors transmit a signal that leads to cell death, and cells bearing them are removed from the repertoire in a process of negative selection (top second panel). T cells that survive selection mature and leave the thymus to circulate in the periphery; they repeatedly leave the blood to migrate through the peripheral lymphoid organs, where they may encounter their specific foreign antigen and become activated (top third panel).

1	in the periphery; they repeatedly leave the blood to migrate through the peripheral lymphoid organs, where they may encounter their specific foreign antigen and become activated (top third panel). Activation leads to clonal expansion and differentiation into effector T cells. Some of these are attracted to sites of infection, where they can kill infected cells or activate macrophages (top fourth panel); others are attracted into B-cell areas, where they help to activate an antibody response (not shown).

1	Precursors commit to the T-cell lineage following Notch signaling and initiate T-cell receptor gene rearrangements Activated T cells proliferate and eliminate infection Mature T cells encounter foreign antigens in the peripheral lymphoid organs and are activated Immature T cells that recognize self MHC receive signals for survival. Those that interact strongly with self antigen are removed from the repertoire T-cell precursor mature T cell dendritic cell macrophage Notch thymic stromal cell thymus lymph node spleen self MHC activates kills gastro-intestinal tract T-cell progenitors develop in the bone marrow and migrate to the thymus where the cells complete their development by rearranging their antigenreceptor genes and undergoing repertoire selection Mature T cells migrate to the peripheral lymphoid organs Activated T cells migrate to sites of infection

1	Fig. 8.16The cellular organization of the human thymus. The thymus, which lies in the midline of the body, above the heart, is made up of several lobules, each of which contains discrete cortical (outer) and medullary (central) regions. As shown in the diagram on the left, the cortex consists of immature thymocytes (dark blue); branched cortical epithelial cells (pale blue), with which the immature cortical thymocytes are closely associated; and scattered macrophages (yellow), which are involved in clearing apoptotic thymocytes. The medulla consists of mature thymocytes (dark blue) and medullary epithelial cells (orange), along with macrophages (yellow) and dendritic cells (yellow) of bone marrow origin. Hassall’s corpuscles are probably also sites of cell degradation. The thymocytes in the outer cortical cell layer are proliferating immature cells, whereas the deeper cortical thymocytes are mainly immature T cells undergoing thymic selection. The photograph shows the equivalent

1	in the outer cortical cell layer are proliferating immature cells, whereas the deeper cortical thymocytes are mainly immature T cells undergoing thymic selection. The photograph shows the equivalent section of a human thymus, stained with hematoxylin and eosin. The cortex is darkly stained, whereas the medulla is lightly stained. The large body in the medulla is a Hassall’s corpuscle. Photograph courtesy of C.J. Howe.

1	The thymic epithelium arises early in embryonic development from endodermderived structures known as the third pharyngeal pouches. These epithelial tissues form a rudimentary thymus, or thymic anlage. This is colonized by cells of hematopoietic origin that give rise to large numbers of thymocytes, which are committed to the T-cell lineage, and to intrathymic dendritic cells. Thymocytes are not simply passengers within the thymus: they influence the arrangement of the thymic epithelial cells on which they depend for survival, inducing the formation of a reticular epithelial structure that surrounds the developing thymocytes (Fig. 8.17).

1	The cellular architecture of the human thymus is illustrated in Fig. 8.16. Bone marrow-derived cells are differentially distributed between the thymic cortex and medulla. The cortex contains only immature thymocytes and scattered macrophages, whereas more mature thymocytes, along with dendritic cells, macrophages, and some B cells, are found in the medulla. As will be discussed below, this organization reflects the different developmental events that occur in these two compartments.

1	The importance of the thymus in immunity was first discovered through experiments on mice; indeed, most of our knowledge of T-cell development in the thymus comes from the mouse. It was found that surgical removal of the thymus (thymectomy) at birth resulted in immunodeficient mice, focusing interest on this organ at a time when the difference between T and B lymphocytes in mammals had not yet been defined. Much evidence, including observations in immunodeficient children, has since confirmed the importance of the thymus in T-cell development. In DiGeorge syndrome in humans and in mice with the nude mutation, the thymus does not form and the affected individual produces B lymphocytes but few T lymphocytes. DiGeorge syndrome is a complex combination of cardiac, facial, endocrine, and immune defects associated with deletions of chromosome 22q11. The nude mutation in mice is due to a defect in the gene for Foxn1, a transcription factor required for terminal epithelial cell

1	and immune defects associated with deletions of chromosome 22q11. The nude mutation in mice is due to a defect in the gene for Foxn1, a transcription factor required for terminal epithelial cell differentiation; the name nude was given to this mutation because it also causes hairlessness. Rare cases of a defect in the human FOXN1 gene (which is on chromosome 17) have been associated with T-cell immunodeficiency, absence of a thymus, congenital alopecia, and nail dystrophy.

1	In mice, the thymus continues to develop for 3–4 weeks after birth, whereas in humans it is fully developed at birth. The rate of T-cell production by the thymus is greatest before puberty. After puberty, the thymus begins to shrink, and the production of new T cells in adults is reduced, although it does continue throughout life. In both mice and humans, removal of the thymus after puberty is not accompanied by any notable loss of T-cell function or numbers. Thus, it seems that once the T-cell repertoire is established, immunity can be sustained without the production of significant numbers of new T cells; the pool of peripheral T cells is instead maintained by long-lived T cells and also by division of some mature T cells. 8-11 Commitment to the T-cell lineage occurs in the thymus following Notch signaling.

1	8-11 Commitment to the T-cell lineage occurs in the thymus following Notch signaling. T lymphocytes develop from a lymphoid progenitor in the bone marrow that also gives rise to B lymphocytes. Some of these progenitors leave the bone marrow and migrate to the thymus. In the thymus, the progenitor cell receives a signal from thymic epithelial cells that is transduced through a receptor called Notch1 to switch on specific genes. Notch signaling is widely used in animal development to specify tissue differentiation; in lymphocyte development, the Notch signal instructs the precursor to commit to the T-cell lineage rather than the B-cell lineage. Notch signaling is required throughout T-cell development and is also thought to help regulate other T-cell lineage choices, including the α:β versus γ:δ choice.

1	Notch signaling in thymic progenitor cells is essential to initiate the T-cellspecific gene expression program and commitment to the T-cell lineage (Fig. 8.18). First, Notch signaling induces the expression of two transcription factors, T-cell factor-1 (TCF1) and GATA3, each of which is required for T-cell development. Together, TCF1 and GATA3 initiate expression of several T-lineage-specific genes, such as those encoding components of the CD3 complex, as well as Rag1, a gene required for T-cell receptor and B-cell receptor gene rearrangements (see Fig. 8.18). However, TCF1 and GATA3 are not sufficient to induce the entire program of T-cell-specific gene expression. A third transcription factor, Bcl11b, is required to induce T-lineage commitment by restricting progenitor cells from adopting alternative fates; this final phase of T-cell commitment is a necessary prerequisite for activating the complete T-cell gene expression program.

1	8-12 T-cell precursors proliferate extensively in the thymus, but most die there. T-cell precursors arriving in the thymus from the bone marrow spend up to a week differentiating there before they enter a phase of intense proliferation. In a young adult mouse the thymus contains about 108 to 2 × 108 thymocytes. About 5 × 107 new cells are generated each day; however, only about 106 to 2 × 106 (roughly 2–4%) of these leave the thymus each day as mature T cells. Despite the disparity between the number of T cells generated in the thymus and the number leaving, the thymus does not continue to grow in size or cell number. This is because about 98% of the thymocytes that develop in the thymus also die in the thymus by apoptosis (see Section 1-14). Cells undergoing apoptosis are recognized and ingested by macrophages, and apoptotic bodies, which are the residual condensed chromatin of apoptotic cells, are seen inside macrophages throughout the thymic cortex (Fig. 8.19). This apparently

1	Fig. 8.17 The epithelial cells of the thymus form a network surrounding developing thymocytes. In this scanning electron micrograph of the thymus, the developing thymocytes (the spherical cells) occupy the interstices of an extensive network of epithelial cells. Photograph courtesy of W. van Ewijk. Fig. 8.18 The stages of α:β T-cell development in the mouse thymus correlate with the program of gene rearrangement, and the expression of cell-surface proteins, signaling proteins, and transcription factors.

1	Lymphoid precursors are triggered to proliferate and become thymocytes committed to the T-cell lineage through interactions with Notch ligands expressed on the thymic stroma. T-cell commitment requires Notch signaling to induce the expression of TCF1 and GATA3, which in turn induce the expression of Bcl11b. This gene expression program begins in the double-negative (DN1) cells that express CD44 and Kit. Cells become irreversibly committed to the T-cell lineage at the subsequent (DN2) stage, which is marked by expression of CD25, the α chain of the IL-2 receptor. After this, the DN2 (CD44+CD25+) cells begin to rearrange the β-chain locus, becoming CD44low and Kitlow as this occurs, and they become DN3 cells. The DN3 cells are arrested in the CD44lowCD25+ stage until they productively rearrange the β-chain locus; the in-frame β chain then pairs with a surrogate chain called pTα to form the pre-T-cell receptor (pre-TCR), which is expressed on the cell surface and triggers entry into the

1	the β-chain locus; the in-frame β chain then pairs with a surrogate chain called pTα to form the pre-T-cell receptor (pre-TCR), which is expressed on the cell surface and triggers entry into the cell cycle. Expression of small amounts of pTα:β on the cell surface in association with CD3 signals the cessation of β-chain gene rearrangement and triggers rapid cell proliferation, which causes the loss of CD25. The cells are then known as DN4 cells. Eventually, the DN4 cells cease to proliferate and CD4 and CD8 are expressed. The small CD4+CD8+ double-positive cells begin efficient rearrangement at the α-chain locus. The cells then express low levels of an α:β T-cell receptor and the associated CD3 complex and are ready for selection. Most cells die by failing to be positively selected or as a consequence of negative selection, but some are selected to mature into CD4 or CD8 single-positive cells and eventually to leave the thymus. Maturation of CD4+CD8+ double-positive cells into CD4 or

1	a consequence of negative selection, but some are selected to mature into CD4 or CD8 single-positive cells and eventually to leave the thymus. Maturation of CD4+CD8+ double-positive cells into CD4 or CD8 single-positive cells is regulated by transcription factors ThPOK and Runx3, respectively. KLF2 is first expressed at the single-positive stage; if it is absent, thymocytes exhibit a defect in emigrating to peripheral lymphoid tissues, due in part to their failure to express receptors involved in trafficking, such as the sphingosine 1-phosphate (S1P) receptor, S1PR1 (see Fig. 8.32). The individual contributions to T-cell development of the other proteins are discussed in the text.

1	Fig. 8.19 Developing T cells that undergo apoptosis are ingested by macrophages in the thymic cortex. The left panel shows a section through the thymic cortex and part of the medulla in which cells have been stained for apoptosis with a red dye. The thymic cortex is to the right in the photograph. Apoptotic cells are scattered throughout the cortex but are rare in the medulla. The right panel shows at higher magnification a section of thymic cortex that has been stained red for apoptotic cells and blue for macrophages. The apoptotic cells can be seen within macrophages. Magnifications: left panel, ×45; right panel, ×164. Photographs courtesy of J. Sprent and C. Surh. profligate waste of thymocytes is a crucial part of T-cell development because it reflects the intensive screening that each thymocyte undergoes for the ability to recognize self peptide:self MHC complexes and for self-tolerance.

1	8-13 Successive stages in the development of thymocytes are marked by changes in cell-surface molecules. Like developing B cells, developing thymocytes pass through a series of distinct stages. These are marked by changes in the status of the T-cell receptor genes and in the expression of the T-cell receptor, and by changes in the expression of cell-surface proteins such as the CD3 complex (see Section 7-7) and the co-receptor proteins CD4 and CD8 (see Section 4-18). These surface changes reflect the state of functional maturation of the cell, and particular combinations of cell-surface proteins are used as markers for T cells at different stages of differentiation. The principal stages are summarized in Fig. 8.20. Two distinct lineages of T cells—α:βand γ:δ, which have different types of T-cell receptor chains—are produced early in T-cell development. Later, α:β T cells develop into two distinct functional subsets—CD4 T cells and CD8 T cells.

1	When progenitor cells first enter the thymus from the bone marrow, they lack most of the surface molecules characteristic of mature T cells, and their receptor genes are not rearranged. These cells give rise to the major population of α:β T cells and the minor population of γ:δ T cells. If injected into the peripheral circulation, these lymphoid progenitors can even give rise to B cells and NK cells, although it is uncertain whether individual thymic progenitor cells retain this multipotency, or whether the progenitor cell population consists of a mixture of cells, only some of which are fully committed to the αβor γδ T-cell lineage.

1	Interactions with the thymic stroma trigger an initial phase of differentiation along the T-cell lineage pathway, followed by cell proliferation and the expression of the first cell-surface molecules specific for T cells, for example, CD2 and (in mice) Thy-1. At the end of this phase, which can last about a week, the thymocytes bear distinctive markers of the T-cell lineage but do not express any of the three cell-surface markers that define mature T cells. These are the CD3:T-cell receptor complex and the co-receptors CD4 or CD8. Because of the absence of CD4 and CD8, such cells are called double-negative thymocytes (see Fig. 8.20).

1	Fig. 8.20 Two distinct lineages of thymocytes are produced in the thymus. CD4, CD8, and T-cell receptor complex molecules (CD3, and the T-cell receptor α and β chains) are important cell-surface molecules for identifying thymocyte subpopulations. The earliest cell population in the thymus does not express any of these proteins, and because these cells do not express CD4 or CD8, they are called ‘double-negative’ thymocytes. These cells include precursors that give rise to two T-cell lineages: the minority population of γ:δ T cells (which lack CD4 or CD8 even when mature), and the majority α:β T-cell lineage. The development of prospective α:β T cells proceeds through stages in which both CD4 and CD8 are expressed by the same cell; these are known as ‘double-positive’ thymocytes. These cells enlarge and divide. Later, they become small resting double-positive cells that express low levels of the T-cell receptor. Most thymocytes die within the thymus after becoming small double-positive

1	enlarge and divide. Later, they become small resting double-positive cells that express low levels of the T-cell receptor. Most thymocytes die within the thymus after becoming small double-positive cells, but those cells whose receptors can interact with self peptide:self MHC molecular complexes lose expression of either CD4 or CD8 and increase the level of expression of the T-cell receptor. The outcome of this process is the ‘single-positive’ thymocytes, which, after maturation, are exported from the thymus as mature single-positive CD4 or CD8 T cells.

1	In the fully developed thymus, only ~60% of the double-negative thymocytes are immature T cells. The double-negative thymocyte pool (about 5% of all thymocytes) also includes two populations of more mature T cells that belong to minority lineages, including T cells expressing γ:δ T-cell receptors (see Section 8-16) and T cells bearing α:β T-cell receptors of very limited diversity (iNKT cells; see Section 6-19). In this and subsequent discussions, we reserve the term ‘double-negative thymocytes’ for the immature thymocytes that do not yet express a complete T-cell receptor molecule. These cells give rise to both γ:δ and α:β T cells (see Fig. 8.20), although most of them develop along the α:β pathway.

1	The α:β pathway is shown in more detail in Fig. 8.18. The double-negative stage can be further subdivided into four stages on the basis of expression of the adhesion molecule CD44, CD25 (the α chain of the IL-2 receptor), and Kit, the receptor for SCF (see Section 8-1). At first, double-negative thymocytes express Kit and CD44 but not CD25 and are called DN1 cells; in these cells, the genes encoding both chains of the T-cell receptor are in the germline configuration. As thymocytes mature, they begin to express CD25 on their surface and are called DN2 cells; later, expression of CD44 and Kit is reduced, and they are called DN3 cells.

1	Rearrangement of the T-cell receptor β-chain locus begins in DN2 cells with some Dβ to Jβ rearrangements and continues in DN3 cells with Vβ to DJβ rearrangement. Cells that fail to make a successful rearrangement of the β-chain locus remain at the DN3 (CD44lowCD25+) stage and soon die, whereas cells that make productive β-chain gene rearrangements and express the β-chain protein lose expression of CD25 once again and progress to the DN4 stage, in which they proliferate. The functional significance of the transient expression of CD25 is unclear: T cells develop normally in mice in which the IL-2 gene has been deleted by gene knockout (see Appendix I, Section A-35). By contrast, Kit is quite important for the development of the earliest double-negative thymocytes, in that mice lacking Kit have a much smaller number of double-negative T cells. In addition, continuous Notch signaling is important for progression through each stage of T-cell development. A second essential factor is IL-7,

1	a much smaller number of double-negative T cells. In addition, continuous Notch signaling is important for progression through each stage of T-cell development. A second essential factor is IL-7, which is produced by the thymic stroma. In the absence of IL-7, IL-7 receptor α, γ-c, or the IL-7 receptor signaling protein Jak3, a severe block in T-cell development occurs in both mice and humans. In fact, the human primary immunodeficiency disease characterized by defects in T cells and NK cells, X-linked SCID (severe combined immunodeficiency disease), is caused by a genetic deficiency leading to the absence of γ-c protein expression.

1	In DN3 thymocytes (see Fig. 8.18), the expressed β chains pair with a surrogate pre-T-cell receptor α chain called pTα (pre-T-cell α), which allows the assembly of a complete pre-T-cell receptor (pre-TCR) that is analogous in structure and function to the pre-B-cell receptor. The pre-TCR is expressed on the cell surface in a complex with the CD3 molecules that provide the signaling components of T-cell receptors (see Section 7-7). As with the preB-cell receptor, the assembly of the CD3:pre-T-cell receptor complex causes constitutive signaling that does not require interaction with a ligand. Recent structural evidence shows that the pre-TCR forms dimers in a manner similar to pre-BCR dimerization. The pTα Ig domain makes two important contacts. It associates with the constant-region Ig domain of the Vβsubunit to form the pre-TCR itself. A distinct surface of the pre-Tα then binds to a Vβ domain from another pre-TCR molecule, forming a bridge between two different pre-TCRs. The region

1	of the Vβsubunit to form the pre-TCR itself. A distinct surface of the pre-Tα then binds to a Vβ domain from another pre-TCR molecule, forming a bridge between two different pre-TCRs. The region of contact with the Vβ involves residues that are highly conserved across many Vβ families. In this way, expression of the pre-TCR induces ligand-independent dimerization, which leads to cell proliferation, the arrest of further β-chain gene rearrangement, and the expression of both CD8 and CD4. These double-positive thymocytes make up the vast majority of thymocytes. Once the large double-positive thymocytes have ceased to proliferate and have become small double-positive cells, the α-chain locus begins to rearrange.

1	thymic cortical epithelial cells and the receptors of developing T cells has a crucial role in positive selection, as we will see later in this chapter. After positive selection, developing T cells migrate from the cortex to the medulla. The medulla contains fewer lymphocytes, and those present are predominantly the newly matured single-positive T cells that will eventually leave the thymus. The medulla plays a role in negative selection. The antigen-presenting cells in this environment include dendritic cells that express co-stimulatory molecules, which are generally absent from the cortex. In addition, specialized medullary epithelial cells present peripheral antigens for the negative selection of T cells reactive for these self antigens. 8-15 T cells with α:βor γ:δ receptors arise from a common progenitor.

1	T cells bearing γ:δ receptors differ from α:β T cells in that they are found primarily in epithelial and mucosal sites and lack expression of the CD4 and CD8 co-receptors; in comparison with α:β T cells, relatively little is known about the ligands recognized by the γ:δ T-cell receptors, which are thought not to be MHC restricted (see Section 4-20). Recall from Section 5-11 that different genetic loci are used to make these two types of T-cell receptors. The γ and δ loci are the first to undergo rearrangement, followed shortly thereafter by the β locus. In addition, the δ locus is contained within the α locus, so rearrangements at the α locus eliminate the δ coding sequences on the chromosome. While the mechanism regulating commitment of individual precursor cells to the α:β versus the γ:δ lineage is still not understood, there is some plasticity in this process. This can be deduced from the pattern of gene rearrangements found in thymocytes and in mature γ:δ and α:β T cells. Mature

1	lineage is still not understood, there is some plasticity in this process. This can be deduced from the pattern of gene rearrangements found in thymocytes and in mature γ:δ and α:β T cells. Mature γ:δ T cells can contain rearranged β-chain genes, although 80% of these are nonproductive, and mature α:β T cells often contain rearranged, but mostly out-of-frame, γ-chain genes.

1	8-16 T cells expressing γ:δ T-cell receptors arise in two distinct phases during development. Although γ:δ T cells arise from the same progenitors as α:β T cells, most mature γ:δ T cells are components of the innate rather than the adaptive immune system. When their maturation in the thymus is complete, the cells have acquired a defined effector function that can be rapidly elicited following their activation. After emigration from the thymus, most γ:δT cells home to mucosal and epithelial sites in the body, and take up stable residence in these locations.

1	In mice, the majority of γ:δ T cells in the body arise during embryonic development and the early neonatal period. In the fetal thymus, the first T cells to develop are γ:δ T cells that all express T-cell receptors assembled from the same Vγ and Vδ regions (Fig 8.22). These cells populate the epidermis; the T cells become wedged among the keratinocytes and adopt a dendritic-like form that has given them the name of dendritic epidermal T cells (dETCs) (Fig. 8.23). DETCs provide surveillance of the skin and respond to infection and injury by producing cytokines and chemokines. These factors induce inflammation to enhance pathogen clearance, and they promote wound healing to repair lesions in the skin. In steady-state conditions, dETCs also produce growth factors that help maintain epidermal growth and survival.

1	Following the dETC cells, a second subset of γ:δ T cells develops in the fetal thymus. These cells home to mucosal epithelia of tissues such as the reproductive tract and the lung, and also to the dermis of the skin. This subset is programmed to produce inflammatory cytokines such as IL-17 when stimulated, and is thought to play a role in responses to infection and injury. Like the dETCs, these IL-17-producing γ:δ T (Tγ:δ-17) cells express T-cell receptors that are essentially invariant, being composed of a single Vγ–Vδ combination. However, the two subsets, dETCs and the fetal Tγ:δ-17 cells, express T-cell receptors that use distinct Vγ gene segments—Vγ5 in the dETCs and Vγ6 in the Tγ:δ-17 cells. As fetal thymocytes do not express the enzyme TdT, there are no N-nucleotides contributing additional diversity at the junctions between V, D, and J gene segments of the T-cell receptors in these two fetally derived

1	V 2–7D1 D2 J2 C V˜4J˜1 C˜1 Stem cells in adult Starting during late fetal development ° ° ° ° ° V 2–7D1 D2 J2 C° ° ° ° ° V˜7J˜ C˜ ˜:° cells home to lymph nodes and spleen and are programmed to secrete IFN-˜ ˜:° cells home to intestinal epithelium and are programmed to secrete IFN-˜ 107 106 105 104 103 15 16 17 18 19 1 2 V1 D2 J2 C V6 J1 C1 C1 Numbers of thymocytes skin epidermis uterus skin, dermis lung spleen, lymph nodes liver intestinal epithelium Days of gestation Birth Weeks of age V1 D2 J2 C V 5 J1 V 2–7D1 D2 J2 C V˜4J˜1 C˜1 Stem cells predominant in fetus and newborn, rare in adult Starting day 17 of development Stem cells in fetus Days 14–18 of development Stem cells in fetus Days 16–19 of development V˜5 V˜6 V˜4 and V˜1 V˜1,2,4 and V˜7 ° ° ° ° ˜ ˜ ˜ ˜ ˜:° thymocytes ° ° ° °˜ ˜ ° ° ° ° ° V°6D 1D 2J 2 C V˜1J˜ C˜ ° ° ° ° ˜:° T cells become established in skin epidermis and are programmed to secrete keratinocyte growth factor, and infammatory cytokines and chemokines ˜:° T cells

1	° ° ° ° V°6D 1D 2J 2 C V˜1J˜ C˜ ° ° ° ° ˜:° T cells become established in skin epidermis and are programmed to secrete keratinocyte growth factor, and infammatory cytokines and chemokines ˜:° T cells become established in mucosal epithelia, including reproductive tract, lung, and skin dermis, and are programmed to secrete IL-17 ˜:° cells home to lymph nodes, spleen, lung, and skin dermis and are programmed to secrete IL-17 ˜:° cells home to lymph nodes, spleen, lung, and liver and are programmed to secrete IL-4 and IFN-˜

1	Fig. 8.22 The rearrangement of T-cell receptor γ and δ genes in the mouse proceeds in waves of cells expressing different Vγ and Vδ gene segments. At about 2 weeks of gestation in the mouse, the Cγ1 locus is expressed with its closest V gene (Vγ5). After a few days, Vγ5-bearing cells decline in numbers in the thymus (first row of panels) and are replaced by cells expressing the next most proximal gene, Vγ6. Both these rearranged γ chains are expressed with the same rearranged δ-chain gene, as shown in the lower panels, and there is little junctional diversity in either the Vγ or the Vδ chain. As a consequence, most of the γ:δ T cells produced in each of these early waves share the same specificity, although the antigen recognized in each case is not known. The Vγ5-bearing cells become established selectively in the epidermis; they are programmed to secrete keratinocyte growth factor and inflammatory cytokines and chemokines. In contrast, Vγ6-bearing cells become established in the

1	established selectively in the epidermis; they are programmed to secrete keratinocyte growth factor and inflammatory cytokines and chemokines. In contrast, Vγ6-bearing cells become established in the lung, the dermis of the skin, and the epithelium of the reproductive tract, and are programmed to secrete IL-17. The next wave of γδ development begins on day 17 of gestation, and produces two different populations. One population rearranges and expresses the Vγ4 chain, which pairs with heterogeneous delta chains. These Vγ4-bearing cells are the second subset of Tγ:δ-17 (IL-17-secreting) cells, and home to lymph nodes, spleen, lung, and the dermis of the skin. The second population in this wave expresses Vγ1, and homes to lymph nodes, spleen, and liver. Some of these cells are paired with Vγ6 chains and are programmed to secrete IL-4 and IFN-γ, and represent γ:δ NKT cells. Finally, the last wave of γ:δ T-cell development begins late during fetal development, and persists into adulthood.

1	chains and are programmed to secrete IL-4 and IFN-γ, and represent γ:δ NKT cells. Finally, the last wave of γ:δ T-cell development begins late during fetal development, and persists into adulthood. This last wave includes a heterogeneous population of cells bearing Vγ1, Vγ2, and Vγ4 chains paired with many different delta chains. These cells home to lymphoid organs and are programmed to secrete IFN-γ. The other population in this last wave are cells bearing the Vγ7 chain paired with heterogeneous delta chains. These γ:δ cells home to the intestinal epithelium and are programmed to secrete IFN-γ as well as antimicrobial compounds. Although γ:δ T cells continue to be produced after birth, at this stage the α:β T-cell lineage becomes the dominant population developing in the thymus.

1	Fig. 8.23 Dendritic epidermal T cells reside within the epithelial layer, forming an interdigitating network with Langerhans cells. This face-on view of a murine epidermal sheet shows Langerhans cells (green) and dendritic epidermal T cells (dETCs; red) forming an interdigitating network within the layers of the epidermis. The epidermal epithelial cells are not visible in this fluorescence image. The branching dendritic-like form of these γ:δ T cells is the source of their name. Although the ligands for all γ:δ T-cell receptors are not known, some γ:δ T cells recognize nonclassical MHC molecules (see Sections 6-16 and 6-17), which can be induced in epithelia by stresses such as UV damage or pathogens. Thus, dETCs may serve as sentinels of such damage, producing cytokines that activate the innate immune response and, in turn, adaptive immunity. Courtesy of Adrian Hayday.

1	subsets of γ:δ T cells. Why certain V, D, and J gene segments are selected for rearrangement at particular times during embryonic development remains incompletely understood.

1	dETCs and the Vγ6-positive Tγ:δ-17 cells develop exclusively from the early wave of hematopoietic stem cells derived from the fetal liver (see Fig. 8.22). Consequently, these two γ:δ T-cell subsets arise only for a brief period of time in the fetal thymus, and then never again. A second phase of γ:δ T-cell development is initiated in the fetal thymus just before birth. This phase persists at a low level in the adult thymus throughout life, and produces several subsets of cells, each with distinct effector functions and tissue homing properties. Like the dETCs and fetal Tγ:δ-17 cells, these later-developing γ:δ T cells can be generally classified by their usage of distinct Vγ–Vδ regions in their T-cell receptors (see Fig. 8.22), although the receptor sequences within each population are more diverse due to the presence of N-region nucleotides added by TdT.

1	One population of these later-developing γ:δ T cells is programmed to secrete IL-17 when activated; these represent a second subset of Tγ:δ-17 cells that express a different Vγ region than do the fetal Tγ:δ-17 cells. Specifically, these later-developing Tγ:δ-17 cells express T-cell receptors using the Vγ4 region. This Tγ:δ-17 subset is found in all lymphoid organs, as well as in the skin dermis and the intestinal epithelium, where the cells provide rapid inflammatory signals in response to bacterial and parasitic infections. In addition, γ:δ T cells using the Vγ7 region in their T-cell receptors also develop in this second phase. The Vγ7-positive γ:δ T cells home specifically to the intestinal epithelium. In that location, the cells are poised to respond to gut microbes that breach the epithelial barrier, and are important producers of antibacterial compounds as well as IFN-γ.

1	In contrast to the γ:δT-cell subsets that reside in barrier tissues such as the skin and intestinal epithelium, γ:δ T cells are also found in lymphoid organs. The majority of the lymphoid-resident γ:δ T cells arise during the late fetal–early neonatal period as well as thereafter, and represent a more diverse population expressing the Vγ1 region. Vγ1-positive T cells are composed of two major groups—an IFN-γplus IL-4-producing subset that homes to the liver as well as several lymphoid organs, and an IFN-γ-producing subset that homes to all lymphoid organs. The former population of cells, which can be identified by their expression of a unique TCRδ chain (Vδ6) that is paired to Vγ1, is remarkably similar to the α:β T-cell receptor-expressing subset of iNKT cells, and the cells are therefore often referred to as γ:δ NKT cells. Unlike the mucosal and epithelial resident γ:δ T-cell populations, whose importance in tissue homeostasis, repair, and innate defense against infections has been

1	often referred to as γ:δ NKT cells. Unlike the mucosal and epithelial resident γ:δ T-cell populations, whose importance in tissue homeostasis, repair, and innate defense against infections has been well established, the functions of γ:δ T cells within secondary lymphoid organs is still not well understood.

1	8-17 Successful synthesis of a rearranged β chain allows the production of a pre-T-cell receptor that triggers cell proliferation and blocks further β-chain gene rearrangement.

1	We now return to the development of α:β T cells. The rearrangement of the βand α-chain loci closely parallels the rearrangement of immunoglobulin heavy-chain and light-chain loci during B-cell development (see Sections 8-2 through 8-5). As shown in Fig. 8.24, the β-chain gene segments rearrange first, with the Dβ gene segments rearranging to Jβ gene segments, and this is followed by Vβ to DJβ rearrangement. If no functional β chain can be synthesized from this rearrangement, the cell will not be able to produce a pre-T-cell receptor and will die. However, unlike B cells with nonproductive heavy-chain gene rearrangements, thymocytes with nonproductive β-chain VDJ rearrangements can be rescued by further rearrangement, which is possible because of the two clusters of Dβ and Jβ gene segments upstream of two Cβ genes (see Fig. 5.13).

1	The likelihood of a productive VDJ join at the β locus is therefore somewhat higher than the 55% chance for a productive immunoglobulin heavy-chain gene arrangement. Once a productive β-chain gene rearrangement has occurred, the β chain is expressed together with the invariant pTα and the CD3 molecules (see Fig. 8.24) and is transported in this complex to the cell surface. The β:pTα complex is a functional pre-T-cell receptor analogous to the μ:VpreB:λ5 pre-Bcell receptor complex in B-cell development (see Section 8-3). Expression of

1	Fig. 8.24 The stages of gene rearrangement in α:β T cells. The sequence of gene rearrangements is shown, together with an indication of the stage at which the events take place and the nature of the cell-surface receptor molecules expressed at each stage. The β-chain locus rearranges first, in CD4–CD8– double-negative thymocytes expressing CD25 and low levels of CD44. As with immunoglobulin heavy-chain genes, D to J gene segments rearrange before V gene segments rearrange to DJ (second and third panels). It is possible to make up to four attempts to generate a productive rearrangement at the β-chain locus, because there are four D gene segments with two sets of J gene segments associated with each TCRβ chain locus (not shown). The productively rearranged gene is expressed initially within the cell and then at low levels on the cell surface. It associates with pTα, a surrogate 33-kDa α chain that is equivalent to λ5 in B-cell development, and this pTα:β heterodimer forms a complex with

1	the cell and then at low levels on the cell surface. It associates with pTα, a surrogate 33-kDa α chain that is equivalent to λ5 in B-cell development, and this pTα:β heterodimer forms a complex with the CD3 chains (fourth panel). The expression of the pre-T-cell receptor signals the developing thymocytes to halt β-chain gene rearrangement and to undergo multiple cycles of division. At the end of this proliferative burst, the CD4 and CD8 molecules are expressed, the cell ceases cycling, and the α chain is now able to undergo rearrangement. The first α-chain gene rearrangement deletes all δ D, J, and C gene segments on the chromosome, although these are retained as a circular DNA, indicating that these are nondividing cells (bottom panel). This permanently inactivates the δ-chain gene. Rearrangements at the α-chain locus can proceed through several cycles, because of the large number of Vα and Jα gene segments, so that productive rearrangements almost always occur.

1	When a functional α chain is produced that pairs efficiently with the β chain, the CD3lowCD4+CD8+ thymocyte is ready to undergo selection for its ability to recognize self peptides in association with self-MHC molecules.

1	the pre-T-cell receptor at the DN3 stage of thymocyte development induces signals that cause the phosphorylation and degradation of RAG-2, thus halting β-chain gene rearrangement and ensuring allelic exclusion at the β locus. These signals also induce the DN4 stage, in which rapid cell proliferation occurs, and eventually the co-receptor proteins CD4 and CD8 are expressed. The pre-T-cell receptor signals constitutively via the cytoplasmic protein kinase Lck, an Src-family tyrosine kinase (see Fig. 7.12), but seems not to require a ligand on the thymic epithelium. Lck subsequently associates with the co-receptor proteins. In mice genetically deficient in Lck, T-cell development is arrested before the CD4+CD8+ double-positive stage, and no α-chain gene rearrangements can be made.

1	The role of the expressed β chain in suppressing further β-locus rearrangement can be demonstrated in mice containing a rearranged TCRβ transgene: these mice express the transgenic β chain on virtually 100% of their T cells, and rearrangement of their endogenous β-chain genes is strongly suppressed. The importance of pTα has been shown in mice deficient in pTα, in which there is a hundredfold decrease in α:β T cells and an absence of allelic exclusion at the β locus.

1	During the proliferation of DN4 cells triggered by expression of the pre-T-cell receptor, the RAG-1 and RAG-2 genes are repressed (see Fig. 8.18). Hence, no rearrangement of the α-chain locus occurs until the proliferative phase ends, at which time RAG-1 and RAG-2 are transcribed again, and the functional RAG-1:RAG-2 complex accumulates. This ensures that each cell in which a β-chain gene has been successfully rearranged gives rise to many CD4+CD8+ thymocytes. Once the cells stop dividing, each of them can independently rearrange its α-chain genes, so that a single functional β chain can be associated with many different α chains in the progeny cells. During the period of α-chain gene rearrangement, α:β T-cell receptors are first expressed and selection by self peptide:self MHC complexes on the thymus cells can begin.

1	The progression of thymocytes from the double-negative to the double-positive and finally to the single-positive stage is accompanied by a distinct pattern of expression of proteins involved in DNA rearrangement, signaling, and T-cell-specific gene expression (see Fig. 8.18). TdT, the enzyme responsible for the insertion of N-nucleotides, is expressed throughout T-cell receptor gene rearrangement; N-nucleotides are found at the junctions of all rearranged α and β genes. Lck and another tyrosine kinase, ZAP-70, are both expressed from an early stage in thymocyte development. As well as its key role in signaling from the pre-T-cell receptor, Lck is also important for γ:δ T-cell development. In contrast, gene knockout studies (see Appendix I, Section A-35) show that ZAP-70, although expressed from the double-negative stage onward, is not essential for pre-T-cell receptor signaling, as double-negative thymocytes also express the related Syk kinase, which is capable of fulfilling this

1	from the double-negative stage onward, is not essential for pre-T-cell receptor signaling, as double-negative thymocytes also express the related Syk kinase, which is capable of fulfilling this role. Instead, ZAP-70 is required later, to promote the development of single-positive thymocytes from double-positive thymocytes; at this stage, Syk is no longer expressed. Fyn, an Src-family kinase similar to Lck, is expressed at increasing levels from the double-positive stage onward. It is not essential for the development of α:β thymocytes as long as Lck is present, but is required for the development of iNKT cells (see Section 8-26).

1	8-18 T-cell α-chain genes undergo successive rearrangements until positive selection or cell death intervenes. The T-cell receptor α-chain genes are comparable to the immunoglobulin κ and λ light-chain genes in that they do not have D gene segments and are rearranged only after their partner receptor chain has been expressed. As with the light-chain genes, repeated attempts at α-chain gene rearrangement are possible, as illustrated in Fig. 8.25. The presence of multiple Vα gene segments, and about 60 Jα gene segments spread over some 80 kilobases of DNA, allows many successive Vα to Jα rearrangements to take place at both α-chain alleles. This means that T cells with an initial nonproductive α-gene rearrangement are much more likely to be rescued by a subsequent rearrangement than are B cells with a nonproductive light-chain gene rearrangement.

1	One key difference between B and T cells is that the final assembly of an immunoglobulin leads to the cessation of gene rearrangement and initiates the further differentiation of the B cell, whereas rearrangement of the Vα gene segments continues in T cells unless there is signaling by a self peptide:self MHC complex that positively selects the receptor (see Section 8-19 below). This means that many T cells have in-frame rearrangements on both chromosomes and so can produce two types of α chains. This is possible because expression of the T-cell receptor is not in itself sufficient to shut off gene rearrangement. Continued rearrangements on both chromosomes can allow several different α chains to be produced successively as well as simultaneously in each developing T cell and to be tested for self peptide:self MHC recognition in partnership with the same β chain. This phase of gene rearrangement lasts for 3 or 4 days in the mouse and ceases only when positive selection occurs as a

1	for self peptide:self MHC recognition in partnership with the same β chain. This phase of gene rearrangement lasts for 3 or 4 days in the mouse and ceases only when positive selection occurs as a consequence of receptor engagement, or when the cell dies. One can predict that if the frequency of positive selection is sufficiently low, roughly one in three mature T cells will express two productively rearranged α chains at the cell surface. This has been confirmed for both human and mouse T cells. Thus, in the strict sense, T-cell receptor α-chain genes are not subject to allelic exclusion.

1	T cells with dual specificity might be expected to give rise to inappropriate immune responses if the cell is activated through one receptor yet can act upon target cells recognized by the second receptor. However, only one of the two receptors is likely to be able to recognize peptide presented by a self MHC molecule, and so the T cell will have only a single functional specificity. This is because once a thymocyte has been positively selected by self peptide:self MHC recognition, α-chain gene rearrangement ceases. Thus, the existence of cells with two α-chain genes productively rearranged and two α chains expressed at the cell surface does not truly challenge the idea that a single functional specificity is expressed by each cell.

1	Fig. 8.25 Multiple successive rearrangement events can rescue nonproductive T-cell receptor α-chain gene rearrangements. The multiplicity of V and J gene segments at the α-chain locus allows successive rearrangement events to ‘leapfrog’ over previously rearranged VJ segments, deleting any intervening gene segments. The α-chain rescue pathway resembles that of the immunoglobulin κ light-chain genes (see Section 8-5), but the number of possible successive rearrangements is greater. α-chain gene rearrangement continues until either a productive rearrangement leads to positive selection or the cell dies. Summary.

1	Summary. The thymus provides a specialized and architecturally organized microenvironment for the development of mature T cells. Precursors of T cells migrate from the bone marrow to the thymus, where they interact with environmental cues, such as ligands for the Notch receptor, that drive commitment to the T lineage. Developing thymocytes develop along one of several T-cell lineages: the most prominent subsets in the thymus are γ:δ T cells, conventional α:β T cells, and α:β T cells with receptors of very limited diversity, such as iNKT cells.

1	T-cell progenitors develop along the γ:δ or the α:β T-cell lineages. Early in ontogeny, the production of γ:δ T cells predominates over α:β T cells, and these cells populate several peripheral tissues, including the skin, the intestine, and other mucosal and epithelial surfaces. These subsets predominantly develop from fetal liver, rather than bone marrow, stem cells. Later, more than 90% of thymocytes express α:β T-cell receptors. In developing thymocytes, the γ, δ, and β genes are the first to rearrange. Cells of the α:β lineage that rearrange a functional beta chain form a pre-T-cell receptor that signals thymocyte proliferation, α-chain gene rearrangement, and CD4 and CD8 expression. Most steps in T-cell development take place in the thymic cortex, whereas the medulla contains mainly mature T cells. Positive and negative selection of T cells.

1	Positive and negative selection of T cells. Up to the stage at which an α:β receptor is produced, T-cell development is independent of MHC proteins or antigen. From this point onward, developmental decisions in the α:β T-cell lineage depend on the interaction of the receptor with peptide:MHC ligands it encounters in the thymus, and we now consider this phase of T-cell development.

1	T-cell precursors committed to the α:β lineage at the DN3 stage undergo vigorous proliferation in the subcapsular region and progress to the DN4 stage. These cells then rapidly transit through an immature CD8 single-positive stage and become double-positive cells that express low levels of the T-cell receptor and both the CD4 and CD8 co-receptors as they move deeper into the thymic cortex. These double-positive cells have a life-span of only about 3–4 days unless they are rescued by engagement of their T-cell receptor. The rescue of double-positive cells from programmed cell death and their maturation into CD4 or CD8 single-positive cells is the process known as positive selection. Only about 10–30% of the T-cell receptors generated by gene rearrangement will be able to recognize self peptide:self MHC complexes and thus function in self MHC-restricted responses to foreign antigens (see Chapter 4); those that have this capability are selected for survival in the thymus. Double-positive

1	MHC complexes and thus function in self MHC-restricted responses to foreign antigens (see Chapter 4); those that have this capability are selected for survival in the thymus. Double-positive cells also undergo negative selection: T cells whose receptors recognize self peptide:self MHC complexes too strongly undergo apoptosis, thus eliminating potentially self-reactive cells. In this section, we examine the interactions between developing double-positive thymocytes and different thymic components and discuss the mechanisms by which these interactions shape the mature T-cell repertoire.

1	8-19 Only thymocytes whose receptors interact with self peptide:self MHC complexes can survive and mature.

1	Early experiments using bone marrow chimeras (see Appendix I, Section A-32) and thymic grafting provided evidence that MHC molecules in the thymus influence the MHC-restricted T-cell repertoire. However, mice transgenic for rearranged T-cell receptor genes provided the first conclusive evidence that the interaction of the T cell with self peptide:self MHC complexes is necessary for the survival of immature T cells and their maturation into naive CD4 or CD8 T cells. For these experiments, the rearranged αand β-chain genes were cloned from a T-cell clone (see Appendix I, Section A-20) whose origin, antigen specificity, and MHC restriction were known. When such genes are introduced into the mouse genome, they are expressed early during thymocyte development. As a consequence of expressing functional transgene-encoded TCRα-and β-chain proteins in developing T cells, the rearrangement of endogenous T-cell receptor genes is inhibited, albeit to different degrees. In general, endogenous

1	functional transgene-encoded TCRα-and β-chain proteins in developing T cells, the rearrangement of endogenous T-cell receptor genes is inhibited, albeit to different degrees. In general, endogenous β-chain gene rearrangement is inhibited completely but that of α-chain genes is inhibited only incompletely. The result is that most of the developing thymocytes in TCR transgenic mouse lines express the T-cell receptor encoded by the transgenes.

1	By introducing T-cell receptor transgenes specific for a known peptide:MHC complex, the effect of allelic variations in MHC molecules on the maturation of thymocytes with receptors of known specificity can be studied directly, without the need for immunization and analysis of effector function. Such studies showed that thymocytes bearing a particular T-cell receptor could develop to the double-positive stage in thymuses that expressed different MHC molecules from those present in the mouse from which the original T-cell clone was isolated. However, these transgenic thymocytes developed into mature CD4 or CD8 single-positive thymocytes only if the thymus expressed the same self MHC molecule as that on which the original T-cell clone was selected (Fig. 8.26).

1	Such experiments also discovered the fate of T cells that fail positive selection. Rearranged receptor genes from a mature T cell specific for a peptide presented by a particular MHC molecule were introduced into a recipient mouse lacking that MHC molecule, and the fate of thymocytes was investigated by staining with antibodies specific for the transgenic receptor. Antibodies against other molecules, such as CD4 and CD8, were used at the same time to mark the stages of T-cell development. It was found that cells that fail to recognize the MHC molecules present on the thymic epithelium never progress further than the double-positive stage and die in the thymus within 3 or 4 days of their last division. 8-20 Positive selection acts on a repertoire of T-cell receptors with inherent specificity for MHC molecules.

1	Positive selection acts on a repertoire of T-cell receptors whose specificity is determined by randomly generated combinations of V, D, and J gene segments (see Section 5-7). Despite this, T-cell receptors exhibit a bias toward recognition of MHC molecules even before positive selection. If the specificity of the unselected repertoire were completely random, only a very small proportion of thymocytes would be expected to recognize any MHC molecule. However, an inherent specificity of T-cell receptors for MHC molecules has been detected by examining mature T cells that represent the unselected receptor repertoire. Such T cells can be produced in vitro from fetal thymuses that lack expression of MHC class I and MHC class II molecules by triggering generalized ‘positive selection’ using antibodies that bind to the Vβ chain of T-cell receptors and to the CD4 co-receptor. When such antibody-selected CD4 T cells are examined, roughly 5% can respond to any one MHC class II genotype. Because

1	that bind to the Vβ chain of T-cell receptors and to the CD4 co-receptor. When such antibody-selected CD4 T cells are examined, roughly 5% can respond to any one MHC class II genotype. Because these cells developed without selection by MHC molecules, this reactivity must reflect an inherent MHC-specificity encoded in the germline V gene segments. This specificity should significantly increase the proportion of receptors that can be positively selected by any individual’s MHC molecules.

1	The germline-encoded reactivity seems to be due to specific amino acids in the CDR1 and CDR2 regions of T-cell receptor Vβ and Vα regions. The CDR1 and CDR2 regions are encoded in the germline V gene segments and are highly

1	Fig. 8.26 Positive selection is demonstrated by the development of T cells expressing rearranged T-cell receptor transgenes. In mice transgenic for rearranged α:β T-cell receptor genes, the maturation of T cells depends on the MHC haplotype expressed in the thymus. If the transgenic mice express the same MHC haplotype in their thymic stromal cells as the mouse from which the rearranged TCRα-chain and TCRβ-chain genes originally developed (both MHCa, top panel), then the T cells expressing the transgenic T-cell receptor will develop from the double-positive stage (pale green) into mature T cells (dark green), in this case mature CD8+ single-positive cells. If the MHCa-restricted TCR transgenes are genetically crossed into a different MHC background (MHCb, yellow, bottom panel), then developing T cells expressing the transgenic receptor will progress to the double-positive stage but will fail to mature further. This failure is due to the absence of an interaction between the transgenic

1	T cells expressing the transgenic receptor will progress to the double-positive stage but will fail to mature further. This failure is due to the absence of an interaction between the transgenic T-cell receptor with MHC molecules on the thymic cortex, and thus no signal for positive selection is delivered, leading to apoptotic death by neglect.

1	Fig. 8.27 The MHC molecules that induce positive selection determine co-receptor specificity. In mice transgenic for T-cell receptors restricted by an MHC class I molecule (top panel), the mature T cells that develop all have the CD8 (red) phenotype. In mice transgenic for receptors restricted by an MHC class II molecule (bottom panel), all mature T cells have the CD4 (blue) phenotype. In both cases, normal numbers of immature, double-positive thymocytes (half blue, half red) are found. The specificity of the T-cell receptor determines the outcome of the developmental pathway, ensuring that the only T cells that mature are those equipped with a co-receptor that is able to bind the same self MHC molecule as the T-cell receptor.

1	variable (see Section 5-8). But among this variability, certain amino acids are conserved and common to many V segments. Analysis of numerous crystal structures has revealed that when the T-cell receptor binds a peptide:MHC complex, specific amino acids of the Vβ region interact with a particular part of the MHC protein. For example, in many human and mouse Vβ regions, the CDR2 has a tyrosine at position 48, and this interacts with a region in the middle of the α1 helix of MHC class I and class II proteins. Two other amino acids commonly found in other Vβ regions (tyrosine at 46 and glutamic acid at 54) interact with the same region of MHC. T cells expressing Vβ genes with mutations at any of these positions showed reduced positive selection, demonstrating that the interaction of such V regions with MHC molecules contributes to T-cell development.

1	8-21 Positive selection coordinates the expression of CD4 or CD8 with the specificity of the T-cell receptor and the potential effector functions of the T cell.

1	At the time of positive selection, the thymocyte expresses both CD4 and CD8 co-receptor molecules. By the end of thymic selection, mature α:β T cells ready for export to the periphery have stopped expressing one of these co-receptors. The majority of these cells belong to the conventional CD4 or CD8 T-cell lineages. Less abundant subsets, such as iNKT cells and a subset of regulatory T cells expressing CD4 and high levels of CD25, also develop in the thymus from CD4+CD8+ cells. Moreover, almost all mature T cells that express CD4 have receptors that recognize peptides bound to self MHC class II molecules and are programmed to become cytokine-secreting helper T cells. In contrast, most of the cells that express CD8 have receptors that recognize peptides bound to self MHC class I molecules and are programmed to become cytotoxic effector cells. Thus, positive selection also determines the cell-surface phenotype and functional potential of the mature T cell, selecting the appropriate

1	and are programmed to become cytotoxic effector cells. Thus, positive selection also determines the cell-surface phenotype and functional potential of the mature T cell, selecting the appropriate co-receptor for efficient antigen recognition and the appropriate program for the T cell’s eventual functional differentiation in an immune response.

1	Experiments with mice transgenic for rearranged T-cell receptor genes show clearly that the specificity of the T-cell receptor for self peptide:self MHC complexes determines which co-receptor a mature T cell will express. If the transgenes encode a T-cell receptor specific for antigen presented by self MHC class I molecules, mature T cells that express the transgenic receptor are CD8 T cells. Similarly, in mice made transgenic for a receptor that recognizes antigen with self MHC class II molecules, mature T cells that express the transgenic receptor are CD4 T cells (Fig. 8.27).

1	The importance of MHC molecules in this selection is illustrated by the human immunodeficiency diseases caused by mutations that lead to an absence of MHC molecules on lymphocytes and thymic epithelial cells. People who lack MHC class II molecules have CD8 T cells but only a few, highly abnormal CD4 T cells; a similar result has been obtained in mice in which MHC class II expression has been eliminated by targeted gene disruption (see Appendix I, Section A-35). Likewise, mice and humans that lack MHC class I molecules lack CD8 T cells. Thus, MHC class II molecules are absolutely required for CD4 T-cell development, whereas MHC class I molecules are similarly required for CD8 T-cell development.

1	In mature T cells, the co-receptor functions of CD8 and CD4 depend on their respective abilities to bind invariant sites on MHC class I and MHC class II molecules (see Section 4-18). Co-receptor binding to an MHC molecule is also required for normal positive selection, as shown for CD4 in the experiment discussed in the next section. In thymocytes, nearly all of the Lck is associated with CD4 and CD8 co-receptors, providing a mechanism to ensure that signaling is initiated only in thymocytes bearing T-cell receptors that bind to MHC molecules. Thus, positive selection depends on engagement of both the antigen receptor and co-receptor by an MHC molecule, and this signal determines the survival of single-positive cells that express only the appropriate co-receptor. Commitment to either the CD4 or CD8 lineage is coordinated with receptor specificity, and it seems that the developing thymocyte integrates signals from both the antigen receptor and the co-receptor. Co-receptor-associated

1	CD4 or CD8 lineage is coordinated with receptor specificity, and it seems that the developing thymocyte integrates signals from both the antigen receptor and the co-receptor. Co-receptor-associated Lck signals are most effectively delivered when CD4 rather than CD8 is engaged as a co-receptor, and these Lck signals play a large part in the decision to become a mature CD4 cell.

1	T-cell receptor signaling regulates this choice of the CD4 versus the CD8 lineage by controlling the expression of two transcription factors, ThPOK and Runx3 (see Fig. 8.18). The role of ThPOK was identified through a naturally occurring loss-of-function mutation in mice that lacked CD4 T-cell development. In mice lacking ThPOK, MHC class II-restricted thymocytes are redirected toward the CD8 lineage. ThPOK is not expressed in pre-selection double-positive thymocytes, but strong T-cell receptor signaling at this stage of development induces its expression. ThPOK, in turn, reinforces its own expression and represses expression of Runx3; together, the expression of ThPOK and absence of Runx3 lead to CD4 commitment and the ability to express cytokine genes typical of CD4 cells. If T-cell signaling is of insufficient strength or duration, however, ThPOK is not induced, and Runx3 is allowed to be expressed. This leads to silencing of CD4 expression, maintenance of CD8 expression, and the

1	is of insufficient strength or duration, however, ThPOK is not induced, and Runx3 is allowed to be expressed. This leads to silencing of CD4 expression, maintenance of CD8 expression, and the expression of genes typical of CD8 T cells, namely, genes that encode proteins involved in target-cell killing.

1	While the majority of double-positive thymocytes that undergo positive selection develop into either CD4 or CD8 single-positive T cells, the thymus also generates less numerous populations of other T-cell subsets with specialized functions; these will be discussed further in Section 8-26. 8-22 Thymic cortical epithelial cells mediate positive selection of developing thymocytes. Thymus transplantation studies indicate that stromal cells are important for positive selection. These cells form a web of cell processes that make close contacts with the double-positive T cells undergoing positive selection (see Fig. 8.17), and T-cell receptors can be seen clustering with MHC molecules at the sites of contact. Direct evidence that thymic cortical epithelial cells mediate positive selection comes from an ingenious manipulation of mice whose MHC class II genes have been eliminated by targeted gene disruption (Fig. 8.28).

1	Fig. 8.28 Thymic cortical epithelial cells mediate positive selection. In the thymus of normal mice (first panels), which express MHC class II molecules on epithelial cells in the thymic cortex (blue) as well as on medullary epithelial cells (orange) and bone marrow-derived cells (yellow), both CD4 (blue) and CD8 (red) T cells mature. Double-positive thymocytes are shown as half red and half blue. The second panels represent mutant mice in which MHC class II expression has been eliminated by targeted gene disruption; in these mice, few CD4 T cells develop, although CD8 T cells develop normally. In MHC class II-negative mice containing an MHC class II transgene engineered so that it is expressed only on the epithelial cells of the thymic cortex (third panels), normal numbers of CD4 T cells mature. In contrast, if a mutant MHC class II molecule with a defective CD4binding site is expressed (fourth panel), positive selection of CD4 T cells does not take place. This indicates that the

1	mature. In contrast, if a mutant MHC class II molecule with a defective CD4binding site is expressed (fourth panel), positive selection of CD4 T cells does not take place. This indicates that the cortical epithelial cells are the critical cell type mediating positive selection and that the MHC class II molecule needs to be able to interact with the CD4 protein.

1	Mutant mice that lack MHC class II molecules do not normally produce CD4 T cells. To test the role of the thymic epithelium in positive selection, an MHC class II gene was placed under the control of a promoter that restricted the gene’s expression to thymic cortical epithelial cells. This was then introduced as a transgene into the MHC class II-mutant mice, and CD4 T-cell development was restored. A variant of this experiment showed that, to promote the development of CD4 T cells, the MHC class II molecule on the thymic cortical epithelium must be able to interact effectively with CD4. Thus, when the MHC class II transgene expressed in the thymus contains a mutation that prevents binding of the MHC to CD4, very few CD4 T cells develop. Equivalent studies of CD8 interaction with MHC class I molecules showed that co-receptor binding is also necessary for the positive selection of CD8 cells.

1	The critical role of the thymic cortical epithelium in positive selection raises the question whether there is anything distinctive about the antigen-presenting properties of these cells. The thymic stromal cells may simply be in closest proximity to the developing thymocytes, as there are very few macrophages and dendritic cells in the cortex to perform the antigen presentation. In addition, however, thymic epithelium differs from other tissues in the expression of key proteases that are involved in MHC class I and II antigen processing (see Section 6-8). Cortical epithelial cells express cathepsin L as opposed to the more widely expressed cathepsin S, and mice deficient in cathepsin L have severely impaired CD4 T-cell development. Thymic epithelial cells from mice lacking cathepsin L exhibit a relatively high proportion of MHC class II molecules on their surface that retain the class II invariant chain-associated peptide (CLIP) (see Fig. 6.11). Cortical epithelial cells also express

1	a relatively high proportion of MHC class II molecules on their surface that retain the class II invariant chain-associated peptide (CLIP) (see Fig. 6.11). Cortical epithelial cells also express a unique proteasome subunit, β5T, whereas other cells express β5 or β5i. Mice deficient in β5T have severely impaired CD8 T-cell development. Because mice that lack either cathepsin L or β5T still have normal levels of MHC on the surface of their thymic cortical cells, it would seem that it is the peptide repertoire displayed by the MHC molecules on cortical epithelial cells that is responsible for altering CD8 T-cell development, although the mechanism is still unclear.

1	8-23 T cells that react strongly with ubiquitous self antigens are deleted in the thymus. When the T-cell receptor of a mature naive T cell is strongly ligated by a peptide:MHC complex displayed on a specialized antigen-presenting cell in a peripheral lymphoid organ, the T cell is activated to proliferate and produce effector T cells. In contrast, when the T-cell receptor of a developing thymocyte is similarly ligated by a self peptide:self MHC complex in the thymus, it dies by apoptosis (Fig. 8.29). The response of immature T cells to stimulation by antigen is the basis of negative selection. Elimination of immature T cells in the thymus prevents their potentially harmful activation later, should they encounter the same self peptides when they are mature T cells.

1	Negative selection has been demonstrated using TCR-transgenic mice expressing T-cell receptors specific for self peptides derived from proteins encoded on the Y chromosome, and thus expressed only in male mice. Thymocytes bearing these receptors disappear from the developing T-cell population in male mice at the double-positive stage of development, and no single-positive cells bearing the transgenic receptors mature. By contrast, in female mice, which lack the male-specific peptide, T cells bearing the transgenic receptors mature normally. Negative selection to male-specific peptides has also been demonstrated in nontransgenic mice and also occurs by deletion of T cells.

1	TCR transgenic mice were very useful for the classic experiments above, but they express a functional T-cell receptor earlier during development than normal mice and have a very high frequency of cells reactive to any particular peptide. A more realistic system for evaluating negative selection involves the transgenic expression of only the β chain of a T-cell receptor reactive to a given peptide antigen. In such mice, the β chain pairs with endogenous α chains, yet the frequency of peptide-reactive T cells is sufficient for detection using peptide:MHC tetramers (see Appendix I, Section A-24). These and other more physiologic approaches showed that clonal deletion can occur at either the double-positive or the single-positive stage, presumably depending on where the T cell encounters the antigen that causes deletion.

1	These experiments illustrate the principle that self peptide:self MHC complexes encountered in the thymus purge the mature T-cell repertoire of cells bearing self-reactive receptors. One obvious problem with this scheme is that many tissue-specific proteins, such as pancreatic insulin, would not be expected to be expressed in the thymus. However, it is now clear that many such ‘tissue-specific’ proteins are expressed by certain stromal cells in the thymic medulla; thus, intrathymic negative selection could apply even to proteins that are otherwise restricted to tissues outside the thymus. The expression of some, but not all, tissue-specific proteins in the thymic medulla is controlled by a gene called AIRE (autoimmune regulator). AIRE is expressed in medullary stromal cells (Fig. 8.30), interacts with many proteins involved in transcription, and seems to lengthen transcripts that would otherwise terminate earlier. Mutations in AIRE give rise to the human autoimmune disease known as

1	interacts with many proteins involved in transcription, and seems to lengthen transcripts that would otherwise terminate earlier. Mutations in AIRE give rise to the human autoimmune disease known as autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) or autoimmune polyglandular syndrome type I, highlighting the important role of intrathymic expression of tissue-specific proteins in maintaining tolerance to self. Negative selection of developing T cells involves interactions with ubiquitous and tissue-restricted self antigens, and can take place in both the thymic cortex and the thymic medulla (see Fig. 8.29).

1	It is unlikely that all possible self proteins are expressed in the thymus. Thus, negative selection in the thymus may not remove all T cells reactive to self antigens that appear exclusively in other tissues or are expressed at different stages

1	Fig. 8.29 Negative selection of thymocytes can occur in the cortex or the medulla. When the T-cell receptor (TCR) on a developing thymocyte is strongly stimulated by recognition of self peptide:self MHC complexes (red cell), the thymocyte is induced to die, a process known as negative selection. Negative selection can occur in the cortex when a CD4+CD8+ double-positive thymocyte has strong reactivity to peptide:MHC complexes found on cortical epithelial cells (left panel). Negative selection can also occur in the medulla when an immature CD4 or CD8 single-positive thymocyte receives strong T-cell receptor signaling following recognition of peptide:MHC complexes on medullary epithelial cells (middle panel) or on bone marrow-derived macrophages or dendritic cells (right panel). AIRE expression in the thymus Fig. 8.30 AIRE is expressed in the medulla of the thymus and promotes the expression of proteins normally expressed in peripheral tissues.

1	AIRE expression in the thymus Fig. 8.30 AIRE is expressed in the medulla of the thymus and promotes the expression of proteins normally expressed in peripheral tissues. Expression of AIRE by thymic medullary cells is limited to the medullary region of the thymus, where it is expressed in a subset of epithelial-like cells. The expression of the thymic medullary epithelial marker MTS10 is shown in red. AIRE expression is shown in green by immunofluorescence, and is present in only a fraction of the medullary epithelial cells. Photograph courtesy of R.K. Chin and Y.-X. Fu. in development. There are, however, several mechanisms operating in the periphery that can prevent mature T cells from responding to tissue-specific antigens; these are discussed in Chapter 15, when we consider the problem of autoimmune responses and their avoidance. 8-24 Negative selection is driven most efficiently by bone marrow-derived antigen-presenting cells.

1	8-24 Negative selection is driven most efficiently by bone marrow-derived antigen-presenting cells. As discussed above, negative selection occurs throughout thymocyte development, both in the thymic cortex and in the medulla, and so is likely to be mediated by antigen presentation by several different cell types (see Fig. 8.29). There does seem to be a hierarchy in the effectiveness of cells in mediating negative selection. At the top are bone marrow-derived dendritic cells and macrophages. These are antigen-presenting cells that also activate mature T cells in peripheral lymphoid tissues, as we shall see in Chapter 9. The self antigens presented by these cells are therefore the most important source of potential autoimmune responses, and T cells responding to such self peptides must be eliminated in the thymus.

1	In addition, both the thymocytes themselves and the thymic epithelial cells can cause the deletion of self-reactive cells. The medullary epithelial cells expressing AIRE, and thus presenting a wide range of self-antigens, are one population that has been shown to directly induce thymocyte negative selection. More generally, in patients undergoing bone marrow transplantation from an unrelated donor, where all the thymic macrophages and dendritic cells are of donor type, negative selection mediated by thymic epithelial cells is of critical importance in maintaining tolerance to the recipient’s own antigens. 8-25 The specificity and/or the strength of signals for negative and positive selection must differ.

1	T cells undergo both positive selection for self MHC restriction and negative selection for self-tolerance by interacting with self peptide:self MHC complexes expressed on stromal cells in the thymus. An unresolved issue is how the interaction of the T-cell receptor with self peptide:self MHC complexes distinguishes between these opposite outcomes. First, more receptor specificities must be positively selected than are negatively selected. Otherwise, all the cells that were positively selected in the thymic cortex would be eliminated by negative selection, and no T cells would ever be produced. Second, the consequences of the interactions that lead to positive and negative selection must differ: cells that recognize self peptide:self MHC complexes on cortical epithelial cells are induced to mature, whereas those whose receptors might confer strong and potentially damaging autoreactivity are induced to die.

1	Currently, the choice between positive and negative selection is thought to hinge on the strength of self peptide:self MHC binding by the T-cell receptor, an idea known as the affinity hypothesis (Fig. 8.31). Low-affinity interactions rescue the cell from death by neglect, leading to positive selection; high-affinity interactions induce apoptosis and thus negative selection. Because more complexes are likely to bind weakly than strongly, this model explains the positive selection of a larger repertoire of cells than are negatively selected. Using T-cell receptor transgenic thymocytes, it was shown that variants of the antigenic peptide could induce positive selection in thymic organ cultures or in vivo. Peptide variants that induced positive selection had a lower affinity for the T-cell receptor than did antigenic peptide. How this quantitative difference in receptor affinity translates into a qualitatively distinct cell fate is still an area of active investigation. Many of the

1	receptor than did antigenic peptide. How this quantitative difference in receptor affinity translates into a qualitatively distinct cell fate is still an area of active investigation. Many of the biochemical signals induced by low-affinity interactions are weaker or of shorter duration than those from high-affinity interactions. However, low-affinity interactions lead to sustained

1	Fig. 8.31 The affinity model of T-cell positive and negative selection. Random TCRα and β chain gene rearrangements generate a large pool of immature thymocytes expressing a varied repertoire of specificities. The T-cell receptors on many of these cells fail to have sufficient binding strength to the self peptide:self MHC complexes on thymic epithelium and so receive no signals. These cells die by neglect. Another fraction of immature thymocytes are positively selected because their T-cell receptors bind with sufficient strength to the self peptide:self MHC complexes on thymic epithelium to generate T-cell receptor-dependent survival signals. From this cohort of positively selected thymocytes, negative selection removes those thymocytes whose receptors have excessively strong reactivity to self peptides complexed with self MHC molecules (resulting in clonal deletion), thereby establishing self-tolerance of the mature T-cell population. A small subset of positively selected cells

1	to self peptides complexed with self MHC molecules (resulting in clonal deletion), thereby establishing self-tolerance of the mature T-cell population. A small subset of positively selected cells receiving signals slightly weaker than those inducing negative selection differentiate into regulatory T cells (Tregs), a process referred to as agonist selection.

1	activation of the protein kinase Erk, whereas high-affinity interactions lead to only transient activation of Erk, suggesting that differential activation of this or other MAPKs might determine the outcome of thymic selection. Indeed, experiments showed that developing T cells need to engage low-affinity ligands for more than 24 hours for positive selection to occur. 8-26 Self-recognizing regulatory T cells and innate T cells develop in the thymus. Additional populations besides the conventional CD4+ and CD8+ α:β T cells discussed above emerge from the thymus; they are numerically minor but functionally important. Two of these subsets, the T cells (see Section 9-23) reg and the iNKT cells (see Section 6.18), have been well studied, and found to each have unique developmental requirements.

1	Thymically derived T cells are a subset of CD4+ T cells that function to main- reg tain self-tolerance. These cells arise from CD4+CD8+ thymocytes, as do conventional T cells. During their maturation, they upregulate the transcription factor FoxP3. T cell development also depends on IL-2 receptor signaling, a cytokine signal that is not required for the development of conventional T cells. The repertoire of T-cell receptors expressed on T cells is thought to be composed of receptors with high affinity for self MHC:self peptide complexes. Evidence supporting this conclusion comes from studies showing that some lines of TCR transgenic mice generate large numbers of T cells when the mice also express the antigen for this T-cell receptor. In addition, studies using mice expressing a fluorescent reporter that monitors T-cell receptor signal strength have shown that T cells express high levels of the fluorescent reporter, both during their development and after their export from the thymus,

1	reporter that monitors T-cell receptor signal strength have shown that T cells express high levels of the fluorescent reporter, both during their development and after their export from the thymus, indicating that they likely express T-cell receptors with high affinity for self. This process of positive selection following high-affinity T-cell receptor interactions with self peptide:self MHC complexes has been termed agonist selection—in other words, agonist selection refers to interactions of a T-cell receptor with a self peptide:self MHC that would normally activate a mature T cell expressing that T-cell receptor.

1	A second specialized subset of T cells that develops from CD4+CD8+ thymocyte precursors is a lineage known as invariant NKT cells (iNKT cells), based on their expression of the NK1.1 receptor commonly found on NK cells. iNKT cells are activated as part of the early response to many infections; they differ from the major lineage of α:β T cells in recognizing CD1 molecules rather than MHC class I or MHC class II molecules (see Section 6-18). Unlike other T cells, iNKT cells require for their development a T-cell receptor interaction with CD1 molecules expressed on thymocytes and a signal through the adaptor protein SAP. iNKT cells, like γ:δ T cells, acquire a defined effector program during their development in the thymus. Therefore, these cells exhibit a memory-cell phenotype when they leave the thymus and

1	After 3–4 days in the medulla, CD4 and CD8 T cells upregulate SIP1 and exit the thymus into the blood blood vessel SIPSIPR1 CD4 and CD8 T cells entering the medulla are not fully mature Fig. 8.32 Thymocyte emigration is induced by signaling through the sphingosine 1-phosphate receptor, S1PR1. CD4 and CD8 single-positive thymocytes that have successfully survived positive and negative selection are found in the medulla but are not yet fully mature. At the termination of the maturation process, which takes 3–4 days, the CD4 and CD8 single-positive thymocytes upregulate the sphingosine 1-phosphate (S1P) receptor, known as S1PR1. S1PR1 is a G-proteincoupled receptor that promotes chemotaxis of the cells toward the ligand S1P. Due to the high levels of S1P in the blood, single-positive thymocytes are induced to leave the thymus by entering the blood, where they become part of the recirculating naive T-cell population.

1	MOVIE 8.1 migrate to peripheral lymphoid tissues and mucosal surfaces. iNKT cells have been suggested to develop in response to ‘agonist’ signaling. Recent studies have revealed that CD1-binding lipid antigens produced by the commensal microbes in the gut are an important source of these agonist ligands, and that the composition of the gut microbiome regulates the development of iNKT cells early in life. Since agonist stimulation of immature T cells is also known to cause clonal deletion, it is not yet clear which activating interactions lead to clonal deletion in the thymus and which lead to selection of Treg cells or the nonconventional iNKT cells. 8-27 The final stage of T-cell maturation occurs in the thymic medulla.

1	After surviving positive and negative selection, thymocytes complete their maturation in the thymic medulla and then emigrate to peripheral lymphoid organs. Their final maturation results in changes to the T-cell receptor signaling machinery. Whereas an immature double-positive or single-positive thymocyte stimulated through the T-cell receptor will undergo apoptosis, a mature single-positive thymocyte responds by proliferating. The final maturation stage takes less than 4 days, and functionally competent T cells then emigrate from the thymus into the bloodstream (Fig. 8.32). Emigration requires recognition of the lipid molecule sphingosine 1-phosphate (S1P) by the G-proteincoupled receptor S1PR1, which is expressed by thymocytes during their final maturation. S1P is present in high concentration in blood and lymph, and mature thymocytes seem to be drawn toward it. Mature thymocytes also express CD62L (L-selectin), a lymph-node homing receptor that facilitates the localization of

1	in blood and lymph, and mature thymocytes seem to be drawn toward it. Mature thymocytes also express CD62L (L-selectin), a lymph-node homing receptor that facilitates the localization of mature naive T cells to peripheral lymphoid organs after their emigration from the thymus.

1	8-28 T cells that encounter sufficient quantities of self antigens for the first time in the periphery are eliminated or inactivated.

1	Many autoreactive T cells are purged during their development in the thymus. As discussed in Section 8-23, this negative selection process is facilitated by the AIRE protein, which promotes the expression of many tissue-specific antigens in thymic medullary epithelial cells. Nonetheless, not all self antigens are expressed in the thymus, and some autoreactive T cells complete their maturation and migrate to the periphery. Our understanding of the fates of autoreactive T cells in the periphery comes mainly from the study of mice transgenic for self-reactive T-cell receptors. In some cases, T cells reacting to self antigens in the periphery are eliminated. This usually follows a brief period of activation and cell division, and so is known as activation-induced cell death. In other cases, the self-reactive cells may be rendered anergic. When studied in vitro, these anergic T cells prove refractory to signals delivered through the T-cell receptor.

1	The question immediately arises: if the encounter of a mature naive lymphocyte with a self antigen leads to cell death or anergy, why does this not also happen to a mature lymphocyte that recognizes a pathogen-derived antigen? The answer is that infection sets up inflammation, which induces the expression of co-stimulatory molecules on the antigen-presenting dendritic cells and the production of cytokines promoting lymphocyte activation. The outcome of an encounter with antigen in these conditions is the activation, proliferation, and differentiation of the lymphocyte to effector-cell status. In the absence of infection or inflammation, dendritic cells still process and present self antigens, but in the absence of co-stimulatory and other signals, any interaction of a mature lymphocyte with its specific antigen seems to result in a tolerance-inducing (tolerogenic) signal from the antigen receptor. Summary.

1	The stages of thymocyte development up to the expression of the pre-T-cell receptor—including the decision between commitment to either the α:β or the γ:δ lineage—are all independent of interactions with peptide:MHC antigens. With the successful rearrangement of α-chain genes and expression of the T-cell receptor, α:β thymocytes undergo further development that is determined by the interactions of their T-cell receptors with self peptides presented by the MHC molecules on the thymic stroma. CD4+CD8+ double-positive thymocytes whose receptors interact with self peptide:self MHC complexes on thymic cortical epithelial cells are positively selected, and will eventually mature into CD4 or CD8 single-positive cells. T cells that react too strongly with self antigens are deleted in the thymus, a process driven by bone marrow-derived antigen-presenting cells and AIRE-expressing epithelial cells in the medullary region of the thymus. The outcome of positive and negative selection is the

1	a process driven by bone marrow-derived antigen-presenting cells and AIRE-expressing epithelial cells in the medullary region of the thymus. The outcome of positive and negative selection is the generation of a mature conventional T-cell repertoire that is both MHC-restricted and self-tolerant. Some non-conventional T-cell lineages undergo ‘agonist’ selection following strong T-cell receptor signaling. Precisely how the recognition of self peptide:self MHC ligands by the T-cell receptor leads to either positive or negative selection remains an unsolved problem.

1	Summary to Chapter 8.

1	In this chapter we have learned about the formation of the B-cell and T-cell lineages from an uncommitted hematopoietic stem cell. The somatic gene rearrangements that generate the highly diverse repertoire of antigen receptors—immunoglobulin for B cells, and the T-cell receptor for T cells— occur in the early stages of the development of T cells and B cells from a common bone marrow-derived lymphoid progenitor. Mammalian B-cell development takes place in fetal liver and, after birth, in the bone marrow; T cells also originate from stem cells in the fetal liver or the bone marrow, but undergo most of their development in the thymus. Much of the somatic recombination machinery, including the RAG proteins that are an essential part of the V(D)J recombinase, is common to both B and T cells. In both B and T cells, gene rearrangements begin with the loci that contain D gene segments, and proceed successively at each locus. The first step in B-cell development is the rearrangement of the

1	In both B and T cells, gene rearrangements begin with the loci that contain D gene segments, and proceed successively at each locus. The first step in B-cell development is the rearrangement of the locus for the immunoglobulin heavy chain, and for T cells the β chain. In each case, the developing cell is allowed to proceed to the next stage of development only if the rearrangement has produced an in-frame sequence that can be translated into a protein expressed on the cell surface: either the pre-B-cell receptor or the pre-T-cell receptor. Cells that do not generate successful rearrangements for both receptor chains die by apoptosis. The course of conventional B-cell development is summarized in Fig. 8.14, and that of α:βT cells in Fig. 8.33.

1	Once a functional antigen receptor has appeared on the cell surface, the lymphocyte is tested in two ways. Positive selection tests for the potential usefulness of the antigen receptor, whereas negative selection removes self-reactive cells from the lymphocyte repertoire, rendering it tolerant to the antigens of the body. Positive selection is particularly important for T cells, because it ensures that only cells bearing T-cell receptors that can recognize antigen in combination with self MHC molecules will continue to mature. Positive selection also coordinates the choice of co-receptor expression. CD4 becomes expressed by T cells harboring MHC class II-restricted receptors, and CD8 by cells harboring MHC class I-restricted receptors. This ensures the optimal use of these receptors in responses to pathogens. For B cells, positive selection seems to occur at the final transition from immature to mature B cells, which occurs in peripheral lymphoid tissues. Tolerance to self antigens is

1	to pathogens. For B cells, positive selection seems to occur at the final transition from immature to mature B cells, which occurs in peripheral lymphoid tissues. Tolerance to self antigens is enforced by negative selection at different stages throughout the development of both B and T cells, and positive selection likewise seems to represent a continuous process.

1	Fig. 8.33 A summary of the development of human α:β T cells. The state of the T-cell receptor genes, the expression of some essential intracellular proteins, and the expression of some cell-surface molecules are shown for successive stages of α:β T-cell development. Note that because the T-cell receptor genes do not undergo further changes during antigen-driven development, only the phases during which they are actively undergoing rearrangement in the thymus are indicated. The antigen-dependent phases of CD4 and CD8 cells are depicted separately, and are detailed in Chapter 9.

1	TERMINALDIFFERENTIATIONTERMINALANTIGENDEPENDENTDIFFERENTIATIONANTIGENDEPENDENTStem cell Early double-negative thymocyte Late double-negative thymocyte Early double-positive thymocyte Late double-positive thymocyte Naive CD4 T cell Memory CD4 T cell Effector CD4 T cell Naive CD8 T cell Memory CD8 T cell Effector CD8 T cell ˜-chain gene rearrangements T cells °-chain gene rearrangements Intra-cellular proteins Surface marker proteinsANTIGENINDEPENDENTpre-T receptor D–J rearranged V–DJ rearranged V–J rearranged Germline Germline CD34? CD2 HSA CD44hi RAG-1 RAG-2 TdT Lck ZAP-70 RAG-1 RAG-2 TdT Lck ZAP-70 RAG-1 RAG-2 Lck ZAP-70 Lck ZAP-70 LKLF Lck ZAP-70 CD25 CD44lo HSA PT˜CD4 CD8 HSA CD4 CD62L CD45RA CD5 CD69 CD4 CD8 HSA CD4 CD45RO CD44 CD4 CD45RO CD44hi Fas FasL (type 1) CD8 CD45RA CD8 CD45RO CD44 FasL Fas CD8 CD44hi TH17: IL-17 TH1: IFN-°TH2: IL-4 IFN-°granzyme perforin Germline Germline T-cell receptor Questions.

1	Questions. 8.1 True or False: B-cell development is not affected in mice that are lacking the cytokine receptor common γ chain (γ-c). 8.2 Fill-in-the-Blanks: B-cell development is regulated by the expression of various transcription factors that enable gene rearrangement and the successful progression into a new developmental stage. For example, during the _________ stage, Rag-1 and Rag-2 expression is induced by __________, which permits the successful D to J rearrangement and then V to DJ rearrangement of the heavy-chain locus. As a consequence, a functional __________ is expressed, and upon signaling, the cell is instructed to perform _________ and progress toward the next developmental step and rearrange the light-chain locus. 8.3 True or False: Self antigen recognition is needed in order to cross-link the pre-B-cell receptor, which in turn allows this complex to signal and permit the transition from pro-B cell to pre-B cell.

1	8.4 Matching: Match the B-cell stage with the proper description: A. Early pro-B cell i. V-DJ rearranging (heavy chain) B. Small pre-B cell ii. D-J rearranging (heavy chain) C. Immature B cell iii. Expressing pre-B-cell receptor D. Late pro-B cell iv. V-J rearranging (light chain) E. Large pre-B cell v. Surface IgM 8.5 Short Answer: How does the process of allelic exclusion prevent the rearrangement of the second heavy-chain locus, and why is this important? 8.6 Short Answer: How can one large pre-B cell give rise to multiple B cells with different antigen specificities? 8.7 Matching: Match the following terms to the appropriate definition: A. Receptor editing i. Result of persistent autoreactivity after failure of successful receptor editing B. Isotypic exclusion ii. Selection of either the κ or the λ light chain C. Clonal deletion iii. Result of a peripheral encounter of a weakly cross-linking or low-valence antigen

1	B. Isotypic exclusion ii. Selection of either the κ or the λ light chain C. Clonal deletion iii. Result of a peripheral encounter of a weakly cross-linking or low-valence antigen D. Anergy iv. Process by which the light-chain locus is rearranged in order to produce a nonautoreactive receptor E. Immunological v. B cells that have affinity for ignorance a self-antigen but for various reasons do not respond to it 8.8 True or False: All CD4 and CD8 double-negative thymocytes are immature T cells. 8.9 Matching: Match the correct expression of CD44 and CD25 and the T-cell receptor locus rearrangement status with the appropriate DN T-cell stage: A. DN1 i. CD44+CD25+, D to J TCRβ-chain locus rearrangement B. DN2 ii. CD44+CD25–, germline T-cell receptor locus C. DN3 iii. CD44lowCD25+, V to DJ β-chain locus rearrangement

1	A. DN1 i. CD44+CD25+, D to J TCRβ-chain locus rearrangement B. DN2 ii. CD44+CD25–, germline T-cell receptor locus C. DN3 iii. CD44lowCD25+, V to DJ β-chain locus rearrangement D. DN4 iv. CD44–CD25–, functional β-chain rearrangement 8.10 Fill-in-the-Blanks: Successful rearrangement of the ______ during the DN__ stage permits the formation of the pre-T-cell receptor, which is analogous in structure and function to the pre-B-cell receptor. The TCRβ chain associates itself with the _______, which allows ligand-independent cross-linking of the pre-T-cell receptor, causing __________, the arrest of further ________ gene rearrangement, and the expression of both _______. As with the B-cell light-chain locus, the _________ can undergo multiple rearrangements to produce a functional protein. 8.11 Matching: Match the following subsets of murine γ:δ T cells with the appropriate description:

1	8.11 Matching: Match the following subsets of murine γ:δ T cells with the appropriate description: A. Dendritic i. Can be divided in two groups: epidermal IFN-γ and IL-4 producing, and IFN-γ T cells producing subsets B. Vγ4+ ii. Cells that home to the reproductive tract, lung, and dermis; upon stimulation these can produce inflammatory cytokines C. Vγ6+ T cells iii. A population of later developing γ:δ T cells programmed to secrete IL-17 when activated, and can be found in all lymphoid organs, as well as the dermis D. Vγ1+ T cells iv. Cells that, as a response to a pathogen or a wound, can induce inflammation, promote wound healing, and produce growth factors; also characterized by their T-cell receptors’ use of the Vγ5 segment E. Vγ7+ T cells v. Specifically home to the intestinal epithelium 8.12 Multiple Choice: Which of the following options correctly describes a difference between the B-cell receptor and the T-cell receptor?

1	A. VDJ rearrangement of the T-cell receptor β chain occurs first in T-cell development, as opposed to the B-cell receptor, which undergoes VDJ rearrangement after light-chain VJ rearrangement. B. T cells do not require the formation of a pre-T-cell receptor in order to advance their development, as opposed to B cells, which require signaling through the pre-B-cell receptor in order to undergo allelic exclusion and continue development. C. Expression of the B-cell receptor stops further light-chain rearrangement and enforces strict allelic exclusion, while expression of the T-cell receptor does not restrict further rearrangements of the alpha chain until there is signaling through peptide:MHC binding, resulting in many T cells that express two different TCRα chains. D. TCRα chains cannot undergo successive rearrangements, as opposed to B-cell receptors, which go through the process of receptor editing.

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1	Zerrahn, J., Held, W., and Raulet, D.H.: The MHC reactivity of the T cell repertoire prior to positive and negative selection. Cell 1997, 88:627–636. 8-21 Positive selection coordinates the expression of CD4 or CD8 with the specificity of the T-cell receptor and the potential effector functions of the T cell. Egawa, T., and Littman, D.R.: ThPOK acts late in specification of the helper T cell lineage and suppresses Runx-mediated commitment to the cytotoxic T cell lineage. Nat. Immunol. 2008, 9:1131–1139. He, X., Xi, H., Dave, V.P., Zhang, Y., Hua, X., Nicolas, E., Xu, W., Roe, B.A., and Kappes, D.J.: The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment. Nature 2005, 433:826–833. Singer, A., Adoro, S., and Park, J.H.: Lineage fate and intense debate: myths, models and mechanisms of CD4versus CD8-lineage choice. Nat. Rev. Immunol. 2008, 8:788–801.

1	Singer, A., Adoro, S., and Park, J.H.: Lineage fate and intense debate: myths, models and mechanisms of CD4versus CD8-lineage choice. Nat. Rev. Immunol. 2008, 8:788–801. von Boehmer, H., Kisielow, P., Lishi, H., Scott, B., Borgulya, P., and Teh, H.S.: The expression of CD4 and CD8 accessory molecules on mature T cells is not random but correlates with the specificity of the α:β receptor for antigen. Immunol. Rev. 1989, 109:143–151. 8-22 Thymic cortical epithelial cells mediate positive selection of developing thymocytes. Cosgrove, D., Chan, S.H., Waltzinger, C., Benoist, C., and Mathis, D.: The thymic compartment responsible for positive selection of CD4+ T cells. Int. Immunol. 1992, 4:707–710. Ernst, B.B., Surh, C.D., and Sprent, J.: Bone marrow-derived cells fail to induce positive selection in thymus reaggregation cultures. J. Exp. Med. 1996, 183:1235–1240.

1	Ernst, B.B., Surh, C.D., and Sprent, J.: Bone marrow-derived cells fail to induce positive selection in thymus reaggregation cultures. J. Exp. Med. 1996, 183:1235–1240. Murata, S., Sasaki, K., Kishimoto, T., Niwa, S.-I., Hayashi, H., Takahama, Y., and Tanaka, K.: Regulation of CD8+ T cell development by thymus-specific proteasomes. Science 2007, 316:1349–1353. Nakagawa, T., Roth, W., Wong, P., Nelson, A., Farr, A., Deussing, J., Villadangos, J.A., Ploegh, H., Peters, C., and Rudensky, A.Y.: Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus. Science 1998, 280:450–453. 8-23 T cells that react strongly with ubiquitous self antigens are deleted in the thymus. Anderson, M.S., Venanzi, E.S., Klein, L., Chen, Z., Berzins, S.P., Turley, S.J., von Boehmer, H., Bronson, R., Dierich, A., Benoist, C., et al.: Projection of an immunological self shadow within the thymus by the aire protein. Science 2002, 298:1395–1401.

1	Kishimoto, H., and Sprent, J.: Negative selection in the thymus includes semimature T cells. J. Exp. Med. 1997, 185:263–271. Zal, T., Volkmann, A., and Stockinger, B.: Mechanisms of tolerance induction in major histocompatibility complex class II-restricted T cells specific for a blood-borne self antigen. J. Exp. Med. 1994, 180:2089–2099. 8-24 Negative selection is driven most efficiently by bone marrow-derived antigen-presenting cells. Anderson, M.S., and Su, M.A.: Aire and T cell development. Curr. Opin. Immunol. 2011, 23:198–206. McCaughtry, T.M., Baldwin, T.A., Wilken, M.S., and Hogquist, K.A.: Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla. J. Exp. Med. 2008, 205:2575–2584. Sprent, J., and Webb, S.R.: Intrathymic and extrathymic clonal deletion of T cells. Curr. Opin. Immunol. 1995, 7:196–205. Webb, S.R., and Sprent, J.: Tolerogenicity of thymic epithelium. Eur. J. Immunol. 1990, 20:2525–2528.

1	Webb, S.R., and Sprent, J.: Tolerogenicity of thymic epithelium. Eur. J. Immunol. 1990, 20:2525–2528. 8-25 The specificity and/or the strength of signals for negative and positive selection must differ. Alberola-Ila, J., Hogquist, K.A., Swan, K.A., Bevan, M.J., and Perlmutter, R.M.: Positive and negative selection invoke distinct signaling pathways. J. Exp. Med. 1996, 184:9–18. Ashton-Rickardt, P.G., Bandeira, A., Delaney, J.R., Van Kaer, L., Pircher, H.P., Zinkernagel, R.M., and Tonegawa, S.: Evidence for a differential avidity model of T-cell selection in the thymus. Cell 1994, 76:651–663. Bommhardt, U., Scheuring, Y., Bickel, C., Zamoyska, R., and Hunig, T.: MEK activity regulates negative selection of immature CD4+CD8+ thymocytes. J. Immunol. 2000, 164:2326–2337. Hogquist, K.A., Jameson, S.C., Heath, W.R., Howard, J.L., Bevan, M.J., and Carbone, F.R.: T-cell receptor antagonist peptides induce positive selection. Cell 1994, 76:17–27.

1	Hogquist, K.A., Jameson, S.C., Heath, W.R., Howard, J.L., Bevan, M.J., and Carbone, F.R.: T-cell receptor antagonist peptides induce positive selection. Cell 1994, 76:17–27. 8-26 Self-recognizing regulatory T cells and innate T cells develop in the thymus. Jordan, M.S., Boesteanu, A., Reed, A.J., Petrone, A.L., Holenbeck, A.E., Lerman, M.A., Naji, A., and Caton, A.J.: Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2001, 2:301–306. Moran, A.E., and Hogquist, K.A.: T-cell receptor affinity in thymic development. Immunology 2012, 135:261–267. Zheng, Y., and Rudensky, A.Y.: Foxp3 in control of the regulatory T cell lineage. Nat. Immunol. 2007, 8:457–462. 8-27 The final stage of T-cell maturation occurs in the thymic medulla.

1	Zheng, Y., and Rudensky, A.Y.: Foxp3 in control of the regulatory T cell lineage. Nat. Immunol. 2007, 8:457–462. 8-27 The final stage of T-cell maturation occurs in the thymic medulla. Matloubian, M., Lo, C.G., Cinamon, G., Lesneski, M.J., Xu, Y., Brinkmann, V., Allende, M.L., Proia, R.L., and Cyster, J.G.: Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 2004, 427:355–360. Zachariah, M.A., and Cyster, J.G.: Neural crest-derived pericytes promote egress of mature thymocytes at the corticomedullary junction. Science 2010, 328:1129–1135. 8-28 T cells that encounter sufficient quantities of self antigens for the first time in the periphery are eliminated or inactivated. Fink, P.J., and Hendricks, D.W.: Post-thymic maturation: young T cells assert their individuality. Nat. Rev. Immunol. 2011, 11:544–549.

1	Fink, P.J., and Hendricks, D.W.: Post-thymic maturation: young T cells assert their individuality. Nat. Rev. Immunol. 2011, 11:544–549. Steinman, R.M., and Nussenzweig, M.C.: Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proc. Natl Acad. Sci. USA 2002, 99:351–358. Xing, Y., and Hogquist, K.A.: T-cell tolerance: central and peripheral. Cold Spring Harb. Perspect. Biol. 2012, 4.pii:a006957

1	An adaptive immune response is initiated when a pathogen overwhelms innate defense mechanisms. As the pathogen replicates and antigen accumulates, sensor cells of the innate immune system become activated to trigger the adaptive immune response. While some infections may be dealt with solely by innate immunity, as discussed in Chapters 2 and 3, host defense against most pathogens, almost by definition, requires recruitment of adaptive immunity. This is shown by the immunodeficiency syndromes that are associated with failure of particular parts of the adaptive immune response; these will be discussed in Chapter 13. In the next three chapters, we will learn how the adaptive immune response involving antigen-specific T cells and B cells is initiated and deployed. T-cell responses that lead to cellular immunity will be considered first, in this chapter; and B-cell responses that lead to antibody-mediated, or humoral, immunity will be considered in Chapter 10. In Chapter 11 we will

1	lead to cellular immunity will be considered first, in this chapter; and B-cell responses that lead to antibody-mediated, or humoral, immunity will be considered in Chapter 10. In Chapter 11 we will consider the dynamics of T-cell and B-cell responses in the context of their integration with innate immunity and how this culminates in one of the most important features of adaptive immunity—immunological memory.

1	Once T cells have completed their primary development in the thymus, they enter the bloodstream. On reaching a secondary lymphoid organ, they leave the blood to migrate through the lymphoid tissue, returning via the lymphatics to the bloodstream to recirculate between blood and secondary lymphoid tissues. Mature recirculating T cells that have not yet encountered their specific antigens are known as naive T cells. To participate in an adaptive immune response, a naive T cell must meet its specific antigen, presented to it as a peptide:MHC complex on the surface of an antigen-presenting cell, and be induced to proliferate and differentiate into progeny with new activities that contribute to removal of antigen. These progeny cells are called effector T cells and, unlike naive T cells, perform their functions as soon as they encounter development and function of secondary lymphoid organs—sites for the initiation of adaptive immune responses.

1	priming of naive T cells by pathogen-activated dendritic cells. General properties of effector T cells and their cytokines. T-cell-mediated cytotoxicity. their specific antigen on other cells—generally without requirement for further differentiation. Because of their requirement to recognize peptide antigens presented by MHC molecules, all effector T cells act on other host cells, not on the pathogen itself. The cells on which effector T cells act will be referred to as their target cells.

1	On recognizing antigen, naive T cells differentiate into several functional classes of effector T cells that are specialized for different activities. CD8 T cells recognize pathogen peptides presented by MHC class I molecules, and naive CD8 T cells differentiate into cytotoxic effector T cells that recognize and kill infected cells. CD4 T cells have a more flexible repertoire of effector activities. After recognizing pathogen peptides presented by MHC class II molecules, naive CD4 T cells can differentiate down distinct pathways that generate effector subsets with different immunological functions. The main CD4 effector subsets are TH1, TH2, TH17, and TFH, which activate their target cells; and regulatory T cells, or Treg cells, which inhibit the extent of immune activation.

1	Effector T cells differ from their naive precursors in ways that equip them to respond quickly and efficiently when they encounter specific antigen on target cells. Among the changes that occur are alterations in the expression of surface molecules that alter the patterns of migration of effector T cells, directing them to exit the secondary lymphoid tissues and move to sites of inflammation where pathogens have entered, or to B-cell zones within secondary lymphoid tissues, where they help generate pathogen-specific antibodies. The interactions with target cells in these sites are mediated both by direct T-cell–target cell contact and the release of cytokines, which can act locally on target cells; and at a distance to orchestrate the clearance of antigen. Some of the effector functions of T cells will be considered in this chapter; others will be discussed in Chapters 10 and 11 in the context of T-cell help for B cells and heightened activation of effector cells of the innate immune

1	T cells will be considered in this chapter; others will be discussed in Chapters 10 and 11 in the context of T-cell help for B cells and heightened activation of effector cells of the innate immune system.

1	The activation and clonal expansion of a naive T cell on its initial encounter with antigen is often called priming, to distinguish this process from the responses of effector T cells to antigen on their target cells and the responses of primed memory T cells. The initiation of adaptive immunity is one of the most compelling narratives in immunology. As we will learn, the activation of naive T cells is controlled by a variety of signals. The primary signal that a naive T cell must recognize is antigen in the form of a peptide:MHC complex on the surface of a specialized antigen-presenting cell, as discussed in Chapter 6. Activation of the naive T cell also requires that it recognize co-stimulatory molecules that are displayed by antigen-presenting cells. Finally, cytokines that control differentiation into different types of effector cells are delivered to the activated naive T cell. All these events are set in motion by earlier signals that arise from the initial detection of the

1	differentiation into different types of effector cells are delivered to the activated naive T cell. All these events are set in motion by earlier signals that arise from the initial detection of the pathogens by the innate immune system. Microbe-derived signals are delivered to cells of the innate immune system by receptors such as the Toll-like receptors (TLRs), which recognize microbe-associated molecular patterns, or MAMPs, that signify the presence of nonself (see Chapters 2 and 3). As we will see in this chapter, these signals are essential to activate antigen-presenting cells so that they are able, in turn, to activate naive T cells.

1	By far the most important antigen-presenting cells in the activation of naive T cells are dendritic cells, whose major function is to ingest and present antigen. Tissue dendritic cells take up antigen at sites of infection and are activated as part of the innate immune response. This induces their migration to local lymphoid tissue and their maturation into cells that are highly effective at presenting antigen to recirculating naive T cells. In the first part of this chapter we will consider the development and organization of secondary lymphoid tissues and discuss how naive T cells and dendritic cells meet in these sites to initiate adaptive immunity. Development and function of secondary lymphoid organs—sites for the initiation of adaptive immune responses.

1	As discussed in Chapter 8, the primary lymphoid organs—the thymus and bone marrow—are the tissue sites where antigenic receptor repertoires of T and B cells, respectively, are selected. Adaptive immune responses are initiated in the secondary lymphoid organs—lymph nodes, spleen, and the mucosa-associated lymphoid tissues (MALTs) such as the Peyer’s patches in the gut. The architecture of these tissues is similar throughout the body and is structured to provide a crossroads for the interaction of rare clonal precursors of recirculating T and B cells with their cognate antigens—whether delivered by dendritic cells in the case of T cells, or as free antigens in the case of B cells. In view of the rarity of naive T cells that recognize a specific peptide:MHC complex—roughly 50–500 cells in the entire immune repertoire of approximately 100 million T cells in the mouse—and the large area over which an infectious agent can invade, the antigens derived from the pathogen, or in some instances

1	entire immune repertoire of approximately 100 million T cells in the mouse—and the large area over which an infectious agent can invade, the antigens derived from the pathogen, or in some instances the pathogen itself, must be brought from sites of entry to secondary lymphoid organs to facilitate their recognition by lymphocytes. In this part of the chapter we shall first consider the development and structure of secondary lymphoid organs that enable these interactions. We shall then discuss how naive T cells are directed to exit the blood and enter the lymphoid organs. This will be followed by considering how dendritic cells pick up antigen and travel to local lymphoid organs, where they can both present antigen to T cells and activate them.

1	9-1 T and B lymphocytes are found in distinct locations in secondary lymphoid tissues. The various secondary lymphoid organs are organized roughly along the same lines (see Chapter 1), with distinct areas in which B cells and T cells are concentrated—the B-cell and T-cell zones. They also contain macrophages, dendritic cells, and nonleukocyte stromal cells. In the case of the spleen, which is specialized for the capture of antigens that enter the bloodstream, the lymphoid tissue component is called the white pulp (Fig. 9.1).

1	Fig. 9.1 Secondary lymphoid tissues serve as anatomical crossroads for interactions of antigens and lymphocytes. secondary lymphoid tissues are specialized to serve as sites that facilitate interactions between lymphocytes and antigens. In lymph nodes (upper panel), antigen (denoted by red dots) is delivered in lymph either free or as cargo of dendritic cells that have taken up the antigen in the tissues drained by the lymph node. The antigen is conducted via afferent lymphatics to the subcapsular sinus, from which it is delivered to T-cell zones, where T cells can recognize it on the surface of dendritic cells; or, in the case of B cells, it is detected as a free antigen at the border of the T-cell zone and B-cell follicles. T and B cells enter the lymph node via high endothelial venules (hevs) in T-cell zones, and then diverge into T-cell and B-cell zones. In the spleen (middle panel), antigen is delivered via arterioles that branch from the central arteriole to the marginal sinus,

1	(hevs) in T-cell zones, and then diverge into T-cell and B-cell zones. In the spleen (middle panel), antigen is delivered via arterioles that branch from the central arteriole to the marginal sinus, which is the boundary between the white pulp and red pulp, with which the marginal sinus communicates. In the marginal sinus, antigen can be taken up by marginal zone B cells, macrophages, or dendritic cells, which can transport antigen into either T-cell zones (periarteriolar lymphoid sheath, or paLs) or B-cell follicles. T and B cells enter the spleen via the same route as antigen, and leave the marginal sinus to travel to either the paLs or the B-cell follicles. In the intestines (lower panel), antigens are transported from the lumen via the microfold or M cells—specialized epithelium that overlays peyer’s patches—to dendritic cells that reside in the subepithelial dome. antigen-loaded dendritic cells are then surveyed by T cells in T-cell zones, and if the antigens they bear are not

1	overlays peyer’s patches—to dendritic cells that reside in the subepithelial dome. antigen-loaded dendritic cells are then surveyed by T cells in T-cell zones, and if the antigens they bear are not recognized by T cells locally, the dendritic cells can migrate to mesenteric lymph nodes to be further surveyed. as for lymph nodes, T and B cells enter the peyer’s patches via hevs in the T-cell zones.

1	Each area of white pulp is demarcated from the red pulp by a marginal sinus, a vascular network that is formed from branches of the central arteriole. Circulating T and B cells are initially delivered to the marginal sinus, which is a highly organized region of cells that is specialized for the capture of blood-borne antigens or intact microbes, such as viruses and bacteria. It is rich in macrophages and contains a unique population of B cells, the marginal zone B cells, which do not recirculate. Pathogens reaching the bloodstream are efficiently trapped in the marginal zone by macrophages, and it could be that marginal zone B cells are adapted to provide the first responses to such pathogens.

1	From the marginal sinus, T and B cells migrate centrally toward the central arteriole, where they bifurcate into T-cell zones that are clustered around the central arteriole—the so-called periarteriolar lymphoid sheath (PALS)— and B-cell zones, or follicles, that are located more peripherally. Some follicles may contain germinal centers, in which B cells involved in an adaptive immune response are proliferating and undergoing somatic hypermutation (see Section 1-16). The antigen-driven production of germinal centers will be described in detail when we consider B-cell responses in Chapter 10.

1	Other types of cells are found within the B-cell and T-cell areas. The B-cell zone contains a network of follicular dendritic cells (FDCs), which are concentrated mainly in the area of the follicle most distant from the central arteriole. FDCs have long processes that are in contact with B cells. FDCs are a distinct type of cell from the dendritic cells we encountered previously (see Section 1-3), in that they are not leukocytes and are not derived from bone marrow precursors; in addition, they are not phagocytic and do not express MHC class II proteins. FDCs are specialized for the capture of antigen in the form of immune complexes—complexes of antigen, antibody, and complement. The immune complexes are not internalized but remain intact on the surface of the FDC for prolonged periods of time, where the antigen can be recognized by B cells. FDCs are also important in the development of B-cell follicles.

1	T-cell zones contain a network of bone marrow-derived dendritic cells, sometimes known as interdigitating dendritic cells from the way in which their processes interweave among T cells. There are two major subtypes of these dendritic cells, distinguished by characteristic cell-surface proteins: one expresses the α chain of CD8, whereas the other is CD8-negative but expresses CD11b:CD18, an integrin that is also expressed by macrophages.

1	As in the spleen, the T cells and B cells in lymph nodes are organized into discrete T-cell and B-cell areas (see Fig. 9.1). B-cell follicles have a similar structure and composition to those in the spleen and are located just under the outer capsule of the lymph node. T-cell zones surround the follicles in the paracortical areas. Unlike the spleen, lymph nodes have connections to both the blood system and the lymphatic system. Lymph conducted to lymph nodes by afferent lymphatic vessels enters into the subcapsular space, which is also known as the marginal sinus, and brings in antigen and antigen-bearing dendritic cells from the tissues. T and B cells enter the lymph node via specialized blood vessels called high endothelial venules (HEVs) that are found in T-cell zones, as will be discussed further in Section 9-3.

1	The mucosa-associated lymphoid tissues (MALTs) are associated with the body’s epithelial surfaces, which provide physical barriers against infection. Peyer’s patches are part of the MALT and are lymph node-like structures interspersed at intervals just beneath the gut epithelium. They have B-cell follicles and T-cell zones (see Fig. 9.1), and the epithelium overlying them contains specialized M cells that are adapted to channel antigens and pathogens directly from the gut lumen to the underlying lymphoid tissue (see Section 1-16 and Chapter 12). Peyer’s patches and similar tissue present in the tonsils provide specialized sites where B cells can become committed to the synthesis of IgA. The mucosal immune system is discussed in more detail in Chapter 12. 9-2 The development of secondary lymphoid tissues is controlled by lymphoid tissue inducer cells and proteins of the tumor necrosis factor family.

1	Before discussing how T cells and B cells become partitioned into their respective zones in secondary lymphoid organs, we shall briefly look at how these organs develop in the first place. Lymphatic vessels are formed during embryonic development from endothelial cells that originate in blood vessels. Some endothelial cells in the early venous system begin to express the homeobox transcription factor Prox1. These cells bud from the vein, migrate away, and reassociate to form a parallel network of lymphatic vessels. Mice lacking Prox1 have normal arteries and veins, but fail to form a lymphatic system, showing this factor to be critical in establishing the identity of lymphatic endothelium. As the lymphatic vessels form, hematopoietic cells called lymphoid tissue inducer (LTi) cells arise in the fetal liver and are carried in the bloodstream to sites of prospective lymph nodes and Peyer’s patches. LTi cells initiate the formation of lymph nodes and Peyer’s patches by interacting with

1	in the fetal liver and are carried in the bloodstream to sites of prospective lymph nodes and Peyer’s patches. LTi cells initiate the formation of lymph nodes and Peyer’s patches by interacting with stromal cells and inducing the production of cytokines and chemokines, which recruit other lymphoid cells to these sites. Members of the tumor necrosis factor (TNF)/ TNF receptor (TNFR) family of cytokines turn out to be critically involved in the interactions between LTi cells and stromal cells.

1	The role of this family of cytokines in the formation of secondary lymphoid organs has been demonstrated in a series of studies involving knockout mice in which either the TNF-family ligand or its receptor was inactivated (Fig. 9.2). These knockout mice have complicated phenotypes, which is partly due to the fact that individual TNF-family proteins can bind to multiple receptors and, conversely, many receptors can bind more than one ligand. In addition, it seems clear that there is some overlapping function or cooperation between TNF-family proteins. Nonetheless, some general conclusions can be drawn.

1	Lymph-node development depends on the expression of TNF-family proteins known as the lymphotoxins (LTs), and different types of lymph nodes depend on signals from different LTs. LT-α3, a soluble homotrimer of the LT-α chain, supports the development of cervical and mesenteric lymph nodes, and possibly lumbar and sacral lymph nodes. All these lymph nodes drain mucosal sites. LT-α3 probably exerts its effects by binding to TNFR-I. The membrane-bound heterotrimer consisting of two molecules of LT-α and one molecule of the distinct transmembrane protein LT-β (that is, LT-α2:β1), often known as LT-β, binds only to the LT-β receptor and supports the development of all the other lymph nodes. Peyer’s patches also do not form in the absence of LT-β. The effects of the LT knockouts are not reversible in adult animals; there are certain critical developmental periods during which the absence or inhibition of these LT-family proteins will permanently prevent the development of lymph nodes and

1	in adult animals; there are certain critical developmental periods during which the absence or inhibition of these LT-family proteins will permanently prevent the development of lymph nodes and Peyer’s patches.

1	LTi cells express LT-β, which engages the LT-β receptors on stromal cells in the prospective lymphoid site, activating the non-canonical NFκB pathway

1	Fig. 9.2 The role of TNF family members in the development of peripheral lymphoid organs. The role of TnF family members in the development of peripheral lymphoid organs has been deduced mainly from the study of knockout mice deficient in one or more TnF-family ligands or receptors. some receptors bind more than one ligand, and some ligands bind more than one receptor, complicating elucidation of the effects of their deletion. (note that receptors are named for the first ligand known to bind them.) The defects are organized here with respect to the two main receptors, TnFr-I and the LT-β receptor, and their ligands, TnF-α and the lymphotoxins (LTs). note that in some cases, the losses of individual ligands out of several that bind the same receptor lead to different respective phenotypes, as indicated in the figure. This is due to the ability of the different ligands to bind different sets of receptors. The LT-α protein chain contributes to two distinct ligands, the trimer LT-α3 and

1	as indicated in the figure. This is due to the ability of the different ligands to bind different sets of receptors. The LT-α protein chain contributes to two distinct ligands, the trimer LT-α3 and the heterodimer LT-α2:β1, each of which acts through a distinct receptor. In general, signaling through the LT-β receptor is required for lymph-node and follicular dendritic cell (FdC) development and for normal splenic architecture, whereas signaling through TnFr-I is also required for FdCs and normal splenic architecture but not for lymph-node development.

1	Fig. 9.3 The development of secondary lymphoid organs is orchestrated by chemokines. The cellular organization of lymphoid organs is initiated by stromal cells and vascular endothelial cells, which secrete the chemokine CCL21 (first panel). dendritic cells with CCr7—a receptor for CCL21—are attracted to the site of the developing lymph node by CCL21 (second panel); it is not known whether at the earliest stages of lymph-node development immature dendritic cells enter from the bloodstream or via the lymphatics, as they do later in life. once in the lymph node, the dendritic cells express the chemokine CCL19, which is also bound by CCr7. Together, the chemokines secreted by stromal cells and dendritic cells attract T cells to the developing lymph node (third panel). The same combination of chemokines also attracts B cells into the developing lymph node (fourth panel). The B cells are able to either induce the differentiation of the nonleukocyte FdCs (which are a lineage distinct from

1	chemokines also attracts B cells into the developing lymph node (fourth panel). The B cells are able to either induce the differentiation of the nonleukocyte FdCs (which are a lineage distinct from the bone marrow-derived dendritic cells) or direct their recruitment into the lymph node. once present, the FdCs secrete CXCL13, a chemokine that is a chemoattractant for B cells. The production of CXCL13 drives the organization of B cells into discrete B-cell areas (follicles) around the FdCs and contributes to the further recruitment of B cells from the circulation into the lymph node (fifth panel).

1	(see Section 7-23). This induces the stromal cells to express adhesion molecules and chemokines such as CXCL13 (B-lymphocyte chemokine, BLC), which in turn recruits more LTi cells, which have receptors for these molecules, eventually generating large clusters of cells that will become lymph nodes or Peyer’s patches. The chemokines also attract cells such as lymphocytes and other hematopoietic-lineage cells with appropriate receptors to populate the forming lymphoid organ. The principles, and even some of the molecules, underlying the development of secondary lymphoid organs in the fetus are very similar to those that maintain the organization of lymphoid organs in the adult, as we shall see in the next section.

1	Although the spleen will develop in mice deficient in any of the known TNF or TNFR family members, its architecture will be abnormal in many of these mutants (see Fig. 9.2). LT (most probably the membrane-bound LT-β) is required for the normal segregation of T-cell and B-cell zones in the spleen. TNF-α, binding to TNFR-I, also contributes to the organization of the white pulp: when TNF-α signals are disrupted, B cells surround T-cell zones in a ring rather than forming discrete follicles, and the marginal zones are not well defined.

1	Perhaps the most important role of TNF-α and TNFR-I in lymphoid organ development is in the development of FDCs, as these cells are lacking in mice with knockouts of either TNF-α or TNFR-I (see Fig. 9.2). The knockout mice do have lymph nodes and Peyer’s patches, because they express LTs, but these structures lack FDCs. LT-β is also required for FDC development: mice that cannot form LT-β or signal through its receptor lack normal FDCs in the spleen and any residual lymph nodes. Unlike the disruption of lymph-node development, the disorganized lymphoid architecture in the spleen is reversible if the missing TNF-family member is restored. B cells are the likely source of the LT-β, because normal B cells can restore FDCs and follicles when transferred to RAG-deficient recipients (which lack lymphocytes). 9-3 T and B cells are partitioned into distinct regions of secondary lymphoid tissues by the actions of chemokines.

1	Circulating T and B cells seed secondary lymphoid tissues from the blood by a common route, but are then directed into their respective compartments under the control of distinct chemokines that are produced by both stromal cells and bone marrow-derived cells resident in the Tand B-cell zones (Fig. 9.3). The localization of T cells into T-cell zones involves two chemokines, CCL19 (MIP-3β) and CCL21 (secondary lymphoid chemokine, SLC). Both of these bind the receptor CCR7, which is expressed by T cells; mice that lack CCR7 do not form normal T-cell zones and have impaired primary immune responses. CCL21 is produced by stromal cells of T-cell zones in secondary lymphoid tissues, and is displayed on endothelial cells of high endothelial venules (HEVs). Another source of CCL21 is interdigitating dendritic cells, which also produce CCL19 and are prominent in T-cell zones. Indeed, dendritic cells themselves express CCR7 and will localize to secondary lymphoid tissues even in RAG-deficient

1	dendritic cells, which also produce CCL19 and are prominent in T-cell zones. Indeed, dendritic cells themselves express CCR7 and will localize to secondary lymphoid tissues even in RAG-deficient mice, which lack lymphocytes and therefore defined T-cell zones. Thus, during normal lymph-node development, the T-cell zone might be organized first through the attraction of dendritic cells and T cells by CCL21 produced by stromal cells. This organization would then be reinforced by CCL21 and CCL19 secreted by resident dendritic cells, which in turn attract more T cells and migratory dendritic cells.

1	CXCL13B cells induce the differentiation of follicular dendritic cells, which in turn secrete the chemokine CXCL13 to attract more B cells B cells are initially attracted into the developing lymph node by the same chemokines

1	Like T cells, circulating B cells express CCR7, which initially directs them into the lymph node across HEVs. Because they also constitutively express the chemokine receptor CXCR5, they are then attracted to the follicles by the ligand for this receptor, CXCL13. The most likely source of CXCL13 is the FDC, possibly along with other follicular stromal cells. This is reminiscent of the expression of CXCL13 by stromal cells during the formation of the lymph node (Section 9-2). B cells are, in turn, the source of the LT that is required for the development of FDCs, which is reminiscent of LTi cells expressing the LT required to activate stromal cells. The reciprocal dependence of B cells and FDCs, and LTis and stromal cells, illustrates the complex web of interactions that organizes secondary lymphoid tissues. A subset of CD4 T cells called T follicular helper, or TFH, cells can also express CXCR5 following their activation by antigen, allowing them to enter B-cell follicles to

1	secondary lymphoid tissues. A subset of CD4 T cells called T follicular helper, or TFH, cells can also express CXCR5 following their activation by antigen, allowing them to enter B-cell follicles to participate in the formation of germinal centers (see Chapter 10).

1	9-4 Naive T cells migrate through secondary lymphoid tissues, sampling peptide:MHC complexes on dendritic cells.

1	Naive T cells perpetually circulate from the bloodstream into lymph nodes, spleen, and mucosa-associated lymphoid tissues and back to the blood (see Fig. 1.21). This allows them to contact thousands of dendritic cells every day and sample the peptide:MHC complexes on the surfaces of these cells. Because of their high rates of recirculation and their concentration in T-cell zones where incoming dendritic cells dwell, each T cell has a high probability of encountering antigens derived from any pathogen that has set up an infection anywhere in the body (Fig. 9.4). Within hours of their arrival, naive T cells that do not encounter their specific antigen exit from the lymphoid tissue and reenter the bloodstream, where they continue to recirculate—via the efferent lymphatics in lymph nodes or MALTs, or directly back to the blood in the spleen, which has no connection with the lymphatic system.

1	When a naive T cell recognizes its specific antigen on the surface of an activated dendritic cell, however, it ceases to migrate. It remains in the T-cell zone,

1	Fig. 9.4 Naive T cells encounter antigen during their recirculation through peripheral lymphoid organs. naive T cells recirculate through peripheral lymphoid organs, such as a lymph node (shown here), entering from the arterial blood via the specialized vascular endothelium of high endothelial venules (hevs). entry into the lymph node is regulated by chemokines (not shown) that direct the T cells’ migration through the hev wall and into the paracortical areas, where the T cells encounter mature dendritic cells (top panel). Those T cells shown in green do not encounter their specific antigen; they receive a survival signal through their interaction with self peptide:self MhC complexes and IL-7, and leave the lymph node through the lymphatics to return to the circulation (second panel). T cells shown in blue encounter their specific antigen on the surface of mature dendritic cells; they lose their ability to exit from the node and become activated to proliferate and to differentiate

1	T cells shown in blue encounter their specific antigen on the surface of mature dendritic cells; they lose their ability to exit from the node and become activated to proliferate and to differentiate into effector T cells (third panel). after several days, these antigen-specific effector T cells regain the expression of receptors needed to exit from the node, leave via the efferent lymphatics, and enter the circulation in greatly increased numbers (bottom panel).

1	20 4 6 8 Emigration of effector T cells Time after viral infection (days) Antigen-speciÿc T cells are detained transiently in the lymph node, where they become activated ActivationTrapping Number of antigen-speciÿc cells in efferent lymph

1	Fig. 9.5 Trapping and activation of antigen-specific naive T cells in lymphoid tissue. naive T cells entering the lymph node from the blood encounter antigen-presenting dendritic cells in T-cell zones. T cells that recognize their specific antigen bind stably to the dendritic cells and are activated through their T-cell receptors, resulting in their retention within the lymph node as they develop into effector T cells. By 5 days after the arrival of antigen, activated effector T cells are leaving the lymph node in large numbers via the efferent lymphatics. Lymphocyte recirculation and recognition are so effective that all the naive T cells in the peripheral circulation specific for a particular antigen can be trapped by that antigen in one node within 2 days.

1	Fig. 9.6 Lymphocyte entry into a lymph node from the blood occurs in distinct stages involving the activity of adhesion molecules, chemokines, and chemokine receptors. naive T cells are induced to roll along the surface of a high endothelial venule (hev) by the interactions of selectins expressed by the T cells with vascular addressins on the endothelial cell membranes. Chemokines present at the hev surface activate receptors on the T cell, and chemokine receptor signaling leads to an increase in the affinity of integrins on the T cell for the adhesion molecules expressed on the hev. This induces strong adhesion. after adhesion, the T cells follow gradients of chemokines to pass through the hev wall into the paracortical region of the lymph node.

1	where it proliferates for several days, undergoing clonal expansion and differentiation to give rise to effector T cells and memory cells of identical antigen specificity. At the end of this period, most effector T cells exit the lymphoid organ and reenter the bloodstream, through which they migrate to the sites of infection (see Chapter 11). Some effector T cells that are fated to interact with B cells migrate instead to B-cell zones, where they participate in the germinal center response (see Chapter 10).

1	The efficiency with which T cells screen antigen-presenting cells in lymph nodes is very high, as can be seen by the rapid trapping of antigen-specific T cells in a single lymph node containing antigen; within 48 hours, all antigen-specific T cells in the body can be trapped in the lymph node draining a site of antigen injection (Fig. 9.5). Such efficiency is crucial for the initiation of an adaptive immune response, as only one naive T cell in 105–106 is likely to be specific for a particular antigen, and adaptive immunity depends on the activation and expansion of these rare cells. 9-5 Lymphocyte entry into lymphoid tissues depends on chemokines and adhesion molecules.

1	Migration of naive T cells into secondary lymphoid tissues depends on their binding to high endothelial venules (HEVs) through cell–cell interactions that are not antigen-specific but are governed by cell-adhesion molecules. The main classes of adhesion molecules involved in lymphocyte interactions are the selectins, the integrins, members of the immunoglobulin superfamily, and some mucin-like molecules (see Fig. 3.30). Entry of lymphocytes into lymph nodes occurs in distinct stages that include initial rolling of lymphocytes along the endothelial surface, activation of integrins, firm adhesion, and transmigration or diapedesis across the endothelial layer into the paracortical areas, the T-cell zones (Fig. 9.6). These stages are regulated by a coordinated interplay of adhesion molecules and chemokines that resembles the recruitment of leukocytes to sites of inflammation (see Chapter 3). Adhesion molecules have fairly broad roles in immune responses, being involved not only in

1	and chemokines that resembles the recruitment of leukocytes to sites of inflammation (see Chapter 3). Adhesion molecules have fairly broad roles in immune responses, being involved not only in lymphocyte migration but also in interactions between naive T cells and antigen-presenting cells (see Section 9-14).

1	The selectins (Fig. 9.7) are important for specifically guiding leukocytes to particular tissues, a phenomenon known as leukocyte homing. L-selectin (CD62L) is expressed on leukocytes, whereas P-selectin (CD62P) and E-selectin (CD62E) are expressed on vascular endothelium (see Section 3-18). L-selectin on naive T cells guides their exit from the blood into secondary lymphoid tissues by initiating a light attachment to the wall of the HEV that results in the T cells’ rolling along the endothelial surface (see Fig. 9.6). P-selectin and E-selectin are expressed on the vascular endothelium at sites of infection, and serve to recruit effector cells into the infected tissue. Selectins are cell-surface molecules with a common core structure and are distinguished from each other by the presence of different lectin-like domains in their extracellular portion. The lectin domains bind to particular sugar groups, and each selectin binds to a cell-surface carbohydrate. L-selectin binds to the

1	of different lectin-like domains in their extracellular portion. The lectin domains bind to particular sugar groups, and each selectin binds to a cell-surface carbohydrate. L-selectin binds to the carbohydrate moiety—sulfated sialyl-LewisX—of mucin-like molecules called vascular addressins, which are expressed on the surface of vascular endothelial cells. Two of these addressins, CD34 and GlyCAM-1 (see Fig. 9.7), are expressed on high endothelial venules in lymph nodes. A third, MAdCAM-1, is expressed on endothelium in mucosae, and guides lymphocyte entry into mucosal lymphoid tissue such as the Peyer’s patches in the gut.

1	The interaction between L-selectin and the vascular addressins is responsible for the specific homing of naive T cells to lymphoid organs. On its own, however, it does not enable the cell to cross the endothelial barrier into the lymphoid tissue. This requires the concerted action of chemokines and integrins. 9-6 Activation of integrins by chemokines is responsible for the entry of naive T cells into lymph nodes.

1	9-6 Activation of integrins by chemokines is responsible for the entry of naive T cells into lymph nodes. Naive T cells rolling on the endothelium of HEVs via selectins require two additional types of cell-adhesion molecules to enter secondary lymphoid organs— integrins, and members of the immunoglobulin superfamily. Integrins bind tightly to their ligands after receiving signals that induce a change in their conformation. Signaling by chemokines activates integrins on leukocytes to bind tightly to the vascular wall in preparation for the migration of the leukocytes into sites of inflammation (see Section 3-18). Similarly, chemokines present at the luminal surface of the HEV activate integrins expressed on naive T cells during migration into lymphoid organs (see Fig. 9.6).

1	An integrin molecule consists of a large α chain that pairs noncovalently with a smaller β chain. There are several integrin subfamilies, broadly defined by their common β chains. We will be concerned here chiefly with the leukocyte integrins, which have a common β2 chain paired with distinct α chains (Fig. 9.8). All T cells express the integrin αL:β2 (CD11a:CD18), better known as leukocyte functional antigen-1 (LFA-1). It enables migration of both naive and effector T cells out of the blood. This integrin is also present on macrophages and neutrophils, and is involved in their recruitment to sites of infection (see Section 3-18). LFA-1 is also important in the adhesion of both naive and effector T cells to their target cells. Nevertheless, T-cell responses can be normal in individuals genetically lacking the β2 integrin chain and hence all β2 integrins,

1	Fig. 9.7 L-selectin binds to mucinlike vascular addressins. L-selectin is expressed on naive T cells and recognizes carbohydrate motifs. Its binding to sulfated sialyl-LewisX moieties on the vascular addressins Cd34 and GlyCaM-1 on hevs binds the lymphocyte weakly to the endothelium. The relative importance of Cd34 and GlyCaM-1 in this interaction is unclear. Cd34 has a transmembrane anchor and is expressed in appropriately glycosylated form only on hev cells, although it is found in other forms on other endothelial cells. GlyCaM-1 is expressed on hevs but has no transmembrane region and may be secreted into the hevs. The addressin MadCaM-1 is expressed on mucosal endothelium and guides lymphocytes to mucosal lymphoid tissue. The configuration shown represents mouse MadCaM-1, which contains an Iga-like domain closest to the cell membrane; human MadCaM-1 has an elongated mucin-like domain and lacks the Iga-like domain.

1	Fig. 9.8 Integrins are important in T-lymphocyte adhesion. Integrins are heterodimeric proteins containing a β chain, which defines the class of integrin, and an α chain, which defines the different integrins within a class. The α chain is larger than the β chain and contains binding sites for divalent cations that may be important in signaling. LFa-1 (integrin αL:β2) is expressed on all leukocytes. It binds ICaMs and is important in cell migration and in the interactions of T cells with antigen-presenting cells (apCs) or target cells; it is expressed at higher levels on effector T cells than on naive T cells. Lymphocyte peyer’s patch adhesion molecule (LpaM-1, or integrin α4:β7) is expressed by a subset of naive T cells and contributes to lymphocyte entry into mucosal lymphoid tissues by supporting adhesive interactions with vascular addressin MadCaM-1. vLa-4 (integrin α4:β1) is expressed strongly after T-cell activation. It binds to vCaM-1 on activated endothelium and is important

1	supporting adhesive interactions with vascular addressin MadCaM-1. vLa-4 (integrin α4:β1) is expressed strongly after T-cell activation. It binds to vCaM-1 on activated endothelium and is important for recruiting effector T cells into sites of infection.

1	Fig. 9.9 Immunoglobulin superfamily adhesion molecules involved in leukocyte interactions. adhesion molecules of the immunoglobulin superfamily bind to adhesion molecules of various types, including integrins (LFa-1 and vLa-4) and other immunoglobulin superfamily members [the Cd2–Cd58 (LFa-3) interaction]. These interactions have a role in lymphocyte migration, homing, and cell–cell interactions; see Fig. 3.24 for the other molecules listed here. including LFA-1. This is probably because T cells also express other adhesion molecules, including the immunoglobulin superfamily member CD2 and β1 integrins, which may compensate for the absence of LFA-1. Expression of the β1 integrins increases significantly at a late stage in T-cell activation, and they are thus often called VLAs, for very late activation antigens; they are important in directing effector T cells to inflamed target tissues.

1	At least five members of the immunoglobulin superfamily are especially important in T-cell activation (Fig. 9.9). Three very similar intercellular adhesion molecules (ICAMs)—ICAM-1, ICAM-2, and ICAM-3—all bind to the T-cell integrin LFA-1. ICAM-1 and ICAM-2 are expressed on endothelium as well as on antigen-presenting cells, and binding to these molecules enables lymphocytes to migrate through blood vessel walls. ICAM-3 is expressed only on naive T cells and is thought to have an important role in the adhesion of T cells to antigen-presenting cells by binding to LFA-1 expressed on dendritic cells. The two remaining immunoglobulin superfamily adhesion molecules, CD58 (formerly known as LFA-3) on the antigen-presenting cell and CD2 on the T cell, bind to each other; this interaction synergizes with that of ICAM-1 or ICAM-2 with LFA-1.

1	As discussed above in the context of lymphoid tissue development (see Section 9-3), naive T cells are specifically attracted into the T-cell zones of secondary lymphoid tissues by chemokines. The chemokines bind to proteoglycans in the extracellular matrix and high endothelial venule wall, forming a chemical gradient, and are recognized by receptors on the naive T cell. The extravasation of naive T cells is prompted by the chemokine CCL21, which is expressed by vascular high endothelial cells and the stromal cells of lymphoid tissues, as well as by dendritic cells that reside in T-cell zones. It binds to the chemokine receptor CCR7 on naive T cells, stimulating activation of the intracellular receptor-associated G-protein subunit Gαi. The resulting intracellular signaling rapidly increases the affinity of integrin binding (see Section 3-18).

1	Fig. 9.10 Lymphocytes in the blood enter lymphoid tissue by to the activation of LFa-1. This causes the T cell to bind tightly crossing the walls of high endothelial venules. The first step is to ICaM-1 on the endothelial cell, allowing migration across the the binding of L-selectin on the lymphocyte to sulfated carbohydrates endothelium. as in the case of neutrophil migration (see Fig. 3.31), (sulfated sialyl-LewisX) of GlyCaM-1 and Cd34 on the hev. Local matrix metalloproteinases on the lymphocyte surface (not shown) chemokines such as CCL21 bound to a proteoglycan matrix on the enable the lymphocyte to penetrate the basement membrane. hev surface stimulate chemokine receptors on the T cell, leading

1	The entry of a naive T cell into a lymph node is shown in detail in Fig. 9.10. Initial rolling of the T cell along the surface of HEVs is mediated by L-selectin. Recognition of CCL21 on the endothelial surface of the HEV by CCR7 on the T cell causes LFA-1 to become activated, increasing its affinity for ICAM-2 and ICAM-1. ICAM-2 is expressed constitutively on all endothelial cells, whereas in the absence of inflammation, ICAM-1 is expressed only on the high endothelial cells of secondary lymphoid tissues. The organization of LFA-1 molecules in the T-cell membrane is also altered by chemokine stimulation, such that they become concentrated in areas of cell–cell contact. This produces stronger binding, which arrests the T cell on the endothelial surface and thus enables it to enter the lymphoid tissue.

1	Once naive T cells have arrived in the T-cell zone via high endothelial venules, CCR7 directs their retention in this location, as they are attracted to dendritic cells that produce CCL21 and CCL19 in the T-cell zone. The naive T cells scan the surfaces of dendritic cells for specific peptide:MHC complexes, and if they find their antigen and bind to it, they are trapped in the lymph node. If they are not activated by antigen, naive T cells soon leave the lymph node (see Fig. 9.4). 9-7 The exit of T cells from lymph nodes is controlled by a chemotactic lipid.

1	9-7 The exit of T cells from lymph nodes is controlled by a chemotactic lipid. T cells exit from a lymph node via the cortical sinuses, which lead into the medullary sinus and then the efferent lymphatic vessel. The egress of T cells from secondary lymphoid organs involves the lipid molecule sphingosine 1-phosphate (S1P) (Fig. 9.11). This lipid has chemotactic activity and signaling properties similar to those of chemokines, in that the receptors for S1P are G-protein-coupled receptors. A concentration gradient of S1P between the lymphoid tissues and lymph or blood acts to draw unactivated naive T cells expressing an S1P receptor away from the lymphoid tissues and back into circulation.

1	Fig. 9.11 The egress of lymphocytes from lymphoid tissue is mediated by a sphingosine 1-phosphate (S1P) gradient. The level of sphingosine 1-phosphate (s1p) within lymphoid tissue is low compared with efferent lymph, thereby forming an s1p gradient (indicated by shading). The s1p receptor 1 (s1pr1) expressed on naive T cells is responsive to the s1p gradient. In the absence of antigen recognition, s1pr1 signaling promotes T-cell egress from the T-cell zones into the efferent lymphatic vessel. T cells activated by an antigen-expressing dendritic cell upregulate Cd69, which causes a decrease in s1pr1 expression and retention in the T-cell zone. effector T cells eventually reexpress s1pr1 as Cd69 expression decreases, and thereby egress from the lymph node. FTY720 inhibits T-cell egression by downmodulating expression of s1pr1 by ligand-induced internalization and by s1pr1-mediated closure of egress ports on the endothelium by enhancement of junctional contacts (not shown).

1	T cells activated by antigen in lymphoid organs downregulate the surface expression of the S1P receptor, S1PR1, for several days. This loss of S1PR1 surface expression is caused by CD69, a surface protein whose expression is induced by T-cell receptor signaling and which acts to internalize S1PR1. During this period, T cells cannot respond to the S1P gradient and do not exit the lymphoid organ. After several days of proliferation, as T-cell activation wanes, CD69 expression decreases and S1PR1 reappears on the surface of effector T cells, allowing them to migrate out of the lymphoid tissue in response to the S1P gradient.

1	The regulation of the exit of both naive and effector lymphocytes from secondary lymphoid organs by S1P is the basis for a new kind of potential immunosuppressive drug, FTY720 (fingolimod). FTY720 inhibits immune responses by preventing lymphocytes from returning to the circulation, thereby sequestering them in lymphoid tissues and causing rapid onset of lymphopenia (a lack of lymphocytes in the blood). In vivo, FTY720 becomes phosphorylated and mimics S1P as an agonist at S1P receptors. Phosphorylated FTY720 may inhibit lymphocyte exit by effects on endothelial cells that increase tight junction formation and close exit portals, or by chronic activation of S1P receptors, leading to inactivation and downregulation of the receptor. 9-8 T-cell responses are initiated in secondary lymphoid organs by activated dendritic cells.

1	9-8 T-cell responses are initiated in secondary lymphoid organs by activated dendritic cells. Secondary lymphoid organs were first shown to be important in the initiation of adaptive immune responses by ingenious experiments in which a flap of skin was isolated from the body wall so that it had blood circulation but no lymphatic drainage. Antigen placed in the flap did not elicit a T-cell response, showing that T cells do not become sensitized in the infected tissue itself. Rather, pathogens and their products must be transported to lymphoid tissues. Antigens introduced directly into the bloodstream are picked up by antigen-presenting cells in the spleen. Pathogens infecting other sites, such as a skin wound, are transported in lymph via lymphatic vessels and trapped in the lymph nodes nearest

1	Fig. 9.13 Antigen-presenting cells are distributed by type in specific areas of the lymph node. dendritic cells are found throughout the cortex of the lymph node in the T-cell areas. Mature dendritic cells are by far the strongest activators of naive T cells, and can present antigens from many types of pathogens, such as bacteria or viruses as shown here. Macrophages are distributed throughout the lymph node but are concentrated mainly in the marginal sinus, where the afferent lymph collects before percolating through the lymphoid tissue, and also in the medullary cords, where the efferent lymph collects before passing via the efferent lymphatics into the blood. B cells are found mainly in the follicles and can contribute to neutralizing soluble antigens such as toxins.

1	Dendritic cells can be activated via their TLRs and other pathogen-recognition receptors (see Chapter 3), by tissue damage, or by cytokines produced during the inflammatory response. Activated dendritic cells migrate to the lymph node and express the co-stimulatory molecules that are required, in addition to antigen, for the activation of naive T cells. In the lymphoid tissues, these dendritic cells present antigen to naive T lymphocytes and prime antigen-specific T cells to divide and mature into effector cells that reenter the circulation.

1	Macrophages, which are found in most tissues including lymphoid tissue, and B cells, which are located primarily in lymphoid tissue, can be similarly activated by pathogen-recognition receptors to express co-stimulatory molecules and act as antigen-presenting cells. The distribution of dendritic cells, macrophages, and B cells in a lymph node is shown schematically in Fig. 9.13. Only these three cell types express co-stimulatory molecules required to efficiently activate T cells, and they express these molecules only when activated in the context of infection. However, these cells activate T-cell responses in distinct ways. Dendritic cells take up, process, and present antigens from all types of sources, and are present mainly in the T-cell areas where they drive the initial clonal expansion and differentiation of naive T cells into effector T cells. By contrast, B cells and macrophages specialize in processing and presenting soluble antigens and antigens from intracellular pathogens,

1	and differentiation of naive T cells into effector T cells. By contrast, B cells and macrophages specialize in processing and presenting soluble antigens and antigens from intracellular pathogens, respectively; they interact mainly with effector CD4 T cells already primed by dendritic cells to recruit helper functions of those T cells.

1	9-9 Dendritic cells process antigens from a wide array of pathogens. Dendritic cells primarily arise from myeloid progenitors within the bone marrow (see Fig. 1.3). They emerge from the bone marrow to migrate via the blood to tissues throughout the body, or directly to secondary lymphoid organs. There are two major classes of dendritic cells: conventional dendritic cells, and plasmacytoid dendritic cells (Fig. 9.14). The cell-surface markers and subset-specific transcription factors that distinguish these two classes, and the interferon-producing functions of plasmacytoid dendritic cells in the innate

1	Fig. 9.14 Conventional and plasmacytoid dendritic cells have different roles in the immune response. Mature conventional dendritic cells (left panel) are primarily concerned with the activation of naive T cells. There are several subsets of conventional dendritic cells, but these all process antigen efficiently, and when they are mature they express MhC proteins and co-stimulatory molecules for priming naive T cells. The cell-surface proteins expressed by the mature dendritic cell are described in the text. Immature dendritic cells lack many of the cell-surface molecules shown here but have numerous surface receptors that recognize pathogen molecules, including most of the Toll-like receptors (TLrs). plasmacytoid dendritic cells (right panel) are sentinels primarily for viral infections, and secrete large amounts of class I interferons. This category of dendritic cell is less efficient in priming naive T cells, but they express the intracellular receptors TLr-7 and TLr-9 for sensing

1	and secrete large amounts of class I interferons. This category of dendritic cell is less efficient in priming naive T cells, but they express the intracellular receptors TLr-7 and TLr-9 for sensing viral infections.

1	immune response, are discussed in Chapter 3. In this chapter we shall focus on the role of conventional dendritic cells in the adaptive immune response— presenting antigens to and activating naive T cells.

1	Conventional dendritic cells are abundant at barrier tissue sites, such as the intestines, lung, and skin, where they are in close contact with surface epithelia. They are also present in most solid organs such as the heart and kidneys. In the absence of infection or tissue injury, dendritic cells have low levels of co-stimulatory molecules, and so are not yet equipped to stimulate naive T cells. Like macrophages, dendritic cells are very active in ingesting antigens by phagocytosis using complement receptors and Fc receptors (which recognize the constant regions of antibodies in antigen:antibody complexes), and C-type lectins, which recognize carbohydrates and on dendritic cells include the mannose receptor, DEC 205, langerin, and Dectin-1. Other extracellular antigens are taken up nonspecifically by the process of macropinocytosis, in which large volumes of surrounding fluid are engulfed. In this way microbes that have evolved strategies to escape recognition by phagocytic

1	up nonspecifically by the process of macropinocytosis, in which large volumes of surrounding fluid are engulfed. In this way microbes that have evolved strategies to escape recognition by phagocytic receptors, such as bacteria with thick polysaccharide capsules, can be ingested. The versatility in pathways for antigen uptake enables dendritic cells to present antigens from virtually any type of microbe, including fungi, parasites, viruses, and bacteria (Fig. 9.15). Uptake of extracellular antigens by these pathways directs them into the endocytic pathway, where they are processed and presented on MHC class II molecules (see Chapter 6) for recognition by CD4 T cells.

1	A second route of antigen handling by dendritic cells occurs when antigen directly enters the cytosol, for example, through viral infection. Dendritic cells are directly susceptible to infection by some viruses, which enter the cytoplasm by binding to cell-surface molecules that act as entry receptors. Viral proteins synthesized in the cytoplasm of dendritic cells are processed in the proteasome and presented on the cell surface as peptides loaded onto MHC class I molecules after transport into the endoplasmic reticulum, as in any other type of virus-infected cell (see Chapter 6). This enables dendritic cells to present antigen to and activate naive CD8 T cells, which then differentiate into cytotoxic effector CD8 T cells that recognize and kill any virus-infected cell.

1	Fig. 9.15 The different routes by which dendritic cells can take presentation to Cd8 T cells (third panel). It is possible, however, for up, process, and present protein antigens. uptake of antigens exogenous antigens taken into the endocytic pathway to be delivered into the endocytic system, either by receptor-mediated phagocytosis into the cytosol for eventual delivery to MhC class I molecules for or by macropinocytosis, is considered to be the major route for presentation to Cd8 T cells, a process called cross-presentation delivering peptides to MhC class II molecules for presentation to (fourth panel). Finally, it seems that antigens can be transmitted from Cd4 T cells (first two panels). production of antigens in the cytosol, one dendritic cell to another, particularly for presentation to Cd8 for example, as a result of viral infection, is thought to be the T cells, although the details of this route are still unclear (fifth panel). major route for delivering peptides to MhC

1	to Cd8 for example, as a result of viral infection, is thought to be the T cells, although the details of this route are still unclear (fifth panel). major route for delivering peptides to MhC class I molecules for

1	Uptake of extracellular virus particles or virus-infected cells into the endocytic pathway by macropinocytosis or phagocytosis can also result in the presentation of viral peptides on MHC class I molecules. This phenomenon, known as cross-presentation, is an alternative to the usual cytosolic pathway for MHC class I antigen processing and is discussed in Section 6-5. Here, viral antigens that enter dendritic cells via endocytic or phagocytic vesicles may be diverted to the cytosol for proteasomal degradation and transferred to the endoplasmic reticulum for loading onto MHC class I molecules. The result is that viruses that do not directly infect dendritic cells can stimulate the activation of CD8 T cells. Cross-presentation is performed most efficiently by a subset of conventional dendritic cells that is specialized for stimulating T-cell responses to intracellular pathogens (see Section 6-5). Any viral infection can therefore lead to the generation of cytotoxic effector CD8 T cells,

1	cells that is specialized for stimulating T-cell responses to intracellular pathogens (see Section 6-5). Any viral infection can therefore lead to the generation of cytotoxic effector CD8 T cells, whether the virus can directly infect dendritic cells or not. In addition, viral peptides presented on the dendritic cell’s MHC class II molecules activate naive CD4 T cells, which leads to the production of effector CD4 T cells that stimulate the production of antiviral antibodies by B cells and produce cytokines that enhance the immune response.

1	In some cases, such as infections with herpes simplex or influenza viruses, the dendritic cells that migrate to the lymph nodes from peripheral tissues may not be the same cells that finally present antigen to naive T cells. In herpes simplex infection, for example, dendritic cells residing in the skin capture antigen and transport it to the draining lymph nodes (Fig. 9.16). There, some antigen is transferred to resident CD8α-positive dendritic cells, which are the dominant dendritic cells responsible for priming naive CD8 T cells in this infection.

1	Fig. 9.16 Langerhans cells take up antigen in the skin, migrate differentiate into mature dendritic cells that can no longer ingest to the peripheral lymphoid organs, and present foreign antigen but have co-stimulatory activity. now they can prime both antigens to T cells. Langerhans cells (yellow) are one type of naive Cd8 and Cd4 T cells. In the case of some viral infections, for immature dendritic cell that resides in the epidermis. They ingest example, with herpes simplex virus, some dendritic cells arriving from antigen in various ways but have no co-stimulatory activity (first the site of infection seem able to transfer antigen to resident dendritic panel). In the presence of infection, they take up antigen locally cells (orange) in the lymph nodes (third panel) for presentation of and then migrate to the lymph nodes (second panel). There they class I MhC-restricted antigens to naive Cd8 T cells (fourth panel).

1	This type of transfer means that antigens from viruses that infect but rapidly kill dendritic cells can still be presented by uninfected dendritic cells that have been activated via their TLRs and can take up the dying dendritic cells and cross-present this material. cells induces their migration to lymphoid organs and enhances antigen processing.

1	A critical step in the induction of adaptive immunity is the activation of dendritic cell maturation. When an infection occurs, dendritic cells capture pathogens by means of phagocytic receptors or macropinocytosis, and then activate responses to these pathogens through pattern recognition receptors such as TLRs (Fig. 9.17, top panel). Multiple members of the TLR family are expressed on tissue dendritic cells and are thought to be involved in detecting and signaling the presence of the various classes of pathogens (see Fig. 3.16). In humans, conventional dendritic cells express all known TLRs except for TLR-9, which is, however, expressed by plasmacytoid dendritic cells along with TLR-1 and TLR-7, and other TLRs to a lesser degree. In addition to the pattern recognition receptors described in Chapter 3, several of the phagocytic receptors used by dendritic cells to take up pathogens also provide maturation signals. Examples include the lectin DC-SIGN, which binds mannose and fucose

1	in Chapter 3, several of the phagocytic receptors used by dendritic cells to take up pathogens also provide maturation signals. Examples include the lectin DC-SIGN, which binds mannose and fucose residues present on a wide range of pathogens; and Dectin-1, which recognizes β-1,3-linked glucans found in fungal cell walls (see Fig. 3.2). Other receptors that can bind pathogens, such as receptors for complement, or phagocytic receptors such as the mannose receptor, may contribute to dendritic cell activation as well as to phagocytosis.

1	TLR signaling results in a significant alteration in the chemokine receptors expressed by dendritic cells, which facilitates their migration into secondary lymphoid tissues. This change in dendritic cell behavior is often called licensing, as the cells are now embarked on the program of differentiation that will enable them to activate T cells. TLR signaling induces expression of the receptor CCR7, which makes the activated dendritic cells sensitive to the chemokine CCL21 produced by lymphoid tissue and induces their migration through the lymphatics and into the local lymphoid tissues. Whereas T cells must cross the wall of high endothelial venules to leave the blood and reach the T-cell zones, dendritic cells entering via the afferent lymphatics migrate directly into the T-cell zones from the marginal sinus.

1	dendritic cells originate from bone marrow progenitors and migrate via the blood, from which they enter and populate most tissues, including peripheral lymphoid tissues, into which they can make direct entry. entry to particular tissues is based on the particular chemokine receptors they express: CCr1, CCr2, CCr5, CCr6, CXCr1, and CXCr2 (not all shown here, for simplicity). Tissue-resident dendritic cells are highly phagocytic via receptors such as dectin-1, deC 205, dC-sIGn, and langerin, and are actively macropinocytic, but they do not express co-stimulatory molecules. They carry most of the different types of Toll-like receptors (TLrs; see the text). at sites of infection, dendritic cells are exposed to pathogens, leading to activation of their TLrs (top panel). TLr signaling causes the dendritic cells to become activated (‘licensed’), which involves induction of the chemokine receptor CCr7 (second panel). TLr signaling also increases the processing of antigens taken up into

1	the dendritic cells to become activated (‘licensed’), which involves induction of the chemokine receptor CCr7 (second panel). TLr signaling also increases the processing of antigens taken up into phagosomes. dendritic cells expressing CCr7 are sensitive to CCL19 and CCL21, which direct them to the draining lymphoid tissue (third panel). CCL19 and CCL21 provide further maturation signals, which result in higher levels of co-stimulatory B7 molecules and MhC molecules. By the time they arrive in the draining lymph node, conventional dendritic cells have become powerful activators of naive T cells but are no longer phagocytic. They express B7.1, B7.2, and high levels of MhC class I and class II molecules, as well as high levels of the adhesion molecules ICaM-1, ICaM-2, LFa-1, and Cd58 (bottom panel).

1	CCL21 signaling through CCR7 not only induces the migration of dendritic cells into lymphoid tissue, but it also contributes to their enhanced antigen-presenting function (see Fig. 9.17, third panel). By the time activated dendritic cells arrive within lymphoid tissues, they are no longer able to engulf antigens by phagocytosis or macropinocytosis. They instead express very high levels of long-lived MHC class I and MHC class II molecules, which enable them to stably present peptides from pathogens already taken up and processed. Of equal importance, they express high levels of co-stimulatory molecules on their surface. There are two main co-stimulatory molecules: the structurally related transmembrane glycoproteins B7.1 (CD80) and B7.2 (CD86), which deliver co-stimulatory signals by interacting with receptors on naive T cells (see Section 7-21). Activated dendritic cells also express very high levels of adhesion molecules, including DC-SIGN, and they secrete the chemokine CCL19, which

1	with receptors on naive T cells (see Section 7-21). Activated dendritic cells also express very high levels of adhesion molecules, including DC-SIGN, and they secrete the chemokine CCL19, which specifically attracts naive T cells. Together, these properties enable the dendritic cell to stimulate strong responses in naive T cells (see Fig. 9.17, bottom panel).

1	Despite their enhanced presentation of pathogen-derived antigens, activated dendritic cells also present some self peptides, which could present a problem for the maintenance of self-tolerance. The T-cell receptor repertoire has, however, been purged of receptors that recognize self peptides presented in the thymus (see Chapter 8), so that T-cell responses against most ubiquitous self antigens are avoided. In addition, dendritic cells in the lymphoid tissues that have not been activated by infection will bear self-peptide:MHC complexes on their surface, derived from the breakdown of their own proteins and tissue proteins present in the extracellular fluid. Because these cells do not express the appropriate co-stimulatory molecules, however, they do not have the same capacity to activate naive T cells as do activated dendritic cells. Although the details are still unclear, the presentation of self peptides by lymph node-resident, or ‘unlicensed,’ dendritic cells may induce an

1	naive T cells as do activated dendritic cells. Although the details are still unclear, the presentation of self peptides by lymph node-resident, or ‘unlicensed,’ dendritic cells may induce an alternative program of activation in naive T cells that favors immune regulation rather than immune activation.

1	Intracellular degradation of pathogens reveals pathogen components other than peptides that trigger dendritic cell activation. For example, bacterial or viral DNA containing unmethylated CpG dinucleotide motifs induces the rapid activation of plasmacytoid dendritic cells as a consequence of recognition of the DNA by TLR-9, which is present in intracellular vesicles (see Fig. 3.10). Exposure to unmethylated DNA activates NFκB and mitogen-activated protein kinase (MAPK) signaling pathways (see Figs. 7.19–7.21), leading to the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-18, and interferon (IFN)-α and IFN-β by dendritic cells. In turn, these cytokines act on the dendritic cells themselves to augment the expression of co-stimulatory molecules. Heat-shock proteins are another internal bacterial constituent that can activate the antigen-presenting function of dendritic cells. Similarly, some viruses are recognized by TLRs inside the dendritic cell via double-stranded RNA

1	internal bacterial constituent that can activate the antigen-presenting function of dendritic cells. Similarly, some viruses are recognized by TLRs inside the dendritic cell via double-stranded RNA produced during viral replication.

1	The induction of co-stimulatory activity in antigen-presenting cells by common microbial constituents is believed to allow the immune system to distinguish antigens borne by infectious agents from antigens associated with innocuous proteins, including self proteins. Indeed, many foreign proteins do not induce an immune response when injected on their own because they fail to induce co-stimulatory activity in antigen-presenting cells. When such protein antigens are mixed with bacteria, however, they become immunogenic, because the bacteria induce the essential co-stimulatory activity in cells that ingest the protein. Bacteria or bacterial components used in this way are known as adjuvants (see Appendix I, Section A-1). We will see in Chapter 15 how self proteins mixed with bacterial adjuvants can induce autoimmune disease, illustrating the crucial importance of the regulation of co-stimulatory activity in the discrimination of self from nonself.

1	interferons and may act as helper cells for antigen presentation by conventional dendritic cells.

1	Plasmacytoid dendritic cells are thought to act as sentinels in early defense against viral infection on the basis of their expression of TLRs and the intracellular nucleic acid-sensing RIG-I-like helicases, and their high production of antiviral type I interferons (see Sections 3-10 and 3-22). For several reasons, they are not thought to be involved in a major way in the antigen-specific activation of naive T cells. Plasmacytoid dendritic cells express fewer MHC class II and co-stimulatory molecules on their surface, and they process antigens less efficiently than conventional dendritic cells. In addition, unlike conventional dendritic cells, plasmacytoid dendritic cells do not cease the synthesis and recycling of MHC class II molecules after being activated. This means that they rapidly recycle their surface MHC II molecules and so cannot present pathogen-derived peptide:MHC complexes to T cells for extended periods, as conventional dendritic cells do.

1	Plasmacytoid dendritic cells may, however, act as helper cells for antigen presentation by conventional dendritic cells. This activity was revealed by studies in mice infected with the intracellular bacterium Listeria monocytogenes. Normally, IL-12 made by conventional dendritic cells induces CD4 T cells to produce abundant IFN-γ, which helps macrophages kill the bacteria. When plasmacytoid dendritic cells were experimentally eliminated, IL-12 production by conventional dendritic cells decreased, and the mice became susceptible to Listeria. It appears that plasmacytoid dendritic cells interact with conventional dendritic cells to sustain IL-12 production. Activation of plasmacytoid dendritic cells through TLR-9 induces the expression of CD40 ligand (CD40L or CD154), a TNF-family transmembrane cytokine, which binds to CD40, a TNF-family receptor that is expressed by activated conventional dendritic cells. This interaction enables conventional dendritic cells to sustain production of

1	cytokine, which binds to CD40, a TNF-family receptor that is expressed by activated conventional dendritic cells. This interaction enables conventional dendritic cells to sustain production of the pro-inflammatory cytokine IL-12, strengthening the IL-12-induced production of IFN-γ by T cells. Plasmacytoid dendritic cells can also produce IL-12 themselves, although in smaller amounts than conventional dendritic cells do.

1	9-12 Macrophages are scavenger cells that can be induced by pathogens to present foreign antigens to naive T cells.

1	The two other cell types that can act as antigen-presenting cells to T cells are macrophages and B cells, although there is an important distinction between the function of antigen presentation by these cells and that of dendritic cells. It is unlikely that macrophages and B cells present antigen to activate naive T cells. Rather, these cells present antigen to T cells that have already been primed by conventional dendritic cells as a means to recruit the effector, or ‘helper,’ functions of T cells that, in turn, provide signals to enhance their own effector functions. In this way, naive B cells that are activated by antigen bound to their surface immunoglobulin receptor present peptides derived from that antigen to elicit help from effector T cells in order to differentiate into immunoglobulin-secreting cells. And, as we learned in Chapter 3, while many microorganisms that enter the body are engulfed and destroyed by phagocytes, which provide an innate, antigen-nonspecific first line

1	cells. And, as we learned in Chapter 3, while many microorganisms that enter the body are engulfed and destroyed by phagocytes, which provide an innate, antigen-nonspecific first line of defense against infection, some pathogens have developed mechanisms to avoid elimination by innate immunity, such as resisting the killing properties of phagocytes. Macrophages that have ingested microorganisms but have failed to destroy them can use antigen presentation to recruit the adaptive immune response to enhance their microbicidal activities, as we will discuss further in Chapter 11.

1	Resting macrophages have few or no MHC class II molecules on their surface and do not express B7. The expression of both MHC class II molecules and B7 is induced by the ingestion of microorganisms and recognition of their microbe-associated molecular patterns (MAMPs). Macrophages, like dendritic cells, have a variety of pattern recognition receptors that recognize microbial surface components (see Chapter 3). Receptors such as Dectin-1, scavenger receptors, and complement receptors take up microorganisms into phagosomes, where they are degraded to produce peptides for presentation, while recognition of pathogen components via TLRs triggers intracellular signaling that contributes to the expression of MHC class II molecules and B7. However, unlike conventional dendritic cells, tissue-resident macrophages are generally nonmigratory; they do not traffic to T-cell zones of lymphoid tissues when activated by pathogens. It is thus likely that increased expression of MHC class II molecules

1	macrophages are generally nonmigratory; they do not traffic to T-cell zones of lymphoid tissues when activated by pathogens. It is thus likely that increased expression of MHC class II molecules and co-stimulatory molecules by activated macrophages is more important for locally amplifying T-cell responses already initiated by dendritic cells. This appears to be important for the maintenance and functioning of effector or memory T cells that enter a site of infection.

1	In addition to residing in tissues, macrophages are found in lymphoid organs (see Fig. 9.13). They are present in many areas of the lymph node, including the marginal sinus, where the afferent lymph enters the lymphoid tissue, and in the medullary cords, where the efferent lymph collects before flowing into the blood. However, they are largely sequestered from T-cell zones and are inefficient activators of naive T cells. Rather, their main function in lymphoid tissues appears to be the ingestion of microbes and particulate antigens to prevent them from entering the blood. They are also important scavengers of apoptotic lymphocytes.

1	Macrophages in other sites also continuously scavenge dead or dying cells, which are rich sources of self antigens, so it is particularly important that they should not activate naive T cells. The Kupffer cells of the liver sinusoids and the macrophages of the splenic red pulp, in particular, remove large numbers of dying cells from the blood daily. Kupffer cells express little MHC class II and no TLR-4, the receptor that signals the presence of bacterial LPS. Thus, although they generate large amounts of self peptides in their endosomes, these macrophages are not likely to elicit an immune response. 9-13 B cells are highly efficient at presenting antigens that bind to their surface immunoglobulin.

1	B cells are uniquely adapted to bind specific soluble molecules through their membrane-bound antigenic receptor, or B-cell receptor (BCR), the antigen-binding component of which is membrane-associated IgM, which is highly efficient at internalizing the bound molecules by receptor-mediated endocytosis. If the antigen contains a protein component, the B cell will process the internalized protein to peptide fragments and then display the fragments as peptide:MHC class II complexes. Through this mechanism B cells are able to take up and present even low concentrations of specific antigen to T cells. B cells also constitutively express high levels of MHC class II molecules, and so high levels of specific peptide:MHC class II complexes appear on the B-cell surface (Fig. 9.18). As we will see in Chapter 10, this pathway of antigen presentation allows the B cell to specifically interact with a CD4 T cell that has been previously activated by the same antigen as a mechanism to receive signals

1	Chapter 10, this pathway of antigen presentation allows the B cell to specifically interact with a CD4 T cell that has been previously activated by the same antigen as a mechanism to receive signals from the T cell to drive the B cell's differentiation into an antibody-producing cell.

1	B cells do not constitutively express co-stimulatory molecules, but, as with dendritic cells and macrophages, they can be induced by various microbial constituents to express B7 molecules. In fact, B7.1 was first identified as a protein on B cells activated by LPS, and B7.2 is predominantly expressed by B cells in vivo. Soluble protein antigens are not abundant during infections; most natural antigens, such as bacteria and viruses, are particulate, and many soluble bacterial toxins act by binding to cell surfaces and so are present only at low concentrations in solution. Some natural immunogens enter the body as soluble molecules, however; examples are bacterial toxins, anticoagulants injected by blood-sucking insects, snake venoms, and many allergens. Nevertheless, it is unlikely that B cells are important in priming naive T cells to soluble antigens in natural immune responses. Tissue dendritic cells can take up soluble antigens by macropinocytosis, and although they cannot

1	that B cells are important in priming naive T cells to soluble antigens in natural immune responses. Tissue dendritic cells can take up soluble antigens by macropinocytosis, and although they cannot concentrate these antigens as antigen-specific B cells do, dendritic cells are more likely to encounter a naive T cell with the appropriate antigen specificity than are the extremely limited number of antigen-specific B cells. The chances of a B cell encountering a T cell that can recognize the peptide antigens it displays are greatly increased once a naive T cell has been detained in lymphoid tissue by finding its antigen on the surface of a dendritic cell and has undergone clonal expansion.

1	High density of speciÿc antigen fragments are presented at the B-cell surface Speciÿc antigen is efÿciently internalized by receptor-mediated endocytosis Fig. 9.18 B cells can use their surface immunoglobulin to present specific antigen very efficiently to T cells. The three types of antigen-presenting cells are compared in Fig. 9.19. In each of these cell types the expression of co-stimulatory activity is controlled so as to provoke responses against pathogens while avoiding immunization against self.

1	surface immunoglobulin allows B cells to bind and internalize specific antigen very efficiently, especially if the antigen is present as a soluble protein, as most toxins are. The internalized antigen is processed in intracellular vesicles, where it binds to MhC class II molecules. The vesicles are transported to the cell surface, where the foreign-peptide:MhC class II complexes can be recognized by T cells. When the protein antigen is not specific for the B-cell receptor, its internalization is inefficient and only a few fragments of such proteins are subsequently presented at the B-cell surface (not shown).

1	Fig. 9.19 The properties of the various antigen-presenting cells. dendritic cells, macrophages, and B cells are the main cell types involved in the presentation of foreign antigens to T cells. These cells vary in their means of antigen uptake, MhC class II expression, co-stimulator expression, the type of antigen they present effectively, their locations in the body, and their surface adhesion molecules (not shown). antigen presentation by dendritic cells is primarily involved in activating naive T cells for expansion and differentiation. Macrophages and B cells present antigen primarily to receive specific help from effector T cells in the form of cytokines or surface molecules. Summary.

1	Summary. An adaptive immune response is generated when naive T cells contact activated antigen-presenting cells in the secondary lymphoid organs. These tissues have a specialized architecture that facilitates efficient interaction between circulating lymphocytes and their target antigens. The formation and organization of the peripheral lymphoid organs are controlled by proteins of the TNF family and their receptors (TNFRs). Lymphoid tissue inducer (LTi) cells expressing lymphotoxin-β (LT-β) interact with stromal cells expressing the LT-β receptor in the developing embryo to induce chemokine production, which in turn initiates formation of the lymph nodes and Peyer’s patches. Similar interactions between lymphotoxin-expressing B cells and TNFR-Iexpressing follicular dendritic cells (FDCs) establish the normal architecture of the spleen and lymph nodes. B and T cells are partitioned into distinct areas within lymphoid tissue by specific chemokines.

1	To ensure that rare antigen-specific T cells survey the body effectively for pathogen-bearing antigen-presenting cells, T cells continuously recirculate through the lymphoid organs and thus can sample antigens brought in by antigen-presenting cells from many different tissue sites. The migration of naive T cells into lymphoid organs is guided by the chemokine receptor CCR7, which binds CCL21 that is produced by stromal cells in the T-cell zones of secondary lymphoid tissues and is displayed on the specialized endothelium of HEVs. L-selectin expressed by naive T cells initiates their rolling along the specialized surfaces of high endothelial venules, where contact with CCL21 induces a switch in the integrin LFA-1 expressed by T cells to a configuration with affinity for the ICAM-1 expressed on the venule endothelium. This initiates strong adhesion, diapedesis, and migration of the T cells into the T-cell zone. There, naive T cells meet antigen-bearing dendritic cells, of which there

1	on the venule endothelium. This initiates strong adhesion, diapedesis, and migration of the T cells into the T-cell zone. There, naive T cells meet antigen-bearing dendritic cells, of which there are two main populations: conventional dendritic cells, and plasmacytoid dendritic cells. Conventional dendritic cells continuously survey secondary tissues for invading pathogens and are the dendritic cells responsible for activating naive T cells. Contact with pathogens delivers signals to dendritic cells via TLRs and other receptors that accelerate antigen processing and the production of for-eign-peptide:self MHC complexes. TLR signaling also induces expression of CCR7 by dendritic cells, which directs their migration to T-cell zones of secondary lymphoid organs, where they encounter and activate naive T cells.

1	Macrophages and B cells can also process particulate or soluble antigens from pathogens to be presented as peptide:MHC complexes to T cells. However, whereas antigen presentation to naive T cells is uniquely mediated by dendritic cells, antigen presentation by macrophages and B cells enables the latter two cell types to recruit the effector activities of previously activated antigen-specific T cells. For example, as discussed in Chapter 11, by presenting antigens of ingested pathogens, macrophages recruit help from IFN-γ-producing CD4 T cells to augment their intracellular killing of these pathogens. Presentation of antigens by B cells recruits help from T cells to stimulate antibody production and class switching, a topic discussed further in Chapter 10. In all three types of antigen-presenting cells, the expression of co-stimulatory molecules is activated in response to signals from receptors that also function in innate immunity to signal the presence of infectious agents.

1	Priming of naive T cells by pathogen-activated dendritic cells. T-cell responses are initiated when a mature naive CD4 or CD8 T cell encounters an activated antigen-presenting cell displaying the appropriate peptide:MHC ligand. We will now describe the generation of effector T cells from naive T cells. The activation and differentiation of naive T cells, often called priming, is distinct from the later responses of effector T cells to antigen on their target cells, and from the responses of primed memory T cells to subsequent encounters with the same antigen. Priming of naive CD8 T cells generates cytotoxic T cells capable of directly killing pathogen-infected cells. CD4 cells develop into a diverse array of effector cell types depending on the nature of the signals they receive during priming. CD4 effector activity can also include cytotoxicity, but more frequently it involves the secretion of a set of cytokines, which direct target cells toward a more pathogen-specific response.

1	9-14 Cell-adhesion molecules mediate the initial interaction of naive T cells with antigen-presenting cells. As they migrate through the cortical region of the lymph node, naive T cells bind transiently to each antigen-presenting cell that they encounter. Activated dendritic cells bind naive T cells very efficiently through interactions between LFA-1 and CD2 on the T cell and ICAM-1, ICAM-2, and CD58 on the dendritic cell (Fig. 9.20). Perhaps because of this synergy, the precise role of each adhesion molecule has been difficult to distinguish. People lacking LFA-1 can have normal T-cell responses, and this also seems to be true for genetically engineered mice lacking CD2, suggesting substantial redundancy in the function of these molecules.

1	The transient binding of naive T cells to antigen-presenting cells is crucial in providing time for a T cell to sample large numbers of MHC molecules for the presence of its cognate antigenic peptide. In those rare cases in which a naive T cell recognizes its peptide:MHC ligand, signaling through the T-cell receptor induces a conformational change in LFA-1 that greatly increases its affinity for ICAM-1 and ICAM-2. This conformational change is the same as that induced by signaling through CCR7 during the migration of naive T cells into a secondary lymphoid organ (see Section 9-6). The change in LFA-1 stabilizes the association between the antigen-specific T cell and the antigen-presenting cell (Fig. 9.21). The association can persist for several days, during which time the naive T cell proliferates and its progeny, which can also adhere to the antigen-presenting cell, differentiate into effector T cells.

1	Most encounters of T cells with antigen-presenting cells do not, however, result in the recognition of an antigen. In this case, the T cell must be able to separate efficiently from the antigen-presenting cell so that it can continue to migrate through the lymphoid tissue, eventually exiting to reenter the blood and continue circulating. Dissociation, like stable binding, may also involve signaling between the T cell and the antigen-presenting cells, but little is known of its mechanism. Conformational change in LFA-1 increases afÿnity and prolongs cell–cell contact Subsequent binding of T-cell receptors signals LFA-1 Fig. 9.21 Transient adhesive interactions between T cells and antigen-presenting cells are stabilized by specific antigen recognition.

1	Fig. 9.21 Transient adhesive interactions between T cells and antigen-presenting cells are stabilized by specific antigen recognition. Fig. 9.20 Cell-surface molecules of the immunoglobulin superfamily are important in the interactions of lymphocytes with antigen-presenting cells. In the initial encounter of T cells with antigen-presenting cells, Cd2 binding to Cd58 on the antigen-presenting cell synergizes with LFa-1 binding to ICaM-1 and ICaM-2. LFa-1 is the αL:β2 integrin heterodimer Cd11a:Cd18. ICaM-1 and ICaM-2 are also known as Cd54 and Cd102, respectively. MOVIE XX.X When a T cell binds to its specific ligand on an antigen-presenting cell, intracellular signaling through the T-cell receptor (TCr) induces a conformational change in LFa-1 that causes it to bind with higher affinity to ICaMs on the antigen-presenting cell. The T cell shown here is a Cd4 T cell.

1	on resting T cells, the β and γ chains are expressed constitutively. They bind IL-2 with moderate affinity. activation of T cells induces the synthesis of the α chain and the formation of the high-affinity heterotrimeric receptor. The β and γ chains show similarities in amino acid sequence to cell-surface receptors for growth hormone and prolactin, each of which also regulates cell growth and differentiation.

1	Fig. 9.22 Three kinds of signals are involved in activation of naive T cells by antigen-presenting cells. Binding of the foreign-peptide:self MhC complex by the T-cell receptor and, in this example, a Cd4 co-receptor transmits a signal (arrow 1) to the T cell that antigen has been encountered. effective activation of naive T cells requires a second signal (arrow 2), the co-stimulatory signal, to be delivered by the same antigen-presenting cell (apC). In this example, Cd28 on the T cell encountering B7 molecules on the antigen-presenting cell delivers signal 2, whose net effect is the increased survival and proliferation of the T cell that has received signal 1. ICos and various members of the TnF receptor family may also provide co-stimulatory signals. For Cd4 T cells in particular, different pathways of differentiation produce subsets of effector T cells that carry out different effector responses, depending on the nature of a third signal (arrow 3) delivered by the

1	particular, different pathways of differentiation produce subsets of effector T cells that carry out different effector responses, depending on the nature of a third signal (arrow 3) delivered by the antigen-presenting cell. Cytokines are commonly, but not exclusively, involved in directing this differentiation.

1	9-15 Antigen-presenting cells deliver multiple signals for the clonal expansion and differentiation of naive T cells.

1	When discussing the activation of naive T cells, it is useful to consider at least three different types of signals (Fig. 9.22). The first signal is generated from the interaction of a specific peptide:MHC complex with the T-cell receptor. Engagement of the T-cell receptor with its specific peptide antigen is essential for activating a naive T cell. However, even if the co-receptor—CD4 or CD8—is also ligated, this does not, on its own, stimulate the T cell to fully proliferate and differentiate into effector T cells. Expansion and differentiation of naive T cells involve at least two other kinds of signals: co-stimulatory signals that promote the survival and expansion of the T cells, and cytokines that direct T-cell differentiation into one of the different subsets of effector T cells. Additional signals, such as Notch ligands, can contribute to the effector differentiation of naive T cells, although these signals appear to be of lesser importance than those of lineage-specifying

1	Additional signals, such as Notch ligands, can contribute to the effector differentiation of naive T cells, although these signals appear to be of lesser importance than those of lineage-specifying cytokines.

1	The best-characterized co-stimulatory molecules are the B7 molecules. These homodimeric members of the immunoglobulin superfamily are found exclusively on the surfaces of cells, such as dendritic cells, that stimulate naive T-cell proliferation (see Section 9-8). The receptor for B7 molecules on the T cell is CD28, a member of the immunoglobulin superfamily (see Section 7-21). Ligation of CD28 by B7 molecules is necessary for the optimal clonal expansion of naive T cells; targeted deficiency of B7 molecules or experimental blockade of the binding of B7 molecules to CD28 has been shown to inhibit T-cell responses. 9-16 CD28-dependent co-stimulation of activated T cells induces expression of interleukin-2 and the high-affinity IL-2 receptor.

1	Naive T cells are found as small resting cells with condensed chromatin and scanty cytoplasm, and they synthesize little RNA or protein. On activation, they reenter the cell cycle and divide rapidly to produce large numbers of progeny as they undergo antigen-driven differentiation. Unlike effector T cells, which can produce a diversity of cytokines depending on the mature effector phenotype, naive T cells primarily produce interleukin-2 (IL-2) when activated. Based on in vitro studies, IL-2 was long thought to be required for the proliferation of naive T cells. However, in vivo studies indicate that while IL-2 can augment T-cell proliferation and survival, in many cases it is dispensable and other functions of IL-2 might be more important. In particular, IL-2 is essential for the maintenance of regulatory T cells, which do not produce their own IL-2 when activated. IL-2 also appears to affect the balance of effector and memory T cells that develop in a primary response to antigen, as

1	of regulatory T cells, which do not produce their own IL-2 when activated. IL-2 also appears to affect the balance of effector and memory T cells that develop in a primary response to antigen, as will be discussed in Chapter 11.

1	The initial encounter with specific antigen in the presence of a co-stimulatory signal triggers entry of the T cell into the G1 phase of the cell cycle; at the same time, it also induces the synthesis of IL-2 along with the α chain of the IL-2 receptor (also known as CD25). The IL-2 receptor is composed of three chains: α, β, and γ (Fig. 9.23). Prior to activation, naive T cells express a form of the receptor composed only of the βand γ chains, which has only moderate affinity for IL-2 binding. Within hours of activation, naive T cells upregulate the expression of CD25. Association of CD25 with the βand γ heterodimer creates a receptor with a much higher affinity for IL-2, allowing the T cell to respond to very low concentrations of IL-2. In contrast to naive T cells, regulatory T, or T , cells constitutively express

1	In contrast to naive T cells, regulatory T, or T , cells constitutively express CD25, and thus the high-affinity, trimeric form of the IL-2 receptor (see Fig. 9.23). As is discussed later (see Section 9-23), it is thought that by expressing the high-affinity form of the IL-2 receptor, T cells can outcompete T cells that express only the low-affinity form of the receptor for binding of the limited quantities of IL-2 that are available early in the response to antigen. In this way, T cells act as a ‘sink’ for IL-2 to limit its availability to other cells.

1	However, once activated naive T cells have upregulated CD25, they form the high-affinity receptor and compete with T cells for binding of IL-2. The bind- ing of IL-2 by these activated naive T cells triggers signaling that supports their activation and differentiation, and can enhance their proliferation (Fig. 9.24). T cells activated in this way can divide up to four times a day for several days, allowing one precursor cell to give rise to thousands of clonal progeny that all bear the same antigenic receptor.

1	Antigen recognition by the T-cell receptor induces the synthesis or activation of the transcription factors NFAT, AP-1, and NFκB, which bind to the promoter region of the IL-2 gene in naive T cells to activate its transcription (see Sections 7-14 and 7-16). Co-stimulation through CD28 contributes to the production of IL-2 in at least three ways. First, CD28 signaling activates PI 3-kinase, which increases production of the AP-1 and NFκB transcription factors, thereby increasing the transcription of IL-2 mRNA. However, the mRNAs of many cytokines, including IL-2, are very short-lived because of an ‘instability’ sequence (AUUUAUUUA) in the 3' untranslated region. CD28 signaling prolongs the lifetime of an IL-2 mRNA molecule by inducing the expression of proteins that block the activity of the instability sequence, resulting in increased translation and more IL-2 protein. Finally, PI 3-kinase helps activate the protein kinase Akt (see Section 7-17), which generally promotes cell growth

1	the instability sequence, resulting in increased translation and more IL-2 protein. Finally, PI 3-kinase helps activate the protein kinase Akt (see Section 7-17), which generally promotes cell growth and survival, increasing the total production of IL-2 by activated T cells.

1	9-17 Additional co-stimulatory pathways are involved in T-cell activation. Once a naive T cell is activated, it expresses a number of proteins in addition to CD28 that contribute to sustaining or modifying the co-stimulatory signal. These other co-stimulatory receptors generally belong to either the CD28 or the TNF receptor family.

1	CD28-related proteins are expressed on activated T cells and modify the co-stimulatory signal as the T-cell response develops. One such protein is the inducible co-stimulator (ICOS), which binds a ligand known as ICOSL (ICOS ligand, or B7-H2), a structural relative of B7.1 and B7.2. ICOSL is produced on activated dendritic cells, monocytes, and B cells. Although ICOS resembles CD28 in driving T-cell proliferation, it does not induce IL-2 but seems to regulate the expression of other cytokines, such as IL-4 and IFN-γ, made by CD4 T-cell subsets. ICOS is particularly important for enabling CD4 T cells to function as helper cells for B-cell responses such as isotype switching. ICOS is expressed on T cells in germinal centers within lymphoid follicles, and mice lacking ICOS fail to develop germinal centers and have severely diminished antibody responses.

1	Another receptor for B7 molecules is CTLA-4 (CD152), which is related in sequence to CD28. CTLA-4 binds B7 molecules about 20 times more avidly Fig. 9.24 Activated T cells secrete and respond to IL-2. activation of naive T cells induces the expression and secretion of IL-2 and the expression of high-affinity IL-2 receptors. IL-2 binds to the high-affinity IL-2 receptors to enhance T-cell growth and differentiation. CTLA-4 binds B7 more avidly than does CD28 and delivers inhibitory signals to activated T cells activated T cell antigen-presenting cell MHC class II CD4 + TCRCTLA-4 B7.1

1	CTLA-4 binds B7 more avidly than does CD28 and delivers inhibitory signals to activated T cells activated T cell antigen-presenting cell MHC class II CD4 + TCRCTLA-4 B7.1 Fig. 9.25 CTLA-4 is an inhibitory receptor for B7 molecules. naive T cells express Cd28, which delivers a co-stimulatory signal on binding B7 molecules (see Fig. 9.22), thereby driving the survival and expansion of the T cells. activated T cells express increased levels of CTLa-4 (Cd152), which has a higher affinity than Cd28 for B7 molecules and thus binds most or all of the B7 molecules. CTLa-4 thereby serves to regulate the proliferative phase of the T-cell response.

1	than CD28, but its effect is to inhibit, rather than activate, the T cell (Fig. 9.25). CTLA-4 does not contain an ITIM motif, and it is suggested to inhibit T-cell activation by competing with CD28 for interaction with B7 molecules expressed by antigen-presenting cells. Activation of naive T cells induces the surface expression of CTLA-4, making activated T cells less sensitive than naive T cells to stimulation by the antigen-presenting cell, thereby restricting IL-2 production. Thus, binding of CTLA-4 to B7 molecules is essential for limiting the proliferative response of activated T cells to antigen and B7. This was confirmed by producing mice with a disrupted CTLA-4 gene; such mice develop a fatal disorder characterized by a massive overgrowth of lymphocytes. Antibodies that block CTLA-4 from binding to B7 molecules markedly increase T cell-dependent immune responses.

1	Several TNF-family molecules also deliver co-stimulatory signals. These all seem to function by activating NFκB through a TRAF-dependent pathway (see Section 7-23). The binding of CD70 on dendritic cells to its constitutively expressed CD27 receptor on naive T cells delivers a potent co-stimulatory signal to T cells early in the activation process. The receptor CD40 on dendritic cells binds to CD40 ligand expressed on T cells, initiating two-way signaling that transmits activating signals to the T cell and also induces the dendritic cell to express increased B7, thus stimulating further T-cell proliferation. The role of the CD40–CD40 ligand pair in sustaining a T-cell response is demonstrated in mice lacking CD40 ligand; when these mice are immunized, the clonal expansion of responding T cells is curtailed at an early stage. The T-cell molecule 4-1BB (CD137) and its ligand 4-1BBL, which is expressed on activated dendritic cells, macrophages, and B cells, make up another pair of

1	T cells is curtailed at an early stage. The T-cell molecule 4-1BB (CD137) and its ligand 4-1BBL, which is expressed on activated dendritic cells, macrophages, and B cells, make up another pair of TNF-family co-stimulators. The effects of this interaction are also bidirectional, with both the T cell and the antigen-presenting cell receiving activating signals; this type of interaction is sometimes referred to as the T-cell:antigen-presenting cell dialog. Another co-stimulatory receptor and its ligand, OX40 and OX40L, are expressed on activated T cells and dendritic cells, respectively. Mice deficient in OX40 show reduced CD4 T-cell proliferation in response to viral infection, indicating that OX40 has a role in sustaining ongoing T-cell responses by enhancing T-cell survival and proliferation.

1	9-18 Proliferating T cells differentiate into effector T cells that do not require co-stimulation to act.

1	During the 4–5 days of rapid cell division that follow naive T-cell activation, T cells differentiate into effector T cells that acquire the ability to synthesize molecules required for their specialized helper or cytotoxic functions when they re-encounter their specific antigen. Effector T cells undergo additional changes that distinguish them from naive T cells. One of the most important is in their activation requirements: once a T cell has differentiated into an effector cell, encounter with its specific antigen results in immune attack without the need for co-stimulation (Fig. 9.26). This distinction is particularly easy to understand for CD8 cytotoxic T cells, which must be able to act on any cell infected with a virus, whether or not the infected cell can express co-stimulatory molecules. However, this feature is also important for the effector function of CD4 cells, as effector CD4 T cells must be able to activate B cells and macrophages that have taken up antigen even if

1	molecules. However, this feature is also important for the effector function of CD4 cells, as effector CD4 T cells must be able to activate B cells and macrophages that have taken up antigen even if these cells are not initially expressing co-stimulatory molecules.

1	Changes are also seen in the cell-adhesion molecules and receptors expressed by effector T cells. They lose cell-surface L-selectin and therefore cease to recirculate through lymph nodes. Instead, they express glycans that serve as ligands for Pand E-selectins (for example, P-selectin glycoprotein-1, or PSGL-1), which are upregulated on inflamed vascular endothelial cells and

1	Fig. 9.26 Effector T cells can respond to their target cells effector cell status (central panel). once the cells have differentiated without co-stimulation. a naive T cell that recognizes antigen on into effector T cells, any encounter with specific antigen triggers the surface of an antigen-presenting cell and receives the required their effector actions without the need for co-stimulation. Thus, as two signals (arrows 1 and 2, left panel) becomes activated, and it illustrated here, a cytotoxic T cell can kill any virus-infected target both secretes and responds to IL-2. IL-2 signaling enhances clonal cells, including those that do not express co-stimulatory molecules. expansion and contributes to the differentiation of the T cells to allow effector T cells to roll on the blood vessels at sites of inflammation. They also express higher levels of LFA-1 and CD2 than do naive T cells, as well as the integrin VLA-4, which allows them to bind to vascular endothelium bearing the adhesion

1	at sites of inflammation. They also express higher levels of LFA-1 and CD2 than do naive T cells, as well as the integrin VLA-4, which allows them to bind to vascular endothelium bearing the adhesion molecule VCAM-1, which is also expressed on the inflamed endothelium. This allows effector T cells to exit the bloodstream and enter sites of infection, where they orchestrate the local immune response. These changes in the T-cell surface are summarized in Fig. 9.27, and will be discussed further in Chapter 11.

1	Fig. 9.27 Activation of T cells changes the expression of several cell-surface molecules. The example here is a Cd4 T cell. resting naive T cells express L-selectin, through which they home to lymph nodes, but express relatively low levels of other adhesion molecules such as Cd2 and LFa-1. upon activation, expression of L-selectin ceases and, instead, expression of ligands for pand e-selectins are induced (e.g., psGL-1), which allow the activated T cells to roll on pand e-selectins expressed on endothelium at sites of inflammation. Increased amounts of the integrin LFa-1 are also produced, which is activated to bind its ligands, ICaM-1 and ICaM-2. a newly expressed integrin called vLa-4, which allows T cells to arrest on inflamed vascular endothelium, ensures that activated T cells enter peripheral tissues at sites where they are likely to encounter infection. activated T cells also have on their surface a higher density of the adhesion molecule Cd2, increasing the avidity of their

1	peripheral tissues at sites where they are likely to encounter infection. activated T cells also have on their surface a higher density of the adhesion molecule Cd2, increasing the avidity of their interaction with potential target cells, as well as a higher density of the adhesion molecule Cd44. By alternative splicing of the rna transcript of the Cd45 gene, a change occurs in the isoform of Cd45 that is expressed, with activated T cells expressing the Cd45ro isoform, which associates with the T-cell receptor and Cd4. This change makes the T cell more sensitive to stimulation by low concentrations of peptide:MhC complexes. Finally, the sphingosine 1-phosphate receptor 1(s1pr1) is expressed by resting naive T cells, allowing the egress from lymphoid tissues of cells that do not become activated (see Fig. 9.11). downregulation of s1pr1 for several days after activation prevents T-cell egress during the period of proliferation and differentiation. after several days, it is expressed

1	activated (see Fig. 9.11). downregulation of s1pr1 for several days after activation prevents T-cell egress during the period of proliferation and differentiation. after several days, it is expressed again, allowing effector cells to exit from the lymphoid tissues.

1	9-19 CD8 T cells can be activated in different ways to become cytotoxic effector cells. Naive T cells fall into two large classes, of which one carries the co-receptor CD8 on its surface and the other bears the co-receptor CD4. CD8 T cells all differentiate into CD8 cytotoxic T cells (sometimes called cytotoxic lymphocytes, or CTLs), which kill their target cells (Fig. 9.28). They are important in the defense against intracellular pathogens, especially viruses. Virus-infected cells display fragments of viral proteins as peptide:MHC class I complexes on their surface, and these are recognized by cytotoxic T lymphocytes.

1	Perhaps because the effector actions of these cells are so destructive, naive CD8 T cells require more co-stimulatory activity to drive them to become activated effector cells than do naive CD4 T cells. This requirement can be met in two ways. The simplest is priming by activated dendritic cells, which have high intrinsic co-stimulatory activity. In some viral infections, dendritic cells become sufficiently activated to directly induce CD8 T cells to produce the IL-2 required for their differentiation into cytotoxic effector cells, without help from CD4 T cells. This property of dendritic cells has been exploited to generate cytotoxic T-cell responses against tumors, as we will see in Chapter 16.

1	In the majority of viral infections, however, CD8 T-cell activation requires additional help, which is provided by CD4 effector T cells. CD4 T cells that recognize related antigens presented by the antigen-presenting cell can amplify the activation of naive CD8 T cells by further activating the antigen-presenting cell (Fig. 9.29). B7 expressed by the dendritic cell first activates the CD4 T cells to express IL-2 and CD40 ligand (see Sections 9-16 and 9-17). CD40 ligand binds CD40 on the dendritic cell, delivering an additional signal that increases the expression of B7 and 4-1BBL by the dendritic cell, which in turn provides additional co-stimulation to the naive CD8 T cell. The IL-2 produced by activated CD4 T cells also acts to promote effector CD8 T-cell differentiation. 9-20 CD4 T cells differentiate into several subsets of functionally different effector cells.

1	9-20 CD4 T cells differentiate into several subsets of functionally different effector cells. In contrast with CD8 T cells, CD4 T cells differentiate into several subsets of effector T cells that orchestrate different immune functions. The main functional subsets are TH1 (T helper 1), TH2, TH17, T follicular helper (TFH), and regulatory T (Treg) cells. The TH1, TH2, and TH17 subsets are elicited by different classes of pathogens and are defined on the basis of the different combinations of cytokines that they secrete (Fig. 9.30). These subsets cooperate with different innate cells of the myelomonocytic series and with innate lymphoid cells (ILCs) to form integrated ‘immune modules’ specialized for the clearance of the different classes of pathogens (see Fig. 3.37). One or the other of these subsets will typically become predominant as an immune response progresses, especially in persistent infections, autoimmunity, or allergies. As

1	Fig. 9.29 Most CD8 T-cell responses require CD4 T cells. Cd8 T cells recognizing antigen on weakly co-stimulatory cells may become activated only in the presence of Cd4 T cells interacting with the same antigen-presenting cell (apC). This happens mainly by an effector Cd4 T cell recognizing antigen on the antigen-presenting cell and being triggered to induce increased levels of co-stimulatory activity on the antigen-presenting cell. The Cd4 T cells also produce abundant IL-2 and thus help drive Cd8 T-cell proliferation. This may in turn activate the Cd8 T cell to make its own IL-2.

1	Fig. 9.30 Subsets of CD4 effector T cells are specialized to provide help to different target cells for the eradication of different classes of pathogens. unlike Cd8 T cells, which act directly on infected target cells to eliminate pathogens, Cd4 T cells typically enhance the effector functions of other cells that eradicate pathogens—whether cells of the innate immune system, or, in the case of TFh cells, antigen-specific B cells. Th1 cells (first panels) produce cytokines, such as IFn-γ, which activate macrophages, enabling them to destroy intracellular microorganisms more efficiently. Th2 cells (second panels) produce cytokines that recruit and activate eosinophils (IL-5) and mast cells and basophils (IL-4), and promote enhanced barrier immunity at mucosal surfaces (IL-13) to eradicate helminths. Th17 cells (third panels) secrete IL-17-family cytokines that induce local epithelial and stromal cells to produce chemokines that recruit neutrophils to sites of infection. Th17 cells also

1	Th17 cells (third panels) secrete IL-17-family cytokines that induce local epithelial and stromal cells to produce chemokines that recruit neutrophils to sites of infection. Th17 cells also produce IL-22, which along with IL-17 can activate epithelial cells at the barrier site to produce antimicrobial peptides that kill bacteria. TFh cells (fourth panels) form cognate interactions with naive B cells through linked recognition of antigen and traffic to B-cell follicles, where they promote the germinal center response. TFh cells produce cytokines characteristic of other subsets and participate in type 1, 2, and 3 responses that are recruited against different types of pathogens. TFh cells producing IFn-γ activate B cells to produce strongly opsonizing antibodies belonging to certain IgG subclasses (IgG1 and IgG3 in humans, and their homologs, IgG2a and IgG2b, in the mouse) in type 1 responses. Those TFh cells producing IL-4 drive B cells to differentiate and produce immunoglobulin Ige,

1	(IgG1 and IgG3 in humans, and their homologs, IgG2a and IgG2b, in the mouse) in type 1 responses. Those TFh cells producing IL-4 drive B cells to differentiate and produce immunoglobulin Ige, which arms mast cells and basophils for granule release in type 2 responses. TFh cells that produce IL-17 appear to be important for generating opsonizing antibodies directed against extracellular pathogens in the context of type 3/Th17 immunity. regulatory T cells (right panels) generally suppress T-cell and innate immune cell activity and help prevent the development of autoimmunity during immune responses.

1	we shall discuss further in Chapter 11, the functional features of these T-cell subsets parallel in many ways those of innate lymphoid cells (ILCs), which, although lacking antigenic receptors, produce many of the same patterns of effector cytokines or cytotoxins. The first CD4 T-cell subsets to be distinguished were the TH1 and TH2 subsets, hence their names. TH1 cells are characterized by the production of IFN-γ, whereas TH2 cells are characterized by the production of IL-4, IL-5, and IL-13. TH17 cells are so named because they produce the cytokines IL-17A and IL-17F; they also produce IL-22. TFH cells develop in concert with TH1, TH2, or TH17 cells to help B cells generate class-switched immunoglobulins of different isotypes, which are targeted to different innate immune effector cells by the array of Fc receptors they display. Treg cells have immunoregulatory function and promote tolerance to, rather than clearance of, the antigens they recognize.

1	TH1 cells help to eradicate infections by microbes that can survive or replicate within macrophages. Examples include certain viruses, protozoans, and intracellular bacteria, such as the mycobacteria that cause tuberculosis and leprosy. These bacteria are phagocytosed by macrophages in the usual way but can evade the intracellular killing mechanisms described in Chapter 3. If a TH1 cell recognizes bacterial antigens displayed on the surface of an infected macrophage, it will activate the macrophage further through the release of IFN-γ, which enhances the macrophage’s microbicidal activity to kill ingested bacteria. Type 1 responses also promote B-cell class switching that favors production of opsonizing IgG antibodies, such as IgG2a in mouse. We shall describe the macrophage-activating functions of TH1 cells in more detail in Chapter 11.

1	TH2 cells help to control infections by extracellular parasites, particularly helminths, by promoting responses mediated by eosinophils, mast cells, and IgE. In particular, cytokines produced as part of a type 2 response are required for class switching of B cells to produce IgE, the primary role of which is to fight parasitic infections. IgE is also the antibody responsible for allergies and asthma, making TH2 differentiation of additional medical interest.

1	The third major effector subset of CD4 T cells is TH17. TH17 cells are typically induced in response to extracellular bacteria and fungi, and amplify neutrophilic responses that help to clear such pathogens (see Fig. 9.30). TH17, or type 3, responses also promote B-cell class switching to opsonizing IgG2 and IgG3 antibodies. Cytokines produced by TH17 cells, including IL-17 and IL-22, are also important in activating barrier epithelial cells in the gastrointestinal, respiratory, and urogenital tracts and the skin, to produce antimicrobial peptides that resist microbial invasion.

1	In contrast to TH1, TH2, or TH17 cells, TFH cells contribute to the eradication of most classes of pathogens through their unique role in providing help to B cells to promote germinal center responses—irrespective of the pattern of immune response with which they are associated. Thus, TFH cells are elicited in the context of either type 1, type 2, or type 3 responses, where they play a central role in the development of distinct patterns of class-switched antibodies. TFH cells are identified mainly by their expression of certain markers, such as CXCR5 and PD-1, and their localization to lymphoid follicles.

1	Prior to the discovery of TFH cells, a point of controversy had been the role of CD4 T effector subsets in providing B-cell help. Although it was originally implied that this was primarily the function of TH2 cells, it is now thought that the TFH cell, rather than TH1, TH2, or TH17 cells, is the primary effector T cell that provides B-cell help for high-affinity antibody production in lymphoid follicles. Nevertheless, TFH cells develop as a component of type 1, 2, or 3 responses, and share production of some of the same lineage-defining cytokines of TH1, TH2, and TH17 cells to drive the differentiation of naive B cells to alternative patterns of isotype switching. This explains how, in the course of an infection, B cells can receive help to switch to IgE through the presence of ‘TH2’ cytokines, or switch to other isotypes such as IgG2a through the presence of ‘TH1’ cytokines. Thus, while the developmental relationship of TFH to other CD4 subsets is still a matter of active research,

1	or switch to other isotypes such as IgG2a through the presence of ‘TH1’ cytokines. Thus, while the developmental relationship of TFH to other CD4 subsets is still a matter of active research, TFH cells appear to represent a distinct branch of effector T cells that remain within the lymphoid tissues and are specialized for providing B-cell help. We will return to the helper functions of TFH cells in more detail in Chapters 10 and 11.

1	All the effector T cells described above are involved in activating their target cells to make responses that help clear pathogens from the body. Other CD4 T cells have a different function. These are called regulatory T cells, or Treg cells, because their function is to suppress T-cell responses rather than activate them. Thus, Treg cells are involved in limiting the immune response and preventing autoimmunity. Two main subsets of regulatory T cells are currently recognized. One subset becomes committed to a regulatory fate while still in the thymus, and is known as natural, or thymically derived, T cells (nT and tT , respectively; see Section 8-26). The other subset of T cells differenti ates from naive CD4 T cells in the periphery under the influence of particular environmental conditions. This group is known as induced, or peripherally derived, T cells (iT and pT , respectively). These cells will be discussed further in Section 9-23.

1	9-21 Cytokines induce the differentiation of naive CD4 T cells down distinct effector pathways.

1	Having briefly noted the types and functions of CD4 T-cell subsets, we will now consider how they are derived from naive T cells. The fate of the progeny of a naive CD4 T cell is largely defined during the initial priming period and is regulated by signals provided by the local environment, whether by the priming antigen-presenting cell or other innate immune cells that have been activated by a pathogen. As noted previously, the principal determinants of the developmental fate of naive CD4 T cells are the combination and balance of lineage-specifying cytokines, which are integrated with TCR and co-stimulatory signaling during priming. The five main subsets into which naive CD4 T cells may develop—TH1, TH2, TH17, TFH, and induced regulatory T cells (iTreg cells)—are associated with distinct signals that induce their formation, different transcription factors that drive their differentiation, and unique cytokines and surface markers that define their identity (Figs. 9.31 and 9.32).

1	TH1 development is induced when there is a predominance of the cytokines IFN-γand IL-12 during the early stages of naive T-cell activation. As described in Section 3-16, many key cytokines, including IFN-γ and IL-12, stimulate the JAK–STAT intracellular signaling pathway, resulting in the activation of specific gene networks. Different members of the JAK and STAT families are activated by different cytokines. Each of the effector pathways is dependent on a distinct pattern of STAT activation downstream of the lineage-specifying cytokines to program a unique transcription factor network that defines the gene-expression profile of mature effector T cells (see Fig. 9.32). For TH1 development, STAT1 and STAT4 are critical and are sequentially activated by interferons (type 1—IFN-α and IFN-β; or type 2—IFN-γ) and IL-12, respectively, which are produced by innate immune cells early during infection. Activated group 1 ILCs, such as NK cells, may also be an important source of IFN-γ. Finally,

1	type 2—IFN-γ) and IL-12, respectively, which are produced by innate immune cells early during infection. Activated group 1 ILCs, such as NK cells, may also be an important source of IFN-γ. Finally, TH1 cells themselves may provide IFN-γ, thus reinforcing the signal for the differentiation of more TH1 cells through a positive feedback loop.

1	Fig. 9.31 Cytokines are the principal determinants of alternative programs of CD4 T-cell effector differentiation.

1	antigen-presenting cells, principally dendritic cells, as well as other innate immune cells can provide various cytokines that induce the development of naive Cd4 T cells into distinct subsets. The environmental conditions, such as the exposure to various pathogens, determine which cytokines innate sensor cells will produce. Th1 cells differentiate in response to sequential IFn-γ and IL-12 signaling, whereas Th2 cells differentiate in response to IL-4. IL-6 produced by dendritic cells acts with transforming growth factor-β (TGF-β) to induce differentiation of Th17 cells, which upregulate expression of the IL-23 receptor and become responsive to IL-23. TFh cells also require IL-6 for their development, although it is not currently understood what additional signals might induce their differentiation from naive precursors. When pathogens are absent, the presence of TGF-β and IL-2, and the lack of IL-6, favor the development of induced T cells.

1	Fig. 9.32 Different members of the STAT family of transcription factors act immediately downstream of cytokines that determine CD4 T-cell subset development. With the exception of TGF-β, which participates in both Th17 and iTreg development, each of the cytokines that specify the development of distinct effector cells activates different members of the sTaT family of transcription factors. Th1 cell differentiation is dependent on sequential activation of sTaT1 and sTaT4 by binding of IFn-γ and IL-12 to their respective receptors on antigen-activated naive Cd4 T cells. Both of these sTaT factors participate in the induction of T-bet expression, which then cooperates with the sTaTs to program Th1 differentiation. Th2 cell differentiation is dependent on sTaT6 activation downstream of IL-4 receptor signaling. sTaT6 acts to increase GaTa3 expression, which cooperates with sTaT6 to program Th2 differentiation. IL-6 activates sTaT3, which, in concert with TGF-β, participates in the

1	IL-4 receptor signaling. sTaT6 acts to increase GaTa3 expression, which cooperates with sTaT6 to program Th2 differentiation. IL-6 activates sTaT3, which, in concert with TGF-β, participates in the induction of rorγt expression and Th17 differentiation. IL-23, which acts later in Th17 differentiation, also activates sTaT3 to sustain and amplify the Th17 program. The programming of TFh cell differentiation by sTaT factors is not fully understood, although sTaT3 actions upstream of Bcl-6 expression are essential. activation of sTaT5 by IL-2 is important in iTreg differentiation and acts upstream of Foxp3 expression.

1	The activation of STAT1 by interferon induces in activated naive CD4 T cells induces the expression of another transcription factor, T-bet, which switches on the genes for IFN-γ and the inducible component of the IL-12 receptor, IL-12Rβ2 (the other component of the receptor, IL-12Rβ1, is already expressed on naive T cells). These T cells are now committed to becoming TH1 cells, and can be further activated by IL-12 produced by dendritic cells and macrophages to induce STAT4 signaling. STAT4 further upregulates T-bet expression and completes TH1 programming. Due to its central role in programming TH1 development, T-bet is sometimes referred to as a ‘master regulator’ of TH1 cell differentiation.

1	TH2 development requires IL-4. When an antigen-activated naive T cell encounters IL-4, its receptor activates STAT6, which promotes expression of the transcription factor GATA3. GATA3 is a powerful activator of the genes encoding several cytokines produced by TH2 cells, such as IL-4 and IL-13. GATA3 also induces its own expression, thereby stabilizing TH2 differentiation via cell-intrinsic positive feedback. The initial source of IL-4 that triggers a TH2 response has long been debated. Eosinophils, basophils, and mast cells are each attractive possibilities because they can produce abundant IL-4 when activated by chitin, a polysaccharide present in helminth parasites, as well as in insects and crustaceans, which induces TH2 responses. In mice treated with chitin, eosinophils and basophils are recruited into tissues and are activated to produce IL-4. In humans, group 2 ILCs can also produce IL-4, suggesting that these cells might contribute to TH2 differentiation, although this is

1	are recruited into tissues and are activated to produce IL-4. In humans, group 2 ILCs can also produce IL-4, suggesting that these cells might contribute to TH2 differentiation, although this is unproven. Clearly, there are several innate immune cells that might contribute IL-4 for TH2 development, and the cellular source might differ, contingent on the inciting antigen. Similar to the positive feedback for TH1 cell development provided by IFN-γ produced by activated TH1 cells, IL-4 produced by activated TH2 cells may amplify TH2 development from naive T-cell precursors.

1	TH17 cells arise when the cytokines IL-6 and transforming growth factor (TGF)-β predominate during naive CD4 T-cell activation (see Figs. 9.31 and 9.32). Development of TH17 cells requires the actions of STAT3, which is activated by IL-6 signaling. Developing TH17 cells express the receptor for the cytokine IL-23, rather than the IL-12 receptor typical of TH1 cells, and the expansion and further development of TH17 effector activity seem to require IL-23, similar to the requirement for IL-12 in effective TH1 responses (see Figs.

1	9.31 and 9.32). The signature transcription factor, or master regulator, of TH17 cell differentiation is RORγt, a nuclear hormone receptor that is central to stabilizing the development of TH17 cells. The source of IL-6 and TGF-β required for TH17 cell differentiation is primarily derived from innate immune cells activated by microbial products. Unlike TH1 or TH2 cells, TH17 cells do not appear to directly induce further TH17 cell development from naive CD4 T cells via positive feedback, as they do not produce IL-6. However, IL-17 produced by TH17 cells appears to enhance IL-6 production by innate immune cells and provide an indirect mechanism for reinforcing TH17 differentiation from naive precursors.

1	upon antigen recognition in secondary lymphoid tissues, and not the thymus. They develop when naive T cells are activated in the presence of the cytokine transforming growth factor-β (TGF-β) and in the absence of IL-6 and other pro-inflammatory cytokines. Thus, it is the presence or absence of IL-6 that determines whether TGF-β co-signaling leads to the development of immunosuppressive Treg cells or of TH17 cells, which promote inflammation and the generation of immunity (Fig. 9.33). The generation of IL-6 by innate immune cells is regulated by the presence or absence of pathogens, with pathogen products tending to stimulate its production. In the absence of pathogens, IL-6 production is low, favoring differentiation of the immunosuppressive Treg cells and so preventing unwanted immune responses. Like nT cells, iT cells are distinguished by expression of the transcription factor FoxP3 and cell-surface CD25, and appear to be functionally equivalent to nT cells. Both iT and nT cells

1	responses. Like nT cells, iT cells are distinguished by expression of the transcription factor FoxP3 and cell-surface CD25, and appear to be functionally equivalent to nT cells. Both iT and nT cells themselves can produce TGF-β, as well as IL-10, which act in an inhibitory manner to suppress immune responses and inflammation, and may act to support further iTreg differentiation.

1	TFH cells, unlike the subsets described above, have not been produced efficiently in vitro, and so the requirements for their differentiation are not yet clearly established. IL-6 seems to be important for TFH development, but much remains to be learned about the control of this subset. One transcription factor important for TFH development is Bcl-6, which is required for the expression of CXCR5, the receptor for the chemokine CXCL13, which is produced by the stromal cells of the B-cell follicle. CXCR5 is essential for TFH localization in follicles, and is not expressed by other effector T-cell subsets. TFH cells also express ICOS, the ligand for which is expressed abundantly by B cells. ICOS seems crucial for the helper activity of TFH cells, because mice lacking ICOS show a severe defect in T-cell-dependent antibody responses. In addition to production of low amounts of cytokines characteristic of the effector T-cell subsets with which they develop in parallel (for example, IFN-γ,

1	in T-cell-dependent antibody responses. In addition to production of low amounts of cytokines characteristic of the effector T-cell subsets with which they develop in parallel (for example, IFN-γ, IL-4, or IL-17), and which promote different patterns of B-cell class switching, TFH cells produce high amounts of IL-21, a cytokine that supports the proliferation and differentiation of B cells into antibody-producing plasma cells.

1	9-22 CD4 T-cell subsets can cross-regulate each other’s differentiation through the cytokines they produce.

1	The various subsets of effector CD4 T cells each have very different functions. For the immune response to efficiently control different types of pathogens it must orchestrate a coordinated effector response that is dominated by one of these subsets. A principal means of achieving this is through the distinct ensemble of cytokines that are produced by the different subsets. Importantly, some of these same cytokines also participate in positive and negative feedback loops that control the differentiation of effector T cells from naive precursors, thereby providing a mechanism to promote one pattern of effector response while suppressing others. For example, both IFN-γ (produced by TH1 cells) and IL-4 (produced by TH2 cells) potently inhibit TH17 development, promoting TH1 or TH2 development, respectively (Fig. 9.34). Similarly, there is cross-regulation between TH1 and TH2 cells. IL-4 produced by TH2 cells potently inhibits TH1 development. Conversely, IFN-γ, a product of TH1 cells,

1	respectively (Fig. 9.34). Similarly, there is cross-regulation between TH1 and TH2 cells. IL-4 produced by TH2 cells potently inhibits TH1 development. Conversely, IFN-γ, a product of TH1 cells, can inhibit the proliferation of TH2 cells (see Fig. 9.34). TGF-β produced by T cells inhibits the development of both TH1 and TH2 cells. In this way, cytokines produced by effector T cells reinforce the differentiation of their own kind from naive precursor cells.

1	TH1 cells generate copious amounts of IFN-γ when they recognize antigen on a target cell, thus reinforcing the signal for the differentiation of more TH1 cells through a positive feedback loop. In this way, recognition of a particular type

1	Fig. 9.33 A shared requirement for TGF-β in the differentiation of iTreg and TH17 cells provides a developmental link that reflects their complementary roles in promoting mutualism with the microbiota. a major site for the deployment of iT and Th17 cells is mucosal tissues, particularly the intestines, where the immune system must cope with an extraordinarily high density of microbial organisms that comprise the microbiota. While the microbiota provides its host with important metabolic functions, it also represents a potential threat as some of its constituents are opportunistic pathogens that can cause serious infections if they breach the mucosal barrier. as an adaptation to restrain untoward inflammation directed against the microbiota while retaining the capacity to mount a host-protective immune response should barrier breach occur, the developmental balance between iTreg cells, which suppress inflammatory responses against the microbiota, and Th17 cells, which promote

1	host-protective immune response should barrier breach occur, the developmental balance between iTreg cells, which suppress inflammatory responses against the microbiota, and Th17 cells, which promote host-protective inflammatory responses, is determined by the balance between production of the vitamin a metabolite all-trans retinoic acid (at-ra) and production of the pro-inflammatory cytokine IL-6 by mucosal dendritic cells. at homeostasis, antigens derived from the microbiota are presented by a specialized subset of resident dendritic cells that produce at-ra, but no IL-6. however, when antigens are recognized in the context of TLr-stimulating signals, at-ra production is suppressed in favor of IL-6, thereby favoring the development of Th17 effector cells.

1	under homeostatic conditions, TGF-β produced by T cells represses Th1 and Th2 responses in order to promote T development. under inflammatory conditions that favor IL-6 production, TGF-β production by T cells similarly inhibits the activation of Th1 or Th2 responses (upper panels) in order to facilitate the development of Th17 cells, which otherwise would be potently inhibited by IFn-γ or IL-4. Conversely, if signals are present to induce Th1 or Th2 cells, the cytokines IFn-γ or IL-4 produced by them can override the effect of IL-6 and inhibit Th17 development (lower center panel). IFn-γ produced by Th1 cells blocks the growth of Th2 cells (right panels). on the other hand, IL-4 produced by Th2 cells dominantly prevents Th1 cell development in favor of Th2 (left panels). although not shown, all T-cell subsets can produce IL-10 under conditions of chronic antigen stimulation, which inhibits the production of IL-12, IL-4, and IL-23 by dendritic cells and macrophages, thereby suppressing

1	all T-cell subsets can produce IL-10 under conditions of chronic antigen stimulation, which inhibits the production of IL-12, IL-4, and IL-23 by dendritic cells and macrophages, thereby suppressing the development and/or maintenance of Th1, Th2, and Th17 cells.

1	Fig. 9.34 The subsets of CD4 T cells each produce cytokines that can negatively regulate the development or effector activity of other subsets. IL-4 or IFN-˜can inhibit development of TH17 cells IL-4IFN-˜TH17IL-4IFN-˜IL-4 acts to inhibit differentiation of TH1 cells IFN-˜acts on TH2 cells to inhibit proliferation TH1TH2IL-4TGF-°TGF-°IFN-˜IFN-˜Activated TH2 cells secrete IL-4 Activated TH1 cells secrete IFN-˜TH2TregTH1Treg cells suppress the differentiation and proliferation of TH1 and TH2 cells of pathogen by the innate immune system initiates a chain reaction that links the innate response to the adaptive immune response, which in turn amplifies the innate response. Thus, certain intracellular bacterial infections (for example, mycobacteria and Listeria) induce dendritic cells and macrophages to produce IL-12, favoring the emergence of TH1 effector cells. TH1 cells, in turn, promote enhanced macrophage activation that clears these intracellular pathogens.

1	The adverse consequences of inappropriate cross-regulation of effector T-cell responses by cytokines have been demonstrated in a number of infectious models in mice. Such studies reinforce the notion that induction of the appropriate effector CD4 T-cell subset is crucial for pathogen clearance, and show that subtle differences in CD4 T-cell responses can have a significant impact on the outcome of infection. One example of this is the murine model of infection by the protozoan parasite Leishmania major, which requires a TH1 response and activation of macrophages for clearance. C57BL/6 mice produce TH1 cells that protect the animal by activating infected macrophages to kill

1	L. major. In BALB/c mice infected with L. major, however, CD4 T cells fail to differentiate into TH1 cells; instead, they become TH2 cells, which are unable to activate macrophages to inhibit Leishmania growth. This difference seems to result from a population of memory T cells that are specific for gut-derived antigens but cross-react with an antigen, LACK (Leishmania analog of the receptors of activated C kinase), expressed by the Leishmania parasite. These memory cells are present in both strains of mice, but for unknown reasons they produce IL-4 in BALB/c mice but not in C57BL/6 mice. In BALB/c mice, the small amount of IL-4 secreted by these memory cells during Leishmania infection drives new Leishmania-specific CD4 T cells to become TH2 cells instead of TH1 cells, leading to failure of pathogen elimination and death. The preferential development of TH2 rather than TH1 cells in BALB/c mice can be reversed if IL-4 is blocked early during infection by anti-IL-4 antibody, but this

1	of pathogen elimination and death. The preferential development of TH2 rather than TH1 cells in BALB/c mice can be reversed if IL-4 is blocked early during infection by anti-IL-4 antibody, but this treatment is ineffective after a week or so of infection, demonstrating the crucial importance of cytokines early in developmental decisions made by naive T cells (Fig. 9.35).

1	9-23 Regulatory CD4 T cells are involved in controlling adaptive immune responses. Regulatory T cells play a central role in preventing autoreactive immune responses and fall into different groups that are defined by their different developmental origins and functions. Natural T-regulatory (nT ) cells develop in the thymus (see Section 8-26) and are CD4-positive cells that constitutively express CD25 and high levels of the L-selectin receptor CD62L and of CTLA-4. Induced T (iT ) cells arise in the periphery from naive CD4 T cells and also express CD25 and CTLA-4 (see Section 9-20). Collectively, T cells represent about 5–10% of the CD4 T cells in circuation. A hallmark of both natural and induced T cells is expression of the transcription factor FoxP3, which, among other actions, interferes with the interaction between AP-1 and NFAT at the IL-2 gene promoter, preventing transcriptional activation of the gene and production of IL-2.

1	ventional α:β T-cell receptors and are selected in the thymus by high-affinity binding to MHC molecules containing self-peptides. It is not currently known whether they are activated to express their regulatory function in the periphery by the same self ligands that selected them in the thymus or by other self or non-self antigens. Multiple mechanisms appear to contribute to the ability of T cells to inhibit responses of other T cells, but principal among these are interactions with antigen-presenting cells that interfere with the capacity of antigen-presenting cells to provide activating signals. The expression of high levels of CTLA-4 on the surface of natural T cells is thought to permit them to compete for B7 expressed by antigen-presenting cells, thereby preventing adequate co-stimulation of naive T cells. Indeed, it has been proposed that CTLA4 expressed on T cells can physically remove B7 molecules from the surface of antigen-presenting cells, thereby depleting them of

1	of naive T cells. Indeed, it has been proposed that CTLA4 expressed on T cells can physically remove B7 molecules from the surface of antigen-presenting cells, thereby depleting them of co-stimulatory activity. Similarly, by expressing CD25, and thus the high-affinity receptor for IL-2, and lacking the ability to produce IL-2, T cells appear to sequester IL-2 from naive T cells, which lack CD25 expression until fully activated.

1	Other functions of T cells are mediated by their production of immunosup pressive cytokines. TGF-β produced by T cells can inhibit T-cell prolifera tion (see Fig. 9.34). IL-10, which is produced by T cells late in an immune response, inhibits the expression of MHC molecules and co-stimulatory molecules by antigen-presenting cells. As a means of limiting the responses of effector T cells, IL-10 also inhibits the production of pro-inflammatory cytokines by antigen-presenting cells. For example, IL-10 potently inhibits the production of IL-12 and IL-23 by antigen-presenting cells and thus impairs their ability to promote the differentiation and maintenance of TH1 and TH17 cells, respectively. The critical role of T cells in immune regulation is highlighted by several autoimmune syndromes (described in Chapter 15) that are caused by deficiency in different aspects of T cell function.

1	Although they differentiate in secondary lymphoid tissues after export from the thymus, induced T cells also express FoxP3 and share most of the phenotypic and functional features of natural T cells. A major function of iT cells is the prevention of inflammatory immune responses to the commensal microbiota, particularly microbes resident in mucosal tissues such as the intestines. Here, iT cells appear to be the dominant source of IL-10, deficiency of which causes inflammatory bowel disease, an immune-mediated disease of the intestines characterized by chronic reactivity against antigens of the intestinal microbiota (see also Section 15-23). As will be discussed in more detail in Chapter 12, the differentiation of induced T cells in the intestines is favored by the presence of antigen-presenting cells that produce retinoic acid, which is derived from vitamin A. Retinoic acid produced by intestinal dendritic cells acts with TGF-β to induce T differentiation while suppressing the

1	cells that produce retinoic acid, which is derived from vitamin A. Retinoic acid produced by intestinal dendritic cells acts with TGF-β to induce T differentiation while suppressing the differentiation of TH17 cells (see Fig. 9.33). The antagonistic balance of retinoic acid and IL-6 therefore

1	Fig. 9.35 The development of CD4 T-cell subsets can be manipulated by altering the cytokines acting during the early stages of infection. elimination of infection with the intracellular protozoan parasite Leishmania major requires a Th1 response, because IFn-γ is needed to activate the macrophages that provide protection. BaLB/c mice are normally susceptible to L. major because they generate a Th2 response to the pathogen. This is because they produce IL-4 early during infection and this induces naive T cells to develop into the Th2 lineage (see the text). Treatment of BaLB/c mice with neutralizing anti-IL-4 antibodies at the beginning of infection inhibits this IL-4 and prevents the diversion of naive T cells toward the Th2 lineage; these mice develop a protective Th1 response. controls the differentiation of induced Treg cells and TH17 cells, respectively, in the intestinal mucosa-associated lymphoid tissues (MALT).

1	controls the differentiation of induced Treg cells and TH17 cells, respectively, in the intestinal mucosa-associated lymphoid tissues (MALT). CD4 T cells that lack FoxP3 expression but produce immunosuppressive cytokines characteristic of Treg cells have also been described. One such population, referred to as TR1 cells, has been defined largely by their production of IL-10, but absence of expression of FoxP3. However, we now recognize that many different cells, including TH1, TH2, TH17, and B cells, can produce IL-10 under certain circumstances, such as during chronic responses to persistent antigen. Therefore, it is uncertain whether TR1 cells represent a distinct subset of T cells, and if so, whether they have unique functions in immune regulation. Summary.

1	Summary. The crucial first step in adaptive immunity is the activation, or priming, of naive antigen-specific T cells by antigen-presenting cells within the lymphoid tissues through which they constantly circulate. The most distinctive feature of antigen-presenting cells is the expression of cell-surface co-stimulatory molecules, of which the B7 molecules are the most important. Naive T cells will respond to antigen only when the antigen-presenting cell presents both a specific antigen to the T-cell receptor and a B7 molecule to CD28 on the T cell. This dual requirement for both receptor ligation and co-stimulation by the same antigen-presenting cell helps to prevent naive T cells from responding to self antigens on tissue cells, which lack co-stimulatory activity.

1	Activation of naive T cells leads to their proliferation and differentiation into effector T cells, the critical event in most adaptive immune responses. Various combinations of cytokines regulate the type of effector T cell that develops in response to antigen. In turn, the cytokines present during primary T-cell activation are influenced by the innate immune system. Once an expanded clone of T cells acquires effector function, its progeny can act on any target cell that displays antigen on its surface. Effector T cells have a variety of functions. CD8 cytotoxic T cells recognize virus-infected cells and kill them. TH1 effector cells promote the activation of macrophages to enhance their killing of intracellular pathogens. TH2 cells promote mucosal barrier immunity against pathogens, such as helminths, requiring the effector activities of cells such as eosinophils and mast cells for their elimination. The elimination of certain types of bacteria and fungi is orchestrated by TH17

1	such as helminths, requiring the effector activities of cells such as eosinophils and mast cells for their elimination. The elimination of certain types of bacteria and fungi is orchestrated by TH17 cells, particularly at barrier sites, where they recruit neutrophils to sites of infection and promote the production of antimicrobial peptides by epithelial cells. TFH cells are specialized for interactions with B cells and localization to the B-cell follicle and germinal centers, where they provide help for antibody production and isotype switching. Regulatory CD4 T-cell subsets restrain the immune response by preventing the activation of self-reactive naive T cells by antigen-presenting cells and producing inhibitory cytokines that limit the effector responses of other T-cell subsets.

1	General properties of effector T cells and their cytokines. T-cell effector functions involve the interaction of an effector T cell with a target cell displaying specific antigen. Effector proteins expressed by the T cell, whether cell-associated (for example, CD40L) or secreted (for example, cytokines), are focused on the target by mechanisms that are activated by antigen recognition. The focusing mechanism is common to all types of effector T cells, whereas their effector actions depend on the type of effector T cell that is engaged. 9-24 Effector T-cell interactions with target cells are initiated by antigen-nonspecific cell-adhesion molecules.

1	Once an effector T cell has completed its differentiation in the lymphoid tissue, it must find target cells that are displaying the peptide:MHC complex that it recognizes. TFH cells encounter their B-cell targets without leaving the lymphoid tissue. However, most other effector T cells emigrate from their site of activation in lymphoid tissues and enter the blood, either directly if primed by antigen in the spleen or via the efferent lymphatics and thoracic duct if primed in lymph nodes. Because of the cell-surface changes that have occurred during their differentiation, effector T cells can now migrate into tissues, particularly at sites of infection. They are guided to these sites by changes in the adhesion molecules expressed on the endothelium of the local blood vessels as a result of infection, and by local chemotactic factors, as will be discussed further in Chapter 11.

1	The initial binding of an effector T cell to its target, like that of a naive T cell to an antigen-presenting cell, is an antigen-nonspecific interaction mediated by LFA-1 and CD2. The levels of LFA-1 and of CD2 are twoto fourfold higher on effector T cells than on naive T cells, and so effector T cells can bind efficiently to target cells that have less ICAM and CD58 on their surface than do antigen-presenting cells. This interaction is transient unless recognition of antigen on the target cell by the T-cell receptor triggers an increase in the affinity of the T-cell’s LFA-1 for its ligands. The T cell then binds more tightly to its target and remains bound long enough to release its effector molecules. Effector CD4 T cells, which activate macrophages or induce B cells to secrete antibody, have to switch on new genes and synthesize new proteins to carry out their effector actions and so must maintain contact with their targets for relatively long periods. Cytotoxic T cells, by

1	have to switch on new genes and synthesize new proteins to carry out their effector actions and so must maintain contact with their targets for relatively long periods. Cytotoxic T cells, by contrast, can be observed under the microscope attaching to and dissociating from successive targets relatively rapidly as they kill them (Fig. 9.36). Killing of the target, or some local change in the T cell, allows the effector T cell to detach and address new targets. How CD4 effector T cells disengage from their antigen-negative targets is not known, although evidence suggests that CD4 binding to MHC class II molecules without engagement of the T-cell receptor provides a signal for the cell to detach.

1	their targets to regulate signaling and to direct the release of effector molecules. When binding to their specific antigenic peptide:self MHC complexes or to self peptide:self MHC complexes, the T-cell receptors and their associated co-receptors cluster at the site of cell–cell contact, forming what is called the supramolecular activation complex (SMAC) or the immunological synapse. Other cell-surface molecules also cluster here. For example, the

1	Fig. 9.36 Interactions of T cells with their targets initially involve nonspecific adhesion molecules. The major initial interaction is between LFa-1 on the T cell, illustrated here as a cytotoxic Cd8 T cell, and ICaM-1 or ICaM-2 on the target cell (top panel). This binding allows the T cell to remain in contact with the target cell and to scan its surface for the presence of specific peptide:MhC complexes. If the target cell does not carry the specific antigen, the T cell disengages (second panel) and can scan other potential targets until it finds the specific antigen (third panel). signaling through the T-cell receptor increases the strength of the adhesive interactions, prolonging the contact between the two cells and stimulating the T cell to deliver its effector molecules. The T cell then disengages (bottom panel). Chapter 9: T-Cell-Mediated Immunity

1	Chapter 9: T-Cell-Mediated Immunity Outer ring (red) pSMAC LFA-1:ICAM-1 talin TCR, CD4, CD28 peptide:MHC CD8, PKC-˜Inner circle (green) cSMAC Organization of the immunological synapse T cell dendritic cell pSMAC cSMAC

1	Fig. 9.37 The area of contact between an effector T cell and another cell forms an immunological synapse. a confocal fluorescence micrograph of the area of contact between a Cd4 T cell and an antigen-presenting cell (apC) (as viewed through one of the cells) is shown. proteins in the contact area between the T cell and the apC form a structure called the immunological synapse, also known as the supramolecular activation complex (sMaC), which is organized into two distinct regions: the outer, or peripheral sMaC (psMaC), indicated by the red ring; and the inner, or central sMaC (csMaC), indicated in bright green. The csMaC is enriched in the T-cell receptor (TCr), Cd4, Cd8, Cd28, Cd2, and pKC-ε. The psMaC is enriched for the integrin LFa-1 and the cytoskeletal protein talin. photograph courtesy of a. Kupfer.

1	tight binding of LFA-1 to ICAM-1 induced by ligation of the T-cell receptor creates a molecular seal that surrounds the T-cell receptor and its co-receptor (Fig. 9.37). In some cases, the contact surface organizes into two zones: a central zone known as the central supramolecular activation complex (cSMAC) and an outer zone known as the peripheral supramolecular activation complex (pSMAC). The cSMAC contains most of the signaling proteins known to be important in T-cell activation. The pSMAC is notable mainly for the presence of the LFA-1 and the cytoskeletal protein talin, which connects LFA-1 to the actin cytoskeleton (see Section 3-18). The immunological synapse is not a static structure as implied by Fig. 9.37, but is quite dynamic. T-cell receptors move from the periphery into the cSMAC, where they undergo endocytosis through ubiquitin-mediated degradation involving the E3 ligase Cbl (see Section 7-5). Because T-cell receptors are being degraded in the cSMAC, signaling is

1	the cSMAC, where they undergo endocytosis through ubiquitin-mediated degradation involving the E3 ligase Cbl (see Section 7-5). Because T-cell receptors are being degraded in the cSMAC, signaling is actually weaker there than in the peripheral contact areas, where microclusters of T-cell receptors are being formed and are highly active (see Section 7-8).

1	Clustering of the T-cell receptors signals a reorientation of the cytoskeleton that polarizes the effector cell and focuses the release of effector molecules at the site of contact with the target cell. This is illustrated for a cytotoxic T cell in Fig. 9.38. An important intermediary in the effect of T-cell signaling on the cytoskeleton is the Wiskott–Aldrich syndrome protein (WASp), defects in which result in the inability of T cells to become polarized, among other effects, and cause an immune deficiency syndrome for which the protein is named (see Sections 7-19 and 13-6). Activation and recruitment of WASp by T-cell receptor signaling is mediated by the adaptor protein Vav (see Section 7-19). Polarization starts with the local reorganization of the cortical actin cytoskeleton at the site of contact; this in turn leads to the reorientation of the microtubule-organizing center (MTOC), the center from which the microtubule cytoskeleton is produced, and reorientation of the Golgi

1	site of contact; this in turn leads to the reorientation of the microtubule-organizing center (MTOC), the center from which the microtubule cytoskeleton is produced, and reorientation of the Golgi apparatus (GA), through which most proteins destined for secretion travel. In the cytotoxic T cell, the cytoskeletal reorientation focuses exocytosis of the preformed cytotoxic granules at the site of T-cell contact with its target cell. The polarization of a T cell also focuses the secretion of newly synthesized effector molecules induced by ligation of the T-cell receptor. For example, the secreted cytokine IL-4, which is the principal effector molecule of TH2 cells, is confined and concentrated at the site of contact with the target cell.

1	Thus, the T-cell receptor controls the delivery of effector signals in three ways: it induces tight binding of effector cells to their target cells to create a narrow space in which effector molecules can be concentrated; it focuses delivery of effector molecules at the site of contact by inducing a reorientation of the secretory apparatus of the effector cell; and it triggers the synthesis and/or release of the effector molecules. All these mechanisms contribute to targeting actions of effector molecules onto the cell bearing specific antigen. Effector T-cell activity is thus highly selective for appropriate target cells, even though effector molecules themselves are not antigen-specific. 9-26 The effector functions of T cells are determined by the array of effector molecules that they produce.

1	9-26 The effector functions of T cells are determined by the array of effector molecules that they produce. The effector molecules produced by effector T cells fall into two broad classes: cytotoxins, which are stored in specialized cytotoxic granules and released by CD8 cytotoxic T cells (see Fig. 9.38), and cytokines and related membrane-associated proteins, which are synthesized de novo by all effector T cells. Cytotoxins are the principal effector molecules of cytotoxic T cells and are discussed in Section 9-31. Their release in particular must be tightly regulated because they are not specific: they can penetrate the lipid bilayer and trigger apoptosis in any cell. By contrast, CD4 effector T cells act mainly through the production of cytokines and membrane-associated proteins, and their actions are largely restricted to cells bearing MHC class II molecules and expressing receptors for these proteins.

1	The main effector molecules of T cells are summarized in Fig. 9.39. The cytokines are a diverse group of proteins and we will review them briefly before discussing the T-cell cytokines and their actions. Secreted cytokines and membrane-associated molecules often act in concert to mediate these effects. 9-27 Cytokines can act locally or at a distance. Cytokines are small soluble proteins secreted by cells that can alter the behavior or properties of the secreting cell itself (autocrine actions) or of another cell (paracrine actions). Cytokines are produced by many cell types in addition to those of the immune system. We have already introduced the families of

1	Fig. 9.38 The cellular polarization of T cells during specific antigen recognition allows effector molecules to be focused on the antigen-bearing target cell. The example illustrated here is a Cd8 cytotoxic T cell. Cytotoxic T cells contain specialized lysosomes called cytotoxic granules (shown in red in the left panels), which contain cytotoxic proteins. Initial binding to a target cell through adhesion molecules does not have any effect on the location of the cytotoxic granules. Binding of the T-cell receptor causes the T cell to become polarized: reorganization within the cortical actin cytoskeleton at the site of contact aligns the microtubule-organizing center (MToC), which in turn aligns the secretory apparatus, including the Golgi apparatus (Ga), toward the target cell. proteins stored in cytotoxic granules derived from the Golgi are then directed specifically onto the target cell. The photomicrograph in panel a shows an unbound, isolated cytotoxic T cell. The microtubule

1	stored in cytotoxic granules derived from the Golgi are then directed specifically onto the target cell. The photomicrograph in panel a shows an unbound, isolated cytotoxic T cell. The microtubule cytoskeleton is stained in green and the cytotoxic granules in red. note how the granules are dispersed throughout the T cell. panel b depicts a cytotoxic T cell bound to a (larger) target cell. The granules are now clustered at the site of cell–cell contact in the bound T cell. The electron micrograph in panel c shows the release of granules from a cytotoxic T cell. panels a and b courtesy of G. Griffiths. panel c courtesy of e. podack.

1	Fig. 9.39 The different types of effector T-cell subsets produce different effector molecules. Cd8 T cells are predominantly killer T cells that recognize peptide:MhC class I complexes. They release perforin (which helps deliver granzymes into the target cell) and granzymes (which are pro-proteases that are activated intracellularly to trigger apoptosis in the target cell), and often also produce the cytokine IFn-γ. They also carry the membrane-bound effector molecule Fas ligand (Cd178). When this binds to Fas (Cd95) on a target cell it activates apoptosis in the Fas-bearing cell. The various functional subsets of Cd4 T cells recognize peptide:MhC class II complexes. Th1 cells are specialized to activate macrophages that are infected by or have ingested pathogens; they secrete IFn-γ to activate the infected cell, as well as other effector molecules. They can express membrane-bound Cd40 ligand and/or Fas ligand.

1	Cd40 ligand triggers activation of the target cell, whereas Fas ligand triggers the death of Fas-bearing targets, and so which molecule is expressed strongly influences Th1 function. Th2 cells are specialized for promoting immune responses to parasites and also promote allergic responses. They provide help in B-cell activation and secrete the B-cell growth factors IL-4, IL-5, IL-9, and IL-13. The principal membrane-bound effector molecule expressed by Th2 cells is Cd40 ligand, which binds to Cd40 on B cells and induces B-cell proliferation and isotype switching (see Chapter 10). Th17 cells produce members of the IL-17 family and IL-22, and promote acute inflammation by helping to recruit neutrophils to sites of infection. T cells produce inhibitory cytokines such as IL-10 and TGF-β that may act at a distance, but also exert inhibitory actions such as sequestration of B7 and IL-2, which act via cell–cell interactions.

1	cytokines and their receptors that are important in innate and adaptive immunity in Chapters 3 and 7 (see Sections 3-15 and 7-1). Here we are concerned with cytokines that mediate the effector functions of T cells. Many cytokines produced by T cells are given the name interleukin (IL) followed by a number. The cytokines produced by T cells are shown in Fig. 9.40, and a more comprehensive list of cytokines of immunological interest is in Appendix III. Although many cytokines can have diverse biological effects when tested in vitro, targeted disruption of the genes for cytokines and cytokine receptors in mice (see Appendix I, Section A-35) has helped to clarify their physiological roles.

1	Binding of the T-cell receptor orchestrates the polarized release of cytokines so that they are concentrated at the site of contact with the target cell (see Section 9-25). Furthermore, most of the soluble cytokines have local actions that synergize with those of the membrane-bound effector molecules. The effect of all these molecules is therefore combinatorial, and, because the membrane-bound effectors can bind only to receptors on an interacting cell, this is another mechanism by which selective effects of cytokines are focused on the target cell. The effects of some cytokines are further confined to target cells by tight regulation of their synthesis: the synthesis of IL-2, IL-4, and IFN-γ, for example, is controlled by mRNA instability (see Section 9-16), so that their secretion by T cells does not continue after the interaction with a target cell has ended.

1	Some cytokines have distant effects. IL-3 and GM-CSF (see Fig. 9.39) are released by TH1, TH2, and TH17 cells and act on bone marrow cells to stimulate the production of macrophages and granulocytes, which are important innate effector cells in both antibodyand T-cell-mediated immunity. IL-3 and GM-CSF also stimulate the production of dendritic cells from bone marrow precursors. IL-17A and IL-17F produced by TH17 cells act primarily on stromal cells, activating them to produce G-CSF, which enhances production

1	Fig. 9.40 The nomenclature and functions of well-defined many effects through what is called a ‘cytokine network.’ ↑ increase; T-cell cytokines. each cytokine has multiple activities on different ↓, decrease; CTL, cytotoxic lymphocyte; nK cells, natural killer cells; cell types. Major activities of effector cytokines are highlighted in CsF, colony-stimulating factor; IBd, inflammatory bowel disease; no, red. The mixture of cytokines secreted by a given cell type produces nitric oxide. of neutrophils by the bone marrow. TH2 cells produce IL-5, which stimulates bone marrow production of eosinophils. Whether a given cytokine effect is local or more distant is likely to reflect the amounts released, the degree to which this release is focused on the target cell, and the stability of the cytokine in vivo. 9-28 T cells express several TNF-family cytokines as trimeric proteins that are usually associated with the cell surface.

1	9-28 T cells express several TNF-family cytokines as trimeric proteins that are usually associated with the cell surface. Most effector T cells express members of the TNF family as membrane-associated proteins on the cell surface. These include TNF-α, the lymphotoxins (LTs), Fas ligand (CD178), and CD40 ligand, the latter two always being cell-surface associated. TNF-α is made by T cells in soluble and membrane-associated forms and assembles into a homotrimer. Secreted LT-α is a homotrimer, but in its membrane-bound form, LT-α is linked to a third, transmembrane member of this family called LT-β to form heterotrimers, called simply LT-β (see Section 9-2). The receptors for TNF-α and LT-α, TNFR-I and TNFR-II, form homotrimers when bound to their ligands. The trimeric structure is characteristic of all members of the TNF family, and the ligand-induced trimerization of their receptors seems to be the critical event in initiating signaling.

1	Fas ligand and CD40 ligand bind respectively to the transmembrane proteins Fas (CD95) and CD40 on target cells. Fas contains a ‘death’ domain in its cytoplasmic tail, and binding of Fas by Fas ligand induces death by apoptosis in the Fas-bearing cell (see Fig. 11.22). Other TNFR-family members, including TNFR-I, are also associated with death domains and can also induce apoptosis. Thus, TNF-α and LT-α can induce apoptosis by binding to TNFR-I.

1	CD40 ligand is particularly important for CD4 T-cell effector function; its expression is induced on TH1, TH2, TH17, and TFH cells, and it delivers activating signals to B cells and innate immune cells through CD40. The cytoplasmic tail of CD40 lacks a death domain; instead, it is linked downstream to proteins called TRAFs (TNF-receptor-associated factors). CD40 is involved in the activation of B cells and macrophages; the ligation of CD40 on B cells promotes growth and isotype switching, whereas CD40 ligation on macrophages induces them to secrete higher amounts of pro-inflammatory cytokines (for example, TNF-α) and become receptive to much lower concentrations of IFN-γ. Deficiency in CD40 ligand expression is associated with immunodeficiency, as we will learn in Chapter 13. Summary.

1	Interactions between effector T cells and their targets are initiated by transient antigen-nonspecific adhesion. T-cell effector functions are elicited only when peptide:MHC complexes on the surface of the target cell are recognized by the receptor on an effector T cell. This recognition event triggers the effector T cell to adhere more strongly to the antigen-bearing target cell and to release its effector molecules directly at the target cell, leading to the activation or death of the target. The immunological consequences of antigen recognition by an effector T cell are determined largely by the set of effector molecules that the T cell produces on binding a specific target cell. CD8 cytotoxic T cells store preformed cytotoxins in specialized cytotoxic granules whose release is tightly focused at the site of contact with the infected target cell, thus killing it without killing any uninfected cells nearby. Cytokines and members of the TNF family of membrane-associated effector

1	focused at the site of contact with the infected target cell, thus killing it without killing any uninfected cells nearby. Cytokines and members of the TNF family of membrane-associated effector proteins are synthesized de novo by most effector T cells. Membrane-associated effector molecules can deliver signals only to an interacting cell bearing the appropriate receptor, whereas soluble cytokines can act on cytokine receptors expressed locally on the target cell, or on other cells at a distance. The actions of cytokines and membrane-associated effector molecules through their specific receptors, together with the effects of the cytotoxins released by CD8 cells, account for most of the effector functions of T cells.

1	T-cell-mediated cytotoxicity.

1	All viruses, and some bacteria, multiply in the cytoplasm of infected cells; indeed, a virus is a highly sophisticated parasite that has no biosynthetic or metabolic apparatus of its own and, in consequence, can replicate only inside cells. Although susceptible to antibody-mediated clearance before they enter cells, once they enter cells these pathogens are not accessible to antibodies and can be eliminated only by the destruction or modification of the infected cells in which they replicate. This role in host defense is largely filled by CD8 cytotoxic T cells, although TH1 cells may also acquire cytotoxic capacities. The crucial role of cytotoxic T cells in limiting such infections is seen in the increased susceptibility of animals artificially depleted of these T cells, or of mice or humans that lack the MHC class I molecules that present antigen to CD8 T cells. The elimination of infected cells without the destruction of healthy tissue requires the cytotoxic mechanisms of CD8 T

1	humans that lack the MHC class I molecules that present antigen to CD8 T cells. The elimination of infected cells without the destruction of healthy tissue requires the cytotoxic mechanisms of CD8 T cells to be both powerful and accurately targeted.

1	9-29 Cytotoxic T cells induce target cells to undergo programmed cell death via extrinsic and intrinsic pathways of apoptosis.

1	To deprive cytosolic pathogens of their cellular host, cytotoxic T cells target the infected host cells for death. Cells can die in various ways. Physical or chemical injury, such as the deprivation of oxygen that occurs in heart muscle during a heart attack or membrane damage with antibody and complement, leads to cell disintegration or necrosis. This form of cell death is often accompanied by local inflammation and stimulates a wound healing response. The other form of cell death is known as programmed cell death, which can occur by apoptosis or autophagy. Apoptosis is a regulated process that is induced either by specific extracellular signals or by the lack of signals required for survival, and proceeds by a series of cellular events that include plasma membrane blebbing, changes in the distribution of membrane lipids, and enzymatic fragmentation of chromosomal DNA. A hallmark of apoptosis is the fragmentation of nuclear DNA into pieces 200 base pairs long through the activation

1	the distribution of membrane lipids, and enzymatic fragmentation of chromosomal DNA. A hallmark of apoptosis is the fragmentation of nuclear DNA into pieces 200 base pairs long through the activation of nucleases that cleave the DNA between nucleosomes. As described in Chapter 6, autophagy is the process of degrading senescent or abnormal proteins and organelles. In autophagic cell death, large vacuoles degrade cellular organelles before the condensation and destruction of the nucleus that is characteristic of apoptosis.

1	Cytotoxic T cells kill by inducing their targets to undergo apoptosis (Fig. 9.41). Two general pathways are involved in signaling apoptotic cell death. One, called the extrinsic pathway of apoptosis, is mediated by the activation of so-called death receptors by extracellular ligands. Engagement of ligand stimulates apoptosis in receptor-bearing cells. The other pathway is known as the intrinsic or mitochondrial pathway of apoptosis and is induced in response to noxious stimuli (for example, ultraviolet irradiation or chemotherapeutic drugs), or lack of the growth factors required for survival. Common to both pathways is the activation of specialized proteases called aspartic acid-specific cysteine proteases, or caspases, which were introduced in Chapter 3 for their role in processing the cytokines IL-1 and IL-18 to their mature forms.

1	Fig. 9.41 Cytotoxic CD8 T cells can induce apoptosis in target cells. specific recognition of peptide:MhC complexes on a target cell (top panels) by a cytotoxic Cd8 T cell (CTL) leads to the death of the target cell by apoptosis. Cytotoxic T cells can recycle to kill multiple targets. each killing requires the same series of steps, including receptor binding and the directed release of cytotoxic proteins stored in granules. The process of apoptosis is shown in the micrographs (bottom panels), where panel a shows a healthy cell with a normal nucleus. early in apoptosis (panel b) the chromatin becomes condensed (red) and, although the cell sheds membrane vesicles, the integrity of the cell membrane is retained, in contrast to the necrotic cell in the upper part of the same field. In late stages of apoptosis (panel c), the cell nucleus (middle cell) is very condensed, no mitochondria are visible, and the cell has lost much of its cytoplasm and membrane through the shedding of vesicles.

1	of apoptosis (panel c), the cell nucleus (middle cell) is very condensed, no mitochondria are visible, and the cell has lost much of its cytoplasm and membrane through the shedding of vesicles. photographs (×3500) courtesy of r. Windsor and e. hirst.

1	Like many other proteases, caspases are synthesized as inactive pro-enzymes, in this case, pro-caspases, in which the catalytic domain is inhibited by an adjacent pro-domain. Pro-caspases are activated by other caspases that cleave the protein to release the inhibitory pro-domain. There are two classes of caspases involved in the apoptotic pathway: initiator caspases promote apoptosis by cleaving and activating other caspases; effector caspases initiate the cellular changes associated with apoptosis. The extrinsic pathway uses two related initiator caspases, caspase 8 and caspase 10, whereas the intrinsic pathway uses caspase 9. Both pathways use caspases 3, 6, and 7 as effector caspases. The effector caspases cleave a variety of proteins that are critical for cellular integrity and also activate enzymes that promote the death of the cell. For example, they cleave and degrade nuclear proteins that are required for the structural integrity of the nucleus, and activate the endonucleases

1	enzymes that promote the death of the cell. For example, they cleave and degrade nuclear proteins that are required for the structural integrity of the nucleus, and activate the endonucleases that fragment the chromosomal DNA.

1	Cytotoxic T cells can induce target-cell death by either the extrinsic or the intrinsic apoptotic pathway. The extrinsic pathway is mediated by expression of FasL and TNF-α or LT-α, receptors for which (Fas, or CD95, and TNFR-I) are expressed by other cells of the immune system, as well as non-immune-system cells. Because the distribution of these receptors is somewhat restricted, cytotoxic T cells have acquired a more universal mechanism for inducing cell death in antigen-specific targets: the directional release of cytotoxic granules that activate the intrinsic pathway of apoptosis. When cytotoxic T cells are mixed with target cells and rapidly brought into contact by centrifugation, they can induce antigen-specific target cells to die within 5 minutes, although death can take hours to become fully evident. The rapidity of this response reflects the release of preformed effector molecules that are delivered to the target cell. In addition to killing the host cell, the apoptotic

1	to become fully evident. The rapidity of this response reflects the release of preformed effector molecules that are delivered to the target cell. In addition to killing the host cell, the apoptotic mechanism may also act directly on cytosolic pathogens. For example, the nucleases that are activated in apoptosis to destroy cellular DNA can also degrade viral DNA. This prevents the assembly of virions and the release of infectious virus, which could otherwise infect nearby cells. Other enzymes activated in the course of apoptosis may destroy nonviral cytosolic pathogens. Apoptosis is therefore preferable to necrosis as a means of killing infected cells; in cells dying by necrosis, intact pathogens are released from the dead cell, and these can continue to infect healthy cells or parasitize the macrophages that ingest them.

1	9-30 The intrinsic pathway of apoptosis is mediated by the release of cytochrome c from mitochondria. Apoptosis by the intrinsic pathway is triggered by the release of cytochrome c from mitochondria, which triggers the activation of caspases. Once in the cytoplasm, cytochrome c binds to a protein called Apaf-1 (apoptotic protease activating factor-1), stimulating its oligomerization to form the apoptosome. The apoptosome then recruits an initiator caspase, pro-caspase 9, aggregation of which promotes its self-cleavage and frees its catalytic domain to activate effector caspases (Fig. 9.42). The release of cytochrome c is controlled by interactions between members of the Bcl-2 family of proteins. The Bcl-2 family of proteins is defined by the presence of one or more Bcl-2 homology (BH) domains and can be divided into two general groups: members that promote apoptosis, and members that

1	When programmed cell death is induced, the mitochondria swell and leak, releasing cytochrome c, which binds to and induces a conformational change in Apaf-1 caspase 3 cleaved ICAD Apaf-1:cytochrome ccomplex self assembles into an apoptosome, which recruits and activates multiple copies of pro-caspase 9, which in turn activates pro-caspase 3 Caspase 3 cleaves ICAD, releasing CAD to enter the nucleus and cleave DNA pro-caspase 3 pro-caspase 3 cleavage CAD Apaf-1

1	Fig. 9.42 In the intrinsic pathway, cytochrome c release from apaf-1. The resultant conformational change that ensues in apaf-1 mitochondria induces formation of the apoptosome, which induces self-assembly of the multimeric apoptosome, which recruits activates pro-caspase 9 to initiate programmed cell death. pro-caspase 9 (third panel). Clustering of pro-caspase 9 by the In normal cells, cytochrome c is confined to the mitochondria apoptosome activates it, allowing it to cleave downstream caspases, (first panel). however, during stimulation of the intrinsic pathway, such as caspase 3; this results in the activation of enzymes such as the mitochondria swell, allowing the cytochrome c to leak out ICad, which can cleave dna (fourth panel). into the cytosol (second panel), where cytochrome c is bound by Fig. 9.43 General scheme of intrinsic pathway regulation by the Bcl-2 family of proteins. extracellular apoptotic stimuli activate a group of pro-apoptotic (sentinel) proteins. sentinel

1	c is bound by Fig. 9.43 General scheme of intrinsic pathway regulation by the Bcl-2 family of proteins. extracellular apoptotic stimuli activate a group of pro-apoptotic (sentinel) proteins. sentinel proteins can function either to block the protection provided by pro-survival, protector proteins or to directly activate pro-apoptotic, executioner proteins. In mammalian cells, apoptosis is mediated by the executioner proteins Bax, Bak, and Bok. In normal cells, these proteins are prevented from acting by the protector proteins (Bcl-2, Bcl-XL, and Bcl-W). The release of activated executioner proteins causes the release of cytochrome c and subsequent cell death, as shown in Fig. 9.42.

1	Apoptotic stimuli Sentinels Bad, Bil, Bid, Bim, p53, PUMA, NOXA Death Protectors Bcl-2, Bcl-XL, Bcl-W Executioners Bax, Bak, Bok inhibit apoptosis (Fig. 9.43). Pro-apoptotic Bcl-2 family members, such as Bax, Bak, and Bok (referred to as executioners), bind to mitochondrial membranes and can directly cause cytochrome c release. How they do this is still not known, but they may form pores in the membranes.

1	The anti-apoptotic Bcl-2 family members are induced by stimuli that promote cell survival. The best known of the anti-apoptotic proteins is Bcl-2 itself. The Bcl2 gene was first identified as an oncogene in a B-cell lymphoma, and its overexpression in tumors makes the cells more resistant to apoptotic stimuli and thus more likely to progress to an invasive cancer. Other members of the inhibitory family include Bcl-XL and Bcl-W. Anti-apoptotic proteins function by binding to the mitochondrial membrane to block the release of cytochrome c. The precise mechanism of inhibition is not clear, but they may function by directly blocking the function of the pro-apoptotic family members.

1	A second family of pro-apoptotic Bcl-2 family members are termed 'sentinels' and are activated by apoptotic stimuli. Once activated, these proteins, which include Bad, Bid, and PUMA, can either act to block the activity of the antiapoptotic proteins or act directly to stimulate the activity of the executioner pro-apoptotic proteins. 9-31 Cytotoxic effector proteins that trigger apoptosis are contained in the granules of CD8 cytotoxic T cells.

1	The principal mechanism of cytotoxic T-cell action is the calcium-dependent release of specialized cytotoxic granules upon recognition of antigen on the surface of a target cell. Cytotoxic granules are modified lysosomes that contain at least three distinct classes of cytotoxic effector proteins that are expressed specifically in cytotoxic T cells: perforin, granzymes, and granulysin (Fig. 9.44). These proteins are stored in cytotoxic granules in an active form, but conditions within the granules prevent their actions until after release. Perforin acts by forming pores in, or perforating, the target-cell plasma membrane, which both causes direct damage to the target cell and forms a conduit through which other contents of cytotoxic granules are delivered into the cytosol of the target cell. Granzymes, of which there are 5 in humans and 10 in the mouse, activate apoptosis once delivered to the target-cell cytosol via pores formed by perforin. Granulysin, which is expressed in humans

1	Granzymes, of which there are 5 in humans and 10 in the mouse, activate apoptosis once delivered to the target-cell cytosol via pores formed by perforin. Granulysin, which is expressed in humans but not in mice, has antimicrobial activity and at high concentrations is also able to induce apoptosis in target cells. Cytotoxic granules also contain the proteoglycan serglycin, which acts as a scaffold, forming a complex with perforin and the granzymes.

1	Both perforin and granzymes are required for effective target-cell killing. In cytotoxic cells that lack granzymes, the presence of perforin alone can kill target cells, but large numbers of cytotoxic cells are needed because the killing is very inefficient. In contrast, cytotoxic T cells from mice lacking perforin are unable to kill other cells, due to the lack of a mechanism to deliver granzymes into the target cell.

1	Granzymes trigger apoptosis in the target cell both by directly activating caspases and by damaging mitochondria, which also activates caspases. The two most abundant granzymes are granzymes A and B. Granzyme A triggers cell death by caspase-independent mitochondrial damage, through mechanisms that are not completely understood. Granzyme B, like the caspases, cleaves proteins after aspartic acid residues and activates caspase 3, thereby activating a caspase proteolytic cascade, which eventually activates the caspase-activated deoxyribonuclease (CAD) by cleaving an inhibitory protein (ICAD) that binds to and inactivates CAD. This nuclease is believed to be the enzyme that degrades DNA in target cells (Fig. 9.45). Granzyme B also targets mitochondria to activate the intrinsic apoptotic pathway; it cleaves the protein BID (for BH3-interacting domain death agonist protein), either directly recognition of its antigen on a virus-infected cell by a cytotoxic Cd8 T cell induces the T cell to

1	it cleaves the protein BID (for BH3-interacting domain death agonist protein), either directly recognition of its antigen on a virus-infected cell by a cytotoxic Cd8 T cell induces the T cell to release the contents of its cytotoxic granules in a directed fashion. perforin and granzymes, in a complex with the proteoglycan serglycin, are delivered to the membrane of the target cell (top panel). By an unknown mechanism, perforin directs the entry of the granule contents into the cytosol of the target cell without apparent pore formation, and the introduced granzymes then act on specific intracellular targets such as the proteins BId and pro-caspase 3 (second panel). either directly or indirectly, the granzymes cause the cleavage of BId into truncated BId (tBId) and the cleavage of pro-caspase 3 into an active caspase (third panel). tBId acts on mitochondria to release cytochrome c into the cytosol. This promotes apoptosis by inducing the formation of the apoptosome that activates

1	3 into an active caspase (third panel). tBId acts on mitochondria to release cytochrome c into the cytosol. This promotes apoptosis by inducing the formation of the apoptosome that activates procaspase 9, which in turn further amplifies caspase 3 activation. activated caspase 3 targets ICad to release caspase-activated dnase (Cad), which fragments the dna (bottom panel).

1	or indirectly by activated caspase 3, causing disruption of the mitochondrial outer membrane and the release from the mitochondrial intermembrane space of pro-apoptotic molecules such as cytochrome c. As discussed above (Section 9-30), cytochrome c is central to amplification of the intrinsic apoptotic cascade, as it initiates assembly of the apoptosome with Apaf-1, which in turn activates the initiator caspase 9. Thus, granzyme B acts directly to activate the effector caspase 3, and indirectly to activate the initiator caspase 9.

1	Cells undergoing programmed cell death are rapidly ingested by phagocytic cells, which recognize a change in the cell membrane: phosphatidylserine, which is normally found only in the inner leaflet of the membrane, replaces phosphatidylcholine as the predominant phospholipid in the outer leaflet. The ingested cell is broken down and completely digested by the phagocyte without the induction of co-stimulatory proteins. Thus, apoptosis is normally an immunologically ‘quiet’ process; that is, apoptotic cells do not normally contribute to or stimulate immune responses. 9-32 Cytotoxic T cells are selective serial killers of targets expressing a specific antigen.

1	When cytotoxic T cells are offered a mixture of equal amounts of two target cells, one bearing a specific antigen and the other not, they kill only the target cell bearing the specific antigen. The ‘innocent bystander’ cells and the cytotoxic T cells themselves are not killed. The cytotoxic T cells are probably not killed because release of the cytotoxic effector molecules is highly polarized. As we saw in Fig. 9.38, cytotoxic T cells orient their Golgi apparatus and microtubule-organizing center to focus secretion on the point of contact with a target cell. Granule movement toward the point of contact is shown in Fig. 9.46. Cytotoxic T cells attached to several different target cells reorient their secretory apparatus toward each cell in turn and kill them one by one, strongly suggesting that the mechanism whereby cytotoxic mediators are released allows attack at only one point of contact at any one time. The narrowly focused action of CD8 cytotoxic T cells allows them to kill single

1	that the mechanism whereby cytotoxic mediators are released allows attack at only one point of contact at any one time. The narrowly focused action of CD8 cytotoxic T cells allows them to kill single infected cells in a tissue without creating widespread tissue damage (Fig. 9.47) and is of crucial importance in tissues where cell regeneration does not occur, as with the neurons of the central nervous system, or is very limited, as in the pancreatic islets.

1	Cytotoxic T cells can kill their targets rapidly because they store preformed cytotoxic proteins in forms that are inactive in the environment of the cytotoxic granule. Cytotoxic proteins are synthesized and loaded into the granules soon after the first encounter of a naive cytotoxic precursor T cell with its specific antigen. Ligation of the T-cell receptor similarly induces de novo synthesis of perforin and granzymes in effector CD8 T cells, so that the supply of cytotoxic granules is replenished. This makes it possible for a single CD8 T cell to kill a series of targets in succession. Fig. 9.45 Perforin, granzymes, and serglycin are released from cytotoxic granules and deliver granzymes into the cytosol of target cells to induce apoptosis.

1	Fig. 9.45 Perforin, granzymes, and serglycin are released from cytotoxic granules and deliver granzymes into the cytosol of target cells to induce apoptosis. Release of cytochrome cinto cytosol activates apoptosis, and CAD induces DNA fragmentation Truncated BID (tBID) disrupts mitochondrial outer membrane, and activated caspase 3 cleaves ICAD, releasing caspase-activated DNase (CAD) Granzyme B is delivered into the cytosol of the infected cell via pores formed by perforin, and targets BID and pro-caspase 3 pro-caspase 3BID BAX BAD Engagement of TCR by peptide:MHC complex causes directed release of perforin and granzymes complexed with serglycin cytotoxic T cell virus-infected cell perforin cytotoxic granulegranzymeserglycin MHC TCR caspase 3 tBID cytochrome cCAD cleaved ICAD DNA

1	Fig. 9.46 Effector molecules are released from T-cell granules in a highly polar fashion. The granules of cytotoxic T cells can be labeled with fluorescent dyes, allowing the granules to be seen under the microscope and their movements to be followed by time-lapse photography. here we show a series of pictures taken during the interaction of a cytotoxic T cell with a target cell, which is eventually killed. In the top panel, at time 0, the T cell (upper right) has just made contact with a target cell (diagonally below). at this time, the granules of the T cell, labeled with a red fluorescent dye, are distant from the point of contact. In the second panel, after 1 minute has elapsed, the granules have begun to move toward the target cell, a move that has essentially been completed in the third panel, after 4 minutes. after 40 minutes, in the last panel, the granule contents have been released into the space between the T cell and the target, which has begun to undergo apoptosis (note

1	third panel, after 4 minutes. after 40 minutes, in the last panel, the granule contents have been released into the space between the T cell and the target, which has begun to undergo apoptosis (note the fragmented nucleus). The T cell will now disengage from the target cell, whereupon it can go on to recognize and kill other targets. photographs courtesy of G. Griffiths.

1	9-33 Cytotoxic T cells also act by releasing cytokines.

1	Inducing apoptosis in target cells is the main way in which CD8 cytotoxic T cells eliminate infection. However, most CD8 cytotoxic T cells also release the cytokines IFN-γ, TNF-α, and LT-α, which contribute to host defense in other ways. IFN-γ inhibits viral replication directly, and induces the increased expression of MHC class I molecules and of other proteins that are involved in peptide loading of these newly synthesized MHC class I molecules in infected cells. This increases the chance that infected cells will be recognized as target cells for cytotoxic attack. IFN-γ also activates macrophages, recruiting them to sites of infection, where they serve both as effector cells and as antigen-presenting cells. TNF-α and LT-α can synergize with IFN-γ in macrophage activation via TNFR-II, and can kill some target cells through their interaction with TNFR-I, which can induce apoptosis (see Sections 9-28 and 9-29). Thus, effector CD8 cytotoxic T cells act in a variety of ways to limit the

1	can kill some target cells through their interaction with TNFR-I, which can induce apoptosis (see Sections 9-28 and 9-29). Thus, effector CD8 cytotoxic T cells act in a variety of ways to limit the spread of cytosolic pathogens.

1	Summary.

1	Effector CD8 cytotoxic T cells are essential in host defense against pathogens that reside in the cytosol: most commonly these will be viruses. These cytotoxic T cells can kill any cell harboring such pathogens by recognizing foreign peptides that are transported to the cell surface bound to MHC class I molecules. CD8 cytotoxic T cells perform their killing function by releasing three types of preformed cytotoxic proteins: granzymes, which use multiple mechanisms to induce apoptosis in any type of target cell; perforin, which acts in the delivery of granzymes into the target cell; and granulysin, which has antimicrobial activity and is pro-apoptotic. These properties allow the cytotoxic T cell to attack and destroy virtually any cell infected with a cytosolic pathogen. The membrane-bound Fas ligand, expressed by CD8 and some CD4 T cells, may also induce apoptosis by binding to Fas, which is expressed on some target cells. However, this pathway is less important in most infections than

1	ligand, expressed by CD8 and some CD4 T cells, may also induce apoptosis by binding to Fas, which is expressed on some target cells. However, this pathway is less important in most infections than that mediated by cytotoxic granules. CD8 cytotoxic T cells also produce IFN-γ, which inhibits viral replication and is an important inducer of MHC class I molecule expression and macrophage activation. Cytotoxic T cells kill infected targets with great precision, sparing adjacent normal cells. This precision is crucial in minimizing tissue damage while allowing the eradication of infected cells.

1	Summary to Chapter 9.

1	An adaptive immune response is initiated when naive T cells encounter specific antigen on the surface of an antigen-presenting cell in T-cell zones of secondary lymphoid tissues. In most cases, the antigen-presenting cells responsible for activating naive T cells, and inducing their clonal expansion, are conventional dendritic cells that express the co-stimulatory molecules B7.1 and B7.2. Conventional dendritic cells not only reside in lymphoid tissues, but they also survey the periphery, where they encounter pathogens, take up antigen at sites of infection, become activated through innate recognition, and migrate to local lymphoid tissue. The dendritic cell may become a potent direct activator of naive T cells, or it may transfer antigen to dendritic cells resident in secondary lymphoid organs for cross-presentation to naive CD8 T cells. Plasmacytoid dendritic cells contribute to rapid responses against viruses by producing type I interferons. Activated T cells produce IL-2, which is

1	for cross-presentation to naive CD8 T cells. Plasmacytoid dendritic cells contribute to rapid responses against viruses by producing type I interferons. Activated T cells produce IL-2, which is important in modulating early proliferation and differentiation of T cells; various other signals drive the differentiation of several types of effector T cells, which primarily act by releasing mediators directly onto their target cells. This triggering of effector T cells by peptide:MHC complexes occurs independently of co-stimulation, so that any infected target cell can be activated or destroyed by an effector T cell. CD8 cytotoxic T cells kill target cells infected with cytosolic pathogens, thus removing sites of pathogen replication. CD4 T cells can become specialized effectors that in turn promote distinct arms of the immune response by targeting different innate and adaptive immune cells for enhanced effector function: macrophages (TH1); eosinophils, basophils, and mast cells (TH2);

1	promote distinct arms of the immune response by targeting different innate and adaptive immune cells for enhanced effector function: macrophages (TH1); eosinophils, basophils, and mast cells (TH2); neutrophils (TH17); or B cells (TFH). Thus, effector T cells control virtually all known effector mechanisms of the adaptive and innate immune response. In addition, subsets of CD4 regulatory T cells are produced that help control and limit immune responses by suppressing T-cell activity.

1	Fig. 9.47 Cytotoxic T cells kill target cells bearing specific antigen while sparing neighboring uninfected cells. all the cells in a tissue are susceptible to killing by the cytotoxic proteins of armed effector Cd8 T cells, but only infected cells are killed. specific recognition by the T-cell receptor identifies which target cell to kill, and the polarized release of the cytotoxic granules (not shown) ensures that neighboring cells are spared. T cell recognizes infected cell Infected cell is programmed for death Neighboring uninfected cells are not killed Questions. 9.1 Multiple Choice: Which of the following statements is true? A. development of the arterial and venous system is regulated by the homeobox transcription factor prox1. B. arteries deliver lymphotoxin to the non-hematopoietic stromal LTi cell to induce lymph-node development. C. Lymphotoxin-α3 signaling represses nFκB to induce chemokines such as CXCL13.

1	B. arteries deliver lymphotoxin to the non-hematopoietic stromal LTi cell to induce lymph-node development. C. Lymphotoxin-α3 signaling represses nFκB to induce chemokines such as CXCL13. D. Lymphotoxin-α3 binds TnFr-I and supports development of the cervical and mesenteric lymph nodes.

1	C. Lymphotoxin-α3 signaling represses nFκB to induce chemokines such as CXCL13. D. Lymphotoxin-α3 binds TnFr-I and supports development of the cervical and mesenteric lymph nodes. 9.2 Fill-in-the-Blanks: T and B cells are distributed to the secondary lymphoid organs through the blood. These are then directed to their respective compartments as instructed by chemokines. For example, CCL21 is secreted by _________ of the T-cell zone in the spleen and displayed by the _______in the lymph nodes. signaling of this chemokine as well as _______through CCr7 directs the T cells into the respective T-cell zone. In contrast, _______ is the ligand for CXCr5, which is secreted by __________ and attracts B cells to the ______. T cells can also respond to CXCL13 as a subset of T cells expresses _______, which allows them to enter the B-cell follicle and participate in the formation of the germinal center.

1	9.3 Multiple Choice: Which of the following correctly describes events necessary for naive T-cell entry into the lymph node? A. CCr7 signaling induces Gαi, which results in lowered affinity for integrin binding. B. upregulation of s1p receptor on naive T cells promotes migration into the lymph node. C. rolling in the hev exposes the T cell to CCL21, which activates LFa-1 and promotes migration. D. MadCaM-1 expression on the hev interacts with Cd62L on the T cell and promotes migration into the lymph node. 9.4 Short Answer: In some cases, hsv or influenza viruses infect antigen-presenting cells from peripheral tissues that do not present the viral antigens to naive T cells. how is the immune system able to develop an adaptive immune response to such pathogens? 9.5 True or False: TLr stimulation induces CCr7 expression in the dendritic cells, which promotes migration to the lymph node through the bloodstream.

1	9.5 True or False: TLr stimulation induces CCr7 expression in the dendritic cells, which promotes migration to the lymph node through the bloodstream. 9.6 Matching: Classify each of the following activation signatures as a conventional dendritic cell (cdC) or plasmacytoid dendritic cell (pdC) pathogen response. ___1. production of CCL18 ___2. Continuous MhC recycling upon activation ___3. expression of dC-sIGn ___4. expression of Cd80 and Cd86 ___5. Cd40L expression upon TLr-9 stimulation 9.7 Short Answer: how does the process of antigen presentation differ among B cells, dendritic cells, and macrophages in the context of an immune response? 9.8 Multiple Choice: Which of the following is a common consequence of TCr and CCr7 signaling? A. Integrin activation B. positive selection C. Th1 induction D. Th2 induction 9.9 Multiple Choice: Which of the following describes a mechanism by which Cd28 signaling can increase IL-2 production?

1	B. positive selection C. Th1 induction D. Th2 induction 9.9 Multiple Choice: Which of the following describes a mechanism by which Cd28 signaling can increase IL-2 production? A. Cd28 signaling induces the expression of proteins that stabilize the IL-2 mrna sequence. B. pI 3-kinase inhibits akt, supporting IL-2 production by cell cycle arrest. C. pI 3-kinase suppresses the production of ap-1 and nFκB, thereby increasing IL-2 production. 9.10 True or False: In the majority of viral infections, Cd8 T-cell activation requires Cd4 T-cell help. 9.11 Matching: Match each Cd4 T cell subset-specific secreted cytokine with its respective effector function. A. IL-17 i. eradication of intracellular infections B. IL-4 ii. response to extracellular bacteria C. IFn-γ iii. Control of extracellular parasites

1	A. IL-17 i. eradication of intracellular infections B. IL-4 ii. response to extracellular bacteria C. IFn-γ iii. Control of extracellular parasites D. IL-10 iv. suppression of T-cell responses 9.12 Matching: The following cytokines drive Cd4 Th subset effector differentiation. Match each with its respective subset-specific transcription factor. A. IFn-γ i. rorγt B. IL-4 ii. Foxp3 C. IL-6 and TGF-β iii. T-bet D. TGF-β iv. GaTa3 9.13 Multiple Choice: Which of the following statements is false? A. TCr signaling is strongest at the csMaC. B. Cb1, an e3 ligase, mediates degradation of TCrs in the csMaC. C. Cytoskeletal reorganization directs effector molecule release at the immunological synapse. D. Integrins such as LFa-1 associate in the sMaC. 9.14 Fill-in-the-Blanks: For each of the following sentences, fill in the blanks with the best word selected from the list below. not all words will be used; each word should be used only once.

1	9.14 Fill-in-the-Blanks: For each of the following sentences, fill in the blanks with the best word selected from the list below. not all words will be used; each word should be used only once. Cd8 T cells can specifically mediate the destruction of infected or malignant cells. In order to do this, Cd8 T cells induce _______ cell death, which can be induced in two different ways. First, Cd8 T cells possess ligands such as _______, _______, or _______ that can induce the _______ apoptosis pathway. In contrast, cell death can also be induced through an intrinsic pathway. To initiate this mechanism, _______ are released, which allow the entrance of granzymes into the cell. once the granzymes have gained access to the cell’s cytoplasm, these can cleave and activate _______, which in turn cleaves _______, allowing _______ to degrade dna. Granzyme B also cleaves _______, and as a consequence disrupts the mitochondrial membrane, allowing for release of _______ and formation of the _______.

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1	Hehlgans, T., and Mannel, D.N.: The TNF–TNF receptor system. Biol. Chem. 2002, 383:1581–1585. Ware, C.F.: Network communications: lymphotoxins, LIGHT, and TNF. Annu. Rev. Immunol. 2005, 23:787–819. 9-29 Cytotoxic T cells induce target cells to undergo programmed cell death via extrinsic and intrinsic pathways of apoptosis. Aggarwal, B.B.: Signalling pathways of the TNF superfamily: a double-edged sword. Nat. Rev. Immunol. 2003, 3:745–756. Ashton-Rickardt, P.G.: The granule pathway of programmed cell death. Crit. Rev. Immunol. 2005, 25:161–182. Bishop, G.A.: The multifaceted roles of TRAFs in the regulation of B-cell function. Nat. Rev. Immunol. 2004, 4:775–786. Green, D.R., Droin, N., and Pinkoski, M.: Activation-induced cell death in T cells. Immunol. Rev. 2003, 193:70–81. Russell, J.H., and Ley, T.J.: Lymphocyte-mediated cytotoxicity. Annu. Rev. Immunol. 2002, 20:323–370.

1	Russell, J.H., and Ley, T.J.: Lymphocyte-mediated cytotoxicity. Annu. Rev. Immunol. 2002, 20:323–370. Siegel, R.M.: Caspases at the crossroads of immune-cell life and death. Nat. Rev. Immunol. 2006, 6:308–317. Wallin, R.P., Screpanti, V., Michaelsson, J., Grandien, A., and Ljunggren, H.G.: Regulation of perforin-independent NK cell-mediated cytotoxicity. Eur. J. Immunol. 2003, 33:2727–2735. 9-30 The intrinsic pathway of apoptosis is mediated by the release of cytochrome c from mitochondria. Borner, C.: The Bcl-2 protein family: sensors and checkpoints for life-ordeath decisions. Mol. Immunol. 2003, 39:615–647. Bratton, S.B., and Salvesen, G.S.: Regulation of the Apaf-1-caspase-9 apoptosome. J. Cell Sci. 2010, 123:3209–3214. Chowdhury, D., and Lieberman, J.: Death by a thousand cuts: granzyme pathways of programmed cell death. Annu. Rev. Immunol. 2008, 26:389–420.

1	Chowdhury, D., and Lieberman, J.: Death by a thousand cuts: granzyme pathways of programmed cell death. Annu. Rev. Immunol. 2008, 26:389–420. Hildeman, D.A., Zhu, Y., Mitchell, T.C., Kappler, J., and Marrack, P.: Molecular mechanisms of activated T cell death in vivo. Curr. Opin. Immunol. 2002, 14:354–359. Strasser, A.: The role of BH3-only proteins in the immune system. Nat. Rev. Immunol. 2005, 5:189–200. 9-31 Cytotoxic effector proteins that trigger apoptosis are contained in the granules of CD8 cytotoxic T cells. Barry, M., Heibein, J.A., Pinkoski, M.J., Lee, S.F., Moyer, R.W., Green, D.R., and Bleackley, R.C.: Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid. Mol. Cell Biol. 2000, 20:3781–3794. Grossman, W.J., Revell, P.A., Lu, Z.H., Johnson, H., Bredemeyer, A.J., and Ley, T.J.: The orphan granzymes of humans and mice. Curr. Opin. Immunol. 2003, 15:544–552.

1	Grossman, W.J., Revell, P.A., Lu, Z.H., Johnson, H., Bredemeyer, A.J., and Ley, T.J.: The orphan granzymes of humans and mice. Curr. Opin. Immunol. 2003, 15:544–552. Lieberman, J.: The ABCs of granule-mediated cytotoxicity: new weapons in the arsenal. Nat. Rev. Immunol. 2003, 3:361–370. Pipkin, M.E., and Lieberman, J.: Delivering the kiss of death: progress on understanding how perforin works. Curr. Opin. Immunol. 2007, 19:301–308. Yasukawa, M., Ohminami, H., Arai, J., Kasahara, Y., Ishida, Y., and Fujita, S.: Granule exocytosis, and not the Fas/Fas ligand system, is the main pathway of cytotoxicity mediated by alloantigen-specific CD4+ as well as CD8+ cytotoxic T lymphocytes in humans. Blood 2000, 95:2352–2355. 9-32 Cytotoxic T cells are selective and serial killers of targets expressing a specific antigen. Stinchcombe, J.C., and Griffiths, G.M.: Secretory mechanisms in cell-mediated cytotoxicity. Annu. Rev. Cell Dev. Biol. 2007, 23:495–517.

1	Stinchcombe, J.C., and Griffiths, G.M.: Secretory mechanisms in cell-mediated cytotoxicity. Annu. Rev. Cell Dev. Biol. 2007, 23:495–517. Veugelers, K., Motyka, B., Frantz, C., Shostak, I., Sawchuk, T., and Bleackley, R.C.: The granzyme B-serglycin complex from cytotoxic granules requires dynamin for endocytosis. Blood 2004, 103:3845–3853. 9-33 Cytotoxic T cells also act by releasing cytokines. Amel-Kashipaz, M.R., Huggins, M.L., Lanyon, P., Robins, A., Todd, I., and Powell, R.J.: Quantitative and qualitative analysis of the balance between type 1 and type 2 cytokine-producing CD8– and CD8+ T cells in systemic lupus erythematosus. J. Autoimmun. 2001, 17:155–163. Dobrzanski, M.J., Reome, J.B., Hollenbaugh, J.A., and Dutton, R.W.: Tc1 and Tc2 effector cell therapy elicit long-term tumor immunity by contrasting mechanisms that result in complementary endogenous type 1 antitumor responses. J. Immunol. 2004, 172:1380–1390.

1	Prezzi, C., Casciaro, M.A., Francavilla, V., Schiaffella, E., Finocchi, L., Chircu, L.V., Bruno, G., Sette, A., Abrignani, S., and Barnaba, V.: Virus-specific CD8+ T cells with type 1 or type 2 cytokine profile are related to different disease activity in chronic hepatitis C virus infection. Eur. J. Immunol. 2001, 31:894–906. The Humoral Immune Response

1	Many pathogens multiply in the body’s extracellular spaces, and even intracellular pathogens can spread by moving through the extracellular fluids. The extracellular spaces are protected by the humoral immune response, in which antibodies produced by B cells act to destroy extracellular microorganisms and their products, and prevent the spread of intracellular infections. As we introduced in Section 1-20, antibodies contribute to immunity in three main ways: neutralization, opsonization, and complement activation (Fig. 10.1). Antibodies can bind to pathogens and prevent their ability to enter and infect cells, and therefore are thus said to neutralize the pathogen; antibodies may also bind bacterial toxins, preventing their action or ability to enter cells. Antibodies also facilitate opsonization, the uptake of the pathogens by phagocytes, by binding to Fc receptors through their constant regions (C regions). Finally, antibodies bound to pathogens can activate proteins of the

1	opsonization, the uptake of the pathogens by phagocytes, by binding to Fc receptors through their constant regions (C regions). Finally, antibodies bound to pathogens can activate proteins of the classical pathway of the complement system, as we described in Chapter 2. This can increase opsonization by placing other complement proteins onto the pathogen’s surface, help recruit phagocytic cells to the site of infection, and activate the membrane-attack complex, which can directly lyse certain microorganisms by forming pores in their membranes. The choice of which effector mechanisms are used is influenced by the heavy-chain isotype of the antibodies produced, which determines their class (see Section 5-12).

1	In the first part of this chapter, we describe the interactions of naive B cells with antigen and with helper T cells that lead to the activation of B cells and antibody production. Some microbial antigens can provoke antibody production without T-cell help, but activation of naive B cells by antigens usually involves help from T follicular helper (TFH) cells (see Section 9-20). Activated B cells then differentiate into antibody-secreting plasma cells and memory B cells. Most antibody responses undergo a process called affinity maturation, in which antibodies of greater affinity for their target antigen are produced by the somatic hypermutation of antibody variable-region (V-region) genes. We examine the molecular mechanism of somatic hypermutation and its immunological consequences, as well as class switching—a process that generates the different classes of antibodies that confer functional diversity on the antibody response. Both affinity maturation and class switching occur only

1	well as class switching—a process that generates the different classes of antibodies that confer functional diversity on the antibody response. Both affinity maturation and class switching occur only in B cells and require T-cell help. In the second part of the chapter, we introduce the distributions and functions of various classes of antibody, in particular those secreted into mucosal sites. In the third part of the chapter, we discuss in detail how the Fc region of the antibody engages various effector mechanisms to contain and eliminate infections. Like the T-cell response, the humoral immune response produces immunological memory, and this is discussed in Chapter 11.

1	Fig. 10.1 Antibodies mediate the humoral immune response through neutralization, opsonization, and complement activation. After being secreted by plasma cells, antibodies protect the host from infection in three main ways. They can inhibit the toxic effects or infectivity of pathogens or their products by binding to them, a process called neutralization (top panel). When bound to pathogens, the antibody’s Fc region can bind to Fc receptors on accessory cells, such as macrophages and neutrophils, helping these cells to ingest and kill the pathogen. This process is called opsonization (middle panel). Antibodies can trigger complement by activating C1, the first step in the classical complement pathway. Deposition of complement proteins enhances opsonization and can also directly kill certain bacterial cells by activating the membrane-attack complex (bottom panel). B-cell activation by antigen and helper T cells. The distributions and functions of immunoglobulin classes.

1	B-cell activation by antigen and helper T cells. The distributions and functions of immunoglobulin classes. The destruction of antibody-coated pathogens via Fc receptors.

1	Fig. 10.2 A second signal is required for B-cell activation by either thymus-dependent or thymus-independent antigens. The first signal (indicated as 1) required for B-cell activation is delivered through its antigen receptor (BCR) and activates several pathways as described in Chapter 7. Signaling by the BCR is enhanced by the co-receptors CD21 and CD19, which interact with C3b on opsonized microbial surfaces. For thymus-dependent antigens (first panel), a second signal (indicated as 2) is delivered by a helper T cell (TFH) that recognizes degraded fragments of the antigen as peptides bound to MHC class II molecules on the B-cell surface. CD40L on the TFH cell binds to CD40 on the B cell, activating the non-canonical NFκB signaling pathway via NFκB-inducing kinase (NIK). This induces expression of pro-survival genes such as Bcl-2 (see Section 7-17). For thymus-independent antigens (second panel), a second signal can be delivered through Toll-like receptors that recognize

1	induces expression of pro-survival genes such as Bcl-2 (see Section 7-17). For thymus-independent antigens (second panel), a second signal can be delivered through Toll-like receptors that recognize antigen-associated TLR ligands, such as bacterial lipopolysaccharide (LPS) or bacterial DNA, as described in Chapter 3.

1	B-cell activation by antigen and helper T cells.

1	The surface immunoglobulin that serves as the B-cell receptor (BCR) plays two roles in B-cell activation in response to pathogens. Like the antigen receptor on T cells, the BCR initiates a signaling cascade upon binding antigens derived from the microbe. In addition, the BCR can deliver the antigen to intracellular sites for antigen processing, so that antigenic peptides bound to MHC class II molecules can be returned to the B-cell surface. These peptide:MHC class II complexes are recognized by antigen-specific helper T cells that have already differentiated in response to the same pathogen. The effector T cells express surface molecules and cytokines that help the B cell to proliferate and to differentiate into antibody-secreting cells and into memory B cells, and a structure called the germinal center (see Section 10-6) is formed during an intermediate phase of the antibody response, before the emergence of longterm plasma cells that generate antibody or of memory B cells. Some

1	the germinal center (see Section 10-6) is formed during an intermediate phase of the antibody response, before the emergence of longterm plasma cells that generate antibody or of memory B cells. Some microbial antigens can activate B cells directly in the absence of T-cell help, and the ability of B cells to respond directly to these antigens provides a rapid response to many important pathogens. However, the fine tuning of antibody responses to increase the affinity of the antibody for the antigen and the switching to most immunoglobulin classes other than IgM depend on the interaction of antigen-stimulated B cells with helper T cells and other cells in the peripheral lymphoid organs. Thus, antibodies induced by microbial antigens alone tend to have lower affinity and to be less functionally versatile than those induced with T-cell help.

1	10-1 Activation of B cells by antigen involves signals from the B-cell receptor and either TFH cells or microbial antigens. As we learned in Chapter 8, activation of naive T cells requires signals derived from the T-cell receptor as well as co-stimulatory signals provided by professional antigen-presenting cells. Similarly, in addition to signals derived from the B-cell receptor, naive B cells also require accessory signals that can arise either from a helper T cell or, in some cases, directly from microbial constituents (Fig. 10.2). B-cell activation by antigen and helper T cells.

1	Protein antigens alone are unable to induce antibody responses in animals or humans who lack T cells, and they are therefore known as thymus-dependent or TD antigens, and typically involve antigen-specific T-cell help. The T cells involved are TFH cells that reside in the lymphoid tissues and are not fully differentiated TH1, TH2, or TH17 effector cells. To receive T-cell help, the B cell must display antigen on its surface in a form that a T cell can recognize. This occurs when antigen bound by surface immunoglobulin on the B cell is internalized and degraded within the B cell and peptides derived from it are returned to the cell surface in a complex with MHC class II molecules (see Fig. 10.2, first panel). When the TFH cell recognizes these peptide:MHC complexes, it provides the B cell with signals that favor survival and induce proliferation. These signals include the activation of CD40 on B cells by TFH expression of its ligand, CD40L (CD154), and production of various cytokines

1	with signals that favor survival and induce proliferation. These signals include the activation of CD40 on B cells by TFH expression of its ligand, CD40L (CD154), and production of various cytokines by TFH cells, including IL-21 (Fig. 10.3). CD40 signaling activates the non-canonical NFκB pathway (see Section 7-23) and enhances B-cell survival by inducing the expression of anti-apoptotic molecules such as Bcl-2. IL-21 signaling activates STAT3 and enhances cellular proliferation and differentiation into plasma cells and memory B cells. Other cytokines produced by TFH cells include IL-6, TGF-β, IFN-γ, and IL-4, which provide signals that can regulate the type of antibody produced, as we will see in Section 10-12. These cytokines are also made by other differentiated effector subsets (described in Chapter 9), but TFH cells are distinct from these. For example, TFH cells transcribe the IL-4 gene using regulatory elements that are independent of the transcription factors GATA-3 and STAT6,

1	in Chapter 9), but TFH cells are distinct from these. For example, TFH cells transcribe the IL-4 gene using regulatory elements that are independent of the transcription factors GATA-3 and STAT6, which are responsible for IL-4 production by TH2 cells.

1	While B-cell responses to protein antigens rely on help from T cells, some microbial constituents can induce antibody production in the absence of helper T cells. These microbial antigens are known as thymus-independent or TI antigens because they can induce antibody responses in individuals who have no T lymphocytes. Such antigens are typically highly repetitive molecules, such as the polysaccharides of bacterial cell walls, and can cross-link the BCR on B cells. In such cases, a second signal can be derived from direct recognition of a common microbial constituent such as LPS that can activate TLR signaling in the B cell (see Fig. 10.2, second panel), activating the NFκB pathway, as described in Chapter 3. Thymus-independent antibody responses provide some protection against extracellular bacteria, and we will return to them later.

1	Further differentiation can proceed to the germinal center, resting memory cells, or antibody-secreting plasma cells B-cell proliferation generates plasmablasts that form the primary focus TFH B cell AP-1 NFAT NF˜B Bcl-2 STAT3 germinal center B cell plasmacell memoryB cell various targets Antigen recognition by TFH cells induces signals that activate B cells IL-21 IL-6 TGF-°LFN-˛IL-4 STAT3 CD40 CD40L MHCII TCR

1	Fig. 10.3 TFH cells provide several signals that activate B cells which activates the transcription factor STAT3 to enhance B-cell and control their subsequent differentiation. After antigen proliferation and survival. TFH cells can also produce cytokines that binding to the B-cell receptor delivers the first signal for B-cell will regulate isotype switching (see Section 10-12). After receiving activation (not shown), the TFH cell delivers additional signals these signals, activated B cells begin to proliferate (second panel), when it recognizes a peptide:MHC class II complex on the B-cell enter the germinal center, and eventually become plasma cells or surface (first panel). Besides expression of CD40 ligand, the TFH cell memory B cells (third panel). secretes several important cytokines. Included among them is IL-21, 10-2 Linked recognition of antigen by T cells and B cells promotes robust antibody responses.

1	B-cell activation by antigens on microbial surfaces can be greatly stimulated by the concurrent deposition of complement on these pathogens. The B-cell co-receptor complex contains the cell-surface proteins CD19, CD21, and CD81 (see Fig. 7.27). When CD21, or complement receptor 2 (CR2), binds to the complement fragments C3d and C3dg that are deposited on microbial surfaces (see Section 2-13), it is brought near to the activated B-cell receptor bound to the same surface. CD21 and CD19 are associated with each other, and CD19 becomes phosphorylated by the activated B-cell receptor. This recruits PI 3-kinase, which then stimulates several downstream pathways, enhancing proliferation, differentiation, and antibody production (see Fig. 10.2, arrow 1). This effect is shown dramatically when mice are immunized with the experimental antigen hen egg-white lysozyme that is coupled to three linked molecules of C3dg. In this case the dose of modified lysozyme needed to induce antibody in the

1	mice are immunized with the experimental antigen hen egg-white lysozyme that is coupled to three linked molecules of C3dg. In this case the dose of modified lysozyme needed to induce antibody in the absence of added adjuvant is as little as 1/10,000 of that needed with the unmodified lysozyme.

1	For T-dependent antibody responses, the T cells involved are activated by the same antigen as is recognized by the B cells; this is called linked recognition. However, the peptide recognized by the TFH cell is likely to differ from the protein epitope recognized by the B cell’s antigen receptor. Natural antigens, such as viruses and bacteria, contain multiple proteins and carry both protein and carbohydrate epitopes. For linked recognition to occur, the peptide recognized by the T cell must be physically associated with the antigen recognized by the B cell’s receptor, so that the B cell can take up and present the appropriate peptide to the T cell. For example, a B cell that recognizes an epitope on a viral coat protein will internalize the complete virus particle. The B cell can degrade multiple viral proteins into peptides for display on MHC class II molecules on the B-cell surface. CD4 T cells specific for such viral peptides may have been activated by dendritic cells earlier in

1	multiple viral proteins into peptides for display on MHC class II molecules on the B-cell surface. CD4 T cells specific for such viral peptides may have been activated by dendritic cells earlier in the infection, and some will have differentiated into TFH cells. When these TFH cells are activated by B cells presenting their peptide, they are stimulated to provide specific signals that help B cells to generate antibodies against the viral coat protein (Fig. 10.4).

1	Linked recognition relies on the concentration of the appropriate peptide for presentation by MHC class II molecules on the B-cell surface. B cells whose B-cell receptor binds a particular antigen are 10,000 times more efficient at displaying peptide fragments of that antigen on their MHC class II molecules than are B cells that process the antigen through macropinocytosis alone. Linked recognition was originally discovered through studies of the production of antibodies against haptens, which are small chemical groups that cannot elicit antibody responses on their own (see Appendix I, Section A-1). But haptens that are coupled to a carrier protein become immunogenic—known as the hapten carrier effect—for two reasons. The protein can carry multiple hapten groups, allowing it to cross-link B-cell receptors. Also, T cells that are activated against peptides of the carrier protein can become TFH cells and strengthen the antibody response to the hapten. Accidental coupling of a hapten to

1	B-cell receptors. Also, T cells that are activated against peptides of the carrier protein can become TFH cells and strengthen the antibody response to the hapten. Accidental coupling of a hapten to a protein is responsible for the allergic responses shown by many people to the antibiotic penicillin, which reacts with host proteins to form a coupled hapten that can stimulate an antibody response, as we will learn in Chapter 14.

1	Linked recognition works to preserve self-tolerance, since autoreactive antibodies will arise only if self-reactive TFH and self-reactive B cells are present at the same time. This is discussed further in Chapter 15. Vaccine design can take advantage of linked recognition, as in the vaccine used to immunize infants against Haemophilus influenzae type b (see Section 16-26). 10-3 B cells that encounter their antigens migrate toward the lymphoid tissues.

1	10-3 B cells that encounter their antigens migrate toward the lymphoid tissues. The frequency of naive lymphocytes specific for a particular antigen is extremely low (less than 1 in 10,000). Thus, the chance of a random encounter between a T and a B cell with the same antigen specificity should be less than 1 in 108, making it remarkable that B cells ever interact with TFH cells with similar antigen specificity. For these reasons, linked recognition requires a precise regulation of the migration of activated B and T cells—orchestrated by several sets of ligands and receptors—into specific locations within the lymphoid tissues, which serves to increase the chances of a productive interaction (Fig. 10.5).

1	Naive T cells and B cells express the sphingosine 1-phosphate receptor, S1PR1, which they use to egress from the peripheral lymphoid tissues (see Section 9-7). However, before they exit, they are retained and initially occupy two distinct zones, the T-cell areas and the primary lymphoid follicles (or B-cell areas or B-cell zones), respectively (see Figs 1.18–1.20). These zones are established by different patterns of chemokine receptor expression and chemokine production. Naive T cells express the chemokine receptor CCR7, and localize to zones where its ligands, CCL19 and CCL21, are highly expressed by stromal cells and dendritic cells (see Section 9-3). Circulating naive B cells express CXCR5, and when they migrate into lymphoid tissues, they enter the primary lymphoid follicles, where the chemokine CXCL13 is abundant. Within the follicle, stromal cells and a specialized cell type, the follicular dendritic cell (FDC), secrete CXCL13. The FDC is a nonphagocytic cell of

1	where the chemokine CXCL13 is abundant. Within the follicle, stromal cells and a specialized cell type, the follicular dendritic cell (FDC), secrete CXCL13. The FDC is a nonphagocytic cell of nonhematopoietic origin that bears numerous long processes; it functions by trapping antigen using complement receptors on its cell surface for access by B cells in the follicle.

1	Once in the follicle, naive B cells encounter the soluble TNF-family cytokine BAFF (see Section 8-8), which is secreted by FDCs, stromal cells, and dendritic cells and which acts as a survival factor for B cells. BAFF can act through three

1	Fig. 10.4 T cells and B cells must recognize antigens contained within the same molecular complex in order to interact. In this example, an internal viral protein harbors a peptide epitope (shown as red) that is presented by MHC class II molecules and is recognized by a CD4 T cell. The virus also harbors a native epitope on an external viral coat protein (shown as blue) that is recognized by the surface immunoglobulin on a B cell. If the virus is captured and presented by a dendritic cell, a peptide-specific CD4 T cell (blue) becomes activated (top left panel), whereas nonspecific T cells (green) remain inactive. If the virus is recognized by a specific B cell (top right panel), peptides derived from internal viral proteins are processed and presented by MHC class II molecules. When the activated T cell recognizes its peptide on this B cell (bottom panel), the T cell will deliver various accessory signals to the B cell that promote antibody production against the coat protein. This

1	activated T cell recognizes its peptide on this B cell (bottom panel), the T cell will deliver various accessory signals to the B cell that promote antibody production against the coat protein. This process is known as linked recognition.

1	Fig. 10.5 Antigen-binding B cells meet T cells at the border between the T-cell area and a B-cell follicle in secondary lymphoid tissues. Antigens enter the spleen from the blood and collect in T-cell zones and follicles (first panel). Naive CCR7-positive T cells and CXCR5-positive B cells migrate to distinct regions where the chemokines CCL19 and CCL21, or CXCL13 and 7α, 25-hydroxycholesterol (7α, 25-HC), respectively, are being produced (second panel). If a B cell encounters its antigen, either on a follicular dendritic cell (FDC) or a macrophage, it increases expression of CCR7 and migrates toward the border with the T-cell zone (third panel). T cells activated by antigen-presenting dendritic cells induce expression of CXCR5 and migrate to this same border, where linked recognition induces further B-cell proliferation. After 2 to 3 days, B cells reduce expression of CCR7, but retain EBI2 and migrate in response to 7α, 25-HC to the outer follicle and interfollicular regions (fourth

1	further B-cell proliferation. After 2 to 3 days, B cells reduce expression of CCR7, but retain EBI2 and migrate in response to 7α, 25-HC to the outer follicle and interfollicular regions (fourth panel). After another day or so, some B cells cluster in the interfollicular regions near the red pulp, proliferate, and differentiate into plasmablasts, forming a primary focus with terminal differentiation into antibody-secreting plasma cells. T cells that retain EBI2 expression may remain in the follicle and induce Bcl-6 expression to become TFH cells that participate with B cells there to form a germinal center reaction.

1	Some B cells migrate to form a primary focus and differentiate into plasmablasts, while some T cells induce Bcl-6 and become TFH cells TFH primary focus interfollicular zone CCL21 CXCL13 7˜,25-HC resting T cells resting B cells FDC dendritic cell Interactions with T cells sustain EBI2 expression on B cells, which move to outer follicular and interfollicular regions Activated B cells induce CCR7 and EBI2, and T cells induce CXCR5, and both cells migrate to follicular and interfollicular regions Before activation, resting B cells express CXCR5 and reside in follicles and T cells express CCR7 and reside in T-cell zones activated B cells activated T cells receptors, but its major actions in promoting survival are through BAFF-R (Fig. 10.6). BAFF-R signals through TRAF3 (see Section 3-7) to activate the non-canonical NFκB pathway, as described for CD40 (see Fig. 7.31), and, like CD40 signaling, induces expression of Bcl-2. Two other receptors for BAFF are TACI and BCMA, although BAFF has a

1	the non-canonical NFκB pathway, as described for CD40 (see Fig. 7.31), and, like CD40 signaling, induces expression of Bcl-2. Two other receptors for BAFF are TACI and BCMA, although BAFF has a relatively low affinity for BCMA. TACI and BCMA also bind the related cytokine APRIL, and they signal through TRAF2, 5, and 6 to induce signaling pathways involved in B-cell activation.

1	Antigens derived from microorganisms and viruses are transported into lymph nodes via the afferent lymph, and into the spleen via the blood. Opsonized antigens bearing C3b or C3dg accumulate in the B-cell follicles because they are trapped by complement receptors CR1 and CR2 expressed on the surface of FDCs. Opsonized particulate antigens can also be taken up by specialized macrophages residing in the subcapsular sinus (SCS) of lymph nodes and the marginal sinus of the spleen, regions that are both adjacent to the B-cell

1	BAFF (B-cell activating factor, also called B-lymphocyte stimulator, or BLyS) and APRIL (a proliferation-inducing ligand) are both members of the TNF superfamily of cytokines. They are initially produced as membrane-bound trimers by several cell types. BAFF is produced by FDCs and other cells in the B-cell follicle, where it supports B-cell survival. Its main receptor, BAFF-R, signals in a manner similar to CD40 (see Fig. 7.31) through TRAF3 and NIK to activate both the non-canonical NFκB pathway, leading to the RelB:p52 transcription factor, and the canonical p50:p65 NFκB pathway. BAFF also binds to the receptors TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) and BCMA (B-cell maturation antigen), although its affinity for the latter is relatively weak. These receptors activate the canonical NFκB pathway. The low endocytic and Antigen preservation by

1	Opsonized antigens entering lymph degradative activity of subcapsular macrophages nodes from afferent lymphatics bind subcapsular macrophages also allows antigen to be to the complement receptors on the preserves the antigens transported into the follicle surface of macrophages present in trapped on their surfaces, to become localized on the the subcapsular sinus allowing B cells to surface of follicular follicles (Fig. 10.7). These macrophages seem to retain the antigen on their surface rather than ingesting and degrading it. These antigens can then be sampled and carried by antigen-specific follicular B cells. B cells of any antigen specificity could also acquire antigen from these macrophages via their complement receptors and transport it within the follicle. In the spleen, marginal zone B cells shuttle between that site and the follicle, carrying antigen trapped in the marginal zone for deposition on FDCs. SCS macrophages can also actively function to restrict the dissemination

1	zone B cells shuttle between that site and the follicle, carrying antigen trapped in the marginal zone for deposition on FDCs. SCS macrophages can also actively function to restrict the dissemination of infection. In mice, infection of these macrophages in lymph nodes by vesicular stomatitis virus (VSV), a relative of rabies virus, triggers the cells to produce interferon and to recruit plasmacytoid dendritic cells (pDCs). Type I interferon produced by pDCs restricts further viral spread, which would otherwise eventually pass on to the central nervous system.

1	After a naive follicular B cell first encounters specific antigen displayed by FDCs or macrophages, within a few hours it will become positioned in the outer follicles of lymphoid tissue close to the sites where antigen enters the lymph node or spleen. This positioning is orchestrated by the B cell’s expression of a chemokine receptor, EBI2 (GPR183), whose ligands are oxysterols such as 7α, 25-dihydroxycholesterol. The precise source of these ligands is still unclear, but they are abundant in the outer follicular and interfollicular regions. After sampling antigens there for 6 hours to 1 day, the B cell induces expression of CCR7, which functions together with EBI2 to distribute activated B cells along the interface between the B-cell follicle and the T-cell zone, where CCL21 is expressed.

1	During an immune response, T cells are activated within the T-cell zones by dendritic cells. When naive T cells are activated, some will proliferate, differentiate into effector cells, downregulate expression of S1P1, and exit the lymphoid tissue. However, others will induce expression of CXCR5 and migrate to the border with the B-cell follicle. There, T cells can encounter B cells activated during the same response, increasing the chance that they might recognize linked antigens presented by activated B cells that have recently moved to this location (see Fig. 10.5).

1	Fig. 10.7 Opsonized antigens are captured and preserved by subcapsular sinus macrophages. Macrophages residing in the lymph node subcapsular sinus (SCS) express complement receptors 1 and 2 (CR1 and CR2, respectively), are poorly endocytic, and have reduced levels of lysosomal enzymes compared with macrophages in the medulla. Opsonized antigen arriving from the afferent lymphatics binds to CR1 and CR2 on the surface of SCS macrophages. Instead of being completely degraded by these macrophages, some antigen is retained on the cell surface, where it can be presented and transferred to the surface of follicular B cells. B cells are then able to transport the antigen into the follicle, where it can be trapped on the surfaces of follicular dendritic cells.

1	The SLAM receptor family members SLAM, Ly108, and CD84 are expressed on T cells and B cells and mediate homotypic interactions that lead to adhesion between cells. SLAM can also enhance signaling by the T-cell receptor to augment production of cytokines such as IL-21 that help B cells. The SLAM-associated molecule SAP is a signaling adapter that is required for one SLAM receptor to sustain binding with another. T cells initially express SAP at low levels that are insufficient for sustained adhesion between T and B cells. Fully differentiated TFH cells express high levels of the transcription factor Bcl-6, which induces higher levels of SAP expression. This level is sufficient to sustain cell–cell interactions and allow for the delivery of CD40L and cytokine signals to B cells. 10-4 T cells express surface molecules and cytokines that activate B cells, which in turn promote TFH-cell development.

1	When TFH cells encounter an activating peptide presented by B cells, the TFH cells respond by expressing receptors and cytokines that in turn activate B cells. As mentioned above, the induced expression of CD40L on TFH cells activates CD40 on B cells to increase B-cell survival, and also induces B-cell expression of co-stimulatory molecules, especially those of the B7 family. Activated T cells also express CD30 ligand (CD30L), which binds to CD30 expressed on B cells and promotes B-cell activation. Mice lacking CD30 show reduced proliferation of activated B cells in lymphoid follicles and weaker secondary humoral responses than normal. TFH cells also secrete several cytokines that regulate B-cell proliferation and antibody production. Primary among these is IL-21, which is produced early in immune responses by TFH cells and which activates the transcription factor STAT3 in B cells to support proliferation and differentiation. IL-21 exerts similar autocrine effects on TFH cells. Later

1	in immune responses by TFH cells and which activates the transcription factor STAT3 in B cells to support proliferation and differentiation. IL-21 exerts similar autocrine effects on TFH cells. Later in the antibody response, TFH cells will produce other cytokines, such as IL-4 and IFN-γ, that are characteristic of the other T helper subsets (described in Chapter 9). These will impact B-cell differentiation, particularly class switching, as we discuss later.

1	The ability of TFH cells to successfully deliver these signals to B cells depends on intimate contact between these cells. Specific adhesion molecules, including several Ig superfamily receptors of the SLAM (signaling lymphocyte activation molecule) family, are involved that prolong and stabilize cell–cell contact. TFH cells and B cells both express SLAM (CD150), CD84, and Ly108, which promote cell adhesion through homotypic binding interactions (Fig. 10.8). The cytoplasmic regions of these SLAM family receptors all interact with an adaptor protein, SAP (SLAM-associated protein), which is expressed highly by TFH cells and which is necessary for prolonging cell–cell contact mediated by these receptors. The SAP gene is inactivated in X-linked lymphoproliferative syndrome, which is associated with a T-cell and NK-cell lymphoproliferative disorder and with a defect in antibody production due to failed interactions between TFH cells and B cells in the germinal center, discussed below. The

1	with a T-cell and NK-cell lymphoproliferative disorder and with a defect in antibody production due to failed interactions between TFH cells and B cells in the germinal center, discussed below. The regulated migration of activated B cells and TFH cells to the same location in the peripheral lymphoid organ increases the chance that linked recognition will occur and deliver appropriate help for B-cell differentiation. Antigen-stimulated B cells that fail to interact with T cells that recognize the same antigen die within 24 hours.

1	This first interaction between T and B cells not only provides important help to B cells, but also influences T-cell differentiation by signals provided by the B cell. Activated B cells express ICOSL, a member of the B7 family of co-stimulatory molecules and a ligand for ICOS (inducible co-stimulatory protein), which is expressed by T cells. This Tand B-cell interaction, provided by linked recognition, activates ICOS signaling in T cells and is important for the completion of TFH differentiation (see Section 7-21), leading to induction of the transcription factors Bcl-6 and c-Maf. These transcription factors are required for SAP production and the consequent sustained contact between B and TFH cells. 10-5 Activated B cells differentiate into antibody-secreting plasmablasts and plasma cells.

1	After their initial encounter, B cells that have received T-cell help migrate from the follicle border to continue to proliferate and differentiate. Two to three days after activation, B cells begin to decrease expression of CCR7 and to increase expression of EBI2 (see Fig. 10.5). Decreased expression of CCR7 causes B cells to move away from the boundary with the T-cell zone: EBI2 directs their migration back to the interfollicular regions and the subcapsular sinus in the lymph nodes, or, in the spleen, to the splenic bridging channels, a region between the T-cell area and the red pulp. Here, some B cells will form an emerging aggregate of differentiating B cells called the primary focus, which in lymph nodes is located in the medullary cords, where lymph drains out of the node, and in the spleen can be seen as extrafollicular foci in the splenic red pulp. Primary foci are apparent by about 5 days after an infection or immunization with an antigen not previously encountered.

1	B cells proliferate in the primary focus for several days, and this constitutes the first phase of the primary humoral immune response. Some of these proliferating B cells differentiate into antibody-synthesizing plasmablasts in the primary focus. Not all B cells activated by the initial interaction with TFH cells will move into the primary focus. Some will migrate into the lymphoid follicle, where they may eventually differentiate into plasma cells, as described below. Plasmablasts are cells that have begun to secrete antibody, yet are still dividing and express many of the characteristics of activated B cells that allow their interaction with T cells. After a few more days, the plasmablasts in the primary focus stop dividing and may eventually die. Subsequently, long-lived plasma cells will develop and migrate to the bone marrow, where they will continue antibody production. Since many long-lived plasma cells are generated long after the primary focus has dissipated, it is likely

1	will develop and migrate to the bone marrow, where they will continue antibody production. Since many long-lived plasma cells are generated long after the primary focus has dissipated, it is likely that they do not arise directly from plasmablasts in the primary focus, but rather from B cells that entered the germinal center reaction.

1	The properties of resting B cells, plasmablasts, and plasma cells are compared in Fig. 10.9. The differentiation of a B cell into a plasma cell is accompanied by many morphological changes that reflect a commitment to the production of large amounts of secreted antibody, which can constitute up to 20% of all the protein synthesized by a plasma cell. Plasmablasts and plasma cells have a prominent perinuclear Golgi apparatus and an extensive rough endoplasmic reticulum that is rich in immunoglobulin molecules that are being synthesized and exported into the lumen of the endoplasmic reticulum for secretion. Plasmablasts have relatively large numbers of B-cell receptors on the cell surface, whereas plasma cells have many fewer. This low level of surface immunoglobulin on plasma cells may still be physiologically important, since their survival seems to be determined in part by their ability to continue to bind antigen. Plasmablasts still express B7 co-stimulatory molecules and MHC class

1	be physiologically important, since their survival seems to be determined in part by their ability to continue to bind antigen. Plasmablasts still express B7 co-stimulatory molecules and MHC class II molecules; by contrast, plasma cells turn down the expression of MHC class II molecules. Nevertheless, T cells still provide important signals for plasma-cell differentiation and survival, such as IL-6 and CD40 ligand.

1	Fig. 10.9 Plasma cells secrete antibody at a high rate but can no longer respond to antigen. Resting naive B cells have membrane-bound immunoglobulin (usually IgM and IgD) and MHC class II molecules on their surface. Although their V genes do not carry somatic mutations, B cells can take up antigen and present it to helper T cells. The T cells in return induce the B cells to proliferate and to undergo isotype switching and somatic hypermutation, but B cells do not secrete significant amounts of antibody during this period. Plasmablasts have an intermediate phenotype. They secrete antibody but retain substantial surface immunoglobulin and MHC class II molecules and so can continue to take up and present antigen to T cells. Plasmablasts early in the immune response and those activated by T-independent antigens have usually not undergone somatic hypermutation and class switching, and therefore secrete IgM. Plasma cells are terminally differentiated cells that secrete antibodies. Plasma

1	antigens have usually not undergone somatic hypermutation and class switching, and therefore secrete IgM. Plasma cells are terminally differentiated cells that secrete antibodies. Plasma cells have very low levels of surface immunoglobulin but can express MHC class II molecules and may suppress TFH activity in a negative feedback pathway while differentiating. Early in the immune response they differentiate from unswitched activated B cells and secrete IgM; later in the response they derive from activated B cells that entered the germinal center reaction and underwent class switching and somatic hypermutation. Plasma cells have lost the ability to change the class of their antibody or undergo further somatic hypermutation.

1	Naive B cells travel to the lymph node via the bloodstream and leave via the efferent lymph Plasma cells migrate to the medullary cords or leave via the efferent lymphatics HEVfollicle germinal center primary focus B cells that encounter antigen in the follicle form a primary focus. Some proliferating B cells migrate into the follicle to form a germinal center Plasma cells migrate to the bone marrow bone marrow stromal cell Recent evidence indicates that even the low level of MHC class II molecules expressed on plasma cells functions to present cognate antigen to TFH cells, and acts to suppress IL-21 production and Bcl-6 expression, thus acting as a feedback pathway to regulate ongoing B-cell responses. While some plasma cells survive for only days to a few weeks after their final differentiation, others are very long lived and account for the persistence of antibody responses. 10-6 The second phase of a primary B-cell immune response proliferate to form germinal centers.

1	10-6 The second phase of a primary B-cell immune response proliferate to form germinal centers. Not all B cells activated by TFH cells will migrate to the outer follicle and eventually establish a primary focus. Instead, some move into a primary lymphoid follicle together with their associated TFH cells (Fig. 10.10), where they continue to proliferate and ultimately form a germinal center; follicles with germinal centers are also called secondary lymphoid follicles. Downregulating EBI2 by B cells appears to favor germinal center formation. In mice lacking EBI2 expression in B cells, antigen-activated B cells remain near the border with the T-cell zone and are able to form germinal centers, but generate fewer plasmablasts.

1	Germinal centers are composed mainly of proliferating B cells, but antigen-specific T cells make up about 10% of germinal center lymphocytes and provide indispensable help to the B cells. The germinal center is an area of active cell division that forms within a surrounding region of resting B cells in the primary follicle. Proliferating germinal center B cells displace the resting B cells toward the periphery of the follicle, forming a mantle zone of resting cells around the two distinguishable areas of activated B cells, called the light zone and the dark zone (Fig. 10.11, left panel). The germinal center grows in size as the immune response proceeds, and then shrinks and finally disappears when the infection is cleared. Germinal centers are present for about 3–4 weeks after initial antigen exposure.

1	The primary focus and the germinal center reaction differ in the quality of antibody that they produce. Plasmablasts, germinal center B cells, and early memory B cells begin to emerge during the first 4–5 days of an immune response. Plasmablasts in primary foci primarily secrete antibodies of the IgM isotype that offer some immediate protection. In contrast, B cells in the germinal center reaction undergo several processes that produce antibodies that are more effective in eliminating infections. These processes include somatic hypermutation, which alters the V regions of immunoglobulin genes (see below), and which enables a process called affinity maturation, which selects for the survival of mutated B cells that have a high affinity for the antigen.

1	Fig. 10.10 Activated B cells form germinal centers in lymphoid follicles. Activation of B cells in a lymph node is shown here. Top panel: naive circulating B cells enter lymph nodes from the blood via high endothelial venules (HEV) and are attracted by chemokines into the primary lymphoid follicle; if these B cells do not encounter antigen in the follicle, they leave via the efferent lymphatic vessel. Second panel: B cells that have bound antigen move to the border with the T-cell area, where they may encounter activated helper T cells specific for the same antigen; these T cells interact with the B cells and activate them to start proliferation and differentiation into plasmablasts. Some B cells activated at the T-cell–B-cell border migrate to form a primary focus of antibody-secreting plasmablasts in the interfollicular regions (spleen) or medullary cords (lymph nodes), whereas others move back into the follicle, where they continue to proliferate and form a germinal center.

1	plasmablasts in the interfollicular regions (spleen) or medullary cords (lymph nodes), whereas others move back into the follicle, where they continue to proliferate and form a germinal center. Germinal centers are sites of sustained B-cell proliferation and differentiation. Follicles in which germinal centers have formed are known as secondary follicles. Within the germinal center, B cells begin their differentiation into either antibody-secreting plasma cells or memory B cells. Third and fourth panels: plasma cells leave the germinal center and migrate to the medullary cords, or leave the lymph node altogether via the efferent lymphatics and migrate to the bone marrow.

1	Schematic representation of a follicle Cyclic reentry of cells into the dark zone with a germinal center is dependent on reexpression of CXCR4 on centrocytes Fig. 10.11The structure of a germinal center. The germinal center is a specialized microenvironment in which B-cell proliferation, somatic hypermutation, and selection for strength of antigen binding all occur. Closely packed centroblasts, which express CXCR4 and CXCR5, form the ‘dark zone’ of the germinal center; the less densely packed ‘light zone’ contains centrocytes, which express only CXCR5. Stromal cells in the dark zone produce CXCL12, which attracts the CXCR4-expressing centroblasts. Cyclic reentry describes the process by which B cells can lose and gain expression of CXCR4 and thus move from the light zone to the dark zone and back again.

1	In addition, class switching allows the selected B cells to produce antibodies with a variety of effector functions. These B cells will differentiate either into plasma cells that secrete higher-affinity and class-switched antibody in the latter part of the primary immune response, or into memory B cells as described in Chapter 11.

1	B cells in the germinal center divide rapidly, every 6–8 hours. Initially, these rapidly proliferating B cells, called centroblasts, express the chemokine receptors CXCR4 and CXCR5 but markedly reduce their expression of surface immunoglobulin, particularly of IgD. Centroblasts proliferate in the dark zone of the germinal center, named for its densely packed appearance (Fig. 10.12). Stromal cells in the dark zone produce CXCL12 (SDF-1), a ligand for CXCR4 that acts to retain centroblasts in this region. As time goes on, some centroblasts reduce their rate of cell division, enter the growth phase, pausing at the G2/M phase of the cell cycle, reduce CXCR4 expression, and begin to produce higher levels of surface immunoglobulin. These B cells are termed centrocytes. The loss of CXCR4 allows centrocytes to move into the light zone, a less densely packed area containing abundant FDCs that produce the chemokine CXCL13 (BLC), a ligand for CXCR5 (see Fig. 10.11, right panel). The B cells

1	centrocytes to move into the light zone, a less densely packed area containing abundant FDCs that produce the chemokine CXCL13 (BLC), a ligand for CXCR5 (see Fig. 10.11, right panel). The B cells proliferate in the light zone, but to a lesser extent than in the dark zone.

1	Fig. 10.12Germinal centers are sites of intense cell proliferation and cell death. The photomicrograph (first panel) shows a section through a human tonsillar germinal center. Closely packed centroblasts, seen in the lower part of this photomicrograph, form the so-called dark zone of the germinal center. Above this region is the less densely packed light zone. The second panel shows immunofluorescent staining of a germinal center. B cells are found in the dark zone, light zone, and mantle zone. Proliferating cells are stained green for Ki67, an antigen expressed in nuclei of dividing cells, revealing the rapidly proliferating centroblasts in the dark zone. The dense network of follicular dendritic cells, stained red, mainly occupies the light zone. Centrocytes in the light zone proliferate to a lesser degree than centroblasts. Small recirculating B cells occupy the mantle zone at the edge of the B-cell follicle. Large masses of CD4 T cells, stained blue, can be seen in the T-cell

1	to a lesser degree than centroblasts. Small recirculating B cells occupy the mantle zone at the edge of the B-cell follicle. Large masses of CD4 T cells, stained blue, can be seen in the T-cell zones, which separate the follicles. There are also significant numbers of T cells in the light zone of the germinal center; CD4 staining in the dark zone is associated mainly with CD4-positive phagocytes, that digest B cells that die there. Photographs courtesy of I. MacLennan.

1	Light micrograph of germinal center (high power) Germinal center stained to show T cells, follicular dendritic cells, and proliferating B cells MOVIE 10.1

1	MOVIE 10.1 Fig. 10.13 The primary antibody repertoire is diversified by three processes that modify the rearranged immunoglobulin gene. First panel: the primary antibody repertoire is initially composed of IgM-containing variable regions (red) produced by V(D)J recombination and constant regions (blue) from the μ gene segment. The range of reactivity of this primary repertoire can be further modified by somatic hypermutation, by class switch recombination at the immunoglobulin loci, and in some species by gene conversion (not shown). Second panel: somatic hypermutation results in mutations (shown as black lines) being introduced into the heavy-chain and light-chain V regions (red), altering the affinity of the antibody for its antigen. Third panel: in class switch recombination, the initial μ heavy-chain C regions (blue) are replaced by heavy-chain regions of another isotype (shown as yellow), modifying the effector activity of the antibody but not its antigen specificity.

1	hypermutation, and cells with mutations that improve affinity for antigen are selected. Somatic hypermutation introduces mutations that change anywhere from one to a few amino acids in the immunoglobulin, producing closely related B-cell clones that differ subtly in specificity and antigen affinity (Fig. 10.13). These mutations in the V genes are initiated by an enzyme called activation-induced cytidine deaminase, or AID, which is expressed only by germinal center B cells. Before describing the enzymatic mechanisms initiated by AID, we first present a general overview of this process in which random mutations can improve antibody affinity.

1	The immunoglobulin V-region genes accumulate mutations at a rate of about one base pair change per 103 base pairs per cell division, while the rate of mutations in the rest of the cell’s DNA is much lower: around one base pair change per 1010 base pairs per cell division. Somatic hypermutation also affects some DNA flanking the rearranged V gene, but does not generally extend into the C-region exons. Since each V region is encoded by about 360 base pairs and about three out of every four base changes will alter the amino acid encoded, there is about a 50% chance during each B-cell division that a mutation will occur to the receptor.

1	The point mutations accumulate in a stepwise manner as the descendants of each B cell proliferate in the germinal center to form B-cell clones (Fig. 10.14). An altered receptor can affect the ability of a B cell to bind antigen and thus will affect the fate of the B cell in the germinal center. Most mutations have a negative impact on the ability of the B-cell receptor to bind the original antigen, by preventing the correct folding of the immunoglobulin molecule or by blocking the complementarity-determining regions from binding antigen. Detrimental mutations may alter conserved framework regions (see Fig. 4.7) and disrupt basic immunoglobulin structure. Cells that harbor such detrimental mutations are eliminated by apoptosis in a process of negative selection, either because they can no longer make a functional B-cell receptor or because they cannot take up antigen as well as sibling B cells (Fig. 10.15). Germinal centers are filled with apoptotic B cells that are quickly engulfed by

1	make a functional B-cell receptor or because they cannot take up antigen as well as sibling B cells (Fig. 10.15). Germinal centers are filled with apoptotic B cells that are quickly engulfed by macrophages, giving rise to the characteristic tingible body macrophages. These contain dark-staining nuclear debris in their cytoplasm. Negative selection is implied by the relative scarcity of amino acid replacements in the framework regions, reflecting the loss of cells that had mutated any one of the many residues that are critical for immunoglobulin V-region folding. This process prevents rapidly dividing B cells from expanding to numbers that would overwhelm the lymphoid tissues. Less frequently, mutations may improve the affinity of a B-cell receptor for antigen, and these mutations will be selectively expanded (see Fig. 10.15) because the cells expressing receptors with such mutations will have an increased survival rate compared with low-affinity cells. Positive selection is evident in

1	expanded (see Fig. 10.15) because the cells expressing receptors with such mutations will have an increased survival rate compared with low-affinity cells. Positive selection is evident in the accumulation of numerous amino acid replacements in the complementarity-determining regions, which determine antibody specificity

1	Fig. 10.14 Somatic hypermutation introduces mutations into the rearranged immunoglobulin variable (V) regions that improve antigen binding. The process of somatic hypermutation can be tracked by sequencing immunoglobulin V regions from hybridomas (clones of antibody-producing cells; see Appendix I, Section A-7) established at different time points after the experimental immunization of mice. The result of one experiment is depicted here. Each V region sequenced is represented by a horizontal line. The complementarity-determining regions CDR1, CDR2, and CDR3 are shown by pink shading. Mutations that change the amino acid sequence are represented by red bars. Within a few days of immunization, the V regions within a particular clone of responding B cells begin to acquire mutations, and over the course of the next week more mutations accumulate (top panels). B cells whose V regions have accumulated deleterious mutations and can no longer bind antigen die. B cells whose V regions have

1	the course of the next week more mutations accumulate (top panels). B cells whose V regions have accumulated deleterious mutations and can no longer bind antigen die. B cells whose V regions have acquired mutations that improve the affinity of the B-cell receptor for antigen are able to compete more effectively for antigen, and receive signals that drive their proliferation and expansion. The antibodies they produce also have this improved affinity. This process of mutation and selection can continue in the lymph node germinal center through multiple cycles in response to secondary and tertiary immune responses elicited by further immunization with the same antigen (center and bottom panels). In this way, the antigen-binding efficiency of the antibody response is improved over time.

1	and affinity (see Fig. 10.14), a process we discuss in the next section. The result of selection for enhanced binding to antigen is that the nucleotide changes that alter amino acid sequences, and thus protein structure, tend to be clustered in the CDRs of the immunoglobulin V-region genes, whereas silent, or neutral, mutations that preserve amino acid sequence and do not alter protein structure are scattered throughout the V region.

1	Fig. 10.15 Selection for high-affinity mutants in the germinal center relies on help provided by TFH cells. After activated B cells interact with TFH cells at the follicle border, they migrate to germinal centers (GCs), where the following events depicted here occur. In the dark zone of the GC, somatic hypermutation alters the immunoglobulin V regions (first panel). In some B cells (yellow), the mutated B-cell receptor (BCR) will have low or no affinity for the antigen, while in other B cells (orange) the mutated BCR affinity may be higher. After exiting the dark zone, the B cells with higher-affinity BCRs will capture antigen (red) trapped on follicular dendritic cells (FDCs) and then process and present it on MHC class II molecules (second panel). B cells with low-affinity BCRs will fail to capture and present antigen. B cells that present linked antigen epitopes to TFH cells will receive help through CD40L and IL-21, which promote survival and proliferation. B cells that lack

1	fail to capture and present antigen. B cells that present linked antigen epitopes to TFH cells will receive help through CD40L and IL-21, which promote survival and proliferation. B cells that lack antigen on MHC class II molecules receive no help and will eventually die (third panel). Some of the proliferating B cells undergo repeated cycles of entry to the dark zone, mutation, and selection (fourth panel), and other progeny B cells undergo differentiation to either memory B cells or plasma cells (not shown).

1	Fig. 10.16 Antigens are trapped in immune complexes that bind to the surface of follicular dendritic cells. Radiolabeled antigen localizes to, and persists in, lymphoid follicles of draining lymph nodes (see the light micrograph and the schematic representation (middle panel), showing a germinal center in a lymph node). The intense dark staining shows the localization in the germinal center of radiolabeled antigen that had been injected 3 days previously. The antigen is in the form of antigen:antibody:complement complexes bound to Fc receptors and to complement receptors CR1 or CR2 on the surface of the follicular dendritic cell (FDC), as depicted in the right-hand panel and inset. These complexes are not internalized, as such antigen can persist in this form for long periods. Photograph courtesy of J. Tew. 10-8 Positive selection of germinal center B cells involves contact with TFH cells and CD40 signaling.

1	Selection of B cells with improved affinity for antigen occurs in increments. It was originally discovered in vitro that resting B cells could be kept alive by simultaneously cross-linking their B-cell receptors and ligating their cell-surface CD40. In vivo these signals are delivered by antigen and by TFH cells, respectively. The details of selection in the germinal center have become more clear recently from in vivo two-photon microscopic studies (see Appendix I, A-10) that show that positive selection of a B cell depends on the B cell’s ability to take up antigen, and to receive signals delivered by TFH cells. It is thought that somatic hypermutation occurs in the centroblasts in the dark zone; when a centroblast reduces its rate of proliferation and becomes a centrocyte, it increases the number of B-cell receptors on its surface and moves to the light zone, where there are abundant FDCs. Antigen can be trapped and stored for long periods in the form of immune complexes on FDCs

1	the number of B-cell receptors on its surface and moves to the light zone, where there are abundant FDCs. Antigen can be trapped and stored for long periods in the form of immune complexes on FDCs (Fig. 10.16 and Fig. 10.17). The centrocyte’s ability to bind antigen determines its relative ability to acquire

1	Fig. 10.17 Immune complexes bound to FDCs form iccosomes, which are released and can be taken up by B cells in the germinal center. FDCs have a prominent cell body and many dendritic processes. Immune complexes, bound to complement receptors and Fc receptors on the FDC surface, become clustered, forming prominent ‘beads’ along the dendrites (a). An intermediate form of FDC is shown, which has both straight filiform dendrites and others that are becoming beaded. These beads are shed from the cell as iccosomes (immune complex-coated bodies), which can bind to a B cell in the germinal center (b) and be taken up by it (c). In panels b and c, the iccosome has been formed with immune complexes containing horseradish peroxidase, which is electron-dense and therefore appears dark in the transmission electron micrographs. Photographs courtesy of A.K. Szakal.

1	antigen, in competition with the other clonally related centrocytes harboring different mutations. Centrocytes whose receptors bind antigen better will capture and present more peptides on their surface MHC class II molecules. TFH cells in the germinal center recognize these peptides and, as before, are activated to deliver signals to the B cell that promote survival. Centrocytes whose mutations reduce antigen-binding affinity will take up less antigen, and so will receive weaker survival signals from TFH cells. Successful B cells will reexpress CXCR4 and return to the dark zone, where they will undergo additional rounds of division, in effect becoming centroblasts again. Germinal center B cells that fail to acquire sufficient antigen from FDCs to engage TFH cells will become apoptotic and be lost. This process of B-cell migration within the germinal center is known as the cyclic reentry model (see Fig. 10.11, right panel). In this way, the affinity and specificity of B cells are

1	and be lost. This process of B-cell migration within the germinal center is known as the cyclic reentry model (see Fig. 10.11, right panel). In this way, the affinity and specificity of B cells are continually refined during the germinal center response, through affinity maturation (see Section 10-6). The selection process can be quite stringent: although 50–100 B cells may seed the germinal center, most of them leave no progeny, and by the time the germinal center reaches maximum size, it is typically composed of the descendants of only one or a few B cells.

1	In the germinal center, TFH cells and B cells interact to deliver signals that are important for both cells (see Section 10-4). Mice that lack ICOS are deficient in the germinal center reaction and have severely reduced class-switched antibody responses due to defective TFH-cell function. CD40 signaling in B cells is activated by CD40L on TFH cells and increases expression of the survival molecule Bcl-XL, a relative of Bcl-2. These interactions also include signaling by SLAM family receptors through the adapter protein SAP, as discussed above. Two-photon intravital microscopy has revealed that mice lacking the SLAM receptor CD84 have reduced numbers of conjugates between antigen-specific T cells and B cells in germinal centers, and these mice also have a reduced humoral response to antigen. 10-9 Activation-induced cytidine deaminase (AID) introduces mutations into genes transcribed in B cells.

1	10-9 Activation-induced cytidine deaminase (AID) introduces mutations into genes transcribed in B cells. Now that we have discussed the cellular processes involved in somatic hypermutation and affinity maturation, we will delve into the details of the mutation process itself. The enzyme AID is important for both somatic hyper-mutation and class switch recombination, as mice lacking AID have defects in both processes. People with mutations in the AID gene that inactivate the enzyme—that is, have activation-induced cytidine deaminase deficiency, or AID deficiency—also lack both somatic hypermutation and class switching. This condition leads to the production of predominantly IgM antibodies and the absence of affinity maturation, a syndrome known as hyper IgM type 2 immunodeficiency (discussed in Chapter 13).

1	AID is related to enzymes that deaminate cytosine to uracil in making nucleotide precursors for RNA and DNA synthesis. Its closest homolog, APOBEC1 (apolipoprotein B mRNA editing catalytic polypeptide 1), is an RNA-editing enzyme that deaminates cytosine in the context of RNA. However, AID fulfills its activity in antibody gene diversification by acting on cytosine in the DNA of the immunoglobulin locus. When AID deaminates cytidine residues in the immunoglobulin V regions, somatic hypermutation is initiated; when cytidine residues in switch regions are deaminated, class switch recombination is initiated.

1	AID can deaminate cytidine residues in single-stranded DNA but not double-stranded DNA (Fig. 10.18). For AID to act, AID target genes are typically being transcribed, so that the DNA double helix is temporarily unwound. Since AID is expressed only in germinal center B cells, targeting of the immunoglobulin genes takes place only in these cells and in the actively transcribed rearranged The activity of AID, which is expressed only in B cells, requires access to the cytidine side chain of a single-stranded DNA molecule (first panel), which is normally prevented by the hydrogen bonding in double-stranded DNA. AID initiates a nucleophilic attack on the exposed cytosine ring (second panel), which is resolved by the deamination of the cytidine to form uridine (third panel). Fig. 10.19 AID initiates DNA lesions whose repair leads to somatic hypermutation, class switch recombination, or gene conversion.

1	When AID converts a cytidine (C) to uridine (U) in the DNA of an immunoglobulin gene, the final mutation produced depends on which repair pathways are used. Somatic hypermutation can result from either the mismatch repair (MSH2/6) pathway combined with error-prone polymerase activity of Polη, or the base-excision repair (UNG) pathway. Acting together, these can generate point mutations at and around the site of the original C:G pair. REV1 is a DNA repair enzyme that can synthesize DNA, or recruit other enzymes that can synthesize DNA, over the abasic sites in damaged DNA. REV1 itself will insert only C opposite the abasic site, but it can help recruit other polymerases that can also insert A, G, and T. The end result is insertion of a random nucleotide at the C:G residues where AID initially acted. Both class switch recombination and gene conversion require the formation of a single-strand break in the DNA. A single-strand break is formed when apurinic/apyrimidinic endonuclease 1

1	acted. Both class switch recombination and gene conversion require the formation of a single-strand break in the DNA. A single-strand break is formed when apurinic/apyrimidinic endonuclease 1 (APE1) removes a damaged residue from the DNA as part of the repair process (see Fig. 10.20, bottom two panels). In class switch recombination, single-strand breaks made in two of the so-called switch regions upstream of the C-region genes are converted to double-strand breaks. The cell’s machinery for repairing double-strand breaks, which is very similar to the later stages of V(D)J recombination, then rejoins the DNA ends in a way that leads to a recombination event in which a different C-region gene is brought adjacent to the rearranged V region. Gene conversion results from the broken DNA strand using homologous sequences flanking the immunoglobulin gene as a template for repair DNA synthesis, thus replacing part of the gene with new sequences.

1	V regions where RNA polymerase generates transient single-stranded regions. Somatic hypermutation does not occur in loci that are not being actively transcribed. Rearranged VH and VL genes are mutated even if they are ‘nonproductive’ rearrangements and are not being expressed as protein, as long as they are being transcribed. Some actively transcribed genes in B cells besides those for immunoglobulins can also be affected by the somatic mutation process, but at a much lower rate. 10-10 Mismatch and base-excision repair pathways contribute to somatic hypermutation following initiation by AID.

1	The uridine produced by AID represents a dual lesion in DNA; not only is uridine foreign to normal DNA, but it is now a mismatch with the guanosine nucleoside on the opposite DNA strand. The presence of uridine in DNA can trigger several types of DNA repair—including the mismatch repair and the base-excision repair pathways—which further alter the DNA sequence. The various repair processes lead to different mutational outcomes (Fig. 10.19). In the mismatch repair pathway, the presence of uridine is detected by the mismatch repair proteins MSH2 and MSH6 (MSH2/6). They recruit nucleases that remove the complete uridine nucleotide along with several adjacent nucleotides from the damaged DNA strand. This is followed by a fill-in ‘patch repair’ by a DNA polymerase; unlike the process in all other cells, in B cells this DNA synthesis is error-prone and tends to introduce mutations at nearby A:T base pairs.

1	The initial steps in the base-excision repair pathway are shown in Fig. 10.20. In this pathway, the enzyme uracil-DNA glycosylase (UNG) removes the uracil base from the uridine to create an abasic site in the DNA. If no further modification is made, this will result at the next round of DNA replication in the random insertion of a nucleotide opposite the abasic site by DNA polymerase, leading to mutation. The action of UNG may, however, be followed by the action of another enzyme, apurinic/apyrimidinic endonuclease 1 (APE1), which excises the abasic residue to create a single-strand discontinuity (known as a single-strand nick) in the DNA at the site of the original cytidine. Repair of the single-strand nick proceeding through double-strand breaks may result in gene conversion. Gene conversion is not used in the diversification of immunoglobulin genes in humans and mice, but is of importance in some other mammals and in birds.

1	Fig. 10.20 The base-excision repair pathway produces single-strand nicks in DNA by the sequential actions of AID, uracil-DNA glycosylase (UNG), and apurinic/ apyrimidinic endonuclease 1 (APE1). Double-stranded DNA (first panel) can be made accessible to AID by transcription that unwinds the DNA helix locally (second panel). AID, which is specifically expressed in activated B cells, converts cytidine residues to uridines (third panel). The ubiquitous base-excision repair enzyme UNG can then remove the uracil ring from uridine, creating an abasic site (fourth panel). The repair endonuclease APE1 then cuts the sugar–phosphate DNA backbone next to the abasic residue (fifth panel), thereby forming a single-strand nick in the DNA (sixth panel). APE1 does not excise ribose to form a single-strand nick in DNA, but rather cuts the DNA backbone to yield a 5ʹ-deoxyribosephosphate terminus that is then removed by, for example, DNA polymerase b.

1	Somatic hypermutation involves both mutation at the original cytidines targeted by AID and mutation at nearby non-cytidine nucleotides. If the original U:G mismatch is recognized by UNG, then an abasic site will be generated in the DNA (see Fig. 10.19). If no further modification is made to this site, it can be replicated without instructive base pairing from the template strand by a class of error-prone ‘translesion’ DNA polymerases that normally repair gross damage to DNA, such as that caused by ultraviolet (UV) radiation. These polymerases can incorporate any nucleotide into the new DNA strand opposite the abasic site, and after a further round of DNA replication this can result in a stable mutation at the site of the original C:G base pair.

1	In the mismatch repair pathway in B cells, but not in other cell types, the DNA lesion is repaired by error-prone DNA polymerases rather than by more accurate polymerases that faithfully copy the undamaged template strand. Individuals with a defect in the translesion polymerase Polη have relatively fewer mutations than usual at A:T, but not at C:G, in their hypermutated immunoglobulin V regions. This fact suggests that Polη is the repair polymerase involved in this pathway of somatic hypermutation. These individuals also have a form of xeroderma pigmentosum, a condition resulting from the inability of their cells to repair DNA damage caused by UV radiation. 10-11 AID initiates class switching to allow the same assembled VH exon to be associated with different CH genes in the course of an immune response.

1	VH exon to be associated with different CH genes in the course of an immune response. All the progeny of a particular B cell activated in an immune response will express the same VH gene that was generated during its development in the bone marrow, although the gene may be modified by somatic hypermutation. In contrast, that B cell’s progeny may express several different C-region isotypes as the cells mature and proliferate during the immune response. The first antigen receptors expressed by B cells are IgM and IgD, and the first antibody produced in an immune response is always IgM. Later in the immune response, the same assembled V region may be expressed in IgG, IgA, or IgE antibodies. This change is known as class switching (or isotype switching), and, unlike the expression of IgD, it involves irreversible DNA recombination. It is stimulated in the course of an immune response by external signals such as cytokines released by TFH cells.

1	Switching from IgM to the other immunoglobulin classes occurs only after MOVIE 5.2 B cells have been stimulated by antigen. It is achieved through class switch recombination, which is a type of nonhomologous DNA recombination that is guided by stretches of repetitive DNA known as switch regions. Switch regions lie in the intron between the JH gene segments and the Cμ gene, and at equivalent sites upstream of the genes for each of the other heavy-chain isotypes, with the exception of the δ gene, which does not require DNA rearrangement for its expression (Fig. 10.21, first panel). When a B cell switches from the coexpression of IgM and IgD to the expression of another subtype, DNA recombination occurs between Sμ and the S region immediately upstream of

1	Fig. 10.21 Class switching involves recombination between specific switch signals. The top panel shows the organization of a rearranged immunoglobulin heavy-chain locus before class switching. Second panel: this figure illustrates switching between the μ and ε isotypes in the mouse heavy-chain locus. Switch regions (S) are repetitive DNA sequences that guide class switching and are found upstream of each of the immunoglobulin C-region genes, with the exception of the δ gene. Switching is guided by the initiation of transcription by RNA polymerase (shaded circle) through these regions from promoters (shown as arrows) located upstream of each S. Due to the repetitive sequences, RNA polymerase can stall within the S regions, allowing these regions to serve as substrates for AID, and subsequently for UNG and APE1. Third panel: these enzymes introduce a high density of single-strand nicks into the non-template DNA strand and the template strand. Staggered nicks are converted to

1	subsequently for UNG and APE1. Third panel: these enzymes introduce a high density of single-strand nicks into the non-template DNA strand and the template strand. Staggered nicks are converted to double-strand breaks by a mechanism that is not yet understood. Fourth panel: these breaks are then recognized by the cell’s double-strand break repair machinery, which involves DNA-PKcs, Ku proteins, and other repair proteins. Bottom two panels: the two switch regions, in this case Sμ and Sε, are brought together by the repair proteins, and class switching is completed by excision of the intervening region of DNA (including Cμ and Cδ) and ligation of the Sμ and Sε regions.

1	the new constant-region gene. In such a recombination event, the Cμ coding regions and the entire intervening DNA between Cμ and the S region undergoing rearrangement are deleted. Figure 10.21 illustrates switching from Cμ to Cε in the mouse. All switch recombination events produce genes that can encode a functional protein, because the switch sequences lie in introns and therefore cannot cause frameshift mutations. The enzyme AID initiates class switch recombination, and acts only on regions of DNA being transcribed. Certain properties of the switch region sequences

1	Fig. 10.22 RNA processed from switch region introns interacts with AID and guides its activity. Top panel: promoters upstream of each switch region initiate transcription by RNA polymerase upstream of a rearranged VH gene, as in the case of Cμ, shown here, or a noncoding exon for all other constant regions. In all cases, the switch region itself lies within an intron upstream of the exons encoding the constant regions. This intronic switch region RNA is removed from the primary RNA transcript by splicing at specific splice acceptor and donor sites. Middle panel: after splicing, the switch region RNA is further processed and its repetitive elements allow the formation of putative G-quadruplex structures. Evidence indicates that these RNAs are able to bind AID, as implied in the cartoon. Bottom panel: the RNA acts as a guide to bring AID to the switch region by the ability of the G-quadruplex to hybridize with the original DNA template strand from which it was transcribed.

1	promote the accessibility to AID when they are being transcribed. Each switch region consists of many repeats of a G-rich sequence element on the non-template strand. For example, Sμ consists of about 150 repeats of the sequence (GAGCT)n(GGGGGT), where n is usually 3 but can be as many as 7. The sequences of the other switch regions (Sγ, Sα, and Sε) differ in exact sequence but all contain repeats of the GAGCT and GGGGGT sequences. It appears that movement of RNA polymerase through this highly repetitive region is occasionally halted—called polymerase stalling. This may be caused by bubble-like structures, called R-loops, that form when the transcribed RNA displaces the non-template strand of the DNA double helix (see Fig. 10.21, third panel) due to having many G residues in tandem on one strand.

1	Polymerase stalling seems closely connected with the recruitment of AID to specific switch regions being transcribed. A multisubunit RNA processing/ degradation complex, the RNA exosome, associates with AID and accumulates on transcribed switch regions, and the protein Spt5 associates with the stalled polymerase; both are necessary for AID to generate double-stranded breaks. Recent evidence indicates that AID is selectively guided to the transcribed switch by an additional mechanism. After an RNA polymerase has completed transcription of one RNA template, the intron RNA harboring the switch region is spliced out. This RNA is processed to generate an RNA structure, called a G-quadruplex, that is based on the G-rich repetitive element of the switch region (Fig. 10.22). This G-quadruplex serves a dual purpose, both binding to AID and also associating with the switch region from which it was transcribed, based on its sequence complementarity. Thus the G-quadruplex guides AID to the

1	serves a dual purpose, both binding to AID and also associating with the switch region from which it was transcribed, based on its sequence complementarity. Thus the G-quadruplex guides AID to the appropriate switch region, where particular palindromic sequences, such as AGCT, act as good substrates to allow its cytidine deaminase activity to act on both strands concurrently. In this way, the G-quadruplex functions in a manner similar to the synthetic guide RNAs that deliver the Cas9 endonuclease to specific genomic regions, as described in Appendix I, Section A-35).

1	Following the generation of double-stranded breaks in switch regions, general cellular mechanisms for repairing these breaks lead to the nonhomologous recombination between switch regions that results in class switching (see Fig. 10.21, fourth and fifth panels). The ends to be joined are brought together by the alignment of repetitive sequences common to the different switch regions, and rejoining of the DNA ends then leads to excision of all DNA between the two switch regions and the formation of a chimeric region at the junction. Loss of AID completely blocks class switching, but deficiency of UNG in both mice and humans severely impairs class switching, suggesting sequential actions of AID and UNG in generating DNA breaks. Joining of DNA ends is probably mediated by classic nonhomologous end joining (as in V(D) J recombination) as well as by a poorly understood alternative end-joining pathway. Class switching is sometimes impaired in the disease ataxia telangiectasia, which is

1	end joining (as in V(D) J recombination) as well as by a poorly understood alternative end-joining pathway. Class switching is sometimes impaired in the disease ataxia telangiectasia, which is caused by mutations in the DNA-PKcs-family kinase ATM, a known DNA repair protein. The role of ATM in class switching is not yet entirely clear, however.

1	10-12 Cytokines made by TFH cells direct the choice of isotype for class switching in T-dependent antibody responses. Now that we understand the general mechanisms that control DNA rearrangements of class switching, we are ready to explain how a particular heavy-chain is selected during an immune response. It is the choice of antibody isotype that ultimately determines the effector function of antibodies, and we will see that this choice is largely controlled by the cytokines that are produced by TFH cells in the germinal center reaction.

1	As discussed above, interactions between germinal center B cells and TFH cells are essential for class switching to occur. The required interactions occur through the interplay of CD40 on B cells with CD40 ligand on activated helper T cells. Genetic deficiency of CD40 ligand greatly reduces class switching and causes abnormally high levels of plasma IgM, a condition known as hyper IgM syndrome. People with this defect lack antibodies of classes other than IgM and exhibit severe humoral immunodeficiency, manifested as repeated infections with common bacterial pathogens. Much of the IgM in hyper IgM syndromes may be induced by thymus-independent antigens on the pathogens that chronically infect these patients. Nevertheless, people with CD40 ligand deficiency can make IgM antibodies in response to thymus-dependent antigens, which indicates that in the B-cell response, CD40L–CD40 interac tions are most important in enabling a sustained response that includes class switching and affinity

1	to thymus-dependent antigens, which indicates that in the B-cell response, CD40L–CD40 interac tions are most important in enabling a sustained response that includes class switching and affinity maturation, rather than in the initial activation of B cells.

1	The selection of the particular C region for class switch recombination is not random but is regulated by the cytokines produced by TFH cells and other cells during the immune response. Different cytokines preferentially induce switching to different isotypes (Fig. 10.23). Cytokines induce class switching in part by inducing the production of RNA transcripts through the switch regions that lie 5ʹ to each heavy-chain C gene segment. When activated B cells are exposed to IL-4, for example, transcription from promoters that lie upstream of the switch regions of Cγ1 and Cε can be detected a day or two before switching occurs. This will make it possible for switch to occur to either of these two heavy-chain C genes, but in any particular germinal center B cell, recombination will occur in only one. In the example of class switching shown in Fig. 10.21, transcription through the Sε regions caused the rearrangement between the Sμ and Sε regions, making the IgE isotype antibody. This results

1	one. In the example of class switching shown in Fig. 10.21, transcription through the Sε regions caused the rearrangement between the Sμ and Sε regions, making the IgE isotype antibody. This results because IL-4 signaling activates the transcription factor STAT6, which initiates transcription of the Iε promoter upstream of the Sε region. Other cytokines activate other promoters upstream of other switch regions to produce other antibody classes. TFH cells also produce IL-21, which promotes switching to IgG1 and IgG3. Transforming growth factor (TGF)-β induces switching to IgG2b (Cγ2b) and IgA (Cα). IL-5 promotes switching to IgA, and interferon (IFN)-γ induces switching to IgG2a and IgG3.

1	The individual cytokines induce (violet) or inhibit (red) the production of certain antibody classes. Much of the inhibitory effect is probably the result of directed switching to a different class. The actions of IL-21 on class switching are regulated by IL-4. These data are drawn from experiments with mouse cells. Fig. 10.23 Different cytokines induce switching to different antibody classes. InducesInhibitsInhibits Induces Induces InducesInduces Induces Induces Induces Induces Inhibits Inhibits Inhibits Inhibits Inhibits Inhibits IgEIgG2aIgG3IgM IgG1 IgG2b IgACytokines IL-4 IL-5 IFN-˜TGF-°Role of cytokines in regulating expression of antibody classes Augments production IL-21 10-13 B cells that survive the germinal center reaction eventually differentiate into either plasma cells or memory cells.

1	When B cells have undergone affinity maturation and class switching, some eventually exit from the light zone and start to differentiate into plasma cells that produce large amounts of antibody. In B cells, the transcription factors Pax5 and Bcl-6 inhibit the expression of transcription factors required for plasma-cell differentiation, and both Pax5 and Bcl-6 are downregulated when the B cell starts differentiating. The transcription factor IRF4 then induces the expression of BLIMP-1, a transcriptional repressor that switches off genes required for B-cell proliferation, class switching, and affinity maturation. B cells in which BLIMP-1 is induced become plasma cells; they cease proliferating, increase the synthesis and secretion of immunoglobulins, and change their cell-surface properties. Plasma cells downregulate CXCR5 and upregulate CXCR4 and α4:β1 integrins so that they can leave the germinal centers and home to peripheral tissues.

1	Some plasma cells deriving from germinal centers in lymph nodes or spleen migrate to the bone marrow, where a subset live for a long period, whereas others migrate to the medullary cords in lymph nodes or splenic red pulp. B cells that have been activated in germinal centers in mucosal tissues, and which are predominantly switched to IgA production, stay within the mucosal system. A splice variant of XBP1 (X-box binding protein 1) is expressed in plasma cells and helps to regulate their secretory capacity. Plasma cells in bone marrow receive signals from stromal cells that are essential for their survival, and they can be very long lived, whereas plasma cells in the medullary cords or red pulp are not long lived. XBP1 is also required for plasma cells to colonize bone marrow successfully. Plasma cells in the bone marrow are the source of long-lasting high-affinity class-switched antibody.

1	Other germinal center B cells differentiate into memory B cells. Memory B cells are long-lived descendants of cells that were once stimulated by antigen and had proliferated in the germinal center. They divide very slowly if at all; they express surface immunoglobulin but secrete no antibody, or do so only at a low rate. Because the precursors of some memory B cells arise from the germinal center reaction, memory B cells can inherit the genetic changes that occur there, including somatic hypermutation and the gene rearrangements that result in a class switch. The signals that control which path of differentiation a B cell takes are still being investigated. We will briefly return to memory B cells in Chapter 11. 10-14 Some antigens do not require T-cell help to induce B-cell responses.

1	10-14 Some antigens do not require T-cell help to induce B-cell responses. Humans and mice with T-cell deficiencies are able to produce antibodies against thymus-independent (TI) antigens, which we introduced in Section 10-1. These antigens include certain bacterial polysaccharides, polymeric proteins, and lipopolysaccharides, which are able to stimulate naive B cells in the absence of T-cell help. These nonprotein bacterial products cannot elicit classical T-cell responses, yet they induce antibody responses in normal individuals. In addition, there are TI antigens that are not derived from bacteria; these include plant-derived mitogens and lectins, viral antigens, and superantigens, and some parasite-derived antigens.

1	Thymus-independent antigens fall into two classes, TI-1 and TI-2, which activate B cells by two different mechanisms. TI-1 antigens rely on activity that can directly induce B-cell division without T-cell help. We now understand that TI-1 antigens contain molecules that cause the proliferation and differentiation of most B cells regardless of their antigen specificity; this is known as polyclonal activation (Fig. 10.24, top panels). TI-1 antigens are therefore

1	Fig. 10.24 TI-1 antigens induce polyclonal B-cell responses at high concentrations, and antigen-specific antibody responses at low concentrations. At high antigen concentration, the signal delivered by the B-cell-activating moiety of TI-1 antigens is sufficient to induce B-cell proliferation and antibody secretion in the absence of specific antigen binding to surface immunoglobulin. Thus, all B cells respond (top panels). At low concentration, only B cells specific for the TI-1 antigen bind enough of it to focus its B-cell activating properties onto the B cell; this gives a specific antibody response to epitopes on the TI-1 antigen (lower panels).

1	often called B-cell mitogens, a mitogen being a substance that induces cells to undergo mitosis. For example, LPS and bacterial DNA are both TI-1 antigens because they activate TLRs expressed by B cells (see Section 3-5) and can act as a mitogen. Naive murine B cells express most TLRs constitutively, but naive human B cells do not express high levels of most TLRs until they receive stimulation through the B-cell receptor. So by the time a B cell has been stimulated by antigen through its B-cell receptor, it is likely to express several TLRs and be responsive to stimulation by TLR ligands that accompany the antigens. Thus, when B cells are exposed to concentrations of TI-1 antigens that are 103–105 times lower than those used for polyclonal activation, only those B cells whose B-cell receptors specifically bind the TI-1 antigen become activated. At these low concentrations, amounts of TI-1 antigen sufficient for B-cell activation can only be concentrated on the B-cell surface with the

1	specifically bind the TI-1 antigen become activated. At these low concentrations, amounts of TI-1 antigen sufficient for B-cell activation can only be concentrated on the B-cell surface with the aid of this specific binding (see Fig. 10.24, bottom panels). B-cell responses to TI-1 antigens in the early stages of an infection may be important in defense against several extracellular pathogens, but they do not lead to affinity maturation or memory B cells, both of which require antigen-specific T-cell help.

1	The second class of thymus-independent antigens—TI-2 antigens—consists of molecules that have highly repetitive structures, such as bacterial capsular polysaccharides. These contain no intrinsic B-cell-stimulating activity. Whereas TI-1 antigens can activate both immature and mature B cells, TI-2 antigens can activate only mature B cells; immature B cells, as we saw in Section 8-6, are inactivated by encounter with repetitive epitopes. Infants and young children up to about 5 years of age do not make fully effective antibody responses against polysaccharide antigens, and this might be because most of their B cells are immature.

1	Responses to several TI-2 antigens are prominently made by marginal zone B cells, a subset of nonrecirculating B cells that line the border of the splenic white pulp, and by B-1 cells (see Section 8-9). Marginal zone B cells are rare at birth and accumulate with age; they might therefore be responsible for most physiological TI-2 responses, which increase in efficiency with age. TI-2 antigens probably act by simultaneously cross-linking a critical number of B-cell receptors on the surface of antigen-specific mature B cells (Fig. 10.25, left

1	Fig. 10.25 B-cell activation by TI-2 antigens requires, or is greatly enhanced by, cytokines. Multiple cross-linking of the B-cell receptor by TI-2 antigens can lead to IgM antibody production (left panels), but there is evidence that in addition cytokines greatly augment these responses, and also lead to isotype switching (right panels). It is not clear where such cytokines originate, but one possibility is that dendritic cells, which may be able to bind the antigen through innate immune-system receptors on their surface and so present it to the B cells, secrete a soluble TNF-family cytokine called BAFF, which can activate class switching by the B cell.

1	lgGlgMActivated dendritic cells release a cytokine, BAFF, that augments production of antibody against TI-2 antigens and induces class switching TI-2 antigens alone can signal B cells to produce IgM antibody BBBAFFIgMIgM12 panels). Dendritic cells and macrophages can provide co-stimulatory signals for activation of B cells by TI-2 antigens. One of these co-stimulatory signals is BAFF, which can be secreted by dendritic cells and interacts with the receptor TACI on the B cell (see Fig. 10.25, right panels). The density of TI-2 antigen epitopes is critical; excessive cross-linking of B-cell receptors renders mature B cells unresponsive or anergic, as in immature B cells, while too low a density may be insufficient for activation.

1	An important class of TI-2 antigens arises during infection by capsulated bacteria. Many common extracellular bacterial pathogens are surrounded by a polysaccharide capsule that enables them to resist ingestion by phagocytes. The bacteria not only escape direct destruction by phagocytes but also avoid stimulating T-cell responses against bacterial peptides presented by macrophages. IgM antibodies rapidly produced against the capsular polysaccharide independent of peptide-specific T-cell help will coat the bacteria, promoting their ingestion and destruction by phagocytes early in the infection.

1	Not all antibodies against bacterial polysaccharides are produced strictly through this TI-2 mechanism. We mentioned earlier the importance of antibodies against the capsular polysaccharide of Haemophilus influenzae type b in protective immunity to this bacterium. The immunodeficiency disease Wiskott–Aldrich syndrome is caused by defects in T cells that impair their interaction with B cells (see Chapter 13). Patients with Wiskott–Aldrich syndrome respond poorly to protein antigens, but, unexpectedly, also fail to make IgM and IgG antibody against polysaccharide antigens and are highly susceptible to infection with encapsulated bacteria such as H. influenzae. The failure to make IgM seems to be due in part to greatly reduced development of the marginal zone of the spleen, which contains B cells responsible for making much of the ‘natural’ IgM antibody against ubiquitous carbohydrate antigens. Thus, IgM and IgG antibodies induced by TI-2 antigens are likely to be an important part of

1	B cells responsible for making much of the ‘natural’ IgM antibody against ubiquitous carbohydrate antigens. Thus, IgM and IgG antibodies induced by TI-2 antigens are likely to be an important part of the humoral immune response in many bacterial infections, and in humans at least, the production of class-switched antibodies to TI-2 antigens might normally rely on some degree of T-cell help.

1	As well as producing IgM, TI responses can include switching to certain other antibody classes, such as IgG3 in the mouse. This is probably the result of help from dendritic cells (see Fig. 10.25, right panels), which provide secreted cytokines such as BAFF and membrane-bound signals to proliferating plasmablasts as they respond to TI antigens. The distinguishing features of thymus-dependent, TI-1, and TI-2 antibody responses are summarized in Fig. 10.26. Summary.

1	Summary. B-cell activation by many antigens requires both binding of the antigen by the B-cell surface immunoglobulin—the B-cell receptor—and interaction of the B cell with antigen-specific helper T cells. Helper T cells recognize peptide fragments derived from the antigen internalized by the B cells and displayed by the B cells as peptide:MHC class II complexes. Follicular helper T cells stimulate B cells by conjugation in germinal centers, with binding of CD40 ligand on the T cell to CD40 on the B cell, and by their release of cytokines, such as IL-21. Activated B cells also express molecules, such as ICOSL, that can stimulate T cells. The initial interaction between B and T cells occurs at the border of the T-cell and B-cell areas of secondary lymphoid tissue, to which antigen-activated helper T cells and B cells migrate in response to chemokines. Further interactions between T cells and B cells continue after migration into the follicle and the formation of a germinal center.

1	Fig. 10.26 Properties of different classes of antigen that elicit antibody responses. Some data indicate a minor role for T cells in antibody responses to TI-2 antigens; robust responses to TI-2 antigens can be observed in T-cell-deficient mice.

1	T cells induce a phase of vigorous B-cell proliferation in the germinal center reaction and direct the differentiation of clonally expanded B cells into either antibody-secreting plasma cells or memory B cells. Immunoglobulin genes expressed in B cells are diversified in the germinal center reaction by somatic hypermutation and class switching, initiated by activation-induced cytidine deaminase (AID). Unlike V(D)J recombination, these processes occur only in B cells. Somatic hypermutation diversifies the V region through the introduction of point mutations that are selected for providing greater affinity for the antigen as the immune response proceeds. Class switching does not affect the V region but increases the functional diversity of immunoglobulins by replacing the Cμ region in the immunoglobulin gene, which is first expressed with another heavy-chain C region to produce IgG, IgA, or IgE antibodies. Class switching provides antibodies with the same antigen specificity but

1	the immunoglobulin gene, which is first expressed with another heavy-chain C region to produce IgG, IgA, or IgE antibodies. Class switching provides antibodies with the same antigen specificity but distinct effector capacities. The switching to different antibody isotypes is regulated by cytokines released from helper T cells. Some nonprotein antigens stimulate B cells in the absence of linked recognition by peptide-specific helper T cells. Responses to these thymus-independent antigens are accompanied by only limited class switching and do not induce memory B cells. However, such responses have a crucial role in host defense against pathogens whose surface antigens cannot elicit peptide-specific T-cell responses.

1	The distributions and functions of immunoglobulin classes.

1	Extracellular pathogens can invade most sites within the body, and so antibodies must be equally widely distributed to combat them. Most classes of antibodies are distributed by diffusion from their site of synthesis, but specialized transport mechanisms are required to deliver antibodies across the epithelial surfaces lining the mucosa of organs such as the lungs and intestine. The particular heavy-chain isotype of the antibody can either limit antibody diffusion or engage specific transporters that deliver the antibody across epithelia. This part of the chapter describes these mechanisms and the antibody classes that use them to enter compartments of the body where their particular effector functions are appropriate. Here we restrict our discussion to the protective functions of antibodies that result solely from their binding to pathogens, and in the next part of the chapter, we discuss the effector cells and molecules that are specifically engaged by different antibody classes.

1	10-15 Antibodies of different classes operate in distinct places and have distinct effector functions. Pathogens most commonly enter the body across the epithelial barriers of the mucosa lining the respiratory, digestive, and urogenital tracts, or through damaged skin. Less often, insects, wounds, or hypodermic needles introduce microorganisms directly into the blood. Antibodies protect all the body’s mucosal surfaces, tissues, and blood from such infections; these antibodies serve to neutralize the pathogen or promote its elimination before it can establish a significant infection.

1	The different classes of antibodies (see Fig. 5.19) are adapted to function in different compartments of the body. Their functional activities and distributions are listed in Fig. 10.27. Because a given V region can become associated with any C region through class switching, the progeny of a single B cell can produce antibodies that share the same specificity yet provide all of the protective functions appropriate for each body compartment. All naive B cells express cell-surface IgM and IgD. IgM is the first antibody secreted by activated B cells but is less than 10% of the immunoglobulin found in plasma. Little IgD antibody is produced at any time, while IgE contributes a small but biologically important part of the immune response. IgG and IgA are the predominant antibody classes. IgE contributes a small but biologically important part of the response. The overall predominance of IgG is also due in part to its longer lifetime in the plasma (see Fig. 5.20).

1	The major effector functions of each class (+++) are shaded in dark red, whereas lesser functions (++) are shown in dark pink, and very minor functions (+) in pale pink. The distributions are marked similarly, with actual average levels in serum being shown in the bottom row. IgA has two subclasses, IgA1 and IgA2. The IgA column refers to both. *IgG2 can act as an opsonin in the presence of an Fc receptor of the appropriate allotype, found in about 50% of people of Caucasian descent.

1	IgM antibodies are produced first in a humoral immune response and tend to be of low affinity. However, IgM molecules form pentamers that are stabilized by a single J-chain molecule (see Fig. 5.23) and have 10 antigen-binding sites, conferring higher overall avidity when binding to multivalent antigens such as bacterial capsular polysaccharides. This higher avidity of the pentamer compensates for the low affinity of the individual antigen-binding site within the IgM monomers. Because of the large size of the pentamers, IgM is found mainly in the bloodstream and, to a lesser extent, in the lymph, rather than in intercellular spaces within tissues. The pentameric structure of IgM makes it especially effective in activating the complement system, as we will see in the last part of this chapter. IgM hexamers can also form, and these fix complement much more efficiently than pentamers, possibly because C1q is also a hexamer. However, the in vivo role of IgM hexamers in protecting against

1	IgM hexamers can also form, and these fix complement much more efficiently than pentamers, possibly because C1q is also a hexamer. However, the in vivo role of IgM hexamers in protecting against infections has not been fully established.

1	Infection of the bloodstream has serious consequences unless it is controlled quickly, and the rapid production of IgM and its efficient activation of the complement system are important in controlling such infections. Some IgM is produced by conventional B cells that have not undergone class switching, but most is produced by B-1 cells residing in the peritoneal cavity and pleural spaces and by marginal zone B cells of the spleen. These cells secrete antibodies against commonly encountered carbohydrate antigens, including those of bacteria, and do not require T-cell help; they therefore provide a preformed repertoire of IgM antibodies in blood and body cavities that can recognize invading pathogens (see Section 8-9).

1	Antibodies of the other classes—IgG, IgA, and IgE—are smaller, and diffuse easily out of the blood into the tissues. IgA can form dimers (see Fig. 5.23), but IgG and IgE are always monomeric. The affinity of the individual antigen-binding sites for their antigen is therefore critical for the effectiveness of these antibodies, and most of the B cells expressing these classes have been selected in the germinal centers for their increased affinity for antigen after somatic hypermutation. IgG4 is the least abundant of the IgG subclasses, but has the unusual ability to form hybrid antibodies. One IgG4 heavy chain and attached light chain can split from the original heavy-chain dimer and reassociate with a different IgG4 heavy chain–light chain pair, forming a bivalent IgG4 antibody with two distinct antigen specificities.

1	IgG is the principal class of antibody in blood and extracellular fluid, whereas IgA is the principal class in secretions, the most important being those from the epithelia lining the intestinal and respiratory tracts. IgG efficiently opsonizes pathogens for engulfment by phagocytes and activates the complement system, but IgA is a less potent opsonin and a weak activator of complement. IgG operates mainly in the tissues, where accessory cells and molecules are available, whereas dimeric IgA operates mainly on epithelial surfaces, where complement and phagocytes are not normally present; therefore IgA functions chiefly as a neutralizing antibody. Monomeric IgA can be produced by plasma cells that differentiate from class-switched B cells in lymph nodes and spleen, and it acts as a neutralizing antibody in extracellular spaces and in the blood. This monomeric IgA is predominantly of the subclass IgA1; the ratio of IgA1 to IgA2 in the blood is 10:1. The IgA antibodies produced by plasma

1	antibody in extracellular spaces and in the blood. This monomeric IgA is predominantly of the subclass IgA1; the ratio of IgA1 to IgA2 in the blood is 10:1. The IgA antibodies produced by plasma cells in the gut are dimeric and predominantly of subclass IgA2; the ratio of IgA2 to IgA1 in the gut is 3:2.

1	Finally, IgE antibody is present only at very low levels in blood or extracellular fluid, but is bound avidly by receptors on mast cells that are found just beneath the skin and mucosa and along blood vessels in connective tissue. Antigen binding to this cell-associated IgE triggers mast cells to release powerful chemical mediators that induce reactions such as coughing, sneezing, and vomiting, which in turn can expel infectious agents, as discussed later in this chapter. 10-16 Polymeric immunoglobulin receptor binds to the Fc regions of IgA and IgM and transports them across epithelial barriers.

1	In the mucosal immune system, IgA-secreting plasma cells are found predominantly in the lamina propria, which lies immediately below the basement membrane of many surface epithelia. From there the IgA antibodies can be transported across the epithelium to its external surface, for example to the lumen of the gut or of the bronchi (Fig. 10.28). IgA antibody synthesized in the lamina propria is secreted as a dimeric IgA molecule associated with a single J chain. This polymeric form of IgA binds specifically to a receptor called the polymeric immunoglobulin receptor (pIgR), which is present on the basolateral surfaces of the overlying epithelial cells. When the pIgR has bound a molecule of dimeric IgA, the complex is internalized and carried in a transport vesicle through the cytoplasm of the epithelial cell to its luminal surface. This process is called transcytosis. IgM also binds to the pIgR and can be secreted into the gut by the same mechanism. Upon reaching the luminal surface of

1	epithelial cell to its luminal surface. This process is called transcytosis. IgM also binds to the pIgR and can be secreted into the gut by the same mechanism. Upon reaching the luminal surface of the enterocyte, the antibody is released into the mucous layer covering the gut lining by proteolytic cleavage of the extracellular domain of the pIgR. The cleaved extracellular domain of the pIgR is known as secretory component (frequently abbreviated to SC) and remains associated with the antibody. Secretory component is bound to the part of the Fc region of IgA that contains the binding site for the Fcα receptor I, which is why secretory IgA does not bind to this receptor. Secretory component serves several physiological roles. It binds to mucins in mucus, acting as ‘glue’ to bind secreted IgA to the mucous layer on the luminal surface of the gut epithelium, where the antibody binds and neutralizes gut pathogens and their toxins (see Fig. 10.28). Secretory component also protects the

1	IgA to the mucous layer on the luminal surface of the gut epithelium, where the antibody binds and neutralizes gut pathogens and their toxins (see Fig. 10.28). Secretory component also protects the antibodies against cleavage by gut enzymes.

1	The principal sites of IgA synthesis and secretion are the gut, the respiratory epithelium, the lactating breast, and various other exocrine glands such as the salivary and tear glands. It is believed that the primary functional role of IgA antibodies is to protect epithelial surfaces from infectious agents, just as IgG antibodies protect the extracellular spaces inside tissues. By binding bacteria, virus particles, and toxins, IgA antibodies prevent the attachment of bacteria and viruses to epithelial cells and the uptake of toxins, and provide the first line of defense against a wide variety of pathogens. IgA is also thought to have an additional role in the gut, that of regulating the gut microbiota (see Chapter 12). The alveolar spaces in the lower respiratory tract lack the thicker mucosal layer characteristic of the upper respiratory tract, because efficient gas diffusion would be impeded by a mucous layer covering the alveolar epithelium. IgG can rapidly transudate into these

1	mucosal layer characteristic of the upper respiratory tract, because efficient gas diffusion would be impeded by a mucous layer covering the alveolar epithelium. IgG can rapidly transudate into these spaces and is the major isotype responsible for protection there.

1	Fig. 10.28 Dimeric IgA is the major class of antibody present in the lumen of the gut. IgA is synthesized by plasma cells in the lamina propria and transported into the lumen of the gut through epithelial cells at the base of the crypts. Dimeric IgA binds to the layer of mucus overlying the gut epithelium and acts as an antigen-specific barrier to pathogens and toxins in the gut lumen The structure of a molecule of FcRn (blue) is shown bound to one chain of the Fc portion of IgG (red), at the interface of the Cγ2 and Cγ3 domains, with the Cγ2 region at the top. The β2-microglobulin component of the FcRn is green. The dark-blue structure attached to the Fc portion of IgG is a carbohydrate chain, reflecting glycosylation. FcRn transports IgG molecules across the placenta in humans and also across the gut in rats and mice. It also has a role in maintaining the levels of IgG in adults. Although only one molecule of FcRn is shown binding to the Fc portion, it is thought that it takes two

1	across the gut in rats and mice. It also has a role in maintaining the levels of IgG in adults. Although only one molecule of FcRn is shown binding to the Fc portion, it is thought that it takes two molecules of FcRn to capture one molecule of IgG. Courtesy of

1	Fig. 10.29 The neonatal Fc receptor (FcRn) binds to the Fc portion of IgG. P. Björkman. 10-17 The neonatal Fc receptor carries IgG across the placenta and prevents IgG excretion from the body.

1	Newborn infants are especially vulnerable to infection, having had no previous exposure to the microbes in the environment they enter at birth. IgA antibodies are secreted in breast milk and are transferred to the gut of the newborn infant, where they provide protection from newly encountered bacteria until the infant can synthesize its own protective antibody. IgA is not the only protective antibody that a mother passes on to her baby. Maternal IgG is transported across the placenta directly into the bloodstream of the fetus during intrauterine life; human babies at birth have as high levels of plasma IgG as their mothers, and with the same range of antigen specificities. The selective transport of IgG from mother to fetus is due to an IgG transport protein in the placenta, FcRn (neonatal Fc receptor), which is closely related in structure to MHC class I molecules. Despite this similarity, FcRn binds IgG quite differently from the binding of peptide to MHC class I molecules, because

1	Fc receptor), which is closely related in structure to MHC class I molecules. Despite this similarity, FcRn binds IgG quite differently from the binding of peptide to MHC class I molecules, because its peptide-binding groove is occluded. It binds to the Fc portion of IgG molecules (Fig. 10.29). Two molecules of FcRn bind one molecule of IgG, bearing it across the placenta. Maternal IgG is ingested by the newborn animal from its mother’s milk and colostrum, the protein-rich fluid secreted by the early postnatal mammary gland. In this case, FcRn transports the IgG from the lumen of the neonatal gut into the blood and tissues. Interestingly, FcRn is also found in adults in the gut and liver and on endothelial cells. Its function in adults is to maintain the levels of IgG in plasma, which it does by binding antibody, endocytosing it, and recycling it to the blood, thus preventing its excretion from the body.

1	By means of these specialized transport systems, mammals are supplied from birth with antibodies against pathogens common in their environments. As they mature and make their own antibodies of all isotypes, these are distributed selectively to different sites in the body (Fig. 10.30). Thus, throughout life, class switching and the distribution of antibody classes throughout the body provide effective protection against infection in extracellular spaces. 10-18 High-affinity IgG and IgA antibodies can neutralize toxins and block the infectivity of viruses and bacteria.

1	Pathogens can cause damage to a host by producing toxins or by infecting cells directly, and antibodies can protect by blocking both of these actions. Many bacteria cause disease by secreting toxins that damage or disrupt the function of the host’s cells (Fig. 10.31). To affect cells, many toxins consist of separate domains for exerting toxicity and for binding to specific cell-surface receptors by which they enter cells. Antibodies that bind a toxin’s receptor-binding site can prevent cell entry and protect cells from attack (Fig. 10.32). Antibodies that act in this way to neutralize toxins are referred to as neutralizing antibodies. Most toxins are active at nanomolar concentrations: a single molecule of diphtheria toxin can kill a cell. To neutralize toxins, therefore, antibodies must be able to diffuse into the tissues and bind the toxin rapidly and with high affinity. The ability of IgG antibodies to diffuse easily throughout the

1	Fig. 10.30 Immunoglobulin classes are selectively distributed in the body. IgG and IgM predominate in blood (shown here for simplicity by IgM and IgG in the heart), whereas IgG and monomeric IgA are the major antibodies in extracellular fluid within the body. Dimeric IgA predominates in secretions across epithelia, including breast milk. The fetus receives IgG from the mother by transplacental transport. IgE is found mainly associated with mast cells just beneath epithelial surfaces (especially of the respiratory tract, gastrointestinal tract, and skin). The brain is normally devoid of immunoglobulin.

1	Fig. 10.31 Many common diseases are caused by bacterial toxins. The toxins shown here are all exotoxins—proteins secreted by the bacteria. High-affinity IgG and IgA antibodies protect against these toxins. Bacteria also have nonsecreted endotoxins, such as lipopolysaccharide, which are released when the bacterium dies and may also mediate pathogenesis of disease. Host responses to exotoxins are more complex because the innate immune system has receptors for some endotoxins, such as TLR-4 (see Chapter 3). extracellular fluid, and their high affinity for antigen once affinity maturation has taken place, make them the principal antibodies that neutralize toxins in tissues. High-affinity IgA antibodies similarly neutralize toxins at the mucosal surfaces of the body. Dissociation of toxin releases its active chain, which poisons cell Antibody protects cell by blocking binding of toxin Toxin binds to cellular receptors Endocytosis of toxin:receptor complexes

1	Dissociation of toxin releases its active chain, which poisons cell Antibody protects cell by blocking binding of toxin Toxin binds to cellular receptors Endocytosis of toxin:receptor complexes Fig. 10.32 Neutralization of toxins by IgG antibodies protects molecule binds a cell-surface receptor, which enables the molecule cells from damage. The damaging effects of many bacteria are to be internalized. Another part of the toxin molecule then enters the due to the toxins they produce (see Fig. 10.31). These toxins are cytoplasm and poisons the cell. Antibodies that inhibit toxin binding usually composed of several distinct moieties. One part of the toxin can prevent, or neutralize, these effects.

1	Diphtheria and tetanus toxins are two bacterial toxins in which the toxic and receptor-binding functions are on separate protein chains. It is therefore possible to immunize individuals, usually as infants, with modified toxin molecules in which the toxic chain has been denatured. These modified toxins, called toxoids, lack toxic activity but retain the receptor-binding site. Thus, immunization with the toxoid induces neutralizing antibodies that protect against the native toxin.

1	Some insect or animal venoms are so toxic that a single exposure can cause severe tissue damage or death. For these the adaptive immune response is too slow to be protective. Exposure to these venoms is a rare event, and protective vaccines have not been developed for use in humans. Instead, neutralizing antibodies are generated by immunizing other species, such as horses, with insect and snake venoms to produce anti-venom antibodies, or antivenins. The antivenins are injected into exposed individuals to protect them against the toxic effects of the venom. Transfer of antibodies in this way is known as passive immunization (see Appendix I, Section A-30).

1	Animal viruses infect cells by binding to a particular cell-surface receptor. These are often cell-type-specific proteins that determine which cells a virus can infect, or its tropism. Many antibodies that neutralize viruses do so by directly blocking the binding of virus to surface receptors (Fig. 10.33). The hemagglutinin of influenza virus, for example, binds to terminal sialic acid residues on the carbohydrates of glycoproteins present on epithelial cells of the respiratory tract. It is known as hemagglutinin because it recognizes and binds to similar sialic acid residues on chicken red blood cells and agglutinates these red blood cells. Antibodies against the hemagglutinin can prevent infection by the influenza virus. Such antibodies are called virus-neutralizing antibodies, and, as with the neutralization of toxins, high-affinity IgA and IgG antibodies are particularly important. However, antibodies can also neutralize viruses by interfering with the fusion mechanisms used to

1	with the neutralization of toxins, high-affinity IgA and IgG antibodies are particularly important. However, antibodies can also neutralize viruses by interfering with the fusion mechanisms used to enter the cell’s cytoplasm after binding to surface receptors.

1	Many bacteria have cell-surface molecules called adhesins that enable them to bind to the surface of host cells. This adherence is crucial to the ability of these bacteria to cause disease, whether they subsequently enter the cell, as do Salmonella species, or remain attached to the cell surface as extracellular pathogens (Fig. 10.34). Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, has a cell-surface protein known as pilin that enables the bacterium to adhere to the epithelial cells of the urinary and reproductive tracts and is essential to its infectivity. Antibodies against pilin can inhibit this adhesive reaction and prevent infection.

1	IgA antibodies secreted onto the mucosal surfaces of the intestinal, respiratory, and reproductive tracts are particularly important in inhibiting the colonization of these surfaces by pathogens and in preventing infection of the epithelial cells. Adhesion of bacteria to cells within tissues can also contribute to pathogenesis, and IgG antibodies against adhesins protect tissues from damage in much the same way as IgA antibodies protect mucosal surfaces.

1	Fig. 10.33 Viral infection of cells can be blocked by neutralizing antibodies. For a virus to multiply within a cell, it must introduce its genes into the cell. The first step in entry is usually the binding of the virus to a receptor on the cell surface. For enveloped viruses, as shown in the figure, entry into the cytoplasm requires fusion of the viral envelope and the cell membrane. For some viruses this fusion event takes place on the cell surface (not shown); for others it can occur only within the more acidic environment of endosomes, as shown here. Non-enveloped viruses must also bind to receptors on cell surfaces, but they enter the cytoplasm by disrupting endosomes. Antibodies bound to viral surface proteins neutralize the virus, inhibiting either its initial binding to the cell or its subsequent entry. 10-19 Antibody:antigen complexes activate the classical pathway of complement by binding to C1q.

1	10-19 Antibody:antigen complexes activate the classical pathway of complement by binding to C1q. Chapter 2 introduced the complement system as an essential component of innate immunity. Complement activation can proceed in the absence of antibody via the lectin pathway through the actions of mannose-binding lectin (MBL) and ficolins. But complement is also an important effector of antibody responses via the classical pathway. The different pathways of complement activation converge to coat pathogen surfaces or antigen:antibody complexes with covalently attached complement fragment C3b, which acts as an opsonin to promote uptake and removal by phagocytes. In addition, the terminal complement components can form a membrane-attack complex that damages some bacteria.

1	In the classical pathway, complement activation is triggered by C1, a complex of C1q and the serine proteases C1r and C1s (see Section 2-7). Complement activation is initiated when antibodies that are attached to the surface of a pathogen then bind to C1 via C1q (Fig. 10.35). C1q can be bound by either Fig. 10.34 Antibodies can prevent the attachment of bacteria to cell surfaces. Many bacterial infections require an interaction between the bacterium and a cell-surface receptor. This is particularly true for infections of mucosal surfaces. The attachment process involves very specific molecular interactions between bacterial adhesins and their receptors on host cells; antibodies against bacterial adhesins can block such infections.

1	Fig. 10.35 The classical pathway of complement activation is initiated by the binding of C1q to antibody on a pathogen surface. When a molecule of IgM binds several identical epitopes on a pathogen surface, it is bent into the ‘staple’ conformation, which allows the globular heads of C1q to bind to the Fc regions of IgM (left panels). Multiple molecules of IgG bound on the surface of a pathogen allow the binding of a single molecule of C1q to two or more Fc regions (right panels). In both cases, the binding of C1q to the Fc regions induces a conformational change that activates the associated C1r, which becomes an active enzyme that cleaves the pro-enzyme C1s, generating a serine protease that initiates the classical complement cascade (see Chapter 2). Fig. 10.36 The two conformations of IgM. The left panel shows the planar conformation of soluble IgM; the right panel shows the ‘staple’ conformation of IgM bound to a bacterial flagellum. Photographs (×760,000) courtesy of K.H. Roux.

1	IgM or IgG antibodies, but, because of the structural requirements of binding to C1q, neither of these antibody classes can activate complement in solution; the complement reactions are initiated only when the antibodies are already bound to multiple sites on a cell surface, normally that of a pathogen.

1	Each globular head of a C1q molecule can bind to one Fc region, and binding of two or more heads activates the C1 complex. In plasma, the pentameric IgM molecule has a planar conformation that does not bind C1q (Fig. 10.36, left panel); however, binding to the surface of a pathogen deforms the IgM pen-tamer so that it looks like a staple (see Fig. 10.36, right panel), and this distortion exposes binding sites for the C1q heads. As mentioned in Section 10-15, IgM hexamers can also form but comprise less than 5% of total serum IgM. Hexameric IgM activates complement about 20 times more efficiently than its pentameric form, possibly because C1q is also a hexamer. The in vivo role of IgM hexamers in protecting against infections has not been fully established, and it has even been suggested that IgM hexamers are too reactive and may be harmful.

1	Although C1q binds with low affinity to some subclasses of IgG in solution, the binding energy required for C1q activation is achieved only when a single molecule of C1q can bind two or more IgG molecules that are held within 30–40 nm of each other as a result of binding antigen. This requires that multiple molecules of IgG be bound to a single pathogen or to an antigen in solution. For this reason, IgM is much more efficient than IgG in activating complement. The binding of C1q to a single bound IgM molecule, or to two or more bound IgG molecules (see Fig. 10.35), leads to activation of the protease activity of C1r, triggering the complement cascade. 10-20 Complement receptors and Fc receptors both contribute to removal of immune complexes from the circulation.

1	Fc receptors confer the distinct effector functions to the various antibody isotypes by interacting with their Fc regions. One such function is the clearance from the circulation of antigen:antibody complexes (immune complexes), which can include toxins, or debris from dead host cells and microorganisms, bound by neutralizing antibodies. Immune complexes can be cleared by the binding of the antibody’s Fc region to Fc receptors expressed on various phagocytic cells in tissues. This clearance is also helped by complement activation (described in the last section), which occurs when the Fc region activates C1q. The deposition of C4b and C3b onto the immune complex aids clearance by binding to complement receptor 1 (CR1) on the surface of erythrocytes (see Section 2-13 for a description of the different types of complement receptors). The erythrocytes transport the bound complexes of antigen, antibody, and complement to the liver and spleen. Here, macrophages bearing CR1 and Fc

1	CR1 on the erythrocyte surface has an important role in the clearance of immune complexes from the circulation. Immune complexes bind to CR1 on erythrocytes, which transport them to the liver and spleen, where they are removed by macrophages expressing receptors for both Fc and bound complement components. receptors remove the complexes from the erythrocyte surface without destroying the cell, and then degrade the complexes (Fig. 10.37). Even larger aggregates of particulate antigen, such as bacteria, viruses, and cell debris, can be coated with complement, picked up by erythrocytes, and transported to the spleen for destruction.

1	Complement-coated immune complexes that are not removed from the circulation tend to deposit in the basement membranes of small blood vessels, most notably those of the renal glomerulus, where the blood is filtered to form urine. Immune complexes that pass through the basement membrane of the glomerulus bind to CR1 present on the renal podocytes, cells that lie beneath the basement membrane. The functional significance of these receptors in the kidney is unknown; however, they have an important role in the pathology of some autoimmune diseases. In the autoimmune disease systemic lupus erythematosus (SLE) (see Section 15-16), excessive levels of circulating immune complexes lead to their deposition in large amounts on the podocytes, damaging the glomerulus; kidney failure is the principal danger in this disease. The strongest genetic risk factor for SLE is C1q deficiency, although this is very rare. Mutations in complement receptors 2 and 3 and the Fc receptor FcγRIIIa are also

1	danger in this disease. The strongest genetic risk factor for SLE is C1q deficiency, although this is very rare. Mutations in complement receptors 2 and 3 and the Fc receptor FcγRIIIa are also associated with increased susceptibility to develop lupus, implying the involvement of both complement receptors and FcR pathways in clearing immune complexes.

1	Antigen:antibody complexes can also be a cause of pathology in patients with deficiencies in the early components of complement (C1, C2, and C4). These deficiencies result in the classical complement pathway not being activated properly, and immune complexes not being cleared effectively because they do not become tagged with complement. These patients also suffer tissue damage as a result of immune-complex deposition, especially in the kidneys. Summary.

1	The T-cell-dependent antibody response begins with IgM secretion but quickly progresses to the production of additional antibody classes. Each class is specialized both in its localization in the body and in the functions it can perform. IgM antibodies are found mainly in blood; they are pentameric in structure. IgM is specialized to activate complement efficiently upon binding antigen and to compensate for the low affinity of a typical IgM antigen-binding site. IgG antibodies are usually of higher affinity and are found in blood and in extracellular fluid, where they can neutralize toxins, viruses, and bacteria, opsonize them for phagocytosis, and activate the complement system. IgA antibodies are synthesized as monomers, which enter blood and extracellular fluids, or they are secreted as dimeric molecules by plasma cells in the lamina propria of various mucosal tissues. IgA dimers are selectively transported across the epithelial layer into sites such as the lumen of the gut, where

1	dimeric molecules by plasma cells in the lamina propria of various mucosal tissues. IgA dimers are selectively transported across the epithelial layer into sites such as the lumen of the gut, where they neutralize toxins and viruses and block the entry of bacteria across the intestinal epithelium. Most IgE antibody is bound to the surface of mast cells that reside mainly just below the body surface; antigen binding to this IgE triggers local defense reactions. Antibodies can defend the body against extracellular pathogens and their toxic products in several ways. The simplest is by direct interactions with pathogens or their products, for example, by binding to the active sites of toxins and neutralizing them or by blocking their ability to bind to host cells through specific receptors. When antibodies of the appropriate isotype bind to antigens, they can activate the classical pathway of complement, which leads

1	Fig. 10.37 Erythrocyte CR1 helps to clear immune complexes from the circulation. Small antigen:antibody complexes form in the circulation FcRIn the spleen and liver, phagocytic cells remove the immune complexes from the erythrocyte surface Complement receptor CR1 on erythrocytes binds the immune complexes via bound C3b CR1 to the elimination of the pathogen by the various mechanisms described in Chapter 2. Soluble immune complexes of antigen and antibody also fix complement and are cleared from the circulation via complement receptors on red blood cells. The destruction of antibody-coated pathogens via Fc receptors.

1	The neutralization of toxins, viruses, or bacteria by high-affinity antibodies can protect against infection but does not, on its own, solve the problem of how to remove the pathogens and their products from the body. Moreover, many pathogens cannot be neutralized by antibody and must be destroyed by other means. Many pathogen-specific antibodies do not bind to neutralizing targets on pathogen surfaces and thus need to be linked to other effector mechanisms to play their part in host defense. We have already seen how the binding of antibody to antigen can activate complement. Another important defense mechanism is the activation of a variety of accessory effector cells bearing receptors called Fc receptors because they are specific for the Fc portion of antibodies. These receptors facilitate the phagocytosis of antibody-bound extracellular pathogens by macrophages, dendritic cells, and neutrophils. Other, nonphagocytic cells of the immune system—NK cells, eosinophils, basophils, and

1	the phagocytosis of antibody-bound extracellular pathogens by macrophages, dendritic cells, and neutrophils. Other, nonphagocytic cells of the immune system—NK cells, eosinophils, basophils, and mast cells (see Fig. 1.8)—are triggered to secrete stored mediators when their Fc receptors are engaged by antibody-coated pathogens. These mechanisms maximize the effectiveness of all antibodies regardless of where they bind.

1	10-21 The Fc receptors of accessory cells are signaling receptors specific for immunoglobulins of different classes. The Fc receptors are a family of cell-surface molecules that bind the Fc portion of immunoglobulins. Each member of the Fc family recognizes immunoglobulin of one or a few closely related heavy-chain isotypes through a recognition domain on the α chain of the Fc receptor. Most Fc receptors are themselves members of the immunoglobulin gene superfamily. Different cell types bear different sets of Fc receptors, and the isotype of the antibody thus determines which types of cells will be engaged in a given response. The different Fc receptors, the cells that express them, and their specificities for different antibody classes are shown in Fig. 10.38.

1	Most Fc receptors function as part of a multisubunit complex. Only the α chain is required for antibody recognition; the other chains are required for transport of the receptor to the cell surface and for signal transduction when an Fc region is bound. Some Fcγ receptors, the Fcα receptor I, and the high-affinity receptor for IgE (FcεRI) all use a γ chain for signaling. This chain, which is closely related to the ζ chain of the T-cell receptor complex (see Section 7-7), associates noncovalently with the Fc-binding α chain. The human FcγRII-A is a single-chain receptor in which the cytoplasmic domain of the α chain replaces the function of the γ chain. FcγRII-B1 and FcγRII-B2 are also single-chain receptors, but function as inhibitory receptors because they contain an ITIM that engages the inositol 5ʹ-phosphatase SHIP (see Section 7-25). The most prominent function of Fc receptors is the activation of accessory cells to attack pathogens, but they also contribute in other ways to immune

1	inositol 5ʹ-phosphatase SHIP (see Section 7-25). The most prominent function of Fc receptors is the activation of accessory cells to attack pathogens, but they also contribute in other ways to immune responses. For example, FcγRII-B receptors negatively regulate the activities of B cells, mast cells, macrophages, and neutrophils by adjusting the threshold at which immune complexes will activate these cells. Fc receptors expressed by dendritic cells enable them to ingest antigen:antibody complexes efficiently and thus process these antigens and present their peptides to T cells.

1	ReceptorFc˜RI Fc°RI (CD89) Cell type Binding Structure Macrophages Neutrophils Eosinophils IgG1 108 M–1 2 × 106 M–1 2 × 106 M–1 1) IgG1=IgG3 2) IgG4 3) IgG2 Order of afÿnity Macrophages Neutrophils Eosinophils Platelets Langerhans cells Macrophages Neutrophils Eosinophils B cells Mast cells IgG1 IgG1 IgG1 ITIM ITIM IgG1=IgG3 IgA1=IgA2 NK cells Eosinophils Macrophages Neutrophils Mast cells IgG1 Mast cells Basophils IgE IgA1, IgA2 IgA, IgM Fc˛RII-A (CD32) Fc˛RIII (CD16) Fc˛RI (CD64) Fc˛RII-B2 (CD32) Fc˛RII-B1 (CD32) or Effect of ligation Uptake Stimulation Activation of respiratory burst Induction of killing Uptake Granule release (eosinophils) Uptake Inhibition of stimulation No uptake Inhibition of stimulation Induction of killing (NK cells) Uptake Induction of killing UptakeSecretion of granules Macrophages Eosinophils† Neutrophils Macrophages B cells Eosinophils B cells ˜ ° 72 kDa ° 40 kDa ˜-like domain ° 50–70 kDa ˜or ˝ Fc°/˝RFc˜RII (CD23) ° 45 kDa ˙ 33 kDa ˜ 9 kDa ˜ 9 kDa ° 55–75

1	granules Macrophages Eosinophils† Neutrophils Macrophages B cells Eosinophils B cells ˜ ° 72 kDa ° 40 kDa ˜-like domain ° 50–70 kDa ˜or ˝ Fc°/˝RFc˜RII (CD23) ° 45 kDa ˙ 33 kDa ˜ 9 kDa ˜ 9 kDa ° 55–75 kDa ° 70 kDa N lectin domain trimer Degranulation IgE 1) IgG1 2) IgG3=IgG2* 3) IgG4 1) IgG1=IgG3 2) IgG4 3) IgG2 2 × 106 M–1 1) IgG1=IgG3 2) IgG4 3) IgG2 1) IgM 2) IgA 5 × 105 M–1 3 × 109 M–12–7 × 107 M–1 (trimer) 2–7 × 106 M–1 (monomer) 1010 M–1 107 M–1

1	Fig. 10.38 Distinct receptors for the Fc region of the glycosylphosphatidylinositol membrane anchor without γ chains, different immunoglobulin classes are expressed on different whereas in NK cells it is a transmembrane molecule associated with accessory cells. The subunit structure and binding properties of γ chains. The FcγRII-B1 differs from the FcγRII-B2 by the presence these receptors and the cell types expressing them are shown. All of an additional exon in the intracellular region (indicated by yellow are immunoglobulin superfamily members except FcεRII, which triangle). This exon prevents the FcγRII-B1 from being internalized is a lectin and can form trimers. The exact chain composition after cross-linking. The binding affinities are taken from data on of any receptor can vary from one cell type to another. For human receptors. *Only some allotypes of FcγRII-A bind IgG2. †In example, FcγRIII in neutrophils is expressed as a molecule with a eosinophils, the molecular weight of

1	one cell type to another. For human receptors. *Only some allotypes of FcγRII-A bind IgG2. †In example, FcγRIII in neutrophils is expressed as a molecule with a eosinophils, the molecular weight of the CD89α chain is 70–100 kDa.

1	Antibody-coated viruses that enter the cytoplasm are cleared by a system that employs a novel class of Fc receptor called TRIM21 (tripartite motif-containing 21) that is expressed by a variety of immune and nonimmune cell types. TRIM21 is a cytosolic IgG receptor that has a higher affinity for IgG than any other Fc receptor, and it also has E3 ligase activity. When a virus that has bound IgG enters the cytoplasm, TRIM21 attaches to the antibody and uses its E3 ligase activity to ubiquitinate viral proteins. This leads to proteasomal degradation of virions in the cytosol before translation of virally encoded genes can occur. 10-22 Fc receptors on phagocytes are activated by antibodies bound to the surface of pathogens and enable the phagocytes to ingest and destroy pathogens.

1	10-22 Fc receptors on phagocytes are activated by antibodies bound to the surface of pathogens and enable the phagocytes to ingest and destroy pathogens. The most important Fc-bearing cells in humoral immune responses are the phagocytic cells of the monocytic and myelocytic lineages, particularly macrophages and neutrophils. Many bacteria are directly recognized, ingested, and destroyed by phagocytes, and these bacteria are not pathogenic in normal individuals. However, some bacterial pathogens have polysaccharide capsules, a large structure that lies outside the bacterial cell membrane and resists direct engulfment by phagocytes. Such pathogens become susceptible to phagocytosis only when they are coated with antibodies and complement that engage the Fcγ or Fcα receptors and the complement receptor CR1 on phagocytic cells, triggering bacterial uptake (Fig. 10.39). The stimulation

1	Macrophage membranes fuse, creating a membrane-enclosed vesicle, the phagosome Lysosomes fuse with these vesicles, delivering enzymes that degrade the bacterium When C3b binds to CR1 and antibody binds to Fc receptor, bacteria are phagocytosed Bacterium is coated with complement and IgG antibody FcreceptorsC3bbacteriummacrophagelysosomeCR1

1	Fig. 10.39 Fc and complement receptors on phagocytes trigger the uptake and degradation of antibody-coated bacteria. Many bacteria resist phagocytosis by macrophages and neutrophils. Antibodies bound to these bacteria, however, enable the bacteria to be ingested and degraded through the interaction of the multiple Fc domains arrayed on the bacterial surface with Fc receptors on the phagocyte surface. Antibody coating also induces activation of the complement system and the binding of complement components to the bacterial surface. These can interact with complement receptors (for example, CR1) on the phagocyte. Fc receptors and complement receptors synergize in inducing phagocytosis. Bacteria coated with IgG antibody and complement are therefore more readily ingested than those coated with IgG alone. Binding of Fc and complement receptors signals the phagocyte to increase the rate of phagocytosis, to fuse lysosomes with phagosomes, and to increase its bactericidal activity.

1	No activation of macrophage, no destruction of bacterium Activation of macrophage, leading to phagocytosis and destruction of bacterium Free immunoglobulin does not cross-link Fc receptors Aggregation of immunoglobulin on bacterial surface allows cross-linking of Fc receptors Fc receptors macrophage bacterium of phagocytosis by complement-coated antigens binding to complement receptors is particularly important early in the immune response, before isotype-switched antibodies have been made. Capsular polysaccharides belong to the TI-2 class of thymus-independent antigens, and can therefore stimulate the early production of IgM antibodies, which are very effective at activating the complement system. IgM binding to encapsulated bacteria thus triggers the opsonization of these bacteria by complement and their prompt ingestion and destruction by phagocytes bearing complement receptors. Recently, Fcα/μR was discovered as a receptor that binds both IgA and IgM. Fcα/μR is expressed primarily

1	and their prompt ingestion and destruction by phagocytes bearing complement receptors. Recently, Fcα/μR was discovered as a receptor that binds both IgA and IgM. Fcα/μR is expressed primarily on macrophages and B cells in the lamina propria of the intestine and in germinal centers. It is thought to have a role in the endocytosis of IgM antibody complexed with bacteria such as Staphylococcus aureus.

1	Phagocyte activation can initiate an inflammatory response that causes tissue damage, and so Fc receptors on phagocytes must be able to distinguish antibody molecules bound to a pathogen from the much larger number of free antibody molecules that are not bound to anything. This distinction is made possible by the aggregation of antibodies that occurs when they bind to multimeric antigens or to multivalent particulate antigens such as viruses and bacteria. Individual Fc receptors on a cell surface bind monomers of free antibody with low affinity, but when presented with an antibody-coated particle, the simultaneous binding by multiple Fc receptors results in binding of high avidity, and this is the principal mechanism by which bound antibodies are distinguished from free immunoglobulin (Fig. 10.40). The result is that Fc receptors enable cells to detect pathogens via the antibody molecules bound to them. Fc receptors therefore give phagocytic cells that lack intrinsic specificity the

1	10.40). The result is that Fc receptors enable cells to detect pathogens via the antibody molecules bound to them. Fc receptors therefore give phagocytic cells that lack intrinsic specificity the ability to identify and remove specific pathogens and their products from the extracellular spaces.

1	Fig. 10.40 Bound antibody is distinguishable from free immunoglobulin by its state of aggregation. Free immunoglobulin molecules bind most Fc receptors with very low affinity and cannot cross-link Fc receptors. Antigen-bound immunoglobulin, however, binds to Fc receptors with high avidity because several antibody molecules that are bound to the same surface bind to multiple Fc receptors on the surface of the accessory cell. This Fc receptor cross-linking sends a signal to activate the cell bearing it. With Fc receptors that have ITIMs, the result is inhibition.

1	Phagocytosis is greatly enhanced by interactions between the molecules coating an opsonized microorganism and receptors on the phagocyte surface. When an antibody-coated pathogen binds to Fcγ receptors, for example, the cell surface of the phagocyte extends around the surface of the pathogen through successive binding of the Fcγ receptors to the antibody Fc regions bound to the pathogen. This is an active process that is triggered by the stimulation of the Fcγ receptors. Phagocytosis leads to enclosure of the pathogen (or particle) in an acidified cytoplasmic vesicle—the phagosome. This then fuses with one or more lysosomes to generate a phagolysosome; lysosomal enzymes are released into the vesicle interior, where they destroy the bacterium (see Fig. 10.39). The process of intracellular killing by phagocytes was described in more detail in Chapter 3.

1	Some particles are too large for a phagocyte to ingest; parasitic worms are one example. In this case the phagocyte attaches to the surface of the antibody-coated parasite via its Fcγ, Fcα, or Fcε receptors, and the contents of the secretory granules or lysosomes of the phagocyte are released by exocytosis. The contents are discharged directly onto the surface of the parasite and damage it. Thus, stimulation of Fcγ and Fcα receptors can trigger either the internalization of external particles by phagocytosis or the externalization of internal vesicles by exocytosis. The principal leukocytes involved in the destruction of bacteria are macrophages and neutrophils, whereas large parasites such as helminths are usually attacked by eosinophils (Fig. 10.41), nonphagocytic cells that can bind antibody-coated parasites via several different Fc receptors, including the low-affinity Fcε receptor for IgE, CD23 (see Fig. 10.38). Cross-linking of these receptors by antibody-coated surfaces

1	bind antibody-coated parasites via several different Fc receptors, including the low-affinity Fcε receptor for IgE, CD23 (see Fig. 10.38). Cross-linking of these receptors by antibody-coated surfaces activates the eosinophil to release its granule contents, which include proteins toxic to parasites (see Fig. 14.10). Cross-linking by antigen of IgE bound to the high-affinity FcεRI on mast cells and basophils also results in exocytosis of their granule contents, as we describe below.

1	10-23 Fc receptors activate NK cells to destroy antibody-coated targets.

1	Virus-infected cells are usually destroyed by T cells that recognize virus-derived peptides bound to cell-surface MHC molecules. Cells infected by some viruses also signal the presence of intracellular infection by expressing on their surface proteins, such as viral envelope proteins, that can be recognized by antibodies originally produced against the virus particle. Host cells with antibodies bound to them can be killed by a specialized non-T, non-B cell of the lymphoid lineage called a natural killer cell (NK cell), which we met in Chapter 3. NK cells are large cells with prominent intracellular granules and make up a small fraction of peripheral blood lymphocytes. Although belonging to the lymphoid lineage, NK cells express a limited repertoire of invariant receptors recognizing a range of ligands that are induced on abnormal cells, such as those infected with viruses; NK cells are considered to be part of innate immunity (see Section 3-25). On recognition of a ligand, the NK cell

1	of ligands that are induced on abnormal cells, such as those infected with viruses; NK cells are considered to be part of innate immunity (see Section 3-25). On recognition of a ligand, the NK cell kills the target cell directly without the need for antibody. Although first discovered for their ability to kill some tumor cells, NK cells play an important role in innate immunity in the early stages of virus infection.

1	As well as this innate function, NK cells can recognize and destroy antibody-coated target cells in a process called antibody-dependent cell-mediated cytotoxicity (ADCC). This is triggered when antibody bound to the surface of a cell interacts with Fc receptors on the NK cell (Fig. 10.42). NK cells express the receptor FcγRIII (CD16), which recognizes the IgG1 and IgG3 subclasses. The killing mechanism is analogous to that of cytotoxic T cells, involving the release of cytoplasmic granules containing perforin and granzymes (see Section 9-31). ADCC has been shown to have a role in the defense against Fig. 10.41 Eosinophils attacking a schistosome larva in the presence of serum from an infected patient.

1	Fig. 10.41 Eosinophils attacking a schistosome larva in the presence of serum from an infected patient. Large parasites, such as worms, cannot be ingested by phagocytes; however, when the worm is coated with antibody, eosinophils can attack it through binding via their Fc receptors for IgG and IgA. Similar attacks on large targets can be mounted by other Fc receptor-bearing cells. These cells release the toxic contents of their granules directly onto the target, a process known as exocytosis. Photograph courtesy of A. Butterworth.

1	Fig. 10.42 Antibody-coated target cells can be killed by cells encounter cells coated with IgG antibody, they rapidly kill the NK cells in antibody-dependent cell-mediated cytotoxicity target cell. ADCC is only one way in which NK cells can contribute to (ADCC). NK cells (see Chapter 3) are large granular non-T, non-B host defense. lymphoid cells that have FcγRIII (CD16) on their surface. When these infection by viruses, and represents another mechanism by which antibodies can direct an antigen-specific attack by an effector cell that itself lacks specificity for antigen. 10-24 Mast cells and basophils bind IgE antibody via the high-affinity Fcε receptor.

1	10-24 Mast cells and basophils bind IgE antibody via the high-affinity Fcε receptor. When pathogens cross epithelial barriers and establish a local focus of infection, the host must mobilize its defenses and direct them to the site of pathogen growth. One way in which this is achieved is to activate the cells known as mast cells. Mast cells are large cells containing distinctive cytoplasmic granules that contain a mixture of chemical mediators, including histamine, that act rapidly to make local blood vessels more permeable. Mast cells have a distinctive appearance after staining with the dye toluidine blue that makes them readily identifiable in tissues (see Fig. 1.8). They are found in particularly high concentrations in vascularized connective tissues just beneath epithelial surfaces, including the submucosal tissues of the gastrointestinal and respiratory tracts and the dermis of the skin.

1	Mast cells have Fc receptors specific for IgE (FcεRI) and IgG (FcγRIII), and can be activated to release their granules and to secrete lipid inflammatory mediators and cytokines via antibody bound to these receptors. Most Fc receptors bind stably to the Fc regions of antibodies only when the antibodies have themselves bound antigen, and cross-linking of multiple Fc receptors is needed for strong binding. In contrast, FcεRI binds IgE antibody monomers with a very high affinity—approximately 1010 M–1. Thus, even at the low levels of circulating IgE present in normal individuals, a substantial portion of the total IgE is bound to the FcεRI on mast cells in tissues and on circulating basophils.

1	Although mast cells are usually stably associated with bound IgE, this on its own does not activate them, nor will the binding of monomeric antigen to the IgE. Mast-cell activation occurs only when the bound IgE is cross-linked by multivalent antigens. This signal activates the mast cell to release the contents of its granules, which occurs in seconds (Fig. 10.43), to synthesize and release lipid mediators such as prostaglandin D2 and leukotriene C4, and to secrete cytokines such as TNF-α, thereby initiating a local inflammatory response. Degranulation also releases stored histamine, which increases local blood flow and vascular permeability; this quickly leads to an accumulation Resting mast cell has granules that contain histamine and other infammatory mediators Multivalent antigen cross-links bound IgE antibody, causing release of granule contents Fc˜RI IgE antibody Resting mast cell Activated mast cell

1	Fig. 10.43 IgE antibody cross-linking on mast-cell surfaces leads to a rapid release of inflammatory mediators. Mast cells are large cells found in connective tissue and can be distinguished by their secretory granules, which contain many inflammatory mediators. They bind stably to monomeric IgE antibodies through the very high-affinity receptor FcεRI. Antigen cross-linking of the bound IgE antibody molecules triggers rapid degranulation, releasing inflammatory mediators into the surrounding tissue. These mediators trigger local inflammation, which recruits cells and proteins required for host defense to sites of infection. These cells are also triggered during allergic reactions when allergens bind to IgE on mast cells. Photographs courtesy of A.M. Dvorak.

1	of fluid and blood proteins, including antibodies, in the surrounding tissue. Shortly afterward there is an influx of blood-borne cells such as neutrophils and, later, monocytes, eosinophils, and effector lymphocytes. This influx can last from a few minutes to a few hours and produces an inflammatory response at the site of infection. Thus, mast cells are part of the front-line host defenses against pathogens that enter the body across epithelial barriers. They are also of medical importance because of their involvement in IgE-mediated allergic responses, which are discussed in Chapter 14. In allergic responses, mast cells are activated in the way described above by exposure to normally innocuous antigens (allergens), such as pollen, to which the individual has previously mounted a sensitizing immune response that produces allergen-specific IgE. 10-25 IgE-mediated activation of accessory cells has an important role in resistance to parasite infection.

1	Mast cells are thought to serve at least three important functions in host defense. First, their location near body surfaces allows them to recruit both pathogen-specific elements, such as antigen-specific lymphocytes, and nonspecific effector elements, such as neutrophils, macrophages, basophils, and eosinophils, to sites where infectious agents are most likely to enter the internal milieu. Second, the inflammation they cause increases the flow of lymph from sites of antigen deposition to the regional lymph nodes, where naive lymphocytes are first activated. Third, the ability of mast-cell products to trigger muscular contraction can contribute to the physical expulsion of pathogens from the lungs or the gut. Mast cells respond rapidly to the binding of antigen to surface-bound IgE antibodies, and their activation leads to the initiation of an inflammatory response and the recruitment and activation of basophils and eosinophils, which contribute further to the inflammatory response

1	and their activation leads to the initiation of an inflammatory response and the recruitment and activation of basophils and eosinophils, which contribute further to the inflammatory response (see Chapter 14). There is increasing evidence that such IgE-mediated responses are crucial to defense against parasite infestation.

1	A role for mast cells in the clearance of parasites is suggested by the accumulation of mast cells in the intestine, known as mastocytosis, that accompanies helminth infection, and by observations in W/WV mutant mice, which have a profound mast-cell deficiency caused by a mutation in the gene c-kit. These mutant mice show impaired clearance of the intestinal nematodes Trichinella spiralis and Strongyloides species. Clearance of Strongyloides is even more impaired in W/WV mice that lack IL-3 and so also fail to produce basophils. Thus, both mast cells and basophils seem to contribute to defense against these helminth parasites.

1	Other evidence points to the importance of IgE antibodies and eosinophils in the defense against parasites. Infection with certain types of multicellular parasites, particularly helminths, is strongly associated with the production of IgE antibodies and the presence of abnormally large numbers of eosinophils (eosinophilia) in blood and tissues. Furthermore, experiments in mice show that depletion of eosinophils by polyclonal anti-eosinophil antisera increases the severity of infection with the parasitic helminth Schistosoma mansoni. Eosinophils seem to be directly responsible for helminth destruction; examination of infected tissues shows degranulated eosinophils adhering to helminths, and experiments in vitro have shown that eosinophils can kill S. mansoni in the presence of anti-schistosome IgG or IgA antibodies (see Fig. 10.41).

1	The role of IgE, mast cells, basophils, and eosinophils can also be seen in resistance to the feeding of blood-sucking ixodid ticks. Skin at the site of a tick bite has degranulated mast cells and an accumulation of degranulated basophils and eosinophils, an indicator of recent activation. Subsequent resistance to feeding by these ticks develops after the first exposure, suggesting a specific immunological mechanism. Mice deficient in mast cells show no such acquired resistance to ticks, and in guinea pigs the depletion of either basophils or eosinophils by specific polyclonal antibodies also reduces resistance to tick feeding. Finally, experiments in mice showed that resistance to ticks is mediated by specific IgE antibody. Thus, many clinical studies and experiments support a role for this system of IgE bound to the high-affinity FcεRI in host resistance to pathogens that enter across epithelia or exoparasites such as ticks that breach it. Summary.

1	Antibody-coated pathogens are recognized by effector cells through Fc receptors that bind to an array of constant regions (Fc portions) provided by the pathogen-bound antibodies. Binding activates the cell and triggers destruction of the pathogen, through either phagocytosis, granule release, or both. Fc receptors comprise a family of proteins, each of which recognizes immunoglobulins of particular isotypes. Fc receptors on macrophages and neutrophils recognize the constant regions of IgG or IgA antibodies bound to a pathogen and trigger the engulfment and destruction of such bacteria. Binding to the Fc receptor also induces the production of microbicidal agents in the intracellular vesicles of the phagocyte. Eosinophils are important in the elimination of parasites too large to be engulfed; they bear Fc receptors specific for the constant region of IgG, as well as receptors for IgE; aggregation of these receptors triggers the release of toxic substances onto the surface of the

1	engulfed; they bear Fc receptors specific for the constant region of IgG, as well as receptors for IgE; aggregation of these receptors triggers the release of toxic substances onto the surface of the parasite. NK cells, tissue mast cells, and blood basophils also release their granule contents when their Fc receptors are engaged. The high-affinity receptor for IgE is expressed constitutively by mast cells and basophils. It differs from other Fc receptors in that it can bind free monomeric antibody, thus enabling an immediate response to pathogens at their site of first entry into the tissues. When IgE bound to the surface of a mast cell is aggregated by binding to antigen, it triggers the release of histamine and many other mediators that increase the blood flow to sites of infection; it thereby recruits antibodies and effector cells to these sites. Mast cells are found principally below epithelial surfaces of the skin and beneath the basement membrane of the digestive and respiratory

1	recruits antibodies and effector cells to these sites. Mast cells are found principally below epithelial surfaces of the skin and beneath the basement membrane of the digestive and respiratory tracts. Their activation by innocuous substances is responsible for many of the symptoms of acute allergic reactions, as will be described in Chapter 14.

1	Summary to Chapter 10.

1	The humoral immune response to infection involves the production of antibody by plasma cells derived from B lymphocytes, the binding of this antibody to the pathogen, and the elimination of the pathogen by phagocytic cells and molecules of the humoral immune system. The production of antibody usually requires the action of helper T cells specific for a peptide fragment of the antigen recognized by the B cell, a phenomenon called linked recognition. An activated B cell first moves to the T-zone–B-zone boundary in secondary lymphoid tissues, where it may encounter its cognate T cell and begin to proliferate. Some B cells become plasmablasts, while others move to the germinal center, where somatic hypermutation and class switch recombination take place. B cells that bind antigen with the highest affinity are selected for survival and further differentiation, leading to affinity maturation of the antibody response. Cytokines made by helper T cells direct class switching, leading to the

1	affinity are selected for survival and further differentiation, leading to affinity maturation of the antibody response. Cytokines made by helper T cells direct class switching, leading to the production of antibody of various classes that can be distributed to various body compartments.

1	IgM antibodies are produced early in an infection by conventional B cells and are also made in the absence of infection by subsets of nonconventional B cells in particular locations (as natural antibodies). IgM has a major role in protecting against infection in the bloodstream, whereas isotypes secreted later in an adaptive immune response, such as IgG, diffuse into the tissues. Antigens that have highly repeating antigenic determinants and that contain mitogens— called TI antigens—can elicit IgM and some IgG independently of T-cell help, and this provides an early protective immune response. Multimeric IgA is produced in the lamina propria and is transported across epithelial surfaces, whereas IgE is made in small amounts and binds avidly to receptors on the surface of basophils and mast cells.

1	Antibodies that bind with high affinity to critical sites on toxins, viruses, and bacteria can neutralize them. However, pathogens and their products are destroyed and removed from the body largely through uptake into phagocytes and degradation inside these cells. Antibodies that coat pathogens bind to Fc receptors on phagocytes, which are thereby triggered to engulf and destroy the pathogen. Binding of antibody C regions to Fc receptors on other cells leads to the exocytosis of stored mediators; this is particularly important in parasite infections, in which Fcε-expressing mast cells are triggered by the binding of antigen to IgE antibody to release inflammatory mediators directly onto parasite surfaces. Antibodies can also initiate the destruction of pathogens by activating the complement system. Complement components can opsonize pathogens for uptake by phagocytes, and recruit phagocytes to sites of infection. Receptors for complement components and Fc receptors often synergize in

1	system. Complement components can opsonize pathogens for uptake by phagocytes, and recruit phagocytes to sites of infection. Receptors for complement components and Fc receptors often synergize in activating the uptake and destruction of pathogens and immune complexes. Thus, the humoral immune response is targeted to the infecting pathogen through the production of specific antibody; however, the effector actions of that antibody are determined by its heavy-chain isotype.

1	Questions. 10.1 Multiple Choice: Which of the following is not an antibody effector function? A. Opsonization B. Neutralization C. Complement activation D. Linked recognition E. NK-cell cytotoxicity F. Mast-cell degranulation 10.2 Short Answer: The Haemophilus influenzae type b (Hib) vaccine was initially composed only of the polysaccharide capsule of the organism, but this failed to mount potent antibody responses. Directly conjugating the Hib polysaccharide to a tetanus or diphtheria toxoid, however, yielded very potent antibody responses to Hib, and is the current vaccine formulation. Indicate which immunological phenomenon is taken advantage of by conjugating the Hib capsule-derived polysaccharide to a toxoid, and how it works to elicit a potent antibody response.

1	10.3 Matching: During T-dependent antibody responses, numerous receptor/ligand interactions and cytokine signaling events occur between TFH cells and activated B cells. For the following list of surface receptors/ligands and cytokines, indicate whether they are produced by T cells (T), B cells (B), both (TB), or neither (N) in this context. A. IL-21 B. ICOSL C. CD40L D. CD30L E. Peptide:MHC II F. CCL21 G. SLAM 10.4 Matching: Match the human disease to the associated genetic defect. A. X-linked i. Translesion polymerase lymphoproliferative disorder Polη B. Hyper IgM type 2 ii. ATM (a DNA-PKcsimmunodeficiency family kinase) C. Xeroderma pigmentosum iii. SLAM-associated protein (SAP) D. Ataxia telangiectasia iv. Activation-induced cytidine deaminase (AID) 10.5 Matching: Indicate whether the following properties apply to IgA, IgD, IgE, IgG, and/or IgM. A. First produced during humoral response B. Monomeric (predominantly) C. Dimeric (predominantly)

1	A. First produced during humoral response B. Monomeric (predominantly) C. Dimeric (predominantly) D. Pentameric (predominantly) E. Contains a J chain F. Capable of eliciting complement deposition G. Most abundant in mucosal surfaces and secretions H. Low-affinity I. Bound onto mast cells J. Binds to polymeric immunoglobulin receptor (pIgR) K. Binds the neonatal Fc receptor (FcRn) 10.6 Short Answer: How is TRIM21, a novel class of Fc receptor, different from other Fc receptors? 10.7 Multiple Choice: Which of the following functions is not elicited by antibody binding to Fcγ receptors? A. Antibody-dependent cell-mediated cytotoxicity (ADCC) via NK cells B. Phagocytosis by neutrophils C. Mast-cell degranulation D. Downregulation of B-cell activity E. Ingestion of immune complexes by dendritic cells 10.8 Multiple Choice: Which of the following is a false statement?

1	C. Mast-cell degranulation D. Downregulation of B-cell activity E. Ingestion of immune complexes by dendritic cells 10.8 Multiple Choice: Which of the following is a false statement? A. Naive B-cell survival in follicles is dependent on BAFF, which signals through BAFF-R, TACI, and BCMA to induce Bcl-2 expression. B. Subcapsular sinuses of lymph nodes and marginal sinuses of the spleen are functionally similar areas filled with specialized macrophages that retain but do not digest antigens. C. ICOS signaling in T cells is essential for their completion of TFH differentiation and expression of the transcription factors Bcl-6 and c-Maf. D. Both plasmablasts and plasma cells express B7 co-stimulatory molecules, MHC class II molecules, and high levels of B-cell receptors. E. TFH cells determine the choice of isotype for class switching in T-dependent antibody responses.

1	E. TFH cells determine the choice of isotype for class switching in T-dependent antibody responses. 10.9 True or False: Germinal centers contain a light and a dark zone. In the light zone, B cells proliferate extensively and are called centroblasts. They are maintained there by CXCL12–CXCR4 chemokine signaling and undergo somatic hypermutation leading to affinity maturation and class switching. In the dark zone, B cells cease proliferation and are called centrocytes. Here, they are maintained by CXCL13–CXCR5 chemokine signaling, express higher levels of B-cell receptor, and interact extensively with TFH cells. 10.10 Multiple Choice: Choose the correct statement: A. R-loops are structures formed during somatic hypermutation that promote accessibility of the immunoglobulin V regions to AID. B. APE1 removes deaminated cytosine to create an abasic residue that results in the random insertion of a base during the next round of DNA replication.

1	B. APE1 removes deaminated cytosine to create an abasic residue that results in the random insertion of a base during the next round of DNA replication. C. Frameshift mutations during class switch recombination do not occur because switch regions lie in introns. D. The error-prone MSH2/6 polymerase repairs DNA lesions and causes mutations that promote somatic hypermutation. 10.11Fill-in-the-Blanks: Fc receptors diversify the effector functions of the distinct antibody isotypes. Most Fc receptors can bind the Fc regions of antibodies with _________ affinity. In contrast, FcεRI binds with ________ affinity. Multivalent antigen-bound IgE can bind _________ in mast cells and cause release of lipid mediators such as _________ and _________. Mast cells also degranulate in response to cross-linking of the FC receptor-bound IgE, which causes release of __________, and as a consequence local blood flow and __________ are increased, initiating an inflammatory response. General references.

1	General references. Batista, F.D., and Harwood, N.E.: The who, how and where of antigen presentation to B cells. Nat. Rev. Immunol. 2009, 9:15–27. Nimmerjahn, F., and Ravetch, J.V.: Fcγ receptors as regulators of immune responses. Nat. Rev. Immunol. 2008, 8:34–47. Rajewsky, K.: Clonal selection and learning in the antibody system. Nature 1996, 381:751–758. Section references. 10-1 Activation of B cells by antigen involves signals from the B-cell receptor and either TFH cells or microbial antigens. Crotty, S.: T follicular helper cell differentiation, function, and roles in disease. Immunity 2014, 41:529–542. Maglione, P.J., Simchoni, N., Black, S., Radigan, L., Overbey, J.R., Bagiella, E., Bussel, J.B., Bossuyt, X., Casanova, J.L., Meyts, I., et al.: IRAK-4and MyD88 deficiencies impair IgM responses against T-independent bacterial antigens. Blood 2014, 124:3561–3571. Pasare, C., and Medzhitov, R.: Control of B-cell responses by Toll-like receptors. Nature 2005, 438:364–368.

1	Pasare, C., and Medzhitov, R.: Control of B-cell responses by Toll-like receptors. Nature 2005, 438:364–368. Vijayanand, P., Seumois, G., Simpson, L.J., Abdul-Wajid, S., Baumjohann, D., Panduro, M., Huang, X., Interlandi, J., Djuretic, I.M., Brown, D.R., et al.: Interleukin-4 production by follicular helper T cells requires the conserved Il4 enhancer hypersensitivity site V. Immunity 2012, 36:175–187. 10-2 Linked recognition of antigen by T cells and B cells promotes robust antibody responses. Barrington, R.A., Zhang, M., Zhong, X., Jonsson, H., Holodick, N., Cherukuri, A., Pierce, S.K., Rothstein, T.L., and Carroll, M.C.: CD21/CD19 coreceptor signaling promotes B cell survival during primary immune responses. J. Immunol. 2005, 175:2859–2867.

1	Eskola, J., Peltola, H., Takala, A.K., Kayhty, H., Hakulinen, M., Karanko, V., Kela, E., Rekola, P., Ronnberg, P.R., Samuelson, J.S., et al.: Efficacy of Haemophilus influenzae type b polysaccharide-diphtheria toxoid conjugate vaccine in infancy. N.Engl. J. Med. 1987, 317:717–722. Kalled, S.L.: Impact of the BAFF/BR3 axis on B cell survival, germinal center maintenance and antibody production. Semin. Immunol. 2006, 18:290–296. Mackay, F., and Browning, J.L.: BAFF: a fundamental survival factor for B cells. Nat. Rev. Immunol. 2002, 2:465–475. Mackay, F., and Schneider, P.: Cracking the BAFF code. Nat. Rev. Immunol. 2009, 9:491–502. MacLennan, I.C.M., Gulbranson-Judge, A., Toellner, K.M., Casamayor-Palleja, M., Chan, E., Sze, D.M.Y., Luther, S.A., and Orbea, H.A.: The changing preference of T and B cells for partners as T-dependent antibody responses develop. Immunol. Rev. 1997, 156:53–66.

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1	Hogan, S.P., Rosenberg, H.F., Moqbel, R., Phipps, S., Foster, P.S., Lacy, P., Kay, A.B., and Rothenberg, M.E.: Eosinophils: biological properties and role in health and disease. Clin. Exp. Allergy 2008, 38:709–750. Karakawa, W.W., Sutton, A., Schneerson, R., Karpas, A., and Vann, W.F.: Capsular antibodies induce type-specific phagocytosis of capsulated Staphylococcus aureus by human polymorphonuclear leukocytes. Infect. Immun. 1986, 56:1090–1095. 10-23 Fc receptors activate NK cells to destroy antibody-coated targets. Chung, A.W., Rollman, E., Center, R.J., Kent, S.J., and Stratov, I.: Rapid degranulation of NK cells following activation by HIV-specific antibodies. J. Immunol. 2009, 182:1202–1210. Lanier, L.L., and Phillips, J.H.: Evidence for three types of human cytotoxic lymphocyte. Immunol. Today 1986, 7:132. Leibson, P.J.: Signal transduction during natural killer cell activation: inside the mind of a killer. Immunity 1997, 6:655–661.

1	Leibson, P.J.: Signal transduction during natural killer cell activation: inside the mind of a killer. Immunity 1997, 6:655–661. Sulica, A., Morel, P., Metes, D., and Herberman, R.B.: Ig-binding receptors on human NK cells as effector and regulatory surface molecules. Int. Rev. Immunol. 2001, 20:371–414. Takai, T.: Multiple loss of effector cell functions in FcRγ-deficient mice. Int. Rev. Immunol. 1996, 13:369–381. 10-24 Mast cells and basophils bind IgE antibody via the high-affinity Fcε receptor. Beaven, M.A., and Metzger, H.: Signal transduction by Fc receptors: the FcεRI case. Immunol. Today 1993, 14:222–226. Kalesnikoff, J., Huber, M., Lam, V., Damen, J.E., Zhang, J., Siraganian, R.P., and Krystal, G.: Monomeric IgE stimulates signaling pathways in mast cells that lead to cytokine production and cell survival. Immunity 2001, 14:801–811. Sutton, B.J., and Gould, H.J.: The human IgE network. Nature 1993, 366:421–428.

1	Sutton, B.J., and Gould, H.J.: The human IgE network. Nature 1993, 366:421–428. 10-25 IgE-mediated activation of accessory cells has an important role in resistance to parasite infection. Capron, A., Riveau, G., Capron, M., and Trottein, F.: Schistosomes: the road from host-parasite interactions to vaccines in clinical trials. Trends Parasitol. 2005, 21:143–149. Grencis, R.K.: Th2-mediated host protective immunity to intestinal nematode infections. Philos. Trans. R. Soc. Lond. B 1997, 352:1377–1384. Grencis, R.K., Else, K.J., Huntley, J.F., and Nishikawa, S.I.: The in vivo role of stem cell factor (c-kit ligand) on mastocytosis and host protective immunity to the intestinal nematode Trichinella spiralis in mice. Parasite Immunol. 1993, 15:55–59.

1	Kasugai, T., Tei, H., Okada, M., Hirota, S., Morimoto, M., Yamada, M., Nakama, A., Arizono, N., and Kitamura, Y.: Infection with Nippostrongylus brasiliensis induces invasion of mast cell precursors from peripheral blood to small intestine. Blood 1995, 85:1334–1340. Ushio, H., Watanabe, N., Kiso, Y., Higuchi, S., and Matsuda, H.: Protective immunity and mast cell and eosinophil responses in mice infested with larval Haemaphysalis longicornis ticks. Parasite Immunol. 1993, 15:209–214. Integrated Dynamics of Innate and Adaptive Immunity

1	Throughout this book we have examined the separate ways in which the innate and the adaptive immune responses protect against invading pathogens. In this chapter, we consider how the cells and molecules of the immune system work as an integrated defense system to eliminate or control different types of infectious agents, and also how the adaptive immune system provides long-lasting protective immunity. In Chapters 2 and 3, we saw how innate immunity is brought into play in the earliest phases of an infection and is probably sufficient to prevent colonization of the body by most of the microorganisms encountered in the environment. We also introduced innate lymphoid cells (ILCs), which, although lacking antigen-specific receptors, share overlapping developmental and functional features with effector CD4 T-cell subsets and cytotoxic CD8 T cells and act early during infection to generate distinct types of immune responses that target specific types of pathogens. Unlike naive T and B

1	with effector CD4 T-cell subsets and cytotoxic CD8 T cells and act early during infection to generate distinct types of immune responses that target specific types of pathogens. Unlike naive T and B cells, ILCs reside in barrier tissues, such as the intestinal and respiratory mucosae, where they are poised to respond rapidly to pathogens to impair or eliminate their spread.

1	However, most pathogens have developed strategies to evade innate immune defenses and establish a focus of infection. In these circumstances, the innate immune response sets the scene for the induction of an adaptive immune response, which is orchestrated by signals that emanate from innate sensor cells and is coordinated with innate effector cells to bring about pathogen clearance. In the primary immune response, which occurs against a pathogen encountered for the first time, ILCs respond to innate sensor cells to mount a rapid response over the first few hours to days of pathogen invasion. Concurrent with this response, clonal expansion and differentiation of naive lymphocytes into effector T cells and antibody-secreting B cells is initiated and guided by innate sensor cells and ILCs. However, the adaptive response requires several days to weeks to fully mature, largely due to the rarity of antigen-specific precursor cells. Following expansion and differentiation in the secondary

1	However, the adaptive response requires several days to weeks to fully mature, largely due to the rarity of antigen-specific precursor cells. Following expansion and differentiation in the secondary lymphoid tissues, effector T cells migrate to sites of infection and, along with pathogen-specific antibodies, enhance the effector functions of innate immune cells, and, in most cases, effectively target the pathogen for elimination (Fig. 11.1).

1	4. Memory phase 3. Effector phase 1. Establishment of infection 2. Inductive phase Level of microorganism Threshold level of antigen to activate adaptive immune response Duration of infection Integration of innate and adaptive immunity in response to specific types of pathogens. Effector T cells augment the effector functions of innate immune cells. Immunological memory.

1	Effector T cells augment the effector functions of innate immune cells. Immunological memory. Fig. 11.1 The course of a typical acute infection that is cleared by an adaptive immune response. 1. The infectious agent colonizes and its numbers increase as it replicates. The innate response is initiated immediately following detection of the pathogen. 2. When numbers of the pathogen exceed the threshold dose of antigen required for an adaptive response, the response is initiated; the pathogen continues to grow, restrained by responses of the innate immune system. At this stage, immunological memory also starts to be induced. 3. After 4–7 days, effector cells and molecules of the adaptive response start to clear the infection. 4. When the infection has been cleared and the dose of antigen has fallen below the response threshold, the response ceases, but antibody, residual effector cells, and immunological memory provide lasting protection against reinfection in most cases.

1	During this period, specific immunological memory is also established by adaptive immune cells. This ensures a rapid reinduction of antigen-specific antibody and effector T cells on encounter with the same pathogen during a secondary immune response, thus providing long-lasting and often lifelong protection against the pathogen. Immunological memory is discussed in the last part of the chapter. Memory responses differ in several ways from primary responses. We discuss the reasons for this, and what is known about how immunological memory is maintained. Integration of innate and adaptive immunity in response to specific types of pathogens.

1	The immune response is a dynamic process, and both its nature and its intensity change over time. It begins with the antigen-independent responses of innate immunity and becomes both more focused on the pathogen and more powerful as the antigen-specific adaptive immune response matures. The character of the response differs contingent on the type of pathogen. Different types of pathogens (for example, intracellular and extracellular bacteria, viruses, helminthic parasites, and fungi) elicit different types of immune response (for example, type 1, 2, or 3), so that the most effective immune response is induced for effective elimination of the pathogen. The innate immune system not only anticipates and initiates the adaptive Tand B-cell responses, but continues to provide effector cells and reinforcing pathways of the different types of immunity throughout infection. Early in the response, different subsets of innate lymphoid cells (ILCs) are activated by cytokines produced by innate

1	reinforcing pathways of the different types of immunity throughout infection. Early in the response, different subsets of innate lymphoid cells (ILCs) are activated by cytokines produced by innate sensor cells. This early response acts to constrain pathogen entry at the initial site of infection to prevent dissemination while the adaptive response develops. However, the more sensitive and specific actions of effector T cells and class-switched, affinity-matured antibodies are often required for the complete elimination of infection, or sterilizing immunity. In this part of the chapter, we provide an overview of how the different phases of an immune response are orchestrated in space and time and then discuss how distinct cytokines from innate sensor cells activate different innate lymphoid cell subsets to restrain pathogen invasion and direct pathogen-specific defenses while the adaptive response is developing.

1	11-1 The course of an infection can be divided into several distinct phases. Although some of the microbe-associated molecular patterns (MAMPs) of different types of pathogens are shared, others are not, and these differences underlie the induction of distinct patterns of innate and adaptive immunity that can be broadly grouped into type 1, type 2, and type 3 responses, as will be discussed below. Irrespective of the inciting pathogen and the pattern of immune response it provokes, however, the tempo of the host response is similar and can be broken down into various stages (Fig. 11.1, and see Fig. 3.38).

1	In the first stage of infection, a new host is exposed to infectious particles either shed by an infected individual or present in the environment. The numbers, route, mode of transmission, and stability of an infectious agent outside the host determine its infectivity. The first contact with a new host occurs through an epithelial surface, such as the skin or the mucosal surfaces of the respiratory, gastrointestinal, or urogenital tracts. After making contact, an infectious agent must establish a focus of infection. It must either adhere to the epithelial surface and colonize it, or penetrate it to replicate in the tissues (Fig. 11.2). Bites by arthropods (insects and ticks) and wounds breach the epidermal barrier and help some microorganisms gain entry through the skin. Integration of innate and adaptive immunity in response to specific types of pathogens.

1	Integration of innate and adaptive immunity in response to specific types of pathogens. Fig. 11.2 Infections and the responses to them can be divided antigen and antigen-loaded dendritic cells to lymphatics (second into a series of stages. These are illustrated here for a pathogenic panel) and thence to local lymph nodes (third panel). This leads to microorganism (red) entering across a wound in an epithelium. The an adaptive immune response in the lymph node that involves the microorganism first adheres to epithelial cells and then invades activation and further differentiation of B cells and T cells with the beyond the epithelium into underlying tissues (first panel). A local eventual production of antibody and effector T cells, which clear the innate immune response helps to contain the infection, and delivers infection (fourth panel).

1	Only when a microorganism has successfully established a focus of infection in the host does disease occur (see Fig. 11.2). With few exceptions, little damage will be caused unless the pathogen spreads from the original focus or secretes toxins that spread to other parts of the body. Extracellular pathogens spread by direct extension of the infection through the lymphatics or the bloodstream. Spread into the bloodstream usually occurs only after the lymphatic system has been overwhelmed. Obligate intracellular pathogens spread from cell to cell; they do so either by direct transmission from one cell to the next or by release into the extracellular fluid and reinfection of both adjacent and distant cells. Facultative intracellular pathogens can do the same after a period of survival in the extracellular environment. In contrast, some of the bacteria that cause gastroenteritis exert their effects without spreading into the tissues. They establish a site of infection on the luminal

1	the extracellular environment. In contrast, some of the bacteria that cause gastroenteritis exert their effects without spreading into the tissues. They establish a site of infection on the luminal surface of the epithelium lining the gut and cause pathology by damaging the epithelium or by secreting toxins that cause damage either in situ or after crossing the epithelial barrier and entering the circulation.

1	The establishment of a focus of infection in tissues and the response of the innate immune system produce changes in the immediate environment. Many microorganisms are repelled or kept in check at this stage by innate defenses, which are triggered by stimulation of the various germlineencoded pattern recognition receptors expressed by innate sensor cells—such as epithelial cells, tissue-resident mast cells, macrophages, and dendritic cells (see Chapters 2 and 3). Cytokines and chemokines produced by pathogen-activated innate sensor cells initiate local inflammation, and also activate ILCs. These responses are activated within minutes to hours, and are sustained for at least several days. The inflammatory response is induced through activation of the endothelium of local post-capillary venules (see Fig. 3.31). This leads to the recruitment of circulating innate effector cells, particularly neutrophils and monocytes, thereby increasing the numbers of phagocytes available for microbe

1	venules (see Fig. 3.31). This leads to the recruitment of circulating innate effector cells, particularly neutrophils and monocytes, thereby increasing the numbers of phagocytes available for microbe clearance. As monocytes enter tissue and become activated, additional inflammatory cells are attracted into the infected tissue so that the inflammatory response is maintained and reinforced. Leakiness of the inflamed endothelium also leads to the influx of serum proteins, including complement, activation of which in a primary infection occurs mainly via the alternative and lectin pathways (see Fig. 2.15). This results in the production of the anaphylatoxins C3a and C5a, which further activate the vascular endothelium; and C3b, which opsonizes microbes for more effective clearance by recruited phagocytes. This early phase of the inflammatory response is largely nonspecific for the type of pathogen.

1	Concordant with the production of pro-inflammatory cytokines such as TNF-α, which activate nonspecific inflammation, innate sensor cells produce additional cytokines that differentially activate specific subsets of ILCs within the first hours of an infection. This is due to the expression of unique MAMPs—or unique combinations of MAMPs—by different types of pathogens, which elicit different patterns of cytokines from innate sensor cells. This has important consequences in directing the type of immune response that will be mounted against the pathogen, as subsets of ILCs are differentially activated to produce their own effector cytokines and chemokines contingent on the pattern of cytokines produced by innate sensor cells (Fig. 11.3). The products of activated ILCs amplify and coordinate local innate responses that are better tailored to resist specific types of pathogens, and also alter the recruitment and maturation of different myelomonocytic innate effector cells (that is,

1	local innate responses that are better tailored to resist specific types of pathogens, and also alter the recruitment and maturation of different myelomonocytic innate effector cells (that is, granulocytes like neutrophils, eosinophils, and basophils, or monocytes) at the site of infection. Cytokines produced by ILCs may also guide the development of naive T cells into distinct effector subsets (for example, TH1, TH2, or TH17 cells)—either directly by acting on naive T cells themselves or indirectly by modulating the activation of dendritic cells that migrate to regional lymphoid tissues to prime naive T cells. In this way, ILCs perform an important bridging function during the first few days of an immune response, by both providing for innate defense and influencing the type of adaptive response that follows.

1	Adaptive immunity is triggered when an infection eludes or overwhelms the innate defense mechanisms and generates a threshold level of antigen (see Fig. 11.1). Adaptive immune responses are then initiated in the local lymphoid Normal mice and humans Mice and humans lacking innate immunity (PMN– , MAC–) SCID or RAG-defcient mice and humans (PMN+ , MAC+ , T/B–)

1	Integration of innate and adaptive immunity in response to specific types of pathogens. 449 tissue, in response to antigens presented by dendritic cells activated during the course of the innate immune response (see Fig. 11.2, second and third panels). Antigen-specific effector T cells and antibody-secreting B cells are generated by clonal expansion and differentiation over several days, during which innate responses orchestrated by ILCs ‘buy time’ for the adaptive response to mature. Within several days of infection, antigen-specific T cells and then antibodies are released into the blood, and from there can enter the site of infection (see Fig. 11.2, fourth panel). Adaptive responses are more powerful because antigen-specific targeting of innate effector mechanisms can eliminate pathogens more precisely. For example, antibodies can activate complement to directly kill pathogens; they can opsonize pathogens for enhanced phagocytosis; and they can arm Fc-bearing innate effector cells

1	more precisely. For example, antibodies can activate complement to directly kill pathogens; they can opsonize pathogens for enhanced phagocytosis; and they can arm Fc-bearing innate effector cells for release of microbicidal factors or recruit the cytocidal actions of natural killer (NK) cells—these last capabilities known as antibody-dependent cell-mediated cytotoxicity (ADCC). Effector CD8 T cells can directly kill antigen-bearing target cells via similar cytocidal cytotoxic actions, and effector CD4 T cells can directly release cytokines onto macrophages to enhance their microbicidal actions.

1	Resolution of an infection typically involves complete clearance of the pathogen, and thus the source of antigens, over the course of days to weeks, following which most effector lymphocytes die—a stage known as clonal contraction (see Section 11-16). What remain are long-lived antibody-producing plasma cells that sustain circulating antibodies for months to years, and small numbers of memory B and T cells that may also persist for years, poised for an accelerated adaptive response in the event of future encounters with the same pathogen. Thus, in addition to clearing the infectious agent, an effective adaptive immune response prevents reinfection. For some infectious agents, this protection is essentially absolute, whereas for others infection is only reduced or attenuated on reexposure to the pathogen.

1	It is not known how many infections are dealt with solely by the nonadaptive mechanisms of innate immunity, because such infections are eliminated early and produce little in the way of symptoms or pathology. Innate immunity does, however, seem to be essential for effective host defense, as shown by the progression of infection in mice that lack components of innate immunity but have an intact adaptive immune system (Fig. 11.4). Conversely, many infections can be curbed—but not cleared—in the absence of adaptive immunity.

1	After many types of infection, little or no residual pathology follows an effective primary adaptive response. In some cases, however, the infection itself, or the response it induces, causes significant tissue damage. In yet other cases, such as infection with cytomegalovirus or Mycobacterium tuberculosis, the pathogen is contained but not eliminated, and can persist in a latent form. If the adaptive immune response is later weakened, as it is in acquired immune deficiency syndrome (AIDS), these pathogens may resurface to cause virulent systemic infections. We will focus on the strategies used by certain pathogens to evade or subvert adaptive immunity, and thereby establish a persistent, or chronic, infection, in Chapter 13. 11-2 The effector mechanisms that are recruited to clear an infection depend on the infectious agent.

1	11-2 The effector mechanisms that are recruited to clear an infection depend on the infectious agent. Most infections ultimately engage both Tand B-cell-mediated adaptive immunity, and in many cases both are helpful in clearing or containing the pathogen and setting up protective immunity. However, the relative importance of the different effector mechanisms, and the effective classes of antibody involved, vary with different pathogens. An emerging concept is that there are different types of immune responses that are focused on the activation of distinct immune effector modules (see Section 1-19). In each type of immune response, a specific collection of innate and adaptive mechanisms act together Duration of infection

1	Duration of infection Fig. 11.4 The time course of infection in normal and immunodeficient mice and humans. The red curve shows the rapid growth of microorganisms in the absence of innate immunity, when macrophages (MAC) and polymorphonuclear leukocytes (PMN) are lacking. The green curve shows the course of infection in mice and humans that have innate immunity but have no T or B lymphocytes and so lack adaptive immunity. The yellow curve shows the normal course of an infection in immunocompetent mice or humans.

1	to eliminate a specific type of pathogen. Each effector module includes subsets of innate sensor cells, ILCs, effector T cells, and antibody isotypes, which coordinate with subsets of circulating or tissue-resident myelomonocytic cells whose microbicidal functions they recruit and enhance (Fig. 11.5). Circulating myelomonocytic cells are important innate effector cells that are targeted for heightened functions by ILCs, effector T cells, and antibodies following their recruitment into sites of infection. In their order of abundance in circulating blood, these include neutrophils, monocytes (which enter inflamed tissues and differentiate into activated macrophages), eosinophils, and basophils. Tissue-resident mast cells, which share many functions with basophils, are also targeted for heightened function.

1	It appears that each of the three major ILC and effector CD4 T-cell subsets (ILC1/ILC2/ILC3 and TH1/TH2/TH17, respectively) evolved to enhance and coordinate the functions of, and integrate adaptive immunity with, different arms of the myelomonocytic pathway for optimal eradication of different classes of pathogens: monocyte and macrophages are enhanced by TH1 cells; eosinophils, basophils, and mast cells by TH2 cells; and neutrophils by TH17 cells. The three major types of immune responses are controlled by cytokine and chemokine networks, as discussed below. Fig. 11.5 Integration of ILCs, T-cell subsets, and innate effector cells into immune effector modules. The major inductive and effector cytokines, and transcription factors (e.g., ID2, T-bet, GATA3, RORγt, and AHR), that are associated with each effector module are shown. See text for details.

1	Type 1 responses are characterized by the actions of group 1 ILCs (ILC1), TH1 cells, opsonizing IgG isotypes (for example, IgG1 and IgG2), and macrophages in response to intracellular pathogens, including intracellular bacteria, viruses, and parasites (see Fig. 11.5). Type 2 responses are characterized by the actions of group 2 ILCs (ILC2), TH2 cells, IgE, and the innate effector cells eosinophils, basophils, and tissue mast cells, the latter two of which are armed for function by IgE bound to surface Fcε receptors. Type 2 responses are induced by, and target, multicellular parasites, or helminths. Type 3 responses are characterized by the actions of group 3 ILCs (ILC3), TH17 cells, opsonizing IgG isotypes, and neutrophils in response to extracellular bacteria and fungi. It is the activation of different subsets of ILCs early in the innate response that sets the stage for polarized type 1, type 2, or type 3 responses. Unlike the effector CD4 T cells with which they share overlapping

1	of different subsets of ILCs early in the innate response that sets the stage for polarized type 1, type 2, or type 3 responses. Unlike the effector CD4 T cells with which they share overlapping functional features, ILCs do not require priming and differentiation in order to acquire their effector functions; hence they are able to respond rapidly to amplify the activities of resident and recruited innate effector cells. Here we will consider in more detail the induction and actions of ILC subsets, as these responses precede and are integrated with adaptive T-cell responses.

1	As discussed in Chapter 3, ILC1s and related NK cells are characterized by their production of IFN-γ in response to IL-12 and IL-18 produced by pathogen-activated dendritic cells and macrophages. Functionally, ILC1s and NK cells most closely resemble TH1 cells and CTLs, respectively. ILC1s lack the cytolytic granules that are characteristic of NK cells and CTLs, and appear to promote clearance of intracellular pathogens through their activation of infected macrophages by the release of IFN-γ. Thus, through their production of IL-12 and IL-18, macrophages can rapidly induce ILC1 production of IFN-γ, which acts back on the macrophage to induce its heightened killing of intracellular pathogens several days prior to the development and recruitment of TH1 cells. Moreover, the production of IFN-γ by ILC1s may contribute to the early polarization of TH1 cells, linking the effector function of these cells to the induction of the TH1 cell response that follows. Similarly, the rapid induction

1	by ILC1s may contribute to the early polarization of TH1 cells, linking the effector function of these cells to the induction of the TH1 cell response that follows. Similarly, the rapid induction of the cytolytic activity of NK cells enables the killing of a range of pathogen-infected cells, through the recognition of surface molecules that are expressed on the target cells (see Section 3-23), in advance of the antigen-driven development and deployment of cytolytic CD8 T cells. Also, similar to the effect on TH1 cells of IFN-γ production by ILC1s, the production of IFN-γ by activated NK cells may contribute to the enhanced differentiation of cytolytic CD8 T cells.

1	ILC2s that reside in mucosal tissues are preferentially activated by the cytokines thymic stromal lymphopoietin (TSLP), a STAT5-activating cytokine, and IL-33 or IL-25, each of which are produced in response to helminths. These cytokines are primarily produced by epithelial cells that sense molecular patterns common to helminths, such as chitin, a polysaccharide polymer of β-1,4-N-acetylglucosamine that is a widespread constituent of helminths, the exoskeletons of insects, and some fungi. Activated ILC2s rapidly produce large amounts of IL-13 and IL-5; IL-13 stimulates mucus production by goblet cells in the epithelium and mucosal smooth muscle contractions that facilitate worm expulsion; and IL-5 stimulates the production and activation of eosinophils that can kill worms. Unlike TH2 cells, with which they share functional features, ILC2s appear to produce little or no IL-4 in vivo, suggesting that they might not directly promote TH2 differentiation. However, eosinophils and basophils

1	with which they share functional features, ILC2s appear to produce little or no IL-4 in vivo, suggesting that they might not directly promote TH2 differentiation. However, eosinophils and basophils that are recruited by chemokines produced by ILC2s are activated to produce IL-4 in response to the IL-5 and IL-13 produced by ILC2s, possibly providing an indirect mechanism by which TH2 differentiation is directed by ILC2s. Moreover, IL-13 produced by ILC2s appears to regulate the activation and migration to regional lymphoid tissues of dendritic cells that promote TH2 differentiation, although it is unclear whether these dendritic cells can also produce IL-4.

1	ILC3s play a critical early role in defense against extracellular bacteria and fungi at barrier tissues. Similarly toTH17 cells, ILC3s are responsive to IL-23 and IL-1β; these cytokines elicit the production of IL-17 and IL-22, which promote early type 3 responses. IL-17 is a pro-inflammatory cytokine that acts on a variety of cells, including stromal cells, epithelial cells, and myeloid cells, to stimulate the production of other pro-inflammatory cytokines (for example, IL-6 and IL-1β), hematopoietic growth factors (G-CSF and GM-CSF), and chemokines that recruit neutrophils and monocytes. IL-22 acts on epithelial cells to induce their production of antimicrobial peptides (AMPs) and promote enhanced barrier integrity. As with other ILCs, the cytokines produced by ILC3s act indirectly via IL-6 and IL-1β in a positive feedback loop to enhance type 3 responses by increasing local production of IL-23 and IL-1β. Through induction of elevated IL-6, IL-1β, and IL-23, ILC3s may also promote

1	IL-6 and IL-1β in a positive feedback loop to enhance type 3 responses by increasing local production of IL-23 and IL-1β. Through induction of elevated IL-6, IL-1β, and IL-23, ILC3s may also promote the differentiation of TH17 cells in mucosal lymphoid tissues, where they can be found in substantial numbers.

1	In a further parallel with effector CD4 T cells, an important feature of ILCs is that they ‘license’ other innate immune cells for killing or expulsion of microbes, but they do not do so themselves. Instead, myelomonocytic cells, and even cells of the mucosal epithelium, are the agents of ILCs and effector CD4 T cells; they are recruited and/or activated through the pro-inflammatory cytokines and chemokines that these lymphoid cells produce. An exception is NK cells, which, like effector CD8 T cells, directly kill target cells that harbor intracellular pathogens. As we will discuss below, because of their ability to focus effector cytokines on antigen-bearing target cells and to induce B-cell maturation and the production of class-switched antibodies, effector CD4 T cells provide an additional layer of licensing of innate effector cells that increases their lethality and ability to achieve microbial clearance. Summary.

1	Summary. Integration of the innate and adaptive immune responses is required for effective protection of the host against pathogenic microorganisms. The responses of the innate immune system act early to restrain pathogens while simultaneously helping to initiate the adaptive immune response, which takes a longer time to fully develop. Different types of pathogens cause the activation of different patterns of cytokine production by innate sensor cells. This, in turn, promotes the activation of different patterns of innate lymphoid cells (ILCs), which recruit innate effector cells to sites of infection and contribute to the differentiation of parallel programs of CD4 T-cell differentiation. Coordination of the induction of different immune effector modules that are composed of related subsets of ILCs, innate effector cells, CD4 effector T cells, and class-switched antibodies underlies the different types of immunity directed against different types of pathogens.

1	Effector T cells augment the effector functions of innate immune cells.

1	In Chapter 9 we described how dendritic cells loaded with their antigen cargo migrate away from the infected tissue through the lymphatics to enter secondary lymphoid tissues, where they initiate the adaptive immune response. We discussed how CD8 T cells are primed to become cytotoxic effectors that are specialized for killing infected target cells that express MHC class I molecules. We also saw how transcription factor networks activated by specific cytokines direct the differentiation of naive CD4 T cells into distinct classes of CD4 effector T cells—TH1, TH2, and TH17 (see Fig. 9.31). In Chapter 10 we discussed the specialized role of TFH cells, which engage antigen-bearing B cells to control antibody class switching and B-cell maturation in germinal centers in the context of type 1, type 2, and type 3 responses. We now turn our attention to the specialized roles of subsets of effector CD4 T cells that emigrate from secondary lymphoid tissues after their differentiation to

1	1, type 2, and type 3 responses. We now turn our attention to the specialized roles of subsets of effector CD4 T cells that emigrate from secondary lymphoid tissues after their differentiation to orchestrate the functions of innate immune cells at sites of infection.

1	As discussed in the preceding sections, the pattern of cytokines produced by the innate immune response during the early course of infection is determined by how the microorganism influences the behavior of innate sensor cells and the different subsets of ILCs they engage. The local inflammatory conditions produced by these interactions have a major impact on how T cells differentiate during their initial contact with dendritic cells, thus determining the subsets of effector T cells that are generated (see Chapter 9). In turn, the recruitment of effector T cells to sites of infection sustains and amplifies innate effector cell responses initiated by ILCs through effector mechanisms that require antigen-specific recognition, whether through cell–cell contact between CD4 or CD8 T cells and cell targets bearing their cognate antigens, or via pathogen-specific antibodies. In this part of the chapter, we will discuss how the differentiation of effector CD4 T cells generated during the

1	and cell targets bearing their cognate antigens, or via pathogen-specific antibodies. In this part of the chapter, we will discuss how the differentiation of effector CD4 T cells generated during the adaptive immune response alters their expression of surface receptors and causes them to leave secondary lymphoid tissues and home to sites of infection. We will then consider how TH1, TH2, and TH17 cells interact with innate immune cells at sites of infection to bring about the clearance of the specific pathogens that elicit their development and recruitment. Finally, we will consider how the primary effector response is terminated as the pathogen is eliminated.

1	11-3 Effector T cells are guided to specific tissues and sites of infection by changes in their expression of adhesion molecules and chemokine receptors.

1	When naive T cells differentiate into effector T cells, changes occur in the expression of specific surface molecules that redirect their trafficking from T-cell zones into B-cell zones, in the case of TFH cells, or from lymphoid to nonlymphoid tissues, in the case of other effector T cells. During the 3–5 days required for differentiation of naive T cells into effector T cells within secondary lymphoid tissues, marked changes occur in the expression of these trafficking molecules, including alterations in the display of selectins and their ligands, integrins, and chemokine receptors. As we will see, some of these changes are generic, and are common to all CD4 and CD8 effector T cells. Others are tissue-specific, facilitating the recruitment of T cells back to the tissues in which they were primed. Yet others are T-cell subset-specific, particularly the patterns of chemokine receptor expression, which are important in directing TFH cells to germinal centers, where they provide help to

1	primed. Yet others are T-cell subset-specific, particularly the patterns of chemokine receptor expression, which are important in directing TFH cells to germinal centers, where they provide help to developing B cells, or in directing TH1, TH2, and TH17 cells to the same tissue sites as the myelomonocytic cells whose effector functions they will recruit and enhance.

1	Naive CD4 T cells that are activated by antigen and become TFH cells acquire expression of CXCR5 and lose expression of CCR7 and S1PR1, the receptor for the chemotactic lipid sphingosine 1-phosphate (see Section 9-7). The constitutive expression of CXCL13 by follicular dendritic cells establishes a gradient that attracts developing TFH cells first to the border of the T-cell zone with a B-cell follicle, where they can interact with B cells that present their cognate antigen, and then into the B-cell follicle, where they provide help to germinal center B cells. Unlike TFH cells, other effector CD4 and CD8 T cells must leave the lymphoid tissue in which they have developed to interact with myelomonocytic cells at sites of infection in nonlymphoid tissues. The exit of effector T cells is induced by their loss of CCR7 and their reexpression of S1PR1. S1PR1 is normally rapidly downregulated by CD69 following antigenic stimulation

1	Fig. 11.6 Effector T cells change their surface molecules, allowing them to home to sites of infection. Naive T cells home to lymph nodes through the binding of L-selectin to sulfated carbohydrates displayed by various proteins, such as CD34 and GlyCAM-1 (not shown), on the high endothelial venule (HEV, upper panel). After encounter with antigen, many of the differentiated effector T cells lose expression of L-selectin, leave the lymph node about 4–5 days later, and now express the integrin VLA-4 and increased levels of LFA-1 (not shown). These bind to VCAM-1 and ICAM-1, respectively, on peripheral vascular endothelium at sites of inflammation (lower panel). On differentiating into effector cells, T cells also alter their splicing of the mRNA encoding the cell-surface protein CD45. The CD45RO isoform expressed by effector T cells lacks one or more exons that encode extracellular domains present in the CD45RA isoform expressed by naive T cells, and somehow makes effector T cells more

1	isoform expressed by effector T cells lacks one or more exons that encode extracellular domains present in the CD45RA isoform expressed by naive T cells, and somehow makes effector T cells more sensitive to stimulation by specific antigen.

1	of naive T cells in order to retain the developing effector cells in the lymphoid tissue as they undergo differentiation and clonal expansion (see Section 9-6). Most effector T cells also shed L-selectin, which mediates rolling on the high endothelial venules of secondary lymphoid tissues such as lymph nodes, in favor of P-selectin glycoprotein ligand-1 (PSGL-1), a homodimeric sialoglycoprotein that is the major ligand for tethering and rolling on the Pand E-selectin expressed by activated endothelial cells at sites of inflammation (Fig. 11.6). In contrast to granulocytes and monocytes, which constitutively express glycosyltransferases necessary for biosynthesis of selectin ligands, T cells express these enzymes only after effector T-cell development. Effector differentiation induces expression of the glycosyltransferase α1,3-fucosyltransferase VII (FucT-VII), a key enzyme required for both Pand E-selectin ligand generation. Thus, although PSGL-1 is expressed by both naive and

1	expression of the glycosyltransferase α1,3-fucosyltransferase VII (FucT-VII), a key enzyme required for both Pand E-selectin ligand generation. Thus, although PSGL-1 is expressed by both naive and effector T cells, it is appropriately glycosylated for selectin binding only by effector T cells.

1	The expression of other adhesion molecules such as integrins that are important for the recruitment of effector T cells to inflamed tissues is also increased (see Fig. 11.6). Naive T cells mainly express LFA-1 (αLβ2), which is retained on effector T cells as they develop from naive T-cell precursors. However, LFA-1 is not the only integrin that these cells express. Effector T cells also synthesize the integrin α4:β1, or VLA-4, which binds to the adhesion molecule VCAM-1, a member of the immunoglobulin superfamily related to ICAM-1. When T cells are activated by chemokine signaling, VLA-4 is altered so that it can bind to VCAM-1 with greater affinity, similar to chemokine-induced binding of activated LFA-1 to ICAM-1 (see Section 3-18). Thus, chemokines activate VCAM-1 to bind VLA-4 on vascular endothelial cells near sites of inflammation, allowing extravasation of effector T cells. Although both VCAM-1 and ICAM-1 are expressed on activated endothelial cell surfaces, there appears to be

1	endothelial cells near sites of inflammation, allowing extravasation of effector T cells. Although both VCAM-1 and ICAM-1 are expressed on activated endothelial cell surfaces, there appears to be preferential utilization of one of the two adhesion pairs in some inflamed tissue vascular beds: recruitment of effector T cells is more dependent on VLA-4 in some tissues, and more dependent on LFA-1 in others.

1	Induction of expression of some adhesion molecules is compartmentalized so that effector T cells primed within lymphoid compartments of those tissues home back to them, whether during an active immune response or at homeostasis. Thus, the site of priming appears to imprint effector T cells with the ability to traffic to particular tissues. This is achieved by the expression of adhesion molecules that bind selectively to tissue-specific addressins. In this context, the adhesion molecules are often known as homing receptors (Fig. 11.7). As we shall see in Chapter 12, dendritic cells that prime T cells in the gut-associated lymphoid tissues (GALT) induce expression of the α4:β7 integrin, which binds to the mucosal vascular addressin MAdCAM-1 that is constitutively expressed by endothelial cells of blood vessels within the gut mucosa (see Fig. 11.7, lower left panel).

1	T cells primed in the GALT also express specific chemokine receptors that bind chemokines produced constitutively—and specifically—by the gut epithelium. Thus, at homeostasis CCR9 expressed on T cells primed in lymphoid Intestinal epithelial cells express CCL25, which binds CCR9 on gut-homing effector T cell CCL25CCR9˜4:°7integrinCCL25bloodvesselLymphocytes homing to the small intestine bind MAdCAM-1 and the chemokine CCL25 on vascular endothelium laminapropriaintestinalepitheliumCCR9MAdCAM-1˜4:°7

1	Fig. 11.7 Skinand gut-homing T cells use particular combinations of integrins and chemokines to migrate specifically to their target tissues. α4β7 expressed on circulating lymphocytes primed in gut-associated lymphoid tissues initally binds MAdCAM-1 (upper left panel), then uses CCR9 to move along a CCL25 chemokine gradient to traverse the endothelium and migrate to the intestinal epothelium (upper right panel). Similarly, circulating lymphocytes primed in lymph nodes draining the skin bind to the endothelium lining a cutaneous blood vessel by interactions between cutaneous lymphocyte antigen (CLA) and constitutively expressed E-selectin on the endothelial cells (lower left panel). The adhesion is strengthened by an interaction between lymphocyte chemokine receptor CCR4 and the endothelial chemokine CCL17. Once through the endothelium, effector T lymphocytes are attracted to keratinocytes of the epidermis by the chemokine CCL27, which binds to the receptor CCR10 on lymphocytes (lower

1	chemokine CCL17. Once through the endothelium, effector T lymphocytes are attracted to keratinocytes of the epidermis by the chemokine CCL27, which binds to the receptor CCR10 on lymphocytes (lower right panel).

1	tissues of the small intestine recruits those T cells back to the lamina propria subjacent to the epithelium of the small intestine along a CCL25 gradient (see Fig. 11.7, upper right panel). In contrast, T cells primed in skin-draining lymph nodes preferentially home back to the skin. They are induced to express the adhesion molecule cutaneous lymphocyte antigen (CLA), an isoform of PSGL-1 that differs in its pattern of glycosylation and binds to E-selectin on cutaneous vascular endothelium (see Fig. 11.7, lower panels). CLA-expressing T lymphocytes also express the chemokine receptors CCR4 and CCR10, which bind sequentially the chemokines CCL17 (TARC) and CCL27 (CTACK), respectively, which are present at highest levels in cutaneous blood vessels and the epidermis. Because these tissue-homing chemokines are produced at steady state, they are referred to as homeostatic chemokines. They are analogous to chemokines produced constitutively in lymphoid tissues at steady state, such as

1	chemokines are produced at steady state, they are referred to as homeostatic chemokines. They are analogous to chemokines produced constitutively in lymphoid tissues at steady state, such as CCL19 and CCL21, that direct CCR7-bearing naive T cells along a gradient from the endothelium of HEVs to T-cell zones (Fig. 11.8). Homeostatic chemokines are to be contrasted with inflammatory chemokines, which are elicited in the context of infection to recruit circulating immune cells to sites of inflammation.

1	Chemokines are classified into four families on the basis of structural differences: CXCL, CCL, XCL, and CXC3CL. Chemokines can also be classified as pro-inflammatory (red), homeostatic (green), and mixed function (yellow). Chemokines bind to a subfamily of seven-transmembrane G-protein-coupled receptors, which are classified as CXCR, CCR, XCR, and CX3CR on the basis of the class of chemokines they bind. Many, but not all, of the chemokine-chemokine receptor networks that coordinate immune modules are represented here. The connection between receptors and cell types on which they are expressed is indicated by the ‘circuit’ representation of lines and connecting nodes. To connect chemokines and their receptors to target cells, follow a horizontal line and turn on a vertical one at each node; the rhomboids (diamond shapes) link vertical lines to the cell type. Note that most chemokine receptors can bind multiple chemokines. Modified from Mantovani et al., Nat. Rev. Immunol. 2006,

1	node; the rhomboids (diamond shapes) link vertical lines to the cell type. Note that most chemokine receptors can bind multiple chemokines. Modified from Mantovani et al., Nat. Rev. Immunol. 2006, 6:907-918.

1	Fig. 11.8 Chemokine networks coordinate the interactions of innate and adaptive immune-cell populations.

1	In addition to the general and tissue-specific changes in trafficking molecules induced during effector T-cell differentiation, there is subset-specific expression of chemokine receptors that accompanies the loss of CCR7 expression. This results in distinct patterns of chemokine receptor expression by TH1, TH2, and TH17 cells that guide their differential recruitment to sites of inflammation contingent on the local patterns of inflammatory chemokines induced by the innate immune response to different types of pathogens (Fig. 11.9). For example, TH1 cells express CCR5, which is also expressed on monocytes that mature into macrophages as they enter the inflammatory site. Thus, both TH1 cells and the innate effector cells whose effector functions they enhance are recruited to the same tissue site by the same chemokines (see Fig. 11.8). As is the case for many other chemokine receptors, CCR5 has multiple ligands (CCL3, CCL4, CCL5, and CCL8), which may be induced by different cellular

1	site by the same chemokines (see Fig. 11.8). As is the case for many other chemokine receptors, CCR5 has multiple ligands (CCL3, CCL4, CCL5, and CCL8), which may be induced by different cellular sources and by different pathogens targeted by type 1 immunity. Some of these are produced by activated macrophages themselves following their recruitment to the inflammatory site. This provides a feed-forward mechanism by which the emerging innate response is amplified and then contributes to the recruitment of TH1 cells, which then provide antigen-dependent ‘help’ to further activate the macrophages, as we discuss in the next section. TH1 cells also express CXCR3, which is shared by NK cells and cytotoxic CD8 T cells. In response to CXCR3 ligands— CXCL9 and CXCL10—these cells are recruited to the same inflammatory site to coordinate the cell-mediated killing of targets infected by intracellular patho gens, such as Listeria monocytogenes, or certain viruses.

1	TH2 and TH17 cells display different patterns of inflammatory chemokine receptors, some of which, like those expressed by TH1 cells, are shared with the myelomonocytic cells with which they interact in inflamed tissues (see Figs. 11.8 and 11.9). The shared expression pattern of chemokine receptors by innate and adaptive effector cells represents an important mechanism for the spatiotemporal coordination and integration of immune effector modules in response to different types of pathogen (see Fig. 11.8). Thus, the local release of cytokines and chemokines at the site of infection has far-reaching consequences. In addition to recruiting granulocytes and monocytes, which constitutively express their specific complement of chemokine receptors while in circulation, changes induced in the blood vessel walls also enable newly generated effector T lymphocytes to enter infected tissues. Once in the tissue, recruited T cells produce T helper cell type–specific cytokines that further increase

1	vessel walls also enable newly generated effector T lymphocytes to enter infected tissues. Once in the tissue, recruited T cells produce T helper cell type–specific cytokines that further increase specific chemokine production by innate immune cells in an additional feed-forward mechanism that results in further effector T cell and innate effector cell trafficking into the tissue. Because the cytokines that differentially drive local production of effector module-specific chemokines are similarly produced by ILCs, this represents another major function of ILCs in coordinating the early polarization of pathogen-specific responses.

1	11-4 Pathogen-specific effector T cells are enriched at sites of infection as adaptive immunity progresses.

1	In the early stage of the adaptive immune response, only a minority of the effector T cells that enter infected tissues will be specific for pathogen. This is because activation of the endothelium of local blood vessels by inflammatory cytokines induces expression of selectins, integrin ligands, and chemokines that can recruit any circulating effector or memory T cell that expresses the appropriate trafficking receptors, irrespective of its antigenic specificity. However, specificity of the reaction is rapidly increased as the number of pathogen-specific T cells increases and recognition of antigen within the inflamed tissue retains them there. Although the precise mechanisms controlling retention of antigen-activated effector T cells in the inflamed tissue are not entirely understood, it is thought that the same mechanisms that retain antigen-activated naive T cells within secondary lymphoid tissues during effector

1	Fig. 11.9 The expression of adhesion and chemokine receptors is altered during effector T cell differentiation. During a primary immune response, specific cytokines derived from the innate immune system (indicated along the three diverging arrows) and unique master transcription factors (T-bet, GATA3, and RORγt) direct naive CD4 T cells to differentiate into TH1, TH2, or TH17 effector cells. Effector T cells of each subset lose expression of L-selectin (CD62L) and CCR7, and express characteristic chemokine receptors. Fig. 11.10 TH1 cells activate macrophages to become highly microbicidal. When an effector TH1 cell specific for a bacterial peptide contacts an infected macrophage, the T cell is induced to secrete the macrophage-activating factor IFN-γ and to express CD40 ligand. Together these newly synthesized TH1 proteins activate the macrophage.

1	T-cell development might be at play. This includes a role for the S1P pathway, although other chemokine signals may participate. By the peak of an adaptive immune response, after several days of clonal expansion and differentiation, a large fraction of the recruited T cells will be specific for the infecting pathogen.

1	Effector T cells that enter tissues but do not recognize their cognate antigen are not retained there. They either undergo apoptosis locally or enter the afferent lymphatics and migrate to the draining lymph nodes and eventually return to the bloodstream. Thus, T cells in the afferent lymph that drains tissues are memory or effector T cells, which characteristically express the CD45RO isoform of the cell-surface molecule CD45 and lack L-selectin (see Fig. 11.6). Effector T cells and some memory T cells have similar trafficking phenotypes, as we discuss later (see Section 11-22), and both seem to be committed to migration through, and in some cases, retention within, barrier tissues that are the primary sites of infection. In addition to allowing effector T cells to clear all sites of infection, this pattern of migration allows them to contribute, along with memory cells, to protecting the host against reinfection with the same pathogen.

1	11-5 TH1 cells coordinate and amplify the host response to intracellular pathogens through classical activation of macrophages.

1	Type 1 responses (see Fig. 11.5) are important for the eradication of those pathogens that have evolved mechanisms to survive and replicate within macrophages—for example, viruses, and bacterial and protozoan pathogens that can survive inside macrophage intracellular vesicles. In the case of viruses, a TH1 response is generally involved in helping to activate the CD8 cytotoxic T cells that will recognize virus-infected cells and destroy them (see Chapter 9). TFH cells that differentiate in type 1 responses induce the production of subclasses of IgG antibodies that neutralize virus particles in the blood and extracellular fluid. In the case of intracellular bacteria such as mycobacteria and Salmonella, and of protozoa such as Leishmania and Toxoplasma, which all take up residence inside macrophages, the role of TH1 cells is to activate macrophages to heighten their microbicidal function (Fig 11.10).

1	Pathogens of all types are ingested by macrophages from the extracellular fluid, and are often destroyed without the need for additional macrophage activation. In several clinically important infections, such as those caused by mycobacteria, ingested pathogens are not killed, and can even set up a chronic infection in macrophages and incapacitate them. Such microorganisms are able to maintain themselves in the hostile environment of phagosomes— shielded from the effects of both antibodies and cytotoxic T cells—by inhibiting the fusion of phagosomes and lysosomes, or by preventing the acidification required to activate lysosomal proteases. Nevertheless, peptides derived from such microorganisms can be displayed by MHC class II molecules on the macrophage surface, where they are recognized by antigen-specific effector TH1 cells. The TH1 cell is stimulated to synthesize membrane-associated proteins and soluble cytokines that enhance the macrophage’s antimicrobial defenses and enable it

1	by antigen-specific effector TH1 cells. The TH1 cell is stimulated to synthesize membrane-associated proteins and soluble cytokines that enhance the macrophage’s antimicrobial defenses and enable it to either eliminate the pathogen or control its growth and spread. This boost to antimicrobial mechanisms is known as ‘classical’ macrophage activation, the result of which is the so-called classically-activated, or M1, macrophage (Fig. 11.11).

1	Macrophages require two main signals for classical activation, and effector TH1 cells can deliver both. One signal is the cytokine IFN-γ; the other, CD40L, sensitizes the macrophage to respond to IFN-γ (see Fig. 11.10). TH1 cells also secrete lymphotoxin, which can substitute for CD40 ligand in M1 macrophage activation. The M1 macrophage is a potent antimicrobial effector cell. Phagosomes fuse with lysosomes, and microbicidal reactive oxygen and Fig. 11.11 Macrophages activated by TH1 cells undergo changes that greatly increase their antimicrobial effectiveness and amplify the immune response.

1	Fig. 11.11 Macrophages activated by TH1 cells undergo changes that greatly increase their antimicrobial effectiveness and amplify the immune response. Activated macrophages increase their expression of CD40 and of TNF receptors, and are stimulated to secrete TNF-α. This autocrine stimulus synergizes with IFN-γ secreted by TH1 cells to induce classical, or M1, macrophage activation characterized by the production of nitric oxide (NO) and superoxide (O2–). The macrophage also upregulates its B7 molecules in response to binding to CD40 ligand on the T cell, and increases its expression of MHC class II molecules in response to IFN-γ, thus allowing further activation of resting CD4 T cells.

1	nitrogen species are generated, as described in Section 3-2. When TH1 cells stimulate macrophages through these molecules, the M1 macrophage also secretes TNF-α, further stimulating macrophages through the TNFR-I, the same receptor activated by LT-α. TNF receptor signalling seems to be required to maintain the viability of the macrophage in this setting; in mice lacking TNFR-I (see Section 9-28), infection by Mycobacterium avium, an opportunistic intracellular pathogen that does not normally cause disease, leads to excessive apoptosis of macrophages that results in the release and dissemination of the pathogen before it can be killed within the infected macrophage. CD8 T cells also produce IFN-γ and can activate macrophages presenting antigens derived from cytosolic proteins on MHC class I molecules. Macrophages can also be made more sensitive to IFN-γ by very small amounts of bacterial LPS, and this latter pathway may be particularly important when CD8 T cells are the primary source

1	I molecules. Macrophages can also be made more sensitive to IFN-γ by very small amounts of bacterial LPS, and this latter pathway may be particularly important when CD8 T cells are the primary source of the IFN-γ.

1	In addition to increased intracellular killing, TH1 cells induce other changes in macrophages that help to amplify the adaptive immune response against intracellular pathogens. These changes include an increase in the number of MHC class II molecules, B7 molecules, CD40, and TNF receptors on the M1 macrophage surface (see Figs. 11.10 and 11.11), making the cell more effective at presenting antigen to T cells, and more responsive to CD40 ligand and TNF-α. In addition, M1 macrophages secrete IL-12, which increases the amount of IFN-γ produced by ILC1s and TH1 cells. This also promotes the differentiation of activated naive CD4 T cells into TH1 effector cells, and naive CD8 T cells into cytotoxic effectors (see Sections 9-20 and 9-18).

1	Another important function of TH1 cells is the recruitment of additional phagocytic cells to sites of infection. TH1 cells recruit macrophages by two mechanisms (Fig. 11.12). First, they make the hematopoietic growth factors IL-3 and GM-CSF, which stimulate the production of new monocytes in the bone marrow. Second, the TNF-α and lymphotoxin secreted by TH1 cells at sites of infection change the surface properties of endothelial cells so that monocytes adhere to them. Chemokines such as CCL2, which are induced by TH1 cells at inflammatory sites, direct the migration of monocytes through the vascular endothelium and into the infected tissue, where they differentiate into macrophages (see Section 3-17). Cytokines and chemokines secreted by M1 macrophages themselves are also important in recruiting other monocytes to sites of infection. Collectively, these TH1-mediated effects provide a positive feedback loop that amplifies and sustains type 1 responses until the pathogen is controlled

1	other monocytes to sites of infection. Collectively, these TH1-mediated effects provide a positive feedback loop that amplifies and sustains type 1 responses until the pathogen is controlled or eliminated.

1	Certain intravesicular bacteria, including some mycobacteria and Listeria monocytogenes, escape from phagocytic vesicles and enter the cytoplasm, where they are no longer susceptible to the microbicidal actions of activated macrophages. Their presence can, however, be detected by cytotoxic CD8 T cells. The pathogens released when macrophages are killed by these CTLs can be killed in the extracellular environment by antibody-mediated mechanisms, or can be phagocytosed by freshly recruited macrophages. In this circumstance, the provision of TH1-mediated ‘help’ for the development of CTLs, such as the provision of IL-2, may play an important role in coordinating the TH1 and CTL responses.

1	Fig. 11.12 The immune response to intracellular bacteria is differrentiation and potentiates the release of other cytokines. IL-3 coordinated by activated TH1 cells. The activation of TH1 cells and GM-CSF stimulate the production of new monocytes by acting by infected macrophages results in the synthesis of cytokines that on hematopoietic stem cells in the bone marrow. New macrophages both induce M1 macrophage and coordinate the immune response are recruited to the site of infection by the actions of secreted to intracellular pathogens. IFN-γ and CD40 ligand synergize in TNF-α, LT-α, and other cytokines on vascular endothelium, which activating the macrophage, which allows it to kill engulfed pathogens. signal monocytes to leave the bloodstream and enter the tissues Chronically infected macrophages lose the ability to kill intracellular where they become macrophages. A chemokine with monocyte bacteria, and membrane-bound Fas ligand or LT-β produced by chemotactic activity (CCL2)

1	macrophages lose the ability to kill intracellular where they become macrophages. A chemokine with monocyte bacteria, and membrane-bound Fas ligand or LT-β produced by chemotactic activity (CCL2) signals monocytes to migrate into sites the TH1 cell can kill these macrophages, releasing the engulfed of infection and accumulate there. Thus, the TH1 cell coordinates bacteria, which are taken up and killed by fresh macrophages. In a macrophage response that is highly effective in destroying this way, IFN-γ and LT-β synergize in the removal of intracellular intracellular infectious agents. bacteria. IL-2 produced by TH1 cells augments effector T-cell 11-6 Activation of macrophages by TH1 cells must be tightly regulated to avoid tissue damage.

1	As discussed in Chapter 9, distinguishing features of effector T cells are their capacity for antigen-induced activation of effector functions without the requirement for co-stimulation, and also their efficient delivery of effector molecules through polarized secretion or expression of cytokines and cell-surface molecules—often through formation of an immunological synapse with an antigen-bearing cell (see Section 9-25). After a TH1 cell recognizes its cognate antigen expressed by a macrophage, the secretion of effector molecules requires several hours. TH1 cells must therefore adhere to their target cells far longer than do cytotoxic CD8 T cells. Similarly to cytotoxic T cells, the secretory machinery of the TH1 cell becomes oriented toward the site of contact with the macrophage and newly synthesized cytokines are secreted there (see Fig. 9.38). CD40 ligand also seems to be delivered to the same contact site. So although all macrophages have receptors for IFN-γ, the infected

1	and newly synthesized cytokines are secreted there (see Fig. 9.38). CD40 ligand also seems to be delivered to the same contact site. So although all macrophages have receptors for IFN-γ, the infected macrophage that presents antigen to the TH1 cell is far more likely to become activated than nearby uninfected macrophages.

1	In addition to more efficiently focusing activating signals on infected macrophages, the antigen-specific induction of macrophage activation may play an important role in limiting tissue injury. By targeting only infected macrophages through MHC:peptide recognition, TH1 cells minimize ‘collateral damage’ that might otherwise result to normal components of the inflamed tissue: oxygen radicals, NO, and proteases that are toxic to host cells as well as to the pathogen that is targeted for destruction. Thus, antigen-specific macrophage activation by TH1 cells is a means of deploying this powerful defensive mechanism to maximum effect while minimizing local tissue damage. In this regard, it is notable that although ILC1s are also producers of IFN-γ, they lack antigen receptors that can focus the cytokine on infected macrophages for more efficient activation. It is not yet known whether ILC1 cells have other mechanisms with which to direct IFN-γ onto macrophages, or whether they play a more

1	cytokine on infected macrophages for more efficient activation. It is not yet known whether ILC1 cells have other mechanisms with which to direct IFN-γ onto macrophages, or whether they play a more limited role in macrophage activation, but the IFN-γ they produce is important in indirectly enhancing the local inflammatory response.

1	11-7 Chronic activation of macrophages by TH1 cells mediates the formation of granulomas to contain intracellular pathogens that cannot be cleared.

1	Some intracellular pathogens, most notably Mycobacterium tuberculosis, are sufficiently resistant to the microbicidal effects of activated macrophages that they are incompletely eliminated by a type 1 response. This gives rise to chronic, low-level infection that requires an ongoing TH1 response to prevent pathogen proliferation and spread. In this circumstance, chronic coordination between TH1 cells and macrophages underlies the formation of the immunological reaction called the granuloma, in which microbes are held in check within a central area of macrophages surrounded by activated lymphocytes (Fig. 11.13). A characteristic feature of granulomas is the fusion of several macrophages to form multinucleated giant cells, which can be found at the border of the central focus of activated macrophages and the lymphocytes that surround them and which appear to have heightened antimicrobial activity. A granuloma serves to ‘wall off’ pathogens that resist destruction. In tuberculosis, the

1	macrophages and the lymphocytes that surround them and which appear to have heightened antimicrobial activity. A granuloma serves to ‘wall off’ pathogens that resist destruction. In tuberculosis, the centers of large granulomas can become isolated and the cells there die, probably from a combination of lack of oxygen and the cytotoxic effects of activated macrophages. As the dead tissue in the center resembles cheese, this process is called ‘caseous’ necrosis. Thus, the chronic activation of TH1 cells can cause significant pathology. The absence of the TH1 response, however, leads to the more serious consequence of death from disseminated infection, which is now seen frequently in patients with AIDS and concomitant mycobacterial infection.

1	11-8 Defects in type 1 immunity reveal its important role in the elimination of intracellular pathogens. In mice whose gene for IFN-γor CD40 ligand has been deleted by gene targeting, classical macrophage activation is impaired; consequently, the animals succumb to sublethal doses of Mycobacterium, Salmonella, and Leishmania species. Classical (M1) macrophage activation is also crucial in controlling vaccinia virus. However, although IFN-γ and CD40 ligand are probably the most important effector molecules synthesized by TH1 cells, the immune response to pathogens that proliferate in macrophage vesicles is complex, and other cytokines secreted by TH1 cells may also be crucial (see Fig. 11.12).

1	The depletion of CD4 T cells in people with HIV/AIDS causes ineffective TH1 responses that can lead to the dissemination of microbes that are normally cleared by macrophages. This is the case with the opportunist fungal pathogen Pneumocystis jirovecii (see also Chapter 13). The lungs of healthy people are kept clear of P. jirovecii by phagocytosis and intracellular killing by alveolar macrophages. Pneumonia caused by P. jirovecii is, however, a frequent cause of death in people with AIDS. In the absence of CD4 T cells, phagocytosis of P. jirovecii and intracellular killing by lung macrophages are impaired, and the Granuloma multi-nucleated giant cell epithelioid cell T cells mycobacteria IFN-˜Partial removal of live M. tuberculosis TH1

1	Fig. 11.13 Granulomas form when an intracellular pathogen or its constituents cannot be completely eliminated. When mycobacteria (red) resist the effects of macrophage activation, a characteristic localized inflammatory response called a granuloma develops. This consists of a central core of infected macrophages. The core may include multinucleate giant cells, which are fused macrophages, surrounded by large macrophages often called epithelioid cells, but in granulomas caused by mycobacteria the core usually becomes necrotic. Mycobacteria can persist in the cells of the granuloma. The central core is surrounded by T cells, many of which are CD4-positive. The exact mechanisms by which this balance is achieved, and how it breaks down, are unknown. Granulomas, as seen in the bottom panel, also form in the lungs and elsewhere in a disease known as sarcoidosis, which may be caused by inapparent mycobacterial infection. Photograph courtesy of J. Orrell.

1	Panel a: the whipworm Trichuris trichiura is a helminth parasite that lives partly embedded in intestinal epithelial cells. This scanning electron micrograph of mouse colon shows the head of the parasite buried in an epithelial cell and its posterior lying free in the lumen. Panel b: a cross-section of crypts from the colon of a mouse infected with T. trichiura shows the markedly increased production of mucus by goblet cells in the intestinal epithelium. The mucus is seen as large droplets in vesicles inside the goblet cells and stains dark blue with periodic acid–Schiff reagent. Magnification ×400. pathogen colonizes the surface of the lung epithelium and invades lung tissue. The requirement for CD4 T cells seems to be due, at least in part, to a requirement for the macrophage-activating cytokines IFN-γ and TNF-α produced by TH1 cells. 11-9 TH2 cells coordinate type 2 responses to expel intestinal helminths and repair tissue injury.

1	Type 2 immunity is directed against parasitic helminths: roundworms (nematodes) and two types of flatworms—tapeworms (cestodes) and flukes (trematodes). Unlike microbial pathogens, or ‘micropathogens’ (bacteria, viruses, fungi, and protozoa), which replicate rapidly and can overwhelm host defenses by sheer numbers, most helminths do not replicate in their mammalian host. Moreover, helminths are multicellular; they are metazoan ‘macropathogens’ that are far too large—ranging in size from approximately 1 mm to over 1 meter—to be engulfed by host phagocytic cells, and therefore require very different strategies for host defense. In the developing world, the intestines of virtually all animals and humans are colonized by helminth parasites (Fig. 11.14). Many of these infections may be cleared rapidly by the generation of an effective type 2 response, although often the host response is successful in reducing worm burden, but not in completely clearing the parasite, resulting in chronic

1	rapidly by the generation of an effective type 2 response, although often the host response is successful in reducing worm burden, but not in completely clearing the parasite, resulting in chronic disease. In these circumstances, the parasite persists for long periods despite the host’s attempts to expel it, and causes disease by competing with the host for nutrients, or by causing local tissue injury.

1	Irrespective of the type of helminth involved, or its site of host entry, the host adaptive response is orchestrated by TH2 cells (Fig. 11.15; see also Fig. 9.30). The TH2 response is induced by the actions of worm products on a variety of different innate cells: epithelial cells, ILC2 cells, mast cells, and dendritic cells. Dendritic cells required for the presentation of helminth antigens to naive CD4 T cells appear to be activated by IL-13 produced by ILC2 cells and innate cytokines, such as epithelium-derived TSLP, which repress the development of TH1and TH17-inducing dendritic cells in favor of dendritic cells that promote TH2 cell differentiation. The initial source of the IL-4 required for TH2 cell differentiation appears to be context-specific and redundant. Thus, although several cell types have been proposed as the source, including iNKT cells, mast cells, and basophils, none of these has been proven to be essential.

1	The development of TH2 cells in draining lymphoid tissues is followed by their exodus to sites of helminth invasion, where they enhance the recruitment and function of circulating type 2 innate effector cells—eosinophils, basophils, tissue mast cells, and macrophages. Like TH1 and TH17 cells, TH2 cells express a distinct complement of chemokine receptors that is shared with the circulating innate effector cells with which they interact, thus selectively guiding them to sites of ongoing type 2 responses (see Figs. 11.8 and 11.9). CCR3 and CCR4 are expressed both by TH2 cells and by eosinophils and basophils, as is CRTH2, the ligand for which is prostaglandin D2, a lipid mediator that is produced by activated tissue mast cells. Ligands for CCR3 (for example, the eotaxins CCL11, CCL24, and CCL26) are produced by multiple innate immune cells in tissue sites of helminth infection and are induced by IL-4 and IL-13 signaling. Hence, ILC2 cells, TH2 cells, and eosinophils and basophils can

1	CCL26) are produced by multiple innate immune cells in tissue sites of helminth infection and are induced by IL-4 and IL-13 signaling. Hence, ILC2 cells, TH2 cells, and eosinophils and basophils can each amplify the recruitment of other type 2 cells through this chemokine network.

1	Although the TH2 effector response can coordinate the direct killing of some worms by enhancing innate effector cell functions, a major focus of the antihelminth response is expelling the worms and limiting the tissue damage they cause when they invade the host—functions that are both mediated by type 2 cytokines. IL-13 directly enhances the production of mucus by goblet cells, activates smooth muscle cells in mucosal tissues for hypermotility, and increases the migration and turnover of epithelial cells in the mucosa (see Fig. 11.15, first panel). In the intestines, which are the most common site of worm infestation, each of these actions is a critical component of the host response, as it helps to eliminate parasites that have attached to the epithelium and decreases the surface area available for colonization.

1	The response to helminths generates high levels of IgE antibody, induced by IL-4-producing TFH cells that develop in concert with TH2 cells (see Section 9-20). IgE binds to Fcεreceptors expressed by mast cells, eosinophils, and basophils, which arms them for antigen-specific recognition and activation. Type 2 adaptive responses also promote production of IgG1, which is recognized by macrophages and engages them in the type 2 response. IL-4 and IL-13 produced by TH2 cells also result in the differentiation of alternatively activated macrophages (also called M2 macrophages). Unlike classically activated, M1 macrophages, which differentiate after interaction with TH1 cells and are potent activators of inflammation (see Fig. 11.10), M2 macrophages participate in worm killing and expulsion, and also promote tissue remodeling and repair (see Fig. 11.15). A major difference between M1 and M2 macrophages is their different metabolism of arginine to produce antipathogen products. Whereas M1

1	and also promote tissue remodeling and repair (see Fig. 11.15). A major difference between M1 and M2 macrophages is their different metabolism of arginine to produce antipathogen products. Whereas M1 macrophages express iNOS, which produces the potent intracellular microbicide nitric oxide (NO) (see Section 3-2), M2 macrophages express arginase-1, which produces ornithine and proline from arginine. Along with other factors, ornithine increases the contractility of mucosal smooth muscle and promotes tissue remodeling and repair (see Fig. 11.15). Through a mechanism that is unclear, ornithine has also been found to be directly toxic to IgG-coated larvae of certain helminths. Because helminths that have invaded tissues are too large to be ingested by macrophages, the targeted release of toxic mediators directly onto the worm by antibody-dependent cell-mediated cytotoxicity (ADCC) enables macrophages, as well as eosinophils (see below), to attack these large extracellular pathogens.

1	Macrophages activated by TH2 cells also appear to be important in walling off invading worms, as well as repairing tissue damage caused as worms migrate through host tissues. These ‘tissue repair’ functions of M2 macrophages are dependent on secreted factors important in tissue remodeling and include the stimulation of collagen production, formation of which requires proline that is generated by arginase-1 activity. Moreover, TH2-activated macrophages can also induce the formation of granulomas that entrap worm larvae in tissues. In this regard, antigen-specific macrophage activation by TH2 cells has non-redundant function in type 2 responses. Although ILC2 cells, and innate effector cells, may promote M2 macrophage activation via IL-13, they are unable to sustain this response. Thus, in several models of worm infection, antihelminth responses are considerably impaired in RAG-deficient or T-cell-depleted mice, demonstrating that sustained alternative activation of macrophages requires

1	models of worm infection, antihelminth responses are considerably impaired in RAG-deficient or T-cell-depleted mice, demonstrating that sustained alternative activation of macrophages requires TH2 cells.

1	The IL-5 produced by TH2 cells and ILC2 cells recruits and activates eosinophils (see Fig. 11.15), which have direct toxic effects on worms by releasing cytotoxic molecules stored in their secretory granules, such as major basic protein (MBP). In addition to Fcε receptors that arm them for degranulation with IgE, eosinophils bear Fc receptors for IgG and can mediate ADCC against IgGcoated parasites (see Fig. 10.38). They also express the Fcα receptor (CD89) and degranulate in response to stimulation by secretory IgA.

1	IL-3 and IL-9 produced by TH2 cells in the mucosae recruit, expand, and activate a specialized population of mast cells known as mucosal mast cells (see Fig. 11.15). The innate cytokines IL-25 and IL-33 also activate mucosal mast cells early in a response to helminths. Mucosal mast cells differ from their counterparts in other tissues by having only small numbers of IgE receptors and producing very little histamine. When activated by cytokines, or by the binding of worm antigens to receptor-bound IgE, mucosal mast cells release large amounts of preformed inflammatory mediators that are stored in secretory granules: prostaglandins, leukotrienes, and several proteases, including the mucosal mast cell protease (MMCP-1), which can degrade the epithelial tight junctions to increase permeability and fluid flow into the mucosal lumen. Together, the mast-cell-derived mediators increase vascular and epithelial permeability, increase intestinal motility, stimulate the production of mucus by

1	and fluid flow into the mucosal lumen. Together, the mast-cell-derived mediators increase vascular and epithelial permeability, increase intestinal motility, stimulate the production of mucus by goblet cells, and induce leukocyte recruitment, all of which contribute to the ‘weep and sweep’ response that helps to expel parasites from the host.

1	11-10 TH17 cells coordinate type 3 responses to enhance the clearance of extracellular bacteria and fungi. The subset of effector T cells generated in response to infection by extracellular bacteria and fungi is TH17. At homeostasis, TH17 cells are deployed almost exclusively in the intestinal mucosa, where they contribute to the mutualistic relationship between the host and the intestinal microbiota—which is composed of extracellular bacteria and some fungi. However, they are also critical for defense against pathogenic extracellular bacteria and fungi that invade at barrier sites, as well as components of the normal microbiota that may enter the host when epithelial barrier function is compromised, whether as the result of trauma or pathogenic infection. In these settings, a principal function of TH17 cells is the orchestration of type 3 responses, in which neutrophils are the principal type of innate effector cell.

1	As discussed in Chapter 9, the development of TH17 cells is induced by the combined actions of TGF-β and the pro-inflammatory cytokines IL-6, IL-1, and IL-23 (see Fig. 9.31). The latter is preferentially produced by CD103+CD11b+ conventional dendritic cells that recognize MAMPs produced by extracellular bacteria, such as flagellin, which is recognized by TLR5; or MAMPs produced by fungi, such as β-glucan polymers of glucose expressed by yeast and fungi that are recognized by Dectin-1. As for TH1 and TH2 cells, the egress of TH17 cells from secondary lymphoid tissues is associated with altered chemokine expression: primarily the induction of CCR6, the ligand for which (CCL20) is produced by activated epithelial cells in mucosal tissues and skin, as well as TH17 cells themselves and ILC3 cells (see Figs. 11.8 and 11.9).

1	TH17 cells are stimulated to release IL-17A and IL-17F when they encounter antigen at sites of infection (Fig. 11.16). A primary effect of these cytokines is the enhanced production and recruitment of neutrophils. The receptor for TH17 IL-17 and IL-22 produced by TH17 cells induce the production of antimicrobial peptides by epithelial cells IL-17 IL-22 antimicrobial peptides IL-22 produced by TH17 increases epithelial cell turnover Increased epithelial cell division and shedding impairs bacterial colonization Direct killing or growth inhibition of bacteria attached to the epithelium turnover IL-22

1	Fig. 11.16 The immune response to extracellular bacteria produce cytokines that activate local epithelial and stromal cells to and some fungi is coordinated by activated TH17 cells. TH17 coordinate the immune response to extracellular bacteria and some cells activated by antigen-bearing macrophages and dendritic cells types of fungi. in barrier tissues (e.g., intestinal or respiratory mucosa, and skin) IL-17 produced by TH17 cells activates stromal cells and myeloid cells to produce G-CSF, which stimulates neutrophil production in bone marrow IL-17IL-17 G-CSF stromal cell myeloid cell bone marrow neutrophil production Increases numbers of circulating neutrophils to sustain supply of short-lived innate effectors at infection site

1	Increases numbers of circulating neutrophils to sustain supply of short-lived innate effectors at infection site Fig. 11.17 The cytokines IL-12 and IL-23 have a component in common, as do their receptors. The heterodimeric cytokines IL-12 and IL-23 both contain the p40 subunit, and the receptors for IL-12 and IL-23 have the IL-12Rβ1 subunit in common, which binds the p40 subunit. IL-12 signaling primarily activates the transcriptional activator STAT4, which increases IFN-γ production. IL-23 primarily activates STAT3, but also activates STAT4 weakly (not shown). Both cytokines augment the activity and proliferation of the CD4 subsets that express receptors for them; TH1 cells express IL-12R, and TH17 cells express primarily IL-23R, but can also express low levels of IL-12R (not shown). Mice deficient in p40 lack expression of both of these cytokines, and manifest immune defects as a result of deficiencies in both TH1 and TH17 activities.

1	IL-17A and IL-17F is expressed widely on cells such as fibroblasts, epithelial cells, and keratinocytes. IL-17 induces these cells to secrete various cytokines, including IL-6, which amplifies the TH17 response, and the hematopoietic factor granulocyte colony-stimulating factor (G-CSF), which increases the production of neutrophils by the bone marrow. IL-17 also stimulates production of the chemokines CXCL8 and CXCL2, the receptors for which (CXCR1 and CXCR2) are uniquely expressed by neutrophils (see Fig. 11.8). Thus, one important action of IL-17 at sites of infection is to induce local cells to secrete cytokines and chemokines that attract neutrophils.

1	TH17 cells also produce IL-22, a member of the IL-10 family that acts cooperatively with IL-17 to induce the expression by epithelial cells of antimicrobial proteins (see Fig. 11.16). These include β-defensins and the C-type lectins RegIIIβ and RegIIIγ, all of which can directly kill bacteria (see Section 2-4). IL-22 and IL-17 can also induce epithelial cells to produce metal-binding proteins that are bacteriostatic and fungistatic. Lipocalin-2 limits iron availability to bacterial pathogens; S100A8 and S100A9 are two antimicrobial peptides that heterodimerize to form the antimicrobial protein calprotectin, which sequesters zinc and manganese from microbes. Many of these antimicrobial agents are also produced by neutrophils recruited to the site of infection; calprotectin has been reported to comprise up to a third of the cytosolic protein of neutrophils. IL-22 also stimulates the proliferation and shedding of epithelial cells as a mechanism to deprive bacteria and fungi of a

1	reported to comprise up to a third of the cytosolic protein of neutrophils. IL-22 also stimulates the proliferation and shedding of epithelial cells as a mechanism to deprive bacteria and fungi of a ‘foothold’ for colonization at epithelial surfaces. While ILC3 cells in barrier tissues respond rapidly to pathogens to produce IL-22, pathogen-specific TH17 cells have been shown to amplify and sustain the production of IL-22 at sites of infection.

1	As in type 1 and type 2 responses, integration of innate and adaptive effector cells in the type 3 response is mediated in large part by the production of pathogen-specific antibodies that opsonize extracellular bacteria and fungi for destruction by neutrophils, macrophages, and complement. TFH cells that develop coordinately with TH17 cells promote the production of high-affinity IgG and IgA antibodies by plasma cells that can express CCR6 and thereby localize to sites of type 3 responses in barrier tissues, where they can arm neutrophils and macrophages ‘on-site.’ Antibodies are the principal immune reactants that clear primary infections by common extracellular bacteria that elicit type 3 responses, such as Staphylococcus aureus and Streptococcus pneumoniae. 11-11 Differentiated effector T cells continue to respond to signals as they carry out their effector functions.

1	11-11 Differentiated effector T cells continue to respond to signals as they carry out their effector functions. The commitment of CD4 T cells to distinct lineages of effector cells occurs in peripheral lymphoid tissues, such as lymph nodes. However, the effector activities of these cells are not defined simply by the signals received in the lymphoid tissues. Evidence suggests that there is continuous regulation of the expansion and the effector activities of differentiated CD4 cells, in particular those of TH17 and TH1 cells, once they enter sites of infection.

1	As noted in Chapter 9, commitment of naive T cells to become TH17 cells is triggered by exposure to TGF-β and IL-6; commitment to become TH1 cells is initially triggered by IFN-γ. These initial conditions are not, however, sufficient to generate complete or effective TH17 or TH1 responses. In addition, each T-cell subset also requires stimulation by another cytokine: IL-23 in the case of TH17 cells, and IL-12 in the case of TH17 cells. IL-23 and IL-12 are closely related in structure; each is a heterodimer and they have a subunit in common (Fig. 11.17). IL-23 is composed of one p40 (IL-12 p40) and one p19 (IL-23 p19) subunit, whereas IL-12 has the p40 (IL-12 p40) subunit and a unique p35 (IL-12 p35) subunit. Committed TH17 cells express a receptor for IL-23, and, as will be discussed below, low levels of the receptor for IL-12. TH1 cells express the receptor for IL-12. The receptors for IL-12 and IL-23 are also related. They have a common subunit, IL-12Rβ1, which is expressed by naive

1	low levels of the receptor for IL-12. TH1 cells express the receptor for IL-12. The receptors for IL-12 and IL-23 are also related. They have a common subunit, IL-12Rβ1, which is expressed by naive T cells. Upon receipt of differentiating cytokine signals, developing TH17 cells synthesize IL-23R, the inducible component of the mature IL-23 receptor heterodimer;

1	TH1 cells from mice cured of infection with L. major are transferred into Rag2– or IL-12 p40–defcient mice, which are then injected with L. major TH1 cells synthesize IL-12Rβ2, the inducible component of the mature IL-12 receptor.

1	TH1 cells synthesize IL-12Rβ2, the inducible component of the mature IL-12 receptor. IL-23 and IL-12 amplify the activities of TH17 and TH1 cells, respectively. Like many other cytokines, they both act through the JAK–STAT intracellular signaling pathway (see Fig. 9.32). IL-23 signaling primarily activates the intracellular transcriptional activator STAT3, but also activates STAT4. In contrast, IL-12 strongly activates STAT4, with minimal activation of STAT3. IL-23 does not initiate the commitment of naive CD4 T cells to become TH17 cells, but it does stimulate their expansion and contributes to their maintenance. Many in vivo responses that depend on IL-17 are diminished in the absence of IL-23. For example, mice lacking the IL-23-specific subunit p19 show decreased production of IL-17A and IL-17F in the lung after infection by Klebsiella pneumoniae.

1	IL-12 regulates the effector activity of committed TH1 cells at sites of infection. Studies of two different pathogens have shown that the initial differentiation of TH1 cells is not sufficient for protection, and that continuous signals are required. Mice deficient in IL-12 p40 can resist initial infection by Toxoplasma gondii as long as IL-12 is administered continuously. If IL-12 is administered during the first 2 weeks of infection, p40-deficient mice survive the initial infection and establish a latent chronic infection characterized by cysts containing the pathogen. When IL-12 administration is stopped, however, these mice gradually reactivate the latent cysts and the animals eventually die of toxoplasmic encephalitis. IFN-γ production by pathogen-specific T cells decreases in the absence of IL-12 but can be restored by IL-12 administration. Similarly, the adoptive transfer of differentiated TH1 cells from mice cured of Leishmania major protects RAG-deficient mice infected by L.

1	of IL-12 but can be restored by IL-12 administration. Similarly, the adoptive transfer of differentiated TH1 cells from mice cured of Leishmania major protects RAG-deficient mice infected by L. major, but cannot protect IL-12 p40-deficient mice (Fig. 11.18). Together, these experiments indicate that TH1 cells continue to respond to signals during an infection, and that continuous IL-12 is needed to sustain the effectiveness of differentiated TH1 cells against at least some pathogens.

1	11-12 Effector T cells can be activated to release cytokines independently of antigen recognition.

1	As we have seen, a central paradigm in adaptive immunity is the requirement for antigen recognition by the cognate receptors of naive lymphocytes to induce their differentiation into mature effector cells. However, effector T cells also acquire the ability to be activated by pairs of cytokines, independently of antigen recognition by their T-cell receptor. The cytokine pairs that mediate this ‘noncognate’ function of differentiated effector cells appear to be the same as those that activate the ILC subset that parallels each T-cell subset (Fig. 11.19). And in each case, the pair of stimulating cytokines includes one cytokine that activates a receptor that signals via a STAT factor, and one that activates a receptor that signals via NFκB—typically a member of the IL-1 receptor family. Thus, for both TH1 cells and ILC1 cells, stimulation by IL-12 (STAT4) plus IL-18 induces production of IFN-γ. Similarly, stimulation of TH2 and ILC2 cells by TSLP (STAT5) plus IL-33 produces IL-5 and

1	Thus, for both TH1 cells and ILC1 cells, stimulation by IL-12 (STAT4) plus IL-18 induces production of IFN-γ. Similarly, stimulation of TH2 and ILC2 cells by TSLP (STAT5) plus IL-33 produces IL-5 and IL-13, and both TH17 and ILC3 cells stimulated by IL-23 (STAT3) plus IL-1 produce IL-17 and IL-22. In this way, mature effector CD4 T cells acquire innate-like functional properties that allow them to amplify different types of immune responses without the requirement for antigen recognition. Note that in the case of type 1 and type 3 cells, the IL-1 family member involved (IL-18 and IL-1, respectively) is produced by inflammasome activation in myeloid cells. Conversely, IL-33,

1	TH1 cells protect Rag2-defcient mice, but mice lacking the IL-12 p40 subunit show progressive growth of the parasite Fig. 11.18 Continuous IL-12 is necessary for resistance to pathogens requiring TH1 responses. Mice that have eliminated an infection with Leishmania major and have generated TH1 cells specific to the pathogen were used as a source of T cells that were adoptively transferred either into Rag2-deficient mice, which lack T cells and B cells and cannot control L. major infection but can produce IL-12, or into mice lacking IL-12 p40, which cannot produce IL-12. On subsequent infection of the Rag2-deficient mice, lesions did not enlarge because the transferred TH1 cells conferred immunity. But despite the fact that the transferred cells were already differentiated TH1 cells, they did not confer resistance to IL-12 p40-deficient mice, which lacked a source of IL-12 to support TH1 function.

1	Fig. 11.19 Effector T cells can be activated to release cytokines independently of antigen recognition. Analogous to ILCs, effector T cells can be stimulated to produce effector cytokines independently of T-cell receptor signaling via the coordinate actions of pairs of cytokines. which activates type 2 responses, is inactivated by the inflammasome, indicating another basis for counterregulation between type 2 and type 1 or type 3 immune responses. Although the precise role of noncognate activation of effector T cells by cytokines is not clearly defined, it may provide a mechanism by which tissue-resident memory T cells could be rapidly recruited in recall, or memory, responses (see Section 11-22). 11-13 Effector T cells demonstrate plasticity and cooperativity that enable adaptation during anti-pathogen responses.

1	Thus far we have discussed subsets of effector CD4 T cells as though they are intrinsically stable, such that their functional phenotype is unchanging after their development. Similarly, we have discussed the different types of immunity as though they are unimodal, that is, only one type of response is recruited to clear a given pathogen. Although this is often the case, it is not always so. Just as the pathogens can modify their tactics to evade destruction, so too can the elicited effector T cells adapt in order to clear the host of these pathogens. Adaptation can occur by flexibility in the programming of individual T cells, referred to as T-cell plasticity, wherein effector T cells can transition into different cytokine phenotypes contingent on changes in the local inflammatory environment. It can also occur as a result of cooperation between different subsets of T cells. Plasticity applies to cells of the same clonal origin and identical antigenic specificity, whereas cooperation

1	It can also occur as a result of cooperation between different subsets of T cells. Plasticity applies to cells of the same clonal origin and identical antigenic specificity, whereas cooperation applies to cells that develop from different clonal precursors and target different antigens, typically at different stages of an infection.

1	Although some degree of plasticity has been experimentally demonstrated for each of the major effector CD4 subsets, it appears to be most prevalent in type 3 responses. It is common for TH17 cells to deviate, or be ‘reprogrammed,’ into TH1-type cells (Fig. 11.20). This was originally discovered using cytokine reporter mice, in which TH17 cells that express IL-17F could be identified and isolated based on their expression of a cell-associated reporter molecule controlled by the Il17f gene. When TH17 cells isolated using the reporter were restimulated in the presence of the TH1-polarizing cytokine IL-12, their progeny rapidly lost expression of IL-17 and acquired expression of IFN-γ. Moreover, repetitive restimulation of TH17 cells with the TH17 lineage cytokine IL-23 could lead to a subset of progeny that also acquired features of TH1 cells. In both cases, reprogramming of TH17 cells into TH1 cells required expression of the TH1-associated transcription factor T-bet and loss of the

1	of progeny that also acquired features of TH1 cells. In both cases, reprogramming of TH17 cells into TH1 cells required expression of the TH1-associated transcription factor T-bet and loss of the TH17-associated transcription factor RORγt, both of which were contingent on activation of STAT4 by the IL-12 and IL-23 receptors. Thus, TH17 cells deficient for either T-bet or STAT4 failed to demonstrate transition into TH1 cells, or ‘TH17 cell plasticity.’

1	An example of the importance of effector T-cell plasticity and cooperativity between subsets is found in host protection against facultative intracellular bacterial pathogens, such as Salmonella, which, unlike obligate extracellular bacteria, have also evolved mechanisms to survive within macrophages that are not activated by IFN-γ. Early in infection, Salmonella can colonize the intestinal epithelium similarly to other enteric Gram-negative pathogens. During this period, a TH17 response dominates, resulting in a robust IL-17induced influx of neutrophils that engulf extracellular bacteria and IL-22induced release of antimicrobial proteins that restrain bacterial growth in the intestinal lumen. During this intestinal phase of infection, much of the T-cell response appears to be directed against antigenic epitopes within bacterial flagellins, which are potent activators of TLR5. Activation of this innate sensor promotes IL-23 expression by CD11b+ classical dendritic cells in the

1	against antigenic epitopes within bacterial flagellins, which are potent activators of TLR5. Activation of this innate sensor promotes IL-23 expression by CD11b+ classical dendritic cells in the intestine, and thereby induces a type 3 immune response. Flagellin-specific TH1cells also emerge during the early intestinal phase of infection, and may arise from TH17 cell precursors as a result of plasticity. To escape destruction by macrophages activated for intracellular killing by these ‘ex-TH17’ TH1 cells, Salmonella simultaneously downregulates the expression of flagellin and begins synthesizing new proteins, such as SseI and SseJ, that allow it to suppress intracellular killing within macrophages. This allows Salmonella to both evade detection by flagellin-specific T cells and use the host macrophage as a safe haven to shield it from extracellular killing—at least temporarily—as the infection spreads systemically.

1	During the systemic phase of the infection, the T-cell response shifts to become focused on those antigens that enable the intracellular lifestyle of the pathogen. Some of these newly expressed antigens appear to activate cytosolic sensors within CD8α+ classical dendritic cells, which produce IL-12 to activate pathogen-specific TH1 cells and a type 1 response. The pathogen can now be cleared by TH1-induced macrophage activation directed against these newly expressed antigens. Because the anti-pathogen response now includes both type 3 and type 1 immunity to different sets of antigens that the bacterium requires for its extracellular and intracellular lifestyles, Salmonella is deprived of a niche for its survival and is cleared from the host. 11-14 Integration of celland antibody-mediated immunity is critical for protection against many types of pathogens.

1	The type of effector T cell or antibody required for host protection depends on the infectious strategy and lifestyle of the pathogen. As we learned in Chapter 9, cytotoxic T cells are important in destroying virus-infected cells, and in some viral diseases they are the predominant class of lymphocytes present in the blood during a primary infection. Nevertheless, the role of antibodies in clearing viruses from the body and preventing them from establishing another infection can be essential. Ebola virus causes a hemorrhagic fever and is one of the most lethal viruses known, but patients who do survive are protected and asymptomatic if they become infected again. In both the initial and recurrent infection, a strong, rapid antiviral IgG response against the virus is essential for survival. The antibody response clears the virus from the bloodstream and gives the patient time to activate cytotoxic T cells. This antibody response does not occur in infections that prove fatal; the virus

1	The antibody response clears the virus from the bloodstream and gives the patient time to activate cytotoxic T cells. This antibody response does not occur in infections that prove fatal; the virus continues to replicate, and even though there is activation of T cells, the disease progresses.

1	Cytotoxic T cells are also required for the destruction of cells infected with some intracellular bacterial pathogens, such as Rickettsia (the causative agent of typhus) or Listeria, which can escape from phagocytic vesicles to avoid the killing mechanisms of activated macrophages. In contrast, mycobacteria, which resist phagolysosomal killing and can live inside macrophage vesicles, are mainly kept in check by TH1 cells, which activate infected macrophages to kill the bacteria. Nevertheless, antibodies are induced in these infections and can contribute to pathogen killing when organisms are released from dying phagocytes, and are important in resistance to reinfection.

1	In many cases the most efficient protective immunity is mediated by neutralizing antibodies that can prevent pathogens from establishing an infection, and most of the established vaccines against acute childhood viral infections work primarily by inducing protective antibodies. Effective immunity against polio virus, for example, requires preexisting antibody, because the virus rapidly infects motor neurons and destroys them unless it is immediately neutralized by antibody and prevented from spreading within the body. In polio, specific IgA on mucosal epithelial surfaces also neutralizes the virus before it enters the tissues. Thus, protective immunity can involve effector mechanisms (IgA in this case) that do not operate in the elimination of the primary infection.

1	Fig. 11.20 Plasticity of CD4+ T-cell subsets. There is a hierarchy of stability of effector and regulatory CD4 T cells. Naive CD4+ T cells are multipotent, whereas TH1 and TH2 cells appear to be relatively stable, or at ‘ground state’; that is, they are highly resistant to transitioning into other effector cell phenotypes. iT cells and TH17 cells are less stable, and can transition into other subsets depending on prevailing cytokines. When acted on by IL-6 and IL-1, iT cells can transition into TH17 cells, or when acted on by IL-12 can become TH1 cells. TH17 cells acted on by IL-12 can transition into TH1 cells. Notably, the transitions of iT cells into TH17 cells, and TH17 cells into TH1 cells, appear to be unidirectional, or irreversible. Developing TH2 cells (left) repress expression of the inducible component of the IL-12 receptor (IL-12Rβ) and are unresponsive to IL-12; iTreg, TH17, and TH1 subsets (right) remain responsive to IL-12.

1	11-15 Primary CD8 T-cell responses to pathogens can occur in the absence of CD4 T-cell help. Many CD8 T-cell responses are deficient in the absence of help from CD4 T cells (see Section 9-19). In such circumstances, CD4 T-cell help is required to activate dendritic cells for them to become able to stimulate a complete CD8 T-cell response, an activity that has been described as licensing of the antigen-presenting cell (see Section 9-10). Licensing involves the induction of co-stimulatory molecules such as B7, CD40, and 4-1BBL on the dendritic cell, which can then deliver signals that fully activate naive CD8 T cells (see Fig. 9.29). Licensing enforces a requirement for dual recognition of an antigen by the immune system by both CD4 and CD8 T cells, which provides a useful safeguard against autoimmunity. Dual recognition is also seen in the cooperation between T cells and B cells for antibody generation (see Chapter 10). However, not all CD8 T-cell responses require such help.

1	Some infectious agents, such as the intracellular Gram-positive bacterium Listeria monocytogenes and the Gram-negative bacterium Burkholderia pseudomallei, appear to be able to directly license dendritic cells to induce primary CD8 T-cell responses without requirement for CD4 T-cell help (Fig. 11.21). Primary CD8 T-cell responses to L. monocytogenes were examined in mice that were genetically deficient in MHC class II molecules and thus lacked CD4 T cells (see Section 11-23). The numbers of CD8 T cells specific for a particular antigen expressed by the pathogen were measured by using tetrameric peptide:MHC complexes, or peptide:MHC tetramers (see Appendix I, Section A-24), which can identify CD4 or CD8 T cells on the basis of the antigenic specificity of their T-cell receptors. On day 7 of infection, wild-type mice and mice lacking CD4 T cells showed equivalent expansion, and

1	Fig. 11.21 Naive CD8 T cells can be activated directly by potent antigen-presenting cells through their T-cell receptor or through the action of cytokines. Left panels: naive CD8 T cells that encounter peptide:MHC class I complexes on the surface of dendritic cells expressing high levels of co-stimulatory molecules as a result of the inflammatory environment produced by some pathogens (upper left panel) are activated to proliferate in response, eventually differentiating into cytotoxic CD8 T cells (lower left panel). Right panels: activated dendritic cells also produce the cytokines IL-12 and IL-18, whose combined effect on CD8 T cells rapidly induces the production of IFN-γ (upper right panel). This activates macrophages for the destruction of intracellular bacteria and can promote antiviral responses in other cells (lower right panel).

1	equivalent cytotoxic capacity, of pathogen-specific CD8 T cells. Mice lacking CD4 T cells cleared the initial infection by L. monocytogenes as effectively as wild-type mice. These experiments clearly show that protective responses can be generated by pathogen-specific CD8 T cells without CD4 T-cell help. However, as we will see later, the nature of the CD8 memory response is different and is diminished in the absence of CD4 T-cell help. Naive CD8 T cells can also undergo ‘bystander’ activation by IL-12 and IL-18 to produce IFN-γ very early during infection (see Fig. 11.21). Mice infected with

1	Naive CD8 T cells can also undergo ‘bystander’ activation by IL-12 and IL-18 to produce IFN-γ very early during infection (see Fig. 11.21). Mice infected with L. monocytogenes or B. pseudomallei rapidly produce a strong IFN-γ response, which is essential for their survival. The source of this IFN-γ seems to be both NK cells and naive CD8 T cells, which begin to secrete it within the first few hours after infection. This is believed to be too soon for any significant expansion of pathogen-specific CD8 T cells, which would initially be too rare to contribute in an antigen-specific manner. The production of IFN-γ by both NK and CD8 T cells at this early time can be blocked experimentally by antibodies against IL-12 and IL-18, suggesting that these cytokines are responsible. These experiments indicate that naive CD8 T cells can contribute nonspecifically in a kind of innate defense, one not requiring CD4 T cells, in response to early signals of infection.

1	11-16 Resolution of an infection is accompanied by the death of most of the effector cells and the generation of memory cells.

1	When an infection is effectively repelled by the adaptive immune system, two things occur. First, the actions of effector cells remove the pathogen and, with them, the antigens that originally stimulated their differentiation. Second, in the absence of antigen, most effector T cells undergo ‘death by neglect,’ removing themselves by apoptosis. The resulting ‘clonal contraction’ of effector T cells appears to be due both to the loss of pro-survival cytokines that are produced by antigenic stimulation, such as IL-2, and to the loss of expression of receptors for these cytokines. CD25, the IL-2 receptor component that mediates high-affinity binding, is transiently upregulated on antigen-activated T cells, but then declines, thus limiting IL-2 signaling in the absence of antigenic restimulation. Also, as is discussed in Section 11-21, most effector T cells lose expression of the specific component of the IL-7 receptor IL-7Rα (CD127) soon after activation. Like IL-2 signaling, IL-7

1	Also, as is discussed in Section 11-21, most effector T cells lose expression of the specific component of the IL-7 receptor IL-7Rα (CD127) soon after activation. Like IL-2 signaling, IL-7 signaling activates STAT5, which promotes the expression of anti-apoptotic survival factors such as Bcl-2. Effector cells that lose responsiveness to IL-2 and IL-7 lose Bcl-2 and express Bim, which is a pro-apoptotic factor that acts via the intrinsic, or mitochondrial, pathway of apoptosis that leads to assembly of the apoptosome (see Sections 9-29 and 9-30).

1	While many effector T cells die from the loss of survival signals and the activation of the Bim-mediated intrinsic pathway of apoptosis, effector T-cell death can also occur via the extrinsic pathway of apoptosis that is activated by signaling via members of the TNF receptor superfamily, particularly Fas (CD95) (Fig. 11.22). Activation of the extrinsic pathway (or death receptor pathway) leads to the formation of the death-inducing signaling complex (DISC). The first step in Fas-mediated formation of the DISC is binding of the trimeric FasL, which results in the trimerization of Fas. This causes the death domains of Fas to bind to the death domain of FADD (Fas-associated via death domain), an adaptor protein introduced in Section 3-25. FADD contains a death domain and an additional domain called a death effector domain (DED) that can bind to DEDs present in other proteins. When FADD is recruited to Fas, the DED of FADD then recruits the initiator caspases pro-caspase 8 and pro-caspase

1	called a death effector domain (DED) that can bind to DEDs present in other proteins. When FADD is recruited to Fas, the DED of FADD then recruits the initiator caspases pro-caspase 8 and pro-caspase 10 via interaction with a DED in the pro-caspases. The high local concentration of these caspases in association with activated receptors allows the caspases to cleave themselves, resulting in their activation. Once activated, caspases 8 and 10 are released from the receptor complex and can activate the downstream

1	Fig. 11.22 Binding of Fas ligand to Fas initiates the extrinsic panel). FADD also contains a domain called a death effector domain pathway of apoptosis. The cell-surface receptor Fas contains (DED) that allows it to recruit pro-caspase 8 or pro-caspase 10, (not a so-called death domain (DD) in its cytoplasmic tail. When Fas shown), which also contains a DED domain (right panel). Clustered ligand (FasL) binds Fas, this trimerizes the receptor (left panel). The pro-caspase 8 activates itself to release an active caspase into the adaptor protein FADD (also known as MORT-1) also contains a death cytoplasm (not shown). domain and can bind to the clustered death domains of Fas (center effector caspases that induce apoptosis. Loss-of-function mutations in Fas lead to the increased survival of lymphocytes and are one cause of the disease autoimmune lymphoproliferative syndrome (ALPS). This disease can also be due to mutations in FasL and in caspase 10.

1	The relative contributions of the Bimand Fas-mediated apoptotic pathways to effector T-cell loss depend on the infectious agent, but they appear to be complementary mechanisms, as mice with specific deficiencies of Bim or Fas have milder defects in T-cell clearance than mice with deficiencies of both. Thus, the two pathways appear to be nonredundant; what aspects of infection contribute to the dominance of one mechanism over the other in response to different pathogens is unclear. Irrespective of whether their death is induced by the intrinsic or extrinsic pathway, dying T cells are rapidly cleared by phagocytes that recognize on their surface the membrane lipid phosphatidylserine. This lipid is normally found only on the inner surface of the plasma membrane, but in apoptotic cells it rapidly redistributes to the outer surface, where it can be recognized by specific receptors on many cells. Thus, not only is the pathogen removed at the end of infection, but most of the

1	cells it rapidly redistributes to the outer surface, where it can be recognized by specific receptors on many cells. Thus, not only is the pathogen removed at the end of infection, but most of the pathogen-specific effector cells are also removed. Some of the pathogen-specific effector cells survive, however, and provide the basis for memory T-cell and B-cell responses, as will be discussed in the next section.

1	Summary. CD4 T cells develop in response to, and subsequently amplify and sustain, innate immune responses that are induced by pathogens. Pathogen antigens are transported to local lymphoid organs by migrating dendritic cells and are presented to antigen-specific naive T cells that continuously recirculate through the lymphoid organs. T-cell priming and the differentiation of effector T cells occurs here, and the effector T cells either leave the lymphoid organ to provide cell-mediated immunity at sites of infection in the tissues or remain in the lymphoid organ to participate in humoral immunity by activating antigen-binding B cells. Distinct types of CD4 T cells develop in response to infection by different types of pathogens, and their development is influenced largely by the cytokines produced by innate sensor cells and ILCs that are activated early in the response.

1	Effector CD4 T cells serve to amplify and enhance early innate responses orchestrated by ILCs, while TFH cells that develop in concert with each subset of effector T cells direct the production of high-affinity antibodies that arm innate effector cells for heightened pathogen elimination. TH1 responses promote the development and activation of classical, M1 macrophages to protect against intracellular pathogens. TH2 responses are directed against infections by parasites such as helminths, and promote the development and activation of alternative, M2 macrophages and the recruitment of eosinophils and basophils to sites of infection. TH17 cells are integral to the clearance of extracellular bacteria and fungi, by orchestrating sustained neutrophil recruitment and production of antimicrobial peptides by epithelial cells of barrier tissues such as the intestines, lungs, and skin. CD8 T cells have an important role in protective immunity, especially in protecting the host against infection

1	peptides by epithelial cells of barrier tissues such as the intestines, lungs, and skin. CD8 T cells have an important role in protective immunity, especially in protecting the host against infection by viruses and intracellular infections by Listeria and other microbial pathogens that have special means for entering the host cell’s cytoplasm. Primary CD8 T-cell responses to pathogens usually require CD4 T-cell help, but can occur in response to some pathogens without such help. The patterns of anti-pathogenic response are not fixed, and effector T cells retain plasticity for adapting their response as pathogens alter their survival strategy in response to pressure from the immune system. Ideally, the adaptive immune response eliminates the infectious agent, at which time the expanded clonal populations of effector T cells contract, retaining only small populations of long-lived memory cells that provide the host with a state of protective immunity against reinfection with the same

1	populations of effector T cells contract, retaining only small populations of long-lived memory cells that provide the host with a state of protective immunity against reinfection with the same pathogen.

1	Immunological memory.

1	In this part of the chapter, we will examine how long-lasting protective immunity is maintained after an infection is successfully eliminated. Immunological memory is perhaps the most important consequence of an adaptive immune response, because it enables the immune system to respond more rapidly and effectively to pathogens that have been encountered previously, and prevents them from causing disease. Memory responses, called secondary immune responses, tertiary immune responses, and so on, depending on the number of exposures to antigen, also differ qualitatively from primary responses. This is particularly clear for B-cell responses, in that antibodies made during secondary and subsequent responses exhibit distinct characteristics, such as higher affinity to antigen compared with antibodies made during the primary response. Memory T-cell responses can also be distinguished qualitatively from the responses of naive or effector T cells, in terms of location, trafficking patterns,

1	made during the primary response. Memory T-cell responses can also be distinguished qualitatively from the responses of naive or effector T cells, in terms of location, trafficking patterns, and effector functions.

1	11-17 Immunological memory is long lived after infection or vaccination. Most children in developed countries are now vaccinated against measles virus; before vaccination was widespread, most were naturally exposed to this virus and developed an acute, unpleasant, and potentially dangerous illness. Whether through vaccination or infection, children exposed to the virus acquire long-term protection from measles, lasting for most people for the whole of their life. The same is true of many other acute infectious diseases (see Chapter 16): this state of protection is a consequence of immunological memory.

1	Because smallpox has been eradicated, recall responses measured in people who were vaccinated for smallpox can be taken to represent true memory in the absence of reinfection. After smallpox vaccination, antibody levels show an early peak with a period of rapid decay, which is followed by long-term maintenance that shows no significant decay. CD4 and CD8 T-cell memory is long lived but gradually decays, with a half-life in the range of 8–15 years.

1	The basis of immunological memory has been difficult to explore experimentally. Although the phenomenon was first recorded by the ancient Greeks and has been exploited routinely in vaccination programs for more than 200 years, it was recognized only within the last 30 years that this memory phenomenon reflects a small population of specialized memory cells that are formed during the adaptive immune response and can persist in the absence of the antigen that originally induced them. This mechanism of maintaining memory is consistent with the finding that only individuals who were themselves previously exposed to a given infectious agent are immune. The idea that immunological memory does not depend on repeated exposure to infection as a result of contact with other infected individuals has been supported by observations made of populations of people living on remote islands. In that setting, a virus such as measles can cause an epidemic, infecting all people living on the island at

1	has been supported by observations made of populations of people living on remote islands. In that setting, a virus such as measles can cause an epidemic, infecting all people living on the island at that time, after which the virus disappears for many years. On reintroduction from outside the island, the virus does not affect the original population but causes disease in those people born since the first epidemic.

1	The duration of immunological memory has been estimated by examining responses in people who received vaccinia, the virus used to immunize against smallpox (Fig. 11.23). Because smallpox was eradicated in 1978, it is presumed that their responses represent true immunological memory and are not due to restimulation from time to time by the smallpox virus. One study found strong vaccinia-specific CD4 and CD8 T-cell memory responses as long as 75 years after the original immunization, and from the strength of these responses it was estimated that the memory response had an approximate half-life of between 8 and 15 years. Half-life represents the time required for a response to diminish to 50% of its original strength. By contrast with T-cell memory, titers of antivirus antibody remained stable, almost without measurable decline.

1	These findings show that immunological memory need not be maintained by repeated exposure to infectious virus. Instead, it is likely that memory is sustained by long-lived antigen-specific lymphocytes that were induced by the original exposure and that persist until a second encounter with the pathogen. Although most of the memory cells are in a resting state, a small percentage of memory cells are dividing at any one time. It appears that this turnover is maintained by cytokines, such as IL-7 and IL-15, produced either constitutively or during antigen-specific immune responses directed at other, non-cross-reactive antigens. The number of memory cells for a given antigen is highly regulated, persisting with a relatively long half-life balanced by cell proliferation and cell death.

1	Immunological memory can be measured experimentally in various ways. Adoptive transfer assays (see Appendix I, Section A-30) of lymphocytes from animals immunized with simple, nonliving antigens have been favored for such studies, because the antigen cannot proliferate. In these experiments, the existence of memory cells is measured purely in terms of the transfer of specific responsiveness from an immunized, or ‘primed,’ animal to a nonimmunized recipient, as tested by a subsequent immunization with the antigen. Animals that received memory cells have a faster and more robust response to antigen challenge than do controls that did not receive cells, or that received cells from nonimmune donors.

1	Experiments like these have shown that when an animal is first immunized with a protein antigen, functional helper T-cell memory against that antigen appears abruptly and reaches a maximum after 5 days or so. Functional antigen-specific B-cell memory appears some days later, then enters a phase of cell proliferation and selection in lymphoid tissues. By 1 month after immunization, memory B cells are present at their maximum level. These levels of memory cells are then maintained, with little alteration, for the lifetime of the animal. It is important to recognize that the functional memory elicited in these experiments can be due to the precursors of memory cells as well as the memory cells themselves. These precursors are probably activated T cells and B cells, some of whose progeny will later differentiate into memory cells. Thus, precursors to memory cells can appear very shortly after immunization, even though resting memory-type lymphocytes may not yet have developed.

1	In the following sections we look in more detail at the changes that occur in lymphocytes after antigen priming and lead to the development of resting memory lymphocytes, and discuss the mechanisms that might account for these changes. 11-18 Memory B-cell responses are more rapid and have higher affinity for antigen compared with responses of naive B cells.

1	Immunological memory in B cells can be examined in vitro by isolating B cells from immunized or unimmunized mice and restimulating them with antigen in the presence of helper T cells specific for the same antigen (Fig. 11.24). B cells from immunized mice produce responses that differ both quantitatively and qualitatively compared with naive B cells from unimmunized mice. B cells that respond to the antigen increase in frequency by up to 100-fold after their initial priming in the primary immune response. Further, due to the process of affinity maturation (described in Chapter 10), antibodies produced by B cells of immunized mice typically have higher affinity for antigen than antibodies produced by unprimed B lymphocytes. The response from immunized mice is due to memory B cells that arise in the primary response. Memory B cells can arise from the germinal center reaction during a primary response, and may have undergone isotype switching and somatic mutations there. But memory B

1	arise in the primary response. Memory B cells can arise from the germinal center reaction during a primary response, and may have undergone isotype switching and somatic mutations there. But memory B cells can also arise independently of the germinal center reaction from short-lived plasma cells produced in the primary response. In either case, they circulate through the blood and take up residence in the spleen and lymph nodes. Memory B cells express some markers that distinguish them from naive B cells and plasma cells. One marker of memory B cells is simply

1	Fig. 11.24 The generation of secondary antibody responses from memory B cells is distinct from the generation of the primary antibody response. These responses can be studied and compared by isolating B cells from immunized and unimmunized donor mice, and stimulating them in culture in the presence of antigen-specific effector T cells. The primary response usually consists of antibody molecules made by plasma cells derived from a quite diverse population of precursor B cells specific for different epitopes of the antigen and with receptors that have a range of affinities for the antigen. The antibodies are of relatively low affinity overall, with few somatic mutations. The secondary response derives from a far more limited population of high-affinity B cells, which have, however, undergone significant clonal expansion. Their receptors and antibodies are of high affinity for the antigen and show extensive somatic mutation. The overall effect is that although there is usually only a

1	significant clonal expansion. Their receptors and antibodies are of high affinity for the antigen and show extensive somatic mutation. The overall effect is that although there is usually only a 10to 100-fold increase in the frequency of activatable B cells after priming, the quality of the antibody response is radically altered, in that these precursors induce a far more intense and effective response.

1	10,000 100 10 1 0.1 0.01

1	Fig. 11.25 Both the affinity and the amount of antibody increase with repeated immunization. The upper panel shows the increase in antibody concentration with time after a primary (1°), followed by a secondary (2°) and a tertiary (3°), immunization; the lower panel shows the increase in the affinity of the antibodies (affinity maturation). Affinity maturation is seen largely in IgG antibody (as well as in IgA and IgE, which are not shown) coming from mature B cells that have undergone isotype switching and somatic hypermutation to yield higher-affinity antibodies. The blue shading represents IgM on its own, the yellow shading IgG, and the green shading the presence of both IgG and IgM. Although some affinity maturation occurs in the primary antibody response, most arises in later responses to repeated antigen injections. Note that these graphs are on a logarithmic scale; it would otherwise be impossible to represent the overall increase of around 1 millionfold in the concentration of

1	to repeated antigen injections. Note that these graphs are on a logarithmic scale; it would otherwise be impossible to represent the overall increase of around 1 millionfold in the concentration of specific IgG antibody from its initial level.

1	the expression of a switched surface immunoglobulin isotype, compared with naive B cells that express surface IgM and IgD. In contrast, plasma cells have very low surface immunoglobulin altogether. In humans, a marker of memory B cells is CD27, a member of the TNF receptor family that is also expressed by naive T cells and binds the TNF-family ligand CD70, which is expressed by dendritic cells (see Section 9-17).

1	A primary antibody response is characterized by the initial rapid production of IgM and a slightly delayed IgG response due to time required for class switching (Fig. 11.25). The secondary antibody response is characterized in its first few days by the production of relatively small amounts of IgM antibody and much larger amounts of IgG antibody, with some IgA and IgE. At the beginning of the secondary response, antibodies are made by memory B cells that were generated in the primary response and have already switched from IgM to another isotype, and that express IgG, IgA, or IgE on their surface. Memory B cells express somewhat higher levels of MHC class II molecules and the co-stimulatory ligand B7.1 than do naive B cells. This helps memory B cells acquire and present antigen more efficiently to TFH cells compared with naive B cells, and to TFH cells through the B7.1 receptor CD28, so that they can in turn help in antibody production so it begins earlier after antigen exposure

1	efficiently to TFH cells compared with naive B cells, and to TFH cells through the B7.1 receptor CD28, so that they can in turn help in antibody production so it begins earlier after antigen exposure compared with primary responses. The secondary response is characterized by a more vigorous and earlier generation of plasma cells than in the primary response, thus accounting for the almost immediate abundant production of IgG (see Fig. 11.25).

1	during secondary immune responses. During a secondary exposure to infection, antibodies persisting from a primary immune response are available immediately to bind the pathogen and mark it for degradation by complement or by phagocytes. If the antibody can completely neutralize the pathogen, a secondary immune response may not occur. Otherwise, excess antigens will bind to receptors on B cells and initiate a secondary response in the peripheral lymphoid organs. Memory B cells recirculate through the same secondary lymphoid compartments as naive B cells, principally the follicles of the spleen, lymph nodes, and the Peyer’s patches of the gut mucosa. B cells with the highest avidity for antigen are activated first. Thus memory B cells, which have been selected previously for their avidity to antigen, make up a substantial component of the secondary response.

1	Besides responding more rapidly, memory B cells can reenter germinal centers during secondary immune responses and undergo additional somatic hypermutation and affinity maturation, as described in Sections 10-6 through 10-8. As in primary responses, secondary B-cell responses begin at the interface between the T-cell and B-cell zones, where memory B cells that have acquired antigen can present peptide:MHC class II complexes to helper T cells. This interaction initiates proliferation of both the B cells and T cells.

1	Reactivated memory B cells that have not yet undergone differentiation into plasma cells migrate into the follicle and become germinal center B cells, undergoing additional rounds of proliferation and somatic hypermutation before differentiating into antibody-secreting plasma cells. Since B cells with the higher-affinity antigen receptors will more efficiently acquire and present antigen to antigen-specific TFH cells in the germinal center, the affinity of the antibodies produced during secondary and tertiary responses rises progressively (see Fig. 10.14). increased frequency relative to their frequency as naive T cells.

1	increased frequency relative to their frequency as naive T cells. Until relatively recently, analysis of T-cell memory relied on assays of T-cell function rather than a direct identification of antigen-specific memory T cells. Some assays of T-cell effector function, such as providing help to B cells or macrophages, can take several days to perform. Because of this, such assays are not optimal for distinguishing memory T cells from preexisting effector cells, since memory cells can be reactivated during the time frame of the assay. This is a problem particularly for effector CD4 T cells, but does not apply as much for effector CD8 T cells, which can program a target cell for lysis in 5 minutes. In contrast, memory CD8 T cells need more time than this to be reactivated to become cytotoxic, so that the actions of memory CD8 T cells will appear much later than those of preexisting effector cells.

1	Examining T-cell memory has been made easier by the development of MHC tetramers (see Appendix I, Section A-24). Before MHC tetramers, effector and memory responses were studied using naive T cells from mice carrying specific T-cell receptor (TCR) transgenes. Such TCR-transgenic T cells could be uniquely identified by antibodies to their rearranged T-cell receptors, but were not part of the host’s natural T-cell repertoire. MHC tetramers measure the in vivo frequency of all clones with a given antigen specificity, but do not distinguish between different T-cell clones of the same specificity. MHC tetramers were generated for MHC class I molecules first, but are now also available for some MHC class II molecules, allowing the study of both CD8 and CD4 T cells both in normal mice and in humans.

1	MHC tetramers have allowed a direct analysis of the formation of memory T cells. In the example shown in Fig. 11.26, T-cell responses to infection by the intracellular bacteria Listeria monocytogenes are analyzed with MHC class II tetramers specific to the toxin listeriolysin O (LLO). In the naive T-cell repertoire of the mouse, there are approximately 100 LLO-specific CD4 T cells, which undergo 1000-fold expansion into effector T cells during the Fig. 11.26 Generation of memory T cells expansion phase of 6 days after infection. When the infection is eliminated, after an infection. After an infection, in this case with an attenuated strain of Listeria

1	LLOp:I-Ab+ 106 106 cells 105 105 104 104 103 103 102 102 101 monocytogenes, the number of T cells specific for the listeriolysin (LLO) toxin increases dramatically and then falls back to give a sustained low level of memory T cells. T-cell responses are detected by binding of an MHC tetramer consisting of an LLO peptide bound by I-Ab. The left panel shows the primary response of CD4 T cells that are LLO-specific, and the right panel shows the contraction and memory phase. Approximately 100 T cells in the naive T-cell repertoire expand to about 100,000 effector cells by day 7, and then contract to about 7000 memory cells by day 25. These memory cells then slowly decay to 500 cells by day 450. Data courtesy of Marc Jenkins.

1	a slower contraction phase follows in which these T cells are reduced by about 100-fold within a few weeks. This leaves a population of memory T cells present at a frequency of about 10-fold higher than in the naive repertoire, and this population persists with a half-life of about 60 days. 11-21 Memory T cells arise from effector T cells that maintain sensitivity to IL-7 or IL-15.

1	11-21 Memory T cells arise from effector T cells that maintain sensitivity to IL-7 or IL-15. Naive and memory T cells can be distinguished by differences in their expression of various cell-surface proteins, by their distinct responses to stimuli, and by their expression of certain genes. Overall, memory cells continue to express many markers of activated T cells, such as phagocytic glycoprotein-1 (Pgp1, CD44), but they stop expressing other activation markers, such as CD69. Memory T cells express more Bcl-2, a protein that promotes cell survival and may be responsible for their long half-life. Figure 11.27 lists several molecules by which naive, effector, and memory T cells can be distinguished.

1	Fig. 11.27 Expression of many proteins alters when naive T cells become memory T cells. Proteins that are expressed differently in naive T cells, effector T cells, and memory T cells include adhesion molecules, which govern interactions with antigen-presenting cells and endothelial cells; chemokine receptors, which affect migration to lymphoid tissues and sites of inflammation; proteins and receptors that promote the survival of memory cells; and proteins that are involved in effector functions, such as granzyme B. Some changes also increase the sensitivity of the memory T cell to antigen stimulation. Many of the changes that occur in memory T cells are also seen in effector cells, but some, such as expression of the cell-surface proteins CD25 and CD69, are specific to effector T cells; others, such as expression of the survival factor Bcl-2, are limited to long-lived memory T cells. This list represents a general picture that applies to both CD4 and CD8 T cells in mice and humans,

1	others, such as expression of the survival factor Bcl-2, are limited to long-lived memory T cells. This list represents a general picture that applies to both CD4 and CD8 T cells in mice and humans, but some details that may differ between these sets of cells have been omitted for simplicity.

1	Days after transfer 1470 0 Mice infected with LCMV generate a primary CD8 response; some effector cells express high levels of IL-7R˜, while others do not Only transfer of the IL-7R˜hi-CD8 T cells into naive mice led to robust expansion of antigen-specifc CD8 cells after secondary challenge LCMV TCR-transgenic mouse naive mice antigen challenge Number of antigen-specifcCD8 T cells antigen challenge CD8 IL-7R˜hi cells transfer transfer IL-7R˜lo cells

1	Fig. 11.28 Expression of the IL-7 receptor (IL- 7Rα) indicates which CD8 effector T cells can generate robust memory responses. Mice expressing a T-cell receptor (TCR) transgene specific for a viral antigen from lymphocytic choriomeningitis virus (LCMV) were infected with the virus, and effector cells were collected on day 11. Effector CD8 T cells expressing high levels of IL-7Rα (IL-7Rαhi, blue) were separated and transferred into one group of naive mice, and effector CD8 T cells expressing low IL-7Rα (IL-7Rαlo, green) were transferred into another group. Three weeks after transfer, the mice were challenged with a bacterium engineered to express the original viral antigen, and the numbers of responding transferred T cells (detected by their expression of the transgenic TCR) were measured at various times after challenge. Only the transferred IL-7Rαhi effector cells could generate a robust expansion of CD8 T cells after the secondary challenge.

1	Among the important markers of memory T cells is the α subunit of the IL-7 receptor (IL-7Rαor CD127). Naive T cells express IL-7Rα, but it is rapidly lost upon activation and is not expressed by most effector T cells. For example, the experiment shown in Fig. 11.28 examines mice infected with lymphocytic choriomeningitis virus (LCMV). Around day 7 after infection, a small population of approximately 5% of CD8 effector T cells expressed high levels of IL-7Rα. Adoptive transfer of these IL-7Rαhi cells, but not IL-7Rαlo effector T cells, could provide functional CD8 T-cell memory to uninfected mice. This experiment suggests that memory T cells arise from effector T cells that maintain or reexpress IL-7Rα, perhaps because they compete more effectively for the survival signals delivered by IL-7.

1	The homeostatic mechanisms governing the survival of memory T cells also differ from those for naive T cells. Memory T cells divide more frequently than naive T cells, and their expansion is controlled by a shift in the balance between proliferation and cell death. As illustrated in Fig. 11.29, naive T cells require contact with self peptide:self MHC complexes in addition to cytokine stimulation for their long-term survival in the periphery (see Fig. 9.4). As with naive cells, the survival of memory T cells requires signaling by the receptors for the cytokines IL-7 and IL-15. IL-7 is required for the survival of both CD4 and CD8 memory T cells. In addition, IL-15 is critical for the long-term survival and proliferation of CD8 memory T cells under normal conditions. It also appears that memory T cells are less dependent on contact with self peptide:self MHC than naive T cells and are more sensitive to cytokines.

1	Memory T cells still require contact with peptide:MHC complexes to become reactivated during a secondary encounter with pathogen, but are also more sensitive to restimulation by antigen than are naive T cells. Furthermore, they more quickly and more vigorously produce several cytokines such as IFN-γ, TNF-α, and IL-2 in response to such stimulation. A similar progression occurs for T cells in humans after immunization with a vaccine against yellow fever virus.

1	For their survival in the periphery, naive T cells require periodic stimulation with the cytokines IL-7 and IL-15 and with self-antigens presented by self MHC molecules. On priming with its specific antigen, a naive T cell divides and differentiates. Most of the progeny differentiate into relatively short-lived effector cells that have lost expression of the IL-7 receptor (yellow), but some effector cells retain or reexpress the receptor and become long-lived memory T cells. These memory cells can be maintained by IL-7 and IL-15 and are less dependent on contact with self peptide:self MHC complexes for survival compared with naive T cells. However, contact with self antigens may be necessary for some memory T cells to keep up their numbers in the memory pool, but this may vary between different clones and is the subject of ongoing investigation. 11-22 Memory T cells are heterogeneous and include central memory, effector memory, and tissue-resident subsets.

1	Changes in other cell-surface proteins that occur on memory CD4 T cells after exposure to antigen are significant (see Fig. 11.27). L-selectin (CD62L) is the homing receptor that directs T cells into secondary lymphoid tissues, and it is lost by effector cells and most memory CD4 T cells. CD44 is a receptor for hyaluronic acid and other ligands expressed in peripheral tissues, and it is induced on effector and memory T cells. The change in expression of these two molecules helps memory T cells migrate from the blood into the peripheral tissues rather than migrating directly into lymphoid tissues, as would naive T cells. Different isoforms of CD45, a cell-surface protein tyrosine phosphatase expressed on all hematopoietic cells, are useful in distinguishing naive from effector and memory T cells. The CD45RO isoform is produced because of changes in the alternative splicing of exons that encode the CD45 extracellular domain and identifies effector and memory cells, although it is

1	T cells. The CD45RO isoform is produced because of changes in the alternative splicing of exons that encode the CD45 extracellular domain and identifies effector and memory cells, although it is unclear what functional consequences this change may impose. Some surface receptors, such as CD25, the α subunit of the IL-2 receptor, are expressed on activated effector cells, but not memory cells; however, they can be reexpressed when memory cells are again reactivated by antigen and become effector T cells.

1	Memory T cells are heterogeneous, and both CD4 and CD8 T cells are classified into three major subsets. Each type exhibits a distinct pattern of receptors, for example, for different chemokines and adhesion molecules, and shows different activation characteristics (Fig. 11.30). Central memory T cells (TCM) express the chemokine receptor CCR7, which allows their recirculation to be similar to that of naive T cells and allows them to traffic through the T-cell zones of peripheral lymphoid tissues. Central memory cells are very sensitive to cross-linking of their T-cell receptors and rapidly express CD40 ligand in response; however, they are relatively slower compared with other memory subsets to acquire effector functions such as production of cytokines early after restimulation. Central memory cells primarily migrate from the blood, into the secondary lymphoid organs, then into the lymphatic system and back into the blood, a route very similar to the migration pattern of naive T cells.

1	cells primarily migrate from the blood, into the secondary lymphoid organs, then into the lymphatic system and back into the blood, a route very similar to the migration pattern of naive T cells. By contrast, effector memory T cells (TEM) lack the chemokine receptor CCR7, but express high levels of β1 and β2 integrins, and so are specialized for rapidly entering inflamed tissues. They also express receptors for inflammatory chemokines and can rapidly mature into effector T cells and secrete large amounts of IFN-γ, IL-4, and IL-5 early after restimulation. Effector memory T cells migrate from the blood primarily into peripheral nonlymphoid tissues, then through the lymphatic system and finally into secondary lymphoid tissues. There they can reenter the lymphatic system and reach the blood again. In contrast to central and effector memory cells, tissue-resident memory T cells (TRM) comprise a substantial fraction of memory T cells that do not migrate, but rather take up long-term

1	blood again. In contrast to central and effector memory cells, tissue-resident memory T cells (TRM) comprise a substantial fraction of memory T cells that do not migrate, but rather take up long-term residency in various epithelial sites (Fig. 11.31). Like TEM cells, TRM cells lack CCR7 but express other chemokine receptors (for example, CXCR3, CCR9) that allow migration into peripheral tissues such as the dermis or the lamina propria of the intestine. In these sites, TRM cells induce CD69, which

1	CD45RO CCR3 CCR5 Effector memory cells lack CCR7 and migrate to tissues CCR7 Central memory cells express CCR7 and remain in lymphoid tissue CD45RO Most effector cells die after a few days Some effector cells may become quiescent memory cells Memory cells derive directly from some effector T cells CD45RO IL-7R˜Effector T cells differentiate, secrete cytokines, and express cytokine receptors IL-4 IL-2 perforin FasL Naive T cell sees antigen T dendritic cell CD45RACCR7 Fig. 11.30 T cells differentiate into central memory and effector memory subsets, which are distinguished by the expression of the chemokine receptor CCR7.

1	Fig. 11.30 T cells differentiate into central memory and effector memory subsets, which are distinguished by the expression of the chemokine receptor CCR7. Quiescent memory cells bearing the characteristic CD45RO surface protein can arise from activated effector cells (right half of diagram) or directly from activated naive T cells (left half of diagram). Two types of quiescent memory T cells can derive from the primary T-cell response: central memory cells and effector memory cells. Central memory cells express CCR7 and remain in peripheral lymphoid tissues after restimulation. Memory cells of the other type—effector memory cells—mature rapidly into effector T cells after restimulation, and secrete large amounts of IFN-γ, IL-4, and IL-5. They do not express the receptor CCR7, but express receptors (CCR3 and CCR5) for inflammatory chemokines.

1	reduces S1PR expression, thereby promoting retention in tissues. TRM cells, particularly CD8 TRM cells, enter and reside within the epithelium. TGF-βproduction by epithelial cells induces TRM cells to express the integrin αE:β7, which binds E-cadherin expressed by epithelium and is required for TRM retention. The distinction between TCM, TEM, and TRM memory populations has been made both in humans and in the mouse. However, each subset itself is not strictly a homogeneous population. For example, within the CCR7-expressing TCM cells, there are cells with differing expression of other markers, particularly chemokine receptors. A subset of the CCR7-positive TCM cells also express CXCR5, similarly to TFH cells, although it is not yet clear whether these memory cells can provide help to B cells in the germinal center.

1	On stimulation by antigen, TCM cells rapidly lose expression of CCR7 and differentiate into TEM cells. TEM cells are also heterogeneous in the chemokine receptors they express, and have been classified according to chemokine receptors typical of TH1 cells (CCR5), TH17 cells (CCR6), and TH2 cells (CCR4). Central memory cells do not appear committed to particular effector lineages,

1	Fig. 11.31 Tissue-resident memory T cells are a major immune compartment that surveys peripheral tissues for reinfection by pathogens. After activation and priming in lymphoid tissues, activated CD8 and CD4 T cells enter the blood and enter tissues in response to various chemokines, as shown here for entry into the dermis, guided by expression of CXCR3. The reexpression of CD69 by T cells caused by antigen or other unknown signals leads to decreased S1PR1 surface expression, promoting retention of these cells in the dermis. In response to TGF-β, some cells induce integrin αE:β7 (CD103), which binds E-cadherin expressed by epithelial cells, promoting entry and retention of T cells in the epidermis, where many CD8 TRM cells reside. Recent estimates indicate that TRM cells may outnumber the recirculating T cells that migrate through the body.

1	and even effector memory cells are not fully committed to the TH1, TH17, or TH2 lineage, although there is some correlation between their eventual output of TH1, TH17, or TH2 cells and the chemokine receptors expressed. Further stimulation with antigen seems to drive the differentiation of effector memory cells gradually into the distinct effector T-cell lineages. 11-23 CD4 T-cell help is required for CD8 T-cell memory and involves CD40 and IL-2 signaling.

1	There is experimental evidence that CD4 T cells play an important role in programming optimal CD8 T-cell memory. In the experiment shown in Fig. 11.32, the primary and memory CD8 T-cell responses were compared between wild-type mice and mice that lack expression of MHC class II, and which therefore have a defect in CD4 T cells. In this experiment, the CD8 T-cell response was measured against a protein, ovalbumin, carried by an experimental strain of Listeria monocytogenes. After 7 days of infection, both types of mice showed equivalent expansion and activity of antigen-specific CD8 effector T cells. But mice with a defect in CD4 T cells generated much weaker secondary responses, characterized by the presence of far fewer expanding memory CD8 T cells after a secondary challenge. These results imply a role for CD4 T cells either in the initial programming of CD8 T cells or during the secondary memory response.

1	Further experiments suggest that this CD4 T-cell help is necessary for the initial programming of naive CD8 T cells. Memory CD8 T cells that developed in the absence of CD4 help were transferred into wild-type mice. After transfer, the recipient mice were challenged again, whereupon the CD8 T cells showed a reduced ability to proliferate even though the recipient mice expressed MHC class II. This result indicates that CD4 T-cell help is required during the priming of CD8 T cells and not simply at the time of secondary responses.

1	Fig. 11.32 CD4 T cells are required for the development of functional CD8 memory T cells. Mice that do not express MHC class II molecules (MHC II–/–) fail to develop CD4 T cells. Wild-type and MHC II–/– mice were infected with Listeria monocytogenes expressing the model antigen ovalbumin (LM-OVA). After 7 days, the number of OVA-specific CD8 T cells can be measured by using specific MHC tetramers that contain an OVA peptide, and therefore bind to T-cell receptors that react with this antigen. After 7 days of infection, mice lacking CD4 T cells were found to have the same number of OVA-specific CD8 T cells as wild-type mice. However, when mice were allowed to recover for 60 days—a time during which memory T cells could develop—and were then re-challenged with LM-OVA, the mice lacking CD4 T cells failed to expand CD8 memory cells specific to OVA, whereas there was a strong CD8 memory response in the wild-type mice.

1	This requirement for CD4 help in CD8 memory generation has also been demonstrated by experiments in which CD4 T cells were depleted by treatment with antibody or in which mice were deficient in the CD4 gene. The mechanism underlying this requirement for CD4 T cells is not completely understood. It may involve two types of signals received by the CD8 T cell— those received through CD40 and those received through the IL-2 receptor. CD8 T cells that do not express CD40 are unable to generate memory T cells. Although many cells could potentially express the CD40 ligand needed to stimulate CD40, it is most likely that CD4 T cells are the source of this signal.

1	The requirement for IL-2 signaling in programming CD8 memory was discovered by using CD8 T cells that were unable to respond to IL-2 because of a genetic deficiency in the IL-2Rα subunit. Because IL-2Rα signaling is required for the development of T cells, mice lacking IL-2Rα develop a lymphopro liferative disorder. However, this disorder does not develop in mice that are mixed bone marrow chimeras harboring both wild-type and IL-2Rα-deficient cells, and these chimeras can be used to study the behavior of IL-2Rα-deficient cells. When these chimeric mice were infected with LCMV and their responses were tested, memory CD8 responses were found to be defective specifically in the T cells lacking IL-2Rα.

1	The experiment shown in Fig. 11.33 indicates that, distinct from their effect in programming naive CD8 T cells, CD4 T cells also provide help in maintaining the number of CD8 memory T cells. In this case, CD8 memory T cells that had been programmed in normal mice were transferred into immunologically naive mice that either expressed or lacked MHC class II. Transfer of CD8 memory cells into mice lacking MHC class II resulted in a more rapid decrease in the number of memory CD8 T cells in comparison with a similar transfer

1	Fig. 11.33 CD4 T cells promote the maintenance of CD8 memory cells. The dependence of memory CD8 T cells on CD4 T cells is shown by the different lifetimes of the memory cells after their transfer into host mice that either have normal CD4 T cells (wildtype) or lack CD4 T cells (MHC II–/–). In the absence of MHC class II proteins, CD4 T cells fail to develop in the thymus. When CD8 memory T cells specific for LCMV were isolated from donor mice 35 days after infection with the virus and transferred into these hosts, memory cells were maintained only in mice that had CD4 T cells. The basis for this action of CD4 T cells is not yet clear, but has implications for conditions such as HIV/AIDS in which the number of CD4 T cells is diminished.

1	into wild-type mice. In addition, CD8 effector cells transferred into mice lacking MHC class II had a relative impairment of CD8 effector functions. These experiments imply that CD4 T cells activated by MHC class II-expressing antigen-presenting cells during an immune response have a significant impact on the quantity and quality of the CD8 T-cell response, even when they are not needed for the initial CD8 T-cell activation. CD4 T cells help to program naive CD8 T cells to be able to generate memory T cells, help to promote efficient effector activity, and help to maintain memory T-cell numbers. 11-24 In immune individuals, secondary and subsequent responses are mainly attributable to memory lymphocytes.

1	11-24 In immune individuals, secondary and subsequent responses are mainly attributable to memory lymphocytes. In the normal course of an infection, a pathogen proliferates to a level sufficient to elicit an adaptive immune response and then stimulates the production of antibodies and effector T cells that eliminate the pathogen from the body. Most of the effector T cells then die, and antibody levels gradually decline, because the antigens that elicited the response are no longer present at the level needed to sustain it. We can think of this as feedback inhibition of the response. Memory T and B cells remain, however, and maintain a heightened ability to mount a response to a recurrence of infection with the same pathogen.

1	Antibody and memory lymphocytes remaining in an immunized individual can have the effect of reducing the activation of naive B and T cells on a subsequent encounter with the same antigen. In fact, passively transferring antibody to a naive recipient can be used to inhibit naive B-cell responses to that same antigen. This phenomenon has been put to practical use to prevent Rh– mothers from making an immune response to an Rh+ fetus, which can result in hemolytic disease of the newborn (see Appendix I, Section A-6). If anti-Rh antibody is given to the mother before she is first exposed to her child’s Rh+ red blood cells, her response will be inhibited. The mechanism of this suppression is likely to involve the antibody-mediated clearance and destruction of fetal red blood cells that have entered the mother, thus preventing naive B cells and T cells from mounting an immune response. Presumably, the anti-Rh antibody is in excess over antigen, so that not only is antigen eliminated, but

1	entered the mother, thus preventing naive B cells and T cells from mounting an immune response. Presumably, the anti-Rh antibody is in excess over antigen, so that not only is antigen eliminated, but immune complexes are not formed to stimulate naive B cells through Fc receptors. Memory B-cell responses are, however, not inhibited by antibody, so the Rh– mothers at risk must be identified and treated before a primary response has occurred. Because of their high affinity for antigen and alterations in their B-cell receptor signaling requirements, memory B cells are much more sensitive to the small amounts of antigen that cannot be efficiently cleared by the passive anti-Rh antibody. The ability of memory B cells to be activated to produce antibody, even when exposed to preexisting antibody, also allows secondary antibody responses to occur in individuals who are already immune.

1	These suppressive mechanisms might also explain a phenomenon called original antigenic sin. This term was coined to describe the tendency of people to make antibodies only against the epitopes expressed on the first influenza virus variant to which they are exposed, even in subsequent infections with viral variants that have additional, highly immunogenic epitopes (Fig. 11.34). Antibodies against the original virus will tend to suppress responses of naive B cells specific for the new epitopes. This might benefit the host by using only those B cells that can respond most rapidly and effectively to the virus. This pattern is broken only if the person is exposed to an influenza virus that lacks all epitopes seen in the original infection, because now no preexisting antibodies bind the virus, and naive B cells are able to respond.

1	A similar suppression of naive T-cell responses by antigen-specific memory T cells can occur in the setting of infection by lymphocytic choriomeningitis 1000 100 10 A B C D E F G Response to epitope Response to epitope Response to epitope A B C D E F A B C D Percentagenormal response 1000 100 10 Percentagenormal response 1000 100 10 Percentagenormal response Same individual at 20 years infected with a new variant in˜uenza virus makes antibody only against epitopes shared with original virus, not against epitopes shared with the variant encountered at age 5 years Same individual at 5 years infected with a variant in˜uenza virus makes antibody only against the epitopes shared with the original virus Individual at 2 years infected with in˜uenza virus makes antibody against all epitopes present on the virus

1	Fig. 11.34 When individuals who have been infected with one influenza virus responds preferentially to those epitopes shared variant of influenza virus are infected with a second or third with the original virus, and makes a smaller than normal response variant, they make antibodies only against epitopes that were to new epitopes on the virus (center panel). Even at age 20 years, present on the initial virus. A child infected for the first time with this commitment to respond to epitopes shared with the original an influenza virus at 2 years of age makes a response to all epitopes virus, and the subnormal response to new epitopes, is retained (right (left panel). At age 5 years, the same child exposed to a different panel). This phenomenon is called ‘original antigenic sin.’ virus (LCMV) in the mouse or dengue virus in humans. Mice that were primed with one strain of LCMV responded to a subsequent infection with a second strain of LCMV by expanding CD8 T cells that reacted to antigens

1	the mouse or dengue virus in humans. Mice that were primed with one strain of LCMV responded to a subsequent infection with a second strain of LCMV by expanding CD8 T cells that reacted to antigens specific for the first strain. However, this type of effect was not observed when responses to variable ovalbumin antigenic epitopes were examined in the setting of recurrent infections using the bacterial pathogen Listeria monocytogenes, suggesting that the suppression caused by ‘original antigenic sin’ does not occur in all immune responses.

1	Summary.

1	Protective immunity against reinfection is one of the most important consequences of adaptive immunity, and results from the establishment of populations of long-lived memory B cells and memory T cells. These antigen-specific memory cells arise from the populations of lymphocytes that expand dramatically during the primary infection, and that survive at higher frequencies than in the naive lymphocyte repertoires. Both their increased frequency and their capacity to respond more rapidly to restimulation to the same antigen contribute to protective immunity, which can be transferred to naive recipients by memory B and T cells. Memory lymphocytes are maintained by their expression of receptors for cytokines, such as IL-7 and IL-15, that provide survival signals. Memory B cells can be distinguished by changes in their immunoglobulin genes because of isotype switching and somatic hypermutation, and secondary and subsequent immune responses are characterized by antibodies with increasing

1	by changes in their immunoglobulin genes because of isotype switching and somatic hypermutation, and secondary and subsequent immune responses are characterized by antibodies with increasing affinity for the antigen. The advent of receptor-specific reagents—MHC tetramers—has allowed for the direct analysis of the expansion and differentiation of effector and memory T cells. We now recognize that T-cell memory is complex, and memory T cells are quite heterogeneous, having central memory, effector memory, and tissue-resident memory subtypes. While CD8 T cells can generate effective primary responses in the absence of help from CD4 T cells, it is becoming clear that CD4 T cells have an integral role in regulating CD8 T-cell memory. These issues will be critical in understanding, for example, how to design effective vaccines for diseases such as HIV/AIDS.

1	Summary to Chapter 11.

1	Vertebrates resist infection by pathogenic microorganisms in several ways. The innate defenses can act immediately and may succeed in repelling the infection, but if not, they are followed by a series of induced early responses that help to contain the infection as adaptive immunity develops. These first two phases of the immune response rely on recognizing the presence of infection by using the nonclonotypic receptors of the innate immune system. They are summarized in Fig. 11.35 and covered in detail in Chapter 3. Several specialized subsets of immune cells, which can be viewed as intermediates between innate and adaptive immunity, act next and include the innate lymphoid cells, or ILCs, which are rapid responders to cytokines produced by innate sensor cells and can help to bias the CD4 T-cell response toward parallel subsets of effector T cells; and NK cells, which can be recruited to lymph nodes and secrete IFN-γ, and thus promote a TH1 response. The third phase of an immune

1	CD4 T-cell response toward parallel subsets of effector T cells; and NK cells, which can be recruited to lymph nodes and secrete IFN-γ, and thus promote a TH1 response. The third phase of an immune response is the adaptive immune response (see Fig. 11.35), which is mounted in the peripheral lymphoid tissue that serves the particular site of infection and takes several days to develop, because T and B lymphocytes must encounter their specific antigen, proliferate, and differentiate into effector cells. T-cell dependent B-cell responses cannot be initiated until antigen-specific TFH cells have had a chance to proliferate and differentiate. Once an adaptive immune response has occurred, the antibodies and effector T cells are dispersed via the circulation and recruited into the infected tissues; the infection is usually controlled and the pathogen is contained or eliminated. The final effector mechanisms used to clear an infection depend on the type of infectious agent, and in most cases

1	the infection is usually controlled and the pathogen is contained or eliminated. The final effector mechanisms used to clear an infection depend on the type of infectious agent, and in most cases they are the same as those employed in the early phases of immune defense; only the recognition mechanism changes and is now more selective (see Fig. 11.35).

1	Fig. 11.35 The components of the three phases of the immune response against different classes of microorganisms. The mechanisms of innate immunity that operate in the first two phases of the immune response are described in Chapters 2 and 3, and thymus-independent (T-independent) B-cell responses are covered in Chapter 10. The early phases contribute to the initiation of adaptive immunity, and they influence the functional character of the antigen-specific effector T cells and antibodies that appear on the scene in the late phase of the response. There are striking similarities in the effector mechanisms at each phase of the response; the main change is in the recognition structure used.

1	An effective adaptive immune response leads to a state of protective immunity. This state consists of the presence of effector cells and molecules produced in the initial response, and of immunological memory. Immunological memory is manifested as a heightened ability to respond to pathogens that have previously been encountered and successfully eliminated. Memory T and B lymphocytes have the property of being able to transfer immune memory to naive recipients. The mechanisms that maintain immunological memory include certain cytokines, such as IL-7 and IL-15, as well as homeostatic interactions between the T-cell receptors on memory cells with self peptide:self MHC complexes. The artificial induction of protective immunity, which includes immunological memory, by vaccination is the most outstanding accomplishment of immunology in the field of medicine. The understanding of how this is accomplished is now catching up with its practical success. However, as we will see in Chapter 13,

1	outstanding accomplishment of immunology in the field of medicine. The understanding of how this is accomplished is now catching up with its practical success. However, as we will see in Chapter 13, many pathogens do not induce protective immunity that completely eliminates the pathogen, so we will need to learn what prevents this before we can prepare effective vaccines against these pathogens.

1	Questions. 11.1 True or False: The immune response is a dynamic process that initiates with an antigen-independent response, which becomes more focused and powerful as it develops antigen specificity. Once the adaptive immune system develops, a single type of response is capable of eliminating any type of pathogen. 11.2 Multiple Choice: Which statement is incorrect? A. IL-12 and IL-18 production by macrophages and dendritic cells induces IFN-γ secretion by ILC1 to induce heightened killing of intracellular pathogens. B. ILC3s are activated by thymic stromal lymphopoietin (TSLP), which activates STAT5 and induces IL-17 production. C. Molecular patterns common to helminths activate IL-33 and IL-25 production, which in turn activates ILC2s to induce mucus production by goblet cells and mucosal smooth muscle contraction. D. ILC3-derived IL-22 acts on epithelial cells to induce production of antimicrobial peptides and promotes an enhanced barrier integrity.

1	D. ILC3-derived IL-22 acts on epithelial cells to induce production of antimicrobial peptides and promotes an enhanced barrier integrity. 11.3 Matching: Match the following proteins with their effect on T-cell migration. A. CXCR5 _______ i. Interacts with Pand E-selectin, expressed at activated endothelial cells B. PSGL-1 _______ ii. CXCL13 binding attracts TFH cells to the B-cell follicle C. FucT-VII _______ iii. Interacts with VCAM-1 to initiate extravasation of the effector T cells

1	B. PSGL-1 _______ ii. CXCL13 binding attracts TFH cells to the B-cell follicle C. FucT-VII _______ iii. Interacts with VCAM-1 to initiate extravasation of the effector T cells D. VLA-4 _______ iv. Necessary for the production of Pand E-selectin 11.4 Fill-in-the-Blanks: Expression of selective adhesion molecules among effector T cells helps compartmentalize their distribution. For example, T cells that are primed in the GALT induce the expression of the __________ integrin, which binds to ___________, which is constitutively expressed by the gut mucosal endothelial cells. These T cells also express the chemokine receptor ______, which attracts T cells to the lamina propria subjacent to the small intestine epithelium via a _________ gradient. This compartmentalization capacity is not unique to the gut and can be observed in other organs such as the skin. For example, expression of a glycosylated form of PSGL-1, ______, binds ________ on cutaneous vascular endothelium.

1	11.5 Multiple Choice: Which of the following statements incorrectly describes TH1 macrophage activation? A. CD40 ligand sensitizes the macrophage to respond to IFN-γ B. LT-α can substitute for CD40 ligand in macrophage activation C. TNFR-I activation is antagonized by activated TH1 cells D. Macrophages are sensitized to IFN-γ by small amounts of bacterial LPS 11.6 Short Answer: How do M2 macrophages stimulate collagen production in order to promote tissue repair? 11.7 Multiple Choice: Which of the following is not true concerning type 3 responses? A. The primary innate effector cells are neutrophils, which are recruited by CXCL8 and CXCL2 and have an increased output due to G-CSF and GM-CSF B. At homeostasis, TH17 cells are present almost exclusively in the intestinal mucosa C. IL-17 is the central cytokine D. IL-22 is produced to induce antimicrobial peptide production and epithelial cell proliferation and shedding of natural killer cells

1	C. IL-17 is the central cytokine D. IL-22 is produced to induce antimicrobial peptide production and epithelial cell proliferation and shedding of natural killer cells E. IL-23 initiates the commitment of naive CD4+ T cells to the TH17 fate 11.8 Multiple Choice: Which of the following pathogens is able to induce a robust CD8+ T-cell response independent of CD4+ T-cell help? A. Streptococcus pneumoniae B. Lymphocytic choriomeningitis virus (LCMV) C. Listeria monocytogenes D. Staphylococcus aureus E. Salmonella

1	A. Streptococcus pneumoniae B. Lymphocytic choriomeningitis virus (LCMV) C. Listeria monocytogenes D. Staphylococcus aureus E. Salmonella F. Toxoplasma 11.9 Fill-in-the-Blanks: During an immune response to a pathogen, activated T cells express ______, a high-affinity IL-2 receptor component, and lose expression of ________, an IL-7 receptor component. The activated cells also generate different isoforms of _________, a protein tyrosine phosphatase expressed by all hematopoietic cells. Effector and central memory cells develop, and these can be distinguished by high expression of ___________________ in the former and ________ in the latter. The survival of both CD4+ and CD8+ memory T cells is dependent on _________, although the survival of CD8+ memory T cells is additionally dependent on _________. Section references. 11-1 The course of an infection can be divided into several distinct phases.

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1	11.10 True or False: CD27 is a marker of naive B cells as well as memory T cells. 11.11 Short Answer: How is inflammasome activation able to help induce type 1 and type 3 responses while blunting type 2 responses? 11.12 Matching: Match the cytokine to the downstream STAT. Walker, J.A., Barlow, J.L., and McKenzie, A.N.J.: Innate lymphoid cells—how did we miss them? Nat. Rev. Immunol. 2013, 13:75–87. 11-3 Effector T cells are guided to specific tissues and sites of infection by changes in their expression of adhesion molecules and chemokine receptors. Griffith, J.W., Sokol, C.L., and Luster, A.D.: Chemokines and chemokine receptors: positioning cells for host defense and immunity. Annu. Rev. Immunol. 2014, 32:659–702. Hidalgo, A., Peired, A.J., Wild, M.K., Vestweber, D., and Frenette, P.S.: Complete identification of E-selectin ligands on neutrophils reveals distinct functions of PSGL-1, ESL-1, and CD44. Immunity 2007, 26:477–489.

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1	Fridman, W.H.: Regulation of B cell activation and antigen presentation by Fc receptors. Curr. Opin. Immunol. 1993, 5:355–360. Klenerman, P., and Zinkernagel, R.M.: Original antigenic sin impairs cytotoxic T lymphocyte responses to viruses bearing variant epitopes. Nature 1998, 394:482–485. Mongkolsapaya, J., Dejnirattisai, W., Xu, X.N., Vasanawathana, S., Tangthawornchaikul, N., Chairunsri, A., Sawasdivorn, S., Duangchinda, T., Dong, T., Rowland-Jones, S., et al.: Original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever. Nat. Med. 2003, 9:921–927. Pollack, W., Gorman, J.G., Freda, V.J., Ascari, W.Q., Allen, A.E., and Baker, W.J.: Results of clinical trials of RhoGAm in women. Transfusion 1968, 8:151–153. Zehn, D., Turner, M.J., Lefrançois, L., and Bevan, M.J.: Lack of original antigenic sin in recall CD8+ T cell responses. J. Immunol. 2010, 184:6320–6326. The Mucosal Immune System

1	Zehn, D., Turner, M.J., Lefrançois, L., and Bevan, M.J.: Lack of original antigenic sin in recall CD8+ T cell responses. J. Immunol. 2010, 184:6320–6326. The Mucosal Immune System Adaptive immune responses are typically initiated in the peripheral lymph nodes that drain the infected tissues. While most internal tissues are free of active microbial growth, the skin and the various mucosae lining organs that directly contact the external world will have continuous encounters with environmental microbes. These surfaces are where most pathogens invade. In this chapter, we will discuss the specialized features of the immune system that serves these mucosal surfaces—the mucosal immune system.

1	The mucosal immune system, in particular, that of the gut, may well have been the first part of the vertebrate adaptive immune system to evolve, possibly linked to the need to deal with the vast populations of commensal bacteria that coevolved with the vertebrates. Organized lymphoid tissues and immunoglobulin antibodies are first found in vertebrates in the gut of primitive cartilaginous fishes, and two important central lymphoid organs—the thymus and the avian bursa of Fabricius—derive from the embryonic intestine. Fish also have a primitive form of secretory antibody that protects their body surface and may be the forerunner of IgA in mammals. It has therefore been suggested that the mucosal immune system represents the original vertebrate immune system, and that the spleen and lymph nodes are later specializations The nature and structure of the mucosal immune system.

1	The first line of defense against invasion by potential pathogens and commensal microorganisms is the thin layer of epithelium that covers all these surfaces. However, the epithelium can be breached relatively easily, and so its barrier function needs to be supplemented by defenses provided by the cells and molecules of the mucosal immune system. The innate defenses of mucosal tissues, such as antimicrobial peptides and cells bearing invariant pathogen-recognition receptors, are described in Chapters 2 and 3. In this chapter we concentrate on the adaptive mucosal immune system, highlighting only those innate responses that are of particular importance to our discussion. Many of the anatomical and immunological principles underlying the mucosal immune system apply to all its constituent tissues; here we will use the intestine as our example, and the reader is referred to the general references at the end of this chapter for further details of the other sites.

1	12-1 The mucosal immune system protects the internal surfaces of the body. The mucosal immune system comprises the internal body surfaces that are lined by a mucus-secreting epithelium—the gastrointestinal tract, the upper and lower respiratory tract, the urogenital tract, and the middle ear. It also includes the exocrine glands associated with these organs, such as the conjunctivae and lacrymal glands of the eye, the salivary glands, and the lactating breast (Fig. 12.1). The mucosal surfaces represent an enormous area to The nature and structure of the mucosal immune system. The mucosal response to infection and regulation of mucosal immune responses.

1	The nature and structure of the mucosal immune system. The mucosal response to infection and regulation of mucosal immune responses. respiratory tract gastrointestinal tract urogenital tract conjunctiva esophagus stomach intestine sinus middle ear trachea lungs oral cavity lachrymal gland salivary gland Mucosal tissues of the human body mammary gland kidney uterus bladder vagina Fig. 12.1 The mucosal immune system. The tissues of the mucosal immune system are the lymphoid organs and cells associated with the intestine, respiratory tract, and urogenital tract, as well as the oral cavity, pharynx, middle ear, and the glands associated with these tissues, such as the salivary glands and lachrymal glands. The lactating breast is also part of the mucosal immune system. be protected. The human small intestine, for instance, has a surface area of almost 400 m2, which is 200 times that of the skin.

1	be protected. The human small intestine, for instance, has a surface area of almost 400 m2, which is 200 times that of the skin. The mucosal immune system forms the largest part of the body’s immune tissues, containing approximately three-quarters of all lymphocytes and producing the majority of immunoglobulin in healthy individuals. It is also exposed continuously to antigens and other materials entering from the environment. When compared with lymph nodes and spleen (which in this chapter we will call the systemic immune system), the mucosal immune system has many unique and unusual features (Fig. 12.2).

1	Fig. 12.2 Distinctive features of the mucosal immune system. The mucosal immune system is bigger, encounters a wider range of antigens, and encounters them much more frequently than the rest of the immune system—what we call in this chapter the systemic immune system. This is reflected in distinctive anatomical features, specialized mechanisms for the uptake of antigen, and unusual effector and regulatory responses that are designed to prevent unwanted immune responses to food and other innocuous antigens. The nature and structure of the mucosal immune system. Worldwide deaths annually from mucosal infections Acute respiratory infections (4 million) Diarrheal diseases (2.2 million) Tuberculosis (1.5 million) HIV/AIDS (2 million) Measles (400,000) Hepatitis B (103,000*) Whooping cough (294,000) Roundworm and hookworm (6000) 0 4 million3 million2 million1 million

1	Because of their physiological functions in gas exchange (the lungs), food absorption (the gut), sensory activities (eyes, nose, mouth, and throat), and reproduction (uterus and vagina), the mucosal surfaces are thin and permeable barriers to the interior of the body. The importance of these tissues to life means that effective defense mechanisms are essential to protect them from invasion. Equally significant is that their fragility and permeability create obvious vulnerability to infection, and it is not surprising that the vast majority of infectious agents invade the human body by these routes (Fig. 12.3). Diarrheal diseases, acute respiratory infections, pulmonary tuberculosis, measles, whooping cough, and worm infestations continue to be the major causes of death throughout the world, especially in infants in developing countries. To these must be added the human immunodeficiency virus (HIV), a pathogen whose natural route of entry via a mucosal surface is often overlooked, as

1	especially in infants in developing countries. To these must be added the human immunodeficiency virus (HIV), a pathogen whose natural route of entry via a mucosal surface is often overlooked, as well as other sexually transmitted infections such as syphilis.

1	The mucosal surfaces are also portals of entry for a vast array of foreign antigens that are not pathogenic. This is best seen in the gut, which is exposed to enormous quantities of food proteins—an estimated 30–35 kg per year per person. At the same time, the healthy large intestine is colonized by at least a thousand species of bacteria that live in symbiosis with their host and are known as commensal microorganisms, or the microbiota. These bacteria are present at levels of at least 1012 organisms per milliliter in the colon contents, making them the most numerous cells in the body by a factor of 10. Substantial populations of viruses and fungi are also found in the healthy intestine. In normal circumstances these organisms do no harm, and many are beneficial to their hosts, having important metabolic functions, as well as being essential for normal immune function. The other mucosal surfaces are also colonized by substantial populations of resident commensal organisms (Fig. 12.4).

1	As food proteins and the microbiota contain many foreign antigens, they are capable of being recognized by the adaptive immune system. Generating protective immune responses against these harmless agents would, however, be inappropriate and wasteful. Indeed, aberrant immune responses of this kind are now believed to be the cause of some relatively common diseases, including celiac disease (caused by a response to the wheat protein gluten; discussed in Chapter 14) and inflammatory bowel diseases such as Crohn’s disease (a response to commensal bacteria). As we shall see, the intestinal mucosal immune system has evolved means of distinguishing harmful pathogens from antigens in food and the normal microbiota. Similar issues are faced at other mucosal surfaces, such as the respiratory tract and female genital

1	Fig. 12.3 Mucosal infections comprise one of the biggest health problems worldwide. Most of the pathogens that cause death throughout the world either are those of mucosal surfaces or enter the body through these routes. Respiratory infections are caused by numerous bacteria (such as Streptococcus pneumoniae and Haemophilus influenzae, which cause pneumonia; and Bordetella pertussis, the cause of whooping cough) and viruses (such as influenza and respiratory syncytial virus). Diarrheal diseases are caused by both bacteria (such as the cholera bacterium Cholera vibrio) and viruses (such as rotaviruses). The human immunodeficiency virus (HIV) that causes AIDS enters through the mucosa of the urogenital tract or is secreted into breast milk and passed from mother to child in this way. The bacterium Mycobacterium tuberculosis, which causes tuberculosis, also enters through the respiratory tract. Measles manifests itself as a systemic disease, but it originally enters via the oral/

1	The bacterium Mycobacterium tuberculosis, which causes tuberculosis, also enters through the respiratory tract. Measles manifests itself as a systemic disease, but it originally enters via the oral/ respiratory route. Hepatitis B is also a sexually transmitted virus. Finally, parasitic worms inhabiting the intestine cause chronic debilitating disease and premature death. Most of these deaths, especially those from acute respiratory and diarrheal diseases, occur in children under 5 years old in the developing world, and there are still no effective vaccines against many of these pathogens. Numbers shown are the most recent estimated figures available (The Global Burden of Disease: 2004 Update. World Health Organization, 2008). *Does not include deaths from liver cancer or cirrhosis resulting from chronic infection.

1	PC2 (4.4%)

1	Fig. 12.4Composition of the commensal microbiota at (Actinobacteria), Bacteroides fragilis (Bacteroidetes), and Escherichia different mucosal surfaces in healthy humans. Panel a: coli (Proteobacteria). Panel b: principal component analysis of the the different sizes of the pie charts for different sites reflect the microbiomes isolated from the indicated human tissues, plotting number of distinct bacterial species typically present at those the first and second principal components. The primary component sites. The colon contains the greatest number of different species of microbiome variation is due to body area and accounts for (over 1000 as estimated from individual surveys). The color key 13% of the variation in microbial identity between samples taken indicates the four bacterial phyla that contain the majority of from these sites. a, adapted from Dethlefsen, L., et al.: Nature commensal species. Ubiquitous commensal bacteria include 2007, 449:811‑818. b, from Huttenhower, C., et

1	that contain the majority of from these sites. a, adapted from Dethlefsen, L., et al.: Nature commensal species. Ubiquitous commensal bacteria include 2007, 449:811‑818. b, from Huttenhower, C., et al.: Nature 2012, Lactobacillus and Clostridium spp. (Firmicutes), Bifidobacterium spp. 486:207–214.

1	tract. Here, protective immunity against pathogens is essential, but many of the antigens entering these tissues are also harmless, being derived from commensal organisms, pollen, other innocuous environmental material, and, in the lower urogenital tract, seminal fluid. The fetus is a further important source of foreign antigen encountered by the normal mucosal immune system to which immune responses must be controlled. 12-2 Cells of the mucosal immune system are located both in anatomically defined compartments and scattered throughout mucosal tissues.

1	12-2 Cells of the mucosal immune system are located both in anatomically defined compartments and scattered throughout mucosal tissues. Lymphocytes and other immune-system cells such as macrophages and dendritic cells are found throughout the intestinal tract, both in organized tissues and scattered throughout the surface epithelium of the mucosa and in the underlying layer of connective tissue called the lamina propria. The organized secondary lymphoid tissues in the gut comprise a group of organs known as the gut-associated lymphoid tissues (GALT), together with the draining mesenteric and caudal lymph nodes (Fig. 12.5). The GALT and the mesenteric lymph nodes have the anatomically compartmentalized structure typical of peripheral lymphoid organs, and are sites at which immune responses are initiated. The cells scattered throughout the epithelium and the lamina propria comprise the effector cells of the local immune response.

1	The intestinal mucosa of the small intestine to mesenteric lymph node isolated lymphoid follicle Intestinal lumen Peyer’s patch subepithelial dome crypt follicle (B cells) T-cell area lamina propria lamina propria lymphocyte epithelium intraepithelial lymphocyte lymphatic villus Scattered lymphoid cells Organized lymphoid tissues Intestinal lymphocytes are found in organized tissues where immune responses are induced, and scattered throughout the intestine, where they carry out effector functions Fig. 12.5 Gut-associated lymphoid tissues and lymphocyte populations.

1	is made up of fingerlike processes (villi) covered by a thin layer of epithelial cells (red) that are responsible for digestion of food and absorption of nutrients. These epithelial cells are replaced continually by new cells that derive from stem cells in the crypts. The tissue layer underlying the epithelium is called the lamina propria, and is colored pale yellow throughout this chapter. Lymphocytes are found in several discrete compartments in the intestine, with the organized lymphoid tissues such as Peyer’s patches and isolated lymphoid follicles forming what is known as the gut‑associated lymphoid tissues (GALT). These tissues lie in the wall of the intestine itself, separated from the contents of the intestinal lumen by the single layer of epithelium. The draining lymph nodes for the gut are the mesenteric lymph nodes, which are connected to Peyer’s patches and the intestinal mucosa by afferent lymphatic vessels and are the largest lymph nodes in the body. Together, these

1	for the gut are the mesenteric lymph nodes, which are connected to Peyer’s patches and the intestinal mucosa by afferent lymphatic vessels and are the largest lymph nodes in the body. Together, these organized tissues are the sites of antigen presentation to T cells and B cells and are responsible for the induction phase of immune responses. Peyer’s patches and mesenteric lymph nodes contain discrete T‑cell areas (blue) and B‑cell follicles (yellow), while the isolated follicles comprise mainly B cells. Many lymphocytes are found scattered throughout the mucosa outside the organized lymphoid tissues: these are effector cells—effector T cells and antibody‑secreting plasma cells, as well as innate lymphoid cells (ILCs). Effector lymphocytes are found both in the epithelium and in the lamina propria. Lymphatics also drain from the lamina propria to the mesenteric lymph nodes.

1	The GALT comprises the Peyer’s patches, which are present only in the small intestine; isolated lymphoid follicles (ILF), which are found throughout the intestine; the appendix (in humans); and the tonsils and adenoids in the throat. The palatine tonsils, adenoids, and lingual tonsils are large aggregates of lymphoid tissue covered by a layer of squamous epithelium and form a ring, known as Waldeyer’s ring, at the back of the mouth around the entrance of the gut and airways (Fig. 12.6). They often become extremely enlarged in childhood because of recurrent infections, and in the past were frequently removed as a result. A reduced IgA response to oral polio vaccination has been seen in individuals who have had their tonsils and adenoids removed. The tonsils and adenoids form a ring of lymphoid tissues, Waldeyer’s ring, around the entrance of the gut and airway adenoid palatine tonsil lingual tonsil tongue

1	The tonsils and adenoids form a ring of lymphoid tissues, Waldeyer’s ring, around the entrance of the gut and airway adenoid palatine tonsil lingual tonsil tongue Fig. 12.6 A ring of lymphoid organs called Waldeyer’s ring surrounds the entrance to the intestine and respiratory tract. The adenoids lie at either side of the base of the nose, while the palatine tonsils lie at either side of the back of the oral cavity. The lingual tonsils are discrete lymphoid organs on the base of the tongue. The micrograph shows a section through an inflamed human tonsil, where the areas of organized lymphoid tissue are covered by a layer of squamous epithelium (at top of photo). The surface contains deep crevices (crypts) that increase the surface area but can easily become sites of infection. Hematoxylin and eosin staining. Magnification ×100. Fig. 12.7 Transport of antigens by M cells facilitates antigen presentation.

1	The first panel illustrates the passage of antigen through M cells in the follicle‑associated epithelium of Peyer’s patches. M cells have convoluted basal membranes that form ‘pockets’ within the epithelial layer, allowing close contact with lymphocytes and other cells. This favors the local transport of antigens that have been taken up from the intestine by the M cells and their delivery to dendritic cells for antigen presentation. The top right micrograph of a Peyer’s patch stained with fluorescently labeled antibodies shows epithelial cells (cytokeratin, dark blue), with M‑cell pockets inferred from the presence of T cells (CD3, red) and B cells (CD20, green). At bottom right, Peyer’s patch follicle epithelium shows CX3CR1expressing myeloid cells (green), which include some dendritic cells, interacting with M cells identified by expression of peptidoglycan recognition protein‑S (red) and apical staining for the UEA‑1 lectin (cyan). Some CX3CR1‑expressing cells extend processes into

1	interacting with M cells identified by expression of peptidoglycan recognition protein‑S (red) and apical staining for the UEA‑1 lectin (cyan). Some CX3CR1‑expressing cells extend processes into the M cells (arrows). Top right, micrograph from Espen, S., et al.: Immunol. Today 1999, 20:141–151. Bottom right, micrograph from Wang et al.: J. Immunol. 2011, 187:5277–5285.

1	The Peyer’s patches of the small intestine, the lymphoid tissue of the appendix, and the isolated lymphoid follicles are located within the intestinal wall. Peyer’s patches are important sites for the initiation of immune responses in the gut. Visible to the naked eye, they have a distinctive appearance, forming dome-like aggregates of lymphoid cells that project into the intestinal lumen (see Fig. 1.24). There are 100–200 Peyer’s patches in the human small intestine. They are much richer in B cells than the systemic peripheral lymphoid organs, each Peyer’s patch consisting of a large number of B-cell follicles with germinal centers, with small T-cell areas between and immediately below the follicles (see Fig. 12.5). The subepithelial dome area lies immediately beneath the epithelium and is rich in dendritic cells, T cells, and B cells. Separating the lymphoid tissues from the gut lumen is a layer of follicle-associated epithelium. This contains conventional intestinal epithelial

1	and is rich in dendritic cells, T cells, and B cells. Separating the lymphoid tissues from the gut lumen is a layer of follicle-associated epithelium. This contains conventional intestinal epithelial cells known as enterocytes and a smaller number of specialized epithelial cells called microfold cells (M cells), as shown in Fig. 1.24. M-cell development is controlled by local B cells and RANK ligand (RANKL), which is a member of the tumor necrosis factor (TNF) superfamily like CD40L (see Section 7-23). Unlike the enterocytes that make up most of the intestinal epithelium, M cells have a folded luminal surface instead of microvilli and do not secrete digestive enzymes or mucins, and so lack the thick layer of surface mucus (the glycocalyx) found covering conventional epithelial cells (see Fig. 1.24). They are therefore directly exposed to microorganisms and particles within the gut lumen and are the preferred route by which antigens such as microbes enter the Peyer’s patch from the

1	Fig. 1.24). They are therefore directly exposed to microorganisms and particles within the gut lumen and are the preferred route by which antigens such as microbes enter the Peyer’s patch from the lumen (Fig. 12.7). The follicle-associated epithelium also contains lymphocytes and dendritic cells.

1	Several thousand isolated lymphoid follicles can be identified microscopically throughout the small and large intestines, but they are more frequent in the large intestine, correlating with the load of local microorganisms. Like Peyer’s patches, these follicles have an epithelium containing M cells that lies over the organized lymphoid tissue. However, they contain mainly B cells and develop only after birth in response to antigen stimulation due to colonization of the gut by commensal microorganisms. Peyer’s patches, in contrast, are already present in the fetal gut, although their full development is not completed until after birth. In the mouse gut, isolated lymphoid follicles seem to arise from small aggregates in the intestinal wall called cryptopatches, which contain dendritic cells and lymphoid tissue inducer (LTi) cells (see Section 9-2). Cryptopatches have not yet been identified in the human gut. Peyer’s patches and isolated lymphoid follicles are connected by lymphatics to

1	and lymphoid tissue inducer (LTi) cells (see Section 9-2). Cryptopatches have not yet been identified in the human gut. Peyer’s patches and isolated lymphoid follicles are connected by lymphatics to the draining lymph nodes.

1	The tissues of the small intestine drain to the mesenteric lymph nodes, which are located in the connective tissue that tethers the intestine to the rear wall of the abdomen. These are the largest lymph nodes in the body and play a crucial role in initiating and shaping immune responses to intestinal antigens. The mucosal surface and lymphoid aggregates of the large intestine drain to part of the mesenteric lymph node and to a separate node known as the caudal lymph node, found close to the bifurcation of the aorta. The mesenteric lymph nodes and Peyer’s patches differentiate independently of the systemic immune system during fetal development, and their development involves distinct chemokines and receptors of the tumor necrosis factor (TNF) family (see Section 9-2). The differences between the GALT and the systemic lymphoid organs are thus imprinted early in life.

1	In some species such as mice, isolated lymphoid follicles are also found in the lining of the nose, and in the wall of the upper respiratory tract; those in the nose are called nasal-associated lymphoid tissues (NALT), while those in the upper respiratory tract are known as bronchus-associated lymphoid tissues (BALT). The term mucosa-associated lymphoid tissues (MALT) is sometimes used to refer collectively to all such tissues found in mucosal organs, although defined organized lymphoid tissues are not found in the nose or respiratory tract in adult humans unless infection is present. 12-3 The intestine has distinctive routes and mechanisms of antigen uptake.

1	Antigens present at mucosal surfaces must be transported across an epithelial barrier before they can stimulate the mucosal immune system. Peyer’s patches and isolated lymphoid follicles are highly adapted for the uptake of antigen from the intestinal lumen. The M cells in the follicle-associated epithelium are continually taking up molecules and particles from the gut lumen by endocytosis or phagocytosis (see Fig. 12.7). For several bacteria this may involve specific recognition of the bacterial FimH protein found in type 1 pili by a glycoprotein (GP2) on the M cell. This material is transported through the interior of the cell in membrane-bound vesicles to the basal cell membrane, where it is released into the extracellular space—a process known as transcytosis. Because M cells lack a glycocalyx and so are much more accessible than enterocytes, a number of pathogens target M cells to gain access to the subepithelial space, even though they then find themselves in the heart of the

1	glycocalyx and so are much more accessible than enterocytes, a number of pathogens target M cells to gain access to the subepithelial space, even though they then find themselves in the heart of the intestinal adaptive immune system. These include Salmonella enterica serotype Typhi, the causative agent of typhoid fever; other Salmonella enterica serotypes, which are major causes of bacterial food poisoning; Shigella species that cause dysentery; and Yersinia pestis, which causes plague. Poliovirus, reoviruses, some retroviruses such as HIV, and prions such as the causal agent of scrapie follow the same entry route. After entry into the M cell, bacteria produce proteins that reorganize the M-cell cytoskeleton in a manner that encourages their transcytosis.

1	The basal cell membrane of an M cell is extensively folded, forming a pocket that encloses lymphocytes and which makes close contacts with local myeloid cells, including dendritic cells (see Fig. 12.7). Macrophages and dendritic cells take up the transported material released from the M cells and process it for presentation to T lymphocytes. Local dendritic cells are in a favorable position to acquire gut antigens, and they are recruited toward, or even into, the follicle-associated epithelium in response to chemokines that are released constitutively by the epithelial cells. The chemokines include CCL20 (MIP-3α) and CCL9 (MIP-1γ), which bind to the receptors CCR6 and CCR1, respectively, on dendritic cells (see Appendix IV for a listing of chemokines and their receptors). The antigen-loaded dendritic cells then migrate from the dome region to the T-cell areas of the Peyer’s patch, where they meet naive, antigen-specific T cells. Together, the dendritic cells and primed T cells then

1	dendritic cells then migrate from the dome region to the T-cell areas of the Peyer’s patch, where they meet naive, antigen-specific T cells. Together, the dendritic cells and primed T cells then activate B cells and initiate class switching to IgA. All these processes— the uptake of antigen by M cells, the migration of dendritic cells into the epithelial layer, the production of chemokines, and the subsequent migration of dendritic cells into T-cell areas—are markedly increased in the presence of pathogenic organisms and their products due to the ligation of pattern recognition receptors on epithelial cells and immune cells (see Section 3-5). Similar processes also underlie the induction of immune responses in isolated lymphoid follicles of the gut and in the MALT of other mucosal surfaces.

1	12-4 The mucosal immune system contains large numbers of effector lymphocytes even in the absence of disease. In addition to the organized lymphoid organs, mucosal surfaces such as the gut and lung contain enormous numbers of lymphocytes and other leukocytes scattered throughout the tissue. Most of the scattered lymphocytes have the appearance of cells that have been activated by antigen, and they comprise the effector T cells and plasma cells of the mucosal immune system. In the intestine, effector cells are found in two main compartments: the epithelium and the lamina propria (see Fig. 12.5).

1	These tissues are quite distinct in immunological terms, despite being separated by only a thin layer of basement membrane. The lymphoid component of the epithelium consists mainly of lymphocytes, which in the small intestine are virtually all CD8 T cells. The lamina propria contains many types of immune cells, including IgA-producing plasma cells, conventional CD4 and CD8 T cells with effector and memory phenotypes, innate lymphoid cells, dendritic cells, macrophages, and mast cells. T cells in the lamina propria of the small intestine express the integrin α4:β7 and the chemokine receptor CCR9 (Fig. 12.8), which attracts them into the tissue from the bloodstream. Intraepithelial lymphocytes (IELs) are mostly CD8 T cells and express either the conventional

1	Fig. 12.8 Priming of naive T cells and the redistribution of effector T cells in the intestinal immune system. Naive T cells carry the chemokine receptor CCR7 and L‑selectin, which direct their entry into Peyer’s patches via high endothelial venules (HEVs). In the T‑cell area they encounter antigen that has been transported into the lymphoid tissue by M cells and is presented by local dendritic cells. During activation, and under the selective control of gut‑derived dendritic cells, the T cells lose L‑selectin and acquire the chemokine receptor CCR9 and the integrin α4:β7. After activation, but before full differentiation, the primed T cells exit from the Peyer’s patch via the draining lymphatics, passing through the mesenteric lymph node to enter the thoracic duct. The thoracic duct empties into the bloodstream, delivering the activated T cells back to the wall of the small intestine. Here T cells bearing CCR9 and α4:β7 are attracted specifically to leave the bloodstream and enter

1	into the bloodstream, delivering the activated T cells back to the wall of the small intestine. Here T cells bearing CCR9 and α4:β7 are attracted specifically to leave the bloodstream and enter the lamina propria of the villus.

1	bloodvessellaminapropriaCCR10CCR9MAdCAM-1˜4:°7Gut-homing effector T cells bind MAdCAM-1 on endothelium largeintestinesmallintestineendotheliumepitheliumCCL25E-cadherin˜E:°7CCL28Gut epithelial cells express chemokines specifc for gut-homing T cells Fig. 12.9 Molecular control of intestine-specific homing of lymphocytes. α:β form of CD8 or the CD8α:α homodimer, which may act to dampen T-cell activation. These IELs express CCR9 and the integrin αE:β7 (CD103), which binds to E-cadherin on epithelial cells (Fig. 12.9). By contrast, in the lamina propria it is CD4 T cells that predominate.

1	The healthy intestinal mucosa therefore displays many characteristics of a chronic inflammatory response—namely, the presence of numerous effector lymphocytes and other leukocytes in the tissues. The presence of such large numbers of effector cells would be unusual for a healthy nonlymphoid tissue, but in the gut does not necessarily signify infection. Rather, it is the local response to the myriad innocuous antigens normally present at mucosal surfaces, and is essential for maintaining the beneficial symbiosis between host and microbiota. It involves a balanced generation of effector and regulatory T cells, but, when required, can be refocused to produce a full adaptive immune response to invading pathogens. 12-5 The circulation of lymphocytes within the mucosal immune system is controlled by tissue-specific adhesion molecules and chemokine receptors.

1	The entry of effector lymphocytes into the mucosa results from changes in their homing characteristics as they become activated. Naive T cells and B cells circulating in the bloodstream are not predetermined as to which compartment of the immune system they will end up in, and they enter Peyer’s patches and mesenteric lymph nodes through high endothelial venules (HEVs) (see Fig. 9.4). As in the systemic immune system, this process is controlled largely by the chemokines CCL21 and CCL19, which are released from the lymphoid tissues and bind the receptor CCR7 on naive lymphocytes. In the Peyer’s patches, this is assisted by binding of the mucosal vascular addressin MAdCAM-1 on HEVs to the L-selectin expressed on naive T cells. CXCR5 responding to CXCL13 produced in B-cell follicles is also important for recruitment of naive B cells to Peyer’s patches and isolated lymphoid follicles of the intestine. As in other secondary lymphoid tissues, if the naive lymphocytes do not see their

1	also important for recruitment of naive B cells to Peyer’s patches and isolated lymphoid follicles of the intestine. As in other secondary lymphoid tissues, if the naive lymphocytes do not see their antigen, they exit via the lymphatics and return to the bloodstream. If they encounter antigen in the GALT, the lymphocytes become activated and lose expression of CCR7 and L-selectin. This means that they have lost their ability to home to secondary lymphoid organs, because they cannot enter them via the HEVs (see Section 9-5).

1	Left panel: T and B lymphocytes primed by antigen in the Peyer’s patches or mesenteric lymph nodes arrive as effector lymphocytes in the bloodstream supplying the intestinal wall (see Fig. 12.8). The lymphocytes express the integrin α4:β7, which binds specifically to MAdCAM‑1 expressed selectively on the endothelium of blood vessels in mucosal tissues. This provides the adhesion signal needed for the emigration of cells into the lamina propria. Right panel: if primed in the small intestine, the effector lymphocytes also express the chemokine receptor CCR9, which allows them to respond to CCL25 (yellow circles) produced by epithelial cells of the small intestine; this enhances selective recruitment. Effector lymphocytes that have been primed in the large intestine do not express CCR9 but instead express CCR10. CCR10 may respond to CCL28 (blue circles) produced by colon epithelial cells to fulfill a similar function. Lymphocytes destined to enter the epithelial layer stop expressing the

1	express CCR10. CCR10 may respond to CCL28 (blue circles) produced by colon epithelial cells to fulfill a similar function. Lymphocytes destined to enter the epithelial layer stop expressing the α4:β7 integrin and instead express the αE:β7 integrin. The receptor for this is E‑cadherin on the epithelial cells. These interactions may help keep lymphocytes in the epithelium once they have entered it.

1	Lymphocytes that have been activated in the mucosal lymphoid organs then travel to the mucosa, where they accumulate as effector cells. Although some T and B lymphocytes initially activated in Peyer’s patches may migrate directly into adjacent parts of the lamina propria, most leave via the lymphatics, pass through mesenteric lymph nodes, and eventually end up in the thoracic duct. From there they circulate in the bloodstream (see Fig. 12.8) and selectively reenter the intestinal lamina propria via small blood vessels. Antigen-specific naive B cells in the follicular areas of Peyer’s patches undergo isotype switching from IgM to IgA production there, but they only differentiate fully into IgAproducing plasma cells once they have returned to the lamina propria. As a result, plasma cells are rarely found in Peyer’s patches, and this is also true of effector T cells, which also only differentiate fully after arrival in the mucosa.

1	Gut-specific homing by antigen-stimulated T and B cells is determined in large part by the expression of the adhesion molecule α4:β7 integrin on the lymphocytes. This binds to the mucosal vascular addressin MAdCAM-1, found on the endothelial cells that line the blood vessels within the gut wall (see Fig. 12.9). Lymphocytes originally primed in the gut are also lured back as a result of tissue-specific expression of chemokines by the gut epithelium. In the case of the small intestine, CCL25 (TECK) produced constitutively by epithelial cells is a ligand for the receptor CCR9 expressed on gut-homing T cells and B cells. Within the intestine there seems to be regional specialization of chemokine expression, as CCL25 is not expressed outside the small intestine and CCR9 is not required for migration of lymphocytes to the colon. However, the colon, lactating mammary gland, and salivary glands express CCL28 (MEC, mucosal epithelial chemokine), which is a ligand for the receptor CCR10 on

1	migration of lymphocytes to the colon. However, the colon, lactating mammary gland, and salivary glands express CCL28 (MEC, mucosal epithelial chemokine), which is a ligand for the receptor CCR10 on gut-primed lymphocytes and attracts IgA-producing B lymphoblasts to these tissues. The addressins and chemokine receptors involved in migration of activated lymphocytes to other mucosal surfaces are unknown.

1	Under most normal circumstances, only lymphocytes that first encounter antigen in a gut-associated secondary lymphoid organ are induced to express gut-specific homing receptors and integrins. As we shall see in the next sections, these molecules are induced or ‘imprinted’ on T lymphocytes by intestinal dendritic cells during antigen presentation. In contrast, dendritic cells from nonmucosal lymphoid tissues induce lymphocytes to express other adhesion molecules and chemokine receptors—for example, α4:β1 integrin (VLA-4), which binds VCAM-1; cutaneous lymphocyte antigen (CLA), which binds E-selectin; and the chemokine receptor CCR4—which direct them to tissues such as the skin (see Section 11-3). The tissue-specific consequences of lymphocyte priming in the GALT explain why effective vaccination against intestinal infections requires immunization by a mucosal route, because other routes, such as subcutaneous or intramuscular immunization, do not involve dendritic cells with the correct

1	against intestinal infections requires immunization by a mucosal route, because other routes, such as subcutaneous or intramuscular immunization, do not involve dendritic cells with the correct imprinting properties.

1	12-6 Priming of lymphocytes in one mucosal tissue may induce protective immunity at other mucosal surfaces.

1	Not all parts of the mucosal immune system exploit the same tissue-specific chemokines, allowing localized compartmentalization of lymphocyte recirculation within the system. Thus, effector T and B cells primed in lymphoid organs draining the small intestine (mesenteric lymph nodes and Peyer’s patches) are most likely to return to the small intestine; similarly, those primed in the respiratory tract migrate most efficiently back to the respiratory mucosa. This homing is obviously useful in returning antigen-specific effector cells to the mucosal organ in which they will be most effective in fighting an infection or in controlling immune responses against foreign proteins and commensals. Nevertheless, some lymphocytes that have been primed in the GALT, for example, can also recirculate as effector cells to other mucosal tissues such as the respiratory tract, urogenital tract, and lactating breast. This overlap between mucosal recirculation routes gave rise to the idea of a common

1	as effector cells to other mucosal tissues such as the respiratory tract, urogenital tract, and lactating breast. This overlap between mucosal recirculation routes gave rise to the idea of a common mucosal immune system, which is distinct from other parts of the immune system. Although this is now understood to be an oversimplification, it does have important implications for vaccine development, because it may enable immunization by one mucosal route to be used to protect against infection at another mucosal surface. An important example of this is the induction of IgA antibody production in the lactating breast by natural infection or vaccination of mucosal surfaces such as the intestine. This is because the vasculature of the lactating breast expresses MAdCAM-1 and the phenomenon is a crucial means of generating protective immunity that can be transmitted to infants by passive transfer of the antibodies in milk. A further example has been shown in experimental animals, in which

1	a crucial means of generating protective immunity that can be transmitted to infants by passive transfer of the antibodies in milk. A further example has been shown in experimental animals, in which nasal immunization has a special ability to prime immune responses in the urogenital tract against HIV. The mechanisms behind this are unknown.

1	12-7 Distinct populations of dendritic cells control mucosal immune responses.

1	As elsewhere, dendritic cells are important for initiating and shaping immune responses in mucosal tissues, and are located both in secondary mucosal lymphoid organs and scattered throughout the mucosal surfaces. Within Peyer’s patches, dendritic cells are found in two main areas. In the subepithelial dome region, dendritic cells can acquire antigen from M cells (Fig. 12.10). Both of the major subtypes of dendritic cells are present in the intestine (see Sections 6-5 and 9-1). In mice, the most abundant subset of dendritic cells in the Peyer’s patch expresses CD11b (αM integrin) and, when activated, tends to produce IL-23. This promotes development of TH17 cells and stimulates ILC3 cells, both of which produce IL-17 and IL-22 (see Sections 3-23 and 11-2). These dendritic cells express CCR6, the receptor for CCL20 produced by follicle-associated epithelial cells. In resting conditions, they reside beneath the epithelium and produce IL-10 in response to antigen uptake, maintaining a

1	CCR6, the receptor for CCL20 produced by follicle-associated epithelial cells. In resting conditions, they reside beneath the epithelium and produce IL-10 in response to antigen uptake, maintaining a noninflammatory environment. However, during infection by a pathogen such as Salmonella, dendritic cells are rapidly recruited into the epithelial layer of the Peyer’s patch in response to the CCL20 that is released in increased quantities by epithelial cells in the presence of the bacteria. Bacterial products also activate the dendritic cells to express co-stimulatory molecules, allowing them to induce pathogen-specific naive T cells to differentiate into effector cells. Also in the T-cell area of Peyer’s patches are the less abundant CD11b-negative subset of dendritic cells, whose development requires the factor BATF3 and which produce the cytokine IL-12 (see Sections 6-5 and 9-9). CD11b-expressing dendritic cells are protective in many intestinal infections.

1	Dendritic cells are also abundant in the wall of the small intestine outside Peyer’s patches, mainly in the lamina propria. These sample antigens from the lumen and surrounding tissue, and they spend a relatively short time in the intestine before migrating in afferent lymph to the draining mesenteric lymph node, where they present antigen to naive T cells. As elsewhere, migration of dendritic cells depends on the chemokine receptor CCR7 (see Fig. 9.17). It is estimated that 5–10% of the mucosal dendritic cell population emigrates to the mesenteric lymph nodes every day in the resting intestine, allowing constant delivery of antigens from the intestinal surface to T cells. In the absence of infection or inflammation, the encounter between migrating dendritic cells and naive T cells in mesenteric lymph nodes results in the generation of antigen-specific FoxP3+ regulatory T cells that express the gut-homing molecules CCR9 and integrin α4:β7 described above (see Section 12-4). These

1	mesenteric lymph nodes results in the generation of antigen-specific FoxP3+ regulatory T cells that express the gut-homing molecules CCR9 and integrin α4:β7 described above (see Section 12-4). These ‘primed’ Treg cells then leave the lymph node, return to the wall of the small intestine, and suppress the production of inflammatory responses to harmless antigens in food.

1	Fig. 12.10 Capture of antigens from the intestinal lumen by mononuclear cells in the lamina propria. Top row, first panel: soluble antigens such as food proteins can be transported directly across or between enterocytes, or taken up by M cells in the surface epithelium outside Peyer’s patches. Second panel: enterocytes can capture and internalize antigen:antibody complexes by means of the neonatal Fc receptor (FcRn) on their surface and transport them across the epithelium by transcytosis. Lamina propria dendritic cells express FcRn and other Fc receptors and capture and internalize the complexes. Third panel: an enterocyte infected with an intracellular pathogen undergoes apoptosis and is phagocytosed by a dendritic cell. Lower rows, left panels: mononuclear cells can extend cellular processes between the cells of the epithelium without disturbing its integrity. These cells, now thought to be macrophages, may internalize antigen and pass it to neighboring dendritic cells for

1	processes between the cells of the epithelium without disturbing its integrity. These cells, now thought to be macrophages, may internalize antigen and pass it to neighboring dendritic cells for presentation to T cells. The micrograph shows mononuclear cells stained for CD11c (green) in the lamina propria of a villus of mouse small intestine. The epithelium appears black but its luminal (outer) surface is shown by the white line. A cell process stretches between two epithelial cells and extends its tip into the lumen of the intestine. Magnification ×200. Center panels: mucus‑secreting goblet cells can transport soluble antigens to lamina propria dendritic cells. The micrograph shows the soluble marker dextran (purple) being transported across goblet cells (white in the bottom right panel) in the epithelium (nuclei stained blue) to underlying dendritic cells (stained for CD11c in green). Scale bar 10 μm. Right panels: dendritic cells (purple) may enter the epithelial layer and capture

1	in the epithelium (nuclei stained blue) to underlying dendritic cells (stained for CD11c in green). Scale bar 10 μm. Right panels: dendritic cells (purple) may enter the epithelial layer and capture bacteria before returning to the lamina propria. Dendritic cells and macrophages remaining in the lamina propria are stained blue or green. Scale bar 10 μm. Bottom left, micrograph from Niess, J.H., et al.: Science 2005, 307:254–258. Bottom center, micrograph from McDole, J.R., et al.: Nature 2012, 483:345–349. Bottom right, micrograph from Farache et al.: Immunity 2013, 38:581–595.

1	Both the generation of Treg cells and their expression of gut-homing molecules require that the dendritic cells produce retinoic acid, which is derived from the metabolism of dietary vitamin A through the action of retinal dehydrogenases. Retinoic acid is also produced by stromal cells in the mesenteric lymph node, further enhancing the effects of the migratory dendritic cells. Retinoic acid-producing dendritic cells are also found in Peyer’s patches, and may also be important for generating regulatory T cells either in the Peyer’s patch itself, or after they migrate to the mesenteric lymph node. The induction of regulatory T cells in intestinal tissues is assisted by transforming growth factor-β (TGF-β), which is produced by dendritic cells. Migratory populations of dendritic cells that continuously take up local antigens in the tissue and transport them to the draining lymph nodes are also found in the large intestine and other mucosal surfaces such as the lung. Although it is

1	that continuously take up local antigens in the tissue and transport them to the draining lymph nodes are also found in the large intestine and other mucosal surfaces such as the lung. Although it is believed dendritic cells from these tissues are also involved in maintaining tolerance to harmless materials such as commensal bacteria, they do not produce retinoic acid, and it is not clear how they influence T-cell differentiation and homing.

1	The dendritic cells in the intestinal lamina propria also include the two major subsets described above. Collectively, the properties of intestinal dendritic cells result in a dominantly tolerogenic environment that prevents unnecessary and damaging reactions to foods and commensal microorganisms. The anti-inflammatory behavior of mucosal dendritic cells in the healthy gut is promoted by factors that are constitutively produced in the mucosal environment. These include thymic stromal lymphopoietin (TSLP), TGF-β produced by dendritic and epithelial cells, prostaglandin PGE2 produced by stromal cells, and IL-10 produced by intestinal macrophages and CD4 T cells. Retinol stored in the liver and delivered into the small intestine via the bile provides an additional source for the local generation of retinoic acid for conditioning dendritic cells in the wall of the small intestine. 12-8 Macrophages and dendritic cells have different roles in mucosal immune responses.

1	12-8 Macrophages and dendritic cells have different roles in mucosal immune responses. The lamina propria of the healthy intestine contains the largest population of macrophages in the body. Like dendritic cells, they express CD11c and class II MHC, but unlike dendritic cells in this site, they lack CD103 expression, but express FcγR1 (CD64; see Fig. 10.38) and CX3CR1, the receptor for CX3CL1 (fractalkine). Macrophages are also unable to migrate from the intestine to the draining lymph nodes and cannot present antigen to naive T cells. Unlike many other tissue-resident macrophages, such as those in the brain or liver, which develop from embryonic precursors (see Section 3-1), those in the intestine require constant replenishment by blood monocytes.

1	Macrophages are important for maintaining a healthy intestine. They are positioned immediately under the epithelium and are highly phagocytic, and thus ideally suited to ingest and degrade any microbes that penetrate across the epithelial barrier. They can also clear away dying epithelial cells, which are found in large numbers in the intestine, an inevitable consequence of such a rapidly dividing tissue. However, unlike macrophages in other parts of the body, intestinal macrophages do not produce significant quantities of inflammatory cytokines or reactive oxygen or nitrogen species in response to phagocytosis or exposure to stimuli such as bacteria or TLR ligands. This is because they produce large amounts of IL-10 constitutively, allowing them to limit inflammation while acting as powerful scavengers. Macrophage-derived IL-10 also contributes to maintaining antigen-specific tolerance in the mucosa, as it is needed to sustain the survival and secondary expansion of FoxP3+ Treg cells

1	scavengers. Macrophage-derived IL-10 also contributes to maintaining antigen-specific tolerance in the mucosa, as it is needed to sustain the survival and secondary expansion of FoxP3+ Treg cells that have migrated back to the intestine after being primed by tolerogenic dendritic cells in the lymph node. Indeed, they have features of both these populations, and their functions are specifically adapted to the conditions of their local environment. Thus macrophages and dendritic cells play distinct but complementary roles in the steady-state intestine. Migratory dendritic cells carry out the initial priming and shaping of T-cell responses in secondary lymphoid organs, and sessile macrophages scavenge cellular debris and microbes and may tune the activity of already primed T cells in the mucosa itself.

1	12-9 Antigen-presenting cells in the intestinal mucosa acquire antigen by a variety of routes.

1	The total surface area provided by M cells in the epithelium of Peyer’s patches for antigen to the intestinal immune system is limited, and the lamina propria itself is covered by an intact epithelium. Various additional mechanisms have been proposed to explain how antigen crosses the epithelium to gain access to macrophages and dendritic cells (see Fig. 12.10). Soluble antigens such as food proteins might be transported across epithelial cells or between gaps formed in the epithelium where dying cells are being shed. Alternatively, M cells may reside in the surface epithelium of the mucosa outside Peyer’s patches. Some intestinal bacteria, such as enteropathogenic and enterohemolytic strains of E. coli, have specialized means of attaching to and invading epithelial cells, allowing them to enter the underlying lamina propria directly. Antigen from the lumen can be delivered to lamina propria dendritic cells by uptake of antibody-coated antigens by epithelial cells expressing the

1	to enter the underlying lamina propria directly. Antigen from the lumen can be delivered to lamina propria dendritic cells by uptake of antibody-coated antigens by epithelial cells expressing the neonatal Fc receptor (FcRn). Antigen derived from apoptotic epithelial cells may be processed by cross-presenting dendritic cells (see Section 6-5) for induction of immune responses against enteric viruses, such as rotaviruses, which cause diarrheal disease because of their specialized ability to infect enterocytes (see Fig. 12.10).

1	Macrophages in the lamina propria may also participate in local antigen uptake by sending transepithelial dendrites that extend between epithelial cells and reach the lumen to sample bacteria (see Fig. 12.10). Lamina propria macrophages have also been reported to take up soluble antigen from the lumen, passing it on to dendritic cells for subsequent presentation to T cells. Some experiments also suggest that dendritic cells or macrophages might make their way into the lumen to acquire antigens such as bacteria, before returning with them to the lamina propria. 12-10 Secretory IgA is the class of antibody associated with the mucosal immune system.

1	12-10 Secretory IgA is the class of antibody associated with the mucosal immune system. The dominant class of antibody in the mucosal immune system is IgA, which is produced locally by plasma cells in the mucosal wall. The nature of IgA differs between the two main compartments in which it is found—the blood and mucosal secretions. IgA in the blood is mainly in the form of a monomer (mIgA) that is produced in the bone marrow by plasma cells derived from B cells activated in lymph nodes. In mucosal tissues, IgA is produced almost exclusively as a polymer, usually as a dimer, in which the two immunoglobulin monomers are linked by a J chain (see Section 5-16).

1	The naive B-cell precursors of the IgA-secreting mucosal plasma cells are activated in Peyer’s patches and mesenteric lymph nodes. Class switching of activated B cells to IgA is controlled by the cytokine TGF-β. In the human gut, this class switching is entirely T-cell dependent and occurs only in organized lymphoid tissues, where follicular helper T cells (TFH) instruct B cells by the same mechanisms as described in Chapter 10. The subsequent expansion and differentiation of IgA-switched B cells are driven by IL-5, IL-6, IL-10, and IL-21. Upward of 75,000 IgA-producing plasma cells are present in the normal human intestine, and 3–4 g of IgA is secreted by the mucosal tissues each day, and is the major immunoglobulin class produced there. This continuous production of large quantities of IgA occurs in the absence of pathogenic invasion and is driven almost entirely by recognition of the commensal microbiota.

1	In humans, monomeric and dimeric IgA are both found as two isotypes, IgA1 and IgA2. The ratio of IgA1 to IgA2 varies markedly depending on the tissue, being about 10:1 in the blood and upper respiratory tract, about 3:2 in the small intestine, and 2:3 in the colon. Some common pathogens of the respiratory mucosa (such as Haemophilus influenzae) and the genital mucosa (such as Neisseria gonorrhoeae) produce proteolytic enzymes that can cleave IgA1, whereas IgA2 is resistant to cleavage. The higher proportion of plasma cells secreting IgA2 in the large intestine might result because the high density of commensal microorganisms at this site drives the production of cytokines that cause selective class switching. In mice, only one IgA isotype is found, and it is most closely similar to IgA2 in humans.

1	After activation and differentiation, the resulting IgA-expressing B lymphoblasts express the mucosal homing integrin α4:β7, as well as the chemokine receptors CCR9 and CCR10, and localize to mucosal tissues by the mechanisms discussed above. Once in the lamina propria, the B cells undergo final differentiation into plasma cells, which synthesize IgA dimers and secrete them into the subepithelial space (Fig. 12.11). To reach its target antigen in the gut lumen, the IgA has to be transported across the epithelium by the polymeric immunoglobulin receptor (pIgR), which we introduced in Section 10-16. pIgR is expressed constitutively on the basolateral surfaces of the immature epithelial cells located at the base of the intestinal crypts and binds covalently to the Fc portion of J-chain-linked polymeric immunoglobulins such as dimeric IgA and pentameric IgM, and it transports the antibody by transcytosis to the luminal surface of the epithelium, where it is released by proteolytic

1	polymeric immunoglobulins such as dimeric IgA and pentameric IgM, and it transports the antibody by transcytosis to the luminal surface of the epithelium, where it is released by proteolytic cleavage of the extracellular domain of the receptor. Part of the cleaved pIgR remains associated with the IgA and is known as secretory component (frequently abbreviated to SC). The resulting antibody is protected from proteolytic cleavage and is referred to as secretory IgA (SIgA).

1	In some animals there is a second route of IgA secretion into the intestine— the hepatobiliary route. Dimeric IgA that has not bound pIgR is taken up into venules in the lamina propria, which drain intestinal blood to the liver via the portal vein. In the liver these small veins (sinusoids) are lined by an endothelium that allows the antibodies access to underlying hepatocytes, which have pIgR on their surface. IgA is taken up into the hepatocytes and transported by transcytosis into an adjacent bile duct. In this way, secretory IgA can be delivered directly into the upper small intestine via the common bile duct. This hepatobiliary route allows dimeric IgA to eliminate antigens that have invaded the lamina propria and have been bound there by IgA. Although highly efficient in rats and other rodents, this route does not seem to be of great significance in humans and other primates, in whom hepatocytes do not express pIgR.

1	IgA secreted into the gut lumen binds to the layer of mucus coating the epithelial surface via carbohydrate determinants in secretory component. There it is involved in preventing invasion by pathogenic organisms and, just as important, it also has a crucial role in maintaining the homeostatic balance

1	Fig. 12.11 Transcytosis of IgA antibody across epithelia is mediated by the polymeric Ig receptor (pIgR), a specialized transport protein. Most IgA antibody is synthesized in plasma cells lying just beneath epithelial basement membranes of the gut, the respiratory epithelia, the tear and salivary glands, and the lactating mammary gland. The IgA dimer linked by a J chain diffuses across the basement membrane and is bound by the pIgR on the basolateral surface of the epithelial cell. The bound complex undergoes transcytosis, by which it is transported in a vesicle across the cell to the apical surface. There the pIgR is cleaved, leaving the extracellular IgA‑binding component bound to the IgA molecule as the so‑called secretory component. Although not shown, carbohydrate on the secretory component binds to mucins in mucus and holds the IgA at the epithelial surface. The residual piece of the pIgR is nonfunctional and is degraded. IgA is transported across epithelia in this way into the

1	binds to mucins in mucus and holds the IgA at the epithelial surface. The residual piece of the pIgR is nonfunctional and is degraded. IgA is transported across epithelia in this way into the lumina of several organs that are in contact with the external environment.

1	Fig. 12.12 Mucosal IgA has several functions in epithelial surfaces. First panel: IgA adsorbs on the layer of mucus covering the epithelium, where it can neutralize pathogens and their toxins, preventing their access to tissues and inhibiting their functions. Second panel: antigen internalized by the epithelial cell can meet and be neutralized by IgA in endosomes. Third panel: toxins or pathogens that have reached the lamina propria encounter pathogen‑specific IgA there, and the resulting complexes are reexported into the lumen across the epithelial cell as the dimeric IgA is secreted. Fourth panel: antigen bound to secretory IgA in the lumen can bind via carbohydrate residues on the Fc portion of IgA to Dectin‑1 on M cells in Peyer’s patches and be transported to underlying dendritic cells. Binding of the IgA‑containing complex to DC‑SIGN on the dendritic cells induces them to produce anti‑inflammatory IL‑10.

1	between the host and the commensal microbiota. It does this in a number of ways (Fig. 12.12). First, it inhibits microbial adherence to the epithelium, its ability to bind bacteria being assisted by the unusually wide and flexible angle between the Fab pieces of the IgA molecule, particularly the IgA1 isotype (see Section 5-12), allowing very efficient bivalent binding to large antigens such as bacteria. Secretory IgA can also neutralize microbial toxins or enzymes.

1	In addition to its activities in the lumen, IgA can neutralize bacterial lipopolysaccharide and viruses it encounters within endosomes inside epithelial cells, as well as across the epithelial barrier in the lamina propria after bacteria and viruses have penetrated there. The resulting IgA:antigen complexes are then reexported into the gut lumen, from where they are excreted from the body (see Fig. 12.12). Complexes containing dimeric IgA formed in the lamina propria can also be excreted via the hepatobiliary route described above. In addition to enabling the elimination of antigens, the formation of IgA:antigen complexes can enhance the uptake of luminal antigen by M cells and local dendritic cells, via binding of carbohydrate residues on IgA to lectin receptors such as Dectin-1 and DC-SIGN. As well as these antigen-specific effects, secretory IgA can restrict the entry of bacteria in a nonspecific manner. This is because the high carbohydrate content of the Fc part of the IgA heavy

1	As well as these antigen-specific effects, secretory IgA can restrict the entry of bacteria in a nonspecific manner. This is because the high carbohydrate content of the Fc part of the IgA heavy chain allows it to act as a decoy for receptors that bacteria use to bind carbohydrates on the epithelial surface. Secretory IgA has little capacity to activate the classical pathway of complement or to act as an opsonin, and so does not induce inflammation. Uptake of IgA:antigen complexes by dendritic cells also induces these cells to produce anti-inflammatory IL-10. Together these properties mean that IgA can limit the penetration of microbes into the mucosa without risking inflammatory damage to these fragile tissues, something that would be potentially harmful in the intestine. For the same reasons, secretory IgA is crucial to the beneficial symbiosis between an individual and gut commensal bacteria (see Section 12-20).

1	12-11 T-independent processes can contribute to IgA production in some species.

1	In mice, unlike humans, a significant proportion of intestinal IgA is derived from T-cell-independent B-cell activation and class switching. This depends on activation of the innate immune system by the products of commensal microbes and may result from the direct interaction of B cells with conventional dendritic cells and follicular dendritic cells in solitary lymphoid follicles. This antibody production seems to involve lymphocytes of the B-1 subset (see Section 8-9), which arise from precursor B cells in the peritoneal cavity and migrate to the intestinal wall in response to microbial constituents such as lipopolysaccharide. Once in the mucosa, TGF-β-dependent class switching to IgA occurs under the influence of local factors, including IL-6, retinoic acid, and BAFF and APRIL (see Fig. 10.6), which bind to TACI on B cells substituting for signals otherwise supplied by CD4 helper T cells (see Section 10-1). Intestinal epithelial cells can produce BAFF and APRIL, while local

1	Fig. 10.6), which bind to TACI on B cells substituting for signals otherwise supplied by CD4 helper T cells (see Section 10-1). Intestinal epithelial cells can produce BAFF and APRIL, while local eosinophils may contribute by producing APRIL, IL-6, and TGF-β. Other myeloid cells may produce nitric oxide (NO) and TNF-α, both of which assist in the processing and activation of TGF-β.

1	The IgA antibodies produced in these T-cell-independent responses are of limited diversity and of generally low affinity, with little evidence of somatic hypermutation. They are nevertheless an important source of ‘natural’ antibodies directed at commensal bacteria. As yet, there is little evidence for this source of IgA in humans, in whom all secretory IgA responses involve somatic hypermutation and seem to be T-cell dependent. The enzyme activation-induced cytidine deaminase (AID), which is required for class switching (see Chapter 5), cannot be detected in human intestinal lamina propria, indicating that class switching is unlikely to occur there. Nevertheless, its occurrence in lamina propria B cells in mice may offer a glimpse into the evolutionary history of specific antibody responses in the mucosa, and might indicate pathways that could be activated when T-cell-dependent IgA production is compromised in humans, as it is in AIDS. Nonetheless, it is likely that secondary

1	responses in the mucosa, and might indicate pathways that could be activated when T-cell-dependent IgA production is compromised in humans, as it is in AIDS. Nonetheless, it is likely that secondary reactivation of IgA-committed B lymphoblasts occurs in the lamina propria for full differentiation of plasma cells, which likely involves production by myeloid and epithelial cells of APRIL, BAFF, and other mediators 12-12 IgA deficiency is relatively common in humans but may be compensated for by secretory IgM.

1	Selective deficiency of IgA production is the commonest primary immune deficiency in humans, occurring in about 1 in 500 to 700 individuals in populations of Caucasian origin, although it is somewhat rarer in other ethnic groups. The most frequent genetic mutation that has been identified in this condition is in the TACI receptor for BAFF. A slightly higher incidence of respiratory infections, atopy (a tendency for allergic reactions to harmless environmental antigens), and autoimmune disease has been reported in older people with IgA deficiency. However, most individuals with IgA deficiency are not overly susceptible to infections unless there is also a deficiency in IgG2 production. The dispensability of IgA probably reflects the ability of IgM to replace IgA as the predominant antibody in secretions, and increased numbers of IgM-producing plasma cells are indeed found in the intestinal mucosa of IgA-deficient people. Because IgM is a J-chain-linked polymer, IgM produced in the gut

1	in secretions, and increased numbers of IgM-producing plasma cells are indeed found in the intestinal mucosa of IgA-deficient people. Because IgM is a J-chain-linked polymer, IgM produced in the gut mucosa is bound efficiently by the pIgR and is transported across epithelial cells into the gut lumen as secretory IgM. The importance of this backup mechanism has been shown in knockout mice. Animals lacking IgA alone have a normal phenotype, but those lacking the pIgR are susceptible to mucosal infections. They also show increased penetration of commensal bacteria into tissues and a consequent systemic immune response to these bacteria. Genetic absence of the pIgR has never been reported in humans, suggesting that such a defect is lethal.

1	12-13 The intestinal lamina propria contains antigen-experienced T cells and populations of unusual innate lymphoid cells. Most of the T cells in the healthy lamina propria have been activated by dendritic cells and express markers of effector or memory T cells, such as CD45RO in humans, and express gut-homing markers such as CCR9 and α4:β7 integrin, as well as receptors for pro-inflammatory chemokines such as CCL5 (RANTES). The T-cell population of the lamina propria has a ratio of CD4 to CD8 T cells of 3:1 or more, similar to that in systemic lymphoid tissues.

1	Lamina propria CD4 T cells secrete large amounts of cytokines such as interferon (IFN)-γ, IL-17, and IL-22, even in the absence of overt inflammation. This likely reflects the constant state of immune recognition of the microbiota and other environmental antigens that takes place in the intestine, and their importance is underlined by the frequent opportunistic infections of the intestine that occur in individuals lacking CD4 T cells, such as those with HIV infection (see Section 13-24). Effector TH17 cells are prominent in the intestinal mucosa, and their products are important components of local immune defense. IL-17 is needed for full expression of the poly-immunoglobulin receptor involved in secretion of IgA into the lumen, while IL-22 stimulates intestinal epithelial cells to produce antimicrobial peptides that help maintain epithelial barrier integrity. Effector CD8 T cells are also present in the normal lamina propria and are capable of both cytokine production and cytotoxic

1	antimicrobial peptides that help maintain epithelial barrier integrity. Effector CD8 T cells are also present in the normal lamina propria and are capable of both cytokine production and cytotoxic activity when a protective immune response to a pathogen is required.

1	In any other situation, the presence of such large numbers of differentiated effector T cells would suggest the presence of a pathogen and likely would lead to inflammation. The fact that it does not in the healthy lamina propria is because the generation of TH1, TH17, and cytotoxic T cells is balanced by the presence of substantial numbers of IL-10-producing regulatory T cells. In the small intestine, these are mostly FoxP3-negative, whereas in the colon, FoxP3positive T cells dominate. Many of the inducible T cells recognize antigens derived from organisms within the microbiota.

1	The healthy lamina propria also contains many innate lymphoid cells (ILCs) (see Sections 1-19 and 9-20). The ILC3 subset is prominent in both human and mouse intestinal mucosa. Mature ILC3s produce IL-17 and IL-22, and some express the NK-cell receptors NKp44 and NKp46. Their development is controlled by the aryl hydrocarbon receptor and the transcription factor RORγT (see Section 9-21). ILC3s are present in secondary lymphoid organs in the intestine and are important for their lymphoid tissue development there. In response to IL-23 secreted by local dendritic cells, ILC3s produce IL-22, which stimulates the epithelium to generate antimicrobial peptides that promote local defense against bacterial and fungal pathogens in the intestine. During the course of inflammatory diseases, ILC3s can acquire the ability to produce IFN-γ in response to IL-12, and combined with their production of IL-17, this endows them with significant pathological properties. IL-5 and IL-13 produced by ILC2s

1	can acquire the ability to produce IFN-γ in response to IL-12, and combined with their production of IL-17, this endows them with significant pathological properties. IL-5 and IL-13 produced by ILC2s form an important layer of T-cell-independent responses to helminth parasites in the intestine, and an equivalent population is involved in allergic reactions in the respiratory tract.

1	CD1-restricted iNKT cells (see Section 6-18) and mucosal invariant T (MAIT) cells (see Section 6-19) are also present in the lamina propria, and account for 2–3% of lamina propria T cells in human small intestine. MAIT cells express an invariant TCRα chain paired with a limited range of TCRβ chain and recognize metabolites of vitamin B derived mainly from the riboflavin metabolism pathway in microbes presented by MR1. 12-14 The intestinal epithelium is a unique compartment of the immune system.

1	12-14 The intestinal epithelium is a unique compartment of the immune system. We have already introduced the fact that there are abundant intraepithelial lymphocytes (IELs) present in intestine. In the healthy small intestine, there are 10–15 lymphocytes for every 100 epithelial cells, making the IELs one of the single largest populations of lymphocytes in the body (Fig. 12.13). More than 90% of the IELs in the small intestine are T cells, and around 80% of these carry CD8, in complete contrast to the lymphocytes in the lamina propria. IELs are also present in the large intestine, although there are fewer of them relative to the number of epithelial cells and the proportion of CD4 T cells is greater than in the small intestine.

1	Like the lymphocytes in the lamina propria, most IELs have an activated appearance even in the absence of infection by a pathogen, and they contain intracellular granules containing perforin and granzymes, like those in conventional effector CD8 cytotoxic T cells. However, the T-cell receptors of most CD8 IELs show evidence of oligoclonality, with restricted use of V(D)J gene segments, an indication that they may expand locally in response to a relatively small number of antigens. The IELs of the small intestine express the chemokine receptor CCR9, and the αE:β7 integrin (CD103), which interacts with E-cadherin expressed on epithelial cells and assists their retention in the epithelium (see Fig.12.9).

1	Fig.12.13 Intraepithelial lymphocytes. The epithelium of the are CD8 T cells. Magnification ×400. The electron micrograph on small intestine contains a large population of lymphocytes known the right shows that the IELs lie between epithelial cells (EC) on the as intraepithelial lymphocytes (IELs; left panel). The micrograph in basement membrane (BM) separating the lamina propria (LP) from the center is of a section through human small intestine in which the epithelium. One IEL can be seen having crossed the basement CD8 T cells have been stained brown with a peroxidase‑labeled membrane into the epithelium, leaving a trail of cytoplasm in its monoclonal antibody. Most of the lymphocytes in the epithelium wake. Magnification ×8000.

1	There are two main subsets of CD8 intraepithelial T cells—type a (‘inducible’) and type b (‘natural’)—identified based on which form of CD8 is expressed. The relative proportions of the subsets vary with age, strain (in mice), and number of bacteria in the intestine. Type a (inducible) IELs express α:β T-cell receptors and the CD8α:β heterodimer. They are derived from naive CD8 T cells that were activated by antigen in the Peyer’s patches or mesenteric lymph nodes, and they function as conventional class I MHC-restricted cytotoxic T cells, killing virus-infected cells, for example (Fig. 12.14, top panels). They also secrete effector cytokines such as IFN-γ.

1	Type a IELs (top panels) are conventional CD8 cytotoxic T cells that recognize peptides derived from viruses or other intracellular pathogens bound to classical MHC class I molecules on infected epithelial cells. Type a IELs express an α:β T‑cell receptor and the CD8α:β heterodimer co‑receptor. Type b IELs carrying the CD8α:α homodimer (bottom panels) recognize MIC‑A and MIC‑B using the receptor NKG2D and are activated by IL‑15. Human epithelial cells that have been stressed by infection or altered cell growth or by a toxic peptide from the protein α‑gliadin (a component of gluten) upregulate expression of the nonclassical MHC class I molecules MIC‑A and MIC‑B and produce IL‑15. Both types of IELs can kill by the release of perforin and granzyme. Apoptosis of epithelial cells can also be induced by the binding of Fas ligand on the T cell to Fas on the epithelial cell.

1	Type b (natural) CD8 IELs can express either an α:β or a γ:δ T-cell receptor, but are distinguished by their expression of the CD8α:α homodimer. The γ:δ T cells in the intestine are enriched for particular Vγ and Vδ genes and are distinct from those found in other tissues (see Fig. 8.23). Some of the α:β receptors expressed by IELs bind nonconventional ligands, including those presented by MHC class Ib molecules (see Section 6-17). Type b IELs also express molecules typical of natural killer cells, such as the activating C-type lectin NKG2D, which binds to the two MHC-like molecules MIC-A and MIC-B. These are induced on intestinal epithelial cells in response to cellular injury, stress, or ligation of TLRs (see Section 6-16). The injured cells can then be recognized and killed by the IELs, a process that is enhanced by the production of IL-15 by the damaged epithelial cells. Like innate immune cells, type b IELs constitutively express genes associated with inflammation, such as the

1	a process that is enhanced by the production of IL-15 by the damaged epithelial cells. Like innate immune cells, type b IELs constitutively express genes associated with inflammation, such as the production of high levels of cytotoxic molecules, NO, and pro-inflammatory cytokines and chemokines. Their role in the gut may be the rapid recognition and elimination of epithelial cells that express an abnormal phenotype as a result of stress or infection (see Fig. 12.14, bottom panels). Type b IELs are also thought to be important in aiding the repair of the mucosa after inflammatory damage: they stimulate the release of antimicrobial peptides, thus helping to remove the source of the inflammation; and they release cytokines such as keratinocyte growth factor, which promotes epithelial barrier function, and TGF-β, which assists tissue repair, as well as inhibiting local inflammatory reactions.

1	Type b IELs are kept in check by their co-expression of signaling inhibitors, including the immunomodulatory cytokine TGF-β and inhibitory receptors like those found on NK cells. The importance of these control processes is shown by the fact that inappropriate or excess activation of type b IELs may give rise to disease. For example, increased numbers of IELs expressing a γ:δ T-cell receptor are found in celiac disease, which is caused by an abnormal immune response to the wheat protein gluten (see Section 14-17). MIC-Adependent cytotoxic activity of intraepithelial T cells contributes to the intestinal damage in this condition, as certain components of gluten can stimulate the production of IL-15 by epithelial cells and increase expression of MIC-A. These processes lead to killing of epithelial cells by the activated IELs, as described above (see Fig. 12.14, bottom panels).

1	The origin and development of type b IELs has been controversial and is unexplored in humans. Unlike type a IELs, many type b IELs expressing an α:β T-cell receptor seem not to have undergone conventional positive and negative selection (see Chapter 8), and express apparently autoreactive T-cell receptors. The absence of the CD8α:βheterodimer, however, means that these T cells have low affinity for conventional peptide:MHC complexes, because the CD8β chain binds more strongly than the CD8α chain to classical MHC molecules. Type b α:β T-cell receptor-expressing IELs therefore cannot act as self-reactive effector cells. This low affinity for self MHC molecules is also probably the reason that these cells escape negative selection in the thymus. Rather, they appear to develop via a process of so-called agonist selection, in which late double-negative/early double-positive T cells are positively selected in the thymus by unknown ligands and are released immediately to the intestine. Here

1	so-called agonist selection, in which late double-negative/early double-positive T cells are positively selected in the thymus by unknown ligands and are released immediately to the intestine. Here they mature and are induced to express the CD8α:α homodimer under the influence of TGF-β produced by epithelial cells. Nonclassical MHC molecules expressed on the intestinal epithelium are also important for the maturation of these type b IELs. One example of this kind of selection molecule is the thymus leukemia antigen (TL), another nonclassical MHC class I molecule (see Fig. 6.26) found in certain mouse strains which does not present peptides. TL is expressed by intestinal epithelial cells and directly binds CD8α:α with high affinity.

1	Type b IELs expressing a γ:δ T-cell receptor also develop via agonist selection in the thymus, as part of the programmed wave of γ:δ T-cell development (see Fig. 8.23). The expression of this receptor is driven by specific ligands in the thymus and endows these cells with the specific ability to migrate to the intestinal epithelium, where they may be further programmed by the same agonist ligand.

1	The local differentiation events involved in the development of type b IELs require the presence of the cytokine IL-15, which is produced in response to the microbiota and ‘trans-presented’ to IELs in a complex with the IL-15 receptor present on epithelial cells. Type b IEL development is dependent on the aryl hydrocarbon receptor (AhR), a transcription factor activated by various environmental ligands that are derived from brassica and other dietary vegetables. Mice that lack the AhR have reduced numbers of ILC3 and type b IELs and show abnormalities in epithelial barrier repair, reinforcing the view that these unusual lymphocytes play important roles in the innate immune response to local materials in the intestine. Summary.

1	The mucosal tissues of the body such as the intestine and respiratory tract are continuously exposed to enormous amounts of different antigens, which can be either pathogenic invaders or harmless materials such as foods and commensal organisms. Potential immune responses to this antigen load are controlled by a distinct compartment of the immune system, the mucosal immune system, which is the largest in the body. Its unique features include distinctive routes and processes for the uptake and presentation of antigens, exploitation of M cells to transport antigens across the epithelium of Peyer’s patches, and retinoic acid-producing dendritic cells that imprint the T and B cells they activate with gut-homing properties. Dendritic cells also favor the generation of FoxP3-positive Treg cells in the normal gut. Tissue-resident intestinal macrophages contribute to these regulatory processes by phagocytosing antigens without causing inflammation, due to their production of IL-10. Lymphocytes

1	the normal gut. Tissue-resident intestinal macrophages contribute to these regulatory processes by phagocytosing antigens without causing inflammation, due to their production of IL-10. Lymphocytes primed in the mucosa-associated lymphoid tissues acquire specific homing receptors, allowing them to redistribute preferentially back to mucosal surfaces as effector cells. The adaptive immune response in mucosal tissues is characterized by the production of secretory dimeric IgA, and by the presence of distinct populations of memory/effector T cells in the epithelium and lamina propria. CD4 T cells in the lamina propria produce pro-inflammatory cytokines such as IL-17 and IFN-γ even in the absence of overt infection, but this is normally balanced by the presence of IL-10-producing Treg cells. IELs exhibit cytolytic activity and other innate functions that help maintain a healthy epithelial barrier.

1	The mucosal response to infection and regulation of mucosal immune responses. The major role of the mucosal immune response is defense against infectious agents, which include all forms of microorganisms from viruses to multicellular parasites. This means that the host must be able to generate a wide spectrum of immune responses tailored to meet the challenge of individual pathogens; unsurprisingly, many microbes have evolved means of adapting to and subverting the host response. To ensure an adequate response to pathogens, the mucosal immune system needs to be able to recognize and respond to any foreign antigen, but it must not produce the same effector response to a harmless antigen (from food or commensals) as it would to a pathogen. A major role of the mucosal immune system is to balance these competing demands, and how it does this will be the focus of this part of the chapter.

1	12-15 Enteric pathogens cause a local inflammatory response and the development of protective immunity. Despite the array of innate immune mechanisms in the gut, and stiff competition from the indigenous microbiota, the gut is a frequent site of infection by a wide variety of pathogenic organisms. These include many viruses; enteric bacteria such as Vibrio, Salmonella, and Shigella species; protozoans such as Entamoeba histolytica; and multicellular helminth parasites such as tapeworms and pinworms. These pathogens cause disease in many ways, and as elsewhere in the body, the key to generating protective immunity is the activation of appropriate aspects of the innate immune system.

1	The effector mechanisms of the innate immune system can themselves eliminate most intestinal infections rapidly and without significant spread of the infection beyond the intestine. The essential features of these responses in epithelial surfaces are discussed in Section 2-2 and here we highlight only aspects that are unique or unusual to the intestine. Of these, the most important involve the epithelial cells themselves (Fig. 12.15). The tight junctions between

1	NF˜BTLRNODsCCL2CCL1IL-6CXCL1CXCL8IL-1IL-1IL-18I˜BTLR-5CCL20defensinstightjunctionslysosomephagosomephagophorecaspaseNOD-2TLRs, NOD1, and NOD2 activate NF˜B, inducing the epithelial cell to express in˜ammatory cytokines, chemokines, and other mediators. These recruit and activate neutrophils, macrophages, and dendritic cells Bacteria are recognized by TLRs on cell surface or in intracellular vesicles Bacteria or their products directly entering the cytosol are recognized by NOD1 and NOD2 Activation of in˜ammasome induces production of IL-1, IL-18 which activate myeloid cells and increase barrier integrity Bacteria in cytoplasm or escaping from phagosome are taken into forming autophagosome and destroyed after fusion with lysosome Intracellular infection triggers formation of in˜ammasome Destruction of bacteria in autophagosome

1	Fig. 12.15 Epithelial cells have a crucial role in innate defense against pathogens. TLRs are present in intracellular vesicles or on the basolateral or apical surfaces of epithelial cells, where they recognize different components of invading bacteria. Cytoplasmic NOD1 and NOD2 detect cell‑wall peptides from bacteria. TLRs and NODs activate NFκB (see Fig. 3.15), inducing epithelial cells to produce CXCL8, CXCL1 (GROα), CCL1, and CCL2, which attract neutrophils and macrophages, CCL20, which attracts dendritic cells, and IL‑1 and IL‑6 that activate macrophages. Many types of cell damage can activate inflammasome (see Section 3‑9) that activates pro‑caspase 1 and produces IL‑1 and IL‑18. Bacteria that invade the epithelial‑cell cytoplasm or escape into the cytosol from phagosomes can induce autophagy. The organisms become ubiquitinated, leading to the recruitment of adaptor proteins that attract the phagophore, forming an autophagosome. Fusion with lysosomes then leads to destruction of

1	autophagy. The organisms become ubiquitinated, leading to the recruitment of adaptor proteins that attract the phagophore, forming an autophagosome. Fusion with lysosomes then leads to destruction of the cargo within the autophagosome. NOD2 can also trigger autophagosome formation by binding directly to adaptor proteins, including the Crohn’s disease‑associated molecule ATGL16L1.

1	Typhimurium adheres to and enters M cells, which it then kills by causing apoptosis (top left). It then can infect macrophages and gut epithelial cells. TLR‑5 expressed by the epithelial basal membrane can bind salmonella flagellin, activating the NFκB pathway. After uptake by macrophages in the lamina propria, invasive Salmonella induces caspase 1 activation, promoting production of IL‑1 and IL‑8. CXCL8 is also produced by the infected macrophages, and together these mediators recruit and activate neutrophils (lower left panel). Salmonellae can also invade gut epithelial cells directly by adherence of fine threadlike protrusions on the luminal epithelial surface called fimbriae (top middle panel). The cell processes extended between epithelial cells by mononuclear phagocytes may be infected by salmonellae in the lumen and thus effectively breach the epithelial layer (top right panel). Dendritic cells in lamina propria may become infected from infected macrophages and carry them to

1	by salmonellae in the lumen and thus effectively breach the epithelial layer (top right panel). Dendritic cells in lamina propria may become infected from infected macrophages and carry them to the draining mesenteric lymph node to prime the adaptive immune response (lower right panel). If containment processes in the lymph node fail, Salmonella can invade beyond the intestine and its lymphoid tissues and enter the bloodstream to cause systemic infection.

1	Fig. 12.16 Salmonella enterica serovar Typhimurium is an important cause of food poisoning and penetrates the epithelial layer in three ways. Salmonella

1	If defenses fail, salmonellae can enter the bloodstream and cause a systemic infection neutrophil caspase 1 activation IL-18 CXCL8 IL-1˜Chemokines and cytokines produced by macrophages recruit neutrophils out of blood vessels and activate them Dendritic cells loaded with bacterial antigens acquired directly or from macrophages travel to the mesenteric lymph node via afferent lymphatics and provoke an adaptive immune response Salmonellae enter and kill M cells, and then infect macrophages and epithelial cells Salmonellae invade the luminal surface of epithelial cells Salmonellae enter phagocytic cells that are sampling the gut luminal contents M cell TLR-5 gut lumen these cells form a barrier that is normally impermeable to macromolecules and invaders. The constant production of new epithelial cells from stem cells in the crypts also allows the barrier to be repaired rapidly after mechanical damage or loss of cells. Nonetheless, pathogens have acquired mechanisms to gain entry through

1	cells from stem cells in the crypts also allows the barrier to be repaired rapidly after mechanical damage or loss of cells. Nonetheless, pathogens have acquired mechanisms to gain entry through these barriers; some entry mechanisms used by salmonella are shown in Fig. 12.16, and those used by shigella in Fig. 12.17.

1	Epithelial cells also bear TLRs on both their apical and basal surfaces, from which they can sense bacteria in the gut lumen and those that have penetrated across the epithelium. In addition, epithelial cells carry TLRs in intracellular vacuoles that can detect intracellular pathogens or extracellular pathogens and their products that have been internalized by endocytosis. Epithelial cells also have intracellular sensors, described in Chapter 3, and can react when microorganisms or their products enter the cytoplasm. These sensors include the nucleotide-binding oligomerization domain (NOD) proteins NOD1 and NOD2 (see Section 3-8 and Fig. 3.17). NOD1 recognizes a diaminopimelic acid-containing peptide that is found only in the cell walls of Gram-negative bacteria. NOD2 recognizes a muramyl dipeptide found in the peptidoglycans of most bacteria, and epithelial cells defective in NOD2 are less resistant to infection by intracellular bacteria. Mice lacking NOD2 also show increased

1	muramyl dipeptide found in the peptidoglycans of most bacteria, and epithelial cells defective in NOD2 are less resistant to infection by intracellular bacteria. Mice lacking NOD2 also show increased translocation of bacteria across the epithelium and out of Peyer’s patches. A defect in recognition of the commensal microbiota by NOD2 also seems to be important in Crohn’s disease, as up to 25% of patients carry a mutation in the NOD2 gene that renders the NOD2 protein nonfunctional.

1	Shigella cell-wall peptides bind and oligomerize NOD1, activating the NF˜B pathway RIPK2 I˜K I˜B NF˜B NOD1 Ligation of TLRs or NOD proteins in epithelial cells stimulates the production of cytokines, such as IL-1 and IL-6, and the production of chemokines. The chemokines include CXCL8, which is a potent neutrophil chemoattractant, and CCL2, CCL3, CCL4, and CCL5, which attract monocytes, eosinophils, and T cells out of the blood. Stimulated epithelial cells also increase their production of the chemokine CCL20, which attracts immature dendritic cells toward the epithelial surface (see Sections 12-4 and 12-7).

1	Epithelial cells also express members of the intracellular NOD-like receptor (NLR) family, including gNLRP3, NLRC4, and NLRP6, that can form inflammasomes (see Fig. 12.15). As described in Section 3-9, formation of an inflammasome leads to activation of caspase 1, which cleaves pro-IL-1 and pro-IL-18 to produce the active cytokines (see Fig. 3.19). Both these cytokines contribute to epithelial defense against bacterial invasion by promoting barrier integrity, but can cause tissue damage if present for long periods.

1	One mechanism recently recognized as important for epithelial defense against infection is autophagy, which we discussed in Section 6-6 for its relationship to antigen processing. In this process, a crescent-shaped double-membrane fragment in the cytoplasm, called the isolation membrane, or phagophore, engulfs various cytoplasmic contents to form a complete vesicle, the autophagosome, which fuses with lysosomes to degrade the contents (see Fig. 12.15). When autophagy is disrupted, bacteria cannot be contained effectively, and epithelial cells become stressed. This can lead to increased penetration of bacteria into the body and to NFκB-mediated inflammation. Autophagy is promoted by the NOD1 and NOD2 intracellular bacterial sensors. As with NOD2, mutations in the autophagy-related genes ATG16L1 and IRGM1 are associated with susceptibility to Crohn’s disease in humans.

1	Certain specialized populations of epithelial cells have particularly important roles in innate immune defense of the intestine. Paneth cells are found only in the small intestine, where they produce antimicrobial peptides such as RegIIIγ and defensins when exposed to IL-22 released by CD4 TH17 cells or ILC3s. They can also respond directly to microbes, as they express TLRs and NODs and they are highly autophagic. Defects in Paneth cell function lead to reduced bacterial defense and are believed to be important in susceptibility to inflammatory bowel disease in humans. Goblet cells are a further kind of specialized epithelial cell and produce mucus in response to cytokines derived from CD4 TH2 cells or ILC2s. Mucus consists of a complex mixture of highly charged glycoproteins (mucins) and forms an essential component of immune defense in all mucosal surfaces. Its density, charge, and stickiness

1	Fig. 12.17 Shigella flexneri infects intestinal epithelial cells to cause bacterial dysentery. Shigella flexneri binds to M cells and is translocated beneath the gut epithelium (first panel). The bacteria infect intestinal epithelial cells from their basal surface and are released into the cytoplasm (second panel). Muramyl tripeptides containing diaminopimelic acid in the cell walls of the shigellae bind to and oligomerize the protein NOD1. Oligomerized NOD1 binds the serine/threonine kinase RIPK2 and activates the NFκB pathway (see Fig. 3.17), leading to the transcription of genes for chemokines and cytokines (third panel). Activated epithelial cells release the chemokine CXCL8, which acts as a neutrophil chemoattractant (fourth panel). IκB, inhibitor of NFκB; IκK, IκB kinase.

1	mean that it presents a formidable barrier to invasion, by trapping microbes and other particles. At the same time, it acts as a scaffolding to retain IgA antibodies and antimicrobial peptides that have been secreted into the lumen across the epithelium. Mucus is also slippery in nature, meaning that trapped materials can then be expelled easily by normal peristaltic movements. In the intestine, there are two layers of mucus, an outer loose layer and a much denser inner layer, found mostly in the large intestine. Although bacteria can penetrate the loose layer of mucus, they are normally kept away from the surface of the epithelial cells by the inner dense layer, and defects in this structure compromise antimicrobial defense. As we have discussed, the intestinal mucosa is also rich in cells of the innate immune system that can respond rapidly to infection. These include macrophages, eosinophils, mast cells, ILCs, MAIT cells, NKT cells, and γ:δ T cells.

1	12-16 Pathogens induce adaptive immune responses when innate defenses have been breached.

1	If pathogenic bacteria and viruses gain access to the subepithelial space, they may interact with TLRs on inflammatory cells in the underlying tissue. Together with the cascade of inflammatory mediators released by epithelial cells, this dramatically alters the environment of the mucosa and changes the behavior of local antigen-presenting cells such as dendritic cells. As described in Section 9-8, activated dendritic cells will express high levels of co-stimulatory molecules and cytokines such as IL-1, IL-6, IL-12, and IL-23, and promote development of effector T cells. Dendritic cells activated in Peyer’s patches migrate to the T-cell-dependent areas of the patch, whereas dendritic cells that encounter antigen in the lamina propria migrate to the mesenteric lymph node under control of CCR7. The effector T cells activated in these ways acquire gut-homing molecules such as α4:β7 and CCR9 due to the presence of retinoic acid, ensuring that they return to the gut wall to encounter the

1	CCR7. The effector T cells activated in these ways acquire gut-homing molecules such as α4:β7 and CCR9 due to the presence of retinoic acid, ensuring that they return to the gut wall to encounter the invading organisms. Similarly, IgA-producing B lymphocytes are generated in Peyer’s patches and mesenteric lymph nodes, generating plasma cells that accumulate in the lamina propria. IgA secretion into the lumen is enhanced in response to infection because pIgR expression is enhanced by TLR ligands and pro-inflammatory cytokines. In some infections, IgG antibodies can now be found in intestinal secretions, but these are derived from serum and require invading organisms to reach systemic immune tissues.

1	The activated myeloid cells found in the inflamed mucosa also contribute to sustaining the functions of effector T and B cells after their arrival in the mucosa. IL-1 and IL-6 produced by recently arrived monocytes are important for maintaining the survival and function of local TH17 cells. Pro-inflammatory myeloid cells also produce mediators such as IL-6, TNF-α, and nitric oxide that help drive IgA switching and secondary expansion of mucosal B cells. 12-17 Effector T-cell responses in the intestine protect the function of the epithelium.

1	Once activated, the effector T cells that accumulate in the intestine behave much like their counterparts elsewhere in the body, producing cytokines and generating cytolytic activity as appropriate to the pathogen. What is different is that the aim of the protective immune response in the intestine is tailored to preserving the integrity and function of the epithelial barrier. This is achieved in a number of ways, depending on the nature of the pathogen. In virus infections, CD8 cytotoxic T cells among intraepithelial lymphocytes kill infected epithelial cells (see Fig 12.14), triggering their replacement by uninfected cells derived from the rapidly dividing stem cells in the crypts. A similar process can occur during other forms of protective immune responses, with cytokines from CD4 effector T cells directly stimulating epithelial cell division. This forces the replacement of infected cells and generates a moving target for organisms that are attempting to attach to the surface of

1	T cells directly stimulating epithelial cell division. This forces the replacement of infected cells and generates a moving target for organisms that are attempting to attach to the surface of the epithelium. An example of a cytokine of this kind is IL-13, produced by TH2 cells (and ILC2s) during parasitic infections. In addition to its ability to stimulate antimicrobial peptide production by Paneth cells, IL-22 produced by TH17 cells contributes to defense against extracellular bacteria and fungi by enhancing the tight junctions between epithelial cells that keep the barrier intact. Mucus is crucially important in protecting the epithelial barrier, and its production by goblet cells is enhanced by CD4 T-cell-derived cytokines such as IL-13 and IL-22, as well as by products of mast cells and other innate effector cells recruited by T cells. Finally, these mediators and others can enhance the peristaltic action of the intestine and its outward secretion of fluid, washing out pathogens

1	and other innate effector cells recruited by T cells. Finally, these mediators and others can enhance the peristaltic action of the intestine and its outward secretion of fluid, washing out pathogens within the lumen of the intestine. Together these processes aim to generate a hostile and unstable environment for the pathogen, reducing its ability to invade and damage the epithelial barrier.

1	12-18 The mucosal immune system must maintain tolerance to harmless foreign antigens.

1	Antigens within food and commensal bacteria normally do not induce an inflammatory immune response, despite the lack of central tolerance to them (Fig. 12.18). The mucosal immune system’s environment is inherently tolerogenic, and this is a barrier to the development of nonliving vaccines, which need to overcome local regulatory mechanisms. Food proteins are not digested completely in the intestine; significant amounts are absorbed into the body in an immunologically relevant form. The default response to oral administration of a protein antigen is the development of a phenomenon known as oral tolerance. This is a form of peripheral tolerance that renders the systemic and mucosal immune systems relatively unresponsive to the same antigen. It can be demonstrated experimentally in mice by feeding them a foreign protein such as ovalbumin (Fig. 12.19). When the animals are then challenged with the antigen by a nonmucosal route, such as injection into the skin, the immune response one

1	by feeding them a foreign protein such as ovalbumin (Fig. 12.19). When the animals are then challenged with the antigen by a nonmucosal route, such as injection into the skin, the immune response one would expect is blunted. This suppression of systemic immune responses is long lasting and is antigen specific: responses to other antigens are not affected. A similar suppression of subsequent immune responses is observed after the administration of proteins into the respiratory tract, giving rise to the concept of mucosal tolerance, as the usual response to such antigens is delivered via a mucosal surface. Systemic T-cell responses can also be inhibited by feeding humans protein antigens that they have not encountered previously.

1	Oral tolerance can impact all aspects of the peripheral immune response, including T-cell-dependent effector responses and IgE production. Effector T-cell responses in the mucosa are also downregulated in oral tolerance, although low levels of secretory IgA antibodies directed at food proteins can be found in healthy humans, but do not lead to inflammation.

1	Various mechanisms are likely to account for oral tolerance to protein antigens, including anergy, deletion of antigen-specific T cells, and the generation of regulatory T cells induced in the mesenteric lymph node to become gut-homing, antigen-specific FoxP3-positive Treg cells via the production of retinoic acid and TGF-β by migratory dendritic cells (see Section 12-7). Although it is known that all these events are also essential for the suppression of systemic immune responses, the mechanisms responsible for this link between the mucosal and peripheral immune system are not yet understood. At times, oral tolerance can fail, as is believed to occur in celiac disease (discussed in more detail in Section 14-17) or peanut allergies (discussed in Sections 14-10 and 14-12).

1	The intestinal immune system generates protective immunity against antigens that are presented during infections by pathogenic organisms. IgA antibodies are produced locally, serum IgG and IgA are made, and the appropriate effector T cells are activated in the intestine and elsewhere. When the antigen is encountered again, there is effective memory, ensuring rapid protection. Antigens from food proteins induce tolerance locally and systemically, with little or no IgA antibody production. T cells are not activated, and subsequent responses to challenge are suppressed. In the case of commensal bacteria, there may be some local IgA production, but no primary systemic antibody responses, and effector T cells are not activated. On day 7, the mice are injected with ovalbumin plus adjuvant to stimulate an effective immune response Mice are fed either ovalbumin or a control mixture

1	On day 7, the mice are injected with ovalbumin plus adjuvant to stimulate an effective immune response Mice are fed either ovalbumin or a control mixture Fig. 12.19 Tolerance to antigens can be experimentally generated by oral administration. Mice are fed for 2 weeks with 25 mg of either ovalbumin, the experimental protein, or a second protein as a control. Seven days later, the mice are immunized subcutaneously with ovalbumin plus an adjuvant, and after 2 weeks, the serum antibodies and T‑cell function are measured. Mice that were fed ovalbumin have a lower ovalbumin‑specific systemic immune response than those fed the control protein. Although mucosal tolerance can be used to avoid inflammatory disease in experimental animal models of type 1 diabetes mellitus, arthritis, and encephalomyelitis, clinical trials in humans have been less successful, and have been superseded by other therapies, such as monoclonal antibodies, that we will discuss in greater detail in Chapter 16.

1	12-19 The normal intestine contains large quantities of bacteria that are required for health.

1	The surfaces of the healthy body are colonized by large numbers of microorganisms, collectively referred to as the microbiota, or microbiome, composed mostly of bacteria, but also archaea, viruses, fungi, and protozoa . The intestine is the largest source of these organisms, although all the other mucosal tissues harbor their own, distinct populations of microbes. We each harbor more than 1000 species of commensal bacteria in our intestine, and they are present in greatest numbers in the colon and lower ileum. As many of the species cannot be grown in culture, their exact numbers and identities are only now being established by high-throughput sequencing techniques. In humans, there are several major phyla of bacteria, plus the archaea—in descending order, Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Archaea. There are at least 1014 of these microorganisms that collectively weigh about 1 kg. The intestinal microbiota normally exists in a mutually beneficial, or

1	Proteobacteria, Actinobacteria, and Archaea. There are at least 1014 of these microorganisms that collectively weigh about 1 kg. The intestinal microbiota normally exists in a mutually beneficial, or symbiotic, relationship known as mutualism that has been established in humans over many millennia and has coevolved with vertebrates throughout their history. As a result, the populations of these microbes found in different groups of animals are distinctive and are highly adapted to their individual host species.

1	The microbiota has an essential role in maintaining health. Its members assist in the metabolism of dietary constituents such as cellulose, as well as degrade toxins and produce essential cofactors such as vitamin K1. Short-chain fatty acids (SCFAs), such as acetate, propionate, and especially butyrate, produced by anaerobic metabolism of dietary carbohydrates by commensal bacteria are an essential source of energy for colonic enterocytes through their entry as substrates into the tricarboxylic acid (TCA) cycle. Surgical procedures, such as ileostomy, that remove the normal fecal flow to the colon can cause a syndrome called diversion colitis, in which enterocytes starved of SCFAs undergo inflammation and necrosis. Providing SCFAs to the affected colon segment can reverse this condition. Another important property of commensal organisms is that they interfere with the ability of pathogenic bacteria to colonize and invade the gut, partly by competing for space and nutrients. They can

1	Another important property of commensal organisms is that they interfere with the ability of pathogenic bacteria to colonize and invade the gut, partly by competing for space and nutrients. They can also directly inhibit the pro-inflammatory signaling pathways that pathogens stimulate in epithelial cells and that are needed for invasion. Perturbations in the balance between the various species of bacteria present in the microbiota (dysbiosis) have been found to increase susceptibility to a variety of diseases (see Sections 12-21 and 12-22).

1	The protective role of the commensal microbiota is dramatically illustrated by the adverse effects of broad-spectrum antibiotics. These antibiotics can kill large numbers of commensal gut bacteria, thereby creating an ecological niche for bacteria that would not otherwise be able to compete successfully. One example of a bacterium that grows in the antibiotic-treated gut and can cause a severe infection is Clostridium difficile (Fig. 12.20). This organism is an increasing problem in countries where broad-spectrum antibiotic use is prevalent, as it produces toxins that cause severe diarrhea and mucosal injury. Restoring the normal microbiota by a transplant of feces from healthy individuals can be used to treat C. difficile infection.

1	The importance of the local defense mechanisms against commensal bacteria for health is shown by experiments in animals that lack one or more of the factors involved. For instance, mice without secretory antibodies have increased numbers of commensal bacteria that have penetrated the intestinal mucosa and have disseminated beyond its draining lymphoid tissues. The composition of the microbiota in these mice is also altered, with increased numbers of bacteria, but decreased species diversity. Similar dysbiosis has been described in mice lacking FoxP3+ regulatory T cells or eosinophils. while preventing inflammation without compromising the ability to react to invaders.

1	Despite their beneficial effects, commensal bacteria are a potential threat, as is shown when the integrity of the intestinal epithelium is damaged. In these circumstances, normally innocuous gut bacteria, such as nonpathogenic E. coli, can cross the mucosa, invade the bloodstream, and cause fatal systemic infection. Therefore, the immune system in the intestine has to mount some form of response to control commensal microbes (Fig. 12.21). Since inappropriate reactions may lead to chronic inflammation and damage to the intestine, the immune system must balance the recognition and response to commensal bacteria with the cost of damaging tissues from inflammation. Commensal bacteria elicit antigen-specific responses that maintain the local balance between host and microbiota and are largely confined to the intestine itself. Unlike soluble food antigens, commensal bacteria do not induce a state of systemic immune unresponsiveness, and when these organisms enter the bloodstream, they can

1	confined to the intestine itself. Unlike soluble food antigens, commensal bacteria do not induce a state of systemic immune unresponsiveness, and when these organisms enter the bloodstream, they can stimulate a normal primary systemic immune response.

1	Recognition of the microbiota by the adaptive immune system is dependent on the uptake and intracellular transport of organisms by local dendritic cells that remain in Peyer’s patches or migrate no further than the mesenteric lymph node (see Fig. 12.21), which acts to prevent wider dissemination of the microbiota. Because commensal microbes are noninvasive, dendritic cells are not fully activated and induce a finely balanced response, comprising secretory IgA antibodies that enter intestinal secretions and are directed at commensal bacteria. Up to 75% of commensal organisms living in the lumen appear to be coated by IgA (see Fig. 12.21), limiting their adherence to and penetration of the epithelium. In addition, coating of the microbiota with SIgA can alter their gene expression. Many of the large numbers of fully differentiated TH1 and

1	Fig. 12.20 Infection by Clostridium difficile. Treatment with antibiotics causes massive death of the commensal bacteria that normally colonize the colon. This allows pathogenic bacteria to proliferate and to occupy an ecological niche that is normally occupied by harmless commensal bacteria. Clostridium difficile is an example of a pathogen producing toxins that can cause severe bloody diarrhea in patients treated with antibiotics.

1	Fig. 12.21 Several local processes ensure peaceful homeostasis between host and microbiota. Commensal bacteria in the lumen gain access to the immune system via M cells. Antigens are taken up by dendritic cells in Peyer’s patches and isolated follicles under noninflammatory conditions (left panel). Presentation of these antigens generates IgA‑switched B cells that localize in the lamina propria as IgA‑producing plasma cells (right panel). IgA then binds commensal bacteria, altering their gene expression, limiting their access to the epithelium, and blocking their binding to the surface. Interference with penetration of the epithelium is assisted by the presence of thick layers of mucus, which also contain mucin glycoproteins that have antibacterial properties. In addition, stimulation of pattern recognition receptors on Paneth cells induces the production of antimicrobial peptides such as RegIIIγ and defensins (see Section 2‑4), which are also stimulated by IL‑22 derived from TH17 CD4

1	recognition receptors on Paneth cells induces the production of antimicrobial peptides such as RegIIIγ and defensins (see Section 2‑4), which are also stimulated by IL‑22 derived from TH17 CD4 T cells and ILC3s. IL‑22 also tightens the epithelial barrier. Phagocytic macrophages found immediately under the epithelium can ingest and kill bacteria that penetrate the surface.

1	TH17 cells found in healthy intestine are also directed at the microbiota. While these cells produce mediators that can assist bacterial clearance by macrophages and epithelial cells, they come with the risk of producing inflammation and collateral damage. This does not occur, because of IL-10 produced by T cells and FoxP3+ regulatory T cells present in the mucosa. TH17 and FoxP3+ regulatory T cells in the intestine can enter germinal centers in Peyer’s patches and acquire the functions of follicular helper T cells, leading to selective IgA switching.

1	The endotoxin present on commensal bacteria also seems unusually sensitive to neutralization by gut enzymes such as alkaline phosphatase, leading to weaker immune activation. If commensal bacteria do cross the epithelium in small numbers, their lack of virulence factors means they cannot resist uptake and killing by phagocytic cells, and they are rapidly destroyed. In contrast to what happens in other tissues, ingestion of commensal bacteria in the intestine does not lead to inflammation. If macrophages cannot respond to the inhibitory effects of IL-10, intestinal inflammation develops spontaneously. Eosinophils in the healthy intestine assist antigen-specific IgA switching by producing APRIL, IL-6, and TGF-β when exposed to commensal microbes (see Section 12-11). Thus, commensal organisms associate with the mucosal surface without invading or provoking inflammation. This symbiosis involves many innate and adaptive immune effector cells that are usually associated with chronic

1	associate with the mucosal surface without invading or provoking inflammation. This symbiosis involves many innate and adaptive immune effector cells that are usually associated with chronic inflammation but in the intestine create a state sometimes referred to as physiological inflammation.

1	12-21 The intestinal microbiota plays a major role in shaping intestinal and systemic immune function. Commensal bacteria and their products play an essential role in normal development of the immune system. This effect is illustrated in germ-free, or gnotobiotic, mice, in which there is no colonization of the gut by microorganisms. These animals have marked reductions in the size of all lymphoid organs, low serum immunoglobulin levels, fewer mature T cells, and markedly reduced immune responses, especially TH1 and TH17 responses. Such mice are prone to make TH2-type responses such as IgE antibodies and are more susceptible Fig 12.22 Effects of the microbiota on disease and systemic immune function.

1	Fig 12.22 Effects of the microbiota on disease and systemic immune function. The presence and composition of the microbiota have many downstream consequences for the function of the immune system and other body tissues, some of which may be secondary to the events in the mucosa, while others may reflect the ability of products of intestinal microbes to enter the circulation. The microbiota is also known to have many effects on susceptibility to a wide range of diseases in humans and experimental animals.

1	to certain immunological diseases such as type 1 diabetes. In the intestine, Peyer’s patches do not develop normally and isolated lymphoid follicles are absent. Germ-free mice also have severely reduced numbers of T lymphocytes and ILCs in the lamina propria and epithelium, nearly absent IgA-secreting plasma cells, and reduced mediators of local immunity, such as antimicrobial peptides, retinoic acid, IL-7, IL-22, IL-25, IL-33, and TSLP. In contrast, invariant NKT cells (iNKTs) are more abundant in the germ-free intestine, perhaps contributing to the TH2 bias seen in germ-free animals.

1	The effects of the intestinal microbiota extend far beyond the intestine (Fig. 12.22). For example, several autoimmune diseases are more frequent in germ-free animals. The germ-free state greatly increases the severity of symptoms in a genetic model of type 1 diabetes. The composition of the microbiota influences susceptibility to many different immunological diseases, metabolic disorders such as obesity, cancer, cardiovascular disease, and even psychiatric disorders. The basis for these associations is unclear and few individual commensal species have been identified in disease susceptibility. However, some affected individuals have unusual compositions of the major bacterial species that normally make up the microbiota, a form of dysbiosis, as we saw in Section 12-19. In experimental models, disease susceptibility can be conferred by transferring intestinal bacteria between affected and unaffected animals, supporting the idea that the change in microbiota is a causal factor, rather

1	disease susceptibility can be conferred by transferring intestinal bacteria between affected and unaffected animals, supporting the idea that the change in microbiota is a causal factor, rather than being secondary to preexisting disease. This observation underlies the use of probiotics, which are particular mixtures of live bacteria and yeast that are considered beneficial. Their use may manipulate the intestinal microbiota to prevent disease and promote health, although much remains to be understood about their potential benefits.

1	Many different mechanisms are probably involved in the effects of the microbiota (Fig. 12.23). Ligation of TLRs and NLRs is undoubtedly important for many of the local effects on epithelial cells and myeloid cells. Flagellin present on many intestinal bacterial species can stimulate TLR-5 on mucosal CD11b-expressing dendritic cells, inducing the production of IL-6 and IL-23 and favoring TH17 and IgA responses. There are also examples of individual bacterial species that have specific effects on immune function. Colonization of mice with segmented filamentous bacteria (SFB) enhances IgA production, accumulation of IELs, and the number of intestinal effector TH17 T cells (see Fig. 12.23). Conversion of dietary tryptophan by lactobacilli into kynurenine metabolites can activate the AhR (see Section 12-14) and enhance IL-22 production by ILC3. Polysaccharide A (PSA) from Bacteroides fragilis drives the differentiation of Treg cells in a TLR-2-dependent manner. Also, several Clostridium

1	Section 12-14) and enhance IL-22 production by ILC3. Polysaccharide A (PSA) from Bacteroides fragilis drives the differentiation of Treg cells in a TLR-2-dependent manner. Also, several Clostridium species stimulate the preferential generation of FoxP3+ regulatory T cells in the colon, perhaps by promoting a TGF-β-rich environment and by producing SCFAs. The mechanism by which SCFAs directly alter immune cell function is currently unclear. As yet, few specific organisms have been identified to explain the effects of dysbiosis on human disease, although certain E coli species, collectively called enteroadherent Escherichia coli, have been found to be prevalent in patients with Crohn’s disease. Recent studies have also shown increased abundance of Prevotella copri in a number of patients with newly diagnosed rheumatoid arthritis, but much more work needs to be done to confirm these associations and to find if there are similar effects in other diseases.

1	Fig 12.23 The microbiota tune local and systemic immune responses. The microbiota has local and distant effects on immune function, although only a few individual organisms and mechanisms have been identified. Segmented filamentous bacteria (SFB) potently induce SFB‑specific TH17 cells, perhaps by inducing epithelial cells to produce serum amyloid A (SAA) protein that may act on dendritic cells. Bacterial flagellin favors TH17 and IgA responses by stimulating TLR‑5 on mucosal CD11b‑expressing dendritic cells. The microbiota is also needed for the presence of isolated lymphoid follicles and ILCs, especially ILC3 cells, but inhibits the accumulation of invariant NKT cells (iNKTs). Besides providing energy to colonic enterocytes, butyrate and other SCFAs may also act to drive the generation of FoxP3+ Tregs, although the molecular mechanism is still unclear. Clostridia also induce production of TGF‑β by epithelial cells. The polysaccharide antigen (PSA) from Bacteroides fragilis

1	of FoxP3+ Tregs, although the molecular mechanism is still unclear. Clostridia also induce production of TGF‑β by epithelial cells. The polysaccharide antigen (PSA) from Bacteroides fragilis stimulates the preferential generation of regulatory T cells, possibly by binding to TLR‑2 on CD4+ T cells. Unidentified members of the microbiota are needed to maintain the production of TSLP, IL‑25, IL‑33, and RA.

1	12-22 Full immune responses to commensal bacteria provoke intestinal disease.

1	Elegant experiments in the 1990s led to the now generally accepted idea that potentially aggressive T cells that can respond to commensal bacteria are present in normal animals but are usually kept in check by active regulation (Fig. 12.24). If these regulatory mechanisms fail, unrestricted immune responses to commensal bacteria can lead to inflammatory bowel diseases such as Crohn’s disease. Many genes that are associated with susceptibility to Crohn’s disease in humans encode proteins that regulate innate immunity. When these regulatory processes fail, systemic immune responses are generated against antigens from commensal bacterial, such as flagellin. T-cell responses are also generated in the mucosa, leading to severe intestinal damage. IL-23 is important in this process, promoting differentiation of TH17 effector cells. IL-23 and IL-12 in concert can also induce inflammatory TH1 responses in the intestine, with some CD4 effector T cells found to produce both IFN-γ and IL-17 under

1	of TH17 effector cells. IL-23 and IL-12 in concert can also induce inflammatory TH1 responses in the intestine, with some CD4 effector T cells found to produce both IFN-γ and IL-17 under these circumstances. These experimental results are consistent with clinical evidence for a linkage between polymorphisms in the IL-23 receptor and Crohn’s disease in humans. In all experimental models, the intestinal damage depends on the presence of commensal bacteria, can be prevented by treatment with antibiotics, and does not occur in germ-free animals.

1	Patients with Crohn’s disease and the related disorder ulcerative colitis exhibit dysbiosis and harbor unusual populations of intestinal microbiota. However, with the exception of enteroadherent Escherichia coli mentioned above, no individual species of commensal bacteria have yet been proven to be responsible for causing the damage. There is also experimental evidence that local responses to certain pathogenic viruses or parasites such as Toxoplasma gondii may trigger bystander activation of effector T cells specific to commensal organisms and produce persistent inflammation. Summary.

1	The immune system in the mucosa has to distinguish between potential pathogens and harmless antigens, generating strong effector responses to pathogens but remaining unresponsive to foods and commensals. Food proteins induce an active form of immunological tolerance in the systemic and mucosal immune systems; this tolerance may be mediated by regulatory T cells producing IL-10 and/or TGF-β. Commensal bacteria are also recognized by the immune system, but this is limited to the mucosa and its draining lymphoid tissues because commensal antigens are presented to T cells by semi-mature dendritic cells that migrate from the intestinal wall to draining mesenteric lymph nodes. This results in active mucosal tolerance and the production of local IgA antibodies that restrict colonization by the microorganisms, but ‘ignorance’ of these antigens by the systemic immune system. Because commensal bacteria have many beneficial effects for the host, these immunoregulatory processes are important in

1	but ‘ignorance’ of these antigens by the systemic immune system. Because commensal bacteria have many beneficial effects for the host, these immunoregulatory processes are important in allowing the bacteria to coexist with the immune system. When the normal regulatory processes break down, local dendritic cells become fully activated and induce differentiation of naive T cells into effector T cells in the mesenteric lymph node. This is important for protective immunity against pathogens, but when it occurs under the wrong circumstances, it can lead to inflammatory diseases such as Crohn’s disease or celiac disease. As a consequence of these competing, but interacting, needs of the immune response, the intestine normally has the appearance of physiological inflammation, which helps maintain normal function of the gut and immune system. This process is driven mostly by the need to control the intestinal microbiota without eliminating it completely or causing damaging inflammation, and

1	normal function of the gut and immune system. This process is driven mostly by the need to control the intestinal microbiota without eliminating it completely or causing damaging inflammation, and results in the coordinated production of IgA, activation of regulatory and effector T cells, and several innate immune responses. Abnormalities in the host response can alter the composition and behavior of the microbiota, while changes in the microbiota can also influence the development and outcome of many diseases outside the intestine.

1	Summary to Chapter 12. The mucosal immune system is a large and complex apparatus that has a crucial role in health, not just by protecting physiologically vital organs but also by helping to regulate the tone of the entire immune system and prevent disease. The peripheral lymphoid organs focused on by most immunologists may be a recent specialization of an original template that evolved in mucosal tissues. The mucosal surfaces of the body are highly vulnerable to infection

1	Fig. 12.24 T cells with the potential to produce inflammation in response to commensal bacteria are present in normal animals, but are controlled by regulatory T cells. Transfer of unseparated CD4+ T cells from a normal mouse into an immunodeficient mouse, such as one lacking the rag gene (rag –/–), will lead to reconstitution of the CD4+ T‑cell compartment. However if ‘naive’ CD4+ T cells (CD4+CD45RBhi) are purified and transferred, the host mice develop severe inflammation of the colon. This can be prevented by co‑transferring the CD4+CD25+ FoxP3+ T cells that were removed during the purification of the naive CD4+ T‑cell population. The effects of these regulatory T cells are blocked by neutralizing TGF‑β in vivo and are also dependent on IL‑10. The intestinal inflammation caused by naive CD4+ T cells requires the presence of the microbiota, as it is prevented in germ‑free mice, or by treatment with antibiotics. These experiments demonstrate that some CD4+ T cells in normal animals

1	CD4+ T cells requires the presence of the microbiota, as it is prevented in germ‑free mice, or by treatment with antibiotics. These experiments demonstrate that some CD4+ T cells in normal animals are capable of provoking inflammatory responses against the intestinal microbiota, but that these are normally held in check by regulatory T cells. Micrographs from Powrie, F., et al.: J. Exp Med. 1996, 183:2669–2674.

1	and possess a complex array of innate and adaptive mechanisms of immunity. The adaptive immune system of the mucosa-associated lymphoid tissues differs from that of the rest of the peripheral lymphoid system in several respects: the immediate juxtaposition of mucosal epithelium and lymphoid tissue; diffuse lymphoid tissue as well as more organized lymphoid organs; specialized antigen uptake mechanisms and distinctive dendritic cells and macrophages; the predominance of activated/memory lymphocytes and distinctive innate lymphoid cells (ILCs) even in the absence of infection; the production of dimeric secretory IgA as the predominant antibody; and the downregulation of immune responses to innocuous antigens such as food antigens and commensal microorganisms. No systemic immune response can normally be detected to these antigens. In contrast, pathogenic microorganisms induce strong protective responses. The key factor in the decision between tolerance and the development of powerful

1	can normally be detected to these antigens. In contrast, pathogenic microorganisms induce strong protective responses. The key factor in the decision between tolerance and the development of powerful adaptive immune responses is the context in which antigen is presented to T lymphocytes in the mucosal immune system. When there is no inflammation, presentation of antigen to T cells by dendritic cells induces the differentiation of regulatory T cells. By contrast, pathogenic microorganisms crossing the mucosa induce an inflammatory response in the tissues, which stimulates the maturation of antigen-presenting cells and their expression of co-stimulatory molecules, thus favoring a protective T-cell response. This decision-making process is controlled mostly by the way in which specialized dendritic cells react to their environment before migrating to present antigen to naive T cells. The mutualistic loop formed between the host immune response and the local microbiota plays a central

1	dendritic cells react to their environment before migrating to present antigen to naive T cells. The mutualistic loop formed between the host immune response and the local microbiota plays a central role in maintaining health and in the development of disease.

1	Questions. 12.1 Multiple Choice: Which of the following is an incorrect statement? A. Microfold cells have a folded luminal surface and possess a thick layer of mucus that allows the entry of microbes to Peyer’s patches. B. Microfold cells recognize several bacterial proteins by GP2 and release the material to the extracellular space by a process called transcytosis. C. Gut‑associated lymphoid tissues attract dendritic cells trough chemokines such as CCL20 and CCL9. D. Pathogens such as Yersinia pestis and Shigella target microfold cells to gain access to the subepithelial space. 12.2 True or False: Intraepithelial lymphocytes are mostly CD4 T cells, in contrast to the lamina propria, where CD8 T cells predominate. 12.3 Matching: Match each chemokine or chemokine receptor to its tissue homing function. A. CXCL13 i. Recruitment of lymphocytes to the colon, lactating mammary gland, and salivary glands 12.4 Multiple Choice: Which of the following is a correct statement?

1	A. CXCL13 i. Recruitment of lymphocytes to the colon, lactating mammary gland, and salivary glands 12.4 Multiple Choice: Which of the following is a correct statement? A. CD11b+ dendritic cells stimulate ILC3s and are the main source for IL‑12 in Peyer’s patches. B. CD11b– dendritic cells require BATF3 for their development. C. Retinoic acid production by naive T cells is required for dendritic cells for the generation of Treg cells. D. CCL20 prevents entrance of dendritic cells into the epithelial layer of Peyer’s patches. 12.5 Short Answer: IgA:antigen complexes can be reexported to the gut lumen in order to enhance pathogen excretion from the organism. In contrast, the formation of IgA:antigen complexes can also enhance the uptake of luminal antigen. How can uptake of antigen be beneficial to the organism?

1	12.6 Short Answer: Large amounts of IgA are produced by intestinal B cells and plasma cells and secreted into the lumen as a means to keep the microbiota in check and prevent invasion from pathogens, yet most individuals with IgA deficiency are not overly susceptible to infections. Explain why this is the case. 12.7 Multiple Choice: Which of the following best describes intraepithelial lymphocytes (IELs)? A. Express CCR9 and α4β7 integrin B. Express CCR9 and αEβ7 integrin (CD103) C. Have a CD4 to CD8 T cell ratio of 3:1 D. Contain CD4+ T cells that produce IFN‑γ, IL‑17, and IL‑22 E. Consist of 90% T cells, 80% of which express CD8 as an α:α homodimer or an α:β heterodimer F. A and C G. B and E H. A, C, and D 12.8 Multiple Choice: Which of the following cell types depend on aryl hydrocarbon receptor expression for proper development? A. Type b intraepithelial lymphocyte (EIL) B. ILC1 C. B cell D. Macrophage E. ILC2

1	A. Type b intraepithelial lymphocyte (EIL) B. ILC1 C. B cell D. Macrophage E. ILC2 F. Neutrophil 12.9 Matching: Match the human disease with the C. Antibiotic treatment that eliminates iii. Inflammatory the bulk of commensal flora allowing bowel disease a particular species to overgrow and (Crohn’s produce toxins that lead to severe disease and diarrhea and mucosal injury ulcerative colitis) D. Hyperactive immune responses to iv. Diversion colitis commensal bacteria due to defects in innate immunity genes 12.10 True or False: Lamina propria CD4+ T cells secrete large amounts of cytokines such as IFN‑γ, IL‑17, and IL‑22 only in response to pathogens and inflammatory insults. 12.11 True or False: Most Tregs in the small intestine do not express FoxP3. General references. Hooper, L.V., Littman, D.R., and Macpherson, A.J.: Interactions between the microbiota and the immune system. Science 2012, 336:1268–1273.

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1	Sokol, H., Conway, K.L., Zhang, M., Choi, M., Morin, B., Cao, Z., Villablanca, E.J., Li, C., Wijmenga, C., Yun, S.H., et al: Card9 mediates intestinal epithelial cell restitution, T-helper 17 responses, and control of bacterial infection in mice. Gastroenterology 2013, 145:591–601. Sonnenberg, G.F., Fouser, L.A., and Artis, D.: Functional biology of the IL-22IL-22R pathway in regulating immunity and inflammation at barrier surfaces. Adv. Immunol. 2010, 107:1-29. Turner, J.E., Stockinger, B., and Helmby, H.: IL-22 mediates goblet cell hyperplasia and worm expulsion in intestinal helminth infection. PLoS Patho. 2013, 9:e1003698. 12-18 The mucosal immune system must maintain tolerance to harmless foreign antigens.

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1	Huang, G., Wang, Y., and Chi, H.: Control of T cell fates and immune tolerance by p38α signaling in mucosal CD103+ dendritic cells. J. Immunol. 2013, 191:650–659. Mowat, A.M., Strobel, S., Drummond, H.E., and Ferguson, A.: Immunological responses to fed protein antigens in mice. I. Reversal of oral tolerance to ovalbumin by cyclophosphamide. Immunology 1982, 45:105–113. 12-19 The normal intestine contains large quantities of bacteria that are required for health. preventing inflammation without compromising the ability to react to invaders. Arpaia, N., Campbell, C., Fan, X., Dikiy, S., van der Veeken, J., deRoos, P., Liu, H., Cross, J.R., Pfeffer, K., Coffer, P.J., et al.: Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature 2013, 504:451–455.

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1	Failures of Host Defense Mechanisms In the normal course of an infection, the infectious agent first triggers an innate immune response. The foreign antigens of the infectious agent, enhanced by signals from innate immune cells, then induce an adaptive immune response that ultimately clears the infection and establishes a state of protective immunity. This does not always happen, however. In this chapter we examine circumstances in which there are failures of host defense against infectious agents, whether due to immune defects in an abnormal host, as occurs in immunodeficiency, or to the evasion or subversion of immune defenses in normal hosts by pathogens. Finally, we will consider the special case in which the immune defenses of a normal host are impaired by one infectious agent that leads to more generalized susceptibility to infection, as occurs in the acquired immune deficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV).

1	In the first part of the chapter, we examine primary, or inherited, immunodeficiency diseases, in which host defense fails due to an inherited defect in a gene that results in the elimination or impaired function of one or more components of the immune system, leading to heightened susceptibility to infection with particular classes of pathogens. Immunodeficiency diseases caused by defects in T or Blymphocyte development, phagocyte function, and complement components have all been discovered. In the second part of the chapter, we briefly consider mechanisms by which pathogens evade or subvert specific components of the immune response to avoid elimination, socalled immune evasion. In the last part of the chapter, we consider how persistent infection by HIV leads to AIDS, an example of secondary, or acquired, immunodeficiency. The study of circumstances and mechanisms by which the immune system can fail has already contributed greatly to our understanding of host defense mechanisms

1	secondary, or acquired, immunodeficiency. The study of circumstances and mechanisms by which the immune system can fail has already contributed greatly to our understanding of host defense mechanisms and, in the longer term, might help to provide new methods of controlling or preventing infectious diseases, including AIDS.

1	immunodeficiency diseases. evasion and subversion of immune defenses. acquired immune deficiency syndrome. Immunodeficiency diseases.

1	Immunodeficiencies occur when one or more components of the immune system are defective; immunodeficiencies are classified as primary (inherited, or congenital) or secondary (acquired). Primary immunodeficiencies are caused by inherited mutations in any of a large number of genes that are involved in or control immune responses. Well over 150 primary immunodeficiencies have now been described that affect the development of immune cells, their function, or both. Clinical features of these disorders are therefore highly variable, although a common feature is recurrent and often overwhelming infections in very young children. In contrast, secondary immunodeficiencies are acquired as a consequence of other diseases, or are secondary to environmental factors such as starvation, or are an adverse consequence of medical intervention. Some forms of immunodeficiency principally affect immuneregulatory pathways. Defects of this type can lead to allergy, abnormal proliferation of lymphocytes,

1	consequence of medical intervention. Some forms of immunodeficiency principally affect immuneregulatory pathways. Defects of this type can lead to allergy, abnormal proliferation of lymphocytes, autoimmunity, and certain types of cancer, and will be discussed in other chapters. Here, we will mainly focus on those immunodeficiencies that predispose to infection.

1	Primary immunodeficiencies can be classified on the basis of the components of the immune system involved. However, because of the integration of many aspects of immune defense, defects in one component of the immune system can impact the function of others. Therefore, primary defects in innate immunity can lead to defects in adaptive immunity, and vice versa. Nevertheless, it is instructive to consider immune defects in the context of the major types of immunity affected, as these can lead to distinct patterns of infection and clinical disease. By examining which infectious diseases accompany a particular immunodeficiency, we gain insights into components of the immune system that are important in the response to particular agents. The inherited immunodeficiencies also reveal how interactions between different immune cell types contribute to the immune response and to the development of T and B lymphocytes. Finally, these inherited diseases can lead us to the defective gene, often

1	between different immune cell types contribute to the immune response and to the development of T and B lymphocytes. Finally, these inherited diseases can lead us to the defective gene, often revealing new information about the molecular basis of immune processes and providing the necessary information for diagnosis, genetic counseling, and eventually the possibility of gene therapy for cure.

1	13-1 A history of repeated infections suggests a diagnosis of immunodeficiency. Patients with immune deficiency are usually detected clinically by a history of recurrent infection, often by the same or similar pathogens. The type of infection is a guide to which part of the immune system is deficient. Recurrent infection by pyogenic, or pusforming, bacteria suggests a defect in antibody, complement, or phagocyte function, reflecting the role of these parts of the immune system in defense against such infections. Alternatively, a history of persistent fungal skin infection, such as cutaneous candidiasis, or recurrent viral infections suggests a defect in host defense mediated by T lymphocytes. 13-2 Primary immunodeficiency diseases are caused by inherited gene defects.

1	13-2 Primary immunodeficiency diseases are caused by inherited gene defects. Before the advent of antibiotics, most individuals with inherited immune defects died in infancy or early childhood because of their susceptibility to particular classes of pathogens. Such cases were not easily identified, because many normal infants also died of infection. Most of the gene defects that cause inherited immunodeficiencies are recessive, and many are caused by mutations in genes on the X chromosome. As males have only one X chromosome, all males who inherit an X chromosome carrying a defective gene will be affected by the disease. In contrast, female carriers with one defective X chromosome are usually healthy. Gene knockout techniques in mice (see Appendix I, Section A35) have created many immunodeficient states that are adding rapidly to our knowledge of the contribution of individual proteins to normal immune function. Nevertheless, immunodeficiency diseases.

1	human immunodeficiency diseases remain the best source of insight into the normal pathways of defense against infectious diseases. For example, deficiencies of antibody, of complement, or of phagocytic function each increase the risk of infection by certain types of bacteria. This reflects the fact that the normal pathway of host defense against such bacteria is the binding of antibody followed by the fixation of complement, which allows the opsonized bacteria to be taken up by phagocytic cells and killed. Breaking any of the links in this chain of events causes a similar immunodeficient state.

1	Immunodeficiencies also teach us about the redundancy of defense mechanisms against infectious disease. By chance, the first person to be reported with a hereditary deficiency of complement (C2 deficiency) was a healthy immunologist. This teaches us that there are multiple protective immune mechanisms against infection, such that a defect in one component of immunity might be compensated for by other components. Thus, although there is abundant evidence that complement deficiency increases susceptibility to pyogenic infection, not every human with complement deficiency suffers from recurrent infections. Examples of immunodeficiency diseases are listed in Fig. 13.1. None is very common (a selective deficiency in IgA being the most frequently reported), and some are extremely rare. These diseases are described in subsequent sections, and we have grouped the diseases according to where the specific causal defect lies in the adaptive or innate immune systems.

1	13-3 Defects in T-cell development can result in severe combined immunodeficiencies. The developmental pathways leading to circulating naive T cells and B cells are summarized in Fig. 13.2. Patients with defects in Tcell development are highly susceptible to a broad range of infectious agents. This demonstrates the central role of Tcell differentiation and maturation in adaptive immune responses to virtually all antigens. Because such patients exhibit neither Tcelldependent antibody responses nor cellmediated immune responses, and thus cannot develop immunological memory, they are said to suffer from severe combined immunodeficiency (SCID).

1	Xlinked SCID (XSCID) is the most frequent form of SCID and is caused by mutations in the gene IL2RG on the human X chromosome, which encodes the interleukin2 receptor (IL2R) common gamma chain (γc). γc is required in all receptors of the IL2 cytokine family (IL2, IL4, IL7, IL9, IL15, and IL21). Patients with XSCID thus have defects in signaling of all IL2family cytokines, and, owing to the defects in IL7 and IL15, their T cells and NK cells fail to develop normally (see Fig. 13.2). Bcell numbers, on the other hand, are normal, but due to absence of Tcell help, their function is not. XSCID patients are overwhelmingly male; in females who are carriers of the mutation, Tcell and NKcell progenitors in which Xinactivation has preserved the wildtype IL2RG allele progress through development to establish a normal mature immune repertoire. XSCID is known as the ‘bubble boy disease’ after a boy with XSCID who lived in a protective bubble for more than a decade before he died from complications

1	a normal mature immune repertoire. XSCID is known as the ‘bubble boy disease’ after a boy with XSCID who lived in a protective bubble for more than a decade before he died from complications of a bone marrow transplant. A clinically and immunologically indistinguishable type of SCID is associated with an inactivating mutation in the kinase Jak3 (see Section 81), which physically associates with γc and transduces signaling through γcchain cytokine receptors. This autosomal recessive mutation also impairs the development of T and NK cells, but the development of B cells is unaffected.

1	Other immunodeficiencies in mice have pinpointed more precisely the roles of individual cytokines and their receptors in Tcell and NKcell development. For example, mice with targeted mutations in the βc gene (IL2RB) defined a key role for IL15 as a growth factor for the development of NK cells, as well as Fig. 13.1 Human immunodeficiency syndromes. the specific gene defect, the consequence for the immune system, and the resulting disease susceptibilities are listed for some common and some rare human immunodeficiency syndromes. Severe combined immunodeficiency (SCiD) can be due to many different defects, as summarized in Fig. 13.2 and described in the text. aiD, activation-induced cytidine deaminase; atM, ataxia telangiectasiamutated protein; eBV, epstein–Barr virus; iKK, inhibitor of κ B kinase; Stat3, signal transducer and activator of transcription 3; taP, transporters associated with antigen processing; unG, uracil-Dna glycosylase.

1	a role for the cytokine in Tcell maturation and trafficking. Mice with targeted mutations in IL15 itself or the α chain of its receptor also have no NK cells and relatively normal Tcell development, but they show a more specific Tcell defect, primarily limited to impaired maintenance of memory CD8 T cells.

1	Humans with a deficiency of the IL7 receptor α chain have no T cells but normal levels of NK cells, illustrating that IL7 signaling, while essential for Tcell development, is not essential for the development of NK cells (see Fig. 13.2). Interestingly, mice with a genetargeted deficiency of the IL7R share with humans a deficiency of T cells, but also lack B cells, which is not the case in humans. This illustrates the speciesspecific role of certain cytokines, and provides a cautionary note against extrapolating findings from mice to humans. In humans and mice whose T cells show defective production of IL2 after receptor stimulation, most Tcell development itself is normal, although there is impaired development of FoxP3+ Treg cells that predisposes to immuneregulatory abnormalities and autoimmunity (see Chapter 15). The more limited effects of individual cytokine signaling defects are in contrast to the global defects in T and NKcell development in patients with XSCID.

1	TCRpre-TCR CD8 CD4 pro-T cell pro-B cell pre-B cell immature B cell B1 B cell natural killer cell B2 B cell MZ B cell pro-NK CLPHSC pre-T cell double-positive T cell CD8 T cell thymus Bone marrow CD4 T cell CD8 T cell CD4 T cell pre-BCR BCR IgD CD3˜, CD3°, CD3˛TAP1, TAP2, tapasin CIITA, RFXANK, RFX5, RFXAP ˝H, ˙5, Igˆ, IgˇBLNK ZAP-70 AIRERAG-1, RAG-2 Artemis, DNA-PKc, DNA ligase IV CD45 BTK IL-7RˆADA Periphery ˇc, Jak3 °c SCID SCID XSCID MHC class I defciency MHC class II defciency B-cell defciency B-cell defciency SCID-like immunodefciency APECEDSCID Omenn syndrome RS-SCID SCID SCID SCID X-linked agammaglobulinemia Fig. 13.2 Defects in T-cell and B-cell development that cause immunodeficiency. the pathways leading to circulating naive t cells and B cells are shown here. Mutations in genes that encode the proteins (indicated in red boxes) are known to cause human immunodeficiency diseases. BCR, B-cell receptor; ClP, common lymphoid progenitor; HSC, hematopoietic stem cell; MZ B cell,

1	that encode the proteins (indicated in red boxes) are known to cause human immunodeficiency diseases. BCR, B-cell receptor; ClP, common lymphoid progenitor; HSC, hematopoietic stem cell; MZ B cell, marginal zone B cell; pre-BCR, pre-B-cell receptor; pre-tCR, pre-t-cell receptor; RS-SCiD, radiation-sensitive SCiD; SCiD, severe combined immunodeficiency; tCR, t-cell receptor; XSCiD, X-linked SCiD. immunodeficiency can also be caused by mutations in genes in the thymic epithelium that impair thymic development, and thus t-cell development.

1	As in all serious Tcell deficiencies, patients with XSCID do not make effective antibody responses to most antigens, although their B cells seem normal. Most, but not all, naive IgMpositive B cells from female carriers of XSCID have inactivated the defective X chromosome rather than the normal one (see Section 133), showing that Bcell development is affected by, but is not wholly dependent on, the γc chain. Mature memory B cells that have undergone class switching have inactivated the defective X chromosome almost without exception. This might reflect the fact that the γc chain is also part of the receptor for IL21, which is important for the maturation of classswitched B cells (see Section 104). 13-4 SCID can also be due to defects in the purine salvage pathway.

1	Variants of autosomal recessive SCID that arise from defects in enzymes of the salvage pathway of purine synthesis include adenosine deaminase (ADA) deficiency (see Fig. 13.2) and purine nucleotide phosphorylase (PNP) deficiency. ADA catalyzes the conversion of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively, and its deficiency results in the accumulation of deoxyadenosine and its precursor, Sadenosylhomocysteine, which are toxic to developing T and B cells. PNP catalyzes the conversion of inosine and guanosine to hypoxanthine and guanine, respectively. PNP deficiency, which is a rarer form of SCID, also causes the accumulation of toxic precursors but affects developing T cells more severely than B cells. In both diseases, the development of lymphopenia, or decreased numbers of lymphocytes, is progressive after birth, resulting in profound lymphopenia within the first few years of life. Because both enzymes are housekeeping proteins expressed by many cell types,

1	numbers of lymphocytes, is progressive after birth, resulting in profound lymphopenia within the first few years of life. Because both enzymes are housekeeping proteins expressed by many cell types, the immune deficiency associated with each of these inherited defects is part of a broader clinical syndrome.

1	13-5 Defects in antigen receptor gene rearrangement can result in SCID.

1	Another group of autosomally inherited defects leading to SCID is caused by failures of DNA rearrangement in developing lymphocytes. Mutations in either the RAG1 or RAG2 gene that result in nonfunctional proteins cause arrest of lymphocyte development at the pro to preTcell and Bcell transitions because of a failure of V(D)J recombination (see Fig. 13.2). Thus, there is a complete lack of both T cells and B cells in these patients. Because the effects of RAG deficiencies are limited to lymphocytes that undergo antigen gene rearrangement, NKcell development is not impaired. There are other children with hypomorphic mutations (which cause reduced, but not absent, function) in either RAG1 or RAG2 who can make a small amount of functional RAG protein, allowing limited V(D)J recombination. This latter group includes patients with a distinctive and severe disease called Omenn syndrome, which, in addition to increased susceptibility to multiple opportunistic infections, has clinical features

1	latter group includes patients with a distinctive and severe disease called Omenn syndrome, which, in addition to increased susceptibility to multiple opportunistic infections, has clinical features very similar to graftversushost disease characterized by rashes, eosinophilia, diarrhea, and enlargement of the lymph nodes (see Section 1536). Normal or increased numbers of activated T cells are found in these children. An explanation for this phenotype is that low levels of RAG activity allow some limited Tcell receptor gene recombination. No B cells are found, however, suggesting that B cells have more stringent requirements for RAG activity. Due to the limited number of Tcell receptors that are successfully rearranged, the repertoire of T cells is highly restricted in patients with Omenn syndrome, and there is activation and clonal expansion of the limited number of specificities present. The clinical features strongly suggest that these peripheral T cells are autoreactive and are

1	syndrome, and there is activation and clonal expansion of the limited number of specificities present. The clinical features strongly suggest that these peripheral T cells are autoreactive and are responsible for the graftversushost phenotype. In addition to Omenn syndrome, which is manifested very early in life, other forms of immunodeficiency have also been associated with reduced but not absent RAG activity, and are often characterized by granulomatous disease that is not evident until late childhood or adolescence.

1	A subset of patients with autosomal recessive SCID are characterized by an abnormal sensitivity to ionizing radiation. They produce very few mature B and T cells because there is a failure of DNA rearrangement in their developing lymphocytes; only rare VJ or VDJ joints are seen, and most of these are abnormal. This type of SCID is due to defects in ubiquitous DNA repair proteins involved in repairing DNA doublestrand breaks, which are generated not only during antigen receptor gene rearrangement (see Section 55) but also by ionizing radiation. Owing to the increased radiosensitivity in these patients, this class of SCID is called radiationsensitive SCID (RSSCID) to distinguish it from SCID due to lymphocytespecific defects. Defects in the genes for Artemis, DNA proteinkinase catalytic subunit (DNAPKcs), and DNA ligase IV cause RSSCID (see Fig. 13.2). Because defects in repair of DNA breaks increase the risk of translocations during cell division that can lead to malignant

1	subunit (DNAPKcs), and DNA ligase IV cause RSSCID (see Fig. 13.2). Because defects in repair of DNA breaks increase the risk of translocations during cell division that can lead to malignant transformation, patients with RSSCID variants are also more likely to develop cancer.

1	13-6 Defects in signaling from T-cell antigen receptors can cause severe immunodeficiency.

1	Several gene defects have been described that interfere with signaling through the Tcell receptor (TCR) and thus block the activation of T cells early in thymic development. Patients with mutations in the CD3δ, CD3ε, or CD3ζ chains of the CD3 complex have defective preTcell receptor signaling and fail to progress to the doublepositive stage of thymic development (see Fig. 13.2), resulting in SCID. Another lymphocyte signaling defect that leads to severe immunodeficiency is caused by mutations in the tyrosine phosphatase CD45. Humans and mice with CD45 deficiency show a marked reduction in peripheral Tcell numbers and also abnormal Bcell maturation. Severe immunodeficiency also occurs in patients who make a defective form of the cytosolic protein tyrosine kinase ZAP70, which transmits signals from the Tcell receptor (see Section 77). CD4 T cells emerge from the thymus in normal numbers, whereas CD8 T cells are absent. However, the CD4 T cells that mature fail to respond to stimuli that

1	from the Tcell receptor (see Section 77). CD4 T cells emerge from the thymus in normal numbers, whereas CD8 T cells are absent. However, the CD4 T cells that mature fail to respond to stimuli that normally activate the cells through the Tcell receptor.

1	Wiskott–Aldrich syndrome (WAS), which is caused by a defect in the WAS gene on the X chromosome that encodes WAS protein (WASp), has shed new light on the molecular basis of signaling and immune synapse formation between various cells in the immune system. Although the disease also affects platelets and was first described as a bloodclotting disorder, it also causes immunodeficiency that is characterized by reduced Tcell numbers, defective NKcell cytotoxicity, and a failure of antibody responses (see Section 719). WASp is expressed in all hematopoietic cell lineages and is a key regulator of lymphocyte and platelet development and function through its transduction of receptormediated signals that induce reorganization of the cytoskeleton (see Section 925). Several signaling pathways downstream of the Tcell receptor are known to activate WASp (see Section 7.19). Activation of WASp in turn activates the Arp2/3 complex, which is essential for initiating actin polymerization that is

1	downstream of the Tcell receptor are known to activate WASp (see Section 7.19). Activation of WASp in turn activates the Arp2/3 complex, which is essential for initiating actin polymerization that is critical for immune synapse formation and the polarized release of effector molecules by T cells. In patients with WAS, and in mice whose Was gene has been knocked out, T cells fail to respond normally to Tcell receptor crosslinking. It has also recently been suggested that WASp is required for the suppressive function of natural Treg cells, and this may help explain why patients with WASp are susceptible to autoimmune diseases.

1	13-7 Genetic defects in thymic function that block T-cell development result in severe immunodeficiencies. A disorder of thymic development associated with SCID and a lack of body hair has been known for many years in mice; the mutant strain is descriptively named nude (see Section 810). A small number of children have been described with the same phenotype. In both mice and humans this syndrome is caused by mutations in the gene FOXN1, which encodes a transcription factor selectively expressed in skin and thymus. FOXN1 is necessary for the differentiation of thymic epithelium and the formation of a functional thymus. In patients with a mutation in FOXN1, the lack of thymic function prevents normal Tcell development. Bcell development is normal in individuals with the mutation, yet Bcell responses are deficient because of the lack of T cells, and the response to nearly all pathogens is profoundly impaired.

1	DiGeorge syndrome is another disorder in which the thymic epithelium fails to develop normally, resulting in SCID. The genetic abnormality underlying this complex developmental disorder is a deletion within one copy of chromosome 22. The deletion varies between 1.5 and 5 megabases in size, with the smallest deletion that causes the syndrome containing approximately 24 genes. The relevant gene within this interval is TBX1, which encodes the transcription factor Tbox 1. DiGeorge syndrome is caused by the deletion of a single copy of this gene, such that patients with this disorder are haploinsufficient for TBX1. Without the proper inductive thymic environment, T cells cannot mature, and both cellmediated immunity and Tcelldependent antibody production are impaired. Patients with this syndrome have normal levels of serum immunoglobulin but an absence of, or incomplete development of, the thymus and parathyroid glands, with varying degrees of Tcell immunodeficiency.

1	Defects in the expression of MHC molecules can lead to severe immunodeficiency as a result of effects on the positive selection of T cells in the thymus (see Fig. 13.2). Individuals with bare lymphocyte syndrome lack expression of all MHC class II molecules; the disease is now called MHC class II deficiency. Because the thymus lacks MHC class II molecules, CD4 T cells cannot be positively selected and few develop. The antigenpresenting cells in these individuals also lack MHC class II molecules and so the few CD4 T cells that do develop cannot be stimulated by antigen. MHC class I expression is normal, and CD8 T cells develop normally. However, such people suffer from severe immunodeficiency, illustrating the central importance of CD4 T cells in adaptive immunity to most pathogens.

1	MHC class II deficiency is caused not by mutations in the MHC genes themselves but by mutations in one of several genes encoding generegulatory proteins that are required for the transcriptional activation of MHC class II genes. Four complementing gene defects (known as groups A, B, C, and D) have been defined in patients who fail to express MHC class II molecules, indicating that the products of at least four different genes are required for the normal expression of these proteins. Genes corresponding to each complementation group have been identified: the MHC class II transactivator, or CIITA, is mutated in group A, and the genes RFXANK, RFX5, and RFXAP are mutated in groups B, C, and D, respectively (see Fig. 13.2). These last three encode proteins that are components of a multimeric complex, RFX, which is involved in the control of gene transcription. RFX binds a DNA sequence named an Xbox, which is present in the promoter region of all MHC class II genes.

1	A more limited immunodeficiency, associated with chronic respiratory bacterial infections and skin ulceration with vasculitis, has been observed in a small number of patients who have almost no cellsurface MHC class I molecules—a condition known as MHC class I deficiency. In contrast to those with MHC class II deficiency, affected individuals have normal levels of mRNA encoding MHC class I molecules and normal production of MHC class I proteins, but very few of the proteins reach the cell surface. This condition is due either to mutations in TAP1 or TAP2, which encode the subunits of the peptide transporter responsible for transporting peptides generated in the cytosol into the endoplasmic reticulum, where they are loaded into nascent MHC I molecules, or to mutations in TAPBP, which encodes tapasin, another component of the peptide transporter complex (see Section 64). Although the reduction in MHC class I molecules on the surface of thymic epithelial cells results in reduced numbers

1	tapasin, another component of the peptide transporter complex (see Section 64). Although the reduction in MHC class I molecules on the surface of thymic epithelial cells results in reduced numbers of CD8 T cells (see Fig. 13.2), people with MHC class I deficiency are not abnormally susceptible to viral infections. This is surprising given the key role of MHC class I presentation and of cytotoxic CD8 T cells in combating viral infections. There is, however, evidence for TAPindependent pathways for the presentation of certain peptides by MHC class I molecules, and the clinical phenotype of TAP1and TAP2deficient patients indicates that these pathways can compensate to allow sufficient development and function of CD8 T cells to control viruses.

1	Some defects in thymic cells lead to a phenotype with other effects besides those of immunodeficiency. The gene AIRE encodes a transcription factor that enables thymic epithelial cells to express many self proteins and so to mediate efficient negative selection. Defects in AIRE lead to a complex syndrome called APECED (autoimmune polyendocrinopathycandidiasisectodermal dystrophy), which is characterized by autoimmunity, developmental defects, and immunodeficiency (see Section 823, and Chapter 15). in antibody production that cause an inability to clear extracellular bacteria and some viruses.

1	In addition to inherited defects in proteins that are crucial to both Tcell and Bcell development, such as RAG1 and RAG2, defects that are specific to Bcell development have also been identified (see Fig. 13.2). Patients with these defects are characterized by an inability to cope with extracellular bacteria and some viruses whose efficient clearance requires specific antibodies. Pyogenic bacteria, such as staphylococci and streptococci, have polysaccharide capsules that are not directly recognized by the receptors on macrophages and neutrophils that stimulate phagocytosis. The bacteria escape elimination by the innate immune response and are successful extracellular pathogens, but can be cleared by an adaptive immune response. Opsonization by antibody and complement enables phagocytes to ingest and destroy the bacteria (see Section 1022). The principal effect of deficiencies in antibody production is therefore a failure to control infections by pyogenic bacteria. Susceptibility to

1	to ingest and destroy the bacteria (see Section 1022). The principal effect of deficiencies in antibody production is therefore a failure to control infections by pyogenic bacteria. Susceptibility to some viral infections, notably those caused by enteroviruses, is also increased because of the importance of antibodies in neutralizing viruses that enter the body through the gut.

1	The first description of an immunodeficiency disease was Ogden C. Bruton’s account, in 1952, of the failure of a male child to produce antibody. Because inheritance of this condition is Xlinked and is characterized by the absence of immunoglobulin in the serum (agammaglobulinemia), it was called Bruton’s Xlinked agammaglobulinemia (XLA) (see Fig. 13.2). Since then, autosomal recessive variants of agammaglobulinemia have been described. Infants with these diseases are usually identified as a result of recurrent infections with pyogenic bacteria, such as Streptococcus pneumoniae, and enteroviruses. In this regard, it should be noted that normal infants have a transient deficiency in immunoglobulin production in the first 3–12 months of life. The newborn infant has antibody levels comparable to those of the mother because of the transplacental transport of maternal IgG (see Section 1017). As this IgG is catabolized, antibody levels gradually decrease until the infant begins to produce

1	to those of the mother because of the transplacental transport of maternal IgG (see Section 1017). As this IgG is catabolized, antibody levels gradually decrease until the infant begins to produce significant amounts of its own IgG at about 6 months (Fig. 13.3). Thus, IgG levels are quite low between the ages of 3 months and 1 year. This can lead to a period of heightened susceptibility to infection, especially in premature babies, who begin with lower levels of maternal IgG and also reach immune competence later after birth. Because of the transient protection afforded

1	Fig. 13.3 Immunoglobulin levels in newborn infants fall to low levels at about 6 months of age. Babies are born with high levels of maternal igG, which is actively transported across the placenta from the mother during gestation. after birth, the production of igM starts almost immediately; the production of igG, however, does not begin for about 6 months, during which time the total level of igG falls as the maternally acquired igG is catabolized. thus, igG levels are low from about the age of 3 months to 1 year, which can lead to susceptibility to disease. newborn infants by maternal antibodies, XLA is typically detected several months after birth, when maternal antibody levels in the infant have declined.

1	The defective gene in XLA encodes a protein tyrosine kinase called BTK (Bruton’s tyrosine kinase), which is a member of the Tec family of kinases that transduce signals through the preBcell receptor (preBCR; see Section 720). As discussed in Section 83, the preBcell receptor is composed of successfully rearranged μ heavy chains complexed with the surrogate light chain composed of λ5 and VpreB, and with the signaltransducing subunits Igα and Igβ. Stimulation of the preBcellreceptor recruits cytoplasmic proteins, including BTK, which convey signals required for the proliferation and differentiation of preB cells. In the absence of BTK function, Bcell maturation is largely arrested at the preBcell stage (see Fig. 13.2; see also Section 83), resulting in profound Bcell deficiency and agammaglobulinemia. Some B cells do mature, however, perhaps as a result of compensation by other Tec kinases.

1	During embryonic development, females randomly inactivate one of their two X chromosomes. Because BTK is required for Blymphocyte development, only cells in which the normal allele of BTK is active develop into mature B cells. Thus, in all B cells in female carriers of a mutant BTK gene, the active X chromosome is the normal one and the abnormal X chromosome is inactivated. This fact allowed female carriers of XLA to be identified even before the nature of the BTK protein was known. In contrast, the active X chromosomes in the T cells and macrophages of carriers are an equal mixture of normal and BTK mutant X chromosomes. Nonrandom Xinactivation only in B cells shows conclusively that the BTK gene is required for the development of B cells but not the other cell types, and that BTK must act in the B cells themselves rather than in stromal or other cells required for Bcell development (Fig. 13.4).

1	Autosomal recessive deficiencies in other components of the preBCR also block early Bcell development and cause severe Bcell deficiency and congenital agammaglobulinemia similar to that of XLA. These disorders are much rarer than XLA, and include mutations in the genes that encode the μ heavy chain (IGHM), which is the second most common cause of agammaglobulinemia; λ5 (IGLL1); and Igα (CD79A) and Igβ (CD79B) (see Fig. 13.2). Mutations that cripple the Bcell receptor signaling adaptor, Bcell linker protein (encoded by BLNK), also cause the arrest of early Bcell development that results in selective Bcell deficiency.

1	Patients with pure Bcell defects resist many pathogens other than pyogenic bacteria. Fortunately, the latter can be suppressed with antibiotics and with monthly infusions of human immunoglobulin collected from a large pool of donors. Because there are antibodies against many common pathogens in this pooled immunoglobulin, it serves as a fairly successful shield against infection. T-cell activation and function that lead to abnormal antibody responses.

1	T-cell activation and function that lead to abnormal antibody responses. After their development in the bone marrow or thymus, B and T cells require antigendriven activation and differentiation to mount effective immune responses. Analogous to defects in early Tcell development, defects in Tcell activation and differentiation that occur after thymic selection have an impact on both cellmediated immunity and antibody responses (Fig. 13.5). Defects specific to the activation and differentiation of B cells can impair their ability to undergo class switching to IgG, IgA, and IgE while leaving cellmediated immunity largely intact. Depending on where in the process of T or Bcell differentiation these defects occur, the characteristics of the immune deficiency that results can be either profound or relatively circumscribed.

1	A common feature of patients with defects that affect Bcell class switching is hyperIgM syndrome (see Fig. 13.5). These patients have normal Band Tcell development and normal or high serum levels of IgM, but make very limited antibody responses against antigens that require Tcell help. Thus immunoglobulin isotypes other than IgM and IgD are produced only in trace amounts. This renders these patients highly susceptible to infection with extracellular pathogens. Several causes for hyperIgM syndromes have been distinguished, and these have helped to elucidate the pathways that are essential for normal classswitch recombination and somatic hypermutation in B cells. Defects have been found in both Tcell helper function and in the B cells themselves.

1	in X-linked agammaglobulinemia (Xla), a protein tyrosine kinase of the tec family called BtK, which is encoded on the X chromosome, is defective. in normal individuals, B-cell development proceeds through a stage in which the pre-B-cell receptor, consisting of μλ5:VpreB (see Section 8-3), transduces a signal via BtK, triggering further B-cell development. in males with Xla, no signal can be transduced and, although the pre-B-cell receptor is expressed, the B cells develop no further. in female mammals, including humans, one of the two X chromosomes in each cell is permanently inactivated early in development. Because the choice of which chromosome to inactivate is random, half of the pre-B cells in a carrier female will have inactivated the chromosome with the wild-type BTK gene, meaning that they can express only the defective BTK gene and cannot develop further. therefore, in the carrier, mature B cells always have the nondefective X chromosome active. this is in sharp contrast to

1	that they can express only the defective BTK gene and cannot develop further. therefore, in the carrier, mature B cells always have the nondefective X chromosome active. this is in sharp contrast to all other cell types, which have the nondefective X chromosome active in only half of their cells. nonrandom X-chromosome inactivation in a particular cell lineage is a clear indication that the product of the X-linked gene is required for the development of cells of that lineage. it is also sometimes possible to identify the stage at which the gene product is required, by detecting the point in development at which X-chromosome inactivation develops bias. using this kind of analysis, one can identify carriers of X-linked traits such as Xla without needing to know the nature of the mutant gene.

1	Fig. 13.5 Defects in T-cell and B-cell activation and differentiation cause immunodeficiencies. the pathways leading to activation and differentiation of naive t cells and B cells are shown here. the protein products of genes known to be mutated in the relevant human immunodeficiency diseases are indicated in red boxes. BCR, B-cell receptor; CViDs, common variable immunodeficiencies; tCR, t-cell receptor. note that the defect in cytoskeletal function in Wiskott–aldrich syndrome (WaS) affects immune-cell function at many steps in this schema, and is not included in the figure for the sake of clarity.

1	Fig. 13.6 Patients with CD40 ligand deficiency are unable to activate their B cells fully. lymphoid tissues in patients with CD40 ligand deficiency, which manifests as a hyper-igM syndrome, are devoid of germinal centers (top panel), unlike a normal lymph node (bottom panel). B-cell activation by t cells is required both for isotype switching and for the formation of germinal centers, where extensive B-cell proliferation takes place. Photographs courtesy of R. Geha and a. Perez-atayde. TCRCD4 activated CD4 T cell mature B cell plasma cells AID, UNG, TACI, CD19, ICOS resting macrophage activated macrophage lgA lgE lgG IL-12, intracellular killing CD40L, NEMO CD40 mutated genes not known BCR IgD B-cell intrinsic hyper-IgM syndromes or CVIDs IgA defciency X-linked hyper-IgM syndromes hyper-IgM syndromes

1	The most common form of hyperIgM syndrome is Xlinked hyperIgM syndrome, or CD40 ligand deficiency, which is caused by mutations in the gene encoding CD40 ligand (CD154) (see Fig. 13.5). CD40 ligand is normally expressed on activated T cells, enabling them to engage the CD40 protein on antigenpresenting cells, including B cells, dendritic cells, and macrophages (see Section 104). In males with CD40 ligand deficiency, B cells are normal, but in the absence of engagement of CD40, their B cells do not undergo isotype switching or initiate the formation of germinal centers (Fig. 13.6). These patients therefore have severe reductions in circulating levels of all antibody isotypes except IgM and are highly susceptible to infections by pyogenic extracellular bacteria.

1	Because CD40 signaling is also required for the activation of dendritic cells and macrophages for optimal production of IL12, which is important for the production of IFNγ by TH1 cells and NK cells, patients with CD40 ligand deficiency also have defects in type 1 immunity and thus manifest a form of combined immunodeficiency. Inadequate crosstalk between T cells and dendritic cells via CD40L–CD40 interaction can lead to lower levels of costimulatory molecules on dendritic cells, thus impairing their ability to stimulate naive T cells (see Section 917). These patients are therefore susceptible to infections by extracellular pathogens that require classswitched antibodies, such as pyogenic bacteria, but also have defects in the clearance of intracellular pathogens, such as mycobacteria, and are particularly prone to opportunistic infections by Pneumocystis jirovecii, which is normally killed by activated macrophages.

1	A similar syndrome has been identified in patients with mutations in two other genes. Not unexpectedly, one is the gene encoding CD40, mutations in which have been found in a few patients with an autosomal recessive variant of hyperIgM syndrome (see Fig. 13.5). In another form of Xlinked hyperIgM syndrome, known as NEMO deficiency, mutations occur in the gene encoding the protein NEMO (‘NFκB essential modulator’; also known as IKKγ, a subunit of the kinase IKK), which is an essential component of the intracellular signaling pathway downstream of CD40 that leads to activation of the transcription factor NFκB (see Fig. 3.15). This group of hyperIgM syndromes shows that mutations at different points in the CD40L–CD40 signaling pathway result in a similar combined immunodeficiency syndrome. In view of the role of NFκB signaling in many other pathways, NEMO deficiency results in additional immune dysfunction beyond its impairment of Bcell class switching (see Section 1315), as well as

1	In view of the role of NFκB signaling in many other pathways, NEMO deficiency results in additional immune dysfunction beyond its impairment of Bcell class switching (see Section 1315), as well as nonimmune manifestations, including abnormalities of the skin.

1	Other variants of hyperIgM syndrome are due to intrinsic defects in the process of Bcell classswitch recombination. Patients having these defects are susceptible to severe extracellular bacterial infections, but because Tcell differentiation and function are spared, they do not show increased susceptibility to intracellular pathogens or opportunistic agents such as P. jirovecii. One classswitching defect is due to mutations in the gene for activationinduced cytidine deaminase (AID), which is required for both somatic hypermutation and class switching (see Section 107). Patients with autosomally inherited defects in the AID gene (AICDA) fail to switch antibody isotype and also have greatly reduced somatic hypermutation (see Fig. 13.5). Immature B cells accumulate in abnormal germinal centers, causing enlargement of the lymph nodes and spleen. Another variant of Bcellintrinsic hyperIgM syndrome was identified recently in a small number of patients with an autosomal recessive defect in

1	causing enlargement of the lymph nodes and spleen. Another variant of Bcellintrinsic hyperIgM syndrome was identified recently in a small number of patients with an autosomal recessive defect in the DNA repair enzyme uracilDNA glycosylase (UNG; see Section 1010), which is also involved in class switching; these patients have normal AID function and normal somatic hypermutation, but defective class switching.

1	Other examples of predominantly antibody deficiency include the most common forms of primary immunodeficiency, referred to as common variable immunodeficiencies (CVIDs). CVIDs are a clinically and genetically heterogeneous group of disorders that typically go undiagnosed until late childhood or adulthood, because the immune deficiency is relatively mild. Unlike other causes of immunoglobulin deficiency, patients with CVID can have defects in immunoglobulin production that are limited to one or more isotypes (see Fig. 13.5). IgA deficiency, the most common primary immunodeficiency, exists in both sporadic and familial forms, and both autosomal recessive and autosomal dominant inheritance have been described. The etiology of IgA deficiency in most patients is not understood, and these patients are asymptomatic. In IgAdeficient patients who do develop recurrent infections, an associated defect in one of the IgG subclasses is often found.

1	A small minority of CVID patients have mutations in the transmembrane protein TACI (TNFlike receptor transmembrane activator and CAML interactor), which is encoded by the gene TNFRSF13B. TACI is the receptor for the cytokines BAFF and APRIL, which are produced by T cells, dendritic cells, and macrophages, and which can provide costimulatory and survival signals for Bcell activation and class switching (see Section 103). Other patients with selective deficiencies in IgG subclasses have also been described. Bcell numbers are typically normal in these patients, but serum levels of the affected immunoglobulin isotype are depressed. Although some of these patients have recurrent bacterial infections, as in IgA deficiency, many are asymptomatic. CVID patients with other defects that affect immunoglobulin class switching have been identified. Included in this group are patients with inherited defects in CD19, which is a component of the Bcell coreceptor (see Fig. 13.5). A genetic defect that

1	class switching have been identified. Included in this group are patients with inherited defects in CD19, which is a component of the Bcell coreceptor (see Fig. 13.5). A genetic defect that has been linked to a small percentage of people with CVID is deficiency of the costimulatory molecule ICOS. As described in Section 917, ICOS is upregulated on T cells when they are activated. The effects of ICOS deficiency have confirmed its essential role in Tcell help for the later stages of Bcell differentiation, including class switching and the formation of memory cells.

1	The final immunodeficiency to be considered in this section is hyperIgE syndrome (HIES), also called Job’s syndrome. This disease is characterized by recurrent skin and pulmonary infections caused by pyogenic bacteria, chronic mucocutaneous candidiasis (noninvasive fungal infection of the skin and mucosal surfaces), very high serum concentrations of IgE, and chronic eczematous dermatitis or skin rash. HIES is inherited in an autosomal recessive or dominant pattern, with the latter manifesting skeletal and dental abnormalities not found in the recessive variant. The inherited defect in the autosomal dominant variant of HIES is in the transcription factor STAT3, which is activated downstream of several cytokine receptors, including those for IL6, IL22, and IL23, and which is central to the differentiation of TH17 cells and activation of ILC3 cells. STAT3 signaling activated by IL6 and IL22 is also important for enhanced antimicrobial resistance of epithelial cells of the skin and

1	the differentiation of TH17 cells and activation of ILC3 cells. STAT3 signaling activated by IL6 and IL22 is also important for enhanced antimicrobial resistance of epithelial cells of the skin and mucosal barriers. Because differentiation of TH17 cells is deficient in these patients, the recruitment of neutrophils normally orchestrated by the TH17 response is also defective, as is production of IL22, an important cytokine in activating epithelial cell production of antimicrobial peptides. This is thought to underlie the impaired defense against extracellular bacteria and fungi at barrier epithelia, such as the skin and mucosae. The cause of the elevated IgE is not understood, but it might be due to an abnormal accentuation of skin and mucosal TH2 responses as a result of TH17 deficiency. In an autosomal recessive variant of HIES, the mutation is in the gene that encodes the protein DOCK8 (dedicator of cytokinesis 8), the function of which in immune cells is poorly characterized.

1	In an autosomal recessive variant of HIES, the mutation is in the gene that encodes the protein DOCK8 (dedicator of cytokinesis 8), the function of which in immune cells is poorly characterized. However, because DOCK8 is thought to play a broader role in Tcell function, as well as NKcell function, this variant of HIES is distinguished from that caused by STAT3 defects by the additional occurrence of opportunistic infections and recurrent cutaneous viral infections (for example, herpes simplex), as well as allergic and autoimmune manifestations.

1	agents are pinpointed by genetic deficiencies of cytokine pathways central to type 1/TH1 and type 3/TH17 responses.

1	Inherited defects in cytokines that are involved in the development and function of different effector Tcell subsets have been defined, as have defects in the receptors or the signaling pathways through which they act. In contrast to the Tcell immunodeficiencies considered above, here we consider those deficiencies that do not have major defects in antibody production. A small number of families have been discovered with individuals who suffer from persistent and sometimes fatal attacks by intracellular pathogens normally restrained by type 1 immunity, especially Mycobacterium, Salmonella, and Listeria species. These microbes are specialized for survival within macrophages, and their eradication requires enhanced microbicidal activities induced by IFNγ produced by type 1 cells: NK cells, ILC1 cells, and TH1 cells (see Section 112). Accordingly, susceptibility to these infections is conferred by a variety of inherited mutations that impair or abolish the function of IL12 or IFNγ, key

1	ILC1 cells, and TH1 cells (see Section 112). Accordingly, susceptibility to these infections is conferred by a variety of inherited mutations that impair or abolish the function of IL12 or IFNγ, key cytokines in the development and functions of type 1 cells (Fig. 13.7). Patients with mutations in the genes encoding the p40 subunit of IL12 (IL12B), the IL12 receptor β1 chain (IL12RB1), and the two subunits (R1 and R2) of the IFNγ receptor (IFNGR1 and IFNGR2) have been identified. Although affected individuals have heightened susceptibility to the more virulent M. tuberculosis, they suffer more frequently from the nontuberculous, or atypical, strains of mycobacteria, such as Mycobacterium avium, likely due to the greater prevalence of atypical strains in the environment. They may also develop disseminated infection after vaccination with Mycobacterium bovis bacillus Calmette–Guérin (BCG), the strain of M. bovis that is used as a live vaccine against M. tuberculosis. Because the IL12 p40

1	disseminated infection after vaccination with Mycobacterium bovis bacillus Calmette–Guérin (BCG), the strain of M. bovis that is used as a live vaccine against M. tuberculosis. Because the IL12 p40 subunit is shared by IL12 and IL23, IL12 p40 deficiency results in a broader infectious disease risk due to defective type 1 and type 3 (TH17) functions (see Fig. 13.7).

1	Fig. 13.7 Inherited defects in effector cytokine pathways also, because Stat1 is activated is by the receptors of type ii that impair type 1/TH1 and type 3/TH17 immunity. Shown are interferon (iFn-γ) and type i interferon (iFn-α and iFn-β, not shown), pathways in il-12, il-23, and iFn-γ signaling for which inherited defects in Stat1 result in impaired antibacterial and antiviral immune defects have been described. note that defects in il-12p40 defense, whereas deficiency in either of the iFn-γ receptor subunits (p40) and il-12Rβ1 result in impaired function of both ilC1 cells/ (iFn-γR1 or iFn-γR2) primarily results in impaired defense against nK cells/tH1 cells and ilC3/tH17 cells because these subunits intracellular bacteria. are shared, respectively, by il-12 and il-23 and their receptors. Similarly, deficiency of the IL12Rβ1 chain, which is shared by the IL12 and IL23 receptors, results in broader susceptibilities than the deficiencies of IFNγ or its receptor.

1	Similarly, deficiency of the IL12Rβ1 chain, which is shared by the IL12 and IL23 receptors, results in broader susceptibilities than the deficiencies of IFNγ or its receptor. Autosomal recessive lossoffunction mutations in STAT1 impair IFNγ receptor signaling and are also associated with increased susceptibility to infections by mycobacterial and other intracellular bacteria (see Fig. 13.7). However, due to its shared function in IFNα receptor signaling in response to IFNα and IFNβ (type I interferons), patients with STAT1 defects are also susceptible to viral infections. Interestingly, patients with partial loss of function of STAT1 have been identified who are susceptible to mycobacterial infections, but not viral infections, suggesting a more stringent requirement of STAT1 in protection against the former.

1	In addition to the TH17related defects described above for STAT3deficient HIES (see Section 139), other defects in cytokinemediated functions of this pathway have been identified that lack the hyperIgE component (Fig. 13.8). Whereas heightened susceptibility to intracellular bacteria is a feature common to immunodeficiencies that impair type 1 immunity, heightened susceptibility to infections by Candida spp. and pyogenic bacteria, particularly

1	C. albicans and S. aureus, respectively, is characteristic of these type 3 deficiencies. This reflects the specialized function of TH17 and ILC3 cells in barrier defense against fungi and extracellular bacteria. Inherited deficiencies in IL17F and IL17RA, the shared receptor component for homo and heterodimeric IL17F–IL17A ligands, confer susceptibility to these infectious agents and thus identify a key role for IL17 cytokines in host defense against them. A similar susceptibility to chronic mucocutaneous candidiasis and pyogenic bacteria has been found in patients with autosomal dominant, gainoffunction mutations in STAT1. Because TH17 cell development is impaired by STAT1 signaling downstream of several cytokine receptors (for example, type I and type II IFN receptors), individuals with these mutations have impaired type 3 defenses. Note that this is in contrast to patients with lossoffunction STAT1 mutations, who are predisposed to intracellular bacterial infections due to

1	with these mutations have impaired type 3 defenses. Note that this is in contrast to patients with lossoffunction STAT1 mutations, who are predisposed to intracellular bacterial infections due to defective type 1 immunity.

1	In addition to inherited defects in effector cytokine genes, some forms of immunodeficiency result in the production of neutralizing autoantibodies against these cytokines. This results in infectious risks similar to those caused by primary cytokine deficiencies. Most patients with APECED syndrome (caused by defects in the AIRE gene; see Section 137) develop chronic mucocutaneous candidiasis that is due to the development of autoantibodies against IL17A, IL17F, and/or IL22. Patients with neutralizing autoantibodies against IFNγ who have impaired protection against atypical mycobacteria have also been reported, although the basis for this is unknown. 13-11 Inherited defects in the cytolytic pathway of lymphocytes can cause uncontrolled lymphoproliferation and inflammatory responses to viral infections.

1	Cytolytic granules are formed from components of late endosomes and lysosomes. Once formed, there are multiple steps in the exocytosis of cytolytic granules from cytotoxic cells and their delivery to target cells. The importance of the cytolytic pathway in immune regulation is highlighted by inherited defects that impair key steps in either the formation or the exocytosis of cytolytic granules (Fig. 13.9). These result in a severe and often fatal condition, known as hemophagocytic lymphohistiocytic (HLH) syndrome, which manifests as uncontrolled activation and expansion of CD8 T lymphocytes and macrophages that infiltrate multiple organs, causing tissue necrosis and organ failure. The hyperactive immune response is thought to reflect the inability of cytotoxic cells to destroy infected targets, and possibly themselves, following an initiating viral infection, particularly by a herpes family virus such as Epstein–Barr virus (EBV). In this regard it is notable that despite the impaired

1	and possibly themselves, following an initiating viral infection, particularly by a herpes family virus such as Epstein–Barr virus (EBV). In this regard it is notable that despite the impaired release of cytolytic granules, the release of IFNγ by CTLs and NK cells of patients with this disorder is not typically impaired, which contributes to the heightened activity of macrophages and associated inflammatory disorder caused by an increased release of proinflammatory cytokines such TNF, IL6, and macrophage colonystimulating factor (MCSF). The activated macrophages phagocytose blood cells, including erythrocytes and leukocytes, which gives the syndrome its name.

1	There are multiple autosomal recessive variants of HLH, also referred to as familial hemophagocytic lymphohistiocytosis (FHL), that are distinguished by the protein in the cytolytic pathway that is affected (see Fig. 13.9). Examples include inherited deficiencies of the cytolytic granule protein perforin, which is required for pore formation in the target cell (and when defective is associated with the syndrome FHL2); of the priming protein, Munc134 (FHL3); of syntaxin 11 (FHL4), a member of the SNARE family of proteins that mediate membrane fusion; and of Munc182 (FHL5), a protein involved in the reorganization of SNARE proteins to activate the fusion process. Because components of the biogenesis and exocytosis of cytolytic granules are shared with other secretory vesicles, such as lysosomes, additional immune defects can occur in affected individuals, as can nonimmune defects. In particular, a subset of the immunodeficiencies that affect cytolytic granule function are also

1	as lysosomes, additional immune defects can occur in affected individuals, as can nonimmune defects. In particular, a subset of the immunodeficiencies that affect cytolytic granule function are also characterized by partial loss of skin pigmentation. This is due to defects in vesicular transport proteins that are also required for the formation or exocytosis of melanosomes, organelles that store the skin pigment melanin in melanocytes. Examples of these immunodeficiencies include Chediak–Higashi syndrome, caused by mutations in a protein, CHS1, which regulates lysosomal trafficking, and Griscelli syndrome, caused by mutations in a small GTPase, RAB27a (see Fig. 13.9), which is integral to the tethering of certain vesicles, including cytolytic granules, to cytoskeletal structures to enable their intracellular trafficking.

1	In patients with Chediak–Higashi syndrome, abnormal giant lysosomes and granules accumulate in T lymphocytes, myeloid cells, platelets, and melanocytes. The hair is typically a metallic silver color, vision is poor because of abnormalities in retinal pigment cells, and platelet dysfunction causes increased bleeding. Because the phagocytes of these patients have defective phagolysosomal fusion, the patients experience defective killing of intracellular and extracellular pathogens in addition to defective cytolytic activity of CTLs and NK cells. Affected children therefore suffer early in life from severe, recurrent infections by a range of bacteria and fungi. This is typically followed by development of hemophagocytic lymphohistiocytosis, often triggered by viral infection such as EBV, leading to an accelerated phase of the disease. Three variants of Griscelli syndrome have been identified, each caused by a distinct gene defect: in the type 2 variant (RAB27A mutation), the defect

1	leading to an accelerated phase of the disease. Three variants of Griscelli syndrome have been identified, each caused by a distinct gene defect: in the type 2 variant (RAB27A mutation), the defect results in both immunodeficiency and pigment abnormalities; in types 1 and 3, only the pigment abnormalities are found. Although the immune defects in children

1	Fig. 13.9 Defects in components involved in the exocytosis of cytotoxic granules cause familial hemophagocytic lymphohistiocytosis (FHL) syndromes. Following antigen recognition, there is polarization of perforin-containing cytotoxic granules of the Ctl toward the target cell at the site of immunological synapse formation. Cytotoxic granules are transported along microtubules to the plasma membrane, where they dock through a RaB27a-dependent interaction. Docked vesicles are primed by Munc13-4-mediated triggering of a conformational change in syntaxin 11, which is part of a large SnaRe (soluble N-ethylmaleimide-sensitive factor accessory protein receptor) complex connecting the docked vesicle with the plasma membrane. through the actions of Munc18-2, a fusion reaction is initiated via the syntaxin 11-containing SnaRe complex that releases the contents of the cytotoxic granules into the synapse-bounded intercellular space, allowing perforin-mediated pore formation in the target-cell

1	syntaxin 11-containing SnaRe complex that releases the contents of the cytotoxic granules into the synapse-bounded intercellular space, allowing perforin-mediated pore formation in the target-cell plasma membrane. the sites in the exocytic pathway that are affected by inherited defects in each of the proteins highlighted in red are indicated, as is the associated familial hemophagocytic lymphohistiocytosis syndrome.

1	with type 2 Griscelli syndrome have much in common with those of Chediak– Higashi syndrome, their myeloid cells lack the giant intracellular granules that are characteristic of the latter. 13-12 X-linked lymphoproliferative syndrome is associated with fatal infection by Epstein–Barr virus and with the development of lymphomas.

1	In some primary immunodeficiencies, there is unique susceptibility to a single pathogen. Such is the case for two rare Xlinked immunodeficiencies with a similar lymphoproliferative defect that results from vulnerability to a herpes family virus, the Epstein–Barr virus (EBV), albeit via independent mechanisms. EBV specifically infects B cells and typically causes a selflimiting infection in normal individuals due to active control of the virus by the actions of NK cells, NKT cells, and cytotoxic T cells with specificity for B cells expressing EBV antigens. Following the development of immunity to EBV, the virus is not completely cleared, but is maintained in a latent state in infected B cells (see Section 1324). In the presence of certain types of immunodeficiency, however, this control can break down, resulting in overwhelming EBV infection (severe infectious mononucleosis) that is accompanied by unrestrained proliferation of EBVinfected B cells and cytotoxic T cells,

1	this control can break down, resulting in overwhelming EBV infection (severe infectious mononucleosis) that is accompanied by unrestrained proliferation of EBVinfected B cells and cytotoxic T cells, hypogammaglobulinemia (low levels of circulating immunoglobulins), and the potential for the development of lethal, nonHodgkin’s Bcell lymphomas. These occur in the rare immunodeficiency Xlinked lymphoproliferative (XLP) syndrome. XLP syndrome results from mutations in one of two Xlinked genes: the SH2 domaincontaining gene 1A (SH2D1A), which encodes SAP [signaling lymphocyte activation molecule (SLAM)associated protein], or the Xlinked inhibitor of apoptosis gene (XIAP).

1	In XLP1, which accounts for approximately 80% of patients with this syndrome, the defect in SAP results in defective coupling of immunecell receptors of the SLAM family with the Srcfamily tyrosine kinase Fyn in T cells, NKT cells, and NK cells (Fig. 13.10). SLAM family members interact through homotypic or heterotypic binding to modulate the outcome of interactions between T cells and antigenpresenting cells and between NK cells and their target cells. In the absence of SAP, ineffective EBVspecific cytotoxic Tcell and NKcell responses are made, and there is severe deficiency of NKT cells, indicating that SAP has a nonredundant role in the control of EBV infection and the development of NKT cells. There is unchecked proliferation of EBVreactive cytotoxic T cells and NK cells that results in systemic macrophage activation, inflammation, and hemophagocytic features similar to those that occur in immunodeficiencies caused by defects in the cytolytic pathway (see

1	Fig.13.10 X-linked lymphoproliferative disease (XLP) is caused by inherited defects in SAP and XIAP, resulting in abnormal SLAM and TNF family receptor signaling, respectively. SlaM is an immune receptor family, members of which are expressed by t cells, B cells, natural killer (nK) cells, dendritic cells, and macrophages. Signaling is initiated via homotypic or heterotypic interactions between family members. SlaM signaling recruits the Src homology 2 (SH2) domain-containing factor SaP, which recognizes tyrosine-based motifs in the cytoplasmic domain of SlaM to activate the Src-related tyrosine kinase Fyn (left upper panel). Fyn then phosphorylates additional tyrosine residues of SlaM to recruit additional signaling components. Mutation in SaP in patients with XlP1 (right upper panel) disrupts activation of Fyn and SlaM signaling, thereby impairing tand nK-cell cytotoxicity, which results in severe epstein–Barr virus (eBV) infections and lymphoma. Defective SlaM signaling also

1	disrupts activation of Fyn and SlaM signaling, thereby impairing tand nK-cell cytotoxicity, which results in severe epstein–Barr virus (eBV) infections and lymphoma. Defective SlaM signaling also impairs upregulation of inducible t-cell co-stimulator (iCOS) expression in tFH cells, which results in impaired antibody responses. activation of apoptosis-inducing caspases by tnF family receptors, such as Fas, is normally inhibited by XiaP (lower panel). XiaP interacts with both initiator caspases (8 and 9) and executioner caspases (3 and 7) through its baculoviral inhibitory repeat (BiR) domain to inhibit their actions. in XlP2 patients, XiaP is defective, resulting in abnormal regulation of caspase activation that leads to the complex clinical phenotype that includes lymphoproliferation and defective control of eBV.

1	Section 1311). Moreover, defective SLAM signaling between TFH cells and B cells of XLP1 patients causes impaired Tdependent antibody responses and hypogammaglobulinemia. Defects in the XIAP protein, which normally binds the TNFreceptorassociated factors TRAF1 and TRAF2 and inhibits the activation of apoptosisinducing caspases (see Section 723), lead to a similar Xlinked syndrome called XLP2 (see Fig. 13.10). XIAP deficiency results in the enhanced apoptosis and turnover of activated T cells and NK cells. Paradoxically, this leads to a lymphoproliferative phenotype similar to XLP1, the basis of which is not well understood. As in XLP1, there is also severe depletion of NKT cells, indicating that, like SAP, XIAP is required for the normal maintenance of these cells. As in XLP1, there is also defective control of EBV infection in XLP2, although this is less prominent. The exact reason for the impaired suppression of EBV latency in these immunodeficiencies remains to be defined.

1	13-13 Immunodeficiency is caused by inherited defects in the development of dendritic cells. Our understanding of the diversity and function of dendritic cells has been advanced from studies of mice with genetargeted deletion of transcription factors that result in the loss of subsets of these cells, and from the defects in protection against specific pathogens that result as a consequence of the loss of these subsets. In humans, where study of the development and function of dendritic cells is more challenging, the identification of primary immune deficiencies that result from defects in genes that encode the transcription factors GATA2 and IRF8 has begun to provide insights into the relative roles of these cells in different species.

1	An autosomal dominant mutation of GATA2 has been identified in the largest group of patients with an inherited deficiency of dendritic cells. Affected individuals develop a progressive loss of all subsets of dendritic cells (conventional and plasmacytoid) and monocytes, as well as reduced numbers of B and NK lymphoid cells, a condition that has been termed DCML deficiency. Although Tcell numbers are normal in these patients, their function becomes impaired as dendritic cells are lost. The loss of products of several hematopoietic lineages, but not all, suggests redundancy of the function of GATA2 in unaffected lineages. The basis for the progressive decline in products of the affected lineages is unknown, but is thought to reflect a role for GATA2 in maintaining stemcell progenitors that seed these populations. Given the deficiency of all dendritic and monocyte cells, the immune defects in these individuals are diverse, as are pathogen susceptibilities. These patients also have a

1	seed these populations. Given the deficiency of all dendritic and monocyte cells, the immune defects in these individuals are diverse, as are pathogen susceptibilities. These patients also have a substantial risk of hematologic malignancies.

1	Two inherited defects in interferon regulatory factor 8 (IRF8) are the first to be described that are associated with specific defects in the development of dendritic cells. In both variants, the mutation is in the DNAbinding domain of the transcription factor. In an autosomal recessive form, there is loss of monocytes and all types of circulating dendritic cells; all conventional and plasmacytoid dendritic cells are absent. Because dendritic cells are the primary antigenpresenting cells for naive T cells, their deficiency results in impaired development of effector T cells, and patients with these defects are susceptible to a range of severe opportunistic infections early in life, including those caused by intracellular bacteria, viruses, and fungi. They also develop a striking increase in the number of circulating immature granulocytes; this is thought to be due to diversion of myeloid precursors down the granulocytic pathway in the absence of the monocyte–dendritic cell

1	increase in the number of circulating immature granulocytes; this is thought to be due to diversion of myeloid precursors down the granulocytic pathway in the absence of the monocyte–dendritic cell developmental pathway. In contrast, in patients with autosomal dominant inheritance of a dominantnegative mutant allele of IRF8, there is a less severe phenotype, one that is characterized by a more selective deficiency of the CD1cpositive subset of dendritic cells (thought to be the equivalent of the CD11bpositive subset of mouse

1	Fig. 13.11 Defects in complement components are associated with susceptibility to certain infections and the accumulation of immune complexes. Defects in the early components of the alternative pathway and in C3 lead to susceptibility to extracellular pathogens, particularly pyogenic bacteria. Defects in the early components of the classical pathway predominantly affect the processing of immune complexes (see Section 10-20) and the clearance of apoptotic cells, leading to immune-complex disease. Deficiency of mannose-binding lectin (MBl), the recognition molecule of the mannose-binding lectin pathway, is associated with bacterial infections, mainly in early childhood. Defects in the membrane-attack components are associated only with susceptibility to strains of Neisseria species, the causative agents of meningitis and gonorrhea, implying that the effector pathway is important chiefly in defense against these organisms.

1	dendritic cells). This results in heightened susceptibility to intracellular bacteria, particularly atypical Mycobacterium spp., without the myeloproliferative syndrome seen in patients with the autosomal recessive variant. and tissue damage.

1	The diseases discussed so far are mainly due to disturbances of the adaptive immune system. In the next few sections we look at some immunodeficiency diseases that affect cells and molecules of the innate immune system. We start with the complement system, which can be activated by any of three pathways that converge on the cleavage and activation of complement component C3, allowing it to bind covalently to pathogen surfaces, where it acts as an opsonin (discussed in Chapter 2). Not surprisingly, the spectrum of infections associated with complement deficiencies overlaps substantially with that seen in patients with deficiencies in antibody production. In particular, there is increased susceptibility to extracellular bacteria that require opsonization by antibody and/or complement for efficient clearance by phagocytes (Fig. 13.11). Defects in the activation of C3 by any of the three pathways, as well as defects in C3 itself, are associated with increased susceptibility to infection

1	clearance by phagocytes (Fig. 13.11). Defects in the activation of C3 by any of the three pathways, as well as defects in C3 itself, are associated with increased susceptibility to infection by a range of pyogenic bacteria, including S. pneumoniae, emphasizing the role of C3 as a central effector that promotes the phagocytosis and clearance of capsulated bacteria.

1	In contrast, defects in the membraneattack components of complement (C5C9) downstream of C3 activation have more limited effects, and result almost exclusively in susceptibility to Neisseria species. A similar susceptibility to Neisseria species is found in patients with defects in the alternative complement pathway components factor D and properdin, indicating that defense against these bacteria, which can survive intracellularly, is largely mediated via antibodyindependent extracellular lysis by the membraneattack complex. Data from largepopulation studies in Japan, where endemic N. meningitidis infection is rare, show that the risk each year of infection with this organism is approximately 1 in 2,000,000 to a normal person. This compares with a risk of 1 in 200 to a person in the same population with an inherited deficiency of one of the membraneattack complex proteins—a 10,000fold increase in risk.

1	Membrane-attack components C5 C6 C7 C8 C9 Deÿciency leads to infection with Neisseria spp. only C3b deposition C3 Deÿciency leads to infection with pyogenic bacteria and Neisseria spp. Sometimes immune-complex disease C3 convertase Deÿciency leads to immune-complex disease Deÿciency leads to infection with pyogenic bacteria and Neisseria spp. but no immune-complex disease Deÿciency of MBL leads to bacterial infections, mainly in childhood MBL MASP1 MASP2 C2 C4 C1 C2 C4 Factor D Factor P CLASSICAL PATHWAY ALTERNATIVE PATHWAY MBL PATHWAY

1	The early components of the classical complement pathway are particularly important for the elimination of immune complexes (discussed in Section 1020) and apoptotic cells, which can cause significant pathology in autoimmune diseases such as systemic lupus erythematosus. This aspect of inherited complement deficiency is discussed in Chapter 15. Deficiencies in mannosebinding lectin (MBL), which initiates complement activation independently of antibody (see Section 26), are relatively common (found in 5% of the population). MBL deficiency may be associated with a mild immunodeficiency that results in an increased incidence of bacterial infection in early childhood. A similar phenotype is found in patients with defects in the gene that encodes the MBLassociated serine protease2 (MASP2).

1	Another set of complementrelated diseases is caused by defects in complementcontrol proteins (Fig. 13.12). Deficiencies in decayaccelerating factor (DAF) or protectin (CD59), membraneassociated control proteins that protect the surfaces of the body’s cells from complement activation, lead to destruction of red blood cells, resulting in the disease paroxysmal nocturnal hemoglobinuria, as discussed in Section 216. Deficiencies in soluble complementregulatory proteins such as factor I and factor H have various outcomes. Homozygous factor I deficiency is a rare defect that results in uncontrolled activity of the alternative pathway C3 convertase, leading to a de facto C3 deficiency (see Section 216). Deficiencies in MCP, factor I, or factor H can also cause a condition known as atypical hemolytic–uremic syndrome, so called because it leads to lysis of red blood cells (hemolysis) and impaired kidney function (uremia).

1	A striking consequence of the loss of a complementregulatory protein is seen in patients with C1inhibitor defects, which cause the syndrome known as hereditary angioedema (HAE) (see Section 216). Deficiency of C1 inhibitor results in a failure to regulate both the blood clotting and complement activation pathways, leading to excessive production of vasoactive mediators that cause fluid accumulation in the tissues (edema) and local laryngeal swelling that can result in suffocation. 13-15 Defects in phagocytic cells permit widespread bacterial infections. Deficiencies in phagocyte numbers or function can be associated with severe immunodeficiency; indeed, a total absence of neutrophils is incompatible with survival in a normal environment. Phagocyte immunodeficiencies can be grouped into four general types: deficiencies in phagocyte production, phagocyte adhesion, phagocyte activation, and phagocyte killing of microorganisms (Fig. 13.13). We consider each in turn.

1	Inherited deficiencies of neutrophil production (neutropenias) are classified either as severe congenital neutropenia (SCN) or cyclic neutropenia. In severe congenital neutropenia, which can be inherited as a dominant or recessive trait, the neutrophil count is persistently less than 0.5 ×109 per liter of blood (normal numbers are 3 ×109 to 5.5 × 109 per liter). Cyclic neutropenia is characterized by fluctuation in neutrophil numbers from near normal to very low or undetectable with an approximate cycle time of 21 days, resulting in periodicity of infectious risk. The most common causes of SCN are sporadic or autosomal dominant mutations of the gene that encodes neutrophil elastase (ELA2), a component of the azurophilic, or primary, granules involved in the degradation of phagocytosed microbes. Altered targeting of defective elastase 2 to granules causes apoptosis of developing myelocytes and a developmental block at the promyelocyte–myelocyte stage. Some mutations of ELA2 cause

1	Fig. 13.13 Defects in phagocytic cells are associated with persistence of bacterial infection. Defects in neutrophil development caused by congenital neutropenias result in profound defects in antibacterial defense. impairment of the leukocyte integrins with a common β2 subunit (CD18) or defects in the selectin ligand sialyl-lewisX, prevent phagocytic cell adhesion and migration to sites of infection (leukocyte adhesion deficiency). inability to transmit signals through toll-like receptors (tlRs), due to defects in MyD88 or iRaK4, for example, impairs the proximal sensing of many infectious agents by innate immune cells. the respiratory burst is defective in chronic granulomatous disease, glucose-6-phosphate dehydrogenase (G6PD) deficiency, and myeloperoxidase deficiency. in chronic granulomatous disease, infections persist because macrophage activation is defective, leading to chronic stimulation of CD4 t cells and hence to granulomas. Vesicle fusion in phagocytes is defective in

1	disease, infections persist because macrophage activation is defective, leading to chronic stimulation of CD4 t cells and hence to granulomas. Vesicle fusion in phagocytes is defective in Chediak–Higashi syndrome. these diseases illustrate the critical role of phagocytes in removing and killing pathogenic bacteria.

1	cyclic neutropenia; how the mutant elastase causes a 21day cycle in neutropenia is still a mystery. A rare autosomal dominant form of SCN is caused by mutations in the oncogene GFI1, which encodes a transcriptional repressor that acts on ELA2. This finding arose from the unexpected observation that mice lacking the protein Gfi1 are neutropenic due to overexpression of Ela2.

1	Autosomal recessive forms of SCN have also been identified. Deficiency of the mitochondrial protein HAX1 leads to increased apoptosis in developing myeloid cells, resulting in a severe neutropenia referred to as Kostmann’s disease. The heightened sensitivity of developing neutrophils to apoptosis is highlighted by SCN associated with genetic defects in glucose metabolism. Patients with recessive mutations in the genes encoding the glucose6phosphatase catalytic subunit 3 (G6PC3) or the glucose6phosphate translocase 1 (SLC37A4) also demonstrate increased apoptosis during granulocyte development that results in neutropenia. Acquired neutropenia associated with chemotherapy, malignancy, or aplastic anemia is also associated with a similar spectrum of severe pyogenic bacterial infections. Finally, neutropenia can also be a feature of other primary immunodeficiency diseases, including CD40 ligand deficiency, CVID, XLA, Wiskott–Aldrich syndrome, and GATA2 deficiency. Some patients exhibit

1	neutropenia can also be a feature of other primary immunodeficiency diseases, including CD40 ligand deficiency, CVID, XLA, Wiskott–Aldrich syndrome, and GATA2 deficiency. Some patients exhibit formation of autoantibodies that lead to accelerated destruction of neutrophils.

1	Defects in the migration of phagocytic cells to extravascular sites of infection can cause serious immunodeficiency. Leukocytes reach such sites by emigrating from blood vessels in a tightly regulated process (see Fig. 3.31). Deficiencies in the molecules involved in each stage of this process can prevent neutrophils and macrophages from penetrating infected tissues, and are referred to as leukocyte adhesion deficiencies (LADs). Deficiency in the leukocyte integrin common β2 subunit CD18, which is a component of LFA1, MAC1, and p150:95, prevents the migration of leukocytes into an infected site by abolishing the cells’ ability to adhere tightly to the endothelium. Because it was the first LAD to be characterized, it is now referred to as type 1 LAD, or LAD1, and is the most common LAD variant. Reduced rolling of leukocytes on the endothelium has been described in rare patients who lack the sialylLewisX antigen owing to a deficiency in the GDPfucosespecific transporter that is involved

1	Reduced rolling of leukocytes on the endothelium has been described in rare patients who lack the sialylLewisX antigen owing to a deficiency in the GDPfucosespecific transporter that is involved in the biosynthesis of sialylLewisX and other fucosylated ligands for the selectins. This is referred to as type 2 LAD or LAD2. LAD3 results from deficiency of Kindlin3, a protein involved in the induction of the highaffinity binding state of β integrins required for firm adhesion. Each variant of LAD has an autosomal recessive pattern of inheritance and causes severe, lifethreatening bacterial or fungal infections early in life that are characterized by impaired wound healing and, in pyogenic bacterial infections, the absence of pus formation. The infections that occur in these patients are resistant to antibiotic treatment. LAD3 is also associated with defects in platelet aggregation that cause increased bleeding.

1	A key step in the activation of innate immune cells, including phagocytes, is their recognition of microbeassociated molecular patterns through Tolllike receptors (TLRs; see Section 35). Several primary immunodeficiencies have been identified that are caused by defects in intracellular signaling components of TLRs. Signaling through all TLRs except TLR3 requires the adaptor protein MyD88, which recruits and activates the kinases IRAK4 and IRAK1, which are required for downstream activation of the NFκB and MAP kinase pathways (see Section 37). Autosomal recessive mutations in the genes encoding MyD88 or IRAK4 have a similar phenotype: recurrent, severe peripheral and invasive infections by pyogenic bacteria that elicit little inflammation, a situation known as a ‘cold’ infection. Note that many of the signaling functions of MyD88 and IRAK4 molecules are shared with IL1 family receptors. Thus, at least part of the immune defect in patients with inherited abnormalities in these molecules

1	many of the signaling functions of MyD88 and IRAK4 molecules are shared with IL1 family receptors. Thus, at least part of the immune defect in patients with inherited abnormalities in these molecules may be attributed to aberrant signaling by IL1 family members. Note also that NEMO deficiency, which impairs Bcell class switching (see Section 139), also impairs TLR and IL1 receptor family signaling through its block of normal NFκB activation. Immunodeficiency associated with defects in NEMO therefore affects both adaptive and innate immune function. Interestingly, increased viral infections are not typical in patients with MyD88 mutations, despite this protein’s role in signaling by each of the nucleic acidsensing TLRs except TLR3 (for example, TLR7, TLR8, and TLR9). This indicates that activation of interferon regulating factors (IRFs) that induce interferon responses downstream of these TLRs remains intact despite the defects in MyD88.

1	Remarkably, of the ten TLRs found in humans, defects in only one—TLR3— have been linked to immunodeficiency. While defects in other TLRs have been identified (for example, TLR5), they have not been associated with an overt immunodeficiency phenotype, likely reflecting a substantial level of redundancy. On the other hand, patients with hemizygous (dominant) and homozygous (recessive) mutations in the gene encoding TLR3, which senses doublestranded RNA, typically have recurrent herpes simplex virus1 (HSV1) infections of the central nervous system (herpes simplex encephalitis) due to impaired production of type I interferons by cells of the nervous system. Those with inherited deficiencies in molecules involved in TLR3 signaling (for example, TRIF, TRAF3, or TBK1) are similarly susceptible to HSV1 encephalitis, as are patients with mutations in the TLRtransport protein UNC93B1, which is required for the transport of TLR3 from the endoplasmic reticulum to the endolysosome. Interestingly,

1	HSV1 encephalitis, as are patients with mutations in the TLRtransport protein UNC93B1, which is required for the transport of TLR3 from the endoplasmic reticulum to the endolysosome. Interestingly, leukocytes from these patients have no defect in their response to TLR3 ligands or HSV1, indicating redundancy of TLR3 function in these cells, but not those of the central nervous system. Similarly, these patients show only a limited predisposition to other viral infections, implying the existence of TLR3independent protection against most other types of viral infection.

1	Genetic defects that affect signaling by pattern recognition receptors (PRRs) other than TLRs have been described. CARD9 is an adaptor molecule involved in signaling downstream of Ctype lectin receptors expressed on myeloid cells. Dectin1, Dectin2, and macrophageinducible Ctype lectin (MINCLE) each recognize fungalassociated molecular patterns that signal through CARD9 to induce secretion of proinflammatory cytokines, including IL6 and IL23 (see Section 31). Autosomal recessive CARD9 deficiency results in impaired TH17 cell responses to fungi, with the result that patients with this deficiency, like patients with inborn errors of IL17 immunity (for example, IL17RA deficiency and IL17F deficiency; see Section 1310), suffer chronic mucocutaneous candidiasis. However, in addition, these patients can suffer infections by dermatophytes, which are ubiquitous filamentous fungi that normally cause common superficial skin and nail infections, such as tinea pedis (athlete’s foot).

1	Fig. 13.14 The autoinflammatory diseases.

1	Most of the other known defects in phagocytic cells affect their ability to ingest microbes and destroy them once ingested (see Fig. 13.13). Patients with chronic granulomatous disease (CGD) are highly susceptible to bacterial and fungal infections and form granulomas as a result of an inability to kill bacteria ingested by phagocytes (see Fig. 11.13). The defect in this case is in the production of reactive oxygen species (ROS) such as the superoxide anion (see Section 32). Discovery of the molecular defect in this disease gave weight to the idea that these agents killed bacteria directly; this notion has since been challenged by the finding that the generation of ROS is not itself sufficient to kill target microorganisms. It is now thought that ROS cause an influx of K+ ions into the phagocytic vacuole, increasing the pH to the optimal level for the action of microbicidal peptides and proteins, which are the key agents in killing the invading microorganism.

1	Genetic defects affecting any of the constituent proteins of the NADPH oxidase expressed in neutrophils and monocytes (see Section 32) can cause chronic granulomatous disease. Patients with the disease have chronic bacterial infections, which in some cases lead to the formation of granulomas. Deficiencies in glucose6phosphate dehydrogenase (G6PD) and myeloperoxidase (MPO) also impair intracellular bacterial killing and lead to a similar, although less severe, phenotype. 13-16 Mutations in the molecular regulators of inflammation can ‘autoinflammatory disease.’

1	There are a small number of diseases in which mutations in genes that control the life, death, and activities of inflammatory cells are associated with severe inflammatory disease. Although they do not lead to immunodeficiency, we have included them in this chapter because they are singlegene defects affecting a crucial aspect of innate immunity—the inflammatory response. These conditions represent a failure of the normal mechanisms that limit inflammation, and are known as autoinflammatory diseases: they can lead to inflammation even in the absence of infection (Fig. 13.14). Familial Mediterranean fever (FMF) is characterized by episodic attacks of severe inflammation, which can occur at various sites throughout the body and are associated with fever, an acutephase response (see Section 318), and severe malaise. The pathogenesis of FMF was a mystery until its cause was discovered to be mutations in the gene MEFV, which encodes a protein called pyrin, to reflect its association with

1	318), and severe malaise. The pathogenesis of FMF was a mystery until its cause was discovered to be mutations in the gene MEFV, which encodes a protein called pyrin, to reflect its association with fever. Pyrin and pyrin domaincontaining proteins are involved in pathways that lead to the apoptosis of inflammatory cells, and in pathways that inhibit the secretion of proinflammatory cytokines such as IL1β. It is proposed that an absence of functional pyrin leads to unregulated cytokine activity and defective apoptosis, resulting in a failure to control inflammation. In mice, an absence of pyrin causes increased sensitivity to lipopolysaccharide and a defect in macrophage apoptosis. A disease with similar clinical manifestations, known as TNFreceptor associated periodic syndrome (TRAPS), is caused by mutations in quite a different gene, that encoding the TNFα receptor TNFRI. Patients with TRAPS have reduced levels of TNFRI, which lead to increased levels of proinflammatory TNFα in the

1	caused by mutations in quite a different gene, that encoding the TNFα receptor TNFRI. Patients with TRAPS have reduced levels of TNFRI, which lead to increased levels of proinflammatory TNFα in the circulation because it is not regulated by binding to this receptor. The disease responds to therapeutic blockade with antiTNF agents such as etanercept, a soluble TNF receptor developed primarily to treat patients with rheumatoid arthritis (see Section 168). Mutations in the gene encoding PSTPIP1 (prolineserinethreonine phosphataseinteracting protein 1), which interacts with pyrin, are associated with another dominantly inherited autoinflammatory syndrome—pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA). The mutations increase binding of pyrin to PSTPIP1, and it has been proposed that the interaction sequesters pyrin and limits its normal regulatory function.

1	The episodic autoinflammatory diseases Muckle–Wells syndrome and familial cold autoinflammatory syndrome (FCAS) are clearly linked to the inappropriate stimulation of inflammation, because they are due to mutations in NLRP3, a component of the ‘inflammasome’ that normally senses cell damage and stress as a result of infection (see Section 39). The mutations lead to the activation of NLRP3 in the absence of such stimuli and the unregulated production of proinflammatory cytokines. Patients with these dominantly inherited syndromes present with episodes of fever—which is induced by exposure to cold in the case of FCAS—as well as urticarial rash, joint pains, and conjunctivitis. Mutations in NLRP3 are also associated with the autoinflammatory disorder chronic infantile neurologic cutaneous and articular syndrome (CINCA), in which short, recurrent fever episodes are common, although severe arthropathic, neurologic, and dermatologic symptoms predominate. Both pyrin and NLRP3 are

1	and articular syndrome (CINCA), in which short, recurrent fever episodes are common, although severe arthropathic, neurologic, and dermatologic symptoms predominate. Both pyrin and NLRP3 are predominantly expressed in leukocytes and in cells that act as innate barriers to pathogens, such as intestinal epithelial cells. The stimuli that modulate pyrin and related molecules include inflammatory cytokines and stressrelated changes in cells. Indeed, Muckle–Wells syndrome responds dramatically to the drug anakinra, an antagonist of the receptor for IL1.

1	13-17 Hematopoietic stem cell transplantation or gene therapy can be useful to correct genetic defects.

1	It is frequently possible to correct the defects in lymphocyte development that lead to SCID and some other immunodeficiency phenotypes by replacing the defective component, generally by hematopoietic stem cell (HSC) transplantation (see Section 1536). The main difficulties in these therapies result from human leukocyte antigen (HLA) polymorphism. To be useful, the graft must share some HLA alleles with the host. As we learned in Section 821, the HLA alleles expressed by the thymic epithelium determine which T cells can be positively selected. When HSCs are used to restore immune function to individuals with a normal thymic stroma, both the T cells and the antigenpresenting cells are derived from the graft. Therefore, unless the graft shares at least some HLA alleles with the recipient, the T cells that are selected on host thymic epithelium cannot be activated by graftderived antigenpresenting cells (Fig. 13.15).

1	MHCb-restricted T cells can be activated by MHCa×b APC, and recognize infected MHCb cells Donor cells undergo selection on MHCb in the recipient thymus Hematopoietic stem cell transplant. One MHC allele shared MHCa×bmacrophageAPCMHCbTcells Fig. 13.15 The donor and the recipient of a hematopoietic stem cell (HSC) graft must share at least some MHC molecules to restore immune function.

1	an HSC transplant from a genetically different donor is illustrated in which the donor marrow cells share some MHC molecules with the recipient. the shared MHC type is designated ‘b’ and illustrated in blue; the MHC type of the donor HSCs that is not shared is designated ‘a’ and shown in yellow. in the recipient, developing donor lymphocytes are positively selected on MHCb on thymic epithelial cells and negatively selected by the recipient’s stromal epithelial cells and at the corticomedullary junction by encounter with dendritic cells derived from both the donor HSCs and residual recipient dendritic cells. the negatively selected cells are shown as apoptotic cells. the donor-derived antigen-presenting cells (aPCs) in the periphery can activate t cells that recognize MHCb molecules; the activated t cells can then recognize the recipient’s infected MHCbbearing cells. Host-versus-graft response. Graft failure

1	Host-versus-graft response. Graft failure Fig. 13.16 Grafting of bone marrow can be used to correct immunodeficiencies caused by defects in lymphocyte maturation, but two problems can arise. First, if there are mature t cells in the bone marrow, they can attack cells of the host by recognizing their MHC antigens, causing graft-versus-host disease (top panel). this can be prevented by t-cell depletion of the donor bone marrow (center panel). Second, if the recipient has competent t cells, these can attack the bone marrow stem cells (bottom panel). this causes failure of the graft by the usual mechanism of transplant rejection (see Chapter 15).

1	There is also a danger that mature, postthymic T cells that contaminate donor HSCs prepared from the peripheral blood or bone marrow might recognize the host as foreign and attack it, causing graftversushost disease (GVHD) (Fig. 13.16, top panel). This can be overcome by depleting the donor graft of mature T cells. For immunodeficiency diseases other than SCID where there are residual T cells and NK cells in the recipient, some form of myeloablative treatment (destruction of the bone marrow, typically using cytotoxic drugs) is usually carried out before transplantation, both to generate space for engraftment of the transplanted HSCs and to minimize the threat of hostversusgraft disease (HVGD) (see Fig. 13.16, third panel). The intensity of the myeloablative regimen is dependent on the nature of the immunodeficiency. For diseases where persistence of the patient’s cells can be tolerated, engraftment of only a fraction of donor HSCs is sufficient for cure and nonmyeloablative

1	on the nature of the immunodeficiency. For diseases where persistence of the patient’s cells can be tolerated, engraftment of only a fraction of donor HSCs is sufficient for cure and nonmyeloablative chemotherapy may suffice prior to HSC transplantation. In other conditions such as XLP, which require complete elimination of the patient’s blood cells and full engraftment of donor cells, more intensive (myeloablative) chemotherapy may be required.

1	Because many specific gene defects that cause inherited immunodeficiencies have been identified, an alternative therapeutic approach is somatic gene therapy. This strategy involves the isolation of HSCs from the patient’s bone marrow or peripheral blood, introduction of a normal copy of the defective gene with the use of a viral vector, and reinfusion of the corrected stem cells into the patient. Initially, retroviral vectors were used for gene therapy trials, but were ceased due to severe complications in some patients. Although the genetic defect was corrected in patients with Xlinked SCID, CGD, and WAS who received this treatment, some patients developed leukemia due to insertion of the retrovirus within a protooncogene. The inability to control the site in the genome in which retrovirally encoded genes insert and the use of viral vectors with strong promoters that can transactivate neighboring genes is therefore problematic. More recently, the use of selfinactivating retroviral

1	encoded genes insert and the use of viral vectors with strong promoters that can transactivate neighboring genes is therefore problematic. More recently, the use of selfinactivating retroviral and lentiviral vectors for gene correction has shown promise as a means of avoiding this complication. Also, a technique for the generation of induced pluripotent stem cells (iPS cells) from a patient’s own somatic cells has been demonstrated. Forced expression of a set of transcription factors can reprogram somatic cells to become pluripotent progenitors that can give rise to HSCs. This approach offers the promise of ‘repairing’ specific defective genes in patientderived stem cells by gene targeting ex vivo before reinfusion, but is not yet established. Until better methods for introduction of corrected genes into selfrenewing stem cells are identified, allogeneic HSC transplantation will remain the mainstay of treatment for many primary immunodeficiencies.

1	13-18 Noninherited, secondary immunodeficiencies are major predisposing causes of infection and death.

1	The primary immunodeficiencies have taught us much about the biology of specific proteins of the immune system. Fortunately, these conditions are rare. In contrast, secondary immunodeficiency is relatively common. Malnutrition devastates many populations around the world, and a major feature of malnutrition is secondary immunodeficiency. This particularly affects cellmediated immunity, and death in famines is frequently caused by infection. Measles, which itself causes immunosuppression, is an important cause of death in malnourished children. In the developed world, measles is an unpleasant illness but major complications are uncommon. In contrast, measles in the malnourished has a high mortality. Tuberculosis is another important infection in the malnourished. In mice, protein starvation causes immunodeficiency by affecting antigenpresenting cell function, but in humans it is not clear how malnourishment specifically affects immune responses. Links between the endocrine and immune

1	causes immunodeficiency by affecting antigenpresenting cell function, but in humans it is not clear how malnourishment specifically affects immune responses. Links between the endocrine and immune systems may provide part of the answer.

1	Adipocytes (fat cells) produce the hormone leptin, levels of which are related directly to the amount of fat present in the body; leptin levels therefore fall during starvation when fat is consumed. Both mice and humans with genetic leptin deficiency have reduced Tcell responses, and in mice the thymus atrophies. In both starved mice and those with inherited leptin deficiency, these abnormalities can be reversed by the administration of leptin. Secondary immunodeficiency states are also associated with hematopoietic tumors such as leukemia and lymphomas. Myeloproliferative diseases, such as leukemia, can be associated with deficiencies of neutrophils (neutropenia) or an excess of immature myeloid progenitors that lack functional properties of mature neutrophils, either of which increases susceptibility to bacterial and fungal infections. Destruction or invasion of peripheral lymphoid tissue by lymphomas or metastases from other cancers can promote opportunistic infections.

1	Congenital asplenia (a rare inherited absence of the spleen), surgical removal of the spleen, and destruction of spleen function by certain diseases are associated with a lifelong predisposition to overwhelming infection by S. pneumoniae, graphically illustrating the role of mononuclear phagocytic cells within the spleen in the clearance of this organism from blood. Patients who have lost spleen function should be vaccinated against pneumococcal infection and are often recommended to take prophylactic antibiotics throughout their life.

1	Secondary immunodeficiency is also a complication of certain medical therapies. A major complication of cytotoxic drugs used to treat cancer is immunosuppression and increased susceptibility to infection. Many of these drugs kill all dividing cells, including normal cells of the bone marrow and lymphoid systems. Infection is thus one of the major side effects of cytotoxic drug therapy. Immune suppression to induce host tolerance of solid organ allografts, such as kidney or heart transplants, also carries a substantial increased risk for infection and even for malignancy. The recent introduction of biologic therapies for some forms of autoimmunity has led to an increased risk of infection because of their immunosuppressive effects. For example, administration of antibodies that block TNFα in patients with rheumatoid arthritis or other forms of autoimmunity has been associated with infrequent, but increased, instances of infectious complications. Summary.

1	Genetic defects can occur in almost any molecule involved in the immune response. These defects give rise to characteristic deficiency diseases, which, although rare, provide much information about the development and functioning of the immune system in normal humans. Inherited immunodeficiencies illustrate the vital role of the adaptive immune response and T cells in particular, without which both cellmediated and humoral immunity fail. They have provided information about the separate roles of B lymphocytes in humoral immunity and of T lymphocytes in cellmediated immunity, the importance of phagocytes and complement in humoral and innate immunity, and the specific functions of a growing number of cellsurface or signaling molecules in the adaptive immune response. There are also some inherited immune disorders whose causes we still do not understand. The study of these diseases will undoubtedly teach us more about the normal immune response and its control. Acquired defects in the

1	immune disorders whose causes we still do not understand. The study of these diseases will undoubtedly teach us more about the normal immune response and its control. Acquired defects in the immune system, the secondary immunodeficiencies, are much more common than the primary, inherited immunodeficiencies. In the next sections we briefly consider general mechanisms by which successful pathogens evade or subvert immune defenses and then detail how extreme subversion of the immune system by one pathogen, the human immunodeficiency virus (HIV), has created a major pandemic characterized by the acquired immune deficiency syndrome (AIDS) in affected individuals.

1	Evasion and subversion of immune defenses.

1	In the previous section, we learned how specific defects in immune pathways lead to infection, often by microbes that would normally be defeated by a healthy immune system. These ‘opportunistic’ infections often dominate the clinical expression of inherited immunodeficiencies because the causative organisms are ubiquitous and abundant in the environment. A minority of microbes are true pathogens that can infect those with normal immune defenses. A defining feature of pathogens is their ability to avoid immune destruction by components of the innate and adaptive immune systems, at least long enough to replicate in the infected host and spread to new hosts. At one end of the spectrum are pathogens that establish an acute infection, replicate quickly, and find a new host before they are cleared by a successful immune response; at the other are pathogens that establish chronic infections, persisting long term in the host while evading elimination by immune defenses. Successful pathogens

1	by a successful immune response; at the other are pathogens that establish chronic infections, persisting long term in the host while evading elimination by immune defenses. Successful pathogens use different strategies to achieve these ends, and over millions of years of coevolution with their hosts have evolved a remarkable diversity of strategies for avoiding detection and destruction by the immune system, often employing several strategies that subvert immunity at multiple points. The antiimmune strategies employed by pathogens are as sophisticated as the immune system itself. They must be for any pathogens to achieve success against the diverse strategies that vertebrates have evolved to ensure host protection.

1	Viruses, bacteria, and protozoan (unicellular) or metazoan (multicellular) parasites can all act as pathogens. While fungi and helminthic (metazoan) parasites are major causes of common skin infections and intestinal worm infestation, respectively, they do not typically cause lifethreatening infections in normal people and will not be considered further here. In contrast, a select number of viruses, bacteria, and protozoan parasites are the major causes of morbidity and mortality caused by infectious agents. AIDS, tuberculosis, and malaria, caused by the human immunodeficiency virus (HIV), Mycobacterium tuberculosis, and Plasmodium falciparum, respectively, are the three largest infectious disease threats to humans; each of these pathogens infects over 100 million people worldwide and kills 1 to 2 million people annually. Although the strategies for survival within—and propagation between—hosts differ for each type of pathogen, many of the innate and adaptive immune mechanisms

1	and kills 1 to 2 million people annually. Although the strategies for survival within—and propagation between—hosts differ for each type of pathogen, many of the innate and adaptive immune mechanisms employed to thwart the pathogen are the same. Here we briefly consider the lifestyles of, and the principal immune responses elicited by, different types of pathogens, and the strategies the pathogens employ to evade or subvert the immune system.

1	strategies to avoid detection by pattern recognition receptors and destruction by antibody, complement, and antimicrobial peptides.

1	Extracellular bacterial pathogens replicate outside of host cells, whether on the surfaces of barrier tissues they colonize (for example, gastrointestinal or respiratory tract), or in tissue spaces or blood following invasion across barrier epithelia. Both Gramnegative and positive species are pathogenic, and, as discussed in Section 1110, they typically elicit type 3 immunity, which orchestrates neutrophilic responses, the development of opsonizing and complementfixing antibodies, and the production of antimicrobial peptides by barrier epithelial cells and immune cells that clear these microbes from barrier epithelia and prevent their invasion. Some of the MAMPS expressed by Gramnegative and positive bacteria are distinct, but share similar immunecell activating properties. Gramnegative pathogens contain LPS, a potent activator of TLR4, in their outer cell membrane, whereas the cell wall of Grampositive pathogens contains peptidoglycans, which activate TLR2 and NOD1 and NOD2. One

1	pathogens contain LPS, a potent activator of TLR4, in their outer cell membrane, whereas the cell wall of Grampositive pathogens contains peptidoglycans, which activate TLR2 and NOD1 and NOD2. One strategy of immune evasion used by these pathogens is therefore shielding of surface MAMPs to avoid their detection by pattern recognition receptors on immune cells (Fig. 13.17). Several Gramnegative pathogens modify the lipid A core of LPS with carbohydrates and other moieties that interfere with TLR4 binding. Indeed, some bacteria produce variants of lipid A that act as TLR4 antagonists rather than agonists. Select Grampositive pathogens have evolved mechanisms to modulate peptidoglycan recognition by NODs, or to produce hydrolases that degrade peptidoglycan.

1	Fig. 13.17 Mechanisms used by bacteria to subvert the host immune system. listed are examples of immune evasion/subversion mechanisms used by different strains of extracellular and intracellular bacterial pathogens. examples of the strains of bacteria that employ each mechanism are listed in the far right column (e.g., Streptococcus pneumoniae, Porphyromonus gingivalis, Pseudomonas aeruginosa, Brucella abortus, Yersinia pestis). Fig. 13.18 Host defense against Streptococcus pneumoniae is type specific. the different strains of S. pneumoniae have antigenically distinct capsular polysaccharides. the capsule prevents effective phagocytosis until the bacterium is opsonized by specific antibody and complement, allowing phagocytes to destroy it. antibody against one type of

1	S. pneumoniae does not cross-react with the other types, so an individual immune to one type has no protective immunity to a subsequent infection with a different type. an individual must generate a new adaptive immune response each time he or she is infected with a different type of S. pneumoniae.

1	A limited number of Grampositive pathogens can also shield their outer cell membrane with a thick carbohydrate capsule. In addition to inhibiting recognition of peptidoglycan and activation of the alternative pathway of complement, the capsule prevents antibody and complement deposition on the bacterial surface, thereby avoiding direct damage by the membraneattack complex of the complement cascade. The capsule also impairs clearance of the pathogen by phagocytes (see Fig. 13.17). In the case of Streptococcus pneumoniae, an important cause of bacterial pneumonia, the carbohydrate capsule also serves as a platform for antigenic variation to modulate the expression of surface antigenic epitopes recognized by antibody. Over 90 known types of S. pneumoniae are distinguished by differences in the structure of their polysaccharide capsules. The different types are distinguished by using specific antibodies as reagents in serological tests and so are often known as serotypes. Infection with

1	the structure of their polysaccharide capsules. The different types are distinguished by using specific antibodies as reagents in serological tests and so are often known as serotypes. Infection with one serotype can lead to typespecific immunity, which protects against reinfection with that type but not with a different serotype. Thus, from the point of view of the adaptive immune system, each serotype of S. pneumoniae represents a distinct organism, with the result that essentially the same pathogen can cause disease many times in the same individual (Fig. 13.18). Similarly, antigenic variation mediated by DNA rearrangement also occurs in bacteria and helps to account for the success of enteropathogenic E. coli, or of Neisseria species, which cause gonorrhea and meningitis. Fimbriae or pili are expressed on the bacterial surface and used for attachment to hostcell surfaces and are major antigenic targets for antibodymediated blockade of bacterial attachment and colonization. The

1	or pili are expressed on the bacterial surface and used for attachment to hostcell surfaces and are major antigenic targets for antibodymediated blockade of bacterial attachment and colonization. The gene locus encoding the expressed Neisseria pilus (pilE) can undergo recombination with partial pilin genes stored in ‘silent’ (pilS) loci to generate a constantly shifting pilus for display on the bacterial surface. By constantly changing the expressed pilus, the bacterium evades antibodymediated immune clearance.

1	Among other antiimmune strategies used by extracellular pathogens are mechanisms to inactivate the C3 convertase of the complement cascade; the expression of Fcbinding proteins that block functional antibody binding to the bacterium (for example, Protein A); and the decoration of the bacterial surface with host inhibitors of complement (for example, factor H). These bacteria have also evolved mechanisms to defeat antimicrobial peptides (AMPs; for example, defensins and cathelicidins). These small cationic and amphipathic peptides have significant antimicrobial activity by inserting into negatively charged cell membranes to generate pores that lyse the bacterium. Pathogens can alter their membrane composition to minimize AMP binding, and can produce proteases that degrade the AMPs.

1	An unusual feature of Gramnegative pathogens, including both extracellular and intracellular bacteria, is their capacity to inject immune modulatory bacterial proteins directly into host cells via specialized structures: the type III and type IV secretion systems (T3SS and T4SS, respectively) (Fig. 13.19). These needlelike structures, or injectisomes, assemble on the bacterial surface and provide a conduit through which bacterial proteins are secreted directly into the cytosol of target cells. A range of bacterial virulence factors that aid in subverting the host immune response are delivered via this mechanism, including bacterial factors that block signaling cascades central to the inflammatory response: NFκB and MAP kinases. Among the most remarkable of these are the Yersinia outer proteins (Yops) produced by Yersinia pestis, the causative agent of bubonic plague. Secretion of several of these factors (for example, YopH, YopE, YopO, and YopT) into phagocytes disrupts the actin

1	proteins (Yops) produced by Yersinia pestis, the causative agent of bubonic plague. Secretion of several of these factors (for example, YopH, YopE, YopO, and YopT) into phagocytes disrupts the actin cytoskeleton, which is essential for phagocytosis. The essential roles played by T3SS or T4SS in immune subversion by a number of Gramnegative pathogens are demonstrated by the loss of pathogenicity of mutant bacteria lacking components of these structures.

1	13-20 Intracellular bacterial pathogens can evade the immune system by seeking shelter within phagocytes.

1	To avoid the major effectors directed against extracellular bacteria—complement and antibodies—some bacterial pathogens have evolved specialized mechanisms for surviving within macrophages, using these phagocytes as their primary host cell, as well as a vehicle for dissemination within the host. This ‘Trojan horse’ strategy is achieved by three general strategies: blockade of phagosome–lysosome fusion; escape from the phagosome into the cytosol; and resistance to killing mechanisms within the phagolysosome. Mycobacterium tuberculosis, for example, is taken up by macrophages but prevents the fusion of the phagosome with the lysosome, protecting itself from the bactericidal actions of the lysosomal contents. Other microorganisms, such as the bacterium Listeria monocytogenes, escape from the phagosome into the cytoplasm of the macrophage, where they multiply. They then spread to adjacent cells in the tissue without emerging into the extracellular environment. They do this by hijacking

1	phagosome into the cytoplasm of the macrophage, where they multiply. They then spread to adjacent cells in the tissue without emerging into the extracellular environment. They do this by hijacking the cytoskeletal protein actin, which assembles into filaments at the rear of the bacterium. The actin filaments drive the bacteria forward into vacuolar projections to adjacent cells; the vacuoles are then lysed by the listeria, releasing the bacteria into the cytoplasm of the adjacent cell. Moreover, Listeria has been shown to induce the formation of bacteriacontaining blebs on the surface of infected cells. These blebs which express phosphatidyl serine on the outer membrane leaflet. This membrane phospholipid is normally restricted to the inner membrane leaflet, and when exposed on the outer membrane leaflet is normally recognized by phagocytes as a signal for the uptake of apoptotic cell debris. In this way, Listeria is delivered directly to phagocytic cells, thereby avoiding attack by

1	membrane leaflet is normally recognized by phagocytes as a signal for the uptake of apoptotic cell debris. In this way, Listeria is delivered directly to phagocytic cells, thereby avoiding attack by antibodies.

1	Following uptake, Salmonella species use a type III secretion system (see Fig. 13.19) to secrete effectors such as SifA into the host cytosol and membranes in order to alter the composition of the Salmonellacontaining vacuole so as to avoid destruction. Remarkably, Salmonella can also inject factors that delay the apoptotic death of host macrophages, prolonging the phagocytes’ lifespans until their bacterial cargo can be delivered to new cellular hosts. Other actions of intracellular bacteria counter reactive oxygen species or microbicidal peptides delivered into the phagolysosome by the ingesting phagocyte. Fig. 13.19 Pathogenic bacteria use specialized secretion systems to inject effector molecules into host cells.

1	Fig. 13.19 Pathogenic bacteria use specialized secretion systems to inject effector molecules into host cells. a number of pathogenic Gram-negative bacteria use a complex, needle-like protein assembly—a type iii or iV secretion system, or injectisome—to inject virulence proteins into target cells to compromise host defenses and establish infection. these ‘nanoinjectors’ are assembled from more than 20 proteins, and are composed of a base that spans the two bacterial membranes, a needle that is anchored in the base and is formed by the polymerization of repeating α-helical subunits, and a tip complex that serves as a docking structure for the translocon, which penetrates the host-cell membrane to allow bacterial effector proteins to pass into the host cell.

1	Fig. 13.20 T-cell and macrophage responses to Mycobacterium leprae are sharply different in the two polar forms of leprosy. infection with M. leprae, whose cells stain as small dark red dots in the photographs, can lead to two very different forms of disease (top panels). in tuberculoid leprosy (left), growth of the organism is well controlled by tH1-like cells that activate infected macrophages. the tuberculoid lesion contains granulomas and is inflamed, but the inflammation is local and causes only local effects, such as peripheral nerve damage. in lepromatous leprosy (right), infection is widely disseminated and the bacilli grow uncontrolled in macrophages; in the late stages of disease there is major damage to connective tissues and to the peripheral nervous system. there are several intermediate stages between these two polar forms (not shown). the lower panel shows northern blots demonstrating that the cytokine patterns in the two polar forms of the disease are sharply

1	several intermediate stages between these two polar forms (not shown). the lower panel shows northern blots demonstrating that the cytokine patterns in the two polar forms of the disease are sharply different, as shown by the analysis of Rna isolated from lesions of four patients with lepromatous leprosy and four patients with tuberculoid leprosy. Cytokines typically produced by tH2 cells (il-4, il-5, and il-10) dominate in the lepromatous form, whereas cytokines produced by tH1 cells (il-2, iFn-γ, and tnF-β) dominate in the tuberculoid form. it therefore seems that tH1-like cells predominate in tuberculoid leprosy, and tH2-like cells in lepromatous leprosy. iFn-γ would be expected to activate macrophages, enhancing the killing of M. leprae, whereas il-4 can actually inhibit the induction of bactericidal activity in macrophages. Photographs courtesy of G. Kaplan; cytokine patterns courtesy of R.l. Modlin.

1	As a tradeoff for their intracellular lifestyle, intracellular bacteria risk activation of immune effectors that target these pathogens: NK cells and T cells. As discussed in Section 115, a major function of the type 1 immune response is activation of NK cells and TH1 cells that activate phagocytes for enhanced intracellular killing by secretion of IFNγ or expression of CD40L. Additionally, those intracellular pathogens that have evolved mechanisms for escape of the phagosome, such as Listeria, generate cytosolic peptides that feed the MHC class I antigen presentation pathway and thus induce cytotoxic Tcell responses that target their host cell for destruction. In leprosy, a disease caused by skin and peripheral nerve infection with Mycobacterium leprae, effective host defense requires macrophage activation by NK and TH1 cells (Fig. 13.20).

1	Cytokine patterns in leprosy lesions TNF-˜IFN-°IL-2 IL-4 IL-5 IL-10 Lepromatous TH2 cytokinesTH1 cytokines Lepromatous Tuberculoid Tuberculoid Lepromatous leprosy Organisms show ˜orid growth in macrophages High infectivity Hypergammaglobulinemia Disseminated infection. Bone, cartilage, and diffuse nerve damage Low or absent T-cell responsiveness. No response to M. leprae antigens Infection with Mycobacterium leprae can result in different clinical forms of leprosy There are two polar forms, tuberculoid and lepromatous leprosy, but several intermediate forms also exist Normal T-cell responsiveness. Speciÿc response to M. leprae antigens Tuberculoid leprosy Organisms present at low to undetectable levels Low infectivity Normal serum immunoglobulin levels Granulomas and local in˜ammation. Peripheral nerve damage

1	Like M. tuberculosis, M. leprae is able to persist and grow in macrophage vesicles and is normally restrained, but not cleared, by a type 1 host response. In patients who mount normal type 1 immune responses, few live bacteria are found, little antibody is produced, and, although skin and peripheral nerves are damaged by the inflammatory responses associated with macrophage activation, the disease progresses slowly and the patients typically survive. Because of its similarities to tuberculosis, this variant is called tuberculoid leprosy. This is in contrast to lepromatous leprosy, in which type 1 responses to M. leprae are deficient and an ineffective type 2 response is mounted instead. This results in abundant growth of the bacterium in macrophages and gross tissue destruction that is eventually fatal, if untreated. Although high levels of antibacterial antibodies are produced in patients with lepromatous leprosy, probably due to the high bacterial load, the antibodies are

1	that is eventually fatal, if untreated. Although high levels of antibacterial antibodies are produced in patients with lepromatous leprosy, probably due to the high bacterial load, the antibodies are ineffective at controlling infection because they do not reach the intracellular bacteria.

1	13-21 Immune evasion is also practiced by protozoan parasites. Most common protozoan parasites, such as Plasmodium and Trypanosoma species, have complex life cycles, part of which occurs in humans and part of which occurs in an intermediate host, such as an arthropod vector (for example, mosquitoes, flies, or ticks). The route of transmission of these organisms by their intermediate host is unusual, in that the normal barriers to infection are bypassed when the infectious agent is directly delivered to blood by a bite or the taking of a blood meal. Thus, many of the normal innate immune defenses associated with barrier function are completely bypassed during infection. Further, the most successful of these organisms have developed complex and varied immune evasion strategies that often result in ‘hideandseek’ chronic infections characterized by episodic disease manifestations, despite their eliciting antibody and cellmediated adaptive immune responses.

1	As described above for some bacterial pathogens (see Section 1319), Trypanosoma brucei, a causative agent of trypanosomiasis, or sleeping sickness, has evolved a remarkable capacity for antigenic variation to evade the antibody response elicited in infected humans. The trypanosome is coated with a single type of glycoprotein, the variantspecific glycoprotein (VSG), which elicits a potent protective antibody response that rapidly clears most of the parasites. The trypanosome genome, however, contains about 1000 VSG genes, each encoding a protein with distinct antigenic properties. A VSG gene is expressed by being placed into the active expression site in the parasite genome. Only one VSG gene is expressed at a time, and it can be changed by a gene rearrangement that places a new VSG gene into the expression site (Fig. 13.21). So, under the selective pressure of an effective host antibody response, the few trypanosomes within the population that express a different VSG escape

1	VSG gene into the expression site (Fig. 13.21). So, under the selective pressure of an effective host antibody response, the few trypanosomes within the population that express a different VSG escape elimination and multiply to cause a recurrence of disease (see Fig. 13.21, bottom panel). Antibodies are then made against the new VSG, and the whole cycle is repeated, resulting in periods of active and quiescent disease. The chronic cycles of antigen clearance lead to immunecomplex damage and

1	Fig. 13.21 Antigenic variation in trypanosomes allows them to escape immune surveillance. the surface of a trypanosome is covered with a variant-specific glycoprotein (VSG). each trypanosome has about 1000 genes encoding different VSGs, but only the gene in a specific expression site within the telomere at one end of the chromosome is active. although several genetic mechanisms have been observed for changing the VSG gene expressed, the usual mechanism is gene conversion. an inactive gene, which is not at the telomere, is copied and transposed into the telomeric expression site, where it becomes active. When an individual is first infected, antibodies are raised against the VSG initially expressed by the trypanosome population. a small number of trypanosomes spontaneously switch their VSG gene to a new type, and although the host antibody eliminates the initial variant, the new variant is unaffected. as the new variant grows, the whole sequence of events is repeated.

1	inflammation, and eventually to neurological damage, resulting finally in the coma that gives sleeping sickness its name. The cycles of evasive action make trypanosome infections very difficult for the immune system to defeat, and they are a major health problem in Africa.

1	Caused by Plasmodium species, malaria is another serious and widespread disease. Like Trypanosoma brucei, Plasmodium species vary their antigens to avoid elimination by the immune system. In addition, plasmodia undergo different parts of their life cycle in different cellular hosts within humans. Initial infection is by the sporozoite form of the organism, which is transmitted by the bite of an infected mosquito and targets hepatocytes in the liver. Here, the organism replicates rapidly to produce merozoites that burst from infected hepatocytes to infect circulating red blood cells. Thus, as the immune system focuses it efforts on the eradication of the parasite in the liver, the parasite morphs and escapes to its second cellular host, red blood cells. And because red blood cells are the only cells in the body that lack MHC class I molecules, antigens produced by the merozoites within infected red blood cells escape detection by CD8 T cells, preventing cytotoxic destruction of the

1	only cells in the body that lack MHC class I molecules, antigens produced by the merozoites within infected red blood cells escape detection by CD8 T cells, preventing cytotoxic destruction of the infected cells. This represents one of the most elegant adaptations to evade cellmediated immunity.

1	Immune subversion is also practiced by protozoan parasites. Leishmania major, which is transmitted to dermal tissues of humans by the bite of sandflies, is an obligate intracellular parasite that replicates within tissue macrophages. As is true of other intracellular pathogens that reside within phagocytic vesicles, eradication of L. major is dependent on a type 1 immune response. Through mechanisms that are incompletely understood, L. major specifically inhibits the production of IL12 by its macrophage host, thereby inhibiting the production of IFNγ by NK cells and inhibiting the differentiation and function of TH1 cells. In addition, L. major has been shown to actively induce IL10producing Treg cells that suppress clearance of the infection. 13-22 RNA viruses use different mechanisms of antigenic variation to keep a step ahead of the adaptive immune system.

1	Viruses are both the simplest and most diverse of pathogens. They can replicate only within living cells, relying on host cellular machinery to replicate and propagate themselves. As obligate intracellular pathogens, they activate intracellular PRRs that sense viral genetic material and provoke cytolytic immune responses by innate and adaptive immune cells—NK cells and CD8 T cells, respectively. They also induce type I interferon responses, which activate cellintrinsic mechanisms to limit viral replication in both infected and uninfected cells. Although many cells produce type I interferons, plasmacytoid dendritic cells are innate sensor cells that are specialized for high levels of type I interferon production early in viral infections and, along with NK cells, play a central role in early antiviral host defense before the adaptive response matures. The latter involves all arms of adaptive immunity: induction of TH1 cells that provide help for production of opsonizing and

1	role in early antiviral host defense before the adaptive response matures. The latter involves all arms of adaptive immunity: induction of TH1 cells that provide help for production of opsonizing and complementfixing virusspecific antibodies that block viral entry into uninfected cells and activate complement to destroy enveloped viruses; and cytolytic CD8 T cells, which destroy virally infected cells and produce IFNγ.

1	The strategies used by viruses to defeat immune defenses are as varied as the pathogens themselves. However, some general strategies relate to the type of viral genome. RNA viruses must replicate their genomes using RNA polymerases, which lack the proofreading ability of DNA polymerase. A consequence of this is that RNA viruses have a greater rate of mutation than DNA viruses, with the practical consequence that RNA viruses cannot support large genomes. However, this also affords them opportunities for rapidly altering antigenic epitopes targeted by the adaptive immune system as a mechanism for immune evasion. Further, some RNA viruses have segmented genomes, which lend themselves to reassortment during viral replication. Both of these mechanisms are used by influenza virus, a common seasonal viral pathogen that causes acute infections and has been responsible for several major pandemics. At any one time, a single virus type is responsible for most cases of influenza throughout the

1	viral pathogen that causes acute infections and has been responsible for several major pandemics. At any one time, a single virus type is responsible for most cases of influenza throughout the world. The human population gradually develops protective immunity to this virus type, chiefly through the production of neutralizing antibody directed against the viral hemagglutinin, the main surface protein of the influenza virus. Because the virus is rapidly cleared from immune individuals, it might be in danger of running out of potential hosts were it not able to use both mutation mechanisms to alter its antigenic type (Fig. 13.22).

1	The first of these, caused by point mutations in the genes encoding the two major viral surface glycoproteins—hemagglutinin and neuraminidase—is called antigenic drift. Every 2–3 years a variant flu virus arises with mutations that allow it to evade neutralization by the antibodies present in the population. Other mutations may affect epitopes in viral proteins that are recognized by T cells, particularly CD8 cytotoxic T cells, with the consequence that cells infected with the mutant virus also escape destruction. People immune to the old flu virus are thus susceptible to the new variant, but because the changes in the viral proteins are relatively minor, there is still some crossreaction with antibodies and memory T cells produced against the previous variant, and most of the population still has some level of immunity. Thus, epidemics resulting from antigenic drift are typically mild.

1	Antigenic changes in influenza virus that result from reassortment of the segmented RNA genome are known as antigenic shift, and result in major changes in the hemagglutinin expressed by the virus. Antigenic shifts cause global pandemics of severe disease, often with substantial mortality, because the new hemagglutinin is recognized poorly, if at all, by antibodies and T cells

1	Fig. 13.22 Two types of variation allow repeated infection with type A influenza virus. neutralizing antibody that mediates protective immunity is directed at the viral surface protein hemagglutinin (H), which is responsible for viral binding to and entry into cells. antigenic drift (left panels) involves the emergence of point mutants with altered binding sites for protective antibodies on the hemagglutinin. the new virus can grow in a host that is immune to the previous strain of virus, but as t cells and some antibodies can still recognize epitopes that have not been altered, the new variants cause only mild disease in previously infected individuals. antigenic shift (right panels) is a rare event involving the reassortment of the segmented Rna viral genomes of two different influenza viruses, probably in a bird or a pig. these antigen-shifted viruses have large changes in their hemagglutinin, and therefore t cells and antibodies produced in earlier infections are not protective.

1	viruses, probably in a bird or a pig. these antigen-shifted viruses have large changes in their hemagglutinin, and therefore t cells and antibodies produced in earlier infections are not protective. these shifted strains cause severe infection that spreads widely, causing the influenza pandemics that occur every 10–50 years. there are eight Rna molecules in each viral genome, but for simplicity only three are shown.

1	directed against the previous variant. Antigenic shift is due to reassortment of the segmented RNA genome of the human influenza virus and animal influenza viruses in an animal host, in which the hemagglutinin gene from the animal virus replaces the hemagglutinin gene in the human virus (see Fig. 13.22).

1	Hepatitis C virus (HCV) is an RNA virus that can cause both acute and chronic infections of the liver. It is the most common cause of bloodborne chronic infection in the United States, and the leading viral cause of liver cirrhosis. As with influenza virus, HCV has a high capacity for mutation of immune epitopes that allow it to evade elimination. However, unlike influenza, the viral surface glycoprotein responsible for binding of HCV to hepatocytes (E2, which binds CD81) presents a difficult target against which to produce effective neutralizing antibodies, due both to its heavy glycosylation in the region of CD81 binding and its high rate of mutation. Antibody responses against HCV are therefore of limited effectiveness. Similarly, high rates of mutation of Tcell epitopes select for escape variants of HCV that evade cytolytic Tcell responses. Finally, there is evidence that HCV also expresses factors that subvert the function of dendritic cells, thereby impairing the induction of

1	variants of HCV that evade cytolytic Tcell responses. Finally, there is evidence that HCV also expresses factors that subvert the function of dendritic cells, thereby impairing the induction of Tcell immunity.

1	13-23 DNA viruses use multiple mechanisms to subvert NK-cell and CTL responses.

1	Of all the pathogens, DNA viruses that can establish chronic infections have evolved the greatest diversity of mechanisms for subverting or escaping immune defenses. Unlike RNA viruses, DNA viruses have relatively low mutation rates and are thus less able to employ antigenic variation to evade immune defenses. However, because their lower rate of mutation allows them to support much larger genomes, these viruses have been able to accommodate a remarkable number of viral genes encoding proteins that can subvert nearly every aspect of antiviral defense. In the case of poxvirus, adenovirus, and especially herpesviruses, all of them large DNA viruses that will be our focus here, over 50% of the genome can be dedicated to immune evasionrelated genes. Further, some of these viruses, particularly the herpesviruses, have evolved mechanisms that allow them to enter a state known as latency, in which the virus is not actively replicated. In the latent state, the virus does not cause disease;

1	the herpesviruses, have evolved mechanisms that allow them to enter a state known as latency, in which the virus is not actively replicated. In the latent state, the virus does not cause disease; however, because no viral peptides are produced to load MHC class I molecules that signal the virus’s presence to cytolytic T cells, it cannot be eliminated, and can establish lifelong infections. As we will discuss in Section 1324, latent infections can be reactivated, resulting in recurrent illness. Of the eight types of herpesvirus that infect humans, at least one of the five most common types—herpes simplex virus (HSV)1 and 2 (both of which can cause labial and genital herpes), Epstein–Barr virus (EBV, which causes infectious mononucleosis), varicellazoster (which causes chickenpox and shingles), and cytomegalovirus (CMV)—infects nine out of ten people, and typically establishes lifelong latency. Here we highlight major mechanisms by which these viruses succeed (Fig. 13.23).

1	Central to the longterm survival of the DNA viruses is evasion of CTLs and NK cells. The presentation of viral peptides by MHC class I molecules at a cell surface signals CD8 T cells to kill the infected cell. Many of the large DNA viruses evade immune recognition by producing proteins called immunoevasins, which prevent the appearance of viral peptide:MHC class I complexes on the infected cell (Fig. 13.24). Indeed, at least one viral inhibitor of every key step in the processing and presentation of peptide:MHC class I complexes has been described. Some immunoevasins block peptide entry into the endoplasmic reticulum by targeting the TAP transporter (Fig. 13.25, left panel). Viral proteins can also prevent peptide:MHC complexes from reaching the cell surface by retaining MHC class I molecules in the endoplasmic reticulum (see Fig. 13.25, middle panel). Several viral proteins catalyze the degradation of newly synthesized MHC class I molecules by a process known as dislocation, which

1	in the endoplasmic reticulum (see Fig. 13.25, middle panel). Several viral proteins catalyze the degradation of newly synthesized MHC class I molecules by a process known as dislocation, which initiates the pathway normally used to degrade misfolded endoplasmic reticulum proteins by directing them back into the cytosol (Fig. 13.25, right panel). By preventing the formation of stably assembled/folded peptide:MHC class I complexes, these viral proteins divert the peptide:MHC class I complexes into the ERassociated degradation (ERAD) pathway for disposal. Through these multiple mechanisms, viral factors impair or completely block the presentation of viral peptides to CTLs. The actions of viral inhibitors are not limited to the MHC class I pathway, as viral inhibitors of the class II processing pathway have also been described; these inhibitors ultimately target CD4 T cells. Finally, as many viruses target cells other than dendritic cells, their antigens come to the attention of CD8 T

1	pathway have also been described; these inhibitors ultimately target CD4 T cells. Finally, as many viruses target cells other than dendritic cells, their antigens come to the attention of CD8 T cells via crosspresentation. Viral mechanisms that interfere with this pathway are not well described, although it is known that because the viruses are not required to persist in dendritic cells, the viruses can block recognition and destruction of their cellular hosts even after primed CTL effectors have been generated.

1	In addition to their role in the acute innate response to viral infection, a major function of NK cells is the recognition and cytolysis of cells that have downregulated MHC class I molecules as a foil to pathogen attempts to evade detection by CTLs. Accordingly, viruses that target the MHC class I pathway have also evolved mechanisms to repress the cytolytic activity of NK cells. Strategies here include, but are not limited to, expression of viral homologs of MHC class I that engage killer inhibitory receptors (KIRs) and leukocyte inhibitory receptors (LIRs). For example, human CMV produces a homolog of HLA class I called Fig. 13.24 Immunoevasins produced by viruses interfere with the processing of antigens that bind to MHC class I molecules. UL18, which binds LIR1 on NK cells and provides an inhibitory signal that blocks NKcell cytolysis. Viral products have been defined that also antagonize activating receptors on NK cells, as well as inhibit NKcell effector pathways.

1	DNA viruses have evolved mechanisms to subvert additional functions of the immune system. The mechanisms used include the expression of viral homologs of cytokines or chemokines and their receptors, or viral proteins

1	Fig. 13.25 The peptide-loading complex in the endoplasmic reticulum is targeted by viral immunoevasins. the left panel shows blockade of peptide entry to the endoplasmic reticulum (eR). the cytosolic iCP47 protein from herpes simplex virus (HSV)-1 prevents peptides from binding to taP in the cytosol, whereas the uS6 protein from human CMV interferes with the atP-dependent transfer of peptides through taP. the middle panel shows the retention of MHC class i molecules in the eR by the adenovirus e19 protein. this binds certain MHC molecules and retains them in the eR through an eR-retention motif, at the same time competing with tapasin to prevent association with taP and peptide loading. the right panel shows how the murine herpes virus mK3 protein, an e3ubiquitin ligase, targets newly synthesized MHC class i molecules. mK3 associates with tapasin:taP complexes and directs the addition of ubiquitin subunits with K48 linkages (see Section 7-5) to the cytoplasmic tail of the MHC class i

1	MHC class i molecules. mK3 associates with tapasin:taP complexes and directs the addition of ubiquitin subunits with K48 linkages (see Section 7-5) to the cytoplasmic tail of the MHC class i molecule. the polyubiquitination of the cytoplasmic tail of MHC initiates the process of degradation of the MHC molecule by the proteasomal pathway.

1	that bind cytokines or their receptors to inhibit their actions. As type I and II interferons are major effector cytokines in antiviral defense, diverse viral strategies are centered on the inhibition of this family of cytokines, whether by production of decoy receptors or inhibitory binding proteins, inhibition of JAK/STAT signaling by IFN receptors, inhibition of cytokine transcription, or interference with transcription factors induced by IFNs. Some DNA viruses also produce antagonists of the proinflammatory cytokines IL1, IL18, and TNFα, among others. Viral homologs of immunosuppressive cytokines are also produced. CMV impairs antiviral responses by producing a homolog of the cytokine IL10, called cmvIL10, which downregulates the production of several proinflammatory cytokines by immune cells, including IFNγ, IL12, IL1, and TNFα, to promote tolerogenic rather than immunogenic adaptive responses to viral antigens.

1	Several viruses also interfere with chemokine responses by producing either decoy chemokine receptors or chemokine homologs that interfere with natural ligandinduced signaling through chemokine receptors. Collectively, herpesviruses and poxviruses produce over 40 viral homologs of receptors belonging to the seven transmembranespanning Gproteincoupled chemokine receptor (vGPCR) superfamily. Finally, CMV has been shown to promote chronic infection that is associated with ‘exhaustion’ of antiviral CD8 T cells. CD8 T cells induced in this setting are characterized by expression of an inhibitory receptor of the CD28 superfamily, the programmed death1 (PD1) receptor (see Section 724), activation of which by its ligand PDL1 suppresses CD8 Tcell effector function. Blockade of the PDL1–PD1 interaction restores antiviral CD8 effector function and decreases the viral load, indicating that ongoing activation of this pathway is involved in impaired viral clearance. A similar mechanism has been

1	restores antiviral CD8 effector function and decreases the viral load, indicating that ongoing activation of this pathway is involved in impaired viral clearance. A similar mechanism has been implicated in RNA viruses that can establish chronic infections, such as hepatitis C virus (HCV). Suffice it to say that the range of strategies that viruses have evolved to subvert immune clearance mechanisms is quite remarkable, and the discovery of these mechanisms continues to have a major impact on our understanding of host–pathogen relationships.

1	13-24 Some latent viruses persist in vivo by ceasing to replicate until immunity wanes.

1	As mentioned in the previous section, a major class of viral agents that cause latent infections in humans are the herpesviruses, large, enveloped DNA viruses that are characterized by their ability to establish lifelong infections. While we have considered a number of strategies by which these viruses subvert immunity, they have also evolved mechanisms to maintain their genome within the nucleus of infected cells indefinitely without replicating. In contrast to an actively lytic, or productive, phase of the viral life cycle, wherein the virus replicates and lyses its cellular host, herpesviruses can establish latency, or a lysogenic phase, by expression of a small region of their genome called the latency associated transcript (LAT). In addition to suppressing the transcription of the remaining viral genome, the LAT produces factors that interfere with apoptotic death of the host cell, both interfering with immune mechanisms that might clear the cell and prolonging the cell’s

1	the remaining viral genome, the LAT produces factors that interfere with apoptotic death of the host cell, both interfering with immune mechanisms that might clear the cell and prolonging the cell’s lifespan—and that of the viral genome it harbors. An example is herpes simplex virus (HSV), the cause of cold sores, which infects epithelial cells and then spreads to sensory neurons serving the infected area. An effective immune response controls the epithelial infection, but the virus persists in a latent state in the sensory neurons. Factors such as sunlight, bacterial infection, or hormonal changes reactivate the virus, which then travels down the axons of the sensory neuron and reinfects the epithelial tissues (Fig. 13.26). At this point, the immune response again becomes active and controls the local infection by killing the epithelial cells, producing a new sore. This cycle can be repeated many times.

1	Fig. 13.26 Persistence and reactivation of herpes simplex virus infection. the initial infection in the skin is cleared by an effective immune response, but residual infection persists in sensory neurons such as those of the trigeminal ganglion, whose axons innervate the lips. When the virus is reactivated, usually by some environmental stress and/or alteration in immune status, the skin in the area served by the nerve is reinfected from virus in the ganglion and a new cold sore results. this process can be repeated many times.

1	There are two reasons why the sensory neuron remains infected: first, the virus is quiescent and generates few virusderived peptides to present on MHC class I molecules; second, neurons carry very low levels of MHC class I molecules, which makes it harder for CD8 cytotoxic T cells to recognize infected neurons and attack them. The low level of MHC class I expression is beneficial because it reduces the risk that neurons, which have a limited capacity for regeneration, will be targeted inappropriately by cytotoxic T cells. It does, however, make neurons attractive as cellular reservoirs for persistent infections. Herpesviruses often enter latency. Herpes zoster (varicellazoster), which causes chickenpox, remains latent in one or a few dorsal root ganglia after the acute illness is over, and can be reactivated by stress or immunosuppression. It then spreads down the nerve and reinfects the skin to cause the disease herpes zoster, or shingles, which is marked by the reappearance of the

1	can be reactivated by stress or immunosuppression. It then spreads down the nerve and reinfects the skin to cause the disease herpes zoster, or shingles, which is marked by the reappearance of the classic varicella rash in the area of skin served by the infected dorsal root. Unlike herpes simplex, which reactivates frequently, herpes zoster usually reactivates only once in a lifetime in an immunocompetent host.

1	Yet another herpesvirus, the Epstein–Barr virus (EBV), establishes a persistent infection in most individuals. EBV enters latency in B cells after a primary infection that often passes without being diagnosed. In a minority of infected individuals, the initial acute infection of B cells is more severe, causing the disease known as infectious mononucleosis or glandular fever. EBV infects B cells by binding to CR2 (CD21), a component of the Bcell coreceptor complex, and to MHC class II molecules. In the primary infection, most of the infected cells proliferate and produce virus, leading in turn to the proliferation of antigenspecific T cells and the excess of mononuclear white cells in the blood that gives the disease its name. Virus is released from the B cells, destroying them in the process, and virus can be recovered from saliva. The infection is eventually controlled by virusspecific CD8 cytotoxic T cells, which kill the infected proliferating B cells. A fraction of memory B

1	process, and virus can be recovered from saliva. The infection is eventually controlled by virusspecific CD8 cytotoxic T cells, which kill the infected proliferating B cells. A fraction of memory B lymphocytes become latently infected, however, and EBV remains quiescent in these cells.

1	These two forms of infection are accompanied by quite different patterns of expression of viral genes. EBV has a large DNA genome encoding more than 70 proteins. Many of these are required for viral replication and are expressed by the replicating virus, providing a source of viral peptides by which infected cells can be recognized. In a latent infection, in contrast, the virus survives within the host B cells without replicating, and a very limited set of viral proteins is expressed. One of these is the Epstein–Barr nuclear antigen 1 (EBNA1), which is needed to maintain the viral genome. EBNA1 interacts with the proteasome (see Section 62) to prevent its own degradation into peptides that would otherwise elicit a Tcell response.

1	Latently infected B cells can be isolated by culturing B cells from individuals who have apparently cleared their EBV infection: in the absence of T cells, latently infected cells retaining the EBV genome become transformed into socalled immortal cell lines, the equivalent of tumorigenesis in vitro. Infected B cells occasionally undergo malignant transformation in vivo, giving rise to a Bcell lymphoma called Burkitt’s lymphoma. In this lymphoma, expression of the peptide transporters TAP1 and TAP2 is downregulated (see Section 63), and so cells are unable to process endogenous antigens for presentation on HLA class I molecules (the human MHC class I). This deficiency provides one explanation for how these tumors escape attack by CD8 cytotoxic T cells. Patients with acquired and inherited immunodeficiencies of Tcell function have an increased risk of developing EBVassociated lymphomas, presumably as a result of a failure of immune surveillance.

1	The viruses hepatitis B (HBV, a DNA virus) and hepatitis C (HCV, an RNA virus) infect the liver and cause acute and chronic hepatitis, liver cirrhosis, and in some cases hepatocellular carcinoma. Immune responses probably have an important role in the clearance of both types of hepatitis infection, but in many cases HBV and HCV set up a chronic infection. Although HCV mainly infects the liver during the early stage of a primary infection, the virus subverts the adaptive immune response by interfering with dendriticcell activation and maturation. This leads to inadequate activation of CD4 T cells and a consequent lack of TH1 cell differentiation, which is thought to be responsible for the infection becoming chronic, most probably because of the lack of CD4 Tcell help to activate naive CD8 cytotoxic T cells. There is evidence that the decrease in levels of viral antigen seen after antiviral treatment improves CD4 Tcell help and allows the restoration of cytotoxic CD8 Tcell function and

1	cytotoxic T cells. There is evidence that the decrease in levels of viral antigen seen after antiviral treatment improves CD4 Tcell help and allows the restoration of cytotoxic CD8 Tcell function and memory CD8 Tcell function. The delay in dendriticcell maturation caused by HCV is thought to synergize with another property of the virus that helps it to evade an immune response: the RNA polymerase that the virus uses to replicate its genome lacks proofreading capacity. This contributes to a high viral mutation rate and thus a change in its antigenicity, which allows it to evade adaptive immunity.

1	Summary. Infectious agents can cause recurrent or persistent disease by avoiding normal host defense mechanisms or by subverting them to promote their own replication. There are many different ways of evading or subverting the immune response. Antigenic variation, latency, resistance to immune effector mechanisms, and suppression of the immune response all contribute to persistent and medically important infections. In some cases the immune response is part of the problem: some pathogens use immune activation to spread infection, and others would not cause disease if it were not for the immune response. Each of these mechanisms teaches us something about the nature of the immune response and its weaknesses, and each requires a different medical approach to prevent or to treat infection. Acquired immune deficiency syndrome.

1	Acquired immune deficiency syndrome. The most extreme example of immune subversion by a pathogen is the acquired immune deficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV). The disease is characterized by a progressive loss of CD4 T cells, which, when they have become sufficiently depleted, results in high susceptibility to opportunistic infections and certain malignancies. The earliest documented case of HIV infection in humans to date was reported in a sample of serum from Kinshasa (Democratic Republic of the Congo) that was stored in 1959. It was not until 1981, however, that the first cases of AIDS were officially reported. Because the disease seemed to be spread by contact with body fluids, the cause was suspected to be a new virus, and by 1983 the causative agent, HIV, was isolated and identified.

1	There are at least two types of HIV—HIV1 and HIV2—that are closely related. While both types are transmitted by sexual contact and bloodborne exposure (for example, blood transfusion, shared needles), HIV1 replicates to higher viral loads in blood and is therefore more easily transmitted; HIV1 has a high rate of transmission from mother to child, which is uncommon in HIV2. Although disease is indistinguishable in patients who progress to AIDS, HIV1 progresses to AIDS more rapidly and with greater incidence than HIV2. HIV1 is therefore by far the most prevalent cause of AIDS worldwide. And while both HIV1 and HIV2 are endemic to West Africa, HIV2 is rarely found elsewhere. Both viruses seem to have originally spread to humans from other primate species in Africa. Viral genome sequencing of isolates suggests that the primate precursor of HIV1, simian immunodeficiency virus (SIV), has passed

1	Fig. 13.27 Phylogenetic origins of HIV-1 and HIV-2. HiV-1 shows marked genetic variability and is classified on the basis of genomic sequence into four major groups: M (main), O (outlier), n (non-M, non-O), and P (non-M, non-n, non-O), which are further diversified into subtypes, or clades, that are designated by the letters a to K. in different parts of the world, different subtypes predominate. Phylogenetic analyses of chimpanzee simian immunodeficiency virus (SiVcpz), gorilla SiV (SiVgor), and HiV-1 sequences demonstrate that the four groups of HiV-1 (M, n, O, and P) originated from four independent cross-species transmission events: two transfers of SiVcpzPtt from central chimpanzees (subspecies Pan troglodytes troglodytes, or Ptt) gave rise to HiV-1 groups M and n, while two transfers of SiVgor from western lowland gorillas (subspecies Gorilla gorilla gorilla) gave rise to HiV-1 groups O and P. Similarly, separate zoonotic transmissions of SiVsmm from sooty mangabey monkeys to

1	of SiVgor from western lowland gorillas (subspecies Gorilla gorilla gorilla) gave rise to HiV-1 groups O and P. Similarly, separate zoonotic transmissions of SiVsmm from sooty mangabey monkeys to humans are responsible for at least nine different lineages of HiV-2 (groups a–H and a newly described lineage, u). SiVstm and SiVmac resulted from experimental infections of stump-tailed macaques and rhesus macaques with SiVsmm, respectively. abbreviations: cpzPts, chimpanzee Pan troglodytes schweinfurthii; cpzPtt, chimpanzee Pan troglodytes troglodytes; mac, macaque; SiV, simian immunodeficiency virus; smm, sooty mangabey monkey; stm, stump-tailed macaque. Figure courtesy of Drs. Beatrice Hahn and Gerald learn.

1	to humans on at least four independent occasions from chimpanzees or western lowland gorillas, whereas HIV2 originated in the sooty mangabey (Fig. 13.27). The best estimate is that the most prevalent of the four major variants of HIV1, group M (responsible for ~99% of HIV1 infections worldwide), was transmitted to humans from chimpanzees in the first half of the twentieth century; transmission of group O also dates to the early twentieth century, whereas the two other HIV1 variants (groups N and P) appear to have been transmitted more recently. As is true for other zoonotic infections where there has been insufficient time for pathogen and host to coevolve to an equilibrium that attenuates virulence, SIV is generally less pathogenic in its nonhuman primate host than is HIV in its human host. Thus, whereas development of AIDS is nearly universal in HIV1infected humans that do not receive treatment, development of AIDS in SIVinfected nonhuman primates is considerably more variable, with

1	Thus, whereas development of AIDS is nearly universal in HIV1infected humans that do not receive treatment, development of AIDS in SIVinfected nonhuman primates is considerably more variable, with some primates failing to develop disease at all.

1	HIV infection does not immediately cause AIDS. Without treatment, the average time to development of AIDS following infection of adults is several years. The long delay between infection and development of symptomatic immune deficiency reflects the unusual tropism of the virus for CD4 T cells of the immune system, as well as the nature of the immune response to the virus. HIV is now pandemic, and despite great strides in treatment and prevention that have followed from a greater understanding of the pathogenesis and epidemiology of the disease, 1.6 million people died of AIDSrelated causes in 2012 and an estimated 35.3 million are infected by HIV worldwide, presaging the death of many from AIDS for years to come (Fig. 13.28). In subSaharan Africa, which accounts for over twothirds of the global incidence, 1 in every 20 adults is infected. Indeed, HIV/AIDS has emerged as the most deadly single infectious agent in the short time since its identification as a new human pathogen.

1	of the global incidence, 1 in every 20 adults is infected. Indeed, HIV/AIDS has emerged as the most deadly single infectious agent in the short time since its identification as a new human pathogen. Nevertheless, there is cause for optimism: the incidence of new cases of HIV infections worldwide has declined annually since its peak in 1997, and the number of annual deaths from HIV/AIDS has steadily declined since its peak in the mid2000s. Among the regions with the most rapid declines in incidence of new infections is subSaharan Africa. Still, there are focal areas of increasing incidence (for example, Eastern Europe and Central Asia).

1	13-25 HIV is a retrovirus that establishes a chronic infection that slowly progresses to AIDS.

1	HIV is an enveloped RNA virus whose structure is shown in Fig. 13.29. Each virus particle, or virion, is decorated with two viral envelope proteins that are used by the virus to infect target cells, and contains two copies of an RNA genome and numerous copies of viral enzymes that are required to establish infection in the cellular host. HIV is an example of a retrovirus, so named because the viral genome must be transcribed from RNA into DNA in the infected cell—the reverse (retro) of the usual pattern of transcription—by the viral reverse transcriptase enzyme. This generates a DNA intermediate that is integrated into the hostcell chromosomes to enable viral replication. RNA transcripts produced from the integrated viral DNA serve both as mRNAs to direct the synthesis of viral proteins and later as the RNA genomes of new viral particles. These escape from the cell by budding from the plasma membrane, each enclosed in a membrane envelope.

1	Worldwide totals 35.3 million/2.3 million/1.6 million total cases in 2012 new cases in 2012 deaths in 2012 Sub-Saharan Africa 25 million/1.6 million/1.2 million Caribbean 250,000/12,000/11,000 South and South-east Asia 3.9 million/270,000/220,000 Latin America 1.5 million/86,000/52,000 Eastern Europe and Central Asia 1.3 million/130,000/91,000 East Asia 800,000/270,000/220,000 North America 1.3 million/48,000/20,000 Western Europe 860,000/29,000/17,000 North Africa and Middle East 260,000/32,000/17,000 Oceania 51,000/2100/1200 Fig. 13.28 The incidence of new HIV infection is increasing more slowly in many regions of the world, but AIDS is still a major disease burden. the number of individuals living with HiV/aiDS is large and continues to grow, but the number of new infections in 2012 decreased by over one-third since the peak of the epidemic. Worldwide, in 2012, it is estimated that there were around 35.3 million individuals infected with HiV, including some 2.4 million new cases,

1	by over one-third since the peak of the epidemic. Worldwide, in 2012, it is estimated that there were around 35.3 million individuals infected with HiV, including some 2.4 million new cases, and approximately 1.6 million deaths from aiDS, a decrease of 30% since the peak in 2005. new infections in children have declined approximately 50% since 2001, with 260,000 new cases in 2012. (AIDS Epidemic Update, unaiDS/World Health Organization, 2013.)

1	HIV belongs to a group of retroviruses called the lentiviruses, from the Latin lentus, meaning slow, because of the gradual course of the diseases that they cause. These viruses persist and continue to replicate for many years before causing overt signs of disease. In the case of HIV, the virus targets cells of the immune system itself, producing an initial acute infection that is controlled to the point that infection is not apparent, but rarely leading to an immune response that can prevent ongoing replication of the virus. Thus, although the initial acute infection does seem to be controlled by the immune system, HIV establishes latency within cells of the immune system and continues to replicate and infect new cells for many years. As will be discussed below, this ultimately exhausts the immune system, resulting in immune deficiency, or AIDS, that leads to opportunistic infections and/or malignancy that cause death.

1	Fig. 13.29 The virion of human immunodeficiency virus (HIV). the virus illustrated is HiV-1, the leading cause of aiDS. the virion is roughly spherical and measures 120 nm in diameter, or about 60 times smaller than the t cells it infects. the three viral enzymes that are packaged in the virion—reverse transcriptase, integrase, and protease—are shown schematically in the viral capsid. in reality, many molecules of these enzymes are contained in each virion. Photograph courtesy of H. Gelderblom. gp120env RNA genome envelope protease (p10) integrase (p32) nucleocapsid reverse transcriptase (p64) lipid membrane MHC proteins matrix proteins gp41 p17gag gp120/gp41 complex, or viral spike 13-26 HIV infects and replicates within cells of the immune system.

1	A defining characteristic of HIV is its ability to infect and replicate within activated cells of the immune system. Three immune cell types are the primary targets of HIV infection: CD4 T cells, macrophages, and dendritic cells. Of these, CD4 T cells support the great majority of viral replication. HIV’s ability to enter particular cell types, known as its cellular tropism, is determined by the expression of specific receptors for the virus on the surface of those cells. HIV enters cells by means of a complex of two noncovalently associated viral glycoproteins, gp120 and gp41, which form trimers in the viral envelope. The gp120 subunits of trimeric gp120/gp40 complexes bind with high affinity to the cellsurface molecule CD4, which is expressed on CD4 T cells, and to a lesser extent on subsets of dendritic cells and macrophages. Before fusion and entry of the virus, gp120 must also bind a coreceptor on the host cell. The major coreceptors are the chemokine receptors CCR5 and CXCR4.

1	subsets of dendritic cells and macrophages. Before fusion and entry of the virus, gp120 must also bind a coreceptor on the host cell. The major coreceptors are the chemokine receptors CCR5 and CXCR4. While CCR5 is predominantly expressed on subsets of effector memory CD4 T cells, dendritic cells, and macrophages, CXCR4 is expressed primarily by naive and central memory CD4 T cells. As we will discuss below, the particular chemokine coreceptor bound by a given viral particle is of importance in the transmission of HIV between individuals and its propagation within an infected person. After binding CD4, gp120 undergoes a conformational change that exposes a highaffinity site that is bound by the coreceptor. This, in turn, causes gp41 to unfold and insert a portion of its structure (fusion peptide) into the plasma membrane of the target cell, inducing fusion of the viral envelope with the cell’s plasma membrane. This allows the viral nucleocapsid, composed of the viral genome and

1	peptide) into the plasma membrane of the target cell, inducing fusion of the viral envelope with the cell’s plasma membrane. This allows the viral nucleocapsid, composed of the viral genome and associated viral proteins, to enter the hostcell cytoplasm (Fig. 13.30).

1	Once the virus has entered cells, it replicates similarly to other retroviruses. Reverse transcriptase transcribes the viral RNA into a complementary DNA (cDNA) copy. The viral cDNA, which encodes nine genes (Fig. 13.31), is then integrated into the hostcell genome by viral integrase, which recognizes and partially cleaves repetitive DNA sequences, called long terminal repeats (LTRs), that reside at each end of the viral genome. LTRs are required for the integration of the provirus into the hostcell DNA and contain binding sites for generegulatory proteins that control expression of the viral genes. The integrated cDNA copy is known as the provirus.

1	Like other retroviruses, the HIV genome is small, with three major genes— gag, pol, and env. The gag gene encodes the structural proteins of the viral nucleocapsid core, pol encodes the enzymes involved in viral replication, and env encodes the viral envelope glycoproteins. The gag and pol mRNAs are translated to give polyproteins—long polypeptide chains that are then cleaved by the viral protease (encoded by pol) into individual functional proteins. Thus, pol alone encodes the virion’s three major enzymes that are required for viral replication: reverse transcriptase, integrase, and viral protease. The product of the env gene, gp160, has to be cleaved by a hostcell protease into gp120 and gp41, which are then assembled as trimers in the viral envelope. HIV has six other, smaller, regulatory genes encoding proteins that affect viral replication and infectivity in various ways. Two of these, Tat and Rev, perform regulatory functions that are essential early in the viral replication

1	genes encoding proteins that affect viral replication and infectivity in various ways. Two of these, Tat and Rev, perform regulatory functions that are essential early in the viral replication cycle. The remaining four—Nef, Vif, Vpr, and Vpu—are essential for efficient virus production in vivo.

1	HIV can complete its replication cycle in the host cell to produce progeny virus, or, like other retroviruses and herpesviruses, establish a latent infection in which the provirus remains quiescent. What determines whether infection of a cell results in latency or a productive infection is unclear, but is thought to be related to the activation state of the cell infected. As we will discuss in the next section, transcription of the provirus following integration is initiated by host transcription factors, which are induced by immunecell activation.

1	Viral cDNA enters nucleus and is integrated into host DNA Reverse transcriptase copies viral RNA genomes into double-stranded cDNA Viral envelope fuses with cell membrane allowing viral genome to enter the cell Virus particle binds to CD4 and co-receptor on T cell nucleus CD4reverse transcriptase viral RNA genomegp41 gp120 viral cDNA provirus chromosomal DNA cytoplasm co-receptor cell membrane The late proteins Gag, Pol, and Env are translated and assembled into virus particles, which bud from the cell Tat ampliÿes transcription of viral RNA. Rev increases transport of singly spliced or unspliced viral RNA to cytoplasm RNA transcripts are multiply spliced, allowing translation of early genes tat and rev T-cell activation induces low-level transcription of provirus gp160PolGagTatNFB˜Rev

1	Fig. 13.30 The life cycle of HIV. top row: the virus binds to CD4 using gp120, which is altered by CD4 binding so that it now also binds a chemokine receptor that acts as a co-receptor for viral entry. this binding releases gp41, which causes fusion of the viral envelope with the cell membrane and release of the viral core into the cytoplasm. Once in the cytoplasm, the viral core releases the Rna genome, which is reverse-transcribed into double-stranded cDna using the viral reverse transcriptase. the double-stranded cDna migrates to the nucleus in association with the viral integrase and the Vpr protein and is integrated into the cell genome, becoming a provirus. Bottom row: activation of CD4 t cells induces the expression of the transcription factors nFκB and nFat, which bind to the proviral ltR and initiate transcription of the HiV genome. the first viral transcripts are extensively processed, producing spliced mRnas encoding several regulatory proteins, including tat and Rev. tat

1	ltR and initiate transcription of the HiV genome. the first viral transcripts are extensively processed, producing spliced mRnas encoding several regulatory proteins, including tat and Rev. tat both enhances transcription from the provirus and binds to the Rna transcripts, stabilizing them in a form that can be translated. Rev binds the Rna transcripts and transports them to the cytosol. as levels of Rev increase, less extensively spliced and unspliced viral transcripts are transported out of the nucleus. the singly spliced and unspliced transcripts encode the structural proteins of the virus, and unspliced transcripts, which are also the new viral genomes, are packaged with these proteins to form many new virus particles.

1	Thus, infection of a cell that becomes dormant soon after infection might favor viral latency, whereas infection of activated cells favors productive viral replication. This has important consequences in the case of CD4 T cells, which, unlike macrophages and dendritic cells, are very longlived and can provide a reservoir of latent HIV provirus that can be activated when the T cells are reactivated, even years after initial infection. Because macrophages and dendritic cells in tissues are shortlived cells that do not divide, latency in these

1	Fig. 13.31 The genomic organization of HIV. like all retroviruses, HiV-1 has an Rna genome flanked by long terminal repeats (ltRs) involved in viral integration and in regulation of transcription of the viral genome. the genome can be read in three frames, and several of the viral genes overlap in different reading frames. this allows the virus to encode many proteins in a small genome. the three main protein products—Gag, Pol, and env—are synthesized by all infectious retroviruses. the known functions of the different genes and their products are listed. the products of gag, pol, and env are known to be present in the mature viral particle, together with the viral Rna. the mRnas for tat, Rev, and nef proteins are produced by splicing of viral transcripts, so their genes are split in the viral genome. in the case of nef, only one exon, shown in yellow, is translated.

1	Gene product/function Group-speciÿc antigen gag pol env tat rev vif Polymerase Envelope Transactivator Regulator of viral expression Viral infectivity vpr Viral protein R vpu Viral protein U nef Negative-regulation factor Gene pol env tat LTR LTR gag Core proteins and matrix proteins Reverse transcriptase, protease, and integrase enzymes Transmembrane glycoproteins. gp120 binds CD4 and CCR5; gp41 is required for virus fusion and internalization Positive regulator of transcription Allows export of unspliced and partially spliced transcripts from nucleus Affects particle infectivity Transport of DNA to nucleus. Augments virion production. Cell-cycle arrest Promotes intracellular degradation of CD4 and enhances release of virus from cell membrane Augments viral replication in vivo and in vitro. Decreases CD4, MHC class I and II expression nefrev tat rev vif vpr vpu host cells would be shortlived. Thus, longlived latency of HIV is primarily a consequence of the tropism of the virus for

1	Decreases CD4, MHC class I and II expression nefrev tat rev vif vpr vpu host cells would be shortlived. Thus, longlived latency of HIV is primarily a consequence of the tropism of the virus for CD4 T cells. The combined features of tropism for CD4 T cells and activationdependent transcription of the provirus are central to the pathogenesis of HIV and its characteristic progressive depletion of CD4 T cells that leads to AIDS.

1	13-27 Activated CD4 T cells are the major source of HIV replication.

1	HIV provirus requires activation of the host cell to complete its replication cycle and produce infectious virions that can infect other cells. This is due to a requirement for transactivation of proviral gene expression by transcription factors of the host cell. Two host transcription factors can initiate transcription of the viral genome: NFκB and NFAT. Both of these factors require cellular activation for their translocation to the nucleus, where they bind DNA and induce gene transcription (see Sections 714 and 716). While NFκB is expressed in all of the immune cells infected by HIV, NFAT is primarily activated in CD4 T cells, enabling transactivation of the provirus by an additional factor in this cell host. This, coupled with the fact that CD4 T cells are longlived and abundant in immune tissues, contributes to CD4 T cells being the major cellular source for HIV replication. Here we consider the mechanism by which transcription of the HIV provirus is regulated in CD4 T cells.

1	As discussed in Sections 714 and 716, activation of T cells by antigen induces activation and nuclear translocation of NFAT and NFκB; activation of effectormemory T cells by cytokines can also activate NFκB in the absence of antigen (Section 1112). Thus, in addition to antigendependent activation of HIV provirus transcription by NFAT and NFκB, provirus might be activated independently of Tcell receptor stimulation in memory CD4 T cells via NFκB alone, as it is in infected macrophages and dendritic cells. Binding of NFAT and NFκB initiates transcription of viral RNA by binding to promoters in the proviral LTR. The viral transcript is spliced in various ways to produce mRNAs for translation of viral proteins (see Fig. 13.26).

1	At least two of the viral proteins—Tat and Rev—serve to enhance production of the viral genome (see Fig. 13.30). Tat binds to a transcriptional activation region (TAR) in the 5ʹ LTR. This recruits cellular cyclin T1 and its partner, cyclindependent kinase 9 (CDK9), to form a complex that phosphorylates RNA polymerase and enhances its ability to generate fulllength transcripts of the viral genome. In this way, Tat provides a positive feedback circuit for amplification of productive viral replication. Rev is important for shuttling unspliced viral RNA transcripts out of the nucleus by binding to a specific viral RNA sequence, the Rev response element (RRE). Eukaryotic cells have mechanisms to prevent the export from the cell nucleus of incompletely spliced mRNA transcripts. This could pose a problem for the retrovirus, which is dependent on export of unspliced mRNA species that encode the full complement of viral proteins, as well as the viral RNA genome. While export of a fully spliced

1	problem for the retrovirus, which is dependent on export of unspliced mRNA species that encode the full complement of viral proteins, as well as the viral RNA genome. While export of a fully spliced mRNA transcript that encodes Tat and Rev occurs early after viral infection by means of the normal host cellular mechanisms of mRNA export, the export of later, unspliced viral transcripts requires Rev to prevent their destruction by the host cell.

1	The success of viral replication also depends on the proteins Nef, Vif, Vpu, and Vpr. These viral products appear to have evolved to defeat host immune mechanisms of viral clearance, as well as antiviral restriction factors—host cellular proteins that function in a cellautonomous manner to inhibit replication of retroviruses. Nef (negative regulation factor) performs multiple critical functions in the viral life cycle. It acts early in the viral life cycle to sustain Tcell activation and the establishment of a persistent state of HIV infection, in part by lowering the threshold for Tcell receptor signaling and downregulating expression of the inhibitory costimulatory receptor CTLA4. Combined, these actions result in greater and more sustained Tcell activation that promotes viral replication. Nef also contributes to immune evasion of infected cells by downregulating expression of MHC class I and class II molecules, making actively infected cells less likely to induce an antiviral

1	Nef also contributes to immune evasion of infected cells by downregulating expression of MHC class I and class II molecules, making actively infected cells less likely to induce an antiviral immune response or be killed by cytotoxic T cells. Nef also promotes the clearance of surface CD4 molecules, which otherwise would bind to the virion during budding and interfere with virion release. Vif (viral infectivity factor) acts to overcome a cytidine deaminase called APOBEC, which catalyzes the conversion of deoxycytidine to deoxyuridine in reversetranscribed viral cDNA, thereby destroying its ability to encode viral proteins. Vpu (viral protein U) is unique to HIV1 and variants of SIV, and is required to overcome a cellular factor called tetherin, which inserts into both the plasma membrane of the host cell and the envelope of the mature virion to block its release. The function of Vpr (viral protein R) is not fully understood, but it appears to target the restriction factor SAMHD1, a

1	of the host cell and the envelope of the mature virion to block its release. The function of Vpr (viral protein R) is not fully understood, but it appears to target the restriction factor SAMHD1, a cellular protein that inhibits HIV1 infection in myeloid cells and quiescent CD4 T cells by limiting the intracellular pool of deoxynucleotides (dNTPs) available for viral cDNA synthesis by reverse transcriptase.

1	13-28 There are several routes by which HIV is transmitted and establishes infection. Infection with HIV occurs after the transfer of body fluids from an infected person to an uninfected one. HIV infection is most commonly spread by sexual intercourse. Transmission by the exchange of contaminated needles used for intravenous drug delivery or by the therapeutic use of infected blood or blood products also occurs, although the latter route of transmission has largely been eliminated in countries where blood products are routinely screened for HIV. An important route of virus transmission is from an infected mother to child, which can occur in utero, during birth, or through breast milk. Rates of transmission from an untreated infected mother to her child vary (from about 15% to 45%), depending on the viral load in the mother and whether the mother breastfeeds the child, as breastfeeding increases the risk of transmission.

1	The use of antiretroviral drugs to decrease maternal viral load during pregnancy dramatically reduces the transmission rate to the child (see Section 1335).

1	The virus can be transmitted as free infectious particles or via infected cells for which the virus has tropism (for example, CD4 T cells and macrophages). Infected cells are found in blood, but can also be present in semen or vaginal secretions, as well as breast milk; free virus is present in blood, semen, vaginal fluid, or mother’s milk. As we discuss in the next section, HIV virions can differentially express gp120 variants that bind either CCR5 or CXCR4, thereby influencing the cell types infected. In the genital and gastrointestinal mucosae, which are the dominant sites of primary infection by sexual transmission, HIV virions establish infection initially in a small number of mucosal immune cells that express CCR5—effector memory CD4 T cells, dendritic cells, and macrophages. The virus replicates locally in these cells before spreading via T cells or dendritic cells (mucosal macrophages are nonmigratory) to lymph nodes draining the mucosa. The lymphoid compartment of mucosal

1	virus replicates locally in these cells before spreading via T cells or dendritic cells (mucosal macrophages are nonmigratory) to lymph nodes draining the mucosa. The lymphoid compartment of mucosal tissues is enriched for TH1 and TH17 cells, which express CCR5 (naive T cells and TH2 cells do not), so initial viral replication is favored in these subsets of CD4 T cells. After accelerated replication in regional lymph nodes, where there is a high concentration of CD4 T cells, the virus disseminates widely via the bloodstream, and gains broader access to the gutassociated lymphoid tissues (GALT), where the highest number of CD4 T cells in the body reside.

1	13-29 HIV variants with tropism for different co-receptors play different roles in transmission and progression of disease. To establish infection in a new host, HIV must make contact with a CD4expressing immune cell. The cell type targeted is determined by the affinity of viral gp120 for the different chemokine coreceptors: CCR5 or CXCR4. Accordingly, the two major tropism variants of HIV are referred to as ‘R5’ and ‘X4,’ respectively. Because CCR5 dominates on CD4expressing immune cells resident at the major sites of viral transmission—sites that are constantly exposed to commensal microbes and thus harbor large numbers of activated immune cells (mucosal tissues of the female and male genital tracts or rectum for sexual transmission; upper gastrointestinal tract for mothertochild transmission)—CCR5tropic R5 strains of virus are typically required for transmission, and dominate early in infection.

1	Before HIV can contact CD4expressing immune cells in the genital and intestinal mucosae, it must traverse the epithelium of these tissues. Here, the CCR5tropic variants of the virus also have an advantage. Infection occurs across two types of epithelium: the stratified, or multilayered, squamous epithelium that lines the mucosae of the vagina, foreskin of the penis, ectocervix, rectum, oropharynx, and esophagus; or the singlelayered columnar epithelium lining endocervix, rectum, and upper GI tract. Epithelial cells of the rectum and endocervix can express CCR5 and have been shown to selectively translocate R5, but not X4, HIV variants through the epithelial monolayer. Other molecules expressed by epithelial cells also participate; gp120binding glycosphingolipids expressed by epithelial cells of the vaginal or ectocervical mucosae also foster transcytosis of virus across the epithelium. The rate at which virus can transit epithelial barriers to establish infection is fast. SIV virus

1	cells of the vaginal or ectocervical mucosae also foster transcytosis of virus across the epithelium. The rate at which virus can transit epithelial barriers to establish infection is fast. SIV virus has been shown to penetrate the cervicovaginal epithelium within 30 to 60 minutes of exposure.

1	In addition to direct transcytosis across epithelial cells, the interdigitating processes of dendritic cells that ramify between epithelial cells provide an avenue for HIV to traverse the epithelium. A complex ferrying mechanism seems to transfer HIV picked up by dendritic cells to CD4 T cells in lymphoid tissue. HIV can attach to dendritic cells by the binding of viral gp120 to Ctype lectin receptors such as langerin (CD207), the mannose receptor (CD206), and DCSIGN. A portion of the bound virus is rapidly taken up into vacuoles, where it can remain for days in an infectious state. In this way the virus is protected and remains stable until it encounters a susceptible CD4 T cell, whether in the local mucosal environment or after being carried to draining lymphoid tissue (Fig. 13.32). Finally, at some mucosal sites, CCR5expressing CD4 T cells reside within the epithelium (intraepithelial T cells), and have been shown to be sites of early viral replication. Thus, HIV can infect CD4 T

1	at some mucosal sites, CCR5expressing CD4 T cells reside within the epithelium (intraepithelial T cells), and have been shown to be sites of early viral replication. Thus, HIV can infect CD4 T cells either directly or via dendritic cells that interact with CD4 T cells.

1	HIV is internalized into early endosomes Dendritic cells that have migrated to lymph nodes transfer HIV to CD4 T cells Intraepithelial dendritic cells bind HIV using DC-SIGN Fig. 13.32 Dendritic cells can initiate infection by transporting HIV from mucosal surfaces to lymphoid tissue. During the acute phase of infection, which typically lasts for several weeks and is characterized by an influenzalike illness, there is rapid replication of the virus, primarily in CCR5expressing CD4 T cells (Fig. 13.33). This period is marked by an abundance of virus circulating in the blood (viremia) and the rapid decline of CCR5expressing CD4 T cells, the latter due primarily to the

1	HiV adheres to the surface of intraepithelial dendritic cells by the binding of viral gp120 to DC-SiGn (left panel). it gains access to dendritic cells at sites of mucosal injury or possibly to dendritic cells that have protruded between epithelial cells to sample the external world; HiV can also bind directly to some epithelial cells and is transported across them to subepithelial dendritic cells (not shown). Dendritic cells internalize HiV virions into mildly acidic early endosomes and migrate to lymphoid tissue (center panel). HiV virions are translocated back to the cell surface, and when the dendritic cell encounters CD4 t cells in a secondary lymphoid tissue, the HiV is transferred to the t cell (right panel).

1	Fig. 13.33 The typical course of untreated infection with HIV. other symptoms become more frequent as the CD4 t-cell count in the first few weeks are typified by an acute influenza-like viral illness, peripheral blood falls, starting at about 500 cells · μl–1. the disease sometimes called seroconversion disease, with high titers of virus in then enters the symptomatic phase. When CD4 t-cell counts fall the blood. an adaptive immune response follows, which controls the below 200 cells · μl–1, the patient is said to have aiDS. note that acute illness and largely restores levels of CD4 t cells but does not CD4 t-cell counts are measured for clinical purposes in cells per eradicate the virus. this is the asymptomatic phase, which typically microliter (cells · μl–1), rather than cells per milliliter (cells · ml–1), the lasts 5–10 years without treatment. Opportunistic infections and unit used elsewhere in this book.

1	extensive death of CD4 T cells in the GALT that are killed by viral cytopathic effects (macrophages and dendritic cells appear more resistant to lysis by replicating virus). The depletion of immune cells in the gut might compound the rapid production of virus in the GALT by fostering increased immunecell activation due to barrier breakdown and translocation of constituents of the microbiota. Because of the high viral titers and preponderance of R5 strains during the acute phase of infection, the risk of viral transmission to uninfected contacts during this time is especially high.

1	The acute phase and its high viremia reside in virtually all patients once an adaptive immune response is established (see Fig. 13.33). Cytolytic CD8 T cells specific for viral antigens develop and kill HIVinfected cells, and virusspecific antibodies become detectable in the serum of those infected (seroconversion). The development of a CTL response results in early control of the virus, resulting in a sharp drop in viral titers and a rebound of CD4 Tcell counts. The level of virus that persists in blood plasma at this stage of infection, referred to as the viral set point, is usually a good indicator of future disease progression. At this point, the disease transitions to a clinically latent, or asymptomatic, phase marked by low viremia and slowly declining CD4 Tcell numbers, typically over several years. During this time the virus continues to actively replicate, but it is held in check, principally by HIVspecific CD8 T cells and antibodies.

1	Under strong selective pressure brought by the antiviral immune response, there is selection for HIV escape mutants that are no longer detected by adaptive immune cells. This gives rise to many different viral variants in a single infected person and to even broader variation within the population as a whole. Late in infection, in approximately 50% of cases, the dominant viral type switches from R5 to X4 variants that infect T cells via CXCR4 coreceptors. This is followed by a rapid decline in CD4 Tcell count and progression to AIDS. The exact mechanism by which this shift in viral tropism leads to accelerated loss of CD4 T cells is unknown. On balance, then, R5 variants appear critical for transmission of the virus from infected to uninfected individuals, whereas X4 variants that emerge under selective pressure exerted by the antiviral immune response contribute to the progression of disease within an infected individual.

1	13-30 A genetic deficiency of the co-receptor CCR5 confers resistance to HIV infection.

1	Evidence for the importance of CCR5 in transmission of HIV infection has come from studies of individuals with a high risk of exposure to HIV1 who remain seronegative. Lymphocytes and macrophages from these people are resistant to HIV infection in cultures inoculated with HIV. The resistance of these individuals to HIV infection is explained by discovery that they are homozygous for a nonfunctional variant of CCR5 called Δ32, caused by a 32basepair deletion from the coding region that leads to a frameshift mutation and a truncated protein. The frequency of this mutant allele in Caucasians is high at 0.09 (that is, about 10% of the population are heterozygous carriers of the allele and about 1% are homozygous). The mutant allele has not been found in Japanese or in black Africans from Western or Central Africa. Whether heterozygous deficiency of CCR5 provides some protection against infection by HIV is controversial, but it appears to contribute to a modest, if any, reduction in

1	Western or Central Africa. Whether heterozygous deficiency of CCR5 provides some protection against infection by HIV is controversial, but it appears to contribute to a modest, if any, reduction in progression rates. In addition to the structural polymorphism of the gene, variations in the promoter region of the CCR5 gene have been associated with different rates of disease progression. The high incidence of the CCR5Δ32 allele in Caucasians predating the HIV pandemic suggests selection for this variant in a past epidemic. Both smallpox and bubonic plague have been put forward as possible selective agents, but this is as yet unproven.

1	13-31 An immune response controls but does not eliminate HIV.

1	Infection with HIV generates an immune response that contains the virus but only very rarely, if ever, eliminates it. A timecourse of the response of various adaptive immune elements to HIV in adults is shown, together with the levels of infectious virus in plasma, in Fig. 13.34. As was noted earlier, in the acute phase, virusmediated cytopathicity results in a substantial depletion of CD4 T cells, particularly in mucosal tissues. There is a good initial recovery of Tcell numbers and transition to the asymptomatic phase of disease as the immune response develops and curbs viral replication (see Fig. 13.33). However, replication of the virus persists, and, after a variable period lasting from a few months to more than 20 years, the CD4 Tcell numbers fall too low to maintain effective immunity, and AIDS develops (defined as less than 200 CD4 T cells per microliter in peripheral blood). Several factors conspire to progressively deplete CD4 T cells until they can no longer maintain

1	immunity, and AIDS develops (defined as less than 200 CD4 T cells per microliter in peripheral blood). Several factors conspire to progressively deplete CD4 T cells until they can no longer maintain immunity: destruction by cytotoxic lymphocytes directed against HIVinfected cells, immune activation (direct and indirect) that induces activation of latent virus, ongoing viral cytopathic effects, and insufficient Tcell regeneration in the thymus. In this section we consider in turn the roles of CD8 cytotoxic T cells, CD4 T cells, antibodies, and soluble factors in mounting the immune response to HIV infection that initially contains the infection but ultimately fails.

1	Studies of peripheral blood cells from infected individuals reveal cytotoxic T cells specific for viral peptides that can kill infected cells in vitro. In vivo, cytotoxic T cells traffic to sites of HIV replication, where they are thought to kill many productively infected cells before any infectious virus is released, thereby containing viral load at the quasistable levels that are characteristic of the asymptomatic period. Evidence for the clinical importance of the control of HIVinfected cells by CD8 cytotoxic T cells comes from studies relating the numbers and activity of CD8 T cells to viral load. There is also direct evidence from experiments in macaques infected with SIV that CD8 cytotoxic T cells control retrovirusinfected cells; treatment of infected animals with monoclonal antibodies that remove CD8 T cells is rapidly followed by a large increase in viral load.

1	In addition to direct cytotoxicity mediated by recognition of cells infected with virus, a variety of factors produced by CD4, CD8, and NK cells are important in antiviral immunity. Chemokines that bind CCR5, such as CCL5, CCL3, and CCL4, are released at the site of infection by CD8 T cells and inhibit virus spread by competing with R5 strains of HIV1 for the engagement of coreceptor CCR5, whereas factors still unknown compete with R4 strains for binding to CXCR4. Cytokines such as IFNα and IFNγ may also be involved in controlling virus spread. Evidence shows that in addition to being a major target for HIV infection, CD4 T cells also have an important role in the host response to HIVinfected cells.

1	Evidence shows that in addition to being a major target for HIV infection, CD4 T cells also have an important role in the host response to HIVinfected cells. Fig. 13.34 The immune response to HIV. infectious virus is present at relatively low levels in the peripheral blood of infected individuals during a prolonged asymptomatic phase, during which the virus is replicated persistently in lymphoid tissues. During this period, CD4 t-cell counts gradually decline (see Fig. 13.33), although antibodies and CD8 cytotoxic t cells directed against the virus remain at high levels. two different antibody responses are shown in the figure, one to the envelope protein (env) of HiV, and one to the core protein p24. eventually, the levels of antibody and HiV-specific cytotoxic t lymphocytes (Ctls) also decline, and there is a progressive increase in infectious HiV in the peripheral blood.

1	An inverse correlation is found between the strength of CD4 Tcell proliferative responses to HIV antigen and viral load. In addition, the type of effector CD4 Tcell response mounted against the virus appears important. There is an inverse correlation with viral load and control of acute infection in patients whose CD4 T cells express greater TH1 type activity, including production of IFNγ and granzyme B. Moreover, CD4 T cells from patients who do not progress to AIDS long after infection by HIV show strong antiviral proliferative responses. Finally, early treatment of acutely infected individuals with antiretroviral drugs is associated with a recovery in CD4 proliferative responses to HIV antigens. If antiretroviral therapy is stopped, the CD4 responses persist in some of these people and are associated with reduced levels of viremia. However, infection continues to persist in these patients and immunological control of the infection will ultimately fail. If CD4 Tcell responses are

1	are associated with reduced levels of viremia. However, infection continues to persist in these patients and immunological control of the infection will ultimately fail. If CD4 Tcell responses are essential for the control of HIV infection, then the fact that HIV is tropic for these cells and kills them may explain the inability of the host immune response to control the infection in the long term.

1	Antibodies against HIV proteins are generated early in the course of infection, but, like T cells, are ultimately unable to clear the virus. As for viral Tcell epitopes, the virus shows a high capacity for generating escape mutants under the selective pressure of the antibody response. Two aspects of the antibody response appear to be important: (1) generating neutralizing antibodies against gp120 and gp41 envelope viral antigens in order to block viral attachment or entry into CD4positive target cells, and (2) generating nonneutralizing antibodies that target infected cells for antibodydependent cellular cytotoxicity (ADCC). Although neutralizing antibodies are eventually produced in nearly all who are HIVinfected, the relative inaccessibility of viral epitopes that bind CD4 and chemokine coreceptors hampers the development of such antibodies for a prolonged period (typically months); this buys the virus time to generate escape mutants before the neutralizing antibodies can be

1	coreceptors hampers the development of such antibodies for a prolonged period (typically months); this buys the virus time to generate escape mutants before the neutralizing antibodies can be produced. Indeed, the generation of socalled broadly neutralizing antibodies, which can block infection by multiple viral strains, is typically found in those with high viral titers, emphasizing the fact that these antibodies cannot significantly modify established disease. Analyses of effective neutralizing antibodies to HIV indicate that they have undergone extensive somatic hypermutation that is rarely induced before the first year following infection. Nevertheless, the passive administration of some antibodies against HIV can protect experimental animals from mucosal infection by HIV, offering hope that an effective vaccine might be developed that could prevent new infections.

1	In contrast to neutralizing antibodies, which develop late in infection and appear to play a modest role in restraining viral replication, there is growing evidence that nonneutralizing antibodies that recruit ADCC by NK cells, macrophages, and neutrophils develop early in infections and are important in restraining viral replication in concert with the actions of cytolytic CD8 T cells. Again, however, the high rate of mutability of the virus allows it to stay a step ahead and persist. The mutations that occur as HIV replicates can allow resulting virus variants to escape recognition by CTLs or antibodies, and are important in contributing to the longterm failure of the immune system to contain the infection. An immune response is often dominated by T or B cells specific for particular epitopes—immunodominant epitopes—and mutations in immunodominant HIV peptides presented by MHC class I molecules have been found, as have mutations in the epitopes targeted by neutralizing and

1	epitopes—immunodominant epitopes—and mutations in immunodominant HIV peptides presented by MHC class I molecules have been found, as have mutations in the epitopes targeted by neutralizing and nonneutralizing antibodies. Mutant peptides have been found to inhibit T cells responsive to the wildtype epitope, thus allowing both the mutant and wildtype viruses to survive.

1	While the immune response to HIV is ultimately unsuccessful, its importance in restraining disease progression is clear. This is perhaps best exemplified in the tragic case of children infected with HIV perinatally, in whom the course of disease is much more fulminant than in adults. This reflects a poor immune response to the virus in the acute phase of infection due to immaturity of the neonatal immune system, as well as infection by a viral strain that has already evaded an immune system that is genetically close to that of the child. In essence, the poor immune response results in the lack of a latent phase, leading rapidly to AIDS. 13-32 Lymphoid tissue is the major reservoir of HIV infection.

1	In view of the active, ongoing immune response to HIV infections and the advent of antiretroviral therapies that efficiently blunt viral replication (see Section 1335), it is important to identify the reservoirs that allow the virus to persist. Although HIV load and turnover are usually measured in terms of the RNA present in virions in the blood, the major reservoir of HIV infection appears to be lymphoid tissue. Here, in addition to infected CD4 T cells, macrophages, and dendritic cells, HIV is also trapped in the form of immune complexes on the surface of follicular dendritic cells in germinal centers. These cells are not themselves infected but may act as a reservoir of infective virions that can persist for months, if not longer. Although tissue macrophages and dendritic cells seem able to harbor replicating HIV without being killed by it, these cells are shortlived and are not thought to be major reservoirs of latent infection. However, they appear to be important in spreading

1	able to harbor replicating HIV without being killed by it, these cells are shortlived and are not thought to be major reservoirs of latent infection. However, they appear to be important in spreading virus to other tissues, such as the brain, where infected cells in the central nervous system may contribute to the virus’s longterm persistence.

1	From studies of patients receiving antiretroviral treatment, it is estimated that more than 95% of the virus that can be detected in the plasma is derived from productively infected CD4 T cells that have a very short halflife—about 2 days. Virusproducing CD4 T cells are found in the Tcell areas of lymphoid tissues, and these T cells are thought to succumb to infection while being activated in an immune response. Latently infected CD4 memory T cells that become reactivated by antigen also produce virus that can spread to other activated CD4 T cells. In addition to cells that are productively or latently infected, a further large population of cells is infected by defective proviruses, which do not produce infectious virus. Unfortunately, latently infected CD4 memory T cells have an extremely long mean halflife of around 44 months. This means that drug therapy that effectively eliminates viral replication would have to be administered for over 70 years to completely clear the virus.

1	long mean halflife of around 44 months. This means that drug therapy that effectively eliminates viral replication would have to be administered for over 70 years to completely clear the virus. Practically, then, infected patients will never be able to eliminate an HIV infection, and require treatment for life.

1	13-33 Genetic variation in the host can alter the rate of disease progression.

1	It became clear early in the HIV/AIDS pandemic that the course of the disease could vary widely. Indeed, while nearly all untreated HIVinfected individuals progress to AIDS and ultimately die from opportunistic infections or cancer, not all do. A small percentage of people exposed to the virus seroconvert, but do not seem to have progressive disease. Their CD4 Tcell counts and other measures of immune competence are maintained for decades without antiretroviral therapy. Among these longterm nonprogressors, one subgroup, called elite controllers, have unusually low levels of circulating virus (undetectable by standard clinical assays, despite ongoing lowlevel viral replication) and represent approximately 1 in 300 infected individuals. They are being studied intensively to discover how they are able to control their infection. A second group consists of individuals who engage in highrisk behaviors that repeatedly expose them to infection yet remain virus and diseasefree. Although

1	they are able to control their infection. A second group consists of individuals who engage in highrisk behaviors that repeatedly expose them to infection yet remain virus and diseasefree. Although evidence of prior HIV infection has been reported in such individuals, it is unclear whether these individuals were ever truly infected with infectious virus or were exposed to highly attenuated or defective strains unable e, an effect that acts early in progression to aiDS; l, acts late in aiDS progression; ?, plausible mechanism of action with no direct support. From O’Brien, S.J., and nelson, G.W.: Nat. Genet. 36:565–574. Reprinted with permission from Macmillan Publishers ltd. © 2004.

1	to successfully establish infection. In any case, study of these individuals is of considerable interest as it could provide a better understanding of how the host immune response might better control the virus and define what genetic factors might predispose to a protective host response. It might also provide mechanistic insights that could guide development of better vaccines.

1	Although genetic variation in the virus itself can affect the outcome of infection, a growing number of host gene variants are being defined that impact the rate of progression of HIV infection toward AIDS. The implementation of genomewide association studies (GWAS) and, more recently, better highthroughput tools to define individual genetic variation (for example, exome and wholegenome sequencing) are accelerating discovery of genetic variations that distinguish highly susceptible and resistant individuals (Fig. 13.35). As discussed in Section 1330, one of the clearest cases of host genetic variation affecting HIV infection is a mutant allele of CCR5, CCR5Δ32, that when homozygous effectively blocks HIV1 infection, and when heterozygous may slow AIDS progression. Genetic polymorphisms in the HLA class I locus, particularly in HLA-B and HLA-C alleles, are another major factor in determining disease progression and are currently the strongest predictors of HIV control. Evidence from

1	in the HLA class I locus, particularly in HLA-B and HLA-C alleles, are another major factor in determining disease progression and are currently the strongest predictors of HIV control. Evidence from GWAS has mapped polymorphisms to the peptidebinding groove of HLA class I molecules as key determinants defining disease progression. Polymorphisms outside of the peptidebinding groove, as well as those in noncoding regions that control the expression levels of HLA molecules, are also implicated. The HLA class I alleles HLA-B57, HLA-B27, and HLA-B13, among others, are associated with a better prognosis, whereas HLA-B35 and HLA-B07 are associated with more rapid disease progression. Homozygosity of HLA class I alleles (HLA-A, HLA-B, and HLA-C) is also associated with more rapid progression, presumably because the Tcell response to infection is less diverse. Remarkably, one of the strongest associations with viral control is a singlenucleotide polymorphism (SNP) 35 kb upstream of the HLA-C

1	because the Tcell response to infection is less diverse. Remarkably, one of the strongest associations with viral control is a singlenucleotide polymorphism (SNP) 35 kb upstream of the HLA-C locus; this polymorphism confers greater immune control that correlates with increased expression levels of HLAC—with the greater control presumably being due to enhanced presentation of viral peptides to CD8 T cells. Certain polymorphisms of the killercell immunoglobulinlike receptors (KIRs) present on NK cells (see Section 326), in particular the receptor KIR3DS1 in combination with certain alleles of HLA-B, also delay the progression to AIDS. Mutations that affect the production of cytokines such as IFNγ and IL10 have also been implicated in the restriction of HIV progression.

1	Fig. 13.35 Genes that influence progression to AIDS in humans.

1	Effect Mechanism of action Allele ModeGene HIV entry Cytokine anti-HIV Acquired immunity, cell-mediated Acquired immunity, innate Recessive Prevents infection Knockout of CCR5 expression Recessive Accelerates AIDS (E) Increases CCR5 expression CCR5 ˜32 P1 Dominant Delays AIDS Interacts with and reduces CXCR4 I64CCR2 Homozygous Accelerates AIDS Decreases breadth of HLA class I epitope recognition A, B, C Accelerates AIDS Defects CD8-mediated T-cell clearance of HIV-1 B*35-Px HLA Codominant Delays AIDS Delays HIV-1 escape B*27 B*57 Dominant Accelerates AIDS Decreases CCL5 expression In1.1cCCL5 Recessive Delays AIDS (L) Impedes CCR5–CXCR4 transition (?) 3´ACXCL12 Dominant Accelerates P. jirovecii pneumonia (L) Alters T-cell activations (?)E3KCXCR6 Dominant Enhances infection Stimulates immune response (?)H7CCL2-CCL7-CCL11 Dominant Accelerates AIDS (E) –179TIFNG Epistatic with HLA-Bw4 Delays AIDS Clears HIV+, HLA– cells (?) 3DS1KIR3DS1 Dominant Limits infection Decreases IL-10 expression

1	response (?)H7CCL2-CCL7-CCL11 Dominant Accelerates AIDS (E) –179TIFNG Epistatic with HLA-Bw4 Delays AIDS Clears HIV+, HLA– cells (?) 3DS1KIR3DS1 Dominant Limits infection Decreases IL-10 expression 5´AIL10 Accelerates AIDS Dominant Prevents lymphoma (L) Decreases available CCR5 Delays AIDS Genes that infuence progression to AIDS 13-34 The destruction of immune function as a result of HIV infection leads to increased susceptibility to opportunistic infection and eventually to death.

1	When CD4 Tcell numbers decline below a critical level, cellmediated immunity is lost, and infections with a variety of opportunistic microbes appear (Fig. 13.36). Typically, resistance is lost early to oral Candida species and to M. tuberculosis, which cause thrush (oral candidiasis) and tuberculosis, respectively. Later, patients suffer from shingles (caused by the activation of latent herpes zoster), from aggressive EBVinduced Bcell lymphomas, and from Kaposi sarcoma, a tumor of endothelial cells that probably results both from a response to cytokines produced in the infection and a herpesvirus called Kaposi sarcomaassociated herpesvirus (KSHV, or HHV8). Since the earliest recognition of AIDS, pneumonia caused by P. jirovecii (previously called P. carinii) has been the most common opportunistic infection; it was typically fatal before effective antifungal therapy was introduced. Coinfection by hepatitis C virus is common and associated with more rapid progression of hepatitis. In

1	infection; it was typically fatal before effective antifungal therapy was introduced. Coinfection by hepatitis C virus is common and associated with more rapid progression of hepatitis. In the final stages of AIDS, infection with cytomegalovirus or a member of the Mycobacterium avium group of bacteria is more prominent. It is important to note that not all patients with AIDS get all of these infections or tumors, and there are other tumors and infections that are less prominent but still significant. Figure 13.36 lists the most common opportunistic infections and tumors, which are typically controlled until the CD4 Tcell count drops toward zero.

1	infections are the major cause of death in aiDS, the most prominent being respiratory infection with P. jirovecii and mycobacteria. Host defense against most of these pathogens requires effective macrophage activation by CD4 t cells or effective cytotoxic t cells. Opportunistic pathogens are present in the normal environment, but cause severe disease primarily in immunocompromised hosts, such as aiDS patients and cancer patients. aiDS patients are also susceptible to several rare cancers, such as Kaposi sarcoma [associated with human herpesvirus 8 (HHV8)] and various lymphomas, suggesting that immune surveillance of the causative herpesviruses by t cells can normally prevent such tumors (see Chapter 16). 13-35 Drugs that block HIV replication lead to a rapid decrease in titer of infectious virus and an increase in CD4 T cells. Studies with drugs that block HIV replication indicate that the virus is repli cating rapidly at all phases of infection, including the asymptomatic phase.

1	Studies with drugs that block HIV replication indicate that the virus is repli cating rapidly at all phases of infection, including the asymptomatic phase. Three viral proteins in particular have been the target of drugs aimed at arrest ing viral replication. These are viral reverse transcriptase, which is required for synthesis of the provirus; viral integrase, which is required for insertion of the viral provirus into the host genome; and viral protease, which cleaves viral polyproteins to produce virion proteins and viral enzymes. Reverse tran scriptase is inhibited by nucleoside analogs such as zidovudine (AZT), which was the first antiHIV drug to be licensed in the United States. Inhibitors of reverse transcriptase, integrase, and protease prevent infection of uninfected Therapy with antiretroviral drugs (% of patient days)

1	Therapy with antiretroviral drugs (% of patient days) Fig. 13.37 The mortality of patients with advanced HIV infection fell in the United States in parallel with the introduction of combination antiretroviral drug therapy. the graph shows the number of deaths, expressed each calendar quarter as the deaths per 100 person-years. Figure based on data from F. Palella.

1	Fig. 13.38 The time-course of reduction of HIV circulating in the blood during drug treatment. the production of new HiV particles can be arrested for prolonged periods by treatment using combinations of protease inhibitors and viral reversetranscriptase inhibitors. after the initiation of such treatment, virus production is curtailed as the infected cells die and no new cells are infected. the half-life of virus decay occurs in three phases. in the first phase the half-life (t1/2) is about 2 days, reflecting the half-life of productively infected CD4 t cells; this phase lasts about 2 weeks, during which time viral production declines cells; cells that are already infected can continue to produce virions because, once the provirus is established, reverse transcriptase and integrase are not needed to make new virus particles, and while the viral protease acts at a very late maturation step of the virus, inhibition of the protease does not prevent virus from being released. However, in

1	needed to make new virus particles, and while the viral protease acts at a very late maturation step of the virus, inhibition of the protease does not prevent virus from being released. However, in all cases, further cycles of infection by released virions are blocked, and replication is therefore prevented.

1	The introduction of combination therapy with a cocktail of viral protease inhibitors and nucleoside analogs, also known as highly active antiretroviral therapy (HAART), dramatically reduced mortality and morbidity in patients with advanced HIV infection in the United States between 1995 and 1997 (Fig. 13.37). Many patients treated with HAART show a rapid and dramatic reduction in viremia, eventually maintaining levels of HIV RNA close to the limit of detection (50 copies per ml of plasma) for a long period (Fig. 13.38). It is unclear how the virus particles are removed so rapidly from the circulation after the initiation of HAART. It seems most likely that they are opsonized by specific antibody and complement and removed by cells of the mononuclear phagocyte system. Opsonized HIV particles may also be trapped in lymphoid follicles on the surface of follicular dendritic cells.

1	HAART is also accompanied by a slow but steady increase in CD4 T cells, despite the fact that many other compartments of the immune system remain compromised. Three complementary mechanisms have been established for the recovery in CD4 Tcell numbers. The first is a redistribution of CD4 T memory cells from lymphoid tissues into the circulation as viral replication as the lymphocytes that were productively infected at the onset of treatment die. Released virus is rapidly cleared from the circulation, where it has a half-life of 6 hours, and there is a decrease in virus levels in plasma of more than 95% during this first phase. the second phase lasts for about 6 months; the virus now has a half-life of about 2 weeks. During this phase, virus is released from infected macrophages and from resting, latently infected CD4 t cells stimulated to divide and develop productive infection. it is thought that there is then a third phase of unknown length that results from the reactivation of

1	resting, latently infected CD4 t cells stimulated to divide and develop productive infection. it is thought that there is then a third phase of unknown length that results from the reactivation of integrated provirus in memory t cells and other long-lived reservoirs of infection. this reservoir of latently infected cells might remain present for many years. Measurement of this phase of viral decay is impossible at present because viral levels in plasma are below detectable levels (dotted line). Data Months on treatment courtesy of G.M. Shaw.

1	is controlled; this occurs within weeks of starting treatment. The second is a reduction in the abnormal levels of immune activation as HIV infection is controlled; this is associated with reduced killing of infected CD4 T cells by cytotoxic T lymphocytes. The third is much slower and is caused by the emergence of new naive T cells from the thymus, which is indicated by the presence of Tcell receptor excision circles (TRECs) in these laterarriving cells (see Section 59).

1	Although HAART is effective at inhibiting HIV replication, thereby preventing the progression to AIDS and greatly decreasing transmission by those infected, it is ineffective at eradicating all viral stores. Cessation of HAART therefore leads to a rapid rebound of virus multiplication, so that patients require treatment indefinitely. This, coupled with the sideeffects and high cost of HAART, have stimulated investigation into other targets to block viral replication (Fig. 13.39) as well as ways of eliminating viral reservoirs to eradicate infection permanently. New classes of antiHIV replication drugs include viral entry inhibitors, which block the binding of gp120 to CCR5 or block viral fusion by inhibiting gp41; and viral integrase inhibitors, which block the insertion of the reversetranscribed viral genome into the host DNA. Another approach under development is to enhance the activity of HIV restriction factors, including APOBEC (see Section 1327) and TRIM 5α. APOBEC causes

1	viral genome into the host DNA. Another approach under development is to enhance the activity of HIV restriction factors, including APOBEC (see Section 1327) and TRIM 5α. APOBEC causes extensive mutation of newly formed HIV cDNA to destroy its coding and replicative capacity, and TRIM 5α limits HIV1 infections by targeting the viral nucleocapsid and preventing the uncoating and release of viral RNA after it enters cells.

1	Given the success of HAART in blocking active viral replication, the inability of existing therapies to purge reservoirs of latently infected cells has become the greatest barrier to a cure. To overcome this, strategies are being considered that would induce viral replication in latently infected cells in combination with measures to enhance immune clearance of virus and infected cells. Examples of ways to activate latent virus include the administration of cytokines that activate viral transcription and replication (for example, IL2, IL6, and TNFα), or the use of agents that target epigenetic modifiers, such as histone deacetylase (HDAC) inhibitors, that can activate latent provirus. To date, however, no clinical trial using agents that target latent viral reservoirs has shown a significant reduction in viral load over that gained from HAART alone. Indeed, it was recently discovered that the activation of viral replication in latently infected cells is intrinsically stochastic, so

1	reduction in viral load over that gained from HAART alone. Indeed, it was recently discovered that the activation of viral replication in latently infected cells is intrinsically stochastic, so that many immune cells harboring latent provirus will fail to activate viral replication in any given cycle of cellular activation. This adaptation of HIV to avoid elimination of latently infected cells could represent a formidable barrier to strategies aimed at ‘flushing out’ latent virus so that it can be eliminated.

1	Viral entry inhibitors Viral integrase inhibitors Virus assembly Protease inhibitors Reverse transcriptase inhibitors. Nucleoside analogs and non-nucleoside analogs interrupt transcription of viral RNA into viral cDNA Fig. 3.39 Possible targets for interference with the HIV life and budding of infectious virions. as yet, only drugs that inhibit cycle. in principle, HiV could be attacked by therapeutic drugs at reverse transcriptase and protease action have been developed. multiple points in its life cycle: virus entry, reverse transcription of viral Combination therapy using different kinds of drugs is more effective Rna, insertion of viral cDna into cellular Dna by the viral integrase, than using a single drug. cleavage of viral polyproteins by the viral protease, and assembly

1	An alternative strategy for cure has been highlighted in a single HIV patient who underwent hematopoietic stemcell transplantation (HSCT) for treatment of leukemia in Berlin (hence referred to as the Berlin patient). By using a stemcell donor who was homozygous for the CCR5Δ32 coreceptor mutation, the patient was reconstituted with immune cells resistant to viral propagation. The patient’s CD4 Tcell counts rebounded and he was found to be free of any evidence of HIV infection (or leukemia) following cessation of antiretroviral therapy posttransplant. He has remained so for more than 5 years, suggesting that he has been cured of infection. In view of the large number of infected individuals worldwide, the risk of complications with HSCT, and rarity of HLAmatched CCR5deletant donors, this will never be a practical approach for cure at the population level. Moreover, there is a risk of progression or reinfection posttransplant by CXCR4tropic viral variants. This outcome does, however,

1	will never be a practical approach for cure at the population level. Moreover, there is a risk of progression or reinfection posttransplant by CXCR4tropic viral variants. This outcome does, however, dramatically establish that eradication of a latency reservoir (in this case by inductive chemoirradiation therapy for leukemia) combined with blockade of viral replication—whether by genetic or therapymediated interventions— might achieve a permanent cure.

1	13-36 In the course of infection HIV accumulates many mutations, which can result in the outgrowth of drug-resistant variants.

1	The rapid replication of HIV, with the generation of 109 to 1010 virions every day, is coupled with a mutation rate of approximately 3 × 10–5 substitutions per nucleotide per cycle of replication, and thus leads to the generation of many variants of HIV in a single infected patient in the course of a day. This high mutation rate arises from the errorprone nature of retroviral replication and poses a formidable challenge to the immune system. Reverse transcriptase lacks the proofreading mechanisms of cellular DNA polymerases, and the RNA genomes of retroviruses are copied into DNA with relatively low fidelity. Thus, although primary infection is typically established by a single founder virus, numerous variants of HIV, called quasispecies, rapidly develop within an infected individual. This phenomenon was first recognized in HIV and has since proved to be common to all lentiviruses.

1	As a consequence of its high variability, HIV rapidly develops resistance to antiviral drugs, much as it develops escape mutants that evade Tcell recognition (see Section 1331). When drug is administered, viral variants with mutations that confer resistance emerge and multiply until the previous levels of virus are regained. Resistance to some viral protease inhibitors requires only a single mutation and appears after only a few days (Fig. 13.40); resistance to some inhibitors of reverse transcriptase develops in a similarly short time. In contrast, resistance to the nucleoside analog zidovudine takes months to develop, as it requires three or four mutations in the viral reverse transcriptase. Because of the relatively rapid appearance of resistance to antiHIV drugs, successful drug treatment has typically depended on combination therapy, where

1	Fig. 13.40 Resistance of HIV to protease inhibitors develops rapidly. after the administration of a single protease inhibitor drug to a patient with HiV there is a precipitous fall in plasma levels of viral Rna, with a half-life of about 2 days (top panel). this is accompanied by an initial rise in the number of CD4 t cells in peripheral blood (center panel). Within days of starting the drug, mutant drug-resistant variants can be detected in plasma (bottom panel) and in peripheral blood lymphocytes. after only 4 weeks of treatment, viral Rna levels and CD4 lymphocyte levels have returned to their original pre-drug levels, and 100% of plasma HiV is present as the drug-resistant mutant. the chance of simultaneous resistance mutations in multiple HIV proteins is virtually nil. Nevertheless, monotherapy with newer generation antiretroviral agents has proven effective in patients with low viral loads at the onset of treatment.

1	13-37 Vaccination against HIV is an attractive solution but poses many difficulties. Although the effectiveness of HAART in restraining HIV replication has profoundly altered the natural history and transmission rates of HIV infection, a safe and effective vaccine for the prevention of HIV infection and AIDS remains the ultimate goal. Ideally, an effective vaccine would elicit both broadly neutralizing antibodies that block viral entry into target cells (that is, antigp120) and effective cytolytic Tcell responses, to both prevent and control HIV infection, respectively. However, no such vaccine has yet been developed, and its attainment is fraught with difficulties that have not been faced in the development of vaccines against other diseases.

1	The main problem is the nature of the infection itself, featuring a virus that directly undermines the central component of adaptive immunity—the CD4 T cell—and that proliferates and mutates extremely rapidly to cause sustained infection in the face of strong cytotoxic Tcell and antibody responses. The development of vaccines that could be administered to patients already infected, to boost immune responses and prevent progression to AIDS, has been considered, as have prophylactic vaccines that would prevent initial infection. The development of therapeutic vaccination in those already infected would be extremely difficult. As discussed in the previous section, HIV evolves in individual patients by the selective proliferation of mutant viruses that escape recognition by antibodies and cytotoxic T cells. The ability of the virus to persist in latent form as a transcriptionally silent provirus invisible to the immune system might also prevent even an immunized person from clearing an

1	T cells. The ability of the virus to persist in latent form as a transcriptionally silent provirus invisible to the immune system might also prevent even an immunized person from clearing an infection once it has been established.

1	There has been more hope for prophylactic vaccination to prevent new infection. But even here, the lack of protection of the normal immune response and the sheer scale of sequence diversity among HIV strains in the infected population—there are currently thousands of different HIV strains circulating in the human population—remain significant challenges. Patients infected with one strain of virus do not seem to be resistant to closely related strains, and cases of superinfection, where two strains simultaneously infect the same cell, have also been described. This is compounded by the intrinsic difficulty in generating broadly neutralizing antibodies against HIV envelope glycoproteins (see Section 1331). Further, there remains uncertainty over what form protective immunity to HIV might take. It is now felt that induction of both effective antibody and Tcell responses will be required to achieve protective immunity, although which epitopes might provide the best targets and how best to

1	It is now felt that induction of both effective antibody and Tcell responses will be required to achieve protective immunity, although which epitopes might provide the best targets and how best to induce them remain undefined. Finally, the time from conception to design to performance of complete clinical trials of vaccines against HIV takes years, slowing the rate of progress; to date, few major clinical vaccine trials have been completed, and those have failed.

1	However, against this pessimistic background, progress has been made and there remains hope that successful vaccines might yet be developed. Various strategies are being tried in an attempt to develop vaccines against HIV, varying among delivery of recombinant HIV proteins, plasmid DNA vaccination with HIV genes (see Section 1630), delivery of HIV genes in viral vectors, or combinations thereof. Many successful vaccines against other viral diseases contain a live attenuated strain of the virus, which raises an immune response but does not cause disease (see Section 1623). There are substantial difficulties in the development of live attenuated vaccines against HIV, not least the worry of recombination between vaccine strains and wildtype viruses that would lead to a reversion to virulence. An alternative approach is the use of other viruses, such as vaccinia or adenovirus, to deliver and express HIV genes that elicit Band Tcell responses against HIV antigens. Because these viral

1	An alternative approach is the use of other viruses, such as vaccinia or adenovirus, to deliver and express HIV genes that elicit Band Tcell responses against HIV antigens. Because these viral vectors have already demonstrated safety in other human vaccination studies, they have been obvious choices for initial trials. Recently, there has been encouraging, albeit limited, success with this type of approach in combination with boosts using recombinant gp120. Delivery of HIV gag, pol, and env genes via a canarypox viral vector followed by boosts with HIV gp120 was shown to reduce the risk of infection in a modest but significant number of highrisk vaccine recipients. This represents the first demonstration of any degree of efficacy in a large HIV vaccine trial to date. Perhaps as important, data from this study provided insights into the type of immune response that correlated with protection, indicating that the induction of non neutralizing antibodies that elicit ADCC (for example,

1	data from this study provided insights into the type of immune response that correlated with protection, indicating that the induction of non neutralizing antibodies that elicit ADCC (for example, IgG3 isotype) might provide protection. Because neutralizing antibodies against HIV have proven so difficult to elicit, this provides encouragement that they might not be required. Further, a study that used a cytomegalovirus (CMV) vector to deliver SIV genes to rhesus monkeys showed that potent CTL responses were induced. Although these CTL responses did not prevent infection by a pathogenic SIV strain, they did result in clearance of virus in about half of the monkeys vaccinated after its systemic spread. This unprecedented result suggests that the viral vector used for delivery of HIV genes—in this case a vector that produces HIV antigens for prolonged periods after vaccination—might play an important role in the type and amplitude of the antiviral CD8 Tcell response elicited, and

1	genes—in this case a vector that produces HIV antigens for prolonged periods after vaccination—might play an important role in the type and amplitude of the antiviral CD8 Tcell response elicited, and protection might be achieved by an effective Tcell response alone. Additional studies will be needed to determine whether combination vaccines that elicit the appropriate non neutralizing antibodies and robust CD8 Tcell responses might achieve protection even in the absence of neutralizing antibodies.

1	In addition to the biological obstacles to developing effective HIV vaccines, there are difficult ethical issues. It would be unethical to conduct a vaccine trial without trying at the same time to minimize the exposure of a vaccinated population to the virus itself. The effectiveness of a vaccine can, however, only be assessed in a population in which the exposure rate to the virus is high enough to assess whether vaccination protects against infection. This means that initial vaccine trials might have to be conducted in countries where the incidence of infection is very high and public health measures have not yet succeeded in reducing the spread of HIV. 13-38 Prevention and education are important in controlling the spread of HIV and AIDS.

1	The spread of HIV can be prevented if precautions are taken by those already infected and those who are uninfected but at risk for exposure. The advent of HAART represents a major advance in blocking the transmission of HIV from infected people due to its ability to greatly reduce viral titers in body fluids. However, most who are infected with HIV do not have access to HAART, as it is expensive and requires lifelong treatment, and many of those infected are unaware that they carry the virus. Even where HAART is unavailable, access to regular screening for those at risk is critical to inform those infected so they can take measures to avoid passing the virus to others. This, in turn, requires strict confidentiality and mutual trust. A barrier to the control of HIV is reluctance of individuals to find out whether they are infected, especially as one of the consequences of a positive HIV test is stigmatization by society. Here, education becomes an important component of the prevention

1	to find out whether they are infected, especially as one of the consequences of a positive HIV test is stigmatization by society. Here, education becomes an important component of the prevention strategy, both to remove the stigma and to provide guidance on how transmission of the virus can be prevented.

1	Precautions that can be taken by uninfected individuals are relatively inexpensive and involve measures to protect against contact with body fluids, such as semen, blood, blood products, or milk, from people who are infected. It has been repeatedly demonstrated that this is sufficient to prevent infection, as exemplified by healthcare workers who care for AIDS patients for long periods without seroconversion or signs of infection. The routine use of condoms greatly reduces the risk of HIV transmission, as does restraint from breastfeeding by infected mothers of newborns. Male circumcision also reduces transmission rates, as the foreskin is a major site of viral entry in uncircumcised males. Additional measures that have been considered include the use of microbicidal gels or suppositories, improvements in which have led to products that show promise in recent trials. Some of these agents can also reduce the transmission of other sexually transmitted diseases (for example, genital

1	improvements in which have led to products that show promise in recent trials. Some of these agents can also reduce the transmission of other sexually transmitted diseases (for example, genital herpes) that increase the risk of HIV transmission. Finally, there is increasing interest in the prophylactic use of antiretroviral drugs (referred to as preexposure prophylaxis, or PrEP), which are administered either topically or orally to individuals at high risk for contracting HIV. To date, two reverse transcriptase inhibitors have shown efficacy in trials, and combined daily use of both drugs taken orally has demonstrated over 90% reduction in the risk of HIV infection. Moreover, use of antiretroviral therapy immediately postexposure—for example, in hospital workers exposed to contaminated blood by accidental needlestick—substantially reduces the risk of acquiring HIV. One concern with this approach is the risk of developing drug resistance in those who do contract HIV while on PrEP,

1	blood by accidental needlestick—substantially reduces the risk of acquiring HIV. One concern with this approach is the risk of developing drug resistance in those who do contract HIV while on PrEP, particularly in individuals with poor adherence to the dosing regimen. Although the significance of this risk has yet to be established, it remains an issue. Nevertheless, testing of new PrEP strategies based on additional antiretrovirals or of longacting formulations that reduce the risk of poor compliance represents areas of considerable promise.

1	Summary.

1	Infection with the human immunodeficiency virus (HIV) is the cause of acquired immune deficiency syndrome (AIDS). Although substantial gains in curbing the rate of spread of HIV have been made, this worldwide epidemic continues to spread, especially through heterosexual contact in lessdeveloped countries. HIV is an enveloped retrovirus that replicates in cells of the immune system. Viral entry requires the presence of CD4 and a particular chemokine receptor, and the viral cycle is dependent on transcription factors found in activated T cells. Infection with HIV causes a loss of CD4 T cells and an acute viremia that rapidly subsides as cytotoxic Tcell responses develop, but HIV infection is not eliminated by this immune response. HIV establishes a state of persistent infection in which the virus is continually replicating in newly infected cells. Current treatment consists of combinations of antiviral drugs that block viral replication and cause a rapid decrease in virus levels and a

1	virus is continually replicating in newly infected cells. Current treatment consists of combinations of antiviral drugs that block viral replication and cause a rapid decrease in virus levels and a slow increase in CD4 Tcell counts. The main effect of HIV infection is the destruction of CD4 T cells, which occurs through the direct cytopathic effects of HIV infection and through killing by CD8 cytotoxic T cells. As CD4 Tcell counts wane, the body becomes progressively more susceptible to opportunistic infection. Eventually, most untreated HIVinfected individuals develop AIDS and die; however, a small minority, socalled longterm nonprogressors, remain healthy for many years with no apparent ill effects of infection. We hope to be able to learn from these people how infection with HIV can be controlled. The existence of these people, and of others who seem to have been naturally immunized against infection, gives hope that it will be possible to develop effective vaccines against HIV.

1	Summary to Chapter 13.

1	Whereas most infections elicit protective immunity, successful pathogens have developed some means of at least partly resisting the immune response and can cause serious, sometimes persistent, disease. Some individuals have inherited deficiencies in different components of the immune system, making them highly susceptible to certain classes of infectious agents. Persistent infections and inherited immunodeficiency diseases illustrate the importance of innate and adaptive immunity in effective host defense, and present ongoing challenges for immunological research. The human immunodeficiency virus (HIV), which leads to acquired immune deficiency syndrome (AIDS), combines the characteristics of a persistent infectious agent with the ability to create immunodeficiency in its human host, a combination that is usually slowly lethal to the patient. The key to fighting new pathogens such as HIV is to increase our understanding of the basic properties of the immune system and its role in

1	a combination that is usually slowly lethal to the patient. The key to fighting new pathogens such as HIV is to increase our understanding of the basic properties of the immune system and its role in combating infection.

1	Questions. 13.1 Matching: Match the following gene defects with the associated primary immunodeficiency. 13.2 True or False: individuals with mutations in the genes encoding the il-12 p40 subunit are susceptible not only to pathogens such as M. tuberculosis that require a tH1 response, but type 3 (tH17) responses are also affected. 13.3 Short Answer: name two genetic defects that lead to the absence of CD8+ t cells with CD4+ t cells preserved, and one genetic defect that leads to the absence of CD4+ t cells with CD8+ t cells preserved. 13.4 Short Answer: Both CD40l deficiency and aiD deficiency cause hyper-igM syndrome, but t-cell function is severely impaired in CD40l deficiency and preserved in aiD deficiency. Why? 13.5 True or False: Common variable immunodeficiency (CViD) severely impairs both t-cell and antibody responses. 13.6 Multiple Choice: Which of the following hereditary immune disorders does not have an autoimmune or autoinflammatory phenotype?

1	13.6 Multiple Choice: Which of the following hereditary immune disorders does not have an autoimmune or autoinflammatory phenotype? A. autoimmune polyendocrinopathy-candidiasisectodermal dystrophy (aPeCeD), caused by defects in B. Familial Mediterranean fever (FMF), caused by pyrin mutations C. Ommen syndrome, caused by RAG1 or RAG2 hypomorphic mutations D. Wiskott–aldrich syndrome (WaS), caused by WaS deficiency E. Hyper-ige syndrome (also called Job’s syndrome), caused by Stat3 or DOCK8 mutations

1	D. Wiskott–aldrich syndrome (WaS), caused by WaS deficiency E. Hyper-ige syndrome (also called Job’s syndrome), caused by Stat3 or DOCK8 mutations F. Chronic granulomatous disease (CGD), caused by production of reactive oxygen species in phagocytes 13.7 Multiple Choice: Pyogenic bacteria are protected by polysaccharide capsules against recognition by receptors on macrophages and neutrophils. antibody-dependent opsonization is one of the mechanisms utilized by phagocytes to ingest and destroy these bacteria. Which of the following diseases or deficiencies directly affects a mechanism by which the immune system controls infection by these pathogens? A. il-12 p40 deficiency B. Defects in AIRE C. WaSp deficiency D. Defects in C3 13.8 Multiple Choice: Defects in which of the following genes have a phenotype similar to defects in ELA2, the gene that encodes neutrophil elastase? A. GFI1 B. CD55 (encodes DaF) C. CD59

1	A. GFI1 B. CD55 (encodes DaF) C. CD59 D. XIAP 13.9 Matching: Match each protein to the associated 13.10 Multiple Choice: Which of the following pathogens primarily evade(s) the immune system by antigenic variation? A. influenza a virus B. Herpes simplex virus-1 C. Cytomegalovirus D. Trypanosoma brucei E. Plasmodium falciparum F. Hepatitis B virus 13.11 Human immunodeficiency virus (HiV) produces various immunoevasins. One of these, nef, is exceptionally pleiotropic and a major target of CD8+ t-cell responses. Which of the following is not one of the functions of nef? A. inhibition of the restriction factor SaMHD1 B. MHC class i downregulation General references. Alcami, A., and Koszinowski, U.H.: Viral mechanisms of immune evasion. Immunol. Today 2000, 21:447–455. De Cock, K.M., Mbori-Ngacha, D., and Marum, E.: Shadow on the continent: public health and HIV/AIDS in Africa in the 21st century. Lancet 2002, 360:67–72.

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1	Carcelain, G., Debre, P., and Autran, B.: Reconstitution of CD4+ T lymphocytes in HIV-infected individuals following antiretroviral therapy. Curr. Opin. Immunol. 2001, 13:483–488. Farber, J.M., and Berger, E.A.: HIV’s response to a CCR5 inhibitor: I’d rather tighten than switch! Proc. Natl Acad. Sci. USA 2002, 99:1749–1751. Ho, D.D.: Perspectives series: host/pathogen interactions. Dynamics of HIV-1 replication in vivo. J. Clin. Invest. 1997, 99:2565–2567. Kordelas, L.,Verheyen, J., and Esser, S.: Shift of HIV tropism in stem-cell transplantation with CCR5 Delta32 mutation. N. Engl. J. Med. 2014, 371:880–882. Lundgren, J.D., and Mocroft, A.: The impact of antiretroviral therapy on AIDS and survival. J. HIV Ther. 2006, 11:36–38. Perelson, A.S., Essunger, P., Cao, Y.Z., Vesanen, M., Hurley, A., Saksela, K., Markowitz, M., and Ho, D.D.: Decay characteristics of HIV-1-infected compartments during combination therapy. Nature 1997, 387:188–191.

1	Wei, X., Ghosh, S.K., Taylor, M.E., Johnson, V.A., Emini, E.A., Deutsch, P., Lifson, J.D., Bonhoeffer, S., Nowak, M.A., Hahn, B.H., et al.: Viral dynamics in human immunodeficiency virus type 1 infection. Nature 1995, 373:117–122. 13-36 In the course of infection HIV accumulates many mutations, which can result in the outgrowth of drug-resistant variants. Condra, J.H., Schleif, W.A., Blahy, O.M., Gabryelski, L.J., Graham, D.J., Quintero, J.C., Rhodes, A., Robbins, H.L., Roth, E., Shivaprakash, M., et al.: In vivo emergence of HIV-1 variants resistant to multiple protease inhibitors. Nature 1995, 374:569–571. Finzi, D., and Siliciano, R.F.: Viral dynamics in HIV-1 infection. Cell 1998, 93:665–671. Katzenstein, D.: Combination therapies for HIV infection and genomic drug resistance. Lancet 1997, 350:970–971.

1	Katzenstein, D.: Combination therapies for HIV infection and genomic drug resistance. Lancet 1997, 350:970–971. Moutouh, L., Corbeil, J., and Richman, D.D.: Recombination leads to the rapid emergence of HIV 1 dually resistant mutants under selective drug pressure. Proc. Natl Acad. Sci. USA 1996, 93:6106–6111. 13-37 Vaccination against HIV is an attractive solution but poses many difficulties. Baba, T.W., Liska, V., Hofmann-Lehmann, R., Vlasak, J., Xu, W., Ayehunie, S., Cavacini, L.A., Posner, M.R., Katinger, H., Stiegler, G., et al.: Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian– human immunodeficiency virus infection. Nat. Med. 2000, 6:200–206. Barouch, D.H.: The quest for an HIV-1 vaccine--moving forward. N. Engl. J. Med. 2013, 369:2073–2076.

1	Barouch, D.H.: The quest for an HIV-1 vaccine--moving forward. N. Engl. J. Med. 2013, 369:2073–2076. Barouch, D.H., Kunstman, J., Kuroda, M.J., Schmitz, J.E., Santra, S., Peyerl, F.W., Krivulka, G.R., Beaudry, K., Lifton, M.A., Gorgone, D.A., et al.: Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes. Nature 2002, 415:335–339. Isitman, G., Stratov, I., and Kent, S.J.: Antibody-dependent cellular cytotoxicity and Nk cell-driven immune escape in HIV Infection: Implications for HIV vaccine development. Adv. Virol. 2012, 212:637208. Letvin, N.L.: Progress and obstacles in the development of an AIDS vaccine. Nat. Rev. Immunol. 2006, 6:930–939. McMichael, A.J., and Koff, W.C.: Vaccines that stimulate T cell immunity to HIV-1: the next step. Nat. Immunol. 2014, 15:319–322. 13-38 Prevention and education are important in controlling the spread of HIV and AIDS.

1	13-38 Prevention and education are important in controlling the spread of HIV and AIDS. Coates, T.J., Aggleton, P., Gutzwiller, F., Des-Jarlais, D., Kihara, M., Kippax, S., Schechter, M., and van-den-Hoek, J.A.: HIV prevention in developed countries. Lancet 1996, 348:1143–1148. Decosas, J., Kane, F., Anarfi, J.K., Sodji, K.D., and Wagner, H.U.: Migration and AIDS. Lancet 1995, 346:826–828. Dowsett, G.W.: Sustaining safe sex: sexual practices, HIV and social context. AIDS 1993, 7 Suppl. 1:S257–S262. Kirby, M.: Human rights and the HIV paradox. Lancet 1996, 348:1217–1218. Nelson, K.E., Celentano, D.D., Eiumtrakol, S., Hoover, D.R., Beyrer, C., Suprasert, S., Kuntolbutra, S., and Khamboonruang, C.: Changes in sexual behavior and a decline in HIV infection among young men in Thailand. N. Engl. J. Med. 1996, 335:297–303. Weniger, B.G., and Brown, T.: The march of AIDS through Asia. N. Engl. J. Med. 1996, 335:343–345.

1	Weniger, B.G., and Brown, T.: The march of AIDS through Asia. N. Engl. J. Med. 1996, 335:343–345. The adaptive immune response is a critical component of host defense against infection and is essential for normal health. Antigens not associated with infectious agents sometimes elicit adaptive immune responses, and this can cause disease. One circumstance in which this happens is when harmful immunologically mediated hypersensitivity reactions known generally as allergic reactions occur in response to inherently harmless ‘environmental’ antigens such as pollen, food, and drugs.

1	Historically, hypersensitivity reactions due to immunological responses were classified by Gell and Coombs into four broad types, of which type I hypersensitivity reactions represented immediate-type allergic reactions mediated by IgE antibodies, with mast-cell activation the major final effector mechanism. Type II and III hypersensitivity responses were defined as those that were driven by antigen-specific IgG antibodies, the final effector mechanism being complement (type II) or FcR-bearing cellular effectors (type III). Finally, type IV hypersensitivity responses were depicted as being driven by cellular effectors, including lymphocytes and a variety of myeloid cell types. While the Gell and Coombs classification system provides an effective framework for understanding the mechanisms underlying some prototypic immunologic reactions, it is now becoming clear that most normal and pathologic host immune responses involve both the humoral and cellular arms of the immune system, and

1	underlying some prototypic immunologic reactions, it is now becoming clear that most normal and pathologic host immune responses involve both the humoral and cellular arms of the immune system, and that the definitions provided in Chapter 11 for types 1, 2, and 3 immune response modules provide a more thorough mechanistic context for understanding disease pathogenesis, including allergic responses (see Fig. 11.5). In most allergic reactions, such as those to food, pollen, or house dust, reactions occur because the individual has become sensitized to an innocuous anti-gen—the allergen—by producing IgE antibodies against it. This is usually a result of the formation of an unwanted type 2 immune response to the allergen. Subsequent exposure to the allergen triggers the activation of IgE-binding cells, chiefly mast cells and basophils, in the exposed tissue, leading to a series of responses that are characteristic of this type of allergic reaction. In hay fever (allergic

1	of IgE-binding cells, chiefly mast cells and basophils, in the exposed tissue, leading to a series of responses that are characteristic of this type of allergic reaction. In hay fever (allergic rhinoconjunctivitis), for example, symptoms occur when allergenic proteins leached out of grass or weed pollen grains come into contact with the mucous membrane of the nose and eyes. In contrast, other hypersensitivity disorders, such as allergic contact dermatitis, serum sickness, or celiac disease, are not dependent on IgE antibodies and represent unwanted immune responses driven by IgG antibodies and/or cellular immune responses.

1	We are all exposed regularly to common environmental agents that can cause allergic reactions in some individuals. While most of the population does not develop clinically significant allergic reactions to the majority of potential allergens, in some surveys over half of the population shows an allergic response to at least one substance in the environment. Some individuals manifest allergic responses to multiple common antigens. A predisposition to become IgE-sensitized to environmental allergens is called atopy, and later in the chapter we discuss the various factors—both genetic and environmental— that may contribute to this predisposition. Genetic factors clearly play a role in predisposing an individual to IgE-mediated allergic disease. If both parents are atopic, a child has a 40–60% chance of developing an IgE-mediated allergy, whereas the risk is much lower, on the order of 10%, if neither parent is atopic. IgE and IgE-mediated allergic diseases.

1	IgE and IgE-mediated allergic diseases. Effector mechanisms in IgE-mediated allergic reactions. Non-IgE-mediated allergic diseases. Fig. 14.1 IgE-mediated reactions to extrinsic antigens. All IgE-mediated responses involve mast-cell degranulation, but the symptoms experienced by the patient can be very different depending, for example, on whether the allergen is injected directly into the bloodstream, is eaten, or comes into contact with the mucosa of the ocular or respiratory tract.

1	IgE is prominent in the defense against extracellular parasitic organisms, especially helminths and protozoa (see Section 11-9). These parasitic organisms are prevalent in developing nations, but most serum IgE in developed nations is directed against innocuous antigens, sometimes causing allergic symptoms (Fig. 14.1). Almost half the population of North America and Europe is sensitized to one or more common environmental antigens, and, although rarely life-threatening, allergic diseases initiated by contact with a specific allergen can cause much distress and lost time from school and work. The burden of allergic diseases in the Western world is considerable, with their prevalence having more than doubled in the past 20 years. Consequently, most clinical and scientific attention paid to IgE has been directed toward its pathologic roles in allergic disease rather than its protective capacity. Until the last decade, developing countries in Africa and the Middle East reported a

1	paid to IgE has been directed toward its pathologic roles in allergic disease rather than its protective capacity. Until the last decade, developing countries in Africa and the Middle East reported a relatively low prevalence of allergy; however, this situation is rapidly changing, probably as a result of Western-style modernization.

1	In this chapter we first consider the mechanisms that favor the sensitization of an individual to an allergen, resulting in the production of antigen-specific IgE. We then describe the IgE-mediated allergic reaction itself—the pathological consequences of the interaction between allergen and the IgE bound to the high-affinity Fcε receptor on mast cells and basophils. Finally, we consider the causes and consequences of other types of immunological hypersensitivity reaction. IgE and IgE-mediated allergic diseases. Immediate hypersensitivity reactions are those allergic reactions caused by activation of mast cells and basophils by multivalent antigen bridging IgE bound to their cell surfaces. IgE differs from other antibody isotypes in being predominantly localized in the tissues, where it is tightly bound to the surfaces IgE and IgE-mediated allergic diseases.

1	Fig. 14.2 Sensitization to an inhaled allergen. Der p 1 is a (third panel), and this IgE becomes bound to Fc receptors on common respiratory allergen that is found in fecal pellets of the resident submucosal mast cells. On a subsequent encounter with house dust mite. When an atopic individual first encounters Der p 1, Der p 1, the allergen binds to the mast cell-bound IgE, triggering subepithelial dendritic cells ingest the allergenic protein and traffic mast-cell activation and the release of mast-cell granule contents, to the draining lymph node, where TH2 cells specific for Der p 1 are which cause the symptoms of the allergic reaction (last panel). produced (first and second panels). Interaction of these T cells with Der p 1 is a protease that cleaves occludin, a protein that helps to Der p 1-specific B cells leads to the production of class-switched maintain epithelial tight junctions; the enzymatic activity of Der p 1 is plasma cells producing Der p 1-specific IgE in the mucosal

1	Der p 1-specific B cells leads to the production of class-switched maintain epithelial tight junctions; the enzymatic activity of Der p 1 is plasma cells producing Der p 1-specific IgE in the mucosal tissues thought to help it pass through the epithelium.

1	of mast cells and some other cell types through the high-affinity IgE receptor FcεRI (see Section 10-24). Binding of antigen to IgE cross-links the high-affinity IgE receptors, causing the release of chemical mediators from the mast cells that can lead to allergic disease (Fig. 14.2). How an initial antibody response to environmental antigens comes to be dominated by IgE production in atopic individuals is still being worked out. In this part of the chapter we describe the current understanding of the factors that contribute to this process. 14-1 Sensitization involves class switching to IgE production on first contact with an allergen.

1	To produce an allergic reaction against a given antigen, an individual must first be exposed to the antigen under conditions that result in the production of IgE antibodies. Allergic symptoms occur when an individual who has been sensitized in this fashion has subsequent exposure to the antigen. Exposure can lead to different clusters of symptoms, characterized by the tissues that are most prominently affected. The most common forms of allergic response in developed countries are to airborne allergens, causing symptoms that affect predominantly the nasal passages (allergic rhinitis), the eyes (allergic conjunctivitis), or the lower airways and lungs (asthma). Ingested allergens can lead to food allergy, sometimes affecting only the gastrointestinal tract (for example, eosinophilic esophagitis), but not infrequently involving locations distant from the site of antigen entry. Reactions that occur at locations distant from the site of entry of the challenging antigen are considered to be

1	but not infrequently involving locations distant from the site of antigen entry. Reactions that occur at locations distant from the site of entry of the challenging antigen are considered to be systemic reactions, and are thought to occur because of spread of the antigen throughout the body via the blood circulation. Systemic reactions can be limited to a single distant organ, causing hives (also called urticaria) when they target the skin, wheezing (or bronchospasms) when they involve the lungs, and life-threatening lowering of the blood pressure when they target the vascular system. Serious systemic reactions are designated by the term anaphylaxis. It is not known why sensitization with a particular allergen in one individual leads to local reactions at the time of allergen challenge, whereas sensitization with the same allergen can yield anaphylaxis in another individual. In fact, even in a single individual, a challenge that usually yields only a mild local reaction can be

1	whereas sensitization with the same allergen can yield anaphylaxis in another individual. In fact, even in a single individual, a challenge that usually yields only a mild local reaction can be followed at the time of another challenge by a severe systemic reaction.

1	Atopic individuals often develop sensitization to many different antigens, and can express multiple forms of allergic symptoms, which depend on the route and quantity of allergen—for example, atopic eczema that develops in childhood in response to sensitization to food antigens is followed in a sizable proportion of those individuals developing allergic rhinitis and/or asthma in response to airborne allergens. This progression of allergic responses in some individuals from atopic eczema in childhood to allergic rhinitis and eventually to asthma in later life has been termed the atopic march. Allergic reactions in non-atopic people, in contrast, are predominantly due to sensitization to one specific allergen, such as bee venom or a drug such as penicillin, and can develop at any time of life. It is important to remember, however, that not all encounters with a potential allergen will lead to sensitization, and not all sensitizations will lead to a symptomatic allergic response, even in

1	It is important to remember, however, that not all encounters with a potential allergen will lead to sensitization, and not all sensitizations will lead to a symptomatic allergic response, even in atopic individuals.

1	The immune response leading to IgE production in response to antigen is driven by two main groups of signals that together are typical of type 2 immune reactions. The first consists of signals that favor the differentiation of naive T cells to a TH2 phenotype. The second comprises TH2 cytokines and co-stimulatory signals that stimulate B cells to switch to the production of IgE. As described in Section 9-21, the fate of a naive CD4 T cell responding to an antigenic peptide presented by a dendritic cell is determined by the cytokines it is exposed to before and during this response, and by the intrinsic properties of the antigen, the antigen dose, and the route of presentation. Exposure to IL-4, IL-5, IL-9, and IL-13 favors the development of TH2 cells, whereas exposure to IFN-γ and IL-12 (and its relative IL-27) favors TH1-cell development.

1	Immune defenses against multicellular parasites are found mainly at the sites of parasite entry, namely, under the skin and in the mucosal tissues of the airways and the gut. Cells of the innate and adaptive immune systems at these sites are specialized to secrete cytokines that promote a type 2 response to parasitic infection. In the presence of an invading parasite, dendritic cells taking up antigens in these tissues migrate to regional lymph nodes, where they tend to drive antigen-specific naive CD4 T cells to become effector TH2 cells. TH2 cells themselves secrete IL-4, IL-5, IL-9, and IL-13, thus maintaining an environment in which further differentiation of TH2 cells is favored. The cytokine IL-33, which can be produced by activated mast cells and by damaged or injured epithelial cells, also contributes to amplification of the TH2 response. IL-33 can act directly on TH2 cells via the IL-33 receptors that these cells express. Allergic responses against common environmental

1	cells, also contributes to amplification of the TH2 response. IL-33 can act directly on TH2 cells via the IL-33 receptors that these cells express. Allergic responses against common environmental antigens are normally avoided because mucosal dendritic cells that encounter antigen in the absence of danger signals such as those provoked by microbial infection generally induce naive CD4 T cells to differentiate into antigen-specific regulatory T cells (T cells). The T cells suppress T-cell responses and contribute to a state of tolerance to the antigen (see Section 12-8) rather than allowing the production of effector or helper cells that might support the production of an allergic response.

1	The cytokines and chemokines produced by TH2 cells both amplify the TH2 response and stimulate the class switching of activated B cells to IgE production. As we saw in Chapter 10, IL-4 or IL-13 provides the first signal that switches B cells to IgE production. IL-4 and IL-13 acting on T and B lymphocytes activate the Janus-family tyrosine kinases Jak1 and Jak3 (see Section 7-20), ultimately leading to phosphorylation (and thereby activation) of the transcriptional regulator STAT6. Mice lacking functional IL-4, IL-13, or STAT6 have impaired TH2 responses and an impaired ability to switch to production of IgE, demonstrating the key importance of these cytokines and their signaling pathways in the IgE response. The second signal for IgE production is a co-stimulatory interaction between CD40 ligand on the T-cell surface and CD40 on the B-cell surface. This interaction is essential for all antibody class switching. Patients with a genetic deficiency of CD40 ligand produce no IgG, IgA, or

1	ligand on the T-cell surface and CD40 on the B-cell surface. This interaction is essential for all antibody class switching. Patients with a genetic deficiency of CD40 ligand produce no IgG, IgA, or IgE, and display a hyper-IgM syndrome phenotype (see Section 13-9).

1	Murine mast cells and basophils can also produce the signals that drive IgE production by B cells. Mast cells and basophils express FcεRI, and when they are activated by antigen cross-linking their FcεRI-bound IgE, they express cell-surface CD40 ligand and secrete IL-4. Similar data exist for human basophils that have also been primed by inflammatory stimuli (Fig. 14.3). Like TH2 cells, they can induce class switching and IgE production by B cells. Generally, class switching to IgE occurs in lymph nodes (secondary lymphoid organs) that drain the site of antigen entry or in inducible lymphoid follicles (also called tertiary lymphoid tissues) that form in mucosal and other tissues at sites of persistent inflammation. The potential for formation in mucosal tissues of tertiary lymphoid follicles with germinal centers containing B cells that have switched to production of IgE means that mast cells or basophils can amplify the B-cell response close to the site of the allergic reaction. One

1	with germinal centers containing B cells that have switched to production of IgE means that mast cells or basophils can amplify the B-cell response close to the site of the allergic reaction. One goal of therapy for allergies is to block this amplification process and thus prevent allergic reactions from becoming self-sustaining.

1	In humans, the IgE response, once initiated, can also be amplified by the capture of IgE by Fcε receptors on dendritic cells. Some populations of human immature dendritic cells—for example, the Langerhans cells of the skin— express surface FcεRI in an inflammatory setting, and once anti-allergen IgE antibodies have been produced, they can bind to these receptors. The bound IgE forms a highly effective trap for allergen, which is then efficiently processed by the dendritic cell for presentation to naive T cells, thus maintaining and reinforcing the TH2 response to the allergen. Eosinophils have also been reported to express IgE receptors, but this is still controversial. Eosinophils may act as antigen-presenting cells to T cells in a standard fashion after upregulation of eosinophil MHC class II and co-stimulatory molecules; however, this probably occurs in tissues where activated T cells have migrated rather than in lymph nodes where naive T cells are primed by dendritic cells.

1	14-2 Although many types of antigens can cause allergic sensitization, proteases are common sensitizing agents. Most airborne allergens are relatively small, highly soluble proteins that are carried on dry particles such as pollen grains or mite feces (Fig. 14.4). On contact with the mucus-covered epithelia of the eyes, nose, or airways, the soluble allergen is eluted from the particle and diffuses into the mucosa, where it can be picked up by dendritic cells and provoke sensitization (see Fig. 14.2). At mucosal surfaces, allergens are typically presented to the immune system at low concentrations. It has been estimated that the maximum exposure of a person to the common pollen allergens in ragweed (Ambrosia species) does not exceed 1 μg per year. It is thought that low-dose sensitization favors formation of a strong TH2 response. Consequently, these minute doses of allergen can provoke irritating and even life-threatening TH2-driven IgE antibody responses in atopic individuals.

1	Antigen exposures that lead to allergic responses do not always involve such low doses of antigen, especially at other tissue sites. For example, bee venom is a frequent cause of allergic sensitization, and individual bee stings result in the injection into the skin of 20–75 μg of bee venom (1 to 2 orders of magnitude more than the total dose of ragweed antigen that is inhaled into the airways). In the case of food allergy, ingestion of many grams of an allergenic food into the gastrointestinal tract over prolonged periods of time can lead to sensitization. Sensitization can also occur in response to small or large doses of injected antigens. For example, before the introduction of recombinant

1	Top panel: IgE secreted by plasma cells binds to the high-affinity IgE receptor on basophils (illustrated here) and mast cells. Bottom panel: when the surface-bound IgE is cross-linked by antigen, these cells express CD40 ligand (CD40L) and secrete IL-4, which in turn binds to IL-4 receptors (IL-4R) on the activated B cell. Together with ligation of B-cell CD40 by basophil CD40L, this activates class switching by the B cell and the production of more IgE. These interactions can occur in vivo at the site of allergen-triggered inflammation, for example, in bronchus-associated lymphoid tissue.

1	human insulin, individuals with diabetes could develop allergy to porcine insulin, usually administered in doses of 1–2 milligrams per injection. In contrast, doses of penicillin-type drugs (including cephalosporins and other β-lactam-containing antibiotics) that can lead to sensitization when administered by intramuscular or intravenous injection are usually in the 1to 2-gram per injection range. Considerable effort has been directed toward identifying physical, chemical, or functional characteristics that might be common for all allergens, but no common characteristics of all allergens have emerged. Thus, it appears that in a susceptible host, essentially any antigenic molecule can elicit an allergic response.

1	While any type of molecule appears to be able to elicit an allergic response, the search for common features of allergenic molecules has demonstrated that some clinically significant allergens are proteases. One ubiquitous protease allergen is the cysteine protease Der p 1, which is present in the feces of the house dust mite Dermatophagoides pteronyssinus. Der p 1 provokes allergic reactions in about 20% of the North American population. This enzyme has been found to cleave occludin, a protein component of intercellular tight junctions in the airway mucosa. This reveals one possible reason for the allergenicity of certain enzymes. By destroying the integrity of the tight junctions between epithelial cells, Der p 1 may gain abnormal access to subepithelial antigen-presenting cells (see Fig. 14.2). The tendency of proteases to induce IgE production is highlighted by individuals with Netherton’s syndrome (Fig. 14.5), which is characterized by high levels of IgE and multiple allergies.

1	14.2). The tendency of proteases to induce IgE production is highlighted by individuals with Netherton’s syndrome (Fig. 14.5), which is characterized by high levels of IgE and multiple allergies. This disease is caused by a mutation in SPINK5 (serine protease inhibitor Kazaltype 5), which encodes the serine protease inhibitor LEKTI (lymphoepithelial Kazal type-related inhibitor). LEKTI is expressed in the most differentiated viable layer of the skin (the granular cell layer), just internal to the cornified cell layer of the epidermis. Absence of LEKTI in Netherton’s syndrome results in overly active epidermal kallikreins, proteases that can cleave desmosomes in the skin, leading to keratinocyte shedding and disturbed skin barrier function. Overly active kallikrein 5 leads to overexpression in the skin of TNF-α, ICAM-1, IL-8, and thymic stromal lymphopoietin (TSLP). TSLP is a major agonist of allergic manifestations in the skin, and is essential for the development of both the

1	in the skin of TNF-α, ICAM-1, IL-8, and thymic stromal lymphopoietin (TSLP). TSLP is a major agonist of allergic manifestations in the skin, and is essential for the development of both the eczematous skin lesions and the allergic manifestations (including food allergy) seen in Netherton’s syndrome. Additionally, LEKTI is thought to inhibit the proteases released by bacteria such as Staphylococcus aureus. This may be of special significance in the eczematous process, since a very large fraction of individuals with chronic eczema show persistent colonization with S. aureus and resolution of the eczema is facilitated by elimination of the Staphylococcus, in addition to suppression of the inflammatory response.

1	The observation that loss-of-function mutations in a protease inhibitor in Netherton’s syndrome led to the development of multiple allergies provides additional support for the possibility that protease inhibitors might be novel therapeutic targets in some allergic disorders. Furthermore, the cysteine protease papain, derived from the papaya fruit, is used as a meat. Papain can

1	Fig. 14.5 Netherton’s syndrome illustrates the association of proteases with the development of high levels of IgE and allergy. This 26-year-old man with Netherton’s syndrome, caused by a deficiency in the protease inhibitor SPINK5, had persistent erythroderma (redness of the skin), recurrent infections of the skin and other tissues, and multiple food allergies associated with high serum IgE levels. In the top photograph, large erythematous plaques covered with scales and erosions are visible over the upper trunk. The lower panel shows a section through the skin of the same patient. Note the psoriasis-like hyperplasia of the epidermis. Neutrophils are also present in the epidermis. In the dermis, a perivascular infiltrate is evident. Although not discernible at this magnification, the infiltrate contains both mononuclear cells and neutrophils. Source: Sprecher, E., et al.: Clin. Exp. Dermatol. 2004, 29:513–517.

1	cause allergic reactions in workers preparing the enzyme. Allergies caused by environmental allergens present in the workplace are called occupational allergies. Although Der p 1 and papain are potent allergens, not all allergens are enzymes. In fact, two allergens identified from filarial worms are enzyme inhibitors, and, in general, most allergenic pollen-derived proteins do not seem to possess enzymatic activity.

1	Knowledge of the identity of allergenic proteins can be important to public health and can have economic significance, as illustrated by the following cautionary tale. Some years ago, the gene for 2S albumin from Brazil nuts, a protein that is rich in methionine and cysteine, was transferred by genetic engineering into soybeans intended for animal feed. This was done to improve the nutritional value of soybeans, which are intrinsically poor in these sulfur-containing amino acids. This experiment led to the discovery that 2S albumin is the major Brazil nut allergen. Injection of extracts of the genetically modified soybeans into the epidermis triggered an allergic skin response in people allergic to Brazil nuts. As there could be no guarantee that the modified soybeans could be kept out of the human food chain if they were produced on a large scale, development of this genetically modified food was abandoned.

1	14-3 Genetic factors contribute to the development of IgE-mediated allergic disease. Susceptibility to development of allergic disease has both genetic and environmental components. In studies performed in Western industrialized countries, up to 40% of the test population shows an exaggerated tendency to mount IgE responses to a wide variety of common environmental allergens. Atopic individuals often develop two or more allergic diseases such as allergic rhinoconjunctivitis, allergic asthma, or allergic eczema. Individuals who manifest all three of these disorders are said to express the atopic triad.

1	Genome-wide association studies (GWASs) have uncovered more than 40 susceptibility genes for the allergic skin condition atopic eczema (also known as atopic dermatitis) and for allergic asthma (Fig. 14.6). Some of the susceptibility genes are common to both atopic eczema and allergic asthma, suggesting that some aspects of the atopic diathesis (that is, predisposition) are governed by similar genetic factors regardless of the organs that are the targets of the allergic response. For example, specific alleles at the IL-33 receptor and IL-13 loci show strong association with both allergic asthma and atopic eczema. This sharing of genetic risk alleles by allergic asthma and atopic eczema is consistent with the finding that these two disorders are commonly found together in atopic families, with some family members manifesting both disorders, while other family members may manifest only atopic eczema or allergic asthma, but not both. There are, however, alleles of many genes (especially

1	with some family members manifesting both disorders, while other family members may manifest only atopic eczema or allergic asthma, but not both. There are, however, alleles of many genes (especially genes that regulate skin-barrier function) that show linkage to atopic eczema without enhancing the risk for allergic asthma or allergic rhinoconjunctivitis, indicating that other genetic factors contribute importantly to the phenotype of allergic responsiveness any individual may express. In addition, there are many ethnic differences in the susceptibility genes for a given allergic disease. Several of the chromosome regions associated with allergy or asthma are also associated with the inflammatory disease psoriasis and with autoimmune diseases, suggesting that these loci contain genes that are involved in exacerbating inflammation.

1	One candidate susceptibility gene for both allergic asthma and atopic eczema resides at chromosome 11q12–13 and encodes the β subunit of the high-affinity IgE receptor FcεRI. Another region of the genome associated with allergic disease, 5q31–33, contains at least four types of candidate genes that might be responsible for increased susceptibility. First, there is a cluster of tightly linked genes for cytokines that enhance IgE class switching, eosinophil Fig. 14.6 Susceptibility loci for asthma. Loci that have shown linkage on the basis of GWAS or targeted gene analysis are listed, separated into genes that are expressed in epithelial cells of the airways, genes that regulate the differentiation and/or function of CD4 T cells and ILC2s, and genes with other miscellaneous or unknown functions.

1	survival, and mast-cell proliferation, all of which help to produce and maintain an IgE-mediated allergic response. This cluster includes the genes for IL-3, IL-4, IL-5, IL-9, IL-13, and granulocyte–macrophage colony-stimulating factor (GM-CSF). In particular, genetic variation in the promoter region of the gene encoding IL-4 has been associated with raised IgE levels in atopic individuals. The variant promoter directs increased expression of a reporter gene in experimental systems and thus might produce increased IL-4 in vivo. Atopy has also been associated with a gain-of-function mutation of the α subunit of the IL-4 receptor, with the mutation causing increased signaling after ligation of the receptor.

1	A second set of genes in this region of chromosome 5 belongs to the TIM family (for T cell, immunoglobulin domain, and mucin domain). The genes in this set encode three T-cell-surface proteins (Tim-1, -2, and -3) and one protein expressed primarily on antigen-presenting cells (Tim-4). In mice, Tim-3 protein is specifically expressed on TH1 cells and negatively regulates TH1 responses, whereas Tim-2 (and to a lesser extent Tim-1) is preferentially expressed in TH2 cells and negatively regulates them. Mouse strains that carry different variants of the Tim genes differ both in their susceptibility to allergic inflammation of the airways and in the production of IL-4 and IL-13 by their T cells. Although no homolog of the mouse Tim-2 gene has been found in humans, inherited variation in the three human TIM genes has been correlated with airway hyperreactivity or hyperresponsiveness. In this condition, contact not only with allergen but also with nonspecific irritants causes airway

1	the three human TIM genes has been correlated with airway hyperreactivity or hyperresponsiveness. In this condition, contact not only with allergen but also with nonspecific irritants causes airway narrowing (bronchoconstriction), with wheezy breathlessness similar to that seen in asthma. The third candidate susceptibility gene in this part of the genome encodes p40, one of the two subunits of IL-12 and IL-23. These cytokines promote TH1 and TH17 responses, and genetic variation in p40 expression that could cause reduced production of IL-12 and IL-23 was found to be associated with more severe asthma. A fourth candidate susceptibility gene which encodes the β-adrenergic receptor is also located in this region. Variation in this receptor might be associated with alteration in smooth muscle responsiveness to endogenous and pharmacological ligands.

1	The detection of multiple potential susceptibility genes illustrates a common challenge in identifying the genetic basis of complex disease traits. Relatively small regions of the genome, identified as containing genes for altered disease susceptibility, may contain many good candidates, judging by their known physiological activities. Identifying the gene, or genes, that truly lead to expression of the disease may require studies of several very large populations of patients and controls. For chromosome 5q31–33, for example, it is still too early to know how important each of the different polymorphisms is in the complex genetics of atopy.

1	A second type of inherited variation in IgE responses is linked to the HLA class II region (the human MHC class II region), and it affects responses to specific allergens, rather than a general susceptibility to atopy. IgE production in response to particular allergens is associated with certain HLA class II alleles, implying that particular peptide:MHC combinations might favor a strong TH2 response; for example, IgE responses to several ragweed pollen allergens are associated with haplotypes containing the HLA class II allele DRB1*1501. Many people are therefore generally predisposed to make TH2 responses and are specifically predisposed to respond to some allergens more than others. Allergic responses to drugs such as penicillin were originally thought to show no association with HLA class II or with the presence or absence of atopy. Recent studies, however, have shown evidence that some drugs can interact with specific HLA alleles in a way that changes the structure of peptide

1	class II or with the presence or absence of atopy. Recent studies, however, have shown evidence that some drugs can interact with specific HLA alleles in a way that changes the structure of peptide antigens bound in the groove of the HLA molecule so that these altered peptides elicit an autoimmune-type reaction. An example of this is the binding of the seizure medication carbamazepine with HLA-B15:02 and peptide bound in this HLA-B allele. The immune response to this carbamazepine:peptide:HLA-B complex can lead to development of toxic epidermal necrolysis, a severe immune-mediated skin reaction in which widespread skin loss occurs due to necrosis, leaving the skin looking scalded.

1	There are also likely to be genes that affect only particular aspects of allergic disease. In asthma, for example, there is evidence that different genes affect at least three aspects of the disease—IgE production, the inflammatory response, and clinical responses to particular treatments. On chromosome 20, polymorphism of the gene encoding ADAM33, a metalloproteinase expressed by bronchial smooth muscle cells and lung fibroblasts, has been associated with asthma and bronchial hyperreactivity. This is likely to be an example of genetic variation in the pulmonary inflammatory response and in the pathological anatomical changes that occur in the airways (airway remodeling). In the skin, filaggrin importantly contributes to normal skin barrier function by binding keratin molecules into the lipid envelope of cornifying keratinocytes. Loss-of-function mutations in the gene encoding filaggrin lead to the development of eczema. Through unknown mechanisms, filaggrin mutations also can

1	the lipid envelope of cornifying keratinocytes. Loss-of-function mutations in the gene encoding filaggrin lead to the development of eczema. Through unknown mechanisms, filaggrin mutations also can contribute to the development of asthma. Almost half of people in the United States who suffer from severe eczema have at least one mutated filaggrin allele. Between 7 and 10% of Caucasians carry a loss-of-function mutation in filaggrin, and the frequency of this mutation is considerably higher in individuals with asthma.

1	14-4 Environmental factors may interact with genetic susceptibility to cause allergic disease. Studies of susceptibility suggest that environmental factors and genetic variation each account for about 50% of the risk of developing atopy. The prevalence of atopic allergic diseases, and of asthma in particular, is increasing in economically advanced regions of the world, and this is likely due to the impact of changes in certain environmental factors on individuals with genetic backgrounds that predispose them toward atopy. Interestingly, although the incidence of asthma is lower in economically underdeveloped regions of Africa, Americans of African ancestry show increased asthma frequency and severity compared with Americans not of African ancestry. This shows a clear impact of environment on the expressivity of genetic influences.

1	The prevalence of atopy and especially allergic asthma has been steadily increasing in the developed world for the past 50–60 years. One hypothesis for this steady increase is changes in exposure to infectious diseases in early childhood as our population has moved increasingly from rural into urban environments. This shift has meant less early-life exposure to microorganisms associated with farm animals and microorganisms in the soil. This change in exposure is thought to lead to alterations in the intestinal microbiota, which performs an important immunomodulatory function (discussed in Chapter 12). Changes in exposure to ubiquitous microorganisms as a possible cause of increased atopy was first suggested in 1989, ultimately giving rise to the hygiene hypothesis (Fig. 14.7). The original proposition was that less hygienic environments, particularly those encountered in less developed rural settings, predispose to infections early in childhood, which help to protect against the

1	proposition was that less hygienic environments, particularly those encountered in less developed rural settings, predispose to infections early in childhood, which help to protect against the development of atopy and allergic asthma. It was originally proposed that the protective effect might be due to mechanisms that skew immune responses away from the production of TH2 cells (and their associated cytokines, which dispose toward IgE production) and toward the production of TH1 cells. This would impede responses that favor the production of IgE and promote responses that suppress class switching to IgE.

1	Suggesting that this interpretation was overly simplistic was the strong negative correlation between infection by helminths (such as hookworms and schistosomes) and the development of allergic disease. A study in Venezuela showed that children treated for a prolonged period with antihelminthic agents had

1	Fig. 14.7 Genes, the environment, and atopic allergic diseases. Both inherited and environmental factors are important determinants of the likelihood of developing allergic disease. Many genes are known to influence the development of asthma (see Fig. 14.6). The postulate of the ‘hygiene hypothesis’ is that exposure to some infections and to common environmental microorganisms in infancy and childhood drives the immune system toward a general state of non-atopy. In contrast, children who have a genetic susceptibility to atopy and who live in an environment with low exposure to infectious disease and environmental microorganisms, or who received multiple courses of antibiotics in infancy and early childhood, are thought not to develop efficient immunoregulatory mechanisms and to be most susceptible to the development of atopic allergic disease.

1	a higher prevalence of atopy than did untreated and heavily parasitized children. As helminths provoke a strong TH2-mediated IgE response, this seemed inconsistent with the hygiene hypothesis.

1	One possible explanation for this apparent inconsistency suggests that all types of infection might protect against the development of atopy because the host responses they elicit include the production of cytokines such as IL-10 and TGF-β, perhaps as part of the homeostatic responses that occur as the infection is being controlled. IL-10 and TGF-β suppress the formation of both TH1 and TH2 responses, and IL-10 suppresses TH17 responses (see Sections 9-21 and 9-23). A large proportion of allergic reactions are initiated by antigens that enter though mucosal surfaces such as the respiratory or intestinal epithelium. As described in Chapter 12, the human mucosal immune system has evolved mechanisms of regulating responses to commensal flora and environmental antigens (such as food antigens) that involve the generation of IL-10/TGF-βproducing Treg cells. The idea underlying the current version of the hygiene hypothesis is that decreased early exposure to common microbial pathogens and

1	that involve the generation of IL-10/TGF-βproducing Treg cells. The idea underlying the current version of the hygiene hypothesis is that decreased early exposure to common microbial pathogens and commensals in some way makes the body less efficient at producing these Treg cells, thus increasing the risk of making an allergic response to a common environmental antigen.

1	In support of a role for disturbed immunoregulatory pathways in susceptibility to asthma is evidence that exposure to certain types of childhood infection, with the important exception of some respiratory infections that we consider below, helps to protect against the development of allergic disease. Younger children from families with three or more older siblings and children aged less than 6 months who are exposed to other children in daycare facilities— situations linked to a greater exposure to infections—appear to be partially protected against atopy and asthma. Additionally, children with early-life exposure to a farm or ones having a dog are also somewhat protected against development of atopy and asthma, presumably because of their exposure to farmor pet-associated microbes. Furthermore, early colonization of the gut by commensal bacteria such as lactobacilli and bifidobacteria, or infection by gut pathogens such as Toxoplasma gondii or Helicobacter pylori, is associated with

1	early colonization of the gut by commensal bacteria such as lactobacilli and bifidobacteria, or infection by gut pathogens such as Toxoplasma gondii or Helicobacter pylori, is associated with a reduced prevalence of allergic disease. There is also emerging evidence that, conversely, repeated exposure to antibiotics in early life increases the risk of developing asthma.

1	A history of infection with hepatitis A virus also seems to have a negative association with atopy. A possible explanation for this association is that the human counterpart of the murine Tim-1 protein (see Section 14-3) is the cellular receptor for hepatitis A virus (designated HAVCR1). The infection of T cells by hepatitis A virus could thus directly influence their differentiation and cytokine production, limiting the development of an IgE-generating response.

1	In contrast to these negative associations between childhood infection and the development of atopy and asthma is evidence that children who have had attacks of bronchiolitis associated with respiratory syncytial virus (RSV) infection are more prone to developing asthma later on. Children hospitalized with RSV infection have a skewed ratio of cytokine production away from IFN-γ toward IL-4, presumably increasing their likelihood of developing TH2 responses and increased production of IgE. This effect of RSV appears to depend on age at first infection. Experimental infection of neonatal mice with RSV was followed by lower increases in the production of IFN-γcompared with mice that received experimental RSV infection at 4 or 8 weeks of age. When the mice were rechallenged at 12 weeks of age with RSV infection, animals that had been primarily infected as neonates had more severe lung inflammation than animals first infected at 4 or 8 weeks of age.

1	Other environmental factors that might contribute to the increase in atopic disease are changes in diet, allergen exposure, atmospheric pollution, and tobacco smoke. Pollution has been blamed for an increase in the prevalence of nonallergic cardiopulmonary diseases such as chronic bronchitis, but an association with allergic disease has been more difficult to demonstrate. There is, however, increasing evidence for an interaction between allergens and pollution, particularly in genetically susceptible individuals. Diesel exhaust particles are the best-studied pollutant in this context; they increase IgE production 20to 50-fold when combined with allergen, with an accompanying shift to TH2 cytokine production. Reactive oxidant chemicals such as ozone are generated as a result of such pollution, and individuals less able to deal with this onslaught may be at increased risk of allergic disease.

1	Genes that might be governing this aspect of susceptibility are GSTP1 and GSTM1, members of the glutathione-S-transferase superfamily that are important in preventing oxidant stress. Individuals who were allergic to ragweed pollen and who carried particular variant alleles of these genes showed an increased airway hyperreactivity when challenged with the allergen-plus-diesel exhaust particles, compared with the allergen alone. A study in Mexico City on the effects of atmospheric ozone levels on atopic children with allergic asthma also found that the children carrying the null allele of GSTM1 were more susceptible than noncarriers to airway hyperreactivity when exposed to given levels of ozone. Underscoring the potential for reactive oxygen species such as ozone and superoxide to contribute to asthma exacerbation, studies using mice indicate that airway myeloid cells that produce high levels of super-oxide worsen antigen-induced airway hyperreactivity. Inhibitors of NADPH oxidase,

1	to asthma exacerbation, studies using mice indicate that airway myeloid cells that produce high levels of super-oxide worsen antigen-induced airway hyperreactivity. Inhibitors of NADPH oxidase, required for the production of superoxide, reduce antigen-induced airway hyperreactivity in sensitized and challenged animals, whereas adoptive transfer of superoxide-producing myeloid cells into the airways of sensitized and challenged mice causes marked exacerbation of hyperresponsiveness.

1	14-5 Regulatory T cells can control allergic responses.

1	The observation that treatment of peripheral blood mononuclear cells (consisting primarily of lymphocytes and monocytes) from atopic individuals with anti-CD3 and anti-CD28 stimulates production of substantial quantities of TH2 cytokines, whereas similar treatment of cells from non-atopic individuals does not, suggests that circulating leukocytes in atopic individuals have been previously stimulated in a fashion that programs them to generate type 2 responses. An increasing number of studies suggest that regulatory mechanisms that normally serve to suppress overly aggressive type 2 responses are also abnormal in subjects with atopy. When peripheral blood CD4+CD25+ Treg cells from atopic individuals are co-cultured with polyclonally activated CD4+ T cells, they are less effective at suppressing TH2 cytokine production compared with similar Treg cells from non-atopic individuals, and this defect is even more pronounced during the pollen season. More evidence for a role for Treg cells in

1	TH2 cytokine production compared with similar Treg cells from non-atopic individuals, and this defect is even more pronounced during the pollen season. More evidence for a role for Treg cells in atopy comes from mice deficient in the transcription factor FoxP3, the master switch for producing both natural (thymus-derived) and some types of induced Treg cells. These mice develop several manifestations of atopy, including increased numbers of blood eosinophils and increased levels of circulating IgE, as well as spontaneous allergic airway inflammation. Manipulation of the Treg pathway can ameliorate experimental asthmatic inflammation in mice. Increasing expression of the anti-inflammatory enzyme indoleamine 2,3-dioxygenase (IDO) by treatment with IFN-γ or by unmethylated CpG DNA can induce the generation or activation of Treg cells. Induction of IDO activity in resident dendritic cells in the lung by stimulation with CpG DNA enhances Treg activity and ameliorates experimental asthma in

1	the generation or activation of Treg cells. Induction of IDO activity in resident dendritic cells in the lung by stimulation with CpG DNA enhances Treg activity and ameliorates experimental asthma in mice. These findings suggest that therapies aiming to enhance Treg function could be beneficial in asthma and other atopic disorders. Other immunoregulatory molecules that might serve as immunotherapeutic agents for treatment of asthma include the cytokines IL-35 and IL-27, which, like IL-10, can inhibit TH2 responses. Alternatively, blockade of the cytokine IL-31 is anticipated to be therapeutically beneficial since IL-31 promotes TH2-driven inflammation.

1	Summary.

1	Allergens are generally innocuous antigens that commonly provoke an IgE antibody response in susceptible individuals. Such antigens normally enter the body at very low doses by diffusion across mucosal surfaces and trigger a type 2 immune response. The differentiation of naive allergen-specific T cells into TH2 cells is favored by cytokines such as IL-4 and IL-13. Allergen-specific TH2 cells producing IL-4 and IL-13 drive allergen-specific B cells to produce IgE. The specific IgE produced in response to the allergen binds to the high-affinity receptor for IgE on mast cells and basophils. IgE production can be amplified by these cells because, upon activation, they produce IL-4 and express CD40 ligand. The tendency to IgE overproduction is influenced by both genetic and environmental factors. Once IgE has been produced in response to an allergen, reexposure to the allergen triggers an allergic response. We describe the mechanism and pathology of the allergic responses themselves in the

1	Once IgE has been produced in response to an allergen, reexposure to the allergen triggers an allergic response. We describe the mechanism and pathology of the allergic responses themselves in the next part of the chapter.

1	Effector mechanisms in IgE-mediated allergic reactions.

1	Allergic reactions are triggered when allergens cross-link preformed IgE bound to the high-affinity receptor FcεRI on mast cells. Mast cells line external mucosal surfaces and serve to alert the immune system to local infection. Once activated, they induce inflammatory reactions by secreting pharmacological mediators such as histamine stored in preformed granules and by synthesizing prostaglandins, leukotrienes, and platelet-activating factor from the plasma membrane. They also release various cytokines and chemokines after activation. In the case of an allergic reaction, they provoke unpleasant reactions to innocuous antigens that are not associated with invading pathogens that need to be expelled. The consequences of IgE-mediated mast-cell activation depend on the dose of antigen and its route of entry; symptoms range from the swollen eyes and rhinitis associated with contact of pollen with the conjunctiva of the eye and the nasal epithelium, to the life-threatening circulatory

1	its route of entry; symptoms range from the swollen eyes and rhinitis associated with contact of pollen with the conjunctiva of the eye and the nasal epithelium, to the life-threatening circulatory collapse that occurs in anaphylaxis (Fig. 14.8). The immediate reaction caused by mast-cell degranulation is followed, to a greater or lesser extent depending on the disease, by a more sustained inflammation, which is due to the recruitment of other effector leukocytes, notably TH2 lymphocytes, eosinophils, and basophils.

1	Fig. 14.8 Mast-cell activation has different effects on different tissues. Increased ˜uid in tissues causing increased ˜ow of lymph to lymph nodes, increased cells and protein in tissues, increased effector response in tissues Hypotension potentially leading to anaphylactic shock Congestion and blockage of airways (wheezing, coughing, phlegm) Swelling and mucus secretion in nasal passages Ocular itching Sneezing Expulsion of gastrointestinal tract contents (diarrhea, vomiting) Decreased airway diameter, increased mucus secretion Eyes, nasal passages, and airways Mast-cell activation and granule release Increased ˜uid secretion, increased peristalsis Increased blood ˜ow, increased permeability Gastrointestinal tract Blood vessels 14-6 Most IgE is cell-bound and engages effector mechanisms of the immune system by pathways different from those of other antibody isotypes.

1	Antibodies engage effector cells such as mast cells by binding to receptors specific for their Fc constant regions. Most antibodies engage Fc receptors only after their antigen-binding sites have bound specific antigen, forming an immune complex of antigen and antibody. IgE is an exception, because it is captured by the high-affinity Fcε receptor (FcεRI) in the absence of bound antigen. This means that, unlike other antibodies, which are found mainly in body fluids, IgE is mostly found fixed on cells that carry this receptor—mast cells in tissues, and basophils in the circulation and at sites of inflammation. The ligation of the cell-bound IgE antibody by specific multivalent antigen triggers the activation of these cells at the sites of antigen entry into the tissues. The release from these activated mast cells of inflammatory lipid mediators, cytokines, and chemokines at sites of IgE-triggered reactions recruits eosinophils and basophils to augment the allergic response. It also

1	these activated mast cells of inflammatory lipid mediators, cytokines, and chemokines at sites of IgE-triggered reactions recruits eosinophils and basophils to augment the allergic response. It also recruits TH2 cells, which can then mount a local type 2 cellular response.

1	There are two types of IgE-binding Fc receptors. The first, FcεRI, expressed on mast cells and basophils, is a high-affinity receptor of the immunoglobulin superfamily (see Section 10-24). When IgE bound to this receptor is cross-linked by specific antigen, it transduces an activating signal through the receptor-bound Lyn tyrosine kinase, which phosphorylates ITAMs on the intracellular domain of the receptor. This recruits and activates the amplifying tyrosine kinase Syk, which phosphorylates and activates a broad range of downstream effector pathways. High levels of IgE, such as those that exist in people with allergic diseases or parasite infections, can result in a marked increase in FcεRI on the surface of mast cells, an enhanced sensitivity of such cells to activation by low concentrations of specific antigen, and a markedly increased, IgE-dependent release of chemical mediators and cytokines.

1	The second IgE receptor, FcεRII, usually known as CD23, is a C-type lectin and is structurally unrelated to FcεRI; it binds IgE with low affinity. CD23 is present on many cell types, including B cells, activated T cells, monocytes, eosinophils, platelets, follicular dendritic cells, and some thymic epithelial cells. This receptor was thought to be crucial for the regulation of IgE levels, but mouse strains in which the gene for CD23 has been inactivated still develop relatively normal polyclonal IgE responses. Nevertheless, CD23 does seem to be involved in enhancing IgE antibody levels in some situations. Responses against a specific antigen are known to be increased in the presence of the antigen complexed with IgE, but such enhancement fails to occur in mice that lack the gene for CD23. This has been interpreted to indicate that CD23 on antigen-presenting cells has a role in the capture of antigen that is complexed with IgE.

1	14-7 Mast cells reside in tissues and orchestrate allergic reactions. When Paul Ehrlich described mast cells found in the mesentery of rabbits, he called them Mastzellen (‘fattened cells’). Like basophils, mast cells contain granules rich in acidic proteoglycans that take up basic dyes. Mast cells are derived from hematopoietic stem cells but mature locally, often residing near surfaces exposed to pathogens and allergens, such as mucosal tissues and the connective tissues surrounding blood vessels. Mucosal mast cells differ in some of their properties from submucosal or connective tissue mast cells, but both can be involved in allergic reactions. Fig. 14.9 Molecules released by activated mast cells. Mast cells release a wide variety of biologically active proteins and other chemical mediators. The enzymes and toxic mediators listed in the first two rows are released from the preformed granules. The cytokines, chemokines, and lipid mediators are mostly synthesized after activation.

1	The major factors for mast-cell growth and development include stem-cell factor (the ligand for the receptor tyrosine kinase Kit), IL-3, and TH2-associated cytokines such as IL-4 and IL-9. Mice with defective Kit lack differentiated mast cells, and although they produce IgE, they cannot make IgE-mediated inflammatory responses. This shows that such responses depend almost exclusively on mast cells. Mast-cell activation depends on the activation of phosphatidylinositol 3-kinase (PI 3-kinase) in mast cells by Kit, and pharmacological inactivation of the p110δ isoform of PI 3-kinase has been shown to protect mice against allergic responses. Inhibitors of the amplifying tyrosine kinase Syk are also showing promise as blockers of IgE-dependent mast-cell responses.

1	Mast cells express FcεRI constitutively on their surface and are activated when antigens cross-link IgE bound to these receptors (see Fig. 10.43). A relatively low level of allergen is sufficient to trigger degranulation. There are many mast-cell precursors in tissues, and they can rapidly differentiate into mature mast cells in conditions of allergic inflammation, thus aiding the continuation of the allergic response. Mast-cell degranulation begins within seconds of antigen binding, releasing an array of preformed and newly generated inflammatory mediators (Fig. 14.9). Granule contents include the short-lived vasoactive amine histamine, serine esterases, and proteases such as chymase and tryptase.

1	Histamine has four known receptors through which it acts—H1 through H4, each a G-protein-coupled receptor. Histamine acts via the H1 receptor on local blood vessels to cause an immediate increase in local blood flow and vessel permeability. This leads to edema and local inflammation. Histamine is also a major stimulus for itching and sneezing, by virtue of its activation of neural receptors. Acting through the H1 receptor on dendritic cells, histamine can increase antigen-presenting capacity and TH1 cell priming; acting through the H1 receptor on T cells, it can enhance TH1 cell proliferation and IFN-γproduction. By acting through H2, H3, and H4 receptors on a variety of leukocytes and tissue cells, histamine participates in atopic dermatitis, chronic urticaria, and several autoimmune disorders.

1	Human mast cells are classified into subtypes on the basis of their protease content and tissue location. Mast cells in mucosal epithelia express tryptase as their primary serine protease. These cells are designated MCT. Mast cells in the submucosa and other connective tissues predominantly express chymase, tryptase, carboxypeptidase A, and cathepsin G and are designated MCCT. The proteases released by the mast cells activate matrix metalloproteinases, which break down extracellular matrix proteins, causing tissue disintegration and damage. These proteases can exert beneficial effects such as degrading snake and bee venoms, thus helping to suppress allergic responses to these agents.

1	Following activation through FcεRI, in addition to releasing preformed mediators such as histamine and serine proteases that are stored in their intracellular granules, mast cells also synthesize de novo and release chemokines, cytokines, and lipid mediators—prostaglandins, leukotrienes, thromboxanes (collectively called eicosanoids), and platelet-activating factor. MCT and MCCT mast cells, for example, produce the cytokine IL-4, which helps perpetuate type 2 immune responses. These secreted products contribute to both acute and chronic inflammation. The lipid mediators, in particular, can act both rapidly and persistently to cause smooth muscle contraction, increased vascular permeability, and the secretion of mucus, as well as induce the influx and activation of leukocytes, which contribute to allergic inflammation.

1	Eicosanoids derive mainly from the membrane-associated fatty acid arachidonic acid. This is cleaved from membrane phospholipids by phospholipase A2, which is activated at the plasma membrane as a result of cell activation. Arachidonic acid can be modified by either of two pathways to give rise to lipid mediators. Modification via the cyclooxygenase pathway produces the prostaglandins and thromboxanes, whereas leukotrienes are produced via the lipoxygenase pathway. Prostaglandin D2 is the major prostaglandin produced by mast cells and recruits TH2 cells, eosinophils, and basophils, all of which express its receptor (PTGDR). Prostaglandin D2 is critical to the development of allergic diseases such as asthma, and polymorphisms in the PTGDR gene have been linked to an increased risk of developing asthma. The leukotrienes, especially C4, D4, and E4, are also important in sustaining inflammatory responses in tissues. Nonsteroidal anti-inflammatory drugs such as aspirin and ibuprofen exert

1	asthma. The leukotrienes, especially C4, D4, and E4, are also important in sustaining inflammatory responses in tissues. Nonsteroidal anti-inflammatory drugs such as aspirin and ibuprofen exert their effects by preventing prostaglandin production. They inhibit the cyclooxygenases that act on arachidonic acid to form the ring structure present in prostaglandins.

1	Large amounts of the cytokine tumor necrosis factor (TNF)-α are also released by mast cells after activation. Some comes from stores in the granules; some is newly synthesized by the activated mast cells. TNF-α activates endothelial cells, resulting in increased expression of adhesion molecules, which in turn promotes the influx of pro-inflammatory leukocytes and lymphocytes into the affected tissue (see Chapter 3). Additionally, mast-cell TNF-α contributes importantly to the influx of leukocytes into regional lymph nodes in response to microbial infection of peripheral tissues.

1	Through the action of all of these mediators, IgE-mediated mast-cell activation orchestrates a broad inflammatory cascade that is amplified by the recruitment of several types of leukocytes including eosinophils, basophils, TH2 lymphocytes, and B cells. The biological role of this reaction in normal host immunity is as a defense against parasite infection (see Section 10-25). In an allergic reaction, however, the acute and chronic inflammatory reactions triggered by mast-cell activation have important pathophysiological consequences, as seen in the diseases associated with allergic responses to environmental antigens. The role of mast cells is not, however, limited to IgE-driven pro-inflammatory responses. Increasingly, mast cells are also considered to have a role in immunoregulation. They can be stimulated by neuropeptides such as substance P and by TLR ligands. In response to multiple stimuli, they can secrete the immunosuppressive cytokine IL-10, suppressing

1	Fig. 14.10 Eosinophils secrete a range of highly toxic granule proteins and other inflammatory mediators. As for mast cells (see Fig. 14.9), enzymes and toxic mediators released by eosinophils are largely stored preformed in granules. In contrast, cytokines, chemokines, and lipid mediators are largely synthesized after eosinophil activation. T-cell responses. Conversely, interactions between mast cells and regulatory T cells can prevent mast-cell degranulation. 14-8 Eosinophils and basophils cause inflammation and tissue damage in allergic reactions.

1	Eosinophils are granulocytic leukocytes that originate in bone marrow. They are so called because their granules, which contain arginine-rich basic proteins, are colored bright orange by the acidic stain eosin. In healthy humans, these cells represent less than 6% of the leukocytes in the circulation; most eosinophils are found in tissues, especially in the connective tissue immediately underneath respiratory, gut, and urogenital epithelium, implying a likely role for these cells in defense against invading organisms at these sites. They possess numerous cell-surface receptors, including receptors for cytokines (such as IL-5), Fcγ and Fcα receptors, and the complement receptors CR1 and CR3, through which they can be activated and stimulated to degranulate. For example, parasites coated with IgG, C3b, or IgA can cause eosinophil degranulation. In allergic tissue reactions, the large concentrations of IL-5, IL-3, and GM-CSF that are typically present are likely to contribute to

1	with IgG, C3b, or IgA can cause eosinophil degranulation. In allergic tissue reactions, the large concentrations of IL-5, IL-3, and GM-CSF that are typically present are likely to contribute to degranulation.

1	When activated, eosinophils express two kinds of effector function. First, they can release highly toxic granule proteins and free radicals, which can kill microorganisms and parasites but also can cause significant damage to host tissues in allergic reactions (Fig. 14.10). Second, they can synthesize chemical mediators, including prostaglandins, leukotrienes, and cytokines. These amplify the inflammatory response by activating epithelial cells and by recruiting and activating more eosinophils and leukocytes. In chronic inflammatory responses, eosinophils can contribute to airway tissue remodeling.

1	What were later to be defined as eosinophils were observed in the 19th century in the first pathological description of fatal status asthmaticus (an episode of severe asthma that does not respond to treatment and leads to respiratory failure and death), but the precise role of these cells in allergic disease generally is still unclear. In allergic tissue reactions, for example, those that lead to chronic asthma, mast-cell degranulation, and TH2 activation cause eosinophils to accumulate in large numbers and to become activated. Among other things, eosinophils secrete TH2-type cytokines and in vitro can promote the apoptosis of TH1 cells by their expression of IDO and consequent production of kynurenine, which acts on the TH1 cells. Their apparent promotion of TH2cell expansion may thus be partly due to a relative reduction in TH1-cell numbers. The continued presence of eosinophils is characteristic of chronic allergic inflammation, and eosinophils are thought to be major contributors

1	partly due to a relative reduction in TH1-cell numbers. The continued presence of eosinophils is characteristic of chronic allergic inflammation, and eosinophils are thought to be major contributors to tissue damage. However, the observation that eosinophils accumulate at sites where there are high levels of cell turnover and considerable local stem-cell activity bolsters a growing consensus that eosinophils play an important role in restoring tissue homeostasis after infection and other types of tissue damage.

1	The activation and degranulation of eosinophils is strictly regulated, because their inappropriate activation is harmful to the host. The first level of control acts on the production of eosinophils by the bone marrow. Few eosinophils are produced in the absence of infection or other immune stimulation. But when TH2 cells are activated, cytokines they produce such as IL-5 and GM-CSF increase the production of eosinophils in the bone marrow and their release into the circulation. However, transgenic animals overexpressing IL-5 have increased numbers of eosinophils (eosinophilia) in the circulation but not in their tissues, indicating that the migration of eosinophils from the circulation into tissues is regulated separately, by a second set of controls. The key molecules in this case are the CC chemokines that have been named eotaxins because of their specificity for eosinophils: CCL11 (eotaxin 1), CCL24 (eotaxin 2), and CCL26 (eotaxin 3).

1	The eotaxin receptor on eosinophils, CCR3, is quite promiscuous and binds other CC chemokines, including CCL5, CCL7, and CCL13, which also induce eosinophil chemotaxis and activation. Identical or similar chemokines stimulate mast cells and basophils. For example, eotaxins attract basophils and cause their degranulation. TH2 cells also carry the receptor CCR3 and migrate toward eotaxins.

1	Basophils are also present at the site of an inflammatory reaction, and growth factors for basophils are very similar to those for eosinophils; they include IL-3, IL-5, and GM-CSF. There is evidence for reciprocal control of the maturation of the stem-cell population into basophils or eosinophils. For example, TGF-β in the presence of IL-3 suppresses eosinophil differentiation and enhances that of basophils. Basophils are normally present in very low numbers in the circulation and seem to have a similar role to that of eosinophils in defense against pathogens. Like eosinophils, they are recruited to the sites of IgE-mediated allergic reactions. Basophils express high-affinity FcεRI on their cell surfaces and so have IgE bound. On activation by antigen binding to IgE or by cytokines, they release histamine from their granules and also produce IL-4 and IL-13.

1	Eosinophils, mast cells, and basophils can interact with each other. Eosinophil degranulation releases major basic protein (see Fig. 14.10), which in turn causes the degranulation of mast cells and basophils. This effect is augmented by any of the cytokines that affect eosinophil and basophil growth, differentiation, and activation, such as IL-3, IL-5, and GM-CSF. 14-9 IgE-mediated allergic reactions have a rapid onset but can also lead to chronic responses. Under laboratory conditions, the clinical response of a sensitized individual to challenge by intradermal allergen or inhalation of allergen can be divided into an ‘immediate reaction’ and a ‘late-phase reaction’ (Fig. 14.11). The immediate Fig. 14.11 Allergic reactions in response to test antigens can be divided into an immediate response and a late-phase response.

1	Left panel: the response to an inhaled antigen can be divided into early and late responses. An asthmatic response in the lungs with narrowing of the airways caused by the constriction of bronchial smooth muscle and development of edema can be measured as a fall in the peak expiratory flow rate (PEFR). The immediate response peaks within minutes after antigen inhalation and then subsides, returning to near baseline PEFR. Six to eight hours after antigen challenge, there can be a late-phase response that also results in a fall in the PEFR. The immediate response is caused by the direct effects on blood vessels, nerves, and smooth muscle of rapidly metabolized mediators such as histamine and lipid mediators released by mast cells. The late-phase response is caused by the continued production of these mediators, by the production of vasoactive compounds that dilate blood vessels, and by recruitment of lymphocytes and myeloid cells, which together lead to the production of edema. Right

1	of these mediators, by the production of vasoactive compounds that dilate blood vessels, and by recruitment of lymphocytes and myeloid cells, which together lead to the production of edema. Right panel: a wheal-and-flare allergic reaction develops within a minute or two of intradermal injection of antigen and lasts for up to 30–60 minutes. The more widespread edematous response characteristic of the late phase develops approximately 6 hours later and can persist for up to 2 or 3 days. The photograph shows an intradermal skin challenge with allergen resulting in a wheal-and-flare (early-phase) reaction observed 15 minutes after allergen challenge (left) and a late-phase reaction occurring 6 hours after challenge (right). The allergen was grass pollen extract. Photograph courtesy of S.R. Durham.

1	reaction is due to IgE-mediated mast-cell activation and starts within seconds of allergen exposure. It is the result of the actions of histamine, prostaglandins, and other preformed or rapidly synthesized mediators released by mast cells. These mediators cause a rapid increase in vascular permeability, resulting in visible edema and reddening of the skin (in a skin response) and airway narrowing as result of edema and the constriction of smooth muscle (in an airway response). In the skin, histamine acting on H1 receptors on local blood vessels causes an immediate increase in vascular permeability, which leads to extravasation of fluid and edema. Histamine also acts on H1 receptors on local nerve endings, leading to reflex vasodilation of cutaneous blood vessels and local reddening of the skin. The resulting skin lesion is called a wheal-andflare reaction (see Fig. 14.11, right panel).

1	Whether a late-phase reaction occurs depends on allergen dose and on aspects of the cellular immune activation that are difficult to quantify. At doses of intradermally administered allergen that are deemed safe for skin testing of subjects with allergic asthma, for example, a late reaction occurs in about 50% of individuals who show an immediate response (see Fig. 14.11, right panel). The late reaction peaks between 3 and 9 hours after antigen challenge, and in skin tests becomes obvious as a much increased area and degree of edema (see Fig. 14.11, right panel) that can persist for 24 hours or longer. The late-phase reaction is caused by the continued synthesis and release of inflammatory mediators by mast cells, especially vasoactive mediators such as calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor (VEGF), which cause vasodilation and vascular leakage that result in edema and the recruitment of eosinophils, basophils, monocytes, and lymphocytes. The

1	(CGRP) and vascular endothelial growth factor (VEGF), which cause vasodilation and vascular leakage that result in edema and the recruitment of eosinophils, basophils, monocytes, and lymphocytes. The importance of this cellular influx is shown by the ability of glucocorticoid medications to block the late-phase response through their inhibition of cell recruitment, whereas glucocorticoids do not block the immediate response. A late-phase reaction can also occur after aerosol exposure to allergen, and is characterized by a second phase of airway narrowing with sustained edema and cellular infiltration into the peribronchial spaces (see Fig. 14.11, left panel).

1	In patients with a clinical history of allergic disease, allergists use the immediate response to help assess and confirm sensitization, and to determine which allergens are responsible. Minute amounts of potential allergens are introduced into the skin by a skin prick—one site for each allergen—and if the individual is sensitive to any of the allergens tested, a wheal-and-flare reaction will occur at the site within a few minutes (see Fig. 14.11, right panel). Although the reaction after the administration of such small amounts of allergen is usually very localized, there is a small risk of inducing anaphylaxis. Another standard test for allergy is to measure the circulating concentration of IgE antibody specific for a particular allergen in a sandwich ELISA (see Appendix I, Section A-4).

1	The late-phase reaction described above occurs under controlled experimental conditions to a single, relatively high dose of allergen and so does not reflect all the effects of long-term natural exposure. In IgE-mediated allergic diseases, a long-term consequence of allergen exposure can be chronic allergic inflammation, which consists of a persistent type 2 immune response with a dominant cellular quality that is driven by TH2 lymphocytes, basophils, eosinophils, and macrophages. These chronic reactions contribute importantly to serious long-term illnesses, such as chronic asthma. In long-standing asthma, for example, the cytokines released by TH2 cells and vasoactive mediators such as calcitonin gene-related peptide and vascular endothelial growth factor result in persistent edema, which results in persistent narrowing of the airways. They can also lead to airway tissue remodeling, which changes the bronchial tissue via smooth muscle hypertrophy (an increase in the size of the

1	which results in persistent narrowing of the airways. They can also lead to airway tissue remodeling, which changes the bronchial tissue via smooth muscle hypertrophy (an increase in the size of the muscle cells) and hyperplasia (an increase in the number of cells), subepithelial deposition of collagen, and often goblet cell hyperplasia. Although TH2 cytokines appear to predominate in this chronic phase of asthma, TH1 cytokines (such as IFN-γ) and TH17 cytokines (IL-17, IL-21, and IL-22) can also participate.

1	In the natural situation, the clinical symptoms produced by an IgE-mediated allergic reaction depend critically on several variables: the amount of allergen-specific IgE present, the route by which the allergen is introduced, the dose of allergen, and most probably some underlying defect in barrier function in the particular tissue or organ affected. The outcomes produced by different combinations of allergen dose and route of entry are summarized in Fig. 14.12. When exposure to allergen in a sensitized individual triggers an allergic reaction, both the immediate and the chronic effects are focused on the site at which mast-cell degranulation occurs and they involve the recruitment of many soluble and cellular components of effector pathways. 14-10 Allergen introduced into the bloodstream can cause anaphylaxis.

1	If allergen is introduced directly into the bloodstream, for example, by a bee or wasp sting, or is rapidly absorbed into the bloodstream from the gut in a sensitized individual, connective-tissue mast cells associated with blood vessels throughout the body can become immediately activated, resulting in a widespread release of histamine and other mediators that causes the systemic reaction called anaphylaxis. The symptoms of anaphylaxis can range in severity from mild urticaria (hives) to fatal anaphylactic shock (see Fig. 14.12, first and last panels). Acute urticaria is a response to foreign allergens that are delivered to the skin via the systemic blood circulation. Activation of mast cells in the skin by allergen causes them to release histamine, which in turn causes itchy, red swellings all over the body—a disseminated version of the wheal-and-flare reaction. Although acute urticaria is commonly caused by an IgE-mediated reaction against an allergen, the causes of chronic

1	swellings all over the body—a disseminated version of the wheal-and-flare reaction. Although acute urticaria is commonly caused by an IgE-mediated reaction against an allergen, the causes of chronic urticaria, in which the urticarial rash persists or recurs over long periods, remain incompletely defined. Some cases of chronic urticaria are caused by autoantibodies

1	Connective tissue mast cells (MCCT) Mucosal mast cells (MCC) Route of allergen entry Intravenous Subcutaneous Inhalation Ingestion Widespread release of histamine, Local release of histamine which acts on blood vessels to causes wheal-and-fare reaction. increase permeability, leading to Airborne or topical allergens hives (low dose, ˜g or less) or penetrating skin can also be a anaphylactic shock (medium to high cause of atopic eczema dose, milligrams to grams)

1	Fig. 14.12The route of administration of allergen determines the type of IgE-mediated allergic reaction that results. There are two main anatomical distributions of mast cells: those associated with vascularized connective tissues and called connective tissue mast cells (MCCT), and those found in submucosal layers of the gut and respiratory tract and called mucosal mast cells (MCC). In an allergic individual, all of these mast cells are coated through their cell-surface Fcε receptors with IgE directed against specific allergens. The response to an allergen then depends on which mast cells are activated. Allergen in the bloodstream (intravenous) activates connective tissue mast cells throughout the body, resulting in the systemic release of histamine and other mediators. Entry of allergen through the skin activates local connective tissue mast cells, leading to a local inflammatory reaction. After an experimental skin

1	Allergic rhinitis (upper airway), Contraction of intestinal smooth muscle caused by increased mucus induces vomiting. Outfow of fuid into production and nasal irritation. gut causes diarrhea. Antigen diffuses

1	Asthma (lower airway) due to into blood vessels and is widely contraction of bronchial smooth muscle disseminated, causing urticaria (hives), and increased mucus secretion anaphylaxis, or atopic eczema prick with allergen or bite from an insect to which the individual is sensitized, this manifests as a wheal-and-flare reaction. In atopic individuals, airborne or topically applied allergens that penetrate the skin may lead to atopic eczema. Inhaled allergen that penetrates respiratory mucosal epithelia activates mainly mucosal mast cells, causing increased secretion of mucus by the mucosal epithelium and irritation in the nasal mucosa, leading to allergic rhinitis—or to asthma if constriction of smooth muscle in the lower airways occurs. Ingested allergen penetrates the gut epithelium, causing vomiting due to intestinal smooth muscle contraction and diarrhea due to outflow of fluid across the gut epithelium. Food allergens can also be disseminated in the bloodstream, causing widespread

1	vomiting due to intestinal smooth muscle contraction and diarrhea due to outflow of fluid across the gut epithelium. Food allergens can also be disseminated in the bloodstream, causing widespread urticaria (hives) when the allergen reaches the skin, or they may cause eczema.

1	against either the α chain of FcεRI or against IgE itself, and thus can be considered a form of autoimmunity. Interaction of the autoantibody with the receptor triggers mast-cell degranulation, with resulting urticaria. In some patients, treatment with omalizumab (a therapeutic monoclonal anti-IgE antibody) leads to resolution of hives, demonstrating a role for IgE in these individuals, even though the antigen that elicited the IgE often cannot be identified. In anaphylactic shock, a widespread increase in vascular permeability and smooth muscle contraction results from a massive release of histamine and other mast celland basophil-derived mediators such as leukotrienes.

1	The consequences are a catastrophic reduction of blood pressure, culminating in hypotensive shock, (a condition in which low blood pressure leads to inadequate supply of blood to vital organs, often leading to death), and constriction of the airways, culminating in respiratory failure. The most common causes of anaphylaxis are allergic reactions to wasp and bee stings, ingested or injected medications, or allergic responses to foods in sensitized individuals. For example, anaphylaxis in individuals allergic to peanuts is relatively common. Severe anaphylactic shock can be rapidly fatal if untreated, but can usually be controlled by the immediate injection of epinephrine, which via stimulation of β-adrenergic receptors causes relaxation of airway smooth muscles, and via stimulation of α-adrenergic receptors reverses the life-threatening cardiovascular effects.

1	Systemic allergic reactions can occur following repeated treatment with many classes of drugs. A relatively common inducer of IgE-mediated allergic reaction is penicillin and other drugs that share aspects of its structure and immunological reactivity. In people who have developed IgE antibodies against penicillin, injection of the drug can cause anaphylaxis and even death. While administration of oral penicillin to an allergic individual can also cause anaphylaxis, the symptoms after oral ingestion are usually less severe and very rarely result in death. One of the reasons that penicillin is particularly prone to inducing allergic reactions is that it acts as a hapten (see Appendix I, Section A-1); it is a small molecule with a highly reactive β-lactam ring that is crucial for its antibacterial activity. This ring reacts with amino groups on host proteins to form covalent conjugates. When penicillin is ingested or injected, it forms conjugates with self proteins, and the

1	its antibacterial activity. This ring reacts with amino groups on host proteins to form covalent conjugates. When penicillin is ingested or injected, it forms conjugates with self proteins, and the penicillin-modified self peptides are recognized as foreign and elicit a host immune response. A large proportion of individuals who are treated with intravenous penicillin develop IgG antibodies against the drug, but these usually cause no symptoms. In some individuals, self proteins conjugated with penicillin provoke a TH2 response that activates penicillin-binding B cells to produce IgE antibody against the penicillin hapten. Thus, penicillin acts both as the B-cell antigen and, by modifying self peptides, as the T-cell antigen. When penicillin is injected intravenously into an allergic individual, the penicillin-modified proteins can cross-link IgE molecules on tissue mast cells and circulating basophils and thus cause anaphylaxis. Great care should be taken to avoid giving a drug to

1	the penicillin-modified proteins can cross-link IgE molecules on tissue mast cells and circulating basophils and thus cause anaphylaxis. Great care should be taken to avoid giving a drug to patients with a past history of allergy to that drug or a close structural relative.

1	As is true for individuals with sensitivity to inhalant allergens, patients with a past history of anaphylactic-type reactions to penicillin or other β-lactam antibiotics can be evaluated by skin-prick testing. A positive skin test, manifested by the formation of a wheal-and-flare reaction at the site of the test, is associated with a substantial risk of developing an anaphylactic reaction when treated with therapeutic doses of the drug. 14-11 Allergen inhalation is associated with the development of rhinitis and asthma.

1	The respiratory tract is an important route of allergen entry (see Fig. 14.12, third panels). Many atopic people react to airborne allergens with an IgEmediated allergic reaction known as allergic rhinitis. This results from the activation of mucosal mast cells beneath the nasal epithelium by allergens such as pollens that, when they contact the epithelium, release their soluble protein contents, which then diffuse across the mucous membranes of the nasal passages. Allergic rhinitis is characterized by intense itching and sneezing; local edema leading to blocked nasal passages; a nasal discharge, which is typically rich in eosinophils; and irritation of the nasal mucosa as a result of histamine release. A similar reaction to airborne allergens deposited on the conjunctiva of the eye is called allergic conjunctivitis. Allergic rhinitis and conjunctivitis are commonly caused by environmental allergens that are present only during certain seasons of the year. For example, hay fever

1	is called allergic conjunctivitis. Allergic rhinitis and conjunctivitis are commonly caused by environmental allergens that are present only during certain seasons of the year. For example, hay fever (known

1	Fig. 14.13 Histologic evidence of chronic inflammation in the airways of an asthmatic patient. Panel a shows a section through a bronchus of a patient who died of asthma; there is almost total occlusion of the airway by a mucus plug. In panel b, a close-up view of the bronchial wall shows injury to the epithelium lining the bronchus, accompanied by a dense inflammatory infiltrate. Although not discernable at this magnification, the infiltrate includes eosinophils, neutrophils, and lymphocytes. Photographs courtesy of T. Krausz.

1	clinically as seasonal allergic rhinoconjunctivitis) is caused by a variety of allergens, including certain grass and tree pollens. Symptoms in the late summer or autumn are commonly caused by weed pollen, such as that of ragweed, or the spores of fungi such as Alternaria. Ubiquitous allergens such as Fel d 1 in cat dander, Der p 1 in the feces of house dust mites, and Bla g 1 in cockroach can be a cause of year-round, or perennial, allergic rhinoconjunctivitis.

1	A more serious IgE-mediated respiratory disease is allergic asthma, which is triggered by allergen-induced activation of submucosal mast cells in the lower airways. This can lead within seconds to bronchial constriction and an increased secretion into the airways of fluid and mucus, making breathing more difficult by trapping inhaled air in the lungs. Patients with allergic asthma usually need treatment, and severe asthmatic attacks can be life threatening. The same allergens that cause allergic rhinitis and conjunctivitis commonly cause asthma attacks. For example, respiratory arrest caused by severe attacks of asthma in the summer or autumn has been associated with the inhalation of Alternaria spores.

1	Chronic allergen exposure leads to an important feature of asthma, namely, chronic inflammation of the airways, which is characterized by the continued presence of increased numbers of pathologic lymphocytes, eosinophils, neutrophils, basophils, and other leukocytes (Fig. 14.13). The concerted actions of these cells cause airway hyperreactivity and remodeling—a thickening of the airway walls due to hyperplasia and hypertrophy of the smooth muscle layer, with the eventual development of fibrosis. Fibrotic remodeling leads to a permanent narrowing of the airways, and is responsible for many of the clinical manifestations of chronic allergic asthma. In chronic asthmatics, a general hyperreactivity of the airways to nonimmunological stimuli such as perfumes or volatile irritants also often develops.

1	It has become apparent that there are many different phenotypic subtypes of asthma. These subtypes are being recognized because patients differ widely in responsiveness to different therapies, in the nature of the inflammatory cell infiltrates that are present in their airways, and in the molecular signature of inflammatory mediators that can be recovered from the airways. Many investigators refer to these subtypes as asthma ‘endotypes.’ The expectation is that classification of patients’ asthmas by endotype will elucidate differences in the underlying pathophysiology of their disease and will improve therapeutic outcomes by permitting their therapy to be matched to the underlying molecular disorder that is leading to symptoms. Some of the most common endotypes include common allergic asthma, exercise-induced asthma, neutrophil-predominant (as opposed to eosinophil-predominant) asthma, and steroid-resistant severe asthma. The fundamental driver of the allergic response in allergic

1	asthma, exercise-induced asthma, neutrophil-predominant (as opposed to eosinophil-predominant) asthma, and steroid-resistant severe asthma. The fundamental driver of the allergic response in allergic asthma is thought to be pathologically activated TH2 cells, and eosinophils and basophils are prominent in the inflammatory infiltrates in the lungs. In severe, steroid-resistant asthma, TH17 cells appear to play a larger role, and neutrophils are prominent in the inflammatory infiltrates. TH17 cells also appear to be major inducers of the asthmatic syndrome allergic bronchopulmonary aspergillosis (ABPA). Other endotypes are characterized by the participation of additional leukocyte subsets and different effector-cell populations. The endotype of asthma of any individual patient is thought to be the result of the specific conditions under which the individual was sensitized to allergen and the specific predisposition of the individual based on inherited genetic factors and environmentally

1	be the result of the specific conditions under which the individual was sensitized to allergen and the specific predisposition of the individual based on inherited genetic factors and environmentally determined epigenetic factors.

1	For the following discussion of mechanisms of asthma, we will focus on the most common endotype, common allergic asthma. In patients with allergic asthma, allergen challenge can cause activation of mast cells in an antigen-specific, IgE-dependent fashion, leading to mast-cell mediator release. Allergens can also stimulate the airway epithelium directly, through TLRs and other damage receptors, to release IL-25 and IL-33. These cytokines can lead to the activation of submucosal type 2 innate lymphoid cells (ILC2s), inducing them to release IL-4, IL-5, IL-9, and IL-13. At the same time, bronchial epithelial cells can produce at least two of the chemokine ligands—CCL5 and CCL11—that bind to the receptor CCR3 expressed on TH2 cells, macrophages, eosinophils, and basophils. Thus, these chemokines, together with the products of activated ILC2s, enhance the type 2 response by attracting more TH2 cells and eosinophils to the damaged lungs. The direct effects of ILC2and TH2 cell-derived

1	together with the products of activated ILC2s, enhance the type 2 response by attracting more TH2 cells and eosinophils to the damaged lungs. The direct effects of ILC2and TH2 cell-derived cytokines and chemokines on airway smooth muscle cells and fibroblasts lead to the apoptosis of epithelial cells and airway remodeling. The remodeling is induced in part by the production of TGF-β, which has numerous effects on the epithelium, ranging from inducing apoptosis to stimulating cell proliferation. The direct action of additional TH2-type cytokines such as IL-9 and IL-13 on airway epithelial cells may also have a dominant role in another major feature of chronic allergic asthma, the induction of goblet-cell metaplasia, which is the increased differentiation of epithelial cells into goblet cells, and a consequent increase in mucus secretion. CD1d-restricted invariant NKT cells (iNKTs, a type of innate-like lymphocyte; see Sections 3-27, 6-18, and 8-26) also seem to have an important role

1	and a consequent increase in mucus secretion. CD1d-restricted invariant NKT cells (iNKTs, a type of innate-like lymphocyte; see Sections 3-27, 6-18, and 8-26) also seem to have an important role in the development of airway hyperreactivity, whether allergen-induced or nonspecific, and this function can be enhanced by the cooperation of ILC2s. Animal models of asthma have shown that airway hyperreactivity is exacerbated by the presence of iNKT cells. Additionally, in mouse models, superoxide-producing myeloid lineage regulatory cells also appear to play pathological roles in the establishment of airway hyperreactivity.

1	Mice do not naturally develop asthma, but a disease resembling human asthma develops in mice that lack the transcription factor T-bet. This transcription factor is required for TH1 differentiation (see Section 9-21). When T-bet is absent, T-cell responses are skewed toward the TH2 phenotype. T-bet-deficient mice have increased levels of the TH2 cytokines IL-4, IL-5, and IL-13, and develop airway inflammation involving lymphocytes and eosinophils (Fig. 14.14). T-bet+/+ T-bet–/– Normal lung biopsy Airway in˜ammation with lymphocytes and eosinophils Normal airway Airway remodeling with increased collagen deposited around airway Fig. 14.14 Mice lacking the transcription factor T-bet develop allergic airway inflammation and T-cell responses polarized toward TH2.

1	Fig. 14.14 Mice lacking the transcription factor T-bet develop allergic airway inflammation and T-cell responses polarized toward TH2. T-bet binds to the promoter of the gene encoding IL-2 and is present in TH1 but not TH2 cells. Mice with a gene-targeted deletion of T-bet (T-bet–/–) manifest impaired TH1 responses, and show spontaneous differentiation of TH2 cells and development of an asthma-like phenotype in the lungs. Left-hand panels: lung and airways in normal mice. Right-hand panels: T-betdeficient mice show lung inflammation, with lymphocytes and eosinophils around the airway and blood vessels (top) and airway remodeling with increased collagen around the airway (bottom). Photographs courtesy of L. Glimcher.

1	They also develop nonspecific airway hyperreactivity to nonimmunological stimuli, similar to what is seen in human asthma. These changes occur in the absence of any exogenous inflammatory stimulus and show that, in extreme circumstances, a genetic imbalance toward TH2 responses can cause allergic disease. The availability of a large number of genetically deficient mouse strains has permitted testing of the roles of many inflammatory effector cells and cytokines in this experimental model, providing hypotheses that are now being tested in human asthma.

1	Although allergic asthma is initially driven by a response to a specific allergen, the subsequent chronic inflammation seems to be perpetuated even in the apparent absence of ongoing exposure to allergen. The airways become characteristically hyperreactive, and factors other than antigen can trigger asthma attacks. Asthmatics characteristically show hyperresponsiveness to environmental chemical irritants such as cigarette smoke and sulfur dioxide. Viral agents, especially rhinovirus, or, to a smaller extent, bacterial respiratory tract infections can also exacerbate the disease. Both irritant agents and the infectious agents can induce IL-25 and IL-33 release from airway epithelial cells, leading to activation of ILC2s and exacerbation of the chronic asthmatic inflammation. The significance of viral augmentation of the asthmatic response is evident from the fact that rhinovirus infection is one of the main causes of hospitalizations for asthma and is associated with the majority of

1	of viral augmentation of the asthmatic response is evident from the fact that rhinovirus infection is one of the main causes of hospitalizations for asthma and is associated with the majority of deaths from asthma.

1	14-12 Allergy to particular foods causes systemic reactions as well as symptoms limited to the gut. Adverse reactions to particular foods are common, but only some are due to an immune reaction. ‘Food allergy’ can be classified into IgE-mediated allergic reactions, non-IgE-mediated food allergy (celiac disease, discussed in Section 14-17), idiosyncrasies, and food intolerance. Idiosyncrasies are abnormal responses to particular foods whose cause is unknown but which can provoke symptoms resembling those of an allergic response. Food intolerances are nonimmune adverse reactions due mainly to metabolic deficits, such as intolerance of cow’s milk due to the inability to digest lactose.

1	IgE-mediated food allergies affect about 1–4% of American and European adults and are slightly more frequent in children (around 5%). About 25% of food allergy in children is due to peanuts, and peanut allergy is increasing in incidence. Figure 14.15 lists risk factors for developing IgE-mediated food allergy. IgE-mediated food allergy can manifest itself in a variety of ways, ranging from a swelling of the lips and oral tissue on contact with the allergen, to gastrointestinal cramping, diarrhea, or vomiting. Local gastrointestinal symptoms are due to activation of mucosal mast cells, leading to transepithelial fluid loss and smooth muscle contractions. Food allergens that subsequently reach the bloodstream can lead to urticaria, asthma, and, in the most severe cases, systemic anaphylaxis that can lead to cardiovascular collapse (see Section 14-10). Certain foods, most importantly peanuts, tree nuts, and shellfish, are particularly associated with severe anaphylaxis. Around 150 deaths

1	that can lead to cardiovascular collapse (see Section 14-10). Certain foods, most importantly peanuts, tree nuts, and shellfish, are particularly associated with severe anaphylaxis. Around 150 deaths occur each year in the United States as a result of a severe allergic reaction to food, with peanut and tree nut allergies accounting for most of the deaths. Peanut allergy is a significant public health problem, especially in schools, where children may be unwittingly exposed to peanuts, which are present in many foods. Recent studies offer hope for reducing the incidence of severe food allergy. In one study, infants with severe eczema who were at high risk for developing peanut allergy were randomly assigned either to be fed peanuts regularly, starting between ages 4 and 11 months, or to be on a peanut-avoidance diet for 5 years. At the age of 5, the children who had consumed peanuts showed more than a threefold reduction in the frequency of peanut allergy; the reduction was associated

1	on a peanut-avoidance diet for 5 years. At the age of 5, the children who had consumed peanuts showed more than a threefold reduction in the frequency of peanut allergy; the reduction was associated with decreased production of peanut-specific IgE. This suggests that deliberate introduction into the diet of allergen at the appropriate time to at-risk individuals may suppress the development of food allergy.

1	Of interest is that one of the characteristic features of food allergens is a high degree of resistance to digestion by pepsin in the stomach. This allows them to reach the mucosal surface of the small intestine as intact allergens. Cases of IgE-mediated food allergies arising in small numbers of previously unaffected adults who were taking antacids or proton-pump inhibitors for ulcers or acid reflux have been proposed to be due to impaired digestion of potential allergens in the less acidic stomach conditions produced by these medications. 14-13 IgE-mediated allergic disease can be treated by inhibiting the effector pathways that lead to symptoms or by desensitization techniques that aim at restoring biological tolerance to the allergen.

1	Most of the current drugs that are used to treat allergic disease either treat only the symptoms—examples of such drugs are antihistamines and β-agonists—or are general anti-inflammatory or immunosuppressive drugs such as the corticosteroids (Fig. 14.16). Treatment is largely palliative, rather than curative, and the drugs often need to be taken throughout life. Anaphylactic reactions are treated with epinephrine, which stimulates the re-formation of endothelial Fig. 14.16 Approaches to the treatment of allergic disease. Examples of treatments in current clinical use for allergic reactions are listed in the top half of the table, with approaches under investigation listed below.

1	tight junctions, promotes the relaxation of constricted bronchial smooth muscle, and stimulates the heart. Antihistamines that target the H1 receptor reduce the symptoms that follow the release of histamine from mast cells in allergic rhinoconjunctivitis and IgE-triggered urticaria. In urticaria, for example, the relevant H1 receptors include those on blood vessels and unmyelinated nerve fibers in the skin. Anticholinergic drugs bronchodilate constricted airways and reduce respiratory secretions. Antileukotriene drugs act as antagonists of leukotriene receptors on smooth muscle, endothelial cells, and mucous-gland cells, and are also used to relieve the symptoms of allergic rhinoconjunctivitis and asthma. Inhaled bronchodilators that act on β-adrenergic receptors to relax constricted muscle relieve acute asthma attacks. In chronic allergic disease it is extremely important to treat and prevent the chronic inflammatory injury to tissues, and regular use of inhaled corticosteroids is

1	relieve acute asthma attacks. In chronic allergic disease it is extremely important to treat and prevent the chronic inflammatory injury to tissues, and regular use of inhaled corticosteroids is now recommended in persistent asthma to help suppress inflammation. Topical corticosteroids are used to suppress the chronic inflammatory changes seen in eczema.

1	A new type of allergy suppressive therapy that is beginning to gain significant use is blockade of IgE function by treatment with monoclonal anti-IgE antibodies, omalizumab being an example. This antibody binds the Fc portion of IgE at the same site that binds the FcεRI on basophils and mast cells. The portion of the Fc domain of IgE that binds to the low-affinity IgE receptor (FcεRII) that is expressed on a variety of leukocytes other than basophils and mast cells is different from the domain that binds the high-affinity FcεRI; omalizumab by steric hindrance blocks binding of IgE to the low-affinity receptor as well as to FcεRI. Prevention of binding of IgE to its receptors on basophils results in downregulation of these receptors on these cells, making them less easily activated by exposure to allergens. Omalizumab also appears to act in chronic allergic asthma to reduce IgE-mediated antigen trapping and presentation by dendritic cells, thus preventing the activation of new

1	by exposure to allergens. Omalizumab also appears to act in chronic allergic asthma to reduce IgE-mediated antigen trapping and presentation by dendritic cells, thus preventing the activation of new allergen-specific TH2 cells. Altogether, these actions lead to suppression of the late-phase response to allergen challenge (see Section 14-9). The antibody is administered by subcutaneous injections once every 2 to 4 weeks. This treatment has been shown to be highly effective for patients with chronic urticaria and also appears to be effective in individuals with severe chronic asthma. Of special interest is that in studies of children with moderate to severe asthma who were treated for 4 years with omalizumab, most remained symptom free without any anti-asthma treatment, suggesting that the anti-IgE therapy modified the natural history of the disease.

1	Another, more routinely used approach that aims to permanently eliminate the allergic response is allergen desensitization. This form of immunotherapy aims to restore the patient’s ability to tolerate exposure to the allergen. Patients are desensitized by injection with escalating doses of allergen, starting with tiny amounts. The mechanism by which desensitization occurs is not definitively established, but for most successfully desensitized patients, the procedure results in a change in the antibody response from one that is IgE predominant to one dominated by an IgG subclass. Successful desensitization appears to depend on the induction of Treg cells secreting IL-10 and/or TGF-β, which skew the response away from IgE production (see Section 14-4). For example, beekeepers exposed to repeated stings (paralleling the therapeutic desensitization process) are often naturally protected from severe allergic reactions such as anaphylaxis through a mechanism that involves IL-10-secreting T

1	stings (paralleling the therapeutic desensitization process) are often naturally protected from severe allergic reactions such as anaphylaxis through a mechanism that involves IL-10-secreting T cells. Similarly, specific allergen immunotherapy for sensitivity to insect venom and airborne allergens induces the increased production of IL-10 and in some cases TGF-β, as well as the production of IgG isotypes, particularly IgG4, an isotype selectively promoted by IL-10. Recent evidence shows that desensitization is also associated with a reduction in the numbers of inflammatory cells at the site of the allergic reaction. A potential complication of the desensitization approach is that in spite of starting with extremely small doses of allergen, some patients can experience an IgE-mediated allergic response, sometimes including bronchospasms. Thus, many physicians feel that allergen immunotherapy is contraindicated in patients with severe asthma. For patients who experience resolution of

1	response, sometimes including bronchospasms. Thus, many physicians feel that allergen immunotherapy is contraindicated in patients with severe asthma. For patients who experience resolution of their allergy symptoms during allergen immunotherapy, weekly or every other week injections are continued for 3 years, and then the therapy is discontinued. In approximately half of patients treated in this fashion, symptoms do not recur following cessation of the injections. These patients experience durable ability to tolerate the allergen without symptoms. Recent studies suggest that administration of immunotherapy via the sublingual route is equally or more effective than administration by subcutaneous injection, offering the possibility of less expensive and perhaps more effective immunotherapy in the future.

1	When a patient is allergic to a drug that is essential for treatment of a disease (such as an antibiotic, insulin, or a chemotherapeutic agent), it is often possible to achieve a state of temporary acute desensitization by treating the individual with progressively increasing doses of the drug, starting at a very low dose that causes no allergic symptoms and increasing the dose every half hour until the therapeutic dose is reached. It is common for individuals undergoing drug desensitization to manifest mild to moderate allergic symptoms (itching, urticaria, mild wheezing) at some time during the procedure. If this occurs, the physician reduces the dose to the previous tolerated dose and then advances the dose again. This procedure is thought to lead to subclinical activation of mast cells and basophils that have been sensitized with IgE against the drug, inducing them to gradually release their intracellular mediators at a rate that does not cause severe symptoms; eventually all of

1	and basophils that have been sensitized with IgE against the drug, inducing them to gradually release their intracellular mediators at a rate that does not cause severe symptoms; eventually all of the cell-bound IgE is consumed by this process, leaving insufficient IgE available to cause an allergic reaction when subsequent therapeutic doses are administered. In order to maintain the desensitized state, the patient must receive daily therapeutic doses of the drug. If treatment is interrupted, then newly formed mast cells and basophils can be charged with newly secreted drug-specific IgE and can accumulate at levels sufficient to yield a new anaphylactic reaction.

1	An alternative, and still experimental, immunotherapy approach is a vaccination strategy using allergen coupled to oligodeoxynucleotides rich in unmethylated CpG. The oligonucleotide mimics the CpG motifs in bacterial DNA and strongly promotes TH1 responses while suppressing TH2 responses. This appears to be useful for chronic treatment of an antigen-specific allergic response, but is not effective for acute desensitization.

1	A further approach to the treatment of allergic disease may be to block the recruitment of eosinophils to sites of allergic inflammation. The eotaxin receptor CCR3 is a potential target in this context. In experimental animals, the production of eosinophils in bone marrow and their exit into the circulation is reduced by blocking IL-5 action. Anti-IL-5 antibody (mepolizumab) is of benefit in treating human patients with the hypereosinophilic syndrome, in which chronic overproduction of eosinophils causes severe organ damage. Clinical trials of anti-IL-5 treatment of asthma, however, show that, in practice, any beneficial effect is likely to be limited to a small subset of asthma patients with prednisone-dependent eosinophilic asthma; in these patients, IL-5 blockade seems to reduce the number of asthma attacks when the corticosteroid dose is reduced. Summary.

1	The allergic response to innocuous antigens reflects the pathophysiological aspects of a defensive immune response whose physiological role is to protect against helminth parasites. It is triggered by the binding of antigen to IgE antibodies bound to the high-affinity IgE receptor FcεRI on mast cells and basophils. Mast cells are strategically distributed beneath the mucosal surfaces of the body and in connective tissue. Antigen cross-linking the IgE on the surface of mast cells causes them to release large amounts of inflammatory mediators. The resulting inflammation can be divided into early events that are characterized by short-lived mediators such as histamine, and later events that involve leukotrienes, cytokines, and chemokines, which recruit and activate eosinophils, basophils, and other leukocytes. This response can evolve into chronic inflammation, which is characterized by the presence of effector T cells and eosinophils, and is most clearly seen in chronic allergic asthma.

1	Non-IgE-mediated allergic diseases. In this part of the chapter we focus on immunological hypersensitivity responses involving IgG antibodies and type 1 or type 3 immune responses that involve antigen-specific TH1 or TH17 cells or CD8 T cells. These effector arms of the immune response occasionally react with noninfectious antigens to produce acute or chronic allergic reactions. Although the mechanisms initiating the various forms of hypersensitivity are different, much of the pathology is due to the same immunological effector mechanisms. in susceptible individuals occur by binding of the drug to the surface of circulating blood cells.

1	in susceptible individuals occur by binding of the drug to the surface of circulating blood cells. Antibody-mediated destruction of red blood cells (hemolytic anemia) or platelets (thrombocytopenia) can be caused by some drugs, including the β-lactam antibiotics penicillin and cephalosporin. In these reactions, the drug binds covalently to the cell surface and is a target for anti-drug IgG antibodies that cause destruction of the cell. The anti-drug antibodies are made in only a minority of people, and it is not clear why these individuals make them. The cell-bound antibody triggers the clearance of the cell from the circulation, predominantly by tissue macrophages in the spleen, which bear Fcγ receptors. can follow the administration of large quantities of poorly catabolized antigens.

1	can follow the administration of large quantities of poorly catabolized antigens. Hypersensitivity reactions can arise following treatment with soluble antigens such as animal antisera. The pathology is caused by the deposition of antigen:antibody aggregates, or immune complexes, in particular tissues and sites. Immune complexes are generated in all antibody responses, but their pathogenic potential is determined, in part, by their size and by the amount, affinity, and isotype of the responding antibody. Larger aggregates fix complement and are readily cleared from the circulation by the mononuclear phagocyte system. However, the small complexes that form when antigen is in excess tend to be deposited in blood vessel walls. There they can ligate Fc receptors on leukocytes, leading to leukocyte activation and tissue injury.

1	A local hypersensitivity reaction called an Arthus reaction (Fig. 14.17) can be triggered in the skin of sensitized individuals who possess IgG antibodies against the sensitizing antigen. When antigen is injected into the skin, circulating IgG antibody that has diffused into the skin forms immune complexes locally. The immune complexes bind Fc receptors such as FcγRIII on mast cells and other leukocytes, generating a local inflammatory response and increased vascular permeability. Fluid and cells, especially polymorphonuclear leukocytes, then enter the site of inflammation from local blood vessels. The immune complexes also activate complement, leading to the production

1	C5a Local in˜ammation, increased ˜uid and protein release, phagocytosis, and blood vessel occlusion 1–2 hours Locally injected antigen in immune individual with IgG antibody Activation of Fc˜RIII on mast cells induces their degranulation Local immune-complex formation activates complement. C5a binds to and sensitizes the mast cell to respond to immune complexes

1	Fig. 14.17 The deposition of immune complexes in tissues activation of complement, and the complement component C5a causes a local inflammatory response known as an Arthus contributes to sensitizing the mast cell to respond to immune reaction. In individuals who have already made IgG antibody against complexes. As a result of mast-cell activation, inflammatory cells an antigen, the same antigen injected into the skin forms immune invade the site, and blood vessel permeability and blood flow are complexes with IgG antibody that has diffused out of the capillaries. increased. Platelets also accumulate inside the vessel at the site, Because the dose of antigen is low, the immune complexes are ultimately leading to vessel occlusion. If the reaction is severe, all formed only close to the site of injection, where they activate mast these changes can lead to tissue necrosis. cells bearing Fcγ receptors (FcγRIII). The immune complex induces of the complement fragment C5a. This is a key

1	site of injection, where they activate mast these changes can lead to tissue necrosis. cells bearing Fcγ receptors (FcγRIII). The immune complex induces of the complement fragment C5a. This is a key participant in the inflammatory reaction because it interacts with C5a receptors on leukocytes to activate these cells and attract them to the site of inflammation (see Section 2-5). Both C5a and FcγRIII have been shown to be required for the experimental induction of an Arthus reaction in the lung by macrophages in the walls of the alveoli, and they are probably required for the same reaction induced by mast cells in the skin and the synovial linings of joints. Recruitment and activation of C5a receptor-bearing leukocytes leads to tissue injury, sometimes resulting in frank necrosis.

1	A systemic reaction known as serum sickness can result from the injection of large quantities of a poorly catabolized foreign antigen. This illness was so named because it frequently followed the administration of therapeutic horse antiserum. In the pre-antibiotic era, antiserum made by immunizing horses with Streptococcus pneumoniae was often used to treat pneumococcal pneumonia; the specific anti-pneumococcal antibodies in the horse serum would help the patient to clear the infection. In much the same way, antivenin (serum from horses immunized with snake venoms) is still used today as a source of neutralizing antibodies to treat people suffering from the bites of poisonous snakes. Serum sickness occurs 7–10 days after the injection of horse serum, an inter Drug-induced Serum val that corresponds to the time required to mount an IgG-switched primary

1	Serum sickness occurs 7–10 days after the injection of horse serum, an inter Drug-induced Serum val that corresponds to the time required to mount an IgG-switched primary Sickness immune response against the foreign horse serum antigens. The clinical features of serum sickness are chills, fever, rash, arthritis, and sometimes glomerulonephritis (inflammation of the glomeruli of the kidneys). Urticaria is a prominent feature of the rash, implying a role for histamine derived from mast-cell degranulation. In this case, the mast-cell degranulation is triggered by the ligation of cell-surface FcγRIII by IgG-containing immune complexes and by the anaphylatoxins C3a and C5a released due to complement activation by these complexes. fever, vasculitis, arthritis, nephritisforeign serum injectionLevelinplasmaTime (days) foreign serum proteins antigen:antibody complexes antibody against foreign serum proteins

1	fever, vasculitis, arthritis, nephritisforeign serum injectionLevelinplasmaTime (days) foreign serum proteins antigen:antibody complexes antibody against foreign serum proteins Fig. 14.18 Serum sickness is a classic example of a transient immune complex-mediated syndrome. An injection of a foreign protein, such as horse antitoxin, leads to an anti-horse serum antibody response. These antibodies form immune complexes with the circulating foreign proteins. The complexes are deposited in small blood vessels and activate complement and phagocytes, inducing fever and inflammatory lesions in blood vessels in the skin and connective tissues (vasculitis), in the kidney (nephritis), and in joints (arthritis). All these effects are transient and resolve when the foreign protein is cleared.

1	The course of serum sickness is illustrated in Fig. 14.18. The onset of disease coincides with the development of antibodies against the abundant soluble proteins in the foreign serum; these antibodies form immune complexes with their antigens throughout the body. The immune complexes fix complement and can bind to and activate leukocytes bearing Fc and complement receptors; these leukocytes in turn cause widespread tissue damage. The formation of immune complexes causes clearance of the foreign antigen, so serum sickness is usually a self-limiting disease. Serum sickness after a second dose of antigen follows the kinetics of a secondary antibody response (see Section 10-14), with symptoms typically appearing within a day or two.

1	With the increasing clinical use of humanized monoclonal antibodies (such as anti-TNF-α used for the treatment of rheumatoid arthritis), cases of serum sickness are observed, fortunately rarely, in settings where the attempt to humanize the monoclonal antibody was not successful for selected patients because they produce uncommon Ig allotypes. In these individuals, symptoms are generally mild, and one of the most significant features of the anti-monoclonal antibody response is more rapid clearance of the antibody from the circulation, leading to reduction of its therapeutic effects.

1	Pathological immune-complex deposition is seen in other situations in which antigen persists. One is when an adaptive antibody response fails to clear the infecting pathogen, as occurs in subacute bacterial endocarditis or chronic viral hepatitis. In these situations, the replicating pathogen is continuously generating new antigen in the presence of a persistent antibody response, with the consequent formation of abundant immune complexes. These are deposited within small blood vessels and result in injury in many tissues and organs, including the skin, kidneys, and nerves.

1	Immune-complex disease also occurs when inhaled allergens provoke IgG rather than IgE antibody responses, perhaps because they are present at relatively high levels in the air. When a person is reexposed to high doses of such allergens, immune complexes form in the walls of alveoli in the lungs. This leads to the accumulation of fluid, protein, and cells in the alveolar wall, slowing blood:air exchange of O2 and CO2, compromising lung function. This type of reaction is more likely to occur in occupations such as farming, in which there is repeated exposure to hay dust or mold spores, and the resulting disease is known as farmer’s lung. If exposure to antigen is sustained, the lining of the lungs can be permanently damaged. 14-16 Hypersensitivity reactions can be mediated by TH1 cells and CD8 cytotoxic T cells.

1	Unlike the immediate hypersensitivity reactions, which are mediated by antibodies, cellular hypersensitivity reactions such as delayed-type hypersensitivity reactions are mediated by antigen-specific effector T cells. We have already seen the involvement of TH2 effector cells and the cytokines they produce in the chronic response of IgE-initiated allergic reactions. Here we consider the hypersensitivity diseases caused by TH1 and CD8 cytotoxic T cells (Fig. 14.19). These cells function in hypersensitivity in essentially the same way as when they respond to a pathogen (described in Chapter 9), and the responses can be transferred between experimental animals by purified T cells or cloned T-cell lines. Much of the chronic inflammation seen in some of the allergic diseases described earlier is due to cellular hypersensitivity reactions mediated by antigen-specific TH1 cells acting in concert with TH2 cells.

1	The prototypic delayed-type hypersensitivity reaction is the Mantoux test— the standard tuberculin test that is used to determine whether an individual has previously been infected with Mycobacterium tuberculosis. In the Mantoux test, small amounts of tuberculin—a complex extract of peptides and carbohydrates derived from M. tuberculosis—are injected intradermally. In people who have been exposed to the bacterium, either by infection or by immunization with the BCG vaccine (an attenuated form of M. tuberculosis), a local T-cell-mediated inflammatory reaction evolves over 24–72 hours. The response is caused by TH1 cells, which enter the site of antigen injection, recognize complexes of peptide:MHC class II molecules on antigen-presenting cells, and release inflammatory cytokines such as IFN-γ, TNF-α, and lymphotoxin. These stimulate the expression of adhesion molecules on endothelium and increase local blood vessel permeability, allowing plasma and accessory cells to enter the site,

1	IFN-γ, TNF-α, and lymphotoxin. These stimulate the expression of adhesion molecules on endothelium and increase local blood vessel permeability, allowing plasma and accessory cells to enter the site, thus causing a visible swelling (Fig. 14.20). Each of these phases takes several hours and so the fully developed response only appears 24–48 hours after challenge. The cytokines produced by the activated TH1 cells and their actions are shown in Fig. 14.21.

1	Very similar reactions are observed in allergic contact dermatitis (also called contact hypersensitivity), which is an immune-mediated local inflammatory reaction in the skin caused by direct skin contact with certain antigens. It is important to note that not all contact dermatitis is immune-mediated and allergic in nature; it can also be caused by direct damage to the skin by irritant or toxic chemicals. 24–72 hours A TH1 effector cell recognizes antigen and releases cytokines, which act on vascular endothelium Recruitment of phagocytes and plasma to site of antigen injection causes visible lesion Antigen is injected into subcutaneous tissue and processed by local antigen-presenting cells

1	Fig. 14.19 Cellular hypersensitivity reactions. These responses are mediated by T cells and require 3–5 days or more to develop. They can be grouped into three syndromes, according to the route by which antigen passes into the body. In delayed-type hypersensitivity the antigen is injected into the skin; in contact hypersensitivity it is absorbed into the skin; and in gluten-sensitive enteropathy it is absorbed by the gut. In contact hypersensitivity, vesicles commonly form. They represent accumulations of fluid in small blister-like lesions at the level of the basement membrane between the dermis and epidermis. Their formation at this location is probably the result of antigen penetrating the epidermis, accumulating at the basement membrane, and inducing a local inflammatory response with edema fluid. DNFB (dinitrofluorobenzene) is a sensitizing agent that can cause contact hypersensitivity. MOVIE 14.1

1	MOVIE 14.1 Fig. 14.20 The stages of a delayed-type hypersensitivity reaction. The first phase involves uptake, processing, and presentation of the antigen by local antigen-presenting cells. In the second phase, TH1 cells that have been primed by a previous exposure to the antigen migrate into the site of injection and become activated. Because these specific cells are rare, and because there is little inflammation to attract cells into the site, it can take several hours for a T cell of the correct specificity to arrive. These cells release mediators that activate local endothelial cells, which recruit an inflammatory cell infiltrate dominated by macrophages and cause the accumulation of fluid, serum proteins, and more leukocytes, thus producing a visible lesion.

1	Antigen in the local tissues is internalized and processed by antigen-presenting cells and presented on MHC class II molecules. Antigen-specific TH1 cells that recognize the antigen:MHC complexes locally at the site of antigen injection release chemokines and cytokines that recruit macrophages and other leukocytes to the site. Antigen presentation by the newly recruited macrophages then amplifies the response. T cells can also affect local blood vessels through the release of TNF-α and lymphotoxin (LT), and stimulate the production of macrophages through the release of IL-3 and GM-CSF. TH1 cells activate macrophages through the release of IFN-γ and TNF-α, and kill macrophages and other sensitive cells through the cell-surface expression of the Fas ligand. Contact Sensitivity to Poison Ivy Fig. 14.21 The delayed-type hypersensitivity response is directed by chemokines and cytokines released by antigen-stimulated TH1 cells.

1	Contact Sensitivity to Poison Ivy Fig. 14.21 The delayed-type hypersensitivity response is directed by chemokines and cytokines released by antigen-stimulated TH1 cells. TNF-˜ and LT Chemokines lL-3/GM-CSFIFN-˛ Stimulate monocyte production by bone marrow stem cells Recruit macrophages and other leukocytes to site of antigen deposition Induces expression of vascular adhesion molecules. Activates macrophages, increasing release of infammatory mediators Cause local tissue destruction. Cooperate with IFN-˜to increase expression of adhesion molecules on local blood vessels Antigen is processed by tissue macrophages and stimulates TH1 cells cytokines cytotoxins chemokines TH1

1	Allergic contact dermatitis can be caused by the activation of CD4 or CD8 T cells, depending on the pathway by which the antigen is processed. Typical antigens that cause allergic contact dermatitis are highly reactive small molecules that can easily penetrate intact skin, especially if they cause itching that leads to scratching and its consequent injury to the skin barrier. These chemicals then react with self proteins, creating haptenated proteins that can be proteolytically processed in antigen-presenting cells to haptenated peptides capable of being presented by MHC molecules and recognized by T cells as foreign antigens. As with other allergic responses, there are two phases to a cutaneous allergic response: sensitization and elicitation. During the sensitization phase, Langerhans cells in the epidermis and dendritic cells in the dermis take up and process antigen, and migrate to regional lymph nodes, where they activate T cells (see Fig. 9.13) with the consequent production of

1	in the epidermis and dendritic cells in the dermis take up and process antigen, and migrate to regional lymph nodes, where they activate T cells (see Fig. 9.13) with the consequent production of memory T cells, which localize in the dermis. In the elicitation phase, a subsequent exposure to the sensitizing chemical leads to antigen presentation to memory T cells in the dermis, with the release of T-cell cytokines such as IFN-γ and IL-17. This stimulates the keratinocytes of the epidermis to release IL-1, IL-6, TNF-α, GM-CSF, the chemokine CXCL8, and the interferon-inducible chemokines CXCL11 (IP-9), CXCL10 (IP-10), and CXCL9 (Mig, a monokine induced by IFN-γ). These cytokines and chemokines enhance the inflammatory response by inducing the migration of monocytes into the lesion and their maturation into macrophages, and by attracting more T cells (Fig. 14.22).

1	The rash produced by contact with the poison ivy plant (Fig. 14.23) is a common example of allergic contact dermatitis in the United States and is caused by a CD8 T-cell response to urushiol oil (a mixture of pentadecacatechols) in the plant. These chemicals are lipid-soluble and so can cross the cell membrane and attach to intracellular proteins. The modified proteins are recognized by the immunoproteasome, and following cleavage, they are translocated into the endoplasmic reticulum and delivered to the cell surface bound to MHC class I molecules. CD8 T cells recognizing the peptides cause damage either by killing the eliciting cell or by secreting cytokines such as IFN-γ.

1	Fig. 14.22 Elicitation of a delayed-type hypersensitivity TH1 cells (which were primed in lymph nodes as a result of prior response to a contact-sensitizing agent. A contact-sensitizing antigen exposure). The activated TH1 cells then secrete cytokines agent is a small, highly reactive molecule that can penetrate intact such as IFN-γ that stimulate keratinocytes to secrete additional skin. It binds covalently as a hapten to a variety of endogenous cytokines and chemokines, which in turn attract monocytes and proteins, altering their structures so they become antigenic. These induce their maturation into activated tissue macrophages, further modified proteins are internalized, processed by Langerhans cells contributing to inflammatory lesions like those caused by poison ivy (the major antigen-presenting cells of skin), and presented to effector (see Fig. 14.23). NO, nitric oxide.

1	The ability of CD4 T cells to mediate contact hypersensitivity responses is established by experimental exposure to the strong sensitizing chemical picryl chloride. Picryl chloride modifies extracellular self proteins by haptenation. These haptenated proteins can then be proteolytically processed by APCs, yielding haptenated peptides that bind to self-MHC class II molecules and are recognized by TH1 cells. When sensitized TH1 cells recognize these complexes, they produce extensive inflammation by activating macrophages (see Fig. 14.22). Common clinical features of allergic contact hypersensitivity responses are erythema of the affected skin; development of a dermal and epidermal infiltrate consisting of monocytes, macrophages, lymphocytes, scant neutrophils, and mast cells; formation of intraepidermal abscesses; and vesicles (blisterlike collections of edema fluid between the dermis and epidermis).

1	Some insect proteins also elicit a delayed-type hypersensitivity response. One example of this in the skin is a severe reaction to mosquito bites. Instead of a small itchy bump, people allergic to proteins in mosquito saliva can develop an immediate hypersensitivity reaction such as urticaria and swelling or, much more rarely, anaphylactic shock (see Section 14-10). Some allergic individuals subsequently develop a delayed reaction (consisting of a late-phase response) that can include profound swelling that can involve an entire limb.

1	Contact hypersensitivity responses to divalent cations such as nickel have also been observed. These divalent cations can alter the conformation or the peptide binding of MHC class II molecules, and thus provoke a T-cell response. In humans, nickel can also bind to the receptor TLR-4 and produce a pro-inflammatory signal. Sensitization to nickel is widespread as a result of prolonged contact with nickel-containing items such as jewelry, buttons, and clothing fasteners, but some countries now have standards that specify that such products must have non-nickel coatings, and this is reducing the prevalence of nickel allergy in those countries.

1	Finally, although this section has focused on the role of TH1 and cytotoxic T cells in inducing cellular hypersensitivity reactions, there is evidence that antibody and complement might also have a role. Mice deficient in B cells, antibody, or complement show impaired contact hypersensitivity reactions. In particular, IgM antibodies (produced in part by B1 cells), which activate the complement cascade, facilitate the initiation of these reactions. Fig. 14.23 Blistering skin lesions on the hand of a patient with allergic contact dermatitis caused by poison ivy. Photograph courtesy of R. Geha.

1	Photograph courtesy of R. Geha. Fig. 14.24 The pathological features of celiac disease. Left: the surface of the normal small intestine is folded into fingerlike villi, which provide an extensive surface for nutrient absorption. Right: the local immune response against the food protein α-gliadin, a prominent component of wheat, oat, and barley gluten, leads to massive infiltration of the lamina propria (in the deeper, inner portion of the villi) with CD4 T cells, plasma cells, macrophages, and smaller numbers of other leukocytes, ultimately leading to destruction of the villi. In parallel, there is lengthening and increased mitotic activity in the underlying crypts, where new epithelial cells are produced. Because the villi contain all the mature epithelial cells that digest and absorb foodstuffs, their loss can result in life-threatening malabsorption and diarrhea. Photographs courtesy of Allan Mowat. 14-17 Celiac disease has features of both an allergic response and autoimmunity.

1	Celiac disease is a chronic condition of the upper small intestine caused by an immune response directed at gluten, a complex of proteins present in wheat, oats, and barley. Elimination of gluten from the diet restores normal gut function, but to date no approach for desensitization to gluten has been developed, so gluten ingestion must be avoided throughout life. The pathology of celiac disease is characterized by the loss of the slender, fingerlike villi formed by the intestinal epithelium (a condition termed villous atrophy), together with an increase in the size of the sites in which epithelial cells are renewed (crypt hyperplasia) (Fig. 14.24). These pathological changes result in the loss of the mature epithelial cells that cover the villi and normally absorb and digest food, and are accompanied by severe inflammation of the intestinal wall, with increased numbers of T cells, macrophages, and plasma cells in the lamina propria, as well as increased numbers of lymphocytes in the

1	accompanied by severe inflammation of the intestinal wall, with increased numbers of T cells, macrophages, and plasma cells in the lamina propria, as well as increased numbers of lymphocytes in the epithelial layer. Gluten seems to be the only food component that provokes intestinal inflammation in this way, a property that reflects gluten’s ability to stimulate both innate and specific immune responses in genetically susceptible individuals. The incidence of celiac disease has increased fourfold in the past 60 years, correlating with changes in baking practice that include adding large amounts of extra gluten to dough to decrease the time required for the dough to rise and to improve the texture.

1	Celiac disease shows an extremely strong genetic predisposition, with more than 95% of patients expressing the HLA-DQ2 class II MHC allele. In mono-zygotic twins, if one twin develops it, there is an 80% probability that the other will, but only a 10% concordance is observed in dizygotic twins. Nevertheless, most individuals expressing HLA-DQ2 do not develop celiac disease despite the almost universal presence of gluten in the Western diet. Thus, other genetic or environmental factors must make important contributions to susceptibility.

1	Peptides naturally produced from gluten do not bind to MHC class II molecules FasIFN-˜An enzyme, tissue transglutaminase (tTG), modiÿes the peptides so they now can be processed and bind to the MHC class II molecules The bound peptide activates gluten-speciÿc CD4 T cells FasLtTGThe activated T cells can kill mucosal epithelial cells by binding Fas. They also secrete IFN-˜, which activates the epithelial cell to produce cytokines and chemokines that recruit other in°ammatory cells Fig. 14.25 Molecular basis of immune recognition of gluten in celiac disease. After the digestion of gluten by gut digestive enzymes, deamidation of epitopes by tissue transglutaminase renders the gluten more susceptible to being readily processed by local antigen-presenting cells, ultimately leading to its binding to HLA-DQ molecules and priming of the immune system.

1	Most evidence indicates that celiac disease requires the aberrant priming of IFN-γ-producing CD4 T cells by antigenic peptides present in α-gliadin, one of the major proteins in gluten. It is generally accepted that only a limited number of peptides can provoke an immune response leading to celiac disease. This is likely due to the unique structure of the peptide-binding groove of the HLA-DQ2 molecule. The key step in the immune recognition of α-gliadin is the deamidation of its peptides by the enzyme tissue transglutaminase (tTG), which converts selected glutamine residues to negatively charged glutamic acid. Only peptides containing negatively charged residues in certain positions bind strongly to HLA-DQ2, and thus the transamination reaction promotes the formation of peptide:HLA-DQ2 complexes, which can activate antigen-specific CD4 T cells (Fig. 14.25). Activated gliadin-specific CD4 T cells accumulate in the lamina propria, producing IFN-γ, a cytokine that when present in this

1	complexes, which can activate antigen-specific CD4 T cells (Fig. 14.25). Activated gliadin-specific CD4 T cells accumulate in the lamina propria, producing IFN-γ, a cytokine that when present in this location leads to intestinal inflammation.

1	Celiac disease is entirely dependent on the presence of the foreign antigen, gluten. It is not associated with a specific immune response against self antigens in the tissue—the intestinal epithelium—that is damaged during the immune response. Thus, celiac disease is not a classical autoimmune disease. But it does have some features of autoimmunity. Autoantibodies against tissue transglutaminase are found in all patients with celiac disease; indeed, the presence of serum IgA antibodies against this enzyme is used as a sensitive and specific test for the disease. Interestingly, no tTG-specific T cells have been found, and it has been proposed that gluten-reactive T cells provide help to B cells that are reactive to tissue transglutaminase. In support of this hypothesis, gluten can complex with the enzyme and therefore could be taken up and presented by tTG-reactive B cells (Fig. 14.26). There is no evidence, however, that these autoantibodies contribute directly to tissue damage.

1	Fig. 14.26 A hypothesis to explain antibody production against tissue transglutaminase (tTG) in the absence of T cells specific for tTG in celiac patients. tTG-reactive B cells endocytose gluten–tTG complexes and present gluten peptides to the gluten-specific T cells. The stimulated T cells can now provide help to these B cells, which produce autoantibodies against tTG. Gluten peptides activate mucosal epithelial cells to express MIC molecules MIC IL1R IL1 NKG2D CD8 T cell (IEL) Intraepithelial lymphocytes (IELs) express NKG2D, which binds to MIC molecules and activates the IELs to kill the epithelial cell

1	Fig. 14.27 The activation of cytotoxic T cells by the innate immune system in celiac disease. Gluten peptides can induce the expression of the MHC class Ib molecules MIC-A and MIC-B on gut epithelial cells and the synthesis and release of IL-1 from these cells. Intraepithelial lymphocytes (IELs), many of which are CD8 cytotoxic T cells, recognize the MIC proteins via the receptor NKG2D, which, together with the co-stimulator IL-1, activates the IELs to kill the MIC-bearing cells, leading to destruction of the gut epithelium.

1	Chronic T-cell responses against food proteins are normally prevented by the development of oral tolerance (see Section 12-18). Why this breaks down in patients with celiac disease is unknown. The properties of the HLA-DQ2 molecule provide a partial explanation, but there must be additional factors because most HLA-DQ2-positive individuals do not develop celiac disease, and the high concordance rates in monozygotic twins indicate a role for additional genetic factors. The incidence of celiac disease is especially high in individuals with trisomy 21 (Down syndrome), approximately sixfold higher than in the normal population, underscoring the impact of genetic factors on disease prevalence. Polymorphisms in the gene for CTLA-4 or in other immunoregulatory genes have been suggested to be associated with susceptibility. There could also be differences in how individuals digest gliadin in the intestine, so that differing amounts survive for deamidation and presentation to T cells.

1	The gluten protein also seems to have several properties that contribute to pathogenesis. As well as its relative resistance to digestion, there is mounting evidence that some gliadin-derived peptides stimulate the innate immune system by inducing the release of IL-15 by intestinal epithelial cells. This process is antigen-nonspecific and involves peptides that cannot be bound by HLA-DQ2 molecules or recognized by CD4 T cells. IL-15 release leads to the activation of dendritic cells in the lamina propria, as well as the upregulation of MIC-A expression by epithelial cells. CD8 T cells in the mucosal epithelium can be activated via their NKG2D receptors, which recognize MIC-A, and they can kill MIC-A-expressing epithelial cells via these same NKG2D receptors (Fig. 14.27). Triggering of these innate immune responses by α-gliadin may create some intestinal damage on its own and also induce some of the co-stimulatory events necessary for initiating an antigen-specific CD4 T-cell response

1	innate immune responses by α-gliadin may create some intestinal damage on its own and also induce some of the co-stimulatory events necessary for initiating an antigen-specific CD4 T-cell response to other parts of the α-gliadin molecule. The ability of gluten to stimulate both innate and adaptive immune responses may thus explain its unique ability to induce celiac disease.

1	Summary.

1	Non-IgE-mediated immunological hypersensitivity reflects normal immune mechanisms that are inappropriately directed against innocuous antigens or inflammatory stimuli. It comprises both immediate-type and delayed-type reactions. Immediate-type reactions are due to the binding of specific IgG antibodies to allergen-modified cell surfaces, as in drug-induced hemolytic anemia, or to the formation of immune complexes of antibodies bound to poorly catabolized antigens, as occurs in serum sickness. Cellular hypersensitivity reactions mediated by TH1 cells and cytotoxic T cells develop more slowly than immediate-type reactions. The TH1-mediated hypersensitivity reaction in the skin provoked by mycobacterial tuberculin is used to diagnose previous exposure to Mycobacterium tuberculosis. The allergic reaction to poison ivy is due to the recognition and destruction by cytotoxic T cells of skin cells modified by a plant molecule, and to cytotoxic T-cell cytokines. These T-cell-mediated responses

1	reaction to poison ivy is due to the recognition and destruction by cytotoxic T cells of skin cells modified by a plant molecule, and to cytotoxic T-cell cytokines. These T-cell-mediated responses require the induced synthesis of effector molecules and develop over 1–10 days.

1	Summary to Chapter 14.

1	In susceptible individuals, immune responses to otherwise innocuous antigens can produce allergic reactions upon reexposure to the same antigen. Most allergic reactions involve the production of IgE antibody against common environmental allergens. Some people are intrinsically prone to making IgE antibodies against many allergens, and such people are said to be atopic. IgE production is driven by antigen-specific TH2 cells; the response is skewed toward TH2 by an array of chemokines and cytokines that engage specific signaling pathways, including signals that activate ILC2 cells in submucosal tissues at sites of antigen entry. The IgE produced binds to the high-affinity IgE receptor FcεRI on mast cells and basophils. Specific effector T cells, mast cells, and eosinophils, in combination with TH1 and TH2 cytokines and chemokines, orchestrate chronic allergic inflammation, which is the major cause of the chronic morbidity of asthma. Failure to regulate these responses can occur at many

1	TH1 and TH2 cytokines and chemokines, orchestrate chronic allergic inflammation, which is the major cause of the chronic morbidity of asthma. Failure to regulate these responses can occur at many levels of the immune system, including defects in regulatory T cells. Increasingly successful processes for suppressing allergic responses and reestablishing the ability to tolerate the sensitizing antigen are being developed, raising the hope of reducing the prevalence of allergic disorders. Antibodies of certain isotypes and various antigen-specific effector T cells contribute to allergic hypersensitivity to other antigens.

1	Questions. 14.1 True or False: Only TH2 cells can initiate the chain of signals needed to induce B cells to class-switch to IgE. 14.2 Multiple Choice: Which of the following has not been associated with genetic susceptibility to both allergic asthma and atopic eczema? A. β subunit of FcεRI B. GM-CSF C. IL-3 D. IL-4 E. IFN-γ 14.3 Different factors affect our susceptibility to allergic diseases. Which of the following is a false statement? A. Environmental factors rarely contribute to the development of allergic disease. B. The prevalence of atopy has been steadily increasing in the developed world. C. Individuals with variant alleles of GSTP1 and GSTM1 have higher susceptibility to increased airway hyperactivity. D. Children less than 6 months old who are exposed to other children in day care appear to be partially protected against asthma. 14.4 True or False: Like other antibodies, IgE is mainly found in body fluids.

1	14.4 True or False: Like other antibodies, IgE is mainly found in body fluids. 14.5 Matching: Match the following options with the best 14.6 Which of the following statements is true? A. Connective tissue mast cells do not participate in the initiation of an anaphylactic reaction. B. Epinephrine should be avoided in patients suffering from anaphylactic shock as it may worsen the patient’s condition. C. During anaphylactic shock, blood vessels lose permeability, and high blood pressure leads to death. D. Penicillin can modify self proteins, causing an immune response with IgE production in some individuals that can lead to anaphylaxis upon re-encountering the drug. 14.7 Multiple Choice: Hypersensitivity reactions can cause pathology through the deposition of immune complexes. Which of the following is a mechanism by which immune complexes can be pathogenic? (More than one may apply.) ___ A. Immune complexes deposit in blood vessel walls.

1	___ B. IgE is cross-linked on the surface of mast cells and basophils, leading to activation. ___ C. Fc receptor ligation leads to leukocyte activation and tissue injury. ___ D. The complement system is activated, leading to the production of anaphylatoxin C5a. ___ E. CD8+ T cells are stimulated to secrete IL-4. 14.8 Fill-in-the-Blanks: There are two phases to a cutaneous allergic response: ______ and ______. The first phase is characterized by activation of T cells by skin antigen-presenting cells called _________, while the second phase invokes release of chemokines and cytokines by __________ upon subsequent antigen exposure. 14.9 Matching: Match each allergic reaction with the corresponding immune process.

1	14.9 Matching: Match each allergic reaction with the corresponding immune process. ___ A. Arthus reaction i. Formation of local immune complexes caused by IgG antibodies acting against an antigen in previously sensitized individuals ___ B. Poison ivy rash ii. Systemic reaction to injection of large quantities of foreign antigen, primarily IgG-mediated 14.10 Multiple Choice: Which of the following is a false statement? A. The tuberculin test illustrates the prototypic delayed-type hypersensitivity reaction. B. TH1 cells are not directly involved in delayed-type hypersensitivity reactions. General references. Fahy, J.V.: Type 2 inflammation in asthma – present in most, absent in many. Nat. Rev. Immunol. 2015, 15:57–65. Holgate, S.T.: Innate and adaptive immune responses in asthma. Nat. Med. 2012, 18:673–683.

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1	Arndt, P.A.: Drug-induced immune hemolytic anemia: the last 30 years of changes. Immunohematology 2014, 30:44–54. Greinacher, A., Potzsch, B., Amiral, J., Dummel, V., Eichner, A., and Mueller Eckhardt, C.: Heparin-associated thrombocytopenia: isolation of the antibody and characterization of a multimolecular PF4–heparin complex as the major antigen. Thromb. Haemost. 1994, 71:247–251. Semple, J.W., and Freedman, J.: Autoimmune pathogenesis and autoimmune hemolytic anemia. Semin. Hematol. 2005, 42:122–130. 14-15 Systemic disease caused by immune-complex formation can follow the administration of large quantities of poorly catabolized antigens. Bielory, L., Gascon, P., Lawley, T.J., Young, N.S., and Frank, M.M.: Human serum sickness: a prospective analysis of 35 patients treated with equine anti-thymocyte globulin for bone marrow failure. Medicine (Baltimore) 1988, 67:40–57.

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1	We have already seen how undesirable adaptive immune responses can be elicited by environmental antigens, and how this can cause serious disease in the form of allergic and atopic reactions (see Chapter 14). In this chapter, we examine responses to other medically important categories of antigens—those expressed by the body’s own cells and tissues, by the commensal microbiota, or by transplanted organs. The responses to self antigens or antigens associated with the microbiota that lead to tissue damage and disease are broadly referred to as autoimmunity—although, strictly speaking, disease-causing immune responses to the commensal microbiota are a form of xenoimmunity because the organisms from which the antigens derive are foreign and are not encoded by the human genome. Nevertheless, here we will consider immune-mediated disease directed against the commensal microbiota to be part of the extended spectrum of autoimmune diseases, because the microbiota can be regarded as being part

1	we will consider immune-mediated disease directed against the commensal microbiota to be part of the extended spectrum of autoimmune diseases, because the microbiota can be regarded as being part of a ‘superorganism’ made up of host and commensal microbiota together. The response to nonself antigens on transplanted organs is called allograft rejection.

1	The gene rearrangements that occur during lymphocyte development in the central lymphoid organs inevitably result in the generation of some lymphocytes with affinity for self antigens. Such lymphocytes are normally removed from the repertoire or held in check by a variety of mechanisms. These generate a state of self-tolerance in which an individual’s immune system does not attack the normal tissues of the body. Autoimmunity represents a breakdown or failure of the mechanisms of self-tolerance. Therefore, we first revisit the mechanisms that keep the lymphocyte repertoire self-tolerant and see how these may fail. We then discuss a selection of autoimmune diseases that illustrate the various pathogenic mechanisms by which autoimmunity can damage the body. How genetic and environmental factors predispose to or trigger autoimmunity are then considered. In the remaining part of the chapter, we discuss the adaptive immune responses to nonself tissue antigens that cause transplant

1	factors predispose to or trigger autoimmunity are then considered. In the remaining part of the chapter, we discuss the adaptive immune responses to nonself tissue antigens that cause transplant rejection.

1	The making and breaking of self-tolerance. As we learned in Chapter 8, the immune system takes advantage of surrogate markers of self and nonself to identify and delete potentially self-reactive lymphocytes. Despite this, some self-reactive lymphocytes escape elimination and can subsequently be activated to cause autoimmunity. In addition, many lymphocytes with some degree of self-reactivity can also respond to foreign antigens; therefore, if all weakly self-reactive lymphocytes were eliminated, the function of the immune system would be impaired. 15-1 A critical function of the immune system is to discriminate self from nonself. The immune system has very powerful effector mechanisms that can eliminate a variety of pathogens. Early in the study of immunity it was realized that these could, if turned against the host, cause severe tissue damage. The concept The making and breaking of self‑tolerance. Autoimmune diseases and pathogenic mechanisms.

1	The making and breaking of self‑tolerance. Autoimmune diseases and pathogenic mechanisms. The genetic and environmental basis of autoimmunity. Responses to alloantigens and transplant rejection. Fig. 15.1 Some common autoimmune diseases. The diseases listed are among the most common autoimmune diseases and will be used as examples in this part of the chapter. They are listed in order of prevalence.

1	of autoimmunity was first presented at the beginning of the 20th century by Paul Ehrlich, who described it as ‘horror autotoxicus.’ Autoimmune responses resemble normal immune responses to pathogens in that they are specifically activated by antigens—in this case self antigens, or autoantigens—and give rise to autoreactive effector cells and antibodies, called autoantibodies, against the self antigen. When dysregulated reactions to self tissues occur they cause a variety of chronic syndromes called autoimmune diseases. These syndromes are quite varied in their severity, their tissue distribution, and effector mechanisms that are critical in causing tissue damage (Fig. 15.1).

1	Collectively, autoimmune disorders affect approximately 5% of the populations of Western countries, and their incidence is on the rise. Nevertheless, their relative individual rarity indicates that the immune system has evolved multiple mechanisms to prevent self-injury. The most fundamental principle underlying these mechanisms is the discrimination of self from nonself, but this discrimination is not easy to achieve. B cells recognize the three-dimensional shape of an epitope, but an epitope presented by a pathogen can be indistinguishable from one originating in humans. Similarly, short peptides derived from the processing of pathogen antigens can be identical to self peptides. So how does a lymphocyte know what ‘self’ really is if there are no unique molecular signatures of self?

1	The first mechanism proposed for distinguishing between self and nonself was that recognition of antigen by an immature lymphocyte leads to a negative signal causing lymphocyte death or inactivation. Thus, ‘self’ was thought to comprise molecules recognized by a lymphocyte shortly after it began to express its antigen receptor. Indeed, this is an important mechanism of inducing self-tolerance in lymphocytes developing in the thymus and bone marrow. The tolerance induced at this stage is known as central tolerance (see Chapter 8). Newly formed lymphocytes are especially sensitive to inactivation by strong signals through their antigen receptor, whereas the same signals activate mature lymphocytes in the periphery. The making and breaking of self‑tolerance.

1	Tolerance induced to antigens recognized after lymphocytes have left the central or primary lymphoid organs is known as peripheral tolerance. An antigenic quality that correlates with self in the periphery is recognition in the absence of ‘danger’ signals that are produced by the innate immune system as a result of tissue damage or infection. Nearly all cells in the body become senescent and die, and many cells routinely undergo turnover at steady state (for example, hematopoietic cells and epithelial cells of the intestines and skin). Typically, this occurs by programmed cell death, or apoptosis. In contrast to cell death that results from physical or microbial injury, which generate damageor microbe-associated molecular patterns (DAMPs and MAMPs, respectively), death of senescent cells by apoptosis releases signals to tissue phagocytes that generally promote an anti-inflammatory response and repress presentation of antigens in an activating form. Thus, self antigens recognized in

1	by apoptosis releases signals to tissue phagocytes that generally promote an anti-inflammatory response and repress presentation of antigens in an activating form. Thus, self antigens recognized in the context of normal, or physiologic, cellular turnover fail to induce pro-inflammatory cytokines (for example, IL-6 or IL-12) and co-stimulatory molecules (for example, B7.1) that would otherwise induce naive T cells to undergo effector differentiation. In these circumstances, the encounter of a naive lymphocyte with a self antigen may lead to no signal at all, or such an encounter can promote the development of regulatory lymphocytes that suppress the development of damaging effector responses. The removal of apoptotic cells by phagocytes is thus important for maintaining tissue homeostasis and activating programs in antigen-presenting cells that promote immunological tolerance. Some of the same mechanisms appear to be involved in the induction of tolerance to antigens of the commensal

1	and activating programs in antigen-presenting cells that promote immunological tolerance. Some of the same mechanisms appear to be involved in the induction of tolerance to antigens of the commensal microbiota in the intestines, where recognition of bacterial antigens typically does not generate inflammation unless there is associated tissue damage.

1	Thus, several clues are used to distinguish self from nonself ligands: encounter with the ligand when the lymphocyte is immature, recognition of antigen in the context of antigen-presenting cells that have received tolerizing signals from recognition of homeostatic cell turnover signals, and binding of ligand in the absence of inflammatory cytokines or co-stimulatory signals. All of these mechanisms are error-prone because none of them distinguishes a self ligand from a foreign one at the molecular level. The immune system therefore has several additional mechanisms for controlling autoimmune responses should they start. 15-2 Multiple tolerance mechanisms normally prevent autoimmunity.

1	The mechanisms that normally prevent autoimmunity may be thought of as a succession of checkpoints. Each checkpoint is partially effective in preventing antiself responses, and together the checkpoints act synergistically to provide efficient protection against autoimmunity without inhibiting the ability of the immune system to mount effective responses to pathogens. Central tolerance mechanisms eliminate newly formed, strongly autoreactive lymphocytes. On the other hand, mature self-reactive lymphocytes that do not sense self strongly in the central lymphoid organs—because their cognate self antigens are not expressed there, for example—may be killed or inactivated in the periphery. The principal mechanisms of peripheral tolerance are anergy (functional unresponsiveness), suppression by T cells, induction of T cell development instead of effector T-cell development (functional deviation), and deletion of lymphocytes from the repertoire due to activation-induced cell death. In

1	by T cells, induction of T cell development instead of effector T-cell development (functional deviation), and deletion of lymphocytes from the repertoire due to activation-induced cell death. In addition, some antigens are sequestered in organs that are not normally accessible to the immune system (Fig. 15.2).

1	Each checkpoint strikes a balance between preventing autoimmunity and not impairing immunity too greatly, and in combination, all the checkpoints provide an effective overall defense against autoimmune disease. It is relatively The different ways in which the immune system prevents activation of and damage caused by autoreactive lymphocytes are listed, along with the specific mechanism and where such tolerance predominantly occurs. Autoimmunity Immune dysregulation Genetic factors Infection and environmental exposure Fig. 15.3 Requirements for the development of autoimmune disease. In genetically predisposed individuals, autoimmunity may be triggered as a result of the failure of intrinsic tolerance mechanisms and/or environmental triggers such as infection.

1	easy to find isolated breakdowns of one or even more layers of protection, even in healthy individuals. Thus, activation of autoreactive lymphocytes does not necessarily equal autoimmune disease. In fact, a low level of autoreactivity is physiologic and crucial to normal immune function. Autoantigens help to form the repertoire of mature lymphocytes, and the survival of naive T cells and B cells in the periphery requires continuous exposure to autoantigens (see Chapter 8). Autoimmune disease develops only if enough safeguards are overcome to lead to a sustained reaction to self that includes the generation of effector cells and molecules that destroy tissues. Although the mechanisms by which this occurs are incompletely understood, autoimmunity is thought to result from a combination of genetic susceptibility, breakdown in natural tolerance mechanisms, and environmental triggers such as infections (Fig. 15.3).

1	15-3 Central deletion or inactivation of newly formed lymphocytes is the first checkpoint of self-tolerance. Central tolerance mechanisms, which remove strongly autoreactive lymphocytes, are the first and most important checkpoints in self-tolerance (see Chapter 8). Without them, the immune system would be strongly self-reactive, and lethal autoimmunity would occur early in life. It is unlikely that peripheral tolerance mechanisms would be sufficient to compensate for the failure to remove self-reactive lymphocytes during primary development. Indeed, no known autoimmune diseases are attributable to complete failure of these mechanisms, although some are associated with a partial failure of central tolerance.

1	For a long time it was thought that many self antigens were not expressed in the thymus or bone marrow, and that peripheral mechanisms must be the only way of generating tolerance to them. It is now clear that many (but not all) tissue-specific antigens, such as insulin, are expressed in the thymus by either thymic epithelial cells in the medulla or a CD8α+ subset of dendritic cells, and thus self-tolerance against these antigens can be generated centrally. How these ‘peripheral’ genes are turned on ectopically in the thymus is not yet completely worked out, but an important clue has been found. A single transcription factor, AIRE (for autoimmune regulator), is thought to be responsible for turning on many peripheral genes in the thymus (see Section 8-23). The AIRE gene is defective in patients with a rare inherited form of autoimmunity—APECED (autoimmune polyendocrinopathy–candidiasis– ectodermal dystrophy), also known as autoimmune polyglandular syndrome type 1 (APS-1)—that leads to

1	with a rare inherited form of autoimmunity—APECED (autoimmune polyendocrinopathy–candidiasis– ectodermal dystrophy), also known as autoimmune polyglandular syndrome type 1 (APS-1)—that leads to destruction of multiple endocrine tissues, including insulin-producing pancreatic islets, and to fungal infections, particularly candidiasis. Mice engineered to lack the AIRE gene fail to express many peripheral genes in the thymus and develop a similar syndrome. This links AIRE to the expression of these genes, and the antigens they encode, indicating that an inability to express these antigens in the thymus leads to autoimmune disease (Fig. 15.4). The autoimmunity that accompanies AIRE deficiency takes time to develop and does not always affect all potential organ targets. So as well as emphasizing the importance of central tolerance, this disease shows that other layers of tolerance control have important roles.

1	In the absence of AIRE, T cells reactive to tissue-specifc antigens mature and leave the thymus In the thymus, T cells arise capable of recognizing tissue-speciÿc antigens Under control of the AIRE protein, thymic medullary cells express tissue-speciÿc proteins, leading to deletion of tissue-reactive T cells 15-4 Lymphocytes that bind self antigens with relatively low affinity activated.

1	Most circulating lymphocytes have a low affinity for self antigens but make no effector response to them, and may be considered ‘ignorant’ of self (see Section 8-6). Such ignorant but latently self-reactive cells can be recruited into autoimmune responses if their threshold for activation is exceeded by co-activating factors. One such stimulus is infection. Naive T cells with low affinity for a ubiquitous self-antigen can become activated if they encounter an activated dendritic cell presenting that antigen and expressing high levels of co-stimulatory signals or pro-inflammatory cytokines as a result of the presence of infection.

1	A situation in which normally ignorant lymphocytes may be activated is when their autoantigens are also ligands for Toll-like receptors (TLRs). These receptors are usually considered to be specific for microbe-associated molecular patterns (see Section 3-5), but some of these patterns can be found among self molecules. An example is unmethylated CpG sequences in DNA that are recognized by TLR-9. Unmethylated CpG is normally much more common in bacterial than mammalian DNA, but is enriched in apoptotic mammalian cells. In a scenario of extensive cell death coupled with inadequate clearance of apoptotic fragments, B cells specific for chromatin components can internalize CpG sequences via their B-cell receptors. These sequences can be recognized by TLR-9 intracellularly, leading to a co-stimulatory signal that activates the previously ignorant anti-chromatin B cell (Fig. 15.5). B cells activated in this way produce anti-chromatin autoantibodies and also can act as antigen-presenting

1	signal that activates the previously ignorant anti-chromatin B cell (Fig. 15.5). B cells activated in this way produce anti-chromatin autoantibodies and also can act as antigen-presenting cells for autoreactive T cells. Ribonucleoprotein complexes containing uridine-rich RNA have similarly been shown to activate

1	Fig. 15.4 The ‘autoimmune regulator’ gene AIRE promotes the expression of some tissue-specific antigens in thymic medullary cells, causing the deletion of immature thymocytes that can react to these antigens. Although the thymus expresses many genes, and thus self proteins, common to all cells, it is not obvious how antigens that are specific to specialized tissues, such as retina or ovary (first panel), gain access to the thymus to promote the negative selection of immature autoreactive thymocytes. It is now known that a gene called AIRE promotes the expression of many tissue‑specific proteins in thymic medullary cells. Some developing thymocytes will be able to recognize these tissue‑specific antigens (second panel). Peptides from these proteins are presented to the developing thymocytes as they undergo negative selection in the thymus (third panel), causing deletion of these cells. In the absence of AIRE, this deletion does not occur; instead, the autoreactive thymocytes mature and

1	as they undergo negative selection in the thymus (third panel), causing deletion of these cells. In the absence of AIRE, this deletion does not occur; instead, the autoreactive thymocytes mature and are exported to the periphery (fourth panel), where they could cause autoimmune disease. Indeed, people and mice that lack expression of AIRE develop an autoimmune syndrome called APECED, or autoimmune polyendocrinopathy–candidiasis– ectodermal dystrophy.

1	Fig. 15.5 Self antigens that are recognized by Toll-like receptors can activate autoreactive B cells by providing co-stimulation. The receptor TLR‑9 promotes the activation of B cells that produce antibodies specific for DNA, a common autoantibody in the autoimmune disease systemic lupus erythematosus (SLE) (see Fig. 15.1). Although B cells with strong affinity for DNA are eliminated in the bone marrow, some DNA‑specific B cells with lower affinity escape and persist in the periphery but are not normally activated. Under some conditions and in genetically susceptible individuals, the concentration of DNA may increase, leading to the ligation of enough B‑cell receptors to initiate activation of these B cells. B cells signal through their receptor (left panel) but also take up the DNA (center panel) and deliver it to an endosomal compartment (right panel). Here the DNA has access to TLR‑9, which recognizes DNA that is enriched in unmethylated CpG DNA sequences. Such CpG‑enriched

1	(center panel) and deliver it to an endosomal compartment (right panel). Here the DNA has access to TLR‑9, which recognizes DNA that is enriched in unmethylated CpG DNA sequences. Such CpG‑enriched sequences are much more common in microbial than eukaryotic DNA and normally this allows TLR‑9 to distinguish pathogens from self. DNA in apoptotic mammalian cells is enriched in unmethylated CpG, however, and the DNA‑specific B cell will also concentrate this self‑DNA in the endosomal compartment. This would provide sufficient ligands to activate TLR‑9, potentiating the activation of the DNA‑specific B cells and leading to the production of autoantibodies against DNA.

1	B cells with specifcity for DNA bind soluble fragments of DNA, sending a signal through the B-cell receptor The cross-linked B-cell receptor is internalized with the bound DNA molecule GC-rich fragments from the internalized DNA bind to TLR-9 in an endosomal compartment, sending a co-stimulatory signal TLR-9 naive B cells through binding by TLR-7 or TLR-8. Autoantibodies against DNA, chromatin, and ribonucleoproteins are produced in the autoimmune disease systemic lupus erythematosus (SLE), and this appears to be one mechanism by which self-reactive B cells are stimulated to produce them.

1	Another mechanism by which ignorant lymphocytes can be drawn into action is by the changing of the availability or form of self antigens. Some antigens are normally intracellular and not encountered by lymphocytes, but they may be released as a result of massive tissue injury or inflammation. These antigens can then activate ignorant T and B cells, leading to autoimmunity. This can occur after myocardial infarction, when an autoimmune response is detectable some days after the release of cardiac antigens. Such reactions are typically transient and cease when the autoantigens have been removed; however, when clearance mechanisms are inadequate, they can continue, causing clinical autoimmune disease.

1	Additionally, some autoantigens are present in great quantity but are usually in a nonimmunogenic form. IgG is a good example, as there are large quantities of it in blood and extracellular fluids. B cells specific for the IgG constant region are not usually activated, because IgG is monomeric and cannot cross-link the B-cell receptor. However, when immune complexes form following infection or immunization, enough IgG is in multivalent form to evoke a response from otherwise ignorant B cells. The anti-IgG autoantibody they produce is called rheumatoid factor because it is often present in rheumatoid arthritis. Again, this response is normally short-lived, as long as the immune complexes are cleared rapidly.

1	A unique situation can occur when activated B cells undergo somatic hypermutation in germinal centers (see Section 10-7), resulting in some activated B cells increasing their affinity for a self antigen or becoming newly self-reactive (Fig. 15.6). There seems, however, to be a mechanism to control germinal center B cells that have acquired affinity for self. In this case, if a hypermutated self-reactive B cell encounters strong cross-linking of its B-cell receptor in the germinal center, it undergoes apoptosis rather than further proliferation. 15-5 Antigens in immunologically privileged sites do not induce immune attack but can serve as targets.

1	15-5 Antigens in immunologically privileged sites do not induce immune attack but can serve as targets. Foreign tissue grafts placed in some body sites do not elicit immune responses. For instance, grafts placed in the brain and anterior chamber of the eye do not induce rejection. Such locations are termed immunologically privileged sites (Fig. 15.7). It was originally believed that immunological privilege arose from the failure of antigens to leave privileged sites and induce immune responses. Subsequent studies have shown that antigens do leave these sites and interact with T cells. However, instead of eliciting an effector immune response, they induce a tolerogenic response that does not injure the tissue.

1	Immunologically privileged sites seem to be unusual in three ways. First, communication between the privileged site and the body is atypical in that extracellular fluid does not pass through conventional lymphatics, although proteins placed in these sites do leave and can have immunological effects. Privileged sites are generally surrounded by tissue barriers that exclude naive lymphocytes. The brain, for example, is guarded by the blood–brain barrier. Second, soluble factors that affect the course of an immune response are produced in privileged sites. The anti-inflammatory transforming growth factor (TGF)-β seems to be particularly important in this regard. Under homeostatic conditions, antigens recognized in concert with TGF-β tend to induce Treg responses, rather than pro-inflammatory TH17 responses, which are induced by TGF-β in the presence of IL-6 (see Section 9-21). Third, the expression of Fas ligand in immunologically privileged sites may provide a further level of

1	TH17 responses, which are induced by TGF-β in the presence of IL-6 (see Section 9-21). Third, the expression of Fas ligand in immunologically privileged sites may provide a further level of protection by inducing the apoptosis of Fas-bearing effector lymphocytes that enter these sites.

1	Paradoxically, antigens sequestered in immunologically privileged sites are often targets of autoimmune attack; for example, brain and spinal cord autoantigens such as myelin basic protein are targeted in the autoimmune disease multiple sclerosis, a chronic inflammatory demyelinating disease of the central nervous system (see Fig. 15.1). Thus, the tolerance normally shown to these antigens cannot be due to previous deletion of the self-reactive T cells. In experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis, mice become diseased only when they are immunized with myelin antigens and adjuvants, which cause infiltration of the central nervous system with antigen-specific TH17 and TH1 cells that induce a local inflammatory response that damages nerve tissue.

1	Thus, some antigens expressed in immunologically privileged sites induce neither tolerance nor lymphocyte activation in normal circumstances, but if auto-reactive lymphocytes are activated elsewhere, these autoantigens can become targets for autoimmune attack. Likely, T cells specific for antigens sequestered in immunologically privileged sites are in a state of immunological ignorance. Further evidence comes from the eye disease sympathetic ophthalmia (Fig. 15.8). If one eye is ruptured by a blow or other trauma, an autoimmune response to eye proteins can occur, although this happens only rarely. Once the response is induced, it often attacks both eyes. Immunosuppression—and, rarely, removal of the damaged eye, the source of antigen—is required to preserve vision in the undamaged eye.

1	Unsurprisingly, effector T cells can enter immunologically privileged sites when such sites become infected. Effector T cells can enter most tissues after activation (see Chapter 11), but accumulation of cells is seen only when antigen is recognized in the site, triggering the production of cytokines that alter tissue barriers. 15-6 Autoreactive T cells that express particular cytokines may be nonpathogenic or may suppress pathogenic lymphocytes.

1	15-6 Autoreactive T cells that express particular cytokines may be nonpathogenic or may suppress pathogenic lymphocytes. As described in Chapter 9, CD4 T cells can differentiate into various types of effector lineages, namely, TH1, TH2, and TH17 cells. These effector subsets evolved to control different types of infections and orchestrate distinct types of responses, as reflected in their different effects on antigen-presenting cells, B cells, and innate cells such as macrophages, eosinophils, and neutrophils (see Chapters 9–11). A similar paradigm holds true for autoimmunity: certain T-cell-mediated autoimmune diseases such as type 1 diabetes mellitus (see Fig. 15.1) depend on TH1 cells to cause disease, whereas others, such as psoriasis (an autoimmune disease of the skin), depend on TH17 cells.

1	Fig. 15.6 Elimination of autoreactive B cells in germinal centers. During somatic hypermutation in germinal centers (top panel), B cells with autoreactive B‑cell receptors can arise. Ligation of these receptors by soluble autoantigen (center panel) induces apoptosis of the autoreactive B cell by signaling through the B‑cell antigen receptor in the absence of helper T cells (bottom panel). Fig. 15.8 Damage to an immunologically privileged site can induce an autoimmune response. In the disease sympathetic ophthalmia, trauma to one eye releases the sequestered eye antigens into the surrounding tissues, making them accessible to T cells. The effector cells that are elicited attack the traumatized eye, and also infiltrate and attack the healthy eye. Thus, although the sequestered antigens do not induce a response by themselves, if a response is induced elsewhere they can serve as targets for attack.

1	In murine models of diabetes, when cytokines were infused to influence T-cell differentiation or when knockout mice predisposed to TH2 differentiation were studied, the development of diabetes was inhibited. In some cases, potentially pathogenic T cells specific for pancreatic islet-cell components, and expressing TH2 instead of TH1 cytokines, actually suppressed disease caused by TH1 cells of the same specificity. So far, attempts to control human autoimmune disease by switching cytokine profiles from one effector cell type to another (for example, TH1 to TH2), a procedure termed immune modulation, have not proved successful. Another subset of CD4 T cells, Treg cells, might prove to be more important in the prevention of autoimmune disease, and efforts to deviate effector to regulatory T-cell responses are being studied as a novel therapy for autoimmunity. 15-7 Autoimmune responses can be controlled at various stages by regulatory T cells.

1	15-7 Autoimmune responses can be controlled at various stages by regulatory T cells. Autoreactive cells that escape the tolerance-inducing mechanisms described above can still be regulated so that they do not cause disease. This regulation takes two forms: the first is extrinsic, and is mediated by regulatory T cells that act on activated T cells and antigen-presenting cells; the second is intrinsic, and has its basis in limits on the size and duration of immune responses that are programmed into lymphocytes themselves. We shall first discuss the role of regulatory T cells (introduced in Chapter 9).

1	Tolerance due to regulatory lymphocytes is distinguished from other forms of self-tolerance by the fact that Treg cells have the potential to suppress self-reactive lymphocytes that recognize antigens different from those recognized by the Treg cell (Fig. 15.9). This type of tolerance is known as regulatory tolerance. The key feature of regulatory tolerance is that regulatory cells can suppress autoreactive lymphocytes that recognize a variety of different self antigens, as long as the antigens are from the same tissue or are presented by the same antigen-presenting cell. As discussed in Chapter 9, two general types of regulatory T cells have been defined experimentally. ‘Natural’ Treg (nTreg) cells are programmed in the thymus to express the transcription factor FoxP3 in response to self antigens. When activated by the same antigens in peripheral tissues, nTreg cells inhibit other self-reactive T cells that recognize antigens in the same tissue to prevent their differentiation into

1	antigens. When activated by the same antigens in peripheral tissues, nTreg cells inhibit other self-reactive T cells that recognize antigens in the same tissue to prevent their differentiation into effector T cells or prevent their effector function. ‘Induced’ T (iT ) cells also express FoxP3 but develop in peripheral immune tissues in response to antigens recognized in the presence of TGF-β but in the absence of pro-inflammatory cytokines. Giving animals large amounts of self antigen orally, which induces so-called oral tolerance (see Section 12-18), can sometimes lead to unresponsiveness to these antigens when given by other routes, and can prevent autoimmune disease. Oral tolerance is routinely generated to antigens such as food antigens and is accompanied by the generation of iTreg cells in the gut-draining mesenteric lymph nodes. These cells are known to suppress immune responses to the given antigen in the gut itself, but how the suppression in the rest of the peripheral immune

1	in the gut-draining mesenteric lymph nodes. These cells are known to suppress immune responses to the given antigen in the gut itself, but how the suppression in the rest of the peripheral immune system is achieved is unclear. Many investigators have hypothesized that iTreg cells could have therapeutic potential for the treatment of autoimmune disease if they could be isolated or induced to differentiate and then be infused into patients.

1	The importance of FoxP3—and the Treg cells whose development and function it controls—in the maintenance of immune tolerance is evident from the fact that humans and mice that carry mutations in the gene for FoxP3 rapidly develop severe, systemic autoimmunity (discussed in Section 15-21). A protective role for FoxP3-expressing Treg cells has been demonstrated in several autoimmune syndromes in mice, including diabetes, EAE, SLE, and inflammation of the large intestine, or colon (colitis). Experiments in mouse models of these diseases have established that FoxP3+ Treg cells actively suppress disease in the normal immune system, as depletion of these cells results in multi-organ autoimmune disease. Treg cells have also been shown to prevent or ameliorate other immunopathologic syndromes, such as graft-versus-host disease and graft rejection, which are described later in this chapter.

1	The importance of regulatory T cells has been demonstrated in several human autoimmune diseases. For example, in some patients with multiple sclerosis or with autoimmune polyglandular syndrome type 2 (a rare syndrome in which two or more autoimmune diseases occur simultaneously), the suppressive activity of FoxP3+ Treg cells is defective, although their numbers are normal. Thus, Treg cells have an important role in preventing autoimmunity, and a variety of functional defects in these cells may lead to autoimmunity. FoxP3-expressing Treg cells are not the only type of regulatory lymphocyte that has been identified. For example, FoxP3-negative regulatory T cells characterized by their production of IL-10 are enriched in the intestinal tissues, where they may suppress inflammatory bowel disease (IBD) through an IL-10dependent mechanism. The developmental origins of these cells are not currently understood.

1	Almost every type of lymphocyte has been shown to display regulatory activity in some circumstance. Even B cells can regulate experimentally induced autoimmune syndromes, including collagen-induced arthritis (CIA) and EAE. This activity is probably mediated in a similar way to that of regulatory CD4 T cells, with the secretion of cytokines that inhibit proliferation and differentiation of effector T cells.

1	In addition to the extrinsic regulation of autoreactive T and B cells by regulatory cells, lymphocytes have intrinsic proliferation and survival limits that can help restrict autoimmune as well as normal responses (see Section 11-16). This is illustrated by the development of spontaneous autoimmunity that is caused by mutations in pathways that control apoptosis, such as the Bcl-2 pathway or the Fas pathway (see Section 7-23); mutations in these pathways lead to spontaneous autoimmunity. This form of autoimmunity provides evidence that autoreactive cells are normally generated but are then eliminated by apoptosis. This seems to be an important mechanism for both Tand B-cell tolerance.

1	periphery thymus periphery Cytokines (IL-10 and TGF-˜) produced by Treg cells inhibit other self-reactive T cells TGF-˜Regulatory tolerance T cell specifc for self antigen recognized in thymus becomes a natural regulatory T cell (nTreg) T cell specifc for self or commensal microbiota antigen recognized in presence of TGF-˜becomes an induced regulatory T cell (iTreg) Fig. 15.9 Tolerance mediated by regulatory T cells can inhibit multiple autoreactive T cells that all recognize antigens from the same tissue.

1	Fig. 15.9 Tolerance mediated by regulatory T cells can inhibit multiple autoreactive T cells that all recognize antigens from the same tissue. Specialized autoreactive natural regulatory T (nTreg) cells develop in the thymus in response to stimulation by self antigens that is too weak to cause deletion but is greater than required for simple positive selection (upper left panel). Regulatory T cells can also be induced from naive self‑reactive T cells in the periphery if the naive T cell recognizes its antigen and is activated in the presence of the cytokine TGF‑β (upper right panel). The lower panel shows how regulatory T cells, both natural and induced, can inhibit other self‑reactive T cells. If regulatory T cells encounter their self antigen on an antigen‑presenting cell, they secrete inhibitory cytokines such as IL‑10 and TGF‑β that inhibit all surrounding autoreactive T cells, regardless of their precise autoantigen specificity. Summary.

1	Discrimination between self and nonself is imperfect, partly because a proper balance must be struck between preventing autoimmune disease and preserving immune competence. Self-reactive lymphocytes always exist in the natural immune repertoire but are not often activated. In autoimmune diseases, however, these cells become activated by autoantigens. If activation persists, autoreactive effector lymphocytes are generated and cause disease. The immune system has a remarkable set of mechanisms that work together to prevent autoimmune disease (see Fig. 15.2). This collective action means that each mechanism need not work perfectly nor apply to every possible self-reactive cell. Self-tolerance begins during lymphocyte development, when autoreactive T cells in the thymus and B cells in the bone marrow are deleted or, in the case of CD4 T cells, give rise to a subpopulation of self antigen-reactive FoxP3+ ‘natural’ or ‘thymic’ Treg cells that suppress autoimmune responses after exiting the

1	marrow are deleted or, in the case of CD4 T cells, give rise to a subpopulation of self antigen-reactive FoxP3+ ‘natural’ or ‘thymic’ Treg cells that suppress autoimmune responses after exiting the thymus. Mechanisms of peripheral tolerance, such as anergy and deletion, or the extrathymic production of ‘induced’ or ‘peripheral’ Treg cells, complement these central tolerance mechanisms for antigens that are not expressed in the thymus or bone marrow. Weakly self-reactive lymphocytes are not removed in the primary lymphoid tissues (thymus and bone marrow), as deletion of weakly autoreactive cells would impose too great a limitation on the immune repertoire, resulting in impaired immune responses to pathogens. Instead, weakly self-reactive cells are suppressed only if they are activated in the periphery, and the mechanisms that suppress them include inhibition by T cells, which are themselves autoreactive, although not pathogenic. T cells can inhibit self-reactive lymphocytes if the

1	in the periphery, and the mechanisms that suppress them include inhibition by T cells, which are themselves autoreactive, although not pathogenic. T cells can inhibit self-reactive lymphocytes if the regulatory cells are targeting autoantigens located in the same vicinity as the autoantigens to which the self-reactive lymphocytes respond. This allows regulatory cells to home to and suppress sites of autoimmune inflammation. A final mechanism that controls autoimmunity is the natural tendency of immune responses to be self-limited; intrinsic programs in activated lymphocytes make them prone to apoptosis. Activated lymphocytes also acquire sensitivity to external apoptosis-inducing signals, such as those mediated by Fas.

1	Autoimmune diseases and pathogenic mechanisms.

1	Here we describe some common clinical autoimmune syndromes, and ways in which loss of self-tolerance can generate self-reactive lymphocytes that cause tissue damage. The mechanisms of pathogenesis resemble in many ways those that target invading pathogens. Damage by autoantibodies, mediated through the complement and Fc receptor systems, has an important role in some diseases, such as SLE. Similarly, cytotoxic T cells directed at self tissues destroy them much as they would virus-infected cells; this is one way by which pancreatic β cells are destroyed in diabetes. However, unlike most pathogens, self proteins are not typically eliminated, so that, with rare exceptions—such as the islet cells in the pancreas—the response persists chronically. Some pathogenic mechanisms are unique to autoimmunity, such as antibodies against cell-surface receptors that affect their function, as in myasthenia gravis. In this part of the chapter we describe the pathogenic mechanisms of some major

1	to autoimmunity, such as antibodies against cell-surface receptors that affect their function, as in myasthenia gravis. In this part of the chapter we describe the pathogenic mechanisms of some major autoimmune diseases.

1	15-8 Specific adaptive immune responses to self antigens can cause autoimmune disease.

1	In certain genetically susceptible strains of experimental animals, autoimmune disease can be induced by the injection of ‘self’ tissues that were taken from a genetically identical animal and mixed with strong adjuvants (see Appendix I, Section A-1). This shows directly that autoimmunity can be provoked by inducing a specific adaptive immune response to self antigens. Such experimental systems highlight the importance of the activation of other components of the immune system, primarily dendritic cells, by bacteria contained in the adjuvant. There are drawbacks to the use of such animal models for the study of autoimmunity, however. In humans and genetically autoimmune-prone animals, autoimmunity usually arises spontaneously: that is, we do not know what events initiate the immune response to self that leads to autoimmune disease. By studying the patterns of autoantibodies and particular tissues affected, it has been possible to identify some of the self antigens that are targets of

1	to self that leads to autoimmune disease. By studying the patterns of autoantibodies and particular tissues affected, it has been possible to identify some of the self antigens that are targets of autoimmune disease, although it has yet to be proven that the immune reaction was initiated in response to these same antigens.

1	Some autoimmune disorders may be triggered by infectious agents that express an epitope resembling a self antigen found in a tissue and that lead to sensitization of the patient against that tissue. There is, however, also evidence from animal models of autoimmunity that some autoimmune disorders are caused by internal dysregulation of the immune system without the apparent participation of infectious agents. 15-9 Autoimmunity can be classified into either organ-specific or systemic disease.

1	The classification of disease is an uncertain science, especially in the absence of a precise understanding of causative mechanisms. This is well illustrated by the difficulty in classifying autoimmune diseases. From a clinical perspective it is often useful to distinguish between the following two major patterns of autoimmunity: diseases restricted to specific organs of the body, known as ‘organ-specific’ autoimmune diseases; and those in which many tissues of the body are affected, the ‘systemic’ autoimmune diseases. In both types, disease has a tendency to become chronic because, with a few notable exceptions (for example, Hashimoto’s thyroiditis), autoantigens are rarely cleared from the body. Some autoimmune diseases seem to be dominated by the pathogenic effects of a particular immune effector pathway, either autoantibodies or effector T cells. However, both of these pathways often contribute to the overall pathogenesis.

1	In organ-specific diseases, autoantigens from one or a few organs are targeted, and disease is limited to those organs. Examples include Hashimoto’s thyroiditis and Graves’ disease, which both predominantly affect the thyroid gland; and type 1 diabetes, which is caused by immune attack on insulin-producing pancreatic β cells. Examples of systemic autoimmune disease are SLE and primary Sjögren’s syndrome, in which tissues as diverse as the skin, kidneys, and brain may all be affected (Fig. 15.10).

1	The autoantigens recognized in these two categories of disease are themselves organ-specific and systemic, respectively. Thus, Graves’ disease is characterized by the production of antibodies against the thyroid-stimulating hormone (TSH) receptor, Hashimoto’s thyroiditis by antibodies against thyroid peroxidase, and type 1 diabetes by anti-insulin antibodies. By contrast, SLE is characterized by the presence of antibodies against antigens that are ubiquitous and abundant in every cell of the body, such as chromatin and the proteins of the pre-mRNA splicing machinery—the spliceosome complex. A strict separation of diseases into organ-specific and systemic categories does, however, break down to some extent, because not all autoimmune diseases can be usefully classified in this manner. For example, autoimmune hemolytic anemia, in which red blood cells are destroyed, sometimes occurs

1	Diseases that tend to occur in clusters are grouped in single boxes. Clustering is defined as more than one disease affecting a single patient or different members of a family. Not all autoimmune diseases can be classified according to this scheme. For example, autoimmune hemolytic anemia can occur in isolation or in association with systemic lupus erythematosus. as a solitary entity and could be classified as an organ-specific disease. In other circumstances it can occur in conjunction with SLE as part of a systemic autoimmune disease.

1	A prevalent variant of chronic inflammatory disease is inflammatory bowel disease (IBD), which includes two main clinical entities—Crohn’s disease (discussed later in this chapter) and ulcerative colitis. We discuss IBD in this chapter because it has many features of an autoimmune disease, even though it is not primarily targeted against self-tissue antigens. Instead, the targets of the dysregulated immune response in IBD are antigens derived from the commensal microbiota resident in the intestines. Strictly speaking, therefore, IBD is an outlier among autoimmune diseases in that the immune response is not directed against ‘self’ antigens; rather, it is directed against microbial antigens of the resident, or ‘self,’ microbiota. Nevertheless, features of immune tolerance breakdown are also seen in IBD, and, as with the organ-specific autoimmune diseases, the tissue destruction wrought by the aberrant immune response is primarily localized to a single organ—the intestines.

1	15-10 Multiple components of the immune system are typically recruited in autoimmune disease. Immunologists have long been concerned with which parts of the immune system are important in different autoimmune syndromes, because this can be useful in understanding disease etiology and developing therapies. In myasthenia gravis, for example, autoantibodies produced against the acetylcholine receptor block receptor function at the neuromuscular junction, resulting in a syndrome of muscle weakness. In other autoimmune conditions, antibodies in the form of immune complexes are deposited in tissues and cause tissue damage as a consequence of the inflammation that results from complement activation and ligation of Fc receptors on inflammatory cells.

1	Relatively common autoimmune diseases in which effector T cells seem to be the main destructive agents include type 1 diabetes, psoriasis, IBD, and multiple sclerosis. In these diseases, T cells recognize self peptides or peptides derived from the commensal microbiota that are complexed with self MHC molecules. The damage in such diseases is caused by T cells recruiting and activating myeloid cells of the innate immune system to cause local inflammation, or by direct T-cell damage to tissue cells.

1	When disease can be transferred from a diseased individual to a healthy one by transferring autoantibodies and/or self-reactive T cells, this both confirms that the disease is autoimmune in nature and also proves the involvement of the transferred material in the pathological process. In myasthenia gravis, serum from affected patients can transfer symptoms to animal recipients, thus proving the pathogenic role of the anti-acetylcholine autoantibodies (Fig. 15.11). Similarly, in the animal model disease EAE, T cells from affected animals can transfer disease to normal animals (Fig. 15.12).

1	Fig. 15.11 Identification of autoantibodies that can transfer disease in patients with myasthenia gravis. Autoantibodies from the serum of patients with myasthenia gravis immunoprecipitate the acetylcholine receptor from lysates of skeletal muscle cells (right‑hand panels). Because the antibodies can bind to both the murine and the human acetylcholine receptor, they can transfer disease when injected into mice (bottom panel). This experiment demonstrates that the antibodies are pathogenic. However, to be able to produce antibodies, the same patients should also have CD4 T cells that respond to a peptide derived from the acetylcholine receptor. To detect them, T cells from patients with myasthenia gravis are isolated and grown in the presence of the acetylcholine receptor plus antigen‑presenting cells of the correct MHC type (left‑hand panels). T cells specific for epitopes of the acetylcholine receptor are stimulated to proliferate and can thus be detected.

1	Disease can be transmitted by transfer of T cells from affected animal Mice injected with myelin basic protein and complete Freund's adjuvant develop EAE and are paralyzed The disease is mediated by TH17 and TH1 cells specifc for myelin basic protein TNF-˜IFN-°paralysisparalysisMouse after induction of EAE (left), compared with normal healthy mouse IL-17TH17TH1

1	Fig. 15.12 T cells specific for myelin basic protein mediate can also cause these disease symptoms. Inflammation of the brain inflammation of the brain in experimental autoimmune and paralysis are mediated by TH1 and TH17 cells specific for MBP. encephalomyelitis (EAE). This disease is produced in experimental Cloned MBP‑specific TH1 cells can transfer symptoms of EAE to animals by injecting them with isolated spinal cord homogenized in naive recipients provided that the recipients carry the correct MHC complete Freund’s adjuvant. EAE is due to an inflammatory reaction allele. In this system it has therefore proved possible to identify the in the brain that causes a progressive paralysis affecting first the tail peptide:MHC complex recognized by the TH1 clones that transfer and hind limbs (as shown in the mouse on the left of the photograph, disease. Other purified components of the myelin sheath can also compared with a healthy mouse on the right) before progressing induce the

1	limbs (as shown in the mouse on the left of the photograph, disease. Other purified components of the myelin sheath can also compared with a healthy mouse on the right) before progressing induce the symptoms of EAE, so there is more than one autoantigen to forelimb paralysis and eventual death. One of the autoantigens in this disease. Photograph from Wraith, D., et al.: Cell 1989, identified in the spinal cord homogenate is myelin basic protein 59:247–255. With permission from Elsevier. (MBP). Immunization with MBP alone in complete Freund’s adjuvant

1	Pregnancy can demonstrate a role for antibodies in disease, as IgG antibodies, but not T cells, can cross the placenta (see Section 10-15). For some autoimmune diseases (Fig. 15.13), transmission of autoantibodies across the placenta leads to disease in the fetus or neonate (Fig. 15.14). This provides proof in humans that autoantibodies cause some of the symptoms of autoimmunity. The symptoms of disease in the newborn infant typically disappear rapidly as

1	Fig. 15.13 Some autoimmune diseases that can be transferred across the placenta by pathogenic IgG autoantibodies. These diseases are caused mostly by autoantibodies against cell‑surface or tissue‑matrix molecules. This suggests that an important factor determining whether an autoantibody that crosses the placenta causes disease in the fetus or newborn baby is the accessibility of the antigen to the autoantibody. Autoimmune congenital heart block is caused by fibrosis of the developing cardiac conducting tissue, which expresses abundant Ro antigen. Ro protein is a constituent of an intracellular small cytoplasmic ribonucleoprotein. It is not yet known whether it is expressed at the cell surface of cardiac conducting tissue to act as a target for autoimmune tissue injury. Nevertheless, autoantibody binding leads to tissue damage and results in slowing of the heart rate (bradycardia).

1	Fig. 15.14 Antibody-mediated autoimmune diseases can is little lasting damage because the symptoms disappear along with appear in the infants of affected mothers as a consequence the maternal antibody. In Graves’ disease, the symptoms are caused of transplacental antibody transfer. In pregnant women, IgG by antibodies against the thyroid‑stimulating hormone receptor antibodies cross the placenta and accumulate in the fetus before (TSHR). Children of mothers making thyroid‑stimulating antibody are birth (see Fig. 10.30). Babies born to mothers with IgG‑mediated born with hyperthyroidism, but this can be corrected by replacing autoimmune disease therefore frequently show symptoms similar to the plasma with normal plasma (plasmapheresis), thus removing the those of the mother in the first few weeks of life. Fortunately, there maternal antibody.

1	the maternal antibody is catabolized, but in some cases the antibodies cause organ injury before they are removed, such as damage to the conducting tissue of the heart in babies of mothers with SLE or Sjögren’s syndrome. Antibody clearance can be accelerated by exchange of the infant’s blood or plasma (plasmapheresis), although this is not useful after permanent injury has occurred. Figure 15.15 lists a selection of autoimmune diseases, along with the parts of the immune response that contribute to their pathogenesis. However, although the diseases noted above are clear examples that a particular effector function can drive disease, most autoimmune diseases are not caused solely by a single effector pathway. It is more useful to consider autoimmune responses, like immune responses to pathogens, as engaging the integrated immune system and typically involving T, B, and innate immune cells. Indeed,

1	Pathogenic Present, but role unclear Pathogenic Present, but role unclear T cells B cells AntibodyDisease Pathogenic Help for antibody Pathogenic Present antigen to T cells Present antigen to T cells Help for antibody Antibody secretion Pathogenic Present antigen to T cells Systemic lupus erythematosus Type 1 diabetes Myasthenia gravis Multiple sclerosis Autoimmune diseases involve all aspects of the immune response Fig. 15.15 Autoimmune diseases involve all aspects of the immune response.

1	Fig. 15.15 Autoimmune diseases involve all aspects of the immune response. Although some autoimmune diseases have traditionally been thought to be mediated by B cells or T cells, it is useful to consider that, typically, all aspects of the immune system have a role. For four important autoimmune diseases, the figure lists the roles of T cells, B cells, and antibody. In some diseases, such as SLE, T cells can have multiple roles such as helping B cells to make autoantibody and directly promoting tissue damage. B cells can have two roles as well—presenting autoantigens to stimulate T cells and secreting pathogenic autoantibodies.

1	although autoimmunity research has traditionally focused on identification of the antigen specificity and effector subclass of autoreactive T and B cells, experimental evidence shows that cells of the innate immune system—particularly phagocytic myeloid cells—are critical in mediating tissue damage in most autoimmune diseases. Innate lymphoid cells (ILCs) have also been found in autoimmune lesions, especially those at barrier surfaces. However, the exact role of ILCs, and whether they may be good therapeutic targets in autoimmune disorders, is currently unclear. feedback from inflammation, inability to clear the self antigen, and a broadening of the autoimmune response.

1	feedback from inflammation, inability to clear the self antigen, and a broadening of the autoimmune response. When normal immune responses are engaged to destroy a pathogen, the typical outcome is elimination of the foreign invader, after which the immune response ceases, accompanied by mass extinction of most effector cells and persistence of a small cohort of memory lymphocytes (see Chapter 11). In autoimmunity, however, the self antigen cannot be easily eliminated, because it is in vast excess or is ubiquitous (as is, for example, chromatin). Thus, a very important mechanism for limiting an immune response is abrogated in many autoimmune diseases.

1	In general, autoimmune diseases are characterized by an early activation phase with the involvement of only a few autoantigens, followed by a chronic stage. The constant presence of autoantigen leads to chronic inflammation. This leads to the release of more autoantigens as a result of tissue damage, and this breaks an important barrier to autoimmunity known as ‘sequestration,’ by which many self antigens are normally kept apart from the immune system. It also leads to the attraction of nonspecific effector cells such as macrophages and neutrophils that respond to the release of cytokines and chemokines from injured tissues (Fig. 15.16). The result is a continuing and evolving self-destructive process.

1	B cell is activated by a T cell specifc for self peptide B cells differentiate into plasma cells, secreting large amounts of self antigen-specifc antibody Circulating B cell binds self antigens released from injured cells At sites of injury, self antigen-specifc antibody initiates an infammatory response, causing more cell injury More B cells bind self antigens, amplifying the cycle of tissue damage Fig. 15.16 Autoantibody-mediated inflammation can lead to the release of autoantigens from damaged tissues, which in turn promotes further activation of autoreactive B cells. Autoantigens, particularly intracellular ones that are targets in SLE, stimulate B cells only when released from dying cells (first panel). The result is the activation of autoreactive T and B cells and the eventual secretion of autoantibodies (second and third panels).

1	These autoantibodies can mediate tissue damage through a variety of effector functions (see Chapter 10), resulting in the further death of cells (fourth panel). A positive feedback loop is established because these additional autoantigens recruit and activate additional autoreactive B cells (fifth panel), which in turn can start the cycle over again, as shown in the first panel.

1	Progression of the autoimmune response is often accompanied by recruitment of new clones of lymphocytes reactive to new epitopes on the initiating autoantigen, as well as new autoantigens. This phenomenon is known as epitope spreading, and is important in perpetuating and amplifying disease. As seen in Chapter 10, activated B cells can internalize cognate antigens by receptor-mediated endocytosis via their antigen receptor, process them, and present the derived peptides to T cells. Epitope spreading can occur in several ways. Because antibody-bound antigens can be more efficiently presented, self antigens that are normally present in concentrations too low to activate naive cell processing of the internalized autoantigen can reveal novel, previously hidden, peptide epitopes called cryptic epitopes that the B cell can then present to T cells. Autoreactive T cells responding to these ‘new’ epitopes will provide help to any B cells presenting these peptides, recruiting additional B-cell

1	epitopes that the B cell can then present to T cells. Autoreactive T cells responding to these ‘new’ epitopes will provide help to any B cells presenting these peptides, recruiting additional B-cell clones to the autoimmune reaction, with the consequent production of a greater variety of autoantibodies. In addition, on binding and internalizing specific antigen via their B-cell receptor, B cells will also internalize any other molecules closely associated with that antigen. By these routes, B cells can act as antigen-presenting cells for peptides derived from antigens completely different from the original autoantigen that initiated the autoimmune reaction.

1	The autoantibody response in SLE initiates these mechanisms of epitope spreading. In this disease, autoantibodies against both the protein and DNA components of chromatin are found. Figure 15.17 shows how autoreactive B cells specific for DNA can recruit autoreactive T cells specific for histone proteins, another component of chromatin, into the autoimmune response. In turn, these T cells provide help not only to the original DNA-specific B cells but also to histone-specific B cells, resulting in the production of both anti-DNA and anti-histone antibodies. Another autoimmune disease in which epitope spreading is linked to the progression of disease is pemphigus vulgaris, which is characterized by severe blistering of the skin and mucosal membranes. It is caused by autoantibodies against desmogleins, a type of cadherin present in cell junctions (desmosomes) that hold cells of the epidermis together (Fig. 15.18). Binding of

1	Fig. 15.17 Epitope spreading occurs when B cells specific for various components of a complex antigen are stimulated by an autoreactive helper T cell of a single specificity. In patients with SLE, an ever‑broadening immune response is made against nucleoprotein antigens such as nucleosomes, which consist of histones and DNA and are released from dying and disintegrating cells. The upper panel shows how the emergence of a single clone of autoreactive CD4 T cells can lead to a diverse B‑cell response to nucleosome components. The T cell in the center is specific for a particular peptide (red) from the linker histone H1, which is present on the surface of the nucleosome. The B cells at the top are specific for epitopes on the surface of a nucleosome, on H1 and DNA, respectively, and thus bind and endocytose intact nucleosomes, process the constituents, and present the H1 peptide to the helper T cell. Such B cells will be activated to make antibodies, which in the case of the DNA‑specific

1	and endocytose intact nucleosomes, process the constituents, and present the H1 peptide to the helper T cell. Such B cells will be activated to make antibodies, which in the case of the DNA‑specific B cell will be anti‑DNA antibodies. The B cell at the bottom right is specific for an epitope on histone H2, which is hidden inside the intact nucleosome and is thus inaccessible to the B‑cell receptor. This B cell does not bind the nucleosome and does not become activated by the H1‑specific helper T cell. A B cell specific for another type of nucleoprotein particle, the ribosome (which is composed of RNA and specific ribosomal proteins), will not bind nucleosomes (bottom left) and will not be activated by the T cell. In reality a T cell interacts with one B cell at a time, but different members of the same T‑cell clone will interact with B cells of different specificity. The lower panel shows the broadening of the T‑cell response to the nucleosome. The H1‑specific B cell in the center has

1	of the same T‑cell clone will interact with B cells of different specificity. The lower panel shows the broadening of the T‑cell response to the nucleosome. The H1‑specific B cell in the center has processed an intact nucleosome and is presenting a variety of nucleosome‑derived peptide antigens on its MHC class II molecules. This B cell can activate a T cell specific for any of these peptide antigens, which will include those from the internal histones H2, H3, and H4 as well as those from H1. This H1‑specific B cell will not activate T cells specific for peptide antigens of ribosomes because ribosomes do not contain histones.

1	autoantibodies to the extracellular domains of these adhesion molecules causes dissociation of the junctions and dissolution of the affected tissue. Pemphigus vulgaris usually starts with lesions in the oral and genital mucosa; only later does the skin become involved. In the mucosal stage, only autoantibodies against certain epitopes on desmoglein Dsg-3 are found, and these antibodies seem unable to cause skin blistering. Progression to the skin disease is associated both with intramolecular epitope spreading within Dsg-3, which gives rise to autoantibodies that can cause deep skin blistering, and with intermolecular epitope spreading to another desmoglein, Dsg-1, which is more abundant in the epidermis. Dsg-1 is also the autoantigen in a less severe variant of the disease, pemphigus foliaceus. In that disease, the autoantibodies first produced against Dsg-1 cause no damage, and disease appears only after autoantibodies are made against epitopes on parts of the protein involved in

1	In that disease, the autoantibodies first produced against Dsg-1 cause no damage, and disease appears only after autoantibodies are made against epitopes on parts of the protein involved in the adhesion of epidermal cells.

1	15-12 Both antibody and effector T cells can cause tissue damage in autoimmune disease. The manifestations of autoimmune disease are caused by effector mechanisms of the immune system being directed at the body’s own tissues. As discussed previously, the response can be amplified and maintained by the constant supply of new autoantigen. An exception to this rule is type 1 diabetes, in which the autoimmune response destroys most or all of the target cells. This leads to a failure to produce sufficient insulin to maintain glucose homeostasis, resulting in the symptoms of diabetes.

1	Historically, the mechanisms of tissue injury in autoimmunity have been classified according to a scheme adopted for ‘hypersensitivity’ reactions that were defined in the early 1960s, prior to a more modern understanding of immune mechanisms (Fig. 15.19; also see introduction to Chapter 14). We now recognize that the dominant types of immunity that are orchestrated for the clearance of different types of pathogens are the same ones that become dysregulated in autoimmunity, and that both B and T cells, as well as effector cells of the innate immune system, contribute—even in cases where a particular type of response (for example, autoantibody-mediated cellular injury) predominates in causing tissue damage. The antigen, or group of antigens, against which the autoimmune response is directed, and the mechanism by which the antigen-bearing tissue is damaged, together determine the pathology and clinical expression of the disease.

1	Type 2 immune responses mediated by IgE (previously referred to as type I hypersensitivity) typically cause allergic or atopic inflammatory disease (see Chapter 14) and play no major part in most forms of autoimmunity. By contrast, autoimmunity that damages tissues by autoantibodies—whether by binding of IgG or IgM to autoantigens located on cell surfaces or extracellular matrix (type II hypersensitivity) or by tissue localization of immune complexes composed of soluble autoantigens and their cognate autoantibodies (type III hypersensitivity)—often appears to be linked to dysregulated type 3 (TH17) or type 1 (TH1) immunity, or to T-cell-independent generation of IgM-producing B cells. Because antibody-mediated injury can target a specific cell or tissue type (for example, autoimmune thyroiditis), or it can result in immune complexes that are deposited in specific vascular beds (for example, rheumatoid arthritis), disease can be organ-specific or systemic. In some forms of

1	thyroiditis), or it can result in immune complexes that are deposited in specific vascular beds (for example, rheumatoid arthritis), disease can be organ-specific or systemic. In some forms of autoimmunity, such as SLE, autoantibodies cause damage by both of these mechanisms. Finally, several organ-specific autoimmune diseases are due to a type 1 response in which TH1 cells and/or cytotoxic T cells directly cause tissue damage (type IV hypersensitivity; for example, type 1 diabetes); alternatively, some such diseases are due to a type 3 response in which TH17 cells promote inflammation at barrier tissues (for example, psoriasis or Crohn’s disease).

1	Antibodies made against Dsg-1 and Dsg-3 unzip the adhesive interactions in desmosomes EC1EC2EC3EC4EC5Dsg-3 EC1EC2EC3EC4EC5Dsg-1 keratinocyte extracellular matrix early epitope targeted in mucosal stage of disease late epitope targeted in skin stage of disease Different B-cell epitopes on desmogleins are targeted by autoantibodies Fig. 15.18 Pemphigus vulgaris is a skin-blistering disease caused by autoantibodies specific for desmoglein.

1	Fig. 15.18 Pemphigus vulgaris is a skin-blistering disease caused by autoantibodies specific for desmoglein. An adhesion molecule in the cell junctions that hold keratinocytes together, desmoglein is a cell‑surface protein with five extracellular domains (EC1–EC5; upper panel). Early in the autoimmune response, antibodies are made against the EC5 domain of shed desmoglein‑3 (Dsg‑3), but do not cause disease. However, in time, intra‑and intermolecular epitope spreading occurs and IgG antibodies are made against the EC1 and EC2 domains of Dsg‑3 and Dsg‑1. These autoantibodies can inhibit adhesion of desmoglein in desmosomes (lower panel), and thereby interfere with the physiological adhesive interactions of desmoglein that are necessary for maintaining skin integrity. Consequently, the antibodies cause the outer layers of the skin to separate, producing blisters.

1	Autoimmune diseases can be grouped according to the predominant type of immune response and the mechanism by which it damages tissues. In many autoimmune diseases, several immunopathogenic mechanisms operate in parallel. This is illustrated here for rheumatoid arthritis, which appears in more than one category of immunopathogenic mechanism.

1	In most autoimmune diseases, however, several mechanisms of pathogenesis operate. Thus, helper T cells are almost always required for the production of pathogenic autoantibodies. Reciprocally, B cells often have an important role in the maximal activation of T cells that mediate tissue damage or help auto-antibody production. In type 1 diabetes and rheumatoid arthritis, for example, both T-celland antibody-mediated pathways cause tissue injury. SLE is an example of autoimmunity that was previously thought to be mediated solely by antibodies and immune complexes but is now known to have a component of T-cell-mediated pathogenesis. Moreover, in virtually all autoimmune diseases, innate immune cells contribute to inflammation and antibodyor T-cell-mediated tissue injury. We will first examine how autoantibodies cause tissue damage, then consider self-reactive T-cell responses and their role in autoimmunity. 15-13 Autoantibodies against blood cells promote their destruction.

1	15-13 Autoantibodies against blood cells promote their destruction. IgG or IgM responses to antigens located on the surface of blood cells lead to the rapid destruction of these cells. An example of this is autoimmune hemolytic anemia, in which antibodies against self antigens on red blood cells trigger destruction of the cells, leading to anemia. This can occur in two ways (Fig. 15.20). Red cells with bound IgG or IgM antibody can be rapidly cleared from the circulation by interaction with Fc or complement receptors, respectively, on cells of the mononuclear–macrophage phagocytic system, particularly in the spleen. Alternatively, the autoantibody-sensitized red blood cells can be lysed by formation of the membrane-attack complex of complement. In autoimmune thrombocytopenic purpura, autoantibodies against the GpIIb:IIIa fibrinogen receptor or other platelet-specific surface antigens can cause thrombocytopenia (a depletion of platelets), which can in turn cause hemorrhage.

1	Lysis of nucleated cells by complement is less common because these cells are better defended by complement-regulatory proteins, which protect cells against immune attack by interfering with the activation of complement components (see Section 2-15). Nevertheless, circulating nucleated cells targeted by autoantibodies are still destroyed by cells of the mononuclear phagocytic system or NK cells via antibody-dependent cell-mediated cytotoxicity (ADCC). Autoantibodies against neutrophils, for example, cause neutropenia, which increases susceptibility to infection with pyogenic bacteria. In all these cases, accelerated clearance of autoantibody-sensitized cells is the cause of their depletion. One therapeutic approach to this type of autoimmunity is removal of the spleen, the organ in which the main clearance of red cells, platelets, and leukocytes occurs. Another is the administration of large quantities of nonspecific IgG (termed IVIG, for intravenous immunoglobulin), which among other

1	main clearance of red cells, platelets, and leukocytes occurs. Another is the administration of large quantities of nonspecific IgG (termed IVIG, for intravenous immunoglobulin), which among other mechanisms inhibits the Fc receptor-mediated uptake of antibody-coated cells and activates inhibitory Fc receptors to suppress production of inflammatory mediators by myeloid cells.

1	15-14 The fixation of sublytic doses of complement to cells in tissues stimulates a powerful inflammatory response. The binding of IgG and IgM antibodies to cells in tissues causes inflammatory injury by a variety of mechanisms. One of these is the fixation of complement. Although nucleated cells are relatively resistant to lysis by complement, the assembly of sublytic amounts of the membrane-attack complex on their surface provides a powerful activating stimulus. Depending on the cell type, this interaction can cause cytokine release, a respiratory burst, or the mobilization of membrane phospholipids to generate arachidonic acid—the precursor of prostaglandins and leukotrienes, lipid mediators of inflammation.

1	Most cells in tissues are fixed in place, and innate and adaptive immune cells are attracted to them by chemoattractant molecules. One such molecule is the complement fragment C5a, which is released as a result of complement activation triggered by autoantibody binding. Other chemoattractants, such as leukotriene B4, can be released by the autoantibody-targeted cells. Inflammatory leukocytes are further activated by binding to autoantibody Fc regions and fixed complement C3 fragments on cells. Tissue injury can result from the products of the activated leukocytes and by antibody-dependent cellular cytotoxicity mediated by NK cells (see Section 10-23). A probable example of this type of autoimmunity is Hashimoto’s thyroiditis, in which autoantibodies against tissue-specific antigens are found at extremely high levels for prolonged periods. Direct T-cell-mediated cytotoxicity, which we discuss later, is probably also important in this disease.

1	Fig. 15.20 Antibodies specific for cell-surface antigens can destroy cells. In autoimmune hemolytic anemias, red blood cells (erythrocytes) coated with IgG autoantibodies against a cell‑surface antigen (upper panel) are rapidly cleared from the circulation by uptake by Fc receptor‑bearing macrophages located primarily in the spleen (lower left panel). Erythrocytes coated with IgM autoantibodies fix C3 and are cleared by CR1‑bearing macrophages. The binding of certain rare autoantibodies that fix complement extremely efficiently causes the formation of the membrane‑attack complex on the red cells, leading to intravascular hemolysis (lower right panel).

1	Fig. 15.21 Feedback regulation of thyroid hormone production is disrupted in Graves’ disease. Graves’ disease is caused by autoantibodies specific for the receptor for thyroid‑stimulating hormone (TSH). Normally, thyroid hormones are produced in response to TSH and limit their own production by inhibiting the production of TSH by the pituitary (left panels). In Graves’ disease, the autoantibodies are agonists for the TSH receptor and therefore stimulate the production of thyroid hormones (right panels). The thyroid hormones inhibit TSH production in the normal way but do not affect production of the autoantibody; the excessive thyroid hormone production induced in this way causes hyperthyroidism. 15-15 Autoantibodies against receptors cause disease by stimulating or blocking receptor function.

1	15-15 Autoantibodies against receptors cause disease by stimulating or blocking receptor function. Autoimmune disease can occur when autoantibody binds a cell-surface receptor. Antibody binding to a receptor can either stimulate the receptor or block stimulation by the natural ligand. In Graves’ disease, autoantibodies against the thyroid-stimulating hormone (TSH) receptor on thyroid cells stimulate excessive production of thyroid hormone. The production of thyroid hormone is normally controlled by feedback regulation; high levels of thyroid hormone inhibit the release of TSH by the pituitary. In Graves’ disease, feedback inhibition fails because the autoantibody continues to stimulate the TSH receptor in the absence of TSH, and the patient produces chronically elevated levels of thyroid hormone (‘hyperthyroidism’; Fig. 15.21).

1	In myasthenia gravis, autoantibodies against the α chain of the nicotinic acetylcholine receptor present at neuromuscular junctions in skeletal muscle cells can block stimulation of muscle contraction. The antibodies are believed to drive internalization and degradation of the receptor (Fig. 15.22). Fig. 15.22 Autoantibodies inhibit receptor function in myasthenia gravis. In normal circumstances, acetylcholine released from stimulated motor neurons at the neuromuscular junction binds to acetylcholine receptors on skeletal muscle cells, triggering muscle contraction (upper panel). Myasthenia gravis is caused by autoantibodies against the α subunit of the receptor for acetylcholine. These autoantibodies bind to the receptor without activating it and also cause receptor internalization and degradation (lower panel). As the number of receptors on the muscle is decreased, the muscle becomes less responsive to acetylcholine.

1	Fig. 15.23 Autoimmune diseases caused by autoantibodies against cell-surface receptors. These antibodies produce different effects depending on whether they are agonists (which stimulate the receptor) or antagonists (which inhibit it). Note that different autoantibodies against the insulin receptor can either stimulate or inhibit signaling. Patients with myasthenia gravis develop potentially fatal progressive weakness as a result of their disease. Diseases caused by autoantibodies that act as agonists or antagonists on cell-surface receptors are listed in Fig. 15.23. 15-16 Autoantibodies against extracellular antigens cause inflammatory injury.

1	15-16 Autoantibodies against extracellular antigens cause inflammatory injury. Antibody responses to extracellular matrix molecules are infrequent, but can be very damaging. In Goodpasture’s syndrome, antibodies are formed against the α3 chain of basement membrane collagen (type IV collagen). These antibodies bind to the basement membranes of renal glomeruli (Fig. 15.24) and, in some cases, to the basement membranes of pulmonary alveoli, causing a rapidly fatal disease if untreated. The autoantibodies bound to basement membrane ligate Fcγreceptors on innate effector cells, such as monocytes and neutrophils, leading to their activation. These cells, in turn, release chemokines that attract a further influx of monocytes and neutrophils into glomeruli, causing severe tissue injury. The autoantibodies also cause a local activation of complement, which amplifies tissue injury.

1	Immune complexes are produced when there is an antibody response to a soluble antigen. Normally, such complexes cause little tissue damage because they are cleared efficiently by red blood cells that bear complement receptors and by phagocytes of the mononuclear phagocytic system that have both complement and Fc receptors. This clearance system can, however, fail in circumstances where the production of immune complexes exceeds the capacity Fig. 15.24 Autoantibodies reacting with glomerular basement membrane cause the inflammatory glomerular disease known as Goodpasture’s syndrome.

1	Upper two panels: schematic of antibody‑mediated damage to a glomerulus of the kidney. The autoantibody binds to type IV collagen within the basement membrane of the glomerular capillaries, causing complement activation and recruitment and activation of neutrophils and monocytes. Third panel: section of renal glomerulus in biopsy taken from the kidney of a patient with Goodpasture’s syndrome. The glomerulus is stained for IgG deposition by immunofluorescence. Anti‑glomerular basement membrane antibody (stained green) is deposited in a linear fashion along the glomerular basement membrane. Bottom panel: silver staining of a section through a renal glomerulus shows that the glomerulus capillaries (G) are compressed by the formation of a crescent (C), composed of proliferating epithelial cells and an influx of neutrophils (N) and monocytes (M), that have filled the urinary (Bowman's) space surrounding the glomerular capillaries.

1	monocyte or dendritic cell autoreactive B-cell survival Increased autoantibodies BAFF BAFF-R BCMA TACI dsDNA immune complex ssRNA apoptotic cellnecrotic cell IFN-˜IFN-˜plasmacytoid dendritic cell MyD88TLR-7 TLR-9 Nucleic acid-containing immune complexes generated from dying cells activate plasmacytoid DCs to produce IFN-˜IFN-˜stimulates myeloid cells to produce BAFF, which enhances the survival and autoantibody production of autoreactive B cells Fig. 15.25 Defective clearance of nucleic acid-containing immune complexes activates overproduction of BAFF and type I interferons that can cause SLE.

1	of the normal clearance mechanisms, or when there are deficiencies in normal clearance mechanisms. An example of the former is serum sickness (see Section 14-15), which is caused by the injection of large amounts of serum proteins or by small-molecule drugs binding to serum proteins and acting as haptens. Serum sickness is a transient disease, lasting until the immune complexes have been cleared. Similarly, normal clearance mechanisms can be overwhelmed in chronic infections, such as bacterial endocarditis, in which the immune response to bacteria lodged on a cardiac valve is incapable of clearing the infection. The persistent release of bacterial antigens from the valve infection in the presence of a strong antibacterial antibody response causes widespread immune-complex injury to small blood vessels in organs such as the kidney and the skin. Other chronic infections, such as hepatitis C infection, can lead to the production of cryoglobulins and the condition mixed essential

1	small blood vessels in organs such as the kidney and the skin. Other chronic infections, such as hepatitis C infection, can lead to the production of cryoglobulins and the condition mixed essential cryoglobulinemia, in which immune complexes are deposited in joints and tissues. Alternatively, there can be inherited impairment of mechanisms that contribute to normal clearance of immune complexes; such impairment may be caused by reduced expression of or functional defects in specific components of either complement or its receptors, or in Fc receptors, each of which occurs in subsets of patients with SLE.

1	Indeed, SLE can result from either the overproduction or the defective clearance of immune complexes, or both, at multiple levels (Fig. 15.25). In this disease, there is chronic IgG antibody production directed at ubiquitous self antigens present in nucleated cells, leading to a wide range of autoantibodies against common cellular constituents. The main antigens are three types of intracellular nucleoprotein particles—the nucleosome subunits of chromatin, the spliceosome, and a small cytoplasmic ribonucleoprotein complex containing two proteins known as Ro and La (named after the first two letters of the surnames of the two patients in whom autoantibodies against these proteins were discovered). For these autoantigens to participate in immune-complex formation, they must become extracellular. The autoantigens of SLE are exposed on dead and dying cells released from injured tissues.

1	In SLE, large quantities of antigen are available, so large amounts of small immune complexes are produced continuously and are deposited in the walls of small blood vessels in the renal glomerular basement membrane, joints, and other organs (Fig. 15.26). This leads to the activation of phagocytic cells through their Fc receptors. A hereditary deficiency of some complement proteins, specifically those for C1q, C2, and C4, is strongly associated with the development of SLE in humans. C1q, C2, and C4 are early components in the classical complement pathway, which is important in antibody-mediated clearance of apoptotic cells and immune complexes (see Chapter 2). If apoptotic cells and immune complexes are not cleared, the chance that their antigens will activate low-affinity self-reactive lymphocytes in the periphery is increased. The consequent tissue damage releases more nucleoprotein complexes, which in turn form more immune complexes. During this process, autoreactive T cells also

1	lymphocytes in the periphery is increased. The consequent tissue damage releases more nucleoprotein complexes, which in turn form more immune complexes. During this process, autoreactive T cells also become activated, although much less is known about their specificity. Animal models for SLE cannot be initiated without the help of T cells, and T cells can also be directly pathogenic, forming part of the cellular infiltrates in the skin and kidney. As discussed in the next section,

1	In SLE, it is believed that antibody:nucleic acid immune complexes containing, for example, ssRNA or dsDNA from dead cells, are bound by FcγRIIa (green rods) on plasmacytoid dendritic cells. The Fc receptor‑bound ssRNA and dsRNA are delivered to endosomes, where they activate TLR‑7 and TLR‑9, respectively, to induce IFN‑α production (upper panel). IFN‑α increases BAFF production by monocytes and dendritic cells, and BAFF interacts with receptors on B cells. Excess BAFF can increase autoreactive B‑cell survival, leading to increased autoantibody production (lower panel). T cells contribute to autoimmune disease in two ways: by helping B cells make antibodies, analogous to a normal T-dependent immune response; and by direct effector functions as they infiltrate and destroy target tissues. 15-17 T cells specific for self antigens can cause direct tissue injury and sustain autoantibody responses.

1	Traditionally, it has been more difficult for a number of reasons to demonstrate the existence of autoreactive T cells than the presence of autoantibodies. First, autoreactive human T cells cannot transfer disease to experimental animals because T-cell recognition is MHC-restricted. Second, autoantibodies can be used to stain self tissues to reveal distribution of the autoantigen, whereas T cells cannot be used in the same way. However, the use of fluorophore-labeled peptide–MHC tetramers (see Appendix 1, Section A-24) that can stain antigen-specific T cells for flow cytometry is now providing a means to both identify and track autoreactive T cells in vivo in autoimmune diseases. Furthermore, there is already strong evidence for the involvement of autoreactive T cells in many autoimmune diseases. In type 1 diabetes, for example, the insulin-producing βcells of the pancreatic islets are selectively destroyed by cytotoxic T cells. This is borne out by the finding that in the rare cases

1	In type 1 diabetes, for example, the insulin-producing βcells of the pancreatic islets are selectively destroyed by cytotoxic T cells. This is borne out by the finding that in the rare cases in which patients with diabetes were transplanted with half a pancreas from an identical twin donor, the βcells in the grafted tissue were rapidly and selectively destroyed by the recipient’s T cells. Recurrence of disease can be prevented by the immunosuppressive drug cyclosporin A (see Chapter 16), which inhibits T-cell activation.

1	Autoantigens recognized by CD4 T cells can be identified by adding cells or tissues containing autoantigens to cultures of blood mononuclear cells, and testing for recognition by CD4 cells derived from an autoimmune patient. If the autoantigen is present, it should be effectively recognized by autoreactive CD4 T cells. The identification of autoantigenic peptides is particularly difficult in autoimmune diseases in which CD8 T cells have a role, because autoantigens recognized by CD8 T cells are not effectively presented in such cultures. Peptides presented by MHC class I molecules must usually be made by the target cells themselves (see Chapter 6); intact cells of target tissue from the patient must therefore be used to study autoreactive CD8 T cells that cause tissue damage. Conversely, the pathogenesis of the disease can itself give clues to the identity of the antigen in some CD8 T-cell-mediated diseases. For example, in type 1 diabetes, the insulin-producing β cells seem to be

1	the pathogenesis of the disease can itself give clues to the identity of the antigen in some CD8 T-cell-mediated diseases. For example, in type 1 diabetes, the insulin-producing β cells seem to be specifically targeted and destroyed by CD8 T cells (Fig. 15.27). This suggests that a protein unique to β cells is the source of the peptide recognized by the pathogenic CD8 T cells. Studies in the NOD (non-obese diabetic) mouse model of type 1 diabetes have shown that peptides from insulin itself are recognized by pathogenic CD8 T cells, confirming insulin as one of the principal autoantigens in this model of diabetes.

1	Multiple sclerosis (MS) is a T-cell-mediated neurologic disease caused by a destructive immune response against central nervous system myelin antigens, Fig. 15.26 Deposition of immune complexes in the renal glomeruli causes renal failure in systemic lupus erythematosus (SLE). Panel a: a section through a renal glomerulus from a patient with SLE, showing that the deposition of immune complexes has caused thickening of the glomerular basement membrane, seen as the clear ‘canals’ running through the glomerulus. Panel b: a similar section stained with fluorescent antiimmunoglobulin, revealing immunoglobulin deposits in the basement membrane. In panel c, the immune complexes are seen under the electron microscope as dense protein deposits between the glomerular basement membrane and the renal epithelial cells. Polymorphonuclear neutrophilic leukocytes are also present, attracted by the deposited immune complexes. Photographs courtesy of H.T. Cook and M. Kashgarian.

1	Fig. 15.27 Selective destruction of pancreatic β cells in type 1 diabetes indicates that the autoantigen is produced in β cells and recognized on their surface. In type 1 diabetes there is highly specific destruction of insulin‑producing β cells in the pancreatic islets of Langerhans, sparing other islet cell types (α and δ). This is shown schematically in the upper panels. In the lower panels, islets from normal (left) and diabetic (right) mice are stained for insulin (brown), which shows the β cells, and for glucagon (black), which shows the α cells. Note the lymphocytes infiltrating the islet in the diabetic mouse (right) and the selective loss of the β cells (brown), whereas the α cells (black) are spared. The characteristic morphology of the islet is also disrupted with the loss of the β cells. Photographs courtesy of I. Visintin.

1	˜ cell˛ cell˝ cellThe islets of Langerhans contain several cell types secreting distinct hormones. Each cell expresses different tissue-speciÿc proteins glucagoninsulinsomatostatinGlucagon and somatostatin are still produced by the ˜and °cells, but no insulin can be made In type 1 diabetes an effector T cell recognizes peptides from a ˛-cell speciÿc protein and kills the ˛cell CTL including myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG) (Fig. 15.28). MS takes its name from the hard (sclerotic) lesions, or plaques, that develop in the white matter of the central nervous system. These lesions show dissolution of the myelin sheath that normally surrounds nerve cell axons, along with inflammatory infiltrates of lymphocytes and macrophages, particularly surrounding blood vessels. Patients with MS may develop a variety of neurological symptoms, including muscle weakness, ataxia, blindness, and paralysis. Normally, few lymphocytes cross

1	particularly surrounding blood vessels. Patients with MS may develop a variety of neurological symptoms, including muscle weakness, ataxia, blindness, and paralysis. Normally, few lymphocytes cross the blood–brain barrier, but if this barrier breaks down, activated CD4 T cells specific for myelin antigens and expressing α4:β1 integrin can bind vascular cell adhesion molecules (VCAMs) on activated endothelium (see Section 11-3), enabling the T cells to migrate out of the blood vessel. There they reencounter their specific autoantigen presented by MHC class II molecules on infiltrating macrophages or microglial cells (phagocytic macrophage-like cells resident in the central nervous system). Inflammation causes increased vascular permeability, and the site becomes heavily infiltrated by TH17 and TH1 effector CD4 T cells, which produce IL-17, IFN-γ, and GM-CSF. Cytokines and chemokines produced by these effector T cells in turn recruit and activate myeloid cells that exacerbate

1	by TH17 and TH1 effector CD4 T cells, which produce IL-17, IFN-γ, and GM-CSF. Cytokines and chemokines produced by these effector T cells in turn recruit and activate myeloid cells that exacerbate inflammation, resulting in further recruitment of T cells, B cells, and innate immune cells to the lesion. Autoreactive B cells produce autoantibodies against myelin antigens with help from T cells. These combined activities lead to demyelination and interference with neuronal function.

1	The clinical course of MS both mirrors what is seen in other autoimmune diseases and also displays how the tissue specificity of such conditions affects their progression. Most MS patients experience a disease course characterized by acute attacks (relapse) followed by a reduction in disease activity (remission) that may last for months or years. This relapsing–remitting course is characteristic of many autoimmune diseases (besides MS, Crohn’s disease and rheumatoid arthritis, among others), in terms of both the symptoms patients experience and the degree of immune-cell infiltration into the affected organ. Not only are the triggers of relapses not always clear, but the events leading to spontaneous disease remission—even when the autoantigen is still present

1	Infammatory reaction occurs in the brain due to mast-cell activation, complement activation, antibodies, and cytokines in the organ—remain to be discovered. Furthermore, the relapsing–remitting nature of diseases such as MS makes conducting clinical trials around these disorders especially difficult, as they must be performed over relatively long periods of time to ensure a therapy is effective in preventing relapses and disability.

1	Ultimately, often after decades, most MS patients change from a relapsing– remitting disease course to ‘secondary progressive’ MS. In this phase patients begin to undergo a steady neurologic decline without overt periods of remission, and for many patients their disease becomes less responsive to the therapies that effectively target the adaptive immune system in relapsing–remitting MS. The reasons for this are unclear, though it has been suggested that the long-term relapsing–remitting course ultimately exhausts the central nervous system’s regenerative capacity, leading to chronic neurodegeneration. Further, prolonged disease may allow immune cells and activated microglia to remain behind the blood–brain barrier, continuing to promote neuronal damage without the need for continuous recruitment of large numbers of inflammatory cells from the periphery.

1	Rheumatoid arthritis (RA) is a chronic disease characterized by inflammation of the synovium (the thin lining of a joint). As disease progresses, the inflamed synovium invades and damages the cartilage; this is followed by bone erosion (Fig. 15.29), leading to chronic pain, loss of function, and disability. RA was first considered an autoimmune disease driven by B cells producing anti-IgG autoantibodies called rheumatoid factor (see Section 15-4). However, the identification of rheumatoid factor in some healthy individuals, and its absence in some patients with rheumatoid arthritis, suggested that more complex mechanisms orchestrate this pathology. The discovery that RA has an association with particular class II HLA-DR genes of the MHC suggested that T cells are also involved in the pathogenesis of this disease. In RA, as in MS, most data from humans and mouse models indicate that, at least early in disease development, autoreactive TH17 cells become activated. Autoreactive T cells

1	of this disease. In RA, as in MS, most data from humans and mouse models indicate that, at least early in disease development, autoreactive TH17 cells become activated. Autoreactive T cells provide help to B cells to produce arthritogenic antibodies. The activated TH17 cells also produce cytokines that recruit neutrophils and monocytes/macrophages, which, along with endothelial cells and synovial fibroblasts, are stimulated to produce more pro-inflammatory cytokines such as TNF-α, IL-1, or chemokines (CXCL8, CCL2), and finally matrix metalloproteinases, which are responsible for tissue destruction. IL-17A, which has been found in high concentrations in the synovium and synovial fluid of RA patients, can induce expression of the ligand for receptor activator of NFκB (RANKL), which stimulates the differentiation of osteoclast precursors into mature osteoclasts that resorb bone in affected joints. Although we do not yet know how RA starts, mouse models have shown that both T cells and B

1	the differentiation of osteoclast precursors into mature osteoclasts that resorb bone in affected joints. Although we do not yet know how RA starts, mouse models have shown that both T cells and B cells are needed to initiate disease. Interestingly, interrupting

1	Fig. 15.28 The pathogenesis of multiple sclerosis. At sites of inflammation, activated T cells autoreactive for brain antigens can cross the blood–brain barrier and enter the brain, where they reencounter their antigens on microglial cells and secrete cytokines such as IFN‑γ. The production of T‑cell and macrophage cytokines exacerbates the inflammation and induces a further influx of blood cells (including macrophages, dendritic cells, and B cells) and blood proteins (such as complement) into the affected site. Mast cells also become activated. The individual roles of these components in demyelination and loss of neuronal function are still not well understood. CNS, central nervous system.

1	osteoclast joint cartilage MMPs attack tissues. Activation of bone-destroying osteoclasts by RANK ligand results in joint destruction cytokines fbroblasts IL-6 RANK ligandMMP TNF-˜Cytokines induce production of MMP and RANK ligand by fbroblasts Unknown trigger sets up initial focus of infammation in synovial membrane, attracting leukocytes into the tissue Autoreactive CD4 T cells activate macrophages, resulting in production of pro-infammatory cytokines and sustained infammation

1	Fig. 15.29 The pathogenesis of rheumatoid arthritis. Inflammation of the synovial membrane, initiated by some unknown trigger, attracts autoreactive lymphocytes and macrophages to the inflamed tissue. Autoreactive effector CD4 T cells activate macrophages, and pro‑inflammatory cytokines such as IL‑1, IL‑6, IL‑17, and TNF‑α are produced. Fibroblasts activated by cytokines produce matrix metalloproteinases (MMPs), which contribute to tissue destruction. The TNF family cytokine RANK ligand, expressed by T cells and fibroblasts in the inflamed joint, is the primary activator of bone‑destroying osteoclasts. Antibodies against several joint proteins are also produced (not shown), but their role in pathogenesis is uncertain. this complex cascade at multiple levels—including therapeutic antibodies against cytokines (TNF-α), B cells, and T-cell activation—have all been successful in treating the symptoms of the disease (discussed in Section 16-8).

1	Studies of the targets of autoantibodies in RA have yielded insights into how this disease develops, and have also identified a more global mechanism by which self proteins may be seen as foreign in other autoimmune conditions. During inflammation, the amino acid arginine can be converted into citrulline, and this change may result in structural alterations of the self protein that cause the immune system to now view it as nonself (Fig. 15.30). Experimental models have shown that antibodies against these altered proteins can be pathogenic, and diagnostic tests for anti-citrullinated protein antibodies (ACPAs) are highly specific for RA. Interestingly, smoking—long known as the most important environmental risk factor for RA development—has been associated with APCAs in patients with HLA risk alleles, suggesting that this tolerance-breaking mechanism may be an important node in the gene–environment interactions that lead to autoimmunity. Finally, other post-translational modifications

1	alleles, suggesting that this tolerance-breaking mechanism may be an important node in the gene–environment interactions that lead to autoimmunity. Finally, other post-translational modifications (oxidation, glycosylation) of self proteins in the periphery have now been shown to stimulate Tand B-cell responses in other autoimmune diseases.

1	Summary.

1	Autoimmune diseases can be broadly classified into those that affect a specific organ and those that affect tissues throughout the body. Organ-specific autoimmune diseases include type 1 diabetes, multiple sclerosis, Graves’ disease, and Crohn’s disease. In each case the effector functions target autoantigens that are restricted to particular organs—insulin-producing β cells of the pancreas (type 1 diabetes), the myelin sheathing on axons in the central nervous system (multiple sclerosis), and the thyroid-stimulating hormone receptor (Graves’ disease)—or, in the case of Crohn’s disease, components of the intestinal microbiota. In contrast, systemic diseases such as systemic lupus erythematosus (SLE) cause inflammation in multiple tissues because their autoantigens, which include chromatin and ribonucleoproteins, are found in most cells of the body. In some organ-specific diseases, immune destruction of the target tissue and the unique self antigens it expresses leads to cessation of

1	and ribonucleoproteins, are found in most cells of the body. In some organ-specific diseases, immune destruction of the target tissue and the unique self antigens it expresses leads to cessation of autoimmune activity, but systemic diseases tend to be chronically active if untreated, because their autoantigens cannot be cleared. Another way of classifying autoimmune diseases is according to the effector functions that are most important in pathogenesis. It is becoming clear, however, that many diseases once thought to be mediated solely by one effector function actually involve several. In this way, autoimmune diseases resemble pathogen-directed immune responses, which typically elicit the activities of multiple effectors—adaptive and innate.

1	For a disease to be classified as autoimmune, the tissue damage must be shown to be caused by the adaptive immune response to self antigens. Autoinflammatory reactions directed against the commensal microbiota of the intestines, such as those seen in inflammatory bowel diseases (IBDs), are a special case in that the target antigens are not strictly ‘self,’ but are derived from the ‘extended self’ of the intestinal microbiota. IBD, nevertheless, shares immunopathogenic features with other autoimmune diseases. The most convincing proof that the immune response is causal in autoimmunity is the transfer of disease by transferring the active component of the immune response to an appropriate recipient. Autoimmune diseases are mediated by autoreactive lymphocytes and their soluble products, pro-inflammatory cytokines, and autoantibodies responsible for inflammation and tissue injury. A few autoimmune diseases are caused by antibodies that bind to cell-surface receptors, causing either

1	pro-inflammatory cytokines, and autoantibodies responsible for inflammation and tissue injury. A few autoimmune diseases are caused by antibodies that bind to cell-surface receptors, causing either excess activity or inhibition of receptor function. In some diseases, transplacental passage of IgG autoantibodies can cause disease in the fetus and neonate. T cells can be involved directly in inflammation or cellular destruction, and they are typically required to initiate and sustain an autoantibody response. Similarly, B cells are important antigen-presenting cells for sustaining autoantigen-specific T-cell responses and causing epitope spreading. In spite of our knowledge of the mechanisms of tissue damage and the therapeutic approaches that this information has engendered, it remains to be determined how autoimmune responses are induced.

1	The genetic and environmental basis of autoimmunity. Given the complex mechanisms that exist to prevent autoimmunity, it is not surprising that autoimmune diseases are the result of multiple factors, both genetic and environmental. We first discuss the genetic basis of autoimmunity, attempting to understand how genetic defects perturb various tolerance mechanisms. Genetic defects alone are not, however, always sufficient to cause autoimmune disease. Environmental factors also play a part, although these factors are poorly understood. As we shall see, genetic and environmental factors together can overcome tolerance mechanisms and result in disease. 15-18 Autoimmune diseases have a strong genetic component.

1	15-18 Autoimmune diseases have a strong genetic component. It is increasingly clear that some individuals are genetically predisposed to autoimmunity. Perhaps the clearest demonstration of this is found in inbred mouse strains that are prone to various types of autoimmune diseases. Mice of the NOD strain are very likely to get diabetes, with female mice becoming diabetic faster than males (Fig. 15.31). For reasons that are still unclear, many autoimmune diseases are more common in females than in males (see Fig. 15.37 below), with some disorders (SLE and MS) showing a high degree of sexual dimorphism. Autoimmune diseases in humans also have a genetic component. Some autoimmune diseases, including type 1 diabetes, run in families, suggesting a role for genetic susceptibility. Most convincingly, if one identical (monozygotic) twin is affected, the other twin is quite likely to be affected as well, whereas concordance of disease is much less in nonidentical (dizygotic) twins.

1	Environmental influences are also clearly involved. For example, although most members of a colony of NOD mice develop diabetes, they do so at different ages. Moreover, disease onset often differs from one animal colony to the next, even though all the mice are genetically identical. Thus, environmental variables must be, in part, determining the rate of diabetes development in genetically susceptible individuals. Particularly striking is the importance of the intestinal microbiota in the development of IBD in mice that Fig. 15.30 The enzyme peptidyl arginine deiminase converts the arginine residues of tissue proteins to citrulline. In tissues stressed by wounds or infection, peptidyl arginine deiminase (PAD) activity is induced. By converting arginine residues to citrulline, PAD destabilizes proteins and makes them more susceptible to degradation. It also introduces novel B‑cell and T‑cell epitopes into tissue proteins that can stimulate an autoimmune response.

1	Fig. 15.31 Sex differences in the incidence of autoimmune disease. Many autoimmune diseases are more common in females than males, as illustrated here by the cumulative incidence of diabetes in a population of diabetes‑prone NOD mice. Females (red line) get diabetes at a much younger age than do males, indicating their greater predisposition. Data kindly provided by S. Wong. Fig. 15.32 Defects in cytokine production or signaling that can lead to autoimmunity. Some of the signaling pathways involved in autoimmunity have been identified by genetic analysis, mainly in animal models. The effects of overexpression or underexpression of some of the cytokines and intracellular signaling molecules involved are listed here (see the text for further discussion).

1	Incidence of diabetes in NOD mice (%) are genetically predisposed to develop intestinal inflammation. Treatment with broad-spectrum antibiotics that reduce or eliminate many components of the commensal flora can delay or eliminate disease onset, and raising susceptible mice under germ-free conditions (i.e., without a microbiota) eliminates disease. Conversely, certain intestinal microbes—such as segmented filamentous bacteria (SFB)—present in some mouse colonies promote intestinal TH17 responses that have been linked to intestinal inflammation. Although analogous organisms in humans have not been clearly identified, human studies suggest that components of the microbiota may predispose genetically susceptible individuals to autoimmune disease. For instance, although Crohn’s disease incidence in susceptible monozygotic twins is much higher than in dizygotic twins, the concordance rate is not 100%. The explanation for incomplete concordance could lie in variability in the intestinal

1	in susceptible monozygotic twins is much higher than in dizygotic twins, the concordance rate is not 100%. The explanation for incomplete concordance could lie in variability in the intestinal microbiota, epigenetic differences, or factors yet to be defined.

1	15-19 Genomics-based approaches are providing new insight into the immunogenetic basis of autoimmunity.

1	Since the advent of gene knockout technology in mice (see Appendix I, Section A-35), many genes encoding immune system proteins have been experimentally disrupted. Several strains of mice that have been generated show signs of autoimmunity, including autoantibodies and infiltration of organs by T cells. The study of these mice has expanded our knowledge of the pathways that contribute to autoimmunity, and therefore their induced mutations might be candidates for identifying naturally occurring mutations. These mutations likely affect genes that encode cytokines, co-receptors, molecules involved in antigen-signaling cascades, co-stimulatory molecules, proteins involved in apoptosis, and proteins that clear antigen or antigen:antibody complexes. A number of cytokines and signaling proteins implicated in autoimmune disease are listed in Fig. 15.32. Other targeted or mutant genes with autoimmune phenotypes in mice are listed in Fig. 15.33, as are their corresponding human counterparts,

1	implicated in autoimmune disease are listed in Fig. 15.32. Other targeted or mutant genes with autoimmune phenotypes in mice are listed in Fig. 15.33, as are their corresponding human counterparts, where known.

1	Cytokine, receptor, or intracellular signal ResultDefect Defects in cytokine production or signaling that can lead to autoimmunity TNF-˜TNF-˜In˜ammatory bowel disease, arthritis, vasculitis IL-2, IL-7, IL-2R In˜ammatory bowel disease STAT4 In˜ammatory bowel disease IL-23R In˜ammatory bowel disease, psoriasis IL-10, IL-10R, STAT3 In˜ammatory bowel disease TGF-°Ubiquitous underexpression leads to in˜ammatory bowel disease. Underexpression speciÿcally in T cells leads to SLE IL-3 Demyelinating syndrome IFN-˛Overexpression in skin leads to SLE SLE IL-1 receptor agonist Arthritis Overexpression Underexpression Fig. 15.33 Categories of genetic defects that lead to autoimmune syndromes.

1	Fig. 15.33 Categories of genetic defects that lead to autoimmune syndromes. Many genes have been identified in which mutations predispose to autoimmunity in humans and animal models. These are best understood by the type of process affected by the genetic defect. A list of such genes (or the related protein product) is given here, organized by process (see the text for further discussion). In some cases, the same gene has been identified in mice and humans. In other cases, different genes affecting the same mechanism are implicated in mice and humans. The smaller number of human genes identified so far undoubtedly reflects the difficulty of identifying the genes responsible in outbred human populations.

1	In humans, genetic susceptibility to autoimmune disorders has been recently assessed by large-scale genome-wide association studies (GWASs), which look for a correlation between disease frequency and genetic variants (typically single-nucleotide polymorphisms, or SNPs). Such studies typically involve thousands of patients with a given autoimmune disease diagnosis and Fig. 15.34 Manhattan plot depicting risk alleles from genome-wide association studies (GWASs) of Crohn’s disease. The plot highlights selected gene loci identified by analyses of single nuclear polymorphisms (SNPs) with highly significant disease associations in patients with Crohn’s disease compared with healthy controls (see also Section 15‑23). The height of the peaks reflects the statistical significance of the association. The dotted line indicates the threshold for significant associations (5 × 10–08). Figure courtesy of John Rioux and Ben Weaver.

1	healthy controls in order to identify highly significant associations. Example results from GWASs that identify candidate genes linked to Crohn's disease are shown in the ‘Manhattan’ plot in Fig. 15.34. These plots are so named because they resemble a profile view of skyscrapers in the Manhattan skyline. Here, genomic coordinates are located on the x-axis, with the negative logarithm of the P-value of the association being given on the y-axis, and each assayed SNP is represented by a dot. Thus, the variants with the greatest disease association are the ‘tallest skyscrapers’ on the plot. Using this approach, hundreds of significant variants have been identified for multiple autoimmune diseases, suggesting that genetic susceptibility to autoimmune disease in humans may be due to a combination of susceptibility alleles at multiple loci.

1	Analyses of GWASs from multiple autoimmune diseases indicate that certain immune pathways—most notably those involved in T-cell activation and function—are common to multiple different forms of autoimmunity. For example, type 1 diabetes, Graves’ disease, Hashimoto’s thyroiditis, rheumatoid arthritis, and multiple sclerosis all show genetic association with the CTLA4 locus on chromosome 2. The cell-surface protein CTLA-4 is produced by activated T cells and is an inhibitory receptor for B7 co-stimulatory molecules (see Section 9-17). Similarly, many of the most common autoimmune disorders have been linked to central factors involved in the development and function of the TH17 and TH1 immune pathways (Fig. 15.35).

1	Despite confirming much of our knowledge from experimental immunology, these studies have also revealed our ignorance of gene-regulatory mechanisms that predispose to human disease. For instance, the vast majority of risk alleles identified to date (>80%) are not contained within exons (the protein-coding regions of genes), and many variants reside kilobases away from immunologically relevant genes. Understanding how genetic variation at these noncoding sequences in the genome can contribute to disease is a very active area of research. Recent evidence using computational algorithms, coupled to transcriptional and epigenetic profiling of human immune-cell populations,

1	TH1 cell TH17 cell IL-12 production IL-23 production IFN-˜production IFN-˜IL-17 and IL-22 production IL-17, IL-22 IL-12R°2IL-12R°1 IL-12 IL-12p40IL-12p35 IL-23R CCR6 IL-12R°1 IL-23 IL-12p19IL-12p40 Ankylosing spondylitis Infammatory bowel disease Psoriasis Multiple sclerosis Type 1 diabetes Rheumatoid arthritis JAK2TYK2 STAT4 JAK2TYK2 STAT3 1 2 3 4 5 Systemic lupus erythematosus Ulcerative colitis 7 8 6 Dendritic cell NF˛B Dectin-1 CARD9 PTGER4 °-glucan 1, 2 3 2 22 1, 2, 3 1, 2, 3, 4 1, 2, 3, 44, 7, 8 1, 2, 3, 4, 5, 6, 7 2, 6, 7, 8 2, 3, 4 1, 2, 3, 4, 5, 6, 7 4, 7, 8 1, 2, 4 Fig. 15.35 Associations of components of the IL-12R and IL-23R response pathways with autoimmune diseases.

1	Fig. 15.35 Associations of components of the IL-12R and IL-23R response pathways with autoimmune diseases. Multiple components of the interleukin‑12 (IL‑12R) and ‑23 (IL‑23R) receptor response pathways show significant genome‑wide associations with a broad range of immune‑mediated diseases; that is, these components map within genomic intervals that are associated with the respective disease in genome‑wide association studies. Although this figure shows these components in the conventional context of T helper 1 (TH1) and TH17 lymphocytes, it is now recognized that they are widely expressed in innate lymphoid cells and the specific cell type may vary from phenotype to phenotype. Adapted from Parkes M. et al.: Nat. Rev. Genetics 2013, 14:661. With permission from Macmillan Publishers Ltd.

1	suggests that many of the causal variants are located within critical gene-regulatory elements that control gene expression in immune cells (for example enhancers). Many of these gene-regulatory elements are utilized by effector or regulatory T cells following their activation, further confirming T-cell activation as a key event in the etiology of autoimmune disorders. Ultimately, a deeper understanding of how these variants contribute to disease will require new techniques to experimentally mimic and manipulate risk alleles, either singly or in combination, in order to fully elucidate how they affect the biology of immune-cell populations relevant to disease.

1	Despite our current ignorance of how most common genetic variants predispose to (or protect from) autoimmune disorders, several other approaches have begun to shed light on the genetic mechanisms of disease. These include the study of mutations that cause overt alterations in molecules regulating tolerance or the innate immune system; the study of patients with rare, monogenic defects of immune tolerance; and investigations into how certain HLA alleles predispose to disease by their ability to present certain self antigens. We will briefly explore each of these in the following sections. 15-20 Many genes that predispose to autoimmunity fall into categories that affect one or more tolerance mechanisms.

1	15-20 Many genes that predispose to autoimmunity fall into categories that affect one or more tolerance mechanisms. Many of the genes identified as predisposing to autoimmunity can be classified as affecting autoantigen availability and clearance; apoptosis; signaling thresholds; cytokine expression or signaling; co-stimulatory molecules or their receptors; or regulatory T cells (see Figs. 15.32 and 15.33).

1	Genes that control antigen availability and clearance are important both centrally, in the thymus, and in the periphery. In the thymus, genes that control expression of self proteins influence tolerance in developing lymphocytes. In the periphery, hereditary deficiency of some proteins can predispose to autoimmunity—for example, deficiency of early components of the complement cascade is associated with the development of SLE (see Section 15-16). Genes that control apoptosis, such as FAS, are important in regulating the duration and vigor of immune responses. Failure to regulate immune responses properly causes excessive destruction of self tissues, releasing autoantigens. In addition, because clonal deletion and anergy are not absolute, immune responses can include some self-reactive cells. As long as their numbers are limited by apoptotic mechanisms, they may not necessarily cause autoimmune disease, but they could cause a problem if apoptosis is not properly regulated.

1	One of the largest categories of mutations associated with autoimmunity pertains to signals that control lymphocyte activation. These include mutations in co-stimulatory molecules, inhibitory Fc receptors, and inhibitory receptors containing ITIMs, such as PD-1 and CTLA-4 (see Section 15-19). Another subset contains mutations in proteins involved in signal transduction through the antigen receptor itself. Mutations that affect signaling intensity in either direction—making signaling more or less sensitive—can result in autoimmunity. A decrease in sensitivity in the thymus, for example, can lead to a failure of negative selection and thereby to autoreactivity in the periphery. In contrast, increasing receptor sensitivity in the periphery can lead to greater and prolonged activation, resulting in an exaggerated immune response with the side effect of autoimmunity. Additionally, mutations that affect the expression or signaling of cytokines and co-stimulatory molecules have been linked

1	in an exaggerated immune response with the side effect of autoimmunity. Additionally, mutations that affect the expression or signaling of cytokines and co-stimulatory molecules have been linked to autoimmunity. A final subset comprises mutations effecting Treg-cell development or function, such as FoxP3 mutations (see Section 15-21).

1	15-21 Monogenic defects of immune tolerance. Predisposition to most of the common autoimmune diseases is due to the combined effects of multiple genes, but there are some monogenic autoimmune diseases (Fig. 15.36). Here, the mutant allele confers a very high risk of disease to the individual, but the overall impact on the population is minimal because these variants are rare. The existence of monogenic autoimmune disease was first observed in mutant mice in which the inheritance of an autoimmune syndrome followed a pattern consistent with a single-gene defect. Such alleles are usually recessive or X-linked. For example, the disease APECED is a recessive autoimmune disease caused by a defect in the gene AIRE (see Section 15-3).

1	Two monogenic autoimmune syndromes have been linked to defects in regulatory T cells. The X-linked recessive autoimmune syndrome IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) is typically caused by missense mutations in the gene encoding the transcription factor FoxP3, which is key in the differentiation and function of some types of Treg cells (see Section 9-21). This disease is characterized by severe allergic inflammation, autoimmune polyendocrinopathy, secretory diarrhea, hemolytic anemia, and thrombocytopenia, and usually leads to early death. Despite mutation of the FOXP3 gene, the number of FoxP3+ Treg cells in the blood of individuals with IPEX is comparable to the number in healthy individuals; however, the

1	Human disease Mouse mutant or knockoutGene Single-gene traits associated with autoimmunity APECED (APS-1) Knockout Mechanism of autoimmunity Decreased expression of self antigens in the thymus, resulting in defective negative selection of self-reactive T cells AIRE IPEX Knockout and mutation (scurfy) Decreased function of CD4 CD25 regulatory T cells FOXP3 ALPS lpr/lpr; gld/gld mutants Failure of apoptotic death of self-reactive B and T cells FAS SLE Knockout Defective clearance of immune complexes and apoptotic cellsC1q IBD Hypomorph Defective autophagy/clearance of bacteria by innate cells in intestines ATG16L1 IBD Knockout Defective IL-10 signaling; impaired anti-infammatory responseIL10RA, IL10RB Type 1 diabetes None Decreased expression of insulin in thymus; impaired negative selectionINS Association with Graves’ disease, type 1 diabetes, and others Knockout Failure of T-cell anergy and reduced activation threshold of self-reactive T cells CTLA4

1	Fig. 15.36 Single-gene traits associated with autoimmunity. APS‑1, autoimmune polyglandular syndrome 1; IPEX, immune Listed are examples of monogenic disorders that cause dysregulation, polyendocrinopathy, enteropathy, X‑linked syndrome; autoimmunity in humans. Mice with targeted deletions (knockout) ALPS, autoimmune lymphoproliferative syndrome. The lpr mutation or spontaneous mutations (for example, lpr/lpr) in homologous in mice affects the gene for Fas, whereas the gld mutation affects genes have similar disease characteristics and are useful models the gene for FasL. Adapted from J.D. Rioux and A.K. Abbas: Nature for the study of the pathogenic basis for these disorders. APECED, 435:584–589. With permission from Macmillan Publishers Ltd. autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy; suppressive function normally displayed by these cells is impaired. A spontaneous frameshift mutation in the mouse Foxp3 gene (the scurfy mutation) that results in loss of the

1	dystrophy; suppressive function normally displayed by these cells is impaired. A spontaneous frameshift mutation in the mouse Foxp3 gene (the scurfy mutation) that results in loss of the DNA-binding domain of FoxP3 or complete knockout of Foxp3 leads to an analogous systemic autoimmune disease, in this case associated with the absence of FoxP3+ Treg cells.

1	Autoimmunity caused by defective development and survival of Treg cells also results from mutation of CD25, the high-affinity chain of the IL-2 receptor complex that is constitutively expressed by Treg cells (see Section 9-16). Because deficiency of CD25 affects the development and function of effector T cells as well, in addition to autoimmunity, patients affected by this mutation suffer multiple immunological deficiencies and susceptibility to infections. These findings further confirm the importance of Treg cells in the regulation of the immune system.

1	An interesting case of a monogenic autoimmune disease is autoimmune lymphoproliferative syndrome (ALPS), a systemic autoimmune syndrome caused by mutations in the gene encoding Fas. Fas is normally present on the surface of activated T and B cells, and when ligated by Fas ligand, it signals the Fas-bearing cell to undergo apoptosis (see Section 11-16). In this way it functions to limit the extent of immune responses. Mutations that eliminate or inactivate Fas lead to a massive accumulation of lymphocytes, especially T cells, and in mice, to the production of large quantities of pathogenic autoantibodies and a disease that resembles SLE. A mutation leading to this autoimmune syndrome was first observed in the MRL mouse strain and named lpr, for lymphoproliferation; it was subsequently identified as a mutation in Fas. The study of human patients with the rare autoimmune lymphoproliferative syndrome, which is similar to the syndrome in the MRL/lpr mice, led to the identification of FAS

1	as a mutation in Fas. The study of human patients with the rare autoimmune lymphoproliferative syndrome, which is similar to the syndrome in the MRL/lpr mice, led to the identification of FAS as the mutant gene responsible for most of these cases (see Fig. 15.36).

1	Fig. 15.37 Associations of HLA and sex with susceptibility to autoimmune disease. The ‘relative risk’ for an HLA allele in an autoimmune disease is calculated by comparing the observed number of patients carrying the HLA allele with the number that would be expected, given the prevalence of the HLA allele in the general population. For type 1 insulin‑dependent diabetes mellitus, the association is in fact with the HLA‑DQ gene, which is tightly linked to the DR genes but is not detectable by serotyping. Some diseases show a significant bias in the sex ratio; this is taken to imply that sex hormones are involved in pathogenesis. Consistent with this, the difference in the sex ratio in these diseases is greatest between menarche and menopause, when levels of such hormones are highest. Healthy controls Diabetes DR3/x (24%) DR4/x (17.5%) DR2/x (30.3%) DR3/4 (2.5%) (25.7%) DR4/x (27%) (4%) DR3/4 (39%) DR3/x (30%) DRx/x DRx/x

1	Healthy controls Diabetes DR3/x (24%) DR4/x (17.5%) DR2/x (30.3%) DR3/4 (2.5%) (25.7%) DR4/x (27%) (4%) DR3/4 (39%) DR3/x (30%) DRx/x DRx/x Autoimmune diseases caused by single genes are rare, but are of great interest, as the mutations causing them identify important pathways that normally prevent the development of autoimmune responses. 15-22 MHC genes have an important role in controlling susceptibility to autoimmune disease. Among genetic loci that contribute to autoimmunity, susceptibility to autoimmune disease has so far been most consistently associated with MHC genotype (Fig. 15.37), particularly MHC class II alleles, thus implicating CD4 T cells in their etiology. The development of experimental diabetes or arthritis in transgenic mice expressing specific human HLA antigens strongly suggests that particular MHC alleles confer disease susceptibility.

1	As in genome-wide association studies (GWASs), association of MHC with disease is identified by comparing the frequencies of MHC alleles in patients with the disease with the frequencies in the normal population. For type 1 diabetes, this approach demonstrated an association with the HLA-DR3 and HLA-DR4 alleles, identified by serotyping (Fig. 15.38). Such studies also showed that the MHC class II allele HLA-DR2 has a dominant protective

1	Fig. 15.38 Population studies show association of susceptibility to type 1 diabetes with HLA genotype. The HLA genotypes (determined by serotyping) of patients with diabetes (lower panel) are not representative of those found in the general population (upper panel). Almost all patients with diabetes express HLA‑DR3 and/or HLA‑DR4, and HLA‑DR3/DR4 heterozygosity is greatly overrepresented in diabetics compared with controls. These alleles are linked tightly to HLA‑DQ alleles that confer susceptibility to type 1 diabetes. By contrast, HLA‑DR2 protects against the development of diabetes and is found only extremely rarely in patients with diabetes. The small letter x represents any allele other than DR2, DR3, or DR4. Family studies of HLA haplotypes in type 1 diabetes

1	Family studies of HLA haplotypes in type 1 diabetes Fig. 15.39 Family studies show strong linkage of susceptibility to type 1 diabetes with HLA genotype. In families in which two or more siblings have type 1 diabetes, it is possible to compare the HLA genotypes of affected siblings. Affected siblings share two HLA haplotypes much more frequently than would be expected if the HLA genotype did not influence disease susceptibility.

1	effect: individuals carrying HLA-DR2, even in association with one of the susceptibility alleles, rarely develop diabetes. It has also been shown that two siblings affected with the same autoimmune disease are far more likely than expected to share the same MHC haplotypes (Fig. 15.39). As HLA genotyping has become more exact through DNA sequencing, disease associations originally discovered by serotyping have been defined more precisely. For example, the association between type 1 diabetes and the DR3 and DR4 alleles is now known to be due to their tight genetic linkage to DQβ alleles that confer susceptibility to the disease. Indeed, susceptibility is most closely associated with polymorphisms at a particular position in the DQβ amino acid sequence that affect the peptide-binding cleft of MHC class II (Fig. 15.40). The diabetes-prone NOD strain of mice also has a serine residue polymorphism at that same position in the homologous mouse MHC class II molecule, known as I-Ag7.

1	The association of MHC genotype with autoimmune disease is not surprising; associations can be explained by a simple model in which susceptibility to an autoimmune disease is determined by differences in the ability of different allelic variants of MHC molecules to present autoantigenic peptides to autoreactive T cells. This would be consistent with what we know of T-cell involvement in particular diseases. In diabetes, for example, there are associations with both MHC class I and MHC class II alleles, consistent with the finding that both CD8 and CD4 T cells mediate the autoimmune response. An alternative hypothesis emphasizes the role of MHC alleles in shaping the T-cell receptor repertoire (see Chapter 8). This hypothesis proposes that self peptides associated with certain MHC molecules may drive the positive selection of developing thymocytes that are specific for particular autoantigens. Such

1	Fig. 15.40 Amino acid changes in the sequence of an MHC class II protein correlate with susceptibility to and protection from diabetes. The HLA‑DQβ1 chain contains an aspartic acid (Asp) residue at position 57 in most people; in Caucasoid populations, patients with type 1 diabetes (T1DM) more often have valine, serine, or alanine at this position instead, as well as other differences. Asp 57, shown in red on the backbone structure of the DQβ chain in the top panel, forms a salt bridge (shown in green in the center panel) to an arginine residue (shown in pink) in the adjacent α chain (gray). The change to an uncharged residue (for example, alanine, shown in yellow in the bottom panel) disrupts this salt bridge, altering the stability of the DQ molecule. The non‑obese diabetic (NOD) strain of mice, which develops spontaneous diabetes, shows a similar substitution of serine for aspartic acid at position 57 of the homologous I‑Aβ chain, and NOD mice transgenic for β chains with Asp 57

1	of mice, which develops spontaneous diabetes, shows a similar substitution of serine for aspartic acid at position 57 of the homologous I‑Aβ chain, and NOD mice transgenic for β chains with Asp 57 have a marked reduction in diabetes incidence. Courtesy of C. Thorpe.

1	Fig. 15.41 Crohn’s disease results from a breakdown of the normal homeostatic mechanisms that limit inflammatory responses to the gut microbiota. The innate and adaptive immune systems normally cooperate to limit inflammatory responses to intestinal bacteria through a combination of mechanisms: a mucus layer produced by goblet cells; tight junctions between the intestinal epithelial cells; antimicrobial peptides released from epithelial cells and Paneth cells; and induction of Treg cells that inhibit effector CD4 T‑cell development and promote the production of IgA antibodies that are transported into the intestinal lumen, where they inhibit translocation of intestinal bacteria (not shown). In individuals with impaired homeostatic mechanisms, dysregulated TH1‑and TH17‑cell responses to the intestinal microbiota can result, generating disease‑causing chronic inflammation. Crohn’s disease susceptibility genes of innate immunity include NOD2 and the autophagy genes ATG16L1 and IRGM. A

1	the intestinal microbiota can result, generating disease‑causing chronic inflammation. Crohn’s disease susceptibility genes of innate immunity include NOD2 and the autophagy genes ATG16L1 and IRGM. A major susceptibility gene that affects adaptive immune cells is IL23R, which is expressed by TH17 cells (see also Fig. 15.34).

1	autoantigenic peptides might be expressed at too low a level or bind too poorly to MHC molecules to drive thymic negative selection, but might be present at a sufficient level or bind strongly enough to drive positive selection. This hypothesis is supported by observations that I-Ag7, the MHC class II molecule in NOD mice, binds many peptides very poorly and may therefore be less effective in driving thymic negative selection. 15-23 Genetic variants that impair innate immune responses can predispose to T-cell-mediated chronic inflammatory disease.

1	As noted earlier in this chapter, Crohn’s disease (CD) is one of the two major types of inflammatory bowel disease. CD is thought to result from abnormal hyperresponsiveness of CD4 T cells to antigens of the commensal gut microbiota, rather than to true self antigens. Dysregulation of TH17 and TH1 cells is thought to be pathogenic. Disease can result from a failure of mucosal innate immune mechanisms to sequester luminal bacteria from the adaptive immune system, from T-cell-intrinsic defects that cause heightened effector responses, or from failure of Treg cells to suppress microbiota-reactive TH17 and TH1 cells (Fig. 15.41). Patients with CD have episodes of severe inflammation that commonly affect the terminal ileum, with or without involvement of the colon—hence the alternative name ‘regional ileitis’ for this disease—but any part of the gastrointestinal tract can be involved. The disease is characterized by chronic inflammation and granulomatous lesions in the mucosa and

1	name ‘regional ileitis’ for this disease—but any part of the gastrointestinal tract can be involved. The disease is characterized by chronic inflammation and granulomatous lesions in the mucosa and sub-mucosa of the intestine. Genetic analysis of patients with CD and their families has identified a growing list of disease-susceptibility genes (see Fig. 15.34). One of the earliest to be identified was NOD2 (also known as CARD15), which is expressed predominantly in monocytes, dendritic cells, and the Paneth cells of the small intestine, and is involved in recognition of microbial antigens as part of the innate immune response (see Section 3-8). Mutations and rare polymorphic variants in NOD2 are strongly associated with CD. Mutations in the same gene are also the cause of a dominantly inherited granulomatous disease named Blau syndrome, in which granulomas typically develop in the skin, eyes, and joints. Whereas CD results from a loss of function of NOD2, it is thought that Blau

1	inherited granulomatous disease named Blau syndrome, in which granulomas typically develop in the skin, eyes, and joints. Whereas CD results from a loss of function of NOD2, it is thought that Blau syndrome results from a gain of function.

1	goblet cell intestinal epithelial cells homeostatic dendritic cell TregTH17TH1 anti-infammatory activated phagocytic cell macrophage auto-phagosome (ATG16L1 IRGM) Paneth cell antimicrobial peptides NOD2 bacteriaintestinal lumen mucus layer intestinal lamina propria pro-infammatory IL-6, TGF-˜IL-1˜, IL-23 TGF-˜RAIL-12

1	NOD2 is an intracellular receptor for the muramyl dipeptide derived from bacterial peptidoglycan, and its stimulation activates the transcription factor NFκB and the expression of genes encoding pro-inflammatory cytokines and chemokines (see Section 3-8 and Fig. 12.15). In Paneth cells—specialized intestinal epithelial cells in the base of the small intestinal crypts—activation of NOD2 stimulates the release of granules containing antimicrobial peptides that help sequester commensal bacteria to the intestinal lumen, away from the adaptive immune system. Mutant forms of NOD2 that have lost this function limit this innate antibacterial response, thereby predisposing the individual to heightened effector CD4 T-cell responses to the commensal microbiota and consequent chronic intestinal inflammation (see Section 12-22).

1	In addition to NOD2, other deficiencies in innate immunity have been identified in patients with CD, including defective CXCL8 production and neutrophil accumulation, which can synergize with NOD2 defects to promote intestinal inflammation. Thus, compound defects in innate immunity and the regulation of inflammation may act synergistically to promote immunopathology in CD. GWASs have identified other susceptibility genes for CD that may be linked to impaired innate immune functions (see Fig. 15.34). Defects in two genes (ATG16L1 and IRGM) that contribute to autophagy have been linked to CD, suggesting that other mechanisms that impair clearance of commensal bacteria might predispose to chronic intestinal inflammation. Autophagy, or the digestion of a cell’s cytoplasm by its own lysosomes, is important in the turnover of damaged cellular organelles and proteins; autophagy also has a role in antigen processing and presentation (see Section 6-9), and contributes to the clearance of some

1	is important in the turnover of damaged cellular organelles and proteins; autophagy also has a role in antigen processing and presentation (see Section 6-9), and contributes to the clearance of some phagocytosed bacteria.

1	While defects in important pathways of the innate immune system contribute to CD, genes that regulate the adaptive immune response have also been associated with susceptibility. Most notably, there are variants of the gene for the IL-23 receptor (IL23R) that predispose to disease, consistent with heightened TH17 responses in diseased tissues. Collectively, the growing number of susceptibility genes that confer increased risk for CD point to abnormal regulation of homeostatic innate and adaptive immune responses to the intestinal microbiota as a common factor. 15-24 External events can initiate autoimmunity.

1	The geographic distribution of autoimmune diseases reveals a heterogeneous distribution among different continents, countries, and ethnic groups. For example, the incidence of disease in the Northern Hemisphere seems to decrease from north to south. This gradient is particularly prominent for diseases such as multiple sclerosis and type 1 diabetes in Europe, where the incidence is greater in the northern countries than in Mediterranean regions. Numerous epidemiologic and genetic associations suggest that this may be partly related to levels of vitamin D. The active form of vitamin D is formed in the skin in response to sunlight—which is less intense and less available in northern latitudes—and has numerous immunoregulatory functions that affect cells of the innate and adaptive immune systems, including suppression of TH17 cell development. Studies have also shown an increased incidence of autoimmunity in more developed countries, the basis of which is unknown.

1	Besides vitamin D levels, there are numerous other nongenetic factors contributing to these geographic variations, including socioeconomic status and diet. The contribution of nongenetic factors to disease is exemplified in genetically identical mice, which develop autoimmunity at different rates and severity. There is an emerging appreciation for the diversity of the commensal microbiota having a role in contributing to autoimmune disease—including extraintestinal disease—reflecting the importance of the interplay of the microbiome with the innate and adaptive immune systems in shaping the

1	Fig. 15.42 Infectious agents could break self-tolerance in several different ways. Left panels: because some antigens are sequestered from the circulation, either behind a tissue barrier or within the cell, an infection that breaks cell and tissue barriers might expose hidden antigens. Right panels: molecular mimicry might result in infectious agents inducing either T‑ or B‑cell responses that can cross‑react with self antigens.

1	systemic immune response. Finally, exposure to infections and environmental toxins may be factors that help trigger autoimmunity. However, it should be noted that epidemiological and clinical studies over the past century have also shown a negative correlation between exposure to some types of infections in early life and the development of allergy and autoimmune diseases. This ‘hygiene hypothesis’ proposes that a lack of infection during childhood may affect the regulation of the immune system in later life, leading to a greater likelihood of allergic and autoimmune responses (see Section 14-4). 15-25 Infection can lead to autoimmune disease by providing an environment that promotes lymphocyte activation.

1	15-25 Infection can lead to autoimmune disease by providing an environment that promotes lymphocyte activation. How might pathogens contribute to autoimmunity? While an infection is in progress, inflammatory mediators released from activated antigen-presenting cells and lymphocytes and the increased expression of co-stimulatory molecules can affect so-called bystander cells—lymphocytes that are not themselves specific for the antigens of the infectious agent. Self-reactive lymphocytes can become activated in these circumstances, particularly if tissue destruction by the infection leads to an increase in the availability of the self antigen (Fig. 15.42, left panels). Furthermore, pro-inflammatory cytokines, such as IL-1 and IL-6, impair the suppressive activity of regulatory T cells, allowing self-reactive naive T cells to become activated to differentiate into effector T cells that can initiate an autoimmune response.

1	The perpetuation or exacerbation of autoimmune disease by viral or bacterial infections has been shown in experimental animal models. For example, the severity of type 1 diabetes in NOD mice is exacerbated by Coxsackie virus B4 infection, which leads to inflammation, tissue damage, the release of sequestered islet antigens, and the generation of autoreactive T cells.

1	We discussed earlier the ability of self ligands such as unmethylated CpG DNA and RNA to directly activate autoreactive B cells via their TLRs and thus break self-tolerance (see Section 15-4 and Fig. 15.25). Microbial ligands for TLRs may also promote autoimmunity by stimulating dendritic cells and macrophages to produce large quantities of cytokines that cause local inflammation and help stimulate already activated autoreactive T and B cells. This mechanism might be relevant to the flare-ups of inflammation that follow infection in patients with autoimmune vasculitis associated with anti-neutrophil cytoplasmic antibodies.

1	One example of how TLR ligands can induce local inflammation derives from an animal model of arthritis in which injection of bacterial CpG DNA, which is recognized by TLR-9, into the joints of mice induces an arthritis characterized by macrophage infiltration. These macrophages express chemokine receptors on their surface and produce large amounts of CC chemokines, which promote leukocyte recruitment to the site of injection. and self molecules can lead to antiself responses and autoimmune disease.

1	and self molecules can lead to antiself responses and autoimmune disease. Infection with certain pathogens is associated with autoimmune sequelae. Some pathogens express antigens that resemble host molecules, a phenomenon called molecular mimicry. In such cases, antibodies produced against a pathogen epitope may cross-react with a self molecule (see Fig. 15.42, right panels). Such structures do not necessarily have to be identical: it is sufficient that they be similar enough to be recognized by the same antibody. Molecular mimicry may also activate autoreactive T cells and result in an attack on self tissues if a processed peptide from a pathogen antigen is similar to a host peptide. A model system to demonstrate molecular mimicry has been generated by using transgenic mice expressing a viral antigen in the pancreas.

1	Inject LCMV into mouse. NP-speciÿc CD8 T cells are activated by LCMV infection Activated CD8 T cells inÿltrate islets and kill ˜ cells expressing NP; this results in diabetes Make transgenic mice that express NP only in the pancreatic ˜ cells pancreas NP is expressed only in ˜ cells and provokes no T-cell response Normally, there is no response to this virus-derived ‘self’ antigen, but upon infection with the virus that was the source of the transgenic antigen, mice develop diabetes, because the virus activates T cells that are cross-reactive with the ‘self’ viral antigen (Fig.15.43).

1	One might wonder why these self-reactive lymphocytes have not been deleted or inactivated by the usual mechanisms of self-tolerance. One reason is that lower-affinity self-reactive B and T cells are not removed efficiently and are present in the naive lymphocyte repertoire as ignorant lymphocytes (see Section 15-4). Pathogens may provide substantially higher local doses of the eliciting antigen in an immunogenic form, whereas normally it would be relatively unavailable to lymphocytes. Some examples of autoimmune syndromes thought to involve molecular mimicry are the rheumatic fever that sometimes follows streptococcal infection, and the reactive arthritis that can occur after enteric infection.

1	Once self-reactive lymphocytes have been activated by such a mechanism, their effector functions can destroy tissues. Autoimmunity of this type is sometimes transient, and remits when the inciting pathogen is eliminated. This is the case in the autoimmune hemolytic anemia that follows mycoplasma infection. The anemia ensues when antibodies against the pathogen cross-react with an antigen on red blood cells, leading to hemolysis (see Section 15-13). The autoantibodies disappear when the patient recovers from the infection. Sometimes, however, the autoimmunity persists well beyond the initial infection. This is true in some cases of rheumatic fever (Fig. 15.44), which occasionally follows a sore throat, scarlet fever, or local skin infections (impetigo) Streptococcal cell wall stimulates antibody response Some antibodies cross-react with heart valve tissue, causing rheumatic fever plasma cell heart antibodies in blood bacteria

1	Streptococcal cell wall stimulates antibody response Some antibodies cross-react with heart valve tissue, causing rheumatic fever plasma cell heart antibodies in blood bacteria Fig. 15.43 Virus infection can break tolerance to a transgenic viral protein expressed in pancreatic β cells. Mice made transgenic for the lymphocytic choriomeningitis virus (LCMV) nucleoprotein under the control of the rat insulin promoter express the nucleoprotein in their pancreatic β cells but do not respond to this protein and therefore do not develop an autoimmune diabetes. However, if the transgenic mice are infected with LCMV, a potent antiviral cytotoxic T‑cell response is elicited, and this kills the β cells, leading to diabetes. It is thought that infectious agents can sometimes elicit T‑cell responses that cross‑react with self peptides (a process known as molecular mimicry) and that this could cause autoimmune disease in a similar way.

1	Fig. 15.44 Antibodies against streptococcal cell-wall antigens cross-react with antigens on heart tissue. The immune response to the bacteria produces antibodies against various epitopes of the bacterial cell surface. Some of these antibodies (yellow) cross‑react with the heart valves, whereas others (blue) do not. An epitope in the heart (orange) is structurally similar, but not identical, to a bacterial epitope (red).

1	caused by Streptococcus pyogenes. The similarity of epitopes on streptococcal antigens to self epitopes leads to antibody-mediated, and possibly T-cellmediated, damage to a variety of tissues, including heart valves and the kidney. Although the tissue injury is typically transient, especially with antibiotic treatment, it can become chronic. Similarly, Lyme disease, an infection with the spirochete Borrelia burgdorferi, can be followed by late-developing autoimmunity (Lyme arthritis). In this case, the mechanism is not entirely clear, but it is likely to involve cross-reactivity of pathogen and host components, leading to autoimmunity. 15-27 Drugs and toxins can cause autoimmune syndromes.

1	Perhaps the clearest evidence of external causative agents in human autoimmunity comes from the effects of certain drugs, which elicit autoimmune reactions in a small proportion of patients. Procainamide, a drug used to treat heart arrhythmias, is notable for inducing autoantibodies similar to those in SLE, although these are rarely pathogenic. Several drugs are associated with the development of autoimmune hemolytic anemia, in which autoantibodies against surface components of red blood cells attack and destroy these cells (see Section 15-13). Toxins in the environment can also cause autoimmunity. When heavy metals, such as gold or mercury, are administered to susceptible strains of mice, a predictable autoimmune syndrome, including the production of autoantibodies, ensues. The extent to which heavy metals promote autoimmunity in humans is debatable, but the animal models show that environmental factors such as toxins could have roles in certain syndromes.

1	The mechanisms by which drugs and toxins cause autoimmunity are uncertain. For some drugs it is thought that they react chemically with self proteins and form derivatives that the immune system recognizes as foreign. The immune response to these haptenated self proteins can lead to inflammation, complement deposition, destruction of tissue, and finally immune responses to the original self proteins. 15-28 Random events may be required for the initiation of autoimmunity.

1	15-28 Random events may be required for the initiation of autoimmunity. Although scientists and physicians would like to attribute the onset of ‘spontaneous’ diseases to some specific cause, this may not always be possible. There might not be one virus or bacterium, or even any understandable pattern of events that precedes the onset of autoimmune disease. The chance encounter in the peripheral lymphoid tissues of a few autoreactive B and T cells that can interact with each other, at just the moment when an infection is providing pro-inflammatory signals, may be all that is needed. This could be a rare event, but in a susceptible individual such events could be more frequent and/or more difficult to control.

1	Thus, the onset or incidence of autoimmunity can seem to be random. Genetic predisposition represents, in part, an increased chance of occurrence of this random event. This view, in turn, could explain why many autoimmune diseases appear in early adulthood or later, after enough time has elapsed to permit low-frequency events to occur. It may also explain why, after certain kinds of aggressive therapies, the disease eventually recurs after a long interval of remission. Summary.

1	The specific causes of most autoimmune diseases are not known. Genetic risk factors, including particular alleles of MHC class II molecules and polymorphisms or mutations of other genes, have been identified, but many individuals with genetic variants that predispose to a particular autoimmune disease do not get the disease. Epidemiological studies of genetically identical populations of animals have highlighted the role of environmental factors in the initiation of autoimmunity, but although environmental factors have a strong influence on disease, they are not well understood. Some toxins and drugs are known to cause autoimmunity, but their role in the common autoimmune diseases is unclear. Similarly, some autoimmune syndromes can follow viral or bacterial infections. Pathogens can promote autoimmunity by causing nonspecific inflammation and tissue damage, and can sometimes elicit responses to self proteins if they express molecules resembling self, a phenomenon known as molecular

1	autoimmunity by causing nonspecific inflammation and tissue damage, and can sometimes elicit responses to self proteins if they express molecules resembling self, a phenomenon known as molecular mimicry. More research is needed to define specific contributions of environmental factors to autoimmune diseases. It may prove that for most diseases no single environmental trigger that induces disease will be found, but rather a combination of triggers, or even stochastic, or chance, events, will have important roles.

1	Responses to alloantigens and transplant rejection.

1	Although transplantation of tissues to replace diseased organs has emerged as an important medical therapy, adaptive immune responses to the grafted tissues are a major impediment. Rejection is caused by immune responses to alloantigens on the graft, which are proteins that vary from individual to individual within a species and are therefore perceived as foreign by the recipient. When tissues containing nucleated cells are transplanted, T-cell responses to the highly polymorphic MHC molecules almost always trigger a response against the grafted organ. Matching the MHC type of the donor and the recipient increases the success rate of grafts, but perfect matching is possible only when donor and recipient are related, and even in these cases, genetic differences at other loci can still trigger rejection, although less severely. Nevertheless, advances in immunosuppression and transplantation medicine now mean that the precise matching of tissues for transplantation is no longer the major

1	rejection, although less severely. Nevertheless, advances in immunosuppression and transplantation medicine now mean that the precise matching of tissues for transplantation is no longer the major factor in graft survival. In blood transfusion, the earliest and most common tissue transplant, MHC matching is not necessary for routine transfusions, because red blood cells and platelets express small amounts of MHC class I molecules and do not express MHC class II molecules; thus, they are not usually T-cell targets. However, antibodies made against platelet MHC class I molecules can be a problem when repeated transfusions of platelets are required. Blood must be matched for ABO and Rh blood group antigens to avoid the rapid destruction of mismatched red blood cells by antibodies in the recipient (see Appendix I, Sections A-5 and A-7), but because there are only four major ABO types and two Rh types, this is a relatively easy form of tissue matching.

1	In this part of the chapter we examine the immune response to tissue grafts and also ask why such responses do not reject the one foreign tissue graft that is tolerated routinely—the mammalian fetus. 15-29 Graft rejection is an immunological response mediated primarily by T cells. The basic rules of tissue grafting were first elucidated by skin transplantation between inbred strains of mice. Skin can be grafted with 100% success between different sites on the same animal or person (an autograft), or between genetically identical animals or people (a syngeneic graft). However, when skin is grafted between unrelated or allogeneic individuals (an allograft), the graft initially survives but is then rejected about 10–13 days after grafting

1	Fig. 15.45 Skin graft rejection is the result of a T-cell-mediated a second‑set rejection, and the accelerated response is MHC‑anti-graft response. Grafts that are syngeneic are permanently specific; skin from a second donor of the same MHC type is rejected accepted (first panels), but grafts differing at the MHC are rejected equally fast, whereas skin from an MHC‑different donor is rejected about 10–13 days after grafting (first‑set rejection, second panels). in a first‑set pattern (not shown). Naive mice that are given T cells When a mouse is grafted for a second time with skin from the same from a sensitized donor behave as if they had already been grafted donor, it rejects the second graft faster (third panels). This is called (final panels).

1	(Fig. 15.45). This response is called an acute rejection, and it depends on a T-cell response, because skin grafted onto nude mice, which lack T cells, is not rejected. The ability to reject skin can be restored to nude mice by the adoptive transfer of normal T cells. When a recipient that has previously rejected a graft is regrafted with skin from the same donor, the second graft is rejected more rapidly (6–8 days) in an accelerated rejection (see Fig. 15.45). Skin from a third-party donor grafted onto the same recipient at the same time does not show this faster response but follows a first-set rejection course. The rapid course of second-set rejection can also be transferred to new recipients by T cells from the initial recipient, showing that second-set rejection is caused by a memory-type response (see Chapter 11) from clonally expanded and primed T cells specific for the donor skin.

1	Immune responses are the major barrier to effective tissue transplantation, destroying grafted tissue by an adaptive immune response to its foreign proteins. These responses can be mediated by either CD8 or CD4 T cells, or both. Antibodies can also contribute to second-set rejection of tissue grafts. 15-30 Transplant rejection is caused primarily by the strong immune response to nonself MHC molecules.

1	15-30 Transplant rejection is caused primarily by the strong immune response to nonself MHC molecules. Antigens that differ between members of the same species are known as alloantigens, and an immune response against such antigens is known as an alloreactive response. When donor and recipient differ at the MHC, an alloreactive immune response is directed at the nonself allogeneic MHC molecule or molecules on the graft. In most tissues these are predominantly MHC class I antigens. Once a recipient has rejected a graft of a particular MHC type, any further graft bearing the same nonself MHC will be rapidly rejected in a second response. The frequency of T cells specific for any nonself MHC molecule is relatively high, making differences at MHC loci the most potent trigger of initial graft rejections (see Section 6-13); indeed, the MHC was originally so named because of its central role in graft rejection.

1	Once it became clear that recognition of nonself MHC molecules was a major determinant of graft rejection, a considerable amount of effort was put into MHC matching of recipient and donor. Today, with advances in immunosuppression, MHC matching has become largely irrelevant for most allografts, although it remains important for bone marrow transplantation, for reasons that are discussed in Section 15-36. Even a perfect match at the MHC locus, known as the HLA locus in humans, does not prevent rejection reactions. Grafts between HLA-identical siblings will invariably incite a rejection reaction, albeit more slowly than an unmatched graft, unless donor and recipient are identical twins. This reaction is the result of differences between antigens from non-MHC proteins that also vary between individuals.

1	Thus, unless donor and recipient are identical twins, all graft recipients must be given immunosuppressive drugs chronically to prevent rejection. Indeed, the current success of clinical transplantation of solid organs is more the result of advances in immunosuppressive therapy (see Chapter 16) than of improved tissue matching. The limited supply of cadaveric organs, coupled with the urgency of identifying a recipient once a donor organ becomes available, means that accurate matching of tissue types is achieved only rarely, with the notable exception of matched-sibling donation of kidneys. 15-31 In MHC-identical grafts, rejection is caused by peptides from other alloantigens bound to graft MHC molecules.

1	15-31 In MHC-identical grafts, rejection is caused by peptides from other alloantigens bound to graft MHC molecules. When donor and recipient are identical at the MHC but differ at other genetic loci, graft rejection is not as rapid, but left unchecked it will still destroy the graft (Fig. 15.46). This is the reason that grafts between HLA-identical siblings would be rejected without immunosuppressive treatment. MHC class I and II molecules bind and present a large selection of peptides derived from self proteins made in the cell, and if these proteins are polymorphic, then different peptides will be produced from them in different members of a species. Such proteins can also be recognized as minor histocompatibility antigens

1	Fig. 15.46 Even complete matching at the MHC does not ensure graft survival. Although syngeneic grafts are not rejected (left panels), MHC‑identical grafts from donors that differ at other loci (minor H antigen loci) are rejected (right panels), albeit more slowly than MHC‑disparate grafts (center panels). Percentage 100 of grafts surviving

1	Percentage 100 of grafts surviving Fig. 15.47 Minor H antigens are peptides derived from polymorphic cellular proteins bound to MHC class I molecules. Self proteins are routinely digested by proteasomes within the cell’s cytosol, and peptides derived from them are delivered to the endoplasmic reticulum, where they can bind to MHC class I molecules and be delivered to the cell surface. If a polymorphic protein differs between the graft donor (shown in red on the left) and the recipient (shown in blue on the right), it can give rise to an antigenic peptide (red on the donor cell) that can be recognized by the recipient’s T cells as nonself and elicit an immune response. Such antigens are the minor H antigens.

1	(Fig. 15.47). One set of proteins that induce minor histocompatibility responses are encoded on the male Y chromosome. Responses induced by these proteins are known collectively as H-Y. As Y chromosome-specific genes are not expressed in females, female anti-male responses occur; however, male anti-female responses do not occur, because both sexes express X-chromosome genes. One H-Y antigen has been identified in mice and humans as peptides from a protein encoded by the gene Smcy. An X-chromosome homolog of Smcy (or Kdm5d), called Smcx (or Kdm5c), does not contain these peptide sequences, which are therefore expressed uniquely in males. Most minor histocompatibility antigens are encoded by autosomal genes and their identity is largely unknown, although an increasing number have now been identified at the genetic level.

1	The response to minor histocompatibility antigens is in many ways analogous to the response to viral infection. However, whereas an antiviral response eliminates only infected cells, a large fraction of cells in the graft express minor histocompatibility antigens, and thus the graft is destroyed in the response against these antigens. Given the virtual certainty of mismatches in minor histocompatibility antigens between two individuals, and the potency of the reactions they incite, it is understandable that successful transplantation requires the use of powerful immunosuppressive drugs. 15-32 There are two ways of presenting alloantigens on the transplanted donor organ to the recipient’s T lymphocytes.

1	15-32 There are two ways of presenting alloantigens on the transplanted donor organ to the recipient’s T lymphocytes. Before naive alloreactive T cells can develop into effector T cells that cause rejection, they must be activated by antigen-presenting cells that express both the allogeneic MHC and co-stimulatory molecules. Organ grafts carry with them antigen-presenting cells of donor origin, sometimes called passenger leukocytes, and these are an important stimulus to alloreactivity. This route for sensitization of the recipient to a graft seems to involve donor antigen-presenting cells leaving the graft and migrating to secondary lymphoid tissues of the recipient, including the spleen and lymph nodes, where they can activate those host T cells that bear the corresponding T-cell receptors. Because the lymphatic drainage of solid organ allografts is interrupted by transplantation, migration of donor antigen-presenting cells occurs via the

1	Fig. 15.48 Acute rejection of a kidney graft through the direct pathway of allorecognition. Donor dendritic cells in the graft (in this case a kidney) carry complexes of donor HLA molecules and donor peptides on their surfaces. The dendritic cells are carried via the blood to secondary lymphoid organs (a lymph node is illustrated here), where they move to the T‑cell areas. Here, they activate the recipient’s T lymphocytes, whose receptors can bind specifically to the complexes of allogeneic donor HLA (both class I and class II) in combination with donor peptides. After activation, the effector T cells travel in the blood to the grafted organ, where they attack cells that display the peptide:HLA molecule complexes for which the T cells are specific.

1	blood, not lymphatics. The activated alloreactive effector T cells can then circulate to the graft, which they attack directly (Fig. 15.48). This recognition pathway is known as direct allorecognition (Fig. 15.49, upper panel). Indeed, if the grafted tissue is depleted of antigen-presenting cells by treatment with antibodies or by prolonged incubation, rejection occurs only after a much longer time. A second mechanism of allograft recognition leading to graft rejection is the uptake of allogeneic proteins by the recipient’s own antigen-presenting cells and their presentation to T cells by self MHC molecules. This is known as indirect allorecognition (see Fig. 15.49, lower panel). Peptides derived from both the foreign MHC molecules themselves and minor histocompatibility antigens can be presented by indirect allorecognition.

1	Direct allorecognition is thought to be largely responsible for acute rejection, especially when MHC mismatches mean that the frequency of directly alloreactive recipient T cells is high. Furthermore, a direct cytotoxic T-cell attack on graft cells can be made only by T cells that recognize the graft MHC molecules directly. Nonetheless, T cells with specificity for alloantigens presented on self MHC can contribute to graft rejection by activating macrophages, which cause tissue injury and fibrosis. T cells with indirect allospecificity are also likely to be important in the development of an antibody response to a graft. Antibodies produced against nonself antigens from the same species are known as alloantibodies.

1	Fig. 15.49 Direct and indirect pathways of allorecognition contribute to graft rejection. Dendritic cells from an organ graft stimulate both the direct and indirect pathways of allorecognition when they travel from the graft to secondary lymphoid tissues. The upper panel shows how the allogeneic HLA class I and II allotypes of donor type on a donor dendritic cell (donor DC) will interact directly with the T‑cell receptors of alloreactive CD4 and CD8 T cells of the recipient (direct allorecognition). The lower panel shows how the death of the same antigen‑presenting cell produces membrane vesicles containing the allogeneic HLA class I and II allotypes, which are then endocytosed by the recipient’s dendritic cells (recipient DC). Peptides derived from the donor’s HLA molecules (yellow) can then be presented by the recipient’s HLA molecules (orange) to peptide‑specific T cells (indirect allorecognition). Presentation by HLA class II molecules to CD4 T cells is shown here. Peptides

1	can then be presented by the recipient’s HLA molecules (orange) to peptide‑specific T cells (indirect allorecognition). Presentation by HLA class II molecules to CD4 T cells is shown here. Peptides derived from donor HLA can also be presented by recipient HLA class I molecules to CD8 T cells (not shown).

1	Graft failure dead graft Antibodies against donor blood group antigens bind vascular endothelium of graft, initiating an in˜ammatory response that occludes blood vessels Graft becomes engorged and purple-colored because of hemorrhage Fig. 15.50 Preexisting antibody against donor graft antigens can cause hyperacute graft rejection. Prior to transplantation, some recipients have made antibodies that react with donor ABO or HLA class I antigens. When the donor organ is grafted into such a recipient, these antibodies bind to the vascular endothelium in the graft, initiating the complement and clotting cascades. Blood vessels in the graft become obstructed by clots and leak, causing hemorrhage of blood into the graft. The graft becomes engorged, turns purple from the presence of deoxygenated blood, and dies. 15-33 Antibodies that react with endothelium cause hyperacute graft rejection.

1	Antibody responses are an important potential cause of graft rejection. Preexisting alloantibodies against blood group antigens and polymorphic MHC antigens can cause rapid rejection of transplanted organs in a complement-dependent reaction that can occur within minutes of transplantation. This type of reaction is known as hyperacute graft rejection. Most grafts that are transplanted routinely in clinical medicine are vascularized organ grafts linked directly to the recipient’s circulation. In some cases the recipient may have preexisting antibodies against donor graft antigens. Antibodies of the ABO type can bind to all tissues, not just red blood cells. In addition, antibodies against other antigens can be produced in response to a previous transplant or a blood transfusion. All such preexisting antibodies can cause rapid rejection of vascularized grafts because they react with antigens on the vascular endothelial cells of the graft and initiate the complement and blood clotting

1	preexisting antibodies can cause rapid rejection of vascularized grafts because they react with antigens on the vascular endothelial cells of the graft and initiate the complement and blood clotting cascades. The vessels of the graft become blocked, or thrombosed, causing its rapid destruction. Such grafts become engorged and purple-colored from hemorrhaged blood, which becomes deoxygenated (Fig. 15.50). This problem can be avoided by ABO-matching as well as cross-matching donor and recipient. Cross-matching involves determining whether the recipient has antibodies that react with the white blood cells of the donor. Antibodies of this type, when found, have hitherto been considered a serious contraindication to transplantation of most solid organs, because in the absence of any treatment they lead to near-certain hyperacute rejection.

1	For reasons that are incompletely understood, some transplanted organs, particularly the liver, are less susceptible to this type of injury, and can be transplanted despite ABO incompatibilities. In addition, the presence of donor-specific MHC alloantibodies and a positive cross-match are no longer considered an absolute contraindication for transplantation, as treatment with intravenous immunoglobulin has been successful in a proportion of patients in whom antibodies against the donor tissue were already present.

1	A similar problem prevents routine use of animal organs—xenografts—in transplantation. If xenografts could be used, it would circumvent a limitation in organ replacement therapy: the shortage of donor organs. Pigs have been suggested as a source of organs for xenografting, but most humans have antibodies that react with a ubiquitous cell-surface carbohydrate antigen (α-Gal) of other mammalian species, including pigs. When pig xenografts are placed in humans, these antibodies trigger hyperacute rejection by binding graft endothelial cells and initiating complement and clotting cascades. The problem of hyperacute rejection is exacerbated in xenografts because complement-regulatory proteins such as CD59, DAF (CD55), and MCP (CD46) (see Section 2-16) work less efficiently across a species barrier. A recent step toward xenotransplantation has been the development of transgenic pigs expressing human DAF as well as pigs that lack α-Gal. These approaches might one day reduce or eliminate

1	barrier. A recent step toward xenotransplantation has been the development of transgenic pigs expressing human DAF as well as pigs that lack α-Gal. These approaches might one day reduce or eliminate hyperacute rejection in xenotransplantation.

1	15-34 Late failure of transplanted organs is caused by chronic injury to the graft. The success of immunosuppression means that about 90% of cadaveric kidney grafts are still functioning a year after transplantation. There has, however, been little improvement in rates of long-term graft survival: the half-life for functional survival of renal allografts remains about 8 years. Although traditionally the late failure of a transplanted organ has been termed chronic rejection, it is typically difficult to determine whether the cause of chronic allograft injury involves specific immune alloreactivity, nonimmune injury, or both.

1	The pattern of chronic injury to transplanted organs is variable, depending on the tissue. A major component of late failure of vascularized transplanted organs is a chronic reaction called chronic allograft vasculopathy, which is a prominent cause of injury in heart and kidney allografts. This is charac-terized by concentric arteriosclerosis of graft blood vessels, which leads to hypoperfusion of the graft and its eventual fibrosis and atrophy (Fig. 15.51). Multiple mechanisms may contribute to this form of vascular injury, although the major cause is thought to be recurring, subclinical acute rejection events, whether due to the development of allospecific antibodies reactive to the vascular endothelium of the graft (so-called donor-specific antibodies), or to allograft-reactive effector T cells, or both. Some forms of immunosuppressive therapy (for example, calcineurin inhibitors such as cyclosporin) also cause vascular injury, although this is typically more limited to very small

1	T cells, or both. Some forms of immunosuppressive therapy (for example, calcineurin inhibitors such as cyclosporin) also cause vascular injury, although this is typically more limited to very small arteries and causes a different pattern of injury, referred to as arteriolar hyalinosis, that is marked by proteinaceous deposits that narrow the vascular lumen. In transplanted livers, chronic rejection is associated with loss of bile ducts, the so-called ‘vanishing bile duct syndrome,’ whereas in transplanted lungs, the major cause of late organ failure is accumulation of scar tissue in the bron-chioles, termed bronchiolitis obliterans. Alloreactive responses can occur months to years after transplantation, and may be associated with gradual loss of graft function that is hard to detect clinically. Other important causes of chronic graft dysfunction include: ischemia– reperfusion injury, which can promote sterile inflammatory signals at the time of grafting due to the restoration of blood

1	Other important causes of chronic graft dysfunction include: ischemia– reperfusion injury, which can promote sterile inflammatory signals at the time of grafting due to the restoration of blood flow after a period of poor perfusion of the organ to be transplanted; viral infections that emerge as a result of immunosuppression; and recurrence of the same disease in the allograft that destroyed the original organ. Irrespective of etiology, chronic allograft injury is typically irreversible and progressive, ultimately leading to complete failure of allograft function. 15-35 A variety of organs are transplanted routinely in clinical medicine. Three major advances have made it possible to use organ transplantation rou-tinely in the clinic. First, surgical techniques for performing organ replace-ment have advanced to the point where such surgeries are now relatively routine in most major medical centers. Second, networks of transplantation centers have been organized to procure healthy

1	have advanced to the point where such surgeries are now relatively routine in most major medical centers. Second, networks of transplantation centers have been organized to procure healthy organs that become available from cadaveric donors. Third, the use of powerful immunosuppressive drugs Endothelial injury enables immune effectors to enter the wall of the artery and to in˜ict increasing damage

1	Fig. 15.51 Chronic rejection in the blood vessels of a transplanted kidney. Left‑hand panel: chronic rejection is initiated by the interaction of anti‑HLA class I alloantibodies with blood vessels of the transplanted organ. Antibodies bound to endothelial cells recruit Fc receptor‑bearing monocytes and neutrophils. Right‑hand panel: accumulating damage leads to thickening of the internal elastic lamina and to infiltration of the underlying intima with smooth muscle cells, macrophages, granulocytes, alloreactive T cells, and antibodies. The net effect is to narrow the lumen of the blood vessel and create a chronic inflammation that intensifies tissue remodeling. Eventually the vessel becomes obstructed, ischemic, and fibrotic.

1	Rabbit anti‑thymoglobulin and anti‑CD52 monoclonal antibody (alemtuzumab) are used to deplete T cells and other leukocytes before transplantation. Anti‑CD3 monoclonal antibody prevents the generation of signaling by the T‑cell receptor complex, whereas cyclosporin and tacrolimus interfere with the translocation of nuclear factor of activated T cells (NFAT) to the nucleus by inhibiting calcineurin. The CTLA‑4–Fc fusion protein belatacept binds B7 and prevents the generation of co‑stimulation via CD28. Basiliximab, an anti‑CD25 antibody, binds to the high‑affinity IL‑2 receptor on partially activated T cells and prevents IL‑2 signaling. Sirolimus interferes with activation of the mTOR cascade, which is required for differentiation of effector T cells. Azathioprine and mycophenolate inhibit the replication and proliferation of activated T cells.

1	that inhibit T-cell activation, thereby limiting the development of anti-allograft effector T cells and antibodies, has markedly increased graft survival rates (Fig. 15.52). The different organs or tissues that are frequently transplanted and allograft survival rates are listed in Fig. 15.53. The most frequently transplanted solid organ is the kidney, the organ first successfully transplanted between identical twins in the 1950s. Transplantation of the cornea is even more frequent; this tissue is a special case because it is not vascularized, and corneal grafts between unrelated people are usually successful without immunosuppression.

1	Many problems other than graft rejection are associated with organ transplantation. First, donor organs are difficult to obtain. Second, the disease that destroyed the transplant recipient’s organ might also destroy the graft, as in the destruction of pancreatic β cells in autoimmune diabetes. Third, the immunosuppression required to prevent graft rejection increases the risk of cancer and infection. The problems most amenable to scientific solution are the development of more effective means of immunosuppression that prevent rejection with minimal impairment of more generalized immunity, the induction of graft-specific tolerance, and the development of xenografts as a practical solution to organ availability.

1	The numbers of organ and tissue grafts performed in the United States in 2014 are shown. HSC, hematopoietic stem cells (includes bone marrow, peripheral blood HSCs, and cord blood transplants). *Number of grafts includes multiple organ grafts (for example, kidney and pancreas, or heart and lung). For solid organs, 5‑year survival of the transplanted graft is based on transplants performed between 2002 and 2007. Data from the United Network for Organ Sharing. #Kidney survival listed (81.4%) is for kidneys from living donors; 5‑year survival for cadaveric donor transplants is 69.1%. †Pancreas survival listed (53.4%) is when transplanted alone; 5‑year survival when transplanted with a kidney is 73.5%. ** Includes autologous and allogeneic transplants. ‡Successful HSC engraftment is assessed within weeks of transplant, not years. Nearly all solid organ grafts (e.g., kidney, heart) require long‑term immunosuppression. 15-36 The converse of graft rejection is graft-versus-host disease.

1	15-36 The converse of graft rejection is graft-versus-host disease. Transplantation of hematopoietic stem cells (HSCs) from peripheral blood, bone marrow, or fetal cord blood is a successful therapy for some tumors derived from hematopoietic cells, such as certain leukemias and lymphomas. By replacing genetically defective stem cells with normal donor ones, HSC transplantation can also be used to cure some primary immunodeficiencies (see Chapter 13) and other inherited blood cell disorders, such as severe forms of thalassemia. In leukemia therapy, the recipient’s bone marrow, the source of the leukemia, must first be destroyed by a combination of irradiation and aggressive cytotoxic chemotherapy.

1	One of the major complications of allogeneic HSC transplantation is graftversus-host disease (GVHD), in which mature donor T cells present in preparations of HSCs recognize the tissues of the recipient as foreign, causing a severe inflammatory disease in multiple tissues, but particularly involving the skin, intestines, and liver and characterized by rashes, diarrhea, and liver dysfunction (Fig. 15.54). Because the consequences of GVHD are particularly aggressive when there is mismatch of MHC class I or class II antigens, HLA matching between donor and recipient is more critical than in solid organ transplantation. Most transplants are therefore undertaken only when the donor and recipient are HLA-matched siblings or, less frequently, when there is an HLA-matched unrelated donor. Therefore, GVHD mostly occurs in the context of disparities between minor histocompatibility antigens, so immunosuppression must be used in every HSC transplant.

1	The presence of alloreactive donor T cells can be demonstrated experimentally by the mixed lymphocyte reaction (MLR), in which lymphocytes from a potential donor are mixed with irradiated lymphocytes from the recipient. If the donor lymphocytes contain naive T cells that recognize alloantigens on the recipient lymphocytes, they will proliferate or kill the recipient target cells (Fig. 15.55). However, the limitation of the MLR in the selection of HSC donors is that the test does not accurately quantify alloreactive T cells. A more accurate test is a version of the limiting-dilution assay (see Appendix I, Section A-21), which precisely counts the frequency of alloreactive T cells. Effector CD4 and CD8 T cells enter host tissues and cause injury T cells circulate in blood to secondary lymphoid tissues. Alloreactive cells interact with dendritic cells and proliferate efferentlymphskinlymphnodebloodvesselAllogeneic hematopoietic cell transplant contains mature and memory T cells

1	Fig. 15.54 Graft-versus-host disease is due to donor T cells in the graft that attack the recipient’s tissues. After bone marrow transplantation, any mature donor CD4 and CD8 T cells present in the graft that are specific for the recipient’s HLA allotypes become activated in secondary lymphoid tissues. Effector CD4 and CD8 T cells move into the circulation and preferentially enter and attack tissues of the graft recipient, particularly epithelial cells of the skin, intestines, and liver that have been damaged by the conditioning regimen of chemotherapy and irradiation prior to transplantation.

1	Fig. 15.55 The mixed lymphocyte reaction (MLR) can be used to detect histoincompatibility. Peripheral blood mononuclear cells, which include lymphocytes and monocytes, are isolated from the two individuals to be tested. The cells from the person who serves as the stimulator (yellow) are first irradiated to prevent their proliferation. Then they are mixed with the cells from the other person, who serves as the responder (blue), and cultured for 5 days (top panel). In the culture, responder lymphocytes are stimulated by allogeneic HLA class I and II molecules expressed by the stimulator’s monocytes and the dendritic cells that differentiate from the monocytes. The stimulated lymphocytes proliferate and differentiate into effector cells. Five days after mixing, the culture is assessed for T‑cell proliferation (bottom left panel), which is due to CD4 T cells recognizing HLA class II differences, and for cytotoxic T cells (bottom right panel) produced in response to HLA class I

1	for T‑cell proliferation (bottom left panel), which is due to CD4 T cells recognizing HLA class II differences, and for cytotoxic T cells (bottom right panel) produced in response to HLA class I differences. The mixed lymphocyte reaction is instrumental in distinguishing MHC class II from MHC class I.

1	Although GVHD is harmful to the recipient of an HSC transplant, it can have beneficial effects that are crucial to the success of the therapy. Much of the therapeutic effect of HSC transplantation for leukemia can be due to a graft-versus-leukemia effect, in which the donor T cells in the allogeneic preparations of HSCs recognize minor histocompatibility antigens expressed by the leukemic cells and kill the leukemic cells. One of the treatment options for suppressing the development of GVHD is the elimination of mature T cells from the preparations of donor HSCs in vitro before transplantation, thereby removing alloreactive T cells. Those T cells that subsequently mature from the donor marrow in vivo in the recipient are tolerant to the recipient’s antigens. Although the elimination of GVHD has benefits for the patient, there is an increased risk of leukemic relapse, which provides strong evidence in support of the graft-versus-leukemia effect.

1	Immunodeficiency is another complication of donor T-cell depletion. Because most of the recipient’s T cells are destroyed by the combination of chemotherapy and irradiation used to treat the recipient before transplant, donor T cells are the major source for reconstituting a mature T-cell repertoire early after transplant. This is particularly true in adults, who have poor residual thymic function and therefore a limited ability to repopulate their T-cell repertoire from T-cell precursors. Thus, if too many T cells are depleted from the graft, transplant recipients experience, and can die from, opportunistic infections. The need to balance the beneficial effects of the graft-versus-leukemia effect and immunocompetence with the adverse effects of GVHD caused by donor T cells has spawned much research. One particularly promising approach is to prevent donor T cells from reacting with recipient antigens that they could meet shortly after the transplant. This is accomplished by depleting

1	research. One particularly promising approach is to prevent donor T cells from reacting with recipient antigens that they could meet shortly after the transplant. This is accomplished by depleting the recipient’s antigen-presenting cells. Here, the donor T cells are not activated during the initial inflammation that accompanies the transplant, and thereafter they do not promote GVHD. However, it is unclear whether there would be a graft-versus-leukemia effect in this context.

1	15-37 Regulatory T cells are involved in alloreactive immune responses.

1	As in all immune responses, regulatory T cells are thought to have an important immunoregulatory role in the alloreactive immune responses involved in graft rejection. Experiments on the transplantation of allogeneic HSCs in mice have thrown some light on this question. Here, depletion of CD25+ Treg cells in either the recipient or the HSC graft before transplantation accelerated the onset of GVHD and subsequent death. In contrast, supplementing the graft with either fresh or ex vivo expanded Treg cells delayed, or even prevented, death from GVHD, with similar results in early human studies. Also, treatment with a low dose of IL-2, which is thought to preferentially expand Treg cells, has shown positive effects in preventing GVHD. Similar observations have been made in experimental mouse models of solid organ transplantation, where the transfer of either naturally occurring or induced Treg cells significantly delayed allograft rejection. These experiments suggest that enriching or

1	models of solid organ transplantation, where the transfer of either naturally occurring or induced Treg cells significantly delayed allograft rejection. These experiments suggest that enriching or generating Treg cells in preparations of donor HSCs might provide a possible therapy for GVHD in the future.

1	Another class of regulatory T cells, CD8+CD28– T cells, have an anergic phenotype and are thought to maintain T-cell tolerance indirectly by inhibiting the capacity of antigen-presenting cells to activate CD4+ T cells. These cells have been isolated from transplant patients, and can be distinguished from alloreactive CD8 T cells because they do not display cytotoxic activity against donor cells and express high levels of the inhibitory killer receptor CD94 (see Section 3-25). This suggests that CD8+CD28– T cells interfere with the activation of antigen-presenting cells and have a role in the maintenance of transplant tolerance. 15-38 The fetus is an allograft that is tolerated repeatedly.

1	15-38 The fetus is an allograft that is tolerated repeatedly. All of the transplants discussed so far are the result of advances in modern medicine. However, one ‘foreign’ tissue that is repeatedly grafted and tolerated is the mammalian fetus. The fetus carries paternal MHC and minor histocompatibility antigens that differ from those of the mother (Fig. 15.56), and yet a mother can successfully bear many children expressing the same nonself proteins derived from the father. The mysterious lack of fetal rejection has consistently puzzled immunologists, and no comprehensive explanation has yet emerged. One problem is that acceptance of the fetal allograft is so much the norm that it is difficult to study the mechanism that prevents rejection; if the mechanism for rejecting the fetus is rarely activated, how can one analyze the mechanisms that control it?

1	The mechanisms contributing to ‘fetomaternal tolerance’ are likely multifactorial and redundant. Although it has been proposed that the fetus is simply not recognized as foreign, women who have borne children often make antibodies directed against the father’s MHC and red blood cell antigens. However, the placenta, which is a fetus-derived tissue, seems to sequester the fetus from the mother’s T cells. The outer layer of the placenta—the interface between fetal and maternal tissues—is the trophoblast. This does not express MHC class II molecules, and expresses only low levels and a restricted subset of MHC class I molecules, making it resistant to direct alloantigen recognition by maternal T cells. Tissues lacking MHC class I expression are, however, vulnerable to attack by NK cells (see Section 3-25). The trophoblast might be protected from attack by NK cells by the expression of a nonclassical and minimally polymorphic HLA class I molecule, HLA-G, which has been shown to inhibit NK

1	Section 3-25). The trophoblast might be protected from attack by NK cells by the expression of a nonclassical and minimally polymorphic HLA class I molecule, HLA-G, which has been shown to inhibit NK killing.

1	The placenta may also inhibit the mother’s T cells by an active mechanism of nutrient depletion. The enzyme indoleamine 2,3-dioxygenase (IDO) is expressed at a high level by cells at the maternal–fetal interface. This enzyme depletes the essential amino acid tryptophan at this site, and T cells starved of tryptophan show reduced responsiveness. Inhibition of IDO in pregnant mice, using the inhibitor 1-methyltryptophan, causes rapid rejection of allogeneic, but not syngeneic, fetuses.

1	The cytokine milieu at the maternal–fetal interface also contributes to fetal tolerance. Both the uterine epithelium and the trophoblast secrete TGF-β and IL-10. This combination of cytokines suppresses the development of effector T cells in favor of iTreg cells (see Section 9-23). Regulatory T cells are increased during pregnancy, including iTreg cells in the placenta. These cells are important for suppressing responses to the fetus in mice, as iTreg deficiency promotes fetal resorption—the equivalent of spontaneous abortion in humans—as does induction of TH1-inducing cytokines (for example, IFN-γ and IL-12). Provocatively, a regulatory element that controls FoxP3 expression in iTreg

1	Fig. 15.56 The fetus is an allograft that is not rejected. With very few exceptions, the mother and father in human families have different HLA types (top panel). When the mother becomes pregnant she carries for 9 months a fetus that expresses one HLA haplotype of maternal origin (pink) and one HLA haplotype of paternal origin (blue) (bottom panel). Although the paternal HLA class I and II molecules expressed by the fetus are alloantigens against which the mother’s immune system has the potential to respond, the fetus does not provoke such a response during pregnancy and is protected from preexisting alloreactive antibodies or T cells. Even when the mother bears several children to the same father, no sign of immunological rejection is seen.

1	cells, but is not required for FoxP3 expression by nTreg cells, has been found only in placental mammals. This suggests that iTreg cells might have evolved to play an important role in maternal–fetal tolerance. Finally, stromal cells of the specialized maternal uterine tissue that directly interfaces with the placenta— the decidua—appear to repress the local expression of key T cell-attracting chemokines. Collectively, then, both maternal and fetal factors contribute to the formation of an immunologically privileged site akin to other sites of local immune suppression that allow prolonged acceptance of tissue grafts, such as the eye (see Section 15-5). Summary.

1	Summary. Clinical transplantation is now an everyday reality, its success built on MHC matching, immunosuppressive drugs, and advances in surgical techniques. However, even accurate MHC matching does not prevent graft rejection; other genetic differences between host and donor can result in allogeneic proteins whose peptides are presented by MHC molecules on the grafted tissue, and responses to these can lead to rejection. Because we lack the ability to specifically suppress the response to the graft without compromising host defense, most transplants require generalized immunosuppression of the recipient that can increase the risk of cancer and infection. The fetus is a natural allograft that must be accepted for the species to survive. A better understanding of tolerance to the fetus could ultimately provide insights for inducing specific allograft tolerance in transplantation. Summary to Chapter 15.

1	Ideally, the effector functions of the immune system would be targeted only to foreign pathogens and never to self tissues. In practice, because foreign and self proteins are chemically similar, strict discrimination between self and nonself is impossible. Yet the immune system maintains tolerance to self. This is accomplished by layers of regulation, all of which use surrogate markers to distinguish self from nonself to properly direct the immune response. When these mechanisms break down, autoimmune disease can result. Minor breaches of single regulatory barriers probably occur every day but are quelled by the effects of other regulatory layers; thus, tolerance operates at the level of the overall immune system. For disease to occur, multiple layers of tolerance have to be overcome and the effect needs to be chronic. These layers begin with central tolerance in the bone marrow and thymus, and include peripheral mechanisms such as anergy, cytokine deviation, and regulatory T cells.

1	the effect needs to be chronic. These layers begin with central tolerance in the bone marrow and thymus, and include peripheral mechanisms such as anergy, cytokine deviation, and regulatory T cells. Sometimes immune responses do not occur simply because the antigens are not available, as in immune sequestration.

1	Perhaps because of selective pressure to mount effective immune responses to pathogens, the dampening of immune responses to promote self-tolerance is limited and prone to failure. Genetic predisposition has an important role in determining which individuals will develop an autoimmune disease. Environmental forces also have a significant role, because even identical twins are not always both affected by the same autoimmune disease. Influences from the environment include infections, toxins, and chance events.

1	When self-tolerance is broken and autoimmune disease ensues, the effector mechanisms are quite similar to those employed in responses to pathogens. Although the details vary from disease to disease, both antibody and T cells can be involved. Much has been learned about immune responses made to tissue antigens by examining the response to nonself transplanted organs and tissues; lessons learned in the study of graft rejection apply to autoimmunity and vice versa. Transplantation has brought on syndromes of rejection that are in many ways similar to autoimmune disease, but the targets are either major or minor histocompatibility antigens. T cells are the main effectors in graft rejection and graft-versus-host disease, although antibodies can also contribute.

1	For each of the undesirable responses discussed here, the question is how to control the response without adversely affecting protective immunity to infection. The answer may lie in a more complete understanding of the regulation of the immune response, especially the suppressive mechanisms important in tolerance. The deliberate control of the immune response is examined further in Chapter 16. Questions. 15.1 True or False: Inflammatory bowel disease—Crohn's disease and ulcerative colitis—is a disease in which the adaptive immune system causes tissue damage in response to self antigens. 15.2 Matching: Match the following monogenic autoimmune diseases with the associated defective gene. 15.3 Multiple Choice: Which of the following statements is incorrect? A. The autoantibodies induced by procainamide, a drug widely used to treat abnormal heart rhythms, are similar to the autoantibodies that characterize systemic lupus erythematosus.

1	A. The autoantibodies induced by procainamide, a drug widely used to treat abnormal heart rhythms, are similar to the autoantibodies that characterize systemic lupus erythematosus. B. Inflammatory mediators released during the course of an infection can lead to activation of self‑reactive lymphocytes and thus cause an autoimmune response. C. Crohn’s disease and Blau syndrome are both strongly associated with loss‑of‑function mutations in NOD2, among other causes. D. ATG16L1 and IRGM are genes that contribute to autophagy under normal circumstances, and defects in these have been linked to Crohn’s disease. 15.4 Multiple Choice: Which of the following options correctly describe a transplantation scenario? A. A syngeneic skin graft from a young mouse is rejected by an adult mouse. B. An allogeneic skin graft from a male mouse is not rejected by a female mouse. C. A syngeneic skin graft from a male mouse is rejected by a female mouse.

1	B. An allogeneic skin graft from a male mouse is not rejected by a female mouse. C. A syngeneic skin graft from a male mouse is rejected by a female mouse. D. A skin autograft is rejected 3 weeks after transplantation. 15.5 Short Answer: How can graft‑versus‑host disease (GVHD) be of benefit to patients with leukemia? 15.6 Multiple Choice: Which of the following options incorrectly describes a mechanism used to prevent fetal rejection? A. High expression of 2,3‑dioxygenase (IDO), which starves T cells of tryptophan B. Absence of MHC class II expression and low levels of MHC class I expression by the trophoblast C. Downregulation of HLA‑G expression by the trophoblast D. Secretion of TGF‑β and IL‑10 by the uterine epithelium and the trophoblast. 15.7 Multiple Choice: Which of the following is not a mechanism by which immunologically privileged sites maintain tolerance? A. Exclusion of effector T cells during infection

1	15.7 Multiple Choice: Which of the following is not a mechanism by which immunologically privileged sites maintain tolerance? A. Exclusion of effector T cells during infection B. Tissue barriers that exclude naive lymphocytes (for example, the blood–brain barrier) C. Anti‑inflammatory cytokine production (for example, TGF‑β) D. Expression of Fas ligand to induce apoptosis of Fas‑bearing effector lymphocytes E. Decreased communication via conventional lymphatics 15.8 Multiple Choice: Which of the following is not a mechanism of peripheral tolerance? A. Anergy B. Negative selection C. Induction of Tregs D. Deletion E. Suppression by Tregs 15.9 Short Answer: The phenomenon of epitope spreading occurs in systemic lupus erythematosus (SLE), where anti‑DNA autoantibodies are present and may progress to the production of anti‑histone antibodies. Describe mechanistically how this occurs.

1	15.10 Short Answer: Autoimmune polyendocrinopathy– candidiasis–ectodermal dysplasia (APECED) is caused by defects in the transcription factor AIRE, which result in impaired expression of peripheral genes and reduced negative selection (that is, impaired central tolerance). Patients afflicted with APECED suffer destruction of multiple endocrine tissues and exhibit impaired antifungal immunity. However, these autoimmune phenomena take time to develop and do not develop in all potential organ targets in all patients. Explain why this is the case.

1	15.11 Fill-in-the-Blanks: Autoantibodies that develop in certain autoimmune disorders can act as either antagonists or agonists, depending on whether they inhibit or stimulate a function. In _______________, autoantibodies against the _______________ receptor block its function in the neuromuscular junction, resulting in a syndrome of muscle weakness. Another example is _________________, where autoantibodies against the __________________ receptor stimulate excessive production of thyroid hormone. 15.12 Matching: Match the autoimmune disease with its pathophysiology: A. Rheumatoid arthritis i. Chronic hepatitis C infection leading to production of immune complexes that deposit in joints and tissues Section references. 15-1 A critical function of the immune system is to discriminate self from nonself. Ehrlich, P., and Morgenroth, J.: On haemolysins, in Himmelweit, F. (ed): The Collected Papers of Paul Ehrlich. London, Pergamon, 1957, 246–255.

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1	15-23 Genetic variants that impair innate immune responses can predispose to T cell-mediated chronic inflammatory disease. Cadwell, K., Liu, J.Y., Brown, S.L., Miyoshi, H., Loh, J., Lennerz, J.K., Kishi, C., Kc, W., Carrero, J.A., Hunt, S., et al.: A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature 2008, 456:259–263. Eckmann, L., and Karin, M.: NOD2 and Crohn’s disease: loss or gain of function? Immunity 2005, 22:661–667. Xavier, R.J., and Podolsky, D.K.: Unravelling the pathogenesis of inflammatory bowel disease. Nature 2007, 448:427–434. 15-24 External events can initiate autoimmunity. Klareskog, L., Padyukov, L., Ronnelid, J., and Alfredsson, L.: Genes, environment and immunity in the development of rheumatoid arthritis. Curr. Opin. Immunol. 2006 18:650–655.

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1	Sfriso, P., Ghirardello, A., Botsios, C., Tonon, M., Zen, M., Bassi, N., Bassetto, F., and Doria, A.: Infections and autoimmunity: the multifaceted relationship. J. Leukocyte Biol. 2010, 87:385–395. Takeuchi, O., and Akira, S.: Pattern recognition receptors and inflammation. Cell 2010, 140:805–820. 15-26 Cross-reactivity between foreign molecules on pathogens and self molecules can lead to anti-self responses and autoimmune disease. Barnaba, V., and Sinigaglia, F.: Molecular mimicry and T cell-mediated autoimmune disease. J. Exp. Med. 1997, 185:1529–1531. Rose, N.R.: Infection, mimics, and autoimmune disease. J. Clin. Invest. 2001, 107:943–944. Rose, N.R., Herskowitz, A., Neumann, D.A., and Neu, N.: Autoimmune myocarditis: a paradigm of post-infection autoimmune disease. Immunol. Today 1988, 9:117–120. 15-27 Drugs and toxins can cause autoimmune syndromes. 15-28 Random events may be required for the initiation of autoimmunity.

1	15-27 Drugs and toxins can cause autoimmune syndromes. 15-28 Random events may be required for the initiation of autoimmunity. Eisenberg, R.A., Craven, S.Y., Warren, R.W., and Cohen, P.L.: Stochastic control of anti-Sm autoantibodies in MRL/Mp-lpr/lpr mice. J. Clin. Invest. 1987, 80:691–697. Yoshida, S., and Gershwin, M.E.: Autoimmunity and selected environmental factors of disease induction. Semin. Arthritis Rheum. 1993, 22:399–419. 15-29 Graft rejection is an immunological response mediated primarily by T cells. Cornell, L.D., Smith, R.N., and Colvin, R.B.: Kidney transplantation: mechanisms of rejection and acceptance. Annu. Rev. Pathol. 2008, 3:189–220. Wood, K.J., and Goto, R.: Mechanisms of rejection: current perspectives. Transplantation. 2012, 93:1–10. 15-30 Transplant rejection is caused primarily by the strong immune response to nonself MHC molecules.

1	15-30 Transplant rejection is caused primarily by the strong immune response to nonself MHC molecules. Macdonald, W.A., Chen, Z., Gras, S., Archbold, J.K., Tynan, F.E., Clements, C.S., Bharadwaj, M., Kjer-Nielsen, L., Saunders, P.M., Wilce, M.C.J., et al.: T cell allorecognition via molecular mimicry. Immunity 2009, 31:897–908. Macedo, C., Orkis, E.A., Popescu, I., Elinoff, B.D., Zeevi, A., Shapiro, R., Lakkis, F.G., and Metes, D.: Contribution of naive and memory T-cell populations to the human alloimmune response. Am. J. Transplant. 2009, 9:2057–2066. Opelz, G., and Wujciak, T.: The influence of HLA compatibility on graft survival after heart transplantation. The Collaborative Transplant Study. N. Engl. J. Med. 1994, 330:816–819. 15-31 In MHC-identical grafts, rejection is caused by peptides from other alloantigens bound to graft MHC molecules.

1	15-31 In MHC-identical grafts, rejection is caused by peptides from other alloantigens bound to graft MHC molecules. Dierselhuis, M., and Goulmy, E.: The relevance of minor histocompatibility antigens in solid organ transplantation. Curr Opin Organ Transplant. 2009, 14:419–425. den Haan, J.M., Meadows, L.M., Wang, W., Pool, J., Blokland, E., Bishop, T.L., Reinhardus, C., Shabanowitz, J., Offringa, R., Hunt, D.F., et al.: The minor histocompatibility antigen HA-1: a diallelic gene with a single amino acid polymorphism. Science 1998, 279:1054–1057. Mutis, T., Gillespie, G., Schrama, E., Falkenburg, J.H., Moss, P., and Goulmy, E.: Tetrameric HLA class I-minor histocompatibility antigen peptide complexes demonstrate minor histocompatibility antigen-specific cytotoxic T lymphocytes in patients with graft-versus-host disease. Nat. Med. 1999, 5:839–842. 15-32 There are two ways of presenting alloantigens on the transplanted donor organ to the recipient’s T lymphocytes.

1	15-32 There are two ways of presenting alloantigens on the transplanted donor organ to the recipient’s T lymphocytes. Jiang, S., Herrera, O., and Lechler, R.I.: New spectrum of allorecognition pathways: implications for graft rejection and transplantation tolerance. Curr. Opin. Immunol. 2004, 16:550–557. Safinia, N., Afzali, B., Atalar, K., Lombardi, G., and Lechler, R.I.: T-cell alloimmunity and chronic allograft dysfunction. Kidney Int. 2010, 78(Suppl 119):S2–S12. Lakkis, F.G., Arakelov, A., Konieczny, B.T., and Inoue, Y.: Immunologic ignorance of vascularized organ transplants in the absence of secondary lymphoid tissue. Nat. Med. 2000, 6:686–688. 15-33 Antibodies that react with endothelium cause hyperacute graft rejection. Griesemer, A., Yamada, K., and Sykes, M.: Xenotransplantation: immunological hurdles and progress toward tolerance. Immunol. Rev. 2014, 258:241–258.

1	Griesemer, A., Yamada, K., and Sykes, M.: Xenotransplantation: immunological hurdles and progress toward tolerance. Immunol. Rev. 2014, 258:241–258. Montgomery, R.A., Cozzi, E., West, L.J., and Warren, D.S.: Humoral immunity and antibody-mediated rejection in solid organ transplantation. Semin. Immunol. 2011, 23:224–234. Williams, G.M., Hume, D.M., Hudson, R.P., Jr, Morris, P.J., Kano, K., and Milgrom, F.: ‘Hyperacute’ renal-homograft rejection in man. N. Engl. J. Med. 1968, 279:611–618. 15-34 Late failure of transplanted organs is caused by chronic injury to the graft. Smith, R.N., and Colvin, R.B.: Chronic alloantibody mediated rejection. Semin. Immunol. 2012, 24:115–121. Libby, P., and Pober, J.S.: Chronic rejection. Immunity 2001, 14:387–397. 15-35 A variety of organs are transplanted routinely in clinical medicine.

1	15-35 A variety of organs are transplanted routinely in clinical medicine. Ekser, B., Ezzelarab, M., Hara, H., van der Windt, D.J., Wijkstrom, M., Bottino, R., Trucco, M., and Cooper, D.K.C.: Clinical xenotransplantation: the next medical revolution? Lancet 2012, 379:672–683. Lechler, R.I., Sykes, M., Thomson, A.W., and Turka, L.A.: Organ transplantation—how much of the promise has been realized? Nat. Med. 2005, 11:605–613. Ricordi, C., and Strom, T.B.: Clinical islet transplantation: advances and immunological challenges. Nat. Rev. Immunol. 2004, 4:259–268. 15-36 The converse of graft rejection is graft-versus-host disease. Blazar, B.R., Murphy, W.J., and Abedi, M.: Advances in graft-versus-host disease biology and therapy. Nat. Rev. Immunol. 2012, 12:443–458. Shlomchik, W.D.: Graft-versus-host disease. Nat. Rev. Immunol. 2007, 7:340–352. 15-37 Regulatory T cells are involved in alloreactive immune responses.

1	15-37 Regulatory T cells are involved in alloreactive immune responses. Ferrer, I.R., Hester, J., Bushell, A., and Wood, K.J.: Induction of transplantation tolerance through regulatory cells: from mice to men. Immunol. Rev. 2014, 258:102–116. Qin, S., Cobbold, S.P., Pope, H., Elliott, J., Kioussis, D., Davies, J., and Waldmann, H.: “Infectious” transplantation tolerance. Science 1993, 259:974–977. Tang, Q., and Bluestone, J.A.: Regulatory T-cell therapy in transplantation: moving to the clinic. Cold Spring Harb. Perspect. Med. 2013, 3:1–15. 15-38 The fetus is an allograft that is tolerated repeatedly. Erlebacher, A.: Mechanisms of T cell tolerance towards the allogeneic fetus. Nat. Rev. Immunol. 2013, 13:23–33. Samstein, R.M., Josefowicz, S.Z., Arvey, A., Treuting, P.M., and Rudensky A.Y.: Extrathymic generation of regulatory T cells in placental mammals mitigates maternal-fetal conflict. Cell 2012, 150:29–38. Manipulation of the Immune Response

1	In this chapter, we consider the various ways in which the immune system can be manipulated to suppress unwanted immune responses in the form of autoimmunity, allergy, and graft rejection, or to stimulate protective immune responses. Intentional manipulations of the immune system date back over 500 years to the use of variolation as a measure to protect against smallpox. In the late 1800s these measures advanced greatly with the development of numerous vaccines and antisera against other infectious agents. Later progress, against unwanted immune responses, came with the introduction of a number of now-conventional pharmaceutical agents; although these allow only relatively nonspecific control over unwanted immune reactions, they remain an important component of clinical medicine. More recently, these standard therapeutics have been joined by so-called biological therapeutics, or biologics, which are artificially produced versions of natural products, such as hormones, cytokines, and

1	these standard therapeutics have been joined by so-called biological therapeutics, or biologics, which are artificially produced versions of natural products, such as hormones, cytokines, and monoclonal antibodies, or derivatives of these such as engineered fusion proteins. These biologics possess extraordinary specificity, and though some have been used for decades, such as the hormone insulin in patients with type 1 diabetes, recent advances in cell biology and engineering have allowed for the introduction of a broad array of new biologics that allow for very precise manipulation of the immune system. Finally, long-standing efforts to deploy the power of the adaptive immune system against tumors have made major advances, and biologics that target negative regulators of immunity to stimulate protective responses against cancers have made a significant impact in clinical medicine. The categories of agents used to manipulate immune responses are listed in Fig. 16.1. This chapter will

1	protective responses against cancers have made a significant impact in clinical medicine. The categories of agents used to manipulate immune responses are listed in Fig. 16.1. This chapter will discuss these approaches, beginning with pharmaceutical agents used in clinical practice. In the first part of the chapter, we focus on efforts to relieve unwanted immune responses, and on advances in cancer treatment based on immune-system therapies. In the last part of the chapter, we discuss current vaccination strategies against infectious diseases, and consider how a more rational approach to the design and development of vaccines promises to increase their efficacy and widen their usefulness and application.

1	Treatment of unwanted immune responses. Unwanted immune responses occur in many settings, such as autoimmune disease, transplant rejection, and allergy, which present different therapeutic challenges. The goal of treatment in all cases is to avoid tissue damage and prevent the disruption of tissue function. Some unwanted immune responses can be anticipated so that preventive measures may be taken, as in the case of allograft rejection. Other unwanted responses may be undetectable until after they become established, as is the case with autoimmune or allergic reactions. The relative difficulty of suppressing established immune responses is seen in animal models of autoimmunity, in which treatments that could have prevented induction of the disease are generally unable to halt it once it is established. Treatment of unwanted immune responses. Using the immune response to attack tumors. Fighting infectious diseases with vaccination.

1	Treatment of unwanted immune responses. Using the immune response to attack tumors. Fighting infectious diseases with vaccination. categories (Fig. 16.2). There are the powerful anti-inflammatory drugs of the corticosteroid family such as prednisone, the cytotoxic drugs such as azathioprine and cyclophosphamide, and the noncytotoxic fungal and bacterial derivatives such as cyclosporin A, tacrolimus (FK506 or fujimycin), and rapamycin (sirolimus), which inhibit intracellular signaling pathways within T lymphocytes. Finally, a recently introduced drug, fingolimod, interferes with signaling by the sphingosine 1-phosphate receptor that controls the egress of B and T cells from lymphoid organs, thus preventing effector lymphocytes from reaching peripheral tissues. Most of these drugs exert broad inhibition of the immune system, and suppress helpful as well as harmful responses. Opportunistic infection is therefore a common complication of immunosuppressive drug therapy.

1	Newer treatments attempt to target the aspects of the immune response that cause tissue damage, such as cytokine action, while avoiding wholesale immunosuppression, but even these therapeutic agents can affect important components of the response to infectious disease. The most immediate way of inhibiting a particular part of the immune response is via highly specific antibodies, usually directed against specific proteins expressed and/or secreted by immune cells. Approaches of this type that were experimental at the time of previous editions of this book are now part of established medical practice. Anticytokine monoclonal antibodies, such as the drug infliximab (anti-TNF-α) used in the treatment of rheumatoid arthritis, can neutralize local excesses of cytokines or chemokines or target natural cellular regulatory mechanisms to inhibit unwanted immune responses. Proteins besides antibodies are also in use to control immune responses, an example being abatacept, a fusion protein

1	natural cellular regulatory mechanisms to inhibit unwanted immune responses. Proteins besides antibodies are also in use to control immune responses, an example being abatacept, a fusion protein consisting of the Fc region of an immunoglobulin fused to the extracellular domain of CTLA-4. Abatacept reduces co-stimulation of T cells by binding to B7 molecules and blocking their interactions with CD28, and it is currently used to treat patients with rheumatoid arthritis who fail to respond to antiTNF-α therapy.

1	16-1 Corticosteroids are powerful anti-inflammatory drugs that alter the transcription of many genes. Corticosteroid drugs are powerful anti-inflammatory and immunosuppressive agents that are used widely to attenuate the harmful effects of autoimmune or allergic immune responses (see Chapters 14 and 15), as well as responses against transplanted organs. Corticosteroids are derivatives of the glucocorticoid family of steroid hormones that play a crucial role in maintaining the body’s homeostasis; one of the most widely used is prednisone, a synthetic version of the hormone cortisol. Corticosteroids cross the cell’s plasma Treatment of unwanted immune responses.

1	Treatment of unwanted immune responses. membrane and bind to intracellular receptors of the nuclear receptor family. Activated glucocorticoid receptors are transported to the nucleus, where they bind directly to DNA and interact with other transcription factors to regulate as many as 20% of the genes expressed in leukocytes. The response to steroid therapy is complex, given the large number of genes regulated in leukocytes and in other tissues. With respect to immunosuppression, corticosteroids exert multiple anti-inflammatory effects, which are briefly summarized in Fig. 16.3.

1	Corticosteroids target the pro-inflammatory functions of monocytes and macrophages and reduce the number of CD4 T cells. They can induce the expression of certain anti-inflammatory genes, such as AnxaI, which encodes a protein inhibitor of phospholipase A2 and thereby prevents this enzyme from generating pro-inflammatory prostaglandins and leukotrienes (see Sections 3-3 and 14-7). Conversely, corticosteroids can also suppress the expression of pro-inflammatory genes, including those encoding the cytokines IL-1β and TNF-α.

1	The therapeutic effects of corticosteroid drugs are due to their presence at much higher concentrations than the natural concentration of glucocorticoid hormones, causing exaggerated responses with both toxic and beneficial effects. Adverse effects include fluid retention, weight gain, diabetes, bone mineral loss, and thinning of the skin, requiring a careful balance to be maintained between beneficial and harmful effects. These drugs can also decrease in effectiveness over time. Despite these drawbacks, inhaled corticosteroids have proven highly beneficial in the treatment of chronic asthma (see Section 14-13). In the treatment of autoimmunity or allograft rejection, in which high doses of oral corticosteroids are needed to be effective, they are most often administered in combination with other immunosuppressant drugs to keep the corticosteroid dose and side-effects to a minimum. These other drugs include cytotoxic agents that act as immunosuppressants by killing rapidly dividing

1	with other immunosuppressant drugs to keep the corticosteroid dose and side-effects to a minimum. These other drugs include cytotoxic agents that act as immunosuppressants by killing rapidly dividing lymphocytes, and drugs that more specifically target lymphocyte signaling pathways.

1	16-2 Cytotoxic drugs cause immunosuppression by killing dividing cells and have serious side-effects.

1	The three cytotoxic drugs most commonly used as immunosuppressants are azathioprine, cyclophosphamide, and mycophenolate. These drugs interfere with DNA synthesis, and their major pharmacological action is on tissues in which cells are continually dividing. Developed originally to treat cancer, these drugs were found to be immunosuppressive after observations that they were cytotoxic to dividing lymphocytes. Azathioprine also interferes with CD28 co-stimulation in T cells, thus promoting T-cell apoptosis (see Section 7-24). The use of these compounds is, however, limited by their toxic effects on all tissues in which cells are dividing, such as the skin, gut lining, and bone marrow. Effects include decreased immune function, as well as anemia, leukopenia, thrombocytopenia, damage to intestinal epithelium, hair loss, and fetal death or injury. As a result of their toxicity, these drugs are used at high doses only when the aim is to eliminate all dividing lymphocytes, as in the

1	to intestinal epithelium, hair loss, and fetal death or injury. As a result of their toxicity, these drugs are used at high doses only when the aim is to eliminate all dividing lymphocytes, as in the treatment of lymphoma and leukemia; in these cases, treated patients require subsequent bone marrow transplantation to restore their hematopoietic function. When used to treat unwanted immune responses such as autoimmune conditions, they are used at lower doses and in combination with other drugs such as corticosteroids.

1	Azathioprine is converted in vivo to the purine analog 6-thioguanine (6-TG), which is metabolized to 6-thioinosinic acid. This competes with inosine monophosphate, blocking the de novo synthesis of adenosine monophosphate and guanosine monophosphate, thus inhibiting DNA synthesis. 6-TG is also incorporated into the DNA in place of guanine, and accumulation of

1	Fig. 16.3 Anti-inflammatory effects of corticosteroid therapy. Corticosteroids regulate the expression of many genes, with a net anti-inflammatory effect. First, they reduce the production of inflammatory mediators, including cytokines, prostaglandins, and nitric oxide (NO). Second, they inhibit inflammatory cell migration to sites of inflammation by inhibiting the expression of adhesion molecules. Third, corticosteroids promote the death by apoptosis of leukocytes. The layers of complexity are illustrated by the actions of annexin-1 (originally identified as a factor induced by corticosteroids and named lipocortin), which has been shown to participate in all of the effects of corticosteroids listed on the right. NOS, NO synthase.

1	MOVIE 16.1 6-TG increases the DNA’s sensitivity to mutations induced by the ultraviolet radiation in sunlight. Thus, patients treated with azathioprine have the longterm side-effect of increased risk of skin cancer. Azathioprine also generates 6-thioguanine triphosphate (6-thio-GTP), which in T cells binds to the small GTPase Rac1 in place of GTP and suppresses its activity. Signaling from CD28 co-stimulation requires Rac1, and T cells therefore do not receive the anti-apoptotic signals from co-stimulation and instead undergo apoptosis. Mycophenolate mofetil, the 2-morpholinoethyl ester of mycophenolic acid, is the newest addition to the family of cytotoxic immunosuppressive drugs; it works in a similar fashion to azathioprine. It is metabolized to mycophenolic acid, which inhibits the enzyme inosine monophosphate dehydrogenase, thus blocking the de novo synthesis of guanosine monophosphate.

1	Azathioprine and mycophenolate are less toxic than cyclophosphamide, which is metabolized to phosphoramide mustard, which alkylates DNA. Cyclophosphamide is a member of the nitrogen mustard family of compounds, which were originally developed as chemical weapons. It has a range of highly toxic effects including inflammation of and hemorrhage from the bladder, known as hemorrhagic cystitis, and induction of bladder neoplasia. 16-3 Cyclosporin A, tacrolimus, rapamycin, and JAK inhibitors with various T-cell signaling pathways.

1	Three noncytotoxic alternatives to the cytotoxic drugs are available as immunosuppressants and are widely used to treat transplant recipients. These are cyclosporin A, tacrolimus (previously known as FK506), and rapamycin (also known as sirolimus). Cyclosporin A is a cyclic decapeptide derived from a soil fungus found in Norway, Tolypocladium inflatum. Tacrolimus is a macrolide compound from the filamentous bacterium Streptomyces tsukabaensis, found in Japan; macrolides are compounds that contain a many-membered lactone ring to which is attached one or more deoxysugars. Rapamycin, another macrolide, is derived from Streptomyces hygroscopicus, found on Easter Island (‘Rapa Nui’ in Polynesian—hence the name of the drug). All three compounds exert their pharmacological effects by binding to members of a family of intracellular proteins known as the immunophilins, forming complexes that interfere with signaling pathways important for the clonal expansion of lymphocytes.

1	As explained in Section 7-14, cyclosporin A and tacrolimus block T-cell proliferation by inhibiting the phosphatase activity of the Ca2+-activated protein phosphatase calcineurin, which is required for the activation of the transcription factor NFAT. Both drugs reduce the expression of several cytokine genes that are normally induced on T-cell activation (Fig. 16.4), including the gene encoding interleukin (IL)-2, which is an important growth factor for T cells (see Section 9-16). Cyclosporin A and tacrolimus inhibit T-cell proliferation in response to either specific antigens or allogeneic cells, and are used extensively in medical practice to prevent the rejection of allogeneic organ grafts. Although the major immunosuppressive effects of both drugs are probably the result of inhibition of T-cell proliferation, they also act on other cells and have a large variety of other immunological effects (see Fig. 16.4).

1	These two drugs inhibit calcineurin by first binding to an immunophilin molecule; cyclosporin A binds to the cyclophilins, and tacrolimus to the FK-binding proteins (FKBPs). Immunophilins are peptidyl-prolyl cis–trans isomerases, but their isomerase activity is not relevant to the immunosuppressive activity of the drugs that bind them. Rather, the immunophilin:drug complexes bind and inhibit the Ca2+-activated serine/threonine phosphatase calcineurin. In a normal immune response, the increase in intracellular calcium ions in response to T-cell receptor signaling activates the calcium-binding protein Fig. 16.4 Cyclosporin A and tacrolimus inhibit lymphocyte and some granulocyte responses.

1	Fig. 16.4 Cyclosporin A and tacrolimus inhibit lymphocyte and some granulocyte responses. T lymphocyte Reduced expression of IL-2, IL-3, IL-4, GM-CSF, TNF-˜ Reduced proliferation following decreased IL-2 production Reduced Ca2+-dependent exocytosis of granule-associated serine esterases Inhibition of antigen-driven apoptosis calmodulin; calmodulin then activates calcineurin (see Fig. 7.18). Binding of the immunophilin:drug complex to calcineurin prevents the latter’s activation by calmodulin; the bound calcineurin is unable to dephosphorylate and activate NFAT (Fig. 16.5). Calcineurin is found in other cells besides T cells, but its levels in T cells are much lower than in other tissues. T cells are therefore particularly susceptible to the inhibitory effects of these drugs.

1	Cyclosporin A and tacrolimus are effective immunosuppressants but are not problem-free. As with the cytotoxic agents, they affect all immune responses indiscriminately. This can be countered by carefully varying the dose of drug given during the course of a response. During organ transplantation, for example, high doses are required during the time of grafting, but once the graft has become established the dose can be decreased in order to allow useful protective immune responses while maintaining adequate suppression of the residual response to the grafted tissue. This balance is difficult to achieve and requires careful monitoring of the patient. These drugs also have effects on many different tissues and thus can have broad side-effects, such as injury to kidney tubule epithelial cells. Finally, treatment with these drugs is relatively expensive, because they are complex natural products that must be taken for long periods. Nevertheless, at present they are the immunosuppressants

1	Finally, treatment with these drugs is relatively expensive, because they are complex natural products that must be taken for long periods. Nevertheless, at present they are the immunosuppressants of choice in clinical transplantation, and they are also being tested in a variety of autoimmune diseases, especially those that, like graft rejection, are mediated by T cells.

1	Rapamycin has a different mode of action from either cyclosporin A or tacrolimus. Like tacrolimus, rapamycin binds to the FKBP family of immunophilins, but the rapamycin:immunophilin complex does not inhibit calcineurin activity. Instead, it inhibits a serine/threonine kinase known as mTOR (mammalian target of rapamycin), which is involved in regulating cell growth and proliferation (see Section 7-17). The mTOR pathway can be activated by different upstream signaling pathways, including the Ras/MAPK pathway and

1	Fig. 16.5 Cyclosporin A and tacrolimus inhibit T-cell activation by interfering with the serine/threonine-specific phosphatase calcineurin. As shown in the upper panel, signaling via T-cell receptor-associated tyrosine kinases leads to opening of calciumrelease-activated calcium (CRAC) channels in the plasma membrane. This increases the concentration of Ca2+ in the cytoplasm and promotes calcium binding to the regulatory protein calmodulin (see Fig. 7.18). Calmodulin is activated by binding Ca2+ and can then target many downstream effector proteins such as the phosphatase calcineurin. Binding by calmodulin activates calcineurin to dephosphorylate the transcription factor NFAT (see Section 7-14), which then enters the nucleus and transcribes genes that are required for T-cell activation to progress. As shown in the lower panel, when cyclosporin A or tacrolimus or both are present, they form complexes with their immunophilin targets, cyclophilin and FK-binding protein, respectively.

1	to progress. As shown in the lower panel, when cyclosporin A or tacrolimus or both are present, they form complexes with their immunophilin targets, cyclophilin and FK-binding protein, respectively. These complexes bind to calcineurin, preventing it from becoming activated by calmodulin, and thereby preventing the dephosphorylation of NFAT.

1	Fig. 16.6 Rapamycin inhibits cell growth and proliferation by selectively blocking activation of the kinase mTOR by Raptor. Rapamycin binds FK-binding protein (FKBP), the same immunophilin protein that binds to tacrolimus (FK506). The rapamycin:FKBP complex does not inhibit calcineurin, but instead blocks one of two complexes that activate mTOR, a large kinase that regulates many metabolic pathways. mTOR is activated downstream of various growth factor signaling pathways, and becomes associated with either of two proteins, Raptor (regulatory associated protein of mTOR) and Rictor (rapamycininsensitive companion of mTOR). The complex with Raptor, mTORC1, promotes cell proliferation, translation of proteins, and autophagy, and the complex with Rictor, mTORC2, influences cell adhesion and migration by controlling the actin cytoskeleton. The rapamycin:FKBP complex acts to inhibit the Raptorassociated mTORC1, and thereby selectively reduces cell growth and proliferation.

1	the PI 3-kinase pathway. These pathways activate AKT, which phosphorylates and inactivates a regulatory complex called TSC. This complex normally acts as an inhibitor of the small GTPase Rheb; after TSC is phosphorylated, Rheb is free to activate mTOR (see Fig. 7.22). Two distinct mTOR complexes can be formed, mTORC1 and mTORC2, which are controlled by two regulatory proteins called Raptor and Rictor, respectively, and which activate different downstream cellular pathways (Fig. 16.6). Rapamycin appears to inhibit only the mTORC1 complex, as the rapamycin:FKBP complex selectively inhibits the Raptor-dependent pathway that regulates this complex (see Fig. 16.6). Blockade of this pathway markedly reduces T-cell proliferation, arresting cells in the G1 phase of the cell cycle and promoting apoptosis. Rapamycin inhibits lymphocyte proliferation driven by growth factors such as IL-2, IL-4, and IL-6, and increases the number of regulatory T cells, perhaps because these cells use different

1	apoptosis. Rapamycin inhibits lymphocyte proliferation driven by growth factors such as IL-2, IL-4, and IL-6, and increases the number of regulatory T cells, perhaps because these cells use different signaling pathways from those of effector T cells. Rapamycin also selectively reduces the outgrowth of effector T cells while apparently enhancing the formation of memory T cells. Because of this, the use of rapamycin to augment T-cell memory induced by vaccines is being considered.

1	One recently introduced drug manipulates immune responses by regulating the migration of immune effector cells to the sites of a graft or of autoimmune disease. In Section 9-7, we described how emigration of lymphocytes out of the lymphoid tissues requires recognition of the lipid molecule sphingosine 1-phosphate (S1P) by the G-protein-coupled receptor S1PR1. Fingolimod (FTY720), a sphingosine 1-phosphate analog, is a relatively newer drug that causes the retention of effector lymphocytes in lymphoid organs, thus preventing these cells from mediating their effector activities in target tissues. Fingolimod was approved in 2010 for treatment of the autoimmune disease multiple sclerosis, and has shown promise in the treatment of kidney graft rejection and asthma.

1	Cytokines activate many aspects of the immune response, and many cytokine receptors use Janus kinases (JAKs) in signal transduction (see Section 3-16). The four JAK family members, JAK1, JAK2, JAK3, and TYK2, bind and phosphorylate the cytoplasmic regions of cytokine receptors and initiate the activation of different STAT transcription factors. Selective JAK inhibitors have been developed over the last decade that can block the kinase activity of one or more members of this family. Since different JAKs binds to different cytokine receptors, JAK inhibitors, or Jakinibs, can therefore exert potentially specific effects on the quality of T-cell development. Two Jakinibs are now approved for use in treating inflammatory diseases and are being investigated for their application in cancer. For example, tofacitinib inhibits JAK3, interfering with signaling by IL-2 and IL-4, and somewhat more weakly JAK1, interfering with signaling by IL-6. Tofacitinib is approved for treatment of rheumatoid

1	example, tofacitinib inhibits JAK3, interfering with signaling by IL-2 and IL-4, and somewhat more weakly JAK1, interfering with signaling by IL-6. Tofacitinib is approved for treatment of rheumatoid arthritis. Ruxolitinib inhibits JAK1 and JAK2 and has been approved for treating myelofibrosis, an abnormal proliferation of bone marrow progenitor cells that causes fibrosis.

1	used to eliminate lymphocyte subsets or to inhibit lymphocyte function.

1	All the drugs discussed so far exert a general inhibition on immune responses and can have severe side effects, but antibodies can act in a more specific manner and with less direct toxicity. The initial therapeutic use of antibodies extends back to the late 1800s with the development of equine sera for treatment of diphtheria and tetanus. Today, intravenous immunoglobulin (IVIG), a collection of polyvalent IgG antibodies pooled from many blood donors, is still widely used as a treatment for various primary and acquired immune deficiencies. It is also used in some acute infections, where it likely works by providing antibodies that may neutralize certain pathogens or their toxins. Finally, IVIG is also used to treat certain autoimmune and inflammatory diseases, such as immune thrombocytopenia and Kawasaki disease. In these cases, IVIG exerts an immunomodulatory effect that seems to operate through interactions with inhibitory Fc receptors that inhibit immune-cell activation.

1	The relatively recent expansion in the use of antibodies as therapeutic agents has extended their function from targeting pathogens to targeting components of the immune system itself in order to achieve a specific regulatory result. For example, the potential of antibodies to eliminate unwanted lymphocytes is demonstrated by anti-lymphocyte globulin, a preparation of polyclonal immunoglobulin from rabbits (and previously horses) immunized with human lymphocytes, which has been used for many years to treat episodes of acute graft rejection. Anti-lymphocyte globulin does not, however, discriminate between useful lymphocytes and those responsible for the unwanted responses and therefore leads to global immunosuppression. Foreign immunoglobulins are also highly antigenic in humans, and the large doses of anti-lymphocyte globulin used in therapy often cause a condition called serum sickness, resulting from the formation of immune complexes of the animal immunoglobulin and human antibodies

1	doses of anti-lymphocyte globulin used in therapy often cause a condition called serum sickness, resulting from the formation of immune complexes of the animal immunoglobulin and human antibodies against it (see Section 14-15).

1	Anti-lymphocyte globulin is nevertheless still used to treat acute rejection, and this has stimulated the quest for monoclonal antibodies (see Appendix I, Section A-7) that would achieve more specifically targeted effects. One such antibody is alemtuzumab (marketed as Campath-1H), which is directed at the cell-surface protein CD52 expressed by most lymphocytes. It has similar actions to anti-lymphocyte globulin, causing long-standing lymphopenia. It is also used to eliminate cancer cells in the treatment of chronic lymphocytic leukemia.

1	Immunosuppressive monoclonal antibodies act by one of two general mechanisms. Some, such as alemtuzumab, trigger the destruction of lymphocytes in vivo and are referred to as depleting antibodies, whereas others are nondepleting and act by blocking the function of their target protein without killing the cell that bears it. Depleting monoclonal IgG antibodies bind to lymphocytes and target them to macrophages and NK cells, which bear Fc receptors and kill the lymphocytes by phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC), respectively. Complement-mediated lysis may also play a part in lymphocyte destruction. 16-5 Antibodies can be engineered to reduce their immunogenicity in humans.

1	16-5 Antibodies can be engineered to reduce their immunogenicity in humans. A major impediment to therapy with monoclonal antibodies in humans has been that these antibodies are most readily made by immunizing nonhuman species, such as the mouse, to generate antibodies of the desired specificity (see Appendix I, Section A-7). Humans may develop an antibody response against such nonhuman antibodies, since aggregated forms of foreign antibodies can be immunogenic. Such a reaction not only interferes with the therapeutic actions of the antibodies, but also leads to allergic reactions, and, if treatment is continued, may result in anaphylaxis (see Section 14-10). Once a patient has made a response to an antibody, it can no longer be used for future treatment. This problem can, in principle, be avoided by making antibodies that are not recognized as foreign by the human immune system, a process called humanization.

1	Various approaches have been tried to humanize antibodies. The variable regions encoding the antigen-recognition determinants from a murine antibody can be spliced onto the Fc regions of human IgG by gene manipulation. Antibodies of this type are called chimeric antibodies. However, this approach leaves regions within the murine variable regions that could potentially induce

1	Fig. 16.7 Monoclonal antibodies used to treat human diseases can be engineered to decrease immunogenicity but maintain their antigen specificity. Antibodies that are derived fully from mice, named with the suffix -omab, are immunogenic in humans. This causes patients to generate antibodies against them, limiting their usefulness over time. This immunogenicity can be reduced by making chimeric antibodies in which the V regions from the mouse are spliced onto human antibody constant regions; such antibodies are named with the suffix -ximab. Humanization is the process of splicing in just the complementarity-determining regions from the mouse antibody, further reducing immunogenicity; humanized antibodies are named with the suffix -zumab. New techniques now allow fully human (-umab) monoclonal antibodies to be derived, which are the least immunogenic type of antibody currently used for treating humans.

1	immune responses (Fig. 16.7). Genetically engineered mice that harbor human immunoglobulin genes inserted into their immunoglobulin locus represent one way that human antibodies may be obtained from the immunization of mice. Newer methods are aimed at generating fully human monoclonal antibodies directly from human cells through the use of viral transformation of human primary B-cell lines or antibody-secreting plasmablasts, or by generating human B-cell hybridomas.

1	Monoclonal antibodies belong to a new class of therapeutic compounds called biologics, which includes other natural proteins such as anti-lymphocyte globulin, cytokines, protein fragments, and even whole cells, which are used, for example, in the adoptive transfer of T cells in cancer immunotherapy. Many monoclonal antibodies have been, or are in the process of being, approved for clinical use by the US Food and Drug Administration (Fig. 16.8), and a systematic naming process identifies the type of antibody. Murine monoclonal antibodies are designated by the suffix -omab, such as muromomab (originally called OKT3), a murine antibody against CD3. Chimeric antibodies in which the entire variable region is spliced into human constant regions have the suffix -ximab, such as basiliximab, an anti-CD25 antibody approved for the treatment of transplantation rejection. Humanized antibodies in which the murine hypervariable regions have been spliced into a human antibody have the suffix -zumab,

1	antibody approved for the treatment of transplantation rejection. Humanized antibodies in which the murine hypervariable regions have been spliced into a human antibody have the suffix -zumab, as in alemtuzumab and natalizumab (Tysabri). The latter is directed against the α4 integrin subunit, and is used to treat multiple sclerosis and Crohn’s disease. Antibodies derived entirely from human sequences have the suffix -umab, as in adalimumab, an antibody derived from phage display that binds TNF-α; it is used to treat several autoimmune diseases.

1	16-6 Monoclonal antibodies can be used to prevent allograft rejection.

1	Antibodies specific for various physiological targets are being used, or are under investigation, to prevent the rejection of transplanted organs by inhibiting the development of harmful inflammatory and cytotoxic responses. For example, alemtuzumab, discussed in Section 16-4, is licensed for the treatment of certain leukemias but is also used in both solid-organ and bone marrow transplantation. In solid-organ transplantation, alemtuzumab may be given to the recipient around the time of transplantation to remove mature T lymphocytes from the circulation. In bone marrow transplantation, alemtuzumab can be used in vitro to deplete donor bone marrow of mature T cells before its infusion into a recipient, or used in vivo to treat the recipient following infusion. Elimination of mature T cells from donor bone marrow is very effective at reducing the incidence of graft-versus-host disease (see Section 15-36). In this disease, the T lymphocytes in the donor bone marrow recognize the

1	T cells from donor bone marrow is very effective at reducing the incidence of graft-versus-host disease (see Section 15-36). In this disease, the T lymphocytes in the donor bone marrow recognize the recipient as foreign and mount a damaging response, causing rashes, diarrhea, and hepatitis, which can occasionally be fatal. Bone marrow transplantation is also used as a treatment for leukemia, as T cells in the graft can have a so-called graft-versus-leukemia effect where they recognize the leukemic cells as foreign and destroy them. It was originally thought that elimination of mature donor

1	Fig. 16.8 Monoclonal antibodies developed for immunotherapy. A substantial fraction of pharmaceuticals currently under development are antibodies, and additions to this list, current as of this writing, are under development and in clinical trials. T cells during such procedures would be disadvantageous, as the antileukemic action of the donor T cells could be lost, but this seems to not be the case when alemtuzumab is used as the depleting agent. Specific antibodies directed against T cells have been used to treat episodes of graft rejection that occur after transplantation. The murine antibody muromomab (OKT3) targets the CD3 complex and leads to T-cell immunosuppression by inhibiting signaling through the T-cell receptor. It has been used clinically in solid-organ transplantation but is often associated with a dangerous side-effect, namely, the stimulation of pro-inflammatory cytokine release, and its use is declining. This cytokine release is related to muromomab’s intact

1	Fc region, which can activate Fc receptors via cross-linking and activate the cells that bear these receptors. In the antibody called teplizumab, or OKT3γ1 (Ala-Ala), amino acids 234 and 235 in the human IgG1 Fc region have been changed to alanines, and this antibody no longer stimulates cytokine release. Two other antibodies, daclizumab and basiliximab, approved for treating kidney transplant rejection, are directed against CD25 (a subunit of the IL-2 receptor) and reduce T-cell activation, presumably by blocking the growth-promoting signals delivered by IL-2.

1	A primate model of kidney transplant rejection showed promising effects for a humanized monoclonal antibody against the CD40 ligand expressed by T cells (see Section 9-17). A possible mechanism of protection by this antibody is blockade of the activation of dendritic cells by helper T cells that recognize donor antigens. Only preliminary studies of anti-CD40 ligand antibodies have been performed in humans. One antibody was associated with thromboembolic complications and was withdrawn; a different anti-CD40 ligand antibody was administered to patients with the autoimmune disease systemic lupus erythematosus (SLE) without significant complications, but also with little evidence of efficacy.

1	In experimental models, monoclonal antibodies against other targets have also had some success in preventing graft rejection, including nondepleting antibodies that bind the CD4 co-receptor or the co-stimulatory receptor CD28 on lymphocytes. Similarly, abatacept, a soluble recombinant CTLA-4–Ig fusion protein which binds to the co-stimulatory B7 molecules on antigen-presenting cells and prevents their interaction with CD28 on T cells, is approved for the treatment of rheumatoid arthritis. 16-7 Depletion of autoreactive lymphocytes can treat autoimmune disease.

1	In addition to their use in preventing transplantation rejection, monoclonal antibodies can be used to treat certain autoimmune diseases, and the different immune mechanisms targeted are discussed in the next few sections. We start by discussing the use of depleting and nondepleting antibodies to remove lymphocytes nonspecifically. The anti-CD20 monoclonal antibody rituximab was originally developed to treat B-cell lymphomas, but has also been tried in treating certain autoimmune diseases. By ligating CD20, rituximab (Rituxan, MabThera) transduces a signal that induces lymphocyte apoptosis and depletes B cells for several months. Certain autoimmune diseases are believed to involve autoantibody-mediated pathogenesis. There is evidence for the efficacy of rituximab in some patients with autoimmune hemolytic anemia, SLE, rheumatoid arthritis, or type II mixed cryoglobulinemia, all of which have autoantibodies as a part of their clinical presentation. Although CD20 is not expressed on

1	autoimmune hemolytic anemia, SLE, rheumatoid arthritis, or type II mixed cryoglobulinemia, all of which have autoantibodies as a part of their clinical presentation. Although CD20 is not expressed on antibody-producing plasma cells, their B-cell precursors are targeted by anti-CD20, resulting in a substantial reduction in the short-lived, but not the long-lived, plasma-cell population.

1	Alemtuzumab, discussed above for its use in treating leukemia and in transplant rejection, has shown some beneficial effect in studies of small numbers of patients with multiple sclerosis. However, immediately after its infusion, most multiple sclerosis patients suffered a frightening, although fortunately brief, flare-up of their illness, illustrating another potential complication of antibody therapy. Alemtuzumab was acting as intended, killing cells by complementand Fc-dependent mechanisms. However, it also stimulated the release of cytokines, including TNF-α, interferon (IFN)-γ, and IL-6, which transiently block nerve conduction in nerve fibers previously affected by demyelination. This caused the transient but dramatic exacerbation of symptoms. Nevertheless, alemtuzumab may be useful at early stages of the disease, when the inflammatory response is maximal, but this has yet to be determined.

1	Fig. 16.9 Anti-inflammatory effects of anti-TNF-α therapy and objective parameters of disease activity (as measured by pain in rheumatoid arthritis. The clinical course of 24 patients was score and swollen-joint count, respectively) and in the systemic followed for 4 weeks after treatment with either a placebo or a inflammatory acute-phase response, measured as a fall in the monoclonal antibody against TNF-α at a dose of 10 mg • kg–1. The concentration of the acute-phase C-reactive protein. Data courtesy antibody therapy was associated with a reduction in both subjective of R.N. Maini.

1	Treating patients suffering from rheumatoid arthritis or multiple sclerosis by using anti-CD4 antibodies has been tried, but with disappointing results. In controlled studies, the antibodies showed only small therapeutic effects but caused depletion of T lymphocytes from peripheral blood for more than 6 years after treatment. The likely explanation for the failure seems to be that these antibodies failed to delete already primed CD4 T cells secreting pro-inflammatory cytokines, and may thus have missed their target. This cautionary tale shows that it is possible to deplete large numbers of lymphocytes and yet completely fail to kill the cells that matter. 16-8 Biologics that block TNF-α, IL-1, or IL-6 can alleviate autoimmune diseases.

1	16-8 Biologics that block TNF-α, IL-1, or IL-6 can alleviate autoimmune diseases. Anti-inflammatory therapy either can attempt to eliminate an autoimmune response altogether, as with immunosuppressive drugs or depleting antibodies, or it can try to reduce the tissue injury caused by the immune response. This second category of treatment is called immunomodulatory therapy, and is illustrated by the use of conventional anti-inflammatory agents such as aspirin, nonsteroidal anti-inflammatory drugs, or low-dose corticosteroids. A newer avenue of immunomodulatory therapy using biologics is illustrated by several FDA-approved antibodies that block the activity of powerful pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6.

1	Anti-TNF therapy was the first specific biological therapeutic to enter the clinic. Anti-TNF-α antibodies induced striking remissions in rheumatoid arthritis (Fig. 16.9) and reduced inflammation in Crohn’s disease, an inflammatory bowel disease (see Section 15-23). Two types of established biologics are used to antagonize TNF-α in clinical practice. The first type comprises the antiTNF-α antibodies, such as infliximab and adalimumab, which bind to TNF-α and block its activity. The second type is a recombinant human TNF receptor (TNFR) subunit p75–Fc fusion protein called etanercept, which also binds TNF-α, neutralizing its activity. These are extremely potent anti-inflammatory agents, and the number of diseases in which they have been shown to be effective is growing as further clinical trials are performed. In addition to rheumatoid arthritis, the rheumatic diseases ankylosing spondylitis, psoriatic arthropathy, and juvenile idiopathic arthritis (other than the systemic-onset subset)

1	are performed. In addition to rheumatoid arthritis, the rheumatic diseases ankylosing spondylitis, psoriatic arthropathy, and juvenile idiopathic arthritis (other than the systemic-onset subset) respond well to blockade of TNF-α, and this treatment is now routine in many cases.

1	An illustration of the importance of TNF-α in defending against infection is the observation that TNF-α blockade carries a small but increased risk of patients developing serious infections, including tuberculosis (see Section 3-20). AntiTNF-α therapy has not been successful in all diseases. TNF-α blockade as a treatment for experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, led to amelioration of the disease, but in human patients with multiple sclerosis treated with anti-TNF-α, relapses became more frequent, possibly because of an increase in T-cell activation.

1	Antibodies and recombinant proteins against the pro-inflammatory cytokine IL-1 and its receptor have not proved as effective as TNF-α blockade for treating rheumatoid arthritis in humans, despite being equally powerful in animal models of arthritis. An antibody against the cytokine IL-1 has been licensed for clinical use against the hereditary autoinflammatory disease Muckle– Wells syndrome (see Section 13-9), and blockade of the IL-1β receptor by the recombinant protein anakinra (Kineret) has also proven useful in adults with moderate to severe rheumatoid arthritis. Another cytokine antagonist in clinical use is the humanized antibody tocilizumab; by virtue of being directed against the IL-6 receptor, it blocks the effects of the pro-inflammatory cytokine IL-6. This seems to be as effective as anti-TNF-α in patients with rheumatoid arthritis and also shows promise in treating systemic-onset juvenile idiopathic arthritis, an autoinflammatory condition.

1	Interferon (IFN)-β (Avonex) is used to treat diseases of viral origin based on its ability to enhance immunity, but is also effective in treating multiple sclerosis, attenuating its course and severity and reducing the occurrence of relapses. Until recently, it was unclear how IFN-β could reduce rather than enhance immunity. In Section 3-9, we described the inflammasome, in which innate sensors of the NLR family activate caspase 1 to cleave the IL-1 pro-protein into the active form of the cytokine (see Fig. 3.19). We now know that IFN-β acts at two levels to reduce IL-1 production. It inhibits the activity of the NALP3 (NLRP3) and NLRP1 inflammasomes and also reduces expression of the IL-1 pro-protein, reducing the substrate available to caspase 1. Thus, IFN-β limits the production of a powerful pro-inflammatory cytokine, which may explain its observed effects on the symptoms of multiple sclerosis.

1	16-9 Biologic agents can block cell migration to sites of inflammation and reduce immune responses.

1	Effector lymphocytes expressing the integrin α4:β1 (VLA-4) bind to VCAM-1 on endothelium in the central nervous system, while those expressing integrin α4:β7 (lamina propria-associated molecule 1) bind to MAdCAM-1 on the endothelium in the gut. The humanized monoclonal antibody natalizumab is specific for the α4 integrin subunit and binds both VLA-4 and α4:β7, preventing their interaction with their ligands (Fig. 16.10). This antibody has shown therapeutic benefit in placebo-controlled trials in patients with Crohn’s disease or with multiple sclerosis. The early signs that this treatment could be successful illustrate the fact that disease depends on the continuing emigration of lymphocytes, monocytes, and macrophages from the circulation into the tissues of the brain in multiple sclerosis, and into the gut wall in Crohn’s disease. However, blockade of α4:β1 integrin is not specific and, like anti-TNF therapy, could lead to reduced defense against infection. Rarely patients treated

1	and into the gut wall in Crohn’s disease. However, blockade of α4:β1 integrin is not specific and, like anti-TNF therapy, could lead to reduced defense against infection. Rarely patients treated with natalizumab have developed progressive multifocal leukoencephalopathy (PML), an opportunistic infection caused by the JC virus. This led to the temporary withdrawal of natalizumab from the market in 2005, but in June 2006 it was again allowed to be prescribed for multiple sclerosis and for Crohn’s disease.

1	A similar problem with multifocal leukoencephalopathy led to the withdrawal from the market in the United States and Europe in 2009 of another anti-integrin antibody, efalizumab; this drug targets the αL subunit CD11a and had shown promise in treating psoriasis, an inflammatory skin disease driven primarily by T cells that produce pro-inflammatory cytokines. 16-10 Blockade of co-stimulatory pathways that activate lymphocytes can be used to treat autoimmune disease.

1	16-10 Blockade of co-stimulatory pathways that activate lymphocytes can be used to treat autoimmune disease. Blocking co-stimulatory pathways, as noted above in connection with the prevention of transplantation rejection (see Section 16-6), has also been applied to autoimmune diseases. For example, CTLA-4–Ig (abatacept) blocks the interaction of the B7 expressed by antigen-presenting cells with the CD28 expressed by T cells. This drug is approved for the treatment of rheumatoid arthritis, and also seems to be beneficial in treating psoriasis. When CTLA-4–Ig was given to patients with psoriasis, there was an improvement in the psoriatic rash and histological evidence of loss of activation of keratinocytes, T cells, and dendritic cells within the damaged skin.

1	Another co-stimulatory pathway that has been targeted in psoriasis is the interaction between the adhesion molecule CD2 on T cells and CD58 (LFA-3) on antigen-presenting cells. A recombinant CD58–IgG1 fusion protein, called alefacept (Amevive), inhibits the interaction between CD2 and CD58, and is now a routine and effective treatment for psoriasis. Although memory T cells are targeted by this therapy, responses to vaccination such as antitetanus remain intact. 16-11 Some commonly used drugs have immunomodulatory properties.

1	16-11 Some commonly used drugs have immunomodulatory properties. Certain existing medications, such as the statins and angiotensin-converting enzyme (ACE) inhibitors widely used in the prevention and treatment of cardiovascular disease, can also modulate the immune response in experimental animals. Statins are very widely prescribed drugs that block the enzyme 3-hydroxy-3-methylglutaryl-co-enzyme A (HMG-CoA) reductase, thereby reducing cholesterol levels. They also reduce the increased level of expression of MHC class II molecules in some autoimmune diseases. These effects may be due to an alteration in the cholesterol content of membranes, thereby influencing lymphocyte signaling. In animal models, these drugs also seem to cause T cells to switch from a more pathogenic TH1 response to a more protective TH2 response, although whether this occurs in human patients is not clear.

1	The hormone vitamin D3, essential for bone and mineral homeostasis, also exerts immunomodulatory effects. It decreases IL-12 production by dendritic cells and leads to a decrease in IL-2 and IFN-γ production by CD4 T cells, and

1	Fig. 16.10 Treatment with an anti-α4 integrin humanized monoclonal antibody reduces relapses in multiple sclerosis. Left panel: interaction between α4:β1 integrin (VLA-4) on lymphocytes and macrophages and VCAM-1 expressed on endothelial cells permits the adhesion of these cells to brain endothelium. This facilitates the migration of these cells into the plaques of inflammation in multiple sclerosis. Center panel: the monoclonal antibody natalizumab (blue) binds to the α4 chain of the integrin and blocks adhesive interactions between lymphocytes and monocytes and VCAM-1 on endothelial cells, thus preventing the cells from entering the tissue and exacerbating the inflammation. The future of this treatment is unclear because of the development of a rare infection as a side-effect (see the text). Right panel: the number of new lesions detected on magnetic resonance imaging (MRI) of the brain is greatly reduced in patients treated with natalizumab compared with a placebo. Data from

1	the text). Right panel: the number of new lesions detected on magnetic resonance imaging (MRI) of the brain is greatly reduced in patients treated with natalizumab compared with a placebo. Data from Miller, D.H., et al.:

1	N. Engl. J. Med. 2003, 348:15–23. protective effects have been demonstrated in a variety of animal models of autoimmunity, such as EAE (see Section 15-5) and diabetes, as well as in transplantation. The major drawback of vitamin D3 is that its immunomodulatory effects are seen only at dosages that would lead to hypercalcemia and bone resorption in humans. There is a major search under way for structural analogs of vitamin D3 that retain the immunomodulatory effects but do not cause hypercalcemia. 16-12 Controlled administration of antigen can be used to manipulate the nature of an antigen-specific response.

1	16-12 Controlled administration of antigen can be used to manipulate the nature of an antigen-specific response. In some diseases, the target antigen of an unwanted immune response can be identified. It can then be possible to use the antigen itself, rather than drugs or antibodies, to treat the disease, because the manner of antigen presentation can alter the immune response and reduce or eliminate its pathogenic features. As discussed in Section 14-13, this principle has been applied with some success to the treatment of allergies caused by an IgE response to very low doses of antigen. Repeated treatment of allergic individuals with increasing doses of allergen seems to divert the allergic response to one dominated by T cells that favor the production of IgG and IgA antibodies from B cells. These antibodies are thought to desensitize the patient by binding the small amounts of allergen normally encountered and preventing it from binding to IgE.

1	There has been considerable interest in using peptide antigens to suppress pathogenic responses in T-cell-mediated autoimmune disease. The type of CD4 T-cell response induced by a peptide depends on the way in which it is presented to the immune system (see Section 9-18). For instance, peptides given orally tend to induce regulatory T cells through production of transforming growth factor (TGF)-β, but do not induce TH1 cells or systemic antibody production (see Section 12-14). Indeed, experiments in animals indicate that oral antigens can protect against induced autoimmune disease. Diseases resembling multiple sclerosis or rheumatoid arthritis can be induced in mice by the injection of myelin basic protein (MBP) or collagen type II, respectively, in Freund’s complete adjuvant (see Section 16-29). Oral administration of MBP or type II collagen inhibits the development of these diseases in animals, but oral administration of the whole protein in patients with multiple sclerosis or

1	16-29). Oral administration of MBP or type II collagen inhibits the development of these diseases in animals, but oral administration of the whole protein in patients with multiple sclerosis or rheumatoid arthritis has had marginal therapeutic effects. Similarly, no protective effect was found in a large study that examined whether giving low-dose parenteral insulin could delay the onset of diabetes in people at high risk of developing the disease.

1	Other approaches using antigen to shift the autoimmune T-cell response to a less damaging TH2 response have been more effective in humans. The peptide drug glatiramer acetate (Copaxone) is approved for treating multiple sclerosis, in which it may reduce relapse rates by up to 30%. It is a polymer consisting of the four amino acids glutamic acid, alanine, tyrosine, and lysine in ratios that mimic their composition in MBP, and it induces a TH2-type protective response. A more refined strategy uses altered peptide ligands (APLs), in which amino acid substitutions have been made in specific amino acids in an antigenic peptide that are at the T-cell receptor contact positions. APLs can be designed to act as partial agonists or antagonists, or to induce regulatory T cells. But despite the success of APLs in treating EAE in mice, a trial of these peptides for multiple sclerosis in some patients led to exacerbated disease or to allergic reaction associated with a vigorous TH2 response, and

1	of APLs in treating EAE in mice, a trial of these peptides for multiple sclerosis in some patients led to exacerbated disease or to allergic reaction associated with a vigorous TH2 response, and their value in humans remains to be seen.

1	Summary. Treatments for unwanted immune responses, such as graft rejection, autoimmunity, or allergic reactions, include conventional drugs—anti-inflammatory, cytotoxic, and immunosuppressive drugs—as well as biologic agents such as monoclonal antibodies and immunomodulatory proteins. Anti-inflammatory drugs, of which the most potent are the corticosteroids, have a broad spectrum of actions and a wide range of toxic side-effects. Their dose must be carefully controlled, and they are normally used in combination with either cytotoxic or immunosuppressive drugs. The cytotoxic drugs work by killing dividing cells and thereby prevent lymphocyte proliferation, but they suppress all immune responses indiscriminately and also kill other types of dividing cells. The immunosuppressive drugs, such as cyclosporin A and rapamycin, interfere in specific signaling pathways and are generally less toxic, but are more expensive and still suppress the immune response somewhat indiscriminately.

1	Several types of biologic agents are now established in the clinic for treating transplant rejection and autoimmune diseases (Fig. 16.11). Many monoclonal antibodies have been approved for human use that deplete lymphocytes either generally or selectively, or inhibit lymphocyte activation through receptor blockade, or prevent lymphocyte migration into tissues. Immunomodulatory agents also include monoclonal antibodies or fusion proteins that inhibit the inflammatory actions of TNF-α, a triumph of immunotherapy. 16.2 and 16.8 can act in one of three general ways. First (red), they can act by depleting cells from inflammatory sites, causing global cell-specific depletion, or blocking integrin interactions, thereby inhibiting lymphocyte trafficking. Second (blue), agents may block specific cellular interactions or inhibit various co-stimulatory pathways. Third (green), agents may target the terminal effector mechanisms, such as the neutralization of various pro-inflammatory cytokines.

1	Fig. 16.12 Tumor rejection antigens are specific to individual tumors. Mice immunized with irradiated tumor cells and challenged with viable cells of the same tumor can, in some cases, reject a lethal dose of that tumor (left panels). This is the result of an immune response to tumor rejection antigens. If the immunized mice are challenged with viable cells of a different tumor, there is no protection and the mice die (right panels). Using the immune response to attack tumors.

1	Cancer is one of the three leading causes of death in industrialized nations, the others being infectious disease and cardiovascular disease. As treatments for infectious diseases and the prevention of cardiovascular disease continue to improve, and the average life expectancy increases, cancer is likely to become the most common fatal disease in these countries. Cancers are caused by the uncontrolled growth of the progeny of transformed cells. A major problem in treating cancer is controlling metastasis, or the spread of cancerous cells from one part of the body to unconnected parts. Curing cancer therefore requires that all the malignant cells be removed or destroyed without killing the patient. An attractive way of achieving this would be to induce an immune response against the tumor that would discriminate between the cells of the tumor and their normal-cell counterparts, in the same way that vaccination against a viral or bacterial pathogen induces a specific immune response

1	that would discriminate between the cells of the tumor and their normal-cell counterparts, in the same way that vaccination against a viral or bacterial pathogen induces a specific immune response that provides protection only against that pathogen. Immunological approaches to the treatment of cancer have been attempted for more than a century, but it is only in the past decade that immunotherapy of cancer has shown real promise. An important conceptual advance has been the integration of conventional approaches such as surgery or chemotherapy, which substantially reduce tumor load, with immunotherapy.

1	16-13 The development of transplantable tumors in mice led to the discovery of protective immune responses to tumors. The finding that tumors could be induced in mice after treatment with chemical carcinogens or irradiation, coupled with the development of inbred strains of mice, made it possible to undertake the key experiments that led to the discovery of immune responses to tumors. These tumors could be transplanted between mice, and the experimental study of tumor rejection has generally been based on the use of such tumors. If they bear MHC molecules foreign to the mice into which they are transplanted, the tumor cells are readily recognized and destroyed by the immune system, a fact that was exploited to develop the first MHC-congenic strains of mice. Specific immunity to tumors must therefore be studied within inbred strains, so that host and tumor can be matched for their MHC type.

1	In mice, transplantable tumors exhibit a variable pattern of growth when injected into syngeneic recipients. Most tumors grow progressively and eventually kill the host. However, if mice are injected with irradiated tumor cells that cannot grow, they are frequently protected against subsequent injection with a normally lethal dose of viable cells of the same tumor (Fig. 16.12). There seems to be a spectrum of immunogenicity among transplantable tumors: injections of irradiated tumor cells seem to induce varying degrees of protective immunity against a challenge injection of viable tumor cells at a distant site. These protective effects are not seen in T-cell-deficient mice but can be conferred by adoptive transfer of T cells from immune mice, showing the need for T cells to mediate these effects.

1	These observations indicate that the tumors express antigens that can become targets of a tumor-specific T-cell response that rejects the tumor. These tumor rejection antigens are expressed by experimentally induced murine tumors (in which they are often termed tumor-specific transplantation antigens), and are usually specific for an individual tumor. Thus, immunization with irradiated tumor cells from one tumor usually protects a syngeneic mouse from challenge with live cells from that same tumor, but not from challenge with a different syngeneic tumor (see Fig. 16.12). 16-14 Tumors are ‘edited’ by the immune system as they evolve and can escape rejection in many ways.

1	Paul Ehrlich, who received the 1908 Nobel Prize for his work in immunology, was perhaps the first to propose that the immune system could be used to treat established tumors, suggesting that the molecules we call antibodies might be used to deliver toxins to cancer cells. In the 1950s, Frank MacFarlane Burnet, recipient of the 1960 Nobel Prize, and Lewis Thomas formulated the ‘immune surveillance’ hypothesis, according to which cells of the immune system would detect and destroy tumor cells. Since then, it has become clear that the relationship between the immune system and cancer is considerably more complex, and this hypothesis has been modified to consider three phases of tumor growth. The first is the ‘elimination phase,’ in which the immune system recognizes and destroys potential tumor cells—the phenomenon previously referred to as immune surveillance (Fig. 16.13). If elimination is not completely successful, what follows is an ‘equilibrium phase,’ in which tumor cells undergo

1	cells—the phenomenon previously referred to as immune surveillance (Fig. 16.13). If elimination is not completely successful, what follows is an ‘equilibrium phase,’ in which tumor cells undergo changes or mutations that aid their survival as a result of the selection pressure imposed by the immune system. During the equilibrium phase, a process known as cancer immunoediting continuously shapes the properties of the tumor cells that survive. In the final ‘escape phase,’ tumor cells that have acquired the ability to elude the attentions of the immune system and grow unimpeded become clinically detectable.

1	Mice with targeted gene deletions or treated with antibodies to remove specific components of innate and adaptive immunity have provided the best evidence that immune surveillance influences the development of certain types of tumors. For example, mice lacking perforin, part of the killing mechanism of NK cells and CD8 cytotoxic T cells (see Section 9-31), show an increased frequency of lymphomas—tumors of the lymphoid system. Strains of mice lacking the RAG and STAT1 proteins, thus being deficient in both adaptive and certain innate immune mechanisms, develop gut epithelial and breast tumors. Mice lacking T lymphocytes expressing γ:δ receptors show markedly increased susceptibility to skin tumors induced by the topical application of carcinogens, illustrating a role for intraepithelial γ:δ T cells (see Section 6-20) in surveying and killing abnormal epithelial cells. Both IFN-γ and IFN-α/β are important in the elimination of tumor cells, either directly or indirectly through their

1	γ:δ T cells (see Section 6-20) in surveying and killing abnormal epithelial cells. Both IFN-γ and IFN-α/β are important in the elimination of tumor cells, either directly or indirectly through their actions on other cells. Studies of the various effector cells of the immune system show that γ:δ T cells are a major source of IFN-γ, which may explain their importance in the removal of cancer cells.

1	Fig. 16.13 Malignant cells can be controlled by immune surveillance. Some types of tumor cells are recognized by a variety of immune-system cells, which can eliminate them (left panel). If the tumor cells are not completely eliminated, variants occur that eventually escape the immune system and proliferate to form a tumor.

1	According to the immunoediting hypothesis, those tumor cells that survive the equilibrium phase have acquired additional changes, either due to additional mutations or from selection during the equilibrium phase, that prevent their elimination by the immune system. In an immunocompetent individual, the equilibrium phase of the immune response continually removes tumor cells, delaying tumor growth; if the immune system is compromised, the equilibrium phase quickly turns into escape, as no tumor cells at all are removed. An excellent clinical example to support the presence of the equilibrium phase is the occurrence of cancer in recipients of organ transplants. One study reported the development of melanoma between 1 and 2 years after transplantation in two patients who had received kidneys from the same donor, a patient who had had successfully treated malignant melanoma 16 years before her death. Presumably, melanoma cells, which are known to spread easily to other organs, were

1	from the same donor, a patient who had had successfully treated malignant melanoma 16 years before her death. Presumably, melanoma cells, which are known to spread easily to other organs, were present in the donor kidneys at the time of transplantation but were in equilibrium phase with the immune system. If so, this would indicate that the melanoma cells had not been killed off completely by the immunocompetent immune system of the donor, but instead had simply been held in check. Because the recipients’ immune systems were immunosuppressed to prevent graft rejection, the melanoma cells were released from equilibrium and began to divide rapidly and spread to other parts of the body.

1	Another situation in which the suppression of immunity can lead to tumor development is in post-transplant lymphoproliferative disorder, which can occur when patients are immunosuppressed after, for example, solid-organ transplantation. It usually takes the form of a B-cell expansion driven by Epstein–Barr virus (EBV) in which the B cells can undergo mutations and become malignant. Here, antiviral immunity functions as a form of cancer immunosurveillance, as it normally eliminates the EBV that leads to B-cell transformation.

1	Tumors can avoid stimulating an immune response or can evade it when it occurs by means of numerous mechanisms, which are summarized in Fig. 16.14. Spontaneous tumors may initially lack mutations that produce new tumor-specific antigens that elicit T-cell responses (see Fig. 16.14, first panel). And even when a tumor-specific antigen is expressed and is taken up and presented by antigen-presenting cells (APCs), if co-stimulatory signals are absent the antigen-presenting cell will tend to tolerize any antigen-specific naive T cells rather than activating them (see Fig. 16.14, second panel). How long such tumors are treated as ‘self’ is unclear. Recent sequencing of entire tumor genomes reveals that mutations may generate as many as 10–15 unique antigenic peptides that could be recognized as ‘foreign’ by T cells. In addition, cellular transformation is frequently associated with induction of MHC class Ib proteins (such as MIC-A and MIC-B) that are ligands for NKG2D, thus allowing tumor

1	‘foreign’ by T cells. In addition, cellular transformation is frequently associated with induction of MHC class Ib proteins (such as MIC-A and MIC-B) that are ligands for NKG2D, thus allowing tumor recognition by NK cells (see Section 6-17). But cancer cells tend to be genetically unstable, so that clones that are not recognized by an immune response may be able to escape elimination.

1	Some tumors, such as colon and cervical cancers, lose the expression of a particular MHC class I molecule, perhaps through immune selection by T cells specific for a peptide presented by that MHC class I molecule (see Fig. 16.14, third panel). In experimental studies, when a tumor loses expression of all MHC class I molecules (Fig. 16.15), it can no longer be recognized by cytotoxic T cells, although it might become susceptible to NK cells (Fig. 16.16). Tumors that lose only one MHC class I molecule might be able to avoid recognition by specific CD8 cytotoxic T cells while still remaining resistant to NK cells, conferring a selective advantage in vivo. Tumors also seem to be able to evade immune attack by creating a micro-environment that is generally immunosuppressive (see Fig. 16.14, fourth panel). Many tumors make immunosuppressive cytokines. Transforming growth factor-β (TGF-β) was first identified in the culture supernatant of a

1	Fig. 16.14 Tumors can avoid immune recognition in a variety tumors may be eliminated. The genetic instability of tumors allows of ways. First panel: tumors can have low immunogenicity. Tumors antigenic change, part of an equilibrium phase, during which tumor may lack antigens recognized by T cells, may have lost one or more cells lacking immunogenic antigens can expand. Fourth panel: tumors MHC molecules, or may express inhibitory molecules such as PD-L1 often produce molecules, such as TGF-β, IL-10, IDO, or PD-L1, that that repress T-cell function. Second panel: tumor-specific antigens suppress immune responses directly or recruit regulatory T cells that may be cross-presented by dendritic cells without co-stimulatory can secrete immunosuppressive cytokines. Fifth panel: tumor cells signals, inducing a tolerant state in T cells. Third panel: tumors can can secrete molecules such as collagen that form a physical barrier initially express antigens to which the immune system responds.

1	inducing a tolerant state in T cells. Third panel: tumors can can secrete molecules such as collagen that form a physical barrier initially express antigens to which the immune system responds. Such around the tumor, preventing lymphocyte access.

1	tumor (hence its name), and, as we have seen, it tends to suppress the inflammatory T-cell responses and cell-mediated immunity that are needed to control tumor growth. Recall that TGF-β induces the development of inducible regulatory T cells (Treg cells; see Section 9-21), which have been found in a variety of cancers and might expand specifically in response to tumor antigens. In mouse models, removal of Treg cells increases resistance to cancer, whereas their transfer into a Treg-negative recipient allows cancer cells to more greatly proliferate.

1	The microenvironments of some tumors also contain populations of myeloid cells, collectively called myeloid-derived suppressor cells (MDSCs), which can inhibit T-cell activation within the tumor. MDSCs may be heterogeneous, comprising both monocytic and polymorphonuclear cells, and are incompletely characterized at present. Several tumors of different tissue origins, such as melanoma, ovarian carcinoma, and B-cell lymphoma, have also been shown to produce the immunosuppressive cytokine IL-10, which can reduce dendritic cell activity and inhibit T-cell activation.

1	Some tumors express cell-surface proteins that directly inhibit immune responses (see Fig. 16.14, fourth panel). For example, some tumors express programmed death ligand-1 (PD-L1), a B7 family member and ligand for the inhibitory receptor PD-1 expressed by activated T cells (see Section 7-24). Furthermore, tumors can produce enzymes that act to suppress local immune responses. The enzyme indoleamine 2,3-dioxygenase (IDO) catabolizes tryptophan, an essential amino acid, in order to produce the immunosuppressive metabolite kynurenine. IDO seems to function in maintaining a balance between immune responses and tolerance during infections, but can be induced during the equilibrium phase of tumor development. Finally, tumor cells can produce materials such as collagen that create a physical barrier to interaction with cells of the immune system (see Fig. 16.14, last panel). Fig. 16.15 Loss of MHC class I expression in a prostatic carcinoma.

1	Fig. 16.15 Loss of MHC class I expression in a prostatic carcinoma. Some tumors can evade immune surveillance by a loss of expression of MHC class I molecules, preventing their recognition by CD8 T cells. Shown is a section of a human prostate cancer that has been stained with a peroxidase-conjugated antibody against HLA class I molecules. The brown stain that represents HLA class I expression is restricted to infiltrating lymphocytes and tissue stromal cells. The tumor cells that occupy most of the section show no staining. Photograph courtesy of G. Stamp.

1	Fig. 16.16 Tumors that lose expression of all MHC class I molecules as a mechanism of escape from immune surveillance are more susceptible to being killed by NK cells. Regression of transplanted tumors is largely due to the actions of cytotoxic T lymphocytes (CTLs), which recognize novel peptides bound to MHC class I antigens on the surface of the cell (left panels). NK cells have inhibitory receptors that bind MHC class I molecules, so variants of the tumor that have low levels of MHC class I, although less sensitive to CD8 cytotoxic T cells, become susceptible to NK cells (center panels). Although nude mice lack T cells, they have higher than normal levels of NK cells, and so tumors that are sensitive to NK cells grow less well in nude mice than in normal mice. Transfection with MHC class I genes can restore both resistance to NK cells and susceptibility to CD8 cytotoxic T cells (right panels). The bottom panels show scanning electron micrographs of NK cells attacking leukemia

1	class I genes can restore both resistance to NK cells and susceptibility to CD8 cytotoxic T cells (right panels). The bottom panels show scanning electron micrographs of NK cells attacking leukemia cells. The NK cell is the smaller cell on the left in both photographs. Left panel: shortly after binding to the target cell, the NK cell has put out numerous microvillous extensions and established a broad zone of contact with the leukemia cell. Right panel: 60 minutes after mixing, long microvillous processes can be seen extending from the NK cell (bottom left) to the leukemia cell and there is extensive damage to the leukemia cell; the plasma membrane has rolled up and fragmented. Photographs reprinted from Herberman, R., and Callewaert, D: Mechanisms of Cytotoxicity by Natural Killer Cells, 1985, with permission from Elsevier.

1	16-15 Tumor rejection antigens can be recognized by T cells and form the basis of immunotherapies.

1	The tumor rejection antigens recognized by the immune system are peptides of tumor-cell proteins that are presented to T cells on MHC molecules. These peptides become the targets of a tumor-specific T-cell response even though they can also be present on normal tissues. For instance, strategies to induce immunity to the relevant antigens in melanoma patients can induce vitiligo, an autoimmune destruction of pigmented cells in healthy skin. Several categories of tumor rejection antigens can be distinguished (Fig. 16.17). One consists strictly of tumor-specific antigens that result from point mutations or gene rearrangements that occur during oncogenesis and that affect a particular gene product. Point mutations in the gene for a particular protein may alter the epitope for T cells by altering the specific residues in a peptide that is already able to bind to MHC class I molecules, or they may allow some mutant peptides to bind de novo to MHC class I molecules. These peptides are often

1	the specific residues in a peptide that is already able to bind to MHC class I molecules, or they may allow some mutant peptides to bind de novo to MHC class I molecules. These peptides are often referred to as neoepitopes as they are newly immunogenic versions of normal proteins. Either change may evoke a new T-cell response against the tumor. In Band T-cell tumors, which are derived from single clones of lymphocytes,

1	The molecules listed here have all been shown to be recognized by cytotoxic T lymphocytes raised from patients with the tumor type listed. a special class of tumor-specific antigen comprises the unique rearranged antigen receptor expressed by the clone. However, not all mutated peptides may be properly processed or be able to associate with MHC molecules and thus ensure that they stimulate an effective response.

1	The second category of tumor rejection antigens is the cancer-testis antigens. These are proteins encoded by genes that are normally expressed only in male germ cells in the testis. Male germ cells do not express MHC molecules, and therefore peptides from these molecules are not normally presented to T lymphocytes. Tumor cells show widespread abnormalities of gene expression, including the activation of genes encoding cancer-testis antigens, such as the melanoma-associated antigens (MAGE) (see Fig. 16.17). When expressed by tumor cells, peptides derived from these ‘germ-cell’ proteins can now be presented to T cells by MHC class I molecules. Therefore, these proteins are effectively tumor-specific in their expression as antigens. Perhaps the cancer-testis antigen best characterized immunologically is NY-ESO-1 (New York esophageal squamous cell carcinoma-1), which is highly immunogenic and is expressed by a variety of human tumors, including melanomas.

1	The third category is the ‘differentiation antigens’ encoded by genes that are expressed only in particular types of tissues. Examples of these are the differentiation antigens expressed in melanocytes and melanoma cells, which include several proteins in the pathways that produce the black pigment melanin, and the CD19 antigen expressed by B cells. The fourth category consists of antigens that are strongly overexpressed in tumor cells compared with their normal counterparts. An example is HER-2/neu (also known as c-Erb-2), which is a receptor tyrosine kinase homologous to the epidermal growth factor receptor. HER-2/neu is overexpressed in many adenocarcinomas, including breast and ovarian cancers, where it is associated with a poor clinical prognosis. CD8 T lymphocytes have been found infiltrating solid tumors overexpressing HER-2/neu but are not capable of destroying such tumors in vivo. The fifth category of tumor rejection antigens consists of molecules that display abnormal

1	infiltrating solid tumors overexpressing HER-2/neu but are not capable of destroying such tumors in vivo. The fifth category of tumor rejection antigens consists of molecules that display abnormal post-translational modifications. An example is underglycosylated mucin, MUC-1, which is expressed by several tumors, including breast and pancreatic cancers. The sixth category consists of novel proteins that are generated when one or more introns are retained in the mRNA transcribed from a gene, which occurs in melanoma. Proteins encoded by viral oncogenes comprise the seventh category of tumor rejection antigens. These oncoviral proteins can have a critical role in the oncogenic process and, because they are foreign, they can evoke a T-cell response. Examples are the human papilloma virus type 16 proteins E6 and E7, which are expressed in cervical carcinoma (see Section 16-18).

1	In melanoma, tumor-specific antigens were discovered by culturing irradiated tumor cells with autologous lymphocytes, a technique known as the mixed lymphocyte–tumor cell culture. From such cultures, cytotoxic T cells were identified that were reactive against melanoma peptides and would kill tumor cells bearing the relevant tumor-specific antigen. Such studies have revealed that melanomas carry at least five different antigens that can be recognized by cytotoxic T lymphocytes. It seems that cytotoxic T lymphocytes reactive against melanoma antigens are not effective in vivo, perhaps due to deficient priming and insufficient effector function, or to downstream resistance mechanisms. However, melanoma-specific T cells can be isolated from peripheral blood, lymph nodes, or directly from lymphocytes infiltrating the tumor and propagated in vitro. These T cells do not recognize the products of mutant proto-oncogenes or tumor suppressor genes, but instead recognize antigens derived from

1	infiltrating the tumor and propagated in vitro. These T cells do not recognize the products of mutant proto-oncogenes or tumor suppressor genes, but instead recognize antigens derived from the protein products of other mutant genes or from normal proteins that are now displayed on tumor cells at levels detectable by T cells for the first time. Cancer-testis antigens such as the melanoma MAGE antigens discussed above probably represent early developmental antigens reexpressed in the process of tumorigenesis. Only a minority of melanoma patients have T cells reactive to the MAGE antigens, indicating that these antigens either are not expressed or are not immunogenic in most cases.

1	The most common melanoma antigens are peptides from the enzyme tyrosinase or from three other proteins—gp100, MART1, and gp75. These are differentiation antigens specific to the melanocyte lineage. It is likely that over-expression of these antigens in tumor cells leads to an abnormally high density of specific peptide:MHC complexes and it is this that makes them immunogenic. Although tumor rejection antigens are usually presented as peptides complexed with MHC class I molecules, the enzyme tyrosinase has also been shown to stimulate CD4 T-cell responses in some melanoma patients. This is likely because it is ingested and presented by cells expressing MHC class II molecules. Both CD4 and CD8 T cells are likely to be important in achieving immunological control of tumors. CD8 cells can kill the tumor cells directly, while CD4 T cells have a role in the activation of CD8 cytotoxic T cells and the establishment of memory. CD4 T cells may also kill tumor cells by means of the cytokines

1	the tumor cells directly, while CD4 T cells have a role in the activation of CD8 cytotoxic T cells and the establishment of memory. CD4 T cells may also kill tumor cells by means of the cytokines that they secrete, such as TNF-α.

1	Other potential tumor rejection antigens include the products of mutated cellular oncogenes or tumor suppressors, such as Ras and p53, and also fusion proteins, such as the Bcr–Abl tyrosine kinase that results from the chromosomal translocation (t9;22) found in chronic myeloid leukemia (CML). When present on CML cells, the HLA class I molecule HLA-A*0301 can display a peptide derived from the fusion site between Bcr and Abl. This peptide was detected by a powerful technique known as ‘reverse immunogenetics,’ in which endogenous peptides were eluted from the MHC binding groove and their sequence was determined by highly sensitive mass spectrometry. This technique has identified HLA-bound peptides from other tumor antigens, such as the MART1 and gp100 tumor antigens of melanomas, as well as candidate peptide sequences for vaccination against infectious diseases.

1	T cells specific for the Bcr–Abl fusion peptide can be identified in peripheral blood from patients with CML by using tetramers of HLA-A*0301 carrying the peptide as specific ligands for the antigen-specific T-cell receptor (see Section 7-24). Cytotoxic T lymphocytes specific for this and other tumor antigens can be selected in vitro by using peptides derived from the mutated or fused portions of these oncogenic proteins; these cytotoxic T cells are able to recognize and kill tumor cells.

1	After a bone marrow transplant to treat CML, mature lymphocytes from the bone marrow donor infused into the patient can help to eliminate any residual tumor. This technique is known as donor lymphocyte infusion (DLI). At present, it is not clear to what extent the clinical response is due to a graftversus-host effect, in which the donor lymphocytes are responding to general alloantigens expressed on the leukemia cells (see Section 15-36), or whether a specific antileukemic response is important. It is encouraging that it has been possible to separate T lymphocytes in vitro that mediate either a graft-versushost effect or a graft-versus-leukemia effect. The ability to prime the donor cells against leukemia-specific peptides offers the prospect of enhancing the antileukemic effect while minimizing the risk of graft-versus-host disease. 16-16 T cells expressing chimeric antigen receptors are an effective treatment in some leukemias.

1	16-16 T cells expressing chimeric antigen receptors are an effective treatment in some leukemias. Adoptive T-cell therapy involves ex vivo expansion of tumor-specific T cells to large numbers and the infusion of those T cells into patients. Cells are expanded in vitro by various methods, such as treatment with IL-2, anti-CD3 antibodies, and allogeneic antigen-presenting cells to provide a co-stimulatory signal. Adoptive T-cell therapy is made more effective when the patient is immunosuppressed before treatment and IL-2 is then administered systemically. Another approach that has excited much interest is the use of retroviral vectors to transfer tumor-specific T-cell receptor (TCR) genes into patients’ T cells before reinfusion. This can have long-lasting effects as a result of the ability of T cells to become memory cells, and there is no requirement for histocompatibility because the transfused cells are derived from the patient.

1	Another form of adoptive immunotherapy also uses retroviruses to introduce genes into a patient’s T cells, but involves expressing a novel type of receptor, known as a chimeric antigen receptor (CAR). CARs are fusion receptors that contain extracellular antigen-specific domains fused to intracellular domains that provide signals for activation and co-stimulation. These receptors are introduced into T cells via retroviral vectors to produce so-called CAR T cells. This approach differs from conventional adoptive T-cell therapies as the use of a CAR allows the T cell’s target specificity to be almost any molecule recognizable by an antibody rather than just peptide:MHC complexes. Recently this approach was used to target CD19 as a tumor rejection antigen in treating acute lymphocytic leukemia (ALL), an aggressive cancer of transformed B cells (Fig. 16.18). The CAR used in this case had an extracellular domain of an antibody that recognizes human CD19. The intracellular domain had three

1	(ALL), an aggressive cancer of transformed B cells (Fig. 16.18). The CAR used in this case had an extracellular domain of an antibody that recognizes human CD19. The intracellular domain had three ITAMs from the ζ chain of the T-cell receptor CD3 complex (see Chapter 7) fused with a co-stimulation domain from 4-1BB, a member of the TNF receptor superfamily. These CART-19 transduced T cells were expanded in vitro and transferred into a patient. The results of this case combined with others have demonstrated that CD8 T cells expressing CART-19 (see Fig. 16.18) are effective at achieving complete clinical remissions in many patients with ALL. This approach is not without its side-effects, however, as it also eliminates normal B cells in patients and they therefore require treatment with IVIG.

1	16-17 Monoclonal antibodies against tumor antigens, alone or linked to toxins, can control tumor growth. Using monoclonal antibodies to destroy tumors requires that a tumor-specific antigen be expressed on the tumor’s cell surface, so that antibody can direct Fig. 16.18 Chimeric antigen receptors (CARs) expressed in T cells can confer antitumor specificity to a patient’s lymphocytes. Bottom panel: a chimeric antigen receptor, CART-19, is composed of an extracellular single-chain antibody that binds to CD19 that is fused to intracellular signaling domains from 4-1BB and the CD3ζ chain. Top row of panels: a lentivirus, a type of retrovirus, is used to express the gene encoding CART-19 in the T cells harvested from a patient diagnosed with ALL. After in vitro activation and expansion, the transfected CART-19 cells are infused into the patient and exert cytotoxic actions against CD19-expressing tumor cells as well as nontransformed B cells. T cells are harvested from the blood of a

1	the transfected CART-19 cells are infused into the patient and exert cytotoxic actions against CD19-expressing tumor cells as well as nontransformed B cells. T cells are harvested from the blood of a patient with a B-cell tumor A retrovirus encoding an anti-CD19 CAR infects T cells that are activated with antibodies to CD3 and CD28 Infected T cells express an anti-CD19 CAR T cells are infused into patient to mediate antitumor activity CD19 anti-CD19 Anti-CD19 CAR 4-1BB signaling domain Anti-CD19 CAR chimeric receptor tumor cell T cell T cell CD3˜ chain ITAMs VH TCR CD28 CD3 VL

1	Fig. 16.19 Monoclonal antibodies that recognize tumor-specific antigens have been used to help eliminate tumors. Tumor-specific antibodies of the correct isotypes can lyse tumor cells by recruiting effector cells such as NK cells and activating them via their Fc receptors (left panels). Another strategy has been to couple the antibody to a powerful toxin (center panels). When the antibody binds to the tumor cell and is endocytosed, the toxin is released from the antibody and can kill the tumor cell. If the antibody is coupled to a radioisotope (right panels), binding of the antibody to a tumor cell will deliver a dose of radiation sufficient to kill the tumor cell. In addition, nearby tumor cells could also receive a lethal radiation dose, even though they do not bind the antibody. Antibody fragments have started to replace whole antibodies for coupling to toxins or radioisotopes.

1	the activity of a cytotoxic cell, toxin, or even radioactive nuclide specifically to the tumor (Fig. 16.19). Some of the cell-surface molecules targeted in clinical trials are shown in Fig. 16.20, and some of these treatments have now been licensed. Striking improvements in survival have been reported for breast cancer patients treated with the monoclonal antibody trastuzumab (Herceptin), which targets the receptor HER-2/neu. This receptor is overexpressed in about one-quarter of breast cancer patients and is associated with a poor prognosis.

1	Herceptin is thought to act by blocking the binding of the natural ligand (so far unidentified) to this receptor, and by downregulating the level of expression of the receptor. The effects of this antibody can be enhanced when it is combined with conventional chemotherapy. Beyond blocking a growth signal for tumor cells, experiments in mice suggest that some of trastuzumab’s antitumor effects also involve innate and adaptive immune responses, such as directing ADCC or inducing antitumor T-cell responses. A monoclonal antibody that has yielded excellent results in the treatment of non-Hodgkin’s B-cell lymphoma is the anti-CD20 antibody rituximab, which triggers the apoptosis of B cells upon binding to CD20 on their surface (see Section 16-7). ADCC may be another mechanism by which rituximab acts, as its clinical efficacy has been linked to polymorphisms in activating Fc receptors.

1	Technical problems with monoclonal antibodies as therapeutic agents include inefficient killing of cells after binding the monoclonal antibody, inefficient penetration of the antibody into the tumor mass (which can be improved by using small antibody fragments), and soluble target antigens mopping up the antibody. The efficiency of killing can be enhanced by linking the antibody to a toxin, producing a reagent called an immunotoxin (see Fig. 16.19): two favored toxins are ricin A chain and Pseudomonas toxin. The antibody must be internalized to allow cleavage of the toxin from the antibody in the endocytic compartment, permitting the freed toxin chain to penetrate and kill the cell. Toxins coupled to native antibodies have had limited success in cancer therapy, but fragments of antibodies such as single-chain Fv molecules (see Section 4-3) show more promise. An example of a successful immunotoxin is a recombinant Fv anti-CD22 antibody fused to a fragment of Pseudomonas toxin, which

1	such as single-chain Fv molecules (see Section 4-3) show more promise. An example of a successful immunotoxin is a recombinant Fv anti-CD22 antibody fused to a fragment of Pseudomonas toxin, which induced complete remissions in two-thirds of a group of patients with a type of B-cell leukemia known as hairy-cell leukemia in whom the disease was resistant to conventional chemotherapy.

1	Monoclonal antibodies can also be conjugated to chemotherapeutic drugs or to radioisotopes. In the case of a drug-linked antibody, the binding of the antibody to a cell-surface antigen concentrates the drug to the site of the tumor. After internalization, the drug is released in the endosomes and exerts its cytostatic or cytotoxic effect. For example, the antibody trastuzumab has been linked to the cytotoxic agent mertansine, a drug that inhibits the assembly of microtubules, in a conjugate called T-DM1. Since HER-2 is overexpressed only in cancer cells, T-DM1 selectively delivers the toxin specifically to tumor cells.

1	Fig. 16.21 An effective vaccine against human papilloma virus (HPV) induces antibodies that protect against HPV infection. Serotype 16 of HPV (HPV-16) is highly associated with the development of cervical cancer. In a clinical trial, 755 healthy uninfected women were immunized with a vaccine generated from highly purified noninfectious ‘viruslike particles’ (VLP) consisting of the capsid protein L1 of HPV-16 and formulated with an alum adjuvant (in this case aluminum hydroxyphosphate sulfate). In comparison with the very low titers of antibody in placebo-treated uninfected women (green line), or women previously infected with HPV who received placebo (blue line), the women treated with the viruslike particle vaccine (red line) developed high titers of antibody against the L1 capsid protein. None of these immunized women subsequently became infected by HPV-16. An anti-HPV vaccine marketed as Gardasil is now available and recommended for use in girls and young women as a protection from

1	None of these immunized women subsequently became infected by HPV-16. An anti-HPV vaccine marketed as Gardasil is now available and recommended for use in girls and young women as a protection from cervical cancer caused by HPV serotypes 6, 11, 16, and 18. mMU, milli-Merck units.

1	The HPV-16 vaccine induces high titers of specifc antibody that persist long after vaccination 1000 uninfected recipients of placebo recipients previously infected with HPV-16 placebo recipients not previously infected with HPV-16 Another drug–antibody conjugate, brentuximab vedotin, links an anti-CD30 antibody with a different microtubule inhibitor and is approved for certain forms of relapsed lymphomas.

1	A variation on this approach is to link an antibody to an enzyme that metabolizes a nontoxic pro-drug to the active cytotoxic drug, a technique known as antibody-directed enzyme/pro-drug therapy (ADEPT). With this technique, a small amount of enzyme localized by the antibody can generate much larger amounts of active cytotoxic drug in the immediate vicinity of tumor cells. Monoclonal antibodies linked to radioisotopes (see Fig. 16.19) have been successfully used to treat refractory B-cell lymphoma, using anti-CD20 antibodies linked to yttrium-90 (ibritumomab tiuxetan). These approaches have the advantage of also killing neighboring tumor cells, because the released drug or radioactive emissions can affect cells adjacent to those that bind the antibody. Monoclonal antibodies coupled to γ-emitting radioisotopes have also been used successfully to image tumors for the purposes of diagnosis and monitoring tumor spread.

1	16-18 Enhancing the immune response to tumors by vaccination holds promise for cancer prevention and therapy. In addition to CAR T cells and monoclonal antibody-based therapies, there are two other major approaches to cancer immunotherapy. Cancer vaccines are based on the idea that tumors are intrinsically poorly immunogenic, and the vaccine should act to supply the immunogenicity. A second approach, called checkpoint blockade, which we will discuss in the next section, is based on the idea that the immune system has been primed but is held in check by tolerance mechanisms, which can be blocked by therapeutic interventions.

1	Many cancers are associated with viral infections, and vaccines that prevent these infections can reduce cancer risk. A major breakthrough in anticancer therapy occurred in 2005 with the completion of a clinical trial involving 12,167 women that tested a vaccine against human papilloma virus (HPV). This trial showed that a recombinant vaccine against HPV was 100% effective in preventing cervical cancer caused by two key HPV strains, HPV-16 and HPV-18, which are associated with 70% of cervical cancers. The vaccine most likely prevents infection of cervical epithelium by HPV through the induction of anti-HPV antibodies (Fig. 16.21). Although this trial showed the potential of vaccines to prevent cancer, attempts to use vaccines to treat existing tumors have been less effective. In the case of HPV, certain types of vaccines that have increased immunogenicity for eliciting T-cell responses are beginning to show effectiveness in treating existing intraepithelial neoplasia caused by the

1	of HPV, certain types of vaccines that have increased immunogenicity for eliciting T-cell responses are beginning to show effectiveness in treating existing intraepithelial neoplasia caused by the virus. Similarly, the majority of liver cancers are associated with chronic hepatitis caused by several viruses. The vaccine against hepatitis B can reduce primary liver cancer due to this virus, although it will not protect against cancers caused by infections by other viruses such as hepatitis C.

1	Vaccines based on tumor antigens are, in principle, the ideal approach to T-cell-mediated cancer immunotherapy, but they are difficult to develop. For HPV, the relevant antigens are known. For most spontaneous tumors, however, relevant peptides of tumor rejection antigens may not be the same in different patients’ tumors and may be presented only by particular MHC alleles. This means that an effective tumor vaccine must include a range of tumor antigens. It is also clear that cancer vaccines for therapy should be used only when the tumor burden is low, such as after adequate surgery and chemotherapy.

1	The sources of antigens for cell-based cancer vaccines are the individual patients’ tumors removed at surgery. These vaccines are prepared by mixing either irradiated tumor cells or tumor extracts with killed bacteria such as Bacille Calmette–Guérin (BCG) or Corynebacterium parvum, which act as adjuvants to enhance the immunogenicity of the tumor antigens (see Appendix I, Section A-41). Vaccination using BCG adjuvants has had variable results in the past, but renewed interest is based on recent appreciation of their interaction with Toll-like receptors (TLRs). Stimulation of TLR-4 by BCG and other ligands has been tested against melanoma and other solid tumors. CpG DNA, which binds to TLR-9, has also been used to increase the immunogenicity of cancer vaccines. In cases where candidate tumor rejection antigens have been identified, for example, in melanomas, experimental vaccination strategies have included the use of whole proteins, peptide vaccines based on sequences recognized by

1	rejection antigens have been identified, for example, in melanomas, experimental vaccination strategies have included the use of whole proteins, peptide vaccines based on sequences recognized by cytotoxic T lymphocytes and helper T lymphocytes (either administered alone or presented by the patient’s own dendritic cells), and recombinant viruses encoding these peptide epitopes.

1	The potency of dendritic cells in activating T-cell responses provides the rationale for yet another antitumor vaccination strategy. The use of antigen-loaded dendritic cells to stimulate therapeutically useful cytotoxic T-cell responses to tumors has now undergone clinical trials in humans with cancer. One such vaccine, sipuleucel-T (Provenge), was recently approved for treatment of metastatic prostate cancer. In this therapy, a patient’s monocytes are extracted from peripheral blood and cultured with a fusion protein containing the antigen prostatic acid phosphatase (PAP), which is expressed by most prostate cancers, and the cytokine granulocyte–macrophage colony-stimulating factor (GM-CSF), which induces monocytes to undergo maturation to monocyte-derived dendritic cells. The resulting cells are reinfused into the patient to induce an immune response specific to the PAP antigen. This treatment reduced the risk of death by 22% and improved survival by about 4 months relative to a

1	cells are reinfused into the patient to induce an immune response specific to the PAP antigen. This treatment reduced the risk of death by 22% and improved survival by about 4 months relative to a placebo group. Other methods in clinical trials include loading dendritic cells ex vivo with DNA encoding the tumor antigen or with mRNA derived from tumor cells, and the use of apoptotic or necrotic tumor cells as sources of antigens.

1	16-19 Checkpoint blockade can augment immune responses to existing tumors.

1	Other approaches to tumor immunotherapy attempt to strengthen the natural immune responses against a tumor by one of two approaches: by making the tumor itself more immunogenic, or by relieving the normal inhibitory mechanisms that regulate these responses. The first kind of approach has been explored by inducing the expression of co-stimulatory molecules, such as B7 molecules, on tumor cells and then using these cells to activate tumor-specific naive T cells. Similarly, tumor cells may be transfected with the gene encoding granulocyte–macrophage colony-stimulating factor (GM-CSF) in order to induce the maturation of tumor-proximal monocytes into monocytederived dendritic cells. Once these cells have differentiated and capture antigens from the tumor, they may migrate to the local lymph nodes and activate tumor-specific T cells. No approved therapies have yet emerged from these approaches. In mice, B7-transfected cells seem less potent than the monoctye-derived dendritic cells

1	lymph nodes and activate tumor-specific T cells. No approved therapies have yet emerged from these approaches. In mice, B7-transfected cells seem less potent than the monoctye-derived dendritic cells differentiated by GM-CSF in inducing antitumor responses. This may be because molecules in addition to B7 act in priming naive T cells and these molecules may be expressed only by specific types of cross-presenting dendritic cells.

1	Another approach to cancer immunotherapy is called checkpoint blockade, which attempts to interfere with the normal inhibitory signals that regulate lymphocytes. Immune responses are controlled by several positive and negative immunological checkpoints. A positive checkpoint for T cells is controlled by the B7 co-stimulatory receptors expressed by professional antigen-presenting cells such as dendritic cells, as discussed earlier. Negative immunological checkpoints are provided by inhibitory receptors such as CTLA-4 and PD-1.

1	CTLA-4 imposes a critical checkpoint for potentially autoreactive T cells by binding to B7 molecules on dendritic cells and delivering a negative signal that must be overcome by other signals before T cells can become activated. Blocking CTLA-4 with antibodies may therefore lower the threshold for T-cell activation. Some evidence also indicates that anti-CTLA-4 antibodies may augment immune responses by eliminating regulatory T cells, which express CTLA-4 on their surface. Whatever the mechanism, the absence of this checkpoint causes self-reactive T cells that are normally held in check to become activated instead and produce a multi-tissue autoimmune reaction, as seen in CTLA-4-deficient mice.

1	Since checkpoint blockade relies on activation of the patient’s own immune system against tumors, its effects are not immediately evident, presenting a challenge in evaluating clinical responses to such therapy. Guidelines for evaluating clinical responses were based on the immediate effects of chemotherapeutic drugs or radiation, whereas checkpoint blockade requires more time for reversing immune inhibition and expanding tumor-specific T cells that then exert their effects within the tumor. Once such issues were considered, it became possible to design clinical trials that could document the effects of checkpoint blockade used in combination with traditional anticancer therapies.

1	Checkpoint blockade based on the anti-CTLA-4 antibody ipilimumab has now been shown to be effective in treating metastatic melanoma and recently received FDA approval for this indication. Patients with metastatic melanoma who were treated with ipilimumab showed an increase in the number and activity of T cells recognizing NY-ESO-1, a cancer-testis antigen expressed by melanoma. Overall only about 15% of patients exhibited a response to ipilimumab, but the treatment appeared to induce long-term remission in responding patients. One side-effect of ipilimumab in these patients seemed to be an increased risk of autoimmune phenomena, in agreement with the role of CTLA-4 in maintaining tolerance of self-reactive T cells.

1	Another checkpoint involves the inhibitory receptor PD-1 and its ligands PD-L1 and PD-L2. PD-L1 is expressed on a wide variety of human tumors; in renal cell carcinoma, PD-L1 expression is associated with a poor prognosis. In mice, transfection of the gene encoding PD-L1 into tumor cells increased their growth in vivo and reduced their susceptibility to lysis by cytotoxic T cells. These effects were reversed by an antibody against PD-L1. In humans, the anti-PD-1 antibody pembrolizumab has been shown to be effective in previously treated melanoma patients, giving a nearly 30% response rate. It is FDA-approved for use following treatment with ipilimumab, or in patients with a BRAF mutation after treatment with ipilimumab and a B-raf inhibitor. Another anti-PD-1 antibody, nivolumab, is also approved for treatment of metastatic melanoma and is being considered for use in treatment of Hodgkin’s lymphoma. Ongoing clinical trials are evaluating checkpoint blockade using antibodies to PD-L1

1	approved for treatment of metastatic melanoma and is being considered for use in treatment of Hodgkin’s lymphoma. Ongoing clinical trials are evaluating checkpoint blockade using antibodies to PD-L1 and PD-L2.

1	Summary.

1	Some tumors elicit specific immune responses that suppress or modify their growth. Tumors evade or suppress these responses in several ways, passing through various stages of a process known as immunological editing. Understanding how the immune system promotes and prevents cancer growth has led to new therapies now deployed in the clinic. The possibility of eradicating cervical cancer, for example, has been brought closer by the development of an effective vaccine against specific strains of cancer-causing human papilloma virus. Monoclonal antibodies have also been successfully developed for tumor immunotherapy in several cases, such as an anti-CD20 antibody used to treat B-cell lymphoma. Attempts are also being made to develop vaccines incorporating peptides designed to generate effective cytotoxic and helper T-cell responses. CAR T cells engineered to recognize CD19 expressed on B cells can be an effective treatment for acute lymphocytic leukemia. Checkpoint blockade strategies for

1	cytotoxic and helper T-cell responses. CAR T cells engineered to recognize CD19 expressed on B cells can be an effective treatment for acute lymphocytic leukemia. Checkpoint blockade strategies for CTLA-4 and PD-1 have been approved for treating melanoma, and related strategies are being developed for other biologic targets to stimulate antitumor immune responses or block inhibitory mechanisms that suppress such responses. One vaccine using dendritic cells that present tumor antigens has been approved for treating prostate cancer. A current trend in cancer therapy has been to incorporate immunotherapy with other traditional anticancer treatments to take advantage of the specificity and power of the immune system.

1	Fighting infectious diseases with vaccination.

1	The two most important contributions to public health in the past 100 years—sanitation and vaccination—have markedly decreased deaths from infectious disease, and yet infectious diseases remain the leading cause of death worldwide. Modern immunology itself grew from the success of Edward Jenner’s and Louis Pasteur’s vaccines against smallpox and chicken cholera, respectively, and its greatest triumph has been the global eradication of smallpox, announced by the World Health Organization in 1979. A global campaign to eradicate polio is now well under way. With the past decade’s tremendous progress in basic immunology, particularly in understanding innate immunity, there is now great hope that vaccines for other major infectious diseases, including malaria, tuberculosis, and HIV, are within reach. The vision of the current generation of vaccine scientists is to elevate their art to the level of modern drug design; to move it from an empiric practice to a true ‘pharmacology of the immune

1	reach. The vision of the current generation of vaccine scientists is to elevate their art to the level of modern drug design; to move it from an empiric practice to a true ‘pharmacology of the immune system.’

1	The goal of vaccination is the generation of long-lasting and protective immunity. Throughout this book, we have illustrated how the innate and the adaptive immune systems collaborate in the face of infection to eliminate pathogens and generate protective immunity with immunological memory. Indeed, a single infection is often (but not always) sufficient to generate protective immunity to a pathogen. Recognition of this important relationship was recorded more than 2000 years ago in accounts of the Peloponnesian War, during which two successive outbreaks of plague struck Athens. The Greek historian Thucydides noted that people who had survived infection during the first outbreak were not susceptible to infection during the second.

1	The recognition of this type of relationship perhaps prompted the practice of variolation against smallpox, in which an inoculation of a small amount of dried material from a smallpox pustule was used to produce a mild infection that was then followed by long-lasting protection against reinfection. Smallpox itself has been recognized in medical literature for more than 1000 years; variolation seems to have been practiced in India and China many centuries before its introduction into the West (some time in the 1400s–1500s), and it was familiar to Jenner. However, infection after variolation was not always mild: fatal smallpox ensued in about 3% of cases, which would not meet modern criteria of drug safety. It seems there was some recognition that milkmaids exposed to a bovine virus similar to smallpox—cowpox—seemed protected from smallpox infection, and there is even one historical account suggesting that cowpox inoculation had been tried before Jenner. However, Jenner’s achievement

1	to smallpox—cowpox—seemed protected from smallpox infection, and there is even one historical account suggesting that cowpox inoculation had been tried before Jenner. However, Jenner’s achievement was not only the realization that infection with cowpox would provide protective immunity against smallpox in humans without the risk of significant disease, but its experimental proof by the intentional variolation of people whom he had previously vaccinated. He named the process vaccination (from vacca, Latin for cow), and Pasteur, in his honor, extended the term

1	Fig. 16.22 Diseases for which effective vaccines are still needed. *Current measles vaccines are effective but heat-sensitive, which makes their use difficult in tropical countries; heat stability is being improved. Mortality data are the most recent estimated figures available (2014). Global Health Estimates 2000–2012. World Health Organization, June 2014. to the stimulation of protection against other infectious agents. Humans are not a natural host of cowpox, which establishes only a brief and limited subcutaneous infection. But the cowpox virus contains antigens that stimulate an immune response that cross-reacts with smallpox antigens and thereby confers protection against the human disease. Since the early 20th century, the virus used to vaccinate against smallpox has been vaccinia virus, which is related to both cowpox and smallpox, but whose origin is obscure.

1	As we will see, many current vaccines offer protection by inducing the formation of neutralizing antibodies. However, that statement contains a hidden tautology; pathogens for which current vaccines are effective may also be pathogens for which antibodies are sufficient for protection. For several major pathogens—malaria, tuberculosis, and HIV—even a robust antibody response is not fully protective. The elimination of these pathogens requires additional effector activities, such as the generation of strong and durable cell-mediated immunity, which are not efficiently generated by current vaccine technologies. These are the issues that face modern vaccine scientists. 16-20 Vaccines can be based on attenuated pathogens or material from killed organisms.

1	16-20 Vaccines can be based on attenuated pathogens or material from killed organisms. Vaccine development in the early part of the 20th century followed two empirical approaches. The first was the search for attenuated organisms with reduced pathogenicity, which would stimulate protective immunity but not cause disease. This approach continues into the present with the design of genetically attenuated pathogens in which desirable mutations are introduced into the organism by recombinant DNA technologies. This idea is being applied to important pathogens, such as malaria, for which vaccines are currently unavailable, and may be important in the future for designing vaccines for influenza and HIV.

1	The second approach was the development of vaccines based on killed organisms and, subsequently, on purified components of organisms that would be as effective as live whole organisms. Killed vaccines were desirable because any live vaccine, including vaccinia, can cause lethal systemic infection in immunosuppressed people. Evolving from this approach were vaccines based on the conjugation of purified antigens, as described for Haemophilus influenzae (see Section 16-27). This approach continues with the addition of ‘reverse immunogenetics’ (see Section 16-15) to identify candidate peptide antigens for T cells and with strategies to use ligands that activate TLRs or other innate sensors as adjuvants to enhance responses to simple antigens.

1	Immunization with such approaches is now considered so safe and so important that most states in the United States require all children to be immunized against several potentially deadly diseases. These include the the viral diseases measles, mumps, and polio, for which live-attenuated vaccines are used, as well as against tetanus (caused by Clostridium tetani), diphtheria (caused by Corynebacterium diphtheriae), and whooping cough (caused by Bordetella pertussis), for which vaccines composed of inactivated toxins or toxoids prepared from the respective bacteria are used. More recently, a vaccine has become available against H. influenzae type b (HiB), one of the causative agents of meningitis, as well as two vaccines for childhood diarrhea caused by rotaviruses, and, as described in Section 16-18, a vaccine for preventing HPV infection for protection against cervical cancer. Most vaccines are given to children within the first year of life. The vaccines against measles, mumps, and

1	16-18, a vaccine for preventing HPV infection for protection against cervical cancer. Most vaccines are given to children within the first year of life. The vaccines against measles, mumps, and rubella (MMR), against chickenpox (varicella), and against influenza, when recommended, are usually given between the ages of 1 and 2 years.

1	Impressive as these accomplishments are, there are still many diseases for which we lack effective vaccines (Fig. 16.22). For many pathogens, natural infection does not seem to generate protective immunity, and infections become chronic or recurrent. In many infections of this type, such as malaria, tuberculosis, and HIV, antibodies are insufficient to prevent reinfection and to eliminate the pathogen, and cell-mediated immunity instead seems to be more important in limiting the pathogen, but alone is still insufficient to provide full immunity. It is not the absence of an immune response to the pathogen that is the problem, but rather that this response does not clear the pathogen, eliminate pathogenesis, or prevent reinfection.

1	Another obstacle is that even when a vaccine such as that against measles can be used effectively in developed countries, technical and economic problems can prevent its widespread use in developing countries, where mortality from these diseases is still high. For example, simple costs of storage and deployment can be significant barriers to the use of existing vaccines in poorer countries. Therefore, the development of vaccines therefore remains an important goal of immunology, and the latter half of the 20th century saw a shift to a more rational approach based on a detailed molecular understanding of microbial pathogenicity, analysis of the protective host response to pathogenic organisms, and an understanding of the regulation of the immune system to generate effective Tand B-lymphocyte responses. 16-21 Most effective vaccines generate antibodies that prevent the damage caused by toxins or that neutralize the pathogen and stop infection.

1	16-21 Most effective vaccines generate antibodies that prevent the damage caused by toxins or that neutralize the pathogen and stop infection. Although the requirements for generating protective immunity vary with the nature of the infecting organism, many effective vaccines currently work by inducing antibodies against the pathogen. For many pathogens, including extracellular organisms and viruses, antibodies can provide protective immunity. This is not the case for all pathogens, unfortunately; some may require additional cell-mediated immune responses such as those mediated by CD8 T cells.

1	Effective protective immunity against some microorganisms requires the presence of preexisting antibody at the time of infection, either to prevent the damage caused by the pathogen or to prevent reinfection by the pathogen altogether. The first case is illustrated by vaccines against tetanus and diphtheria, whose clinical manifestations of infection are due to the effects of extremely powerful exotoxins (see Fig. 10.31). Preexisting antibody against the exotoxin is necessary to provide a defense against these diseases. Indeed, the tetanus exotoxin is so powerful that the tiny amount that can cause disease may be insufficient to lead to a protective immune response; consequently, even survivors of tetanus require vaccination to be protected against the risk of subsequent attack.

1	The second way in which antibodies can protect is by preventing infection a second time by the same pathogen, as in the case of certain viral infections. While CD8 T cells are able to kill already virally infected cells during an infection, antibodies are able to prevent infection of cells by the virus in the first place. This action is called neutralization. The ability of an antibody to neutralize a pathogen may depend on its affinity, its isotype subclass, complement, and the activity of phagocytic cells. For example, preexisting antibodies are required to protect against the polio virus, which infects critical host cells within a short period after entering the body and is not easily controlled by T lymphocytes once intracellular infection has been established. Vaccines against seasonal influenza virus provide protection in this same manner, by inducing antibodies that reduce the chance of a second infection by the same strain of influenza. For many viruses, antibodies produced by

1	influenza virus provide protection in this same manner, by inducing antibodies that reduce the chance of a second infection by the same strain of influenza. For many viruses, antibodies produced by an infection or by vaccination can neutralize the virus and prevent further spread of infection, but this is not always the case. In HIV infection, despite the generation of antibodies that can bind to surface viral epitopes, most of these antibodies fail to neutralize the virus. In addition, HIV has many different strains, or clades, and most vaccines based on HIV proteins do not induce antibodies that neutralize all clades, presenting a challenge for effective vaccine design. However, a recent clinical trial suggests that boosting previously vaccinated subjects with protein 5–7 years after immunization may induce some antibodies with crossclade activity.

1	Immune responses to infectious agents usually involve antibodies directed at multiple epitopes, and only some of these antibodies, if any, confer protection. The particular T-cell epitopes recognized can also affect the nature of the response. In Section 10-2, we described linked recognition, in which antigen-specific B cells and T cells provide mutually activating signals, leading to affinity maturation and isotype switching that may be required for neutralization. This process requires that an appropriate peptide epitope for T cells be presented by the B cells, and typically that the T-cell epitope be contained within the region of protein epitope recognized by the B cell, a fact that must be considered in modern vaccine design. Indeed, the predominant epitope recognized by T cells after vaccination with respiratory syncytial virus induces a vigorous inflammatory response but fails to elicit neutralizing antibodies and thus causes pathology without protection.

1	16-22 Effective vaccines must induce long-lasting protection while being safe and inexpensive.

1	A successful vaccine must possess several features in addition to its ability to provoke a protective immune response (Fig. 16.23). First, it must be safe. Vaccines must be given to huge numbers of people, relatively few of whom are likely to die of, or sometimes even catch, the disease that the vaccine is designed to prevent. This means that even a low level of toxicity is unacceptable. Second, the vaccine must be able to produce protective immunity in a very high proportion of the people to whom it is given. Third, particularly in poorer countries where it is impracticable to give regular ‘booster’ vaccinations to dispersed rural populations, a successful vaccine must generate long-lived immunological memory. This means that the vaccine must prime both B and T lymphocytes. Fourth, vaccines must be very cheap if they are to be administered to large populations. Vaccines are one of the most cost-effective measures in health care, but this benefit is eroded as the cost per dose rises.

1	Another benefit of an effective vaccination program is the ‘herd immunity’ that it confers on the general population. By lowering the number of susceptible members of a population, vaccination decreases the natural reservoir of infected individuals in that population and so reduces the probability of transmission of infection. Thus, even unvaccinated members will be protected because their individual chance of encountering the pathogen is decreased. However, the herd immunity effect is seen only at relatively high levels of vaccination within a population; for mumps, the necessary level is estimated to be around 80%, and below this level sporadic epidemics can occur. This is illustrated by a marked increase in mumps in the United Kingdom in 2004-2005 in young adults as a result of the variable use in the mid-1990s of a measles/ rubella vaccine rather than the combined MMR, as the combined vaccine was in short supply at that time.

1	16-23 Live-attenuated viral vaccines are usually more potent than ‘killed’ vaccines and can be made safer by the use of recombinant DNA technology.

1	Most antiviral vaccines currently in use consist of either live attenuated or inactivated viruses. Inactivated, or ‘killed,’ viral vaccines consist of viruses treated so that they are unable to replicate. Inactivated viruses therefore cannot produce proteins in the cytosol of infected cells, so peptides from the viral antigens are not presented by MHC class I molecules. Thus, CD8 T cells are neither efficiently generated nor needed with killed virus vaccines. Live-attenuated viral vaccines are generally far more potent: they elicit a greater number of effector mechanisms, including the activation of CD4 T cells and cytotoxic CD8 T cells. CD4 T cells help in shaping the antibody response, which is important for a vaccine’s subsequent protective effect. Cytotoxic CD8 T cells provide protection while infection by the virus itself is under way, and, if maintained, may contribute to protective memory. Attenuated viral vaccines include the routine childhood vaccines in use for polio,

1	while infection by the virus itself is under way, and, if maintained, may contribute to protective memory. Attenuated viral vaccines include the routine childhood vaccines in use for polio, measles, mumps, rubella, and varicella. Other attenuated live viral vaccines that are licensed for special circumstances or for use in high-risk populations include influenza, poxvirus (vaccinia), and yellow fever virus.

1	Traditionally, attenuation is achieved by growing the virus in cultured cells. Viruses are usually selected for preferential growth in nonhuman cells and, in the course of selection, become less able to grow in human cells (Fig. 16.24). Because these attenuated strains replicate poorly in human hosts, they induce immunity but not disease. Although attenuated virus strains contain multiple mutations in genes encoding several of their proteins, it might be possible for a pathogenic virus strain to reemerge by a further series of mutations. For example, the type 3 Sabin polio vaccine strain differs from a wild-type progenitor strain at only 10 of 7429 nucleotides. On extremely rare occasions, reversion of the vaccine to a neurovirulent strain can occur, causing paralytic disease in the unfortunate recipient.

1	Attenuated viral vaccines can also pose particular risks to immunodeficient recipients, in whom they often behave as virulent opportunistic infections. Immunodeficient infants who are vaccinated with live attenuated polio virus before their inherited immunoglobulin deficiencies have been diagnosed are at risk because they cannot clear the virus from their gut, and there is therefore an increased chance that mutations associated with the continuing uncontrolled replication of the virus in the gut will revert the virus to a virulent form and lead to fatal paralytic disease. An empirical approach to attenuation is still in use but might be superseded by two new approaches that use recombinant DNA technology. One is the isolation and in vitro mutagenesis of specific viral genes. The mutated genes are used to replace the wild-type genes in a reconstituted virus genome, and this

1	Fig. 16.24 Viruses are traditionally attenuated by selecting in cells of a different species, until it grows only poorly in human for growth in nonhuman cells. To produce an attenuated virus, cells. The adaptation is a result of mutation, usually a combination the virus must first be isolated by growing it in cultured human cells. of several point mutations. It is usually difficult to tell which of the The adaptation to growth in cultured human cells can cause some mutations in the genome of an attenuated viral stock are critical to attenuation in itself; the rubella vaccine, for example, was made in attenuation. An attenuated virus will grow poorly in the human host this way. In general, however, the virus is then adapted to growth and will therefore produce immunity but not disease.

1	Resulting virus is viable, immunogenic but avirulent. It can be used as a vaccine Isolate virulence gene Isolate pathogenic virus Mutate virulence gene Delete virulence gene receptor-binding protein virulence core proteins Fig. 16.25 Attenuation can be achieved more rapidly and reliably with recombinant DNA techniques. If a gene in the virus that is required for virulence but not for growth or immunogenicity can be identified, this gene can be either multiply mutated (left lower panel) or deleted from the genome (right lower panel) by using recombinant DNA techniques. This procedure creates an avirulent (nonpathogenic) virus that can be used as a vaccine. The mutations in the virulence gene are usually large, so that it is very difficult for the virus to revert to the wild type. deliberately attenuated virus can then be used as a vaccine (Fig. 16.25). The advantage of this approach is that mutations can be engineered so that reversion to wild type is virtually impossible.

1	Such an approach might be useful in developing live influenza vaccines. As described in Chapter 13, the influenza virus can reinfect the same host several times, because it undergoes antigenic shift and thus predominantly escapes the original immune response. A weak protection conferred by previous infections with a different subtype of influenza is observed in adults, but not in children, and is called heterosubtypic immunity. The current approach to vaccination against influenza is to use a killed virus vaccine that is reformulated annually on the basis of the prevalent strains of virus. The vaccine is moderately effective, reducing mortality in elderly people and illness in healthy adults. The ideal influenza vaccine would be an attenuated live organism that matched the prevalent virus strain. This could be created by first introducing a series of attenuating mutations into the gene encoding a viral polymerase protein, PB2. The mutated gene segment from the attenuated virus could

1	strain. This could be created by first introducing a series of attenuating mutations into the gene encoding a viral polymerase protein, PB2. The mutated gene segment from the attenuated virus could then be substituted for the wild-type gene in a virus carrying the relevant hemagglutinin and neuraminidase antigen variants of the current epidemic or pandemic strain. Alternatively, broadly neutralizing antibodies that block the receptor-binding domain of the hemagglutinin can be generated in humans and could be used as a universal vaccine. Public attention has recently been directed toward the possibility of a flu pandemic caused by the H5N1 avian flu strain. This strain can be passed between birds and humans and is associated with a high mortality rate; however, a pandemic would occur only if human-to-human transmission could occur. A live-attenuated vaccine would be used only if a pandemic occurred, because to give it beforehand would introduce new influenza virus genes that might

1	if human-to-human transmission could occur. A live-attenuated vaccine would be used only if a pandemic occurred, because to give it beforehand would introduce new influenza virus genes that might recombine with existing influenza viruses.

1	genetically attenuated parasites (GAPs). Similar approaches have been used for bacterial vaccine development. The most important example of an attenuated vaccine is that of BCG, which is quite effective at protecting against serious disseminated tuberculosis in children, but is not protective against adult pulmonary tuberculosis. The current BCG vaccine, which remains the most widely used vaccine in the world, was obtained from a pathogenic isolate of Mycobacterium bovis and passaged in a laboratory at the beginning of the 20th century. Since then, several genetically diverse strains of BCG have evolved. The level of protection afforded by the BCG vaccine is extremely variable, ranging from none in some countries, such as Malawi, to 50–80% in the UK.

1	Considering that tuberculosis remains one of the biggest killers worldwide, there is an urgent need for a new vaccine. Two recombinant BCG (rBCG) vaccines intended to prevent infection in unexposed individuals recently passed Phase I clinical trials. One was engineered to overexpress an immunodominant antigen of M. tuberculosis, to engender greater specificity toward the human pathogen. The second expresses the pore-forming protein listeriolysin from L. monocytogenes to induce the passage of BCG antigens from phagosomes into the cytoplasm and allow cross-presentation (see Section 6-5) on MHC class I molecules, thereby stimulating BCG-specific cytotoxic T cells. A similar approach is being used to generate new vaccines for malaria. Analysis of different stages of Plasmodium falciparum, the major cause of fatal malaria, identified genes that are selectively expressed in sporozoites within the mosquito’s salivary gland, where they first become infectious for human

1	Fig. 16.26 Genetically attenuated parasites can be engineered sporozoites with targeted disruption of key genes [for example, p52 as live vaccines to provide protective immunity. Top panel: and p36 (p52–/p36–), or uis3 (uis3–)], the sporozoites circulate in the wild-type Plasmodium sporozoites transmitted through the bite of bloodstream and mimic an early infection but cannot establish a an infected mosquito enter the bloodstream and are carried to the productive infection in the liver. The mice do, however, produce an liver, where they infect hepatocytes. Each sporozoite multiplies in the immune response against the sporozoites and are protected against liver, killing the infected cell and releasing thousands of merozoites, a subsequent infection by wild-type sporozoites. the next stage in infection. Bottom panels: in mice immunized with hepatocytes. Deletion of two such genes from the P. falciparum genome rendered sporozoites incapable of establishing a blood-stage infection in

1	in infection. Bottom panels: in mice immunized with hepatocytes. Deletion of two such genes from the P. falciparum genome rendered sporozoites incapable of establishing a blood-stage infection in mice, yet capable of inducing an immune response that protected mice from subsequent infection by wild-type P. falciparum. This protection was dependent on CD8 T cells, and to some extent on IFN-γ, indicating that cell-mediated immunity is important for protection against this parasite (Fig. 16.26). This highlights once again the importance of being able to generate vaccines that are capable of inducing strong cell-mediated immunity.

1	16-25 The route of vaccination is an important determinant of success. The ideal vaccination induces host defense at the point of entry of the infectious agent. Stimulation of mucosal immunity is therefore an important goal for vaccination against the many organisms that enter through mucosal surfaces. Still, most vaccines are given by injection. This route has several disadvantages. Injections are painful and unpopular, reducing vaccine uptake, and they are expensive, requiring needles, syringes, and a trained injector. Mass vaccination by injection is laborious. There is also the immunological drawback that injection may not be the most effective way of stimulating an appropriate immune response because it does not mimic the usual route of entry of the majority of pathogens against which vaccination is directed. Many important pathogens infect mucosal surfaces or enter the body through mucosal surfaces. Examples include respiratory microorganisms such as

1	Many important pathogens infect mucosal surfaces or enter the body through mucosal surfaces. Examples include respiratory microorganisms such as B. pertussis, rhinoviruses, and influenza viruses, and enteric microorganisms such as Vibrio cholerae, Salmonella typhi, enteropathogenic Escherichia coli, and Shigella. Intranasally administered live-attenuated vaccine against influenza virus induces mucosal antibodies, which are more effective than systemic antibodies in the control of upper respiratory tract infection. However, the systemic antibodies induced by injection are effective in controlling lower respiratory tract disease, which is responsible for the severe morbidity and mortality due to this disease. Thus, a realistic goal of any pandemic influenza vaccine is to prevent the lower respiratory tract disease but accept the fact that mild illness will not be prevented.

1	The power of the mucosal approach is illustrated by the effectiveness of live-attenuated polio vaccines. The Sabin oral polio vaccine consists of three attenuated polio virus strains and is highly immunogenic. Moreover, just as polio itself can be transmitted by fecal contamination of public swimming pools and other failures of hygiene, the vaccine can be transmitted from one individual to another by the fecal–oral route. Infection with Salmonella likewise stimulates a powerful mucosal and systemic immune response.

1	Presentation of soluble protein antigens by the oral route often results in tolerance, which is important given the enormous load of food-borne and airborne antigens presented to the gut and respiratory tract (see Chapter 12). Nonetheless, the mucosal immune system responds to and eliminates mucosal infections that enter by the oral route, such as pertussis, cholera, and polio. The proteins from these microorganisms that stimulate immune responses are therefore of special interest. One group of powerfully immunogenic proteins at mucosal surfaces is a group of bacterial toxins that have the property of binding to eukaryotic cells and being resistant to proteases. A recent finding of potential practical importance is that certain of these proteins, such as the

1	E. coli heat-labile toxin and pertussis toxin, have adjuvant properties that are retained even when the parent molecule has been engineered to eliminate its toxic properties. These molecules can be used as adjuvants for oral or nasal vaccines. In mice, nasal insufflation of either of these mutant toxins together with tetanus toxoid resulted in the development of protection against lethal challenge with tetanus toxin. 16-26 Bordetella pertussis vaccination illustrates the importance of the perceived safety of a vaccine.

1	16-26 Bordetella pertussis vaccination illustrates the importance of the perceived safety of a vaccine. The history of vaccination against the bacterium that causes whooping cough, Bordetella pertussis, illustrates the challenges of developing and disseminating an effective vaccine, as well as the public appeal of acellular conjugate vaccines over attenuated live organisms. At the beginning of the 20th century, whooping cough killed about 0.5% of American children under the age of 5 years. In the early 1930s, a trial of a killed, whole bacterial cell vaccine on the Faroe Islands provided evidence of a protective effect. In the United States, systematic use of a whole-cell vaccine in combination with diphtheria and tetanus toxoids (the DTP vaccine) during the 1940s resulted in a decline in the annual infection rate from 200 to fewer than 2 cases per 100,000 of the population. First vaccination with DTP was typically given at the age of 3 months.

1	Whole-cell pertussis vaccine causes side-effects, typically redness, pain, and swelling at the site of the injection; less commonly, vaccination is followed by high temperature and persistent crying. Very rarely, fits and a short-lived sleepiness or a floppy unresponsive state ensue. During the 1970s, widespread concern developed after several anecdotal observations that encephalitis leading to irreversible brain damage might very rarely follow pertussis vaccination. In Japan, in 1972, about 85% of children were given the pertussis vaccine, and fewer than 300 cases of whooping cough and no deaths were reported. As a result of two deaths after vaccination in Japan in 1975, the use of DTP was temporarily suspended and then reintroduced with the first vaccination at 2 years of age rather than at 3 months. In 1979, there were about 13,000 cases of whooping cough and 41 deaths. The possibility that pertussis vaccine very rarely causes severe brain damage has been studied extensively, and

1	at 3 months. In 1979, there were about 13,000 cases of whooping cough and 41 deaths. The possibility that pertussis vaccine very rarely causes severe brain damage has been studied extensively, and expert consensus is that pertussis vaccine is not a primary cause of brain injury. There is no doubt that there is greater morbidity from whooping cough than from the vaccine.

1	The public and medical perception that whole-cell pertussis vaccination might be unsafe provided a powerful incentive to develop safer pertussis vaccines. Study of the natural immune response to B. pertussis showed that infection induced antibodies against four components of the bacterium— pertussis toxin, filamentous hemagglutinin, pertactin, and fimbrial antigens. Immunization of mice with these antigens in purified form protected them against challenge with pertussis. This has led to the development of acellular pertussis vaccines, all of which contain purified pertussis toxoid—that is, toxin inactivated by chemical treatment, for example with hydrogen peroxide or formaldehyde, or more recently by genetic engineering of the toxin. Some pertussis vaccines also contain filamentous hemagglutinin, pertactin, and/or fimbrial antigens, either alone or in any combination of the three. Current evidence shows that these vaccines are probably as effective as whole-cell pertussis vaccine

1	pertactin, and/or fimbrial antigens, either alone or in any combination of the three. Current evidence shows that these vaccines are probably as effective as whole-cell pertussis vaccine while lacking its common minor side-effects. The acellular vaccine is more expensive, however, thus restricting its use in poorer countries.

1	The history of pertussis vaccination illustrates that, first and foremost, vaccines must be extremely safe and free of side-effects; second, the public and medical profession must perceive the vaccine to be safe; and third, careful study of the nature of the protective immune response can lead to acellular vaccines that are safer than whole-cell vaccines but still as effective. Still, public concerns about vaccination remain high. Unwarranted fears of a link between the combined live-attenuated MMR vaccine and autism saw the uptake of MMR vaccine in England fall from a peak of 92% of children in 1995–1996 to 84% in 2001–2002. Small clustered outbreaks of measles and mumps in London since 2002 illustrate the importance of maintaining high uptake of vaccine to maintain herd immunity. 16-27 Conjugate vaccines have been developed as a result of linked recognition between T and B cells.

1	16-27 Conjugate vaccines have been developed as a result of linked recognition between T and B cells. Many bacteria, including Neisseria meningitidis (meningococcus), Streptococcus pneumoniae (pneumococcus), and H. influenzae, have an outer capsule composed of polysaccharides that are speciesand type-specific for particular strains of the bacterium. The most effective defense against these microorganisms is opsonization of the polysaccharide coat with antibody. The aim of vaccination for these organisms is therefore to elicit antibodies against the polysaccharide capsules of the bacteria. However, effective acellular vaccines cannot be made from a single isolated constituent of a microorganism, since generation of an effective antibody response requires the participation of several types of cells, and this fact has led to the development of conjugate vaccines (Fig. 16.27).

1	Capsular polysaccharides can be harvested from bacterial growth medium and, because they are T-cell-independent antigens (see Section 10-1), they can be used on their own as vaccines. However, young children under the age Fig. 16.27 Conjugate vaccines take advantage of linked recognition to boost B-cell responses against polysaccharide antigens. The Hib vaccine against Haemophilus influenzae type b is a conjugate of bacterial polysaccharide and the tetanus toxoid protein. The B cell recognizes and binds the polysaccharide, internalizes and degrades the whole conjugate, and then displays toxoid-derived peptides on surface MHC class II molecules. Helper T cells generated in response to earlier vaccination against the toxoid recognize the complex on the B-cell surface and activate the B cell to produce anti-polysaccharide antibody. This antibody can then protect against infection with H. influenzae type b. Chapter 16: Manipulation of the Immune Response

1	Fig. 16.28 The effect of vaccination against group C Neisseria meningitidis (meningococcus) on the number of cases of group B and group C meningococcal disease in England and Wales. Meningococcal infection affects roughly 5 in 100,000 people a year in the UK, with groups B and C meningococci accounting for almost all the cases. Before the introduction of the meningitis C vaccine, group C disease was the second most common cause of meningococcal disease, accounting for about 40% of cases. Group C disease now accounts for less than 10% of cases, with group B disease accounting for more than 80% of cases. After the introduction of the vaccine, there was a significant decrease in the number of laboratory-confirmed cases of group C disease in all age groups. The impact was greatest in the immunized groups, with reductions of more than 90% in these groups. An impact has also been seen in the unimmunized age groups, with a reduction of about 70%, suggesting that this vaccine has had a herd

1	groups, with reductions of more than 90% in these groups. An impact has also been seen in the unimmunized age groups, with a reduction of about 70%, suggesting that this vaccine has had a herd immunity effect.

1	Number of cases

1	Number of cases of 2 years cannot make good T-cell-independent antibody responses and cannot be vaccinated effectively with polysaccharide (PS) vaccines. An efficient way of overcoming this problem is to conjugate bacterial polysaccharides chemically to protein carriers (see Fig. 16.27). This carrier protein provides peptides that can be recognized by antigen-specific T cells, thus converting a T-cell-independent response into a T-cell-dependent antipolysaccharide antibody response. Using this approach, various conjugate vaccines have been developed against H. influenzae type b, an important cause of serious childhood chest infections and meningitis, and against N. meningitidis serogroup C, an important cause of meningitis, and these are now widely applied. The success of the latter vaccine in the United Kingdom is illustrated in Fig. 16.28, which shows that the incidence of meningitis C has been markedly reduced in comparison with meningitis B, against which there is currently no

1	in the United Kingdom is illustrated in Fig. 16.28, which shows that the incidence of meningitis C has been markedly reduced in comparison with meningitis B, against which there is currently no vaccine. Endemic meningitis B is due to diverse serogroup B strains, so an ideal vaccine would target the group B capsular polysaccharide. Unfortunately, group B polysaccharide is identical to some polysialyl polysaccharides on human cells, and is poorly immunogenic due to tolerance of these self antigens. Some strategies to chemically modify the group B polysaccharide for use in a conjugate vaccine have been considered, but a major focus in group B meningococcal vaccine development has been to instead direct immunity against noncapsular antigens, which will be generally effective against endemic disease.

1	16-28 Peptide-based vaccines can elicit protective immunity, but they require adjuvants and must be targeted to the appropriate cells and cell compartment to be effective.

1	Another vaccine-development strategy that does not require the whole organism, whether killed or attenuated, identifies the T-cell peptide epitopes that stimulate protective immunity. Candidate peptides can be identified in two ways: in one, overlapping peptides from immunogenic proteins are systematically synthesized and their ability to stimulate protective immunity is tested; alternatively, a reverse immunogenetic approach (see Section 16-15) can be used to predict potential peptide epitopes from a genome sequence. The latter approach has been applied to malaria by using the complete sequence of the Plasmodium falciparum genome. The starting point was the association between the human MHC class I molecule HLA-B53 and resistance to cerebral malaria, a relatively infrequent, but usually fatal, complication of infection by P. falciparum. It was thought that HLA-B53 might protect against cerebral malaria because it could present peptides that are particularly good at activating naive

1	fatal, complication of infection by P. falciparum. It was thought that HLA-B53 might protect against cerebral malaria because it could present peptides that are particularly good at activating naive cytotoxic T lymphocytes. Peptides eluted from HLA-B53 frequently contain a proline as the second of their nine amino acids. On the basis of this information, reverse genetic analysis identified candidate protective peptides from four proteins of P. falciparum expressed in the early phase of hepatocyte infection—an important phase of infection to target in an effective immune response. One of the candidate peptides, from liver stage antigen-1, has been shown to be recognized by cytotoxic T cells when bound to HLA-B53 and may be a useful peptide for use in vaccination.

1	Peptide-based vaccines, although promising, have several drawbacks. First, a particular peptide may not bind to all the MHC molecules present in the population. Because humans are highly polymorphic in the MHC, a large panel of protective peptides would be needed for coverage of most individuals. Second, some direct exchange of short peptides on MHC molecules can occur without physiological antigen processing. If the required antigenic peptides load directly onto MHC molecules on cells other than dendritic cells, this may induce tolerance in T cells rather than stimulating immunity. Third, exogenous proteins and peptides delivered by a synthetic vaccine are efficiently processed for presentation by MHC class II molecules, but require ‘cross-presentation’ in specific types of dendritic cells to be loaded onto MHC class I molecules (see Section 6-5). Directing peptide-based vaccines to such cells may enhance vaccine efficacy.

1	Recent peptide-based vaccines have already shown promise in human clinical trials. Patients with established vulvar intraepithelial neoplasia, an early form of vulvar cancer caused by human papilloma virus (HPV), were treated with a vaccine consisting of long peptides covering the entire length of two oncoproteins of HPV-16—E6 and E7—and delivered in an oil-in-water emulsion as adjuvant. By using very long peptides, around 100 amino acids in length, multiple candidate peptide epitopes can be delivered that may also be presented by different MHC alleles. These peptides seem to be too long for direct exchange with peptides on cell surfaces and require processing by dendritic cells in order to be loaded onto MHC class I molecules. This vaccine induced complete clinical remission in one-quarter of the patients, and about half of the treated patients showed significant clinical responses that correlated with in vitro evidence of enhanced cell-mediated immunity.

1	16-29 Adjuvants are important for enhancing the immunogenicity of vaccines, but few are approved for use in humans. Vaccines based on peptides or purified proteins require additional components to mimic how real infections activate immunity. Such components of a vaccine are known as adjuvants, which are defined as substances that enhance the immunogenicity of antigens (see Appendix I, Section A-41). For example, tetanus toxoid is not immunogenic in the absence of adjuvants, and so tetanus toxoid vaccines contain inorganic aluminum salts (alum) in the form of noncrystalline gels that bind polyvalently to the toxoid by ionic interactions. Pertussis toxin has adjuvant properties in its own right and, when given mixed as a toxoid with tetanus and diphtheria toxoids, not only protects against whooping cough but also acts as an additional adjuvant for the other two toxoids. This mixture makes up the DTP triple vaccine given to infants in the first year of life.

1	The antigenic components and adjuvants in a vaccine are not approved for use on their own; they are approved only in the context of the specific vaccine in which they are formulated. At present, alum is the only adjuvant that is approved by the FDA in the United States for use in marketed human vaccines, although some other adjuvant–vaccine combinations are undergoing clinical trials. Alum is the common name for certain inorganic aluminum salts, of which aluminum hydroxide and aluminum phosphate are most frequently used as adjuvants. In Europe, in addition to the alum adjuvants, an oil (squalene)-in-water emulsion called MF-59 is used as an adjuvant in a formulation of influenza vaccine and is undergoing evaluation in clinical trials. As we described in Section 3-9, alum seems to act as an adjuvant by stimulating one of the innate immune system’s bacterial sensor mechanisms, NLRP3, thus activating the inflammasome and the inflammatory reactions that are a prerequisite for an effective

1	adjuvant by stimulating one of the innate immune system’s bacterial sensor mechanisms, NLRP3, thus activating the inflammasome and the inflammatory reactions that are a prerequisite for an effective adaptive immune response.

1	Several other adjuvants are widely used experimentally in animals but are not approved for use in humans. Many of these are sterile constituents of bacteria, particularly of their cell walls. Freund’s complete adjuvant is an oil-in-water emulsion containing killed mycobacteria. The peptidoglycan muramyl dipeptide and the glycolipid trehalose dimycolate (TDM) found in mycobacterial cell walls contain much of the adjuvant activity of the whole killed organism. Other bacterial adjuvants include killed B. pertussis, bacterial polysaccharides, bacterial heat-shock proteins, and bacterial DNA. Many of these adjuvants cause quite marked inflammation and so are not suitable for use in vaccines for humans.

1	Many adjuvants seem to work by triggering the innate viral and bacterial sensor pathways in APCs, via TLRs and proteins of the NOD-like receptor family such as NLRP3 (see Chapter 3), and thereby activating them to initiate an adaptive immune response. The TLR-4 agonist lipopolysaccharide (LPS) has adjuvant effects, but its use is limited by its toxicity. Small amounts of injected LPS can induce a state of shock and systemic inflammation that mimics Gram-negative sepsis, raising the question whether its adjuvant effect can be separated from its toxic effects. Monophosphoryl lipid A, an LPS derivative and TLR-4 ligand, partly achieves this, retaining adjuvant effects but being associated with much lower toxicity than LPS. Both unmethylated CpG DNA, which activates TLR-9, and imiquimod, a small-molecule drug that acts as a TLR-7 agonist, can provide adjuvant activity experimentally, but neither is approved as an adjuvant in human vaccines.

1	16-30 Protective immunity can be induced by DNA-based vaccination.

1	Surprisingly, when bacterial plasmids were used to express proteins in vivo for gene therapy, some were found to stimulate an immune response. Later, it was found that DNA encoding a viral immunogen, when injected intramuscularly in mice, induced antibody responses and cytotoxic T cells that could protect against subsequent infection from the live virus. This response does not seem to damage the muscle tissue, is safe and effective, and, because it uses only a single microbial gene or a stretch of DNA encoding sets of antigenic peptides, does not carry the risk of active infection. This procedure is termed DNA vaccination, and can be carried out in various ways. In one, DNA coated onto minute metal particles can be administered by a gene gun, so that particles penetrate the skin and potentially some underlying muscle, but other approaches, such as electroporation, are also possible. Because of DNA’s stability, DNA vaccination is suitable for mass immunization. One problem with

1	potentially some underlying muscle, but other approaches, such as electroporation, are also possible. Because of DNA’s stability, DNA vaccination is suitable for mass immunization. One problem with DNA-based vaccines, however, is that they are comparatively weak. Mixing in plasmids that encode cytokines such as IL-12, IL-23, or GM-CSF makes immunization with genes encoding protective antigens much more effective. In DNA vaccination, the antigen is produced by cells that are directly transfected, such as skin or muscle, but CD8 T-cell activation requires cross-presentation of the antigen by dendritic cells. Current approaches are identifying how best to transfect DNA into these dendritic cell populations. DNA vaccines are being tested in human trials for malaria, influenza, HIV infection, and breast cancer.

1	16-31 Vaccination and checkpoint blockade may be useful in controlling existing chronic infections. There are many chronic diseases in which infection persists because of a failure of the immune system to eliminate disease. Such infections can be divided into two groups: those in which there is an obvious immune response that fails to eliminate the organism, and those that seem to be invisible to the immune system and evoke a barely detectable immune response.

1	In the first category, the immune response is often partly responsible for the pathogenic effects. Infection by the helminth Schistosoma mansoni is associated with a powerful TH2-type response, characterized by high levels of IgE, circulating and tissue eosinophilia, and a harmful fibrotic response to schistosome ova in the liver, leading to hepatic fibrosis. Other common parasites, such as Plasmodium and Leishmania species, also cause damage in many patients because they are not eliminated effectively by the immune response. The mycobacterial agents of tuberculosis and leprosy cause a persistent intracellular infection; a TH1 response helps to contain these infections but also causes granuloma formation and tissue necrosis (see Fig. 11.13).

1	Among viruses, hepatitis B and hepatitis C infections are commonly followed by a persistent viral burden and hepatic injury, resulting in eventual death from hepatitis or from hepatocellular carcinoma. Infection with HIV, as we have seen in Chapter 13, also persists despite an ongoing immune response. In a preliminary trial involving HIV-infected patients, dendritic cells derived from the patients’ own bone marrow were loaded with chemically inactivated HIV. After immunization with the loaded cells, a robust T-cell response to HIV was observed in some patients that was associated with the production of IL-2 and IFN-γ (Fig. 16.29). Viral load in these patients was reduced by 80%,

1	Fig. 16.29 Vaccination with dendritic cells loaded with HIV substantially reduces viral load and generates T-cell immunity. Left panel: viral load is shown for a weak and transient response to treatment (pink); the red bar represents individuals who made a strong and durable response. Right panel: CD4 T-cell IL-2 and interferon-γ production for individuals who made a weak or strong response. The production of both these cytokines, indicating T-cell activity, correlates with the response to treatment.

1	Viral load weak response strong response weak response strong response weak response strong response IFN-˜(after 112 days of treatment) IL-2 Effect of dendritic-cell vaccination for HIV on immune function and virus production 0 1 2 3 4 5 0 10,000 20,000 30,000 40,000 50,000 Percentage of HIV-specifc CD4 cells expressing cytokine and in almost half of these patients the suppression of viremia lasted for more than a year. Nonetheless, these responses were not sufficient to eliminate the HIV infection.

1	In the second category of chronic infection, which is predominantly viral, the immune response fails to clear the infection because of the relative invisibility of the infectious agent to the immune system. A good example is herpes simplex virus type 2, which is transmitted venereally, becomes latent in nerve tissue, and causes genital herpes, which is frequently recurrent. The invisibility of this virus seems to be caused by a viral protein, ICP-47, that binds to the TAP complex (see Section 6-3) and inhibits peptide transport into the endoplasmic reticulum in infected cells. Thus, viral peptides are not presented to the immune system by MHC class I molecules. A similar example in this category of chronic infection is genital warts, caused by certain papilloma viruses that evoke very little immune response, particularly a cell-mediated response. As discussed previously, the results of a recent clinical trial showed that using long-peptide vaccines against HPV-16 was effective in

1	little immune response, particularly a cell-mediated response. As discussed previously, the results of a recent clinical trial showed that using long-peptide vaccines against HPV-16 was effective in increasing the strength of cell-mediated immune responses to viral antigens, and in reducing or eliminating precancerous lesions associated with the HPV infection (see Section 16-28). These results are a positive indication that vaccines directed at increasing cell-mediated responses to other pathogens may be similarly effective.

1	Summary.

1	Vaccination is arguably the greatest success of immunology, having eradicated or virtually eliminated several human diseases. It is the single most successful manipulation of the immune system so far, because it takes advantage of the immune system’s natural specificity and inducibility. But important human infectious diseases remain that lack effective vaccines. Most effective vaccines are based on attenuated live microorganisms, but such vaccines carry some risk and are potentially lethal to immunosuppressed or immunodeficient individuals. New techniques are being developed to generate genetically attenuated pathogens for use as vaccines, particularly for malaria and tuberculosis. While most current viral vaccines are based on live attenuated virus, many bacterial vaccines are based on components of the microorganism, including components of the toxins that it produces. Protective responses to carbohydrate antigens, which in very young children do not provoke lasting immunity, can

1	components of the microorganism, including components of the toxins that it produces. Protective responses to carbohydrate antigens, which in very young children do not provoke lasting immunity, can be enhanced by conjugation of the carbohydrate to a protein. Vaccines based on peptides, particularly very long peptides, are just emerging from the experimental stage and are beginning to be tested in humans. A vaccine’s immunogenicity often depends on adjuvants that can help, directly or indirectly, to activate antigen-presenting cells that are necessary for the initiation of immune responses. Adjuvants activate these cells by engaging the innate immune system and providing ligands for TLRs and other innate sensors on antigen-presenting cells. The development of oral vaccines is particularly important for stimulating immunity to the many pathogens that enter through the mucosa.

1	Summary to Chapter 16.

1	One of the great future challenges in immunology is to be able to control the immune system so that unwanted immune responses can be suppressed and desirable responses elicited. Current methods of suppressing unwanted responses rely, to a great extent, on drugs that suppress adaptive immunity indiscriminately and are thus inherently flawed. We have seen in this book that the immune system can suppress its own responses in an antigen-specific manner and that, by studying these endogenous regulatory events, it has been possible to devise strategies to manipulate specific responses while sparing general immune competence. New treatments, including many monoclonal antibodies, have emerged as clinically important therapies to selectively suppress the responses that lead to allergy, autoimmunity, or the rejection of grafted organs. Similarly, as we understand more about tumors and infectious agents, better strategies to mobilize the immune system against cancer and infection are becoming

1	or the rejection of grafted organs. Similarly, as we understand more about tumors and infectious agents, better strategies to mobilize the immune system against cancer and infection are becoming possible. To achieve all this, we need to learn more about the induction of immunity and the biology of the immune system, and to apply what we have learned to human disease.

1	Questions. 16.1 Multiple Choice: Which of the following immunomodulators has a similar mechanism to azathioprine? A. Mycophenolate B. Cyclophosphamide C. Abatacept D. Rapamycin 16.2 Matching: Match the following immunomodulating antibodies with their respective mechanism of action. 16.3 True or False: Chimeric antigen receptor (CAR) T cells are cells that have been retrovirally transduced with a tumor-specific T-cell receptor in order to treat a leukemia. 16.4 Multiple Choice: Which statement is false? A. The vaccine Provenge is prepared using the patient’s own antigen-loaded dendritic cells to induce therapeutic antitumor T-cell responses. B. Clinical trials of vaccines against HPV-16 and HPV18 (associated with 70% of cervical cancers) were 100% effective in preventing cervical cancers caused by these viruses.

1	B. Clinical trials of vaccines against HPV-16 and HPV18 (associated with 70% of cervical cancers) were 100% effective in preventing cervical cancers caused by these viruses. C. Cell-based cancer vaccines can use the patient’s tumor as a source of antigens. In order to enhance immunogenicity these can be mixed with adjuvants such as CpG, which binds to TLR-7. 16.5 Multiple Choice: Which of the following treatments against cancer is a checkpoint blockade therapy? (One or more may apply.) A. Ipilimumab (anti-CTLA-4 antibody) B. Trastuzumab (anti-HER-2/neu antibody) C. Rituximab (anti-CD20 antibody) D. Pembrolizumab (anti-PD-1 antibody) E. Sipuleucel-T (patient’s dendritic cells cultured with prostatic acid phosphatase tumor antigen and GM-CSF and reinfused into patient) 16.6 True or False: Chimeric antigen receptor (CAR) T cells can recognize other target molecules besides peptide:MHC complexes.

1	16.7 Matching: Classify the currently used vaccines of the following organisms as live-attenuated (A), toxin-based (T), killed (K), or conjugate polysaccharide (P). A. ___ Corynebacterium diphtheriae B. ___ H. influenzae type B C. ___ Measles/mumps/rubella (MMR) D. ___ Bacille Calmette–Guérin (BCG) E. ___ Influenza A virus F. ___ Sabin polio vaccine 16.8 Fill-in-the-Blanks: Vaccines have exhibited many phenomena that are beneficial and can be exploited. For example, when an antibody response against a bacterial polysaccharide is desired, it is conjugated to a protein to exploit the phenomenon of ____________________, thus ensuring T-dependent antibody responses. In addition, vaccines may protect against different subtypes of virus, as in the case of influenza, a phenomenon called _____________ immunity. When enough people in a population are vaccinated, ______ immunity is achieved, where even unvaccinated individuals are indirectly protected from infection.

1	16.9 Short Answer: Explain the three main drawbacks of peptide-based vaccines. 16.10 True or False: All routes of vaccination successfully elicit virtually identical immune responses. 16.11 Matching: Match the adjuvant to the immune receptor it stimulates. A. Alum i. TLR-9 B. Freund’s complete adjuvant ii. TLR-4 C. Lipopolysaccharide iii. NLRP3 D. DNA iv. NOD2 E. Imiquimod v. TLR-7/8 General references. Maus, M.V., Fraietta, J.A., Levine, B.L., Kalos, M., Zhao, Y., and June, C.H.: Adoptive immunotherapy for cancer or viruses. Annu Rev Immunol. 2014, 32:189–225. Feldmann, M.: Translating molecular insights in autoimmunity into effective therapy. Annu. Rev. Immunol. 2009, 27:1–27. Kappe, S.H., Vaughan, A.M., Boddey, J.A., and Cowman, A.F.: That was then but this is now: malaria research in the time of an eradication agenda. Science 2010, 328:862–866. Kaufmann, S.H.: Future vaccination strategies against tuberculosis: thinking outside the box. Immunity 2010, 33:567–577.

1	Kaufmann, S.H.: Future vaccination strategies against tuberculosis: thinking outside the box. Immunity 2010, 33:567–577. Korman, A.J., Peggs, K.S., and Allison, J.P.: Checkpoint blockade in cancer immunotherapy. Adv. Immunol. 2006, 90:297–339. Section references. 16-1 Corticosteroids are powerful anti-inflammatory drugs that alter the transcription of many genes. Kampa, M., and Castanas, E.: Membrane steroid receptor signaling in normal and neoplastic cells. Mol. Cell. Endocrinol. 2006, 246:76–82. Löwenberg, M., Verhaar, A.P., van den Brink, G.R., and Hommes, D.W.: Glucocorticoid signaling: a nongenomic mechanism for T-cell immunosuppression. Trends Mol. Med. 2007, 13:158–163. Rhen, T., and Cidlowski, J.A.: Antiinflammatory action of glucocorticoids— new mechanisms for old drugs. N. Engl. J. Med. 2005, 353:1711–1723. Barnes, P.,J.: Glucocorticosteroids: current and future directions. Br. J. Pharmacol. 2011, 163:29–43.

1	Barnes, P.,J.: Glucocorticosteroids: current and future directions. Br. J. Pharmacol. 2011, 163:29–43. 16-2 Cytotoxic drugs cause immunosuppression by killing dividing cells and have serious side-effects. Aarbakke, J., Janka-Schaub, G., and Elion, G.B.: Thiopurine biology and pharmacology. Trends Pharmacol. Sci. 1997, 18:3–7. Allison, A.C., and Eugui, E.M.: Mechanisms of action of mycophenolate mofetil in preventing acute and chronic allograft rejection. Transplantation 2005, 802 Suppl.: S181–S190. O’Donovan, P., Perrett, C.M., Zhang, X., Montaner, B., Xu, Y.Z., Harwood, C.A., McGregor, J.M.,Walker, S.L., Hanaoka, F., and Karran, P.: Azathioprine and UVA light generate mutagenic oxidative DNA damage. Science 2005, 309:1871–1874. Taylor, A.L., Watson, C.J., and Bradley, J.A.: Immunosuppressive agents in solid organ transplantation: mechanisms of action and therapeutic efficacy. Crit. Rev. Oncol. Hematol. 2005, 56:23–46.

1	Taylor, A.L., Watson, C.J., and Bradley, J.A.: Immunosuppressive agents in solid organ transplantation: mechanisms of action and therapeutic efficacy. Crit. Rev. Oncol. Hematol. 2005, 56:23–46. Zhu, L.P., Cupps, T.R., Whalen, G., and Fauci, A.S.: Selective effects of cyclophosphamide therapy on activation, proliferation, and differentiation of human B cells. J. Clin. Invest. 1987, 79:1082–1090. 16-3 Cyclosporin A, tacrolimus, rapamycin and JAK inhibitors are effective immunosuppressive agents that interfere with various T-cell signaling pathways. Araki, K., Turner, A.P., Shaffer, V.O., Gangappa, S., Keller, S.A., Bachmann, M.F., Larsen, C.P., and Ahmed, R.: mTOR regulates memory CD8 T-cell differentiation. Nature 2009, 460:108–112. Battaglia, M., Stabilini, A., and Roncarolo, M.G.: Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. Blood 2005, 105:4743–4748.

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1	Kim, S.J., Park, Y., and Hong, H.J.: Antibody engineering for the development of therapeutic antibodies. Mol. Cell 2005, 20:17–29. Liu, X.Y., Pop, L.M., and Vitetta, E.S.: Engineering therapeutic monoclonal antibodies. Immunol. Rev. 2008, 222:9–27. Smith, K., Garman, L., Wrammert, J., Zheng, N.Y., Capra, J.D., Ahmed, R., and Wilson, P.C.: Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat. Protocols 2009, 4:372–384. Traggiai, E., Becker, S., Subbarao, K., Kolesnikova, L., Uematsu, Y., Gismondo, M.R., Murphy, B.R., Rappuoli, R., and Lanzavecchia, A.: An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat. Med. 2004, 10:871–875. Winter, G., Griffiths, A.D., Hawkins, R.E., and Hoogenboom, H.R.: Making antibodies by phage display technology. Annu. Rev. Immunol. 1994, 12:433–455. 16-6 Monoclonal antibodies can be used to prevent allograft rejection.

1	16-6 Monoclonal antibodies can be used to prevent allograft rejection. Kirk, A.D., Burkly, L.C., Batty, D.S., Baumgartner, R.E., Berning, J.D., Buchanan, K., Fechner, J.H., Jr, Germond, R.L., Kampen, R.L., Patterson, N.B., et al.: Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates. Nat. Med. 1999, 5:686–693. Li, X.C., Strom, T.B., Turka, L.A., and Wells, A.D.: T-cell death and transplantation tolerance. Immunity 2001, 14:407–416. Londrigan, S.L., Sutherland, R.M., Brady, J.L., Carrington, E.M., Cowan, P.J., d’Apice,A.J.,O’Connell,P.J.,Zhan,Y.,and Lew,A.M.: In situ protection against islet allograft rejection by CTLA4Ig transduction. Transplantation 2010, 90:951–957. Masharani, U.B., and Becker, J.: Teplizumab therapy for type 1 diabetes. Expert Opin. Biol. Ther. 2010, 10:459–465.

1	Masharani, U.B., and Becker, J.: Teplizumab therapy for type 1 diabetes. Expert Opin. Biol. Ther. 2010, 10:459–465. Pham, P.T., Lipshutz, G.S., Pham, P.T., Kawahji, J., Singer, J.S., and Pham, P.C.: The evolving role of alemtuzumab (Campath-1H) in renal transplantation. Drug Des. Dev. Ther. 2009, 3:41–49. Sageshima, J., Ciancio, G., Chen, L., and Burke, G.W.: Anti-interleukin-2 receptor antibodies—basiliximab and daclizumab—for the prevention of acute rejection in renal transplantation. Biologics 2009, 3:319–336. 16-7 Depletion of autoreactive lymphocytes can treat autoimmune disease. Coiffier, B., Lepage, E., Briere, J., Herbrecht, R., Tilly, H., Bouabdallah, R., Morel, P., Van Den Neste, E., Salles, G., Gaulard, P., et al.: CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N. Engl. J. Med. 2002, 346:235–242. References.

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1	16-17 Monoclonal antibodies against tumor antigens, alone or linked to toxins, can control tumor growth. Bradley,A.M., Devine, M., and DeRemer, D.: Brentuximab vedotin: an anti-CD30 antibody-drug conjugate. Am. J. Health Syst. Pharm. 2013, 70:589–597. Hortobagyi, G.N.: Trastuzumab in the treatment of breast cancer. N. Engl. J. Med. 2005, 353:1734–1736. Kreitman, R.J., Wilson, W.H., Bergeron, K., Raggio, M., Stetler-Stevenson, M., FitzGerald, D.J., and Pastan, I.: Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia. N. Engl. J. Med. 2001, 345:241–247. Park, S., Jiang, Z., Mortenson, E.D., Deng, L., Radkevich-Brown, O., Yang, X., Sattar, H., Wang, Y., Brown, N.K., Greene, M., et al.: The therapeutic effect of antiHER2/neu antibody depends on both innate and adaptive immunity. Cancer Cell 2010, 18:160–170.

1	Tol, J., and Punt, C.J.: Monoclonal antibodies in the treatment of metastatic colorectal cancer: a review. Clin. Ther. 2010, 32:437–453. Veeramani, S., Wang, S.Y., Dahle, C., Blackwell, S., Jacobus, L., Knutson, T., Button, A., Link, B.K., and Weiner, G.J.: Rituximab infusion induces NK activation in lymphoma patients with the high-affinity CD16 polymorphism. Blood 2011, 118:3347–3349. Verma, S., Miles, D., Gianni, L., Krop, I.E., Welslau, M., Baselga, J., Pegram, M., Oh, D.Y., Diéras, V., Guardino, E., et al.: Trastuzumab emtansine for HER2-positive advanced breast cancer. N. Engl. J. Med. 2012, 367:1783–1791. Weiner, L.M., Dhodapkar MV, and Ferrone S.: Monoclonal antibodies for cancer immunotherapy. Lancet 2009, 373:1033–1040. Weng, W.K., and Levy, R.: Genetic polymorphism of the inhibitory IgG Fc receptor FcgammaRIIb is not associated with clinical outcome in patients with follicular lymphoma treated with rituximab. Leuk. Lymphoma 2009, 50:723–727.

1	16-18 Enhancing the immune response to tumors by vaccination holds promise for cancer prevention and therapy. Kantoff, P.W., Higano, C.S., Shore, N.D., Berger, E.R., Small, E.J., Penson, D.F., Redfern, C.H., Ferrari, A.C., Dreicer, R., Sims, R.B., et al.: Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N. Engl. J. Med. 2010, 363:411–422. Kenter, G.G., Welters, M.J., Valentijn, A.R., Lowik, M.J., Berends-van der Meer, D.M., Vloon, A.P., Essahsah, F., Fathers, L.M., Offringa, R., Drijfhout, J.W., et al.: Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N.Engl. J. Med. 2009, 361:1838–1847. References. Mao, C., Koutsky, L.A., Ault, K.A., Wheeler, C.M., Brown, D.R., Wiley, D.J., Alvarez, F.B., Bautista, O.M., Jansen, K.U., and Barr, E.: Efficacy of human papillomavirus-16 vaccine to prevent cervical intraepithelial neoplasia: a randomized controlled trial. Obstet. Gynecol. 2006, 107:18–27.

1	Palucka, K., Ueno, H., Fay, J., and Banchereau, J.: Dendritic cells and immunity against cancer. J. Intern. Med. 2011, 269:64–73. Vambutas, A., DeVoti, J., Nouri, M., Drijfhout, J.W., Lipford, G.B., Bonagura, V.R., van der Burg, S.H., and Melief, C.J.: Therapeutic vaccination with papillomavirus E6 and E7 long peptides results in the control of both established virus-induced lesions and latently infected sites in a pre-clinical cottontail rabbit papillomavirus model. Vaccine 2005, 23:5271–5280. 16-19 Checkpoint blockade can augment immune responses to existing tumors. Ansell, S.M., Lesokhin, A.M., Borrello, I., Halwani, A., Scott, E.C., Gutierrez, M., Schuster, S,J,, Millenson, M.M., Cattry, D., Freeman, G.J., et al.: PD-1 blockade with nivolumab in relapsed or refractory Hodgkin's lymphoma. N. Engl. J. Med. 2015, 372:311–319.

1	Bendandi, M., Gocke, C.D., Kobrin, C.B., Benko, F.A., Sternas, L.A., Pennington, R., Watson, T.M., Reynolds, C.W., Gause, B.L., Duffey, P.L., et al.: Complete molecular remissions induced by patient-specific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma. Nat. Med. 1999, 5:1171–1177. Egen, J.G., Kuhns, M.S., and Allison, J.P.: CTLA-4: new insights into its biological function and use in tumor immunotherapy. Nat. Immunol. 2002, 3:611–618. Hamid, O., Robert, C., Daud, A., Hodi, F.S., Hwu, W.J., Kefford, R., Wolchok, J.D., Hersey, P., Joseph, R.W., Weber, J.S., et al.: Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N. Engl. J. Med. 2013, 369:134–144. Hodi, F.S., O'Day, S.J., McDermott, D.F., Weber, R.W., Sosman, J.A., Haanen, J.B., Gonzalez, R., Robert, C., Schadendorf, D., Hassel, J.C. et al.: Improved survival with ipilimumab in patients with metastatic melanoma. N. Engl. J. Med 2010; 363:711–723

1	Li, Y., Hellstrom, K.E., Newby, S.A., and Chen, L.: Costimulation by CD48 and B7–1 induces immunity against poorly immunogenic tumors. J. Exp. Med. 1996, 183:639–644. Phan, G.Q., Yang, J.C., Sherry, R.M., Hwu, P., Topalian, S.L., Schwartzentruber, D.J., Restifo, N.P., Haworth, L.R., Seipp, C.A., Freezer, L.J., et al.: Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma. Proc. Natl Acad. Sci. USA 2003, 100:8372–8377. Yuan, J., Gnjatic, S., Li, H., Powel, S., Gallardo, H.F., Ritter, E., Ku, G.Y., Jungbluth, A.A., Segal, N.H., Rasalan, T.S., et al.: CTLA-4 blockade enhances polyfunctional NY-ESO-1 specific T cell responses in metastatic melanoma patients with clinical benefit. Proc. Natl Acad. Sci. USA 2008, 105:20410–20415. 16-20 Vaccines can be based on attenuated pathogens or material from killed organisms.

1	16-20 Vaccines can be based on attenuated pathogens or material from killed organisms. Anderson, R.M., Donnelly, C.A., and Gupta, S.: Vaccine design, evaluation, and community-based use for antigenically variable infectious agents. Lancet 1997, 350:1466–1470. Dermer, P., Lee, C., Eggert, J., and Few, B.: A history of neonatal group B streptococcus with its related morbidity and mortality rates in the United States. J. Pediatr. Nurs. 2004, 19:357–363. Rabinovich, N.R., McInnes, P., Klein, D.L., and Hall, B.F.: Vaccine technologies: view to the future. Science 1994, 265:1401–1404. 16-21 Most effective vaccines generate antibodies that prevent the damage caused by toxins or that neutralize the pathogen and stop infection. Levine, M.M., and Levine, O.S.: Influence of disease burden, public perception, and other factors on new vaccine development, implementation, and continued use. Lancet 1997, 350:1386–1392.

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1	Gupta, R.K., Best, J., and MacMahon, E.: Mumps and the UK epidemic 2005. BMJ 2005, 330:1132–1135. Hviid, A., Rubin, S., and Mlemann, K.: Mumps. Lancet 2008, 371:932–944. Magnan, S., and Drake, M.: The effectiveness of vaccination against influenza in healthy, working adults. N. Engl. J. Med. 1995, 333:889–893. 16-23 Live-attenuated viral vaccines are usually more potent than ‘killed’ vaccines and can be made safer by the use of recombinant DNA technology. Mueller, S.N., Langley, W.A., Carnero, E., García-Sastre, A., and Ahmed, R.: Immunization with live attenuated influenza viruses that express altered NS1 proteins results in potent and protective memory CD8+ T-cell responses. J. Virol. 2010, 84:1847–1855. Murphy, B.R., and Collins, P.L.: Live-attenuated virus vaccines for respiratory syncytial and parainfluenza viruses: applications of reverse genetics. J. Clin. Invest. 2002, 110:21–27.

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1	attenuated parasites (GAPs). Grode, L., Seiler, P., Baumann, S., Hess, J., Brinkmann, V., Nasser Eddine, A., Mann, P., Goosmann, C., Bandermann, S., Smith, D., et al.: Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette–Guérin mutants that secrete listeriolysin. J. Clin. Invest. 2005, 115:2472–2479. Guleria, I., Teitelbaum, R., McAdam, R.A., Kalpana, G., Jacobs, W.R., Jr, and Bloom, B.R.: Auxotrophic vaccines for tuberculosis. Nat. Med. 1996, 2:334–337. Labaied, M., Harupa, A., Dumpit, R.F., Coppens, I., Mikolajczak, S.A., and Kappe, S.H.: Plasmodium yoelii sporozoites with simultaneous deletion of P52 and P36 are completely attenuated and confer sterile immunity against infection. Infect. Immun. 2007, 75:3758–3768.

1	Martin, C.: The dream of a vaccine against tuberculosis; new vaccines improving or replacing BCG? Eur. Respir. J. 2005, 26:162–167. Thaiss, C.A., and Kaufmann, S.H.: Toward novel vaccines against tuberculosis: current hopes and obstacles. Yale J. Biol. Med. 2010, 83:209–215. Vaughan, A.M., Wang, R., and Kappe, S.H.: Genetically engineered, attenuated whole-cell vaccine approaches for malaria. Hum. Vaccines 2010, 6:1–8. 16-25 The route of vaccination is an important determinant of success. Amorij, J.P., Hinrichs, W.Lj., Frijlink, H.W., Wilschut, J.C., and Huckriede, A.: Needle-free influenza vaccination. Lancet Infect. Dis. 2010, 10:699–711. Belyakov, I.M., and Ahlers, J.D.: What role does the route of immunization play in the generation of protective immunity against mucosal pathogens? J. Immunol. 2009, 183:6883–6892.

1	Belyakov, I.M., and Ahlers, J.D.: What role does the route of immunization play in the generation of protective immunity against mucosal pathogens? J. Immunol. 2009, 183:6883–6892. Douce, G., Fontana, M., Pizza, M., Rappuoli, R., and Dougan, G.: Intranasal immunogenicity and adjuvanticity of site-directed mutant derivatives of cholera toxin. Infect. Immun. 1997, 65:2821–2828. Dougan, G., Ghaem-Maghami, M., Pickard, D., Frankel, G., Douce, G., Clare, S., Dunstan, S., and Simmons, C.: The immune responses to bacterial antigens encountered in vivo at mucosal surfaces. Phil. Trans. R. Soc. Lond. B 2000, 355:705–712. Eriksson, K., and Holmgren, J.: Recent advances in mucosal vaccines and adjuvants. Curr. Opin. Immunol. 2002, 14:666–672. 16-26 Bordetella pertussis vaccination illustrates the importance of the perceived safety of a vaccines. Decker, M.D., and Edwards, K.M.: Acellular pertussis vaccines. Pediatr. Clin. North Am. 2000, 47:309–335.

1	Decker, M.D., and Edwards, K.M.: Acellular pertussis vaccines. Pediatr. Clin. North Am. 2000, 47:309–335. Madsen, K.M., Hviid, A., Vestergaard, M., Schendel, D., Wohlfahrt, J., Thorsen, P., Olsen, J., and Melbye, M.: A population-based study of measles, mumps, and rubella vaccination and autism. N. Engl. J. Med. 2002, 347:1477–1482. Mortimer, E.A.: Pertussis vaccines, in Plotkin, S.A., and Mortimer, E.A. (eds): Vaccines, 2nd ed. Philadelphia, W.B. Saunders Co., 1994. Poland, G.A.: Acellular pertussis vaccines: new vaccines for an old disease. Lancet 1996, 347:209–210. 16-27 Conjugate vaccines have been developed as a result of linked recognition between T and B cells. Berry, D.S., Lynn, F., Lee, C.H., Frasch, C.E., and Bash, M.C.: Effect of O acetylation of Neisseria meningitidis serogroup A capsular polysaccharide on development of functional immune responses. Infect. Immun. 2002, 70:3707–3713.

1	Brer, M., Dull, P.M., Rappuoli, R., and Costantino, P.: Chemistry of a new investigational quadrivalent meningococcal conjugate vaccine that is immunogenic at all ages. Vaccine 2009, 27:5574–5580. Levine, O.S., Knoll, M.D., Jones, A., Walker, D.G., Risko, N., and Gilani, Z.: Global status of Haemophilus influenzae type b and pneumococcal conjugate vaccines: evidence, policies, and introductions. Curr. Opin. Infect. Dis. 2010, 23:236–241. Peltola, H., Kilpi, T., and Anttila, M.: Rapid disappearance of Haemophilus influenzae type b meningitis after routine childhood immunisation with conjugate vaccines. Lancet 1992, 340:592–594. Rappuoli, R.: Conjugates and reverse vaccinology to eliminate bacterial meningitis. Vaccine 2001, 19:2319–2322. 16-28 Peptide-based vaccines can elicit protective immunity, but they require adjuvants and must be targeted to the appropriate cells and cell compartment to be effective.

1	16-28 Peptide-based vaccines can elicit protective immunity, but they require adjuvants and must be targeted to the appropriate cells and cell compartment to be effective. Alonso, P.L., Sacarlal, J., Aponte, J.J., Leach, A., Macete, E., Aide, P., Sigauque, B., Milman, J., Mandomando, I., Bassat, Q., et al.: Duration of protection with RTS, S/AS02A malaria vaccine in prevention of Plasmodium falciparum disease in Mozambican children: single-blind extended follow-up of a randomised controlled trial. Lancet 2005, 366:2012–2018. Berzofsky, J.A.: Epitope selection and design of synthetic vaccines. Molecular approaches to enhancing immunogenicity and cross-reactivity of engineered vaccines. Ann. N.Y. Acad. Sci. 1993, 690:256–264. Davenport, M.P., and Hill, A.V.: Reverse immunogenetics: from HLA-disease associations to vaccine candidates. Mol. Med. Today 1996, 2:38–45. Hill, A.V.: Pre-erythrocytic malaria vaccines: towards greater efficacy. Nat. Rev. Immunol. 2006, 6:21–32.

1	Hill, A.V.: Pre-erythrocytic malaria vaccines: towards greater efficacy. Nat. Rev. Immunol. 2006, 6:21–32. Hoffman, S.L., Rogers, W.O., Carucci, D.J., and Venter, J.C.: From genomics to vaccines: malaria as a model system. Nat. Med. 1998, 4:1351–1353. Ottenhoff, T.H., Doherty, T.M., Dissel, J.T., Bang, P., Lingnau, K., Kromann, I., and Andersen, P.: First in humans: a new molecularly defined vaccine shows excellent safety and strong induction of long-lived Mycobacterium tuberculosis-specific Th1-cell like responses. Hum. Vaccin. 2010, 6:1007–1015. Zwaveling, S., Ferreira Mota, S.C., Nouta, J., Johnson, M., Lipford, G.B., Offringa, R., van der Burg, S.H., and Melief, C.J.: Established human papillomavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides. J. Immunol. 2002, 169:350–358. 16-29 Adjuvants are important for enhancing the immunogenicity of vaccines, but few are approved for use in humans.

1	16-29 Adjuvants are important for enhancing the immunogenicity of vaccines, but few are approved for use in humans. Coffman, R.L., Sher, A., and Seder, R.A.: Vaccine adjuvants: putting innate immunity to work. Immunity 2010, 33:492–503. Hartmann, G., Weiner, G.J., and Krieg, A.M.: CpG DNA: a potent signal for growth, activation, and maturation of human dendritic cells. Proc. Natl Acad. Sci. USA 1999, 96:9305–9310. Palucka, K., Banchereau, J., and Mellman, I.: Designing vaccines based on biology of human dendritic cell subsets. Immunity 2010, 33:464–478. Persing, D.H., Coler, R.N., Lacy, M.J., Johnson, D.A., Baldridge, J.R., Hershberg, R.M., and Reed, S.G.: Taking toll: lipid A mimetics as adjuvants and immunomodulators. Trends Microbiol. 2002, 10:S32–S37. Pulendran, B.: Modulating vaccine responses with dendritic cells and Toll-like receptors. Immunol. Rev. 2004, 199:227–250. Takeda, K., Kaisho, T., and Akira, S.: Toll-like receptors. Annu. Rev. Immunol. 2003, 21:335–376.

1	Takeda, K., Kaisho, T., and Akira, S.: Toll-like receptors. Annu. Rev. Immunol. 2003, 21:335–376. 16-30 Protective immunity can be induced by DNA-based vaccination. Donnelly, J.J., Ulmer, J.B., Shiver, J.W., and Liu, M.A.: DNA vaccines. Annu. Rev. Immunol. 1997, 15:617–648. Gurunathan, S., Klinman, D.M., and Seder, R.A.: DNA vaccines: immunology, application, and optimization. Annu. Rev. Immunol. 2000, 18:927–974. Li, L., Kim, S., Herndon, J.M., Goedegebuure, P., Belt, B.A., Satpathy, A.T., Fleming, T.P., Hansen, T.H., Murphy, K.M., and Gillanders, W.E.: Cross-dressed CD8α+/CD103+ dendritic cells prime CD8+ T cells following vaccination. Proc. Natl Acad. Sci. USA 2012, 109:12716–12721. Nchinda, G., Kuroiwa, J., Oks, M., Trumpfheller, C., Park, C.G., Huang, Y., Hannaman, D., Schlesinger, S.J., Mizenina, O., Nussenzweig, M.C., et al.: The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells. J. Clin. Invest. 2008, 118:1427–1436.

1	Wolff, J.A., and Budker, V.: The mechanism of naked DNA uptake and expression. Adv. Genet. 2005, 54:3–20. 16-31 Vaccination and checkpoint blockade may be useful in controlling existing chronic infections. Burke, R.L.: Contemporary approaches to vaccination against herpes simplex virus. Curr. Top. Microbiol. Immunol. 1992, 179:137–158. Grange, J.M., and Stanford, J.L.: Therapeutic vaccines. J. Med. Microbiol. 1996, 45:81–83. Hill, A., Jugovic, P., York, I., Russ, G., Bennink, J., Yewdell, J., Ploegh, H., and Johnson, D.: Herpes simplex virus turns off the TAP to evade host immunity. Nature 1995, 375:411–415. Lu, W., Arraes, L.C., Ferreira, W.T., and Andrieu, J.M.: Therapeutic dendritic-cell vaccine for chronic HIV-1 infection. Nat. Med. 2004, 10:1359–1365. Modlin, R.L.: Th1–Th2 paradigm: insights from leprosy. J. Invest. Dermatol. 1994, 102:828–832.

1	Modlin, R.L.: Th1–Th2 paradigm: insights from leprosy. J. Invest. Dermatol. 1994, 102:828–832. Plebanski, M., Proudfoot, O., Pouniotis, D., Coppel, R.L., Apostolopoulos, V., and Flannery, G.: Immunogenetics and the design of Plasmodium falciparum vaccines for use in malaria-endemic populations. J. Clin. Invest. 2002, 110:295–301. Reiner, S.L., and Locksley, R.M.: The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 1995, 13:151–177. Stanford, J.L.: The history and future of vaccination and immunotherapy for leprosy. Trop. Geogr. Med. 1994, 46:93–107. The Immunologist's Toolbox A-1. Immunization.

1	The Immunologist's Toolbox A-1. Immunization. Natural immune responses are normally directed at antigens borne by pathogenic microorganisms. The immune system can also be induced to respond to simple nonliving antigens, and experimental immunologists have focused on the responses to these simple antigens in developing our understanding of the immune response. The deliberate induction of an immune response is known as immunization. Experimental immunizations are routinely carried out by injecting the test antigen into the animal or human subject. The route, dose, and form in which antigen is administered can profoundly affect whether a response occurs and the type of response that is produced. The induction of protective immune responses against common microbial pathogens in humans is often called vaccination, although this term originally referred to the induction of immune responses against smallpox by immunizing with the cross-reactive virus, vaccinia.

1	To determine whether an immune response has occurred and to follow its course, the immunized individual is monitored for the appearance of immune reactants. Immune responses to most antigens include soluble factors, such as cytokines and specific antibodies, and cellular responses, such as the generation of specific effector T cells. Monitoring the cytokine and antibody responses usually involves the analysis of relatively crude preparations of antiserum (plural: antisera). Serum is the fluid phase of clotted blood, which, if taken from an individual immunized against a particular antigen, is called antiserum. To study immune responses mediated by T cells, blood lymphocytes or cells from lymphoid organs such as the spleen are tested; T-cell responses are more commonly studied in experimental animals than in humans.

1	Antisera generated by immunization with even the simplest antigen will contain many different antibody molecules that bind to the immunogen in slightly different ways. In addition, antisera contain many antibodies that do not bind at all to the immunizing antigen, because they were present in the individual prior to immunization. These nonspecific antibodies often lead to technical difficulties in using antisera for detecting an immunogen. To circumvent this problem, antibodies that bind to the immunogen can be purified by affinity chromatography using immobilized antigen (see Section A-3). Alternatively, these problems can be avoided by making monoclonal antibodies (see Section A-7).

1	Any substance that can elicit an immune response is said to be immunogenic and is called an immunogen. There is a clear operational distinction between an immunogen and an antigen. Immunogens are substances that elicit an adaptive immune response, whereas an antigen is defined as any substance that can bind to a specific antibody. All antigens therefore have the potential to elicit specific antibodies; however, not all antigens are immunogenic. An example of this distinction is evident when considering protein antigens. In spite of the fact that antibodies against proteins are of enormous utility in experimental biology and medicine, purified proteins are not generally immunogenic. This is because purified proteins lack microbial-associated molecular patterns (MAMPs), and therefore do not elicit an innate immune response. To provoke an immune response to a purified protein, the protein must be administered together with an adjuvant (see below).

1	Certain properties of antigens that favor the initiation of an adaptive immune response have been defined by studying antibody responses to simple natural proteins such as hen egg-white lysozyme, to synthetic polypeptide antigens, and to small organic molecules of simple structure. The study of antibody responses to small organic molecules, such as phenyl arsonates and nitrophenyls, was essential in defining early immunological principles. These molecules do not provoke antibodies when injected by themselves. However, antibodies can be raised against them if the molecule is attached covalently, by simple chemical reactions, to a protein carrier. Such small molecules were termed haptens (from the Greek haptein, to fasten) by the immunologist Karl Landsteiner, who first studied them in the early 20th century. He found that animals immunized with a hapten–carrier conjugate produced three distinct sets of antibodies (Fig. A.1). One set comprised hapten-specific antibodies that reacted

1	early 20th century. He found that animals immunized with a hapten–carrier conjugate produced three distinct sets of antibodies (Fig. A.1). One set comprised hapten-specific antibodies that reacted with the same hapten on any carrier, as well as with free hapten. The second set of antibodies was specific for the carrier protein, as shown by their ability to bind both the hapten-modified and unmodified carrier protein. Finally, some antibodies reacted only with the specific conjugate of hapten and carrier used for immunization. Landsteiner’s studies focused primarily on the antibody response to the hapten, as these small molecules could be synthesized in many closely related forms. He observed that antibodies raised against a particular hapten bind that hapten but, in general, fail to bind even very closely related chemical structures. The binding of haptens by anti-hapten antibodies has played an important part in defining the precision of antigen binding by antibody molecules.

1	bind even very closely related chemical structures. The binding of haptens by anti-hapten antibodies has played an important part in defining the precision of antigen binding by antibody molecules. Anti-hapten antibodies are also important medically because they mediate allergic reactions to penicillin and other compounds that elicit antibody responses when they attach to self proteins (see Section 14-10).

1	Fig. A.1 Antibodies can be elicited by small chemical groups called haptens only when the hapten is linked to an immunogenic protein carrier. Following immunization with a hapten–carrier conjugate, three types of antibodies are produced. One set (blue) binds the carrier protein alone and is called carrier-specific. One set (red) binds to the hapten on any carrier or to free hapten in solution and is called hapten-specific. One set (purple) binds only the specific conjugate of hapten and carrier used for immunization, apparently binding to sites at which the hapten joins the carrier, and is called conjugate-specific. The amount of antibody of each type in this serum is shown schematically in the graphs at the bottom; note that the original antigen binds more antibody than the sum of anti-hapten and anti-carrier antibodies as a result of the additional binding of conjugate-specific antibody.

1	The route by which antigen is administered affects both the magnitude and the type of response obtained. The most common routes by which antigen is introduced experimentally or as a vaccine into the body are into tissue by subcutaneous (s.c.) injection into the fatty layer just below the dermis or by intradermal (i.d.) or intramuscular (i.m.) injection; directly into the bloodstream by intravenous (i.v.) injection or transfusion; into the gastrointestinal tract by oral administration; and into the respiratory tract by intranasal (i.n.) administration or inhalation.

1	Antigens injected subcutaneously generally elicit strong responses, most probably because the antigen is taken up by resident dendritic cells in the skin and efficiently presented in local lymph nodes, and so this is the method most commonly used when the object of the experiment is to elicit specific antibodies or T cells against a given antigen. Antigens injected or transfused directly into the bloodstream tend to induce immune unresponsiveness or tolerance unless they bind to host cells or are in the form of aggregates that are readily taken up by antigen-presenting cells.

1	Antigen administration via the gastrointestinal tract is used mostly in the study of allergy. It has distinctive effects, frequently eliciting a local antibody response in the intestinal lamina propria, while producing a systemic state of tolerance that manifests as a diminished response to the same antigen if subsequently administered in immunogenic form elsewhere in the body (see Chapter 12). This ‘split tolerance’ may be important in avoiding allergy to antigens in food, because the local response prevents food antigens from entering the body, while the inhibition of systemic immunity helps to prevent the formation of IgE antibodies, which are the cause of such allergies (see Chapter 14).

1	The immune response to an antigen is also influenced by the dose of immunogen administered. Below a certain threshold dose, most proteins do not elicit any immune response. Above the threshold dose, there is a gradual increase in the response as the dose of antigen is increased, until a broad plateau level is reached, followed by a decline at very high antigen doses (Fig. A.2). In general, secondary and subsequent immune responses occur at lower antigen doses and achieve higher plateau values, which is a sign of immunological memory. Most proteins are poorly immunogenic or nonimmunogenic when administered by themselves. Strong adaptive immune responses to protein antigens almost always require that the antigen be injected in a mixture known as an adjuvant. An adjuvant is any substance that enhances the immunogenicity of substances mixed with it. Commonly used adjuvants are listed in Fig. A.3.

1	Adjuvants generally enhance immunogenicity in two different ways. First, adjuvants convert soluble protein antigens into particulate material, which is more readily ingested by phagocytic antigen-presenting cells such as macrophages and dendritic cells. For example, the antigen can be adsorbed on particles of the adjuvant (such as alum), be made particulate by emulsification in mineral oils, or be incorporated into the colloidal particles of immune stimulatory complexes (ISCOMs). Second, and more important, adjuvants contain PAMPs that elicit a strong innate immune response. When taken up by phagocytic cells, the PAMPs in the adjuvant stimulate inflammatory cytokine production and induce the activation of the antigen-presenting cell. The activated antigen-presenting cells upregulate abundant levels of co-stimulatory molecules that are important for activating T cells. Activated antigen-presenting cells also upregulate high levels of MHC class I and class II proteins, plus many of the

1	levels of co-stimulatory molecules that are important for activating T cells. Activated antigen-presenting cells also upregulate high levels of MHC class I and class II proteins, plus many of the proteins important for efficient antigen processing and presentation (see Section 3-12). Due to the strong local inflammatory responses induced by adjuvants that contain PAMPs, most of the adjuvants commonly used in experimental animals are not approved for use in humans.

1	The typical antigen dose–response curve shown here illustrates both the influence of dose on a primary antibody response (amounts of antibody produced expressed in arbitrary units) and the effect of the dose used for priming on a secondary antibody response elicited by a dose of antigen of 103 arbitrary mass units. Very low doses of antigen do not cause an immune response at all. Slightly higher doses seem to inhibit specific antibody production, an effect known as low-zone tolerance. Above these doses there is a steady increase in the response with antigen dose until an optimum response is reached; this response persists across a broad range of doses. Very high doses of antigen also inhibit immune responsiveness to a subsequent challenge, a phenomenon known as high-zone tolerance.

1	Fig. A.3 Common adjuvants and their use. Adjuvants are mixed with the antigen and usually render it particulate, which helps to retain the antigen in the body and promotes uptake by macrophages. Most adjuvants include bacteria or bacterial components that stimulate macrophages and dendritic cells, aiding in the induction of the immune response. ISCOMs are small micelles of the detergent Quil A; when viral proteins are placed in these micelles, they apparently fuse with the antigen-presenting cell, allowing the antigen to enter the cytosol. Thus, the antigen-presenting cell can stimulate a response to the viral protein, much as a virus infecting these cells would stimulate an antiviral response. Vaccines designed to elicit responses to purified proteins often include compounds that stimulate pattern recognition receptors, such as Toll-like receptors (TLRs), NOD-like receptors (NLRs), or C-type lectin receptors.

1	Nevertheless, some human vaccines naturally contain microbial antigens that can also act as effective adjuvants. For example, purified constituents of the bacterium Bordetella pertussis, which is the causative agent of whooping cough, are used as both antigen and adjuvant in the triplex DPT (diphtheria, pertussis, tetanus) vaccine. In addition, modified TLR ligands, such as monophosphoryl lipid A, a derivative of LPS, or poly(I):poly(C12U), a derivative of polyI:C, are currently included as components of several human vaccines. A-2 Antibody responses.

1	A-2 Antibody responses. B cells contribute to adaptive immunity by secreting antibodies, and the response of B cells to an injected immunogen is usually measured by analyzing the specific antibody produced in a humoral immune response. This is most conveniently achieved by assaying the antibody that accumulates in the fluid phase of the blood, or plasma; such antibodies are known as circulating antibodies. Circulating antibody is usually measured by collecting blood, allowing it to clot, and then isolating the serum from the clotted blood. The amount and characteristics of the antibody in the resulting antiserum are then determined using the assays described below. Because assays for antibody were originally conducted using antisera from immune individuals, they are commonly referred to as serological assays, and the use of antibodies in such testing is often called serology.

1	The most important characteristics of an antibody response are the specificity, amount, isotype or class, and affinity of the antibodies produced. The specificity determines the ability of the antibody to distinguish the immunogen from other antigens. The amount of antibody can be determined in many different ways and is a function of the number of responding B cells, their rate of antibody synthesis, and the persistence of the antibody after production. The persistence of an antibody in the plasma and extracellular fluid bathing the tissues is determined mainly by its isotype or class (see Sections 5-12 and 10-14); each isotype has a different half-life in vivo. The isotypic composition of an antibody response also determines the biological functions these antibodies can perform and the sites in which antibody will be found. Finally, the strength of binding of the antibody to its antigen in terms of a single antigen-binding site binding to a monovalent antigen is termed its affinity;

1	in which antibody will be found. Finally, the strength of binding of the antibody to its antigen in terms of a single antigen-binding site binding to a monovalent antigen is termed its affinity; the total binding strength of a molecule with more than one binding site is called its avidity. Binding strength is important: the higher the affinity of the antibody for its antigen, the less antibody is required to eliminate the antigen, because antibodies with higher affinity will bind at lower antigen concentrations. All these parameters of the humoral immune response help to determine the capacity of that response to protect the host from infection.

1	A-3 Affinity chromatography.

1	The specificity of antigen:antibody binding interactions can be exploited for the purification of a specific antigen from a complex mixture, or alternatively, for the purification of specific antibodies from antiserum containing a mixture of different antibodies. The technique employed is called affinity chromatography (Fig. A.4). For purification of an antigen, antigen-specific antibodies are bound, often covalently, to small, chemically reactive beads, which are loaded into a column. The antigen mixture is allowed to pass over the beads. The specific antigen binds; all the other components in the mixture can then be washed away. The specific antigen is then eluted, typically by lowering the pH to 2.5 or raising it to greater than 11. Antibodies bind stably under physiological conditions of salt concentration, temperature, and pH, but the binding is reversible because the bonds are noncovalent. Affinity chromatography can also be used to purify antibodies from complex antisera by

1	of salt concentration, temperature, and pH, but the binding is reversible because the bonds are noncovalent. Affinity chromatography can also be used to purify antibodies from complex antisera by using beads coated with specific antigen. The technique is known as affinity chromatography because it separates molecules on the basis of their affinity for one another.

1	A-4 Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and competitive inhibition assay.

1	Radioimmunoassay (RIA) and enzymelinked immunosorbent assay (ELISA) are direct binding assays for antibody (or antigen); both work on the same principle, but the means of detecting specific binding is different. Radioimmunoassays are commonly used to measure the levels of hormones in blood and tissue fluids, while ELISA assays are frequently used in viral diagnostics, for example, in detecting cases of infection with the human immunodeficiency virus (HIV), which is the cause of AIDS. For both of these methods, one needs a pure preparation of a known antigen or antibody, or both, in order to standardize the assay. We will describe the assay that is used to determine the amount of a specific antigen in a sample, for instance, the amount of HIV p24 protein in a patient’s serum. For this, a preparation of pure antibody specific for the antigen is required. One can also use RIA or ELISA to determine the amount of specific antibody in a mixture, such as serum; in this case, a preparation of

1	of pure antibody specific for the antigen is required. One can also use RIA or ELISA to determine the amount of specific antibody in a mixture, such as serum; in this case, a preparation of pure antigen is needed as a starting point.

1	For the determination of antigen concentration using RIA, pure antibody against the antigen is radioactively labeled, usually with 125I; for ELISA, an enzyme is linked chemically to the antibody. The unlabeled component, which in this case would be the solution containing an unknown amount of antigen, is attached to a solid support, such as the wells of a plastic multiwell plate, which will adsorb a certain amount of any protein. Following this, the labeled antibody

1	Fig. A.4 Affinity chromatography uses antigen–antibody binding to purify antigens or antibodies. To purify a specific antigen from a complex mixture of molecules, a monoclonal antibody is attached to an insoluble matrix, such as chromatography beads, and the mixture of molecules is passed over the matrix. The specific antibody binds the antigen of interest; other molecules are washed away. Specific antigen is then eluted by altering the pH, which can usually disrupt antibody– antigen bonds. Antibodies can be purified in the same way on beads coupled to antigen (not shown). Add anti-A antibody covalently linked to enzyme Wash away unbound antibody sample 1 (antigen A) sample 2 (antigen B) Enzyme makes colored product from added colorless substrate Measure absorbance of light by colored product Fig. A.5 The principle of the enzyme-linked immunosorbent assay (ELISA).

1	Fig. A.5 The principle of the enzyme-linked immunosorbent assay (ELISA). To detect antigen A, purified antibody specific for antigen A is linked chemically to an enzyme. The samples to be tested are coated onto the surface of plastic wells, to which they bind nonspecifically; residual sticky sites on the plastic are blocked by adding irrelevant proteins (not shown). The labeled antibody is then added to the wells under conditions that prevent nonspecific binding, so that only binding to antigen A causes the labeled antibody to be retained on the surface. Unbound labeled antibody is removed from all wells by washing, and bound antibody is detected by an enzyme-dependent color-change reaction. This assay allows arrays of wells known as microtiter plates to be read in fiberoptic multichannel spectrometers, greatly speeding the assay. Modifications of this basic assay allow antibody or antigen in unknown samples to be measured as shown in Figs. A.6 and A.25.

1	is added to the well and allowed to bind to the unlabeled antigen under conditions in which nonspecific adsorption is blocked, and any unbound antibody and other proteins are washed away. Antibody binding in RIA is measured directly in terms of the amount of radioactivity retained by the coated wells, whereas in ELISA the binding is detected by a reaction that converts a substrate into a reaction product of a different color (Fig. A.5). The color change can be read directly in the reaction tray, making data collection very easy and providing a quantitative measurement of reaction product concentration; furthermore, ELISA also avoids the hazards of radioactivity. This makes ELISA the preferred method for most direct-binding assays. In a variation of this assay, labeled antiimmunoglobulin antibodies can also be used in RIA or ELISA as a second layer, following the binding of unlabeled antibody to unlabeled antigen-coated plates. The use of such a second layer amplifies the signal,

1	antibodies can also be used in RIA or ELISA as a second layer, following the binding of unlabeled antibody to unlabeled antigen-coated plates. The use of such a second layer amplifies the signal, because at least two molecules of the labeled anti-immunoglobulin antibody are able to bind to each unlabeled antibody. As mentioned above, RIA and ELISA can also be carried out in reverse when the goal is to determine the amount of antibody in a solution; in this case, unlabeled antibody is adhered to the plates, labeled antigen is added, and the amount of labeled antigen bound after washing is measured.

1	A modification of ELISA known as a capture or sandwich ELISA (or more generally as an antigencapture assay) is commonly used to detect secreted products such as cytokines. Rather than the antigen being directly attached to a plastic plate, antigen-specific antibodies are bound to the plate. These are able to bind antigen with high affinity, and thus concentrate it on the surface of the plate, even with antigens that are present in very low concentrations in the initial mixture. A separate labeled antibody that recognizes a different epitope from that recognized by the immobilized first antibody is then used to detect the bound antigen.

1	Another variant of the antigen-capture assay, often referred to as a multiplex assay, has been developed to allow quantitation of multiple antigens in a single sample. This technique is often utilized to examine the levels of multiple cytokines in clinical serum samples, or in sera from experimental animals, cases in which it is not feasible to assess each cytokine of interest individually. For this type of assay, small microspheres are differentially labeled with fluorescent dyes that can be distinguished based on their distinct emission spectra. Microspheres labeled with a given fluorescent dye are conjugated to antibodies specific for one antigen, for instance, a single cytokine. The microspheres— up to 100 different microspheres with unique identifiers—are added to the sample to capture the antigen. Bound antigen is then detected using a second antibody that binds the antigen at a distinct site. This second antibody is conjugated to a different fluorescent dye, and the magnitude

1	the antigen. Bound antigen is then detected using a second antibody that binds the antigen at a distinct site. This second antibody is conjugated to a different fluorescent dye, and the magnitude of its fluorescence is a measure of the quantity of bound antigen. The machine that performs this multiplex analysis, the Luminex® analyzer, then measures the amount of fluorescence associated with each differentially labeled microsphere.

1	These assays illustrate two crucial aspects of all serological assays. First, at least one of the reagents must be available in a pure, detectable form in order to obtain quantitative information. Second, there must be a means of separating the bound fraction of the labeled reagent from the unbound, free fraction so that the percentage of specific binding can be determined. Normally, this separation is achieved by having the unlabeled partner trapped on a solid support. Labeled molecules that do not bind can then be washed away, leaving just the labeled partner that has bound. In Fig. A.5, the unlabeled antigen is attached to the well and the labeled antibody is trapped by binding to it. The separation of bound reagent from the free fraction is an essential step in every assay that uses antibodies.

1	RIA and ELISA do not allow one to measure directly the amount of antigen or antibody in a sample of unknown composition, because both depend on the binding of a pure labeled antigen or antibody. There are various ways around Add labeled antigen A Add labeled antigen A + unlabeled antigen 100 50 1 2 0 standard curve Anti-A antibody bound Wash away unbound antigen Count bound label Known concentration of antigen A Mixture containing low concentration of antigen A Amount of competitor added Percent control binding Compete with standard curve curve 1 less inhibition curve 2 Fig. A.6 Competitive inhibition assay for antigen in unknown samples.

1	Fig. A.6 Competitive inhibition assay for antigen in unknown samples. A fixed amount of unlabeled antibody is attached to a set of wells, and a standard reference preparation of a labeled antigen is bound to it. Unlabeled standard or test samples are then added in various amounts and the displacement of labeled antigen is measured, generating characteristic inhibition curves. A standard curve is obtained by using known amounts of unlabeled antigen identical to that used as the labeled species, and comparison with this curve allows the amount of antigen in unknown samples to be calculated. The green line on the graph represents a sample lacking any substance that reacts with anti-A antibodies.

1	this problem, one of which is to use a competitive inhibition assay, as shown in Fig. A.6. In this type of assay, the presence and amount of a particular antigen in an unknown sample are determined by the antigen’s ability to compete with a labeled reference antigen for binding to an antibody attached to a plastic well. A standard curve is first constructed by adding various amounts of a known, unlabeled standard preparation; the assay can then measure the amount of antigen in unknown samples by comparison with the standard. The competitive binding assay can also be used for measuring antibody in a sample of unknown composition by attaching the appropriate antigen to the plate and measuring the ability of the test sample to inhibit the binding of a labeled specific antibody. A-5 Hemagglutination and blood typing.

1	A-5 Hemagglutination and blood typing. The direct measurement of antibody binding to antigen is used in most quantitative serological assays. However, some important assays are based on the ability of antibody binding to alter the physical state of the antigen to which the antibody binds. These secondary interactions can be detected in a variety of ways. For instance, when the antigen is displayed on the surface of a large particle such as a bacterium, antibodies can cause the bacteria to clump, or agglutinate. The same principle applies to the reactions used in blood typing, only here the target antigens are on the surface of red blood cells and the clumping reaction caused by antibodies against them is called hemagglutination (from the Greek haima, blood).

1	Fig. A.7 Hemagglutination is used to type blood groups and match compatible donors and recipients for blood transfusion. Common gut bacteria bear antigens that are similar or identical to blood-group antigens, and these stimulate the formation of antibodies against these antigens in individuals who do not bear the corresponding antigen on their own red blood cells (left column); thus, type O individuals, who lack A and B, have both anti-A and anti-B antibodies, whereas type AB individuals have neither. The pattern of agglutination of the red blood cells of a transfusion donor or recipient with anti-A and anti-B antibodies reveals the individual’s ABO blood group. Before transfusion, the serum of the recipient is also tested for antibodies that agglutinate the red blood cells of the donor, and vice versa, a procedure called a cross-match, which may detect potentially harmful antibodies against other blood groups that are not part of the ABO system.

1	Hemagglutination is used to determine the ABO blood group of blood donors and transfusion recipients. Clumping or agglutination is induced by antibodies or agglutinins called anti-A or anti-B that bind to the A or B blood-group substances, respectively (Fig. A.7). These blood-group antigens are arrayed in many copies on the surface of the red blood cell, causing the cells to agglutinate when cross-linked by antibodies. Because hemagglutination involves the cross-linking of blood cells by the simultaneous binding of antibody molecules to identical antigens on different cells, this reaction also demonstrates that each antibody molecule must have at least two identical antigen-binding sites. A-6 Coombs tests and the detection of rhesus incompatibility.

1	Coombs tests use anti-immunoglobulin antibodies to detect the antibodies that cause hemolytic disease of the newborn, or erythroblastosis fetalis. Anti-immunoglobulin antibodies were first developed by Robin Coombs, and the test for this disease is still called the Coombs test. Hemolytic disease of the newborn occurs when a mother makes IgG antibodies specific for the rhesus or Rh bloodgroup antigen expressed on the red blood cells of her fetus. Rh-negative mothers make these antibodies when they are exposed at delivery to Rh-positive fetal red blood cells bearing the paternally inherited Rh antigen. During subsequent pregnancies, these antibodies are transported across the placenta to the fetus. This normal process is generally beneficial, as it protects newborn infants against infection. However, IgG anti-Rh antibodies coat the fetal red blood cells, which are then destroyed by phagocytic cells in the liver, causing a hemolytic anemia in the fetus and newborn infant.

1	Rh antigens are widely spaced on the red blood cell surface, and so the IgG anti-Rh antibodies do not bind in the correct conformation to fix complement and so do not cause lysis of red blood cells in vitro. Furthermore, for reasons that are not fully understood, antibodies against Rh antigens do not agglutinate red blood cells, unlike antibodies against the ABO blood-group antigens. Thus, detecting anti-Rh antibodies was difficult until anti-human immunoglobulin antibodies were developed. With these, maternal IgG antibodies bound to the fetal red blood cells can be detected after washing the cells to remove unbound immunoglobulin that is present in the fetal serum. Adding anti-human immunoglobulin antibodies against the washed fetal red blood cells agglutinates any cells to which maternal antibodies are bound. This is the direct Coombs test (Fig. A.8), so called because it directly detects antibody bound to the surface of the fetal red blood cells. An indirect Coombs test is used to

1	antibodies are bound. This is the direct Coombs test (Fig. A.8), so called because it directly detects antibody bound to the surface of the fetal red blood cells. An indirect Coombs test is used to detect nonagglutinating anti-Rh antibody in maternal serum: the serum is first incubated with Rh-positive red blood cells, which bind the anti-Rh antibody, after which the antibody-coated cells are washed to remove unbound immunoglobulin and are then agglutinated with antiimmunoglobulin antibody (see Fig. A.8). The indirect Coombs test allows Rh incompatibilities that might lead to hemolytic disease of the newborn to be detected, and this knowledge allows the disease to be prevented (see Section 15-10). The Coombs test is also commonly used to detect antibodies against drugs that bind to red blood cells and cause hemolytic anemia.

1	A-7 Monoclonal antibodies.

1	The antibodies generated in a natural immune response or after immunization in the laboratory are a mixture of molecules of different specificities and affinities. Some of this heterogeneity results from the production of antibodies that bind to different epitopes on the immunizing antigen, but even antibodies directed at a single antigenic determinant such as a hapten can be markedly heterogeneous, as shown by isoelectric focusing. In this technique, proteins are separated on the basis of their isoelectric point, the pH at which their net charge is zero. By electrophoresing proteins in a pH gradient for long enough, each molecule migrates along the pH gradient until it reaches the pH at which it is neutral and is thus concentrated (focused) at that point. When antiserum containing anti-hapten antibodies is treated in this way and then transferred to a solid support such as nitrocellulose paper, the anti-hapten antibodies can be detected by their ability to bind labeled hapten. The

1	antibodies is treated in this way and then transferred to a solid support such as nitrocellulose paper, the anti-hapten antibodies can be detected by their ability to bind labeled hapten. The binding of antibodies of various isoelectric points to the hapten shows that even antibodies that bind the same antigenic determinant can be heterogeneous.

1	Antisera are valuable for many biological purposes but they have certain inherent disadvantages that relate to the heterogeneity of the antibodies they contain. First, each antiserum is different from all other antisera, even if raised in a genetically identical animal by using the identical preparation of antigen and the same immunization protocol. Second, antisera can be produced in only limited volumes, and thus it is impossible to use the identical serological reagent in a long or complex series of experiments or clinical tests. Finally, even antibodies purified by affinity chromatography (see Section A-3) can include minor populations of antibodies that give unexpected cross-reactions, which confound the analysis of experiments. To avoid these problems, and to harness the full potential of antibodies, it was necessary to develop a way of making an unlimited supply of antibody molecules of homogeneous structure and known specificity. This has been achieved through the production

1	of antibodies, it was necessary to develop a way of making an unlimited supply of antibody molecules of homogeneous structure and known specificity. This has been achieved through the production of monoclonal antibodies from cultures of hybrid antibody-forming cells or, more recently, by genetic engineering.

1	Starting in the 1950s, biochemists in search of a homogeneous preparation of antibody that they could subject to detailed chemical analysis turned to proteins produced by patients with multiple myeloma, a common tumor of plasma cells. It was known that antibodies are normally produced by plasma cells, and because this disease is associated with the presence of large amounts of a homogeneous gamma globulin called a myeloma protein in the patient’s serum, it seemed likely that myeloma proteins would serve as models for

1	Fig. A.8 The Coombs direct and indirect anti-globulin tests for antibody against red blood cell antigens. An Rh– mother of an Rh+ fetus can become immunized to fetal red blood cells that enter the maternal circulation at the time of delivery. In a subsequent pregnancy with an Rh+ fetus, IgG anti-Rh antibodies can cross the placenta and damage the fetal red blood cells. In contrast to anti-Rh antibodies, maternal anti-ABO antibodies are of the IgM isotype and cannot cross the placenta, and so do not cause harm. Anti-Rh antibodies do not agglutinate red blood cells, but their presence on the fetal red blood cell surface can be shown by washing away unbound immunoglobulin and then adding antibody against human immunoglobulin, which agglutinates the antibody-coated cells. Anti-Rh antibodies can be detected in the mother’s serum in an indirect Coombs test; the serum is incubated with Rh+ red blood cells, and once the antibody has bound, the red blood cells are treated as in the direct

1	can be detected in the mother’s serum in an indirect Coombs test; the serum is incubated with Rh+ red blood cells, and once the antibody has bound, the red blood cells are treated as in the direct Coombs test.

1	Fig. A.9 The production of monoclonal antibodies. Mice are immunized with antigen A and given an intravenous booster immunization 3 days before they are killed, in order to produce a large population of spleen cells secreting specific antibody. Spleen cells die after a few days in culture. To produce a continuous source of antibody they are fused with immortal myeloma cells by using polyethylene glycol (PEG) to produce a hybrid cell line called a hybridoma. The myeloma cells are selected beforehand to ensure that they are not secreting antibody themselves and that they lack the enzyme hypoxanthine:guanine phosphoribosyl transferase (HGPRT); without this enzyme, unfused myeloma cells are sensitive to the hypoxanthine–aminopterin–thymidine (HAT) medium, which is used to select hybrid cells. The HGPRT gene contributed by the spleen cell allows hybrid cells to survive in the HAT medium, and only hybrid cells can grow continuously in culture, because of the malignant potential contributed

1	The HGPRT gene contributed by the spleen cell allows hybrid cells to survive in the HAT medium, and only hybrid cells can grow continuously in culture, because of the malignant potential contributed by the myeloma cells combined with the finite life-span of unfused spleen cells. Unfused myeloma cells and unfused spleen cells therefore die in the HAT medium, as shown here by cells with dark, irregular nuclei. Individual hybridomas are obtained by single cell dilution and then screened for antibody production, and single clones that make antibody of the desired specificity can be isolated and grown. The cloned hybridoma cells are grown in bulk culture to produce large amounts of antibody. As each hybridoma is descended from a single cell, all the cells of a hybridoma cell line make the same antibody molecule, which is thus called a monoclonal antibody.

1	normal antibody molecules. Thus, much of the early knowledge of antibody structure came from studies on myeloma proteins. These studies showed that monoclonal antibodies could be obtained from immortalized plasma cells. However, the antigen specificity of most myeloma proteins was unknown, which limited their usefulness as objects of study or as immunological tools.

1	This problem was solved by Georges Köhler and César Milstein, who devised a technique for producing a homogeneous population of antibodies of known antigenic specificity. They did this by fusing spleen cells from an immunized mouse to cells of a mouse myeloma to produce hybrid cells that both proliferated indefinitely and secreted antibody specific for the antigen used to immunize the spleen cell donor. The spleen cell provides the ability to make specific antibody, while the myeloma cell provides the ability to grow indefinitely in culture and secrete immunoglobulin continuously. By using a myeloma cell partner that produces no antibody proteins itself, the antibody produced by the hybrid cells comes only from the immune spleen cell partner. After fusion, the hybrid cells are selected using drugs that kill the myeloma parental cell, while the unfused parental spleen cells have a limited life-span and soon die, so that only hybrid myeloma cell lines, or hybridomas, survive. Those

1	using drugs that kill the myeloma parental cell, while the unfused parental spleen cells have a limited life-span and soon die, so that only hybrid myeloma cell lines, or hybridomas, survive. Those hybridomas producing antibody of the desired specificity are then identified and cloned by regrowing the cultures from single cells (Fig. A.9). Because each hybridoma is a clone derived from fusion with a single B cell, all the antibody molecules it produces are identical in structure, including their antigen-binding site and isotype. Such antibodies are called monoclonal antibodies. This technology has revolutionized the use of antibodies by providing a limitless supply of antibody of a single and known specificity. Monoclonal antibodies are now used in most serological assays, as diagnostic probes, and as therapeutic agents. So far, however, only mouse monoclonals are routinely produced in this way, and efforts to use the same approach to make human monoclonal antibodies have met with

1	probes, and as therapeutic agents. So far, however, only mouse monoclonals are routinely produced in this way, and efforts to use the same approach to make human monoclonal antibodies have met with limited success. ‘Fully human’ therapeutic monoclonal antibodies are currently made by using phage display technology (described in Section A-8), by using recombinant DNA technology to clone and express antibody genes from human plasma cells (see Section A-9), or by immunizing transgenic mice (see Section A-34) carrying human antibody genes.

1	A-8 Phage display libraries for antibody V-region production. In a technique for producing antibody-like molecules, gene segments encoding the antigen-binding variable, or V, domains of antibodies are fused to genes encoding the coat protein of a bacteriophage. Bacteriophages Construct fusion protein of V region with a bacteriophage coat protein Cloning a random population of variable regions gives rise to a mixture of bacteriophages— a phage display library Select phages with desired V regions by specifc binding to antigen VH antibody protein DNA Isolate population of genes encoding antibody variable regions VH VL VL Fig. A.10 The production of antibodies by genetic engineering.

1	Short primers to consensus sequences in heavyand light-chain variable (V) regions of immunoglobulin genes are used to generate a library of heavyand light-chain V-region DNAs by PCR, with spleen DNA as the starting material. These heavyand light-chain V-region genes are cloned randomly into filamentous phages such that each phage expresses one heavy-chain and one light-chain V region as a surface fusion protein with antibody-like properties. The resulting phage display library is multiplied in bacteria, and the phages are then bound to a surface coated with antigen. The unbound phages are washed away; the bound phages are recovered, multiplied in bacteria, and again bound to antigen. After a few cycles, only specific high-affinity antigen-binding phages are left. These can be used like antibody molecules, or their V genes can be recovered and engineered into antibody genes to produce genetically engineered antibody molecules (not shown). This technology may replace the hybridoma

1	antibody molecules, or their V genes can be recovered and engineered into antibody genes to produce genetically engineered antibody molecules (not shown). This technology may replace the hybridoma technology for producing monoclonal antibodies, and has the advantage that humans can be used as the source of DNA.

1	containing such gene fusions are used to infect bacteria, and the resulting phage particles have coats that express the antibody-like fusion protein, with the antigen-binding domain displayed on the outside of the bacteriophages. A collection of recombinant phages, each displaying a different antigen-binding domain on the surface, is known as a phage display library. In much the same way that antibodies specific for a particular antigen can be isolated from a complex mixture by affinity chromatography (see Section A-3), phages expressing antigen-binding domains specific for a particular antigen can be isolated by selecting the phages in the library for binding to that antigen. The phage particles that bind are recovered and used to infect fresh bacteria. Each phage isolated in this way will produce a monoclonal antigen-binding particle analogous to a monoclonal antibody (Fig. A.10). The genes encoding the antigen-binding site, which are unique to each phage, can then be recovered from

1	produce a monoclonal antigen-binding particle analogous to a monoclonal antibody (Fig. A.10). The genes encoding the antigen-binding site, which are unique to each phage, can then be recovered from the phage DNA and used to construct genes for a complete antibody molecule by joining them to parts of immunoglobulin genes that encode the invariant parts of an antibody. When these reconstructed antibody genes are introduced into a suitable host-cell line, such as the non-antibody-producing myeloma cells used for hybridomas, the transfected cells can secrete antibodies with all the desirable characteristics of monoclonal antibodies produced from hybridomas.

1	A-9 Generation of human monoclonal antibodies from vaccinated individuals.

1	In some cases, human monoclonal antibodies can be made by cloning the rearranged antibody heavyand light-chain gene sequences from plasma cells isolated from vaccinated individuals. Based on the expression of cell-surface molecules such as CD27 and CD38, human plasma cells can be isolated from the peripheral blood of individuals who were immunized approximately 1 week earlier. Individual plasma cells are sorted into wells of microtiter plates, and the antibody heavyand light-chain variable-region sequences are cloned from each cell by PCR. These sequences are then inserted into constructs that recreate the full-length antibody heavyand light-chain genes, and the paired heavyand light-chain vectors are introduced into an immortalized human cell line. Human cells are then screened to identify those secreting antibody proteins that bind to the immunizing antigen. These immortalized cells become a permanent source of the specific human antibody protein.

1	A-10 Microscopy and imaging using fluorescent dyes.

1	Because antibodies bind stably and specifically to their corresponding antigen, they are invaluable as probes for identifying a particular molecule in cells, tissues, or biological fluids. Antibody molecules can be used to locate their target molecules accurately in single cells or in tissue sections by a variety of different labeling techniques. When the antibody itself, or the anti-immunoglobulin antibody used to detect it, is labeled with a fluorescent dye (a fluorochrome or fluorophore) and then detected by microscopy, the technique is known as immunofluorescence microscopy. As in all serological techniques, the antibody binds stably to its antigen, allowing unbound antibody to be removed by thorough washing. Because antibodies against proteins recognize the surface features of the native, folded protein, the native structure of the protein being sought usually needs to be preserved, either by using only the most gentle chemical fixation techniques or by using frozen tissue

1	the native, folded protein, the native structure of the protein being sought usually needs to be preserved, either by using only the most gentle chemical fixation techniques or by using frozen tissue sections that are fixed only after the antibody reaction has been performed. Some antibodies, however, bind proteins even if they are denatured, and such antibodies will bind specifically even to protein in fixed tissue sections.

1	The fluorescent dye can be covalently attached directly to the specific antibody; however, the bound antibody is more commonly detected by fluorescently labeled anti-immunoglobulin, a technique known as indirect immunofluorescence. The dyes chosen for immunofluorescence are excited by light of one wavelength, usually blue or green, and emit light of a different wavelength in the visible spectrum. The most commonly used fluorochromes are fluorescein, which emits green light; Texas Red and peridinin chlorophyll protein (PerCP), which emit red light; and rhodamine and phycoerythrin (PE), which emit orange/red light (Fig. A.11). By using selective filters, only the light coming from the fluorochrome used is detected in the fluorescence microscope (Fig. A.12). Although Albert Coons first devised this technique to

1	Fig. A.12 Immunofluorescence microscopy. Antibodies labeled with a fluorescent dye such as fluorescein (green triangle) are used to reveal the presence of their corresponding antigens in cells or tissues. The stained cells are examined using a microscope that exposes them to blue or green light to excite the fluorescent dye. The excited dye emits light at a characteristic wavelength, which is captured by viewing the sample through a selective filter. This technique is applied widely in biology to determine the location of molecules in cells and tissues. Different antigens can be detected in tissue sections by labeling antibodies with dyes of distinctive color. Here, antibodies against the protein glutamic acid decarboxylase (GAD) coupled to a green dye are shown to stain the β cells of pancreatic islets of Langerhans. The α cells do not make this enzyme and are labeled with antibodies against the hormone glucagon coupled with an orange fluorescent dye. GAD is an important antigen in

1	islets of Langerhans. The α cells do not make this enzyme and are labeled with antibodies against the hormone glucagon coupled with an orange fluorescent dye. GAD is an important antigen in type 1 diabetes, a disease in which the insulin-secreting β cells of the islets of Langerhans are destroyed by an immune attack on self tissues (see Chapter 15). Photograph courtesy of M. Solimena and P. De Camilli.

1	identify the plasma cell as the source of antibody, it can be used to detect the distribution of any protein. By attaching different dyes to different antibodies, the distribution of two or more molecules can be determined in the same cell or tissue section (see Fig. A.12).

1	The development of the confocal fluorescent microscope, which uses computer-aided techniques to produce ultrathin optical sections of a cell or tissue, gives very high resolution (sub-micrometer) fluorescence microscopy without the need for elaborate sample preparation. The light source for excitation (a laser) is focused onto a particular plane in the specimen, and the emitted light is refocused through a ‘pinhole’ so that only light from the desired plane reaches the detector, thus removing out-of-focus emissions from above or below the plane. This gives a sharper image than conventional fluorescence microscopy, and a three-dimensional picture can be built up from successive optical sections taken along the ‘vertical’ axis. Confocal microscopy can be used on fixed cells stained with fluorescently tagged antibodies or on living cells expressing proteins tagged with naturally fluorescent proteins. The first of these fluorescent proteins to come into wide use was green fluorescent

1	fluorescently tagged antibodies or on living cells expressing proteins tagged with naturally fluorescent proteins. The first of these fluorescent proteins to come into wide use was green fluorescent protein (GFP), isolated from the jellyfish Aequorea victoria. The list of fluorescent proteins in routine use now includes those emitting red, blue, cyan, or yellow fluorescence. By using cells transfected with genes encoding different fusion proteins, it has been possible to visualize the redistribution of T-cell receptors, co-receptors, adhesion molecules, and other signaling molecules, such as CD45, that takes place when a T cell makes contact with a target cell (see Fig. 9.37).

1	Confocal microscopy, however, can penetrate only around 80 μm into a tissue, and at the wavelengths typically used for excitation, the source light will soon bleach the fluorescent label and damage the specimen. This means that the technique is not suitable for imaging a live specimen over a period of time sufficient, for example, to track the movements of cells in a tissue. The more recently developed technique of twophoton scanning fluorescence microscopy overcomes some of these limitations. In this technology, ultrashort pulses of laser light of much longer wavelength (and thus with photons of lower energy) are used for excitation, and two of these lower-energy photons arriving nearly simultaneously are required to excite the fluorophore. Excitation will therefore occur in only a very small region at the focus of the microscope, where the beam of light is most intense, and so fluorescence emission will be restricted to the plane of focus, producing a sharp, high-contrast image. The

1	region at the focus of the microscope, where the beam of light is most intense, and so fluorescence emission will be restricted to the plane of focus, producing a sharp, high-contrast image. The longer-wavelength light (typically in the near infrared) is also less damaging to living tissue than the blue and ultraviolet wavelengths typically used in confocal microscopy, and so imaging can be carried out over a longer period. More of the emitted light is collected than in confocal microscopy, and because single photons scattering within the tissue cannot cause fluorescence and consequent background haze, imaging to greater depths (several hundred micrometers) is possible. Like confocal microscopy, two-photon microscopy produces thin optical sections from which a three-dimensional image can be built up.

1	To track the movements of molecules or cells over time, confocal or two-photon microscopy is combined with timelapse video imaging using sensitive digital cameras. In immunology, time-lapse two-photon fluorescence imaging has been particularly valuable for tracking the movements of individual T cells and B cells expressing fluorescent proteins in intact lymphoid organs and observing where they interact (see Chapter 10). A-11 Immunoelectron microscopy.

1	A-11 Immunoelectron microscopy. Antibodies can be used to detect the intracellular location of structures or particular proteins at high resolution by electron microscopy, a technique known as immunoelectron microscopy. Antibodies against the required antigen are labeled with gold particles and then applied to ultrathin sections, which are then examined in the transmission electron microscope. Antibodies labeled with gold particles of different diameters enable two or more proteins to be studied simultaneously (see Fig. 6.12). The difficulty with this technique is in staining the ultrathin section adequately, because few molecules of antigen are present in each section. A-12 Immunohistochemistry.

1	An alternative to immunofluorescence (see Section A-10) for detecting a protein in tissue sections is immunohistochemistry, in which the specific antibody is chemically coupled to an enzyme that converts a colorless substrate into a colored reaction product in situ. The localized deposition of the colored product where antibody has bound can be directly observed under a light microscope. The antibody binds stably to its antigen, allowing unbound antibody to be removed by thorough washing. This method of detecting bound antibody is analogous to ELISA (see Section A-4) and frequently uses the same coupled enzymes, the difference in detection being primarily that in immunohistochemistry the colored products are insoluble and precipitate at the site where they are formed. Horseradish peroxidase and alkaline phosphatase are the two enzymes most commonly used in these applications. Horseradish peroxidase oxidizes the substrate diaminobenzidine to produce a brown precipitate, while alkaline

1	and alkaline phosphatase are the two enzymes most commonly used in these applications. Horseradish peroxidase oxidizes the substrate diaminobenzidine to produce a brown precipitate, while alkaline phosphatase can produce red or blue dyes depending on the substrates used; a common substrate is 5-bromo-4-chloro-3-indolyl phosphate plus nitroblue tetrazolium (BCIP/NBT), which gives rise to a dark blue or purple stain. As with immunofluorescence, the native structure of the protein being sought usually needs to be preserved so that it will be recognized by the antibody. Tissues are fixed by the most gentle chemical fixation techniques, or frozen tissue sections are used that are fixed only after the antibody reaction has been performed.

1	A-13 Immunoprecipitation and co-immunoprecipitation.

1	To raise antibodies against membrane proteins and other cellular structures that are difficult to purify, mice are often immunized with whole cells or crude cell extracts. Antibodies against the individual molecules are then obtained by using these immunized mice to produce hybridomas making monoclonal antibodies (see Section A-7) that bind to the cell type used for immunization. To characterize the molecules identified by the antibodies, cells of the same type are labeled with radioisotopes and dissolved in nonionic detergents that disrupt cell membranes but do not interfere with antigen–antibody interactions. This allows the labeled protein to be isolated by binding to the antibody in a reaction known as immunoprecipitation. The antibody is usually attached to a solid support, such as the beads that are used in affinity chromatography (see Section A-3), or to Protein A, a protein derived from the cell wall of Staphylococcus aureus that binds tightly to the Fc region of IgG

1	such as the beads that are used in affinity chromatography (see Section A-3), or to Protein A, a protein derived from the cell wall of Staphylococcus aureus that binds tightly to the Fc region of IgG antibodies. Cells can be labeled in two main ways for immunoprecipitation analysis. All the proteins in a cell can be labeled metabolically by growing the cell in a medium containing radioactive amino acids that are then incorporated into cellular proteins (Fig. A.13). Alternatively, one can label only the cell-surface proteins by radioiodination under conditions that prevent iodine from crossing the plasma membrane and labeling proteins inside the cell, or by a reaction that labels only membrane proteins with biotin, a small molecule that is detected readily by labeled avidin, a protein found in egg whites that binds biotin with very high affinity.

1	Once the labeled proteins have been isolated by the antibody, they can be characterized in several ways. The most common is polyacrylamide gel electrophoresis (PAGE) of the proteins after they have been dissociated from antibody in the strong ionic detergent sodium dodecyl sulfate (SDS), a technique

1	Fig. A.13 Cellular proteins reacting with an antibody can be dissociates it from the antibody and also coats the protein with a characterized by immunoprecipitation of labeled cell lysates. strong negative charge, allowing it to migrate according to its size All actively synthesized cellular proteins can be labeled metabolically in polyacrylamide gel electrophoresis (PAGE). The positions of the by incubating cells with radioactive amino acids (shown here for labeled proteins are determined by autoradiography using X-ray methionine; 35S-Met), or one can label just the cell-surface proteins film. This technique of SDS-PAGE can be used to determine the by using radioactive iodine in a form that cannot cross the cell molecular weight and subunit composition of a protein. Patterns of membrane or by a reaction with the small molecule biotin, detected protein bands observed with metabolic labeling are usually more by its reaction with labeled avidin (not shown). Cells are lysed with complex

1	or by a reaction with the small molecule biotin, detected protein bands observed with metabolic labeling are usually more by its reaction with labeled avidin (not shown). Cells are lysed with complex than those revealed by radioiodination, owing to the detergent and individual labeled cell-associated proteins can be presence of precursor forms of the protein (right panel). The mature precipitated with a monoclonal antibody attached to beads. After form of a surface protein can be identified as being the same size as unbound proteins have been washed away, the bound protein that detected by surface iodination or biotinylation (not shown). is eluted in the detergent sodium dodecyl sulfate (SDS), which generally abbreviated as SDSPAGE. SDS binds relatively homogeneously to proteins, conferring a charge that allows the electrophoretic field to drive protein migration through the gel. The rate of migration is controlled mainly by protein size (see Fig. A.13). Proteins of differing charges

1	a charge that allows the electrophoretic field to drive protein migration through the gel. The rate of migration is controlled mainly by protein size (see Fig. A.13). Proteins of differing charges can be separated using isoelectric focusing (see Section A-7). This technique can be combined with SDS-PAGE in a procedure known as twodimensional gel electrophoresis. For this, the immunoprecipitated protein is eluted in urea, a nonionic solubilizing agent, and run on an isoelectric focusing gel in a narrow tube of polyacrylamide. This first-dimensional isoelectric focusing gel is then placed across the top of an SDS-PAGE slab gel, which is then run vertically to separate the proteins by molecular weight (Fig. A.14). Two-dimensional gel electrophoresis is a powerful technique that allows many hundreds of proteins in a complex mixture to be distinguished from one another.

1	Immunoprecipitation and the related technique of immunoblotting (see Section A-14) are useful for determining the molecular weight and isoelectric point of a protein as well as its abundance, distribution, and whether, for example, it undergoes changes in molecular weight and isoelectric point as a result of processing within the cell. Coimmunoprecipitation is an extension of the immunoprecipitation technique and is used to determine whether a given protein interacts physically

1	Fig. A.14 Two-dimensional gel electrophoresis of MHC class II molecules. Proteins in mouse spleen cells have been labeled metabolically (see Fig. A.13), precipitated with a monoclonal antibody against the mouse MHC class II molecule H2-A, and separated by isoelectric focusing in one direction and SDS-PAGE in a second direction at right angles to the first (hence the term two-dimensional gel electrophoresis). This allows one to distinguish molecules of the same molecular weight on the basis of their charge. The separated proteins are detected by autoradiography. The MHC class II molecules are composed of two chains, α and β, and in the different MHC class II molecules these have different isoelectric points (compare upper and lower panels). The MHC genotype of mice is indicated by lowercase superscripts (k, p). Actin, a common contaminant, is marked a. Photographs courtesy of J.F. Babich.

1	with another given protein. Cell extracts containing the presumed interaction complex are first immunoprecipitated with antibody against one of the proteins. The material isolated by this means is then tested for the presence of the other protein by immunoblotting with a specific antibody against the second protein. A-14 Immunoblotting (Western blotting).

1	Like immunoprecipitation (see Section A-13), immunoblotting is used for identifying the presence of a given protein in a cell lysate, but it avoids the problem of having to label large quantities of cells with radioisotopes. Unlabeled cells are placed in detergent to solubilize all cell proteins, and the lysate is run on SDS-PAGE to separate the proteins (see Section A-13). The size-separated proteins are then transferred from the gel to a stable support such as a nitrocellulose membrane. Specific proteins are detected by treatment with antibodies able to react with SDS-solubilized proteins (mainly those that react with denatured sequences); the bound antibodies are revealed by anti-immunoglobulin antibodies labeled with an enzyme. The term Western blotting as a synonym for immunoblotting arose because the comparable technique for detecting specific DNA sequences is known as Southern blotting, after Edwin Southern, who devised it, which in turn provoked the name ‘Northern’ for blots

1	arose because the comparable technique for detecting specific DNA sequences is known as Southern blotting, after Edwin Southern, who devised it, which in turn provoked the name ‘Northern’ for blots of size-separated RNA, and ‘Western’ for blots of size-separated proteins. Western blots have many applications in basic research and clinical diagnosis. They are often used to test sera for the presence of antibodies against specific proteins, for example, to detect antibodies against different constituents of HIV (Fig. A.15).

1	A-15 Use of antibodies in the isolation and characterization of multiprotein complexes by mass spectrometry. Many of the proteins that function in immune cells are components of multi-protein complexes. This is the case for cell-surface receptors, such as the T-cell and B-cell antigen receptors and most cytokine receptors, as well as intracellular proteins involved in signal transduction, gene expression, and cell death. Antibodies that bind to one member of such a complex can be used to identify the other members of the complex by a process of co-immunoprecipitation followed by Western blotting or mass spectrometry.

1	A mass spectrometer can provide extremely precise measurements of the masses of constituents in a preparation of molecules. To identify unknown proteins in a sample, such as that acquired by co-immunoprecipitation, the sample often is first subjected to one-dimensional SDS-PAGE or two-dimensional gel electrophoresis (see Section A-13) to separate the proteins in the complex for individual analysis. Gel slices are excised and treated with a proteolytic enzyme, such as trypsin, to digest the protein into a series of peptides that can be easily eluted from the gel. The peptide mixture is then introduced into the mass spectrometer, which ionizes the peptides, transfers them to the gas phase, and then separates them under high vacuum by subjecting them to a magnetic field. The separation is based on the mass/charge (m/z)

1	Fig. A.15 Western blotting is used to identify antibodies against the human immunodeficiency virus (HIV) in serum from infected individuals. The virus is dissociated into its constituent proteins by treatment with the detergent SDS, and its proteins are separated using SDS-PAGE. The separated proteins are transferred to a nitrocellulose sheet and reacted with the test serum. Anti-HIV antibodies in the serum bind to the various HIV proteins and are detected with enzyme-linked anti-human immunoglobulin, which deposits colored material from a colorless substrate. This general methodology will detect any combination of antibody and antigen and is used widely, although the denaturing effect of SDS means that the technique works most reliably with antibodies that recognize the antigen when it is denatured.

1	Fig. A.16 Characterization of multiprotein complexes by mass on differences in their mass to charge (m/z) ratio by subjecting spectrometry. Following immunoprecipitation of a multiprotein them under high vacuum to a magnetic field. A detector collects complex using antibodies specific for one component of the information on the signal intensity for each peptide ion and displays complex, the individual proteins are separated by gel electrophoresis. the information as a histogram. This histogram, usually referred to An individual band representing one protein is isolated and digested as a spectrum, is compared with a database containing potential with a protease such as trypsin. The digested protein sample is cleavage sites for the proteolytic enzyme used in all known protein injected into the mass spectrometer, which ionizes the peptides, sequences, allowing for identification (ID) of the protein in the sample. transfers them to the gas phase, and then separates them based ratio of

1	the mass spectrometer, which ionizes the peptides, sequences, allowing for identification (ID) of the protein in the sample. transfers them to the gas phase, and then separates them based ratio of each ion, and a detector collects information on the signal intensity for each ion and displays the data as a histogram (Fig. A.16). This histogram, usually referred to as a spectrum, can be compared with a database containing potential cleavage sites (for the proteolytic enzyme used) in all known protein sequences. Due to the precision of these measurements, and the information derived from multiple peptides comprising the initial protein, the spectrum can often be unambiguously assigned to a unique protein in the database.

1	Modern multidimensional mass spectrometers (MS/MS) allow peptide ions to be sequenced as well as analyzed by their mass. In these instruments, peptide ions separated in one sector are fragmented in a second sector by collision with other molecules (often an inert gas such as N2), with the resultant fragments separated in a third section (Fig. A.17). Fragmentation occurs primarily in the

1	Fig. A.17 Determining the amino acid sequence of a peptide by multidimensional mass spectrometry (MS/MS). Multidimensional mass spectrometers (MS/MS) consist of two mass spectrometers linked in tandem but with an interceding middle sector that fragments ions. In the first sector, the first mass spectrometer separates peptide ions, as shown in Fig. A.16. Each peptide ion from this first separation is then fragmented in the middle sector of the apparatus by collision with other molecules (often an inert gas such as N2). Since fragmentation occurs primarily in the peptide backbone, a mixture of fragments is generated in which the fragments each differ by one amino acid residue. The resultant fragments are then separated in the second mass spectrometer, the final sector. The sequence of the peptide can be read directly from the second mass spectrum. The order of amino acid residues in the peptide can be deduced because of the exquisite precision of the measurements of each ion together

1	peptide can be read directly from the second mass spectrum. The order of amino acid residues in the peptide can be deduced because of the exquisite precision of the measurements of each ion together with knowledge of the exact mass of each possible amino acid residue.

1	peptide backbone, allowing the sequence of the peptide to be read directly from the mass spectrum of the mixture of fragments. In place of gel electrophoresis, a liquid chromatograph can be employed upstream of the mass analyzers (LC-MS/MS), to provide additional separation of peptides prior to mass separation and allow a very complex mixture of thousands of peptides to be sequenced in a single run. It was this latter technique that played an essential role in the early studies that identified the repertoire of peptides bound to MHC molecules on the surface of antigen-presenting cells (see Chapter 6). A-16 Isolation of peripheral blood lymphocytes by density-gradient fractionation.

1	The first step in studying lymphocytes is to isolate them so that their behavior can be analyzed in vitro. Human lymphocytes can be isolated most readily from peripheral blood by density centrifugation over a step gradient consisting of a mixture of the carbohydrate polymer Ficoll-Hypaque™ and the dense iodine-containing compound metrizamide. A step gradient is made by preparing a solution of Ficoll-Hypaque at a precise density (1.077 g/liter for human cells) and placing a layer of this solution at the bottom of a centrifuge tube. A sample of heparinized blood mixed with saline (heparin prevents clotting) is carefully layered on top of the Ficoll-Hypaque solution. Following centrifugation for about 30 minutes, the components of the blood have separated based on their densities. The upper layer contains the blood plasma and platelets, which remain in the top layer during the short centrifugation. Red blood cells and granulocytes have a higher density than the Ficoll-Hypaque solution

1	layer contains the blood plasma and platelets, which remain in the top layer during the short centrifugation. Red blood cells and granulocytes have a higher density than the Ficoll-Hypaque solution and collect at the bottom of the tube. The resulting population, called peripheral blood mononuclear cells (PBMCs), collects at the interface between the blood and the Ficoll-Hypaque layers and consists mainly of lymphocytes and monocytes (Fig. A.18). Although this population is readily accessible, it is not necessarily representative of the lymphoid system, because only recirculating lymphocytes can be isolated from blood.

1	The ‘normal’ ranges in the numbers of the different types of white blood cells in blood, along with the normal ranges in concentrations of the various antibody classes, are given in Fig. A.19. A-17 Isolation of lymphocytes from tissues other than blood. In experimental animals, and occasionally in humans, lymphocytes are isolated from lymphoid organs, such as spleen, thymus, bone marrow, lymph nodes, or mucosal-associated lymphoid tissues—in humans, most commonly the palatine tonsils (see Fig. 12.6). A specialized population of lymphocytes resides in surface epithelia; these cells are isolated by fractionating the Diluted anticoagulated blood (left panel) is layered over Ficoll-Hypaque™ and centrifuged. Red blood cells and polymorphonuclear leukocytes or granulocytes are denser and travel through the Ficoll-Hypaque™, while mononuclear cells consisting of lymphocytes together with some monocytes band over it and can be recovered at the interface (right panel).

1	Fig. A.18 Peripheral blood mononuclear cells can be isolated from whole blood by Ficoll-Hypaque™ centrifugation. centrifugation diluted blood Ficoll-HypaqueTM (specifc gravity =1.078) Fig. A.19 The major cellular and humoral components of human blood. Human blood contains B cells, T cells, and myeloid cells, as well as high concentrations of antibodies and complement proteins. epithelial layer after its detachment from the basement membrane. Finally, in situations where local immune responses are prominent, lymphocytes can be isolated from the site of the response itself. For example, in order to study the autoimmune reaction that is thought to be responsible for rheumatoid arthritis, an inflammatory response in joints, lymphocytes are isolated from the fluid aspirated from the inflamed joint space.

1	Lasercapture microdissection is a technique used to isolate specific populations of cells from an intact tissue sample or histological specimen after visualizing the cells by light microscopy. The cells of interest can be ‘captured’ by placing a polymer over the sample on the microscope slide, and using an infrared laser to melt the polymer onto the sample in discrete locations. Once completed, the polymer–cell composite can be lifted and DNA, RNA, or proteins can be isolated from the dissected cells (Fig. A.20). A variant of this approach makes use of an ultraviolet (UV) laser, rather than infrared. In this case, the UV laser acts as a molecular cutting tool, and can be used to cut away or even ablate the unwanted portions of the tissue, leaving intact the area of interest. A-18 Flow cytometry and FACS analysis.

1	A-18 Flow cytometry and FACS analysis. An immensely powerful tool for defining and enumerating populations of immune cells is the flow cytometer, which detects and counts individual cells passing in a stream through a laser beam. A flow cytometer equipped to separate the identified cells is called a fluorescenceactivated cell sorter (FACS). These instruments are used to study the properties of cell subsets that are identified by using monoclonal antibodies against cell-surface or intracellular proteins. Individual cells within a mixed population are first labeled by

1	Fig. A.20 Laser-capture microdissection. Specific populations of cells from an intact tissue sample or histological specimen can be isolated after visualizing the cells by light microscopy. A polymer called the transfer film is placed over the sample on the microscope slide, and an infrared laser is used to melt the polymer onto the sample in discrete locations. The polymer–cell composite is then lifted and the cells of interest are isolated. DNA, RNA, or proteins can be prepared from the dissected cells.

1	treatment with specific monoclonal antibodies coupled to fluorescent dyes, or by specific antibodies followed by fluorescently tagged anti-immunoglobulin antibodies. The mixture of labeled cells is then suspended in a much larger volume of saline and forced through a small nozzle, creating a fine stream of liquid composed of droplets, each containing a single cell. As each cell passes through a laser beam it scatters the laser light, and any dye molecules bound to the cell are excited and fluoresce. Sensitive photomultiplier tubes detect both the scattered light, which gives information about the size and granularity of the cell, and the fluorescence emissions, which give information about the binding of the labeled monoclonal antibodies and hence about the expression of cell-surface or intracellular proteins by each cell (Fig. A.21).

1	In the cell sorter, the signals passed back to the computer are used to generate an electric charge, which is passed from the nozzle through the liquid stream at the precise time that the stream breaks up into droplets, each containing no more than a single cell; droplets containing a charge can then be deflected from the main stream of droplets as they pass between plates of opposite charge, so that positively charged droplets are attracted to a negatively charged plate, and vice versa. Once deflected, droplets containing cells are collected in tubes. In this way, specific subpopulations of cells, distinguished by the binding of the labeled antibody, can be purified from a mixed population of cells. Alternatively, to deplete a population of cells, the same fluorochrome can be used to label different antibodies directed at marker proteins expressed by the various undesired cell types. The cell sorter can be used to direct labeled cells to a waste channel, retaining only the unlabeled

1	different antibodies directed at marker proteins expressed by the various undesired cell types. The cell sorter can be used to direct labeled cells to a waste channel, retaining only the unlabeled cells.

1	When cells are labeled with a single fluorescent antibody, the data from a flow cytometer are usually displayed in the form of a one-dimensional histogram of fluorescence intensity versus cell numbers. If two or more antibodies are used, each coupled to a different fluorescent dye, then the data are usually

1	Fig. A.21 Flow cytometers allow individual cells to be identified by their cell-surface antigens and to be sorted. Cells to be analyzed by flow cytometry are first labeled with fluorescent dyes (top panel). Direct labeling uses dye-coupled antibodies specific for cell-surface antigens (as shown here), while indirect labeling uses a dye-coupled immunoglobulin to detect unlabeled cell-bound antibody. The cells are forced through a nozzle in a single-cell stream that passes through a laser beam (second panel). Photomultiplier tubes (PMTs) detect the scattering of light, which is a sign of cell size and granularity, as well as emissions from the different fluorescent dyes. This information is analyzed by computer (CPU). By examining a large number of cells, the proportion of cells with a specific set of characteristics can be determined and levels of expression of various molecules on these cells can be measured. The lower part of the figure shows how these data can be represented, the

1	set of characteristics can be determined and levels of expression of various molecules on these cells can be measured. The lower part of the figure shows how these data can be represented, the example in this case being the expression of two surface immunoglobulins, IgM and IgD, on a sample of B cells from a mouse spleen. The two immunoglobulins have been labeled with different-colored dyes. When the expression of just one type of molecule is to be analyzed (IgM or IgD), the data are usually displayed as a histogram, as in the left-hand panels. Histograms display the distribution of cells expressing a single measured parameter (for example, size, granularity, fluorescence intensity). When two or more parameters are measured for each cell (IgM and IgD), various types of two-dimensional plots can be used to display the data, as shown in the right-hand panel. All four plots represent the same data, and in each case, the horizontal axis represents intensity of IgM fluorescence, and the

1	can be used to display the data, as shown in the right-hand panel. All four plots represent the same data, and in each case, the horizontal axis represents intensity of IgM fluorescence, and the vertical axis the intensity of IgD fluorescence. Two-color plots provide more information than histograms; they allow recognition, for example, of cells that are ‘bright’ for both colors, ‘dull’ for one and bright for the other, dull for both, negative for both, and so on. For example, the cluster of dots in the extreme lower left portions of the plots represents cells that do not express either immunoglobulin, and are mostly T cells. The standard dot plot (upper left) places a single dot for each cell whose fluorescence is measured. This format works well for identifying cells that lie outside the main groups, but tends to saturate in areas containing a large number of cells of the same type. A second means of presenting these data is the color dot plot (lower left), which uses color density

1	main groups, but tends to saturate in areas containing a large number of cells of the same type. A second means of presenting these data is the color dot plot (lower left), which uses color density to indicate high-density areas. A contour plot (upper right) draws 5% ‘probability’ contours, with contour lines drawn to indicate each successive 5% of the population; this format provides the best monochrome visualization of regions of high and low density. The lower right plot is a 5% probability contour map, which also shows outlying cells as dots.

1	displayed in the form of a two-dimensional scatter diagram or as a contour diagram, where the fluorescence of one dye-labeled antibody is plotted against that of a second, with the result that a population of cells labeled with one antibody can be further subdivided by its labeling with the second antibody (see Fig. A.21). By examining large numbers of cells, flow cytometry can give quantitative data on the percentage of cells bearing different proteins, such as surface immunoglobulin, which characterizes B cells; the T-cell receptor-associated molecules known as CD3; and the CD4 and CD8 co-receptor proteins that distinguish the major T-cell subsets. Likewise, FACS analysis has been instrumental in defining stages in the early development of B and T cells. This technology also played a vital role in the early identification of AIDS as a disease in which T cells bearing CD4 are depleted selectively (see Chapter 13). Advances in FACS technology permit progressively more antibodies

1	a vital role in the early identification of AIDS as a disease in which T cells bearing CD4 are depleted selectively (see Chapter 13). Advances in FACS technology permit progressively more antibodies labeled with distinct fluorescent dyes to be used at the same time. For experiments aimed at cell analysis, rather than cell sorting, machines with four lasers that can simultaneously measure 18 different fluorescent dyes are currently available. However, FACS analysis is constrained by the spectral properties of the fluorescent dyes used for coupling to antibodies, and this technology may have reached its limit.

1	Dot plots IgD IgD IgM IgM Contour maps standard 5% probability color density 0.1 1 10 100 1000 0.1 1 10 100 1000 0.1 1 10 100 1000 0.1 1 10 100 1000 0.1 1 10 100 10000.1 1 10 100 1000 1000 100 10 1 0.1 1000 100 10 1 0.1 Analysis of cells stained with labeled antibodies forward scatter side scatter red PMT green photomultiplier tube (PMT) Laser stream of fuid containing antibody-labeled cells CPU Mixture of cells is labeled with fuorescent antibody with outliers The magnetic ÿeld is removed, releasing the coupled cells When a magnetic ÿeld is applied, the coupled cells stick to the iron wool; unlabeled cells are washed out N N Heterogeneous population of lymphocytes is mixed with antibodies coupled to paramagnetic particles or beads and poured over an iron wool mesh

1	An alternative to FACS is a technology based on detecting heavy-metal atoms that are coupled to antibodies. Cell populations labeled with heavy metal-coupled antibodies are analyzed on a machine called a CyTOF™, which combines liquid fluidics with a mass spectrometer. As each cell is analyzed, the quantity of each heavy metal associated with that cell, and thus the abundance of the target of each antibody, is measured. In total, this technology is estimated to have the capability to measure 100 distinct heavy metals, greatly expanding the range of analysis that is currently possible by FACS. However, with this technique, the cells are destroyed by the ionization process required for the mass spectrometry analysis, so the CyTOF cannot function as a cell sorter. A-19 Lymphocyte isolation using antibody-coated magnetic beads.

1	Although FACS is superb for isolating small numbers of cells in pure form, mechanical means of separating cells are preferable when large numbers of lymphocytes must be prepared quickly. A powerful and efficient way of isolating lymphocyte populations is to couple paramagnetic beads to monoclonal antibodies that recognize distinguishing cell-surface molecules. These antibody-coated beads are mixed with the cells to be separated and are run through a column containing material that attracts the paramagnetic beads when the column is placed in a strong magnetic field. Cells binding the magnetically labeled antibodies are retained; cells lacking the appropriate surface molecule can be washed away (Fig. A.22). The retained cells are recovered by removing the column from the magnetic field. In this case, the bound cells are positively selected for expression of the particular cell-surface molecule, while the unbound cells are negatively selected for its absence.

1	A-20 Isolation of homogeneous T-cell lines. The analysis of specificity and effector function of T cells depends heavily on the study of monoclonal populations of T lymphocytes. These can be obtained in four distinct ways—T-cell hybrids, cloned T-cell lines, T-cell tumors, and

1	Fig. A.22 Lymphocyte subpopulations can be separated physically by using antibodies coupled to paramagnetic particles or beads. A mouse monoclonal antibody specific for a particular cell-surface molecule is coupled to paramagnetic particles or beads. It is mixed with a heterogeneous population of lymphocytes and poured over an iron wool mesh in a column. A magnetic field is applied so that the antibody-bound cells stick to the iron wool while cells that have not bound antibody are washed out; these cells are said to be negatively selected for lack of the molecule in question. The bound cells are released by removing the magnetic field; they are said to be positively selected for presence of the antigen recognized by the antibody.

1	limiting-dilution culture. By analogy with B-cell hybridomas (see Section A-7), normal T cells proliferating in response to specific antigen can be fused to malignant T-cell lymphoma lines to generate Tcell hybrids. The hybrids express the receptor of the normal T cell, but proliferate indefinitely owing to the cancerous state of the lymphoma parent. T-cell hybrids can be cloned to yield a population of cells all having the same T-cell receptor. When stimulated by their specific antigen, these cells release cytokines such as the T-cell growth factor interleukin-2 (IL-2), and the production of cytokines is used as an assay to assess the antigen specificity of the T-cell hybrid.

1	T-cell hybrids are excellent tools for the analysis of T-cell specificity, because they grow readily in suspension culture. However, they cannot be used to analyze the regulation of specific T-cell proliferation in response to antigen because they are continually dividing. T-cell hybrids also cannot be transferred into an animal to test for function in vivo because they would give rise to tumors. Functional analysis of T-cell hybrids is also confounded by the fact that the malignant partner cell affects their behavior in functional assays. Therefore, the regulation of T-cell growth and the effector functions of T cells must be studied using Tcell clones. These are clonal cell lines of a single T-cell type and antigen specificity, which are derived from cultures of heterogeneous T cells, called Tcell lines, whose growth is dependent on periodic restimulation with specific antigen and, frequently, on the addition of T-cell growth factors (Fig. A.23). T-cell clones also require periodic

1	Tcell lines, whose growth is dependent on periodic restimulation with specific antigen and, frequently, on the addition of T-cell growth factors (Fig. A.23). T-cell clones also require periodic restimulation with antigen and are more tedious to grow than T-cell hybrids, but because their growth depends on specific antigen recognition, they maintain antigen specificity, which is often lost in T-cell hybrids. Cloned T-cell lines can be used for studies of effector function both in vitro and in vivo. In addition, the proliferation of T cells, a critical aspect of clonal selection, can be characterized only in cloned T-cell lines, where such growth is dependent on antigen recognition. Thus, both types of monoclonal T-cell lines, T-cell hybrids and antigen-dependent

1	T-cell clones, have valuable applications in experimental studies. Studies of human T cells have relied largely on T-cell clones because a suitable fusion partner for making T-cell hybrids has not been identified. However, a human T-cell lymphoma line, called Jurkat, has been characterized extensively because it secretes IL-2 when its antigen receptor is cross-linked with anti-receptor monoclonal antibodies. This simple assay system has yielded much information about signal transduction in T cells. One of the Jurkat cell line’s most interesting features, shared with T-cell hybrids, is that it stops growing when its antigen receptor is cross-linked. This has allowed mutants lacking the receptor or having defects in signal transduction pathways to be selected simply by culturing the cells with anti-receptor antibody and selecting those that continue to grow. Thus, T-cell tumors, T-cell hybrids, and cloned T-cell lines all have valuable applications in experimental immunology.

1	Finally, primary T cells from any source can be isolated as single, antigen-specific cells by limiting dilution (see Section A-21) rather than by first establishing a mixed population of T cells in culture as a T-cell line and then deriving clonal subpopulations. During the growth of T-cell lines, particular T-cell clones can come to dominate the cultures and give a false picture of the number and specificities in the original sample. Direct cloning of primary T cells avoids this artifact. A-21 Limiting-dilution culture. On many occasions it is important to know the frequency of antigen-specific lymphocytes, especially T cells, in order to measure the efficiency with which an individual responds to a particular antigen, for example, or the degree to which specific immunological memory has been established. There are a

1	The T cells are placed into culture with antigen-presenting cells and antigen. Antigen-speciÿc T cells proliferate, while T cells that do not recognize the antigen do not proliferate Fig. A.23 Production of cloned T-cell lines. T cells from an immunized donor, comprising a mixture of cells with different specificities, are activated with antigen and antigen-presenting cells. Single responding cells are cultured by limiting dilution (see Section A-21) in the T-cell growth factor IL-2, which selectively stimulates the responding cells to proliferate. From these single cells, cloned lines specific for antigen are identified and can be propagated by culture with antigen, antigen-presenting cells, and IL-2.

1	Fig. A.24 The frequency of specific lymphocytes can be determined using limiting-dilution assay. Various numbers of lymphoid cells from normal or immunized mice are added to individual culture wells and stimulated with antigen and antigen-presenting cells (APCs) or polyclonal mitogen and added growth factors. After several days, the wells are tested for a specific response to antigen, such as cytotoxic killing of target cells. Each well that initially contained a specific T cell will make a response to its target, and from the Poisson distribution one can determine that when 37% of the wells are negative, each well contained, on average, one specific T cell at the beginning of the culture. In the example shown, for the unimmunized mouse 37% of the wells are negative when 160,000 T cells have been added to each well; thus the frequency of antigen-specific T cells is 1 in 160,000. When the mouse is immunized, 37% of the wells are negative when only 1100 T cells have been added; hence

1	have been added to each well; thus the frequency of antigen-specific T cells is 1 in 160,000. When the mouse is immunized, 37% of the wells are negative when only 1100 T cells have been added; hence the frequency of specific T cells after immunization is 1 in 1100, an increase in responsive cells of 150-fold.

1	number of methods for doing this, either by detecting the cells directly by the specificity of their receptor, or by detecting activation of the cells to provide some particular function, such as cytokine secretion or cytotoxicity.

1	The response of a lymphocyte population is a measure of the overall response, but the frequency of lymphocytes able to respond to a given antigen can be determined by limitingdilution culture. This assay makes use of the Poisson distribution, a statistical function that describes how objects are distributed at random. For instance, when a sample of heterogeneous T cells is distributed equally into a series of culture wells, some wells will receive no T cells specific for a given antigen, some will receive one specific T cell, some two, and so on. The T cells in the wells are activated with specific antigen, antigen-presenting cells, and growth factors. After allowing several days for their growth and differentiation, the cells in each well are tested for a response to antigen, such as cytokine release or the ability to kill specific target cells (Fig. A.24). The assay is replicated with different numbers of T cells in the samples. The logarithm of the proportion of wells in which

1	cytokine release or the ability to kill specific target cells (Fig. A.24). The assay is replicated with different numbers of T cells in the samples. The logarithm of the proportion of wells in which there is no response is plotted against the number of cells initially added to each well. If cells of one type, typically antigen-specific T cells because of their rarity, are the only limiting factor for obtaining a response, then a straight line is obtained. From the Poisson distribution, it is known that there is, on average, one antigen-specific cell per well when the proportion of negative wells is 37%. Thus, the frequency of antigen-specific cells in the population equals the reciprocal of the number of cells added to each well when 37% of the wells are negative. After priming, the frequency of specific cells goes up substantially, reflecting the antigen-driven proliferation of antigen-specific cells. The limiting-dilution assay can also be used to measure the frequency of B cells

1	of specific cells goes up substantially, reflecting the antigen-driven proliferation of antigen-specific cells. The limiting-dilution assay can also be used to measure the frequency of B cells that can make antibody against a given antigen.

1	Fig. A.25 The frequency of cytokine-secreting T cells can be determined by the ELISPOT assay. The ELISPOT assay is a variant of the ELISA assay in which antibodies bound to a plastic surface are used to capture cytokines secreted by individual T cells. Usually, cytokine-specific antibodies are bound to the surface of a plastic tissue-culture well and the unbound antibodies are removed (top panel). Activated T cells are then added to the well and settle onto the antibody-coated surface (second panel). If a T cell is secreting the appropriate cytokine, this will then be captured by the antibody molecules on the plate surrounding the T cell (third panel). After a period of time the T cells are removed, and the presence of the specific cytokine is detected using an enzyme-labeled second antibody specific for the same cytokine. Where this antibody binds, a colored reaction product can be formed (fourth panel). Each T cell that originally secreted the cytokine gives rise to a single spot of

1	specific for the same cytokine. Where this antibody binds, a colored reaction product can be formed (fourth panel). Each T cell that originally secreted the cytokine gives rise to a single spot of color, hence the name of the assay. The results of such an ELISPOT assay for T cells secreting IFN-γ in response to different stimuli are shown in the last panel. In this example, T cells from a stem-cell transplant recipient were treated with a control peptide (top two panels) or a peptide from cytomegalovirus (bottom two panels). As can be seen, there are a greater number of spots in the bottom two panels, a clear indication that the patient’s T cells are able to respond to the viral peptide and produce IFN-γ. Photographs courtesy of S. Nowack.

1	A-22 ELISPOT assay.

1	A modification of the ELISA antigen-capture assay (see Section A-4), called the ELISPOT assay, is a powerful tool for measuring the frequency of T-cell responses and also provides information about the cytokines produced. Populations of T cells are stimulated with the antigen of interest, and are then allowed to settle onto a plastic plate coated with antibodies against the cytokine that is to be assayed (Fig. A.25). If an activated T cell is secreting that cytokine, the cytokine is captured by the antibody on the plastic plate. After a period of time the cells are removed, and a second antibody against the cytokine is added to the plate to reveal a circle (‘spot’) of bound cytokine surrounding the position of each activated T cell; it is these circles that give the ELISPOT assay its name. By counting each spot and knowing the number of T cells originally added to the plate, one can easily calculate the frequency of T cells secreting that particular cytokine. ELISPOT can also be used

1	By counting each spot and knowing the number of T cells originally added to the plate, one can easily calculate the frequency of T cells secreting that particular cytokine. ELISPOT can also be used to detect specific antibody secretion by B cells, in this case by using antigen-coated surfaces to trap specific antibody and labeled anti-immunoglobulin to detect the bound antibody.

1	A-23 Identification of functional subsets of T cells based on cytokine production or transcription factor expression.

1	One problem with the detection of cytokine production on a single-cell level is that the cytokines are secreted by the T cells into the surrounding medium, and any association with the originating cell is lost. Three methods have been devised that allow the cytokine profile produced by individual cells to be determined. The first, that of intracellular cytokine staining (Fig. A.26), relies on the use of metabolic poisons that inhibit protein export from the cell. The cytokine thus accumulates within the endoplasmic reticulum and vesicular network of the cell. If the cells are subsequently fixed and rendered permeable by the use of mild detergents, antibodies can gain access to these intracellular compartments and detect the cytokine. The T cells can be stained for other markers simultaneously, and thus the frequency of IL-10-producing CD25+ CD4 T cells, for example, can be easily obtained.

1	A second method, which has the advantage that the cells being analyzed are not killed in the process, is called cytokine capture. This technique uses hybrid antibodies, in which the two separate heavyand light-chain pairs from different antibodies are combined to give a mixed antibody molecule in which Cytokine-speciÿc antibodies are bound to the surface of a plastic well Activated T cells are added to the well. These T cells are a mixture of cells with different effector functions Cytokine secreted by some activated T cells is captured by the bound antibody The captured cytokine is revealed by a second cytokine-speciÿc antibody, which is coupled to an enzyme, giving rise to a spot of insoluble colored precipitate

1	Fig. A.26 Cytokine-secreting cells can be identified by intracellular cytokine staining. Fluorochrome-labeled antibodies can be used to detect the cytokines secreted by activated T cells after the cytokine molecules have been allowed to accumulate inside the cell. The accumulation of cytokine molecules to a high enough concentration for efficient detection is achieved by treating the activated T cells with inhibitors of protein export. In such treated cells, proteins destined to be secreted are instead retained within the endoplasmic reticulum (left panel). These treated cells are then fixed, to cross-link the proteins inside the cell and in the cell membranes, so that they are not lost when the cell is permeabilized by dissolving the cell membrane in a mild detergent (center panel). Fluorochromelabeled antibodies can now enter the permeabilized cell and bind to the cytokines inside the cell (right panel). Cells labeled in this way can also be labeled with antibodies that bind to

1	Fluorochromelabeled antibodies can now enter the permeabilized cell and bind to the cytokines inside the cell (right panel). Cells labeled in this way can also be labeled with antibodies that bind to cell-surface proteins to determine which subsets of T cells are secreting particular cytokines.

1	Cytokine-speciÿc antibodies penetrate the cell to bind the intracellular cytokine molecules The cell is ÿxed and permeabilized with mild detergents Activated T cells are treated with an inhibitor that blocks protein export, allowing cytokines to accumulate in the ER the two antigen-binding sites recognize different ligands (Fig. A.27). In the bispecific antibodies used to detect cytokine production, one of the antigen-binding sites is specific for a T-cell surface marker, while the other is specific for the cytokine in question. The bispecific antibody binds to the T cells through the binding site for the cell-surface marker, leaving the cytokine-binding site free. If that T cell is secreting the particular cytokine, it is captured by the bound antibody before it diffuses away from the surface of the cell. It can then be detected by adding to the cells a fluorochrome-labeled second antibody specific for the cytokine.

1	A third method for identifying which T cells in a population produce a particular cytokine utilizes cytokine gene reporter mice. In these lines of mice, a cDNA clone encoding a readily detectable protein (the ‘reporter’ protein) is inserted into the 3ʹ untranslated region of the targeted cytokine gene downstream of a sequence known as an internal ribosome entry site (IRES). The IRES sequence allows translation of the reporter protein from the same mRNA as that encoding the cytokine; thus, the reporter protein is produced only when the cytokine mRNA is expressed (Fig. A.28). Common reporter proteins for this application are fluorescent proteins, such as green fluorescent protein (GFP). In fact, the GFP commonly used for this purpose contains

1	Fig. A.27 Hybrid antibodies containing cell-specific and cytokine-specific binding sites can be used to assay cytokine secretion by living cells and to purify cells secreting particular cytokines. Hybrid antibodies can be made by mixing together heavyand light-chain pairs from antibodies of different specificities, for example, an antibody against an MHC class I molecule and an antibody specific for a cytokine such as IL-4 (first panel). The hybrid antibodies are then added to a population of activated T cells, and bind to each cell via the MHC class I binding arm (second panel). If some of the cells in the population are secreting the appropriate cytokine, IL-4, this is captured by the cytokine-specific arm of the hybrid antibody (third panel). The presence of the cytokine can then be revealed, for example, by using a fluorochrome-labeled second antibody specific for the same cytokine but binding to a different site from the one used by the hybrid antibody (last panel). The labeled

1	for example, by using a fluorochrome-labeled second antibody specific for the same cytokine but binding to a different site from the one used by the hybrid antibody (last panel). The labeled cells are analyzed by flow cytometry or are isolated using a fluorescence-activated cell sorter (FACS). Alternatively, the second cytokine-specific antibody can be coupled to magnetic beads, and the cytokine-producing cells isolated magnetically.

1	An IRES element and the eGFP coding sequences are inserted downstream of the cytokine gene stop codon a point mutation that greatly improves its spectral properties for experimental purposes. This version of GFP is commonly referred to as ‘enhanced GFP,’ or ‘eGFP’ for short. eGFP can be detected by FACS or by fluorescent microscopy using the settings designed to detect the commonly used fluorescent dye FITC. Due to broad utility of these fluorescent proteins, a host of GFP derivatives have been developed by genetic engineering of the original GFP protein. Each derivative has distinct fluorescent properties and therefore can be uniquely identified, allowing these proteins to be used in combination with each other to provide information about multiple cytokines at the same time (Fig. A.29).

1	Several of these techniques for measuring cytokine expression by T-cell subsets have been adapted to examine the transcription factors expressed by T cells and other lymphocytes, providing an alternative method for identifying functional lymphocyte subsets. In one approach, antibodies specific for lineage-defining transcription factors are used to label permeabilized cells. As was described above for the intracellular cytokine staining assay, the cells can then be examined by flow cytometry or immunofluorescence microscopy. Lines of reporter mice have also been generated, in which the gene locus encoding a transcription factor is modified to express a fluorescent protein, such as eGFP. For both of these approaches, the advantage of using transcription factors to identify lymphocyte subsets is that there is no need to stimulate the cells prior to antibody staining or to assessing reporter protein expression, as lineage-defining transcription factors are constitutively expressed by the

1	is that there is no need to stimulate the cells prior to antibody staining or to assessing reporter protein expression, as lineage-defining transcription factors are constitutively expressed by the cells. Therefore, this approach is more widely used for the identification of T-cell and other lymphocyte subsets in intact tissues by microscopy.

1	Fig. A.28 Cytokine-expressing cells can be tracked in vivo using cytokine gene knock-in reporter mice. To identify cells expressing a specific cytokine in intact animals, the locus encoding the cytokine is modified by homologous recombination (see Fig. A.44 and Section A-35). An internal ribosome entry site (IRES) and the gene for a fluorescent protein such as eGFP are inserted 3ʹ of the last exon of the cytokine gene, downstream of the cytokine protein stop codon and upstream of the mRNA transcription termination and polyadenylation signal (the poly-A site). The IRES element allows the ribosome to initiate translation of a second protein-coding sequence at an internal site on the mRNA. When the modified locus is transcribed and spliced to form the mature mRNA, both the intact cytokine protein and the fluorescent reporter protein (e.g., eGFP) are produced from the same transcript. This allows the identification and characterization of cytokine-expressing cells, such as by flow

1	protein and the fluorescent reporter protein (e.g., eGFP) are produced from the same transcript. This allows the identification and characterization of cytokine-expressing cells, such as by flow cytometry, based on the detection of eGFP.

1	Fig. A.29 Fluorescent proteins are available in a rainbow of colors. Derivatives of GFP and a red-fluorescent coral protein can generate eight different fluorescent colors. A beach scene is drawn with bacterial strains expressing each fluorescent protein. Courtesy of Roger Tsien. A-24 Identification of T-cell receptor specificity using peptide:MHC tetramers. 10,000 1000 100 10 1 1 10 100 HLA-A2 + CMV-speciÿc tetramer staining CD8 staining 1000 10,000 Peptide:MHC tetramers are bound by T cells expressing receptors of the appropriate speciÿcity streptavidin MHC class I The peptide:MHC tetramer is made from recombinant MHC molecules with speciÿc peptides, bound to streptavidin via biotin

1	For many years, the ability to identify antigen-specific T cells directly through their receptor specificity eluded immunologists. Foreign antigen could not be used directly to identify T cells because, unlike B cells, T cells do not recognize antigen alone but rather the complexes of peptide fragments of antigen bound to self MHC molecules. Moreover, the affinity of interaction between the T-cell receptor and the peptide:MHC complex is in practice so low that attempts to label T cells with their specific peptide:MHC complexes routinely failed. The breakthrough in labeling antigen-specific T cells came with the idea of making multimers of the peptide:MHC complex, so as to increase the avidity of the interaction.

1	Peptides can be biotinylated using the bacterial enzyme BirA, which recognizes a specific amino acid sequence. Recombinant MHC molecules containing this target sequence are used to make peptide:MHC complexes, which are then biotinylated. Avidin, or its bacterial counterpart streptavidin, contains four sites that bind biotin with extremely high affinity. Mixing the biotinylated peptide:MHC complex with avidin or streptavidin results in the formation of a peptide:MHC tetramer—four specific peptide:MHC complexes bound to a single molecule of streptavidin (Fig. A.30). Routinely, the streptavidin moiety is labeled with a fluorochrome to allow detection of those T cells capable of binding the peptide:MHC tetramer.

1	Peptide:MHC tetramers have been used to identify populations of antigen-specific T cells in, for example, patients with acute Epstein–Barr virus infections (infectious mononucleosis), showing that up to 80% of the peripheral T cells in infected individuals can be specific for a single peptide:MHC complex. They have also been used to follow responses over timescales of years in individuals with HIV or, in the example we show, cytomegalovirus infections. These reagents have also been important in identifying the cells responding, for example, to nonclassical MHC class I molecules such as HLA-E or HLA-G, in both cases showing that these nonclassical molecules are recognized by subsets of NK receptors.

1	Fig. A.30 Peptide:MHC complexes coupled to streptavidin to form tetramers are able to stain antigen-specific T cells. Peptide:MHC tetramers are formed from recombinant refolded peptide:MHC complexes containing a single defined peptide epitope. MHC molecules that contain biotin can be chemically synthesized, but usually the recombinant MHC heavy chain is linked to a bacterial biotinylation sequence, a target for the Escherichia coli enzyme BirA, which is used to add a single biotin group to the MHC molecule. Streptavidin is a tetramer, each subunit having a single binding site for biotin; hence the streptavidin:peptide:MHC complex creates a tetramer of peptide:MHC complexes (top panel). Although the affinity between the T-cell receptor and its peptide:MHC ligand is too low for a single complex to bind stably to a T cell, the tetramer, by being able to make a more avid interaction with multiple peptide:MHC complexes binding simultaneously, is able to bind to T cells whose receptors are

1	to bind stably to a T cell, the tetramer, by being able to make a more avid interaction with multiple peptide:MHC complexes binding simultaneously, is able to bind to T cells whose receptors are specific for the particular peptide:MHC complex (middle panel). Routinely, the streptavidin molecules are coupled to a fluorochrome, so that the binding to T cells can be monitored by flow cytometry. In the example shown in the bottom panel, T cells have been stained simultaneously with antibodies specific for CD3 and CD8, and with a tetramer of HLA-A2 molecules containing a cytomegalovirus peptide. Only the CD3+ cells are shown, with the staining of CD8 displayed on the vertical axis and the tetramer staining displayed along the horizontal axis. The CD8– cells (mostly CD4+), on the bottom left of the figure, show no specific tetramer staining, while the bulk of the CD8+ cells, on the top left, likewise show no tetramer staining. However, a discrete population of tetramer positive CD8+ cells,

1	the figure, show no specific tetramer staining, while the bulk of the CD8+ cells, on the top left, likewise show no tetramer staining. However, a discrete population of tetramer positive CD8+ cells, at the top right of the panel, comprising some 5% of the total CD8+ cells, can clearly be seen. Data courtesy of G. Aubert.

1	A-25 Biosensor assays for measuring the rates of association and dissociation of antigen receptors for their ligands.

1	Two of the important questions that are always asked of any receptor–ligand interaction are, What is the strength of binding, or affinity, of the interaction, and, What are the rates of association and dissociation? These parameters are generally assessed using purified preparations of proteins. For receptors that are integral membrane proteins in their native state, soluble forms of the proteins are prepared, usually by truncating the proteins to eliminate their membrane-spanning domains. With these purified proteins, binding rates can be measured by following the binding of ligands to receptors immobilized on gold-plated glass slides, using a phenomenon known as surface plasmon resonance (SPR) to detect the binding (Fig. A.31). A full explanation of surface plasmon resonance is beyond the scope of this textbook, as it is based on advanced physical and quantum mechanical principles. In brief, it relies on the total internal reflection of a beam of light from the surface of a

1	beyond the scope of this textbook, as it is based on advanced physical and quantum mechanical principles. In brief, it relies on the total internal reflection of a beam of light from the surface of a gold-coated glass slide. As the light is reflected, some of its energy excites electrons in the gold coating and these excited electrons are affected by the electric field of any molecules binding to the surface of the glass coating. The more molecules that bind to the surface, the greater is the effect on the excited electrons, and this in turn affects the reflected light beam. The reflected light thus becomes a sensitive measure of the number of atoms bound to the gold surface of the slide.

1	If a purified receptor is immobilized on the surface of the gold-coated glass slide, to make a biosensor ‘chip,’ and a solution containing the ligand is streamed over that surface, the binding of ligand to the receptor can be followed until it reaches equilibrium (see Fig. A.31). If the ligand is then washed out, dissociation of ligand from the receptor can easily be followed and the The ligand to be tested, in this case peptide:MHC complexes, is immobilized on a special gold-plated surface Soluble T-cell receptors are allowed to ˜ow over the surface and bind to the peptide:MHC complexes Receptor binding reaches equilibrium Unbound receptors are washed away; continued washing removes receptors as they dissociate from the peptide:MHC complexes Ru Time Time Time Ru Ru

1	Fig. A.31 Measurement of receptor–ligand interactions can be made in real time using a biosensor. Biosensors are able to measure the binding of molecules on the surface of gold-plated glass chips through the indirect effects of the binding on the total internal reflection of a beam of polarized light at the surface of the chip. Changes in the angle and intensity of the reflected beam are measured in ‘resonance units’ (Ru) and plotted against time in what is termed a ‘sensorgram.’ Depending on the exact nature of the receptor–ligand pair to be analyzed, either the receptor or the ligand can be immobilized on the surface of the chip. In the example shown, peptide:MHC complexes are immobilized on such a surface (first panel). T-cell receptors in solution are now allowed to flow over the surface, and to bind to the immobilized peptide:MHC complexes (second panel). As the T-cell receptors bind, the sensorgram (inset panel below the main panel) reflects the increasing amount of protein

1	surface, and to bind to the immobilized peptide:MHC complexes (second panel). As the T-cell receptors bind, the sensorgram (inset panel below the main panel) reflects the increasing amount of protein bound. As the binding reaches either saturation or equilibrium (third panel), the sensorgram shows a plateau, as no more protein binds. At this point, unbound receptors can be washed away. With continued washing, bound receptors now start to dissociate and are removed in the flow of the washing solution (last panel). The sensorgram now shows a declining curve, reflecting the rate at which receptor and ligand dissociation occurs.

1	Fig. A.32 Polyclonal mitogens, many of plant origin, stimulate lymphocyte proliferation in tissue culture. Many of these mitogens are used to test the ability of lymphocytes in human peripheral blood to proliferate. Cells are first incubated with a fluorescent dye such as carboxyfluorescein succinimidyl ester (CFSE). This dye enters the cell and, once in the cytosol, becomes covalently coupled to lysine residues on cellular proteins. Each time the cell divides, the amount of CFSE is diluted by one-half, as each daughter cell inherits one-half of the CFSE-labeled proteins. Cell division can then be analyzed by flow cytometry, where a histogram of CFSE fluorescence displays a series of peaks, each of which represents cells that have undergone a fixed number of divisions. Under optimal conditions, this assay is capable of detecting up to 7–8 cell divisions, after which CFSE fluorescence can no longer be measured.

1	dissociation rate calculated. A new solution of the ligand at a different concentration can then be streamed over the chip and the binding once again measured. The affinity of binding can be calculated in a number of ways in this type of assay. Most simply, the ratio of the rates of association and dissociation will give an estimate of the affinity, but more accurate estimates can be obtained from the measurements of the binding at different concentrations of ligand. From measurements of binding at equilibrium, a Scatchard plot will give a measurement of the affinity of the receptor–ligand interaction. A-26 Assays of lymphocyte proliferation.

1	To function in adaptive immunity, rare antigen-specific lymphocytes must proliferate extensively before they differentiate into functional effector cells in order to generate sufficient numbers of effector cells of a particular specificity. Thus, the analysis of induced lymphocyte proliferation is a central issue in their study. It is, however, difficult to detect the proliferation of normal lymphocytes in response to specific antigen because only a minute proportion of cells will be stimulated to divide. Great impetus was given to the field of lymphocyte culture by the finding that certain substances induce many or all lymphocytes of a given type to proliferate. These substances are referred to collectively as polyclonal mitogens because they induce mitosis in lymphocytes of many different specificities or clonal origins. T and B lymphocytes are stimulated by different polyclonal mitogens (Fig. A.32). Polyclonal mitogens seem to trigger essentially the same growth response mechanisms

1	specificities or clonal origins. T and B lymphocytes are stimulated by different polyclonal mitogens (Fig. A.32). Polyclonal mitogens seem to trigger essentially the same growth response mechanisms as antigen. Lymphocytes normally exist as resting cells in the G0 phase of the cell cycle. When stimulated with polyclonal mitogens, they rapidly enter the G1 phase and progress through the cell cycle. In most studies, lymphocyte proliferation is most simply measured by the incorporation of 3H-thymidine into DNA. This assay is used clinically for assessing the ability of lymphocytes from patients with suspected immunodeficiencies to proliferate in response to a nonspecific stimulus.

1	An alternative to the use of a radioisotope to measure lymphocyte proliferation is to use a fluorescent assay that can be performed by FACS. For this assay, the lymphocytes are incubated with a fluorescent dye such as carboxyfluorescein succinimidyl ester (CFSE). This dye enters the cell and, once in the cytosol, becomes covalently coupled to lysine residues on cellular proteins. Each time the cell divides, the amount of CFSE is cut in half, as each daughter cell inherits one-half of the CFSE-labeled proteins. When a population of dividing cells is analyzed by FACS, peaks of CFSE fluorescence can be detected, each of which represents cells that have undergone a fixed number of divisions (Fig. A.33). This assay is capable of detecting up to 7–8 cell divisions, after which CFSE fluorescence can no longer be measured.

1	Culture cells with antigens A or B antigen A no antigen antigen B Measure T-cell proliferation antigen A antigen B no antigen 53 Proliferation Days of culture After 5–10 days, remove cells from lymph node Immunize with antigen A Once lymphocyte culture had been optimized using the proliferative response to polyclonal mitogens as an assay, it became possible to detect antigen-specific T-cell proliferation in culture by measuring 3H-thymidine uptake in response to an antigen to which the T-cell donor had previously been immunized (Fig. A.34). This is the assay most commonly used for assessing T-cell responses after immunization, but it reveals little about the functional capabilities of the responding T cells. These must be ascertained by functional assays, as outlined in Sections A-28 and A-29. A-27 Measurements of apoptosis.

1	A-27 Measurements of apoptosis. Apoptotic cells can be detected by a procedure known as TUNEL (TdTdependent dUTP–biotin nick end labeling) staining. In this technique, the 3ʹends of the DNA fragments generated in apoptotic cells are labeled with biotin-coupled uridine by using the enzyme terminal deoxynucleotidyl transferase (TdT). The biotin label is then detected with enzyme-tagged streptavidin, which binds to biotin. When the colorless substrate of the enzyme is added to a tissue section or cell culture, it produces a colored precipitate only in cells that have undergone apoptosis (Fig. A.35).

1	Additional methods are often used to detect apoptosis of cells in experimental animals. One simple method is to incubate cells with a fluorescently labeled preparation of the protein Annexin V. This protein has a high affinity for a specific membrane phospholipid, phosphatidylserine (PS). In healthy cells, PS is found exclusively on the inner leaflet of the plasma membrane, and is therefore inaccessible to extracellular incubation with Annexin V. When cells are undergoing apoptosis, the PS is transported to the outer cell surface, where it can be bound by the fluorescently labeled Annexin V, which is then detected

1	Fig. A.34 Antigen-specific T-cell proliferation is used frequently as an assay for T-cell responses. T cells from mice or humans that have been immunized with an antigen (A) proliferate when they are exposed to antigen A and antigen-presenting cells but not when cultured with unrelated antigens to which the hosts have not been immunized (antigen B). Proliferation can be measured by incorporation of 3H-thymidine into the DNA of actively dividing cells. Antigen-specific proliferation is a hallmark of specific CD4 T-cell immunity.

1	Fig. A.35 In the TUNEL assay, fragmented DNA is labeled are added (second panel). The biotinylated DNA can be detected by by terminal deoxynucleotidyl transferase (TdT) to reveal using streptavidin, which binds to biotin, coupled to enzymes that apoptotic cells. When cells undergo programmed cell death, or convert a colorless substrate into a colored insoluble product (third apoptosis, their DNA becomes fragmented (first panel). The enzyme panel). Cells stained in this way can be detected by light microscopy, TdT is able to add nucleotides to the ends of DNA fragments; most as shown in the photograph of apoptotic cells (stained red) in the commonly in this assay, biotin-labeled nucleotides (usually dUTP) thymic cortex. Photograph courtesy of R. Budd and J. Russell.

1	by FACS (Fig. A.36). Annexin V staining is often combined with a viability dye such as propidium iodide (PI) or 7-aminoactinomycin D (7-AAD). These two dyes fluoresce when bound to DNA, but are unable to enter viable or apoptotic cells prior to their loss of membrane integrity. Therefore, when combined with Annexin V, cells in the early stages of apoptosis can be identified as Annexin V-positive but PI/7-AAD-negative, whereas cells in the late stages of apoptosis are both Annexin V-positive and PI/7-AAD-positive. An additional assay that provides a sensitive means of detecting apoptotic cells by FACS analysis is based on the detection of activated caspase 3, a cysteine protease that functions in the execution phase of the apoptotic cell death program. Caspase 3 is initially synthesized by cells in an inactive precursor form called a pro-caspase. When cells are undergoing apoptosis, pro-caspase 3 is cleaved into two subunits that dimerize to form the active enzyme. Antibodies have been

1	cells in an inactive precursor form called a pro-caspase. When cells are undergoing apoptosis, pro-caspase 3 is cleaved into two subunits that dimerize to form the active enzyme. Antibodies have been generated that detect the active form of caspase 3, but not pro-caspase 3, and fluorescently coupled versions of these antibodies can be used to detect apoptotic cells that have been fixed and permeabilized (Fig A.37). A-28 Assays for cytotoxic T cells. Activated CD8 T cells generally kill any cells that display the specific pep-tide:MHC class I complex they recognize. Thus, CD8 T-cell function can be determined using the simplest and most rapid T-cell bioassay—the killing of a target cell by a cytotoxic T cell. This is usually detected in a 51Cr-release assay. Live cells will take up, but do not spontaneously release, radioactively labeled sodium chromate, Na2 51CrO4. When these labeled cells are killed, the radio-active chromate is released and its presence in the supernatant of

1	but do not spontaneously release, radioactively labeled sodium chromate, Na2 51CrO4. When these labeled cells are killed, the radio-active chromate is released and its presence in the supernatant of mixtures of target cells and cytotoxic T cells can be measured (Fig. A.38). In a similar assay, proliferating target cells such as tumor cells can be labeled with 3H-thymidine, which is incorporated into the replicating DNA. On attack by a cytotoxic T cell, pro-caspase 3/7 active caspase 3/7 pro-caspase 9 caspase 9 cytochrome c Apoptosis Apaf-1 active Apaf-1 Apoptotic stimuli activate caspase 3/7 Specifc antibody binds only to active caspase 3/7 Fig. A.37 Detection of apoptotic cells by intracellular staining for active caspases. An early event in the apoptotic process is the release of cytochrome c from mitochondria. Cytochrome c binds to Apaf-1, leading to the cleavage of pro-caspase 9 into active caspase 9. Caspase 9 then cleaves pro-caspase 3 and pro-caspase 7 to yield their active

1	c from mitochondria. Cytochrome c binds to Apaf-1, leading to the cleavage of pro-caspase 9 into active caspase 9. Caspase 9 then cleaves pro-caspase 3 and pro-caspase 7 to yield their active forms, ‘executioner’ caspases that promote cell death. Antibodies that recognize the active caspase 3 or caspase 7, but not the pro-caspase forms of these enzymes, will detect permeabilized cells undergoing apoptosis. Fig. A.36 Detection of apoptotic cells with Annexin V. In healthy cells, the membrane phospholipid phosphatidylserine is oriented with its polar headgroup facing the cytosolic face of the plasma membrane. When cells undergo apoptosis, the enzyme responsible for maintaining phosphatidylserine polarity, called flippase, is no longer active. As a result, phosphatidylserine becomes randomly oriented, with many molecules exposing their polar head groups on the extracellular face of the plasma membrane. The protein Annexin V binds tightly to the exposed phosphatidylserine and, if

1	randomly oriented, with many molecules exposing their polar head groups on the extracellular face of the plasma membrane. The protein Annexin V binds tightly to the exposed phosphatidylserine and, if fluorescently labeled, can be used to detect apoptotic cells by FACS. healthy cell In an apoptotic cell phosphatidylserine appears on the outer leafet of the plasma membrane and will bind to Annexin V In a healthy cell phosphatidylserine is confned to the inner leafet of the plasma membrane phosphatidyl-ethanolamine Annexin V phosphatidyl serine phosphatidyl choline apoptotic cell

1	Label target cells with Na2 51CrO4 Killed cells release radioactive chromium Add cytotoxic T cells to labeled target cells Fig. A.38 Cytotoxic T-cell activity is often assessed by chromium release from labeled target cells. Target cells are labeled with radioactive chromium as Na251CrO4, washed to remove excess radioactivity, and exposed to cytotoxic T cells. Cell destruction is measured by the release of radioactive chromium into the medium, detectable within 4 hours of mixing target cells with T cells. the DNA of the target cells is rapidly fragmented and retained in the filtrate, while large, unfragmented DNA is collected on a filter, and one can measure either the release of these fragments or the retention of 3H-thymidine in chromosomal DNA. These assays provide a rapid, sensitive, and specific measure of the activity of cytotoxic T cells.

1	An alternative to these in vitro cytotoxicity assays is to measure target-cell killing by cytotoxic T cells in intact experimental animals. This assay is generally performed with mice that have been infected with a pathogen known to induce a strong cytotoxic T-cell response, such as a virus. Target cells are incubated with the antigenic peptide, which will bind to MHC class I on the target-cell surface. These cells are then incubated with a low concentration of the fluorescent dye CFSE (see Section A-26). A control population of cells that is not given the antigenic peptide is incubated with a high concentration of CFSE, allowing these cells to be distinguished from the antigen-bearing target cells. The two cell populations are mixed 1:1 and injected into the experimental animals. Four hours later, spleen cells are recovered from the animals and analyzed by FACS, allowing the specific target-cell lysis to be calculated from the ratio of the two CFSE-labeled cell populations (Fig.

1	hours later, spleen cells are recovered from the animals and analyzed by FACS, allowing the specific target-cell lysis to be calculated from the ratio of the two CFSE-labeled cell populations (Fig. A.39).

1	Target cells and control cells are labeled with different concentrations of dye, mixed 1:1, and injected into mice

1	Fig. A.39 Assay for cytotoxic T-cell activity using CFSE-labeled target cells. To detect cytotoxic T-cell activity in intact experimental animals, mice that have been infected with a virus are injected with a mixture of target cells labeled with the fluorescent dye CFSE. One group of target cells is pre-incubated with a viral peptide that binds to MHC class I on the target cells; this group of cells is labeled with a low concentration of CFSE. A second group of cells is incubated with a control (nonviral) peptide and is labeled with a high concentration of CFSE. The two groups of cells are mixed together in a 1:1 ratio, and injected into the infected mice. After 4 hours, the mice are sacrificed and the target cells are recovered and analyzed by flow cytometry. Examination of the ratio of the two target-cell populations provides a measure of specific lysis of viral peptide-coated target cells. A-29 Assays for CD4 T cells.

1	A-29 Assays for CD4 T cells. CD4 T-cell functions usually involve the activation rather than the killing of cells bearing specific antigen. The activating effects of CD4 T cells on B cells or macrophages are mediated in large part by cytokines, which are released by the T cell when it recognizes antigen. Thus, CD4 T-cell function is usually studied by measuring the type and amount of cytokine released. Because different effector T cells release different amounts and types of cytokines, one can learn about the effector potential of a T cell by measuring the proteins it produces.

1	Cytokines can be detected by their activity in biological assays of cell growth, where the cytokines serve either as growth factors or as growth inhibitors. A more specific assay is a modification of ELISA known as a capture or sandwich ELISA (see Section A-4). In this assay, the cytokine is characterized by its ability to act as a bridge between two monoclonal antibodies reacting with different epitopes on the cytokine molecule. Cytokine-secreting cells can also be detected by ELISPOT (see Section A-22).

1	Sandwich ELISA and ELISPOT avoid a major problem of cytokine bioassays, namely, the ability of different cytokines to stimulate the same response in a bioassay. Bioassays must always be confirmed by inhibition of the response with neutralizing monoclonal antibodies specific for the cytokine. Another way of identifying cells actively producing a given cytokine is to stain them with a fluorescently tagged anti-cytokine monoclonal antibody, and then identify and count them by FACS (see Section A-23).

1	A quite different approach to detecting cytokine production is to determine the presence and amount of the relevant cytokine mRNA in stimulated T cells. This can be done for single cells by in situ hybridization and for cell populations by the reverse transcriptase–polymerase chain reaction (RT–PCR). Reverse transcriptase is an enzyme used by certain RNA viruses, such as HIV, to convert an RNA genome into a DNA copy, or cDNA. In RT–PCR, mRNA is isolated from cells and cDNA copies are made in vitro using reverse transcriptase. The desired cDNA is then selectively amplified by PCR by using sequence-specific primers. When the products of the reaction are subjected to electrophoresis on an agarose gel, the amplified DNA can be visualized as a band of a specific size. The amount of amplified cDNA sequence will be proportional to its representation in the mRNA; stimulated T cells actively producing a particular cytokine will produce large amounts of that particular mRNA and will thus give

1	cDNA sequence will be proportional to its representation in the mRNA; stimulated T cells actively producing a particular cytokine will produce large amounts of that particular mRNA and will thus give correspondingly large amounts of the selected cDNA on RT–PCR. The level of cytokine mRNA in the original tissue is usually determined by comparison with the outcome of RT–PCR on the mRNA produced by a so-called ‘housekeeping gene’ expressed by all cells.

1	A-30 Transfer of protective immunity.

1	Protective immunity to a pathogen may involve humoral immunity, cell-mediated immunity, or both. For studies in experimental animals such as inbred mice, the nature of protective immunity can be determined by transferring serum or lymphoid cells from an immunized donor animal to an unimmunized syngeneic recipient (that is, a genetically identical animal of the same inbred strain). If protection against infection can be conferred by the transfer of serum, the immunity is provided by circulating antibodies and is called humoral immunity. Transfer of immunity by antiserum or purified antibodies provides immediate protection against many pathogens and against toxins such as those of tetanus and snake venom (Fig. A.40). However, although protection is immediate, it is temporary, lasting only so long as the transferred antibodies remain active in the recipient’s body. This type of transfer is therefore called passive immunization, to distinguish it from active immunization with antigen,

1	so long as the transferred antibodies remain active in the recipient’s body. This type of transfer is therefore called passive immunization, to distinguish it from active immunization with antigen, which can provide lasting immunity. A disadvantage of passive

1	Fig. A.40 In vivo assay for the presence of protective immunity after vaccination in animals. Mice are injected with the test vaccine, such as a heat-killed pathogen, or a control such as saline solution. Different groups are then challenged with lethal or pathogenic doses of the test pathogen or with an unrelated pathogen as a specificity control (not shown). Unimmunized animals die or become severely infected (left panel). Successful vaccination is seen as specific protection of immunized mice against infection with the test pathogen. This is called active immunity, and the process is called active immunization (middle panel). If this immune protection can be transferred to a normal syngeneic recipient with serum from an immune donor, then immunity is mediated by antibodies; such immunity is called humoral immunity and the process is called passive immunization (right panel). If immunity can be transferred only by infusing lymphoid cells from the immune donor into a normal syngeneic

1	is called humoral immunity and the process is called passive immunization (right panel). If immunity can be transferred only by infusing lymphoid cells from the immune donor into a normal syngeneic recipient, then the immunity is called cell-mediated immunity and the transfer process is called adoptive transfer or adoptive immunization (not shown). Passive immunity is short-lived, because antibody is eventually catabolized, but adoptively transferred immunity is mediated by immune cells, which can survive and provide longer-lasting immunity.

1	immunization is that the recipient may become immunized to the antiserum used to transfer immunity. Horse or sheep sera are the usual sources of anti-snake venoms used in humans, and repeated administration can lead either to serum sickness (see Section 14-5) or, if the recipient becomes allergic to the foreign serum, to anaphylaxis (see Section 14-10).

1	Protection against many diseases cannot be transferred with serum but can be transferred by lymphoid cells from immunized donors. The transfer of lymphoid cells from an immune donor to a normal syngeneic recipient is called adoptive transfer or adoptive immunization, and the immunity transferred is called adoptive immunity. Immunity that can be transferred only with lymphoid cells is called cellmediated immunity. Such cell transfers must be between genetically identical donors and recipients, such as members of the same inbred strain of mouse, so that the donor lymphocytes are not rejected by the recipient and do not attack the recipient’s tissues. Adoptive transfer of immunity is used clinically in humans in experimental approaches to cancer therapy or as an adjunct to bone marrow transplantation; in these cases, the patient’s own T cells, or the T cells of the bone marrow donor, are given. A-31 Adoptive transfer of lymphocytes.

1	A-31 Adoptive transfer of lymphocytes. Ionizing radiation from X-ray or gamma-ray sources kills lymphoid and other immune cells at doses that spare the other tissues of the body. This makes it possible to eliminate immune function in a recipient animal before CD45.1 CD45.2 After transfer into CD45.2 recipient, CD45.1 cells can be identifed by ˜ow cytometry Cells isolated from one congenic mouse line express CD45.1

1	CD45.1 CD45.2 After transfer into CD45.2 recipient, CD45.1 cells can be identifed by ˜ow cytometry Cells isolated from one congenic mouse line express CD45.1 Fig. A.41 Adoptive transfer of congenically marked cells. Hematopoietic cells can be transferred between genetically identical (or nearly identical) mice. The transferred cells, usually a minority population in the recipient, are identified based on expression of an allelic variant of an abundant cell-surface receptor. One common receptor used for this purpose is CD45, which has two alleles that can be distinguished by allele-specific antibodies. When cells from a CD45.1+ mouse are transferred into mice of the identical strain (save for their expression of CD45.2), the donor-cell population can easily be identified by antibody staining followed by flow cytometry or immunofluorescence microscopy.

1	attempting to restore immune function by adoptive transfer, and allows the effect of the adoptively transferred cells to be studied in the absence of other lymphoid cells. James Gowans originally used this technique to prove the role of the lymphocyte in immune responses. He showed that all active immune responses could be transferred to irradiated recipients by small lymphocytes from immunized donors.

1	One common use of adoptive transfer assays takes advantage of the availability of T-cell receptor or B-cell receptor transgenic mice (see Section A-34). In this case, the adoptively transferred lymphocytes are a homogeneous population with a fixed antigen specificity. These cells can be transferred into unmanipulated recipient animals of the same inbred strain without the need to deplete the host immune system, and their ability to respond to immunization or challenge by infection can be monitored. One advantage of this experimental strategy is that relatively small numbers of antigen-specific T cells or B cells can be transferred; after dilution by the recipient’s lymphocyte population, the responses of these cells can be examined in the environment of a normal immune response carried out by the host’s immune system. Commonly, the transferred cells are ‘marked’ with an allelic variant of an abundant cell-surface receptor, such as CD45 (Fig. A.41). When the donor lymphocytes express

1	by the host’s immune system. Commonly, the transferred cells are ‘marked’ with an allelic variant of an abundant cell-surface receptor, such as CD45 (Fig. A.41). When the donor lymphocytes express one allelic variant of CD45 and the recipient cells express a different variant, the transferred cells can easily be distinguished from the host cells by staining with an antibody that binds to one variant of CD45, but not the other. When two strains of mice are genetically identical with the exception of a single gene, they are said to be congenic. In the example above, the donor strain and the recipient strain are referred to as ‘CD45 congenics’; it should be noted, however, that in the case where one strain is a T-cell receptor or B-cell receptor transgenic line, this terminology is not completely accurate, as the presence of the transgenic DNA as a genetic difference is conveniently ignored. Such adoptive transfer studies are a cornerstone in the study of the intact immune system. They

1	accurate, as the presence of the transgenic DNA as a genetic difference is conveniently ignored. Such adoptive transfer studies are a cornerstone in the study of the intact immune system. They have provided a rapid and convenient means of determining the effects of many gene deficiencies, such as those in cell-surface receptors, transcription factors, cytokines, and cell survival/cell death genes, on the ability of T cells or B cells to mount protective immune responses.

1	A-32 Hematopoietic stem-cell transfers.

1	All cells of hematopoietic origin can be eliminated by treatment with high doses of γ radiation or X rays, allowing replacement of the entire hematopoietic system, including lymphocytes, by transfusion of donor bone marrow or purified hematopoietic stem cells from another animal. The resulting animals are called radiation bone marrow chimeras, from the Greek word chimera, a mythical animal that had the head of a lion, the tail of a serpent, and the body of a goat. This technique is used experimentally to examine the development of immune-cell lineages, as opposed to their effector functions, and has been particularly important in studying T-cell development. Essentially the same technique is used in humans to replace the hematopoietic system when it fails, as in aplastic anemia or after nuclear accidents, or to eradicate the bone marrow and replace it with normal marrow in the treatment of certain cancers. In humans, bone marrow is the main source of hematopoietic stem cells, but they

1	accidents, or to eradicate the bone marrow and replace it with normal marrow in the treatment of certain cancers. In humans, bone marrow is the main source of hematopoietic stem cells, but they are increasingly being obtained from peripheral blood after the donor has been treated with hematopoietic growth factors such as GM-CSF, or from umbilical cord blood, which is rich in such stem cells (see Chapter 15).

1	A-33 In vivo administration of antibodies. Antibodies administered to intact experimental animals, or to humans, provide a potent means of manipulating the immune system. Depending on the target molecule recognized by the antibody, and the intrinsic properties of each antibody, in vivo antibody administration can either inhibit the function of the target molecule or, in some cases, lead to the elimination of a cell population that expresses the target molecule.

1	In animal models, antibodies targeting an individual cytokine have been used to inhibit that cytokine’s function during an otherwise intact immune response. Experiments of this type provided some of the first evidence that the cytokine IL-12 provides an important signal in polarizing CD4+ T cells into the TH1 lineage following infection with an intracellular protozoan. This approach has also been used with great success in humans. One of the most common treatments for the inflammatory autoimmune disease rheumatoid arthritis (see Chapter 16) is the administration of an antibody that binds to the cytokine TNF-α; in this case, the inhibition of TNF-α activity provides patients with relief from the symptoms of joint inflammation. The great utility of this antibody therapy has led to the development of related strategies for inhibiting cytokine actions in vivo. One successful approach has been to create a hybrid protein that has the ligand-binding domain of the cytokine’s receptor fused to

1	of related strategies for inhibiting cytokine actions in vivo. One successful approach has been to create a hybrid protein that has the ligand-binding domain of the cytokine’s receptor fused to the constant-region domains (Fc) of an antibody heavy chain (Fig. A.42). This Fc-fusion protein acquires the stability and long half-life of an antibody, but has the binding properties of the cytokine receptor. When given in vivo, the Fc-fusion protein binds to the cytokine, thereby interfering with the cytokine’s ability to stimulate its receptor on immune cells. As an example, the Fc-fusion protein containing the TNF-receptor ligand-binding domain has also been used as an effective treatment for patients with rheumatoid arthritis.

1	Antibody administration can also be used to augment the immune response by interfering with T-cell surface receptors, such as CTLA-4 or PD-1. When engaged by their ligands, these receptors normally function to downregulate the immune response. From experiments in mice, antibodies that bind to and inhibit these receptors have been found to enhance immune responses to tumors, leading in some cases to tumor eradication. Currently, these strategies are being tested in humans for a variety of tumor types, and the initial results have shown great promise.

1	In vivo administration of antibodies can also be used to deplete specific cell populations. The efficiency with which a given antibody functions for in vivo depletion is quite variable, as the mechanism relies on a process known as antibodydependent cellmediated cytotoxicity (ADCC) (see Section 10-23 and Fig. 10.36). When a cell is coated with antibodies, it becomes a target of natural killer (NK) cells that express the Fc receptor known as CD16 or FcγRIII. The cross-linking of FcγRIII induces the NK cell to kill the antibody-coated target cell. While FcγRIII is a receptor for IgG, it does not bind with equal affinity to all IgG subtypes; thus the efficiency of ADCC after administration of a given antibody is determined by its ability to cross-link FcγRIII and induce NK cell killing. Common uses for this technique include the depletion of CD4+ T cells with an antibody to CD4, or the depletion of CD8+ T cells with an antibody to CD8. In human patients undergoing organ transplantation,

1	uses for this technique include the depletion of CD4+ T cells with an antibody to CD4, or the depletion of CD8+ T cells with an antibody to CD8. In human patients undergoing organ transplantation, T cells are transiently depleted by administering an antibody to the CD3 component of the T-cell receptor complex. This produces a severe, but temporary, state of immunosuppression during the early stages of post-transplantation. As with all in vivo antibody depletion regimens, the depleted cell population gradually returns as cells of that subset are replenished by ongoing lymphocyte development.

1	Fig. A.42 In vivo administration of antibodies is an effective therapeutic. The cytokine TNF-α contributes to chronic inflammation in a range of conditions, including rheumatoid arthritis, by binding to and triggering signaling of the TNF receptor (TNFR). To treat these conditions, a fusion protein consisting of the constant-region domains of human IgG1 is fused to the extracellular portion of the TNFR, creating a therapeutic known as etanercept. When administered to patients, this fusion protein is effective at binding TNF-α and preventing it from triggering TNFR signaling, thereby reducing inflammation.

1	Fig. A.43 The function and expression of genes can be studied in vivo by using transgenic mice. DNA encoding a protein of interest, here the mouse MHC class II protein Eα, is purified and microinjected into the male pronuclei of fertilized eggs. The eggs are then implanted into pseudopregnant female mice. The resulting offspring are screened for the presence of the transgene in their cells, and positive mice are used as founders that transmit the transgene to their offspring, establishing a line of transgenic mice that carry one or more extra genes. The function of the Eα gene used here is tested by breeding the transgene into C57BL/6 mice that carry an inactivating mutation in their endogenous Eα gene. A-34 Transgenic mice.

1	A-34 Transgenic mice. The function of genes has traditionally been studied by observing the effects of spontaneous mutations in whole organisms and, more recently, by analyzing the effects of targeted mutations in cultured cells. The advent of gene cloning and in vitro mutagenesis now makes it possible to produce specific mutations in whole animals. Mice with extra copies or altered copies of a gene in their genome can be generated by transgenesis, which is now a well-established procedure. To produce transgenic mice, a cloned gene is introduced into the mouse genome by microinjection into the male pronucleus of a fertilized egg, which is then implanted into the uterus of a pseudopregnant female mouse. In some of the eggs, the injected DNA becomes integrated randomly into the genome, giving rise to a mouse that has an extra genetic element of known structure, the transgene (Fig. A.43).

1	This technique allows one to study the impact of a newly discovered gene on development, to identify the regulatory regions of a gene required for its normal tissue-specific expression, to determine the effects of its overexpression or its expression in inappropriate tissues, and to find out the impact of mutations on gene function. Transgenic mice have been particularly useful in studying the role of T-cell and B-cell receptors in lymphocyte development, as described in Chapter 8, and in providing a source of primary T and B lymphocytes of known antigen specificity for adoptive transfer studies (see Section A-31). This utility is largely due to the fact that expression of the transgene-encoded T-cell and B-cell receptors preempts the rearrangement and expression of the endogenous antigen receptor genes during T-cell and B-cell development, respectively, thereby generating homogeneous populations of cells bearing a unique antigen receptor of known specificity.

1	A-35 Gene knockout by targeted disruption. In many cases, the functions of a particular gene can be fully understood only if a mutant animal that does not express the gene can be obtained. Whereas genes used to be discovered through the identification of mutant phenotypes, it is now far more common to discover and isolate the normal gene and then determine its function by replacing it in vivo with a defective copy. This procedure is known as gene knockout, and it has been made possible by two developments: a powerful strategy to select for targeted mutation by homologous recombination, and the development of continuously growing lines of embryonic stem cells (ES cells). These are embryonic cells that, on implantation into a blastocyst, can give rise to all cell lineages in a chimeric mouse.

1	The technique of gene targeting takes advantage of the phenomenon known as homologous recombination (Fig. A.44). Cloned copies of the target gene are altered to make them nonfunctional and are then introduced into the ES cell, where they recombine with the homologous gene in the cell’s genome, replacing the normal gene with a nonfunctional copy. Homologous recombination is a rare event in mammalian cells, and thus a powerful selection strategy is required to detect those cells in which it has occurred. Most commonly, the introduced gene construct has its sequence disrupted by an inserted antibiotic-resistance gene such as that for neomycin resistance (neor). If this construct undergoes homologous recombination with the endogenous copy of the gene, the endogenous gene is disrupted but the antibiotic-resistance gene remains functional, allowing cells that have incorporated the gene to be selected in culture for resistance to the neomycin-like drug G418. However, antibiotic resistance on

1	gene remains functional, allowing cells that have incorporated the gene to be selected in culture for resistance to the neomycin-like drug G418. However, antibiotic resistance on its own shows only that the cells have taken up and integrated the neomycin-resistance gene. To be able to select for those cells in which homologous recombination has occurred, the ends of the construct usually carry the thymidine kinase gene from the herpes simplex virus (HSV-tk). Cells that incorporate DNA randomly usually retain the entire DNA construct including HSV-tk, whereas homologous recombination between the construct and cellular DNA, the desired result, involves the exchange of homologous DNA sequences so that the nonhomologous HSV-tk genes at the ends of the construct are eliminated. Cells carrying HSV-tk are killed by the antiviral drug ganciclovir, and so cells with homologous recombinations have the unique feature of being resistant to both neomycin and ganciclovir, allowing them to be

1	HSV-tk are killed by the antiviral drug ganciclovir, and so cells with homologous recombinations have the unique feature of being resistant to both neomycin and ganciclovir, allowing them to be selected efficiently when these drugs are added to the cultures (see Fig. A.44).

1	DNA is introduced into cell Target gene interrupted by insertion of neomycin-resistance gene (neor) neorDNA fails to integrate Cell is killed by ganciclovir DNA integrates at random site on genome. Cell expresses both neorand HSV-tk Cell expresses neomycin resistance but not HSV-tk, so is not killed by either G418 or ganciclovir Homologous recombination replaces wild-type ˜2 -microglobulin gene with interrupted copy Cell is killed by G418 (a neomycin analog)

1	To knock out a gene in vivo, it is necessary only to disrupt one copy of the cellular gene in an ES cell. These ES cells are then injected into a blastocyst, which is reimplanted into the uterus. The cells carrying the disrupted gene become incorporated into the developing embryo and contribute to all tissues of the resulting chimeric offspring, including those of the germline. The mutated gene can therefore be transmitted to some of the offspring of the original chimera, and further breeding to homozygosity of the mutant gene produces mice that completely lack the expression of that particular gene product (Fig. A.45). The effects of the absence of the gene’s function can then be

1	Fig. A.44 The deletion of specific genes can be accomplished by homologous recombination. When pieces of DNA are introduced into cells, they can integrate into cellular DNA in two different ways. If they randomly insert into sites of DNA breaks, the whole piece is usually integrated, often in several copies. However, extrachromosomal DNA can also undergo homologous recombination with the cellular copy of the gene, in which case only the central, homologous region is incorporated into cellular DNA. Inserting a selectable marker gene such as resistance to neomycin (neor) into the coding region of a gene does not prevent homologous recombination, and it achieves two goals. First, any cell that has integrated the injected DNA is protected from the neomycin-like antibiotic G418. Second, when the gene recombines with homologous cellular DNA, the neor gene disrupts the coding sequence of the modified cellular gene. Homologous recombinants can be discriminated from random insertions if the

1	the gene recombines with homologous cellular DNA, the neor gene disrupts the coding sequence of the modified cellular gene. Homologous recombinants can be discriminated from random insertions if the gene encoding herpes simplex virus thymidine kinase (HSV-tk) is placed at one or both ends of the DNA construct, which is often known as a ‘targeting construct’ because it targets the cellular gene. In random DNA integrations, HSV-tk is retained. HSV-tk renders the cell sensitive to the antiviral agent ganciclovir. However, as HSV-tk is not homologous to the target DNA, it is lost from homologous recombinants. Thus, cells that have undergone homologous recombination are uniquely resistant to both G418 and ganciclovir, and survive in a mixture of the two antibiotics. The presence of the disrupted gene has to be confirmed by Southern blotting or by PCR using primers in the neor gene and in cellular DNA lying outside the region used in the targeting construct. By using two different

1	disrupted gene has to be confirmed by Southern blotting or by PCR using primers in the neor gene and in cellular DNA lying outside the region used in the targeting construct. By using two different resistance genes, one can disrupt the two cellular copies of a gene, making a deletion mutant (not shown).

1	Fig. A.45 Gene knockout in embryonic stem cells enables mutant mice to be produced. Specific genes can be inactivated by homologous recombination in cultures of embryonic stem cells (ES cells). Homologous recombination is performed as described in Fig. A.44. In this example, the gene encoding β2-microglobulin in ES cells is disrupted by homologous recombination with a targeting construct. Only a single copy of the gene needs to be disrupted. ES cells in which homologous recombination has taken place are injected into mouse blastocysts. If the mutant ES cells give rise to germ cells in the resulting chimeric mice (striped in the figure), the mutant gene can be transferred to their offspring. By breeding the mutant gene to homozygosity, offspring can be tested to determine if a mutant phenotype is generated. In this case, the homozygous mutant mice lack MHC class I molecules on their cells, because MHC class I molecules have to pair with β2-microglobulin for surface expression. The

1	is generated. In this case, the homozygous mutant mice lack MHC class I molecules on their cells, because MHC class I molecules have to pair with β2-microglobulin for surface expression. The β2-microglobulin-deficient mice can then be bred with mice transgenic for subtler mutants of the deleted gene, allowing the effect of such mutants to be tested in vivo.

1	studied. In addition, the parts of the gene that are essential for its function can be identified by determining whether function can be restored by introducing different mutated copies of the gene back into the genome by transgenesis. The manipulation of the mouse genome by gene knockout and transgenesis has revolutionized our understanding of the role of individual genes in lymphocyte development and function. Because the most commonly used ES cells are derived from a poorly characterized strain of mice known as strain 129, the analysis of the function of a gene knockout often requires extensive back-crossing to another strain. One can track the presence of the mutant copy of the gene by the presence of the neor gene. After sufficient back-crossing, the mice are intercrossed to produce mutants on a stable genetic background.

1	A problem with gene knockouts arises when the function of the gene is essential for the survival of the animal; in such cases the gene is termed a recessive lethal gene, and homozygous animals cannot be produced. To study the function of such a gene, tissue-specific or developmentally regulated gene deletion can be employed. This strategy makes use of the DNA sequences and enzymes used by bacteriophage P1 to excise itself from a host cell’s genome. Integrated bacteriophage P1 DNA is flanked by recombination signal sequences called loxP sites. A recombinase, Cre, recognizes these sites, cuts the DNA, and joins the two ends, thus excising the intervening DNA in the form of a circle. This mechanism can be adapted to allow the deletion of specific genes in a transgenic animal only in certain tissues or at certain times in development. First, loxP sites flanking a gene, or perhaps flanking just a single exon, are introduced by homologous recombination (Fig. A.46). Usually, the introduction

1	or at certain times in development. First, loxP sites flanking a gene, or perhaps flanking just a single exon, are introduced by homologous recombination (Fig. A.46). Usually, the introduction of these sequences into flanking or intronic DNA does not disrupt the normal function of the gene. Next, mice containing such loxP mutant genes are mated with mice made transgenic for Cre recombinase that has been placed under the control of a tissue-specific or inducible promoter. When the Cre recombinase is active, either in the appropriate tissue or when induced, it excises the DNA between the inserted loxP sites, thus inactivating the gene or exon. Thus, for example, using a T-cell-specific promoter to drive expression of the Cre recombinase, a gene can be deleted only in T cells while remaining functional in all other cells of the animal. This extremely powerful genetic technique was used to demonstrate the importance of B-cell receptors in B-cell survival.

1	Recently, a new technology has been developed for inducing specific gene disruptions in mice; it is known as the CRISPR/Cas9 system. This technique is adapted from a bacterial system that uses an RNA-based strategy to generate double-stranded DNA breaks in the genomes of invading pathogens or plasmids, a form of bacterial immunity. The Cas9 gene encodes an endonuclease; this has been modified for use in eukaryotic cells by incorporating a nuclear localization signal into the protein-coding sequence of the enzyme. To target mutations to a particular gene, a synthetic guide RNA is produced that incorporates a short sequence (~20 nucleotides) homologous to the gene Animals containing the loxP sites are made transgenic for the Cre protein, expressed from a tissue-specifc promoter only in certain cells, e.g., lymphocytes When expressed, the Cre protein excises the DNA between the loxP sites, deleting the gene only in specifc cell types

1	Fig. A.46 The P1 bacteriophage recombination system times during development (center panel). In the cells in which the Cre can be used to eliminate genes in particular cell lineages. protein is expressed, it recognizes the loxP sequences and excises The P1 bacteriophage protein Cre excises DNA that is bounded the DNA lying between them (right panel). Thus, individual genes can by recombination signal sequences called loxP sequences. be deleted only in certain cell types or only at certain times. In this These sequences can be introduced at either end of a gene by way, genes that are essential for the normal development of a mouse homologous recombination (left panel). Animals carrying genes can be analyzed for their function in the developed animal and/or in flanked by loxP can also be made transgenic for the gene encoding specific cell types. Genes are shown as boxes, RNA as squiggles, the Cre protein, which is placed under the control of a tissue-specific and proteins as colored

1	made transgenic for the gene encoding specific cell types. Genes are shown as boxes, RNA as squiggles, the Cre protein, which is placed under the control of a tissue-specific and proteins as colored balls. promoter so that it is expressed only in certain cells or only at certain being targeted along with sequences that bind the Cas9 enzyme. The guide RNA recruits Cas9 to the genomic location, where the endonuclease will produce a double-stranded DNA break (Fig. A.47). When this break is repaired

1	Fig. A.47 Genetic engineering using the bacterial CRISPR/ Cas9 system. Genetic engineering can be targeted to a specific gene locus in cells by using two components, the bacterial Cas9 enzyme and a guide RNA (left panel). The guide RNA is a single-stranded RNA that contains two regions of sequence in tandem, the first with homology to the gene being targeted and a second recognized by the Cas9 enzyme. The guide RNA targets the enzyme to the homologous genomic region, promoting double-stranded DNA cleavage by the Cas9 endonuclease 3–4 nucleotides upstream of the protospacer adjacent motif (PAM) sequence (right panel). The PAM sequence required by the Cas9 endonuclease is the dinucleotide GG (CC on the other strand). When the double-stranded DNA break is repaired by the nonhomologous end-joining pathway, small deletions and/or point mutations are introduced into the target gene, often leading to loss of gene function. To induce a specific sequence replacement in the target gene, cells

1	pathway, small deletions and/or point mutations are introduced into the target gene, often leading to loss of gene function. To induce a specific sequence replacement in the target gene, cells are provided with a template DNA, in addition to Cas9 and the guide RNA. This template is a double-stranded DNA sequence homologous to the target gene, but containing specific nucleotide changes. In the presence of this template, cells will repair the Cas9-mediated double-stranded cleavage using homologous recombination, rather than nonhomologous end-joining, thereby replacing the original sequence with the sequence provided in the template DNA.

1	by the nonhomologous end-joining DNA repair pathway, small insertions or deletions are commonly introduced, leading to a disruption of the original sequence.

1	This powerful technique can be used to generate homozygous gene deficiencies in cultured cells and cell lines, but importantly, it can also be used as a single-step means of generating homozygous mutant mice. For this latter purpose, RNA molecules encoding Cas9 are mixed with guide RNAs and injected into single-cell mouse zygotes, using the same technique as that used to generate transgenic mouse lines (see Fig. A.43). Due to the efficiency of the CRISPR/Cas9 system, these embryos frequently harbor mutations on both alleles of the targeted gene. Thus, after transplantation of the embryos into foster mothers, the pups born from these embryos are already homozygous for the targeted gene, without the need for lengthy mouse breeding. A refinement of this technique has been developed that allows specific nucleotide changes to be introduced into the targeted gene, rather than the random changes resulting from nonhomologous end-joining. This is accomplished by introducing a DNA

1	that allows specific nucleotide changes to be introduced into the targeted gene, rather than the random changes resulting from nonhomologous end-joining. This is accomplished by introducing a DNA oligonucleotide into the fertilized mouse zygotes along with the Cas9 and guide RNAs. The oligonucleotide contains the desired nucleotide changes flanked by sequences homologous to the targeted gene. When this oligonucleotide is present, the double-strand DNA break introduced by Cas9 is preferentially repaired by a homology-directed process that replaces the damaged DNA with the sequences from the oligonucleotide (see Fig. A.47).

1	A-36 Knockdown of gene expression by RNA interference (RNAi).

1	In some cases, the function of a gene can be assessed by reducing, or even eliminating, the expression of that gene in specific cells. This can be accomplished by harnessing a system known as RNA interference, or RNAi, that is present in many eukaryotic cell types. When small double-stranded RNA molecules (referred to as small interfering RNAs, or siRNAs) are introduced into cells, the two RNA strands will be separated and one will bind to an enzyme complex known as RISC (RNA-induced silencing complex). The bound siRNA targets the RISC complex to the mRNA to which it has homology, leading either to translation arrest or to degradation of the mRNA, and thus to silencing of the gene (Fig. A.48). For cells that are not easily transfected with siRNA molecules directly, such as primary lymphocytes and myeloid cells, gene silencing can be implemented by using recombinant viruses. In this case, genes encoding small hairpin RNAs (shRNAs) are introduced into viral vectors that can be packaged

1	and myeloid cells, gene silencing can be implemented by using recombinant viruses. In this case, genes encoding small hairpin RNAs (shRNAs) are introduced into viral vectors that can be packaged into infectious viral particles. The shRNAs encode small RNAs that form a double-stranded hairpin structure; these hairpins are processed by enzymes in the cell to generate the siRNAs needed for gene silencing (see Fig. A.48). Since many primary hematopoietic cell types are readily transduced with recombinant viruses, such as retroviruses and lentiviruses, shRNAs can be effectively used to silence genes in these cell types.

1	Fig. A.48 Knockdown of gene expression using the RNAi pathway. Small double-stranded RNA molecules with homology to an mRNA transcript will target the mRNA for degradation or translation arrest. This pathway is initiated by expression of a short hairpin RNA (shRNA), which can be produced from an expression vector that is introduced into cells, or by the direct transfection of cells with small double-stranded RNA molecules called siRNA. shRNA molecules are processed by the enzyme Dicer to generate siRNA duplexes. The siRNA duplexes bind to the RISC complex, which separates the two RNA strands, retaining the noncoding strand of the siRNA. This noncoding strand targets the siRNA–RISC complex to the mRNA, leading to mRNA degradation or translation termination. Compiled by Daniel DiToro, Carson Moseley, and Jeff Singer, University of Alabama at Birmingham. Data based on CD designations made at the 9th Workshop on Human Leukocyte Differentiation Antigens.

1	Compiled by Daniel DiToro, Carson Moseley, and Jeff Singer, University of Alabama at Birmingham. Data based on CD designations made at the 9th Workshop on Human Leukocyte Differentiation Antigens. * May function as dimers Compiled by Robert Schreiber, Washington University School of Medicine, St Louis, and Daniel DiToro, Carson Moseley, and Jeff Singer, University of Alabama at Birmingham. Chromosome locations are for humans. Chemokines for which there is no human homolog are listed with the mouse chromosome. † ELR refers to the three amino acids that precede the first cysteine residue of the CXC motif. If these amino acids are Glu-Leu-Arg (i.e. ELR+), then the chemokine is chemotactic for neutrophils; if they are not (ELR–) then the chemokine is chemotactic for lymphocytes Compiled by Joost Oppenheim, National Cancer Institute, NIH. Emil von Behring (1854–1917) discovered antitoxin antibodies with Shibasaburo Kitasato.

1	Compiled by Joost Oppenheim, National Cancer Institute, NIH. Emil von Behring (1854–1917) discovered antitoxin antibodies with Shibasaburo Kitasato. Baruj Benacerraf (1920–2011) discovered immune response genes and collaborated in the first demonstration of MHC restriction. Bruce Beutler (1957–) discovered the role of the Toll-like receptor in innate immunity in mice. Jules Bordet (1870–1961) discovered complement as a heat-labile component in normal serum that would enhance the antimicrobial potency of specific antibodies. Ogden C. Bruton (1908–2003) documented the first description of an immunodeficiency disease describing the failure of a male child to produce antibody. Because inheritance of this condition is X-linked and is characterized by the absence of immunoglobulin in the serum (agammaglobulinemia), it was called Bruton’s X-linked agammaglobulinemia.

1	Frank MacFarlane Burnet (1899–1985) proposed the first generally accepted clonal selection hypothesis of adaptive immunity. Robin Coombs (1921–2006) first developed anti-immunoglobulin antibodies to detect the antibodies that cause hemolytic disease of the newborn. The test for this disease is still called the Coombs test. Jean Dausset (1916–2009) was an early pioneer in the study of the human major histocompatibility complex or HLA. Peter Doherty (1940–) and Rolf Zinkernagel (1944–) showed that antigen recognition by T cells is MHC-restricted, thereby establishing the biological role of the proteins encoded by the major histocompatibility complex and leading to an understanding of antigen processing and its importance in the recognition of antigen by T cells. Gerald Edelman (1929–2014) made crucial discoveries about the structure of immunoglobulins, including the first complete sequence of an antibody molecule.

1	Gerald Edelman (1929–2014) made crucial discoveries about the structure of immunoglobulins, including the first complete sequence of an antibody molecule. Paul Ehrlich (1854–1915) was an early champion of humoral theories of immunity, and proposed a famous side-chain theory of antibody formation that bears a striking resemblance to current thinking about surface receptors. James Gowans (1924–) discovered that adaptive immunity is mediated by lymphocytes, focusing the attention of immunologists on these small cells. Jules Hoffman (1941–) discovered the role of the Toll-like receptor in innate immunity in Drosophila melanogaster. Michael Heidelberger (1888–1991) developed the quantitative precipitin assay, ushering in the era of quantitative immunochemistry.

1	Michael Heidelberger (1888–1991) developed the quantitative precipitin assay, ushering in the era of quantitative immunochemistry. Charles A. Janeway, Jr. (1945–2003) recognized the importance of co-stimulation for initiating adaptive immune responses. He predicted the existence of receptors of the innate immune system that would recognize pathogen-associated molecular patterns and would signal activation of the adaptive immune system. His laboratory discovered the first mammalian Toll-like receptor that had this function. He was also the principal original author of this textbook. Edward Jenner (1749–1823) described the successful protection of humans against smallpox infection by vaccination with cowpox or vaccinia virus. This founded the field of immunology.

1	Edward Jenner (1749–1823) described the successful protection of humans against smallpox infection by vaccination with cowpox or vaccinia virus. This founded the field of immunology. Niels Jerne (1911–1994) developed the hemolytic plaque assay and several important immunological theories, including an early version of clonal selection, a prediction that lymphocyte receptors would be inherently biased to MHC recognition, and the idiotype network. Shibasaburo Kitasato (1852–1931) discovered antibodies in collaboration with Emil von Behring. Robert Koch (1843–1910) defined the criteria needed to characterize an infectious disease, known as Koch’s postulates. Georges Kler (1946–1995) pioneered monoclonal antibody production from hybrid antibody-forming cells with César Milstein. Karl Landsteiner (1868–1943) discovered the ABO blood group antigens. He also carried out detailed studies of the specificity of antibody binding using haptens as model antigens.

1	Karl Landsteiner (1868–1943) discovered the ABO blood group antigens. He also carried out detailed studies of the specificity of antibody binding using haptens as model antigens. Peter Medawar (1915–1987) used skin grafts to show that tolerance is an acquired characteristic of lymphoid cells, a key feature of clonal selection theory. Èlie Metchnikoff (1845–1916) was the first champion of cellular immunology, focusing his studies on the central role of phagocytes in host defense. César Milstein (1927–2002) pioneered monoclonal antibody production with Georges Köhler. Ray Owen (1915–2014) discovered that genetically different twin calves with a common placenta, thus sharing placental blood circulation, were immunologically tolerant to one another’s tissues. Louis Pasteur (1822–1895) was a French microbiologist and immunologist who validated the concept of immunization first studied by Jenner. He prepared vaccines against chicken cholera and rabies.

1	Louis Pasteur (1822–1895) was a French microbiologist and immunologist who validated the concept of immunization first studied by Jenner. He prepared vaccines against chicken cholera and rabies. Rodney Porter (1917–1985) worked out the polypeptide structure of the antibody molecule, laying the groundwork for its analysis by protein sequencing. Ignác Semmelweis (1818–1865) German-Hungarian physician who first determined a connection between hospital hygiene and an infectious disease, puerperal fever, and consequently introduced antisepsis into medical practice. George Snell (1903–1996) worked out the genetics of the murine major histocompatibility complex and generated the congenic strains needed for its biological analysis, laying the groundwork for our current understanding of the role of the MHC in T-cell biology.

1	Tomio Tada (1934–2010) first formulated the concept of the regulation of the immune response by ‘suppressor T cells’ in the 1970s, from indirect experimental evidence. The existence of such cells could not be verified at the time and the concept became discredited, but Tada was vindicated when researchers in the 1980s identified the cells now called ‘regulatory T cells.’ Susumu Tonegawa (1939–) discovered the somatic recombination of immunological receptor genes that underlies the generation of diversity in human and murine antibodies and T-cell receptors. Jg Tschopp (1951-2011) contributed to the delineation of the complement system and T-cell cytolytic mechanisms, and made seminal contributions to the fields of apoptosis and innate immunity, in particular by discovering the inflammasome. Don C. Wiley (1944–2001) solved the first crystal structure of an MHC I protein, providing a startling insight into how T cells recognize their antigen in the in the context of MHC molecules.

1	Don C. Wiley (1944–2001) solved the first crystal structure of an MHC I protein, providing a startling insight into how T cells recognize their antigen in the in the context of MHC molecules. Fig. 1.1 reproduced courtesy of Yale University, Harvey Cushing/John Hay Whitney Medical Library. Fig. 1.4 second panel from Tilney, L.G., Portnoy, D.A.: Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. J. Cell. Biol. 1989, 109:1597–1608. With permission from Rockefeller University Press. Fig. 1.24 photographs from Mowat, A., Viney, J.: The anatomical basis of intestinal immunity. Immunol. Rev. 1997, 156:145–166. Fig. 1.34 photographs from Kaplan, G., et al.: Efficacy of a cell-mediated reaction to the purified protein derivative of tuberculin in the disposal of Mycobacterium leprae from human skin. PNAS 1988, 85:5210–5214.

1	Chapter 2 Fig. 2.7 top panel from Button, B., et al.: A periciliary brush promotes the lung health by separating the mucus layer from airway epithelia. Science 2012, 337:937–941. With permission from AAAS. Fig. 2.12 micrograph adapted from Mukherjee, S., et al.: Antibacterial membrane attack by a pore-forming intestinal C-type lectin. Nature 2014, 505:103–107. Fig. 2.35 photographs reproduced with permission from Bhakdi, S., et al.: Functions and relevance of the terminal complement sequence. Blut 1990, 60:309–318. © 1990 Springer-Verlag.

1	Functions and relevance of the terminal complement sequence. Blut 1990, 60:309–318. © 1990 Springer-Verlag. Chapter 3 Fig. 3.12 structure reprinted with permission from Jin, M.S., et al.: Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a triacylated lipopeptide. Cell 2007, 130:1071–82. © 2007 with permission from Elsevier. Fig. 3.13 structure reprinted with permission from Macmillan Publishers Ltd. Park, B.S., et al.: The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature 2009, 458:1191–1195. Fig. 3.34 model structure reprinted with permission from Macmillan Publishers Ltd. Emsley, J., et al.: Structure of pentameric human serum amyloid P component. Nature 1994, 367: 338–345.

1	Chapter 4 Fig. 4.5 photograph from Green, N.M.: Electron microscopy of the immunoglobulins. Adv. Immunol. 1969, 11:1–30. © 1969 with permission from Elsevier. Fig. 4.15 and Fig. 4.24 model structures from Garcia, K.C., et al.: An αβ T cell receptor structure at 2.5 Å and its orientation in the TCR-MHC complex. Science 1996, 274:209–219. Reprinted with permission from AAAS.

1	Fig. 6.6 reprinted with permission from Macmillan Publishers Ltd. Whitby, F.G., et al.: Structural basis for the activation of 20S proteasomes by 11S regulators. Nature 2000, 408:115–120. Fig. 6.7 bottom panel from Velarde, G., et al.: Three-dimensional structure of transporter associated with antigen processing (TAP) obtained by single particle image analysis. J. Biol. Chem. 2001 276:46054–46063. © 2001 ASBMB. Fig. 6.22 structures from Mitaksov, V.E., & Fremont, D.: Structural definition of the H-2Kd peptide-binding motif. J. Biol. Chem. 2006, 281:10618–10625. © 2006 American Society of Biochemistry and Molecular Biology. Fig. 6.25 molecular model reprinted with permission from Macmillan Publishers Ltd. Fields, B.A., et al.: Crystal structure of a T-cell receptor β-chain complexed with a superantigen. Nature 1996, 384:188–192.

1	Fig. 8.19 photographs reprinted with permission from Macmillan Publishers Ltd. Surh, C. D., Sprent, J.: T-cell apoptosis detected in situ during positive and negative selection in the thymus. Nature 1994, 372:100–103. Fig. 9.12 ﬂuorescent micrographs reprinted with permission from Macmillan Publishers Ltd. Pierre, P., Turley, S.J., et al.: Development regulation of MHC class II transport in mouse dendritic cells. Nature 1997, 388:787–792. Fig. 9.38 panel c from Henkart, P.A., & Martz, E. (eds): Second International Workshop on Cell Mediated Cytotoxicity. © 1985 Kluwer/Plenum Publishers. With kind permission of Springer Science and Business Media.

1	Chapter 10 Fig. 10.17 left panel from Szakal, A.K., et al.: Isolated follicular dendritic cells: cytochemical antigen localization, Nomarski, SEM, and TEM morphology. J. Immunol. 1985, 134:1349–1359. © 1985 The American Association of Immunologists. Fig. 10.17 center and right panels from Szakal, A.K., et al.: Microanatomy of lymphoid tissue during humoral immune responses: structure function relationships. Ann. Rev. Immunol. 1989, 7:91–109. © 1989 Annual Reviews www.annualreviews.org. Chapter 12

1	Fig. 12.4 adapted by permission from Macmillan Publishers Ltd. Dethlefsen, L., McFall-Ngai, M., Relman, D.A.: An ecological and evolutionary perspective on human–microbe mutualism and disease. Nature 2007, 449:811–818. © 2007. Fig. 12.10 bottom left micrograph from Niess, J.H., et al.: CX3CR1mediated dendritic cell access to the intestinal lumen and bacterial clearance. Science 2005, 307:254–258. Reprinted with permission from AAAS. Fig. 12.10 bottom center micrograph from McDole, J.R., et al.: Goblet cells deliver luminal antigen to CD103+ DCs in the small intestine. Nature 2012, 483: 345–9. With permission from Macmillan Publishers Ltd. Fig. 12.10 bottom right micrograph from Farache, J., et al.: Luminal bacteria recruit CD103+ dendritic cells into the intestinal epithelium to sample bacterial antigens for presentation. Immunity 2013, 38: 581–95. With permission from Elsevier.

1	Chapter 13 Fig. 13.20 top left photograph from Kaplan, G., Cohn, Z.A.:The immunobiology of leprosy. Int. Rev. Exp. Pathol. 1986, 28:45–78. © 1986 with permission from Elsevier. Fig. 13.37 based on data from Palella, F.J., et al.: Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N. Engl. J. Med. 1998, 338:853–860. Fig. 13.40 adapted by permission from Macmillan Publishers Ltd. Wei, X., et al.: Viral dynamics in human immunodeficiency virus type 1 infection. Nature 1995, 373:117–122.

1	Chapter 14 Fig. 14.5 top photograph from Sprecher, E., et al.: Deleterious mutations in SPINK5 in a patient with congenital ichthyosiform erythroderma: molecular testing as a helpful diagnostic tool for Netherton syndrome. Clin. Exp. Dermatol. 2004, 29:513–517. Fig. 14.14 photographs from Finotto, S., et al: Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet. Science 2002, 295:336–338. Reprinted with permission from AAAS. Fig. 14.24 left photograph from Mowat, A.M., Viney, J.L.: The anatomical basis of intestinal immunity. Immunol. Rev. 1997 156:145–166. Chapter 16 Fig. 16.16 photographs are reprinted from Herberman, R., & Callewaert, D. (eds): Mechanisms of Cytotoxicity by Natural Killer Cells, © 1985 with permission from Elsevier. -omab Suffix applied to fully murine monoclonal antibodies used for human therapies. -umab Suffix applied to fully human monoclonal antibodies used for human therapies.

1	-omab Suffix applied to fully murine monoclonal antibodies used for human therapies. -umab Suffix applied to fully human monoclonal antibodies used for human therapies. -ximab Suffix applied to chimeric (i.e., mouse/human) monoclonal antibodies used for human therapies. -zumab Suffix applied to humanized monoclonal antibodies used for human therapies. 12/23 rule Phenomenon wherein two gene segments of an immunoglobulin or T-cell receptor can be joined only if one recognition signal sequence has a 12-base-pair spacer and the other has a 23-basepair spacer. α:β heterodimer The dimer of one α and one β chain that makes up the antigen-recognition portion of an α:β T-cell receptor. α:β T-cell receptors See T-cell receptor. α4:β1 integrin (VLA-4, CD49d/CD29) See integrins. Properties of individual CD antigens can be found in Appendix II. α-defensins A class of antimicrobial peptides produced by neutrophils and the Paneth cells of the intestine.

1	α-defensins A class of antimicrobial peptides produced by neutrophils and the Paneth cells of the intestine. α-galactoceramide (α-GalCer) An immunogenic glycolipid originally extracted from marine sponges but actually produced by various bacteria that is a ligand presented by CD1 to invariant NKT (iNKT) cells. 2B4 A receptor belonging to the signaling lymphocyte activation molecule (SLAM) family expressed by NK cells, which binds to CD48, another SLAM receptor. These signal through SAP and Fyn to promote survival and proliferation. 19S regulatory caps Multisubunit component of the proteasome that functions to capture ubiquitinated proteins for degradation in the catalytic core. 20S catalytic core Multisubunit component of proteasome responsible for protein degradation. abatacept An Fc fusion protein containing the CTLA-4 extracellular domain used in treating rheumatoid arthritis that blocks co-stimulation of T cells by binding B7 molecules.

1	abatacept An Fc fusion protein containing the CTLA-4 extracellular domain used in treating rheumatoid arthritis that blocks co-stimulation of T cells by binding B7 molecules. accelerated rejection The more rapid rejection of a second graft after rejection of the first graft. It was one of the pieces of evidence that showed that graft rejection was due to an adaptive immune response. accessory effector cells Cells that aid in an adaptive immune response but are not involved in specific antigen recognition. They include phagocytes, neutrophils, mast cells, and NK cells. acellular pertussis vaccines A formulation of pertussis used for vaccination containing chemically inactivated antigens, including pertussis toxoid. acquired immune deficiency syndrome (AIDS) A disease caused by infection with the human immunodeficiency virus (HIV-1). AIDS occurs when an infected patient has lost most of his or her CD4 T cells, so that infections with opportunistic pathogens occur.

1	activating receptors On NK cells, a receptor whose stimulation results in activation of the cell’s cytotoxic activity. activation-induced cell death A process by which autoreactive T cells are induced to die if they complete thymic maturation and migrate to the periphery. activation-induced cytidine deaminase (AID) Enzyme that initiates somatic hypermutation and isotype switching by deaminating DNA directly at cytosine in immunoglobulin V regions or switch regions. Loss of AID activity in patients leads to loss of both activities, causing hyper IgM and lack of affinity maturation. activator protein 1 (AP-1) A transcription factor formed as one of the outcomes of intracellular signaling by antigen receptors of lymphocytes. active immunization Immunization with antigen to provoke adaptive immunity.

1	active immunization Immunization with antigen to provoke adaptive immunity. acute desensitization An immunotherapeutic technique for rapidly inducing temporary tolerance to, for example, an essential drug such as insulin or penicillin in a person who is allergic to it. Also called rapid desensitization. When performed properly, can produce symptoms of mild to moderate anaphylaxis. acute phase In reference to HIV infection, the period that occurs soon after a person becomes infected. It is characterized by an inﬂuenza-like illness, abundant virus in the blood, and a decrease in the number of circulating CD4 T cells. acute-phase proteins Proteins with innate immune function whose production is increased in the presence of an infection (the acute-phase response). They circulate in the blood and participate in early phases of host defense against infection. An example is mannose-binding lectin.

1	acute-phase response A change in the proteins present in the blood that occurs during the early phases of an infection. It includes the production of acute-phase proteins, many of which are produced in the liver. acute rejection The rejection of a tissue or organ graft from a genetically unrelated donor that occurs within 10–13 days of transplantation unless prevented by immunosuppressant treatment. adaptive immunity Immunity to infection conferred by an adaptive immune response. adaptors Nonenzymatic proteins that form physical links between members of a signaling pathway, particularly between a receptor and other signaling proteins. They recruit members of the signaling pathway into functional protein complexes. ADCC See antibody-dependent cell-mediated cytotoxicity. adenoids Paired mucosa-associated lymphoid tissues located in the nasal cavity.

1	ADCC See antibody-dependent cell-mediated cytotoxicity. adenoids Paired mucosa-associated lymphoid tissues located in the nasal cavity. adenosine deaminase (ADA) deficiency An inherited defect characterized by nonproduction of the enzyme adenosine deaminase, which leads to the accumulation of toxic purine nucleosides and nucleotides in cells, resulting in the death of most developing lymphocytes within the thymus. It is a cause of severe combined immunodeficiency. adhesins Cell-surface proteins on bacteria that enable them to bind to the surfaces of host cells. adipose differentiation related protein A protein that functions in the maintenance and storage of neutral lipid droplets in many types of cells. adjuvant Any substance that enhances the immune response to an antigen with which it is mixed.

1	adjuvant Any substance that enhances the immune response to an antigen with which it is mixed. afferent lymphatic vessels Vessels of the lymphatic system that drain extracellular ﬂuid from the tissues and carry antigen, macrophages, and dendritic cells from sites of infection to lymph nodes or other peripheral lymphoid organs. affinity The strength of binding of one molecule to another at a single site, such as the binding of a monovalent Fab fragment of antibody to a monovalent antigen. Cf. avidity. affinity hypothesis Hypothesis that proposes how the choice between negative selection and positive selection of T cells in the thymus is made, according to the strength of self-peptide:MHC binding by the T-cell receptor. Low-affinity interactions rescue the cell from death by neglect, leading to positive selection; high-affinity interactions induce apoptosis and thus negative selection.

1	affinity maturation The increase in affinity for their specific antigen of the antibodies produced as an adaptive immune response progresses. This phenomenon is particularly prominent in secondary and subsequent immunizations. agammaglobulinemia An absence of antibodies in the blood. See also X-linked agammaglobulinemia (XLA). age-related macular degeneration A leading cause of blindness in the elderly, for which some single-nucleotide polymorphisms (SNPs) in the factor H genes confer an increased risk. agnathan paired receptors resembling Ag receptors (APARs) Multigene family of genes containing immunoglobulin domains present in hagfish and lamprey, that possibly represent ancestral predecessors of mammalian antigen receptors. agnathans A class of vertebrate comprising jawless fish lacking adaptive immunity based on the RAG-mediated V(D)J recombination, but possessing a distinct system of adaptive immunity based on somatically assembled VLRs.

1	agonist selection A process by which T cells are positively selected in the thymus by their interaction with relatively high-affinity ligands. AID See activation-induced cytidine deaminase. AIDS See acquired immune deficiency syndrome. AIM2 (absent in melanoma 2) A member of PYHIN subfamily of NLR (NOD-like receptor) family containing an N-terminal HIN domain. It activates caspase 1 in response to viral double-stranded DNA. AIRE Gene encoding a protein (autoimmune regulator) that is involved in the expression of numerous genes by thymic medullary epithelial cells, enabling developing T cells to be exposed to self proteins characteristic of other tissues, thereby promoting tolerance to these proteins. Deficiency of AIRE leads to an autoimmune disease, APECED.

1	airway hyperreactivity, hyperresponsiveness The condition in which the airways are pathologically sensitive to both immunological (allergens) and nonimmunological stimuli, such as cold air, smoke, or perfumes. This hyperreactivity usually is present in chronic asthma. airway tissue remodeling A thickening of the airway walls that occurs in chronic asthma due to hyperplasia and hypertrophy of the smooth muscle layer and mucus glands, with the eventual development of fibrosis. Often results in an irreversible decrease of lung function. Akt Serine/threonine kinase activated downstream of PI3 kinase with numerous downstream targets involved in cell growth and survival, including activation of the mTOR pathways. alefacept Recombinant CD58–IgG1 fusion protein that blocks CD2 binding by CD58 used in treatment for psoriasis. alemtuzumab Antibody to CD52 used for lymphocyte depletion, such as for T-cell depletion during bone marrow allografts used in treating chronic myeloid leukemia.

1	alemtuzumab Antibody to CD52 used for lymphocyte depletion, such as for T-cell depletion during bone marrow allografts used in treating chronic myeloid leukemia. allele A variant form of a gene; many genes occur in several (or more) different forms within the general population. See also heterozygous, homozygous, polymorphism. allelic exclusion In a heterozygous individual, the expression of only one of the two alternative alleles of a particular gene. In immunology, the term describes the restricted expression of the individual chains of the antigen receptor genes, such that each individual lymphocyte produces immunoglobulin or T-cell receptors of a single antigen specificity. allergen Any antigen that elicits an allergic reaction.

1	allergen Any antigen that elicits an allergic reaction. allergen desensitization An immunotherapeutic technique that aims either to change an allergic immune response to a symptom-free non-allergic response, or to develop immunologic tolerance to an allergen that has been causing unpleasant clinical symptoms. The procedure involves exposing an allergic individual to increasing doses of allergen. allergic asthma An allergic reaction to inhaled antigen, which causes constriction of the bronchi, increased production of airway mucus, and difficulty in breathing. allergic conjunctivitis An allergic reaction involving the conjunctiva of the eye that occurs in sensitized individuals exposed to airborne allergens. It is usually manifested together with nasal allergy symptoms as allergic rhinoconjunctivitis or hay fever.

1	allergic contact dermatitis A largely T-cell-mediated immunological hypersensitivity reaction manifested by a skin rash at the site of contact with the allergen. Often the stimulus is a chemical agent, for example urushiol oil from the leaves of the poison ivy plant, which can haptenate normal host molecules to render them allergenic. allergic reaction A specific response to an innocuous environmental antigen, or allergen, that is caused by sensitized B or T cells. Allergic reactions can be caused by various mechanisms, but the most common is the binding of allergen to IgE bound to mast cells, which causes the cells to release histamine and other biologically active molecules that cause the signs and symptoms of asthma, hay fever, and other common allergic responses. allergic rhinitis An allergic reaction in the nasal mucosa that causes excess mucus production, nasal itching, and sneezing.

1	allergic rhinitis An allergic reaction in the nasal mucosa that causes excess mucus production, nasal itching, and sneezing. allergy The state in which a symptomatic immune reaction is made to a normally innocuous environmental antigen. It involves the interaction between the antigen and antibody or primed T cells produced by earlier exposure to the same antigen. alloantibodies Antibodies produced against antigens from a genetically nonidentical member of the same species. alloantigens Antigens from another genetically nonidentical member of the same species. allogeneic Describes two individuals or two mouse strains that differ at genes in the MHC. The term can also be used for allelic differences at other loci. allograft A transplant of tissue from an allogeneic (genetically nonidentical) donor of the same species. Such grafts are invariably rejected unless the recipient is immunosuppressed.

1	allograft A transplant of tissue from an allogeneic (genetically nonidentical) donor of the same species. Such grafts are invariably rejected unless the recipient is immunosuppressed. allograft rejection The immunologically mediated rejection of grafted tissues or organs from a genetically nonidentical donor. It is due chieﬂy to recognition of nonself MHC molecules on the graft. alloreactivity The recognition by T cells of MHC molecules other than self. Such responses are also called alloreactions or alloreactive responses. altered peptide ligands (APLs) Peptides in which amino acid substitutions have been made in T-cell receptor contact positions that affect their binding to the receptor. alternative pathway A form of complement activation that is initiated by spontaneous hydrolysis of C3 and which uses factor B and factor D to form the unique C3 convertase C3bBb. alternatively activated macrophages See M2 macrophages.

1	alternatively activated macrophages See M2 macrophages. alum Inorganic aluminum salts (for example aluminum phosphate and aluminum hydroxide); they act as adjuvants when mixed with antigens and are one of the few adjuvants permitted for use in humans. amphipathic Describes molecules that have a positively charged (or hydrophilic) region separated from a hydrophobic region. anakinra A recombinant IL-1 receptor antagonist (IL-1RA) used to block IL-1 receptor activation and used in treating rheumatoid arthritis. anaphylactic shock See anaphylaxis. anaphylatoxins Pro-inﬂammatory complement fragments C5a and C3a released by cleavage during complement activation. They are recognized by specific receptors, and recruit ﬂuid and inﬂammatory cells to the site of their release.

1	anaphylaxis A rapid-onset and systemic allergic reaction to antigen, for example to insect venom injected directly into the bloodstream, or to foods such as peanuts. Severe systemic reactions can be potentially fatal due to circulatory collapse and suffocation from tracheal swelling. It usually results from antigens binding to IgE bound by Fcε receptors on mast cells, leading to systemic release of inﬂammatory mediators. anchor residues Specific amino acid residues in antigenic peptides that determine peptide binding specificity to MHC class I molecules. Anchor residues for MHC class II molecules exist but are less obvious than for MHC class I. anergy A state of nonresponsiveness to antigen. People are said to be anergic when they cannot mount delayed-type hypersensitivity reactions to a test antigen, whereas T cells and B cells are said to be anergic when they cannot respond to their specific antigen under optimal conditions of stimulation.

1	ankylosing spondylitis Inﬂammatory disease of the spine leading to vertebral fusion strongly associated with HLA-B27. antibody A protein that binds specifically to a particular substance—called its antigen. Each antibody molecule has a unique structure that enables it to bind specifically to its corresponding antigen, but all antibodies have the same overall structure and are known collectively as immunoglobulins. Antibodies are produced by differentiated B cells (plasma cells) in response to infection or immunization, and bind to and neutralize pathogens or prepare them for uptake and destruction by phagocytes. antibody combining site See antigen-binding site. antibody-dependent cell-mediated cytotoxicity (ADCC) The killing of antibody-coated target cells by cells with Fc receptors that recognize the constant region of the bound antibody. Most ADCC is mediated by NK cells that have the Fc receptor FcγRIII on their surface.

1	antibody-directed enzyme/pro-drug therapy (ADEPT) Treatment in which an antibody is linked to an enzyme that metabolizes a nontoxic pro-drug to the active cytotoxic drug. antibody repertoire The total variety of antibodies in the body of an individual. antigen Any molecule that can bind specifically to an antibody or generate peptide fragments that are recognized by a T-cell receptor. antigen-binding site The site at the tip of each arm of an antibody that makes physical contact with the antigen and binds it noncovalently. The antigen specificity of the site is determined by its shape and the amino acids present. antigenic determinant That portion of an antigenic molecule that is bound by the antigen-binding site of a given antibody or antigen receptor; it is also known as an epitope.

1	antigenic determinant That portion of an antigenic molecule that is bound by the antigen-binding site of a given antibody or antigen receptor; it is also known as an epitope. antigenic drift The process by which inﬂuenza virus varies genetically in minor ways from year to year. Point mutations in viral genes cause small differences in the structure of the viral surface antigens. antigenic shift A radical change in the surface antigens of inﬂuenza virus, caused by reassortment of their segmented genome with that of another inﬂuenza virus, often from an animal. antigenic variation Alterations in surface antigens that occur in some pathogens (such as African trypanosomes) from one generation to another, which allows them to evade preexisting antibodies. antigen presentation The display of antigen on the surface of a cell in the form of peptide fragments bound to MHC molecules. T cells recognize antigen when it is presented in this way.

1	antigen presentation The display of antigen on the surface of a cell in the form of peptide fragments bound to MHC molecules. T cells recognize antigen when it is presented in this way. antigen-presenting cells (APCs) Highly specialized cells that can process antigens and display their peptide fragments on the cell surface together with other, co-stimulatory, proteins required for activating naive T cells. The main antigen-presenting cells for naive T cells are dendritic cells, macrophages, and B cells. antigen processing The intracellular degradation of foreign proteins into peptides that can bind to MHC molecules for presentation to T cells. All protein antigens must be processed into peptides before they can be presented by MHC molecules. antigen receptor The cell-surface receptor by which lymphocytes recognize antigen. Each individual lymphocyte bears receptors of a single antigen specificity.

1	antigen receptor The cell-surface receptor by which lymphocytes recognize antigen. Each individual lymphocyte bears receptors of a single antigen specificity. anti-lymphocyte globulin Antiserum raised in another species against human T cells. It is used in the temporary suppression of immune responses in transplantation. antimicrobial enzymes Enzymes that kill microorganisms by their actions. An example is lysozyme, which digests bacterial cell walls. antimicrobial peptides, antimicrobial proteins Amphipathic peptides or proteins secreted by epithelial cells and phagocytes that kill a variety of microbes nonspecifically, mainly by disrupting cell membranes. Antimicrobial peptides in humans include the defensins, the cathelicidins, the histatins, and RegIIIγ.

1	antiserum The ﬂuid component of clotted blood from an immune individual that contains antibodies against the antigen used for immunization. An antiserum contains a mixture of different antibodies that all bind the antigen, but which each have a different structure, their own epitope on the antigen, and their own set of cross-reactions. This heterogeneity makes each antiserum unique. antivenin Antibody raised against the venom of a poisonous snake or other organism and which can be used as an immediate treatment for the bite to neutralize the venom. aorta-gonad-mesonephros (AGM) An embryonic region in which hematopoietic cells arise during development. AP-1 A heterodimeric transcription factor formed as one of the outcomes of intracellular signaling via the antigen receptors of lymphocytes and the TLRs of cells of innate immunity. Most often, contains one Fos-family member and one Jun-family member. AP-1 mainly activates the expression of genes for cytokines and chemokines.

1	APECED See autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. An RNA editing enzyme that deaminates cytidine to uracil in certain mRNAs, such as apolipoprotein B, and which is related to the enzyme AID involved in somatic hypermutation and isotype switching. apoptosis A form of cell death common in the immune system, in which the cell activates an internal death program. It is characterized by nuclear DNA degradation, nuclear degeneration and condensation, and the rapid phagocytosis of cell remains. Proliferating lymphocytes experience high rates of apoptosis during their development and during immune responses. apoptosome A large, multimeric protein structure that forms in the process of apoptosis when cytochrome c is released from mitochondria and binds Apaf-1. A heptamer of cytochrome c-Apaf-1 heterodimers assembles into wheel-like structure that binds and activates procaspase-9, an initiator caspase, to initiate the caspase cascade.

1	appendix A gut-associated lymphoid tissue located at the beginning of the colon. APRIL A TNF family cytokine related to BAFF that binds the receptors TACI and BCMA on B cells to promote survival and regulate differentiation. apurinic/apyrimidinic endonuclease 1 (APE1) A DNA repair endonuclease involved in class switch recombination. Artemis An endonuclease involved in the gene rearrangements that generate functional immunoglobulin and T-cell receptor genes. Arthus reaction A local skin reaction that occurs when a sensitized individual with IgG antibodies against a particular antigen is challenged by injection of the antigen into the dermis. Immune complexes of the antigen with IgG antibodies in the extracellular spaces in the dermis activate complement and phagocytic cells to produce a local inﬂammatory response.

1	aryl hydrocarbon receptor (AhR) A basic helix-loop-helix transcription factor that is activated by various aromatic ligands including, famously, dioxin. It functions in the normal activity of several types of immune cells including some ILCs and IELs. ASC (PYCARD) An adaptor protein containing pyrin and CARD domains involved in activating caspase 1 in the inﬂammasome. asymptomatic phase In reference to HIV infection, period in which the infection is being partly held in check and no symptoms occur; it may last for many years. ataxia telangiectasia (ATM) A disease characterized by a staggering gait and multiple disorganized blood vessels, and often accompanied by clinical immunodeficiency. It is caused by defects in the ATM protein, which is involved in DNA repair pathways that are also used in V(D)J recombination and class-switch recombination.

1	atopic march The clinical observation that it is common for children with atopic eczema to later develop allergic rhinitis and/or asthma. atopy A genetically based increased tendency to produce IgE-mediated allergic reactions against innocuous substances. ATP-binding cassette (ABC) A large family of proteins containing a particular domain for nucleotide-binding that includes many transporters, such as TAP1 and TAP2, but also various NOD members. attenuation The process by which human or animal pathogens are modified by growth in culture so that they can grow in their host and induce immunity without producing serious clinical disease. atypical hemolytic uremic syndrome A condition characterized by damage to platelets and red blood cells and inﬂammation of the kidneys that is caused by uncontrolled complement activation in individuals with inherited deficiencies in complement regulatory proteins. autoantibodies Antibodies specific for self antigens.

1	autoantibodies Antibodies specific for self antigens. autoantigens A self antigen to which the immune system makes a response. autocrine Describes a cytokine or other biologically active molecule acting on the cell that produces it. autograft A graft of tissue from one site to another on the same individual. autoimmune disease Disease in which the pathology is caused by adaptive immune responses to self antigens. autoimmune hemolytic anemia A pathological condition with low levels of red blood cells (anemia), which is caused by autoantibodies that bind red blood cell surface antigens and target the red blood cell for destruction. autoimmune lymphoproliferative syndrome (ALPS) An inherited syndrome in which a defect in the Fas gene leads to a failure in normal apoptosis, causing unregulated immune responses, including autoimmune responses.

1	autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) A disease characterized by a loss of tolerance to self antigens, caused by a breakdown of negative selection in the thymus. It is due to defects in the gene AIRE, which encodes a transcriptional regulatory protein that enables many self antigens to be expressed by thymic medullary epithelial cells. Also called autoimmune polyglandular syndrome type I. autoimmune thrombocytopenic purpura An autoimmune disease in which antibodies against platelets are made. Antibody binding to platelets causes them to be taken up by cells with Fc receptors and complement receptors, resulting in a decrease in platelet count that leads to purpura (bleeding). autoimmunity Adaptive immunity specific for self antigens. autoinflammatory diseases Diseases due to unregulated inﬂammation in the absence of infection; they can have a variety of causes, including inherited genetic defects.

1	autoinflammatory diseases Diseases due to unregulated inﬂammation in the absence of infection; they can have a variety of causes, including inherited genetic defects. autophagosome A double bilayer membrane structure that functions in macroautophagy by engulfing cytoplasmic contents and fusing with lysosomes. autophagy The digestion and breakdown by a cell of its own organelles and proteins in lysosomes. It may be one route by which cytosolic proteins can be processed for presentation on MHC class II molecules. avidity The sum total of the strength of binding of two molecules or cells to one another at multiple sites. It is distinct from affinity, which is the strength of binding of one site on a molecule to its ligand. avoidance Mechanisms that prevent a host's exposure to microbes, such as anatomic barriers or particular behaviors.

1	avoidance Mechanisms that prevent a host's exposure to microbes, such as anatomic barriers or particular behaviors. azathioprine A powerful cytotoxic drug that is converted to its active form in vivo, which then kills rapidly proliferating cells, including proliferating lymphocytes; it is used as an immunosuppressant to treat autoimmune disease and in transplantation. B-1 B cells A class of atypical, self-renewing B cells (also known as CD5 B cells) found mainly in the peritoneal and pleural cavities in adults and considered part of the innate rather than the adaptive immune system. They have a much less diverse antigen-receptor repertoire than conventional B cells and are the major source of natural antibody.

1	B7 molecules, B7.1 and B7.2 Cell-surface proteins on specialized antigen-presenting cells such as dendritic cells, which are the major co-stimulatory molecules for T cells. B7.1 (CD80) and B7.2 (CD86) are closely related members of the immunoglobulin superfamily and both bind to the CD28 and CTLA-4 proteins on T cells. β1i (LMP2), β2i (MECL-1), β5i (LMP7) Alternative proteasome subunits that replace the constitutive catalytic subunits β1, β2, and β5 that are induced by interferons and produce the immunoproteasome. β5t Alternative proteasome subunit expressed by thymic epithelial cells that substitutes for β5 to produce the thymoproteasome involved in generating peptides encountered by thymocytes during development. β-defensins Antimicrobial peptides made by virtually all multicellular organisms. In mammals they are produced by the epithelia of the respiratory and urogenital tracts, skin, and tongue.

1	β-defensins Antimicrobial peptides made by virtually all multicellular organisms. In mammals they are produced by the epithelia of the respiratory and urogenital tracts, skin, and tongue. β sandwich A secondary protein structure composed of two β sheets that fold such that one lies over the other, as in an immunoglobulin fold. β sheets A secondary protein structure composed of β strands stabilized by noncovalent interactions between backbone amide and carbonyl groups. In ‘parallel’ β sheets, the adjacent strands run in the same direction; in ‘antiparallel’ β sheets, adjacent strands run in opposite directions. Immunoglobulin domains are made up of two antiparallel β sheets arranged in the form of a β barrel. β strands A secondary protein structure in which the polypeptide backbone of several consecutive amino acids is arranged in a ﬂat, or planar, conformation, and often illustrated as an arrow.

1	β strands A secondary protein structure in which the polypeptide backbone of several consecutive amino acids is arranged in a ﬂat, or planar, conformation, and often illustrated as an arrow. β2-microglobulin The light chain of the MHC class I proteins, encoded outside the MHC. It binds noncovalently to the heavy or α chain. B and T lymphocyte attenuator (BTLA) An inhibitory CD28-related receptor expressed by B and T lymphocytes that interacts with the herpes virus entry molecule (HVEM), a member of the TNF receptor family. bacteria A vast kingdom of unicellular prokaryotic microorganisms, some species of which cause infectious diseases in humans and animals, while others make up most of the body’s commensal microbiota. Disease-causing bacteria may live in the extracellular spaces, or inside cells in vesicles or in the cytosol. BAFF B-cell activating factor belonging to the TNF family that binds the receptors BAFF-R and TACI to promote B cell survival.

1	BAFF B-cell activating factor belonging to the TNF family that binds the receptors BAFF-R and TACI to promote B cell survival. BAFF-R Receptor for BAFF that can activate canonical and non-canonical NF-κB signaling and promote survival of B cells. bare lymphocyte syndrome See MHC class I deficiency, MHC class II deficiency. base-excision repair Type of DNA repair that can lead to mutation and that is involved in somatic hypermutation and class switching in B cells. basiliximab Antibody to human CD25 used to block IL-2 receptor signaling in T cells for treatment of rejection in renal transplantation. basophils Type of white blood cell containing granules that stain with basic dyes. It is thought to have a function similar to mast cells. BATF3 A transcription factor expressed in dendritic cells belonging to the AP1 family, which includes many other factors such as c-Jun and Fos.

1	BATF3 A transcription factor expressed in dendritic cells belonging to the AP1 family, which includes many other factors such as c-Jun and Fos. B cells, B lymphocytes One of the two types of antigen-specific lymphocytes responsible for adaptive immune responses, the other being the T cells. The function of B cells is to produce antibodies. B cells are divided into two classes. Conventional B cells have highly diverse antigen receptors and are generated in the bone marrow throughout life, emerging to populate the blood and lymphoid tissues. B-1 cells have much less diverse antigen receptors and form a population of self-renewing B cells in the peritoneal and pleural cavities.

1	B-cell antigen receptor, B-cell receptor (BCR) The cell-surface receptor on B cells for specific antigen. It is composed of a transmembrane immunoglobulin molecule (which recognizes antigen) associated with the invariant Igα and Igβ chains (which have a signaling function). On activation by antigen, B cells differentiate into plasma cells producing antibody molecules of the same antigen specificity as this receptor. B-cell co-receptor A transmembrane signaling receptor on the B-cell surface composed of the proteins CD19, CD81, and CD21 (complement receptor 2), which binds complement fragments on bacterial antigens also bound by the B-cell receptor. Co-ligation of this complex with the B-cell receptor increases responsiveness to antigen about 100-fold.

1	B-cell co-receptor complex A transmembrane signaling receptor on the B-cell surface composed of the proteins CD19, CD81, and CD21 (complement receptor 2), which binds complement fragments on bacterial antigens also bound by the B-cell receptor. Co-ligation of this complex with the B-cell receptor increases responsiveness to antigen about 100-fold. B-cell mitogens Any substance that nonspecifically causes B cells to proliferate. Bcl-2 family Family of intracellular proteins that includes members that promote apoptosis (Bax, Bak, and Bok) and members that inhibit apoptosis (Bcl-2, Bcl-W, and Bcl-XL). Bcl-6 A transcriptional repressor that opposes differentiation of B cells into plasma cells. BCMA Receptor of the TNFR superfamily that binds APRIL. Bcr–Abl tyrosine kinase Constitutively active tyrosine kinase fusion protein caused by a chromosomal translocation—the Philadelphia chromosome— between Bcr with the Abl tyrosine kinase genes associated with chronic myeloid leukemia.

1	BDCA-2 (blood dendritic cell antigen 2) A C-type lectin expressed selectively as a receptor on the surface of human plasmacytoid dendritic cells. Berlin patient A man with HIV who was treated in Berlin with a hematopoietic stem cell (HSC) transplant from a donor deficient in a co-receptor for the virus (CCR5) for an unrelated illness (leukemia). He is thought to be cured of HIV infection, and is one of the only known patients in which the virus is thought to be completely eliminated, a so-called ‘sterilizing’ cure. biologics therapy Medical treatments comprising natural proteins such as antibodies and cytokines, and antisera or whole cells. Blau syndrome An inherited granulomatous disease caused by gain-offunction mutations in the NOD2 gene. BLIMP-1 A transcriptional repressor that promotes B-cell differentiation into plasma cells and suppresses proliferation, and further class switching and affinity maturation. BLNK B-cell linker protein. See SLP-65.

1	BLNK B-cell linker protein. See SLP-65. bone marrow The tissue where all the cellular elements of the blood—red blood cells, white blood cells, and platelets—are initially generated from hematopoietic stem cells. The bone marrow is also the site of further B-cell development in mammals and the source of stem cells that give rise to T cells on migration to the thymus. Thus, bone marrow transplantation can restore all the cellular elements of the blood, including the cells required for adaptive immune responses. booster immunization See secondary immunization. bradykinin A vasoactive peptide that is produced as a result of tissue damage and acts as an inﬂammatory mediator. broadly neutralizing antibodies Antibodies that block viral infection by multiple strains. In reference to HIV, these are antibodies that block binding of the virus to CD4 and/or chemokine co-receptors.

1	bronchus-associated lymphoid tissue (BALT) Organized lymphoid tissue found in the bronchi in some animals. Adult humans do not normally have such organized lymphoid tissue in the respiratory tract, but it may be present in some infants and children. Bruton’s tyrosine kinase (Btk) A Tec-family tyrosine kinase important in B-cell receptor signaling. Btk is mutated in the human immunodeficiency disease X-linked agammaglobulinemia. Bruton’s X-linked agammaglobulinemia See X-linked agammaglobulinemia. bursa of Fabricius Lymphoid organ associated with the gut that is the site of B-cell development in chickens. butyrate A short chain fatty acid produced abundantly by anaerobic digestion of carbohydrates in the intestine by commensals that can inﬂuence host cells in several ways, acting as an energy source for enterocytes and as an inhibitor of histone deacetylases.

1	C1 complex, C1 Protein complex activated as the first step in the classical pathway of complement activation, composed of C1q bound to two molecules each of the proteases C1r and C1s. Binding of a pathogen or antibody to C1q activates C1r, which cleaves and activates C1s, which cleaves C4 and C2. C1 inhibitor (C1INH) An inhibitor protein for C1 that binds and inactivates C1r:C1s enzymatic activity. Deficiency in C1INH causes hereditary angioedema through production of vasoactive peptides that cause subcutaneous and laryngeal swelling. C2 Complement protein of the classical and lectin pathways that is cleaved by the C1 complex to yield C2b and C2a. C2a is an active protease that forms part of the classical C3 convertase C4bC2a. C3 Complement protein on which all complement activation pathways converge. C3 cleavage forms C3b, which can bind covalently to microbial surfaces, where it promotes destruction by phagocytes.

1	C3 Complement protein on which all complement activation pathways converge. C3 cleavage forms C3b, which can bind covalently to microbial surfaces, where it promotes destruction by phagocytes. C3 convertase Enzyme complex that cleaves C3 to C3b and C3a on the surface of a pathogen. The C3 convertase of the classical and lectin pathways is formed from membrane-bound C4b complexed with the protease C2a. The alternative pathway C3 convertase is formed from membrane-bound C3b complexed with the protease Bb. C3(H2O)Bb See fluid-phase C3 convertase. C3a See anaphylatoxins. C3b See C3. C3b2Bb The C5 convertase of the alternative pathway of complement activation. C3bBb The C3 convertase of the alternative pathway of complement activation. C3dg Breakdown product of iC3b that remains attached to the microbial surface, where it can bind complement receptor CR2. C3f A small fragment of C3b that is removed by factor I and MCP to leave iC3b on the microbial surface.

1	C3f A small fragment of C3b that is removed by factor I and MCP to leave iC3b on the microbial surface. C4 Complement protein of the classical and lectin pathways. C4 is cleaved by C1s to C4b, which forms part of the classical C3 convertase. C4b-binding protein (C4BP) A complement-regulatory protein that inactivates the classical pathway C3 convertase formed on host cells by displacing C2a from the C4bC2a complex. C4BP binds C4b attached to host cells, but cannot bind C4b attached to pathogens. C4b2a C3 convertase of the classical and lectin pathways of complement activation. C4b2a3b C5 convertase of the classical and lectin pathways of complement activation. C5 convertase Enzyme complex that cleaves C5 to C5a and C5b. C5a See anaphylatoxins. C5a receptor The cell-surface receptor for the pro-inﬂammatory C5a fragment of complement, present on macrophages and neutrophils. C5b Fragment of C5 that initiates the formation of the membrane-attack complex (MAC).

1	C5b Fragment of C5 that initiates the formation of the membrane-attack complex (MAC). C5L2 (GPR77) Non-signaling decoy receptor for C5a expressed by phagocytes. C6, C7, C8, C9 Complement proteins that act with C5b to form the membrane-attack complex, producing a pore that leads to lysis of the target cell. calcineurin A cytosolic serine/threonine phosphatase with a crucial role in signaling via the T-cell receptor. The immunosuppressive drugs cyclosporin A and tacrolimus inactivate calcineurin, suppressing T-cell responses. calmodulin Calcium-binding protein that is activated by binding Ca2+; it is then able to bind to and regulate the activity of a wide variety of enzymes. calnexin A chaperone protein in the endoplasmic reticulum (ER) that binds to partly folded members of the immunoglobulin superfamily of proteins and retains them in the ER until folding is complete.

1	calnexin A chaperone protein in the endoplasmic reticulum (ER) that binds to partly folded members of the immunoglobulin superfamily of proteins and retains them in the ER until folding is complete. calprotectin A complex of heterodimers of the antimicrobial peptides S100A8 and S100A9, which sequester zinc and manganese from microbes. Produced in abundance by neutrophils, and in lesser amounts by macrophages and epithelial cells. calreticulin A chaperone protein in the endoplasmic reticulum that, together with ERp57 and tapasin, forms the peptide-loading complex that loads peptides onto newly synthesized MHC class I molecules. cancer immunoediting A process that occurs during the development of a cancer when it is acquiring mutations that favor its survival and escape from immune responses, such that cancer cells with these mutations are selected for survival and growth.

1	cancer-testis antigens Proteins expressed by cancer cells that are normally expressed only in male germ cells in the testis. capping A process occurring in the nucleus in which the modified purine 7-methylguanosine is added to the 5ʹ phosphate of the first nucleotide of the RNA transcript. capsular polysaccharides See capsulated bacteria. capsulated bacteria Referring to bacteria surrounded by a polysaccharide shell that resists actions of phagocytes, resulting in pus formation at the site of infection. Also called pyogenic (pus-forming) bacteria. carboxypeptidase N (CPN) A metalloproteinase that inactivates C3a and C5a. CPN deficiency causes a condition of recurrent angioedema. cardiolipin A lipid found in many bacteria and in the inner mitochondrial membrane that is a ligand recognized by some human γ:δ T cells. caspase 8 An initiator caspase activated by various receptors that activates the process of apoptosis.

1	caspase 8 An initiator caspase activated by various receptors that activates the process of apoptosis. caspase 11 This caspase is homologous to human capsase 4 and 5. Its expression is induced by TLR signaling. Intracellular LPS can directly activate it, leading to pyroptosis. caspase recruitment domain (CARD) A protein domain present in some receptor tails that can dimerize with other CARD-domain-containing proteins, including caspases, thus recruiting them into signaling pathways. caspases A family of cysteine proteases that cleave proteins at aspartic acid residues. They have important roles in apoptosis and in the processing of cytokine pro-polypeptides. cathelicidins Family of antimicrobial peptides that in humans has one member. cathelin A cathepsin L inhibitor. cathepsins A family of proteases using cysteine at their active site that frequently function in processing antigens taken into the vesicular pathway.

1	cathelin A cathepsin L inhibitor. cathepsins A family of proteases using cysteine at their active site that frequently function in processing antigens taken into the vesicular pathway. CC chemokines One of the two main classes of chemokines, distinguished by two adjacent cysteines (C) near the amino terminus. They have names CCL1, CCL2, etc. See Appendix IV for a list of individual chemokines. CCL9 (MIP-1γ) Chemokine made by follicle-associated epithelial cells and binds CCR6, recruiting activated T and B cells, NK cells, and dendritic cells into GALT. CCL19 Chemokine made by dendritic cells and stromal cells in T-cell zones of lymph nodes that binds CCR7 and functions to attract naive T cells. CCL20 Chemokine made by follicle-associated epithelial cells and binds CCR6, recruiting activated T and B cells, NK cells, and dendritic cells into GALT.

1	CCL20 Chemokine made by follicle-associated epithelial cells and binds CCR6, recruiting activated T and B cells, NK cells, and dendritic cells into GALT. CCL21 Chemokine made by dendritic cells and stromal cells in T cell zones of lymph nodes that binds CCR7 and functions to attract naive T cells. CCL25 (TECK) Chemokine made by small-intestinal epithelial cells that binds CCR9 to recruit gut-homing T and B cells. CCL28 (MEC, mucosal epithelial chemokine) Chemokine made by colonic intestinal cells, salivary gland, and lactating mammary gland cells that binds CCR10 to recruit B lymphocytes producing IgA into these tissues. CCR1 Chemokine receptor expressed by neutrophils, monocytes, B cells, and dendritic cells, that binds several chemokines, including CCL6 and CCL9. CCR6 Chemokine receptor expressed by follicular and marginal zone B cells and dendritic cells that binds CCL20.

1	CCR6 Chemokine receptor expressed by follicular and marginal zone B cells and dendritic cells that binds CCL20. CCR7 Chemokine receptor expressed by all naive T and B cells, and some memory T and B cells, such as central memory T cells, that binds CCL19 and CCL21 made by dendritic cells and stromal cells in lymphoid tissues. CCR9 Chemokine receptor expressed by dendritic cells, T cells, and thymocytes, and some γ:δT cells, that binds CCL25 that mediates recruitment of gut-homing cells. CCR10 Chemokine receptor expressed by many cells that binds CCL27 and CCL28 that mediates intestinal recruitment of IgA-producing B lymphocytes. CD1 Small family of MHC class I-like proteins that are not encoded in the MHC and can present glycolipid antigens to CD4 T cells. CD3 complex The invariant proteins CD3γ, δ, and ε, and the dimeric ζ chains, which form the signaling complex of the T-cell receptor. Each of them contains one or more ITAM signaling motifs in their cytoplasmic tails.

1	CD4 The co-receptor for T-cell receptors that recognize peptide antigens bound to MHC class II molecules. It binds to the lateral face of the MHC molecule. CD8 The co-receptor for T-cell receptors that recognize peptide antigens bound to MHC class I molecules. It binds to the lateral face of the MHC molecule. CD11b (αM integrin) Integrin expressed by macrophages and some dendritic cells that functions with β2 integrin (CD18) as complement receptor 3 (CR3). CD19 See B-cell co-receptor. CD21 Another name for complement receptor 2 (CR2). See also B-cell co-receptor. CD22 An inhibitory receptor on B cells that binds sialic acid-modified glycoproteins commonly found on mammalian cells and contains an ITIM motif in its cytoplasmic tail. CD23 The low-affinity Fc receptor for IgE.

1	CD23 The low-affinity Fc receptor for IgE. CD25 Also known as IL-2 receptorα (IL-2Rα), this is the high-affinity component of the IL-2 receptor, which also includes IL-2Rβ and the common γ chain. It is upregulated by activated T cells and is constitutively expressed by Treg cells to confer responsiveness to IL-2. CD27 A TNF receptor-family protein constitutively expressed on naive T cells that binds CD70 on dendritic cells and delivers a potent co-stimulatory signal to T cells early in the activation process. CD28 An activating receptor on T cells that binds to the B7 co-stimulatory molecules present on specialized antigen-presenting cells such as dendritic cells. CD28 is the major co-stimulatory receptor on naive T cells. CD30, CD30 ligand CD30 on B cells and CD30 ligand (CD30L) on helper T cells are co-stimulatory molecules involved in stimulating the proliferation of antigen-activated naive B cells.

1	CD30, CD30 ligand CD30 on B cells and CD30 ligand (CD30L) on helper T cells are co-stimulatory molecules involved in stimulating the proliferation of antigen-activated naive B cells. CD31 A cell-adhesion molecule found both on lymphocytes and at endothelial cell junctions. CD31–CD31 interactions are thought to enable leukocytes to leave blood vessels and enter tissues. CD40, CD40 ligand CD40 on B cells and CD40 ligand (CD40L, CD154) on activated helper T cells are co-stimulatory molecules whose interaction is required for the proliferation and class switching of antigen activated naive B cells. CD40 is also expressed by dendritic cells, and here the CD40–CD40L interaction provides co-stimulatory signals to naive T cells.

1	CD40 ligand deficiency An immunodeficiency disease in which little or no IgG, IgE, or IgA antibody is produced and even IgM responses are deficient, but serum IgM levels are normal to high. It is due to a defect in the gene encoding CD40 ligand (CD154), which prevents class switching from occurring. Also known as X-linked hyper IgM syndrome, reﬂecting location of gene that encodes CD40L on the X chromosome and phenotype of elevated IgM antibody relative to other immunoglobulins. CD44 Also known as phagocytic glycoprotein-1 (Pgp1), CD44 is a cell-surface glycoprotein expressed by naive lymphocytes and upregulated on activated T cells. It is a receptor for hyaluronic acid and functions in cell–cell and cell–extracellular matrix adhesion. High expression of CD44 is used as a marker for effector and memory T cells.

1	CD45 A transmembrane tyrosine phosphatase found on all leukocytes. It is expressed in different isoforms on different cell types, including the different subtypes of T cells. Also called leukocyte common antigen, it is a generic marker for hematopoietically derived cells, with the exception of erythrocytes. CD45RO An alternatively spliced variant of CD45 that serves as a marker for memory T cells. CD48 See 2B4. CD59, protectin Cell-surface protein that protects host cells from complement damage by blocking binding of C9 to the C5b678 complex, thus preventing MAC formation. CD69 A cell-surface protein that is rapidly expressed by antigen-activated T cells. It acts to down-modulate the expression of the sphingosine 1 phosphate receptor 1 (S1PR1), thereby retaining activated T cells within T-cell zones of secondary lymphoid tissues as they divide and differentiate into effector T cells.

1	CD70 The ligand for CD27 that is expressed on activated dendritic cells and delivers a potent co-stimulatory signal to T cells early in the activation process. CD81 See B-cell co-receptor. CD84 See SLAM (signaling lymphocyte activation molecule). CD86 (B7-2) A transmembrane protein of the immunoglobulin superfamily that is expressed on antigen-presenting cells and binds to CD28 expressed by T cells. CD94 A C-type lectin that is a subunit of the KLR-type receptors of NK cells. CD103 Integrin αE:β7, a cell-surface marker on a subset of dendritic cells in the gastrointestinal tract that are involved in inducing tolerance to antigens from food and the commensal microbiota. CD127 Also known as IL-7 receptor α (IL-7Rα), which pairs with the common γ chain of the IL-2 receptor family to form the IL-7 receptor. It is expressed by naive T cells and a subset of memory T cells to support their survival.

1	celiac disease A chronic condition of the upper small intestine caused by an immune response directed at gluten, a complex of proteins present in wheat, oats, and barley. The gut wall becomes chronically inﬂamed, the villi are destroyed, and the gut’s ability to absorb nutrients is compromised. cell-adhesion molecules Cell-surface proteins of several different types that mediate the binding of one cell to other cells or to extracellular matrix proteins. Integrins, selectins, and members of the immunoglobulin gene superfamily (such as ICAM-1) are among the cell-adhesion molecules important in the operation of the immune system. cell-mediated immune responses An adaptive immune response in which antigen-specific effector T cells have the main role. The immunity to infection conferred by such a response is called cell-mediated immunity. A primary cell-mediated immune response is the T-cell response that occurs the first time a particular antigen is encountered.

1	cellular hypersensitivity reactions A hypersensitivity reaction mediated largely by antigen-specific T lymphocytes. cellular immunology The study of the cellular basis of immunity. central lymphoid organs, central lymphoid tissues The sites of lymphocyte development; in humans, these are the bone marrow and thymus. B lymphocytes develop in bone marrow, whereas T lymphocytes develop within the thymus from bone marrow-derived progenitors. Also called the primary lymphoid organs. central memory T cells (TCM) Memory lymphocytes that express CCR7 and recirculate between blood and secondary lymphoid tissues similarly to naive T cells. They require restimulation in secondary lymphoid tissues to become fully mature effector T cells. central tolerance Immunological tolerance to self antigens that is established while lymphocytes are developing in central lymphoid organs. Cf. peripheral tolerance.

1	central tolerance Immunological tolerance to self antigens that is established while lymphocytes are developing in central lymphoid organs. Cf. peripheral tolerance. centroblasts Large, rapidly dividing activated B cells present in the dark zone of germinal centers in follicles of peripheral lymphoid organs. centrocytes Small B cells that derive from centroblasts in the germinal centers of follicles in peripheral lymphoid organs; they populate the light zone of the germinal center. cGAS (cyclic GAMP synthase) A cytosolic enzyme that is activated by double-stranded DNA to form cyclic guanosine monophosphate-adenosine monophoshate. See cyclic dinucleotides (CDNs). checkpoint blockade Approach to tumor therapy that attempts to interfere with the normal inhibitory signals that regulate lymphocytes.

1	checkpoint blockade Approach to tumor therapy that attempts to interfere with the normal inhibitory signals that regulate lymphocytes. Chediak–Higashi syndrome A defect in phagocytic cell function caused by a defect in a protein involved in intracellular vesicle fusion. Lysosomes fail to fuse properly with phagosomes, and killing of ingested bacteria is impaired. chemokines Small chemoattractant protein that stimulates the migration and activation of cells, especially phagocytic cells and lymphocytes. Chemokines have a central role in inﬂammatory responses. Properties of individual chemokines are listed in Appendix IV. chemotaxis Cellular movement occurring in response to chemical signals in the environment. chimeric antigen receptor (CAR) Engineered fusion proteins composed of extracellular antigen-specific receptors (e.g., single-chain antibody) and intracellular signaling domains that activate and co-stimulate, expressed in T cells for use in cancer immunotherapy.

1	chronic allograft vasculopathy Chronic damage that can lead to late failure of transplanted organs. Arteriosclerosis of graft blood vessels leads to hypoperfusion of the graft and its eventual fibrosis and atrophy. chronic granulomatous disease (CGD) An immunodeficiency in which multiple granulomas form as a result of defective elimination of bacteria by phagocytic cells. It is caused by defects in the NADPH oxidase system of enzymes that generate the superoxide radical involved in bacterial killing. An autoinﬂammatory disease due to defects in the gene NLRP3, one of the components of the inﬂammasome. chronic rejection Late failure of a transplanted organ, which can be due to immunological or nonimmunological causes. CIIV An early endocytic compartment containing MHC class II molecules in dendritic cells.

1	CIIV An early endocytic compartment containing MHC class II molecules in dendritic cells. class I cytokine receptors A group of receptors for the hematopoietin superfamily of cytokines. These include receptors using the common γ chain for IL-2, IL-4, IL-7, IL-15, and IL-21, and a common β chain for GM-CSF, IL-3, and IL-5. class II-associated invariant chain peptide (CLIP) A peptide of variable length cleaved from the invariant chain (Ii) by proteases. It remains associated with the MHC class II molecule in an unstable form until it is removed by the HLA-DM protein. class II cytokine receptors A group of heterodimeric receptors for a family of cytokines that includes interferon (IFN)-α, IFN-β, IFN-γ, and IL-10.

1	class II cytokine receptors A group of heterodimeric receptors for a family of cytokines that includes interferon (IFN)-α, IFN-β, IFN-γ, and IL-10. class switching, class switch recombination A somatic gene recombination process in activated B cells that replaces one heavy-chain constant region with one of a different isotype, switching the isotype of antibodies from IgM to the production of IgG, IgA, or IgE. This affects the antibody effector functions but not their antigen specificity. Also known as isotype switching. Cf. somatic hypermutation. classes The class of an antibody is defined by the type of heavy chain it contains. There are five main antibody classes: IgA, IgD, IgM, IgG, and IgE, containing heavy chains α, δ, μ, γ, and ε, respectively. The IgG class has several subclasses. See also isotypes. classical C3 convertase The complex of activated complement components C4b2a, which cleaves C3 to C3b on pathogen surfaces in the classical pathway of complement activation.

1	classical C3 convertase The complex of activated complement components C4b2a, which cleaves C3 to C3b on pathogen surfaces in the classical pathway of complement activation. classical MHC class I genes MHC class I genes whose proteins function by presenting peptide antigens for recognition by T cells. Cf. nonclassical MHC class Ib. classical monocyte The major form of monocyte in circulation capable of recruitment to sites of inﬂammation and differentiation into macrophages. classical pathway The complement-activation pathway that is initiated by C1 binding either directly to bacterial surfaces or to antibody bound to the bacteria, thus ﬂagging the bacteria as foreign. See also alternative pathway, lectin pathway. classically activated macrophage See M1 macrophages. cleavage stimulation factor A multi-subunit protein complex involved in the modification of the 3ʹ end of pre-messenger RNA for the addition of the polyadenine (polyA) tail.

1	cleavage stimulation factor A multi-subunit protein complex involved in the modification of the 3ʹ end of pre-messenger RNA for the addition of the polyadenine (polyA) tail. clonal deletion The elimination of immature lymphocytes when they bind to self antigens, which produces tolerance to self as required by the clonal selection theory of adaptive immunity. Clonal deletion is the main mechanism of central tolerance and can also occur in peripheral tolerance. clonal expansion The proliferation of antigen-specific lymphocytes in response to antigenic stimulation that precedes their differentiation into effector cells. It is an essential step in adaptive immunity, allowing rare antigen-specific cells to increase in number so that they can effectively combat the pathogen that elicited the response.

1	clonal selection theory The central paradigm of adaptive immunity. It states that adaptive immune responses derive from individual antigen-specific lymphocytes that are self-tolerant. These specific lymphocytes proliferate in response to antigen and differentiate into antigen-specific effector cells that eliminate the eliciting pathogen, and into memory cells to sustain immunity. The theory was formulated by Macfarlane Burnet and in earlier forms by Niels Jerne and David Talmage. clone A population of cells all derived from the same progenitor cell. clonotypic Describes a feature unique to members of a clone. For example, the distribution of antigen receptors in the lymphocyte population is said to be clonotypic, as the cells of a given clone all have identical antigen receptors. Clostridium difficile Gram-positive anaerobic toxogenic spore-forming bacterium frequently associated with severe colitis following treatment with certain broad-spectrum antibiotics.

1	Clostridium difficile Gram-positive anaerobic toxogenic spore-forming bacterium frequently associated with severe colitis following treatment with certain broad-spectrum antibiotics. c-Maf A transcription factor acting in the development of TFH cells. coagulation system A collection of proteases and other proteins in the blood that trigger blood clotting when blood vessels are damaged. coding joint DNA join formed by the imprecise joining of a V gene segment to a (D)J gene segment during recombination of the immunoglobulin or T-cell receptor genes. It is the joint retained in the rearranged gene. Cf. signal joint. codominant Describes the situation in which the two alleles of a gene are expressed in roughly equal amounts in the heterozygote. Most genes show this property, including the highly polymorphic MHC genes. collectins A family of calcium-dependent sugar-binding proteins (lectins) containing collagen-like sequences. An example is mannose-binding lectin (MBL).

1	collectins A family of calcium-dependent sugar-binding proteins (lectins) containing collagen-like sequences. An example is mannose-binding lectin (MBL). combinatorial diversity The diversity among antigen receptors generated by combining separate units of genetic information, comprising two types. First, receptor gene segments are joined in many different combinations to generate diverse receptor chains; second, two different receptor chains (heavy and light in immunoglobulins; α and β, or γ and δ, in T-cell receptors) are combined to make the antigen-recognition site. commensal microbiota, commensal microorganisms Microorganisms (predominantly bacteria) that normally live harmlessly in symbiosis with their host (for example the gut bacteria in humans and other animals). Many commensals confer a positive benefit on their host in some way. common β chain A transmembrane polypeptide (CD131) that is a common subunit for receptor of the cytokines IL-3, IL-5, and GM-CSF.

1	common β chain A transmembrane polypeptide (CD131) that is a common subunit for receptor of the cytokines IL-3, IL-5, and GM-CSF. common γ chain (γc) A transmembrane polypeptide chain (CD132) that is common to a subgroup of cytokine receptors. common lymphoid progenitor (CLP) Stem cell that can give rise to all the types of lymphocytes with the exception of innate lymphoid cells (ILCs). common mucosal immune system The mucosal immune system as a whole, the name reﬂecting the fact that lymphocytes that have been primed in one part of the mucosal system can recirculate as effector cells to other parts of the mucosal system. common myeloid progenitor (CMP) Stem cells that can give rise to the myeloid cells of the immune system—macrophages, granulocytes, mast cells, and dendritic cells of the innate immune system. This stem cell also gives rise to megakaryocytes and red blood cells.

1	common variable immunodeficiencies (CVIDs) A relatively common deficiency in antibody production in which only one or a few isotypes are affected. It can be due to a variety of genetic defects. complement A set of plasma proteins that act together as a defense against pathogens in extracellular spaces. The pathogen becomes coated with complement proteins that facilitate its removal by phagocytes and that can also kill certain pathogens directly. Activation of the complement system can be initiated in several different ways. See classical pathway, alternative pathway, lectin pathway. complement activation The activation of the normally inactive proteins of the complement system that occurs on infection. See classical pathway, alternative pathway, lectin pathway. complement proteins See C1, C2, C3, etc..

1	complement proteins See C1, C2, C3, etc.. complement receptors (CRs) Cell-surface proteins of various types that recognize and bind complement proteins that have become bound to an antigen such as a pathogen. Complement receptors on phagocytes enable them to identify and bind pathogens coated with complement proteins, and to ingest and destroy them. See CR1, CR2, CR3, CR4, CRIg, and the C1 complex. complement regulatory proteins Proteins that control complement activity and prevent complement from being activated on the surfaces of host cells. complement system A set of plasma proteins that act together as a defense against pathogens in extracellular spaces. The pathogen becomes coated with complement proteins that facilitate its removal by phagocytes and that can also kill certain pathogens directly. Activation of the complement system can be initiated in several different ways. See classical pathway, alternative pathway, lectin pathway.

1	complementarity-determining regions (CDRs) Parts of the V domains of immunoglobulins and T-cell receptors that determine their antigen specificity and make contact with the specific ligand. The CDRs are the most variable part of antigen receptor, and contribute to the diversity of these proteins. There are three such regions (CDR1, CDR2, and CDR3) in each V domain. conformational epitopes, discontinuous epitopes Antigenic structure (epitope) on a protein antigen that is formed from several separate regions in the sequence of the protein brought together by protein folding. Antibodies that bind conformational epitopes bind only native folded proteins. conjugate vaccines Antibacterial vaccines made from bacterial capsular polysaccharides bound to proteins of known immunogenicity, such as tetanus toxoid. constant Ig domains (C domains) Type of protein domain that makes up the constant regions of each chain of an immunoglobulin molecule.

1	constant Ig domains (C domains) Type of protein domain that makes up the constant regions of each chain of an immunoglobulin molecule. constant region, C region That part of an immunoglobulin or a T-cell receptor that is relatively constant in amino acid sequence between different molecules. Also known as the Fc region in antibodies. The constant region of an antibody determines its particular effector function. Cf. variable region. continuous epitope, linear epitope Antigenic structure (epitope) in a protein that is formed by a single small region of amino acid sequence. Antibodies that bind continuous epitopes can bind to the denatured protein. The epitopes detected by T cells are continuous. Also called a linear epitope. conventional (or classical) dendritic cells (cDCs) The lineage of dendritic cells that mainly participates in antigen presentation to, and activation of, naive T cells. Cf. plasmacytoid dendritic cells.

1	co-receptors Cell-surface protein that increases the sensitivity of a receptor to its ligand by binding to associated ligands and participating in signaling. The antigen receptors on T cells and B cells act in conjunction with co-receptors, which are either CD4 or CD8 on T cells, and a co-receptor complex of three proteins, one of which is the complement receptor CR2, on B cells. cortex The outer part of a tissue or organ; in lymph nodes it refers to the follicles, which are mainly populated by B cells. corticosteroids Family of drugs related to natural steroids such as cortisone. Corticosteroids can kill lymphocytes, especially developing thymocytes, inducing apoptotic cell death. They are medically useful anti-inﬂammatory and immunosuppressive agents. co-stimulatory molecules Cell-surface proteins on antigen-presenting cells that deliver co-stimulatory signals to naive T cells. Examples are the B7 molecules on dendritic cells, which are ligands for CD28 on naive T cells.

1	co-stimulatory receptors Cell-surface receptors on naive lymphocytes through which the cells receive signals additional to those received through the antigen receptor, and which are necessary for the full activation of the lymphocyte. Examples are CD30 and CD40 on B cells, and CD27 and CD28 on T cells. CR1 (CD35) A receptor expressed by phagocytic cells that binds to C3b. It stimulates phagocytosis and inhibits C3 convertase formation on host-cell surfaces. CR2 (CD21) Complement receptor that is part of the B-cell co-receptor complex. It binds to antigens coated with breakdown products of C3b, especially C3dg, and, by cross-linking the B-cell receptor, enhances sensitivity to antigen at least 100-fold. It is also the receptor used by the Epstein–Barr virus to infect B cells.

1	CR3 (CD11b:CD18) Complement receptor 3. A β2 integrin that acts both as an adhesion molecule and as a complement receptor. CR3 on phagocytes binds iC3b, a breakdown product of C3b on pathogen surfaces, and stimulates phagocytosis. CR4 (CD11c:CD18) A β2 integrin that acts both as an adhesion molecule and as a complement receptor. CR4 on phagocytes binds iC3b, a breakdown product of C3b on pathogen surfaces, and stimulates phagocytosis. CRAC channel Channels in the lymphocyte plasma membrane that open to let calcium ﬂow into the cell during the response of the cell to antigen. Channel opening is induced by release of calcium from the endoplasmic reticulum. C-reactive protein An acute-phase protein that binds to phosphocholine, a constituent of the surface C-polysaccharide of the bacterium Streptococcus pneumoniae and of many other bacteria, thus opsonizing them for uptake by phagocytes. CRIg (complement receptor of the immunoglobulin family)

1	CRIg (complement receptor of the immunoglobulin family) A complement receptor that binds to inactivated forms of C3b. Crohn’s disease Chronic inﬂammatory bowel disease thought to result from an abnormal overresponsiveness to the commensal gut microbiota. cross-matching A test used in blood typing and histocompatibility typing to determine whether donor and recipient have antibodies against each other’s cells that might interfere with successful transfusion or grafting. cross-presentation The process by which extracellular proteins taken up by dendritic cells can give rise to peptides presented by MHC class I molecules. It enables antigens from extracellular sources to be presented by MHC class I molecules and activate CD8 T cells.

1	cross-priming Activation of CD8 T cells by dendritic cells in which the antigenic peptide presented by MHC class I molecules is derived from an exogenous protein (i.e., by cross-presentation), rather than produced within the dendritic cells directly. Cf. direct presentation. cryptdins α-Defensins (antimicrobial peptides) made by the Paneth cells of the small intestine. cryptic epitopes Any epitope that cannot be recognized by a lymphocyte receptor until the antigen has been broken down and processed. cryptopatches Aggregates of lymphoid tissue in the gut wall that are thought to give rise to isolated lymphoid follicles. CstF-64 Subunit of cleavage stimulation factor that favors polyadenylation at pAS leading to the secreted form of IgM. C-terminal Src kinase (Csk) A kinase that phosphorylates the C-terminal tyrosine of Src-family kinases in lymphocytes, thus inactivating them.

1	C-terminal Src kinase (Csk) A kinase that phosphorylates the C-terminal tyrosine of Src-family kinases in lymphocytes, thus inactivating them. CTLA-4 A high-affinity inhibitory receptor on T cells for B7 molecules; its binding inhibits T-cell activation. C-type lectins Large class of carbohydrate-binding proteins that require Ca2+ for binding, including many that function in innate immunity. cutaneous lymphocyte antigen (CLA) A cell-surface molecule that is involved in lymphocyte homing to the skin in humans. CVIDs See common variable immunodeficiencies. CX3CR1 Chemokine receptor expressed by monocytes, macrophages, NK cells, and activated T cells that binds CXCL1 (Fractalkine). CXC chemokines One of the two main classes of chemokines, distinguished by a Cys-X-Cys (CXC) motif near the amino terminus. They have names CXCL1, CXCL2, etc. See Appendix IV for a list of individual chemokines.

1	CXCL12 (SDF-1) Chemokine produced by stromal cells in the dark zone of the germinal center that binds CXCR4 expressed by centroblasts. CXCL13 Chemokine produced in the follicle and the light zone of the germinal center that binds CXCR5 expressed on circulating B cells and centrocytes. CXCR5 A chemokine receptor expressed by circulating B cells and activated T cells that binds the chemokine CXCL13 and directs cell migration into the follicle. cyclic dinucleotides (CDNs) Cyclic dimers of guanylate and/or adenylate monophosphate that are produced by various bacteria as second messengers and detected by STING. cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP or cGAMP) See cyclic dinucleotides (CDNs).

1	cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP or cGAMP) See cyclic dinucleotides (CDNs). cyclic neutropenia A dominantly inherited disease in which neutrophil numbers ﬂuctuate from near normal to very low or absent, with an approximate cycle time of 21 days. This is in contrast to severe congenital neutropenia (SCN), in which the inherited defect results in persistently low neutrophil numbers. cyclic reentry model An explanation of the behavior of B cells in lymphoid follicles, proposing that activated B cells in germinal centers lose and gain expression of the chemokine receptor CXCR4 and thus move from the light zone to the dark zone and back again under the inﬂuence of the chemokine CXCL12. cyclophilins A family of prolylisomerases that affect protein folding, that also bind cyclosporin A to produce a complex that associates with calcineurin, preventing its activation by calmodulin.

1	cyclophilins A family of prolylisomerases that affect protein folding, that also bind cyclosporin A to produce a complex that associates with calcineurin, preventing its activation by calmodulin. cyclophosphamide A DNA alkylating agent that is used as an immunosuppressive drug. It acts by killing rapidly dividing cells, including lymphocytes proliferating in response to antigen. cyclosporin A (CsA) A powerful noncytotoxic immunosuppressive drug that inhibits signaling from the T-cell receptor, preventing T-cell activation and effector function. It binds to cyclophilin, and the complex formed binds to and inactivates the phosphatase calcineurin. cystic fibrosis Disease caused by defect in CFTR gene, leading to abnormally thick mucus and causing serious recurrent infections of the lung. cytidine deaminase activity (CDA) An enzymatic activity exhibited by AID-APOBEC family proteins of agnathan species that may mediate rearrangement and assembly of complete VLR genes.

1	cytidine deaminase activity (CDA) An enzymatic activity exhibited by AID-APOBEC family proteins of agnathan species that may mediate rearrangement and assembly of complete VLR genes. cytokines Proteins made by a cell that affect the behavior of other cells, particularly immune cells. Cytokines made by lymphocytes are often called interleukins (abbreviated IL). Cytokines and their receptors are listed in Appendix III. Cf. chemokines. cytomegalovirus UL16 protein A nonessential glycoprotein of cytomegalovirus that is recognized by innate receptors expressed by NK cells. cytosol One of several major compartments within cells containing elements such as the cytoskeleton, and mitochondria, and separated by membranes from distinct compartments such as the nucleus and vesicular system. cytotoxic T cells T cells that can kill other cells, typically CD8 T cells defending against intracellular pathogens that live or reproduce in the cytosol, but in some cases also CD4 T cells.

1	cytotoxic T cells T cells that can kill other cells, typically CD8 T cells defending against intracellular pathogens that live or reproduce in the cytosol, but in some cases also CD4 T cells. daclizumab Antibody to human CD25 used to block IL-2 receptor signaling in T cells for treatment of rejection in renal transplantation. DAG See diacylglycerol. damage-associated molecular patterns (DAMPs) See pathogen-associated molecular patterns (PAMPs). DAP10, DAP12 Signaling chains containing ITAMS that are associated with the tails of some activating receptors on NK cells. dark zone See germinal center. DC-SIGN A lectin on the dendritic-cell surface that binds ICAM-3 with high affinity. DDX41 (DEAD box polypeptide 41) A candidate DNA sensor of the RLR family that appears to signal through the STING pathway.

1	DDX41 (DEAD box polypeptide 41) A candidate DNA sensor of the RLR family that appears to signal through the STING pathway. death effector domain (DED) Protein-interaction domain originally discovered in proteins involved in programmed cell death or apoptosis. As part of the intracellular domains of some adaptor proteins, death domains are involved in transmitting pro-inﬂammatory and/or pro-apoptotic signals. death-inducing signaling complex (DISC) A multi-protein complex that is formed by signaling through members of the ‘death receptor’ family of apoptosis-inducing cellular receptors, such as Fas. It activates the caspase cascade to induce apoptosis. decay-accelerating factor (DAF or CD55) A cell-surface protein that protects cells from lysis by complement. Its absence causes the disease paroxysmal nocturnal hemoglobinuria. Dectin-1 A phagocytic receptor on neutrophils and macrophages that recognizes β-1,3-linked glucans, which are common components of fungal cell walls.

1	Dectin-1 A phagocytic receptor on neutrophils and macrophages that recognizes β-1,3-linked glucans, which are common components of fungal cell walls. defective ribosomal products (DRiPs) Peptides translated from introns in improperly spliced mRNAs, translations of frameshifts, or improperly folded proteins, which are recognized and tagged by ubiquitin for degradation by the proteasome. defensins See α-defensins, β-defensins. delayed-type hypersensitivity reactions A form of cell-mediated immunity elicited by antigen in the skin stimulating sensitized Th1 CD4 lymphocytes and CD8 lymphocytes. It is called delayed-type hypersensitivity because the reaction appears hours to days after antigen is injected. Referred to as type IV hypersensitivity in the historic Gell and Coombs classification.

1	dendritic cells Bone marrow-derived cells found in most tissues, including lymphoid tissues. There are two main functional subsets. Conventional dendritic cells take up antigen in peripheral tissues, are activated by contact with pathogens, and travel to the peripheral lymphoid organs, where they are the most potent stimulators of T-cell responses. Plasmacytoid dendritic cells can also take up and present antigen, but their main function in an infection is to produce large amounts of the antiviral interferons as a result of pathogen recognition through receptors such as TLRs. Both these types of dendritic cells are distinct from the follicular dendritic cell that presents antigen to B cells in lymphoid follicles. dendritic epidermal T cells (dETCs) A specialized class of γ:δ T cells found in the skin of mice and some other species, but not humans. They express Vγ5:Vδ1 and may interact with ligands such as Skint-1 expressed by keratinocytes.

1	dephosphorylation The removal of a phosphate group from a molecule, usually a protein. depleting antibodies Immunosuppressive monoclonal antibodies that trigger the destruction of lymphocytes in vivo. They are used for treating episodes of acute graft rejection. diacyl and triacyl lipoproteins Ligands for the Toll-like receptors TLR1:TLR2 and TLR2:TLR6. diacylglycerol A lipid intracellular signaling molecule formed from membrane inositol phospholipids that are cleaved by the action of phospholipase C-γ after the activation of many different receptors. The diacylglycerol stays in the membrane and activates protein kinase C and RasGRP, which further propagate the signal. diapedesis The movement of blood cells, particularly leukocytes, from the blood across blood vessel walls into tissues. differentiation antigens Referring to a category of genes with restricted expression patterns that can be targeted as antigens by immunotherapies in treatment of cancers.

1	differentiation antigens Referring to a category of genes with restricted expression patterns that can be targeted as antigens by immunotherapies in treatment of cancers. DiGeorge syndrome Recessive genetic immunodeficiency disease in which there is a failure to develop thymic epithelium. Parathyroid glands are also absent and there are anomalies in the large blood vessels. direct allorecognition Host recognition of a grafted tissue that involves donor antigen-presenting cells leaving the graft, migrating via the lymph to regional lymph nodes, and activating host T cells bearing the corresponding T-cell receptors. direct presentation The process by which proteins produced within a given cell give rise to peptides presented by MHC class I molecules. This may refer to antigen-presenting cells, such as dendritic cells, or to nonimmune cells that will become the targets of CTLs.

1	dislocation In reference to viral defense mechanisms, the degradation of newly synthesized MHC class I molecules by viral proteins. disseminated intravascular coagulation (DIC) Blood clotting occurring simultaneously in small vessels throughout the body in response to disseminated TNF-α, which leads to the massive consumption of clotting proteins, so that the patient’s blood cannot clot appropriately. Seen in septic shock. diversion colitis Inﬂammation and necrosis of intestinal enterocytes following surgical diversion of normal ﬂow of fecal contents due to impaired metabolism resulting from loss of short-chain fatty acids derived from microbiota. diversity gene segment (DH) Short DNA sequences that form a join between the V and J gene segments in rearranged immunoglobulin heavy-chain genes and in T-cell receptor βand δ-chain genes. See gene segments.

1	DN1, DN2, DN3, DN4 Substages in the development of CD4+CD8+ double-positive T cells in the thymus. Rearrangement of the TCRβ-chain locus starts at DN2 and is completed by DN4. DNA-dependent protein kinase (DNA-PK) Protein kinase in the DNA repair pathway involved in the rearrangement of immunoglobulin and T-cell receptor genes. DNA ligase IV Enzyme responsible for joining the DNA ends to produce the coding joint during V(D)J recombination. DNA transposons Genetic elements encoding their own transposase that can insert themselves into and excise themselves from the DNA genomes of a host. DNA vaccination Vaccination by introduction into skin and muscle of DNA encoding the desired antigen; the expressed protein can then elicit antibody and T-cell responses. donor lymphocyte infusion (DLI) Transfer of mature lymphocytes (i.e., T cells) from donor into patients during bone marrow transplantation for cancer treatment to help eliminate residual tumor.

1	donor lymphocyte infusion (DLI) Transfer of mature lymphocytes (i.e., T cells) from donor into patients during bone marrow transplantation for cancer treatment to help eliminate residual tumor. double-negative thymocytes Immature T cells in the thymus that lack expression of the two co-receptors CD4 and CD8 and represent the progenitors to the remaining T cells developing in the thymus. In a normal thymus, these represent about 5% of thymocytes. double-positive thymocytes Immature T cells in the thymus that are characterized by expression of both the CD4 and the CD8 co-receptor proteins. They represent the majority (about 80%) of thymocytes and are the progenitors to the mature CD4 and CD8 T cells. double-strand break repair (DSBR) A nonhomologous end joining pathway of DNA repair used in the completion of isotype switching. double-stranded RNA (dsRNA) A chemical structure that is a replicative intermediate of many viruses that is recognized by TLR-3.

1	double-stranded RNA (dsRNA) A chemical structure that is a replicative intermediate of many viruses that is recognized by TLR-3. Down syndrome cell adhesion molecule (Dscam) See Dscam. DR4, DR5 Members of the TNFR superfamily expressed by many cell types that can be activated by the TRAIL to induce apoptosis. draining lymph nodes A lymph node downstream of a site of infection that receives antigens and microbes from the site via the lymphatic system. Draining lymph nodes often enlarge enormously during an immune response and can be palpated; they were originally called swollen glands. Dscam A member of the immunoglobulin superfamily that in insects is thought to opsonize invading bacteria and aid their engulfment by phagocytes. It can be made in a multiplicity of different forms as a result of alternative splicing.

1	dysbiosis Altered balance of microbial species comprising the microbiota resulting from a variety of causes (e.g., antibiotics, genetic disorders) and frequently associated with outgrowth of pathogenic organisms such as Clostridium difficile. dysregulated self Refers to changes that take place in infected or malignant cells that alter expression of various surface receptors that can be detected by the innate immune system. E3 ligase An enzymatic activity that directs the transfer of a ubiquitin molecule from an E2 ubiquitin-conjugating enzyme onto a specific protein target. early-onset sarcoidosis Disease associated with activating NOD2 mutations characterized by inﬂammation in tissues such as liver. early pro-B cell See pro-B cells. EBI2 (GPR183) A chemokine receptor that binds oxysterols and regulates B-cell movement to the outer follicular and interfollicular regions during early phases of B-cell activation in lymphoid tissues.

1	E-cadherin Integrin expressed by epithelial cells important in forming the adherens junctions between adjacent cells. edema Swelling caused by the entry of ﬂuid and cells from the blood into the tissues; it is one of the cardinal features of inﬂammation. effector caspases Intracellular proteases that are activated as a result of an apoptotic signal and mediate the cellular changes associated with apoptosis. To be distinguished from initiator caspases, which act upstream of effector caspases to initiate the caspase cascade. effector CD4 T cells The subset of differentiated effector T cells carrying the CD4 co-receptor molecule, which includes the TH1, TH2, TH17, and regulatory T cells.

1	effector CD4 T cells The subset of differentiated effector T cells carrying the CD4 co-receptor molecule, which includes the TH1, TH2, TH17, and regulatory T cells. effector lymphocytes The cells that differentiate from naive lymphocytes after initial activation by antigen and can then mediate the removal of pathogens from the body without further differentiation. They are distinct from memory lymphocytes, which must undergo further differentiation to become effector lymphocytes. effector mechanisms Those processes by which pathogens are destroyed and cleared from the body. Innate and adaptive immune responses use most of the same effector mechanisms to eliminate pathogens. effector memory T cells (TEM) Memory lymphocytes that recirculate between blood and peripheral tissues and are specialized for rapid maturation into effector T cells after restimulation with antigen in non-lymphoid tissues.

1	effector modules This term refers to a set of immune mechanisms, either cell-mediated and humoral, innate or adaptive, that act together in the elimination of a particular category of pathogen. effector T lymphocytes The T cells that perform the functions of an immune response, such as cell killing and cell activation, that clear the infectious agent from the body. There are several different subsets, each with a specific role in an immune response. electrostatic interactions Chemical interaction occurring between charged atoms, as in the charged amino acid side chains and an ion in a salt bridge. elimination phase Stage of anti-tumor immune response that detects and eliminates cancer cells, also called immune surveillance. elite controllers A subset of HIV-infected long-term non-progressors who have clinically undetectable levels of virus without antiretroviral therapy.

1	elite controllers A subset of HIV-infected long-term non-progressors who have clinically undetectable levels of virus without antiretroviral therapy. ELL2 A transcription elongation factor that favors the polyadenylation at pAS leading to the secreted form of IgM. endocrine Describes the action of a biologically active molecule such as a hormone or cytokine that is secreted by one tissue into the blood and acts on a distant tissue. Cf. autocrine, paracrine. endogenous pyrogens Cytokines that can induce a rise in body temperature. endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) Enzyme in the endoplasmic reticulum that trims polypeptides to a size at which they can bind to MHC class I molecules. A system of enzymes in the endoplasmic reticulum that recognizes incompletely or misfolded proteins and assures their eventual degradation.

1	A system of enzymes in the endoplasmic reticulum that recognizes incompletely or misfolded proteins and assures their eventual degradation. endosteum The region in bone marrow adjacent to the inner surface of the bone; hematopoietic stem cells are initially located there. endothelial activation The changes that occur in the endothelial walls of small blood vessels as a result of inﬂammation, such as increased permeability and the increased production of cell-adhesion molecules and cytokines. endothelial cell Cell type that forms the endothelium, the epithelium of a blood vessel wall. endothelial protein C receptor (EPCR) A nonclassical MHC class I protein induced on endothelial cells that can interact with the blood coagulation factor XIV (protein C) and can be recognized by some γ:δ T cells. endothelium The epithelium that forms the walls of blood capillaries and the lining of larger blood vessels.

1	endothelium The epithelium that forms the walls of blood capillaries and the lining of larger blood vessels. endotoxins Toxins derived from bacterial cell walls released by damaged cells. They can potently induce cytokine synthesis and in large amounts can cause a systemic reaction called septic shock or endotoxic shock. enteroadherent Escherichia coli Referring to multiple strains of E. coli capable of attachment to, and infection and destruction of cells of the intestinal microvilli, causing colitis and diarrheagenic diseases. eomesodermin A transcription factor involved in development and function of certain types of NK cells, ILCs, and CD8 T cells. eosinophilia An abnormally large number of eosinophils in the blood. eosinophils A type of white blood cell containing granules that stain with eosin. It is thought to be important chieﬂy in defense against parasitic infections, but is also medically important as an effector cell in allergic reactions.

1	eotaxins CC chemokines that act predominantly on eosinophils, including CCL11 (eotaxin 1), CCL24 (eotaxin 2), and CCL26 (eotaxin 3). epitope A site on an antigen recognized by an antibody or an antigen receptor. T-cell epitopes are short peptide bound to MHC molecules. B-cell epitopes are typically structural motifs on the surface of the antigen. Also called an antigenic determinant. epitope spreading Increase in diversity of responses to autoantigens as the response persists, as a result of responses being made to epitopes other than the original one. equilibrium phase Stage of anti-tumor immune response when immunoediting allows the immune response to continuously shape the antigenic character of cancer cells. Erk Extracellular signal-related kinase, a protein kinase that is the MAPK for one module of the T-cell receptor signaling pathway. Erk also functions in other receptors in other cell types.

1	Erk Extracellular signal-related kinase, a protein kinase that is the MAPK for one module of the T-cell receptor signaling pathway. Erk also functions in other receptors in other cell types. ERp57 A chaperone protein involved in loading peptide onto MHC class I molecules in the endoplasmic reticulum. error-prone ‘translesion’ DNA polymerases A DNA polymerase operates during DNA repair, such as Polη which can repair a basic lesion by incorporating untemplated nucleotides into the newly formed DNA strand. escape mutants Mutants of pathogens that are changed in such a way that they can evade the immune response against the original pathogen. escape phase Final stage of anti-tumor immune response when immunoediting has removed the expression of antigenic targets such that the cancer cells are no loner detected by the immune system. E-selectin See selectins.

1	E-selectin See selectins. etanercept Fc fusion protein containing the p75 subunit of the TNF receptor that neutralizes TNF-α used for treatment of rheumatoid arthritis and other inﬂammatory diseases. eukaryotic initiation factor 2 (eIF2α) Subunit of eukaryotic initiation factor that helps form the preinitiation complex that begins protein translation from mRNA. When it is phosphorylated by PKR, protein translation is suppressed. eukaryotic initiation factor 3 (eIF3) Multisubunit complex that acts in formation of the 43S preinitiation complex. It can bind interferon-induced transmembrane (IFIT) proteins which thereby suppress translation of viral proteins. exogenous pyrogen Any substance originating outside the body that can induce fever, such as the bacterial lipopolysaccharide LPS. Cf. endogenous pyrogens. exotoxins A protein toxin produced and secreted by a bacterium.

1	exotoxins A protein toxin produced and secreted by a bacterium. experimental autoimmune encephalomyelitis (EAE) An inﬂammatory disease of the central nervous system that develops after mice are immunized with neural antigens in a strong adjuvant. extrachromosomal DNA DNA not contained within chromosomes, such as the circular DNA produced by V(D)J recombination occurring between RSSs in the same chromosomal orientation and is eventually lost from the cell. extravasation The movement of cells or ﬂuid from within blood vessels into the surrounding tissues. extrinsic pathway of apoptosis A pathway triggered by extracellular ligands binding to specific cell-surface receptors (death receptors) that signal the cell to undergo programmed cell death (apoptosis).

1	Fab fragment Antibody fragment composed of a single antigen-binding arm of an antibody without the Fc region, produced by cleavage of IgG by the enzyme papain. It contains the complete light chain plus the amino-terminal variable region and first constant region of the heavy chain, held together by an interchain disulfide bond. F(abʹ)2 fragment Antibody fragment composed of two linked antigen-binding arms (Fab fragments) without the Fc regions, produced by cleavage of IgG with the enzyme pepsin. factor B Protein in the alternative pathway of complement activation, in which it is cleaved to Ba and an active protease, Bb, the latter binding to C3b to form the alternative pathway C3 convertase, C3bBb. factor D A serine protease in the alternative pathway of complement activation, which cleaves factor B into Ba and Bb. factor H Complement-regulatory protein in plasma that binds C3b and competes with factor B to displace Bb from the convertase.

1	factor H Complement-regulatory protein in plasma that binds C3b and competes with factor B to displace Bb from the convertase. factor H binding protein (fHbp) A protein produced by the pathogen Neisseria meningitidis that recruits factor H to its membrane, thereby inactivating C3b deposited on its surface, and evading destruction by complement. factor I Complement-regulatory protease in plasma that cleaves C3b to the inactive derivative iC3b, thus preventing the formation of a C3 convertase. factor I deficiency A genetically determined lack of the complement-regulatory protein factor I. This results in uncontrolled complement activation, so that complement proteins rapidly become depleted. Those with the deficiency suffer repeated bacterial infections, especially with ubiquitous pyogenic bacteria. factor P Plasma protein released by activated neutrophils that stabilizes the C3 convertase C3bBb of the alternative pathway.

1	factor P Plasma protein released by activated neutrophils that stabilizes the C3 convertase C3bBb of the alternative pathway. familial cold autoinflammatory syndrome (FCAS) An episodic autoinﬂammatory disease caused by mutations in the gene NLRP3, encoding NLRP3, a member of the NOD-like receptor family and a component of the inﬂammasome. The symptoms are induced by exposure to cold. familial hemophagocytic lymphohistiocytosis (FHL) A family of progressive and potentially lethal inﬂammatory diseases caused by an inherited deficiency of one of several proteins involved in the formation or release of cytolytic granules. Large numbers of polyclonal CD8-positive T cells accumulate in lymphoid and other organs, and this is associated with activated macrophages that phagocytose blood cells, including erythrocytes and leukocytes.

1	familial Mediterranean fever (FMF) A severe autoinﬂammatory disease, inherited as an autosomal recessive disorder. It is caused by mutation in the gene (MEFV) that encodes the protein pyrin, which is expressed in granulocytes and monocytes. In patients with this disorder, defective pyrin is thought to spontaneously activate inﬂammasomes. farmer’s lung A hypersensitivity disease caused by the interaction of IgG antibodies with large amounts of an inhaled antigen in the alveolar wall of the lung, causing alveolar wall inﬂammation and compromising respiratory gas exchange. Fc fragment, Fc region The carboxy-terminal halves of the two heavy chains of an IgG molecule disulfide-bonded to each other by the residual hinge region. It is produced by cleavage of IgG by papain. In the complete antibody this portion is often called the Fc region.

1	Fc receptors Family of cell-surface receptors that bind the Fc portions of different immunoglobulins: Fcγ receptors bind IgG, for example, and Fcε receptors bind IgE. FCAS See familial cold autoinflammatory syndrome. FcεRI The high affinity receptor for the Fc region of IgE. Expressed primarily on the surface of mast cells and basophils. When multivalent antigen interacts with IgE that is bound to FcεRI and cross-links nearby receptors, it causes activation of the receptor-bearing cell. FcγR1 (CD64) Fc receptor highly expressed by monocytes and macrophages that has the highest affinity of the Fc receptors for IgG. FcγRIIB-1 An inhibitory receptor on B cells that recognizes the Fc portion of IgG antibodies. FcγRIIB-1 contains an ITIM motif in its cytoplasmic tail.

1	FcγRIIB-1 An inhibitory receptor on B cells that recognizes the Fc portion of IgG antibodies. FcγRIIB-1 contains an ITIM motif in its cytoplasmic tail. FcγRIII Cell-surface receptors that bind the Fc portion of IgG molecules. Most Fcγ receptors bind only aggregated IgG, allowing them to discriminate bound antibody from free IgG. Expressed variously on phagocytes, B lymphocytes, NK cells, and follicular dendritic cells, the Fcγ receptors have a key role in humoral immunity, linking antibody binding to effector cell functions. FcRn (neonatal Fc receptor) Neonatal Fc receptor, a receptor that transports IgG from mother to fetus across the placenta, and across other epithelia such as the epithelium of the gut. FHL See familial hemophagocytic lymphohistiocytosis. fibrinogen-related proteins (FREPs) Members of the immunoglobulin superfamily that are thought to have a role in innate immunity in the freshwater snail Biomphalaria glabrata.

1	fibrinogen-related proteins (FREPs) Members of the immunoglobulin superfamily that are thought to have a role in innate immunity in the freshwater snail Biomphalaria glabrata. ficolins Carbohydrate-binding proteins that can initiate the lectin pathway of complement activation. They are members of the collectin family and bind to the N-acetylglucosamine present on the surface of some pathogens. fingolimod Small-molecule immunosuppressive drug that interferes with the actions of sphingosine, leading to retention of effector T cells in lymphoid organs. FK506 See tacrolimus. FK-binding proteins (FKBPs) Group of prolyl isomerases related to the cyclophilins and bind the immunosuppressive drug FK506 (tacrolimus). flagellin A protein that is the major constituent of the ﬂagellum, the tail-like structure used in bacterial locomotion. TLR-5 recognizes intact ﬂagellin protein that has dissociated from the ﬂagellum.

1	fluid-phase C3 convertase Short-lived alternative pathway C3 convertase, C3(H20)Bb, that is continually produced at a low level in the plasma that can initiate activation of the alternative pathway of complement. fMet-Leu-Phe (fMLF) receptor A pattern recognition receptor for the peptide fMet-Leu-Phe, which is specific to bacteria, on neutrophils and macrophages. fMet-Leu-Phe acts as a chemoattractant. folic acid A B vitamin, derivatives of folic acid produced by various bacteria can be bound by the nonclassical MHC class Ib protein MR1 for recognition by MAIT cells. follicle-associated epithelium Specialized epithelium separating the lymphoid tissues of the gut wall from the intestinal lumen. As well as enterocytes it contains microfold cells, through which antigens enter the lymphoid organs from the gut. follicles An area of predominantly B cells in a peripheral lymphoid organ, such as a lymph node, which also contains follicular dendritic cells.

1	follicles An area of predominantly B cells in a peripheral lymphoid organ, such as a lymph node, which also contains follicular dendritic cells. follicular B cells The majority population of long-lived recirculating conventional B cells found in the blood, the spleen, and the lymph nodes. Also known as B-2 B cells. follicular dendritic cell (FDC) A cell type of uncertain origin in B-cell follicles of peripheral lymphoid organs that captures antigen:antibody complexes using non-internalized Fc receptors and presents them to B cells for internalization and processing during the germinal center reaction. follicular helper T cell (TFH) Type of effector CD4 T cell that resides in lymphoid follicles and provides help to B cells for antibody production. framework regions Relatively invariant regions that provide a protein scaffold for the hypervariable regions in the V domains of immunoglobulins and T-cell receptors.

1	framework regions Relatively invariant regions that provide a protein scaffold for the hypervariable regions in the V domains of immunoglobulins and T-cell receptors. Freund’s complete adjuvant Emulsion of oil and water containing killed mycobacteria used to enhance immune responses to experimental antigens. fungi A kingdom of single-celled and multicellular eukaryotic organisms, including the yeasts and molds, that can cause a variety of diseases. Immunity to fungi is complex and involves both humoral and cell-mediated responses. Fyn See Src-family tyrosine kinases. γ:δ T cells Subset of T lymphocytes bearing a T-cell receptor composed of the antigen-recognition chains, γ and δ, assembled in a γ:δ heterodimer. γ:δ T-cell receptors Antigen receptor carried by a subset of T lymphocytes that is distinct from the α:βT-cell receptor. It is composed of a γ and a δ chain, which are produced from genes that undergo gene rearrangement.

1	γ-glutamyl diaminopimelic acid (iE-DAP) A product of degradation of the peptidoglycan of Gram-negative bacteria. It is sensed by NOD1. GAP See GTPase-activating proteins. GEFs See guanine nucleotide exchange factors. gene rearrangement The process of somatic recombination of gene segments in the immunoglobulin and T-cell receptor genetic loci to produce a functional gene. This process generates the diversity found in immunoglobulin and T-cell receptor variable regions.

1	gene segments Sets of short DNA sequences at the immunoglobulin and T-cell receptor loci that encode different regions of the variable domains of antigen receptors. Gene segments of each type are joined together by somatic recombination to form a complete variable-domain exon. There are three types of gene segments: V gene segments encode the first 95 amino acids, D gene segments (in heavy-chain and TCRα chain loci only) encode about 5 amino acids, and J gene segments encode the last 10–15 amino acids of the variable domain. There are multiple copies of each type of gene segment in the germline DNA, but only one of each type is joined together to form the variable domain. genetic locus The site of a gene on a chromosome. In the case of the genes for the immunoglobulin and T-cell receptor chains, the term locus refers to the complete collection of gene segments and C-region genes for the given chain.

1	genome-wide association studies (GWASs) Genetic association studies in the general population that look for a correlation between disease frequency and variant alleles by scanning the genomes of many people for the presence of informative single-nucleotide polymorphisms (SNPs). germ-free mice Mice that are raised in the complete absence of intestinal and other microorganisms. Such mice have very depleted immune systems, but they can respond virtually normally to any specific antigen, provided it is mixed with a strong adjuvant. germinal center Sites of intense B-cell proliferation and differentiation that develop in lymphoid follicles during an adaptive immune response. Somatic hypermutation and class switching occur in germinal centers. germline theory An excluded hypothesis that antibody diversity was encoded by a separate germline gene for each antibody, known not to be true for most vertebrates, although cartilaginous fishes do have some rearranged V regions in the germline.

1	glycosylphosphatidylinositol (GPI) tail A glycolipid modification of proteins that can allow attachment to host membranes without the requirement of a transmembrane protein domain. gnathostomes The class of jawed vertebrates comprising most fish and all mammals. These possess an adaptive immunity based on the RAG-mediated V(D)J recombination. gnotobiotic mice See germ-free mice. goblet cells Specialized epithelial cells located in many sites throughout the body responsible for mucus production; important in protection of the epithelium. Goodpasture’s syndrome An autoimmune disease in which autoantibodies against type IV collagen (found in basement membranes) are produced, causing extensive inﬂammation in kidneys and lungs. gout Disease caused by monosodium urate crystals deposited in the cartilaginous tissues of joints, causing inﬂammation. Urate crystals activate the NLRP3 inﬂammasome, which induces inﬂammatory cytokines.

1	G proteins Intracellular GTPases that act as molecular switches in signaling pathways. They bind GTP to induce their active conformation, which is lost when GTO is hydrolyzed to GDP. There are two kinds of G proteins: the heterotrimeric (α, β, γ subunits) receptor-associated G proteins, and the small G proteins, such as Ras and Raf, which act downstream of many transmembrane signaling events. G-protein-coupled receptors (GPCRs) A large class of seven-span transmembrane cell-surface receptors that associate with intracellular heterotrimeric G proteins after ligand binding, and signal by activation of the G protein. Important examples are the chemokine receptors. G-quadruplex A structure formed from G-rich regions of DNA in which four guanine bases form a planar hydrogen-bonded network, or guanine tetrad, that can further stack on other guanine tetrads. G-quadruplexes processed from intronic switch region RNA may target AID back to the switch regions during isotype switching.

1	graft rejection See allograft rejection. graft-versus-host disease (GVHD) An attack on the tissues of the recipient by mature T cells in a bone marrow graft from a nonidentical donor, which can cause a variety of symptoms; sometimes these are severe. graft-versus-leukemia effect A beneficial side-effect of bone marrow grafts given to treat leukemia, in which mature T cells in the graft recognize minor histocompatibility antigens or tumor-specific antigens on the recipient’s leukemic cells and attack them. Gram-negative bacteria Bacteria that fail to retain crystal violet stain following alcohol wash due to a thin peptidoglycan layer. Gram-negative binding proteins (GNBPs) Proteins that act as the pathogen-recognition proteins in the Toll pathway of immune defense in Drosophila.

1	Gram-negative binding proteins (GNBPs) Proteins that act as the pathogen-recognition proteins in the Toll pathway of immune defense in Drosophila. granulocyte-macrophage stimulating factor (GM-CSF) A cytokine involved in the growth and differentiation of cells of the myeloid lineage, including dendritic cells, monocytes and tissue macrophages, and granulocytes. granulocytes White blood cells with multilobed nuclei and cytoplasmic granules. They comprise the neutrophils, eosinophils, and basophils. Also known as polymorphonuclear leukocytes. granuloma A site of chronic inﬂammation usually triggered by persistent infectious agents such as mycobacteria or by a nondegradable foreign body. Granulomas have a central area of macrophages, often fused into multinucleate giant cells, surrounded by T lymphocytes.

1	Grass A serine protease of Drosophila that functions downstream of peptidoglycan-recognition proteins (PGRPs) and Gram-negative binding proteins (GNBPs) to initiate the proteolytic cascade leading to Toll activation. Graves’ disease An autoimmune disease in which antibodies against the thyroid-stimulating hormone receptor cause overproduction of thyroid hormone and thus hyperthyroidism. Griscelli syndrome An inherited immunodeficiency disease that affects the pathway for secretion of lysosomes. It is caused by mutations in a small GTPase Rab27a, which controls the movement of vesicles within cells. group 1 ILCs (ILC1s) The subtype of innate lymphoid cells (ILCs) characterized by IFN-γ production. GTPase-activating proteins (GAP) Regulatory proteins that accelerate the intrinsic GTPase activity of G proteins and thus facilitate the conversion of G proteins from the active (GTP-bound) state to the inactive (GDP-bound) state.

1	guanine nucleotide exchange factors (GEFs) Proteins that can remove the bound GDP from G proteins, thus allowing GTP to bind and activate the G protein. gut-associated lymphoid tissues (GALT) Lymphoid tissues associated with the gastrointestinal tract, comprising Peyer’s patches, the appendix, and isolated lymphoid follicles found in the intestinal wall, where adaptive immune responses are initiated, and by lymphatics to mesenteric lymph nodes. GVHD See graft-versus-host disease. H-2 locus, H-2 genes The major histocompatibility complex of the mouse. Haplotypes are designated by a lower-case superscript, as in H-2b. H-2DM See HLA-DM. H-2O See HLA-DO. H2-M3 A nonclassical MHC class Ib protein in mice that can bind and present peptides having an N-formylated amino terminus for recognition by CD8 T cells. H5N1 avian flu A highly pathogenic inﬂuenza subtype responsible for ‘bird ﬂu’.

1	H5N1 avian flu A highly pathogenic inﬂuenza subtype responsible for ‘bird ﬂu’. haploinsufficient Describes the situation in which the presence of only one normal allele of a gene is not sufficient for normal function. hapten carrier effect Antibody production against a small chemical group, the hapten, following its attachment to a carrier protein for which an immune response has been generated. haptens Any small molecule that can be recognized by a specific antibody but cannot by itself elicit an immune response. A hapten must be chemically linked to a protein molecule to elicit antibody and T-cell responses. Hashimoto’s thyroiditis An autoimmune disease characterized by persistent high levels of antibody against thyroid-specific antigens. These antibodies recruit NK cells to the thyroid, leading to damage and inﬂammation.

1	heavy chain, H chain One of the two types of protein chain in an immunoglobulin molecule, the other being called the light chain. There are several different classes, or isotypes, of heavy chain (α,δ, ε, γ, and μ), each of which confers a distinctive functional activity on the antibody molecule. Each immunoglobulin molecule contains two identical heavy chains. heavy-chain-only IgGs (hcIgGs) Antibodies produced by some camelid species composed of heavy-chain dimers without an associated light chain that retain antigen binding capacity. heavy-chain variable region (VH) Referring to the V region of the heavy chain of an immunoglobulin. helicard See MDA-5. helper CD4 T cells, helper T cells Effector CD4 T cells that stimulate or ‘help’ B cells to make antibody in response to antigenic challenge. TH2, TH1, and the TFH subsets of effector CD4 T cells can perform this function.

1	hemagglutinin (HA) Substances that can cause hemagglutination, such as human antibodies that recognize the ABO blood group antigens on red blood cells, or the inﬂuenza virus hemagglutinin, a glycoprotein that functions in viral fusion with endosome membranes. hematopoietic stem cells (HSCs) Type of pluripotent cell in the bone marrow that can give rise to all the different blood cell types. hematopoietin superfamily Large family of structurally related cytokines that includes growth factors and many interleukins with roles in both adaptive and innate immunity. hemochromatosis protein A protein expressed by intestinal epithelial cells that regulates iron uptake and transport by interacting with the transferrin receptor to decrease its affinity for iron-loaded transferrin.

1	hemolytic disease of the newborn A severe form of Rh hemolytic disease in which maternal anti-Rh antibody enters the fetus and produces a hemolytic anemia so severe that the fetus has mainly immature erythroblasts in the peripheral blood. hemophagocytic lymphohistiocytic (HLH) syndrome A dysregulated expansion of CD8-positive lymphocytes that is associated with macrophage activation. The activated macrophages phagocytose blood cells, including erythrocytes and leukocytes. hepatobiliary route Route whereby mucosally produced dimeric IgA enters the portal veins in the lamina propria, is transported to the liver, and reaches the bile duct by transcytosis. This pathway is not of great significance in humans. heptamer The conserved seven-nucleotide DNA sequence in the recombination signal sequences (RSSs) ﬂanking gene segments in the immunoglobulin and T-cell receptor loci.

1	heptamer The conserved seven-nucleotide DNA sequence in the recombination signal sequences (RSSs) ﬂanking gene segments in the immunoglobulin and T-cell receptor loci. HER-2/neu A receptor tyrosine kinase overexpressed in many cancers, particularly breast cancer, that is the target of trastuzumab (Herceptin) used in its treatment. herd immunity Protection conferred to unvaccinated individuals in a population produced by vaccination of others and reduction in the natural reservoir for infection. hereditary angioedema (HAE) A genetic deficiency of the C1 inhibitor of the complement system. In the absence of C1 inhibitor, spontaneous activation of the complement system can cause diffuse ﬂuid leakage from blood vessels, the most serious consequence of which is swelling of the larynx, leading to suffocation. hereditary hemochromatosis A disease caused by defects in the HFE gene characterized by abnormally high retention of iron in the liver and other organs.

1	hereditary hemochromatosis A disease caused by defects in the HFE gene characterized by abnormally high retention of iron in the liver and other organs. herpes virus entry molecule (HVEM) See B and T lymphocyte attenuator. heterosubtypic immunity Immune protection against a pathogen conferred by infection with a distinct strain, typically with reference to different inﬂuenza A serotypes. heterotrimeric G proteins See G proteins. heterozygous Describes individuals that have two different alleles of a given gene, one inherited from the mother and one from the father. HFE See hemochromatosis protein. high endothelial cells, high endothelial venules (HEV) Specialized small venous blood vessels in lymphoid tissues. Lymphocytes migrate from the blood into lymphoid tissues by attaching to the high endothelial cells in the walls of the venules and squeezing between them.

1	highly active antiretroviral therapy (HAART) A combination of drugs that is used to control HIV infection. It comprises nucleoside analogs that prevent reverse transcription, and drugs that inhibit the viral protease. hinge region The ﬂexible domain that joins the Fab arms to the Fc piece in an immunoglobulin. The ﬂexibility of the hinge region in IgG and IgA molecules allows the Fab arms to adopt a wide range of angles, permitting binding to epitopes spaced variable distances apart. HIP/PAP An antimicrobial C-type lectin secreted by intestinal cells in humans. Also known as RegIIIα. histamine A vasoactive amine stored in mast-cell granules. Histamine released by antigen binding to IgE antibodies bound to mast cells causes the dilation of local blood vessels and the contraction of smooth muscle, producing some of the symptoms of IgE-mediated allergic reactions. Antihistamines are drugs that counter histamine action.

1	histatins Antimicrobial peptides constitutively produced by the parotid, sublingual, and submandibular glands in the oral cavity. Active against pathogenic fungi such as Cryptococcus neoformans and Candida albicans. HIV See human immunodeficiency virus. HLA The genetic designation for the human MHC. Individual loci are designated by upper-case letters, as in HLA-A, and alleles are designated by numbers, as in HLA-A*0201. HLA-DM An invariant MHC protein resembling MHC class II in humans that is involved in loading peptides onto MHC class II molecules. A homologous protein in mice is called H-2M, or sometimes H2-DM. HLA-DO An invariant MHC class II molecule that binds HLA-DM, inhibiting the release of CLIP from MHC class II molecules in intracellular vesicles. A homologous protein in mice is called H-2O or H2-DO. homeostatic chemokines Chemokines that are produced at steady-state to direct the localization of immune cells to lymphoid tissues.

1	homeostatic chemokines Chemokines that are produced at steady-state to direct the localization of immune cells to lymphoid tissues. homing The direction of a lymphocyte into a particular tissue. homing receptors Receptors on lymphocytes for chemokines, cytokines, and adhesion molecules specific to particular tissues, and which enable the lymphocyte to enter that tissue. homozygous Describes individuals that have two identical alleles of a given gene, inherited separately from each parent. host-versus-graft disease (HVGD) Another name for the allograft rejection reaction. The term is used mainly in relation to bone marrow transplantation when immune cells of the host recognize and destroy transplanted bone marrow or hematopoietic stem cells (HSCs).

1	human immunodeficiency virus (HIV) The causative agent of the acquired immune deficiency syndrome (AIDS). HIV is a retrovirus of the lentivirus family that selectively infects macrophages and CD4 T cells, leading to their slow depletion, which eventually results in immunodeficiency. There are two major strains of the virus, HIV-1 and HIV-2, of which HIV-1 causes most disease worldwide. HIV-2 is endemic to West Africa but is spreading. human leukocyte antigen (HLA) See HLA. humanization The genetic engineering of mouse hypervariable loops of a desired specificity into otherwise human antibodies for use as therapeutic agents. Such antibodies are less likely to cause an immune response in people treated with them than are wholly mouse antibodies. humoral Referring to effector proteins in the blood or body ﬂuids, such as antibodies in adaptive immunity, or complement proteins in innate immunity.

1	humoral Referring to effector proteins in the blood or body ﬂuids, such as antibodies in adaptive immunity, or complement proteins in innate immunity. humoral immunity, humoral immune response Immunity due to proteins circulating in the blood, such as antibodies (in adaptive immunity) or complement (in innate immunity). Adaptive humoral immunity can be transferred to unimmunized recipients by the transfer of serum containing specific antibody. HVGD See host-versus-graft disease. hydrophobic interaction Chemical interaction occurring between nearby hydrophobic moieties typically excluding water molecules. 21-hydroxylase An enzyme of non-immune function but encoded in the MHC locus required for normal cortisol synthesis by the adrenal gland. 3-hydroxy-3-methylglutaryl-co-enzyme A (HMG-CoA) reductase Rate-limiting enzyme in the production of cholesterol and a target of cholesterol-lowering drugs such as the statins.

1	3-hydroxy-3-methylglutaryl-co-enzyme A (HMG-CoA) reductase Rate-limiting enzyme in the production of cholesterol and a target of cholesterol-lowering drugs such as the statins. hygiene hypothesis A hypothesis first proposed in 1989 that reduced exposure to ubiquitous environmental microorganisms was a cause of the increased frequency of patients with allergies observed over the course of the midto late-20th century. hyper IgE syndrome (HIES) Also called Job’s syndrome. A disease characterized by recurrent skin and pulmonary infections and high serum concentrations of IgE. hyper IgM syndrome A group of genetic diseases in which there is overproduction of IgM antibody, among other symptoms. They are due to defects in various genes for proteins involved in class switching such as CD40 ligand and the enzyme AID. See activation-induced cytidine deaminase, CD40 ligand deficiency. hyper IgM type 2 immunodeficiency See activation-induced cytidine deaminase.

1	hyper IgM type 2 immunodeficiency See activation-induced cytidine deaminase. hyperacute graft rejection Immediate rejection reaction caused by preformed natural antibodies that react against antigens on the transplanted organ. The antibodies bind to endothelium and trigger the blood-clotting cascade, leading to an engorged, ischemic graft and rapid death of the organ. hypereosinophilic syndrome Disease associated with an overproduction of eosinophils. hypervariable regions See complementarity-determining regions. hypomorphic mutations Applied to mutations that result in reduced gene function. IκB A cytoplasmic protein that constitutively associates with the NFκB homodimer, composed of p50 and p65 subunits. When IκB is phosphorylated by activated IKK (IκB kinase), IκB becomes degraded and allows the NFκB dimer to be released as an active transcription factor. IκB kinase (IKK) See IKK. iC3b Inactive complement fragment produced by cleavage of C3b.

1	IκB kinase (IKK) See IKK. iC3b Inactive complement fragment produced by cleavage of C3b. ICAMs ICAM-1, ICAM-2, ICAM-3. Cell-adhesion molecules of the immunoglobulin superfamily that bind to the leukocyte integrin CD11a:CD18 (LFA-1). They are crucial in the binding of lymphocytes and other leukocytes to antigen-presenting cells and endothelial cells. ICOS (inducible co-stimulatory) A CD28-related co-stimulatory receptor that is induced on activated T cells and can enhance T-cell responses. It binds a co-stimulatory ligand known as ICOSL (ICOS ligand), which is distinct from the B7 molecules. ICOSL See ICOS. IFI16 (IFN-γ-inducible protein 16) A member of the PYHIN subfamily of NLR (NOD-like receptor) family containing an N-terminal HIN domain. It activates the STING pathway in response to double-stranded DNA.

1	IFIT (IFN-induced protein with tetratricoid repeats) A small family of host proteins induced by interferons that regulate protein translation during infection in part by interactions with eIF3. IFITM (interferon-induced transmembrane protein) A small family of host transmembrane proteins induced by interferons that function in the cell's vesicular compartment to restrain various steps in viral replication. IFN-α, IFN-β Antiviral cytokines produced by a wide variety of cells in response to infection by a virus, and which also help healthy cells resist viral infection. They act through the same receptor, which signals through a Janus-family tyrosine kinase. Also known as the type I interferons. IFN-γ A cytokine of the interferon structural family produced by effector CD4 TH1 cells, CD8 T cells, and NK cells. Its primary function is the activation of macrophages, and it acts through a different receptor from that of the type I interferons.

1	IFN-γ-induced lysosomal thiol reductase (GILT) An enzyme present in the endosomal compartment of many antigen-presenting cells that denatures disulfide bonds to facilitate the degradation and processing of proteins. IFN-λ Also called type III interferons, this family includes IL-28A, IL-28B, and IL-29, which bind a common receptor expressed by a limited set of epithelial tissues. IFN-λ receptor Receptor composed of a unique IL-28Rα subunit and the β subunit of the IL-10 receptor that recognizes IL-28A, IL-28B, and IL-29. Igα, Igβ See B-cell receptor. IgA Immunoglobulin class composed of α heavy chains that can occur in a monomeric and a polymeric (mainly dimeric) form. Polymeric IgA is the main antibody secreted by mucosal lymphoid tissues.

1	IgA Immunoglobulin class composed of α heavy chains that can occur in a monomeric and a polymeric (mainly dimeric) form. Polymeric IgA is the main antibody secreted by mucosal lymphoid tissues. IgA deficiency The class of immunoglobulin characterized by α heavy chains. It is the most common type of immunodeficiency. It can occur in a monomeric and a polymeric (mainly dimeric) form. Polymeric IgA is the main antibody secreted by mucosal lymphoid tissues. IgD Immunoglobulin class composed of δ heavy chains that appears as surface immunoglobulin on mature B cells. IgE Immunoglobulin class composed of ε heavy chains that acts in defense against parasite infections and in allergic reactions. IgG Immunoglobulin class composed of γ heavy chains that is the most abundant class of immunoglobulin in the plasma. IgM Immunoglobulin class composed of μ heavy chains that is the first to appear on B cells and the first to be secreted. IgNAR See immunoglobulin new antigen receptor.

1	IgM Immunoglobulin class composed of μ heavy chains that is the first to appear on B cells and the first to be secreted. IgNAR See immunoglobulin new antigen receptor. IgW Type of heavy-chain isotype present in cartilaginous fishes composed of six immunoglobulin domains. IKK The IkB kinase, IKK, is a multisubunit protein complex composed of IKKα, IKKβ, and IKKγ (or NEMO). IKKε A kinase that interacts with TBK1 (TANK-binding kinase 1) in the phosphorylation of IRF3 downstream of TLR-3 signaling. IL-1 family One of four major families of cytokines, this family contains 11 cytokines that are structurally similar to IL-1α, and are largely pro-inﬂammatory in function. IL-1β A cytokine produced by active macrophages that has many effects in the immune response, including the activation of vascular endothelium, activation of lymphocytes, and the induction of fever.

1	IL-1β A cytokine produced by active macrophages that has many effects in the immune response, including the activation of vascular endothelium, activation of lymphocytes, and the induction of fever. IL-6 Interleukin-6, a cytokine produced by activated macrophages and which has many effects, including lymphocyte activation, the stimulation of antibody production, and the induction of fever. IL-7 receptor (IL-7Rα) See CD127. IL-21 A cytokine produced by T cells (e.g.,TFH cells) that activates STAT3 and promotes survival and proliferation, particularly germinal center B cells. ILC1 A subset of innate lymphoid cells characterized by production of IFN-γ. ILCs (innate lymphoid cells) These are a class of innate immune cells having overlapping characteristics with T cells but lacking an antigen receptor. They arise in several groups, ILC1, ILC2, ILC3, and NK cells, which exhibit properties roughly similar to TH1, TH2, TH17, and CD8 T cells.

1	Imd (immunodeficiency) signaling pathway A defense against Gram-negative bacteria in insects that results in the production of antimicrobial peptides such as diptericin, attacin, and cecropin. imiquimod Drug (aldara) approved for treatment of basal cell carcinoma, genital warts, and actinic keratoses known to activate TLR-7, although not approved as an adjuvant for vaccines. immature B cells B cells that have rearranged a heavyand a light-chain V-region gene and express surface IgM, but have not yet matured sufficiently to express surface IgD as well. immediate hypersensitivity reactions Allergic reactions that occur within seconds to minutes of encounter with antigen, caused largely by activation of mast cells or basophils.

1	immediate hypersensitivity reactions Allergic reactions that occur within seconds to minutes of encounter with antigen, caused largely by activation of mast cells or basophils. immune complexes Complexes formed by the binding of antibody to its cognate antigen. Activated complement proteins, especially C3b, are often bound in immune complexes. Large immune complexes form when sufficient antibody is available to cross-link multivalent antigen; these are cleared by cells of the reticuloendothelial system that bear Fc receptors and complement receptors. Small, soluble immune complexes form when antigen is in excess; these can be deposited in small blood vessels and damage them. immune evasion Mechanisms used by pathogens to avoid detection and/ or elimination by host immune defenses. immune modulation The deliberate attempt to change the course of an immune response, for example by altering the bias toward TH1 or TH2 dominance.

1	immune modulation The deliberate attempt to change the course of an immune response, for example by altering the bias toward TH1 or TH2 dominance. immune surveillance The recognition, and in some cases the elimination, of tumor cells by the immune system before they become clinically detectable. immune system The tissues, cells, and molecules involved in innate immunity and adaptive immunity. immunodeficiency diseases Any inherited or acquired disorder in which some aspect or aspects of host defense are absent or functionally defective. immunodominant Describes epitopes in an antigen that are preferentially recognized by T cells, such that T cells specific for those epitopes come to dominate the immune response. immunoevasins Viral proteins that prevent the appearance of peptide:MHC class I complexes on the infected cell, thus preventing the recognition of virus-infected cells by cytotoxic T cells.

1	immunoevasins Viral proteins that prevent the appearance of peptide:MHC class I complexes on the infected cell, thus preventing the recognition of virus-infected cells by cytotoxic T cells. immunogenic Any molecule that, on its own, is able to elicit an adaptive immune response on injection into a person or animal. immunoglobulin (Ig) The protein family to which antibodies and B-cell receptors belong. immunoglobulin A (IgA) See IgA. immunoglobulin D (IgD) See IgD. immunoglobulin domain Protein domain first described in antibody molecules but present in many proteins. immunoglobulin E (IgE) See IgE. immunoglobulin fold The tertiary structure of an immunoglobulin domain, comprising a sandwich of two β sheets held together by a disulfide bond. immunoglobulin G (IgG) See IgG. immunoglobulin-like domain (Ig-like domain) Protein domain structurally related to the immunoglobulin domain.

1	immunoglobulin G (IgG) See IgG. immunoglobulin-like domain (Ig-like domain) Protein domain structurally related to the immunoglobulin domain. immunoglobulin-like proteins Proteins containing one or more immunoglobulin-like domains, which are protein domains structurally similar to those of immunoglobulins. immunoglobulin M (IgM) See IgM. immunoglobulin new antigen receptor (IgNAR) A form of heavy-chainonly Ig molecule made by shark species. immunoglobulin repertoire The variety of antigen-specific immunoglobulins (antibodies and B-cell receptors) present in an individual. Also known as the antibody repertoire. immunoglobulin superfamily Large family of proteins with at least one Ig or Ig-like domain, many of which are involved in antigen recognition and cell– cell interaction in the immune system and other biological systems.

1	immunological ignorance A form of self-tolerance in which reactive lymphocytes and their target antigen are both detectable within an individual, yet no autoimmune attack occurs. immunological memory The ability of the immune system to respond more rapidly and more effectively on a second encounter with an antigen. Immunological memory is specific for a particular antigen and is long-lived. immunological synapse The highly organized interface that develops between a T cell and the target cell it is in contact with, formed by T-cell receptors binding to antigen and cell-adhesion molecules binding to their counterparts on the two cells. Also known as the supramolecular adhesion complex. immunological tolerance See tolerance.

1	immunological tolerance See tolerance. immunologically privileged sites Certain sites in the body, such as the brain, that do not mount an immune response against tissue allografts. Immunological privilege can be due both to physical barriers to cell and antigen migration and to the presence of immunosuppressive cytokines. immunology The study of all aspects of host defense against infection and also of the adverse consequences of immune responses. immunomodulatory therapy Treatments that seek to modify an immune response in a beneficial way, for example to reduce or prevent an autoimmune or allergic response. immunophilins See cyclophilins, FK-binding proteins. immunoproteasome A form of proteasome found in cells exposed to interferons. It contains three subunits that are different from the normal proteasome.

1	immunoproteasome A form of proteasome found in cells exposed to interferons. It contains three subunits that are different from the normal proteasome. immunoreceptor tyrosine-based activation motif (ITAM) Sequence motifs in the signaling chains of receptors, such as antigen receptors on lymphocytes, that are the site of tyrosine phosphorylation after receptor activation, leading to recruitment of other signaling proteins. immunoreceptor tyrosine-based inhibition motif (ITIM) Sequence motifs in the signaling chains of inhibitory receptors that are sites of tyrosine phosphorylation, leading to inhibitory signaling, such as through recruitment of phosphatases that remove phosphate groups added by tyrosine kinases. immunoreceptor tyrosine-based switch motif (ITSM) A sequence motif present in the cytoplasmic tails of some inhibitor receptors.

1	immunoreceptor tyrosine-based switch motif (ITSM) A sequence motif present in the cytoplasmic tails of some inhibitor receptors. immunotoxin Antibodies that are chemically coupled to toxic proteins usually derived from plants or microbes. The antibody targets the toxin moiety to the required cells. indirect allorecognition Recognition of a grafted tissue that involves the uptake of allogeneic proteins by the recipient’s antigen-presenting cells and their presentation to T cells by self MHC molecules. indoleamine 2,3-dioxygenase (IDO) Enzyme expressed by immune cells and some tumors that catabolizes tryptophan into kynurenine metabolites that can have immunosuppressive functions. induced pluripotent stem cells (iPS cells) Pluripotent stem cells that are derived from adult somatic cells by the introduction of a cocktail of transcription factors.

1	induced pluripotent stem cells (iPS cells) Pluripotent stem cells that are derived from adult somatic cells by the introduction of a cocktail of transcription factors. infectious mononucleosis The common form of infection with the Epstein–Barr virus. It consists of fever, malaise, and swollen lymph nodes. Also called glandular fever. inflammasome A pro-inﬂammatory protein complex that is formed after stimulation of the intracellular NOD-like receptors. Production of an active caspase in the complex processes cytokine proproteins into active cytokines. inflammation General term for the local accumulation of ﬂuid, plasma proteins, and white blood cells that is initiated by physical injury, infection, or a local immune response. inflammatory bowel disease (IBD) General name for a set of inﬂammatory conditions in the gut, such as Crohn’s disease and colitis, that have an immunological component.

1	inflammatory bowel disease (IBD) General name for a set of inﬂammatory conditions in the gut, such as Crohn’s disease and colitis, that have an immunological component. inflammatory cells Cells such as macrophages, neutrophils, and effector TH1 lymphocytes that invade inﬂamed tissues and contribute to the inﬂammation. inflammatory chemokines Chemokines that are produced in response to infection or injury to direct the localization of immune cells to sites of inﬂammation. inflammatory inducers Chemical structures that indicate the presence of invading microbes or cellular damage, such as bacterial lipopolysaccharides, extracellular ATP, or urate crystals. inflammatory mediators Chemicals such as cytokines produced by immune cells that act on target cells to promote defense against microbes. inflammatory monocytes An activated form of monocyte producing a variety of pro-inﬂammatory cytokines. inflammatory response See inflammation.

1	inflammatory monocytes An activated form of monocyte producing a variety of pro-inﬂammatory cytokines. inflammatory response See inflammation. infliximab Chimeric antibody to TNF-α used in the treatment of inﬂammatory diseases, such as Crohn's disease and rheumatoid arthritis. inherited immunodeficiency diseases See primary immunodeficiencies. inhibitory receptors On NK cells, receptors whose stimulation results in suppression of the cell’s cytotoxic activity. initiator caspases Proteases that promote apoptosis by cleaving and activating other caspases. iNKT See invariant NKT cells.

1	initiator caspases Proteases that promote apoptosis by cleaving and activating other caspases. iNKT See invariant NKT cells. innate immunity The various innate resistance mechanisms that are encountered first by a pathogen, before adaptive immunity is induced, such as anatomical barriers, antimicrobial peptides, the complement system, and macrophages and neutrophils carrying nonspecific pathogen-recognition receptors. Innate immunity is present in all individuals at all times, does not increase with repeated exposure to a given pathogen, and discriminates between groups of similar pathogens, rather than responding to a particular pathogen. Cf. adaptive immunity. innate lymphoid cells (ILCs) See ILCs. innate recognition receptors General term for a large group of proteins that recognize many different inﬂammatory inducers and that are encoded in the germline and do not need gene rearrangement in somatic cells to be expressed.

1	inositol 1,4,5-trisphosphate (IP3) A soluble second messenger produced by the cleavage of membrane inositol phospholipids by phospholipase C-γ. It acts on receptors in the endoplasmic reticulum membrane, resulting in the release of stored Ca2+ into the cytosol. integrin Heterodimeric cell-surface proteins involved in cell–cell and cell–matrix interactions. They are important in adhesive interactions between lymphocytes and antigen-presenting cells and in lymphocyte and leukocyte adherence to blood vessel walls and migration into tissues. integrin α4:β7 Integrin binding to VCAM-1, MAdCAM-1, and fibronectin and expressed by various cells, such as IELs, that traffic to intestinal lamina propria. intercellular adhesion molecules (ICAMs) See ICAMs. interdigitating dendritic cells See dendritic cells.

1	intercellular adhesion molecules (ICAMs) See ICAMs. interdigitating dendritic cells See dendritic cells. interferon regulatory factor (IRF) A family of nine transcription factors that regulate a variety of immune responses. For example, IRF3 and IRF7 are activated as a result of signaling from some TLRs. Several IRFs promote expression of the genes for type I interferons. interferon stimulated genes (ISGs) A category of gene induced by interferons, which include many that promote innate defense against pathogens, such as oligoadenylate synthetase, PKR, and the Mx, IFITs, and IFITM proteins. interferon-α receptor (IFNAR) This receptor recognizes IFN-α and IFN-β to activate STAT1 and STAT2 and induce expression of many ISGs. interferon-induced transmembrane protein (IFITM) See IFITM. interferon-producing cells (IPCs) See plasmacytoid dendritic cells.

1	interferon-induced transmembrane protein (IFITM) See IFITM. interferon-producing cells (IPCs) See plasmacytoid dendritic cells. interferons (IFNs) Several related families of cytokines originally named for their interference of viral replication. IFN-α and IFN-β are antiviral in their effects; IFN-γ has other roles in the immune system. intergenic control regions Sites in non-coding regions of genes that control their expression and rearrangement by interactions with transcription factors and chromatin-modifying proteins. interleukin (IL) A generic name for cytokines produced by leukocytes. The more general term cytokine is used in this book, but the term interleukin is used in the naming of specific cytokines such as IL-2. Some key interleukins are listed in the glossary under their abbreviated names, for example IL-1β and IL-2. Cytokines are listed in Appendix III.

1	intraepithelial lymphocytes (IELs) Lymphocytes present in the epithelium of mucosal surfaces such as the gut. They are predominantly T cells, and in the gut are predominantly CD8 T cells. intrathymic dendritic cells See dendritic cells. intrinsic pathway of apoptosis Signaling pathway that mediates apoptosis in response to noxious stimuli including UV irradiation, chemotherapeutic drugs, starvation, or lack of the growth factors required for survival. It is initiated by mitochondrial damage. Also called the mitochondrial pathway of apoptosis. invariant chain (Ii, CD74) A polypeptide that binds in the peptide-binding cleft of newly synthesized MHC class II proteins in the endoplasmic reticulum and blocks other peptides from binding there. It is degraded in the endosome, allowing for loading of antigenic peptides there.

1	invariant NKT cells (iNKT cells) A type of innate-like lymphocyte that carries a T-cell receptor with an invariant α chain and a β chain of limited diversity that recognizes glycolipid antigens presented by CD1 MHC class Ib molecules. This cell type also carries the surface marker NK1.1, which is usually associated with NK cells. IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. A very rare inherited condition in which CD4 CD25 regulatory T cells are lacking as a result of a mutation in the gene for the transcription factor FoxP3, leading to the development of autoimmunity. ipilimumab Antibody to human CTLA-4 used to treat melanoma, and first checkpoint blockade immunotherapy.

1	ipilimumab Antibody to human CTLA-4 used to treat melanoma, and first checkpoint blockade immunotherapy. Ir (immune response) genes An archaic term for genetic polymorphisms controlling the intensity of the immune response to a particular antigen, now known to result from allelic differences in MHC molecules, especially MHC class II molecules, that inﬂuence binding of particular peptides. IRAK1, IRAK4 Protein kinases that are part of the intracellular signaling pathways leading from TLRs. IRAK4 deficiency An immunodeficiency characterized by recurrent bacterial infections, caused by inactivating mutations in the IRAK4 gene that result in a block in TLR signaling. IRF9 A member of the IRF family of transcription factors that interacts with activated STAT1 and STAT2 to form the complex called ISGF3, which induces transcription of many ISGs.

1	IRF9 A member of the IRF family of transcription factors that interacts with activated STAT1 and STAT2 to form the complex called ISGF3, which induces transcription of many ISGs. IRGM3 A protein that functions in the maintenance and storage of neutral lipid droplets in many types of cells in association with adipose differentiation related protein. irradiation-sensitive SCID (IR-SCID) A type of severe combined immunodeficiency due to mutations in DNA repair proteins, such as Artemis, that causes abnormal sensitivity to ionizing radiation and defects in V(D)J recombination. ISGF3 See IRF9. isoforms Different forms of the same protein, for example the different forms encoded by different alleles of the same gene. isolated lymphoid follicles (ILF) A type of organized lymphoid tissue in the gut wall that is composed mainly of B cells. isolation membrane See phagophore.

1	isolated lymphoid follicles (ILF) A type of organized lymphoid tissue in the gut wall that is composed mainly of B cells. isolation membrane See phagophore. isotype The designation of an immunoglobulin chain in respect of the type of constant region it has. Light chains can be of either κ or λ isotype. Heavy chains can be of μ, δ, γ, α, or ε isotype. The different heavy-chain isotypes have different effector functions and determine the class and functional properties of antibodies (IgM, IgD, IgG, IgA, and IgE, respectively). isotype switching See class switching. isotypic exclusion Describes the use of one or other of the light-chain isotypes, κ or λ, by a given B cell or antibody. JAK inhibitors (Jakinibs) Small molecule kinase inhibitors with relative selectivity for one or more of the JAK kinases.

1	JAK inhibitors (Jakinibs) Small molecule kinase inhibitors with relative selectivity for one or more of the JAK kinases. Janus kinase (JAK) family Enzymes of the JAK–STAT intracellular signaling pathways that link many cytokine receptors with gene transcription in the nucleus. The kinases phosphorylate STAT proteins in the cytosol, which then move to the nucleus and activate a variety of genes. J chain Small polypeptide chain made by B cells that attaches to polymeric immunoglobulins IgM and IgA by disulfide bonds, and is essential for formation of the binding site for the polymeric immunoglobulin receptor. JNK See Jun kinase. Job’s syndrome See hyper IgE syndrome.

1	JNK See Jun kinase. Job’s syndrome See hyper IgE syndrome. joining gene segment, J gene segment Short DNA sequences that encode the J regions of immunoglobulin and T-cell receptor variable domains. In a rearranged light-chain, TCRα, or TCRγ genes, the J gene segment is joined to a V gene segment. In a rearranged heavy-chain, TCRβ, or TCRδ locus, a J gene segment is joined to a D gene segment. Jun kinase A protein kinase that phosphorylates the transcription factor c-Jun, enabling it to bind to c-Fos to form the AP-1 transcription factor. junctional diversity The variability in sequence present in antigen-specific receptors that is created during the process of joining V, D, and J gene segments and which is due to imprecise joining and insertion of nontemplated nucleotides at the joins between gene segments. κ chain One of the two classes or isotypes of immunoglobulin light chains.

1	κ chain One of the two classes or isotypes of immunoglobulin light chains. K63-linkages In polyubiquitin chains, the covalent ligation of lysine 63 amino group of one ubiquitin protein with the carboxy terminus of a second ubiquitin. This type of linkage is most associated with activation of signaling by formation of a scaffold recognized by signaling adaptors such as TAB1/2. killer cell immunoglobulin-like receptors (KIRs) Large family of receptors present on NK cells, through which the cells’ cytotoxic activity is controlled. The family contains both activating and inhibitory receptors. killer cell lectin-like receptors (KLRs) Large family of receptors present on NK cells, through which the cells’ cytotoxic activity is controlled. The family contains both activating and inhibitory receptors.

1	kinase suppressor of Ras A scaffold protein in the Raf–MEK1–Erk MAPkinase cascade that binds to all three members following antigen receptor signaling to facilitate their interactions and to accelerate the signaling cascade. kinin system An enzymatic cascade of plasma proteins that is triggered by tissue damage to produce several inﬂammatory mediators, including the vasoactive peptide bradykinin. Kostmann’s disease A form of severe congenital neutropenia, an inherited condition in which the neutrophil count is low. In Kostmann’s disease, this is due to a deficiency of the mitochondrial protein HAX1, which leads to apoptosis of developing myeloid cells and persistent neutropenia. KSR See kinase suppressor of Ras. Ku A DNA repair protein required for immunoglobulin and T-cell receptor gene rearrangement. Kupffer cells Phagocytes lining the hepatic sinusoids; they remove debris and dying cells from the blood, but are not known to elicit immune responses.

1	Kupffer cells Phagocytes lining the hepatic sinusoids; they remove debris and dying cells from the blood, but are not known to elicit immune responses. kynurenine metabolites Various compounds derived from tryptophan through the actions of the enzymes indolamine-2,3-dioxygenase (IDO) or tryptophan-2,3-dioxygenase (TDO) expressed in various immune cells or the liver. λ chain One of the two classes or isotypes of immunoglobulin light chains. λ5 See surrogate light chain. L-selectin Adhesion molecule of the selectin family found on lymphocytes. L-selectin binds to CD34 and GlyCAM-1 on high endothelial venules to initiate the migration of naive lymphocytes into lymphoid tissue. lamellar bodies Lipid-rich secretory organelles in keratinocytes and lung pneumocytes that release β-defensins into the extracellular space. lamina propria A layer of connective tissue underlying a mucosal epithelium. It contains lymphocytes and other immune-system cells.

1	lamina propria A layer of connective tissue underlying a mucosal epithelium. It contains lymphocytes and other immune-system cells. large pre-B cell Stage of B-cell development immediately after the pro-B cell, in which the cell expresses the pre-B-cell receptor and undergoes several rounds of division. LAT See linker for activation of T cells. late-phase reaction Allergic reactions that occurs several hours after initial encounter with an antigen. Thought to be manifestations of recruitment of multiple leukocyte subsets to the site of allergen exposure. late pro-B cell Stage in B-cell development in which VH to DJH joining occurs. latency A state in which a virus infects a cell but does not replicate. Lck An Src-family tyrosine kinase that associates with the cytoplasmic tails of CD4 and CD8 and phosphorylates the cytoplasmic tails of the T-cell receptor signaling chains, thus helping to activate signaling from the T-cell receptor complex once antigen has bound.

1	lectin A carbohydrate-binding protein. lectin pathway Complement activation pathway that is triggered by mannose-binding lectins (MBLs) or ficolins bound to bacteria. lentiviruses A group of retroviruses that include the human immunodeficiency virus, HIV-1. They cause disease after a long incubation period. lethal factor An endopeptidase produced by Bacillus anthracis that cleaves NLRP1, inducing cell death within the infected cell, typically a macrophage. leucine-rich repeat (LRR) Protein motifs that are repeated in series to form, for example, the extracellular portions of Toll-like receptors. leukocyte A white blood cell. Leukocytes include lymphocytes, polymorphonuclear leukocytes, and monocytes.

1	leukocyte A white blood cell. Leukocytes include lymphocytes, polymorphonuclear leukocytes, and monocytes. leukocyte adhesion deficiencies (LADs) A class of immunodeficiency diseases in which the ability of leukocytes to enter sites infected by extracellular pathogens is affected, imparing elimination of infection. There are several different causes, including a deficiency of the common β chain of the leukocyte integrins. leukocyte adhesion deficiency type 2 Disease causes by defects in the production of sulfated sialyl-LewisX that prevent neutrophils from interacting with Pand E-selectin, eliminating their ability to migrate properly to sites of infection.

1	leukocyte functional antigens (LFAs) Cell-adhesion molecules on leukocytes that were initially defined using monoclonal antibodies. LFA-1 is a β2 integrin; LFA-2 (now usually called CD2) is a member of the immunoglobulin superfamily, as is LFA-3 (now called CD58). LFA-1 is particularly important in T-cell adhesion to endothelial cells and antigen-presenting cells. leukocyte receptor complex (LRC) A large cluster of immunoglobulin-like receptor genes that includes the killer cell immunoglobulin-like receptor (KIR) genes. leukocytosis The presence of increased numbers of leukocytes in the blood. It is commonly seen in acute infection. leukotrienes Lipid mediators of inﬂammation that are derived from arachidonic acid. They are produced by macrophages and other cells. LFA-1 See leukocyte functional antigens. LGP2 A member of the RLR family, it cooperates with RIG-I and MDA-5 in the recognition of viral RNA.

1	LFA-1 See leukocyte functional antigens. LGP2 A member of the RLR family, it cooperates with RIG-I and MDA-5 in the recognition of viral RNA. licensing The activation of a dendritic cell so that it is able to present antigen to naive T cells and activate them. light chain, L chain The smaller of the two types of polypeptide chains that make up an immunoglobulin molecule. It consists of one V and one C domain, and is disulfide-bonded to the heavy chain. There are two classes, or isotypes, of light chain, known as κ and λ, which are produced from separate genetic loci. light-chain variable region (VL) Referring to the V region of the light chain of an immunoglobulin. light zone See germinal center. lingual tonsils Paired masses of organized peripheral lymphoid tissue situated at the base of the tongue, in which adaptive immune responses can be initiated. They are part of the mucosal immune system. See also palatine tonsils.

1	linked recognition The rule that for a helper T cell to be able to activate a B cell, the epitopes recognized by the B cell and the helper T cell have to be derived from the same antigen (that is, they must originally have been physically linked). linker for activation of T cells A cytoplasmic adaptor protein with several tyrosines that become phosphorylated by the tyrosine kinase ZAP-70. It helps to coordinate downstream signaling events in T-cell activation. LIP10 A cleaved fragment of invariant chain retaining the transmembrane segments that remains bound to MHC class II proteins and helps target the complex to the endosome. LIP22 The initial cleaved fragment of invariant chain bound to MHC class II molecules. lipid bodies Storage organelles rich in neutral lipids within the cytoplasm. lipocalin-2 An antimicrobial peptide produced in abundance by neutrophils and mucosal epithelial cells that inhibits bacterial and fungal growth by limiting availability of iron.

1	lipocalin-2 An antimicrobial peptide produced in abundance by neutrophils and mucosal epithelial cells that inhibits bacterial and fungal growth by limiting availability of iron. lipopeptide antigens A diverse set of antigens derived from microbial lipids typically presented by nonclassical MHC class Ib molecules such as CD1 molecules to invariant T-cell populations, including iNKT cells. lipopolysaccharide (LPS) The surface lipopolysaccharide of Gram-negative bacteria, which stimulates TLR-4 on macrophages and dendritic cells. lipoteichoic acids Components of bacterial cell walls that are recognized by Toll-like receptors. long-term non-progressors HIV-infected individuals who mount an immune response that controls viral loads such that they do not progress to AIDS despite the absence of antiretroviral therapy. See also elite controllers. LPS-binding protein Protein in blood and extracellular ﬂuid that binds bacterial lipopolysaccharide (LPS) shed from bacteria.

1	LPS-binding protein Protein in blood and extracellular ﬂuid that binds bacterial lipopolysaccharide (LPS) shed from bacteria. Ly49 receptors A family of C-type lectins expressed by mouse, but not human, NK cells. These can be either activating or inhibitory in function. Ly49a See Ly49 receptors. Ly49H See Ly49 receptors. Ly108 See SLAM. lymph The extracellular ﬂuid that accumulates in tissues and is drained by lymphatic vessels that carry it through the lymphatic system to the thoracic duct, which returns it to the blood. lymph nodes A type of peripheral lymphoid organ present in many locations throughout the body where lymphatic vessels converge. lymphatic system The system of lymph-carrying vessels and peripheral lymphoid tissues through which extracellular ﬂuid from tissues passes before it is returned to the blood via the thoracic duct. lymphatic vessels, lymphatics Thin-walled vessels that carry lymph.

1	lymphatic vessels, lymphatics Thin-walled vessels that carry lymph. lymphoblast A lymphocyte that has enlarged after activation and has increased its rate of RNA and protein synthesis, but is not yet fully differentiated. lymphocyte A class of white blood cells that bear variable cell-surface receptors for antigen and are responsible for adaptive immune responses. There are two main types—B lymphocytes (B cells) and T lymphocytes (T cells)—which mediate humoral and cell-mediated immunity, respectively. On antigen recognition, a lymphocyte enlarges to form a lymphoblast and then proliferates and differentiates into an antigen-specific effector cell. lymphocyte receptor repertoire All the highly variable antigen receptors carried by B and T lymphocytes. lymphoid Describes tissues composed mainly of lymphocytes.

1	lymphocyte receptor repertoire All the highly variable antigen receptors carried by B and T lymphocytes. lymphoid Describes tissues composed mainly of lymphocytes. lymphoid organs Organized tissues characterized by very large numbers of lymphocytes interacting with a nonlymphoid stroma. The central, or primary, lymphoid organs, where lymphocytes are generated, are the thymus and bone marrow. The main peripheral, or secondary, lymphoid organs, in which adaptive immune responses are initiated, are the lymph nodes, spleen, and mucosa-associated lymphoid organs such as tonsils and Peyer’s patches. lymphoid tissue Tissue composed of large numbers of lymphocytes. lymphoid tissue inducer (LTi) cells Cells of the blood lineage, which arise in the fetal liver and are carried in the blood to sites where they will form lymph nodes and other peripheral lymphoid organs. lymphopenia Abnormally low levels of lymphocytes in the blood.

1	lymphopenia Abnormally low levels of lymphocytes in the blood. lymphopoiesis The differentiation of lymphoid cells from a common lymphoid progenitor. lymphotoxins (LTs) Cytokines of the tumor necrosis factor (TNF) family that are directly cytotoxic for some cells. They occur as trimers of LT-α chains (LT-α3) and heterotrimers of LT-α and LT-β chains (LT-α2:β1). lysogenic phase The phase of the viral life cycle in which the virus genome integrates into the host cell genome but remains dormant, employing mechanisms to avoid destroying its cellular host. lysozyme Antimicrobial enzyme that degrades bacterial cell walls. lytic phase, productive phase The phase of the viral life cycle in which there is active viral replication followed by destruction of the infected host cell as the virus escapes to infect new target cells.

1	M1 macrophages The name sometimes given to ‘classically’ activated macrophages, which develop in the context of type 1 responses and have pro-inﬂammatory properties. M2 macrophages The name sometimes given to ‘alternatively’ activated macrophages, which develop in in the context of type 2 responses (e.g., parasite infection) and promote tissue remodeling and repair. macroautophagy The engulfment by a cell of large quantities of its own cytoplasm, which is then delivered to the lysosomes for degradation. macrophages Large mononuclear phagocytic cells present in most tissues that have many functions, such as scavenger cells, pathogen-recognition cells, production of pro-inﬂammatory cytokines. Macrophages arise both embryonically and from bone marrow precursors throughout life.

1	macropinocytosis A process in which large amounts of extracellular ﬂuid are taken up into an intracellular vesicle. This is one way in which dendritic cells can take up a wide variety of antigens from their surroundings. MAdCAM-1 Mucosal cell-adhesion molecule-1. A mucosal addressin that is recognized by the lymphocyte surface proteins L-selectin and VLA-4, enabling the specific homing of lymphocytes to mucosal tissues. MAIT cells See mucosal associated invariant T cells. major basic protein Protein released by activated eosinophils that acts on mast cells and basophils to cause their degranulation. major histocompatibility complex (MHC) A cluster of genes on human chromosome 6 that encodes a set of membrane glycoproteins called the MHC molecules. The MHC also encodes proteins involved in antigen processing and other aspects of host defense. The genes for the MHC molecules are the most polymorphic in the human genome, having large numbers of alleles at the various loci.

1	MAL An adaptor protein that associates with MyD88 in signaling by TLR-2/1, TLR-2/6, and TLR-4. mannose-binding lectin (MBL) Mannose-binding protein present in the blood. It can opsonize pathogens bearing mannose on their surfaces and can activate the complement system via the lectin pathway, an important part of innate immunity. mannose receptor (MR) A receptor on macrophages that is specific for mannose-containing carbohydrates that occur on the surfaces of pathogens but not on host cells. mantle zone A rim of B lymphocytes that surrounds lymphoid follicles.

1	mantle zone A rim of B lymphocytes that surrounds lymphoid follicles. Mantoux test A screening test for tuberculosis in which a sterile-filtered glycerol extract of Mycobacterium tuberculosis bacilli (Tb) is injected intradermally and the result is read 48–72 hours later. Induration, firm swelling caused by infiltration into the skin of inﬂammatory cells, can indicate previous exposure to Tb, either prior vaccination or current infection of M. tuberculosis. Generally, induration at the site of injection greater than 10 mm in diameter indicates the need for additional tests to assess whether infection with Tb is present. MAP kinase (MAPK) See mitogen-activated protein kinase. MARCO (macrophage receptor with a collagenous structure) See scavenger receptor). marginal sinus A blood-filled vascular network that branches from the central arteriole and demarcates each area of white pulp in the spleen.

1	marginal sinus A blood-filled vascular network that branches from the central arteriole and demarcates each area of white pulp in the spleen. marginal zone Area of lymphoid tissue lying at the border of the white pulp in the spleen. marginal zone B cells A unique population of B cells found in the spleen marginal zones; they do not circulate and are distinguished from conventional B cells by a distinct set of surface proteins. MASP-1, MASP-2, MASP-3 Serine proteases of the classical and lectin pathway of complement activation that bind to C1q, ficolins, and mannosebinding lectin, and function in their activation to cleave C4.

1	mast cells A large granule-rich cell found in connective tissues throughout the body, most abundantly in the submucosal tissues and the dermis. The granules store bioactive molecules including the vasoactive amine histamine, which are released on mast-cell activation. Mast cells are thought to be involved in defenses against parasites and they have a crucial role in allergic reactions. mastocytosis The overproduction of mast cells. mature B cell B cell that expresses IgM and IgD on its surface and has gained the ability to respond to antigen. MAVS (mitochondrial antiviral signaling protein) A CARD-containing adaptor protein attached to the outer mitochondrial membrane that signals downstream of RIG-I and MDA-5 to activate IRF3 and NFκB in response to viral infection. MBL-associated serine proteases See MASP-1, MASP-2, MASP-3. M cells Specialized epithelial cell type in the intestinal epithelium over Peyer’s patches, through which antigens and pathogens enter from the gut.

1	M cells Specialized epithelial cell type in the intestinal epithelium over Peyer’s patches, through which antigens and pathogens enter from the gut. MD-2 Accessory protein for TLR-4 activity. MDA-5 (melanoma differentiation-associated 5, also helicard) This protein contains an RNA helicase-like domain similar to RIG-I, and senses double-stranded RNA for detection of intracellular viral infections. medulla The central or collecting point of an organ. The thymic medulla is the central area of each thymic lobe, rich in bone marrow-derived antigen-presenting cells and the cells of a distinctive medullary epithelium. The medulla of the lymph node is a site of macrophage and plasma cell concentration through which the lymph ﬂows on its way to the efferent lymphatics. MEK1 A MAPK kinase in the Raf–MEK1–Erk signaling module, which is a part of a signaling pathway in lymphocytes leading to activation of the transcription factor AP-1.

1	MEK1 A MAPK kinase in the Raf–MEK1–Erk signaling module, which is a part of a signaling pathway in lymphocytes leading to activation of the transcription factor AP-1. melanoma-associated antigens (MAGE) Heterogeneous group of proteins of diverse or unknown functions characterized by restricted expression limited to tumors (i.e., melanoma) or testis germ cells. membrane associated ring finger (C3HC4) 1, MARCH-1 An E3 ligase expressed in B cells, dendritic cells, and macrophages that induces the constitutive degradation of MHC class II molecules, regulating their steady-state expression. membrane attack Effector pathway of complement based on formation of the membrane-attack complex (MAC). membrane-attack complex (MAC) Protein complex composed of C5b to C9 that assembles a membrane-spanning hydrophilic pore on pathogen surfaces, causing cell lysis.

1	membrane-attack complex (MAC) Protein complex composed of C5b to C9 that assembles a membrane-spanning hydrophilic pore on pathogen surfaces, causing cell lysis. membrane cofactor of proteolysis (MCP or CD46) A complement regulatory protein, a host-cell membrane protein that acts in conjunction with factor I to cleave C3b to its inactive derivative iC3b and thus prevent convertase formation. membrane immunoglobulin (mIg) Transmembrane immunoglobulin present on B cells; it is the B-cell receptor for antigen. memory B cells See memory cells. memory cells B and T lymphocytes that mediate immunological memory. They are more sensitive than naive lymphocytes to antigen and respond rapidly on reexposure to the antigen that originally induced them. mesenteric lymph nodes Lymph nodes located in the connective tissue (mesentery) that tethers the intestine to the rear wall of the abdomen. They drain the GALT.

1	mesenteric lymph nodes Lymph nodes located in the connective tissue (mesentery) that tethers the intestine to the rear wall of the abdomen. They drain the GALT. metastasis Spread of a tumor from its original location to distant organs of the body by traveling through the blood or lymphatics or by direct extension. 2ʹ-O-methyltransferase (MTase) An enzyme that transfers a methyl group to the 2ʹ hydroxyl of the first and second ribose groups in mRNA. Viruses that acquire MTase can produce cap-1 and cap-2 on their transcripts and thereby evade restriction by IFIT1. MF-59 A proprietary adjuvant based on squaline and water used in Europe and Canada in conjunction with inﬂuenza vaccine. MHC class I See MHC class I molecules. MHC class I deficiency An immunodeficiency disease in which MHC class I molecules are not present on the cell surface, usually as a result of an inherited deficiency of either TAP-1 or TAP-2.

1	MHC class I deficiency An immunodeficiency disease in which MHC class I molecules are not present on the cell surface, usually as a result of an inherited deficiency of either TAP-1 or TAP-2. MHC class I molecules Polymorphic cell-surface proteins encoded in the MHC locus and expressed on most cells. They present antigenic peptides generated in the cytosol to CD8 T cells, and also bind the co-receptor CD8. MHC class II See MHC class II molecules. MHC class II compartment (MIIC) The cellular vesicles in which MHC class II molecules accumulate, encounter HLA-DM, and bind antigenic peptides, before migrating to the surface of the cell. MHC class II deficiency A rare immunodeficiency disease in which MHC class II molecules are not present on cells as a result of various inherited defects. Patients are severely immunodeficient and have few CD4 T cells.

1	MHC class II molecules Polymorphic cell-surface proteins encoded in the MHC locus are expressed primarily on specialized antigen-presenting cells. They present antigenic peptides derived from internalized extracellular pathogens to CD4 T cells and also bind the co-receptor CD4. MHC class II transactivator (CIITA) Protein that activates transcription of MHC class II genes. Defects in the CIITA gene are one cause of MHC class II deficiency. MHC haplotype A set of alleles in the MHC that is inherited unchanged (that is, without recombination) from one parent. MHC molecules Highly polymorphic cell-surface proteins encoded by MHC class I and MHC class II genes involved in presentation of peptide antigens to T cells. They are also known as histocompatibility antigens. MHC restriction The fact that a peptide antigen can only be recognized by a given T cell if it is bound to a particular self MHC molecule. MHC restriction is a consequence of events that occur during T-cell development.

1	MIC-A, MIC-B MHC class Ib proteins that are induced by stress, infection, or transformation in many cell types and are recognized by NKG2D. microautophagy The continuous internalization of the cytosol into the vesicular system. microbial glycolipids Diverse class of antigens frequently presented by CD1 molecules to iNKT cells. microbiome See commensal microorganisms. microbiota See commensal microorganisms. microclusters Assemblies of small numbers of T-cell receptors that may be involved in the initiation of T-cell receptor activation by antigen in naive T cells. microfold cells See M cells. microglial cells An embryonically derived form of tissue macrophage in the central nervous system that is dependent on IL-34 for local self-renewal throughout life. minor histocompatibility antigens Peptides of polymorphic cellular proteins bound to MHC molecules that can lead to graft rejection when they are recognized by T cells.

1	minor histocompatibility antigens Peptides of polymorphic cellular proteins bound to MHC molecules that can lead to graft rejection when they are recognized by T cells. minor lymphocyte stimulating (Mls) antigens An old term referring to non-MHC antigens responsible for unusually strong T cell responses to cells from different strains of mice, now known to be superantigens encoded by endogenous retroviruses. mismatch repair A type of DNA repair that causes mutations and is involved in somatic hypermutation and class switching in B cells. missing self Refers to the loss of cell-surface molecules that engage with inhibitory receptors on NK cells, resulting in NK-cell activation.

1	missing self Refers to the loss of cell-surface molecules that engage with inhibitory receptors on NK cells, resulting in NK-cell activation. mitogen-activated protein kinases (MAPKs) A series of protein kinases that become phosphorylated and activated on cellular stimulation by a variety of ligands, and lead to new gene expression by phosphorylating key transcription factors. The MAPKs are part of many signaling pathways, especially those leading to cell proliferation, and have different names in different organisms. mixed essential cryoglobulinemia Disease due to the production of cryoglobulins (cold-precipitable immunoglobulins), sometimes in response to chronic infections such as hepatitis C, which can lead to the deposition of immune complexes in joints and tissues.

1	mixed lymphocyte reaction (MLR) A test for histocompatibility in which lymphocytes from donor and recipient are cultured together. If the two people are histoincompatible, the recipient’s T cells recognize the allogeneic MHC molecules on the cells of the other donor as ‘foreign’ and proliferate. molecular mimicry The similarity between some pathogen antigens and host antigens, such that antibodies and T cells produced against the former also react against host tissues. This similarity may be the cause of some autoimmunity. monoclonal antibodies Antibodies produced by a single clone of B lymphocytes, so that they are all identical. monocyte Type of white blood cell with a bean-shaped nucleus; it is a precursor of tissue macrophages. monomorphic Describes a gene that occurs in only one form. Cf. polymorphic.

1	monocyte Type of white blood cell with a bean-shaped nucleus; it is a precursor of tissue macrophages. monomorphic Describes a gene that occurs in only one form. Cf. polymorphic. motheaten A mutation in the SHP-1 protein phosphatase that impairs the function of some inhibitory receptors, such as Ly49, resulting in over-activation of various cells, including NK cells. Mice with this mutation have a 'motheaten' appearance due to chronic inﬂammation. MR1 A 'non-classical' MHC class Ib molecule that binds certain folic acid metabolites produced by bacteria for recognition by mucosal associated invariant T (MAIT) cells. MRE11A (meitotic recombination 11 homolog a) A protein involved in DNA damage and repair mechanisms that also recognizes cytoplasmic dsDNA and can activate the STING pathway. MSH2, MSH6 Mismatch repair proteins that detect uridine and recruit nucleases to remove the damaged and several adjacent nucleotides.

1	MSH2, MSH6 Mismatch repair proteins that detect uridine and recruit nucleases to remove the damaged and several adjacent nucleotides. mTOR (mammalian target of rapamycin) Serine/threonine kinase that functions in regulating numerous aspects of cell metabolism and function in complex with regulatory proteins Raptor or Rictor. The Raptor/mTOR complex (mTORC1) is inhibited by the immunosuppressive drug rapamycin. mTORC1, mTORC2 Active complexes of mTOR formed with the regulatory proteins Raptor and Rictor, respectively. mucins Highly glycosylated cell-surface proteins. Mucin-like molecules are bound by L-selectin in lymphocyte homing. Muckle–Wells syndrome An inherited episodic autoinﬂammatory disease caused by mutations in the gene encoding NLRP3, a component of the inﬂammasome.

1	Muckle–Wells syndrome An inherited episodic autoinﬂammatory disease caused by mutations in the gene encoding NLRP3, a component of the inﬂammasome. mucosal associated invariant T cells (MAIT) Primarily γ:δT cells with limited diversity present in the mucosal immune system that respond to bacterially derived folate derivates presented by the nonclassical MHC class Ib molecule MR1. mucosa-associated lymphoid tissue (MALT) Generic term for all organized lymphoid tissue found at mucosal surfaces, in which an adaptive immune response can be initiated. It comprises GALT, NALT, and BALT (when present). mucosal epithelia Mucus-coated epithelia lining the body’s internal cavities that connect with the outside (such as the gut, airways, and vaginal tract).

1	mucosal epithelia Mucus-coated epithelia lining the body’s internal cavities that connect with the outside (such as the gut, airways, and vaginal tract). mucosal immune system The immune system that protects internal mucosal surfaces (such as the linings of the gut, respiratory tract, and urogenital tracts), which are the site of entry for virtually all pathogens and other antigens. See also mucosa-associated lymphoid tissue. mucosal mast cells Specialized mast cells present in mucosa. They produce little histamine but large amounts of prostaglandins and leukotrienes. mucosal tolerance The suppression of specific systemic immune responses to an antigen by the previous administration of the same antigen by a mucosal route. mucus Sticky solution of proteins (mucins) secreted by goblet cells of internal epithelia, forming a protective layer on the epithelial surface.

1	mucus Sticky solution of proteins (mucins) secreted by goblet cells of internal epithelia, forming a protective layer on the epithelial surface. multiple sclerosis A neurological autoimmune disease characterized by focal demyelination in the central nervous system, lymphocytic infiltration in the brain, and a chronic progressive course. multipotent progenitor cells (MPPs) Bone marrow cells that can give rise to both lymphoid and myeloid cells but are no longer self-renewing stem cells. muramyl dipeptide (MDP) A component of the peptidoglycan of most bacteria that is recognized by the intracellular sensor NOD2. muromomab A mouse antibody against human CD3 used to treat transplant rejection; this was the first monoclonal antibody approved as a drug in humans. mutualism A symbiotic relationship between two organisms in which both benefit, such as the relationship between a human and its normal resident (commensal) gut microorganisms.

1	mutualism A symbiotic relationship between two organisms in which both benefit, such as the relationship between a human and its normal resident (commensal) gut microorganisms. Mx (myxoma resistant) proteins Interferon-inducible proteins required for cellular resistance to inﬂuenza virus replication. myasthenia gravis An autoimmune disease in which autoantibodies against the acetylcholine receptor on skeletal muscle cells cause a block in neuromuscular junctions, leading to progressive weakness and eventually death. mycophenolate An inhibitor of the synthesis of guanosine monophosphate that acts as a cytotoxic immunosuppressive drug. It acts by killing rapidly dividing cells, including lymphocytes proliferating in response to antigen. mycophenolate mofetil Pro-drug used in cancer treatment that is metabolized to mycophenolate, and inhibitor of inosine monophosphate dehydrogenase, thereby impairing guanosine monophosphate, and thus DNA, synthesis.

1	MyD88 An adaptor protein that functions in signaling by all TLR proteins except TLR3. myeloid Refers to the lineage of blood cells that includes all leukocytes except lymphocytes. myeloid-derived suppressor cells (MDSCs) Cells in tumors that can inhibit T-cell activation within the tumor. myelomonocytic series Innate immune cells derived from myelomonocytic bone marrow precursors, including neutrophils, basophils, eosinophils, monocytes, and dendritic cells. NADPH oxidase Multicomponent enzyme complex that is assembled and activated in the phagolysosome membrane in stimulated phagocytes. It generates superoxide in an oxygen-requiring reaction called the respiratory burst. NAIP2 An NLR protein that, together with NLRC4, recognizes the PrgJ protein of the Salmonella typhimurium type III injection system to activate an inﬂammasome pathway in response to infection.

1	NAIP2 An NLR protein that, together with NLRC4, recognizes the PrgJ protein of the Salmonella typhimurium type III injection system to activate an inﬂammasome pathway in response to infection. NAIP5 An NLR protein that, together with NLRC4, recognizes intracellular ﬂagellin to activate an inﬂammasome pathway in response to infection. naive lymphocytes T cells or B cells that have undergone normal development in the thymus or the bone marrow but have not yet been activated by foreign (or self) antigens. naive T cells Lymphocytes that have never encountered their specific antigen and thus have never responded to it, as distinct from effector and memory lymphocytes.

1	naive T cells Lymphocytes that have never encountered their specific antigen and thus have never responded to it, as distinct from effector and memory lymphocytes. nasal-associated lymphoid tissue (NALT) Organized lymphoid tissues found in the upper respiratory tract. In humans, NALT consists of Waldeyer’s ring, which includes the adenoids, palatine, and lingual tonsils, plus other similarly organized lymphoid tissue located around the pharynx. It is part of the mucosal immune system. natalizumab Humanized antibody to α4 integrin used to treat Crohn's disease and multiple sclerosis. It blocks lymphocytes' adhesion to endothelium, impairing their migration into tissues. natural antibodies Antibodies produced by the immune system in the apparent absence of any infection. They have a broad specificity for self and microbial antigens, can react with many pathogens, and can activate complement.

1	natural cytotoxicity receptors (NCRs) Activating receptors on NK cells that recognize infected cells and stimulate cell killing by the NK cell. natural interferon-producing cells See plasmacytoid dendritic cells. natural killer (NK) cell A type of ILC that is important in innate immunity to viruses and other intracellular pathogens, and in antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells express activating and inhibitory receptors, but not the antigen-specific receptors of T or B cells. necrosis The process of cell death that occurs in response to noxious stimuli, such as nutrient deprivation, physical injury, or infection. To be distinguished from apoptosis, in which the cell activates an internal, or intrinsic, program of death, such as occurs in immune cells as a result of deficiency of cell survival signals.

1	negative selection The process by which self-reactive thymocytes are deleted from the repertoire during T-cell development in the thymus. Autoreactive B cells undergo a similar process in bone marrow. NEMO See IKK. NEMO deficiency See X-linked hypohidrotic ectodermal dysplasia and immunodeficiency. neoepitopes Type of tumor rejection antigen created by mutations in protein that can be presented by self-MHC molecules to T cells. neonatal Fc receptor (FcRn) See FcRn. neuraminidase An inﬂuenza virus protein that cleaves sialic acid from host cells to allow viral detachment, a common antigenic determinant, and target of antiviral neuraminidase inhibitors. neutralization Inhibition of the infectivity of a virus or the toxicity of a toxin molecule by the binding of antibodies. neutralizing antibodies Antibodies that inhibit the infectivity of a virus or the toxicity of a toxin. neutropenia Abnormally low levels of neutrophils in the blood.

1	neutralizing antibodies Antibodies that inhibit the infectivity of a virus or the toxicity of a toxin. neutropenia Abnormally low levels of neutrophils in the blood. neutrophil The most numerous type of white blood cell in human peripheral blood. Neutrophils are phagocytic cells with a multilobed nucleus and granules that stain with neutral stains. They enter infected tissues and engulf and kill extracellular pathogens. neutrophil elastase Proteolytic enzyme stored in the granules of neutrophils that is involved in the processing of antimicrobial peptides. neutrophil extracellular traps (NETs) A meshwork of nuclear chromatin that is released into the extracellular space by neutrophils undergoing apoptosis at sites of infection, serving as a scaffold that traps extracellular bacteria to enhance their phagocytosis by other phagocytes.

1	NFκB A heterodimeric transcription factor activated by the stimulation of Toll-like receptors and also by antigen receptor signaling composed of p50 and p65 subunits. NFAT See nuclear factor of activated T cells. Nfil3 A transcription factor important during the development of several types of immune cells including certain types of NK cells. NHEJ See nonhomologous end joining. nitric oxide A reactive molecular gas species produced by cells— particularly macrophages—during infection, that is toxic to bacteria and intracellular microbes. nivolumab Human anti-PD-1 antibody used for checkpoint blockade in treatment of metastatic melanoma. NK receptor complex (NKC) A cluster of genes that encode a family of receptors on NK cells. NKG2 Family of C-type lectins that supply one of the subunits of KLR-family receptors on NK cells. NKG2D Activating C-type lectin receptor on NK cells, cytotoxic T cells, and γ:δT cells that recognizes the stress-response proteins MIC-A and MIC-B.

1	NKG2D Activating C-type lectin receptor on NK cells, cytotoxic T cells, and γ:δT cells that recognizes the stress-response proteins MIC-A and MIC-B. NLRC4 An NLR family member that cooperates with NAIP2 and NAIP5. NLRP family A group of 14 NOD-like receptor (NLR) proteins that contain a pyrin domain and function in the formation of a signaling complex called the inﬂammasome. NLRP3 A member of the family of intracellular NOD-like receptor proteins that have pyrin domains. It acts as a sensor of cellular damage and is part of the inﬂammasome. Sometimes called NALP3. N-nucleotides Nontemplated nucleotides inserted by the enzyme terminal deoxynucleotidyl transferase into the junctions between gene segments of T-cell receptor and immunoglobulin heavy-chain V regions during gene segment joining. Translation of these N-regions markedly increases the diversity of these receptor chains.

1	NOD subfamily A subgroup of NLR proteins that contain a CARD domain which is used for activation of downstream signaling. NOD1, NOD2 Intracellular proteins of the NOD subfamily that contain a leucine-rich repeat (LRR) domain that binds components of bacterial cell walls to activate the NFκB pathway and initiate inﬂammatory responses. NOD-like receptors (NLRs) Large family of proteins containing a nucleotide-oligomerization domain (NOD) associated with various other domains, and whose general function is the detection of microbes and of cellular stress. nonamer Conserved nine-nucleotide DNA sequence in the recombination signal sequences (RSSs) ﬂanking gene segments in the immunoglobulin and T-cell receptor loci. non-canonical inflammasome An alternate form of the inﬂammasome that is independent of caspase 1, but instead relies on caspase 11 (mice) or caspases 4 or 5 (human).

1	non-canonical inflammasome An alternate form of the inﬂammasome that is independent of caspase 1, but instead relies on caspase 11 (mice) or caspases 4 or 5 (human). non-canonical NFκB pathway A pathway for NFκB activation that is distinct from the one activated by antigen receptor stimulation. This pathway leads to activation of the NFκB-inducing kinase, NIK, which phosphorylates and activates IκB kinase α (IKKα) inducing cleavage of the NFκB precursor protein p100 to form the active p52 subunit. nonclassical MHC class Ib genes A class of proteins encoded within the MHC that are related to the MHC class I molecules but are not highly polymorphic and present a restricted set of antigens. non-depleting antibodies Immunosuppressive antibodies that block the function of target proteins on cells without causing the cells to be destroyed. nonhomologous end joining (NHEJ) DNA repair pathway that directly ligates double-stranded DNA breaks without use of a homologous template.

1	nonhomologous end joining (NHEJ) DNA repair pathway that directly ligates double-stranded DNA breaks without use of a homologous template. nonproductive rearrangements Rearrangements of T-cell receptor or immunoglobulin gene segments that cannot encode a protein because the coding sequences are in the wrong translational reading frame. nonreceptor kinase Cytoplasmic protein kinases that associate with the intracellular tails of signaling receptors and help generate the signal but are not an intrinsic part of the receptor itself. non-structural protein 1 (NS1) An inﬂuenza A virus protein that inhibits TRIM25, an intermediate signaling protein downstream of the viral sensors RIG-I and MDA-5, thereby promoting evasion of innate immunity. nuclear factor of activated T cells A family of transcription factors that are activated in response to increased cytoplasmic calcium following antigen receptor signaling in lymphocytes.

1	nuclear factor of activated T cells A family of transcription factors that are activated in response to increased cytoplasmic calcium following antigen receptor signaling in lymphocytes. nucleotide-binding oligomerization domain (NOD) A type of conserved domain originally recognized in ATP-binding cassette (ABC) transporters present present in a large number of proteins, but which also mediates protein homooligomerization. nude A mutation in mice that results in hairlessness and defective formation of the thymic stroma, so that mice homozygous for this mutation have no mature T cells. NY-ESO-1 A particular highly immunogenic cancer-testis antigen expressed by many types of human tumors including melanoma. occupational allergies An allergic reaction induced to an allergen to which someone is habitually exposed in their work.

1	occupational allergies An allergic reaction induced to an allergen to which someone is habitually exposed in their work. oligoadenylate synthetase Enzyme produced in response to stimulation of cells by interferon. It synthesizes unusual nucleotide polymers, which in turn activate a ribonuclease that degrades viral RNA. Omenn syndrome A severe immunodeficiency disease characterized by defects in either of the RAG genes. Affected individuals make small amounts of functional RAG protein, allowing a small amount of V(D)J recombination. opsonization The coating of the surface of a pathogen by antibody and/or complement that makes it more easily ingested by phagocytes. oral tolerance The suppression of specific systemic immune responses to an antigen by the prior administration of the same antigen by the oral (enteric) route.

1	oral tolerance The suppression of specific systemic immune responses to an antigen by the prior administration of the same antigen by the oral (enteric) route. original antigenic sin The tendency of humans to make antibody responses to those epitopes shared between the first strain of a virus they encounter and subsequent related viruses, while ignoring other highly immunogenic epitopes on the second and subsequent viruses. p50 See NFκB. p65 See NFκB. PA28 proteasome-activator complex A multisubunit protein complex induced by interferon-γ that takes the place of the 19S regulatory cap of the proteasome and increases the rate of peptides exiting from the proteasome catalytic core. palatine tonsils Paired masses of organized peripheral lymphoid tissues located on each side of the throat, and in which an adaptive immune response can be generated. They are part of the mucosal immune system.

1	Paneth cells Specialized epithelial cells at the base of the crypts in the small intestine that secrete antimicrobial peptides. papain A protease that cleaves the IgG antibody molecule on the amino-terminal side of disulfide linkages, producing two Fab fragments and one Fc fragment. paracortical areas The T-cell area of lymph nodes. paracrine Describes a cytokine or other biologically active molecule acting on cells near to those that produce it. parasites Organisms that obtain sustenance from a live host. In immunology, it refers to worms and protozoa, the subject matter of parasitology. paroxysmal nocturnal hemoglobinuria A disease in which complement regulatory proteins are defective, so that activation of complement binding to red blood cells leads to episodes of spontaneous hemolysis. passive immunization The injection of antibody or immune serum into a naive recipient to provide specific immunological protection. Cf. active immunization.

1	passive immunization The injection of antibody or immune serum into a naive recipient to provide specific immunological protection. Cf. active immunization. pathogen Microorganism that typically causes disease when it infects a host. pathogen-associated molecular patterns (PAMPs) Molecules specifically associated with groups of pathogens that are recognized by cells of the innate immune system. pathogenesis The origin or cause of the pathology of a disease. pathogenic microorganisms Microorganism that typically causes disease when it infects a host. patrolling monocyte A form of circulating monocyte that adheres to and surveys the vascular endothelium, distinguished from classical monocytes by its low expression of Ly6C. pattern recognition receptors (PRRs) Receptors of the innate immune system that recognize common molecular patterns on pathogen surfaces.

1	pattern recognition receptors (PRRs) Receptors of the innate immune system that recognize common molecular patterns on pathogen surfaces. PD-1 Programmed death-1, a receptor on T cells that when bound by its ligands, PD-L1 and PD-L2, inhibits signaling from the antigen receptor. PD-1 contains an ITIM motif in its cytoplasmic tail. Target of cancer therapies aimed at stimulating T-cell responses to tumors. PD-L1 (programmed death ligand-1, B7-H1) Transmembrane receptor that binds to the inhibitory receptor PD-1. PD-L1 is expressed on many cell types and is upregulated by inﬂammatory cytokines. PD-L2 (programmed death ligand-2, B7-DC) Transmembrane receptor that binds to the inhibitory receptor PD-1; mainly expressed on dendritic cells. PECAM See CD31. pembrolizumab Human anti-PD-1 antibody used for checkpoint blockade in treatment of metastatic melanoma. pemphigus vulgaris An autoimmune disease characterized by severe blistering of the skin and mucosal membranes.

1	pemphigus vulgaris An autoimmune disease characterized by severe blistering of the skin and mucosal membranes. pentameric IgM Major form of the IgM antibodies produced by the action of J chain resulting in higher avidity for antigens. pentraxin A family of acute-phase proteins formed of five identical subunits, to which C-reactive protein and serum amyloid protein belong. pepsin A protease that cleaves several sites on the carboxy-terminal side of the disulfide linkages, producing the F(abʹ)2 fragment and several fragments of the Fc region. peptide-binding cleft The longitudinal cleft in the top surface of an MHC molecule into which the antigenic peptide is bound. Sometimes called the peptide-binding groove. peptide editing In the context of antigen processing and presentation, the removal of unstably bound peptides from MHC class II molecules by HLA-DM.

1	peptide editing In the context of antigen processing and presentation, the removal of unstably bound peptides from MHC class II molecules by HLA-DM. peptide-loading complex (PLC) A protein complex in the endoplasmic reticulum that loads peptides onto MHC class I molecules. peptide:MHC tetramers Four specific peptide:MHC complexes bound to a single molecule of ﬂuorescently labeled streptavidin, which are used to identify populations of antigen-specific T cells. peptidoglycan A component of bacterial cell walls that is recognized by certain receptors of the innate immune system. peptidoglycan-recognition proteins (PGRPs) A family of Drosophila proteins that bind peptidoglycans from bacterial cell walls that serve to initiate the proteolytic cascade of the TOLL pathway. periarteriolar lymphoid sheath (PALS) Part of the inner region of the white pulp of the spleen; it contains mainly T cells.

1	periarteriolar lymphoid sheath (PALS) Part of the inner region of the white pulp of the spleen; it contains mainly T cells. peripheral lymphoid organs, peripheral lymphoid tissues The lymph nodes, spleen, and mucosa-associated lymphoid tissues, in which adaptive immune responses are induced, as opposed to the central lymphoid organs, in which lymphocytes develop. They are also called secondary lymphoid organs and tissues. peripheral tolerance Tolerance acquired by mature lymphocytes in the peripheral tissues, as opposed to central tolerance, which is acquired by immature lymphocytes during their development. Peyer’s patches Organized peripheral lymphoid organs under the epithelium in the small intestine, especially the ileum, and in which an adaptive immune response can be initiated. They contain lymphoid follicles and T-cell areas. They are part of the gut-associated lymphoid tissues (GALT). phagocyte oxidase See NADPH oxidase. phagocytic glycoprotein-1 (Pgp1) See CD44.

1	phagocyte oxidase See NADPH oxidase. phagocytic glycoprotein-1 (Pgp1) See CD44. phagocytosis The internalization of particulate matter by cells by a process of engulfment, in which the cell membrane surrounds the material, eventually forming an intracellular vesicle (phagosome) containing the ingested material. phagolysosome Intracellular vesicle formed by the fusion of a phagosome (containing ingested material) and a lysosome, and in which the ingested material is broken down. phagophore A crescent-shaped double-membrane cytoplasmic structure. phagosome Intracellular vesicle formed when particulate material is ingested by a phagocyte. phosphatidylinositol 3-kinase (PI 3-kinase) Enzyme involved in intracellular signaling pathways. It phosphorylates the membrane lipid phosphatidylinositol 3,4-bisphosphate (PIP2) to form phosphatidylinositol 3,4,5-trisphosphate (PIP3), which can recruit signaling proteins containing pleckstrin homology (PH) domains to the membrane.

1	phosphatidylinositol kinases Enzymes that phosphorylate the inositol headgroup on membrane lipids to produce phosphorylated derivatives that have a variety of functions in intracellular signaling. phospholipase C-γ (PLC-γ) Key enzyme in intracellular signaling pathways leading from many different receptors. It is activated by membrane recruitment and tyrosine phosphorylation following receptor ligation, and cleaves membrane inositol phospholipids into inositol trisphosphate and diacylglycerol. phosphorylation Addition of a phosphate group to a molecule, usually a protein, catalyzed by enzymes called kinases. phycoerythrin A light harvesting protein pigment made by algae and used in conjunction with ﬂow cytometry, it can also be recognized as a ligand by some γ:δ T-cell receptors.

1	phycoerythrin A light harvesting protein pigment made by algae and used in conjunction with ﬂow cytometry, it can also be recognized as a ligand by some γ:δ T-cell receptors. physiological inflammation The state of the normal healthy intestine, whose wall contains large numbers of effector lymphocytes and other cells. It is thought to be the result of continual stimulation by commensal organisms and food antigens. pi-cation interactions Chemical interaction between a cation (e.g., Na+) and the pi-electron system of an aromatic moiety. pilin An adhesin of Neisseria gonorrhoeae allowing attachment to and infection of epithelial cells of urinary and reproductive tracts. PIP2 Phosphatidylinositol 3,4-bisphosphate, a membrane-associated phospholipid that is cleaved by phospholipase C-γ to give the signaling molecules diacylglycerol and inositol trisphosphate and is phosphorylated by PI3-kinase to generate PIP3.

1	PIP3 Phosphatidylinositol 3,4,5-trisphosphate, a membrane-associated phospholipid that can recruit intracellular signaling molecules containing pleckstrin homology (PH) domains to the membrane. PKR Serine/threonine kinase activated by IFN-α and IFN-β. It phosphorylates the eukaryotic protein synthesis initiation factor eIF-2, inhibiting translation and thus contributing to the inhibition of viral replication. plasma cells Terminally differentiated activated B lymphocytes. Plasma cells are the main antibody-secreting cells of the body. They are found in the medulla of the lymph nodes, in splenic red pulp, in bone marrow, and in mucosal tissues. plasmablasts A B cell in a lymph node that already shows some features of a plasma cell. plasmacytoid dendritic cells (pDCs) A distinct lineage of dendritic cells that secrete large amounts of interferon on activation by pathogens and their products via receptors such as Toll-like receptors. Cf. conventional dendritic cells.

1	platelet-activating factor (PAF) A lipid mediator that activates the blood clotting cascade and several other components of the innate immune system. pluripotent Typically referring to the capacity of a progenitor cell to generate all possible lineages of an organ system. P-nucleotides Short palindromic nucleotide sequences formed between gene segments of the rearranged V-region gene generated by the asymmetric opening of the hairpin intermediate during RAG-mediated rearrangement. Polη An error-prone, 'translesion', DNA polymerase involved in repairing DNA damage caused by UV radiation and in somatic hypermutation. polyclonal activation The activation of lymphocytes by a mitogen regardless of antigen specificity, leading to the activation of clones of lymphocytes of multiple antigen specificities. polygenic Containing several separate loci encoding proteins of identical function; applied to the MHC. Cf. polymorphic.

1	polygenic Containing several separate loci encoding proteins of identical function; applied to the MHC. Cf. polymorphic. polymerase stalling The halting of RNA polymerase during the transcription of a gene at locations within the gene locus, known to be a regulated process, and involved in mechanisms of isotype switching. polymeric immunoglobulin receptor (pIgR) The receptor for polymeric immunoglobulins IgA and IgM on basolateral surfaces of mucosal and glandular epithelial cells that transports IgA (or IgM) into secretions. polymorphic Existing in a variety of different forms; applied to a gene, occurring in a variety of different alleles. polymorphism Applied to genes, variability at a gene locus in which all variants occur at a frequency greater than 1%. polymorphonuclear leukocytes See granulocytes.

1	polymorphism Applied to genes, variability at a gene locus in which all variants occur at a frequency greater than 1%. polymorphonuclear leukocytes See granulocytes. polysaccharide capsules A distinct structure in some bacteria—both Gram-negative and Gram-positive—that lies outside cell membrane and cell wall that can prevent direct phagocytosis by macrophages without the aid of antibody or complement. polyubiquitin chains Polymers of ubiquitin covalently linked from lysine residues within one ubiquitin monomer to the carboxy terminus of a second ubiquitin. PorA Outer membrane protein of Neisseria meningitidis that binds C4BP, thereby inactivating C3b deposited on its surface. positive selection A process occurring in the thymus in which only those developing T cells whose receptors can recognize antigens presented by self MHC molecules can mature.

1	positive selection A process occurring in the thymus in which only those developing T cells whose receptors can recognize antigens presented by self MHC molecules can mature. post-transplant lymphoproliferative disorder B-cell expansion driven by Epstein–Barr virus (EBV) in which the B cells can undergo mutations and become malignant. This can occur when patients are immunosuppressed after, for example, solid organ transplantation. pre-B-cell receptor Receptor produced by pre-B cells that includes an immunoglobulin heavy chain, as well as surrogate light-chain proteins, Igα and Igβ signaling subnits. Signaling through this receptor induces the pre-B cell to enter the cell cycle, to turn off the RAG genes, to degrade the RAG proteins, and to expand by several cell divisions.

1	pre-T-cell receptor Receptor protein produced by developing T lymphocytes at the pre-T-cell stage. It is composed of TCRβ chains that pair with a surrogate α chain called pTα (pre-T-cell α), and is associated with the CD3 signaling chains. Signaling through this receptor induces pre-T-cell proliferation, expression of CD4 and CD8, and cessation of TCR β chain rearrangement. prednisone A synthetic steroid with potent anti-inﬂammatory and immunosuppressive activity used in treating acute graft rejection, autoimmune disease, and lymphoid tumors. PREX1 A guanine exchange factor (GEF) activated downstream of small G proteins in response to activation of GPCRs such as the fMLP or C5a receptor. PrgJ A protein component of the Salmonella typhimurium type III secretion system inner rod used by the bacterium to infect eukaryotic cells. This protein is detected by NLR proteins NAIP2 and NLRC4.

1	PrgJ A protein component of the Salmonella typhimurium type III secretion system inner rod used by the bacterium to infect eukaryotic cells. This protein is detected by NLR proteins NAIP2 and NLRC4. primary focus Site of early antibody production by plasmablasts in medullary cords of lymph nodes that precedes the germinal center reaction and differentiation of plasma cells. primary granules Granules in neutrophils that correspond to lysosomes and contain antimicrobial peptides such as defensins and other antimicrobial agents. primary immune response The adaptive immune response that follows the first exposure to a particular antigen. primary immunization See priming. primary immunodeficiencies A lack of immune function that is caused by a genetic defect. primary lymphoid follicles Aggregates of resting B lymphocytes in peripheral lymphoid organs. Cf. secondary lymphoid follicle. primary lymphoid organs See central lymphoid organs.

1	primary lymphoid follicles Aggregates of resting B lymphocytes in peripheral lymphoid organs. Cf. secondary lymphoid follicle. primary lymphoid organs See central lymphoid organs. priming The first encounter with a given antigen, which generates the primary adaptive immune response. pro-B cells A stage in B-lymphocyte development in which cells have displayed B-cell surface marker proteins but have not yet completed heavy-chain gene rearrangement. pro-caspase 1 The inactive pro-form of caspase 1 that is part of the NLRP3 inﬂammasome. pro-inflammatory Tending to induce inﬂammation. profilin An actin-binding protein that sequesters monomeric actin. Protozoan profilins contain sequences recognized by TLR-11 and TLR-12. programmed cell death See apoptosis. progressive multifocal leukoencephalopathy (PML) Disease in immunocompromised patients caused by opportunisitic infection by JC virus, for example as a consequence of immunotherapy.

1	progressive multifocal leukoencephalopathy (PML) Disease in immunocompromised patients caused by opportunisitic infection by JC virus, for example as a consequence of immunotherapy. propeptides Inactive precursor form of a polypeptide or peptide, which requires proteolytic processing to produce the active peptide. properdin See factor P. prostaglandins Lipid products of the metabolism of arachidonic acid that have a variety of effects on tissues, including activities as inﬂammatory mediators. prostatic acid phosphatase (PAP) Enzyme expressed by prostate cancer cells used as tumor rejection antigen in the vaccine Sipuleucel-T (Provenge). proteasome A large intracellular multisubunit protease that degrades proteins, producing peptides. protein inhibitors of activated STAT (PIAS) A small family of proteins that inhibit STAT family transcription factors.

1	protein inhibitors of activated STAT (PIAS) A small family of proteins that inhibit STAT family transcription factors. protein-interaction domains, protein-interaction modules Protein domains, usually with no enzymatic activity themselves, that have binding specificity for particular sites (such as phosphorylated tyrosines, proline-rich regions, or membrane phospholipids) on other proteins or cellular structures. protein kinase C-θ (PKC-θ) A serine/threonine kinase that is activated by diacylglycerol as part of the signaling pathways from the antigen receptor in lymphocytes. protein kinases Enzymes that add phosphate groups to proteins at particular amino acid residues: tyrosine, threonine, or serine. protein phosphatases Enzymes that remove phosphate groups from proteins phosphorylated on tyrosine, threonine, or serine residues by protein kinases. proteolytic subunits β1, β2, β5 Constitutive components of the proteasome's catalytic chamber.

1	proteolytic subunits β1, β2, β5 Constitutive components of the proteasome's catalytic chamber. provirus The DNA form of a retrovirus when it is integrated into the host-cell genome, where it can remain transcriptionally inactive for long periods. P-selectin See selectins. P-selectin glycoprotein ligand-1 (PSGL-1) Protein expressed by activated effector T cells that is a ligand for P-selectin on endothelial cells, and may enable activated T cells to enter all tissues in small numbers. pseudo-dimeric peptide:MHC complexes Hypothetical complexes containing one antigen peptide:MHC molecule and one self peptide:MHC molecule on the surface of the antigen-presenting cell, which have been proposed to initiate T-cell activation. pseudogenes Gene elements that have lost the ability to encode a functional protein but that are retained in the genome and may continue to be transcribed normally.

1	pseudogenes Gene elements that have lost the ability to encode a functional protein but that are retained in the genome and may continue to be transcribed normally. psoriasis Chronic autoimmune disease thought to be driven by T cells manifested in skin, but which can also involve nails and joints (psoriatic arthropathy). psoriatic arthropathy See psoriasis. pTα See pre-T-cell receptor. purine nucleotide phosphorylase (PNP) deficiency An enzyme defect that results in severe combined immunodeficiency. The deficiency of PNP causes an intracellular accumulation of purine nucleosides, which are toxic to developing T cells. purinergic receptor P2X7 An ATP-activated ion channel that allows potassium efﬂux from cells when activated, which can trigger inﬂammasome activation in response to excessive extracellular ATP.

1	purinergic receptor P2X7 An ATP-activated ion channel that allows potassium efﬂux from cells when activated, which can trigger inﬂammasome activation in response to excessive extracellular ATP. pus Thick yellowish-white liquid typically found at sites of infection with some types of extracellular bacteria, which is composed of the remains of dead neutrophils and other cells. pus-forming bacteria Capsulated bacteria that result in pus formation at the site of infection. Also called pyogenic (pus-forming) bacteria. PYHIN A family of four intracellular sensor proteins containing an H inversion (HIN) domain in place of the LRR domain found in most other NLR proteins. The HIN domain functions in recognition of cytoplasmic dsDNA. Examples are AIM2 and IFI16. pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) Autoinﬂammatory syndrome that is caused by mutations in a protein that interacts with pyrin. pyogenic bacteria See pus-forming bacteria.

1	pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) Autoinﬂammatory syndrome that is caused by mutations in a protein that interacts with pyrin. pyogenic bacteria See pus-forming bacteria. pyrin One of several protein interaction domains, structurally related to but distinct from CARD, TIR, DD, and DED domains. pyroptosis A form of programmed cell death that is associated with abundant pro-inﬂammatory cytokines such as IL-1β and IL-18 produced through inﬂammasome activation. Qa-1 determinant modifiers (Qdm) A class of peptides derived from the leader peptides of various HLA class I molecules that can be bound by the human HLA-E and murine Qa-1 proteins, and then are recognized by the inhibitory NKG2A:CD94 receptor. quasi-species The different genetic forms of certain RNA viruses that are formed by mutation during the course of an infection. Rac See Rho family small GTPase proteins.

1	quasi-species The different genetic forms of certain RNA viruses that are formed by mutation during the course of an infection. Rac See Rho family small GTPase proteins. radiation-sensitive SCID (RS-SCID) Severe combined immunodeficiency due to a defect in DNA repair pathways, which renders cells unable to perform V(D)J recombination and unable to repair radiation-induced double-strand breaks. RAE1 (retinoic acid early inducible 1) protein family Several murine MHC class Ib proteins; these are orthologs of human RAET1 family proteins, including H60 and MULT1, and are ligands for murine NKG2D. RAET1 A family of 10 MHC class Ib proteins that are ligands for NKG2D, and includes several UL16-binding proteins (ULBPs). Raf A protein kinase in the Raf–MEK1–Erk signaling cascade that is the first protein kinase in the pathway, and is activated by the small GTPase Ras.

1	Raf A protein kinase in the Raf–MEK1–Erk signaling cascade that is the first protein kinase in the pathway, and is activated by the small GTPase Ras. RAG-1, RAG-2 Proteins encoded by the recombination-activating genes RAG-1 and RAG-2, which form a dimer that initiates V(D)J recombination. rapamycin An immunosuppressant drug that blocks intracellular signaling pathways involving the serine/threonine kinase mammalian target of rapamycin (mTOR) required for the inhibition of apoptosis and T-cell expansion. Also called sirolimus. Raptor See mTORC1. Ras A small GTPase with important roles in intracellular signaling pathways, including those from lymphocyte antigen receptors. reactive oxygen species (ROS) Superoxide anion (O2–) and hydrogen peroxide (H2O2), produced by phagocytic cells such as neutrophils and macrophages after ingestion of microbes, and which help kill the ingested microbes.

1	rearrangement by inversion In V(D)J recombination, the rearrangement of gene segments having RSS elements in an opposing orientation, leading to retention. receptor editing The replacement of a light or heavy chain of a self-reactive antigen receptor on immature B cells with a newly rearranged chain that does not confer autoreactivity. receptor-mediated endocytosis The internalization into endosomes of molecules bound to cell-surface receptors. receptor serine/threonine kinases Receptors that have an intrinsic serine/threonine kinase activity in their cytoplasmic tails. receptor tyrosine kinases Receptors that have an intrinsic tyrosine kinase activity in their cytoplasmic tails. recombination signal sequences (RSSs) DNA sequences at one or both ends of V, D, and J gene segments that are recognized by the RAG-1:RAG-2 recombinase. They consist of a conserved heptamer and nonamer element separated by 12 or 23 base pairs.

1	red pulp The nonlymphoid area of the spleen in which red blood cells are broken down. RegIIIγ An antimicrobial protein of the C-type lectin family, produced by Paneth cells in the gut in mice. regulatory T cells Effector CD4 T cells that inhibit T-cell responses and are involved in controlling immune reactions and preventing autoimmunity. Several different subsets have been distinguished, notably the natural regulatory T-cell lineage that is produced in the thymus, and the induced regulatory T cells that differentiate from naive CD4 T cells in the periphery in certain cytokine environments. regulatory tolerance Tolerance due to the actions of regulatory T cells. Relish A distinct member of the Drosophila NFκB transcription factor family that induces the expression of several antimicrobial peptides in response to Gram-negative bacteria. resistance A general immune strategy aimed at reducing or eliminating pathogens; compare with avoidance and tolerance.

1	resistance A general immune strategy aimed at reducing or eliminating pathogens; compare with avoidance and tolerance. respiratory burst An oxygen-requiring metabolic change in neutrophils and macrophages that have taken up opsonized particles, such as complementor antibody-coated bacteria, by phagocytosis. It leads to the production of toxic metabolites that are involved in killing the engulfed microorganisms. restriction factors Host proteins that act in a cell-autonomous manner to inhibit the replication of retroviruses such as HIV. retinoic acid Signaling molecule derived from vitamin A with many roles in the body. It is thought to be involved in the induction of immunological tolerance in the gut. retrotranslocation complex The return of endoplasmic reticulum proteins to the cytosol.

1	retrotranslocation complex The return of endoplasmic reticulum proteins to the cytosol. retrovirus A single-stranded RNA virus that uses the viral enzyme reverse transcriptase to transcribe its genome into a DNA intermediate that integrates into the host-cell genome to undergo viral replication. reverse transcriptase Viral RNA-dependent DNA polymerase that is found in retroviruses and transcribes the viral genomic RNA into DNA during the life cycle of retroviruses (such as HIV). Rheb A small GTPase that activates mTOR when in its GTP-bound form, and is inactivated by a GTPase-activating protein (GAP) complex TSC1/2. rheumatic fever Disease caused by antibodies elicited by infection with some Streptococcus species. These antibodies cross-react with kidney, joint, and heart antigens. rheumatoid arthritis (RA) A common inﬂammatory joint disease that is probably due to an autoimmune response.

1	rheumatoid arthritis (RA) A common inﬂammatory joint disease that is probably due to an autoimmune response. rheumatoid factor An anti-IgG antibody of the IgM class first identified in patients with rheumatoid arthritis, but which is also found in healthy individuals. Rho See Rho family small GTPase proteins. Rho family small GTPase proteins Several distinct small GTPase family members that regulate the actin cytoskeleton in response to signaling through various receptors. Examples: Rac, Rho, and Cdc42. Rictor See mTORC2. RIG-I (retinoic acid-inducible gene I) See RIG-I-like receptors (RLRs). RIG-I-like receptors (RLRs) A small family of intracellular viral sensors that use a carboxy terminal RNA helicase-like domain in detection of various forms of viral RNA. These signal through MAVS to activate antiviral immunity. Examples include RIG-I, MDA-5, and LGP2.

1	RIP2 A CARD domain containing serine-threonine kinase that functions in signaling by NOD proteins to activate the NFκB transcription factor. Riplet An E3 ubiquitin ligase involved in signaling by RIG-I and MDA-5 for the activation of MAVS. rituximab A chimeric antibody to CD20 used to eliminate B cells in treatment of non-Hodgkin's lymphoma. R-loops A structure formed when transcribed RNA displaces the nontemplate strand of the DNA double helix at switch regions in the immunoglobulin constant-region gene cluster. R-loops are thought to promote class switch recombination. RNA exosome A multisubunit complex involved in processing and editing of RNA. ruxolitinib An inhibitor of JAK1 and JAK2 approved for treatment of myelofibrosis.

1	RNA exosome A multisubunit complex involved in processing and editing of RNA. ruxolitinib An inhibitor of JAK1 and JAK2 approved for treatment of myelofibrosis. S1PR1 A G protein-coupled receptor expressed on circulating lymphocytes that binds the chemotactic phospholipid, sphingosine 1-phosphate, which forms a chemotactic gradient that promotes the egress of non-activated lymphocytes out of secondary lymphoid tissues into the efferent lymphatics and blood. See also CD69. SAP (SLAM-associated protein) An intracellular adaptor protein involved in signaling by SLAM (signaling lymphocyte activation molecule). Inactivating mutations in this gene cause X-linked lymphoproliferative (XLP) syndrome. scaffolds Adaptor-type proteins with multiple binding sites, which bring together specific proteins into a functional signaling complex.

1	scaffolds Adaptor-type proteins with multiple binding sites, which bring together specific proteins into a functional signaling complex. scavenger receptors Receptors on macrophages and other cells that bind to numerous ligands, such as bacterial cell-wall components, and remove them from the blood. The Kupffer cells in the liver are particularly rich in scavenger receptors. Includes SR-A I, SR-A II, and MARCO. scid Mutation in mice that causes severe combined immunodeficiency. It was eventually found to be due to mutation of the DNA repair protein DNA-PK. SCID See severe combined immunodeficiency. seasonal allergic rhinoconjunctivitis IgE-mediated allergic rhinitis and conjunctivitis caused by exposure to specific seasonally occurring antigens, for example grass or weed pollens. Commonly called hay fever.

1	Sec61 A multisubunit transmembrane protein pore complex that resides in the membrane of the endoplasmic reticulum and allows peptides to be translocated from the ER lumen into the cytoplasm. second messengers Small molecules or ions (such as Ca2+) that are produced in response to a signal; they act to amplify the signal and carry it to the next stage within the cell. Second messengers generally act by binding to and modifying the activities of enzymes. secondary granules Type of granule in neutrophils that stores certain antimicrobial peptides. secondary immune response The immune response that occurs in response to a second exposure to an antigen. In comparison with the primary response, it starts sooner after exposure, produces greater levels of antibody, and produces class-switched antibodies. It is generated by the reactivation of memory lymphocytes.

1	secondary immunization A second or booster injection of an antigen, given some time after the initial immunization. It stimulates a secondary immune response. secondary immunodeficiencies Deficiencies in immune function that are a consequence of infection (e.g., HIV infection), other diseases (e.g., leukemia), malnutrition, etc.. secondary lymphoid follicle A follicle containing a germinal center of proliferating activated B cells during an ongoing adaptive immune response. secondary lymphoid organs See peripheral lymphoid organs. secondary lymphoid tissues See peripheral lymphoid organs. secretory component (SC) Fragment of the polymeric immunoglobulin receptor that remains after cleavage and is attached to secreted IgA after transport across epithelial cells. secretory IgA (SIgA) Polymeric IgA antibody (mainly dimeric) containing bound J chain and secretory component. It is the predominant form of immunoglobulin in most human secretions.

1	secretory IgA (SIgA) Polymeric IgA antibody (mainly dimeric) containing bound J chain and secretory component. It is the predominant form of immunoglobulin in most human secretions. secretory phospholipase A2 Antimicrobial enzyme present in tears and saliva and also secreted by the Paneth cells of the gut. segmented filamentous bacteria (SFB) Referring to commensal Gram-positive Firmicute species and members of the Clostridiaceae family that adhere to the intestinal wall of rodents and several other species that induce TH17 and IgA responses. selectins Family of cell-adhesion molecules on leukocytes and endothelial cells that bind to sugar moieties on specific glycoproteins with mucin-like features. self antigens The potential antigens on the tissues of an individual, against which an immune response is not usually made except in the case of autoimmunity. self-tolerance The failure to make an immune response against the body’s own antigens.

1	self-tolerance The failure to make an immune response against the body’s own antigens. sensitization The acute adaptive immune response made by susceptible individuals on first exposure to an allergen. In some of these individuals, subsequent exposure to the allergen will provoke an allergic reaction. sensitized In allergy, describes an individual who has made an IgE response on initial encounter with an environmental antigen and who manifests IgE-producing memory B cells. Subsequent allergen exposure can elicit an allergic response. sepsis Bacterial infection of the bloodstream. This is a very serious and frequently fatal condition. septic shock Systemic shock reaction that can follow infection of the bloodstream with endotoxin-producing Gram-negative bacteria. It is caused by the systemic release of TNF-α and other cytokines. Also called endotoxic shock.

1	sequence motif A pattern of nucleotides or amino acids shared by different genes or proteins that often have related functions. serine protease inhibitor (serpin) Class of proteins that inhibit various proteases, originally referring to those specific to serine proteases. seroconversion The phase of an infection when antibodies against the infecting agent are first detectable in the blood. serotypes Name given to a strain of bacteria, or other pathogen, that can be distinguished from other strains of the same species by specific antibodies. serum sickness A usually self-limiting immunological hypersensitivity reaction originally seen in response to the therapeutic injection of large amounts of foreign serum (now most usually evoked by the injection of drugs such as penicillin). It is caused by the formation of immune complexes of the antigen and the antibodies formed against it, which become deposited in the tissues, especially the kidneys.

1	severe combined immunodeficiency (SCID) Type of immune deficiency (due to various causes) in which both B-cell (antibody) and T-cell responses are lacking; it is fatal if not treated. severe congenital neutropenia (SCN) An inherited condition in which the neutrophil count is persistently extremely low. This is in contrast to cyclic neutropenia, in which neutrophil numbers ﬂuctuate from near normal to very low or absent, with an approximate cycle time of 21 days. SH2 (Src homology 2) domain See Src-family protein tyrosine kinases. shear-resistant rolling The capacity of neutrophils to maintain attached to the vascular endothelium under high rates of ﬂow—or shear—enabled by specialized plasma membrane extensions called slings. shingles Disease caused when herpes zoster virus (the virus that causes chickenpox) is reactivated later in life in a person who has had chickenpox.

1	shingles Disease caused when herpes zoster virus (the virus that causes chickenpox) is reactivated later in life in a person who has had chickenpox. SHIP (SH2-containing inositol phosphatase) An SH2-containing inositol phosphatase that removes the phosphate from PIP3 to produce PIP2. shock The potentially fatal circulatory collapse caused by the systemic actions of cytokines such as TNF-α. SHP (SH2-containing phosphatase) An SH2-containing protein phosphatase. signal joint The noncoding joint formed in DNA by the recombination of RSSs during V(D)J recombination. Cf. coding joint. signal peptide The short N-terminal peptide sequence responsible for directing newly synthesized proteins into the secretory pathway. signal transducers and activators of transcription (STATs) See Janus kinase (JAK) family.

1	signal transducers and activators of transcription (STATs) See Janus kinase (JAK) family. signaling scaffold A configuration of proteins and modifications, such as phosphorylation or ubiquitination, that facilitates signaling by binding various enzymes and their substrates. single-chain antibody Referring to the heavy-chain-only IgGs produced by camelids or shark species that lack the light chain present in conventional antibodies. single-nucleotide polymorphisms (SNPs) Positions in the genome that differ by a single base between individuals. single-positive thymocytes A mature T cell that expresses either the CD4 or the CD8 co-receptor, but not both. single-stranded RNA (ssRNA) Normally confined to the nucleus and cytoplasm, this normal molecular form serves as a ligand for TLR-7, TLR-8, and TLR-9 when it is present in endosomes, as during parts of a viral life cycle.

1	sipuleucel-T (Provenge) Cell-based immunotherapy used to treat prostate cancer that combined prostatic acid phosphatase as a tumor rejection antigen presented by dendritic cells derived from a patient's monocytes. sirolimus See rapamycin. Sjögren’s syndrome An autoimmune disease in which exocrine glands, particularly the lacrimal glands of the eyes and salivary glands of the mouth, are damaged by the immune system. This results in dry eyes and mouth. Skint-1 A transmembrane immunoglobulin superfamily member expressed by thymic stromal cells and keratinocytes that is required for the development of dendritic epidermal T cells, which are a type of γ:δ T cell. SLAM (signaling lymphocyte activation molecule) A family of related cell-surface receptors that mediate adhesion between lymphocytes, that includes SLAM, 2B4, CD84, Ly106, Ly9, and CRACC. slings See shear-resistant rolling.

1	slings See shear-resistant rolling. SLP-65 A scaffold protein in B cells that recruits proteins involved in the intracellular signaling pathway from the antigen receptor. Also called BLNK. SLP-76 A scaffold protein involved in the antigen-receptor signaling pathway in lymphocytes. small G proteins Single-subunit G proteins, such as Ras, that act as intracellular signaling molecules downstream of many transmembrane signaling events. Also called small GTPases. small pre-B cell Stage in B-cell development immediately after the large pre-B cell in which cell proliferation ceases and light-chain gene rearrangement commences. somatic diversification theories Gerenal hypotheses proposing that the immunoglobulin repertoire was formed from a small number of V genes that diversified in somatic cells. Cf. germline theory.

1	somatic diversification theories Gerenal hypotheses proposing that the immunoglobulin repertoire was formed from a small number of V genes that diversified in somatic cells. Cf. germline theory. somatic DNA recombination DNA recombination that takes place in somatic cells (to distinguish it from the recombination that takes place during meiosis and gamete formation). somatic gene therapy The introduction of functional genes into somatic cells to treat disease. somatic hypermutation Mutations in V-region DNA of rearranged immunoglobulin genes that produce variant immunoglobulins, some of which bind antigen with a higher affinity. These mutations affect only somatic cells and are not inherited through germline transmission. spacer See 12/23 rule. sphingolipids A class of membrane lipid containing sphingosine (2-amino4-octadecene-1,3-diole), an amino alcohol with unsaturated 18-hydrocarbon chain.

1	spacer See 12/23 rule. sphingolipids A class of membrane lipid containing sphingosine (2-amino4-octadecene-1,3-diole), an amino alcohol with unsaturated 18-hydrocarbon chain. sphingosine 1-phosphate (S1P) A phospholipid with chemotactic activity that controls the egress of T cells from lymph nodes. sphingosine 1-phosphate receptor (S1P1) A G-protein-coupled receptor activated by sphingosine 1-phosphate, a lipid mediator in the blood that regulates several physiologic processes, including the trafficking of naive lymphocytes from tissues into the blood. spleen An organ in the upper left side of the peritoneal cavity containing a red pulp, involved in removing senescent blood cells, and a white pulp of lymphoid cells that respond to antigens delivered to the spleen by the blood. S-protein (vitronectin) Plasma protein that binds incompletely formed MAC complexes, such as C5b67, preventing bystander complement damage to host membranes.

1	S-protein (vitronectin) Plasma protein that binds incompletely formed MAC complexes, such as C5b67, preventing bystander complement damage to host membranes. Spt5 A transcription elongation factor required for isotype switching in B cells that functions in associatioin with RNA polymerase to enable recruitment of AID to its targets in the genome. SR-A I, SR-A II See scavenger receptors. Src-family protein tyrosine kinases Receptor-associated protein tyrosine kinases characterized by Src-homology protein domains (SH1, SH2, and SH3). The SH1 domain contains the kinase, the SH2 domain can bind phosphotyrosine residues, and the SH3 domain can interact with proline-rich regions in other proteins. In T cells and B cells they are involved in relaying signals from the antigen receptor. staphylococcal complement inhibitor (SCIN) Staphylococcal protein that inhibits the activity of the classical and alternative C3 convertases, promoting the evasion of destruction by complement.

1	staphylococcal complement inhibitor (SCIN) Staphylococcal protein that inhibits the activity of the classical and alternative C3 convertases, promoting the evasion of destruction by complement. staphylococcal enterotoxins (SEs) Secreted toxins produced by some staphylococci, which cause food poisoning and also stimulate many T cells by binding to MHC class II molecules and the Vβ domain of certain T-cell receptors, acting as superantigens. staphylococcal protein A (Spa) Staphylococcal protein that blocks the binding of the antibody Fc region with C1, thereby preventing complement activation. staphylokinase Staphylococcal protease that cleaves immunoglobulins bound to its surface, thereby preventing complement activation. STAT (signal transducers and activators of transcription) A family of seven transcription factors activated by many cytokine and growth factor receptors. STAT3 See STAT. STAT6 See STAT. statins Drug inhibitors of HMG-CoA reductase used to lower cholesterol.

1	STAT3 See STAT. STAT6 See STAT. statins Drug inhibitors of HMG-CoA reductase used to lower cholesterol. sterile injury Damage to tissues due to trauma, ischemia, metabolic stress, or autoimmunity, bearing many immune features similar to infection. sterilizing immunity An immune response that completely eliminates a pathogen. STIM1 A transmembrane protein that acts as a Ca2+ sensor in the endoplasmic reticulum. When Ca2+ is depleted from the endoplasmic reticulum, STIM1 is activated and induces opening of plasma membrane CRAC channels. STING (stimulator of interferon genes) A dimeric protein complex in the cytoplasm anchored to the ER membrane that functions in intracellular sensing for infection. It is activated by specific cyclic di-nucleotides to activate TBK1, which phosphorylates IRF3 to induce transcription of type I interferon genes. stress-induced self See dysregulated self.

1	stress-induced self See dysregulated self. stromal cells The nonlymphoid cells in central and peripheral lymphoid organs that provide soluble and cell-bound signals required for lymphocyte development, survival, and migration. subcapsular sinus (SCS) The site of lymphatic entry in lymph nodes lined by phagocytes, including subcapsular macrophages which capture particulate and opsonized antigens draining in from tissues. sulfated sialyl-LewisX A sulfated tetrasaccharide carbohydrate structure attached to many cell surface proteins, it binds the P-selectin and E-selectin molecules on the surface of cells, such as neutrophils, that mediate interactions with the endothelium. superoxide dismutase (SOD) An enzyme that converts the superoxide ion produced in the phagolysosome into hydrogen peroxide, a substrate for further reactive antimicrobial metabolites.

1	superoxide dismutase (SOD) An enzyme that converts the superoxide ion produced in the phagolysosome into hydrogen peroxide, a substrate for further reactive antimicrobial metabolites. suppressor of cytokine signaling (SOCS) Regulatory protein that interacts with JAK kinases to inhibit signaling by activated receptors. supramolecular activation complex (SMAC) Organized structure that forms at the point of contact between a T cell and its target cell, in which the ligand-bound antigen receptors are co-localized with other cell-surface signaling and adhesion molecules. Also known as supramolecular adhesion complex. surface immunoglobulin (sIg) The membrane-bound immunoglobulin that acts as the antigen receptor on B cells. surfactant proteins A and D (SP-A and SP-D) Acute-phase proteins that help protect the epithelial surfaces of the lung against infection.

1	surfactant proteins A and D (SP-A and SP-D) Acute-phase proteins that help protect the epithelial surfaces of the lung against infection. surrogate light chain A protein in pre-B cells, made up of two subunits, VpreB and λ5, that can pair with a full-length immunoglobulin heavy chain and the Igα and Igβ signaling subunits and signals for pre-B-cell differentiation. switch regions Genomic regions, several kilobases in length each, located between the JH region and the heavy-chain Cμ genes, or in equivalent positions upstream of other C-region genes (except Cδ), containing hundreds of G-rich repeated sequences that function in class switch recombination. Syk A cytoplasmic tyrosine kinase found in B cells that acts in the signaling pathway from the B-cell antigen receptor. symbiotic Relationship between two agents, typically diverse species, that confers benefits to both. sympathetic ophthalmia Autoimmune response that occurs in the other eye after one eye is damaged.

1	sympathetic ophthalmia Autoimmune response that occurs in the other eye after one eye is damaged. syngeneic graft A graft between two genetically identical individuals. It is accepted as self. systemic immune system Name sometimes given to the lymph nodes and spleen to distinguish them from the mucosal immune system. systemic lupus erythematosus (SLE) An autoimmune disease in which autoantibodies against DNA, RNA, and proteins associated with nucleic acids form immune complexes that damage small blood vessels, especially in the kidney. T10, T22 Murine MHC class Ib genes expressed by activated lymphocytes and recognized by a subset of γ:δ T cells. T lymphocytes (T cells) See T cell. TAB1, TAB2 An adaptor complex that binds K63 linked polyubiquitin chains. TAB1/2 complex with TAK1, targeting TAK1 to signaling scaffolds where it phosphorylates substrates such as IKKα.

1	TAB1, TAB2 An adaptor complex that binds K63 linked polyubiquitin chains. TAB1/2 complex with TAK1, targeting TAK1 to signaling scaffolds where it phosphorylates substrates such as IKKα. TACE (TNF-α-converting enzyme) A protease responsible for cleavage of the membrane-associated form of TNF-α, allowing cytokine release into its soluble form that can enter the systemic circulation. TACI A receptor for BAFF expressed on B cells that activates the canonical NFκB pathway. tacrolimus An immunosuppressant polypeptide drug that binds FKBPs and inactivates T cells by inhibiting calcineurin, thus blocking activation of the transcription factor NFAT. Also called FK506. TAK1 A serine-threonine kinase that is activated by phosphorylation by the IRAK complex, and that activates downstream targets such as IKKβ and MAPKs.

1	TAK1 A serine-threonine kinase that is activated by phosphorylation by the IRAK complex, and that activates downstream targets such as IKKβ and MAPKs. talin An intracellular protein involved in the linkage of activated integrins, such as LFA-1, to the cytoskeleton to allow changes in cellular motility and migration, such as in the diapedesis of neutrophils across the vascular endothelium. TAP1, TAP2 Transporters associated with antigen processing. ATP-binding cassette proteins that form a heterodimeric TAP-1:TAP-2 complex in the endoplasmic reticulum membrane, through which short peptides are transported from the cytosol into the lumen of the endoplasmic reticulum, where they associate with MHC class I molecules. tapasin TAP-associated protein. A key molecule in the assembly of MHC class I molecules; a cell deficient in this protein has only unstable MHC class I molecules on the cell surface.

1	tapasin TAP-associated protein. A key molecule in the assembly of MHC class I molecules; a cell deficient in this protein has only unstable MHC class I molecules on the cell surface. Tbet A transcription factor active in many immune cell types but most typically associated with ILC1 and TH1 function. TBK1 (TANK-binding kinase) A serine-threonine kinase activated during signaling by TLR-3 and MAVS and serving to phosphorylate and activate IRF3 for induction of type I interferon gene expression. T cell, T lymphocyte One of the two types of antigen-specific lymphocytes responsible for adaptive immune responses, the other being the B cells.

1	T cell, T lymphocyte One of the two types of antigen-specific lymphocytes responsible for adaptive immune responses, the other being the B cells. T cells are responsible for the cell-mediated adaptive immune reactions. They originate in the bone marrow but undergo most of their development in the thymus. The highly variable antigen receptor on T cells is called the T-cell receptor and recognizes a complex of peptide antigen bound to MHC molecules on cell surfaces. There are two main lineages of T cells: those carrying α:β receptors and those carrying γ:δ receptors. Effector T cells perform a variety of functions in an immune response, acting always by interacting with another cell in an antigen-specific manner. Some T cells activate macrophages, some help B cells produce antibody, and some kill cells infected with viruses and other intracellular pathogens. T-cell antigen receptor See T-cell receptor.

1	T-cell antigen receptor See T-cell receptor. T-cell areas Regions of peripheral lymphoid organs that are enriched in naive T cells and are distinct from the follicles. They are the sites at which adaptive immune responses are initiated. T-cell plasticity Flexibility in the developmental programming of CD4 T cells such that effector T-cell subsets are not irreversibly fixed in their function or the transcriptional networks that underpin those functions. T-cell receptor (TCR) The cell-surface receptor for antigen on T lymphocytes. It consists of a disulfide-linked heterodimer of the highly variable α and β chains in a complex with the invariant CD3 and ζ proteins, which have a signaling function. T cells carrying this type of receptor are often called α:βT cells. An alternative receptor made up of variable γ and δ chains is expressed with CD3 and ζ on a subset of T cells. T-cell receptor α (TCRα) and β (TCRβ) The two chains of the α:β T-cell receptor.

1	T-cell receptor α (TCRα) and β (TCRβ) The two chains of the α:β T-cell receptor. T-cell receptor excision circles (TRECs) Circular DNA fragments excised from the chromosome during V(D)J recombination in developing thymoctytes that are transiently retained in T cells that have recently left the thymus. T-cell zones See T-cell areas. T-DM1 An antibody-drug conjugate combining trastuzumab (Herceptin) with mertansine used to treat recurrent metastatic breast cancer previously treated with a different trastuzumab drug conjugate. TdT See terminal deoxynucleotidyl transferase. TEPs See thioester-containing proteins. terminal deoxynucleotidyl transferase (TdT) Enzyme that inserts nontemplated N-nucleotides into the junctions between gene segments in T-cell receptor and immunoglobulin V-region genes during their assembly.

1	tertiary immune responses Adaptive immune response provoked by a third injection of the same antigen. It is more rapid in onset and stronger than the primary response. T follicular helper (TFH) cell An effector T cell found in lymphoid follicles that provides help to B cells for antibody production and class switching. TH1 A subset of effector CD4 T cells characterized by the cytokines they produce. They are mainly involved in activating macrophages but can also help stimulate B cells to produce antibody. TH2 A subset of effector CD4 T cells that are characterized by the cytokines they produce. They are involved in stimulating B cells to produce antibody, and are often called helper CD4 T cells. TH17 A subset of CD4 T cells that are characterized by production of the cytokine IL-17. They help recruit neutrophils to sites of infection.

1	TH17 A subset of CD4 T cells that are characterized by production of the cytokine IL-17. They help recruit neutrophils to sites of infection. thioester-containing proteins Homologs of complement component C3 that are found in insects and are thought to have some function in insect innate immunity. thioredoxin (TRX) A set of sensor proteins normally bound to thioredoxininteracting protein (TXNIP). Oxidative stress causes thioredoxin to release TXNIP, which can mediate downstream actions. thioredoxin-interacting protein (TXNIP) See thioredoxin. thymectomy Surgical removal of the thymus. thymic anlage The tissue from which the thymic stroma develops during embryogenesis. thymic cortex The outer region of each thymic lobule in which thymic progenitor cells (thymocytes) proliferate, rearrange their T-cell receptor genes, and undergo thymic selection, especially positive selection on thymic cortical epithelial cells.

1	thymic stroma The epithelial cells and connective tissue of the thymus that form the essential microenvironment for T-cell development. thymic stromal lymphopoietin (TSLP) Thymic stroma-derived lymphopoietin. A cytokine thought to be involved in promoting B-cell development in the embryonic liver. It is also produced by mucosal epithelial cells in response to helminthic infections, and promotes type 2 immune responses through its actions on macrophages, ILC2s, and TH2 cells. thymocytes Developing T cells when they are in the thymus. The majority are not functionally mature and are unable to mount protective T-cell responses. thymoproteasome Specialized form of the proteasome composed of a unique subunit, β 5t, that replaces β 5i (LMP7) and associates with β 1i and β 2i in the catalytic chamber. thymus A central lymphoid organ, in which T cells develop, situated in the upper part of the middle of the chest, just behind the breastbone.

1	thymus A central lymphoid organ, in which T cells develop, situated in the upper part of the middle of the chest, just behind the breastbone. thymus-dependent antigens (TD) Antigens that elicit responses only in individuals that have T cells. thymus-independent antigens (TI) Antigens that can elicit antibody production without the involvement of T cells. There are two types of TI antigens: the TI-1 antigens, which have intrinsic B-cell activating activity, and the TI-2 antigens, which activate B cells by having multiple identical epitopes that cross-link the B-cell receptor. thymus leukemia antigen (TL) Nonclassical MHC class Ib molecule expressed by intestinal epithelial cells and a ligand for CD8α:α. TI-1 antigens See thymus-independent antigens. TI-2 antigens See thymus-independent antigens. tickover The low-level generation of C3b continually occurring in the blood in the absence of infection.

1	TI-2 antigens See thymus-independent antigens. tickover The low-level generation of C3b continually occurring in the blood in the absence of infection. tingible body macrophages Phagocytic cells engulfing apoptotic B cells, which are produced in large numbers in germinal centers at the height of an adaptive immune response. TIR (for Toll–IL-1 receptor) domain Domain in the cytoplasmic tails of the TLRs and the IL-1 receptor, which interacts with similar domains in intracellular signaling proteins. tissue-resident memory T cells (TRM) Memory lymphocytes that do not migrate after taking up residence in barrier tissues, where they are retained long term. They appear to be specialized for rapid effector function after restimulation with antigen or cytokines at sites of pathogen entry. TLR-1 Cell-surface Toll-like receptor that acts in a heterodimer with TLR-2 to recognize lipoteichoic acid and bacterial lipoproteins.

1	TLR-1 Cell-surface Toll-like receptor that acts in a heterodimer with TLR-2 to recognize lipoteichoic acid and bacterial lipoproteins. TLR-2 Cell-surface Toll-like receptor that acts in a heterodimer with either TLR-1 or TLR-6 to recognize lipoteichoic acid and bacterial lipoproteins. TLR-3 Endosomal Toll-like receptor that recognizes double-stranded viral RNA. TLR-4 Cell-surface Toll-like receptor that, in conjunction with the accessory proteins MD-2 and CD14, recognizes bacterial lipopolysaccharide and lipoteichoic acid. TLR-5 Cell-surface Toll-like receptor that recognizes the ﬂagellin protein of bacterial ﬂagella. TLR-6 Cell-surface Toll-like receptor that acts in a heterodimer with TLR-2 to recognize lipoteichoic acid and bacterial lipoproteins. TLR-7 Endosomal Toll-like receptor that recognizes single-stranded viral RNA. TLR-8 Endosomal Toll-like receptor that recognizes single-stranded viral RNA.

1	TLR-7 Endosomal Toll-like receptor that recognizes single-stranded viral RNA. TLR-8 Endosomal Toll-like receptor that recognizes single-stranded viral RNA. TLR-9 Endosomal Toll-like receptor that recognizes DNA containing unmethylated CpG. TLR-11, TLR-12 Mouse Toll-like receptor that recognizes profilin and profilin-like proteins. TNF family Cytokine family, the prototype of which is tumor necrosis factor-α (TNF or TNF-α). It contains both secreted (for example TNF-α and lymphotoxin) and membrane-bound (for example CD40 ligand) members. An autoinﬂammatory disease characterized by recurrent, periodic episodes of inﬂammation and fever caused by mutations in gene that encodes TNF receptor I. The defective TNFR-I proteins fold abnormally and accumulate in cells in such a way that they spontaneously activate production of TNF-α. See also familial Mediterranean fever.

1	TNF receptors Family of cytokine receptors which includes some that lead to apoptosis of the cell on which they are expressed (for example Fas and TNFR-I), whereas others lead to activation. tocilizumab Humanized anti-IL-6 receptor antibody used in treating rheumatoid arthritis. tofacitinib An inhibitor of JAK3 and JAK1 used to treat rheumatoid arthritis and under investigation in other inﬂammatory disorders. tolerance The failure to respond to an antigen. Tolerance to self antigens is an essential feature of the immune system; when tolerance is lost, the immune system can destroy self tissues, as happens in autoimmune disease. tolerant Describes the state of immunological tolerance, in which the individual does not respond to a particular antigen. tolerogenic Describes an antigen or type of antigen exposure that induces tolerance. Toll Receptor protein in Drosophila that activates the transcription factor NFκB, leading to the production of antimicrobial peptides.

1	Toll Receptor protein in Drosophila that activates the transcription factor NFκB, leading to the production of antimicrobial peptides. Toll-like receptors (TLRs) Innate receptors on macrophages, dendritic cells, and some other cells, that recognize pathogens and their products, such as bacterial lipopolysaccharide. Recognition stimulates the receptor-bearing cells to produce cytokines that help initiate immune responses. tonsils See lingual tonsils, palatine tonsils. toxic shock syndrome A systemic toxic reaction caused by the massive production of cytokines by CD4 T cells activated by the bacterial superantigen toxic shock syndrome toxin-1 (TSST-1), which is secreted by Staphylococcus aureus. toxic shock syndrome toxin-1 (TSST-1) See toxic shock syndrome. toxoids Inactivated toxins that are no longer toxic but retain their immunogenicity so that they can be used for immunization.

1	toxic shock syndrome toxin-1 (TSST-1) See toxic shock syndrome. toxoids Inactivated toxins that are no longer toxic but retain their immunogenicity so that they can be used for immunization. TRAF3 An E3 ligase that produces a K63 polyubiquitin signaling scaffold in TLR-3 signaling to induce type I interferon gene expression. TRAF6 (tumor necrosis factor receptor-associated factor 6) An E3 ligase that produces a K63 polyubiquitin signaling scaffold in TLR-4 signaling to activate the NFκB pathway. A member of the TNF cytokine family expressed on the cell surface of some cells, such as NK cells, that induces cell death in target cells by ligation of the 'death' receptors DR4 and DR5. TRAM An adaptor protein that pairs with TRIF in signaling by TLR-4. transcytosis The active transport of molecules, such as secreted IgA, through epithelial cells from one face to the other.

1	TRAM An adaptor protein that pairs with TRIF in signaling by TLR-4. transcytosis The active transport of molecules, such as secreted IgA, through epithelial cells from one face to the other. Transib A superfamily of transposable elements identified computationally and proposed to date back more than 500 million years and to have given rise to transposons in diverse species. transitional immunity Referring to the recognition by some adaptive immune system (e.g., MAIT, γ:δT cells) of non-peptide ligands expressed as a consequence of infection, such as various MHC class Ib molecules. transitional stages Defined stages in the development of immature B cells into mature B cells in the spleen, after which the B cell expresses B-cell co-receptor component CD21. transporters associated with antigen processing-1 and -2 (TAP1 and TAP2) See TAP1, TAP2.

1	transporters associated with antigen processing-1 and -2 (TAP1 and TAP2) See TAP1, TAP2. transposase An enzyme capable of cutting DNA and allowing integration and excision of transposable genetic elements into or from the genome of a host. trastuzumab Humanized antibody to HER-2/neu used in treatment of breast cancer. TRECs See T-cell receptor excision circles. TRIF An adaptor protein that alone is involved in signaling by TLR-3, and that when paired with TRAM, functions in signaling by TLR-4. TRIKA1 A complex of the E2 ubiquitin ligase UBC13 and cofactor Uve1A, that interacts with TRAF6 in forming the K63 polyubiquitin signaling scaffold in TLR signaling downstream of MyD88. TRIM21 (tripartite motif-containing 21) A cytosolic Fc receptor and E3 ligase that is activated by IgG and can ubiquitinate viral proteins after an antibody-coated virus enters the cytoplasm. TRIM25 An E3 ubiquitin ligase involved in signaling by RIG-I and MDA-5 for the activation of MAVS.

1	TRIM25 An E3 ubiquitin ligase involved in signaling by RIG-I and MDA-5 for the activation of MAVS. tropism The characteristic of a pathogen that describes the cell types it will infect. TSC Protein complex that acts as a GTPase-activating protein (GAP) for Rheb in its non-phosphorylated state. TSC is inactivated when phosphorylated by Akt. TSLP Thymic stroma-derived lymphopoietin. A cytokine thought to be involved in promoting B-cell development in the embryonic liver. tumor necrosis factor-α See TNF family. tumor rejection antigens Antigens on the surface of tumor cells that can be recognized by T cells, leading to attack on the tumor cells. TRAs are peptides of mutant or overexpressed cellular proteins bound to MHC class I molecules on the tumor-cell surface.

1	type 1 diabetes mellitus Disease in which the β cells of the pancreatic islets of Langerhans are destroyed so that no insulin is produced. The disease is believed to result from an autoimmune attack on the β cells. It is also known as insulin-dependent diabetes mellitus (IDDM), because the symptoms can be ameliorated by injections of insulin. type 1 immunity Class of effector activities aimed at elimination of intracellular pathogens. type 2 immunity Class of effector activities aimed at elimination of parasites and promoting barrier and mucosal immunity. type 3 immunity Class of effector activities aimed at elimination of extracellular pathogens such as bacteria and fungi. type I interferons The antiviral interferons IFN-α and IFN-β. type II interferon The antiviral interferon IFN-γ. type III secretion system (T3SS) Specialized appendage of Gram-negative bacteria used to aid infection of eukaryotic cells by direct secretion of effector proteins into their cytoplasm.

1	type III secretion system (T3SS) Specialized appendage of Gram-negative bacteria used to aid infection of eukaryotic cells by direct secretion of effector proteins into their cytoplasm. tyrosinase Enzyme in melanin synthesis pathway and frequently a tumor rejection antigen in melanoma. tyrosine phosphatases Enzymes that remove phosphate groups from phosphorylated tyrosine residues on proteins. See also CD45. tyrosine protein kinases Enzymes that specifically phosphorylate tyrosine residues in proteins. They are critical in the signaling pathways that lead to Tand B-cell activation. UBC13 See TRIKA1. ubiquitin A small protein that can be attached to other proteins and functions as a protein interaction module or to target them for degradation by the proteasome. ubiquitin ligase Enzyme that attaches ubiquitin covalently to exposed lysine residues on the surfaces of other proteins.

1	ubiquitin ligase Enzyme that attaches ubiquitin covalently to exposed lysine residues on the surfaces of other proteins. ubiquitin–proteasome system (UPS) A quality control system in the cell that involves K48-linked ubiquitination of target proteins that are then recognized by the proteasome for degradation. ubiquitination The process of attachment of one or many subunits of ubiquitin to a target protein, which can mediate either degradation by the proteasome, or formation of scaffolds used for signaling, depending on the nature of the linkages. UL16-binding proteins, or ULBPs See RAET1. ULBP4 See RAET1. ulcerative colitis One of the two major types of inﬂammatory bowel disease thought to result from an abnormal overresponsiveness to the commensal gut microbiota. See also Crohn’s disease. UNC93B1 A mutlipass transmembrane protein that is necessary for the normal transport of TLR-3, TLR-7, and TLR-9 from the ER, where they are assembled, to the endosome, where they function.

1	UNC93B1 A mutlipass transmembrane protein that is necessary for the normal transport of TLR-3, TLR-7, and TLR-9 from the ER, where they are assembled, to the endosome, where they function. unmethylated CpG dinucleotides While mammalian genomes have heavily methylated the cytosine within CpG sequences, unmethylated CpG is more typically characteristic of bacterial genomes, and is recognized by TLR-9 when encountered in the endosomal compartment. uracil-DNA glycosylase (UNG) Enzyme that removes uracil bases from DNA in a DNA repair pathway that can lead to somatic hypermutation, class switch recombination or gene conversion. urticaria The technical term for hives, which are red, itchy skin wheals usually brought on by an allergic reaction. Uve1A See TRIKA1. Vα Variable region from the TCRα chain. Vβ Variable region from the TCRβ chain.

1	Uve1A See TRIKA1. Vα Variable region from the TCRα chain. Vβ Variable region from the TCRβ chain. vaccination The deliberate induction of adaptive immunity to a pathogen by injecting a dead or attenuated (nonpathogenic) live form of the pathogen or its antigens (a vaccine). variability plot A measure of the difference between the amino acid sequences of different variants of a given protein. The most variable proteins known are antibodies and T-cell receptors. variable Ig domains (V domains) The amino-terminal protein domain of the polypeptide chains of immunoglobulins and T-cell receptor, which is the most variable part of the chain. variable lymphocyte receptors (VLRs) Nonimmunoglobulin LRR-containing variable receptors and secreted proteins expressed by the lymphocyte-like cells of the lamprey. They are generated by a process of somatic gene rearrangement.

1	variable region The region of an immunoglobulin or T-cell receptor that is formed of the amino-terminal domains of its component polypeptide chains. These are the most variable parts of the molecule and contain the antigen-binding sites. variolation The intentional inhalation of or skin infection with material taken from smallpox pustules of an infected person for the purpose of deriving protective immunity. VCAM-1 An adhesion molecule expressed by vascular endothelium at sites of inﬂammation; it binds the integrin VLA-4, which allows effector T cells to enter sites of infection. V(D)J recombinase A multiprotein complex containing RAG-1 and RAG-2, as well as other proteins involved in cellular DNA repair. V(D)J recombination The process exclusive to developing lymphocytes in vertebrates, that recombines different gene segments into sequences encoding complete protein chains of immunoglobulins and T-cell receptors.

1	vesicular compartments One of several major compartments within cells, composed of the endoplasmic reticulum, Golgi, endosomes, and lysosomes. V gene segments Gene segments in immunoglobulin and T-cell receptor loci that encode the first 95 amino acids or so of the protein chain. There are multiple different V gene segments in the germline genome. To produce a complete exon encoding a V domain, one V gene segment must be rearranged to join up with a J or a rearranged DJ gene segment. viral entry inhibitors Drugs that inhibit the entry of HIV into its host cells. viral integrase inhibitors Drugs that inhibit the action of the HIV integrase, so that the virus cannot integrate into the host-cell genome. viral protease Enzyme encoded by the human immunodeficiency virus that cleaves the long polyprotein products of the viral genes into individual proteins.

1	viral protease Enzyme encoded by the human immunodeficiency virus that cleaves the long polyprotein products of the viral genes into individual proteins. viral set point In human immunodeficiency virus infection, the level of HIV virions persisting in the blood after the acute phase of infection has passed. virus Pathogen composed of a nucleic acid genome enclosed in a protein coat. Viruses can replicate only in a living cell, because they do not possess the metabolic machinery for independent life. virus-neutralizing antibodies Antibodies that block the ability of a virus to establish infection of cells. vitronectin See S-protein. VLRs See variable lymphocyte receptors. VpreB See surrogate light chain. WAS See Wiskott–Aldrich syndrome. WASp The protein defective in patients with Wiskott–Aldrich syndrome. When activated, WASp promotes actin polymerization. Weibel–Palade bodies Granules within endothelial cells that contain P-selectin.

1	Weibel–Palade bodies Granules within endothelial cells that contain P-selectin. wheal-and-flare reaction A skin reaction observed in an allergic individual when an allergen to which the individual has been sensitized is injected into the dermis. It consists of a raised area of skin containing edema ﬂuid, and a spreading, red, itchy inﬂammatory reaction around it. white pulp The discrete areas of lymphoid tissue in the spleen. Wiskott–Aldrich syndrome (WAS) An immunodeficiency disease characterized by defects in the cytoskeleton of cells due to a mutation in the protein WASp, which is involved in interactions with the actin cytoskeleton. Patients with this disease are highly susceptible to infections with pyogenic bacteria due to defects in T-follicular helper cell interactions with B cells.

1	XBP1 (X-box binding protein 1) A transcription factor that induces genes required for optimal protein secretion by plasma cells, and is part of the unfolded protein response. XBP1 mRNA is spliced from an inactive to an active form by signals produced by ER stress. XCR1 A chemokine receptor selectively expressed by a subset of dendritic cells that are specialized for cross-presentation whose development requires the transcription factor BATF3. xenografts Grafted organs taken from a different species than the recipient. xenoimmunity In the context of immune-mediated disease, refers to immunity directed against foreign antigens of non-human species, such as bacteria-derived antigens of the commensal microbiota that are targets in inﬂammatory bowel disease (IBD). xeroderma pigmentosum Several autosomal recessive diseases caused by defects in repair of ultraviolet light-induced DNA damage. Defects in Polη cause type V xeroderma pigmentosum. xid See X-linked immunodeficiency.

1	xid See X-linked immunodeficiency. X-linked agammaglobulinemia (XLA) A genetic disorder in which B-cell development is arrested at the pre-B-cell stage and no mature B cells or antibodies are formed. The disease is due to a defect in the gene encoding the protein tyrosine kinase Btk, which is encoded on the X chromosome. X-linked hyper IgM syndrome See CD40 ligand deficiency. A syndrome with some features resembling hyper IgM syndrome. It is caused by mutations in the protein NEMO, a component of the NFκB signaling pathway. Also called NEMO deficiency. X-linked immunodeficiency An immunodeficiency disease in mice due to defects in the protein tyrosine kinase Btk. Shares the gene defect with X-linked agammaglobulinemia in humans, but leads to a milder B cell defect than seen in the human disease.

1	X-linked lymphoproliferative (XLP) syndrome Rare immunodeficiency diseases that result from mutations in the gene SH2D1A (XLP1) or XIAP (XLP2). Boys with this deficiency typically develop overwhelming Epstein–Barr virus infection during childhood, and sometimes lymphomas. An immunodeficiency disease in which T-cell development fails at an early intrathymic stage and no production of mature T cells or T-cell dependent antibody occurs. It is due to a defect in a gene that encodes the γc chain shared by the receptors for several different cytokines. XLP See X-linked lymphoproliferative syndrome. XRCC4 A protein that functions in NHEJ DNA repair by interacting with DNA ligase IV and Ku70/80 at double-strand breaks. ζ chain One of the signaling chains associated with the T-cell receptor that has three ITAM motifs in its cytoplasmic tail.

1	ζ chain One of the signaling chains associated with the T-cell receptor that has three ITAM motifs in its cytoplasmic tail. ZAP-70 (ζ-chain-associated protein) A cytoplasmic tyrosine kinase found in T cells that binds to the phosphorylated ζ chain of the T-cell receptor and is a key enzyme in signaling T-cell activation. ZFP318 A spliceosome protein expressed in mature and activated B cells, but not immature B cells, that favors splicing from the rearranged VDJ exon of immunoglobulin heavy chain to the Cδ exon, thereby promoting expression of surface IgD. zoonotic Describes a disease of animals that can be transmitted to humans. zymogens An inactive form of an enzyme, usually a protease, that must be modified in some way, for example by selective cleavage of the protein chain, before it can become active.

1	xxxviii its related products in new formats. Kim Davis, as Associate Managing We are privileged to have compiled this 19th edition and areEditor, has adeptly ensured that the complex production of this multi-enthusiastic about all that it offers our readers. We learned much in the authored textbook proceeded smoothly and efficiently. Dominik Pucek process of editing Harrison’s and hope that you will find this edition a oversaw the production of the new procedural videos and Priscilla uniquely valuable educational resource. Beer expertly oversaw the production of our extensive DVD content. The Editors Jeffrey Herzich ably served as production manager for this new edition.

1	Chapter 1 The Practice of Medicine the practice of Medicine The Editors THE PHYSICIAN IN THE TWENTY-FIRST CENTURY No greater opportunity, responsibility, or obligation can fall to the lot of a human being than to become a physician. In the care of the suf-1 part 1: General Considerations in Clinical Medicine fering, [the physician] needs technical skill, scientific knowledge, and human understanding.… Tact, sympathy, and understanding are expected of the physician, for the patient is no mere collection of symp toms, signs, disordered functions, damaged organs, and disturbed emotions. [The patient] is human, fearful, and hopeful, seeking relief, help, and reassurance. —Harrison’s Principles of Internal Medicine, 1950

1	—Harrison’s Principles of Internal Medicine, 1950 The practice of medicine has changed in significant ways since the first edition of this book appeared more than 60 years ago. The advent of molecular genetics, molecular and systems biology, and molecular pathophysiology; sophisticated new imaging techniques; and advances in bioinformatics and information technology have contributed to an explosion of scientific information that has fundamentally changed the way physicians define, diagnose, treat, and attempt to prevent disease. This growth of scientific knowledge is ongoing and accelerating.

1	The widespread use of electronic medical records and the Internet have altered the way doctors practice medicine and access and exchange information (Fig. 1-1). As today’s physicians strive to integrate copious amounts of scientific knowledge into everyday practice, it is critically important that they remember two things: first, that the ultimate goal of medicine is to prevent disease and treat patients; and second, that despite more than 60 years of scientific advances since the first edition of this text, cultivation of the intimate relationship between physician and patient still lies at the heart of successful patient care.

1	Deductive reasoning and applied technology form the foundation for the solution to many clinical problems. Spectacular advances in biochemistry, cell biology, and genomics, coupled with newly developed imaging techniques, allow access to the innermost parts of the cell and provide a window into the most remote recesses of the body. Revelations about the nature of genes and single cells have opened a portal for formulating a new molecular basis for the physiology of systems. Increasingly, physicians are learning how subtle changes in many different genes can affect the function of cells and organisms. Researchers are deciphering the complex mechanisms by which genes are regulated. Clinicians have developed a new appreciation of the role of stem cells in normal tissue function; in the development of cancer, degenerative diseases, and other disorders; and in the treatment of certain diseases. Entirely new areas of research, including studies of the human microbiome, have become important

1	of cancer, degenerative diseases, and other disorders; and in the treatment of certain diseases. Entirely new areas of research, including studies of the human microbiome, have become important in understanding both health and disease. The knowledge gleaned from the science of medicine continues to enhance physicians’ understanding of complex disease processes and provide new approaches to treatment and prevention. Yet skill in the most sophisticated applications of laboratory technology and in the use of the latest therapeutic modality alone does not make a good physician.

1	When a patient poses challenging clinical problems, an effective physician must be able to identify the crucial elements in a complex history and physical examination; order the appropriate laboratory, imaging, and diagnostic tests; and extract the key results from densely populated computer screens to determine whether to treat or to “watch.” As the number of tests increases, so does the likelihood that some incidental finding, completely unrelated to the clinical problem at hand, will be uncovered. Deciding whether a clinical clue is worth pursuing or should be dismissed as a “red herring” and weighing whether a proposed test, preventive measure, or treatment entails a greater risk than the disease itself are essential judgments that a skilled clinician must make many times each day. This combination of medical knowledge, intuition, experience, and judgment defines the art of medicine, which is as necessary to the practice of medicine as is a sound scientific base.

1	CLINICAL SKILLS History-Taking The written history of an illness should include all the facts of medical significance in the life of the patient. Recent events should be given the most attention. Patients should, at some early point, have the opportunity to tell their own story of the illness without frequent interruption and, when appropriate, should receive expressions of interest, encouragement, and empathy from the physician. Any event related by a patient, however trivial or seemingly irrelevant, may provide the key to solving the medical problem. In general, only patients who feel comfortable with the physician will offer complete information; thus putting the patient at ease to the greatest extent possible contributes substantially to obtaining an adequate history.

1	An informative history is more than an orderly listing of symptoms. By listening to patients and noting the way in which they describe their symptoms, physicians can gain valuable insight. Inflections of voice, facial expression, gestures, and attitude (i.e., “body language”) may offer important clues to patients’ perception of their symptoms. Because patients vary in their medical sophistication and ability to recall facts, the reported medical history should be corroborated whenever possible. The social history also can provide important insights into the types of diseases that should be considered. The family history not only identifies rare Mendelian disorders within a family but often reveals risk factors for common disorders, such as coronary heart disease, hypertension, and asthma. A thorough family history may require input from multiple relatives to ensure completeness and accuracy; once recorded, it can be updated readily. The process of history-taking provides an

1	asthma. A thorough family history may require input from multiple relatives to ensure completeness and accuracy; once recorded, it can be updated readily. The process of history-taking provides an opportunity to observe the patient’s behavior and to watch for features to be pursued more thoroughly during the physical examination.

1	The very act of eliciting the history provides the physician with an opportunity to establish or enhance the unique bond that forms the basis for the ideal patient-physician relationship. This process helps the physician develop an appreciation of the patient’s view of the illness, the patient’s expectations of the physician and the health care system, and the financial and social implications of the illness for the patient. Although current health care settings may impose time constraints on patient visits, it is important not to rush the history-taking. A hurried approach may lead patients to believe that what they are relating is not of importance to the physician, and thus they may withhold relevant information. The confidentiality of the patient-physician relationship cannot be overemphasized.

1	Physical Examination The purpose of the physical examination is to identify physical signs of disease. The significance of these objective indications of disease is enhanced when they confirm a functional or structural change already suggested by the patient’s history. At times, however, physical signs may be the only evidence of disease.

1	The physical examination should be methodical and thorough, with consideration given to the patient’s comfort and modesty. Although attention is often directed by the history to the diseased organ or part of the body, the examination of a new patient must extend from head to toe in an objective search for abnormalities. Unless the physical examination is systematic and is performed consistently from patient to patient, important segments may be omitted inadvertently. The results of the examination, like the details of the history, should be recorded at the time they are elicited—not hours later, when they are subject to the distortions of memory. Skill in physical diagnosis is acquired with experience, but it is not merely technique that determines success in eliciting signs of disease. The detection of a few scattered petechiae, a faint diastolic murmur, or a small mass in the abdomen is not a question of keener eyes and ears or more sensitive fingers but of a mind alert to those

1	The detection of a few scattered petechiae, a faint diastolic murmur, or a small mass in the abdomen is not a question of keener eyes and ears or more sensitive fingers but of a mind alert to those findings. Because physical findings can change with time, the physical examination should be repeated as frequently as the clinical situation warrants.

1	FIgURE 1-1 Woodcuts from Johannes de Ketham’s Fasciculus Medicinae, the first illustrated medical text ever printed, show methods of information access and exchange in medical practice during the early Renaissance. Initially published in 1491 for use by medical students and practitioners, Fasciculus Medicinae appeared in six editions over the next 25 years. Left: Petrus de Montagnana, a well-known physician and teacher at the University of Padua and author of an anthology of instructive case studies, consults medical texts dating from antiquity up to the early Renaissance. Right: A patient with plague is attended by a physician and his attendants. (Courtesy, U.S. National Library of Medicine.)

1	Given the many highly sensitive diagnostic tests now available (particularly imaging techniques), it may be tempting to place less emphasis on the physical examination. Indeed, many patients are seen by consultants after a series of diagnostic tests have been performed and the results are known. This fact should not deter the physician from performing a thorough physical examination since important clinical findings may have escaped detection by the barrage of prior diagnostic tests. The act of examining (touching) the patient also offers an opportunity for communication and may have reassuring effects that foster the patient-physician relationship.

1	diagnostic Studies Physicians rely increasingly on a wide array of laboratory tests to solve clinical problems. However, accumulated laboratory data do not relieve the physician from the responsibility of carefully observing, examining, and studying the patient. It is also essential to appreciate the limitations of diagnostic tests. By virtue of their impersonal quality, complexity, and apparent precision, they often gain an aura of certainty regardless of the fallibility of the tests themselves, the instruments used in the tests, and the individuals performing or interpreting the tests. Physicians must weigh the expense involved in laboratory procedures against the value of the information these procedures are likely to provide. Single laboratory tests are rarely ordered. Instead, physicians generally request “batteries” of multiple tests, which often prove useful.

1	Single laboratory tests are rarely ordered. Instead, physicians generally request “batteries” of multiple tests, which often prove useful. For example, abnormalities of hepatic function may provide the clue to nonspecific symptoms such as generalized weakness and increased fatigability, suggesting a diagnosis of chronic liver disease. Sometimes a single abnormality, such as an elevated serum calcium level, points to a particular disease, such as hyperparathyroidism or an underlying malignancy.

1	The thoughtful use of screening tests (e.g., measurement of low-density lipoprotein cholesterol) may be of great value. A group of laboratory values can conveniently be obtained with a single specimen at relatively low cost. Screening tests are most informative when they are directed toward common diseases or disorders and when their results indicate whether other useful—but often costly—tests or interventions are needed. On the one hand, biochemical measurements, together with simple laboratory determinations such as blood count, urinalysis, and erythrocyte sedimentation rate, often provide a major clue to the presence of a pathologic process. On the other hand, the physician must learn to evaluate occasional screening-test abnormalities that do not necessarily connote significant disease. An in-depth workup after the report of an isolated laboratory abnormality in a person who is otherwise well is almost invariably wasteful and unproductive. Because so many tests are performed

1	An in-depth workup after the report of an isolated laboratory abnormality in a person who is otherwise well is almost invariably wasteful and unproductive. Because so many tests are performed routinely for screening purposes, it is not unusual for one or two values to be slightly abnormal. Nevertheless, even if there is no reason to suspect an underlying illness, tests yielding abnormal results ordinarily are repeated to rule out laboratory error. If an abnormality is confirmed, it is important to consider its potential significance in the context of the patient’s condition and other test results.

1	The development of technically improved imaging studies with greater sensitivity and specificity proceeds apace. These tests provide remarkably detailed anatomic information that can be a pivotal factor in medical decision-making. Ultrasonography, a variety of isotopic scans, CT, MRI, and positron emission tomography have supplanted older, more invasive approaches and opened new diagnostic vistas. In light of their capabilities and the rapidity with which they can lead to a diagnosis, it is tempting to order a battery of imaging studies. All physicians have had experiences in which imaging studies revealed findings that led to an unexpected diagnosis. Nonetheless, patients must endure each of these tests, and the added cost of unnecessary testing is substantial. Furthermore, investigation of an unexpected abnormal finding may be associated with risk and/or expense and may lead to the diagnosis of an irrelevant or incidental problem. A skilled physician must learn to use these powerful

1	of an unexpected abnormal finding may be associated with risk and/or expense and may lead to the diagnosis of an irrelevant or incidental problem. A skilled physician must learn to use these powerful diagnostic tools judiciously, always considering whether the results will alter management and benefit the patient.

1	PRINCIPLES oF PATIENT CARE Evidence-Based Medicine Evidence-based medicine refers to the making of clinical decisions that are formally supported by data, preferably data derived from prospectively designed, randomized, controlled clinical trials. This approach is in sharp contrast to anecdotal experience, which is often biased. Unless they are attuned to the importance of using larger, more objective studies for making decisions, even the most experienced physicians can be influenced to an undue extent by recent encounters with selected patients. Evidence-based medicine has become an increasingly important part of routine medical practice and has led to the publication of many practice guidelines.

1	Practice guidelines Many professional organizations and government agencies have developed formal clinical-practice guidelines to aid physicians and other caregivers in making diagnostic and therapeutic decisions that are evidence-based, cost-effective, and most appropriate to a particular patient and clinical situation. As the evidence base of medicine increases, guidelines can provide a useful framework for managing patients with particular diagnoses or symptoms. Clinical guidelines can protect patients— particularly those with inadequate health care benefits—from receiving substandard care. These guidelines also can protect conscientious caregivers from inappropriate charges of malpractice and society from the excessive costs associated with the overuse of medical resources. There are, however, caveats associated with clinical-practice guidelines since they tend to oversimplify the complexities of medicine. Furthermore, groups with different perspectives may develop divergent

1	are, however, caveats associated with clinical-practice guidelines since they tend to oversimplify the complexities of medicine. Furthermore, groups with different perspectives may develop divergent recommendations regarding issues as basic as the need for screening of women in their forties by mammography or of men over age 50 by serum prostate-specific antigen (PSA) assay. Finally, guidelines, as the term implies, do not—and cannot be expected to—account for the uniqueness of each individual and his or her illness. The physician’s challenge is to integrate into clinical practice the useful recommendations offered by experts without accepting them blindly or being inappropriately constrained by them.

1	Medical decision-Making Medical decision-making is an important responsibility of the physician and occurs at each stage of the diagnostic and therapeutic process. The decision-making process involves the ordering of additional tests, requests for consultations, and decisions about treatment and predictions concerning prognosis. This process requires an in-depth understanding of the pathophysiology and natural history of disease. As discussed above, medical decision-making should be evidence-based so that patients derive full benefit from the available scientific knowledge. Formulating a differential diagnosis requires not only a broad knowledge base but also the ability to assess the relative probabilities of various diseases. Application of the scientific method, including hypothesis formulation and data collection, is essential to the process of accepting or rejecting a particular diagnosis. Analysis of the differential diagnosis is an iterative process. As new information or test

1	and data collection, is essential to the process of accepting or rejecting a particular diagnosis. Analysis of the differential diagnosis is an iterative process. As new information or test results are acquired, the group of disease processes being considered can be contracted or expanded appropriately.

1	Despite the importance of evidence-based medicine, much medical decision-making relies on good clinical judgment, an attribute that is difficult to quantify or even to assess qualitatively. Physicians must use their knowledge and experience as a basis for weighing known factors, along with the inevitable uncertainties, and then making a sound judgment; this synthesis of information is particularly important when a relevant evidence base is not available. Several quantitative tools may be invaluable in synthesizing the available information, including diagnostic tests, Bayes’ theorem, and multivariate statistical models. Diagnostic tests serve to reduce uncertainty about an individual’s diagnosis or prognosis and help the physician decide how best to manage that individual’s condition. The battery of diagnostic tests complements the history and the physical examination. The accuracy of a particular test is ascertained by determining its sensitivity (true-positive rate) and specificity

1	battery of diagnostic tests complements the history and the physical examination. The accuracy of a particular test is ascertained by determining its sensitivity (true-positive rate) and specificity (true-negative rate) as well as the predictive value of a positive and a negative result. Bayes’ theorem uses information on a test’s sensitivity and specificity, in conjunction with the pretest probability of a diagnosis, to determine mathematically the posttest probability of the diagnosis. More complex clinical problems can be approached with multivariate statistical models, which generate highly accurate information even when multiple factors are acting individually or together to affect disease risk, progression, or response to treatment. Studies comparing the performance of statistical models with that of expert clinicians have documented equivalent accuracy, although the models tend to be more consistent. Thus, multivariate statistical models may be particularly helpful to less

1	models with that of expert clinicians have documented equivalent accuracy, although the models tend to be more consistent. Thus, multivariate statistical models may be particularly helpful to less experienced clinicians. See Chap. 3 for a more thorough discussion of decision-making in clinical medicine.

1	Electronic Medical Records Both the growing reliance on computers and the strength of information technology now play central roles in medicine. Laboratory data are accessed almost universally through computers. Many medical centers now have electronic medical records, computerized order entry, and bar-coded tracking of medications. Some of these systems are interactive, sending reminders or warning of potential medical errors.

1	Electronic medical records offer rapid access to information that is invaluable in enhancing health care quality and patient safety, including relevant data, historical and clinical information, imaging studies, laboratory results, and medication records. These data can be used to monitor and reduce unnecessary variations in care and to provide real-time information about processes of care and clinical outcomes. Ideally, patient records are easily transferred across the health care system. However, technologic limitations and concerns about privacy and cost continue to limit broad-based use of electronic health records in many clinical settings. As valuable as it is, information technology is merely a tool and can never replace the clinical decisions that are best made by the physician. Clinical knowledge and an understanding of a patient’s needs, supplemented by quantitative tools, still represent the best approach to decision-making in the practice of medicine.

1	Evaluation of outcomes Clinicians generally use objective and readily measurable parameters to judge the outcome of a therapeutic intervention. These measures may oversimplify the complexity of a clinical condition as patients often present with a major clinical problem in the context of multiple complicating background illnesses. For example, a patient may present with chest pain and cardiac ischemia, but with a background of chronic obstructive pulmonary disease and renal insufficiency. For this reason, outcome measures such as mortality, length of hospital stay, or readmission rates are typically risk-adjusted. An important point is that patients usually seek medical attention for subjective reasons; they wish to obtain relief from pain, to preserve or regain function, and to enjoy life. The components of a patient’s health status or quality of life can include bodily comfort, capacity for physical activity, personal and professional function, sexual function, cognitive function,

1	The components of a patient’s health status or quality of life can include bodily comfort, capacity for physical activity, personal and professional function, sexual function, cognitive function, and overall perception of health. Each of these important areas can be assessed through structured interviews or specially designed questionnaires. Such assessments provide useful parameters by which a physician can judge patients’ subjective views of their disabilities and responses to treatment, particularly in chronic illness. The practice of medicine requires consideration and integration of both objective and subjective outcomes.

1	Chapter 1 The Practice of Medicine Women’s Health and disease Although past epidemiologic studies and clinical trials have often focused predominantly on men, more recent studies have included more women, and some, like the Women’s Health Initiative, have exclusively addressed women’s health issues. Significant sex-based differences exist in diseases that afflict both men and women. Much is still to be learned in this arena, and ongoing studies should enhance physicians’ understanding of the mechanisms underlying these differences in the course and outcome of certain diseases. For a more complete discussion of women’s health, see Chap. 6e.

1	Care of the Elderly The relative proportion of elderly individuals in the populations of developed nations has grown considerably over the past few decades and will continue to grow. The practice of medicine is greatly influenced by the health care needs of this growing demographic group. The physician must understand and appreciate the decline in physiologic reserve associated with aging; the differences in appropriate doses, clearance, and responses to medications; the diminished responses of the elderly to vaccinations such as those against influenza; the different manifestations of common diseases among the elderly; and the disorders that occur commonly with aging, such as depression, dementia, frailty, urinary incontinence, and fractures. For a more complete discussion of medical care for the elderly, see Chap. 11 and Part 5, Chaps. 93e and 94e.

1	Errors in the delivery of Health Care A 1999 report from the Institute of Medicine called for an ambitious agenda to reduce medical error rates and improve patient safety by designing and implementing fundamental changes in health care systems. Adverse drug reactions occur in at least 5% of hospitalized patients, and the incidence increases with the use of a large number of drugs. Whatever the clinical situation, it is the physician’s responsibility to use powerful therapeutic measures wisely, with due regard for their beneficial actions, potential dangers, and cost. It is the responsibility of hospitals and health care organizations to develop systems to reduce risk and ensure patient safety. Medication errors can be reduced through the use of ordering systems that rely on electronic processes or, when electronic options are not available, that eliminate misreading of handwriting. Implementation of infection control systems, enforcement of hand-washing protocols, and careful

1	processes or, when electronic options are not available, that eliminate misreading of handwriting. Implementation of infection control systems, enforcement of hand-washing protocols, and careful oversight of antibiotic use can minimize the complications of nosocomial infections. Central-line infection rates have been dramatically reduced at many centers by careful adherence of trained personnel to standardized protocols for introducing and maintaining central lines. Rates of surgical infection and wrong-site surgery can likewise be reduced by the use of standardized protocols and checklists. Falls by patients can be minimized by judicious use of sedatives and appropriate assistance with bed-to-chair and bed-to-bathroom transitions. Taken together, these and other measures are saving thousands of lives each year.

1	The Physician’s Role in Informed Consent The fundamental principles of medical ethics require physicians to act in the patient’s best interest and to respect the patient’s autonomy. These requirements are particularly relevant to the issue of informed consent. Patients are required to sign a consent form for essentially any diagnostic or therapeutic procedure. Most patients possess only limited medical knowledge and must rely on their physicians for advice. Communicating in a clear and understandable manner, physicians must fully discuss the alternatives for care and explain the risks, benefits, and likely consequences of each alternative. In every case, the physician is responsible for ensuring that the patient thoroughly understands these risks and benefits; encouraging questions is an important part of this process. This is the very definition of informed consent. Full, clear explanation and discussion of the proposed procedures and treatment can greatly mitigate the fear of the

1	important part of this process. This is the very definition of informed consent. Full, clear explanation and discussion of the proposed procedures and treatment can greatly mitigate the fear of the unknown that commonly accompanies hospitalization. Excellent communication can also help alleviate misunderstandings in situations where complications of intervention occur. Often the patient’s understanding is enhanced by repeatedly discussing the issues in an unthreatening and supportive way, answering new questions that occur to the patient as they arise.

1	Special care should be taken to ensure that a physician seeking a patient’s informed consent has no real or apparent conflict of interest involving personal gain. The Approach to grave Prognoses and death No circumstance is more distressing than the diagnosis of an incurable disease, particularly when premature death is inevitable. What should the patient and family be told? What measures should be taken to maintain life? What can be done to maintain the quality of life?

1	Honesty is absolutely essential in the face of a terminal illness. The patient must be given an opportunity to talk with the physician and ask questions. A wise and insightful physician uses such open communication as the basis for assessing what the patient wants to know and when he or she wants to know it. On the basis of the patient’s responses, the physician can assess the right tempo for sharing information. Ultimately, the patient must understand the expected course of the disease so that appropriate plans and preparations can be made. The patient should participate in decision-making with an understanding of the goal of treatment (palliation) and its likely effects. The patient’s religious beliefs must be taken into consideration. Some patients may find it easier to share their feelings about death with their physician, who is likely to be more objective and less emotional, than with family members.

1	The physician should provide or arrange for emotional, physical, and spiritual support and must be compassionate, unhurried, and open. In many instances, there is much to be gained by the laying on of hands. Pain should be controlled adequately, human dignity maintained, and isolation from family and close friends avoided. These aspects of care tend to be overlooked in hospitals, where the intrusion of life-sustaining equipment can detract from attention to the whole person and encourage concentration instead on the life-threatening disease, against which the battle ultimately will be lost in any case. In the face of terminal illness, the goal of medicine must shift from cure to care in the broadest sense of the term. Primum succurrere, first hasten to help, is a guiding principle. In offering care to a dying patient, a physician must be prepared to provide information to family members and deal with their grief and sometimes their feelings of guilt or even anger. It is important for

1	care to a dying patient, a physician must be prepared to provide information to family members and deal with their grief and sometimes their feelings of guilt or even anger. It is important for the doctor to assure the family that everything reasonable has been done. A substantial problem in these discussions is that the physician often does not know how to gauge the prognosis. In addition, various members of the health care team may offer different opinions. Good communication among providers is essential so that consistent information is provided to patients. This is especially important when the best path forward is uncertain. Advice from experts in palliative and terminal care should be sought whenever necessary to ensure that clinicians are not providing patients with unrealistic expectations. For a more complete discussion of endof-life care, see Chap. 10.

1	The significance of the intimate personal relationship between physician and patient cannot be too strongly emphasized, for in an extraordinarily large number of cases both the diagnosis and treatment are directly dependent on it. One of the essential qualities of the clinician is interest in humanity, for the secret of the care of the patient is in caring for the patient. —Francis W. Peabody, October 21, 1925, Lecture at Harvard Medical School

1	Physicians must never forget that patients are individual human beings with problems that all too often transcend their physical complaints. They are not “cases” or “admissions” or “diseases.” Patients do not fail treatments; treatments fail to benefit patients. This point is particularly important in this era of high technology in clinical medicine. Most patients are anxious and fearful. Physicians should instill confidence and offer reassurance but must never come across as arrogant or patronizing. A professional attitude, coupled with warmth and openness, can do much to alleviate anxiety and to encourage patients to share all aspects of their medical history. Empathy and compassion are the essential features of a caring physician. The physician needs to consider the setting in which an illness occurs—in terms not only of patients themselves but also of their familial, social, and cultural backgrounds. The ideal patient-physician relationship is based on thorough knowledge of the

1	an illness occurs—in terms not only of patients themselves but also of their familial, social, and cultural backgrounds. The ideal patient-physician relationship is based on thorough knowledge of the patient, mutual trust, and the ability to communicate.

1	The dichotomy of Inpatient and outpatient Internal Medicine The hospital environment has changed dramatically over the last few decades. Emergency departments and critical care units have evolved to identify and manage critically ill patients, allowing them to survive formerly fatal diseases. At the same time, there is increasing pressure to reduce the length of stay in the hospital and to manage complex disorders in the outpatient setting. This transition has been driven not only by efforts to reduce costs but also by the availability of new outpatient technologies, such as imaging and percutaneous infusion catheters for long-term antibiotics or nutrition, minimally invasive surgical procedures, and evidence that outcomes often are improved by minimizing inpatient hospitalization.

1	In these circumstances, two important issues arise as physicians cope with the complexities of providing care for hospitalized patients. On the one hand, highly specialized health professionals are essential to the provision of optimal acute care in the hospital; on the other, these professionals—with their diverse training, skills, responsibilities, experiences, languages, and “cultures”—need to work as a team.

1	In addition to traditional medical beds, hospitals now encompass multiple distinct levels of care, such as the emergency department, procedure rooms, overnight observation units, critical care units, and palliative care units. A consequence of this differentiation has been the emergence of new trends, including specialties (e.g., emergency medicine and end-of-life care) and the provision of in-hospital care by hospitalists and intensivists. Most hospitalists are board-certified internists who bear primary responsibility for the care of hospitalized patients and whose work is limited entirely to the hospital setting. The shortened length of hospital stay that is now standard means that most patients receive only acute care while hospitalized; the increased complexities of inpatient medicine make the presence of a generalist with specific training, skills, and experience in the hospital environment extremely beneficial. Intensivists are board-certified physicians who are further

1	medicine make the presence of a generalist with specific training, skills, and experience in the hospital environment extremely beneficial. Intensivists are board-certified physicians who are further certified in critical care medicine and who direct and provide care for very ill patients in critical care units. Clearly, then, an important challenge in internal medicine today is to ensure the continuity of communication and information flow between a patient’s primary care doctor and these physicians who are in charge of the patient’s hospital care. Maintaining these channels of communication is frequently complicated by patient “handoffs”—i.e., from the outpatient to the inpatient environment, from the critical care unit to a general medicine floor, and from the hospital to the outpatient environment. The involvement of many care providers in conjunction with these transitions can threaten the traditional one-to-one relationship between patient and primary care physician. Of course,

1	environment. The involvement of many care providers in conjunction with these transitions can threaten the traditional one-to-one relationship between patient and primary care physician. Of course, patients can benefit greatly from effective collaboration among a number of health care professionals; however, it is the duty of the patient’s principal or primary physician to provide cohesive guidance through an illness. To meet this challenge, primary care physicians must be familiar with the techniques, skills, and objectives of specialist physicians and allied health professionals who care for their patients in the hospital. In addition, primary care doctors must ensure that their patients will benefit from scientific advances and from the expertise of specialists when they are needed both in and out of the hospital. Primary care physicians can also explain the role of these specialists to reassure patients that they are in the hands of the physicians best trained to manage an acute

1	in and out of the hospital. Primary care physicians can also explain the role of these specialists to reassure patients that they are in the hands of the physicians best trained to manage an acute illness. However, the primary care physician should retain ultimate responsibility for making major decisions about diagnosis and treatment and should assure patients and their families that decisions are being made in consultation with these specialists by a physician who has an overall and complete perspective on the case.

1	A key factor in mitigating the problems associated with multiple care providers is a commitment to interprofessional teamwork. Despite the diversity in training, skills, and responsibilities among health care professionals, common values need to be reinforced if patient care is not to be adversely affected. This component of effective medical care is widely recognized, and several medical schools have integrated interprofessional teamwork into their curricula. The evolving concept of the “medical home” incorporates team-based primary care with linked subspecialty care in a cohesive environment that ensures smooth transitions of care cost-effectively.

1	Appreciation of the Patient’s Hospital Experience The hospital is an 5 intimidating environment for most individuals. Hospitalized patients find themselves surrounded by air jets, buttons, and glaring lights; invaded by tubes and wires; and beset by the numerous members of the health care team—hospitalists, specialists, nurses, nurses’ aides, physicians’ assistants, social workers, technologists, physical therapists, medical students, house officers, attending and consulting physicians, and many others. They may be transported to special laboratories and imaging facilities replete with blinking lights, strange sounds, and unfamiliar personnel; they may be left unattended at times; and they may be obligated to share a room with other patients who have their own health problems. It is little wonder that a patient’s sense of reality may be compromised. Physicians who appreciate the hospital experience from the patient’s perspective and who make an effort to develop a strong relationship

1	wonder that a patient’s sense of reality may be compromised. Physicians who appreciate the hospital experience from the patient’s perspective and who make an effort to develop a strong relationship within which they can guide the patient through this experience may make a stressful situation more tolerable.

1	Trends in the delivery of Health Care: A Challenge to the Humane Physician Many trends in the delivery of health care tend to make medical care impersonal. These trends, some of which have been mentioned already, include (1) vigorous efforts to reduce the escalating costs of health care; (2) the growing number of managed-care programs, which are intended to reduce costs but in which the patient may have little choice in selecting a physician or in seeing that physician consistently; (3) increasing reliance on technological advances and computerization for many aspects of diagnosis and treatment; and (4) the need for numerous physicians to be involved in the care of most patients who are seriously ill.

1	In light of these changes in the medical care system, it is a major challenge for physicians to maintain the humane aspects of medical care. The American Board of Internal Medicine, working together with the American College of Physicians–American Society of Internal Medicine and the European Federation of Internal Medicine, has published a Charter on Medical Professionalism that underscores three main principles in physicians’ contract with society: (1) the primacy of patient welfare, (2) patient autonomy, and (3) social justice. While medical schools appropriately place substantial emphasis on professionalism, a physician’s personal attributes, including integrity, respect, and compassion, also are extremely important. Availability to the patient, expression of sincere concern, willingness to take the time to explain all aspects of the illness, and a nonjudgmental attitude when dealing with patients whose cultures, lifestyles, attitudes, and values differ from those of the physician

1	to take the time to explain all aspects of the illness, and a nonjudgmental attitude when dealing with patients whose cultures, lifestyles, attitudes, and values differ from those of the physician are just a few of the characteristics of a humane physician. Every physician will, at times, be challenged by patients who evoke strongly negative or positive emotional responses. Physicians should be alert to their own reactions to such patients and situations and should consciously monitor and control their behavior so that the patient’s best interest remains the principal motivation for their actions at all times.

1	An important aspect of patient care involves an appreciation of the patient’s “quality of life,” a subjective assessment of what each patient values most. This assessment requires detailed, sometimes intimate knowledge of the patient, which usually can be obtained only through deliberate, unhurried, and often repeated conversations. Time pressures will always threaten these interactions, but they should not diminish the importance of understanding and seeking to fulfill the priorities of the patient.

1	EXPANdINg FRoNTIERS IN MEdICAL PRACTICE The Era of “omics”: genomics, Epigenomics, Proteomics, Microbiomics, Metagenomics, Metabolomics, Exposomics . . . In the spring of 2003, announcement of the complete sequencing of the human genome officially ushered in the genomic era. However, even before that landmark accomplishment, the practice of medicine had been evolving as a result of the insights into both the human genome and the genomes of a wide variety of microbes. The clinical implications of these insights are illustrated by the complete genome sequencing of H1N1 influenza virus in 2009 and the rapid identification of H1N1 influenza as a potentially fatal pandemic illness, with swift development and dissemination of an effective protective vaccine. Today, gene expression profiles are being

1	Chapter 1 The Practice of Medicine used to guide therapy and inform prognosis for a number of diseases, the use of genotyping is providing a new means to assess the risk of certain diseases as well as variations in response to a number of drugs, and physicians are better understanding the role of certain genes in the causality of common conditions such as obesity and allergies. Despite these advances, the use of complex genomics in the diagnosis, prevention, and treatment of disease is still in its early stages. The task of physicians is complicated by the fact that phenotypes generally are determined not by genes alone but by the interplay of genetic and environmental factors. Indeed, researchers have just begun to scratch the surface of the potential applications of genomics in the practice of medicine.

1	Rapid progress also is being made in other areas of molecular medicine. Epigenomics is the study of alterations in chromatin and histone proteins and methylation of DNA sequences that influence gene expression. Every cell of the body has identical DNA sequences; the diverse phenotypes a person’s cells manifest are the result of epigenetic regulation of gene expression. Epigenetic alterations are associated with a number of cancers and other diseases. Proteomics, the study of the entire library of proteins made in a cell or organ and its complex relationship to disease, is enhancing the repertoire of the 23,000 genes in the human genome through alternate splicing, post-translational processing, and posttranslational modifications that often have unique functional consequences. The presence or absence of particular proteins in the circulation or in cells is being explored for diagnostic and disease-screening applications. Microbiomics is the study of the resident microbes in humans and

1	or absence of particular proteins in the circulation or in cells is being explored for diagnostic and disease-screening applications. Microbiomics is the study of the resident microbes in humans and other mammals. The human haploid genome has ~20,000 genes, while the microbes residing on and in the human body comprise over 3–4 million genes; the contributions of these resident microbes are likely to be of great significance with regard to health status. In fact, research is demonstrating that the microbes inhabiting human mucosal and skin surfaces play a critical role in maturation of the immune system, in metabolic balance, and in disease susceptibility. A variety of environmental factors, including the use and overuse of antibiotics, have been tied experimentally to substantial increases in disorders such as obesity, metabolic syndrome, atherosclerosis, and immune-mediated diseases in both adults and children. Metagenomics, of which microbiomics is a part, is the genomic study of

1	in disorders such as obesity, metabolic syndrome, atherosclerosis, and immune-mediated diseases in both adults and children. Metagenomics, of which microbiomics is a part, is the genomic study of environmental species that have the potential to influence human biology directly or indirectly. An example is the study of exposures to microorganisms in farm environments that may be responsible for the lower incidence of asthma among children raised on farms. Metabolomics is the study of the range of metabolites in cells or organs and the ways they are altered in disease states. The aging process itself may leave telltale metabolic footprints that allow the prediction (and possibly the prevention) of organ dysfunction and disease. It seems likely that disease-associated patterns will be sought in lipids, carbohydrates, membranes, mitochondria, and other vital components of cells and tissues. Finally, exposomics refers to efforts to catalogue and capture environmental exposures such as

1	in lipids, carbohydrates, membranes, mitochondria, and other vital components of cells and tissues. Finally, exposomics refers to efforts to catalogue and capture environmental exposures such as smoking, sunlight, diet, exercise, education, and violence, which together have an enormous impact on health. All of this new information represents a challenge to the traditional reductionist approach to medical thinking. The variability of results in different patients, together with the large number of variables that can be assessed, creates difficulties in identifying preclinical disease and defining disease states unequivocally. Accordingly, the tools of systems biology and network medicine are being applied to the enormous body of information now obtainable for every patient and may eventually provide new approaches to classifying disease. For a more complete discussion of a complex systems approach to human disease, see Chap. 87e.

1	The rapidity of these advances may seem overwhelming to practicing physicians. However, physicians have an important role to play in ensuring that these powerful technologies and sources of new information are applied with sensitivity and intelligence to the patient. Since “omics” are evolving so rapidly, physicians and other health care professionals must continue to educate themselves so that they can apply this new knowledge to the benefit of their patients’ health and wellbeing. Genetic testing requires wise counsel based on an understanding of the value and limitations of the tests as well as the implications of their results for specific individuals. For a more complete discussion of genetic testing, see Chap. 84.

1	The globalization of Medicine Physicians should be cognizant of diseases and health care services beyond local boundaries. Global travel has implications for disease spread, and it is not uncommon for diseases endemic to certain regions to be seen in other regions after a patient has traveled to and returned from those regions. In addition, factors such as wars, the migration of refugees, and climate change are contributing to changing disease profiles worldwide. Patients have broader access to unique expertise or clinical trials at distant medical centers, and the cost of travel may be offset by the quality of care at those distant locations. As much as any other factor influencing global aspects of medicine, the Internet has transformed the transfer of medical information throughout the world. This change has been accompanied by the transfer of technological skills through telemedicine and international consultation—for example, regarding radiologic images and pathologic specimens.

1	world. This change has been accompanied by the transfer of technological skills through telemedicine and international consultation—for example, regarding radiologic images and pathologic specimens. For a complete discussion of global issues, see Chap. 2.

1	Medicine on the Internet On the whole, the Internet has had a very positive effect on the practice of medicine; through personal computers, a wide range of information is available to physicians and patients almost instantaneously at any time and from anywhere in the world. This medium holds enormous potential for the delivery of current information, practice guidelines, state-of-the-art conferences, journal content, textbooks (including this text), and direct communications with other physicians and specialists, expanding the depth and breadth of information available to the physician regarding the diagnosis and care of patients. Medical journals are now accessible online, providing rapid sources of new information. By bringing them into direct and timely contact with the latest developments in medical care, this medium also serves to lessen the information gap that has hampered physicians and health care providers in remote areas.

1	Patients, too, are turning to the Internet in increasing numbers to acquire information about their illnesses and therapies and to join Internet-based support groups. Patients often arrive at a clinic visit with sophisticated information about their illnesses. In this regard, physicians are challenged in a positive way to keep abreast of the latest relevant information while serving as an “editor” as patients navigate this seemingly endless source of information, the accuracy and validity of which are not uniform.

1	A critically important caveat is that virtually anything can be published on the Internet, with easy circumvention of the peer-review process that is an essential feature of academic publications. Both physicians and patients who search the Internet for medical information must be aware of this danger. Notwithstanding this limitation, appropriate use of the Internet is revolutionizing information access for physicians and patients and in this regard represents a remarkable resource that was not available to practitioners a generation ago.

1	Public Expectations and Accountability The general public’s level of knowledge and sophistication regarding health issues has grown rapidly over the last few decades. As a result, expectations of the health care system in general and of physicians in particular have risen. Physicians are expected to master rapidly advancing fields (the science of medicine) while considering their patients’ unique needs (the art of medicine). Thus, physicians are held accountable not only for the technical aspects of the care they provide but also for their patients’ satisfaction with the delivery and costs of care.

1	In many parts of the world, physicians increasingly are expected to account for the way in which they practice medicine by meeting certain standards prescribed by federal and local governments. The hospitalization of patients whose health care costs are reimbursed by the government and other third parties is subjected to utilization review. Thus, a physician must defend the cause for and duration of a patient’s hospitalization if it falls outside certain “average” standards. Authorization for reimbursement increasingly is based on documentation of the nature and complexity of an illness, as reflected by recorded elements of the history and physical examination. A growing “pay-forperformance” movement seeks to link reimbursement to quality of care. The goal of this movement is to improve standards of health care and contain spiraling health care costs. In many parts of the United States, managed (capitated) care contracts with insurers have replaced traditional fee-for-service care,

1	standards of health care and contain spiraling health care costs. In many parts of the United States, managed (capitated) care contracts with insurers have replaced traditional fee-for-service care, placing the onus of managing the cost of all care directly on the providers and increasing the emphasis on preventive strategies. In addition, physicians are expected to give evidence of their current competence through mandatory continuing education, patient record audits, maintenance of certification, and relicensing.

1	Medical Ethics and New Technologies The rapid pace of technological advance has profound implications for medical applications that go far beyond the traditional goals of disease prevention, treatment, and cure. Cloning, genetic engineering, gene therapy, human–computer interfaces, nanotechnology, and use of designer drugs have the potential to modify inherited predispositions to disease, select desired characteristics in embryos, augment “normal” human performance, replace failing tissues, and substantially prolong life span. Given their unique training, physicians have a responsibility to help shape the debate on the appropriate uses of and limits placed on these new techniques and to consider carefully the ethical issues associated with the implementation of such interventions.

1	The Physician as Perpetual Student From the time doctors graduate from medical school, it becomes all too apparent that their lot is that of the “perpetual student” and that the mosaic of their knowledge and experiences is eternally unfinished. This realization is at the same time exhilarating and anxiety-provoking. It is exhilarating because doctors can apply constantly expanding knowledge to the treatment of their patients; it is anxiety-provoking because doctors realize that they will never know as much as they want or need to know. Ideally, doctors will translate the latter feeling into energy through which they can continue to improve themselves and reach their potential as physicians. It is the physician’s responsibility to pursue new knowledge continually by reading, attending conferences and courses, and consulting colleagues and the Internet. This is often a difficult task for a busy practitioner; however, a commitment to continued learning is an integral part of being a

1	conferences and courses, and consulting colleagues and the Internet. This is often a difficult task for a busy practitioner; however, a commitment to continued learning is an integral part of being a physician and must be given the highest priority.

1	The Physician as Citizen Being a physician is a privilege. The capacity to apply one’s skills for the benefit of one’s fellow human beings is a noble calling. The doctor–patient relationship is inherently unbalanced in the distribution of power. In light of their influence, physicians must always be aware of the potential impact of what they do and say and must always strive to strip away individual biases and preferences to find what is best for the patient. To the extent possible, physicians should also act within their communities to promote health and alleviate suffering. Meeting these goals begins by setting a healthy example and continues in taking action to deliver needed care even when personal financial compensation may not be available. A goal for medicine and its practitioners is to strive to provide the means by which the poor can cease to be unwell.

1	A goal for medicine and its practitioners is to strive to provide the means by which the poor can cease to be unwell. Learning Medicine It has been a century since the publication of the Flexner Report, a seminal study that transformed medical education and emphasized the scientific foundations of medicine as well as the acquisition of clinical skills. In an era of burgeoning information and access to medical simulation and informatics, many schools are implementing new curricula that emphasize lifelong learning and the acquisition of competencies in teamwork, communication skills, system-based practice, and professionalism. These and other features of the medical school curriculum provide the foundation for many of the themes highlighted in this chapter and are expected to allow physicians to progress, with experience and learning over time, from competency to proficiency to mastery.

1	At a time when the amount of information that must be mastered to practice medicine continues to expand, increasing pressures both within and outside of medicine have led to the implementation of restrictions on the amount of time a physician-in-training can spend in the hospital. Because the benefits associated with continuity of medical care and observation of a patient’s progress over time were thought to be outstripped by the stresses imposed on trainees by long hours and by the fatigue-related errors they made in caring for patients, strict limits were set on the number of patients that trainees could be responsible for at one time, the number of new patients they could evaluate in a day on call, and the number of hours they could spend in the hospital. In 1980, residents in medicine worked in the hospital more than 90 hours per week on average. In 1989, their hours were restricted to no more than 80 per week. Resident physicians’ hours further decreased by ~10% between 1996 and

1	worked in the hospital more than 90 hours per week on average. In 1989, their hours were restricted to no more than 80 per week. Resident physicians’ hours further decreased by ~10% between 1996 and 2008, and in 2010 the Accreditation Council for Graduate Medical Education further restricted (i.e., to 16 hours per shift) consecutive in-hospital duty hours for first-year residents. The impact of these changes is still being assessed, but the evidence that medical errors have decreased as a consequence is sparse. An unavoidable by-product of fewer hours at work is an increase in the number of “handoffs” of patient responsibility from one physician to another. These transfers often involve a transition from a physician who knows the patient well, having evaluated that individual on admission, to a physician who knows the patient less well. It is imperative that these transitions of responsibility be handled with care and thoroughness, with all relevant information exchanged and

1	admission, to a physician who knows the patient less well. It is imperative that these transitions of responsibility be handled with care and thoroughness, with all relevant information exchanged and acknowledged.

1	Research, Teaching, and the Practice of Medicine The word doctor is derived from the Latin docere, “to teach.” As teachers, physicians should share information and medical knowledge with colleagues, students of medicine and related professions, and their patients. The practice of medicine is dependent on the sum total of medical knowledge, which in turn is based on an unending chain of scientific discovery, clinical observation, analysis, and interpretation. Advances in medicine depend on the acquisition of new information through research, and improved medical care requires the transmission of that information. As part of their broader societal responsibilities, physicians should encourage patients to participate in ethical and properly approved clinical investigations if these studies do not impose undue hazard, discomfort, or inconvenience. However, physicians engaged in clinical research must be alert to potential conflicts of interest between their research goals and their

1	do not impose undue hazard, discomfort, or inconvenience. However, physicians engaged in clinical research must be alert to potential conflicts of interest between their research goals and their obligations to individual patients. The best interests of the patient must always take priority.

1	To wrest from nature the secrets which have perplexed philosophers in all ages, to track to their sources the causes of disease, to correlate the vast stores of knowledge, that they may be quickly available for the prevention and cure of disease—these are our ambitions. —William Osler, 1849–1919 Paul Farmer, Joseph Rhatigan WHY gLoBAL HEALTH?

1	Global health is not a discipline; it is, rather, a collection of problems. Some scholars have defined global health as the field of study and practice concerned with improving the health of all people and achieving health equity worldwide, with an emphasis on addressing transnational problems. No single review can do much more than identify the leading problems in applying evidence-based medicine in settings of great poverty or across national boundaries. However, this is a moment of opportunity: only recently, persistent epidemics, improved metrics, and growing interest have been matched by an unprecedented investment in addressing the health problems of poor people in the developing world. To ensure that this opportunity is not wasted, the facts need to be laid out for specialists and laypeople alike. This chapter introduces the major international bodies that address health problems; identifies the more significant barriers to improving the health of people who to date have not,

1	laypeople alike. This chapter introduces the major international bodies that address health problems; identifies the more significant barriers to improving the health of people who to date have not, by and large, had access to modern medicine; and summarizes population-based data on the most common health problems faced by people living in poverty. Examining specific problems—notably HIV/AIDS (Chap. 226) but also tuberculosis (TB, Chap. 202), malaria (Chap. 248), and key “noncommunicable” chronic diseases (NCDs)—helps sharpen the discussion of barriers to prevention, diagnosis, and care as well as the means of overcoming them. This chapter closes by discussing global health equity, drawing on notions of social justice that once were central to international public health but had fallen out of favor during the last decades of the twentieth century.

1	Concern about health across national boundaries dates back many centuries, predating the Black Plague and other pandemics. One of the first organizations founded explicitly to tackle cross-border health issues was the Pan American Sanitary Bureau, which was formed in 1902 by 11 countries in the Americas. The primary goal of what later became the Pan American Health Organization was the control of infectious diseases across the Americas. Of special concern was yellow fever, which had been running a deadly course through much of South and Central America and halted the construction of the Panama Canal. In 1948, the United Nations formed the first truly global health institution: the World Health Organization (WHO). In 1958, under the aegis of the WHO and in line with a long-standing focus on communicable diseases that cross borders, leaders in global health initiated the effort that led to what some see as the greatest success in international health: the eradication of smallpox.

1	on communicable diseases that cross borders, leaders in global health initiated the effort that led to what some see as the greatest success in international health: the eradication of smallpox. Naysayers were surprised when the smallpox eradication campaign, which engaged public health officials throughout the world, proved successful in 1979 despite the ongoing Cold War.

1	At the International Conference on Primary Health Care in Alma-Ata (in what is now Kazakhstan) in 1978, public health officials from around the world agreed on a commitment to “Health for All by the Year 2000,” a goal to be achieved by providing universal access to primary health care worldwide. Critics argued that the attainment of this goal by the proposed date was impossible. In the ensuing years, a strategy for the provision of selective primary health care emerged that included four inexpensive interventions collectively known as GOBI: growth monitoring, oral rehydration, breast-feeding, and immunizations for diphtheria, whooping cough, tetanus, polio, TB, and measles. GOBI later was expanded to GOBI-FFF, which also included female education, food, and family planning. Some public health figures saw GOBI-FFF as an interim strategy to achieve “health for all,” but others criticized it as a retreat from the bolder commitments of Alma-Ata.

1	The influence of the WHO waned during the 1980s. In the early 1990s, many observers argued that, with its vastly superior financial resources and its close—if unequal—relationships with the governments of poor countries, the World Bank had eclipsed the WHO as the most important multilateral institution working in the area of health. One of the stated goals of the World Bank was to help poor countries identify “cost-effective” interventions worthy of public funding and international support. At the same time, the World Bank encouraged many of those nations to reduce public expenditures in health and education in order to stimulate economic growth as part of (later discredited) structural adjustment programs whose restrictions were imposed as a condition for access to credit and assistance through international financial institutions such as the World Bank and the International Monetary Fund. There was a resurgence of many diseases, including malaria, trypanosomiasis, and

1	assistance through international financial institutions such as the World Bank and the International Monetary Fund. There was a resurgence of many diseases, including malaria, trypanosomiasis, and schistosomiasis, in Africa. TB, an eminently curable disease, remained the world’s leading infectious killer of adults. Half a million women per year died in childbirth during the last decade of the twentieth century, and few of the world’s largest philanthropic or funding institutions focused on global health equity.

1	HIV/AIDS, first described in 1981, precipitated a change. In the United States, the advent of this newly described infectious killer marked the culmination of a series of events that discredited talk of “closing the book” on infectious diseases. In Africa, which would emerge as the global epicenter of the pandemic, HIV disease strained TB control programs, and malaria continued to take as many lives as ever. At the dawn of the twenty-first century, these three diseases alone killed nearly 6 million people each year. New research, new policies, and new funding mechanisms were called for. The past decade has seen the rise of important multilateral global health financing institutions such as the Global Fund to Fight AIDS, Tuberculosis, and Malaria; bilateral efforts such as the U.S. President’s Emergency Plan for AIDS Relief (PEPFAR); and private philanthropic organizations such as the Bill & Melinda Gates Foundation. With its 193 member states and 147 country offices, the WHO remains

1	Emergency Plan for AIDS Relief (PEPFAR); and private philanthropic organizations such as the Bill & Melinda Gates Foundation. With its 193 member states and 147 country offices, the WHO remains important in matters relating to the cross-border spread of infectious diseases and other health threats. In the aftermath of the epidemic of severe acute respiratory syndrome in 2003, the WHO’s International Health Regulations—which provide a legal foundation for that organization’s direct investigation into a wide range of global health problems, including pandemic influenza, in any member state—were strengthened and brought into force in May 2007.

1	Even as attention to and resources for health problems in poor countries grow, the lack of coherence in and among global health institutions may undermine efforts to forge a more comprehensive and effective response. The WHO remains underfunded despite the ever-growing need to engage a wider and more complex range of health issues. In another instance of the paradoxical impact of success, the rapid growth of the Gates Foundation, which is one of the most important developments in the history of global health, has led some foundations to question the wisdom of continuing to invest their more modest resources in this field. This indeed may be what some have called “the golden age of global health,” but leaders of major organizations such as the WHO, the Global Fund, the United Nations Children’s Fund (UNICEF), the Joint United Nations Programme on HIV/AIDS (UNAIDS), PEPFAR, and the Gates Foundation must work together to design an effective architecture that will make the most of

1	Children’s Fund (UNICEF), the Joint United Nations Programme on HIV/AIDS (UNAIDS), PEPFAR, and the Gates Foundation must work together to design an effective architecture that will make the most of opportunities to link new resources for and commitments to global health equity with the emerging understanding of disease burden and unmet need. To this end, new and old players in global health must invest heavily in discovery (relevant basic science), development of new tools (preventive, diagnostic, and therapeutic), and modes of delivery that will ensure the equitable provision of health products and services to all who need them.

1	Political and economic concerns have often guided global health interventions. As mentioned, early efforts to control yellow fever were tied to the completion of the Panama Canal. However, the precise nature of the link between economics and health remains a matter for debate. Some economists and demographers argue that improving the health status of populations must begin with economic development; others maintain that addressing ill health is the starting point for development in poor countries. In either case, investment in health care, especially the control of communicable diseases, should lead to increased productivity. The question is where to find the necessary resources to start the predicted “virtuous cycle.”

1	During the past two decades, spending on health in poor countries has increased dramatically. According to a study from the Institute for Health Metrics and Evaluation (IHME) at the University of Washington, total development assistance for health worldwide grew to $28.2 billion in 2010—up from $5.6 billion in 1990. In 2010, the leading contributors included U.S. bilateral agencies such as PEPFAR, the Global Fund, nongovernmental organizations (NGOs), the WHO, the World Bank, and the Gates Foundation. It appears, however, that total development assistance for health plateaued in 2010, and it is unclear whether growth will continue in the upcoming decade.

1	To reach the United Nations Millennium Development Goals, which include targets for poverty reduction, universal primary education, and gender equality, spending in the health sector must be increased above the 2010 levels. To determine by how much and for how long, it is imperative to improve the ability to assess the global burden of disease and to plan interventions that more precisely match need.

1	Refining metrics is an important task for global health: only recently have there been solid assessments of the global burden of disease. The first study to look seriously at this issue, conducted in 1990, laid the foundation for the first report on Disease Control Priorities in Developing Countries and for the World Bank’s 1993 World Development Report Investing in Health. Those efforts represented a major advance in the understanding of health status in developing countries. Investing in Health has been especially influential: it familiarized a broad audience with cost-effectiveness analysis for specific health interventions and with the notion of disability-adjusted life years (DALYs). The DALY, which has become a standard measure of the impact of a specific health condition on a population, combines absolute years of life lost and years lost due to disability for incident cases of a condition. (See Fig. 2-1 and Table 2-1 for an analysis of the global disease burden by DALYs.)

1	In 2012, the IHME and partner institutions began publishing results from the Global Burden of Diseases, Injuries, and Risk Factors Study 2010 (GBD 2010). GBD 2010 is the most comprehensive effort to date to produce longitudinal, globally complete, and comparable estimates of the burden of diseases, injuries, and risk factors. This report reflects the expansion of the available data on health in the poorest countries and of the capacity to quantify the impact of specific conditions on a population. It measures current levels and recent trends in all major diseases, injuries, and risk factors among 21 regions and for 20 age groups and both sexes. The GBD 2010 team revised and improved the health-state severity weight system, collated published data, and used household surveys to enhance the breadth and accuracy of disease burden data. As analytic methods and data quality improve, important trends can be identified in a comparison of global disease burden estimates from 1990 to 2010.

1	Of the 52.8 million deaths worldwide in 2010, 24.6% (13 million) were due to communicable diseases, maternal and perinatal conditions, and nutritional deficiencies—a marked decrease compared with figures for 1990, when these conditions accounted for 34% of global mortality. Among the fraction of all deaths related to communicable diseases, maternal and perinatal conditions, and nutritional deficiencies, 76% occurred in sub-Saharan Africa and southern Asia. While the proportion of deaths due to these conditions has decreased significantly in the past decade, there has been a dramatic rise in the number of deaths from NCDs, which constituted the top five causes of death in 2010. The leading cause of death among adults in 2010 was ischemic heart disease, accounting for 7.3 million deaths (13.8% of total deaths) worldwide. In high-income countries ischemic heart disease accounted for 17.9% of total deaths, and in developing (lowand middle-income) countries it accounted for 10.1%. It is

1	of total deaths) worldwide. In high-income countries ischemic heart disease accounted for 17.9% of total deaths, and in developing (lowand middle-income) countries it accounted for 10.1%. It is noteworthy that ischemic heart disease was responsible for just 2.6% of total deaths in sub-Saharan Africa (Table 2-2). In second place—causing 11.1% of global mortality—was cerebrovascular disease, which accounted for 9.9% of deaths in high-income countries, 10.5% in developing countries, and 4.0% in sub-Saharan Africa. Although the third leading cause of death in high-income countries was lung cancer (accounting for 5.6% of all deaths), this condition did not figure among the top 10 causes in lowand middle-income countries. Among the 10 leading causes of death in sub-Saharan Africa, 6 were infectious diseases, with malaria and HIV/AIDS ranking as the dominant contributors to disease burden. In high-income countries, however, only one infectious disease—lower respiratory infection—ranked among

1	diseases, with malaria and HIV/AIDS ranking as the dominant contributors to disease burden. In high-income countries, however, only one infectious disease—lower respiratory infection—ranked among the top 10 causes of death.

1	The GBD 2010 found that the worldwide mortality figure among children <5 years of age dropped from 16.39 million in 1970 to 11.9 million in 1990 and to 6.8 million in 2010—a decrease that surpassed predictions. Of childhood deaths in 2010, 3.1 million (40%) occurred in the neonatal period. About one-third of deaths among children <5 years old occurred in southern Asia and almost one-half in sub-Saharan Africa; <1% occurred in high-income countries.

1	The global burden of death due to HIV/AIDS and malaria was on an upward slope until 2004; significant improvements have since been documented. Global deaths from HIV infection fell from 1.7 million in 2006 to 1.5 million in 2010, while malaria deaths dropped from 1.2 million to 0.98 million over the same period. Despite these improvements, malaria and HIV/AIDS continue to be major burdens in particular regions, with global implications. Although it has a minor impact on mortality outside sub-Saharan Africa and Southeast Asia, malaria is the eleventh leading cause of death worldwide. HIV infection ranked thirty-third in global DALYs in 1990 but was the fifth leading cause of disease burden in 2010, with sub-Saharan Africa bearing the vast majority of this burden (Fig. 2-1).

1	The world’s population is living longer: global life expectancy has increased significantly over the past 40 years from 58.8 years in 1970 to 70.4 years in 2010. This demographic change, accompanied by the fact that the prevalence of NCDs increases with age, is dramatically shifting the burden of disease toward NCDs, which have surpassed communicable, maternal, nutritional, and neonatal causes. By 2010, 65.5% of total deaths at all ages and 54% of all DALYs were due to NCDs. Increasingly, the global burden of disease comprises conditions and injuries that cause disability rather than death.

1	Worldwide, although both life expectancy and years of life lived in good health have risen, years of life lived with disability have also increased. Despite the higher prevalence of diseases common in older populations (e.g., dementia and musculoskeletal disease) in developed and high-income countries, best estimates from 2010 reveal that disability resulting from cardiovascular diseases, chronic respiratory diseases, and the long-term impact of communicable diseases was greater in lowand middle-income countries. In most developing countries, people lived shorter lives and experienced disability and poor health for a greater proportion of their lives. Indeed, 50% of the global burden of disease occurred in southern Asia and sub-Saharan Africa, which together account for only 35% of the world’s population.

1	Clear disparities in burden of disease (both communicable and noncommunicable) across country income levels are strong indicators that poverty and health are inherently linked. Poverty remains one of the most important root causes of poor health worldwide, and the global burden of poverty continues to be high. Among the 6.7 billion people alive in 2008, 19% (1.29 billion) lived on less than $1.25 a day— one standard measurement of extreme poverty—and another 1.18 billion lived on $1.25 to $2 a day. Approximately 600 million children—more than 30% of those in low-income countries—lived in extreme poverty in 2005. Comparison of national health indicators with gross domestic product per capita among nations shows a clear relationship between higher gross domestic product and better health, with only a few outliers. Numerous studies have also documented the link between poverty and health within nations as well as across them.

1	The GBD 2010 study found that the three leading risk factors for global disease burden in 2010 were (in order of frequency) high blood pressure, tobacco smoking (including secondhand smoke), and alcohol use—a substantial change from 1990, when childhood undernutrition was ranked first. Though ranking eighth in 2010, childhood undernutrition remains the leading risk factor for death worldwide among children <5 years of age. In an era that has seen obesity become a major health concern in many developed countries—and the sixth leading risk factor worldwide—the persistence of undernutrition is surely cause for great consternation. Low body weight is still the dominant risk factor for disease burden in sub-Saharan Africa. Inability to feed the hungry reflects many years of failed development projects and must be addressed as a problem of the highest priority. Indeed, no health care initiative, however generously funded, will be effective without adequate nutrition.

1	In a 2006 publication that examined how specific diseases and injuries are affected by environmental risk, the WHO estimated that roughly one-quarter of the total global burden of disease, one-third of the global disease burden among children, and 23% of all deaths were due to modifiable environmental factors. Many of these factors lead to deaths from infectious diseases; others lead to deaths Communicable, maternal, neonatal, and nutritional disorders

1	FIgURE 2-1 Global DALY (disability-adjusted life year) ranks for the top causes of disease burden in 1990 and 2010. COPD, chronic obstructive pulmonary disease. (Reproduced with permission from C Murray et al: Disability-adjusted life years [DALYs] for 291 diseases and injuries in 21 regions, 1990–2010: A systematic analysis for the Global Burden of Disease Study 2010. Lancet 380:2197–2223, 2012.) from malignancies. Etiology and nosology are increasingly difficult to parse. As much as 94% of diarrheal disease, which is linked to unsafe drinking water and poor sanitation, can be attributed to environmental factors. Risk factors such as indoor air pollution due to use of solid fuels, exposure to secondhand tobacco smoke, and outdoor air pollution account for 20% of lower respiratory infections in developed countries and for as many as 42% of such infections in developing countries. Various forms of unintentional injury and malaria top the list of health problems to which environmental

1	in developed countries and for as many as 42% of such infections in developing countries. Various forms of unintentional injury and malaria top the list of health problems to which environmental factors contribute. Some 4 million children die every year from causes related to unhealthy environments, and the number of infant deaths due to environmental factors in developing countries is 12 times that in developed countries.

1	The second edition of Disease Control Priorities in Developing Countries, published in 2006, is a document of great breadth and ambition, providing cost-effectiveness analyses for more than 100 interventions and including 21 chapters focused on strategies for strengthening health systems. Cost-effectiveness analyses that compare relatively equivalent interventions and facilitate the best choices under constraint are necessary; however, these analyses are often based on an incomplete knowledge of cost and evolving evidence of effectiveness. As both resources and objectives for global health grow, cost-effectiveness analyses (particularly those based on older evidence) must not hobble the increased worldwide commitment to providing resources and accessible health care services to all who need them. This is why we use the term global health equity. To illustrate these points, it 11taBLe 2-1 LeadInG Causes of dIsease Burden, 2010 Percent of

1	Percent of Disease or Injury DALYs (Millions) Total DALYs Disease or Injury DALYs (Millions) Total DALYs 1 Ischemic heart disease 129.8 5.2 2 Lower respiratory infections 115.2 4.7 3 Cerebrovascular disease 102.2 4.1 4 Diarrheal disease 89.5 3.6 5 HIV/AIDS 81.5 3.3 6 Malaria 82.7 3.3 7 Low back pain 80.7 3.2 8 Preterm birth complications 77.0 3.1 9 COPD 76.8 3.1 10 Road injury 75.5 3.1 1 Lower respiratory infections 109.0 5.2 2 Diarrheal disease 88.0 4.2 3 Ischemic heart disease 85.5 4.1 4 Malaria 82.7 3.9 5 Cerebrovascular disease 79.4 3.8 6 HIV/AIDS 77.0 3.7 7 Preterm birth complications 74.4 3.5 8 Road injury 66.2 3.2 9 COPD 65.6 3.1 10 Low back pain 58.4 2.8 1 Ischemic heart disease 21.8 8.2 2 Low back pain 17.0 6.4 3 Cerebrovascular disease 11.3 4.2 4 Major depressive disorder 9.7 3.7 5 Lung cancer 9.2 3.5 6 COPD 8.6 3.2 7 Other musculoskeletal disorders 8.2 3.1 8 Diabetes mellitus 7.3 2.8 9 Neck pain 7.2 2.7 10 Falls 6.8 2.5 1 Malaria 76.6 13.3 2 HIV/AIDS 57.8 10.1 3 Lower

1	3.7 5 Lung cancer 9.2 3.5 6 COPD 8.6 3.2 7 Other musculoskeletal disorders 8.2 3.1 8 Diabetes mellitus 7.3 2.8 9 Neck pain 7.2 2.7 10 Falls 6.8 2.5 1 Malaria 76.6 13.3 2 HIV/AIDS 57.8 10.1 3 Lower respiratory infections 43.5 7.6 4 Diarrheal diseases 39.2 6.8 5 Protein-energy malnutrition 22.3 3.9 6 Preterm birth complications 20.0 3.5 7 Neonatal sepsis 18.9 3.3 8 Meningitis 16.3 2.8 9 Neonatal encephalopathy 14.9 2.6 10 Road injury 13.9 2.5 aThe term developing countries refers to lowand middle-income economies. See data.worldbank.org/about/country-classifications. bThe World Bank classifies high-income countries as those whose gross national income per capita is $12,476 or more. See data.worldbank.org/about/country-classifications. Abbreviations: COPD, chronic obstructive pulmonary disease; DALYs, disability-adjusted life years. Source: Institute for Health Metrics and Evaluation, University of Washington (2013). Data are available through

1	COPD, chronic obstructive pulmonary disease; DALYs, disability-adjusted life years. Source: Institute for Health Metrics and Evaluation, University of Washington (2013). Data are available through www.healthmetricsandevaluation.org/gbd/visualizations/country.

1	taBLe 2-2 LeadInG Causes of death WorLdWIde, 2010 Deaths Percent of

1	Deaths Percent of Disease or Injury (Millions) Total Deaths Disease or Injury (Millions) Total Deaths 1 Ischemic heart disease 7.3 13.3 2 Cerebrovascular disease 5.9 11.1 3 COPD 2.9 5.5 4 Lower respiratory infections 2.8 5.3 5 Lung cancer 1.5 2.9 6 HIV/AIDS 1.5 2.8 7 Diarrheal diseases 1.4 2.7 8 Road injury 1.3 2.5 9 Diabetes 1.3 2.4 10 Tuberculosis 1.2 2.3 1 Cerebrovascular disease 4.2 10.5 2 Ischemic heart disease 4.0 10.1 3 COPD 2.4 6.1 4 Lower respiratory infections 2.3 5.9 5 Diarrheal diseases 1.4 3.6 6 HIV/AIDS 1.4 3.4 7 Malaria 1.2 2.9 8 Road injury 1.2 2.9 9 Tuberculosis 1.1 2.9 10 Diabetes 1.0 2.6 1 Ischemic heart disease 1.6 17.9 2 Cerebrovascular disease 0.9 9.9 3 Lung cancer 0.5 5.6 4 Lower respiratory infections 0.4 4.7 5 COPD 0.4 4.5 6 Alzheimer’s and other dementias 0.4 4.0 7 Colon and rectum cancers 0.3 3.3 8 Diabetes 0.2 2.6 9 Other cardiovascular and circulatory diseases 0.2 2.5 10 Chronic kidney disease 0.2 2.0 1 Malaria 1.1 12.7 2 HIV/AIDS 1.0 12.0 3 Lower

1	0.4 4.0 7 Colon and rectum cancers 0.3 3.3 8 Diabetes 0.2 2.6 9 Other cardiovascular and circulatory diseases 0.2 2.5 10 Chronic kidney disease 0.2 2.0 1 Malaria 1.1 12.7 2 HIV/AIDS 1.0 12.0 3 Lower respiratory infections 0.8 9.3 4 Diarrheal diseases 0.5 6.6 5 Cerebrovascular disease 0.3 4.0 6 Protein-energy malnutrition 0.3 4.0 7 Tuberculosis 0.3 3.6 8 Road injury 0.2 2.8 9 Preterm birth complications 0.2 2.8 10 Meningitis 0.2 2.6 aThe term developing countries refers to lowand middle-income economies. See data.worldbank.org/about/country-classifications. bThe World Bank classifies high-income countries as those whose gross national income per capita is $12,476 or more. See data.worldbank.org/about/country-classifications. Abbreviation: COPD, chronic obstructive pulmonary disease. Source: Institute for Health Metrics and Evaluation, University of Washington (2013). Data available through www.healthmetricsandevaluation.org/gbd/visualizations/country.

1	is instructive to look to HIV/AIDS, which in the course of the last three decades has become the world’s leading infectious cause of adult death. Chapter 226 provides an overview of the HIV epidemic in the world today. Here the discussion will be limited to HIV/AIDS in the developing world. Lessons learned from tackling HIV/AIDS in resource-constrained settings are highly relevant to discussions of other chronic diseases, including NCDs, for which effective therapies have been developed. Approximately 34 million people in all countries worldwide were living with HIV infection in 2011; more than 8 million of those in low-and middle-income countries were receiving antiretroviral therapy (ART)—a number representing a 20-fold increase over the corresponding figure for 2003. By the end of 2011, 54% of people eligible for treatment were receiving ART. (It remains to be seen how many of these people are receiving ART regularly and with the requisite social support.)

1	In the United States, the availability of ART has transformed HIV/ AIDS from an inescapably fatal destruction of cell-mediated immunity into a manageable chronic illness. In high-income countries, improved ART has prolonged life by an estimated average of 35 years per patient—up from 6.8 years in 1993 and 24 years in 2006. This success rate exceeds that obtained with almost any treatment for adulthood cancer or for complications of coronary artery disease. In developing countries, treatment has been offered broadly only since 2003, and only in 2009 did the number of patients receiving treatment exceed 40% of the number who needed it. Before 2003, many arguments were raised to justify not moving forward rapidly with ART programs for people living with HIV/AIDS in resource-limited settings. The standard litany included the price of therapy compared with the poverty of the patient, the complexity of the intervention, the lack of infrastructure for laboratory monitoring, and the lack of

1	The standard litany included the price of therapy compared with the poverty of the patient, the complexity of the intervention, the lack of infrastructure for laboratory monitoring, and the lack of trained health care providers. Narrow cost-effectiveness arguments that created false dichotomies—prevention or treatment rather than both—too often went unchallenged. As a cumulative result of these delays in the face of health disparities, old and new, there were millions of premature deaths.

1	Disparities in access to HIV treatment gave rise to widespread moral indignation and a new type of health activism. In several middle-income countries, including Brazil, public programs have helped bridge the access gap. Other innovative projects pioneered by international NGOs in diverse settings such as Haiti and Rwanda have established that a simple approach to ART that is based on intensive community engagement and support can achieve remarkable results (Fig. 2-2).

1	During the past decade, the availability of ART has increased sharply in the lowand middle-income countries that have borne the greatest burden of the HIV/AIDS pandemic. In 2000, very few people living with HIV/AIDS in these nations had access to ART, whereas by 2011, as stated above, 8 million people, a majority of those deemed eligible, in these countries were receiving ART. This scale-up was made possible by a number of developments: a staggering drop in the cost of ART, the development of a standardized approach to treatment, substantial investments by funders, and the political commitment of governments to make ART available. Civil-society AIDS activists spurred many of these efforts.

1	Starting in the early 2000s, a combination of factors, including work by the Clinton Foundation HIV/AIDS Initiative and Médecins Sans Frontières, led to the availability of generic ART medications. While first-line ART cost more than $10,000 per patient per year in 2000, first-line regimens in lowand middle-income countries are now available for less than $100 per year. At the same time, fixed-dose combination drugs that are easier to administer have become more widely available.

1	Also around this time, the WHO began advocating a public health approach to the treatment of people with AIDS in resource-limited settings. This approach, derived from models of care pioneered by the NGO Partners In Health and other groups, proposed standard first-line treatment regimens based on a simple five-drug formulary, with a more complex (and more expensive) set of second-line options in reserve. Clinical protocols were standardized, and intensive training packages for health professionals and community health workers were developed and implemented in many countries. These efforts were supported by new funding from the World Bank, the Global Fund, and PEPFAR. In 2003, lack of access to ART was declared a global public health emergency by the WHO and UNAIDS, and those two agencies launched the “3 by 5 initiative,” setting an ambitious target: to have 3 million people in developing countries on treatment by the end of 2005. Worldwide funding for HIV/AIDS treatment increased

1	launched the “3 by 5 initiative,” setting an ambitious target: to have 3 million people in developing countries on treatment by the end of 2005. Worldwide funding for HIV/AIDS treatment increased dramatically during this period, rising from $300 million in 1996 to over $15 billion in 2010.

1	Many countries set corresponding national targets and have worked to integrate ART into their national AIDS programs and health systems and to harness the synergies between HIV/AIDS treatment and prevention activities. Further lessons with implications for policy and action have come from efforts now under way among lower-income countries. Rwanda provides an example: Over the past decade, mortality from HIV disease has fallen by >78% as the country—despite its relatively low gross national income (Fig. 2-3)—has provided almost universal access to ART. The reasons for this success include strong national leadership, evidence-based policy, cross-sector collaboration, community-based care, and a deliberate focus on a health system approach that embeds HIV/AIDS treatment and prevention in the primary health care service delivery platform. As we will discuss later in this chapter, these principles can be applied to other conditions, including NCDs.

1	Chapter 202 provides a concise overview of the pathophysiology and treatment of TB. In 2011, an estimated 12 million people were living with active TB, and 1.4 million died from it. The disease is closely linked to HIV infection in much of the world: of the 8.7 million estimated new cases of TB in 2011, 1.2 million occurred among people living with HIV. Indeed, a substantial proportion of the TB resurgence FIgURE 2-2 An HIV/TB-co-infected patient in Rwanda before (left ) and after (right ) 6 months of treatment. 100% infection control in hospitals and clinics is associated with explosive and lethal epidemics due to these strains and that patients may be infected with multiple strains.

1	100% infection control in hospitals and clinics is associated with explosive and lethal epidemics due to these strains and that patients may be infected with multiple strains. Gross domestic product per capita, 2009 (log) Namibia Swaziland Zambia Gabon South Africa Senegal Benin MaliEthiopia Malawi ChadEritrea Niger Liberia Madagascar Somalia Djibouti Sudan Burundi D.R. Congo Congo Angola Mauritius United Nations 2010 target $100 0% 20% 40% 60% $1,000 $10,000 FIgURE 2-3 Antiretroviral therapy (ART) coverage in sub-Saharan Africa, 2009.

1	Estimated ART coverage, 2009 registered in southern Africa is attributed to HIV co-infection. Even before the advent of HIV, however, it was estimated that fewer than one-half of all cases of TB in developing countries were ever diagnosed, much less treated. Primarily because of the common failure to diagnose and treat TB, international authorities devised a single strategy to reduce the burden of disease. In the early 1990s, the World Bank, the WHO, and other international bodies promoted the DOTS strategy (directly observed therapy using short-course isoniazidand rifampinbased regimens) as highly cost-effective. Passive case-finding of smear-positive patients was central to the strategy, and an uninterrupted drug supply was, of course, deemed necessary for cure.

1	DOTS was clearly effective for most uncomplicated cases of drug-susceptible TB, but a number of shortcomings were soon identified. First, the diagnosis of TB based solely on sputum smear microscopy— a method dating from the late nineteenth century—is not sensitive. Many cases of pulmonary TB and all cases of exclusively extrapulmonary TB are missed by smear microscopy, as are most cases of active disease in children. Second, passive case-finding relies on the availability of health care services, which is uneven in the settings where TB is most prevalent. Third, patients with multidrug-resistant TB (MDR-TB) are by definition infected with strains of Mycobacterium tuberculosis resistant to isoniazid and rifampin; thus exclusive reliance on these drugs is unwarranted in settings in which drug resistance is an established problem.

1	The crisis of antibiotic resistance registered in U.S. hospitals is not confined to the industrialized world or to common bacterial infections. The great majority of patients sick with and dying from TB are afflicted with strains susceptible to all first-line drugs. In some settings, however, a substantial minority of patients with TB are infected with M. tuberculosis strains resistant to at least one first-line anti-TB drug. A 2012 article in a leading journal reported that, in China, 10% of all patients with TB and 26% of all previously treated patients were sick with MDR strains of M. tuberculosis. Most of these cases were the result of primary transmission. To improve DOTS-based responses to MDR-TB, global health authorities adopted DOTS-Plus, which adds the diagnostics and drugs necessary to manage drug-resistant disease. Even as DOTS-Plus was being piloted in resource-constrained settings, however, new strains of extensively drug-resistant (XDR) M. tuberculosis (resistant to

1	to manage drug-resistant disease. Even as DOTS-Plus was being piloted in resource-constrained settings, however, new strains of extensively drug-resistant (XDR) M. tuberculosis (resistant to isoniazid and rifampin, any fluoroquinolone, and at least one injectable second-line drug) had already threatened the success of TB control programs in beleaguered South Africa, for example, where high rates of HIV infection have led to a doubling of TB incidence over the last decade. Despite the poor capacity for detection of MDRand XDR-TB in most resource-limited settings, an estimated 630,000 cases of MDR-TB were thought to occur in 2011. Approximately 9% of these drug-resistant cases were caused by XDR strains. It is clear that poor

1	TUBERCULoSIS ANd AIdS AS CHRoNIC dISEASES: LESSoNS LEARNEd

1	Strategies effective against MDR-TB have implications for the management of drug-resistant HIV infection and even drug-resistant malaria, which, through repeated infections and a lack of effective therapy, has become a chronic disease in parts of Africa (see “Malaria,” below). Equatorial As new therapies, whether for TB or for hepatitis C infec-Guinea tion, become available, many of the problems encountered in the past will recur. Indeed, examining AIDS and TB as chronic diseases—instead of simply communicable diseases—makes it possible to draw a number of conclusions, many of them pertinent to global health in general. First, the chronic infections discussed here are best treated with multidrug regimens to which the infecting strains are susceptible. This is true of chronic infections due to many bacteria, fungi, parasites, or viruses; even acute infections such as those caused by Plasmodium spe cies are not reliably treated with a single drug.

1	Second, charging fees for AIDS prevention and care poses insurmountable problems for people living in poverty, many of whom are unable to pay even modest amounts for services or medications. Like efforts to battle airborne TB, such services might best be seen as a public good promoting public health. Initially, a subsidy approach will require sustained donor contributions, but many African countries have set targets for increased national investments in health—a pledge that could render ambitious programs sustainable in the long run, as the Rwanda experience suggests. Meanwhile, as local investments increase, the price of AIDS care is decreasing. The development of generic medications means that ART can now cost <$0.25 per day; costs continue to decrease.

1	Third, the effective scale-up of pilot projects requires strengthening and sometimes rebuilding of health care systems, including those charged with delivering primary care. In the past, the lack of health care infrastructure has been cited as a barrier to providing ART in the world’s poorest regions; however, AIDS resources, which are at last considerable, may be marshaled to rebuild public health systems in sub-Saharan Africa and other HIV-burdened regions—precisely the settings in which TB is resurgent.

1	Fourth, the lack of trained health care personnel, most notably doctors and nurses, in resource-poor settings must be addressed. This personnel deficiency is invoked as a reason for the failure to treat AIDS in poor countries. In what is termed the brain drain, many physicians and nurses emigrate from their home countries to pursue opportunities abroad, leaving behind health systems that are understaffed and ill equipped to deal with the epidemic diseases that ravage local populations. The WHO recommends a minimum of 20 physicians and 100 nurses per 100,000 persons, but recent reports from that organization and others confirm that many countries, especially in sub-Saharan Africa, fall far short of those target numbers. Specifically, more than one-half of those countries register fewer than 10 physicians per 100,000 population. In contrast, the United States and Cuba register 279 and 596 doctors per 100,000 population, respectively. Similarly, the majority of sub-Saharan African

1	than 10 physicians per 100,000 population. In contrast, the United States and Cuba register 279 and 596 doctors per 100,000 population, respectively. Similarly, the majority of sub-Saharan African countries do not have even half of the WHO-recommended minimum number of nurses. Further inequalities in health care staffing exist within countries. Rural–urban disparities in health care personnel mirror disparities of both wealth and health. For instance, nearly 90% of Malawi’s population lives in rural areas, but more than 95% of clinical officers work at urban facilities, and 47% of nurses work at tertiary care facilities. Even community health workers trained to provide first-line services to rural populations often transfer to urban districts.

1	One reason doctors and nurses leave sub-Saharan Africa and other resource-poor areas is that they lack the tools to practice their trade there. Funding for “vertical” (disease-specific) programs can be used not only to strengthen health systems but to recruit and train physicians and nurses to underserved regions where they, in turn, can help to train and then work with community health workers in supervising care for patients with AIDS and many other diseases within their communities. Such training should be undertaken even where physicians are abundant, since close community-based supervision represents the highest standard of care for chronic disease, whether in developing or developed countries. The United States has much to learn from Rwanda.

1	Fifth, the barriers to adequate health care and patient adherence that are raised by extreme poverty can be removed only with the deployment of “wrap-around services”: food supplements for the hungry, help with transportation to clinics, child care, and housing. Extreme poverty makes it difficult for many patients to comply with therapy for chronic diseases, whether communicable or not. Indeed, poverty in its many dimensions is far and away the greatest barrier to the scale-up of treatment and prevention programs. In many rural regions of Africa, hunger is the major coexisting condition in patients with AIDS or TB, and those consumptive diseases cannot be treated effectively without adequate caloric intake.

1	Finally, there is a need for a renewed basic-science commitment to the discovery and development of vaccines; more reliable, less expensive diagnostic tools; and new classes of therapeutic agents. This need applies not only to the three leading infectious killers—against none of which is there an effective vaccine—but also to most other neglected diseases of poverty.

1	Chapter 248 reviews the etiology, pathogenesis, and clinical treatment of malaria, the world’s third-ranking infectious killer. Malaria’s human cost is enormous, with the highest toll among children—especially African children—living in poverty. In 2010, there were ~219 million cases of malaria, and the disease is thought to have killed 660,000 people; 86% of these deaths (~568,000) occurred among children <5 years old. The poor disproportionately experience the burden of malaria: more than 80% of estimated malaria deaths occur in just 14 countries, and mortality rates are highest in sub-Saharan Africa. The Democratic Republic of the Congo and Nigeria account for more than 40% of total estimated malaria deaths globally.

1	Microeconomic analyses focusing on direct and indirect costs estimate that malaria may consume >10% of a household’s annual income. A study in rural Kenya shows that mean direct-cost burdens vary between the wet and dry seasons (7.1% and 5.9% of total household expenditure, respectively) and that this proportion is >10% in the poorest households in both seasons. A Ghanaian study that categorized the population by income group highlighted the regressive nature of this cost: responding to malaria consumed only 1% of a wealthy family’s income but 34% of a poor household’s income.

1	Macroeconomic analyses estimate that malaria may reduce the per capita gross national product of a disease-endemic country by 50% relative to that of a non-malaria-endemic country. The causes of this drag include impaired cognitive development of children, decreased schooling, decreased savings, decreased foreign investment, and restriction of worker mobility. In light of this enormous cost, it is little wonder that an important review by the economists Sachs and Malaney concludes that “where malaria prospers most, human societies have prospered least.”

1	Rolling Back Malaria In part because of differences in vector distribution and climate, resource-rich countries offer few blueprints for malaria control and treatment that are applicable in tropical (and resource-poor) settings. In 2001, African heads of state endorsed the WHO Roll Back Malaria (RBM) campaign, which prescribes strategies appropriate for sub-Saharan African countries. In 2008, the RBM partnership launched the Global Malaria Action Plan (GMAP). This strategy integrates prevention and care and calls for an avoidance of single-dose regimens and an awareness of existing drug resistance. The GMAP recommends a number of key tools to reduce malaria-related morbidity and mortality rates: the use of insecticide-treated bed nets (ITNs), indoor residual spraying, and artemisinin-based combination therapy (ACT) as well as intermittent preventive treatment during pregnancy, prompt diagnosis, and other vector control measures such as larviciding and environmental management.

1	InsectIcIde-treated bed nets ITNs are an efficacious and cost-effective public health intervention. A meta-analysis of controlled trials in seven sub-Saharan African countries indicates that parasitemia prevalence is reduced by 24% among children <5 years of age who sleep under ITNs compared with that among those who do not. Even untreated nets reduce malaria incidence by one-quarter. On an individual level, the utility of ITNs extends beyond protection from malaria. Several studies suggest that ITNs reduce all-cause mortality among children under age 5 to a greater degree than can be attributed to the reduction in malarial disease alone. Morbidity (specifically that due to anemia), which predisposes children to diarrheal and respiratory illnesses and pregnant women to the delivery of low-birth-weight infants, also is reduced in populations using ITNs. In some areas, ITNs offer a supplemental benefit by preventing transmission of lymphatic filariasis, cutaneous leishmaniasis, Chagas’

1	infants, also is reduced in populations using ITNs. In some areas, ITNs offer a supplemental benefit by preventing transmission of lymphatic filariasis, cutaneous leishmaniasis, Chagas’ disease, and tick-borne relapsing fever. At the community level, investigators suggest that the use of an ITN in just one household may reduce the number of mosquito bites in households up to a hundred meters away by reducing mosquito density. The cost of ITNs per DALY saved—estimated at $29—makes ITNs a good-value public health investment.

1	The WHO recommends that all individuals living in malaria-endemic areas sleep under protective ITNs. About 140 million long-lasting ITNs were distributed in high-burden African countries in 2006–2008, and rates of household ownership of ITNs in high-burden countries increased to 31%. Although the RBM partnership has seen modest success, the WHO’s 2009 World Malaria Report states that the percentage of children <5 years of age using an ITN (24%) remains well below the World Health Assembly’s target of 80%. Limited success in scaling up ITN coverage reflects the inadequately acknowledged economic barriers that prevent the destitute sick from gaining access to critical preventive technologies and the challenges faced in designing and implementing effective delivery platforms for these products. In other words, this is a delivery failure rather than a lack of knowledge of how best to reduce malaria deaths.

1	Indoor resIdual sprayIng Indoor residual spraying is one of the most common interventions for preventing the transmission of malaria in endemic areas. Vector control using insecticides approved by the WHO, including DDT, can effectively reduce or even interrupt malaria transmission. However, studies have indicated that spraying is effective in controlling malaria transmission only if most (~80%) of the structures in the targeted community are treated. Moreover, since a successful program depends on well-trained spraying teams as well as on effective monitoring and planning, indoor residual spraying is difficult to employ and is often reliant on health systems with a strong infrastructure. Regardless of the limitations of indoor residual spraying, the WHO recommends its use in combination with ITNs. Neither intervention alone is sufficient to prevent transmission of malaria entirely.

1	artemIsInIn-based combInatIon therapy The emergence and spread of chloroquine resistance have increased the need for antimalarial combination therapy. To limit the spread of resistance, the WHO now recommends that only ACT (as opposed to artemisinin monotherapy) be used for uncomplicated falciparum malaria. Like that of other antimalarial interventions, the use of ACT has increased in the last few years, but coverage rates remain very low in several countries in sub-Saharan Africa. The RBM partnership has invested significantly in measures to enhance access to ACT by facilitating its delivery through the public health sector and developing innovative funding mechanisms (e.g., the Affordable Medicines Facility—malaria) that reduce its cost significantly so that ineffective monotherapies can be eliminated from the market.

1	In the last several years, resistance to antimalarial medicines and insecticides has become an even larger problem than in the past. In 2009, confirmation of artemisinin resistance was reported. Although the WHO has called for an end to the use of artemisinin monotherapy, the marketing of such therapies continues in many countries. Ongoing use of artemisinin monotherapy increases the likelihood of drug resistance, a deadly prospect that will make malaria far more difficult to treat. Between 2001 and 2011, global malaria deaths were reduced by an estimated 38%, with reductions of ≥50% in 10 African countries as well as in most endemic countries in other regions. Again the experience in Rwanda is instructive: from 2005 to 2011, malaria deaths dropped by >85% for the same reasons mentioned earlier in recounting that nation’s successes in battling HIV.

1	Meeting the challenge of malaria control will continue to require careful study of appropriate preventive and therapeutic strategies in the context of an increasingly sophisticated molecular understanding of pathogen, vector, and host. However, an appreciation of the economic and social devastation wrought by malaria—like that inflicted by diarrhea, AIDS, and TB—on the most vulnerable populations should heighten the level of commitment to critical analysis of ways to implement proven strategies for prevention and treatment.

1	Funding from the Global Fund, the Gates Foundation, the World Bank’s International Development Association, and the U.S. President’s Malaria Initiative, along with leadership from public health authorities, is critical to sustain the benefits of prevention and treatment. Building on the growing momentum of the last decade with adequate financial support, innovative strategies, and effective tools for prevention, diagnosis, and treatment, we may one day achieve the goal of a world free of malaria.

1	Although the burden of communicable diseases—especially HIV infection, TB, and malaria—still accounts for the majority of deaths in resource-poor regions such as sub-Saharan Africa, 63% of all deaths worldwide in 2008 were held to be due to NCDs. Although we will use this term to describe cardiovascular diseases, cancers, diabetes, and chronic lung diseases, this usage masks important distinctions. For instance, two significant NCDs in low-income countries, rheumatic heart disease (RHD) and cervical cancer, represent the chronic sequelae of infections with group A Streptococcus and human papillomavirus, respectively. It is in these countries that the burden of disease due to NCDs is rising most rapidly. Close to 80% of deaths attributable to NCDs occur in lowand middle-income countries, where 86% of the global population lives. The WHO reports that ~25% of global NCD-related deaths take place before the age of 60—a figure representing ~5.7 million people and exceeding the total number

1	86% of the global population lives. The WHO reports that ~25% of global NCD-related deaths take place before the age of 60—a figure representing ~5.7 million people and exceeding the total number of deaths due to AIDS, TB, and malaria combined. In almost all high-income countries, the WHO reported that NCD deaths accounted for ~70% of total deaths in 2008. By 2020, NCDs will account for 80% of the global burden of disease and for 7 of every 10 deaths in developing countries. The recent increase in resources for and attention to communicable diseases is both welcome and long overdue, but developing countries are already carrying a “double burden” of communicable and noncommunicable diseases.

1	diabetes, Cardiovascular disease, and Cancer: A global Perspective In contrast to TB, HIV infection, and malaria—diseases caused by single pathogens that damage multiple organs—cardiovascular diseases reflect injury to a single organ system downstream of a variety of insults, both infectious and noninfectious. Some of these insults result from rapid changes in diet and labor conditions. Other insults are of a less recent vintage. The burden of cardiovascular disease in low-income countries represents one consequence of decades of neglect of health systems. Furthermore, cardiovascular research and investment have long focused on the ischemic conditions that are increasingly common in highand middle-income countries. Meanwhile, despite awareness of its health impact in the early twentieth century, cardiovascular damage in response to infection and malnutrition has fallen out of view until recently.

1	The misperception of cardiovascular diseases as a problem primarily of elderly populations in middleand high-income countries has contributed to the neglect of these diseases by global health institutions. Even in Eastern Europe and Central Asia, where the collapse of the Soviet Union was followed by a catastrophic surge in cardiovascular disease deaths (mortality rates from ischemic heart disease nearly doubled between 1991 and 1994 in Russia, for example), the modest flow of overseas development assistance to the health sector focused on the communicable causes that accounted for <1 in 20 excess deaths during that period.

1	dIabetes The International Diabetes Federation reports that the number of diabetic patients in the world is expected to increase from 366 million in 2011 to 552 million by 2030. Already, a significant proportion of diabetic patients live in developing countries where, because those affected are far more frequently between ages 40 and 59, the complications of microand macrovascular disease take a far greater toll. Globally, these complications are a major cause of disability and reduced quality of life. A high fasting plasma glucose level alone ranks seventh among risks for disability and is the sixth leading risk factor for global mortality. The GBD 2010 estimates that diabetes accounted for 1.28 million deaths in 2010, with almost 80% of those deaths occurring in lowand middle-income countries.

1	Predictions of an imminent rise in the share of deaths and disabilities due to NCDs in developing countries have led to calls for preventive policies to improve diet, increase exercise, and restrict tobacco use, along with the prescription of multidrug regimens for persons at high-level vascular risk. Although this agenda could do much to prevent pandemic NCD, it will do little to help persons with established heart disease stemming from nonatherogenic pathologies.

1	cardIovascular dIsease Because systemic investigation of the causes of stroke and heart failure in sub-Saharan Africa has begun only recently, little is known about the impact of elevated blood pressure in this portion of the continent. Modestly elevated blood pressure in the absence of tobacco use in populations with low rates of obesity may confer little risk of adverse events in the short run. In contrast, persistently elevated blood pressure above 180/110 goes largely undetected, untreated, and uncontrolled in this part of the world. In the cohort of men assessed in the Framingham Heart Study, the prevalence of blood pressures above 210/120—severe hypertension—declined from 1.8% in the 1950s to 0.1% by the 1960s with the introduction of effective antihypertensive agents. Although debate continues about appropriate screening strategies and treatment thresholds, rural health centers staffed largely by nurses must quickly gain access to essential antihypertensive medications.

1	The epidemiology of heart failure reflects inequalities in risk factor prevalence and in treatment. The reported burden of this condition has remained unchanged since the 1950s, but the causes of heart failure and the age of the people affected vary across the globe. Heart failure as a consequence of pericardial, myocardial, endocardial, or valvular injury accounts for as many as 5% of all medical admissions to hospitals around the world. In high-income countries, coronary artery disease and hypertension among the elderly account for most cases of heart failure. For example, in the United States, coronary artery disease is present in 60% of patients with heart failure and hypertension in 70%. Among the world’s poorest 1 billion people, however, heart failure reflects poverty-driven exposure of children and young adults to rheumatogenic strains of streptococci and cardiotropic microorganisms (e.g., HIV, Trypanosoma cruzi, enteroviruses, M. tuberculosis), untreated high blood pressure,

1	of children and young adults to rheumatogenic strains of streptococci and cardiotropic microorganisms (e.g., HIV, Trypanosoma cruzi, enteroviruses, M. tuberculosis), untreated high blood pressure, and nutrient deficiencies. The mechanisms underlying other causes of heart failure common in these populations—such as idiopathic dilated cardiomyopathy, peripartum cardiomyopathy, and endomyocardial fibrosis—remain unclear.

1	In stark contrast to the extraordinary lengths to which clinicians in wealthy countries will go to treat ischemic cardiomyopathy, little attention has been paid to young patients with nonischemic cardiomyopathies in resource-poor settings. Nonischemic cardiomyopathies, such as those due to hypertension, RHD, and chronic lung disease, account for >90% of cases of cardiac failure in sub-Saharan Africa and include poorly understood entities such as peripartum cardiomyopathy (which has an incidence in rural Haiti of 1 per 300 live births) and HIV-associated cardiomyopathy. Multidrug regimens that include beta blockers, angiotensin-converting enzyme inhibitors, and other agents can dramatically reduce mortality risk and improve quality of life for these patients. Lessons learned in the scale-up of chronic care for HIV infection and TB may be illustrative as progress is made in establishing the means to deliver heart-failure therapies.

1	Some of the lessons learned from the chronic infections discussed above are, of course, relevant to cardiovascular disease, especially those classified as NCDs but caused by infectious pathogens. Integration of prevention and care remains as important today as in 1960 when Paul Dudley White and his colleagues found little evidence of myocardial infarction in the region near the Albert Schweitzer Hospital in Lambaréné, Gabon, but reported that “the high prevalence of mitral stenosis is astonishing…. We believe strongly that it is a duty to help bring to these sufferers the benefits of better penicillin prophylaxis and of cardiac surgery when indicated. The same responsibility exists for those with correctable congenital cardiovascular defects.”

1	RHD affects more than 15 million people worldwide, with more than 470,000 new cases each year. Among the 2.4 million annual cases of pediatric RHD, an estimated 42% occur in sub-Saharan Africa. This disease, which may cause endocarditis or stroke, leads to more than 345,000 deaths per year—almost all occurring in developing countries. Researchers in Ethiopia have reported annual death rates as high as 12.5% in rural areas. In part because the prevention of RHD has not advanced since the disease’s disappearance in wealthy countries, no part of sub-Saharan Africa has eradicated RHD despite examples of success in Costa Rica, Cuba, and some Caribbean nations. A survey of acute heart failure among adults in sub-Saharan Africa showed that ~14.3% of these cases were due to RHD.

1	Strategies to eliminate rheumatic heart disease may depend on active case-finding, with confirmation by echocardiography, among high-risk groups as well as on efforts to expand access to surgical interventions among children with advanced valvular damage. Partnerships between established surgical programs and areas with limited or nonexistent facilities may help expand the capacity to provide life-saving interventions to patients who otherwise would die early and painfully. A long-term goal is the establishment of regional centers of excellence equipped to provide consistent, accessible, high-quality services.

1	Clinicians from tertiary care centers in sub-Saharan Africa and elsewhere have continued to call for prevention and treatment of the cardiovascular conditions of the poor. The reconstruction of health services in response to pandemic infectious disease offers an opportunity to identify and treat patients with organ damage and to undertake the prevention of cardiovascular and other chronic conditions of poverty.

1	cancer Cancers account for ~5% of the global burden of disease. Low-and middle-income countries accounted for more than two-thirds of the 12.6 million cases and 7.6 million deaths due to cancer in 2008. By 2030, annual mortality from cancer will increase by 4 million—with developing countries experiencing a sharper increase than developed nations. “Western” lifestyle changes will be responsible for the increased incidence of cancers of the breast, colon, and prostate among populations in lowand middle-income countries, but historic realities, sociocultural and behavioral factors, genetics, and poverty itself also will have a profound impact on cancer-related mortality and morbidity rates. At least 2 million cancer cases per year—18% of the global cancer burden—are attributable to infectious causes, which are responsible for <10% of cancers in developed countries but account for up to 20% of all malignancies in lowand middle-income countries. Infectious causes of cancer such as human

1	causes, which are responsible for <10% of cancers in developed countries but account for up to 20% of all malignancies in lowand middle-income countries. Infectious causes of cancer such as human papillomavirus, hepatitis B virus, and Helicobacter pylori will continue to have a much larger impact in developing countries. Environmental and dietary factors, such as indoor air pollution and high-salt diets, also contribute to increased rates of certain cancers (e.g., lung and gastric cancers). Tobacco use (both smoking and chewing) is the most important source of increased mortality rates from lung and oral cancers. In contrast to decreasing tobacco use in many developed countries, the number of smokers is growing in developing countries, especially among women and young persons.

1	For many reasons, outcomes of malignancies are far worse in developing countries than in developed nations. As currently funded, overstretched health systems in poor countries are not capable of early detection; the majority of patients already have incurable malignancies at diagnosis. Treatment of cancers is available for only a very small number of mostly wealthy citizens in the majority of poor countries, and, even when treatment is available, the range and quality of services are often substandard. Yet this need not be the future. Only a decade ago, MDR-TB and HIV infection were considered untreatable in settings of great poverty. The feasibility of creating innovative programs that reduce technical and financial barriers to the provision of care for treatable malignancies among the world’s poorest populations is now clear (Fig. 2-4). Several middle-income countries, including Mexico, have expanded publicly funded cancer care to reach poorer populations. This commitment of

1	the world’s poorest populations is now clear (Fig. 2-4). Several middle-income countries, including Mexico, have expanded publicly funded cancer care to reach poorer populations. This commitment of resources has dramatically improved outcomes for cancers, from childhood leukemia to cervical cancer.

1	Prevention of Noncommunicable diseases False debates, including those pitting prevention against care, continue in global health and reflect, in part, outmoded paradigms or a partial understanding of disease burden and etiology as well as the dramatic variations in risk within a single nation. Moreover, debates are sometimes politicized as a result of vested interests. For example, in 2004, the WHO released its Global Strategy on Diet, Physical Activity, and Health, which focused on the population-wide promotion of healthy diet and regular physical activity in an effort to reduce the growing global problem of obesity. Passing this strategy at the World Health Assembly proved difficult because of strong opposition from the food industry and from a number of WHO member states, including the United States. Although globalization has had many positive effects, one negative effect has been the growth in both developed and developing countries of well-financed lobbies that have aggressively

1	United States. Although globalization has had many positive effects, one negative effect has been the growth in both developed and developing countries of well-financed lobbies that have aggressively promoted unhealthy dietary changes and increased consumption of alcohol and tobacco. Foreign direct investment in tobacco, beverage, and food products in developing countries reached $90.3 billion in 2010— a figure nearly 490 times greater than the $185 million spent during that year to address NCDs by bilateral funding agencies, the WHO, the World Bank, and all other sources of development assistance for health combined. Investment in curbing NCDs remains disproportionately low despite the WHO’s 2008–2013 Action Plan for the Global Strategy for the Prevention and Control of Noncommunicable Diseases.

1	FIgURE 2-4 An 11-year-old Rwandan patient with embryonal rhabdomyosarcoma before (left) and after (right) 48 weeks of chemotherapy plus surgery. Five years later, she is healthy with no evidence of disease.

1	The WHO estimates that 80% of all cases of cardiovascular disease and type 2 diabetes as well as 40% of all cancers can be prevented through healthier diets, increased physical activity, and avoidance of tobacco. These estimates mask large local variations. Although some evidence indicates that population-based measures can have some impact on these behaviors, it is sobering to note that increasing obesity levels have not been reversed in any population. Tobacco avoidance may be the most important and most difficult behavioral modification of all. In the twentieth century, 100 million people worldwide died of tobacco-related diseases; it is projected that more than 1 billion people will die of these diseases in the twenty-first century, with the vast majority of those deaths in developing countries. The WHO’s 2003 Framework Convention on Tobacco Control represented a major advance, committing all of its signatories to a set of policy measures shown to reduce tobacco consumption.

1	countries. The WHO’s 2003 Framework Convention on Tobacco Control represented a major advance, committing all of its signatories to a set of policy measures shown to reduce tobacco consumption. Today, ~80% of the world’s 1 billion smokers live in lowand middle-income countries. If trends continue, tobacco-related deaths will increase to 8 million per year by 2030, with 80% of those deaths in lowand middle-income countries.

1	The WHO reports that some 450 million people worldwide are affected by mental, neurologic, or behavioral problems at any given time and that ~877,000 people die by suicide every year. Major depression is the leading cause of years lost to disability in the world today. One in four patients visiting a health service has at least one mental, neurologic, or behavioral disorder, but most of these disorders are neither diagnosed nor treated. Most lowand middle-income countries devote <1% of their health expenditures to mental health.

1	Increasingly effective therapies exist for many of the major causes of mental disorders. Effective treatments for many neurologic diseases, including seizure disorders, have long been available. One of the greatest barriers to delivery of such therapies is the paucity of skilled personnel. Most sub-Saharan African countries have only a handful of psychiatrists, for example; most of them practice in cities and are unavailable within the public sector or to patients living in poverty.

1	Among the few patients who are fortunate enough to see a psychiatrist or neurologist, fewer still are able to adhere to treatment regimens: several surveys of already diagnosed patients ostensibly receiving daily therapy have revealed that, among the poor, multiple barriers prevent patients from taking their medications as prescribed. In one study from Kenya, no patients being seen in an epilepsy clinic had therapeutic blood levels of anti-seizure medications, even though all had had these drugs prescribed. Moreover, many patients had no detectable blood levels of these agents. The same barriers that prevent the poor from having reliable access to insulin or ART prevent them from benefiting from antidepressant, antipsychotic, and antiepileptic agents. To alleviate this problem, some authorities are proposing the training of health workers to provide community-based adherence support, counseling services, and referrals for patients in need of mental health services. One such program

1	are proposing the training of health workers to provide community-based adherence support, counseling services, and referrals for patients in need of mental health services. One such program instituted in Goa, India, used “lay” counselors and resulted in a significant reduction in symptoms of common mental disorders among the target population.

1	World Mental Health: Problems and Priorities in Low-Income Countries still offers a comprehensive analysis of the burden of mental, behavioral, and social problems in low-income countries and relates the mental health consequences of social forces such as violence, dislocation, poverty, and disenfranchisement of women to current economic, political, and environmental concerns. In the years since this report was published, however, a number of pilot projects designed to deliver community-based care to patients with chronic mental illness have been launched in settings as diverse as Goa, India; Banda Aceh, Indonesia; rural China; post-earthquake Haiti; and Fiji. Some of these programs have been school-based and have sought to link prevention to care. CoNCLUSIoN: ToWARd A SCIENCE oF IMPLEMENTATIoN

1	Public health strategies draw largely on quantitative methods— epidemiology, biostatistics, and economics. Clinical practice, including the practice of internal medicine, draws on a rapidly expanding knowledge base but remains focused on individual patient care; clinical interventions are rarely population-based. But global health equity depends on avoiding the false debates of the past: neither public health nor clinical approaches alone are adequate to address the problems of global health. There is a long way to go before evidence-based internal medicine is applied effectively among the world’s poor. Complex infectious diseases such as HIV/AIDS and TB have proved difficult but not impossible to manage; drug resistance and lack of effective health systems have complicated such work. Beyond what is usually termed “communicable diseases”—i.e., in the arena of chronic diseases such as cardiovascular disease and mental illness—global health is a nascent endeavor. Efforts to address any

1	what is usually termed “communicable diseases”—i.e., in the arena of chronic diseases such as cardiovascular disease and mental illness—global health is a nascent endeavor. Efforts to address any one of these problems in settings of great scarcity need to be integrated into broader efforts to strengthen failing health systems and alleviate the growing personnel crisis within these systems.

1	Such efforts must include the building of “platforms” for care delivery that are robust enough to incorporate new preventive, diagnostic, and therapeutic technologies rapidly in response to changes both in the burden of disease and in the needs not met by dominant paradigms and systems of health delivery. Academic medical centers have tried to address this “know–do” gap as new technologies are introduced and assessed through clinical trials, but the reach of these institutions into settings of poverty is limited in rich and poor countries alike. When such centers link their capacities effectively to the public institutions charged with the delivery of health care to the poor, great progress can be made.

1	For these reasons, scholarly work and practice in the field once known as “international health” and now often designated “global health equity” are changing rapidly. That work is still informed by the tension between clinical practice and population-based interventions, between analysis and action, and between prevention and care. Once metrics are refined, how might they inform efforts to lessen premature morbidity and mortality rates among the world’s poor? As in the nineteenth century, human rights perspectives have proved helpful in turning attention to the problems of the destitute sick; such perspectives may also inform strategies for delivering care equitably.

1	A number of university hospitals are developing training programs for physicians with an interest in global health. In medical schools across the United States and in other wealthy countries, interest in global health has exploded. One study has shown that more than 25% of medical students take part in at least one global health experience prior to graduation. Half a century or even a decade ago, such high levels of interest would have been unimaginable. An estimated 12 million people die each year simply because they live in poverty. An absolute majority of these premature deaths occur in Africa, with the poorer regions of Asia not far behind. Most of these deaths occur because the world’s poorest do not have access to the fruits of science. They include deaths from vaccine-preventable illness, deaths during childbirth, deaths from infectious diseases that might be

1	General Considerations in Clinical Medicine Preventing such a future is the most important goal of global health. decision-Making in Clinical Medicine Daniel B. Mark, John B. Wong INTRodUCTIoN To a medical student who requires hours to collect a patient’s history, 3 cured with access to antibiotics and other essential medicines, deaths from malaria that would have been prevented by bed nets and access to therapy, and deaths from waterborne illnesses. Other excess mortality is attributable to the inadequacy of efforts to develop new preventive, diagnostic, and therapeutic tools. Those funding the discovery and development of new tools typically neglect the concurrent need for strategies to make them available to the poor. Indeed, some would argue that the biggest challenge facing those who seek to address this outcome gap is the lack of practical means of distribution in the most heavily affected regions.

1	The development of tools must be followed quickly by their equitable distribution. When new preventive and therapeutic tools are developed without concurrent attention to delivery or implementation, one encounters what are sometimes termed perverse effects: even as new tools are developed, inequalities of outcome—lower morbidity and mortality rates among those who can afford access, with sustained high morbidity and mortality among those who cannot—will grow in the absence of an equity plan to deliver the tools to those most at risk.

1	perform a physical examination, and organize that information into a coherent presentation, an experienced clinician’s ability to decide on a diagnosis and management plan in minutes may seem extraordinary. What separates the master clinician from the novice is an elusive quality called “expertise.” The first part of this chapter provides an overview of our current understanding of expertise in clinical reasoning, what it is, and how it can be developed.

1	The proper use of diagnostic tests and the integration of the results into the patient’s clinical assessment may also be equally bewildering to students. Hoping to hit the unknown diagnostic target, novice medical practitioners typically apply a “shotgun” approach to testing. The expert, in contrast, usually focuses her testing strategy to specific diagnostic hypotheses. The second part of the chapter reviews basic statistical concepts useful for interpreting diagnostic tests and quantitative tools useful for clinical decision-making. Evidence-based medicine (EBM) constitutes the integration of the best available research evidence with clinical judgment as applied to the care of individual patients. The third part of the chapter provides an overview of the tools of EBM.

1	BRIEF INTRodUCTIoN To CLINICAL REASoNINg Clinical Expertise Defining “clinical expertise” remains surprisingly difficult. Chess has an objective ranking system based on skill and performance criteria. Athletics, similarly, have ranking systems to distinguish novices from Olympians. But in medicine, after physicians complete training and pass the boards, no further tests or benchmarks identify those who have attained the highest levels of clinical performance. Of course, physicians often consult a few “elite” clinicians for their “special problem-solving prowess” when particularly difficult or obscure cases have baffled everyone else. Yet despite their skill, even master clinicians typically cannot explain their exact processes and methods, thereby limiting the acquisition and dissemination of the expertise used to achieve their impressive results. Furthermore, clinical virtuosity appears not to be generalizable, e.g., an expert on hypertrophic cardiomyopathy may be no better (and

1	of the expertise used to achieve their impressive results. Furthermore, clinical virtuosity appears not to be generalizable, e.g., an expert on hypertrophic cardiomyopathy may be no better (and possibly worse) than a first-year medical resident at diagnosing and managing a patient with neutropenia, fever, and hypotension.

1	Broadly construed, clinical expertise includes not only cognitive dimensions and the integration of verbal and visual cues or information but also complex fine-motor skills necessary for invasive and noninvasive procedures and tests. In addition, “the complete package” of expertise in medicine includes the ability to communicate effectively with patients and work well with members of the medical team. Research on medical expertise remains relatively sparse overall, with most of the work focused on diagnostic reasoning, and much less work focused on treatment decisions or the technical skills involved in the performance of procedures. Thus, in this chapter, we focus primarily on the cognitive elements of clinical reasoning.

1	Because clinical reasoning takes place in the heads of doctors, it is therefore not readily observable, making it obviously difficult to study. One method of research on reasoning asks doctors to “think out loud” as they receive increments of clinical information in a manner meant to simulate a clinical encounter. Another research approach has focused on how doctors should reason diagnostically rather than on how they actually do reason. Much of what is known about clinical reasoning comes from empirical studies of nonmedical problem-solving behavior. Because of the diverse perspectives contributing to this area, with important contributions from cognitive psychology, sociology, medical education, economics, informatics, and decision sciences, no single integrated model of clinical reasoning exists, and not infrequently, different terms and models describe similar phenomena.

1	Intuitive versus Analytic Reasoning A contemporary model of reasoning, dual-process theory distinguishes two general systems of cognitive processes. Intuition (System 1) provides rapid effortless judgments from memorized associations using pattern recognition and other simplifying “rules of thumb” (i.e., heuristics). For example, a very simple pattern that could be useful in certain situations is “African-American women plus hilar adenopathy equals sarcoid.” Because no effort is involved in recalling the pattern, typically, the clinician is unable to say how those judgments were formulated. In contrast, analysis (System 2), the other form of reasoning in the dual-process model, is slow, methodical, deliberative, and effortful. These are, of course, idealized extremes of the cognitive continuum. How these systems interact in different decision problems, how experts use them differently from novices, and when their use can lead to errors in judgment remain the subject of considerable

1	How these systems interact in different decision problems, how experts use them differently from novices, and when their use can lead to errors in judgment remain the subject of considerable study and debate.

1	Pattern recognition is a complex cognitive process that appears largely effortless. One can recognize people’s faces, the breed of a dog, or an automobile model without necessarily being able to say what specific features prompted the recognition. Analogously, experienced clinicians often recognize familiar diagnosis patterns quickly. In the absence of an extensive stored repertoire of diagnostic patterns, students (as well as more experienced clinicians operating outside their area of expertise) often use the more laborious System 2 analytic approach along with more intensive and comprehensive data collection to reach the diagnosis. The following three brief scenarios of a patient with hemoptysis demonstrate three distinct patterns:

1	The following three brief scenarios of a patient with hemoptysis demonstrate three distinct patterns: A 46-year-old man presents to his internist with a chief complaint of hemoptysis. An otherwise healthy nonsmoker, he is recovering from an apparent viral bronchitis. This presentation pattern suggests that the small amount of blood-streaked sputum is due to acute bronchitis, so that a chest x-ray provides sufficient reassurance that a more serious disorder is absent. In the second scenario, a 46-year-old patient who has the same chief complaint but with a 100-pack-year smoking history, a productive morning cough, and episodes of blood-streaked sputum fits the pattern of carcinoma of the lung. Consequently, along with the chest x-ray, the physician obtains a sputum cytology examination and refers this patient for a chest computed tomography (CT) scan.

1	In the third scenario, a 46-year-old patient with hemoptysis who immigrated from a developing country has an echocardiogram as well, because the physician hears a soft diastolic rumbling murmur at the apex on cardiac auscultation, suggesting rheumatic mitral stenosis and possibly pulmonary hypertension.

1	Although rapid, pattern recognition used without sufficient reflection can result in premature closure: mistakenly concluding that one already knows the correct diagnosis and therefore failing to complete the data collection that would demonstrate the lack of fit of the initial pattern selected. For example, a 45-year-old man presents with a 3-week history of a “flulike” upper respiratory infection (URI) including symptoms of dyspnea and a productive cough. On the basis of the presenting complaints, the clinician uses a “URI assessment form” to improve the quality and efficiency of care by standardizing the information gathered. After quickly acquiring the requisite structured examination components and noting in particular the absence of fever and a clear chest examination, the physician prescribes medication for acute bronchitis and sends the patient home with the reassurance that his illness was not serious. Following a sleepless night with significant dyspnea, the patient develops

1	prescribes medication for acute bronchitis and sends the patient home with the reassurance that his illness was not serious. Following a sleepless night with significant dyspnea, the patient develops nausea and vomiting and collapses. He presents to the emergency department in cardiac arrest and is unable to be resuscitated. His autopsy shows a posterior wall myocardial infarction and a fresh thrombus in an atherosclerotic right coronary artery. What went wrong? The clinician had decided, based on the patient’s appearance, even before starting the history, that the patient’s complaints were not serious. Therefore, he felt confident that he could perform an abbreviated and focused examination by using the URI assessment protocol rather than considering the broader range of possibilities and performing appropriate tests to confirm or refute his initial hypotheses. In particular, by concentrating on the URI, the clinician failed to elicit the full dyspnea history, which would have

1	and performing appropriate tests to confirm or refute his initial hypotheses. In particular, by concentrating on the URI, the clinician failed to elicit the full dyspnea history, which would have suggested a far more serious disorder, and he neglected to search for other symptoms that could have directed him to the correct diagnosis.

1	Heuristics, also referred to as cognitive shortcuts or rules of thumb, are simplifying decision strategies that ignore part of the data available so as to provide an efficient path to the desired judgment. They are generally part of the intuitive system tools. Two major research programs have come to different conclusions about the value of heuristics in clinical judgment. The “heuristics and biases” program focused on understanding how heuristics in problem solving could be biased by testing the numerical intuition of psychology undergraduates against the rules of statistics. In contrast, the “fast and frugal heuristics” research program explored how and when decision makers’ reliance on simple heuristics can produce good decisions. Although many heuristics have relevance to clinical reasoning, only four will be mentioned here.

1	When assessing a particular patient, clinicians often weigh the similarity of that patient’s symptoms, signs, and risk factors against those of their mental representations of the diagnostic hypotheses being considered. In other words, among the diagnostic possibilities, clinicians identify the diagnosis for which the patient appears to be a representative example. Analogous to pattern recognition, this cognitive shortcut is called the representativeness heuristic. However, physicians using the representativeness heuristic can reach erroneous conclusions if they fail to consider the underlying prevalence (i.e., the prior, or pretest, probabilities) of the two competing diagnoses that could explain the patient’s symptoms. Consider a patient with hypertension and headache, palpitations, and diaphoresis. Inexperienced clinicians might judge pheochromocytoma to be quite likely based on the representativeness heuristic with this classic symptom triad suggesting pheochromocytoma. Doing so

1	diaphoresis. Inexperienced clinicians might judge pheochromocytoma to be quite likely based on the representativeness heuristic with this classic symptom triad suggesting pheochromocytoma. Doing so would be incorrect given that other causes of hypertension are much more common than pheochromocytoma, and this triad of symptoms can occur in patients who do not have pheochromocytoma. Less experience with a particular diagnosis and with the breadth of presentations (e.g., diseases that affect multiple organ systems such as sarcoid) may also lead to errors.

1	A second commonly used cognitive shortcut, the availability heuristic, involves judgments based of how easily prior similar cases or outcomes can be brought to mind. For example, an experienced clinician may recall 20 elderly patients seen over the last few years who presented with painless dyspnea of acute onset and were found to have acute myocardial infarction (MI). A novice clinician may spend valuable time seeking a pulmonary cause for the symptoms before considering and then confirming the cardiac diagnosis. In this situation, the patient’s clinical pattern does not fit the most common pattern of acute MI, but experience with this atypical presentation, along with the ability to recall it, directs the physician to the diagnosis.

1	Errors with the availability heuristic arise from several sources of recall bias. Rare catastrophes are likely to be remembered with a clarity and force disproportionate to their likelihood for future diagnosis— for example, a patient with a sore throat eventually found to have leukemia or a young athlete with leg pain eventually found to have a sarcoma—and those publicized in the media or that are recent experiences are, of course, easier to recall and therefore more influential on clinical judgments.

1	The third commonly used cognitive shortcut, the anchoring heuristic (also called conservatism or stickiness), involves estimating a probability of disease (the anchor) and then insufficiently adjusting that probability up or down (compared with Bayes’ rule) when interpreting new data about the patient, i.e., sticking to their initial diagnosis. For example, a clinician may still judge the probability of coronary artery disease (CAD) to be high after a negative exercise thallium test and proceed to cardiac catheterization (see “Measures of Disease Probability and Bayes’ Rule,” below).

1	The fourth heuristic states that clinicians should use the simplest explanation possible that will account adequately for the patient’s symptoms or findings (Occam’s razor or, alternatively, the simplicity heuristic). Although this is an attractive and often used principle, it is important to remember that no biologic basis for it exists. Errors from the simplicity heuristic include premature closure leading to the neglect of unexplained significant symptoms or findings.

1	Even experienced physicians use analytic reasoning processes (System 2) when the problem they face is recognized to be complex or to involve important unfamiliar elements or features. In such situations, clinicians proceed much more methodically in what has been referred to as the hypothetico-deductive model of reasoning. From the outset, expert clinicians working analytically generate, refine, and discard diagnostic hypotheses. The hypotheses drive questions asked during history taking and may change based on the working hypotheses of the moment. Even the physical examination is focused by the working hypotheses. Is the spleen enlarged? How big is the liver? Is it tender? Are there any palpable masses or nodules? Each question must be answered (with the exclusion of all other inputs) before the examiner can move on to the next specific question. Each diagnostic hypothesis provides testable predictions and sets a context for the next question or step to follow. For example, if the

1	before the examiner can move on to the next specific question. Each diagnostic hypothesis provides testable predictions and sets a context for the next question or step to follow. For example, if the enlarged and quite tender liver felt on physical examination is due to acute hepatitis (the hypothesis), certain specific liver function tests should be markedly elevated (the prediction). If the tests come back normal, the hypothesis may have to be discarded or substantially modified.

1	Negative findings often are neglected but are as important as positive ones because they often reduce the likelihood of the diagnostic hypotheses under consideration. Chest discomfort that is not provoked or worsened by exertion in an active patient reduces the likelihood that chronic ischemic heart disease is the underlying cause. The absence of a resting tachycardia and thyroid gland enlargement reduces the likelihood of hyperthyroidism in a patient with paroxysmal atrial fibrillation.

1	The acuity of a patient’s illness may override considerations of prevalence and the other issues described above. “Diagnostic imperatives” recognize the significance of relatively rare but potentially catastrophic diagnoses if undiagnosed and untreated. For example, clinicians are taught to consider aortic dissection routinely as a possible cause of acute severe chest discomfort. Even though the typical history of dissection differs from that of MI, dissection is far less prevalent, so diagnosing dissection remains challenging unless it is explicitly and routinely considered as a diagnostic imperative (Chap. 301). If the clinician fails to elicit any of the characteristic features of dissection by history and finds equivalent blood pressures in both arms and no pulse deficits, he may feel comfortable discarding the aortic dissection hypothesis. If, however, the chest x-ray shows a possible widened mediastinum, the hypothesis may be reinstated and an appropriate imaging test ordered

1	comfortable discarding the aortic dissection hypothesis. If, however, the chest x-ray shows a possible widened mediastinum, the hypothesis may be reinstated and an appropriate imaging test ordered (e.g., thoracic CT scan, transesophageal echocardiogram) to evaluate more fully. In nonacute situations, the prevalence of potential alternative diagnoses should play a much more prominent role in diagnostic hypothesis generation.

1	Cognitive scientists studying the thought processes of expert clinicians have observed that clinicians group data into packets, or “chunks,” that are stored in short-term or “working memory” and manipulated to generate diagnostic hypotheses. Because short-term memory can typically retain only 5–9 items at a time, the number of packets that can be actively integrated into hypothesis-generating activities is similarly limited. For this reason, the cognitive shortcuts discussed above play a key role in the generation of diagnostic hypotheses, many of which are discarded as rapidly as they are formed (thereby demonstrating that the distinction between analytic and intuitive reasoning is an arbitrary and simplistic, but nonetheless useful, representation of cognition).

1	Research into the hypothetico-deductive model of reasoning has had surprising difficulty identifying the elements of the reasoning process that distinguish experts from novices. This has led to a shift from examining the problem-solving process of experts to analyzing the organization of their knowledge. For example, diagnosis may be based on the resemblance of a new case to prior individual instances (exemplars). Experts have a much larger store of memorized cases, for example, visual long-term memory in radiology. However, clinicians do not simply rely on literal recall of specific cases but have constructed elaborate conceptual networks of memorized information or models of disease to aid in arriving at their conclusions. That is, expertise involves an increased ability to connect symptoms, signs, and risk factors to one another in meaningful ways; relate those findings to possible diagnoses; and identify the additional information necessary to confirm the diagnosis.

1	No single theory accounts for all the key features of expertise in medical diagnosis. Experts have more knowledge about more things and a larger repertoire of cognitive tools to employ in problem solving than do novices. One definition of expertise highlights the ability to make powerful distinctions. In this sense, expertise involves a working knowledge of the diagnostic possibilities and what features distinguish one disease from another. Memorization alone is insufficient. Memorizing a medical textbook would not make one an expert. But having access to detailed and specific relevant information is critically important. Clinicians of the past primarily accessed their own remembered experience. Clinicians of the future will be able to access the experience of large numbers of clinicians using electronic tools, but, as with the memorized textbook, the data alone will not create an instant expert. The expert adds these data to an extensive internalized database of knowledge and

1	using electronic tools, but, as with the memorized textbook, the data alone will not create an instant expert. The expert adds these data to an extensive internalized database of knowledge and experience not available to the novice (and nonexpert).

1	Despite all the work that has been done to understand expertise, in medicine and other disciplines, it remains uncertain whether there is any didactic program that can accelerate the progression from novice to expert or from experienced clinician to master clinician. Deliberate effortful practice (over an extended period of time, sometimes said to be 10 years or 10,000 practice hours) and personal coaching are two strategies that are often used outside medicine (e.g., music, athletics, chess) to promote expertise. Their use in developing medical expertise and maintaining or enhancing it has not yet been adequately explored.

1	The modern ideal of medical therapeutic decision making is to “personalize” the recommendation. In the abstract, personalizing treatment involves combining the best available evidence about what works with an individual patient’s unique features (e.g., risk factors) and his or her preferences and health goals to craft an optimal treatment recommendation with the patient. Operationally, there are two different and complementary levels of personalization possible: individualizing the evidence for the specific patient based on relevant clinical and other characteristics, and personalizing the patient interaction by incorporating their values, often referred to as shared decision-making, which is critically important, but falls outside the scope of this chapter.

1	Individualizing the evidence about therapy does not mean relying on physician impressions of what works based on personal experience. Because of small sample sizes and rare events, the chance of drawing erroneous causal inferences from one’s own clinical experience is very high. For most chronic diseases, therapeutic effectiveness is only demonstrable statistically in patient populations. It would be incorrect to infer with any certainty, for example, that treating a hypertensive patient with angiotensin-converting enzyme (ACE) inhibitors necessarily prevented a stroke from occurring during treatment, or that an untreated patient would definitely have avoided a stroke had he or she been treated. For many chronic diseases, a majority of patients will remain event free regardless of treatment choices; some will have events regardless of which treatment is selected; and those who avoided having an event through treatment cannot be individually identified. Blood pressure lowering, a

1	choices; some will have events regardless of which treatment is selected; and those who avoided having an event through treatment cannot be individually identified. Blood pressure lowering, a readily observable surrogate endpoint, does not have a tightly coupled relationship with strokes prevented. Consequently, demonstrating therapeutic effectiveness cannot rely simply on observing the outcome of an individual patient but should instead be based on large groups of patients carefully studied and properly analyzed.

1	Therapeutic decision making, therefore, should be based on the best available evidence from clinical trials and well-done outcome studies. Authoritative, well-done clinical practice guidelines that synthesize such evidence offer readily available, reliable, and trustworthy information relevant to many treatment decisions clinicians face. However, all guidelines recognize that their “one size fits all” recommendations may not apply to individual patients. Increased attention is now being paid to understand how best to adjust group-level clinical evidence of treatment harms and benefits to account for the absolute level of risks faced by subgroups and even individual patients, using, for example, validated clinical risk scores.

1	More than a decade of research on variations in clinician practice patterns has shed much light on the forces that shape clinical decisions. These factors can be grouped conceptually into three overlapping categories: (1) factors related to physicians’ personal characteristics and practice style, (2) factors related to the practice setting, and (3) factors related to economic incentives.

1	Factors Related to Practice Style To ensure that necessary care is provided at a high level of quality, physicians fulfill a key role in medical care by serving as the patient’s agent. Factors that influence performance in this role include the physician’s knowledge, training, and experience. Clearly, physicians cannot practice EBM (described later in the chapter) if they are unfamiliar with the evidence. As would be expected, specialists generally know the evidence in their field better than do generalists. Beyond published evidence and practice guidelines, a major set of influences on physician practice can be subsumed under the general concept of “practice style.” The practice style serves to define norms of clinical behavior. Beliefs about effectiveness of different therapies and preferred patterns of diagnostic test use are examples of different facets of a practice style. The physician beliefs that drive these different practice styles may be based on personal experience,

1	preferred patterns of diagnostic test use are examples of different facets of a practice style. The physician beliefs that drive these different practice styles may be based on personal experience, recollection, and interpretation of the available medical evidence. For example, heart failure specialists are much more likely than generalists to achieve target doses of ACE inhibitor therapy in their heart failure patients because they are more familiar with what the targets are (as defined by large clinical trials), have more familiarity with the specific drugs (including adverse effects), and are less likely to overreact to foreseeable problems in therapy such as a rise in creatinine levels or asymptomatic hypotension.

1	Beyond the patient’s welfare, physician perceptions about the risk of a malpractice suit resulting from either an erroneous decision or a bad outcome may drive clinical decisions and create a practice referred to as defensive medicine. This practice involves using tests and therapies with very small marginal benefit, ostensibly to preclude future criticism should an adverse outcome occur. Without any conscious awareness of a connection to the risk of litigation, however, over time such patterns of care may become accepted as part of the practice norm, thereby perpetuating their overuse, e.g., annual cardiac exercise testing in asymptomatic patients.

1	Practice Setting Factors Factors in this category relate to the physical resources available to the physician’s practice and the practice environment. Physician-induced demand is a term that refers to the repeated observation that once medical facilities and technologies are made available to physicians, they will use them. Other environmental factors that can influence decision-making include the local availability of specialists for consultations and procedures; “high-tech” advanced imaging or procedure facilities such as MRI machines and proton beam therapy centers; and fragmentation of care.

1	Economic Incentives Economic incentives are closely related to the other two categories of practice-modifying factors. Financial issues can exert both stimulatory and inhibitory influences on clinical practice. In general, physicians are paid on a fee-for-service, capitation, or salary basis. In fee-for-service, physicians who do more get paid more, thereby encouraging overuse, consciously or unconsciously. When fees are reduced (discounted reimbursement), doctors tend to increase the number of services provided to maintain revenue. Capitation, in contrast, provides a fixed payment per patient per year to encourage physicians to consider a global population budget in managing individual patients and ideally reducing the use of interventions with small marginal benefit. In contrast to inexpensive preventive services, however, this type of incentive is more likely to affect expensive interventions. To discourage volume-based excessive utilization, fixed salary compensation plans pay

1	preventive services, however, this type of incentive is more likely to affect expensive interventions. To discourage volume-based excessive utilization, fixed salary compensation plans pay physicians the same regardless of the clinical effort expended, but may provide an incentive to see fewer patients.

1	Despite the great technological advances in medicine over the last century, uncertainty remains a key challenge in all aspects of medical decision-making. Compounding this challenge is the massive information overload that characterizes modern medicine. Today’s clinician needs access to close to 2 million pieces of information to practice medicine. According to one estimate, doctors subscribe to an average of seven journals, representing over 2500 new articles each year. Of course, to be useful, this information must be sifted for applicability to and then integrated with patient-specific data. Although computers appear to offer an obvious solution both for information management and for quantification of medical care uncertainties, many practical problems must be solved before computerized decision support can be routinely incorporated into the clinical reasoning process in a way that demonstrably improves the quality of care. For the present, understanding the nature of diagnostic

1	decision support can be routinely incorporated into the clinical reasoning process in a way that demonstrably improves the quality of care. For the present, understanding the nature of diagnostic test information can help clinicians become more efficient users of such data. The next section reviews important concepts related to diagnostic testing.

1	dIAgNoSTIC TESTINg: MEASURES oF TEST ACCURACY The purpose of performing a test on a patient is to reduce uncertainty about the patient’s diagnosis or prognosis in order to facilitate optimal management. Although diagnostic tests commonly are thought of as laboratory tests (e.g., blood count) or procedures (e.g., colonoscopy or bronchoscopy), any technology that changes a physician’s understanding of the patient’s problem qualifies as a diagnostic test. Thus, even the history and physical examination can be considered a form of diagnostic test. In clinical medicine, it is common to reduce the results of a test to a dichotomous outcome, such as positive or negative, normal or abnormal. Although this simplification ignores useful information (such as the degree of abnormality), such simplification does make it easier to demonstrate the fundamental principles of test interpretation discussed below.

1	The accuracy of diagnostic tests is defined in relation to an accepted “gold standard,” which defines the presumably true state of the patient (Table 3-1). Characterizing the diagnostic performance of a new test requires identifying an appropriate population (ideally, patients in whom the new test would be used) and applying both the new and the gold standard tests to all subjects. Biased estimates of test performance may occur from using an inappropriate population or from incompletely applying the gold standard test. By comparing the two tests, the characteristics of the new test are determined. The sensitivity or true-positive rate of the new test is the proportion of patients with disease (defined by the gold standard) who have a positive (new) test. This measure reflects how well the new test identifies patients with disease. The proportion of patients with disease who have a negative test is the false-negative rate and is calculated as 1 – sensitivity. Among patients without

1	the new test identifies patients with disease. The proportion of patients with disease who have a negative test is the false-negative rate and is calculated as 1 – sensitivity. Among patients without disease, the proportion who have a negative test is the specificity, or true-negative rate. This measure reflects how well the new test correctly identifies patients without disease. Among patients without disease, the proportion who have a positive test is the false-positive rate, calculated as 1 – specificity. A perfect test would have a sensitivity of 100% and a specificity of 100% and would completely distinguish patients with disease from those without it.

1	Calculating sensitivity and specificity requires selection of a threshold value or cut point above which the test is considered “positive.” Making the cut point “stricter” (e.g., raising it) lowers sensitivity but improves specificity, whereas making it “laxer” (e.g., lowering it) raises sensitivity but lowers specificity. This dynamic trade-off between more accurate identification of subjects with disease versus those without disease is often displayed graphically as a receiver operating characteristic (ROC) curve (Fig. 3-1) by plotting sensitivity (y axis) versus 1 – specificity (x axis). Each point on the curve represents a potential cut point with an associated sensitivity and specificity value. The area under the ROC curve often is used as a quantitative measure of the information content of a test. Values range from 0.5 (no diagnostic information from testing at all; the test is equivalent to flipping a coin) to 1.0 (perfect test). The choice of cut point should depend on the

1	content of a test. Values range from 0.5 (no diagnostic information from testing at all; the test is equivalent to flipping a coin) to 1.0 (perfect test). The choice of cut point should depend on the relative harms and benefits of treatment for those without versus those with disease. For example, if treatment was safe with substantial benefit, then choosing a high-sensitivity cut point (upper right of the ROC curve) for a low-risk test may be appropriate (e.g., phenylketonuria in newborns), but if treatment had substantial risk for harm, then choosing a high-specificity cut point (lower left of the ROC curve) may be appropriate (e.g., amniocentesis that may lead to therapeutic abortion of a normal fetus). The choice of cut point may also depend on the likelihood of disease, with low likelihoods placing a greater emphasis on the harms of treating false-positive tests and higher likelihoods placing a greater emphasis on missed benefit by not treating false-negative tests.

1	Unfortunately, there are no perfect tests. After every test is completed, the true disease state of the patient remains uncertain. Quantifying this residual uncertainty can be done with Bayes’ rule, which provides a simple way to calculate the likelihood of disease after a test result or posttest probability from three parameters: the pretest probability of disease, the test sensitivity, and the test specificity. The pretest probability is a quantitative estimate of the likelihood of the diagnosis before the test is performed and is usually the prevalence of the disease in the underlying population although occasionally it can be the disease 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 illustrates a trade-off that occurs between improved test sensitivity (accurate detection of patients with disease) and improved test specificity (accurate detection of patients without disease), because the test value defining when the test turns from “negative” to

1	(accurate detection of patients with disease) and improved test specificity (accurate detection of patients without disease), because the test value defining when the test turns from “negative” to “positive” is varied. A 45° line would indicate a test with no predictive value (sensitivity = specificity at every test value). The area under each ROC curve is a measure of the information content of the test. Thus, a larger ROC area signifies increased diagnostic accuracy.

1	incidence. For some common conditions, such as CAD, nomograms and statistical models generate estimates of pretest probability that account for history, physical examination, and test findings. The posttest probability (also called the predictive value of the test) is a revised statement of the likelihood of the diagnosis, accounting for both pretest probability and test results. For the likelihood of disease following a positive test (i.e., positive predictive value), Bayes’ rule is calculated as: For example, with a pretest probability of 0.50 and a “positive” diagnostic test result (test sensitivity = 0.90 and specificity = 0.90): 0.50× 0.90 Posttest probability = 0.50× 0.90+ (1− 0.50)0.10 × = 0.90

1	Posttest probability = 0.50× 0.90+ (1− 0.50)0.10 × = 0.90 The term predictive value often is used as a synonym for the posttest probability. Unfortunately, clinicians commonly misinterpret reported predictive values as intrinsic measures of test accuracy. Studies of diagnostic tests compound the confusion by calculating predictive values on the same sample used to measure sensitivity and specificity. Since all posttest probabilities are a function of the prevalence of disease in the tested population, such calculations may be misleading unless the test is applied subsequently to populations with the same disease prevalence. For these reasons, the term predictive value is best avoided in favor of the more informative posttest probability following a positive or a negative test result.

1	The nomogram version of Bayes’ rule (Fig. 3-2) helps us to conceptually understand how it estimates the posttest probability of disease. In this nomogram, the impact of the diagnostic test result is summarized by the likelihood ratio, which is defined as the ratio of the probability of a given test result (e.g., “positive” or “negative”) in a patient with disease to the probability of that result in a patient without disease, thereby providing a measure of how well the test distinguishes those with from those without disease.

1	For a positive test, the likelihood ratio positive is calculated as the ratio of the true-positive rate to the false-positive rate (or sensitivity/ [1 – specificity]). For example, a test with a sensitivity of 0.90 and a specificity of 0.90 has a likelihood ratio of 0.90/(1 – 0.90), or 9. Thus, for this hypothetical test, a “positive” result is nine times more likely in a patient with the disease than in a patient without it. Most tests in medicine have likelihood ratios for a positive result between 1.5 and 20. Higher values are associated with tests that more substantially increase the posttest likelihood of disease. A very high likelihood ratio positive (exceeding 10) usually implies high specificity, so a positive high-specificity test helps “rule in” disease. If sensitivity is excellent but specificity is less so, the likelihood ratio will be reduced substantially (e.g., with a 90% sensitivity but a 55% specificity, the likelihood ratio is 2.0).

1	For a negative test, the corresponding likelihood ratio negative is the ratio of the false-negative rate to the true-negative rate (or [1 – sensitivity]/ specificity). Lower likelihood ratio values more substantially lower the posttest likelihood of disease. A very low likelihood ratio negative (falling below 0.10) usually implies high sensitivity, so a negative high-sensitivity test helps “rule out” disease. The hypothetical test considered above with a sensitivity of 0.9 and a specificity of 0.9 would have a likelihood ratio for a negative test result of (1 – 0.9)/0.9, or 0.11, meaning that a negative result is about one-tenth as likely in patients with disease than in those without disease (or 10 times more likely in those without disease than in those with disease).

1	Consider two tests commonly used in the diagnosis of CAD: an exercise treadmill test and an exercise single-photon emission CT (SPECT) myocardial perfusion imaging test (Chap. 270e). Meta-analysis has shown that a positive treadmill ST-segment response has an average sensitivity of 66% and an average specificity of 84%, yielding a likelihood ratio of 4.1 (0.66/[1 – 0.84]) (consistent with small discriminatory ability because it falls between 2 and 5). For a patient with a 10% pretest probability of CAD, the posttest probability of disease after a positive result rises to only about 30%. If a patient with a pretest probability of CAD of 80% has a positive test result, the posttest probability of disease is about 95%.

1	In contrast, exercise SPECT myocardial perfusion test is more accurate for CAD. For simplicity, assume that the finding of a reversible exercise-induced perfusion defect has both a sensitivity and a specificity of 90%, yielding a likelihood ratio for a positive test of 9.0 (0.90/ [1 – 0.90]) (consistent with moderate discriminatory ability because it falls between 5 and 10). For the same 10% pretest probability patient, a positive test raises the probability of CAD to 50% (Fig. 3-2). However, despite the differences in posttest probabilities between these two tests (30% versus 50%), the more accurate test may not improve diagnostic likelihood enough to change patient management (e.g., decision to refer to cardiac catheterization) because the more accurate test has only moved the physician from being fairly certain that the patient did not have CAD to a 50:50 chance of disease. In a patient with a pretest probability of 80%, exercise SPECT test raises the posttest probability to 97%

1	from being fairly certain that the patient did not have CAD to a 50:50 chance of disease. In a patient with a pretest probability of 80%, exercise SPECT test raises the posttest probability to 97% (compared with 95% for the exercise treadmill). Again, the more accurate test does not provide enough improvement in posttest confidence to alter management, and neither test has improved much on what was known from clinical data alone.

1	In general, positive results with an accurate test (e.g., likelihood ratio positive 10) when the pretest probability is low (e.g., 20%) do not move the posttest probability to a range high enough to rule in disease (e.g., 80%). In screening situations, pretest probabilities are often particularly low because patients are asymptomatic. In such cases, specificity becomes particularly important. For example, in screening first-time female blood donors without risk factors for HIV, a positive test raised the likelihood of HIV to only 67% despite a specificity of 99.995% because the prevalence was 0.01%. Conversely, with a high pretest 0.1 99 0.1 0.5 98 0.2 0.5 95 0.5 0.5 0.5 40 0.2 0.2 0.1 0.05 0.1 0.05 5 0.02 0.02 70 0.01 0.01 0.5 95 0.5 0.2 98 0.2 0.1 99 0.1 Pretest Likelihood Posttest Pretest Likelihood Posttest Probability, % Ratio Probability, % Probability, % Ratio Probability, %

1	FIgURE 3-2 Nomogram version of Bayes’ rule used to predict the posttest probability of disease (right-hand scale) using the pretest probability of disease (left-hand scale) and the likelihood ratio for a positive test (middle scale). See text for information on calculation of likelihood ratios. To use, place a straight edge connecting the pretest probability and the likelihood ratio and read off the posttest probability. The right-hand part of the figure illustrates the value of a positive exercise treadmill test (likelihood ratio 4, green line) and a positive exercise thallium single-photon emission computed tomography perfusion study (likelihood ratio 9, broken yellow line) in a patient with a pretest probability of coronary artery disease of 50%. (Adapted from Centre for Evidence-Based Medicine: Likelihood ratios. Available at http://www.cebm.net/ index.aspx?o=1043.) probability, a negative test may not rule out disease adequately if it is not sufficiently sensitive. Thus, the

1	Medicine: Likelihood ratios. Available at http://www.cebm.net/ index.aspx?o=1043.) probability, a negative test may not rule out disease adequately if it is not sufficiently sensitive. Thus, the largest change in diagnostic likelihood following a test result occurs when the clinician is most uncertain (i.e., pretest probability between 30% and 70%). For example, if a patient has a pretest probability for CAD of 50%, a positive exercise treadmill test will move the posttest probability to 80% and a positive exercise SPECT perfusion test will move it to 90% (Fig. 3-2).

1	As presented above, Bayes’ rule employs a number of important simplifications that should be considered. First, few tests have only positive or negative results, and many tests provide multiple outcomes (e.g., ST-segment depression and exercise duration with exercise testing). Although Bayes’ rule can be adapted to this more detailed test result format, it is computationally more complex to do so. Similarly, when multiple tests are performed, the posttest probability may be used as the pretest probability to interpret the second test. However, this simplification assumes conditional independence—that is, that the results of the first test do not affect the likelihood of the second test result—and this is often not true.

1	Finally, it has long been asserted that sensitivity and specificity are prevalence-independent parameters of test accuracy, and many texts still make this statement. This statistically useful assumption, however, is clinically simplistic. A treadmill exercise test, for example, has a sensitivity in a population of patients with one-vessel CAD of around 30%, whereas its sensitivity in patients with severe three-vessel CAD approaches 80%. Thus, the best estimate of sensitivity to use in a particular decision may vary, depending on the severity of disease in the local population. A hospitalized, symptomatic, or referral population typically has a higher prevalence of disease and, in particular, a higher prevalence of more advanced disease than does an outpatient population. Consequently, test sensitivity will likely be higher in hospitalized patients, and test specificity higher in outpatients.

1	Bayes’ rule, while illustrative as presented above, provides an unrealistically simple solution to most problems a clinician faces. Predictions based on multivariable statistical models, however, can more accurately address these more complex problems by accounting for specific patient characteristics. In particular, these models explicitly account for multiple possibly overlapping pieces of patient-specific information and assign a relative weight to each on the basis of its unique contribution to the prediction in question. For example, a logistic regression model to predict the probability of CAD considers all the relevant independent factors from the clinical examination and diagnostic testing and their significance instead of the limited data that clinicians can manage in their heads or with Bayes’ rule. However, despite this strength, prediction models are usually too complex computationally to use without a calculator or computer (although this limitation may be overcome once

1	or with Bayes’ rule. However, despite this strength, prediction models are usually too complex computationally to use without a calculator or computer (although this limitation may be overcome once medicine is practiced from a fully computerized platform).

1	To date, only a handful of prediction models have been validated properly (for example, Wells criteria for pulmonary embolism) (Table 3-2). The importance of independent validation in a population separate from the one used to develop the model cannot be overstated. An unvalidated prediction model should be viewed with the skepticism appropriate for any new drug or medical device that has not had rigorous clinical trial testing. When statistical models have been compared directly with expert clinicians, they have been found to be more consistent, as would be expected, but not significantly more accurate. Their biggest promise, then, may be in helping less-experienced clinicians identify critical discriminating patient characteristics and become more accurate in their predictions.

1	Over the last 40 years, many attempts have been made to develop computer systems to aid clinical decision-making and patient management. Conceptually attractive because computers offer ready access to the vast information available to today’s physicians, they may also support management decisions by making accurate predictions of outcome, simulating the whole decision process, or providing algorithmic guidance. Computer-based predictions using Bayesian or statistical regression models inform a clinical decision but do not actually reach a “conclusion” or “recommendation.” Artificial intelligence systems attempt to simulate or replace human reasoning with a computer-based analogue. To date, such approaches have achieved only limited success. Reminder or protocol-directed systems do not make predictions but use existing algorithms, such as guidelines, to guide clinical practice. In general, however, decision support systems have had little impact on practice. Reminder systems, although

1	predictions but use existing algorithms, such as guidelines, to guide clinical practice. In general, however, decision support systems have had little impact on practice. Reminder systems, although not yet in widespread use, have shown the most promise, particularly in correcting drug dosing and promoting adherence to guidelines. Checklists, as used by pilots for example, have garnered recent support as an approach to avoid or reduce errors.

1	Compared with the decision support methods discussed above, decision analysis represents a prescriptive approach to decision making in the face of uncertainty. Its principal application is risk, abundant uncertainty, trade-offs in the outcomes emphasizing a role for preferences, or absence of evidence due to an idiosyncratic feature. For a public health example, Fig. 3-3 displays a decision tree to evaluate strategies for screening for HIV infection. Infected individuals who are unaware of their illness cause up to of the initial diagnosis because of delayed diagnosis. Early identification offers the opportunity to prevent progression to AIDS through CD4 count and viral load monitoring and combination antiretroviral therapy and to reduce spread by reducing risky injection or sexual behaviors.

1	In 2003, the Centers for Disease Control and Prevention (CDC) proposed that routine universal HIV testing should be incorporated into standard adult medical care and, in part, cited a decision analysis model comparing HIV screening with usual care. Assuming a 1% prevalence of unidentified HIV infection in the population, routine screening of a cohort of 43-year-old men and women increased life expectancy by 5.5 days and lifetime costs by $194 per person screened, yielding an incremental cost-effectiveness ratio for screening versus usual care of $15,078 per quality-adjusted life-year (the additional cost to society to increase population health by 1 year of perfect health). Factors that influenced the results included assumptions about the effectiveness of behavior modification on subsequent sexual behavior, the benefits of early therapy for HIV infection, and the prevalence and incidence of HIV infection in the population targeted. This model, which required over 75 separate data

1	sexual behavior, the benefits of early therapy for HIV infection, and the prevalence and incidence of HIV infection in the population targeted. This model, which required over 75 separate data points, provided novel insights into a public health problem in the absence of a randomized clinical trial and helped weigh the pros and cons of such a health policy recommendation. Although such models have been developed for selected clinical problems, their benefit and application to individual real-time clinical management have yet to be demonstrated.

1	High-quality medical care begins with accurate diagnosis. Recently, diagnostic errors have been re-envisioned: the old view was that they were caused by a lack of sufficient skill of an individual clinician; the new view is that they represent a quality of care patient-safety problem traceable to breakdowns in the health care system. Whether this conceptual shift will lead to new ways to improve diagnosis is uncertain. An annual rate of diagnostic errors of 10–15%, possibly leading to 40,000 deaths in the United States, is commonly cited, but these figures are imprecise.

1	Solutions to the “diagnostic errors as a system of care problem” have focused on system-level approaches, such as decision support and other tools integrated into electronic medical records. The use of checklists has been proposed as a means of reducing some of the cognitive errors discussed earlier in the chapter, such as premature closure. Although checklists have been shown to be useful in certain medical contexts, such as operating rooms and intensive care units, their value in preventing diagnostic errors that lead to patient adverse events remains to be shown.

1	Clinical medicine is defined traditionally as a practice combining medical knowledge (including scientific evidence), intuition, and judgment in the care of patients (Chap. 1). EBM updates this construct by placing much greater emphasis on the processes by which clinicians gain knowledge of the most up-to-date and relevant clinical research 20,000 new cases of HIV infection annually FIgURE 3-3 Basic structure of decision model used to evaluate strategies for screen-in the United States, and about 40% of HIV-ing for HIV in the general population. HAART, highly active antiretroviral therapy. positive patients progress to AIDS within a year (Provided courtesy of G. Sanders, with permission.) to determine for themselves whether medical interventions alter the disease course and improve the length or quality of life. The meaning of practicing EBM becomes clearer through an examination of its four key steps: 1. Formulating the management question to be answered 2.

1	Formulating the management question to be answered 2. Searching the literature and online databases for applicable research data 3. Appraising the evidence gathered with regard to its validity and relevance 4. Integrating this appraisal with knowledge about the unique aspects of the patient (including the patient’s preferences about the possible outcomes) The process of searching the world’s research literature and appraising the quality and relevance of studies thus identified can be quite time-consuming and requires skills and training that most clinicians do not possess. Thus, identifying recent systematic overviews of the problem in question (Table 3-3) may offer the best starting point for most EBM searches.

1	Generally, the EBM tools listed in Table 3-3 provide access to research information in one of two forms. The first, primary research reports, is the original peer-reviewed research work that is published in medical journals and accessible through MEDLINE in abstract form. However, without training in using MEDLINE, quickly and efficiently locating reports that are on point in a huge sea of irrelevant or unhelpful citations may be difficult, and important studies could also be missed. The second form, systematic reviews, is the highest level of evidence in the hierarchy because it comprehensively summarizes the available evidence on a particular topic up to a certain date. To avoid the potential biases in review articles, predefined explicit search strategies and inclusion and exclusion criteria are used to find all of the relevant scientific research and grade its quality. The prototype for this kind of resource is the Cochrane Database of Systematic Reviews. When appropriate, a

1	criteria are used to find all of the relevant scientific research and grade its quality. The prototype for this kind of resource is the Cochrane Database of Systematic Reviews. When appropriate, a meta-analysis quantitatively summarizes the systematic review findings. The next two sections explicate the major types of clinical research reports available in the literature and the process of aggregating those data into meta-analyses.

1	SoURCES oF EVIdENCE: CLINICAL TRIALS ANd REgISTRIES The notion of learning from observation of patients is as old as medicine itself. Over the last 50 years, physicians’ understanding of how best to turn raw observation into useful evidence has evolved considerably. Case reports, personal anecdotal experience, and small single-center case series are now recognized as having severe limitations in validity and generalizability, and although they may generate hypotheses or be the first reports of adverse events, they have no role in formulating modern standards of practice. The major tools used to develop reliable evidence consist of the randomized clinical trial and the large observational registry. A registry or database typically is focused on a disease or syndrome (e.g., cancer, CAD, heart failure), a clinical procedure (e.g., bone marrow transplantation, coronary revascularization), or an administrative process (e.g., claims data used for billing and reimbursement).

1	By definition, in observational data, the investigator does not control patient care. Carefully collected prospective observational data, however, can achieve a level of evidence quality approaching that of major clinical trial data. At the other end of the spectrum, data collected retrospectively (e.g., chart review) are limited in form and content to what previous observers recorded, which may not include the specific research data being sought, e.g., claims data. Advantages of observational data include the inclusion of a broader population as encountered in practice than is typically represented in clinical trials because of their restrictive inclusion and exclusion criteria. In addition, observational data provide primary evidence for research questions when a randomized trial cannot be performed. For example, it would be difficult to randomize patients to test diagnostic or therapeutic strategies that are unproven but widely accepted in practice, and it would be unethical to

1	be performed. For example, it would be difficult to randomize patients to test diagnostic or therapeutic strategies that are unproven but widely accepted in practice, and it would be unethical to randomize based on sex, racial/ethnic group, socioeconomic status, or country of residence or to randomize patients to a potentially harmful intervention, such as smoking or deliberately overeating to develop obesity.

1	A well-done prospective observational study of a particular management strategy differs from a well-done randomized clinical trial most importantly by its lack of protection from treatment selection bias. The use of observational data to compare diagnostic or therapeutic strategies assumes that sufficient uncertainty exists in clinical practice to ensure that similar patients will be managed differently by different physicians. In short, the analysis assumes that a sufficient element of randomness (in the sense of disorder rather than in the formal statistical sense) exists in clinical management. In such cases, statistical models attempt to adjust for important imbalances to “level the playing field” so that a fair comparison among treatment options can be made. When management is clearly not random (e.g., all eligible left main CAD patients are referred for coronary bypass surgery), the problem may be too confounded (biased) for statistical correction, and observational data may not

1	not random (e.g., all eligible left main CAD patients are referred for coronary bypass surgery), the problem may be too confounded (biased) for statistical correction, and observational data may not provide reliable evidence.

1	In general, the use of concurrent controls is vastly preferable to that of historical controls. For example, comparison of current surgical management of left main CAD with left main CAD patients treated medically during the 1970s (the last time these patients were routinely treated with medicine alone) would be extremely misleading because “medical therapy” has substantially improved in the interim. MEDLINE National Library of Medicine database with cita-www.nlm.nih.gov Free via Internet. tions back to 1966.

1	MEDLINE National Library of Medicine database with cita-www.nlm.nih.gov Free via Internet. tions back to 1966. Randomized controlled clinical trials include the careful prospective design features of the best observational data studies but also include the use of random allocation of treatment. This design provides the best protection against measured and unmeasured confounding due to treatment selection bias (a major aspect of internal validity). However, the randomized trial may not have good external validity (generalizability) if the process of recruitment into the trial resulted in the exclusion of many patients seen in clinical practice.

1	Consumers of medical evidence need to be aware that randomized trials vary widely in their quality and applicability to practice. The process of designing such a trial often involves many compromises. For example, trials designed to gain U.S. Food and Drug Administration (FDA) approval for an investigational drug or device must fulfill regulatory requirements that may result in a trial population and design that differs substantially from what practicing clinicians would find most useful.

1	The Greek prefix meta signifies something at a later or higher stage of development. Meta-analysis is research that combines and summarizes the available evidence quantitatively. Although occasionally used to examine nonrandomized studies, meta-analysis is used most typically to summarize all randomized trials examining a particular therapy. Ideally, unpublished trials should be identified and included to avoid publication bias (i.e., missing “negative” trials that may not be published). Furthermore, the best meta-analyses obtain and analyze individual patient-level data from all trials rather than working only the summary data in published reports of each trial. Nonetheless, not all published meta-analyses yield reliable evidence for a particular problem, so their methodology should be scrutinized carefully to ensure proper study design and analysis. The results of a well-done meta-analysis are likely to be most persuasive if they include at least several large-scale, properly

1	be scrutinized carefully to ensure proper study design and analysis. The results of a well-done meta-analysis are likely to be most persuasive if they include at least several large-scale, properly performed randomized trials. Meta-analysis can especially help detect benefits when individual trials are inadequately powered (e.g., the benefits of streptokinase thrombolytic therapy in acute MI demonstrated by ISIS-2 in 1988 were evident by the early 1970s through meta-analysis). However, in cases in which the available trials are small or poorly done, meta-analysis should not be viewed as a remedy for the deficiency in primary trial data.

1	Meta-analyses typically focus on summary measures of relative treatment benefit, such as odds ratios or relative risks. Clinicians also should examine what absolute risk reduction (ARR) can be expected from the therapy. A useful summary metric of absolute treatment benefit is the number needed to treat (NNT) to prevent one adverse outcome event (e.g., death, stroke). NNT is simply 1/ARR. For example, if a hypothetical therapy reduced mortality rates over a 5-year follow-up by 33% (the relative treatment benefit) from 12% (control arm) to 8% (treatment arm), the ARR would be 12% – 8% = 4%, and the NNT would be 1/0.04, or 25. Thus, it would be necessary to treat 25 patients for 5 years to prevent 1 death. If the hypothetical treatment was applied to a lower-risk population, say, with a 6% 5-year mortality, the 33% relative treatment benefit would reduce absolute mortality by 2% (from 6% to 4%), and the NNT for the same therapy in this lower-risk group of patients would be 50. Although

1	5-year mortality, the 33% relative treatment benefit would reduce absolute mortality by 2% (from 6% to 4%), and the NNT for the same therapy in this lower-risk group of patients would be 50. Although not always made explicit, comparisons of NNT estimates from different studies should account for the duration of follow-up used to create each estimate.

1	According to the 1990 Institute of Medicine definition, clinical practice guidelines are “systematically developed statements to assist practitioner and patient decisions about appropriate health care for specific clinical circumstances.” This definition emphasizes several crucial features of modern guideline development. First, guidelines are created by using the tools of EBM. In particular, the core of the development process is a systematic literature search followed by a review of the relevant peer-reviewed literature. Second, guidelines usually are focused on a clinical disorder (e.g., adult diabetes, stable angina pectoris) or a health care intervention (e.g., cancer screening). Third, the primary objective of guidelines is to improve the quality of medical care by identifying care practices that should be routinely implemented, based on high-quality evidence and high benefit-to-harm ratios for the interventions. Guidelines are intended to “assist” decision-making, not to define

1	that should be routinely implemented, based on high-quality evidence and high benefit-to-harm ratios for the interventions. Guidelines are intended to “assist” decision-making, not to define explicitly what decisions should be made in a particular situation, in part because evidence alone is never sufficient for clinical decision-making (e.g., deciding whether to intubate and administer antibiotics for pneumonia in a terminally ill individual, in an individual with dementia, or in an otherwise healthy 30-year-old mother).

1	Guidelines are narrative documents constructed by expert panels whose composition often is determined by interested professional organizations. These panels vary in the degree to which they represent all relevant stakeholders. The guideline documents consist of a series of specific management recommendations, a summary indication of the quantity and quality of evidence supporting each recommendation, an assessment of the benefit-to-harm ratio for the recommendation, and a narrative discussion of the recommendations. Many recommendations simply reflect the expert consensus of the guideline panel because literature-based evidence is absent. The final step in guideline construction is peer review, followed by a final revision in response to the critiques provided. To improve the reliability and trustworthiness of guidelines, the Institute of Medicine has made methodologic recommendations for guideline development.

1	Guidelines are closely tied to the process of quality improvement in medicine through their identification of evidence-based best practices. Such practices can be used as quality indicators. Examples include the proportion of acute MI patients who receive aspirin upon admission to a hospital and the proportion of heart failure patients with a depressed ejection fraction treated with an ACE inhibitor.

1	In this era of EBM, it is tempting to think that all the difficult decisions practitioners face have been or soon will be solved and digested into practice guidelines and computerized reminders. However, EBM provides practitioners with an ideal rather than a finished set of tools with which to manage patients. Moreover, even with such evidence, it is always worth remembering that the response to therapy of the “average” patient represented by the summary clinical trial outcomes may not be what can be expected for the specific patient sitting in front of a physician in the clinic or hospital. In addition, meta-analyses cannot generate evidence when there are no adequate randomized trials, and most of what clinicians confront in practice will never be thoroughly tested in a randomized trial. For the foreseeable future, excellent clinical reasoning skills, experience supplemented by well-designed quantitative tools, and a keen appreciation for the role of individual patient preferences

1	For the foreseeable future, excellent clinical reasoning skills, experience supplemented by well-designed quantitative tools, and a keen appreciation for the role of individual patient preferences in their health care will continue to be of paramount importance in the practice of clinical medicine.

1	Katrina Armstrong, Gary J. Martin

1	A primary goal of health care is to prevent disease or detect it early enough that intervention will be more effective. Tremendous progress has been made toward this goal over the last 50 years. Screening tests are available for many common diseases and encompass biochemical (e.g., cholesterol, glucose), physiologic (e.g., blood pressure, growth curves), radiologic (e.g., mammogram, bone densitometry), and cytologic (e.g., Pap smear) approaches. Effective preventive interventions have resulted in dramatic declines in mortality from many diseases, particularly infections. Preventive interventions include counseling about risk behaviors, vaccinations, medications, and, in some relatively uncommon settings, surgery. Preventive services (including screening tests, preventive interventions, and counseling) are different than other medical interventions because they are proactively administered to healthy individuals instead of in response to a symptom, sign, or diagnosis. Thus, the

1	and counseling) are different than other medical interventions because they are proactively administered to healthy individuals instead of in response to a symptom, sign, or diagnosis. Thus, the decision to recommend a screening test or preventive intervention requires a particularly high bar of evidence that testing and intervention are both practical and effective.

1	must be extremely low risk to have an acceptable benefit-to-harm ratio, the ability to target individuals who are more likely to develop disease could enable the application of a wider set of potential approaches and increase efficiency. Currently, there are many types of data that can predict disease incidence in an asymptomatic individual. Genomic data have received the most attention to date, at least in part because mutations in high-penetrance genes have clear implications for preventive care (Chap. 84). Women with mutations in either BRCA1 or BRCA2, the two major breast cancer susceptibility genes identified to date, have a markedly increased risk (5to 20-fold) of breast and ovarian cancer. Screening and prevention recommendations include prophylactic oophorectomy and breast magnetic resonance imaging (MRI), both of which are considered to incur too much harm for women at average cancer risk. Some women opt for prophylactic mastectomy to dramatically reduce their breast cancer

1	resonance imaging (MRI), both of which are considered to incur too much harm for women at average cancer risk. Some women opt for prophylactic mastectomy to dramatically reduce their breast cancer risk. Although the proportion of common disease explained by high-penetrance genes appears to be relatively small (5–10% of most diseases), mutations in rare, moderate-penetrance genes, and variants in low-penetrance genes, also contribute to the prediction of disease risk. The advent of affordable whole exome/whole genome sequencing is likely to speed the dissemination of these tests into clinical practice and may transform the delivery of preventive care.

1	Other forms of “omic” data also have the potential to provide important predictive information, including proteomics and metabolomics. These fields are earlier in development and have yet to move into clinical practice. Imaging and other clinical data may also be integrated into a risk-stratified paradigm as evidence grows about the predictive ability of these data and the feasibility of their collection. Of course, all of these data may also be helpful in predicting the risk of harms from screening or prevention, such as the risk of a false-positive mammogram. To the degree that this information can be incorporated into personalized screening and prevention strategies, it could also improve delivery and efficiency.

1	In addition to advances in risk prediction, there are several other factors that are likely to promote the importance of screening and prevention in the near term. New imaging modalities are being developed that promise to detect changes at the cellular and subcellular levels, greatly increasing the probability that early detection improves outcomes. The rapidly growing understanding of the biologic pathways underlying initiation and progression of many common diseases has the potential to transform the development of preventive interventions, including chemoprevention. Furthermore, screening and prevention offer the promise of both improving health and sparing the costs of disease treatment, an issue that has gained national attention with the continued growth in health care costs.

1	This chapter will review the basic principles of screening and prevention in the primary care setting. Recommendations for specific disorders such as cardiovascular disease, diabetes, and cancer are provided in the chapters dedicated to those topics. The basic principles of screening populations for disease were published by the World Health Organization in 1968 (Table 4-1). In general, screening is most effective when applied to relatively common disorders that carry a large disease burden (Table 4-2). The five leading causes of mortality in the United States are heart diseases, malignant neoplasms, accidents, cerebrovascular diseases, and chronic obstructive pulmonary disease. Thus, many screening strategies are targeted at these conditions. From a global health perspective, these conditions are priorities, but malaria, malnutrition, AIDS, tuberculosis, and violence also carry a heavy disease burden (Chap. 2). challenging for some common diseases. For example, although

1	challenging for some common diseases. For example, although Alzheimer’s disease is the sixth leading cause of death in the prInCIpLes of sCreenInG The condition should be an important health problem. There should be a treatment for the condition. Facilities for diagnosis and treatment should be available. There should be a latent stage of the disease. There should be a test or examination for the condition. The test should be acceptable to the population. The natural history of the disease should be adequately understood. There should be an agreed policy on whom to treat. The cost of finding a case should be balanced in relation to overall medical expenditure.

1	United States, there are no curative treatments and no evidence that early treatment improves outcomes. Lack of facilities for diagnosis and treatment is a particular challenge for developing countries and may change screening strategies, including the development of “see and treat” approaches such as those currently used for cervical cancer screening in some countries. A long latent or preclinical phase where early treatment increases the chance of cure is a hallmark of many cancers; for example, polypectomy prevents progression to colon cancer. Similarly, early identification of hypertension or hyperlipidemia allows therapeutic interventions that reduce the long-term risk of cardiovascular or cerebrovascular events. In contrast, lung cancer screening has historically proven more challenging because most tumors are not curable by the time they can be detected on a chest x-ray. However, the length of the preclinical phase also depends on the level of resolution of the screening test,

1	because most tumors are not curable by the time they can be detected on a chest x-ray. However, the length of the preclinical phase also depends on the level of resolution of the screening test, and this situation changed with the development of chest computed tomography (CT). Low-dose chest CT scanning can detect tumors earlier and was recently demonstrated to reduce lung cancer mortality by 20% in individuals who had at least a 30-pack-year history of smoking. The short interval between the ability to detect disease on a screening test and the development of incurable disease also contributes to the limited effectiveness of mammography screening in reducing breast cancer mortality among premenopausal women. Similarly, the early detection of prostate cancer may not lead to a difference in the mortality rate because the disease is often indolent and competing morbidities, such as coronary artery disease, may ultimately cause mortality (Chap. 100). This uncertainty about the natural

1	in the mortality rate because the disease is often indolent and competing morbidities, such as coronary artery disease, may ultimately cause mortality (Chap. 100). This uncertainty about the natural history is also reflected in the controversy about treatment of prostate cancer, further contributing to the challenge of screening in this disease. Finally, screening programs can incur significant economic costs that must be considered in the context of the available resources and alternative strategies for improving health outcomes.

1	Because screening and preventive interventions are recommended to asymptomatic individuals, they are held to a high standard for demonstrating a favorable risk-benefit ratio before implementation. In general, the principles of evidence-based medicine apply to demonstrating the efficacy of screening tests and preventive interventions, where randomized controlled trials (RCTs) with mortality outcomes are the gold standard. However, because RCTs are often not feasible, observational studies, such as case-control designs, have been used to assess the effectiveness of some interventions such as colorectal cancer screening. For some strategies, such as cervical cancer screening, the only data available are ecologic data demonstrating dramatic declines in mortality. Chapter 4 Screening and Prevention of Disease

1	Irrespective of the study design used to assess the effectiveness of screening, it is critical that disease incidence or mortality is the primary endpoint rather than length of disease survival. This is important because lead time bias and length time bias can create the appearance of an improvement in disease survival from a screening test when there is no actual effect. Lead time bias occurs because screening identifies a case before it would have presented clinically, thereby creating the perception that a patient lived longer after diagnosis simply by moving the date of diagnosis earlier rather than the date of death later. Length time bias occurs because screening is more likely to identify slowly progressive disease than rapidly progressive disease. Thus, within a fixed period of time, a screened population will have a greater proportion of these slowly progressive cases and will appear to have better disease survival than an unscreened population.

1	A variety of endpoints are used to assess the potential gain from screening and preventive interventions. 1.

1	1. The absolute and relative impact of screening on disease incidence or mortality. The absolute difference in disease incidence or mortality between a screened and nonscreened group allows the comparison of size of the benefit across preventive services. A meta-analysis of Swedish mammography trials (ages 40–70) found that ~1.2 fewer women per 1000 would die from breast cancer if they were screened over a 12-year period. By comparison, ~3 lives per 1000 would be saved from colon cancer in a population (ages 50–75) screened with annual fecal occult blood testing (FOBT) over a 13-year period. Based on this analysis, colon cancer screening may actually save more women’s lives than does mammography. However, the relative impact of FOBT (30% reduction in colon cancer death) is similar to the relative impact of mammography (14–32% reduction in breast cancer death), emphasizing the importance of both relative and absolute comparisons. 2.

1	2. The number of subjects screened to prevent disease or death in one individual. The inverse of the absolute difference in mortality is the number of subjects who would need to be screened or receive a preventive intervention to prevent one death. For example, 731 women ages 65–69 would need to be screened by dual-energy x-ray absorptiometry (DEXA) (and treated appropriately) to prevent one hip fracture from osteoporosis. 3.

1	3. Increase in average life expectancy for a population. Predicted increases in life expectancy for various screening and preventive interventions are listed in Table 4-3. It should be noted, however, that the increase in life expectancy is an average that applies to a population, not to an individual. In reality, the vast majority of the population does not derive any benefit from a screening test or preventive intervention. A small subset of patients, however, will benefit greatly. For example, Pap smears do not benefit the 98% of women who never develop cancer of the cervix. However, for the 2% who would have developed cervical cancer, Pap smears may add as much as 25 years to their lives. Some studies suggest that a 1-month gain of life expectancy is a reasonable goal for a population-based screening or prevention strategy.

1	Just as with most aspects of medical care, screening and preventive interventions also incur the possibility of adverse outcomes. These adverse outcomes include side effects from preventive medications and vaccinations, false-positive screening tests, overdiagnosis of disease from screening tests, anxiety, radiation exposure from some screening tests, and discomfort from some interventions and screening tests. The risk of side effects from preventive medications is analogous to the use of medications in therapeutic settings and is considered in the Food and Drug Administration (FDA) approval process. Side effects from currently recommended vaccinations are primarily limited to discomfort and minor immune reactions. However, the concern about associations between vaccinations and serious adverse outcomes continues to limit the acceptance of many vaccinations despite the lack of data supporting the causal nature of these associations.

1	The possibility of a false-positive test occurs with nearly all screening tests, although the definition of what constitutes a false-positive Mammography: Women, 40–50 years 0–5 days Women, 50–70 years 1 month Pap smears, age 18–65 2–3 months Getting a 35-year-old smoker to quit 3–5 years

1	Beginning regular exercise for a 40-year-old 9 months–2 years man (30 min, 3 times a week) result often varies across settings. For some tests such as screening mammography and screening chest CT, a false-positive result occurs when an abnormality is identified that is not malignant, requiring either a biopsy diagnosis or short-term follow-up. For other tests such as Pap smears, a false-positive result occurs because the test identifies a wide range of potentially premalignant states, only a small percentage of which would ever progress to an invasive cancer. This risk is closely tied to the risk of overdiagnosis in which the screening test identifies disease that would not have presented clinically in the patient’s lifetime. Assessing the degree of overdiagnosis from a screening test is very difficult given the need for long-term follow-up of an unscreened population to determine the true incidence of disease over time. Recent estimates suggest that as much as 15–25% of breast

1	is very difficult given the need for long-term follow-up of an unscreened population to determine the true incidence of disease over time. Recent estimates suggest that as much as 15–25% of breast cancers identified by mammography screening and 15–37% of prostate cancers identified by prostate-specific antigen testing may never have presented clinically. Screening tests also have the potential to create unwarranted anxiety, particularly in conjunction with false-positive findings. Although multiple studies have documented increased anxiety through the screening process, there are few data suggesting this anxiety has long-term adverse consequences, including subsequent screening behavior. Screening tests that involve radiation (e.g., mammography, chest CT) add to the cumulative radiation exposure for the screened individual. The absolute amount of radiation is very small from any of these tests, but the overall impact of repeated exposure from multiple sources is still being

1	exposure for the screened individual. The absolute amount of radiation is very small from any of these tests, but the overall impact of repeated exposure from multiple sources is still being determined. Some preventive interventions (e.g., vaccinations) and screening tests (e.g., mammography) may lead to discomfort at the time of administration, but again, there is little evidence of long-term adverse consequences.

1	The decision to implement a population-based screening and prevention strategy requires weighing the benefits and harms, including the economic impact of the strategy. The costs include not only the expense of the intervention but also time away from work, downstream costs from false-positive results or adverse events, and other potential harms. Cost-effectiveness is typically assessed by calculating the cost per year of life saved, with adjustment for the quality of life impact of different interventions and disease states (i.e., quality-adjusted life-year). Typically, strategies that cost <$50,000 to $100,000 per quality-adjusted year of life saved are considered “cost-effective” (Chap. 3).

1	The U.S. Preventive Services Task Force (USPSTF) is an independent panel of experts in preventive care that provides evidence-based recommendations for screening and preventive strategies based on an assessment of the benefit-to-harm ratio (Tables 4-4 and 4-5). Because there are multiple advisory organizations providing recommendations for preventive services, the agreement among the organizations varies across the different services. For example, all advisory groups support screening for hyperlipidemia and colorectal cancer, whereas consensus is lower for breast cancer screening among women in their 40s and almost nonexistent for prostate cancer screening. Because the guidelines are only updated periodically, differences across advisory organizations may also reflect the data that were available when the guideline was issued. For example, multiple organizations have recently issued recommendations supporting lung cancer screening among heavy smokers based on the results of the

1	were available when the guideline was issued. For example, multiple organizations have recently issued recommendations supporting lung cancer screening among heavy smokers based on the results of the National Lung Screening Trial (NLST) published in 2011, whereas the USPSTF did not review lung cancer screening until 2014.

1	29taBLe 4-4 sCreenInG tests reCoMMended By the u.s. preventIve servICes task forCe for averaGe-rIsk aduLts Abbreviations: DEXA, dual-energy x-ray absorptiometry; HCV, hepatitis C virus; HPV, human papillomavirus; PCR, polymerase chain reaction. Source: Adapted from the U.S. Preventive Services Task Force 2013. http://www.uspreventiveservicestaskforce.org/adultrec.htm.

1	For many screening tests and preventive interventions, the balance Although informed consent is important for all aspects of mediof benefits and harms may be uncertain for the average-risk population cal care, shared decision-making may be a particularly important but more favorable for individuals at higher risk for disease. Although approach to decisions about preventive services when the benefit-toage is the most commonly used risk factor for determining screening harm ratio is uncertain for a specific population. For example, many and prevention recommendations, the USPSTF also recommends expert groups, including the USPSTF, recommend an individualized some screening tests in populations with other risk factors for the dis-discussion about prostate cancer screening, because the decision-ease (e.g., syphilis). In addition, being at increased risk for the disease making process is complex and relies heavily on personal issues. often supports initiating screening at an earlier age

1	(e.g., syphilis). In addition, being at increased risk for the disease making process is complex and relies heavily on personal issues. often supports initiating screening at an earlier age than that recom-Some men may decline screening, whereas others may be more will-mended for the average-risk population. For example, when there is ing to accept the risks of an early detection strategy. Recent analysis a significant family history of breast or colon cancer, it is prudent to suggests that many men may be better off not screening for prostate initiate screening 10 years before the age at which the youngest family cancer because watchful waiting was the preferred strategy when member was diagnosed with cancer. quality-adjusted life-years were considered. Another example of

1	Chapter 4 Screening and Prevention of Disease shared decision-making involves the choice of techniques for colon cancer screening (Chap. 100). In controlled studies, the use of annual FOBT reduces colon cancer deaths by 15–30%. Flexible sigmoidoscopy reduces colon cancer deaths by ~60%. Colonoscopy offers the same benefit as or greater benefit than flexible sigmoidoscopy, but its use incurs additional costs and risks. These screening procedures have not been compared directly in the same population, but the estimated cost to society is similar: $10,000–25,000 per year of life saved. Thus, although one patient may prefer the ease of preparation, less time disruption, and the lower risk of flexible sigmoidoscopy, others may prefer the sedation and thoroughness of colonoscopy.

1	In considering the impact of preventive services, it is important to recognize that tobacco and alcohol use, diet, and exercise constitute the vast majority of factors that influence preventable deaths in developed countries. Perhaps the single greatest preventive health care measure is to help patients quit smoking (Chap. 470). However, efforts in these areas frequently involve behavior changes (e.g., weight loss, exercise, seat belts) or the management of addictive conditions (e.g., tobacco and alcohol use) that are often recalcitrant to intervention. Although these are challenging problems, evidence strongly supports the role of counseling by health care providers (Table 4-6) in effecting health behavior change. Educational campaigns, public policy changes, and community-based interventions have also proven to be important parts of a strategy for addressing these factors in some settings. Although the USPSTF found that the evidence was conclusive to recommend a relatively small set

1	have also proven to be important parts of a strategy for addressing these factors in some settings. Although the USPSTF found that the evidence was conclusive to recommend a relatively small set of counseling activities, counseling in areas such as physical activity and injury prevention (including seat belts and bicycle and motorcycle helmets) has become a routine part of primary care practice.

1	The implementation of disease prevention and screening strategies in practice is challenging. A number of techniques can assist physicians with the delivery of these services. An appropriately configured electronic health record can provide reminder systems that make it easier for physicians to track and meet guidelines. Some systems give patients secure access to their medical records, providing an additional means Alcohol and drug use 467, 468e

1	Alcohol and drug use 467, 468e Sexually transmitted infections 163, 226 to enhance adherence to routine screening. Systems that provide nurses and other staff with standing orders are effective for smoking prevention and immunizations. The Agency for Healthcare Research and Quality and the Centers for Disease Control and Prevention have developed flow sheets and electronic tools as part of their “Put Prevention into Practice” program (http://www.uspreventiveservicestaskforce.org/ tools.htm). Many of these tools use age categories to help guide implementation. Age-specific recommendations for screening and counseling are summarized in Table 4-7.

1	Many patients see a physician for ongoing care of chronic illnesses, and this visit provides an opportunity to include a “measure of prevention” for other health problems. For example, a patient seen for management of hypertension or diabetes can have breast cancer screening incorporated into one visit and a discussion about colon cancer screening at the next visit. Other patients may respond more favorably to a clearly defined visit that addresses all relevant screening and prevention interventions. Because of age or comorbidities, it may be appropriate with some patients to abandon certain screening and prevention activities, although there are fewer data about when to “sunset” these services. For many screening tests, the benefit of screening does not accrue until 5 to 10 years of follow-up, and there are generally few data to support continuing screening for most diseases past age 75. In addition, for patients with advanced diseases and limited life expectancy, there is

1	of follow-up, and there are generally few data to support continuing screening for most diseases past age 75. In addition, for patients with advanced diseases and limited life expectancy, there is considerable benefit from shifting the focus from screening procedures to the conditions and interventions more likely to affect quality and length of life.

1	mitted disease; UV, ultraviolet. principles of Clinical pharmacology Dan M. Roden Drugs are the cornerstone of modern therapeutics. Nevertheless, it is well recognized among physicians and in the lay community that the 5 Leading Causes of Age Group Age-Specific Mortality Screening Prevention Interventions to Consider for Each Specific Population Note: The numbers in parentheses refer to areas of risk in the mortality column affected by the specified intervention. Abbreviations: AAA, abdominal aortic aneurysm; ATV, all-terrain vehicle; HPV, human papillomavirus; MMR, measles-mumps-rubella; PSA, prostate-specific antigen; STD, sexually trans-

1	Chapter 5 Principles of Clinical Pharmacology outcome of drug therapy varies widely among individuals. While this variability has been perceived as an unpredictable, and therefore inevitable, accompaniment of drug therapy, this is not the case. The goal of this chapter is to describe the principles of clinical pharmacology that can be used for the safe and optimal use of available and new drugs.

1	Drugs interact with specific target molecules to produce their beneficial and adverse effects. The chain of events between administration of a drug and production of these effects in the body can be divided into two components, both of which contribute to variability in drug actions. The first component comprises the processes that determine drug delivery to, and removal from, molecular targets. The resulting description of the relationship between drug concentration and time is termed pharmacokinetics. The second component of variability in drug action comprises the processes that determine variability in drug actions despite equivalent drug delivery to effector drug sites. This description of the relationship between drug concentration and effect is termed pharmacodynamics. As discussed further below, pharmacodynamic variability can arise as a result of variability in function of the target molecule itself or of variability in the broad biologic context in which the drug-target

1	further below, pharmacodynamic variability can arise as a result of variability in function of the target molecule itself or of variability in the broad biologic context in which the drug-target interaction occurs to achieve drug effects.

1	Two important goals of the discipline of clinical pharmacology are (1) to provide a description of conditions under which drug actions vary among human subjects; and (2) to determine mechanisms underlying this variability, with the goal of improving therapy with available drugs as well as pointing to new drug mechanisms that may be effective in the treatment of human disease. The first steps in the discipline were empirical descriptions of the influence of disease on drug actions and of individuals or families with unusual sensitivities to adverse drug effects. These important descriptive findings are now being replaced by an understanding of the molecular mechanisms underlying variability in drug actions. Thus, the effects of disease, drug coadministration, or familial factors in modulating drug action can now be reinterpreted as variability in expression or function of specific genes whose products determine pharmacokinetics and pharmacodynamics. Nevertheless, it is often the

1	drug action can now be reinterpreted as variability in expression or function of specific genes whose products determine pharmacokinetics and pharmacodynamics. Nevertheless, it is often the personal interaction of the patient with the physician or other health care provider that first identifies unusual variability in drug actions; maintained alertness to unusual drug responses continues to be a key component of improving drug safety.

1	Unusual drug responses, segregating in families, have been recognized for decades and initially defined the field of pharmacogenetics. Now, with an increasing appreciation of common and rare polymorphisms across the human genome, comes the opportunity to reinterpret descriptive mechanisms of variability in drug action as a consequence of specific DNA variants, or sets of variants, among individuals. This approach defines the field of pharmacogenomics, which may hold the opportunity of allowing practitioners to integrate a molecular understanding of the basis of disease with an individual’s genomic makeup to prescribe personalized, highly effective, and safe therapies.

1	Drug therapy is an ancient feature of human culture. The first treatments were plant extracts discovered empirically to be effective for indications like fever, pain, or breathlessness. This symptom-based empiric approach to drug development was supplanted in the twentieth century by identification of compounds targeting more fundamental biologic processes such as bacterial growth or elevated blood pressure; the term “magic bullet,” coined by Paul Ehrlich to describe the search for effective compounds for syphilis, captures the essence of the hope that understanding basic biologic processes will lead to highly effective new therapies. An integral step in modern drug development follows identification of a chemical lead with biologic activity with increasingly sophisticated medicinal chemistry-based structural modifications to develop compounds with specificity for the chosen target, lack of “off-target” effects, and pharmacokinetic properties suitable for human use (e.g., consistent

1	structural modifications to develop compounds with specificity for the chosen target, lack of “off-target” effects, and pharmacokinetic properties suitable for human use (e.g., consistent bioavailability, long elimination half-life, no high-risk pharmacokinetic features described further below).

1	A common starting point for contemporary drug development is basic biologic discovery that implicates potential target molecules: examples of such target molecules include HMG-CoA reductase or the BRAF V600E mutation in many malignant melanomas. The development of compounds targeting these molecules has not only revolutionized treatment for diseases such as hypercholesterolemia or malignant melanoma, but has also revealed new biologic features of disease. Thus, for example, initial spectacular successes with vemurafenib (which targets BRAF V600E) were followed by near-universal tumor relapse, strongly suggesting that inhibition of this pathway alone would be insufficient for tumor control. This reasoning, in turn, supports a view that many complex diseases will not lend themselves to cure by target ing a single magic bullet, but rather single drugs or combinations will a symptom and those designed to prolong useful life. An increasing emphasis on the principles of evidence-based

1	cure by target ing a single magic bullet, but rather single drugs or combinations will a symptom and those designed to prolong useful life. An increasing emphasis on the principles of evidence-based medicine and techniques such as large clinical trials and meta-analyses have defined benefits of drug therapy in broad patient populations. Establishing the balance between risk and benefit is not always simple. An increasing body of evidence supports the idea, with which practitioners are very familiar, that individual patients may display responses that are not expected from large population studies and often have comorbidities that typically exclude them from large clinical trials. In addition, therapies that provide symptomatic benefits but shorten life may be entertained in patients with serious and highly symptomatic diseases such as heart failure or cancer. These considerations illustrate the continuing, highly personal nature of the relationship between the prescriber and the

1	with serious and highly symptomatic diseases such as heart failure or cancer. These considerations illustrate the continuing, highly personal nature of the relationship between the prescriber and the patient.

1	Adverse Effects Some adverse effects are so common and so readily associated with drug therapy that they are identified very early during clinical use of a drug. By contrast, serious adverse effects may be sufficiently uncommon that they escape detection for many years after a drug begins to be widely used. The issue of how to identify rare but serious adverse effects (that can profoundly affect the benefit-risk perception in an individual patient) has not been satisfactorily resolved. Potential approaches range from an increased understanding of the molecular and genetic basis of variability in drug actions to expanded postmarketing surveillance mechanisms. None of these have been completely effective, so practitioners must be continuously vigilant to the possibility that unusual symptoms may be related to specific drugs, or combinations of drugs, that their patients receive.

1	Therapeutic Index Beneficial and adverse reactions to drug therapy can be described by a series of dose-response relations (Fig. 5-1). Well-tolerated drugs demonstrate a wide margin, termed the therapeutic ratio, therapeutic index, or therapeutic window, between the doses required to produce a therapeutic effect and those producing toxicity. In cases where there is a similar relationship between plasma drug concentration and effects, monitoring plasma concentrations can be a highly effective aid in managing drug therapy by enabling concentrations to be maintained above the minimum required to produce an effect and below the concentration range likely to produce toxicity. Such monitoring has been widely used to guide therapy with specific agents, such as certain antiarrhythmics, anticonvulsants, and antibiotics. Many of the principles in clinical pharmacology and examples need to attack multiple pathways whose perturbation results in disease.

1	The use of combination therapy in settings such as hypertension, tuber culosis, HIV infection, and many cancers highlights potential for such a “systems biology” view of drug therapy. It is true across all cultures and diseases that factors such as compliance, genetic variants affecting pharmacokinetics or pharmacodynamics, and drug interactions contribute to drug responses. In addition, cultureor ancestry-specific factors play a role. For example, the frequency of specific genetic variants modulating drug responses often varies by ancestry, as discussed later. Cost issues or cultural factors may determine the likelihood that specific drugs, drug combinations, or over-the-counter (OTC) remedies are prescribed. The broad principles of clinical pharmacology enunciated here can be used to analyze the mechanisms underlying successful or unsuccessful therapy with any drug. INdICATIoNS FoR dRUg THERAPY: RISK VERSUS BENEFIT

1	INdICATIoNS FoR dRUg THERAPY: RISK VERSUS BENEFIT It is self-evident that the benefits of drug therapy should outweigh the risks. Benefits fall into two broad categories: those designed to alleviate

1	FIgURE 5-1 The concept of a therapeutic ratio. Each panel illustrates the relationship between increasing dose and cumulative probability of a desired or adverse drug effect. Top. A drug with a wide therapeutic ratio, i.e., a wide separation of the two curves. Bottom. A drug with a narrow therapeutic ratio; here, the likelihood of adverse effects at therapeutic doses is increased because the curves are not well separated. Further, a steep dose-response curve for adverse effects is especially undesirable, as it implies that even small dosage increments may sharply increase the likelihood of toxicity. When there is a definable relationship between drug concentration (usually measured in plasma) and desirable and adverse effect curves, concentration may be substituted on the abscissa. Note that not all patients necessarily demonstrate a therapeutic response (or adverse effect) at any dose, and that some effects (notably some adverse effects) may occur in a dose-independent fashion.

1	outlined below, which can be applied broadly to therapeutics, have been developed in these arenas. The processes of absorption, distribution, metabolism, and excretion—collectively termed drug disposition—determine the concentration of drug delivered to target effector molecules.

1	The processes of absorption, distribution, metabolism, and excretion—collectively termed drug disposition—determine the concentration of drug delivered to target effector molecules. When a drug is administered orally, subcutaneously, intramuscularly, rectally, sublingually, or directly into desired sites of action, the amount of drug actually entering the systemic circulation may be less than with the intravenous route (Fig. 5-2A). The fraction of drug available to the systemic circulation by other routes is termed bioavailability. Bioavailability may be <100% for two main reasons: (1) absorption is reduced, or (2) the drug undergoes metabolism or elimination prior to entering the systemic circulation. Occasionally, the administered drug formulation is inconsistent or has degraded with time; for example, the anticoagulant dabigatran degrades rapidly (over weeks) once exposed to air, so the amount administered may be less than prescribed.

1	When a drug is administered by a nonintravenous route, the peak concentration occurs later and is lower than after the same dose given by rapid intravenous injection, reflecting absorption from the site of administration (Fig. 5-2). The extent of absorption may be reduced because a drug is incompletely released from its dosage form, undergoes destruction at its site of administration, or has physicochemical properties such as insolubility that prevent complete absorption from its site of administration. Slow absorption rates are deliberately designed into “slow-release” or “sustained-release” drug formulations in order to minimize variation in plasma concentrations during the interval between doses. “First-Pass” Effect When a drug is administered orally, it must traverse the intestinal epithelium, the portal venous system, and the liver prior

1	FIgURE 5-2 Idealized time-plasma concentration curves after a single dose of drug. A. The time course of drug concentration after an instantaneous IV bolus or an oral dose in the one-compartment model shown. The area under the time-concentration curve is clearly less with the oral drug than the IV, indicating incomplete bioavailability. Note that despite this incomplete bioavailability, concentration after the oral dose can be higher than after the IV dose at some time points. The inset shows that the decline of concentrations over time is linear on a log-linear plot, characteristic of first-order elimination, and that oral and IV drugs have the same elimination (parallel) time course. B. The decline of central compartment concentration when drug is distributed both to and from a peripheral compartment and eliminated from the central compartment. The rapid initial decline of concentration reflects not drug elimination but distribution.

1	FIgURE 5-3 Mechanism of presystemic clearance. After drug enters the enterocyte, it can undergo metabolism, excretion into the intestinal lumen, or transport into the portal vein. Similarly, the hepatocyte may accomplish metabolism and biliary excretion prior to the entry of drug and metabolites to the systemic circulation. (Adapted by permission from DM Roden, in DP Zipes, J Jalife [eds]: Cardiac Electrophysiology: From Cell to Bedside, 4th ed. Philadelphia, Saunders, 2003. Copyright 2003 with permission from Elsevier.) to entering the systemic circulation (Fig. 5-3). Once a drug enters the enterocyte, it may undergo metabolism, be transported into the portal vein, or be excreted back into the intestinal lumen. Both excretion into the intestinal lumen and metabolism decrease systemic bioavailability. Once a drug passes this enterocyte barrier, it may also be taken up into the hepatocyte, where bioavailability can be further limited by metabolism or excretion into the bile. This

1	bioavailability. Once a drug passes this enterocyte barrier, it may also be taken up into the hepatocyte, where bioavailability can be further limited by metabolism or excretion into the bile. This elimination in intestine and liver, which reduces the amount of drug delivered to the systemic circulation, is termed presystemic elimination, presystemic extraction, or first-pass elimination.

1	Drug movement across the membrane of any cell, including enterocytes and hepatocytes, is a combination of passive diffusion and active transport, mediated by specific drug uptake and efflux molecules. One widely studied drug transport molecule is P-glycoprotein, the product of the MDR1 gene. P-glycoprotein is expressed on the apical aspect of the enterocyte and on the canalicular aspect of the hepatocyte (Fig. 5-3). In both locations, it serves as an efflux pump, limiting availability of drug to the systemic circulation. P-glycoprotein–mediated drug efflux from cerebral capillaries limits drug brain penetration and is an important component of the blood-brain barrier.

1	Drug metabolism generates compounds that are usually more polar and, hence, more readily excreted than parent drug. Metabolism takes place predominantly in the liver but can occur at other sites such as kidney, intestinal epithelium, lung, and plasma. “Phase I” metabolism involves chemical modification, most often oxidation accomplished by members of the cytochrome P450 (CYP) monooxygenase superfamily. CYPs that are especially important for drug metabolism are presented in Table 5-1, and each drug may be a substrate for one or more of these enzymes. “Phase II” metabolism involves conjugation of specific endogenous compounds to drugs or their metabolites. The enzymes aInhibitors affect the molecular pathway, and thus may affect substrate. bClinically important genetic variants described; see Table 5-2. Note: A listing of CYP substrates, inhibitors, and inducers is maintained at http://medicine .iupui.edu/flockhart/table.htm.

1	Note: A listing of CYP substrates, inhibitors, and inducers is maintained at http://medicine .iupui.edu/flockhart/table.htm. that accomplish phase II reactions include glucuronyl-, acetyl-, sulfo-, and methyltransferases. Drug metabolites may exert important pharmacologic activity, as discussed further below. Clinical Implications of Altered Bioavailability Some drugs undergo near-complete presystemic metabolism and, thus, cannot be administered orally. Nitroglycerin cannot be used orally because it is completely extracted prior to reaching the systemic circulation. The drug is, therefore, used by the sublingual or transdermal routes, which bypass presystemic metabolism.

1	Some drugs with very extensive presystemic metabolism can still be administered by the oral route, using much higher doses than those required intravenously. Thus, a typical intravenous dose of verapamil is 1–5 mg, compared to the usual single oral dose of 40–120 mg. Administration of low-dose aspirin can result in exposure of cyclooxygenase in platelets in the portal vein to the drug, but systemic sparing because of first-pass aspirin deacylation in the liver. This is an example of presystemic metabolism being exploited to therapeutic advantage.

1	Most pharmacokinetic processes, such as elimination, are first-order; that is, the rate of the process depends on the amount of drug present. Elimination can occasionally be zero-order (fixed amount eliminated per unit time), and this can be clinically important (see “Principles of Dose Selection”). In the simplest pharmacokinetic model (Fig. 5-2A), a drug bolus (D) is administered instantaneously to a central compartment, from which drug elimination occurs as a first-order process. Occasionally, central and other compartments correspond to physiologic spaces (e.g., plasma volume), whereas in others they are simply mathematical functions used to describe drug disposition. The first-order nature of drug elimination leads directly to the relationship describing drug concentration (C) at any time (t) following the bolus:

1	C = D • e(0.69/− tt1/2) where V c is the volume of the compartment into which drug is delivered and t1/2 is elimination half-life. As a consequence of this relationship, a plot of the logarithm of concentration versus time is a straight line (Fig. 5-2A, inset). Half-life is the time required for 50% of a first-order process to be complete. Thus, 50% of drug elimination is achieved after one drug-elimination half-life, 75% after two, 87.5% after three, etc. In practice, first-order processes such as elimination are near-complete after four–five half-lives.

1	In some cases, drug is removed from the central compartment not only by elimination but also by distribution into peripheral compartments. In this case, the plot of plasma concentration versus time after a bolus may demonstrate two (or more) exponential components (Fig. 5-2B). In general, the initial rapid drop in drug concentration represents not elimination but drug distribution into and out of peripheral tissues (also first-order processes), while the slower component represents drug elimination; the initial precipitous decline is usually evident with administration by intravenous but not by other routes. Drug concentrations at peripheral sites are determined by a balance between drug distribution to and redistribution from those sites, as well as by elimination. Once distribution is near-complete (four–five distribution half-lives), plasma and tissue concentrations decline in parallel.

1	Clinical Implications of Half-Life Measurements The elimination half-life not only determines the time required for drug concentrations to fall to near-immeasurable levels after a single bolus, it is also the sole determinant of the time required for steady-state plasma concentrations to be achieved after any change in drug dosing (Fig. 5-4). This applies to the initiation of chronic drug therapy (whether by multiple oral doses or by continuous intravenous infusion), a change in chronic drug dose or dosing interval, or discontinuation of drug. Steady state describes the situation during chronic drug administration when the amount of drug administered per unit time equals drug eliminated per unit time. With a continuous intravenous infusion, plasma concentrations at steady state are stable, while with chronic oral drug administration, plasma concentrations vary during the dosing interval but the time-concentration profile between dosing intervals is stable (Fig. 5-4).

1	In a typical 70-kg human, plasma volume is ∼3 L, blood volume is ∼5.5 L, and extracellular water outside the vasculature is ∼20 L. The volume of distribution of drugs extensively bound to plasma proteins but not to tissue components approaches plasma volume; warfarin is one such example. By contrast, for drugs highly bound to tissues, the volume of distribution can be far greater than any physiologic space. For example, the volume of distribution of digoxin and tricyclic antidepressants is hundreds of liters, obviously exceeding total-body volume. Such drugs are not readily removed by dialysis, an important consideration in overdose. Initiation of therapy Change of chronic therapy ChangedosingDose = D * 10th dose Dose = 2•D Dose = 2•D Dose = 0.5•D Discontinue drug Loading dose + dose = D Concentration

1	Initiation of therapy Change of chronic therapy ChangedosingDose = D * 10th dose Dose = 2•D Dose = 2•D Dose = 0.5•D Discontinue drug Loading dose + dose = D Concentration FIgURE 5-4 Drug accumulation to steady state. In this simulation, drug was administered (arrows) at intervals = 50% of the elimination half-life. Steady state is achieved during initiation of therapy after ∼5 elimination half-lives, or 10 doses. A loading dose did not alter the eventual steady state achieved. A doubling of the dose resulted in a doubling of the steady state but the same time course of accumulation. Once steady state is achieved, a change in dose (increase, decrease, or drug discontinuation) results in a new steady state in ∼5 elimination half-lives. (Adapted by permission from DM Roden, in DP Zipes, J Jalife [eds]: Cardiac Electrophysiology: From Cell to Bedside, 4th ed. Philadelphia, Saunders, 2003. Copyright 2003 with permission from Elsevier.)

1	Clinical Implications of drug distribution In some cases, pharmacologic effects require drug distribution to peripheral sites. In this instance, the time course of drug delivery to and removal from these sites determines the time course of drug effects; anesthetic uptake into the central nervous system (CNS) is an example. loadIng doses For some drugs, the indication may be so urgent that administration of “loading” dosages is required to achieve rapid elevations of drug concentration and therapeutic effects earlier than with chronic maintenance therapy (Fig. 5-4). Nevertheless, the time required for true steady state to be achieved is still determined only by the elimination half-life.

1	rate of Intravenous admInIstratIon Although the simulations in Fig. 5-2 use a single intravenous bolus, this is usually inappropriate in practice because side effects related to transiently very high concentrations can result. Rather, drugs are more usually administered orally or as a slower intravenous infusion. Some drugs are so predictably lethal when infused too rapidly that special precautions should be taken to prevent accidental boluses. For example, solutions of potassium for intravenous administration >20 mEq/L should be avoided in all but the most exceptional and carefully monitored circumstances. This minimizes the possibility of cardiac arrest due to accidental increases in infusion rates of more concentrated solutions.

1	Transiently high drug concentrations after rapid intravenous administration can occasionally be used to advantage. The use of midazolam for intravenous sedation, for example, depends upon its rapid uptake by the brain during the distribution phase to produce sedation quickly, with subsequent egress from the brain during the redistribution of the drug as equilibrium is achieved. Similarly, adenosine must be administered as a rapid bolus in the treatment of reentrant supraventricular tachycardias (Chap. 276) to prevent elimination by very rapid (t1/2 of seconds) uptake into erythrocytes and endothelial cells before the drug can reach its clinical site of action, the atrioventricular node.

1	Clinical Implications of Altered Protein Binding Many drugs circulate in the plasma partly bound to plasma proteins. Since only unbound (free) drug can distribute to sites of pharmacologic action, drug response is related to the free rather than the total circulating plasma drug concentration. In chronic kidney or liver disease, protein binding may be decreased and thus drug actions increased. In some situations (myocardial infarction, infection, surgery), acute phase reactants transiently increase drug binding and thus decrease efficacy. These changes assume the greatest clinical importance for drugs that are highly protein-bound since even a small change in protein binding can result in large changes in free drug; for example, a decrease in binding from 99% to 98% doubles the free drug concentration from 1% to 2%. For some drugs (e.g., phenytoin), monitoring free rather than total drug concentrations can be useful.

1	Drug elimination reduces the amount of drug in the body over time. An important approach to quantifying this reduction is to consider that drug concentrations at the beginning and end of a time period are unchanged and that a specific volume of the body has been “cleared” of the drug during that time period. This defines clearance as volume/ time. Clearance includes both drug metabolism and excretion. Clinical Implications of Altered Clearance While elimination half-life determines the time required to achieve steady-state plasma concentration (C ss), the magnitude of that steady state is determined by clearance (Cl) and dose alone. For a drug administered as an intravenous infusion, this relationship is: C = dosing rate/Cl or dosing rate = Cl . C When drug is administered orally, the average plasma concentration within a dosing interval (C ) replaces C , and the dosage (dose avg,ss ss per unit time) must be increased if bioavailability (F) is less than 1:

1	Dose/time = Cl . C /F avg,ss Genetic variants, drug interactions, or diseases that reduce the activity of drug-metabolizing enzymes or excretory mechanisms lead to decreased clearance and, hence, a requirement for downward dose adjustment to avoid toxicity. Conversely, some drug interactions and genetic variants increase the function of drug elimination pathways, and hence, increased drug dosage is necessary to maintain a therapeutic effect. Metabolites may produce effects similar to, overlapping with, or distinct from those of the parent drug. Accumulation of the major metabolite of procainamide, N-acetylprocainamide (NAPA), likely accounts for marked QT prolongation and torsades des pointes ventricular tachycardia (Chap. 276) during therapy with procainamide. Neurotoxicity during therapy with the opioid analgesic meperidine is likely due to accumulation of normeperidine, especially in renal disease.

1	Prodrugs are inactive compounds that require metabolism to generate active metabolites that mediate the drug effects. Examples include many angiotensin-converting enzyme (ACE) inhibitors, the angiotensin receptor blocker losartan, the antineoplastic irinotecan, the anti-estrogen tamoxifen, the analgesic codeine (whose active metabolite morphine probably underlies the opioid effect during codeine administration), and the antiplatelet drug clopidogrel. Drug metabolism has also been implicated in bioactivation of procarcinogens and in generation of reactive metabolites that mediate certain adverse drug effects (e.g., acetaminophen hepatotoxicity, discussed below).

1	When plasma concentrations of active drug depend exclusively on a single metabolic pathway, any condition that inhibits that pathway (be it disease-related, genetic, or due to a drug interaction) can lead to dramatic changes in drug concentrations and marked variability in drug action. This problem of high-risk pharmacokinetics is especially pronounced in two settings. First, variability in bioactivation of a prodrug can lead to striking variability in drug action; examples include decreased CYP2D6 activity, which prevents analgesia by codeine, and decreased CYP2C19 activity, which reduces the antiplatelet effects of clopidogrel. The second setting is drug elimination that relies on

1	Chapter 5 Principles of Clinical Pharmacology a single pathway. In this case, inhibition of the elimination pathway by genetic variants or by administration of inhibiting drugs leads to marked elevation of drug concentration and, for drugs with a narrow therapeutic window, an increased likelihood of dose-related toxicity. Individuals with loss-of-function alleles in CYP2C9, responsible for metabolism of the active S-enantiomer of warfarin, appear to be at increased risk for bleeding. When drugs undergo elimination by multi-ple-drug metabolizing or excretory pathways, absence of one pathway (due to a genetic variant or drug interaction) is much less likely to have a large impact on drug concentrations or drug actions.

1	PRINCIPLES oF PHARMACodYNAMICS The onset of drug Action For drugs used in the urgent treatment of acute symptoms, little or no delay is anticipated (or desired) between the drug-target interaction and the development of a clinical effect. Examples of such acute situations include vascular thrombosis, shock, or status epilepticus.

1	For many conditions, however, the indication for therapy is less urgent, and a delay between the interaction of a drug with its pharmacologic target(s) and a clinical effect is clinically acceptable. Common pharmacokinetic mechanisms that can contribute to such a delay include slow elimination (resulting in slow accumulation to steady state), uptake into peripheral compartments, or accumulation of active metabolites. Another common explanation for such a delay is that the clinical effect develops as a downstream consequence of the initial molecular effect the drug produces. Thus, administration of a proton pump inhibitor or an H2-receptor blocker produces an immediate increase in gastric pH but ulcer healing that is delayed. Cancer chemotherapy similarly produces delayed therapeutic effects.

1	drug Effects May Be disease Specific A drug may produce no action or a different spectrum of actions in unaffected individuals compared to patients with underlying disease. Further, concomitant disease can complicate interpretation of response to drug therapy, especially adverse effects. For example, high doses of anticonvulsants such as phenytoin may cause neurologic symptoms, which may be confused with the underlying neurologic disease. Similarly, increasing dyspnea in a patient with chronic lung disease receiving amiodarone therapy could be due to drug, underlying disease, or an intercurrent cardiopulmonary problem. Thus, the presence of chronic lung disease may argue against the use of amiodarone.

1	While drugs interact with specific molecular receptors, drug effects may vary over time, even if stable drug and metabolite concentrations are maintained. The drug-receptor interaction occurs in a complex biologic milieu that can vary to modulate the drug effect. For example, ion channel blockade by drugs, an important anticonvulsant and anti-arrhythmic effect, is often modulated by membrane potential, itself a function of factors such as extracellular potassium or local ischemia. Receptors may be upor downregulated by disease or by the drug itself. For example, β-adrenergic blockers upregulate β-receptor density during chronic therapy. While this effect does not usually result in resistance to the therapeutic effect of the drugs, it may produce severe agonist-mediated effects (such as hypertension or tachycardia) if the blocking drug is abruptly withdrawn.

1	The desired goal of therapy with any drug is to maximize the likelihood of a beneficial effect while minimizing the risk of adverse effects. Previous experience with the drug, in controlled clinical trials or in postmarketing use, defines the relationships between dose or plasma concentration and these dual effects (Fig. 5-1) and has important implications for initiation of drug therapy: 1. The target drug effect should be defined when drug treatment is started. With some drugs, the desired effect may be difficult to measure objectively, or the onset of efficacy can be delayed for weeks or months; drugs used in the treatment of cancer and psychiatric disease are examples. Sometimes a drug is used to treat a symptom, such as pain or palpitations, and here it is the patient who will report whether the selected dose is effective. In yet other settings, such as anticoagulation or hypertension, the desired response can be repeatedly and objectively assessed by simple clinical or laboratory

1	the selected dose is effective. In yet other settings, such as anticoagulation or hypertension, the desired response can be repeatedly and objectively assessed by simple clinical or laboratory tests.

1	2. The nature of anticipated toxicity often dictates the starting dose. If side effects are minor, it may be acceptable to start chronic therapy at a dose highly likely to achieve efficacy and down-titrate if side effects occur. However, this approach is rarely, if ever, justified if the anticipated toxicity is serious or life-threatening; in this circumstance, it is more appropriate to initiate therapy with the lowest dose that may produce a desired effect. In cancer chemotherapy, it is common practice to use maximum-tolerated doses. 3. The above considerations do not apply if these relationships between dose and effects cannot be defined. This is especially relevant to some adverse drug effects (discussed in further detail below) whose development are not readily related to drug dose. 4. If a drug dose does not achieve its desired effect, a dosage increase is justified only if toxicity is absent and the likelihood of serious toxicity is small.

1	4. If a drug dose does not achieve its desired effect, a dosage increase is justified only if toxicity is absent and the likelihood of serious toxicity is small. Failure of Efficacy Assuming the diagnosis is correct and the correct drug is prescribed, explanations for failure of efficacy include drug interactions, noncompliance, or unexpectedly low drug dosage due to administration of expired or degraded drug. These are situations in which measurement of plasma drug concentrations, if available, can be especially useful. Noncompliance is an especially frequent problem in the long-term treatment of diseases such as hypertension and epilepsy, occurring in ≥25% of patients in therapeutic environments in which no special effort is made to involve patients in the responsibility for their own health. Multidrug regimens with multiple doses per day are especially prone to noncompliance.

1	Monitoring response to therapy, by physiologic measures or by plasma concentration measurements, requires an understanding of the relationships between plasma concentration and anticipated effects. For example, measurement of QT interval is used during treatment with sotalol or dofetilide to avoid marked QT prolongation that can herald serious arrhythmias. In this setting, evaluating the electrocardiogram at the time of anticipated peak plasma concentration and effect (e.g., 1–2 h postdose at steady state) is most appropriate. Maintained high vancomycin levels carry a risk of nephrotoxicity, so dosages should be adjusted on the basis of plasma concentrations measured at trough (predose). Similarly, for dose adjustment of other drugs (e.g., anticonvulsants), concentration should be measured at its lowest during the dosing interval, just prior to a dose at steady state (Fig. 5-4), to ensure a maintained therapeutic effect.

1	Concentration of drugs in Plasma as a guide to Therapy Factors such as interactions with other drugs, disease-induced alterations in elimination and distribution, and genetic variation in drug disposition combine to yield a wide range of plasma levels in patients given the same dose. Hence, if a predictable relationship can be established between plasma drug concentration and beneficial or adverse drug effect, measurement of plasma levels can provide a valuable tool to guide selection of an optimal dose, especially when there is a narrow range between the plasma levels yielding therapeutic and adverse effects. Monitoring is commonly used with certain types of drugs including many anticonvulsants, antirejection agents, antiarrhythmics, and antibiotics. By contrast, if no such relationship can be established (e.g., if drug access to important sites of action outside plasma is highly variable), monitoring plasma concentration may not provide an accurate guide to therapy (Fig. 5-5A).

1	The common situation of first-order elimination implies that average, maximum, and minimum steady-state concentrations are related linearly to the dosing rate. Accordingly, the maintenance dose may be adjusted on the basis of the ratio between the desired and measured concentrations at steady state; for example, if a doubling of the steady-state plasma concentration is desired, the dose should be doubled. This does not apply to drugs eliminated by zero-order kinetics (fixed amount per unit time), where small dosage increases will produce disproportionate increases in plasma concentration; examples include phenytoin and theophylline. Normal P-glycoprotein function and increased drug levels are associated 37 with adverse effects, drug dosages must 5 be reduced in patients with renal dysfunc tion to avoid toxicity. The antiarrhythmics renal excretion and carry a risk of QT pro-

1	Chapter 5 Principles of Clinical Pharmacology longation and arrhythmias if doses are not reduced in renal disease. In end-stage renal disease, sotalol has been given as 40 mg after dialysis (every second day), compared to the usual daily dose, 80–120 mg every 12 h. The extensive hepatic metabolism, so that renal Time failure has little effect on its plasma concentration. However, its metabolite, norme-Decreased P-glycoprotein function peridine, does undergo renal excretion, accumulates in renal failure, and probably accounts for the signs of CNS excitation, such as irritability, twitching, and seizures, that appear when multiple doses of meperi dine are administered to patients with renal disease. Protein binding of some drugs (e.g., phenytoin) may be altered in uremia, so measuring free drug concentration may be desirable.

1	In non-end-stage renal disease, changes in renal drug clearance are generally proportional to those in creatinine clear- ance, which may be measured directly or estimated from the serum creatinine (Chap. 333e). This estimate, coupled with the knowledge of how much drug is normally excreted renally versus nonrenally, allows an estimate of the dose adjustment required. In practice, most decisions involving dosing adjustment in patients with renal failure use published recommended adjustments in dosage or dosing interval based on the severity of renal dysfunction indicated by creatinine clearance. Any such modification of dose is a first approximation and should be followed by and clinical observation to further opti mize therapy for the individual patient. FIgURE 5-5 A. The efflux pump P-glycoprotein excludes drugs from the endothelium of capillaries in the brain and so constitutes a key element of the blood-brain barrier. Thus, reduced

1	FIgURE 5-5 A. The efflux pump P-glycoprotein excludes drugs from the endothelium of capillaries in the brain and so constitutes a key element of the blood-brain barrier. Thus, reduced P-glycoprotein function (e.g., due to drug interactions or genetically determined variability in gene transcription) increases penetration of substrate drugs into the brain, even when plasma LIVER dISEASE concentrations are unchanged. B. The graph shows an effect of a β1-receptor polymorphism on Standard tests of liver function are not use-receptor function in vitro. Patients with the hypofunctional variant (red) may display lesser heart-ful in adjusting doses in diseases like heparate slowing or blood pressure lowering on exposure to a receptor blocking agent. titis or cirrhosis. First-pass metabolism may

1	An increase in dosage is usually best achieved by changing the drug dose but not the dosing interval (e.g., by giving 200 mg every 8 h instead of 100 mg every 8 h). However, this approach is acceptable only if the resulting maximum concentration is not toxic and the trough value does not fall below the minimum effective concentration for an undesirable period of time. Alternatively, the steady state may be changed by altering the frequency of intermittent dosing but not the size of each dose. In this case, the magnitude of the fluctuations around the average steady-state level will change—the shorter the dosing interval, the smaller the difference between peak and trough levels.

1	Renal excretion of parent drug and metabolites is generally accomplished by glomerular filtration and by specific drug transporters. If a drug or its metabolites are primarily excreted through the kidneys decrease, leading to increased oral bioavail ability as a consequence of disrupted hepatocyte function, altered liver architecture, and portacaval shunts. The oral bioavailability for high first-pass drugs such as morphine, meperidine, midazolam, and nifedipine is almost doubled in patients with cirrhosis, compared to those with normal liver function. Therefore, the size of the oral dose of such drugs should be reduced in this setting.

1	Under conditions of decreased tissue perfusion, the cardiac output is redistributed to preserve blood flow to the heart and brain at the expense of other tissues (Chap. 279). As a result, drugs may be distributed into a smaller volume of distribution, higher drug concentrations will be present in the plasma, and the tissues that are best perfused (the brain and heart) will be exposed to these higher concentrations, resulting in increased CNS or cardiac effects. As well, decreased perfusion of the kidney and liver may impair drug clearance. Another consequence of severe heart failure is decreased gut perfusion, which may reduce drug absorption and, thus, lead to reduced or absent effects of orally administered therapies.

1	In the elderly, multiple pathologies and medications used to treat them result in more drug interactions and adverse effects. Aging also results in changes in organ function, especially of the organs involved in drug disposition. Initial doses should be less than the usual adult dosage and should be increased slowly. The number of medications, and doses per day, should be kept as low as possible. Even in the absence of kidney disease, renal clearance may be reduced by 35–50% in elderly patients. Dosages should be adjusted on the basis of creatinine clearance. Aging also results in a decrease in the size of, and blood flow to, the liver and possibly in the activity of hepatic drug-metabolizing enzymes; accordingly, the hepatic clearance of some drugs is impaired in the elderly. As with liver disease, these changes are not readily predicted.

1	Elderly patients may display altered drug sensitivity. Examples include increased analgesic effects of opioids, increased sedation from benzodiazepines and other CNS depressants, and increased risk of bleeding while receiving anticoagulant therapy, even when clotting parameters are well controlled. Exaggerated responses to cardiovascular drugs are also common because of the impaired responsiveness of normal homeostatic mechanisms. Conversely, the elderly display decreased sensitivity to β-adrenergic receptor blockers.

1	Adverse drug reactions are especially common in the elderly because of altered pharmacokinetics and pharmacodynamics, the frequent use of multidrug regimens, and concomitant disease. For example, use of long half-life benzodiazepines is linked to the occurrence of hip fractures in elderly patients, perhaps reflecting both a risk of falls from these drugs (due to increased sedation) and the increased incidence of osteoporosis in elderly patients. In population surveys of the noninstitutionalized elderly, as many as 10% had at least one adverse drug reaction in the previous year. identify and validate DNA variants contributing to variable drug actions.

1	identify and validate DNA variants contributing to variable drug actions. Candidate gene Studies in Pharmacogenetics Most studies to date have used an understanding of the molecular mechanisms modulating drug action to identify candidate genes in which variants could explain variable drug responses. One very common scenario is that variable drug actions can be attributed to variability in plasma drug concentrations. When plasma drug concentrations vary widely (e.g., more than an order of magnitude), especially if their distribution is non-unimodal as in Fig. 5-6, variants in single genes controlling drug concentrations often contribute. In this case, the most obvious candidate genes are those responsible for drug metabolism and elimination. Other candidate genes are those encoding the target molecules with which drugs interact to produce their effects or molecules modulating that response, including those involved in disease pathogenesis.

1	genome-Wide Association Studies in Pharmacogenomics The field has also had some success with “unbiased” approaches such as genome-wide association (GWA) (Chap. 82), particularly in identifying single variants associated with high risk for certain forms of drug toxicity (Table 5-2). GWA studies have identified variants in the HLA-B locus that are associated with high risk for severe skin rashes during treatment with the anticonvulsant carbamazepine and the antiretroviral abacavir. A GWA study of simvastatin-associated myopathy identified a single non-coding single nucleotide polymorphism (SNP) in SLCO1B1, encoding OATP1B1, a drug transporter known to modulate simvastatin might be associated with variable drug levels and hence, effect, was 2 mutant alleles 1–2 wild-type alleles Duplication: >2 wild-type alleles Poor metabolizers (PMs) Ultrarapid metabolizers (UMs) APopulation frequency TimeUMEMPMUMEMPMConcentrationB

1	While most drugs used to treat disease in children are the same are those in adults, there are few studies that provide solid data to guide dosing. different rates after birth, and disease mechanisms may be different in children. In practice, doses are adjusted for size (weight or body surface area) as a first approximation unless age-specific data are available. (SEE ALSo CHAPS. 82 ANd 84) The concept that genetically deter advanced at the end of the nineteenth century, and the examples of familial clustering of unusual drug responses were noted in the mid-twentieth century. A goal of traditional Mendelian genetics is to identify DNA variants associated with a distinct phenotype in multiple related family members (Chap. 84). However, it is unusual for a drug response phenotype to be accurately measured in more than one family member, let alone across a kindred. Thus, non-family-based approaches are generally used to

1	FIgURE 5-6 A. CYP2D6 metabolic activity was assessed in 290 subjects by administration of a test dose of a probe substrate and measurement of urinary formation of the CYP2D6-generated metabolite. The heavy arrow indicates a clear antimode, separating poor metabolizer subjects (PMs, red), with two loss-of-function CYP2D6 alleles, indicated by the intron-exon structures below the chart. Individuals with one or two functional alleles are grouped together as extensive metabolizers (EMs, green). Also shown are ultra-rapid metabolizers (UMs), with 2–12 functional copies of the gene (gray), displaying the greatest enzyme activity. (Adapted from M-L Dahl et al: J Pharmacol Exp Ther 274:516, 1995.) B. These simulations show the predicted effects of CYP2D6 genotype on disposition of a substrate drug. With a single dose (left), there is an inverse “gene-dose” relationship between the number of active alleles and the areas under the time-concentration curves (smallest in UM subjects; highest in

1	With a single dose (left), there is an inverse “gene-dose” relationship between the number of active alleles and the areas under the time-concentration curves (smallest in UM subjects; highest in PM subjects); this indicates that clearance is greatest in UM subjects. In addition, elimination half-life is longest in PM subjects. The right panel shows that these single dose differences are exaggerated during chronic therapy: steady-state concentration is much higher in PM subjects (decreased clearance), as is the time required to achieve steady state (longer elimination half-life).

1	Gene Drugs Effect of Genetic Variantsa Chapter 5 Principles of Clinical Pharmacology K-ras mutation Panitumumab, cetuximab Lack of efficacy with KRAS mutation Philadelphia Busulfan, dasatinib, nilotinib, Decreased efficacy in Philadelphia chromosome–negative chronic chromosome imatinib myelogenous leukemia aDrug effect in homozygotes unless otherwise specified. Note: EM, extensive metabolizer (normal enzymatic activity); PM, poor metabolizer (homozygote for reduced or loss of function allele); UM, ultra-rapid metabolizer (enzymatic activity much greater than normal, e.g., with gene duplication, Fig. 5-6). Further data at U.S. Food and Drug Administration: http://www.fda.gov/Drugs/ ScienceResearch/ResearchAreas/Pharmacogenetics/ucm083378.htm; or Pharmacogenetics Research Network/Knowledge Base: http://www.pharmgkb.org.

1	uptake into the liver, which accounts for 60% of myopathy risk. GWA gENETIC VARIANTS AFFECTINg PHARMACoKINETICS approaches have also implicated interferon variants in antileukemic Clinically important genetic variants have been described in multiple responses and in response to therapy in hepatitis C. Ribavirin, used molecular pathways of drug disposition (Table 5-2). A distinct multi-as therapy in hepatitis C, causes hemolytic anemia, and this has been modal distribution of drug disposition (as shown in Fig. 5-6) argues for linked to variants in ITPA, encoding inosine triphosphatase. a predominant effect of variants in a single gene in the metabolism of that substrate. Individuals with two alleles (variants) encoding for nonfunctional protein make up one group, often termed poor metabolizers (PM phenotype); for some genes, many variants can produce such a loss of function, complicating the use of genotyping in clinical practice. Individuals with one functional allele make up a second

1	(PM phenotype); for some genes, many variants can produce such a loss of function, complicating the use of genotyping in clinical practice. Individuals with one functional allele make up a second (intermediate metabolizers) and may or may not be distinguishable from those with two functional alleles (extensive metabolizers, EMs). Ultra-rapid metabolizers with especially high enzymatic activity (occasionally due to gene duplication; Fig. 5-6) have also been described for some traits. Many drugs in widespread use can inhibit specific drug disposition pathways (Table 5-1), and so EM individuals receiving such inhibitors can respond like PM patients (phenocopying). Polymorphisms in genes encoding drug uptake or drug efflux transporters may be other contributors to variability in drug delivery to target sites and, hence, in drug effects.

1	CYP Variants Members of the CYP3A family (CYP3A4, 3A5) metabolize the greatest number of drugs in therapeutic use. CYP3A4 activity is highly variable (up to an order of magnitude) among individuals, but the underlying mechanisms are not well understood. In whites, but not African Americans, there is a common loss-of-function polymorphism in the closely related CYP3A5 gene. Decreased efficacy of the antirejection agent tacrolimus in African-American subjects has been attributed to more rapid elimination due to relatively greater CYP3A5 activity. A lower risk of vincristine-associated neuropathy has been reported in CYP3A5 “expressers.”

1	CYP2D6 is second to CYP3A4 in the number of commonly used drugs that it metabolizes. CYP2D6 activity is polymorphically distributed, with about 7% of Europeanand African-derived populations (but very few Asians) displaying the PM phenotype (Fig. 5-6). Dozens of loss-of-function variants in the CYP2D6 gene have been described; the PM phenotype arises in individuals with two such alleles. In addition, ultra-rapid metabolizers with multiple functional copies of the CYP2D6 gene have been identified.

1	Codeine is biotransformed by CYP2D6 to the potent active metabolite morphine, so its effects are blunted in PMs and exaggerated in ultra-rapid metabolizers. In the case of drugs with beta-blocking properties metabolized by CYP2D6, greater signs of beta blockade (e.g., bronchospasm, bradycardia) are seen in PM subjects than in EMs. This can be seen not only with orally administered beta blockers such as metoprolol and carvedilol, but also with ophthalmic timolol and with the sodium channel–blocking antiarrhythmic propafenone, a CYP2D6 substrate with beta-blocking properties. Ultra-rapid metabolizers may require very high dosages of tricyclic antidepressants to achieve a therapeutic effect and, with codeine, may display transient euphoria and nausea due to very rapid generation of morphine. Tamoxifen undergoes CYP2D6-mediated biotransformation to an active metabolite, so its efficacy may be in part related to this polymorphism. In addition, the widespread use of selective serotonin

1	Tamoxifen undergoes CYP2D6-mediated biotransformation to an active metabolite, so its efficacy may be in part related to this polymorphism. In addition, the widespread use of selective serotonin reuptake inhibitors (SSRIs) to treat tamoxifen-related hot flashes may also alter the drug’s effects because many SSRIs, notably fluoxetine and paroxetine, are also CYP2D6 inhibitors.

1	The PM phenotype for CYP2C19 is common (20%) among Asians and rarer (2–3%) in European-derived populations. The impact of polymorphic CYP2C19-mediated metabolism has been demonstrated with the proton pump inhibitor omeprazole, where ulcer cure rates with “standard” dosages were much lower in EM patients (29%) than in PMs (100%). Thus, understanding the importance of this polymorphism would have been important in developing the drug, and knowing a patient’s CYP2C19 genotype should improve therapy. CYP2C19 is responsible for bioactivation of the antiplatelet drug clopidogrel, and several large studies have documented decreased efficacy (e.g., increased myocardial infarction after placement of coronary stents) among Caucasian subjects with reduction of function alleles. In addition, some studies suggest that omeprazole and possibly other proton pump inhibitors phenocopy this effect.

1	There are common variants of CYP2C9 that encode proteins with loss of catalytic function. These variant alleles are associated with increased rates of neurologic complications with phenytoin, hypoglycemia with glipizide, and reduced warfarin dose required to maintain stable anticoagulation. The angiotensin-receptor blocker losartan is a prodrug that is bioactivated by CYP2C9; as a result, PMs and those receiving inhibitor drugs may display little response to therapy.

1	Transferase Variants One of the most extensively studied phase II polymorphisms is the PM trait for thiopurine S-methyltransferase (TPMT). TPMT bioinactivates the antileukemic drug 6-mercaptopurine. Further, 6-mercaptopurine is itself an active metabolite of the immunosuppressive azathioprine. Homozygotes for alleles encoding the inactive TPMT (1 in 300 individuals) predictably exhibit severe and potentially fatal pancytopenia on standard doses of azathioprine or 6-mercaptopurine. On the other hand, homozygotes for fully functional alleles may display less anti-inflammatory or antileukemic effect with the drugs.

1	N-acetylation is catalyzed by hepatic N-acetyl transferase (NAT), which represents the activity of two genes, NAT-1 and NAT-2. Both enzymes transfer an acetyl group from acetyl coenzyme A to the drug; polymorphisms in NAT-2 are thought to underlie individual differences in the rate at which drugs are acetylated and thus define “rapid acetylators” and “slow acetylators.” Slow acetylators make up ∼50% of Europeanand African-derived populations but are less common among Asians. Slow acetylators have an increased incidence of the drug-induced lupus syndrome during procainamide and hydralazine therapy and of hepatitis with isoniazid. Induction of CYPs (e.g., by rifampin) also increases the risk of isoniazid-related hepatitis, likely reflecting generation of reactive metabolites of acetylhydrazine, itself an isoniazid metabolite.

1	Individuals homozygous for a common promoter polymorphism that reduces transcription of uridine diphosphate glucuronosyltransferase (UGT1A1) have benign hyperbilirubinemia (Gilbert’s syndrome; Chap. 358). This variant has also been associated with diarrhea and increased bone marrow depression with the antineoplastic prodrug irinotecan, whose active metabolite is normally detoxified by UGT1A1mediated glucuronidation. The antiretroviral atazanavir is a UGT1A1 inhibitor, and individuals with the Gilbert’s variant develop higher bilirubin levels during treatment.

1	Multiple polymorphisms identified in the β2-adrenergic receptor appear to be linked to specific phenotypes in asthma and congestive heart failure, diseases in which β2-receptor function might be expected to determine prognosis. Polymorphisms in the β2-receptor gene have also been associated with response to inhaled β2-receptor agonists, while those in the β1-adrenergic receptor gene have been associated with variability in heart rate slowing and blood pressure lowering (Fig. 5-5B). In addition, in heart failure, a common polymorphism in the β1-adrenergic receptor gene has been implicated in variable clinical outcome during therapy with the investigational beta blocker bucindolol. Response to the 5-lipoxygenase inhibitor zileuton in asthma has been linked to polymorphisms that determine the expression level of the 5-lipoxygenase gene.

1	Drugs may also interact with genetic pathways of disease to elicit or exacerbate symptoms of the underlying conditions. In the porphyrias, CYP inducers are thought to increase the activity of enzymes proximal to the deficient enzyme, exacerbating or triggering attacks (Chap. 430). Deficiency of glucose-6-phosphate dehydrogenase (G6PD), most often in individuals of African, Mediterranean, or South Asian descent, increases the risk of hemolytic anemia in response to the antimalarial primaquine (Chap. 129) and the uric acid–lowering agent rasburicase, which do not cause hemolysis in patients with normal amounts of the enzyme. Patients with mutations in the ryanodine receptor, which controls intracellular calcium in skeletal muscle and other tissues, may be asymptomatic until exposed to certain general anesthetics, which trigger the rare syndrome of malignant hyperthermia. Certain antiarrhythmics and other drugs can produce marked QT prolongation and torsades des pointes (Chap. 276), and

1	general anesthetics, which trigger the rare syndrome of malignant hyperthermia. Certain antiarrhythmics and other drugs can produce marked QT prolongation and torsades des pointes (Chap. 276), and in some patients, this adverse effect represents unmasking of previously sub-clinical congenital long QT syndrome. Up to 50% of the variability in steady-state warfarin dose requirement is attributable to polymorphisms in the promoter of VKORC1, which encodes the warfarin target, and in the coding region of CYP2C9, which mediates its elimination.

1	Tumor and Infectious Agent genomes The actions of drugs used to treat infectious or neoplastic disease may be modulated by variants in these nonhuman germline genomes. Genotyping tumors is a rapidly evolving approach to target therapies to underlying mechanisms and to avoid potentially toxic therapy in patients who would derive no benefit (Chap. 101e). Trastuzumab, which potentiates anthracycline-related cardiotoxicity, is ineffective in breast cancers that do not express the herceptin receptor. Imatinib targets a specific tyrosine kinase, BCR-Abl1, that is generated by the translocation that creates the Philadelphia chromosome typical of chronic myelogenous leukemia (CML). BCR-Abl1 is not only active but may be central to the pathogenesis of CML; its use in BCR-Abl1-positive tumors has resulted in remarkable antitumor efficacy. Similarly, the anti–epidermal growth factor receptor (EGFR) antibodies cetuximab and panitumumab appear especially effective in colon cancers in which K-ras,

1	in remarkable antitumor efficacy. Similarly, the anti–epidermal growth factor receptor (EGFR) antibodies cetuximab and panitumumab appear especially effective in colon cancers in which K-ras, a G protein in the EGFR pathway, is not mutated. Vemurafenib does not inhibit wild-type BRAF but is active against the V600E mutant form of the kinase.

1	The description of genetic variants linked to variable drug responses naturally raises the question of if and how to use this information in practice. Indeed, the U.S. Food and Drug Administration (FDA) now incorporates pharmacogenetic data into information (“package inserts”) meant to guide prescribing. A decision to adopt pharmacogenetically guided dosing for a given drug depends on multiple factors. The most important are the magnitude and clinical importance of the genetic effect and the strength of evidence linking genetic variation to variable drug effects (e.g., anecdote versus post-hoc analysis of clinical trial data versus randomized prospective clinical trial). The evidence can be strengthened if statistical arguments from clinical trial data are complemented by an understanding of underlying physiologic mechanisms. Cost versus expected benefit may also be a factor.

1	When the evidence is compelling, alternate therapies are not available, and there are clear recommendations for dosage adjustment in subjects with variants, there is a strong argument for deploying genetic testing as a guide to prescribing. The association between HLA-B*5701 and severe skin toxicity with abacavir is an example. In other situations, the arguments are less compelling: the magnitude of the genetic effect may be smaller, the consequences may be less serious, alternate therapies may be available, or the drug effect may be amenable to monitoring by other approaches. Ongoing clinical trials are addressing the utility of preprescription genotyping in large populations exposed to drugs with known pharmacogenetic variants (e.g., warfarin). Importantly, technological advances are now raising the possibility of inexpensive whole genome sequencing. Incorporating a patient’s whole genome sequence into their electronic medical record would allow the information to be accessed as

1	now raising the possibility of inexpensive whole genome sequencing. Incorporating a patient’s whole genome sequence into their electronic medical record would allow the information to be accessed as needed for many genetic and pharmacogenetic applications, and the argument has been put forward that this approach would lower logistic barriers to use of pharmacogenomic variant data in prescribing. There are multiple issues (e.g., economic, technological, and ethical) that need to be addressed if such a paradigm is to be adopted (Chap. 82). While barriers to bringing genomic and pharmacogenomic information to the bedside seem daunting, the field is very young and evolving rapidly. Indeed, one major result of understanding the role of genetics in drug action has been improved screening of drugs during the development process to reduce the likelihood of highly variable metabolism or unanticipated toxicity.

1	Drug interactions can complicate therapy by increasing or decreasing the action of a drug; interactions may be based on changes in drug disposition or in drug response in the absence of changes in drug levels. Interactions must be considered in the differential diagnosis of any unusual response occurring during drug therapy. Prescribers should recognize that patients often come to them with a legacy of drugs acquired during previous medical experiences, often with multiple physicians who may not be aware of all the patient’s medications. A meticulous drug history should include examination of the patient’s medications and, if necessary, calls to the pharmacist to identify prescriptions. It should also address the use of agents not often volunteered during questioning, such as OTC drugs, health food supplements, and topical agents such as eye drops. Lists of interactions are available from a number of electronic sources. While it is unrealistic to expect the practicing physician to

1	health food supplements, and topical agents such as eye drops. Lists of interactions are available from a number of electronic sources. While it is unrealistic to expect the practicing physician to memorize these, certain drugs consistently run the risk of generating interactions, often by inhibiting or inducing specific drug elimination pathways. Examples are presented below and in Table 5-3. Accordingly, when these drugs are started or stopped, prescribers must be especially alert to the possibility of interactions.

1	Gastrointestinal absorption can be reduced if a drug interaction results in drug binding in the gut, as with aluminum-containing antacids, kaolin-pectin suspensions, or bile acid sequestrants. Drugs such as histamine H2-receptor antagonists or proton pump inhibitors that alter gastric pH may decrease the solubility and hence absorption of weak bases such as ketoconazole.

1	Expression of some genes responsible for drug elimination, notably CYP3A and MDR1, can be markedly increased by inducing drugs, such as rifampin, carbamazepine, phenytoin, St. John’s wort, and glutethimide, and by smoking, exposure to chlorinated insecticides such as DDT (CYP1A2), and chronic alcohol ingestion. Administration of inducing agents lowers plasma levels, and thus effects, over 2–3 weeks as gene expression is increased. If a drug dose is stabilized in the presence of an inducer that is subsequently stopped, major toxicity can occur as clearance returns to preinduction levels and drug concentrations rise. Individuals vary in the extent to which drug metabolism can be induced, likely through genetic mechanisms. Interactions that inhibit the bioactivation of prodrugs will decrease drug effects (Table 5-1).

1	Interactions that inhibit the bioactivation of prodrugs will decrease drug effects (Table 5-1). Interactions that decrease drug delivery to intracellular sites of action can decrease drug effects: tricyclic antidepressants can blunt the antihypertensive effect of clonidine by decreasing its uptake into adrenergic neurons. Reduced CNS penetration of multiple HIV protease inhibitors (with the attendant risk of facilitating viral replication in a sanctuary site) appears attributable to P-glycoprotein-mediated exclusion of the drug from the CNS; indeed, inhibition of P-glycoprotein has been proposed as a therapeutic approach to enhance drug entry to the CNS (Fig. 5-5A).

1	The most common mechanism here is inhibition of drug elimination. In contrast to induction, new protein synthesis is not involved, and the effect develops as drug and any inhibitor metabolites accumulate (a function of their elimination half-lives). Since shared substrates of a single enzyme can compete for access to the active site of the protein, many CYP substrates can also be considered inhibitors. However, some drugs are especially potent as inhibitors (and occasionally may not even be substrates) of specific drug elimination pathways, and so it is in the use of these agents that clinicians must be most alert to the potential for interactions (Table 5-3). Commonly implicated interacting drugs of this type include amiodarone, cimetidine, Chapter 5 Principles of Clinical Pharmacology

1	Chapter 5 Principles of Clinical Pharmacology Antacids Reduced absorption Bile acid sequestrants Proton pump inhibitors Altered gastric pH H2-receptor blockers Rifampin Induction of CYPs and/or P-glycoprotein Carbamazepine Barbiturates Phenytoin St. John’s wort Glutethimide Nevirapine (CYP3A; CYP2B6) Tricyclic antidepressants Inhibitors of CYP2D6 Fluoxetine Quinidine Cimetidine Inhibitor of multiple CYPs Ketoconazole, itraconazole Inhibitor of CYP3A Erythromycin, clarithromycin Calcium channel blockers Ritonavir Amiodarone Inhibitor of many CYPs and of P-glycoprotein

1	Amiodarone Inhibitor of many CYPs and of P-glycoprotein Gemfibrozil (and other fibrates) CYP3A inhibition Quinidine P-glycoprotein inhibition Amiodarone Verapamil Cyclosporine Itraconazole Erythromycin Phenylbutazone Inhibition of renal tubular transport Probenecid Salicylates erythromycin and some other macrolide antibiotics (clarithromycin but not azithromycin), ketoconazole and other azole antifungals, the antiretroviral agent ritonavir, and high concentrations of grapefruit juice (Table 5-3). The consequences of such interactions will depend on the drug whose elimination is being inhibited (see “The Concept of High-Risk Pharmacokinetics,” above). Examples include CYP3A inhibitors increasing the risk of cyclosporine toxicity or of rhabdomyolysis with some HMG-CoA reductase inhibitors (lovastatin, simvastatin, atorvastatin, but not pravastatin), and P-glycoprotein inhibitors increasing the risk of toxicity with digoxin therapy or of bleeding with the thrombin inhibitor dabigatran.

1	These interactions can occasionally be exploited to therapeutic benefit. The antiviral ritonavir is a very potent CYP3A4 inhibitor that is sometimes added to anti-HIV regimens, not because of its antiviral effects but because it decreases clearance, and hence increases efficacy, of other anti-HIV agents. Similarly, calcium channel blockers have been deliberately coadministered with cyclosporine to reduce its clearance and thus its maintenance dosage and cost. Phenytoin, an inducer of many systems, including CYP3A, inhibits CYP2C9. CYP2C9 metabolism of losartan to its active metabolite is inhibited by phenytoin, with potential loss of antihypertensive effect. Decreased concentration and effects of methadone, dabigatran Increased effect of many β blockers Decreased codeine effect; possible decreased tamoxifen effect

1	Decreased concentration and effects of methadone, dabigatran Increased effect of many β blockers Decreased codeine effect; possible decreased tamoxifen effect Increased concentration and effects of phenytoin Increased concentration and toxicity of some HMG-CoA reductase inhibitors cyclosporine, cisapride, terfenadine (now withdrawn) Increased concentration and effects of indinavir (with ritonavir) Decreased clearance and dose requirement for cyclosporine (with calcium channel blockers) Azathioprine and 6-mercaptopurine toxicity Decreased clearance (risk of toxicity) for quinidine Rhabdomyolysis when co-prescribed with some HMG-CoA reductase inhibitors Risk of toxicity with P-glycoprotein substrates (e.g., digoxin, dabigatran) Increased risk of methotrexate toxicity with salicylates Grapefruit (but not orange) juice inhibits CYP3A, especially at inhibition may increase the risk of adverse effects (e.g., cyclosporine, fruit juice.

1	Grapefruit (but not orange) juice inhibits CYP3A, especially at inhibition may increase the risk of adverse effects (e.g., cyclosporine, fruit juice. CYP2D6 is markedly inhibited by quinidine, a number of neurolep tic drugs (chlorpromazine and haloperidol), and the SSRIs fluoxetine and paroxetine. The clinical consequences of fluoxetine’s interaction with CYP2D6 substrates may not be apparent for weeks after the drug is started, because of its very long half-life and slow generation of a CYP2D6-inhibiting metabolite. 6-Mercaptopurine is metabolized not only by TPMT but also by xanthine oxidase. When allopurinol, an inhibitor of xanthine oxidase, is administered with standard doses of azathioprine or 6-mercaptopu rine, life-threatening toxicity (bone marrow suppression) can result.

1	A number of drugs are secreted by the renal tubular transport sys tems for organic anions. Inhibition of these systems can cause excessive drug accumulation. Salicylate, for example, reduces the renal clearance of methotrexate, an interaction that may lead to methotrexate toxicity. Renal tubular secretion contributes substantially to the elimination of penicillin, which can be inhibited (to increase its therapeutic effect) by probenecid. Similarly, inhibition of the tubular cation transport system by cimetidine decreases the renal clearance of dofetilide. Drugs may act on separate components of a common process to generate effects greater than either has alone. Antithrombotic therapy with combinations of antiplatelet agents (glycoprotein IIb/IIIa inhibitors, aspirin, clopidogrel) and anticoagulants (warfarin, heparins) is often used in the treatment of vascular disease, although such combinations carry an increased risk of bleeding.

1	Nonsteroidal anti-inflammatory drugs (NSAIDs) cause gastric ulcers, and in patients treated with warfarin, the risk of upper gastrointestinal bleeding is increased almost threefold by concomitant use of an NSAID. Indomethacin, piroxicam, and probably other NSAIDs antagonize the antihypertensive effects of β-adrenergic receptor blockers, diuretics, ACE inhibitors, and other drugs. The resulting elevation in blood pressure ranges from trivial to severe. This effect is not seen with aspirin and sulindac but has been found with the cyclooxygenase 2 (COX-2) inhibitor celecoxib.

1	Torsades des pointes ventricular tachycardia during administration of QT-prolonging antiarrhythmics (quinidine, sotalol, dofetilide) occurs much more frequently in patients receiving diuretics, probably reflecting hypokalemia. In vitro, hypokalemia not only prolongs the QT interval in the absence of drug but also potentiates drug block of ion channels that results in QT prolongation. Also, some diuretics have direct electrophysiologic actions that prolong QT. The administration of supplemental potassium leads to more frequent and more severe hyperkalemia when potassium elimination is reduced by concurrent treatment with ACE inhibitors, spironolactone, amiloride, or triamterene.

1	The pharmacologic effects of sildenafil result from inhibition of the phosphodiesterase type 5 isoform that inactivates cyclic guanosine monophosphate (GMP) in the vasculature. Nitroglycerin and related nitrates used to treat angina produce vasodilation by elevating cyclic GMP. Thus, coadministration of these nitrates with sildenafil can cause profound hypotension, which can be catastrophic in patients with coronary disease. Sometimes, combining drugs can increase overall efficacy and/or reduce drug-specific toxicity. Such therapeutically useful interactions are described in chapters dealing with specific disease entities.

1	The beneficial effects of drugs are coupled with the inescapable risk of untoward effects. The morbidity and mortality from these adverse effects often present diagnostic problems because they can involve every organ and system of the body and may be mistaken for signs of underlying disease. As well, some surveys have suggested that drug therapy for a range of chronic conditions such as psychiatric disease or hypertension does not achieve its desired goal in up to half of treated patients; thus, the most common “adverse” drug effect may be failure of efficacy.

1	Adverse reactions can be classified in two broad groups. One type results from exaggeration of an intended pharmacologic action of the drug, such as increased bleeding with anticoagulants or bone marrow suppression with antineoplastics. The second type of adverse reaction ensues from toxic effects unrelated to the intended pharmacologic actions. The latter effects are often unanticipated (especially with new drugs) and frequently severe and may result from recognized as well as previously undescribed mechanisms.

1	Drugs may increase the frequency of an event that is common in a general population, and this may be especially difficult to recognize; an excellent example is the increase in myocardial infarctions with the COX-2 inhibitor rofecoxib. Drugs can also cause rare and serious adverse effects, such as hematologic abnormalities, arrhythmias, severe skin reactions, or hepatic or renal dysfunction. Prior to regulatory approval and marketing, new drugs are tested in relatively few patients who tend to be less sick and to have fewer concomitant diseases than those patients who subsequently receive the drug therapeutically.

1	Because of the relatively small number of patients studied in clinical trials and the selected nature of these patients, rare adverse effects are generally not detected prior to a drug’s approval; indeed, if they are detected, the new drugs are generally not approved. Therefore, physicians need to be cautious in the prescription of new drugs and alert for the appearance of previously unrecognized adverse events.

1	Elucidating mechanisms underlying adverse drug effects can assist development of safer compounds or allow a patient subset at especially high risk to be excluded from drug exposure. National adverse reaction reporting systems, such as those operated by the FDA (suspected adverse reactions can be reported online at http://www.fda.gov/safety/ medwatch/default.htm) and the Committee on Safety of Medicines in Great Britain, can prove useful. The publication or reporting of a newly recognized adverse reaction can in a short time stimulate many similar such reports of reactions that previously had gone unrecognized.

1	Occasionally, “adverse” effects may be exploited to develop an entirely new indication for a drug. Unwanted hair growth during minoxidil treatment of severely hypertensive patients led to development of the drug for hair growth. Sildenafil was initially developed as an antianginal, but its effects to alleviate erectile dysfunction not only led to a new drug indication but also to increased understanding of the role of type 5 phosphodiesterase in erectile tissue. These examples further reinforce the concept that prescribers must remain vigilant to the possibility that unusual symptoms may reflect unappreciated drug effects.

1	Some 25–50% of patients make errors in self-administration of prescribed medicines, and these errors can be responsible for adverse drug effects. Similarly, patients commit errors in taking OTC drugs by not reading or following the directions on the containers. Health care providers must recognize that providing directions with prescriptions does not always guarantee compliance. In hospitals, drugs are administered in a controlled setting, and patient compliance is, in general, ensured. Errors may occur nevertheless— the wrong drug or dose may be given or the drug may be given to the wrong patient—and improved drug distribution and administration systems are addressing this problem.

1	Patients receive, on average, 10 different drugs during each hospitalization. The sicker the patient, the more drugs are given, and there is a corresponding increase in the likelihood of adverse drug reactions. When <6 different drugs are given to hospitalized patients, the probability of an adverse reaction is ∼5%, but if >15 drugs are given, the probability is >40%. Retrospective analyses of ambulatory patients have revealed adverse drug effects in 20%. Serious adverse reactions are also well-recognized with “herbal” remedies and OTC compounds; examples include kava-associated hepatotoxicity, L-tryptophan-associated eosinophilia-myalgia, and phenylpropanolamine-associated stroke, each of which has caused fatalities. A small group of widely used drugs accounts for a disproportionate number of reactions. Aspirin and other NSAIDs, analgesics, digoxin, anticoagulants, diuretics, antimicrobials, glucocorticoids, antineoplastics, and hypoglycemic agents account for 90% of reactions.

1	Drugs or more commonly reactive metabolites generated by CYPs can covalently bind to tissue macromolecules (such as proteins or DNA) to cause tissue toxicity. Because of the reactive nature of these metabolites, covalent binding often occurs close to the site of production, typically the liver. The most common cause of drug-induced hepatotoxicity is acetaminophen overdosage (Chap. 361). Normally, reactive metabolites are detoxified by combining with hepatic glutathione. When glutathione becomes depleted, the metabolites bind instead to hepatic protein, with resultant hepatocyte damage. The hepatic necrosis produced by the ingestion of acetaminophen can be prevented or attenuated by the administration of substances such as N-acetylcysteine that reduce the binding of electrophilic metabolites to hepatic proteins. The risk of acetaminophen-related hepatic necrosis is increased in patients receiving drugs such as phenobarbital or phenytoin, which increase

1	Chapter 5 Principles of Clinical Pharmacology the rate of drug metabolism, or ethanol, which exhausts glutathione stores. Such toxicity has even occurred with therapeutic dosages, so patients at risk through these mechanisms should be warned. Most pharmacologic agents are small molecules with low molecular weights (<2000) and thus are poor immunogens. Generation of an immune response to a drug therefore usually requires in vivo activation and covalent linkage to protein, carbohydrate, or nucleic acid.

1	Drug stimulation of antibody production may mediate tissue injury by several mechanisms. The antibody may attack the drug when the drug is covalently attached to a cell and thereby destroy the cell. This occurs in penicillin-induced hemolytic anemia. Antibody-drug-antigen complexes may be passively adsorbed by a bystander cell, which is then destroyed by activation of complement; this occurs in quinineand quinidine-induced thrombocytopenia. Heparin-induced thrombocytopenia arises when antibodies against complexes of platelet factor 4 peptide and heparin generate immune complexes that activate platelets; thus, the thrombocytopenia is accompanied by “paradoxical” thrombosis and is treated with thrombin inhibitors. Drugs or their reactive metabolites may alter a host tissue, rendering it antigenic and eliciting autoantibodies. For example, hydralazine and procainamide (or their reactive metabolites) can chemically alter nuclear material, stimulating the formation of antinuclear

1	it antigenic and eliciting autoantibodies. For example, hydralazine and procainamide (or their reactive metabolites) can chemically alter nuclear material, stimulating the formation of antinuclear antibodies and occasionally causing lupus erythematosus. Drug-induced pure red cell aplasia (Chap. 130) is due to an immune-based drug reaction.

1	Serum sickness (Chap. 376) results from the deposition of circulating drug-antibody complexes on endothelial surfaces. Complement activation occurs, chemotactic factors are generated locally, and an inflammatory response develops at the site of complex entrapment. Arthralgias, urticaria, lymphadenopathy, glomerulonephritis, or cerebritis may result. Foreign proteins (vaccines, streptokinase, therapeutic antibodies) and antibiotics are common causes. Many drugs, particularly antimicrobial agents, ACE inhibitors, and aspirin, can elicit anaphylaxis with production of IgE, which binds to mast cell membranes. Contact with a drug antigen initiates a series of biochemical events in the mast cell and results in the release of mediators that can produce the characteristic urticaria, wheezing, flushing, rhinorrhea, and (occasionally) hypotension.

1	Drugs may also elicit cell-mediated immune responses. Topically administered substances may interact with sulfhydryl or amino groups in the skin and react with sensitized lymphocytes to produce the rash characteristic of contact dermatitis. Other types of rashes may also result from the interaction of serum factors, drugs, and sensitized lymphocytes.

1	The manifestations of drug-induced diseases frequently resemble those of other diseases, and a given set of manifestations may be produced by different and dissimilar drugs. Recognition of the role of a drug or drugs in an illness depends on appreciation of the possible adverse reactions to drugs in any disease, on identification of the temporal relationship between drug administration and development of the illness, and on familiarity with the common manifestations of the drugs. A suspected adverse drug reaction developing after introduction of a new drug naturally implicates that drug; however, it is also important to remember that a drug interaction may be responsible. Thus, for example, a patient on a chronic stable warfarin dose may develop a bleeding complication after introduction of amiodarone; this does not reflect a direct reaction to amiodarone but rather its effect to inhibit warfarin metabolism. Many associations between particular drugs and specific reactions have been

1	of amiodarone; this does not reflect a direct reaction to amiodarone but rather its effect to inhibit warfarin metabolism. Many associations between particular drugs and specific reactions have been described, but there is always a “first time” for a novel association, and any drug should be suspected of causing an adverse effect if the clinical setting is appropriate.

1	Illness related to a drug’s intended pharmacologic action is often more easily recognized than illness attributable to immune or other mechanisms. For example, side effects such as cardiac arrhythmias in patients receiving digitalis, hypoglycemia in patients given insulin, or bleeding in patients receiving anticoagulants are more readily related to a specific drug than are symptoms such as fever or rash, which may be caused by many drugs or by other factors. Electronic listings of adverse drug reactions can be useful. However, exhaustive compilations often provide little sense of perspective in terms of frequency and seriousness, which can vary considerably among patients.

1	Eliciting a drug history from each patient is important for diagnosis. Attention must be directed to OTC drugs and herbal preparations as well as to prescription drugs. Each type can be responsible for adverse drug effects, and adverse interactions may occur between OTC drugs and prescribed drugs. Loss of efficacy of oral contraceptives or cyclosporine with concurrent use of St. John’s wort (a P-glycoprotein inducer) is an example. In addition, it is common for patients to be cared for by several physicians, and duplicative, additive, antagonistic, or synergistic drug combinations may therefore be administered if the physicians are not aware of the patients’ drug histories. Every physician should determine what drugs a patient has been taking, for the previous month or two ideally, before prescribing any medications. Medications stopped for inefficacy or adverse effects should be documented to avoid pointless and potentially dangerous reexposure. A frequently overlooked source of

1	prescribing any medications. Medications stopped for inefficacy or adverse effects should be documented to avoid pointless and potentially dangerous reexposure. A frequently overlooked source of additional drug exposure is topical therapy; for example, a patient complaining of bronchospasm may not mention that an ophthalmic beta blocker is being used unless specifically asked. A history of previous adverse drug effects in patients is common. Since these patients have shown a predisposition to drug-induced illnesses, such a history should dictate added caution in prescribing new drugs.

1	Laboratory studies may include demonstration of serum antibody in some persons with drug allergies involving cellular blood elements, as in agranulocytosis, hemolytic anemia, and thrombocytopenia. For example, both quinine and quinidine can produce platelet agglutination in vitro in the presence of complement and the serum from a patient who has developed thrombocytopenia following use of this drug. Biochemical abnormalities such as G6PD deficiency, serum pseudocholinesterase level, or genotyping may also be useful in diagnosis, often after an adverse effect has occurred in the patient or a family member.

1	Once an adverse reaction is suspected, discontinuation of the suspected drug followed by disappearance of the reaction is presumptive evidence of a drug-induced illness. Confirming evidence may be sought by cautiously reintroducing the drug and seeing if the reaction reappears. However, that should be done only if confirmation would be useful in the future management of the patient and if the attempt would not entail undue risk. With concentration-dependent adverse reactions, lowering the dosage may cause the reaction to disappear, and raising it may cause the reaction to reappear. When the reaction is thought to be allergic, however, readministration of the drug may be hazardous, since anaphylaxis may develop.

1	If the patient is receiving many drugs when an adverse reaction is suspected, the drugs likeliest to be responsible can usually be identified; this should include both potential culprit agents as well as drugs that alter their elimination. All drugs may be discontinued at once or, if this is not practical, discontinued one at a time, starting with the ones most suspect, and the patient observed for signs of improvement. The time needed for a concentration-dependent adverse effect to disappear depends on the time required for the concentration to fall below the range associated with the adverse effect; that, in turn, depends on the initial blood level and on the rate of elimination or metabolism of the drug. Adverse effects of drugs with long half-lives or those not directly related to serum concentration may take a considerable time to disappear.

1	Modern clinical pharmacology aims to replace empiricism in the use of drugs with therapy based on in-depth understanding of factors that determine an individual’s response to drug treatment. Molecular pharmacology, pharmacokinetics, genetics, clinical trials, and the educated prescriber all contribute to this process. No drug response should ever be termed idiosyncratic; all responses have a mechanism whose understanding will help guide further therapy with that drug or successors. This rapidly expanding understanding of variability in drug actions makes the process of prescribing drugs increasingly daunting for the practitioner. However, fundamental principles should guide this process: The benefits of drug therapy, however defined, should always outweigh the risk. The smallest dosage necessary to produce the desired effect should be used. The number of medications and doses per day should be minimized.

1	The smallest dosage necessary to produce the desired effect should be used. The number of medications and doses per day should be minimized. Although the literature is rapidly expanding, accessing it is becoming easier; electronic tools to search databases of literature and unbiased opinion will become increasingly commonplace. Genetics play a role in determining variability in drug response and may become a part of clinical practice. Electronic medical record and pharmacy systems will increasingly incorporate prescribing advice, such as indicated medications not used; unindicated medications being prescribed; and potential dosing errors, drug interactions, or genetically determined drug responses. Prescribers should be particularly wary when adding or stopping specific drugs that are especially liable to provoke interactions and adverse reactions. Prescribers should use only a limited number of drugs, with which they are thoroughly familiar.

1	Prescribers should use only a limited number of drugs, with which they are thoroughly familiar. of death in women. In 1997, the majority of U.S. women surveyed 6e-1 thought that cancer (35%) rather than heart disease (30%) was the leading cause of death in women (Fig. 6e-2). In 2012, these percep- tions were reversed, with 56% of U.S. women surveyed recognizing

1	The National Institutes of Health’s Office of Research on Women’s Health celebrated its twentieth anniversary in 2010 with a new strategic plan recognizing the study of the biologic basis of sex differences as a distinct scientific discipline. It has become clear that both sex chromosomes and sex hormones contribute to these differences. Indeed, it is recommended that the term sex difference be used for biologic processes that differ between males and females and the term gender difference be used for features related to social influences. The clinical discipline of women’s health emphasizes greater attention to patient education and involvement in disease prevention and medical decision-making and has become a model for patient-centered health care. DISEASE RISK: REALITY AND PERCEPTION

1	DISEASE RISK: REALITY AND PERCEPTION The leading causes of death are the same in women and men: (1) heart disease, and (2) cancer (Table 6e-1; Fig. 6e-1). The leading cause of cancer death, lung cancer, is the same in both sexes. Breast cancer is the second leading cause of cancer death in women, but it causes about 60% fewer deaths than does lung cancer. Men are substantially more likely to die from suicide and accidents than are women.

1	Women’s risk for many diseases increases at menopause, which occurs at a median age of 51.4 years. In the industrialized world, women spend one-third of their lives in the postmenopausal period. Estrogen levels fall abruptly at menopause, inducing a variety of physiologic and metabolic responses. Rates of cardiovascular disease (CVD) increase and bone density begins to decrease rapidly after menopause. In the United States, women live on average about 5 years longer than men, with a life expectancy at birth in 2011 of 81.1 years compared with 76.3 years in men. Elderly women outnumber elderly men, so that age-related conditions such as hypertension have a female preponderance. However, the difference in life expectancy between men and women has decreased an average of 0.1 year every year since its peak of 7.8 years in 1979. If this convergence in mortality figures continues, it is projected that mortality rates will be similar by 2054.

1	Public awareness campaigns have resulted in a marked increase in the percentage of U.S. women knowing that CVD is the leading cause that heart disease rather than cancer (24%) was the leading cause of death in women (Fig. 6e-2). Although awareness of heart disease has improved substantially among black and Hispanic women over this time period, these groups were 66% less likely than white women to recognize that heart disease is the leading cause of death in women.

1	Nevertheless, women younger than 65 years still consider breast cancer to be their leading health risk, despite the fact that death rates from breast cancer have been falling since the 1990s. In any specific decade of life, a woman’s risk for breast cancer never exceeds 1 in 34. Although a woman’s lifetime risk of developing breast cancer if she lives past 85 years is about 1 in 9, it is much more likely that she will die from CVD than from breast cancer. In other words, many elderly women have breast cancer but die from other causes. Similarly, a minority of women are aware that lung cancer is the leading cause of cancer death in women. Physicians are also less likely to recognize women’s risk for CVD. Even in 2012, only 21% of U.S. women surveyed reported that their physicians had counseled them about their risk for heart disease. These misconceptions are unfortunate as they perpetuate inadequate attention to modifiable risk factors such as dyslipidemia, hypertension, and cigarette

1	them about their risk for heart disease. These misconceptions are unfortunate as they perpetuate inadequate attention to modifiable risk factors such as dyslipidemia, hypertension, and cigarette smoking.

1	(See also Chap. 448) Alzheimer’s disease (AD) affects approximately twice as many women as men. Because the risk for AD increases with age, part of this sex difference is accounted for by the fact that women live longer than men. However, additional factors probably contribute to the increased risk for AD in women, including sex differences in brain size, structure, and functional organization. There is emerging evidence for sex-specific differences in gene expression, not only for genes on the X and Y chromosomes but also for some autosomal genes. Estrogens have pleiotropic genomic and nongenomic effects on the central nervous system, including neurotrophic actions in key areas involved in cognition and memory. Women with AD have lower endogenous estrogen levels than do women without AD. These observations have led to the hypothesis that estrogen is neuroprotective. Deaths anD perCentage of totaL Deaths for the LeaDing Causes of Death By sex in the uniteD states in 2010

1	Deaths anD perCentage of totaL Deaths for the LeaDing Causes of Death By sex in the uniteD states in 2010 Cause of Death Rank Deaths Deaths, % Rank Deaths Deaths, % Note: Category titles beginning with “other” or “all other” are not ranked when determining the leading causes of death. Source: Data from Centers for Disease Control and Prevention: National Vital Statistics Reports, Vol. 61, No. 4, May 8, 2013, Table 12, http://www.cdc.gov/nchs/data/nvsr/nvsr61/nvsr61_04.pdf. Rates per 100,000 Rates per 100,000 20–24 25–29 30–34 35–39 40–44 45–49 50–54 55–59 60–64 65–69 70–74 75–79 80–84 >85 Age, years Age, years Ca lung, trachea, bronhus

1	Ca lung, trachea, bronhus FIgURE 6e-1 Death rates per 100,000 population for 2007 by 5-year age groups in U.S. women. Note that the scale of the y axis is increased in the graph on the right compared with that on the left. Accidents and HIV/AIDS are the leading causes of death in young women 20–34 years of age. Accidents, breast cancer, and ischemic heart disease (IHD) are the leading causes of death in women 35–49 years of age. IHD becomes the leading cause of death in women beginning at age 50 years. In older women, IHD remains the leading cause of death, cerebrovascular disease becomes the second leading cause of death, and lung cancer is the leading cause of cancer-related deaths. At age 85 years and beyond, Alzheimer’s disease (AD) becomes the third leading cause of death. Ca, cancer; CLRD, chronic lower respiratory disease; DM, diabetes mellitus. (Data adapted from Centers for Disease Control and Prevention, http://www.cdc.gov/nchs/data/dvs/MortFinal2007_WorkTable210R.pdf.)

1	Some studies have suggested that estrogen administration improves cognitive function in nondemented postmenopausal women as well as in women with AD, and several observational studies have suggested that postmenopausal hormone therapy (HT) may decrease the risk of AD. However, HT placebo-controlled trials have found no improvement in disease progression or cognitive function in women with AD. Further, the Women’s Health Initiative Memory Study (WHIMS), an ancillary study in the Women’s Health Initiative (WHI), found no benefit compared with placebo of estrogen alone [combined continuous equine estrogen (CEE), 0.625 mg daily] or estrogen with progestin [CEE, 0.625 mg daily, and medroxyprogesterone acetate (MPA), 2.5 mg daily] on cognitive function or the development of dementia in women ≥65 years. Indeed, there was a significantly increased risk for both dementia and mild cognitive impairment in women receiving HT. However, preliminary findings from the Kronos Early Estrogen Prevention

1	years. Indeed, there was a significantly increased risk for both dementia and mild cognitive impairment in women receiving HT. However, preliminary findings from the Kronos Early Estrogen Prevention Study (KEEPS), a randomized clinical trial of early initiation of HT after menopause that compared CEE 0.45 mg daily, 50 μg of weekly transdermal estradiol (both estrogen arms included cyclic oral micronized progesterone 200 mg daily for 12 days each month), or placebo, found no adverse effects on cognitive function.

1	(See also Chap. 293) There are major sex differences in CVD, the leading cause of death in men and women in developed countries. A greater number of U.S. women than men die annually of CVD and stroke. Deaths from CVD have decreased markedly in men since 1980, whereas CVD deaths only began to decrease substantially in women beginning in 2000. However, in middle-aged women, the prevalence rates of both coronary heart disease (CHD) and stroke have increased in the 1999–2004 National Health and Nutrition Survey (NHANES) compared to the 1988–1994 NHANES, whereas prevalence rates have decreased or remained unchanged, respectively, in men. These increases were paralleled by an increasing prevalence of abdominal obesity and other components of metabolic syndrome in women. Sex steroids have major effects on the cardiovascular system and lipid metabolism. Estrogen increases high-density lipoprotein (HDL)

1	Sex steroids have major effects on the cardiovascular system and lipid metabolism. Estrogen increases high-density lipoprotein (HDL) FIgURE 6e-2 Changes in perceived leading causes of death among women surveyed in 1997 compared with those surveyed in 2012.

1	FIgURE 6e-2 Changes in perceived leading causes of death among women surveyed in 1997 compared with those surveyed in 2012. In 1997, cancer was cited as the leading cause of death in women, not heart disease. In 2012, this trend had reversed. The rate of awareness that heart disease is the leading cause of death in women was significantly higher in 2012 (56% vs 30%, p <.001) than in 1997. (Data adapted from L Mosca et al: Circulation 127:1254, 2013.) and lowers low-density lipoprotein (LDL), whereas androgens have the opposite effect. Estrogen has direct vasodilatory effects on the vascular endothelium, enhances insulin sensitivity, and has antioxidant and anti-inflammatory properties. There is a striking increase in CHD after both natural and surgical menopause, suggesting that endogenous estrogens are cardioprotective. Women also have longer QT intervals on electrocardiograms, and this increases their susceptibility to certain arrhythmias.

1	CHD presents differently in women, who are usually 10–15 years older than their male counterparts and are more likely to have comorbidities such as hypertension, congestive heart failure, and diabetes mellitus (DM). In the Framingham study, angina was the most common initial symptom of CHD in women, whereas myocardial infarction (MI) was the most common initial presentation in men. Women more often have atypical symptoms such as nausea, vomiting, indigestion, and upper back pain. Although awareness that heart disease is the leading cause of death in women has nearly doubled over the last 15 years, women remain less aware that its symptoms are often atypical, and they are less likely to contact 9-1-1 when they experience such symptoms.

1	Women with MI are more likely to present with cardiac arrest or cardiogenic shock, whereas men are more likely to present with ventricular tachycardia. Further, younger women with MI are more likely to die than are men of similar age. However, this mortality gap has decreased substantially in recent years because younger women have experienced greater improvements in survival after MI than men (Fig. 6e-3). The improvement in survival is due largely to a reduction in comorbidities, suggesting a greater attention to modifiable risk factors in women.

1	Nevertheless, physicians are less likely to suspect heart disease in women with chest pain and less likely to perform diagnostic and therapeutic cardiac procedures in women. Women are less likely to receive therapies such as angioplasty, thrombolytic therapy, coronary artery bypass grafts (CABGs), beta blockers, and aspirin. There are also sex differences in outcomes when women with CHD do receive therapeutic interventions. Women undergoing CABG surgery have more advanced disease, a higher perioperative mortality rate, less relief of angina, and less graft patency; however, 5and 10-year survival rates are similar. Women undergoing percutaneous transluminal coronary angioplasty have lower rates of initial angiographic and clinical success than men, but they also have a lower rate of restenosis and a better long-term outcome. Women may benefit less and have more frequent serious bleeding complications from thrombolytic therapy compared with men. Factors such as older age, more comorbid

1	and a better long-term outcome. Women may benefit less and have more frequent serious bleeding complications from thrombolytic therapy compared with men. Factors such as older age, more comorbid conditions,

1	FIgURE 6e-3 Hospital mortality rates in men and women for acute myocardial infarction (MI) in 1994–1995 compared with 2004–2006. Women younger than age 65 years had substantially greater mortality than men of similar age in 1994–1995. Mortality rates declined markedly for both sexes across all age groups in 2004–2006 compared with 1994–1995. However, there was a more striking decrease in mortality in women younger than age 75 years compared with men of similar age. The mortality rate reduction was largest in women less than age 55 years (52.9%) and lowest in men of similar age (33.3%). (Data adapted from V Vaccarino et al: Arch Intern Med 169:1767, 2009.) smaller body size, and more severe CHD in women at the time of 6e-3 events or procedures account in part for the observed sex differences.

1	Elevated cholesterol levels, hypertension, smoking, obesity, low HDL cholesterol levels, DM, and lack of physical activity are important risk factors for CHD in both men and women. Total triglyceride levels are an independent risk factor for CHD in women but not in men. Low HDL cholesterol and DM are more important risk factors for CHD in women than in men. Smoking is an important risk factor for CHD in women—it accelerates atherosclerosis, exerts direct negative effects on cardiac function, and is associated with an earlier age of menopause. Cholesterol-lowering drugs are equally effective in men and women for primary and secondary prevention of CHD. However, because of perceptions that women are at lower risk for CHD, they receive fewer interventions for modifiable risk factors than do men. In contrast to men, randomized trials showed that aspirin was not effective in the primary prevention of CHD in women; it did significantly reduce the risk of ischemic stroke.

1	The sex differences in CHD prevalence, beneficial biologic effects of estrogen on the cardiovascular system, and reduced risk for CHD in observational studies led to the hypothesis that HT was cardioprotective. However, the WHI, which studied more than 16,000 women on CEE plus MPA or placebo and more than 10,000 women with hysterectomy on CEE alone or placebo, did not demonstrate a benefit of HT for the primary or secondary prevention of CHD. In addition, CEE plus MPA was associated with an increased risk for CHD, particularly in the first year of therapy, whereas CEE alone neither increased nor decreased CHD risk. Both CEE plus MPA and CEE alone were associated with an increased risk for ischemic stroke.

1	In the WHI, there was a suggestion of a reduction in CHD risk in women who initiated HT closer to menopause. This finding suggests that the time at which HT is initiated is critical for cardioprotection. According to this “timing” hypothesis, HT has differential effects, depending on the stage of atherosclerosis; adverse effects are seen with advanced, unstable lesions. A recent study using data from the Danish Osteoporosis Prevention Study (DOPS), an open-label randomized trial of triphasic oral estradiol compared with no treatment in recently menopausal or perimenopausal women (a cyclic oral synthetic progestin, norethisterone acetate, was added in women who had a uterus), found significantly reduced mortality and CVD after 10 years of HT. However, DOPS was designed to investigate HT for the primary prevention of osteoporotic bone fractures, and CVD outcomes were not prespecified endpoints. Further, there were relatively few CVD events in the study groups.

1	KEEPS was designed to directly test the “timing” hypothesis. Seven hundred twenty-seven recently menopausal women age 42–58 years (mean 52.7 years) were randomized to oral CEE (lower dose than WHI), transdermal estradiol, or placebo for 4 years; both estrogen arms included oral cyclical micronized progesterone (see above section on AD for dosing details). There were no significant beneficial or deleterious effects on the progression of atherosclerosis by computed tomography assessment of coronary artery calcification in either HT arm. Adverse events including stroke, MI, venous thromboembolism, and breast cancer were not increased in the HT arms compared with the placebo arm. There were improvements in hot flashes, night sweats, mood, sexual function, and bone density in the HT arms. This relatively small study does not suggest that early HT administration, transdermally or orally, reduces atherosclerosis. However, the study suggests that short-term HT may be safely administered for

1	relatively small study does not suggest that early HT administration, transdermally or orally, reduces atherosclerosis. However, the study suggests that short-term HT may be safely administered for symptom relief in recently menopausal women. HT is discussed further in Chap. 413.

1	(See also Chap. 417) Women are more sensitive to insulin than men are. Despite this, the prevalence of type 2 DM is similar in men and women. There is a sex difference in the relationship between endogenous androgen levels and DM risk. Higher bioavailable testosterone levels are associated with increased risk in women, whereas lower bioavailable testosterone levels are associated with increased risk in men. Polycystic ovary syndrome and gestational DM—common conditions in premenopausal women—are associated with a significantly increased risk for type 2 DM. Premenopausal women with DM lose the cardioprotective effect of female sex and have rates of CHD identical to those in males. These women have impaired endothelial function and reduced coronary vasodilatory responses, which may predispose to cardiovascular complications. Among individuals with DM, women have a greater risk for MI than do men. Women with DM are more likely to have left ventricular hypertrophy. Women with DM receive

1	to cardiovascular complications. Among individuals with DM, women have a greater risk for MI than do men. Women with DM are more likely to have left ventricular hypertrophy. Women with DM receive less aggressive treatment for modifiable CHD risk factors than men with DM. In the WHI, CEE plus MPA significantly reduced the incidence of DM, whereas with CEE alone, there was only a trend toward decreased DM incidence.

1	(See also Chap. 298) After age 60, hypertension is more common in U.S. women than in men, largely because of the high prevalence of hypertension in older age groups and the longer survival of women. Isolated systolic hypertension is present in 30% of women >60 years old. Sex hormones affect blood pressure. Both normotensive and hypertensive women have higher blood pressure levels during the follicular phase than during the luteal phase. In the Nurses’ Health Study, the relative risk of hypertension was 1.8 in current users of oral contraceptives, but this risk is lower with the newer low-dose contraceptive preparations. HT is not associated with hypertension. Among secondary causes of hypertension, there is a female preponderance of renal artery fibromuscular dysplasia.

1	The benefits of treatment for hypertension have been dramatic in both women and men. A meta-analysis of the effects of hypertension treatment, the Individual Data Analysis of Antihypertensive Intervention Trial, found a reduction of risk for stroke and for major cardiovascular events in women. The effectiveness of various antihypertensive drugs appears to be comparable in women and men; however, women may experience more side effects. For example, women are more likely to develop cough with angiotensin-converting enzyme inhibitors.

1	(See also Chap. 377e) Most autoimmune disorders occur more commonly in women than in men; they include autoimmune thyroid and liver diseases, lupus, rheumatoid arthritis (RA), scleroderma, multiple sclerosis (MS), and idiopathic thrombocytopenic purpura. However, there is no sex difference in the incidence of type 1 DM, and ankylosing spondylitis occurs more commonly in men. Women may be more resistant to bacterial infections than men. Sex differences in both immune responses and adverse reactions to vaccines have been reported. For example, there is a female preponderance of postvaccination arthritis.

1	Adaptive immune responses are more robust in women than in men; this may be explained by the stimulatory actions of estrogens and the inhibitory actions of androgens on the cellular mediators of immunity. Consistent with an important role for sex hormones, there is variation in immune responses during the menstrual cycle, and the activity of certain autoimmune disorders is altered by castration or pregnancy (e.g., RA and MS may remit during pregnancy). Nevertheless, the majority of studies show that exogenous estrogens and progestins in the form of HT or oral contraceptives do not alter autoimmune disease incidence or activity. Exposure to fetal antigens, including circulating fetal cells that persist in certain tissues, has been speculated to increase the risk of autoimmune responses. There is clearly an important genetic component to autoimmunity, as indicated by the familial clustering and HLA association of many such disorders. X chromosome genes also contribute to sex differences

1	is clearly an important genetic component to autoimmunity, as indicated by the familial clustering and HLA association of many such disorders. X chromosome genes also contribute to sex differences in immunity. Indeed, nonrandom X chromosome inactivation may be a risk factor for autoimmune diseases.

1	(See also Chap. 226) Women account for almost 50% of the 34 million persons infected with HIV-1 worldwide. AIDS is an important cause of death in younger women (Fig. 6e-1). Heterosexual contact with an at-risk partner is the fastest-growing transmission category, and women are more susceptible to HIV infection than are men. This increased susceptibility is accounted for in part by an increased prevalence of sexually transmitted diseases in women. Some studies have suggested that hormonal contraceptives may increase the risk of HIV transmission. Progesterone has been shown to increase susceptibility to infection in nonhuman primate models of HIV. Women are also more likely to be infected by multiple variants of the virus than are men. Women with HIV have more rapid decreases in their CD4 cell counts than do men. Compared with men, HIV-infected women more frequently develop candidiasis, but Kaposi’s sarcoma is less common than it is in men. Women have more adverse reactions, such as

1	cell counts than do men. Compared with men, HIV-infected women more frequently develop candidiasis, but Kaposi’s sarcoma is less common than it is in men. Women have more adverse reactions, such as lipodystrophy, dyslipidemia, and rash, with antiretroviral therapy than do men. This observation is explained in part by sex differences in the pharmacokinetics of certain antiretroviral drugs, resulting in higher plasma concentrations in women.

1	(See also Chap. 416) The prevalence of both obesity (body mass index ≥30 kg/m2) and abdominal obesity (waist circumference ≥88 cm in women) is higher in U.S. women than in men. However, between 1999 and 2008, the prevalence of obesity increased significantly in men but not in women. The prevalence of abdominal obesity increased over this time period in both sexes. More than 80% of patients who undergo bariatric surgery are women. Pregnancy and menopause are risk factors for obesity.

1	There are major sex differences in body fat distribution. Women characteristically have gluteal and femoral or gynoid pattern of fat distribution, whereas men typically have a central or android pattern. Women have more subcutaneous fat than men. In women, endogenous androgen levels are positively associated with abdominal obesity, and androgen administration increases visceral fat. In contrast, there is an inverse relationship between endogenous androgen levels and abdominal obesity in men. Further, androgen administration decreases visceral fat in these obese men. The reasons for these sex differences in the relationship between visceral fat and androgens are unknown. Studies in humans also suggest that sex steroids play a role in modulating food intake and energy expenditure.

1	In men and women, abdominal obesity characterized by increased visceral fat is associated with an increased risk for CVD and DM. Obesity increases a woman’s risk for certain cancers, in particular postmenopausal breast and endometrial cancer, in part because adipose tissue provides an extragonadal source of estrogen through aromatization of circulating adrenal and ovarian androgens, especially the conversion of androstenedione to estrone. Obesity increases the risk of infertility, miscarriage, and complications of pregnancy.

1	(See also Chap. 425) Osteoporosis is about five times more common in postmenopausal women than in age-matched men, and osteoporotic hip fractures are a major cause of morbidity in elderly women. Men accumulate more bone mass and lose bone more slowly than do women. Sex differences in bone mass are found as early as infancy. Calcium intake, vitamin D, and estrogen all play important roles in bone formation and bone loss. Particularly during adolescence, calcium intake is an important determinant of peak bone mass. Vitamin D deficiency is surprisingly common in elderly women, occurring in >40% of women living in northern latitudes. Receptors for estrogens and androgens have been identified in bone. Estrogen deficiency is associated with increased osteoclast activity and a decreased number of bone-forming units, leading to net bone loss. The aromatase enzyme, which converts androgens to estrogens, is also present in bone. Estrogen is an important determinant of bone mass in men (derived

1	bone-forming units, leading to net bone loss. The aromatase enzyme, which converts androgens to estrogens, is also present in bone. Estrogen is an important determinant of bone mass in men (derived from the aromatization of androgens) as well as in women.

1	On average, women have lower body weights, smaller organs, a higher percentage of body fat, and lower total-body water than men. There are also important sex differences in drug action and metabolism that are not accounted for by these differences in body size and composition. Sex steroids alter the binding and metabolism of a number of drugs. Further, menstrual cycle phase and pregnancy can alter drug action. Two-thirds of cases of drug-induced torsades des pointes, a rare, life-threatening ventricular arrhythmia, occur in women because they have a longer, more vulnerable QT interval. These drugs, which include certain antihistamines, antibiotics, antiarrhythmics, and antipsychotics, can prolong cardiac repolarization by blocking cardiac voltage-gated potassium channels. Women require lower doses of neuroleptics to control schizophrenia. Women awaken from anesthesia faster than do men given the same doses of anesthetics. Women also take more medications than men, including

1	require lower doses of neuroleptics to control schizophrenia. Women awaken from anesthesia faster than do men given the same doses of anesthetics. Women also take more medications than men, including over-the-counter formulations and supplements. The greater use of medications combined with these biologic differences may account for the reported higher frequency of adverse drug reactions in women than in men.

1	(See also Chap. 466) Depression, anxiety, and affective and eating disorders (bulimia and anorexia nervosa) are more common in women than in men. Epidemiologic studies from both developed and developing nations consistently find major depression to be twice as common in women as in men, with the sex difference becoming evident in early adolescence. Depression occurs in 10% of women during pregnancy and in 10–15% of women during the postpartum period. There is a high likelihood of recurrence of postpartum depression with subsequent pregnancies. The incidence of major depression diminishes after age 45 years and does not increase with the onset of menopause. Depression in women appears to have a worse prognosis than does depression in men; episodes last longer, and there is a lower rate of spontaneous remission. Schizophrenia and bipolar disorders occur at equal rates in men and women, although there may be sex differences in symptoms.

1	Both biologic and social factors account for the greater prevalence of depressive disorders in women. Men have higher levels of the neurotransmitter serotonin. Sex steroids also affect mood, and fluctuations during the menstrual cycle have been linked to symptoms of premenstrual syndrome. Sex hormones differentially affect the hypothalamic-pituitary-adrenal responses to stress. Testosterone appears to blunt cortisol responses to corticotropin-releasing hormone. Both low and high levels of estrogen can activate the hypothalamic-pituitaryadrenal axis.

1	(See also Chap. 38) There are striking sex differences in sleep and its disorders. During sleep, women have an increased amount of slow-wave activity, differences in timing of delta activity, and an increase in the number of sleep spindles. Testosterone modulates neural control of breathing and upper airway mechanics. Men have a higher prevalence of sleep apnea. Testosterone administration to hypogonadal men as well as to women increases apneic episodes during sleep. Women with the hyperandrogenic disorder polycystic ovary syndrome have an increased prevalence of obstructive sleep apnea, and apneic episodes are positively correlated with their circulating testosterone levels. In contrast, progesterone accelerates breathing, and in the past, progestins were used for treatment of sleep apnea.

1	(See also Chaps. 467 and 470) Substance abuse is more common in men than in women. However, one-third of Americans who suffer from alcoholism are women. Women alcoholics are less likely 6e-5 to be diagnosed than men. A greater proportion of men than women seek help for alcohol and drug abuse. Men are more likely to go to an alcohol or drug treatment facility, whereas women tend to approach a primary care physician or mental health professional for help under the guise of a psychosocial problem. Late-life alcoholism is more common in women than in men. On average, alcoholic women drink less than alcoholic men but exhibit the same degree of impairment. Blood alcohol levels are higher in women than in men after drinking equivalent amounts of alcohol, adjusted for body weight. This greater bioavailability of alcohol in women is due to both the smaller volume of distribution and the slower gastric metabolism of alcohol secondary to lower activity of gastric alcohol dehydrogenase than is

1	bioavailability of alcohol in women is due to both the smaller volume of distribution and the slower gastric metabolism of alcohol secondary to lower activity of gastric alcohol dehydrogenase than is the case in men. In addition, alcoholic women are more likely to abuse tranquilizers, sedatives, and amphetamines. Women alcoholics have a higher mortality rate than do nonalcoholic women and alcoholic men. Women also appear to develop alcoholic liver disease and other alcohol-related diseases with shorter drinking histories and lower levels of alcohol consumption. Alcohol abuse also poses special risks to a woman, adversely affecting fertility and the health of the baby (fetal alcohol syndrome). Even moderate alcohol use increases the risk of breast cancer, hypertension, and stroke in women.

1	More men than women smoke tobacco, but this sex difference continues to decrease. Women have a much larger burden of smoking-related disease. Smoking markedly increases the risk of CVD in premenopausal women and is also associated with a decrease in the age of menopause. Women who smoke are more likely to develop chronic obstructive pulmonary disease and lung cancer than men and at lower levels of tobacco exposure. Postmenopausal women who smoke have lower bone density than women who never smoked. Smoking during pregnancy increases the risk of preterm deliveries and low birth weight infants.

1	More than one in three women in the United States have experienced rape, physical violence, and/or stalking by an intimate partner. Adult women are much more likely to be raped by a spouse, ex-spouse, or acquaintance than by a stranger. Domestic or intimate partner violence is a leading cause of death among young women. Domestic violence may be an unrecognized feature of certain clinical presentations, such as chronic abdominal pain, headaches, and eating disorders, in addition to more obvious manifestations such as trauma. Intimate partner violence is an important risk factor for depression, substance abuse, and suicide in women. Screening instruments can accurately identify women experiencing intimate partner violence. Such screening by health care providers is acceptable to women in settings ensuring adequate privacy and safety.

1	Women’s health is now a mature discipline, and the importance of sex differences in biologic processes is well recognized. There has been a striking reduction in the excess mortality rate from MI in younger women. Nevertheless, ongoing misperceptions about disease risk, not only among women but also among their physicians, result in inadequate attention to modifiable risk factors. Research into the fundamental mechanisms of sex differences will provide important biologic insights. Further, those insights will have an impact on both women’s and men’s health. the use of performance-enhancing drugs to increase muscularity and 7e-1 lean appearance. Although menopause in women has been the subject of intense investigation for more than five decades, the issues that are Shalender Bhasin, Shehzad Basaria specific to men’s health are just beginning to gain the attention that they deserve because of their high prevalence and impact on overall health, well-being, and quality of life.

1	The emergence of men’s health as a distinct discipline within internal medicine is founded on the evidence that men and women differ across their life span in their susceptibility to disease, in the clinical manifestations of the disease, and in their response to treatment. Furthermore, men and women weigh the health consequences of illness differently and have different motivations for seeking care. Men and women experience different types of disparities in access to health care services and in the manner in which health care is delivered to them because of a complex array of socioeconomic and cultural factors. Attitudinal and institutional barriers to accessing care, fear and embarrassment due to the perception by some that it is not manly to seek medical help, and reticence on the part of patients and physicians to discuss issues related to sexuality, drug use, and aging have heightened the need for programs tailored to address the specific health needs of men.

1	Sex differences in disease prevalence, susceptibility, and clinical manifestations of disease were discussed in Chap. 6e (“Women’s Health”). It is notable that the two leading causes of death in both men and women—heart disease and cancer—are the same. However, men have a higher prevalence of neurodevelopmental and degenerative disorders; substance abuse disorders, including the use of performance-enhancing drugs and alcohol dependence; diabetes; and cardiovascular disease; and women have a higher prevalence of autoimmune disorders, depression, rheumatologic disorders, and osteoporosis. Men are substantially more likely to die from accidents, suicides, and homicides than women. Among men 15–34 years of age, unintentional injuries, homicides, and suicides account for over three-fourths of all deaths. Among men 35–64 years of age, heart disease, cancer, and unintentional injuries are the leading causes of death. Among men 65 years of age or older, heart disease, cancer, lower

1	of all deaths. Among men 35–64 years of age, heart disease, cancer, and unintentional injuries are the leading causes of death. Among men 65 years of age or older, heart disease, cancer, lower respiratory tract infections, and stroke are the major causes of death.

1	The biologic bases of sex differences in disease susceptibility, progression, and manifestation remain incompletely understood and are likely multifactorial. Undoubtedly, sex-specific differences in the genetic architecture and circulating sex hormones influence disease phenotype; additionally, epigenetic effects of sex hormones during fetal life, early childhood, and pubertal development may imprint sexual and nonsexual behaviors, body composition, and disease susceptibility. Reproductive load and physiologic changes during pregnancy, including profound hormonal and metabolic shifts and microchimerism (transfer of cells from the mother to the fetus and from the fetus to the mother), may affect disease susceptibility and disease severity in women. Sociocultural norms of child-rearing practices, societal expectations of gender roles, and the long-term economic impact of these practices and gender roles also may affect disease risk and its clinical manifestation. The trajectories of

1	societal expectations of gender roles, and the long-term economic impact of these practices and gender roles also may affect disease risk and its clinical manifestation. The trajectories of age-related changes in sex hormones (SEE CHAP. 411)

1	A number of studies have established that testosterone concentrations decrease with advancing age. This age-related decline starts in the third decade of life and progresses thereafter (Fig. 7e-1). Low total and bioavailable testosterone concentrations are associated with decreased skeletal muscle mass and strength, higher visceral fat mass, insulin resistance, and increased risk of coronary artery disease and mortality (Table 7e-1). Most studies suggest that these symptoms and signs develop with total testosterone levels below 320 ng/dL and free testosterone levels below 64 pg/mL in older men. Testing for low testosterone in older men should be limited to those with symptoms or signs attributable to androgen deficiency.

1	Testosterone therapy of healthy older men with low testosterone increases lean body mass, grip strength, and self-reported physical function (Fig. 7e-2). Testosterone therapy also increases vertebral but not femoral bone mineral density. In men with sexual dysfunction and low testosterone levels, testosterone therapy improves libido, but effects on erectile function and response to selective phosphodiesterase inhibitors are variable (Chap. 67). As discussed in Chap. 411, there is concern that testosterone therapy may stimulate the growth of prostate cancers.

1	Sexual Dysfunction (See Chap. 67) Various forms of sexual dysfunction are a major motivating factor for men seeking care at men’s health clinics. The landmark descriptions of the human sexual response cycle by Masters and Johnson, demonstrating that men and women display predictable physiologic responses after sexual stimulation, provided the basis for rational classification of human sexual disorders. Accordingly, sexual disorders have been classified into four categories depending on phase of sexual response cycle in which the abnormality exists: 1. 2. 3. 4. Disorders of pain Classification of the patient’s disorder into these categories is important because the etiologic factors, diagnostic tests, and therapeutic

1	2. 3. 4. Disorders of pain Classification of the patient’s disorder into these categories is important because the etiologic factors, diagnostic tests, and therapeutic Total testosterone (ng/dL) vs. Age (y) during the reproductive and postreproductive years vary substantially between men and women and may influence the sex differences in the temporal evolution of age-related conditions such as osteoporosis, breast cancer, and autoimmune disease.

1	In a reflection of the growing attention to issues related to men’s health, health clinics focused on the health problems of men are being established with increasing frequency. Although the major threats to men’s health have not changed—heart disease, cancer, and uninten tional injury continue to dominate the list of major medical causes of morbidity and mortality in men—the men who attend men’s health clinics do so largely for sexual, reproductive, and urologic health concerns involving common conditions such as androgen deficiency syndromes, age-related decline in testosterone levels, sexual dysfunc-FIGURE 7e-1 Age-related decline in total testosterone levels. Total tion, muscle dysmorphia and anabolic-androgenic steroid use, lower testosterone levels measured using liquid chromatography tandem urinary tract symptoms, and medical complications of prostate cancer mass spectrometry in men of the Framingham Heart Study (FHS), the therapy, which are the focus of this chapter.

1	tandem urinary tract symptoms, and medical complications of prostate cancer mass spectrometry in men of the Framingham Heart Study (FHS), the therapy, which are the focus of this chapter. Additionally, new catego-European Male Aging Study (EMAS), and the Osteoporotic Fractures ries of body image disorders have emerged in men that had not been in Men Study (MrOS). (Reproduced with permission from S Bhasin et al: J recognized until the 1980s, such as body dysmorphia syndrome and Clin Endocrinol Metab 96:2430, 2011.) assoCiation of testosterone levels with outCoMes in older Men 1. Positively associated with: mineral density, bone geometry, and volumetric bone mineral 2. Negatively associated with: 3. Not associated with: strategies vary for each class of sexual disorder. Historically, the classification and nomenclature for sexual disorders used criteria identified in the Diagnostic and Statistical Manual of Mental Disorders (DSM), based on the erroneous belief that sexual disorders in

1	and nomenclature for sexual disorders used criteria identified in the Diagnostic and Statistical Manual of Mental Disorders (DSM), based on the erroneous belief that sexual disorders in men are largely psychogenic in their origin. However, the recognition of erectile dysfunction as a manifestation of systemic disease and the availability of easy-to-use oral selective phosphodiesterase-5 inhibitors have placed sexual disorders in men within the purview of the primary care provider.

1	MUSCLE DYSMORPHIA SYNDROME IN MEN: A BODY IMAGE DISORDER Muscle dysmorphia is a form of body image disorder characterized by a pathologic preoccupation with muscularity and leanness. The men with muscle dysmorphia express a strong desire to be more muscular and lean. These men describe shame and embarrassment about their body size and shape and often report adverse symptoms such as dissatisfaction with appearance, preoccupation with bodybuilding and muscularity, and functional impairment. Patients with muscle dysmorphia also report higher rates of mood and anxiety disorders, as well as obsessive and compulsive behaviors. These men often experience impairment of social and occupational functioning.

1	Patients with muscle dysmorphia syndrome—nearly all men—are almost always engaged in weightlifting and body building and are more likely to use performance-enhancing drugs, especially anabolic-androgenic steroids. Muscle dysmorphia disorder predisposes men to an increased risk of disease due to the combined interactive effects of the intensity of physical exercise, the use of performance-enhancing drugs, and other lifestyle factors associated with weightlifting and the use of performance-enhancing drugs. No randomized trials of any treatment modalities have been conducted; anecdotally, behavioral and cognitive therapies have been tried with varying degrees of success.

1	Anabolic-Androgenic Steroid Abuse by Athletes and Recreational Body-Builders The illicit use of anabolic-androgenic steroids (AAS) to enhance athletic performance first surfaced in the 1950s among powerlifters and spread rapidly to other sports and to professional as well as high school athletes and recreational bodybuilders. In the early 1980s, the use of AAS spread beyond the athletic community into the general population. As many as 3 million Americans, most of them men, have likely used these compounds. Most AAS users are not athletes, but rather recreational weightlifters who use these drugs to look lean and more muscular.

1	FIGURE 7e-2 The effects of testosterone therapy on body composition, muscle strength, bone mineral density, and sexual function in intervention trials. The point estimates and the associated 95% confidence intervals are shown. A. The effects of testos terone therapy on lean body mass, grip strength, and fat mass in a meta-analysis of randomized trials. (Data derived from S Bhasin et al: Nat Clin Pract Endocrinol Metab 2:146, 2006.) B. The effects of testoster analysis of randomized trials. (Data derived from a meta-analysis by MJ Tracz et al: J Clin Endocrinol Metab 91:2011, 2006.) C. The effects of testosterone therapy on measures of sexual function in men with baseline testosterone less than 10 nmol/L (290 ng/dL). (Data derived from a meta-analysis by AM Isidori et al: Clin Endocrinol (Oxf) 63:381, 2005.) (Reproduced with permission from M Spitzer et al: Nat Rev Endocrinol 9:414, 2013.)

1	The most commonly used AAS include testosterone esters, nandrolone, stanozolol, methandienone, and methenolone. AAS users generally use increasing doses of multiple steroids in a practice known as stacking.

1	The adverse effects of long-term AAS abuse remain poorly understood. Most of the information about the adverse effects of AAS has emerged from case reports, uncontrolled studies, or clinical trials that used replacement doses of testosterone (Table 7e-2). Of note, AAS users may administer 10–100 times the replacement doses of testosterone over many years, making it unjustifiable to extrapolate from trials using replacement doses. A substantial fraction of AAS users also use other drugs that are perceived to be muscle-building or performance-enhancing, such as growth hormone; erythropoiesisstimulating agents; insulin; and stimulants such as amphetamine, clenbuterol, cocaine, ephedrine, and thyroxine; and drugs perceived to reduce adverse effects such as human chorionic gonadotropin, aromatase inhibitors, or estrogen antagonists. The men who abuse AAS are Abbreviation: HPT axis, hypothalamic-pituitary-testicular axis.

1	Abbreviation: HPT axis, hypothalamic-pituitary-testicular axis. Source: Modified with permission from HG Pope Jr et al: Adverse health consequences of performance-enhancing drugs: an endocrine society scientific statement. Endocr Rev 35:341, 2014. more likely to engage in other high-risk behaviors than nonusers. The adverse events associated with AAS use may be due to AAS themselves, concomitant use of other drugs, high-risk behaviors, and host characteristics that may render these individuals more susceptible to AAS use or to other high-risk behaviors. The high rates of mortality and morbidities observed in AAS users are alarming. The risk of death among elite powerlifters has been reported to be fivefold greater than in age-matched men from the general population. The causes of death among powerlifters included suicides, myocardial infarction, hepatic coma, and non-Hodgkin’s lymphoma.

1	Numerous reports of cardiac death among young AAS users raise concerns about the adverse cardiovascular effects of AAS. High doses of AAS may induce proatherogenic dyslipidemia, increase thrombosis risk via effects on clotting factors and platelets, induce vasospasm through their effects on vascular nitric oxide, and induce myocardial hypertrophy and fibrosis. Replacement doses of testosterone, when administered parenterally, are associated with only a small decrease in high-density lipoprotein (HDL) cholesterol and little or no effect on total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglyceride levels. In contrast, supraphysiologic doses of testosterone and orally administered, 17-α-alkylated, nonaromatizable AAS are associated with marked reductions in HDL cholesterol and increases in LDL cholesterol.

1	Long-term AAS use may be associated with myocardial hypertrophy and fibrosis as well as shortening of QT intervals. AAS use suppresses LH and FSH secretion and inhibits endogenous testosterone production and spermatogenesis. Consequently, stopping AAS may be associated with sexual dysfunction, fatigue, infertility, and depressive symptoms.

1	In some AAS users, hypothalamic-pituitary-testicular axis suppres-7e-3 sion may last more than a year, and in a few individuals, complete recovery may not occur. The symptoms of androgen deficiency during AAS withdrawal may cause some men to revert back to using AAS, leading to continued use and AAS dependence. As many as 30% of AAS users develop a syndrome of AAS dependence, characterized by long-term AAS use, despite adverse medical and psychiatric effects. Supraphysiologic doses of testosterone may also impair insulin sensitivity, predisposing to diabetes. Elevated liver enzymes, cholestatic jaundice, hepatic neoplasms, and peliosis hepatis have been reported with oral 17-α-alkylated AAS. AAS use may cause muscle hypertrophy without compensatory adaptations in tendons, ligaments, and joints, thus increasing the risk of tendon and joint injuries. AAS use is associated with acne, baldness, and increased body hair.

1	Unsafe injection practices, high-risk behaviors, and increased rates of incarceration render AAS users at increased risk of HIV and hepatitis B and C. In one survey, nearly 1 in 10 gay men had injected AAS or other substances, and AAS users were more likely to report high-risk unprotected anal sex than other men. Some AAS users develop hypomanic and manic symptoms during AAS exposure (irritability, aggressiveness, reckless behavior, and occasional psychotic symptoms, sometimes associated with violence) and major depression (sometimes associated with suicidality) during AAS withdrawal. Users may also develop other forms of illicit drug use, which may be potentiated or exacerbated by AAS. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: AAS users generally mistrust physicians and seek medical help infrequently; when they do seek medical help, it is often for the treatment of AAS withdrawal syndrome, infertility, gynecomastia, or other medical or psychiatric complications of AAS use. The suspicion of AAS use should be raised by increased hemoglobin and hematocrit levels; suppressed luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels; low HDL cholesterol; and low testicular volume and sperm density in a person who looks highly muscular (Table 7e-3). A combination of these findings and a self-report of AAS use by the patient, which usually can be elicited by a tactful interview, are often sufficient to establish a diagnosis in clinical practice.

1	Accredited laboratories use gas chromatography and mass spectrometry or liquid chromatography and mass spectrometry to detect AAS abuse. In recent years, the availability of high-resolution mass spectrometry and tandem mass spectrometry has further improved the sensitivity of detecting AAS abuse. Illicit testosterone use is most often detected by the urinary testosterone-toepitestosterone ratio and further confirmed by the use of the 13C:12C deteCtion of the use of anaboliC-androgeniC steroids Clinical indicators that should raise suspicion of anabolic-androgenic Detection of anabolic-androgenic steroids LC-MS/MS analysis of urine Detection of exogenous testosterone use Isotope ratio mass spectrometry analysis to detect differences in 13C:12C ratio in exogenous and endogenous testosterone Abbreviations: FSH, follicle-stimulating hormone; LC-MS/MS, liquid chromatography and tandem mass spectrometry; LH, luteinizing hormone.

1	Abbreviations: FSH, follicle-stimulating hormone; LC-MS/MS, liquid chromatography and tandem mass spectrometry; LH, luteinizing hormone. ratio in testosterone by the use of isotope ratio combustion mass spectrometry. Exogenous testosterone administration increases urinary testosterone glucuronide excretion and, consequently, the testosterone-to-epitestosterone ratio. Ratios above 4 suggest exogenous testosterone use but can also reflect genetic variation. Genetic variations in the uridine diphosphoglucuronyl transferase 2B17 (UGT2B17), the major enzyme for testosterone glucuronidation, affect the testosterone-to-epitestosterone ratio. Synthetic testosterone has a lower 13C:12C ratio than endogenously produced testosterone, and these differences can be detected by isotope ratio combustion mass spectrometry.

1	The nonathlete weightlifters who abuse AAS rarely seek medical treatment and do not typically view these drugs and the associated lifestyle as deleterious to their health. In turn, many internists erroneously view AAS abuse as largely a problem of cheating in competitive sports, whereas, in fact, most AAS users are not athletes. Also, physicians often have a poor understanding of the factors motivating the use of these performance-enhancing drugs, the long-term health effects of AAS, and the associated psychopathologies that may affect treatment choices.

1	In addition to treating the underlying body dysmorphia disorder that motivates the use of these drugs, the treatment should be directed at the symptoms or the condition for which the patient seeks therapy, such as infertility, sexual dysfunction, gynecomastia, or depressive symptoms. Accordingly, therapy may include some combination of cognitive and behavioral therapy for the muscle dysmorphia syndrome, antidepressant therapy for depression, selective phosphodiesterase-5 inhibitors for erectile dysfunction, selective estrogen receptor modulators or aromatase inhibitors to reactivate the hypothalamic-pituitary-testicular axis, or hCG to restore testosterone levels. Clomiphene citrate, a partial estrogen receptor agonist, administered in a dose of 25–50 mg on alternate days, can increase LH and FSH levels and restore testosterone levels in a vast majority of men with AAS withdrawal syndrome. However, the recovery of sexual function during clomiphene administration is variable despite

1	LH and FSH levels and restore testosterone levels in a vast majority of men with AAS withdrawal syndrome. However, the recovery of sexual function during clomiphene administration is variable despite improvements in testosterone levels. Anecdotally, other aromatase inhibitors, such as anastrozole, have also been used. hCG, administered by intramuscular injections of 750–1500 IU three times each week, can raise testosterone levels into the normal range. Some patients may not respond to either clomiphene or hCG therapy, raising the possibility of irreversible long-term toxic effects of AAS on Leydig cell function.

1	Lower urinary tract symptoms (LUTS) in men include storage symptoms (urgency, daytime and nighttime frequency, and urgency incontinence), voiding disturbances (slow or intermittent stream, difficulty in initiating micturition, straining to void, pain or discomfort during the passage of urine, and terminal dribbling), or postmicturition symptoms (a sense of incomplete voiding after passing urine and postmicturition dribble). The overactive bladder syndrome refers to urgency with or without urgency incontinence, usually with urinary frequency and nocturia, and is often due to detrusor muscle overactivity. LUTS have historically been attributed to benign prostatic hyperplasia, although it has become apparent that the pathophysiologic mechanisms of LUTS are complex and multifactorial and may include structural or functional abnormalities of the bladder, bladder neck, prostate, distal sphincter mechanism, and urethra, as well as abnormalities in the neural control to the lower urinary

1	may include structural or functional abnormalities of the bladder, bladder neck, prostate, distal sphincter mechanism, and urethra, as well as abnormalities in the neural control to the lower urinary tract. A presumptive diagnosis of benign prostatic hyperplasia should be made only in men with LUTS who have demonstrable evidence of prostate enlargement and obstruction based on the size of the prostate. Diuretics, antihistamines, antidepressants, and other medications that have anticholinergic properties can cause or exacerbate LUTS in older men. The intensity of LUTS symptoms tends to fluctuate over time.

1	LUTS is highly prevalent in older men, affecting nearly 50% of men over the age of 65 and 70% of men over the age of 80. LUTS adversely affects quality of life because of its impact on sleep, ability to perform activities of daily living, and depressive symptoms. LUTS is often associated with erectile dysfunction. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: Medical evaluation should include assessment of the symptom severity using the International Prostate Symptom Score and, in some patients, a frequency-volume chart. The impact of LUTS on sleep and activities of daily living and quality of life should be evaluated. Evaluation should also include verification of medications that may contribute to LUTS, digital prostate examination, neurologic examination focused on perineum and lower extremities, urinalysis, fasting blood glucose, electrolytes, creatinine, and prostate-specific antigen (PSA). Urodynamic studies are not required in most patients but are recommended when invasive surgical therapies are being considered.

1	Men who have mild symptoms can be reassured and followed. Men with mild to moderate LUTS can be treated effectively using α-adrenergic antagonists, phosphodiesterase-5 (PDE5) inhibitors, steroid 5α-reductase inhibitors, or anticholinergic agents alone or in combination. Selective α-adrenergic antagonists are typically the first line of therapy. In men with probable benign prostate obstruction with gland enlargement and LUTS, therapy using a steroid 5a-reductase inhibitor, such as finasteride or dutasteride, for 1 or more years improves urinary symptoms and flow rate and reduces prostatic volume. Long-term treatment with 5α-reductase inhibitors can reduce progression to acute urinary retention and need for prostate surgery. Combined administration of a steroid 5α-reductase inhibitor and α1-adrenergic blocker can rapidly improve urinary symptoms and reduce the relative risk of acute urinary retention and surgery. PDE5 inhibitors, when administered chronically alone or in combination

1	α1-adrenergic blocker can rapidly improve urinary symptoms and reduce the relative risk of acute urinary retention and surgery. PDE5 inhibitors, when administered chronically alone or in combination with α-adrenergic blockers, are effective in improving LUTS and erectile dysfunction through their effects on nitric oxide– cyclic guanosine monophosphate (cGMP) in the bladder, urethra, and prostate. PDE5 inhibitors do not improve urinary flow parameters. Anticholinergic drugs are used for the treatment of overactive bladder in men with prominent urgency symptoms and no evidence of elevated postvoid residual urine. Surgery is indicated when medical therapy fails or if symptoms progress despite medical therapy.

1	Prostate cancer is the most common malignancy in American men, accounting for 29% of all diagnosed cancers and approximately 13% of all cancer deaths; its incidence is on the rise, partly due to increased screening with PSA. In 2013, approximately 233,000 new cases of prostate cancer were diagnosed in the United States and there were 29,480 deaths related to prostate cancer. The majority of these men have low-grade, organ-confined prostate cancer and excellent prospects of long-term survival. Substantial improvement in survival in men with prostate cancer has focused attention on the high prevalence of sexual dysfunction, physical dysfunction, and low vitality, which are important contributors to poor quality of life among patients treated for prostate cancer. The pathophysiology of these symptoms after radical prostatectomy is multifactorial, but denervation and androgen deficiency are important contributors to these symptoms.

1	Androgen deficiency is common in men with prostate cancer. Testosterone levels decline with age, and men with prostate cancer are at risk of having low testosterone levels simply by virtue of their age.

1	However, total and free testosterone levels are even lower in men with prostate cancer, who have undergone prostatectomy, when compared with age-matched controls without cancer. Androgen deficiency in men with prostate cancer is associated with distressing symptoms such as fatigue, sexual dysfunction, hot flushes, mobility limitation, and decreased physical function. Even with a bilateral nerve-sparing procedure, more than 50% of men develop sexual dysfunction after surgery. Although there is some recovery of sexual function with passage of time, 40–50% of men undergoing radical prostatectomy find their sexual performance to be problematic 18 months after surgery. Sexual performance problems are a source of psychosocial distress in men with localized prostate cancer. In addition to its causal contribution to distressing symptoms, androgen deficiency in men with prostate cancer increases the risk of bone fractures, diabetes, coronary heart disease, and frailty.

1	Testosterone Therapy in Men with History of Prostate Cancer A history of prostate cancer has historically been considered a contraindication for testosterone therapy. This guidance is based on observations that testosterone promotes the growth of metastatic prostate cancer. Metastatic prostate cancer generally regresses after orchidectomy and androgen deprivation therapy. Androgen receptor signaling plays a central role in maintaining growth of normal prostate and prostate cancer. PSA levels are lower in hypogonadal men and increase after testosterone therapy. Prostate volume is lower in hypogonadal men and increases after testosterone therapy to levels seen in age-matched controls.

1	However, the role of testosterone in prostate cancer is complex. Epidemiologic studies have not revealed a consistent relationship between serum testosterone and prostate cancer. In a landmark randomized trial, testosterone therapy of older men with low testosterone did not affect intraprostatic androgen levels or the expression of androgen-dependent prostatic genes. The suppression of circulating testosterone levels by a gonadotropin-releasing hormone (GnRH) antagonist also does not affect intraprostatic androgen concentrations. Open-label trials and retrospective analyses of testosterone therapy in men with prostate cancer, who have undergone radical prostatectomy 7e-5 and have undetectable PSA levels after radical prostatectomy, have found very low rates of PSA recurrence. Even in men with high-grade prostatic intraepithelial neoplasia (HGPIN)—a group at high risk of developing prostate cancer—testosterone therapy for 1 year did not increase PSA or rates of prostate cancer.

1	After radical prostatectomy, in the absence of residual cancer, PSA becomes undetectable within a month. An undetectable PSA after radical prostatectomy is a good indicator of biochemical recurrence-free survival at 5 years. Therefore, men with organ-confined prostate cancer (pT2), Gleason score ≤6, and a preoperative PSA of <10 ng/mL, who have had undetectable PSA levels (<0.1 ng/mL) for >2 years after radical prostatectomy, have very low risk of disease recurrence (<0.5% at 10 years) and may be considered for testosterone therapy on an individualized basis. If testosterone therapy is instituted, it should be associated with careful monitoring of PSA levels and done in consultation with a urologist.

1	In patients with prostate cancer and distant metastases, androgen deprivation therapy (ADT) improves survival. In patients with locally advanced disease, ADT in combination with external-beam radiation or as an adjuvant therapy (after prostatectomy and pelvic lymphadenectomy) also has been shown to improve survival. However, ADT is being increasingly used as primary therapy in men with localized disease and in men encountering biochemical recurrence without clear evidence of survival advantage. Because most men with prostate cancer die of conditions other than their primary malignancy, recognition and management of these adverse effects is paramount.

1	Profound hypogonadism resulting from ADT is associated with sexual dysfunction, vasomotor symptoms, gynecomastia, decreased muscle mass and strength, frailty, increased fat mass, anemia, fatigue, bone loss, loss of body hair, depressive symptoms, and reduced quality of life. Diabetes and cardiovascular disease have recently been added to the list of these complications (Fig. 7e-3). Treatment with GnRH Any fracture (1.54) Shahinian et al. 2005, NEJM Fracture requiring hospitalization (1.66) Diabetes (1.44) Keating et al. 2006, JCO Myocardial infarction (1.11) Peripheral vascular disease (1.16) Keating et al. 2006, JCO Coronary heart disease (1.16) Hu et al. 2012, Eur Urol Sudden death (1.16)

1	Keating et al. 2006, JCO Myocardial infarction (1.11) Peripheral vascular disease (1.16) Keating et al. 2006, JCO Coronary heart disease (1.16) Hu et al. 2012, Eur Urol Sudden death (1.16) FIGURE 7e-3 Adverse cardiometabolic and skeletal effects of androgen deprivation therapy (ADT) in men receiving ADT for prostate cancer. Administration of ADT has been associated with increased risk of thromboembolic events, fractures, and diabetes. Some, but not all, studies have reported increased risk of cardiovascular events in men receiving ADT. (Data on relative risk were derived from VB Shahinian et al: N Engl J Med 352:154, 2005; NL Keating et al: J Clin Oncol 24:4448, 2006; and JC Hu et al: Eur Urol 61:1119, 2012.) 1. Weigh the risks and benefits of ADT and whether intermittent ADT is a feasible and safe option. 2. Perform a baseline assessment including fasting glucose, plasma lipids, blood pressure, bone mineral density, and FRAX® score.

1	2. Perform a baseline assessment including fasting glucose, plasma lipids, blood pressure, bone mineral density, and FRAX® score. 3. Optimize calcium and vitamin D intake, encourage structured physical activity and exercise, and consider pharmacologic therapy in men with a previous minimal trauma fracture and those with a 10-year risk of a major osteoporotic fracture >20%, unless contraindicated. 4. Monitor body weight, fasting glucose, plasma lipids, blood pressure, and bone mineral density, and encourage smoking cessation and physical activity. 5. In men who are receiving ADT and who experience bothersome hot flushes, as indicated by sleep disturbance or interference with work or activities of daily living, consider initial therapy with venlafaxine. If in effective, add medroxyprogesterone acetate. 6. In men who experience painful breast enlargement, consider therapy with an estrogen receptor antagonist, such as tamoxifen.

1	6. In men who experience painful breast enlargement, consider therapy with an estrogen receptor antagonist, such as tamoxifen. agonists in men with prostate cancer is associated with rapid induction of insulin resistance, hyperinsulinemia, and a significant increase in the risk of incident diabetes. Metabolic syndrome is prevalent in over 50% of men undergoing long-term ADT. Some but not all studies have reported an increased risk of cardiovascular events, death due to cardiovascular events, and peripheral vascular disease in men undergoing ADT. Men receiving ADT are also at increased risk of thromboembolic events. The rates of acute kidney injury are higher in men currently receiving ADT than in men not receiving ADT; the increased risk appears to be particularly associated with the use of combined regimens of a GnRH agonist plus an antiandrogen. ADT also is associated with substantially increased risk of osteoporosis and bone fractures. APPROACH TO THE PATIENT:

1	The benefits of ADT in treating nonmetastatic prostate cancer should be carefully weighed against the risks of ADT-induced adverse events (Table 7e-4). If ADT is medically indicated, consider whether intermittent ADT is a feasible option. Men being considered for ADT should undergo assessment of cardiovascular, diabetes, and fracture risk; this assessment may include measurement of blood glucose, plasma lipids, and bone mineral density (BMD) by dual-energy x-ray absorptiometry. Institute measures to prevent bone loss, including physical activity, adequate calcium and vitamin D intake, and pharmacologic therapy in men with a previous minimal trauma fracture and those with a 10-year risk of a major osteoporotic fracture >20%, unless contraindicated. Men with prostate cancer who are receiving ADT should be monitored for weight gain and diabetes. Encourage lifestyle interventions, including physical activity and exercise, and attention to weight, blood pressure, lipid profile, blood

1	ADT should be monitored for weight gain and diabetes. Encourage lifestyle interventions, including physical activity and exercise, and attention to weight, blood pressure, lipid profile, blood glucose, and smoking cessation, to reduce the risk of cardiometabolic complications. In randomized trials, medroxyprogesterone, cyproterone acetate, and the selective serotonin reuptake inhibitor venlafaxine have been shown to be more efficacious than placebo in alleviating hot flushes. The side effects of these medications, including increased appetite and weight gain with medroxyprogesterone, gynecomastia with estrogenic compounds, and dry mouth with venlafaxine, should be weighed against their relative efficacy. Acupuncture, soy products, vitamin E, and herbal medicines have been used empirically for the treatment of vasomotor symptoms without clear evidence of efficacy. Gynecomastia can be prevented by local radiation therapy or the use of an antiestrogen or an aromatase inhibitor; these

1	for the treatment of vasomotor symptoms without clear evidence of efficacy. Gynecomastia can be prevented by local radiation therapy or the use of an antiestrogen or an aromatase inhibitor; these therapies are effective in alleviating pain and tenderness but are less effective in reducing established gynecomastia.

1	Chapter 8 Medical Disorders During Pregnancy Medical disorders during pregnancy Robert L. Barbieri, John T. Repke Each year, approximately 4 million births occur in the United States, and more than 130 million births occur worldwide. A significant 8 proportion of births are complicated by medical disorders. In the past, many medical disorders were contraindications to pregnancy. Advances in obstetrics, neonatology, obstetric anesthesiology, and medicine have increased the expectation that pregnancy will result in a positive outcome for both mother and fetus despite most of these conditions. A successful pregnancy requires important physiologic adaptations, such as a marked increase in cardiac output. Medical problems that interfere with the physiologic adaptations of pregnancy increase the risk for poor pregnancy outcome; conversely, in some instances, pregnancy may adversely impact an underlying medical disorder.

1	(See also Chap. 298) In pregnancy, cardiac output increases by 40%, with most of the increase due to an increase in stroke volume. Heart rate increases by ~10 beats/min during the third trimester. In the second trimester, systemic vascular resistance decreases, and this decline is associated with a fall in blood pressure. During pregnancy, a blood pressure of 140/90 mmHg is considered to be abnormally elevated and is associated with an increase in perinatal morbidity and mortality. In all pregnant women, the measurement of blood pressure should be performed in the sitting position, because the lateral recumbent position may result in a blood pressure lower than that recorded in the sitting position. The diagnosis of hypertension requires the measurement of two elevated blood pressures at least 6 h apart. Hypertension during pregnancy is usually caused by preeclampsia, chronic hypertension, gestational hypertension, or renal disease.

1	Approximately 5–7% of all pregnant women develop preeclampsia, the new onset of hypertension (blood pressure >140/90 mmHg) and proteinuria (either a 24 hour urinary protein >300 mg/24 h, or a protein-creatinine ratio ≥0.3) after 20 weeks of gestation. Although the precise pathophysiology of preeclampsia remains unknown, recent studies show excessive placental production of antagonists to both vascular epithelial growth factor (VEGF) and transforming growth factor β (TGF-β). These antagonists to VEGF and TGF-β disrupt endothelial and renal glomerular function resulting in edema, hypertension, and proteinuria. The renal histological feature of preeclampsia is glomerular endotheliosis. Glomerular endothelial cells are swollen and encroach on the vascular lumen. Preeclampsia is associated with abnormalities of cerebral circulatory autoregulation, which increase the risk of stroke at mildly and moderately elevated blood pressures. Risk factors for the development of preeclampsia include

1	abnormalities of cerebral circulatory autoregulation, which increase the risk of stroke at mildly and moderately elevated blood pressures. Risk factors for the development of preeclampsia include nulliparity, diabetes mellitus, a history of renal disease or chronic hypertension, a prior history of preeclampsia, extremes of maternal age (>35 years or <15 years), obesity, antiphospholipid antibody syndrome, and multiple gestation. Low-dose aspirin (81 mg daily, initiated at the end of the first trimester) may reduce the risk of preeclampsia in pregnant women at high risk of developing the disease.

1	In December, 2013 The American College of Obstetricians and Gynecologists issued a report summarizing the findings and recommendations of their Task Force on Hypertension in Pregnancy. With respect to preeclampsia several pertinent revisions to the diagnostic criteria were made including: proteinuria is no longer an absolute requirement for making the diagnosis; the terms mild and severe preeclampsia have been replaced, and the disease is now termed preeclampsia either with or without severe features; removal of fetal growth restriction as a defining criterion for severe preeclampsia.

1	Preeclampsia with severe features is the presence of new-onset hypertension and proteinuria accompanied by end-organ damage. Features may include severe elevation of blood pressure (>160/110 mmHg), evidence of central nervous system (CNS) dysfunction (headaches, blurred vision, seizures, coma), renal dysfunction (oliguria or creatinine >1.5 mg/dL), pulmonary edema, hepatocellular injury (serum alanine aminotransferase level more than twofold the upper limit of normal), hematologic dysfunction (platelet count <100,000/L or disseminated intravascular coagulation [DIC]). The HELLP syndrome (hemolysis, elevated liver enzymes, low platelets) is a special subtype of severe preeclampsia and is a major cause of morbidity and mortality in this disease. Platelet dysfunction and coagulation disorders further increase the risk of stroke.

1	Preeclampsia resolves within a few weeks after delivery. For pregnant women with preeclampsia prior to 37 weeks of gestation, delivery reduces the mother’s morbidity but exposes the fetus to the risk of premature birth. The management of preeclampsia is challenging because it requires the clinician to balance the health of the mother and fetus simultaneously. In general, prior to term, women with mild preeclampsia without severe features may be managed conservatively with limited physical activity, although bed rest is not recommended, close monitoring of blood pressure and renal function, and careful fetal surveillance. For women with preeclampsia with severe features, delivery is recommended unless the patient is eligible for expectant management in a tertiary hospital setting. Expectant management of preeclampsia with severe features remote from term affords some benefits for the fetus but significant risks for the mother.

1	The definitive treatment of preeclampsia is delivery of the fetus and placenta. For women with preeclampsia with severe features, aggressive management of blood pressures >160/110 mmHg reduces the risk of cerebrovascular accidents. IV labetalol or hydralazine is most commonly used to acutely manage severe hypertension in preeclampsia; labetalol is associated with fewer episodes of maternal hypotension. Oral nifedipine and labetalol are commonly used to manage hypertension in pregnancy. Elevated arterial pressure should be reduced slowly to avoid hypotension and a decrease in blood flow to the fetus. Angiotensin-converting enzyme (ACE) inhibitors as well as angiotensin-receptor blockers should be avoided in the second and third trimesters of pregnancy because of their adverse effects on fetal development.

1	Magnesium sulfate is the preferred agent for the prevention and treatment of eclamptic seizures. Large, randomized clinical trials have demonstrated the superiority of magnesium sulfate over phenytoin and diazepam in reducing the risk of seizure and, possibly, the risk of maternal death. Magnesium may prevent seizures by interacting with N-methyl-D-aspartate (NMDA) receptors in the CNS. Given the difficulty of predicting eclamptic seizures on the basis of disease severity, once the decision to proceed with delivery is made, most patients carrying a diagnosis of preeclampsia should be treated with magnesium sulfate. Women who have had preeclampsia appear to be at increased risk of cardiovascular and renal disease later in life. Pregnancy complicated by chronic essential hypertension is associated with intrauterine growth restriction and increased perinatal mortality.

1	Pregnant women with chronic hypertension are at increased risk for superimposed preeclampsia and abruptio placentae. Women with chronic hypertension should have a thorough prepregnancy evaluation, both to identify remediable causes of hypertension and to ensure that the prescribed antihypertensive agents (e.g., ACE inhibitors, angiotensin-receptor blockers) are not associated with an adverse outcome of pregnancy. α-Methyldopa, labetalol, and nifedipine are the most commonly used medications for the treatment of chronic hypertension in pregnancy. The target blood pressure is in the range of 130–150 mmHg systolic and 80–100 mmHg diastolic. Should hypertension worsen during pregnancy, baseline evaluation of renal function (see below) is necessary to help differentiate the effects of chronic hypertension from those of superimposed preeclampsia. There are no convincing data that the treatment of mild chronic hypertension improves perinatal outcome.

1	The development of elevated blood pressure during pregnancy or in the first 24 h post-partum in the absence of preexisting chronic hypertension or proteinuria is referred to as gestational hypertension. Mild gestational hypertension that does not progress to preeclampsia has not been associated with adverse pregnancy outcome or adverse long-term prognosis.

1	(See also Chaps. 333 and 341) Normal pregnancy is characterized by an increase in glomerular filtration rate and creatinine clearance. This increase occurs secondary to a rise in renal plasma flow and increased glomerular filtration pressures. Patients with underlying renal disease and hypertension may expect a worsening of hypertension during pregnancy. If superimposed preeclampsia develops, the additional endothelial injury results in a capillary leak syndrome that may make management challenging. In general, patients with underlying renal disease and hypertension benefit from aggressive management of blood pressure. Preconception counseling is also essential for these patients so that accurate risk assessment and medication changes can occur prior to pregnancy. In general, a prepregnancy serum creatinine level <133 μmol/L (<1.5 mg/dL) is associated with a favorable prognosis. When renal disease worsens during pregnancy, close collaboration between the internist and the

1	a prepregnancy serum creatinine level <133 μmol/L (<1.5 mg/dL) is associated with a favorable prognosis. When renal disease worsens during pregnancy, close collaboration between the internist and the maternal-fetal medicine specialist is essential so that decisions regarding delivery can be weighed to balance the sequelae of prematurity for the neonate versus long-term sequelae for the mother with respect to future renal function.

1	(See also Chaps. 283–286) Valvular heart disease is the most common cardiac problem complicating pregnancy.

1	Mitral Stenosis This is the valvular disease most likely to cause death during pregnancy. The pregnancy-induced increase in blood volume, cardiac output, and tachycardia can increase the transmitral pressure gradient and cause pulmonary edema in women with mitral stenosis. Women with moderate to severe mitral stenosis who are planning pregnancy and have either symptomatic disease or pulmonary hypertension should undergo valvuloplasty prior to conception. Pregnancy associated with long-standing mitral stenosis may result in pulmonary hypertension. Sudden death has been reported when hypovolemia occurs. Careful control of heart rate, especially during labor and delivery, minimizes the impact of tachycardia and reduced ventricular filling times on cardiac function. Pregnant women with mitral stenosis are at increased risk for the development of atrial fibrillation and other tachyarrhythmias. Medical management of severe mitral stenosis and atrial fibrillation with digoxin and beta

1	mitral stenosis are at increased risk for the development of atrial fibrillation and other tachyarrhythmias. Medical management of severe mitral stenosis and atrial fibrillation with digoxin and beta blockers is recommended. Balloon valvulotomy can be carried out during pregnancy. The immediate postpartum period is a time of particular concern secondary to rapid volume shifts. Careful monitoring of cardiac and fluid status should be observed.

1	Mitral Regurgitation and Aortic Regurgitation and Stenosis The pregnancy-induced decrease in systemic vascular resistance reduces the risk of cardiac failure with these conditions. As a rule, mitral valve prolapse does not present problems for the pregnant patient, and aortic stenosis, unless very severe, is well tolerated. In the most severe cases of aortic stenosis, limitation of activity or balloon valvuloplasty may be indicated.

1	(See also Chap. 282) Reparative surgery has markedly increased the number of women with surgically repaired congenital heart disease. Maternal morbidity and mortality are greater among these women than among those without surgical repairs. When pregnant, these patients should be jointly managed by a cardiologist and an obstetrician familiar with these problems. The presence of a congenital cardiac lesion in the mother increases the risk of congenital cardiac disease in the newborn. Prenatal screening of the fetus for congenital cardiac disease with ultrasound is recommended. Atrial or ventricular septal defect is usually well tolerated during pregnancy in the absence of pulmonary hypertension, provided that the woman’s prepregnancy cardiac status is favorable. Use of air filters on IV sets during labor and delivery in patients with intracardiac shunts is recommended.

1	Supraventricular tachycardia (Chap. 276) is a common cardiac complication of pregnancy. Treatment is the same as in the nonpregnant patient, and fetal tolerance of medications such as adenosine and calcium channel blockers is acceptable. When necessary, pharmacologic or electric cardioversion may be performed to improve cardiac performance and reduce symptoms. This intervention is generally well tolerated by mother and fetus. Peripartum cardiomyopathy (Chap. 287) is an uncommon disorder of pregnancy associated with myocarditis, and its etiology remains unknown. Treatment is directed toward symptomatic relief and improvement of cardiac function. Many patients recover completely; others are left with progressive dilated cardiomyopathy. Recurrence in a subsequent pregnancy has been reported, and women who do not have normal baseline left-ventricular function after an episode of peripartum cardiomyopathy should be counseled to avoid pregnancy.

1	SPECIFIC HIgH-RISK CARdIAC LESIoNS Marfan Syndrome (See also Chap. 427) This autosomal dominant disease is associated with a high risk of maternal morbidity. Approximately 15% of pregnant women with Marfan syndrome develop a major cardiovascular manifestation during pregnancy, with almost all women surviving. An aortic root diameter <40 mm is associated with a favorable outcome of pregnancy. Prophylactic therapy with beta blockers has been advocated, although large-scale clinical trials in pregnancy have not been performed. Ehlers-Danlos syndrome (EDS) may be associated with premature labor, and in type IV EDS there is increased risk of organ or vascular rupture that may cause death.

1	Pulmonary Hypertension (See also Chap. 304) Maternal mortality in the setting of severe pulmonary hypertension is high, and primary pulmonary hypertension is a contraindication to pregnancy. Termination of pregnancy may be advisable in these circumstances to preserve the life of the mother. In the Eisenmenger syndrome, i.e., the combination of pulmonary hypertension with right-to-left shunting due to congenital abnormalities (Chap. 282), maternal and fetal deaths occur frequently. Systemic hypotension may occur after blood loss, prolonged Valsalva maneuver, or regional anesthesia; sudden death secondary to hypotension is a dreaded complication. Management of these patients is challenging, and invasive hemodynamic monitoring during labor and delivery is recommended in severe cases. In patients with pulmonary hypertension, vaginal delivery is less stressful hemodynamically than cesarean section, which should be reserved for accepted obstetric indications.

1	(See also Chap. 300) A hypercoagulable state is characteristic of pregnancy, and deep venous thrombosis (DVT) occurs in about 1 in 500 pregnancies. In pregnant women, most unilateral DVTs occur in the left leg because the left iliac vein is compressed by the right iliac artery and the uterus compresses the inferior vena cava. Pregnancy is associated with an increase in procoagulants such as factors V and VII and a decrease in anticoagulant activity, including proteins C and S. Pulmonary embolism is one of the most common causes of maternal death in the United States. Activated protein C resistance caused by the factor V Leiden mutation increases the risk for DVT and pulmonary embolism during pregnancy. Approximately 25% of women with DVT during pregnancy carry the factor V Leiden allele. Additional genetic mutations associated with DVT during pregnancy include the prothrombin G20210A mutation (heterozygotes and homozygotes) and the methylenetetrahydrofolate reductase C677T mutation

1	Additional genetic mutations associated with DVT during pregnancy include the prothrombin G20210A mutation (heterozygotes and homozygotes) and the methylenetetrahydrofolate reductase C677T mutation (homozygotes).

1	Aggressive diagnosis and management of DVT and suspected pulmonary embolism optimize the outcome for mother and fetus. In general, all diagnostic and therapeutic modalities afforded the non-pregnant patient should be utilized in pregnancy except for D-dimer measurement, in which values are elevated in normal pregnancy. Anticoagulant therapy with low-molecular-weight heparin (LMWH) or unfractionated heparin is indicated in pregnant women with DVT. LMWH may be associated with an increased risk of epidural hematoma in women receiving an epidural anesthetic in labor. Four weeks prior to anticipated delivery, LMWH should be switched to unfractionated heparin. Warfarin therapy is contraindicated in the first trimester due to its association with fetal chondrodysplasia punctata. In the second and third trimesters, warfarin may cause fetal optic atrophy and mental retardation. When DVT occurs in the postpartum period, LMWH therapy for 7–10 days may be followed by warfarin therapy for 3–6

1	and third trimesters, warfarin may cause fetal optic atrophy and mental retardation. When DVT occurs in the postpartum period, LMWH therapy for 7–10 days may be followed by warfarin therapy for 3–6 months. Warfarin is not contraindicated in breast-feeding women. For women at moderate or high risk of DVT who have a cesarean delivery, mechanical and/or pharmacologic prophylaxis is warranted.

1	(See also Chaps. 417–419) In pregnancy, the fetoplacental unit induces major metabolic changes, the purpose of which is to shunt glucose and amino acids to the fetus while the mother uses ketones and triglycerides to fuel her metabolic needs. These metabolic changes are accompanied by maternal insulin resistance caused in part by placental production of steroids, a growth hormone variant, and placental lactogen. Although pregnancy has been referred to as a state of “accelerated starvation,” it is better characterized as “accelerated ketosis.” In pregnancy, after an overnight fast, plasma glucose is lower by 0.8–1.1 mmol/L (15–20 mg/dL) than in the nonpregnant state. This difference is due to the use of glucose by the fetus. In early pregnancy, fasting may result in circulating glucose concentrations in the range of 2.2 mmol/L (40 mg/dL) and may be associated with symptoms of hypoglycemia. In contrast to the decrease in maternal glucose concentration, plasma hydroxybutyrate and

1	concentrations in the range of 2.2 mmol/L (40 mg/dL) and may be associated with symptoms of hypoglycemia. In contrast to the decrease in maternal glucose concentration, plasma hydroxybutyrate and acetoacetate levels rise to two to four times normal after a fast.

1	Pregnancy complicated by diabetes mellitus is associated with higher maternal and perinatal morbidity and mortality rates. Preconception counseling and treatment are important for the diabetic patient contemplating pregnancy and can reduce the risk of congenital malformations and improve pregnancy outcome. Folate supplementation reduces the incidence of fetal neural tube defects, which occur with greater frequency in fetuses of diabetic mothers. In addition, optimizing glucose control during key periods of organogenesis reduces other congenital anomalies, including sacral agenesis, caudal dysplasia, renal agenesis, and ventricular septal defect.

1	Once pregnancy is established, glucose control should be managed more aggressively than in the nonpregnant state. In addition to dietary changes, this enhanced management requires more frequent blood glucose monitoring and often involves additional injections of insulin or conversion to an insulin pump. Fasting blood glucose levels should be maintained at <5.8 mmol/L (<105 mg/dL), with avoidance of values >7.8 mmol/L (140 mg/dL). Commencing in the third trimester, regular surveillance of maternal glucose control as well as assessment of fetal growth (obstetric sonography) and fetoplacental oxygenation (fetal heart rate monitoring or biophysical profile) optimize pregnancy outcome. Pregnant diabetic patients without vascular disease are at greater risk for delivering a macrosomic fetus, and attention to fetal growth via clinical and ultrasound examination is important. Fetal macrosomia is associated with an increased risk of maternal and fetal birth trauma, including permanent newborn

1	attention to fetal growth via clinical and ultrasound examination is important. Fetal macrosomia is associated with an increased risk of maternal and fetal birth trauma, including permanent newborn Erb’s palsy. Pregnant women with diabetes have an increased risk of developing preeclampsia, and those with vascular disease are at greater risk for developing intrauterine growth restriction, which is associated with an increased risk of fetal and neonatal death. Excellent pregnancy outcomes in patients with diabetic nephropathy and proliferative retinopathy have been reported with aggressive glucose control and intensive maternal and fetal surveillance.

1	As pregnancy progresses, glycemic control may become more difficult to achieve due to an increase in insulin resistance. Because of delayed pulmonary maturation of the fetuses of diabetic mothers, early delivery should be avoided unless there is biochemical evidence of fetal lung maturity. In general, efforts to control glucose and avoid preterm delivery result in the best overall outcome for both mother and newborn. Preterm delivery is generally performed only for the usual obstetric indications (e.g., preeclampsia, fetal growth restriction, non-reassuring fetal testing) or for worsening maternal renal or active proliferative retinopathy.

1	Gestational diabetes occurs in approximately 4% of pregnancies. All pregnant women should be screened for gestational diabetes unless they are in a low-risk group. Women at low risk for gestational diabetes are those <25 years of age; those with a body mass index <25 kg/m2, no maternal history of macrosomia or gestational diabetes, and no diabetes in a first-degree relative; and those who are not members of a high-risk ethnic group (African American, Hispanic, Native American). A typical two-step strategy for establishing the diagnosis of gestational diabetes involves administration of a 50-g oral glucose challenge with a single serum glucose measurement at 60 min. If the plasma glucose is <7.8 mmol/L (<130 mg/dL), the test is considered normal. Plasma glucose >7.8 mmol/L (>130 mg/dL) warrants administration of a 100-g oral glucose challenge with plasma glucose measurements obtained in the fasting state and at 1, 2, and 3 h. Normal plasma glucose concentrations at these time points

1	administration of a 100-g oral glucose challenge with plasma glucose measurements obtained in the fasting state and at 1, 2, and 3 h. Normal plasma glucose concentrations at these time points are <5.8 mmol/L (<105 mg/dL), 10.5 mmol/L (190 mg/dL), 9.1 mmol/L (165 mg/dL), and 8.0 mmol/L (145 mg/dL), respectively. Some centers have adopted more sensitive criteria, using values of <5.3 mmol/L (<95 mg/dL), <10 mmol/L (<180 mg/dL), <8.6 mmol/L (<155 mg/dL), and <7.8 mmol/L (<140 mg/dL) as the upper norms for a 3-h glucose tolerance test. Two elevated glucose values indicate a positive test. Adverse pregnancy outcomes for mother and fetus appear to increase with glucose as a continuous variable; thus it is challenging to define the optimal threshold for establishing the diagnosis of gestational diabetes.

1	Pregnant women with gestational diabetes are at increased risk of stillbirth, preeclampsia, and delivery of infants who are large for their gestational age, with resulting birth lacerations, shoulder dystocia, and birth trauma including brachial plexus injury. These fetuses are at risk of hypoglycemia, hyperbilirubinemia, and polycythemia. Tight control of blood sugar during pregnancy and labor can reduce these risks.

1	Treatment of gestational diabetes with a two-step strategy—dietary intervention followed by insulin injections if diet alone does not adequately control blood sugar [fasting glucose <5.6 mmol/L (<100 mg/dL) and 2-h postprandial glucose <7.0 mmol/L (<126 mg/dL)]— is associated with a decreased risk of birth trauma for the fetus. Oral hypoglycemic agents such as glyburide and metformin have become more commonly utilized for managing gestational diabetes refractory to nutritional management, but many experts favor insulin therapy. For women with gestational diabetes, there is a 40% risk of being diagnosed with diabetes within the 10 years after the index pregnancy. In women with a history of gestational diabetes, exercise, weight loss, and treatment with metformin reduce the risk of developing diabetes. All women with a history of gestational diabetes should be counseled about prevention strategies and evaluated regularly for diabetes.

1	(See also Chap. 416) Pregnant women who are obese have an increased risk of stillbirth, congenital fetal malformations, gestational diabetes, preeclampsia, urinary tract infections, post-date delivery, and cesarean delivery. Women contemplating pregnancy should attempt to attain a healthy weight prior to conception. For morbidly obese women who have not been able to lose weight with lifestyle changes, bariatric surgery may result in weight loss and improve pregnancy outcomes. Following bariatric surgery, women should delay conception for 1 year to avoid pregnancy during an interval of rapid metabolic changes. (See also Chap. 405) In pregnancy, the estrogen-induced increase in thyroxine-binding globulin increases circulating levels of total T3 and total T4. The normal range of circulating levels of free T4, free T3, and thyroid-stimulating hormone (TSH) remain unaltered by pregnancy.

1	The thyroid gland normally enlarges during pregnancy. Many physiologic adaptations to pregnancy may mimic subtle signs of hyperthyroidism. Maternal hyperthyroidism occurs at a rate of ~2 per 1000 pregnancies and is generally well tolerated by pregnant women. Clinical signs and symptoms should alert the physician to the occurrence of this condition. Hyperthyroidism in pregnancy is most commonly caused by Graves’ disease, but autonomously functioning nodules and gestational trophoblastic disease should also be considered. Although pregnant women are able to tolerate mild hyperthyroidism without adverse sequelae, more severe hyperthyroidism can cause spontaneous abortion or premature labor, and thyroid storm is associated with a significant risk of maternal death.

1	Testing for hypothyroidism using TSH measurements before or early in pregnancy may be warranted in symptomatic women and in women with a personal or family history of thyroid disease. With use of this case-finding approach, about 30% of pregnant women with mild hypothyroidism remain undiagnosed, leading some to recommend universal screening. Children born to women with an elevated serum TSH (and a normal total thyroxine) during pregnancy may have impaired performance on neuropsychologic tests.

1	Methimazole crosses the placenta to a greater degree than propylthiouracil and has been associated with fetal aplasia cutis. However, propylthiouracil can be associated with liver failure. Some experts recommend propylthiouracil in the first trimester and methimazole thereafter. Radioiodine should not be used during pregnancy, either for scanning or for treatment, because of effects on the fetal thyroid. In emergent circumstances, additional treatment with beta blockers may be necessary. Hyperthyroidism is most difficult to control in the first trimester of pregnancy and easiest to control in the third trimester.

1	The goal of therapy for hypothyroidism is to maintain the serum TSH in the normal range, and thyroxine is the drug of choice. During pregnancy, the dose of thyroxine required to keep the TSH in the normal range rises. In one study, the mean replacement dose of thyroxine required to maintain the TSH in the normal range was 0.1 mg daily before pregnancy and increased to 0.15 mg daily during pregnancy. Since the increased thyroxine requirement occurs as early as the fifth week of pregnancy, one approach is to increase the thyroxine dose by 30% (two additional pills weekly) as soon as pregnancy is diagnosed and then adjust the dose by serial measurements of TSH. Pregnancy has been described as a state of physiologic anemia. Part of the reduction in hemoglobin concentration is dilutional, but iron and folate deficiencies are major causes of correctable anemia during pregnancy.

1	In populations at high risk for hemoglobinopathies (Chap. 127), hemoglobin electrophoresis should be performed as part of the prenatal screen. Hemoglobinopathies can be associated with increased maternal and fetal morbidity and mortality. Management is tailored to the specific hemoglobinopathy and is generally the same for both pregnant and nonpregnant women. Prenatal diagnosis of hemoglobinopathies in the fetus is readily available and should be discussed with prospective parents either prior to or early in pregnancy.

1	Thrombocytopenia occurs commonly during pregnancy. The majority of cases are benign gestational thrombocytopenias, but the differential diagnosis should include immune thrombocytopenia (Chap. 140), thrombotic thrombocytopenic purpura, and preeclampsia. Maternal thrombocytopenia may also be caused by DIC, which is a consumptive coagulopathy characterized by thrombocytopenia, prolonged prothrombin time (PT) and activated partial thromboplastin time (aPTT), elevated fibrin degradation products, and a low fibrinogen concentration. Several catastrophic obstetric events are associated with the development of DIC, including retention of a dead fetus, sepsis, abruptio placentae, and amniotic fluid embolism.

1	Headache appearing during pregnancy is usually due to migraine (Chap. 21), a condition that may worsen, improve, or be unaffected by pregnancy. A new or worsening headache, particularly if associated with visual blurring, may signal eclampsia (above) or pseudotumor cerebri (benign intracranial hypertension); diplopia due to a sixth-nerve palsy suggests pseudotumor cerebri (Chap. 39). The risk of seizures in patients with epilepsy increases in the postpartum period but not consistently during pregnancy; management is discussed in Chap. 445. The risk of stroke is generally thought to increase during pregnancy because of a hypercoagulable state; however, studies suggest that the period of risk occurs primarily in the postpartum period and that both ischemic and hemorrhagic strokes may occur at this time. Guidelines for use of heparin therapy are summarized above (see “Deep Venous Thrombosis and Pulmonary Embolism”); warfarin is teratogenic and should be avoided.

1	The onset of a new movement disorder during pregnancy suggests chorea gravidarum, a variant of Sydenham’s chorea associated with rheumatic fever and streptococcal infection (Chap. 381); the chorea may recur with subsequent pregnancies. Patients with preexisting multiple sclerosis (Chap. 458) experience a gradual decrease in the risk of relapses as pregnancy progresses and, conversely, an increase in attack risk during the postpartum period. Disease-modifying agents, including interferon β, should not be administered to pregnant multiple sclerosis patients, but moderate or severe relapses can be safely treated with pulse glucocorticoid therapy. Finally, certain tumors, particularly pituitary adenoma and meningioma (Chap. 403), may manifest during pregnancy because of accelerated growth, possibly driven by hormonal factors.

1	Peripheral nerve disorders associated with pregnancy include Bell’s palsy (idiopathic facial paralysis) (Chap. 459), which is approximately threefold more likely to occur during the third trimester and immediate postpartum period than in the general population. Therapy with glucocorticoids should follow the guidelines established for non-pregnant patients. Entrapment neuropathies are common in the later stages of pregnancy, presumably as a result of fluid retention. Carpal tunnel syndrome (median nerve) presents first as pain and paresthesia in the hand (often worse at night) and later with weakness in the thenar muscles. Treatment is generally conservative; wrist splints may be helpful, and glucocorticoid injections or surgical section of the carpal tunnel can usually be postponed. Meralgia paresthetica (lateral femoral cutaneous nerve entrapment) consists of pain and numbness in the lateral aspect of the thigh without weakness. Patients are usually reassured to learn that these

1	paresthetica (lateral femoral cutaneous nerve entrapment) consists of pain and numbness in the lateral aspect of the thigh without weakness. Patients are usually reassured to learn that these symptoms are benign and can be expected to remit spontaneously after the pregnancy has been completed. Restless leg syndrome is the most common peripheral nerve and movement disorder in pregnancy. Disordered iron metabolism is the suspected etiology. Management is expectant in most cases.

1	Up to 90% of pregnant women experience nausea and vomiting during the first trimester of pregnancy. Hyperemesis gravidarum is a severe form that prevents adequate fluid and nutritional intake and may require hospitalization to prevent dehydration and malnutrition. Crohn’s disease may be associated with exacerbations in the second and third trimesters. Ulcerative colitis is associated with disease exacerbations in the first trimester and during the early postpartum period. Medical management of these diseases during pregnancy is similar to management in the nonpregnant state (Chap. 351).

1	Exacerbation of gallbladder disease is common during pregnancy. In part, this aggravation may be due to pregnancy-induced alteration in the metabolism of bile and fatty acids. Intrahepatic cholestasis of pregnancy is generally a third-trimester event. Profound pruritus may accompany this condition, and it may be associated with increased fetal mortality. Placental bile salt deposition may contribute to progressive uteroplacental insufficiency. Therefore, regular fetal surveillance should be undertaken once the diagnosis of intrahepatic cholestasis is made, and delivery should be planned once the fetus reaches about 37 weeks of gestation. Favorable results with ursodiol have been reported.

1	Acute fatty liver is a rare complication of pregnancy. Frequently confused with the HELLP syndrome (see “Preeclampsia” above) and severe preeclampsia, the diagnosis of acute fatty liver of pregnancy may be facilitated by imaging studies and laboratory evaluation. Acute fatty liver of pregnancy is generally characterized by markedly increased serum levels of bilirubin and ammonia and by hypoglycemia. Management of acute fatty liver of pregnancy is supportive; recurrence in subsequent pregnancies has been reported. All pregnant women should be screened for hepatitis B. This information is important for pediatricians after delivery of the infant. All infants receive hepatitis B vaccine. Infants born to mothers who are carriers of hepatitis B surface antigen should also receive hepatitis B immune globulin as soon after birth as possible and preferably within the first 72 h. Screening for hepatitis C is recommended for individuals at high risk for exposure.

1	Other than bacterial vaginosis, the most common bacterial infections during pregnancy involve the urinary tract (Chap. 162). Many pregnant women have asymptomatic bacteriuria, most likely due to stasis caused by progestational effects on ureteral and bladder smooth muscle and later in pregnancy due to compression effects of the enlarging uterus. In itself, this condition is not associated with an adverse outcome of pregnancy. However, if asymptomatic bacteriuria is left untreated, symptomatic pyelonephritis may occur. Indeed, ~75% of pregnancy-associated pyelonephritis cases are the result of untreated asymptomatic bacteriuria. All pregnant women should be screened with a urine culture for asymptomatic bacteriuria at the first prenatal visit. Subsequent screening with nitrite/leukocyte esterase strips is indicated for high-risk women, such as those with sickle cell trait or a history of urinary tract infections. All women with positive screens should be treated. Pregnant women who

1	esterase strips is indicated for high-risk women, such as those with sickle cell trait or a history of urinary tract infections. All women with positive screens should be treated. Pregnant women who develop pyelonephritis need careful monitoring, including inpatient IV antibiotic administration due to the elevated risk of urosepsis and acute respiratory distress syndrome in pregnancy.

1	Abdominal pain and fever during pregnancy create a clinical dilemma. The diagnosis of greatest concern is intrauterine amniotic infection. While amniotic infection most commonly follows rupture of the membranes, this is not always the case. In general, antibiotic therapy is not recommended as a temporizing measure in these circumstances. If intrauterine infection is suspected, induced delivery with concomitant antibiotic therapy is generally indicated. Intrauterine amniotic infection is most often caused by pathogens such as Escherichia coli and group B Streptococcus (GBS). In high-risk patients at term or in preterm patients, routine intrapartum prophylaxis of GBS disease is recommended. Penicillin G and ampicillin are the drugs of choice. In penicillin-allergic patients with a low risk of anaphylaxis, cefazolin is recommended. If the patient is at high risk of anaphylaxis, vancomycin is recommended. If the organism is known to be sensitive to clindamycin, this antibiotic may be

1	of anaphylaxis, cefazolin is recommended. If the patient is at high risk of anaphylaxis, vancomycin is recommended. If the organism is known to be sensitive to clindamycin, this antibiotic may be used. For the reduction of neonatal morbidity due to GBS, universal screening of pregnant women for GBS between 35 and 37 weeks of gestation, with intrapartum antibiotic treatment of infected women, is recommended.

1	Postpartum infection is a significant cause of maternal morbidity and mortality. Postpartum endomyometritis is more common after cesarean delivery than vaginal delivery and develops in 2% of women after elective repeat cesarean section and in up to 10% after emergency cesarean section following prolonged labor. To reduce the risk of endomyometritis, prophylactic antibiotics should be given to all patients undergoing cesarean section, and administration 30–60 min prior to skin incision is preferable to administration at the time of umbilical cord clamping. As most cases of postpartum endomyometritis are polymicrobial, broad-spectrum antibiotic coverage with a penicillin, an aminoglycoside, and metronidazole is recommended (Chap. 201). Most cases resolve within 72 h. Women who do not respond to antibiotic treatment for postpartum endomyometritis should be evaluated for septic pelvic thrombophlebitis. Imaging studies may be helpful in establishing the diagnosis, which is primarily a

1	to antibiotic treatment for postpartum endomyometritis should be evaluated for septic pelvic thrombophlebitis. Imaging studies may be helpful in establishing the diagnosis, which is primarily a clinical diagnosis of exclusion. Patients with septic pelvic thrombophlebitis generally have tachycardia out of proportion to their fever and respond rapidly to IV administration of heparin.

1	All pregnant patients are screened prenatally for gonorrhea and chlamydial infections, and the detection of either should result in prompt treatment. Ceftriaxone and azithromycin are the agents of choice (Chaps. 181 and 213). VIRAL INFECTIoNS Influenza (See also Chap. 224) Pregnant women with influenza are at increased risk of serious complications and death. All women who are pregnant or plan to become pregnant in the near future should receive inactivated influenza vaccine. The prompt initiation of antiviral treatment is recommended for pregnant women in whom influenza is suspected. Treatment can be reconsidered once the results of high-sensitivity tests are available. Prompt initiation of treatment lowers the risk of admission to an intensive care unit and death.

1	Cytomegalovirus Infection The most common cause of congenital viral infection in the United States is cytomegalovirus (CMV) (Chap. 219). As many as 50–90% of women of childbearing age have antibodies to CMV, but only rarely does CMV reactivation result in neonatal infection. More commonly, primary CMV infection during pregnancy creates a risk of congenital CMV. No currently accepted treatment of CMV infection during pregnancy has been demonstrated to protect the fetus effectively. Moreover, it is difficult to predict which fetus will sustain a life-threatening CMV infection. Severe CMV disease in the newborn is characterized most often by petechiae, hepatosplenomegaly, and jaundice. Chorioretinitis, microcephaly, intracranial calcifications, hepatitis, hemolytic anemia, and purpura may also develop. CNS involvement, resulting in the development of psychomotor, ocular, auditory, and dental abnormalities over time, has been described.

1	Rubella (See also Chap. 230e) Rubella virus is a known teratogen; first-trimester rubella carries a high risk of fetal anomalies, though the risk significantly decreases later in pregnancy. Congenital rubella may be diagnosed by percutaneous umbilical-blood sampling with the detection of IgM antibodies in fetal blood. All pregnant women and all women of childbearing age should be tested for their immune status to rubella. All nonpregnant women who are not immune to rubella should be vaccinated. The incidence of congenital rubella in the United States is extremely low.

1	Herpesvirus Infection (See also Chap. 216) The acquisition of genital herpes during pregnancy is associated with spontaneous abortion, prematurity, and congenital and neonatal herpes. A cohort study of pregnant women without evidence of previous herpesvirus infection demonstrated that ~2% acquired a new herpesvirus infection during the pregnancy. Approximately 60% of the newly infected women had no clinical symptoms. Infection occurred with equal frequency in all three trimesters. If herpesvirus seroconversion occurred early in pregnancy, the risk of transmission to the newborn was very low. In women who acquired genital herpes shortly before delivery, the risk of transmission was high. The risk of active genital herpes lesions at term can be reduced by prescribing acyclovir for the last 4 weeks of pregnancy to women who have had their first episode of genital herpes during the pregnancy.

1	Herpesvirus infection in the newborn can be devastating. Disseminated neonatal herpes carries with it high mortality and morbidity rates from CNS involvement. It is recommended that pregnant women with active genital herpes lesions at the time of presentation in labor be delivered by cesarean section. Parvovirus Infection (See also Chap. 221) Parvovirus infection (caused by human parvovirus B19) may occur during pregnancy. It rarely causes sequelae, but susceptible women infected during pregnancy may be at risk for fetal hydrops secondary to erythroid aplasia and profound anemia.

1	HIV Infection (See also Chap. 226) The predominant cause of HIV infection in children is transmission of the virus from mother to newborn during the perinatal period. All pregnant women should be screened for HIV infection. Factors that increase the risk of mother-to-newborn transmission include high maternal viral load, low maternal CD4+ T cell count, prolonged labor, prolonged duration of membrane rupture, and the presence of other genital tract infections, such as syphilis or herpes. Prior to the widespread use of antiretroviral treatment, the perinatal transmission rate was in the range of 20%. In women with a good response to antiretroviral treatment, the transmission rate is about 1%. Measurement of maternal plasma HIV RNA copy number guides the decision for vaginal versus cesarean delivery. For women with <1000 copies of plasma HIV RNA/ml who are receiving combination antiretroviral therapy, the risk of transmission to the newborn is approximately 1% regardless of mode of

1	delivery. For women with <1000 copies of plasma HIV RNA/ml who are receiving combination antiretroviral therapy, the risk of transmission to the newborn is approximately 1% regardless of mode of delivery or duration of membrane rupture. These women may elect to attempt a vaginal birth following the spontaneous onset of labor. For women with a viral load of ≥1000 copies/ml prior to 38 weeks of gestation, a scheduled prelabor cesarean at 38 weeks is recommended to reduce the risk of HIV transmission to the newborn. To reduce the risk of mother-to-newborn transmission, women with >400 copies of HIV RNA/ml should be treated during the intrapartum interval with zidovudine. All newborns of HIV-infected mothers should be treated with zidovudine for 6 months after birth. Women who are HIV-positive may transmit the virus through their breast milk. In developed countries, HIV-infected mothers are advised not to breast-feed.

1	(See also Chap. 148) For rubella-nonimmune individuals contemplating pregnancy, measles-mumps-rubella vaccine should be administered, ideally at least 3 months prior to conception but otherwise in the immediate postpartum period. In addition, pregnancy is not a contraindication for vaccination against influenza, tetanus, diphtheria, and pertussis (Tdap), and these vaccines are recommended for appropriate individuals.

1	Maternal death is defined as death occurring during pregnancy or within 42 days of completion of pregnancy from a cause related to or aggravated by pregnancy, but not due to accident or incidental causes. From 1935 to 2007, the U.S. maternal death rate decreased from nearly 600/100,000 births to 12.7/100,000 births. There are significant health disparities in the maternal mortality rate, with the highest rates among non-Hispanic black women. In 2007, maternal mortality rates (per 100,000) by race were 10.5 among non-Hispanic white women, 8.9 among Hispanic women, and 28.4 among non-Hispanic black women. The most common causes of maternal death in the United States today are pulmonary embolism, obstetric hemorrhage, hypertension, sepsis, cardiovascular conditions (including peripartum cardiomyopathy), and ectopic pregnancy. As stated above, the maternal mortality rate in the United States is about 12.7/100,00 births. In some countries in sub-

1	As stated above, the maternal mortality rate in the United States is about 12.7/100,00 births. In some countries in sub- Saharan Africa and southern Asia, the maternal mortality rate is about 500/100,000 live births. The most common cause of maternal death in these countries is maternal hemorrhage. The high maternal death rates are due in part to inadequate contraceptive and family-planning services, an insufficient number of skilled birth attendants, and difficulty in accessing birthing centers and emergency obstetrical care units. Maternal death is a global public-health tragedy that could be mitigated with the application of modest resources.

1	With improved diagnostic and therapeutic modalities as well as advances in the treatment of infertility, more patients with medical complications will be seeking and will require complex obstetric care. Improved outcomes of pregnancy in these women will be best attained by a team of internists, maternal-fetal medicine (high-risk obstetrics) specialists, and anesthesiologists assembled to counsel these patients about the risks of pregnancy and to plan their treatment prior to conception. The importance of preconception counseling cannot be overstated. It is the responsibility of all physicians caring for women in the reproductive age group to assess their patients’ reproductive plans as part of their overall health evaluation. 9 Medical evaluation of the surgical patient Wei C. Lau, Kim A. Eagle

1	Medical evaluation of the surgical patient Wei C. Lau, Kim A. Eagle Cardiovascular and pulmonary complications continue to account for major morbidity and mortality in patients undergoing noncardiac surgery. Emerging evidence-based practices dictate that the internist should perform an individualized evaluation of the surgical patient to provide an accurate preoperative risk assessment and stratification that will guide optimal perioperative risk-reduction strategies. This chapter reviews cardiovascular and pulmonary preoperative risk assessment, targeting intermediateand high-risk patients with the goal of improving outcome. It also reviews perioperative management and prophylaxis of diabetes mellitus, endocarditis, and venous thromboembolism. Simple, standardized preoperative screening questionnaires, such as the one shown in Table 9-1, have been developed for the purpose of identifying patients at intermediate or high risk who may benefit 1. Age, weight, height 2.

1	Age, weight, height 2. Are you: Female and 55 years of age or older or male and 45 years of age of older? If yes, are you 70 years of age or older? 3. Do you take anticoagulant medications (“blood thinners”)? 4. Do you have or have you had any of the following heart-related conditions? Heart disease Heart attack within the last 6 months Angina (chest pain) 5. Do you have or have you ever had any of the following? Rheumatoid arthritis Kidney disease Liver disease Diabetes 6. Do you get short of breath when you lie flat? 7. Are you currently on oxygen treatment? 8. Do you have a chronic cough that produces any discharge or fluid? 9. Do you have lung problems or diseases? 10. Have you or any blood member of your family ever had a problem other than nausea with any anesthesia? If yes, describe: 11.

1	9. Do you have lung problems or diseases? 10. Have you or any blood member of your family ever had a problem other than nausea with any anesthesia? If yes, describe: 11. If female, is it possible that you are pregnant? Pregnancy test: Please list date of last menstrual period: aUniversity of Michigan Health System patient information report. Patients who answer yes to any of questions 2–9 should receive a more detailed clinical evaluation. Source: Adapted from KK Tremper, P Benedict: Anesthesiology 92:1212, 2000; with permission.

1	Source: Adapted from KK Tremper, P Benedict: Anesthesiology 92:1212, 2000; with permission. from a more detailed clinical evaluation. Evaluation of such patients for surgery should always begin with a thorough history and physical examination and with a 12-lead resting electrocardiogram (ECG), in accordance with the American College of Cardiology/American Heart Association (ACC/AHA) guidelines. The history should focus on symptoms of occult cardiac or pulmonary disease. The urgency of the surgery should be determined, as true emergency procedures are associated with unavoidably higher morbidity and mortality risk. Preoperative laboratory testing should be carried out only for specific clinical conditions, as noted during clinical examination. Thus, healthy patients of any age who are undergoing elective surgical procedures without coexisting medical conditions should not require any testing unless the degree of surgical stress may result in unusual changes from the baseline state.

1	A stepwise approach to cardiac risk assessment and stratification in patients undergoing noncardiac surgery is illustrated in Fig. 9-1. Assessment of exercise tolerance in the prediction of in-hospital perioperative risk is most helpful in patients who self-report worsening exercise-induced cardiopulmonary symptoms; those who may benefit from noninvasive or invasive cardiac testing regardless of a scheduled surgical procedure; and those with known coronary artery disease (CAD) or with multiple risk factors who are able to exercise. For predicting perioperative events, poor exercise tolerance has been defined as the inability to walk four blocks or climb two flights of stairs at a normal pace or to meet a metabolic equivalent (MET) level of 4 (e.g., carrying objects of 15–20 lb or playing golf or doubles tennis) because of the development of dyspnea, angina, or excessive fatigue (Table 9-2).

1	Previous studies have compared several cardiac risk indices. The American College of Surgeons’ National Surgical Quality Improvement Chapter 9 Medical Evaluation of the Surgical Patient SurgeryCoronary revascularizationwithin 5 yearsIf yes, and no recurrentsymptoms2SurgeryCoronary revascularization within 5 years If yes, and no recurrent symptoms2 Recent coronary evaluation3If no, or recurrent symptomsRecent coronary evaluation3 If no, or recurrent symptoms No Clinical assessment --Age >70, <4 METs --Signs of CHF, AS --EKG changes ischemic or infarct

1	Clinical assessment --Age >70, <4 METs --Signs of CHF, AS --EKG changes ischemic or infarct SurgerySurgeryPositive stress testNon invasive cardiac testInitiate and/orcontinue optimalpreventive medicaltherapy treatmentNegative stress testIdentify, initiate treatment inpatients requiring preventiveor continue long termmedical preventive therapySurgery Surgery Positive stress test Coronary revascularization ACC/AHA guidelines Non invasive cardiac test Initiate and/or continue optimal preventive medical therapy treatment Negative stress test Identify, initiate treatment in patients requiring preventive or continue long term medical preventive therapy Poor functional capacity, history of angina Yes No

1	FIgURE 9-1 Composite algorithm for cardiac risk assessment and stratification in patients undergoing noncardiac surgery. Stepwise clinical evaluation: [1] emergency surgery; [2] prior coronary revascularization; [3] prior coronary evaluation; [4] clinical assessment; [5] RCRI; [6] risk modification strategies. Preventive medical therapy = beta blocker and statin therapy. RCRI, revised cardiac risk index. (Adapted from LA Fleisher et al: Circulation 116:1971, 2007, with permission.) Program prospective database has identified five predictors of perioperative myocardial infarction (MI) and cardiac arrest based on increasing age, American Society of Anesthesiologists class, type of surgery, dependent functional status, and abnormal serum creatinine level. However, given its accuracy and simplicity, the revised cardiac risk assessMent of CardIaC rIsk By funCtIonaL status

1	Higher • Has difficulty with adult activities of daily living • Cannot walk four blocks or up two flights of stairs or does not meet a MET level of 4 active: easily does vigorous tasks Lower • Performs regular vigorous exercises Source: From LA Fleisher et al: Circulation 116:1971, 2007.

1	index (RCRI) (Table 9-3) is favored. The RCRI relies on the presence or absence of six identifiable predictive factors: high-risk surgery, ischemic heart disease, congestive heart failure, cerebrovascular disease, diabetes mellitus, and renal dysfunction. Each of these predictors is assigned one point. The risk of major cardiac events—defined as myocardial infarction, pulmonary edema, ventricular fibrillation or primary cardiac arrest, and complete heart block—can then be predicted. Based on the presence of none, one, two, three, or more of these clinical predictors, the rate of development of one of these four major cardiac events is estimated to be 0.4, 0.9, 7, and 11%, respectively (Fig. 9-2). An RCRI score of 0 signifies a 0.4–0.5% risk of cardiac events; RCRI 1, 0.9–1.3%; RCRI 2, 4–7%; and RCRI ≥3, 9–11%. The clinical utility of the RCRI is to identify patients with three or more predictors who are at very high risk (≥11%) for cardiac complications and who may benefit from

1	2, 4–7%; and RCRI ≥3, 9–11%. The clinical utility of the RCRI is to identify patients with three or more predictors who are at very high risk (≥11%) for cardiac complications and who may benefit from further risk stratification with noninvasive cardiac testing or initiation of preoperative preventive medical management.

1	History of myocardial infarction Current angina considered to be ischemic Requirement for sublingual nitroglycerin Positive exercise test Pathological Q-waves on ECG History of PCI and/or CABG with current angina considered to be ischemic Left ventricular failure by physical examination History of paroxysmal nocturnal dyspnea History of pulmonary edema S3 gallop on cardiac auscultation Bilateral rales on pulmonary auscultation Pulmonary edema on chest x-ray History of transient ischemic attack History of cerebrovascular accident Treatment with insulin Abbreviations: CABG, coronary artery bypass grafting; ECG, electrocardiogram; PCI, percutaneous coronary interventions. Source: Adapted from TH Lee et al: Circulation 100:1043, 1999.

1	Abbreviations: CABG, coronary artery bypass grafting; ECG, electrocardiogram; PCI, percutaneous coronary interventions. Source: Adapted from TH Lee et al: Circulation 100:1043, 1999. There is little evidence to support widespread application of preoperative noninvasive cardiac testing for all patients undergoing major surgery. Rather, a discriminative approach based on clinical risk categorization appears to be both clinically useful and cost-effective. There is potential benefit in identifying asymptomatic but high-risk patients, such as those with left main or left main–equivalent CAD or those with three-vessel CAD and poor left ventricular function, who may benefit from coronary revascularization (Chap. 293). However, evidence does not support aggressive attempts to identify patients at intermediate risk who have asymptomatic but advanced coronary artery disease, in whom coronary revascularization appears to offer little advantage over medical therapy.

1	RCRI 0 1 2 ˜3 Event Rate 0.50% 1.30% 6.00% 11% An RCRI score ≥3 in patients with severe myocardial ischemia on stress testing should lead to consideration of coronary revascularization prior to noncardiac surgery. Noninvasive cardiac testing is most appropriate if it is anticipated that, in the event of a strongly positive test, a patient will meet guidelines for coronary angiography and coronary revascularization. Pharmacologic stress tests are more useful than exercise testing in patients with functional limitations. Dobutamine echocardiography and persantine, adenosine, or dobutamine nuclear perfusion testing (Chap. 270e) have excellent negative predictive values (near 100%) but poor positive predictive values (<20%) in the identification of patients at risk for perioperative MI or death. Thus, a negative study is reassuring, but a positive study is a relatively weak predictor of a “hard” perioperative cardiac event.

1	RISK ModIFICATIoN: PREVENTIVE STRATEgIES To REdUCE CARdIAC RISK Perioperative Coronary Revascularization Currently, potential options for reducing perioperative cardiovascular risk include coronary artery revascularization and/or perioperative preventive medical therapies (Chap. 293). Prophylactic coronary revascularization with either coronary artery bypass grafting (CABG) or percutaneous coronary intervention (PCI) provides no shortor midterm survival benefit for patients without left main CAD or three-vessel CAD in the presence of poor left ventricular systolic function and is not recommended for patients with stable CAD before noncardiac surgery. Although PCI is associated with lower procedural risk than is CABG in the perioperative setting, the placement of a coronary artery stent soon before noncardiac surgery may increase the risk of bleeding during surgery if dual antiplatelet therapy (aspirin and thienopyridine) is administered; moreover, stent placement shortly before

1	soon before noncardiac surgery may increase the risk of bleeding during surgery if dual antiplatelet therapy (aspirin and thienopyridine) is administered; moreover, stent placement shortly before noncardiac surgery increases the perioperative risk of MI and cardiac death due to stent thrombosis if such therapy is withdrawn prematurely (Chap. 296e). It is recommended that, if possible, noncardiac surgery be delayed 30–45 days after placement of a bare metal coronary stent and for 365 days after a drug-eluting stent. For patients who must undergo noncardiac surgery early (>14 days) after PCI, balloon angioplasty without stent placement appears to be a reasonable alternative because dual antiplatelet therapy is not necessary in such patients. One recent clinical trial further suggests that after 6 months, bare metal and drug eluting stents may not pose a threat.

1	perIoperatIve preventIve medIcal therapIes The goal of perioperative preventive medical therapies with β-adrenergic antagonists, HMG-CoA reductase inhibitors (statins), antiplatelet agents, and α2 agonists is to reduce perioperative adrenergic stimulation, ischemia, and inflammation, which are triggered during the perioperative period. β-adrenergIc antagonIsts The use of perioperative beta blockade should be based on a thorough assessment of a patient’s perioperative clinical and surgery-specific cardiac risk (RCRI ≥2). For patients with or without mild to moder- Chapter 9 Medical Evaluation of the Surgical Patient 10% 15% 4–7 9–11 0% 5%Risk of cardiac events0.9–1.30.4–0.5

1	Chapter 9 Medical Evaluation of the Surgical Patient 10% 15% 4–7 9–11 0% 5%Risk of cardiac events0.9–1.30.4–0.5 The POISE trial highlights the importance of a clear risk-and-benefit assessment, with careful initiation and titration to therapeutic efficacy of preoperative beta blockers in patients undergoing noncardiac surgery. A recent meta-analysis which included the POISE study further supports that excessive beta blocker dosing is, in fact, harmful. The ACC/AHA guidelines recommend the following: (1) Beta blockers should be continued in patients with active cardiac conditions who are undergoing surgery and are receiving beta blockers. (2) Beta blockers titrated to heart rate and blood pressure are probably recommended for patients undergoing vascular surgery who are at high cardiac risk defined by CAD or cardiac ischemia on preoperative testing.

1	(3) Beta blockers are reasonable for high-risk patients (RCRI ≥2) who undergo vascular surgery. (4) Beta blockers are reasonable for patients with known CAD or high risk (RCRI ≥2) who undergo intermediate-risk surgery. (5) Nondiscriminant administration of high-dose beta blockers without dose titration to effectiveness is contraindicated for patients who have never been treated with a beta blocker.

1	hmg-coa reductase InhIbItors (statIns) A number of prospective and retrospective studies support the perioperative prophylactic use of statins for reduction of cardiac complications in patients with established atherosclerosis. The ACC/AHA Guidelines support the protective efficacy of perioperative statins on cardiac complications in intermediate risk patients undergoing major noncardiac surgery. For patients undergoing noncardiac surgery and currently taking statins, statin therapy should be continued to reduce perioperative cardiac risk. Statins are reasonable for patients undergoing vascular surgery with or without clinical risk factors (RCRI ≥1). angIotensIn-convertIng enzyme (ace) InhIbItors Evidence supports the discontinuation of ACE inhibitors and angiotensin receptor blockers for 24 h prior to noncardiac surgery due to adverse circulatory effects after induction of anesthesia.

1	oral antIplatelet agents Evidence-based recommendations regarding perioperative use of aspirin and/or thienopyridine to reduce cardiac risk currently lack clarity. A substantial increase in perioperative bleeding and in the need for transfusion in patients receiving dual antiplatelet therapy has been observed. The discontinuation of thienopyridine and aspirin for 5–7 days prior to major surgery to minimize the risk of perioperative bleeding and transfusion must be balanced with the potential increased risk of an acute coronary syndrome and of subacute stent thrombosis in patients with recent coronary stent implantation. If clinicians elect to withhold antiplatelet agents prior to surgery, these agents should be restarted as soon as possible postoperatively.

1	α2 agonIsts Several prospective and retrospective meta-analyses of perioperative α2 agonists (clonidine and mivazerol) demonstrated a reduction of cardiac death rates among patients with known coronary artery disease who underwent noncardiac surgery. α2 agonists thus may be considered for perioperative control of hypertension in patients with known coronary artery disease or an RCRI score ≥2. calcIum channel blockers Evidence is lacking to support the use of calcium channel blockers as a prophylactic strategy to decrease perioperative risk in major noncardiac surgery.

1	anesthetIcs Mortality risk is low with safe delivery of modern anesthesia, especially among low-risk patients undergoing low-risk surgery (Table 9-4). Inhaled anesthetics have predictable circulatory and respiratory effects: all decrease arterial pressure in a dose-dependent manner by reducing sympathetic tone and causing systemic vasodilation, myocardial depression, and decreased cardiac output. Inhaled anesthetics also cause respiratory depression, with diminished responses to both hypercapnia and hypoxemia, in a dose-dependent manner; in addition, these agents have a variable effect on heart rate. Prolonged residual neuromuscular blockade also increases the risk of postoperative pulmonary complications due to reduction in functional residual lung capacity, loss of diaphragmatic and intercostal muscle function, atelectasis, and arterial hypoxemia from ventilation-perfusion mismatch.

1	Higher • Emergent major operations, especially in the elderly • Prolonged surgery associated with large fluid shift and/or • Prostate surgery Lower • Eye, skin, and superficial surgery Source: From LA Fleisher et al: Circulation 116:1971, 2007, with permission. Several meta-analyses have shown that rates of pneumonia and respiratory failure are lower among patients receiving neuroaxial anesthesia (epidural or spinal) rather than general anesthesia (inhaled). However, there were no significant differences in cardiac events between the two approaches. Evidence from a meta-analysis of randomized controlled trials supports postoperative epidural analgesia for >24 h for the purpose of pain relief. However, the risk of epidural hematoma in the setting of systemic anticoagulation for venous thromboembolism prophylaxis (see below) and postoperative epidural catheterization must be considered.

1	Perioperative pulmonary complications occur frequently and lead to significant morbidity and mortality. The guidelines from the American College of Physicians recommend the following: 1. All patients undergoing noncardiac surgery should be assessed for risk of pulmonary complications (Table 9-5). 2. Patients undergoing emergency or prolonged (3to 4-h) surgery; aortic aneurysm repair; vascular surgery; major abdominal, thoracic, neurologic, head, or neck surgery; and general anesthesia 1. Upper respiratory tract infection: cough, dyspnea 2. 3. 4. 5. American Society of Anesthesiologists Class ≥2 6. 7. 8. Serum albumin <3.5 g/dL 9. 10. Impaired sensorium (confusion, delirium, or mental status changes) 11. 12. 13. 14. Spirometry threshold before lung resection a. FEV1 <2 L b. MVV <50% of predicted c. PEF <100 L or 50% predicted value d. PCO2 ≥45 mmHg e. PO2 ≤50 mmHg

1	12. 13. 14. Spirometry threshold before lung resection a. FEV1 <2 L b. MVV <50% of predicted c. PEF <100 L or 50% predicted value d. PCO2 ≥45 mmHg e. PO2 ≤50 mmHg Abbreviations: FEV1, forced expiratory volume in 1 s; MVV, maximal voluntary ventilation; PEF, peak expiratory flow rate; PCO2, partial pressure of carbon dioxide; PO2, partial pressure of oxygen. Source: A Qaseem et al: Ann Intern Med 144:575-80. Modified from GW Smetana et al: Ann Intern Med 144:581, 2006, and from DN Mohr et al: Postgrad Med 100:247, 1996. • Cessation of smoking for at least 8 weeks before and until at least 10 days bronchodilator and/or steroid therapy, when indicated of infection and secretion, when indicated reduction, when appropriate duration of anesthesia of long-acting neuromuscular blocking drugs, when indicated of aspiration and maintenance of optimal bronchodilation • Optimization of inspiratory capacity maneuvers, with attention to: Mobilization of secretions

1	Mobilization of secretions Encouragement of coughing Selective use of a nasogastric tube Adequate pain control without excessive narcotics Source: From VA Lawrence et al: Ann Intern Med 144:596, 2006, and WF Dunn, PD Scanlon: Mayo Clin Proc 68:371, 1993. should be considered to be at elevated risk for postoperative pulmonary complications. 3. Patients at higher risk of pulmonary complications should undergo incentive spirometry, deep-breathing exercises, cough encouragement, postural drainage, percussion and vibration, suctioning and ambulation, intermittent positive-pressure breathing, continuous positive airway pressure, and selective use of a nasogastric tube for postoperative nausea, vomiting, or symptomatic abdominal distention to reduce postoperative risk (Table 9-6). 4.

1	4. Routine preoperative spirometry and chest radiography should not be used routinely for predicting risk of postoperative pulmonary complications but may be appropriate for patients with chronic obstructive pulmonary disease or asthma. 5. Spirometry is of value before lung resection in determining candidacy for coronary artery bypass; however, it does not provide a spirometric threshold for extrathoracic surgery below which the risks of surgery are unacceptable. 6. Pulmonary artery catheterization, administration of total parenteral nutrition (as opposed to no supplementation), or total enteral nutrition has no benefit in reducing postoperative pulmonary complications.

1	(See also Chaps. 417–419) Many patients with diabetes mellitus have significant symptomatic or asymptomatic CAD and may have silent myocardial ischemia due to autonomic dysfunction. Evidence supports intensive perioperative glycemic control to achieve near-normal glucose levels (90–110 mg/dL) rather than moderate glycemic control (120–200 mg/dL), using insulin infusion. This practice must be balanced against the risk of hypoglycemic complications. Oral hypoglycemic agonists should not be given on the morning of surgery. Perioperative hyperglycemia should be treated with IV infusion of short-acting insulin or SC sliding-scale insulin. Patients whose diabetes is diet controlled may proceed to surgery with close postoperative monitoring.

1	(See also Chap. 155) Perioperative prophylactic antibiotics should be administered to patients with congenital or valvular heart disease, prosthetic valves, mitral valve prolapse, or other cardiac abnormalities, in accordance with ACC/AHA practice guidelines. (See also Chap. 300) Perioperative prophylaxis of venous thromboembolism should follow established guidelines of the American College of Chest Physicians. Aspirin is not supported as a single agent for thromboprophylaxis. Low-dose unfractionated heparin (≤5000 units SC bid), low-molecular weight heparin (e.g., enoxaparin, 30 mg bid or 40 mg qd), or a pentasaccharide (fondaparinux, 2.5 mg qd) is appropriate for patients at moderate risk; unfractionated heparin (5000 units SC tid) is appropriate for patients at high risk. Graduated compression stockings and pneumatic compression devices are useful supplements to anticoagulant therapy.

1	In 2010, according to the Centers for Disease Control and Prevention, 2,468,435 individuals died in the United States (Table 10-1). Approximately 73% of all deaths occur in those >65 years of age. The epidemiology of mortality is similar in most developed countries; cardiovascular diseases and cancer are the predominant causes of death, a marked change since 1900, when heart disease caused ~8% of all deaths and cancer accounted for <4% of all deaths. In 2010, the year with the most recent available data, AIDS did not rank among the top 15 causes of death, causing just 8369 deaths. Even among people age 35–44, heart disease, cancer, chronic liver disease, and accidents all cause more deaths than AIDS.

1	It is estimated that in developed countries ~70% of all deaths are preceded by a disease or condition, making it reasonable to plan for dying in the foreseeable future. Cancer has served as the paradigm for terminal care, but it is not the only type of illness with a recognizable and predictable terminal phase. Because heart failure, chronic obstructive pulmonary disease (COPD), chronic liver failure, dementia, and many other conditions have recognizable terminal phases, a systematic approach to end-of-life care should be part of all medical specialties. Many patients with illness-related suffering also can benefit from palliative care regardless of prognosis. Ideally, palliative care should be considered part of comprehensive care for all patients. Palliative care can be improved by coordination between caregivers, doctors, and patients for advance care planning, as well as dedicated teams of physicians, nurses, and other providers.

1	The rapid increases in life expectancy in developed countries over the last century have been accompanied by new difficulties facing individuals, families, and society as a whole in addressing the needs of an aging population. These challenges include both more complicated conditions and technologies to address them at the end of life. The development of technologies that can prolong life without restoring full health has led many Americans to seek out alternative end-of-life care settings and approaches that relieve suffering for those with terminal diseases. Over the last few decades in the United States, a significant change in the site of death has occurred that coincides with patient and family preferences. Nearly 60% of Americans died as inpatients in hospitals in 1980. By 2000, the trend was reversing, with ~31% of Americans dying as hospital inpatients (Fig. 10-1). This shift has been most dramatic for those dying from cancer and COPD and for younger and very old individuals.

1	trend was reversing, with ~31% of Americans dying as hospital inpatients (Fig. 10-1). This shift has been most dramatic for those dying from cancer and COPD and for younger and very old individuals. In the last decade, it has been associated with the increased use of hospice care; in 2008, approximately 39% of all decedents in the United States received such care. Cancer patients currently constitute ~36.9% of hospice users. About 79% of patients receiving hospice care die out of the hospital, and around 42% of those receiving hospice care die in a private residence. In addition, in 2008, for the first time, the American Board of Medical Specialties (ABMS) offered certification in hospice and palliative medicine. With shortening of hospital stays,

1	Number of Deaths Among People Cause of Death Number of Deaths Percentage of Total ≥65 Years of Age Number of Deaths Percentage of Total All deaths Heart disease Malignant neoplasms Chronic lower respiratory diseases Cerebrovascular diseases Accidents Alzheimer’s disease Diabetes mellitus Nephritis, nephritic syndrome, 2,468,435 100 1,798,276 499,331 100 597,689 24.2 477,338 141,362 28.3 574,743 23.3 396,670 142,107 28.5 138,080 5.6 118,031 27,132 5.4 129,476 5.2 109,990 35,846 7.2 120,859 4.9 41,300 11,256 2.3 83,494 3.4 82,616 8859 1.8 69,071 2.8 49,191 4931 1.0 50,476 2.0 41,994 4102 0.8 50,097 2.0 42,846 26,138 5.2 38,364 1.6 6008 3671 0.7 Source: National Center for Health Statistics (data for all age groups from 2010), http://www.cdc.gov/nchs; National Statistics (England and Wales, 2012), http://www.statistics.gov.uk.

1	Source: National Center for Health Statistics (data for all age groups from 2010), http://www.cdc.gov/nchs; National Statistics (England and Wales, 2012), http://www.statistics.gov.uk. many serious conditions are being treated at home or on an outpatient basis. Consequently, providing optimal palliative and end-of-life care requires ensuring that appropriate services are available in a variety of settings, including noninstitutional settings. Central to this type of care is an interdisciplinary team approach that typically encompasses pain and symptom management, spiritual and psychological care for the patient, and support for family caregivers during the patient’s illness and the bereavement period.

1	Terminally ill patients have a wide variety of advanced diseases, often with multiple symptoms that demand relief, and require noninvasive therapeutic regimens to be delivered in flexible care settings. Fundamental to ensuring quality palliative and end-of-life care is a focus on four broad domains: (1) physical symptoms; (2) psychological symptoms; (3) social needs that include interpersonal relationships, caregiving, and economic concerns; and (4) existential or spiritual needs. A comprehensive assessment screens for and evaluates needs in each of these four domains. Goals for care are established in discussions with the patient and/or family, based on the assessment in each of the domains. Interventions then are aimed at improving or managing symptoms and needs. Although physicians are responsible Decedents, % 60.00 50.00 40.00 30.00 20.00 10.00 0.00 FIgURE 10-1 Graph showing trends in the site of death in the last two decades.

1	Decedents, % 60.00 50.00 40.00 30.00 20.00 10.00 0.00 FIgURE 10-1 Graph showing trends in the site of death in the last two decades. , percentage of hospital inpatient deaths; , percentage of decedents enrolled in a hospice. for certain interventions, especially technical ones, and for coordinating the interventions, they cannot be responsible for providing all of them. Because failing to address any one of the domains is likely to preclude a good death, a well-coordinated, effectively communicating interdisciplinary team takes on special importance in end-of-life care. Depending on the setting, critical members of the interdisciplinary team will include physicians, nurses, social workers, chaplains, nurse’s aides, physical therapists, bereavement counselors, and volunteers.

1	ASSESSMENT ANd CARE PLANNINg Comprehensive Assessment Standardized methods for conducting a comprehensive assessment focus on evaluating the patient’s condition in all four domains affected by illness: physical, psychological, social, and spiritual. The assessment of physical and mental symptoms should follow a modified version of the traditional medical history and physical examination that emphasizes symptoms. Questions should aim at elucidating symptoms and discerning sources of suffering and gauging how much those symptoms interfere with the patient’s quality of life. Standardized assessment is critical. Currently, there are 21 symptom assessment instruments for cancer alone. Further research on and validation of these assessment tools, especially taking into account patient perspectives, could improve their effectiveness. Instruments with good psychometric properties that assess a wide range of symptoms include the Memorial Symptom Assessment Scale (MSAS), the Rotterdam Symptom

1	could improve their effectiveness. Instruments with good psychometric properties that assess a wide range of symptoms include the Memorial Symptom Assessment Scale (MSAS), the Rotterdam Symptom Checklist, the Worthing Chemotherapy Questionnaire, and the Computerized Symptom Assessment Instrument. These instruments are long and may be useful for initial clinical or for research assessments. Shorter instruments are useful for patients whose performance status does not permit comprehensive assessments. Suitable shorter instruments include the Condensed Memorial Symptom Assessment Scale, the Edmonton Symptom Assessment System, the

1	M.D. Anderson Symptom Assessment Inventory, and the Symptom Distress Scale. Using such instruments ensures that the assessment is comprehensive and does not focus only on pain and a few other physical symptoms. Invasive tests are best avoided in end-of-life care, and even minimally invasive tests should be evaluated carefully for their benefit-to-burden ratio for the patient. Aspects of the physical examination that are uncomfortable and unlikely to yield useful information can be omitted.

1	Regarding social needs, health care providers should assess the status of important relationships, financial burdens, caregiving needs, and access to medical care. Relevant questions will include the following: How often is there someone to feel close to? How has this illness been for your family? How has it affected your relationships? How much help do you need with things like getting meals and getting around? How much trouble do you have getting the medical care you need? In the area of existential needs, providers should assess distress and the patient’s sense of being emotionally and existentially settled and of finding purpose or meaning. Helpful assessment questions can include the following: How much are you able to find meaning since your illness began? What things are most important to you at this stage? In addition, it can be helpful to ask how the patient perceives his or her care: How much do you feel your doctors and nurses respect you? How clear is the information from

1	to you at this stage? In addition, it can be helpful to ask how the patient perceives his or her care: How much do you feel your doctors and nurses respect you? How clear is the information from us about what to expect regarding your illness? How much do you feel that the medical care you are getting fits with your goals? If concern is detected in any of these areas, deeper evaluative questions are warranted.

1	Communication Especially when an illness is life-threatening, there are many emotionally charged and potentially conflict-creating moments, collectively called “bad news” situations, in which empathic and effective communication skills are essential. Those moments include communicating with the patient and/or family about a terminal diagnosis, the patient’s prognosis, any treatment failures, deemphasizing efforts to cure and prolong life while focusing more on symptom management and palliation, advance care planning, and the patient’s death. Although these conversations can be difficult and lead to tension, research indicates that end-of-life discussions can lead to earlier hospice referrals rather than overly aggressive treatment, benefiting quality of life for patients and improving the bereavement process for families.

1	Just as surgeons plan and prepare for major operations and investigators rehearse a presentation of research results, physicians and health care providers caring for patients with significant or advanced illness can develop a practiced approach to sharing important information and planning interventions. In addition, families identify as important both how well the physician was prepared to deliver bad news and the setting in which it was delivered. For instance, 27% of families making critical decisions for patients in an intensive care unit (ICU) desired better and more private physical space to communicate with physicians, and 48% found having clergy present reassuring.

1	An organized and effective seven-step procedure for communicating bad news goes by the acronym P-SPIKES: (1) prepare for the discussion, (2) set up a suitable environment, (3) begin the discussion by finding out what the patient and/or family understand, (4) determine how they will comprehend new information best and how much they want to know, (5) provide needed new knowledge accordingly, (6) allow for emotional responses, and (7) share plans for the next steps in care. Table 10-2 provides a summary of these steps along with Acronym Steps Aim of the Interaction Preparations, Questions, or Phrases S Setting of the interaction K Knowledge of the condition Mentally prepare for the interaction with the patient and/or family. Ensure the appropriate setting for a serious and potentially emotionally charged discussion. Begin the discussion by establishing the baseline and whether the patient and family can grasp the information.

1	Begin the discussion by establishing the baseline and whether the patient and family can grasp the information. Ease tension by having the patient and family contribute. Discover what information needs the patient and/or family have and what limits they want regarding the bad information. Provide the bad news or other information to the patient and/or family sensitively. Identify the cause of the emotions— e.g., poor prognosis. Empathize with the patient and/or family’s feelings. Explore by asking open-ended questions. Delineate for the patient and the family the next steps, including additional tests or interventions.

1	Empathize with the patient and/or family’s feelings. Explore by asking open-ended questions. Delineate for the patient and the family the next steps, including additional tests or interventions. Review what information needs to be communicated. Plan how you will provide emotional support. Rehearse key steps and phrases in the interaction. Ensure that patient, family, and appropriate social supports are present. Devote sufficient time. Ensure privacy and prevent interruptions by people or beeper. Bring a box of tissues. Start with open-ended questions to encourage participation. Possible phrases to use: What do you understand about your illness? When you first had symptom X, what did you think it might be? What did Dr. X tell you when he or she sent you here? What do you think is going to happen? Possible phrases to use: If this condition turns out to be something serious, do you want to know?

1	Possible phrases to use: If this condition turns out to be something serious, do you want to know? Would you like me to tell you all the details of your condition? If not, who would you like me to talk to? Do not just dump the information on the patient and family. Check for patient and family understanding. Possible phrases to use: I feel badly to have to tell you this, but . . . Unfortunately, the tests showed . . . I’m afraid the news is not good . . . Strong feelings in reaction to bad news are normal. Acknowledge what the patient and family are feeling. Remind them such feelings are normal, even if frightening. Give them time to respond. Remind patient and family you won’t abandon them. Possible phrases to use: I imagine this is very hard for you to hear. You look very upset. Tell me how you are feeling. I wish the news were different. We’ll do whatever we can to help you.

1	I imagine this is very hard for you to hear. You look very upset. Tell me how you are feeling. I wish the news were different. We’ll do whatever we can to help you. It is the unknown and uncertain that can increase anxiety. Recommend a schedule with goals and landmarks. Provide your rationale for the patient and/or family to accept (or reject). If the patient and/or family are not ready to discuss the next steps, schedule a follow-up visit. Source: Adapted from R Buckman: How to Break Bad News: A Guide for Health Care Professionals. Baltimore, Johns Hopkins University Press, 1992. suggested phrases and underlying rationales for each one. Additional research that further considers the response of patients to systematic methods of delivering bad news could build the evidence base for even more effective communication procedures.

1	Continuous goal Assessment Major barriers to ensuring quality palliative and end-of-life care include difficulty providing an accurate prognosis and emotional resistance of patients and their families to accepting the implications of a poor prognosis. There are two practical solutions to these barriers. One is to integrate palliative care with curative care regardless of prognosis. With this approach, palliative care no longer conveys the message of failure, having no more treatments, or “giving up hope.” Fundamental to integrating palliative care with curative therapy is to include continuous goal assessment as part of the routine patient reassessment that occurs at most patient-physician encounters. Alternatively, some practices may find it useful to implement a standard point in the clinical course to address goals of care and advance care planning. For example, some oncology practices ask all patients whose Eastern Cooperative Oncology Group (ECOG) performance status is 3 or

1	clinical course to address goals of care and advance care planning. For example, some oncology practices ask all patients whose Eastern Cooperative Oncology Group (ECOG) performance status is 3 or less—meaning they spend 50% or more of the day in bed—or those who develop metastatic disease about their goals of care and advance care preferences.

1	Goals for care are numerous, ranging from cure of a specific disease, to prolonging life, to relief of a symptom, to delaying the course of an incurable disease, to adapting to progressive disability without disrupting the family, to finding peace of mind or personal meaning, to dying in a manner that leaves loved ones with positive memories. Discernment of goals for care can be approached through a seven-step protocol: (1) ensure that medical and other information is as complete as reasonably possible and is understood by all relevant parties (see above); (2) explore what the patient and/or family are hoping for while identifying relevant and realistic goals; (3) share all the options with the patient and family; (4) respond with empathy as they adjust to changing expectations; (5) make a plan, emphasizing what can be done toward achieving the realistic goals; (6) follow through with the plan; and (7) review and revise the plan periodically, considering at every encounter whether the

1	plan, emphasizing what can be done toward achieving the realistic goals; (6) follow through with the plan; and (7) review and revise the plan periodically, considering at every encounter whether the goals of care should be reviewed with the patient and/or family. Each of these steps need not be followed in rote order, but together they provide a helpful framework for interactions with patients and their families about goals for care. It can be especially challenging if a patient or family member has difficulty letting go of an unrealistic goal. One strategy is to help them refocus on more realistic goals and also suggest that while hoping for the best, it is still prudent to plan for other outcomes as well.

1	Advance Care Planning • practIces Advance care planning is a process of planning for future medical care in case the patient becomes incapable of making medical decisions. A 2010 study of adults 60 or older who died between 2000 and 2006 found that 42% required decision making about treatment in the final days of life but 70% lacked decision-making capacity. Among those lacking decision-making capacity, around one-third did not have advance planning directives. Ideally, such planning would occur before a health care crisis or the terminal phase of an illness. Diverse barriers prevent this. Polls suggest 80% of Americans endorse advance care planning and completing living wills. However, data suggest between 33 and 42% have actually completed one. Other countries have even lower completion rates. Most patients expect physicians to initiate advance care planning and will wait for physicians to broach the subject. Patients also wish to discuss advance care planning with their families.

1	rates. Most patients expect physicians to initiate advance care planning and will wait for physicians to broach the subject. Patients also wish to discuss advance care planning with their families. Yet patients with unrealistic expectations are significantly more likely to prefer aggressive treatments. Fewer than one-third of health care providers have completed advance care planning for themselves. Hence, a good first step is for health care providers to complete their own advance care planning. This makes providers aware of the critical choices in the process and the issues that are especially charged and allows them to tell their patients truthfully that they personally have done advance planning. Lessons from behavioral economics suggest that setting this kind of social norming helps people view completing an advance directive as acceptable and even expected.

1	Steps in effective advance care planning center on (1) introducing the topic, (2) structuring a discussion, (3) reviewing plans that have been discussed by the patient and family, (4) documenting the plans, (5) updating them periodically, and (6) implementing the advance care directives (Table 10-3). Two of the main barriers to advance care planning are problems in raising the topic and difficulty in structuring a succinct discussion. Raising the topic can be done efficiently as a routine matter, noting that it is recommended for all patients, analogous to purchasing insurance or estate planning. Many of the most difficult cases have involved unexpected, acute episodes of brain damage in young individuals.

1	Structuring a focused discussion is a central communication skill. Identify the health care proxy and recommend his or her involvement in the process of advance care planning. Select a worksheet, preferably one that has been evaluated and demonstrated to produce reliable and valid expressions of patient preferences, and orient the patient and proxy to it. Such worksheets exist for both general and disease-specific situations. Discuss with the patient and proxy one scenario as an example to demonstrate how to think about the issues. It is often helpful to begin with a scenario in which the patient is likely to have settled preferences for care, such as being in a persistent vegetative state. Once the patient’s preferences for interventions in this scenario are determined, suggest that the patient and proxy discuss and complete the worksheet for the others. If appropriate, suggest that they involve other family members in the discussion. On a return visit, go over the patient’s

1	the patient and proxy discuss and complete the worksheet for the others. If appropriate, suggest that they involve other family members in the discussion. On a return visit, go over the patient’s preferences, checking and resolving any inconsistencies. After having the patient and proxy sign the document, place it in the medical chart and be sure that copies are provided to relevant family members and care sites. Because patients’ preferences can change, these documents have to be reviewed periodically.

1	types of documents Advance care planning documents are of three broad types. The first includes living wills or instructional directives; these are advisory documents that describe the types of decisions that should direct care. Some are more specific, delineating different scenarios and interventions for the patient to choose from. Among these, some are for general use and others are designed for use by patients with a specific type of disease, such as cancer or HIV. A second type is a less specific directive that provides general statements of not wanting life-sustaining interventions or forms that describe the values that should guide specific discussions about terminal care. These can be problematic because, when critical decisions about specific treatments are needed, they require assessments by people other than the patient of whether a treatment fulfills a particular wish. The third type of advance directive allows the designation of a health care proxy (sometimes also referred

1	by people other than the patient of whether a treatment fulfills a particular wish. The third type of advance directive allows the designation of a health care proxy (sometimes also referred to as a durable attorney for health care), who is an individual selected by the patient to make decisions. The choice is not either/or; a combined directive that includes a living will and designates a proxy is often used, and the directive should indicate clearly whether the specified patient preferences or the proxy’s choice takes precedence if they conflict. The Five Wishes and the Medical Directive are such combined forms. Some states have begun to put into practice a “Physician Orders for Life-Sustaining Treatment (POLST)” paradigm, which builds on communication between providers and patients to include guidance for end-of-life care in a color-coordinated form that follows the patient across treatment settings. The procedures for completing advance care planning documents vary according to

1	include guidance for end-of-life care in a color-coordinated form that follows the patient across treatment settings. The procedures for completing advance care planning documents vary according to state law.

1	A potentially misleading distinction relates to statutory as opposed to advisory documents. Statutory documents are drafted to fulfill relevant state laws. Advisory documents are drafted to reflect the patient’s wishes. Both are legal, the first under state law and the latter under common or constitutional law. legal aspects The U.S. Supreme Court has ruled that patients have a constitutional right to decide about refusing and terminating medical interventions, including life-sustaining interventions, and that mentally incompetent patients can exercise this right by providing “clear and convincing evidence” of their preferences. Because advance care directives permit patients to provide such evidence, commentators agree that Step Goals to be Achieved and Measures to Cover Useful Phrases or Points to Make

1	Step Goals to be Achieved and Measures to Cover Useful Phrases or Points to Make Introducing advance Ask the patient what he or she knows about advance care planning I’d like to talk with you about something I try to discuss with all my care planning and if he or she has already completed an advance care directive. patients. It’s called advance care planning. In fact, I feel that this is such an important topic that I have done this myself. Are you familiar with advance care planning or living wills? Indicate that you as a physician have completed advance care Have you thought about the type of care you would want if you ever planning. became too sick to speak for yourself? That is the purpose of advance care planning. Indicate that you try to perform advance care planning with all There is no change in health that we have not discussed. I am bring-patients regardless of prognosis. ing this up now because it is sensible for everyone, no matter how well or ill, old or young.

1	Explain the goals of the process as empowering the patient and Have many copies of advance care directives available, including ensuring that you and the proxy understand the patient’s preferences. in the waiting room, for patients and families. Provide the patient relevant literature, including the advance care Know resources for state-specific forms (available at directive that you prefer to use. www.nhpco.org). Recommend the patient identify a proxy decision-maker who should attend the next meeting. Structured discus-Affirm that the goal of the process is to follow the patient’s wishes if Use a structured worksheet with typical scenarios. sion of scenarios the patient loses decision-making capacity. Begin the discussion with persistent vegetative state and con-and patient Elicit the patient’s overall goals related to health care. sider other scenarios, such as recovery from an acute event with serious disability, asking the patient about his or her preferences

1	Elicit the patient’s overall goals related to health care. sider other scenarios, such as recovery from an acute event with serious disability, asking the patient about his or her preferences Elicit the patient’s preferences for specific interventions in a few regarding specific interventions, such as ventilators, artificial salient and common scenarios. nutrition, and CPR, and then proceeding to less invasive inter-Help the patient define the threshold for withdrawing and ventions, such as blood transfusions and antibiotics. withholding interventions. Define the patient’s preference for the role of the proxy. Review the patient’s After the patient has made choices of interventions, review them to preferences ensure they are consistent and the proxy is aware of them. Document the Formally complete the advance care directive and have a witness sign it. patient’s preferences Provide a copy for the patient and the proxy.

1	Document the Formally complete the advance care directive and have a witness sign it. patient’s preferences Provide a copy for the patient and the proxy. Insert a copy into the patient’s medical record and summarize in a progress note. Update the directive Periodically, and with major changes in health status, review the directive with the patient and make any modifications. Apply the directive The directive goes into effect only when the patient becomes unable to make medical decisions for himself or herself. Reread the directive to be sure about its content. Discuss your proposed actions based on the directive with the proxy. Abbreviation: CPR, cardiopulmonary resuscitation.

1	they are constitutionally protected. Most commentators believe that a legislation, the Affordable Care Act of 2010, raised substantial con-state is required to honor any clear advance care directive whether or troversy when early versions of the law included Medicare reimburse-not it is written on an “official” form. Many states have enacted laws ment for advance care planning consultations. These provisions were explicitly to honor out-of-state directives. If a patient is not using a withdrawn over accusations that they would lead to the rationing of statutory form, it may be advisable to attach a statutory form to the care for the elderly. advance care directive being used. State-specific forms are readily available free of charge for health care providers and patients and fami- lies through the National Hospice and Palliative Care Organization’s “Caring Connections” website (http://www.caringinfo.org). PHYSICAL SYMPToMS ANd THEIR MANAgEMENT

1	In January 2014, Texas judge R. H. Wallace ruled that a brain dead Great emphasis has been placed on addressing dying patients’ pain. woman who was 23 weeks pregnant should be removed from life sup-Some institutions have made pain assessment a fifth vital sign to port. This was after several months of disagreement between the wom-emphasize its importance. This also has been advocated by large health an’s family and the hospital providing care. The hospital cited Texas care systems such as the Veterans’ Administration and accrediting law that states that life-sustaining treatment must be administered to a bodies such as the Joint Commission. Although this embrace of pain as pregnant woman, but the judge sided with the woman’s family saying the fifth vital sign has been symbolically important, no data document that the law did not apply because the patient was legally dead. that it has improved pain management practices. Although good pal-

1	As of 2013, advance directives are legal in all states and the District liative care requires good pain management, it also requires more. The of Columbia either through state specific legislation, state judicial rul-frequency of symptoms varies by disease and other factors. The most ings, or United States Supreme Court rulings. Many states have their common physical and psychological symptoms among all terminally own statutory forms. Massachusetts and Michigan do not have living ill patients include pain, fatigue, insomnia, anorexia, dyspnea, depreswill laws, although both have health care proxy laws. In 27 states, sion, anxiety, and nausea and vomiting. In the last days of life, terminal the laws state that the living will is not valid if a woman is pregnant. delirium is also common. Assessments of patients with advanced can-However, like all other states except Alaska, these states have enacted cer have shown that patients experienced an average of 11.5 different durable power of

1	of patients with advanced can-However, like all other states except Alaska, these states have enacted cer have shown that patients experienced an average of 11.5 different durable power of attorney for health care laws that permit patients physical and psychological symptoms (Table 10-4). to designate a proxy decision-maker with authority to terminate Evaluations to determine the etiology of these symptoms usually life-sustaining treatments. Only in Alaska does the law prohibit prox-can be limited to the history and physical examination. In some cases, ies from terminating life-sustaining treatments. The health reform radiologic or other diagnostic examinations will provide sufficient benefit in directing optimal palliative care to warrant the risks, potential discomfort, and inconvenience, especially to a seriously ill patient. Only a few of the common symptoms that present difficult management issues will be addressed in this chapter. Additional information on the management of

1	especially to a seriously ill patient. Only a few of the common symptoms that present difficult management issues will be addressed in this chapter. Additional information on the management of other symptoms, such as nausea and vomiting, insomnia, and diarrhea, can be found in Chaps. 54 and 99, Chap. 38, and Chap. 55, respectively.

1	Pain • Frequency The frequency of pain among terminally ill patients varies widely. Substantial pain occurs in 36–90% of patients with advanced cancer. In the SUPPORT study of hospitalized patients with diverse conditions and an estimated survival ≤6 months, 22% reported moderate to severe pain, and caregivers of those patients noted that 50% had similar levels of pain during the last few days of life. A meta-analysis found pain prevalence of 58–69% in studies that included patients characterized as having advanced, metastatic, or terminal cancer; 44–73% in studies that included patients characterized as undergoing cancer treatment; and 21–46% in studies that included posttreatment individuals.

1	etIology Nociceptive pain is the result of direct mechanical or chemical stimulation of nociceptors and normal neural signaling to the brain. It tends to be localized, aching, throbbing, and cramping. The classic example is bone metastases. Visceral pain is caused by nociceptors in gastrointestinal, respiratory, and other organ systems. It is a deep or colicky type of pain classically associated with pancreatitis, myocardial infarction, or tumor invasion of viscera. Neuropathic pain arises from disordered nerve signals. It is described by patients as burning, electrical, or shocklike pain. Classic examples are poststroke pain, tumor invasion of the brachial plexus, and herpetic neuralgia.

1	assessment Pain is a subjective experience. Depending on the patient’s circumstances, perspective, and physiologic condition, the same physical lesion or disease state can produce different levels of reported pain and need for pain relief. Systematic assessment includes eliciting the following: (1) type: throbbing, cramping, burning, etc.; (2) periodicity: continuous, with or without exacerbations, or incident; (3) location; (4) intensity; (5) modifying factors; (6) effects of treatments; (7) functional impact; and (8) impact on patient. Several validated pain assessment measures may be used, such as the Visual Analogue Scale, the Brief Pain Inventory, and the pain component of one of the more comprehensive symptom assessment instruments. Frequent reassessments are essential to assess the effects of interventions.

1	InterventIons Interventions for pain must be tailored to each individual, with the goal of preempting chronic pain and relieving breakthrough pain. At the end of life, there is rarely reason to doubt a patient’s report of pain. Pain medications are the cornerstone of management. If they are failing and nonpharmacologic interventions— including radiotherapy and anesthetic or neurosurgical procedures such as peripheral nerve blocks or epidural medications—are required, a pain consultation is appropriate.

1	Pharmacologic interventions follow the World Health Organization three-step approach involving nonopioid analgesics, mild opioids, and strong opioids, with or without adjuvants (Chap. 18). Nonopioid analgesics, especially nonsteroidal anti-inflammatory drugs (NSAIDs), are the initial treatments for mild pain. They work primarily by inhibiting peripheral prostaglandins and reducing inflammation but also may have central nervous system (CNS) effects. They have a ceiling effect. Ibuprofen, up to a total dose of 1600 mg/d given in four doses of 400 mg each, has a minimal risk of causing bleeding and renal impairment and is a good initial choice. In patients with a history of severe gastrointestinal (GI) or other bleeding, it should be avoided. In patients with a history of mild gastritis or gastroesophageal reflux disease (GERD), acid-lowering therapy such as a proton pump inhibitor should be used. Acetaminophen is an alternative in patients with a history of GI bleeding and can be used

1	gastroesophageal reflux disease (GERD), acid-lowering therapy such as a proton pump inhibitor should be used. Acetaminophen is an alternative in patients with a history of GI bleeding and can be used safely at up to 4 g/d given in four doses of 1 g each. In patients with liver dysfunction due to metastases or other causes and in patients with heavy alcohol use, doses should be reduced.

1	If nonopioid analgesics are insufficient, opioids should be introduced. They work by interacting with µ opioid receptors in the CNS to activate pain-inhibitory neurons; most are receptor antagonists. The mixed agonist/antagonist opioids useful for postacute pain should not be used for the chronic pain in end-of-life care. Weak opioids such as codeine can be used initially. However, if they are escalated and fail to relieve pain, strong opioids such as morphine, 5–10 mg every 4 h, should be used. Nonopioid analgesics should be combined with opioids because they potentiate the effect of opioids.

1	For continuous pain, opioids should be administered on a regular, around-the-clock basis consistent with their duration of analgesia. They should not be provided only when the patient experiences pain; the goal is to prevent patients from experiencing pain. Patients also should be provided rescue medication, such as liquid morphine, for breakthrough pain, generally at 20% of the baseline dose. Patients should be informed that using the rescue medication does not obviate the need to take the next standard dose of pain medication. If after 24 h the patient’s pain remains uncontrolled and recurs before the next dose, requiring the patient to use the rescue medication, the daily opioid dose can be increased by the total dose of rescue medications used by the patient, or by 50% for moderate pain and 100% for severe pain of the standing opioid daily dose.

1	It is inappropriate to start with extended-release preparations. Instead, an initial focus on using short-acting preparations to determine how much is required in the first 24–48 h will allow clinicians to determine opioid needs. Once pain relief is obtained with short-acting preparations, one should switch to extended-release preparations. Even with a stable extended-release preparation regimen, the patient may have incident pain, such as during movement or dressing changes. Short-acting preparations should be taken before such predictable episodes. Although less common, patients may have “end-of-dose failure” with long-acting opioids, meaning that they develop pain after 8 h in the case of an every-12-h medication. In these cases, a trial of giving an every-12-h medication every 8 h is appropriate.

1	Because of differences in opioid receptors, cross-tolerance among opioids is incomplete, and patients may experience different side effects with different opioids. Therefore, if a patient is not experiencing pain relief or is experiencing too many side effects, a change to another opioid preparation is appropriate. When switching, one should begin with 50–75% of the published equianalgesic dose of the new opioid. Unlike NSAIDs, opioids have no ceiling effect; therefore, there is no maximum dose no matter how many milligrams the patient is receiving. The appropriate dose is the dose needed to achieve pain relief. This is an important point for clinicians to explain to patients and families. Addiction or excessive respiratory depression is extremely unlikely in the terminally ill; fear of these side effects should neither prevent escalating opioid medications when the patient is experiencing insufficient pain relief nor justify using opioid antagonists.

1	Opioid side effects should be anticipated and treated preemptively. Nearly all patients experience constipation that can be debilitating (see below). Failure to prevent constipation often results in noncompliance with opioid therapy. Methylnaltrexone is a drug that targets opioid-induced constipation by blocking peripheral opioid receptors but not central receptors for analgesia. In placebo-controlled trials, it has been shown to cause laxation within 24 h of administration. As with the use of opioids, about a third of patients using methylnaltrexone experience nausea and vomiting, but unlike constipation, tolerance develops, usually within a week. Therefore, when one is beginning opioids, an antiemetic such as metoclopramide or a serotonin antagonist often is prescribed prophylactically and stopped after 1 week. Olanzapine also has antinausea properties and can be effective in countering delirium or anxiety, with the advantage of some weight gain.

1	Drowsiness, a common side effect of opioids, also usually abates within a week. During this period, drowsiness can be treated with psychostimulants such as dextroamphetamine, methylphenidate, and modafinil. Modafinil has the advantage of everyday dosing. Pilot reports suggest that donepezil may also be helpful for opiate-induced drowsiness as well as relieving fatigue and anxiety. Metabolites of morphine and most opioids are cleared renally; doses may have to be adjusted for patients with renal failure.

1	Seriously ill patients who require chronic pain relief rarely if ever become addicted. Suspicion of addiction should not be a reason to withhold pain medications from terminally ill patients. Patients and families may withhold prescribed opioids for fear of addiction or dependence. Physicians and health care providers should reassure patients and families that the patient will not become addicted to opioids if they are used as prescribed for pain relief; this fear should not prevent the patient from taking the medications around the clock. However, diversion of drugs for use by other family members or illicit sale may occur. It may be necessary to advise the patient and caregiver about secure storage of opioids. Contract writing with the patient and family can help. If that fails, transfer to a safe facility may be necessary.

1	Tolerance is the need to increase medication dosage for the same pain relief without a change in disease. In the case of patients with advanced disease, the need for increasing opioid dosage for pain relief usually is caused by disease progression rather than tolerance. Physical dependence is indicated by symptoms from the abrupt withdrawal of opioids and should not be confused with addiction.

1	Adjuvant analgesic medications are nonopioids that potentiate the analgesic effects of opioids. They are especially important in the management of neuropathic pain. Gabapentin and pregabalin, calcium channel alpha 2-delta ligands, are now the first-line treatments for neuropathic pain from a variety of causes. Gabapentin is begun at 100–300 mg bid or tid, with 50–100% dose increments every 3 days. Usually 900–3600 mg/d in two or three doses is effective. The combination of gabapentin and nortriptyline may be more effective than gabapentin alone. One potential side effect of gabapentin to be aware of is confusion and drowsiness, especially in the elderly. Pregabalin has the same mechanism of action as gabapentin but is absorbed more efficiently from the GI tract. It is started at 75 mg bid and increased to 150 mg bid. The maximum dose is 225 mg bid. Carbamazepine, a first-generation agent, has been proved effective in randomized trials for neuropathic pain. Other potentially effective

1	and increased to 150 mg bid. The maximum dose is 225 mg bid. Carbamazepine, a first-generation agent, has been proved effective in randomized trials for neuropathic pain. Other potentially effective anticonvulsant adjuvants include topiramate (25–50 mg qd or bid, rising to 100–300 mg/d) and oxcarbazepine (75–300 mg bid, rising to 1200 mg bid). Glucocorticoids, preferably dexamethasone given once a day, can be useful in reducing inflammation that causes pain while elevating mood, energy, and appetite. Its main side effects include confusion, sleep difficulties, and fluid retention. Glucocorticoids are especially effective for bone pain and abdominal pain from distention of the GI tract or liver. Other drugs, including clonidine and baclofen, can be effective in pain relief. These drugs are adjuvants and generally should be used in conjunction with—not instead of—opioids. Methadone, carefully dosed because of its unpredictable half-life in many patients, has activity at the

1	drugs are adjuvants and generally should be used in conjunction with—not instead of—opioids. Methadone, carefully dosed because of its unpredictable half-life in many patients, has activity at the N-methyl-d-aspartamate (NMDA) receptor and is useful for complex pain syndromes and neuropathic pain. It generally is reserved for cases in which first-line opioids (morphine, oxycodone, hydromorphone) are either ineffective or unavailable.

1	Radiation therapy can treat bone pain from single metastatic lesions. Bone pain from multiple metastases can be amenable to radiopharmaceuticals such as strontium-89 and samarium-153. Bisphosphonates (such as pamidronate [90 mg every 4 weeks]) and calcitonin (200 IU intranasally once or twice a day) also provide relief from bone pain but have an onset of action of days. Constipation • Frequency Constipation is reported in up to 87% of patients requiring palliative care.

1	Constipation • Frequency Constipation is reported in up to 87% of patients requiring palliative care. etIology Although hypercalcemia and other factors can cause constipation, it is most frequently a predictable consequence of the use of opioids for the relief of pain and dyspnea and of tricyclic antidepressants, from their anticholinergic effects, and of the inactivity and poor diet that are common among seriously ill patients. If untreated, constipation can cause substantial pain and vomiting and also is associated with confusion and delirium. Whenever opioids and other medications known to cause constipation are used, preemptive treatment for constipation should be instituted.

1	assessment The physician should establish the patient’s previous bowel habits, including the frequency, consistency, and volume. Abdominal and rectal examinations should be performed to exclude impaction or acute abdomen. A number of constipation assessment scales are available, although guidelines issued in the Journal of Palliative Medicine did not recommend them for routine practice. Radiographic assessments beyond a simple flat plate of the abdomen in cases in which obstruction is suspected are rarely necessary.

1	InterventIon Intervention to reestablish comfortable bowel habits and relieve pain and discomfort should be the goals of any measures to address constipation during end-of-life care. Although physical activity, adequate hydration, and dietary treatments with fiber can be helpful, each is limited in its effectiveness for most seriously ill patients, and fiber may exacerbate problems in the setting of dehydration and if impaired motility is the etiology. Fiber is contraindicated in the presence of opioid use. Stimulant and osmotic laxatives, stool softeners, fluids, and enemas are the mainstays of therapy (Table 10-5). In preventing constipation from opioids and other medications, a combination of a laxative and a stool softener (such as senna and docusate) should be used. If after several days of treatment, a bowel movement has not occurred, a rectal examination to remove impacted stool and place a suppository is necessary. For patients with impending bowel obstruction or gastric

1	days of treatment, a bowel movement has not occurred, a rectal examination to remove impacted stool and place a suppository is necessary. For patients with impending bowel obstruction or gastric stasis, octreotide to reduce secretions can be helpful. For patients in whom the suspected mechanism is dysmotility, metoclopramide can be helpful.

1	Nausea • Frequency Up to 70% of patients with advanced cancer have nausea, defined as the subjective sensation of wanting to vomit. etIology Nausea and vomiting are both caused by stimulation at one of four sites: the GI tract, the vestibular system, the chemoreceptor trigger zone (CTZ), and the cerebral cortex. Medical treatments for nausea are aimed at receptors at each of these sites: the GI tract contains mechanoreceptors, chemoreceptors, and 5-hydroxytryptamine type 3 (5-HT3) receptors; the vestibular system probably contains histamine and acetylcholine receptors; and the CTZ contains chemoreceptors, dopamine type 2 receptors, and 5-HT3 receptors. An example of nausea that most likely is mediated by the cortex is anticipatory nausea before a dose of chemotherapy or other noxious stimuli.

1	Specific causes of nausea include metabolic changes (liver failure, uremia from renal failure, hypercalcemia), bowel obstruction, constipation, infection, GERD, vestibular disease, brain metastases, medications (including antibiotics, NSAIDs, proton pump inhibitors, opioids, and chemotherapy), and radiation therapy. Anxiety can also contribute to nausea.

1	InterventIon Medical treatment of nausea is directed at the anatomic and receptor-mediated cause that a careful history and physical examination reveals. When a single specific cause is not found, many advocate beginning treatment with a dopamine antagonist such as haloperidol or prochlorperazine. Prochlorperazine is usually more sedating than haloperidol. When decreased motility is suspected, metoclopramide can be an effective treatment. When inflammation of the GI tract is suspected, glucocorticoids such as dexamethasone are an appropriate treatment. For nausea that follows chemotherapy and radiation therapy, one of the 5-HT3 receptor antagonists (ondansetron, granisetron, dolasetron, palonosetron) is recommended. Studies suggest palonosetron has higher receptor binding affinity and clinical superiority to the other 5-HT3 receptor antagonists. Clinicians should attempt prevention of postchemotherapy nausea rather than provide treatment after the fact. Current clinical guidelines

1	clinical superiority to the other 5-HT3 receptor antagonists. Clinicians should attempt prevention of postchemotherapy nausea rather than provide treatment after the fact. Current clinical guidelines recommend tailoring the strength of treatments to the specific emetic risk posed by a specific chemotherapy drug. When a vestibular cause (such as “motion sickness” or labyrinthitis) is suspected, antihistamines such as meclizine (whose primary side effect is drowsiness) or anticholinergics such as scopolamine can be effective. In anticipatory nausea, a benzodiazepine such as lorazepam is indicated. As with antihistamines, drowsiness and confusion are the main side effects.

1	dyspnea • Frequency Dyspnea is a subjective experience of being short of breath. Frequencies vary among causes of death, but it can affect 80–90% of dying patients with lung cancer, COPD, and heart disease. Dyspnea is among the most distressing physical symptoms and can be even more distressing than pain.

1	assessment As with pain, dyspnea is a subjective experience that may not correlate with objective measures of Po2, Pco2, or respiratory rate. Consequently, measurements of oxygen saturation through pulse oximetry or blood gases are rarely helpful in guiding therapy. Despite the limitations of existing assessment methods, physicians should regularly assess and document patients’ experience of dyspnea and its intensity. Guidelines recommend visual or analogue dyspnea scales to assess the severity of symptoms and the effects of treatment. Potentially reversible or treatable causes of dyspnea include infection, pleural effusions, pulmonary emboli, pulmonary edema, asthma, and tumor encroachment on the airway. However, the risk-versus-benefit ratio of the diagnostic and therapeutic interventions for patients with little time left to live must be considered carefully before one undertakes diagnostic steps. Frequently, the specific etiology cannot be identified, and dyspnea is the

1	for patients with little time left to live must be considered carefully before one undertakes diagnostic steps. Frequently, the specific etiology cannot be identified, and dyspnea is the consequence of progression of the underlying disease that cannot be treated. The anxiety caused by dyspnea and the choking sensation can significantly exacerbate the underlying dyspnea in a negatively reinforcing cycle.

1	InterventIons When reversible or treatable etiologies are diagnosed, they should be treated as long as the side effects of treatment, such as repeated drainage of effusions or anticoagulants, are less burdensome than the dyspnea itself. More aggressive treatments such as stenting a bronchial lesion may be warranted if it is clear that the dyspnea is due to tumor invasion at that site and if the patient and family understand the risks of such a procedure. Usually, treatment will be symptomatic (Table 10-6). A dyspnea scale and careful monitoring should guide dose adjustment. Low-dose opioids reduce the sensitivity of the central respiratory center and the sensation of dyspnea. If patients are not receiving opioids, weak opioids can be initiated; if patients are already receiving opioids, morphine or other strong opioids should be used. Controlled trials do not support the use of nebulized opioids for dyspnea at the end of life. Phenothiazines and chlorpromazine may be helpful when

1	morphine or other strong opioids should be used. Controlled trials do not support the use of nebulized opioids for dyspnea at the end of life. Phenothiazines and chlorpromazine may be helpful when combined with opioids. Benzodiazepines can be helpful if anxiety is present but should be neither used as first-line therapy nor used alone in the treatment of dyspnea. If the patient has a history of COPD or asthma, inhaled bronchodilators and glucocorticoids may be helpful. If the patient has pulmonary edema due to heart failure, diuresis with a medication such as furosemide is indicated. Excess secretions can be dried with scopolamine, transdermally or intravenously. Use of oxygen is controversial. There are conflicting data on its effectiveness for patients with proven hypoxemia. But there is no clear benefit of oxygen compared to room air for nonhypoxemic patients. Noninvasive positive-pressure ventilation using a facemask or nasal plugs may be used for some patients for symptom relief.

1	no clear benefit of oxygen compared to room air for nonhypoxemic patients. Noninvasive positive-pressure ventilation using a facemask or nasal plugs may be used for some patients for symptom relief. For some families and patients, oxygen is distressing; for others, it is reassuring. More general interventions that medical staff can do include sitting the patient upright, removing smoke or other irritants such as perfume, ensuring a supply of fresh air with sufficient humidity, and minimizing other factors that can increase anxiety.

1	Fatigue • Frequency More than 90% of terminally ill patients experience fatigue and/or weakness. Fatigue is one of the most commonly reported symptoms of cancer treatment as well as in the palliative care of multiple sclerosis, COPD, heart failure, and HIV. Fatigue frequently is cited as among the most distressing symptoms.

1	etIology The multiple causes of fatigue in the terminally ill can be categorized as resulting from the underlying disease; from disease-induced factors such as tumor necrosis factor and other cytokines; and from secondary factors such as dehydration, anemia, infection, hypothyroidism, and drug side effects. Apart from low caloric intake, loss of muscle mass and changes in muscle enzymes may play an important role in fatigue of terminal illness. The importance of changes in the CNS, especially the reticular activating system, have been hypothesized based on reports of fatigue in patients receiving cranial radiation, experiencing depression, or having chronic pain in the absence of cachexia or other physiologic changes. Finally, depression and other causes of psychological distress can contribute to fatigue.

1	assessment Like pain and dyspnea, fatigue is subjective. Objective changes, even in body mass, may be absent. Consequently, assessment must rely on patient self-reporting. Scales used to measure fatigue, such as the Edmonton Functional Assessment Tool, the Fatigue Self-Report Scales, and the Rhoten Fatigue Scale, are usually appropriate for research rather than clinical purposes. In clinical practice, a simple performance assessment such as the Karnofsky Performance Status or the ECOG’s question “How much of the day does the patient spend in bed?” may be the best measure. In this 0–4 performance status assessment, 0 = normal activity; 1 = symptomatic without being bedridden; 2 = requiring some, but <50%, bed time; 3 = bedbound more than half the day; and 4 = bedbound all the time. Such a scale allows for assessment over time and correlates with overall disease severity and prognosis. A 2008 review by the European Association of Palliative Care also described several longer assessment

1	scale allows for assessment over time and correlates with overall disease severity and prognosis. A 2008 review by the European Association of Palliative Care also described several longer assessment tools with 9–20 items, including the Piper Fatigue Inventory, the Multidimensional Fatigue Inventory, and the Brief Fatigue Inventory (BFI).

1	InterventIons For some patients, there are reversible causes such as anemia, but for most patients at the end of life, fatigue will not be “cured.” The goal is to ameliorate it and help patients and families adjust expectations. Behavioral interventions should be used to avoid blaming the patient for inactivity and to educate both the family and the patient that the underlying disease causes physiologic changes that produce low energy levels. Understanding that the problem is physiologic and not psychological can help alter expectations regarding the patient’s level of physical activity. Practically, this may mean reducing routine activities such as housework and cooking or social events outside the house and making it acceptable to receive guests lying on a couch. At the same time, institution of exercise regimens and physical therapy can raise endorphins, reduce muscle wasting, and reduce the risk of depression. In addition, ensuring good hydration without worsening edema may help

1	of exercise regimens and physical therapy can raise endorphins, reduce muscle wasting, and reduce the risk of depression. In addition, ensuring good hydration without worsening edema may help reduce fatigue. Discontinuing medications that worsen fatigue may help, including cardiac medications, benzodiazepines, certain antidepressants, or opioids if pain is well-controlled. As end-of-life care proceeds into its final stages, fatigue may protect patients from further suffering, and continued treatment could be detrimental.

1	There are woefully few pharmacologic interventions that target fatigue and weakness. Glucocorticoids can increase energy and enhance mood. Dexamethasone is preferred for its once-a-day dosing and minimal mineralocorticoid activity. Benefit, if any, usually is seen within the first month. Psychostimulants such as dextroamphetamine (5–10 mg PO) and methylphenidate (2.5–5 mg PO) may also enhance energy levels, although a randomized trial did not show methylphenidate beneficial compared with placebo in cancer fatigue. Doses should be given in the morning and at noon to minimize the risk of counterproductive insomnia. Modafinil, developed for narcolepsy, has shown some promise in the treatment of severe fatigue and has the advantage of once-daily dosing. Its precise role in fatigue at the end of life has not been determined. Anecdotal evidence suggests that L-carnitine may improve fatigue, depression, and sleep disruption. Similarly, some studies suggest ginseng can reduce fatigue.

1	PSYCHoLogICAL SYMPToMS ANd THEIR MANAgEMENT depression • Frequency Depression at the end of life presents an apparently paradoxical situation. Many people believe that depression is normal among seriously ill patients because they are dying. People frequently say, “Wouldn’t you be depressed?” However, depression is not a necessary part of terminal illness and can contribute to needless suffering. Although sadness, anxiety, anger, and irritability are normal responses to a serious condition, they are typically of modest intensity and transient. Persistent sadness and anxiety and the physically disabling symptoms that they can lead to are abnormal and suggestive of major depression. Although as many as 75% of terminally ill patients experience emotional distress and depressive symptoms, <30% of terminally ill patients have major depression. Depression is not limited to cancer patients but found in patients with end-stage renal disease, Parkinson’s disease, multiple sclerosis, and other

1	of terminally ill patients have major depression. Depression is not limited to cancer patients but found in patients with end-stage renal disease, Parkinson’s disease, multiple sclerosis, and other terminal conditions.

1	etIology Previous history of depression, family history of depression or bipolar disorder, and prior suicide attempts are associated with increased risk for depression among terminally ill patients. Other symptoms, such as pain and fatigue, are associated with higher rates of depression; uncontrolled pain can exacerbate depression, and depression can cause patients to be more distressed by pain. Many medications used in the terminal stages, including glucocorticoids, and some anticancer agents, such as tamoxifen, interleukin 2, interferon α, and vincristine, also are associated with depression. Some terminal conditions, such as pancreatic cancer, certain strokes, and heart failure, have been reported to be associated with higher rates of depression, although this is controversial. Finally, depression may be attributable to grief over the loss of a role or function, social isolation, or loneliness.

1	assessment Diagnosing depression among seriously ill patients is complicated because many of the vegetative symptoms in the DSM-V (Diagnostic and Statistical Manual of Mental Disorders) criteria for clinical depression—insomnia, anorexia and weight loss, fatigue, decreased libido, and difficulty concentrating—are associated with the dying process itself. The assessment of depression in seriously ill patients therefore should focus on the dysphoric mood, helplessness, hopelessness, and lack of interest and enjoyment and concentration in normal activities. The single questions “How often do you feel downhearted and blue?” (more than a good bit of the time or similar responses) and “Do you feel depressed most of the time?” are appropriate for screening. Visual Analog Scales can also be useful in screening.

1	InterventIons Physicians must treat any physical symptom, such as pain, that may be causing or exacerbating depression. Fostering adaptation to the many losses that the patient is experiencing can also be helpful. Nonpharmacologic interventions, including group or individual psychological counseling, and behavioral therapies such as relaxation and imagery can be helpful, especially in combination with drug therapy.

1	Pharmacologic interventions remain the core of therapy. The same medications are used to treat depression in terminally ill as in non– terminally ill patients. Psychostimulants may be preferred for patients with a poor prognosis or for those with fatigue or opioid-induced somnolence. Psychostimulants are comparatively fast acting, working within a few days instead of the weeks required for selective serotonin reuptake inhibitors (SSRIs). Dextroamphetamine or methylphenidate should be started at 2.5–5.0 mg in the morning and at noon, the same starting doses used for treating fatigue. The dose can be escalated up to 15 mg bid. Modafinil is started at 100 mg qd and can be increased to 200 mg if there is no effect at the lower dose. Pemoline is a nonamphetamine psychostimulant with minimal abuse potential. It is also effective as an antidepressant beginning at 18.75 mg in the morning and at noon. Because it can be absorbed through the buccal mucosa, it is preferred for patients with

1	abuse potential. It is also effective as an antidepressant beginning at 18.75 mg in the morning and at noon. Because it can be absorbed through the buccal mucosa, it is preferred for patients with intestinal obstruction or dysphagia. If it is used for prolonged periods, liver function must be monitored. The psychostimulants can also be combined with more traditional antidepressants while waiting for the antidepressants to become effective and then tapered after a few weeks if necessary. Psychostimulants have side effects, particularly initial anxiety, insomnia, and rarely paranoia, which may necessitate lowering the dose or discontinuing treatment.

1	Mirtazapine, an antagonist at the postsynaptic serotonin receptors, is a promising psychostimulant. It should be started at 7.5 mg before bed. It has sedating, antiemetic, and anxiolytic properties with few drug interactions. Its side effect of weight gain may be beneficial for seriously ill patients; it is available in orally disintegrating tablets.

1	For patients with a prognosis of several months or longer, SSRIs, including fluoxetine, sertraline, paroxetine, citalopram, escitalopram, and fluvoxamine, and serotonin-noradrenaline reuptake inhibitors such as venlafaxine, are the preferred treatment because of their efficacy and comparatively few side effects. Because low doses of these medications may be effective for seriously ill patients, one should use half the usual starting dose for healthy adults. The starting dose for fluoxetine is 10 mg once a day. In most cases, once-a-day dosing is possible. The choice of which SSRI to use should be driven by the patient’s past success or failure with the specific medication, the most favorable side effect profile for that specific agent, and (3) the time it takes to reach steady-state drug levels. For instance, for a patient in whom fatigue is a major symptom, a more activating SSRI (fluoxetine) would be appropriate. For a patient in whom anxiety and sleeplessness are major symptoms, a

1	levels. For instance, for a patient in whom fatigue is a major symptom, a more activating SSRI (fluoxetine) would be appropriate. For a patient in whom anxiety and sleeplessness are major symptoms, a more sedating SSRI (paroxetine) would be appropriate.

1	Atypical antidepressants are recommended only in selected circumstances, usually with the assistance of a specialty consultation. Trazodone can be an effective antidepressant but is sedating and can cause orthostatic hypotension and, rarely, priapism. Therefore, it should be used only when a sedating effect is desired and is often used for patients with insomnia, at a dose starting at 25 mg. In addition to its antidepressant effects, bupropion is energizing, making it useful for depressed patients who experience fatigue. However, it can cause seizures, preventing its use for patients with a risk of CNS neoplasms or terminal delirium. Finally, alprazolam, a benzodiazepine, starting at 0.25–1.0 mg tid, can be effective in seriously ill patients who have a combination of anxiety and depression. Although it is potent and works quickly, it has many drug interactions and may cause delirium, especially among very ill patients, because of its strong binding to the

1	of anxiety and depression. Although it is potent and works quickly, it has many drug interactions and may cause delirium, especially among very ill patients, because of its strong binding to the benzodiazepine–γ-aminobutyric acid (GABA) receptor complex.

1	Unless used as adjuvants for the treatment of pain, tricyclic antidepressants are not recommended. Similarly, monoamine oxidase (MAO) inhibitors are not recommended because of their side effects and dangerous drug interactions. delirium (See Chap. 34) • Frequency In the weeks or months before death, delirium is uncommon, although it may be significantly under-diagnosed. However, delirium becomes relatively common in the hours and days immediately before death. Up to 85% of patients dying from cancer may experience terminal delirium.

1	etIology Delirium is a global cerebral dysfunction characterized by alterations in cognition and consciousness. It frequently is preceded by anxiety, changes in sleep patterns (especially reversal of day and night), and decreased attention. In contrast to dementia, delirium has an acute onset, is characterized by fluctuating consciousness and inattention, and is reversible, although reversibility may be more theoretical than real for patients near death. Delirium may occur in a patient with dementia; indeed, patients with dementia are more vulnerable to delirium.

1	Causes of delirium include metabolic encephalopathy arising from liver or renal failure, hypoxemia, or infection; electrolyte imbalances such as hypercalcemia; paraneoplastic syndromes; dehydration; and primary brain tumors, brain metastases, or leptomeningeal spread of tumor. Commonly, among dying patients, delirium can be caused by side effects of treatments, including radiation for brain metastases, and medications, including opioids, glucocorticoids, anticholinergic drugs, antihistamines, antiemetics, benzodiazepines, and chemotherapeutic agents. The etiology may be multifactorial; e.g., dehydration may exacerbate opioid-induced delirium.

1	assessment Delirium should be recognized in any terminally ill patient with new onset of disorientation, impaired cognition, somnolence, fluctuating levels of consciousness, or delusions with or without agitation. Delirium must be distinguished from acute anxiety and depression as well as dementia. The central distinguishing feature is altered consciousness, which usually is not noted in anxiety, depression, and dementia. Although “hyperactive” delirium characterized by overt confusion and agitation is probably more common, patients also should be assessed for “hypoactive” delirium characterized by sleep-wake reversal and decreased alertness.

1	In some cases, use of formal assessment tools such as the Mini-Mental Status Examination (which does not distinguish delirium from dementia) and the Delirium Rating Scale (which does distinguish delirium from dementia) may be helpful in distinguishing delirium from other processes. The patient’s list of medications must be evaluated carefully. Nonetheless, a reversible etiologic factor for delirium is found in fewer than half of terminally ill patients. Because most terminally ill patients experiencing delirium will be very close to death and may be at home, extensive diagnostic evaluations such as lumbar punctures and neuroradiologic examinations are usually inappropriate.

1	InterventIons One of the most important objectives of terminal care is to provide terminally ill patients the lucidity to say goodbye to the people they love. Delirium, especially with agitation during the final days, is distressing to family and caregivers. A strong determinant of bereavement difficulties is witnessing a difficult death. Thus, terminal delirium should be treated aggressively. At the first sign of delirium, such as day-night reversal with slight changes in mentation, the physician should let the family members know that it is time to be sure that everything they want to say has been said. The family should be informed that delirium is common just before death.

1	If medications are suspected of being a cause of the delirium, unnecessary agents should be discontinued. Other potentially reversible causes, such as constipation, urinary retention, and metabolic abnormalities, should be treated. Supportive measures aimed at providing a familiar environment should be instituted, including restricting visits only to individuals with whom the patient is familiar and eliminating new experiences; orienting the patient, if possible, by providing a clock and calendar; and gently correcting the patient’s hallucinations or cognitive mistakes.

1	Pharmacologic management focuses on the use of neuroleptics and, in the extreme, anesthetics (Table 10-7). Haloperidol remains first-line therapy. Usually, patients can be controlled with a low dose (1–3 mg/d), usually given every 6 h, although some may require as much as 20 mg/d. It can be administered PO, SC, or IV. IM injections should not be used, except when this is the only way to get a patient under control. Newer atypical neuroleptics, such as olanzapine, risperidone, and quetiapine, have shown significant effectiveness in completely resolving delirium in cancer patients. These drugs also have fewer side effects than haloperidol, along with other beneficial effects for terminally ill patients, including antinausea, antianxiety, and weight gain. They are useful for patients with longer anticipated life expectancy because they are less likely to cause dysphoria and have a lower risk of dystonic reactions. Also, because they are metabolized through multiple pathways, they can be

1	anticipated life expectancy because they are less likely to cause dysphoria and have a lower risk of dystonic reactions. Also, because they are metabolized through multiple pathways, they can be used in patients with hepatic and renal dysfunction. Olanzapine has the disadvantage that it is available only orally and that it takes a week to reach steady state. The usual dose is 2.5–5 mg PO bid. Chlorpromazine (10–25 mg every 4–6 h) can be useful if sedation is desired and can be administered IV or PR in addition to PO. Dystonic reactions resulting from dopamine blockade are a side effect of neuroleptics, although they are reported to be rare when these drugs are used to treat terminal delirium. If patients develop dystonic reactions, benztropine should be administered. Neuroleptics may be combined with lorazepam to reduce agitation when the delirium is the result of alcohol or sedative withdrawal.

1	If no response to first-line therapy is seen, a specialty consultation should be obtained with a change to a different medication. If patients fail to improve after a second neuroleptic, sedation with an anesthetic such as propofol or continuous-infusion midazolam may be necessary. By some estimates, at the very end of life, as many as 25% of patients experiencing delirium, especially restless delirium with myoclonus or convulsions, may require sedation. Physical restraints should be used with great reluctance and only when the patient’s violence is threatening to self or others. If they are used, their appropriateness should be reevaluated frequently.

1	Insomnia • Frequency Sleep disorders, defined as difficulty initiating sleep or maintaining sleep, sleep difficulty at least 3 nights a week, or sleep difficulty that causes impairment of daytime functioning, occur in 19–63% of patients with advanced cancer. Some 30–74% of patients with other end-stage conditions, including AIDS, heart disease, COPD, and renal disease, experience insomnia. etIology Patients with cancer may have changes in sleep efficiency such as an increase in stage I sleep. Other etiologies of insomnia are coexisting physical illness such as thyroid disease and coexisting psychological illnesses such as depression and anxiety. Medications, including antidepressants, psychostimulants, steroids, and β agonists, are significant contributors to sleep disorders, as are caffeine and alcohol. Multiple over-the-counter medications contain caffeine and antihistamines, which can contribute to sleep disorders.

1	assessment Assessment should include specific questions concerning sleep onset, sleep maintenance, and early-morning wakening as these will provide clues to the causative agents and to management. Patients should be asked about previous sleep problems, screened for depression and anxiety, and asked about symptoms of thyroid disease. Caffeine and alcohol are prominent causes of sleep problems, and a careful history of the use of these substances should be obtained. Both excessive use and withdrawal from alcohol can be causes of sleep problems.

1	InterventIons The mainstays of intervention include improvement of sleep hygiene (encouragement of regular time for sleep, decreased nighttime distractions, elimination of caffeine and other stimulants and alcohol), intervention to treat anxiety and depression, and treatment for the insomnia itself. For patients with depression who have insomnia and anxiety, a sedating antidepressant such as mirtazapine can be helpful. In the elderly, trazodone, beginning at 25 mg at nighttime, is an effective sleep aid at doses lower than those which cause its antidepressant effect. Zolpidem may have a decreased incidence of delirium in patients compared with traditional benzodiazepines, but this has not been clearly established. When benzodiazepines are prescribed, short-acting ones (such as lorazepam) are favored over longer-acting ones (such as diazepam). Patients who receive these medications should be observed for signs of increased confusion and delirium.

1	SoCIAL NEEdS ANd THEIR MANAgEMENT Financial Burdens • Frequency Dying can impose substantial economic strains on patients and families, causing distress. In the United States, with one of the least comprehensive health insurance systems among the developed countries, ~20% of terminally ill patients and their families spend >10% of family income on health care costs over and above health insurance premiums. Between 10 and 30% of families sell assets, use savings, or take out a mortgage to pay for the patient’s health care costs. Nearly 40% of terminally ill patients in the United States report that the cost of their illness is a moderate or great economic hardship for their family.

1	The patient is likely to reduce and eventually stop working. In 20% of cases, a family member of the terminally ill patient also stops working to provide care. The major underlying causes of economic burden are related to poor physical functioning and care needs, such as the need for housekeeping, nursing, and personal care. More debilitated patients and poor patients experience greater economic burdens.

1	InterventIon This economic burden should not be ignored as a private matter. It has been associated with a number of adverse health outcomes, including preferring comfort care over life-prolonging care as well as consideration of euthanasia or physician-assisted suicide. Economic burdens increase the psychological distress of families and caregivers of terminally ill patients, and poverty is associated with many adverse health outcomes. Importantly, recent studies found that “patients with advanced cancer who reported having end-of-life conversations with physicians had significantly lower health care costs in their final week of life. Higher costs were associated with worse quality of death.” Assistance from a social worker, early on if possible, to ensure access to all available benefits may be helpful. Many patients, families, and health care providers are unaware of options for long-term care insurance, respite care, the Family Medical Leave Act (FMLA), and other sources of

1	may be helpful. Many patients, families, and health care providers are unaware of options for long-term care insurance, respite care, the Family Medical Leave Act (FMLA), and other sources of assistance. Some of these options (such as respite care) may be part of a formal hospice program, but others (such as the FMLA) do not require enrollment in a hospice program.

1	Relationships • Frequency Settling personal issues and closing the narrative of lived relationships are universal needs. When asked if sudden death or death after an illness is preferable, respondents often initially select the former but soon change to the latter as they reflect on the importance of saying goodbye. Bereaved family members who have not had the chance to say goodbye often have a more difficult grief process.

1	InterventIons Care of seriously ill patients requires efforts to facilitate the types of encounters and time spent with family and friends that are necessary to meet those needs. Family and close friends may need to be accommodated with unrestricted visiting hours, which may include sleeping near the patient even in otherwise regimented institutional settings. Physicians and other health care providers may be able to facilitate and resolve strained interactions between the patient and other family members. Assistance for patients and family members who are unsure about how to create or help preserve memories, whether by providing materials such as a scrapbook or memory box or by offering them suggestions and informational resources, can be deeply appreciated. Taking photographs and creating videos can be especially helpful to terminally ill patients who have younger children or grandchildren.

1	Family Caregivers • Frequency Caring for seriously ill patients places a heavy burden on families. Families frequently are required to provide transportation and homemaking as well as other services. Typically, paid professionals such as home health nurses and hospice workers supplement family care; only about a quarter of all caregiving consists of exclusively paid professional assistance. The trend toward more out-of-hospital deaths will increase reliance on families for endof-life care. Increasingly, family members are being called upon to provide physical care (such as moving and bathing patients) and medical care (such as assessing symptoms and giving medications) in addition to emotional care and support.

1	Three-quarters of family caregivers of terminally ill patients are women—wives, daughters, sisters, and even daughters-in-law. Because many are widowed, women tend to be able to rely less on family for caregiving assistance and may need more paid assistance. About 20% of terminally ill patients report substantial unmet needs for nursing and personal care. The impact of caregiving on family caregivers is substantial: both bereaved and current caregivers have a higher mortality rate than that of non-caregiving controls.

1	InterventIons It is imperative to inquire about unmet needs and to try to ensure that those needs are met either through the family or by paid professional services when possible. Community assistance through houses of worship or other community groups often can be mobilized by telephone calls from the medical team to someone the patient or family identifies. Sources of support specifically for family caregivers should be identified through local sources or nationally through groups such as the National Family Caregivers Association (www.nfcacares.org), the American Cancer Society (www.cancer.org), and the Alzheimer’s Association (www.alz.org).

1	EXISTENTIAL NEEdS ANd THEIR MANAgEMENT Frequency Religion and spirituality are often important to dying patients. Nearly 70% of patients report becoming more religious or spiritual when they became terminally ill, and many find comfort in religious or spiritual practices such as prayer. However, ~20% of terminally ill patients become less religious, frequently feeling cheated or betrayed by becoming terminally ill. For other patients, the need is for existential meaning and purpose that is distinct from and may even be antithetical to religion or spirituality. When asked, patients and family caregivers frequently report wanting their professional caregivers to be more attentive to religion and spirituality.

1	assessment Health care providers are often hesitant about involving themselves in the religious, spiritual, and existential experiences of their patients because it may seem private or not relevant to the current illness. But physicians and other members of the care team should be able at least to detect spiritual and existential needs. Screening questions have been developed for a physician’s spiritual history taking. Spiritual distress can amplify other types of suffering and even masquerade as intractable physical pain, anxiety, or depression. The screening questions in the comprehensive assessment are usually sufficient. Deeper evaluation and intervention are rarely appropriate for the physician unless no other member of a care team is available or suitable. Pastoral care providers may be helpful, whether from the medical institution or from the patient’s own community.

1	InterventIons Precisely how religious practices, spirituality, and existential explorations can be facilitated and improve end-of-life care is not well established. What is clear is that for physicians, one main intervention is to inquire about the role and importance of spirituality and religion in a patient’s life. This will help a patient feel heard and help physicians identify specific needs. In one study, only 36% of respondents indicated that a clergy member would be comforting. Nevertheless, the increase in religious and spiritual interest among a substantial fraction of dying patients suggests inquiring of individual patients how this need can be addressed. Some evidence supports specific methods of addressing existential needs in patients, ranging from establishing a supportive group environment for terminal patients to individual treatments emphasizing a patient’s dignity and sources of meaning.

1	legal aspects For centuries, it has been deemed ethical to withhold or withdraw life-sustaining interventions. The current legal consensus in the United States and most developed countries is that patients have a moral as well as constitutional or common law right to refuse medical interventions. American courts also have held that incompetent patients have a right to refuse medical interventions. For patients who are incompetent and terminally ill and who have not completed an advance care directive, next of kin can exercise that right, although this may be restricted in some states, depending how clear and convincing the evidence is of the patient’s preferences. Courts have limited families’ ability to terminate life-sustaining treatments in patients who are conscious, incompetent, but not terminally ill. In theory, patients’ right to refuse medical therapy can be limited by four countervailing interests: (1) preservation of life, (2) prevention of suicide, (3) protection of third

1	not terminally ill. In theory, patients’ right to refuse medical therapy can be limited by four countervailing interests: (1) preservation of life, (2) prevention of suicide, (3) protection of third parties such as children, and (4) preservation of the integrity of the medical profession. In practice, these interests almost never override the right of competent patients and incompetent patients who have left explicit and advance care directives.

1	For incompetent patients who either appointed a proxy without specific indications of their wishes or never completed an advance care directive, three criteria have been suggested to guide the decision to terminate medical interventions. First, some commentators suggest that ordinary care should be administered but extraordinary care could be terminated. Because the ordinary/extraordinary distinction is too vague, courts and commentators widely agree that it should not be used to justify decisions about stopping treatment. Second, many courts have advocated the use of the substituted-judgment criterion, which holds that the proxy decision-makers should try to imagine what the incompetent patient would do if he or she were competent. However, multiple studies indicate that many proxies, even close family members, cannot accurately predict what the patient would have wanted. Therefore, substituted judgment becomes more of a guessing game than a way of fulfilling the patient’s wishes.

1	even close family members, cannot accurately predict what the patient would have wanted. Therefore, substituted judgment becomes more of a guessing game than a way of fulfilling the patient’s wishes. Finally, the best-interests criterion holds that proxies should evaluate treatments by balancing their benefits and risks and select those treatments in which the benefits maximally outweigh the burdens of treatment. Clinicians have a clear and crucial role in this by carefully and dispassionately explaining the known benefits and burdens of specific treatments. Yet even when that information is as clear as possible, different individuals can have very different views of what is in the patient’s best interests, and families may have disagreements or even overt conflicts. This criterion has been criticized because there is no single way to determine the balance between benefits and burdens; it depends on a patient’s personal values. For instance, for some people, being alive even if

1	has been criticized because there is no single way to determine the balance between benefits and burdens; it depends on a patient’s personal values. For instance, for some people, being alive even if mentally incapacitated is a benefit, whereas for others, it may be the worst possible existence. As a matter of practice, physicians rely on family members to make decisions that they feel are best and object only if those decisions seem to demand treatments that the physicians consider not beneficial.

1	practIces Withholding and withdrawing acutely life-sustaining medical interventions from terminally ill patients are now standard practice. More than 90% of American patients die without cardiopulmonary resuscitation (CPR), and just as many forgo other potentially life-sustaining interventions. For instance, in ICUs in the period 1987–1988, CPR was performed 49% of the time, but it was performed only 10% of the time in 1992–1993. On average, 3.8 interventions, such as vasopressors and transfusions, were stopped for each dying ICU patient. However, up to 19% of decedents in hospitals received interventions such as extubation, ventilation, and surgery in the 48 h preceding death. However, practices vary widely among hospitals and ICUs, suggesting an important element of physician preferences rather than objective data.

1	Mechanical ventilation may be the most challenging intervention to withdraw. The two approaches are terminal extubation, which is the removal of the endotracheal tube, and terminal weaning, which is the gradual reduction of the or ventilator rate. One-third of ICU physicians prefer to use the terminal weaning technique, and 13% extubate; the majority of physicians use both techniques. The American Thoracic Society’s 2008 clinical policy guidelines note that there is no single correct process of ventilator withdrawal and that physicians use and should be proficient in both methods but that the chosen approach should carefully balance benefits and burdens as well as patient and caregiver preferences. Physicians’ assessment of patients’ likelihood of survival, their prediction of possible cognitive damage, and patients’ preferences about the use of life support are primary factors in determining the likelihood of withdrawal of mechanical ventilation. Some recommend terminal weaning

1	cognitive damage, and patients’ preferences about the use of life support are primary factors in determining the likelihood of withdrawal of mechanical ventilation. Some recommend terminal weaning because patients do not develop upper airway obstruction and the distress caused by secretions or stridor; however, terminal weaning can prolong the dying process and not allow a patient’s family to be with him or her unencumbered by an endotracheal tube. To ensure comfort for conscious or semiconscious patients before withdrawal of the ventilator, neuromuscular blocking agents should be terminated and sedatives and analgesics administered. Removing the neuromuscular blocking agents permits patients to show discomfort, facilitating the titration of sedatives and analgesics; it also permits interactions between patients and their families. A common practice is to inject a bolus of midazolam (2–4 mg) or lorazepam (2–4 mg) before withdrawal, followed by 5–10 mg of morphine and continuous

1	interactions between patients and their families. A common practice is to inject a bolus of midazolam (2–4 mg) or lorazepam (2–4 mg) before withdrawal, followed by 5–10 mg of morphine and continuous infusion of morphine (50% of the bolus dose per hour) during weaning. In patients who have significant upper airway secretions, IV scopolamine at a rate of 100 μg/h can be administered. Additional boluses of morphine or increases in the infusion rate should be administered for respiratory distress or signs of pain. Higher doses will be needed for patients already receiving sedatives and opioids. Families need to be reassured about treatments for common symptoms after withdrawal of ventilatory support, such as dyspnea and agitation, and warned about the uncertainty of length of survival after withdrawal of ventilatory support: up to 10% of patients unexpectedly survive for 1 day or more after mechanical ventilation is stopped.

1	Beginning in the late 1980s, some commentators argued that physicians could terminate futile treatments demanded by the families of terminally ill patients. Although no objective definition or standard of futility exists, several categories have been proposed. Physiologic futility means that an intervention will have no physiologic effect. Some have defined qualitative futility as applying to procedures that “fail to end a patient’s total dependence on intensive medical care.” Quantitative futility occurs “when physicians conclude (through personal experience, experiences shared with colleagues, or consideration of reported empiric data) that in the last 100 cases, a medical treatment has been useless.” The term conceals subjective value judgments about when a treatment is “not beneficial.” Deciding whether a treatment that obtains an additional 6 weeks of life or a 1% survival advantage confers benefit depends on patients’ preferences and goals. Furthermore, physicians’ predictions

1	Deciding whether a treatment that obtains an additional 6 weeks of life or a 1% survival advantage confers benefit depends on patients’ preferences and goals. Furthermore, physicians’ predictions of when treatments were futile deviated markedly from the quantitative definition. When residents thought CPR was quantitatively futile, more than one in five patients had a >10% chance of survival to hospital discharge. Most studies that purport to guide determinations of futility are based on insufficient data to provide statistical confidence for clinical decision making. Quantitative futility rarely applies in ICU settings. Many commentators reject using futility as a criterion for withdrawing care, preferring instead to consider futility situations as ones that represent conflict that calls for careful negotiation between families and health care providers.

1	In the wake of a lack of consensus over quantitative measures of futility, many hospitals adopted process-based approaches to resolve disputes over futility and enhance communication with patients and surrogates, including focusing on interests and alternatives rather than opposing positions and generating a wide range of options. Some hospitals have enacted “unilateral do not resuscitate (DNR)” policies to allow clinicians to provide a DNR order in cases in which consensus cannot be reached with families and medical opinion is that resuscitation would be futile if attempted. This type of a policy is not a replacement for careful and patient communication and negotiation but recognizes that agreement cannot always be reached. Over the last 15 years, many states, such as Texas, Virginia, Maryland, and California, have enacted so-called medical futility laws that provide physicians a “safe harbor” from liability if they refuse a patient or family’s request for life-sustaining

1	Maryland, and California, have enacted so-called medical futility laws that provide physicians a “safe harbor” from liability if they refuse a patient or family’s request for life-sustaining interventions. For instance, in Texas when a disagreement about terminating interventions between the medical team and the family has not been resolved by an ethics consultation, the hospital is supposed to try to facilitate transfer of the patient to an institution willing to provide treatment. If this fails after 10 days, the hospital and physician may unilaterally withdraw treatments determined to be futile. The family may appeal to a state court. Early data suggest that the law increases futility consultations for the ethics committee and that although most families concur with withdrawal, about 10–15% of families refuse to withdraw treatment. Approximately 12 cases have gone to court in Texas in the 7 years since the adoption of the law. As of 2007, there had been 974 ethics committee

1	about 10–15% of families refuse to withdraw treatment. Approximately 12 cases have gone to court in Texas in the 7 years since the adoption of the law. As of 2007, there had been 974 ethics committee consultations on medical futility cases and 65 in which committees ruled against families and gave notice that treatment would be terminated. Treatment was withdrawn for 27 of those patients, and the remainder were transferred to other facilities or died while awaiting transfer.

1	Euthanasia and physician-assisted suicide are defined in Table 10-8. Terminating life-sustaining care and providing opioid medications to manage symptoms have long been considered ethical by the medical profession and legal by courts and should not be confused with euthanasia or physician-assisted suicide.

1	legal aspects Euthanasia is legal in the Netherlands, Belgium, and Luxembourg. It was legalized in the Northern Territory of Australia in 1995, but that legislation was repealed in 1997. Euthanasia is not legal in any state in the United States. With certain conditions, in Switzerland, a layperson can legally assist suicide. In the United States, physician-assisted suicide is legal in four states: Oregon, Vermont, and Washington State by legislation and Montana by court ruling. In jurisdictions where physician-assisted suicide is legal, physicians wishing to prescribe the necessary medication must fulfill multiple criteria and complete processes that include a waiting period. In other countries and all other states in the United States, physician-assisted suicide and euthanasia are illegal explicitly or by common law.

1	practIces Fewer than 10–20% of terminally ill patients actually consider euthanasia and/or physician-assisted suicide for themselves. In the Netherlands and Oregon, >70% of patients using these interventions are dying of cancer; in Oregon, in 2013, just 1.2% of physician-assisted suicide cases involved patients with HIV/AIDS and 7.2% involved patients with amyotrophic lateral sclerosis. In the Netherlands, the share of deaths attributable to euthanasia or physician-assisted suicide declined from around 2.8% of all deaths in 2001 to around 1.8% in 2005. In 2013, the last year with complete data, around 71 patients in Oregon (just 0.2% of all deaths) died by physician-assisted suicide, although this may be an underestimate. In Washington State, between March 2009 (when the law allowing physician-assisted suicide went into force) and December 2009, 36 individuals died from prescribed lethal doses.

1	Pain is not a primary motivator for patients’ requests for or interest in euthanasia and/or physician-assisted suicide. Fewer than 25% of all patients in Oregon cite inadequate pain control as the reason for desiring physician-assisted suicide. Depression, hopelessness, and, more profoundly, concerns about loss of dignity or autonomy or being a burden on family members appear to be primary factors motivating a desire for euthanasia or physician-assisted suicide. Over 75% cite loss of autonomy or dignity and inability to engage in enjoyable activities as the reason for wanting physician-assisted suicide. About 40% cite being a burden on family. A study from the Netherlands showed that depressed terminally ill cancer patients were four times more likely

1	Voluntary active Intentionally administering medica-Netherlands, euthanasia tions or other interventions to cause Belgium the patient’s death with the patient’s informed consent euthanasia tions or other interventions to cause the patient’s death when the patient was competent to consent but did not—e.g., the patient may not have been asked Passive euthanasia Withholding or withdrawing life-Everywhere sustaining medical treatments from a patient to let him or her die (terminating life-sustaining treatments)

1	Passive euthanasia Withholding or withdrawing life-Everywhere sustaining medical treatments from a patient to let him or her die (terminating life-sustaining treatments) Physician-assisted A physician provides medications or Oregon, suicide other interventions to a patient with Netherlands, the understanding that the patient Belgium, can use them to commit suicide Switzerland to request euthanasia and confirmed that uncontrolled pain was not associated with greater interest in euthanasia. Interestingly, despite the importance of emotional distress in motivating requests for euthanasia and physician-assisted suicide, few patients receive psychiatric care. For instance, in Oregon, only 5.9% of patients have been referred for psychiatric evaluation.

1	Euthanasia and physician-assisted suicide are no guarantee of a painless, quick death. Data from the Netherlands indicate that in as many as 20% of cases technical and other problems arose, including patients waking from coma, not becoming comatose, regurgitating medications, and experiencing a prolonged time to death. Data from Oregon indicate that between 1997 and 2013, 22 patients (~5%) regurgitated after taking prescribed medication, 1 patient awaked, and none experienced seizures. Problems were significantly more common in physician-assisted suicide, sometimes requiring the physician to intervene and provide euthanasia.

1	Whether practicing in a setting where euthanasia is legal or not, over a career, 12–54% of physicians receive a request for euthanasia or physician-assisted suicide from a patient. Competency in dealing with such a request is crucial. Although challenging, the request can also provide a chance to address intense suffering. After receiving a request for euthanasia and/or physician-assisted suicide, health care providers should carefully clarify the request with empathic, open-ended questions to help elucidate the underlying cause for the request, such as “What makes you want to consider this option?” Endorsing either moral opposition or moral support for the act tends to be counterproductive, giving an impression of being judgmental or of endorsing the idea that the patient’s life is worthless. Health care providers must reassure the patient of continued care and commitment. The patient should be educated about alternative, less controversial options, such as symptom management and

1	Health care providers must reassure the patient of continued care and commitment. The patient should be educated about alternative, less controversial options, such as symptom management and withdrawing any unwanted treatments and the reality of euthanasia and/or physician-assisted suicide, because the patient may have misconceptions about their effectiveness as well as the legal implications of the choice. Depression, hopelessness, and other symptoms of psychological distress as well as physical suffering and economic burdens are likely factors motivating the request, and such factors should be assessed and treated aggressively. After these interventions and clarification of options, most patients proceed with another approach, declining life-sustaining interventions, possibly including refusal of nutrition and hydration.

1	Most laypersons have limited experiences with the actual dying process and death. They frequently do not know what to expect of the final hours and afterward. The family and other caregivers must be prepared, especially if the plan is for the patient to die at home.

1	Patients in the last days of life typically experience extreme weakness and fatigue and become bedbound; this can lead to pressure sores. The issue of turning patients who are near the end of life, however, must be balanced against the potential discomfort that movement may cause. Patients stop eating and drinking with drying of mucosal membranes and dysphagia. Careful attention to oral swabbing, lubricants for lips, and use of artificial tears can provide a form of care to substitute for attempts at feeding the patient. With loss of the gag reflex and dysphagia, patients may also experience accumulation of oral secretions, producing noises during respiration sometimes called “the death rattle.” Scopolamine can reduce the secretions. Patients also experience changes in respiration with periods of apnea or Cheyne-Stokes breathing. Decreased intravascular volume and cardiac output cause tachycardia, hypotension, peripheral coolness, and livedo reticularis (skin mottling). Patients can

1	of apnea or Cheyne-Stokes breathing. Decreased intravascular volume and cardiac output cause tachycardia, hypotension, peripheral coolness, and livedo reticularis (skin mottling). Patients can have urinary and, less frequently, fecal incontinence. Changes in consciousness and neurologic function generally lead to two different paths to death (Fig. 10-2).

1	Each of these terminal changes can cause patients and families distress, requiring reassurance and targeted interventions (Table 10-9). Informing families that these changes might occur and providing them with an information sheet can help preempt problems and minimize distress. Understanding that patients stop eating because they are dying, not dying because they have stopped eating, can reduce family FIgURE 10-2 Common and uncommon clinical courses in the last days of terminally ill patients. (Adapted from FD Ferris et al: Module 4: Palliative care, in Comprehensive Guide for the Care of Persons with HIV Disease. Toronto: Mt. Sinai Hospital and Casey Hospice, 1995, http://www .cpsonline.info/content/resources/hivmodule/module4complete.pdf.) and caregiver anxiety. Similarly, informing the family and caregivers that the “death rattle” may occur and that it is not indicative of suffocation, choking, or pain can reduce their worry from the breathing sounds.

1	Families and caregivers may also feel guilty about stopping treatments, fearing that they are “killing” the patient. This may lead to demands for interventions, such as feeding tubes, that may be ineffective. In such cases, the physician should remind the family and caregivers about the inevitability of events and the palliative goals. Interventions may prolong the dying process and cause discomfort. Physicians also should emphasize that withholding treatments is both legal and ethical and that the family members are not the cause of the patient’s death. This reassurance may have to be provided multiple times.

1	Hearing and touch are said to be the last senses to stop functioning. Whether this is the case or not, families and caregivers can be encouraged to communicate with the dying patient. Encouraging them to talk directly to the patient, even if he or she is unconscious, and hold the patient’s hand or demonstrate affection in other ways can be an effective way to channel their urge “to do something” for the patient. When the plan is for the patient to die at home, the physician must inform the family and caregivers how to determine that the patient has died. The cardinal signs are cessation of cardiac function and respiration; the pupils become fixed; the body becomes cool; muscles relax; and incontinence may occur. Remind the family and caregivers that the eyes may remain open even after the patient has died because Changes in the Profound fatigue Bedbound with development of pressure ulcers that are prone to infection, malodor, and pain, and joint pain

1	Changes in the Profound fatigue Bedbound with development of pressure ulcers that are prone to infection, malodor, and pain, and joint pain Dysphagia Inability to swallow oral medications needed for palliative care Apnea, Cheyne-Stokes respirations, dyspnea Potential transmission of infectious agents to caregivers Dry mucosal Cracked lips, mouth sores, membranes and candidiasis can also cause pain. Patient is lazy and giving up. Patient is giving up; patient will suffer from hunger and will starve to death. Patient will suffer from thirst and die of dehydration. Patient is choking and suffocating. Patient is suffocating. Patient is dirty, malodorous, and physically repellent. Patient is in horrible pain and going to have a horrible death. Patient may be malodorous or physically repellent. Reassure family and caregivers that terminal fatigue will not respond to interventions and should not be resisted. Use an air mattress if necessary.

1	Reassure family and caregivers that terminal fatigue will not respond to interventions and should not be resisted. Use an air mattress if necessary. Reassure family and caregivers that the patient is not eating because he or she is dying; not eating at the end of life does not cause suffering or death. Forced feeding, whether oral, parenteral, or enteral, does not reduce symptoms or prolong life. Reassure family and caregivers that dehydration at the end of life does not cause suffering because patients lose consciousness before any symptom distress. Intravenous hydration can worsen symptoms of dyspnea by pulmonary edema and peripheral edema as well as prolong dying process.

1	Intravenous hydration can worsen symptoms of dyspnea by pulmonary edema and peripheral edema as well as prolong dying process. Do not force oral intake. Discontinue unnecessary medications that may have been continued, including antibiotics, diuretics, antidepressants, and laxatives. If swallowing pills is difficult, convert essential medications (analgesics, antiemetics, anxiolytics, and psychotropics) to oral solutions, buccal, sublingual, or rectal administration. Reassure the family and caregivers that this is caused by secretions in the oropharynx and the patient is not choking. Reduce secretions with scopolamine (0.2–0.4 mg SC q4h or 1–3 patches q3d). Reposition patient to permit drainage of secretions. Do not suction. Suction can cause patient and family discomfort and is usually ineffective. Reassure family and caregivers that unconscious patients do not experience suffocation or air hunger. Apneic episodes are frequently a premorbid change.

1	Reassure family and caregivers that unconscious patients do not experience suffocation or air hunger. Apneic episodes are frequently a premorbid change. Opioids or anxiolytics may be used for dyspnea. Oxygen is unlikely to relieve dyspneic symptoms and may prolong the dying process. Remind family and caregivers to use universal precautions. Frequent changes of bedclothes and bedding. Use diapers, urinary catheter, or rectal tube if diarrhea or high urine output. not necessarily connote physical pain. Depending on the prognosis and goals of treatment, consider evaluating for causes of delirium and modify medications. Manage symptoms with haloperidol, chlorpromazine, diazepam, or midazolam. Use baking soda mouthwash or saliva preparation q15–30min. Use topical nystatin for candidiasis. Coat lips and nasal mucosa with petroleum jelly q60–90min. Use ophthalmic lubricants q4h or artificial tears q30min.

1	the retroorbital fat pad may be depleted, permitting the orbit to fall posteriorly, which makes it difficult for the eyelids to cover the eyeball. The physician should establish a plan for who the family or caregivers will contact when the patient is dying or has died. Without a plan, they may panic and call 911, unleashing a cascade of unwanted events, from arrival of emergency personnel and resuscitation to hospital admission. The family and caregivers should be instructed to contact the hospice (if one is involved), the covering physician, or the on-call member of the palliative care team. They should also be told that the medical examiner need not be called unless the state requires it for all deaths. Unless foul play is suspected, the health care team need not contact the medical examiner either.

1	Just after the patient dies, even the best-prepared family may experience shock and loss and be emotionally distraught. They need time to assimilate the event and be comforted. Health care providers are likely to find it meaningful to write a bereavement card or letter to the family. The purpose is to communicate about the patient, perhaps emphasizing the patient’s virtues and the honor it was to care for the patient, and to express concern for the family’s hardship. Some physicians attend the funerals of their patients. Although this is beyond any medical obligation, the presence of the physician can be a source of support to the grieving family and provides an opportunity for closure for the physician. Death of a spouse is a strong predictor of poor health, and even mortality, for the surviving spouse. It may be important to alert the spouse’s physician about the death so that he or she is aware of symptoms that might require professional attention.

1	PALLIATIVE CARE SERVICES: HoW ANd WHERE

1	Determining the best approach to providing palliative care to patients will depend on patient preferences, the availability of caregivers and specialized services in close proximity, institutional resources, and reimbursement. Hospice is a leading, but not the only, model of palliative care services. In the United States, a plurality—41.5%—of hospice care is provided in residential homes. In 2012, just over 17% of hospice care was provided in nursing homes. In the United States, Medicare pays for hospice services under Part A, the hospital insurance part of reimbursement. Two physicians must certify that the patient has a prognosis of ≤6 months if the disease runs its usual course. Prognoses are probabilistic by their nature; patients are not required to die within 6 months but rather to have a condition from which half the individuals with it would not be alive within 6 months. Patients sign a hospice enrollment form that states their intent to forgo curative services related to

1	to have a condition from which half the individuals with it would not be alive within 6 months. Patients sign a hospice enrollment form that states their intent to forgo curative services related to their terminal illness, but they can still receive medical services for other comorbid conditions. Patients also can withdraw enrollment and reenroll later; the hospice Medicare benefit can be revoked later to secure traditional Medicare benefits. Payments to the hospice are per diem (or capitated), not fee-for-service. Payments are intended to cover physician services for the medical direction of the care team; regular home care visits by registered nurses and licensed practical nurses; home health aid and homemaker services; chaplain services; social work services; bereavement counseling; and medical equipment, supplies, and medications. No specific therapy is excluded, and the goal is for each therapy to be considered for its symptomatic (as opposed to disease-modifying) effect.

1	and medical equipment, supplies, and medications. No specific therapy is excluded, and the goal is for each therapy to be considered for its symptomatic (as opposed to disease-modifying) effect. Additional clinical care, including services of the primary physician, is covered by Medicare Part B even while the hospice Medicare benefit is in place. The health reform legislation signed into law in March 2010—the Affordable Care Act—directs the Secretary of Health and Human Services to gather data on Medicare hospice reimbursement with the goal of reforming payment rates to account for resource use over an entire episode of care. The legislation also requires additional evaluations and reviews of eligibility for hospice care by hospice physicians or nurses. Finally, the legislation establishes a demonstration project for concurrent hospice care in Medicare, which would test and evaluate allowing patients to remain eligible for regular Medicare during hospice care.

1	By 2012, the mean length of enrollment in a hospice was around 71.8 days, with the median being 18.7 days. Such short stays create barriers to establishing high-quality palliative services in patients’ homes and also place financial strains on hospice providers because the initial assessments are resource intensive. Physicians should initiate early referrals to the hospice to allow more time for patients to receive palliative care.

1	Hospice care has been the main method in the United States for securing palliative services for terminally ill patients. However, efforts rarely the primary focus of carefully developed or widely used outcome measures. Nevertheless, outcomes are as important in end-of-life care as in any other field of medical care. Specific end-of-life care instruments are being developed both for assessment, such as The Brief Hospice Inventory and NEST (needs near the end of life screening tool), and for outcome measures, such as the Palliative Care Outcomes Scale, as well as for prognosis, such as the Palliative Prognostic Index. The field of end-of-life care is entering an era of evidence-based practice and continuous improvement through clinical trials. Clinical problems of aging Luigi Ferrucci, Stephanie Studenski

1	While an in-depth understanding of internal medicine serves as a foundation, proper care of older adults should be complemented by insight into the multidimensional effects of aging on disease manifestations, consequences, and response to treatment. In younger adults, individual diseases tend to have a more distinct pathophysiology with well-defined risk factors; the same diseases in older persons may have a less distinct pathophysiology and are often the result of failed homeostatic mechanisms. Causes and clinical manifestations are less specific and can vary widely between individuals. Therefore, the care of older patients demands an understanding of the effects of aging on human physiology and a broader perspective that incorporates geriatric syndromes, disability, social contexts, and goals of care. For example, care planning for the older patient should account for the substantial portion of the wide variability in life expectancy across individuals of the same age that can be

1	goals of care. For example, care planning for the older patient should account for the substantial portion of the wide variability in life expectancy across individuals of the same age that can be predicted by simple and inexpensive measures such as walking speed. Estimation of the expected remaining years of life can guide recommendations about appropriate preventive and other long-term interventions and can shape discussions about treatment alternatives.

1	(See also Chap. 93e) Population aging emerged as a worldwide phenomenon for the first time in history within the past century. Since aging influences many facets of life, governments and societies—as well as families and communities—now face new social and economic challenges that affect health care. Fig. 11-1 highlights recent and predicted changes in U.S. population structure. are being made to ensure continuity of palliative care across settings and 25000 through time. Palliative care services are becoming available as consultative services and more rarely as palliative care units in hospitals, in day care and other outpatient settings, and in nursing homes. Palliative care consultations for nonhospice patients can be billed as for other consultations under Medicare Part B, the physician reimbursement part.

1	Many believe palliative care should be offered to patients regardless of their prognosis. A patient, his or her family, and physicians should not have to make a “curative versus palliative care” decision because it is rarely possible to make such a decisive switch to embracing mortality. Number of persons in thousands Care near the end of life cannot be measured by most of the available validated outcome measures because palliative care does not consider death a bad outcome. Similarly, the family and patients receiving end-of-life care may not desire the elements elicited in current qualityof-life measurements. Symptom control, enhanced family relationships, and quality of bereavement are difficult to measure and are

1	Many chronic diseases increase in prevalence with age. It is not unusual for older persons to have multiple chronic diseases (Fig. 11-4), although some seem more susceptible than others to co-occurring problems. Functional problems that pose difficulties or require help in performing basic activities of daily living (ADLs) (Table 11-1) increase with age and are more common among women than among

1	Chapter 11 Clinical Problems of Aging men. In recent decades, the age-specific prevalence of disability has declined, especially in the oldest old. Estimated rates are shown in Fig. 11-5 as the percentage of persons who reported severe difficulty or needed help in bathing, and data on other basic ADLs show similar trends. Although the age-specific prevalence of disability is decreasing, the magnitude of this decline is small compared to the overwhelming effect of population aging. Thus, the number of people with disability in the United States and other countries is rapidly expanding. Rates of cognitive impairments, such as memory problems, also increase with aging (Fig. 11-6). Chronic disease and disability lead to increased use of health care resources. Health care expenditures increase with age, increase more with disability, and are highest in the last year of life. However, new medical technologies and expensive medications are greater influences on health care costs than

1	with age, increase more with disability, and are highest in the last year of life. However, new medical technologies and expensive medications are greater influences on health care costs than population aging itself. General practitioners and internists with little specific training in geriatric medicine provide the bulk of care for older persons.

1	Systemic consequences of aging are widespread but can be clustered into four main domains or processes (Fig. 11-7): (1) body composition; (2) balance between energy availability and energy demand; (3) signaling networks that maintain homeostasis; and (4) neurodegeneration. Each domain can be assessed with routine clinical tests, although more detailed research techniques are also available (Table 11-2).

1	Body Composition Profound changes in body composition may be the most evident and inescapable effect of aging (Fig. 11-8). Over the life span, body weight tends to increase through childhood, puberty, and adulthood until late middle age. Weight tends to decline in men between ages 65 and 70 years and in women somewhat later. Lean body mass, composed predominantly of muscle and visceral organs, decreases steadily after the third decade. In muscle, this atrophy is greater in fast-twitch than in slow-twitch fibers. The origin of this change is unknown, but several lines of evidence suggest that progressive loss of motor neurons probably plays an important role. Fat mass tends to increase in middle age and then declines in late life, reflecting the trajectory of weight change. Waist circumference continues to increase across the life span, a pattern suggesting that visceral

1	FIgURE 11-2 Population aging in different geographic regions. (From United Nations World Population Prospects: The 2008 Revision, http://www .un.org/esa/population/publications/wpp2008/wpp2008_highlights.pdf.) The overall number of children has remained relatively stable, but explosive growth has occurred among older populations. The percentage of growth is particularly dramatic among the oldest of the old. For example, the number of persons aged 80–89 years more than tripled between 1960 and 2010 and will increase over tenfold between 1960 and 2050. Women already outlive men by many years, and the sex discrepancy in longevity is projected to increase further in the future.

1	Population aging occurs at different rates in varying geographic regions of the world. Over the past century, Europe, Australia, and North America have had the populations with the greatest proportions of older persons, but the populations of Asia and South America are aging rapidly, and the population structure on these continents will resemble that of “older” countries by around 2050 (Fig. 11-2). Among older persons, the oldest old (those >80 years of age) are the fastest-growing segment of the population (Fig. 11-3), and the pace of population aging is projected to accelerate in most countries over the next 50 years. There is no evidence that the rate of population aging is decreasing. fat, which is responsible for most of the pathologic consequences of obesity, continues to accumulate. In some individuals, fat also accumulates inside muscle, affecting muscle quality and function. With age, fibroconnective tissue tends to increase in many organ systems. In

1	Percentage of population 80+ years old muscle, fibroconnective tissue buildup also affects muscle quality and function. In combination, the loss of muscle mass and quality result in reduced muscle strength, which ultimately affects functional capacity and mobility. Muscle strength declines with aging; this decrease not only affects functional status but also is a strong independent predictor of mortality (Fig. 11-9). Progressive demineralization and architectural modification occur in bone, resulting in a decline of bone strength. Loss of bone strength increases the risk of fracture. Sex differences in the effects of aging on bone mass are due to differences in peak bone mass and the effects of gonadal hormones on bone. Overall, compared with men, women tend to lose bone mass at a younger age and more quickly reach the threshold of low bone strength that increases fracture risk.

1	All of these changes in body composition can be attributed to disruptions in the links between synthesis, degradation, and repair that Years normally serve to remodel tissues. Such changes in body composition FIgURE 11-3 Percentage of the population age >80 years from are influenced not only by aging and illness but also by lifestyle factors 1950 to 2050 in representative nations. The pace of population such as physical activity and diet. Body composition can be approxiaging will accelerate. (From United Nations World Population Prospects: mated in clinical practice on the basis of weight, height, body mass The 2008 Revision, http://www.un.org/esa/population/publications/ index (BMI; weight in kilograms divided by height in meters squared), wpp2008/wpp2008_highlights.pdf.) and waist circumference or, more precisely, with dual-energy x-ray

1	FIgURE 11-4 Prevalence of comorbidity by age group in persons ≥65 years old living in the United States and enrolled in Medicare parts A and B in 1999. (From JL Wolff et al: Arch Intern Med 162:2269, 2002.) Basic Activities of Daily Living: Self-Care Tasks Transferring from bed to chair and back Using the toilet Moving around (as opposed to being bedridden) Instrumental Activities of Daily Living: Not Necessary for Fundamental Functioning, but Permit an Individual to Live Independently in a Percentage with memory impairment Using the telephone aWith the recognition that older persons may not be that technologically savvy since they were not as extensively exposed to technology during their lifetime.)

1	Using the telephone aWith the recognition that older persons may not be that technologically savvy since they were not as extensively exposed to technology during their lifetime.) FIgURE 11-6 Rates of memory impairment in different age groups. The definition of “moderate or severe memory impairment” is 4 or fewer words recalled out of 20. (Source: Health and Retirement Survey. Accessed November 15, 2013, at aoa.gov/agingstatsdotnet/Main_ Site/Data/2000_Documents/healthstatus.aspx.) FIgURE 11-5 Self-reported prevalence of disability (severe difficulty) in bathing/showering between 1992 and 2007, according to age and sex.) (From Medicare Current Beneficiary Survey 1992–2007.)

1	FIgURE 11-5 Self-reported prevalence of disability (severe difficulty) in bathing/showering between 1992 and 2007, according to age and sex.) (From Medicare Current Beneficiary Survey 1992–2007.) Aging Discrepancy in energy production/utilization Changes in body composition Anorexia/ malnutrition Gait disorders/ falls Disability Disease susceptibility Comorbidity Urinary incontinence Decubitus ulcers Sleep disorders Delirium Cognitive impairment Homeostatic dysregulation Neurodegeneration Domains of the aging phenotype Frailty Disease susceptibility, reduced functional reserve, reduced healing capacity, unstable health, failure to thrive Geriatric syndromes FIgURE 11-7 A unifying model of aging, frailty, and the geriatric syndromes.

1	Chapter 11 Clinical Problems of Aging absorptiometry, CT, or MRI. In healthy men and women in their twenties, lean body mass is, on average, 85% of body weight, with roughly 50% of lean mass represented by skeletal muscle. With aging, both the percentage of lean mass and the percentage represented by muscle decline rapidly, and these changes have important health and functional consequences.

1	Balance Between Energy Availability and Energy demand Release of phosphate from ATP provides every living cell with the energy required for life. However, the storage of ATP is only enough for 6 sec; therefore, ATP is constantly resynthesized. Although ATP can be resynthesized by anaerobic glycolysis, most of the energy used in the body is generated through aerobic metabolism. Therefore, energy consumption is usually estimated indirectly by oxygen consumption (indirect calorimetry). There is currently no method to measure true “fitness,” which is the maximal energy that can be produced by an organism over extended periods. Thus, fitness is estimated indirectly from peak oxygen consumption (MVo ), often during a maximal treadmill test. gressively with aging (Fig. 11-10), and the rate of decline is accelerated in persons who are sedentary and in those affected by chronic diseases.

1	gressively with aging (Fig. 11-10), and the rate of decline is accelerated in persons who are sedentary and in those affected by chronic diseases. A large portion of energy is consumed as the “resting metabolic rate” (RMR)—i.e., the amount of energy expended at rest in a neutral temperature environment and in a postabsorptive state. In healthy men and women, RMR declines with aging, and such decline is only partially explained by the parallel decline in the highly metabolically active tissues that make up lean body mass (Fig. 11-11). However, persons with unstable homeostasis due to illness require additional Approach to Assessment Body Composition Energetics Homeostatic Regulation Neurodegeneration Anthropometrics (weight, height, BMI, waist circumference, arm and leg circumference, skin folds) Imaging CT and MRI, DEXA Other Hydrostatic weighing Self-reported questionnaires investigating physical activity, sense of fatigue/exhaustion, exercise tolerance

1	Imaging CT and MRI, DEXA Other Hydrostatic weighing Self-reported questionnaires investigating physical activity, sense of fatigue/exhaustion, exercise tolerance Performance-based tests of physical function Treadmill testing of oxygen consumption during walking Objective measures of physical activity (accelerometers, double-labeled water) Nutritional biomarkers (e.g., vitamins, antioxidants) Baseline levels of biomarkers and hormone levels Inflammatory markers (e.g., ESR, CRP, IL-6, TNF-α) Response to provocative tests, such as oral glucose tolerance test, dexamethasone test, and others Objective assessment of gait, balance, reaction time, coordination Standard neurologic exam, including assessment of global cognitiona MRI, fMRI, PET, and other dynamic imaging techniques aMini Mental State; Montreal Cognitive Assessment.

1	MRI, fMRI, PET, and other dynamic imaging techniques aMini Mental State; Montreal Cognitive Assessment. Abbreviations: BMI, body mass index; CRP, C-reactive protein; DEXA, dual-energy x-ray absorptiometry; ESR, erythrocyte sedimentation rate; fMRI, functional MRI; IL-6, interleukin 6; PET, positron emission tomography; TNFα, tumor necrosis factor α. FIgURE 11-8 Longitudinal changes of weight, body composition, and waist circumference over the life span, estimated in 1167 participants in the Baltimore Longitudinal Study of Aging. Lean body mass (LBM) and fat mass were estimated with dual-energy x-ray absorptiometry. (Source: The Baltimore Longitudinal study of Aging 2010; unpublished data.) Survivors, n = 3680 Non-survivors, n = 3680

1	(Source: The Baltimore Longitudinal study of Aging 2010; unpublished data.) Survivors, n = 3680 Non-survivors, n = 3680 FIgURE 11-9 Cross-sectional differences and longitudinal changes in muscle strength over a 27-year follow-up. Note that persons who died during the follow-up had lower baseline muscle strength. (From T Rantanen et al: J Appl Physiol 85:2047, 1998.) 1.8 1.6 1.4 1.2 1.0 0.8 0.6 FIgURE 11-10 Longitudinal changes in aerobic capacity in participants in the Baltimore Longitudinal Study of Aging. (From JL Fleg: FIgURE 11-11 Changes in resting metabolic rate with aging. Circulation 112:674, 2005.) (Unpublished data from the Baltimore Longitudinal Study of Aging.) taBLe 11-3 horMones that deCrease, reMaIn staBLe, and InCrease WIth aGInG Chapter 11 Clinical Problems of Aging

1	FIgURE 11-12 Longitudinal trajectory of bioavailable testosterone plasma concentration in the Baltimore Longitudinal Study of Aging (BLSA). The plot is based on 584 men who were 50 years or older with a total of 1455 data points. The average follow up for each subject was 3.2 years. (Figure created using unpublished data from the BLSA.) energy for compensatory mechanisms. Indeed, observational studies have demonstrated (1) that older persons with poor health status and substantial morbidity have a higher RMR than healthier individuals of the same age and sex and (2) that a high RMR is an independent risk factor for mortality and may contribute to the weight loss that often accompanies severe illness. Finally, for reasons that are not yet completely clear but certainly involve changes in the biomechanical characteristics of movement, older age, pathology, and physical impairment increase the energy cost of motor activities such as walking. Overall, older individuals with multiple

1	in the biomechanical characteristics of movement, older age, pathology, and physical impairment increase the energy cost of motor activities such as walking. Overall, older individuals with multiple chronic conditions have low available energy levels and require more energy both at rest and during physical activity. Thus, sick older people may consume all their available energy performing the most basic ADLs, and consequent fatigue and restriction may lead to a sedentary existence. Energy status can be assessed clinically by simply asking patients about their perceived level of fatigue during daily activities such as walking or dressing. Energy capacity can be assessed more precisely by exercise tolerance during a walking test or a treadmill test coupled with spirometry.

1	The main signaling pathways that control homeostasis involve hormones, inflammatory mediators, and antioxidants; all are profoundly affected by aging. Sex hormone levels, such as testosterone in men (Fig. 11-12) and estrogen in women, decrease with age, while other hormone systems may change more subtly (Table 11-3). Most aging individuals, even those who remain healthy and fully functional, tend to dysregulation. For example, taken one at a time, levels of testosterone, dehydroepiandrosterone (DHEA), and insulin-like growth factor 1 (IGF-1) do not predict mortality, but in combination they are highly predictive of longevity. This combination effect is especially strong in the setting of congestive heart failure. Similarly, several micronutrients, such as vitamins (especially vitamin D), minerals (selenium and magnesium), and antioxidants (vitamins D and E), also regulate aspects of metabolism. Low levels of these micronutrients have been associated with accelerated aging and a high

1	minerals (selenium and magnesium), and antioxidants (vitamins D and E), also regulate aspects of metabolism. Low levels of these micronutrients have been associated with accelerated aging and a high risk of adverse outcomes. However, except for vitamin D, no clear evidence suggests that supplementation has positive effects on health. Unfortunately, no standard criteria exist that allow the detection and quantification of homeostatic dysregulation as a general phenomenon.

1	Neurodegeneration It was long generally believed that neurons stop reproducing shortly after birth and that their number declines throughout life. However, results from animal models and even some studies in humans suggest that neurogenesis in the hippocampus continues at low levels throughout life. Brain atrophy occurs with aging after the age of 60 years. Atrophy proceeds at varying rates in different parts of the brain (Fig. 11-14) and is often accompanied by an inflammatory response and microglial activation. Age-associated brain atrophy may contribute to age-related declines in cognitive and motor function. Atrophy may also be a factor in some brain diseases that can occur with aging, such as mild cognitive impairment, in which persons have mild but detectable impairments on tests of cognition but no severe disability in daily activities. In mild cognitive impairment, atrophy has been found mostly in the prefrontal cortex and hippocampus, develop a mild proinflammatory state

1	of cognition but no severe disability in daily activities. In mild cognitive impairment, atrophy has been found mostly in the prefrontal cortex and hippocampus, develop a mild proinflammatory state characterized by high levels of proinflammatory markers, including interleukin 6 (IL-6) and C-reactive protein (CRP) (Fig. 11-13). Aging is also thought to be associated with increased oxidative stress damage, either because the production of reactive oxygen species increases or because antioxidant buffers are less effective. Since hormones, inflammatory markers, and antioxidants are integrated into complex signaling networks, levels

1	FIgURE 11-13 Change in interleukin 6 (IL-6) and C-reactive protein (CRP) with aging. Values are expressed as Z-scores to make them comparable. (From L Ferrucci et al: Blood 105:2294, 2005.) 1.5 1 0.5 0 –0.5 –1 20-39 40-49 50-64 65-74 Age groups Age groups 75-84 85+ 20-39 40-49 50-64 65-74 75-84 n of SD from the sex-specific meanMenIL-6 CRP causative factors. Thus, the therapeutic strategy of sin gle-molecule replacement may be ineffective or even 85+counterproductive. The presence of such signaling networks and feedback loops may help explain why single-hormone “replacement therapy” for problems of aging has demonstrated little benefit. The focus of research in this area is now on multiple-hormone 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Five-year decrease in regional cortical size (SD)

1	FIgURE 11-14 Five-year decline in mean volumes of different brain regions, measured in standard deviation (SD) units (Cohen’s d ). The primary visual cortex shows the least average shrinkage, and the prefrontal and inferior parietal cortex and hippocampus show the most average shrinkage. (From N Raz et al: Ann N Y Acad Sci 1097:84, 2007.) in number at a rate of ∼1% per year, starting after the third decade. These larger motor units contribute to reductions in fine-motor control and manual dexterity. Age-related changes also occur in the autonomic nervous system, affecting cardiovascular and splanchnic function.

1	Systemic Changes Coexisting with and Affecting one Another • the phenotype of agIng: the fInal common pathway of systemIc InteractIon While age-related system changes have been described individually, in reality, these changes develop in parallel and affect one another through many feed-forward and feedback loops. Some systemic interactions are well understood, while others are under investigation. For example, body composition interacts with energy balance and signaling. Higher lean body mass increases energy consumption and improves insulin sensitivity and carbohydrate metabolism. Higher fat mass, especially visceral fat mass, is the culprit in the metabolic syndrome and is associated with low testosterone levels, high sex hormone–binding globulin levels, and increased levels of proinflammatory markers such as

1	CRP and IL-6. Altered signaling can affect neurode generation; insulin resistance and adipokines such as leptin and adiponectin are associated with declines in but these findings are not specific and their diagnostic utility is unclear (Fig. 11-15). Other neurophysiologic changes in the brain frequently occur with aging and may contribute to cognitive decline. Functional imaging studies have shown that some older people have diminished coordination between the brain regions responsible for higher-order cognitive functions and that such diminished coordination is correlated with poor cognitive performance. In young healthy individuals, the brain activity associated with executive cognitive functions (e.g., problem-solving, decision-making) is very well localized; in contrast, in healthy older individuals, the pattern of cortical activation is more diffuse. Brain pathology has typically been associated with specific diseases; amyloid plaques and neurofibrillary tangles are considered

1	older individuals, the pattern of cortical activation is more diffuse. Brain pathology has typically been associated with specific diseases; amyloid plaques and neurofibrillary tangles are considered the pathologic hallmarks of Alzheimer’s disease. However, these pathologic markers have been found at autopsy in many older individuals who had normal cognition, as assessed by extensive testing in the year before death.

1	Taken together, trends in brain changes with aging suggest that some neurophysiologic manifestations are compensatory adaptations rather than primary contributors to age-related declines. Because the brain is capable of reorganization and compensation, extensive neurodegeneration may not be clinically evident. Therefore, early detection requires careful testing. Clinically, cortical and subcortical changes are reflected in the high prevalence of “soft,” nonspecific neurologic signs, often reflected in slow and unstable gait, poor balance, and slow reaction times. These movement changes can be elicited more overtly with “dual tasks,” in which a 1050 cognitive function. Combined with loss of motor neurons and dysfunction of the motor unit, a state of inflammation and reduced levels of testosterone and IGF-1 have been linked to accelerated decline of muscle mass and strength. Normal intersystem coordination is also affected by aging. The hypothalamus normally functions as a central

1	testosterone and IGF-1 have been linked to accelerated decline of muscle mass and strength. Normal intersystem coordination is also affected by aging. The hypothalamus normally functions as a central regulator of metabolism and energy use and coordinates physiologic responses of the entire organism through hormonal signaling; aging-related changes in the hypothalamus alter this control. The central nervous system (CNS) also controls adaptive sympathetic/parasympathetic activity, so that age-related CNS degeneration may have implications for autonomic function.

1	The phenotype that results from the aging process is characterized by increased susceptibility to diseases, high risk of multiple coexisting diseases, impaired response to stress (including limited ability to heal or recover after an acute disease), emergence of “geriatric syndromes” (characterized by stereotyped clinical manifestations but multifactorial causes), altered response to treatment, high risk of disability, and loss of personal autonomy with all its psychological and social consequences. In addition, these key aging processes may interfere with the typical pathophysiology of specific diseases, thereby altering expected clinical manifestations and confounding diagnosis. Clinically, patients may present with obvious problems within only one of these domains, but, since systems interact, all four main domains should be evaluated 7.5 cognitive and a motor task are performed simultaneously. In a simple version of a dual task, when an older adult has to stop walking in order to

1	all four main domains should be evaluated 7.5 cognitive and a motor task are performed simultaneously. In a simple version of a dual task, when an older adult has to stop walking in order to talk, an increased risk of falls can be predicted. Poor dual-task per formance has been interpreted as a marker cessing, so that simultaneous processing 7.0 6.5 6.0 5.5 is more constrained. Beyond the brain, 5.0 4.5 the spinal cord also experiences changes after the age of 60 years, including reduced numbers of motor neurons and damage to myelin. The motor neurons that survive plexity and by service to larger motor units. FIgURE 11-15 Longitudinal changes of regional brain volumes in normal aging and mild As motor units become larger, they decline cognitive impairment (MCI). (From I Driscoll et al: Neurology 72:1906, 2009.) and considered potential therapeutic targets. When patients present with obvious problems in multiple main systems affected by aging, they tend toward extreme degrees of

1	al: Neurology 72:1906, 2009.) and considered potential therapeutic targets. When patients present with obvious problems in multiple main systems affected by aging, they tend toward extreme degrees of susceptibility and loss of resilience, a condition that is globally referred to as frailty.

1	bIologIc underpInnIngs of the domaIns of the agIng phenotype The changes that occur with aging encompass multiple physiologic systems. Although they are often described in isolation, they are likely attributable to the progressive dysfunction of a unique mechanism that affects some fundamental housekeeping mechanism of cellular physiology. An important goal of future research is to connect the aging phenotype in humans to theories of aging that have largely been developed from studies in cell or animal models. If the main theories of aging could be operationalized into assessments that are feasible in humans, it would be possible to test the hypothesis that some of these processes are correlated with all the domains of the aging phenotype, above and beyond chronologic age. Review of the biologic theories (hallmarks) of aging provides an excellent template for a working hypothesis that, at least theoretically, could be tested in longitudinal studies. Candidate mechanisms of mammalian

1	biologic theories (hallmarks) of aging provides an excellent template for a working hypothesis that, at least theoretically, could be tested in longitudinal studies. Candidate mechanisms of mammalian aging include genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication.

1	Frailty Frailty has been described as a physiologic syndrome that is characterized by decreased reserve and diminished resistance to stressors, that results from cumulative decline across multiple physiologic systems, and that causes vulnerability to adverse outcomes and a high risk of death. A proposed “phenotype” definition characterized by weight loss, fatigue, impaired grip strength, diminished physical activity, and slow gait has shown good internal consistency and strong predictive validity and has been used in many clinical and epidemiologic studies. An alternative approach, the Frailty Index, assesses cumulative physiologic and functional burden. When combined with a structured clinical assessment (the Comprehensive Geriatric Assessment), the Frailty Index can be applied in clinical settings and has low rates of missing data; it predicts survival in community-dwelling older people as well as survival, length of stay and discharge location in acute-care settings. Regardless of

1	settings and has low rates of missing data; it predicts survival in community-dwelling older people as well as survival, length of stay and discharge location in acute-care settings. Regardless of the definition, an extensive body of literature shows that older persons who are considered frail by any definition have overt changes in the same four main processes: body composition, homeostatic dysregulation, energetic failure, and neurodegeneration—the characteristics of the aging phenotype. A classic clinical case would be an older woman with sarcopenic obesity characterized by increased body fat and decreased muscle (body composition changes); extremely low exercise tolerance and extreme fatigue (energetic failure); high insulin levels, low IGF-1 levels, inadequate intake of calories, and low levels of vitamins D and E and carotenoids (signal dysregulation); and memory problems, slow gait, and unstable balance (neurodegeneration). This woman is likely to exhibit all the manifestations

1	low levels of vitamins D and E and carotenoids (signal dysregulation); and memory problems, slow gait, and unstable balance (neurodegeneration). This woman is likely to exhibit all the manifestations of frailty, including a high risk of multiple diseases, disability, urinary incontinence, falls, delirium, depression, and other geriatric syndromes. It is expected that the biologic process underlying a particular “aging theory” would be more advanced in this woman than would be expected on the basis of chronologic age.

1	A goal of future research in geriatric medicine that has strong potential for clinical translation is to demonstrate that the hypothetical patient described above is biologically older, according to some robust biomarkers of biologic aging, than would be estimated from chronologic age alone. Conceptualizing frailty through the four main underlying processes is a step in this direction that stems from accumulated evidence and recognizes the heterogeneity and dynamic nature of the aging phenotype. Aging is universal but proceeds at highly variable rates, with wide heterogeneity in the emergence of the aging phenotype. Thus, the question is not whether an older patient is frail, but rather whether the severity of frailty is beyond the threshold of clinical and behavioral relevance. Understanding frailty through the lens of four interacting underlying processes also provides an interface with diseases that, like aging itself, affect the aging phenotype. For example, congestive heart

1	frailty through the lens of four interacting underlying processes also provides an interface with diseases that, like aging itself, affect the aging phenotype. For example, congestive heart failure is associated with low energy availability, multiple hormonal derangements, and a proinflammatory state, thereby contributing to frailty severity. Parkinson’s disease provides an example of neurodegeneration that, in an advanced state, affects body composition, energy metabolism, and homeostatic signaling, resulting in a syndrome that closely resembles frailty. Diabetes is especially important to aging and frailty because it harms body composition, energy metabolism, homeostatic dysregulation, and neuronal integrity. Accordingly, a number of studies have found that type 2 diabetes is a strong risk factor for frailty and for many of its consequences. Since disease and aging interact, careful and appropriate treatment of disease is critical to prevent or reduce frailty.

1	CoNSEQUENCES oF AgINg PRoCESSES, THE AgINg PHENoTYPE, ANd FRAILTY While the pathophysiology of frailty is still being elucidated, its consequences have been well characterized in prospective studies. Four main consequences are important for clinical practice: (1) ineffective or incomplete homeostatic response to stress, (2) multiple coexisting diseases (multior comorbidity) and polypharmacy, (3) physical disability, and (4) the so-called geriatric syndromes. We will briefly address each one of these consequences.

1	Low Resistance to Stress Frailty can be considered a progressive loss of reserve in multiple physiologic functions. At an early stage and in the absence of stress, mildly frail older individuals may appear to be normal. However, they have reduced ability to cope with challenges, such as acute diseases, traumas, surgical procedures, or chemotherapy. Acute illness involving a hospital stay is associated with undernutrition and inactivity, which sometimes may be of such magnitude that the residual muscle mass fails to meet the minimal requirement for walking. Even when nutrition is reinstated, energy reserves may be insufficient to adequately rebuild muscle mass. Older persons have a reduced ability to tolerate infections, in part because they are less able than younger people to build a dynamic inflammatory response to vaccination or infectious exposure; thus, infections are more likely to become severe and systemic and to resolve more slowly. In the context of tolerance to stress,

1	dynamic inflammatory response to vaccination or infectious exposure; thus, infections are more likely to become severe and systemic and to resolve more slowly. In the context of tolerance to stress, assessing aspects of frailty can help estimate the individual’s ability to withstand the rigors of aggressive treatments and to respond to interventions aimed at infection as well as the caregiver’s ability to anticipate and prevent complications of hospitalization and generally to estimate prognosis. Accordingly, treatment plans may be adjusted to improve tolerance and safety; bed rest and hospitalization should be used sparingly; and infections should be prevented, anticipated, and managed assertively.

1	Comorbidity and Polypharmacy Older age is associated with high rates of many chronic diseases (Fig. 11-4). Thus, not unexpectedly, the percentage of individuals affected by multiple medical conditions (coor multimorbidity) also increases with age. In frail older individuals, comorbidity occurs at higher rates than would be expected from the combined probability of the component conditions. It is likely that frailty and comorbidity affect each other, so that multiple diseases contribute to frailty and frailty increases susceptibility to diseases.

1	Clinically, patients with multiple conditions present unique diagnostic and treatment challenges. Standard diagnostic criteria may not be informative because there are additional confusing signs and symptoms. A classic example is the coexistence of deficiencies in iron and vitamin B12, creating an apparently normocytic anemia. The risk/ benefit ratio for many medical and surgical treatment options may be reduced in the face of other diseases. Drug treatment planning is made more complex because comorbid diseases may affect the absorption, volume of distribution, protein binding, and, especially, elimination of many drugs, leading to fluctuation in therapeutic levels and increased risk of underor overdosing. Drug excretion is affected by renal and hepatic changes with aging that may not be detectable with the usual clinical tests. Formulas for estimating glomerular filtration rate in older patients are available, whereas the estimation of changes in hepatic excretion remains a

1	not be detectable with the usual clinical tests. Formulas for estimating glomerular filtration rate in older patients are available, whereas the estimation of changes in hepatic excretion remains a challenge.

1	Patients with many diseases are usually prescribed multiple drugs, especially when they are cared for by multiple specialists who do not communicate. The risk of adverse drug reactions, drug–drug interactions, and poor compliance increases geometrically with the number

1	Chapter 11 Clinical Problems of Aging of drugs prescribed and with the severity of frailty. Some general rules to minimize the chances of adverse drug events are as follows: (1) Always ask patients to bring in all medications, including prescription drugs, over-the-counter products, vitamin supplements, and herbal preparations (the “brown bag test”). (2) Screen for unnecessary drugs; those without a clear indication should be discontinued. (3) Simplify the regimen in terms of number of agents and schedules, try to avoid frequent changes, and use single-daily-dose regimens whenever possible. (4) Avoid drugs that are expensive or not covered by insurance whenever possible. (5) Minimize the number of drugs to those that are absolutely essential, and always check for possible interactions. (6) Make sure that the patient or an available caregiver understands the administered regimen, and provide legible written instructions. (7) Schedule periodic medication reviews.

1	disability and Impaired Recovery from Acute-onset disability The prevalence of disability in self-care and home management increases steeply with aging and tends to be higher among women than among men (Fig. 11-5). Physical and cognitive function in older persons reflects overall health status and predicts health care utilization, institutionalization, and mortality more accurately than any other known biomedical measure. Thus, assessment of function and disability and prediction of the risk of disability are cornerstones of geriatric medicine. Frailty, regardless of the criteria used for its definition, is a robust and powerful risk factor for disability. Because of this strong relationship, measures of physical function and mobility have been proposed as standard criteria for frailty. However, disability occurs late in the frailty process, after reserve and compensation are exhausted. Early in the development of frailty, body composition changes, reductions in fitness, homeostatic

1	However, disability occurs late in the frailty process, after reserve and compensation are exhausted. Early in the development of frailty, body composition changes, reductions in fitness, homeostatic deregulation, and neurodegeneration can begin without affecting daily function. As opposed to disability in younger persons, in which the rule is to look for a clear dominant cause, disability in frail older persons is almost always multifactorial. Multiple disrupted aging processes are usually involved, even when the precipitating cause seems unique. Excess fat mass, poor muscle strength, reduced lean body mass, poor fitness, reduced energy efficiency, poor nutritional intake, low circulating levels of antioxidant micronutrients, high levels of proinflammatory markers, objective signs of neurologic dysfunction, and cognitive impairment all contribute to disability. The multifactorial nature of disability in frail older persons reduces the capacity for compensation and interferes with

1	neurologic dysfunction, and cognitive impairment all contribute to disability. The multifactorial nature of disability in frail older persons reduces the capacity for compensation and interferes with functional recovery. For example, a small lacunar stroke that causes problems with balance in a young hypertensive individual can be overcome by standing and walking with the feet further apart, a strategy that requires brain adaptation, strong muscles, and a high energy capacity. The same small lacunar stroke may cause catastrophic disability in an older person already affected by neurodegeneration and weakness, who is less able to compensate. As a consequence, interventions aimed at preventing and reducing disability in older persons should have a dual focus on both the precipitating cause and the systems needed for compensation. In the case of the lacunar stroke, interventions to promote mobility function might include stroke prevention, balance rehabilitation, and strength training.

1	As a rule of thumb, the assessment of contributing causes and the design of intervention strategies for disability in older persons should always consider the four main aging processes that contribute to frailty. One of the most popular approaches to disability measurement is a modification of the International Classification of Impairments, Disabilities and Handicaps (World Health Organization, 1980) proposed by the Institute of Medicine (1992). This classification infers a causal pathway in four steps: pathology (diseases), impairment (the physical manifestation of diseases), functional limitation (global functions such as walking, grasping, climbing stairs), and disability (ability to fulfill social roles in the environment). In practice, the assessment of functional limitation and disability is performed either by (1) self-reported questionnaire concerning the degree of ability to perform basic self-care or more complex ADLs or by (2) performance-based measures of physical

1	and disability is performed either by (1) self-reported questionnaire concerning the degree of ability to perform basic self-care or more complex ADLs or by (2) performance-based measures of physical function that assess specific domains, such as balance, gait, manual dexterity, coordination, flexibility, and endurance. A concise list of standard tools that can be used to assess physical function in older persons is provided in Table 11-4. In 2001, the WHO officially endorsed a new classification system, the International Classification of Functioning, Disability and Health, known more commonly as the ICF. In the ICF, health measures are classified from bodily, individual, and societal perspectives by means of two lists: a list of body functions and structure and a list of domains of activity and participation. Since an individual’s functioning and disability occur in a context, the ICF also includes a list of environmental factors. A detailed list of codes that allow the

1	of activity and participation. Since an individual’s functioning and disability occur in a context, the ICF also includes a list of environmental factors. A detailed list of codes that allow the classification of body functions, activities, and participation is being developed. The ICF system is widely implemented in Europe and is gaining popularity in the United States. Whatever classification system is used, the health care provider should try to identify factors that can be modified to minimize disability. Many of these factors are discussed in this chapter. Important issues related to aging that are not addressed in this chapter but are covered elsewhere include dementia (Chap. 35) and other cognitive disorders including aphasia, memory loss, and other focal cerebral disorders (Chap. 36).

1	geriatric Syndromes The term geriatric syndrome encompasses clinical conditions that are frequently encountered in older persons; have a deleterious effect on function and quality of life; have a multifactorial pathophysiology, often involving systems unrelated to the apparent chief symptom; and are manifested by stereotypical clinical presentations. The list of geriatric syndromes includes incontinence, delirium, falls, pressure ulcers, sleep disorders, problems with eating or feeding, pain, and depressed mood. In addition, dementia and physical disability are sometimes considered to be geriatric syndromes. The term syndrome is somewhat misleading in this context since it is most commonly used to describe a pattern of symptoms and signs that have a single underlying cause. The term geriatric syndromes, by contrast, refers to “multifactorial health conditions that occur when the accumulated effects of impairments in multiple systems render an older person vulnerable to situational

1	syndromes, by contrast, refers to “multifactorial health conditions that occur when the accumulated effects of impairments in multiple systems render an older person vulnerable to situational challenges.” According to this definition, geriatric syndromes reflect the complex interactions between an individual’s vulnerabilities and exposure to stressors or challenges. This definition aligns well with the concept that geriatric syndromes should be considered as phenotypic consequences of frailty and that a limited number of shared risk factors contribute to their etiology. Indeed, in various combinations and frequencies, virtually all geriatric syndromes are characterized by body composition changes, energy gaps, signaling disequilibria, and neurodegeneration. For example, detrusor (bladder) underactivity is a multifactorial geriatric condition that contributes to urinary retention in the frail elderly. It is characterized by detrusor muscle loss, fibrosis, and axonal degeneration. A

1	underactivity is a multifactorial geriatric condition that contributes to urinary retention in the frail elderly. It is characterized by detrusor muscle loss, fibrosis, and axonal degeneration. A proinflammatory state and a lack of estrogen signaling cause bladder muscle loss and detrusor underactivity, while a chronic urinary tract infection may cause detrusor hyperactivity; all of these factors may contribute to urinary incontinence.

1	Because of limited space, only delirium, falls, chronic pain, incontinence, and anorexia are addressed here. Interested readers are referred to textbooks on geriatric medicine for a discussion of other geriatric syndromes.

1	delirium (See also Chap. 34) Delirium is an acute disorder of disturbed attention that fluctuates with time. It affects 15–55% of hospitalized older patients. Delirium has previously been considered to be transient and reversible and a normal consequence of surgery, chronic disease, or infections in older people. Delirium may be associated with a substantially increased risk for dementia and is an independent risk factor for morbidity, prolonged hospitalization, and death. These associations are particularly strong in the oldest old. Fig. 11-16 shows an algorithm for assessment and management of delirium in hospitalized older patients. The clinical presentation of delirium is heterogeneous, but frequent features are (1) a rapid decline in the level of consciousness, with difficulty focusing, shifting, or sustaining attention; (2) cognitive change (rumbling incoherent speech, memory gaps, disorientation, hallucinations) not explained by dementia; and (3) a medical history suggestive of

1	shifting, or sustaining attention; (2) cognitive change (rumbling incoherent speech, memory gaps, disorientation, hallucinations) not explained by dementia; and (3) a medical history suggestive of preexisting cognitive impairment, frailty, and comorbidity. The strongest predisposing factors for delirium are dementia, any other condition associated with chronic or transient neurologic dysfunction (neurologic diseases, dehydration, alcohol consumption, psychoactive drugs), and sensory (visual and hearing) deprivation; these associations suggest that delirium is a condition of brain function susceptibility (neurodegeneration or transient neuronal impairment) that precludes the avoidance of decompensation in the face of a stressful event. Many stressful conditions have been implicated as precipitating factors, including surgery; anesthesia; persistent pain; treatment with opiates, narcotics, or anticholinergics; sleep deprivation, immobilization; hypoxia; malnutrition; and metabolic and

1	factors, including surgery; anesthesia; persistent pain; treatment with opiates, narcotics, or anticholinergics; sleep deprivation, immobilization; hypoxia; malnutrition; and metabolic and electrolyte derangements. Both the occurrence and the severity of delirium can be reduced by anticipatory screening and preventive strategies targeting the precipitating causes. The Confusion Assessment Method is a simple, validated tool for screening in the hospital setting. The three pillars of treatment are (1) immediate identification and treatment of precipitating factors, (2) withdrawal of drugs that may have promoted the onset of delirium, and (3) supportive care, including management of hypoxia, hydration and nutrition, mobilization, and environmental modifications. Whether patients who are cared for in special delirium units have better outcomes than those who are not is still in question. Physical restraints should be avoided because they tend to increase agitation and injury. Whenever

1	for in special delirium units have better outcomes than those who are not is still in question. Physical restraints should be avoided because they tend to increase agitation and injury. Whenever possible, drug treatment should be avoided because it may prolong or aggravate delirium in some cases. The treatment of choice is low-dose haloperidol. It remains difficult to reduce delirium in patients with acute illness or other stressful conditions. Interventions based on dietary supplementation or careful use of pain medications and sedatives in preand postoperative older patients have been only partially successful.

1	Chapter 11 Clinical Problems of Aging FIgURE 11-16 Algorithm depicting assessment and management of delirium in hospitalized older patients. (Modified from SK Inouye: N Engl J Med 354:1157, 2006.) Hospital admission Assess current and recent changes in mental status Monitor mental status Prevention Address risk factors Improve communication Improve environment Early discharge Avoid psychotropic drugs Acute Impaired mental status Cognitive assessment and delirium evaluation Identify and address predisposing and precipitating risk factors Provide supportive care and prevent complications Manage symptoms of delirium Rule out depression, mania and psychosis Delirium confirmed

1	Falls and Balance disorders Unstable gait and falls are serious concerns in the older adult because they lead not only to injury but also to restricted activity, increased health care utilization, and even death. Like all geriatric syndromes, problems with balance and falls tend to be multifactorial and are strongly connected with the disrupted aging systems that contribute to frailty. Poor muscle strength, neural damage in the basal ganglia and cerebellum, diabetes, and peripheral neuropathy are all recognized risk factors for falls. Therefore, evaluation and management require a structured multisystem approach that spans the entire frailty spectrum and beyond. Accordingly, interventions to prevent or reduce instability and falls usually require a mix of medical, rehabilitative, and environmental modification approaches. Guidelines for the evaluation and management of falls, released by the American Geriatrics Society, recommend asking all older adults about falls and perceived gait

1	modification approaches. Guidelines for the evaluation and management of falls, released by the American Geriatrics Society, recommend asking all older adults about falls and perceived gait instability (Fig. 11-17). Patients with a positive history of multiple falls as well as persons who have sustained one or more injurious falls should undergo an evaluation of gait and balance as well as a targeted history and physical examination to detect

1	Recommend fall prevention, education and exercise program that includes balance, gait and coordination training and strength training Ask all patients about falls in the past year No falls One fall past 6 months Gait or balance problem Report >1 fall, or difficulty with gait or balance, or seeking medical attention because of fall Multifactorial fall risk assessment Check for gait or balance problems History of falls Medications Gait and balance Cognition Visual acuity Lower limb joint function Neurological impairment Muscle strength HR and rhythm Postural hypotension Feet and footwear Environmental hazards Intervene with identified risks Modify medications Prescribe individualized exercise program Treat vision impairment Manage postural hypotension Manage HR and rhythm abnormalities Supplement vitamin D Address foot/shoe problems Reduce environmental hazards Education/ training in self-management and behavioral changes

1	FIgURE 11-17 Algorithm depicting assessment and management of falls in older patients. HR, heart rate. (From American Geriatrics Society and British Geriatrics Society: Clinical Practice Guideline for the Prevention of Falls in Older Persons. New York, American Geriatric Society, 2010.) sensory, nervous system, brain, cardiovascular, and musculoskeletal contributors. Interventions depend on the factors identified but often include medication adjustment, physical therapy, and home modifications. Meta-analyses of strategies to reduce the risk of falls have found that multifactorial risk assessment and management as well as individually targeted therapeutic exercise are effective. Supplementation with vitamin D at 800 IU daily may also help reduce falls, especially in older persons with low vitamin D levels.

1	Persistent Pain Pain from multiple sources is the most common symptom reported by older adults in primary care settings and is also common in acute-care, long-term-care, and palliative-care settings. Acute pain and cancer pain are beyond the scope of this chapter. Persistent pain results in restricted activity, depression, sleep disorders, and social isolation and increases the risk of adverse events due to medication. The most common causes of persistent pain are musculoskeletal problems, but neuropathic pain and ischemic pain occur frequently, and multiple concurrent causes are often found. Alterations in mechanical and structural elements of the skeleton commonly lead to secondary problems in other parts of the body, especially soft tissue or myofascial components. A structured history should elicit information about the quality, severity, and temporal patterns of pain. Physical examination should focus on the back and joints, on trigger points and periarticular areas, and on

1	should elicit information about the quality, severity, and temporal patterns of pain. Physical examination should focus on the back and joints, on trigger points and periarticular areas, and on possible evidence of radicular neurologic patterns and peripheral vascular disease. Pharmacologic management should follow standard progressions, as recommended by the World Health Organization (Chap. 18), and adverse effects on the CNS, which are especially likely in this population, must be monitored. For persistent pain, regular analgesic schedules are appropriate and should be combined with nonpharmacologic approaches such as splints, physical exercise, heat, and other modalities. A variety of adjuvant analgesics such as antidepressants and anticonvulsants may be used; again, however, effects on reaction time and alertness may be dose limiting, especially in older persons with cognitive impairment. Joint or soft tissue injections may be helpful. Education of the patient and mutually

1	on reaction time and alertness may be dose limiting, especially in older persons with cognitive impairment. Joint or soft tissue injections may be helpful. Education of the patient and mutually agreed-upon goal setting are important since pain usually is not fully eliminated but rather is controlled to a tolerable level that maximizes function while minimizing adverse effects.

1	Urinary Incontinence Urinary incontinence—the involuntary leakage of urine—is highly prevalent among older persons (especially women) and has a profound negative impact on quality of life. Approximately 50% of American women will experience some form of urinary incontinence over a lifetime. Increasing age, white race, childbirth, obesity, and medical comorbidity are all risk factors for urinary incontinence. The three main clinical forms of urinary incontinence are as follows: (1) Stress incontinence is the failure of the sphincteric mechanism to remain closed when there is a sudden increase in intraabdominal pressure, such as a cough or sneeze. In women this condition is due to insufficient strength of the pelvic floor muscles, while in men it is almost exclusively secondary to prostate surgery. (2) Urge incontinence is the loss of urine accompanied by a sudden sensation of need to urinate and inability to control it and is due to detrusor muscle overactivity (lack of inhibition)

1	surgery. (2) Urge incontinence is the loss of urine accompanied by a sudden sensation of need to urinate and inability to control it and is due to detrusor muscle overactivity (lack of inhibition) caused by loss of neurologic control or local irritation. (3) Overflow incontinence is characterized by urinary dribbling, either constantly or for some period after urination. This condition is due to impaired detrusor contractility (due usually to denervation, for example, in

1	Prevalence of urinary incontinence*

1	FIgURE 11-18 Rates of urge, stress, and mixed incontinence, by age group, in a sample of 3552 women. *Based on a sample of 3553 participants. (From JL Melville et al: Arch Intern Med 165:537, 2005.) diabetes) or bladder outlet obstruction (prostate hypertrophy in men and cystocele in women). Thus, it is not surprising that the pathogenesis of urinary incontinence is connected to the disrupted aging systems that contribute to frailty, body composition changes (atrophy of the bladder and pelvic floor muscle), and neurodegeneration (both central and peripheral nervous systems). Frailty is a strong risk factor for urinary incontinence. Indeed, older women are more likely to have mixed (urge + stress) incontinence than any pure form (Fig. 11-18). In analogy with the other geriatric syndromes, urinary incontinence derives from a predisposing condition superimposed on a stressful precipitating factor. Accordingly, treatment of urinary incontinence should address both. The first line of

1	urinary incontinence derives from a predisposing condition superimposed on a stressful precipitating factor. Accordingly, treatment of urinary incontinence should address both. The first line of treatment is bladder training associated with pelvic muscle exercise (Kegel exercises) that sometimes should be associated with electrical stimulation. Women with possible vaginal or uterine prolapse should be referred to a specialist. Urinary tract infections should be investigated and eventually treated. A long list of medications can precipitate urinary incontinence, including diuretics, antidepressants, sedative hypnotics, adrenergic agonists or blockers, anticholinergics, and calcium channel blockers. Whenever possible, these medications should be discontinued. Until recently, it was believed that oral or local estrogen treatment alleviated the symptoms of urinary incontinence in postmenopausal women, but this notion is now controversial. Antimuscarinic drugs such as tolterodine,

1	believed that oral or local estrogen treatment alleviated the symptoms of urinary incontinence in postmenopausal women, but this notion is now controversial. Antimuscarinic drugs such as tolterodine, darifenacin, and fesoterodine are modestly effective for mixed-etiology incontinence, but all of these drugs can affect cognition and so must be used with caution and with monitoring of cognitive status. In some cases, surgical treatment should be considered. Chronic catheterization has many adverse effects and should be limited to chronic urinary retention that cannot be managed in any other way. Bacteriuria always occurs and should be treated only if it is symptomatic. Bacterial communities isolated from the urine of women with urinary incontinence appear to differ with the type of incontinence; this observation suggests that the bladder microbiota may play a role in urinary incontinence. If so, this microbial population would be a potential target for treatment.

1	Undernutrition and Anorexia There is strong evidence that the healthy mammalian life span is greatly affected by changes in the activity of central nutrient-sensing mechanisms, especially those that involve the rapamycin (mTOR) network. Polymorphic variations in the gene that encodes mTOR in humans are associated with longevity; this association suggests that the role of nutrient signaling in healthy aging may be conserved in humans. Normal aging is associated with a decline in food intake that is more marked in men than in women. To some extent, food intake is reduced because energy demand declines as a result of the combination of a lower level of physical activity, a decline in lean body mass, and slowed rates of protein turnover. Other contributors to decreased food intake include losses of taste sensation, reduced stomach compliance, higher circulating levels of cholecystokinin, and, in men, low testosterone levels associated with increased leptin. When food intake decreases to a

1	of taste sensation, reduced stomach compliance, higher circulating levels of cholecystokinin, and, in men, low testosterone levels associated with increased leptin. When food intake decreases to a level below the reduced energy demand, the result is energy malnutrition.

1	Malnutrition in older persons should be considered a geriatric syndrome because it is the result of intrinsic susceptibility due to aging, complicated by multiple superimposed precipitating causes. Many older individuals tend to consume a monotonous diet that lacks sufficient fresh food, fruits, and vegetables, so that intake of important micronutrients is inadequate. Undernutrition in older people is associated with multiple adverse health consequences, including impaired muscle function, decreased bone mass, immune dysfunction, anemia, reduced cognitive function, poor wound healing, delayed recovery from surgery, and increased risk of falls, disability, and death. Despite these serious potential consequences, undernutrition often remains unrecognized until it is well advanced because weight loss tends to be ignored by both patients and physicians. Muscle wasting is a frequent feature of weight loss and malnutrition that is often associated with loss of subcutaneous fat. The main

1	weight loss tends to be ignored by both patients and physicians. Muscle wasting is a frequent feature of weight loss and malnutrition that is often associated with loss of subcutaneous fat. The main causes of weight loss are anorexia, cachexia, sarcopenia, malabsorption, hypermetabolism, and dehydration, almost always in various combinations. Many of these causes can be detected and corrected. Cancer accounts for only 10–15% of cases of weight loss and anorexia in older people. Other important causes include a recent move to a long-term-care setting, acute illness (often with inflammation), hospitalization with bed rest for as little as 1–2 days, depression, drugs that cause anorexia and nausea (e.g., digoxin and antibiotics), swallowing problems, oral infections, dental problems, gastrointestinal pathology, thyroid and other hormonal problems, poverty, and isolation, with reduced access to food. Weight loss may also result from dehydration, possibly related to excess sweating,

1	pathology, thyroid and other hormonal problems, poverty, and isolation, with reduced access to food. Weight loss may also result from dehydration, possibly related to excess sweating, diarrhea, vomiting, or reduced fluid intake. Early identification is paramount and requires careful weight monitoring. Patients or caregivers should be taught to record weight regularly at home, the patient should be weighed at each clinical encounter, and a record of serial weights should be maintained in the medical record. If malnutrition is suspected, formal assessment should begin with a standardized screening instrument such as the Mini Nutritional Assessment, the Malnutrition Universal Screening Tool, or the Simplified Nutritional Appetite Questionnaire. The Mini Nutritional Assessment includes questions on appetite, timing of eating, frequency of meals, and taste. Its sensitivity and specificity are >75% for future weight loss of ≥5% of body weight in older people. Many nutritional supplements

1	on appetite, timing of eating, frequency of meals, and taste. Its sensitivity and specificity are >75% for future weight loss of ≥5% of body weight in older people. Many nutritional supplements are available, and their use should be initiated early to prevent more severe weight loss and its consequences. When an older patient has malnutrition, the diet should be liberalized and dietary restrictions should be lifted as much as possible. Nutritional supplements should be given between meals to avoid interference with food intake at mealtime. Limited evidence supports the use of any pharmacologic intervention to treat weight loss. The two antianorexic drugs most often prescribed in older persons are megesterol and dronabinol. Both can increase weight; however, the gain is mostly fat, not muscle, and both drugs have serious side effects. Dronabinol is an excellent drug for use in the palliative-care setting. There is little evidence that intentional weight loss in overweight older people

1	and both drugs have serious side effects. Dronabinol is an excellent drug for use in the palliative-care setting. There is little evidence that intentional weight loss in overweight older people prolongs life. Weight loss after the age of 70 should probably be limited to persons with extreme obesity and should always be medically supervised.

1	Common diseases in older adults may have unexpected and atypical clinical features. Most age-related changes in clinical presentation, evolution, and response to treatment are due to interaction of disease pathophysiology with age-related system dysregulation. Some diseases, such as Parkinson’s disease (PD) and diabetes, directly affect aging systems and therefore have a devastating impact on frailty and its consequences. Chapter 11 Clinical Problems of Aging

1	Parkinson’s disease (See also Chap. 449) Most cases of PD begin after the age of 60 years, and the incidence increases up to the age of ∼80 years. Brain aging and PD have long been thought to be related. The nigrostriatal system deteriorates with aging, and many older persons tend to develop a mild form of movement disorder characterized by bradykinesia and stooped posture that mimics mild PD. It is interesting that, in PD, older age at presentation is associated with a more severe and rapid decline in gait, balance, posture, and cognition. These age-related motor and cognitive manifestations of PD tend to be poorly responsive to levodopa or dopamine agonist treatments, especially in the oldest old. In contrast, age at presentation does not correlate with the severity and progression of other classic PD symptoms, such as tremor, rigidity, and bradykinesia, nor does it affect the response of these symptoms to levodopa. The pattern of PD features in older persons suggests that late-life

1	classic PD symptoms, such as tremor, rigidity, and bradykinesia, nor does it affect the response of these symptoms to levodopa. The pattern of PD features in older persons suggests that late-life PD may reflect a failure of the normal cellular compensatory mechanisms in vulnerable brain regions and that this vulnerability is increased by age-related neurodegeneration, making PD symptoms particularly resistant to levodopa treatment. In addition to motor symptoms, older PD patients tend to have reduced muscle mass (sarcopenia), eating disorders, and poor levels of fitness. Accordingly, PD is a powerful risk factor for frailty and its consequences, including disability, comorbidity, falls, incontinence, chronic pain, and delirium. Use of levodopa and dopaminergic agonists by older PD patients requires complex dosing schedules; therefore, slow-release preparations are preferred. Both dopaminergic and anticholinergic agents increase the risk of confusion and hallucinations. Use of

1	patients requires complex dosing schedules; therefore, slow-release preparations are preferred. Both dopaminergic and anticholinergic agents increase the risk of confusion and hallucinations. Use of anticholinergic agents should generally be avoided. For dopaminergic agents, cognitive side effects can be dose limiting.

1	diabetes (See also Chaps. 417–419) Both the incidence and the prevalence of diabetes mellitus increase with aging. Among persons ≥65 years old, the prevalence is ∼12% (with higher figures among African Americans and Hispanics), reflecting the effects of population aging and the obesity epidemic. Diabetes affects all four main aging systems that contribute to frailty. Obesity, especially visceral obesity, is a strong risk factor for insulin resistance, the metabolic syndrome, and diabetes. Diabetes is associated with both reduced muscle mass and accelerated rates of muscle wasting. Diabetic patients have an elevated RMR and a poor degree of fitness. Diabetes is associated with multiple hormone dysregulation, a proinflammatory state, and excess oxidative stress. Finally, diabetes-induced neurodegeneration involves both the central and peripheral nervous systems. Given these characteristics, it is not surprising that patients with diabetes mellitus are more likely to be frail and at high

1	involves both the central and peripheral nervous systems. Given these characteristics, it is not surprising that patients with diabetes mellitus are more likely to be frail and at high risk of developing physical disability, depression, delirium, cognitive impairment, urinary incontinence, injurious falls, and persistent pain. Thus, the assessment of older diabetic patients should always include screening and risk factor evaluation for these conditions.

1	In young and adult patients, the main treatment goal has been strict glycemic control aimed at bringing the hemoglobin A1c level to within normal values (i.e., ≤6%). However, the risk/benefit ratio is optimized by the use of less aggressive glycemic targets. In fact, in the context of a randomized clinical trial, strict glycemic control was associated with a higher mortality rate. Thus, a more reasonable goal for hemoglobin A1c is 7% or slightly below. Treatment goals are altered further in frail older adults who have a high risk of complications of hypoglycemia and a life expectancy of <5 years. In these cases, an even less stringent target (e.g., 7–8%) should be considered, with A1c monitored every 6 or 12 months. Hypoglycemia is particularly difficult to identify in older diabetic patients because autonomic and nervous system symptoms occur at a lower blood sugar level than in younger diabetics, although the metabolic reactions and neurologic injury effects are similar in the two

1	because autonomic and nervous system symptoms occur at a lower blood sugar level than in younger diabetics, although the metabolic reactions and neurologic injury effects are similar in the two age groups. The autonomic symptoms of hypoglycemia are often masked by beta blockers. Frail older adults are at even higher risk for serious hypoglycemia than are healthier, higher-functioning older adults. In older patients with type 2 diabetes, a history of severe hypoglycemic episodes is associated with higher mortality risk, more severe microvascular complications, and greater risk of dementia. Thus, patients with suspected or documented episodes of hypoglycemia, especially those who are frail or disabled, need more liberal glucose-control goals, careful education about hypoglycemia, and close follow-up by the health care provider, possibly in the presence of a caregiver. Chlorpropamide has a prolonged half-life, particularly in older adults, and should be avoided because it is associated

1	follow-up by the health care provider, possibly in the presence of a caregiver. Chlorpropamide has a prolonged half-life, particularly in older adults, and should be avoided because it is associated with a high risk of hypoglycemia. Metformin should be used with caution and only in patients free of severe renal insufficiency. Renal insufficiency should be assessed by a calculated glomerular filtration rate or, in very old patients who have reduced muscle mass, by a direct measure of creatinine clearance from a 24-h urine collection. Lifestyle changes in diet and exercise and a little weight loss can prevent or delay diabetes in high-risk individuals and are substantially more effective than metformin treatment. The risk of type 2 diabetes decreased by 58% in a study of diet and exercise, and this effect was similar at all ages and in all ethnic groups. The risk reduction with standard care plus metformin was 31%.

1	APPRoACH To THE CARE oF oLdER PERSoNS Effects of Altered Pathophysiology and Multimorbidity on Clinical decision-Making The fact that older people are more likely to have atypical manifestations of disease and multiple coexisting conditions has serious consequences for the availability of high-quality evidence for medical practice and clinical decision-making. Randomized clinical trials—the basis for high-quality evidence—have tended to exclude older persons with atypical manifestations of disease, multimorbidity, or functional limitations. Across a wide range of conditions, the average age of a clinical trial participant is 20 years younger than the average age of the population with the condition. Clinical practice guidelines and care-quality metrics are focused on one condition at a time and tend not to consider the impact of comorbid conditions on the safety and feasibility of each set of recommendations. These disease-centric recommendations tend to result in fragmented care.

1	time and tend not to consider the impact of comorbid conditions on the safety and feasibility of each set of recommendations. These disease-centric recommendations tend to result in fragmented care. Therefore, clinical decision-making with regard to an older person with multiple chronic conditions must be based on the weighing of several influential factors, including the patient’s priorities and preferences, potential beneficial and harmful interactions among the several conditions and their treatments, life expectancy, and practical issues such as transportation, or ability to cooperate with the test or treatment.

1	organization of Health Care for older Adults The complex underlying physiology of aging leads to multiple coexisting medical problems and functional consequences that are often chronic, with recurrent exacerbations and remissions. Combined with the social consequences of aging (e.g., widowhood or lack of an available caregiver), these medical and functional factors mandate that older adults must sometimes use non-medical services to meet functional needs. The end result of these medical, functional, and social factors is that older adults use many health care and social support services in a variety of settings. Thus, it is incumbent on the internist, whether a generalist or specialist, to be familiar with the scope of settings and services that are used by their patients.

1	For many settings, Medicare reimbursement requires a medical order based on specific indications, so the hospitalist or referring physician must be familiar with eligibility requirements. Table 11-5 summarizes the types of services and payment sources for common settings of care. Older adults who have experienced new disability during a hospitalization are eligible for rehabilitation services. Inpatient rehabilitation requires at least 3 h per day of active rehabilitative activity and is limited to specific diagnoses. More and more rehabilitative services are provided in postacute settings, where the required intensity of service is less stringent. Postacute settings are also used for complex nursing services such as provision and supervision of long-term parenteral medication use or wound care. Under current policy, Medicare covers postacute care only if there is an eligible medical, nursing, or rehabilitation service. Otherwise, nursing home care is not covered by Medicare and must

1	care. Under current policy, Medicare covers postacute care only if there is an eligible medical, nursing, or rehabilitation service. Otherwise, nursing home care is not covered by Medicare and must be paid for with personal assets until all resources have been consumed, at which time Medicaid coverage becomes available.

1	Medicaid is a state–federal partnership whose greatest single expenditure is nursing home care. Thus, the need for chronic daily assistance with personal care in a nursing home consumes a large part of most state Medicaid budgets as well as personal assets. Accordingly, alternatives to chronic nursing-home care are of great interest to states, patients, and families. Some states have developed Medicaid-funded day-care programs, sometimes based on the Program for All-Inclusive Day programs Medical, surgical, and psychiatric services that cannot be provided in less complex settings Resuscitation, stabilization, triage, disposition Hospital-based residential program providing team-based, physician-supervised, intensive therapeutic rehabilitation for specific diagnoses Chronic, urgent, and preventive services Medical, nursing, and rehabilitative services after hospitalization, often based in hospitals or nursing homes

1	Chronic, urgent, and preventive services Medical, nursing, and rehabilitative services after hospitalization, often based in hospitals or nursing homes Residential program with daily nursing and aide care for persons who are dependent in self-care Residential program with daily aide care and housing for persons who are dependent in household management Nursing and rehabilitative services for episodes of care provided to persons in the community Supervised settings providing nursing and aide care for scheduled hours Medicare, Medicaid, and private insurance Medicare, Medicaid, and private insurance Medicare, Medicaid, and private insurance Medicare, Medicaid, and private insurance Medicare up to 100 days with eligibility requirements Medicaid, private payment, long-term care insurance Medicare, Medicaid Private payment, Medicaid

1	Medicare, Medicaid, and private insurance Medicare up to 100 days with eligibility requirements Medicaid, private payment, long-term care insurance Medicare, Medicaid Private payment, Medicaid Care of the Elderly (PACE) model. In this situation, older adults who are eligible for both Medicare and Medicaid and who are otherwise eligible for chronic nursing-home care can receive coordinated medical and functional services in conjunction with a day-care program.

1	For most older adults, a caregiver must be available to provide assistance on weeknights and weekends. Under current policy, home health services do not provide chronic functional assistance in the home but rather are targeted at episodes of care supplied by medical or rehabilitative services for older adults who are considered home bound. Some community agencies, whether private or public, can provide homemaker and home aide services to assist the home-bound older adult with functional needs, but there may be income requirements or expensive private payment may be needed.

1	Within the past decade, there has been tremendous growth in a broad spectrum of assisted-living settings. Such settings do not offer the degree of 24-h nursing supervision or personal aide care that is provided in traditional nursing homes, although distinctions are becoming blurred. Most assisted-living settings provide meals, medication supervision, and homemaking services, but they often require that residents be capable of transporting themselves to a congregate meal site. Moreover, most of these settings accept only private payment from residents and their families and thus are hard to access for older adults with limited resources. Some states are exploring coverage for lower-cost residential-care services such as family care homes.

1	Models of Care Coordination The complexity and fragmentation of care for older adults results in both increased costs and increased risk of iatrogenic complications such as missed diagnoses, adverse medication events, further worsening of function, and even death. These serious consequences have led to a strong interest in care coordination through teams of providers, with the goals to reduce unnecessary costs and to prevent adverse events. Table 11-6 lists examples of evidence-based models of care coordination that were recommended in a 2009 Chapter 11 Clinical Problems of Aging taBLe 11-6 evIdenCe-Based ModeLs of Care CoordInatIon for oLder patIents (InstItute of MedICIne, 2009) Source: Reproduced with permission from C Boult et al: J Am Geriatr Soc 57:2328, 2009.

1	Institute of Medicine report. While not mentioned as a specific type of team care, modern information technology offers substantial promise in providing consistent, readily available information across settings and providers. All such team programs are targeted at prevention and management of chronic and complex problems. Evidence from clinical trials or quasi-experimental studies supports the benefit of each model, and for some models data are sufficient to support meta-analyses. The evidence for benefit is not always consistent between studies or types of care but includes some support for improved quality of care, quality of life, function, survival, and health care costs and use. Some models of care are disease-specific and focus on common chronic conditions such as diabetes mellitus, congestive heart failure, chronic obstructive pulmonary disease, and stroke. One challenge in the use of these models is that a majority of older adults will have multiple simultaneous conditions and

1	congestive heart failure, chronic obstructive pulmonary disease, and stroke. One challenge in the use of these models is that a majority of older adults will have multiple simultaneous conditions and thus will need services from multiple programs that may not communicate among themselves.

1	Most models of care are difficult to implement in today’s health care system because nonphysician services are not reimbursed, nor is physician effort that is not incorporated into “face-to-face” time. Thus, several models have been developed largely by the Department of Veterans Affairs Health Care System, Medicare Managed Care providers, and other sponsoring agencies. Medicare has developed a series of demonstration projects that can expand the evidence base and serve policy makers. More recently, there has been an effort to promote coordinated care through Accountable Care Organizations and patient-centered “medical homes.” However, the processes and outcomes of such care must evolve from disease-specific indicators to more general markers, such as optimizing functional status, focusing on outcomes that are important to patients, and minimizing inappropriate care.

1	In older adults, prevention tests and interventions are less consistently recommended for all asymptomatic patients. The guidelines fail to address the influence of health status and life expectancy on recommendations, although the benefits of prevention are clearly affected by life expectancy. For example, in most types of cancer, screening provides no benefit in patients with a life expectancy of ≤5 years. More research is needed to build an appropriate evidence base for ageand life expectancy–adapted preventive services. Health behavior modification, especially increasing physical activity and improving nutrition, probably has the greatest potential to promote healthy aging.

1	Osteoporosis: Bone mineral density (BMD) should be measured at least once after the age of 65 years. There is little evidence that regular monitoring of BMD improves the prediction of fractures. Because of limitations in the precision of dual-energy x-ray absorptiometry, the minimal interval between evaluations should be 2–3 years. Hypertension: Blood pressure should be determined at least once a year or more often in patients with hypertension. Diabetes: Serum glucose and hemoglobin A1c should be checked every 3 years or more often in patients who are obese or hypertensive. Lipid disorders: A lipid panel should be done every 5 years or more often in patients with diabetes or any cardiovascular disease. Colorectal cancer: A fecal occult blood test and a sigmoidoscopy or colonoscopy should be done on a regular schedule up to the age of 75 years. No consensus guidelines exist for these tests >75 years of age.

1	Breast cancer: Mammography should be done every 2 years between the ages of 50 and 74 years. No consensus guidelines exist for mammography after the age of 75 years. Cervical cancer: A Pap smear should be done every 3 years up to the age of 65 years. Influenza: Immunize annually. Shingles: Administer herpes zoster vaccine once after the age of 50 years. Pneumonia: Administer pneumococcal vaccine once at the age of 65 years. Myocardial infarction: Prescribe daily aspirin for patients with prevalent cardiovascular disease or with a poor cardiovascular risk profile. Osteoporosis: Prescribe calcium at 1200 mg daily and vitamin D at ≥800 IU daily.

1	Exercise Rates of regular physical activity decrease with age and are lowest in older persons. This situation is unfortunate because increased physical activity has clear benefits in older adults, improving physical function, muscle strength, mood, sleep, and metabolic risk profile. Some studies suggest that exercise can improve cognition and prevent dementia, but this association is still controversial. Exercise programs, both aerobic and strength training, are feasible and beneficial even in very old and frail individuals. Regular, moderate-intensity exercise can reduce the rate of age-associated decline in physical function. The Centers for Disease Control and Prevention recommends that older persons should spend at least 150 min per week in moderate-intensity aerobic activity (e.g., brisk walking) and should engage in muscle-strengthening activities that work all major muscle groups (legs, hips, back, abdomen, chest, shoulders, and arms) at least 2 days a week. In the absence of

1	brisk walking) and should engage in muscle-strengthening activities that work all major muscle groups (legs, hips, back, abdomen, chest, shoulders, and arms) at least 2 days a week. In the absence of contraindications, more intense and prolonged physical activity provides greater benefits. Frail and sedentary persons may need supervision, at least at the start of the exercise program, to avoid falls and exercise-related injuries.

1	Nutrition Older persons are particularly vulnerable to malnutrition, and many problems that affect older patients can be addressed by dietary modification. As mentioned above, nutrient sensing is the major factor associated with differential longevity in several animal models, including mammals. Treatment with rapamycin, the only pharmacologic intervention that has been associated with longevity, affects nutrient sensing. Nevertheless, there are almost no evidence-based guidelines for individualizing dietary modifications based on differing health outcomes in the elderly. Even when guidelines exist, older people tend to be poorly compliant with dietary recommendations. Basic principles of a healthy diet that are also valid for older persons are as follows:

1	Encourage the consumption of fruits and vegetables; they are rich in micronutrients, mineral, and fibers. Whole grains are also a good source of fiber. Keep in mind that some of these foods are costly and thus less accessible to low-income persons. Emphasize that good hydration is essential. Fluid intake should be at least 1000 mL daily. Encourage the use of fat-free and low-fat dairy products, legumes, poultry, and lean meats. Encourage consumption of fish at least once a week, since there is strong epidemiologic evidence that fish consumption is associated with a lowered risk of Alzheimer’s disease. Match intake of energy (calories) to overall energy needs in order to maintain a healthy weight and BMI (20–27). Recommend moderate (5–10%) caloric restriction only when the BMI is >27. Limit consumption of foods with high caloric density, high sugar content, and high salt content. Limit the intake of foods with a high content of saturated fatty acids and cholesterol.

1	Limit consumption of foods with high caloric density, high sugar content, and high salt content. Limit the intake of foods with a high content of saturated fatty acids and cholesterol. Limit alcohol consumption (one drink per day or less). Introduce vitamin D–fortified foods and/or vitamin D supplements into the diet. Older persons who have little exposure to UVB radiation are at risk of vitamin D insufficiency. Make sure that the diet includes adequate food-related intake of magnesium, vitamin A, and vitamin B12.

1	Make sure that the diet includes adequate food-related intake of magnesium, vitamin A, and vitamin B12. Monitor daily protein intake, which, in healthy older persons, should be in the range of 1.0–1.2 g/kg of body weight. Higher daily protein intake (i.e., ≥1.2–1.5 g/kg) is advised for those who are exercising or are affected by chronic diseases, especially if these conditions are associated with chronic inflammation. Older people with severe kidney disease (i.e., an estimated glomerular filtration rate of <30 mL/min per 1.73 m2) who are not on dialysis should limit protein intake. • For constipation, increase dietary fiber intake to 10–25 g/d and fluid intake to 1500 mL/d. A bulk laxative (methylcellulose or psyllium) can be added.

1	Novel Interventions to Modify Aging Processes Aging is a complex process with multiple manifestations at the molecular, cellular, organ, and whole-organism level. The nature of the aging process is still not fully understood, but aging and its effects may be modulated by appropriate interventions. Dietary and genetic alterations can increase healthy life span and prevent the development of dysregulated systems and the aging phenotype in laboratory model organisms. The mechanisms responsible for life span expansion are “food” sensors typically activated in situations of food shortage, such as IGF/insulin and the TOR (target of rapamycin) pathways. Accordingly, a reduction in food intake without malnutrition extends the life span by 10–50% in diverse organisms, from yeasts to rhesus monkeys. Mechanisms that mediate the effects of caloric restriction are under intensive study because they are potential targets for interventions aimed at counteracting the emergence of the aging phenotype

1	Mechanisms that mediate the effects of caloric restriction are under intensive study because they are potential targets for interventions aimed at counteracting the emergence of the aging phenotype and its deleterious effects in humans. For example, resveratrol, a natural compound found in grape skin that mimics some of the effects of dietary restriction, increases longevity and improves health in mice fed a high-fat diet but has little effect on mice fed a standard diet. Other compounds that potentially mimic caloric restriction are being developed and tested. A high prevalence of IGF-1 receptor gene mutation has been found in Ashkenazi Jewish centenarians and in other long-lived individuals, suggesting that the downregulation of IGF-1 signaling may promote human longevity. A 20-year period of 30% dietary restriction applied to adult rhesus monkeys was associated with reduced cardiovascular and cancer morbidity, reduced signs of aging, and greater longevity, although a second such

1	of 30% dietary restriction applied to adult rhesus monkeys was associated with reduced cardiovascular and cancer morbidity, reduced signs of aging, and greater longevity, although a second such study did not find increased longevity. In humans, dietary restriction is effective against obesity and reduces insulin resistance, inflammation, blood pressure, CRP level, and intima-media thickness of the carotid arteries. However, the beneficial effects of dietary restriction in humans are still controversial, and some potential negative effects have not been sufficiently studied. An interesting effect of caloric restriction in humans is mitochondrial biogenesis. Mitochondrial dysfunction has emerged as a potentially important underlying contributor to aging. Reduced expression of mitochondrial genes is a strongly conserved feature of aging across different species. Mitochondria are the machinery for chemical energy production, and brain and muscle are particularly susceptible to defective

1	genes is a strongly conserved feature of aging across different species. Mitochondria are the machinery for chemical energy production, and brain and muscle are particularly susceptible to defective mitochondrial function. Thus, declining mitochondrial function may be a direct cause of at least three of the main dysregulated systems contributing to the phenotype of aging.

1	This chapter has touched on some of the fundamental aspects of human aging, focusing mostly on those that are relevant to the care of older patients. Many aspects of geriatric medicine have not been addressed because of space limitations. Valuable topics not considered include details of comprehensive geriatric assessment, depression and anxiety, hypertension, orthostatic hypotension, dementia, vision and hearing impairment, osteoporosis, palliative care, prostate disorders, foot problems, and women’s health. Some of these topics are treated extensively elsewhere in this text, sometimes with comments on age-specific issues. The universal process of aging is becoming better understood. There appear to be shared underlying cellular and molecular processes that induce widespread dysregulation in key systems. This dysregulation contributes to clinical manifestations of a frailty phenotype and can be used to understand how to evaluate and manage the older patient.

1	We would like to thank our colleagues who provided criticisms and suggestions for improvement of this chapter. We are particularly indebted to Dr. John Morley for his valuable suggestions regarding the section on undernutrition and anorexia. Chapter 11 Clinical Problems of Aging Factors that Increase the Likelihood of Errors Many factors ubiquitous 12e-1 The Safety and Quality of Health in health care systems can increase the likelihood of errors, including fatigue, stress, interruptions, complexity, and transitions. The effects of Care fatigue in other industries are clear, but its effects in health care have David W. Bates

1	Care fatigue in other industries are clear, but its effects in health care have David W. Bates Safety and quality are two of the central dimensions of health care. In recent years it has become easier to measure safety and quality, and it is increasingly clear that performance in both dimensions could be much better. The public is—with good justification—demanding measurement and accountability, and payment for services will increasingly be based on performance in these areas. Thus, physicians must learn about these two domains, how they can be improved, and the relative strengths and limitations of the current ability to measure them.

1	Safety and quality are closely related but do not completely overlap. The Institute of Medicine has suggested in a seminal series of reports that safety is the first part of quality and that the health care system must first and foremost guarantee that it will deliver safe care, although quality is also pivotal. In the end, it is likely that more net clinical benefit will be derived from improving quality than from improving safety, though both are important and safety is in many ways more tangible to the public. The first section of this chapter will address issues relating to the safety of care and the second will cover quality of care.

1	SAFETY IN HEALTH CARE Safety Theory and Systems Theory Safety theory clearly points out that individuals make errors all the time. Think of driving home from the hospital: you intend to stop and pick up a quart of milk on the way home but find yourself entering your driveway without realizing how you got there. Everybody uses low-level, semiautomatic behavior for many activities in daily life; this kind of error is called a slip. Slips occur often during care delivery—e.g., when people intend to write an order but forget because they have to complete another action first. Mistakes, by contrast, are errors of a higher level; they occur in new or nonstereotypic situations in which conscious decisions are being made. An example would be dosing of a medication with which a physician is not familiar. The strategies used to prevent slips and mistakes are often different.

1	Systems theory suggests that most accidents occur as the result of a series of small failures that happen to line up in an individual instance so that an accident can occur (Fig. 12e-1). It also suggests that most individuals in an industry such as health care are trying to do the right thing (e.g., deliver safe care) and that most accidents thus can be seen as resulting from defects in systems. Systems should be designed both to make errors less likely and to identify those that do inevitably occur. Hazards Some holes due to active failures Other holes due to latent conditions (resident "pathogens") Successive layers of defenses, barriers and safeguards

1	FIguRE 12e-1 “Swiss cheese” diagram. Reason argues that most accidents occur when a series of “latent failures” are present in a system and happen to line up in a given instance, resulting in an accident. Examples of latent failures in the case of a fall might be that the unit is unusually busy and the floor happens to be wet. (Adapted from J Reason: BMJ 320:768, 2000; with permission.) been more controversial until recently. For example, the accident rate among truck drivers increases dramatically if they work over a certain number of hours in a week, especially with prolonged shifts. A recent study of house officers in the intensive care unit demonstrated that they were about one-third more likely to make errors when they were on a 24-h shift than when they were on a schedule that allowed them to sleep 8 h the previous night. The American College of Graduate Medical Education has moved to address this issue by putting in place the 80-h workweek. Although this stipulation is a step

1	them to sleep 8 h the previous night. The American College of Graduate Medical Education has moved to address this issue by putting in place the 80-h workweek. Although this stipulation is a step forward, it does not address the most important cause of fatigue-related errors: extended-duty shifts. High levels of stress and heavy workloads also can increase error rates. Thus, in extremely high-pressure situations, such as cardiac arrests, errors are more likely to occur. Strategies such as using protocols in these settings can be helpful, as can simple recognition that the situation is stressful.

1	Interruptions also increase the likelihood of error and occur frequently in health care delivery. It is common to forget to complete an action when one is interrupted partway through it by a page, for example. Approaches that may be helpful in this area include minimizing interruptions and setting up tools that help define the urgency of an interruption. Complexity represents a key issue that contributes to errors. Providers are confronted by streams of data (e.g., laboratory tests and vital signs), many of which provide little useful information but some of which are important and require action or suggest a specific diagnosis. Tools that emphasize specific abnormalities or combinations of abnormalities may be helpful in this area.

1	Transitions between providers and settings are also common in health care, especially with the advent of the 80-h workweek, and generally represent points of vulnerability. Tools that provide structure in exchanging information—for example, when transferring care between providers—may be helpful.

1	The Frequency of Adverse Events in Health Care Most large studies focusing on the frequency and consequences of adverse events have been performed in the inpatient setting; some data are available for nursing homes, but much less information is available about the outpatient setting. The Harvard Medical Practice Study, one of the largest studies to address this issue, was performed with hospitalized patients in New York. The primary outcome was the adverse event: an injury caused by medical management rather than by the patient’s underlying disease. In this study, an event either resulted in death or disability at discharge or prolonged the length of hospital stay by at least 2 days. Key findings were that the adverse event rate was 3.7% and that 58% of the adverse events were considered preventable. Although New York is not representative of the United States as a whole, the study was replicated later in Colorado and Utah, where the rates were essentially similar. Since then, other

1	preventable. Although New York is not representative of the United States as a whole, the study was replicated later in Colorado and Utah, where the rates were essentially similar. Since then, other studies using analogous methodologies have been performed in various developed nations, and the rates of adverse events in these countries appear to be ~10%. Rates of safety issues appear to be even higher in developing and transitional countries; thus, this is clearly an issue of global proportions. The World Health Organization has focused on this area, forming the World Alliance for Patient Safety.

1	In the Harvard Medical Practice Study, adverse drug events (ADEs) were most common, accounting for 19% of all adverse events, and were followed in frequency by wound infections (14%) and technical complications (13%). Almost half of adverse events were associated with a surgical procedure. Among nonoperative events, 37% were ADEs, 15% were diagnostic mishaps, 14% were therapeutic mishaps, 13% were procedure-related mishaps, and 5% were falls. ADEs have been studied more than any other error category. Studies focusing specifically on ADEs have found that they appear to be much more common than was suggested by the Harvard Medical Practice Study, although most other studies use more inclusive criteria. Detection approaches in the research setting include chart review and

1	CHAPTER 12e The Safety and Quality of Health Care the use of a computerized ADE monitor, a tool that explores the database and identifies signals that suggest an ADE may have occurred. Studies that use multiple approaches find more ADEs than does any individual approach, and this discrepancy suggests that the true underlying rate in the population is higher than would be identified by a single approach. About 6–10% of patients admitted to U.S. hospitals experience an ADE. Injuries caused by drugs are also common in the outpatient setting. One study found a rate of 21 ADEs per every 100 patients per year when patients were called to assess whether they had had a problem with one of their medications. The severity level was lower than in the inpatient setting, but approximately one-third of these ADEs were preventable.

1	The period immediately after a patient is discharged from the hospital appears to be very risky. A recent study of patients hospitalized on a medical service found an adverse event rate of 19%; about one-third of those events were preventable, and another one-third were ameliorable (i.e., they could have been made less severe). ADEs were the single leading error category. Prevention Strategies Most work on strategies to prevent adverse events has targeted specific types of events in the inpatient setting, with nosocomial infections and ADEs having received the most attention. Nosocomial infection rates have been reduced greatly in intensive care settings, especially through the use of checklists. For ADEs, several strategies have been found to reduce the medication error rate, although it has been harder to demonstrate that they reduce the ADE rate overall, and no studies with adequate power to show a clinically meaningful reduction have been published.

1	Implementation of checklists to ensure that specific actions are carried out has had a major impact on rates of catheter-associated bloodstream infection and ventilator-associated pneumonia, two of the most serious complications occurring in intensive care units. The checklist concept is based on the premise that several specific actions can reduce the frequency of these issues; when these actions are all taken for every patient, the result has been an extreme reduction in the frequency of the associated complication. These practices have been disseminated across wide areas, in particular in the state of Michigan.

1	Computerized physician order entry (CPOE) linked with clinical decision support reduces the rate of serious medication errors, defined as those that harm someone or have the potential to do so. In one study, CPOE, even with limited decision support, decreased the serious medication error rate by 55%. CPOE can prevent medication errors by suggesting a default dose, ensuring that all orders are complete (e.g., that they include dose, route, and frequency), and checking orders for allergies, drug–drug interactions, and drug–laboratory issues. In addition, clinical decision support can suggest the right dose for a patient, tailoring it to level of renal function and age. In one study, patients with renal insufficiency received the appropriate dose only one-third of the time without decision support, whereas that fraction increased to approximately two-thirds with decision support; moreover, with such support, patients with renal insufficiency were discharged from the hospital half a day

1	support, whereas that fraction increased to approximately two-thirds with decision support; moreover, with such support, patients with renal insufficiency were discharged from the hospital half a day earlier. As of 2009, only ~15% of U.S. hospitals had implemented CPOE, but many plan to do so and will receive major financial incentives for achieving this goal.

1	Another technology that can improve medication safety is bar coding linked with an electronic medication administration record. Bar coding can help ensure that the right patient gets the right medication at the right time. Electronic medication administration records can make it much easier to determine what medications a patient has received. Studies to assess the impact of bar coding on medication safety are under way, and the early results are promising. Another technology to improve medication safety is “smart pumps.” These pumps can be set according to which medication is being given and at what dose; the health care professional will receive a warning if too high a dose is about to be administered.

1	The National Safety Picture Several organizations, including the National Quality Forum and the Joint Commission, have made recommendations for improving safety. In particular, the National Quality Forum has released recommendations to U.S. hospitals about what practices will most improve the safety of care, and all hospitals are expected to implement these recommendations. Many of these practices arise frequently in routine care. One example is “readback,” the practice of recording all verbal orders and immediately reading them back to the physician to verify the accuracy of what was heard. Another is the consistent use of standard abbreviations and dose designations; some abbreviations and dose designations are particularly prone to error (e.g., 7U may be read as 70).

1	Measurement of Safety Measuring the safety of care is difficult and expensive, since adverse events are, fortunately, rare. Most hospitals rely on spontaneous reporting to identify errors and adverse events, but the sensitivity of this approach is very low, with only ~1 in 20 ADEs reported. Promising research techniques involve searching the electronic record for signals suggesting that an adverse event has occurred. These methods are not yet in wide use but will probably be used routinely in the future. Claims data have been used to identify the frequency of adverse events; this approach works much better for surgical care than for medical care and requires additional validation. The net result is that, except for a few specific types of events (e.g., falls and nosocomial infections), hospitals have little idea about the true frequency of safety issues.

1	Nonetheless, all providers have the responsibility to report problems with safety as they are identified. All hospitals have spontaneous reporting systems, and, if providers report events as they occur, those events can serve as lessons for subsequent improvement.

1	Conclusions about Safety It is abundantly clear that the safety of health care can be improved substantially. As more areas are studied closely, more problems are identified. Much more is known about the epidemiology of safety in the inpatient setting than in outpatient settings. A number of effective strategies for improving inpatient safety have been identified and are increasingly being applied. Some effective strategies are also available for the outpatient setting. Transitions appear to be especially risky. The solutions to improving care often entails the consistent use of systematic techniques such as checklists and often involves leveraging of information technology. Nevertheless, solutions will also include many other domains, such human factors techniques, team training, and a culture of safety.

1	Assessment of quality of care has remained somewhat elusive, although the tools for this purpose have increasingly improved. Selection of health care and measurement of its quality are components of a complex process.

1	Quality Theory Donabedian has suggested that quality of care can be categorized by type of measurement into structure, process, and outcome. Structure refers to whether a particular characteristic is applicable in a particular setting—e.g., whether a hospital has a catheterization laboratory or whether a clinic uses an electronic health record. Process refers to the way care is delivered; examples of process measures are whether a Pap smear was performed at the recommended interval or whether an aspirin was given to a patient with a suspected myocardial infarction. Outcome refers to what actually happens—e.g., the mortality rate in myocardial infarction. It is important to note that good structure and process do not always result in a good outcome. For instance, a patient may present with a suspected myocardial infarction to an institution with a catheterization laboratory and receive recommended care, including aspirin, but still die because of the infarction.

1	Quality theory also suggests that overall quality will be improved more in the aggregate if the performance level of all providers is raised rather than if a few poor performers are identified and punished. This view suggests that systems changes are especially likely to be helpful in improving quality, since large numbers of providers may be affected simultaneously. The theory of continuous quality improvement suggests that organizations should be evaluating the care they deliver on an ongoing basis and continually making small changes to improve their individual processes. This approach can be very powerful if embraced over time. and many believe that they will prove to be a key to improving quality, 12e-3 especially if pay-for-performance with sufficient incentives is broadly implemented (see below). Penalties produce provider resentment and are rarely used in health care.

1	Another set of strategies for improving quality involves changing the systems of care. An example would be introducing reminders about which specific actions needed to be taken at a visit for a specific patient—a strategy that has been demonstrated to improve performance in certain situations, such as the delivery of preventive services. Another approach that has been effective is the development of “bundles” or groups of quality measures that can be implemented together with a high degree of fidelity. A number of hospitals have CHAPTER 12e The Safety and Quality of Health Care implemented a bundle for ventilator-associated pneumonia in the FIguRE 12e-2 Plan-Do-Check-Act cycle. This approach can be used to improve a specific process rapidly. First, planning is undertaken, intensive care unit that includes five measures (e.g., ensuring that the head of the bed is elevated). These hospitals have been able to improve performance substantially.

1	Perhaps the most pressing need is to improve the quality of care and several potential improvement strategies are identified. Next, these strategies are evaluated in small “tests of change.” “Checking” for chronic diseases. The Chronic Care Model has been developed by Wagner and colleagues (Fig. 12e-3); it suggests that a combination of entails measuring whether the strategies have appeared to make a strategies is necessary (including self-management support, changes difference, and “acting” refers to acting on the results. A number of specific tools have been developed to help improve in delivery system design, decision support, and information systems) and that these strategies must be delivered by a practice team composed of several providers, not just a physician. Available evidence about the relative efficacy of strategies in reduc process performance. One of the most important is the Plan-Do

1	Available evidence about the relative efficacy of strategies in reduc process performance. One of the most important is the Plan-Do Check-Act cycle (Fig. 12e-2). This approach can be used for “rapid cycle” improvement of a process—e.g., the time that elapses between a diagnosis of pneumonia and administration of antibiotics to the patient. Specific statistical tools, such as control charts, are often used in conjunction to determine whether progress is being made. Because most medical care includes one or many processes, this tool is espe cially important for improvement.

1	Factors Relating to Quality Many factors can decrease the level of quality, including stress to providers, high or low levels of produc tion pressure, and poor systems. Stress can have an adverse effect on quality because it can lead providers to omit important steps, as can a high level of production pressure. Low levels of production pressure sometimes can result in worse quality, as providers may be bored or have little experience with a specific problem. Poor systems can have a tremendous impact on quality, and even extremely dedicated providers typically cannot achieve high levels of performance if they are operating within a poor system. Data about the Current State of Quality A study published by the RAND Corporation in 2006 provided the most complete picture of quality of care delivered in the United States to date. The results were sobering. this general premise. It is especially notable that the outcome was the

1	this general premise. It is especially notable that the outcome was the HbA1c level, as it has generally been much more difficult to improve outcome measures than process measures (such as whether HbA1c was measured). In this meta-analysis, a variety of strategies were effective, but the most effective ones were the use of team changes and the use of a case manager. When cost-effectiveness is considered in addition, it appears likely that an amalgam of strategies will be needed. However, the more expensive strategies, such as the use of case managers, probably will be implemented widely only if pay-for performance takes hold.

1	National State of Quality Measurement In the inpatient setting, quality measurement is now being performed by a very large proportion of hospitals for several conditions, including myocardial infarction, congestive heart failure, pneumonia, and surgical infection prevention; 20 measures are included in all. This is the result of the Hospital Quality Initiative, which represents a collaboration among many entities, Productive interactions Informed, activated patient Prepared, proactive practice team Improved Outcomes Self-management Support Delivery system design Decision support Clinical information systems Community Resources and policies Health System Organization of health care

1	The authors found that, across a wide range of quality parameters, patients in the United States received only 55% of recommended care overall; there was little variation by subtype, with scores of 54% for preventive care, 54% for acute care, and 56% for care of chronic conditions. The authors concluded that, in broad terms, the chances of getting high-quality care in the United States were little better than those of winning a coin flip. Work from the Dartmouth Atlas of Health Care evaluating geographic variation in use and quality of care demonstrates that, despite large variations in utilization, there is no positive correlation between the two variables at the regional level. An array of data demonstrate, however, that providers with larger volumes for specific conditions, especially for surgical conditions, do have better outcomes.

1	Strategies for Improving Quality and Performance A number of specific strategies can be used to improve quality at the individual level, including rationing, education, feedback, incentives, and penalties. Rationing has been effective in some specific areas, such as persuading physicians to prescribe within a formulary, but it generally has been resisted.

1	Education is effective in the short run and is necessary for changing FIguRE 12e-3 The Chronic Care Model, which focuses on improvopinions, but its effect decays fairly rapidly with time. Feedback on ing care for chronic diseases, suggests that (1) delivery of high-quality performance can be given at either the group or the individual level. care requires a range of strategies that must closely involve and Feedback is most effective if it is individualized and is given in close engage the patient and (2) team care is essential. (From EH Wagner et al: temporal proximity to the original events. Incentives can be effective, Eff Clin Pract 1:2, 1998.) including the Hospital Quality Alliance, the Joint Commission, the National Quality Forum, and the Agency for Healthcare Research and Quality. The data are housed at the Center for Medicare and Medicaid Services, which publicly releases performance data on the measures on a website called Hospital Compare

1	Healthcare Research and Quality. The data are housed at the Center for Medicare and Medicaid Services, which publicly releases performance data on the measures on a website called Hospital Compare (www.cms.gov/Medicare/QualityInitiatives-Patient-Assessment-Instruments/HospitalQualityInits/ HospitalCompare.html). These data are reported voluntarily and are available for a very high proportion of the nation’s hospitals. Analyses demonstrate substantial regional variation in quality and important differences among hospitals. Analyses by the Joint Commission for similar indicators reveal that performance on measures by hospitals has improved over time and that, as might be hoped, lower performers have improved more than higher performers.

1	Public Reporting Overall, public reporting of quality data is becoming increasingly common. There are now commercial websites that have quality-related data for most regions of the United States, and these data can be accessed for a fee. Similarly, national data for hospitals are available. The evidence to date indicates that patients have not made much use of such data but that the data have had an important effect on provider and organization behavior. Instead, patients have relied on provider reputation to make choices, partly because little information was available until very recently and the information that was available was not necessarily presented in ways that were easy for patients to access. Many authorities think that, as more information about quality becomes available, it will become increasingly central to patients’ choices about where to access care.

1	Pay-for-Performance Currently, providers in the United States get paid exactly the same amount for a specific service, regardless of the quality of care delivered. The pay-for-performance theory suggests that, if providers are paid more for higher-quality care, they will invest in strategies that enable them to deliver that care. The current key issues in the pay-for-performance debate relate to (1) how effective it is, (2) what levels of incentives are needed, and (3) what perverse consequences are produced. The evidence on effectiveness is fairly limited, although a number of studies are ongoing. With respect to incentive levels, most quality-based performance incentives have accounted for merely 1–2% of total payment in the United States to date. In the United Kingdom, however, 40% of general practitioners’ salaries have been placed at risk according to performance across a wide array of parameters; this approach has been associated with substantial improvements in reported quality

1	practitioners’ salaries have been placed at risk according to performance across a wide array of parameters; this approach has been associated with substantial improvements in reported quality performance, although it is still unclear to what extent this change represents better performance versus better reporting. The potential for perverse consequences exists with any incentive scheme. One problem is that, if incentives are tied to outcomes, there may be a tendency to transfer the sickest patients to other providers and systems. Another concern is that providers will pay too much attention to quality measures with incentives and ignore the rest of the quality picture. The validity of these concerns remains to be determined. Nonetheless, it appears likely that, under health care reform, the use of various pay-for-performance schemes is likely to increase.

1	The safety and quality of care in the United States could be improved substantially. A number of available interventions have been shown to improve the safety of care and should be used more widely; others are undergoing evaluation or soon will be. Quality also could be dramatically better, and the science of quality improvement continues to mature. Implementation of pay-for-performance should make it much easier for organizations to justify investments in improving safety and quality parameters, including health information technology. However, many improvements will also require changing the structure of care—e.g., moving to a more team-oriented approach and ensuring that patients are more involved in their own care. Health care reform is likely to result in increased use of pay-for-performance. Measures of safety are still relatively immature and could be made much more robust; it would be particularly useful if organizations had measures they could use in routine operations to

1	Measures of safety are still relatively immature and could be made much more robust; it would be particularly useful if organizations had measures they could use in routine operations to assess safety at a reasonable cost. Although the quality measures available are more robust than those for safety, they still cover a relatively small proportion of the entire domain of quality, and more measures need to be developed. The public and payers are demanding better information about safety and quality as well as better performance in these areas. The clear implication is that these domains will have to be addressed directly by providers.

1	Primary Care in Lowand Middle-Income Countries Tim Evans, Kumanan Rasanathan The twentieth century witnessed the rise of an unprecedented global health divide. Industrialized or high-income countries experienced 13e rapid improvement in standards of living, nutrition, health, and health care. Meanwhile, in lowand middle-income countries with much less favorable conditions, health and health care progressed much more slowly. The scale of this divide is reflected in the current extremes of life expectancy at birth, with Japan at the high end (83 years) and Sierra Leone at the low end (47 years). This nearly 40-year difference reflects the daunting range of health challenges faced by lowand middle-income countries. These nations must deal not only with a complex mixture of diseases (both infectious and chronic) and illness-promoting conditions but also, and more fundamentally, with the fragility of the foundations underlying good health (e.g., sufficient food, water, sanitation, and

1	infectious and chronic) and illness-promoting conditions but also, and more fundamentally, with the fragility of the foundations underlying good health (e.g., sufficient food, water, sanitation, and education) and of the systems necessary for universal access to good-quality health care. In the last decades of the twentieth century, the need to bridge this global health divide and establish health equity was increasingly recognized. The Declaration of Alma Ata in 1978 crystallized a vision of justice in health, regardless of income, gender, ethnicity, or education, and called for “health for all by the year 2000” through primary health care. While much progress has been made since the declaration, at the end of the first decade and a half of the twenty-first century, much remains to be done to achieve global health equity.

1	This chapter looks first at the nature of the health challenges in low-and middle-income countries that underlie the health divide. It then outlines the values and principles of a primary health care approach, with a focus on primary care services. Next, the chapter reviews the experience of lowand middle-income countries in addressing health challenges through primary care and a primary health care approach. Finally, the chapter identifies how current challenges and global context provide an agenda and opportunities for the renewal of primary health care and primary care.

1	The term primary care has been used in many different ways: to describe a level of care or the setting of the health system, a set of treatment and prevention activities carried out by specific personnel, a set of attributes for the way care is delivered, or an approach to organizing health systems that is synonymous with the term primary health care. In 1996, the U.S. Institute of Medicine encompassed many of these different usages, defining primary care as “the provision of integrated, accessible health care services by clinicians who are accountable for addressing a large majority of personal health care needs, developing a sustained partnership with patients, and practicing in the context of family and community.”1 We use this definition of primary care in this chapter. Primary care performs an essential function for health systems, providing the first point of contact when people seek health care, dealing with most problems, and referring patients onward to other services when

1	performs an essential function for health systems, providing the first point of contact when people seek health care, dealing with most problems, and referring patients onward to other services when necessary. As is increasingly evident in countries of all income levels, without strong primary care, health systems cannot function properly or address the health challenges of the communities they serve.

1	Primary care is only one part of a primary health care approach. The Declaration of Alma Ata, drafted in 1978 at the International Conference on Primary Health Care in Alma Ata (now Almaty in Kazakhstan), identified many features of primary care as being essential to achieving the goal of “health for all by the year 2000.” However, it also identified the need to work across different sectors, address 1Institute of Medicine. Primary Care: America’s Health in a New Era (1996).

1	the social and economic factors that determine health, mobilize the 13e-1 participation of communities in health systems, and ensure the use and development of technology that was appropriate in terms of setting and cost. The declaration drew from the experiences of lowand middle-income countries in trying to improve the health of their people following independence. Commonly, these countries had built hospital-based systems similar to those in high-income countries. This effort had resulted in the development of high-technology services in urban areas while leaving the bulk of the population without access to health care unless they traveled great distances to these urban facilities. Furthermore, much of the population lacked access to basic public health measures. Primary health care efforts aimed to move care closer to where people lived, to ensure their involvement in decisions about their own health care, and to address key aspects of the physical and social environment essential

1	aimed to move care closer to where people lived, to ensure their involvement in decisions about their own health care, and to address key aspects of the physical and social environment essential to health, such as water, sanitation, and education.

1	After the Declaration of Alma Ata, many countries implemented reforms of their health systems based on primary health care. Most progress involved strengthening of primary care services; unexpectedly, however, much of this progress was seen in high-income countries, most of which constructed systems that made primary care available at low or no cost to their entire populations and that delivered the bulk of services in primary care settings. This endeavor also saw the reinforcement of family medicine as a specialty to provide primary care services. Even in the United States (an obvious exception to this trend), it became clear that the populations of states with more primary care physicians and services were healthier than those with fewer such resources.

1	Progress was also made in many lowand middle-income countries. However, the target of “health for all by the year 2000” was missed by a large margin. The reasons were complex but partly entailed a general failure to implement all aspects of the primary health care approach, particularly work across sectors to address social and economic factors that affect health and provision of sufficient human and other resources to make possible the access to primary care attained in high-income countries. Furthermore, despite the consensus in Alma Ata in 1978, the global health community rapidly became fractured in its commitment to the far-reaching measures called for by the declaration. Economic recession tempered enthusiasm for primary health care, and momentum shifted to programs concentrating on a few priority measures such as immunization, oral rehydration, breastfeeding, and growth monitoring for child survival. Success with these initiatives supported the continued movement of health

1	on a few priority measures such as immunization, oral rehydration, breastfeeding, and growth monitoring for child survival. Success with these initiatives supported the continued movement of health development efforts away from the comprehensive approach of primary health care and toward programs that targeted specific public health priorities. This approach was reinforced by the need to address the HIV/AIDS epidemic. By the 1990s, primary health care had fallen out of favor in many global-health policy circles, and lowand middle-income countries were being encouraged to reduce public sector spending on health and to focus on cost-effectiveness analysis to provide a package of health care measures thought to offer the greatest health benefits.

1	Lowand middle-income countries, defined by a per capita gross national income of <$12,476 (U.S.) per person per year, account for >80% of the world’s population. Average life expectancy in these countries lags far behind that in high-income countries: whereas the average life expectancy at birth in high-income countries is 74 years, it is only 68 years in middle-income countries and 58 years in low-income countries. This discrepancy has received growing attention over the past 40 years. Initially, the situation in poor countries was characterized primarily in terms of high fertility and high infant, child, and maternal mortality rates, with most deaths and illnesses attributable to infectious or tropical diseases among remote, largely rural populations. With growing adult (and especially elderly) populations and changing lifestyles linked to global forces of urbanization, a new set of health challenges characterized by chronic diseases, environmental overcrowding, and road traffic

1	elderly) populations and changing lifestyles linked to global forces of urbanization, a new set of health challenges characterized by chronic diseases, environmental overcrowding, and road traffic injuries has emerged rapidly 35 30 25 20 15 10 5 0 Deaths (millions) Intentional injuries Other unintentional injuries Road traffic accidents Other noncommunicable diseases Cancers Cardiovascular disease Maternal, perinatal, and nutritional conditions Other infectious diseases HIV/AIDS, TB, and malaria

1	FIGURE 13e-1 Projections of disease burden to 2030 for high-, middle-, and low-income countries (left, center, and right, respectively). TB, tuberculosis. (Source: World Health Organization: The Global Burden of Disease 2004 Update, 2008.) (Fig. 13e-1). The majority of tobacco-related deaths globally now occur settings such as Cuba and Kerala State in India. Analyses conducted in lowand middle-income countries, and the risk of a child’s dying over the past three decades indeed show that rapid health improve-from a road traffic injury in Africa is more than twice that in Europe. ment is possible in very different contexts. That some countries Hence, lowand middle-income countries in the twenty-first century continue to lag far behind can be understood through a comparison face a full spectrum of health challenges—infectious, chronic, and of regional differences in progress in terms of life expectancy over injury-related—at much higher incidences and prevalences than are this period

1	of health challenges—infectious, chronic, and of regional differences in progress in terms of life expectancy over injury-related—at much higher incidences and prevalences than are this period (Fig. 13e-3). While most regions have made impressive documented in high-income countries and with many fewer resources to address these challenges.

1	Addressing these challenges, however, does 85 2005 not mean simply waiting for economic growth. Analysis of the association between wealth and health across countries reveals that, for any given level of wealth, there is a substantial variation in life expectancy at birth that has persisted despite overall global progress in life expectancy during the past 30 years (Fig. 13e-2). Health status in low-and middle-income countries varies enormously. Nations such as Cuba and Costa Rica have life expectancies and childhood mortality rates similar to or even better than those in high-income countries; in contrast, countries in sub-Saharan Africa and the former Soviet bloc have experienced significant reverses in these health markers in the past 20 years.

1	As Angus Deaton stated in the World Institute for Development Economics Research annual lec ture on September 29, 2006, “People in poor 35 countries are sick not primarily because they are 0 5,000 10,000 15,000 20,000 25,000 30,000 35,000 40,000 poor but because of other social organizational GDP per capita, constant 2000 international $ failures, including health delivery, which are not automatically ameliorated by higher income.” This FIGURE 13e-2 Gross domestic product (GDP) per capita and life expectancy at birth analysis concurs with classic studies of the array in 169 countries, 1975 and 2005. Only outlying countries are named. (Source: World of societal factors explaining good health in poor Health Organization: Primary Health Care: Now More Than Ever. World Health Report 2008.) 52.1 66.9 60.5East Asia and Pacific 70.4 61.1 Caribbean 71.7 50.1 63.2 45.8 46.1 68.1 71.6 78.8

1	FIGURE 13e-3 Regional trends in life expectancy. CEE and CIS, Central and Eastern Europe and the Commonwealth of Independent States; OECD, Organization for Economic Co-operation and Development. (Source: World Health Organization: Closing the Gap in a Generation: Health Equity Through Action on the Social Determinants of Health. Commission on Social Determinants of Health Final Report, 2008.) progress, sub-Saharan Africa and the former Soviet states have seen stagnation and even reversals.

1	As average levels of health vary across regions and countries, so too do they vary within countries (Fig. 13e-4). Indeed, disparities within countries are often greater than those between high-income and low-income countries. For example, if lowand middle-income countries could reduce their overall childhood mortality rate to that of the richest one-fifth of their populations, global childhood mortality could be decreased by 40%. Disparities in health are mostly a result of social and economic factors such as daily living conditions, access to resources, and ability to participate in life-affecting decisions. In most countries, the health care sector actually tends to exacerbate health inequalities (the “inverse-care law”); because of neglect and discrimination, poor and marginalized communities are much less likely to benefit from public health services than those that are better off. Reforming health systems toward people-centered primary care provides an opportunity to reverse

1	communities are much less likely to benefit from public health services than those that are better off. Reforming health systems toward people-centered primary care provides an opportunity to reverse these negative trends.

1	Health services have failed to make their contribution to reducing these pervasive social inequalities by ensuring universal access to existing, scientifically validated, low-cost interventions such as insecticide-treated bed nets for malaria, taxes on cigarettes, short-course chemotherapy for tuberculosis, antibiotic treatment for pneumonia, 13e-3 dietary modification and secondary prevention measures for high blood pressure and high cholesterol levels, and water treatment and oral rehydration therapy for diarrhea. Despite decades of “essential packages” and “basic” health campaigns, the effective implementation of what is already known to work appears (deceptively) to be difficult.

1	Recent analyses have begun to focus on “the how” (as opposed to “the what”) of health care delivery, exploring why health progress is slow and sluggish despite the abundant availability of proven interventions for health conditions in lowand middle-income countries. Three general categories of reasons are being identified: (1) shortfalls in performance of health systems; (2) stratifying social conditions; and (3) skews in science. Specific health problems often require the development of specific health interventions (e.g., tuberculosis requires short-course chemotherapy). However, the delivery of different interventions is often facilitated by a common set of resources or functions: money or financing, trained health workers, and facilities with reliable supplies fit for multiple purposes. Unfortunately, health systems in most low-and middle-income countries are largely dysfunctional at present.

1	In the large majority of lowand middle-income countries, the level of public financing for health is woefully insufficient: whereas high-income countries spend, on average, 7% of the gross domestic product on health, middle-income countries spend <4% and low-income countries <3%. External financing for health through various donor channels has grown significantly over time. While these funds for health are significant (~$20 billion [U.S.] in 2008 for lowand middle-income countries), they represent <2% of total health expenditures in low-and middle-income countries and hence are neither a sufficient nor a long-term solution to chronic underfinancing. In Africa, 70% of health expenditures come from domestic sources. The predominant form of health care financing—charging patients at the point of service—is the least efficient and the most inequitable, tipping millions of households into poverty annually.

1	Health workers, who represent another critical resource, are often inadequately trained and supported in their work. Recent estimates indicate a shortage of >4 million health workers, constituting a crisis that is greatly exacerbated by the migration of health workers from lowand middle-income countries to high-income countries. Sub-Saharan Africa carries 24% of the global disease burden but has only 3% of the health workforce (Fig. 13e-5). The International Organization for Migration estimated in 2006 that there were more Ethiopian physicians practicing in Chicago than in Ethiopia itself. FIGURE 13e-4 A. Mortality of children under 5 years old, by place of residence, in five countries. (Source: Data from the World Health Organization.) B. Full basic immunization coverage (%), by income group. (Source: Primary Health Care: Now More Than Ever. World Health Report 2008.) % of global burden of disease % of global workforce FIGURE 13e-5 Global burden of disease and health workforce.

1	FIGURE 13e-5 Global burden of disease and health workforce. (Source: World Health Organization: Working Together for Health, 2006.) Critical diagnostics and drugs often do not reach patients in need because of supply-chain failures. Moreover, facilities fail to provide safe care: new evidence suggests much higher rates of adverse events among hospitalized patients in lowand middle-income countries than in high-income countries. Weak government planning, regulatory, monitoring, and evaluation capacities are associated with rampant, unregulated commercialization of health services and chaotic fragmentation of these services as donors “push” their respective priority programs. With such fragile foundations, it is not surprising that low-cost, affordable, validated interventions are not reaching those who need them.

1	Health care delivery systems do not exist in a vacuum but rather are embedded in a complex of social and economic forces that often stratify opportunities for health unfairly. Most worrisome are the pervasive forces of social inequality that serve to marginalize populations with disproportionately large health needs (e.g., the urban poor; illiterate mothers). Why should a poor slum dweller with no income be expected to come up with the money for a bus fare needed to travel to a clinic to learn the results of a sputum test for tuberculosis? How can a mother living in a remote rural village and caring for an infant with febrile convulsions find the means to get her child to appropriate care? Shaky or nonexistent social security systems, dangerous work environments, isolated communities with little or no infrastructure, and systematic discrimination against minorities are among the myriad forces with which efforts for more equitable health care delivery must contend.

1	While science has yielded enormous breakthroughs in health in high-income countries, with some spillover to lowand middle-income countries, many important health problems continue to affect primarily lowand middle-income countries whose research and development investments are deplorably inadequate. The past decade has seen growing efforts to right this imbalance with research and development investment for new drugs, vaccines, and diagnostics that effectively cater to the specific health needs of populations in lowand middle-income countries. For example, the Medicines for Malaria Venture has revitalized a previously “dry” pipeline for new malaria drugs. This is but one of many such efforts, but much more needs to be done.

1	As discussed above, the primary constraint on better health in lowand middle-income countries is related less to the availability of health technologies and more to their effective delivery. Underlying these systems and social challenges to greater equity in health is a major bias regarding what constitutes legitimate “science” to improve health equity. The lion’s share of health research financing is channeled toward the development of new technologies—drugs, vaccines, and diagnostics; in contrast, virtually no resources are directed toward research on how health care delivery systems can become more reliable and overcome adverse social conditions. The complexity of systems and social context is such that this issue of delivery requires an enormous investment in terms not only of money but also of scientific rigor, with the development of new research methods and measures and the attainment of greater legitimacy in the mainstream scientific establishment.

1	These common challenges to lowand middle-income countries partly explain the resurgence of interest in the primary health care approach. In some countries (mostly middle-income), significant progress has been made in expanding coverage by health systems based on primary care and even in improving indicators of population health. More countries are embarking on the creation of primary care services despite the challenges that exist, particularly in low-income countries. Even when these challenges are acknowledged, there are many reasons for optimism that lowand middle-income countries can accelerate progress in building primary care.

1	The new millennium has seen a resurgence of interest in primary health care as a means of addressing global health challenges. This interest has been driven by many of the same issues that led to the Declaration of Alma Ata: rapidly increasing disparities in health between and within countries, spiraling costs of health care at a time when many people lack quality care, dissatisfaction of communities with the care they are able to access, and failure to address changes in health threats, especially noncommunicable disease epidemics. These challenges require a comprehensive approach and strong health systems with effective primary care. Global health development agencies have recognized that sustaining gains in public health priorities such as HIV/AIDS requires not only robust health systems but also the tackling of social and economic factors related to disease incidence and progression. Weak health systems have proved a major obstacle to delivering new technologies, such as

1	systems but also the tackling of social and economic factors related to disease incidence and progression. Weak health systems have proved a major obstacle to delivering new technologies, such as antiretroviral therapy, to all who need them. Changing disease patterns have led to a demand for health systems that can treat people as individuals whether or not they present to a health facility with the public health “priority” (e.g., HIV/AIDS or tuberculosis) to which that facility is targeted. We discuss experiences in lowand middle-income countries in relation to primary care in greater detail below. First, we consider the features of primary health care and primary care as currently understood.

1	At the 2009 World Health Assembly (an annual meeting of all countries to discuss the work of the World Health Organization [WHO]), a resolution was passed reaffirming the principles of the Declaration of Alma Ata and the need for national health systems to be based on primary health care. This resolution did not suggest that nothing had changed in the intervening 30 years since the declaration, nor did it dispute that its prescription needed reframing in light of changing public health needs. The 2008 WHO World Health Report describes how a primary health care approach is necessary “now more than ever” to address global health priorities, especially in terms of disparities and new health challenges. As discussed below, this report highlights four broad areas in which reform is required (Fig. 13e-6). One of these areas—the need to organize health care so that it places the needs of people first—essentially relates to the necessity for strong primary care in health systems and what this

1	13e-6). One of these areas—the need to organize health care so that it places the needs of people first—essentially relates to the necessity for strong primary care in health systems and what this requirement entails. The other three areas also relate to primary care. All four areas require action to move health systems in a direction that will reduce disparities and increase the satisfaction of those they serve. The World Health Report’s recommendations present a vision of primary health care that is based on the principles of Alma Ata but that differs from many attempts to implement primary health care in the 1970s and 1980s.

1	Universal Coverage Reforms to Improve Health Equity Despite progress in many countries, most people in the world can receive health care services only if they can pay at the point of service. Disparities in health are caused not only by a lack of access to necessary health FIGURE 13e-6 The four reforms of primary health care renewal.

1	(Source: World Health Organization: Primary Health Care: Now More Than Ever. World Health Report 2008.) services but also by the impact of expenditure on health. More than 100 million people are driven into poverty each year by health care costs, with countless others deterred from accessing services at all. Moving toward prepayment financing systems for universal coverage, which ensure access to a comprehensive package of services according to need without precipitating economic ruin, is therefore emerging as a major priority in lowand middle-income countries. Increasing coverage of health services can be considered in terms of three axes: the proportion of the population covered, the range of services underwritten, and the percentage of costs paid (Fig. 13e-7). Moving toward universal coverage requires ensuring the availability of health care services to all, eliminating barriers to access, and organizing pooled financing mechanisms, such as taxation or insurance, to remove user

1	coverage requires ensuring the availability of health care services to all, eliminating barriers to access, and organizing pooled financing mechanisms, such as taxation or insurance, to remove user fees at the point of service. It also requires measures beyond financing, including expansion of health services in poorly served areas, improvement in the quality of services provided to marginalized communities, and increased coverage of other social services that significantly affect health (e.g., education).

1	Service Delivery Reforms to Make Health Systems People-Centered Health systems have often been organized around the needs of those who provide health care services, such as clinicians and policymakers. The result is a centralization of services or the provision of vertical programs that target single diseases. The principles of primary health Extend to uninsured Depth: which benefits Height: what proportion of the costs is covered? Public expenditure on health are covered? Breadth: who is insured? FIGURE 13e-7 Three ways of moving toward universal coverage. care, including the development of primary care, reorient care around 13e-5 the needs of the people to whom services cater. This “people-centered” approach aims to provide health care that is both more effective and appropriate.

1	The increase in noncommunicable diseases in lowand middle-income countries offers a further stimulus for urgent reform of service delivery to improve chronic disease care. As discussed above, large numbers of people currently fail to receive relatively low-cost interventions that have reduced the incidence of these diseases in high-income countries. Delivery of these interventions requires health systems that can address multiple problems and manage people over a long period within their own communities, yet many lowand middle-income countries are only now starting to adapt and build primary care services that can address noncommunicable diseases and communicable diseases requiring chronic care. Even some countries (e.g., Iran) that have had significant success in reducing communicable diseases and improving child survival have been slow to adapt their health systems to rapidly accelerating noncommunicable disease epidemics.

1	People-centered care requires a safe, comprehensive, and integrated response to the needs of those presenting to health systems, with treatment at the first point of contact or referral to appropriate services. Because no discrete boundary separates people’s needs for health promotion, curative interventions, and rehabilitation services across different diseases, primary care services must address all presenting problems in a unified way. Meeting people’s needs also involves improved communication between patients and their clinicians, who must take the time to understand the impact of the patients’ social context on the problems they present with. This enhanced understanding is made possible by improvements in the continuity of care so that responsibility transcends the limited time people spend in health care facilities. Primary care plays a vital role in navigating people through the health system; when people are referred elsewhere for services, primary care providers must monitor

1	spend in health care facilities. Primary care plays a vital role in navigating people through the health system; when people are referred elsewhere for services, primary care providers must monitor the resulting consultations and perform follow-up. All too often, people do not receive the benefit of complex interventions undertaken in hospitals because they lose contact with the health care system once discharged. Comprehensiveness and continuity of care are best achieved by ensuring that people have an ongoing personal relationship with a care team.

1	Public Policy Reforms to Promote and Protect the Health of Communities

1	Public policies in sectors other than health care are essential to reduce disparities in health and to make progress toward global public health targets. The 2008 final report of the WHO Commission on Social Determinants of Health provides an exhaustive review of the inter-sectoral policies required to address health inequities at the local, national, and global levels. Advances against major challenges such as HIV/AIDS, tuberculosis, emerging pandemics, cardiovascular disease, cancers, and injuries require effective collaboration with sectors such as transport, housing, labor, agriculture, urban planning, trade, and energy. While tobacco control provides a striking example of what is possible if different sectors work together toward health goals, the lack of implementation of many evidence-based tobacco control measures in most countries just as clearly illustrates the difficulties encountered in such intersectoral work and the unrealized potential of public policies to improve

1	tobacco control measures in most countries just as clearly illustrates the difficulties encountered in such intersectoral work and the unrealized potential of public policies to improve health. At the local level, primary care services can help enact health-promoting public policies in other sectors.

1	Leadership Reforms to Make Health Authorities More Responsive The Declaration of Alma Ata emphasized the importance of participation by people in their own health care. In fact, participation is important at all levels of decision-making. Contemporary health challenges require new models of leadership that acknowledge the role of government in reducing disparities in health but that also recognize the many types of organizations that provide health care services. Governments need to guide and negotiate among these different groups, including nongovernmental organizations (NGOs) and the private sector, and to provide strong regulation where necessary. This difficult task requires a massive reinvestment in leadership and governance capacity, especially if action by different sectors is to be effectively implemented. Moreover, disadvantaged groups and other actors are increasingly expecting that their voices and health needs will be included in the 6 decision-making process. The complex

1	effectively implemented. Moreover, disadvantaged groups and other actors are increasingly expecting that their voices and health needs will be included in the 6 decision-making process. The complex landscape for leadership at the national level is mirrored in many ways at the international or global level. The transnational character of health and the increasing interdependence of countries with respect to outbreak diseases, climate change, security, migration, and agriculture place a premium on more effective global health governance.

1	Aspects of the primary health care approach described above, with an emphasis on primary care services, have been implemented to varying degrees in many lowand middle-income countries over the past half-century. As discussed above, some of these experiences inspired and informed the Declaration of Alma Ata, which itself led many more countries to attempt to implement primary health care. This section describes the experiences of a selection of lowand middle-income countries in improving primary care services that have enhanced the health of their populations.

1	Before Alma Ata, few countries had attempted to develop primary health care on a national level. Rather, most focused on expanding primary care services to specific communities (often rural villages), making use of community volunteers to compensate for the absence of facility-based care. In contrast, in the post–World War II period, China invested in primary care on a national scale, and life expectancy doubled within roughly 20 years. The Chinese expansion of primary care services included a massive investment in infrastructure for public health (e.g., water and sanitation systems) linked to innovative use of community health workers. These “barefoot doctors” lived in and expanded care to rural villages. They received a basic level of training that enabled them to provide immunizations, maternal care, and basic medical interventions, including the use of antibiotics. Through the work of the barefoot doctors, China brought low-cost universal basic health care coverage to its entire

1	maternal care, and basic medical interventions, including the use of antibiotics. Through the work of the barefoot doctors, China brought low-cost universal basic health care coverage to its entire population, most of which had previously had no access to these services.

1	In 1982, the Rockefeller Foundation convened a conference to review the experiences of China along with those of Costa Rica, Sri Lanka, and the state of Kerala in India. In all of these locations, good health care at low cost appeared to have been achieved. Despite lower levels of economic development and health spending, all of these jurisdictions, along with Cuba, had health indicators approaching—or in some cases exceeding—those of developed countries. Analysis of these experiences revealed a common emphasis on primary care services, with expansion of care to the entire population free of charge or at low cost, combined with community participation in decision-making about health services and coordinated work in different sectors (especially education) toward health goals. During the three decades since the Rockefeller meeting, some of these countries have built on this progress, while others have experienced setbacks. Recent experiences in developing primary care services show

1	three decades since the Rockefeller meeting, some of these countries have built on this progress, while others have experienced setbacks. Recent experiences in developing primary care services show that the same combination of features is necessary for success. For example, Brazil—a large country with a dispersed population—has made major strides in increasing the availability of health care in the past quarter century. In this millennium, the Brazilian Family Health Program has expanded progressively across the country, with almost all areas now covered. This program provides communities with free access to primary care teams made up of primary care physicians, community health workers, nurses, dentists, obstetricians, and pediatricians. These teams are responsible for the provision of primary care to all people in a specified geographic area—not only those who access health clinics. Moreover, individual community health workers are responsible for a named list of people within the

1	primary care to all people in a specified geographic area—not only those who access health clinics. Moreover, individual community health workers are responsible for a named list of people within the area covered by the primary care team. Problems with access to health care persist in Brazil, especially in isolated areas and urban slums. However, solid evidence indicates that the Family 3.96 –2.08 –4.24 –6.82 –6.97 –6.77 –8.38 –5.64 High HDI Low HDI

1	FIGURE 13e-8 Improvements in childhood mortality following the Family Health Program in Brazil. HDI, Human Development Index; PSF, Program Sae da Família (Family Health Program). (Source: Ministry of Health, Brazil.) Health Program has already contributed to impressive gains in population health, particularly in terms of childhood mortality and health inequities. In fact, this program has already had an especially marked impact on childhood mortality reduction in less developed areas (Fig. 13e-8).

1	Chile has also built on its existing primary care services in the past decade, aiming to improve the quality of care and the extent of coverage in remote areas, above all for disadvantaged populations. This effort has been made in concert with measures aimed at reducing social inequalities and fostering development, including social welfare benefits for families and disadvantaged groups and increased access to early-childhood educational facilities. As in Brazil, these steps have improved maternal and child health and have reduced health inequities. In addition to directly enhancing primary care services, Brazil and Chile have instituted measures to increase both the accountability of health providers and the participation of communities in decision-making. In Brazil, national and regional health assemblies with high levels of public participation are integral parts of the health policy– making process. Chile has instituted a patient’s charter that explicitly specifies the rights of

1	health assemblies with high levels of public participation are integral parts of the health policy– making process. Chile has instituted a patient’s charter that explicitly specifies the rights of patients in terms of the range of services to which they are entitled.

1	Other countries that have made recent progress with primary health care include Bangladesh, one of the poorest countries in the world. Since achieving its independence from Pakistan in 1971, Bangladesh has seen a dramatic increase in life expectancy, and childhood mortality rates are now lower than those in neighboring nations such as India and Pakistan. The expansion of access to primary health care services has played a major role in these achievements. This progress has been spearheaded by a vibrant NGO community that has focused its attention on improving the lives and livelihoods of poor women and their families through innovative and integrated microcredit, education, and primary care programs.

1	The above examples, along with others from the past 30 years in countries such as Thailand, Malaysia, Portugal, and Oman, illustrate how the implementation of a primary health care approach, with a greater emphasis on primary care, has led to better access to health care services—a trend that has not been seen in many other lowand middle-income countries. This trend, in turn, has contributed to improvements in population health and reductions in health inequities. However, as these nations have progressed, other countries have shown how previous gains in primary care can easily be eroded. In sub-Saharan Africa, undermining of primary care services has contributed to catastrophic reversals in health outcomes catalyzed by the HIV/AIDS epidemic. Countries such as Botswana and Zimbabwe implemented primary health care strategies in the 1980s, increasing access to care and making impressive gains in child health. Both countries have since been severely affected by HIV/AIDS, with pronounced

1	Percentage of total health expenditure FIGURE 13e-9 Changes in source of health expenditure in China over the past 40 years. (Source: World Health Organization: Primary Health Care: Now More Than Ever. World Health Report 2008.) decreases in life expectancy. However, Zimbabwe has also seen political turmoil, a decline of health and other social services, and the flight of health personnel, whereas Botswana has maintained primary care services to a greater extent and has managed to organize widespread access to antiretroviral therapy for people living with HIV/AIDS. Zimbabwe’s health situation has therefore become more desperate than that in Botswana.

1	China provides a particularly striking example of how changes in health policy relevant to the organization of health systems (Fig. 13e-9) can have rapid, far-reaching consequences for population health. Even as the 1982 Rockefeller conference was celebrating China’s achievements in primary care, its health system was unraveling. The decision to open up the economy in the early 1980s led to rapid privatization of the health sector and the breakdown of universal health coverage. As a result, by the end of the 1980s, most people, especially the poorer segments of the population, were paying directly out of pocket for health care, and almost no Chinese had insurance—a dramatic transformation. The “barefoot doctor” schemes collapsed, and the population either turned to care paid for at hospitals or simply became unable to access care. This undermining of access to primary care services in the Chinese system and the resulting increase in impoverishment due to illness contributed to the

1	or simply became unable to access care. This undermining of access to primary care services in the Chinese system and the resulting increase in impoverishment due to illness contributed to the stagnation of progress in health in China at the same time that incomes in that country increased at an unprecedented rate. Reversals in primary care have meant that China now increasingly faces health care issues similar to those faced by India. In both countries, rapid economic growth has been linked to lifestyle changes and noncommunicable disease epidemics. The health care systems of the two nations share two negative features that are common when primary care is weak: a disproportionate focus on specialty services provided in hospitals and unregulated commercialization of health services. China and India have both seen expansion of private hospital services that cater to middle-class and urban populations who can afford care; at the same time, hundreds of millions of people in rural areas

1	and India have both seen expansion of private hospital services that cater to middle-class and urban populations who can afford care; at the same time, hundreds of millions of people in rural areas now struggle to access the most basic services. Even in the former groups, a lack of primary care services has been associated with late presentation with illness and with insufficient investment in primary prevention approaches. This neglect of prevention poses a risk of large-scale epidemics of cardiovascular disease, which could endanger continued economic growth. In addition, the health systems of both countries now depend for the majority of their funding on out-of-pocket payments by people when they use services. Thus substantial proportions of the population must sacrifice other essential goods as a result of health expenditure and may even be driven into poverty by this cost. The commercial nature of health services with inadequate or no regulation has also led to the proliferation

1	goods as a result of health expenditure and may even be driven into poverty by this cost. The commercial nature of health services with inadequate or no regulation has also led to the proliferation of charlatan providers, inappropriate care, and pressure for people to pay for expensive and sometimes unnecessary care. Commercial providers have limited incentives to use interventions (including public health measures) that cannot be charged for or that are what the person who is paying can afford.

1	Faced with these problems, China and India have implemented measures to strengthen primary health care. China has increased government funding of health care, has taken steps toward restoring health insurance, and has enacted a target of universal access to primary care services. India has similarly mobilized funding to greatly expand primary care services in rural areas and is now duplicating this process in urban settings. Both countries are increasingly using public resources from their growing economies to fund primary care services.

1	These encouraging trends are illustrative of new opportunities to implement a primary health care approach and strengthen primary care services in lowand middle-income countries. Brazil, India, China, and Chile are being joined by many other lowand middle-income countries, including Indonesia, Mexico, the Philippines, Turkey, Rwanda, Ethiopia, South Africa, and Ghana, in ambitious initiatives mobilizing new resources to move toward universal coverage of health services at affordable cost.

1	Global public health targets will not be met unless health systems are significantly strengthened. More money is currently being spent on health than ever before. In 2005, global health spending totaled $5.1 trillion (U.S.)—double the amount spent a decade earlier. Although most expenditure occurs in high-income countries, spending in many emerging middle-income countries has rapidly accelerated, as has the allocation of monies for this purpose by both governments in and donors to low-income countries. These twin trends—greater emphasis on building health systems based on primary care and allotment of more money for health care—provide opportunities to address many of the challenges discussed above in lowand middle-income countries.

1	Accelerating progress requires a better understanding of how global health initiatives can more effectively facilitate the development of primary care in low-income countries. A review by the WHO Maximizing Positive Synergies Collaborative Group looked at programs funded by the Global Fund to Fight AIDS, Tuberculosis and Malaria; the Global Alliance for Vaccines and Immunisation (GAVI); the U.S. President’s Emergency Plan for AIDS Relief (PEPFAR); and the World Bank (on HIV/AIDS). This group found that global health initiatives had improved access to and quality of the targeted health services and had led to better information systems and more adequate financing. The review also identified the need for better alignment of global health initiatives with other national health priorities and systematic exploitation of potential synergies. If global health initiatives implement programs that work in tandem with other components of national health systems without undermining staffing and

1	systematic exploitation of potential synergies. If global health initiatives implement programs that work in tandem with other components of national health systems without undermining staffing and procurement of supplies, they have the potential to contribute substantially to the capacity of health systems to provide comprehensive primary care services.

1	Even in the aftermath of the global financial crisis, global health initiatives continue to draw significant funding. In 2009, for example, U.S. President Barack Obama announced increasing development assistance from the United States for global health, earmarking $63 billion over the period 2009–2014 for a Global Health Initiative. New funding is also promised through a range of other initiatives focusing particularly on maternal and child health in low-income countries. The general trend is to coordinate this funding in order to reduce fragmentation of national health systems and to concentrate more on strengthening these systems. Comprehensive primary care in low-income countries must inevitably deal with the rapid emergence of chronic diseases and the growing prominence of injury-related health problems; thus, international health development assistance must become more responsive to these needs.

1	Beyond the new streams of funding for health services, other opportunities exist. Increased social participation in health systems can help build primary care services. In many countries, political pressure from community advocates for more holistic and accountable care as well as entrepreneurial initiatives to scale up community-based services through NGOs have accelerated progress in primary care without major increases in funding. Participation of the population in the provision of health care services and in relevant decision-making often drives services to cater to people’s needs as a whole rather than to narrow public health priorities.

1	Participation and innovation can help address critical issues related to the health workforce in lowand middle-income countries by establishing effective people-centered primary care services. Many primary care services do not need to be delivered by a physician or a nurse. Multidisciplinary teams can include paid community workers who have access to a physician if necessary but who can provide a range of health services on their own. In Ethiopia, more than 30,000 community health workers have been trained and deployed to improve access to primary care services, and there is increasing evidence that this measure is contributing to better health outcomes. In India, more than 600,000 community health advocates have been recruited as part of expanded rural primary care services. In Niger, the deployment of community health workers to deliver essential child health interventions (as a component of integrated community case management) has had impressive results in reducing childhood

1	the deployment of community health workers to deliver essential child health interventions (as a component of integrated community case management) has had impressive results in reducing childhood mortality and decreasing disparities. After the Declaration of Alma Ata, experiences with community health workers were mixed, with particular problems about levels of training and lack of payment. Current endeavors are not immune from these concerns. However, with access to physician support and the deployment of teams, some of these concerns may be addressed. Growing evidence from many countries indicates that shifting appropriate tasks to primary care workers who have had shorter, less expensive training than physicians will be essential to address the human resources crisis.

1	Finally, recent improvements in information and communication technologies, particularly mobile phone and Internet systems, have created the potential for systematic implementation of e-health, telemedicine, and improved health data initiatives in lowand middle-income countries. These developments raise the tantalizing possibility that health systems in these countries, which have long lagged behind those in high-income countries but are less encumbered by legacy systems that have proved hard to modernize in many settings, could leapfrog their wealthier counterparts in exploiting these technologies. Although the challenges posed by poor or absent infrastructure and investment in many lowand middle-income countries cannot be underestimated and will need to be addressed to make this possibility a reality, the rapid rollout of mobile networks and their use for health and other social services in many low-income countries where access to fixed telephone lines was previously very limited

1	a reality, the rapid rollout of mobile networks and their use for health and other social services in many low-income countries where access to fixed telephone lines was previously very limited offer great promise in building primary care services in lowand middle-income countries.

1	As concern continues to mount about glaring inequities in global health, there is a growing commitment to redress these egregious shortfalls, as exemplified by global mobilization around the United Nations’ Millennium Development Goals and the early discussions on what targets should build on these goals in the post-2015 era. This commitment begins first and foremost with a clear vision of the fundamental importance of health in all countries, regardless of income. The values of health and health equity are shared across all borders, and primary health care provides a framework for their effective translation across all contexts.

1	The translation of these fundamental values has its roots in four types of reforms that reflect the distinct but interlinked challenges of (re)orienting a society’s resources on the basis of its citizens’ health needs: (1) organizing health care services around the needs of people and communities; (2) harnessing services and sectors beyond health care to promote and protect health more effectively; (3) establishing sustainable and equitable financing mechanisms for universal coverage; and (4) investing in effective leadership of the whole of society. This common primary health care agenda highlights the striking similarity, despite enormous differences in context, in the nature and direction of the reforms that national health systems must undertake to promote greater equity in health. This shared agenda is complemented by the growing reality of global health interconnectedness due, for example, to shared microbial threats, bridging of ethnolinguistic diversity, flows in migrant

1	This shared agenda is complemented by the growing reality of global health interconnectedness due, for example, to shared microbial threats, bridging of ethnolinguistic diversity, flows in migrant health workers, and mobilization of global funds to support the neediest populations. Embracing solidarity in global health while strengthening health systems through a primary health care approach is fundamental to sustained progress in global health.

1	Complementary, alternative, and Integrative health practices Josephine P. Briggs The search for health includes many beliefs and practices that are outside conventional medicine. Physicians are important sources for 14e Acupuncture and acupressure A family of procedures involving stimulation of defined anatomic points, a component of the major Asian medical traditions; most common application involves the insertion and manipulation of thin metallic needles Alexander technique A movement therapy that uses guidance and education to improve posture, movement, and efficient use of muscles for improvement of overall body functioning Guided imagery The use of relaxation techniques followed by the visualization of images, usually calm and peaceful in nature, to invoke specific images to alter neurologic function or physiologic states Hypnosis The induction of an altered state of consciousness characterized by increased responsiveness to suggestion

1	Hypnosis The induction of an altered state of consciousness characterized by increased responsiveness to suggestion Massage Manual therapies that manipulate muscle and connective tissues to promote muscle relaxation, healing, and sense of well-being Meditation A group of practices, largely based in Eastern spiritual traditions, intended to focus or control attention and obtain greater awareness of the present moment, or mindfulness Reflexology Manual stimulation of points on hands or feet that are believed to affect organ function Rolfing/structural integration A manual therapy that attempts to realign the body by deep tissue manipulation of fascia Spinal manipulation A range of manual techniques, employed by chiropractors and osteopaths, for adjustments of the spine to affect neuromuscular function and other health outcomes Tai chi A mind-body practice originating in China that involves slow, gentle movements and sometimes is described as “moving meditation”

1	Tai chi A mind-body practice originating in China that involves slow, gentle movements and sometimes is described as “moving meditation” Therapeutic touch Secular version of the laying on of hands, described as “healing meditation” Yoga An exercise practice, originally East Indian, that combines breathing exercises, physical postures, and meditation Ayurvedic medicine The major East Indian traditional medicine system; treatment includes meditation, diet, exercise, herbs, and elimination regimens (using emetics and diarrheals) Curanderismo A spiritual healing tradition common in Latin American communities that uses ritual cleansing, herbs, and incantations Native American medicine Diverse traditional systems that incorporate chanting, shaman healing ceremonies, herbs, laying on of hands, and smudging (ritual cleansing with smoke from sacred plants) Tibetan medicine A medical system that uses diagnosis by pulse and urine examination; therapies include herbs, diet, and massage

1	Tibetan medicine A medical system that uses diagnosis by pulse and urine examination; therapies include herbs, diet, and massage Traditional Chinese medicine A medical system that uses acupuncture, herbal mixtures, massage, exercise, and diet Unani medicine An East Indian medical system, derived from Persian medicine, practiced primarily in the Muslim community; also called “hikmat” Anthroposophic medicine A spiritually based system of medicine that incorporates herbs, homeopathy, diet, and a movement therapy called eurythmy Chiropractic Chiropractic care involves the adjustment of the spine and joints to alleviate pain and improve general health; primarily used to treat back problems, musculoskeletal complaints, and headaches Homeopathy A medical system with origins in Germany that is based on a core belief in the theory of “like cures like”—compounds that produce certain syndromes, if administered in very diluted solutions, will be curative

1	Naturopathy A clinical discipline that emphasizes a holistic approach to the patient, herbal medications, diet, and exercise; practitioners have degrees as doctors of naturopathy Osteopathy A clinical discipline, now incorporated into mainstream medicine, that historically emphasized spinal manipulative techniques to relieve pain, restore function, and promote overall health information and guidance about health matters, but our patients also rely on a wide range of other sources including family and friends, cultural traditions, alternative practitioners, and increasingly the Internet, popular media, and advertising. It is essential for physicians to understand what patients are doing to seek health, as this understanding is important to harness potential benefits and to help patients avoid harm.

1	The phrase complementary and alternative medicine is used to describe a group of diverse medical and health care systems, practices, and products that have historic origins outside mainstream medicine. Most of these practices are used together with conventional therapies and therefore have been called complementary to distinguish them from 14e-1 alternative practices, those used as a substitute for standard care. Use of dietary supplements; mind-body practices such as acupuncture, massage, meditation, and hypnosis; and care from a traditional healer all fall under this umbrella. Brief definitions for some of the common complementary and alternative health practices are provided in Table 14e-1. Although some complementary health practices are implemented by a complementary health care provider such as a chiropractor, acupuncturist, or naturopathic practitioner, or by a physician, many of these practices are undertaken as “self-care.” Most are paid for out of pocket.

1	In the last decade or so, the terms integrative care and integrative medicine have entered the dialogue. A 2007 national survey conducted by the Centers for Disease Control and Prevention’s National Center for Health Statistics found that 42% of hospices had integrated complementary health practices into the care they provide. Integration of select complementary approaches is also common in Veterans Administration and Department of Defense facilities, particularly as part of management of pain and post-traumatic stress disorder. The term integrative medicine is usually used to refer to a style of practice that places strong emphasis on a holistic approach to patient care while focusing on reduced use of technology. Physicians advocating

1	Chapter 14e Complementary, Alternative, and Integrative Health Practices this approach generally include selected complementary health practices in the care they offer patients, and many have established practice settings that include complementary health practitioners. Although this approach appears to be attractive to many patients, the heavy use of dietary supplements and the weaknesses in the evidence base for a number of the interventions offered in integrative practices continue to attract substantial concern and controversy.

1	Until a decade ago or so, “complementary and alternative medicine” could be defined as practices that are neither taught in medical schools nor reimbursed, but this definition is no longer workable, since medical students increasingly seek and receive some instruction about complementary health practices, and some practices are reimbursed by third-party payers. Another definition, practices that lack an evidence base, is also not useful, since there is a growing body of research on some of these modalities, and some aspects of standard care do not have a strong evidence base.

1	By its nature, the demarcation between mainstream medicine and complementary health practices is porous, varying from culture to culture and over time. Traditional Chinese medicine and the Indian practice of Ayurvedic medicine were once the dominant health teachings in those cultures. Certain health practices that arose as challenges to the mainstream have been integrated gradually into conventional care. Examples include the teachings of Fernand Lamaze that led to the widespread use of relaxation techniques during childbirth, the promotion of lactation counseling by the La Leche League, and the teaching of Cicely Saunders and Elizabeth Kler-Ross that established the hospice movement.

1	The late nineteenth century saw the development of a number of healing philosophies by care providers who were critical of the medicine of the time. Of these, naturopathy and homeopathy, which arose in Germany, and chiropractic and osteopathy, which developed in the United States, have continued to endure. Osteopathic medicine is currently thoroughly integrated into conventional medicine, although the American Medical Association (AMA) labeled it a cult as late as 1960. The other three traditions have remained resolutely separate from mainstream medicine, although chiropractic care is available in some conventional care settings.

1	The first large survey of use of these practices was performed by David Eisenberg and associates in 1993. It surprised the medical community by showing that more than 30% of Americans use complementary or alternative health approaches. Many studies since that time have extended those conclusions. Subsequently, the National Health Interview Survey (NHIS), a large, national survey conducted by the National Center for Health Statistics, a component of the Centers for Disease Control and Prevention, has addressed the use of complementary health practices and largely confirmed those results. The NHIS is a household survey of many kinds of health practices in the civilian population; it uses methods that create a nationally representative sample and has a sample size large enough to permit valid estimates about some subgroups. In 2002, 2007, and 2012, the survey included a set of questions that addressed complementary and alternative health approaches. Information was obtained from 31,000

1	estimates about some subgroups. In 2002, 2007, and 2012, the survey included a set of questions that addressed complementary and alternative health approaches. Information was obtained from 31,000 adults in 2002 and 23,300 adults and 9400 children in 2007. Only preliminary data are available from the 2012 survey. In all three surveys, approximately 40% of adults report using some form of complementary therapy or health practice. In the 2007 study, 38% of adults and 12% of children had used one or more modalities. These surveys yield the estimate that nonvitamin, nonmineral dietary supplements are used by approximately 18% of the population. The most prevalent mind-body practices are relaxation techniques and meditation, chiropractic, and therapeutic massage. Americans are willing to pay for these services; the estimated out-of-pocket expenditure for complementary health practices in 2007 was $34 billion, representing 1.5% of total health expenditures and 11% of out-of-pocket costs.

1	The appeal of unproven complementary health approaches continues to perplex many physicians. Many factors contribute to these choices. Some patients seek out complementary health practitioners because they offer optimism or greater personal attention. For others, alternative approaches reflect a “self-help” approach to health and wellness or satisfy a search for “natural” or less invasive alternatives, since dietary supplements and other natural products are believed, correctly or not, to be inherently healthier and safer than standard pharmaceuticals. In NHIS surveys, the most common health conditions cited by patients for use of complementary health practices involve management of symptoms often poorly controlled by conventional care, particularly back pain and other painful musculoskeletal complaints, anxiety, and insomnia.

1	PRACTITIONER-BASED DISCIPLINES Licensure and Accreditation At present, six fields of complementary health practice—osteopathic manipulation, chiropractic, acupuncture and traditional Chinese medicine, therapeutic massage, naturopathy, and homeopathy—are subject to some form of educational accreditation and state licensure. Accreditation of educational programs is the responsibility of professional organizations or commissions under federal oversight by the Department of Education. Licensure, in contrast, is strictly a state matter, generally determined by state legislatures. Legal recognition establishes public access to therapies even when there is no scientific consensus about their clinical value.

1	Osteopathic Manipulative Therapy Founded in 1892 by the physician Andrew Taylor Still, osteopathic medicine was originally based on the belief that manipulation of soft tissue and bone can correct a wide range of diseases of the musculoskeletal and other organ systems. Over the ensuing century, the osteopathic profession has welcomed increasing integration with conventional medicine. Today, the postgraduate training, practice, credentialing, and licensure of osteopathic physicians are virtually indistinguishable from those of allopathic physicians. Osteopathic medical schools, however, include training in manual therapies, particularly spinal manipulation. Approximately 70% of family practice osteopathic physicians perform manipulative therapies on some of their patients.

1	Chiropractic The practice of chiropractic care, founded by David Palmer in 1895, is the most widespread practitioner-based complementary health practice in the United States. Chiropractic practice emphasizes manual therapies for treatment of musculoskeletal complaints, although the scope of practice varies widely, and in some rural areas, chiropractors may serve a primary care role, due in part to the lack of other providers. According to the NHIS, approximately 8% of Americans receive chiropractic manipulation in a given year.

1	Since the mid-1970s, chiropractors have been licensed in all 50 states and reimbursed by Medicare. Chiropractic educational standards mandate 2 years of undergraduate training, 4 years of training at an accredited school of chiropractic, and in most states, successful completion of a standardized board examination. Postgraduate training is not required. The U.S. Department of Labor estimates that there are 52,000 licensed chiropractors (2010 figure). There is substantial geographic variation, with greater numbers of practitioners and greater use in the midwest, particularly in rural areas, and lower use in the southeast.

1	Historically, the relationship between the medical and chiropractic professions has been strained. Extending through the 1970s, the AMA set forth standards prohibiting physicians consulting or entering into professional relationships with chiropractors, but in 1987, after a decade of complex litigation, the U.S. District Court found the AMA in violation of antitrust laws. An uneasy truce has followed, with continued physician skepticism, but also evidence for robust patient demand and satisfaction.

1	The role of both osteopathic and chiropractic spinal manipulative therapies (SMTs) in back pain management has been the subject of a number of carefully performed trials and many systematic reviews. Conclusions are not consistent, but the most recent guidelines from the American College of Physicians and the American Pain Society conclude that SMT is associated with small to moderate benefit for low-back pain of less than 4 weeks in duration (evidence level B/C) and moderate benefit (evidence level B) for subacute or chronic lowback pain. The evidence of benefit for neck pain is not as extensive, and continued concern that cervical manipulation may occasionally precipitate vascular injury clouds a contentious debate.

1	Naturopathy Naturopathy is a discipline that emerged in central Europe in the nineteenth century as part of the Natural Cure movement and was introduced to the United States in the early twentieth century by Benjamin Lust. Fifteen states currently license naturopathic physicians, with considerable variation in the scope of practice. The naturopathic profession is actively seeking licensure in other states. There are estimated to be approximately 3000 licensed naturopathic physicians in the United States. There is also a robust naturopathy presence in Canada. Conventional and unconventional diagnostic tests and medications are prescribed, with an emphasis on relatively low doses of drugs, herbal medicines, healthy diet, and exercise. While there is some support for success of naturopathic practitioners in motivating healthy behaviors, concern exists about the heavy promotion of dietary supplements, most with little rigorous evidence.

1	Homeopathy Homeopathy was widespread in the United States in the late nineteenth and early twentieth centuries and continues to be a common alternative practice in many European countries, but estimates from the NHIS suggest that less than 1.5% of Americans visit a homeopathic practitioner in any given year. In the United States, licensure as a homeopathic physician is only possible in three states (Arizona, Connecticut, and Nevada) where it is restricted to licensed physicians. The number of practitioners is uncertain, however, because some states include homeopathy within the scope of practice of other fields, including chiropractic and naturopathy, and some practitioners may self-identify as homeopathic practitioners. As discussed below, the regulatory framework for homeopathic remedies differs from other dietary supplements. Homeopathic remedies are widely available and commonly recommended by naturopathic physicians, chiropractors, and other licensed and unlicensed practitioners.

1	Therapeutic Massage The field of therapeutic massage is growing rapidly, as use by the public is increasing. According to U.S. Department of Labor statistics, there are approximately 155,000 licensed massage therapists employed in the United States, and by 2020, this number is projected to grow by 20%. Forty-three states and the District of Columbia currently have laws regulating massage therapy; however, there is little consistency, and in some states, regulation is by town ordinance. States that do provide licensure for massage therapists typically require a minimum of 500 hours of training at an accredited institution, as well as meeting specific continuing education requirements and carrying malpractice insurance. Massage training programs generally are approved by a state board, but some may also be accredited by an independent agency, such as the Commission on Massage Therapy Accreditation (COMTA). The development of regulatory standards for therapeutic massage has not yet

1	but some may also be accredited by an independent agency, such as the Commission on Massage Therapy Accreditation (COMTA). The development of regulatory standards for therapeutic massage has not yet caught up with the evolution of the field or the high demand. Many techniques used are also employed by physical therapists.

1	Acupuncture and Traditional Chinese Medicine A venerable component of traditional Chinese medicine, with a history of use that extends at least 2000 years, acupuncture became better known in the United States in 1971, when New York Times reporter James Reston wrote about how doctors in China used needles to ease his pain after surgery. More than 3 million adults in the United States use acupuncture, according to NHIS data. In a number of European countries, acupuncture is performed primarily by physicians. In the United States, the training and licensure processes for physicians and nonphysicians differ. Currently, acupuncture is licensed in 42 states and the District of Columbia, with licensure standards varying within the scope of practice of each state. Licensure for nonphysicians generally requires 3 years of accredited training and the successful completion of a standardized examination. The main accrediting organization is the Accreditation Commission for Acupuncture and

1	generally requires 3 years of accredited training and the successful completion of a standardized examination. The main accrediting organization is the Accreditation Commission for Acupuncture and Oriental Medicine. Acupuncture is included in doctor of medicine (MD) and doctor of osteopathic medicine (DO) licensure in 31 states, with 11 states requiring additional training for physicians performing acupuncture.

1	Mind-body practices are a large and diverse group of techniques that are administered or taught to others by a trained practitioner or teacher. Examples include acupuncture, massage therapy, meditation, relaxation techniques, spinal manipulation, and yoga. These approaches are being used more frequently in mainstream health care facilities for both patients and health care providers. Mind-body practices such as meditation and yoga are not licensed in any state, and training in those practices is not subject to national accreditation.

1	Americans often turn to complementary approaches for help in managing health conditions associated with physical and psychological pain—especially back pain, headache, musculoskeletal complaints, and functional pain syndromes. Chronic pain management is often refractory to conventional medical approaches, and standard pharmacologic approaches have substantial drawbacks. Health care guidelines of the American Pain Society and other professional organizations recognize the value of certain complementary approaches as adjuncts to pharmacologic management.

1	The evidence base for the effectiveness of these modalities is still relatively incomplete, but a few rigorous examples where there is promise of usefulness and safety include acupuncture for osteoarthritis pain; tai chi for fibromyalgia pan; and massage, yoga, and spinal manipulation for chronic back pain. In addition, new research is shedding light on the effects of meditation and acupuncture on central mechanisms of pain processing and perception and regulation of emotion and attention. Although many unanswered questions remain about these effects, findings are pointing to scientifically plausible mechanisms by which these modalities might yield benefit.

1	DIETARY SUPPLEMENTS Regulation The Dietary Supplements Health and Education Act (DSHEA), passed in 1994, gives authority to the U.S. Food and Drug Administration (FDA) to regulate dietary supplements, but with expectations that differ in many respects from the regulation of drugs or food additives. Purveyors of dietary supplements cannot claim that they prevent or treat any disease. They can, however, claim that they maintain “normal structure and function” of body systems. For example, a product cannot claim to treat arthritis, but it can claim to maintain “normal joint health.” Homeopathic products predate FDA drug regulations and are sold with no requirement that they be proved effective. Although homeopathic products are widely believed to be safe because they are highly dilute, one product, a nasal spray called Zicam, was withdrawn from the market when it was found to produce anosmia, probably because of a significant zinc content. Homeopathic products, and indeed other

1	one product, a nasal spray called Zicam, was withdrawn from the market when it was found to produce anosmia, probably because of a significant zinc content. Homeopathic products, and indeed other complementary health products and practices, also convey the very significant risk that individuals will use them instead of effective conventional modalities.

1	Regulation of advertising and marketing claims is the purview of the Federal Trade Commission (FTC). The FTC does take legal action against promoters or websites that advertise or sell dietary supplements with false or deceptive statements.

1	Inherent Toxicity Although the public may believe that “natural” equates with “safe,” it is abundantly clear that natural products can be toxic. Misidentification of medicinal mushrooms has led to liver failure. Contamination of tryptophan supplements caused the eosinophiliamyalgia syndrome. Herbal products containing particular species of Aristolochia were associated with genitourinary malignancies and interstitial nephritis. In 2013, dietary supplements containing 1,3-dimethylamylamine (DMAA), often touted as a “natural” stimulant, led to cardiovascular problems, including heart attacks. Among the most controversial dietary supplements is Ephedra sinica, or ma huang, a product used in traditional Chinese medicine for short-term treatment of asthma and bronchial congestion. The scientific basis for these indications was revealed when ephedra was shown to contain the ephedrine alkaloids, especially ephedrine and pseudoephedrine. With the promulgation of the DSHEA regulations,

1	basis for these indications was revealed when ephedra was shown to contain the ephedrine alkaloids, especially ephedrine and pseudoephedrine. With the promulgation of the DSHEA regulations, supplements containing ephedra and herbs rich in caffeine sold widely in the U.S. marketplace because of their claims to promote weight loss and enhance athletic

1	Chapter 14e Complementary, Alternative, and Integrative Health Practices performance. Reports of severe and fatal adverse events associated with use of ephedra-containing products led to an evidence-based review of the data surrounding them, and in 2004, the FDA banned their sale in the United States. Another major current concern with dietary supplements is adulteration with pharmacologic active compounds. Multi-ingredient products marketed for weight loss, body building, “sexual health,” and athletic performance are of particular concern. Recent FDA recalls have involved contamination with steroids, diuretics, stimulants, and phosphodiesterase type 5 inhibitors.

1	Herb-Drug Interactions A number of herbal products have potential impact on the metabolism of drugs. This effect was illustrated most compellingly with the demonstration in 2000 that consumption of St. John’s wort interferes with the bioavailability of the HIV protease inhibitor indinavir. Later studies showed its similar interference with metabolism of topoisomerase inhibitors such as irinotecan, with cyclosporine, and with many other drugs. The breadth of interference stems from the ability of hyperforin in St. John’s wort to upregulate expression of the pregnane X receptor, a promiscuous nuclear regulatory factor that promotes the expression of many hepatic oxidative, conjugative, and efflux enzymes involved in drug and food metabolism.

1	Because of the large number of compounds that alter drug metabolism and the large number of agents some patients are taking, identification of all potential interactions can be a daunting task. Several useful Web resources are available as information sources (Table 14e-2). Clearly, attention to this problem is particularly important with drugs with a narrow therapeutic index, such as anticoagulants, antiseizure medications, antibiotics, immunosuppressants, and cancer chemotherapeutic agents.

1	Physicians regularly face difficult challenges in providing patients with advice and education about complementary practices. Of particular concern to all physicians are practices of uncertain safety and practices that raise inappropriate hopes. Cancer therapies, antiaging regimens, weight-loss programs, sexual function, and athletic performance are frequently targeted for excessive claims and irresponsible marketing. A number of Internet resources provide critical tools for patient education (Table 14e-3). Because many complementary health products and practices are used as self-care and because many patients research these approaches extensively on the Internet, directing patients to responsible websites can often be very helpful. http://www.medscape.com/druginfo/druginterchecker?cid=med This website is maintained by WebMD and includes a free drug interaction checker tool that provides information on interactions between two or more drugs, herbals, and/or dietary supplements.

1	This website is maintained by WebMD and includes a free drug interaction checker tool that provides information on interactions between two or more drugs, herbals, and/or dietary supplements. http://naturaldatabase.therapeuticresearch.com This website provides an interactive natural product–drug interaction checker tool that identifies interactions between drugs and natural products, including herbals and dietary supplements. This service is available by subscription. A PDA version is available. http://www.naturalstandard.com/tools/ This website provides an interactive tool for checking drug and herb/supplement interactions. This service is available by subscription. A PDA version is available. Abbreviation: PDA, personal digital assistant. The Cochrane Collaboration Complementary Medicine Reviews

1	Abbreviation: PDA, personal digital assistant. The Cochrane Collaboration Complementary Medicine Reviews This website offers rigorous systematic reviews of mainstream and complementary health interventions using standardized methods. It includes more than 300 reviews of complementary health practices. Complete reviews require institutional or individual subscription, but summaries are available to the public. http://www.cochrane.org/cochrane-reviews MedlinePlus All Herbs and Supplements, A–Z List NLM FAQ: Dietary Supplements, Complementary or Alternative Medicines These National Library of Medicine (NLM) Web pages provide an A–Z database of science-based information on herbal and dietary supplements; basic facts about complementary health practices; and federal government sources on information about using natural products, dietary supplements, medicinal plants, and other complementary health modalities.

1	http://www.nlm.nih.gov/medlineplus/druginfo/herb_All.html http://www.cochrane.org/cochrane-reviews http://www.nlm.nih.gov/medlineplus/dietarysupplements.html National Institutes of Health National Center for Complementary and Alternative Medicine (NCCAM) This National Institutes of Health NCCAM website contains information for consumers and health care providers on many aspects of complementary health products and practices. Downloadable information sheets include short summaries of complementary health approaches, uses and risks of herbal therapies, and advice on wise use of dietary supplements. http://www.nccam.nih.gov Resources for Health Care Providers: http://www.nccam.nih.gov/health/providers NCCAM Clinical Digest e-Newsletter: http://www.nccam.nih.gov/health/ providers/digest Continuing medical education lectures: http://www.nccam.nih.gov/training/ videolectures

1	NCCAM Clinical Digest e-Newsletter: http://www.nccam.nih.gov/health/ providers/digest Continuing medical education lectures: http://www.nccam.nih.gov/training/ videolectures The scientific evidence regarding complementary therapies is fragmentary and incomplete. Nonetheless, in some areas, particularly pain management, it is increasingly possible to perform the kind of rigorous systematic reviews of complementary health approaches that are the cornerstone of evidence-based medicine. A particularly valuable resource in this respect is the Cochrane Collaboration, which has performed more than 300 systematic reviews of complementary health practices. Practitioners will find this a valuable source to answer patient questions. Practice guidelines, particularly for pain management, are also available from several professional organizations. Links to these resources are provided in Table 14e-3.

1	The use of complementary and alternative health practices reflects an active interest in improved health. An array of unproven modalities will always be used by our patients. While some of these choices need to be actively discouraged, many are in fact innocuous and can be accommodated. Some may be genuinely helpful, particularly in the management of troublesome symptoms. The dialogue with patients about complementary health practices is an opportunity to understand patients’ beliefs and expectations and use those insights to help guide health-seeking practices in a constructive way. The late Dr. Stephen Straus contributed this chapter in prior editions, and some material from his chapter has been retained here.

1	The late Dr. Stephen Straus contributed this chapter in prior editions, and some material from his chapter has been retained here. The Economics of Medical Care Joseph P. Newhouse The purpose of this chapter is to explain to physicians how economists think about physicians’ decision-making with regard to the treatment of patients. Economists’ mode of thinking has shaped health care policy and institutions and thus the environment in which physicians 15e practice, not only in the United States but in many other countries as well. It may prove useful for physicians to understand some aspects of the economists’ way of thinking, even if it sometimes seems foreign or uncongenial.

1	Physicians see themselves as professionals and as healers, assisting their patients with their health care needs. When economists are patients, they probably see physicians the same way, but when they view doctors through the lens of economics as a discipline, they see physicians—and their patients as well—as economic agents. In other words, economists are interested in the degree to which physicians and patients respond to various incentives in deciding how to deploy the resources over which they exercise choice. Examples of issues that would concern an economist include how much time physicians devote to seeing a patient; which tests they order; what drugs, if any, they prescribe; whether they recommend a procedure; whether they refer a patient; and whether they admit a patient to the hospital. In addition, patients consider the cost when they make a decision about whether to seek care.

1	To say that economists view physicians and patients as economic agents is not to say that economists consider financial incentives the predominant factor in the decisions that either physicians or patients make about treatment; it is to say only that these incentives have some influence on these decisions. In fact, the role played by financial incentives in medical decision-making may often be dwarfed by the roles played by scientific knowledge, by professional norms and ethics, and by the influence of peers. However, economic policy greatly influences financial incentives, and economists tend to focus on this domain. Their interest stems from fundamental economic questions: What goods and services are produced and consumed? In particular, how much medical care is available, and how much of other goods and services? How is medical care produced? For example, what mix of specific services is used in treating a particular episode of illness? Who receives particular treatments?

1	Physicians in all societies live and function in economic markets, although those markets differ greatly both from the simple competitive markets depicted in introductory economics textbooks and from country to country, depending on national institutions. Many of the differences between actual medical markets and textbook competitive markets cause what economists term market failure, a condition in which some individuals can be made better off without making anyone else worse off. This chapter explains two features of health care financing that cause market failure: selection and moral hazard. A common response to market failure in medical care is what economists refer to as administered prices, which this chapter also describes. Unfortunately, administered prices exact a cost, leading to what economists call regulatory or government failure. All societies seek a balance between market failure and regulatory failure, a topic addressed in this chapter’s conclusion.

1	In the idealized competitive market found in economic textbooks, buyers and sellers have the same knowledge about the goods or services they are buying and selling. When one party knows more—or when goods of different quality are being sold at the same price, which is analytically similar—markets can break down in the following sense: There may be a price at which an equally well informed buyer and seller could make a transaction that would make them both better off, but that transaction does not occur because one party knows more than the other. Hence, both the potential buyer and the potential seller are worse off.

1	The used car market is a classic example of differential information. 15e-1 Owners of used cars (potential sellers) know more about the quality of their cars than do potential buyers. At any specific market price for a certain make and model of car, the only used cars offered will be those whose sellers value them at less than that price. Assuming that quality varies among used cars, those that are offered for sale will differentially be of low quality (“lemons”) relative to the given price. Owners of higher-quality cars may simply hold on to them. If, however, a potential buyer knew that the car was of higher quality, the buyer might be willing to pay enough so that the owner of the higher-quality car would sell. It is for this reason that sellers may offer warranties and guarantees, although these are uncommon (but not unknown) in medical care.

1	The same thing happens when goods of different quality are sold at the supermarket at the same price. Shoppers are happy to take any box of a particular brand of breakfast cereal or any bottle of a particular soft drink on the shelf because the quality of the contents of any box or bottle is the same; however, that is not the case in the produce section, where shoppers will inspect the fruit they pick up to ensure that the apple is not bruised or the banana overly ripe. At the end of the day, it is the bruised apples and overly ripe bananas that are left in the store. In effect, the seller has not used all the available information in pricing the produce, and buyers exploit that information differential.

1	Selection affects markets for individual—and, to some degree, small-group—health insurance in a fashion similar to that seen in the used car market and at the produce stand, but in this case it is the buyer of insurance who has more knowledge than the seller. Individuals who use above-average amounts of care—for example, those with a chronic disease or a strong proclivity to seek care for a symptom—will value health insurance more than will those who are healthy or who for various reasons shun medical care even if they are symptomatic. However, the insurer does not necessarily know the risk of those it insures, and so it gears insurance premiums to an average risk, which in some instances is conditional on certain observable characteristics, such as age. Just as shoppers do not want the bruised apples and used car buyers do not want lemons, many healthy people will not want to buy insurance voluntarily if its price mainly reflects its use by those who are sick. (Healthy but very

1	the bruised apples and used car buyers do not want lemons, many healthy people will not want to buy insurance voluntarily if its price mainly reflects its use by those who are sick. (Healthy but very risk-averse individuals still may be willing to pay premiums well above their expected use.) In an extreme case, healthy people drop out of the insurance pool, premiums rise because the average person left in the pool is sicker, that rise causes still more people to drop out of the pool, premiums rise further, and so forth, until few people remain to buy insurance.

1	For this reason, no developed country relies primarily on voluntary individual insurance to finance health care, although many countries use it in the supplemental insurance market, and selection is, in fact, often a feature of that market. Instead, governments and/or employers provide or heavily subsidize the purchase of either mandated or voluntary health insurance (e.g., in Canada or Germany, the Medicare and Medicaid programs in the United States and the purchase of insurance in exchanges by lower-income persons) or provide health services directly (e.g., the United Kingdom and the U.S. Veterans Health Administration). In addition, governments or third parties administering individual insurance markets with competing insurers may “risk-adjust” payments to insurers; that is, transfer monies from insurers who enroll better risks (as measured by observable features, such as diagnoses that are not used to rate premiums) to insurers who enroll worse risks. This feature is found in the

1	from insurers who enroll better risks (as measured by observable features, such as diagnoses that are not used to rate premiums) to insurers who enroll worse risks. This feature is found in the American Medicare Advantage program and American insurance exchanges as well as in Germany and the Netherlands. The idea is to reduce insurers’ incentives to structure their products in order to appeal to good risks, especially when insurers are making choices about networks and formularies.

1	Moreover, countries that rely on employment-based health insurance, such as the United States and Germany, either mandate taxes to finance that insurance or provide large tax subsidies for its purchase; otherwise, many healthy employees would prefer that the employer give them the money the employer uses to subsidize the insurance as cash wages. Because an employer that offers health insurance will pay lower cash wages than an otherwise equivalent employer that does

1	CHAPTER 15e The Economics of Medical Care not, larger American employers that, before the Affordable Care Act was implemented in 2014, were not required to offer insurance may not, in fact, have offered it if they had many low-wage employees; the reason is that, if they had offered insurance, the cash wage they could afford to pay would have been below the minimum wage. (For the same reason, these employers typically do not offer a pension benefit.) Many low-wage employers, however, are small businesses that might not be viable if they had to subsidize health insurance. As a result, the Affordable Care Act exempted firms with fewer than 50 employees from any penalties if their employees received a public subsidy and purchased insurance in the exchange. Some self-employed individuals or those who work at small firms may belong to a trade association or a professional society through which they can purchase insurance, but because that purchase is voluntary, it is subject to selection.

1	How does this situation affect the practice of medicine? Prior to the Affordable Care Act, individual and small-group insurance policies typically had preexisting condition clauses to protect the insurer against selection—that is, to protect the insurer against a person’s purchasing insurance (or more complete insurance) after that person had been diagnosed with a disease that is expensive to treat. Even though there is now a penalty for remaining uninsured, some individuals still choose to do so, and others purchase insurance with substantial amounts of cost sharing that they may not be able to pay if they become sick. Caring for such patients may give the physician a choice between making do with less than clinically optimal treatment and proceeding in a clinically optimal way but leaving the patient with a large bill and possible bankruptcy—and potentially leaving the physician with bill collection issues or unpaid bills.

1	Selection can arise in a different guise when physicians are reimbursed a fixed amount per patient (i.e., capitation) rather than receiving fee-for-service payments. Depending on the adequacy of any adjustments in the capitated amount for the resources that a specific patient will require (“risk adjustment”), physicians who receive a fixed amount have a financial incentive to avoid caring for sicker patients. Similarly, physicians who receive a capitated amount for their own services but are not financially responsible for hospital care or the services of other physicians may make an excessive number of referrals, just as physicians reimbursed in a fee-for-service arrangement may make too few.

1	The term moral hazard comes from the actuarial literature; it originally referred to the weaker incentives of an insured individual to prevent the loss against which he or she is insured. A classic example is failure of homeowners in areas prone to brush fires to cut the brush around their houses or possibly install fire-resistant shingles on their roofs because of their expectation that insurance will compensate them if their houses burn down. In some lines of insurance, however, moral hazard is not a substantive issue. Persons who buy life insurance on their own lives are not likely to commit suicide so that their heirs can receive the proceeds. Moreover, despite the brush fire example, homeowner’s insurance probably has little moral hazard associated with it because individuals often cannot replace some valued personal items when a house burns down. In short, if moral hazard is negligible, insured persons take appropriate precautions against the potential loss.

1	In the context of health insurance, this classic form of moral hazard refers to potentially reduced incentives to prevent illness, but that is probably not a major issue. Sickness and disease generally imply some pain and suffering, not to mention possibly shortened life expectancy. Because there is no insurance for pain and suffering, individuals have strong incentives to try to remain healthy regardless of how much health insurance they have. Put another way, having better health insurance probably does not alter those incentives much.

1	Instead of weakened incentives to prevent illness, moral hazard in the health insurance context typically refers to the incentives for better-insured individuals to use more medical services. For instance, a patient with back pain or shoulder pain might seek an MRI if it costs him or her little or nothing, even if the physician feels the clinical value of the MRI is negligible. Conversely, the physician may be more cautious in ordering a test that seems likely to produce little information if there are severe financial consequences for the patient.

1	Some of the strongest evidence on this point comes from the randomized RAND Health Insurance Experiment conducted in the late 1970s and early 1980s. Families whose members were under 65 years of age were randomized to insurance plans in which the amount they had to pay when using services (“cost sharing”) varied from nothing (fully insured care) to a large deductible (catastrophic insurance). All the plans capped families’ annual out-of-pocket payments, with a reduced cap for low-income families. Families with complete insurance used ~40% more services in a year than did families with catastrophic insurance, a figure that did not vary much across the six geographically dispersed sites in which the experiment was run. Although these data come from the era before managed care in the United States, subsequent observational studies in this country and elsewhere have largely confirmed the experiment’s findings with respect to the relationship between variations in care use and variations

1	States, subsequent observational studies in this country and elsewhere have largely confirmed the experiment’s findings with respect to the relationship between variations in care use and variations in patient payment at the point of service. The difference among the plans was almost entirely related to the likelihood that a patient would seek care. Once care was sought, there appeared to be little difference in how physicians treated their patients in different plans.

1	One might assume that the additional care provided to fully insured patients would have resulted in improved outcomes, but by and large it did not. In fact, there was little or no difference in average health outcomes among the different health plans, with the important exception that hypertension, especially in patients with low incomes, was better controlled when care was free.

1	A possible explanation for the paucity of beneficial effects attributable to the additional medical services used by fully insured patients lies in the observations that (1) the additional care targeted both problems for which care can be efficacious and those for which it is not and (2) the population in the experiment, which consisted of non-elderly community-dwelling individuals, was mostly healthy. Perhaps the additional two visits and the greater number of hospitalizations when care was free were as likely to lead to poor outcomes as to good outcomes in that population. Certainly, the subsequent literature on quality of care and medical error rates has implied that a good deal of inappropriate care was—and is—provided to patients. For example, more than half of the antibiotics prescribed to the experiment’s participants were prescribed for viral conditions. Moreover, about one-quarter of patients who were hospitalized (in all plans) were admitted for procedures that could have

1	to the experiment’s participants were prescribed for viral conditions. Moreover, about one-quarter of patients who were hospitalized (in all plans) were admitted for procedures that could have been performed equally well outside the hospital, in line with the substantial decrease in hospital use over the last three decades. In short, the additional inappropriate care provided when care was free was not necessarily innocuous; if a mainly healthy person saw a physician, he or she could have been made worse off. The literature on inappropriate care is mostly American in origin, but the finding probably holds elsewhere as well.

1	Finally, patients’ health habits did not change in response to insurance status. This finding is consistent with the intuition that moral hazard does not much affect incentives to prevent illness.

1	Recently, another randomized experiment was conducted in Oregon among low-income, childless adults who were uninsured. Many people who gained insurance coverage in 2014 when the United States implemented the Affordable Care Act are from this group. Some of the uninsured childless adults won a lottery that made them eligible for Medicaid; those who did not win became the comparison group. After ~2 years, the results suggested that the use of services by persons on Medicaid had increased by ~25–35%. Medicaid served its purpose of providing protection against large medical bills; there was an 81% reduction in the proportion of families who spent >30% of their income on medical care, and there were large reductions in both medical debt and borrowing to pay for medical care. Turning to health outcomes, there was a 30% reduction in depression among the uninsured who received Medicaid relative to the comparison group as well as an increase in the numbers of diagnosed diabetics and of

1	health outcomes, there was a 30% reduction in depression among the uninsured who received Medicaid relative to the comparison group as well as an increase in the numbers of diagnosed diabetics and of diabetics taking medication. Although there were no statistically significant changes in measures of blood pressure, lipids, or glycosylated hemoglobin, confidence intervals were sufficiently large that clinically meaningful effects could not be ruled out.

1	In sum, insurance is certainly desirable to protect families against the financial risk of large medical expenses and in some instances to address underuse of valuable medical services (e.g., by a patient with cardiovascular disease who fails to take medications for financial reasons). Thus, the remedy for moral hazard is not to abolish insurance but instead to strike the right balance between financial protection and incentives to seek care. Moreover, it is probably useful to vary the amounts that patients pay out of pocket, depending on the specific service and the patient’s clinical condition. Health outcomes after myocardial infarction, for example, were better among patients who were randomized to have no copayments for statins, beta blockers, angiotensin-converting enzyme inhibitors, and angiotensin receptor blockers than among those who had to pay for these drugs.

1	Because insurers, whether public or private, cannot pay any price that a physician sets, prices in medical markets with widespread insurance are either set administratively or negotiated. In the simple textbook model of a competitive market, prices approximate the cost of production, but this is not necessarily the case when prices are administered. In the traditional American Medicare program, for example, the government sets a take-it-or-leave-it price. Because of the market share represented by the program, the great majority of physicians choose to take the government’s price rather than leave the program. In some countries (e.g., Canada and Germany), medical societies negotiate fees for all physicians in the nation or in a subnational area. In the United States, commercial insurers negotiate fees with individual physicians or groups of physicians.

1	The principal problem with administered prices arises because someone must set them and that person has an imperfect knowledge of cost. If the price that is set departs markedly from incremental cost, distortions inevitably result. Because the price-setter typically has little information about incremental cost, the set price could be, and often is, far from the actual cost. If the regulator sets the price below cost, the service may not be available or, if it is, will have to be cross-subsidized from a profitable service. If the price is set above cost, there may well be excess entry and too many services being offered on too small a scale. A beneficent regulator in theory could approximate a competitive price by trial and error if technology did not change, but that condition clearly does not hold in medical care. Not only do new goods and services appear continually, but physicians often become more skilled at delivering a service that is already available or at developing new

1	does not hold in medical care. Not only do new goods and services appear continually, but physicians often become more skilled at delivering a service that is already available or at developing new tools to deliver that service at a different (and frequently lower) cost. For example, cataract surgery, which took upward of 8 h when first introduced, can now be completed in <30 min.

1	The distortions between price and cost when prices are administered have consequences for the way medical care is produced. There may well be too much capacity in “profitable” areas of medicine, such as cardiac services and sports medicine, and too little in less profitable areas, such as primary care. A fee above incremental cost for a procedure encourages more procedures.

1	Conversely, payment methods that attempt to cover many services with one fixed payment, such as capitation and a per-admission payment, pay nothing for doing more and therefore may result in too few services and in choices by providers to reduce the number of unprofitable patients under their care, much as a hospital may shutter an emergency room if it becomes a magnet for the uninsured. These phenomena also reflect the asymmetry of information between patients and physicians and, in the case of fee-for-service payment, the incentive for insured patients to go along with a recommendation for additional services (“I am pretty sure I know what the problem is, but 15e-3 let’s just carry out this additional test to be sure”).

1	There is good evidence that physicians respond to prices that are set. For example, if there is a general reduction in fees that, other things being equal, would lower practice income, some physicians order more services, whereas the opposite pertains if all fees increase. This behavior is sufficiently well established empirically that the U.S. Medicare program’s actuaries account for it in their estimates of what various changes in fees will cost or save. The outcome is different if the fee for one procedure or service changes and that procedure accounts for a modest proportion of income. In that case, another service can be substituted. For example, if the fee for a mastectomy increases relative to that for breast-conserving surgery, there will be a higher proportion of mastectomies. A particularly striking example of this type of behavior occurred when Medicare sharply reduced the fees it paid oncologists for chemotherapy in 2005. The proportion of lung cancer patients who received

1	striking example of this type of behavior occurred when Medicare sharply reduced the fees it paid oncologists for chemotherapy in 2005. The proportion of lung cancer patients who received chemotherapy rose by 10%. Margins for some chemotherapeutic agents, however, were cut more than those for other agents, and thereafter oncologists made less use of the agents whose margins had fallen more.

1	Negotiated prices may get closer to cost than administered prices that are set, but they are not a panacea. First, negotiated prices may well exceed cost when there is no effective competition among similar physicians in a particular market. Because medical care markets are typically local, there may only be one group or a few groups in any particular specialty in a smaller market, in which case physicians will have considerable market power to obtain more favorable reimbursement. Further increasing physicians’ market power is the fact that many, and probably most, patients are reluctant to change physicians because their current physician knows their medical history, because they are uncertain whether a new physician would be an improvement, and because insurance may shield them from most of the cost differences among physicians. Finally, in the United States, commercial insurers often negotiate fees as a multiple of the Medicare fee schedule; thus, any distortion in administratively

1	of the cost differences among physicians. Finally, in the United States, commercial insurers often negotiate fees as a multiple of the Medicare fee schedule; thus, any distortion in administratively determined relative fees is carried over into negotiated fees. For example, in the Medicare fee schedule, procedures generally are more profitable than cognitive services known as “evaluation and management,” and this difference probably plays a role in the numerical insufficiency of primary care physicians in the United States.

1	One branch of economics—positive economics—seeks to explain actual phenomena without making a judgment about the desirability of those phenomena. Another branch—normative economics—seeks to prescribe what should happen and, in particular, what public policy should be developed to ensure that it happens. The main result of the application of normative economics is that, under certain very special assumptions, competitive markets result in a system in which no one can be made better off without another person’s being made worse off. These assumptions do not hold in medical care, in part because of selection and moral hazard; economists term the result a market failure. By contrast, as the discussion of administered prices in this chapter indicates, even a beneficent regulator will introduce distortions from lack of sufficient information; moreover, there is no guarantee that a regulator will be beneficent, as periodic corruption scandals show. Economists term this phenomenon regulatory

1	distortions from lack of sufficient information; moreover, there is no guarantee that a regulator will be beneficent, as periodic corruption scandals show. Economists term this phenomenon regulatory or government failure. Economists see decisions about the proper form and amount of public intervention and regulation in medical care as a matter of finding the right balance between various types of market failures and various types of regulatory failures—a balance that different societies may choose to strike differently.

1	CHAPTER 15e The Economics of Medical Care Racial and Ethnic Disparities in Health Care Joseph R. Betancourt, Alexander R. Green Over the course of its history, the United States has experienced dramatic improvements in overall health and life expectancy, largely 16e

1	Native American, Alaskan Native 329.8 300 293.7 258.0 228.3 222.3 192.4 179.6 173.2 168.2 155.2 152.7 147.6 141.1 133.0 129.1 115.9 105.9 102.0 94.5 91.9 28.2 12.0 4.0 7.5 3.6 1.5 0 1.9 1.0 0.8 as a result of initiatives in public health, health promotion, disease prevention, and chronic care management. Our ability to prevent, detect, and treat diseases in their early stages has allowed us to target and reduce rates of morbidity and mortality. Despite interventions that have improved the overall health of the majority of Americans, racial and ethnic minorities (blacks, Hispanics/Latinos, Native Americans/ Alaskan Natives, Asian/Pacific Islanders) have benefited less from these advances than whites and have suffered poorer health outcomes from many major diseases, including cardiovascular disease, cancer, and diabetes. Research has revealed that minorities may receive less care and lower-quality care than whites, even when confounders such as stage of presentation, comorbidities, and

1	disease, cancer, and diabetes. Research has revealed that minorities may receive less care and lower-quality care than whites, even when confounders such as stage of presentation, comorbidities, and health insurance are controlled. These differences in quality are called racial and ethnic disparities in health care. Addressing these disparities has taken on greater importance with the significant transformation of the U.S. health care system and the implementation of health care reform and value-based purchasing. The shift toward creating financial incentives and disincentives to achieve quality goals makes focusing on those who receive lower-quality care more important than ever before. This chapter will provide an overview of racial and ethnic disparities in health and health care, identify root causes, and provide key recommendations to address these disparities at both the clinical and health system levels.

1	Minority Americans have poorer health outcomes than whites from preventable and treatable conditions such as cardiovascular disease, diabetes, asthma, cancer, and HIV/AIDS (Fig. 16e-1). Multiple factors contribute to these racial and ethnic disparities in health. First and foremost, social determinants—such as lower socioeconomic status (SES; e.g., lower income, less wealth, and lower educational attainment), inadequate and unsafe housing, and racism—are strongly linked to poor health outcomes. These factors disproportionately impact minority populations. In fact, SES has consistently been found to be one of the strongest predictors of health outcomes. While the mechanisms are complex (i.e., does poverty cause poor health, or does poor health cause poverty?), it is clear that low-SES populations expe-16e-1 rience disparities in health and that low SES is a major factor in racial/ ethnic disparities.

1	Racial/ethnic disparities are documented globally, although their assessment has centered more on the comparison of individuals by SES in other countries than in the United States. Similar to the U.S. pattern, low-SES residents of other nations tend to have poorer health outcomes. It is noteworthy that results are mixed when the health status of nations is compared by SES. High-SES nations such as the United States do not necessarily have health outcomes that correlate with their high expenditures for health care. For example, as of 2011, the United States ranks 34th in the world—just behind Cuba—on basic public health measures�such as infant mortality. This ranking may be due in part to the correlation between wealth distribution and SES rather than just absolute SES. This area of active research is outside the scope of this chapter.

1	Racism has more recently been shown to predict poorer health outcomes. The physiologic impact of the stress imposed by racism (and poverty), including increased cortisol levels, can lead to chronic adverse effects on health. Lack of access to care also takes a significant toll. Uninsured individuals are less likely to have a regular source of care and are more likely to delay seeking care and to go without needed care; this limited access results in avoidable hospitalizations, emergency hospital care, and adverse health outcomes.

1	In addition to racial and ethnic disparities in health, there are racial and ethnic disparities in the quality of care for persons with access to the health care system. For instance, disparities have been found in the treatment of pneumonia (Fig. 16e-2) and congestive heart failure, with blacks receiving less optimal care than whites when hospitalized for these conditions. Moreover, blacks with end-stage renal disease are referred less often to the transplant list than are their white counterparts (Fig. 16e-3). Disparities have been found, for example, in the use of cardiac diagnostic and therapeutic procedures (with blacks being referred less often than whites for cardiac catheterization and bypass grafting), prescription of analgesia for pain control (with blacks and Latinos receiving less pain medication than whites for long-bone fractures and cancer), and surgical treatment of lung cancer (with blacks receiving less curative surgery than whites for non-small-cell lung cancer).

1	less pain medication than whites for long-bone fractures and cancer), and surgical treatment of lung cancer (with blacks receiving less curative surgery than whites for non-small-cell lung cancer). Again, many of these disparities have occurred even when variations in factors such as insurance status, income, age, comorbid conditions, and symptom expression are taken into account. However, one additional factor—disparities in the quality of care provided at the sites where minorities tend to receive care—has been shown to be an important contributor to overall disparities.

1	FIgURE 16e-1 Age-adjusted death rates for selected causes by race and Hispanic origin, 2005. (From the U.S. Census Bureau, 2009.) 81.5 80 76.9 76.8 75.8 74.2 73.3 69.5 74.6 68.7 66.2 FIgURE 16e-2 Recommended hospital care received by Medicare patients with pneumonia, by race/ethnicity, 2006. The reference population consisted of Medicare beneficiaries with pneumonia who were hospitalized. The composite was calculated by averaging the percentage of the population that received each of the five incorporated components of care. (Adapted from Agency for Healthcare Research and Quality: The 2008 National Health Care Disparities Report.)

1	Little progress has been made in addressing racial/ethnic disparities in cardiovascular procedures and other advanced surgical procedures, whereas some progress has been made in eliminating disparities in primary-care process measures. Data from the National Registry of Myocardial Infarction found evidence of continued disparities in guideline-based admission, procedural, and discharge therapy use from 1994 to 2006. Black patients were less likely than white patients to receive percutaneous coronary intervention/coronary artery bypass grafting (PCI/CABG), a disparity that has improved little since 1994. Further, compared with whites, black patients were less likely to receive lipid-lowering medications at discharge, with a gap that has widened since 1998 (Fig. 16e-4). A 2009 study showed that blacks had worse post–myocardial infarction outcomes than whites but that the difference could be explained by site of care and patient factors (such as socioeconomic status and comorbid

1	showed that blacks had worse post–myocardial infarction outcomes than whites but that the difference could be explained by site of care and patient factors (such as socioeconomic status and comorbid conditions).

1	The Centers for Disease Control and Prevention (CDC) analyzed national and state rates of total knee replacement (TKR) for Medicare enrollees for the period 2000–2006, with patients stratified by sex, age, and black or white race. TKR rates overall in the United States 82.2 80.3 68.9 67.9 59.6 57.9 40.6 40 40.3 FIgURE 16e-3 Referral for evaluation at a transplantation center or placement on a waiting list/receipt of a renal transplant within 18 months after the start of dialysis among patients who wanted a transplant, according to race and sex. The reference population consisted of 239 black women, 280 white women, 271 black men, and 271 white men. Racial differences were statistically significant among both the women and the men (p<.0001 for each comparison). (From JZ Ayanian et al: N Engl J Med 341:1661, 1999.)

1	Percentage of patients increased 58%, with similar increases among whites (61%) and blacks (56%). However, the TKR rate for blacks was 37% lower than the rate for whites in 2000 and 39% lower in 2006; i.e., the disparity not only did not improve but even worsened slightly (Fig. 16e-5). More recent data (up to 2010) show no apparent change in these figures. Data for enrollees in Medicare managed-care plans provides evidence for a narrowing in racial disparities between 1997 and 2003 in several “report card” preventive care measures, such as mammography and glucose and cholesterol testing. However, racial disparities in more complex measures, such as glucose control in diabetic patients and cholesterol levels in patients after a heart attack, actually worsened during that interval.

1	The 2012 National Healthcare Disparities Report, released by the Agency for Healthcare Research and Quality, found little improvement in disparities for core measures of quality over the previous decade. In fact, for blacks, Asians, Native Americans/Alaskan Natives, Hispanics, and poor people, the vast majority of core quality measures (87–92%) stayed the same, and a small proportion (2–8%) got worse, including measures of effectiveness, patient safety, and timeliness of care. While a small number of these measures improved, disparities were eliminated in no measured area. This annual report is particularly important, given that most studies of disparities have not been repeated with the same methodology used to document possible trends. Some studies have tracked disparities using specific disease and treatment registries. For example, by 2008, the use of acute and discharge medications for myocardial infarction had largely been equalized among racial and ethnic groups; however,

1	disease and treatment registries. For example, by 2008, the use of acute and discharge medications for myocardial infarction had largely been equalized among racial and ethnic groups; however, African-American and Hispanic patients still experienced longer delays before reperfusion, with door-to-balloon times of <90 min for 83% of white patients as opposed to 75% and 76% of black and Hispanic patients, respectively.

1	The Institute of Medicine (IOM) report Unequal Treatment, released in March 2002, remains the preeminent study of racial and ethnic disparities in health care in the United States. The IOM was charged with assessing the extent of racial/ethnic differences in health care that are not otherwise attributable to known factors, such as access to care. To provide recommendations regarding interventions aimed at eliminating health care disparities, the IOM studied health system, provider, and patient factors. The study found the following: Racial and ethnic disparities in health care exist and, because they are associated with worse health outcomes, are unacceptable. Racial and ethnic disparities in health care occur in the context of (1) broader historic and contemporary social and economic inequality and (2) evidence of persistent racial and ethnic discrimination in many sectors of American life.

1	Many sources—including health systems, health care providers, patients, and utilization managers—may contribute to racial and ethnic disparities in health care. Bias, stereotyping, prejudice, and clinical uncertainty on the part of health care providers may contribute to racial and ethnic disparities in health care. A small number of studies suggest that minority patients may be more likely to refuse treatments, yet these refusal rates are generally small and do not fully explain health care disparities. Unequal Treatment went on to identify a set of root causes that included the following: Health system factors: These include issues related to the complexity of the health care system, the difficulty that minority patients may have in navigating this complex system, and the lack of availability of interpreter services to assist patients with limited English proficiency. In addition, health care systems are generally ill prepared to identify and address disparities.

1	Provider-level factors: These include issues related to the health care provider, including stereotyping, the impact of race/ethnicity on clinical decision-making, and clinical uncertainty due to poor communication. 0.75 1 1.25 1994–1996 0.53 (0.51, 0.54) 1997–1999 0.52 (0.50, 0.53) 2000–2002 0.55 (0.53, 0.56) 2003–2006 0.54 (0.52, 0.56) Whites more likely to receive 0.25 0.5 0.75 1998–1999 0.92 (0.89, 0.96) 2000–2002 0.84 (0.82, 0.86) 2003–2006 0.76 (0.75, 0.81) likely to receive likely to receive 1 1.25 0.25 0.5 0.75 1 1.25

1	Whites more likely to receive 0.25 0.5 0.75 1998–1999 0.92 (0.89, 0.96) 2000–2002 0.84 (0.82, 0.86) 2003–2006 0.76 (0.75, 0.81) likely to receive likely to receive 1 1.25 0.25 0.5 0.75 1 1.25 FIgURE 16e-4 Racial differences in guideline-based treatments for acute myocardial infarction (AMI). The reference population consisted of 2,515,106 patients with AMI admitted to U.S. hospitals between July 1990 and December 2006. CABG, coronary artery bypass grafting; PCI, percutaneous coronary intervention. (From ED Peterson et al: Am Heart J 156:1045, 2008.) • Patient-level factors: These include patients’ mistrust of the health care system leading to refusal of services, poor adherence to treatment, and delay in seeking care. A more detailed analysis of these root causes is presented below.

1	Health System Factors • HEALTH SYSTEM COMPLEXITY Even among persons who are insured and educated and who have a high degree of health literacy, navigating the U.S. health care system can be complicated and confusing. Some individuals may be at higher risk for receiving substandard care because of their difficulty navigating the system’s complexities. These individuals may include those from cultures unfamiliar with the Western model of health care delivery, those with limited English proficiency, those with low health literacy, and those who are mistrustful of the health care system. These individuals may have difficulty knowing how and where to go for a referral to a specialist; how to prepare for a procedure such as a colonoscopy; or how to follow up on an abnormal test result such as a mammogram. Since people of color in the United States tend to be overrepresented among the groups listed above, the inherent complexity of navigating the health care system has been seen as a root

1	a mammogram. Since people of color in the United States tend to be overrepresented among the groups listed above, the inherent complexity of navigating the health care system has been seen as a root cause for racial/ethnic disparities in health care.

1	OTHER HEALTH SYSTEM FACTORS Racial/ethnic disparities are due not only to differences in care provided within hospitals but also to where and

1	FIgURE 16e-5 Racial trends in age-adjusted total knee replacement in Medicaid enrollees from 2000 to 2006. The reference population consisted of Medicaid part A enrollees ≥65 years of age who were not members of a managed-care plan. (From the Centers for Disease Control and Prevention, 2009.) from whom minorities receive their care; i.e., certain specific providers, geographic regions, or hospitals are lower-performing on certain aspects of quality. For example, one study showed that 25% of hospitals cared for 90% of black Medicare patients in the United States and that these hospitals tended to have lower performance scores on certain quality measures than other hospitals. That said, health systems generally are not well prepared to measure, report, and intervene to reduce disparities in care. Few hospitals or health plans stratify their quality data by race/ethnicity or language to measure disparities, and even fewer use data of this type to develop disparity-targeted interventions.

1	care. Few hospitals or health plans stratify their quality data by race/ethnicity or language to measure disparities, and even fewer use data of this type to develop disparity-targeted interventions. Similarly, despite regulations concerning the need for professional interpreters, research demonstrates that many health care organizations and providers fail to routinely provide this service for patients with limited English proficiency. Despite the link between limited English proficiency and health-care quality and safety, few providers or institutions monitor performance for patients in these areas.

1	Provider-Level Factors • PROVIDER–PATIENT COMMUNICATION Significant evidence highlights the impact of sociocultural factors, race, ethnicity, and limited English proficiency on health and clinical care. Health care professionals frequently care for diverse populations with varied perspectives, values, beliefs, and behaviors regarding health and wellbeing. The differences include variations in the recognition of symptoms, thresholds for seeking care, comprehension of management strategies, expectations of care (including preferences for or against diagnostic and therapeutic procedures), and adherence to preventive measures and medications. In addition, sociocultural differences between patient and provider influence communication and clinical decision-making and are especially pertinent: evidence clearly links provider–patient communication to improved patient satisfaction, regimen adherence, and better health outcomes (Fig. 16e-6). Thus, when sociocultural differences between patient

1	clearly links provider–patient communication to improved patient satisfaction, regimen adherence, and better health outcomes (Fig. 16e-6). Thus, when sociocultural differences between patient and provider are not appreciated, explored, understood, or communicated effectively during the medical encounter, patient dissatisfaction, poor adherence, poorer health outcomes, and racial/ethnic disparities in care may result.

1	A survey of 6722 Americans ≥18 years of age is particularly relevant to this important link between provider–patient communication and health outcomes. Whites, blacks, Hispanics/Latinos, and Asian Americans who had made a medical visit in the past 2 years were asked whether they had trouble understanding their doctors; whether they felt the doctors did not listen; and whether they had medical questions they were afraid to ask. The survey found that 19% of all patients experienced one or more of these problems, yet whites experienced them 16% of the time as opposed to 23% of the time for blacks, 33% for Hispanics/Latinos, and 27% for Asian Americans (Fig. 16e-7). How do we link communication to outcomes? Communication FIgURE 16e-6 The link between effective communication and patient satisfaction, adherence, and health outcomes. (From the Institute of Medicine: Unequal Treatment: Confronting Racial and Ethnic Disparities in Health Care. Washington, DC, National Academy Press, 2002.)

1	In addition, in the setting of even a minimal language barrier, provider–patient communication without an interpreter is recognized as a major challenge to effective health care delivery. These communication barriers for patients with limited English proficiency lead to frequent misunderstanding of diagnosis, treatment, and follow-up plans; inappropriate use of medications; lack of informed consent for surgical procedures; high rates of serious adverse events; and a lower-quality health care experience than is provided to patients who speak fluent English. Physicians who have access to trained interpreters report a significantly higher quality of patient–physician communication than physicians who use other methods. Communication issues related to discordant language disproportionately affect minorities and likely contribute to racial/ethnic disparities in health care.

1	CLINICAL DECISION-MAKING Theory and research suggest that variations in clinical decision-making may contribute to racial and ethnic disparities in health care. Two factors are central to this process: clinical uncertainty and stereotyping. First, a doctor’s decision-making process is nested in clinical uncertainty. Doctors depend on inferences about severity based on what they understand about illness and the information obtained from the patient. A doctor caring for a patient whose symptoms he or she has difficulty understanding and whose “signals”—the set of clues and indications that physicians rely on to make clinical decisions—are hard to read may make a decision different from the one that would be made Percent of adults with one or more communication problems* *Problems include understanding doctor, feeling doctor listened, had questions but did not ask.

1	FIgURE 16e-7 Communication difficulties with physicians, by race/ethnicity. The reference population consisted of 6722 Americans ≥18 years of age who had made a medical visit in the previous 2 years and were asked whether they had had trouble understanding their doctors, whether they felt that the doctors had not listened, and whether they had had medical questions they were afraid to ask. (From the Commonwealth Fund Health Care Quality Survey, 2001.) for another patient who presents with exactly the same clinical condition. Given that the expression of symptoms may differ among cultural and racial groups, doctors—the overwhelming majority of whom are white—may understand symptoms best when expressed by patients of their own racial/ethnic groups. The consequence is that white patients may be treated differently from minority patients. Differences in clinical decisions can arise from this mechanism even when the doctor has the same regard for each patient (i.e., is not prejudiced).

1	Second, the literature on social cognitive theory highlights how natural tendencies to stereotype may influence clinical decision-making. Stereotyping can be defined as the way in which people use social categories (e.g., race, gender, age) in acquiring, processing, and recalling information about others. Faced with enormous information loads and the need to make many decisions, people often subconsciously simplify the decision-making process and lessen cognitive effort by using “categories” or “stereotypes” that bundle information into groups or types that can be processed more quickly. Although functional, stereotyping can be systematically biased, as people are automatically classified into social categories based on dimensions such as race, gender, and age. Many people may not be aware of their attitudes, may not consciously endorse specific stereotypes, and paradoxically may consider themselves egalitarian and not prejudiced.

1	Stereotypes may be strongly influenced by the messages presented consciously and unconsciously in society. For instance, if the media and our social/professional contacts tend to present images of minorities as being less educated, more violent, and nonadherent to health care recommendations, these impressions may generate stereotypes that unnaturally and unjustly impact clinical decision-making. As signs of racism, classism, gender bias, and ageism are experienced (consciously or unconsciously) in our society, stereotypes may be created that impact the way doctors manage patients from these groups. On the basis of training or practice location, doctors may develop certain perceptions about race/ethnicity, culture, and class that may evolve into stereotypes. For example, many medical students and residents are often trained—and minorities cared for—in academic health centers or public hospitals located in socioeconomically disadvantaged areas. As a result, doctors may begin to equate

1	and residents are often trained—and minorities cared for—in academic health centers or public hospitals located in socioeconomically disadvantaged areas. As a result, doctors may begin to equate certain races and ethnicities with specific health beliefs and behaviors (e.g., “these patients” engage in risky behaviors, “those patients” tend to be noncompliant) that are more associated with the social environment (e.g., poverty) than with a patient’s racial/ethnic background or cultural traditions. This “conditioning” phenomenon may also be operative if doctors are faced with certain racial/ethnic patient groups who frequently do not choose aggressive forms of diagnostic or therapeutic intervention. The result over time may be that doctors begin to believe that “these patients” do not like invasive procedures; thus they may not offer these procedures as options. A wide range of studies have documented the potential for provider biases to contribute to racial/ethnic disparities in health

1	procedures; thus they may not offer these procedures as options. A wide range of studies have documented the potential for provider biases to contribute to racial/ethnic disparities in health care. For example, one study measured physicians’ unconscious (or implicit) biases and showed that these were related to differences in decisions to provide thrombolysis for a hypothetical black or white patient with a myocardial infarction.

1	It is important to differentiate stereotyping from prejudice and discrimination. Prejudice is a conscious prejudgment of individuals that may lead to disparate treatment, and discrimination is conscious and intentional disparate treatment. All individuals stereotype subconsciously, yet, if left unquestioned, these subconscious assumptions may lead to lower-quality care for certain groups because of differences in clinical decision-making or differences in communication and patientcenteredness. For example, one study tested physicians’ unconscious racial/ethnic biases and showed that patients perceived more biased physicians as being less patient-centered in their communication. What is particularly salient is that stereotypes tend to be activated most in environments where the individual is stressed, multitasking, and under time pressure—the hallmarks of the clinical encounter.

1	Patient-Level Factors Lack of trust has become a major concern for many health care institutions today. For example, an IOM report, To Err Is Human: Building a Safer Health System, documented alarming rates of medical errors that made patients feel vulnerable and less trustful of the U.S. health care system. The increased media and academic attention to problems related to quality of care (and of disparities themselves) has clearly diminished trust in doctors and nurses.

1	Trust is a crucial element in the therapeutic alliance between patient and health care provider. It facilitates open communication and is directly correlated with adherence to the physician’s recommendations and the patient’s satisfaction. In other words, patients who mistrust their health care providers are less satisfied with the care they receive, and mistrust of the health care system greatly affects patients’ use of services. Mistrust can also result in inconsistent care, “doctor-shopping,” self-medication, and an increased demand by patients for referrals and diagnostic tests.

1	On the basis of historic factors such as discrimination, segregation, and medical experimentation, blacks may be especially mistrustful of providers. The exploitation of blacks by the U.S. Public Health Service during the Tuskegee syphilis study from 1932 to 1972 left a legacy of mistrust that persists even today among this population. Other populations, including Native Americans/Alaskan Natives, Hispanics/ Latinos, and Asian Americans, also harbor significant mistrust of the health care system. A national Kaiser Family Foundation survey of 3884 individuals found that 36% of Hispanics and 35% of blacks (compared with 15% of whites) felt they had been treated unfairly in the health care system in the past because of their race/ethnicity. Perhaps even more alarming, 65% of blacks and 58% of Hispanics (compared with 22% of whites) were afraid of being treated unfairly in the future on that basis (Fig. 16e-8).

1	This mistrust may contribute to wariness in accepting or following recommendations, undergoing invasive procedures, or participating in clinical research, and these choices, in turn, may lead to misunderstanding and the perpetuation of stereotypes among health professionals. The publication Unequal Treatment provides a series of recommendations to address racial and ethnic disparities in health care, focusing on a broad set of stakeholders. These recommendations include health system interventions, provider interventions, patient interventions, and general recommendations, which are described in more detail below. Health System Interventions • COLLECTION AND REPORTING OF DATA ON HEALTH CARE ACCESS AND USE, bY PATIENTS’ RACE/ETHNICITY Unequal Treatment found that the appropriate systems to track and monitor racial and ethnic disparities in health care are lacking and that less is known about the disparities affecting minority groups other than African

1	FIgURE 16e-8 Patient perspectives regarding unfair treatment (Tx) based on race/ethnicity. The reference population consisted of 3884 individuals surveyed about how fairly they had been treated in the health care system in the past and how fairly they felt they would be treated in the future on the basis of their race/ethnicity. (From Race, Ethnicity & Medical Care: A Survey of Public Perceptions and Experiences. Kaiser Family Foundation, 2005.)

1	Americans (Hispanics, Asian Americans, Pacific Islanders, Native 16e-5 Americans, and Alaskan Natives). For instance, only in the mid-1980s did the Medicare database begin to collect data on patient groups outside the standard categories of “white,” “black,” and “other.” Federal, private, and state-supported data-collection efforts are scattered and unsystematic, and many health care systems and hospitals still do not collect data on the race, ethnicity, or primary language of enrollees or patients. A survey by Regenstein and Sickler in 2006 found that 78% of 501 U.S. hospitals collected information on race, 50% collected data on ethnicity, and 50% collected data on primary language. However, the information was not collected by standard categories or collection methods and thus was of questionable accuracy. Surveys by America’s Health Insurance Plans in 2003 and 2006 showed that the proportion of enrollees in plans that collected race/ethnicity data of some type increased from 54% to

1	accuracy. Surveys by America’s Health Insurance Plans in 2003 and 2006 showed that the proportion of enrollees in plans that collected race/ethnicity data of some type increased from 54% to 67%; however, the total percentage of plan enrollees whose race/ethnicity and language are recorded is still much lower than these figures.

1	ENCOURAGEMENT OF THE USE OF EVIDENCE-bASED GUIDELINES AND QUALITY IMPROVEMENT Unequal Treatment highlights the subjectivity of clinical decision-making as a potential cause of racial and ethnic disparities in health care by describing how clinicians—despite the existence of well-delineated practice guidelines—may offer (consciously or unconsciously) different diagnostic and therapeutic options to different patients on the basis of their race or ethnicity. Therefore, the widespread adoption and implementation of evidence-based guidelines is a key recommendation in eliminating disparities. For instance, evidence-based guidelines are now available for the management of diabetes, HIV/AIDS, cardiovascular diseases, cancer screening and management, and asthma—all areas where significant disparities exist. As part of ongoing quality-improvement efforts, particular attention should be paid to the implementation of evidence-based guidelines for all patients, regardless of their race and

1	exist. As part of ongoing quality-improvement efforts, particular attention should be paid to the implementation of evidence-based guidelines for all patients, regardless of their race and ethnicity.

1	SUPPORT FOR THE USE OF LANGUAGE INTERPRETATION SERVICES IN THE CLINICAL SETTING As described previously, a lack of efficient and effective interpreter services in a health care system can lead to patient dissatisfaction, to poor comprehension and adherence, and thus to ineffective/lower-quality care for patients with limited English proficiency. Unequal Treatment’s recommendation to support the use of interpretation services has clear implications for delivery of quality health care by improving doctors’ ability to communicate effectively with these patients.

1	INCREASES IN THE PROPORTION OF UNDERREPRESENTED MINORITIES IN THE HEALTH CARE WORKFORCE Data for 2004 from the Association of American Medical Colleges indicate that, of the 72.4% of U.S. physicians whose race and ethnicity are known, Hispanics make up 2.8%, blacks 3.3%, and Native American and Alaskan Natives 0.3%. Furthermore, U.S. national data show that minorities (excluding Asians) compose just 7.5% of medical school faculty. In addition, minority faculty in 2007 were more likely to be at or below the rank of assistant professor, while whites composed the highest proportion of full professors. Despite representing ∼26% of the U.S. population (a number projected to almost double by 2050), minority students are still underrepresented in medical schools. In 2007, matriculants to U.S. medical schools were 7.2% Latino, 6.4% African American, 0.2% Native Hawaiian or Other Pacific Islander, and 0.3% Native American or Alaskan Native. These percentages have decreased or remained the same

1	schools were 7.2% Latino, 6.4% African American, 0.2% Native Hawaiian or Other Pacific Islander, and 0.3% Native American or Alaskan Native. These percentages have decreased or remained the same since 2007. It will be difficult to develop a diverse health-care workforce that can meet the needs of an increasingly diverse population without dramatic changes in the racial and ethnic composition of medical student bodies.

1	Provider Interventions • INTEGRATION OF CROSS-CULTURAL EDUCATION INTO THE TRAINING OF ALL HEALTH CARE PROFESSIONALS The goal of cross-cultural education is to improve providers’ ability to understand, communicate with, and care for patients from diverse backgrounds. Such education focuses on enhancing awareness of sociocultural influences on health beliefs and behaviors and on building skills to facilitate understanding and management of these factors in the medical encounter. Cross-cultural education includes curricula on health care disparities, use of interpreters, and effective communication and negotiation across cultures. These curricula can be incorporated into health-professions training in medical schools, residency programs, and nursing schools and can be offered as a component of continuing education. Despite the importance of this area of education and the attention it has attracted from medical education accreditation bodies, a national survey of senior resident

1	of continuing education. Despite the importance of this area of education and the attention it has attracted from medical education accreditation bodies, a national survey of senior resident physicians by Weissman and colleagues found that up to 28% felt unprepared to deal with cross-cultural issues, including caring for patients who have religious beliefs that may affect treatment, patients who use complementary medicine, patients who have health beliefs at odds with Western medicine, patients who mistrust the health care system, and new immigrants. In a study at one medical school, 70% of fourth-year students felt inadequately prepared to care for patients with limited English proficiency. Efforts to incorporate cross-cultural education into medical education will contribute to improving communication and to providing a better quality of care for all patients.

1	INCORPORATION OF TEACHING ON THE IMPACT OF RACE, ETHNICITY, AND CULTURE ON CLINICAL DECISION-MAKING Unequal Treatment and more recent studies found that stereotyping by health care providers can lead to disparate treatment based on a patient’s race or ethnicity. The Liaison Committee on Medical Education, which accredits medical schools, issued a directive that medical education should include instruction on how a patient’s race, ethnicity, and culture might unconsciously impact communication and clinical decision-making. Patient Interventions Difficulty navigating the health care system and obtaining access to care can be a hindrance to all populations, particularly to minorities. Similarly, lack of empowerment or involvement in the medical encounter by minorities can be a barrier to care. Patients need to be educated on how to navigate the health care system and how best to access care. Interventions should be used to increase patients’ participation in treatment decisions.

1	general Recommendations • INCREASE AWARENESS OF RACIAL/ETHNIC DISPARITIES IN HEALTH CARE Efforts to raise awareness of racial/ethnic health care disparities have done little for the general public but have been fairly successful among physicians, according to a Kaiser Family Foundation report. In 2006, nearly 6 in 10 people surveyed believed that blacks received the same quality of care as whites, and 5 in 10 believed that Latinos received the same quality of care as whites. These estimates are similar to findings in a 1999 survey. Despite this lack of awareness, most people believed that all Americans deserve quality care, regardless of their background. In contrast, the level of awareness among physicians has risen sharply. In 2002, the majority (69%) of physicians said that the health care system “rarely or never” treated people unfairly on the basis of their racial/ethnic background. In 2005, less than one-quarter (24%) of physicians disagreed with the statement that “minority

1	care system “rarely or never” treated people unfairly on the basis of their racial/ethnic background. In 2005, less than one-quarter (24%) of physicians disagreed with the statement that “minority patients generally receive lower-quality care than white patients.” Increasing awareness of racial and ethnic health disparities among health care professionals and the public is an important first step in addressing these disparities. The ultimate goals are to generate discourse and to mobilize action to address disparities at multiple levels, including health policy makers, health systems, and the community.

1	CONDUCT FURTHER RESEARCH TO IDENTIFY SOURCES OF DISPARITIES AND PROMISING INTERVENTIONS While the literature that formed the basis for the findings reported and recommendations made in Unequal Treatment provided significant evidence for racial and ethnic disparities, additional research is needed in several areas. First, most of the literature on disparities focuses on black-versus-white differences; much less is known about the experiences of other minority groups. Improving the ability to collect racial and ethnic patient data should facilitate this process. However, in instances where the necessary systems are not yet in place, racial and ethnic patient data may be collected prospectively in the setting of clinical or health services research to more fully elucidate disparities for other populations. Second, much of the literature on disparities to date has focused on defining areas in which these disparities exist, but less has been done to identify the multiple factors that

1	other populations. Second, much of the literature on disparities to date has focused on defining areas in which these disparities exist, but less has been done to identify the multiple factors that contribute to the disparities or to test interventions to address these factors. There is clearly a need for research that identifies promising practices and solutions to disparities.

1	Individual health care providers can do several things in the clinical encounter to address racial and ethnic disparities in health care. Be Aware that Disparities Exist Increasing awareness of racial and ethnic disparities among health care professionals is an important first step in addressing disparities in health care. Only with greater awareness can care providers be attuned to their behavior in clinical practice and thus monitor that behavior and ensure that all patients receive the highest quality of care, regardless of race, ethnicity, or culture.

1	Practice Culturally Competent Care Previous efforts have been made to teach clinicians about the attitudes, values, beliefs, and behaviors of certain cultural groups—the key practice “dos and don’ts” in caring for “the Hispanic patient” or the “Asian patient,” for example. In certain situations, learning about a particular local community or cultural group, with a goal of following the principles of community-oriented primary care, can be helpful; when broadly and uncritically applied, however, this approach can actually lead to stereotyping and oversimplification of culture, without respect for its complexity.

1	Cultural competence has thus evolved from merely learning information and making assumptions about patients on the basis of their backgrounds to focusing on the development of skills that follow the principles of patient-centered care. Patient-centeredness encompasses the qualities of compassion, empathy, and responsiveness to the needs, values, and expressed preferences of the individual patient. Cultural competence aims to take things a step further by expanding the repertoire of knowledge and skills classically defined as “patient-centered” to include those that are especially useful in cross-cultural interactions (and that, in fact, are vital in all clinical encounters). This repertoire includes effectively using interpreter services, eliciting the patient’s understanding of his or her condition, assessing decision-making preferences and the role of family, determining the patient’s views about biomedicine versus complementary and alternative medicine, recognizing sexual and

1	or her condition, assessing decision-making preferences and the role of family, determining the patient’s views about biomedicine versus complementary and alternative medicine, recognizing sexual and gender issues, and building trust. For example, while it is important to understand all patients’ beliefs about health, it may be particularly crucial to understand the health beliefs of patients who come from a different culture or have a different health care experience. With the individual patient as teacher, the physician can adjust his or her practice style to meet the patient’s specific needs.

1	Avoid Stereotyping Several strategies can allow health care providers to counteract, both systemically and individually, the normal tendency to stereotype. For example, when racially/ethnically/culturally/socially diverse teams in which each member is given equal power are assembled and are tasked to achieve a common goal, a sense of camaraderie develops and prevents the development of stereotypes based on race/ ethnicity, gender, culture, or class. Thus, health care providers should aim to gain experiences working with and learning from a diverse set of colleagues. In addition, simply being aware of the operation of social cognitive factors allows providers to actively check up on or monitor their behavior. Physicians can constantly reevaluate to ensure that they are offering the same things, in the same ways, to all patients. Understanding one’s own susceptibility to stereotyping—and how disparities may result—is essential in providing equitable, high-quality care to all patients.

1	Work to Build Trust Patients’ mistrust of the health care system and of health care providers impacts multiple facets of the medical encounter, with effects ranging from decreased patient satisfaction to delayed care. Although the historic legacy of discrimination can never be erased, several steps can be taken to build trust with patients and to address disparities. First, providers must be aware that mistrust exists and is more prevalent among minority populations, given the history of discrimination in the United States and other countries. Second, providers must reassure patients that they come first, that everything possible will be done to ensure that they always get the best care available, and that their caregivers will serve as their advocates. Third, interpersonal skills and communication techniques that demonstrate honesty, openness, compassion, and respect on the part of the health care provider are essential tools in dismantling mistrust. Finally, patients indicate that

1	techniques that demonstrate honesty, openness, compassion, and respect on the part of the health care provider are essential tools in dismantling mistrust. Finally, patients indicate that trust is built when there is shared, participatory decision-making and the provider makes a concerted effort to understand the patient’s background. When the doctor–patient relationship is reframed as one of solidarity, the patient’s sense of vulnerability can be transformed into one of trust. The successful elimination of disparities requires trust-building interventions and strengthening of this relationship.

1	The issue of racial and ethnic disparities in health care has gained national prominence, both with the release of the IOM report Unequal Treatment and with more recent articles that have confirmed their persistence and explored their root causes. Furthermore, another 16e-7 influential IOM report, Crossing the Quality Chasm, has highlighted the importance of equity—i.e., no variations in quality of care due to personal characteristics, including race and ethnicity—as a central principle of quality. Current efforts in health care reform and transformation, including a greater focus on value (high-quality care and cost-control), will sharpen the nation’s focus on the care of populations who experience low-quality, costly care. Addressing disparities will become a major focus, and there will be many obvious opportunities for interventions to eliminate them. Greater attention to addressing the root causes of disparities will improve the care provided to all patients, not just those who

1	be many obvious opportunities for interventions to eliminate them. Greater attention to addressing the root causes of disparities will improve the care provided to all patients, not just those who belong to racial and ethnic minorities.

1	The authors thank Marina Cervantes for her contributions to this chapter.

1	Ethical Issues in Clinical Medicine Bernard Lo, Christine Grady Twenty-first-century physicians face novel ethical dilemmas that can be perplexing and emotionally draining. For example, electronic medical records, handheld personal devices, and provision of care by interdisciplinary teams all hold the promise of more coordinated and 17e comprehensive care but also raise new concerns about confidentiality, appropriate boundaries of the doctor–patient relationship, and responsibility. Chapter 1 puts the practice of medicine into a professional and historical context. The current chapter presents approaches and principles that physicians can use to address the ethical issues they encounter in their work. Physicians make ethical judgments about clinical situations every day. Traditional professional codes and ethical principles provide instructive guidance for physicians but need to be interpreted and applied to each situation. Physicians need to be prepared for lifelong learning about

1	codes and ethical principles provide instructive guidance for physicians but need to be interpreted and applied to each situation. Physicians need to be prepared for lifelong learning about ethical issues and dilemmas as well as about new scientific and clinical developments. When struggling with difficult ethical issues, physicians may need to reevaluate their basic convictions, tolerate uncertainty, and maintain their integrity while respecting the opinions of others. Discussing perplexing ethical issues with other members of the health care team, ethics consultation services, or the hospital ethics committee can clarify issues and reveal strategies for resolution, including improving communication and dealing with strong or conflicting emotions.

1	Several approaches may be useful for resolving ethical issues. Among these approaches are those based on ethical principles, virtue ethics, professional oaths, and personal values. These various sources of guidance encompass precepts that may conflict in a particular case, leaving the physician in a quandary. In a diverse society, different individuals may turn to different sources of moral guidance. In addition, general moral precepts often need to be interpreted and applied in the context of a particular clinical situation. When facing an ethical challenge, physicians should articulate their concerns and reasoning, discuss and listen to the views of others involved in the case, and call on available resources as needed. Through these efforts, physicians can gain deeper insight into the ethical issues they face and often can reach mutually acceptable resolutions to complex problems.

1	Ethical principles can serve as general guidelines to help physicians determine the right thing to do. Respecting Patients Physicians should always treat patients with respect, which entails understanding patients’ goals, communicating effectively, obtaining informed and voluntary consent for interventions, respecting informed refusals, and protecting confidentiality. Different clinical goals and approaches are often feasible, and interventions can cause both benefit and harm. Individuals place different values on health and medical care and weigh the benefits and risks of medical interventions differently. Generally, the values and informed choices of patients should be respected.

1	OBTAINING INFORMED CONSENT To help patients make informed decisions, physicians should discuss with them the nature of the proposed care; the alternatives; and the risks, benefits, and likely consequences of each option. Informed consent involves more than obtaining signatures on consent forms. Physicians should promote shared decision-making by educating patients, answering their questions, making recommendations, and helping them deliberate. Patients can be overwhelmed by medical jargon, needlessly complicated explanations, or the provision of too much information at once. Patients can make informed decisions only if they receive honest and understandable information. Competent, informed patients may refuse recommended interventions and choose among reasonable alternatives. If patients cannot give consent in an emergency and if delay of treatment while 17e-1 surrogates are contacted will place their lives or health in peril, treatment can be given without informed consent. People

1	cannot give consent in an emergency and if delay of treatment while 17e-1 surrogates are contacted will place their lives or health in peril, treatment can be given without informed consent. People are presumed to want such emergency care unless they have previously indicated otherwise.

1	Respect for patients does not entitle the patients to insist on any care they want. Physicians are not obligated to provide interventions that have no physiologic rationale, that have already failed, or that are contrary to evidence-based practice recommendations, good clinical judgment, or public policies. National policies and laws also dictate certain decisions—e.g., allocating cadaveric organs for transplantation and, in most states, prohibiting physician-assisted suicide.

1	Physicians should disclose and discuss relevant and accurate information about diagnosis, prognosis, and treatment options. To help patients cope with bad news, doctors can adjust the pace of disclosure, offer empathy and hope, provide emotional support, and call on other resources such as spiritual care or social work. Physicians may be tempted to withhold a serious diagnosis, misrepresent it by using ambiguous terms, or limit discussions of prognosis or risks for fear that certain information will make patients anxious or depressed. Providing honest information about clinical situations preserves patients’ autonomy and trust and promotes sound communication with patients and colleagues. However, patients may choose not to receive such information, asking surrogates to make decisions on their behalf, as is common with serious diagnoses in some traditional cultures.

1	AVOIDING DECEPTION Health care providers sometimes consider using lies or deception to obtain benefits for patients. Lying refers to statements known to be false and intended to mislead the listener. Deception includes statements and actions intended to mislead the listener, whether or not they are literally true. For example, a physician might sign a disability form for a patient who does not meet disability criteria. Although motivated by a desire to help the patient, such deception is ethically problematic because it undermines physicians’ credibility and trustworthiness.

1	MAINTAINING CONFIDENTIALITY Maintaining confidentiality is essential in respecting patients’ autonomy and privacy, encourages them to seek treatment and to discuss problems candidly, and prevents discrimination. However, confidentiality may be overridden to prevent serious harm to third parties or to the patient. Exceptions to confidentiality are justified if the risk is serious and probable, if there are no less restrictive measures by which to avert risk, if the adverse effects of overriding confidentiality are minimized, and if these adverse effects are deemed acceptable by society. For example, the law requires physicians to report cases of tuberculosis, sexually transmitted infection, elder or child abuse, and domestic violence.

1	CARING FOR PATIENTS WHO LACK DECISION-MAKING CAPACITY Some patients are not able to make informed decisions because of unconsciousness, dementia, delirium, or other medical conditions. Although only the courts have the legal authority to determine that a patient is incompetent for making medical decisions, in practice, physicians determine when patients lack the capacity to make health care decisions and arrange for surrogates to make decisions for them, without involving the courts. Patients with decision-making capacity can express a choice and appreciate the medical situation; the nature of the proposed care; the alternatives; and the risks, benefits, and consequences of each alternative. Their choices should be consistent with their values and not the result of delusions or hallucinations. Psychiatrists may help assess decision-making capacity in difficult cases. When impairments are fluctuating or reversible, decisions should be postponed if possible until the patient recovers

1	Psychiatrists may help assess decision-making capacity in difficult cases. When impairments are fluctuating or reversible, decisions should be postponed if possible until the patient recovers decision-making capacity.

1	If a patient lacks decision-making capacity, physicians should ask: Who is the appropriate surrogate, and what would the patient want done? Patients may designate someone to serve as their health care proxy or to assume durable power of attorney for health care; such choices should be respected. (See Chap. 10 for further details about advance care planning.) Unless a patient without decision-making capacity has previously designated a health care proxy, physicians usually ask family members to serve as surrogates. Many patients want family members as surrogates, and family members generally have the patient’s best interests at heart. Statutes in most U.S. states delineate a prioritized list of relatives who may serve as surrogates if the patient has not designated a proxy. Surrogates’ decisions should be guided by the patient’s values, goals, and previously expressed preferences. However, it may be appropriate to override previous preferences in favor of the patient’s current best

1	decisions should be guided by the patient’s values, goals, and previously expressed preferences. However, it may be appropriate to override previous preferences in favor of the patient’s current best interests if an intervention is highly likely to provide a significant benefit, if previous statements do not fit the situation well, or if the patient expressed a desire for the surrogate to have leeway in making decisions.

1	ACTING IN THE BEST INTERESTS OF PATIENTS Respect for patients is broader than respecting their autonomy to make informed choices about their medical care and promoting shared decision-making. Physicians should also be compassionate and dedicated and should act in the best interests of their patients.

1	The principle of beneficence requires physicians to act for the patient’s benefit. Patients typically lack medical expertise and may be vulnerable because of their illness. They rely on physicians to provide sound recommendations and to promote their well-being. Physicians encourage such trust and have a fiduciary duty to act in the best interests of the patient, which should prevail over the physicians’ own self-interest or the interests of third parties, such as hospitals or insurers. Physicians’ fiduciary obligations contrast sharply with business relationships, which are characterized by “let the buyer beware,” not by reliance and trust. A related principle, “first do no harm,” forbids physicians to provide ineffective interventions or to act without due care. Although often cited, this precept alone provides only limited guidance because many beneficial interventions pose serious risks. Physicians should prevent unnecessary harm by recommending interventions that maximize benefit

1	precept alone provides only limited guidance because many beneficial interventions pose serious risks. Physicians should prevent unnecessary harm by recommending interventions that maximize benefit and minimize harm.

1	MANAGING CONFLICTS BETWEEN RESPECTING PATIENTS AND ACTING IN THEIR BEST INTERESTS Conflicts can arise when patients’ refusal of interventions thwarts their own goals for care or causes them serious harm. For example, if a young woman with asthma refuses mechanical ventilation for reversible respiratory failure, simple acceptance of this decision by the physician, in the name of respecting autonomy, is morally constricted. Physicians should elicit patients’ expectations and concerns, correct their misunderstandings, and try to persuade them to accept beneficial therapies. If disagreements persist after such efforts, patients’ informed choices and views of their own best interests should prevail. While refusing recommended care does not render a patient incompetent, it may lead the physician to probe further to ensure that the patient has the capacity to make informed decisions.

1	Acting Justly The principle of justice provides guidance to physicians about how to ethically treat patients and to make decisions about allocating important resources, including their own time. Justice in a general sense means fairness: people should receive what they deserve. In addition, it is important to act consistently in cases that are similar in ethically relevant ways. Otherwise, decisions may be arbitrary, biased, and unfair. Justice forbids discrimination in health care based on race, religion, gender, sexual orientation, or other personal characteristics (Chap. 16e).

1	Justice also requires that limited health care resources be allocated fairly. Universal access to medically needed health care remains an unrealized moral aspiration in the United States and much of the rest of the world. Patients without health insurance often cannot afford health care and lack access to safety-net services. Even among insured patients, insurers may deny coverage for interventions recommended by the physician. In this situation, physicians should advocate for patients and try to help them obtain needed care. Doctors might consider—or patients might request—the use of deception to obtain such benefits. However, avoiding deception is a basic ethical guideline that sets limits on advocating for patients.

1	Allocation of health care resources is unavoidable because these resources are limited. Ideally, decisions about allocation are made at the level of public policy, with physician input. For example, the United Network for Organ Sharing (www.unos.org) provides criteria for allocating scarce organs. Ad hoc resource allocation at the bedside is problematic because it may be inconsistent, unfair, and ineffective. Physicians have an important role, however, in avoiding unnecessary interventions. Evidence-based lists of tests and procedures that physicians and patients should question and discuss were developed through the recent initiative Choosing Wisely (http://www.choosingwisely.org/). At the bedside, physicians should act as patient advocates within constraints set by society, reasonable insurance coverage, and evidence-based practice. For example, if a patient’s insurer has a higher copayment for nonformulary drugs, it still may be reasonable for physicians to advocate for

1	insurance coverage, and evidence-based practice. For example, if a patient’s insurer has a higher copayment for nonformulary drugs, it still may be reasonable for physicians to advocate for nonformulary products for good reasons (e.g., when the formulary drugs are ineffective or not tolerated).

1	Virtue ethics focuses on physicians’ character and qualities, with the expectation that doctors will cultivate such virtues as compassion, dedication, altruism, humility, and integrity. Proponents argue that, if such characteristics become ingrained, they help guide physicians in novel situations. Moreover, merely following ethical precepts or principles without these virtues leads to uncaring doctor–patient relationships.

1	Professional oaths and codes are useful guides for physicians. Most physicians take oaths at student white-coat ceremonies and at medical school graduation, and many are members of professional societies that have professional codes. Members of the profession pledge to the public and to their patients that they will be guided by the principles and values in these oaths or codes. Oaths and codes focus physicians on ethical ideals rather than on daily pragmatic concerns. However, professional oaths and codes—even the Hippocratic tradition—have been criticized for lack of patient or public input and the limited role given to patients in making decisions.

1	Personal values, cultural traditions, and religious beliefs are important sources of personal morality that help physicians address ethical issues and cope with the moral distress they may experience in practice. While essential, personal morality is a limited ethical guide in clinical practice. Physicians have role-specific ethical obligations that go beyond their obligations as good people, including the duties to obtain informed consent and maintain confidentiality discussed earlier in this chapter. Furthermore, in a culturally and religiously diverse world, patients and colleagues have personal moral beliefs that commonly differ from their physicians’.

1	Claims of Conscience Some physicians have conscientious objections to providing or referring patients for certain treatments, such as contraception. While physicians should not be asked to violate deeply held moral beliefs or religious convictions, patients need to receive medically appropriate, timely care. Institutions such as clinics and hospitals have a collective duty to provide care that patients need while making reasonable attempts to accommodate health care workers’ conscientious objections—for example, by arranging for another professional to provide the service in question. Patients seeking a relationship with a doctor or health care institution should be notified in advance of any conscientious objections to the provision of specific interventions. Since patients commonly must select providers for insurance purposes, switching providers when a specific service is needed would be burdensome. There are important limits on claims of conscience. Health care workers may not

1	select providers for insurance purposes, switching providers when a specific service is needed would be burdensome. There are important limits on claims of conscience. Health care workers may not insist that patients receive unwanted medical interventions and may not refuse to treat patients because of their race, ethnicity, national origin, gender, or religion. Such discrimination is illegal and violates the physician’s duty to respect patients.

1	Moral Distress Physicians and other health care providers may experience moral distress when they feel they know the ethically correct action to take in a particular situation but are constrained by institutional policies, limited resources, or a position subordinate to the ultimate decision-maker. Moral distress can lead to anger, anxiety, frustration, fatigue, and work dissatisfaction. Discussing complex clinical situations with colleagues and seeking assistance with difficult decisions helps to alleviate moral distress, as does a healthy work environment characterized by open communication and mutual respect.

1	These various sources of guidance contain precepts that may conflict in a particular case, leaving the physician in a quandary. In a diverse society, different individuals may turn to different sources of moral guidance. In addition, general moral precepts often need to be interpreted and applied in the context of a particular clinical situation. When facing an ethical challenge, physicians should articulate their concerns and reasoning, discuss and listen to the views of others involved in the case, and call on available resources as needed. Through these efforts, physicians can gain deeper insight into the ethical issues they face and often reach mutually acceptable resolutions to complex problems. Recent changes in the organization and delivery of health care have led to new ethical challenges for physicians.

1	The Accreditation Council for Graduate Medical Education requires medical students and residents to observe work-hour limitations, which are intended to help prevent physician burnout, reduce mistakes, and create a better balance between work and private life. In addition to continuing controversy over their effectiveness, some ethical concerns are raised by work-hour limitations. One concern is that physicians may develop a shift-worker mentality that undermines their dedication to the well-being of patients. Forced handoffs to colleagues may actually increase the risk of errors, and inflexibility can be detrimental. In some cases, trainees could provide an irreplaceable benefit to a patient or family by going beyond work-hour limits, especially if there is rapport with the patient or family that is not easily transferred to another provider. For example, a resident may want to discuss decisions about life-sustaining interventions or to comfort a family member over a patient’s death

1	that is not easily transferred to another provider. For example, a resident may want to discuss decisions about life-sustaining interventions or to comfort a family member over a patient’s death (Chap. 10). Thus strict adherence to work-hour limits is not always consistent with the ideal of acting for the good of the patient and with compassion. Exceptions to work-hour limits, however, should remain exceptions and should not be allowed to undercut work-hour policies.

1	Physicians’ roles are changing as care is increasingly provided by multidisciplinary teams. The traditional hierarchy in which the physician is the “captain of the ship” may be inappropriate, particularly in areas such as prevention, disease management and its coordination, and patient education. Physicians should respect team members and acknowledge the expertise of those from other disciplinary backgrounds. Team-based care promises to provide more comprehensive and higher-quality care. However, regular communication and planning are critical to avoid diffusion of responsibility and to ensure that someone is accountable for the completion of patient-care tasks.

1	The increasing use of evidence-based practice guidelines and benchmarking of performance raises the overall quality of care. However, practice guideline recommendations may be inappropriate for an individual patient, while another option may provide substantially greater benefits. In such situations, physicians’ duty to act in the patient’s best interests should take priority over benefits to society as a whole. Physicians need to understand practice guidelines, to recognize situations in which exceptions might be reasonable, and to be prepared to justify an exception.

1	With the growing importance of and interest in global health, ods. Typically, physicians gain valuable experience while providing service to patients in need. Such arrangements, however, can raise ethical challenges—for example, because of differences in beliefs 17e-3 about health and illness, expectations regarding health care and the physician’s role, standards of clinical practice, and norms for disclosure of serious diagnoses. Additional dilemmas arise if visiting physicians take on responsibilities beyond their level of training or if donated drugs and equipment are not appropriate to local needs. Visiting physicians and trainees should exercise due diligence in obtaining needed information about the cultural and clinical practices in the host community, should work closely with local professionals and team members, and should be explicit about their skills, knowledge, and limits. In addition, these arrangements can pose risks. The visiting physician may face personal risk from

1	professionals and team members, and should be explicit about their skills, knowledge, and limits. In addition, these arrangements can pose risks. The visiting physician may face personal risk from infectious disease or motor vehicle accident. The host institution incurs administrative and supervisory costs. Advance preparation for these possibilities minimizes harm, distress, and misunderstanding.

1	Increasingly, physicians use social and electronic media to share information with patients and other providers. Social networking may be especially useful in reaching young or otherwise hard-toaccess patients. However, the use of social media, including blogs, social networks, and websites, raises ethical challenges and can have harmful consequences if not approached prudently. Injudicious use of social media can pose risks to patient confidentiality, expose patients to intimate details of physicians’ personal lives, cross professional boundaries, and jeopardize therapeutic relationships. Posts may be considered unprofessional and lead to adverse consequences for a provider’s reputation, safety, or even employment, especially if they express frustration or anger over work incidents, disparage patients or colleagues, use offensive or discriminatory language, reveal highly personal information, or picture a physician intoxicated, using illegal drugs, or in sexually suggestive poses.

1	patients or colleagues, use offensive or discriminatory language, reveal highly personal information, or picture a physician intoxicated, using illegal drugs, or in sexually suggestive poses. Physicians should remember that, in the absence of highly restrictive privacy settings, postings on the Internet in general and on social networking sites in particular are usually permanent and may be accessible to the public, their employers, and their patients. Physicians should separate professional from personal websites, social networking accounts, and blogs and should follow guidelines developed by institutions and professional societies on using social media to communicate with patients.

1	Acting in patients’ best interests may conflict with the physician’s self-interest or the interests of third parties such as insurers or hospitals. From an ethical viewpoint, patients’ interests should be paramount. Even the appearance of a conflict of interest may undermine trust in the profession. Health care providers may be offered financial incentives to improve the quality or efficiency of care. Such pay-for-performance incentives, however, could lead physicians to avoid sicker patients with more complicated cases or to focus on benchmarked outcomes even when such a focus is not in the best interests of an individual patient. In contrast, fee-for-service payments offer physicians incentives to order more interventions than may be necessary or to refer patients to laboratory or imaging facilities in which they have a financial stake. Regardless of financial incentives, physicians should recommend available care that is in the patient’s best interests—no more and no less.

1	Financial relationships between physicians and industry are increasingly scrutinized. Gifts from drug and device companies may create an inappropriate risk of undue influence, induce subconscious feelings of reciprocity, impair public trust, and increase the cost of health care. Many academic medical centers have banned drug-company gifts of pens, notepads, and meals to physicians. Under the new Physician Payment Sunshine Act, companies must disclose publicly the names of physicians to whom they have made payments or transferred material goods and the amount of those payments or transfers. The challenge will be to distinguish payments for scientific consulting and research contracts—which are consistent with professional and academic missions and should be encouraged—from those for promotional speaking and consulting whose goal is to increase sales of company products.

1	Some health care workers, fearing fatal occupational infections, have refused to care for certain patients, such as those with HIV infection or severe acute respiratory syndrome (SARS). Such fears about personal safety need to be acknowledged. Health care institutions should reduce occupational risk by providing proper training, protective equipment, and supervision. To fulfill their mission of helping patients, physicians should provide appropriate care within their clinical expertise, despite some personal risk.

1	Errors are inevitable in clinical medicine, and some errors cause serious adverse events that harm patients. Most errors are caused by lapses of attention or flaws in the system of delivering health care; only a few result from blameworthy individual behavior (Chaps. 3 and 12e). Physicians and students may fear that disclosing errors will damage their careers. However, patients appreciate being told when an error occurs, receiving an apology, and being informed about efforts to prevent similar errors in the future. Physicians and health care institutions show respect for patients by disclosing errors, offering appropriate compensation for harm done, and using errors as opportunities to improve the quality of care. Overall, patient safety is more likely to be improved through a quality improvement approach to errors rather than a punitive one except in cases of gross incompetence, physician impairment, boundary violations, or repeated violations of standard procedures.

1	Physicians’ interest in learning, which fosters the long-term goal of benefiting future patients, may conflict with the short-term goal of providing optimal care to current patients. When trainees learn to carry out procedures on patients, they lack the proficiency of experienced physicians, and patients may experience inconvenience, discomfort, longer procedures, or even increased risk. Although patients’ consent for trainee participation in their care is always important, it is particularly important for intimate examinations, such as pelvic, rectal, breast, and testicular examinations, and for invasive procedures. To ensure patients’ cooperation, some care providers introduce students as physicians or do not tell patients that trainees will be performing procedures. Such misrepresentation undermines trust, may lead to more elaborate deception, and makes it difficult for patients to make informed choices about their care. Patients should be told who is providing care and how

1	undermines trust, may lead to more elaborate deception, and makes it difficult for patients to make informed choices about their care. Patients should be told who is providing care and how trainees are supervised. Most patients, when informed, allow trainees to play an active role in their care.

1	Physicians may hesitate to intervene when colleagues impaired by alcohol abuse, drug abuse, or psychiatric or medical illness place patients at risk. However, society relies on physicians to regulate themselves. If colleagues of an impaired physician do not take steps to protect patients, no one else may be in a position to do so.

1	Clinical research is essential to translate scientific discoveries into beneficial tests and therapies for patients. However, clinical research raises ethical concerns because participants face inconvenience and risks in research that is designed not specifically to benefit them but rather to advance scientific knowledge. Ethical guidelines for researchers require them to obtain informed and voluntary consent from participants and approval from an institutional review board, which determines that risks to participants are acceptable and have been minimized and recommends appropriate additional protections when research includes vulnerable participants. Physicians may be involved as clinical research investigators or may be in a position to refer or recommend clinical trial participation to their patients. Physicians should be critical consumers of clinical research results and keep up with advances that change standards of practice. Courses and guidance on the ethics of clinical

1	to their patients. Physicians should be critical consumers of clinical research results and keep up with advances that change standards of practice. Courses and guidance on the ethics of clinical research are widely available.

1	Pain: Pathophysiology and Management James P. Rathmell, Howard L. Fields The province of medicine is to preserve and restore health and to relieve suffering. Understanding pain is essential to both of these goals. Because pain is universally understood as a signal of disease, it 18 SEC Tion 1 PAin FIguRE 18-1 Components of a typical cutaneous nerve. There are two distinct functional cat-egories of axons: primary afferents with cell bodies in the dorsal root ganglion, and sympathetic postganglionic fibers with cell bodies in the sympathetic ganglion. Primary afferents include those with large-diameter myelinated (Aβ), small-diameter myelinated (Aδ), and unmyelinated (C) axons. All sympathetic postganglionic fibers are unmyelinated.

1	CHAPTER 18 Pain: Pathophysiology and Management is the most common symptom that brings a patient to a physician’s attention. The function of the pain sensory system is to protect the body and maintain homeostasis. It does this by detecting, localizing, and identifying potential or actual tissue-damaging processes. Because different diseases produce characteristic patterns of tissue damage, the quality, time course, and location of a patient’s pain lend important diagnostic clues. It is the physician’s responsibility to provide rapid and effective pain relief.

1	Pain is an unpleasant sensation localized to a part of the body. It is often described in terms of a penetrating or tissue-destructive process (e.g., stabbing, burning, twisting, tearing, squeezing) and/or of a bodily or emotional reaction (e.g., terrifying, nauseating, sickening). Furthermore, any pain of moderate or higher intensity is accompanied by anxiety and the urge to escape or terminate the feeling. These properties illustrate the duality of pain: it is both sensation and emotion. When it is acute, pain is characteristically associated with behavioral arousal and a stress response consisting of increased blood pressure, heart rate, pupil diameter, and plasma cortisol levels. In addition, local muscle contraction (e.g., limb flexion, abdominal wall rigidity) is often present.

1	PERIPHERAL MECHANISMS The Primary Afferent Nociceptor A peripheral nerve consists of the axons of three different types of neurons: primary sensory afferents, motor neurons, and sympathetic postganglionic neurons (Fig. 18-1). The cell bodies of primary sensory afferents are located in the dorsal root ganglia within the vertebral foramina. The primary afferent axon has two branches: one projects centrally into the spinal cord and the other projects peripherally to innervate tissues. Primary afferents are classified by their diameter, degree of myelination, and conduction velocity. The largest diameter afferent fibers, A-beta (Aβ), respond maximally to light touch and/or moving stimuli; they are present primarily in nerves that innervate the skin. In normal individuals, the activity of these fibers does not produce pain. There are two other classes of primary afferent nerve fibers: the small diameter myelinated A-delta (Aδ) and the unmyelinated (C) axons (Fig. 18-1). These fibers are

1	fibers does not produce pain. There are two other classes of primary afferent nerve fibers: the small diameter myelinated A-delta (Aδ) and the unmyelinated (C) axons (Fig. 18-1). These fibers are present in nerves to the skin and to deep somatic and visceral structures. Some tissues, such as the cornea, are innervated only by Aδ and C fiber afferents. Most Aδ and C fiber afferents respond maximally only to intense (painful) stimuli and produce the subjective experience of pain when they are electrically stimulated; this defines them as primary afferent nociceptors (pain receptors). The ability to detect painful stimuli is completely abolished when conduction in Aδ and C fiber axons is blocked.

1	Individual primary afferent nociceptors can respond to several different types of noxious stimuli. For example, most nociceptors respond to heat; intense cold; intense mechanical distortion, such as a pinch; changes in pH, particularly an acidic environment; and application of chemical irritants including adenosine triphosphate (ATP), serotonin, bradykinin, and histamine.

1	Sensitization When intense, repeated, or prolonged stimuli are applied to damaged or inflamed tissues, the threshold for activating primary afferent nociceptors is lowered, and the frequency of firing is higher for all stimulus intensities. Inflammatory mediators such as bradykinin, nerve-growth factor, some prostaglandins, and leukotrienes contribute to this process, which is called sensitization. Sensitization occurs at the level of the peripheral nerve terminal (peripheral sensitization) as well as at the level of the dorsal horn of the spinal cord (central sensitization). Peripheral sensitization occurs in damaged or inflamed tissues, when inflammatory mediators activate intracellular signal transduction in nociceptors, prompting an increase in the production, transport, and membrane insertion of chemically gated and voltage-gated ion channels. These changes increase the excitability of nociceptor terminals and lower their threshold for activation by mechanical, thermal, and

1	insertion of chemically gated and voltage-gated ion channels. These changes increase the excitability of nociceptor terminals and lower their threshold for activation by mechanical, thermal, and chemical stimuli. Central sensitization occurs when activity, generated by nociceptors during inflammation, enhances the excitability of nerve cells in the dorsal horn of the spinal cord. Following injury and resultant sensitization, normally innocuous stimuli can produce pain (termed allodynia). Sensitization is a clinically important process that contributes to tenderness, soreness, and hyperalgesia (increased pain intensity in response to the same noxious stimulus; e.g., moderate pressure causes severe pain). A striking example of sensitization is sunburned skin, in which severe pain can be produced by a gentle slap on the back or a warm shower.

1	Sensitization is of particular importance for pain and tenderness in deep tissues. Viscera are normally relatively insensitive to noxious mechanical and thermal stimuli, although hollow viscera do generate significant discomfort when distended. In contrast, when affected by a disease process with an inflammatory component, deep structures such as joints or hollow viscera characteristically become exquisitely sensitive to mechanical stimulation.

1	A large proportion of Aδ and C fiber afferents innervating viscera are completely insensitive in normal noninjured, noninflamed tissue. That is, they cannot be activated by known mechanical or thermal stimuli and are not spontaneously active. However, in the presence of inflammatory mediators, these afferents become sensitive to mechanical stimuli. Such afferents have been termed silent nociceptors, and their characteristic properties may explain how, under pathologic conditions, the relatively insensitive deep structures can become the source of severe and debilitating pain and tenderness. Low pH, prostaglandins, leukotrienes, and other inflammatory mediators such as bradykinin play a significant role in sensitization.

1	Nociceptor-Induced Inflammation Primary afferent nociceptors also have a neuroeffector function. Most nociceptors contain polypeptide mediators that are released from their peripheral terminals when they are activated (Fig. 18-2). An example is substance P, an 11-amino-acid peptide. Substance P is released from primary afferent nociceptors and has multiple biologic activities. It is a potent vasodilator, degranulates mast cells, is a chemoattractant for leukocytes, and increases the production and release of inflammatory mediators. Interestingly, depletion of substance P from joints reduces the severity of experimental arthritis. Primary afferent nociceptors are not simply passive messengers of threats to tissue injury but also play an active role in tissue protection through these neuroeffector functions.

1	CENTRAL MECHANISMS The Spinal Cord and Referred Pain The axons of primary afferent nociceptors enter the spinal cord via the dorsal root. They terminate in the dorsal horn of the spinal gray matter (Fig. 18-3). The terminals of primary afferent axons contact spinal neurons that transmit the pain signal to brain sites involved in pain perception. When primary afferents are activated by noxious stimuli, they release neurotransmitters from their terminals that excite the spinal cord neurons. The major neurotransmitter released is glutamate, which rapidly excites dorsal horn neurons. Primary afferent nociceptor terminals also release peptides, including substance P and calcitonin gene-related peptide, which produce a slower and longer-lasting excitation of the dorsal horn neurons. The axon of each primary afferent contacts many spinal neurons, and each spinal neuron receives convergent inputs from many primary afferents.

1	The convergence of sensory inputs to a single spinal pain-transmission neuron is of great importance because it underlies the phenomenon of referred pain. All spinal neurons that receive input from the viscera and deep musculoskeletal structures also receive input from the skin. The convergence patterns are determined by the spinal segment of the dorsal root ganglion that supplies the afferent innervation of a structure. For example, the afferents that supply the central diaphragm are derived from the third and fourth cervical dorsal root ganglia. Primary afferents with cell bodies in these same ganglia supply the skin of the shoulder and lower neck. Thus, sensory inputs from both the shoulder skin and the central diaphragm converge on pain-transmission neurons in the third and fourth cervical spinal segments.

1	Because of this convergence and the fact that the spinal neurons are most often activated by inputs from the skin, activity evoked in spinal neurons by input from deep structures is mislocalized by the patient to a place that roughly corresponds with the region of skin innervated by the same spinal segment. Thus, inflammation near the central diaphragm is often reported as shoulder discomfort. This spatial displacement of pain sensation from the site of the injury that produces it is known as referred pain. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 18-2 Events leading to activation, sensitization, and spread of sensitization of primary afferent nociceptor terminals. A. Direct activation by intense pressure and consequent cell damage. Cell damage induces lower pH (H+) and leads to release of potassium (K+) and to synthesis of prostaglandins (PG) and bradykinin (BK). Prostaglandins increase the sensitivity of the terminal to bradykinin and other pain-producing substances. B. Secondary activation. Impulses generated in the stimulated terminal propagate not only to the spinal cord but also into other terminal branches where they induce the release of peptides, including substance P (SP). Substance P causes vasodilation and neurogenic edema with further accumulation of bradykinin (BK). Substance P also causes the release of histamine (H) from mast cells and serotonin (5HT) from platelets.

1	Ascending Pathways for Pain A majority of spinal neurons contacted by primary afferent nociceptors send their axons to the contralateral thalamus. These axons form the contralateral spinothalamic tract, which lies in the anterolateral white matter of the spinal cord, the lateral edge of the medulla, and the lateral pons and midbrain. The spinothalamic pathway is crucial for pain sensation in humans. Interruption of this pathway produces permanent deficits in pain and temperature discrimination. Spinothalamic tract axons ascend to several regions of the thalamus. There is tremendous divergence of the pain signal from these thalamic sites to several distinct areas of the cerebral cortex that subserve different aspects of the pain experience (Fig. 18-4). One of the thalamic projections is to the somatosensory cortex. This projection mediates the purely sensory aspects of pain, i.e., its location, intensity, and quality. Other thalamic neurons project to cortical regions that are linked

1	FIguRE 18-3 The convergence-projection hypothesis of referred pain. According to this hypothesis, visceral afferent nociceptors converge on the same pain-projection neurons as the afferents from the somatic structures in which the pain is perceived. The brain has no way of knowing the actual source of input and mistakenly “projects” the sensation to the somatic structure. to emotional responses, such as the cingulate gyrus and other areas of the frontal lobes, including the insular cortex. These pathways to the frontal cortex subserve the affective or unpleasant emotional dimension of pain. This affective dimension of pain produces suffering and exerts potent control of behavior. Because of this dimension, fear is FIguRE 18-4 Pain transmission and modulatory pathways.

1	FIguRE 18-4 Pain transmission and modulatory pathways. A. Transmission system for nociceptive messages. Noxious stimuli activate the sensitive peripheral ending of the primary afferent nociceptor by the process of transduction. The message is then transmitted over the peripheral nerve to the spinal cord, where it synapses with cells of origin of the major ascending pain pathway, the spinothalamic tract. The message is relayed in the thalamus to the anterior cingulate (C), frontal insular (F), and somatosensory cortex (SS). B. Pain-modulation network. Inputs from frontal cortex and hypothalamus activate cells in the midbrain that control spinal pain-transmission cells via cells in the medulla. a constant companion of pain. As a consequence, injury or surgical lesions to areas of the frontal cortex activated by painful stimuli can diminish the emotional impact of pain while largely preserving the individual’s ability to recognize noxious stimuli as painful.

1	The pain produced by injuries of similar magnitude is remarkably variable in different situations and in different individuals. For example, athletes have been known to sustain serious fractures with only minor pain, and Beecher’s classic World War II survey revealed that many soldiers in battle were unbothered by injuries that would have produced agonizing pain in civilian patients. Furthermore, even the suggestion that a treatment will relieve pain can have a significant analgesic effect (the placebo effect). On the other hand, many patients find even minor injuries (such as venipuncture) frightening and unbearable, and the expectation of pain can induce pain even without a noxious stimulus. The suggestion that pain will worsen following administration of an inert substance can increase its perceived intensity (the nocebo effect).

1	The powerful effect of expectation and other psychological variables on the perceived intensity of pain is explained by brain circuits that modulate the activity of the pain-transmission pathways. One of these circuits has links to the hypothalamus, midbrain, and medulla, and it selectively controls spinal pain-transmission neurons through a descending pathway (Fig. 18-4). Human brain–imaging studies have implicated this painmodulating circuit in the pain-relieving effect of attention, suggestion, and opioid analgesic medications (Fig. 18-5). Furthermore, each of the component structures of the pathway contains opioid receptors and is sensitive to the direct application of opioid drugs. In animals, lesions of this descending modulatory system reduce the analgesic effect of systemically administered opioids such as morphine. Along with the opioid receptor, the component nuclei of this pain-modulating circuit contain endogenous opioid peptides such as the enkephalins and β-endorphin.

1	The most reliable way to activate this endogenous opioid-mediated modulating system is by suggestion of pain relief or by intense emotion directed away from the pain-causing injury (e.g., during severe threat or an athletic competition). In fact, pain-relieving endogenous opioids are released following surgical procedures and in patients given a placebo for pain relief.

1	Pain-modulating circuits can enhance as well as suppress pain. Both pain-inhibiting and pain-facilitating neurons in the medulla project to and control spinal pain-transmission neurons. Because pain-transmission neurons can be activated by modulatory neurons, it is theoretically possible to generate a pain signal with no peripheral noxious stimulus. In fact, human functional imaging studies have demonstrated increased activity in this circuit during migraine headaches. A central circuit that facilitates pain could account for the finding that pain can be induced by suggestion or enhanced by expectation and provides a framework for understanding how psychological factors can contribute to chronic pain.

1	Lesions of the peripheral or central nociceptive pathways typically result in a loss or impairment of pain sensation. Paradoxically, damage to or dysfunction of these pathways can also produce pain. For example, damage to peripheral nerves, as occurs in diabetic neuropathy, or to primary afferents, as in herpes zoster infection, can result in pain that is referred to the body region innervated by the damaged nerves. Pain may also be produced by damage to the central nervous system (CNS), for example, in some patients following trauma or vascular injury to the spinal cord, brainstem, or thalamic areas that contain central nociceptive pathways. Such neuropathic pains are often severe and are typically resistant to standard treatments for pain.

1	Neuropathic pain typically has an unusual burning, tingling, or electric shock–like quality and may be triggered by very light touch. These features are rare in other types of pain. On examination, a sensory deficit is characteristically present in the area of the patient’s pain. Hyperpathia, a greatly exaggerated pain sensation to innocuous CHAPTER 18 Pain: Pathophysiology and Management

1	FIguRE 18-5 Functional magnetic resonance imaging (fMRI) demonstrates placebo-enhanced brain activity in anatomic regions correlating with the opioidergic descending pain control system. Top panel: Frontal fMRI image shows placebo-enhanced brain activity in the dorsal lateral prefrontal cortex (DLPFC). Bottom panel: Sagittal fMRI images show placebo-enhanced responses in the rostral anterior cingulate cortex (rACC), the rostral ventral medullae (RVM), the periaqueductal gray (PAG) area, and the hypothalamus. The placebo-enhanced activity in all areas was reduced by naloxone, demonstrating the link between the descending opioidergic system and the placebo analgesic response. (Adapted with permission from F Eippert et al: Neuron 63:533, 2009.) or mild nociceptive stimuli, is also characteristic of neuropathic pain; patients often complain that the very lightest moving stimulus evokes exquisite pain (allodynia). In this regard, it is of clinical interest that a topical preparation of 5%

1	of neuropathic pain; patients often complain that the very lightest moving stimulus evokes exquisite pain (allodynia). In this regard, it is of clinical interest that a topical preparation of 5% lidocaine in patch form is effective for patients with postherpetic neuralgia who have prominent allodynia.

1	A variety of mechanisms contribute to neuropathic pain. As with sensitized primary afferent nociceptors, damaged primary afferents, including nociceptors, become highly sensitive to mechanical stimulation and may generate impulses in the absence of stimulation. Increased sensitivity and spontaneous activity are due, in part, to an increased concentration of sodium channels in the damaged nerve fiber. Damaged primary afferents may also develop sensitivity to nor-epinephrine. Interestingly, spinal cord pain-transmission neurons cut off from their normal input may also become spontaneously active. PART 2 Cardinal Manifestations and Presentation of Diseases Thus, both CNS and peripheral nervous system hyperactivity contribute to neuropathic pain.

1	Sympathetically Maintained Pain Patients with peripheral nerve injury occasionally develop spontaneous pain in the region innervated by the nerve. This pain is often described as having a burning quality. The pain typically begins after a delay of hours to days or even weeks and is accompanied by swelling of the extremity, periarticular bone loss, and arthritic changes in the distal joints. The pain may be relieved by a local anesthetic block of the sympathetic innervation to the affected extremity. Damaged primary afferent nociceptors acquire adrenergic sensitivity and can be activated by stimulation of the sympathetic outflow. This constellation of spontaneous pain and signs of sympathetic dysfunction following injury has been termed complex regional pain syndrome (CRPS). When this occurs after an identifiable nerve injury, it is termed CRPS type II (also known as posttraumatic neuralgia or, if severe, causalgia). When a similar clinical picture appears without obvious nerve injury,

1	after an identifiable nerve injury, it is termed CRPS type II (also known as posttraumatic neuralgia or, if severe, causalgia). When a similar clinical picture appears without obvious nerve injury, it is termed CRPS type I (also known as reflex sympathetic dystrophy). CRPS can be produced by a variety of injuries, including fractures of bone, soft tissue trauma, myocardial infarction, and stroke (Chap. 446). CRPS type I typically resolves with symptomatic treatment; however, when it persists, detailed examination often reveals evidence of peripheral nerve injury. Although the pathophysiology of CRPS is poorly understood, the pain and the signs of inflammation, when acute, can be rapidly relieved by blocking the sympathetic nervous system. This implies that sympathetic activity can activate undamaged nociceptors when inflammation is present. Signs of sympathetic hyperactivity should be sought in patients with post-traumatic pain and inflammation and no other obvious explanation.

1	The ideal treatment for any pain is to remove the cause; thus, while treatment can be initiated immediately, efforts to establish the underlying etiology should always proceed as treatment begins. Sometimes, treating the underlying condition does not immediately relieve pain. Furthermore, some conditions are so painful that rapid and effective analgesia is essential (e.g., the postoperative state, burns, trauma, cancer, or sickle cell crisis). Analgesic medications are a first line of treatment in these cases, and all practitioners should be familiar with their use. ASPIRIN, ACETAMINOPHEN, AND NONSTEROIDAL ANTI-INFLAMMATORY AgENTS (NSAIDs)

1	ASPIRIN, ACETAMINOPHEN, AND NONSTEROIDAL ANTI-INFLAMMATORY AgENTS (NSAIDs) These drugs are considered together because they are used for similar problems and may have a similar mechanism of action (Table 18-1). All these compounds inhibit cyclooxygenase (COX), and, except for acetaminophen, all have anti-inflammatory actions, especially at higher dosages. They are particularly effective for mild to moderate headache and for pain of musculoskeletal origin.

1	Because they are effective for these common types of pain and are available without prescription, COX inhibitors are by far the most commonly used analgesics. They are absorbed well from the gastrointestinal tract and, with occasional use, have only minimal side effects. With chronic use, gastric irritation is a common side effect of aspirin and NSAIDs and is the problem that most frequently limits the dose that can be given. Gastric irritation is most severe with aspirin, which may cause erosion and ulceration of the gastric mucosa leading to bleeding or perforation. Because aspirin irreversibly acetylates platelet cyclooxygenase and thereby interferes with coagulation of the blood, gastrointestinal bleeding is a particular risk. Older age and history of gastrointestinal disease increase the risks of aspirin and NSAIDs. In addition to the well-known gastrointestinal toxicity of NSAIDs, nephrotoxicity is a significant problem for patients using these drugs on a chronic basis. Patients

1	the risks of aspirin and NSAIDs. In addition to the well-known gastrointestinal toxicity of NSAIDs, nephrotoxicity is a significant problem for patients using these drugs on a chronic basis. Patients at risk for renal insufficiency, particularly those with significant contraction of their intravascular volume as occurs with chronic diuretic use or

1	Generic Name Dose, mg Interval Comments Nonnarcotic analgesics: usual doses and intervals Codeine 30–60 q4h 30–60 q4h Nausea common Oxycodone — 5–10 q4–6h Usually available with acetaminophen or aspirin Morphine 5 q4h 30 q4h Morphine sustained — 15–60 bid to tid Oral slow-release preparation release Hydromorphone 1–2 q4h 2–4 q4h Shorter acting than morphine sulfate Levorphanol 2 q6–8h 4 q6–8h Longer acting than morphine sulfate; absorbed well PO Methadone 5–10 q6–8h 5–20 q6–8h Delayed sedation due to long half-life; therapy should not be initiated with >40 mg/d, and dose escalation should be made no more frequently than every 3 days Meperidine 50–100 q3–4h 300 q4h Poorly absorbed PO; normeperidine is a toxic metabolite; routine use of this agent is 7-day transdermal patch Buprenorphine 0.3 q6–8h CHAPTER 18 Pain: Pathophysiology and Management Anticholinergic Orthostatic Cardiac Ave. Dose, Generic Name 5-HT NE Sedative Potency Potency Hypotension Arrhythmia mg/d Range, mg/d

1	CHAPTER 18 Pain: Pathophysiology and Management Anticholinergic Orthostatic Cardiac Ave. Dose, Generic Name 5-HT NE Sedative Potency Potency Hypotension Arrhythmia mg/d Range, mg/d Oxcarbazepine 300 bid Pregabalin 150–600 aAntidepressants, anticonvulsants, and antiarrhythmics have not been approved by the U.S. Food and Drug Administration (FDA) for the treatment of pain. 1800 mg/d is FDA approved for postherpetic neuralgia. Abbreviations: 5-HT, serotonin; NE, norepinephrine. acute hypovolemia, should be monitored closely. NSAIDs can also increase blood pressure in some individuals. Long-term treatment with NSAIDs requires regular blood pressure monitoring and treatment if necessary. Although toxic to the liver when taken in high doses, acetaminophen rarely produces gastric irritation and does not interfere with platelet function.

1	The introduction of parenteral forms of NSAIDs, ketorolac and diclofenac, extends the usefulness of this class of compounds in the management of acute severe pain. Both agents are sufficiently potent and rapid in onset to supplant opioids for many patients with acute severe headache and musculoskeletal pain. bGabapentin in doses up to

1	There are two major classes of COX: COX-1 is constitutively expressed, and COX-2 is induced in the inflammatory state. COX-2– selective drugs have similar analgesic potency and produce less gastric irritation than the nonselective COX inhibitors. The use of COX-2–selective drugs does not appear to lower the risk of nephrotoxicity compared to nonselective NSAIDs. On the other hand, COX-2–selective drugs offer a significant benefit in the management of acute postoperative pain because they do not affect blood coagulation. Nonselective COX inhibitors are usually contraindicated postoperatively because they impair platelet-mediated blood clotting and are thus associated with increased bleeding at the operative site. COX-2 inhibitors, including celecoxib (Celebrex), are associated with increased cardiovascular risk. It appears that this is a class effect of NSAIDs, excluding aspirin. These drugs are contraindicated in patients in the immediate period after coronary artery bypass surgery

1	cardiovascular risk. It appears that this is a class effect of NSAIDs, excluding aspirin. These drugs are contraindicated in patients in the immediate period after coronary artery bypass surgery and should be used with caution in elderly patients and those with a history of or significant risk factors for cardiovascular disease.

1	Opioids are the most potent pain-relieving drugs currently available. Of all analgesics, they have the broadest range of efficacy and provide the most reliable and effective method for rapid pain relief. Although side effects are common, most are reversible: nausea, vomiting, pruritus, and constipation are the most frequent and bothersome side effects. Respiratory depression is uncommon at standard analgesic doses, but can be life-threatening. Opioid-related side effects can be reversed rapidly with the narcotic antagonist naloxone. Many physicians, nurses, and patients have a certain trepidation about using opioids that is based on an exaggerated fear of addiction. In fact, there is a vanishingly small chance of patients becoming addicted to narcotics as a result of their appropriate medical use. The physician should not hesitate to use opioid analgesics in patients with acute severe pain. Table 18-1 lists the most commonly used opioid analgesics.

1	Opioids produce analgesia by actions in the CNS. They activate pain-inhibitory neurons and directly inhibit pain-transmission neurons. Most of the commercially available opioid analgesics act at the same opioid receptor (μ-receptor), differing mainly in potency, speed of onset, duration of action, and optimal route of administration. Some side effects are due to accumulation of nonopioid metabolites that are unique to individual drugs. One striking example of this is normeperidine, a metabolite of meperidine. At higher doses of meperidine, typically greater than 1 g/d, accumulation of normeperidine can produce hyperexcitability and seizures that are not reversible with naloxone. Normeperidine accumulation is increased in patients with renal failure.

1	The most rapid pain relief is obtained by intravenous administration of opioids; relief with oral administration is significantly slower. Because of the potential for respiratory depression, patients with any form of respiratory compromise must be kept under close observation following opioid administration; an oxygen-saturation monitor may be useful, but only in a setting where the monitor is under constant surveillance. Opioid-induced respiratory depression is typically accompanied by sedation and a reduction in respiratory rate. A fall in oxygen saturation represents a critical level of respiratory depression and the need for immediate intervention to prevent life-threatening hypoxemia. Ventilatory assistance should be maintained until the opioid-induced respiratory depression has resolved. The opioid antagonist naloxone should be readily available whenever opioids are used at high doses or in patients with compromised pulmonary function. Opioid effects are dose-related, and there

1	The opioid antagonist naloxone should be readily available whenever opioids are used at high doses or in patients with compromised pulmonary function. Opioid effects are dose-related, and there is great variability among patients in the doses that relieve pain and produce side effects. Synergistic respiratory depression is common when opioids are administered with other CNS depressants, most commonly the benzodiazepines. Because of this, initiation of therapy requires titration to optimal dose and interval. The most important principle is to provide adequate pain relief. This requires determining whether the drug has adequately relieved the pain and frequent reassessment to determine the optimal interval for dosing. The most common error made by physicians in managing severe pain with opioids is to prescribe an inadequate dose. Because many patients are reluctant to complain, this practice leads to needless suffering. In the absence of sedation at the expected time of peak effect, a

1	is to prescribe an inadequate dose. Because many patients are reluctant to complain, this practice leads to needless suffering. In the absence of sedation at the expected time of peak effect, a physician should not hesitate to repeat the initial dose to achieve satisfactory pain relief.

1	An innovative approach to the problem of achieving adequate pain relief is the use of patient-controlled analgesia (PCA). PCA uses a microprocessor-controlled infusion device that can deliver a baseline continuous dose of an opioid drug as well as preprogrammed PART 2 Cardinal Manifestations and Presentation of Diseases additional doses whenever the patient pushes a button. The patient can then titrate the dose to the optimal level. This approach is used most extensively for the management of postoperative pain, but there is no reason why it should not be used for any hospitalized patient with persistent severe pain. PCA is also used for short-term home care of patients with intractable pain, such as that caused by metastatic cancer.

1	It is important to understand that the PCA device delivers small, repeated doses to maintain pain relief; in patients with severe pain, the pain must first be brought under control with a loading dose before transitioning to the PCA device. The bolus dose of the drug (typically 1 mg of morphine, 0.2 mg of hydromorphone, or 10 μg of fentanyl) can then be delivered repeatedly as needed. To prevent overdosing, PCA devices are programmed with a lockout period after each demand dose is delivered (5–10 min) and a limit on the total dose delivered per hour. Although some have advocated the use of a simultaneous continuous or basal infusion of the PCA drug, this increases the risk of respiratory depression and has not been shown to increase the overall efficacy of the technique.

1	The availability of new routes of administration has extended the usefulness of opioid analgesics. Most important is the availability of spinal administration. Opioids can be infused through a spinal catheter placed either intrathecally or epidurally. By applying opioids directly to the spinal or epidural space adjacent to the spinal cord, regional analgesia can be obtained using relatively low total doses. Indeed, the dose required to produce effective localized analgesia when using morphine intrathecally (0.1–0.3 mg) is a fraction of that required to produce similar analgesia when administered intravenously (5–10 mg). In this way, side effects such as sedation, nausea, and respiratory depression can be minimized. This approach has been used extensively during labor and delivery and for postoperative pain relief following surgical procedures. Continuous intrathecal delivery via implanted spinal drug-delivery systems is now commonly used, particularly for the treatment of

1	and for postoperative pain relief following surgical procedures. Continuous intrathecal delivery via implanted spinal drug-delivery systems is now commonly used, particularly for the treatment of cancer-related pain that would require sedating doses for adequate pain control if given systemically. Opioids can also be given intranasally (butorphanol), rectally, and transdermally (fentanyl and buprenorphine), or through the oral mucosa (fentanyl), thus avoiding the discomfort of frequent injections in patients who cannot be given oral medication. The fentanyl and buprenorphine transdermal patches have the advantage of providing fairly steady plasma levels, which maximizes patient comfort.

1	Recent additions to the armamentarium for treating opioid-induced side effects are the peripherally acting opioid antagonists alvimopan (Entereg) and methylnaltrexone (Rellistor). Alvimopan is available as an orally administered agent that is restricted to the intestinal lumen by limited absorption; methylnaltrexone is available in a subcutaneously administered form that has virtually no penetration into the CNS. Both agents act by binding to peripheral μ-receptors, thereby inhibiting or reversing the effects of opioids at these peripheral sites. The action of both agents is restricted to receptor sites outside of the CNS; thus, these drugs can reverse the adverse effects of opioid analgesics that are mediated through their peripheral receptors without reversing their analgesic effects. Alvimopan has proven effective in lowering the duration of persistent ileus following abdominal surgery in patients receiving opioid analgesics for postoperative pain control. Methylnaltrexone has

1	Alvimopan has proven effective in lowering the duration of persistent ileus following abdominal surgery in patients receiving opioid analgesics for postoperative pain control. Methylnaltrexone has proven effective for relief of opioid-induced constipation in patients taking opioid analgesics on a chronic basis.

1	Opioid and COX Inhibitor Combinations When used in combination, opioids and COX inhibitors have additive effects. Because a lower dose of each can be used to achieve the same degree of pain relief and their side effects are nonadditive, such combinations are used to lower the severity of dose-related side effects. However, fixed-ratio combinations of an opioid with acetaminophen carry an important risk. Dose escalation as a result of increased severity of pain or decreased opioid effect as a result of tolerance may lead to ingestion of levels of acetaminophen that are toxic to the liver. Although acetaminophen-related hepatotoxicity is uncommon, it remains a significant cause for liver failure. Thus, many practitioners have moved away from the use of opioid-acetaminophen combination analgesics to avoid the risk of excessive acetaminophen exposure as the dose of the analgesic is escalated.

1	Managing patients with chronic pain is intellectually and emotionally challenging. The patient’s problem is often difficult or impossible to diagnose with certainty; such patients are demanding of the physician’s time and often appear emotionally distraught. The traditional medical approach of seeking an obscure organic pathology is usually unhelpful. On the other hand, psychological evaluation and behaviorally based treatment paradigms are frequently helpful, particularly in the setting of a multidisciplinary pain-management center. Unfortunately, this approach, while effective, remains largely underused in current medical practice.

1	There are several factors that can cause, perpetuate, or exacerbate chronic pain. First, of course, the patient may simply have a disease that is characteristically painful for which there is presently no cure. Arthritis, cancer, chronic daily headaches, fibromyalgia, and diabetic neuropathy are examples of this. Second, there may be secondary perpetuating factors that are initiated by disease and persist after that disease has resolved. Examples include damaged sensory nerves, sympathetic efferent activity, and painful reflex muscle contraction (spasm). Finally, a variety of psychological conditions can exacerbate or even cause pain.

1	There are certain areas to which special attention should be paid in a patient’s medical history. Because depression is the most common emotional disturbance in patients with chronic pain, patients should be questioned about their mood, appetite, sleep patterns, and daily activity. A simple standardized questionnaire, such as the Beck Depression Inventory, can be a useful screening device. It is important to remember that major depression is a common, treatable, and potentially fatal illness. Other clues that a significant emotional disturbance is contributing to a patient’s chronic pain complaint include pain that occurs in multiple, unrelated sites; a pattern of recurrent, but separate, pain problems beginning in childhood or adolescence; pain beginning at a time of emotional trauma, such as the loss of a parent or spouse; a history of physical or sexual abuse; and past or present substance abuse.

1	On examination, special attention should be paid to whether the patient guards the painful area and whether certain movements or postures are avoided because of pain. Discovering a mechanical component to the pain can be useful both diagnostically and therapeutically. Painful areas should be examined for deep tenderness, noting whether this is localized to muscle, ligamentous structures, or joints. Chronic myofascial pain is very common, and, in these patients, deep palpation may reveal highly localized trigger points that are firm bands or knots in muscle. Relief of the pain following injection of local anesthetic into these trigger points supports the diagnosis. A neuropathic component to the pain is indicated by evidence of nerve damage, such as sensory impairment, exquisitely sensitive skin (allodynia), weakness, and muscle atrophy, or loss of deep tendon reflexes. Evidence suggesting sympathetic nervous system involvement includes the presence of diffuse swelling, changes in skin

1	skin (allodynia), weakness, and muscle atrophy, or loss of deep tendon reflexes. Evidence suggesting sympathetic nervous system involvement includes the presence of diffuse swelling, changes in skin color and temperature, and hypersensitive skin and joint tenderness compared with the normal side. Relief of the pain with a sympathetic block supports the diagnosis, but once the condition becomes chronic, the response to sympathetic blockade is of variable magnitude and duration; the role for repeated sympathetic blocks in the overall management of CRPS is not established.

1	A guiding principle in evaluating patients with chronic pain is to assess both emotional and organic factors before initiating therapy. Addressing these issues together, rather than waiting to address emotional issues after organic causes of pain have been ruled out, improves compliance in part because it assures patients that a psychological evaluation does not mean that the physician is questioning the validity of their complaint. Even when an organic cause for a patient’s pain can be found, it is still wise to look for other factors. For example, a cancer patient with painful bony metastases may have additional pain due to nerve damage and may also be depressed. Optimal therapy requires that each of these factors be looked for and treated.

1	Once the evaluation process has been completed and the likely causative and exacerbating factors identified, an explicit treatment plan should be developed. An important part of this process is to identify specific and realistic functional goals for therapy, such as getting a good night’s sleep, being able to go shopping, or returning to work. A multidisciplinary approach that uses medications, counseling, physical therapy, nerve blocks, and even surgery may be required to improve the patient’s quality of life. There are also some newer, relatively invasive procedures that can be helpful for some patients with intractable pain. These include image-guided interventions such as epidural injection of glucocorticoids for acute radicular pain and radiofrequency treatment of the facet joints for chronic facet-related back and neck pain. For patients with severe and persistent pain that is unresponsive to more conservative treatment, placement of electrodes within the spinal canal overlying

1	chronic facet-related back and neck pain. For patients with severe and persistent pain that is unresponsive to more conservative treatment, placement of electrodes within the spinal canal overlying the dorsal columns of the spinal cord (spinal cord stimulation) or implantation of intrathecal drug-delivery systems has shown significant benefit. The criteria for predicting which patients will respond to these procedures continue to evolve. They are generally reserved for patients who have not responded to conventional pharmacologic approaches. Referral to a multidisciplinary pain clinic for a full evaluation should precede any invasive procedure. Such referrals are clearly not necessary for all chronic pain patients. For some, pharmacologic management alone can provide adequate relief.

1	The tricyclic antidepressants (TCAs), particularly nortriptyline and desipramine (Table 18-1), are useful for the management of chronic pain. Although developed for the treatment of depression, the TCAs have a spectrum of dose-related biologic activities that include analgesia in a variety of chronic clinical conditions. Although the mechanism is unknown, the analgesic effect of TCAs has a more rapid onset and occurs at a lower dose than is typically required for the treatment of depression. Furthermore, patients with chronic pain who are not depressed obtain pain relief with antidepressants. There is evidence that TCAs potentiate opioid analgesia, so they may be useful adjuncts for the treatment of severe persistent pain such as occurs with malignant tumors. Table 18-2 lists some of the painful conditions that respond to TCAs. TCAs are of particular value in the management of neuropathic pain such as occurs in diabetic neuropathy and postherpetic neuralgia, for which there are few

1	painful conditions that respond to TCAs. TCAs are of particular value in the management of neuropathic pain such as occurs in diabetic neuropathy and postherpetic neuralgia, for which there are few other therapeutic options.

1	The TCAs that have been shown to relieve pain have significant side effects (Table 18-1; Chap. 466). Some of these side effects, such as orthostatic hypotension, drowsiness, cardiac conduction delay, memory impairment, constipation, and urinary retention, Rheumatoid arthritisa,b aControlled trials demonstrate analgesia. bControlled studies indicate benefit but not analgesia.

1	Rheumatoid arthritisa,b aControlled trials demonstrate analgesia. bControlled studies indicate benefit but not analgesia. CHAPTER 18 Pain: Pathophysiology and Management are particularly problematic in elderly patients, and several are additive to the side effects of opioid analgesics. The selective serotonin reuptake inhibitors such as fluoxetine (Prozac) have fewer and less serious side effects than TCAs, but they are much less effective for relieving pain. It is of interest that venlafaxine (Effexor) and duloxetine (Cymbalta), which are nontricyclic antidepressants that block both serotonin and norepinephrine reuptake, appear to retain most of the pain-relieving effect of TCAs with a side effect profile more like that of the selective serotonin reuptake inhibitors. These drugs may be particularly useful in patients who cannot tolerate the side effects of TCAs.

1	These drugs are useful primarily for patients with neuropathic pain. Phenytoin (Dilantin) and carbamazepine (Tegretol) were first shown to relieve the pain of trigeminal neuralgia. This pain has a characteristic brief, shooting, electric shock–like quality. In fact, anticonvulsants seem to be particularly helpful for pains that have such a lancinating quality. Newer anticonvulsants, gabapentin (Neurontin) and pregabalin (Lyrica), are effective for a broad range of neuropathic pains. Furthermore, because of their favorable side effect profile, these newer anticonvulsants are often used as first-line agents.

1	The long-term use of opioids is accepted for patients with pain due to malignant disease. Although opioid use for chronic pain of nonmalignant origin is controversial, it is clear that, for many patients, opioids are the only option that produces meaningful pain relief. This is understandable because opioids are the most potent and have the broadest range of efficacy of any analgesic medications. Although addiction is rare in patients who first use opioids for pain relief, some degree of tolerance and physical dependence is likely with long-term use. Furthermore, animal studies suggest that long-term opioid therapy may worsen pain in some individuals. Therefore, before embarking on opioid therapy, other options should be explored, and the limitations and risks of opioids should be explained to the patient. It is also important to point out that some opioid analgesic medications have mixed agonist-antagonist properties (e.g., butorphanol and buprenorphine). From a practical standpoint,

1	to the patient. It is also important to point out that some opioid analgesic medications have mixed agonist-antagonist properties (e.g., butorphanol and buprenorphine). From a practical standpoint, this means that they may worsen pain by inducing an abstinence syndrome in patients who are physically dependent on other opioid analgesics.

1	With long-term outpatient use of orally administered opioids, it is desirable to use long-acting compounds such as levorphanol, methadone, sustained-release morphine, or transdermal fentanyl (Table 18-1). The pharmacokinetic profiles of these drug preparations enable the maintenance of sustained analgesic blood levels, potentially minimizing side effects such as sedation that are associated with high peak plasma levels, and reducing the likelihood of rebound pain associated with a rapid fall in plasma opioid concentration. Although long-acting opioid preparations may provide superior pain relief in patients with a continuous pattern of ongoing pain, others suffer from intermittent severe episodic pain and experience superior pain control and fewer side effects with the periodic use of short-acting opioid analgesics. Constipation is a virtually universal side effect of opioid use and should be treated expectantly. As noted above in the discussion of acute pain treatment, a recent

1	short-acting opioid analgesics. Constipation is a virtually universal side effect of opioid use and should be treated expectantly. As noted above in the discussion of acute pain treatment, a recent advance for patients is the development of peripherally acting opioid antagonists that can reverse the constipation associated with opioid use without interfering with analgesia.

1	Soon after the introduction of a controlled-release oxycodone formulation (OxyContin) in the late 1990s, a dramatic rise in emergency department visits and deaths associated with oxycodone ingestion appeared, focusing public attention on misuse of prescription pain medications. The magnitude of prescription opioid abuse has grown over the last decade, leading the Centers for Disease Control and Prevention to classify prescription opioid analgesic abuse as an epidemic. This appears to be due in large part to individuals using a prescription drug nonmedically, most often an opioid analgesic. PART 2 Cardinal Manifestations and Presentation of Diseases guiDELinES foR SELECTing AnD MoniToRing PATiEnTS RECEiving CHRoniC oPioiD THERAPy (CoT) foR THE TREATMEnT of CHRoniC, nonCAnCER PAin • Conduct a history, physical examination, and appropriate testing, including an assessment of risk of substance abuse, misuse, or addiction.

1	• Consider a trial of COT if pain is moderate or severe, pain is having an adverse impact on function or quality of life, and potential therapeutic benefits outweigh potential harms. • A benefit-to-harm evaluation, including a history, physical examination, and appropriate diagnostic testing, should be performed and documented before and on an ongoing basis during COT. Informed Consent and Use of Management Plans • Informed consent should be obtained. A continuing discussion with the patient regarding COT should include goals, expectations, potential risks, and alternatives to COT. • Consider using a written COT management plan to document patient and clinician responsibilities and expectations and assist in patient education. • Initial treatment with opioids should be considered as a therapeutic trial to determine whether COT is appropriate.

1	• Initial treatment with opioids should be considered as a therapeutic trial to determine whether COT is appropriate. • Opioid selection, initial dosing, and titration should be individualized according to the patient’s health status, previous exposure to opioids, attainment of therapeutic goals, and predicted or observed harms. patients on COT periodically and as warranted by changing circumstances. Monitoring should include documentation of pain intensity and level of functioning, assessments of progress toward achieving therapeutic goals, presence of adverse events, and adherence to prescribed therapies. patients on COT who are at high risk or who have engaged in aberrant drug-related behaviors, clinicians should periodically obtain urine drug screens or other information to confirm adherence to the COT plan of care.

1	• In patients on COT not at high risk and not known to have engaged in aberrant drug-related behaviors, clinicians should consider periodically obtaining urine drug screens or other information to confirm adherence to the COT plan of care. Source: Adapted with permission from R Chou et al: J Pain 10:113, 2009.

1	Source: Adapted with permission from R Chou et al: J Pain 10:113, 2009. Drug-induced deaths have rapidly risen and are now the second leading cause of death in Americans, just behind motor vehicle fatalities. In 2011, the Office of National Drug Control Policy established a multifaceted approach to address prescription drug abuse, including Prescription Drug Monitoring Programs that allow practitioners to determine if patients are receiving prescriptions from multiple providers and use of law enforcement to eliminate improper prescribing practices. This increased scrutiny leaves many practitioners hesitant to prescribe opioid analgesics, other than for brief periods to control pain associated with illness or injury. For now, the choice to begin chronic opioid therapy for a given patient is left to the individual practitioner. Pragmatic guidelines for properly selecting and monitoring patients receiving chronic opioid therapy are shown in Table 18-3.

1	It is important to individualize treatment for patients with neuropathic pain. Several general principles should guide therapy: the first is to move quickly to provide relief, and the second is to minimize drug side effects. For example, in patients with postherpetic neuralgia and significant cutaneous hypersensitivity, topical lidocaine (Lidoderm patches) can provide immediate relief without side effects. Anticonvulsants (gabapentin or pregabalin; see above) or antidepressants (nortriptyline, desipramine, duloxetine, or venlafaxine) can be used as first-line drugs for patients with neuropathic pain. Systemically administered antiarrhythmic drugs such as lidocaine and mexiletine are less likely to be effective; although intravenous infusion of lidocaine can provide analgesia for patients with different types of neuropathic pain, the relief is usually transient, typically lasting just hours after the cessation of the infusion.

1	The oral lidocaine congener mexiletine is poorly tolerated, producing frequent gastrointestinal adverse effects. There is no consensus on which class of drug should be used as a first-line treatment for any chronically painful condition. However, because relatively high doses of anticonvulsants are required for pain relief, sedation is very common. Sedation is also a problem with TCAs but is much less of a problem with serotonin/norepinephrine reuptake inhibitors (SNRIs; e.g., venlafaxine and duloxetine). Thus, in the elderly or in patients whose daily activities require high-level mental activity, these drugs should be considered the first line. In contrast, opioid medications should be used as a secondor third-line drug class. Although highly effective for many painful conditions, opioids are sedating, and their effect tends to lessen over time, leading to dose escalation and, occasionally, a worsening of pain due to physical dependence. Drugs of different classes can be used in

1	are sedating, and their effect tends to lessen over time, leading to dose escalation and, occasionally, a worsening of pain due to physical dependence. Drugs of different classes can be used in combination to optimize pain control.

1	It is worth emphasizing that many patients, especially those with chronic pain, seek medical attention primarily because they are suffering and because only physicians can provide the medications required for pain relief. A primary responsibility of all physicians is to minimize the physical and emotional discomfort of their patients. Familiarity with pain mechanisms and analgesic medications is an important step toward accomplishing this aim.

1	Chest Discomfort David A. Morrow Chest discomfort is among the most common reasons for which patients present for medical attention at either an emergency depart-ment (ED) or an outpatient clinic. The evaluation of nontraumatic chest discomfort is inherently challenging owing to the broad variety 19 of possible causes, a minority of which are life-threatening conditions that should not be missed. It is helpful to frame the initial diagnostic assessment and triage of patients with acute chest discomfort around three categories: (1) myocardial ischemia; (2) other cardiopulmonary causes (pericardial disease, aortic emergencies, and pulmonary conditions); and (3) non-cardiopulmonary causes. Although rapid identification of high-risk conditions is a priority of the initial assessment, strategies that incorporate routine liberal use of testing carry the potential for adverse effects of unnecessary investigations.

1	Chest discomfort is the third most common reason for visits to the ED in the United States, resulting in 6 to 7 million emergency visits each year. More than 60% of patients with this presentation are hospitalized for further testing, and the rest undergo additional investigation in the ED. Fewer than 25% of evaluated patients are eventually diagnosed with acute coronary syndrome (ACS), with rates of 5–15% in most series of unselected populations. In the remainder, the most common diagnoses are gastrointestinal causes (Fig. 19-1), and fewer than 10% are other life-threatening cardiopulmonary conditions. In a large proportion of patients with transient acute chest discomfort, ACS or another acute cardiopulmonary cause is excluded but the cause is not determined. Therefore, the resources and time devoted to the evaluation of chest discomfort in the absence of a severe cause are substantial. Nevertheless, a disconcerting 2–6% of patients with chest discomfort of presumed non-ischemic

1	and time devoted to the evaluation of chest discomfort in the absence of a severe cause are substantial. Nevertheless, a disconcerting 2–6% of patients with chest discomfort of presumed non-ischemic etiology who are discharged from the ED are later deemed to have had a missed myocardial infarction (MI). Patients with a missed diagnosis of MI have a 30-day risk of death that is double that of their counterparts who are hospitalized.

1	The natural histories of ACS, acute pericardial diseases, pulmonary embolism, and aortic emergencies are discussed in Chaps. 288, 294 and 295, 300, and 301, respectively. In a study of more than 350,000 patients with unspecified presumed non-cardiopulmonary chest discomfort, the mortality rate 1 year after discharge was <2% and did not differ significantly from age-adjusted mortality in the general population. The estimated rate of major cardiovascular events through 30 days in patients with acute chest pain who had been stratified as low risk was 2.5% in a large population-based study that excluded patients with ST-segment elevation or definite noncardiac chest pain. The major etiologies of chest discomfort are discussed in this section and summarized in Table 19-1. Additional elements of the history, physical examination, and diagnostic testing that aid in distinguishing these causes are discussed in a later section (see “Approach to the Patient”).

1	Myocardial ischemia causing chest discomfort, termed angina pectoris, is a primary clinical concern in patients presenting with chest symptoms. Myocardial ischemia is precipitated by an imbalance between myocardial oxygen requirements and myocardial oxygen supply, resulting in insufficient delivery of oxygen to meet the heart’s metabolic demands. Myocardial oxygen consumption may be elevated by increases in heart rate, ventricular wall stress, and myocardial contractility, whereas myocardial oxygen supply is determined by coronary Gastrointestinal 42% Ischemic heart disease 31% Chest wall syndrome 28% Pericarditis 4% Pleuritis 2% Pulmonary embolism 2% Lung cancer 1.5% Aortic aneurysm 1% Aortic stenosis 1% Herpes zoster 1% FIguRE 19-1 Distribution of final discharge diagnoses in patients with nontraumatic acute chest pain. (Figure prepared from data in P Fruergaard et al: Eur Heart J 17:1028, 1996.) PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Gastrointenstinal Esophageal reflux 10–60 min Burning Substernal, epigastric Worsened by postprandial recumbency; relieved by antacids Esophageal spasm 2–30 min Pressure, tightness, Retrosternal Can closely mimic angina burning Peptic ulcer Prolonged; 60–90 min Burning Epigastric, substernal Relieved with food or antacids after meals Gallbladder disease Prolonged Aching or colicky Epigastric, right upper May follow meal quadrant; sometimes to the back Neuromuscular Costochondritis Variable Aching Sternal Sometimes swollen, tender, warm over joint; may be reproduced by localized pressure on examination Trauma or strain Usually constant Aching Localized to area of Reproduced by movement or strain palpation Herpes zoster Usually prolonged Sharp or burning Dermatomal distribution Vesicular rash in area of discomfort

1	Herpes zoster Usually prolonged Sharp or burning Dermatomal distribution Vesicular rash in area of discomfort Psychological Emotional and psy-Variable; may be fleeting Variable; often mani-Variable; may be Situational factors may precipitate chiatric conditions or prolonged fests as tightness and retrosternal symptoms; history of panic attacks, dyspnea with feeling of blood flow and coronary arterial oxygen content. When myocardial ischemia is sufficiently severe and prolonged in duration (as little as 20 min), irreversible cellular injury occurs, resulting in MI.

1	Ischemic heart disease is most commonly caused by atheromatous plaque that obstructs one or more of the epicardial coronary arteries. Stable ischemic heart disease (Chap. 293) usually results from the gradual atherosclerotic narrowing of the coronary arteries. Stable angina is characterized by ischemic episodes that are typically precipitated by a superimposed increase in oxygen demand during physical exertion and relieved upon resting. Ischemic heart disease becomes unstable most commonly when rupture or erosion of one or more atherosclerotic lesions triggers coronary thrombosis (Chap. 291e). Unstable ischemic heart disease is classified clinically by the presence or absence of detectable myocardial injury and the presence or absence of ST-segment elevation on the patient’s electrocardiogram (ECG). When acute coronary atherothrombosis occurs, the intracoronary thrombus may be partially obstructive, generally leading to myocardial ischemia in the absence of ST-segment elevation.

1	(ECG). When acute coronary atherothrombosis occurs, the intracoronary thrombus may be partially obstructive, generally leading to myocardial ischemia in the absence of ST-segment elevation. Marked by ischemic symptoms at rest, with minimal activity, or in an accelerating pattern, unstable ischemic heart disease is classified as unstable angina when there is no detectable myocardial injury and as non–ST elevation MI (NSTEMI) when there is evidence of myocardial necrosis (Chap. 294). When the coronary thrombus is acutely and completely occlusive, transmural myocardial ischemia usually ensues, with ST-segment elevation on the ECG and myocardial necrosis leading to a diagnosis of ST elevation MI (STEMI, see Chap. 295).

1	Clinicians should be aware that unstable ischemic symptoms may also occur predominantly because of increased myocardial oxygen demand (e.g., during intense psychological stress or fever) or because of decreased oxygen delivery due to anemia, hypoxia, or hypotension. However, the term acute coronary syndrome, which encompasses unstable angina, NSTEMI, and STEMI, is in general reserved for ischemia precipitated by acute coronary atherothrombosis. In order to guide therapeutic strategies, a standardized system for classification of MI has been expanded to discriminate MI resulting from acute coronary thrombosis (type 1) from MI occurring secondary to other imbalances of myocardial oxygen supply and demand (type 2; see Chap. 294).

1	Other contributors to stable and unstable ischemic heart disease, such as endothelial dysfunction, microvascular disease, and vasospasm, may exist alone or in combination with coronary atherosclerosis and may be the dominant cause of myocardial ischemia in some patients. Moreover, non-atherosclerotic processes, including congenital abnormalities of the coronary vessels, myocardial bridging, coronary arteritis, and radiation-induced coronary disease, can lead to coronary obstruction. In addition, conditions associated with extreme myocardial oxygen demand and impaired endocardial blood flow, such as aortic valve disease (Chap. 301), hypertrophic cardiomyopathy, or idiopathic dilated cardiomyopathy (Chap. 287), can precipitate myocardial ischemia in patients with or without underlying obstructive atherosclerosis.

1	Characteristics of Ischemic Chest Discomfort The clinical characteristics of angina pectoris, often referred to simply as “angina,” are highly similar whether the ischemic discomfort is a manifestation of stable ischemic heart disease, unstable angina, or MI; the exceptions are differences in the pattern and duration of symptoms associated with these syndromes (Table 19-1). Heberden initially described angina as a sense of “strangling and anxiety.” Chest discomfort characteristic of myocardial ischemia is typically described as aching, heavy, squeezing, crushing, or constricting. However, in a substantial minority of patients, the quality of discomfort is extremely vague and may be described as a mild tightness, or merely an uncomfortable feeling, that sometimes is experienced as numbness or a burning sensation. The site of the discomfort is usually retrosternal, but radiation is common and generally occurs down the ulnar surface of the left arm; the right arm, both arms, neck, jaw,

1	or a burning sensation. The site of the discomfort is usually retrosternal, but radiation is common and generally occurs down the ulnar surface of the left arm; the right arm, both arms, neck, jaw, or shoulders may also be involved. These and other characteristics of ischemic chest discomfort pertinent to discrimination from other causes of chest pain are discussed later in this chapter (see “Approach to the Patient”).

1	Stable angina usually begins gradually and reaches its maximal intensity over a period of minutes before dissipating within several minutes with rest or with nitroglycerin. The discomfort typically occurs predictably at a characteristic level of exertion or psychological stress. By definition, unstable angina is manifest by self-limited anginal chest discomfort that is exertional but occurs at increased frequency with progressively lower intensity of physical activity or even at rest. Chest discomfort associated with MI is typically more severe, is prolonged (usually lasting ≥30 min), and is not relieved by rest.

1	Mechanisms of Cardiac Pain The neural pathways involved in ischemic cardiac pain are poorly understood. Ischemic episodes are thought to excite local chemosensitive and mechanoreceptive receptors that, in turn, stimulate release of adenosine, bradykinin, and other substances that activate the sensory ends of sympathetic and vagal afferent fibers. The afferent fibers traverse the nerves that connect to the upper five thoracic sympathetic ganglia and upper five distal thoracic roots of the spinal cord. From there, impulses are transmitted to the thalamus. Within the spinal cord, cardiac sympathetic afferent impulses may converge with impulses from somatic thoracic structures, and this convergence may be the basis for referred cardiac pain. In addition, cardiac vagal afferent fibers synapse in the nucleus tractus solitarius of the medulla and then descend to the upper cervical spinothalamic tract, and this route may contribute to anginal pain experienced in the neck and jaw.

1	OTHER CARDIOPuLMONARY CAuSES Pericardial and Other Myocardial Diseases (See also Chap. 288)

1	Inflammation of the pericardium due to infectious or noninfectious causes can be responsible for acute or chronic chest discomfort. The visceral surface and most of the parietal surface of the pericardium are insensitive to pain. Therefore, the pain of pericarditis is thought to arise principally from associated pleural inflammation and is more common with infectious causes of pericarditis, which typically involve the pleura. Because of this pleural association, the discomfort of pericarditis is usually pleuritic pain that is exacerbated by breathing, coughing, or changes in position. Moreover, owing to the overlapping sensory supply of the central diaphragm via the phrenic nerve with somatic sensory fibers originating in the third to fifth cervical segments, the pain of pleural pericarditis is often referred to the shoulder and neck. Involvement of the pleural surface of the lateral diaphragm can lead to pain in the upper abdomen.

1	Acute inflammatory and other non-ischemic myocardial diseases can also produce chest discomfort. The symptoms of Takotsubo (stress-related) cardiomyopathy often start abruptly with chest pain and shortness of breath. This form of cardiomyopathy, in its most recognizable form, is triggered by an emotionally or physically stressful event and may mimic acute MI because of its commonly associated ECG abnormalities, including ST-segment elevation, and elevated biomarkers of myocardial injury. Observational studies support a predilection for women >50 years of age. The symptoms of acute myocarditis are highly varied. Chest discomfort may either originate with inflammatory injury of the myocardium or be due to severe increases in wall stress related to poor ventricular performance.

1	Diseases of the Aorta (See also Chap. 301) Acute aortic dissection (Fig. 19-1) is a less common cause of chest discomfort but is important because of the catastrophic natural history of certain subsets of cases when recognized late or left untreated. Acute aortic syndromes encompass a spectrum of acute aortic diseases related to disruption of the media of the aortic wall. Aortic dissection involves a tear in the aortic intima, resulting in separation of the media and creation of a separate “false” lumen. A penetrating ulcer has been described as ulceration of an aortic atheromatous plaque that extends through the intima and into the aortic media, with the potential to initiate an intramedial dissection or rupture into the adventitia. Intramural hematoma is an aortic wall hematoma with no demonstrable intimal flap, no radiologically apparent intimal tear, and no false lumen. Intramural hematoma can occur due to either rupture of the vasa vasorum or, less commonly, a penetrating ulcer.

1	Each of these subtypes of acute aortic syndrome typically presents with chest discomfort that is often severe, sudden in onset, and sometimes described as “tearing” in quality. Acute aortic syndromes involving the ascending aorta tend to cause pain in the midline of the anterior chest, whereas descending aortic syndromes most often present with pain in the back. Therefore, dissections that begin in the ascending aorta and extend to the descending aorta tend to cause pain in the front of the chest that extends toward the back, between the shoulder blades. Proximal aortic dissections that involve the ascending aorta (type A in the Stanford nomenclature) are at high risk for major complications that may influence the clinical presentation, including (1) compromise of the aortic ostia of the coronary arteries, resulting in MI; (2) disruption of the aortic valve, causing acute aortic insufficiency; and (3) rupture of the hematoma into the pericardial space, leading to pericardial

1	of the coronary arteries, resulting in MI; (2) disruption of the aortic valve, causing acute aortic insufficiency; and (3) rupture of the hematoma into the pericardial space, leading to pericardial tamponade.

1	Knowledge of the epidemiology of acute aortic syndromes can be helpful in maintaining awareness of this relatively uncommon group of disorders (estimated annual incidence, 3 cases per 100,000 population). Nontraumatic aortic dissections are very rare in the absence of hypertension or conditions associated with deterioration of the elastic or muscular components of the aortic media, including pregnancy, bicuspid aortic disease, or inherited connective tissue diseases, such as Marfan and Ehlers-Danlos syndromes. Although aortic aneurysms are most often asymptomatic, thoracic aortic aneurysms can cause chest pain and other symptoms by compressing adjacent structures. This pain tends to be steady, deep, and occasionally severe. Aortitis, whether of noninfectious or infectious etiology, in the absence of aortic dissection is a rare cause of chest or back discomfort.

1	Pulmonary Conditions Pulmonary and pulmonary-vascular conditions that cause chest discomfort usually do so in conjunction with dyspnea and often produce symptoms that have a pleuritic nature.

1	PULMONARY EMBOLISM (See also Chap. 300) Pulmonary emboli (annual incidence, ~1 per 1000) can produce dyspnea and chest discomfort that is sudden in onset. Typically pleuritic in pattern, the chest discomfort associated with pulmonary embolism may result from (1) involvement of the pleural surface of the lung adjacent to a resultant pulmonary infarction; (2) distention of the pulmonary artery; or (3) possibly, right ventricular wall stress and/or subendocardial ischemia related to acute pulmonary hypertension. The pain associated with small pulmonary emboli is often lateral and pleuritic and is believed to be related to the first of these three possible mechanisms. In contrast, massive pulmonary emboli may cause severe substernal pain that may mimic an MI and that is plausibly attributed to the second and third of these potential mechanisms. Massive or submassive pulmonary embolism may also be associated with syncope, hypotension, and signs of right heart failure. Other typical

1	to the second and third of these potential mechanisms. Massive or submassive pulmonary embolism may also be associated with syncope, hypotension, and signs of right heart failure. Other typical characteristics that aid in the recognition of pulmonary embolism are discussed later in this chapter (see “Approach to the Patient”).

1	PNEUMOTHORAX (See also Chap. 317) Primary spontaneous pneumothorax is a rare cause of chest discomfort, with an estimated annual incidence in the United States of 7 per 100,000 among men and <2 per 100,000 among women. Risk factors include male sex, smoking, family history, and Marfan syndrome. The symptoms are usually sudden in onset, and dyspnea may be mild; thus, presentation to medical attention is sometimes delayed. Secondary spontaneous pneumothorax may occur in patients with underlying lung disorders, such as chronic obstructive pulmonary disease, asthma, or cystic fibrosis, and usually produces symptoms that are more severe. Tension pneumothorax is a medical emergency caused by trapped intrathoracic air that precipitates hemodynamic collapse.

1	Other Pulmonary Parenchymal, Pleural, or Vascular Disease (See also Chaps. 304, 305, and 316) Most pulmonary diseases that produce chest pain, including pneumonia and malignancy, do so because of involvement of the pleura or surrounding structures. Pleurisy is typically described as a knifelike pain that is worsened by inspiration or coughing. In contrast, chronic pulmonary hypertension can manifest as chest pain that may be very similar to angina in its characteristics, suggesting right ventricular myocardial ischemia in some cases. Reactive airways diseases similarly can cause chest tightness associated with breathlessness rather than pleurisy.

1	NON-CARDIOPuLMONARY CAuSES gastrointenstinal Conditions (See also Chap. 344) Gastrointestinal disorders are the most common cause of nontraumatic chest discomfort and often produce symptoms that are difficult to discern from more serious causes of chest pain, including myocardial ischemia. Esophageal disorders, in particular, may simulate angina in the character and location of the pain. Gastroesophageal reflux and disorders of esophageal motility are common and should be considered in the differential diagnosis of chest pain (Fig. 19-1 and Table 19-1). Acid reflux often causes a burning discomfort. The pain of esophageal spasm, in contrast, is commonly an intense, squeezing discomfort that is retrosternal in location and, like angina, may be relieved by nitroglycerin or dihydropyridine calcium channel antagonists. Chest pain can also result from injury to the esophagus, such as a Mallory-Weiss tear or even an esophageal rupture (Boerhaave syndrome) caused by severe vomiting. Peptic

1	calcium channel antagonists. Chest pain can also result from injury to the esophagus, such as a Mallory-Weiss tear or even an esophageal rupture (Boerhaave syndrome) caused by severe vomiting. Peptic ulcer disease is most commonly epigastric in location but can radiate into the chest (Table 19-1).

1	Hepatobiliary disorders, including cholecystitis and biliary colic, may mimic acute cardiopulmonary diseases. Although the pain arising from these disorders usually localizes to the right upper quadrant of the abdomen, it is variable and may be felt in the epigastrium and radiate to the back and lower chest. This discomfort is sometimes referred to the scapula or may in rare cases be felt in the shoulder, suggesting diaphragmatic irritation. The pain is steady, usually lasts several hours, and subsides spontaneously, without symptoms between attacks. Pain resulting from pancreatitis is typically aching epigastric pain that radiates to the back. Musculoskeletal and Other Causes (See also Chap. 393) Chest discomfort can be produced by any musculoskeletal disorder involving the chest wall or the nerves of the chest wall, neck, or upper limbs. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Costochondritis causing tenderness of the costochondral junctions (Tietze’s syndrome) is relatively common. Cervical radiculitis may manifest as a prolonged or constant aching discomfort in the upper chest and limbs. The pain may be exacerbated by motion of the neck. Occasionally, chest pain can be caused by compression of the brachial plexus by the cervical ribs, and tendinitis or bursitis involving the left shoulder may mimic the radiation of angina. Pain in a dermatomal distribution can also be caused by cramping of intercostal muscles or by herpes zoster (Chap. 217). Emotional and Psychiatric Conditions As many as 10% of patients who present to emergency departments with acute chest discomfort have a panic disorder or related condition (Table 19-1). The symptoms may include chest tightness or aching that is associated with a sense of anxiety and difficulty breathing. The symptoms may be prolonged or fleeting.

1	APPROACH TO THE PATIENT:

1	Given the broad set of potential causes and the heterogeneous risk of serious complications in patients who present with acute nontraumatic chest discomfort, the priorities of the initial clinical encounter include assessment of (1) the patient’s clinical stability and (2) the probability that the patient has an underlying cause of the discomfort that may be life-threatening. The high-risk conditions of principal concern are acute cardiopulmonary processes, including ACS, acute aortic syndrome, pulmonary embolism, tension pneumothorax, and pericarditis with tamponade. Among non-cardiopulmonary causes of chest pain, esophageal rupture likely holds the greatest urgency for diagnosis. Patients with these conditions may deteriorate rapidly despite initially appearing well. The remaining population with non-cardiopulmonary conditions has a more favorable prognosis during completion of the diagnostic work-up. A rapid targeted assessment for a serious cardiopulmonary cause is of particular

1	with non-cardiopulmonary conditions has a more favorable prognosis during completion of the diagnostic work-up. A rapid targeted assessment for a serious cardiopulmonary cause is of particular relevance for patients with acute ongoing pain who have presented for emergency evaluation. Among patients presenting in the outpatient setting with chronic pain or pain that has resolved, a general diagnostic assessment is reasonably undertaken (see “Outpatient Evaluation of Chest Discomfort,” below). A series of questions that can be used to structure the clinical evaluation of patients with chest discomfort is shown in Table 19-2.

1	1. Could the chest discomfort be due to an acute, potentially life-threatening condition that warrants urgent evaluation and management? 2. If not, could the discomfort be due to a chronic condition likely to lead to serious complications? 3. If not, could the discomfort be due to an acute condition that warrants specific treatment? 4. If not, could the discomfort be due to another treatable chronic condition? Esophageal reflux Cervical disk disease Esophageal spasm Arthritis of the shoulder or spine Peptic ulcer disease Costochondritis Gallbladder disease Other musculoskeletal disorders Other gastrointestinal conditions Anxiety state Source: Developed by Dr. Thomas H. Lee for the 18th edition of Harrison’s Principles of Internal Medicine.

1	Source: Developed by Dr. Thomas H. Lee for the 18th edition of Harrison’s Principles of Internal Medicine. The evaluation of nontraumatic chest discomfort relies heavily on the clinical history and physical examination to direct subsequent diagnostic testing. The evaluating clinician should assess the quality, location (including radiation), and pattern (including onset and duration) of the pain as well as any provoking or alleviating factors. The presence of associated symptoms may also be useful in establishing a diagnosis.

1	Quality of Pain The quality of chest discomfort alone is never sufficient to establish a diagnosis. However, the characteristics of the pain are pivotal in formulating an initial clinical impression and assessing the likelihood of a serious cardiopulmonary process (Table 19-1), including acs in particular (Fig. 19-2). Pressure or tightness is consistent with a typical presentation of myocardial ischemic pain. Nevertheless, the clinician must remember that some patients with ischemic chest symptoms deny any “pain” but rather complain of dyspnea or a vague sense of anxiety. The severity of the discomfort has poor diagnostic accuracy. It is often helpful to ask about the similarity of the discomfort to previous definite ischemic symptoms. It is unusual for angina to be sharp, as in knifelike, stabbing, or pleuritic; however, patients sometimes use the word “sharp” to convey the intensity of discomfort rather than the quality. Pleuritic discomfort is suggestive of a process involving the

1	stabbing, or pleuritic; however, patients sometimes use the word “sharp” to convey the intensity of discomfort rather than the quality. Pleuritic discomfort is suggestive of a process involving the pleura, including pericarditis, pulmonary embolism, or pulmonary parenchymal processes. Less frequently, the pain of pericarditis or massive pulmonary embolism is a steady severe pressure or aching that can be difficult to discriminate from myocardial ischemia. “Tearing” or “ripping” pain is often described by patients with acute aortic dissection. However, acute aortic emergencies also present commonly with severe, knifelike pain. A burning quality can suggest acid reflux or peptic ulcer disease but may also occur with myocardial ischemia. Esophageal pain, particularly with spasm, can be a severe squeezing discomfort identical to angina.

1	Location of Discomfort A substernal location with radiation to the neck, jaw, shoulder, or arms is typical of myocardial ischemic discomfort. Some patients present with aching in sites of radiated pain as their only symptoms of ischemia. However, pain that is highly localized—e.g., that which can be demarcated by the tip of one finger—is highly unusual for angina. A retrosternal location should prompt consideration of esophageal pain; however, other gastrointestinal conditions usually present with pain that is most intense in the abdomen or epigastrium, with possible radiation into the chest. Angina may also occur in an epigastric location. However, pain that occurs solely above the mandible or below the epigastrium is rarely angina. Severe pain radiating to the back, particularly between the shoulder blades, should prompt consideration of an acute aortic syndrome. Radiation to the trapezius ridge is characteristic of pericardial pain and does not usually occur with angina.

1	Pattern Myocardial ischemic discomfort usually builds over minutes and is exacerbated by activity and mitigated by rest. In contrast, pain that reaches its peak intensity immediately is more suggestive of aortic dissection, pulmonary embolism, or spontaneous pneumothorax. Pain that is fleeting (lasting only a few seconds) is rarely ischemic in origin. Similarly, pain that is constant in intensity for a prolonged period (many hours to days) is unlikely to represent myocardial ischemia if it occurs in the absence of other clinical consequences, such as abnormalities of the ECG, elevation of cardiac biomarkers, or clinical sequelae (e.g., heart failure or hypotension). Both myocardial ischemia and acid reflux may have their onset in the morning, the latter because of the absence of food to absorb gastric acid.

1	Provoking and Alleviating Factors Patients with myocardial ischemic pain usually prefer to rest, sit, or stop walking. However, clinicians should be aware of the phenomenon of “warm-up angina” in which some patients experience relief of angina as they continue at the same or even a greater level of exertion without symptoms (Chap. 293). Alterations in the intensity of pain with changes in position or movement of the upper extremities and neck are less likely with myocardial ischemia and suggest a musculoskeletal etiology. The pain of pericarditis, however, often is worse in the supine position and relieved by sitting upright and leaning forward. Gastroesophageal reflux may be exacerbated by alcohol, some foods, or by a reclined position. Relief can occur with sitting.

1	Radiation to right arm or shoulder Radiation to both arms or shoulders Associated with exertion Radiation to left arm Associated with diaphoresis Associated with nausea or vomiting Worse than previous angina or similar to previous MI Described as pressure Inframammary location Reproducible with palpation Described as sharp Described as positional Described as pleuritic 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 FIguRE 19-2 Association of chest pain characteristics with the probability of acute myocardial infarction (AMI). (Figure prepared from data in CJ Swap, JT Nagurney: JAMA 294:2623, 2005.)

1	Exacerbation by eating suggests a gastrointestinal etiology such as peptic ulcer disease, cholecystitis, or pancreatitis. Peptic ulcer disease tends to become symptomatic 60–90 min after meals. However, in the setting of severe coronary atherosclerosis, redistribution of blood flow to the splanchnic vasculature after eating can trigger postprandial angina. The discomfort of acid reflux and peptic ulcer disease is usually diminished promptly by acid-reducing therapies. In contrast with its impact in some patients with angina, physical exertion is very unlikely to alter symptoms from gastrointestinal causes of chest pain. Relief of chest discomfort within minutes after administration of nitroglycerin is suggestive of but not sufficiently sensitive or specific for a definitive diagnosis of myocardial ischemia. Esophageal spasm may also be relieved promptly with nitroglycerin. A delay of >10 min before relief is obtained after nitroglycerin suggests that the symptoms either are not caused

1	ischemia. Esophageal spasm may also be relieved promptly with nitroglycerin. A delay of >10 min before relief is obtained after nitroglycerin suggests that the symptoms either are not caused by ischemia or are caused by severe ischemia, such as during acute MI.

1	Associated Symptoms Symptoms that accompany myocardial ischemia may include diaphoresis, dyspnea, nausea, fatigue, faintness, and eructations. In addition, these symptoms may exist in isolation as anginal equivalents (i.e., symptoms of myocardial ischemia other than typical angina), particularly in women and the elderly. Dyspnea may occur with multiple conditions considered in the differential diagnosis of chest pain and thus is not discriminative, but the presence of dyspnea is important because it suggests a cardiopulmonary etiology. Sudden onset of significant respiratory distress should lead to consideration of pulmonary embolism and spontaneous pneumothorax. Hemoptysis may occur with pulmonary embolism, or as blood-tinged frothy sputum in severe heart failure but usually points toward a pulmonary parenchymal etiology of chest symptoms. Presentation with syncope or pre-syncope should prompt consideration of hemodynamically significant pulmonary embolism or aortic dissection as

1	a pulmonary parenchymal etiology of chest symptoms. Presentation with syncope or pre-syncope should prompt consideration of hemodynamically significant pulmonary embolism or aortic dissection as well as ischemic arrhythmias. Although nausea and vomiting suggest a gastrointestinal disorder, these symptoms may occur in the setting of MI (more commonly inferior MI), presumably because of activation of the vagal reflex or stimulation of left ventricular receptors as part of the Bezold-Jarisch reflex.

1	Past Medical History The past medical history is useful in assessing the patient for risk factors for coronary atherosclerosis (Chap. 291e) and venous thromboembolism (Chap. 300) as well as for conditions that may predispose the patient to specific disorders. For example, a history of connective tissue diseases such as marfan syndrome should heighten the clinician’s suspicion of an acute aortic syndrome or spontaneous pneumothorax. A careful history may elicit clues about depression or prior panic attacks.

1	In addition to providing an initial assessment of the patient’s clinical stability, the physical examination of patients with chest discomfort can provide direct evidence of specific etiologies of chest pain (e.g., unilateral absence of lung sounds) and can identify potential precipitants of acute cardiopulmonary causes of chest pain (e.g., uncontrolled hypertension), relevant comorbid conditions (e.g., obstructive pulmonary disease), and complications of the presenting syndrome (e.g., heart failure). However, because the findings on physical examination may be normal in patients with unstable ischemic heart disease, an unremarkable physical exam is not definitively reassuring.

1	general The patient’s general appearance is helpful in establishing an initial impression of the severity of illness. Patients with acute MI or other acute cardiopulmonary disorders often appear anxious, uncomfortable, pale, cyanotic, or diaphoretic. Patients who are massaging or clutching their chests may describe their pain with a clenched fist held against the sternum (Levine’s sign). Occasionally, body habitus is helpful—e.g., in patients with Marfan syndrome or the prototypical young, tall, thin man with spontaneous pneumothorax. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Vital Signs Significant tachycardia and hypotension are indicative of important hemodynamic consequences of the underlying cause of chest discomfort and should prompt a rapid survey for the most severe conditions, such as acute MI with cardiogenic shock, massive pulmonary embolism, pericarditis with tamponade, or tension pneumothorax. Acute aortic emergencies usually present with severe hypertension but may be associated with profound hypotension when there is coronary arterial compromise or dissection into the pericardium. Sinus tachycardia is an important manifestation of submassive pulmonary embolism. Tachypnea and hypoxemia point toward a pulmonary cause. The presence of low-grade fever is nonspecific because it may occur with MI and with thromboembolism in addition to infection.

1	Pulmonary Examination of the lungs may localize a primary pulmonary cause of chest discomfort, as in cases of pneumonia, asthma, or pneumothorax. Left ventricular dysfunction from severe ischemia/infarction as well as acute valvular complications of MI or aortic dissection can lead to pulmonary edema, which is an indicator of high risk.

1	Cardiac The jugular venous pulse is often normal in patients with acute myocardial ischemia but may reveal characteristic patterns with pericardial tamponade or acute right ventricular dysfunction (Chaps. 267 and 288). Cardiac auscultation may reveal a third or, more commonly, a fourth heart sound, reflecting myocardial systolic or diastolic dysfunction. Murmurs of mitral regurgitation or a harsh murmur of a ventricular-septal defect may indicate mechanical complications of STEMI. A murmur of aortic insufficiency may be a complication of proximal aortic dissection. Other murmurs may reveal underlying cardiac disorders contributory to ischemia (e.g., aortic stenosis or hypertrophic cardiomyopathy). Pericardial friction rubs reflect pericardial inflammation.

1	Abdominal Localizing tenderness on the abdominal exam is useful in identifying a gastrointestinal cause of the presenting syndrome. Abdominal findings are infrequent with purely acute cardiopulmonary problems, except in the case of underlying chronic cardiopulmonary disease or severe right ventricular dysfunction leading to hepatic congestion. Vascular Pulse deficits may reflect underlying chronic atherosclerosis, which increases the likelihood of coronary artery disease. However, evidence of acute limb ischemia with loss of the pulse and pallor, particularly in the upper extremities, can indicate catastrophic consequences of aortic dissection. Unilateral lower-extremity swelling should raise suspicion about venous thromboembolism.

1	Musculoskeletal Pain arising from the costochondral and chondrosternal articulations may be associated with localized swelling, redness, or marked localized tenderness. Pain on palpation of these joints is usually well localized and is a useful clinical sign, though deep palpation may elicit pain in the absence of costochondritis. Although palpation of the chest wall often elicits pain in patients with various musculoskeletal conditions, it should be appreciated that chest wall tenderness does not exclude myocardial ischemia. Sensory deficits in the upper extremities may be indicative of cervical disk disease.

1	Electrocardiography is crucial in the evaluation of nontraumatic chest discomfort. The ECG is pivotal for identifying patients with ongoing ischemia as the principal reason for their presentation as well as secondary cardiac complications of other disorders. Professional society guidelines recommend that an ECG be obtained within 10 min of presentation, with the primary goal of identifying patients with ST-segment elevation diagnostic of MI who are candidates for immediate interventions to restore flow in the occluded coronary artery. ST-segment depression and symmetric T-wave inversions at least 0.2 mV in depth are useful for detecting myocardial ischemia in the absence of STEMI and are also indicative of higher risk of death or recurrent ischemia. Serial performance of ECGs (every 30–60 min) is recommended in the ED evaluation of suspected ACS. In addition, an ECG with right-sided lead placement should be considered in patients with clinically suspected ischemia and a nondiagnostic

1	min) is recommended in the ED evaluation of suspected ACS. In addition, an ECG with right-sided lead placement should be considered in patients with clinically suspected ischemia and a nondiagnostic standard 12-lead ECG. Despite the value of the resting ECG, its sensitivity for ischemia is poor—as low as 20% in some studies.

1	Abnormalities of the ST segment and T wave may occur in a variety of conditions, including pulmonary embolism, ventricular hypertrophy, acute and chronic pericarditis, myocarditis, electrolyte imbalance, and metabolic disorders. Notably, hyperventilation associated with panic disorder can also lead to nonspecific ST and T-wave abnormalities. Pulmonary embolism is most often associated with sinus tachycardia but can also lead to rightward shift of the ECG axis, manifesting as an S-wave in lead I, with a Q-wave and T-wave in lead III (Chaps. 268 and 300). In patients with ST-segment elevation, the presence of diffuse lead involvement not corresponding to a specific coronary anatomic distribution and PR-segment depression can aid in distinguishing pericarditis from acute MI.

1	(See Chap. 308e) Plain radiography of the chest is performed routinely when patients present with acute chest discomfort and selectively when individuals who are being evaluated as outpatients have subacute or chronic pain. The chest radiograph is most useful for identifying pulmonary processes, such as pneumonia or pneumothorax. Findings are often unremarkable in patients with ACS, but pulmonary edema may be evident. Other specific findings include widening of the mediastinum in some patients with aortic dissection, Hampton’s hump or Westermark’s sign in patients with pulmonary embolism (Chaps. 300 and 308e), or pericardial calcification in chronic pericarditis.

1	Laboratory testing in patients with acute chest pain is focused on the detection of myocardial injury. Such injury can be detected by the presence of circulating proteins released from damaged myocardial cells. Owing to the time necessary for this release, initial biomarkers of injury may be in the normal range, even in patients with STEMI. Because of superior cardiac tissue-specificity compared with creatine kinase MB, cardiac troponin is the preferred biomarker for the diagnosis of MI and should be measured in all patients with suspected ACS at presentation and repeated in 3–6 h. Testing after 6 h is required only when there is uncertainty regarding the onset of pain or when stuttering symptoms have occurred. It is not necessary or advisable to measure troponin in patients without suspicion of ACS unless this test is being used specifically for risk stratification (e.g., in pulmonary embolism or heart failure).

1	The development of cardiac troponin assays with progressively greater analytical sensitivity has facilitated detection of substantially lower blood concentrations of troponin than was previously possible. This evolution permits earlier detection of myocardial injury, enhances the overall accuracy of a diagnosis of MI, and improves risk stratification in suspected ACS. The greater negative predictive value of a negative troponin result with current-generation assays is an advantage in the evaluation of chest pain in the ED. Rapid rule-out protocols that use serial testing and changes in troponin concentration over as short a period as 1–2 h appear promising and remain under investigation. However, with these advantages has come a trade-off: myocardial injury is detected in a larger proportion of patients who have non-ACS cardiopulmonary conditions than with previous, less sensitive assays. This evolution in testing for myocardial necrosis has rendered other aspects of the clinical

1	of patients who have non-ACS cardiopulmonary conditions than with previous, less sensitive assays. This evolution in testing for myocardial necrosis has rendered other aspects of the clinical evaluation critical to the practitioner’s determination of the probability that the symptoms represent ACS. In addition, observation of a change in cardiac troponin concentration between serial samples is useful in discriminating acute causes of myocardial injury from chronic elevation due to underlying structural heart disease, end-stage renal disease, or interfering antibodies. The diagnosis of MI is reserved for acute myocardial injury that is marked by a rising and/or falling pattern—with at least one value exceeding the 99th percentile reference limit—and that is caused by ischemia. Other non-ischemic insults, such as myocarditis, may result in myocardial injury but should not be labeled MI.

1	Other laboratory assessments may include the D-dimer test to aid in exclusion of pulmonary embolism (Chap. 300). Measurement of a B-type natriuretic peptide is useful when considered in conjunction with the clinical history and exam for the diagnosis of heart failure. B-type natriuretic peptides also provide prognostic information regarding patients with ACS and those with pulmonary embolism. Other putative biomarkers of acute myocardial ischemia or ACS, such as myeloperoxidase, have not been adopted in routine use.

1	Multiple clinical algorithms have been developed to aid in decision-making during the evaluation and disposition of patients with acute nontraumatic chest pain. Such decision-aids have been derived on the basis of their capacity to estimate either of two closely related but not identical probabilities: (1) the probability of a final diagnosis of ACS and (2) the probability of major cardiac events during short-term follow-up. Such decision-aids are used most commonly to identify patients with a low clinical probability of ACS who are candidates either for early provocative testing for ischemia or for discharge from the ED. Goldman and Lee developed one of the first such decision-aids, using only the ECG and risk indicators—hypotension, pulmonary rales, and known ischemic heart disease—to categorize patients into four risk categories ranging from a <1% to a >16% probability of a major cardiovascular complication. The Acute Cardiac Ischemia Time-Insensitive Predictive Instrument

1	categorize patients into four risk categories ranging from a <1% to a >16% probability of a major cardiovascular complication. The Acute Cardiac Ischemia Time-Insensitive Predictive Instrument (ACI-TIPI) combines age, sex, chest pain presence, and ST-segment abnormalities to define a probability of ACS. More recently developed decision-aids are shown in Fig. 19-3. Elements common to each of these tools are (1) symptoms typical for ACS; (2) older age; (3) risk factors for or known atherosclerosis; (4) ischemic ECG abnormalities; and (5) elevated cardiac troponin levels. Although, because of very low specificity, the overall diagnostic performance of such decision-aids is poor (area under the receiver operating curve, 0.55–0.65), they can help identify patients with a very low probability of ACS (e.g., <1%). Nevertheless, no such decision-aid (or single clinical factor) is sufficiently sensitive and well validated to use as a sole tool for clinical decision-making.

1	Clinicians should differentiate between the algorithms discussed above and risk scores derived for stratification of prognosis (e.g., the TIMI and GRACE risk scores, Chap. 295) in patients who already have an established diagnosis of ACS. The latter risk scores were not designed to be used for diagnostic assessment.

1	Exercise electrocardiography (“stress testing”) is commonly employed for completion of risk stratification of patients who have undergone an initial evaluation that has not revealed a specific cause of chest discomfort and has identified them as being at low or selectively intermediate risk of ACS. Early exercise testing is safe in patients without high-risk findings after 8–12 h of observation and can assist in refining their prognostic assessment. For example, of low-risk patients who underwent exercise testing in the first 48 h after presentation, those without evidence of ischemia had a 2% rate of cardiac events through 6 months, whereas the rate was 15% among patients with either clear evidence of ischemia or an equivocal result. Patients who are unable to exercise may undergo pharmacological stress testing with either nuclear perfusion imaging or echocardiography. Notably, some experts have deemed the routine use of stress testing for low-risk patients unsupported by direct

1	stress testing with either nuclear perfusion imaging or echocardiography. Notably, some experts have deemed the routine use of stress testing for low-risk patients unsupported by direct clinical evidence and a potentially unnecessary source of cost.

1	Professional society guidelines identify ongoing chest pain as a contraindication to stress testing. In selected patients with persistent pain and nondiagnostic ECG and biomarker data, resting PART 2 Cardinal Manifestations and Presentation of Diseases HEART Score History Highly suspicious Moderately suspicious Slightly suspicious 2 1 0 ECG Significant ST-depression Non-specific abnormality Normal 2 1 0 Age 65 y 45–<65 y <45 y 2 1 0 Risk factors 3 risk factors 1–2 risk factors None 2 1 0 Troponin (serial) 3 × 99th percentile 1–<3 × 99th percentile �99th percentile 2 1 0 TOTAL Low-risk: 0–3 Not low risk: 4 North American Chest Pain Rule High Risk Criteria Y/N Typical symptoms for ischemia ECG: acute ischemic changes Age 50 y Known coronary artery disease Troponin (serial) >99th percentile Low-risk: All No Not Low-risk: Any Yes 20.2Captured as low-risk (%) 4.4 Sensitivity 99.1 100 Specificity 25.7 5.6

1	Sensitivity 99.1 100 Specificity 25.7 5.6 FIguRE 19-3 Examples of decision-aids used in conjunction with serial measurement of cardiac troponin for evaluation of acute chest pain. (Figure prepared from data in SA Mahler et al: Int J Cardiol 168:795, 2013.) myocardial perfusion images can be obtained; the absence of any perfusion abnormality substantially reduces the likelihood of coronary artery disease. In some centers, early myocardial perfusion imaging is performed as part of a routine strategy for evaluating patients at low or intermediate risk of ACS in parallel with other testing. Management of patients with normal perfusion images can be expedited with earlier discharge and outpatient stress testing, if indicated. Those with abnormal rest perfusion imaging, which cannot discriminate between old or new myocardial defects, must undergo additional in-hospital evaluation.

1	Other noninvasive imaging studies of the chest can be used selectively to provide additional diagnostic and prognostic information on patients with chest discomfort. Echocardiography Echocardiography is not necessarily routine in patients with chest discomfort. However, in patients with an uncertain diagnosis, particularly those with nondiagnostic ST elevation, ongoing symptoms, or hemodynamic instability, detection of abnormal regional wall motion provides evidence of possible ischemic dysfunction. Echocardiography is diagnostic in patients with mechanical complications of MI or in patients with pericardial tamponade. Transthoracic echocardiography is poorly sensitive for aortic dissection, although an intimal flap may sometimes be detected in the ascending aorta.

1	CT Angiography (See Chap. 270e) CT angiography is emerging as a modality for the evaluation of patients with acute chest discomfort. Coronary CT angiography is a sensitive technique for detection of obstructive coronary disease, particularly in the proximal third of the major epicardial coronary arteries. CT appears to enhance the speed to disposition of patients with a low-intermediate probability for ACS; its major strength being the negative predictive value of a finding of no significant disease. In addition, contrast-enhanced CT can detect focal areas of myocardial injury in the acute setting as decreased areas of enhancement. At the same time, CT angiography can exclude aortic dissection, pericardial effusion, and pulmonary embolism. Balancing factors in the consideration of the emerging role of coronary CT angiography in low-risk patients are radiation exposure and additional testing prompted by nondiagnostic abnormal results.

1	MRI (See Chap. 270e) Cardiac magnetic resonance (CMR) imaging is an evolving, versatile technique for structural and functional evaluation of the heart and the vasculature of the chest. CMR accurately measures ventricular dimensions and function and can be performed as a modality for pharmacologic stress perfusion imaging. Gadolinium-enhanced CMR can provide early detection of MI, defining areas of myocardial necrosis accurately, and can delineate patterns of myocardial disease that are often useful in discriminating ischemic from non-ischemic myocardial injury. Although usually not practical for the urgent evaluation of acute chest discomfort, CMR can be a useful modality for cardiac structural evaluation of patients with elevated cardiac troponin levels in the absence of definite coronary artery disease. CMR coronary angiography is in its early stages. MRI also permits highly accurate assessment for aortic dissection but is infrequently used as the first test because CT and

1	coronary artery disease. CMR coronary angiography is in its early stages. MRI also permits highly accurate assessment for aortic dissection but is infrequently used as the first test because CT and transesophageal echocardiography are usually more practical.

1	Because of the challenges inherent in reliably identifying the small proportion of patients with serious causes of acute chest discomfort while not exposing the larger number of low-risk patients to unnecessary testing and extended ED or hospital evaluations, many medical centers have adopted critical pathways to expedite the assessment and management of patients with nontraumatic chest pain, often in dedicated chest pain units. Such pathways are generally aimed at (1) rapid identification, triage, and treatment of high-risk cardiopulmonary conditions (e.g., STEMI); (2) accurate identification of low-risk patients who can be safely observed in units with less intensive monitoring, undergo early exercise testing, or be discharged home; and (3) through more efficient and systematic accelerated diagnostic protocols, safe reduction in costs associated with overuse of testing and unnecessary hospitalizations. In some studies, provision of pro-tocol-driven care in chest pain units has

1	diagnostic protocols, safe reduction in costs associated with overuse of testing and unnecessary hospitalizations. In some studies, provision of pro-tocol-driven care in chest pain units has decreased costs and overall duration of hospital evaluation with no detectable excess of adverse clinical outcomes.

1	Chest pain is common in outpatient practice, with a lifetime prevalence of 20–40% in the general population. More than 25% of patients with MI have had a related visit with a primary care physician in the previous month. The diagnostic principles are the same as in the ED. However, the pretest probability of an acute cardiopulmonary cause is significantly lower. Therefore, testing paradigms are less intense, with an emphasis on the history, physical examination, and ECG. Moreover, decision-aids developed for settings with a high prevalence of significant cardiopulmonary disease have lower positive predictive value when applied in the practitioner’s office. However, in general, if the level of clinical suspicion of ACS is sufficiently high to consider troponin testing, the patient should be referred to the ED for evaluation.

1	Abdominal Pain Danny O. Jacobs, William Silen Correctly interpreting acute abdominal pain can be quite challenging. Few clinical situations require greater judgment, because the most catastrophic of events may be forecast by the subtlest of symptoms and signs. In every instance, the clinician must distinguish those conditions 20 that require urgent intervention from those that do not and can best be managed nonoperatively. A meticulously executed, detailed history and physical examination are critically important for focusing the differential diagnosis, where necessary, and allowing the diagnostic evaluation to proceed expeditiously (Table 20-1). The etiologic classification in Table 20-2, although not complete, provides a useful framework for evaluating patients with abdominal pain.

1	The etiologic classification in Table 20-2, although not complete, provides a useful framework for evaluating patients with abdominal pain. The most common causes of abdominal pain on admission are acute appendicitis, nonspecific abdominal pain, pain of urologic origin, and intestinal obstruction. A diagnosis of “acute or surgical abdomen” is not acceptable because of its often misleading and erroneous connotations. Most patients who present with acute abdominal pain will have self-limited disease processes. However, it is important to remember that pain severity does not necessarily correlate with the severity of the underlying condition. The most obvious of “acute abdomens” may SoME KEy CoMPonEnTS of THE PATiEnT’S HiSToRy

1	SoME KEy CoMPonEnTS of THE PATiEnT’S HiSToRy Age Time and mode of onset of the pain Pain characteristics Duration of symptoms Location of pain and sites of radiation Associated symptoms and their relationship to the pain Nausea, emesis, and anorexia Diarrhea, constipation, or other changes in bowel habits Menstrual history not require operative intervention, and the mildest of abdominal pains 103 may herald an urgently correctable lesion. Any patient with abdominal pain of recent onset requires early and thorough evaluation and accurate diagnosis.

1	SOME MECHANISMS OF PAIN ORIgINATINg IN THE ABDOMEN Inflammation of the Parietal Peritoneum The pain of parietal peritoneal inflammation is steady and aching in character and is located directly over the inflamed area, its exact reference being possible because it is transmitted by somatic nerves supplying the parietal peritoneum. The intensity of the pain is dependent on the type and amount of material to which the peritoneal surfaces are exposed in a given time period. For example , the sudden release into the peritoneal cavity of a small quantity of sterile acid gastric juice causes much more pain than the same amount of grossly contaminated neutral feces. Enzymatically active pancreatic juice incites more pain and inflammation than does the same amount of sterile bile containing no potent enzymes. Blood is normally only a mild irritant and the response to urine can be bland, so exposure of blood and urine to the peritoneal cavity may go unnoticed unless it is sudden and massive.

1	enzymes. Blood is normally only a mild irritant and the response to urine can be bland, so exposure of blood and urine to the peritoneal cavity may go unnoticed unless it is sudden and massive. Bacterial contamination, such as may occur with pelvic inflammatory disease or perforated distal intestine, causes low-intensity pain until multiplication causes a significant amount of inflammatory mediators to be released. Patients with perforated upper gastrointestinal ulcers may present entirely differently depending on how quickly gastric juices enter the peritoneal cavity. Thus, the rate at which any inflammatory material irritates the peritoneum is important.

1	The pain of peritoneal inflammation is invariably accentuated by pressure or changes in tension of the peritoneum, whether produced by palpation or by movement such as with coughing or sneezing. The patient with peritonitis characteristically lies quietly in bed, preferring to avoid motion, in contrast to the patient with colic, who may be thrashing in discomfort.

1	Another characteristic feature of peritoneal irritation is tonic reflex spasm of the abdominal musculature, localized to the involved body segment. Its intensity depends on the integrity of the nervous system, the location of the inflammatory process, and the rate at which it develops. Spasm over a perforated retrocecal appendix or perforation into the lesser peritoneal sac may be minimal or absent because of the protective effect of overlying viscera. Catastrophic abdominal emergencies may be associated with minimal or no detectable pain or muscle spasm in obtunded, seriously ill, debilitated, immunosuppressed, or psychotic patients. A slowly developing process also often greatly attenuates the degree of muscle spasm.

1	Obstruction of Hollow Viscera Intraluminal obstruction classically elicits intermittent or colicky abdominal pain that is not as well localized as the pain of parietal peritoneal irritation. However, the absence of cramping discomfort should not be misleading because distention of a hollow viscus may also produce steady pain with only rare paroxysms. Small-bowel obstruction often presents as poorly localized, intermittent periumbilical or supraumbilical pain. As the intestine progressively dilates and loses muscular tone, the colicky nature of the pain may diminish. With superimposed strangulating obstruction, pain may spread to the lower lumbar region if there is traction on the root of the mesentery. The colicky pain of colonic obstruction is of lesser intensity, is commonly located in the infraumbilical area, and may often radiate to the lumbar region.

1	Sudden distention of the biliary tree produces a steady rather than colicky type of pain; hence, the term biliary colic is misleading. Acute distention of the gallbladder usually causes pain in the right upper quadrant with radiation to the right posterior region of the thorax or to the tip of the right scapula, but it is also not uncommonly found near the midline. Distention of the common bile duct often causes epigastric pain that may radiate to the upper lumbar region. Considerable variation is common, however, so that differentiation between these may be impossible. The typical subscapular pain or lumbar radiation is frequently absent. Gradual dilatation of the biliary tree, as can occur with carcinoma of the head of the pancreas, may cause no pain Pain Originating in the Abdomen PART 2 Cardinal Manifestations and Presentation of Diseases

1	Pain Originating in the Abdomen PART 2 Cardinal Manifestations and Presentation of Diseases Mechanical obstruction of hollow viscera Obstruction of the small or large intestine Obstruction of the biliary tree Obstruction of the ureter Abdominal wall Distortion or traction of mesentery Trauma or infection of muscles Distension of visceral surfaces, e.g., by hemorrhage Hepatic or renal capsules or only a mild aching sensation in the epigastrium or right upper quadrant. The pain of distention of the pancreatic ducts is similar to that described for distention of the common bile duct but, in addition, is very frequently accentuated by recumbency and relieved by the upright position.

1	Obstruction of the urinary bladder usually causes dull, low-intensity pain in the suprapubic region. Restlessness without specific complaint of pain may be the only sign of a distended bladder in an obtunded patient. In contrast, acute obstruction of the intravesicular portion of the ureter is characterized by severe suprapubic and flank pain that radiates to the penis, scrotum, or inner aspect of the upper thigh. Obstruction of the ureteropelvic junction manifests as pain near the costovertebral angle, whereas obstruction of the remainder of the ureter is associated with flank pain that often extends into the same side of the abdomen.

1	Vascular Disturbances A frequent misconception is that pain due to intraabdominal vascular disturbances is sudden and catastrophic in nature. Certain disease processes, such as embolism or thrombosis of the superior mesenteric artery or impending rupture of an abdominal aortic aneurysm, can certainly be associated with diffuse, severe pain. Yet, just as frequently, the patient with occlusion of the superior mesenteric artery only has mild continuous or cramping diffuse pain for 2 or 3 days before vascular collapse or findings of peritoneal inflammation appear. The early, seemingly insignificant discomfort is caused by hyperperistalsis rather than peritoneal inflammation. Indeed, absence of tenderness and rigidity in the presence of continuous, diffuse pain (e.g., “pain out of proportion to physical findings”) in a patient likely to have vascular disease is quite characteristic of occlusion of the superior mesenteric artery. Abdominal pain with radiation to the sacral region, flank, or

1	physical findings”) in a patient likely to have vascular disease is quite characteristic of occlusion of the superior mesenteric artery. Abdominal pain with radiation to the sacral region, flank, or genitalia should always signal the possible presence of a rupturing abdominal aortic aneurysm. This pain may persist over a period of several days before rupture and collapse occur.

1	Abdominal Wall Pain arising from the abdominal wall is usually constant and aching. Movement, prolonged standing, and pressure accentuate the discomfort and associated muscle spasm. In the case of hematoma of the rectus sheath, now most frequently encountered in association with anticoagulant therapy, a mass may be present in the lower quadrants of the abdomen. Simultaneous involvement of muscles in other parts of the body usually serves to differentiate myositis of the abdominal wall from other processes that might cause pain in the same region.

1	Pain referred to the abdomen from the thorax , spine, or genitalia may present a vexing diagnostic challenge because diseases of the upper part of the abdominal cavity such as acute cholecystitis or perforated ulcer may be associated with intrathoracic complications. A most important, yet often forgotten, dictum is that the possibility of intra-thoracic disease must be considered in every patient with abdominal pain, especially if the pain is in the upper abdomen.

1	Systematic questioning and examination directed toward detecting myocardial or pulmonary infarction, pneumonia, pericarditis, or esophageal disease (the intrathoracic diseases that most often masquerade as abdominal emergencies) will often provide sufficient clues to establish the proper diagnosis. Diaphragmatic pleuritis resulting from pneumonia or pulmonary infarction may cause pain in the right upper quadrant and pain in the supraclavicular area, the latter radiation to be distinguished from the referred subscapular pain caused by acute distention of the extrahepatic biliary tree. The ultimate decision as to the origin of abdominal pain may require deliberate and planned observation over a period of several hours, during which repeated questioning and examination will provide the diagnosis or suggest the appropriate studies.

1	Referred pain of thoracic origin is often accompanied by splinting of the involved hemithorax with respiratory lag and decrease in excursion more marked than that seen in the presence of intraabdominal disease. In addition, apparent abdominal muscle spasm caused by referred pain will diminish during the inspiratory phase of respiration, whereas it persists throughout both respiratory phases if it is of abdominal origin. Palpation over the area of referred pain in the abdomen also does not usually accentuate the pain and, in many instances, actually seems to relieve it.

1	Thoracic disease and abdominal disease frequently coexist and may be difficult or impossible to differentiate. For example, the patient with known biliary tract disease often has epigastric pain during myocardial infarction, or biliary colic may be referred to the precordium or left shoulder in a patient who has suffered previously from angina pectoris. For an explanation of the radiation of pain to a previously diseased area, see Chap. 18. Referred pain from the spine, which usually involves compression or irritation of nerve roots, is characteristically intensified by certain motions such as cough, sneeze, or strain and is associated with hyperesthesia over the involved dermatomes. Pain referred to the abdomen from the testes or seminal vesicles is generally accentuated by the slightest pressure on either of these organs. The abdominal discomfort experienced is of dull, aching character and is poorly localized.

1	Pain of metabolic origin may simulate almost any other type of intraabdominal disease. Several mechanisms may be at work. In certain instances, such as hyperlipidemia, the metabolic disease itself may be accompanied by an intraabdominal process such as pancreatitis, which can lead to unnecessary laparotomy unless recognized. C1 esterase deficiency associated with angioneurotic edema is often associated with episodes of severe abdominal pain. Whenever the cause of abdominal pain is obscure, a metabolic origin always must be considered. Abdominal pain is also the hallmark of familial Mediterranean fever (Chap. 392).

1	The problem of differential diagnosis is often not readily resolved. The pain of porphyria and of lead colic is usually difficult to distinguish from that of intestinal obstruction, because severe hyperperistalsis is a prominent feature of both. The pain of uremia or diabetes is nonspecific, and the pain and tenderness frequently shift in location and intensity. Diabetic acidosis may be precipitated by acute appendicitis or intestinal obstruction, so if prompt resolution of the abdominal pain does not result from correction of the metabolic abnormalities, an underlying organic problem should be suspected. Black widow spider bites produce intense pain and rigidity of the abdominal muscles and 105 back, an area infrequently involved in intraabdominal disease.

1	Evaluating and diagnosing causes of abdominal pain in immunosuppressed or otherwise immunocompromised patients is very difficult. This includes those who have undergone organ transplantation; who are receiving immunosuppressive treatments for autoimmune diseases, chemotherapy, or glucocorticoids; who have AIDS; and who are very old. In these circumstances, normal physiologic responses may be absent or masked. In addition, unusual infections may cause abdominal pain where the etiologic agents include cytomegalovirus, mycobacteria, protozoa, and fungi. These pathogens may affect all gastrointestinal organs, including the gallbladder, liver, and pancreas, as well as the gastrointestinal tract, causing occult or overtly symptomatic perforations of the latter. Splenic abscesses due to Candida or Salmonella infection should also be considered, especially when evaluating patients with left upper quadrant or left flank pain. Acalculous cholecystitis is a relative common complication in

1	or Salmonella infection should also be considered, especially when evaluating patients with left upper quadrant or left flank pain. Acalculous cholecystitis is a relative common complication in patients with AIDS, where it is often associated with cryptosporidiosis or cytomegalovirus infection.

1	Neutropenic enterocolitis is often identified as a cause of abdominal pain and fever in some patients with bone marrow suppression due to chemotherapy. Acute graft-versus-host disease should be considered. Optimal management of these patients may require meticulous follow-up including serial examinations to be certain that surgical intervention is not required to treat an underlying disease process.

1	Diseases that injure sensory nerves may cause causalgic pain. It has a burning character and is usually limited to the distribution of a given peripheral nerve. Normal nonpainful stimuli such as touch or a change in temperature may be causalgic and may frequently be present even at rest. The demonstration of irregularly spaced cutaneous pain spots may be the only indication that an old nerve injury exists. Even though the pain may be precipitated by gentle palpation, rigidity of the abdominal muscles is absent, and the respirations are not disturbed. Distention of the abdomen is uncommon, and the pain has no relationship to the intake of food.

1	Pain arising from spinal nerves or roots comes and goes suddenly and is of a lancinating type (Chap. 22). It may be caused by herpes zoster, impingement by arthritis, tumors, a herniated nucleus pulposus, diabetes , or syphilis. It is not associated with food intake, abdominal distention, or changes in respiration. Severe muscle spasm, as in the gastric crises of tabes dorsalis, is common but is either relieved or not accentuated by abdominal palpation. The pain is made worse by movement of the spine and is usually confined to a few dermatomes. Hyperesthesia is very common.

1	Pain due to functional causes conforms to none of the aforementioned patterns. Mechanisms of disease are not clearly established. Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by abdominal pain and altered bowel habits. The diagnosis is made on the basis of clinical criteria (Chap. 352) and after exclusion of demonstrable structural abnormalities. The episodes of abdominal pain are often brought on by stress, and the pain varies considerably in type and location. Nausea and vomiting are rare. Localized tenderness and muscle spasm are inconsistent or absent. The causes of IBS or related functional disorders are not known. APPROACH TO THE PATIENT:

1	Few abdominal conditions require such urgent operative intervention that an orderly approach need be abandoned, no matter how ill the patient. Only patients with exsanguinating intraabdominal hemorrhage (e.g., ruptured aneurysm) must be rushed to the operating room immediately, but in such instances, only a few minutes are required to assess the critical nature of the problem. Under these circumstances, all obstacles must be swept aside, adequate venous access for fluid replacement obtained, and the operation begun. Many of these patients have died in the radiology department or the emergency room while awaiting unnecessary examinations such as electrocardiograms or computed tomography (CT) scans. There are no contraindications to operation when massive intraabdominal hemorrhage is present. Fortunately, this situation is relatively rare. This statement does not necessarily apply to patients with intraluminal gastrointestinal hemorrhage, who can often be managed by other means (Chap.

1	Fortunately, this situation is relatively rare. This statement does not necessarily apply to patients with intraluminal gastrointestinal hemorrhage, who can often be managed by other means (Chap. 57). Nothing will supplant an orderly, painstakingly detailed history, which is far more valuable than any laboratory or radiographic examination. This kind of history is laborious and time-consuming, making it not especially popular, even though a reasonably accurate diagnosis can be made on the basis of the history alone in the majority of cases.

1	In cases of acute abdominal pain, a diagnosis is readily established in most instances, whereas success is not so frequent in patients with chronic pain. IBS is one of the most common causes of abdominal pain and must always be kept in mind (Chap. 352). The location of the pain can assist in narrowing the differential diagnosis (Table 20-3); however, the chronological sequence of events in the patient’s history is often more important than the pain’s location. If the examiner is sufficiently open-minded and unhurried, asks the proper questions, and listens, the patient will usually provide the diagnosis. Careful attention should be paid to the extraabdominal regions. Narcotics or analgesics should not be withheld until a definitive diagnosis or a definitive plan has been formulated; obfuscation of the diagnosis by adequate analgesia is unlikely.

1	An accurate menstrual history in a female patient is essential. It is important to remember that normal anatomic relationships can be significantly altered by the gravid uterus. Abdominal and pelvic pain may occur during pregnancy due to conditions that do PART 2 Cardinal Manifestations and Presentation of Diseases Irritable bowel syndrome Psychiatric disease Peritonitis Diabetes Abbreviation: GERD, gastroesophageal reflux disease. not require surgery. Lastly, some otherwise noteworthy laboratory values (e.g., leukocytosis) may represent the normal physiologic changes of pregnancy.

1	In the examination, simple critical inspection of the patient, e.g., of facies, position in bed, and respiratory activity, provides valuable clues. The amount of information to be gleaned is directly proportional to the gentleness and thoroughness of the examiner. Once a patient with peritoneal inflammation has been examined brusquely, accurate assessment by the next examiner becomes almost impossible. Eliciting rebound tenderness by sudden release of a deeply palpating hand in a patient with suspected peritonitis is cruel and unnecessary. The same information can be obtained by gentle percussion of the abdomen (rebound tenderness on a miniature scale), a maneuver that can be far more precise and localizing. Asking the patient to cough will elicit true rebound tenderness without the need for placing a hand on the abdomen. Furthermore, the forceful demonstration of rebound tenderness will startle and induce protective spasm in a nervous or worried patient in whom true rebound

1	the need for placing a hand on the abdomen. Furthermore, the forceful demonstration of rebound tenderness will startle and induce protective spasm in a nervous or worried patient in whom true rebound tenderness is not present. A palpable gallbladder will be missed if palpation is so aggressive that voluntary muscle spasm becomes superimposed on involuntary muscular rigidity. As with history taking, sufficient time should be spent in the examination. Abdominal signs may be minimal but nevertheless, if accompanied by consistent symptoms, may be exceptionally meaningful. Abdominal signs may be virtually or totally absent in cases of pelvic peritonitis, so careful pelvic and rectal examinations are mandatory in every patient with abdominal pain. Tenderness on pelvic or rectal examination in the absence of other abdominal signs can be caused by operative indications such as perforated appendicitis, diverticulitis, twisted ovarian cyst, and many others. Much attention has been paid to the

1	the absence of other abdominal signs can be caused by operative indications such as perforated appendicitis, diverticulitis, twisted ovarian cyst, and many others. Much attention has been paid to the presence or absence of peristaltic sounds, their quality, and their frequency. Auscultation of the abdomen is one of the least revealing aspects of the physical examination of a patient with abdominal pain. Catastrophes such as a strangulating small intestinal obstruction or perforated appendicitis may occur in the presence of normal peristaltic sounds. Conversely, when the proximal part of the intestine above obstruction becomes markedly distended and edematous, peristaltic sounds may lose the characteristics of borborygmi and become weak or absent, even when peritonitis is not present. It is usually the severe chemical peritonitis of sudden onset that is associated with the truly silent abdomen.

1	Laboratory examinations may be valuable in assessing the patient with abdominal pain, yet, with few exceptions, they rarely establish a diagnosis. Leukocytosis should never be the single deciding factor as to whether or not operation is indicated. A white blood cell count >20,000/μL may be observed with perforation of a viscus, but pancreatitis, acute cholecystitis, pelvic inflammatory disease, and intestinal infarction may also be associated with marked leukocytosis. A normal white blood cell count is not rare in cases of perforation of abdominal viscera. The diagnosis of anemia may be more helpful than the white blood cell count, especially when combined with the history.

1	The urinalysis may reveal the state of hydration or rule out severe renal disease, diabetes, or urinary infection. Blood urea nitrogen, glucose, and serum bilirubin levels may be helpful. Serum amylase levels may be increased by many diseases other than pancreatitis, e.g., perforated ulcer, strangulating intestinal obstruction, and acute cholecystitis; thus, elevations of serum amylase do not rule out the need for an operation.

1	Plain and upright or lateral decubitus radiographs of the abdomen may be of value in cases of intestinal obstruction, perforated ulcer, and a variety of other conditions. They are usually unnecessary in patients with acute appendicitis or strangulated external hernias. In rare instances, barium or water-soluble contrast study of the upper part of the gastrointestinal tract may demonstrate partial intestinal obstruction that may elude diagnosis by other means. If there is any question of obstruction of the colon, oral administration of barium sulfate should be avoided. On the other hand, in cases of suspected colonic obstruction (without perforation), a contrast enema may be diagnostic.

1	In the absence of trauma, peritoneal lavage has been replaced as a diagnostic tool by CT scanning and laparoscopy. Ultrasonography has proved to be useful in detecting an enlarged gallbladder or pancreas, the presence of gallstones, an enlarged ovary, or a tubal pregnancy. Laparoscopy is especially helpful in diagnosing pelvic conditions, such as ovarian cysts, tubal pregnancies, salpingitis, and acute appendicitis. Radioisotopic hepatobiliary iminodiacetic acid scans (HIDAs) may help differentiate acute cholecystitis or biliary colic from acute pancreatitis. A CT scan may demonstrate an enlarged pancreas, ruptured spleen, or thickened colonic or appendiceal wall and streaking of the mesocolon or mesoappendix characteristic of diverticulitis or appendicitis.

1	Sometimes, even under the best circumstances with all available aids and with the greatest of clinical skill, a definitive diagnosis cannot be established at the time of the initial examination. Nevertheless, even in the absence of a clear anatomic diagnosis, it may be abundantly clear to an experienced and thoughtful physician and surgeon that operation is indicated on clinical grounds alone. Should that decision be questionable, watchful waiting with repeated questioning and examination will often elucidate the true nature of the illness and indicate the proper course of action.

1	Headache Peter J. Goadsby, Neil H. Raskin Headache is among the most common reasons patients seek medical attention, on a global basis being responsible for more disability than any other neurologic problem. Diagnosis and management are based on a careful clinical approach augmented by an understanding of the 21 anatomy, physiology, and pharmacology of the nervous system pathways mediating the various headache syndromes. This chapter will focus on the general approach to a patient with headache; migraine and other primary headache disorders are discussed in Chap. 447.

1	A classification system developed by the International Headache Society (www.ihs-headache.org/) characterizes headache as primary or secondary (Table 21-1). Primary headaches are those in which headache and its associated features are the disorder in itself, whereas secondary headaches are those caused by exogenous disorders (Headache Classification Committee of the International Headache Society, 2013). Primary headache often results in considerable disability and a decrease in the patient’s quality of life. Mild secondary headache, such as that seen in association with upper respiratory tract infections, is Source: After J Olesen et al: The Headaches. Philadelphia, Lippincott Williams & Wilkins, 2005. common but rarely worrisome. Life-threatening headache is relatively 107 uncommon, but vigilance is required in order to recognize and appropriately treat such patients.

1	common but rarely worrisome. Life-threatening headache is relatively 107 uncommon, but vigilance is required in order to recognize and appropriately treat such patients. Pain usually occurs when peripheral nociceptors are stimulated in response to tissue injury, visceral distension, or other factors (Chap. 18). In such situations, pain perception is a normal physiologic response mediated by a healthy nervous system. Pain can also result when pain-producing pathways of the peripheral or central nervous system (CNS) are damaged or activated inappropriately. Headache may originate from either or both mechanisms. Relatively few cranial structures are pain-producing; these include the scalp, middle meningeal artery, dural sinuses, falx cerebri, and proximal segments of the large pial arteries. The ventricular ependyma, choroid plexus, pial veins, and much of the brain parenchyma are not pain-producing. The key structures involved in primary headache appear to be the following:

1	The key structures involved in primary headache appear to be the following: The large intracranial vessels and dura mater and the peripheral terminals of the trigeminal nerve that innervate these structures The caudal portion of the trigeminal nucleus, which extends into the dorsal horns of the upper cervical spinal cord and receives input from the first and second cervical nerve roots (the trigeminocervical complex) Rostral pain-processing regions, such as the ventroposteromedial thalamus and the cortex The pain-modulatory systems in the brain that modulate input from trigeminal nociceptors at all levels of the pain-processing pathways and influence vegetative functions, such as hypothalamus and brainstem structures

1	The innervation of the large intracranial vessels and dura mater by the trigeminal nerve is known as the trigeminovascular system. Cranial autonomic symptoms, such as lacrimation, conjunctival injection, nasal congestion, rhinorrhea, periorbital swelling, aural fullness, and ptosis, are prominent in the trigeminal autonomic cephalalgias, including cluster headache and paroxysmal hemicrania, and may also be seen in migraine, even in children. These autonomic symptoms reflect activation of cranial parasympathetic pathways, and functional imaging studies indicate that vascular changes in migraine and cluster headache, when present, are similarly driven by these cranial autonomic systems. Moreover, they can often be mistaken for symptoms or signs of cranial sinus inflammation, which is thus overdiagnosed and inappropriately managed. Migraine and other primary headache types are not “vascular headaches”; these disorders do not reliably manifest vascular changes, and treatment outcomes

1	overdiagnosed and inappropriately managed. Migraine and other primary headache types are not “vascular headaches”; these disorders do not reliably manifest vascular changes, and treatment outcomes cannot be predicted by vascular effects. Migraine is a brain disorder and is best understood and managed as such.

1	CLINICAL EVALuATION OF ACuTE, NEW-ONSET HEADACHE The patient who presents with a new, severe headache has a differential diagnosis that is quite different from the patient with recurrent headaches over many years. In new-onset and severe headache, the probability of finding a potentially serious cause is considerably greater than in recurrent headache. Patients with recent onset of pain require prompt evaluation and appropriate treatment. Serious causes to be considered include meningitis, subarachnoid hemorrhage, epidural or subdural hematoma, glaucoma, tumor, and purulent sinusitis. When worrisome symptoms and signs are present (Table 21-2), rapid diagnosis and management are critical.

1	A careful neurologic examination is an essential first step in the evaluation. In most cases, patients with an abnormal examination or a history of recent-onset headache should be evaluated by a computed tomography (CT) or magnetic resonance imaging (MRI) study. As an initial screening procedure for intracranial pathology in this setting, CT and MRI methods appear to be equally sensitive. In some circumstances, a lumbar puncture (LP) is also required, unless a benign etiology can be otherwise established. A general evaluation of acute headache might include cranial arteries by palpation; cervical spine by Pain induced by bending, lifting, cough Pain associated with local tenderness, e.g., region of temporal artery the effect of passive movement of the head and by imaging; the investigation of cardiovascular and renal status by blood pressure monitoring and urine examination; and eyes by funduscopy, intraocular pressure measurement, and refraction.

1	The psychological state of the patient should also be evaluated because a relationship exists between head pain and depression. This is intended to identify comorbidity rather than provide an explanation for the headache, because troublesome headache is seldom simply caused by mood change. Although it is notable that medicines with antidepressant actions are also effective in the prophylactic treatment of both tension-type headache and migraine, each symptom must be treated optimally. Underlying recurrent headache disorders may be activated by pain that follows otologic or endodontic surgical procedures. Thus, pain about the head as the result of diseased tissue or trauma may reawaken an otherwise quiescent migraine syndrome. Treatment of the headache is largely ineffective until the cause of the primary problem is addressed.

1	Serious underlying conditions that are associated with headache are described below. Brain tumor is a rare cause of headache and even less commonly a cause of severe pain. The vast majority of patients presenting with severe headache have a benign cause. The management of secondary headache focuses on diagnosis and treatment of the underlying condition. Acute, severe headache with stiff neck and fever suggests meningitis. LP is mandatory. Often there is striking accentuation of pain with eye movement. Meningitis can be easily mistaken for migraine in that the cardinal symptoms of pounding headache, photophobia, nausea, and vomiting are frequently present, perhaps reflecting the underlying biology of some of the patients. Meningitis is discussed in Chaps. 164 and 165.

1	Meningitis is discussed in Chaps. 164 and 165. Acute, severe headache with stiff neck but without fever suggests subarachnoid hemorrhage. A ruptured aneurysm, arteriovenous malformation, or intraparenchymal hemorrhage may also present with headache alone. Rarely, if the hemorrhage is small or below the foramen magnum, the head CT scan can be normal. Therefore, LP may be required to definitively diagnose subarachnoid hemorrhage. Intracranial hemorrhage is discussed in Chap. 330. Approximately 30% of patients with brain tumors consider headache to be their chief complaint. The head pain is usually nondescript—an intermittent deep, dull aching of moderate intensity, which may worsen with exertion or change in position and may be associated with nausea and vomiting. This pattern of symptoms results from

1	PART 2 Cardinal Manifestations and Presentation of Diseases migraine far more often than from brain tumor. The headache of brain tumor disturbs sleep in about 10% of patients. Vomiting that precedes the appearance of headache by weeks is highly characteristic of posterior fossa brain tumors. A history of amenorrhea or galactorrhea should lead one to question whether a prolactin-secreting pituitary adenoma (or the polycystic ovary syndrome) is the source of headache. Headache arising de novo in a patient with known malignancy suggests either cerebral metastases or carcinomatous meningitis, or both. Head pain appearing abruptly after bending, lifting, or coughing can be due to a posterior fossa mass, a Chiari malformation, or low cerebrospinal fluid (CSF) volume. Brain tumors are discussed in Chap. 118.

1	Brain tumors are discussed in Chap. 118. (See also Chaps. 39 and 385) Temporal (giant cell) arteritis is an inflammatory disorder of arteries that frequently involves the extra-cranial carotid circulation. It is a common disorder of the elderly; its annual incidence is 77 per 100,000 individuals age 50 and older. The average age of onset is 70 years, and women account for 65% of cases. About half of patients with untreated temporal arteritis develop blindness due to involvement of the ophthalmic artery and its branches; indeed, the ischemic optic neuropathy induced by giant cell arteritis is the major cause of rapidly developing bilateral blindness in patients >60 years. Because treatment with glucocorticoids is effective in preventing this complication, prompt recognition of the disorder is important.

1	Typical presenting symptoms include headache, polymyalgia rheumatica (Chap. 385), jaw claudication, fever, and weight loss. Headache is the dominant symptom and often appears in association with malaise and muscle aches. Head pain may be unilateral or bilateral and is located temporally in 50% of patients but may involve any and all aspects of the cranium. Pain usually appears gradually over a few hours before peak intensity is reached; occasionally, it is explosive in onset. The quality of pain is only seldom throbbing; it is almost invariably described as dull and boring, with superimposed episodic stabbing pains similar to the sharp pains that appear in migraine. Most patients can recognize that the origin of their head pain is superficial, external to the skull, rather than originating deep within the cranium (the pain site for migraineurs). Scalp tenderness is present, often to a marked degree; brushing the hair or resting the head on a pillow may be impossible because of pain.

1	deep within the cranium (the pain site for migraineurs). Scalp tenderness is present, often to a marked degree; brushing the hair or resting the head on a pillow may be impossible because of pain. Headache is usually worse at night and often aggravated by exposure to cold. Additional findings may include reddened, tender nodules or red streaking of the skin overlying the temporal arteries, and tenderness of the temporal or, less commonly, the occipital arteries.

1	The erythrocyte sedimentation rate (ESR) is often, although not always, elevated; a normal ESR does not exclude giant cell arteritis. A temporal artery biopsy followed by immediate treatment with prednisone 80 mg daily for the first 4–6 weeks should be initiated when clinical suspicion is high. The prevalence of migraine among the elderly is substantial, considerably higher than that of giant cell arteritis. Migraineurs often report amelioration of their headaches with prednisone; thus, caution must be used when interpreting the therapeutic response. Glaucoma may present with a prostrating headache associated with nausea and vomiting. The headache often starts with severe eye pain. On physical examination, the eye is often red with a fixed, moderately dilated pupil. Glaucoma is discussed in Chap. 39.

1	Glaucoma is discussed in Chap. 39. Primary headaches are disorders in which headache and associated features occur in the absence of any exogenous cause. The most common are migraine, tension-type headache, and the trigeminal autonomic cephalalgias, notably cluster headache. These entities are discussed in detail in Chap. 447. aMay be complicated by medication overuse. bSome patients may have headache >4 h/d. Abbreviations: CNS, central nervous system; SUNA, short-lasting unilateral neuralgiform headache attacks with cranial autonomic symptoms; SUNCT, short-lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing.

1	The broad diagnosis of chronic daily headache (CDH) can be applied when a patient experiences headache on 15 days or more per month. CDH is not a single entity; it encompasses a number of different headache syndromes, both primary and secondary (Table 21-3). In aggregate, this group presents considerable disability and is thus specially dealt with here. Population-based estimates suggest that about 4% of adults have daily or near-daily headache. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: The first step in the management of patients with CDH is to diagnose any secondary headache and treat that problem (Table 21-3). This can sometimes be a challenge where the underlying cause triggers a worsening of a primary headache. For patients with primary headaches, diagnosis of the headache type will guide therapy. Preventive treatments such as tricyclics, either amitriptyline or nortriptyline at doses up to 1 mg/kg, are very useful in patients with CDH arising from migraine or tension-type headache or where the secondary cause has activated the underlying primary headache. Tricyclics are started in low doses (10–25 mg) daily and may be given 12 h before the expected time of awakening in order to avoid excess morning sleepiness. Anticonvulsants, such as topiramate, valproate, flunarizine (not available in the United States), and candesartan are also useful in migraine.

1	The management of medically intractable headache is difficult. Currently there are a number of promising neuromodulatory approaches, such as occipital nerve stimulation, which appears to modulate thalamic processing in migraine, and has also shown promise in chronic cluster headache, short-lasting unilateral neuralgiform headache attacks with cranial autonomic symptoms (SUNA), short-lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing (SUNCT), and hemicrania continua (Chap. 447). Single-pulse transcranial magnetic stimulation is in use in Europe and is approved for migraine with aura in the United States. Other modalities are discussed in Chap. 447.

1	Overuse of analgesic medication for headache can aggravate headache frequency, markedly impair the effect of preventive medicines, and induce a state of refractory daily or near-daily headache called medication-overuse headache. A proportion of patients who stop taking analgesics will experience substantial improvement in the severity and frequency of their headache. However, even after cessation of analgesic use, many patients continue to have headache, although they may feel clinically improved in some way, especially if they have been using opioids or barbiturates regularly. The residual symptoms probably represent the underlying primary headache disorder, and most commonly, this issue occurs in patients prone to migraine.

1	Management of Medication Overuse: Outpatients For patients who overuse medications, it is essential that analgesic use be reduced and eliminated. One approach is to reduce the medication dose by 10% every 1–2 weeks. Immediate cessation of analgesic use is possible for some patients, provided there is no contraindication. Both approaches are facilitated by the use of a medication diary maintained during the month or two before cessation; this helps to identify the scope of the problem. A small dose of a nonsteroidal anti-inflammatory drug (NSAID) such as naproxen, 500 mg bid, if tolerated, will help relieve residual pain as analgesic use is reduced. NSAID overuse is not usually a problem for patients with daily headache when a NSAID with a longer half-life is taken once or twice daily; however, overuse problems may develop with more frequent dosing schedules or shorter acting NSAIDS. Once the patient has substantially reduced analgesic use, a preventive medication should be introduced.

1	overuse problems may develop with more frequent dosing schedules or shorter acting NSAIDS. Once the patient has substantially reduced analgesic use, a preventive medication should be introduced. It must be emphasized that preventives generally do not work in the presence of analgesic overuse. The most common cause of unresponsiveness to treatment is the use of a preventive when analgesics continue to be used regularly. For some patients, discontinuing analgesics is very difficult; often the best approach is to directly inform the patient that some degree of pain is inevitable during this initial period.

1	Management of Medication Overuse: Inpatients Some patients will require hospitalization for detoxification. Such patients have typically failed efforts at outpatient withdrawal or have a significant medical condition, such as diabetes mellitus, which would complicate withdrawal as an outpatient. Following admission to the hospital, acute medications are withdrawn completely on the first day, in the absence of a contraindication. Antiemetics and fluids are administered as required; clonidine is used for opioid withdrawal symptoms. For acute intolerable pain during the waking hours, aspirin, 1 g IV (not approved in United States), is useful. IM chlorpromazine can be helpful at night; patients must be adequately hydrated. Three to 5 days into the admission, as the effect of the withdrawn substance wears off, a course of IV dihydroergotamine (DHE) can be used. DHE, administered every 8 h for 5 consecutive days, can induce a significant remission that allows a preventive treatment to be

1	wears off, a course of IV dihydroergotamine (DHE) can be used. DHE, administered every 8 h for 5 consecutive days, can induce a significant remission that allows a preventive treatment to be established. 5-HT3 antagonists, such as ondansetron or granisetron, or the neurokinin receptor antagonist, aprepitant, may be required with DHE to prevent significant nausea, and domperidone (not approved in the United States) orally or by suppository can be very helpful. Avoiding sedating or otherwise side effect prone antiemetics is helpful.

1	New daily persistent headache (NDPH) is a clinically distinct syndrome; its causes are listed in Table 21-4. aIncludes postinfectious forms. Clinical Presentation The patient with NDPH presents with headache on most if not all days, and the patient can clearly, and often vividly, recall the moment of onset. The headache usually begins abruptly, but onset may be more gradual; evolution over 3 days has been proposed as the upper limit for this syndrome. Patients typically recall the exact day and circumstances of the onset of headache; the new, persistent head pain does not remit. The first priority is to distinguish between a primary and a secondary cause of this syndrome. Subarachnoid hemorrhage is the most serious of the secondary causes and must be excluded either by history or appropriate investigation (Chap. 330).

1	Secondary NDPH • LOw CSF VOLUME HEAdACHE In these syndromes, head pain is positional: it begins when the patient sits or stands upright and resolves upon reclining. The pain, which is occipitofrontal, is usually a dull ache but may be throbbing. Patients with chronic low CSF volume headache typically present with a history of headache from one day to the next that is generally not present on waking but worsens during the day. Recumbency usually improves the headache within minutes, and it can take only minutes to an hour for the pain to return when the patient resumes an upright position.

1	The most common cause of headache due to persistent low CSF volume is CSF leak following LP. Post-LP headache usually begins within 48 h but may be delayed for up to 12 days. Its incidence is between 10 and 30%. Beverages with caffeine may provide temporary relief. Besides LP, index events may include epidural injection or a vigorous Valsalva maneuver, such as from lifting, straining, coughing, clearing the eustachian tubes in an airplane, or multiple orgasms. Spontaneous CSF leaks are well recognized, and the diagnosis should be considered whenever the headache history is typical, even when there is no obvious index event. As time passes from the index event, the postural nature may become less apparent; cases in which the index event occurred several years before the eventual diagnosis have been recognized. Symptoms appear to result from low volume rather than low pressure: although low CSF pressures, typically 0–50 mmH2O, are usually identified, a pressure as high as 140 mmH2O has

1	have been recognized. Symptoms appear to result from low volume rather than low pressure: although low CSF pressures, typically 0–50 mmH2O, are usually identified, a pressure as high as 140 mmH2O has been noted with a documented leak.

1	Postural orthostatic tachycardia syndrome (POTS; Chap. 454) can present with orthostatic headache similar to low CSF volume headache and is a diagnosis that needs consideration in this setting.

1	When imaging is indicated to identify the source of a presumed leak, an MRI with gadolinium is the initial study of choice (Fig. 21-1). A striking pattern of diffuse meningeal enhancement is so typical that in the appropriate clinical context the diagnosis is established. Chiari malformations may sometimes be noted on MRI; in such cases, surgery to decompress the posterior fossa usually worsens the headache. Spinal MRI with T2 weighting may reveal a leak, and spinal MRI may demonstrate spinal meningeal cysts whose role in these syndromes is yet to be elucidated. The source of CSF leakage may be identified by spinal MRI with appropriate sequences, by CT, or increasingly by MR myelography. Less used now, 111In-DTPA CSF studies in the absence of a directly identified site of leakage, may demonstrate early emptying of 111In-DTPA tracer into the bladder or slow progress of tracer across the brain suggesting a CSF leak.

1	Initial treatment for low CSF volume headache is bed rest. For patients with persistent pain, IV caffeine (500 mg in 500 mL of saline administered over 2 h) can be very effective. An electrocardiogram (ECG) to screen for arrhythmia should be performed before administration. It is reasonable to administer at least two infusions of caffeine before embarking on additional tests to identify the source of the CSF leak. Because IV caffeine is safe and can be curative, it spares many patients the need for further investigations. If unsuccessful, an abdominal binder may be helpful. If a leak can be identified, an autologous blood patch is usually curative. A blood patch is also effective for post-LP headache; in this setting, the location is empirically determined to be the site of the LP. In patients with intractable pain, oral theophylline is a useful alternative; however, its effect is less rapid than caffeine. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 21-1 Magnetic resonance image showing diffuse meningeal enhancement after gadolinium administration in a patient with low cerebrospinal fluid (CSF) volume headache.

1	RAISEd CSF PRESSURE HEAdACHE Raised CSF pressure is well recognized as a cause of headache. Brain imaging can often reveal the cause, such as a space-occupying lesion. NDPH due to raised CSF pressure can be the presenting symptom for patients with idiopathic intracranial hypertension (pseudotumor cerebri) without visual problems, particularly when the fundi are normal. Persistently raised intracranial pressure can trigger chronic migraine. These patients typically present with a history of generalized headache that is present on waking and improves as the day goes on. It is generally worse with recumbency. Visual obscurations are frequent. The diagnosis is relatively straightforward when papilledema is present, but the possibility must be considered even in patients without funduscopic changes. Formal visual field testing should be performed even in the absence of overt ophthalmic involvement. Headache on rising in the morning or nocturnal headache is also characteristic of

1	changes. Formal visual field testing should be performed even in the absence of overt ophthalmic involvement. Headache on rising in the morning or nocturnal headache is also characteristic of obstructive sleep apnea or poorly controlled hypertension.

1	Evaluation of patients suspected to have raised CSF pressure requires brain imaging. It is most efficient to obtain an MRI, including an MR venogram, as the initial study. If there are no contraindications, the CSF pressure should be measured by LP; this should be done when the patient is symptomatic so that both the pressure and the response to removal of 20–30 mL of CSF can be determined. An elevated opening pressure and improvement in headache following removal of CSF are diagnostic. Initial treatment is with acetazolamide (250–500 mg bid); the headache may improve within weeks. If ineffective, topiramate is the next treatment of choice; it has many actions that may be useful in this setting, including carbonic anhydrase inhibition, weight loss, and neuronal membrane stabilization, likely mediated via effects on phosphorylation pathways. Severely disabled patients who do not respond to medical treatment require intracranial pressure monitoring and may require shunting.

1	POSTTRAUMATIC HEAdACHE A traumatic event can trigger a headache process that lasts for many months or years after the event. The term trauma is used in a very broad sense: headache can develop following an injury to the head, but it can also develop after an infectious episode, typically viral meningitis, a flulike illness, or a parasitic infection. Complaints of dizziness, vertigo, and impaired memory can accompany the headache. Symptoms may remit after several weeks or persist for months and even years after the injury. Typically the neurologic examination is normal and CT or MRI studies are unrevealing. Chronic subdural hematoma may on occasion mimic this disorder. Posttraumatic headache may also be seen after carotid dissection and subarachnoid hemorrhage and after intracranial surgery. The underlying theme appears to be that a traumatic event involving the pain-producing meninges can trigger a headache process that lasts for many years.

1	OTHER CAUSES In one series, one-third of patients with NDPH reported headache beginning after a transient flulike illness characterized by fever, neck stiffness, photophobia, and marked malaise. Evaluation typically reveals no apparent cause for the headache. There is no convincing evidence that persistent Epstein-Barr virus infection plays a role in NDPH. A complicating factor is that many patients undergo LP during the acute illness; iatrogenic low CSF volume headache must be considered in these cases. TREATMENT Treatment is largely empirical. Tricyclic antidepressants, notably amitriptyline, and anticonvulsants, such as topiramate, valproate, and gabapentin, have been used with reported benefit. The monoamine oxidase inhibitor phenelzine may also be useful in carefully selected patients. The headache usually resolves within 3–5 years, but it can be quite disabling.

1	Most patients with headache will be seen first in a primary care setting. The task of the primary care physician is to identify the very few worrisome secondary headaches from the very great majority of primary and less troublesome secondary headaches (Table 21-2). Absent any warning signs, a reasonable approach is to treat when a diagnosis is established. As a general rule, the investigation should focus on identifying worrisome causes of headache or on gaining confidence if no primary headache diagnosis can be made. After treatment has been initiated, follow-up care is essential to identify whether progress has been made against the headache complaint. Not all headaches will respond to treatment, but, in general, worrisome headaches will progress and will be easier to identify.

1	When a primary care physician feels the diagnosis is a primary headache disorder, it is worth noting that more than 90% of patients who present to primary care with a complaint of headache will have migraine (Chap. 447). In general, patients who do not have a clear diagnosis, have a primary headache disorder other than migraine or tension-type headache, or are unresponsive to two or more standard therapies for the considered headache type should be considered for referral to a specialist. In a practical sense, the threshold for referral is also determined by the experience of the primary care physician in headache medicine and the availability of secondary care options. John W. Engstrom, Richard A. Deyo

1	John W. Engstrom, Richard A. Deyo The importance of back and neck pain in our society is underscored by the following: (1) the cost of back pain in the United States exceeds $100 billion annually; approximately one-third of these costs are direct health care expenses, and two-thirds are indirect costs resulting from loss of wages and productivity; (2) back symptoms are the most common cause of disability in those <45 years; (3) low back pain is the second most common reason for visiting a physician in the United States; and (4) 70% of persons will have back pain at some point in their lives.

1	The anterior spine consists of cylindrical vertebral bodies separated by intervertebral disks and held together by the anterior and posterior longitudinal ligaments. The intervertebral disks are composed of a central gelatinous nucleus pulposus surrounded by a tough cartilagi-111 nous ring, the annulus fibrosis. Disks are responsible for 25% of spinal column length and allow the bony vertebrae to move easily upon each other (Figs. 22-1 and 22-2). Desiccation of the nucleus pulposus and degeneration of the annulus fibrosus increase with age and result in loss of disk height. The disks are largest in the cervical and lumbar regions where movements of the spine are greatest. The anterior spine absorbs the shock of bodily movements such as walking and running and, with the posterior spine, protects the spinal cord and nerve roots in the spinal canal.

1	The posterior spine consists of the vertebral arches and processes. Each arch consists of paired cylindrical pedicles anteriorly and paired lamina posteriorly. The vertebral arch also gives rise to two transverse processes laterally, one spinous process posteriorly, plus two superior and two inferior articular facets. The apposition of a superior and inferior facet constitutes a facet joint. The posterior spine provides an anchor for the attachment of muscles and ligaments. The contraction of muscles attached to the spinous and transverse processes and lamina works like a system of pulleys and levers that results in flexion, extension, and lateral bending movements of the spine.

1	Nerve root injury (radiculopathy) is a common cause of neck, arm, low back, buttock, and leg pain (see Figs. 31-2 and 31-3). The nerve roots exit at a level above their respective vertebral bodies in the cervical region (e.g., the C7 nerve root exits at the C6-C7 level) and below their respective vertebral bodies in the thoracic and lumbar regions (e.g., the T1 nerve root exits at the T1-T2 level). The cervical nerve roots follow a short intraspinal course before exiting. By contrast, because the spinal cord ends at the vertebral L1 or L2 level, the lumbar nerve roots follow a long intraspinal course and can be injured anywhere from the upper lumbar spine to their exit at the intervertebral foramen. For example, disk herniation at the L4-L5 level can produce not only L5 root compression, but also compression of the traversing S1 nerve root (Fig. 22-3). The lumbar nerve roots are mobile in the spinal canal, but eventually pass through the narrow lateral recess of the spinal canal and

1	but also compression of the traversing S1 nerve root (Fig. 22-3). The lumbar nerve roots are mobile in the spinal canal, but eventually pass through the narrow lateral recess of the spinal canal and intervertebral foramen (Figs. 22-2 and 22-3). Neuroimaging of the spine must include both sagittal and axial views to assess possible compression in either the lateral recess or intervertebral foramen.

1	Pain-sensitive structures of the spine include the periosteum of the vertebrae, dura, facet joints, annulus fibrosus of the intervertebral disk, epidural veins and arteries, and the longitudinal ligaments. Disease of these diverse structures may explain many cases of back pain without nerve root compression. Under normal circumstances, the nucleus pulposus of the intervertebral disk is not pain sensitive. APPROACH TO THE PATIENT: Delineating the type of pain reported by the patient is the essential first step. Attention is also focused on identification of risk factors for a serious underlying etiology. The most frequent causes of back pain are radiculopathy, fracture, tumor, infection, or referred pain from visceral structures (Table 22-1). Local pain is caused by injury to pain-sensitive structures that compress or irritate sensory nerve endings. The site of the pain is near the affected part of the back.

1	Local pain is caused by injury to pain-sensitive structures that compress or irritate sensory nerve endings. The site of the pain is near the affected part of the back. Pain referred to the back may arise from abdominal or pelvic viscera. The pain is usually described as primarily abdominal or pelvic, accompanied by back pain and usually unaffected by posture. The patient may occasionally complain of back pain only. Pain of spine origin may be located in the back or referred to the buttocks or legs. Diseases affecting the upper lumbar spine tend to refer pain to the lumbar region, groin, or anterior thighs. Diseases affecting the lower lumbar spine tend to produce pain referred to the buttocks, posterior thighs, calves, or feet. Referred pain can explain pain syndromes that cross multiple dermatomes without evidence of nerve root compression. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 22-1 Vertebral anatomy. (From A Gauthier Cornuelle, DH Gronefeld: Radiographic Anatomy Positioning. New York, McGraw-Hill, 1998; with permission.) Radicular pain is typically sharp and radiates from the low back to a leg within the territory of a nerve root (see “Lumbar Disk Disease,” below). Coughing, sneezing, or voluntary contraction of abdominal muscles (lifting heavy objects or straining at stool) may elicit the radiating pain. The pain may increase in postures that stretch the nerves and nerve roots. Sitting with the leg outstretched places traction on the sciatic nerve and L5 and S1 roots because the nerve passes posterior to the hip. The femoral nerve (L2, L3, and L4 roots) passes anterior to the hip and is not stretched by sitting. FIguRE 22-2 Spinal column. (From A Gauthier Cornuelle, DH Gronefeld: Radiographic Anatomy Positioning. New York, McGraw-Hill, 1998; with permission.)

1	FIguRE 22-2 Spinal column. (From A Gauthier Cornuelle, DH Gronefeld: Radiographic Anatomy Positioning. New York, McGraw-Hill, 1998; with permission.) The description of the pain alone often fails to distinguish between referred pain and radiculopathy, although a burning or electric quality favors radiculopathy. Pain associated with muscle spasm, although of obscure origin, is commonly associated with many spine disorders. The spasms are accompanied by abnormal posture, tense paraspinal muscles, and dull or achy pain in the paraspinal region. Knowledge of the circumstances associated with the onset of back pain is important when weighing possible serious underlying causes for the pain. Some patients involved in accidents or work-related injuries may exaggerate their pain for the purpose of compensation or for psychological reasons.

1	A physical examination that includes the abdomen and rectum is advisable. Back pain referred from visceral organs may be reproduced during palpation of the abdomen (pancreatitis, abdominal aortic aneurysm [AAA]) or percussion over the costovertebral angles (pyelonephritis). The normal spine has a cervical and lumbar lordosis and a thoracic kyphosis. Exaggeration of these normal alignments may result in hyperkyphosis of the thoracic spine or hyperlordosis of the lumbar spine. Inspection may reveal a lateral curvature of the spine (scoliosis). An asymmetry in the prominence of the paraspinal muscles suggests muscle spasm. Spine pain reproduced by palpation over the spinous process reflects injury of the affected vertebrae or adjacent pain-sensitive structures.

1	Forward bending is often limited by paraspinal muscle spasm; the latter may flatten the usual lumbar lordosis. Flexion at the hips is normal in patients with lumbar spine disease, but flexion of the lumbar spine is limited and sometimes painful. Lateral bending to the side opposite the injured spinal element may stretch the damaged tissues, worsen pain, and limit motion. Hyperextension of the spine (with the patient prone or standing) is limited when nerve root compression, facet joint pathology, or other bony spine disease is present. Pain from hip disease may mimic the pain of lumbar spine disease. Hip pain can be reproduced by internal and external rotation at the hip with the knee and hip in flexion or by compressing the heel with the examiner’s palm while the leg is extended (heel percussion sign).

1	The straight leg–raising (SLR) maneuver is a simple bedside test for nerve root disease. With the patient supine, passive flexion of the extended leg at the hip stretches the L5 and S1 nerve roots and 113 CHAPTER 22 Back and Neck Pain 4th Lumbar vertebral body 5th Lumbar vertebral body 4th Lumbar pedicle L4 root Protruded L4-L5 disk L5 Root S1 Root S2 Root Protruded L5-S1 disk FIguRE 22-3 Compression of L5 and S1 roots by herniated disks. (From AH Ropper, MA Samuels: Adams and Victor’s Principles of Neurology, 9th ed. New York, McGraw-Hill, 2009; with permission.) the sciatic nerve. Passive dorsiflexion of the foot during the maneuver adds to the stretch. In healthy individuals, flexion to at least 80° is normally possible without causing pain, although a tight, stretching sensation in the hamstring muscles is common. The SLR test is positive if the maneuver reproduces the patient’s usual back or limb pain. Eliciting the SLR sign in both the supine and sitting positions can help

1	in the hamstring muscles is common. The SLR test is positive if the maneuver reproduces the patient’s usual back or limb pain. Eliciting the SLR sign in both the supine and sitting positions can help determine if the finding is reproducible. The patient may describe pain in the low back, buttocks, posterior thigh, or lower leg, but the key feature is reproduction of the patient’s usual pain.

1	Pain worse at rest or at night Prior history of cancer History of chronic infection (especially lung, urinary tract, skin) History of trauma Incontinence Age >70 years Intravenous drug use Glucocorticoid use History of a rapidly progressive neurologic deficit Unexplained fever Unexplained weight loss Percussion tenderness over the spine Abdominal, rectal, or pelvic mass Internal/external rotation of the leg at the hip; heel percussion sign Straight leg– or reverse straight leg–raising signs Progressive focal neurologic deficit

1	The crossed SLR sign is present when flexion of one leg reproduces the usual pain in the opposite leg or buttocks. In disk herniation, the crossed SLR sign is less sensitive but more specific than the SLR sign. The reverse SLR sign is elicited by standing the patient next to the examination table and passively extending each leg with the knee fully extended. This maneuver, which stretches the L2-L4 nerve roots, lumbosacral plexus, and femoral nerve, is considered positive if the patient’s usual back or limb pain is reproduced. For all of these tests, the nerve or nerve root lesion is always on the side of the pain.

1	The neurologic examination includes a search for focal weakness or muscle atrophy, focal reflex changes, diminished sensation in the legs, or signs of spinal cord injury. The examiner should be alert to the possibility of breakaway weakness, defined as fluctuations in the maximum power generated during muscle testing. Breakaway weakness may be due to pain or a combination of pain and an underlying true weakness. Breakaway weakness without pain is almost always due to a lack of effort. In uncertain cases, electromyography (EMG) can determine if true weakness due to nerve tissue injury is present. Findings with specific lumbosacral nerve root lesions are shown in Table 22-2 and are discussed below. LABORATORY, IMAgINg, AND EMg STuDIES

1	LABORATORY, IMAgINg, AND EMg STuDIES Laboratory studies are rarely needed for the initial evaluation of nonspecific acute (<3 months in duration) low back pain (ALBP). Risk factors for a serious underlying cause and for infection, tumor, or fracture, in particular, should be sought by history and exam. If risk factors are present (Table 22-1), then laboratory studies (complete blood count [CBC], erythrocyte sedimentation rate [ESR], urinalysis) are indicated. If risk factors are absent, then management is conservative (see “Treatment,” below) Computed tomography (CT) scanning is superior to routine x-rays for the detection of fractures involving posterior spine structures, craniocervical and cervicothoracic junctions, C1 and C2 aReverse straight leg–raising sign present—see “Examination of the Back.” bThese muscles receive the majority of innervation from this root.

1	PART 2 Cardinal Manifestations and Presentation of Diseases “Examination of the Back.” vertebrae, bone fragments within the spinal canal, or misalignment. CT scans are increasingly used as a primary screening modality for moderate to severe acute trauma. Magnetic resonance imaging (MRI) or CT myelography is the radiologic test of choice for evaluation of most serious diseases involving the spine. MRI is superior for the definition of soft tissue structures, whereas CT myelography provides optimal imaging of the lateral recess of the spinal canal and is better tolerated by claustrophobic patients. Annual population surveys in the United States suggest that patients with back pain have reported progressively worse functional limitations in recent years, rather than progressive improvements, despite rapid increases in spine imaging, opioid prescribing, injections, and spine surgery. This suggests that more selective use of diagnostic and treatment modalities may be appropriate.

1	Spine imaging often reveals abnormalities of dubious clinical relevance that may alarm clinicians and patients alike and prompt further testing and unnecessary therapy. Both randomized trials and observational studies have suggested such a “cascade effect” of imaging may create a gateway to other unnecessary care. Based in part on such evidence, the American College of Physicians has made parsimonious spine imaging a high priority in its “Choosing Wisely” campaign, aimed at reducing unnecessary care. Successful efforts to reduce unnecessary imaging have typically been multifaceted. Some include physician education by clinical leaders and computerized decision support, to identify any recent relevant imaging tests and require approved indications for ordering an imaging test. Other strategies have included audit and feedback regarding individual rates of ordering and indications, and more rapid access to physical therapy or consultation for patients without imaging indications.

1	When imaging tests are reported, it may be useful to indicate that certain degenerative findings are common in normal, pain-free individuals. In an observational study, this strategy was associated with lower rates of repeat imaging, opioid therapy, and physical therapy referral. Electrodiagnostic studies can be used to assess the functional integrity of the peripheral nervous system (Chap. 442e). Sensory nerve conduction studies are normal when focal sensory loss confirmed by examination is due to nerve root damage because the nerve roots are proximal to the nerve cell bodies in the dorsal root ganglia. Injury to nerve tissue distal to the dorsal root ganglion (e.g., plexus or peripheral nerve) results in reduced sensory nerve signals. Needle EMG complements nerve conduction studies by detecting denervation or reinnervation changes in a myotomal (segmental) Anterior thigh Anterior thigh, knee

1	Anterior thigh Anterior thigh, knee Knee, medial calf Anterolateral thigh Lateral calf, dorsal foot, posterolateral thigh, buttocks Bottom foot, posterior calf, posterior thigh, buttocks distribution. Multiple muscles supplied by different nerve roots and nerves are sampled; the pattern of muscle involvement indicates the nerve root(s) responsible for the injury. Needle EMG provides objective information about motor nerve fiber injury when clinical evaluation of weakness is limited by pain or poor effort. EMG and nerve conduction studies will be normal when sensory nerve root injury or irritation is the pain source.

1	This is a common cause of acute, chronic, or recurrent low back and leg pain (Figs. 22-3 and 22-4). Disk disease is most likely to occur at the L4-L5 or L5-S1 levels, but upper lumbar levels are involved occasionally. The cause is often unknown, but the risk is increased in overweight individuals. Disk herniation is unusual prior to age 20 years and is rare in the fibrotic disks of the elderly. Complex genetic factors may play a role in predisposing some patients to disk disease. The pain may be located in the low back only or referred to a leg, buttock, or hip. A sneeze, cough, or trivial movement may cause the nucleus pulposus to prolapse, pushing the frayed and weakened annulus posteriorly. With severe disk disease, the nucleus may protrude through the annulus (herniation) or become extruded to lie as a free fragment in the spinal canal.

1	The mechanism by which intervertebral disk injury causes back pain is controversial. The inner annulus fibrosus and nucleus pulposus are normally devoid of innervation. Inflammation and production of proinflammatory cytokines within a ruptured nucleus pulposus may trigger or perpetuate back pain. Ingrowth of nociceptive (pain) nerve fibers into inner portions of a diseased disk may be responsible for some chronic “diskogenic” pain. Nerve root injury (radiculopathy) from disk herniation is usually due to inflammation, but lateral herniation may produce compression in the lateral recess or at the intervertebral foramen.

1	A ruptured disk may be asymptomatic or cause back pain, abnormal posture, limitation of spine motion (particularly flexion), a focal neurologic deficit, or radicular pain. A dermatomal pattern of sensory loss or a reduced or absent deep tendon reflex is more suggestive of a specific root lesion than is the pattern of pain. Motor findings (focal weakness, muscle atrophy, or fasciculations) occur less frequently than focal sensory or reflex changes. Symptoms and signs are usually unilateral, but bilateral involvement does occur with large central disk herniations that compress multiple roots or cause inflammation of Lumbar spinal stenosis without or with neurogenic claudication Neoplasms—Metastatic, Hematologic, Primary Bone Tumors Fractures Trauma/falls, motor vehicle accidents Atraumatic fractures: osteoporosis, neoplastic infiltration, osteomyelitis Osteoporosis—hyperparathyroidism, immobility Osteosclerosis (e.g., Paget’s disease)

1	Trauma/falls, motor vehicle accidents Atraumatic fractures: osteoporosis, neoplastic infiltration, osteomyelitis Osteoporosis—hyperparathyroidism, immobility Osteosclerosis (e.g., Paget’s disease) Autoimmune Inflammatory Arthritis Other Causes of Back Pain Referred pain from visceral disease (e.g., abdominal aortic aneurysm) Postural Psychiatric, malingering, chronic pain syndromes nerve roots within the spinal canal. Clinical manifestations of specific nerve root lesions are summarized in Table 22-2. The differential diagnosis covers a variety of serious and treatable conditions, including epidural abscess, hematoma, fracture, or tumor.

1	The differential diagnosis covers a variety of serious and treatable conditions, including epidural abscess, hematoma, fracture, or tumor. FIguRE 22-4 Left L5 radiculopathy. A. Sagittal T2-weighted image on the left reflex changes may occur when spinal stenosis is associreveals disk herniation at the L4-L5 level. B. Axial T1-weighted image shows para-ated with neural foraminal narrowing and radiculopathy. central disk herniation with displacement of the thecal sac medially and the left L5 Severe neurologic deficits, including paralysis and urinerve root posteriorly in the left lateral recess. nary incontinence, occur only rarely.

1	Fever, constant pain uninfluenced by position, sphincter abnormali-115 ties, or signs of spinal cord disease suggest an etiology other than lumbar disk disease. Absence of ankle reflexes can be a normal finding in persons older than age 60 years or a sign of bilateral S1 radiculopathy. An absent deep tendon reflex or focal sensory loss may indicate injury to a nerve root, but other sites of injury along the nerve must also be considered. For example, an absent knee reflex may be due to a femoral neuropathy or an L4 nerve root injury. A loss of sensation over the foot and lateral lower calf may result from a peroneal or lateral sciatic neuropathy or an L5 nerve root injury. Focal muscle atrophy may reflect injury to the anterior horn cells of the spinal cord, a nerve root, peripheral nerve, or disuse.

1	A lumbar spine MRI scan or CT myelogram is necessary to establish the location and type of pathology. Spine MRIs yield exquisite views of intraspinal and adjacent soft tissue anatomy. Bony lesions of the lateral recess or intervertebral foramen are optimally visualized by CT myelography. The correlation of neuroradiologic findings to symptoms, particularly pain, is not simple. Contrast-enhancing tears in the annulus fibrosus or disk protrusions are widely accepted as common sources of back pain; however, studies have found that many asymptomatic adults have similar findings. Asymptomatic disk protrusions are also common and may enhance with contrast. Furthermore, in patients with known disk herniation treated either medically or surgically, persistence of the herniation 10 years later had no relationship to the clinical outcome. In summary, MRI findings of disk protrusion, tears in the annulus fibrosus, or hypertrophic facet joints are common incidental findings that, by themselves,

1	no relationship to the clinical outcome. In summary, MRI findings of disk protrusion, tears in the annulus fibrosus, or hypertrophic facet joints are common incidental findings that, by themselves, should not dictate management decisions for patients with back pain.

1	The diagnosis of nerve root injury is most secure when the history, examination, results of imaging studies, and the EMG are concordant. The correlation between CT and EMG for localization of nerve root injury is between 65 and 73%. Up to one-third of asymptomatic adults have a lumbar disk protrusion detected by CT or MRI scans. Management of lumbar disk disease is discussed below.

1	Cauda equina syndrome (CES) signifies an injury of multiple lumbosacral nerve roots within the spinal canal distal to the termination of the spinal cord at L1-L2. Low back pain, weakness and areflexia in the legs, saddle anesthesia, or loss of bladder function may occur. The problem must be distinguished from disorders of the lower spinal cord (conus medullaris syndrome), acute transverse myelitis (Chap. 456), and Guillain-Barré syndrome (Chap. 460). Combined involvement of the conus medullaris and cauda equina can occur. CES is commonly due to a ruptured lumbosacral intervertebral disk, lumbosacral spine fracture, hematoma within the spinal canal (e.g., following lumbar puncture in patients with coagulopathy), compressive tumor, or other mass lesion. Treatment options include surgical decompression, some times urgently in an attempt to restore or preserve motor or sphincter function, or radiotherapy for metastatic tumors (Chap. 118).

1	Lumbar spinal stenosis (LSS) describes a narrowed lumbar spinal canal and is frequently asymptomatic. Typical is neurogenic claudication, consisting of back and buttock or leg pain induced by walking or standing and relieved by sitting. Symptoms in the legs are usually bilateral. Unlike vascular claudication, symptoms are often provoked by standing without walking. Unlike lumbar disk disease, symptoms are usually relieved by sitting. Patients with neurogenic claudication can often walk much farther when leaning over a shopping cart and can pedal a stationary bike with ease while sitting. These flexed positions increase the anteroposterior spinal canal diameter and reduce intraspinal venous hypertension, resulting in pain relief. Focal weakness, sensory loss, or insufficient evidence to support the routine use of epidural glucocorticoid injections. Surgical therapy is considered when medical therapy does not relieve symptoms sufficiently to allow for resumption of activities of daily

1	the routine use of epidural glucocorticoid injections. Surgical therapy is considered when medical therapy does not relieve symptoms sufficiently to allow for resumption of activities of daily living or when focal neurologic signs are present. Most patients with neurogenic claudication who are treated medically do not improve over time. Surgical management can produce significant relief of back and leg pain within 6 weeks, and pain relief persists for at least 2 years. However, up to one-quarter develop recurrent stenosis at the same spinal level or an adjacent level 7–10 years after the initial surgery; recurrent symptoms usually respond to a second surgical decompression.

1	Neural foraminal narrowing with radiculopathy is a common consequence of osteoarthritic processes that cause lumbar spinal stenosis (Figs. 22-1 and 22-6), including osteophytes, lateral disk protrusion, calcified disk-osteophytes, facet joint hypertrophy, uncovertebral joint hypertrophy (cervical spine), congenitally shortened pedicles, or, frequently, a combination of these processes. Neoplasms (primary or metastatic), fractures, infections (epidural abscess), or hematomas are other considerations. These con-

1	LSS by itself is frequently asymptomatic, and the correlation between ditions can produce unilateral nerve root symptoms or signs due to the severity of symptoms and degree of stenosis of the spinal canal is compression at the intervertebral foramen or in the lateral recess; variable. LSS can be acquired (75%), congenital, or both. Congenital symptoms are indistinguishable from disk-related radiculopathy, but forms (achondroplasia, idiopathic) are characterized by short, thick ped-treatment may differ depending on the specific etiology. The history icles that produce both spinal canal and lateral recess stenosis. Acquired and neurologic examination alone cannot distinguish between these factors that contribute to spinal stenosis include degenerative diseases possibilities. A spine neuroimaging (CT or MRI) procedure is required (spondylosis, spondylolisthesis, scoliosis), trauma, spine surgery, meta-to identify the anatomic cause. Neurologic findings from the examibolic or endocrine

1	(CT or MRI) procedure is required (spondylosis, spondylolisthesis, scoliosis), trauma, spine surgery, meta-to identify the anatomic cause. Neurologic findings from the examibolic or endocrine disorders (epidural lipomatosis, osteoporosis, acro-nation and EMG can help direct the attention of the radiologist to megaly, renal osteodystrophy, hypoparathyroidism), and Paget’s disease. specific nerve roots, especially on axial images. For facet joint hypertro-MRI provides the best definition of the abnormal anatomy (Fig. 22-5). phy, surgical foraminotomy produces long-term relief of leg and back

1	Conservative treatment of symptomatic LSS includes nonsteroidal pain in 80–90% of patients. The usefulness of therapeutic facet joint anti-inflammatory drugs (NSAIDs), acetaminophen, exercise pro-blocks for pain is controversial. Medical causes of lumbar or cervical grams, and symptomatic treatment of acute pain episodes. There is radiculopathy unrelated to anatomic spine disease include infections recesses of high signal FIguRE 22-5 Axial T2-weighted images of the lumbar spine. A. The image shows a normal thecal sac within the lumbar spinal canal. The thecal sac is bright. The lumbar roots are dark punctuate dots in the posterior thecal sac with the patient supine. B. The thecal sac is not well visualized due to severe lumbar spinal canal stenosis, partially the result of hypertrophic facet joints. PART 2 Cardinal Manifestations and Presentation of Diseases Normal right L4-5 intervertebral foramen, L4 root, and high signal

1	PART 2 Cardinal Manifestations and Presentation of Diseases Normal right L4-5 intervertebral foramen, L4 root, and high signal FIguRE 22-6 Right L5 radiculopathy. A. Sagittal T2-weighted image. There is normal high signal around the exiting right L4 nerve root in the right neural foramen at L4-L5; effacement of the high signal in the right L5-S1 foramen is present one level caudal on the right at L5-S1. B. Axial T2-weighted image. The lateral recesses are normal bilaterally; the intervertebral foramen is normal on the left, but severely stenotic on the right. *Severe right L5-S1 foraminal stenosis. (e.g., herpes zoster, Lyme disease), carcinomatous meningitis, and root avulsion or traction (severe trauma).

1	(e.g., herpes zoster, Lyme disease), carcinomatous meningitis, and root avulsion or traction (severe trauma). Spondylosis, or osteoarthritic spine disease, typically occurs in later life and primarily involves the cervical and lumbosacral spine. Patients often complain of back pain that increases with movement, is associated with stiffness, and is better when inactive. The relationship between clinical symptoms and radiologic findings is usually not straightforward. Pain may be prominent when x-ray, CT, or MRI findings are minimal, and prominent degenerative spine disease can be seen in asymptomatic patients. Osteophytes or combined disk-osteophytes may cause or contribute to central spinal canal stenosis, lateral recess stenosis, or neural foraminal narrowing.

1	Spondylolisthesis is the anterior slippage of the vertebral body, pedicles, and superior articular facets, leaving the posterior elements behind. Spondylolisthesis can be associated with spondylolysis, congenital anomalies, degenerative spine disease, or other causes of mechanical weakness of the pars (e.g., infection, osteoporosis, tumor, trauma, prior surgery). The slippage may be asymptomatic or may cause low back pain and hamstring tightness, nerve root injury (the L5 root most frequently), symptomatic spinal stenosis, or CES in severe cases. Tenderness may be elicited near the segment that has “slipped” forward (most often L4 on L5 or occasionally L5 on S1). Focal anterolisthesis or retrolisthesis can occur at any cervical or lumbar level and be the source of neck or low back pain. Plain x-rays with the neck or low back in flexion and extension will reveal the movement at the abnormal spinal segment. Surgery is considered for pain symptoms that do not respond to conservative

1	Plain x-rays with the neck or low back in flexion and extension will reveal the movement at the abnormal spinal segment. Surgery is considered for pain symptoms that do not respond to conservative measures (e.g., rest, physical therapy) and in cases with progressive neurologic deficit, postural deformity, slippage >50%, or scoliosis.

1	Back pain is the most common neurologic symptom in patients with systemic cancer and is the presenting symptom in 20%. The cause is usually vertebral body metastasis but can also result from spread of cancer through the intervertebral foramen (especially with lymphoma), from carcinomatous meningitis, or from metastasis to the spinal cord. Cancer-related back pain tends to be constant, dull, unrelieved by rest, and worse at night. By contrast, mechanical low back pain usually improves with rest. MRI, CT, and CT myelography are the studies of choice when spinal metastasis is suspected. Once a metastasis is found, imaging of the entire spine reveals additional tumor deposits in one-third of patients. MRI is preferred for soft tissue definition, but the most rapidly available imaging modality is best because the patient’s condition may worsen quickly without intervention. Fewer than 5% of patients who are nonambulatory at the time of diagnosis ever regain the ability to walk; thus, early

1	is best because the patient’s condition may worsen quickly without intervention. Fewer than 5% of patients who are nonambulatory at the time of diagnosis ever regain the ability to walk; thus, early diagnosis is crucial. The management of spinal metastasis is discussed in detail in Chap. 118.

1	Vertebral osteomyelitis is often caused by staphylococci, but other bacteria or tuberculosis (Pott’s disease) may be responsible. The primary source of infection is usually the urinary tract, skin, or lungs. Intravenous drug use is a well-recognized risk factor. Whenever pyogenic osteomyelitis is found, the possibility of bacterial endocarditis should be considered. Back pain unrelieved by rest, spine tenderness over the involved spine segment, and an elevated ESR are the most common findings in vertebral osteomyelitis. Fever or an elevated white blood cell count is found in a minority of patients. MRI and CT are sensitive and specific for early detection of osteomyelitis; CT may be more readily available in emergency settings and better tolerated by some patients with severe back pain. The intervertebral disk can also be affected by infection (diskitis) and, very rarely, by tumor.

1	Spinal epidural abscess (Chap. 456) presents with back pain (aggravated by movement or palpation), fever, radiculopathy, or signs of spinal cord compression. The subacute development of two or more of these findings should increase the index of suspicion for spinal epidural abscess. The abscess may track over multiple spinal levels and is best delineated by spine MRI.

1	Lumbar adhesive arachnoiditis with radiculopathy is due to fibrosis 117 following inflammation within the subarachnoid space. The fibrosis results in nerve root adhesions and presents as back and leg pain associated with focal motor, sensory, or reflex changes. Causes of arachnoiditis include multiple lumbar operations, chronic spinal infections (especially tuberculosis in the developing world), spinal cord injury, intrathecal hemorrhage, myelography (rare), intrathecal injections (glucocorticoids, anesthetics, or other agents), and foreign bodies. The MRI shows clumped nerve roots or loculations of cerebrospinal fluid within the thecal sac. Clumped nerve roots may also occur with demyelinating polyneuropathy or neoplastic infiltration. Treatment is usually unsatisfactory. Microsurgical lysis of adhesions, dorsal rhizotomy, dorsal root ganglionectomy, and epidural glucocorticoids have been tried, but outcomes have been poor. Dorsal column stimulation for pain relief has produced

1	lysis of adhesions, dorsal rhizotomy, dorsal root ganglionectomy, and epidural glucocorticoids have been tried, but outcomes have been poor. Dorsal column stimulation for pain relief has produced varying results.

1	A patient complaining of back pain and an inability to move the legs may have a spine fracture or dislocation; with fractures above L1 the spinal cord is at risk for compression. Care must be taken to avoid further damage to the spinal cord or nerve roots by immobilizing the back or neck pending the results of radiologic studies. Vertebral fractures frequently occur in the absence of trauma in association with osteoporosis, glucocorticoid use, osteomyelitis, or neoplastic infiltration. Sprains and Strains The terms low back sprain, strain, and mechanically induced muscle spasm refer to minor, self-limited injuries associated with lifting a heavy object, a fall, or a sudden deceleration such as in an automobile accident. These terms are used loosely and do not clearly describe a specific anatomic lesion. The pain is usually confined to the lower back, and there is no radiation to the buttocks or legs. Patients with paraspinal muscle spasm often assume unusual postures.

1	Traumatic Vertebral Fractures Most traumatic fractures of the lumbar vertebral bodies result from injuries producing anterior wedging or compression. With severe trauma, the patient may sustain a fracture-dislocation or a “burst” fracture involving the vertebral body and posterior elements. Traumatic vertebral fractures are caused by falls from a height, sudden deceleration in an automobile accident, or direct injury. Neurologic impairment is common, and early surgical treatment is indicated. In victims of blunt trauma, CT scans of the chest, abdomen, or pelvis can be reformatted to detect associated vertebral fractures.

1	METABOLIC CAuSES Osteoporosis and Osteosclerosis Immobilization, osteomalacia, the postmenopausal state, renal disease, multiple myeloma, hyperparathyroidism, hyperthyroidism, metastatic carcinoma, or glucocorticoid use may accelerate osteoporosis and weaken the vertebral body, leading to compression fractures and pain. Up to two-thirds of compression fractures seen on radiologic imaging are asymptomatic. The most common nontraumatic vertebral body fractures are due to postmenopausal or senile osteoporosis (Chap. 425). The risk of an additional vertebral fracture at 1 year following a first vertebral fracture is 20%. The presence of fever, weight loss, fracture at a level above T4, or the conditions described above should increase suspicion for a cause other than senile osteoporosis. The sole manifestation of a compression fracture may be localized back or radicular pain exacerbated by movement and often reproduced by palpation over the spinous process of the affected vertebra.

1	Relief of acute pain can often be achieved with acetaminophen or a combination of opioids and acetaminophen. The role of NSAIDs is controversial. Both pain and disability are improved with bracing. Antiresorptive drugs, especially bisphosphonates (e.g., alendronate), have been shown to reduce the risk of osteoporotic fractures and are the preferred treatment to prevent additional fractures. Less than one-third of patients with prior compression fractures are adequately treated for osteoporosis despite the increased risk for future fractures; even fewer at-risk patients without a history of fracture are adequately treated. Given the negative results of sham-controlled studies of percutaneous vertebroplasty (PVP) and of kyphoplasty for osteoporotic 118 compression fractures associated with debilitating pain, these procedures are not routinely recommended. Osteosclerosis, an abnormally increased bone density often due to Paget’s disease, is readily identifiable on routine x-ray studies

1	debilitating pain, these procedures are not routinely recommended. Osteosclerosis, an abnormally increased bone density often due to Paget’s disease, is readily identifiable on routine x-ray studies and can sometimes be a source of back pain. It may be associated with an isolated increase in alkaline phosphatase in an otherwise healthy older person. Spinal cord or nerve root compression can result from bony encroachment. The diagnosis of Paget’s disease as the cause of a patient’s back pain is a diagnosis of exclusion. For further discussion of these bone disorders, see Chaps. 424, 425, and 426e.

1	Autoimmune inflammatory disease of the spine can present with the insidious onset of low back, buttock, or neck pain. Examples include rheumatoid arthritis (Chap 380), ankylosing spondylitis, reactive arthritis, psoriatic arthritis, or inflammatory bowel disease (Chap. 384). Spondylolysis is a bony defect in the vertebral pars interarticularis (a segment near the junction of the pedicle with the lamina); the cause is usually a stress microfracture in a congenitally abnormal segment. It occurs in up to 6% of adolescents. The defect (usually bilateral) is best visualized on plain x-rays, CT scan, or bone scan and is frequently asymptomatic. Symptoms may occur in the setting of a single injury, repeated minor injuries, or during a growth spurt. Spondylolysis is the most common cause of persistent low back pain in adolescents and is often associated with sports-related activities.

1	Scoliosis refers to an abnormal curvature in the coronal (lateral) plane of the spine. With kyphoscoliosis, there is, in addition, a forward curvature of the spine. The abnormal curvature may be congenital due to abnormal spine development, acquired in adulthood due to degenerative spine disease, or occasionally progressive due to neuromuscular disease. The deformity can progress until ambulation or pulmonary function is compromised. Spina bifida occulta is a failure of closure of one or several vertebral arches posteriorly; the meninges and spinal cord are normal. A dimple or small lipoma may overlie the defect. Most cases are asymptomatic and discovered incidentally during an evaluation for back pain.

1	Tethered cord syndrome usually presents as a progressive cauda equina disorder (see below), although myelopathy may also be the initial manifestation. The patient is often a young adult who complains of perineal or perianal pain, sometimes following minor trauma. MRI studies reveal a low-lying conus (below L1 and L2) and a short and thickened filum terminale. Diseases of the thorax, abdomen, or pelvis may refer pain to the posterior portion of the spinal segment that innervates the diseased organ. Occasionally, back pain may be the first and only manifestation. Upper abdominal diseases generally refer pain to the lower thoracic or upper lumbar region (eighth thoracic to the first and second lumbar vertebrae), lower abdominal diseases to the midlumbar region (second to fourth lumbar vertebrae), and pelvic diseases to the sacral region. Local signs (pain with spine palpation, paraspinal muscle spasm) are absent, and little or no pain accompanies routine movements of the spine.

1	Low Thoracic or Lumbar Pain with Abdominal Disease Tumors of the posterior wall of the stomach or duodenum typically produce epigastric pain (Chaps. 109 and 348), but midline back or paraspinal pain may occur if retroperitoneal extension is present. Fatty foods occasionally induce back pain associated with biliary disease. Diseases of the pancreas can produce right or left paraspinal back pain. Pathology in retroperitoneal structures (hemorrhage, tumors, pyelonephritis) can produce paraspinal pain that radiates to the lower abdomen, groin, or anterior thighs. A mass in the iliopsoas region can produce unilateral lumbar pain with radiation toward the groin, labia, or testicle. The sudden appearance of lumbar pain in a patient receiving anticoagulants suggests retroperitoneal hemorrhage. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Isolated low back pain occurs in some patients with a contained rupture of an abdominal aortic aneurysm (AAA). The classic clinical triad of abdominal pain, shock, and back pain occurs in <20% of patients. The typical patient at risk is an elderly male smoker with back pain. The diagnosis may be missed because the symptoms and signs can be nonspecific. Misdiagnoses include nonspecific back pain, diverticulitis, renal colic, sepsis, and myocardial infarction. A careful abdominal examination revealing a pulsatile mass (present in 50–75% of patients) is an important physical finding. Patients with suspected AAA should be evaluated with abdominal ultrasound, CT, or MRI (Chap. 301).

1	Sacral Pain with gynecologic and urologic Disease Pelvic organs rarely cause low back pain, except for gynecologic disorders involving the uterosacral ligaments. The pain is referred to the sacral region. Endometriosis or uterine cancers may invade the uterosacral ligaments. Pain associated with endometriosis is typically premenstrual and often continues until it merges with menstrual pain. Uterine malposition may cause uterosacral ligament traction (retroversion, descensus, and prolapse) or produce sacral pain after prolonged standing.

1	Menstrual pain may be felt in the sacral region sometimes with poorly localized, cramping pain radiating down the legs. Pain due to neoplastic infiltration of nerves is typically continuous, progressive in severity, and unrelieved by rest at night. Less commonly, radiation therapy of pelvic tumors may produce sacral pain from late radiation necrosis of tissue. Low back pain that radiates into one or both thighs is common in the last weeks of pregnancy. Urologic sources of lumbosacral back pain include chronic prostatitis, prostate cancer with spinal metastasis (Chap. 115), and diseases of the kidney or ureter. Lesions of the bladder and testes do not often produce back pain. Infectious, inflammatory, or neoplastic renal diseases may produce ipsilateral lumbosacral pain, as can renal artery or vein thrombosis. Paraspinal lumbar pain may be a symptom of ureteral obstruction due to nephrolithiasis.

1	OTHER CAuSES OF BACK PAIN Postural Back Pain There is a group of patients with nonspecific chronic low back pain (CLBP) in whom no specific anatomic lesion can be found despite exhaustive investigation. These individuals complain of vague, diffuse back pain with prolonged sitting or standing that is relieved by rest. Exercises to strengthen the paraspinal and abdominal muscles are sometimes helpful. Psychiatric Disease CLBP may be encountered in patients who seek financial compensation; in malingerers; or in those with concurrent substance abuse. Many patients with CLBP have a history of psychiatric illness (depression, anxiety states) or childhood trauma (physical or sexual abuse) that antedates the onset of back pain. Preoperative psychological assessment has been used to exclude patients with marked psychological impairments that predict a poor surgical outcome from spine surgery.

1	The cause of low back pain occasionally remains unclear. Some patients have had multiple operations for disk disease but have persistent pain and disability. The original indications for surgery may have been questionable, with back pain only, no definite neurologic signs, or a minor disk bulge noted on CT or MRI. Scoring systems based on neurologic signs, psychological factors, physiologic studies, and imaging studies have been devised to minimize the likelihood of unsuccessful surgery. HEALTH CARE FOR POPuLATIONS OF BACK PAIN PATIENTS: A CLINICAL CARE SYSTEMS VIEW There are increasing pressures to contain health care costs, especially when expensive care is not based on sound evidence. Physicians, patients, the insurance industry, and government providers of health care will need to work together to ensure cost-effective care for patients with back pain.

1	Surveys in the United States indicate that patients with back pain have reported progressively worse functional limitations in recent years, despite rapid increases in spine imaging, opioid prescribing, injections, and spine surgery. This suggests that more selective use of diagnostic and treatment modalities may be appropriate.

1	Spine imaging often reveals abnormalities of dubious clinical relevance that may alarm clinicians and patients and prompt further testing and unnecessary therapy. Both randomized trials and observational studies have suggested a “cascade effect” of imaging, which may create a gateway to other unnecessary care. Based in part on such evidence, the American College of Physicians has made parsimonious spine imaging a high priority in its “Choosing Wisely” campaign, aimed at reducing unnecessary care. Successful efforts to reduce unnecessary imaging have included physician education by clinical leaders, computerized decision support to identify recent imaging tests and eliminate duplication, and requiring an approved indication to order an imaging test. Other strategies have included audit and feedback regarding individual practitioners’ rates of ordering and indications and facilitating rapid access to physical therapy for patients who do not need imaging. When imaging tests are reported,

1	feedback regarding individual practitioners’ rates of ordering and indications and facilitating rapid access to physical therapy for patients who do not need imaging. When imaging tests are reported, it may also be useful to routinely note that some degenerative findings are common in normal, pain-free individuals. In an observational study, this strategy was associated with lower rates of repeat imaging, opioid therapy, and referral for physical therapy.

1	Mounting evidence of morbidities from long-term opioid therapy (including overdose, dependency, addiction, falls, fractures, accident risk, and sexual dysfunction) has prompted efforts to reduce use for chronic pain, including back pain (Chap. 18). Safety may be improved with automated reminders for high doses, early refills, or overlapping opioid and benzodiazepine prescriptions. Greater access to alternative treatments for chronic pain, such as tailored exercise programs and cognitive-behavioral therapy, may also reduce opioid prescribing.

1	The high cost, wide geographic variations, and rapidly increasing rates of spinal fusion surgery have prompted scrutiny over appropriate indications. Some insurance carriers have begun to limit coverage for the most controversial indications, such as low back pain without radiculopathy. Finally, educating patients and the public about the risks of imaging and excessive therapy may be necessary. A successful media campaign in Australia provides a successful model for this approach. ALBP is defined as pain of <3 months in duration. Full recovery can be expected in more than 85% of adults with ALBP without leg pain. Most have purely “mechanical” symptoms (i.e., pain that is aggravated by motion and relieved by rest).

1	The initial assessment excludes serious causes of spine pathology that require urgent intervention including infection, cancer, or trauma. Risk factors for a serious cause of ALBP are shown in Table 22-1. Laboratory and imaging studies are unnecessary if risk factors are absent. CT, MRI, or plain spine films are rarely indicated in the first month of symptoms unless a spine fracture, tumor, or infection is suspected. The prognosis is generally excellent. Many patients do not seek medical care and improve on their own. Even among those seen in primary care, two-thirds report being substantially improved after 7 weeks. This spontaneous improvement can mislead clinicians and researchers about the efficacy of treatment interventions unless subjected to rigorous prospective trials. Many treatments commonly used in the past but now known to be ineffective, including bed rest, lumbar traction, and coccygectomy, have been largely abandoned.

1	Clinicians should reassure patients that improvement is very likely and instruct them in self-care. Education is an important part of treatment. Satisfaction and the likelihood of follow-up increase when patients are educated about prognosis, treatment methods, 119 activity modifications, and strategies to prevent future exacerbations. Patients who report that they did not receive an adequate explanation for their symptoms are likely to request further diagnostic tests. In general, bed rest should be avoided for relief of severe symptoms or kept to a day or two at most. Several randomized trials suggest that bed rest does not hasten the pace of recovery. In general, the best activity recommendation is for early resumption of normal physical activity, avoiding only strenuous manual labor. Possible advantages of early ambulation for ALBP include maintenance of cardiovascular conditioning, improved disk and cartilage nutrition, improved bone and muscle strength, and increased endorphin

1	Possible advantages of early ambulation for ALBP include maintenance of cardiovascular conditioning, improved disk and cartilage nutrition, improved bone and muscle strength, and increased endorphin levels. Specific back exercises or early vigorous exercise have not shown benefits for acute back pain, but may be useful for chronic pain. Use of heating pads or blankets is sometimes helpful.

1	Evidence-based guidelines recommend over-the-counter medicines such as acetaminophen and NSAIDs as first-line options for treatment of ALBP. In otherwise healthy patients, a trial of acetaminophen can be followed by NSAIDs for time-limited periods. In theory, the anti-inflammatory effects of NSAIDs might provide an advantage over acetaminophen to suppress inflammatory changes that accompany many causes of ALBP, but in practice, there is no clinical evidence to support the superiority of NSAIDs. The risk of renal and gastrointestinal toxicity with NSAIDs is increased in patients with preexisting medical comorbidities (e.g., renal insufficiency, cirrhosis, prior gastrointestinal hemorrhage, use of anticoagulants or steroids, heart failure). Skeletal muscle relaxants, such as cyclobenzaprine or methocarbamol, may be useful, but sedation is a common side effect. Limiting the use of muscle relaxants to nighttime only may be an option for patients with back pain that interferes with sleep.

1	There is no good evidence to support the use of opioid analgesics or tramadol as first-line therapy for ALBP. Their use is best reserved for patients who cannot tolerate acetaminophen or NSAIDs or for those with severe refractory pain. As with muscle relaxants, these drugs are often sedating, so it may be useful to prescribe them at nighttime only. Side effects of short-term opioid use include nausea, constipation, and pruritus; risks of long-term opioid use include hypersensitivity to pain, hypogonadism, and dependency. Falls, fractures, driving accidents, and fecal impaction are other risks. Clinical efficacy of opioids beyond 16 weeks of use is unproven. There is no evidence to support use of oral or injected glucocorticoids for ALBP without radiculopathy. Similarly, therapies for neuropathic pain, such as gabapentin or tricyclic antidepressants, are not indicated for ALBP.

1	Nonpharmacologic treatments for ALBP include spinal manipulation, exercise, physical therapy, massage, acupuncture, transcutaneous electrical nerve stimulation, and ultrasound. Spinal manipulation appears to be roughly equivalent to conventional medical treatments and may be a useful alternative for patients who wish to avoid or who cannot tolerate drug therapy. There is little evidence to support the use of physical therapy, massage, acupuncture, laser therapy, therapeutic ultrasound, corsets, or lumbar traction. Although important for chronic pain, back exercises for ALBP are generally not supported by clinical evidence. There is no convincing evidence regarding the value of ice or heat applications for ABLP; however, many patients report temporary symptomatic relief from ice or frozen gel packs, and heat may produce a short-term reduction in pain after the first week. Patients often report improved satisfaction with the care that they receive when they actively participate in the

1	gel packs, and heat may produce a short-term reduction in pain after the first week. Patients often report improved satisfaction with the care that they receive when they actively participate in the selection of symptomatic approaches that are tried.

1	CLBP is defined as pain lasting >12 weeks; it accounts for 50% of total back pain costs. Risk factors include obesity, female gender, older age, prior history of back pain, restricted spinal mobility, pain radiating into a leg, high levels of psychological distress, poor self-rated health, minimal physical activity, smoking, job dissatisfaction, and widespread pain. In general, the same treatments that are 120 recommended for ALBP can be useful for patients with CLBP. In this setting, however, the long-term benefit of opioid therapy or muscle relaxants is less clear. Evidence supports the use of exercise therapy, and this can be one of the mainstays of treatment for CLBP. Effective regimens have generally included a combination of gradually increasing aerobic exercise, strengthening exercises, and stretching exercises. Motivating patients is sometimes challenging, and in this setting, a program of supervised exercise can improve compliance. In general, activity tolerance is the

1	exercises, and stretching exercises. Motivating patients is sometimes challenging, and in this setting, a program of supervised exercise can improve compliance. In general, activity tolerance is the primary goal, while pain relief is secondary. Supervised intensive physical exercise or “work hardening” regimens have been effective in returning some patients to work, improving walking distance, and reducing pain. In addition, some forms of yoga have been evaluated in randomized trials and may be helpful for patients who are interested. A long-term benefit of spinal manipulation or massage for CLBP is unproven. Medications for CLBP may include acetaminophen, NSAIDs, and tricyclic antidepressants. Trials of tricyclics suggest benefit even for patients without evidence of depression. Trials do not support the efficacy of selective serotonin reuptake inhibitors (SSRIs) for CLBP. However, depression is common among patients with chronic pain and should be appropriately treated.

1	Trials do not support the efficacy of selective serotonin reuptake inhibitors (SSRIs) for CLBP. However, depression is common among patients with chronic pain and should be appropriately treated. Cognitive-behavioral therapy is based on evidence that psychological and social factors, as well as somatic pathology, are important in the genesis of chronic pain and disability. Cognitive-behavioral therapy includes efforts to identify and modify patients’ thinking about their pain and disability. A systematic review concluded that such treatments are more effective than a waiting list control group for short-term pain relief; however, long-term results remain unclear. Behavioral treatments may have effects similar in magnitude to exercise therapy. Back pain is the most frequent reason for seeking complementary and alternative treatments. The most common of these for back pain are spinal manipulation, acupuncture, and massage. The role of most complementary and alternative medicine

1	for seeking complementary and alternative treatments. The most common of these for back pain are spinal manipulation, acupuncture, and massage. The role of most complementary and alternative medicine approaches remains unclear. Biofeedback has not been studied rigorously. There is no convincing evidence that either spinal manipulation or transcutaneous electrical nerve stimulation (TENS) is effective in treating CLBP. Rigorous recent trials of acupuncture suggest that true acupuncture is not superior to sham acupuncture, but that both may offer an advantage over routine care. Whether this is due entirely to placebo effects provided even by sham acupuncture is uncertain. Some trials of massage therapy have been encouraging, but this has been less well studied than spinal manipulation or acupuncture. Various injections, including epidural glucocorticoid injections, facet joint injections, and trigger point injections, have been used for treating CLBP. However, in the absence of

1	or acupuncture. Various injections, including epidural glucocorticoid injections, facet joint injections, and trigger point injections, have been used for treating CLBP. However, in the absence of radiculopathy, there is no evidence that these approaches are effective. Injection studies are sometimes used diagnostically to help determine the anatomic source of back pain. The use of discography to provide evidence that a specific disk is the pain generator is not recommended. Pain relief following a glucocorticoid injection into a facet is commonly used as evidence that the facet joint is the pain source; however, the possibility that the response was a placebo effect or due to systemic absorption of the glucocorticoids is difficult to exclude. Another category of intervention for chronic back pain is electrothermal and radiofrequency therapy. Intradiskal therapy has been proposed using both types of energy to thermocoagulate and destroy nerves in the intervertebral disk, using

1	back pain is electrothermal and radiofrequency therapy. Intradiskal therapy has been proposed using both types of energy to thermocoagulate and destroy nerves in the intervertebral disk, using specially designed catheters or electrodes. Current evidence does not support the use of these intradiskal therapies. Radiofrequency denervation is sometimes used to destroy nerves that are thought to mediate pain, and this technique has been used for facet joint pain (with the target nerve being the medial branch of the primary dorsal ramus), for back pain thought to arise from the intervertebral disk (ramus communicans), and radicular back pain (dorsal root ganglia). A few small trials have produced conflicting results for facet joint and diskogenic pain. A trial in patients with chronic radicular pain found no difference between radiofrequency

1	PART 2 Cardinal Manifestations and Presentation of Diseases denervation of the dorsal root ganglia and sham treatment. These interventional therapies have not been studied in sufficient detail to draw conclusions of their value for CLBP.

1	Surgical intervention for CLBP without radiculopathy has been evaluated in a small number of randomized trials, all conducted in Europe. Each of these studies included patients with back pain and a degenerative disk, but no sciatica. Three of the four trials concluded that lumbar fusion surgery was no more effective than highly structured, rigorous rehabilitation combined with cognitive-behavioral therapy. The fourth trial found an advantage of fusion surgery over haphazard “usual care,” which appeared to be less effective than the structured rehabilitation in other trials. Given conflicting evidence, indications for surgery for CLBP without radiculopathy have remained controversial. Both U.S. and British guidelines suggest considering referral for an opinion on spinal fusion for people who have completed an optimal nonsurgical treatment program (including combined physical and psychological treatment) and who have persistent severe back pain for which they would consider surgery.

1	Lumbar disk replacement with prosthetic disks is U.S. Food and Drug Administration approved for uncomplicated patients needing single-level surgery at the L3-S1 levels. The disks are generally designed as metal plates with a polyethylene cushion sandwiched in between. The trials that led to approval of these devices compared them to spine fusion and concluded that the artificial disks were “not inferior.” Serious complications are somewhat more likely with the artificial disk. This treatment remains controversial for CLBP. Intensive multidisciplinary rehabilitation programs may involve daily or frequent physical therapy, exercise, cognitive-behavioral therapy, a workplace evaluation, and other interventions. For patients who have not responded to other approaches, such programs appear to offer some benefit. Systematic reviews suggest that the evidence is limited and benefits are incremental.

1	Some observers have raised concern that CLBP may often be overtreated. For CLBP without radiculopathy, new British guidelines explicitly recommend against use of SSRIs, any type of injection, TENS, lumbar supports, traction, radiofrequency facet joint denervation, intradiskal electrothermal therapy, or intradiskal radiofrequency thermocoagulation. These treatments are also not recommended in guidelines from the American College of Physicians and the American Pain Society. On the other hand, exercise therapy and treatment of depression appear to be useful and underused.

1	A common cause of back pain with radiculopathy is a herniated disk with nerve root impingement, resulting in back pain with radiation down the leg. The term sciatica is used when the leg pain radiates posteriorly in a sciatic or L5/S1 distribution. The prognosis for acute low back and leg pain with radiculopathy due to disk herniation is generally favorable, with most patients showing substantial improvement over months. Serial imaging studies suggest spontaneous regression of the herniated portion of the disk in two-thirds of patients over 6 months. Nonetheless, there are several important treatment options to provide symptomatic relief while this natural healing process unfolds. Resumption of normal activity is recommended. Randomized trial evidence suggests that bed rest is ineffective for treating sciatica as well as back pain alone. Acetaminophen and NSAIDs are useful for pain relief, although severe pain may require short courses of opioid analgesics.

1	Epidural glucocorticoid injections have a role in providing temporary symptom relief for sciatica due to a herniated disk. However, there does not appear to be a benefit in terms of reducing subsequent surgical interventions. Diagnostic nerve root blocks have been advocated to determine if pain originates from a specific nerve root. However, improvement may result even when the nerve root is not responsible for the pain; this may occur as a placebo effect, from a pain-generating lesion located distally along the peripheral nerve, or from effects of systemic absorption. The utility of diagnostic nerve root blocks remains a subject of debate.

1	Surgical intervention is indicated for patients who have progressive motor weakness due to nerve root injury demonstrated on clinical examination or EMG. Urgent surgery is recommended for patients who have evidence of CES or spinal cord compression, generally suggested by bowel or bladder dysfunction, diminished sensation in a saddle distribution, a sensory level on the trunk, and bilateral leg weakness or spasticity.

1	Surgery is also an important option for patients who have disabling radicular pain despite optimal conservative treatment. Sciatica is perhaps the most common reason for recommending spine surgery. Because patients with a herniated disk and sciatica generally experience rapid improvement over a matter of weeks, most experts do not recommend considering surgery unless the patient has failed to respond to 6–8 weeks of maximum nonsurgical management. For patients who have not improved, randomized trials indicate that, compared to nonsurgical treatment, surgery results in more rapid pain relief. However, after the first year or two of follow-up, patients with sciatica appear to have much the same level of pain relief and functional improvement with or without surgery. Thus, both treatment approaches are reasonable, and patient preferences and needs (e.g., rapid return to employment) strongly influence decision making. Some patients will want the fastest possible relief and find surgical

1	are reasonable, and patient preferences and needs (e.g., rapid return to employment) strongly influence decision making. Some patients will want the fastest possible relief and find surgical risks acceptable. Others will be more risk-averse and more tolerant of symptoms and will choose watchful waiting if they understand that improvement is likely in the end.

1	The usual surgical procedure is a partial hemilaminectomy with excision of the prolapsed disk (diskectomy). Fusion of the involved lumbar segments should be considered only if significant spinal instability is present (i.e., degenerative spondylolisthesis). The costs associated with lumbar interbody fusion have increased dramatically in recent years. There are no large prospective, randomized trials comparing fusion to other types of surgical intervention. In one study, patients with persistent low back pain despite an initial diskectomy fared no better with spine fusion than with a conservative regimen of cognitive intervention and exercise. Artificial disks have been in use in Europe for the past decade; their utility remains controversial in the United States.

1	Neck pain, which usually arises from diseases of the cervical spine and soft tissues of the neck, is common. Neck pain arising from the cervical spine is typically precipitated by movement and may be accompanied by focal tenderness and limitation of motion. Many of the prior com-121 ments made regarding causes of low back pain also apply to disorders of the cervical spine. The text below will emphasize differences. Pain arising from the brachial plexus, shoulder, or peripheral nerves can be confused with cervical spine disease (Table 22-4), but the history and examination usually identify a more distal origin for the pain. Cervical spine trauma, disk disease, or spondylosis with intervertebral foraminal narrowing may be asymptomatic or painful and can produce a myelopathy, radiculopathy, or both. The same risk factors for serious causes of low back pain also apply to neck pain with the additional feature that neurologic signs of myelopathy (incontinence, sensory level, spastic legs)

1	or both. The same risk factors for serious causes of low back pain also apply to neck pain with the additional feature that neurologic signs of myelopathy (incontinence, sensory level, spastic legs) may also occur. Lhermitte’s sign, an electrical shock down the spine with neck flexion, suggests involvement of the cervical spinal cord.

1	Trauma to the cervical spine (fractures, subluxation) places the spinal cord at risk for compression. Motor vehicle accidents, violent crimes, or falls account for 87% of cervical spinal cord injuries (Chap. 456). Immediate immobilization of the neck is essential to minimize further spinal cord injury from movement of unstable cervical spine segments. The decision to obtain imaging should be based on the nature of the injury. The NEXUS low-risk criteria established that normally alert patients without palpation tenderness in the midline; intoxication; neurologic deficits; or painful distracting injuries were very unlikely to have sustained a clinically significant traumatic injury to the cervical spine. The Canadian C-spine rule recommends that imaging should be obtained following neck region trauma if the patient is >65 years old or has limb paresthesias or if there was a dangerous mechanism for the injury (e.g., bicycle collision with tree or parked car, fall from height >3 feet or

1	trauma if the patient is >65 years old or has limb paresthesias or if there was a dangerous mechanism for the injury (e.g., bicycle collision with tree or parked car, fall from height >3 feet or five stairs, diving accident). These guidelines are helpful but must be tailored to individual circumstances; for example, patients with advanced osteoporosis, glucocorticoid use, or cancer may warrant imaging after even mild trauma. A CT scan is the diagnostic procedure of choice for detection of acute fractures following severe trauma; plain x-rays can be used for lesser degrees of trauma. When traumatic injury to the vertebral arteries or cervical spinal cord is suspected, visualization by MRI with magnetic resonance angiography is preferred.

1	Whiplash injury is due to rapid flexion and extension of the neck, usually in automobile accidents. The exact mechanism of the injury is unclear. This diagnosis should not be applied to patients with fractures, disk herniation, head injury, focal neurologic findings, or altered consciousness. Up to 50% of persons reporting whiplash injury acutely have persistent neck pain 1 year later. Once personal C5 Biceps Lateral deltoid Rhomboidsa (elbow extends backward with hand on Lateral arm, medial scapula hip) Infraspinatusa (arm rotates externally with elbow flexed at the side)

1	Deltoida (arm raised laterally 30–45° from the side) C6 Biceps Thumb/index finger; Bicepsa (arm flexed at the elbow in supination) Lateral forearm, thumb/index fingers Dorsal hand/lateral forearm Pronator teres (forearm pronated) C7 Triceps Middle fingers Tricepsa (forearm extension, flexed at elbow) Posterior arm, dorsal forearm, dorsal hand Dorsal forearm Wrist/finger extensorsa C8 Finger Palmar surface of little finger Abductor pollicis brevis (abduction of thumb) Fourth and fifth fingers, medial hand and flexors forearm Medial hand and forearm First dorsal interosseous (abduction of index finger) Abductor digiti minimi (abduction of little finger) T1 Finger Axilla and medial arm Abductor pollicis brevis (abduction of thumb) Medial arm, axilla flexors First dorsal interosseous (abduction of index finger) Abductor digiti minimi (abduction of little finger) aThese muscles receive the majority of innervation from this root.

1	122 compensation for pain and suffering was removed from the Australian health care system, the prognosis for recovery at 1 year from whiplash injury improved also. Imaging of the cervical spine is not cost-effective acutely but is useful to detect disk herniations when symptoms persist for >6 weeks following the injury. Severe initial symptoms have been associated with a poor long-term outcome.

1	Herniation of a lower cervical disk is a common cause of pain or tingling in the neck, shoulder, arm, or hand. Neck pain, stiffness, and a range of motion limited by pain are the usual manifestations. Herniated cervical disks are responsible for ~25% of cervical radiculopathies. Extension and lateral rotation of the neck narrow the ipsilateral intervertebral foramen and may reproduce radicular symptoms (Spurling’s sign). In young adults, acute nerve root compression from a ruptured cervical disk is often due to trauma. Cervical disk herniations are usually posterolateral near the lateral recess. Typical patterns of reflex, sensory, and motor changes that accompany cervical nerve root lesions are summarized in Table 22-4. Although the classic patterns are clinically helpful, there are numerous exceptions because there is overlap in sensory function between adjacent nerve roots, symptoms and signs may be evident in only part of the injured nerve root territory, and (3) the location of

1	exceptions because there is overlap in sensory function between adjacent nerve roots, symptoms and signs may be evident in only part of the injured nerve root territory, and (3) the location of pain is the most variable of the clinical features.

1	Osteoarthritis of the cervical spine may produce neck pain that radiates into the back of the head, shoulders, or arms, or may be the source of headaches in the posterior occipital region (supplied by the C2-C4 nerve roots). Osteophytes, disk protrusions, or hypertrophic facet or uncovertebral joints may alone or in combination compress one or several nerve roots at the intervertebral foramina; these causes together account for 75% of cervical radiculopathies. The roots most commonly affected are C7 and C6. Narrowing of the spinal canal by osteophytes, ossification of the posterior longitudinal ligament (OPLL), or a large central disk may compress the cervical spinal cord and produce signs of radiculopathy and myelopathy in combination (myeloradiculopathy). When little or no neck pain accompanies cervical cord involvement, other diagnoses to be considered include amyotrophic lateral sclerosis (Chap. 452), multiple sclerosis (Chap. 458), spinal cord tumors, or syringomyelia (Chap.

1	accompanies cervical cord involvement, other diagnoses to be considered include amyotrophic lateral sclerosis (Chap. 452), multiple sclerosis (Chap. 458), spinal cord tumors, or syringomyelia (Chap. 456). The possibility of cervical spondylosis should be considered even when the patient presents with symptoms or signs in the legs only. MRI is the study of choice to define anatomic abnormalities of soft tissues in the cervical region including the spinal cord, but plain CT is adequate to assess bony spurs, foraminal narrowing, lateral recess stenosis, or OPLL. EMG and nerve conduction studies can localize and assess the severity of nerve root injury.

1	Rheumatoid arthritis (RA) (Chap. 380) of the cervical facet joints produces neck pain, stiffness, and limitation of motion. Synovitis of the atlantoaxial joint (C1-C2; Fig. 22-2) may damage the transverse ligament of the atlas, producing forward displacement of the atlas on the axis (atlantoaxial subluxation). Radiologic evidence of atlantoaxial subluxation occurs in up to 30% of patients with RA. The degree of subluxation correlates with the severity of erosive disease. When subluxation is present, careful assessment is important to identify early signs of myelopathy. Occasional patients develop high spinal cord compression leading to quadriparesis, respiratory insufficiency, and death. Surgery should be considered when myelopathy or spinal instability is present. MRI is the imaging modality of choice. Ankylosing spondylitis can cause neck pain and less commonly atlantoaxial subluxation; surgery may be required to prevent spinal cord compression.

1	Acute herpes zoster can presents as acute posterior occipital or neck pain prior to the outbreak of vesicles. Neoplasms metastatic to the cervical spine, infections (osteomyelitis and epidural abscess), and metabolic bone diseases may be the cause of neck pain, as discussed above PART 2 Cardinal Manifestations and Presentation of Diseases among causes of low back pain. Neck pain may also be referred from the heart with coronary artery ischemia (cervical angina syndrome). The thoracic outlet contains the first rib, the subclavian artery and vein, the brachial plexus, the clavicle, and the lung apex. Injury to these structures may result in postural or movement-induced pain around the shoulder and supraclavicular region, classified as follows.

1	True neurogenic thoracic outlet syndrome (TOS) is an uncommon disorder resulting from compression of the lower trunk of the brachial plexus or ventral rami of the C8 or T1 nerve roots, caused most often by an anomalous band of tissue connecting an elongate transverse process at C7 with the first rib. Pain is mild or may be absent. Signs include weakness and wasting of intrinsic muscles of the hand and diminished sensation on the palmar aspect of the fifth digit. An anteroposterior cervical spine x-ray will show an elongate C7 transverse process (an anatomic marker for the anomalous cartilaginous band), and EMG and nerve conduction studies confirm the diagnosis. Treatment consists of surgical resection of the anomalous band. The weakness and wasting of intrinsic hand muscles typically does not improve, but surgery halts the insidious progression of weakness.

1	Arterial TOS results from compression of the subclavian artery by a cervical rib, resulting in poststenotic dilatation of the artery and in some cases secondary thrombus formation. Blood pressure is reduced in the affected limb, and signs of emboli may be present in the hand. Neurologic signs are absent. Ultrasound can confirm the diagnosis noninvasively. Treatment is with thrombolysis or anticoagulation (with or without embolectomy) and surgical excision of the cervical rib compressing the subclavian artery. Venous TOS is due to subclavian vein thrombosis resulting in swelling of the arm and pain. The vein may be compressed by a cervical rib or anomalous scalene muscle. Venography is the diagnostic test of choice.

1	Disputed TOS accounts for 95% of patients diagnosed with TOS; chronic arm and shoulder pain are prominent and of unclear cause. The lack of sensitive and specific findings on physical examination or specific markers for this condition results in diagnostic uncertainty. The role of surgery in disputed TOS is controversial. Multidisciplinary pain management is a conservative approach, although treatment is often unsuccessful.

1	Pain from injury to the brachial plexus or peripheral nerves of the arm can occasionally mimic referred pain of cervical spine origin including cervical radiculopathy. Neoplastic infiltration of the lower trunk of the brachial plexus may produce shoulder or supraclavicular pain radiating down the arm, numbness of the fourth and fifth fingers or medial forearm, and weakness of intrinsic hand muscles innervated by the ulnar and median nerves. Delayed radiation injury may produce similar findings, although pain is less often present and almost always less severe. A Pancoast tumor of the lung (Chap. 107) is another cause and should be considered, especially when a concurrent Horner’s syndrome is present. Suprascapular neuropathy may produce severe shoulder pain, weakness, and wasting of the supraspinatus and infraspinatus muscles. Acute brachial neuritis is often confused with radiculopathy; the acute onset of severe shoulder or scapular pain is followed typically over days by weakness of

1	and infraspinatus muscles. Acute brachial neuritis is often confused with radiculopathy; the acute onset of severe shoulder or scapular pain is followed typically over days by weakness of the proximal arm and shoulder girdle muscles innervated by the upper brachial plexus. The onset may be preceded by an infection, vaccination, or minor surgical procedure. The long thoracic nerve may be affected resulting in a winged scapula. Brachial neuritis may also present as an isolated paralysis of the diaphragm with or without involvement of other nerves of the upper limb. Recovery may take up to 3 years.

1	Occasional cases of carpal tunnel syndrome produce pain and paresthesias extending into the forearm, arm, and shoulder resembling a C5 or C6 root lesion. Lesions of the radial or ulnar nerve can mimic a radiculopathy at C7 or C8, respectively. EMG and nerve conduction studies can accurately localize lesions to the nerve roots, brachial plexus, or peripheral nerves. For further discussion of peripheral nerve disorders, see Chap. 459.

1	For further discussion of peripheral nerve disorders, see Chap. 459. Pain arising from the shoulder can on occasion mimic pain from the spine. If symptoms and signs of radiculopathy are absent, then the differential diagnosis includes mechanical shoulder pain (tendonitis, bursitis, rotator cuff tear, dislocation, adhesive capsulitis, or rotator cuff impingement under the acromion) and referred pain (subdiaphragmatic irritation, angina, Pancoast tumor). Mechanical pain is often worse at night, associated with local shoulder tenderness and aggravated by passive abduction, internal rotation, or extension of the arm. Pain from shoulder disease may radiate into the arm or hand, but focal neurologic signs (sensory, motor, or reflex changes) are absent.

1	The evidence regarding treatment for neck pain is less complete than that for low back pain, but the approach is remarkably similar in many respects. As with low back pain, spontaneous improvement is the norm for acute neck pain. The usual goals of therapy are to promote a rapid return to normal function and provide symptom relief while healing proceeds. The evidence in support of nonsurgical treatments for whiplash-associated disorders is generally of limited quality and neither supports nor refutes the common treatments used for symptom relief. Gentle mobilization of the cervical spine combined with exercise programs may be beneficial. Evidence is insufficient to recommend for or against the routine use of acupuncture, cervical traction, TENS, ultrasound, diathermy, or massage. Some patients obtain modest relief using a soft neck collar; there is little risk or cost.

1	For patients with neck pain unassociated with trauma, supervised exercise with or without mobilization appears to be effective. Exercises often include shoulder rolls and neck stretches. The evidence for the use of muscle relaxants, analgesics, and NSAIDs in acute and chronic neck pain is of lower quality and less consistent than for low back pain.

1	Low-level laser therapy directed at areas of tenderness, local acupuncture points, or a grid of predetermined points is a controversial approach to the treatment of neck pain. A 2009 meta-analysis suggested that this treatment may provide greater pain relief than sham fever Charles A. Dinarello, Reuven Porat Body temperature is controlled by the hypothalamus. Neurons in both the preoptic anterior hypothalamus and the posterior hypothalamus receive two kinds of signals: one from peripheral nerves that transmit information from warmth/cold receptors in the skin and the other 23 SECTion 2 from the temperature of the blood bathing the region. These two types of signals are integrated by the thermoregulatory center of the hypothalamus to maintain normal temperature. In a neutral temperature environment, the human metabolic rate produces more heat than is necessary to maintain the core body temperature in the range of 36.5–37.5°C (97.7–99.5°F).

1	therapy for both acute and chronic neck pain, but comparison to 123 other conservative and less expensive treatment measures is needed. Although some surgical studies have proposed a role for anterior diskectomy and fusion in patients with neck pain, these studies generally have not been rigorously conducted. A systematic review suggested that there was no valid clinical evidence to support either cervical fusion or cervical disk arthroplasty in patients with neck pain without radiculopathy. Similarly, there is no evidence to support radiofrequency neurotomy or cervical facet injections for neck pain without radiculopathy.

1	The natural history of neck pain with acute radiculopathy due to disk disease is favorable, and many patients will improve without specific therapy. Although there are no randomized trials of NSAIDs for neck pain, a course of NSAIDs, acetaminophen, or both, with or without muscle relaxants, is reasonable as initial therapy. Other nonsurgical treatments are commonly used, including opioid analgesics, oral glucocorticoids, cervical traction, and immobilization with a hard or soft cervical collar. However, there are no randomized trials that establish the effectiveness of these treatments. Soft cervical collars can be modestly helpful by limiting spontaneous and reflex neck movements that exacerbate pain.

1	As for lumbar radiculopathy, epidural glucocorticoids appear to provide short-term symptom relief in cervical radiculopathy, but rigorous studies addressing this question have not been conducted. If cervical radiculopathy is due to bony compression from cervical spondylosis with foraminal narrowing, periodic follow-up to assess for progression is indicated and consideration of surgical decompression is reasonable. Surgical treatment can produce rapid pain relief, although it is unclear whether long-term outcomes are improved over nonsurgical therapy. Indications for cervical disk surgery include a progressive radicular motor deficit, functionally limiting pain that fails to respond to conservative management, or spinal cord compression.

1	Surgical treatments include anterior cervical diskectomy alone, laminectomy with diskectomy, or diskectomy with fusion. The risk of subsequent radiculopathy or myelopathy at cervical segments adjacent to a fusion is ~3% per year and 26% per decade. Although this risk is sometimes portrayed as a late complication of surgery, it may also reflect the natural history of degenerative cervical disk disease.

1	A normal body temperature is ordinarily maintained despite environmental variations because the hypothalamic thermoregulatory center balances the excess heat production derived from metabolic activity in muscle and the liver with heat dissipation from the skin and lungs. According to studies of healthy individuals 18–40 years of age, the mean oral temperature is 36.8° ± 0.4°C (98.2° ± 0.7°F), with low levels at 6 a.m. and higher levels at 4–6 p.m. The maximal normal oral temperature is 37.2°C (98.9°F) at 6 a.m. and 37.7°C (99.9°F) at 4 p.m.; these values define the 99th percentile for healthy individuals. In light of these studies, an a.m. temperature of >37.2°C (>98.9°F) or a p.m. temperature of >37.7°C (>99.9°F) would define a fever. The normal daily temperature variation is typically 0.5°C (0.9°F). However, in some individuals recovering from a febrile illness, this daily variation can be as great as 1.0°C. During a febrile illness, the diurnal variation is usually maintained, but

1	(0.9°F). However, in some individuals recovering from a febrile illness, this daily variation can be as great as 1.0°C. During a febrile illness, the diurnal variation is usually maintained, but at higher, febrile levels. The daily temperature 124 variation appears to be fixed in early childhood; in contrast, elderly individuals can exhibit a reduced ability to develop fever, with only a modest fever even in severe infections. Rectal temperatures are generally 0.4°C (0.7°F) higher than oral readings. The lower oral readings are probably attributable to mouth breathing, which is a factor in patients with respiratory infections and rapid breathing. Lower-esophageal temperatures closely reflect core temperature. Tympanic membrane thermometers measure radiant heat from the tympanic membrane and nearby ear canal and display that absolute value (unadjusted mode) or a value automatically calculated from the absolute reading on the basis of nomograms relating the radiant temperature measured

1	nearby ear canal and display that absolute value (unadjusted mode) or a value automatically calculated from the absolute reading on the basis of nomograms relating the radiant temperature measured to actual core temperatures obtained in clinical studies (adjusted mode). These measurements, although convenient, may be more variable than directly determined oral or rectal values. Studies in adults show that readings are lower with unadjusted-mode than with adjusted-mode tympanic membrane thermometers and that unadjusted-mode tympanic membrane values are 0.8°C (1.6°F) lower than rectal temperatures. In women who menstruate, the a.m. temperature is generally lower in the 2 weeks before ovulation; it then rises by ∼0.6°C (1°F) with ovulation and remains at that level until menses occur. Body temperature can be elevated in the postprandial state. Pregnancy and endocrinologic dysfunction also affect body temperature.

1	Fever is an elevation of body temperature that exceeds the normal daily variation and occurs in conjunction with an increase in the hypothalamic set point (e.g., from 37°C to 39°C). This shift of the set point from “normothermic” to febrile levels very much resembles the resetting of the home thermostat to a higher level in order to raise the ambient temperature in a room. Once the hypothalamic set point is raised, neurons in the vasomotor center are activated and vasoconstriction commences. The individual first notices vasoconstriction in the hands and feet. Shunting of blood away from the periphery to the internal organs essentially decreases heat loss from the skin, and the person feels cold. For most fevers, body temperature increases by 1–2°C. Shivering, which increases heat production from the muscles, may begin at this time; however, shivering is not required if heat conservation mechanisms raise blood temperature sufficiently. Nonshivering heat production from the liver also

1	from the muscles, may begin at this time; however, shivering is not required if heat conservation mechanisms raise blood temperature sufficiently. Nonshivering heat production from the liver also contributes to increasing core temperature. Behavioral adjustments (e.g., putting on more clothing or bedding) help raise body temperature by decreasing heat loss.

1	The processes of heat conservation (vasoconstriction) and heat production (shivering and increased nonshivering thermogenesis) continue until the temperature of the blood bathing the hypothalamic neurons matches the new thermostat setting. Once that point is reached, the hypothalamus maintains the temperature at the febrile level by the same mechanisms of heat balance that function in the afebrile state. When the hypothalamic set point is again reset downward (in response to either a reduction in the concentration of pyrogens or the use of antipyretics), the processes of heat loss through vasodilation and sweating are initiated. Loss of heat by sweating and vasodilation continues until the blood temperature at the hypothalamic level matches the lower setting. Behavioral changes (e.g., removal of clothing) facilitate heat loss.

1	A fever of >41.5°C (>106.7°F) is called hyperpyrexia. This extraordinarily high fever can develop in patients with severe infections but most commonly occurs in patients with central nervous system (CNS) hemorrhages. In the preantibiotic era, fever due to a variety of infectious diseases rarely exceeded 106°F, and there has been speculation that this natural “thermal ceiling” is mediated by neuropeptides functioning as central antipyretics. In rare cases, the hypothalamic set point is elevated as a result of local trauma, hemorrhage, tumor, or intrinsic hypothalamic malfunction. The term hypothalamic fever is sometimes used to describe elevated temperature caused by abnormal hypothalamic function. However, most patients with hypothalamic damage have subnormal, not supra-normal, body temperatures. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Although most patients with elevated body temperature have fever, there are circumstances in which elevated temperature represents not fever but hyperthermia (heat stroke). Hyperthermia is characterized by an uncontrolled increase in body temperature that exceeds the body’s ability to lose heat. The setting of the hypothalamic thermoregulatory center is unchanged. In contrast to fever in infections, hyperthermia does not involve pyrogenic molecules. Exogenous heat exposure and endogenous heat production are two mechanisms by which hyperthermia can result in dangerously high internal temperatures. Excessive heat production can easily cause hyperthermia despite physiologic and behavioral control of body temperature. For example, work or exercise in hot environments can produce heat faster than peripheral mechanisms can lose it. For a detailed discussion of hyperthermia, see Chap. 479e.

1	It is important to distinguish between fever and hyperthermia since hyperthermia can be rapidly fatal and characteristically does not respond to antipyretics. In an emergency situation, however, making this distinction can be difficult. For example, in systemic sepsis, fever (hyperpyrexia) can be rapid in onset, and temperatures can exceed 40.5°C (104.9°F). Hyperthermia is often diagnosed on the basis of the events immediately preceding the elevation of core temperature— e.g., heat exposure or treatment with drugs that interfere with thermoregulation. In patients with heat stroke syndromes and in those taking drugs that block sweating, the skin is hot but dry, whereas in fever the skin can be cold as a consequence of vasoconstriction. Antipyretics do not reduce the elevated temperature in hyperthermia, whereas in fever—and even in hyperpyrexia—adequate doses of either aspirin or acetaminophen usually result in some decrease in body temperature.

1	The term pyrogen (Greek pyro, “fire”) is used to describe any substance that causes fever. Exogenous pyrogens are derived from outside the patient; most are microbial products, microbial toxins, or whole microorganisms (including viruses). The classic example of an exogenous pyrogen is the lipopolysaccharide (endotoxin) produced by all gram-negative bacteria. Pyrogenic products of gram-positive organisms include the enterotoxins of Staphylococcus aureus and the groups A and B streptococcal toxins, also called superantigens. One staphylococcal toxin of clinical importance is that associated with isolates of S. aureus from patients with toxic shock syndrome. These products of staphylococci and streptococci cause fever in experimental animals when injected intravenously at concentrations of 1–10 μg/kg. Endotoxin is a highly pyrogenic molecule in humans: when injected intravenously into volunteers, a dose of 2–3 ng/kg produces fever, leukocytosis, acute-phase proteins, and generalized

1	of 1–10 μg/kg. Endotoxin is a highly pyrogenic molecule in humans: when injected intravenously into volunteers, a dose of 2–3 ng/kg produces fever, leukocytosis, acute-phase proteins, and generalized symptoms of malaise.

1	Cytokines are small proteins (molecular mass, 10,000–20,000 Da) that regulate immune, inflammatory, and hematopoietic processes. For example, the elevated leukocytosis seen in several infections with an absolute neutrophilia is attributable to the cytokines interleukin (IL) 1 and IL-6. Some cytokines also cause fever; formerly referred to as endogenous pyrogens, they are now called pyrogenic cytokines. The pyrogenic cytokines include IL-1, IL-6, tumor necrosis factor (TNF), and ciliary neurotropic factor, a member of the IL-6 family. Interferons (IFNs), particularly IFN-α, also are pyrogenic cytokines; fever is a prominent side effect of IFN-α used in the treatment of hepatitis. Each pyrogenic cytokine is encoded by a separate gene, and each has been shown to cause fever in laboratory animals and in humans. When injected into humans at low doses (10–100 ng/kg), IL-1 and TNF produce fever; in contrast, for IL-6, a dose of 1–10 μg/kg is required for fever production.

1	A wide spectrum of bacterial and fungal products induce the synthesis and release of pyrogenic cytokines. However, fever can be a manifestation of disease in the absence of microbial infection. For example, inflammatory processes, trauma, tissue necrosis, and antigen-antibody complexes induce the production of IL-1, TNF, and/or IL-6; individually or in combination, these cytokines trigger the hypothalamus to raise the set point to febrile levels.

1	During fever, levels of prostaglandin E2 (PGE2) are elevated in hypothalamic tissue and the third cerebral ventricle. The concentrations of PGE2 are highest near the circumventricular vascular organs (organum vasculosum of lamina terminalis)—networks of enlarged capillaries surrounding the hypothalamic regulatory centers. Destruction of these organs reduces the ability of pyrogens to produce fever. Most studies in animals have failed to show, however, that pyrogenic cytokines pass from the circulation into the brain itself. Thus, it appears that both exogenous pyrogens and pyrogenic cytokines interact with the endothelium of these capillaries and that this interaction is the first step in initiating fever—i.e., in raising the set point to febrile levels.

1	The key events in the production of fever are illustrated in Fig. 23-1. Myeloid and endothelial cells are the primary cell types that produce pyrogenic cytokines. Pyrogenic cytokines such as IL-1, IL-6, and TNF are released from these cells and enter the systemic circulation. Although these circulating cytokines lead to fever by inducing the synthesis of PGE2, they also induce PGE2 in peripheral tissues. The increase in PGE2 in the periphery accounts for the nonspecific myalgias and arthralgias that often accompany fever. It is thought that some systemic PGE2 escapes destruction by the lung and gains access to the hypothalamus via the internal carotid. However, it is the elevation of PGE2 in the brain that starts the process of raising the hypothalamic set point for core temperature.

1	There are four receptors for PGE2, and each signals the cell in different ways. Of the four receptors, the third (EP-3) is essential for fever: when the gene for this receptor is deleted in mice, no fever follows the injection of IL-1 or endotoxin. Deletion of the other PGE2 receptor genes leaves the fever mechanism intact. Although PGE2 is essential for fever, it is not a neurotransmitter. Rather, the release of PGE2 from the brain side of the hypothalamic endothelium triggers the PGE2 receptor on glial cells, and this stimulation results in the rapid release of cyclic adenosine 5′-monophosphate (cAMP), which is a neurotransmitter. As shown in Fig. 23-1, the release of cAMP from glial cells activates neuronal endings from the thermoregulatory center that extend into the area. The elevation of cAMP is thought to account for changes in the hypothalamic set point either directly or indirectly (by inducing the release of neurotransmitters). Distinct receptors for microbial products are

1	of cAMP is thought to account for changes in the hypothalamic set point either directly or indirectly (by inducing the release of neurotransmitters). Distinct receptors for microbial products are located on the hypothalamic endothelium. These receptors are called Toll-like receptors and are similar in many ways to IL-1 receptors. IL-1 receptors and Toll-like receptors share the same signaltransducing mechanism. Thus, the direct activation of Toll-like receptors or IL-1 receptors results in PGE2 production and fever.

1	Infection, microbial toxins, mediators of inflammation, immune reactions Microbial toxins Fever Monocytes/macrophages, endothelial cells, others Heat conservation, heat production Hypothalamic endotheliumPyrogenic cytokines IL-1, IL-6, TNF, IFN Elevated thermoregulatory set point Circulation PGE2 Cyclic AMP FIguRE 23-1 Chronology of events required for the induction of fever. AMP, adenosine 5′-monophosphate; IFN, interferon; IL, interleukin; PGE2, prostaglandin E2; TNF, tumor necrosis factor.

1	FIguRE 23-1 Chronology of events required for the induction of fever. AMP, adenosine 5′-monophosphate; IFN, interferon; IL, interleukin; PGE2, prostaglandin E2; TNF, tumor necrosis factor. Cytokines produced in the brain may account for the hyperpyrexia of CNS hemorrhage, trauma, or infection. Viral infections of the CNS induce microglial and possibly neuronal production of IL-1, TNF, and IL-6. In experimental animals, the concentration of a cytokine required to cause fever is several orders of magnitude lower with direct injection into the brain substance or brain ventricles than with systemic injection. Therefore, cytokines produced in the CNS can raise the hypothalamic set point, bypassing the circumventricular organs. CNS cytokines likely account for the hyperpyrexia of CNS hemorrhage, trauma, or infection. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: The chronology of events preceding fever, including exposure to other infected individuals or to vectors of disease, should be ascertained. Electronic devices for measuring oral, tympanic membrane, or rectal temperatures are reliable, but the same site should be used consistently to monitor a febrile disease. Moreover, physicians should be aware that newborns, elderly patients, patients with chronic liver or renal failure, and patients taking glucocorticoids or being treated with an anticytokine may have active infection in the absence of fever due to a blunted febrile response. The workup should include a complete blood count; a differential count should be performed manually or with an instrument sensitive to the identification of juvenile or band forms, toxic granulations, and Döhle bodies, which are suggestive of bacterial infection. Neutropenia may be present with some viral infections.

1	Measurement of circulating cytokines in patients with fever is not helpful since levels of cytokines such as IL-1 and TNF in the circulation often are below the detection limit of the assay or do not coincide with fever. However, in patients with low-grade fevers or possible disease, the most valuable measurements are the C-reactive protein level and the erythrocyte sedimentation rate. These markers of inflammatory processes are particularly helpful in detecting occult disease. Measurement of circulating IL-6 is useful because IL-6 induces C-reactive protein. Acute-phase reactants are discussed in Chap. 325.

1	Patients receiving long-term treatment with anticytokine-based regimens are at a disadvantage because of lowered host defense against infection. Even when the results of tests for latent Mycobacterium tuberculosis infection are negative, active tuberculosis can develop in patients receiving anti-TNF therapy. With the increasing use of anticytokines to reduce the activity of IL-1, IL-6, IL-12, or TNF in patients with Crohn’s disease, rheumatoid arthritis, or psoriasis, the possibility that these therapies blunt the febrile response must be kept in mind. The blocking of cytokine activity has the distinct clinical drawback of lowering the level of host defenses against both routine bacterial and opportunistic infections. The opportunistic infections reported in patients treated with agents that neutralize TNF-α are similar to those reported in the HIV-1-infected population (e.g., a new infection with or reactivation of Mycobacterium tuberculosis, with dissemination).

1	In nearly all reported cases of infection associated with anticytokine therapy, fever is among the presenting signs. However, the extent to which the febrile response is blunted in these patients remains unknown. A similar situation is seen in patients receiving high-dose glucocorticoid therapy or anti-inflammatory agents such as ibuprofen. Therefore, low-grade fever is of considerable concern in patients receiving anticytokine therapies. The physician should conduct an early and rigorous diagnostic evaluation in these patients. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Most fevers are associated with self-limited infections, such as common viral diseases. The use of antipyretics is not contraindicated in these infections: no significant clinical evidence indicates either that antipyretics delay the resolution of viral or bacterial infections or that fever facilitates recovery from infection or acts as an adjuvant to the immune system. In short, treatment of fever and its symptoms with routine antipyretics does no harm and does not slow the resolution of common viral and bacterial infections.

1	However, in bacterial infections, the withholding of antipyretic therapy can be helpful in evaluating the effectiveness of a particular antibiotic, especially in the absence of positive cultures of the infecting organism, and the routine use of antipyretics can mask an inadequately treated bacterial infection. Withholding antipyretics in some cases may facilitate the diagnosis of an unusual febrile disease. Temperature-pulse dissociation (relative bradycardia) occurs in typhoid fever, brucellosis, leptospirosis, some drug-induced fevers, and factitious fever. As stated earlier, in newborns, elderly patients, patients with chronic liver or kidney failure, and patients taking glucocorticoids, fever may not be present despite infection. Hypothermia can develop in patients with septic shock.

1	Some infections have characteristic patterns in which febrile episodes are separated by intervals of normal temperature. For example, Plasmodium vivax causes fever every third day, whereas fever occurs every fourth day with P. malariae. Another relapsing fever is related to Borrelia infection, with days of fever followed by a several-day afebrile period and then a relapse into additional days of fever. In the Pel-Ebstein pattern, fever lasting 3–10 days is followed by afebrile periods of 3–10 days; this pattern can be classic for Hodgkin’s disease and other lymphomas. In cyclic neutropenia, fevers occur every 21 days and accompany the neutropenia. There is no periodicity of fever in patients with familial Mediterranean fever. However, these patterns have limited or no diagnostic value compared with specific and rapid laboratory tests.

1	Recurrent fever is documented at some point in most autoimmune diseases and nearly all autoinflammatory diseases. Although fever can be a manifestation of autoimmune diseases, recurrent fevers are characteristic of autoinflammatory diseases (Table 23-1), including adult and juvenile Still’s disease, familial Mediterranean fever, and hyper-IgD syndrome. In addition to recurrent fevers, neutrophilia and serosal inflammation characterize autoinflammatory diseases. The fevers associated with these illnesses are dramatically reduced aPyogenic arthritis, pyoderma gangrenosum, and acne. by blocking of IL-1β activity. Anticytokines therefore reduce fever in autoimmune and autoinflammatory diseases. Although fevers in autoinflammatory diseases are mediated by IL-1β, patients also respond to antipyretics.

1	The reduction of fever by lowering of the elevated hypothalamic set point is a direct function of reduction of the PGE2 level in the thermoregulatory center. The synthesis of PGE2 depends on the constitutively expressed enzyme cyclooxygenase. The substrate for cyclooxygenase is arachidonic acid released from the cell membrane, and this release is the rate-limiting step in the synthesis of PGE2. Therefore, inhibitors of cyclooxygenase are potent antipyretics. The antipyretic potency of various drugs is directly correlated with the inhibition of brain cyclooxygenase. Acetaminophen is a poor cyclooxygenase inhibitor in peripheral tissue and lacks noteworthy anti-inflammatory activity; in the brain, however, acetaminophen is oxidized by the p450 cytochrome system, and the oxidized form inhibits cyclooxygenase activity. Moreover, in the brain, the inhibition of another enzyme, COX-3, by acetaminophen may account for the antipyretic effect of this agent. However, COX-3 is not found outside

1	cyclooxygenase activity. Moreover, in the brain, the inhibition of another enzyme, COX-3, by acetaminophen may account for the antipyretic effect of this agent. However, COX-3 is not found outside the CNS.

1	Oral aspirin and acetaminophen are equally effective in reducing fever in humans. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen and specific inhibitors of COX-2 also are excellent antipyretics. Chronic, high-dose therapy with antipyretics such as aspirin or any NSAID does not reduce normal core body temperature. Thus, PGE2 appears to play no role in normal thermoregulation. As effective antipyretics, glucocorticoids act at two levels. First, similar to the cyclooxygenase inhibitors, glucocorticoids reduce PGE2 synthesis by inhibiting the activity of phospholipase A2, which is needed to release arachidonic acid from the cell membrane. Second, glucocorticoids block the transcription of the mRNA for the pyrogenic cytokines. Limited experimental evidence indicates that ibuprofen and COX-2 inhibitors reduce IL-1induced IL-6 production and may contribute to the antipyretic activity of NSAIDs.

1	The objectives in treating fever are first to reduce the elevated hypothalamic set point and second to facilitate heat loss. Reducing fever with antipyretics also reduces systemic symptoms of headache, myalgias, and arthralgias. Oral aspirin and NSAIDs effectively reduce fever but can adversely affect platelets and the gastrointestinal tract. Therefore, acetaminophen is preferred as an antipyretic. In children, acetaminophen or oral ibuprofen must be used because aspirin increases the risk of Reye’s syndrome. If the patient cannot take oral antipyretics, parenteral preparations of NSAIDs and rectal suppositories of various antipyretics can be used.

1	Treatment of fever in some patients is highly recommended. Fever increases the demand for oxygen (i.e., for every increase of 1°C over 37°C, there is a 13% increase in oxygen consumption) and can aggravate the condition of patients with preexisting impairment of cardiac, pulmonary, or CNS function. Children with a history of febrile or nonfebrile seizure should be aggressively treated to reduce fever. However, it is unclear what triggers the febrile seizure, and there is no correlation between absolute temperature elevation and onset of a febrile seizure in susceptible children. In hyperpyrexia, the use of cooling blankets facilitates the reduction of temperature; however, cooling blankets should not be used without oral antipyretics. In hyperpyretic patients with CNS disease or trauma (CNS bleeding), reducing core temperature mitigates the detrimental effects of high temperature on the brain. For a discussion of treatment for hyperthermia, see Chap. 479e.

1	For a discussion of treatment for hyperthermia, see Chap. 479e. Diseases with fever and rash may be classified by type of erup-127 fever and Rash tion: centrally distributed maculopapular, peripheral, confluent desquamative erythematous, vesiculobullous, urticaria-like, nodular, Elaine T. Kaye, Kenneth M. Kaye purpuric, ulcerated, or with eschars. Diseases are listed by these cat- The acutely ill patient with fever and rash may present a diagnostic challenge for physicians. However, the distinctive appearance of an eruption in concert with a clinical syndrome can facilitate a prompt diagnosis and the institution of life-saving therapy or critical infection-control interventions. Representative images of many of the rashes discussed in this chapter are included in Chap. 25e. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: A thorough history of patients with fever and rash includes the following relevant information: immune status, medications taken within the previous month, specific travel history, immunization status, exposure to domestic pets and other animals, history of animal (including arthropod) bites, recent dietary exposures, existence of cardiac abnormalities, presence of prosthetic material, recent exposure to ill individuals, and exposure to sexually transmitted diseases. The history should also include the site of onset of the rash and its direction and rate of spread.

1	A thorough physical examination entails close attention to the rash, with an assessment and precise definition of its salient features. First, it is critical to determine what type of lesions make up the eruption. Macules are flat lesions defined by an area of changed color (i.e., a blanchable erythema). Papules are raised, solid lesions <5 mm in diameter; plaques are lesions >5 mm in diameter with a flat, plateau-like surface; and nodules are lesions >5 mm in diameter with a more rounded configuration. Wheals (urticaria, hives) are papules or plaques that are pale pink and may appear annular (ringlike) as they enlarge; classic (nonvasculitic) wheals are transient, lasting only 24 h in any defined area. Vesicles (<5 mm) and bullae (>5 mm) are circumscribed, elevated lesions containing fluid. Pustules are raised lesions containing purulent exudate; vesicular processes such as varicella or herpes simplex may evolve to pustules. Nonpalpable purpura is a flat lesion that is due to

1	fluid. Pustules are raised lesions containing purulent exudate; vesicular processes such as varicella or herpes simplex may evolve to pustules. Nonpalpable purpura is a flat lesion that is due to bleeding into the skin. If <3 mm in diameter, the purpuric lesions are termed petechiae; if >3 mm, they are termed ecchymoses. Palpable purpura is a raised lesion that is due to inflammation of the vessel wall (vasculitis) with subsequent hemorrhage. An ulcer is a defect in the skin extending at least into the upper layer of the dermis, and an eschar (tâche noire) is a necrotic lesion covered with a black crust.

1	Other pertinent features of rashes include their configuration (i.e., annular or target), the arrangement of their lesions, and their distribution (i.e., central or peripheral). For further discussion, see Chaps. 70, 72, and 147.

1	For further discussion, see Chaps. 70, 72, and 147. This chapter reviews rashes that reflect systemic disease, but it does not include localized skin eruptions (i.e., cellulitis, impetigo) that may also be associated with fever (Chap. 156). The chapter is not intended to be all-inclusive, but it covers the most important and most common diseases associated with fever and rash. Rashes are classified herein on the basis of lesion morphology and distribution. For practical purposes, this classification system is based on the most typical disease presentations. However, morphology may vary as rashes evolve, and the presentation of diseases with rashes is subject to many variations (Chap. 72). For instance, the classic petechial rash of Rocky Mountain spotted fever (Chap. 211) may initially consist of blanchable erythematous macules distributed peripherally; at times, however, the rash associated with this disease may not be predominantly acral, or no rash may develop at all.

1	egories in Table 24-1, and many are highlighted in the text. However, for a more detailed discussion of each disease associated with a rash, the reader is referred to the chapter dealing with that specific disease. (Reference chapters are cited in the text and listed in Table 24-1.)

1	(Reference chapters are cited in the text and listed in Table 24-1.) Centrally distributed rashes, in which lesions are primarily truncal, are the most common type of eruption. The rash of rubeola (measles) starts at the hairline 2–3 days into the illness and moves down the body, typically sparing the palms and soles (Chap. 229). It begins as discrete erythematous lesions, which become confluent as the rash spreads. Koplik’s spots (1to 2-mm white or bluish lesions with an erythematous halo on the buccal mucosa) are pathognomonic for measles and are generally seen during the first 2 days of symptoms. They should not be confused with Fordyce’s spots (ectopic sebaceous glands), which have no erythematous halos and are found in the mouth of healthy individuals. Koplik’s spots may briefly overlap with the measles exanthem.

1	Rubella (German measles) also spreads from the hairline downward; unlike that of measles, however, the rash of rubella tends to clear from originally affected areas as it migrates, and it may be pruritic (Chap. 230e). Forchheimer spots (palatal petechiae) may develop but are nonspecific because they also develop in infectious mononucleosis (Chap. 218) and scarlet fever (Chap. 173). Postauricular and suboccipital adenopathy and arthritis are common among adults with rubella. Exposure of pregnant women to ill individuals should be avoided, as rubella causes severe congenital abnormalities. Numerous strains of enteroviruses (Chap. 228), primarily echoviruses and coxsackieviruses, cause nonspecific syndromes of fever and eruptions that may mimic rubella or measles. Patients with infectious mononucleosis caused by Epstein-Barr virus (Chap. 218) or with primary HIV infection (Chap. 226) may exhibit pharyngitis, lymphadenopathy, and a nonspecific maculopapular exanthem.

1	226) may exhibit pharyngitis, lymphadenopathy, and a nonspecific maculopapular exanthem. The rash of erythema infectiosum (fifth disease), which is caused by human parvovirus B19, primarily affects children 3–12 years old; it develops after fever has resolved as a bright blanchable erythema on the cheeks (“slapped cheeks”) with perioral pallor (Chap. 221). A more diffuse rash (often pruritic) appears the next day on the trunk and extremities and then rapidly develops into a lacy reticular eruption that may wax and wane (especially with temperature change) over 3 weeks. Adults with fifth disease often have arthritis, and fetal hydrops can develop in association with this condition in pregnant women.

1	Exanthem subitum (roseola) is caused by human herpesvirus 6 and is most common among children <3 years of age (Chap. 219). As in erythema infectiosum, the rash usually appears after fever has subsided. It consists of 2to 3-mm rose-pink macules and papules that coalesce only rarely, occur initially on the trunk and sometimes on the extremities (sparing the face), and fade within 2 days.

1	Although drug reactions have many manifestations, including urticaria, exanthematous drug-induced eruptions (Chap. 74) are most common and are often difficult to distinguish from viral exanthems. Eruptions elicited by drugs are usually more intensely erythematous and pruritic than viral exanthems, but this distinction is not reliable. A history of new medications and an absence of prostration may help to distinguish a drug-related rash from an eruption of another etiology. Rashes may persist for up to two weeks after administration of the offending agent is discontinued. Certain populations are more prone than others to drug rashes. Of HIV-infected patients, 50–60% develop a rash in response to sulfa drugs; 90% of patients with mononucleosis due to Epstein-Barr virus develop a rash when given ampicillin.

1	Rickettsial illnesses (Chap. 211) should be considered in the evaluation of individuals with centrally distributed maculopapular eruptions. The usual setting for epidemic typhus is a site of war or natural disaster in which people are exposed to body lice. Endemic typhus or leptospirosis (the latter caused by a spirochete) (Chap. 208) may be CHAPTER 24 Fever and Rash PART 2 Cardinal Manifestations and Presentation of Diseases Rash beginning on wrists and ankles and spreading centripetally; appears on palms and soles later in disease; lesion evolution from blanchable macules to petechiae Coincident primary chancre in 10% of cases; copper-colored, scaly papular eruption, diffuse but prominent on palms and soles; rash never vesicular in adults; condyloma latum, mucous patches, and alopecia in some cases Maculopapular eruption; prominent on upper extremities and face, but can also occur on trunk and lower extremities

1	Maculopapular eruption; prominent on upper extremities and face, but can also occur on trunk and lower extremities Tender vesicles, erosions in mouth; 0.25-cm papules on hands and feet with rim of erythema evolving into tender vesicles Target lesions (central erythema surrounded by area of clearing and another rim of erythema) up to 2 cm; symmetric on knees, elbows, palms, soles; spreads centripetally; papular, sometimes vesicular; when extensive and involving mucous membranes, termed EM major Maculopapular eruption over palms, soles, and extremities; tends to be more severe at joints; eruption sometimes becoming generalized; may be purpuric; may desquamate Subacute course: Osler’s nodes (tender pink nodules on finger or toe pads); petechiae on skin and mucosa; splinter hemorrhages. Acute course (e.g., Staphylococcus aureus): Janeway lesions (painless erythematous or hemorrhagic macules, usually on palms and soles) —

1	Tick vector; widespread but more common in southeastern and southwest-central U.S. Aedes aegypti and A. albopictus mosquito bites; primarily in Africa and Indian Ocean region infection; drug intake (i.e., sulfa, phenytoin, penicillin) Rat bite, ingestion of contaminated food Abnormal heart valve (e.g., viridans group streptococci), intravenous drug use Headache, myalgias, abdominal pain; mortality rates up to 40% if untreated Fever, constitutional symptoms Severe polyarticular, migratory arthralgias, especially involving small joints (e.g., hands, wrists, ankles) 50% of patients <20 years old; fever more common in most severe form, EM major, which can be confused with Stevens-Johnson syndrome (but EM major lacks prominent skin sloughing) 180, 181, 221 Chronic meningococcemia, disseminated gonococcal infection,a human parvovirus B19 infectione Infection, drugs, idiopathic causes Streptococcus, Staphylococcus, etc.

1	Chronic meningococcemia, disseminated gonococcal infection,a human parvovirus B19 infectione Infection, drugs, idiopathic causes Streptococcus, Staphylococcus, etc. PART 2 Cardinal Manifestations and Presentation of Diseases disease) Streptococcus (pyrogenic exotoxins A, B, C) shock syndrome Streptococcus (associated with pyrogenic exotoxin A and/or B or certain M types) Staphylococcal toxic S. aureus (toxic shock shock syndrome syndrome toxin 1, enterotoxins B and others) Diffuse blanchable erythema beginning on face and spreading to trunk and extremities; circumoral pallor; “sandpaper” texture to skin; accentuation of linear erythema in skin folds (Pastia’s lines); enanthem of white evolving into red “strawberry” tongue; desquamation in second week Rash similar to scarlet fever (scarlatiniform) or EM; fissuring of lips, strawberry tongue; conjunctivitis; edema of hands, feet; desquamation later in disease When present, rash often scarlatiniform

1	When present, rash often scarlatiniform Diffuse erythema involving palms; pronounced erythema of mucosal surfaces; conjunctivitis; desquamation 7–10 days into illness Most common among children 2–10 years old; usually follows group A streptococcal pharyngitis May occur in setting of severe group A streptococcal infections (e.g., necrotizing fasciitis, bacteremia, pneumonia) Colonization with toxin-producing S. aureus Fever, pharyngitis, headache Cervical adenopathy, pharyngitis, coronary artery vasculitis Multiorgan failure, hypo-tension; mortality rate 30% Fever >39°C (>102°F), hypotension, multiorgan dysfunction 72, 385 Staphylococcal S. aureus, phage scalded-skin syndrome group II Diffuse tender erythema, often with bullae and desquamation; Nikolsky’s sign Diffuse erythema (often scaling) interspersed with lesions of underlying condition

1	Diffuse tender erythema, often with bullae and desquamation; Nikolsky’s sign Diffuse erythema (often scaling) interspersed with lesions of underlying condition Maculopapular eruption (mimicking exanthematous drug rash), sometimes progressing to exfoliative erythroderma; profound edema, especially facial; pustules may occur Erythematous and purpuric macules, sometimes targetoid, or diffuse erythema progressing to bullae, with sloughing and necrosis of entire epidermis; Nikolsky’s sign; involves mucosal surfaces; TEN (>30% epidermal necrosis) is maximal form; SJS involves <10% of epidermis; SJS/TEN overlap involves 10–30% of epidermis Colonization with toxin-producing S. aureus; occurs in children <10 years old (termed Ritter’s disease in neonates) or adults with renal dysfunction Individuals genetically unable to detoxify arene oxides (anticonvulsants), patients with slow Nacetylating capacity (sulfonamides)

1	Individuals genetically unable to detoxify arene oxides (anticonvulsants), patients with slow Nacetylating capacity (sulfonamides) Uncommon among children; more common among patients with HIV infection, SLE, certain HLA types, or slow acetylators Irritability; nasal or con-172 junctival secretions Macules (2–3 mm) evolving into papules, then vesicles (sometimes umbilicated), on an erythematous base (“dewdrops on a rose petal”); pustules then forming and crusting; lesions appearing in crops; may involve scalp, mouth; intensely pruritic Pruritic erythematous follicular, papular, vesicular, or pustular lesions that may involve axillae, buttocks, abdomen, and especially areas occluded by bathing suits; can manifest as tender isolated nodules on palmar or plantar surfaces (the latter designated “Pseudomonas hot-foot syndrome”)

1	Red macules on tongue and palate evolving to papules and vesicles; skin macules evolving to papules, then vesicles, then pustules over 1 week, with subsequent lesion crusting; lesions initially appearing on face and spreading centrifugally from trunk to extremities; differs from varicella in that (1) skin lesions in any given area are at same stage of development and (2) there is a prominent distribution of lesions on face and extremities (including palms, soles) Erythema rapidly followed by hallmark painful grouped vesicles that may evolve into pustules that ulcerate, especially on mucosal surfaces; lesions at site of inoculation: commonly gingivostomatitis for HSV-1 and genital lesions for HSV-2; recurrent disease milder (e.g., herpes labialis does not involve oral mucosa) — Usually affects children; 10% of adults susceptible; most common in late winter and spring; incidence down by 90% in U.S. as a result of varicella vaccination Nonimmune individuals exposed to smallpox

1	Nonimmune individuals exposed to smallpox Malaise; generally mild disease in healthy children; more severe disease with complications in adults and immunocompromised children Earache, sore eyes and/ or throat; fever may be absent; generally self-limited Prodrome of fever, headache, backache, myalgias; vomiting in 50% of cases PART 2 Cardinal Manifestations and Presentation of Diseases Disseminated Vibrio V. vulnificus vulnificus infection Ecthyma gangrenosum P. aeruginosa, other gram-negative rods, fungi

1	PART 2 Cardinal Manifestations and Presentation of Diseases Disseminated Vibrio V. vulnificus vulnificus infection Ecthyma gangrenosum P. aeruginosa, other gram-negative rods, fungi Generalized vesicles that can evolve to pustules and ulcerations; individual lesions similar for VZV and HSV. Zoster cutaneous dissemination: >25 lesions extending outside involved dermatome. HSV: extensive, progressive mucocutaneous lesions that may occur in absence of dissemination, sometimes disseminate in eczematous skin (eczema herpeticum); HSV visceral dissemination may occur with only localized mucocutaneous disease; in disseminated neonatal disease, skin lesions diagnostically helpful when present, but rash absent in a substantial minority of cases Eschar found at site of mite bite; generalized rash involving face, trunk, extremities; may involve palms and soles; <100 papules and plaques (2–10 mm); tops of lesions developing vesicles that may evolve into pustules

1	Tiny sterile nonfollicular pustules on erythematous, edematous skin; begins on face and in body folds, then becomes generalized Indurated plaque evolving into hemorrhagic bulla or pustule that sloughs, resulting in eschar formation; erythematous halo; most common in axillary, groin, perianal regions Patients with immunosuppression, eczema; neonates Appears 2–21 days after start of drug therapy, depending on whether patient has been sensitized Patients with cirrhosis, diabetes, renal failure; exposure by ingestion of contaminated saltwater, seafood Usually affects neutropenic patients; occurs in up to 28% of individuals with Pseudomonas bacteremia Visceral organ involvement (e.g., liver, lungs) in some cases; neonatal disease particularly severe Headache, myalgias, regional adenopathy; mild disease Acute fever, pruritus, leukocytosis Clinical signs of sepsis 164, 216,

1	Headache, myalgias, regional adenopathy; mild disease Acute fever, pruritus, leukocytosis Clinical signs of sepsis 164, 216, Urticarial vasculitis Serum sickness, Erythematous, edematous “urticaria-like” Patients with serum sick-Fever variable; arthralgias/ 385f often due to infec-plaques, pruritic or burning; unlike urticaria: ness (including hepatitis arthritis tion (including typical lesion duration >24 h (up to 5 days) B), connective tissue hepatitis B viral, and lack of complete lesion blanching with disease enteroviral, parasitic), compression due to hemorrhage drugs; connective tissue disease Disseminated infection Fungal infections (e.g., candidiasis, histoplasmosis, cryptococcosis, sporotrichosis, coccidioidomycosis); mycobacteria

1	Disseminated infection Fungal infections (e.g., candidiasis, histoplasmosis, cryptococcosis, sporotrichosis, coccidioidomycosis); mycobacteria Erythema nodosum Infections (e.g., (septal panniculitis) streptococcal, fungal, mycobacterial, yersinial); drugs (e.g., sulfas, penicillins, oral contraceptives); sarcoidosis; idiopathic causes dermatosis) inflammatory bowel disease; pregnancy; malignancy (usually hematologic); drugs (G-CSF) Subcutaneous nodules (up to 3 cm); fluctuance, draining common with mycobacteria; necrotic nodules (extremities, periorbital or nasal regions) common with Aspergillus, Mucor Large, violaceous, nonulcerative, subcutaneous nodules; exquisitely tender; usually on lower legs but also on upper extremities

1	Large, violaceous, nonulcerative, subcutaneous nodules; exquisitely tender; usually on lower legs but also on upper extremities Tender red or blue edematous nodules giving impression of vesiculation; usually on face, neck, upper extremities; when on lower extremities, may mimic erythema nodosum Immunocompromised Features vary with —f hosts (i.e., bone marrow organism transplant recipients, patients undergoing chemotherapy, HIV-infected patients, alcoholics) More common among Arthralgias (50%); features —f girls and women 15–30 vary with associated years old condition

1	More common among Arthralgias (50%); features —f girls and women 15–30 vary with associated years old condition PART 2 Cardinal Manifestations and Presentation of Diseases aSee “Purpuric Eruptions.” bSee “Confluent Desquamative Erythemas.” cIn human granulocytotropic ehrlichiosis or anaplasmosis (caused by Anaplasma phagocytophila; most common in the upper midwestern and northeastern United States), rash is rare. dSee “Viral hemorrhagic fever” under “Purpuric Eruptions” for dengue hemorrhagic fever/dengue shock syndrome. eSee “Centrally Distributed Maculopapular Eruptions.” fSee etiology-specific chapters. gSee “Peripheral Eruptions.” hSee “Vesiculobullous or Pustular Eruptions.” Abbreviations: CNS, central nervous system; DIC, disseminated intravascular coagulation; G-CSF, granulocyte colony-stimulating factor; HLA, human leukocyte antigen.

1	Abbreviations: CNS, central nervous system; DIC, disseminated intravascular coagulation; G-CSF, granulocyte colony-stimulating factor; HLA, human leukocyte antigen. seen in urban environments where rodents proliferate. Outside the United States, other rickettsial diseases cause a spotted-fever syndrome and should be considered in residents of or travelers to endemic areas. Similarly, typhoid fever, a nonrickettsial disease caused by Salmonella typhi (Chap. 190), is usually acquired during travel outside the United States. Dengue fever, caused by a mosquito-transmitted flavivirus, occurs in tropical and subtropical regions of the world (Chap. 233).

1	Some centrally distributed maculopapular eruptions have distinctive features. Erythema migrans, the rash of Lyme disease (Chap. 210), typically manifests as single or multiple annular plaques. Untreated erythema migrans lesions usually fade within a month but may persist for more than a year. Southern tick-associated rash illness (STARI) (Chap. 210) has an erythema migrans–like rash but is less severe than Lyme disease and often occurs in regions where Lyme is not endemic. Erythema marginatum, the rash of acute rheumatic fever (Chap. 381), has a distinctive pattern of enlarging and shifting transient annular lesions.

1	Collagen vascular diseases may cause fever and rash. Patients with systemic lupus erythematosus (Chap. 378) typically develop a sharply defined, erythematous eruption in a butterfly distribution on the cheeks (malar rash) as well as many other skin manifestations. Still’s disease (Chap. 398) presents as an evanescent, salmon-colored rash on the trunk and proximal extremities that coincides with fever spikes.

1	These rashes are alike in that they are most prominent peripherally or begin in peripheral (acral) areas before spreading centripetally. Early diagnosis and therapy are critical in Rocky Mountain spotted fever (Chap. 211) because of its grave prognosis if untreated. Lesions evolve from macular to petechial, start on the wrists and ankles, spread centripetally, and appear on the palms and soles only later in the disease. The rash of secondary syphilis (Chap. 206), which may be generalized but is prominent on the palms and soles, should be considered in the differential diagnosis of pityriasis rosea, especially in sexually active patients. Chikungunya fever (Chap. 233), which is transmitted by mosquito bite in Africa and the Indian Ocean region, is associated with a maculopapular eruption and severe polyarticular small-joint arthralgias. Hand-foot-and-mouth disease (Chap. 228), most commonly caused by coxsackievirus A16, is distinguished by tender vesicles distributed peripherally and

1	and severe polyarticular small-joint arthralgias. Hand-foot-and-mouth disease (Chap. 228), most commonly caused by coxsackievirus A16, is distinguished by tender vesicles distributed peripherally and in the mouth; outbreaks commonly occur within families. The classic target lesions of erythema multiforme appear symmetrically on the elbows, knees, palms, soles, and face. In severe cases, these lesions spread diffusely and involve mucosal surfaces. Lesions may develop on the hands and feet in endocarditis (Chap. 155).

1	These eruptions consist of diffuse erythema frequently followed by desquamation. The eruptions caused by group A Streptococcus or Staphylococcus aureus are toxin-mediated. Scarlet fever (Chap. 173) usually follows pharyngitis; patients have a facial flush, a “strawberry” tongue, and accentuated petechiae in body folds (Pastia’s lines). Kawasaki disease (Chaps. 72 and 385) presents in the pediatric population as fissuring of the lips, a strawberry tongue, conjunctivitis, adenopathy, and sometimes cardiac abnormalities. Streptococcal toxic shock syndrome (Chap. 173) manifests with hypotension, multiorgan failure, and, often, a severe group A streptococcal infection (e.g., necrotizing fasciitis). Staphylococcal toxic shock syndrome (Chap.

1	172) also presents with hypotension and multiorgan failure, but usually only S. aureus colonization—not a severe S. aureus infection—is documented. Staphylococcal scalded-skin syndrome (Chap. 172) is seen primarily in children and in immunocompromised adults. Generalized erythema is often evident during the prodrome of fever and malaise; profound tenderness of the skin is distinctive. In the exfoliative stage, the skin can be induced to form bullae with light lateral pressure (Nikolsky’s sign). In a mild form, a scarlatiniform eruption mimics scarlet fever, but the patient does not exhibit a strawberry tongue or circumoral pallor. In contrast to the staphylococcal scalded-skin syndrome, in which the cleavage plane is superficial in the epidermis, toxic epidermal necrolysis (Chap. 74), a maximal variant of Stevens-Johnson syndrome, involves sloughing of the entire epidermis, resulting in severe disease. Exfoliative erythroderma syndrome (Chaps. 72 and 74) is a serious reaction

1	74), a maximal variant of Stevens-Johnson syndrome, involves sloughing of the entire epidermis, resulting in severe disease. Exfoliative erythroderma syndrome (Chaps. 72 and 74) is a serious reaction associated with systemic toxicity that is often due to eczema, psoriasis, a drug reaction, or mycosis fungoides. Drug rash with eosinophilia and systemic symptoms (DRESS), often due to antiepileptic and antibiotic agents (Chap. 74), initially appears similar to an exanthematous drug reaction but may progress to exfoliative erythroderma; it is accompanied by multi-organ failure and has an associated mortality rate of ~10%.

1	Varicella (Chap. 217) is highly contagious, often occurring in winter or spring. At any point in time, within a given region of the body, varicella lesions are in different stages of development. In immunocompromised hosts, varicella vesicles may lack the characteristic erythematous base or may appear hemorrhagic. Lesions of Pseudomonas “hot-tub” folliculitis (Chap. 189) are also pruritic and may appear similar to those of varicella. However, hot-tub folliculitis generally occurs in outbreaks after bathing in hot tubs or swimming pools, and lesions occur in regions occluded by bathing suits. Lesions of variola (smallpox) (Chap. 261e) also appear similar to those of varicella but are all at the same stage of development in a given region of the body. Variola lesions are most prominent on the face and extremities, while varicella lesions are most prominent on the trunk. Herpes simplex virus infection (Chap. 216) is characterized by hallmark grouped vesicles on an erythematous base.

1	on the face and extremities, while varicella lesions are most prominent on the trunk. Herpes simplex virus infection (Chap. 216) is characterized by hallmark grouped vesicles on an erythematous base. Primary herpes infection is accompanied by fever and toxicity, while recurrent disease is milder. Rickettsialpox (Chap. 211) is often documented in urban settings and is characterized by vesicles followed by pustules. It can be distinguished from varicella by an eschar at the site of the mouse-mite bite and the papule/plaque base of each vesicle. Acute generalized eruptive pustulosis should be considered in individuals who are acutely febrile and are taking new medications, especially anticonvulsant or antimicrobial agents (Chap. 74). Disseminated Vibrio vulnificus infection (Chap. 193) or ecthyma gangrenosum due to Pseudomonas aeruginosa (Chap. 189) should be considered in immunosuppressed individuals with sepsis and hemorrhagic bullae.

1	Individuals with classic urticaria (“hives”) usually have a hypersensitivity reaction without associated fever. In the presence of fever, urticaria-like eruptions are most often due to urticarial vasculitis (Chap. 385). Unlike individual lesions of classic urticaria, which last up to 24 h, these lesions may last 3–5 days. Etiologies include serum sickness (often induced by drugs such as penicillins, sulfas, salicylates, or barbiturates), connective-tissue disease (e.g., systemic lupus erythematosus or Sjren’s syndrome), and infection (e.g., with hepatitis B virus, enteroviruses, or parasites). Malignancy, especially lymphoma, may be associated with fever and chronic urticaria (Chap. 72).

1	In immunocompromised hosts, nodular lesions often represent disseminated infection. Patients with disseminated candidiasis (often due to Candida tropicalis) may have a triad of fever, myalgias, and eruptive nodules (Chap. 240). Disseminated cryptococcosis lesions (Chap. 239) may resemble molluscum contagiosum (Chap. 220e). Necrosis of nodules should raise the suspicion of aspergillosis (Chap. 241) or mucormycosis (Chap. 242). Erythema nodosum presents with exquisitely tender nodules on the lower extremities. Sweet syndrome (Chap. 72) should be considered in individuals with multiple nodules and plaques, often so edematous that they give the appearance of vesicles or bullae. Sweet syndrome may occur in individuals with infection, inflammatory bowel disease, or malignancy and can also be induced by drugs.

1	Acute meningococcemia (Chap. 180) classically presents in children as a petechial eruption, but initial lesions may appear as blanch-able macules or urticaria. Rocky Mountain spotted fever should be considered in the differential diagnosis of acute meningococcemia. Echovirus infection (Chap. 228) may mimic acute meningococcemia; patients should be treated as if they have bacterial sepsis because prompt differentiation of these conditions may be impossible. Large ecchymotic areas of purpura fulminans (Chaps. 180 and 325) reflect severe underlying disseminated intravascular coagulation, which may be due to infectious or noninfectious causes. The lesions of chronic meningococcemia (Chap. 180) may have a variety of morphologies, including petechial. Purpuric nodules may develop on the legs and resemble erythema nodosum but lack its exquisite tenderness. Lesions of disseminated gonococcemia (Chap. 181) are distinctive, sparse, countable hemorrhagic pustules, usually located near joints.

1	and resemble erythema nodosum but lack its exquisite tenderness. Lesions of disseminated gonococcemia (Chap. 181) are distinctive, sparse, countable hemorrhagic pustules, usually located near joints. The lesions of chronic meningococcemia and those of gonococcemia may be indistinguishable in terms of appearance and distribution. Viral hemorrhagic fever (Chaps. 233 and 234) should be considered in patients with an appropriate travel history and a petechial rash. Thrombotic thrombocytopenic purpura (Chaps. 72, 129, and 140) and hemolytic-uremic syndrome (Chaps. 140, 186, and 191) are closely related and are noninfectious causes of fever and petechiae. Cutaneous small-vessel vasculitis (leukocytoclastic vasculitis) typically manifests as palpable purpura and has a wide variety of causes (Chap. 72).

1	The presence of an ulcer or eschar in the setting of a more widespread eruption can provide an important diagnostic clue. For example, the presence of an eschar may suggest the diagnosis of scrub typhus or rickettsialpox (Chap. 211) in the appropriate setting. In other illnesses (e.g., anthrax) (Chap. 261e), an ulcer or eschar may be the only skin manifestation. ChaPter 26 Fever of Unknown Origin Atlas of Rashes Associated with Fever Kenneth M. Kaye, Elaine T. Kaye Given the extremely broad differential diagnosis, the presentation of a patient with fever and rash often poses a thorny diagnostic challenge 25e for even the most astute and experienced clinician. Rapid narrowing of the differential by prompt recognition of a rash’s key features can result in appropriate and sometimes life-saving therapy. This atlas presents high-quality images of a variety of rashes that have an infectious etiology and are commonly associated with fever.

1	CHAPTER 25e Atlas of Rashes Associated with Fever Figure 25e-2 Koplik’s spots, which manifest as white or bluish lesions with an erythematous halo on the buccal mucosa, usually occur in the first 2 days of measles symptoms and may briefly overlap the measles exanthem. The presence of the erythematous halo (arrow indicates one example) differentiates Koplik’s spots from Fordyce’s spots (ectopic sebaceous glands), which occur in the mouths of healthy individuals. (Courtesy of the Centers for Disease Control and Prevention.) Figure 25e-3 In measles, discrete erythematous lesions become confluent on the face and neck over 2–3 days as the rash spreads downward to the trunk and arms, where lesions remain discrete. (Reprinted from K Wolff, RA Johnson: Fitzpatrick’s Color Atlas and Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005.)

1	(Reprinted from K Wolff, RA Johnson: Fitzpatrick’s Color Atlas and Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005.) Figure 25e-1 A. Erythema leading to “slapped cheeks” appearance in erythema infectiosum (fifth disease) caused by parvovirus B19. B. Lacy reticular rash of erythema infectiosum. (Panel A reprinted from K Wolff, RA Johnson: Fitzpatrick’s Color Atlas and Synopsis of Clinical Dermatology, 6th ed. New York, McGraw-Hill, 2009.) PART 2 Cardinal Manifestations and Presentation of Diseases Figure 25e-4 In rubella, an erythematous exanthem spreads from the hairline downward and clears as it spreads. (Courtesy of Stephen E. Gellis, MD; with permission.)

1	Figure 25e-4 In rubella, an erythematous exanthem spreads from the hairline downward and clears as it spreads. (Courtesy of Stephen E. Gellis, MD; with permission.) Figure 25e-7 This exanthematous, drug-induced eruption con-sists of brightly erythematous macules and papules, some of which are confluent, distributed symmetrically on the trunk and extremities. Ampicillin caused this rash. (Reprinted from K Wolff, RA Johnson: Color Atlas and Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005.) Figure 25e-5 Exanthem subitum (roseola) occurs most commonly in young children. A diffuse maculopapular exanthem follows resolu-tion of fever. (Courtesy of Stephen E. Gellis, MD; with permission.) Figure 25e-6 Erythematous macules and papules are apparent on the trunk and arm of this patient with primary HIV infection. (Reprinted from K Wolff, RA Johnson: Color Atlas and Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005.)

1	(Reprinted from K Wolff, RA Johnson: Color Atlas and Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005.) Figure 25e-8 Erythema migrans is the early cutaneous manifestation of Lyme disease and is characterized by erythematous annular patches, often with a central erythematous focus at the tick-bite site. (Reprinted from RP Usatine et al: Color Atlas of Family Medicine, 2nd ed. New York, McGraw-Hill, 2013. Courtesy of Thomas Corson, MD.) Figure 25e-9 Rose spots are evident as erythematous macules on the trunk of this patient with typhoid fever. (Courtesy of the Centers for Disease Control and Prevention.) Figure 25e-10 Systemic lupus erythematosus showing prominent malar erythema and minimal scaling. Involvement of other sun-exposed sites is also common. (Reprinted from K Wolff, RA Johnson: Fitzpatrick’s Color Atlas and Synopsis of Clinical Dermatology, 6th ed. New York, McGraw-Hill, 2009.)

1	Figure 25e-11 Subacute lupus erythematosus on the upper chest, with brightly erythematous and slightly edematous coalescent pap-ules and plaques. (Reprinted from K Wolff, RA Johnson: Fitzpatrick’s Color Atlas and Synopsis of Clinical Dermatology, 6th ed. New York, McGraw-Hill, 2009.) Figure 25e-12 Chronic discoid lupus erythematosus. Violaceous, hyperpigmented, atrophic plaques, often with evidence of follicular plugging (which may result in scarring), are characteristic of this cuta-neous form of lupus. (Reprinted from K Wolff, RA Johnson, AP Saavedra: Fitzpatrick’s Color Atlas and Synopsis of Clinical Dermatology, 7th ed. New York, McGraw-Hill, 2013.) Figure 25e-13 The rash of Still’s disease typically exhibits evanes-cent, erythematous papules that appear at the height of fever on the trunk and proximal extremities. (Courtesy of Stephen E. Gellis, MD; with permission.) CHAPTER 25e Atlas of Rashes Associated with Fever

1	CHAPTER 25e Atlas of Rashes Associated with Fever Figure 25e-14 Impetigo is a superficial group A streptococcal or Staphylococcus aureus infection consisting of honey-colored crusts and erythematous weeping erosions. (Reprinted from K Wolff, RA Johnson: Fitzpatrick’s Color Atlas and Synopsis of Clinical Dermatology, 6th ed. New York, McGraw-Hill, 2009.) PART 2 Cardinal Manifestations and Presentation of Diseases Figure 25e-15 Erysipelas is a group A streptococcal infection of the superficial dermis and consists of well-demarcated, erythematous, edematous, warm plaques. (Reprinted from K Wolff, RA Johnson, AP Saavedra: Fitzpatrick’s Color Atlas and Synopsis of Clinical Dermatology, 7th ed. New York, McGraw-Hill, 2013.) Figure 25e-16 Top: Petechial lesions of Rocky Mountain spotted fever on the lower legs and soles of a young, otherwise healthy patient. Bottom: Close-up of lesions from the same patient. (Courtesy of Lindsey Baden, MD; with permission.)

1	Figure 25e-17 Primary syphilis with firm, nontender chancres. (Courtesy of M. Rein and the Centers for Disease Control and Prevention.) Figure 25e-19 Secondary syphilis commonly affects the palms and soles with scaling, firm, red-brown papules. Figure 25e-18 Secondary syphilis, demonstrating the papulosqua-mous truncal eruption. Figure 25e-21 Mucous patches on the tongue of a patient with secondary syphilis. (Courtesy of Ron Roddy; with permission.) Figure 25e-22 Petechial lesions in a patient with atypical measles. (Courtesy of Stephen E. Gellis, MD; with permission.) CHAPTER 25e Atlas of Rashes Associated with Fever Figure 25e-20 Condylomata lata are moist, somewhat verrucous intertriginous plaques seen in secondary syphilis.

1	CHAPTER 25e Atlas of Rashes Associated with Fever Figure 25e-20 Condylomata lata are moist, somewhat verrucous intertriginous plaques seen in secondary syphilis. Figure 25e-25 Erythema multiforme is characterized by erythematous plaques with a target or iris morphology, sometimes with a vesicle in the center. It usually represents a hypersensitivity reaction to infections (especially herpes simplex virus or Mycoplasma pneumoniae) or drugs. (Reprinted from K Wolff, RA Johnson: Fitzpatrick’s Color Atlas and Synopsis of Clinical Dermatology, 6th ed. New York, McGraw-Hill, 2009.) Figure 25e-23 Tender vesicles and erosions in the mouth of a patient with hand-foot-and-mouth disease. (Courtesy of Stephen E. Gellis, MD; with permission.) PART 2 Cardinal Manifestations and Presentation of Diseases Figure 25e-24 Septic emboli with hemorrhage and infarction due to acute Staphylococcus aureus endocarditis. (Courtesy of Lindsey Baden, MD; with permission.)

1	Figure 25e-24 Septic emboli with hemorrhage and infarction due to acute Staphylococcus aureus endocarditis. (Courtesy of Lindsey Baden, MD; with permission.) Figure 25e-26 Scarlet fever exanthem. Finely punctuated ery-thema has become confluent (scarlatiniform); accentuation of linear erythema in body folds (Pastia’s lines) is seen here. (Reprinted from K Wolff, RA Johnson: Color Atlas and Synopsis of Clinical Dermatology, 6th ed. New York, McGraw-Hill, 2009.) Figure 25e-28 Diffuse erythema and scaling are present in this patient with psoriasis and the exfoliative erythroderma syndrome. (Reprinted from K Wolff, RA Johnson: Color Atlas and Synopsis of Clinical Dermatology, 6th ed. New York, McGraw-Hill, 2009.) Figure 25e-27 Erythema progressing to bullae with result-ing sloughing of the entire thickness of the epidermis occurs in toxic epidermal necrolysis. This reaction was due to a sulfonamide.

1	Figure 25e-27 Erythema progressing to bullae with result-ing sloughing of the entire thickness of the epidermis occurs in toxic epidermal necrolysis. This reaction was due to a sulfonamide. (Reprinted from K Wolff, RA Johnson: Color Atlas and Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005.) Figure 25e-29 This infant with staphylococcal scalded skin syn-drome demonstrates generalized desquamation. (Reprinted from K Wolff, RA Johnson: Color Atlas and Synopsis of Clinical Dermatology, 6th ed. New York, McGraw-Hill, 2009.) CHAPTER 25e Atlas of Rashes Associated with Fever Figure 25e-30 Fissuring of the lips and an erythematous exan-them are evident in this patient with Kawasaki disease. (Courtesy of Stephen E. Gellis, MD; with permission.) PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Figure 25e-31 Numerous varicella lesions at various stages of evolution: vesicles on an erythematous base and umbilicated vesicles, which then develop into crusting lesions. (Courtesy of the Centers for Disease Control and Prevention.) Figure 25e-33 Herpes zoster is seen in this patient taking predni-sone. Grouped vesicles and crusted lesions are seen in the T2 derma-tome on the back and arm (A) and on the right side of the chest (B). (Reprinted from K Wolff, RA Johnson: Color Atlas and Synopsis of Clinical Dermatology, 6th ed. New York, McGraw-Hill, 2009.)

1	(Reprinted from K Wolff, RA Johnson: Color Atlas and Synopsis of Clinical Dermatology, 6th ed. New York, McGraw-Hill, 2009.) Figure 25e-32 Lesions of disseminated zoster at different stages of evolution, including pustules and crusting, similar to varicella. Note nongrouping of lesions, in contrast to herpes simplex or zos-ter. (Reprinted from K Wolff, RA Johnson, AP Saavedra: Color Atlas and Synopsis of Clinical Dermatology, 7th ed. New York, McGraw-Hill, 2013.) 25e-9 CHAPTER 25e Atlas of Rashes Associated with Fever Figure 25e-35 Ecthyma gangrenosum in a neutropenic patient with Pseudomonas aeruginosa bacteremia. Figure 25e-36 Urticaria showing characteristic discrete and confluent, edematous, erythematous papules and plaques. (Reprinted from K Wolff, RA Johnson, AP Saavedra: Color Atlas and Synopsis of Clinical Dermatology, 7th ed. New York, McGraw-Hill, 2013.)

1	Figure 25e-34 Top: Eschar at the site of the mite bite in a patient with rickettsialpox. Middle: Papulovesicular lesions on the trunk of the same patient. Bottom: Close-up of lesions from the same patient. (Reprinted from A Krusell et al: Emerg Infect Dis 8:727, 2002.) Figure 25e-37 Disseminated cryptococcal infection. A liver transplant recipient developed six cutaneous lesions similar to the one shown. Biopsy and serum antigen testing demonstrated Cryptococcus. Important features of the lesion include a benign-appearing fleshy papule with central umbilication resembling molluscum contagiosum. (Courtesy of Lindsey Baden, MD; with permission.) Figure 25e-38 Disseminated candidiasis. Tender, erythematous, nodular lesions developed in a neutropenic patient with leukemia who was undergoing induction chemotherapy. (Courtesy of Lindsey Baden, MD; with permission.)

1	Figure 25e-40 Erythema nodosum is a panniculitis characterized by tender, deep-seated nodules and plaques usually located on the lower extremities. (Courtesy of Robert Swerlick, MD; with permission.) PART 2 Cardinal Manifestations and Presentation of Diseases Figure 25e-39 Disseminated Aspergillus infection. Multiple necrotic lesions developed in this neutropenic patient undergoing hematopoietic stem cell transplantation. The lesion in the photograph is on the inner thigh and is several centimeters in diameter. Biopsy demonstrated infarction caused by Aspergillus fumigatus. (Courtesy of Lindsey Baden, MD; with permission.) Figure 25e-41 Sweet syndrome is an erythematous indurated plaque with a pseudovesicular border. (Courtesy of Robert Swerlick, MD; with permission.) Figure 25e-42 Fulminant meningococcemia with extensive angu-lar purpuric patches. (Courtesy of Stephen E. Gellis, MD; with permission.)

1	Figure 25e-42 Fulminant meningococcemia with extensive angu-lar purpuric patches. (Courtesy of Stephen E. Gellis, MD; with permission.) Figure 25e-43 Erythematous papular lesions are seen on the leg of this patient with chronic meningococcemia (arrow indicates a lesion). Figure 25e-45 Palpable purpuric papules on the lower leg are seen in this patient with cutaneous small-vessel hypersensitivity vasculitis. (Reprinted from K Wolff, RA Johnson: Color Atlas and Synopsis of Clinical Dermatology, 6th ed. New York, McGraw-Hill, 2009.) CHAPTER 25e Atlas of Rashes Associated with Fever Figure 25e-44 Disseminated gonococcemia in the skin is seen as hemorrhagic papules and pustules with purpuric centers in a centrifu-gal distribution. (Courtesy of Daniel M. Musher, MD; with permission.) Figure 25e-46 The thumb of a patient with a necrotic ulcer of tula-remia. (Courtesy of the Centers for Disease Control and Prevention.)

1	Figure 25e-46 The thumb of a patient with a necrotic ulcer of tula-remia. (Courtesy of the Centers for Disease Control and Prevention.) Figure 25e-47 This 50-year-old man developed high fever and massive inguinal lymphadenopathy after a small ulcer healed on his foot. Tularemia was diagnosed. (Courtesy of Lindsey Baden, MD; with permission.) PART 2 Cardinal Manifestations and Presentation of Diseases Figure 25e-48 This painful trypanosomal chancre developed at the site of a tsetse fly bite on the dorsum of the foot. Trypanosoma brucei was diagnosed from an aspirate of the ulcer. (Courtesy of Edward T. Ryan, MD. N Engl J Med 346:2069, 2002; with permission.)

1	Figure 25e-49 Drug reaction with eosinophilia and systemic symptoms/drug-induced hypersensitivity syndrome (DRESS/ DIHS). This patient developed a progressive eruption exhibiting early desquamation after taking phenobarbital. There was also associated lymphadenopathy and hepatomegaly. (Courtesy of Peter Lio, MD; with permission.) Figure 25e-50 Many small, nonfollicular pustules are seen against a background of erythema in this patient with acute generalized erup-tive pustulosis (AGEP). The rash began in body folds and progressed to cover the trunk and face. (Reprinted from K Wolff, RA Johnson: Color Atlas and Synopsis of Clinical Dermatology, 6th ed. New York, McGraw-Hill, 2009.) CHAPTER 25e Atlas of Rashes Associated with Fever

1	CHAPTER 25e Atlas of Rashes Associated with Fever Figure 25e-51 Smallpox is shown with many pustules on the face, becoming confluent (A), and on the trunk (B). Pustules are all in the same stage of development. C. Crusting, healing lesions are noted on the trunk, arms, and hands. (Reprinted from K Wolff, RA Johnson: Color Atlas and Synopsis of Clinical Dermatology, 6th ed. New York, McGraw-Hill, 2009.) CHAPTER 26 Fever of Unknown Origin fever of unknown origin Chantal P. Bleeker-Rovers, Jos W. M. van der Meer DEFINITION Clinicians commonly refer to any febrile illness without an initially obvious etiology as fever of unknown origin (FUO). Most febrile ill-nesses either resolve before a diagnosis can be made or develop distin-26 guishing characteristics that lead to a diagnosis. The term FUO should be reserved for prolonged febrile illnesses without an established etiology despite intensive evaluation and diagnostic testing. This chapter focuses on classic FUO in the adult patient.

1	FUO was originally defined by Petersdorf and Beeson in 1961 as an illness of >3 weeks’ duration with fever of ≥38.3°C (101°F) on two occasions and an uncertain diagnosis despite 1 week of inpatient evaluation. Nowadays, most patients with FUO are hospitalized if their clinical condition requires it, but not for diagnostic purposes only; thus the in-hospital evaluation requirement has been eliminated from the definition. The definition of FUO has been further modified by the exclusion of immunocompromised patients, whose workup requires an entirely different diagnostic and therapeutic approach. For the optimal comparison of patients with FUO in different geographic areas, it has been proposed that the quantitative criterion (diagnosis uncertain after 1 week of evaluation) be changed to a qualitative criterion that requires the performance of a specific list of investigations. Accordingly, FUO is now defined as: 1. Fever >38.3°C (101°F) on at least two occasions 2.

1	Fever >38.3°C (101°F) on at least two occasions 2. Illness duration of ≥3 weeks 3. 4. Diagnosis that remains uncertain after a thorough history-taking, physical examination, and the following obligatory investigations: determination of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) level; platelet count; leukocyte count and differential; measurement of levels of hemoglobin, electrolytes, creatinine, total protein, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, creatine kinase, ferritin, antinuclear antibodies, and rheumatoid factor; protein electrophoresis; urinalysis; blood cultures (n = 3); urine culture; chest x-ray; abdominal ultrasonography; and tuberculin skin test (TST). The range of FUO etiologies has evolved over time as a result of changes in the spectrum of diseases causing FUO, the widespread Percentage of Cases Due to Indicated Cause

1	The range of FUO etiologies has evolved over time as a result of changes in the spectrum of diseases causing FUO, the widespread Percentage of Cases Due to Indicated Cause PART 2 Cardinal Manifestations and Presentation of Diseases use of antibiotics, and the availability of new diagnostic techniques. The proportion of cases caused by intraabdominal abscesses and tumors, for example, has decreased because of earlier detection by CT and ultrasound. In addition, infective endocarditis is a less frequent cause because blood culture and echocardiographic techniques have improved. Conversely, some diagnoses, such as acute HIV infection, were unknown four decades ago. Table 26-1 summarizes the findings of several large studies on

1	FUO conducted over the past 20 years. In general, infection accounts for about 20–25% of cases of FUO in Western countries; next in frequency are neoplasms and noninfectious inflammatory diseases (NIIDs), the latter including “collagen or rheumatic diseases,” vasculitis syndromes, and granulomatous disorders. In geographic areas outside the West, infections are a much more common cause of FUO (43% vs 22%), while the proportions of cases due to NIIDs and neoplasms are similar. Up to 50% of cases caused by infections in patients with FUO outside Western nations are due to tuberculosis, which is a less common cause in the United States and Western Europe. The number of FUO patients diagnosed with NIIDs probably will not decrease in the near future, as fever may precede more typical manifestations or serologic evidence by months in these diseases. Moreover, many NIIDs can be diagnosed only after prolonged observation and exclusion of other diseases.

1	In the West, the percentage of undiagnosed cases of FUO has increased in more recent studies. An important factor contributing to the seemingly high diagnostic failure rate is that a diagnosis is more often being established before 3 weeks have elapsed, given that patients with fever tend to seek medical advice earlier and better diagnostic techniques, such as CT and MRI, are widely available; therefore, only the cases that are more difficult to diagnose continue to meet the criteria for FUO. Furthermore, most patients who have FUO without a diagnosis currently do well, and thus a less aggressive diagnostic approach may be used in clinically stable patients once diseases with immediate therapeutic or prognostic consequences have been ruled out to a reasonable extent. This factor may be especially relevant to patients with recurrent fever who are asymptomatic in between febrile episodes. In patients with recurrent fever (defined as repeated episodes of fever interspersed with

1	may be especially relevant to patients with recurrent fever who are asymptomatic in between febrile episodes. In patients with recurrent fever (defined as repeated episodes of fever interspersed with fever-free intervals of at least 2 weeks and apparent remission of the underlying disease), the chance of attaining an etiologic diagnosis is <50%.

1	The differential diagnosis for FUO is extensive, but it is important to remember that FUO is far more often caused by an atypical presentation of a rather common disease than by a very rare disease. Table 26-2 presents an overview of possible causes of FUO. An atypical presentation of endocarditis, diverticulitis, vertebral osteomyelitis, and extra-pulmonary tuberculosis are the more common infectious disease diagnoses. Q fever and Whipple’s disease are quite rare but should always be kept in mind as a cause of FUO since the presenting symptoms

1	Bacterial, nonspecific Abdominal abscess, adnexitis, apical granuloma, appendicitis, cholangitis, cholecystitis, diverticulitis, endocarditis, endometritis, epidural abscess, infected vascular catheter, infected joint prosthesis, infected vascular prosthesis, infectious arthritis, infective myonecrosis, intracranial abscess, liver abscess, lung abscess, malakoplakia, mastoiditis, mediastinitis, mycotic aneurysm, osteomyelitis, pelvic inflammatory disease, prostatitis, pyelonephritis, pylephlebitis, renal abscess, septic phlebitis, sinusitis, spondylodiscitis, xanthogranulomatous urinary tract infection

1	Bacterial, specific Actinomycosis, atypical mycobacterial infection, bartonellosis, brucellosis, Campylobacter infection, Chlamydia pneumoniae infection, chronic meningococcemia, ehrlichiosis, gonococcemia, legionellosis, leptospirosis, listeriosis, louse-borne relapsing fever (Borrelia recurrentis), Lyme disease, melioidosis (Pseudomonas pseudomallei), Mycoplasma infection, nocardiosis, psittacosis, Q fever (Coxiella burnetii), rickettsiosis, Spirillum minor infection, Streptobacillus moniliformis infection, syphilis, tick-borne relapsing fever (Borrelia duttonii), tuberculosis, tularemia, typhoid fever and other salmonelloses, Whipple’s disease (Tropheryma whipplei), yersiniosis Fungal Aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, Malassezia furfur infection, paracoccidioidomycosis, Pneumocystis jirovecii pneumonia, sporotrichosis, zygomycosis

1	Parasitic Amebiasis, babesiosis, echinococcosis, fascioliasis, malaria, schistosomiasis, strongyloidiasis, toxocariasis, toxoplasmosis, trichinellosis, trypanosomiasis, visceral leishmaniasis Viral Colorado tick fever, coxsackievirus infection, cytomegalovirus infection, dengue, Epstein-Barr virus infection, hantavirus infection, hepatitis (A, B, C, D, E), herpes simplex, HIV infection, human herpesvirus 6 infection, parvovirus infection, West Nile virus infection Systemic rheumatic and autoimmune Ankylosing spondylitis, antiphospholipid syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, Behçet’s diseases disease, cryoglobulinemia, dermatomyositis, Felty syndrome, gout, mixed connective-tissue disease, polymyositis, pseudogout, reactive arthritis, relapsing polychondritis, rheumatic fever, rheumatoid arthritis, Sjögren’s syndrome, systemic lupus erythematosus, Vogt-Koyanagi-Harada syndrome

1	Vasculitis Allergic vasculitis, Churg-Strauss syndrome, giant cell vasculitis/polymyalgia rheumatica, granulomatosis with polyangiitis, hypersensitivity vasculitis, Kawasaki’s disease, polyarteritis nodosa, Takayasu arteritis, urticarial vasculitis Granulomatous diseases Idiopathic granulomatous hepatitis, sarcoidosis

1	Granulomatous diseases Idiopathic granulomatous hepatitis, sarcoidosis Autoinflammatory syndromes Adult-onset Still’s disease, Blau syndrome, CAPSb (cryopyrin-associated periodic syndromes), Crohn’s disease, DIRA (deficiency of the interleukin 1 receptor antagonist), familial Mediterranean fever, hemophagocytic syndrome, hyper-IgD syndrome (HIDS, also known as mevalonate kinase deficiency), juvenile idiopathic arthritis, PAPA syndrome (pyogenic sterile arthritis, pyoderma gangrenosum, and acne), PFAPA syndrome (periodic fever, aphthous stomatitis, pharyngitis, adenitis), recurrent idiopathic pericarditis, SAPHO (synovitis, acne, pustulosis, hyperostosis, osteomyelitis), Schnitzler’s syndrome, TRAPS (tumor necrosis factor receptor–associated periodic syndrome) CHAPTER 26 Fever of Unknown Origin

1	CHAPTER 26 Fever of Unknown Origin Hematologic malignancies Amyloidosis, angioimmunoblastic lymphoma, Castleman’s disease, Hodgkin’s disease, hypereosinophilic syndrome, leukemia, lymphomatoid granulomatosis, malignant histiocytosis, multiple myeloma, myelodysplastic syndrome, myelofibrosis, non-Hodgkin’s lymphoma, plasmacytoma, systemic mastocytosis, vaso-occlusive crisis in sickle cell disease Solid tumors Most solid tumors and metastases can cause fever. Those most commonly causing FUO are breast, colon, hepatocellular, lung, pancreatic, and renal cell carcinomas. Benign tumors Angiomyolipoma, cavernous hemangioma of the liver, craniopharyngioma, necrosis of dermoid tumor in Gardner’s syndrome

1	ADEM (acute disseminated encephalomyelitis), adrenal insufficiency, aneurysms, anomalous thoracic duct, aortic dissection, aortic-enteral fistula, aseptic meningitis (Mollaret’s syndrome), atrial myxoma, brewer’s yeast ingestion, Caroli disease, cholesterol emboli, cirrhosis, complex partial status epilepticus, cyclic neutropenia, drug fever, Erdheim-Chester disease, extrinsic allergic alveolitis, Fabry’s disease, factitious disease, fire-eater’s lung, fraudulent fever, Gaucher’s disease, Hamman-Rich syndrome (acute interstitial pneumonia), Hashimoto’s encephalopathy, hematoma, hypersensitivity pneumonitis, hypertriglyceridemia, hypothalamic hypopituitarism, idiopathic normal-pressure hydrocephalus, inflammatory pseudotumor, Kikuchi’s disease, linear IgA dermatosis, mesenteric fibromatosis, metal fume fever, milk protein allergy, myotonic dystrophy, nonbacterial osteitis, organic dust toxic syndrome, panniculitis, POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal

1	metal fume fever, milk protein allergy, myotonic dystrophy, nonbacterial osteitis, organic dust toxic syndrome, panniculitis, POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, skin changes), polymer fume fever, post–cardiac injury syndrome, primary biliary cirrhosis, primary hyperparathyroidism, pulmonary embolism, pyoderma gangrenosum, retroperitoneal fibrosis, Rosai-Dorfman disease, sclerosing mesenteritis, silicone embolization, subacute thyroiditis (de Quervain’s), Sweet syndrome (acute febrile neutrophilic dermatosis), thrombosis, tubulointerstitial nephritis and uveitis syndrome (TINU), ulcerative colitis

1	Central Brain tumor, cerebrovascular accident, encephalitis, hypothalamic dysfunction Peripheral Anhidrotic ectodermal dysplasia, exercise-induced hyperthermia, hyperthyroidism, pheochromocytoma aThis table includes all causes of FUO that have been described in the literature. bCAPS includes chronic infantile neurologic cutaneous and articular syndrome (CINCA, also known as neonatal-onset multisystem inflammatory disease, or NOMID), familial cold autoinflammatory syndrome (FCAS), and Muckle-Wells syndrome.

1	can be nonspecific. Serologic testing for Q fever, which results from to consideration of infectious diseases such as malaria, leishmaniasis, exposure to animals or animal products, should be performed when histoplasmosis, or coccidioidomycosis. Fever with signs of endocardithe patient lives in a rural area or has a history of heart valve disease, tis and negative blood culture results poses a special problem. Culture-an aortic aneurysm, or a vascular prosthesis. In patients with unex-negative endocarditis may be due to difficult-to-culture bacteria such plained symptoms localized to the central nervous system (CNS), gas-as nutritionally variant bacteria, HACEK organisms (Haemophilus trointestinal tract, or joints, polymerase chain reaction (PCR) testing parainfluenzae, H. paraphrophilus, Aggregatibacter species [actinofor Tropheryma whipplei should be performed. Travel to or (former) mycetemcomitans, aphrophilus], Cardiobacterium species [hominis, residence in tropical countries or

1	Aggregatibacter species [actinofor Tropheryma whipplei should be performed. Travel to or (former) mycetemcomitans, aphrophilus], Cardiobacterium species [hominis, residence in tropical countries or the American Southwest should lead valvarum], Eikenella corrodens, and Kingella kingae; discussed below), 138 Coxiella burnetii (as indicated above), T. whipplei, and Bartonella species. Marantic endocarditis is a sterile thrombotic disease that occurs as a paraneoplastic phenomenon, especially with adenocarcinomas. Sterile endocarditis is also seen in the context of systemic lupus erythematosus and antiphospholipid syndrome. Of the NIIDs, large-vessel vasculitis, polymyalgia rheumatica, sarcoidosis, familial Mediterranean fever, and adult-onset Still’s disease are rather common diagnoses in patients with FUO. The hereditary autoinflammatory syndromes are very rare and usually present in young patients. Schnitzler’s syndrome, which can present at any age, is uncommon but can often be

1	in patients with FUO. The hereditary autoinflammatory syndromes are very rare and usually present in young patients. Schnitzler’s syndrome, which can present at any age, is uncommon but can often be diagnosed easily in a patient with FUO who presents with urticaria, bone pain, and monoclonal gammopathy. Although most tumors can present with fever, malignant lymphoma is by far the most common diagnosis of FUO among the neoplasms. Sometimes the fever even precedes lymphadenopathy detectable by physical examination. Apart from drug-induced fever and exercise-induced hyperthermia, none of the miscellaneous causes of fever is found very frequently in patients with FUO. Virtually all drugs can cause fever, even that commencing after long-term use. Drug-induced fever, including DRESS (drug reaction with eosinophilia and systemic symptoms; Fig. 25e-49), is often accompanied by eosinophilia and also by lymphadenopathy, which can be extensive. More common causes of drug-induced fever are

1	reaction with eosinophilia and systemic symptoms; Fig. 25e-49), is often accompanied by eosinophilia and also by lymphadenopathy, which can be extensive. More common causes of drug-induced fever are allopurinol, carbamazepine, lamotrigine, phenytoin, sulfasalazine, furosemide, antimicrobial drugs (especially sulfonamides, minocycline, vancomycin, β-lactam antibiotics, and isoniazid), some cardiovascular drugs (e.g., quinidine), and some antiretroviral drugs (e.g., nevirapine). Exercise-induced hyperthermia (Chap. 479e) is characterized by an elevated body temperature that is associated with moderate to strenuous exercise lasting from half an hour up to several hours without an increase in CRP level or ESR; typically these patients sweat during the temperature elevation. Factitious fever (fever artificially induced by the patient—for example, by IV injection of contaminated water) should be considered in all patients but is more common among young women in health care professions. In

1	(fever artificially induced by the patient—for example, by IV injection of contaminated water) should be considered in all patients but is more common among young women in health care professions. In fraudulent fever, the patient is normothermic but manipulates the thermometer. Simultaneous measurements at different body sites (rectum, ear, mouth) should rapidly identify this diagnosis. Another clue to fraudulent fever is a dissociation between pulse rate and temperature. Previous studies of FUO have shown that a diagnosis is more likely in elderly patients than in younger age groups. In many cases, FUO in the elderly results from an atypical manifestation of a common disease, among which giant cell arteritis and polymyalgia rheumatica are most frequently involved. Tuberculosis is the most common infectious disease associated with FUO in elderly patients, occurring much more often than in younger patients. As many of these diseases are treatable, it is well worth pursuing the cause of

1	common infectious disease associated with FUO in elderly patients, occurring much more often than in younger patients. As many of these diseases are treatable, it is well worth pursuing the cause of fever in elderly patients.

1	APPROACH TO THE PATIENT: fever of unknown origin PART 2 Cardinal Manifestations and Presentation of Diseases

1	Figure 26-1 shows a structured approach to patients presenting with FUO. The most important step in the diagnostic workup is the search for potentially diagnostic clues (PDCs) through complete and repeated history-taking and physical examination and the obligatory investigations listed above. PDCs are defined as all localizing signs, symptoms, and abnormalities potentially pointing toward a diagnosis. Although PDCs are often misleading, only with their help can a concise list of probable diagnoses be made. The history should include information about the fever pattern (continuous or recurrent) and duration, previous medical history, present and recent drug use, family history, sexual history, country of origin, recent and remote travel, unusual environmental exposures associated with travel or hobbies, and animal contacts. A complete physical examination should be performed, with special attention to the eyes, lymph nodes, temporal arteries, liver, spleen, sites of previous surgery,

1	or hobbies, and animal contacts. A complete physical examination should be performed, with special attention to the eyes, lymph nodes, temporal arteries, liver, spleen, sites of previous surgery, entire skin surface, and mucous membranes. Before further diagnostic tests are initiated, antibiotic and glucocorticoid treatment, which can mask many diseases, should be stopped. For example, blood and other cultures are not reliable when samples are obtained during antibiotic treatment, and the size of enlarged lymph nodes usually decreases during glucocorticoid treatment, regardless of the cause of the lymphadenopathy. Despite the high number of false-positive ultrasounds and the relatively low sensitivity of chest x-rays, the performance of these simple, low-cost diagnostic tests remains obligatory in all patients with FUO in order to separate cases that are caused by easily diagnosed diseases from those that are not. Abdominal ultrasound is preferred to abdominal CT as an obligatory test

1	in all patients with FUO in order to separate cases that are caused by easily diagnosed diseases from those that are not. Abdominal ultrasound is preferred to abdominal CT as an obligatory test because of relatively low cost, lack of radiation burden, and absence of side effects.

1	Only rarely do biochemical tests (beyond the obligatory tests needed to classify a patient’s fever as FUO) lead directly to a definitive diagnosis in the absence of PDCs. The diagnostic yield of immunologic serology other than that included in the obligatory tests is relatively low. These tests more often yield false-positive rather than true-positive results and are of little use without PDCs pointing to specific immunologic disorders. Given the absence of specific symptoms in many patients and the relatively low cost of the test, investigation of cryoglobulins appears to be a valuable screening test in patients with FUO.

1	Multiple blood samples should be cultured in the laboratory long enough to ensure ample growth time for any fastidious organisms, such as HACEK organisms. It is critical to inform the laboratory of the intent to test for unusual organisms. Specialized media should be used when the history suggests uncommon microorganisms, such as Histoplasma or Legionella. Performing more than three blood cultures or more than one urine culture is useless in patients with FUO in the absence of PDCs (e.g., a high level of clinical suspicion of endocarditis). Repeating blood or urine cultures is useful only when previously cultured samples were collected during antibiotic treatment or within 1 week after its discontinuation. FUO with headache should prompt microbiologic examination of cerebrospinal fluid (CSF) for organisms including herpes simplex virus (HSV; especially HSV-2), Cryptococcus neoformans, and Mycobacterium tuberculosis. In CNS tuberculosis, the CSF typically has elevated protein and

1	(CSF) for organisms including herpes simplex virus (HSV; especially HSV-2), Cryptococcus neoformans, and Mycobacterium tuberculosis. In CNS tuberculosis, the CSF typically has elevated protein and lowered glucose concentrations, with a mononuclear pleocytosis. CSF protein levels range from 100 to 500 mg/dL in most patients, the CSF glucose concentration is <45 mg/dL in 80% of cases, and the usual CSF cell count is between 100 and 500 cells/μL.

1	Microbiologic serology should not be included in the diagnostic workup in patients without PDCs for specific infections. A TST is included in the obligatory investigations, but it may yield false-negative results in patients with miliary tuberculosis, malnutrition, or immunosuppression. Although the interferon γ release assay is less influenced by prior vaccination with bacille Calmette-Guérin or by infection with nontuberculous mycobacteria, its sensitivity is similar to that of the TST; a negative TST or interferon γ release assay therefore does not exclude a diagnosis of tuberculosis. Miliary tuberculosis is especially difficult to diagnose. Granulomatous disease in liver or bone marrow biopsy samples, for example, should always lead to a (re)consideration of this diagnosis. If miliary tuberculosis is suspected, liver biopsy for acid-fast smear, culture, and PCR probably still has the highest diagnostic yield; however, biopsies of bone marrow, lymph nodes, or other involved organs

1	tuberculosis is suspected, liver biopsy for acid-fast smear, culture, and PCR probably still has the highest diagnostic yield; however, biopsies of bone marrow, lymph nodes, or other involved organs also can be considered.

1	The diagnostic yield of echocardiography, sinus radiography, radiologic or endoscopic evaluation of the gastrointestinal tract, and bronchoscopy is very low in the absence of PDCs. Therefore, these tests should not be used as screening procedures. After identification of all PDCs retrieved from the history, physical examination, and obligatory tests, a limited list of the most probable diagnoses should be made. Since most investigations are helpful only for patients who have PDCs for the diagnoses sought, further diagnostic procedures should be limited to specific investigations aimed at confirming or excluding diseases on this list. In FUO, the Fever ˜38.3° C (101° F) and illness lasting ˜3 weeks and no known immunocompromised state History and physical examination Stop antibiotic treatment and glucocorticoids Obligatory investigations:

1	Fever ˜38.3° C (101° F) and illness lasting ˜3 weeks and no known immunocompromised state History and physical examination Stop antibiotic treatment and glucocorticoids Obligatory investigations: ESR and CRP, hemoglobin, platelet count, leukocyte count and differential, electrolytes, creatinine, total protein, protein electrophoresis, alkaline phosphatase, AST, ALT, LDH, creatine kinase, antinuclear antibodies, rheumatoid factor, urinalysis, blood cultures (n=3), urine culture, chest x-ray, abdominal ultrasonography, and tuberculin skin test

1	Stable condition: Follow-up for new PDCs Consider NSAID Deterioration: Further diagnostic tests Consider therapeutic trial PDCs present PDCs absent or misleading Exclude manipulation with thermometer Stop or replace medication to exclude drug fever Guided diagnostic tests DIAGNOSIS Cryoglobulin and funduscopy NO DIAGNOSIS FDG-PET/CT (or labeled leukocyte scintigraphy or gallium scan) Scintigraphy abnormal Scintigraphy normal Confirmation of abnormality (e.g., biopsy, culture) Repeat history and physical examination Perform PDC-driven invasive testing DIAGNOSIS NO DIAGNOSIS DIAGNOSIS NO DIAGNOSIS Chest and abdominal CT Temporal artery biopsy (˜55 years) DIAGNOSIS NO DIAGNOSIS CHAPTER 26 Fever of Unknown Origin

1	CHAPTER 26 Fever of Unknown Origin FIguRE 26-1 Structured approach to patients with FUO. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; FDG-PET/CT, 18F-fluorodeoxyglucose positron emission tomography combined with low-dose computed tomography; LDH, lactate dehydrogenase; PDCs, potentially diagnostic clues (all localizing signs, symptoms, and abnormalities potentially pointing toward a diagnosis); NSAID, nonsteroidal anti-inflammatory drug.

1	diagnostic pointers are numerous and diverse but may be missed drug is the cause. In patients without PDCs or with only misleading on initial examination, often being detected only by a very care-PDCs, funduscopy by an ophthalmologist may be useful in the early ful examination performed subsequently. In the absence of PDCs, stage of the diagnostic workup. When the first-stage diagnostic tests the history and physical examination should therefore be repeated do not lead to a diagnosis, scintigraphy should be performed, esperegularly. One of the first steps should be to rule out factitious or cially when the ESR or CRP level is elevated. fraudulent fever, particularly in patients without signs of inflammation in laboratory tests. All medications, including nonprescription Recurrent Fever In patients with recurrent fever, the diagnostic drugs and nutritional supplements, should be discontinued early in workup should consist of thorough history-taking, physical examithe evaluation to

1	In patients with recurrent fever, the diagnostic drugs and nutritional supplements, should be discontinued early in workup should consist of thorough history-taking, physical examithe evaluation to exclude drug fever. If fever persists beyond 72 h nation, and obligatory tests. The search for PDCs should be directed after discontinuation of the suspected drug, it is unlikely that this to clues matching known recurrent syndromes (Table 26-3). Patients

1	PART 2 Cardinal Manifestations and Presentation of Diseases Systemic rheumatic and autoimmune Ankylosing spondylitis, antiphospholipid syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, Behçet’s diseases disease, cryoglobulinemia, gout, polymyositis, pseudogout, reactive arthritis, relapsing polychondritis, systemic lupus erythematosus Vasculitis Churg-Strauss syndrome, giant cell vasculitis/polymyalgia rheumatica, hypersensitivity vasculitis, polyarteritis nodosa, urticarial vasculitis Granulomatous diseases Idiopathic granulomatous hepatitis, sarcoidosis

1	Granulomatous diseases Idiopathic granulomatous hepatitis, sarcoidosis Autoinflammatory syndromes Adult-onset Still’s disease, Blau syndrome, CAPSb (cryopyrin-associated periodic syndrome), Crohn’s disease, DIRA (deficiency of the IL-1 receptor antagonist), familial Mediterranean fever, hemophagocytic syndrome, hyper-IgD syndrome (HIDS, also known as mevalonate kinase deficiency), juvenile idiopathic arthritis, PAPA syndrome (pyogenic sterile arthritis, pyoderma gangrenosum, and acne), PFAPA syndrome (periodic fever, aphthous stomatitis, pharyngitis, adenitis), recurrent idiopathic pericarditis, SAPHO (synovitis, acne, pustulosis, hyperostosis, osteomyelitis), Schnitzler’s syndrome, TRAPS (tumor necrosis factor receptor–associated periodic syndrome) Angioimmunoblastic lymphoma, Castleman’s disease, colon carcinoma, craniopharyngioma, Hodgkin’s disease, non-Hodgkin lymphoma, malignant histiocytosis, mesothelioma

1	Angioimmunoblastic lymphoma, Castleman’s disease, colon carcinoma, craniopharyngioma, Hodgkin’s disease, non-Hodgkin lymphoma, malignant histiocytosis, mesothelioma Adrenal insufficiency, aortic-enteral fistula, aseptic meningitis (Mollaret’s syndrome), atrial myxoma, brewer’s yeast ingestion, cholesterol emboli, cyclic neutropenia, drug fever, extrinsic allergic alveolitis, Fabry’s disease, factitious disease, fraudulent fever, Gaucher’s disease, hypersensitivity pneumonitis, hypertriglyceridemia, hypothalamic hypopituitarism, inflammatory pseudotumor, metal fume fever, milk protein allergy, polymer fume fever, pulmonary embolism, sclerosing mesenteritis

1	Central Hypothalamic dysfunction Peripheral Anhidrotic ectodermal dysplasia, exercise-induced hyperthermia, pheochromocytoma aThis table includes all causes of recurrent fever that have been described in the literature. bCAPS includes chronic infantile neurologic cutaneous and articular syndrome (CINCA, also known as neonatal-onset multisystem inflammatory disease, or NOMID), familial cold autoinflammatory syndrome (FCAS), and Muckle-Wells syndrome.

1	should be asked to return during a febrile episode so that the history, physical examination, and laboratory tests can be repeated during a symptomatic phase. Further diagnostic tests, such as scintigraphic imaging (see below), should be performed only during a febrile episode because abnormalities may be absent between episodes. In patients with recurrent fever lasting >2 years, it is very unlikely that the fever is caused by infection or malignancy. Further diagnostic tests in that direction should be considered only when PDCs for infections, vasculitis syndromes, or malignancy are present or when the patient’s clinical condition is deteriorating.

1	Scintigraphy Scintigraphic imaging is a noninvasive method allowing delineation of foci in all parts of the body on the basis of functional changes in tissues. This procedure plays an important role in the diagnosis of patients with FUO in clinical practice. Conventional scintigraphic methods used in clinical practice are 67Ga-citrate scintigraphy and 111Inor 99mTc-labeled leukocyte scintigraphy. Focal infectious and inflammatory processes can also be detected by several radiologic techniques, such as CT, MRI, and ultrasound. However, because of the lack of substantial pathologic changes in the early phase, infectious and inflammatory foci cannot be detected at this time. Furthermore, distinguishing active infectious or inflammatory lesions from residual changes due to cured processes or surgery remains critical. Finally, CT and MRI routinely provide information only on part of the body, while scintigraphy readily allows whole-body imaging.

1	Fluorodeoxyglucose Positron Emission Tomography 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) has become an established imaging procedure in FUO. FDG accumulates in tissues with a high rate of glycolysis, which occurs not only in malignant cells but also in activated leukocytes, and thus permits the imaging of acute and chronic inflammatory processes. Normal uptake may obscure pathologic foci in the brain, heart, bowel, kidneys, and bladder. In patients with fever, bone marrow uptake is frequently increased in a nonspecific way due to cytokine activation, which upregulates glucose transporters in bone marrow cells. Compared with conventional scintigraphy, FDG-PET offers the advantages of higher resolution, greater sensitivity in chronic low-grade infections, and a high degree of accuracy in the central skeleton. Furthermore, vascular uptake of FDG is increased in patients with vasculitis. The mechanisms responsible for FDG uptake do not allow differentiation among

1	degree of accuracy in the central skeleton. Furthermore, vascular uptake of FDG is increased in patients with vasculitis. The mechanisms responsible for FDG uptake do not allow differentiation among infection, sterile inflammation, and malignancy. However, in patients with FUO, since all of these disorders are causes of FUO, FDG-PET can be used to guide additional diagnostic tests (e.g., targeted biopsies) that may yield the final diagnosis. Improved anatomic resolution by direct integration with CT (FDG-PET/CT) has further improved the accuracy of this modality.

1	Overall rates of helpfulness in final diagnosis of FUO are 40% for FDG-PET and 54% for FDG-PET/CT. In one study, FDG-PET was never helpful in diagnosing FUO in patients with a normal CRP level and a normal ESR. In two prospective studies in patients with FUO, FDG-PET was superior to 67Ga-citrate scintigraphy, with a similar or better diagnostic yield and results that were available within hours instead of days. In one study, the sensitivity of FDGPET was greater than that of 111In-granulocyte scintigraphy (86% vs 20%) in patients with FUO.

1	Although scintigraphic techniques do not directly provide a definitive diagnosis, they often identify the anatomic location of a particular ongoing metabolic process and, with the help of other techniques such as biopsy and culture, facilitate timely diagnosis and treatment. Pathologic FDG uptake is quickly eradicated by treatment with glucocorticoids in many diseases, including vasculitis and lymphoma; therefore, glucocorticoid use should be stopped or postponed until after FDG-PET is performed. Results reported in the literature and the advantages offered by FDG-PET indicate that conventional scintigraphic techniques should be replaced by FDG-PET/CT in the investigation of patients with FUO at institutions where this technique is available. FDG-PET/CT is a relatively expensive procedure whose availability is still limited compared with that of CT and conventional scintigraphy. Nevertheless, FDGPET/CT can be cost-effective in the FUO diagnostic workup if used at an early stage,

1	whose availability is still limited compared with that of CT and conventional scintigraphy. Nevertheless, FDGPET/CT can be cost-effective in the FUO diagnostic workup if used at an early stage, helping to establish an early diagnosis, reducing days of hospitalization for diagnostic purposes, and obviating unnecessary and unhelpful tests.

1	In some cases, more invasive tests are appropriate. Abnormalities found with scintigraphic techniques often need to be confirmed by pathology and/or culture of biopsy specimens. If lymphadenopathy is found, lymph node biopsy is necessary, even when the affected lymph nodes are hard to reach. In the case of skin lesions, skin biopsy should be undertaken. In one study, pulmonary wedge excision, histologic examination of an excised tonsil, and biopsy of the peritoneum were performed in light of PDCs or abnormal FDGPET results and yielded a diagnosis.

1	If no diagnosis is reached despite scintigraphic and PDC-driven histologic investigations or culture, second-stage screening diagnostic tests should be considered (Fig. 26-1). In three studies, the diagnostic yield of screening chest and abdominal CT in patients with FUO was ~20%. The specificity of chest CT was ~80%, but that of abdominal CT varied between 63% and 80%. Despite the relatively limited specificity of abdominal CT and the probably limited additional value of chest CT after normal FDG-PET, chest and abdominal CT may be used as screening procedures at a later stage of the diagnostic protocol because of their noninvasive nature and high sensitivity. Bone marrow aspiration is seldom useful in the absence of PDCs for bone marrow disorders. With addition of FDG-PET, which is very sensitive in detecting lymphoma, carcinoma, and osteomyelitis, the value of bone marrow biopsy as a screening procedure is probably further reduced. Several studies have shown a high prevalence of

1	sensitive in detecting lymphoma, carcinoma, and osteomyelitis, the value of bone marrow biopsy as a screening procedure is probably further reduced. Several studies have shown a high prevalence of giant cell arteritis among patients with FUO, with rates up to 17% among elderly patients. Giant cell arteritis often involves large arteries and in most cases can be diagnosed by FDG-PET. However, temporal artery biopsy is still recommended for patients ≥55 years of age in a later stage of the diagnostic protocol: FDG-PET will not be useful in vasculitis limited to the temporal arteries because of the small diameter of these vessels and the high levels of FDG uptake in the brain that overlies them. In the past, liver biopsies have often been performed as a screening procedure in patients with FUO. In each of two recent studies, liver biopsy as part of the later stage of a screening diagnostic protocol was helpful in only one patient. Moreover, abnormal liver tests are not predictive of a

1	FUO. In each of two recent studies, liver biopsy as part of the later stage of a screening diagnostic protocol was helpful in only one patient. Moreover, abnormal liver tests are not predictive of a diagnostic liver biopsy in FUO. Liver biopsy is an invasive procedure that carries the possibility of complications and even death. Therefore, it should not be used for screening purposes in patients with FUO except in those with PDCs for liver disease.

1	In patients with unexplained fever after all of the above procedures, the last step in the diagnostic workup—with only a marginal diagnostic yield—comes at an extraordinarily high cost in terms of both expense and discomfort for the patient. Repetition of a thorough history-taking and physical examination and review of laboratory results and imaging studies (including those from other hospitals) are recommended. Diagnostic delay often results from a failure to recognize PDCs in the available information. In these patients with persisting FUO, waiting for new PDCs to appear probably is better than ordering more screening investigations. Only when a patient’s condition deteriorates without providing new PDCs should a further diagnostic workup be performed.

1	Empirical therapeutic trials with antibiotics, glucocorticoids, or antituberculous agents should be avoided in FUO except when a patient’s condition is rapidly deteriorating after the aforementioned diagnostic tests have failed to provide a definite diagnosis. Antibiotic or antituberculous therapy may irrevocably diminish the ability to culture fastidious bacteria or mycobacteria. However, hemodynamic instability or neutropenia is a good indication for empirical antibiotic therapy. If the TST is positive or if granulomatous disease is present with anergy and sarcoidosis seems unlikely, a therapeutic trial for tuberculosis should be started. Especially in miliary tuberculosis, it may be very difficult to obtain a rapid diagnosis. If the fever does not respond after 6 weeks of empirical antituberculous treatment, another diagnosis should be considered. COLCHICINE, NONSTEROIDAL ANTI-INFLAMMATORY DRugS, AND gLuCOCORTICOIDS

1	Colchicine is highly effective in preventing attacks of familial Mediterranean fever but is not always effective once an attack is well under way. When familial Mediterranean fever is suspected, the response to colchicine is not a completely reliable diagnostic tool in the acute phase, but with colchicine treatment most patients show remarkable improvements in the frequency and severity of subsequent febrile episodes within weeks to months. If the fever persists and the source remains elusive after completion of the later-stage investigations, supportive treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) can be helpful. The response of adult-onset Still’s disease to NSAIDs is dramatic in some cases. The effects of glucocorticoids on giant cell arteritis and polymyalgia rheumatica are equally impressive. Early empirical trials with glucocorticoids, however, decrease the chances of reaching a diagnosis for which more specific and sometimes life-saving treatment might be more

1	are equally impressive. Early empirical trials with glucocorticoids, however, decrease the chances of reaching a diagnosis for which more specific and sometimes life-saving treatment might be more appropriate, such as malignant lymphoma. The ability of NSAIDs and glucocorticoids to mask fever while permitting the spread of infection or lymphoma dictates that their use should be avoided unless infectious diseases and malignant lymphoma have been largely ruled out and inflammatory disease is probable and is likely to be debilitating or threatening.

1	Interleukin (IL) 1 is a key cytokine in local and systemic inflammation and the febrile response. The availability of specific IL-1-targeting agents has revealed a pathologic role of IL-1-mediated inflammation in a growing list of diseases. Anakinra, a recombinant form of the naturally occurring IL-1 receptor antagonist (IL-1Ra), blocks the activity of both IL-1α and IL-1β. Anakinra is extremely effective in the treatment of many autoinflammatory syndromes, such as familial Mediterranean fever, cryopyrin-associated periodic syndrome, tumor necrosis factor receptor–associated periodic syndrome, hyper-IgD syndrome, and Schnitzler’s syndrome. There is a growing list of other chronic inflammatory disorders in which the reduction of IL-1 activity can be highly effective. A therapeutic trial with anakinra can be considered in patients whose FUO has not been diagnosed after later-stage diagnostic tests. Although most chronic inflammatory conditions without a known basis can be controlled

1	with anakinra can be considered in patients whose FUO has not been diagnosed after later-stage diagnostic tests. Although most chronic inflammatory conditions without a known basis can be controlled with glucocorticoids, monotherapy with IL-1 blockade can provide improved control without the metabolic, immunologic, and gastrointestinal side effects of glucocorticoid administration.

1	CHAPTER 26 Fever of Unknown Origin 142 PROgNOSIS FUO-related mortality rates have continuously declined over recent decades. The majority of fevers are caused by treatable diseases, and the risk of death related to FUO is, of course, dependent on the underlying disease. In a study by our group (Table 26-1), none of 37 FUO patients without a diagnosis died during a follow-up period of at least 6 months; 4 of 36 patients with a diagnosis died during follow-up due to infection (n = 1) or malignancy (n = 3). Other studies have also shown that malignancy accounts for most FUO-related deaths. NonHodgkin’s lymphoma carries a disproportionately high death toll. In nonmalignant FUO, fatality rates are very low. The good outcome in patients without a diagnosis confirms that potentially lethal occult diseases are very unusual and that empirical therapy with antibiotics, antituberculous agents, or glucocorticoids is rarely required in stable patients. In less affluent regions, infectious diseases

1	diseases are very unusual and that empirical therapy with antibiotics, antituberculous agents, or glucocorticoids is rarely required in stable patients. In less affluent regions, infectious diseases are still a major cause of FUO, and outcomes may be different.

1	Cardinal Manifestations and Presentation of Diseases Syncope is a transient, self-limited loss of consciousness due to acute global impairment of cerebral blood flow. The onset is rapid, duration brief, and recovery spontaneous and complete. Other causes of transient loss of consciousness need to be distinguished from syncope; these include seizures, vertebrobasilar ischemia, hypoxemia, and hypoglycemia. A syncopal prodrome (presyncope) is common, although loss of consciousness may occur without any warning symptoms. Typical presyncopal symptoms include dizziness, lightheadedness or faintness, weakness, fatigue, and visual and auditory disturbances. The causes of syncope can be divided into three general categories: (1) neurally mediated syncope (also called reflex or vasovagal syncope), (2) orthostatic hypotension, and (3) cardiac syncope.

1	Neurally mediated syncope comprises a heterogeneous group of functional disorders that are characterized by a transient change in the reflexes responsible for maintaining cardiovascular homeostasis. Episodic vasodilation (or loss of vasoconstrictor tone) and bradycardia occur in varying combinations, resulting in temporary failure of blood pressure control. In contrast, in patients with orthostatic hypotension due to autonomic failure, these cardiovascular homeostatic reflexes are chronically impaired. Cardiac syncope may be due to arrhythmias or structural cardiac diseases that cause a decrease in cardiac output. The clinical features, underlying pathophysiologic mechanisms, therapeutic interventions, and prognoses differ markedly among these three causes.

1	Syncope is a common presenting problem, accounting for approximately 3% of all emergency room visits and 1% of all hospital admissions. The annual cost for syncope-related hospitalization in the United States is ~$2.4 billion. Syncope has a lifetime cumulative incidence of up to 35% in the general population. The peak incidence in the young occurs between ages 10 and 30 years, with a median peak around 15 years. Neurally mediated syncope is the etiology in the vast majority of these cases. In elderly adults, there is a sharp rise in the incidence of syncope after 70 years.

1	In population-based studies, neurally mediated syncope is the most common cause of syncope. The incidence is slightly higher in females than males. In young subjects, there is often a family history in first-degree relatives. Cardiovascular disease due to structural disease or arrhythmias is the next most common cause in most series, particularly in emergency room settings and in older patients. Orthostatic hypotension also increases in prevalence with age because of the reduced baroreflex responsiveness, decreased cardiac compliance, and attenuation of the vestibulosympathetic reflex associated with aging. In the elderly, orthostatic hypotension is substantially more common in institutionalized (54–68%) than community-dwelling (6%) individuals, an observation most likely explained by the greater prevalence of HigH-RiSK fEATuRES inDiCATing HoSPiTALizATion oR inTEnSivE EvALuATion of SynCoPE

1	HigH-RiSK fEATuRES inDiCATing HoSPiTALizATion oR inTEnSivE EvALuATion of SynCoPE Chest pain suggesting coronary ischemia Features of congestive heart failure Moderate or severe valvular disease Moderate or severe structural cardiac disease Electrocardiographic features of ischemia History of ventricular arrhythmias Prolonged QT interval (>500 ms) Repetitive sinoatrial block or sinus pauses Persistent sinus bradycardia Bior trifascicular block or intraventricular conduction delay with QRS duration≥120 ms Atrial fibrillation Nonsustained ventricular tachycardia Family history of sudden death Preexcitation syndromes Brugada pattern on ECG Palpitations at time of syncope Syncope at rest or during exercise predisposing neurologic disorders, physiologic impairment, and vasoactive medication use among institutionalized patients.

1	The prognosis after a single syncopal event for all age groups is generally benign. In particular, syncope of noncardiac and unexplained origin in younger individuals has an excellent prognosis; life expectancy is unaffected. By contrast, syncope due to a cardiac cause, either structural heart disease or primary arrhythmic disease, is associated with an increased risk of sudden cardiac death and mortality from other causes. Similarly, mortality rate is increased in individuals with syncope due to orthostatic hypotension related to age and the associated comorbid conditions (Table 27-1).

1	The upright posture imposes a unique physiologic stress upon humans; most, although not all, syncopal episodes occur from a standing position. Standing results in pooling of 500–1000 mL of blood in the lower extremities and splanchnic circulation. There is a decrease in venous return to the heart and reduced ventricular filling that result in diminished cardiac output and blood pressure. These hemodynamic changes provoke a compensatory reflex response, initiated by the baroreceptors in the carotid sinus and aortic arch, resulting in increased sympathetic outflow and decreased vagal nerve activity (Fig. 27-1). The reflex increases peripheral resistance, venous return to the heart, and cardiac output and thus limits the fall in blood pressure. If this response fails, as is the case chronically in orthostatic hypotension and transiently in neurally mediated syncope, cerebral hypoperfusion occurs.

1	Syncope is a consequence of global cerebral hypoperfusion and thus represents a failure of cerebral blood flow autoregulatory mechanisms.

1	FIguRE 27-1 The baroreflex. A decrease in arterial pressure unloads the baroreceptors—the terminals of afferent fibers of the glossopharyngeal and vagus nerves—that are situated in the carotid sinus and aortic arch. This leads to a reduction in the afferent impulses that are relayed from these mechanoreceptors through the glossopharyngeal and vagus nerves to the nucleus of the tractus solitarius (NTS) in the dorsomedial medulla. The reduced baroreceptor afferent activity produces a decrease in vagal nerve input to the sinus node that is mediated via connections of the NTS to the nucleus ambiguus (NA). There is an increase in sympathetic efferent activity that is mediated by the NTS projections to the caudal ventrolateral medulla (CVLM) (an excitatory pathway) and from there to the rostral ventrolateral medulla (RVLM) (an inhibitory pathway). The activation of RVLM presympathetic neurons in response to hypotension is thus predominantly due to disinhibition. In response to a sustained

1	ventrolateral medulla (RVLM) (an inhibitory pathway). The activation of RVLM presympathetic neurons in response to hypotension is thus predominantly due to disinhibition. In response to a sustained fall in blood pressure, vasopressin release is mediated by projections from the A1 noradrenergic cell group in the ventrolateral medulla. This projection activates vasopressin-synthesizing neurons in the magnocellular portion of the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus. Blue denotes sympathetic neurons, and green denotes parasympathetic neurons. (From R Freeman: N Engl J Med 358:615, 2008.)

1	Myogenic factors, local metabolites, and to a lesser extent autonomic neurovascular control are responsible for the autoregulation of cerebral blood flow (Chap. 330). The latency of the autoregulatory response is 5–10 s. Typically cerebral blood flow ranges from 50 to 60 mL/min per 100 g brain tissue and remains relatively constant over perfusion pressures ranging from 50 to 150 mmHg. Cessation of blood flow for 6–8 s will result in loss of consciousness, while impairment of consciousness ensues when blood flow decreases to 25 mL/min per 100 g brain tissue.

1	From the clinical standpoint, a fall in systemic systolic blood pressure to ~50 mmHg or lower will result in syncope. A decrease in cardiac output and/or systemic vascular resistance—the determinants of blood pressure—thus underlies the pathophysiology of syncope. Common causes of impaired cardiac output include decreased effective circulating blood volume; increased thoracic pressure; massive pulmonary embolus; cardiac bradyand tachyarrhythmias; valvular heart disease; and myocardial dysfunction. Systemic vascular resistance may be decreased by central and peripheral autonomic nervous system diseases, sympatholytic medications, and transiently during neurally mediated syncope. Increased cerebral vascular resistance, most frequently due to hypocarbia induced by hyperventilation, may also contribute to the pathophysiology of syncope.

1	Two patterns of electroencephalographic (EEG) changes occur in syncopal subjects. The first is a “slow-flat-slow” pattern (Fig. 27-2) in which normal background activity is replaced with high-amplitude slow delta waves. This is followed by sudden flattening of the EEG—a cessation or attenuation of cortical activity—followed by the return of slow waves, and then normal activity. A second pattern, the “slow pattern,” is characterized by increasing and decreasing slow wave activity only. The EEG flattening that occurs in the slow-flat-slow pattern is a marker of more severe cerebral hypoperfusion. Despite the presence of myoclonic movements and other motor activity during some syncopal events, EEG seizure discharges are not detected.

1	Neurally mediated (reflex; vasovagal) syncope is the final pathway of a complex central and peripheral nervous system reflex arc. There is a sudden, transient change in autonomic efferent activity with increased parasympathetic outflow, plus sympathoinhibition (the vasodepressor response), resulting in bradycardia, vasodilation, and/or reduced vasoconstrictor tone. The resulting fall in systemic blood pressure can then reduce cerebral blood flow to below the compensatory limits of autoregulation (Fig. 27-3). In order to elicit neutrally mediated syncope, a functioning autonomic nervous system is necessary, in contrast to syncope resulting from autonomic failure (discussed below).

1	FIguRE 27-2 The electroencephalogram (EEG) in vasovagal syncope. A 1-min segment of a tilt-table test with typical vasovagal syncope demonstrating the “slow-flat-slow” EEG pattern. Finger beat-to-beat blood pressure, electrocardiogram (ECG), and selected EEG channels are shown. EEG slowing starts when systolic blood pressure drops to ~50 mmHg; heart rate is then approximately 45 beats/min (bpm). Asystole occurred, lasting about 8 s. The EEG flattens for a similar period, but with a delay. A transient loss of consciousness, lasting 14 s, was observed. There were muscle jerks just before and just after the flat period of the EEG. (Figure reproduced with permission from W Wieling et al: Brain 132:2630, 2009.)

1	Multiple triggers of the afferent limb of the reflex arc can result network within the medulla that integrates the neural impulses and in neutrally mediated syncope. In some situations, these can mediates the vasodepressor-bradycardic response. be clearly defined, e.g., the carotid sinus, the gastrointestinal tract, or the bladder. Often, however, the trigger is less easily recognized Classification of Neurally Mediated Syncope Neurally mediated syncope and the cause is multifactorial. Under these circumstances, it is likely may be subdivided based on the afferent pathway and provocathat different afferent pathways converge on the central autonomic tive trigger. Vasovagal syncope (the common faint) is provoked by intense emotion, pain, and/or orthostatic stress, whereas the situational reflex 120 syncopes have specific localized stimuli125 that provoke the reflex vasodilation and 100 bradycardia that leads to syncope. The

1	PART 2 Cardinal Manifestations and Presentation of Diseases 60 most of these situational reflex syncopes. The afferent trigger may originate in the pulmonary system, gastrointestinal system, urogenital system, heart, and carotid artery (Table 27-2). Hyperventilation leading to hypocarbia and cerebral vaso constriction, and raised intrathoracic pressure that impairs venous return to the 80 heart, play a central role in many of the situational reflex syncopes. The afferent pathway of the reflex arc differs among 40 these disorders, but the efferent response FIguRE 27-3 A. The paroxysmal hypotensive-bradycardic response that is characteristic of neurally mediated syncope. Noninvasive beat-to-beat blood pressure and heart rate are shown over 5 min (from 60 to 360 s) of an upright tilt on a tilt table. B. The same tracing expanded to show 80 s of the episode (from 80 to 200 s). BP, blood pressure; bpm, beats per minute; HR, heart rate.

1	via the vagus and sympathetic pathways is similar. Alternately, neurally mediated syncope may be subdivided based on the predominant efferent pathway. Vasodepressor syncope describes syncope predominantly due to efferent, sympathetic, vasoconstrictor failure; cardioinhibitory syncope describes syncope predominantly associated with bradycardia or asystole due CAuSES of SynCoPE A. Neurally Mediated Syncope

1	CAuSES of SynCoPE A. Neurally Mediated Syncope Vasovagal syncope Provoked fear, pain, anxiety, intense emotion, sight of blood, unpleasant sights and odors, orthostatic stress Situational reflex syncope Pulmonary Cough syncope, wind instrument player’s syncope, weightlifter’s syncope, “mess trick”a and “fainting lark,”b sneeze syncope, airway instrumentation Urogenital Postmicturition syncope, urogenital tract instrumentation, prostatic massage Gastrointestinal Swallow syncope, glossopharyngeal neuralgia, esophageal stimulation, gastrointestinal tract instrumentation, rectal examination, defecation syncope Cardiac Bezold-Jarisch reflex, cardiac outflow obstruction Carotid sinus Carotid sinus sensitivity, carotid sinus massage Ocular Ocular pressure, ocular examination, ocular surgery B. Orthostatic Hypotension

1	B. Orthostatic Hypotension Primary autonomic failure due to idiopathic central and peripheral neurodegenerative diseases—the “synucleinopathies” Lewy body diseases Parkinson’s disease Lewy body dementia Pure autonomic failure Multiple system atrophy (the Shy-Drager syndrome) Secondary autonomic failure due to autonomic peripheral neuropathies Diabetes C. Cardiac Syncope aHyperventilation for ~1 minute, followed by sudden chest compression. bHyperventilation (~20 breaths) in a squatting position, rapid rise to standing, then Valsalva. to increased vagal outflow; and mixed syncope describes syncope in 145 which there are both vagal and sympathetic reflex changes.

1	Features of Neurally Mediated Syncope In addition to symptoms of orthostatic intolerance such as dizziness, lightheadedness, and fatigue, premonitory features of autonomic activation may be present in patients with neurally mediated syncope. These include diaphoresis, pallor, palpitations, nausea, hyperventilation, and yawning. During the syncopal event, proximal and distal myoclonus (typically arrhythmic and multifocal) may occur, raising the possibility of epilepsy. The eyes typically remain open and usually deviate upward. Pupils are usually dilated. Roving eye movements may occur. Grunting, moaning, snorting, and stertorous breathing may be present. Urinary incontinence may occur. Fecal incontinence is very rare. Postictal confusion is also rare, although visual and auditory hallucinations and near death and out-of-body experiences are sometimes reported.

1	Although some predisposing factors and provocative stimuli are well established (for example, motionless upright posture, warm ambient temperature, intravascular volume depletion, alcohol ingestion, hypoxemia, anemia, pain, the sight of blood, venipuncture, and intense emotion), the underlying basis for the widely different thresholds for syncope among individuals exposed to the same provocative stimulus is not known. A genetic basis for neurally mediated syncope may exist; several studies have reported an increased incidence of syncope in first-degree relatives of fainters, but no gene or genetic marker has been identified, and environmental, social, and cultural factors have not been excluded by these studies.

1	Reassurance, avoidance of provocative stimuli, and plasma volume expansion with fluid and salt are the cornerstones of the management of neurally mediated syncope. Isometric counterpressure maneuvers of the limbs (leg crossing or handgrip and arm tensing) may raise blood pressure by increasing central blood volume and cardiac output. By maintaining pressure in the autoregulatory zone, these maneuvers avoid or delay the onset of syncope. Randomized controlled trials support this intervention.

1	Fludrocortisone, vasoconstricting agents, and betaadrenoreceptor antagonists are widely used by experts to treat refractory patients, although there is no consistent evidence from randomized controlled trials for any pharmacotherapy to treat neurally mediated syncope. Because vasodilation is the dominant pathophysiologic syncopal mechanism in most patients, use of a cardiac pacemaker is rarely beneficial. Possible exceptions are older patients (>40 years) in whom syncope is associated with asystole or severe bradycardia and patients with prominent cardioinhibition due to carotid sinus syndrome. In these patients, dual-chamber pacing may be helpful.

1	Orthostatic hypotension, defined as a reduction in systolic blood pressure of at least 20 mmHg or diastolic blood pressure of at least 10 mmHg within 3 min of standing or head-up tilt on a tilt table, is a manifestation of sympathetic vasoconstrictor (autonomic) failure (Fig. 27-4). In many (but not all) cases, there is no compensatory increase in heart rate despite hypotension; with partial autonomic failure, heart rate may increase to some degree but is insufficient to maintain cardiac output. A variant of orthostatic hypotension is “delayed” orthostatic hypotension, which occurs beyond 3 min of standing; this may reflect a mild or early form of sympathetic adrenergic dysfunction. In some cases, orthostatic hypotension occurs within 15 s of standing (so-called “initial” orthostatic hypotension), a finding that may reflect a transient mismatch between cardiac output and peripheral vascular resistance and does not represent autonomic failure.

1	Characteristic symptoms of orthostatic hypotension include lightheadedness, dizziness, and presyncope (near-faintness) occurring in response to sudden postural change. However, symptoms may be 75 74 and inflammatory neuropathies (Chaps. 459 and 460). Less frequently, orthostatic hypotension is associated with the 72 peripheral neuropathies that accompany vitamin B12 deficiency, neurotoxic exposure, HIV and other infections, and 70 porphyria. Patients with autonomic failure and the elderly are susceptible to falls in blood pressure associated with meals. 200 180 The magnitude of the blood pressure fall is exacerbated by large meals, meals high 150 in carbohydrate, and alcohol intake. The PART 2 Cardinal Manifestations and Presentation of Diseases mechanism of postprandial syncope is not fully elucidated.

1	PART 2 Cardinal Manifestations and Presentation of Diseases mechanism of postprandial syncope is not fully elucidated. Orthostatic hypotension is often iatrogenic. Drugs from several classes may lower peripheral resistance (e.g., alphaadrenoreceptor antagonists used to treat hypertension and prostatic hypertrophy; antihypertensive agents of several classes; 60 120180 240 300 360 180190 200210 220 nitrates and other vasodilators; tricyclic A Time (sec) Time (sec) agents and phenothiazines). Iatrogenic

1	B volume depletion due to diuresis and FIguRE 27-4 A. The gradual fall in blood pressure without a compensatory heart rate increase volume depletion due to medical causes that is characteristic of orthostatic hypotension due to autonomic failure. Blood pressure and heart (hemorrhage, vomiting, diarrhea, or rate are shown over 5 min (from 60 to 360 s) of an upright tilt on a tilt table. B. The same tracing decreased fluid intake) may also result in expanded to show 40 s of the episode (from 180 to 220 s). BP, blood pressure; bpm, beats per decreased effective circulatory volume, minute; HR, heart rate.

1	absent or nonspecific, such as generalized weakness, fatigue, cognitive slowing, leg buckling, or headache. Visual blurring may occur, likely due to retinal or occipital lobe ischemia. Neck pain, typically in the suboccipital, posterior cervical, and shoulder region (the “coat-hanger headache”), most likely due to neck muscle ischemia, may be the only symptom. Patients may report orthostatic dyspnea (thought to reflect ventilation-perfusion mismatch due to inadequate perfusion of ventilated lung apices) or angina (attributed to impaired myocardial perfusion even with normal coronary arteries). Symptoms may be exacerbated by exertion, prolonged standing, increased ambient temperature, or meals. Syncope is usually preceded by warning symptoms, but may occur suddenly, suggesting the possibility of a seizure or cardiac cause.

1	Supine hypertension is common in patients with orthostatic hypotension due to autonomic failure, affecting over 50% of patients in some series. Orthostatic hypotension may present after initiation of therapy for hypertension, and supine hypertension may follow treatment of orthostatic hypotension. However, in other cases, the association of the two conditions is unrelated to therapy; it may in part be explained by baroreflex dysfunction in the presence of residual sympathetic outflow, particularly in patients with central autonomic degeneration. Causes of Neurogenic Orthostatic Hypotension Causes of neurogenic orthostatic hypotension include central and peripheral autonomic nervous system dysfunction (Chap. 454). Autonomic dysfunction of other organ systems (including the bladder, bowels, sexual organs, and sudomotor system) of varying severity frequently accompanies orthostatic hypotension in these disorders (Table 27-2).

1	The primary autonomic degenerative disorders are multiple system atrophy (the Shy-Drager syndrome; Chap. 454), Parkinson’s disease (Chap. 449), dementia with Lewy bodies (Chap. 448), and pure autonomic failure (Chap. 454). These are often grouped together as “synucleinopathies” due to the presence of alpha-synuclein, a small protein that precipitates predominantly in the cytoplasm of neurons in the Lewy body disorders (Parkinson’s disease, dementia with Lewy bodies, and pure autonomic failure) and in the glia in multiple system atrophy. Peripheral autonomic dysfunction may also accompany small-fiber peripheral neuropathies such as those seen in diabetes, amyloid, immune-mediated neuropathies, hereditary sensory and autonomic neuropathies (HSAN; particularly HSAN type III, familial dysautonomia), orthostatic hypotension, and syncope.

1	The first step is to remove reversible causes—usually vasoactive medications (Table 454-6). Next, nonpharmacologic interventions should be introduced. These interventions include patient education regarding staged moves from supine to upright; warnings about the hypotensive effects of large meals; instructions about the isometric counterpressure maneuvers that increase intravascular pressure (see above); and raising the head of the bed to reduce supine hypertension. Intravascular volume should be expanded by increasing dietary fluid and salt. If these nonpharmacologic measures fail, pharmacologic intervention with fludrocortisone acetate and vasoconstricting agents such as midodrine, L-dihydroxyphenylserine, and pseudoephedrine should be introduced. Some patients with intractable symptoms require additional therapy with supplementary agents that include pyridostigmine, yohimbine, desmopressin acetate (DDAVP), and erythropoietin (Chap. 454).

1	Cardiac (or cardiovascular) syncope is caused by arrhythmias and structural heart disease. These may occur in combination because structural disease renders the heart more vulnerable to abnormal electrical activity. Arrhythmias Bradyarrhythmias that cause syncope include those due to severe sinus node dysfunction (e.g., sinus arrest or sinoatrial block) and atrioventricular (AV) block (e.g., Mobitz type II, high-grade, and complete AV block). The bradyarrhythmias due to sinus node dysfunction are often associated with an atrial tachyarrhythmia, a disorder known as the tachycardia-bradycardia syndrome. A prolonged pause following the termination of a tachycardic episode is a frequent cause of syncope in patients with the tachycardia-bradycardia syndrome. Medications of several classes may also cause bradyarrhythmias of sufficient severity to cause syncope. Syncope due to bradycardia or asystole is referred to as a Stokes-Adams attack.

1	Ventricular tachyarrhythmias frequently cause syncope. The likelihood of syncope with ventricular tachycardia is in part dependent on the ventricular rate; rates below 200 beats/min are less likely to cause syncope. The compromised hemodynamic function during ventricular tachycardia is caused by ineffective ventricular contraction, reduced diastolic filling due to abbreviated filling periods, loss of AV synchrony, and concurrent myocardial ischemia.

1	Several disorders associated with cardiac electrophysiologic instability and arrhythmogenesis are due to mutations in ion channel subunit genes. These include the long QT syndrome, Brugada syndrome, and catecholaminergic polymorphic ventricular tachycardia. The long QT syndrome is a genetically heterogeneous disorder associated with prolonged cardiac repolarization and a predisposition to ventricular arrhythmias. Syncope and sudden death in patients with long QT syndrome result from a unique polymorphic ventricular tachycardia called torsades des pointes that degenerates into ventricular fibrillation. The long QT syndrome has been linked to genes encoding K+ channel α-subunits, K+ channel β-subunits, voltage-gated Na+ channel, and a scaffolding protein, ankyrin B (ANK2). Brugada syndrome is characterized by idiopathic ventricular fibrillation in association with right ventricular electrocardiogram (ECG) abnormalities without structural heart disease. This disorder is also genetically

1	is characterized by idiopathic ventricular fibrillation in association with right ventricular electrocardiogram (ECG) abnormalities without structural heart disease. This disorder is also genetically heterogeneous, although it is most frequently linked to mutations in the Na+ channel α-subunit, SCN5A. Catecholaminergic polymorphic tachycardia is an inherited, genetically heterogeneous disorder associated with exerciseor stress-induced ventricular arrhythmias, syncope, or sudden death. Acquired QT interval prolongation, most commonly due to drugs, may also result in ventricular arrhythmias and syncope. These disorders are discussed in detail in Chap. 277.

1	Structural Disease Structural heart disease (e.g., valvular disease, myocardial ischemia, hypertrophic and other cardiomyopathies, cardiac masses such as atrial myxoma, and pericardial effusions) may lead to syncope by compromising cardiac output. Structural disease may also contribute to other pathophysiologic mechanisms of syncope. For example, cardiac structural disease may predispose to arrhythmogenesis; aggressive treatment of cardiac failure with diuretics and/or vasodilators may lead to orthostatic hypotension; and inappropriate reflex vasodilation may occur with structural disorders such as aortic stenosis and hypertrophic cardiomyopathy, possibly provoked by increased ventricular contractility.

1	Treatment of cardiac disease depends on the underlying disorder. Therapies for arrhythmias include cardiac pacing for sinus node disease and AV block, and ablation, antiarrhythmic drugs, and cardioverter-defibrillators for atrial and ventricular tachyarrhythmias. These disorders are best managed by physicians with specialized skills in this area. APPROACH TO THE PATIENT: Syncope is easily diagnosed when the characteristic features are present; however, several disorders with transient real or apparent loss of consciousness may create diagnostic confusion.

1	Syncope is easily diagnosed when the characteristic features are present; however, several disorders with transient real or apparent loss of consciousness may create diagnostic confusion. Generalized and partial seizures may be confused with syncope; however, there are a number of differentiating features. Whereas tonic-clonic movements are the hallmark of a generalized seizure, myoclonic and other movements also may occur in up to 90% of syncopal episodes. Myoclonic jerks associated with syncope may be multifocal or generalized. They are typically arrhythmic and of short duration (<30 s). Mild flexor and extensor posturing also may occur. Partial or partial-complex seizures with secondary generalization are usually preceded by an aura, commonly an unpleasant smell; fear; anxiety; abdominal discomfort; or other visceral sensations. These phenomena should be differentiated from the premonitory features of syncope.

1	Autonomic manifestations of seizures (autonomic epilepsy) may provide a more difficult diagnostic challenge. Autonomic seizures have cardiovascular, gastrointestinal, pulmonary, urogenital, pupillary, and cutaneous manifestations that are similar to the premonitory features of syncope. Furthermore, the cardiovascular manifestations of autonomic epilepsy include clinically significant tachycardias and bradycardias that may be of sufficient magnitude to cause loss of consciousness. The presence of accompanying non-autonomic auras may help differentiate these episodes from syncope.

1	Loss of consciousness associated with a seizure usually lasts longer than 5 min and is associated with prolonged postictal drowsiness and disorientation, whereas reorientation occurs almost immediately after a syncopal event. Muscle aches may occur after both syncope and seizures, although they tend to last longer and be more severe following a seizure. Seizures, unlike syncope, are rarely provoked by emotions or pain. Incontinence of urine may occur with both seizures and syncope; however, fecal incontinence occurs very rarely with syncope.

1	Hypoglycemia may cause transient loss of consciousness, typically in individuals with type 1 or type 2 diabetes treated with insulin. The clinical features associated with impending or actual hypoglycemia include tremor, palpitations, anxiety, diaphoresis, hunger, and paresthesias. These symptoms are due to autonomic activation to counter the falling blood glucose. Hunger, in particular, is not a typical premonitory feature of syncope. Hypoglycemia also impairs neuronal function, leading to fatigue, weakness, dizziness, and cognitive and behavioral symptoms. Diagnostic difficulties may occur in individuals in strict glycemic control; repeated hypoglycemia impairs the counterregulatory response and leads to a loss of the characteristic warning symptoms that are the hallmark of hypoglycemia.

1	Patients with cataplexy experience an abrupt partial or complete loss of muscular tone triggered by strong emotions, typically anger or laughter. Unlike syncope, consciousness is maintained throughout the attacks, which typically last between 30 s and 2 min. There are no premonitory symptoms. Cataplexy occurs in 60–75% of patients with narcolepsy. The clinical interview and interrogation of eyewitnesses usually allow differentiation of syncope from falls due to vestibular dysfunction, cerebellar disease, extrapyramidal system dysfunction, and other gait disorders. If the fall is accompanied by head trauma, a postconcussive syndrome, amnesia for the precipitating events, and/or the presence of loss of consciousness may contribute to diagnostic difficulty.

1	Apparent loss of consciousness can be a manifestation of psychiatric disorders such as generalized anxiety, panic disorders, major depression, and somatization disorder. These possibilities should be considered in individuals who faint frequently without prodromal symptoms. Such patients are rarely injured despite numerous falls. There are no clinically significant hemodynamic changes concurrent with these episodes. In contrast, transient loss of consciousness due to vasovagal syncope precipitated by fear, stress, anxiety, and emotional distress is accompanied by hypotension, bradycardia, or both.

1	The goals of the initial evaluation are to determine whether the transient loss of consciousness was due to syncope; to identify the cause; and to assess risk for future episodes and serious harm (Table 27-1). The initial evaluation should include a detailed history, thorough questioning of eyewitnesses, and a complete physical and neurologic examination. Blood pressure and heart rate should be measured in the supine position and after 3 min of standing to determine whether orthostatic hypotension is present. An ECG should be performed if there is suspicion of syncope due to an arrhythmia or underlying cardiac disease. Relevant electrocardiographic abnormalities include bradyarrhythmias or tachyarrhythmias, AV block, ischemia, old myocardial infarction, long QT syndrome, and bundle branch block. This initial assessment will lead to the identification of a cause of syncope in approximately 50% of patients and also allows stratification of patients at risk for cardiac mortality.

1	Laboratory Tests Baseline laboratory blood tests are rarely helpful in identifying the cause of syncope. Blood tests should be performed when specific disorders, e.g., myocardial infarction, anemia, and secondary autonomic failure, are suspected (Table 27-2).

1	Autonomic Nervous System Testing (Chap. 454) Autonomic testing, including tilt-table testing, can be performed in specialized centers. Autonomic testing is helpful to uncover objective evidence of autonomic failure and also to demonstrate a predisposition to neurally mediated syncope. Autonomic testing includes assessments of parasympathetic autonomic nervous system function (e.g., heart rate variability to deep respiration and a Valsalva maneuver), sympathetic cholinergic function (e.g., thermoregulatory sweat response and quantitative sudomotor axon reflex test), and sympathetic adrenergic function (e.g., blood pressure response to a Valsalva maneuver and a tilt-table test with beat-to-beat blood pressure measurement). The hemodynamic abnormalities demonstrated on tilt-table test (Figs. 27-3 and 27-4) may be useful in distinguishing orthostatic hypotension due to autonomic failure from the hypotensive bradycardic response of neurally mediated syncope. Similarly, the tilt-table test

1	27-3 and 27-4) may be useful in distinguishing orthostatic hypotension due to autonomic failure from the hypotensive bradycardic response of neurally mediated syncope. Similarly, the tilt-table test may help identify patients with syncope due to immediate or delayed orthostatic hypotension.

1	Carotid sinus massage should be considered in patients with symptoms suggestive of carotid sinus syncope and in patients over age 50 years with recurrent syncope of unknown etiology. This test should only be carried out under continuous ECG and blood pressure monitoring and should be avoided in patients with carotid bruits, plaques, or stenosis.

1	Cardiac Evaluation ECG monitoring is indicated for patients with a high pretest probability of arrhythmia causing syncope. Patients should be monitored in hospital if the likelihood of a life-threatening arrhythmia is high, e.g., patients with severe structural or coronary artery disease, nonsustained ventricular tachycardia, trifascicular heart block, prolonged QT interval, Brugada syndrome ECG pattern, or family history of sudden cardiac death (Table 27-1). Outpatient Holter monitoring is recommended for patients who experience frequent syncopal episodes (one or more per week), whereas loop recorders, which continually record and erase cardiac rhythm, are indicated for patients with suspected arrhythmias with low risk of sudden cardiac death. Loop recorders may be external (recommended for evaluation of episodes that occur at a frequency of greater than one per month) or implantable (if syncope occurs less frequently).

1	Echocardiography should be performed in patients with a history of cardiac disease or if abnormalities are found on physical examination or the ECG. Echocardiographic diagnoses that may be responsible for syncope include aortic stenosis, hyper-trophic cardiomyopathy, cardiac tumors, aortic dissection, and pericardial tamponade. Echocardiography also has a role in risk stratification based on the left ventricular ejection fraction. Treadmill exercise testing with ECG and blood pressure monitoring should be performed in patients who have experienced syncope during or shortly after exercise. Treadmill testing may help identify exercise-induced arrhythmias (e.g., tachycardia-related AV block) and exercise-induced exaggerated vasodilation.

1	Electrophysiologic studies are indicated in patients with structural heart disease and ECG abnormalities in whom noninvasive investigations have failed to yield a diagnosis. Electrophysiologic studies have low sensitivity and specificity and should only be performed when a high pretest probability exists. Currently, this test is rarely performed to evaluate patients with syncope. Psychiatric Evaluation Screening for psychiatric disorders may be appropriate in patients with recurrent unexplained syncope episodes. Tilt-table testing, with demonstration of symptoms in the absence of hemodynamic change, may be useful in reproducing syncope in patients with suspected psychogenic syncope. PART 2 Cardinal Manifestations and Presentation of Diseases Mark F. Walker, Robert B. Daroff

1	PART 2 Cardinal Manifestations and Presentation of Diseases Mark F. Walker, Robert B. Daroff Dizziness is an imprecise symptom used to describe a variety of sensations that include vertigo, light-headedness, faintness, and imbalance. When used to describe a sense of spinning or other motion, dizziness is designated as vertigo. Vertigo may be physiologic, occurring during or after a sustained head rotation, or it may be pathologic, due to vestibular dysfunction. The term light-headedness is commonly applied to presyncopal sensations due to brain hypoperfusion but also may refer to disequilibrium and imbalance. A challenge to diagnosis is that patients often have difficulty distinguishing among these various symptoms, and the words they choose do not reliably indicate the underlying etiology.

1	There are a number of potential causes of dizziness. Vascular disorders cause presyncopal dizziness as a result of cardiac dysrhythmia, orthostatic hypotension, medication effects, or other causes. Such presyncopal sensations vary in duration; they may increase in severity until loss of consciousness occurs, or they may resolve before loss of consciousness if the cerebral ischemia is corrected. Faintness and syncope, which are discussed in detail in Chap. 27, should always be considered when one is evaluating patients with brief episodes of dizziness or dizziness that occurs with upright posture.

1	Vestibular causes of dizziness (vertigo or imbalance) may be due to peripheral lesions that affect the labyrinths or vestibular nerves or to involvement of the central vestibular pathways. They may be paroxysmal or due to a fixed unilateral or bilateral vestibular deficit. Acute unilateral lesions cause vertigo due to a sudden imbalance in vestibular inputs from the two labyrinths. Bilateral lesions cause imbalance and instability of vision (oscillopsia) when the head moves. Other causes of dizziness include nonvestibular imbalance and gait disorders (e.g., loss of proprioception from sensory neuropathy, parkinsonism) and anxiety.

1	When evaluating patients with dizziness, questions to consider include the following: (1) Is it dangerous (e.g., arrhythmia, transient ischemic attack/stroke)? (2) Is it vestibular? (3) If vestibular, is it peripheral or central? A careful history and examination often provide sufficient information to answer these questions and determine whether additional studies or referral to a specialist is necessary. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: When a patient presents with dizziness, the first step is to delineate more precisely the nature of the symptom. In the case of vestibular disorders, the physical symptoms depend on whether the lesion is unilateral or bilateral, and whether it is acute or chronic and progressive. Vertigo, an illusion of self or environmental motion, implies asymmetry of vestibular inputs from the two labyrinths or in their central pathways that is usually acute. Symmetric bilateral vestibular hypofunction causes imbalance but no vertigo. Because of the ambiguity in patients’ descriptions of their symptoms, diagnosis based simply on symptom characteristics is typically unreliable. The history should focus closely on other features, including whether this is the first attack, the duration of this and any prior episodes, provoking factors, and accompanying symptoms.

1	Dizziness can be divided into episodes that last for seconds, minutes, hours, or days. Common causes of brief dizziness (seconds) include benign paroxysmal positional vertigo (BPPV) and orthostatic hypotension, both of which typically are provoked by changes in head and body position. Attacks of vestibular migraine and Ménière’s disease often last hours. When episodes are of intermediate duration (minutes), transient ischemic attacks of the posterior circulation should be considered, although migraine and a number of other causes are also possible.

1	Symptoms that accompany vertigo may be helpful in distinguishing peripheral vestibular lesions from central causes. Unilateral hearing loss and other aural symptoms (ear pain, pressure, fullness) typically point to a peripheral cause. Because the auditory pathways quickly become bilateral upon entering the brainstem, central lesions are unlikely to cause unilateral hearing loss, unless the lesion lies near the root entry zone of the auditory nerve. Symptoms such as double vision, numbness, and limb ataxia suggest a brainstem or cerebellar lesion. Because dizziness and imbalance can be a manifestation of a variety of neurologic disorders, the neurologic examination is important in the evaluation of these patients. Particular focus should be given to assessment of eye movements, vestibular function, and hearing.

1	The range of eye movements and whether they are equal in each eye should be observed. Peripheral eye movement disorders (e.g., cranial neuropathies, eye muscle weakness) are usually disconjugate (different in the two eyes). One should check pursuit (the ability to follow a smoothly moving target) and saccades (the ability to look back and forth accurately between two targets). Poor pursuit or inaccurate (dysmetric) saccades usually indicates central pathology, often involving the cerebellum. Finally, one should look for spon taneous nystagmus, an involuntary back-and-forth movement of the eyes. Nystagmus is most often of the jerk type, in which a slow drift (slow phase) in one direction alternates with a rapid saccadic movement (quick phase or fast phase) in the opposite direction that resets the position of the eyes in the orbits. Except in the case of acute vestibulopathy (e.g., vestibular neuritis), if primary position nystagmus is easily seen in the light, it is probably due to a

1	the position of the eyes in the orbits. Except in the case of acute vestibulopathy (e.g., vestibular neuritis), if primary position nystagmus is easily seen in the light, it is probably due to a central cause. Two forms of nystagmus that are characteristic of lesions of the cerebellar pathways are vertical nystagmus with downward changes direction with gaze (gaze-evoked nystagmus). By contrast, tagmus. Use of Frenzel eyeglasses (self-illuminated goggles with convex lenses that blur the patient’s vision but allow the examiner to see the eyes greatly magnified) can aid in the detection of peripheral vestibular nystagmus, because they reduce the patient’s ability to use visual fixation to suppress nystagmus. Table 28-1 outlines key find ings that help distinguish peripheral from central causes of vertigo.

1	The most useful bedside test of peripheral vestibular function is the head impulse test, in which the vestibuloocular reflex (VOR) is assessed with small-amplitude (~20 degrees) rapid head rotations. While the patient fixates on a target, the head is rotated to the left or right. If the VOR is deficient, the rotation is followed by a catch up saccade in the opposite direction (e.g., a leftward saccade after a rightward rotation). The head impulse test can identify both uni lateral (catch-up saccades after rotations toward the weak side) and in both directions).

1	All patients with episodic dizziness, especially if provoked by positional change, should be tested with the Dix-Hallpike maneu ver. The patient begins in a sitting position with the head turned fEATuRES of PERiPHERAL AnD CEnTRAL vERTigo • Nystagmus from an acute peripheral lesion is unidirectional, with fast phases beating away from the ear with the lesion. Nystagmus that changes direction with gaze is due to a central lesion. mixed vertical-torsional nystagmus occurs in BPPV, but pure vertical or pure torsional nystagmus is a central sign. from a peripheral lesion may be inhibited by visual fixation, whereas central nystagmus is not suppressed. • Absence of a head impulse sign in a patient with acute prolonged vertigo should suggest a central cause. • Unilateral hearing loss suggests peripheral vertigo. Findings such as diplopia, dysarthria, and limb ataxia suggest a central disorder.

1	• Unilateral hearing loss suggests peripheral vertigo. Findings such as diplopia, dysarthria, and limb ataxia suggest a central disorder. 45 degrees; holding the back of the head, the examiner then lowers the patient into a supine position with the head extended backward by about 20 degrees while watching the eyes. Posterior canal BPPV can be diagnosed confidently if transient upbeating-torsional nystagmus is seen. If no nystagmus is observed after 15–20 s, the patient is raised to the sitting position, and the procedure is repeated with the head turned to the other side. Again, Frenzel goggles may improve the sensitivity of the test.

1	Dynamic visual acuity is a functional test that can be useful in assessing vestibular function. Visual acuity is measured with the head still and when the head is rotated back and forth by the examiner (about 1–2 Hz). A drop in visual acuity during head motion of more than one line on a near card or Snellen chart is abnormal and indicates vestibular dysfunction.

1	The choice of ancillary tests should be guided by the history and examination findings. Audiometry should be performed whenever a vestibular disorder is suspected. Unilateral sensorineural hearing loss supports a peripheral disorder (e.g., vestibular schwannoma). Predominantly low-frequency hearing loss is characteristic of Ménière’s disease. Electronystagmography or videonystagmography includes recordings of spontaneous nystagmus (if present) and measurement of positional nystagmus. Caloric testing assesses the responses of the two horizontal semicircular canals. The test battery often includes recording of saccades and pursuit to assess central ocular motor function. Neuroimaging is important if a central vestibular disorder is suspected. In addition, patients with unexplained unilateral hearing loss or vestibular hypofunction should undergo magnetic resonance imaging (MRI) of the internal auditory canals, including administration of gadolinium, to rule out a schwannoma.

1	Treatment of vestibular symptoms should be driven by the underlying diagnosis. Simply treating dizziness with vestibular suppressant medications is often not helpful and may make the symptoms worse and prolong recovery. The diagnostic and specific treatment approaches for the most commonly encountered vestibular disorders are discussed below. An acute unilateral vestibular lesion causes constant vertigo, nausea, vomiting, oscillopsia (motion of the visual scene), and imbalance. These symptoms are due to a sudden asymmetry of inputs from the two labyrinths or in their central connections, simulating a continuous rotation of the head. Unlike BPPV, continuous vertigo persists even when the head remains still.

1	When a patient presents with an acute vestibular syndrome, the most important question is whether the lesion is central (e.g., a cerebellar or brainstem infarct or hemorrhage), which may be life-threatening, or peripheral, affecting the vestibular nerve or labyrinth (vestibular neuritis). Attention should be given to any symptoms or signs that point to central dysfunction (diplopia, weakness or numbness, dysarthria). The pattern of spontaneous nystagmus, if present, may be helpful (Table 28-1). If the head impulse test is normal, an acute peripheral vestibular lesion is unlikely. A central lesion cannot always be excluded with certainty based on symptoms and examination alone; thus, older patients with vascular risk factors who present with an acute vestibular syndrome should be evaluated for the possibility of stroke even when there are no specific findings that indicate a central lesion.

1	Most patients with vestibular neuritis recover spontaneously, but glucocorticoids can improve outcome if administered within 3 days of symptom onset. Antiviral medications are of no proven benefit and are not typically given unless there is evidence to suggest herpes zoster oticus (Ramsay Hunt syndrome). Vestibular suppressant medications may reduce acute symptoms but should be avoided after the first several days because they may impede central compensation and recovery. Patients should be encouraged to resume a normal level of activity as soon as possible, and directed vestibular rehabilitation therapy may accelerate improvement.

1	150 BENIgN PAROXYSMAL POSITIONAL VERTIgO BPPV is a common cause of recurrent vertigo. Episodes are brief (<1 min and typically 15–20 s) and are always provoked by changes in head position relative to gravity, such as lying down, rolling over in bed, rising from a supine position, and extending the head to look upward. The attacks are caused by free-floating otoconia (calcium carbonate crystals) that have been dislodged from the utricular macula and have moved into one of the semicircular canals, usually the posterior canal. When head position changes, gravity causes the otoconia to move within the canal, producing vertigo and nystagmus. With posterior canal BPPV, the nystagmus beats upward and torsionally (the upper poles of the eyes beat toward the affected lower ear). Less commonly, the otoconia enter the horizontal canal, resulting in a horizontal nystagmus when the patient is lying with either ear down. Superior (also called anterior) canal involvement is rare. BPPV is treated

1	the otoconia enter the horizontal canal, resulting in a horizontal nystagmus when the patient is lying with either ear down. Superior (also called anterior) canal involvement is rare. BPPV is treated with repositioning maneuvers that use gravity to remove the otoconia from the semicircular canal. For posterior canal BPPV, the Epley maneuver (Fig. 28-1) is the most commonly used procedure. For more refractory cases of BPPV, patients can be taught a variant of this maneuver that they can perform alone at home. A demonstration of the Epley maneuver is available online (http:// www.dizziness-and-balance.com/disorders/bppv/bppv.html).

1	Vestibular symptoms occur frequently in migraineurs, sometimes as a headache aura but usually independent of headache. The duration of vertigo may be from minutes to hours, and some patients also experience more prolonged periods of disequilibrium (lasting days to weeks). Motion sensitivity and sensitivity to visual motion (e.g., movies) are common in patients with vestibular migraine. Although data from controlled studies are generally lacking, vestibular migraine typically is treated with medications that are used for prophylaxis of migraine headaches. Antiemetics may be helpful to relieve symptoms at the time of an attack. Attacks of Ménière’s disease consist of vertigo and hearing loss, as well as pain, pressure, and/or fullness in the affected ear. The low-frequency

1	PART 2 Cardinal Manifestations and Presentation of Diseases hearing loss and aural symptoms are key features that distinguish Ménière’s disease from other peripheral vestibulopathies and from vestibular migraine. Audiometry at the time of an attack shows a characteristic asymmetric low-frequency hearing loss; hearing commonly improves between attacks, although permanent hearing loss may eventually occur. Ménière’s disease is thought to be due to excess fluid (endolymph) in the inner ear; hence the term endolymphatic hydrops. Patients suspected of having Ménière’s disease should be referred to an otolaryngologist for further evaluation. Diuretics and sodium restriction are the initial treatments. If attacks persist, injections of gentamicin into the middle ear are typically the next line of therapy. Full ablative procedures (vestibular nerve section, labyrinthectomy) are seldom required.

1	Vestibular schwannomas (sometimes termed acoustic neuromas) and other tumors at the cerebellopontine angle cause slowly progressive unilateral sensorineural hearing loss and vestibular hypofunction. These patients typically do not have vertigo, because the gradual vestibular deficit is compensated centrally as it develops. The diagnosis often is not made until there is sufficient hearing loss to be noticed. The examination will show a deficient response to the head impulse test when the head is rotated toward the affected side. As noted above, patients with unexplained unilateral sensorineural hearing loss or vestibular hypofunction require MRI of the internal auditory canals to look for a schwannoma.

1	Patients with bilateral loss of vestibular function also typically do not have vertigo, because vestibular function is lost on both sides simultaneously, and there is no asymmetry of vestibular input. Symptoms include loss of balance, particularly in the dark, where vestibular input is most critical, and oscillopsia during head movement, such as while walking or riding in a car. Bilateral vestibular hypofunction may be (1) idiopathic and progressive, (2) part of a neurodegenerative disorder, or (3) iatrogenic, due to medication ototoxicity (most commonly

1	FIguRE 28-1 Modified Epley maneuver for treatment of benign paroxysmal positional vertigo of the right (top panels) and left (bottom panels) posterior semicircular canals. Step 1. With the patient seated, turn the head 45 degrees toward the affected ear. Step 2. Keeping the head turned, lower the patient to the head-hanging position and hold for at least 30 s and until nystagmus disappears. Step 3. Without lifting the head, turn it 90 degrees toward the other side. Hold for another 30 s. Step 4. Rotate the patient onto her side while turning the head another 90 degrees, so that the nose is pointed down 45 degrees. Hold again for 30 s. Step 5. Have the patient sit up on the side of the table. After a brief rest, the maneuver should be repeated to confirm successful treatment. (Figure adapted from http://www.dizziness-and-balance.com/ disorders/bppv/movies/Epley-480x640.avi.) gentamicin or other aminoglycoside antibiotics). Other causes include bilateral vestibular schwannomas

1	adapted from http://www.dizziness-and-balance.com/ disorders/bppv/movies/Epley-480x640.avi.) gentamicin or other aminoglycoside antibiotics). Other causes include bilateral vestibular schwannomas (neurofibromatosis type 2), autoimmune disease, superficial siderosis, and meningeal-based infection or tumor. It also may occur in patients with peripheral polyneuropathy; in these patients, both vestibular loss and impaired proprioception may contribute to poor balance. Finally, unilateral processes such as vestibular neuritis and Ménière’s disease may involve both ears sequentially, resulting in bilateral vestibulopathy.

1	Examination findings include diminished dynamic visual acuity (see above) due to loss of stable vision when the head is moving, abnormal head impulse responses in both directions, and a Romberg sign. Responses to caloric testing are reduced. Patients with bilateral vestibular hypofunction should be referred for vestibular rehabilitation therapy. Vestibular suppressant medications should not be used, as they will increase the imbalance. Evaluation by a neurologist is important not only to confirm the diagnosis but also to consider any other associated neurologic abnormalities that may clarify the etiology.

1	Central lesions causing vertigo typically involve vestibular pathways in the brainstem and/or cerebellum. They may be due to discrete lesions, such as from ischemic or hemorrhagic stroke (Chap. 446), demyelination (Chap. 458), or tumors (Chap. 118), or they may be due to neurodegenerative conditions that include the vestibulocerebellum (Chap. 448). Subacute cerebellar degeneration may be due to immune, including paraneoplastic, processes (Chaps. 122 and 450). Table 28-1 outlines important features of the history and examination that help to identify central vestibular disorders. Acute central vertigo is a medical emergency, due to the possibility of life-threatening stroke or hemorrhage. All patients with suspected central vestibular disorders should undergo brain MRI, and the patient should be referred for full neurologic evaluation.

1	Psychological factors play an important role in chronic dizziness. First, dizziness may be a somatic manifestation of a psychiatric condition such as major depression, anxiety, or panic disorder (Chap. 465e). Second, patients may develop anxiety and autonomic symptoms as a consequence or comorbidity of an independent vestibular disorder. One particular form of this has been termed variously phobic postural vertigo, psychophysiologic vertigo, or chronic subjective dizziness. These patients have a chronic feeling (months or longer) of dizziness and disequilibrium, an increased sensitivity to self-motion and visual motion (e.g., movies), and a particular intensification of symptoms when moving through complex visual environments such as supermarkets (visual vertigo). Although there may be a past history of an acute vestibular disorder (e.g., vestibular neuritis), the neurootologic examination and vestibular testing are normal or indicative of a compensated vestibular deficit, indicating

1	past history of an acute vestibular disorder (e.g., vestibular neuritis), the neurootologic examination and vestibular testing are normal or indicative of a compensated vestibular deficit, indicating that the ongoing subjective dizziness cannot be explained by a primary vestibular disorder. Anxiety disorders are particularly common in patients with chronic dizziness and contribute substantially to the morbidity. Thus, treatment with antianxiety medications (selective serotonin reuptake inhibitors [SSRIs]) and cognitive-behavioral therapy may be helpful. Vestibular rehabilitation therapy is also sometimes beneficial. Vestibular suppressant medications generally should be avoided. This condition should be suspected when the patient states, “My dizziness is so bad, I’m afraid to leave my house” (agoraphobia).

1	Table 28-2 provides a list of commonly used medications for suppression of vertigo. As noted, these medications should be reserved for short-term control of active vertigo, such as during the first few days of acute vestibular neuritis, or for acute attacks of Ménière’s disease. They are less helpful for chronic dizziness and, as previously stated, may hinder central compensation. An exception is that benzodiazepines may attenuate psychosomatic dizziness and the associated anxiety, although SSRIs are generally preferable in such patients. Benzodiazepines Diazepam 2.5 mg 1–3 times daily Clonazepam 0.25 mg 1–3 times daily days 4–6; 60 mg daily days 7–9; 40 mg daily days 10–12; 20 mg daily days 13–15; 10 mg daily days 16–18, 20, 22

1	Benzodiazepines Diazepam 2.5 mg 1–3 times daily Clonazepam 0.25 mg 1–3 times daily days 4–6; 60 mg daily days 7–9; 40 mg daily days 10–12; 20 mg daily days 13–15; 10 mg daily days 16–18, 20, 22 Selective serotonin reuptake inhibitorsh aAll listed drugs are approved by the U.S. Food and Drug Administration, but most are not approved for the treatment of vertigo. bUsual oral (unless otherwise stated) starting dose in adults; a higher maintenance dose can be reached by a gradual increase. cFor motion sickness only. dFor benign paroxysmal positional vertigo. eFor Ménière’s disease. fFor vestibular migraine. gFor acute vestibular neuritis (started within 3 days of onset). hFor psychosomatic vertigo.

1	Vestibular rehabilitation therapy promotes central adaptation processes that compensate for vestibular loss and also may help habituate motion sensitivity and other symptoms of psychosomatic dizziness. The general approach is to use a graded series of exercises that progressively challenge gaze stabilization and balance. Jeffrey M. Gelfand, Vanja C. Douglas

1	Fatigue is one of the most common symptoms in clinical medicine. It is a prominent manifestation of a number of systemic, neurologic, and psychiatric syndromes, although a precise cause will not be identified in a substantial minority of patients. Fatigue refers to an inherently subjective human experience of physical and mental weariness, sluggishness, and exhaustion. In the context of clinical medicine, fatigue is most typically and practically defined as difficulty initiating or maintaining voluntary mental or physical activity. Nearly everyone who has ever been ill with a self-limited infection has experienced this near-universal symptomatology, and fatigue is usually brought to medical attention only when it is either of unclear cause or the severity is out of proportion with what would be expected for the associated trigger. Fatigue should be distinguished from muscle weakness, a reduction of neuromuscular power (Chap. 30); most patients complaining of fatigue are not truly weak

1	be expected for the associated trigger. Fatigue should be distinguished from muscle weakness, a reduction of neuromuscular power (Chap. 30); most patients complaining of fatigue are not truly weak when direct muscle power is tested. By definition, fatigue is also distinct from somnolence and dyspnea on exertion, although patients may use the word fatigue to describe those two symptoms. The task facing clinicians when a patient presents with fatigue is to identify an underlying cause if one exists and to develop 152 a therapeutic alliance, the goal of which is to spare patients expensive and fruitless diagnostic workups and steer them toward effective therapy.

1	Variability in the definitions of fatigue and the survey instruments used in different studies makes it difficult to arrive at precise figures about the global burden of fatigue. The point prevalence of fatigue was 6.7% and the lifetime prevalence was 25% in a large National Institute of Mental Health survey of the U.S. general population. In primary care clinics in Europe and the United States, between 10 and 25% of patients surveyed endorsed symptoms of prolonged (present for >1 month) or chronic (present for >6 months) fatigue, but fatigue was the primary reason for seeking medical attention in only a minority of patients. In a community survey of women in India, 12% reported chronic fatigue. By contrast, the prevalence of chronic fatigue syndrome, as defined by the U.S. Centers for Disease Control and Prevention, is low (Chap. 464e).

1	DIFFERENTIAL DIAgNOSIS Psychiatric Disease Fatigue is a common somatic manifestation of many major psychiatric syndromes, including depression, anxiety, and somatoform disorders. Psychiatric symptoms are reported in more than three-quarters of patients with unexplained chronic fatigue. Even in patients with systemic or neurologic syndromes in which fatigue is independently recognized as a manifestation of disease, comorbid psychiatric symptoms or disease may still be an important source of interaction.

1	Neurologic Disease Patients complaining of fatigue often say they feel weak, but upon careful examination, objective muscle weakness is rarely discernible. If found, muscle weakness must then be localized to the central nervous system, peripheral nervous system, neuromuscular junction, or muscle and the appropriate follow-up studies obtained (Chap. 30). Fatigability of muscle power is a cardinal manifestation of some neuromuscular disorders such as myasthenia gravis and can be distinguished from fatigue by finding clinically apparent diminution of the amount of force that a muscle generates upon repeated contraction (Chap. 461). Fatigue is one of the most common and bothersome symptoms reported in multiple sclerosis (MS) (Chap. 458), affecting nearly 90% of patients; fatigue in MS can persist between MS attacks and does not necessarily correlate with magnetic resonance imaging (MRI) disease activity. Fatigue is also increasingly identified as a troublesome feature of many other

1	persist between MS attacks and does not necessarily correlate with magnetic resonance imaging (MRI) disease activity. Fatigue is also increasingly identified as a troublesome feature of many other neurodegenerative diseases, including Parkinson’s disease, central dysautonomias, and amyotrophic lateral sclerosis. Poststroke fatigue is a well-described but poorly understood entity with a widely varying prevalence. Episodic fatigue can be a premonitory symptom of migraine. Fatigue is also a frequent result of traumatic brain injury, often occurring in association with depression and sleep disorders.

1	Sleep Disorders Obstructive sleep apnea is an important cause of excessive daytime sleepiness in association with fatigue and should be investigated using overnight polysomnography, particularly in those with prominent snoring, obesity, or other predictors of obstructive sleep apnea (Chap. 319). Whether the cumulative sleep deprivation that is common in modern society contributes to clinically apparent fatigue is not known (Chap. 38).

1	Endocrine Disorders Fatigue, sometimes in association with true muscle weakness, can be a heralding symptom of hypothyroidism, particularly in the context of hair loss, dry skin, cold intolerance, constipation, and weight gain. Fatigue in association with heat intolerance, sweating, and palpitations is typical of hyperthyroidism. Adrenal insufficiency can also manifest with unexplained fatigue as a primary or prominent symptom, often in association with anorexia, weight loss, nausea, myalgias, and arthralgias; hyponatremia and hyperkalemia may be present at time of diagnosis. Mild hypercalcemia can cause fatigue, which may be relatively vague, whereas severe hypercalcemia can lead

1	PART 2 Cardinal Manifestations and Presentation of Diseases to lethargy, stupor, and coma. Both hypoglycemia and hyperglycemia can cause lethargy, often in association with confusion; chronic diabetes, particularly type 1 diabetes, is also associated with fatigue independent of glucose levels. Fatigue may also accompany Cushing’s disease, hypoaldosteronism, and hypogonadism. Liver and Kidney Disease Both chronic liver failure and chronic kidney disease can cause fatigue. Over 80% of hemodialysis patients complain of fatigue, which makes this one of the most common patient-reported symptoms in chronic kidney disease.

1	Obesity Obesity is associated with fatigue and sleepiness independent of the presence of obstructive sleep apnea. Obese patients undergoing bariatric surgery experience improvement in daytime sleepiness sooner than would be expected if the improvement were solely the result of weight loss and resolution of sleep apnea. A number of other factors common in obese patients are likely contributors as well, including depression, physical inactivity, and diabetes. Malnutrition Although fatigue can be a presenting feature of malnutrition, nutritional status may also be an important comorbidity and contributor to fatigue in other chronic illnesses, including cancer-associated fatigue.

1	Infection Both acute and chronic infections commonly lead to fatigue as part of the broader infectious syndrome. Evaluation for undiagnosed infection as the cause of unexplained fatigue, and particularly prolonged or chronic fatigue, should be guided by the history, physical examination, and infectious risk factors, with particular attention to risk for tuberculosis, HIV, chronic hepatitis B and C, and endocarditis. Infectious mononucleosis may cause prolonged fatigue that persists for weeks to months following the acute illness, but infection with the Epstein-Barr virus is only very rarely the cause of unexplained chronic fatigue. Drugs Many medications, drug use, drug withdrawal, and chronic alcohol use can all lead to fatigue. Medications that are more likely to be causative in this context include antidepressants, antipsychotics, anxiolytics, opiates, antispasticity agents, antiseizure agents, and beta blockers.

1	Cardiovascular and Pulmonary Fatigue is one of the most taxing patient-reported symptoms of congestive heart failure and chronic obstructive pulmonary disease and negatively affects quality of life. Malignancy Fatigue, particularly in association with unexplained unintended weight loss, can be a sign of occult malignancy, but this is only rarely identified as causative in patients with unexplained chronic fatigue in the absence of other telltale signs or symptoms. Cancer-related fatigue is experienced by 40% of patients at time of diagnosis and greater than 80% of patients later in the disease course. Hematologic Chronic or progressive anemia may present with fatigue, sometimes in association with exertional tachycardia and breathlessness. Anemia may also contribute to fatigue in chronic illness. Low serum ferritin in the absence of anemia may also cause fatigue that is reversible with iron replacement.

1	Systemic Inflammatory/Rheumatologic Disorders Fatigue is a prominent complaint in many chronic inflammatory disorders, including systemic lupus erythematosus, polymyalgia rheumatica, rheumatoid arthritis, inflammatory bowel disease, antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis, sarcoidosis, and Sjögren’s syndrome, but is not usually an isolated symptom. Pregnancy Fatigue is very commonly reported by women during all stages of pregnancy and postpartum.

1	Pregnancy Fatigue is very commonly reported by women during all stages of pregnancy and postpartum. Disorders of unclear Cause Chronic fatigue syndrome (Chap. 464e) and fibromyalgia (Chap. 396) incorporate chronic fatigue as part of the syndromic definition when present in association with a number of other inclusion and exclusion criteria, as discussed in detail in their respective chapters. The pathophysiology of each is unknown. Idiopathic chronic fatigue is used to describe the syndrome of unexplained chronic fatigue in the absence of enough additional clinical features to meet the diagnostic criteria for chronic fatigue syndrome. APPROACH TO THE PATIENT:

1	A detailed history focusing on the quality, pattern, time-course, associated symptoms, and alleviating factors of fatigue is critical in defining the syndrome, determining whether fatigue is the appropriate designation, determining whether the symptoms are acute or chronic, and determining whether fatigue is primarily mental, physical, or both in order to direct further evaluation and treatment. The review of systems should attempt to distinguish fatigue from excessive daytime sleepiness, dyspnea on exertion, exercise intolerance, and muscle weakness. The presence of fever, chills, night sweats, or weight loss should raise suspicion for an occult infection or malignancy. A careful review of prescription, over-the-counter, herbal, and recreational drug and alcohol use is mandatory. Circumstances surrounding the onset of symptoms and potential triggers should be investigated. The social history is important, with attention paid to job stress and work hours, the social support network,

1	surrounding the onset of symptoms and potential triggers should be investigated. The social history is important, with attention paid to job stress and work hours, the social support network, and domestic affairs including a screen for intimate partner violence. Sleep habits and sleep hygiene should be questioned. The impact of fatigue on daily functioning is important to understand the patient’s experience and gauge recovery and the success of treatment.

1	The physical examination of patients with fatigue is guided by the history and differential diagnosis. A detailed mental status examination should be performed with particular attention to symptoms of depression and anxiety. A formal neurologic examination is required to determine whether objective muscle weakness is present. This is usually a straightforward exercise, although occasionally patients with fatigue have difficulty sustaining effort against resistance and sometimes report that generating full power requires substantial mental effort. On confrontational testing, they are able to generate full power for only a brief period before suddenly giving way to the examiner. This type of weakness is often referred to as breakaway weakness and may or may not be associated with pain. This is contrasted with weakness due to lesions in the motor tracts or lower motor unit, in which the patient’s resistance can be overcome in a smooth and steady fashion and full power can never be

1	This is contrasted with weakness due to lesions in the motor tracts or lower motor unit, in which the patient’s resistance can be overcome in a smooth and steady fashion and full power can never be generated. Occasionally, a patient may demonstrate fatigable weakness, in which power is full when first tested but becomes weak upon repeat evaluation without interval rest. Fatigable weakness, which usually indicates a problem of neuromuscular transmission, never has the sudden breakaway quality that one occasionally observes in patients with fatigue. If the presence or absence of muscle weakness cannot be determined with the physical examination, electromyography with nerve conductions studies can be a helpful ancillary test.

1	The general physical examination should screen for signs of cardiopulmonary disease, malignancy, lymphadenopathy, organomegaly, infection, liver failure, kidney disease, malnutrition, endocrine abnormalities, and connective tissue disease. Although the diagnostic yield of the general physical examination may be relatively low in the context of evaluation of unexplained chronic fatigue, elucidating the cause of 2% of cases in one prospective analysis, the yield of a detailed neuropsychiatric and mental status evaluation is likely to be much higher, revealing a potential explanation for fatigue in up to 75–80% of patients in some series. Furthermore, the rite of physical examination demonstrates a thorough and systematic approach to the patient’s complaint and helps build trust and a therapeutic alliance.

1	Laboratory testing is likely to identify the cause of chronic fatigue in only about 5% of cases. Beyond a few standard screening tests, laboratory evaluation should be guided by the history and physical examination; extensive testing is more likely to lead to false-positive results that require explanation and unnecessary investigation and should be avoided in lieu of frequent clinical follow-up. A reasonable approach to screening includes a complete blood count with differential (to screen for anemia, infection, and malignancy), electrolytes (including sodium, potassium, and calcium), glucose, renal function, liver function, and thyroid function. Testing for HIV and adrenal function can also be considered. Published guidelines defining chronic fatigue syndrome also recommend an erythrocyte sedimentation rate (ESR) as part of the evaluation for mimics, but unless the value is very high, such nonspecific testing in the absence of other features is unlikely to clarify the situation.

1	sedimentation rate (ESR) as part of the evaluation for mimics, but unless the value is very high, such nonspecific testing in the absence of other features is unlikely to clarify the situation. Routine screening with an antinuclear antibody (ANA) test is also unlikely to be informative in isolation and is frequently positive at low titers in otherwise healthy adults. Additional unfocused studies, such as whole-body imaging scans, are usually not indicated; in addition to their inconvenience, potential risk, and cost, they often reveal unrelated incidental findings that can prolong the workup unnecessarily.

1	The first priority of treatment is to address the underlying disorder or disorders that account for fatigue, because this can be curative in select contexts and palliative in others. Unfortunately, in many chronic illnesses, fatigue may be refractory to traditional disease-modifying therapies, and it is important in such cases to evaluate for other potential contributors, because the cause may be multifactorial. Antidepressant treatment (Chap. 466) may be helpful for treatment of chronic fatigue when symptoms of depression are present and may be most effective in the context of a multimodal approach. However, antidepressants can also cause fatigue and should be discontinued if they are not clearly effective. Cognitive-behavioral therapy has also been demonstrated to be helpful in the context of chronic fatigue syndrome as well as cancer-associated fatigue. Graded exercise therapy in which physical exercise, most typically walking, is gradually increased with attention to target heart

1	of chronic fatigue syndrome as well as cancer-associated fatigue. Graded exercise therapy in which physical exercise, most typically walking, is gradually increased with attention to target heart rates to avoid overexertion, was shown to modestly improve walking times and self-reported fatigue measures in patients in the United Kingdom with chronic fatigue syndrome in the large 2011 randomized controlled PACE trial. Psychostimulants such as amphetamines, modafinil, and armodafinil can help increase alertness and concentration and reduce excessive daytime sleepiness in certain clinical contexts, which may in turn help with symptoms of fatigue in a minority of patients, but they have generally proven to be unhelpful in randomized trials for treating fatigue in posttraumatic brain injury, Parkinson’s disease, and MS.

1	Development of more effective therapy for fatigue is hampered by limited knowledge of the biologic basis of this symptom. Tentative data suggests that proinflammatory cytokines, such as interleukin 1β and tumor necrosis factor α, might mediate fatigue in some patients; thus, cytokine antagonists represent one possible future approach.

1	Acute fatigue significant enough to require medical evaluation is more likely to lead to an identifiable medical, neurologic, or psychiatric cause than unexplained chronic fatigue. Evaluation of unexplained chronic fatigue most commonly leads to diagnosis of a psychiatric condition or remains unexplained. Identification of a previously undiagnosed serious or life-threatening culprit etiology is rare on longitudinal follow-up in patients with unexplained chronic fatigue. Complete resolution of unexplained chronic fatigue is uncommon, at least over the short term, but multidisciplinary treatment approaches can lead to symptomatic improvements that can substantially improve quality of life. neurologic Causes of weakness and Paralysis Michael J. Aminoff Normal motor function involves integrated muscle activity that is modulated by the activity of the cerebral cortex, basal ganglia, cer-30154

1	PART 2 Cardinal Manifestations and Presentation of Diseases ebellum, red nucleus, brainstem reticular formation, lateral vestibular nucleus, and spinal cord. Motor system dysfunction leads to weakness or paralysis, discussed in this chapter, or to ataxia (Chap. 450) or abnormal movements (Chap. 449). Weakness is a reduction in the power that can be exerted by one or more muscles. It must be distinguished from increased fatigability (i.e., the inability to sustain the performance of an activity that should be normal for a person of the same age, sex, and size), limitation in function due to pain or articular stiffness, or impaired motor activity because severe proprioceptive sensory loss prevents adequate feedback information about the direction and power of movements. It is also distinct from bradykinesia (in which increased time is required for full power to be exerted) and apraxia, a disorder of planning and initiating a skilled or learned movement unrelated to a significant motor

1	from bradykinesia (in which increased time is required for full power to be exerted) and apraxia, a disorder of planning and initiating a skilled or learned movement unrelated to a significant motor or sensory deficit (Chap. 36).

1	Paralysis or the suffix “-plegia” indicates weakness so severe that a muscle cannot be contracted at all, whereas paresis refers to less severe weakness. The prefix “hemi-” refers to one-half of the body, “para-” to both legs, and “quadri-” to all four limbs. The distribution of weakness helps to localize the underlying lesion. Weakness from involvement of upper motor neurons occurs particularly in the extensors and abductors of the upper limb and the flexors of the lower limb. Lower motor neuron weakness depends on whether involvement is at the level of the anterior horn cells, nerve root, limb plexus, or peripheral nerve—only muscles supplied by the affected structure are weak. Myopathic weakness is generally most marked in proximal muscles. Weakness from impaired neuromuscular transmission has no specific pattern of involvement. Weakness often is accompanied by other neurologic abnormalities that help indicate the site of the responsible lesion (Table 30-1).

1	Tone is the resistance of a muscle to passive stretch. Increased tone may be of several types. Spasticity is the increase in tone associated with disease of upper motor neurons. It is velocity-dependent, has a sudden release after reaching a maximum (the “clasp-knife” phenomenon), and predominantly affects the antigravity muscles (i.e., upper-limb flexors and lower-limb extensors). Rigidity is hypertonia that is present throughout the range of motion (a “lead pipe” or “plastic” stiffness) and affects flexors and extensors equally; it sometimes has a cogwheel quality that is enhanced by voluntary movement of the contralateral limb (reinforcement). Rigidity occurs with certain extrapyramidal disorders, such as Parkinson’s disease. Paratonia (or gegenhalten) is increased tone that varies irregularly in a manner seemingly related to the degree of relaxation, is present throughout the range of motion, and affects flexors and extensors equally; it usually results from disease of the frontal

1	in a manner seemingly related to the degree of relaxation, is present throughout the range of motion, and affects flexors and extensors equally; it usually results from disease of the frontal lobes. Weakness with decreased tone (flaccidity) or normal tone occurs with disorders of motor units. A motor unit consists of a single lower motor neuron and all the muscle fibers that it innervates.

1	Muscle bulk generally is not affected by upper motor neuron lesions, although mild disuse atrophy eventually may occur. By contrast, atrophy is often conspicuous when a lower motor neuron lesion is responsible for weakness and also may occur with advanced muscle disease.

1	Muscle stretch (tendon) reflexes are usually increased with upper motor neuron lesions, but may be decreased or absent for a variable period immediately after onset of an acute lesion. Hyperreflexia is usually—but not invariably—accompanied by loss of cutaneous reflexes (such as superficial abdominals; Chap. 437) and, in particular, by an extensor plantar (Babinski) response. The muscle stretch reflexes are depressed with lower motor neuron lesions directly involving specific reflex arcs. They generally are preserved in patients with myopathic weakness except in advanced stages, when they sometimes are attenuated. In disorders of the neuromuscular junction, reflex responses may be affected by preceding voluntary activity of affected muscles; such activity may lead to enhancement of initially depressed reflexes in Lambert-Eaton myasthenic syndrome and, conversely, to depression of initially normal reflexes in myasthenia gravis (Chap. 461).

1	The distinction of neuropathic (lower motor neuron) from myopathic weakness is sometimes difficult clinically, although distal weakness is likely to be neuropathic, and symmetric proximal weakness myopathic. Fasciculations (visible or palpable twitch within a muscle due to the spontaneous discharge of a motor unit) and early atrophy indicate that weakness is neuropathic.

1	PATHOgENESIS upper Motor Neuron Weakness Lesions of the upper motor neurons or their descending axons to the spinal cord (Fig. 30-1) produce weakness through decreased activation of lower motor neurons. In general, distal muscle groups are affected more severely than proximal ones, and axial movements are spared unless the lesion is severe and bilateral. Spasticity is typical but may not be present acutely. Rapid repetitive movements are slowed and coarse, but normal rhythmicity is maintained. With corticobulbar involvement, weakness occurs in the lower face and tongue; extraocular, upper facial, pharyngeal, and jaw muscles are typically spared. Bilateral corticobulbar lesions produce a pseudobulbar palsy: dysarthria, dysphagia, dysphonia, and emotional lability accompany bilateral facial weakness and a brisk jaw jerk. Electromyogram (EMG) (Chap. 442e) shows that with weakness of the upper motor neuron type, motor units have a diminished maximal discharge frequency.

1	Lower Motor Neuron Weakness This pattern results from disorders of lower motor neurons in the brainstem motor nuclei and the anterior horn of the spinal cord or from dysfunction of the axons of these neurons as they pass to skeletal muscle (Fig. 30-2). Weakness is due to a decrease in the number of muscle fibers that can be activated through a loss of α motor neurons or disruption of their connections to muscle. Loss of γ motor neurons does not cause weakness but decreases tension on the muscle spindles, which decreases muscle tone and attenuates the stretch reflexes. An absent stretch reflex suggests involvement of spindle afferent fibers. When a motor unit becomes diseased, especially in anterior horn cell diseases, it may discharge spontaneously, producing fasciculations. When α motor neurons or their axons degenerate, the denervated muscle fibers also may discharge spontaneously. These single muscle

1	FIguRE 30-1 The corticospinal and bulbospinal upper motor neuron pathways. Upper motor neurons have their cell bodies in layer V of the primary motor cortex (the precentral gyrus, or Brodmann’s area 4) and in the premotor and supplemental motor cortex (area 6). The upper motor neurons in the primary motor cortex are somatotopically organized (right side of figure).

1	Axons of the upper motor neurons descend through the sub-cortical white matter and the posterior limb of the internal capsule. Axons of the pyramidal or corticospinal system descend through the brainstem in the cerebral peduncle of the midbrain, the basis pontis, and the medullary pyramids. At the cervicomedullary junction, most corticospinal axons decussate into the contralateral corticospinal tract of the lateral spinal cord, but 10–30% remain ipsilateral in the anterior spinal cord. Corticospinal neurons synapse on premotor interneurons, but some—especially in the cervical enlargement and those connecting with motor neurons to distal limb muscles—make direct mono-synaptic connections with lower motor neurons. They innervate most densely the lower motor neurons of hand muscles and are involved in the execution of learned, fine movements. Corticobulbar neurons are similar to corticospinal neurons but innervate brainstem motor nuclei.

1	Bulbospinal upper motor neurons influence strength and tone but are not part of the pyramidal system. The descending ventromedial bulbospinal pathways originate in the tectum of the midbrain (tectospinal pathway), the vestibular nuclei (vestibulospinal pathway), and the reticular formation (reticulospinal pathway). These pathways influence axial and proximal muscles and are involved in the maintenance of posture and integrated movements of the limbs and trunk. The descending ventrolateral bulbospinal pathways, which originate predominantly in the red nucleus (rubrospinal pathway), facilitate distal limb muscles. The bulbospinal system sometimes is referred to as the extrapyramidal upper motor neuron system. In all figures, nerve cell bodies and axon terminals are shown, respectively, as closed circles and forks. FIguRE 30-2 Lower motor neurons are divided into α and γ types.

1	FIguRE 30-2 Lower motor neurons are divided into α and γ types. The larger α motor neurons are more numerous and innervate the extrafusal muscle fibers of the motor unit. Loss of α motor neurons or disruption of their axons produces lower motor neuron weakness. The smaller, less numerous γ motor neurons innervate the intrafusal muscle fibers of the muscle spindle and contribute to normal tone and stretch reflexes. The α motor neuron receives direct excitatory input from corticomotoneurons and primary muscle spindle afferents. The α and γ motor neurons also receive excitatory input from other descending upper motor neuron pathways, segmental sensory inputs, and interneurons. The α motor neurons receive direct inhibition from Renshaw cell interneurons, and other interneurons indirectly inhibit the α and γ motor neurons.

1	A muscle stretch (tendon) reflex requires the function of all the illustrated structures. A tap on a tendon stretches muscle spindles (which are tonically activated by γ motor neurons) and activates the primary spindle afferent neurons. These neurons stimulate the α motor neurons in the spinal cord, producing a brief muscle contraction, which is the familiar tendon reflex. fiber discharges, or fibrillation potentials, cannot be seen but can be recorded with EMG. Weakness leads to delayed or reduced recruitment of motor units, with fewer than normal activated at a particular discharge frequency.

1	Neuromuscular Junction Weakness Disorders of the neuromuscular junctions produce weakness of variable degree and distribution. The number of muscle fibers that are activated varies over time, depending on the state of rest of the neuromuscular junctions. Strength is influenced by preceding activity of the affected muscle. In myasthenia gravis, for example, sustained or repeated contractions of affected muscle decline in strength despite continuing effort (Chap. 461). Thus, fatigable weakness is suggestive of disorders of the neuromuscular junction, which cause functional loss of muscle fibers due to failure of their activation.

1	Myopathic Weakness Myopathic weakness is produced by a decrease in the number or contractile force of muscle fibers activated within motor units. With muscular dystrophies, inflammatory myopathies, or myopathies with muscle fiber necrosis, the number of muscle fibers is reduced within many motor units. On EMG, the size of each motor unit action potential is decreased, and motor units must be recruited more rapidly than normal to produce the desired power. Some myopathies produce weakness through loss of contractile force of muscle fibers or through relatively selective involvement of type II (fast) 156 fibers. These myopathies may not affect the size of individual motor unit action potentials and are detected by a discrepancy between the electrical activity and force of a muscle.

1	Psychogenic Weakness Weakness may occur without a recognizable organic basis. It tends to be variable, inconsistent, and with a pattern of distribution that cannot be explained on a neuroanatomic basis. On formal testing, antagonists may contract when the patient is supposedly activating the agonist muscle. The severity of weakness is out of keeping with the patient’s daily activities.

1	Hemiparesis Hemiparesis results from an upper motor neuron lesion above the midcervical spinal cord; most such lesions are above the foramen magnum. The presence of other neurologic deficits helps localize the lesion. Thus, language disorders, for example, point to a cortical lesion. Homonymous visual field defects reflect either a cortical or a subcortical hemispheric lesion. A “pure motor” hemiparesis of the face, arm, and leg often is due to a small, discrete lesion in the posterior limb of the internal capsule, cerebral peduncle, or upper pons. Some brainstem lesions produce “crossed paralyses,” consisting of ipsilateral cranial nerve signs and contralateral hemiparesis (Chap. 446). The absence of cranial nerve signs or facial weakness suggests that a hemiparesis is due to a lesion in the high cervical spinal cord, especially if associated with the Brown-Séquard syndrome (Chap. 456).

1	Acute or episodic hemiparesis usually results from focal structural lesions, particularly rapidly expanding lesions, or an inflammatory process. Subacute hemiparesis that evolves over days or weeks may relate to subdural hematoma, infectious or inflammatory disorders (e.g., cerebral abscess, fungal granuloma or meningitis, parasitic infection, multiple sclerosis, sarcoidosis), or primary and metastatic neoplasms. AIDS may present with subacute hemiparesis due to toxoplasmosis or primary central nervous system (CNS) lymphoma. Chronic hemiparesis that evolves over months usually is due to a neoplasm or vascular malformation, a chronic subdural hematoma, or a degenerative disease. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Investigation of hemiparesis (Fig. 30-3) of acute origin starts with a computed tomography (CT) scan of the brain and laboratory studies. If the CT is normal, or in subacute or chronic cases of hemiparesis, magnetic resonance imaging (MRI) of the brain and/or cervical spine (including the foramen magnum) is performed, depending on the clinical accompaniments.

1	Paraparesis Acute paraparesis is caused most commonly by an intra-spinal lesion, but its spinal origin may not be recognized initially if the legs are flaccid and areflexic. Usually, however, there is sensory loss in the legs with an upper level on the trunk, a dissociated sensory loss suggestive of a central cord syndrome (Chap. 456), or hyperreflexia in the legs with normal reflexes in the arms. Imaging the spinal cord (Fig. 30-3) may reveal compressive lesions, infarction (proprioception usually is spared), arteriovenous fistulas or other vascular anomalies, or transverse myelitis (Chap. 456). Diseases of the cerebral hemispheres that produce acute paraparesis include anterior cerebral artery ischemia (shoulder shrug also is affected), superior sagittal sinus or cortical venous thrombosis, and acute hydrocephalus.

1	Paraparesis may result from a cauda equina syndrome, for example, after trauma to the low back, a midline disk herniation, or an intraspinal tumor; although the sphincters are often affected, hip flexion often is spared, as is sensation over the anterolateral thighs. Rarely, paraparesis is caused by a rapidly evolving anterior horn cell disease (such as poliovirus or West Nile virus infection), peripheral neuropathy (such as Guillain-Barré syndrome; Chap. 460), or myopathy (Chap. 462e). Subacute or chronic spastic paraparesis is caused by upper motor neuron disease. When associated with lower-limb sensory loss and sphincter involvement, a chronic spinal cord disorder should be considered (Chap. 456). If hemispheric signs are present, a parasagittal meningioma or chronic hydrocephalus is likely. The absence of spasticity in a long-standing paraparesis suggests a lower motor neuron or myopathic etiology.

1	Hemiparesis UMN signs Cerebral signs Proximal Restricted Paraparesis Quadriparesis Monoparesis Distal LMN signs* Alert UMN signs LMN signs* UMN signs LMN signs* EMG and NCS UMN pattern LMN pattern Myopathic pattern Anterior horn, root, or peripheral nerve disease Muscle or neuromuscular junction disease * or signs of myopathy † If no abnormality detected, consider spinal MRI. ‡ If no abnormality detected, consider myelogram or brain MRI. NoYes NoYes DISTRIBUTION OF WEAKNESS Brain CT or MRI† Spinal MRI‡ FIguRE 30-3 An algorithm for the initial workup of a patient with weakness. CT, computed tomography; EMG, electromyography; LMN, lower motor neuron; MRI, magnetic resonance imaging; NCS, nerve conduction studies; UMN, upper motor neuron.

1	Investigations typically begin with spinal MRI, but when upper motor neuron signs are associated with drowsiness, confusion, seizures, or other hemispheric signs, brain MRI should also be performed, sometimes as the initial investigation. Electrophysiologic studies are diagnostically helpful when clinical findings suggest an underlying neuromuscular disorder.

1	Quadriparesis or generalized Weakness Generalized weakness may be due to disorders of the CNS or the motor unit. Although the terms often are used interchangeably, quadriparesis is commonly used when an upper motor neuron cause is suspected, and generalized weakness is used when a disease of the motor units is likely. Weakness from CNS disorders usually is associated with changes in consciousness or cognition and accompanied by spasticity, hyperreflexia, and sensory disturbances. Most neuromuscular causes of generalized weakness are associated with normal mental function, hypotonia, and hypoactive muscle stretch reflexes. The major causes of intermittent weakness are listed in Table 30-2. A patient with generalized fatigability without objective weakness may have the chronic fatigue syndrome (Chap. 464e).

1	ACUTE qUAdRIPARESIS Quadriparesis with onset over minutes may result from disorders of upper motor neurons (such as from anoxia, hypotension, brainstem or cervical cord ischemia, trauma, and systemic metabolic abnormalities) or muscle (electrolyte disturbances, certain inborn errors of muscle energy metabolism, toxins, and periodic paralyses). Onset over hours to weeks may, in addition to these disorders, be due to lower motor neuron disorders such as Guillain-Barré syndrome (Chap. 460). In obtunded patients, evaluation begins with a CT scan of the brain. If upper motor neuron signs are present but the patient is alert, the initial test is usually an MRI of the cervical cord. If weakness is lower motor neuron, myopathic, or uncertain in origin, the clinical approach begins with blood studies to determine the level of muscle enzymes and electrolytes and with EMG and nerve conduction studies.

1	SUBACUTE OR CHRONIC qUAdRIPARESIS Quadriparesis due to upper motor neuron disease may develop over weeks to years from chronic myelopathies, multiple sclerosis, brain or spinal tumors, chronic subdural hematomas, and various metabolic, toxic, and infectious disorders. It may also result from lower motor neuron disease, a chronic neuropathy (in which weakness is often most profound distally), or myopathic weakness (typically proximal). When quadriparesis develops acutely in obtunded patients, evaluation begins with a CT scan of the brain. If upper motor neuron signs have developed acutely but the patient is alert, the initial test is usually CAuSES of EPiSoDiC gEnERALizED wEAKnESS 1. Electrolyte disturbances, e.g., hypokalemia, hyperkalemia, hypercalcemia, hypernatremia, hyponatremia, hypophosphatemia, hypermagnesemia 2. a. b. Metabolic defects of muscle (impaired carbohydrate or fatty acid utilization; abnormal mitochondrial function) 3. Neuromuscular junction disorders a. b.

1	a. b. Metabolic defects of muscle (impaired carbohydrate or fatty acid utilization; abnormal mitochondrial function) 3. Neuromuscular junction disorders a. b. 4. Central nervous system disorders a. Transient ischemic attacks of the brainstem b. c. 5. Lack of voluntary effort a. b. c. an MRI of the cervical cord. When onset has been gradual, disorders 157 of the cerebral hemispheres, brainstem, and cervical spinal cord can usually be distinguished clinically, and imaging is directed first at the clinically suspected site of pathology. If weakness is lower motor neuron, myopathic, or uncertain in origin, laboratory studies to determine the levels of muscle enzymes and electrolytes, and EMG and nerve conduction studies help to localize the pathologic process.

1	Monoparesis Monoparesis usually is due to lower motor neuron disease, with or without associated sensory involvement. Upper motor neuron weakness occasionally presents as a monoparesis of distal and nonantigravity muscles. Myopathic weakness rarely is limited to one limb. ACUTE MONOPARESIS If weakness is predominantly distal and of upper motor neuron type and is not associated with sensory impairment or pain, focal cortical ischemia is likely (Chap. 446); diagnostic possibilities are similar to those for acute hemiparesis. Sensory loss and pain usually accompany acute lower motor neuron weakness; the weakness commonly localizes to a single nerve root or peripheral nerve, but occasionally reflects plexus involvement. If lower motor neuron weakness is likely, evaluation begins with EMG and nerve conduction studies.

1	SUBACUTE OR CHRONIC MONOPARESIS Weakness and atrophy that develop over weeks or months are usually of lower motor neuron origin. When associated with sensory symptoms, a peripheral cause (nerve, root, or plexus) is likely; otherwise, anterior horn cell disease should be considered. In either case, an electrodiagnostic study is indicated. If weakness is of the upper motor neuron type, a discrete cortical (precentral gyrus) or cord lesion may be responsible, and appropriate imaging is performed.

1	Distal Weakness Involvement of two or more limbs distally suggests lower motor neuron or peripheral nerve disease. Acute distal lower-limb weakness results occasionally from an acute toxic polyneuropathy or cauda equina syndrome. Distal symmetric weakness usually develops over weeks, months, or years and, when associated with numbness, is due to peripheral neuropathy (Chap. 459). Anterior horn cell disease may begin distally but is typically asymmetric and without accompanying numbness (Chap. 452). Rarely, myopathies present with distal weakness (Chap. 462e). Electrodiagnostic studies help localize the disorder (Fig. 30-3).

1	Proximal Weakness Myopathy often produces symmetric weakness of the pelvic or shoulder girdle muscles (Chap. 462e). Diseases of the neuromuscular junction, such as myasthenia gravis (Chap. 461), may present with symmetric proximal weakness often associated with ptosis, diplopia, or bulbar weakness and fluctuating in severity during the day. In anterior horn cell disease, proximal weakness is usually asymmetric, but it may be symmetric if familial. Numbness does not occur with any of these diseases. The evaluation usually begins with determination of the serum creatine kinase level and electrophysiologic studies.

1	Weakness in a Restricted Distribution Weakness may not fit any of these patterns, being limited, for example, to the extraocular, hemifacial, bulbar, or respiratory muscles. If it is unilateral, restricted weakness usually is due to lower motor neuron or peripheral nerve disease, such as in a facial palsy. Weakness of part of a limb is commonly due to a peripheral nerve lesion such as an entrapment neuropathy. Relatively symmetric weakness of extraocular or bulbar muscles frequently is due to a myopathy (Chap. 462e) or neuromuscular junction disorder (Chap. 461). Bilateral facial palsy with areflexia suggests Guillain-Barré syndrome (Chap. 460). Worsening of relatively symmetric weakness with fatigue is characteristic of neuromuscular junction disorders. Asymmetric bulbar weakness usually is due to motor neuron disease. Weakness limited to respiratory muscles is uncommon and usually is due to motor neuron disease, myasthenia gravis, or polymyositis/ dermatomyositis (Chap. 388).

1	CHAPTER 30 Neurologic Causes of Weakness and Paralysis numbness, Tingling, and Sensory Loss Michael J. Aminoff Normal somatic sensation reflects a continuous monitoring process, little of which reaches consciousness under ordinary conditions. 31158 PART 2 Cardinal Manifestations and Presentation of Diseases By contrast, disordered sensation, particularly when experienced as painful, is alarming and dominates the patient’s attention. Physicians should be able to recognize abnormal sensations by how they are described, know their type and likely site of origin, and understand their implications. Pain is considered separately in Chap. 18.

1	Abnormal sensory symptoms can be divided into two categories: positive and negative. The prototypical positive symptom is tingling (pins and needles); other positive sensory phenomena include itch and altered sensations that are described as pricking, bandlike, lightning-like shooting feelings (lancinations), aching, knifelike, twisting, drawing, pulling, tightening, burning, searing, electrical, or raw feelings. Such symptoms are often painful. Positive phenomena usually result from trains of impulses generated at sites of lowered threshold or heightened excitability along a peripheral or central sensory pathway. The nature and severity of the abnormal sensation depend on the number, rate, timing, and distribution of ectopic impulses and the type and function of nervous tissue in which they arise. Because positive phenomena represent excessive activity in sensory pathways, they are not necessarily associated with a sensory deficit (loss) on examination.

1	Negative phenomena represent loss of sensory function and are characterized by diminished or absent feeling that often is experienced as numbness and by abnormal findings on sensory examination. In disorders affecting peripheral sensation, at least one-half the afferent axons innervating a particular site are probably lost or functionless before a sensory deficit can be demonstrated by clinical examination. If the rate of loss is slow, however, lack of cutaneous feeling may be unnoticed by the patient and difficult to demonstrate on examination, even though few sensory fibers are functioning; if it is rapid, both positive and negative phenomena are usually conspicuous. Subclinical degrees of sensory dysfunction may be revealed by sensory nerve conduction studies or somatosensory evoked potentials (Chap. 442e). Whereas sensory symptoms may be either positive or negative, sensory signs on examination are always a measure of negative phenomena.

1	Whereas sensory symptoms may be either positive or negative, sensory signs on examination are always a measure of negative phenomena. Paresthesias and dysesthesias are general terms used to denote positive sensory symptoms. The term paresthesias typically refers to tingling or pins-and-needles sensations but may include a wide variety of other abnormal sensations, except pain; it sometimes implies that the abnormal sensations are perceived spontaneously. The more general term dysesthesias denotes all types of abnormal sensations, including painful ones, regardless of whether a stimulus is evident.

1	Another set of terms refers to sensory abnormalities found on examination. Hypesthesia or hypoesthesia refers to a reduction of cutaneous sensation to a specific type of testing such as pressure, light touch, and warm or cold stimuli; anesthesia, to a complete absence of skin sensation to the same stimuli plus pinprick; and hypalgesia or analgesia, to reduced or absent pain perception (nociception). Hyperesthesia means pain or increased sensitivity in response to touch. Similarly, allodynia describes the situation in which a nonpainful stimulus, once perceived, is experienced as painful, even excruciating. An example is elicitation of a painful sensation by application of a vibrating tuning fork. Hyperalgesia denotes severe pain in response to a mildly noxious stimulus, and hyperpathia, a broad term, encompasses all the phenomena described by hyperesthesia, allodynia, and hyperalgesia. With hyperpathia, the threshold for a sensory stimulus is increased and perception is delayed, but

1	a broad term, encompasses all the phenomena described by hyperesthesia, allodynia, and hyperalgesia. With hyperpathia, the threshold for a sensory stimulus is increased and perception is delayed, but once felt, it is unduly painful.

1	Disorders of deep sensation arising from muscle spindles, tendons, and joints affect proprioception (position sense). Manifestations include imbalance (particularly with eyes closed or in the dark), clumsiness of precision movements, and unsteadiness of gait, which are referred to collectively as sensory ataxia. Other findings on examination usually, but not invariably, include reduced or absent joint position and vibratory sensibility and absent deep tendon reflexes in the affected limbs. The Romberg sign is positive, which means that the patient sways markedly or topples when asked to stand with feet close together and eyes closed. In severe states of deafferentation involving deep sensation, the patient cannot walk or stand unaided or even sit unsupported. Continuous involuntary movements (pseudoathetosis) of the outstretched hands and fingers occur, particularly with eyes closed.

1	Cutaneous receptors are classified by the type of stimulus that optimally excites them. They consist of naked nerve endings (nociceptors, which respond to tissue-damaging stimuli, and thermoreceptors, which respond to noninjurious thermal stimuli) and encapsulated terminals (several types of mechanoreceptor, activated by physical deformation of the skin). Each type of receptor has its own set of sensitivities to specific stimuli, size and distinctness of receptive fields, and adaptational qualities.

1	Afferent fibers in peripheral nerve trunks traverse the dorsal roots and enter the dorsal horn of the spinal cord (Fig. 31-1). From there, the polysynaptic projections of the smaller fibers (unmyelinated and small myelinated), which subserve mainly nociception, itch, temperature sensibility, and touch, cross and ascend in the opposite anterior and lateral columns of the spinal cord, through the brainstem, to the ventral posterolateral (VPL) nucleus of the thalamus and ultimately project to the postcentral gyrus of the parietal cortex (Chap. 18). This is the spinothalamic pathway or anterolateral system. The larger fibers, which subserve tactile and position sense and kinesthesia, project rostrally in the posterior and posterolateral columns on the same side of the spinal cord and make their first synapse in the gracile or cuneate nucleus of the lower medulla. Axons of second-order neurons decussate and ascend in the medial lemniscus located medially in the medulla and in the tegmentum

1	first synapse in the gracile or cuneate nucleus of the lower medulla. Axons of second-order neurons decussate and ascend in the medial lemniscus located medially in the medulla and in the tegmentum of the pons and midbrain and synapse in the VPL nucleus; third-order neurons project to parietal cortex as well as to other cortical areas. This large-fiber system is referred to as the posterior column–medial lemniscal pathway (lemniscal, for short). Although the fiber types and functions that make up the spinothalamic and lemniscal systems are relatively well known, many other fibers, particularly those associated with touch, pressure, and position sense, ascend in a diffusely distributed pattern both ipsilaterally and contra-laterally in the anterolateral quadrants of the spinal cord. This explains why a complete lesion of the posterior columns of the spinal cord may be associated with little sensory deficit on examination.

1	Nerve conduction studies and nerve biopsy are important means of investigating the peripheral nervous system, but they do not evaluate the function or structure of cutaneous receptors and free nerve endings or of unmyelinated or thinly myelinated nerve fibers in the nerve trunks. Skin biopsy can be used to evaluate these structures in the dermis and epidermis.

1	The main components of the sensory examination are tests of primary sensation (pain, touch, vibration, joint position, and thermal sensation) (Table 31-1). The examiner must depend on patient responses, and this complicates interpretation. Further, examination may be limited in some patients. In a stuporous patient, for example, sensory examination is reduced to observing the briskness of withdrawal in response to a pinch or another noxious stimulus. Comparison of responses on the two sides of the body is essential. In an alert but uncooperative patient, it may not be possible to examine cutaneous sensation, but some idea of proprioceptive function may be gained by noting the patient’s best performance of movements requiring balance and precision.

1	Primary Sensation The sense of pain usually is tested 159 with a clean pin, which is then discarded. The patient is asked to close the eyes and focus on the pricking or unpleasant quality of the stimulus, not just the pressure or touch sensation elicited. Areas of hypalgesia should be mapped by proceeding radially from the most hypalgesic site. Temperature sensation to both hot and cold is best tested with small containers filled with water of the desired temperature. An alternative way to test cold sensation is to touch a metal object, such as a tuning fork at room temperature, to the skin. For testing warm temperatures, the tuning fork or another metal object may be held under warm water of the desired temperature and then used. The appreciation of both cold and warmth should be tested because different receptors respond to each. Touch usually is tested with a wisp of cotton or a fine camel hair brush, minimizing pressure on the skin. In general, it is better to avoid testing touch

1	because different receptors respond to each. Touch usually is tested with a wisp of cotton or a fine camel hair brush, minimizing pressure on the skin. In general, it is better to avoid testing touch nucleus of on hairy skin because of the profusion of the sensory endings that surround each hair follicle. The patient is MIDBRAIN tested with the eyes closed and should indicate as soon as the stimulus is perceived, indicating its location.

1	Joint position testing is a measure of proprioception. With the patient’s eyes closed, joint position is tested in the distal interphalangeal joint of the great toe and fingers. The digit is held by its sides, distal to the joint Principal sensory nucleus of V PONS being tested, and moved passively while more proximalMedial lemniscus joints are stabilized—the patient indicates the change in position or direction of movement. If errors are made, more proximal joints are tested. A test of proxi-Nucleus of mal joint position sense, primarily at the shoulder, isfuniculus gracilis performed by asking the patient to bring the two index Nucleus of fingers together with arms extended and eyes closed. funiculus cuneatus Normal individuals can do this accurately, with errors Spinothalamic tract of 1 cm or less. Nucleus of

1	funiculus cuneatus Normal individuals can do this accurately, with errors Spinothalamic tract of 1 cm or less. Nucleus of The sense of vibration is tested with an oscillating tuning fork that vibrates at 128 Hz. Vibration is tested over bony points, beginning distally; in the feet, it is tested over the dorsal surface of the distal phalanx of the big toes and at the malleoli of the ankles, and in the hands, it is tested dorsally at the distal phalanx of the fingers. If abnormalities are found, more proximal sites should be examined. Vibratory thresholds at the same site in the patient and the examiner may be compared for control purposes. Quantitative Sensory Testing Effective sensory testing devices are commercially available. Quantitative sen-

1	Quantitative Sensory Testing Effective sensory testing devices are commercially available. Quantitative sen- FIguRE 31-1 The main somatosensory pathways. The spinothalamic tract (pain, sory testing is particularly useful for serial evaluation of thermal sense) and the posterior column–lemniscal system (touch, pressure, joint cutaneous sensation in clinical trials. Threshold testing position) are shown. Offshoots from the ascending anterolateral fasciculus (spino for touch and vibratory and thermal sensation is the thalamic tract) to nuclei in the medulla, pons, and mesencephalon and nuclear most widely used application. terminations of the tract are indicated. (From AH Ropper, MA Samuels: Adams and Victor’s Principles of Neurology, 9th ed. New York, McGraw-Hill, 2009.) Cortical Sensation The most commonly used tests of cortical function are two-point discrimination, touch localization, and bilateral simultaneous stimulation

1	In patients with sensory complaints, testing should begin in the and tests for graphesthesia and stereognosis. Abnormalities of these center of the affected region and proceed radially until sensation is sensory tests, in the presence of normal primary sensation in an alert perceived as normal. The distribution of any abnormality is defined cooperative patient, signify a lesion of the parietal cortex or thalaand compared to root and peripheral nerve territories (Figs. 31-2 mocortical projections. If primary sensation is altered, these cortical and 31-3). Some patients present with sensory symptoms that do discriminative functions usually will be abnormal also. Comparisons not fit an anatomic localization and are accompanied by either no should always be made between analogous sites on the two sides of abnormalities or gross inconsistencies on examination. The exam-the body because the deficit with a specific parietal lesion is likely to iner should consider whether the sensory

1	on the two sides of abnormalities or gross inconsistencies on examination. The exam-the body because the deficit with a specific parietal lesion is likely to iner should consider whether the sensory symptoms are a disguised be unilateral. request for help with psychologic or situational problems. Sensory Two-point discrimination is tested with special calipers, the points examination of a patient who has no neurologic complaints can of which may be set from 2 mm to several centimeters apart and then be brief and consist of pinprick, touch, and vibration testing in the applied simultaneously to the test site. On the fingertips, a normal hands and feet plus evaluation of stance and gait, including the individual can distinguish about a 3-mm separation of points. Romberg maneuver (Chap. 438). Evaluation of stance and gait also Touch localization is performed by light pressure for an instant tests the integrity of motor and cerebellar systems. with the examiner’s fingertip or a wisp of

1	Evaluation of stance and gait also Touch localization is performed by light pressure for an instant tests the integrity of motor and cerebellar systems. with the examiner’s fingertip or a wisp of cotton wool; the patient,

1	CHAPTER 31 Numbness, Tingling, and Sensory Loss Pain Temperature, heat Temperature, cold Touch Joint position Pinprick Warm metal object Cold metal object Cotton wisp, fine brush Tuning fork, 128 Hz Cutaneous nociceptors Cutaneous thermoreceptors for hot Cutaneous thermoreceptors for cold Cutaneous mechanoreceptors, also naked endings Mechanoreceptors, especially pacinian corpuscles Passive movement of specific joints Joint capsule and tendon endings, muscle spindles Large SpTh, also D SpTh SpTh Lem, also D and SpTh Lem, also D Lem, also D PART 2 Cardinal Manifestations and Presentation of Diseases Abbreviations: D, diffuse ascending projections in ipsilateral and contralateral anterolateral columns; Lem, posterior column and lemniscal projection, ipsilateral; SpTh, spinothalamic projection, contralateral.

1	whose eyes are closed, is required to identify the site of touch. Bilateral simultaneous stimulation at analogous sites (e.g., the dorsum of both hands) can be carried out to determine whether the perception of touch is extinguished consistently on one side (extinction or neglect). Graphesthesia refers to the capacity to recognize, with eyes closed, letters or numbers drawn by the examiner’s fingertip on the palm of the hand. Once again, interside comparison is of prime importance. Inability to recognize numbers or letters is termed agraphesthesia.

1	Stereognosis refers to the ability to identify common objects by palpation, recognizing their shape, texture, and size. Common standard objects such as keys, paper clips, and coins are best used. Patients with normal stereognosis should be able to distinguish a dime from a penny and a nickel from a quarter without looking. Patients should feel the object with only one hand at a time. If they are unable to identify it in one hand, it (lumbo-inguinal n.) Lat. cut. n. of thigh Intermed. & med. cut. n’s. of thigh (from femoral n.) Saphenous n. (from femoral n.) Deep peroneal n. (from common peroneal n.) Med. & lat. plantar n’s. (from posttibial n.) Great auricular n. Ant. cut. n. of neck Supraclavicular n’s. Med. cut. n. of arm & intercostobrachial n. Med. cut. n. Dorsal n. of penis Scrotal branch of perineal n. Obturator n. Lat. cut. n. of calf (from common peroneal n.) Superfcial peroneal n. (from common peroneal n.)

1	Med. cut. n. Dorsal n. of penis Scrotal branch of perineal n. Obturator n. Lat. cut. n. of calf (from common peroneal n.) Superfcial peroneal n. (from common peroneal n.) Sural n. (from tibial n.) should be placed in the other for comparison. Individuals who are unable to identify common objects and coins in one hand but can do so in the other are said to have astereognosis of the abnormal hand. Sensory symptoms and signs can result from lesions at many different levels of the nervous system from the parietal cortex to the peripheral sensory receptor. Noting their distribution and nature is the most important way to localize their source. Their extent, configuration, symmetry, quality, and severity are the key observations.

1	Dysesthesias without sensory findings by examination may be difficult to interpret. To illustrate, tingling dysesthesias in an acral distribution (hands and feet) can be systemic in origin, e.g., secondary to hyperventilation, or induced by a medication such as acetazolamide. Greater } occipital nervesLesser n. Great auricular n. Ant. cut. n. of neck C5 C6 T1 Supraclavicular n’s. Post. Axillary n. 3 (circumflex) 4 cut. rami Lat. of cut. Post cut. n. of arm thor.rami (from radial n.) n’s. 8 9 Lower 10 L1 Lat. cut. of arm 11 (from radial n.) 12 Med. (from musculocut n.) S1 cut. n. Post. rami of of lumbar sacral forearm Iliohypo-Radial n. gastric n. & coccygeal n’s. Med. cut. n. of arm & intercostobrachial n. Post. cut. n. of forearm (from radial n.) Lat. cut. n. of forearm Inf. med. cluneal n. Inf. med. n. of thigh Post cut. n. of thigh Lat. cut. n.of calf (from common femoral n.) Saphenous n. (from femoral n.) Superficial peroneal n. (from common peroneal n.)

1	Inf. med. cluneal n. Inf. med. n. of thigh Post cut. n. of thigh Lat. cut. n.of calf (from common femoral n.) Saphenous n. (from femoral n.) Superficial peroneal n. (from common peroneal n.) Sural n. (from tibial n.) Calcanean branches of sural & tibial n’s. Ulnar n. Inf. lat. cluneal n’s. Median n. Obturator n. Med. cut. n. of thigh (from femoral n.) Lat. plantar n. Med. plantar n. Lat. plantar n. Superficial peroneal n. Saphenous n. Calcanean branches of tibial & sural n’s. Sural n. FIguRE 31-2 The cutaneous fields of peripheral nerves. (Reproduced by permission from W Haymaker, B Woodhall: Peripheral Nerve Injuries, 2nd ed. Philadelphia, Saunders, 1953.) FIguRE 31-3 Distribution of the sensory spinal roots on the surface of the body (dermatomes). (From D Sinclair: Mechanisms of Cutaneous Sensation. Oxford, UK, Oxford University Press, 1981; with permission from Dr. David Sinclair.)

1	Distal dysesthesias can also be an early event in an evolving polyneuropathy or may herald a myelopathy, such as from vitamin B12 deficiency. Sometimes distal dysesthesias have no definable basis. In contrast, dysesthesias that correspond in distribution to that of a particular peripheral nerve structure denote a lesion at that site. For instance, dysesthesias restricted to the fifth digit and the adjacent one-half of the fourth finger on one hand reliably point to disorder of the ulnar nerve, most commonly at the elbow.

1	Nerve and Root In focal nerve trunk lesions, sensory abnormalities are readily mapped and generally have discrete boundaries (Figs. 31-2 and 31-3). Root (“radicular”) lesions frequently are accompanied by deep, aching pain along the course of the related nerve trunk. With compression of a fifth lumbar (L5) or first sacral (S1) root, as from a ruptured intervertebral disk, sciatica (radicular pain relating to the sciatic nerve trunk) is a common manifestation (Chap. 22). With a lesion affecting a single root, sensory deficits may be minimal or absent because adjacent root territories overlap extensively.

1	Isolated mononeuropathies may cause symptoms beyond the territory supplied by the affected nerve, but abnormalities on examination typically are confined to appropriate anatomic boundaries. In multiple mononeuropathies, symptoms and signs occur in discrete territories supplied by different individual nerves and—as more nerves are affected—may simulate a polyneuropathy if deficits become confluent. With polyneuropathies, sensory deficits are generally graded, distal, and symmetric in distribution (Chap. 459). Dysesthesias, followed by numbness, begin in the toes and ascend symmetrically. When dysesthesias reach the knees, they usually also have appeared in the fingertips. The process is nerve length–dependent, and the deficit is often described as “stocking-glove” in type. Involvement of both hands and feet also occurs with lesions of the upper cervical cord or the brainstem, but an upper level of the sensory disturbance may then be found on the trunk and other evidence of a central

1	both hands and feet also occurs with lesions of the upper cervical cord or the brainstem, but an upper level of the sensory disturbance may then be found on the trunk and other evidence of a central lesion may be present, such as sphincter involvement or signs of an upper motor neuron lesion (Chap. 30). Although most polyneuropathies are pansensory and affect all modalities of sensation, selective sensory dysfunction according to nerve fiber size may occur. Small-fiber polyneuropa-161 thies are characterized by burning, painful dysesthesias with reduced pinprick and thermal sensation but with sparing of proprioception, motor function, and deep tendon reflexes. Touch is involved variably; when it is spared, the sensory pattern is referred to as exhibiting sensory dissociation. Sensory dissociation may occur also with spinal cord lesions as well as small-fiber neuropathies. Large-fiber polyneuropathies are characterized by vibration and position sense deficits, imbalance, absent tendon

1	may occur also with spinal cord lesions as well as small-fiber neuropathies. Large-fiber polyneuropathies are characterized by vibration and position sense deficits, imbalance, absent tendon reflexes, and variable motor dysfunction but preservation of most cutaneous sensation. Dysesthesias, if present at all, tend to be tingling or bandlike in quality.

1	Sensory neuronopathy (or ganglionopathy) is characterized by widespread but asymmetric sensory loss occurring in a non-lengthdependent manner so that it may occur proximally or distally and in the arms, legs, or both. Pain and numbness progress to sensory ataxia and impairment of all sensory modalities with time. This condition is usually paraneoplastic or idiopathic in origin (Chaps. 122 and 459) or related to an autoimmune disease, particularly Sjögren’s syndrome. Spinal Cord (See also Chap. 456) If the spinal cord is transected, all sensation is lost below the level of transection. Bladder and bowel function also are lost, as is motor function. Lateral hemisection of the spinal cord produces the Brown-Séquard syndrome, with absent pain and temperature sensation contralaterally and loss of proprioceptive sensation and power ipsilaterally below the lesion (see Figs. 31-1 and 456-1).

1	Numbness or paresthesias in both feet may arise from a spinal cord lesion; this is especially likely when the upper level of the sensory loss extends to the trunk. When all extremities are affected, the lesion is probably in the cervical region or brainstem unless a peripheral neuropathy is responsible. The presence of upper motor neuron signs (Chap. 30) supports a central lesion; a hyperesthetic band on the trunk may suggest the level of involvement. A dissociated sensory loss can reflect spinothalamic tract involvement in the spinal cord, especially if the deficit is unilateral and has an upper level on the torso. Bilateral spinothalamic tract involvement occurs with lesions affecting the center of the spinal cord, such as in syringomyelia. There is a dissociated sensory loss with impairment of pinprick and temperature appreciation but relative preservation of light touch, position sense, and vibration appreciation.

1	Dysfunction of the posterior columns in the spinal cord or of the posterior root entry zone may lead to a bandlike sensation around the trunk or a feeling of tight pressure in one or more limbs. Flexion of the neck sometimes leads to an electric shock–like sensation that radiates down the back and into the legs (Lhermitte’s sign) in patients with a cervical lesion affecting the posterior columns, such as from multiple sclerosis, cervical spondylosis, or recent irradiation to the cervical region.

1	Brainstem Crossed patterns of sensory disturbance, in which one side of the face and the opposite side of the body are affected, localize to the lateral medulla. Here a small lesion may damage both the ipsilateral descending trigeminal tract and the ascending spinothalamic fibers subserving the opposite arm, leg, and hemitorso (see “Lateral medullary syndrome” in Fig. 446-10). A lesion in the tegmentum of the pons and midbrain, where the lemniscal and spinothalamic tracts merge, causes pansensory loss contralaterally.

1	Thalamus Hemisensory disturbance with tingling numbness from head to foot is often thalamic in origin but also can arise from the anterior parietal region. If abrupt in onset, the lesion is likely to be due to a small stroke (lacunar infarction), particularly if localized to the thalamus. Occasionally, with lesions affecting the VPL nucleus or adjacent white matter, a syndrome of thalamic pain, also called Déjerine-Roussy syndrome, may ensue. The persistent, unrelenting unilateral pain often is described in dramatic terms. Cortex With lesions of the parietal lobe involving either the cortex or the subjacent white matter, the most prominent symptoms are contralateral hemineglect, hemi-inattention, and a tendency not to use

1	CHAPTER 31 Numbness, Tingling, and Sensory Loss 162 the affected hand and arm. On cortical sensory testing (e.g., two-point discrimination, graphesthesia), abnormalities are often found but primary sensation is usually intact. Anterior parietal infarction may present as a pseudothalamic syndrome with contralateral loss of primary sensation from head to toe. Dysesthesias or a sense of numbness and, rarely, a painful state may also occur.

1	Focal Sensory Seizures These seizures generally are due to lesions in the area of the postcentral or precentral gyrus. The principal symptom of focal sensory seizures is tingling, but additional, more complex sensations may occur, such as a rushing feeling, a sense of warmth, or a sense of movement without detectable motion. Symptoms typically are unilateral; commonly begin in the arm or hand, face, or foot; and often spread in a manner that reflects the cortical representation of different bodily parts, as in a Jacksonian march. Their duration is variable; seizures may be transient, lasting only for seconds, or persist for an hour or more. Focal motor features may supervene, often becoming generalized with loss of consciousness and tonic-clonic jerking. Arthur Asbury authored or co-authored this chapter in earlier editions of this book. PART 2 Cardinal Manifestations and Presentation of Diseases PREVALENCE, MORBIDITY, AND MORTALITY

1	Arthur Asbury authored or co-authored this chapter in earlier editions of this book. PART 2 Cardinal Manifestations and Presentation of Diseases PREVALENCE, MORBIDITY, AND MORTALITY Gait and balance problems are common in the elderly and contribute to the risk of falls and injury. Gait disorders have been described in 15% of individuals older than 65. By age 80 one person in four will use a mechanical aid to assist with ambulation. Among those 85 and older, the prevalence of gait abnormality approaches 40%. In epidemiologic studies, gait disorders are consistently identified as a major risk factor for falls and injury.

1	A substantial number of older persons report insecure balance and experience falls and fear of falling. Prospective studies indicate that 30% of those older than 65 fall each year. The proportion is even higher in frail elderly and nursing home patients. Each year, 8% of individuals older than 75 suffer a serious fall-related injury. Hip fractures result in hospitalization, can lead to nursing home admission, and are associated with an increased mortality risk in the subsequent year. For each person who is physically disabled, there are others whose functional independence is limited by anxiety and fear of falling. Nearly one in five elderly individuals voluntarily restricts his or her activity because of fear of falling. With loss of ambulation, the quality of life diminishes, and rates of morbidity and mortality increase.

1	An upright bipedal gait depends on the successful integration of postural control and locomotion. These functions are widely distributed in the central nervous system. The biomechanics of bipedal walking are complex, and the performance is easily compromised by a neurologic deficit at any level. Command and control centers in the brain-stem, cerebellum, and forebrain modify the action of spinal pattern generators to promote stepping. While a form of “fictive locomotion” can be elicited from quadrupedal animals after spinal transection, this capacity is limited in primates. Step generation in primates is dependent on locomotor centers in the pontine tegmentum, midbrain, and subthalamic region. Locomotor synergies are executed through the reticular formation and descending pathways in the ventromedial spinal cord. Cerebral control provides a goal and purpose for walking and is involved in avoidance of obstacles and adaptation of locomotor programs to context and terrain.

1	Postural control requires the maintenance of the center of mass over the base of support through the gait cycle. Unconscious postural adjustments maintain standing balance: long latency responses are measurable in the leg muscles, beginning 110 milliseconds after a perturbation. Forward motion of the center of mass provides propulsive force for stepping, but failure to maintain the center of mass within stability limits results in falls. The anatomic substrate for dynamic balance has not been well defined, but the vestibular nucleus and midline cerebellum contribute to balance control in animals. Patients with damage to these structures have impaired balance while standing and walking.

1	Standing balance depends on good-quality sensory information about the position of the body center with respect to the environment, support surface, and gravitational forces. Sensory information for postural control is primarily generated by the visual system, the vestibular system, and proprioceptive receptors in the muscle spindles and joints. A healthy redundancy of sensory afferent information is generally available, but loss of two of the three pathways is sufficient to compromise standing balance. Balance disorders in older individuals sometimes result from multiple insults in the peripheral sensory systems (e.g., visual loss, vestibular deficit, peripheral neuropathy) that critically degrade the quality of afferent information needed for balance stability.

1	Older patients with cognitive impairment from neurodegenerative diseases appear to be particularly prone to falls and injury. There is a growing body of literature on the use of attentional resources to manage gait and balance. Walking is generally considered to be unconscious and automatic, but the ability to walk while attending to a cognitive task (dual-task walking) may be compromised in frail elderly individuals with a history of falls. Older patients with deficits in executive function may have particular difficulty in managing the attentional resources needed for dynamic balance when distracted.

1	Disorders of gait may be attributed to frailty, fatigue, arthritis, and orthopedic deformity, but neurologic causes are disabling and important to address. The heterogeneity of gait disorders observed in clinical practice reflects the large network of neural systems involved in the task. Walking is vulnerable to neurologic disease at every level. Gait disorders have been classified descriptively on the basis of abnormal physiology and biomechanics. One problem with this approach is that many failing gaits look fundamentally similar. This overlap reflects common patterns of adaptation to threatened balance stability and declining performance. The gait disorder observed clinically must be viewed as the product of a neurologic deficit and a functional adaptation. Unique features of the failing gait are often overwhelmed by the adaptive response. Some common patterns of abnormal gait are summarized next. Gait disorders can also be classified by etiology (Table 32-1).

1	Etiology No. of Cases Percent Source: Reproduced with permission from J Masdeu, L Sudarsky, L Wolfson: Gait Disorders of Aging. Lippincott Raven, 1997. The term cautious gait is used to describe the patient who walks with an abbreviated stride and lowered center of mass, as if walking on a slippery surface. This disorder is both common and nonspecific. It is, in essence, an adaptation to a perceived postural threat. There may be an associated fear of falling. This disorder can be observed in more than one-third of older patients with gait impairment. Physical therapy often improves walking to the degree that follow-up observation may reveal a more specific underlying disorder.

1	Spastic gait is characterized by stiffness in the legs, an imbalance of muscle tone, and a tendency to circumduct and scuff the feet. The disorder reflects compromise of corticospinal command and overactivity of spinal reflexes. The patient may walk on the toes. In extreme instances, the legs cross due to increased tone in the adductors. Upper motor neuron signs are present on physical examination. Shoes often reflect an uneven pattern of wear across the outside. The disorder may be cerebral or spinal in origin.

1	Myelopathy from cervical spondylosis is a common cause of spastic or spastic-ataxic gait in the elderly. Demyelinating disease and trauma are the leading causes of myelopathy in younger patients. In chronic progressive myelopathy of unknown cause, a workup with laboratory and imaging tests may establish a diagnosis. A family history should suggest hereditary spastic paraplegia (Chap. 452); genetic testing is now available for some of the common mutations responsible for this disorder. Tropical spastic paraparesis related to the retrovirus human T-cell lymphotropic virus 1 (HTLV-1) is endemic in parts of the Caribbean and South America. A structural lesion, such as a tumor or a spinal vascular malformation, should be excluded with appropriate testing. Spinal cord disorders are discussed in detail in Chap. 456.

1	With cerebral spasticity, asymmetry is common, the upper extremities are usually involved, and dysarthria is often an associated feature. Common causes include vascular disease (stroke), multiple sclerosis, and perinatal injury to the nervous system (cerebral palsy). Other stiff-legged gaits include dystonia (Chap. 449) and stiff-person syndrome (Chap. 122). Dystonia is a disorder characterized by sustained muscle contractions resulting in repetitive twisting movements and abnormal posture. It often has a genetic basis. Dystonic spasms can produce plantar flexion and inversion of the feet, sometimes with torsion of the trunk. In autoimmune stiff-person syndrome, exaggerated lordosis of the lumbar spine and overactivation of antagonist muscles restrict trunk and lower-limb movement and result in a wooden or fixed posture.

1	Parkinson’s disease (Chap. 449) is common, affecting 1% of the population >55 years of age. The stooped posture and shuffling gait are characteristic and distinctive features. Patients sometimes accelerate (festinate) with walking, display retropulsion, or exhibit a tendency to turn en bloc. A National Institutes of Health workshop defined freezing of gait as “brief, episodic absence of forward progression of the feet, despite the intention to walk.” Gait freezing occurs in 26% of Parkinson’s patients by the end of 5 years and develops in most such patients eventually. Postural instability and falling occur as the disease progresses; some falls are precipitated by freezing of gait.

1	Freezing of gait is even more common in some Parkinson’s-related neurodegenerative disorders, such as progressive supranuclear palsy, multiple-system atrophy, and corticobasal degeneration. Patients with these disorders frequently present with axial stiffness, postural instability, and a shuffling, freezing gait while lacking the characteristic pill-rolling tremor of Parkinson’s disease. Falls within the first year suggest the possibility of progressive supranuclear palsy. Hyperkinetic movement disorders also produce characteristic and recognizable disturbances in gait. In Huntington’s disease (Chap. 449), the unpredictable occurrence of choreic movements gives the gait a dancing quality. Tardive dyskinesia is the cause of many odd, stereotypic gait disorders seen in patients chronically exposed to antipsychotics and other drugs that block the D2 dopamine receptor.

1	Frontal gait disorder, sometimes known as gait apraxia, is common in the elderly and has a variety of causes. The term is used to describe a shuffling, freezing gait with imbalance and other signs of higher cerebral dysfunction. Typical features include a wide base of support, a short stride, shuffling along the floor, and difficulty with starts and turns. Many patients exhibit a difficulty with gait initiation that is descriptively characterized as the “slipping clutch” syndrome or gait ignition failure. The term lower-body parkinsonism is also used to describe such patients. Strength is generally preserved, and patients are able to make stepping movements when not standing and maintaining their balance at the same time. This disorder is best considered a higher-level motor control disorder, as opposed to an apraxia (Chap. 36).

1	The most common cause of frontal gait disorder is vascular disease, particularly subcortical small-vessel disease. Lesions are frequently found in the deep frontal white matter and centrum ovale. Gait disorder may be the salient feature in hypertensive patients with ischemic lesions of the deep-hemisphere white matter (Binswanger’s disease). The clinical syndrome includes mental changes (variable in degree), dysarthria, pseudobulbar affect (emotional disinhibition), increased tone, and hyperreflexia in the lower limbs. Communicating hydrocephalus in adults also presents with a gait disorder of this type. Other features of the diagnostic triad (mental changes, incontinence) may be absent in the initial stages. MRI demonstrates ventricular enlargement, an enlarged flow void about the aqueduct, and a variable degree of periventricular white-matter change. A lumbar puncture or dynamic test is necessary to confirm hydrocephalus.

1	Disorders of the cerebellum have a dramatic impact on gait and balance. Cerebellar gait ataxia is characterized by a wide base of support, lateral instability of the trunk, erratic foot placement, and decompensation of balance when attempting to walk on a narrow base. Difficulty maintaining balance when turning is often an early feature. Patients are unable to walk tandem heel to toe and display truncal sway in narrow-based or tandem stance. They show considerable variation in their tendency to fall in daily life.

1	Causes of cerebellar ataxia in older patients include stroke, trauma, tumor, and neurodegenerative disease such as multiple-system atrophy (Chaps. 449 and 454) and various forms of hereditary cerebellar degeneration (Chap. 450). A short expansion at the site of the fragile X mutation (fragile X pre-mutation) has been associated with gait ataxia in older men. Alcoholic cerebellar degeneration can be screened by history and often confirmed by MRI. In patients with ataxia, MRI demonstrates the extent and topography of cerebellar atrophy.

1	As reviewed earlier in this chapter, balance depends on high-quality afferent information from the visual and the vestibular systems and proprioception. When this information is lost or degraded, balance during locomotion is impaired and instability results. The sensory ataxia of tabetic neurosyphilis is a classic example. The contemporary equivalent is the patient with neuropathy affecting large fibers. Vitamin B12 deficiency is a treatable cause of large-fiber sensory loss in the spinal cord and peripheral nervous system. Joint position and vibration sense are diminished in the lower limbs. The stance in such patients is destabilized by eye closure; they often look down at their feet when walking and do poorly in the dark. Table 32-2 compares sensory ataxia with cerebellar ataxia and frontal gait disorder. Some frail older patients exhibit a syndrome of imbalance from the combined effect of multiple sensory deficits. Such patients have disturbances in proprioception, vision, and

1	frontal gait disorder. Some frail older patients exhibit a syndrome of imbalance from the combined effect of multiple sensory deficits. Such patients have disturbances in proprioception, vision, and vestibular sense that impair postural support.

1	Patients with neuromuscular disease often have an abnormal gait, occasionally as a presenting feature. With distal weakness (peripheral neuropathy), the step height is increased to compensate for footdrop, and the sole of the foot may slap on the floor during weight acceptance. Neuropathy may be associated with a degree of sensory imbalance, as described earlier. Patients with myopathy or muscular dystrophy more TABLE 32-2 fEATuRES of CEREBELLAR ATAxiA, SEnSoRy ATAxiA, AnD fRonTAL gAiT DiSoRDERS Base of Wide-based Narrow base, Wide-based support Stride Irregular, Regular with Short, shuffling lurching path deviation Romberg test +/− Unsteady, falls +/− Turns Unsteady +/− Hesitant, multistep typically exhibit proximal weakness. Weakness of the hip girdle may result in some degree of excess pelvic sway during locomotion.

1	Turns Unsteady +/− Hesitant, multistep typically exhibit proximal weakness. Weakness of the hip girdle may result in some degree of excess pelvic sway during locomotion. Alcohol intoxication is the most common cause of acute walking difficulty. Chronic toxicity from medications and metabolic disturbances can impair motor function and gait. Mental status changes may be found, and examination may reveal asterixis or myoclonus. Static equilibrium is disturbed, and such patients are easily thrown off balance. Disequilibrium is particularly evident in patients with chronic renal disease and those with hepatic failure, in whom asterixis may impair postural support. Sedative drugs, especially neuroleptics and long-acting benzodiazepines, affect postural control and increase the risk for falls. These disorders are especially important to recognize because they are often treatable.

1	Psychogenic disorders are common in neurologic practice, and the presentation often involves gait. Some patients with extreme anxiety or phobia walk with exaggerated caution with abduction of the arms, as if walking on ice. This inappropriately overcautious gait differs in degree from the gait of the patient who is insecure and making adjustments for imbalance. Depressed patients exhibit primarily slowness, a manifestation of psychomotor retardation, and lack of purpose in their stride. Hysterical gait disorders are among the most spectacular encountered. Odd gyrations of posture with wastage of muscular energy (astasia–abasia), extreme slow motion, and dramatic fluctuations over time may be observed in patients with somatoform disorders and conversion reactions. APPROACH TO THE PATIENT: Slowly Progressive Disorder of gait PART 2 Cardinal Manifestations and Presentation of Diseases

1	APPROACH TO THE PATIENT: Slowly Progressive Disorder of gait PART 2 Cardinal Manifestations and Presentation of Diseases When reviewing the history, it is helpful to inquire about the onset and progression of disability. Initial awareness of an unsteady gait often follows a fall. Stepwise evolution or sudden progression suggests vascular disease. Gait disorder may be associated with urinary urgency and incontinence, particularly in patients with cervical spine disease or hydrocephalus. It is always important to review the use of alcohol and medications that affect gait and balance. Information on localization derived from the neurologic examination can be helpful in narrowing the list of possible diagnoses.

1	Gait observation provides an immediate sense of the patient’s degree of disability. Arthritic and antalgic gaits are recognized by observation, though neurologic and orthopedic problems may coexist. Characteristic patterns of abnormality are sometimes seen, though, as stated previously, failing gaits often look fundamentally similar. Cadence (steps per minute), velocity, and stride length can be recorded by timing a patient over a fixed distance. Watching the patient rise from a chair provides a good functional assessment of balance.

1	Brain imaging studies may be informative in patients with an undiagnosed disorder of gait. MRI is sensitive for cerebral lesions of vascular or demyelinating disease and is a good screening test for occult hydrocephalus. Patients with recurrent falls are at risk for subdural hematoma. As mentioned earlier, many elderly patients with gait and balance difficulty have white matter abnormalities in the periventricular region and centrum semiovale. While these lesions may be an incidental finding, a substantial burden of white matter disease will ultimately impact cerebral control of locomotion. DEFINITION, ETIOLOgY, AND MANIFESTATIONS

1	DEFINITION, ETIOLOgY, AND MANIFESTATIONS Balance is the ability to maintain equilibrium—a state in which opposing physical forces cancel one another out. In physiology, this term is taken to mean the ability to control the center of mass with respect to gravity and the support surface. In reality, people are not consciously aware of their center of mass, but everyone (particularly gymnasts, figure skaters, and platform divers, for example) move so as to manage it. Disorders of balance present as difficulty maintaining posture while standing and walking and as a subjective sense of disequilibrium, which is a form of dizziness. The cerebellum and vestibular system organize antigravity responses needed to maintain an upright posture. These responses are physiologically complex, and the anatomic representation they entail is not well understood. Failure, resulting in disequilibrium, can occur at several levels: cerebellar, vestibular, somatosensory, and higher-level disequilibrium.

1	Patients with cerebellar ataxia do not generally complain of dizziness, though balance is visibly impaired. Neurologic examination reveals a variety of cerebellar signs. Postural compensation may prevent falls early on, but falls are inevitable with disease progression. The progression of neurodegenerative ataxia is often measured by the number of years to loss of stable ambulation. Vestibular disorders (Chap. 28) have symptoms and signs that fall into three categories: (1) vertigo (the subjective inappropriate perception or illusion of movement); (2) nystagmus (involuntary eye movements); and (3) impaired standing balance. Not every patient has all manifestations. Patients with vestibular deficits related to ototoxic drugs may lack vertigo or obvious nystagmus, but their balance is impaired on standing and walking, and they cannot navigate in the dark. Laboratory testing is available to investigate vestibular deficits.

1	Somatosensory deficits also produce imbalance and falls. There is often a subjective sense of insecure balance and fear of falling. Postural control is compromised by eye closure (Romberg’s sign); these patients also have difficulty navigating in the dark. A dramatic example is provided by the patient with autoimmune subacute sensory neuropathy, which is sometimes a paraneoplastic disorder (Chap. 122). Compensatory strategies enable such patients to walk in the virtual absence of proprioception, but the task requires active visual monitoring. Patients with higher-level disorders of equilibrium have difficulty maintaining balance in daily life and may present with falls. Their awareness of balance impairment may be reduced. Patients taking sedating medications are in this category. In prospective studies, dementia and sedating medications substantially increase the risk for falls.

1	Falls are common in the elderly; 30% of people older than 65 who are living in the community fall each year. Modest changes in balance function have been described in fit older individuals as a result of normal aging. Subtle deficits in sensory systems, attention, and motor reaction time contribute to the risk, and environmental hazards abound. Many falls by older adults are episodes of tripping or slipping, often designated mechanical falls. A fall is not a neurologic problem per se, but there are events for which neurologic evaluation is appropriate. It is important to distinguish falls associated with loss of consciousness (syncope, seizure), which require appropriate evaluation and intervention (Chaps. 27 and 445). In most prospective studies, a small subset of individuals experience a large number of fall events. These individuals with recurrent falls often have gait and balance issues that need to be addressed.

1	Fall Patterns: The Event description The history of a fall is often problematic or incomplete, and the underlying mechanism or cause may be difficult to establish in retrospect. The patient and family may have limited information about what triggered the fall. Injuries can complicate the physical examination. While there is no standard nosology of falls, some common clinical patterns may emerge and provide a clue.

1	drop attacks and collapsIng falls Drop attacks are sudden collapsing falls without loss of consciousness. Patients who collapse from lack of postural tone present a diagnostic challenge. Patients may report that their legs just “gave out” underneath them; their families may describe these patients as “collapsing in a heap.” Orthostatic hypotension may be a factor in some such falls, and this possibility should be thoroughly evaluated. Rarely, a colloid cyst of the third ventricle can present with intermittent obstruction of the foramen of Monro, with a consequent drop attack. While collapsing falls are more common among older patients with vascular risk factors, they should not be confused with vertebrobasilar ischemic attacks.

1	topplIng falls Some patients maintain tone in antigravity muscles but fall over like a tree trunk, as if postural defenses had disengaged. There may be a consistent direction to such falls. The patient with cerebellar pathology may lean and topple over toward the side of the lesion. Patients with lesions of the vestibular system or its central pathways may experience lateral pulsion and toppling falls. Patients with progressive supranuclear palsy often fall over backward. Falls of this nature occur in patients with advanced Parkinson’s disease once postural instability has developed.

1	falls due to gaIt freezIng Another fall pattern in Parkinson’s disease and related disorders is the fall due to freezing of gait. The feet stick to the floor and the center of mass keeps moving, resulting in a disequilibrium from which the patient has difficulty recovering. This sequence of events can result in a forward fall. Gait freezing can also occur as the patient attempts to turn and change direction. Similarly, patients with Parkinson’s disease and festinating gait may find their feet unable to keep up and may thus fall forward.

1	falls related to sensory loss Patients with somatosensory, visual, or vestibular deficits are prone to falls. These patients have particular difficulty dealing with poor illumination or walking on uneven ground. They often report subjective imbalance, apprehension, and fear of falling. Deficits in joint position and vibration sense are apparent on physical examination. These patients may be especially responsive to a rehabilitation-based intervention. weakness and fraIlty Patients who lack strength in antigravity muscles have difficulty rising from a chair, tire easily when walking, and have difficulty maintaining their balance after a perturbation. These patients are often unable to get up after a fall and may have to remain on the floor for a prolonged period until help arrives. Deconditioning of this sort is often treatable. Resistance strength training can increase muscle mass and leg strength, even for people in their eighties and nineties.

1	The most productive approach is to identify the high-risk patient prospectively, before there is a serious injury. Patients at particular risk include hospitalized patients with mental status changes, nursing home residents, patients with dementia, and those taking medications that compromise attention and alertness. Patients with Parkinson’s disease and other gait disorders are also at increased risk. (Table 32-3) summarizes a meta-analysis of prospective studies establishing the principal risk factors for falls. It is often possible to address and mitigate some of the major risk factors. Medication overuse may be the most important remediable risk factor for falls. Abbreviations: OR, odds ratio from retrospective studies; RR, relative risk from prospective studies. Source: Reproduced with permission from J Masdeu, L Sudarsky, L Wolfson: Gait Disorders of Aging. Lippincott Raven, 1997.

1	Source: Reproduced with permission from J Masdeu, L Sudarsky, L Wolfson: Gait Disorders of Aging. Lippincott Raven, 1997. Efforts should be made to define the etiology of the gait disorder and the mechanism underlying the falls by a given patient. Orthostatic changes in blood pressure and pulse should be recorded. Rising from a chair and walking should be evaluated for safety. Specific treatment may be possible once a diagnosis is established. Therapeutic intervention is often recommended for older patients at substantial risk for falls, even if no neurologic disease is identified. A home visit to look for environmental hazards can be helpful. A variety of modifications may be recommended to improve safety, including improved lighting and the installation of grab bars and nonslip surfaces.

1	Rehabilitative interventions aim to improve muscle strength and balance stability and to make the patient more resistant to injury. High-intensity resistance strength training with weights and machines is useful to improve muscle mass, even in frail older patients. Improvements realized in posture and gait should translate to reduced risk of falls and injury. Sensory balance training is another approach to improving balance stability. Measurable gains can be made in a few weeks of training, and benefits can be maintained over 6 months by a 10to 20-min home exercise program. This strategy is particularly successful in patients with vestibular and somatosensory balance disorders. A Tai Chi exercise program has been demonstrated to reduce the risk of falls and injury in patients with Parkinson’s disease. Video Library of Gait Disorders Gail Kang, Nicholas B. Galifianakis, Michael D. Geschwind

1	Video Library of Gait Disorders Gail Kang, Nicholas B. Galifianakis, Michael D. Geschwind Problems with gait and balance are major causes of falls, accidents, and resulting disability, especially in later life, and are often harbingers of neurologic disease. Early diagnosis is essential, especially for treatable conditions, because it may permit the institution of prophylactic measures to prevent dangerous falls and also to reverse or ameliorate the underlying cause. In this video, examples of gait disorders due to Parkinson’s disease, other extrapyramidal disorders, and ataxias, as well as other common gait disorders, are presented. CHAPTER 33e Video Library of Gait Disorders

1	CHAPTER 33e Video Library of Gait Disorders Confusion and Delirium S. Andrew Josephson, Bruce L. Miller Confusion, a mental and behavioral state of reduced comprehen-sion, coherence, and capacity to reason, is one of the most common problems encountered in medicine, accounting for a large number of emergency department visits, hospital admissions, and inpatient 34166 consultations. Delirium, a term used to describe an acute confusional state, remains a major cause of morbidity and mortality, costing over $150 billion dollars yearly in health care costs in the United States alone. Despite increased efforts targeting awareness of this condition, delirium often goes unrecognized in the face of evidence that it is usually the cognitive manifestation of serious underlying medical or neurologic illness.

1	A multitude of terms are used to describe patients with delirium, including encephalopathy, acute brain failure, acute confusional state, and postoperative or intensive care unit (ICU) psychosis. Delirium has many clinical manifestations, but is defined as a relatively acute decline in cognition that fluctuates over hours or days. The hallmark of delirium is a deficit of attention, although all cognitive domains—including memory, executive function, visuospatial tasks, and language—are variably involved. Associated symptoms that may be present in some cases include altered sleep-wake cycles, perceptual disturbances such as hallucinations or delusions, affect changes, and autonomic findings that include heart rate and blood pressure instability.

1	Delirium is a clinical diagnosis that is made only at the bedside. Two subtypes have been described—hyperactive and hypoactive—based on differential psychomotor features. The cognitive syndrome associated with severe alcohol withdrawal (i.e., “delirium tremens”) remains the classic example of the hyperactive subtype, featuring prominent hallucinations, agitation, and hyperarousal, often accompanied by life-threatening autonomic instability. In striking contrast is the hypoactive subtype, exemplified by benzodiazepine intoxication, in which patients are withdrawn and quiet, with prominent apathy and psychomotor slowing.

1	This dichotomy between subtypes of delirium is a useful construct, but patients often fall somewhere along a spectrum between the hyperactive and hypoactive extremes, sometimes fluctuating from one to the other. Therefore, clinicians must recognize this broad range of presentations of delirium to identify all patients with this potentially reversible cognitive disturbance. Hyperactive patients are often easily recognized by their characteristic severe agitation, tremor, hallucinations, and autonomic instability. Patients who are quietly hypoactive are more often overlooked on the medical wards and in the ICU.

1	The reversibility of delirium is emphasized because many etiologies, such as systemic infection and medication effects, can be treated easily. The long-term cognitive effects of delirium remain largely unknown. Some episodes of delirium continue for weeks, months, or even years. The persistence of delirium in some patients and its high recurrence rate may be due to inadequate initial treatment of the underlying etiology. In other instances, delirium appears to cause permanent neuronal damage and cognitive decline. Even if an episode of delirium completely resolves, there may be lingering effects of the disorder; a patient’s recall of events after delirium varies widely, ranging from complete amnesia to repeated re-experiencing of the frightening period of confusion, similar to what is seen in patients with posttraumatic stress disorder.

1	An effective primary prevention strategy for delirium begins with identification of patients at high risk for this disorder, including those preparing for elective surgery or being admitted to the hospital. Although no single validated scoring system has been widely accepted as a screen for asymptomatic patients, there are multiple well-established risk factors for delirium. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases The two most consistently identified risks are older age and baseline cognitive dysfunction. Individuals who are over age 65 or exhibit low scores on standardized tests of cognition develop delirium upon hospitalization at a rate approaching 50%. Whether age and baseline cognitive dysfunction are truly independent risk factors is uncertain. Other predisposing factors include sensory deprivation, such as preexisting hearing and visual impairment, as well as indices for poor overall health, including baseline immobility, malnutrition, and underlying medical or neurologic illness.

1	In-hospital risks for delirium include the use of bladder catheterization, physical restraints, sleep and sensory deprivation, and the addition of three or more new medications. Avoiding such risks remains a key component of delirium prevention as well as treatment. Surgical and anesthetic risk factors for the development of postoperative delirium include specific procedures such as those involving cardiopulmonary bypass, inadequate or excessive treatment of pain in the immediate postoperative period, and perhaps specific agents such as inhalational anesthetics.

1	The relationship between delirium and dementia (Chap. 448) is complicated by significant overlap between the two conditions, and it is not always simple to distinguish between them. Dementia and preexisting cognitive dysfunction serve as major risk factors for delirium, and at least two-thirds of cases of delirium occur in patients with coexisting underlying dementia. A form of dementia with parkinsonism, termed dementia with Lewy bodies, is characterized by a fluctuating course, prominent visual hallucinations, parkinsonism, and an attentional deficit that clinically resembles hyperactive delirium; patients with this condition are particularly vulnerable to delirium. Delirium in the elderly often reflects an insult to the brain that is vulnerable due to an underlying neurodegenerative condition. Therefore, the development of delirium sometimes heralds the onset of a previously unrecognized brain disorder.

1	Delirium is common, but its reported incidence has varied widely with the criteria used to define this disorder. Estimates of delirium in hospitalized patients range from 18 to 64%, with higher rates reported for elderly patients and patients undergoing hip surgery. Older patients in the ICU have especially high rates of delirium that approach 75%. The condition is not recognized in up to one-third of delirious inpatients, and the diagnosis is especially problematic in the ICU environment, where cognitive dysfunction is often difficult to appreciate in the setting of serious systemic illness and sedation. Delirium in the ICU should be viewed as an important manifestation of organ dysfunction not unlike liver, kidney, or heart failure. Outside the acute hospital setting, delirium occurs in nearly one-quarter of patients in nursing homes and in 50 to 80% of those at the end of life. These estimates emphasize the remarkably high frequency of this cognitive syndrome in older patients, a

1	nearly one-quarter of patients in nursing homes and in 50 to 80% of those at the end of life. These estimates emphasize the remarkably high frequency of this cognitive syndrome in older patients, a population expected to grow in the upcoming decades.

1	Until recently, an episode of delirium was viewed as a transient condition that carried a benign prognosis. It is now recognized as a disorder with a substantial morbidity rate and increased mortality rate and often represents the first manifestation of a serious underlying illness. Recent estimates of in-hospital mortality rates among delirious patients have ranged from 25 to 33%, a rate similar to that of patients with sepsis. Patients with an in-hospital episode of delirium have a fivefold higher mortality rate in the months after their illness compared with age-matched nondelirious hospitalized patients. Delirious hospitalized patients have a longer length of stay, are more likely to be discharged to a nursing home, and are more likely to experience subsequent episodes of delirium and cognitive decline; as a result, this condition has enormous economic implications.

1	The pathogenesis and anatomy of delirium are incompletely understood. The attentional deficit that serves as the neuropsychological hallmark of delirium has a diffuse localization within the brainstem, thalamus, prefrontal cortex, and parietal lobes. Rarely, focal lesions such as ischemic strokes have led to delirium in otherwise healthy persons; right parietal and medial dorsal thalamic lesions have been reported most commonly, pointing to the importance of these areas to delirium pathogenesis. In most cases, delirium results from widespread disturbances in cortical and subcortical regions rather than a focal neuroanatomic cause. Electroencephalogram (EEG) data in persons with delirium usually show symmetric slowing, a nonspecific finding that supports diffuse cerebral dysfunction.

1	Multiple neurotransmitter abnormalities, proinflammatory factors, and specific genes likely play a role in the pathogenesis of delirium. Deficiency of acetylcholine may play a key role, and medications with anticholinergic properties also can precipitate delirium. Dementia patients are susceptible to episodes of delirium, and those with Alzheimer’s pathology and dementia with Lewy bodies or Parkinson’s disease dementia are known to have a chronic cholinergic deficiency state due to degeneration of acetylcholine-producing neurons in the basal forebrain. Additionally, other neurotransmitters are also likely to be involved in this diffuse cerebral disorder. For example, increases in dopamine can also lead to delirium. Patients with Parkinson’s disease treated with dopaminergic medications can develop a delirium-like state that features visual hallucinations, fluctuations, and confusion.

1	Not all individuals exposed to the same insult will develop signs of delirium. A low dose of an anticholinergic medication may have no cognitive effects on a healthy young adult but produce a florid delirium in an elderly person with known underlying dementia, although even healthy young persons develop delirium with very high doses of anticholinergic medications. This concept of delirium developing as the result of an insult in predisposed individuals is currently the most widely accepted pathogenic construct. Therefore, if a previously healthy individual with no known history of cognitive illness develops delirium in the setting of a relatively minor insult such as elective surgery or hospitalization, an unrecognized underlying neurologic illness such as a neurodegenerative disease, multiple previous strokes, or another diffuse cerebral cause should be considered. In this context, delirium can be viewed as a “stress test for the brain” whereby exposure to known inciting factors such

1	previous strokes, or another diffuse cerebral cause should be considered. In this context, delirium can be viewed as a “stress test for the brain” whereby exposure to known inciting factors such as systemic infection and offending drugs can unmask a decreased cerebral reserve and herald a serious underlying and potentially treatable illness.

1	APPROACH TO THE PATIENT:

1	Because the diagnosis of delirium is clinical and is made at the bedside, a careful history and physical examination are necessary in evaluating patients with possible confusional states. Screening tools can aid physicians and nurses in identifying patients with delirium, including the Confusion Assessment Method (CAM) (Table 34-1); the Organic Brain Syndrome Scale; the Delirium Rating Scale; and, in the ICU, the ICU version of the CAM and the Delirium Detection Score. Using the well-validated CAM, a diagnosis of delirium is made if there is (1) an acute onset and fluctuating course and (2) inattention accompanied by either (3) disorganized thinking or (4) an altered level of consciousness. These scales may not identify the full spectrum of patients with delirium, and all patients who are acutely confused should be presumed delirious regardless of their presentation due to the wide variety of possible clinical features. A course that fluctuates over hours or days and may worsen at

1	are acutely confused should be presumed delirious regardless of their presentation due to the wide variety of possible clinical features. A course that fluctuates over hours or days and may worsen at night (termed sundowning) is typical but not essential for the diagnosis. Observation of the patient usually will reveal an altered level of consciousness or a deficit of attention. Other features that are sometimes present include alteration of sleep-wake cycles, thought disturbances such as hallucinations or delusions, autonomic instability, and changes in affect.

1	It may be difficult to elicit an accurate history in delirious patients who have altered levels of consciousness or impaired attention. Information from a collateral source such as a spouse or another The diagnosis of delirium requires the presence of features 1 and 2 and of either feature 3 or 4. Feature 1. Acute onset and fluctuating course This feature is satisfied by positive responses to the following questions: Is there evidence of an acute change in mental status from the patient’s baseline? Did the (abnormal) behavior fluctuate during the day, that is, tend to come and go, or did it increase and decrease in severity? Feature 2. Inattention This feature is satisfied by a positive response to the following question: Did the patient have difficulty focusing attention, for example, being easily distractible, or have difficulty keeping track of what was being said? Feature 3. Disorganized thinking

1	Feature 3. Disorganized thinking This feature is satisfied by a positive response to the following question: Was the patient’s thinking disorganized or incoherent, such as rambling or irrelevant conversation, unclear or illogical flow of ideas, or unpredictable switching from subject to subject? Feature 4. Altered level of consciousness This feature is satisfied by any answer other than “alert” to the following question: Overall, how would you rate the patient’s level of consciousness: alert (normal), vigilant (hyperalert), lethargic (drowsy, easily aroused), stupor (difficult to arouse), or coma (unarousable)? aInformation is usually obtained from a reliable reporter, such as a family member, caregiver, or nurse. Source: Modified from SK Inouye et al: Clarifying confusion: The Confusion Assessment Method. A new method for detection of delirium. Ann Intern Med 113:941, 1990.

1	Source: Modified from SK Inouye et al: Clarifying confusion: The Confusion Assessment Method. A new method for detection of delirium. Ann Intern Med 113:941, 1990. family member is therefore invaluable. The three most important pieces of history are the patient’s baseline cognitive function, the time course of the present illness, and current medications.

1	Premorbid cognitive function can be assessed through the collateral source or, if needed, via a review of outpatient records. Delirium by definition represents a change that is relatively acute, usually over hours to days, from a cognitive baseline. As a result, an acute confusional state is nearly impossible to diagnose without some knowledge of baseline cognitive function. Without this information, many patients with dementia or depression may be mistaken as delirious during a single initial evaluation. Patients with a more hypoactive, apathetic presentation with psychomotor slowing may be identified as being different from baseline only through conversations with family members. A number of validated instruments have been shown to diagnose cognitive dysfunction accurately using a collateral source, including the modified Blessed Dementia Rating Scale and the Clinical Dementia Rating (CDR). Baseline cognitive impairment is common in patients with delirium. Even when no such history

1	source, including the modified Blessed Dementia Rating Scale and the Clinical Dementia Rating (CDR). Baseline cognitive impairment is common in patients with delirium. Even when no such history of cognitive impairment is elicited, there should still be a high suspicion for a previously unrecognized underlying neurologic disorder.

1	Establishing the time course of cognitive change is important not only to make a diagnosis of delirium but also to correlate the onset of the illness with potentially treatable etiologies such as recent medication changes or symptoms of systemic infection. Medications remain a common cause of delirium, especially compounds with anticholinergic or sedative properties. It is estimated that nearly one-third of all cases of delirium are secondary to medications, especially in the elderly. Medication histories should include all prescription as well as over-the-counter and herbal substances taken by the patient and any recent changes in dosing or formulation, including substitution of generics for brand-name medications.

1	Other important elements of the history include screening for symptoms of organ failure or systemic infection, which often contributes to delirium in the elderly. A history of illicit drug use, alcoholism, or toxin exposure is common in younger delirious patients. Finally, asking the patient and collateral source about other symptoms that may accompany delirium, such as depression, may help identify potential therapeutic targets. The general physical examination in a delirious patient should include careful screening for signs of infection such as fever, tachypnea, pulmonary consolidation, heart murmur, and stiff neck. The patient’s fluid status should be assessed; both dehydration and fluid overload with resultant hypoxemia have been associated with delirium, and each is usually easily rectified. The appearance of the skin can be helpful, showing jaundice in hepatic encephalopathy, cyanosis in hypoxemia, or needle tracks in patients using intravenous drugs.

1	The neurologic examination requires a careful assessment of mental status. Patients with delirium often present with a fluctuating course; therefore, the diagnosis can be missed when one relies on a single time point of evaluation. Some but not all patients exhibit the characteristic pattern of sundowning, a worsening of their condition in the evening. In these cases, assessment only during morning rounds may be falsely reassuring.

1	An altered level of consciousness ranging from hyperarousal to lethargy to coma is present in most patients with delirium and can be assessed easily at the bedside. In a patient with a relatively normal level of consciousness, a screen for an attentional deficit is in order, because this deficit is the classic neuropsychological hallmark of delirium. Attention can be assessed while taking a history from the patient. Tangential speech, a fragmentary flow of ideas, or inability to follow complex commands often signifies an attentional problem. There are formal neuropsychological tests to assess attention, but a simple bedside test of digit span forward is quick and fairly sensitive. In this task, patients are asked to repeat successively longer random strings of digits beginning with two digits in a row, said to the patient at 1-second intervals. Healthy adults can repeat a string of five to seven digits before faltering; a digit span of four or less usually indicates an attentional

1	digits in a row, said to the patient at 1-second intervals. Healthy adults can repeat a string of five to seven digits before faltering; a digit span of four or less usually indicates an attentional deficit unless hearing or language barriers are present, and many patients with delirium have digit spans of three or fewer digits.

1	More formal neuropsychological testing can be helpful in assessing a delirious patient, but it is usually too cumbersome and time-consuming in the inpatient setting. A Mini-Mental State Examination (MMSE) provides information regarding orientation, language, and visuospatial skills; however, performance of many tasks on the MMSE, including the spelling of “world” backward and serial subtraction of digits, will be impaired by delirious patients’ attentional deficits, rendering the test unreliable.

1	The remainder of the screening neurologic examination should focus on identifying new focal neurologic deficits. Focal strokes or mass lesions in isolation are rarely the cause of delirium, but patients with underlying extensive cerebrovascular disease or neurodegenerative conditions may not be able to cognitively tolerate even relatively small new insults. Patients should be screened for other signs of neurodegenerative conditions such as parkinsonism, which is seen not only in idiopathic Parkinson’s disease but also in other dementing conditions such as Alzheimer’s disease, dementia with Lewy bodies, and progressive supranuclear palsy. The presence of multifocal myoclonus or asterixis on the motor examination is nonspecific but usually indicates a metabolic or toxic etiology of the delirium.

1	Some etiologies can be easily discerned through a careful history and physical examination, whereas others require confirmation with laboratory studies, imaging, or other ancillary tests. A large, diverse group of insults can lead to delirium, and the cause in many patients is often multifactorial. Common etiologies are listed in Table 34-2. Prescribed, over-the-counter, and herbal medications all can precipitate delirium. Drugs with anticholinergic properties, narcotics, and benzodiazepines are particularly common offenders, but nearly any compound can lead to cognitive dysfunction in a predisposed patient. Whereas an elderly patient with baseline dementia PART 2 Cardinal Manifestations and Presentation of Diseases CoMMon ETioLogiES of DELiRiuM

1	PART 2 Cardinal Manifestations and Presentation of Diseases CoMMon ETioLogiES of DELiRiuM Prescription medications: especially those with anticholinergic properties, narcotics, and benzodiazepines Drugs of abuse: alcohol intoxication and alcohol withdrawal, opiates, ecstasy, LSD, GHB, PCP, ketamine, cocaine, “bath salts,” marijuana and its synthetic forms Poisons: inhalants, carbon monoxide, ethylene glycol, pesticides Metabolic conditions Electrolyte disturbances: hypoglycemia, hyperglycemia, hyponatremia, hypernatremia, hypercalcemia, hypocalcemia, hypomagnesemia Hypothermia and hyperthermia Pulmonary failure: hypoxemia and hypercarbia Liver failure/hepatic encephalopathy Renal failure/uremia Cardiac failure Vitamin deficiencies: B12, thiamine, folate, niacin Dehydration and malnutrition Anemia Systemic infections: urinary tract infections, pneumonia, skin and soft tissue infections, sepsis CNS infections: meningitis, encephalitis, brain abscess

1	Systemic infections: urinary tract infections, pneumonia, skin and soft tissue infections, sepsis CNS infections: meningitis, encephalitis, brain abscess Endocrine conditions Hyperthyroidism, hypothyroidism Hyperparathyroidism Adrenal insufficiency Cerebrovascular disorders Global hypoperfusion states Hypertensive encephalopathy Focal ischemic strokes and hemorrhages (rare): especially nondominant Seizure-related disorders Nonconvulsive status epilepticus Intermittent seizures with prolonged postictal states Neoplastic disorders Diffuse metastases to the brain Gliomatosis cerebri Carcinomatous meningitis CNS lymphoma Hospitalization Terminal end-of-life delirium Abbreviations: CNS, central nervous system; GHB, γ-hydroxybutyrate; LSD, lysergic acid diethylamide; PCP, phencyclidine.

1	Hospitalization Terminal end-of-life delirium Abbreviations: CNS, central nervous system; GHB, γ-hydroxybutyrate; LSD, lysergic acid diethylamide; PCP, phencyclidine. may become delirious upon exposure to a relatively low dose of a medication, less susceptible individuals may become delirious only with very high doses of the same medication. This observation emphasizes the importance of correlating the timing of recent medication changes, including dose and formulation, with the onset of cognitive dysfunction.

1	In younger patients, illicit drugs and toxins are common causes of delirium. In addition to more classic drugs of abuse, the recent rise in availability of methylenedioxymethamphetamine (MDMA, ecstasy), γ-hydroxybutyrate (GHB), “bath salts,” synthetic cannabis, and the phencyclidine (PCP)-like agent ketamine, has led to an increase in delirious young persons presenting to acute care settings (Chap. 469e). Many common prescription drugs such as oral narcotics and benzodiazepines are often abused and readily available on the street. Alcohol abuse leading to high serum levels causes confusion, but more commonly, it is withdrawal from alcohol that leads to a hyperactive delirium. Alcohol and benzodiazepine withdrawal should be considered in all cases of delirium because even patients who drink only a few servings of alcohol every day can experience relatively severe withdrawal symptoms upon hospitalization.

1	Metabolic abnormalities such as electrolyte disturbances of sodium, calcium, magnesium, or glucose can cause delirium, and mild derangements can lead to substantial cognitive disturbances in susceptible individuals. Other common metabolic etiologies include liver and renal failure, hypercarbia and hypoxemia, vitamin deficiencies of thiamine and B12, autoimmune disorders including central nervous system (CNS) vasculitis, and endocrinopathies such as thyroid and adrenal disorders.

1	Systemic infections often cause delirium, especially in the elderly. A common scenario involves the development of an acute cognitive decline in the setting of a urinary tract infection in a patient with baseline dementia. Pneumonia, skin infections such as cellulitis, and frank sepsis also lead to delirium. This so-called septic encephalopathy, often seen in the ICU, is probably due to the release of proinflammatory cytokines and their diffuse cerebral effects. CNS infections such as meningitis, encephalitis, and abscess are less common etiologies of delirium; however, in light of the high mortality rates associated with these conditions when they are not treated quickly, clinicians must always maintain a high index of suspicion.

1	In some susceptible individuals, exposure to the unfamiliar environment of a hospital itself can lead to delirium. This etiology usually occurs as part of a multifactorial delirium and should be considered a diagnosis of exclusion after all other causes have been thoroughly investigated. Many primary prevention and treatment strategies for delirium involve relatively simple methods to address the aspects of the inpatient setting that are most confusing.

1	Cerebrovascular etiologies of delirium are usually due to global hypoperfusion in the setting of systemic hypotension from heart failure, septic shock, dehydration, or anemia. Focal strokes in the right parietal lobe and medial dorsal thalamus rarely can lead to a delirious state. A more common scenario involves a new focal stroke or hemorrhage causing confusion in a patient who has decreased cerebral reserve. In these individuals, it is sometimes difficult to distinguish between cognitive dysfunction resulting from the new neurovascular insult itself and delirium due to the infectious, metabolic, and pharmacologic complications that can accompany hospitalization after stroke.

1	Because a fluctuating course often is seen in delirium, intermittent seizures may be overlooked when one is considering potential etiologies. Both nonconvulsive status epilepticus and recurrent focal or generalized seizures followed by postictal confusion can cause delirium; EEG remains essential for this diagnosis. Seizure activity spreading from an electrical focus in a mass or infarct can explain global cognitive dysfunction caused by relatively small lesions. It is very common for patients to experience delirium at the end of life in palliative care settings. This condition, sometimes described as terminal restlessness, must be identified and treated aggressively because it is an important cause of patient and family discomfort at the end of life. It should be remembered that these patients also may be suffering from more common etiologies of delirium such as systemic infection.

1	A cost-effective approach to the diagnostic evaluation of delirium allows the history and physical examination to guide further tests. No established algorithm for workup will fit all delirious patients due to the staggering number of potential etiologies, but one stepwise approach is detailed in Table 34-3. If a clear precipitant is STEPwiSE EvALuATion of A PATiEnT wiTH DELiRiuM History with special attention to medications (including over-the-counter and herbals) General physical examination and neurologic examination Complete blood count Electrolyte panel including calcium, magnesium, phosphorus Liver function tests, including albumin Renal function tests

1	Electrocardiogram Arterial blood gas Serum and/or urine toxicology screen (perform earlier in young persons) Brain imaging with MRI with diffusion and gadolinium (preferred) or CT Suspected CNS infection: lumbar puncture after brain imaging Suspected seizure-related etiology: electroencephalogram (EEG) (if high suspicion, should be performed immediately) Second-tier further evaluation Vitamin levels: B12, folate, thiamine Endocrinologic laboratories: thyroid-stimulating hormone (TSH) and free T4; cortisol Serum ammonia Sedimentation rate Autoimmune serologies: antinuclear antibodies (ANA), complement levels; p-ANCA, c-ANCA. consider paraneoplastic serologies Infectious serologies: rapid plasmin reagin (RPR); fungal and viral serologies if high suspicion; HIV antibody Lumbar puncture (if not already performed) Brain MRI with and without gadolinium (if not already performed)

1	Abbreviations: c-ANCA, cytoplasmic antineutrophil cytoplasmic antibody; CNS, central nervous system; CT, computed tomography; MRI, magnetic resonance imaging; p-ANCA, perinuclear antineutrophil cytoplasmic antibody. identified, such as an offending medication, further testing may not be required. If, however, no likely etiology is uncovered with initial evaluation, an aggressive search for an underlying cause should be initiated.

1	Basic screening labs, including a complete blood count, electrolyte panel, and tests of liver and renal function, should be obtained in all patients with delirium. In elderly patients, screening for systemic infection, including chest radiography, urinalysis and culture, and possibly blood cultures, is important. In younger individuals, serum and urine drug and toxicology screening may be appropriate early in the workup. Additional laboratory tests addressing other autoimmune, endocrinologic, metabolic, and infectious etiologies should be reserved for patients in whom the diagnosis remains unclear after initial testing.

1	Multiple studies have demonstrated that brain imaging in patients with delirium is often unhelpful. If, however, the initial workup is unrevealing, most clinicians quickly move toward imaging of the brain to exclude structural causes. A noncontrast computed tomography (CT) scan can identify large masses and hemorrhages but is otherwise unlikely to help determine an etiology of delirium. The ability of magnetic resonance imaging (MRI) to identify most acute ischemic strokes as well as to provide neuroanatomic detail that gives clues to possible infectious, inflammatory, neurodegenerative, and neoplastic conditions makes it the test of choice. Because MRI techniques are limited by availability, speed of imaging, patient cooperation, and contraindications, many clinicians begin with CT scanning and proceed to MRI if the etiology of delirium remains elusive.

1	Lumbar puncture (LP) must be obtained immediately after appropriate neuroimaging in all patients in whom CNS infection is suspected. Spinal fluid examination can also be useful in identifying inflammatory and neoplastic conditions. As a result, LP should be considered in any delirious patient with a negative workup. EEG does not have a routine role in the workup of delirium, but it remains invaluable if seizure-related etiologies are considered. PART 2 Cardinal Manifestations and Presentation of Diseases Management of delirium begins with treatment of the underlying inciting factor (e.g., patients with systemic infections should be given appropriate antibiotics, and underlying electrolyte disturbances judiciously corrected). These treatments often lead to prompt resolution of delirium. Blindly targeting the symptoms of delirium pharmacologically only serves to prolong the time patients remain in the confused state and may mask important diagnostic information.

1	Relatively simple methods of supportive care can be highly effective in treating patients with delirium. Reorientation by the nursing staff and family combined with visible clocks, calendars, and outside-facing windows can reduce confusion. Sensory isolation should be prevented by providing glasses and hearing aids to patients who need them. Sundowning can be addressed to a large extent through vigilance to appropriate sleep-wake cycles. During the day, a well-lit room should be accompanied by activities or exercises to prevent napping. At night, a quiet, dark environment with limited interruptions by staff can assure proper rest. These sleep-wake cycle interventions are especially important in the ICU setting as the usual constant 24-h activity commonly provokes delirium. Attempting to mimic the home environment as much as possible also has been shown to help treat and even prevent delirium. Visits from friends and family throughout the day minimize the anxiety associated with the

1	mimic the home environment as much as possible also has been shown to help treat and even prevent delirium. Visits from friends and family throughout the day minimize the anxiety associated with the constant flow of new faces of staff and physicians. Allowing hospitalized patients to have access to home bedding, clothing, and nightstand objects makes the hospital environment less foreign and therefore less confusing. Simple standard nursing practices such as maintaining proper nutrition and volume status as well as managing incontinence and skin breakdown also help alleviate discomfort and resulting confusion.

1	In some instances, patients pose a threat to their own safety or to the safety of staff members, and acute management is required. Bed alarms and personal sitters are more effective and much less disorienting than physical restraints. Chemical restraints should be avoided, but only when necessary, very-low-dose typical or atypical antipsychotic medications administered on an as-needed basis are effective. The recent association of antipsychotic use in the elderly with increased mortality rates underscores the importance of using these medications judiciously and only as a last resort. Benzodiazepines often worsen confusion through their sedative properties. Although many clinicians still use benzodiazepines to treat acute confusion, their use should be limited to cases in which delirium is caused by alcohol or benzodiazepine withdrawal.

1	In light of the high morbidity associated with delirium and the tremendously increased health care costs that accompany it, development of an effective strategy to prevent delirium in hospitalized patients is extremely important. Successful identification of high-risk patients is the first step, followed by initiation of appropriate interventions. Simple standardized protocols used to manage risk factors for delirium, including sleep-wake cycle reversal, immobility, visual impairment, hearing impairment, sleep deprivation, and dehydration, have been shown to be effective. Recent trials in the ICU have focused both on identifying sedatives, such as dexmedetomidine, that are less likely to lead to delirium in critically ill patients and on developing protocols for daily awakenings in which infusions of sedative medications are interrupted and the patient is reorientated by the staff. All hospitals and health care systems should work toward decreasing the incidence of delirium.

1	William W. Seeley, Bruce L. Miller

1	Dementia, a syndrome with many causes, affects >5 million people in the United States and results in a total annual health care cost between $157 and $215 billion. Dementia is defined as an acquired deterioration in cognitive abilities that impairs the successful performance of activities of daily living. Episodic memory, the ability to recall events specific in time and place, is the cognitive function most commonly lost; 10% of persons age >70 years and 20–40% of individuals age >85 years have clinically identifiable memory loss. In addition to memory, dementia may erode other mental faculties, including language, visuospatial, praxis, calculation, judgment, and problem-solving abilities. Neuropsychiatric and social deficits also arise in many dementia syndromes, manifesting as depression, apathy, anxiety, hallucinations, delusions, agitation, insomnia, sleep disturbances, compulsions, or disinhibition. The clinical course may be slowly progressive, as in Alzheimer’s disease (AD);

1	apathy, anxiety, hallucinations, delusions, agitation, insomnia, sleep disturbances, compulsions, or disinhibition. The clinical course may be slowly progressive, as in Alzheimer’s disease (AD); static, as in anoxic encephalopathy; or may fluctuate from day to day or minute to minute, as in dementia with Lewy bodies. Most patients with AD, the most prevalent form of dementia, begin with episodic memory impairment, although in other dementias, such as frontotemporal dementia, memory loss is not typically a presenting feature. Focal cerebral disorders are discussed in Chap. 36 and illustrated in a video library in Chap. 37e; the pathogenesis of AD and related disorders is discussed in Chap. 448.

1	Dementia syndromes result from the disruption of specific large-scale neuronal networks; the location and severity of synaptic and neuronal loss combine to produce the clinical features (Chap. 36). Behavior, mood, and attention are modulated by ascending noradrenergic, serotonergic, and dopaminergic pathways, whereas cholinergic signaling is critical for attention and memory functions. The dementias differ in the relative neurotransmitter deficit profiles; accordingly, accurate diagnosis guides effective pharmacologic therapy.

1	AD begins in the entorhinal region of the medial temporal lobe, spreads to the hippocampus, and then moves to lateral and posterior temporal and parietal neocortex, eventually causing a more widespread degeneration. Vascular dementia is associated with focal damage in a variable patchwork of cortical and subcortical regions or white matter tracts that disconnect nodes within distributed networks. In keeping with its anatomy, AD typically presents with episodic memory loss accompanied later by aphasia or navigational problems. In contrast, dementias that begin in frontal or subcortical regions, such as frontotemporal dementia (FTD) or Huntington’s disease (HD), are less likely to begin with memory problems and more likely to present with difficulties with judgment, mood, executive control, movement, and behavior.

1	Lesions of frontal-striatal1 pathways produce specific and predictable effects on behavior. The dorsolateral prefrontal cortex has connections with a central band of the caudate nucleus. Lesions of either the caudate or dorsolateral prefrontal cortex, or their connecting white matter pathways, may result in executive dysfunction, manifesting as poor organization and planning, decreased cognitive flexibility, and impaired working memory. The lateral orbital frontal cortex connects with the ventromedial caudate, and lesions of this system cause impulsiveness, distractibility, and disinhibition. The anterior cingulate cortex and adjacent medial prefrontal cortex project to the nucleus accumbens, and interruption of this system produces apathy, poverty of speech, emotional blunting, or even akinetic mutism. All corticostriatal systems also include topographically organized projections through the globus pallidus and thalamus, and damage to these nodes 1The striatum comprises the

1	or even akinetic mutism. All corticostriatal systems also include topographically organized projections through the globus pallidus and thalamus, and damage to these nodes 1The striatum comprises the caudate/putamen.

1	can likewise reproduce the clinical syndrome of cortical or striatal injury. The single strongest risk factor for dementia is increasing age. The prevalence of disabling memory loss increases with each decade over age 50 and is usually associated with the microscopic changes of AD at autopsy. Yet some centenarians have intact memory function and no evidence of clinically significant dementia. Whether dementia is an inevitable consequence of normal human aging remains controversial. The many causes of dementia are listed in Table 35-1. The frequency of each condition depends on the age group under study, access of the group to medical care, country of origin, and perhaps racial or ethnic background. AD is the most common cause of dementia in Western Most Common Causes of Dementia Less Common Causes of Dementia Thiamine (B1): Wernicke’s encephalopathya B12 (subacute combined multifocal leukoencephalopathy) Tuberculosis, fungal, and protozoala Whipple’s diseasea

1	Less Common Causes of Dementia Thiamine (B1): Wernicke’s encephalopathya B12 (subacute combined multifocal leukoencephalopathy) Tuberculosis, fungal, and protozoala Whipple’s diseasea Drug, medication, and narcotic poisoninga Heavy metal intoxicationa Organic toxins degeneration spectrum Multiple sclerosis Adult Down’s syndrome with ALS-parkinsonism-dementia complex of Guam Prion (Creutzfeldt-Jakob and Gerstmann-Sträussler-Scheinker diseases) Miscellaneous Sarcoidosisa Vasculitisa CADASIL, etc. Acute intermittent porphyriaa

1	Metabolic disorders (e.g., Wilson’s and Leigh’s diseases, leukodystrophies, lipid storage diseases, mitochondrial mutations) countries, accounting for more than half of all patients. Vascular dis-171 ease is considered the second most frequent cause for dementia and is particularly common in elderly patients or populations with limited access to medical care, where vascular risk factors are undertreated. Often, vascular brain injury is mixed with neurodegenerative disorders, making it difficult, even for the neuropathologist, to estimate the contribution of cerebrovascular disease to the cognitive disorder in an individual patient. Dementias associated with Parkinson’s disease (PD) (Chap. 449) are common and may develop years after onset of a parkinsonian disorder, as seen with PD-related dementia (PDD), or can occur concurrently with or preceding the motor syndrome, as in dementia with Lewy bodies (DLB). In patients under the age of 65, FTD rivals AD as the most common cause of

1	dementia (PDD), or can occur concurrently with or preceding the motor syndrome, as in dementia with Lewy bodies (DLB). In patients under the age of 65, FTD rivals AD as the most common cause of dementia. Chronic intoxications, including those resulting from alcohol and prescription drugs, are an important and often treatable cause of dementia. Other disorders listed in Table 35-1 are uncommon but important because many are reversible. The classification of dementing illnesses into reversible and irreversible disorders is a useful approach to differential diagnosis. When effective treatments for the neurodegenerative conditions emerge, this dichotomy will become obsolete.

1	In a study of 1000 persons attending a memory disorders clinic, 19% had a potentially reversible cause of the cognitive impairment and 23% had a potentially reversible concomitant condition that may have contributed to the patient’s impairment. The three most common potentially reversible diagnoses were depression, normal pressure hydrocephalus (NPH), and alcohol dependence; medication side effects are also common and should be considered in every patient (Table 35-1).

1	Subtle cumulative decline in episodic memory is a common part of aging. This frustrating experience, often the source of jokes and humor, is referred to as benign forgetfulness of the elderly. Benign means that it is not so progressive or serious that it impairs reasonably successful and productive daily functioning, although the distinction between benign and more significant memory loss can be difficult to make. At age 85, the average person is able to learn and recall approximately one-half of the items (e.g., words on a list) that he or she could at age 18. A measurable cognitive problem that does not seriously disrupt daily activities is often referred to as mild cognitive impairment (MCI). Factors that predict progression from MCI to an AD dementia include a prominent memory deficit, family history of dementia, presence of an apolipoprotein ε4 (Apo ε4) allele, small hippocampal volumes, an AD-like signature of cortical atrophy, low cerebrospinal fluid Aβ, and elevated tau or

1	family history of dementia, presence of an apolipoprotein ε4 (Apo ε4) allele, small hippocampal volumes, an AD-like signature of cortical atrophy, low cerebrospinal fluid Aβ, and elevated tau or evidence of brain amyloid deposition on positron emission tomography (PET) imaging.

1	The major degenerative dementias include AD, DLB, FTD and related disorders, HD, and prion diseases, including Creutzfeldt-Jakob disease (CJD). These disorders are all associated with the abnormal aggregation of a specific protein: Aβ42 and tau in AD; α-synuclein in DLB; tau, TAR DNA-binding protein of 43 kDa (TDP-43), or fused in sarcoma (FUS) in FTD; huntingtin in HD; and misfolded prion protein (PrPsc) in CJD (Table 35-2). APPROACH TO THE PATIENT: Three major issues should be kept at the forefront: (1) What is the best fit for a clinical diagnosis? (2) What component of the dementia syndrome is treatable or reversible? (3) Can the physician help to alleviate the burden on caregivers? A broad overview of the approach to dementia is shown in Table 35-3. The major degenerative dementias can usually be distinguished by the initial symptoms; neuropsychological, neuropsychiatric, and neurologic findings; and neuroimaging features (Table 35-4).

1	The history should concentrate on the onset, duration, and tempo of progression. An acute or subacute onset of confusion may be due to delirium (Chap. 34) and should trigger the search for intoxication, infection, or metabolic derangement. An elderly person aPotentially reversible dementia. Abbreviations: ALS, amyotrophic lateral sclerosis; CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; LBD, Lewy body disease; PDD, Parkinson’s disease dementia. PART 2 Cardinal Manifestations and Presentation of Diseases Abbreviations: AD, Alzheimer’s disease; CJD, Creutzfeldt-Jakob disease; DLB, dementia with Lewy bodies; FTD, frontotemporal dementia.

1	with slowly progressive memory loss over several years is likely to suffer from AD. Nearly 75% of patients with AD begin with memory symptoms, but other early symptoms include difficulty with managing money, driving, shopping, following instructions, finding words, or navigating. Personality change, disinhibition, and weight gain or compulsive eating suggest FTD, not AD. FTD is also suggested by prominent apathy, compulsivity, loss of empathy for others, or progressive loss of speech fluency or single-word comprehension and by a relative sparing of memory and visuospatial abilities. The diagnosis of DLB is suggested by early visual hallucinations; parkinsonism; proneness to delirium or sensitivity to psychoactive medications; rapid eye movement (REM) behavior disorder (RBD; the loss of skeletal muscle paralysis during dreaming); or Capgras syndrome, the delusion that a familiar person has been replaced by an impostor.

1	A history of stroke with irregular stepwise progression suggests vascular dementia. Vascular dementia is also commonly seen in the setting of hypertension, atrial fibrillation, peripheral vascular disease, and diabetes. In patients suffering from cerebrovascular disease, it can be difficult to determine whether the dementia is due to AD, vascular disease, or a mixture of the two because many of the risk factors for vascular dementia, including diabetes, high cholesterol, elevated homocysteine, and low exercise, are also putative risk factors for AD. Moreover, many patients with a major vascular contribution to their dementia lack a history of stepwise decline. Rapid progression with motor rigidity and myoclonus suggests CJD (Chap. 453e). Seizures may indicate strokes or neoplasm but also occur in AD, particularly early-age-of-onset AD. Gait disturbance is common in vascular dementia, PD/DLB, or NPH. A history of high-risk sexual behaviors or intravenous drug use

1	Abbreviations: CT, computed tomography; EEG, electroencephalogram; MRI, magnetic resonance imaging; PET, positron emission tomography; RBC, red blood cell; RPR, rapid plasma reagin (test); SPECT, single-photon emission computed tomography; TSH, thyroid-stimulating hormone; VDRL, Venereal Disease Research Laboratory (test for syphilis). Abbreviations: AD, Alzheimer’s disease; CBD, cortical basal degeneration; CJD, Creutzfeldt-Jakob disease; DLB, dementia with Lewy bodies; FLAIR, fluid-attenuated inversion recovery; FTD, frontotemporal dementia; MND, motor neuron disease; MRI, magnetic resonance imaging; PSP, progressive supranuclear palsy; REM, rapid eye movement.

1	should trigger a search for central nervous system (CNS) infection, especially HIV or syphilis. A history of recurrent head trauma could indicate chronic subdural hematoma, chronic traumatic encephalopathy (a progressive dementia best characterized in contact sport athletes such as boxers and American football players), intracranial hypotension, or NPH. Subacute onset of severe amnesia and psychosis with mesial temporal T2/fluid-attenuated inversion recovery (FLAIR) hyperintensities on magnetic resonance imaging (MRI) should raise concern for paraneoplastic limbic encephalitis, especially in a long-term smoker or other patients at risk for cancer. Related autoimmune conditions, such as voltage-gated potassium channel (VGKC)or N-methyl-d-aspartate (NMDA)-receptor antibody-mediated encephalopathy, can present with a similar tempo and imaging signature with or without characteristic motor manifestations such as myokymia (anti-VGKC) and faciobrachial dystonic seizures (anti-NMDA). Alcohol

1	can present with a similar tempo and imaging signature with or without characteristic motor manifestations such as myokymia (anti-VGKC) and faciobrachial dystonic seizures (anti-NMDA). Alcohol abuse creates risk for malnutrition and thiamine deficiency. Veganism, bowel irradiation, an autoimmune diathesis, a remote history of gastric surgery, and chronic antihistamine therapy for dyspepsia or gastroesophageal reflux predispose to B12 deficiency. Certain occupations, such as working in a battery or chemical factory, might indicate heavy metal intoxication. Careful review of medication intake, especially for sedatives and analgesics, may raise the issue of chronic drug intoxication. An autosomal dominant family history is found in HD and in familial forms of AD, FTD, DLB, or prion disorders. A history of mood disorders, the recent death of a loved one, or depressive signs, such as insomnia or weight loss, raise the possibility of depression-related cognitive impairments.

1	A thorough general and neurologic examination is essential to document dementia, to look for other signs of nervous system involvement, and to search for clues suggesting a systemic disease that might be responsible for the cognitive disorder. Typical AD spares motor systems until later in the course. In contrast, FTD patients often develop axial rigidity, supranuclear gaze palsy, or a motor neuron disease reminiscent of amyotrophic lateral sclerosis (ALS). In DLB, the initial symptoms may include the new onset of a parkinsonian syndrome (resting tremor, cogwheel rigidity, bradykinesia, festinating gait), but DLB often starts with visual hallucinations or dementia. Symptoms referable to the lower brainstem (RBD, gastrointestinal or autonomic problems) may arise years or even decades before parkinsonism or dementia. Corticobasal syndrome (CBS) features asymmetric akinesia and rigidity, dystonia, myoclonus, alien limb phenomena, pyramidal signs, and prefrontal deficits such as nonfluent

1	parkinsonism or dementia. Corticobasal syndrome (CBS) features asymmetric akinesia and rigidity, dystonia, myoclonus, alien limb phenomena, pyramidal signs, and prefrontal deficits such as nonfluent aphasia with or without motor speech impairment, executive dysfunction, apraxia, or a behavioral disorder. Progressive supranuclear palsy (PSP) is associated with unexplained falls, axial rigidity, dysphagia, and vertical gaze deficits. CJD is suggested by the presence of diffuse rigidity, an akinetic-mute state, and prominent, often startle-sensitive myoclonus.

1	Hemiparesis or other focal neurologic deficits suggest vascular dementia or brain tumor. Dementia with a myelopathy and peripheral neuropathy suggests vitamin B12 deficiency. Peripheral neuropathy could also indicate another vitamin deficiency, heavy metal intoxication, thyroid dysfunction, Lyme disease, or vasculitis. Dry, cool skin, hair loss, and bradycardia suggest hypothyroidism. Fluctuating confusion associated with repetitive stereotyped movements may indicate ongoing limbic, temporal, or frontal seizures. In the elderly, hearing impairment or visual loss may produce confusion and disorientation misinterpreted as dementia. Profound bilateral sensorineural hearing loss in a younger patient with short stature or myopathy, however, should raise concern for a mitochondrial disorder.

1	Brief screening tools such as the Mini-Mental State Examination (MMSE), the Montreal Cognitive Assessment (MOCA), and Cognistat can be used to capture dementia and follow progression. None of these tests is highly sensitive to early-stage dementia or discriminates between dementia syndromes. The MMSE is a 30 point test of cognitive function, with each correct answer being scored as 1 point. It includes tests in the areas of: orientation (e.g., identify season/date/month/year/floor/hospital/town/state/ country); registration (e.g., name and restate 3 objects); recall (e.g., remember the same three objects 5 minutes later); and language (e.g., name pencil and watch; repeat “no if ’s and’s or but’s”; follow a 3-step command; obey a written command; and write a sentence and copy a design). In most patients with MCI and some with clinically apparent AD, bedside screening tests may be normal, and a more challenging and comprehensive set of neuropsychological tests will be required. When the

1	most patients with MCI and some with clinically apparent AD, bedside screening tests may be normal, and a more challenging and comprehensive set of neuropsychological tests will be required. When the etiology for the dementia syndrome remains in doubt, a specially tailored evaluation should be performed that includes tasks of working and episodic memory, executive function, language, and visuospatial and perceptual abilities. In AD, the early deficits involve episodic memory, category generation (“name as many animals as you can in 1 minute”), and visuoconstructive ability. Usually deficits in verbal or visual episodic memory are the first neuropsychological abnormalities detected, and tasks that require the patient to recall a long list of words or a series of pictures after a predetermined delay will demonstrate deficits in most patients. In FTD, the earliest deficits on cognitive testing involve executive control or language (speech or naming) function, but some patients lack

1	delay will demonstrate deficits in most patients. In FTD, the earliest deficits on cognitive testing involve executive control or language (speech or naming) function, but some patients lack either finding despite profound social-emotional deficits. PDD or DLB patients have more severe deficits in visuospatial function but do better on episodic memory tasks than patients with AD. Patients with vascular dementia often demonstrate a mixture of executive control and visuospatial deficits, with prominent psychomotor slowing. In delirium, the most prominent deficits involve attention, working memory, and executive function, making the assessment of other cognitive domains challenging and often uninformative.

1	A functional assessment should also be performed to help the physician determine the day-to-day impact of the disorder on the patient’s memory, community affairs, hobbies, judgment, dressing, and eating. Knowledge of the patient’s functional abilities will help the clinician and the family to organize a therapeutic approach.

1	Neuropsychiatric assessment is important for diagnosis, prognosis, and treatment. In the early stages of AD, mild depressive features, social withdrawal, and irritability or anxiety are the most prominent psychiatric changes, but patients often maintain core social graces into the middle or late stages, when delusions, agitation, and sleep disturbance may emerge. In FTD, dramatic personality change with apathy, overeating, compulsions, disinhibition, euphoria, and loss of empathy are early and common. DLB is associated with visual hallucinations, delusions related to person or place identity, RBD, and excessive daytime sleepiness. Dramatic fluctuations occur not only in cognition but also in arousal. Vascular dementia can present with psychiatric symptoms such as depression, anxiety, delusions, disinhibition, or apathy.

1	The choice of laboratory tests in the evaluation of dementia is complex and should be tailored to the individual patient. The physician must take measures to avoid missing a reversible or treatable cause, yet no single treatable etiology is common; thus, a screen must use multiple tests, each of which has a low yield. Cost/benefit ratios are difficult to assess, and many laboratory screening algorithms for dementia discourage multiple tests. Nevertheless, even a test with only a 1–2% positive rate is worth undertaking if the alternative is missing a treatable cause of dementia. Table 35-3 lists most screening tests for dementia. The American Academy of Neurology recommends the routine measurement of a complete blood count, electrolytes, renal and thyroid function, a vitamin B12 level, and a neuroimaging study (computed tomography [CT] or MRI).

1	Neuroimaging studies, especially MRI, help to rule out primary and metastatic neoplasms, locate areas of infarction or inflammation, detect subdural hematomas, and suggest NPH or diffuse white matter disease. They also help to establish a regional pattern of atrophy. Support for the diagnosis of AD includes hippocampal atrophy in addition to posterior-predominant cortical atrophy (Fig. 35-1). Focal frontal, insular, and/or anterior temporal atrophy suggests FTD (Chap. 448). DLB often features less prominent atrophy, with greater involvement of amygdala than hippocampus. In CJD, magnetic resonance (MR) diffusion-weighted imaging reveals restricted diffusion within the cortical ribbon and basal ganglia in most patients. Extensive white matter abnormalities correlate with a vascular etiology (Fig. 35-2). Communicating hydrocephalus with vertex effacement (crowding of dorsal convexity gyri/sulci), gaping Sylvian fissures despite minimal cortical atrophy, and additional features shown in

1	(Fig. 35-2). Communicating hydrocephalus with vertex effacement (crowding of dorsal convexity gyri/sulci), gaping Sylvian fissures despite minimal cortical atrophy, and additional features shown in Fig. 35-3 suggest NPH. Single-photon emission computed tomography (SPECT) and PET scanning show temporal-parietal hypoperfusion or hypometabolism in AD and frontotemporal deficits in FTD, but these changes often reflect atrophy and can therefore be detected with MRI alone in many patients. Recently, amyloid imaging has shown promise for the diagnosis of AD, and Pittsburgh Compound-B (PiB) (not available outside of research settings) and 18F-AV-45 (florbetapir; approved by the U.S. Food and Drug Administration in 2013) are reliable radioligands for detecting brain amyloid associated with amyloid

1	PART 2 Cardinal Manifestations and Presentation of Diseases

1	FIguRE 35-1 Alzheimer’s disease (AD). Axial T1-weighted magnetic resonance images of a healthy 71-year-old (A) and a 64-year-old with AD (C). Note the reduction in medial temporal lobe volume in the patient with AD. Fluorodeoxyglucose positron emission tomography scans of the same individuals (B and D) demonstrate reduced glucose metabolism in the posterior temporoparietal regions bilaterally in AD, a typical finding in this condition. HC, healthy control. (Images courtesy of Gil Rabinovici, University of California, San Francisco and William Jagust, University of California, Berkeley.) angiopathy or neuritic plaques of AD (Fig. 35-4). Because these abnormalities can be seen in cognitively normal older persons, however (~25% of individuals at age 65), amyloid imaging may also detect preclinical or incidental AD in patients lacking an AD-like dementia syndrome. Currently, the main clinical value of amyloid imaging is to exclude AD as the likely cause of dementia in patients who have

1	or incidental AD in patients lacking an AD-like dementia syndrome. Currently, the main clinical value of amyloid imaging is to exclude AD as the likely cause of dementia in patients who have negative scans. Once disease-modifying therapies become available, use of these biomarkers may help to identify treatment

1	FIguRE 35-2 Diffuse white matter disease. Axial fluid-attenuated inversion recovery (FLAIR) magnetic resonance image through the lateral ventricles reveals multiple areas of hyperintensity (arrows) involving the periventricular white matter as well as the corona radiata and striatum. Although seen in some individuals with normal cognition, this appearance is more pronounced in patients with dementia of a vascular etiology.

1	FIguRE 35-3 Normal-pressure hydrocephalus. A. Sagittal T1-weighted magnetic resonance image (MRI) demonstrates dilation of the lateral ventricle and stretching of the corpus callosum (arrows), depression of the floor of the third ventricle (single arrowhead), and enlargement of the aqueduct (double arrowheads). Note the diffuse dilation of the lateral, third, and fourth ventricles with a patent aqueduct, typical of communicating hydrocephalus. B. Axial T2-weighted MRIs demonstrate dilation of the lateral ventricles. This patient underwent successful ventriculoperitoneal shunting. candidates before irreversible brain injury has occurred. In the meantime, the significance of detecting brain amyloid in an asymptomatic elder remains a topic of vigorous investigation. Similarly, MRI perfusion and structural/functional connectivity methods are being explored as potential treatment-monitoring strategies.

1	Lumbar puncture need not be done routinely in the evaluation of dementia, but it is indicated when CNS infection or inflammation are credible diagnostic possibilities. Cerebrospinal fluid (CSF) levels of Aβ42 and tau proteins show differing patterns with the various dementias, and the presence of low Aβ42 and mildly elevated CSF tau is highly suggestive of AD. The routine use of lumbar puncture in the diagnosis of dementia is debated, but the sensitivity and specificity of AD diagnostic measures are not yet high enough to warrant routine use. Formal psychometric testing helps to document the severity of cognitive disturbance, suggest psychogenic causes, and provide a more formal method for following the disease course. Electroencephalogram (EEG) is not routinely used but can help to suggest CJD (repetitive bursts of diffuse high-amplitude sharp waves, or “periodic complexes”) or an underlying nonconvulsive seizure disorder (epileptiform discharges). Brain biopsy (including meninges)

1	CJD (repetitive bursts of diffuse high-amplitude sharp waves, or “periodic complexes”) or an underlying nonconvulsive seizure disorder (epileptiform discharges). Brain biopsy (including meninges) is not advised except to diagnose vasculitis, potentially treatable neoplasms, or unusual infections when the diagnosis is uncertain. Systemic disorders with CNS manifestations, such as sarcoidosis, can usually be confirmed through biopsy of lymph node or solid organ rather than brain. MR angiography should be considered when cerebral vasculitis or cerebral venous thrombosis is a possible cause of the dementia.

1	The major goals of dementia management are to treat reversible causes and to provide comfort and support to the patient and caregivers. Treatment of underlying causes includes thyroid replacement for hypothyroidism; vitamin therapy for thiamine or B12 deficiency or for elevated serum homocysteine; antimicrobials for opportunistic infections or antiretrovirals for HIV; ventricular shunting for NPH; or appropriate surgical, radiation, and/or chemotherapeutic treatment for CNS neoplasms. Removal of cognition-impairing drugs or medications is frequently useful. If the patient’s cognitive complaints stem from a psychiatric disorder, vigorous treatment of this condition should seek to eliminate the cognitive complaint or confirm that it persists despite adequate resolution of the mood or anxiety symptoms. Patients with degenerative diseases may also be depressed or anxious, and those aspects of their condition often respond to therapy. Antidepressants, such as selective serotonin reuptake

1	symptoms. Patients with degenerative diseases may also be depressed or anxious, and those aspects of their condition often respond to therapy. Antidepressants, such as selective serotonin reuptake inhibitors (SSRIs) or serotonin-norepinephrine reuptake inhibitors (SNRIs) (Chap. 465e), which feature anxiolytic properties but few cognitive side effects, provide the mainstay of treatment when necessary. Anticonvulsants are used to control seizures. Levetiracetam may be particularly useful, but there have as yet been no randomized trials for treatment of AD-associated seizures.

1	Agitation, hallucinations, delusions, and confusion are difficult to treat. These behavioral problems represent major causes for nursing home placement and institutionalization. Before treating these behaviors with medications, the clinician should aggressively seek out modifiable environmental or metabolic factors. Hunger, lack of exercise, toothache, constipation, urinary tract or respiratory infection, electrolyte imbalance, and drug toxicity all represent easily correctable causes that can be remedied without psychoactive drugs. Drugs such as phenothiazines and benzodiazepines may ameliorate the behavior problems but have untoward side effects such as sedation, rigidity, dyskinesia, and occasionally paradoxical disinhibition (benzodiazepines). Despite their unfavorable side effect profile, second-generation antipsychotics such as quetiapine (starting dose, 12.5–25 mg daily) can be used for patients with agitation, aggression, and

1	FIguRE 35-4 Positron emission tomography (PET) images obtained with the amyloid-imaging agent Pittsburgh Compound-B ([11C]PIB) in a normal control (left); three different patients with mild cognitive impairment (MCI; center); and a patient with mild Alzheimer’s disease (AD; right). Some MCI patients have control-like levels of amyloid, some have AD-like levels of amyloid, and some have intermediate levels. (Images courtesy of William Klunk and Chester Mathis, University of Pittsburgh.) 176 psychosis, although the risk profile for these compounds is significant. When patients do not respond to treatment, it is usually a mistake to advance to higher doses or to use anticholinergic drugs or sedatives (such as barbiturates or benzodiazepines). It is important to recognize and treat depression; treatment can begin with a low dose of an SSRI (e.g., escitalopram, starting dose 5 mg daily, target dose 5–10 mg daily) while monitoring for efficacy and toxicity. Sometimes apathy, visual

1	treatment can begin with a low dose of an SSRI (e.g., escitalopram, starting dose 5 mg daily, target dose 5–10 mg daily) while monitoring for efficacy and toxicity. Sometimes apathy, visual hallucinations, depression, and other psychiatric symptoms respond to the cholinesterase inhibitors, especially in DLB, obviating the need for other more toxic therapies. Cholinesterase inhibitors are being used to treat AD (donepezil, rivastigmine, galantamine) and PDD (rivastigmine). Recent work has focused on developing antibodies against Aβ42 as a treatment for AD. Although the initial randomized controlled trials failed, there was some evidence for efficacy in the mildest patient groups. Therefore, researchers have begun to focus on patients with very mild disease and asymptomatic individuals at risk for AD, such as those who carry autosomal dominantly inherited genetic mutations or healthy elders with CSF or amyloid imaging biomarker evidence supporting presymptomatic AD. Memantine proves

1	risk for AD, such as those who carry autosomal dominantly inherited genetic mutations or healthy elders with CSF or amyloid imaging biomarker evidence supporting presymptomatic AD. Memantine proves useful when treating some patients with moderate to severe AD; its major benefit relates to decreasing caregiver burden, most likely by decreasing resistance to dressing and grooming support. In moderate to severe AD, the combination of memantine and a cholinesterase inhibitor delayed nursing home placement in several studies, although other studies have not supported the efficacy of adding memantine to the regimen. A proactive strategy has been shown to reduce the occurrence of delirium in hospitalized patients. This strategy includes frequent orientation, cognitive activities, sleep-enhancement measures, vision and hearing aids, and correction of dehydration. Nondrug behavior therapy has an important place in dementia management. The primary goals are to make the patient’s life

1	measures, vision and hearing aids, and correction of dehydration. Nondrug behavior therapy has an important place in dementia management. The primary goals are to make the patient’s life comfortable, uncomplicated, and safe. Preparing lists, schedules, calendars, and labels can be helpful in the early stages. It is also useful to stress familiar routines, walks, and simple physical exercises. For many demented patients, memory for events is worse than their ability to carry out routine activities, and they may still be able to take part in activities such as walking, bowling, dancing, singing, bingo, and golf. Demented patients often object to losing control over familiar tasks such as driving, cooking, and handling finances. Attempts to help or take over may be greeted with complaints, depression, or anger. Hostile responses on the part of the caregiver are counterproductive and sometimes even harmful. Reassurance, distraction, and calm positive statements are more productive in this

1	or anger. Hostile responses on the part of the caregiver are counterproductive and sometimes even harmful. Reassurance, distraction, and calm positive statements are more productive in this setting. Eventually, tasks such as finances and driving must be assumed by others, and the patient will conform and adjust. Safety is an important issue that includes not only driving but controlling the kitchen, bathroom, and sleeping area environments, as well as stairways. These areas need to be monitored, supervised, and made as safe as possible. A move to a retirement complex, assisted-living center, or nursing home can initially increase confusion and agitation. Repeated reassurance, reorientation, and careful introduction to the new personnel will help to smooth the process. Providing activities that are known to be enjoyable to the patient can be of considerable benefit. The clinician must pay special attention to frustration and depression among family members and caregivers. Caregiver

1	that are known to be enjoyable to the patient can be of considerable benefit. The clinician must pay special attention to frustration and depression among family members and caregivers. Caregiver guilt and burnout are common. Family members often feel overwhelmed and helpless and may vent their frustrations on the patient, each other, and health care providers. Caregivers should be encouraged to take advantage of day-care facilities and respite services. Education and counseling about dementia are important. Local and national support groups, such as the Alzheimer’s Association (www.alz.org), can provide considerable help.

1	PART 2 Cardinal Manifestations and Presentation of Diseases Aphasia, Memory Loss, and other M.-Marsel Mesulam The cerebral cortex of the human brain contains approximately 20 billion neurons spread over an area of 2.5 m2. The primary sensory and motor areas constitute 10% of the cerebral cortex. The rest is subsumed by modality-selective, heteromodal, paralimbic, and limbic areas collectively known as the association cortex (Fig. 36-1). The association cortex mediates the integrative processes that subserve cognition, emotion, and comportment. A systematic testing of these mental functions is necessary for the effective clinical assessment of the association cortex and its diseases. According to current thinking, there are no centers for “hearing words,” “perceiving space,” or “storing memories.”

1	FIguRE 36-1 Lateral (top) and medial (bottom) views of the cerebral hemispheres. The numbers refer to the Brodmann cytoarchitectonic designations. Area 17 corresponds to the primary visual cortex, 41–42 to the primary auditory cortex, 1–3 to the primary somatosensory cortex, and 4 to the primary motor cortex. The rest of the cerebral cortex contains association areas. AG, angular gyrus; B, Broca’s area; CC, corpus callosum; CG, cingulate gyrus; DLPFC, dorsolateral prefrontal cortex; FEF, frontal eye fields (premotor cortex); FG, fusiform gyrus; IPL, inferior parietal lobule; ITG, inferior temporal gyrus; LG, lingual gyrus; MPFC, medial prefrontal cortex; MTG, middle temporal gyrus; OFC, orbitofrontal cortex; PHG, parahippocampal gyrus; PPC, posterior parietal cortex; PSC, peristriate cortex; SC, striate cortex; SMG, supramarginal gyrus; SPL, superior parietal lobule; STG, superior temporal gyrus; STS, superior temporal sulcus; TP, temporopolar cortex; W, Wernicke’s area.

1	Cognitive and behavioral functions (domains) are coordinated by intersecting large-scale neural networks that contain interconnected cortical and subcortical components. Five anatomically defined large-scale networks are most relevant to clinical practice: (1) a perisylvian network for language, (2) a parietofrontal network for spatial orientation, (3) an occipitotemporal network for face and object recognition, (4) a limbic network for retentive memory, and (5) a prefrontal network for the executive control of cognition and comportment.

1	The areas that are critical for language make up a distributed network located along the perisylvian region of the left hemisphere. One hub, located in the inferior frontal gyrus, is known as Broca’s area. Damage to this region impairs phonology, fluency, and the grammatical structure of sentences. The location of a second hub, known as Wernicke’s area, is less clearly settled but is traditionally thought to include the posterior parts of the temporal lobe. Cerebrovascular accidents that damage this area interfere with the ability to understand spoken or written sentences as well as the ability to express thoughts through meaningful words and statements. These two hubs are interconnected with each other and with surrounding parts of the frontal, parietal, and temporal lobes. Damage to this network gives rise to language impairments known as aphasia. Aphasia should be diagnosed only when there are deficits in the formal aspects of language, such as word finding, word choice,

1	to this network gives rise to language impairments known as aphasia. Aphasia should be diagnosed only when there are deficits in the formal aspects of language, such as word finding, word choice, comprehension, spelling, or grammar. Dysarthria and mutism do not by themselves lead to a diagnosis of aphasia. In approximately 90% of right-handers and 60% of left-handers, aphasia occurs only after lesions of the left hemisphere.

1	The clinical examination of language should include the assessment of naming, spontaneous speech, comprehension, repetition, reading, and writing. A deficit of naming (anomia) is the single most common finding in aphasic patients. When asked to name a common object, the patient may fail to come up with the appropriate word, may provide a circumlocutious description of the object (“the thing for writing”), or may come up with the wrong word (paraphasia). If the patient offers an incorrect but related word (“pen” for “pencil”), the naming error is known as a semantic paraphasia; if the word approximates the correct answer but is phonetically inaccurate (“plentil” for “pencil”), it is known as a phonemic paraphasia. In most anomias, the patient cannot retrieve the appropriate name when shown an object but can point to the appropriate object when the name is provided by the examiner. This is known as a one-way (or retrieval-based) naming deficit. A two-way (comprehension-based) naming

1	an object but can point to the appropriate object when the name is provided by the examiner. This is known as a one-way (or retrieval-based) naming deficit. A two-way (comprehension-based) naming deficit exists if the patient can neither provide nor recognize the correct name. Spontaneous speech is described as “fluent” if it maintains appropriate output volume, phrase length, and melody or as “nonfluent” if it is sparse and halting and average utterance length is below four words. The examiner also should note the integrity of grammar as manifested by 177 word order (syntax), tenses, suffixes, prefixes, plurals, and possessives. Comprehension can be tested by assessing the patient’s ability to follow conversation, asking yes-no questions (“Can a dog fly?” “Does it snow in summer?”), asking the patient to point to appropriate objects (“Where is the source of illumination in this room?”), or asking for verbal definitions of single words. Repetition is assessed by asking the patient to

1	the patient to point to appropriate objects (“Where is the source of illumination in this room?”), or asking for verbal definitions of single words. Repetition is assessed by asking the patient to repeat single words, short sentences, or strings of words such as “No ifs, ands, or buts.” The testing of repetition with tongue twisters such as “hippopotamus” and “Irish constabulary” provides a better assessment of dysarthria and palilalia than of aphasia. It is important to make sure that the number of words does not exceed the patient’s attention span. Otherwise, the failure of repetition becomes a reflection of the narrowed attention span (working memory) rather than an indication of an aphasic deficit. Reading should be assessed for deficits in reading aloud as well as comprehension. Alexia describes an inability to either read aloud or comprehend single words and simple sentences; agraphia (or dysgraphia) is used to describe an acquired deficit in spelling.

1	Aphasias can arise acutely in cerebrovascular accidents (CVAs) or gradually in neurodegenerative diseases. The syndromes listed in Table 36-1 are most applicable to the former group, where gray matter and white matter at the lesion site are abruptly and jointly destroyed. Progressive neurodegenerative diseases can have cellular, laminar, and regional specificity, giving rise to a different set of aphasias that will be described separately. The syndromes outlined below are idealizations and rarely occur in pure form.

1	Wernicke’s Aphasia Comprehension is impaired for spoken and written words and sentences. Language output is fluent but is highly paraphasic and circumlocutious. Paraphasic errors may lead to strings of neologisms, which lead to “jargon aphasia.” Speech contains few substantive nouns. The output is therefore voluminous but uninformative. For example, a patient attempts to describe how his wife accidentally threw away something important, perhaps his dentures: “We don’t need it anymore, she says. And with it when that was downstairs was my teeth-tick … a … den … dentith … my dentist. And they happened to be in that bag … see? …Where my two … two little pieces of dentist that I use … that I … all gone. If she throws the whole thing away … visit some friends of hers and she can’t throw them away.”

1	Gestures and pantomime do not improve communication. The patient may not realize that his or her language is incomprehensible and may appear angry and impatient when the examiner fails to decipher the meaning of a severely paraphasic statement. In some patients this type of aphasia can be associated with severe agitation and paranoia. The ability to follow commands aimed at axial musculature may be preserved. The dissociation between the failure to understand simple questions (“What is your name?”) in a patient who rapidly closes his or her eyes, sits up, or rolls over when asked to do so is characteristic of Wernicke’s aphasia and helps differentiate it from

1	CHAPTER 36 Aphasia, Memory Loss, and Other Focal Cerebral Disorders 178 deafness, psychiatric disease, or malingering. Patients with Wernicke’s aphasia cannot express their thoughts in meaning-appropriate words and cannot decode the meaning of words in any modality of input. This aphasia therefore has expressive as well as receptive components. Repetition, naming, reading, and writing also are impaired. The lesion site most commonly associated with Wernicke’s aphasia is the posterior portion of the language network. An embolus to the inferior division of the middle cerebral artery, to the posterior temporal or angular branches in particular, is the most common etiology (Chap. 446). Intracerebral hemorrhage, head trauma, and neoplasm are other causes of Wernicke’s aphasia. A coexisting right hemianopia or superior quadrantanopia is common, and mild right nasolabial flattening may be found, but otherwise, the examination is often unrevealing. The paraphasic, neologistic speech in an

1	right hemianopia or superior quadrantanopia is common, and mild right nasolabial flattening may be found, but otherwise, the examination is often unrevealing. The paraphasic, neologistic speech in an agitated patient with an otherwise unremarkable neurologic examination may lead to the suspicion of a primary psychiatric disorder such as schizophrenia or mania, but the other components characteristic of acquired aphasia and the absence of prior psychiatric disease usually settle the issue. Prognosis for recovery of language function is guarded.

1	Broca’s Aphasia Speech is nonfluent, labored, interrupted by many word-finding pauses, and usually dysarthric. It is impoverished in function words but enriched in meaning-appropriate nouns. Abnormal word order and the inappropriate deployment of bound morphemes (word endings used to denote tenses, possessives, or plurals) lead to a characteristic agrammatism. Speech is telegraphic and pithy but quite informative. In the following passage, a patient with Broca’s aphasia describes his medical history: “I see … the dotor, dotor sent me … Bosson. Go to hospital. Dotor … kept me beside. Two, tee days, doctor send me home.”

1	Output may be reduced to a grunt or single word (“yes” or “no”), which is emitted with different intonations in an attempt to express approval or disapproval. In addition to fluency, naming and repetition are impaired. Comprehension of spoken language is intact except for syntactically difficult sentences with a passive voice structure or embedded clauses, indicating that Broca’s aphasia is not just an “expressive” or “motor” disorder and that it also may involve a comprehension deficit in decoding syntax. Patients with Broca’s aphasia can be tearful, easily frustrated, and profoundly depressed. Insight into their condition is preserved, in contrast to Wernicke’s aphasia. Even when spontaneous speech is severely dysarthric, the patient may be able to display a relatively normal articulation of words when singing. This dissociation has been used to develop specific therapeutic approaches (melodic intonation therapy) for Broca’s aphasia. Additional neurologic deficits include right

1	of words when singing. This dissociation has been used to develop specific therapeutic approaches (melodic intonation therapy) for Broca’s aphasia. Additional neurologic deficits include right facial weakness, hemiparesis or hemiplegia, and a buccofacial apraxia characterized by an inability to carry out motor commands involving oropharyngeal and facial musculature (e.g., patients are unable to demonstrate how to blow out a match or suck through a straw). The cause is most often infarction of Broca’s area (the inferior frontal convolution; “B” in Fig. 36-1) and surrounding anterior perisylvian and insular cortex due to occlusion of the superior division of the middle cerebral artery (Chap. 446). Mass lesions, including tumor, intracerebral hemorrhage, and abscess, also may be responsible. When the cause of Broca’s aphasia is stroke, recovery of language function generally peaks within 2 to 6 months, after which time further progress is limited. Speech therapy is more successful than

1	When the cause of Broca’s aphasia is stroke, recovery of language function generally peaks within 2 to 6 months, after which time further progress is limited. Speech therapy is more successful than in Wernicke’s aphasia.

1	Conduction Aphasia Speech output is fluent but contains many phonemic paraphasias, comprehension of spoken language is intact, and repetition is severely impaired. Naming elicits phonemic paraphasias, and spelling is impaired. Reading aloud is impaired, but reading comprehension is preserved. The lesion sites spare the functionality of Broca’s and Wernicke’s areas but may induce a disconnection between the two. Occasionally, a transient Wernicke’s aphasia may rapidly resolve into a conduction aphasia. The paraphasic output in conduction aphasia interferes with the ability to express meaning, but this deficit is not nearly as severe as the one displayed by patients with Wernicke’s aphasia. Associated neurologic signs in conduction aphasia vary according to the primary lesion site. PART 2 Cardinal Manifestations and Presentation of Diseases

1	Transcortical Aphasias: Fluent and Nonfluent Clinical features of fluent (posterior) transcortical aphasia are similar to those of Wernicke’s aphasia, but repetition is intact. The lesion site disconnects the intact core of the language network from other temporoparietal association areas. Associated neurologic findings may include hemianopia. Cerebrovascular lesions (e.g., infarctions in the posterior watershed zone) and neoplasms that involve the temporoparietal cortex posterior to Wernicke’s area are common causes. The features of nonfluent (anterior) transcortical aphasia are similar to those of Broca’s aphasia, but repetition is intact and agrammatism is less pronounced. The neurologic examination may be otherwise intact, but a right hemiparesis also can exist. The lesion site disconnects the intact language network from prefrontal areas of the brain and usually involves the anterior watershed zone between anterior and middle cerebral artery territories or the supplementary motor

1	the intact language network from prefrontal areas of the brain and usually involves the anterior watershed zone between anterior and middle cerebral artery territories or the supplementary motor cortex in the territory of the anterior cerebral artery.

1	global and Isolation Aphasias Global aphasia represents the combined dysfunction of Broca’s and Wernicke’s areas and usually results from strokes that involve the entire middle cerebral artery distribution in the left hemisphere. Speech output is nonfluent, and comprehension of language is severely impaired. Related signs include right hemiplegia, hemisensory loss, and homonymous hemianopia. Isolation aphasia represents a combination of the two transcortical aphasias. Comprehension is severely impaired, and there is no purposeful speech output. The patient may parrot fragments of heard conversations (echolalia), indicating that the neural mechanisms for repetition are at least partially intact. This condition represents the pathologic function of the language network when it is isolated from other regions of the brain. Broca’s and Wernicke’s areas tend to be spared, but there is damage to the surrounding frontal, parietal, and temporal cortex. Lesions are patchy and can be associated

1	other regions of the brain. Broca’s and Wernicke’s areas tend to be spared, but there is damage to the surrounding frontal, parietal, and temporal cortex. Lesions are patchy and can be associated with anoxia, carbon monoxide poisoning, or complete watershed zone infarctions.

1	Anomic Aphasia This form of aphasia may be considered the “minimal dysfunction” syndrome of the language network. Articulation, comprehension, and repetition are intact, but confrontation naming, word finding, and spelling are impaired. Word-finding pauses are uncommon, so language output is fluent but paraphasic, circumlocutious, and uninformative. The lesion sites can be anywhere within the left hemisphere language network, including the middle and inferior temporal gyri. Anomic aphasia is the single most common language disturbance seen in head trauma, metabolic encephalopathy, and Alzheimer’s disease.

1	Pure Word Deafness The most common causes are either bilateral or left-sided middle cerebral artery (MCA) strokes affecting the superior temporal gyrus. The net effect of the underlying lesion is to interrupt the flow of information from the auditory association cortex to the language network. Patients have no difficulty understanding written language and can express themselves well in spoken or written language. They have no difficulty interpreting and reacting to environmental sounds since primary auditory cortex and auditory association areas of the right hemisphere are spared. Because auditory information cannot be conveyed to the language network, however, it cannot be decoded into neural word representations, and the patient reacts to speech as if it were in an alien tongue that cannot be deciphered. Patients cannot repeat spoken language but have no difficulty naming objects. In time, patients with pure word deafness teach themselves lipreading and may appear to have improved.

1	be deciphered. Patients cannot repeat spoken language but have no difficulty naming objects. In time, patients with pure word deafness teach themselves lipreading and may appear to have improved. There may be no additional neurologic findings, but agitated paranoid reactions are common in the acute stages. Cerebrovascular lesions are the most common cause.

1	Pure Alexia Without Agraphia This is the visual equivalent of pure word deafness. The lesions (usually a combination of damage to the left occipital cortex and to a posterior sector of the corpus callosum—the splenium) interrupt the flow of visual input into the language network. There is usually a right hemianopia, but the core language network remains unaffected. The patient can understand and produce spoken language, name objects in the left visual hemifield, repeat, and write. However, the patient acts as if illiterate when asked to read even the simplest sentence because the visual information from the written words (presented to the intact left visual hemifield) cannot reach the language network. Objects in the left hemifield may be named accurately because they activate nonvisual associations in the right hemisphere, which in turn can access the language network through transcallosal pathways anterior to the splenium. Patients with this syndrome also may lose the ability to

1	associations in the right hemisphere, which in turn can access the language network through transcallosal pathways anterior to the splenium. Patients with this syndrome also may lose the ability to name colors, although they can match colors. This is known as a color anomia. The most common etiology of pure alexia is a vascular lesion in the territory of the posterior cerebral artery or an infiltrating neoplasm in the left occipital cortex that involves the optic radiations as well as the crossing fibers of the splenium. Because the posterior cerebral artery also supplies medial temporal components of the limbic system, a patient with pure alexia also may experience an amnesia, but this is usually transient because the limbic lesion is unilateral.

1	Apraxia and Aphemia Apraxia designates a complex motor deficit that cannot be attributed to pyramidal, extrapyramidal, cerebellar, or sensory dysfunction and that does not arise from the patient’s failure to understand the nature of the task. Apraxia of speech is used to designate articulatory abnormalities in the duration, fluidity, and stress of syllables that make up words. Intoning the words may improve articulation. It can arise with CVAs in the posterior part of Broca’s area or in the course of frontotemporal lobar degeneration (FTLD) with tauopathy. Aphemia is a severe form of acute speech apraxia that presents with severely impaired fluency (often mutism). Recovery is the rule and involves an intermediate stage of hoarse whispering. Writing, reading, and comprehension are intact, and so this is not a true aphasic syndrome. CVAs in parts of Broca’s area or subcortical lesions that undercut its connections with other parts of the brain may be present. Occasionally, the lesion

1	and so this is not a true aphasic syndrome. CVAs in parts of Broca’s area or subcortical lesions that undercut its connections with other parts of the brain may be present. Occasionally, the lesion site is on the medial aspects of the frontal lobes and may involve the supplementary motor cortex of the left hemisphere. Ideomotor apraxia is diagnosed when commands to perform a specific motor act (“cough,” “blow out a match”) or pantomime the use of a common tool (a comb, hammer, straw, or toothbrush) in the absence of the real object cannot be followed. The patient’s ability to comprehend the command is ascertained by demonstrating multiple movements and establishing that the correct one can be recognized. Some patients with this type of apraxia can imitate the appropriate movement (when it is demonstrated by the examiner) and show no impairment when handed the real object, indicating that the sensorimotor mechanisms necessary for the movement are intact. Some forms of ideomotor apraxia

1	is demonstrated by the examiner) and show no impairment when handed the real object, indicating that the sensorimotor mechanisms necessary for the movement are intact. Some forms of ideomotor apraxia represent a disconnection of the language network from pyramidal motor systems so that commands to execute complex movements are understood but cannot be conveyed to the appropriate motor areas. Buccofacial apraxia involves apraxic deficits in movements of the face and mouth. Limb apraxia encompasses apraxic deficits in movements of the arms and legs. Ideomotor apraxia almost always is caused by lesions in the left hemisphere and is commonly associated with aphasic syndromes, especially Broca’s aphasia and conduction aphasia. Because the handling of real objects is not impaired, ideomotor apraxia by itself causes no major limitation of daily living activities. Patients with lesions of the anterior corpus callosum can display ideomotor apraxia confined to the left side of the body, a sign

1	by itself causes no major limitation of daily living activities. Patients with lesions of the anterior corpus callosum can display ideomotor apraxia confined to the left side of the body, a sign known as sympathetic dyspraxia. A severe form of sympathetic dyspraxia, known as the alien hand syndrome, is characterized by additional features of motor disinhibition on the left hand. Ideational apraxia refers to a deficit in the sequencing of goal-directed movements in patients who have no difficulty executing the individual components of the sequence. For example, when the patient is asked to pick up a pen and write, the sequence of uncapping the pen, placing the cap at the opposite end, turning the point toward the writing surface, and writing may be disrupted, and the patient may be seen trying to write with the wrong end of the pen or even with the removed cap. These motor sequencing problems usually are seen in the context of confusional states and dementias rather than focal lesions

1	to write with the wrong end of the pen or even with the removed cap. These motor sequencing problems usually are seen in the context of confusional states and dementias rather than focal lesions associated with aphasic conditions. Limb-kinetic apraxia involves clumsiness in the use of tools or objects that cannot be attributed to sensory, pyramidal, extrapyramidal, or cerebellar dysfunction. This condition can emerge in the context of focal premotor cortex lesions or corticobasal 179 degeneration.

1	gerstmann’s Syndrome The combination of acalculia (impairment of simple arithmetic), dysgraphia (impaired writing), finger anomia (an inability to name individual fingers such as the index and thumb), and right-left confusion (an inability to tell whether a hand, foot, or arm of the patient or examiner is on the right or left side of the body) is known as Gerstmann’s syndrome. In making this diagnosis, it is important to establish that the finger and left-right naming deficits are not part of a more generalized anomia and that the patient is not otherwise aphasic. When Gerstmann’s syndrome arises acutely and in isolation, it is commonly associated with damage to the inferior parietal lobule (especially the angular gyrus) in the left hemisphere.

1	Pragmatics and Prosody Pragmatics refers to aspects of language that communicate attitude, affect, and the figurative rather than literal aspects of a message (e.g., “green thumb” does not refer to the actual color of the finger). One component of pragmatics, prosody, refers to variations of melodic stress and intonation that influence attitude and the inferential aspect of verbal messages. For example, the two statements “He is clever.” and “He is clever?” contain an identical word choice and syntax but convey vastly different messages because of differences in the intonation with which the statements are uttered. Damage to right hemisphere regions corresponding to Broca’s area impairs the ability to introduce meaning-appropriate prosody into spoken language. The patient produces grammatically correct language with accurate word choice, but the statements are uttered in a monotone that interferes with the ability to convey the intended stress and affect. Patients with this type of

1	correct language with accurate word choice, but the statements are uttered in a monotone that interferes with the ability to convey the intended stress and affect. Patients with this type of aprosodia give the mistaken impression of being depressed or indifferent. Other aspects of pragmatics, especially the ability to infer the figurative aspect of a message, become impaired by damage to the right hemisphere or frontal lobes.

1	Subcortical Aphasia Damage to subcortical components of the language network (e.g., the striatum and thalamus of the left hemisphere) also can lead to aphasia. The resulting syndromes contain combinations of deficits in the various aspects of language but rarely fit the specific patterns described in Table 36-1. In a patient with a CVA, an anomic aphasia accompanied by dysarthria or a fluent aphasia with hemiparesis should raise the suspicion of a subcortical lesion site. Progressive Aphasias Aphasias caused by major cerebrovascular accidents start suddenly and display maximal deficits at the onset. These are the “classic” aphasias described above. Aphasias caused by neurodegenerative diseases have an insidious onset and relentless progression. The neuropathology can be selective not only for gray matter but also for specific layers and cell types. The clinico-anatomic patterns are therefore different from those described in Table 36-1.

1	CLINICAL PRESENTATION ANd dIAgNOSIS OF PRIMARY PROgRESSIVE APHASIA (PPA) Several neurodegenerative syndromes, such as typical Alzheimer-type (amnestic) and frontal-type (behavioral) dementias, can also undermine language as the disease progresses. In these cases, the aphasia is an ancillary component of the overall syndrome. When a neurodegenerative language disorder arises in relative isolation and becomes the primary concern that brings the patient to medical attention, a diagnosis of PPA is made.

1	LANgUAgE IN PPA The impairments of language in PPA have slightly different patterns from those seen in CVA-caused aphasias. Three major subtypes of PPA can be recognized. The agrammatic variant is characterized by consistently low fluency and impaired grammar but intact word comprehension. It most closely resembles Broca’s aphasia or anterior transcortical aphasia but usually lacks the right hemiparesis or dysarthria and has more profound impairments of grammar. Peak sites of neuronal loss (gray matter atrophy) include the left inferior frontal gyrus where Broca’s area is located. The neuropathology is usually an FTLD with tauopathy but can also be an atypical form of Alzheimer’s disease (AD) pathology. The semantic variant is characterized by preserved fluency and syntax but poor single-word comprehension and profound two-way naming impairments. This

1	CHAPTER 36 Aphasia, Memory Loss, and Other Focal Cerebral Disorders 180 kind of aphasia is not seen with CVAs. It differs from Wernicke’s aphasia or posterior transcortical aphasia because speech is usually informative, repetition is intact, and comprehension of conversation is relatively preserved, as long as the meaning is not too dependent on words that the patient fails to understand. Peak atrophy sites are located in the left anterior temporal lobe, indicating that this part of the brain plays a critical role in the comprehension of words, especially words that denote concrete objects. The neuropathology is frequently an FTLD with abnormal precipitates of the 43-kDa transactive response DNA-binding protein TDP-43. The logopenic variant is characterized by preserved syntax and comprehension but frequent and severe word-finding pauses, anomia, circumlocutions, and simplifications during spontaneous speech. Peak atrophy sites are located in the temporoparietal junction and posterior

1	but frequent and severe word-finding pauses, anomia, circumlocutions, and simplifications during spontaneous speech. Peak atrophy sites are located in the temporoparietal junction and posterior temporal lobe, partially overlapping with traditional location of Wernicke’s area. However, the comprehension impairment of Wernicke’s aphasia is absent, perhaps because the underlying white matter, frequently damaged by cerebrovascular accidents, remains relatively intact in PPA. In contrast to Broca’s aphasia or agrammatic PPA, the interruption of fluency is variable so that speech may appear entirely normal if the patient is allowed to engage in small talk. Logopenic PPA resembles the anomic aphasia of Table 36-1 but usually has longer and more frequent word-finding pauses. Patients may also have poor phrase and word repetition, in which case the aphasia resembles the conduction aphasia in Table 36-1. Of all PPA subtypes, this is the one most commonly associated with the pathology of AD, but

1	poor phrase and word repetition, in which case the aphasia resembles the conduction aphasia in Table 36-1. Of all PPA subtypes, this is the one most commonly associated with the pathology of AD, but FTLD can also be the cause. In addition to these three major subtypes, PPA can also present in the form of pure word deafness or Gerstmann’s syndrome.

1	Adaptive spatial orientation is subserved by a large-scale network containing three major cortical components. The cingulate cortex provides access to a motivational mapping of the extrapersonal space, the posterior parietal cortex to a sensorimotor representation of salient extrapersonal events, and the frontal eye fields to motor strategies for attentional behaviors (Fig. 36-2). Subcortical components of this network include the striatum and the thalamus. Damage to this network can undermine the distribution of attention within the extrapersonal space, giving rise to hemispatial neglect, simultanagnosia and object finding failures. The integration of egocentric (self-centered) with allocentric (object-centered) coordinates can also be disrupted, giving rise to impairments in route finding, the ability to avoid obstacles, and the ability to dress.

1	Contralesional hemispatial neglect represents one outcome of damage to the cortical or subcortical components of this network. The traditional view that hemispatial neglect always denotes a parietal lobe lesion is inaccurate. According to one model of spatial cognition, the right hemisphere directs attention within the entire extrapersonal space, whereas the left hemisphere directs attention mostly within the contra-lateral right hemispace. Consequently, left hemisphere lesions do not give rise to much contralesional neglect because the global attentional mechanisms of the right hemisphere can compensate for the loss of the contralaterally directed attentional functions of the left hemisphere. Right hemisphere lesions, however, give rise to severe contralesional left hemispatial neglect because the unaffected left hemisphere does not contain ipsilateral attentional mechanisms. This model is consistent with clinical experience, which shows that contralesional neglect is more common,

1	because the unaffected left hemisphere does not contain ipsilateral attentional mechanisms. This model is consistent with clinical experience, which shows that contralesional neglect is more common, more severe, and longer lasting after damage to the right hemisphere than after damage to the left hemisphere. Severe neglect for the right hemispace is rare, even in left-handers with left hemisphere lesions.

1	Clinical Examination Patients with severe neglect may fail to dress, shave, or groom the left side of the body; fail to eat food placed on the PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 36-2 Functional magnetic resonance imaging of language and spatial attention in neurologically intact subjects.

1	The red and black areas show regions of task-related significant activation. (Top) The subjects were asked to determine if two words were synonymous. This language task led to the simultaneous activation of the two epicenters of the language network, Broca’s area (B) and Wernicke’s area (W). The activations are exclusively in the left hemisphere. (Bottom) The subjects were asked to shift spatial attention to a peripheral target. This task led to the simultaneous activation of the three epicenters of the attentional network: the posterior parietal cortex (P), the frontal eye fields (F), and the cingulate gyrus (CG). The activations are predominantly in the right hemisphere. (Courtesy of Darren Gitelman, MD; with permission.) left side of the tray; and fail to read the left half of sentences. When asked to copy a simple line drawing, the patient fails to copy detail on the left, and when the patient is asked to write, there is a tendency to leave an unusually wide margin on the left.

1	When asked to copy a simple line drawing, the patient fails to copy detail on the left, and when the patient is asked to write, there is a tendency to leave an unusually wide margin on the left. Two bedside tests that are useful in assessing neglect are simultaneous bilateral stimulation and visual target cancellation. In the former, the examiner provides either unilateral or simultaneous bilateral stimulation in the visual, auditory, and tactile modalities. After right hemisphere injury, patients who have no difficulty detecting unilateral stimuli on either side experience the bilaterally presented stimulus as coming only from the right. This phenomenon is known as extinction and is a manifestation of the sensory-representational aspect of hemispatial neglect. In the target detection task, targets (e.g., A’s) are interspersed with foils (e.g., other letters of the alphabet) on a 21.5to 28.0-cm (8.5 to 11 in.) sheet of paper, and the patient is asked to circle all the targets. A

1	task, targets (e.g., A’s) are interspersed with foils (e.g., other letters of the alphabet) on a 21.5to 28.0-cm (8.5 to 11 in.) sheet of paper, and the patient is asked to circle all the targets. A failure to detect targets on the left is a manifestation of the exploratory (motor) deficit in hemispatial neglect (Fig. 36-3A). Hemianopia is not by itself sufficient to cause the target detection failure because the patient is free to turn the head and eyes to the left. Target detection failures therefore reflect a distortion of spatial attention, not just of sensory input. Some patients with neglect also may deny the existence of hemiparesis and may even deny ownership of the paralyzed limb, a condition known as anosognosia.

1	181 CHAPTER 36 Aphasia, Memory Loss, and Other Focal Cerebral Disorders FIguRE 36-3 A. A 47-year-old man with a large frontoparietal lesion in the right hemisphere was asked to circle all the A’s. Only targets on the right are circled. This is a manifestation of left hemispatial neglect. B. A 70-year-old woman with a 2-year history of degenerative dementia was able to circle most of the small targets but ignored the larger ones. This is a manifestation of simultanagnosia. BÁLINT’S SYNDROME, SIMuLTANAgNOSIA, DRESSINg APRAXIA, CONSTRuCTION APRAXIA, AND ROuTE FINDINg

1	Bilateral involvement of the network for spatial attention, especially its parietal components, leads to a state of severe spatial disorientation known as Bálint’s syndrome. Bálint’s syndrome involves deficits in the orderly visuomotor scanning of the environment (oculomotor apraxia), accurate manual reaching toward visual targets (optic ataxia), and the ability to integrate visual information in the center of gaze with more peripheral information (simultanagnosia). A patient with simultanagnosia “misses the forest for the trees.” For example, a patient who is shown a table lamp and asked to name the object may look at its circular base and call it an ashtray. Some patients with simultanagnosia report that objects they look at may vanish suddenly, probably indicating an inability to look back at the original point of gaze after brief saccadic displacements. Movement and distracting stimuli greatly exacerbate the difficulties of visual perception. Simultanagnosia can occur without the

1	back at the original point of gaze after brief saccadic displacements. Movement and distracting stimuli greatly exacerbate the difficulties of visual perception. Simultanagnosia can occur without the other two components of Bálint’s syndrome.

1	A modification of the letter cancellation task described above can be used for the bedside diagnosis of simultanagnosia. In this modification, some of the targets (e.g., A’s) are made to be much larger than the others (7.5 to 10 cm vs 2.5 cm [3 to 4 in. vs 1 in.] in height), and all targets are embedded among foils. Patients with simultanagnosia display a counterintuitive but characteristic tendency to miss the larger targets (Fig. 36-3B). This occurs because the information needed for the identification of the larger targets cannot be confined to the immediate line of gaze and requires the integration of visual information across multiple fixation points. The greater difficulty in the detection of the larger targets also indicates that poor acuity is not responsible for the impairment of visual function and that the problem is central rather than peripheral. The test shown in Fig. 36-3B is not by itself sufficient to diagnose simultanagnosia because some patients with a frontal

1	of visual function and that the problem is central rather than peripheral. The test shown in Fig. 36-3B is not by itself sufficient to diagnose simultanagnosia because some patients with a frontal network syndrome may omit the large letters, perhaps because they lack the mental flexibility needed to realize that the two types of targets are symbolically identical despite being superficially different.

1	Bilateral parietal lesions can impair the integration of egocentric with allocentric spatial coordinates. One manifestation is dressing apraxia. A patient with this condition is unable to align the body axis with the axis of the garment and can be seen struggling as he or she holds a coat from its bottom or extends his or her arm into a fold of the garment rather than into its sleeve. Lesions that involve the posterior parietal cortex also lead to severe difficulties in copying simple line drawings. This is known as a construction apraxia and is much more severe if the lesion is in the right hemisphere. In some patients with right hemisphere lesions, the drawing difficulties are confined to the left side of the figure and represent a manifestation of hemispatial neglect; in others, there is a more universal deficit in reproducing contours and three-dimensional perspective. Impairments of route finding can be included in this group of disorders, which reflect an inability to orient the

1	a more universal deficit in reproducing contours and three-dimensional perspective. Impairments of route finding can be included in this group of disorders, which reflect an inability to orient the self with respect to external objects and landmarks.

1	Causes of Spatial Disorientation Cerebrovascular lesions and neoplasms in the right hemisphere are common causes of hemispatial neglect. Depending on the site of the lesion, a patient with neglect also may have hemiparesis, hemihypesthesia, and hemianopia on the left, but these are not invariant findings. The majority of these patients display considerable improvement of hemispatial neglect, usually within the first several weeks. Bálint’s syndrome, dressing apraxia, and route finding impairments are more likely to result from bilateral dorsal parietal lesions; common settings for acute onset include watershed infarction between the middle and posterior cerebral artery territories, hypoglycemia, and sagittal sinus thrombosis.

1	A progressive form of spatial disorientation, known as the posterior cortical atrophy syndrome, most commonly represents a variant of AD with unusual concentrations of neurofibrillary degeneration in the parieto-occipital cortex and the superior colliculus. The patient displays a progressive hemispatial neglect or Bálint’s syndrome, usually accompanied by dressing and construction apraxia. The corticobasal syndrome, which can be caused by AD or FTLD pathology, can also lead to a progressive left hemineglect syndrome. Both syndromes can impair route finding. PART 2 Cardinal Manifestations and Presentation of Diseases

1	A patient with prosopagnosia cannot recognize familiar faces, including, sometimes, the reflection of his or her own face in the mirror. This is not a perceptual deficit because prosopagnosic patients easily can tell whether two faces are identical. Furthermore, a prosopagnosic patient who cannot recognize a familiar face by visual inspection alone can use auditory cues to reach appropriate recognition if allowed to listen to the person’s voice. The deficit in prosopagnosia is therefore modality-specific and reflects the existence of a lesion that prevents the activation of otherwise intact multimodal templates by relevant visual input. Prosopagnosic patients characteristically have no difficulty with the generic identification of a face as a face or a car as a car, but may not recognize the identity of an individual face or the make of an individual car. This reflects a visual recognition deficit for proprietary features that characterize individual members of an object class. When

1	the identity of an individual face or the make of an individual car. This reflects a visual recognition deficit for proprietary features that characterize individual members of an object class. When recognition problems become more generalized and extend to the generic identification of common objects, the condition is known as visual object agnosia. A patient with anomia cannot name the object but can describe its use. In contrast, a patient with visual agnosia is unable either to name a visually presented object or to describe its use. Face and object recognition disorders also can result from the simultanagnosia of Bálint’s syndrome, in which case they are known as apperceptive agnosias as opposed to the associative agnosias that result from inferior temporal lobe lesions.

1	The characteristic lesions in prosopagnosia and visual object agnosia of acute onset consist of bilateral infarctions in the territory of the posterior cerebral arteries. Associated deficits can include visual field defects (especially superior quadrantanopias) and a centrally based color blindness known as achromatopsia. Rarely, the responsible lesion is unilateral. In such cases, prosopagnosia is associated with lesions in the right hemisphere, and object agnosia with lesions in the left. Degenerative diseases of anterior and inferior temporal cortex can cause progressive associative prosopagnosia and object agnosia. The combination of progressive associative agnosia and a fluent aphasia is known as semantic dementia. Patients with semantic dementia fail to recognize faces and objects and cannot understand the meaning of words denoting objects. This needs to be differentiated from the semantic type of PPA where there is severe impairment in understanding words that denote objects

1	and cannot understand the meaning of words denoting objects. This needs to be differentiated from the semantic type of PPA where there is severe impairment in understanding words that denote objects and in naming faces and objects but a relative preservation of face and object recognition.

1	Limbic and paralimbic areas (such as the hippocampus, amygdala, and entorhinal cortex), the anterior and medial nuclei of the thalamus, the medial and basal parts of the striatum, and the hypothalamus collectively constitute a distributed network known as the limbic system. The behavioral affiliations of this network include the coordination of emotion, motivation, autonomic tone, and endocrine function. An additional area of specialization for the limbic network and the one that is of most relevance to clinical practice is that of declarative (explicit) memory for recent episodes and experiences. A disturbance in this function is known as an amnestic state. In the absence of deficits in motivation, attention, language, or visuospatial function, the clinical diagnosis of a persistent global amnestic state is always associated with bilateral damage to the limbic network, usually within the hippocampo-entorhinal complex or the thalamus. Damage to the limbic network does not necessarily

1	amnestic state is always associated with bilateral damage to the limbic network, usually within the hippocampo-entorhinal complex or the thalamus. Damage to the limbic network does not necessarily destroy memories but interferes with their conscious recall in coherent form. The individual fragments of information remain preserved despite the limbic lesions and can sustain what is known as implicit memory. For example, patients with amnestic states can acquire new motor or perceptual skills even though they may have no conscious knowledge of the experiences that led to the acquisition of these skills.

1	The memory disturbance in the amnestic state is multimodal and includes retrograde and anterograde components. The retrograde amnesia involves an inability to recall experiences that occurred before the onset of the amnestic state. Relatively recent events are more vulnerable to retrograde amnesia than are more remote and more extensively consolidated events. A patient who comes to the emergency room complaining that he cannot remember his or her identity but can remember the events of the previous day almost certainly does not have a neurologic cause of memory disturbance. The second and most important component of the amnestic state is the anterograde amnesia, which indicates an inability to store, retain, and recall new knowledge. Patients with amnestic states cannot remember what they ate a few hours ago or the details of an important event they may have experienced in the recent past. In the acute stages, there also may be a tendency to fill in memory gaps with inaccurate,

1	they ate a few hours ago or the details of an important event they may have experienced in the recent past. In the acute stages, there also may be a tendency to fill in memory gaps with inaccurate, fabricated, and often implausible information. This is known as confabulation. Patients with the amnestic syndrome forget that they forget and tend to deny the existence of a memory problem when questioned. Confabulation is more common in cases where the underlying lesion also interferes with parts of the frontal network, as in the case of the Wernicke-Korsakoff syndrome or traumatic head injury.

1	A patient with an amnestic state is almost always disoriented, especially to time, and has little knowledge of current news. The anterograde component of an amnestic state can be tested with a list of four to five words read aloud by the examiner up to five times or until the patient can immediately repeat the entire list without an intervening delay. The next phase of the recall occurs after a period of 5 to 10 min during which the patient is engaged in other tasks. Amnestic patients fail this phase of the task and may even forget that they were given a list of words to remember. Accurate recognition of the words by multiple choice in a patient who cannot recall them indicates a less severe memory disturbance that affects mostly the retrieval stage of memory.

1	The retrograde component of an amnesia can be assessed with questions related to autobiographical or historic events. The anterograde component of amnestic states is usually much more prominent than the retrograde component. In rare instances, occasionally associated with temporal lobe epilepsy or herpes simplex encephalitis, the retrograde component may dominate. Confusional states caused by toxic-metabolic encephalopathies and some types of frontal lobe damage lead to secondary memory impairments, especially at the stages of encoding and retrieval, even in the absence of limbic lesions. This sort of memory impairment can be differentiated from the amnestic state by the presence of additional impairments in the attention-related tasks described below in the section on the frontal lobes. CAuSES, INCLuDINg ALZHEIMER’S DISEASE

1	Neurologic diseases that give rise to an amnestic state include tumors (of the sphenoid wing, posterior corpus callosum, thalamus, or medial temporal lobe), infarctions (in the territories of the anterior or posterior cerebral arteries), head trauma, herpes simplex encephalitis, Wernicke-Korsakoff encephalopathy, paraneoplastic limbic encephalitis, and degenerative dementias such as AD and Pick’s disease. The one common denominator of all these diseases is the presence of bilateral lesions within one or more components in the limbic network. Occasionally, unilateral left-sided hippocampal lesions can give rise to an amnestic state, but the memory disorder tends to be transient. Depending on the nature and distribution of the underlying neurologic disease, the patient also may have visual field deficits, eye movement limitations, or cerebellar findings. AD and its prodromal state of mild cognitive impairment (MCI) are the most common causes of progressive memory impairments. The

1	field deficits, eye movement limitations, or cerebellar findings. AD and its prodromal state of mild cognitive impairment (MCI) are the most common causes of progressive memory impairments. The predilection of the entorhinal cortex and hippocampus for early neurofibrillary degeneration by typical AD pathology is responsible for the initially selective impairment of episodic memory. In time, additional impairments in language, attention, and visuospatial skills emerge as the neurofibrillary degeneration spreads to additional neocortical areas.

1	Transient global amnesia is a distinctive syndrome usually seen in late middle age. Patients become acutely disoriented and repeatedly ask who they are, where they are, and what they are doing. The spell is characterized by anterograde amnesia (inability to retain new information) and a retrograde amnesia for relatively recent events that occurred before the onset. The syndrome usually resolves within 24 to 48 h and is followed by the filling in of the period affected by the retrograde amnesia, although there is persistent loss of memory for the events that occurred during the ictus. Recurrences are noted in approximately 20% of patients. Migraine, temporal lobe seizures, and perfusion abnormalities in the posterior cerebral territory have been postulated as causes of transient global amnesia. The absence of associated neurologic findings occasionally may lead to the incorrect diagnosis of a psychiatric disorder.

1	The frontal lobes can be subdivided into motor-premotor, dorsolateral prefrontal, medial prefrontal, and orbitofrontal components. The terms frontal lobe syndrome and prefrontal cortex refer only to the last three of these four components. These are the parts of the cerebral cortex that show the greatest phylogenetic expansion in primates, especially in humans. The dorsolateral prefrontal, medial prefrontal, and orbitofrontal areas, along with the subcortical structures with which they are interconnected (i.e., the head of the caudate and the dorsomedial nucleus of the thalamus), collectively make up a large-scale network that coordinates exceedingly complex aspects of human cognition and behavior.

1	The prefrontal network plays an important role in behaviors that require multitasking and the integration of thought with emotion. Cognitive operations impaired by prefrontal cortex lesions often are referred to as “executive functions.” The most common clinical manifestations of damage to the prefrontal network take the form of two relatively distinct syndromes. In the frontal abulic syndrome, the patient shows a loss of initiative, creativity, and curiosity and displays a pervasive emotional blandness, apathy, and lack of empathy. In the 183 frontal disinhibition syndrome, the patient becomes socially disinhibited and shows severe impairments of judgment, insight, foresight, and the ability to mind rules of conduct. The dissociation between intact intellectual function and a total lack of even rudimentary common sense is striking. Despite the preservation of all essential memory functions, the patient cannot learn from experience and continues to display inappropriate behaviors

1	of even rudimentary common sense is striking. Despite the preservation of all essential memory functions, the patient cannot learn from experience and continues to display inappropriate behaviors without appearing to feel emotional pain, guilt, or regret when those behaviors repeatedly lead to disastrous consequences. The impairments may emerge only in real-life situations when behavior is under minimal external control and may not be apparent within the structured environment of the medical office. Testing judgment by asking patients what they would do if they detected a fire in a theater or found a stamped and addressed envelope on the road is not very informative because patients who answer these questions wisely in the office may still act very foolishly in real-life settings. The physician must therefore be prepared to make a diagnosis of frontal lobe disease based on historic information alone even when the mental state is quite intact in the office examination.

1	The emergence of developmentally primitive reflexes, also known as frontal release signs, such as grasping (elicited by stroking the palm) and sucking (elicited by stroking the lips) are seen primarily in patients with large structural lesions that extend into the premotor components of the frontal lobes or in the context of metabolic encephalopathies. The vast majority of patients with prefrontal lesions and frontal lobe behavioral syndromes do not display these reflexes. Damage to the frontal lobe disrupts a variety of attention-related functions, including working memory (the transient online holding and manipulation of information), concentration span, the scanning and retrieval of stored information, the inhibition of immediate but inappropriate responses, and mental flexibility. Digit span (which should be seven forward and five reverse) is decreased, reflecting poor working memory; the recitation of the months of the year in reverse order (which should take less than 15 s) is

1	span (which should be seven forward and five reverse) is decreased, reflecting poor working memory; the recitation of the months of the year in reverse order (which should take less than 15 s) is slowed as another indication of poor working memory; and the fluency in producing words starting with the letter a, f, or s that can be generated in 1 min (normally ≥12 per letter) is diminished even in nonaphasic patients, indicating an impairment in the ability to search and retrieve information from long-term stores. In “go–no go” tasks (where the instruction is to raise the finger upon hearing one tap but keep it still upon hearing two taps), the patient shows a characteristic inability to inhibit the response to the “no go” stimulus. Mental flexibility (tested by the ability to shift from one criterion to another in sorting or matching tasks) is impoverished; distractibility by irrelevant stimuli is increased; and there is a pronounced tendency for impersistence and perseveration. The

1	one criterion to another in sorting or matching tasks) is impoverished; distractibility by irrelevant stimuli is increased; and there is a pronounced tendency for impersistence and perseveration. The ability for abstracting similarities and interpreting proverbs is also undermined.

1	The attentional deficits disrupt the orderly registration and retrieval of new information and lead to secondary memory deficits. The distinction of the underlying neural mechanisms is illustrated by the observation that severely amnestic patients who cannot remember events that occurred a few minutes ago may have intact if not superior working memory capacity as shown in tests of digit span. CAuSES: TRAuMA, NEOPLASM, AND FRONTOTEMPORAL DEMENTIA

1	CAuSES: TRAuMA, NEOPLASM, AND FRONTOTEMPORAL DEMENTIA The abulic syndrome tends to be associated with damage in dorsolateral or dorsomedial prefrontal cortex, and the disinhibition syndrome with damage in orbitofrontal or ventromedial cortex. These syndromes tend to arise almost exclusively after bilateral lesions. Unilateral lesions confined to the prefrontal cortex may remain silent until the pathology spreads to the other side; this explains why thromboembolic CVA is an unusual cause of the frontal lobe syndrome. Common settings for frontal lobe syndromes include head trauma, ruptured aneurysms, hydrocephalus, tumors (including metastases, glioblastoma, and falx or olfactory groove meningiomas), and focal degenerative diseases. A major clinical form of FTLD known as the behavioral variant of frontotemporal dementia (bvFTD) causes a progressive frontal lobe syndrome. The behavioral changes can range

1	CHAPTER 36 Aphasia, Memory Loss, and Other Focal Cerebral Disorders 184 from apathy to shoplifting, compulsive gambling, sexual indiscretions, remarkable lack of common sense, new ritualistic behaviors, and alterations in dietary preferences, usually leading to increased taste for sweets or rigid attachment to specific food items. In many patients with AD, neurofibrillary degeneration eventually spreads to prefrontal cortex and gives rise to components of the frontal lobe syndrome, but almost always on a background of severe memory impairment. Rarely, the bvFTD syndrome can arise in isolation in the context of an atypical form of AD pathology. Lesions in the caudate nucleus or in the dorsomedial nucleus of the thalamus (subcortical components of the prefrontal network) also can produce a frontal lobe syndrome. This is one reason why the changes in mental state associated with degenerative basal ganglia diseases such as Parkinson’s disease and Huntington’s disease display components of

1	lobe syndrome. This is one reason why the changes in mental state associated with degenerative basal ganglia diseases such as Parkinson’s disease and Huntington’s disease display components of the frontal lobe syndrome. Bilateral multifocal lesions of the cerebral hemispheres, none of which are individually large enough to cause specific cognitive deficits such as aphasia and neglect, can collectively interfere with the connectivity and therefore integrating (executive) function of the prefrontal cortex. A frontal lobe syndrome is therefore the single most common behavioral profile associated with a variety of bilateral multifocal brain diseases, including metabolic encephalopathy, multiple sclerosis, and vitamin B12 deficiency, among others. Many patients with the clinical diagnosis of a frontal lobe syndrome tend to have lesions that do not involve prefrontal cortex but involve either the subcortical components of the prefrontal network or its connections with other parts of the

1	a frontal lobe syndrome tend to have lesions that do not involve prefrontal cortex but involve either the subcortical components of the prefrontal network or its connections with other parts of the brain. To avoid making a diagnosis of “frontal lobe syndrome” in a patient with no evidence of frontal cortex disease, it is advisable to use the diagnostic term frontal network syndrome, with the understanding that the responsible lesions can lie anywhere within this distributed network. A patient with frontal lobe disease raises potential dilemmas in differential diagnosis: the abulia and blandness may be misinterpreted as depression, and the disinhibition as idiopathic mania or acting out. Appropriate intervention may be delayed while a treatable tumor keeps expanding.

1	Part 2 Cardinal Manifestations and Presentation of Diseases Brain damage may cause a dissociation between feeling states and their expression so that a patient who may superficially appear jocular could still be suffering from an underlying depression that needs to be treated. If neuroleptics become absolutely necessary for the control of agitation, atypical neuroleptics are preferable because of their lower extrapyramidal side effects. Treatment with neuroleptics in elderly patients with dementia requires weighing the potential benefits against the potentially serious side effects.

1	Spontaneous improvement of cognitive deficits due to acute neurologic lesions is common. It is most rapid in the first few weeks but may continue for up to 2 years, especially in young individuals with single brain lesions. Some of the initial deficits appear to arise from remote dysfunction (diaschisis) in parts of the brain that are interconnected with the site of initial injury. Improvement in these patients may reflect, at least in part, a normalization of the remote dysfunction. Other mechanisms may involve functional reorganization in surviving neurons adjacent to the injury or the compensatory use of homologous structures, e.g., the right superior temporal gyrus with recovery from Wernicke’s aphasia. Cognitive rehabilitation procedures have been used in the treatment of higher cortical deficits. There are few controlled studies, but some show a benefit of rehabilitation in the recovery from hemispatial neglect and aphasia. Determining driving competence is challenging,

1	cortical deficits. There are few controlled studies, but some show a benefit of rehabilitation in the recovery from hemispatial neglect and aphasia. Determining driving competence is challenging, especially in the early stages of dementing diseases. The diagnosis of a neurodegenerative disease is not by itself sufficient for asking the patient to stop driving. An on-the-road driving test and reports from family members may help time decisions related to this very important activity.

1	Some of the deficits described in this chapter are so complex that they may bewilder not only the patient and family but also the physician. It is imperative to carry out a systematic clinical evaluation to characterize the nature of the deficits and explain them in lay terms to the patient and family. An enlightened approach to patients with damage to the cerebral cortex requires an understanding of the principles that link neural networks to higher cerebral functions in health and disease. Primary Progressive Aphasia, Memory Loss, and Other Focal Cerebral Disorders Maria Luisa Gorno-Tempini, Jennifer Ogar, Joel Kramer, Bruce L. Miller, Gil Rabinovici, Maria Carmela Tartaglia 37e

1	Primary Progressive Aphasia, Memory Loss, and Other Focal Cerebral Disorders Maria Luisa Gorno-Tempini, Jennifer Ogar, Joel Kramer, Bruce L. Miller, Gil Rabinovici, Maria Carmela Tartaglia 37e Language and memory are essential human functions. For the experienced clinician, the recognition of different types of language and memory disturbances often provides essential clues to the anatomic localization and diagnosis of neurologic disorders. This video illustrates classic disorders of language and speech (including the aphasias), memory (the amnesias), and other disorders of cognition that are commonly encountered in clinical practice. CHAPTER 37e Primary Progressive Aphasia, Memory Loss, and Other Focal Cerebral Disorders PART 2 Cardinal Manifestations and Presentation of Diseases

1	CHAPTER 37e Primary Progressive Aphasia, Memory Loss, and Other Focal Cerebral Disorders PART 2 Cardinal Manifestations and Presentation of Diseases Sleep Disorders Charles A. Czeisler, Thomas E. Scammell, Clifford B. Saper Disturbed sleep is among the most frequent health complaints that physicians encounter. More than one-half of adults in the United States experience at least intermittent sleep disturbance, and only 30% 38 of adult Americans report consistently obtaining a sufficient amount of sleep. The Institute of Medicine has estimated that 50–70 million Americans suffer from a chronic disorder of sleep and wakefulness, which can adversely affect daytime functioning as well as physical and mental health. Over the last 20 years, the field of sleep medicine has emerged as a distinct specialty in response to the impact of sleep disorders and sleep deficiency on overall health.

1	Given the opportunity, most healthy young adults will sleep 7–8 h per night, although the timing, duration, and internal structure of sleep vary among individuals. In the United States, adults tend to have one consolidated sleep episode each night, although in some cultures sleep may be divided into a mid-afternoon nap and a shortened night sleep. This pattern changes considerably over the life span, as infants and young children sleep considerably more than older people. The stages of human sleep are defined on the basis of characteristic patterns in the electroencephalogram (EEG), the electrooculogram (EOG—a measure of eye-movement activity), and the surface electromyogram (EMG) measured on the chin, neck, and legs. The continuous recording of these electrophysiologic parameters to define sleep and wakefulness is termed polysomnography.

1	Polysomnographic profiles define two basic states of sleep: (1) rapid eye movement (REM) sleep and (2) non–rapid eye movement (NREM) sleep. NREM sleep is further subdivided into three stages: N1, N2, and N3, characterized by increasing arousal threshold and slowing of the cortical EEG. REM sleep is characterized by a low-amplitude, mixed-frequency EEG similar to that of NREM stage N1 sleep. The EOG shows bursts of rapid eye movements similar to those seen during eyes-open wakefulness. EMG activity is absent in nearly all skeletal muscles, reflecting the brainstem-mediated muscle atonia that is characteristic of REM sleep.

1	Normal nocturnal sleep in adults displays a consistent organization from night to night (Fig. 38-1). After sleep onset, sleep usually progresses through NREM stages N1–N3 sleep within 45–60 min. NREM stage N3 sleep (also known as slow-wave sleep) predominates in the first third of the night and comprises 15–25% of total nocturnal sleep time in young adults. Sleep deprivation increases the rapidity of sleep onset and both the intensity and amount of slow-wave sleep. The first REM sleep episode usually occurs in the second hour of sleep. NREM and REM sleep alternate through the night with an average period of 90–110 min (the “ultradian” sleep cycle). Overall, in a healthy young adult, REM sleep constitutes 20–25% of total sleep, and NREM stages N1 and N2 constitute 50–60%.

1	Age has a profound impact on sleep state organization (Fig. 38-1). N3 sleep is most intense and prominent during childhood, decreasing with puberty and across the second and third decades of life. N3 sleep declines during adulthood to the point where it may be completely absent in older adults. The remaining NREM sleep becomes more fragmented, with many more frequent awakenings from NREM sleep. It is the increased frequency of awakenings, rather than a decreased ability to fall back asleep, that accounts for the increased wakefulness during the sleep episode in older people. While REM sleep may account for 50% of total sleep time in infancy, the percentage falls off sharply over the first postnatal year as a mature REM-NREM cycle develops; thereafter, REM sleep occupies about 25% of total sleep time.

1	Sleep deprivation degrades cognitive performance, particularly on tests that require continual vigilance. Paradoxically, older people are less vulnerable to the neurobehavioral performance impairment induced by acute sleep deprivation than young adults, maintaining their reaction time and sustaining vigilance with fewer lapses of attention. However, it is more difficult for older adults to obtain recovery sleep after staying awake all night, as the ability to sleep during the daytime declines with age.

1	After sleep deprivation, NREM sleep is generally recovered first, followed by REM sleep. However, because REM sleep tends to be most prominent in the second half of the night, sleep truncation (e.g., by an alarm clock) results in selective REM sleep deprivation. This may 06.00 08.00 increase REM sleep pressure to the point where the first REM sleep 185 may occur much earlier in the nightly sleep episode. Because several disorders (see below) also cause sleep fragmentation, it is important that the patient have sufficient sleep opportunity (at least 8 h per night) for several nights prior to a diagnostic polysomnogram.

1	There is growing evidence that sleep deficiency in humans may cause glucose intolerance and contribute to the development of diabetes, obesity, and the metabolic syndrome, as well as impaired immune responses, accelerated atherosclerosis, and increased risk of cardiac disease and stroke. For these reasons, the Institute of Medicine declared sleep deficiency and sleep disorders “an unmet public health problem.”

1	Two principal neural systems govern the expression of the sleep and wakefulness. The ascending arousal system, illustrated in green in Fig. 38-2, consists of clusters of nerve cells extending from the upper pons to the hypothalamus and basal forebrain that activate the cerebral cortex, thalamus (which is necessary to relay sensory information to the cortex), and other forebrain regions. The ascending arousal neurons use monoamines (norepinephrine, dopamine, serotonin, and histamine), glutamate, or acetylcholine as neurotransmitters to activate their target neurons. Additional arousal-promoting neurons in the hypothalamus use the peptide neurotransmitter orexin (also known as hypocretin, shown in blue) to reinforce activity in the other arousal cell groups.

1	Damage to the arousal system at the level of the rostral pons and lower midbrain causes coma, indicating that the ascending arousal influence from this level is critical in maintaining wakefulness. Damage to the hypothalamic branch of the arousal system causes profound sleepiness, but usually not coma. Specific loss of the orexin neurons produces the sleep disorder narcolepsy (see below).

1	The arousal system is turned off during sleep by inhibitory inputs from cell groups in the sleep-promoting system, shown in Fig. 38-2 in red. These neurons in the preoptic area, lateral hypothalamus, and pons use γ-aminobutyric acid (GABA) to inhibit the arousal system. Many sleep-promoting neurons are themselves inhibited by inputs from the arousal system. This mutual inhibition between the arousal-and sleep-promoting systems forms a neural circuit akin to what electrical engineers call a “flip-flop switch.” A switch of this type tends to promote rapid transitions between the on (wake) and off (sleep) states, while avoiding intermediate states. The relatively rapid transitions between waking and sleeping states, as seen in the EEG of humans and animals, is consistent with this model.

1	Neurons in the ventrolateral preoptic nucleus, one of the key sleep- promoting sites, are lost during normal human aging, correlating with reduced ability to maintain sleep (sleep fragmentation). The ventrolateral preoptic neurons are also injured in Alzheimer’s disease, which may in part account for the poor sleep quality in those patients. Transitions between NREM and REM sleep appear to be governed by a similar switch in the brainstem. GABAergic REM-Off neurons have been identified in the lower midbrain that inhibit REM-On neurons in the upper pons. The REM-On group contains both GABAergic neurons that inhibit the REM-Off group (thus satisfying the conditions for a REM flip-flop switch) as well as glutamatergic neurons that project widely in the central nervous system (CNS) to cause the key phenomena associated with REM sleep.

1	REM-On neurons that project to the medulla and 00.00 02.00 04.00 containing) interneurons, which in turn hyperpolar-FIguRE 38-1 Wake-sleep architecture. Alternating stages of wakefulness, the three ize the motor neurons, producing the atonia of REM stages of NREM sleep (N1–N3), and REM sleep (solid bars) occur over the course of the sleep. REM-On neurons that project to the forebrain night for representative young and older adult men. Characteristic features of sleep in may be important in producing dreams. older people include reduction of N3 slow-wave sleep, frequent spontaneous awaken-The REM sleep switch receives cholinergic input, ings, early sleep onset, and early morning awakening. NREM, non–rapid eye move-which favors transitions to REM sleep, and monoment; REM, rapid eye movement. (From the Division of Sleep and Circadian Disorders, aminergic (norepinephrine and serotonin) input Brigham and Women’s Hospital.) that prevents REM sleep. As a result, drugs that FIguRE 38-2

1	(From the Division of Sleep and Circadian Disorders, aminergic (norepinephrine and serotonin) input Brigham and Women’s Hospital.) that prevents REM sleep. As a result, drugs that FIguRE 38-2 Relationship of drugs for insomnia with wake-sleep systems. The arousal system in the brain (green) includes monoaminergic, glutamatergic, and cholinergic neurons in the brainstem that activate neurons in the hypothalamus, thalamus, basal forebrain, and cerebral cortex. Orexin neurons (blue) in the hypothalamus, which are lost in narcolepsy, reinforce and stabilize arousal by activating other components of the arousal system. The sleep-promoting system (red) consists of GABAergic neurons in the preoptic area, lateral hypothalamus, and brainstem that inhibit the components of the arousal system, thus allowing sleep to occur. Drugs used to treat insomnia include those that block the effects of arousal system neurotransmitters (green and blue) and those that enhance the effects of γ-aminobutyric

1	allowing sleep to occur. Drugs used to treat insomnia include those that block the effects of arousal system neurotransmitters (green and blue) and those that enhance the effects of γ-aminobutyric acid (GABA) produced by the sleep system (red).

1	increase monoamine tone (e.g., serotonin or norepinephrine reuptake inhibitors) tend to reduce the amount of REM sleep. Damage to the neurons that promote REM sleep atonia can produce REM sleep behavior disorder, a condition in which patients act out their dreams (see below). SLEEP-WAKE CYCLES ARE DRIVEN BY HOMEOSTATIC, ALLOSTATIC, AND CIRCADIAN INPuTS

1	SLEEP-WAKE CYCLES ARE DRIVEN BY HOMEOSTATIC, ALLOSTATIC, AND CIRCADIAN INPuTS The gradual increase in sleep drive with prolonged wakefulness, followed by deeper slow-wave sleep and prolonged sleep episodes, demonstrates that there is a homeostatic mechanism that regulates sleep. The neurochemistry of sleep homeostasis is only partially understood, but with prolonged wakefulness, adenosine levels rise in parts of the brain. Adenosine may act through A1 receptors to directly inhibit many arousal-promoting brain regions. In addition, adenosine promotes sleep through A2a receptors; inhibition of these receptors by caffeine is one of the chief ways in which people fight sleepiness. Other humoral factors, such as prostaglandin D2, have also been implicated in this process. Both adenosine and prostaglandin D2 activate the sleep-promoting neurons in the ventrolateral preoptic nucleus.

1	Allostasis is the physiologic response to a threat that cannot be managed by homeostatic mechanisms (e.g., the presence of physical danger or psychological threat). These stress responses can severely impact the need for and ability to sleep. For example, insomnia is very common in patients with anxiety and other psychiatric disorders. Stress-induced insomnia is even more common, affecting most people at some time in their lives. Positron emission tomography (PET) studies in patients with chronic insomnia show hyperactivation of the components of the ascending arousal system, as well as their targets in the limbic system in the forebrain (e.g., cingulate cortex and amygdala). The limbic areas are not only targets for the arousal system, but they also send excitatory outputs back to the arousal system, which contributes to a vicious cycle of anxiety about wakefulness that makes it more difficult to sleep. Approaches to treating insomnia rely on drugs that either inhibit the output of

1	system, which contributes to a vicious cycle of anxiety about wakefulness that makes it more difficult to sleep. Approaches to treating insomnia rely on drugs that either inhibit the output of the ascending arousal system (green and blue in Fig. 38-2) or potentiate the output of the sleep-promoting system (red in Fig. 38-2). However, behavioral approaches (cognitive behavioral therapy and sleep hygiene) that may reduce forebrain limbic activity at bedtime are often equally or more successful.

1	Sleep is also regulated by a strong circadian timing signal, driven by the suprachiasmatic nuclei (SCN) of the hypothalamus, as described below. The SCN sends outputs to key sites in the hypothalamus, which impose 24-h rhythms on a wide range of behaviors and body systems, including the wake-sleep cycle.

1	The wake-sleep cycle is the most evident of many 24-h rhythms in humans. Prominent daily variations also occur in endocrine, thermoregulatory, cardiac, pulmonary, renal, immune, gastrointestinal, and neurobehavioral functions. At the molecular level, endogenous circadian rhythmicity is driven by self-sustaining transcriptional/ translational feedback loops. In evaluating daily rhythms in humans, it is important to distinguish between diurnal components passively evoked by periodic environmental or behavioral changes (e.g., the increase in blood pressure and heart rate that occurs upon assumption of the upright posture) and circadian rhythms actively driven by an endogenous oscillatory process (e.g., the circadian variations in adrenal cortisol and pineal melatonin secretion that persist across a variety of environmental and behavioral conditions).

1	While it is now recognized that most cells in the body have circadian clocks that regulate diverse physiologic processes, most of these disparate clocks are unable to maintain the synchronization with each other that is required to produce useful 24-h rhythms aligned with the external light-dark cycle. The neurons in the SCN are interconnected with one another in such a way as to produce a near-24-h synchronous rhythm of neural activity that is then transmitted to the rest of the body. Bilateral destruction of the SCN results in a loss of most endogenous circadian rhythms including wake-sleep behavior and rhythms in endocrine and metabolic systems. The genetically determined period of this endogenous neural oscillator, which averages ~24.15 h in humans, is normally synchronized to the 24-h period of the environmental light-dark cycle through direct input from intrinsically photosensitive ganglion cells in the retina to the SCN. Humans are exquisitely sensitive to the resetting effects

1	period of the environmental light-dark cycle through direct input from intrinsically photosensitive ganglion cells in the retina to the SCN. Humans are exquisitely sensitive to the resetting effects of light, particularly the shorter wavelengths (~460–500 nm) of the visible spectrum. Small differences in circadian period contribute to variations in diurnal preference in young adults (with the circadian period shorter in those who typically go to bed and rise earlier compared to those who typically go to bed and wake up later), whereas changes in homeostatic sleep regulation may underlie the age-related tendency toward earlier sleep-wake timing.

1	The timing and internal architecture of sleep are directly coupled to the output of the endogenous circadian pacemaker. Paradoxically, the endogenous circadian rhythm for wake propensity peaks just before the habitual bedtime, whereas that of sleep propensity peaks near the habitual wake time. These rhythms are thus timed to oppose the rise of sleep tendency throughout the usual waking day and the decline of sleep propensity during the habitual sleep episode, respectively. Misalignment of the endogenous circadian pacemaker with the desired wake-sleep cycle can, therefore, induce insomnia, decreased alertness, and impaired performance evident in night-shift workers and airline travelers.

1	Polysomnographic staging of sleep correlates with behavioral changes during specific states and stages. During the transitional state (stage N1) between wakefulness and deeper sleep, individuals may respond to faint auditory or visual signals. Formation of short-term memories is inhibited at the onset of NREM stage N1 sleep, which may explain why individuals aroused from that transitional sleep stage frequently lack situational awareness. After sleep deprivation, such transitions may intrude upon behavioral wakefulness notwithstanding attempts to remain continuously awake (see “Shift-Work Disorder,” below).

1	Awakenings from REM sleep are associated with recall of vivid dream imagery over 80% of the time, especially later in the night. Imagery may also be reported after NREM sleep interruptions. Certain disorders may occur during specific sleep stages and are described below under “Parasomnias.” These include sleepwalking, night terrors, and enuresis (bed wetting), which occur most commonly in children during deep (N3) NREM sleep, and REM sleep behavior disorder, which occurs mainly among older men who fail to maintain full atonia during REM sleep, and often call out, thrash around, or even act out entire dreams.

1	All major physiologic systems are influenced by sleep. Blood pressure and heart rate decrease during NREM sleep, particularly during N3 sleep. During REM sleep, bursts of eye movements are associated with large variations in both blood pressure and heart rate mediated by the autonomic nervous system. Cardiac dysrhythmias may occur selectively during REM sleep. Respiratory function also changes. In 187 comparison to relaxed wakefulness, respiratory rate becomes slower but more regular during NREM sleep (especially N3 sleep) and becomes irregular during bursts of eye movements in REM sleep. Decreases in minute ventilation during NREM sleep are out of proportion to the decrease in metabolic rate, resulting in a slightly higher Pco2.

1	Endocrine function also varies with sleep. N3 sleep is associated with secretion of growth hormone in men, while sleep in general is associated with augmented secretion of prolactin in both men and women. Sleep has a complex effect on the secretion of luteinizing hormone (LH): during puberty, sleep is associated with increased LH secretion, whereas sleep in the postpubertal female inhibits LH secretion in the early follicular phase of the menstrual cycle. Sleep onset (and probably N3 sleep) is associated with inhibition of thyroid-stimulating hormone and of the adrenocorticotropic hormone–cortisol axis, an effect that is superimposed on the prominent circadian rhythms in the two systems.

1	The pineal hormone melatonin is secreted predominantly at night in both dayand night-active species, reflecting the direct modulation of pineal activity by a circuitous neural pathway that links the SCN to the sympathetic nervous system, which innervates the pineal gland. Melatonin secretion does not require sleep, but melatonin secretion is inhibited by ambient light, an effect mediated by the neural connection from the retina to the pineal gland via the SCN. Sleep efficiency is highest when the sleep episode coincides with endogenous melatonin secretion. Administration of exogenous melatonin can hasten sleep onset and increase sleep efficiency when administered at a time when endogenous melatonin levels are low, such as in the afternoon or evening or at the desired bedtime in patients with delayed sleep-wake phase disorder, but it does not increase sleep efficiency if administered when endogenous melatonin levels are elevated. This may explain why melatonin is often ineffective in

1	with delayed sleep-wake phase disorder, but it does not increase sleep efficiency if administered when endogenous melatonin levels are elevated. This may explain why melatonin is often ineffective in the treatment of patients with primary insomnia.

1	Sleep is accompanied by alterations of thermoregulatory function. NREM sleep is associated with an increase in the firing of warm-responsive neurons in the preoptic area and a fall in body temperature; conversely, skin warming without increasing core body temperature has been found to increase NREM sleep. REM sleep is associated with reduced thermoregulatory responsiveness. APPROACH TO THE PATIENT: Patients may seek help from a physician because of: (1) sleepiness or tiredness during the day; (2) difficulty initiating or maintaining sleep at night (insomnia); or (3) unusual behaviors during sleep itself (parasomnias).

1	Obtaining a careful history is essential. In particular, the duration, severity, and consistency of the symptoms are important, along with the patient’s estimate of the consequences of the sleep disorder on waking function. Information from a bed partner or family member is often helpful because some patients may be unaware of symptoms such as heavy snoring or may underreport symptoms such as falling asleep at work or while driving. Physicians should inquire about when the patient typically goes to bed, when they fall asleep and wake up, whether they awaken during sleep, whether they feel rested in the morning, and whether they nap during the day. Depending on the primary complaint, it may be useful to ask about snoring, witnessed apneas, restless sensations in the legs, movements during sleep, depression, anxiety, and behaviors around the sleep episode. The physical exam may provide evidence of a small airway, large tonsils, or a neurologic or medical disorder that contributes to the

1	depression, anxiety, and behaviors around the sleep episode. The physical exam may provide evidence of a small airway, large tonsils, or a neurologic or medical disorder that contributes to the main complaint.

1	It is important to remember that, rarely, seizures may occur exclusively during sleep, mimicking a primary sleep disorder; such sleep-related seizures typically occur during episodes of NREM sleep and may take the form of generalized tonic-clonic movements (sometimes with urinary incontinence or tongue biting) or stereo- PART 2 Cardinal Manifestations and Presentation of Diseases typed movements in partial complex epilepsy (Chap. 445). It is often helpful for the patient to complete a daily sleep log for 1–2 weeks to define the timing and amounts of sleep. When relevant, the log can also include information on levels of alertness, work times, and drug and alcohol use, including caffeine and hypnotics.

1	Polysomnography is necessary for the diagnosis of several disorders such as sleep apnea, narcolepsy, and periodic limb movement disorder. A conventional polysomnogram performed in a clinical sleep laboratory allows measurement of sleep stages, respiratory effort and airflow, oxygen saturation, limb movements, heart rhythm, and additional parameters. A home sleep test usually focuses on just respiratory measures and is helpful in patients with a moderate to high likelihood of having obstructive sleep apnea. The multiple sleep latency test (MSLT) is used to measure a patient’s propensity to sleep during the day and can provide crucial evidence for diagnosing narcolepsy and some other causes of sleepiness. The maintenance of wakefulness test is used to measure a patient’s ability to sustain wakefulness during the daytime and can provide important evidence for evaluating the efficacy of therapies for improving sleepiness in conditions such as narcolepsy and obstructive sleep apnea.

1	Up to 25% of the adult population has persistent daytime sleepiness that impairs an individual’s ability to perform optimally in school, at work, while driving, and in other conditions that require alertness. Sleepy students often have trouble staying alert and performing well in school, and sleepy adults struggle to stay awake and focused on their work. More than half of Americans have fallen asleep while driving. An estimated 1.2 million motor vehicle crashes per year are due to drowsy drivers, causing about 20% of all serious crash injuries and deaths. One needn’t fall asleep to have an accident, as the inattention and slowed responses of drowsy drivers are a major contributor. Reaction time is equally impaired by 24 h of sleep loss as by a blood alcohol concentration of 0.10 g/dL.

1	Identifying and quantifying sleepiness can be challenging. First, patients may describe themselves as “sleepy,” “fatigued,” or “tired,” and the meanings of these words may differ between patients. For clinical purposes, it is best to use the term “sleepiness” to describe a propensity to fall asleep; whereas “fatigue” is best used to describe a feeling of low physical or mental energy but without a tendency to actually sleep. Sleepiness is usually most evident when the patient is sedentary, whereas fatigue may interfere with more active pursuits. Sleepiness generally occurs with disorders that reduce the quality or quantity of sleep or that interfere with the neural mechanisms of arousal, whereas fatigue is more common in inflammatory disorders such as cancer, multiple sclerosis (Chap. 458), fibromyalgia (Chap. 396), chronic fatigue syndrome (Chap. 464e), or endocrine deficiencies such as hypothyroidism (Chap. 405) or Addison’s disease (Chap. 406). Second, sleepiness can affect

1	458), fibromyalgia (Chap. 396), chronic fatigue syndrome (Chap. 464e), or endocrine deficiencies such as hypothyroidism (Chap. 405) or Addison’s disease (Chap. 406). Second, sleepiness can affect judgment in a manner analogous to ethanol, such that patients may have limited insight into the condition and the extent of their functional impairment. Finally, patients may be reluctant to admit that sleepiness is a problem because they may have become unfamiliar with feeling fully alert and because sleepiness is sometimes viewed pejoratively as reflecting poor motivation or bad sleep habits.

1	Table 38-1 outlines the diagnostic and therapeutic approach to the patient with a complaint of excessive daytime sleepiness. To determine the extent and impact of sleepiness on daytime function, it is helpful to ask patients about the occurrence of sleep episodes during normal waking hours, both intentional and unintentional. Specific areas to be addressed include the occurrence of inadvertent sleep episodes while driving or in other safety-related settings, sleepiness while at work or school (and the relationship of sleepiness to work and school performance), and the effect of sleepiness on social and family life. Standardized questionnaires such as the Epworth Sleepiness Scale are often used clinically to measure sleepiness.

1	Eliciting a history of daytime sleepiness is usually adequate, but objective quantification is sometimes necessary. The MSLT measures a patient’s propensity to sleep under quiet conditions. The test is performed after an overnight polysomnogram to establish that the patient has had an adequate amount of good-quality nighttime sleep. The MSLT consists of five 20-min nap opportunities every 2 h across the day. The patient is instructed to try to fall asleep, and the major endpoints are the average latency to sleep and the occurrence of REM sleep during the naps. An average sleep latency across the naps of less than 8 min is considered objective evidence of excessive daytime sleepiness. REM sleep normally occurs only during the nighttime sleep episode, and the occurrence of REM sleep in two or more of the MSLT naps provides support for the diagnosis of narcolepsy.

1	For the safety of the individual and the general public, physicians have a responsibility to help manage issues around driving in patients with sleepiness. Legal reporting requirements vary from state to state, but at a minimum, physicians should inform sleepy patients about their increased risk of having an accident and advise such patients not to drive a motor vehicle until the sleepiness has been treated effectively. This discussion is especially important for professional drivers, and it should be documented in the patient’s medical record. Insufficient sleep is probably the most common cause of excessive daytime sleepiness. The average adult needs 7.5–8 h of sleep, but on Difficulty waking in the morning, rebound sleep on weekends and vacations with improvement in sleepiness Obesity, snoring, hypertension Cataplexy, hypnogogic hallucinations, sleep paralysis Restless legs, kicking movements during sleep

1	Sedating medications, stimulant withdrawal, head trauma, systemic inflammation, Parkinson’s disease and other neurodegenerative disorders, hypothyroidism, encephalopathy weeknights, the average U.S. adult gets only 6.75 h of sleep. Only 30% of the U.S. adult population reports consistently obtaining sufficient sleep. Insufficient sleep is especially common among shift workers, individuals working multiple jobs, and people in lower socioeconomic groups. Most teenagers need ≥9 h of sleep, but many fail to get enough sleep because of circadian phase delay, or social pressures to stay up late coupled with early school start times. Late evening light exposure, television viewing, video-gaming, social media, texting, and smart-phone use often delay bedtimes despite the fixed, early wake times required for work or school. As is typical with any disorder that causes sleepiness, individuals with chronically insufficient sleep may feel inattentive, irritable, unmotivated, and depressed, and

1	required for work or school. As is typical with any disorder that causes sleepiness, individuals with chronically insufficient sleep may feel inattentive, irritable, unmotivated, and depressed, and have difficulty with school, work, and driving. Individuals differ in their optimal amount of sleep, and it can be helpful to ask how much sleep the patient obtains on a quiet vacation when he or she can sleep without restrictions. Some patients may think that a short amount of sleep is normal or advantageous, and they may not appreciate their biological need for more sleep, especially if coffee and other stimulants mask the sleepiness. A 2-week sleep log documenting the timing of sleep and daily level of alertness is diagnostically useful and provides helpful feedback for the patient. Extending sleep to the optimal amount on a regular basis can resolve the sleepiness and other symptoms. As with any lifestyle change, extending sleep requires commitment and adjustments, but the improvements

1	sleep to the optimal amount on a regular basis can resolve the sleepiness and other symptoms. As with any lifestyle change, extending sleep requires commitment and adjustments, but the improvements in daytime alertness make this change worthwhile.

1	Respiratory dysfunction during sleep is a common, serious cause of excessive daytime sleepiness as well as of disturbed nocturnal sleep. At least 24% of middle-aged men and 9% of middle-aged women in the United States have a reduction or cessation of breathing dozens or more times each night during sleep, with 9% of men and 4% of women doing so more than a hundred times per night. These episodes may be due to an occlusion of the airway (obstructive sleep apnea), absence of respiratory effort (central sleep apnea), or a combination of these factors (mixed sleep apnea). Failure to recognize and treat these conditions appropriately may lead to impairment of daytime alertness, increased risk of sleep-related motor vehicle crashes, depression, hypertension, myocardial infarction, diabetes, stroke, and increased mortality. Sleep apnea is particularly prevalent in overweight men and in the elderly, yet it is estimated to go undiagnosed in most affected individuals. This is unfortunate

1	stroke, and increased mortality. Sleep apnea is particularly prevalent in overweight men and in the elderly, yet it is estimated to go undiagnosed in most affected individuals. This is unfortunate because several effective treatments are available. Readers are referred to Chap. 319 for a comprehensive review of the diagnosis and treatment of patients with sleep apnea.

1	Narcolepsy is characterized by difficulty sustaining wakefulness, poor regulation of REM sleep, and disturbed nocturnal sleep. All patients with narcolepsy have excessive daytime sleepiness. This sleepiness is often severe, but in some, it is mild. In contrast to patients of the face or neck. Narcolepsy is one of the more common causes of 189 chronic sleepiness and affects about 1 in 2000 people in the United States. Narcolepsy typically begins between age 10 and 20; once established, the disease persists for life.

1	Narcolepsy is caused by loss of the hypothalamic neurons that produce the orexin neuropeptides (also known as hypocretins). Research in mice and dogs first demonstrated that a loss of orexin signaling due to null mutations of either the orexin neuropeptides or one of the orexin receptors causes sleepiness and cataplexy nearly identical to that seen in people with narcolepsy. Although genetic mutations rarely cause human narcolepsy, researchers soon discovered that patients with narcolepsy had very low or undetectable levels of orexins in their cerebrospinal fluid, and autopsy studies showed a nearly complete loss of the orexin-producing neurons in the hypothalamus. The orexins normally promote long episodes of wakefulness and suppress REM sleep, and thus, loss of orexin signaling results in frequent intrusions of sleep during the usual waking episode, with REM sleep and fragments of REM sleep at any time of day (Fig. 38-3).

1	Extensive evidence suggests that an autoimmune process likely causes this selective loss of the orexin-producing neurons. Certain human leukocyte antigens (HLAs) can increase the risk of autoimmune disorders (Chap. 373e), and narcolepsy has the strongest known HLA association. HLA DQB1*06:02 is found in about 90% of people with narcolepsy, whereas it occurs in only 12–25% of the general population. Researchers now hypothesize that in people with DQB1*06:02, an immune response against influenza, Streptococcus, or other infections may also damage the orexin-producing neurons through a process of molecular mimicry. This mechanism may account for the 8to 12-fold increase in new cases of narcolepsy among children in Europe who received a particular brand of H1N1 influenza A vaccine (Pandemrix). On rare occasions, narcolepsy can occur with neurologic disorders such as tumors or strokes that directly damage the orexin-producing neurons in the hypothalamus or their projections.

1	On rare occasions, narcolepsy can occur with neurologic disorders such as tumors or strokes that directly damage the orexin-producing neurons in the hypothalamus or their projections. Diagnosis Narcolepsy is most commonly diagnosed by the history of chronic sleepiness plus cataplexy or other symptoms. Many disorders can cause feelings of weakness, but with true cataplexy, patients will describe definite functional weakness (e.g., slurred speech, dropping a cup, slumping into a chair) that has consistent emotional triggers such as heartfelt mirth when laughing at a great joke, happy surprise at unexpectedly seeing a friend, or intense anger. Cataplexy occurs in about half of all narcolepsy patients and is diagnostically very helpful because it occurs in almost no other disorder. In contrast, occasional hypnagogic hallucinations and sleep paralysis occur in about 20% of the general population, and these symptoms are not as diagnostically specific.

1	When narcolepsy is suspected, the diagnosis should be firmly established with a polysomnogram followed by an MSLT. The 00:00 04:00 08:00 12:00 16:00 00:00 04:00 08:00 12:00 16:00 Clock time paralysis). With severe cataplexy, an indi-FIguRE 38-3 Polysomnographic recordings of a healthy individual and a patient with vidual may be laughing at a joke and then narcolepsy. The individual with narcolepsy enters rapid eye movement (REM) sleep quickly at suddenly collapse to the ground, immobile night and has moderately fragmented sleep. During the day, the healthy subject stays awake but awake for 1–2 min. With milder epi-from 8:00 AM until midnight, but the patient with narcolepsy dozes off frequently, with many sodes, patients may have mild weakness daytime naps that include REM sleep.

1	190 polysomnogram helps rule out other possible causes of sleepiness such as sleep apnea, and the MSLT provides essential, objective evidence of sleepiness plus REM sleep dysregulation. Across the five naps of the MSLT, most patients with narcolepsy will fall asleep in less than 8 min on average, and they will have episodes of REM sleep in at least two of the naps. Abnormal regulation of REM sleep is also manifested by the appearance of REM sleep within 15 min of sleep onset at night, which is rare in healthy individuals sleeping at their habitual bedtime. Stimulants should be stopped 1 week before the MSLT and antidepressants should be stopped 3 weeks prior, because these medications can affect the MSLT. In addition, patients should be encouraged to obtain a fully adequate amount of sleep each night for the week prior to the test to eliminate any effects of insufficient sleep.

1	The treatment of narcolepsy is symptomatic. Most patients with narcolepsy feel more alert after sleep, and they should be encouraged to get adequate sleep each night and to take a 15to 20-min nap in the afternoon. This nap may be sufficient for some patients with mild narcolepsy, but most also require treatment with wake-promoting medications. Modafinil is used quite often because it has fewer side effects than amphetamines and a relatively long half-life; for most patients, 200–400 mg each morning is very effective. Methylphenidate (10–20 mg bid) or dextroamphetamine (10 mg bid) are often effective, but sympathomimetic side effects, anxiety, and the potential for abuse can be concerns. These medications are available in slow-release formulations, extending their duration of action and allowing easier dosing. Sodium oxybate (gamma hydroxybutyrate) is given twice each night and is often very valuable in improving alertness, but it can produce excessive sedation, nausea, and confusion.

1	Cataplexy is usually much improved with antidepressants that increase noradrenergic or serotonergic tone because these medications strongly suppress REM sleep and cataplexy. Venlafaxine (37.5–150 mg each morning) and fluoxetine (10–40 mg each morning) are often quite effective. The tricyclic antidepressants, such as protriptyline (10–40 mg/d) or clomipramine (25–50 mg/d) are potent suppressors of cataplexy, but their anticholinergic effects, including sedation and dry mouth, make them less attractive.1 Sodium oxybate, given at bedtime and 3–4 h later, is also very helpful in reducing cataplexy. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Insomnia is the complaint of poor sleep and usually presents as difficulty initiating or maintaining sleep. People with insomnia are dissatisfied with their sleep and feel that it impairs their ability to function well in work, school, and social situations. Affected individuals often experience fatigue, decreased mood, irritability, malaise, and cognitive impairment.

1	Chronic insomnia, lasting more than 3 months, occurs in about 10% of adults and is more common in women, older adults, people of lower socioeconomic status, and individuals with medical, psychiatric, and substance abuse disorders. Acute or short-term insomnia affects over 30% of adults and is often precipitated by stressful life events such as a major illness or loss, change of occupation, medications, and substance abuse. If the acute insomnia triggers maladaptive behaviors such as increased nocturnal light exposure, frequently checking the clock, or attempting to sleep more by napping, it can lead to chronic insomnia.

1	Most insomnia begins in adulthood, but many patients may be predisposed and report easily disturbed sleep predating the insomnia, suggesting that their sleep is lighter than usual. Clinical studies and animal models indicate that insomnia is associated with activation 1No antidepressant has been approved by the U.S. Food and Drug Administration (FDA) for treating narcolepsy.

1	during sleep of brain areas normally active only during wakefulness. The polysomnogram is rarely used in the evaluation of insomnia, as it typically confirms the patient’s subjective report of long latency to sleep and numerous awakenings but usually adds little new information. Many patients with insomnia have increased fast (beta) activity in the EEG during sleep; this fast activity is normally present only during wakefulness, which may explain why some patients report feeling awake for much of the night. The MSLT is rarely used in the evaluation of insomnia because, despite their feelings of low energy, most people with insomnia do not easily fall asleep during the day, and on the MSLT, their average sleep latencies are usually longer than normal.

1	Many factors can contribute to insomnia, and obtaining a careful history is essential so one can select therapies targeting the underlying factors. The assessment should focus on identifying predisposing, precipitating, and perpetuating factors. Psychophysiologic Factors Many patients with insomnia have negative expectations and conditioned arousal that interfere with sleep. These individuals may worry about their insomnia during the day and have increasing anxiety as bedtime approaches if they anticipate a poor night of sleep. While attempting to sleep, they may frequently check the clock, which only heightens anxiety and frustration. They may find it easier to sleep in a new environment rather than their bedroom, as it lacks the negative associations.

1	Inadequate Sleep Hygiene Patients with insomnia sometimes develop counterproductive behaviors that contribute to their insomnia. These can include daytime napping that reduces sleep drive at night; an irregular sleep-wake schedule that disrupts their circadian rhythms; use of wake-promoting substances (e.g., caffeine, tobacco) too close to bedtime; engaging in alerting or stressful activities close to bedtime (e.g., arguing with a partner, work-related emailing and texting while in bed, sleeping with a smartphone or tablet at the bedside); and routinely using the bedroom for activities other than sleep or sex (e.g., TV, work), so the bedroom becomes associated with arousing or stressful feelings.

1	Psychiatric Conditions About 80% of patients with psychiatric disorders have sleep complaints, and about half of all chronic insomnia occurs in association with a psychiatric disorder. Depression is classically associated with early morning awakening, but it can also interfere with the onset and maintenance of sleep. Mania and hypomania can disrupt sleep and often are associated with substantial reductions in the total amount of sleep. Anxiety disorders can lead to racing thoughts and rumination that interfere with sleep and can be very problematic if the patient’s mind becomes active midway through the night. Panic attacks can occur during sleep and need to be distinguished from other parasomnias. Insomnia is common in schizophrenia and other psychoses, often resulting in fragmented sleep, less deep NREM sleep, and sometimes reversal of the day-night sleep pattern.

1	Medications and Drugs of Abuse A wide variety of psychoactive drugs can interfere with sleep. Caffeine, which has a half-life of 6–9 h, can disrupt sleep for up to 8–14 h, depending on the dose, variations in metabolism, and an individual’s caffeine sensitivity. Insomnia can also result from use of prescription medications too close to bedtime (e.g., theophylline, stimulants, antidepressants, glucocorticoids). Conversely, withdrawal of sedating medications such as alcohol, narcotics, or benzodiazepines can cause insomnia. Alcohol taken just before bed can shorten sleep latency, but it often produces rebound insomnia 2–3 h later as it wears off. This same problem with sleep maintenance can occur with short-acting benzodiazepines such as alprazolam.

1	Medical Conditions A large number of medical conditions disrupt sleep. Pain from rheumatologic disorders or a painful neuropathy commonly disrupts sleep. Some patients may sleep poorly because of respiratory conditions such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, congestive heart failure, or restrictive lung disease, and some of these disorders are worse at night in bed due to circadian variations in airway resistance and postural changes that can result in paroxysmal nocturnal dyspnea. Many women experience poor sleep with the hormonal changes of menopause. Gastroesophageal reflux is also a common cause of difficulty sleeping.

1	Neurologic Disorders Dementia (Chap. 35) is often associated with poor sleep, probably due to a variety of factors, including napping during the day, altered circadian rhythms, and perhaps a weakened output of the brain’s sleep-promoting mechanisms. In fact, insomnia and nighttime wandering are some of the most common causes for institutionalization of patients with dementia, because they place a larger burden on caregivers. Conversely, in cognitively intact elderly men, fragmented sleep and poor sleep quality are associated with subsequent cognitive decline. Patients with Parkinson’s disease may sleep poorly due to rigidity, dementia, and other factors. Fatal familial insomnia is a very rare neurodegenerative condition caused by mutations in the prion protein gene, and although insomnia is a common early symptom, most patients present with other obvious neurologic signs such dementia, myoclonus, dysarthria, or autonomic dysfunction.

1	Treatment of insomnia improves quality of life and can promote long-term health. With improved sleep, patients often report less daytime fatigue, improved cognition, and more energy. Treating the insomnia can also improve the comorbid disease. For example, management of insomnia at the time of diagnosis of major depression often improves the response to antidepressants and reduces the risk of relapse. Sleep loss can heighten the perception of pain, so a similar approach is warranted in acute and chronic pain management. The treatment plan should target all putative contributing factors: establish good sleep hygiene, treat medical disorders, use behavioral therapies for anxiety and negative conditioning, and use pharmacotherapy and/or psychotherapy for psychiatric disorders. Behavioral therapies should be the first-line treatment, followed by judicious use of sleep-promoting medications if needed.

1	If the history suggests that a medical or psychiatric disease contributes to the insomnia, then it should be addressed by, for example, treating the pain, improving breathing, and switching or adjusting the timing of medications.

1	Attention should be paid to improving sleep hygiene and avoiding counterproductive, arousing behaviors before bedtime. Patients should establish a regular bedtime and wake time, even on weekends, to help synchronize their circadian rhythms and sleep patterns. The amount of time allocated for sleep should not be more than their actual total amount of sleep. In the 30 min before bedtime, patients should establish a relaxing “wind-down” routine that can include a warm bath, listening to music, meditation, or other relaxation techniques. The bedroom should be off-limits to computers, televisions, radios, smartphones, videogames, and tablets. Once in bed, patients should try to avoid thinking about anything stressful or arousing such as problems with relationships or work. If they cannot fall asleep within 20 min, it often helps to get out of bed and read or listen to relaxing music in dim light as a form of distraction from any anxiety, but artificial light, including light from a

1	fall asleep within 20 min, it often helps to get out of bed and read or listen to relaxing music in dim light as a form of distraction from any anxiety, but artificial light, including light from a television, cell phone, or computer, should be avoided, because light itself suppresses melatonin secretion and is arousing.

1	Table 38-2 outlines some of the key aspects of good sleep hygiene to improve insomnia. CBT uses a combination of the techniques above plus additional methods to improve insomnia. A trained therapist may use cognitive psychology techniques to reduce excessive worrying about sleep and to reframe faulty beliefs about the insomnia and its daytime consequences. The therapist may also teach the patient relaxation Helpful Behaviors Behaviors to Avoid Use the bed only for sleep and sex Avoid behaviors that interfere with sleep physiology, including: • If you cannot sleep within 20 min, get out of bed and read or do • Napping, especially after 3:00 PM other relaxing activities in dim light • Attempting to sleep too early before returning to bed Make quality sleep a priority In the 2–3 h before bedtime, avoid: • Go to bed and get up at the same • Heavy eating (comfortable bed, bedroom quiet

1	Make quality sleep a priority In the 2–3 h before bedtime, avoid: • Go to bed and get up at the same • Heavy eating (comfortable bed, bedroom quiet Develop a consistent bedtime When trying to fall asleep, avoid: routine. For example: • Prepare for sleep with 20–30 min of • Thinking about life issues relaxation (e.g., soft music, medita • Reviewing events of the day tion, yoga, pleasant reading) techniques, such as progressive muscle relaxation or meditation, to reduce autonomic arousal, intrusive thoughts, and anxiety. If insomnia persists after treatment of these contributing factors, pharmacotherapy is often used on a nightly or intermittent basis. A variety of sedatives can improve sleep.

1	If insomnia persists after treatment of these contributing factors, pharmacotherapy is often used on a nightly or intermittent basis. A variety of sedatives can improve sleep. Antihistamines, such as diphenhydramine, are the primary active ingredient in most over-the-counter sleep aids. These may be of benefit when used intermittently, but often produce rapid tolerance and can produce anticholinergic side effects such as dry mouth and constipation, which limit their use, particularly in the elderly.

1	Benzodiazepine receptor agonists (BzRAs) are an effective and well-tolerated class of medications for insomnia. BzRAs bind to the GABAA receptor and potentiate the postsynaptic response to GABA. GABAA receptors are found throughout the brain, and BzRAs may globally reduce neural activity and may enhance the activity of specific sleep-promoting GABAergic pathways. Classic BzRAs include lorazepam, triazolam, and clonazepam, whereas newer agents such as zolpidem and zaleplon have more selective affinity for the α1 subunit of the GABAA receptor.

1	Specific BzRAs are often chosen based on the desired duration of action. The most commonly prescribed agents in this family are zaleplon (5–20 mg), with a half-life of 1–2 h; zolpidem (5–10 mg) and triazolam (0.125–0.25 mg), with half-lives of 2–4 h; eszopiclone (1–3 mg), with a half-life of 5–8 h; and temazepam (15–30 mg), with a half-life of 8–20 h. Generally, side effects are minimal when the dose is kept low and the serum concentration is minimized during the waking hours (by using the shortest-acting effective agent). For chronic insomnia, intermittent use is recommended, unless the consequences of untreated insomnia outweigh concerns regarding chronic use.

1	The heterocyclic antidepressants (trazodone, amitriptyline,2 and doxepin) are the most commonly prescribed alternatives to BzRAs due to their lack of abuse potential and lower cost. Trazodone (25–100 mg) is used more commonly than the tricyclic antidepressants, because it has a much shorter half-life (5–9 h) and less anticholinergic activity. Medications for insomnia are now among the most commonly prescribed medications, but they should be used cautiously. All sedatives increase the risk of injurious falls and confusion in the elderly, and therefore if needed, these medications should be used at the lowest effective dose. Morning sedation can interfere with driving and judgment, and when selecting a medication, one should 2Trazodone and amitriptyline have not been approved by the FDA for treating insomnia.

1	192 consider the duration of action. Benzodiazepines carry a risk of addiction and abuse, especially in patients with a history of alcohol or sedative abuse. Like alcohol, some sleep-promoting medications can worsen sleep apnea. Sedatives can also produce complex behaviors during sleep, such as sleep walking and sleep eating, although this seems more likely at higher doses. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Patients with restless legs syndrome (RLS) report an irresistible urge to move the legs. Many patients report a creepy-crawly or unpleasant deep ache within the thighs or calves, and those with more severe RLS may have discomfort in the arms as well. For most patients with RLS, these dysesthesias and restlessness are much worse in the evening and first half of the night. The symptoms appear with inactivity and can make sitting still in an airplane or when watching a movie a miserable experience. The sensations are temporarily relieved by movement, stretching, or massage. This nocturnal discomfort usually interferes with sleep, and patients may report daytime sleepiness as a consequence. RLS is very common, affecting 5–10% of adults and is more common in women and older adults.

1	A variety of factors can cause RLS. Iron deficiency is the most common treatable cause, and iron replacement should be considered if the ferritin level is less than 50 ng/mL. RLS can also occur with peripheral neuropathies and uremia and can be worsened by pregnancy, caffeine, alcohol, antidepressants, lithium, neuroleptics, and antihistamines. Genetic factors contribute to RLS, and polymorphisms in a variety of genes (BTBD9, MEIS1, MAP2K5/LBXCOR, and PTPRD) have been linked to RLS, although as yet, the mechanism through which they cause RLS remains unknown. Roughly one-third of patients (particularly those with an early age of onset) have multiple affected family members.

1	RLS is treated by addressing the underlying cause such as iron deficiency if present. Otherwise, treatment is symptomatic, and dopamine agonists are used most frequently. Agonists of dopamine D2/3 receptors such as pramipexole (0.25–0.5 mg q7PM) or ropinirole (0.5–4 mg q7PM) are considered first-line agents. Augmentation is a worsening of RLS such that symptoms begin earlier in the day and can spread to other body regions, and it can occur in about 25% of patients taking dopamine agonists. Other possible side effects of dopamine agonists include nausea, morning sedation, and increases in rewarding behavior such as gambling and sex. Opioids, benzodiazepines, pregabalin, and gabapentin may also be of therapeutic value. Most patients with restless legs also experience periodic limb movement disorder, although the reverse is not the case.

1	Periodic limb movement disorder (PLMD) involves rhythmic twitches of the legs that disrupt sleep. The movements resemble a triple flex-ion reflex with extensions of the great toe and dorsiflexion of the foot for 0.5 to 5.0 s, which recur every 20–40 s during NREM sleep, in episodes lasting from minutes to hours. PLMD is diagnosed by a polysomnogram that includes recordings of the anterior tibialis and sometimes other muscles. The EEG shows that the movements of PLMD frequently cause brief arousals that disrupt sleep and can cause insomnia and daytime sleepiness. PLMD can be caused by the same factors that cause RLS (see above), and the frequency of leg movements improves with the same medications as used for RLS, including dopamine agonists. Recent genetic studies identified polymorphisms associated with RLS/PLMD, suggesting that they may have a common pathophysiology.

1	Parasomnias are abnormal behaviors or experiences that arise from or occur during sleep. A variety of parasomnias can occur during NREM sleep, from brief confusional arousals to sleepwalking and night terrors. The presenting complaint is usually related to the behavior itself, but the parasomnias can disturb sleep continuity or lead to mild impairments in daytime alertness. Two main parasomnias occur in REM sleep: REM sleep behavior disorder (RBD) and nightmares.

1	Sleepwalking (Somnambulism) Patients affected by this disorder carry out automatic motor activities that range from simple to complex. Individuals may walk, urinate inappropriately, eat, exit the house, or drive a car with minimal awareness. Full arousal may be difficult, and occasional individuals may respond to attempted awakening with agitation or violence. Sleepwalking arises from NREM stage N3 sleep, usually in the first few hours of the night, and the EEG usually shows the slow cortical activity of deep NREM sleep even when the patient is moving about. Sleepwalking is most common in children and adolescents, when these sleep stages are most robust. About 15% of children have occasional sleepwalking, and it persists in about 1% of adults. Episodes are usually isolated but may be recurrent in 1–6% of patients. The cause is unknown, although it has a familial basis in roughly one-third of cases. Sleepwalking can be worsened by insufficient sleep, which subsequently causes an

1	recurrent in 1–6% of patients. The cause is unknown, although it has a familial basis in roughly one-third of cases. Sleepwalking can be worsened by insufficient sleep, which subsequently causes an increase in deep NREM sleep; alcohol; and stress. These should be addressed if present. Small studies have shown some efficacy of antidepressants and benzodiazepines; relaxation techniques and hypnosis can also be helpful. Patients and their families should improve home safety (e.g., replace glass doors, remove low tables to avoid tripping) to minimize the chance of injury if sleepwalking occurs.

1	Sleep Terrors This disorder occurs primarily in young children during the first few hours of sleep during NREM stage N3 sleep. The child often sits up during sleep and screams, exhibiting autonomic arousal with sweating, tachycardia, large pupils, and hyperventilation. The individual may be difficult to arouse and rarely recalls the episode on awakening in the morning. Treatment usually consists of reassuring the parents that the condition is self-limited and benign, and like sleepwalking, it may improve by avoiding insufficient sleep.

1	Sleep Bruxism Bruxism is an involuntary, forceful grinding of teeth during sleep that affects 10–20% of the population. The patient is usually unaware of the problem. The typical age of onset is 17–20 years, and spontaneous remission usually occurs by age 40. Sex distribution appears to be equal. In many cases, the diagnosis is made during dental examination, damage is minor, and no treatment is indicated. In more severe cases, treatment with a tooth guard is necessary to prevent tooth injury. Stress management or, in some cases, biofeedback can be useful when bruxism is a manifestation of psychological stress. There are anecdotal reports of benefit with benzodiazepines.

1	Sleep Enuresis Bedwetting, like sleepwalking and night terrors, is another parasomnia that occurs during sleep in the young. Before age 5 or 6 years, nocturnal enuresis should be considered a normal feature of development. The condition usually improves spontaneously by puberty, has a prevalence in late adolescence of 1–3%, and is rare in adulthood. Treatment consists of bladder training exercises and behavioral therapy. Symptomatic pharmacotherapy is usually accomplished in adults with desmopressin (0.2 mg qhs), oxybutynin chloride (5 mg qhs), or imipramine (10–25 mg qhs). Important causes of nocturnal enuresis in patients who were previously continent for 6–12 months include urinary tract infections or malformations, cauda equina lesions, emotional disturbances, epilepsy, sleep apnea, and certain medications.

1	REM Sleep Behavior Disorder (RBD) RBD (Video 38-2) is distinct from other parasomnias in that it occurs during REM sleep. The patient or the bed partner usually reports agitated or violent behavior during sleep, and upon awakening, the patient can often report a dream that accompanied the movements. During normal REM sleep, nearly all skeletal muscles are paralyzed, but in patients with RBD, the polysomnogram often shows limb movements during REM sleep, lasting for seconds to minutes. The movements can be dramatic, and it is not uncommon for the patient or the bed partner to be injured.

1	RBD primarily afflicts older men, and most either have or will develop a neurodegenerative disorder. In longitudinal studies of RBD, half of the patients developed a synucleinopathy such as Parkinson’s disease (Chap. 449) or dementia with Lewy bodies (Chap. 448), or occasionally multiple system atrophy (Chap. 454), within 12 years, and over 80% developed a synucleinopathy by 20 years. RBD can occur in patients taking antidepressants, and in some, these medications may unmask this early indicator of neurodegeneration. Synucleinopathies probably cause neuronal loss in brainstem regions that regulate muscle atonia during REM sleep, and loss of these neurons permits movements to break through during REM sleep. RBD also occurs in about 30% of patients with narcolepsy, but the underlying cause is probably different, as they seem to be at no increased risk of a neurodegenerative disorder.

1	Many patients with RBD have sustained improvement with clonazepam (0.5–2.0 mg qhs).3 Melatonin at doses up to 9 mg nightly may also prevent attacks. A subset of patients presenting with either insomnia or hypersomnia may have a disorder of sleep timing rather than sleep generation. Disorders of sleep timing can be either organic (i.e., due to an abnormality of circadian pacemaker[s]) or environmental/behavioral (i.e., due to a disruption of environmental synchronizers). Effective therapies aim to entrain the circadian rhythm of sleep propensity to an appropriate phase.

1	Delayed Sleep-Wake Phase Disorder Delayed sleep-wake phase disorder (DSWPD) is characterized by: (1) reported sleep onset and wake times intractably later than desired; (2) actual sleep times at nearly the same clock hours daily; and (3) if conducted at the habitual delayed sleep time, essentially normal sleep on polysomnography (except for delayed sleep onset). Patients with DSWPD exhibit an abnormally delayed endogenous circadian phase, which can be assessed by measuring, in a dimly lit environment, the onset of secretion of the endogenous circadian rhythm of pineal melatonin in either the blood or saliva, as light suppresses melatonin secretion. Dim-light melatonin onset (DLMO) in DSWPD patients typically occurs later in the evening than normal, which is about 8:00–9:00 pm (i.e., about 1–2 h before habitual bedtime). Patients tend to be young adults. The delayed circadian phase could be due to: (1) an abnormally long, genetically determined intrinsic period of the endogenous

1	about 1–2 h before habitual bedtime). Patients tend to be young adults. The delayed circadian phase could be due to: (1) an abnormally long, genetically determined intrinsic period of the endogenous circadian pacemaker; (2) reduced phase-advancing capacity of the pacemaker; (3) slower rate of buildup of homeostatic sleep drive during wakefulness; or (4) an irregular prior sleep-wake schedule, characterized by frequent nights when the patient chooses to remain awake while exposed to artificial light well past midnight (for personal, social, school, or work reasons). In most cases, it is difficult to distinguish among these factors, as patients with either a behaviorally induced or biologically driven circadian phase delay may both exhibit a similar circadian phase delay in DLMO, making it difficult for both to fall asleep at the desired hour. DSWPD is a self-perpetuating condition that can persist for years and may not respond to attempts to reestablish normal bedtime hours. Treatment

1	difficult for both to fall asleep at the desired hour. DSWPD is a self-perpetuating condition that can persist for years and may not respond to attempts to reestablish normal bedtime hours. Treatment methods involving phototherapy with blue-enriched light during the morning hours and/or melatonin administration in the evening hours show promise in these patients, although the relapse rate is high. Patients with this circadian rhythm sleep disorder can be distinguished from those who have sleep-onset insomnia because DSWPD patients show late onset of dim-light melatonin secretion.

1	Advanced Sleep-Wake Phase Disorder Advanced sleep-wake phase disorder (ASWPD) is the converse of DSWPD. Most commonly, this syndrome occurs in older people, 15% of whom report that they cannot sleep past 5:00 am, with twice that number complaining that they wake up too early at least several times per week. Patients with ASWPD are sleepy during the evening hours, even in social settings. Sleep-wake timing in ASWPD patients can interfere with a normal social life. Patients with this circadian rhythm sleep disorder can be distinguished from those who have early wakening due to insomnia because ASWPD patients show early onset of dim-light melatonin secretion. In addition to age-related ASWPD, an early-onset familial variant of this condition has also been reported. In two families in which ASWPD was inherited in an autosomal dominant pattern, the syndrome was 3No medications have been approved by the FDA for the treatment of RBD.

1	due to missense mutations in a circadian clock component (in the 193 casein kinase binding domain of PER2 in one family, and in casein kinase I delta in the other) that altered the circadian period. Patients with ASWPD may benefit from bright-light and/or blue enriched phototherapy during the evening hours to reset the circadian pacemaker to a later hour.

1	Non-24-h Sleep-Wake Rhythm Disorder Non-24-h sleep-wake rhythm disorder (N24SWRD) can occur when the primary synchronizing input (i.e., the light-dark cycle) from the environment to the circadian pacemaker is compromised (as occurs in many blind people with no light perception) or when the maximal phase-advancing capacity of the circadian pacemaker cannot accommodate the difference between the 24-h geophysical day and the intrinsic period of the patient’s circadian pacemaker, resulting in loss of entrainment to the 24-h day. Rarely, self-selected exposure to artificial light may, in some sighted patients, inadvertently entrain the circadian pacemaker to a >24-h schedule. Affected patients with N24SWRD have difficulty maintaining a stable phase relationship between the output of the pacemaker and the 24-h day. Such patients typically present with an incremental pattern of successive delays in sleep propensity, progressing in and out of phase with local time. When the N24SWRD patient’s

1	and the 24-h day. Such patients typically present with an incremental pattern of successive delays in sleep propensity, progressing in and out of phase with local time. When the N24SWRD patient’s endogenous circadian rhythms are out of phase with the local environment, nighttime insomnia coexists with excessive daytime sleepiness. Conversely, when the endogenous circadian rhythms are in phase with the local environment, symptoms remit. The interval between symptomatic phases may last several weeks to several months in N24SWRD, depending on the period of the underlying nonentrained rhythm and the 24-h day. Nightly low-dose (0.5 mg) melatonin administration may improve sleep and, in some cases, induce synchronization of the circadian pacemaker.

1	Shift-Work Disorder More than 7 million workers in the United States regularly work at night, either on a permanent or rotating schedule. Many more begin the commute to work or school between 4:00 am and 7:00 am, requiring them to commute and then work during the time of day that they would otherwise be asleep. In addition, each week, millions of “day” workers and students elect to remain awake at night or awaken very early in the morning to work or study to meet work or school deadlines, drive long distances, compete in sporting events, or participate in recreational activities. Such schedules can result in both sleep loss and misalignment of circadian rhythms with respect to the sleep-wake cycle.

1	The circadian timing system usually fails to adapt successfully to the inverted schedules required by overnight work or the phase advance required by early morning (4:00 am to 7:00 am) start times. This leads to a misalignment between the desired work-rest schedule and the output of the pacemaker and to disturbed daytime sleep in most individuals. Excessive work hours (per day or per week), insufficient time off between consecutive days of work or school, and transmeridian travel may be contributing factors. Sleep deficiency, increased length of time awake prior to work, and misalignment of circadian phase produce decreased alertness and performance, increased reaction time, and increased risk of performance lapses, thereby resulting in greater safety hazards among night workers and other sleep-deprived individuals. Sleep disturbance nearly doubles the risk of a fatal work accident. Long-term night shift workers have higher rates of breast, colorectal, and prostate cancer and of

1	other sleep-deprived individuals. Sleep disturbance nearly doubles the risk of a fatal work accident. Long-term night shift workers have higher rates of breast, colorectal, and prostate cancer and of cardiac, gastrointestinal, and reproductive disorders. The World Health Organization has added night-shift work to its list of probable carcinogens.

1	Sleep onset begins in local brain regions before gradually sweeping over the entire brain as sensory thresholds rise and consciousness is lost. A sleepy individual struggling to remain awake may attempt to continue performing routine and familiar motor tasks during the transition state between wakefulness and stage N1 sleep, while unable to adequately process sensory input from the environment. Motor vehicle operators who fail to heed the warning signs of sleepiness are especially vulnerable to sleep-related accidents, as sleep processes can intrude involuntarily upon the waking brain, causing catastrophic consequences. Such sleep-related attentional failures typically last only seconds but are known on occasion to persist for longer durations.

1	194 There is a significant increase in the risk of sleep-related, fatal-to-thedriver highway crashes in the early morning and late afternoon hours, coincident with bimodal peaks in the daily rhythm of sleep tendency. Resident physicians constitute another group of workers at greater risk for accidents and other adverse consequences of lack of sleep and misalignment of the circadian rhythm. Recurrent scheduling of resident physicians to work shifts of ≥24 consecutive hours impairs psychomotor performance to a degree that is comparable to alcohol intoxication, doubles the risk of attentional failures among intensive care unit resident physicians working at night, and significantly increases the risk of serious medical errors in intensive care units, including a fivefold increase in the risk of serious diagnostic mistakes. Some 20% of hospital resident physicians report making a fatigue-related mistake that injured a patient, and 5% admit making a fatigue-related mistake that resulted in

1	of serious diagnostic mistakes. Some 20% of hospital resident physicians report making a fatigue-related mistake that injured a patient, and 5% admit making a fatigue-related mistake that resulted in the death of a patient. Moreover, working for >24 consecutive hours increases the risk of percutaneous injuries and more than doubles the risk of motor vehicle crashes on the commute home. For these reasons, in 2008, the Institute of Medicine concluded that the practice of scheduling resident physicians to work for more than 16 consecutive hours without sleep is hazardous for both resident physicians and their patients. From 5 to 15% of individuals scheduled to work at night or in the early morning hours have much greater-than-average difficulties remaining awake during night work and sleeping during the day; these individuals are diagnosed with chronic and severe shift-work disorder (SWD). Patients with this disorder have a level of excessive sleepiness during work at night or in the

1	during the day; these individuals are diagnosed with chronic and severe shift-work disorder (SWD). Patients with this disorder have a level of excessive sleepiness during work at night or in the early morning and insomnia during day sleep that the physician judges to be clinically significant; the condition is associated with an increased risk of sleep-related accidents and with some of the illnesses associated with night-shift work. Patients with chronic and severe SWD are profoundly sleepy at work. In fact, their sleep latencies during night work average just 2 min, comparable to mean daytime sleep latency durations of patients with narcolepsy or severe sleep apnea.

1	Caffeine is frequently used by night workers to promote wakefulness. However, it cannot forestall sleep indefinitely, and it does not shield users from sleep-related performance lapses. Postural changes, exercise, and strategic placement of nap opportunities can sometimes temporarily reduce the risk of fatigue-related performance lapses. Properly timed exposure to blue-enriched light or bright white light can directly enhance alertness and facilitate more rapid adaptation to night-shift work. Modafinil (200 mg) or armodafinil (150 mg) 30–60 min before the start of each night shift is an effective treatment for the excessive sleepiness during night work in patients with SWD. Although treatment with modafinil or armodafinil significantly improves performance and reduces sleep propensity and the risk of lapses of attention during night work, affected patients remain excessively sleepy.

1	Fatigue risk management programs for night shift workers should promote education about sleep, increase awareness of the hazards associated with sleep deficiency and night work, and screen for common sleep disorders. Work schedules should be designed to minimize: (1) exposure to night work; (2) the frequency of shift rotations; (3) the number of consecutive night shifts; and (4) the duration of night shifts. PART 2 Cardinal Manifestations and Presentation of Diseases

1	Jet Lag Disorder Each year, more than 60 million people fly from one time zone to another, often resulting in excessive daytime sleepiness, sleep-onset insomnia, and frequent arousals from sleep, particularly in the latter half of the night. The syndrome is transient, typically lasting 2–14 d depending on the number of time zones crossed, the direction of travel, and the traveler’s age and phase-shifting capacity. Travelers who spend more time outdoors at their destination reportedly adapt more quickly than those who remain in hotel rooms, presumably due to brighter (outdoor) light exposure. Avoidance of antecedent sleep loss and obtaining naps on the afternoon prior to overnight travel can reduce the difficulties associated with extended wakefulness. Laboratory studies suggest that low doses of melatonin can enhance sleep efficiency, but only if taken when endogenous melatonin concentrations are low (i.e., during the biologic daytime).

1	In addition to jet lag associated with travel across time zones, many patients report a behavioral pattern that has been termed social jet lag, in which bedtimes and wake times on weekends or days off occur 4–8 h later than during the week. Such recurrent displacement of the timing of the sleep-wake cycle is common in adolescents and young adults and is associated with sleep-onset insomnia, poorer academic performance, increased risk of depressive symptoms, and excessive daytime sleepiness.

1	Prominent circadian variations have been reported in the incidence of acute myocardial infarction, sudden cardiac death, and stroke, the leading causes of death in the United States. Platelet aggregability is increased in the early morning hours, coincident with the peak incidence of these cardiovascular events. Recurrent circadian disruption combined with chronic sleep deficiency, such as occurs during night-shift work, is associated with increased plasma glucose concentrations after a meal due to inadequate pancreatic insulin secretion. Night shift workers with elevated fasting glucose have an increased risk of progressing to diabetes. Blood pressure of night workers with sleep apnea is higher than that of day workers. A better understanding of the possible role of circadian rhythmicity in the acute destabilization of a chronic condition such as atherosclerotic disease could improve the understanding of its pathophysiology.

1	Diagnostic and therapeutic procedures may also be affected by the time of day at which data are collected. Examples include blood pressure, body temperature, the dexamethasone suppression test, and plasma cortisol levels. The timing of chemotherapy administration has been reported to have an effect on the outcome of treatment. In addition, both the toxicity and effectiveness of drugs can vary with time of day. For example, more than a fivefold difference has been observed in mortality rates following administration of toxic agents to experimental animals at different times of day. Anesthetic agents are particularly sensitive to time-of-day effects. Finally, the physician must be aware of the public health risks associated with the ever-increasing demands made by the 24/7 schedules in our round-the-clock society. John W. Winkelman, MD, PhD and Gary S. Richardson, MD contributed to this chapter in the prior edition and some material from that chapter has been retained here.

1	John W. Winkelman, MD, PhD and Gary S. Richardson, MD contributed to this chapter in the prior edition and some material from that chapter has been retained here. VIDEO 38-1 A typical episode of severe cataplexy. The patient is joking and then falls to the ground with an abrupt loss of muscle tone. The electromyogram recordings (four lower traces on the right) show reductions in muscle activity during the period of paralysis. The electroencephalogram (top two traces) shows wakefulness throughout the episode. (Video courtesy of Giuseppe Plazzi, University of Bologna.) VIDEO 38-2 Typical aggressive movements in rapid eye movement (REM) sleep behavior disorder. (Video courtesy of Dr. Carlos Schenck, University of Minnesota Medical School.)

1	Disorders of the Eye Jonathan C. Horton THE HuMAN VISuAL SYSTEM The visual system provides a supremely efficient means for the rapid assimilation of information from the environment to aid in the guid-ance of behavior. The act of seeing begins with the capture of images focused by the cornea and lens on a light-sensitive membrane in the back of the eye called the retina. The retina is actually part of the brain, banished to the periphery to serve as a transducer for the conversion of patterns of light energy into neuronal signals. Light is absorbed by pigment in two types of photoreceptors: rods and cones. In the human retina there are 100 million rods and 5 million cones. The rods oper-ate in dim (scotopic) illumination. The cones function under daylight (photopic) conditions. The cone system is specialized for color percep-tion and high spatial resolution. The majority of cones are within the macula, the portion of the retina that serves the central 10° of vision. In the middle of

1	system is specialized for color percep-tion and high spatial resolution. The majority of cones are within the macula, the portion of the retina that serves the central 10° of vision. In the middle of the macula a small pit termed the fovea, packed exclu-sively with cones, provides the best visual acuity. Photoreceptors hyperpolarize in response to light, activating bipo-lar, amacrine, and horizontal cells in the inner nuclear layer. After processing of photoreceptor responses by this complex retinal circuit, the flow of sensory information ultimately converges on a final com-mon pathway: the ganglion cells. These cells translate the visual image impinging on the retina into a continuously varying barrage of action potentials that propagates along the primary optic pathway to visual centers within the brain. There are a million ganglion cells in each retina and hence a million fibers in each optic nerve. Ganglion cell axons sweep along the inner surface of the retina in the nerve fiber

1	within the brain. There are a million ganglion cells in each retina and hence a million fibers in each optic nerve. Ganglion cell axons sweep along the inner surface of the retina in the nerve fiber layer, exit the eye at the optic disc, and travel through the optic nerve, optic chiasm, and optic tract to reach targets in the brain. The majority of fibers synapse on cells in the lateral geniculate body, a thalamic relay station. Cells in the lateral geniculate body project in turn to the primary visual cortex. This afferent retinoge-niculocortical sensory pathway provides the neural substrate for visual perception. Although the lateral geniculate body is the main target of the retina, separate classes of ganglion cells project to other subcorti-cal visual nuclei involved in different functions. Ganglion cells that mediate pupillary constriction and circadian rhythms are light sensi-tive owing to a novel visual pigment, melanopsin. Pupil responses are mediated by input to the pretectal

1	Ganglion cells that mediate pupillary constriction and circadian rhythms are light sensi-tive owing to a novel visual pigment, melanopsin. Pupil responses are mediated by input to the pretectal olivary nuclei in the midbrain. The pretectal nuclei send their output to the Edinger-Westphal nuclei, which in turn provide parasympathetic innervation to the iris sphinc-ter via an interneuron in the ciliary ganglion. Circadian rhythms are timed by a retinal projection to the suprachiasmatic nucleus. Visual orientation and eye movements are served by retinal input to the supe-rior colliculus. Gaze stabilization and optokinetic reflexes are governed by a group of small retinal targets known collectively as the brainstem accessory optic system. The eyes must be rotated constantly within their orbits to place and maintain targets of visual interest on the fovea. This activity, called foveation, or looking, is governed by an elaborate efferent motor sys-tem. Each eye is moved by six extraocular

1	to place and maintain targets of visual interest on the fovea. This activity, called foveation, or looking, is governed by an elaborate efferent motor sys-tem. Each eye is moved by six extraocular muscles that are supplied by cranial nerves from the oculomotor (III), trochlear (IV), and abducens (VI) nuclei. Activity in these ocular motor nuclei is coordinated by pontine and midbrain mechanisms for smooth pursuit, saccades, and gaze stabilization during head and body movements. Large regions of the frontal and parietooccipital cortex control these brainstem eye movement centers by providing descending supranuclear input. 39 SECTion 4

1	DiSoRDERS of EyES, EARS, noSE, AnD THRoAT In approaching a patient with reduced vision, the first step is to decide whether refractive error is responsible. In emmetropia, parallel rays from infinity are focused perfectly on the retina. Sadly, this condition is enjoyed by only a minority of the population. In myopia, the globe is too long, and light rays come to a focal point in front of the retina. Near objects can be seen clearly, but distant objects require a diverging lens in front of the eye. In hyperopia, the globe is too short, and hence a converging lens is used to supplement the refractive power of the eye. In astigmatism, the corneal surface is not perfectly spherical, necessitating a cylindrical corrective lens. As an alternative to eyeglasses or contact lenses, refractive error can be corrected by performing laser in situ keratomileusis (LASIK) or photorefractive keratectomy (PRK) to alter the curvature of the cornea.

1	With the onset of middle age, presbyopia develops as the lens within the eye becomes unable to increase its refractive power to accommodate on near objects. To compensate for presbyopia an emmetropic patient must use reading glasses. A patient already wearing glasses for distance correction usually switches to bifocals. The only exception is a myopic patient, who may achieve clear vision at near simply by removing glasses containing the distance prescription. Refractive errors usually develop slowly and remain stable after adolescence, except in unusual circumstances. For example, the acute onset of diabetes mellitus can produce sudden myopia because of lens edema induced by hyperglycemia. Testing vision through a pinhole aperture is a useful way to screen quickly for refractive error. If visual acuity is better through a pinhole than it is with the unaided eye, the patient needs refraction to obtain best corrected visual acuity.

1	The Snellen chart is used to test acuity at a distance of 6 m (20 ft). For convenience, a scale version of the Snellen chart called the Rosenbaum card is held at 36 cm (14 in.) from the patient (Fig. 39-1). All subjects should be able to read the 6/6 m (20/20 ft) line with each eye using their refractive correction, if any. Patients who need reading glasses because of presbyopia must wear them for accurate testing with the Rosenbaum card. If 6/6 (20/20) acuity is not present in each eye, the deficiency in vision must be explained. If it is worse than 6/240 (20/800), acuity should be recorded in terms of counting fingers, hand motions, light perception, or no light perception. Legal blindness is defined by the Internal Revenue Service as a best corrected acuity of 6/60 (20/200) or less in the better eye or a binocular visual field subtending 20° or less. For driving the laws vary by state, but most states require a corrected acuity of 6/12 (20/40) in at least one eye for unrestricted

1	the better eye or a binocular visual field subtending 20° or less. For driving the laws vary by state, but most states require a corrected acuity of 6/12 (20/40) in at least one eye for unrestricted privileges. Patients with a homonymous hemianopia should not drive.

1	The pupils should be tested individually in dim light with the patient fixating on a distant target. There is no need to check the near response if the pupils respond briskly to light, because isolated loss of constriction (miosis) to accommodation does not occur. For this reason, the ubiquitous abbreviation PERRLA (pupils equal, round, and reactive to light and accommodation) implies a wasted effort with the last step. However, it is important to test the near response if the light response is poor or absent. Light-near dissociation occurs with neurosyphilis (Argyll Robertson pupil), with lesions of the dorsal midbrain (Parinaud’s syndrome), and after aberrant regeneration (oculomotor nerve palsy, Adie’s tonic pupil). CHAPTER 39 Disorders of the Eye PART 2 Cardinal Manifestations and Presentation of Diseases

1	CHAPTER 39 Disorders of the Eye PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 39-1 The Rosenbaum card is a miniature, scale version of the Snellen chart for testing visual acuity at near. When the visual acuity is recorded, the Snellen distance equivalent should bear a nota-tion indicating that vision was tested at near, not at 6 m (20 ft), or else the Jaeger number system should be used to report the acuity.

1	An eye with no light perception has no pupillary response to direct light stimulation. If the retina or optic nerve is only partially injured, the direct pupillary response will be weaker than the consensual pupillary response evoked by shining a light into the healthy fellow eye. A relative afferent pupillary defect (Marcus Gunn pupil) can be elicited with the swinging flashlight test (Fig. 39-2). It is an extremely useful sign in retrobulbar optic neuritis and other optic nerve diseases, in which it may be the sole objective evidence for disease. In bilateral optic neuropathy, no afferent pupil defect is present if the optic nerves are affected equally.

1	Subtle inequality in pupil size, up to 0.5 mm, is a fairly common finding in normal persons. The diagnosis of essential or physiologic anisocoria is secure as long as the relative pupil asymmetry remains constant as ambient lighting varies. Anisocoria that increases in dim light indicates a sympathetic paresis of the iris dilator muscle. The triad of miosis with ipsilateral ptosis and anhidrosis constitutes Horner’s syndrome, although anhidrosis is an inconstant feature. Brainstem stroke, carotid dissection, and neoplasm impinging on the sympathetic chain occasionally are identified as the cause of Horner’s syndrome, but most cases are idiopathic. Anisocoria that increases in bright light suggests a parasympathetic palsy. The first concern is an oculomotor nerve paresis. This possibility is excluded if the eye movements are full and the patient has no ptosis or diplopia. Acute pupillary dilation (mydriasis) can result from

1	FIguRE 39-2 Demonstration of a relative afferent pupil defect (Marcus Gunn pupil) in the left eye, done with the patient fixating on a distant target. A. With dim background lighting, the pupils are equal and relatively large. B. Shining a flashlight into the right eye evokes equal, strong constriction of both pupils. C. Swinging the flashlight over to the damaged left eye causes dilation of both pupils, although they remain smaller than in A. Swinging the flashlight back over to the healthy right eye would result in symmetric constriction back to the appearance shown in B. Note that the pupils always remain equal; the damage to the left retina/optic nerve is revealed by weaker bilateral pupil constriction to a flashlight in the left eye compared with the right eye. (From P Levatin: Arch Ophthalmol 62:768, 1959. Copyright © 1959 American Medical Association. All rights reserved.) damage to the ciliary ganglion in the orbit. Common mechanisms are infection (herpes zoster, influenza),

1	Ophthalmol 62:768, 1959. Copyright © 1959 American Medical Association. All rights reserved.) damage to the ciliary ganglion in the orbit. Common mechanisms are infection (herpes zoster, influenza), trauma (blunt, penetrating, surgical), and ischemia (diabetes, temporal arteritis). After denervation of the iris sphincter the pupil does not respond well to light, but the response to near is often relatively intact. When the near stimulus is removed, the pupil redilates very slowly compared with the normal pupil, hence the term tonic pupil. In Adie’s syndrome a tonic pupil is present, sometimes in conjunction with weak or absent tendon reflexes in the lower extremities. This benign disorder, which occurs predominantly in healthy young women, is assumed to represent a mild dysautonomia. Tonic pupils are also associated with Shy-Drager syndrome, segmental hypohidrosis, diabetes, and amyloidosis. Occasionally, a tonic pupil is discovered incidentally in an otherwise completely normal,

1	Tonic pupils are also associated with Shy-Drager syndrome, segmental hypohidrosis, diabetes, and amyloidosis. Occasionally, a tonic pupil is discovered incidentally in an otherwise completely normal, asymptomatic individual. The diagnosis is confirmed by placing a drop of dilute (0.125%) pilocarpine into each eye. Denervation hypersensitivity produces pupillary constriction in a tonic pupil, whereas the normal pupil shows no response. Pharmacologic dilatation from accidental or deliberate instillation of anticholinergic agents (atropine, scopolamine drops) into the eye also can produce pupillary mydriasis. In this situation, normal strength (1%) pilocarpine causes no constriction.

1	Both pupils are affected equally by systemic medications. They are small with narcotic use (morphine, heroin) and large with anticholinergics (scopolamine). Parasympathetic agents (pilocarpine, demecarium bromide) used to treat glaucoma produce miosis. In any patient with an unexplained pupillary abnormality, a slit-lamp examination is helpful to exclude surgical trauma to the iris, an occult foreign body, perforating injury, intraocular inflammation, adhesions (synechia), angle-closure glaucoma, and iris sphincter rupture from blunt trauma.

1	Eye movements are tested by asking the patient, with both eyes open, to pursue a small target such as a penlight into the cardinal fields of gaze. Normal ocular versions are smooth, symmetric, full, and maintained in all directions without nystagmus. Saccades, or quick refixation eye movements, are assessed by having the patient look back and forth between two stationary targets. The eyes should move rapidly and accurately in a single jump to their target. Ocular alignment can be judged by holding a penlight directly in front of the patient at about 1 m. If the eyes are straight, the corneal light reflex will be centered in the middle of each pupil. To test eye alignment more precisely, the cover test is useful. The patient is instructed to look at a small fixation target in the distance. One eye is covered suddenly while the second eye is observed. If the second eye shifts to fixate on the target, it was misaligned. If it does not move, the first eye is uncovered and the test is

1	One eye is covered suddenly while the second eye is observed. If the second eye shifts to fixate on the target, it was misaligned. If it does not move, the first eye is uncovered and the test is repeated on the second eye. If neither eye moves the eyes are aligned orthotropically. If the eyes are orthotropic in primary gaze but the patient complains of diplopia, the cover test should be performed with the head tilted or turned in whatever direction elicits diplopia. With practice, the examiner can detect an ocular deviation (heterotropia) as small as 1–2° with the cover test. In a patient with vertical diplopia, a small deviation can be difficult to detect and easy to dismiss. The magnitude of the deviation can be measured by placing a prism in front of the misaligned eye to determine the power required to neutralize the fixation shift evoked by covering the other eye. Temporary press-on plastic Fresnel prisms, prism eyeglasses, or eye muscle surgery can be used to restore binocular

1	power required to neutralize the fixation shift evoked by covering the other eye. Temporary press-on plastic Fresnel prisms, prism eyeglasses, or eye muscle surgery can be used to restore binocular alignment.

1	Stereoacuity is determined by presenting targets with retinal disparity separately to each eye by using polarized images. The most popular office tests measure a range of thresholds from 800–40 seconds of arc. Normal stereoacuity is 40 seconds of arc. If a patient achieves this level of stereoacuity, one is assured that the eyes are aligned orthotropically and that vision is intact in each eye. Random dot stereograms have no monocular depth cues and provide an excellent screening test for strabismus and amblyopia in children.

1	The retina contains three classes of cones, with visual pigments of differing peak spectral sensitivity: red (560 nm), green (530 nm), and blue (430 nm). The red and green cone pigments are encoded on the X chromosome, and the blue cone pigment on chromosome 7. Mutations of the blue cone pigment are exceedingly rare. Mutations of the red and green pigments cause congenital X-linked color blindness in 8% of males. Affected individuals are not truly color blind; rather, they differ from normal subjects in the way they perceive color and how they combine primary monochromatic lights to match a particular color. Anomalous trichromats have three cone types, but a mutation in one cone pigment (usually red or green) causes a shift in peak spectral sensitivity, altering the proportion of primary colors required to achieve a color match. Dichromats have only two cone types and therefore will accept a color match based on only two primary colors. 197 Anomalous trichromats and dichromats have

1	colors required to achieve a color match. Dichromats have only two cone types and therefore will accept a color match based on only two primary colors. 197 Anomalous trichromats and dichromats have 6/6 (20/20) visual acuity, but their hue discrimination is impaired. Ishihara color plates can be used to detect red-green color blindness. The test plates contain a hidden number that is visible only to subjects with color confusion from red-green color blindness. Because color blindness is almost exclusively X-linked, it is worth screening only male children.

1	The Ishihara plates often are used to detect acquired defects in color vision, although they are intended as a screening test for congenital color blindness. Acquired defects in color vision frequently result from disease of the macula or optic nerve. For example, patients with a history of optic neuritis often complain of color desaturation long after their visual acuity has returned to normal. Color blindness also can result from bilateral strokes involving the ventral portion of the occipital lobe (cerebral achromatopsia). Such patients can perceive only shades of gray and also may have difficulty recognizing faces (prosopagnosia). Infarcts of the dominant occipital lobe sometimes give rise to color anomia. Affected patients can discriminate colors but cannot name them.

1	Vision can be impaired by damage to the visual system anywhere from the eyes to the occipital lobes. One can localize the site of the lesion with considerable accuracy by mapping the visual field deficit by finger confrontation and then correlating it with the topographic anatomy of the visual pathway (Fig. 39-3). Quantitative visual field mapping is performed by computer-driven perimeters that present a target of variable intensity at fixed positions in the visual field (Fig. 39-3A). By generating an automated printout of light thresholds, these static perimeters provide a sensitive means of detecting scotomas in the visual field. They are exceedingly useful for serial assessment of visual function in chronic diseases such as glaucoma and pseudotumor cerebri.

1	The crux of visual field analysis is to decide whether a lesion is before, at, or behind the optic chiasm. If a scotoma is confined to one eye, it must be due to a lesion anterior to the chiasm, involving either the optic nerve or the retina. Retinal lesions produce scotomas that correspond optically to their location in the fundus. For example, a superior-nasal retinal detachment results in an inferior-temporal field cut. Damage to the macula causes a central scotoma (Fig. 39-3B).

1	Optic nerve disease produces characteristic patterns of visual field loss. Glaucoma selectively destroys axons that enter the superotemporal or inferotemporal poles of the optic disc, resulting in arcuate scotomas shaped like a Turkish scimitar, which emanate from the blind spot and curve around fixation to end flat against the horizontal meridian (Fig. 39-3C). This type of field defect mirrors the arrangement of the nerve fiber layer in the temporal retina. Arcuate or nerve fiber layer scotomas also result from optic neuritis, ischemic optic neuropathy, optic disc drusen, and branch retinal artery or vein occlusion. Damage to the entire upper or lower pole of the optic disc causes an altitudinal field cut that follows the horizontal meridian (Fig. 39-3D). This pattern of visual field loss is typical of ischemic optic neuropathy but also results from retinal vascular occlusion, advanced glaucoma, and optic neuritis.

1	About half the fibers in the optic nerve originate from ganglion cells serving the macula. Damage to papillomacular fibers causes a cecocentral scotoma that encompasses the blind spot and macula (Fig. 39-3E). If the damage is irreversible, pallor eventually appears in the temporal portion of the optic disc. Temporal pallor from a cecocentral scotoma may develop in optic neuritis, nutritional optic neuropathy, toxic optic neuropathy, Leber’s hereditary optic neuropathy, Kjer’s dominant optic atrophy, and compressive optic neuropathy. It is worth mentioning that the temporal side of the optic disc is slightly paler than the nasal side in most normal individuals. Therefore, it sometimes can be difficult to decide whether the temporal pallor visible on fundus examination represents a pathologic change. Pallor of the nasal rim of the optic disc is a less equivocal sign of optic atrophy.

1	At the optic chiasm, fibers from nasal ganglion cells decussate into the contralateral optic tract. Crossed fibers are damaged more by CHAPTER 39 Disorders of the Eye Monocular prechiasmal field defects: PART 2 Cardinal Manifestations and Presentation of Diseases Normal field Central scotoma Nerve-fiber bundle Altitudinal Cecocentral Enlarged blind-spot right eye (arcuate) scotoma scotoma scotoma with peripheral constriction Binocular chiasmal or postchiasmal field defects: Homonymous hemianopia with macular sparing

1	Binocular chiasmal or postchiasmal field defects: Homonymous hemianopia with macular sparing FIguRE 39-3 Ventral view of the brain, correlating patterns of visual field loss with the sites of lesions in the visual pathway. The visual fields overlap partially, creating 120° of central binocular field flanked by a 40° monocular crescent on either side. The visual field maps in this figure were done with a computer-driven perimeter (Humphrey Instruments, Carl Zeiss, Inc.). It plots the retinal sensitivity to light in the central 30° by using a gray scale format. Areas of visual field loss are shown in black. The examples of common monocular, prechiasmal field defects are all shown for the right eye. By convention, the visual fields are always recorded with the left eye’s field on the left and the right eye’s field on the right, just as the patient sees the world.

1	compression than are uncrossed fibers. As a result, mass lesions of The insidious development of a bitemporal hemianopia often goes the sellar region cause a temporal hemianopia in each eye. Tumors unnoticed by the patient and will escape detection by the physician anterior to the optic chiasm, such as meningiomas of the tuberculum unless each eye is tested separately. sella, produce a junctional scotoma characterized by an optic neu-It is difficult to localize a postchiasmal lesion accurately, because ropathy in one eye and a superior-temporal field cut in the other eye injury anywhere in the optic tract, lateral geniculate body, optic radia(Fig. 39-3G). More symmetric compression of the optic chiasm by a tions, or visual cortex can produce a homonymous hemianopia (i.e., pituitary adenoma (see Fig. 403-1), meningioma, craniopharyngioma, a temporal hemifield defect in the contralateral eye and a matching glioma, or aneurysm results in a bitemporal hemianopia (Fig. 39-3H). nasal

1	adenoma (see Fig. 403-1), meningioma, craniopharyngioma, a temporal hemifield defect in the contralateral eye and a matching glioma, or aneurysm results in a bitemporal hemianopia (Fig. 39-3H). nasal hemifield defect in the ipsilateral eye) (Fig. 39-3I). A unilateral postchiasmal lesion leaves the visual acuity in each eye unaffected, although the patient may read the letters on only the left or right half of the eye chart. Lesions of the optic radiations tend to cause poorly matched or incongruous field defects in each eye. Damage to the optic radiations in the temporal lobe (Meyer’s loop) produces a superior quadrantic homonymous hemianopia (Fig. 39-3J), whereas injury to the optic radiations in the parietal lobe results in an inferior quadrantic homonymous hemianopia (Fig. 39-3K). Lesions of the primary visual cortex give rise to dense, congruous hemianopic field defects. Occlusion of the posterior cerebral artery supplying the occipital lobe is a common cause of total homonymous

1	of the primary visual cortex give rise to dense, congruous hemianopic field defects. Occlusion of the posterior cerebral artery supplying the occipital lobe is a common cause of total homonymous hemianopia. Some patients with hemianopia after occipital stroke have macular sparing, because the macular representation at the tip of the occipital lobe is supplied by collaterals from the middle cerebral artery (Fig. 39-3L). Destruction of both occipital lobes produces cortical blindness. This condition can be distinguished from bilateral prechiasmal visual loss by noting that the pupil responses and optic fundi remain normal.

1	RED OR PAINFuL EYE Corneal Abrasions Corneal abrasions are seen best by placing a drop of fluorescein in the eye and looking with the slit lamp, using a cobalt-blue light. A penlight with a blue filter will suffice if a slit lamp is not available. Damage to the corneal epithelium is revealed by yellow fluorescence of the exposed basement membrane underlying the epithelium. It is important to check for foreign bodies. To search the conjunctival fornices, the lower lid should be pulled down and the upper lid everted. A foreign body can be removed with a moistened cotton-tipped applicator after a drop of a topical anesthetic such as proparacaine has been placed in the eye. Alternatively, it may be possible to flush the foreign body from the eye by irrigating copiously with saline or artificial tears. If the corneal epithelium has been abraded, antibiotic ointment and a patch should be applied to the eye. A drop of an intermediate-acting cycloplegic such as cyclopentolate hydrochloride 1%

1	tears. If the corneal epithelium has been abraded, antibiotic ointment and a patch should be applied to the eye. A drop of an intermediate-acting cycloplegic such as cyclopentolate hydrochloride 1% helps reduce pain by relaxing the ciliary body. The eye should be reexamined the next day. Minor abrasions may not require patching, antibiotics, or cycloplegia.

1	Subconjunctival Hemorrhage This results from rupture of small vessels bridging the potential space between the episclera and the conjunctiva. Blood dissecting into this space can produce a spectacular red eye, but vision is not affected and the hemorrhage resolves without treatment. Subconjunctival hemorrhage is usually spontaneous but can result from blunt trauma, eye rubbing, or vigorous coughing. Occasionally it is a clue to an underlying bleeding disorder. Pinguecula Pinguecula is a small, raised conjunctival nodule at the temporal or nasal limbus. In adults such lesions are extremely common and have little significance unless they become inflamed (pingueculitis). They are more apt to occur in workers with frequent outdoor exposure. A pterygium resembles a pinguecula but has crossed the limbus to encroach on the corneal surface. Removal is justified when symptoms of irritation or blurring develop, but recurrence is a common problem.

1	Blepharitis This refers to inflammation of the eyelids. The most common form occurs in association with acne rosacea or seborrheic dermatitis. The eyelid margins usually are colonized heavily by staphylococci. Upon close inspection, they appear greasy, ulcerated, and crusted with scaling debris that clings to the lashes. Treatment consists of strict eyelid hygiene, using warm compresses and eyelash scrubs with baby shampoo. An external hordeolum (sty) is caused by staphylococcal infection of the superficial accessory glands of Zeis or Moll located in the eyelid margins. An internal hordeolum occurs after suppurative infection of the oil-secreting meibomian glands within the tarsal plate of the eyelid. Topical antibiotics such as bacitracin/ polymyxin B ophthalmic ointment can be applied. Systemic antibiotics, usually tetracyclines or azithromycin, sometimes are necessary for treatment of meibomian gland inflammation (meibomitis) or chronic, severe blepharitis. A chalazion is a

1	Systemic antibiotics, usually tetracyclines or azithromycin, sometimes are necessary for treatment of meibomian gland inflammation (meibomitis) or chronic, severe blepharitis. A chalazion is a painless, chronic granulomatous inflammation of a meibomian gland that produces a pealike nodule 199 within the eyelid. It can be incised and drained or injected with glucocorticoids. Basal cell, squamous cell, or meibomian gland carcinoma should be suspected with any nonhealing ulcerative lesion of the eyelids.

1	Dacryocystitis An inflammation of the lacrimal drainage system, dacryocystitis can produce epiphora (tearing) and ocular injection. Gentle pressure over the lacrimal sac evokes pain and reflux of mucus or pus from the tear puncta. Dacryocystitis usually occurs after obstruction of the lacrimal system. It is treated with topical and systemic antibiotics, followed by probing, silicone stent intubation, or surgery to reestablish patency. Entropion (inversion of the eyelid) or ectropion (sagging or eversion of the eyelid) can also lead to epiphora and ocular irritation.

1	Conjunctivitis Conjunctivitis is the most common cause of a red, irritated eye. Pain is minimal, and visual acuity is reduced only slightly. The most common viral etiology is adenovirus infection. It causes a watery discharge, a mild foreign-body sensation, and photophobia. Bacterial infection tends to produce a more mucopurulent exudate. Mild cases of infectious conjunctivitis usually are treated empirically with broad-spectrum topical ocular antibiotics such as sulfacetamide 10%, polymyxin-bacitracin, or a trimethoprim-polymyxin combination. Smears and cultures usually are reserved for severe, resistant, or recurrent cases of conjunctivitis. To prevent contagion, patients should be admonished to wash their hands frequently, not to touch their eyes, and to avoid direct contact with others.

1	Allergic Conjunctivitis This condition is extremely common and often is mistaken for infectious conjunctivitis. Itching, redness, and epiphora are typical. The palpebral conjunctiva may become hypertropic with giant excrescences called cobblestone papillae. Irritation from contact lenses or any chronic foreign body also can induce formation of cobblestone papillae. Atopic conjunctivitis occurs in subjects with atopic dermatitis or asthma. Symptoms caused by allergic conjunctivitis can be alleviated with cold compresses, topical vasoconstrictors, antihistamines, and mast cell stabilizers such as cromolyn sodium. Topical glucocorticoid solutions provide dramatic relief of immune-mediated forms of conjunctivitis, but their long-term use is ill advised because of the complications of glaucoma, cataract, and secondary infection. Topical nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., ketorolac tromethamine) are better alternatives.

1	Keratoconjunctivitis Sicca Also known as dry eye, this produces a burning foreign-body sensation, injection, and photophobia. In mild cases the eye appears surprisingly normal, but tear production measured by wetting of a filter paper (Schirmer strip) is deficient. A variety of systemic drugs, including antihistaminic, anticholinergic, and psychotropic medications, result in dry eye by reducing lacrimal secretion. Disorders that involve the lacrimal gland directly, such as sarcoidosis and Sjögren’s syndrome, also cause dry eye. Patients may develop dry eye after radiation therapy if the treatment field includes the orbits. Problems with ocular drying are also common after lesions affecting cranial nerve V or VII. Corneal anesthesia is particularly dangerous, because the absence of a normal blink reflex exposes the cornea to injury without pain to warn the patient. Dry eye is managed by frequent and liberal application of artificial tears and ocular lubricants. In severe cases the tear

1	blink reflex exposes the cornea to injury without pain to warn the patient. Dry eye is managed by frequent and liberal application of artificial tears and ocular lubricants. In severe cases the tear puncta can be plugged or cauterized to reduce lacrimal outflow.

1	Keratitis Keratitis is a threat to vision because of the risk of corneal clouding, scarring, and perforation. Worldwide, the two leading causes of blindness from keratitis are trachoma from chlamydial infection and vitamin A deficiency related to malnutrition. In the United States, contact lenses play a major role in corneal infection and ulceration. They should not be worn by anyone with an active eye infection. In evaluating the cornea, it is important to differentiate between a superficial infection (keratoconjunctivitis) and a deeper, more serious ulcerative process. The latter is accompanied by greater visual loss, pain, photo-phobia, redness, and discharge. Slit-lamp examination shows disruption of the corneal epithelium, a cloudy infiltrate or abscess in the

1	CHAPTER 39 Disorders of the Eye 200 stroma, and an inflammatory cellular reaction in the anterior chamber. In severe cases, pus settles at the bottom of the anterior chamber, giving rise to a hypopyon. Immediate empirical antibiotic therapy should be initiated after corneal scrapings are obtained for Gram’s stain, Giemsa stain, and cultures. Fortified topical antibiotics are most effective, supplemented with subconjunctival antibiotics as required. A fungal etiology should always be considered in a patient with keratitis. Fungal infection is common in warm humid climates, especially after penetration of the cornea by plant or vegetable material.

1	Herpes Simplex The herpesviruses are a major cause of blindness from keratitis. Most adults in the United States have serum antibodies to herpes simplex, indicating prior viral infection (Chap. 216). Primary ocular infection generally is caused by herpes simplex type 1 rather than type 2. It manifests as a unilateral follicular blepharoconjunctivitis that is easily confused with adenoviral conjunctivitis unless telltale vesicles appear on the periocular skin or conjunctiva. A dendritic pattern of corneal epithelial ulceration revealed by fluorescein staining is pathognomonic for herpes infection but is seen in only a minority of primary infections. Recurrent ocular infection arises from reactivation of the latent herpesvirus. Viral eruption in the corneal epithelium may result in the characteristic herpes dendrite. Involvement of the corneal stroma produces edema, vascularization, and iridocyclitis. Herpes keratitis is treated with topical antiviral agents, cycloplegics, and oral

1	characteristic herpes dendrite. Involvement of the corneal stroma produces edema, vascularization, and iridocyclitis. Herpes keratitis is treated with topical antiviral agents, cycloplegics, and oral acyclovir. Topical glucocorticoids are effective in mitigating corneal scarring but must be used with extreme caution because of the danger of corneal melting and perforation. Topical glucocorticoids also carry the risk of prolonging infection and inducing glaucoma.

1	Herpes Zoster Herpes zoster from reactivation of latent varicella (chickenpox) virus causes a dermatomal pattern of painful vesicular dermatitis. Ocular symptoms can occur after zoster eruption in any branch of the trigeminal nerve but are particularly common when vesicles form on the nose, reflecting nasociliary (V1) nerve involvement (Hutchinson’s sign). Herpes zoster ophthalmicus produces corneal dendrites, which can be difficult to distinguish from those seen in herpes simplex. Stromal keratitis, anterior uveitis, raised intra-ocular pressure, ocular motor nerve palsies, acute retinal necrosis, and postherpetic scarring and neuralgia are other common sequelae. Herpes zoster ophthalmicus is treated with antiviral agents and cycloplegics. In severe cases, glucocorticoids may be added to prevent permanent visual loss from corneal scarring.

1	Episcleritis This is an inflammation of the episclera, a thin layer of connective tissue between the conjunctiva and the sclera. Episcleritis resembles conjunctivitis, but it is a more localized process and discharge is absent. Most cases of episcleritis are idiopathic, but some occur in the setting of an autoimmune disease. Scleritis refers to a deeper, more severe inflammatory process that frequently is associated with a connective tissue disease such as rheumatoid arthritis, lupus erythematosus, polyarteritis nodosa, granulomatosis with polyangiitis (Wegener’s), or relapsing polychondritis. The inflammation and thickening of the sclera can be diffuse or nodular. In anterior forms of scleritis, the globe assumes a violet hue and the patient complains of severe ocular tenderness and pain. With posterior scleritis, the pain and redness may be less marked, but there is often proptosis, choroidal effusion, reduced motility, and visual loss. Episcleritis and scleritis should be treated

1	With posterior scleritis, the pain and redness may be less marked, but there is often proptosis, choroidal effusion, reduced motility, and visual loss. Episcleritis and scleritis should be treated with NSAIDs. If these agents fail, topical or even systemic glucocorticoid therapy may be necessary, especially if an underlying autoimmune process is active.

1	uveitis Involving the anterior structures of the eye, uveitis also is called iritis or iridocyclitis. The diagnosis requires slit-lamp examination to identify inflammatory cells floating in the aqueous humor or deposited on the corneal endothelium (keratic precipitates). Anterior uveitis develops in sarcoidosis, ankylosing spondylitis, juvenile rheumatoid arthritis, inflammatory bowel disease, psoriasis, reactive arthritis, and Behçet’s disease. It also is associated with herpes infections, syphilis, Lyme disease, onchocerciasis, tuberculosis, and leprosy. Although anterior uveitis can occur in conjunction with many diseases, no cause is found to explain the majority of cases. For this reason,

1	PART 2 Cardinal Manifestations and Presentation of Diseases laboratory evaluation usually is reserved for patients with recurrent or severe anterior uveitis. Treatment is aimed at reducing inflammation and scarring by judicious use of topical glucocorticoids. Dilatation of the pupil reduces pain and prevents the formation of synechiae.

1	Posterior uveitis This is diagnosed by observing inflammation of the vitreous, retina, or choroid on fundus examination. It is more likely than anterior uveitis to be associated with an identifiable systemic disease. Some patients have panuveitis, or inflammation of both the anterior and posterior segments of the eye. Posterior uveitis is a manifestation of autoimmune diseases such as sarcoidosis, Behçet’s disease, Vogt-Koyanagi-Harada syndrome, and inflammatory bowel disease. It also accompanies diseases such as toxoplasmosis, onchocerciasis, cysticercosis, coccidioidomycosis, toxocariasis, and histoplasmosis; infections caused by organisms such as Candida, Pneumocystis carinii, Cryptococcus, Aspergillus, herpes, and cytomegalovirus (see Fig. 219-1); and other diseases, such as syphilis, Lyme disease, tuberculosis, cat-scratch disease, Whipple’s disease, and brucellosis. In multiple sclerosis, chronic inflammatory changes can develop in the extreme periphery of the retina (pars

1	Lyme disease, tuberculosis, cat-scratch disease, Whipple’s disease, and brucellosis. In multiple sclerosis, chronic inflammatory changes can develop in the extreme periphery of the retina (pars planitis or intermediate uveitis).

1	Acute Angle-Closure glaucoma This is an unusual but frequently misdiagnosed cause of a red, painful eye. Asian populations have a particularly high risk of angle-closure glaucoma. Susceptible eyes have a shallow anterior chamber because the eye has either a short axial length (hyperopia) or a lens enlarged by the gradual development of cataract. When the pupil becomes mid-dilated, the peripheral iris blocks aqueous outflow via the anterior chamber angle and the intraocular pressure rises abruptly, producing pain, injection, corneal edema, obscurations, and blurred vision. In some patients, ocular symptoms are overshadowed by nausea, vomiting, or headache, prompting a fruitless workup for abdominal or neurologic disease. The diagnosis is made by measuring the intraocular pressure during an acute attack or by performing gonioscopy, a procedure that allows one to observe a narrow chamber angle with a mirrored contact lens. Acute angle closure is treated with acetazolamide (PO or IV),

1	an acute attack or by performing gonioscopy, a procedure that allows one to observe a narrow chamber angle with a mirrored contact lens. Acute angle closure is treated with acetazolamide (PO or IV), topical beta blockers, prostaglandin analogues, α2-adrenergic agonists, and pilocarpine to induce miosis. If these measures fail, a laser can be used to create a hole in the peripheral iris to relieve pupillary block. Many physicians are reluctant to dilate patients routinely for fundus examination because they fear precipitating an angle-closure glaucoma. The risk is actually remote and more than outweighed by the potential benefit to patients of discovering a hidden fundus lesion visible only through a fully dilated pupil. Moreover, a single attack of angle closure after pharmacologic dilatation rarely causes any permanent damage to the eye and serves as an inadvertent provocative test to identify patients with narrow angles who would benefit from prophylactic laser iridectomy.

1	Endophthalmitis This results from bacterial, viral, fungal, or parasitic infection of the internal structures of the eye. It usually is acquired by hematogenous seeding from a remote site. Chronically ill, diabetic, or immunosuppressed patients, especially those with a history of indwelling IV catheters or positive blood cultures, are at greatest risk for endogenous endophthalmitis. Although most patients have ocular pain and injection, visual loss is sometimes the only symptom. Septic emboli from a diseased heart valve or a dental abscess that lodge in the retinal circulation can give rise to endophthalmitis. White-centered retinal hemorrhages known as Roth’s spots (Fig. 39-4) are considered pathognomonic for subacute bacterial endocarditis, but they also appear in leukemia, diabetes, and many other conditions. Endophthalmitis also occurs as a complication of ocular surgery, especially glaucoma filtering, occasionally months or even years after the operation. An occult penetrating

1	and many other conditions. Endophthalmitis also occurs as a complication of ocular surgery, especially glaucoma filtering, occasionally months or even years after the operation. An occult penetrating foreign body or unrecognized trauma to the globe should be considered in any patient with unexplained intraocular infection or inflammation.

1	TRANSIENT OR SuDDEN VISuAL LOSS Amaurosis Fugax This term refers to a transient ischemic attack of the retina (Chap. 446). Because neural tissue has a high rate of metabolism, interruption of blood flow to the retina for more than a few seconds results in transient monocular blindness, a term used interchangeably with amaurosis fugax. Patients describe a rapid fading of vision like a curtain descending, sometimes affecting only a portion of the visual field. Amaurosis fugax usually results from an embolus that becomes stuck within a retinal arteriole (Fig. 39-5). If the embolus breaks up or passes, flow is restored and vision returns quickly to normal without permanent damage. With prolonged interruption of blood flow, the inner retina suffers infarction. Ophthalmoscopy reveals zones of whitened, edematous retina following the distribution of branch retinal arterioles. Complete occlusion of the central retinal artery produces arrest of blood flow and a milky retina with a cherry-red

1	whitened, edematous retina following the distribution of branch retinal arterioles. Complete occlusion of the central retinal artery produces arrest of blood flow and a milky retina with a cherry-red fovea (Fig. 39-6). Emboli are composed of cholesterol (Hollenhorst plaque), calcium, or platelet-fibrin debris. The most common source is an atherosclerotic plaque in the carotid artery or aorta, although emboli also can arise from the heart, especially in patients with diseased valves, atrial fibrillation, or wall motion abnormalities.

1	FIguRE 39-4 Roth’s spot, cotton-wool spot, and retinal hemor-rhages in a 48-year-old liver transplant patient with candidemia from immunosuppression. FIguRE 39-6 Central retinal artery occlusion in a 78-year-old man reducing acuity to counting fingers in the right eye. Note the splinter hemorrhage on the optic disc and the slightly milky appearance to the macula with a cherry-red fovea. In rare instances, amaurosis fugax results from low central retinal artery perfusion pressure in a patient with a critical stenosis of the ipsilateral carotid artery and poor collateral flow via the circle of Willis. In this situation, amaurosis fugax develops when there is a dip in systemic blood pressure or a slight worsening of the carotid stenosis. Sometimes there is contralateral motor or sensory loss, indicating concomitant hemispheric cerebral ischemia.

1	Retinal arterial occlusion also occurs rarely in association with retinal migraine, lupus erythematosus, anticardiolipin antibodies, anticoagulant deficiency states (protein S, protein C, and antithrombin deficiency), pregnancy, IV drug abuse, blood dyscrasias, dysproteinemias, and temporal arteritis. Marked systemic hypertension causes sclerosis of retinal arterioles, splinter hemorrhages, focal infarcts of the nerve fiber layer (cottonwool spots), and leakage of lipid and fluid (hard exudate) into the macula (Fig. 39-7). In hypertensive crisis, sudden visual loss can result from vasospasm of retinal arterioles and retinal ischemia. In addition, acute hypertension may produce visual loss from ischemic swelling of the optic disc. Patients with acute hypertensive retinopathy should be treated by lowering the blood pressure. However, the blood pressure should not be reduced precipitously, because there is a danger of optic disc infarction from sudden hypoperfusion.

1	Impending branch or central retinal vein occlusion can produce prolonged visual obscurations that resemble those described by patients with amaurosis fugax. The veins appear engorged and phlebitic, with numerous retinal hemorrhages (Fig. 39-8). In some patients, venous blood flow recovers spontaneously, whereas others evolve a frank obstruction with extensive retinal bleeding (“blood and thunder” appearance), infarction, and visual loss. Venous occlusion of the retina is often idiopathic, but hypertension, diabetes, and glaucoma CHAPTER 39 Disorders of the Eye FIguRE 39-5 Hollenhorst plaque lodged at the bifurcation of a retinal arteriole proves that a patient is shedding emboli from the carotid artery, great vessels, or heart. FIguRE 39-7 Hypertensive retinopathy with blurred optic disc, scattered hemorrhages, cotton-wool spots (nerve fiber layer infarcts), and foveal exudate in a 62-year-old man with chronic renal failure and a systolic blood pressure of 220.

1	PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 39-10 Retrobulbar optic neuritis is characterized by a nor-mal fundus examination initially, hence the rubric “the doctor sees nothing, and the patient sees nothing.” Optic atrophy develops after severe or repeated attacks. rarely. It is important to biopsy an arterial segment of at least 3 cm and to examine a sufficient number of tissue sections prepared from the specimen.

1	rarely. It is important to biopsy an arterial segment of at least 3 cm and to examine a sufficient number of tissue sections prepared from the specimen. Posterior Ischemic Optic Neuropathy This is an uncommon cause of acute visual loss, induced by the combination of severe anemia and hypotension. Cases have been reported after major blood loss during surgery (especially in patients undergoing cardiac or lumbar spine operations), exsanguinating trauma, gastrointestinal bleeding, and renal dialysis. The fundus usually appears normal, although optic disc swelling develops if the process extends anteriorly far enough to reach the globe. Vision can be salvaged in some patients by prompt blood transfusion and reversal of hypotension.

1	Optic Neuritis This is a common inflammatory disease of the optic nerve. In the Optic Neuritis Treatment Trial (ONTT), the mean age of patients was 32 years, 77% were female, 92% had ocular pain (especially with eye movements), and 35% had optic disc swelling. In most patients, the demyelinating event was retrobulbar and the ocular fundus appeared normal on initial examination (Fig. 39-10), although optic disc pallor slowly developed over subsequent months.

1	Virtually all patients experience a gradual recovery of vision after a single episode of optic neuritis, even without treatment. This rule is so reliable that failure of vision to improve after a first attack of optic neuritis casts doubt on the original diagnosis. Treatment with high-dose IV methylprednisolone (250 mg every 6 h for 3 days) followed by oral prednisone (1 mg/kg per day for 11 days) makes no difference in ultimate acuity 6 months after the attack, but the recovery of visual function occurs more rapidly. Therefore, when visual loss is severe (worse than 20/100), IV followed by PO glucocorticoids are often recommended.

1	For some patients, optic neuritis remains an isolated event. However, the ONTT showed that the 15-year cumulative probability of developing clinically definite multiple sclerosis after optic neuritis is 50%. A brain magnetic resonance (MR) scan is advisable in every patient with a first attack of optic neuritis. If two or more plaques are present on initial imaging, treatment should be considered to prevent the development of additional demyelinating lesions (Chap. 458). This disease usually affects young men, causing gradual, painless, severe central visual loss in one eye, followed weeks to years later by the same process in the other eye. Acutely, the optic disc appears mildly plethoric with surface capillary telangiectasias but no vascular leakage on fluorescein angiography. Eventually optic atrophy ensues. Leber’s optic neuropathy is caused by a point mutation at codon 11778 in the mitochondrial gene encoding nicotinamide adenine dinucleotide

1	FIguRE 39-8 Central retinal vein occlusion can produce massive retinal hemorrhage (“blood and thunder”), ischemia, and vision loss. are prominent risk factors. Polycythemia, thrombocythemia, or other factors leading to an underlying hypercoagulable state should be corrected; aspirin treatment may be beneficial.

1	Anterior Ischemic Optic Neuropathy (AION) This is caused by insufficient blood flow through the posterior ciliary arteries that supply the optic disc. It produces painless monocular visual loss that is sudden in onset, followed sometimes by stuttering progression. The optic disc appears swollen and surrounded by nerve fiber layer splinter hemorrhages (Fig. 39-9). AION is divided into two forms: arteritic and nonarteritic. The nonarteritic form is most common. No specific cause can be identified, although diabetes and hypertension are common risk factors. A crowded disc architecture and small optic cup predispose to the development of nonarteritic AION. No treatment is available. About 5% of patients, especially those age >60, develop the arteritic form of AION in conjunction with giant-cell (temporal) arteritis (Chap. 385). It is urgent to recognize arteritic AION so that high doses of glucocorticoids can be instituted immediately to prevent blindness in the second eye. Symptoms of

1	(temporal) arteritis (Chap. 385). It is urgent to recognize arteritic AION so that high doses of glucocorticoids can be instituted immediately to prevent blindness in the second eye. Symptoms of polymyalgia rheumatica may be present; the sedimentation rate and C-reactive protein level are usually elevated. In a patient with visual loss from suspected arteritic AION, temporal artery biopsy is mandatory to confirm the diagnosis. Glucocorticoids should be started immediately, without waiting for the biopsy to be completed. The diagnosis of arteritic AION is difficult to sustain in the face of a negative temporal artery biopsy, but such cases do occur dehydrogenase (NADH) subunit 4. Additional mutations responsible for the disease have been identified, most in mitochondrial genes that encode proteins involved in electron transport. Mitochondrial mutations that cause Leber’s neuropathy are inherited from the mother by all her children, but usually only sons develop symptoms.

1	FIguRE 39-9 Anterior ischemic optic neuropathy from temporal arteritis in a 67-year-old woman with acute disc swelling, splinter hemorrhages, visual loss, and an erythrocyte sedimentation rate of 70 mm/h. FIguRE 39-11 Optic atrophy is not a specific diagnosis but refers to the combination of optic disc pallor, arteriolar narrowing, and nerve fiber layer destruction produced by a host of eye diseases, especially optic neuropathies.

1	Toxic Optic Neuropathy This can result in acute visual loss with bilateral optic disc swelling and central or cecocentral scotomas. Such cases have been reported to result from exposure to ethambutol, methyl alcohol (moonshine), ethylene glycol (antifreeze), or carbon monoxide. In toxic optic neuropathy, visual loss also can develop gradually and produce optic atrophy (Fig. 39-11) without a phase of acute optic disc edema. Many agents have been implicated as a cause of toxic optic neuropathy, but the evidence supporting the association for many is weak. The following is a partial list of potential offending drugs or toxins: disulfiram, ethchlorvynol, chloramphenicol, amiodarone, monoclonal anti-CD3 antibody, ciprofloxacin, digitalis, streptomycin, lead, arsenic, thallium, d-penicillamine, isoniazid, emetine, sildenafil, tadalafil, vardenafil, and sulfonamides. Deficiency states induced by starvation, malabsorption, or alcoholism can lead to insidious visual loss. Thiamine, vitamin

1	isoniazid, emetine, sildenafil, tadalafil, vardenafil, and sulfonamides. Deficiency states induced by starvation, malabsorption, or alcoholism can lead to insidious visual loss. Thiamine, vitamin B12, and folate levels should be checked in any patient with unexplained bilateral central scotomas and optic pallor.

1	Papilledema This connotes bilateral optic disc swelling from raised intracranial pressure (Fig. 39-12). Headache is a common but not invariable accompaniment. All other forms of optic disc swelling (e.g., 203 from optic neuritis or ischemic optic neuropathy) should be called “optic disc edema”. This convention is arbitrary but serves to avoid confusion. Often it is difficult to differentiate papilledema from other forms of optic disc edema by fundus examination alone. Transient visual obscurations are a classic symptom of papilledema. They can occur in only one eye or simultaneously in both eyes. They usually last seconds but can persist longer. Obscurations follow abrupt shifts in posture or happen spontaneously. When obscurations are prolonged or spontaneous, the papilledema is more threatening. Visual acuity is not affected by papilledema unless the papilledema is severe, longstanding, or accompanied by macular edema and hemorrhage. Visual field testing shows enlarged blind spots

1	Visual acuity is not affected by papilledema unless the papilledema is severe, longstanding, or accompanied by macular edema and hemorrhage. Visual field testing shows enlarged blind spots and peripheral constriction (Fig. 39-3F). With unremitting papilledema, peripheral visual field loss progresses in an insidious fashion while the optic nerve develops atrophy. In this setting, reduction of optic disc swelling is an ominous sign of a dying nerve rather than an encouraging indication of resolving papilledema.

1	Evaluation of papilledema requires neuroimaging to exclude an intracranial lesion. MR angiography is appropriate in selected cases to search for a dural venous sinus occlusion or an arteriovenous shunt. If neuroradiologic studies are negative, the subarachnoid opening pressure should be measured by lumbar puncture. An elevated pressure, with normal cerebrospinal fluid, points by exclusion to the diagnosis of pseudotumor cerebri (idiopathic intracranial hypertension). The majority of patients are young, female, and obese. Treatment with a carbonic anhydrase inhibitor such as acetazolamide lowers intracranial pressure by reducing the production of cerebrospinal fluid. Weight reduction is vital: bariatric surgery should be considered in patients who cannot lose weight by diet control. If vision loss is severe or progressive, a shunt should be performed without delay to prevent blindness. Occasionally, emergency surgery is required for sudden blindness caused by fulminant papilledema.

1	Optic Disc Drusen These are refractile deposits within the substance of the optic nerve head (Fig. 39-13). They are unrelated to drusen of the retina, which occur in age-related macular degeneration. Optic disc drusen are most common in people of northern European descent. Their diagnosis is obvious when they are visible as glittering particles on the surface of the optic disc. However, in many patients they are hidden beneath the surface, producing pseudopapilledema. It is important to recognize optic disc drusen to avoid an unnecessary evaluation for papilledema. Ultrasound or computed tomography (CT) scanning is sensitive for detection of buried optic disc drusen because they contain calcium. In most patients, optic disc drusen are an incidental, innocuous finding, but they can produce visual obscurations. On perimetry they give rise to enlarged blind spots and arcuate CHAPTER 39 Disorders of the Eye

1	CHAPTER 39 Disorders of the Eye FIguRE 39-12 Papilledema means optic disc edema from raised intracranial pressure. This young woman developed acute papill-edema, with hemorrhages and cotton-wool spots, as a rare side effect of treatment with tetracycline for acne. FIguRE 39-13 Optic disc drusen are calcified, mulberry-like deposits of unknown etiology within the optic disc, giving rise to “pseudopapilledema.” 204 scotomas from damage to the optic disc. With increasing age, drusen tend to become more exposed on the disc surface as optic atrophy develops. Hemorrhage, choroidal neovascular membrane, and AION are more likely to occur in patients with optic disc drusen. No treatment is available.

1	Vitreous Degeneration This occurs in all individuals with advancing age, leading to visual symptoms. Opacities develop in the vitreous, casting annoying shadows on the retina. As the eye moves, these distracting “floaters” move synchronously, with a slight lag caused by inertia of the vitreous gel. Vitreous traction on the retina causes mechanical stimulation, resulting in perception of flashing lights. This photopsia is brief and is confined to one eye, in contrast to the bilateral, prolonged scintillations of cortical migraine. Contraction of the vitreous can result in sudden separation from the retina, heralded by an alarming shower of floaters and photopsia. This process, known as vitreous detachment, is a common involutional event in the elderly. It is not harmful unless it damages the retina. A careful examination of the dilated fundus is important in any patient complaining of floaters or photopsia to search for peripheral tears or holes. If such a lesion is found, laser

1	the retina. A careful examination of the dilated fundus is important in any patient complaining of floaters or photopsia to search for peripheral tears or holes. If such a lesion is found, laser application can forestall a retinal detachment. Occasionally a tear ruptures a retinal blood vessel, causing vitreous hemorrhage and sudden loss of vision. On attempted ophthalmoscopy the fundus is hidden by a dark haze of blood. Ultrasound is required to examine the interior of the eye for a retinal tear or detachment. If the hemorrhage does not resolve spontaneously, the vitreous can be removed surgically. Vitreous hemorrhage also results from the fragile neovascular vessels that proliferate on the surface of the retina in diabetes, sickle cell anemia, and other ischemic ocular diseases.

1	Retinal Detachment This produces symptoms of floaters, flashing lights, and a scotoma in the peripheral visual field corresponding to the detachment (Fig. 39-14). If the detachment includes the fovea, there is an afferent pupil defect and the visual acuity is reduced. In most eyes, retinal detachment starts with a hole, flap, or tear in the peripheral retina (rhegmatogenous retinal detachment). Patients with peripheral retinal thinning (lattice degeneration) are particularly vulnerable to this process. Once a break has developed in the retina, liquefied vitreous is free to enter the subretinal space, separating the retina from the pigment epithelium. The combination of vitreous traction on the retinal surface and passage of fluid behind the retina leads inexorably to detachment. Patients with a history of myopia, trauma, or prior cataract extraction are at greatest risk for retinal detachment. The diagnosis is confirmed by ophthalmoscopic examination of the dilated eye.

1	Classic Migraine (See also Chap. 447) This usually occurs with a visual aura lasting about 20 min. In a typical attack, a small central disturbance in the field of vision marches toward the periphery, leaving a transient PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 39-14 Retinal detachment appears as an elevated sheet of retinal tissue with folds. In this patient, the fovea was spared, so acuity was normal, but an inferior detachment produced a superior scotoma.

1	scotoma in its wake. The expanding border of migraine scotoma has a scintillating, dancing, or zigzag edge, resembling the bastions of a fortified city, hence the term fortification spectra. Patients’ descriptions of fortification spectra vary widely and can be confused with amaurosis fugax. Migraine patterns usually last longer and are perceived in both eyes, whereas amaurosis fugax is briefer and occurs in only one eye. Migraine phenomena also remain visible in the dark or with the eyes closed. Generally they are confined to either the right or the left visual hemifield, but sometimes both fields are involved simultaneously. Patients often have a long history of stereotypic attacks. After the visual symptoms recede, headache develops in most patients.

1	Transient Ischemic Attacks Vertebrobasilar insufficiency may result in acute homonymous visual symptoms. Many patients mistakenly describe symptoms in the left or right eye when in fact the symptoms are occurring in the left or right hemifield of both eyes. Interruption of blood supply to the visual cortex causes a sudden fogging or graying of vision, occasionally with flashing lights or other positive phenomena that mimic migraine. Cortical ischemic attacks are briefer in duration than migraine, occur in older patients, and are not followed by headache. There may be associated signs of brainstem ischemia, such as diplopia, vertigo, numbness, weakness, and dysarthria.

1	Stroke Stroke occurs when interruption of blood supply from the posterior cerebral artery to the visual cortex is prolonged. The only finding on examination is a homonymous visual field defect that stops abruptly at the vertical meridian. Occipital lobe stroke usually is due to thrombotic occlusion of the vertebrobasilar system, embolus, or dissection. Lobar hemorrhage, tumor, abscess, and arteriovenous malformation are other common causes of hemianopic cortical visual loss. Factitious (Functional, Nonorganic) Visual Loss This is claimed by hysterics or malingerers. The latter account for the vast majority, seeking sympathy, special treatment, or financial gain by feigning loss of sight. The diagnosis is suspected when the history is atypical, physical findings are lacking or contradictory, inconsistencies emerge on testing, and a secondary motive can be identified. In our litigious society, the fraudulent pursuit of recompense has spawned an epidemic of factitious visual loss.

1	CHRONIC VISuAL LOSS Cataract Cataract is a clouding of the lens sufficient to reduce vision. Most cataracts develop slowly as a result of aging, leading to gradual impairment of vision. The formation of cataract occurs more rapidly in patients with a history of ocular trauma, uveitis, or diabetes mellitus. Cataracts are acquired in a variety of genetic diseases, such as myotonic dystrophy, neurofibromatosis type 2, and galactosemia. Radiation therapy and glucocorticoid treatment can induce cataract as a side effect. The cataracts associated with radiation or glucocorticoids have a typical posterior subcapsular location. Cataract can be detected by noting an impaired red reflex when viewing light reflected from the fundus with an ophthalmoscope or by examining the dilated eye with the slit lamp.

1	The only treatment for cataract is surgical extraction of the opacified lens. Millions of cataract operations are performed each year around the globe. The operation generally is done under local anesthesia on an outpatient basis. A plastic or silicone intraocular lens is placed within the empty lens capsule in the posterior chamber, substituting for the natural lens and leading to rapid recovery of sight. More than 95% of patients who undergo cataract extraction can expect an improvement in vision. In some patients, the lens capsule remaining in the eye after cataract extraction eventually turns cloudy, causing secondary loss of vision. A small opening, called a posterior capsulotomy, is made in the lens capsule with a laser to restore clarity.

1	glaucoma Glaucoma is a slowly progressive, insidious optic neuropathy that usually is associated with chronic elevation of intraocular pressure. After cataract, it is the most common cause of blindness in the world. It is especially prevalent in people of African descent. The mechanism by which raised intraocular pressure injures the optic FIguRE 39-15 Glaucoma results in “cupping” as the neural rim is destroyed and the central cup becomes enlarged and excavated. The cup-to-disc ratio is about 0.8 in this patient. FIguRE 39-16 Age-related macular degeneration consisting of scattered yellow drusen in the macula (dry form) and a crescent of fresh hemorrhage temporal to the fovea from a subretinal neovascu-lar membrane (wet form).

1	CHAPTER 39 Disorders of the Eye nerve is not understood. Axons entering the inferotemporal and superotemporal aspects of the optic disc are damaged first, producing typical nerve fiber bundle or arcuate scotomas on perimetric testing. As fibers are destroyed, the neural rim of the optic disc shrinks and the physiologic cup within the optic disc enlarges (Fig. 39-15). This process is referred to as pathologic “cupping.” The cup-to-disc diameter is expressed as a fraction (e.g., 0.2). The cup-to-disc ratio ranges widely in normal individuals, making it difficult to diagnose glaucoma reliably simply by observing an unusually large or deep optic cup. Careful documentation of serial examinations is helpful. In a patient with physiologic cupping the large cup remains stable, whereas in a patient with glaucoma it expands relentlessly over the years. Observation of progressive cupping and detection of an arcuate scotoma or a nasal step on computerized visual field testing is sufficient to

1	with glaucoma it expands relentlessly over the years. Observation of progressive cupping and detection of an arcuate scotoma or a nasal step on computerized visual field testing is sufficient to establish the diagnosis of glaucoma. Optical coherence tomography reveals corresponding loss of fibers along the arcuate pathways in the nerve fiber layer.

1	About 95% of patients with glaucoma have open anterior chamber angles. In most affected individuals the intraocular pressure is elevated. The cause of elevated intraocular pressure is unknown, but it is associated with gene mutations in the heritable forms. Surprisingly, a third of patients with open-angle glaucoma have an intraocular pressure within the normal range of 10–20 mmHg. For this so-called normal or low-tension form of glaucoma, high myopia is a risk factor.

1	Chronic angle-closure glaucoma and chronic open-angle glaucoma are usually asymptomatic. Only acute angle-closure glaucoma causes a red or painful eye, from abrupt elevation of intraocular pressure. In all forms of glaucoma, foveal acuity is spared until end-stage disease is reached. For these reasons, severe and irreversible damage can occur before either the patient or the physician recognizes the diagnosis. Screening of patients for glaucoma by noting the cup-to-disc ratio on ophthalmoscopy and by measuring intraocular pressure is vital. Glaucoma is treated with topical adrenergic agonists, cholinergic agonists, beta blockers, and prostaglandin analogues. Occasionally, systemic absorption of beta blocker from eyedrops can be sufficient to cause side effects of bradycardia, hypotension, heart block, bronchospasm, or depression. Topical or oral carbonic anhydrase inhibitors are used to lower intraocular pressure by reducing aqueous production. Laser treatment of the trabecular

1	heart block, bronchospasm, or depression. Topical or oral carbonic anhydrase inhibitors are used to lower intraocular pressure by reducing aqueous production. Laser treatment of the trabecular meshwork in the anterior chamber angle improves aqueous outflow from the eye. If medical or laser treatments fail to halt optic nerve damage from glaucoma, a filter must be constructed surgically (trabeculectomy) or a drainage device placed to release aqueous from the eye in a controlled fashion.

1	Macular Degeneration This is a major cause of gradual, painless, bilateral central visual loss in the elderly. It occurs in a nonexudative (dry) form and an exudative (wet) form. Inflammation may be important in both forms of macular degeneration; susceptibility is associated with variants in the gene for complement factor H, an inhibitor of the alternative complement pathway. The nonexudative process begins with the accumulation of extracellular deposits called drusen underneath the retinal pigment epithelium. On ophthalmoscopy, they are pleomorphic but generally appear as small discrete yellow lesions clustered in the macula (Fig. 39-16). With time they become larger, more numerous, and confluent. The retinal pigment epithelium becomes focally detached and atrophic, causing visual loss by interfering with photoreceptor function. Treatment with vitamins C and E, beta-carotene, and zinc may retard dry macular degeneration.

1	Exudative macular degeneration, which develops in only a minority of patients, occurs when neovascular vessels from the choroid grow through defects in Bruch’s membrane and proliferate underneath the retinal pigment epithelium or the retina. Leakage from these vessels produces elevation of the retina, with distortion (metamorphopsia) and blurring of vision. Although the onset of these symptoms is usually gradual, bleeding from a subretinal choroidal neovascular membrane sometimes causes acute visual loss. Neovascular membranes can be difficult to see on fundus examination because they are located beneath the retina. Fluorescein angiography and optical coherence tomography, a technique for acquiring images of the retina in cross-section, are extremely useful for their detection. Major or repeated hemorrhage under the retina from neovascular membranes results in fibrosis, development of a round (disciform) macular scar, and permanent loss of central vision.

1	A major therapeutic advance has occurred with the discovery that exudative macular degeneration can be treated with intraocular injection of antagonists to vascular endothelial growth factor. Bevacizumab, ranibizumab, or aflibercept is administered by direct injection into the vitreous cavity, beginning on a monthly basis. These antibodies cause the regression of neovascular membranes by blocking the action of vascular endothelial growth factor, thereby improving visual acuity.

1	Central Serous Chorioretinopathy This primarily affects males between the ages of 20 and 50 years. Leakage of serous fluid from the choroid causes small, localized detachment of the retinal pigment epithelium and the neurosensory retina. These detachments produce acute or chronic symptoms of metamorphopsia and blurred vision when the macula is involved. They are difficult to visualize with a direct ophthalmoscope because the detached retina is transparent and only slightly elevated. Optical coherence tomography shows fluid beneath the retina, and fluorescein angiography shows dye streaming into the subretinal space. The cause of central serous chorioretinopathy is unknown. Symptoms may resolve spontaneously if the retina reattaches, but recurrent detachment is common. Laser photocoagulation has benefited some patients with this condition. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 39-17 Proliferative diabetic retinopathy in a 25-year-old man with an 18-year history of diabetes, showing neovascular vessels emanating from the optic disc, retinal and vitreous hemorrhage, cot-ton-wool spots, and macular exudate. Round spots in the periphery represent recently applied panretinal photocoagulation.

1	Diabetic Retinopathy A rare disease until 1921, when the discovery of insulin resulted in a dramatic improvement in life expectancy for patients with diabetes mellitus, diabetic retinopathy is now a leading cause of blindness in the United States. The retinopathy takes years to develop but eventually appears in nearly all cases. Regular surveillance of the dilated fundus is crucial for any patient with diabetes. In advanced diabetic retinopathy, the proliferation of neovascular vessels leads to blindness from vitreous hemorrhage, retinal detachment, and glaucoma (Fig. 39-17). These complications can be avoided in most patients by administration of panretinal laser photocoagulation at the appropriate point in the evolution of the disease. For further discussion of the manifestations and management of diabetic retinopathy, see Chaps. 417–419.

1	Retinitis Pigmentosa This is a general term for a disparate group of rod-cone dystrophies characterized by progressive night blindness, visual field constriction with a ring scotoma, loss of acuity, and an abnormal electroretinogram (ERG). It occurs sporadically or in an autosomal recessive, dominant, or X-linked pattern. Irregular black deposits of clumped pigment in the peripheral retina, called bone spicules because of their vague resemblance to the spicules of cancellous bone, give the disease its name (Fig. 39-18). The name is actually a misnomer because retinitis pigmentosa is not an inflammatory process. Most cases are due to a mutation in the gene for rhodopsin, the rod photopigment, or in the gene for peripherin, a glycoprotein located in photoreceptor outer segments. Vitamin A (15,000 IU/d) slightly retards the deterioration of the ERG in patients with retinitis pigmentosa but has no beneficial effect on visual acuity or fields.

1	FIguRE 39-18 Retinitis pigmentosa with black clumps of pigment known as “bone spicules.” The patient had peripheral visual field loss with sparing of central (macular) vision. Leber’s congenital amaurosis, a rare cone dystrophy, has been treated by replacement of the missing RPE65 protein through gene therapy, resulting in modest improvement in visual function. Some forms of retinitis pigmentosa occur in association with rare, hereditary systemic diseases (olivopontocerebellar degeneration, Bassen-Kornzweig disease, Kearns-Sayre syndrome, Refsum’s disease). Chronic treatment with chloroquine, hydroxychloroquine, and phenothiazines (especially thioridazine) can produce visual loss from a toxic retinopathy that resembles retinitis pigmentosa.

1	Epiretinal Membrane This is a fibrocellular tissue that grows across the inner surface of the retina, causing metamorphopsia and reduced visual acuity from distortion of the macula. A crinkled, cellophane-like membrane is visible on the retinal examination. Epiretinal membrane is most common in patients over 50 years of age and is usually unilateral. Most cases are idiopathic, but some occur as a result of hypertensive retinopathy, diabetes, retinal detachment, or trauma. When visual acuity is reduced to the level of about 6/24 (20/80), vitrectomy and surgical peeling of the membrane to relieve macular puckering are recommended. Contraction of an epiretinal membrane sometimes gives rise to a macular hole. Most macular holes, however, are caused by local vitreous traction within the fovea. Vitrectomy can improve acuity in selected cases.

1	Melanoma and Other Tumors Melanoma is the most common primary tumor of the eye (Fig. 39-19). It causes photopsia, an enlarging scotoma, and loss of vision. A small melanoma is often difficult to differentiate from a benign choroidal nevus. Serial examinations are required to document a malignant pattern of growth. Treatment of melanoma is controversial. Options include enucleation, local resection, and irradiation. Metastatic tumors to the eye outnumber primary tumors. Breast and lung carcinomas have a special propensity to spread to the choroid or iris. Leukemia and lymphoma also commonly invade ocular tissues. Sometimes their only sign on eye examination is cellular debris in the vitreous, which can masquerade as a chronic posterior uveitis. Retrobulbar tumor of the optic nerve (meningioma, glioma) or chiasmal tumor (pituitary adenoma, meningioma) produces gradual visual loss with few objective findings except for optic disc pallor. Rarely, sudden expansion of a pituitary adenoma

1	glioma) or chiasmal tumor (pituitary adenoma, meningioma) produces gradual visual loss with few objective findings except for optic disc pallor. Rarely, sudden expansion of a pituitary adenoma from infarction and bleeding (pituitary apoplexy) causes acute retrobulbar visual loss, with headache, nausea, and ocular motor nerve palsies. In any patient with visual field loss or optic atrophy, CT or MR scanning should be considered if the cause remains unknown after careful review of the history and thorough examination of the eye.

1	FIguRE 39-19 Melanoma of the choroid, appearing as an elevated dark mass in the inferior fundus, with overlying hemorrhage. The black line denotes the plane of the optical coherence tomography scan (below) showing the subretinal tumor.

1	When the globes appear asymmetric, the clinician must first decide which eye is abnormal. Is one eye recessed within the orbit (enophthalmos), or is the other eye protuberant (exophthalmos, or proptosis)? A small globe or a Horner’s syndrome can give the appearance of enophthalmos. True enophthalmos occurs commonly after trauma, from atrophy of retrobulbar fat, or from fracture of the orbital floor. The position of the eyes within the orbits is measured by using a Hertel exophthalmometer, a handheld instrument that records the position of the anterior corneal surface relative to the lateral orbital rim. If this instrument is not available, relative eye position can be judged by bending the patient’s head forward and looking down upon the orbits. A proptosis of only 2 mm in one eye is detectable from this perspective. The development of proptosis implies a space-occupying lesion in the orbit and usually warrants CT or MR imaging.

1	graves’ Ophthalmopathy This is the leading cause of proptosis in adults (Chap. 405). The proptosis is often asymmetric and can even appear to be unilateral. Orbital inflammation and engorgement of the extra-ocular muscles, particularly the medial rectus and the inferior rectus, account for the protrusion of the globe. Corneal exposure, lid retraction, conjunctival injection, restriction of gaze, diplopia, and visual loss from optic nerve compression are cardinal symptoms. Graves’ eye disease is a clinical diagnosis, but laboratory testing can be useful. The serum level of thyroid-stimulating immunoglobulins is often elevated. Orbital imaging usually reveals enlarged extraocular eye muscles, but not always. Graves’ ophthalmopathy can be treated with oral prednisone (60 mg/d) for 1 month, followed by a taper over several months. Worsening of symptoms upon glucocorticoid withdrawal is common. Topical lubricants, taping the eyelids closed at night, moisture chambers, and eyelid surgery

1	followed by a taper over several months. Worsening of symptoms upon glucocorticoid withdrawal is common. Topical lubricants, taping the eyelids closed at night, moisture chambers, and eyelid surgery are helpful to limit exposure of ocular tissues. Radiation therapy is not effective. Orbital decompression should be performed for severe, symptomatic exophthalmos or if visual function is reduced by optic nerve compression. In patients with diplopia, prisms or eye muscle surgery can be used to restore ocular alignment in primary gaze.

1	Orbital Pseudotumor This is an idiopathic, inflammatory orbital syndrome that is distinguished from Graves’ ophthalmopathy by the prominent complaint of pain. Other symptoms include diplopia, ptosis, proptosis, and orbital congestion. Evaluation for sarcoidosis, granulomatosis with polyangiitis, and other types of orbital vasculitis or collagen-vascular disease is negative. Imaging often shows swollen eye muscles (orbital myositis) with enlarged tendons. By contrast, in Graves’ ophthalmopathy, the tendons of the eye muscles usually are spared. The Tolosa-Hunt syndrome (Chap. 455) may be regarded as an extension of orbital pseudotumor through the superior orbital fissure into the cavernous sinus. The diagnosis of orbital pseudotumor is difficult. Biopsy of the orbit frequently yields nonspecific evidence of fat infiltration by lymphocytes, plasma cells, and eosinophils. A dramatic response to a therapeutic trial of systemic glucocorticoids indirectly provides the best confirmation of

1	evidence of fat infiltration by lymphocytes, plasma cells, and eosinophils. A dramatic response to a therapeutic trial of systemic glucocorticoids indirectly provides the best confirmation of the diagnosis.

1	Orbital Cellulitis This causes pain, lid erythema, proptosis, conjunctival chemosis, restricted motility, decreased acuity, afferent pupillary defect, fever, and leukocytosis. It often arises from the paranasal sinuses, especially by contiguous spread of infection from the ethmoid sinus through the lamina papyracea of the medial orbit. A history of recent upper respiratory tract infection, chronic sinusitis, thick mucus secretions, or dental disease is significant in any patient with suspected orbital cellulitis. Blood cultures should be obtained, but they are usually negative. Most patients respond to empirical therapy with broad-spectrum IV antibiotics. Occasionally, orbital cellulitis follows an overwhelm-207 ing course, with massive proptosis, blindness, septic cavernous sinus thrombosis, and meningitis. To avert this disaster, orbital cellulitis should be managed aggressively in the early stages, with immediate imaging of the orbits and antibiotic therapy that includes coverage

1	and meningitis. To avert this disaster, orbital cellulitis should be managed aggressively in the early stages, with immediate imaging of the orbits and antibiotic therapy that includes coverage of methicillin-resistant Staphylococcus aureus (MRSA). Prompt surgical drainage of an orbital abscess or paranasal sinusitis is indicated if optic nerve function deteriorates despite antibiotics.

1	Tumors Tumors of the orbit cause painless, progressive proptosis. The most common primary tumors are cavernous hemangioma, lymphangioma, neurofibroma, schwannoma, dermoid cyst, adenoid cystic carcinoma, optic nerve glioma, optic nerve meningioma, and benign mixed tumor of the lacrimal gland. Metastatic tumor to the orbit occurs frequently in breast carcinoma, lung carcinoma, and lymphoma. Diagnosis by fine-needle aspiration followed by urgent radiation therapy sometimes can preserve vision.

1	Carotid Cavernous Fistulas With anterior drainage through the orbit, these fistulas produce proptosis, diplopia, glaucoma, and corkscrew, arterialized conjunctival vessels. Direct fistulas usually result from trauma. They are easily diagnosed because of the prominent signs produced by high-flow, high-pressure shunting. Indirect fistulas, or dural arteriovenous malformations, are more likely to occur spontaneously, especially in older women. The signs are more subtle, and the diagnosis frequently is missed. The combination of slight proptosis, diplopia, enlarged muscles, and an injected eye often is mistaken for thyroid ophthalmopathy. A bruit heard upon auscultation of the head or reported by the patient is a valuable diagnostic clue. Imaging shows an enlarged superior ophthalmic vein in the orbits. Carotid cavernous shunts can be eliminated by intravascular embolization.

1	PTOSIS Blepharoptosis This is an abnormal drooping of the eyelid. Unilateral or bilateral ptosis can be congenital, from dysgenesis of the levator palpebrae superioris, or from abnormal insertion of its aponeurosis into the eyelid. Acquired ptosis can develop so gradually that the patient is unaware of the problem. Inspection of old photographs is helpful in dating the onset. A history of prior trauma, eye surgery, contact lens use, diplopia, systemic symptoms (e.g., dysphagia or peripheral muscle weakness), or a family history of ptosis should be sought. Fluctuating ptosis that worsens late in the day is typical of myasthenia gravis. Examination should focus on evidence for proptosis, eyelid masses or deformities, inflammation, pupil inequality, or limitation of motility. The width of the palpebral fissures is measured in primary gaze to determine the degree of ptosis. The ptosis will be underestimated if the patient compensates by lifting the brow with the frontalis muscle.

1	Mechanical Ptosis This occurs in many elderly patients from stretching and redundancy of eyelid skin and subcutaneous fat (dermatochalasis). The extra weight of these sagging tissues causes the lid to droop. Enlargement or deformation of the eyelid from infection, tumor, trauma, or inflammation also results in ptosis on a purely mechanical basis. Aponeurotic Ptosis This is an acquired dehiscence or stretching of the aponeurotic tendon, which connects the levator muscle to the tarsal plate of the eyelid. It occurs commonly in older patients, presumably from loss of connective tissue elasticity. Aponeurotic ptosis is also a common sequela of eyelid swelling from infection or blunt trauma to the orbit, cataract surgery, or contact lens use.

1	Myogenic Ptosis The causes of myogenic ptosis include myasthenia gravis (Chap. 461) and a number of rare myopathies that manifest with ptosis. The term chronic progressive external ophthalmoplegia refers to a spectrum of systemic diseases caused by mutations of mitochondrial DNA. As the name implies, the most prominent findings are symmetric, slowly progressive ptosis and limitation of eye movements. In general, diplopia is a late symptom because all eye movements are reduced equally. In the Kearns-Sayre variant, retinal pigmentary changes and abnormalities of cardiac conduction develop.

1	CHAPTER 39 Disorders of the Eye 208 Peripheral muscle biopsy shows characteristic “ragged-red fibers.” Oculopharyngeal dystrophy is a distinct autosomal dominant disease with onset in middle age, characterized by ptosis, limited eye movements, and trouble swallowing. Myotonic dystrophy, another autosomal dominant disorder, causes ptosis, ophthalmoparesis, cataract, and pigmentary retinopathy. Patients have muscle wasting, myotonia, frontal balding, and cardiac abnormalities.

1	Neurogenic Ptosis This results from a lesion affecting the innervation to either of the two muscles that open the eyelid: Müller’s muscle or the levator palpebrae superioris. Examination of the pupil helps distinguish between these two possibilities. In Horner’s syndrome, the eye with ptosis has a smaller pupil and the eye movements are full. In an oculomotor nerve palsy, the eye with the ptosis has a larger or a normal pupil. If the pupil is normal but there is limitation of adduction, elevation, and depression, a pupil-sparing oculomotor nerve palsy is likely (see next section). Rarely, a lesion affecting the small, central subnucleus of the oculomotor complex will cause bilateral ptosis with normal eye movements and pupils.

1	The first point to clarify is whether diplopia persists in either eye after the opposite eye is covered. If it does, the diagnosis is monocular diplopia. The cause is usually intrinsic to the eye and therefore has no dire implications for the patient. Corneal aberrations (e.g., keratoconus, pterygium), uncorrected refractive error, cataract, or foveal traction may give rise to monocular diplopia. Occasionally it is a symptom of malingering or psychiatric disease. Diplopia alleviated by covering one eye is binocular diplopia and is caused by disruption of ocular alignment. Inquiry should be made into the nature of the double vision (purely side-by-side versus partial vertical displacement of images), mode of onset, duration, intermittency, diurnal variation, and associated neurologic or systemic symptoms. If the patient has diplopia while being examined, motility testing should reveal a deficiency corresponding to the patient’s symptoms. However, subtle limitation of ocular excursions

1	symptoms. If the patient has diplopia while being examined, motility testing should reveal a deficiency corresponding to the patient’s symptoms. However, subtle limitation of ocular excursions is often difficult to detect. For example, a patient with a slight left abducens nerve paresis may appear to have full eye movements despite a complaint of horizontal diplopia upon looking to the left. In this situation, the cover test provides a more sensitive method for demonstrating the ocular misalignment. It should be conducted in primary gaze and then with the head turned and tilted in each direction. In the above example, a cover test with the head turned to the right will maximize the fixation shift evoked by the cover test.

1	Occasionally, a cover test performed in an asymptomatic patient during a routine examination will reveal an ocular deviation. If the eye movements are full and the ocular misalignment is equal in all directions of gaze (concomitant deviation), the diagnosis is strabismus. In this condition, which affects about 1% of the population, fusion is disrupted in infancy or early childhood. To avoid diplopia, vision is suppressed from the nonfixating eye. In some children, this leads to impaired vision (amblyopia, or “lazy” eye) in the deviated eye.

1	Binocular diplopia results from a wide range of processes: infectious, neoplastic, metabolic, degenerative, inflammatory, and vascular. One must decide whether the diplopia is neurogenic in origin or is due to restriction of globe rotation by local disease in the orbit. Orbital pseudotumor, myositis, infection, tumor, thyroid disease, and muscle entrapment (e.g., from a blowout fracture) cause restrictive diplopia. The diagnosis of restriction is usually made by recognizing other associated signs and symptoms of local orbital disease. Omission of high-resolution orbital imaging is a common mistake in the evaluation of diplopia.

1	Myasthenia gravis (See also Chap. 461) This is a major cause of diplopia. The diplopia is often intermittent, variable, and not confined to any single ocular motor nerve distribution. The pupils are always normal. Fluctuating ptosis may be present. Many patients have a purely ocular form of the disease, with no evidence of systemic muscular weakness. The diagnosis can be confirmed by an IV edrophonium injection, which produces a transient reversal of eyelid or eye muscle PART 2 Cardinal Manifestations and Presentation of Diseases weakness. Blood tests for antibodies against the acetylcholine receptor or the MuSK protein can establish the diagnosis but are frequently negative in the purely ocular form of myasthenia gravis. Botulism from food or wound poisoning can mimic ocular myasthenia. After restrictive orbital disease and myasthenia gravis are excluded, a lesion of a cranial nerve supplying innervation to the extraocular muscles is the most likely cause of binocular diplopia.

1	Oculomotor Nerve The third cranial nerve innervates the medial, inferior, and superior recti; inferior oblique; levator palpebrae superioris; and the iris sphincter. Total palsy of the oculomotor nerve causes ptosis, a dilated pupil, and leaves the eye “down and out” because of the unopposed action of the lateral rectus and superior oblique. This combination of findings is obvious. More challenging is the diagnosis of early or partial oculomotor nerve palsy. In this setting any combination of ptosis, pupil dilation, and weakness of the eye muscles supplied by the oculomotor nerve may be encountered. Frequent serial examinations during the evolving phase of the palsy help ensure that the diagnosis is not missed. The advent of an oculomotor nerve palsy with a pupil involvement, especially when accompanied by pain, suggests a compressive lesion, such as a tumor or circle of Willis aneurysm. Neuroimaging should be obtained, along with a CT or MR angiogram. Occasionally, a catheter

1	when accompanied by pain, suggests a compressive lesion, such as a tumor or circle of Willis aneurysm. Neuroimaging should be obtained, along with a CT or MR angiogram. Occasionally, a catheter arteriogram must be done to exclude an aneurysm.

1	A lesion of the oculomotor nucleus in the rostral midbrain produces signs that differ from those caused by a lesion of the nerve itself. There is bilateral ptosis because the levator muscle is innervated by a single central subnucleus. There is also weakness of the contralateral superior rectus, because it is supplied by the oculomotor nucleus on the other side. Occasionally both superior recti are weak. Isolated nuclear oculomotor palsy is rare. Usually neurologic examination reveals additional signs that suggest brainstem damage from infarction, hemorrhage, tumor, or infection.

1	Injury to structures surrounding fascicles of the oculomotor nerve descending through the midbrain has given rise to a number of classic eponymic designations. In Nothnagel’s syndrome, injury to the superior cerebellar peduncle causes ipsilateral oculomotor palsy and contralateral cerebellar ataxia. In Benedikt’s syndrome, injury to the red nucleus results in ipsilateral oculomotor palsy and contralateral tremor, chorea, and athetosis. Claude’s syndrome incorporates features of both of these syndromes, by injury to both the red nucleus and the superior cerebellar peduncle. Finally, in Weber’s syndrome, injury to the cerebral peduncle causes ipsilateral oculomotor palsy with contra-lateral hemiparesis.

1	In the subarachnoid space the oculomotor nerve is vulnerable to aneurysm, meningitis, tumor, infarction, and compression. In cerebral herniation, the nerve becomes trapped between the edge of the tentorium and the uncus of the temporal lobe. Oculomotor palsy also can result from midbrain torsion and hemorrhages during herniation. In the cavernous sinus, oculomotor palsy arises from carotid aneurysm, carotid cavernous fistula, cavernous sinus thrombosis, tumor (pituitary adenoma, meningioma, metastasis), herpes zoster infection, and the Tolosa-Hunt syndrome.

1	The etiology of an isolated, pupil-sparing oculomotor palsy often remains an enigma even after neuroimaging and extensive laboratory testing. Most cases are thought to result from microvascular infarction of the nerve somewhere along its course from the brainstem to the orbit. Usually the patient complains of pain. Diabetes, hypertension, and vascular disease are major risk factors. Spontaneous recovery over a period of months is the rule. If this fails to occur or if new findings develop, the diagnosis of microvascular oculomotor nerve palsy should be reconsidered. Aberrant regeneration is common when the oculomotor nerve is injured by trauma or compression (tumor, aneurysm). Miswiring of sprouting fibers to the levator muscle and the rectus muscles results in elevation of the eyelid upon downgaze or adduction. The pupil also constricts upon attempted adduction, elevation, or depression of the globe. Aberrant regeneration is not seen after oculomotor palsy from microvascular infarct

1	downgaze or adduction. The pupil also constricts upon attempted adduction, elevation, or depression of the globe. Aberrant regeneration is not seen after oculomotor palsy from microvascular infarct and hence vitiates that diagnosis.

1	Trochlear Nerve The fourth cranial nerve originates in the midbrain, just caudal to the oculomotor nerve complex. Fibers exit the brainstem dorsally and cross to innervate the contralateral superior oblique. The principal actions of this muscle are to depress and intort the globe. A palsy therefore results in hypertropia and excyclotorsion. The cyclotorsion seldom is noticed by patients. Instead, they complain of vertical diplopia, especially upon reading or looking down. The vertical diplopia also is exacerbated by tilting the head toward the side with the muscle palsy and alleviated by tilting it away. This “head tilt test” is a cardinal diagnostic feature.

1	Isolated trochlear nerve palsy results from all the causes listed above for the oculomotor nerve except aneurysm. The trochlear nerve is particularly apt to suffer injury after closed head trauma. The free edge of the tentorium is thought to impinge on the nerve during a concussive blow. Most isolated trochlear nerve palsies are idiopathic and hence are diagnosed by exclusion as “microvascular.” Spontaneous improvement occurs over a period of months in most patients. A base-down prism (conveniently applied to the patient’s glasses as a stick-on Fresnel lens) may serve as a temporary measure to alleviate diplopia. If the palsy does not resolve, the eyes can be realigned by weakening the inferior oblique muscle.

1	Abducens Nerve The sixth cranial nerve innervates the lateral rectus muscle. A palsy produces horizontal diplopia, worse on gaze to the side of the lesion. A nuclear lesion has different consequences, because the abducens nucleus contains interneurons that project via the medial longitudinal fasciculus to the medial rectus subnucleus of the contra-lateral oculomotor complex. Therefore, an abducens nuclear lesion produces a complete lateral gaze palsy from weakness of both the ipsilateral lateral rectus and the contralateral medial rectus. Foville’s syndrome after dorsal pontine injury includes lateral gaze palsy, ipsilateral facial palsy, and contralateral hemiparesis incurred by damage to descending corticospinal fibers. Millard-Gubler syndrome from ventral pontine injury is similar except for the eye findings. There is lateral rectus weakness only, instead of gaze palsy, because the abducens fascicle is injured rather than the nucleus. Infarct, tumor, hemorrhage, vascular

1	except for the eye findings. There is lateral rectus weakness only, instead of gaze palsy, because the abducens fascicle is injured rather than the nucleus. Infarct, tumor, hemorrhage, vascular malformation, and multiple sclerosis are the most common etiologies of brainstem abducens palsy.

1	After leaving the ventral pons, the abducens nerve runs forward along the clivus to pierce the dura at the petrous apex, where it enters the cavernous sinus. Along its subarachnoid course it is susceptible to meningitis, tumor (meningioma, chordoma, carcinomatous meningitis), subarachnoid hemorrhage, trauma, and compression by aneurysm or dolichoectatic vessels. At the petrous apex, mastoiditis can produce deafness, pain, and ipsilateral abducens palsy (Gradenigo’s syndrome). In the cavernous sinus, the nerve can be affected by carotid aneurysm, carotid cavernous fistula, tumor (pituitary adenoma, meningioma, nasopharyngeal carcinoma), herpes infection, and Tolosa-Hunt syndrome.

1	Unilateral or bilateral abducens palsy is a classic sign of raised intracranial pressure. The diagnosis can be confirmed if papilledema is observed on fundus examination. The mechanism is still debated but probably is related to rostral-caudal displacement of the brainstem. The same phenomenon accounts for abducens palsy from Chiari malformation or low intracranial pressure (e.g., after lumbar puncture, spinal anesthesia, or spontaneous dural cerebrospinal fluid leak).

1	Treatment of abducens palsy is aimed at prompt correction of the underlying cause. However, the cause remains obscure in many instances despite diligent evaluation. As was mentioned above for isolated trochlear or oculomotor palsy, most cases are assumed to represent microvascular infarcts because they often occur in the setting of diabetes or other vascular risk factors. Some cases may develop as a postinfectious mononeuritis (e.g., after a viral flu). Patching one eye, occluding one eyeglass lens with tape, or applying a temporary prism will provide relief of diplopia until the palsy resolves. If recovery is incomplete, eye muscle surgery nearly always can realign the eyes, at least in primary position. A patient with an abducens palsy that fails to improve should be reevaluated for an occult etiology (e.g., chordoma, carcinomatous meningitis, carotid cavernous fistula, myasthenia 209 gravis). Skull base tumors are easily missed even on contrast-enhanced neuroimaging studies.

1	Multiple Ocular Motor Nerve Palsies These should not be attributed to spontaneous microvascular events affecting more than one cranial nerve at a time. This remarkable coincidence does occur, especially in diabetic patients, but the diagnosis is made only in retrospect after all other diagnostic alternatives have been exhausted. Neuroimaging should focus on the cavernous sinus, superior orbital fissure, and orbital apex, where all three ocular motor nerves are in close proximity. In a diabetic or immunocompromised host, fungal infection (Aspergillus, Mucorales, Cryptococcus) is a common cause of multiple nerve palsies. In a patient with systemic malignancy, carcinomatous meningitis is a likely diagnosis. Cytologic examination may be negative despite repeated sampling of the cerebrospinal fluid. The cancer-associated Lambert-Eaton myasthenic syndrome also can produce ophthalmoplegia. Giant cell (temporal) arteritis occasionally manifests as diplopia from ischemic palsies of extraocular

1	The cancer-associated Lambert-Eaton myasthenic syndrome also can produce ophthalmoplegia. Giant cell (temporal) arteritis occasionally manifests as diplopia from ischemic palsies of extraocular muscles. Fisher’s syndrome, an ocular variant of Guillain-Barré, produces ophthalmoplegia with areflexia and ataxia. Often the ataxia is mild, and the reflexes are normal. Antiganglioside antibodies (GQ1b) can be detected in about 50% of cases.

1	Supranuclear Disorders of gaze These are often mistaken for multiple ocular motor nerve palsies. For example, Wernicke’s encephalopathy can produce nystagmus and a partial deficit of horizontal and vertical gaze that mimics a combined abducens and oculomotor nerve palsy. The disorder occurs in malnourished or alcoholic patients and can be reversed by thiamine. Infarct, hemorrhage, tumor, multiple sclerosis, encephalitis, vasculitis, and Whipple’s disease are other important causes of supranuclear gaze palsy. Disorders of vertical gaze, especially downward saccades, are an early feature of progressive supranuclear palsy. Smooth pursuit is affected later in the course of the disease. Parkinson’s disease, Huntington’s disease, and olivopontocerebellar degeneration also can affect vertical gaze.

1	The frontal eye field of the cerebral cortex is involved in generation of saccades to the contralateral side. After hemispheric stroke, the eyes usually deviate toward the lesioned side because of the unopposed action of the frontal eye field in the normal hemisphere. With time, this deficit resolves. Seizures generally have the opposite effect: the eyes deviate conjugately away from the irritative focus. Parietal lesions disrupt smooth pursuit of targets moving toward the side of the lesion. Bilateral parietal lesions produce Bálint’s syndrome, which is characterized by impaired eye-hand coordination (optic ataxia), difficulty initiating voluntary eye movements (ocular apraxia), and visuospatial disorientation (simultanagnosia).

1	Horizontal gaze Descending cortical inputs mediating horizontal gaze ultimately converge at the level of the pons. Neurons in the paramedian pontine reticular formation are responsible for controlling conjugate gaze toward the same side. They project directly to the ipsilateral abducens nucleus. A lesion of either the paramedian pontine reticular formation or the abducens nucleus causes an ipsilateral conjugate gaze palsy. Lesions at either locus produce nearly identical clinical syndromes, with the following exception: vestibular stimulation (oculocephalic maneuver or caloric irrigation) will succeed in driving the eyes conjugately to the side in a patient with a lesion of the paramedian pontine reticular formation but not in a patient with a lesion of the abducens nucleus.

1	INTERNUCLEAR OPHTHALMOPLEgIA This results from damage to the medial longitudinal fasciculus ascending from the abducens nucleus in the pons to the oculomotor nucleus in the midbrain (hence, “internuclear”). Damage to fibers carrying the conjugate signal from abducens interneurons to the contralateral medial rectus motoneurons results in a failure of adduction on attempted lateral gaze. For example, a patient with a left internuclear ophthalmoplegia (INO) will have slowed or absent adducting movements of the left eye (Fig. 39-20). A patient CHAPTER 39 Disorders of the Eye PART 2 Cardinal Manifestations and Presentation of Diseases

1	CHAPTER 39 Disorders of the Eye PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 39-20 Left internuclear ophthalmoplegia (INO). A. In primary position of gaze, the eyes appear normal. B. Horizontal gaze to the left is intact. C. On attempted horizontal gaze to the right, the left eye fails to adduct. In mildly affected patients, the eye may adduct partially or more slowly than normal. Nystagmus is usually present in the abducted eye. D. T2-weighted axial magnetic resonance image through the pons showing a demyelinating plaque in the left medial longitudinal fasciculus (arrow).

1	with bilateral injury to the medial longitudinal fasciculus will have bilateral INO. Multiple sclerosis is the most common cause, although tumor, stroke, trauma, or any brainstem process may be responsible. One-and-a-half syndrome is due to a combined lesion of the medial longitudinal fasciculus and the abducens nucleus on the same side. The patient’s only horizontal eye movement is abduction of the eye on the other side.

1	Vertical gaze This is controlled at the level of the midbrain. The neuronal circuits affected in disorders of vertical gaze are not fully elucidated, but lesions of the rostral interstitial nucleus of the medial longitudinal fasciculus and the interstitial nucleus of Cajal cause supra-nuclear paresis of upgaze, downgaze, or all vertical eye movements. Distal basilar artery ischemia is the most common etiology. Skew deviation refers to a vertical misalignment of the eyes, usually constant in all positions of gaze. The finding has poor localizing value because skew deviation has been reported after lesions in widespread regions of the brainstem and cerebellum.

1	PARINAUd’S SYNdROME Also known as dorsal midbrain syndrome, this is a distinct supranuclear vertical gaze disorder caused by damage to the posterior commissure. It is a classic sign of hydrocephalus from aqueductal stenosis. Pineal region or midbrain tumors, cysticercosis, and stroke also cause Parinaud’s syndrome. Features include loss of upgaze (and sometimes downgaze), convergence-retraction nystagmus on attempted upgaze, downward ocular deviation (“setting sun” sign), lid retraction (Collier’s sign), skew deviation, pseudoabducens palsy, and light-near dissociation of the pupils.

1	Nystagmus This is a rhythmic oscillation of the eyes, occurring physiologically from vestibular and optokinetic stimulation or pathologically in a wide variety of diseases (Chap. 28). Abnormalities of the eyes or optic nerves, present at birth or acquired in childhood, can produce a complex, searching nystagmus with irregular pendular (sinusoidal) and jerk features. Examples are albinism, Leber’s congenital amaurosis, and bilateral cataract. This nystagmus is commonly referred to as congenital sensory nystagmus. This is a poor term because even in children with congenital lesions, the nystagmus does not appear until weeks after birth. Congenital motor nystagmus, which looks similar to congenital sensory nystagmus, develops in the absence of any abnormality of the sensory visual system. Visual acuity also is reduced in congenital motor nystagmus, probably by the nystagmus itself, but seldom below a level of 20/200.

1	JERk NYSTAgMUS This is characterized by a slow drift off the target, followed by a fast corrective saccade. By convention, the nystagmus is named after the quick phase. Jerk nystagmus can be downbeat, upbeat, horizontal (left or right), and torsional. The pattern of nystagmus may vary with gaze position. Some patients will be oblivious to their nystagmus. Others will complain of blurred vision or a subjective to-and-fro movement of the environment (oscillopsia) corresponding to the nystagmus. Fine nystagmus may be difficult to see on gross examination of the eyes. Observation of nystagmoid movements of the optic disc on ophthalmoscopy is a sensitive way to detect subtle nystagmus.

1	gAzE-EVOkEd NYSTAgMUS This is the most common form of jerk nystagmus. When the eyes are held eccentrically in the orbits, they have a natural tendency to drift back to primary position. The subject compensates by making a corrective saccade to maintain the deviated eye position. Many normal patients have mild gaze-evoked nystagmus. Exaggerated gaze-evoked nystagmus can be induced by drugs (sedatives, anticonvulsants, alcohol); muscle paresis; myasthenia gravis; demyelinating disease; and cerebellopontine angle, brainstem, and cerebellar lesions. VESTIBULAR NYSTAgMUS Vestibular nystagmus results from dysfunction of the labyrinth (Ménière’s disease), vestibular nerve, or vestibular nucleus in the brainstem. Peripheral vestibular nystagmus often occurs in discrete attacks, with symptoms of nausea and vertigo. There may be associated tinnitus and hearing loss. Sudden shifts in head position may provoke or exacerbate symptoms.

1	the craniocervical junction (Chiari malformation, basilar invagination). It also has been reported in brainstem or cerebellar stroke, lithium or anticonvulsant intoxication, alcoholism, and multiple sclerosis. Upbeat nystagmus is associated with damage to the pontine tegmentum from stroke, demyelination, or tumor. opsoclonus This rare, dramatic disorder of eye movements consists of bursts of consecutive saccades (saccadomania). When the saccades are confined to the horizontal plane, the term ocular flutter is preferred. It can result from viral encephalitis, trauma, or a paraneoplastic effect of neuroblastoma, breast carcinoma, and other malignancies. It has also been reported as a benign, transient phenomenon in otherwise healthy patients. ChaPter 42 Disorders of Smell and Taste

1	ChaPter 42 Disorders of Smell and Taste Use of the Hand-Held Ophthalmoscope Homayoun Tabandeh, Morton F. Goldberg Examination of the living human retina provides a unique opportu-nity for the direct study of nervous, vascular, and connective tissues. 40e Many systemic disorders have retinal manifestations that are valuable for screening, diagnosis, and management of these conditions. Furthermore, retinal involvement in systemic disorders, such as diabetes mellitus, is a major cause of morbidity. Early recognition by ophthalmoscopic screening is a key factor in effective treatment. Ophthalmoscopy has the potential to be one of the most “high-yield” elements of the physical examination. Effective ophthalmoscopy requires a basic understanding of ocular structures and ophthalmoscopic techniques and recognition of abnormal findings.

1	The eye consists of a shell (cornea and sclera), lens, iris diaphragm, ciliary body, choroid, and retina. The anterior chamber is the space between the cornea and the lens, and it is filled with aqueous humor. The space between the posterior aspect of the lens and the retina is filled by vitreous gel. The choroid and the retina cover the posterior two-thirds of the sclera internally. The cornea and the lens form the focusing system of the eye, while the retina functions as the photo-receptor system, translating light to neuronal signals that are in turn transmitted to the brain via the optic nerve and visual pathways. The choroid is a layer of highly vascularized tissue that nourishes the retina and is located between the sclera and the retina. The retinal pigment epithelium (RPE) layer is a monolayer of pigmented cells that are adherent to the overlying retinal photoreceptor cells. RPE plays a major role in retinal photoreceptor metabolism.

1	The important areas that are visible by ophthalmoscopy include the macula, optic disc, retinal blood vessels, and retinal periphery (Fig. 40e-1). The macula is the central part of the retina and is responsible for detailed vision (acuity) and perception of color. The macula is defined clinically as the area of the retina centered on the posterior pole of the fundus, measuring about 5 disc diameters (DD) (7–8 mm) and bordered by the optic disc nasally and the temporal vascular arcades superiorly and inferiorly. Temporally, the macula extends for about 2.5 DD from its center. The fovea, in the central part of the macula, corresponds to the site of sharpest visual acuity. It is approximately 1 DD in size and appears darker in color than the surrounding area. The center of the fovea, the foveola, has a depressed pit-like configuration measuring about 350 μm.

1	The optic disc measures about 1.5 mm and is located about 4 mm (2.5 DD) nasal to the fovea. It contains the central retinal artery and vein as they branch, a central excavation (cup), and a peripheral neural rim. Normally, the cup-to-disc ratio is less than 0.6. The cup is located temporal to the entry of the disc vessels. The normal optic disc is yellow/ pink in color. It has clear and well-defined margins and is in the same plane as the retina (Fig. 40e-2). Pathologic findings include pallor (atrophy), swelling, and enlarged cupping. The equator of the fundus is clinically defined as the area that includes the internal opening of the vortex veins. The peripheral retina extends from the equator anteriorly to the ora serrata.

1	The equator of the fundus is clinically defined as the area that includes the internal opening of the vortex veins. The peripheral retina extends from the equator anteriorly to the ora serrata. FIgURE 40e-1 Diagram showing the landmarks of the normal fundus. The macula is bounded by the superior and inferior vascular arcades and extends for 5 disc diameters (DD) temporal to the optic disc (optic nerve head). The central part of the macula (fovea) is located 2.5 DD temporal to the optic disc. The peripheral fundus is arbitrarily defined as the area extending anteriorly from the opening of the vortex veins to the ora serrata (the juncture between the retina and ciliary body). (Drawing courtesy of Juan R. Garcia. Used with permission from Johns Hopkins University.) CHAPTER 40e Use of the Hand-Held Ophthalmoscope

1	CHAPTER 40e Use of the Hand-Held Ophthalmoscope FIgURE 40e-2 Photograph of a normal left optic disc illustrating branching of the central retinal vein and artery, a physiologic cup, surface capillaries, and distinct margin. The cup is located temporal to the entry of the disc vessels. (From H Tabandeh, MF Goldberg: Retina in Systemic Disease: A Color Manual of Ophthalmoscopy. New York, Thieme, 2009.) There are a number of ways to visualize the retina, including direct ophthalmoscopy, binocular indirect ophthalmoscopy, and slit-lamp biomicroscopy. Most nonophthalmologists prefer direct ophthalmoscopy, performed with a hand-held ophthalmoscope, because the technique is simple to master and the device is very portable. Ophthalmologists often use slit-lamp biomicroscopy and indirect ophthalmoscopy to obtain a more extensive view of the fundus.

1	Direct ophthalmoscopes are simple hand-held devices that include a small light source for illumination, a viewing aperture through which the examiner looks at the retina, and a lens dial used for correction of the examiner’s and the patient’s refractive errors. A more recent design, the PanOptic ophthalmoscope, provides a wider field of view. How to Use a Direct Ophthalmoscope Good alignment is the key. The goal is to align the examiner’s eye with the viewing aperture of the ophthalmoscope, the patient’s pupil, and the area of interest on the retina. Both the patient and the examiner should be in a comfortable position (sitting or lying for the patient, sitting or standing for the examiner). Dilating the pupil and dimming the room lights make the examination easier. Steps for performing direct ophthalmoscopy are summarized in Table 40e-1.

1	The PanOptic ophthalmoscope is a type of direct ophthalmoscope that is designed to provide a wider view of the fundus and has slightly more magnification than the standard direct ophthalmoscope. Steps for using the PanOptic Ophthalmoscope are summarized in Table 40e-2. • Instruct the patient to remove glasses, keep the head straight, and to look steadily at a distant target straight in front. You may keep or remove your own glasses. Position your head at the same level as the patient’s head. • Use your right eye and right hand to examine the patient’s right eye, and use your left eye and left hand to examine the patient’s left eye. the ophthalmoscope light as a pen light, briefly examine the external features of the eye, including lashes, lid margins, conjunctiva, sclera, iris, and pupil shape, size, and reactivity. the ophthalmoscope light into the patient’s pupil at arm’s length and observe the red reflex. Note abnormalities of the red reflex such as an opacity of the media.

1	the ophthalmoscope light into the patient’s pupil at arm’s length and observe the red reflex. Note abnormalities of the red reflex such as an opacity of the media. up a +10 D lens in the lens wheel, while examining the eye from 10 cm, allows magnified viewing of the anterior segment of the eye. the power of the lens in the wheel to zero, and move closer to the patient. Identify the optic disc by pointing the ophthalmoscope about 15° nasally or by following a blood vessel toward the apex of any branching. If the retina is out of focus, turn the lens dial either way, without moving your head. If the disc becomes clearer, keep turning until best focus is achieved; if it becomes more blurred, turn the dial the other way. • Once you visualize the optic nerve, note its shape, size, color, margins, and the cup. Also note the presence of any venous pulsation or surrounding pigment, such as a choroidal or scleral crescent.

1	• Next, examine the macula. The macula is the area between the superior and inferior temporal vascular arcades, and its center is the fovea. You can examine the macula by pointing your ophthalmoscope about 15° temporal to the optic disc. Alternatively, ask the patient to look into the center of the light. Note the foveal reflex and the presence of any hemorrhage, exudate, abnormal blood vessels, scars, deposits, or other abnormalities. • Examine the retinal blood vessels by re-identifying the optic disc and following each of the four main branches away from the disc. The veins are dark red and relatively large. The arteries are narrower and bright red. • Ask the patient to look in the eight cardinal directions to allow you to view the peripheral fundus. In a patient with a well-dilated pupil, it is possible to visualize as far as the equator.

1	• Ask the patient to look in the eight cardinal directions to allow you to view the peripheral fundus. In a patient with a well-dilated pupil, it is possible to visualize as far as the equator. PART 2 Cardinal Manifestations and Presentation of Diseases the ophthalmoscope: Look through the scope at an object that is at least 10 to 15 feet away. Sharpen the image of the object by using the focusing wheel. Set the aperture dial to “small” or home position. the scope on, and adjust the light intensity to “Maximum.” the patient to look straight ahead. Move the ophthalmoscope close to the patient until the eyecup touches the patient’s brow. The eye-cup should be compressed about half its length to optimize the view. the optic disc. the fundus as described in Table 40e-1. Common age-related changes include diminished foveal light reflex, drusen (small yellow subretinal deposits), mild RPE atrophy, and pigment clumping.

1	Retinal hemorrhages may take various shapes and sizes depending on their location within the retina (Figs. 40e-3 and 40e-4). Flame-shaped hemorrhages are located at the level of the superficial nerve fiber layer and represent bleeding from the inner capillary network of the retina. A white-centered hemorrhage is a superficial flame-shaped hemorrhage with an area of central whitening, often representing edema, focal necrosis, or cellular infiltration. Causes of white-centered hemorrhage include bacterial endocarditis and septicemia (Roth spots), lymphoproliferative disorders, diabetes mellitus, hypertension, anemia, and collagen vascular disorders. Dot hemorrhages are small, round, superficial hemorrhages that also originate from the superficial capillary network of the retina. They resemble microaneurysms. Blot hemorrhages are slightly larger in size, dark, and intraretinal. They represent bleeding from the deep capillary network of the retina. Subhyaloid hemorrhages are variable in

1	microaneurysms. Blot hemorrhages are slightly larger in size, dark, and intraretinal. They represent bleeding from the deep capillary network of the retina. Subhyaloid hemorrhages are variable in shape and size and tend to be larger than other types of hemorrhages. They often have a fluid level (“boat-shaped” hemorrhage) and are located within the space between the vitreous and the retina. Subretinal hemorrhages are located deep (external) to the retina. The retinal vessels can be seen crossing over (internal to) such hemorrhages. Subretinal hemorrhages are variable in size and most commonly are caused by choroidal neovascularization (e.g., wet macular degeneration).

1	FIgURE 40e-3 Superficial flame-shaped hemorrhages, dot hem-orrhages, and microaneurysms in a patient with nonproliferative diabetic retinopathy. patientwithchronicleukemia. Conditions associated with retinal hemorrhages include diseases causing retinal microvasculopathy (Table 40e-3), retinitis, retinal macroaneurysm, papilledema, subarachnoid hemorrhage (Terson’s syndrome), Valsalva retinopathy, trauma (ocular injury, head injury, compression injuries of chest and abdomen, shaken baby syndrome, strangulation), macular degeneration, and posterior vitreous detachment. Hyperviscosity states may produce dot and blot hemorrhages, dilated veins (“string of sausages” appearance), optic disc edema, and exudates; similar changes can occur with adaptation to high altitude in mountain climbers.

1	Microaneurysms are outpouchings of the retinal capillaries, appearing as red dots (similar to dot hemorrhages) and measuring 15–50 μm. Microaneurysms have increased permeability and may bleed or leak, resulting in localized retinal hemorrhage or edema. A micro-aneurysm ultimately thromboses and disappears within 3–6 months. Microaneurysms may occur in any condition that causes retinal microvasculopathy (Table 40e-3). microemboli, e.g., talc retinopathy secondary to intravenous drug abuse, septicemia, endocarditis, Purtscher’s retinopathy artery disease, carotid-cavernous fistula, aortic arch syndrome retinopathy, head/neck irradiation

1	Hard exudates are well-circumscribed, shiny, yellow deposits located within the retina. They arise at the margins of areas of retinal edema and indicate increased capillary permeability. Hard exudates contain lipoproteins and lipid-laden macrophages. They may clear spontaneously or following laser photocoagulation, often within 6 months. Hard exudates may occur in isolation or may be scattered throughout the fundus. They may occur in a circular (circinate) pattern centered around an area of leaking microaneurysms. A macular star consists of a radiating, star-shaped pattern of hard exudates that is characteristically seen in severe systemic hypertension and in neuroretinitis associated with cat-scratch disease. Conditions associated with hard exudates include those causing retinal microvasculopathy (Table 40e– 3), papilledema, neuroretinitis such as cat-scratch disease and Lyme disease, retinal vascular lesions (macroaneurysm, retinal capillary hemangioma, Coats’ disease), intraocular

1	(Table 40e– 3), papilledema, neuroretinitis such as cat-scratch disease and Lyme disease, retinal vascular lesions (macroaneurysm, retinal capillary hemangioma, Coats’ disease), intraocular tumors, and wet age-related macular degeneration. Drusen may be mistaken for hard exudates on ophthalmoscopy. Unlike hard exudates, drusen are nonrefractile subretinal deposits with blurred margins. They are usually seen in association with age-related macular degeneration.

1	Cotton-wool spots are yellow/white superficial retinal lesions with indistinct feathery borders measuring 0.25–1 DD in size (Fig. 40e-5). They represent areas of edema within the retinal nerve fiber layer due to focal ischemia. Cotton-wool spots usually resolve spontaneously within 3 months. If the underlying ischemic condition persists, new lesions can develop in different locations. Cotton-wool spots often occur in conjunction with retinal hemorrhages and microaneurysms and represent retinal microvasculopathy caused by a number of systemic conditions (Table 40e-3). They may occur in isolation in HIV retinopathy, systemic lupus erythematosus, anemia, bodily trauma, other systemic conditions (Purtscher’s/Purtscher’s-like retinopathy), and interferon therapy.

1	Retinal neovascular complexes are irregular meshworks of fine blood vessels that grow in response to severe retinal ischemia or chronic inflammation (Fig. 40e-6). They may occur on or adjacent to the optic disc or elsewhere in the retina. Neovascular complexes are very CHAPTER 40e Use of the Hand-Held Ophthalmoscope FIgURE 40e-5 Cotton-wool spots, yellow-white superficial lesions with characteristic feathery borders, in a patient with hypertensive retinopathy. (From H Tabandeh, MF Goldberg: Retina in Systemic Disease: A Color Manual of Ophthalmoscopy. New York, Thieme, 2009.) PART 2 Cardinal Manifestations and Presentation of Diseases FIgURE 40e-6 Optic disc neovascularization in a patient with severeproliferativediabeticretinopathy.Multiplehardexudatesare also present.

1	PART 2 Cardinal Manifestations and Presentation of Diseases FIgURE 40e-6 Optic disc neovascularization in a patient with severeproliferativediabeticretinopathy.Multiplehardexudatesare also present. fragile and have a high risk for hemorrhaging, often causing visual loss. Diseases associated with retinal neovascularization include conditions that cause severe retinal microvasculopathy, especially diabetic and sickle cell retinopathies (Table 40e-3), intraocular tumors, intra-ocular inflammation (sarcoidosis, chronic uveitis), and chronic retinal detachment. Common sources of retinal emboli include carotid artery atheromatous plaque, cardiac valve and septal abnormalities, cardiac arrhythmias, atrial myxoma, bacterial endocarditis, septicemia, fungemia, and intravenous drug abuse.

1	Platelet emboli are yellowish in appearance and conform to the shape of the blood vessel. They usually originate from an atheromatous plaque within the carotid artery and can cause transient loss of vision (amaurosis fugax). Cholesterol emboli, otherwise termed Hollenhorst plaques, are yellow crystalline deposits that are commonly found at the bifurcations of the retinal arteries and may be associated with amaurosis fugax. Calcific emboli have a pearly white appearance, are larger than the platelet and cholesterol emboli, and tend to lodge in the larger retinal arteries in or around the optic disc. Calcific emboli often result in retinal arteriolar occlusion. Septic emboli can cause white-centered retinal hemorrhages (Roth spots), retinal microabscesses, and endogenous endophthalmitis. Fat embolism and amniotic fluid embolism are characterized by multiple small vessel occlusions, typically causing cotton-wool spots and few hemorrhages (Purtscher’s-like retinopathy). Talc embolism

1	Fat embolism and amniotic fluid embolism are characterized by multiple small vessel occlusions, typically causing cotton-wool spots and few hemorrhages (Purtscher’s-like retinopathy). Talc embolism occurs with intravenous drug abuse and is characterized by multiple refractile deposits within the small retinal vessels. Any severe form of retinal artery embolism may result in retinal ischemia and its sequelae, including retinal neovascularization.

1	Cherry red spot at the macula is the term used to describe the dark red appearance of the central foveal area in comparison to the surrounding macular region (Fig. 40e-7). This appearance is most commonly due to a relative loss of transparency of the parafoveal retina resulting from ischemic cloudy swelling or storage of macromolecules within the ganglion cell layer. Diseases associated with a cherry red spot at the macula include central retinal artery occlusion, sphingolipidoses, and mucolipidoses. FIgURE 40e-7 Cherry red spot at the macula and cloudy swelling of the macula in a patient with central retinal artery occlusion due to embolus originating from a carotid artery atheromatous plaque.

1	FIgURE 40e-7 Cherry red spot at the macula and cloudy swelling of the macula in a patient with central retinal artery occlusion due to embolus originating from a carotid artery atheromatous plaque. Retinal crystals appear as fine, refractile, yellow-white deposits. Associated conditions include infantile cystinosis, primary hyperoxaluria, secondary oxalosis, Sjögren-Larson syndrome, intravenous drug abuse (talc retinopathy), and drugs such as tamoxifen, canthaxanthin, nitrofurantoin, methoxyflurane, and ethylene glycol. Crystals may also be seen in primary retinal diseases such as juxtafoveal telangiectasia, gyrate atrophy, and Bietti’s crystalline degeneration. Old microemboli may mimic retinal crystals.

1	Vascular sheathing appears as a yellow-white cuff surrounding a retinal artery or vein (Fig. 40e-8). Diseases associated with retinal vascular sheathing include sarcoidosis, tuberculosis, toxoplasmosis, syphilis, HIV, retinitis (cytomegalovirus, herpes zoster, and herpes simplex), Lyme disease, cat-scratch disease, multiple sclerosis, chronic leukemia, amyloidosis, Behçet’s disease, retinal vasculitis, retinal vascular occlusion, and chronic uveitis. FIgURE 40e-8 Vascular sheathing over the optic disc in a patient with neurosarcoidosis. Retinal detachment is the separation of the retina from the underlying RPE. There are three main types: (1) serous/exudative, (2) tractional, and (3) rhegmatogenous retinal detachment.

1	Retinal detachment is the separation of the retina from the underlying RPE. There are three main types: (1) serous/exudative, (2) tractional, and (3) rhegmatogenous retinal detachment. In serous retinal detachment, the location of the subretinal fluid is position-dependent, characteristically gravitating to the lowermost part of the fundus (shifting fluid sign), and retinal breaks are absent. Diseases associated with serous/exudative retinal detachment include severe systemic hypertension, dural arteriovenous shunt, retinal vascular anomalies, hyperviscosity syndromes, papilledema, posterior uveitis, scleritis, orbital inflammation, and intraocular neoplasms such as choroidal melanoma, choroidal metastasis, lymphoma, and multiple myeloma.

1	Tractional retinal detachment is caused by internal traction on the retina in the absence of a retinal break. The retina in the area of detachment is immobile and concaved internally. Fibrovascular proliferation is a frequent associated finding. Conditions associated with tractional retinal detachment include vascular proliferative retinopathies such as severe proliferative diabetic retinopathy, branch retinal vein occlusion, sickle cell retinopathy, and retinopathy of prematurity. Ocular trauma, proliferative vitreoretinopathy, and intraocular inflammation are other causes of a tractional retinal detachment.

1	Rhegmatogenous retinal detachment is caused by the presence of a retinal break, allowing fluid from the vitreous cavity to gain access to the subretinal space. The surface of the retina is usually convex forward. Rhegmatogenous retinal detachment has a corrugated appearance, and undulates with eye movement. Causes of retinal breaks include posterior vitreous detachment, severe vitreoretinal traction, trauma, intraocular surgery, retinitis, and atrophic holes.

1	Optic disc swelling is abnormal elevation of the optic disc with blurring of its margins (Fig. 40e-9). The term “papilledema” is used to describe swelling of the optic disc secondary to elevation of intra-cranial pressure. In papilledema, the normal venous pulsation at the disc is characteristically absent. The differential diagnosis of optic disc swelling includes papilledema, anterior optic neuritis (papillitis), central retinal vein occlusion, anterior ischemic optic neuropathy, toxic optic neuropathy, hereditary optic neuropathy, neuroretinitis, diabetic papillopathy, hypertension (Fig. 40e-10), respiratory failure, carotid-cavernous fistula, optic disc nerve infiltration (glioma, lymphoma, leukemia, sarcoidosis, and granulomatous infections), ocular hypotony, chronic intraocular inflammation, optic disc drusen (pseudopapilledema), and high hypermetropia (pseudopapilledema). FIgURE 40e-10 Optic disc edema and retinal hemorrhages in a patient with malignant hypertension.

1	FIgURE 40e-10 Optic disc edema and retinal hemorrhages in a patient with malignant hypertension. Choroidal mass lesions appear thickened and may or may not be associated with increased pigmentation. Pigmented mass lesions include choroidal nevus (usually flat), choroidal malignant melanoma (Fig. 40e-11), and melanocytoma. Nonpigmented lesions include amelanotic choroidal melanoma, choroidal metastasis, retinoblastoma, capillary hemangioma, granuloma (e.g., Toxocara canis), choroidal detachment, choroidal hemorrhage, and wet age-related macular degeneration. Other rare tumors that may be visible on ophthalmoscopy include CHAPTER 40e Use of the Hand-Held Ophthalmoscope FIgURE 40e-9 Optic disc swelling in a patient with papilledema due to idiopathic intracranial hypertension. The optic disc is hyper- emic,withindistinctmargins.Superficialhemorrhagesarepresent.

1	FIgURE 40e-9 Optic disc swelling in a patient with papilledema due to idiopathic intracranial hypertension. The optic disc is hyper- emic,withindistinctmargins.Superficialhemorrhagesarepresent. FIgURE 40e-11 Choroidal malignant melanoma. The lesion is highly elevated and pigmented, and has subretinal orange pigment deposits characteristic for malignant melanoma. 40e-6 osteoma, astrocytoma (e.g., tuberous sclerosis), neurilemmoma, and leiomyoma. The differential diagnosis of flat pigmented lesions of the fundus is summarized in Table 40e-4. The appearance of chorioretinal scarring from old Toxoplasma chorioretinitis is shown in Fig. 40e-12. FIgURE 40e-12 Chorioretinal scarring due to old Toxoplasma cho-rioretinitis. The lesion is flat and pigmented. Areas of hypopigmenta-tion are also present.

1	FIgURE 40e-12 Chorioretinal scarring due to old Toxoplasma cho-rioretinitis. The lesion is flat and pigmented. Areas of hypopigmenta-tion are also present. PART 2 Cardinal Manifestations and Presentation of Diseases retinopathy in systemic diseases: Usher’s syndrome, abetalipoproteinemia, Refsum’s disease, Kearns-Sayre syndrome, Alström’s syndrome, Cockayne’s syndrome, Friedreich’s ataxia, mucopolysaccharidoses, paraneoplastic syndrome • Infections: congenital rubella (salt and pepper retinopathy), congenital • Infections: Toxoplasma gondii, Toxocara canis, syphilis, cytomegalovirus, herpes zoster and herpes simplex viruses, west Nile virus, histoplasmosis, parasitic infection • Choroiditis: sarcoidosis, sympathetic ophthalmia, Vogt-Koyanagi-Harada infarct: severe hypertension, sickle cell hemoglobinopathies • Trauma, cryotherapy, laser photocoagulation scars • Drugs: chloroquine/hydroxychloroquine, thioridazine, chlorpromazine, hypertrophy of the retinal pigment epithelium

1	Video Library of Neuro-Ophthalmology Shirley H. Wray The proper control of eye movements requires the coordinated activity of many different anatomic structures in the peripheral and central nervous system, and in turn, manifestations of a diverse array 41e of neurologic and medical disorders are revealed as disorders of eye movement. In this remarkable video collection, an introduction to distinctive eye movement disorders encountered in the context of neuromuscular, paraneoplastic, demyelinating, neurovascular, and neurodegenerative disorders is presented. Cases with Multiple Sclerosis Video 41e-1 Fisher’s One-and-a-Half Syndrome (ID164-2) Video 41e-2 A Case of Ocular Flutter (ID166-2) Video 41e-3 Downbeat Nystagmus and Periodic Alternating Nystagmus (ID168-6) Video 41e-4 Bilateral Internuclear Ophthalmoplegia (ID933-1)

1	Cases with Myasthenia Gravis or Mitochondrial Myopathy Video 41e-5 Unilateral Ptosis: Myasthenia Gravis (Thymic Tumor) (ID163-1) Video 41e-6 Progressive External Ophthalmoplegia (Progressive External Ophthalmoplegia: Mitochondrial Cytopathy) (ID906-2) Cases with Paraneoplastic disease Video 41e-7 Paraneoplastic Upbeat Nystagmus, Cancer of the Pancreas, Positive Anti-Hu Antibody (ID212-3) Video 41e-8 Paraneoplastic Ocular Flutter, Small-Cell Adenocarcinoma of the Lung, Negative Marker (ID936-7) Video 41e-9 Opsoclonus/Flutter, Bilateral Sixth Nerve Palsy, Adenocarcinoma of the Breast, Negative Marker (ID939-8) Cases with Fisher’s Syndrome 41e-1 Video 41e-10 Bilateral Ptosis: Facial Diplegia, Total External Ophthalmoplegia, Positive Anti-GQ1b Antibody (ID944-1) Cases with Vascular disease Video 41e-11 Retinal Emboli (Film or Fundus) (ID16-1) Video 41e-12 Third Nerve Palsy (Microinfarct) (ID939-2)

1	Cases with Vascular disease Video 41e-11 Retinal Emboli (Film or Fundus) (ID16-1) Video 41e-12 Third Nerve Palsy (Microinfarct) (ID939-2) Case with Neurodegenerative disease Video 41e-13 Apraxia of Eyelid Opening (Progressive Supranuclear Palsy) (ID932-3) Case of Thyroid-Associated ophthalmopathy Video 41e-14 Restrictive Orbitopathy of Graves’ Disease, Bilateral Exophthalmos (ID925-4) Case with Wernicke’s encephalopathy Video 41e-15 Bilateral Sixth Nerve Palsies (ID 163-3) Case with the Locked-in-Syndrome Video 41e-16 Ocular Dipping (ID 4-1) The Video Library of Neuro-Ophthalmology shows a number of cases with eye movement disorders. All the clips are taken from Dr. Shirley Wray’s collection on the NOVEL website. To access go to: http://NOVEL.utah.edu/Wray http://Respitory.Countway.Harvard.edu/Wray and/or to her book Shirley H. Wray, MD, PhD, Oxford University Press, 2014. CHAPTER 41e Video Library of Neuro-Ophthalmology Disorders of Smell and Taste

1	Shirley H. Wray, MD, PhD, Oxford University Press, 2014. CHAPTER 41e Video Library of Neuro-Ophthalmology Disorders of Smell and Taste Richard L. Doty, Steven M. Bromley

1	All environmental chemicals necessary for life enter the body by the nose and mouth. The senses of smell (olfaction) and taste (gustation) monitor such chemicals, determine the flavor and palatability of foods and beverages, and warn of dangerous environmental conditions, including fire, air pollution, leaking natural gas, and bacteria-laden foodstuffs. These senses contribute significantly to quality of life and, when dysfunctional, can have untoward physical and psychological consequences. A basic understanding of these senses in health and disease is critical for the physician, because thousands of patients present to doctors’ offices each year with complaints of chemosensory dysfunction. Among the more important recent developments in neurology is the discovery that decreased smell function is among the first signs, if not the first sign, of such neurodegenerative diseases as Parkinson’s disease (PD) and Alzheimer’s disease (AD), signifying their “presymptomatic” phase.

1	ANATOMY AND PHYSIOLOgY Olfactory System Odorous chemicals enter the front of nose during inhalation and active sniffing, as well as the back of the nose (nasopharynx) during deglutition. After reaching the highest recesses of the nasal cavity, they dissolve in the olfactory mucus and diffuse or are actively transported by specialized proteins to receptors located on the cilia of olfactory receptor cells. The cilia, dendrites, cell bodies, and proximal axonal segments of these bipolar cells are located within a unique neuroepithelium covering the cribriform plate, the superior nasal septum, superior turbinate, and sectors of the middle turbinate (Fig. 42-1). Each of the ∼6 million bipolar receptor cells expresses only one of ∼450 receptor protein types, most of which respond to more than a single chemical. When damaged, the receptor cells can be replaced by stem cells near the basement membrane. Unfortunately, such replacement is often incomplete.

1	After coalescing into bundles surrounded by glia-like ensheathing cells (termed fila), the receptor cell axons pass through the cribriform plate to the olfactory bulbs, where they synapse with dendrites of other cell types within the glomeruli (Fig. 42-2). These spherical structures, which make up a distinct layer of the olfactory bulb, are a site of convergence of information, because many more fibers enter than leave them. Receptor cells that express the same type of receptor project to the same glomeruli, effectively making each glomerulus a functional unit. The major projection neurons of the olfactory system—the mitral and tufted cells—send primary dendrites into the glomeruli, connecting not only with the incoming receptor cell axons, but with dendrites of periglomerular cells. The activity of the mitral/tufted cells is modulated by the periglomerular cells, secondary dendrites from other mitral/tufted cells, and granule cells, the most numerous cells of the bulb. The latter

1	activity of the mitral/tufted cells is modulated by the periglomerular cells, secondary dendrites from other mitral/tufted cells, and granule cells, the most numerous cells of the bulb. The latter cells, which are largely GABAergic, receive inputs from central brain structures and modulate the output of the mitral/tufted cells. Interestingly, like the olfactory receptor cells, some cells within the bulb undergo replacement. Thus, neuroblasts formed within the anterior subventricular zone of the brain migrate along the rostral migratory stream, ultimately becoming granule and periglomerular cells.

1	The axons of the mitral and tufted cells synapse within the primary olfactory cortex (POC) (Fig. 42-3). The POC is defined as those cortical structures that receive direct projections from the olfactory bulb, most notably the piriform and entorhinal cortices. Although olfaction is unique in that its initial afferent projections bypass the thalamus, persons with damage to the thalamus can exhibit olfactory deficits, particularly ones of odor identification. Such deficits likely reflect the involvement of thalamic connections between the primary olfactory cortex and the orbitofrontal cortex (OFC), where odor identification occurs. The close anatomic ties between the olfactory system and the CHAPTER 42 Disorders of Smell and Taste FIguRE 42-3 Anatomy of the base of the brain showing the primary olfactory cortex. PART 2 Cardinal Manifestations and Presentation of Diseases

1	CHAPTER 42 Disorders of Smell and Taste FIguRE 42-3 Anatomy of the base of the brain showing the primary olfactory cortex. PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 42-1 Anatomy of the olfactory neural pathways, showing the distribution of olfactory receptors in the roof of the nasal cavity. (Copyright David Klemm, Faculty and Curriculum Support [FACS], Georgetown University Medical Center; used with permission.) amygdala, hippocampus, and hypothalamus help to explain the intimate associations between odor perception and cognitive functions such as memory, motivation, arousal, autonomic activity, digestion, and sex.

1	Taste System Tastants are sensed by specialized receptor cells present within taste buds—small grapefruit-like segmented structures located on the lateral margins and dorsum of the tongue, roof of the mouth, pharynx, larynx, and superior esophagus (Fig. 42-4). Lingual taste buds are imbedded in well-defined protuberances, termed fungi-form, foliate, and circumvallate papillae. After dissolving in a liquid, tastants enter the opening of the taste bud—the taste pore—and bind to receptors on microvilli, small extensions of receptor cells within each taste bud. Such binding changes the electrical potential across the taste cell, resulting in neurotransmitter release onto the first-order taste neurons. Although humans have ∼7500 taste buds, not all harbor

1	FIguRE 42-2 Schematic of the layers and wiring of the olfactory bulb. Each receptor type (red, green, blue) projects to a common glomerulus. The neural activity within each glomerulus is modulated by periglomerular cells. The activity of the primary projection cells, the mitral and tufted cells, is modulated by granule cells, periglomerular cells, and secondary dendrites from adjacent mitral and tufted cells.

1	(From www.med.yale.edu/neurosurg/treloar/index.html.) taste-sensitive cells; some contain only one class of receptor (e.g., cells responsive only to sugars), whereas others contain cells sensitive to more than one class. The number of taste receptor cells per taste bud ranges from zero to well over 100. A small family of three G-proteincoupled receptors (GPCRs), namely T1R1, T1R2, and T1R3, mediate sweet and umami taste sensations. Bitter sensations, on the other hand, depend on T2R receptors, a family of ∼30 GPCRs expressed on cells different from those that express the sweet and umami receptors. T2Rs sense a wide range of bitter substances but do not distinguish among them. Sour tastants are sensed by the PKD2L1 receptor, a member of the transient receptor potential protein (TRP) family. Perception of salty sensations, such as induced by sodium chloride, arises from the entry of Na+ ions into the cells via specialized membrane channels, such as the amiloride-sensitive Na+ channel.

1	Recent studies have found that both bitter and sweet taste-related receptors are also present elsewhere in the body, most notably in the Circumvallate Foliate Fungiform Taste bud Taste pore TRC Taste bud Taste bud FIguRE 42-4 Schematic of the taste bud and its opening (pore), as well as the location of buds on the three major types of papillae: fungi-form (anterior), foliate (lateral), and circumvallate (posterior).

1	alimentary and respiratory tracts. This important discovery generalizes the concept of taste-related chemoreception to areas of the body beyond the mouth and throat, with α-gustducin, the taste-specific G-protein α-subunit, expressed in so-called brush cells found specifically within the human trachea, lung, pancreas, and gallbladder. These brush cells are rich in nitric oxide (NO) synthase, known to defend against xenobiotic organisms, protect the mucosa from acid-induced lesions, and, in the case of the gastrointestinal tract, stimulate vagal and splanchnic afferent neurons. NO further acts on nearby cells, including enteroendocrine cells, absorptive or secretory epithelial cells, mucosal blood vessels, and cells of the immune system. Members of the T2R family of bitter receptors and the sweet receptors of the T1R family have been identified within the gastrointestinal tract and in enteroendocrine cell lines. In some cases, these receptors are important for metabolism, with the T1R3

1	sweet receptors of the T1R family have been identified within the gastrointestinal tract and in enteroendocrine cell lines. In some cases, these receptors are important for metabolism, with the T1R3 receptors and gustducin playing decisive roles in the sensing and transport of dietary sugars from the intestinal lumen into absorptive enterocytes via a sodium-dependent glucose transporter and in regulation of hormone release from gut enteroendocrine cells. In other cases, these receptors may be important for airway protection, with a number of T2R bitter receptors in the motile cilia of the human airway that responded to bitter compounds by increasing their beat frequency. One specific T2R38 taste receptor is expressed in human upper respiratory epithelia and responds to acyl-monoserine lactone quorum-sensing molecules secreted by Pseudomonas aeruginosa and other gram-negative bacteria. Differences in T2R38 functionality, as related to TAS2R38 genotype, correlate with susceptibility to

1	quorum-sensing molecules secreted by Pseudomonas aeruginosa and other gram-negative bacteria. Differences in T2R38 functionality, as related to TAS2R38 genotype, correlate with susceptibility to upper respiratory infections in humans.

1	Taste information is sent to the brain via three cranial nerves (CNs): CN VII (the facial nerve, which involves the intermediate nerve with its branches, the greater petrosal and chorda tympani nerves), CN IX (the glossopharyngeal nerve), and CN X (the vagus nerve) (Fig. 42-5). CN VII innervates the anterior tongue and all of the soft palate, CN IX innervates the posterior tongue, and CN X innervates the laryngeal surface of the epiglottis, larynx, and proximal portion of the esophagus. The mandibular branch of CN V (V3) conveys somatosensory information (e.g., touch, burning, cooling, irritation) to the brain. Although not technically a gustatory nerve, CN V shares primary nerve routes with many of the gustatory nerve fibers and adds temperature, texture,

1	CHAPTER 42 Disorders of Smell and Taste pungency, and spiciness to the taste experience. The chorda tympani nerve is famous for taking a recurrent course through the facial canal in the petrosal portion of the temporal bone, passing through the middle ear, and then exiting the skull via the petrotympanic fissure, where it joins the lingual nerve (a division of CN V) near the tongue. This nerve also carries parasympathetic fibers to the submandibular and sublingual glands, whereas the greater petrosal nerve supplies the palatine glands, thereby influencing saliva production. The axons of the projection cells, which synapse with taste buds, enter the rostral portion of the nucleus of the solitary tract (NTS) FIguRE 42-5 Schematic of the cranial nerves (CNs) that mediate taste function, including the chorda tympani nerve (CN VII), the glos-sopharyngeal nerve (CN IX), and the vagus nerve (CN X).

1	FIguRE 42-5 Schematic of the cranial nerves (CNs) that mediate taste function, including the chorda tympani nerve (CN VII), the glos-sopharyngeal nerve (CN IX), and the vagus nerve (CN X). 214 within the medulla of the brainstem (Fig. 42-5). From the NTS, neurons then project to a division of the ventroposteromedial thalamic nucleus (VPM) via the medial lemniscus. From here, projections are made to the rostral part of the frontal operculum and adjoining insula, a brain region considered the primary taste cortex (PTC). Projections from the PTC then go to the secondary taste cortex, namely the caudolateral OFC. This brain region is involved in the conscious recognition of taste qualities. Moreover, because it contains cells that are activated by several sensory modalities, it is likely a center for establishing “flavor.”

1	The ability to smell is influenced, in everyday life, by such factors as age, gender, general health, nutrition, smoking, and reproductive state. Women typically outperform men on tests of olfactory function and retain normal smell function to a later age than do men. Significant decrements in the ability to smell are present in over 50% of the population between 65 and 80 years of age and in 75% of those 80 years of age and older (Fig. 42-6). Such presbyosmia helps to explain why many elderly report that food has little flavor, a problem that can result in nutritional disturbances. This also helps to explain why a disproportionate number of elderly die in accidental gas poisonings. A relatively complete listing of conditions and disorders that have been associated with olfactory dysfunction is presented in Table 42-1.

1	Aside from aging, the three most common identifiable causes of long-lasting or permanent smell loss seen in the clinic are, in order of frequency, severe upper respiratory infections, head trauma, and chronic rhinosinusitis. The physiologic basis for most head trauma– related losses is the shearing and subsequent scarring of the olfactory fila as they pass from the nasal cavity into the brain cavity. The cribriform plate does not have to be fractured or show pathology for smell loss to be present. Severity of trauma, as indexed by a poor Glasgow Coma Scale score on presentation and the length of posttraumatic amnesia, is associated with higher risk of olfactory impairment. Less than 10% of posttraumatic anosmic patients will recover age-related normal function over time. This increases to nearly 25% of those with less-than-total loss. Upper respiratory infections, such as those associated with the common cold, influenza, pneumonia, or HIV, can directly and permanently harm the

1	to nearly 25% of those with less-than-total loss. Upper respiratory infections, such as those associated with the common cold, influenza, pneumonia, or HIV, can directly and permanently harm the olfactory epithelium by decreasing receptor cell number, damaging cilia on remaining receptor cells, and inducing the replacement of sensory epithelium with respiratory epithelium. The smell loss associated with chronic rhinosinusitis is

1	PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 42-6 Scores on the University of Pennsylvania Smell Identification Test (UPSIT) as a function of subject age and sex. Numbers by each data point indicate sample sizes. Note that women identify odorants better than men at all ages. (From RL Doty et al: Science 226:1421, 1984. Copyright © 1984 American Association for the Advancement of Science.)

1	DiSoRDERS AnD ConDiTionS ASSoCiATED wiTH CoMPRoMiSED oLfACToRy funCTion, AS MEASuRED By oLfACToRy TESTing 22q11 deletion syndrome Liver disease AIDS/HIV infection Lubag disease Adenoid hypertrophy Medications Adrenal cortical insufficiency Migraine Age Multiple sclerosis Alcoholism Multi-infarct dementia Allergies Myasthenia gravis Alzheimer’s disease Narcolepsy with cataplexy Amyotrophic lateral sclerosis (ALS) Neoplasms, cranial/nasal Anorexia nervosa Nutritional deficiencies Asperger’s syndrome Obstructive pulmonary disease Ataxias Obesity Attention deficit/hyperactivity Obsessive compulsive disorder disease Pregnancy Congenital Pseudohypoparathyroidism Cushing’s syndrome Psychopathy Cystic fibrosis Radiation (therapeutic, cranial) Degenerative ataxias REM behavior disorder related to disease severity, with most loss occurring in cases where rhinosinusitis and polyposis are both present. Although systemic glucocorticoid therapy can usually induce short-term functional improvement,

1	disease severity, with most loss occurring in cases where rhinosinusitis and polyposis are both present. Although systemic glucocorticoid therapy can usually induce short-term functional improvement, it does not, on average, return smell test scores to normal, implying that chronic permanent neural loss is present and/ or that short-term administration of systemic glucocorticoids does not completely mitigate the inflammation. It is well established that microinflammation in an otherwise seemingly normal epithelium can influence smell function.

1	A number of neurodegenerative diseases are accompanied by olfactory impairment, including PD, AD, Huntington’s disease, Down’s syndrome, parkinsonism-dementia complex of Guam, dementia with Lewy bodies (DLB), multiple system atrophy, corticobasal degeneration, and frontotemporal dementia; smell loss can also occur in multiple sclerosis (MS) and idiopathic rapid eye movement (REM) behavioral sleep disorder (iRBD). Olfactory impairment in PD often predates the clinical diagnosis by at least 4 years. In staged cases, studies of the sequence of formation of abnormal α-synuclein aggregates and Lewy bodies suggest that the olfactory bulbs may be, along with the dorsomotor nucleus of the vagus, the first site of neural damage in PD. In postmortem studies of patients with very mild “presymptomatic” signs of AD, poorer smell function has been associated with higher levels of AD-related pathology. Smell loss is more marked in patients with early clinical manifestations of DLB than in those with

1	signs of AD, poorer smell function has been associated with higher levels of AD-related pathology. Smell loss is more marked in patients with early clinical manifestations of DLB than in those with mild AD. Interestingly, smell loss is minimal or nonexistent in progressive supranuclear palsy and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism. In MS, the olfactory disturbance varies as a function of the plaque activity within the frontal and temporal lobes.

1	The smell loss seen in iRBD is of the same magnitude as that found in PD. This is of particular interest because patients with iRBD frequently develop PD and hyposmia. There is some evidence that iRBD may actually represent an early associated condition of PD. REM behavior disorder is not only seen in its idiopathic form, but can also be associated with narcolepsy. This led to a recent study of narcoleptic patients with and without REM behavior disorder, which demonstrated that narcolepsy, independent of REM behavior disorder, was associated with impairments in olfactory function. Orexin A, also known as hypocretin-1, is dramatically diminished or undetectable in the cerebrospinal fluid of patients with narcolepsy and cataplexy (Chap. 38). The orexin-containing neurons in the hypothalamus project throughout the entire olfactory system (from the olfactory epithelium to the olfactory cortex), and damage to these orexin-containing projections may be one underlying mechanism for impaired

1	project throughout the entire olfactory system (from the olfactory epithelium to the olfactory cortex), and damage to these orexin-containing projections may be one underlying mechanism for impaired olfactory performance in narcoleptic patients. The administration of intranasal orexin A (hypocretin-1) appears to result in improved olfactory function, supporting the notion that mild olfactory impairment is not only a primary feature of narcolepsy with cataplexy, but that central nervous system orexin deficiency may be a fundamental part of the mechanism for this loss.

1	The majority of patients who present with taste dysfunction exhibit olfactory, not taste, loss. This is because most flavors attributed to taste actually depend on retronasal stimulation of the olfactory receptors during deglutition. As noted earlier, taste buds only mediate basic tastes such as sweet, sour, bitter, salty, and umami. Significant impairment of whole-mouth gustatory function is rare outside of generalized metabolic disturbances or systemic use of some medications, because taste bud regeneration occurs and peripheral damage alone would require the involvement of multiple cranial nerve pathways. Nonetheless, taste can be influenced by (1) the release of foul-tasting materials from the oral cavity from oral medical conditions or appliances (e.g., gingivitis, purulent sialadenitis), (2) transport problems of tastants to the taste buds (e.g., drying of the orolingual mucosa, infections, inflammatory conditions), (3) damage to the taste buds themselves (e.g., local trauma,

1	(2) transport problems of tastants to the taste buds (e.g., drying of the orolingual mucosa, infections, inflammatory conditions), (3) damage to the taste buds themselves (e.g., local trauma, invasive carcinomas), (4) damage to the neural pathways innervating the taste buds (e.g., middle ear infections), (5) damage to central structures (e.g., multiple sclerosis, tumor, epilepsy, stroke), and (6) systemic disturbances of metabolism (e.g., diabetes, thyroid disease, medications). Unlike CN VII, CN IX is relatively protected along its path, although iatrogenic interventions such as tonsillectomy, bronchoscopy, laryngoscopy, endotracheal intubation, and radiation therapy can result in selective injury. CN VII damage commonly results from mastoidectomy, tympanoplasty, and stapedectomy, in some cases inducing persistent metallic sensations. Bell’s palsy (Chap. 455) is one of the most common causes of CN VII injury that results in taste disturbance. On rare occasions, migraines (Chap. 447)

1	cases inducing persistent metallic sensations. Bell’s palsy (Chap. 455) is one of the most common causes of CN VII injury that results in taste disturbance. On rare occasions, migraines (Chap. 447) are associated with a gustatory prodrome or aura, and in some cases, tastants can trigger a migraine attack. Interestingly, dysgeusia occurs in some cases of burning mouth syndrome (BMS; also termed glossodynia or glossalgia), as do dry mouth and thirst. BMS is likely associated with dysfunction of the trigeminal nerve (CN V). Some of the etiologies suggested for this poorly understood syndrome are amenable to treatment, including (1) nutritional deficiencies (e.g., iron, folic acid, B vitamins, zinc), (2) diabetes mellitus (possibly predisposing to oral candidiasis), (3) denture allergy, (4) mechanical irritation from dentures or oral devices, (5) repetitive movements of the mouth (e.g., tongue thrusting, teeth grinding, jaw clenching), (6) tongue ischemia as a result of temporal

1	(4) mechanical irritation from dentures or oral devices, (5) repetitive movements of the mouth (e.g., tongue thrusting, teeth grinding, jaw clenching), (6) tongue ischemia as a result of temporal arteritis, (7) periodontal 215 disease, (8) reflux esophagitis, and (9) geographic tongue.

1	Although both taste and smell can be adversely influenced by pharmacologic agents, drug-related taste alterations are more common. Indeed, over 250 medications have been reported to alter the ability to taste. Major offenders include antineoplastic agents, antirheumatic drugs, antibiotics, and blood pressure medications. Terbinafine, a commonly used antifungal, has been linked to taste disturbance lasting up to 3 years. In a recent controlled trial, nearly two-thirds of individuals taking eszopiclone (Lunesta) experienced a bitter dysgeusia that was stronger in women, systematically related to the time since drug administration, and positively correlated with both blood and saliva levels of the drug. Intranasal use of nasal gels and sprays containing zinc, which are common over-the-counter prophylactics for upper respiratory viral infections, has been implicated in loss of smell function. Whether their efficacy in preventing such infections, which are the most common cause of anosmia

1	prophylactics for upper respiratory viral infections, has been implicated in loss of smell function. Whether their efficacy in preventing such infections, which are the most common cause of anosmia and hyposmia, outweighs their potential detriment to smell function requires study. Dysgeusia occurs commonly in the context of drugs used to treat or minimize symptoms of cancer, with a weighted prevalence from 56–76% depending on the type of cancer treatment. Attempts to prevent taste problems from such drugs using prophylactic zinc sulfate or amifostine have proven to be minimally beneficial. Although antiepileptic medications are occasionally used to treat smell or taste disturbances, the use of topiramate has been reported to result in a reversible loss of an ability to detect and recognize tastes and odors during treatment.

1	As with olfaction, a number of systemic disorders can affect taste. These include chronic renal failure, end-stage liver disease, vitamin and mineral deficiencies, diabetes mellitus, and hypothyroidism (to name a few). In diabetes, there appears to be a progressive loss of taste beginning with glucose and then extending to other sweeteners, salty stimuli, and then all stimuli. Psychiatric conditions can be associated with chemosensory alterations (e.g., depression, schizophrenia, bulimia). A recent review of tactile, gustatory, and olfactory hallucinations demonstrated that no one type of hallucinatory experience is pathognomonic to any given diagnosis.

1	Pregnancy proves to be a unique condition with regard to taste function. There appears to be an increase in dislike and intensity of bitter tastes during the first trimester that may help to ensure that pregnant women avoid poisons during a critical phase of fetal development. Similarly, a relative increase in the preference for salt and bitter in the second and third trimesters may support the ingestion of much needed electrolytes to expand fluid volume and support a varied diet.

1	In most cases, a careful clinical history will establish the probable etiology of a chemosensory problem, including questions about its nature, onset, duration, and pattern of fluctuations. Sudden loss suggests the possibility of head trauma, ischemia, infection, or a psychiatric condition. Gradual loss can reflect the development of a progressive obstructive lesion. Intermittent loss suggests the likelihood of an inflammatory process. The patient should be asked about potential precipitating events, such as cold or flu infections prior to symptom onset, because these often go underappreciated. Information regarding head trauma, smoking habits, drug and alcohol abuse (e.g., intranasal cocaine, chronic alcoholism in the context of Wernicke’s and Korsakoff’s syndromes), exposures to pesticides and other toxic agents, and medical interventions is also informative. A determination of all the medications that the patient was taking before and at the time of symptom onset is important,

1	and other toxic agents, and medical interventions is also informative. A determination of all the medications that the patient was taking before and at the time of symptom onset is important, because many can cause chemosensory disturbances. Comorbid medical conditions associated with smell impairment, such as renal failure, liver disease, hypothyroidism, diabetes, or dementia, should be assessed. Delayed puberty in association with anosmia (with or without midline craniofacial abnormalities, deafness, and renal anomalies) suggests the possibility of Kallmann’s syndrome. Recollection of epistaxis, discharge (clear, purulent, or bloody), nasal obstruction, allergies, and somatic symptoms, including headache or irritation, may have localizing value. Questions related to memory, parkinsonian signs, and seizure activity (e.g., automatisms, blackouts,

1	CHAPTER 42 Disorders of Smell and Taste 216 auras, déjà vu) should be posed. Pending litigation and the possibility of malingering should be considered. Modern forced-choice olfactory tests can detect malingering from improbable responses. Neurologic and otorhinolaryngologic (ORL) examinations, along with appropriate brain and nasosinus imaging, aid in the evaluation of patients with olfactory or gustatory complaints. The neural evaluation should focus on cranial nerve function, with particular attention to possible skull base and intracranial lesions. Visual acuity, field, and optic disc examinations aid in the detection of intracranial mass lesions that induce intracranial pressure (papilledema) and optic atrophy, especially when considering Foster Kennedy syndrome. The ORL examination should thoroughly assess the intranasal architecture and mucosal surfaces. Polyps, masses, and adhesions of the turbinates to the septum may compromise the flow of air to the olfactory receptors,

1	should thoroughly assess the intranasal architecture and mucosal surfaces. Polyps, masses, and adhesions of the turbinates to the septum may compromise the flow of air to the olfactory receptors, because less than a fifth of the inspired air traverses the olfactory cleft in the unobstructed state. Blood tests may be helpful to identify such conditions as diabetes, infection, heavy metal exposure, nutritional deficiency (e.g., vitamin B6 or B12), allergy, and thyroid, liver, and kidney disease. As with other sensory disorders, quantitative sensory testing is advised. Self-reports of patients can be misleading, and a number of patients who complain of chemosensory dysfunction have normal function for their age and gender. Quantitative smell and taste testing provides valid information for worker’s compensation and other legal claims, as well as a way to accurately assess treatment interventions. A number of standardized olfactory and taste tests are commercially available. Most evaluate

1	compensation and other legal claims, as well as a way to accurately assess treatment interventions. A number of standardized olfactory and taste tests are commercially available. Most evaluate the ability of patients to detect and identify odors or tastes. For example, the most widely used of these tests, the 40-item University of Pennsylvania Smell Identification Test (UPSIT), uses norms based on nearly 4000 normal subjects. A determination is made of both absolute dysfunction (i.e., mild loss, moderate loss, severe loss, total loss, probable malingering) and relative dysfunction (percentile rank for age and gender). Although electrophysiologic testing is available at some smell and taste centers (e.g., odor event-related potentials), they require complex stimulus presentation and recording equipment and rarely provide additional diagnostic information. With the exception of electrogustometers, commercially available taste tests have only recently become available. Most use filter

1	equipment and rarely provide additional diagnostic information. With the exception of electrogustometers, commercially available taste tests have only recently become available. Most use filter paper strips impregnated with tastants, so no stimulus preparation is required.

1	Given the various mechanisms by which olfactory and gustatory disturbance can occur, management of patients tends to be condition specific. For example, patients with hypothyroidism, diabetes, or infections often benefit from specific treatments to correct the underlying disease process that is adversely influencing chemoreception. For most patients who present primarily with obstructive/transport loss affecting the nasal and paranasal regions (e.g., allergic rhinitis, polyposis, intranasal neoplasms, nasal deviations), medical and/or surgical intervention is often beneficial. Antifungal and antibiotic treatments may reverse taste problems secondary to candidiasis or other oral infections. Chlorhexidine mouthwash mitigates some salty or bitter dysgeusias, conceivably as a result of its strong positive charge. Excessive dryness of the oral mucosa is a problem with many medications and conditions, and artificial saliva (e.g., Xerolube) or oral pilocarpine treatments may prove

1	of its strong positive charge. Excessive dryness of the oral mucosa is a problem with many medications and conditions, and artificial saliva (e.g., Xerolube) or oral pilocarpine treatments may prove beneficial. Other methods to improve salivary flow include the use of mints, lozenges, or sugarless gum. Flavor enhancers may make food more palatable (e.g., monosodium glutamate), but caution is advised to avoid overusing ingredients containing sodium or sugar, particularly in circumstances when a patient also has underlying hypertension or diabetes. Medications that induce distortions of taste can often be discontinued and replaced with other types of medications or modes of therapy. As mentioned earlier, pharmacologic agents result in taste disturbances much more frequently than smell disturbances, and over 250 medications have been reported to alter the sense of taste. It is important to note, however, that many drug-related effects are long lasting and not reversed by short-term drug

1	and over 250 medications have been reported to alter the sense of taste. It is important to note, however, that many drug-related effects are long lasting and not reversed by short-term drug discontinuance.

1	PART 2 Cardinal Manifestations and Presentation of Diseases

1	A recent study of endoscopic sinus surgery in patients with chronic rhinosinusitis and hyposmia revealed that patients with severe olfactory dysfunction prior to the surgery had a more dramatic and sustained improvement over time compared to patients with more mild olfactory dysfunction prior to intervention. In the case of intranasal and sinus-related inflammatory conditions, such as seen with allergy, viruses, and traumas, the use of intranasal or systemic glucocorticoids may also be helpful. One common approach is to use a tapering course of oral prednisone. The utility of restoring olfaction with either topical or systemic glucocorticoids has been studied. Topical intranasal administration was found to be less effective in general than systemic administration; however, the effects of different nasal administration techniques were not analyzed; for example, intranasal glucocorticoids are more effective if administered in the Moffett’s position (head in the inverted position such as

1	nasal administration techniques were not analyzed; for example, intranasal glucocorticoids are more effective if administered in the Moffett’s position (head in the inverted position such as over the edge of the bed with the bridge of the nose perpendicular to the floor). After head trauma, an initial trial of glucocorticoids may help to reduce local edema and the potential deleterious deposition of scar tissue around olfactory fila at the level of the cribriform plate.

1	Treatments are limited for patients with chemosensory loss or primary injury to neural pathways. Nonetheless, spontaneous recovery can occur. In a follow-up study of 542 patients presenting to our center with smell loss from a variety of causes, modest improvement occurred over an average time period of 4 years in about half of the participants. However, only 11% of the anosmic and 23% of the hyposmic patients regained normal age-related function. Interestingly, the amount of dysfunction present at the time of presentation, not etiology, was the best predictor of prognosis. Other predictors were age and the duration of dysfunction prior to initial testing.

1	A nonblinded study has reported that patients with hyposmia may benefit from smelling strong odors (e.g., eucalyptol, citronella, eugenol, and phyenyl ethyl alcohol) before going to bed and immediately upon awakening each day over the course of several months. The rationale for such an approach comes from animal studies demonstrating that prolonged exposure to odorants can induce increased neural activity within the olfactory bulb. In an uncontrolled study, α-lipoic acid (400 mg/d), an essential cofactor for many enzyme complexes with possible antioxidant effects, was reported to be beneficial in mitigating smell loss following viral infection of the upper respiratory tract; controlled studies are needed to confirm this observation. This agent has also been suggested to be useful in some cases of hypogeusia and BMS.

1	The use of zinc and vitamin A in treating olfactory disturbances is controversial, and there does not appear to be much benefit beyond replenishing established deficiencies. However, zinc has been shown to improve taste function secondary to hepatic deficiencies, and retinoids (bioactive vitamin A derivatives) are known to play an essential role in the survival of olfactory neurons. One protocol in which zinc was infused with chemotherapy treatments suggested a possible protective effect against developing taste impairment. Diseases of the alimentary tract can not only influence chemoreceptive function, but also occasionally influence vitamin B12 absorption. This can result in a relative deficiency of vitamin B12, theoretically contributing to olfactory nerve disturbance. Vitamin B2 (riboflavin) and magnesium supplements are reported in the alternative literature to aid in the management of migraine that, in turn, may be associated with smell dysfunction. Because vitamin D deficiency

1	and magnesium supplements are reported in the alternative literature to aid in the management of migraine that, in turn, may be associated with smell dysfunction. Because vitamin D deficiency is a cofactor of chemotherapy-induced mucocutaneous toxicity and dysgeusia, adding vitamin D3, 1000–2000 units per day, may benefit some patients with smell and taste complaints during or following chemotherapy.

1	A number of medications have reportedly been used with success in ameliorating olfactory symptoms, although strong scientific evidence for efficacy is generally lacking. A report that theophylline improved smell function was uncontrolled and failed to account for the fact that some meaningful improvement occurs without treatment; indeed, the percentage of responders was about the same (∼50%) as that noted by others to show spontaneous improvement over a similar time period. Antiepileptics and some antidepressants (e.g., amitriptyline) have been used to treat dysosmias and smell distortions, particularly following head trauma. Ironically, amitriptyline is also frequently on the list of medications that can ultimately distort smell and taste function, possibly from its anticholinergic effects. A recent study suggests that the use of the centrally acting acetylcholinesterase inhibitor donepezil in AD resulted in improvements on smell identification measures that correlated with overall

1	A recent study suggests that the use of the centrally acting acetylcholinesterase inhibitor donepezil in AD resulted in improvements on smell identification measures that correlated with overall clinician-based impressions of change in dementia severity scores.

1	Alternative therapies, such as acupuncture, meditation, cognitive-behavioral therapy, and yoga, can help patients manage uncomfortable experiences associated with chemosensory disturbance and oral pain syndromes and to cope with the psychosocial stressors surrounding the impairment. Additionally, modification of diet and eating habits is also important. By accentuating the other sensory experiences of a meal, such as food texture, aroma, temperature, and color, one can optimize the overall eating experience for a patient. In some cases, a flavor enhancer like monosodium glutamate (MSG) can be added to foods to increase palatability and encourage intake.

1	Proper oral and nasal hygiene and routine dental care are extremely important ways for patients to protect themselves from disorders of the mouth and nose that can ultimately result in chemosensory disturbance. Patients should be warned not to overcompensate for their taste loss by adding excessive amounts of sugar or salt. Smoking cessation and the discontinuance of oral tobacco use are essential in the management of any patient with smell and/or taste disturbance and should be repeatedly emphasized.

1	A major and often overlooked element of therapy comes from chemosensory testing itself. Confirmation or lack of conformation of loss is beneficial to patients who come to believe, in light of unsupportive family members and medical providers, that they may be “crazy.” In cases where the loss is minor, patients can be informed of the likelihood of a more positive prognosis. Importantly, quantitative testing places the patient’s problem into overall perspective. Thus, it is often therapeutic for an older person to know that, while his or her smell function is not what it used to be, it still falls above the average of his or her peer group. Without testing, many such patients are simply told they are getting old and nothing can be done for them, leading in some cases to depression and decreased self-esteem. Disorders of Hearing Anil K. Lalwani

1	Disorders of Hearing Anil K. Lalwani Hearing loss is one of the most common sensory disorders in humans and can present at any age. Nearly 10% of the adult population has some hearing loss, and one-third of individuals age >65 years have a hearing loss of sufficient magnitude to require a hearing aid.

1	The function of the external and middle ear is to amplify sound to facilitate conversion of the mechanical energy of the sound wave into an electrical signal by the inner ear hair cells, a process called mechanotransduction (Fig. 43-1). Sound waves enter the external auditory canal and set the tympanic membrane (eardrum) in motion, which in turn moves the malleus, incus, and stapes of the middle ear. Movement of the footplate of the stapes causes pressure changes in the fluid-filled inner ear, eliciting a traveling wave in the basilar membrane of the cochlea. The tympanic membrane and the ossicular chain in the middle ear serve as an impedance-matching mechanism, improving the efficiency of energy transfer from air to the fluid-filled inner ear.

1	Stereocilia of the hair cells of the organ of Corti, which rests on the basilar membrane, are in contact with the tectorial membrane and are deformed by the traveling wave. A point of maximal displacement of the basilar membrane is determined by the frequency of the stimulating tone. High-frequency tones cause maximal displacement of the basilar membrane near the base of the cochlea, whereas for low-frequency sounds, the point of maximal displacement is toward 217 the apex of the cochlea. The inner and outer hair cells of the organ of Corti have different innervation patterns, but both are mechanoreceptors. The afferent innervation relates principally to the inner hair cells, and the efferent innervation relates principally to outer hair cells. The motility of the outer hair cells alters the micromechanics of the inner hair cells, creating a cochlear amplifier, which explains the exquisite sensitivity and frequency selectivity of the cochlea.

1	Beginning in the cochlea, the frequency specificity is maintained at each point of the central auditory pathway: dorsal and ventral cochlear nuclei, trapezoid body, superior olivary complex, lateral lemniscus, inferior colliculus, medial geniculate body, and auditory cortex. At low frequencies, individual auditory nerve fibers can respond more or less synchronously with the stimulating tone. At higher frequencies, phase-locking occurs so that neurons alternate in response to particular phases of the cycle of the sound wave. Intensity is encoded by the amount of neural activity in individual neurons, the number of neurons that are active, and the specific neurons that are activated.

1	There is evidence that the right and left ears as well as the central nervous system may process speech asymmetrically. Generally, a sound is processed symmetrically from the peripheral to the central auditory system. However, a “right ear advantage” exists for dichotic listening tasks, in which subjects are asked to report on competing sounds presented to each ear. In most individuals, a perceptual right ear advantage for consonant-vowel syllables, stop consonants, and words also exists. Similarly, whereas central auditory processing for sounds is symmetric with minimal lateral specialization for the most part, speech processing is lateralized. There is specialization of the left auditory cortex for speech recognition and production, and of the right hemisphere for emotional and tonal aspects of speech. Left hemisphere dominance for speech is found in 95–98% of right-handed persons and 70–80% of left-handed persons.

1	Hearing loss can result from disorders of the auricle, external auditory canal, middle ear, inner ear, or central auditory pathways (Fig. 43-2). In general, lesions in the auricle, external auditory canal, or middle ear that impede the transmission of sound from the external environment to the inner ear cause conductive hearing loss, whereas lesions that impair mechanotransduction in the inner ear or transmission of the electrical signal along the eighth nerve to the brain cause sensorineural hearing loss.

1	Conductive Hearing Loss The external ear, the external auditory canal, and the middle ear apparatus is designed to collect and amplify sound and efficiently transfer the mechanical energy of the sound wave to the fluid-filled cochlea. Factors that obstruct the transmission of sound or serve to dampen the acoustical energy result in conductive hearing loss. Conductive hearing loss can occur from obstruction of the external auditory canal by cerumen, debris, and foreign bodies; swelling of the lining of the canal; atresia or neoplasms of the canal; perforations of the tympanic membrane; disruption of the ossicular chain, as occurs with necrosis of the long process of the incus in trauma or infection; otosclerosis; or fluid, scarring, or neoplasms in the middle ear. Rarely, inner ear malformations or pathologies, such as superior semicircular canal dehiscence, lateral semicircular canal dysplasia, incomplete partition of the inner ear, and large vestibular aqueduct, may also be

1	malformations or pathologies, such as superior semicircular canal dehiscence, lateral semicircular canal dysplasia, incomplete partition of the inner ear, and large vestibular aqueduct, may also be associated with conductive hearing loss.

1	Eustachian tube dysfunction is extremely common in adults and may predispose to acute otitis media (AOM) or serous otitis media (SOM). Trauma, AOM, and chronic otitis media are the usual factors responsible for tympanic membrane perforation. While small perforations often heal spontaneously, larger defects usually require surgical intervention. Tympanoplasty is highly effective (>90%) in the repair of tympanic membrane perforations. Otoscopy is usually sufficient to diagnose AOM, SOM, chronic otitis media, cerumen impaction, tympanic membrane perforation, and eustachian tube dysfunction; tympanometry can be useful to confirm the clinical suspicion of these conditions. CHAPTER 43 Disorders of Hearing PART 2 Cardinal Manifestations and Presentation of Diseases

1	CHAPTER 43 Disorders of Hearing PART 2 Cardinal Manifestations and Presentation of Diseases Cholesteatoma, a benign tumor composed of stratified squamous epithelium in the middle ear or mastoid, occurs frequently in adults. This is a slowly growing lesion that destroys bone and normal ear tissue. Theories of pathogenesis include traumatic immigration and invasion of squamous epithelium through a retraction pocket, implantation of squamous epithelia in the middle ear through a perforation or surgery, and metaplasia following chronic infection and irritation. Auricle or pinna External ear ABExternal acoustic canal Tympanic membrane Semicircular canals Vestibulocochlear nerve Cochlea Stapes Incus Malleus Lobe Middle ear Eustachian tube FIguRE 43-1 Ear anatomy. A. Drawing of modified coronal section through external ear and temporal bone, with structures of the middle and inner ear demonstrated. B. High-resolution view of inner ear.

1	On examination, there is often a perforation of the tympanic membrane filled with cheesy white squamous debris. The presence of an aural polyp obscuring the tympanic membrane is highly suggestive of an underlying cholesteatoma. A chronically draining ear that fails to respond to appropriate antibiotic therapy should raise suspicion of a cholesteatoma. Conductive hearing loss secondary to ossicular erosion is common. Surgery is required to remove this destructive process.

1	Hearing Loss History Otologic examination Cerumen impaction TM perforation Cholesteatoma SOM AOM External auditory canal atresia/ stenosis Eustachian tube dysfunction Tympanosclerosis Pure tone and speech audiometry Conductive HL Impedance audiometry Mixed HL SNHL abnormal Impedance audiometry Acute Asymmetric/symmetric Chronic normal Otosclerosis Cerumen impaction Ossicular fixation Cholesteatoma* Temporal bone trauma* Inner ear dehiscence or “third window” AOM SOM TM perforation* Eustachian tube dysfunction Cerumen impaction Cholesteatoma* Temporal bone trauma* Ossicular discontinuity* Middle ear tumor* abnormal normal AOM TM perforation* Cholesteatoma* Temporal bone trauma* Middle ear tumors* glomus tympanicum glomus jugulare Stapes gusher syndrome* Inner ear malformation* Otosclerosis Temporal bone trauma* Inner ear dehiscence or “third window” CNS infection† Tumors† Cerebellopontine angle CNS Stroke† Trauma* Symmetric Asymmetric Inner ear malformation*

1	malformation* Otosclerosis Temporal bone trauma* Inner ear dehiscence or “third window” CNS infection† Tumors† Cerebellopontine angle CNS Stroke† Trauma* Symmetric Asymmetric Inner ear malformation* Presbycusis Noise exposure Radiation therapy MRI/BAER abnormal normal Endolymphatic hydrops Labyrinthitis* Perilymphatic fistula* Radiation therapy Labyrinthitis* Inner ear malformations* Cerebellopontine angle tumors Arachnoid cyst; facial nerve tumor; lipoma; meningioma; vestibular schwannoma Multiple sclerosis† abnormal normal FIguRE 43-2 An algorithm for the approach to hearing loss. AOM, acute otitis media; BAER, brainstem auditory evoked response; CNS, cen-tral nervous system; HL, hearing loss; SNHL, sensorineural hearing loss; SOM, serous otitis media; TM, tympanic membrane. *Computed tomog-raphy scan of temporal bone. †Magnetic resonance imaging (MRI) scan.

1	Conductive hearing loss with a normal ear canal and intact tympanic membrane suggests either ossicular pathology or the presence of “third window” in the inner ear (see below). Fixation of the stapes from otosclerosis is a common cause of low-frequency conductive hearing loss. It occurs equally in men and women and is inherited as an autosomal dominant trait with incomplete penetrance; in some cases, it may be a manifestation of osteogenesis imperfecta. Hearing impairment usually presents between the late teens and the forties. In women, the otosclerotic process is accelerated during pregnancy, and the hearing loss is often first noticeable at this time. A hearing aid or a simple outpatient surgical procedure (stapedectomy) can provide adequate auditory rehabilitation. Extension of otosclerosis beyond the stapes footplate to involve the cochlea (cochlear otosclerosis) can lead to mixed or sensorineural hearing loss. Fluoride therapy to prevent hearing loss from cochlear otosclerosis

1	beyond the stapes footplate to involve the cochlea (cochlear otosclerosis) can lead to mixed or sensorineural hearing loss. Fluoride therapy to prevent hearing loss from cochlear otosclerosis is of uncertain value.

1	Disorders that lead to the formation of a pathologic “third window” in the inner ear can be associated with conductive hearing loss. There are normally two major openings, or windows, that connect the inner ear with the middle ear and serve as conduits for transmission of sound; these are, respectively, the oval and round windows. A third window is formed where the normally hard otic bone surrounding the inner ear is eroded; dissipation of the acoustic energy at the third window is responsible for the “inner ear conductive hearing loss.” The superior semicircular canal dehiscence syndrome resulting from erosion of the otic bone over the superior circular canal can present with conductive hearing loss that mimics otosclerosis. A common symptom is vertigo evoked by loud sounds (Tullio phenomenon), by Valsalva maneuvers that change middle ear pressure, or by applying positive pressure on the tragus (the cartilage anterior to the external opening of the ear canal). Patients with this

1	by Valsalva maneuvers that change middle ear pressure, or by applying positive pressure on the tragus (the cartilage anterior to the external opening of the ear canal). Patients with this syndrome also complain of being able to hear the movement of their eyes and neck. A large jugular bulb or jugular bulb diverticulum can create a “third window” by eroding into the vestibular aqueduct or posterior semicircular canal; the symptoms are similar to those of the superior semicircular canal dehiscence syndrome.

1	Sensorineural Hearing Loss Sensorineural hearing loss results from either damage to the mechanotransduction apparatus of the cochlea or disruption of the electrical conduction pathway from the inner ear to the brain. Thus, injury to hair cells, supporting cells, auditory neurons, or the central auditory pathway can cause sensorineural hearing loss. Damage to the hair cells of the organ of Corti may be caused by intense noise, viral infections, ototoxic drugs (e.g., salicylates, quinine and its synthetic analogues, aminoglycoside antibiotics, loop diuretics such as furosemide and ethacrynic acid, and cancer chemotherapeutic agents such as cisplatin), fractures of the temporal bone, meningitis, cochlear otosclerosis (see above), Ménière’s disease, and aging. Congenital malformations of the inner ear may be the cause of hearing loss in some adults. Genetic predisposition alone or in concert with environmental exposures may also be responsible (see below).

1	Presbycusis (age-associated hearing loss) is the most common cause of sensorineural hearing loss in adults. In the early stages, it is characterized by symmetric, gentle to sharply sloping high-frequency hearing loss (Fig. 43-3). With progression, the hearing loss involves all frequencies. More importantly, the hearing impairment is associated with significant loss in clarity. There is a loss of discrimination for phonemes, recruitment (abnormal growth of loudness), and particular difficulty in understanding speech in noisy environments such as at restaurants and social events. Hearing aids are helpful in enhancing the signal-to-noise ratio by amplifying sounds that are close to the listener. Although hearing aids are able to amplify sounds, they cannot restore the clarity of hearing. Thus, amplification with hearing aids may provide only limited rehabilitation once the word recognition score deteriorates below 50%. Cochlear implants are the treatment of choice when hearing aids prove

1	amplification with hearing aids may provide only limited rehabilitation once the word recognition score deteriorates below 50%. Cochlear implants are the treatment of choice when hearing aids prove inadequate, even when hearing loss is incomplete (see below).

1	Ménière’s disease is characterized by episodic vertigo, fluctuating sensorineural hearing loss, tinnitus, and aural fullness. Tinnitus and/ or deafness may be absent during the initial attacks of vertigo, but it Right 50 dB 64% 55 dBSRT Left 70%Disc. CHAPTER 43 Disorders of Hearing FIguRE 43-3 Presbyacusis or age-related hearing loss. The audiogram shows a moderate to severe downsloping sensorineural hearing loss typical of presbyacusis. The loss of high-frequency hearing is associated with a decreased speech discrimination score; consequently, patients complain of lack of clarity of hearing, especially in a noisy background. HL, hearing threshold level; SRT, speech reception threshold.

1	invariably appears as the disease progresses and increases in severity during acute attacks. The annual incidence of Ménière’s disease is 0.5– 7.5 per 1000; onset is most frequently in the fifth decade of life but may also occur in young adults or the elderly. Histologically, there is distention of the endolymphatic system (endolymphatic hydrops) leading to degeneration of vestibular and cochlear hair cells. This may result from endolymphatic sac dysfunction secondary to infection, trauma, autoimmune disease, inflammatory causes, or tumor; an idiopathic etiology constitutes the largest category and is most accurately referred to as Ménière’s disease. Although any pattern of hearing loss can be observed, typically, low-frequency, unilateral sensorineural hearing impairment is present. Magnetic resonance imaging (MRI) should be obtained to exclude retrocochlear pathology such as a cerebellopontine angle tumor or demyelinating disorder. Therapy is directed toward the control of vertigo.

1	resonance imaging (MRI) should be obtained to exclude retrocochlear pathology such as a cerebellopontine angle tumor or demyelinating disorder. Therapy is directed toward the control of vertigo. A 2-g/d low-salt diet is the mainstay of treatment for control of rotatory vertigo. Diuretics, a short course of glucocorticoids, and intratympanic gentamicin may also be useful adjuncts in recalcitrant cases. Surgical therapy of vertigo is reserved for unresponsive cases and includes endolymphatic sac decompression, labyrinthectomy, and vestibular nerve section. Both labyrinthectomy and vestibular nerve section abolish rotatory vertigo in >90% of cases. Unfortunately, there is no effective therapy for hearing loss, tinnitus, or aural fullness from Ménière’s disease.

1	Sensorineural hearing loss may also result from any neoplastic, vascular, demyelinating, infectious, or degenerative disease or trauma affecting the central auditory pathways. HIV leads to both peripheral and central auditory system pathology and is associated with sensorineural hearing impairment.

1	Primary diseases of the central nervous system can also present with hearing impairment. Characteristically, a reduction in clarity of hearing and speech comprehension is much greater than the loss of the ability to hear pure tone. Auditory testing is consistent with an auditory neuropathy; normal otoacoustic emissions (OAE) and an abnormal auditory brainstem response (ABR) is typical (see below). Hearing loss can accompany hereditary sensorimotor neuropathies and inherited disorders of myelin. Tumors of the cerebellopontine angle such as vestibular schwannoma and meningioma usually present with asymmetric sensorineural hearing loss with greater deterioration of speech understanding than pure tone hearing. Multiple sclerosis may present with acute unilateral or bilateral hearing loss; typically, pure tone testing remains relatively stable while speech understanding 220 fluctuates. Isolated labyrinthine infarction can present with acute hearing loss and vertigo due to a cerebrovascular

1	pure tone testing remains relatively stable while speech understanding 220 fluctuates. Isolated labyrinthine infarction can present with acute hearing loss and vertigo due to a cerebrovascular accident involving the posterior circulation, usually the anterior inferior cerebellar artery; it may also be the heralding sign of impending catastrophic basilar artery infarction (Chap. 446). A finding of conductive and sensory hearing loss in combination is termed mixed hearing loss. Mixed hearing losses are due to pathology of both the middle and inner ear, as can occur in otosclerosis involving the ossicles and the cochlea, head trauma, chronic otitis media, cholesteatoma, middle ear tumors, and some inner ear malformations. Trauma resulting in temporal bone fractures may be associated with conductive, sensorineural, or mixed hearing loss. If the fracture spares the inner ear, there may simply be conductive hearing loss due to rupture of the tympanic membrane or disruption of the ossicular

1	sensorineural, or mixed hearing loss. If the fracture spares the inner ear, there may simply be conductive hearing loss due to rupture of the tympanic membrane or disruption of the ossicular chain. These abnormalities can be surgically corrected. Profound hearing loss and severe vertigo are associated with temporal bone fractures involving the inner ear. A perilymphatic fistula associated with leakage of inner ear fluid into the middle ear can occur and may require surgical repair. An associated facial nerve injury is not uncommon. Computed tomography (CT) is best suited to assess fracture of the traumatized temporal bone, evaluate the ear canal, and determine the integrity of the ossicular chain and the involvement of the inner ear. Cerebrospinal fluid leaks that accompany temporal bone fractures are usually self-limited; the value of prophylactic antibiotics is uncertain. Tinnitus is defined as the perception of a sound when there is no sound in the environment. It may have a

1	bone fractures are usually self-limited; the value of prophylactic antibiotics is uncertain. Tinnitus is defined as the perception of a sound when there is no sound in the environment. It may have a buzzing, roaring, or ringing quality and may be pulsatile (synchronous with the heartbeat). Tinnitus is often associated with either a conductive or sensorineural hearing loss. The pathophysiology of tinnitus is not well understood. The cause of the tinnitus can usually be determined by finding the cause of the associated hearing loss. Tinnitus may be the first symptom of a serious condition such as a vestibular schwannoma. Pulsatile tinnitus requires evaluation of the vascular system of the head to exclude vascular tumors such as glomus jugulare tumors, aneurysms, dural arteriovenous fistulas, and stenotic arterial lesions; it may also occur with SOM. It is most commonly associated with some abnormality of the jugular bulb such as a large jugular bulb or jugular bulb diverticulum.

1	More than half of childhood hearing impairment is thought to be hereditary; hereditary hearing impairment (HHI) can also manifest later in life. HHI may be classified as either nonsyndromic, when hearing loss is the only clinical abnormality, or syndromic, when hearing loss is associated with anomalies in other organ systems. Nearly two-thirds of HHIs are nonsyndromic, and the remaining one-third are syndromic. Between 70 and 80% of nonsyndromic HHI is inherited in an autosomal recessive manner and designated DFNB; another 15–20% is autosomal dominant (DFNA). Less than 5% is X-linked (DFNX) or maternally inherited via the mitochondria.

1	More than 150 loci harboring genes for nonsyndromic HHI have been mapped, with recessive loci outnumbering dominant; numerous genes have now been identified (Table 43-1). The hearing genes fall into the categories of structural proteins (MYH9, MYO7A, MYO15, TECTA, DIAPH1), transcription factors (POU3F4, POU4F3), ion channels (KCNQ4, SLC26A4), and gap junction proteins (GJB2, GJB3, GJB6). Several of these genes, including GJB2, TECTA, and TMC1, cause both autosomal dominant and recessive forms of nonsyndromic HHI. In general, the hearing loss associated with dominant genes has its onset in adolescence or adulthood, varies in severity, and progresses with age, whereas the hearing loss associated with recessive inheritance is congenital and profound. Connexin 26, product of the GJB2 gene, is particularly important because it is responsible for nearly 20% of all cases of childhood deafness; half of genetic deafness in children is GJB2-related. Two frameshift mutations, 35delG and 167delT,

1	particularly important because it is responsible for nearly 20% of all cases of childhood deafness; half of genetic deafness in children is GJB2-related. Two frameshift mutations, 35delG and 167delT, account for >50% of the cases; however, screening for these two mutations alone is insufficient, and sequencing of the entire gene is required to diagnose GJB2-related recessive deafness. The 167delT mutation is highly prevalent in Ashkenazi Jews; ~1 in 1765 individuals in this

1	PART 2 Cardinal Manifestations and Presentation of Diseases population are homozygous and affected. The hearing loss can also vary among the members of the same family, suggesting that other genes or factors influence the auditory phenotype. In addition to GJB2, several other nonsyndromic genes are associated with hearing loss that progresses with age. The contribution of genetics to presbycusis is also becoming better understood. Sensitivity to aminoglycoside ototoxicity can be maternally transmitted through a mitochondrial mutation. Susceptibility to noise-induced hearing loss may also be genetically determined.

1	There are >400 syndromic forms of hearing loss. These include Usher’s syndrome (retinitis pigmentosa and hearing loss), Waardenburg’s syndrome (pigmentary abnormality and hearing loss), Pendred’s syndrome (thyroid organification defect and hearing loss), Alport’s syndrome (renal disease and hearing loss), Jervell and Lange-Nielsen syndrome (prolonged QT interval and hearing loss), neurofibromatosis type 2 (bilateral acoustic schwannoma), and mitochondrial disorders (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes [MELAS]; myoclonic epilepsy and ragged red fibers [MERRF]; progressive external ophthalmoplegia [PEO]) (Table 43-2). APPROACH TO THE PATIENT: Disorders of the Sense of Hearing

1	The goal in the evaluation of a patient with auditory complaints is to determine (1) the nature of the hearing impairment (conductive vs sensorineural vs mixed), (2) the severity of the impairment (mild, moderate, severe, or profound), (3) the anatomy of the impairment (external ear, middle ear, inner ear, or central auditory pathway), and (4) the etiology. The history should elicit characteristics of the hearing loss, including the duration of deafness, unilateral versus bilateral involvement, nature of onset (sudden vs insidious), and rate of progression (rapid vs slow). Symptoms of tinnitus, vertigo, imbalance, aural fullness, otorrhea, headache, facial nerve dysfunction, and head and neck paresthesias should be noted. Information regarding head trauma, exposure to ototoxins, occupational or recreational noise exposure, and family history of hearing impairment may also be important. A sudden onset of unilateral hearing loss, with or without tinnitus, may represent a viral infection

1	or recreational noise exposure, and family history of hearing impairment may also be important. A sudden onset of unilateral hearing loss, with or without tinnitus, may represent a viral infection of the inner ear, vestibular schwannoma, or a stroke. Patients with unilateral hearing loss (sensory or conductive) usually complain of reduced hearing, poor sound localization, and difficulty hearing clearly with background noise. Gradual progression of a hearing deficit is common with otosclerosis, noise-induced hearing loss, vestibular schwannoma, or Ménière’s disease. Small vestibular schwannomas typically present with asymmetric hearing impairment, tinnitus, and imbalance (rarely vertigo); cranial neuropathy, in particular of the trigeminal or facial nerve, may accompany larger tumors. In addition to hearing loss, Ménière’s disease may be associated with episodic vertigo, tinnitus, and aural fullness. Hearing loss with otorrhea is most likely due to chronic otitis media or

1	tumors. In addition to hearing loss, Ménière’s disease may be associated with episodic vertigo, tinnitus, and aural fullness. Hearing loss with otorrhea is most likely due to chronic otitis media or cholesteatoma.

1	Examination should include the auricle, external ear canal, and tympanic membrane. The external ear canal of the elderly is often dry and fragile; it is preferable to clean cerumen with wall-mounted suction or cerumen loops and to avoid irrigation. In examining the eardrum, the topography of the tympanic membrane is more important than the presence or absence of the light reflex. In addition to the pars tensa (the lower two-thirds of the tympanic membrane), the pars flaccida (upper one-third of the tympanic membrane) above the short process of the malleus should also be examined for retraction pockets that may be evidence of chronic eustachian tube dysfunction or cholesteatoma. Insufflation of the ear canal is necessary to assess tympanic membrane mobility and compliance. Careful inspection of the nose, nasopharynx, and upper respiratory tract is indicated. Unilateral serous effusion should prompt a fiberoptic examination of the nasopharynx to exclude neoplasms. Cranial nerves should

1	of the nose, nasopharynx, and upper respiratory tract is indicated. Unilateral serous effusion should prompt a fiberoptic examination of the nasopharynx to exclude neoplasms. Cranial nerves should be evaluated with special attention to facial and trigeminal nerves, which are commonly affected with tumors involving the cerebellopontine angle.

1	Thyroid hormone–binding protein Cytoskeletal protein Potassium channel Gap junction Gap junction Gap junction Class II nonmuscle myosin Cell adhesion molecule Unknown Transmembrane protein Tectorial membrane protein Unknown Developmental gene Cytoskeletal protein Cytoskeletal protein Transcription factor Cytoskeletal protein Cytoskeletal protein Unconventional myosin Developmental gene Vesicular glutamate transporter Transcription factor Transmembrane protein Purinergic receptor Effector of epidermal growth factor– Gap junction Gap junction Cytoskeletal protein Cytoskeletal protein Chloride/iodide transporter Transmembrane protein Transmembrane protein Trafficking of membrane vesicles Transmembrane serine protease

1	The Rinne and Weber tuning fork tests, with a 512-Hz tuning fork, are used to screen for hearing loss, differentiate conductive from sensorineural hearing losses, and confirm the findings of audiologic evaluation. The Rinne test compares the ability to hear by air conduction with the ability to hear by bone conduction. The tines of a vibrating tuning fork are held near the opening of the external auditory canal, and then the stem is placed on the mastoid process; for direct contact, it may be placed on teeth or dentures. The patient is asked to indicate whether the tone is louder by air conduction or bone conduction. Normally, and in the presence of sensorineural hearing loss, a tone is heard louder by air conduction than by bone conduction; however, with conductive hearing loss of ≥30 dB (see “Audiologic Assessment,” below), the bone-conduction stimulus is perceived as louder than the air-conduction stimulus. For the Weber test, the stem of a vibrating tuning fork is placed on

1	Unknown Tectorial membrane protein Gel attachment to nonsensory cell Morphogenesis and cohesion Cytoskeletal protein Reversible S-glutathionylation of CHAPTER 43 Disorders of Hearing the head in the midline and the patient is asked whether the tone is heard in both ears or better in one ear than in the other. With a unilateral conductive hearing loss, the tone is perceived in the affected ear. With a unilateral sensorineural hearing loss, the tone is perceived in the unaffected ear. A 5-dB difference in hearing between the two ears is required for lateralization.

1	LABORATORY ASSESSMENT OF HEARINg Audiologic Assessment The minimum audiologic assessment for hearing loss should include the measurement of pure tone air-conduction and bone-conduction thresholds, speech reception threshold, word recognition score, tympanometry, acoustic reflexes, and acoustic-reflex decay. This test battery provides a screening evaluation of the entire auditory system and allows one to determine whether further PART 2 Cardinal Manifestations and Presentation of Diseases Abbreviations: BOR, branchio-oto-renal syndrome; WS, Waardenburg’s syndrome. differentiation of a sensory (cochlear) from a neural (retrocochlear) hearing loss is indicated.

1	differentiation of a sensory (cochlear) from a neural (retrocochlear) hearing loss is indicated. Pure tone audiometry assesses hearing acuity for pure tones. The test is administered by an audiologist and is performed in a sound-attenuated chamber. The pure tone stimulus is delivered with an audiometer, an electronic device that allows the presentation of specific frequencies (generally between 250 and 8000 Hz) at specific intensities. Airand bone-conduction thresholds are established for each ear. Air-conduction thresholds are determined by presenting the stimulus in air with the use of headphones. Bone-conduction thresholds are determined by placing the stem of a vibrating tuning fork or an oscillator of an audiometer in contact with the head. In the presence of a hearing loss, broad-spectrum noise is presented to the nontest ear for masking purposes so that responses are based on perception from the ear under test.

1	The responses are measured in decibels. An audiogram is a plot of intensity in decibels of hearing threshold versus frequency. A decibel (dB) is equal to 20 times the logarithm of the ratio of the sound pressure required to achieve threshold in the patient to the sound pressure required to achieve threshold in a normal-hearing person. Therefore, a change of 6 dB represents doubling of sound pressure, and a change of 20 dB represents a tenfold change in sound pressure. Loudness, which depends on the frequency, intensity, and duration of a sound, doubles with approximately each 10-dB increase in sound pressure level. Pitch, on the other hand, does not directly correlate with frequency. The perception of pitch changes slowly in the low and high frequencies. In the middle tones, which are important for human speech, pitch varies more rapidly with changes in frequency.

1	Pure tone audiometry establishes the presence and severity of hearing impairment, unilateral versus bilateral involvement, and the type of hearing loss. Conductive hearing losses with a large mass component, as is often seen in middle ear effusions, produce elevation of thresholds that predominate in the higher frequencies. Conductive hearing losses with a large stiffness component, as in fixation of the footplate of the stapes in early otosclerosis, produce threshold elevations in the lower frequencies. Often, the conductive hearing loss involves all frequencies, suggesting involvement of both stiffness and mass. In general, sensorineural hearing losses such as presbycusis affect higher frequencies more than lower frequencies (Fig. 43-3). An exception is Ménière’s disease, which is characteristically associated with low-frequency sensorineural hearing loss. Noise-induced hearing loss has an unusual pattern of hearing impairment in which the loss at 4000 Hz is greater than at higher

1	associated with low-frequency sensorineural hearing loss. Noise-induced hearing loss has an unusual pattern of hearing impairment in which the loss at 4000 Hz is greater than at higher frequencies. Vestibular schwannomas characteristically affect the higher frequencies, but any pattern of hearing loss can be observed.

1	Speech recognition requires greater synchronous neural firing than is necessary for appreciation of pure tones. Speech audiometry tests the clarity with which one hears. The speech reception threshold (SRT) is defined as the intensity at which speech is recognized as a meaningful symbol and is obtained by presenting two-syllable words with an equal accent on each syllable. The intensity at which the patient can repeat 50% of the words correctly is the SRT. Once the SRT is determined, discrimination or word recognition ability is tested by presenting one-syllable words at 25–40 dB above the SRT. The words are phonetically balanced in that the phonemes (speech sounds) occur in the list of words at the same frequency that they occur in ordinary conversational English. An individual with normal hearing or conductive hearing loss can repeat 88–100% of the phonetically balanced words correctly. Patients with a sensorineural hearing loss have variable loss of discrimination. As a general

1	hearing or conductive hearing loss can repeat 88–100% of the phonetically balanced words correctly. Patients with a sensorineural hearing loss have variable loss of discrimination. As a general rule, neural lesions produce greater deficits in discrimination than do cochlear lesions. For example, in a patient with mild asymmetric sensorineural hearing loss, a clue to the diagnosis of vestibular schwannoma is the presence of greater than expected deterioration in discrimination ability. Deterioration in discrimination ability at higher intensities above the SRT also suggests a lesion in the eighth nerve or central auditory pathways.

1	Tympanometry measures the impedance of the middle ear to sound and is useful in diagnosis of middle ear effusions. A tympanogram is the graphic representation of change in impedance or compliance as the pressure in the ear canal is changed. Normally, the middle ear is most compliant at atmospheric pressure, and the compliance decreases as the pressure is increased or decreased (type A); this pattern is seen with normal hearing or in the presence of sensorineural hearing loss. Compliance that does not change with change in pressure suggests middle ear effusion (type B). With a negative pressure in the middle ear, as with eustachian tube obstruction, the point of maximal compliance occurs with negative pressure in the ear canal (type C). A tympanogram in which no point of maximal compliance can be obtained is most commonly seen with discontinuity of the ossicular chain (type Ad). A reduction in the maximal compliance peak can be seen in otosclerosis (type As).

1	During tympanometry, an intense tone elicits contraction of the stapedius muscle. The change in compliance of the middle ear with contraction of the stapedius muscle can be detected. The presence or absence of this acoustic reflex is important in determining the etiology of hearing loss as well as in the anatomic localization of facial nerve paralysis. The acoustic reflex can help differentiate between conductive hearing loss due to otosclerosis and that caused by an inner ear “third window”: it is absent in otosclerosis and present in inner ear conductive hearing loss. Normal or elevated acoustic reflex thresholds in an individual with sensorineural hearing impairment suggest a cochlear hearing loss. An absent acoustic reflex in the setting of sensorineural hearing loss is not helpful in localizing the site of lesion. Assessment of acoustic reflex decay helps differentiate sensory from neural hearing losses. In neural hearing loss, such as with vestibular schwannoma, the reflex

1	in localizing the site of lesion. Assessment of acoustic reflex decay helps differentiate sensory from neural hearing losses. In neural hearing loss, such as with vestibular schwannoma, the reflex adapts or decays with time.

1	OAEs generated by outer hair cells only can be measured with microphones inserted into the external auditory canal. The emissions may be spontaneous or evoked with sound stimulation. The presence of OAEs indicates that the outer hair cells of the organ of Corti are intact and can be used to assess auditory thresholds and to distinguish sensory from neural hearing losses.

1	Evoked Responses Electrocochleography measures the earliest evoked potentials generated in the cochlea and the auditory nerve. Receptor potentials recorded include the cochlear microphonic, generated by the outer hair cells of the organ of Corti, and the summating potential, generated by the inner hair cells in response to sound. The whole nerve action potential representing the composite firing of the first-order neurons can also be recorded during electrocochleography. Clinically, the test is useful in the diagnosis of Ménière’s disease, where an elevation of the ratio of summating potential to action potential is seen.

1	Brainstem auditory evoked responses (BAERs), also known as auditory brainstem responses (ABRs), are useful in differentiating the site of sensorineural hearing loss. In response to sound, five distinct electrical potentials arising from different stations along the peripheral and central auditory pathway can be identified using computer averaging from scalp surface electrodes. BAERs are valuable in situations in which patients cannot or will not give reliable voluntary thresholds. They are also used to assess the integrity of the auditory nerve and brainstem in various clinical situations, including intraoperative monitoring, and in determination of brain death.

1	The vestibular-evoked myogenic potential (VEMP) test elicits a vestibulocolic reflex whose afferent limb arises from acoustically sensitive cells in the saccule, with signals conducted via the inferior vestibular nerve. VEMP is a biphasic, short-latency response recorded from the tonically contracted sternocleidomastoid muscle in response to loud auditory clicks or tones. VEMPs may be diminished or absent in patients with early and late Ménière’s disease, vestibular neuritis, benign paroxysmal positional vertigo, and vestibular schwannoma. On the other hand, the threshold for VEMPs may be lower in cases of superior canal dehiscence, other inner ear dehiscence, and perilymphatic fistula.

1	Imaging Studies The choice of radiologic tests is largely determined by whether the goal is to evaluate the bony anatomy of the external, middle, and inner ear or to image the auditory nerve and brain. Axial and coronal CT of the temporal bone with fine 0.3to 0.6-mm cuts is ideal for determining the caliber of the external auditory canal, integrity of the ossicular chain, and presence of middle ear or mastoid disease; it can also detect inner ear malformations. CT is also ideal for the detection of bone erosion with chronic otitis media and cholesteatoma. Pöschl reformatting in the plane of the superior semicircular canal is required for the identification of dehiscence or absence of bone over the superior semicircular canal. MRI is superior to CT for imaging of retrocochlear pathology such as vestibular schwannoma, meningioma, other lesions of the cerebellopontine angle, demyelinating lesions of the brainstem, and brain tumors. Both CT and MRI are equally capable of identifying inner

1	as vestibular schwannoma, meningioma, other lesions of the cerebellopontine angle, demyelinating lesions of the brainstem, and brain tumors. Both CT and MRI are equally capable of identifying inner ear malformations and assessing cochlear patency for preoperative evaluation of patients for cochlear implantation.

1	In general, conductive hearing losses are amenable to surgical correction, whereas sensorineural hearing losses are usually managed medically. Atresia of the ear canal can be surgically repaired, often with significant improvement in hearing. Tympanic membrane perforations due to chronic otitis media or trauma can be repaired with an outpatient tympanoplasty. Likewise, conductive hearing loss associated with otosclerosis can be treated by stapedectomy, which is successful in >95% of cases. Tympanostomy tubes allow the prompt return of normal hearing in individuals with middle ear 223 effusions. Hearing aids are effective and well tolerated in patients with conductive hearing losses.

1	Patients with mild, moderate, and severe sensorineural hearing losses are regularly rehabilitated with hearing aids of varying configuration and strength. Hearing aids have been improved to provide greater fidelity and have been miniaturized. The current generation of hearing aids can be placed entirely within the ear canal, thus reducing any stigma associated with their use. In general, the more severe the hearing impairment, the larger the hearing aid required for auditory rehabilitation. Digital hearing aids lend themselves to individual programming, and multiple and directional microphones at the ear level may be helpful in noisy surroundings. Because all hearing aids amplify noise as well as speech, the only absolute solution to the problem of noise is to place the microphone closer to the speaker than the noise source. This arrangement is not possible with a self-contained, cosmetically acceptable device. A significant limitation of rehabilitation with a hearing aid is that

1	to the speaker than the noise source. This arrangement is not possible with a self-contained, cosmetically acceptable device. A significant limitation of rehabilitation with a hearing aid is that although it is able to enhance detection of sound with amplification, it cannot restore clarity of hearing that is lost with presbycusis.

1	Patients with unilateral deafness have difficulty with sound localization and reduced clarity of hearing in background noise. They may benefit from a CROS (contralateral routing of signal) hearing aid in which a microphone is placed on the hearing-impaired side and the sound is transmitted to the receiver placed on the contralateral ear. The same result may be obtained with a bone-anchored hearing aid (BAHA), in which a hearing aid clamps to a screw integrated into the skull on the hearing-impaired side. Like the CROS hearing aid, the BAHA transfers the acoustic signal to the contralateral hearing ear, but it does so by vibrating the skull. Patients with profound deafness on one side and some hearing loss in the better ear are candidates for a BICROS hearing aid; it differs from the CROS hearing aid in that the patient wears a hearing aid, and not simply a receiver, in the better ear. Unfortunately, while CROS and BAHA devices provide benefit, they do not restore hearing in the deaf

1	hearing aid in that the patient wears a hearing aid, and not simply a receiver, in the better ear. Unfortunately, while CROS and BAHA devices provide benefit, they do not restore hearing in the deaf ear. Only cochlear implants can restore hearing (see below). Increasingly, cochlear implants are being investigated for the treatment of patients with single-sided deafness; early reports show great promise in not only restoring hearing but also improving sound localization and performance in background noise.

1	In many situations, including lectures and the theater, hearing-impaired persons benefit from assistive devices that are based on the principle of having the speaker closer to the microphone than any source of noise. Assistive devices include infrared and frequency-modulated (FM) transmission as well as an electromagnetic loop around the room for transmission to the individual’s hearing aid. Hearing aids with telecoils can also be used with properly equipped telephones in the same way.

1	In the event that the hearing aid provides inadequate rehabilitation, cochlear implants may be appropriate (Fig. 43-4). Criteria for implantation include severe to profound hearing loss with open-set sentence cognition of ≤40% under best aided conditions. Worldwide, more than 300,000 hearing-impaired individuals have received cochlear implants. Cochlear implants are neural prostheses that convert sound energy to electrical energy and can be used to stimulate the auditory division of the eighth nerve directly. In most cases of profound hearing impairment, the auditory hair cells are lost but the ganglionic cells of the auditory division of the eighth nerve are preserved. Cochlear implants consist of electrodes that are inserted into the cochlea through the round window, speech processors that extract acoustical elements of speech for conversion to electrical currents, and a means of transmitting the electrical energy through the skin. Patients with implants experience sound that helps

1	extract acoustical elements of speech for conversion to electrical currents, and a means of transmitting the electrical energy through the skin. Patients with implants experience sound that helps with speech reading, allows open-set word recognition, and helps in modulating the person’s own voice. Usually, within the first 3–6 months after implantation, adult patients can understand speech without visual cues. With the current generation of multichannel cochlear implants, nearly 75% of patients are able to converse on the telephone.

1	CHAPTER 43 Disorders of Hearing PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 43-4 A cochlear implant is composed of an external microphone and speech processor worn on the ear and a receiver implanted underneath the temporalis muscle. The internal receiver is attached to an electrode that is placed surgically in the cochlea.

1	The U.S. Food and Drug Administration recently approved the first hybrid cochlear implant for the treatment of high-frequency hearing loss. Patients with presbyacusis typically have normal low-frequency hearing, while suffering from high-frequency hearing loss associated with loss of clarity that cannot always be adequately rehabilitated with a hearing aid. However, these patients are not candidates for conventional cochlear implants because they have too much residual hearing. The hybrid implant has been specifically designed for this patient population; it has a shorter electrode than a conventional cochlear implant and can be introduced into the cochlea atraumatically, thus preserving low-frequency hearing. Individuals with a hybrid implant use their own natural low-frequency “acoustic” hearing and rely on the implant for providing “electrical” high-frequency hearing. Patients who have received the hybrid implant perform better on speech testing in both quiet and noisy backgrounds.

1	For individuals who have had both eighth nerves destroyed by trauma or bilateral vestibular schwannomas (e.g., neurofibromatosis type 2), brainstem auditory implants placed near the cochlear nucleus may provide auditory rehabilitation. Tinnitus often accompanies hearing loss. As for background noise, tinnitus can degrade speech comprehension in individuals with hearing impairment. Therapy for tinnitus is usually directed toward minimizing the appreciation of tinnitus. Relief of the tinnitus may be obtained by masking it with background music. Hearing aids are also helpful in tinnitus suppression, as are tinnitus maskers, devices that present a sound to the affected ear that is more pleasant to listen to than the tinnitus. The use of a tinnitus masker is often followed by several hours of inhibition of the tinnitus. Antidepressants have been shown to be beneficial in helping patients cope with tinnitus.

1	Hard-of-hearing individuals often benefit from a reduction in unnecessary noise in the environment (e.g., radio or television) to enhance the signal-to-noise ratio. Speech comprehension is aided by lip reading; therefore, the impaired listener should be seated so that the face of the speaker is well illuminated and easily seen. Although speech should be in a loud, clear voice, one should be aware that in sensorineural hearing losses in general and in hard-ofhearing elderly in particular, recruitment (abnormal perception of loud sounds) may be troublesome. Above all, optimal communication cannot take place without both parties giving it their full and undivided attention.

1	Conductive hearing losses may be prevented by prompt antibiotic therapy of adequate duration for AOM and by ventilation of the middle ear with tympanostomy tubes in middle ear effusions lasting ≥12 weeks. Loss of vestibular function and deafness due to aminoglycoside antibiotics can largely be prevented by careful monitoring of serum peak and trough levels. Some 10 million Americans have noise-induced hearing loss, and 20 million are exposed to hazardous noise in their employment. Noise-induced hearing loss can be prevented by avoidance of exposure to loud noise or by regular use of ear plugs or fluid-filled ear muffs to attenuate intense sound. Table 43-3 lists loudness levels for a variety of

1	Sore Throat, Earache, and upper Respiratory Symptoms Michael A. Rubin, Larry C. Ford, Ralph Gonzales Infections of the upper respiratory tract (URIs) have a tremendous impact on public health. They are among the most common rea-44TABLE 43-3 DECiBEL (LouDnESS) LEvEL of CoMMon EnviRonMEnTAL noiSE Source Decibel (dB) Weakest sound heard 0 Whisper 30 Normal conversation 55–65 City traffic inside car 85 OSHA monitoring requirement begins 90 Jackhammer 95 CHAPTER 44 Sore Throat, Earache, and Upper Respiratory Symptoms Abbreviation: OSHA, Occupational Safety and Health Administration.

1	environmental sounds. High-risk activities for noise-induced hearing loss include use of electrical equipment for wood and metal working and target practice or hunting with small firearms. All internal-combustion and electric engines, including snow and leaf blowers, snowmobiles, outboard motors, and chainsaws, require protection of the user with hearing protectors. Virtually all noise-induced hearing loss is preventable through education, which should begin before the teenage years. Programs for conservation of hearing in the workplace are required by the Occupational Safety and Health Administration (OSHA) whenever the exposure over an 8-h period averages 85 dB. OSHA mandates that workers in such noisy environments have hearing monitoring and protection programs that include a preemployment screen, an annual audiologic assessment, and the mandatory use of hearing protectors. Exposure to loud sounds above 85 dB in the work environment is restricted by OSHA, with halving of allowed

1	screen, an annual audiologic assessment, and the mandatory use of hearing protectors. Exposure to loud sounds above 85 dB in the work environment is restricted by OSHA, with halving of allowed exposure time for each increment of 5 dB above this threshold; for example, exposure to 90 dB is permitted for 8 h; 95 dB for 4 h, and 100 dB for 2 h (Table 43-4).

1	90 8 92 6 95 4 97 3 100 2 102 1.5 105 1 110 0.5 115 ≤0.25 Note: Exposure to impulsive or impact noise should not exceed 140-dB peak sound pressure level. Source: From https://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table= standards&p_id=9735. sons for visits to primary care providers, and although the illnesses are typically mild, their high incidence and transmission rates place them among the leading causes of time lost from work or school. Even though a minority (~25%) of cases are caused by bacteria, URIs are the leading diagnoses for which antibiotics are prescribed on an outpatient basis in the United States. The enormous consumption of antibiotics for these illnesses has contributed to the rise in antibiotic resistance among common community-acquired pathogens such as Streptococcus pneumoniae—a trend that in itself has an enormous influence on public health.

1	Although most URIs are caused by viruses, distinguishing patients with primary viral infection from those with primary bacterial infection is difficult. Signs and symptoms of bacterial and viral URIs are typically indistinguishable. Until consistent, inexpensive, and rapid testing becomes available and is used widely, acute infections will be diagnosed largely on clinical grounds. The judicious use and potential for misuse of antibiotics in this setting pose definite challenges. Nonspecific URIs are a broadly defined group of disorders that collectively constitute the leading cause of ambulatory care visits in the United States. By definition, nonspecific URIs have no prominent localizing features. They are identified by a variety of descriptive names, including acute infective rhinitis, acute rhinopharyngitis/ nasopharyngitis, acute coryza, and acute nasal catarrh, as well as by the inclusive label common cold.

1	The large assortment of URI classifications reflects the wide variety of causative infectious agents and the varied manifestations of common pathogens. Nearly all nonspecific URIs are caused by viruses spanning multiple virus families and many antigenic types. For instance, there are at least 100 immunotypes of rhinovirus (Chap. 223), the most common cause of URI (~30–40% of cases); other causes include influenza virus (three immunotypes; Chap. 224) as well as parainfluenza virus (four immunotypes), coronavirus (at least three immunotypes), and adenovirus (47 immunotypes) (Chap. 223). Respiratory syncytial virus (RSV), a well-established pathogen in pediatric populations, is also a recognized cause of significant disease in elderly and immunocompromised individuals. A host of additional viruses, including some viruses not typically associated with URIs (e.g., enteroviruses, rubella virus, and varicella-zoster virus), account for a small percentage of cases in adults each year.

1	viruses, including some viruses not typically associated with URIs (e.g., enteroviruses, rubella virus, and varicella-zoster virus), account for a small percentage of cases in adults each year. Although new diagnostic modalities (e.g., nasopharyngeal swab for polymerase chain reaction [PCR]) can assign a viral etiology, there are few specific treatment options, and no pathogen is identified in a substantial proportion of cases. A specific diagnostic workup beyond a clinical diagnosis is generally unnecessary in an otherwise healthy adult.

1	The signs and symptoms of nonspecific URI are similar to those of other URIs but lack a pronounced localization to one particular anatomic location, such as the sinuses, pharynx, or lower airway. Nonspecific URI commonly presents as an acute, mild, and self-limited catarrhal syndrome with a median duration of ~1 week (range, 2–10 days). Signs and symptoms are diverse and frequently variable across patients, even when caused by the same virus. The principal 226 signs and symptoms of nonspecific URI include rhinorrhea (with or without purulence), nasal congestion, cough, and sore throat. Other manifestations, such as fever, malaise, sneezing, lymphadenopathy, and hoarseness, are more variable, with fever more common among infants and young children. This varying presentation may reflect differences in host response as well as in infecting organisms; myalgias and fatigue, for example, sometimes are seen with influenza and parainfluenza infections, whereas conjunctivitis may suggest

1	differences in host response as well as in infecting organisms; myalgias and fatigue, for example, sometimes are seen with influenza and parainfluenza infections, whereas conjunctivitis may suggest infection with adenovirus or enterovirus. Findings on physical examination are frequently nonspecific and unimpressive. Between 0.5% and 2% of colds are complicated by secondary bacterial infections (e.g., rhinosinusitis, otitis media, and pneumonia), particularly in higher-risk populations such as infants, elderly persons, and chronically ill or immunosuppressed individuals. Secondary bacterial infections usually are associated with a prolonged course of illness, increased severity of illness, and localization of signs and symptoms, often as a rebound after initial clinical improvement (the “double-dip” sign). Purulent secretions from the nares or throat often are misinterpreted as an indication of bacterial sinusitis or pharyngitis. These secretions, however, can be seen in nonspecific

1	“double-dip” sign). Purulent secretions from the nares or throat often are misinterpreted as an indication of bacterial sinusitis or pharyngitis. These secretions, however, can be seen in nonspecific URI and, in the absence of other clinical features, are poor predictors of bacterial infection.

1	PART 2 Cardinal Manifestations and Presentation of Diseases Antibiotics have no role in the treatment of uncomplicated nonspecific URI, and their misuse facilitates the emergence of antimicrobial resistance; in healthy volunteers, a single course of a commonly prescribed antibiotic like azithromycin can result in macrolide resistance in oral streptococci many months later. In the absence of clinical evidence of bacterial infection, treatment remains entirely symptom based, with use of decongestants and nonsteroidal anti-inflammatory drugs. Clinical trials of zinc, vitamin C, echinacea, and other alternative remedies have revealed no consistent benefit in the treatment of nonspecific URI.

1	Rhinosinusitis refers to an inflammatory condition involving the nasal sinuses. Although most cases of sinusitis involve more than one sinus, the maxillary sinus is most commonly involved; next, in order of frequency, are the ethmoid, frontal, and sphenoid sinuses. Each sinus is lined with a respiratory epithelium that produces mucus, which is transported out by ciliary action through the sinus ostium and into the nasal cavity. Normally, mucus does not accumulate in the sinuses, which remain mostly sterile despite their adjacency to the bacterium-filled nasal passages. When the sinus ostia are obstructed or when ciliary clearance is impaired or absent, the secretions can be retained, producing the typical signs and symptoms of sinusitis. As these secretions accumulate with obstruction, they become more susceptible to infection with a variety of pathogens, including viruses, bacteria, and fungi. Sinusitis affects a tremendous proportion of the population, accounts for millions of

1	they become more susceptible to infection with a variety of pathogens, including viruses, bacteria, and fungi. Sinusitis affects a tremendous proportion of the population, accounts for millions of visits to primary care physicians each year, and is the fifth leading diagnosis for which antibiotics are prescribed. It typically is classified by duration of illness (acute vs. chronic); by etiology (infectious vs. noninfectious); and, when infectious, by the offending pathogen type (viral, bacterial, or fungal).

1	Acute rhinosinusitis—defined as sinusitis of <4 weeks’ duration—constitutes the vast majority of sinusitis cases. Most cases are diagnosed in the ambulatory care setting and occur primarily as a consequence of a preceding viral URI. Differentiating acute bacterial from viral sinusitis on clinical grounds is difficult. Therefore, it is perhaps not surprising that antibiotics are prescribed frequently (in 85–98% of all cases) for this condition.

1	Etiology The ostial obstruction in rhinosinusitis can arise from both infectious and noninfectious causes. Noninfectious etiologies include allergic rhinitis (with either mucosal edema or polyp obstruction), barotrauma (e.g., from deep-sea diving or air travel), and exposure to chemical irritants. Obstruction can also occur with nasal and sinus tumors (e.g., squamous cell carcinoma) or granulomatous diseases (e.g., granulomatosis with polyangiitis, rhinoscleroma), and conditions leading to altered mucus content (e.g., cystic fibrosis) can cause sinusitis through impaired mucus clearance. In ICUs, nasotracheal intubation and nasogastric tubes are major risk factors for nosocomial sinusitis.

1	Viral rhinosinusitis is far more common than bacterial sinusitis, although relatively few studies have sampled sinus aspirates for the presence of different viruses. In the studies that have done so, the viruses most commonly isolated—both alone and with bacteria—have been rhinovirus, parainfluenza virus, and influenza virus. Bacterial causes of sinusitis have been better described. Among community-acquired cases, S. pneumoniae and nontypable Haemophilus influenzae are the most common pathogens, accounting for 50–60% of cases. Moraxella catarrhalis causes disease in a significant percentage (20%) of children but a lesser percentage of adults. Other streptococcal species and Staphylococcus aureus cause only a small percentage of cases, although there is increasing concern about methicillin-resistant

1	S. aureus (MRSA) as an emerging cause. It is difficult to assess whether a cultured bacterium represents a true infecting organism, an insufficiently deep sample (which would not be expected to be sterile), or—especially in the case of previous sinus surgeries—a colonizing organism. Anaerobes occasionally are found in association with infections of the roots of premolar teeth that spread to the adjacent maxillary sinuses. The role of atypical organisms like Chlamydia pneumoniae and Mycoplasma pneumoniae in the pathogenesis of acute sinusitis is unclear. Nosocomial cases commonly are associated with bacteria prevalent in the hospital environment, including S. aureus, Pseudomonas aeruginosa, Serratia marcescens, Klebsiella pneumoniae, and Enterobacter species. Often, these infections are polymicrobial and can involve organisms that are highly resistant to numerous antibiotics. Fungi also are established causes of sinusitis, although most acute cases are in immunocompromised patients and

1	and can involve organisms that are highly resistant to numerous antibiotics. Fungi also are established causes of sinusitis, although most acute cases are in immunocompromised patients and represent invasive, life-threatening infections. The best-known example is rhinocerebral mucormycosis caused by fungi of the order Mucorales, which includes Rhizopus, Rhizomucor, Mucor, Lichtheimia (formerly Mycocladus, formerly Absidia), and Cunninghamella (Chap. 242). These infections classically occur in diabetic patients with ketoacidosis but can also develop in transplant recipients, patients with hematologic malignancies, and patients receiving chronic glucocorticoid or deferoxamine therapy. Other hyaline molds, such as Aspergillus and Fusarium species, also are occasional causes of this disease.

1	Clinical Manifestations Most cases of acute sinusitis present after or in conjunction with a viral URI, and it can be difficult to discriminate the clinical features of one from the other, with timing becoming important in diagnosis (see below). A large proportion of patients with colds have sinus inflammation, although, as previously stated, true bacterial sinusitis complicates only 0.2–2% of these viral infections. Common presenting symptoms of sinusitis include nasal drainage and congestion, facial pain or pressure, and headache. Thick, purulent or discolored nasal discharge is often thought to indicate bacterial sinusitis but also occurs early in viral infections such as the common cold and is not specific to bacterial infection. Other nonspecific manifestations include cough, sneezing, and fever. Tooth pain, most often involving the upper molars, as well as halitosis are occasionally associated with bacterial sinusitis.

1	In acute sinusitis, sinus pain or pressure often localizes to the involved sinus (particularly the maxillary sinus) and can be worse when the patient bends over or is supine. Although rare, manifestations of advanced sphenoid or ethmoid sinus infection can be profound, including severe frontal or retroorbital pain radiating to the occiput, thrombosis of the cavernous sinus, and signs of orbital cellulitis. Acute focal sinusitis is uncommon but should be considered with severe symptoms involving the maxillary sinus and fever, regardless of illness duration. Similarly, patients with advanced frontal sinusitis can present with a condition known as Pott’s puffy tumor, with soft tissue swelling and pitting edema over the frontal bone from a communicating subperiosteal abscess. Life-threatening complications of sinusitis include meningitis, epidural abscess, and cerebral abscess.

1	Patients with acute fungal rhinosinusitis (such as mucormycosis; Chap. 242) often present with symptoms related to pressure effects, particularly when the infection has spread to the orbits and cavernous sinus. Signs such as orbital swelling and cellulitis, proptosis, ptosis, and decreased extraocular movement are common, as is retroor periorbital pain. Nasopharyngeal ulcerations, epistaxis, and headaches are also common, and involvement of cranial nerves V and VII has been described in more advanced cases. Bony erosion may be evident on examination or endoscopy. Often the patient does not appear seriously ill despite the rapidly progressive nature of these infections. Patients with acute nosocomial sinusitis are often critically ill and thus do not manifest the typical clinical features of sinus disease. This diagnosis should be suspected, however, when hospitalized patients with appropriate risk factors (e.g., nasotracheal intubation) develop fever without another apparent cause.

1	Diagnosis Distinguishing viral from bacterial rhinosinusitis in the ambulatory setting is usually difficult because of the relatively low sensitivity and specificity of the common clinical features. One clinical feature that has been used to help guide diagnostic and therapeutic decision-making is illness duration. Because acute bacterial sinusitis is uncommon in patients whose symptoms have lasted <10 days, expert panels now recommend reserving this diagnosis for patients with “persistent” symptoms (i.e., symptoms lasting >10 days in adults or >10–14 days in children) accompanied by the three cardinal signs of purulent nasal discharge, nasal obstruction, and facial pain (Table 44-1). Even among patients who meet these criteria, only 40–50% have true bacterial sinusitis. The use of CT or sinus radiography is not recommended for acute disease, particularly early in the course of illness (i.e., at <10 days) in light of the high prevalence of similar findings among patients with acute

1	sinus radiography is not recommended for acute disease, particularly early in the course of illness (i.e., at <10 days) in light of the high prevalence of similar findings among patients with acute viral rhinosinusitis. In the evaluation of persistent, recurrent, or chronic sinusitis, CT of the sinuses becomes the radiographic study of choice.

1	The clinical history and/or setting often can identify cases of acute anaerobic bacterial sinusitis, acute fungal sinusitis, or sinusitis from noninfectious causes (e.g., allergic rhinosinusitis). In the case of an immunocompromised patient with acute fungal sinus infection, Moderate symptoms Initial therapy: (e.g., nasal purulence/ Amoxicillin, 500 mg PO tid; or congestion or cough) for Amoxicillin/clavulanate, 500/125 mg PO tid or >10 d or Severe symptoms of any Penicillin allergy: duration, including unilateral/focal facial swell-Doxycycline, 100 mg PO bid; or ing or tooth pain Clindamycin, 300 mg PO tid Exposure to antibiotics within 30 d or >30% prevalence of penicillin-resistant Streptococcus pneumoniae: Amoxicillin/clavulanate (extended release), 2000/125 mg PO bid; or An antipneumococcal fluoroquinolone (e.g., moxifloxacin, 400 mg PO daily) Recent treatment failure: Amoxicillin/clavulanate (extended release), 2000 mg PO bid; or

1	An antipneumococcal fluoroquinolone (e.g., moxifloxacin, 400 mg PO daily) Recent treatment failure: Amoxicillin/clavulanate (extended release), 2000 mg PO bid; or An antipneumococcal fluoroquinolone (e.g., moxifloxacin, 400 mg PO daily) aThe duration of therapy is generally 7–10 days (with consideration of a 5-day course), with appropriate follow-up. Severe disease may warrant IV antibiotics and consideration of hospital admission. bAlthough the evidence is not as strong, amoxicillin/clavulanate may be considered for initial use, particularly if local rates of penicillin resistance or β-lactamase production are high.

1	immediate examination by an otolaryngologist is required. Biopsy 227 specimens from involved areas should be examined by a pathologist for evidence of fungal hyphal elements and tissue invasion. Cases of suspected acute nosocomial sinusitis should be confirmed by sinus CT. Because therapy should target the offending organism, a sinus aspirate for culture and susceptibility testing should be obtained, whenever possible, before the initiation of antimicrobial therapy.

1	Most patients with a clinical diagnosis of acute rhinosinusitis improve without antibiotic therapy. The preferred initial approach in patients with mild to moderate symptoms of short duration is therapy aimed at symptom relief and facilitation of sinus drainage, such as with oral and topical decongestants, nasal saline lavage, and—at least in patients with a history of chronic sinusitis or allergies—nasal glucocorticoids. Newer studies have cast doubt on the role of antibiotics and nasal glucocorticoids in acute rhinosinusitis. In one notable double-blind, randomized, placebo-controlled trial, neither antibiotics nor topical glucocorticoids had a significant impact on cure in the study population of patients, the majority of whom had had symptoms for <7 days. Similarly, another high-profile randomized trial comparing antibiotics to placebo in patients with acute rhinosinusitis demonstrated no significant improvement in symptoms by the third day of therapy. Still, antibiotic therapy

1	randomized trial comparing antibiotics to placebo in patients with acute rhinosinusitis demonstrated no significant improvement in symptoms by the third day of therapy. Still, antibiotic therapy can be considered for adult patients whose condition does not improve after 10 days, and patients with more severe symptoms (regardless of duration) should be treated with antibiotics (Table 44-1). However, watchful waiting remains a viable option in many cases.

1	Empirical antibiotic therapy for adults with community-acquired sinusitis should consist of the narrowest-spectrum agent active against the most common bacterial pathogens, including S. pneumoniae and H. influenzae—e.g., amoxicillin or amoxicillin/ clavulanate (with the decision guided by local rates of β-lactamaseproducing H. influenzae). No clinical trials support the use of broader-spectrum agents for routine cases of bacterial sinusitis, even in the current era of drug-resistant S. pneumoniae. For those patients who do not respond to initial antimicrobial therapy, sinus aspiration and/or lavage by an otolaryngologist should be considered. Antibiotic prophylaxis to prevent episodes of recurrent acute bacterial sinusitis is not recommended.

1	Surgical intervention and IV antibiotic administration usually are reserved for patients with severe disease or those with intra-cranial complications such as abscess and orbital involvement. Immunocompromised patients with acute invasive fungal sinusitis usually require extensive surgical debridement and treatment with IV antifungal agents active against fungal hyphal forms, such as amphotericin B. Specific therapy should be individualized according to the fungal species and its susceptibilities as well as the individual patient’s characteristics. Treatment of nosocomial sinusitis should begin with broad-spectrum antibiotics to cover common and often resistant pathogens such as S. aureus and gram-negative bacilli. Therapy then should be tailored to the results of culture and susceptibility testing of sinus aspirates. CHAPTER 44 Sore Throat, Earache, and Upper Respiratory Symptoms

1	CHAPTER 44 Sore Throat, Earache, and Upper Respiratory Symptoms Chronic sinusitis is characterized by symptoms of sinus inflammation lasting >12 weeks. This illness is most commonly associated with either bacteria or fungi, and clinical cure in most cases is very difficult. Many patients have undergone treatment with repeated courses of antibacterial agents and multiple sinus surgeries, increasing their risk of colonization with antibiotic-resistant pathogens and of surgical complications. These patients often have high rates of morbidity, sometimes over many years.

1	In chronic bacterial sinusitis, infection is thought to be due to the impairment of mucociliary clearance from repeated infections rather than to persistent bacterial infection. The pathogenesis of this 228 condition, however, is poorly understood. Although certain conditions (e.g., cystic fibrosis) can predispose patients to chronic bacterial sinusitis, most patients with chronic rhinosinusitis do not have obvious underlying conditions that result in the obstruction of sinus drainage, the impairment of ciliary action, or immune dysfunction. Patients experience constant nasal congestion and sinus pressure, with intermittent periods of greater severity, which may persist for years. CT can be helpful in determining the extent of disease, detecting an underlying anatomic defect or obstructing process (e.g., a polyp), and assessing the response to therapy. Management should involve an otolaryngologist to conduct endoscopic examinations and obtain tissue samples for histologic examination

1	process (e.g., a polyp), and assessing the response to therapy. Management should involve an otolaryngologist to conduct endoscopic examinations and obtain tissue samples for histologic examination and culture. An endoscopy-derived culture not only has a higher yield but also allows direct visualization for abnormal anatomy. Chronic fungal sinusitis is a disease of immunocompetent hosts and is usually noninvasive, although slowly progressive invasive disease is sometimes seen. Noninvasive disease, which typically is associated with hyaline molds such as Aspergillus species and dematiaceous molds such as Curvularia or Bipolaris species, can present as a number of different scenarios. In mild, indolent disease, which usually occurs in the setting of repeated failures of antibacterial therapy, only nonspecific mucosal changes may be seen on sinus CT. Although there is some controversy on this point, endoscopic surgery is usually curative in these cases, with no need for antifungal

1	therapy, only nonspecific mucosal changes may be seen on sinus CT. Although there is some controversy on this point, endoscopic surgery is usually curative in these cases, with no need for antifungal therapy. Another form of disease presents as long-standing, often unilateral symptoms and opacification of a single sinus on imaging studies as a result of a mycetoma (fungus ball) within the sinus. Treatment for this condition also is surgical, although systemic antifungal therapy may be warranted in the rare case in which bony erosion occurs. A third form of disease, known as allergic fungal sinusitis, is seen in patients with a history of nasal polyposis and asthma, who often have had multiple sinus surgeries. Patients with this condition produce a thick, eosinophil-laden mucus with the consistency of peanut butter that contains sparse fungal hyphae on histologic examination. These patients often present with pansinusitis.

1	Treatment of chronic bacterial sinusitis can be challenging and consists primarily of repeated culture-guided courses of antibiotics, sometimes for 3–4 weeks or longer at a time; administration of intranasal glucocorticoids; and mechanical irrigation of the sinus with sterile saline solution. When this management approach fails, sinus surgery may be indicated and sometimes provides significant, albeit short-term, alleviation. Treatment of chronic fungal sinusitis consists of surgical removal of impacted mucus. Recurrence, unfortunately, is common. PART 2 Cardinal Manifestations and Presentation of Diseases Infections of the ear and associated structures can involve both the middle and the external ear, including the skin, cartilage, periosteum, ear canal, and tympanic and mastoid cavities. Both viruses and bacteria are known causes of these infections, some of which result in significant morbidity if not treated appropriately.

1	Infections involving the structures of the external ear are often difficult to differentiate from noninfectious inflammatory conditions with similar clinical manifestations. Clinicians should consider inflammatory disorders as possible causes of external ear irritation, particularly in the absence of local or regional adenopathy. Aside from the more salient causes of inflammation, such as trauma, insect bite, and overexposure to sunlight or extreme cold, the differential diagnosis should include less common conditions such as autoimmune disorders (e.g., lupus or relapsing polychondritis) and vasculitides (e.g., granulomatosis with polyangiitis).

1	Auricular Cellulitis Auricular cellulitis is an infection of the skin overlying the external ear and typically follows minor local trauma. It presents as the typical signs and symptoms of cellulitis, with tenderness, erythema, swelling, and warmth of the external ear (particularly the lobule) but without apparent involvement of the ear canal or inner structures. Treatment consists of warm compresses and oral antibiotics such as cephalexin or dicloxacillin that are active against typical skin and soft tissue pathogens (specifically, S. aureus and streptococci). IV antibiotics such as a first-generation cephalosporin (e.g., cefazolin) or a penicillinase-resistant penicillin (e.g., nafcillin) occasionally are needed for more severe cases, with consideration of MRSA if either risk factors or failure of therapy point to this organism.

1	Perichondritis Perichondritis, an infection of the perichondrium of the auricular cartilage, typically follows local trauma (e.g., piercings, burns, or lacerations). Occasionally, when the infection spreads down to the cartilage of the pinna itself, patients may develop chondritis. The infection may closely resemble auricular cellulitis, with erythema, swelling, and extreme tenderness of the pinna, although the lobule is less often involved in perichondritis. The most common pathogens are P. aeruginosa and S. aureus, although other gram-negative and gram-positive organisms occasionally are involved. Treatment consists of systemic antibiotics active against both P. aeruginosa and S. aureus. An antipseudomonal penicillin (e.g., piperacillin) or a combination of a penicillinase-resistant penicillin and an antipseudomonal quinolone (e.g., nafcillin plus ciprofloxacin) is typically used. Incision and drainage may be helpful for culture and for resolution of infection, which often takes

1	penicillin and an antipseudomonal quinolone (e.g., nafcillin plus ciprofloxacin) is typically used. Incision and drainage may be helpful for culture and for resolution of infection, which often takes weeks. When perichondritis fails to respond to adequate antimicrobial therapy, clinicians should consider a noninfectious inflammatory etiology such as relapsing polychondritis.

1	Otitis Externa The term otitis externa refers to a collection of diseases involving primarily the auditory meatus. Otitis externa usually results from a combination of heat and retained moisture, with desquamation and maceration of the epithelium of the outer ear canal. The disease exists in several forms: localized, diffuse, chronic, and invasive. All forms are predominantly bacterial in origin, with P. aeruginosa and S. aureus the most common pathogens. Acute localized otitis externa (furunculosis) can develop in the outer third of the ear canal, where skin overlies cartilage and hair follicles are numerous. As in furunculosis elsewhere on the body, S. aureus is the usual pathogen, and treatment typically consists of an oral antistaphylococcal penicillin (e.g., dicloxacillin or cephalexin), with incision and drainage in cases of abscess formation.

1	Acute diffuse otitis externa is also known as swimmer’s ear, although it can develop in patients who have not recently been swimming. Heat, humidity, and the loss of protective cerumen lead to excessive moisture and elevation of the pH in the ear canal, which in turn lead to skin maceration and irritation. Infection may then follow; the predominant pathogen is P. aeruginosa, although other gram-negative and gram-positive organisms—and rarely yeasts—have been recovered from patients with this condition. The illness often starts with itching and progresses to severe pain, which is usually elicited by manipulation of the pinna or tragus. The onset of pain is generally accompanied by the development of an erythematous, swollen ear canal, often with scant white, clumpy discharge. Treatment consists of cleansing the canal to remove debris and enhance the activity of topical therapeutic agents—usually hypertonic saline or mixtures of alcohol and acetic acid. Inflammation can also be

1	consists of cleansing the canal to remove debris and enhance the activity of topical therapeutic agents—usually hypertonic saline or mixtures of alcohol and acetic acid. Inflammation can also be decreased by adding glucocorticoids to the treatment regimen or by using Burow’s solution (aluminum acetate in water). Antibiotics are most effective when given topically. Otic mixtures provide adequate pathogen coverage; these preparations usually combine neomycin with polymyxin, with or without glucocorticoids. Systemic antimicrobial agents typically are reserved for severe disease or infections in immunocompromised hosts.

1	Chronic otitis externa is caused primarily by repeated local irritation, most commonly arising from persistent drainage from a chronic middle-ear infection. Other causes of repeated irritation, such as insertion of cotton swabs or other foreign objects into the ear canal, can lead to this condition, as can rare chronic infections such as syphilis, tuberculosis, and leprosy. Chronic otitis externa typically presents as erythematous, scaling dermatitis in which the predominant symptom is pruritus rather than pain; this condition must be differentiated from several others that produce a similar clinical picture, such as atopic dermatitis, seborrheic dermatitis, psoriasis, and dermatomycosis. Therapy consists of identifying and treating or removing the offending process, although successful resolution is frequently difficult.

1	Invasive otitis externa, also known as malignant or necrotizing otitis externa, is an aggressive and potentially life-threatening disease that occurs predominantly in elderly diabetic patients and other immunocompromised persons. The disease begins in the external canal as a soft tissue infection that progresses slowly over weeks to months and often is difficult to distinguish from a severe case of chronic otitis externa because of the presence of purulent otorrhea and an erythematous swollen ear and external canal. Severe, deep-seated otalgia, frequently out of proportion to findings on examination, is often noted and can help differentiate invasive from chronic otitis externa. The characteristic finding on examination is granulation tissue in the posteroinferior wall of the external canal, near the junction of bone and cartilage. If left unchecked, the infection can migrate to the base of the skull (resulting in skull-base osteomyelitis) and onward to the meninges and brain, with a

1	near the junction of bone and cartilage. If left unchecked, the infection can migrate to the base of the skull (resulting in skull-base osteomyelitis) and onward to the meninges and brain, with a high mortality rate. Cranial nerve involvement is seen occasionally, with the facial nerve usually affected first and most often. Thrombosis of the sigmoid sinus can occur if the infection extends to the area. CT, which can reveal osseous erosion of the temporal bone and skull base, can be used to help determine the extent of disease, as can gallium and technetium-99 scintigraphy studies. P. aeruginosa is by far the most common offender, although S. aureus, Staphylococcus epidermidis, Aspergillus, Actinomyces, and some gram-negative bacteria also have been associated with this disease. In all cases, the external ear canal should be cleansed and a biopsy specimen of the granulation tissue within the canal (or of deeper tissues) obtained for culture of the offending organism. IV antibiotic

1	cases, the external ear canal should be cleansed and a biopsy specimen of the granulation tissue within the canal (or of deeper tissues) obtained for culture of the offending organism. IV antibiotic therapy should be given for a prolonged course (6–8 weeks) and directed specifically toward the recovered pathogen. For P. aeruginosa, the regimen typically includes an antipseudomonal penicillin or cephalosporin (e.g., piperacillin or cefepime), often with an aminoglycoside or a fluoroquinolone, the latter of which can even be administered orally given its excellent bioavailability. In addition, antibiotic drops containing an agent active 229 against Pseudomonas (e.g., ciprofloxacin) are usually prescribed and are combined with glucocorticoids to reduce inflammation. Cases of invasive Pseudomonas otitis externa recognized in the early stages can sometimes be treated with oral and otic fluoroquinolones alone, albeit with close follow-up. Extensive surgical debridement, once an important

1	otitis externa recognized in the early stages can sometimes be treated with oral and otic fluoroquinolones alone, albeit with close follow-up. Extensive surgical debridement, once an important component of the treatment approach, is now rarely indicated.

1	In necrotizing otitis externa, recurrence is documented up to 20% of the time. Aggressive glycemic control in diabetics is important not only for effective treatment but also for prevention of recurrence. The role of hyperbaric oxygen has not been clearly established. Otitis media is an inflammatory condition of the middle ear that results from dysfunction of the eustachian tube in association with a number of illnesses, including URIs and chronic rhinosinusitis. The inflammatory response in these conditions leads to the development of a sterile transudate within the middle ear and mastoid cavities. Infection may occur if bacteria or viruses from the nasopharynx contaminate this fluid, producing an acute (or sometimes chronic) illness.

1	Acute Otitis Media Acute otitis media results when pathogens from the nasopharynx are introduced into the inflammatory fluid collected in the middle ear (e.g., by nose blowing during a URI). Pathogenic proliferation in this space leads to the development of the typical signs and symptoms of acute middle-ear infection. The diagnosis of acute otitis media requires the demonstration of fluid in the middle ear (with tympanic membrane [TM] immobility) and the accompanying signs or symptoms of local or systemic illness (Table 44-2).

1	ETIOLOgY Acute otitis media typically follows a viral URI. The causative viruses (most commonly RSV, influenza virus, rhinovirus, and enterovirus) can themselves cause subsequent acute otitis media; more often, they predispose the patient to bacterial otitis media. Studies using tympanocentesis have consistently found S. pneumoniae to be the most important bacterial cause, isolated in up to 35% of cases. H. influenzae (nontypable strains) and M. catarrhalis also are CHAPTER 44 Sore Throat, Earache, and Upper Respiratory Symptoms aDuration (unless otherwise specified): 10 days for patients <6 years old and patients with severe disease; 5–7 days (with consideration of observation only in previously healthy individuals with mild disease) for patients ≥6 years old. bFailure to improve and/or clinical worsening after 48–72 h of observation or treatment. Abbreviation: TM, tympanic membrane. Source: American Academy of Pediatrics Subcommittee on Management of Acute Otitis Media, 2004.

1	230 common bacterial causes of acute otitis media, and concern is increasing with MRSA as an emerging etiologic agent. Viruses, such as those mentioned above, have been recovered either alone or with bacteria in 17–40% of cases. CLINICAL MANIFESTATIONS Fluid in the middle ear is typically demonstrated or confirmed with pneumatic otoscopy. In the absence of fluid, the TM moves visibly with the application of positive and negative pressure, but this movement is dampened when fluid is present. With bacterial infection, the TM can also be erythematous, bulging, or retracted and occasionally can perforate spontaneously. The signs and symptoms accompanying infection can be local or systemic, including otalgia, otorrhea, diminished hearing, and fever. Erythema of the TM is often evident but is nonspecific as it frequently is seen in association with inflammation of the upper respiratory mucosa. Other signs and symptoms occasionally reported include vertigo, nystagmus, and tinnitus.

1	PART 2 Cardinal Manifestations and Presentation of Diseases

1	There has been considerable debate on the usefulness of antibiotics for the treatment of acute otitis media. A higher proportion of treated than untreated patients are free of illness 3–5 days after diagnosis. The difficulty of predicting which patients will benefit from antibiotic therapy has led to different approaches. In the Netherlands, for instance, physicians typically manage acute otitis media with initial observation, administering anti-inflammatory agents for aggressive pain management and reserving antibiotics for high-risk patients, patients with complicated disease, or patients whose condition does not improve after 48–72 h. In contrast, many experts in the United States continue to recommend antibiotic therapy for children <6 months old in light of the higher frequency of secondary complications in this young and functionally immunocompromised population. However, observation without antimicrobial therapy is now the recommended option in the United States for acute

1	of secondary complications in this young and functionally immunocompromised population. However, observation without antimicrobial therapy is now the recommended option in the United States for acute otitis media in children >2 years of age and for mild to moderate disease without middle-ear effusion in children 6 months to 2 years of age. Treatment is typically indicated for patients <6 months old; for children 6 months to 2 years old who have mid-dle-ear effusion and signs/symptoms of middle-ear inflammation; for all patients >2 years old who have bilateral disease, TM perforation, immunocompromise, or emesis; and for any patient who has severe symptoms, including a fever ≥39°C or moderate to severe otalgia (Table 44-2).

1	Because most studies of the etiologic agents of acute otitis media consistently document similar pathogen profiles, therapy is generally empirical except in those few cases in which tympanocentesis is warranted—e.g., cases refractory to therapy and cases in patients who are severely ill or immunodeficient. Despite resistance to penicillin and amoxicillin in roughly one-quarter of S. pneumoniae isolates, one-third of H. influenzae isolates, and nearly all M. catarrhalis isolates, outcome studies continue to find that amoxicillin is as successful as any other agent, and it remains the drug of first choice in recommendations from multiple sources (Table 44-2). Therapy for uncomplicated acute otitis media typically is administered for 5–7 days to patients ≥6 years old; longer courses (e.g., 10 days) should be reserved for patients with severe disease, in whom short-course therapy may be inadequate.

1	A switch in regimen is recommended if there is no clinical improvement by the third day of therapy, given the possibility of infection with a β-lactamase-producing strain of H. influenzae or M. catarrhalis or with a strain of penicillin-resistant S. pneumoniae. Decongestants and antihistamines are frequently used as adjunctive agents to reduce congestion and relieve obstruction of the eustachian tube, but clinical trials have yielded no significant evidence of benefit with either class of agents.

1	Recurrent Acute Otitis Media Recurrent acute otitis media (more than three episodes within 6 months or four episodes within 12 months) generally is due to relapse or reinfection, although data indicate that the majority of early recurrences are new infections. In general, the same pathogens responsible for acute otitis media cause recurrent disease; even so, the recommended treatment consists of antibiotics active against β-lactamase-producing organisms. Antibiotic prophylaxis (e.g., with trimethoprim-sulfamethoxazole [TMP-SMX] or amoxicillin) can reduce recurrences in patients with recurrent acute otitis media by an average of one episode per year, but this benefit is small compared with the high likelihood of colonization with antibiotic-resistant pathogens. Other approaches, including placement of tympanostomy tubes, adenoidectomy, and tonsillectomy plus adenoidectomy, are of questionable overall value in light of the relatively small benefit compared with the potential for

1	placement of tympanostomy tubes, adenoidectomy, and tonsillectomy plus adenoidectomy, are of questionable overall value in light of the relatively small benefit compared with the potential for complications.

1	Serous Otitis Media In serous otitis media (otitis media with effusion), fluid is present in the middle ear for an extended period in the absence of signs and symptoms of infection. In general, acute effusions are self-limited; most resolve in 2–4 weeks. In some cases, however (in particular after an episode of acute otitis media), effusions can persist for months. These chronic effusions are often associated with significant hearing loss in the affected ear. The great majority of cases of otitis media with effusion resolve spontaneously within 3 months without antibiotic therapy. Antibiotic therapy or myringotomy with insertion of tympanostomy tubes typically is reserved for patients in whom bilateral effusion (1) has persisted for at least 3 months and (2) is associated with significant bilateral hearing loss. With this conservative approach and the application of strict diagnostic criteria for acute otitis media and otitis media with effusion, it is estimated that 6–8 million

1	bilateral hearing loss. With this conservative approach and the application of strict diagnostic criteria for acute otitis media and otitis media with effusion, it is estimated that 6–8 million courses of antibiotics could be avoided each year in the United States.

1	Chronic Otitis Media Chronic suppurative otitis media is characterized by persistent or recurrent purulent otorrhea in the setting of TM perforation. Usually, there is also some degree of conductive hearing loss. This condition can be categorized as active or inactive. Inactive disease is characterized by a central perforation of the TM, which allows drainage of purulent fluid from the middle ear. When the perforation is more peripheral, squamous epithelium from the auditory canal may invade the middle ear through the perforation, forming a mass of keratinaceous debris (cholesteatoma) at the site of invasion. This mass can enlarge and has the potential to erode bone and promote further infection, which can lead to meningitis, brain abscess, or paralysis of cranial nerve VII. Treatment of chronic active otitis media is surgical; mastoidectomy, myringoplasty, and tympanoplasty can be performed as outpatient surgical procedures, with an overall success rate of ~80%. Chronic inactive

1	of chronic active otitis media is surgical; mastoidectomy, myringoplasty, and tympanoplasty can be performed as outpatient surgical procedures, with an overall success rate of ~80%. Chronic inactive otitis media is more difficult to cure, usually requiring repeated courses of topical antibiotic drops during periods of drainage. Systemic antibiotics may offer better cure rates, but their role in the treatment of this condition remains unclear.

1	Mastoiditis Acute mastoiditis was relatively common among children before the introduction of antibiotics. Because the mastoid air cells connect with the middle ear, the process of fluid collection and infection is usually the same in the mastoid as in the middle ear. Early and frequent treatment of acute otitis media is most likely the reason that the incidence of acute mastoiditis has declined to only 1.2–2.0 cases per 100,000 person-years in countries with high prescribing rates for acute otitis media. In countries such as the Netherlands, where antibiotics are used sparingly for acute otitis media, the incidence rate of acute mastoiditis is roughly twice that in countries like the United States. However, neighboring Denmark has a rate of acute mastoiditis similar to that in the Netherlands but an antibiotic-prescribing rate for acute otitis media more similar to that in the United States.

1	In typical acute mastoiditis, purulent exudate collects in the mastoid air cells (Fig. 44-1), producing pressure that may result in erosion of the surrounding bone and formation of abscess-like cavities that are usually evident on CT. Patients typically present with pain, erythema, and swelling of the mastoid process along with displacement of the pinna, usually in conjunction with the typical signs and symptoms of acute middle-ear infection. Rarely, patients can develop severe complications if the infection tracks under the periosteum of the temporal bone to cause a subperiosteal abscess, erodes through the mastoid tip to cause a deep neck abscess, or extends posteriorly to cause septic thrombosis of the lateral sinus. FIguRE 44-1 Acute mastoiditis. Axial CT image shows an acute fluid collection within the mastoid air cells on the left.

1	FIguRE 44-1 Acute mastoiditis. Axial CT image shows an acute fluid collection within the mastoid air cells on the left. Purulent fluid should be cultured whenever possible to help guide antimicrobial therapy. Initial empirical therapy usually is directed against the typical organisms associated with acute otitis media, such as S. pneumoniae, H. influenzae, and M. catarrhalis. Patients with more severe or prolonged courses of illness should be treated for infection with S. aureus and gram-negative bacilli (including Pseudomonas). Broad empirical therapy should be narrowed once culture results become available. Most patients can be treated conservatively with IV antibiotics; surgery (cortical mastoidectomy) is reserved for complicated cases and those in which conservative treatment has failed.

1	Oropharyngeal infections range from mild, self-limited viral illnesses to serious, life-threatening bacterial infections. The most common presenting symptom is sore throat—one of the most common reasons for ambulatory care visits by both adults and children. Although sore throat is a symptom in many noninfectious illnesses as well, the overwhelming majority of patients with a new sore throat have acute pharyngitis of viral or bacterial etiology. Millions of visits to primary care providers each year are for sore throat; the majority of cases of acute pharyngitis are caused by typical respiratory viruses. The most important source of concern is infection with group A β-hemolytic Streptococcus (S. pyogenes) that is associated with acute glomerulonephritis and acute rheumatic fever. The risk of rheumatic fever can be reduced by timely penicillin therapy.

1	Etiology A wide variety of organisms cause acute pharyngitis. The relative importance of the different pathogens can only be estimated, since a significant proportion of cases (~30%) have no identified cause. Together, respiratory viruses are the most common identifiable cause 231 of acute pharyngitis, with rhinoviruses and coronaviruses accounting for large proportions of cases (~20% and at least 5%, respectively). Influenza virus, parainfluenza virus, and adenovirus also account for a measurable share of cases, with the former two more seasonal and the latter as part of the more clinically severe syndrome of pharyngoconjunctival fever. Other important but less common viral causes include herpes simplex virus (HSV) types 1 and 2, coxsackievirus A, cytomegalovirus (CMV), and Epstein-Barr virus (EBV). Acute HIV infection can present as acute pharyngitis and should be considered in at-risk populations.

1	Acute bacterial pharyngitis is typically caused by S. pyogenes, which accounts for ~5–15% of all cases of acute pharyngitis in adults; rates vary with the season and with utilization of the health care system. Group A streptococcal pharyngitis is primarily a disease of children 5–15 years of age; it is uncommon among children <3 years old, as is rheumatic fever. Streptococci of groups C and G account for a minority of cases, although these serogroups are nonrheumatogenic. Fusobacterium necrophorum has been increasingly recognized as a cause of pharyngitis in adolescents and young adults and is isolated nearly as often as group A streptococci. This organism is important because of the rare but life-threatening Lemierre’s disease, which is generally associated with F. necrophorum and is usually preceded by pharyngitis (see “Oral Infections,” below). The remaining bacterial causes of acute pharyngitis are seen infrequently (<1% of cases each) but should be considered in appropriate

1	usually preceded by pharyngitis (see “Oral Infections,” below). The remaining bacterial causes of acute pharyngitis are seen infrequently (<1% of cases each) but should be considered in appropriate exposure groups because of the severity of illness if left untreated; these etiologic agents include Neisseria gonorrhoeae, Corynebacterium diphtheriae, Corynebacterium ulcerans, Yersinia enterocolitica, and Treponema pallidum (in secondary syphilis). Anaerobic bacteria also can cause acute pharyngitis (Vincent’s angina) and can contribute to more serious polymicrobial infections, such as peritonsillar or retropharyngeal abscesses (see below). Atypical organisms such as M. pneumoniae and C. pneumoniae have been recovered from patients with acute pharyngitis; whether these agents are commensals or causes of acute infection is debatable.

1	Clinical Manifestations Although the signs and symptoms accompanying acute pharyngitis are not reliable predictors of the etiologic agent, the clinical presentation occasionally suggests one etiology over another. Acute pharyngitis due to respiratory viruses such as rhinovirus or coronavirus usually is not severe and typically is associated with a constellation of coryzal symptoms better characterized as nonspecific URI. Findings on physical examination are uncommon; fever is rare, and tender cervical adenopathy and pharyngeal exudates are not seen. In contrast, acute pharyngitis from influenza virus can be severe and is much more likely to be associated with fever as well as with myalgias, headache, and cough. The presentation of pharyngoconjunctival fever due to adenovirus infection is similar. Since pharyngeal exudate may be present on examination, this condition can be difficult to differentiate from streptococcal pharyngitis. However, adenoviral pharyngitis is distinguished by

1	similar. Since pharyngeal exudate may be present on examination, this condition can be difficult to differentiate from streptococcal pharyngitis. However, adenoviral pharyngitis is distinguished by the presence of conjunctivitis in one-third to one-half of patients. Acute pharyngitis from primary HSV infection can also mimic streptococcal pharyngitis in some cases, with pharyngeal inflammation and exudate, but the presence of vesicles and shallow ulcers on the palate can help differentiate the two diseases. This HSV syndrome is distinct from pharyngitis caused by coxsackievirus (herpangina), which is associated with small vesicles that develop on the soft palate and uvula and then rupture to form shallow white ulcers. Acute exudative pharyngitis coupled with fever, fatigue, generalized lymphadenopathy, and (on occasion) splenomegaly is characteristic of infectious mononucleosis due to EBV or CMV. Acute primary infection with HIV is frequently associated with fever and acute

1	lymphadenopathy, and (on occasion) splenomegaly is characteristic of infectious mononucleosis due to EBV or CMV. Acute primary infection with HIV is frequently associated with fever and acute pharyngitis as well as with myalgias, arthralgias, malaise, and occasionally a nonpruritic maculopapular rash, which may be followed by lymphadenopathy and mucosal ulcerations without exudate.

1	The clinical features of acute pharyngitis caused by streptococci of groups A, C, and G are similar, ranging from a relatively mild illness without many accompanying symptoms to clinically severe cases with profound pharyngeal pain, fever, chills, and abdominal pain. CHAPTER 44 Sore Throat, Earache, and Upper Respiratory Symptoms 232 A hyperemic pharyngeal membrane with tonsillar hypertrophy and exudate is usually seen, along with tender anterior cervical adenopathy. Coryzal manifestations, including cough, are typically absent; when present, they suggest a viral etiology. Strains of S. pyogenes that generate erythrogenic toxin can also produce scarlet fever characterized by an erythematous rash and strawberry tongue. The other types of acute bacterial pharyngitis (e.g., gonococcal, diphtherial, and yersinial) often present as exudative pharyngitis with or without other clinical features. Their etiologies are often suggested only by the clinical history.

1	Diagnosis The primary goal of diagnostic testing is to separate acute streptococcal pharyngitis from pharyngitis of other etiologies (particularly viral) so that antibiotics can be prescribed more efficiently for patients in whom they may be beneficial. The most appropriate standard for the diagnosis of streptococcal pharyngitis, however, has not been established definitively. Throat swab culture is generally regarded as the most appropriate but cannot distinguish between infection and colonization and requires 24–48 h to yield results that vary with technique and culture conditions. Rapid antigen-detection tests offer good specificity (>90%) but lower sensitivity when implemented in routine practice. The sensitivity has also been shown to vary across the clinical spectrum of disease (65–90%). Several clinical prediction systems (Fig. 44-2) can increase the sensitivity of rapid antigen-detection tests to >90% in controlled settings. Since the

1	PART 2 Cardinal Manifestations and Presentation of Diseases Symptoms consistent with viral URI? Risk factors for HIV, gonorrhea? Group A Strep RADT or throat culture Penicillin allergy? No streptococcal testing Test accordingly Symptomatic management • Penicillin G 1.2 million units IM × 1, or • Penicillin VK 250 mg orally QID, or 500 mg orally BID, or • Amoxicillin 500 mg orally BID • Cephalexin 500 mg orally BID or TID (only if non-anaphylactic penicillin allergy), or • Azithromycin† 500 mg orally QD × 5 days, or • Clindamycin 300 mg orally TID Positive NoNegative* NOTE: All treatment durations are for 10 days with appropriate follow-up,unless otherwise specified.Yes Yes Yes No No No *Confirmation of a negative rapid antigen-detection test by a throat culture is not required in adults. †Macrolides do not treat F. necrophorum, a cause of pharyngitis in young adults (see text). Abbreviations: URI, upper respiratory infection; RADT, rapid antigen detection test

1	†Macrolides do not treat F. necrophorum, a cause of pharyngitis in young adults (see text). Abbreviations: URI, upper respiratory infection; RADT, rapid antigen detection test FIguRE 44-2 Algorithm for the diagnosis and treatment of acute pharyngitis. sensitivities achieved in routine clinical practice are often lower, several medical and professional societies continue to recommend that all negative rapid antigen-detection tests in children be confirmed by a throat culture to limit transmission and complications of illness caused by group A streptococci. The Centers for Disease Control and Prevention, the Infectious Diseases Society of America, and the American Academy of Family Physicians do not recommend backup culture when adults have negative results from a highly sensitive rapid antigen-detection test, however, because of the lower prevalence and smaller benefit in this age group.

1	Cultures and rapid diagnostic tests for other causes of acute pharyngitis, such as influenza virus, adenovirus, HSV, EBV, CMV, and M. pneumoniae, are available in many locations and can be used when suspected. The diagnosis of acute EBV infection depends primarily on the detection of antibodies to the virus with a heterophile agglutination assay (monospot slide test) or enzyme-linked immunosorbent assay. Testing for HIV RNA or antigen (p24) should be performed when acute primary HIV infection is suspected. If other bacterial causes are suspected (particularly N. gonorrhoeae, C. diphtheriae, or Y. enterocolitica), specific cultures should be requested since these organisms may be missed on routine throat swab culture.

1	Antibiotic treatment of pharyngitis due to S. pyogenes confers numerous benefits, including a decrease in the risk of rheumatic fever, the primary focus of treatment. The magnitude of this benefit is fairly small, since rheumatic fever is now a rare disease, even among untreated patients. Nevertheless, when therapy is started within 48 h of illness onset, symptom duration is decreased modestly. An additional benefit of therapy is the potential to reduce the transmission of streptococcal pharyngitis, particularly in areas of overcrowding or close contact. Antibiotic therapy for acute pharyngitis is therefore recommended in cases in which S. pyogenes is confirmed as the etiologic agent by rapid antigen-detection test or throat swab culture. Otherwise, antibiotics should be given in routine cases only when another bacterial cause has been identified. Effective therapy for streptococcal pharyngitis consists of either a single dose of IM benzathine penicillin or a full 10-day course of

1	cases only when another bacterial cause has been identified. Effective therapy for streptococcal pharyngitis consists of either a single dose of IM benzathine penicillin or a full 10-day course of oral penicillin (Fig. 44-2).

1	Azithromycin can be used in place of penicillin, although resistance to azithromycin among S. pyogenes strains in some parts of the world (particularly Europe) can prohibit the use of this drug. Newer (and more expensive) antibiotics also are active against streptococci but offer no greater efficacy than the agents mentioned above. Testing for cure is unnecessary and may reveal only chronic colonization. There is no evidence to support antibiotic treatment of group C or G streptococcal pharyngitis or pharyngitis in which mycoplasmas or chlamydiae have been recovered. Cultures can be of benefit because F. necrophorum, an increasingly common cause of bacterial pharyngitis in young adults, is not covered by macrolide therapy. Long-term penicillin prophylaxis (benzathine penicillin G, 1.2 million units IM every 3–4 weeks; or penicillin VK, 250 mg PO bid) is indicated for patients at risk of recurrent rheumatic fever.

1	Treatment of viral pharyngitis is entirely symptom based except in infection with influenza virus or HSV. For influenza, the armamentarium includes the adamantanes amantadine and rimantadine and the neuraminidase inhibitors oseltamivir and zanamivir. Administration of all these agents needs to be started within 48 h of symptom onset to reduce illness duration meaningfully. Among these agents, only oseltamivir and zanamivir are active against both influenza A and influenza B and therefore can be used when local patterns of infection and antiviral resistance are unknown. Oropharyngeal HSV infection sometimes responds to treatment with antiviral agents such as acyclovir, although these drugs are often reserved for immunosuppressed patients.

1	Complications Although rheumatic fever is the best-known com-233 plication of acute streptococcal pharyngitis, the risk of its following acute infection remains quite low. Other complications include acute glomerulonephritis and numerous suppurative conditions, such as peritonsillar abscess (quinsy), otitis media, mastoiditis, sinusitis, bacteremia, and pneumonia—all of which occur at low rates. Although antibiotic treatment of acute streptococcal pharyngitis can prevent the development of rheumatic fever, there is no evidence that it can prevent acute glomerulonephritis. Some evidence supports antibiotic use to prevent the suppurative complications of streptococcal pharyngitis, particularly peritonsillar abscess, which can also involve oral anaerobes such as Fusobacterium. Abscesses usually are accompanied by severe pharyngeal pain, dysphagia, fever, and dehydration; in addition, medial displacement of the tonsil and lateral displacement of the uvula are often evident on examination.

1	are accompanied by severe pharyngeal pain, dysphagia, fever, and dehydration; in addition, medial displacement of the tonsil and lateral displacement of the uvula are often evident on examination. Although early use of IV antibiotics (e.g., clindamycin, penicillin G with metronidazole) may obviate the need for surgical drainage in some cases, treatment typically involves needle aspiration or incision and drainage.

1	Aside from periodontal disease such as gingivitis, infections of the oral cavity most commonly involve HSV or Candida species. In addition to causing painful cold sores on the lips, HSV can infect the tongue and buccal mucosa, causing the formation of irritating vesicles. Although topical antiviral agents (e.g., acyclovir and penciclovir) can be used externally for cold sores, oral or IV acyclovir is often needed for primary infections, extensive oral infections, and infections in immunocompromised patients. Oropharyngeal candidiasis (thrush) is caused by a variety of Candida species, most often C. albicans. Thrush occurs predominantly in neonates, immunocompromised patients (especially those with AIDS), and recipients of prolonged antibiotic or glucocorticoid therapy. In addition to sore throat, patients often report a burning tongue, and physical examination reveals friable white or gray plaques on the gingiva, tongue, and oral mucosa. Treatment, which usually consists of an oral

1	throat, patients often report a burning tongue, and physical examination reveals friable white or gray plaques on the gingiva, tongue, and oral mucosa. Treatment, which usually consists of an oral antifungal suspension (nystatin or clotrimazole) or oral fluconazole, is typically successful. In the uncommon cases of fluconazole-refractory thrush that are seen in some patients with HIV/AIDS, other therapeutic options include oral formulations of itraconazole or voriconazole as well as an IV echinocandin (caspofungin, micafungin, or anidulafungin) or amphotericin B deoxycholate, if needed. In these cases, therapy based on culture and susceptibility test results is ideal.

1	Vincent’s angina, also known as acute necrotizing ulcerative gingivitis or trench mouth, is a unique and dramatic form of gingivitis characterized by painful, inflamed gingiva with ulcerations of the interdental papillae that bleed easily. Since oral anaerobes are the cause, patients typically have halitosis and frequently present with fever, malaise, and lymphadenopathy. Treatment consists of debridement and oral administration of penicillin plus metronidazole, with clindamycin or doxycycline alone as an alternative.

1	Ludwig’s angina is a rapidly progressive, potentially fulminant form of cellulitis that involves the bilateral sublingual and sub-mandibular spaces and that typically originates from an infected or recently extracted tooth, most commonly the lower second and third molars. Improved dental care has reduced the incidence of this disorder substantially. Infection in these areas leads to dysphagia, odynophagia, and “woody” edema in the sublingual region, forcing the tongue up and back with the potential for airway obstruction. Fever, dysarthria, and drooling also may be noted, and patients may speak in a “hot potato” voice. Intubation or tracheostomy may be necessary to secure the airway, as asphyxiation is the most common cause of death. Patients should be monitored closely and treated promptly with IV antibiotics directed against streptococci and oral anaerobes. Recommended agents include ampicillin/sulbactam, clindamycin, or high-dose penicillin plus metronidazole.

1	Postanginal septicemia (Lemierre’s disease) is a rare anaerobic oropharyngeal infection caused predominantly by F. necrophorum. The illness typically starts as a sore throat (most commonly in adolescents and young adults), which may present as exudative tonsillitis or peritonsillar abscess. Infection of the deep pharyngeal tissue allows

1	CHAPTER 44 Sore Throat, Earache, and Upper Respiratory Symptoms 234 organisms to drain into the lateral pharyngeal space, which contains the carotid artery and internal jugular vein. Septic thrombophlebitis of the internal jugular vein can result, with associated pain, dysphagia, and neck swelling and stiffness. Sepsis usually occurs 3–10 days after the onset of sore throat and is often coupled with metastatic infection to the lung and other distant sites. Occasionally, the infection can extend along the carotid sheath and into the posterior mediastinum, resulting in mediastinitis, or it can erode into the carotid artery, with the early sign of repeated small bleeds into the mouth. The mortality rate from these invasive infections can be as high as 50%. Treatment consists of IV antibiotics (clindamycin or ampicillin/sulbactam) and surgical drainage of any purulent collections. The concomitant use of anticoagulants to prevent embolization remains controversial and is sometimes advised,

1	(clindamycin or ampicillin/sulbactam) and surgical drainage of any purulent collections. The concomitant use of anticoagulants to prevent embolization remains controversial and is sometimes advised, with careful consideration of both the risks and the benefits.

1	PART 2 Cardinal Manifestations and Presentation of Diseases Laryngitis is defined as any inflammatory process involving the larynx and can be caused by a variety of infectious and noninfectious processes. The vast majority of laryngitis cases seen in clinical practice in developed countries are acute. Acute laryngitis is a common syndrome caused predominantly by the same viruses responsible for many other URIs. In fact, most cases of acute laryngitis occur in the setting of a viral URI. Etiology Nearly all major respiratory viruses have been implicated in acute viral laryngitis, including rhinovirus, influenza virus, parainfluenza virus, adenovirus, coxsackievirus, coronavirus, and RSV. Acute laryngitis can also be associated with acute bacterial respiratory infections such as those caused by group A Streptococcus or

1	C. diphtheriae (although diphtheria has been virtually eliminated in the United States). Another bacterial pathogen thought to play a role (albeit unclear) in the pathogenesis of acute laryngitis is M. catarrhalis, which has been recovered from nasopharyngeal culture in a significant percentage of cases. Chronic laryngitis of infectious etiology is much less common in developed than in developing countries. Laryngitis due to Mycobacterium tuberculosis is often difficult to distinguish from laryngeal cancer, in part because of the frequent absence of signs, symptoms, and radiographic findings typical of pulmonary disease. Histoplasma and Blastomyces may cause laryngitis, often as a complication of systemic infection. Candida species can cause laryngitis as well, often in association with thrush or esophagitis and particularly in immunosuppressed patients. Rare cases of chronic laryngitis are due to Coccidioides and Cryptococcus.

1	Clinical Manifestations Laryngitis is characterized by hoarseness and also can be associated with reduced vocal pitch or aphonia. As acute laryngitis is caused predominantly by respiratory viruses, these symptoms usually occur in association with other symptoms and signs of URI, including rhinorrhea, nasal congestion, cough, and sore throat. Direct laryngoscopy often reveals diffuse laryngeal erythema and edema, along with vascular engorgement of the vocal folds. In addition, chronic disease (e.g., tuberculous laryngitis) often includes mucosal nodules and ulcerations visible on laryngoscopy; these lesions are sometimes mistaken for laryngeal cancer.

1	Acute laryngitis is usually treated with humidification and voice rest alone. Antibiotics are not recommended except when group A Streptococcus is cultured, in which case penicillin is the drug of choice. The choice of therapy for chronic laryngitis depends on the pathogen, whose identification usually requires biopsy with culture. Patients with laryngeal tuberculosis are highly contagious because of the large number of organisms that are easily aerosolized. These patients should be managed in the same way as patients with active pulmonary disease. The term croup actually denotes a group of diseases collectively referred to as “croup syndrome,” all of which are acute and predominantly viral respiratory illnesses characterized by marked swelling of the subglottic region of the larynx. Croup primarily affects children <6 years old. For a detailed discussion of this entity, the reader should consult a textbook of pediatric medicine.

1	Acute epiglottitis (supraglottitis) is an acute, rapidly progressive form of cellulitis of the epiglottis and adjacent structures that can result in complete—and potentially fatal—airway obstruction in both children and adults. Before the widespread use of H. influenzae type b (Hib) vaccine, this entity was much more common among children, with a peak incidence at ~3.5 years of age. In some countries, mass vaccination against Hib has reduced the annual incidence of acute epiglottitis in children by >90%; in contrast, the annual incidence in adults has changed little since the introduction of Hib vaccine. Because of the danger of airway obstruction, acute epiglottitis constitutes a medical emergency, particularly in children, and prompt diagnosis and airway protection are of the utmost importance.

1	Etiology After the introduction of the Hib vaccine in the mid-1980s, disease incidence among children in the United States declined dramatically. Nevertheless, lack of vaccination or vaccine failure has meant that many pediatric cases seen today are still due to Hib. In adults and (more recently) in children, a variety of other bacterial pathogens have been associated with epiglottitis, the most common being group A Streptococcus. Other pathogens—seen less frequently— include S. pneumoniae, Haemophilus parainfluenzae, and S. aureus (including MRSA). Viruses have not been established as causes of acute epiglottitis.

1	Clinical Manifestations and Diagnosis Epiglottitis typically presents more acutely in young children than in adolescents or adults. On presentation, most children have had symptoms for <24 h, including high fever, severe sore throat, tachycardia, systemic toxicity, and (in many cases) drooling while sitting forward. Symptoms and signs of respiratory obstruction also may be present and may progress rapidly. The somewhat milder illness in adolescents and adults often follows 1–2 days of severe sore throat and is commonly accompanied by dyspnea, drooling, and stridor. Physical examination of patients with acute epiglottitis may reveal moderate or severe respiratory distress, with inspiratory stridor and retractions of the chest wall. These findings diminish as the disease progresses and the patient tires. Conversely, oropharyngeal examination reveals infection that is much less severe than would be predicted from the symptoms—a finding that should alert the clinician to a cause of

1	the patient tires. Conversely, oropharyngeal examination reveals infection that is much less severe than would be predicted from the symptoms—a finding that should alert the clinician to a cause of symptoms and obstruction that lies beyond the tonsils. The diagnosis often is made on clinical grounds, although direct fiberoptic laryngoscopy is frequently performed in a controlled environment (e.g., an operating room) to visualize and culture the typical edematous “cherry-red” epiglottis and facilitate placement of an endotracheal tube. Direct visualization in an examination room (i.e., with a tongue blade and indirect laryngoscopy) is not recommended because of the risk of immediate laryngospasm and complete airway obstruction. Lateral neck radiographs and laboratory tests can assist in the diagnosis but may delay the critical securing of the airway and cause the patient to be moved or repositioned more than is necessary, thereby increasing the risk of further airway compromise. Neck

1	diagnosis but may delay the critical securing of the airway and cause the patient to be moved or repositioned more than is necessary, thereby increasing the risk of further airway compromise. Neck radiographs typically reveal an enlarged edematous epiglottis (the “thumbprint sign,” Fig. 44-3), usually with a dilated hypopharynx and normal subglottic structures. Laboratory tests characteristically document mild to moderate leukocytosis with a predominance of neutrophils. Blood cultures are positive in a significant proportion of cases.

1	Security of the airway is always of primary concern in acute epiglottitis, even if the diagnosis is only suspected. Mere observation for signs of impending airway obstruction is not routinely recommended, particularly in children. Many adults have been managed with observation only since the illness is perceived to be milder in this age group, but some data suggest that this approach may be risky and probably should be reserved only for adult patients who have yet to develop dyspnea or stridor. Once the airway has been secured and specimens of blood and epiglottis tissue have been obtained for culture, treatment with IV antibiotics should be given to cover the most likely organisms, particularly H. influenzae. Because rates of ampicillin resistance in this organism have risen significantly in recent years, therapy with a β-lactam/β-lactamase inhibitor combination or a secondor third-generation cephalosporin is recommended. Typically, ampicillin/sulbactam, cefuroxime, cefotaxime, or

1	in recent years, therapy with a β-lactam/β-lactamase inhibitor combination or a secondor third-generation cephalosporin is recommended. Typically, ampicillin/sulbactam, cefuroxime, cefotaxime, or ceftriaxone is given, with clindamycin and TMP-SMX reserved for patients allergic to β-lactams. Antibiotic therapy should be continued for 7–10 days and should be tailored to the organism recovered in culture. If the household contacts of a patient with H. influenzae epiglottitis include an unvaccinated child under age 4, all members of the household (including the patient) should receive prophylactic rifampin for 4 days to eradicate carriage of H. influenzae.

1	FIguRE 44-3 Acute epiglottitis. In this lateral soft tissue radiograph of the neck, the arrow indicates the enlarged edematous epiglottis (the “thumbprint sign”). Deep neck infections are usually extensions of infection from other primary sites, most often within the pharynx or oral cavity. Many of these infections are life threatening but are difficult to detect at early stages, when they may be more easily managed. Three of the most clinically relevant spaces in the neck are the submandibular (and sublingual) space, the lateral pharyngeal (or parapharyngeal) space, and the retropharyngeal space. These spaces communicate with one another and with other important structures in the head, neck, and thorax, providing pathogens with easy access to areas that include the mediastinum, carotid sheath, skull base, and meninges. Once infection reaches these sensitive areas, mortality rates can be as high as 20–50%.

1	Infection of the submandibular and/or sublingual space typically originates from an infected or recently extracted lower tooth. The result is the severe, life-threatening infection referred to as Ludwig’s angina (see “Oral Infections,” above). Infection of the lateral pharyngeal (or parapharyngeal) space is most often a complication of common infections of the oral cavity and upper respiratory tract, including tonsillitis, peritonsillar abscess, pharyngitis, mastoiditis, and periodontal infection. This space, situated deep in the lateral wall of the pharynx, contains a number of sensitive structures, including the 235 carotid artery, internal jugular vein, cervical sympathetic chain, and portions of cranial nerves IX through XII; at its distal end, it opens into the posterior mediastinum. Involvement of this space with infection can therefore be rapidly fatal. Examination may reveal some tonsillar displacement, trismus, and neck rigidity, but swelling of the lateral pharyngeal wall

1	Involvement of this space with infection can therefore be rapidly fatal. Examination may reveal some tonsillar displacement, trismus, and neck rigidity, but swelling of the lateral pharyngeal wall can easily be missed. The diagnosis can be confirmed by CT. Treatment consists of airway management, operative drainage of fluid collections, and at least 10 days of IV therapy with an antibiotic active against streptococci and oral anaerobes (e.g., ampicillin/ sulbactam). A particularly severe form of this infection involving the components of the carotid sheath (postanginal septicemia, Lemierre’s disease) is described above (see “Oral Infections”). Infection of the retropharyngeal space also can be extremely dangerous, as this space runs posterior to the pharynx from the skull base to the superior mediastinum. Infections in this space are more common among children <5 years old because of the presence of several small retropharyngeal lymph nodes that typically atrophy by age 4 years.

1	superior mediastinum. Infections in this space are more common among children <5 years old because of the presence of several small retropharyngeal lymph nodes that typically atrophy by age 4 years. Infection is usually a consequence of extension from another site of infection—most commonly, acute pharyngitis. Other sources include otitis media, tonsillitis, dental infections, Ludwig’s angina, and anterior extension of vertebral osteomyelitis. Retropharyngeal space infection also can follow penetrating trauma to the posterior pharynx (e.g., from an endoscopic procedure). Infections are commonly polymicrobial, involving a mixture of aerobes and anaerobes; group A β-hemolytic streptococci and S. aureus are the most common pathogens. M. tuberculosis was a common cause in the past but now is rarely involved in the United States.

1	Patients with retropharyngeal abscess typically present with sore throat, fever, dysphagia, and neck pain and are often drooling because of difficulty and pain with swallowing. Examination may reveal tender cervical adenopathy, neck swelling, and diffuse erythema and edema of the posterior pharynx as well as a bulge in the posterior pharyngeal wall that may not be obvious on routine inspection. A soft tissue mass is usually demonstrable by lateral neck radiography or CT. Because of the risk of airway obstruction, treatment begins with securing of the airway, followed by a combination of surgical drainage and IV antibiotic administration. Initial empirical therapy should cover streptococci, oral anaerobes, and S. aureus; ampicillin/sulbactam, clindamycin plus ceftriaxone, or meropenem is usually effective. Complications result primarily from extension to other areas (e.g., rupture into the posterior pharynx may lead to aspiration pneumonia and empyema). Extension may also occur to the

1	effective. Complications result primarily from extension to other areas (e.g., rupture into the posterior pharynx may lead to aspiration pneumonia and empyema). Extension may also occur to the lateral pharyngeal space and mediastinum, resulting in mediastinitis and pericarditis, or into nearby major blood vessels. All these events are associated with a high mortality rate.

1	CHAPTER 45 Oral Manifestations of Disease oral Manifestations of Disease Samuel C. Durso As primary care physicians and consultants, internists are often asked to evaluate patients with disease of the oral soft tissues, teeth, and pharynx. Knowledge of the oral milieu and its unique structures is necessary to guide preventive services and recognize oral manifestations of local or systemic disease (Chap. 46e). Furthermore, internists frequently collaborate with dentists in the care of patients who have a variety of medical conditions that affect oral health or who undergo dental procedures that increase their risk of medical complications.

1	Tooth formation begins during the sixth week of embryonic life and continues through 17 years of age. Teeth start to develop in utero and continue to develop until after the tooth erupts. Normally, all 20 deciduous teeth have erupted by age 3 and have been shed by age 13. Permanent teeth, eventually totaling 32, begin to erupt by age 6 and 236 have completely erupted by age 14, though third molars (“wisdom teeth”) may erupt later. The erupted tooth consists of the visible crown covered with enamel and the root submerged below the gum line and covered with bonelike cementum. Dentin, a material that is denser than bone and exquisitely sensitive to pain, forms the majority of the tooth substance, surrounding a core of myxomatous pulp containing the vascular and nerve supply. The tooth is held firmly in the alveolar socket by the periodontium, supporting structures that consist of the gingivae, alveolar bone, cementum, and periodontal ligament. The periodontal ligament tenaciously binds

1	firmly in the alveolar socket by the periodontium, supporting structures that consist of the gingivae, alveolar bone, cementum, and periodontal ligament. The periodontal ligament tenaciously binds the tooth’s cementum to the alveolar bone. Above this ligament is a collar of attached gingiva just below the crown. A few millimeters of unattached or free gingiva (1–3 mm) overlap the base of the crown, forming a shallow sulcus along the gum-tooth margin.

1	Dental Caries, Pulpal and Periapical Disease, and Complications Dental caries usually begin asymptomatically as a destructive infectious process of the enamel. Bacteria—principally Streptococcus mutans— colonize the organic buffering biofilm (plaque) on the tooth surface. If not removed by brushing or by the natural cleansing and antibacterial action of saliva, bacterial acids can demineralize the enamel. Fissures and pits on the occlusal surfaces are the most frequent sites of early decay. Surfaces between the teeth, adjacent to tooth restorations and exposed roots, are also vulnerable, particularly as individuals age. Over time, dental caries extend to the underlying dentin, leading to cavitation of the enamel. Without management, the caries will penetrate to the tooth pulp, producing acute pulpitis. At this stage, when the pulp infection is limited, the tooth may become sensitive to percussion and to hot or cold, and pain resolves immediately when the irritating stimulus is

1	acute pulpitis. At this stage, when the pulp infection is limited, the tooth may become sensitive to percussion and to hot or cold, and pain resolves immediately when the irritating stimulus is removed. Should the infection spread throughout the pulp, irreversible pulpitis occurs, leading to pulp necrosis. At this later stage, pain can be severe and has a sharp or throbbing visceral quality that may be worse when the patient lies down. Once pulp necrosis is complete, pain may be constant or intermittent, but cold sensitivity is lost.

1	Treatment of caries involves removal of the softened and infected hard tissue and restoration of the tooth structure with silver amalgam, glass ionomer, composite resin, or gold. Once irreversible pulpitis occurs, root canal therapy becomes necessary; removal of the contents of the pulp chamber and root canals is followed by thorough cleaning and filling with an inert material. Alternatively, the tooth may be extracted.

1	Pulpal infection leads to periapical abscess formation, which can produce pain on chewing. If the infection is mild and chronic, a periapical granuloma or eventually a periapical cyst forms, either of which produces radiolucency at the root apex. When unchecked, a periapical abscess can erode into the alveolar bone, producing osteomyelitis; penetrate and drain through the gingivae, producing a parulis (gumboil); or track along deep fascial planes, producing virulent cellulitis (Ludwig’s angina) involving the submandibular space and floor of the mouth (Chap. 201). Elderly patients, patients with diabetes mellitus, and patients taking glucocorticoids may experience little or no pain or fever as these complications develop.

1	Periodontal Disease Periodontal disease and dental caries are the primary causes of tooth loss. Like dental caries, chronic infection of the gingiva and anchoring structures of the tooth begins with formation of bacterial plaque. The process begins at the gum line. Plaque and calculus (calcified plaque) are preventable by appropriate daily oral hygiene, including periodic professional cleaning. Left undisturbed, chronic inflammation can ensue and produce hyperemia of the free and attached gingivae (gingivitis), which then typically bleed with brushing. If this issue is ignored, severe periodontitis can develop, leading to deepening of the physiologic sulcus and destruction of the periodontal ligament. Gingival pockets develop around the teeth. As the periodontium (including the supporting bone) is destroyed, the teeth loosen. A role for chronic inflammation due to chronic periodontal disease in promoting coronary heart disease and stroke has been proposed. Epidemiologic studies have

1	bone) is destroyed, the teeth loosen. A role for chronic inflammation due to chronic periodontal disease in promoting coronary heart disease and stroke has been proposed. Epidemiologic studies have demonstrated a moderate but significant association between chronic periodontal inflammation and atherogenesis, though a causal role remains unproven.

1	PART 2 Cardinal Manifestations and Presentation of Diseases

1	Acute and aggressive forms of periodontal disease are less common than the chronic forms described above. However, if the host is stressed or exposed to a new pathogen, rapidly progressive and destructive disease of the periodontal tissue can occur. A virulent example is acute necrotizing ulcerative gingivitis. Stress and poor oral hygiene are risk factors. The presentation includes sudden gingival inflammation, ulceration, bleeding, interdental gingival necrosis, and fetid halitosis. Localized juvenile periodontitis, which is seen in adolescents, is particularly destructive and appears to be associated with impaired neutrophil chemotaxis. AIDS-related periodontitis resembles acute necrotizing ulcerative gingivitis in some patients and a more destructive form of adult chronic periodontitis in others. It may also produce a gangrene-like destructive process of the oral soft tissues and bone that resembles noma, an infectious condition seen in severely malnourished children in developing

1	in others. It may also produce a gangrene-like destructive process of the oral soft tissues and bone that resembles noma, an infectious condition seen in severely malnourished children in developing nations.

1	Prevention of Tooth Decay and Periodontal Infection Despite the reduced prevalences of dental caries and periodontal disease in the United States (due in large part to water fluoridation and improved dental care, respectively), both diseases constitute a major public health problem worldwide, particularly in certain groups. The internist should promote preventive dental care and hygiene as part of health maintenance. Populations at high risk for dental caries and periodontal disease include those with hyposalivation and/or xerostomia, diabetics, alcoholics, tobacco users, persons with Down syndrome, and those with gingival hyperplasia. Furthermore, patients lacking access to dental care (e.g., as a result of low socioeconomic status) and patients with a reduced ability to provide self-care (e.g., individuals with disabilities, nursing home residents, and persons with dementia or upper-extremity disability) suffer at a disproportionate rate. It is important to provide counseling

1	(e.g., individuals with disabilities, nursing home residents, and persons with dementia or upper-extremity disability) suffer at a disproportionate rate. It is important to provide counseling regarding regular dental hygiene and professional cleaning, use of fluoride-containing toothpaste, professional fluoride treatments, and (for patients with limited dexterity) use of electric toothbrushes and also to instruct persons caring for those who are not capable of self-care. Cost, fear of dental care, and differences in language and culture create barriers that prevent some people from seeking preventive dental services.

1	Developmental and Systemic Disease Affecting the Teeth and Periodontium

1	In addition to posing cosmetic issues, malocclusion, the most common developmental oral problem, can interfere with mastication unless corrected through orthodontic and surgical techniques. Impacted third molars are common and can become infected or erupt into an insufficient space. Acquired prognathism due to acromegaly may also lead to malocclusion, as may deformity of the maxilla and mandible due to Paget’s disease of the bone. Delayed tooth eruption, a receding chin, and a protruding tongue are occasional features of cretinism and hypopituitarism. Congenital syphilis produces tapering, notched (Hutchinson’s) incisors and finely nodular (mulberry) molar crowns. Enamel hypoplasia results in crown defects ranging from pits to deep fissures of primary or permanent teeth. Intrauterine infection (syphilis, rubella), vitamin deficiency (A, C, or D), disorders of calcium metabolism (malabsorption, vitamin D–resistant rickets, hypoparathyroidism), prematurity, high fever, and rare

1	infection (syphilis, rubella), vitamin deficiency (A, C, or D), disorders of calcium metabolism (malabsorption, vitamin D–resistant rickets, hypoparathyroidism), prematurity, high fever, and rare inherited defects (amelogenesis imperfecta) are all causes. Tetracycline, given in sufficiently high doses during the first 8 years of life, may produce enamel hypoplasia and discoloration. Exposure to endogenous pigments can discolor developing teeth; etiologies include erythroblastosis fetalis (green or bluish-black), congenital liver disease (green or yellow-brown), and porphyria (red or brown that fluoresces with ultraviolet light). Mottled enamel occurs if excessive fluoride is ingested during development. Worn enamel is seen with age, bruxism, or excessive acid exposure (e.g., chronic gastric reflux or bulimia). Celiac disease is associated with nonspecific enamel defects in children but not in adults.

1	Total or partial tooth loss resulting from periodontitis is seen with cyclic neutropenia, Papillon-Lefévre syndrome, Chédiak-Higashi syndrome, and leukemia. Rapid focal tooth loosening is most often due to infection, but rarer causes include Langerhans cell histiocytosis, Ewing’s sarcoma, osteosarcoma, and Burkitt’s lymphoma. Early loss of primary teeth is a feature of hypophosphatasia, a rare congenital error of metabolism.

1	Pregnancy may produce gingivitis and localized pyogenic granulomas. Severe periodontal disease occurs in uncontrolled diabetes mellitus. Gingival hyperplasia may be caused by phenytoin, calcium channel blockers (e.g., nifedipine), and cyclosporine, though excellent daily oral care can prevent or reduce its occurrence. Idiopathic familial gingival fibromatosis and several syndrome-related disorders cause similar conditions. Discontinuation of the medication may reverse the drug-induced form, though surgery may be needed to control both of the latter entities. Linear gingival erythema is variably seen in patients with advanced HIV infection and probably represents immune deficiency and decreased neutrophil activity. Diffuse or focal gingival swelling may be a feature of early or late acute myelomonocytic leukemia as well as of other lymphoproliferative disorders. A rare but pathognomonic sign of granulomatosis with polyangiitis is a red-purplish, granular gingivitis (strawberry gums).

1	DISEASES OF THE ORAL MuCOSA Infections Most oral mucosal diseases involve microorganisms (Table 45-1). Pigmented Lesions See Table 45-2. Dermatologic Diseases See Tables 45-1, 45-2, and 45-3 and Chaps. 70–74. Diseases of the Tongue See Table 45-4. HIV Disease and AIDS See Tables 45-1, 45-2, 45-3, and 45-5; Chap. 226; and Fig. 218-3.

1	ulcers Ulceration is the most common oral mucosal lesion. Although there are many causes, the host and the pattern of lesions, including the presence of organ system features, narrow the differential diagnosis (Table 45-1). Most acute ulcers are painful and self-limited. Recurrent aphthous ulcers and herpes simplex account for the majority. Persistent and deep aphthous ulcers can be idiopathic or can accompany HIV/ AIDS. Aphthous lesions are often the presenting symptom in Behçet’s syndrome (Chap. 387). Similar-appearing, though less painful, lesions may occur in reactive arthritis, and aphthous ulcers are occasionally present during phases of discoid or systemic lupus erythematosus (Chap. 382). Aphthous-like ulcers are seen in Crohn’s disease (Chap. 351), but, unlike the common aphthous variety, they may exhibit granulomatous inflammation on histologic examination. Recurrent aphthae are more prevalent in patients with celiac disease and have been reported to remit with elimination of

1	they may exhibit granulomatous inflammation on histologic examination. Recurrent aphthae are more prevalent in patients with celiac disease and have been reported to remit with elimination of gluten.

1	Of major concern are chronic, relatively painless ulcers and mixed red/white patches (erythroplakia and leukoplakia) of >2 weeks’ duration. Squamous cell carcinoma and premalignant dysplasia should be considered early and a diagnostic biopsy performed. This awareness and this procedure are critically important because early-stage malignancy is vastly more treatable than late-stage disease. High-risk sites include the lower lip, floor of the mouth, ventral and lateral tongue, and soft palate–tonsillar pillar complex. Significant risk factors for oral cancer in Western countries include sun exposure (lower lip), tobacco and alcohol use, and human papillomavirus infection. In India and some other Asian countries, smokeless tobacco mixed with betel nut, slaked lime, and spices is a common cause of oral cancer. Rarer causes of chronic oral ulcer, such as tuberculosis, fungal infection, granulomatosis with polyangiitis, and midline granuloma may look identical to carcinoma. Making the

1	cause of oral cancer. Rarer causes of chronic oral ulcer, such as tuberculosis, fungal infection, granulomatosis with polyangiitis, and midline granuloma may look identical to carcinoma. Making the correct diagnosis depends on recognizing other clinical features and performing a biopsy of the lesion. The syphilitic chancre is typically painless and therefore easily missed. Regional lymphadenopathy is invariably present. The syphilitic etiology is confirmed with appropriate bacterial and serologic tests.

1	Disorders of mucosal fragility often produce painful oral ulcers that fail to heal within 2 weeks. Mucous membrane pemphigoid and pemphigus vulgaris are the major acquired disorders. While their clinical features are often distinctive, a biopsy or immunohistochemical examination should be performed to diagnose these entities and to 237 distinguish them from lichen planus and drug reactions. Hematologic and Nutritional Disease Internists are more likely to encounter patients with acquired, rather than congenital, bleeding disorders. Bleeding should stop 15 min after minor trauma and within an hour after tooth extraction if local pressure is applied. More prolonged bleeding, if not due to continued injury or rupture of a large vessel, should lead to investigation for a clotting abnormality. In addition to bleeding, petechiae and ecchymoses are prone to occur at the vibrating line between the soft and hard palates in patients with platelet dysfunction or thrombocytopenia.

1	All forms of leukemia, but particularly acute myelomonocytic leukemia, can produce gingival bleeding, ulcers, and gingival enlargement. Oral ulcers are a feature of agranulocytosis, and ulcers and mucositis are often severe complications of chemotherapy and radiation therapy for hematologic and other malignancies. Plummer-Vinson syndrome (iron deficiency, angular stomatitis, glossitis, and dysphagia) raises the risk of oral squamous cell cancer and esophageal cancer at the postcricoidal tissue web. Atrophic papillae and a red, burning tongue may occur with pernicious anemia. Deficiencies in B-group vitamins produce many of these same symptoms as well as oral ulceration and cheilosis. Consequences of scurvy include swollen, bleeding gums; ulcers; and loosening of the teeth.

1	Most, but not all, oral pain emanates from inflamed or injured tooth pulp or periodontal tissues. Nonodontogenic causes are often overlooked. In most instances, toothache is predictable and proportional to the stimulus applied, and an identifiable condition (e.g., caries, abscess) is found. Local anesthesia eliminates pain originating from dental or periodontal structures, but not referred pains. The most common nondental source of pain is myofascial pain referred from muscles of mastication, which become tender and ache with increased use. Many sufferers exhibit bruxism (grinding of the teeth) secondary to stress and anxiety. Temporomandibular joint disorder is closely related. It affects both sexes, with a higher prevalence among women. Features include pain, limited mandibular movement, and temporomandibular joint sounds. The etiologies are complex; malocclusion does not play the primary role once attributed to it. Osteoarthritis is a common cause of masticatory pain.

1	movement, and temporomandibular joint sounds. The etiologies are complex; malocclusion does not play the primary role once attributed to it. Osteoarthritis is a common cause of masticatory pain. Anti-inflammatory medication, jaw rest, soft foods, and heat provide relief. The temporomandibular joint is involved in 50% of patients with rheumatoid arthritis, and its involvement is usually a late feature of severe disease. Bilateral preauricular pain, particularly in the morning, limits range of motion.

1	Migrainous neuralgia may be localized to the mouth. Episodes of pain and remission without an identifiable cause and a lack of relief with local anesthesia are important clues. Trigeminal neuralgia (tic douloureux) can involve the entire branch or part of the mandibular or maxillary branch of the fifth cranial nerve and can produce pain in one or a few teeth. Pain may occur spontaneously or may be triggered by touching the lip or gingiva, brushing the teeth, or chewing. Glossopharyngeal neuralgia produces similar acute neuropathic symptoms in the distribution of the ninth cranial nerve. Swallowing, sneezing, coughing, or pressure on the tragus of the ear triggers pain that is felt in the base of the tongue, pharynx, and soft palate and may be referred to the temporomandibular joint. Neuritis involving the maxillary and mandibular divisions of the trigeminal nerve (e.g., maxillary sinusitis, neuroma, and leukemic infiltrate) is distinguished from ordinary toothache by the neuropathic

1	involving the maxillary and mandibular divisions of the trigeminal nerve (e.g., maxillary sinusitis, neuroma, and leukemic infiltrate) is distinguished from ordinary toothache by the neuropathic quality of the pain. Occasionally, phantom pain follows tooth extraction. Pain and hyperalgesia behind the ear and on the side of the face in the day or so before facial weakness develops often constitute the earliest symptom of Bell’s palsy. Likewise, similar symptoms may precede visible lesions of herpes zoster infecting the seventh nerve (Ramsey-Hunt syndrome) or trigeminal nerve. Postherpetic neuralgia may follow either condition. Coronary ischemia may produce pain exclusively in the face and jaw; as in typical angina pectoris, this pain is usually reproducible with increased myocardial demand. Aching in several upper molar or

1	CHAPTER 45 Oral Manifestations of Disease 238 TABLE 45-1 vESiCuLAR, BuLLouS, oR uLCERATivE LESionS of THE oRAL MuCoSA Labial vesicles that rupture and crust, and intraoral vesicles that quickly ulcerate; extremely painful; acute gingivitis, fever, malaise, foul odor, and cervical lymphadenopathy; occurs primarily in infants, children, and young adults Eruption of groups of vesicles that may coalesce, then rupture and crust; painful to pressure or spicy foods Skin lesions may be accompanied by small vesicles on oral mucosa that rupture to form shallow ulcers; may coalesce to form large bullous lesions that ulcerate; mucosa may have generalized erythema Unilateral vesicular eruptions and ulceration in linear pattern following sensory distribution of trigeminal nerve or one of its branches

1	Unilateral vesicular eruptions and ulceration in linear pattern following sensory distribution of trigeminal nerve or one of its branches Fatigue, sore throat, malaise, fever, and cervical lymphadenopathy; numerous small ulcers usually appear several days before lymphadenopathy; gingival bleeding and multiple petechiae at junction of hard and soft palates Sudden onset of fever, sore throat, and oropharyngeal vesicles, usually in children <4 years old, during summer months; diffuse pharyngeal congestion and vesicles (1–2 mm), grayish-white surrounded by red areola; vesicles enlarge and ulcerate Fever, malaise, headache with oropharyngeal vesicles that become painful, shallow ulcers; highly infectious; usually affects children under age 10

1	Fever, malaise, headache with oropharyngeal vesicles that become painful, shallow ulcers; highly infectious; usually affects children under age 10 Acute gingivitis and oropharyngeal ulceration, associated with febrile illness resembling mononucleosis and including lymphadenopathy Heals spontaneously in 10–14 days; unless secondarily infected, lesions lasting >3 weeks are not due to primary HSV infection Lasts ∼1 week, but condition may be prolonged if secondarily infected; if severe, topical or oral antiviral treatment may reduce healing time Heals spontaneously in ∼1 week; if severe, topical or oral antiviral treatment may reduce healing time Gradual healing without scarring unless secondarily infected; postherpetic neuralgia is common; oral acyclovir, famciclovir, or valacyclovir reduces healing time and postherpetic neuralgia Oral lesions disappear during convalescence; no treatment is given, though glucocorticoids are indicated if tonsillar swelling compromises the airway

1	Oral lesions disappear during convalescence; no treatment is given, though glucocorticoids are indicated if tonsillar swelling compromises the airway Incubation period of 2–9 days; fever for 1–4 days; recovery uneventful Followed by HIV seroconversion, asymptomatic HIV infection, and usually ultimately by HIV disease Primary HIV infection Lip and oral mucosa (buccal, gingival, lingual mucosa) Mucocutaneous junction of lip, perioral skin Cheek, tongue, gingiva, or palate Oral mucosa, pharynx, tongue Oral mucosa, pharynx, palms, and soles Gingiva, palate, and pharynx PART 2 Cardinal Manifestations and Presentation of Diseases Painful, bleeding gingiva characterized by necrosis and ulceration of gingival papillae and margins plus lymphadenopathy and foul breath Gummatous involvement of palate, jaws, and facial bones; Hutchinson’s incisors, mulberry molars, glossitis, mucous patches, and fissures at corner of mouth

1	Gummatous involvement of palate, jaws, and facial bones; Hutchinson’s incisors, mulberry molars, glossitis, mucous patches, and fissures at corner of mouth Small papule developing rapidly into a large, painless ulcer with indurated border; unilateral lymphadenopathy; chancre and lymph nodes containing spirochetes; serologic tests positive by third to fourth weeks Maculopapular lesions of oral mucosa, 5–10 mm in diameter with central ulceration covered by grayish membrane; eruptions occurring on various mucosal surfaces and skin, accompanied by fever, malaise, and sore throat Gummatous infiltration of palate or tongue followed by ulceration and fibrosis; atrophy of tongue papillae produces characteristic bald tongue and glossitis Most pharyngeal infection is asymptomatic; may produce burning or itching sensation; oropharynx and tonsils may be ulcerated and erythematous; saliva viscous and fetid

1	Most pharyngeal infection is asymptomatic; may produce burning or itching sensation; oropharynx and tonsils may be ulcerated and erythematous; saliva viscous and fetid Painless, solitary, 1to 5-cm, irregular ulcer covered with persistent exudate; ulcer has firm undermined border Debridement and diluted (1:3) peroxide lavage provide relief within 24 h; antibiotics in acutely ill patients; relapse may occur Healing of chancre in 1–2 months, followed by secondary syphilis in 6–8 weeks Lesions may persist from several weeks to a year Gumma may destroy palate, causing complete perforation More difficult to eradicate than urogenital infection, though pharyngitis usually resolves with appropriate antimicrobial treatment Autoinoculation from pulmonary infection is usual; lesions resolve with appropriate antimicrobial therapy 239TABLE 45-1 vESiCuLAR, BuLLouS, oR uLCERATivE LESionS of THE oRAL MuCoSA (CONTINUED) CHAPTER 45 Oral Manifestations of Disease

1	CHAPTER 45 Oral Manifestations of Disease Recurrent aphthous Usually on nonkeratinized Single or clustered painful ulcers with surrounding Lesions heal in 1–2 weeks but may recur monthly or ulcers oral mucosa (buccal and erythematous border; lesions may be 1–2 mm in several times a year; protective barrier with benzolabial mucosa, floor of diameter in crops (herpetiform), 1–5 mm (minor), caine and topical glucocorticoids relieve symptoms; mouth, soft palate, lateral or 5–15 mm (major) systemic glucocorticoids may be needed in severe and ventral tongue) Behçet’s syndrome Oral mucosa, eyes, genita-Multiple aphthous ulcers in mouth; inflammatory Oral lesions often first manifestation; persist several lia, gut, and CNS ocular changes, ulcerative lesions on genitalia; weeks and heal without scarring inflammatory bowel disease and CNS disease

1	Traumatic ulcers Anywhere on oral mucosa; Localized, discrete ulcerated lesions with red Lesions usually heal in 7–10 days when irritant is dentures frequently border; produced by accidental biting of mucosa, removed, unless secondarily infected responsible for ulcers in penetration by foreign object, or chronic irritation vestibule by dentures Squamous cell Any area of mouth, most Red, white, or red and white ulcer with elevated or Invades and destroys underlying tissues; frequently carcinoma commonly on lower lip, indurated border; failure to heal; pain not promi-metastasizes to regional lymph nodes lateral borders of tongue, nent in early lesions and floor of mouth

1	Acute myeloid Gingiva Gingival swelling and superficial ulceration fol-Usually responds to systemic treatment of leukemia; leukemia (usually lowed by hyperplasia of gingiva with extensive occasionally requires local irradiation monocytic) necrosis and hemorrhage; deep ulcers may occur elsewhere on mucosa, complicated by secondary infection Lymphoma Gingiva, tongue, palate, Elevated, ulcerated area that may proliferate rap-Fatal if untreated; may indicate underlying HIV and tonsillar area idly, giving appearance of traumatic inflammation infection Chemical or thermal Any area in mouth White slough due to contact with corrosive Lesion heals in several weeks if not secondarily burns agents (e.g., aspirin, hot cheese) applied locally; infected removal of slough leaves raw, painful surface aSee Table 45-3. Abbreviations: CNS, central nervous system; EM, erythema multiforme; HSV, herpes simplex virus; VZV, varicella-zoster virus.

1	Abbreviations: CNS, central nervous system; EM, erythema multiforme; HSV, herpes simplex virus; VZV, varicella-zoster virus. premolar teeth that is unrelieved by anesthetizing the teeth may point etiology may be neuropathic. Clonazepam, α-lipoic acid, and cognitive to maxillary sinusitis. behavioral therapy have benefited some patients. Some cases associ-

1	Giant cell arteritis is notorious for producing headache, but it may ated with an angiotensin-converting enzyme inhibitor have remitted also produce facial pain or sore throat without headache. Jaw and when treatment with the drug was discontinued. tongue claudication with chewing or talking is relatively common. Tongue infarction is rare. Patients with subacute thyroiditis often DISEASES OF THE SALIVARY gLANDS experience pain referred to the face or jaw before the tenderness of the Saliva is essential to oral health. Its absence leads to dental caries, thyroid gland and transient hyperthyroidism are appreciated. periodontal disease, and difficulties in wearing dental prostheses, mas “Burning mouth syndrome” (glossodynia) occurs in the absence of ticating, and speaking. Its major components, water and mucin, serve an identifiable cause (e.g., vitamin B12 deficiency, iron deficiency, dia-as a cleansing solvent and lubricating fluid. In addition, saliva conbetes mellitus, low-grade

1	water and mucin, serve an identifiable cause (e.g., vitamin B12 deficiency, iron deficiency, dia-as a cleansing solvent and lubricating fluid. In addition, saliva conbetes mellitus, low-grade Candida infection, food sensitivity, or subtle tains antimicrobial factors (e.g., lysozyme, lactoperoxidase, secretory xerostomia) and predominantly affects postmenopausal women. The IgA), epidermal growth factor, minerals, and buffering systems. The

1	PART 2 Cardinal Manifestations and Presentation of Diseases Lichen planus Buccal mucosa, tongue, gingiva, and lips; skin White sponge nevus Oral mucosa, vagina, anal mucosa Smoker’s leukopla-Any area of oral mucosa, kia and smokeless sometimes related to tobacco lesions location of habit Erythroplakia with Floor of mouth com-or without white monly affected in men; patches tongue and buccal tongue, rarely elsewhere Warts (human papil-Anywhere on skin and lomavirus) oral mucosa Striae, white plaques, red areas, ulcers in mouth; purplish papules on skin; may be asymptomatic, sore, or painful; lichenoid drug reactions may look similar Painless white thickening of epithelium; adolescence/early adulthood onset; familial White patch that may become firm, rough, or red-fissured and ulcerated; may become sore and painful but is usually painless Velvety, reddish plaque; occasionally mixed with white patches or smooth red areas

1	Velvety, reddish plaque; occasionally mixed with white patches or smooth red areas Pseudomembranous type (“thrush”): creamy white curdlike patches that reveal a raw, bleeding surface when scraped; found in sick infants, debilitated elderly patients receiving high-dose glucocorticoids or broad-spectrum antibiotics, and patients with AIDS Erythematous type: flat, red, sometimes sore areas in same groups of patients Candidal leukoplakia: nonremovable white thickening of epithelium due to Candida Angular cheilitis: sore fissures at corner of mouth White areas ranging from small and flat to extensive accentuation of vertical folds; found in HIV carriers (all risk groups for AIDS) Single or multiple papillary lesions with thick, white, keratinized surfaces containing many pointed projections; cauliflower lesions covered with normal-colored mucosa or multiple pink or pale bumps (focal epithelial hyperplasia) Protracted; responds to topical glucocorticoids

1	May or may not resolve with cessation of habit; 2% of patients develop squamous cell carcinoma; early biopsy essential High risk of squamous cell cancer; early biopsy essential Responds favorably to antifungal therapy and correction of predisposing causes where possible Course same as for pseudomembranous type Responds to prolonged antifungal therapy Responds to topical antifungal therapy Due to Epstein-Barr virus; responds to high-dose acyclovir but recurs; rarely causes discomfort unless secondarily infected with Candida Lesions grow rapidly and spread; squamous cell carcinoma must be ruled out with biopsy; excision or laser therapy; may regress in HIV-infected patients receiving antiretroviral therapy Type of Change Clinical Features

1	Type of Change Clinical Features Macroglossia Enlarged tongue that may be part of a syndrome found in developmental conditions such as Down syndrome, Simpson-Golabi-Behmel syndrome, or Beckwith-Wiedemann syndrome; may be due to tumor (hemangioma or lymphangioma), metabolic disease (e.g., primary amyloidosis), or endocrine disturbance (e.g., acromegaly or cretinism); may occur when all teeth are removed Fissured (“scro-Dorsal surface and sides of tongue covered by pain Median rhom-Congenital abnormality with ovoid, denuded area in boid glossitis median posterior portion of tongue; may be associated with candidiasis and may respond to antifungal treatment “Geographic” Asymptomatic inflammatory condition of tongue, with tongue (benign rapid loss and regrowth of filiform papillae leading to migratory appearance of denuded red patches “wandering” across glossitis) surface

1	Hairy tongue Elongation of filiform papillae of medial dorsal surface area due to failure of keratin layer of papillae to desquamate normally; brownish-black coloration may be due to staining by tobacco, food, or chromogenic organisms “Strawberry” Appearance of tongue during scarlet fever due to hyper-and “raspberry” trophy of fungiform papillae as well as changes in filiform tongue papillae “Bald” tongue Atrophy may be associated with xerostomia, pernicious anemia, iron-deficiency anemia, pellagra, or syphilis; may be accompanied by painful burning sensation; may be an expression of erythematous candidiasis and respond to antifungal treatment Papules, Candidiasis (hyperplastic and pseudomembranous)a nodules, Ulcers Recurrent aphthous ulcersa Angular cheilitis Squamous cell carcinoma Acute necrotizing ulcerative gingivitisa Necrotizing ulcerative periodontitisa Necrotizing ulcerative stomatitis Non-Hodgkin’s lymphomaa Viral infection (herpes simplex, herpes zoster, cytomegalo

1	Fungal infection (histoplasmosis, cryptococcosis, candidiasis, geotrichosis, aspergillosis) Bacterial infection (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa) common than oral) Zidovudine pigmentation (skin, nails, and occasionally oral mucosa) Addison’s disease Miscellaneous Linear gingival erythemaa aStrongly associated with HIV infection.

1	major salivary glands secrete intermittently in response to autonomic 241 stimulation, which is high during a meal but low otherwise. Hundreds of minor glands in the lips and cheeks secrete mucus continuously throughout the day and night. Consequently, oral function becomes impaired when salivary function is reduced. The sensation of a dry mouth (xerostomia) is perceived when salivary flow is reduced by 50%. The most common etiology is medication, especially drugs with anticholinergic properties but also alpha and beta blockers, calcium channel blockers, and diuretics. Other causes include Sjögren’s syndrome, chronic parotitis, salivary duct obstruction, diabetes mellitus, HIV/AIDS, and radiation therapy that includes the salivary glands in the field (e.g., for Hodgkin’s disease and for head and neck cancer). Management involves the elimination or limitation of drying medications, preventive dental care, and supplementation with oral liquid or salivary substitutes. Sugarless mints or

1	and neck cancer). Management involves the elimination or limitation of drying medications, preventive dental care, and supplementation with oral liquid or salivary substitutes. Sugarless mints or chewing gum may stimulate salivary secretion if dysfunction is mild. When sufficient exocrine tissue remains, pilocarpine or cevimeline has been shown to increase secretions. Commercial saliva substitutes or gels relieve dryness. Fluoride supplementation is critical to prevent caries.

1	Sialolithiasis presents most often as painful swelling but in some instances as only swelling or only pain. Conservative therapy consists of local heat, massage, and hydration. Promotion of salivary secretion with mints or lemon drops may flush out small stones. Antibiotic treatment is necessary when bacterial infection in suspected. In adults, acute bacterial parotitis is typically unilateral and most commonly affects postoperative, dehydrated, and debilitated patients. Staphylococcus aureus (including methicillin-resistant strains) and anaerobic bacteria are the most common pathogens. Chronic bacterial sialadenitis results from lowered salivary secretion and recurrent bacterial infection. When suspected bacterial infection is not responsive to therapy, the differential diagnosis should be expanded to include benign and malignant neoplasms, lymphoproliferative disorders, Sjögren’s syndrome, sarcoidosis, tuberculosis, lymphadenitis, actinomycosis, and granulomatosis with polyangiitis.

1	expanded to include benign and malignant neoplasms, lymphoproliferative disorders, Sjögren’s syndrome, sarcoidosis, tuberculosis, lymphadenitis, actinomycosis, and granulomatosis with polyangiitis. Bilateral nontender parotid enlargement occurs with diabetes mellitus, cirrhosis, bulimia, HIV/AIDS, and drugs (e.g., iodide, propylthiouracil).

1	Pleomorphic adenoma comprises two-thirds of all salivary neoplasms. The parotid is the principal salivary gland affected, and the tumor presents as a firm, slow-growing mass. Although this tumor is benign, its recurrence is common if resection is incomplete. Malignant tumors such as mucoepidermoid carcinoma, adenoid cystic carcinoma, and adenocarcinoma tend to grow relatively fast, depending upon grade. They may ulcerate and invade nerves, producing numbness and facial paralysis. Surgical resection is the primary treatment. Radiation therapy (particularly neutron-beam therapy) is used when surgery is not feasible and as post-resection for certain histologic types with a high risk of recurrence. Malignant salivary gland tumors have a 5-year survival rate of ∼68%.

1	Routine dental care (e.g., uncomplicated extraction, scaling and cleaning, tooth restoration, and root canal) is remarkably safe. The most common concerns regarding care of dental patients with medical disease are excessive bleeding for patients taking anticoagulants, infection of the heart valves and prosthetic devices from hematogenous seeding by the oral flora, and cardiovascular complications resulting from vasopressors used with local anesthetics during dental treatment. Experience confirms that the risk of any of these complications is very low.

1	Patients undergoing tooth extraction or alveolar and gingival surgery rarely experience uncontrolled bleeding when warfarin anticoagulation is maintained within the therapeutic range currently recommended for prevention of venous thrombosis, atrial fibrillation, or mechanical heart valve. Embolic complications and death, however, have been reported during subtherapeutic anticoagulation. Therapeutic anticoagulation should be confirmed before and continued through the procedure. Likewise, low-dose aspirin (e.g., 81–325 mg) can safely be continued. For patients taking aspirin and another antiplatelet medication (e.g., clopidogrel), the decision to continue the second antiplatelet

1	CHAPTER 45 Oral Manifestations of Disease 242 medication should be based on individual consideration of the risks of thrombosis and bleeding. Patients at risk for bacterial endocarditis (Chap. 155) should maintain optimal oral hygiene, including flossing, and have regular professional cleanings. Currently, guidelines recommend that prophylactic antibiotics be restricted to those patients at high risk for bacterial endocarditis who undergo dental and oral procedures involving significant manipulation of gingival or periapical tissue or penetration of the oral mucosa. If unexpected bleeding occurs, antibiotics given within 2 h after the procedure provide effective prophylaxis. Hematogenous bacterial seeding from oral infection can undoubtedly produce late prosthetic-joint infection and therefore requires removal of the infected tissue (e.g., drainage, extraction, root canal) and appropriate antibiotic therapy. However, evidence that late prosthetic-joint infection follows routine dental

1	requires removal of the infected tissue (e.g., drainage, extraction, root canal) and appropriate antibiotic therapy. However, evidence that late prosthetic-joint infection follows routine dental procedures is lacking. For this reason, antibiotic prophylaxis is not recommended before dental surgery for patients with orthopedic pins, screws, and plates. Antibiotic prophylaxis is recommended for patients within the first 2 years after joint replacement who have inflammatory arthropathies, immunosuppression, type 1 diabetes mellitus, previous prosthetic-joint infection, hemophilia, or malnourishment. Concern often arises regarding the use of vasoconstrictors to treat patients with hypertension and heart disease. Vasoconstrictors enhance the depth and duration of local anesthesia, thus reducing the anesthetic dose and potential toxicity. If intravascular injection is avoided, 2% lidocaine with 1:100,000 epinephrine (limited to a total of 0.036 mg of epinephrine) can be used safely in

1	the anesthetic dose and potential toxicity. If intravascular injection is avoided, 2% lidocaine with 1:100,000 epinephrine (limited to a total of 0.036 mg of epinephrine) can be used safely in patients with controlled hypertension and stable coronary heart disease, arrhythmia, or congestive heart failure. Precautions should be taken with patients taking tricyclic antidepressants and nonselective beta blockers because these drugs may potentiate the effect of epinephrine.

1	Elective dental treatments should be postponed for at least 1 month and preferably for 6 months after myocardial infarction, after which the risk of reinfarction is low provided the patient is medically stable (e.g., stable rhythm, stable angina, and no heart failure). Patients who have suffered a stroke should have elective dental care deferred for 6 months. In both situations, effective stress reduction requires good pain control, including the use of the minimal amount of vasoconstrictor necessary to provide good hemostasis and local anesthesia.

1	Bisphosphonate therapy is associated with osteonecrosis of the jaw. However, the risk with oral bisphosphonate therapy is very low. Most patients affected have received high-dose aminobisphosphonate therapy for multiple myeloma or metastatic breast cancer and have undergone tooth extraction or dental surgery. Intraoral lesions, of which two-thirds are painful, appear as exposed yellow-white hard bone involving the mandible or maxilla. Screening tests for determining risk of osteonecrosis are unreliable. Patients slated for aminobisphosphonate therapy should receive preventive dental care that reduces the risk of infection and the need for future dentoalveolar surgery.

1	Halitosis typically emanates from the oral cavity or nasal passages. Volatile sulfur compounds resulting from bacterial decay of food and cellular debris account for the malodor. Periodontal disease, caries, acute forms of gingivitis, poorly fitting dentures, oral abscess, and tongue coating are common causes. Treatment includes correcting poor hygiene, treating infection, and tongue brushing. Hyposalivation can produce and exacerbate halitosis. Pockets of decay in the tonsillar crypts, esophageal diverticulum, esophageal stasis (e.g., achalasia, stricture), sinusitis, and lung abscess account for some instances. A few systemic diseases produce distinctive odors: renal failure (ammoniacal), hepatic (fishy), and ketoacidosis (fruity). Helicobacter pylori gastritis can also produce ammoniacal breath. If a patient presents because of concern about halitosis but no odor is detectable, then pseudohalitosis or halitophobia must be considered.

1	Part 2 Cardinal Manifestations and Presentation of Diseases While tooth loss and dental disease are not normal consequences of aging, a complex array of structural and functional changes that occur with age can affect oral health. Subtle changes in tooth structure (e.g., diminished pulp space and volume, sclerosis of dentinal tubules, and altered proportions of nerve and vascular pulp content) result in the elimination or diminution of pain sensitivity and a reduction in the reparative capacity of the teeth. In addition, age-associated fatty replacement of salivary acini may reduce physiologic reserve, thus increasing the risk of hyposalivation. In healthy older adults, there is minimal, if any, reduction in salivary flow.

1	Poor oral hygiene often results when general health fails or when patients lose manual dexterity and upper-extremity flexibility. This situation is particularly common among frail older adults and nursing home residents and must be emphasized because regular oral cleaning and dental care reduce the incidence of pneumonia and oral disease as well as the mortality risk in this population. Other risks for dental decay include limited lifetime fluoride exposure. Without assiduous care, decay can become quite advanced yet remain asymptomatic. Consequently, much of a tooth—or the entire tooth—can be destroyed before the patient is aware of the process.

1	Periodontal disease, a leading cause of tooth loss, is indicated by loss of alveolar bone height. More than 90% of the U.S. population has some degree of periodontal disease by age 50. Healthy adults who have not had significant alveolar bone loss by the sixth decade of life do not typically experience significant worsening with advancing age.

1	Complete edentulousness with advanced age, though less common than in previous decades, still affects <50% of the U.S. population ffi85 years of age. Speech, mastication, and facial contours are dramatically affected. Edentulousness may also exacerbate obstructive sleep apnea, particularly in asymptomatic individuals who wear dentures. Dentures can improve verbal articulation and restore diminished facial contours. Mastication can also be restored; however, patients expecting dentures to facilitate oral intake are often disappointed. Accommodation to dentures requires a period of adjustment. Pain can result from friction or traumatic lesions produced by loose dentures. Poor fit and poor oral hygiene may permit the development of candidiasis. This fungal infection may be either asymptomatic or painful and is suggested by erythematous smooth or granular tissue conforming to an area covered by the appliance. Individuals with dentures and no natural teeth need regular (annual)

1	asymptomatic or painful and is suggested by erythematous smooth or granular tissue conforming to an area covered by the appliance. Individuals with dentures and no natural teeth need regular (annual) professional oral examinations.

1	Atlas of Oral Manifestations of Disease Samuel C. Durso, Janet A. Yellowitz The health status of the oral cavity is linked to cardiovascular disease, diabetes, and other systemic illnesses. Thus, examining the oral cav-46e Figure 46e-3 Erosive lichen planus. ity for signs of disease is a key part of the physical exam. This chapter presents numerous outstanding clinical photographs illustrating many of the conditions discussed in Chap. 45, Oral Manifestations of Disease. Conditions affecting the teeth, periodontal tissues, and oral mucosa are all represented. CHAPTER 46e Atlas of Oral Manifestations of Disease Figure 46e-1 Gingival overgrowth secondary to calcium channel blocker use. Figure 46e-4 Stevens-Johnson syndrome—reaction to nevirapine. Figure 46e-5 Erythematosus candidiasis under a denture (i.e., the patient should be treated for this fungal infection). Figure 46e-2 Oral lichen planus. Figure 46e-6 Severe periodontitis. Figure 46e-8 Sublingual leukoplakia.

1	Figure 46e-2 Oral lichen planus. Figure 46e-6 Severe periodontitis. Figure 46e-8 Sublingual leukoplakia. Cardinal Manifestations and Presentation of Diseases Figure 46e-9 A. Epulis (gingival hypertrophy) under denture. Figure 46e-7 Angular cheilitis. B. Epulis fissuratum. 46e-3 CHAPTER 46e Figure 46e-13 Healthy mouth. Figure 46e-10 Traumatic lesion inside of cheek. Figure 46e-11 Oral leukoplakia, subtype homogenous leukoplakia. Figure 46e-14 Geographic tongue. Figure 46e-12 Oral carcinoma. Figure 46e-15 Moderate gingivitis. Figure 46e-16 Gingival recession. Figure 46e-19 Root cavity in presence of severe periodontal disease. PART 2 Cardinal Manifestations and Presentation of Diseases Figure 46e-17 Heavy calculus and gingival inflammation. Figure 46e-20 Ulcer on lateral border of tongue —potential carcinoma. Figure 46e-18 Severe gingival inflammation and heavy calculus. Figure 46e-21 Osteonecrosis.

1	Figure 46e-20 Ulcer on lateral border of tongue —potential carcinoma. Figure 46e-18 Severe gingival inflammation and heavy calculus. Figure 46e-21 Osteonecrosis. Figure 46e-22 Severe periodontal disease, missing tooth, very mobile teeth. Figure 46e-23 Salivary stone. Figure 46e-24 A. Calculus. B. Teeth cleaned. Figure 46e-25 Traumatic ulcer. CHAPTER 46e Atlas of Oral Manifestations of Disease Figure 46e-26 Fissured tongue. Figure 46e-27 White coated tongue —likely candidiasis. Dr. Jane Atkinson was a co-author of this chapter in the 17th edition. Some of the materials have been carried over into this edition.

1	47e-1 Dyspnea Richard M. Schwartzstein DYSPNEA The American Thoracic Society defines dyspnea as a “subjective expe-rience of breathing discomfort that consists of qualitatively distinct sensations that vary in intensity. The experience derives from interac-tions among multiple physiological, psychological, social, and environ-mental factors and may induce secondary physiological and behavioral responses.” Dyspnea, a symptom, can be perceived only by the person experiencing it and must be distinguished from the signs of increased work of breathing. MECHANISMS OF DYSPNEA Respiratory sensations are the consequence of interactions between the efferent, or outgoing, motor output from the brain to the ventilatory muscles (feed-forward) and the afferent, or incoming, sensory input from receptors throughout the body (feedback) as well as the integra-tive processing of this information that we infer must be occurring in the brain (Fig. 47e-1). In contrast to painful sensations, which can often

1	throughout the body (feedback) as well as the integra-tive processing of this information that we infer must be occurring in the brain (Fig. 47e-1). In contrast to painful sensations, which can often be attributed to the stimulation of a single nerve ending, dys-pnea sensations are more commonly viewed as holistic, more akin to hunger or thirst. A given disease state may lead to dyspnea by one or more mechanisms, some of which may be operative under some cir-cumstances (e.g., exercise) but not others (e.g., a change in position). 47e SECTion 5 AlTERATionS in CiRCulAToRy AnD RESPiRAToRy FunCTionS ALGORITHM FOR THE INPUTS IN DYSPNEA PRODUCTION

1	FIGuRE 47e-1 Hypothetical model for integration of sensory inputs in the production of dyspnea. Afferent information from the receptors throughout the respiratory system projects directly to the sensory cortex to contribute to primary qualitative sensory experiences and to provide feedback on the action of the ventilatory pump. Afferents also project to the areas of the brain responsible for control of ventilation. The motor cortex, responding to input from the control centers, sends neural messages to the ventilatory muscles and a corollary discharge to the sensory cortex (feed-forward with respect to the instructions sent to the muscles). If the feed-forward and feedback messages do not match, an error signal is generated and the intensity of dyspnea increases. An increasing body of data supports the contribution of affective inputs to the ultimate perception of unpleasant respiratory sensations. (Adapted from MA Gillette, RM Schwartzstein, in SH Ahmedzai, MF Muer [eds]. Supportive

1	supports the contribution of affective inputs to the ultimate perception of unpleasant respiratory sensations. (Adapted from MA Gillette, RM Schwartzstein, in SH Ahmedzai, MF Muer [eds]. Supportive Care in Respiratory Disease. Oxford, UK, Oxford University Press, 2005.)

1	Motor Efferents Disorders of the ventilatory pump—most commonly, increased airway resistance or stiffness (decreased compliance) of the respiratory system—are associated with increased work of breathing or the sense of an increased effort to breathe. When the muscles are weak or fatigued, greater effort is required, even though the mechanics of the system are normal. The increased neural output from the motor cortex is sensed via a corollary discharge, a neural signal that is sent to the sensory cortex at the same time that motor output is directed to the ventilatory muscles.

1	Sensory Afferents Chemoreceptors in the carotid bodies and medulla are activated by hypoxemia, acute hypercapnia, and acidemia. Stimulation of these receptors and of others that lead to an increase in ventilation produce a sensation of “air hunger.” Mechanoreceptors in the lungs, when stimulated by bronchospasm, lead to a sensation of chest tightness. J-receptors, which are sensitive to interstitial edema, and pulmonary vascular receptors, which are activated by acute changes in pulmonary artery pressure, appear to contribute to air hunger. Hyperinflation is associated with the sensation of increased work of breathing, an inability to get a deep breath, or an unsatisfying breath. Metaboreceptors, which are located in skeletal muscle, are believed to be activated by changes in the local biochemical milieu of the tissue active during exercise and, when stimulated, contribute to breathing discomfort.

1	Integration: Efferent-Reafferent Mismatch A discrepancy or mismatch between the feed-forward message to the ventilatory muscles and the feedback from receptors that monitor the response of the ventilatory pump increases the intensity of dyspnea. This mismatch is particularly important when there is a mechanical derangement of the ventilatory pump, as in asthma or chronic obstructive pulmonary disease (COPD). Contribution of Emotional or Affective Factors to Dyspnea Acute anxiety or fear may increase the severity of dyspnea either by altering the interpretation of sensory data or by leading to patterns of breathing that heighten physiologic abnormalities in the respiratory system. In patients with expiratory flow limitation, for example, the increased respiratory rate that accompanies acute anxiety leads to hyperinflation, increased work and effort of breathing, and the sense of an unsatisfying breath.

1	ASSESSING DYSPNEA Quality of Sensation Like pain assessment, dyspnea assessment begins with a determination of the quality of the patient’s discomfort (Table 47e-1). Dyspnea questionnaires or lists of phrases commonly used TAblE 47e-1 ASSoCiATion oF QuAliTATivE DESCRiPToRS, CliniCAl CHARACTERiSTiCS, AnD PATHoPHySiologiC MECHAniSMS oF SHoRTnESS oF bREATH Chest tightness or Asthma, CHF constriction Increased work or effort COPD, asthma, neuroof breathing muscular disease, chest “Air hunger,” need to CHF, PE, COPD, asthma, breathe, urge to breathe pulmonary fibrosis Inability to get a deep Moderate to severe breath, unsatisfying asthma and COPD, pulbreath monary fibrosis, chest Heavy breathing, rapid Sedentary status in breathing, breathing healthy individual or more patient with cardiopul Abbreviations: CHF, congestive heart failure; COPD, chronic obstructive pulmonary disease; PE, pulmonary embolism. Bronchoconstriction, interstitial edema

1	Abbreviations: CHF, congestive heart failure; COPD, chronic obstructive pulmonary disease; PE, pulmonary embolism. Bronchoconstriction, interstitial edema Airway obstruction, neuromuscular disease Increased drive to breathe 47e-2 by patients assist those who have difficulty describing their breathing sensations. Sensory Intensity A modified Borg scale or visual analogue scale can be utilized to measure dyspnea at rest, immediately following exercise, or on recall of a reproducible physical task, such as climbing the stairs at home. An alternative approach is to gain a sense of the patient’s disability by inquiring about what activities are possible. These methods indirectly assess dyspnea and may be affected by nonrespiratory factors, such as leg arthritis or weakness. The Baseline Dyspnea Index and the Chronic Respiratory Disease Questionnaire are commonly used tools for this purpose.

1	Affective Dimension For a sensation to be reported as a symptom, it must be perceived as unpleasant and interpreted as abnormal. Laboratory studies have demonstrated that air hunger evokes a stronger affective response than does increased effort or work of breathing. Some therapies for dyspnea, such as pulmonary rehabilitation, may reduce breathing discomfort, in part, by altering this dimension.

1	Dyspnea most often results from deviations from normal function in the cardiovascular and respiratory systems. These deviations produce breathlessness as a consequence of increased drive to breathe; increased effort or work of breathing; and/or stimulation of receptors in the heart, lungs, or vascular system. Most diseases of the respiratory system are associated with alterations in the mechanical properties of the lungs and/or chest wall, and some stimulate pulmonary receptors. In contrast, disorders of the cardiovascular system more commonly lead to dyspnea by causing gas-exchange abnormalities or stimulating pulmonary and/or vascular receptors (Table 47e-2).

1	Respiratory System Dyspnea • DISEASES OF THE AIRWAYS Asthma and COPD, the most common obstructive lung diseases, are characterized by expiratory airflow obstruction, which typically leads to dynamic hyperinflation of the lungs and chest wall. Patients with moderate to severe disease have both increased resistive and elastic loads (a term that relates to the stiffness of the system) on the ventilatory muscles and experience increased work of breathing. Patients with acute bronchoconstriction also report a sense of tightness, which can exist even when lung function is still within the normal range. These patients are commonly tachypneic; this condition leads to hyperinflation and reduced respiratory system compliance and also limits tidal volume. Both the chest tightness and the tachypnea are probably due to stimulation of pulmonary receptors. Both asthma and COPD may lead to hypoxemia and hypercapnia from ventilation-perfusion (V/Q) mismatch (and diffusion limitation during exercise

1	are probably due to stimulation of pulmonary receptors. Both asthma and COPD may lead to hypoxemia and hypercapnia from ventilation-perfusion (V/Q) mismatch (and diffusion limitation during exercise with emphysema); hypoxemia is much more common than hypercapnia as a consequence of the different ways in which oxygen and carbon dioxide bind to hemoglobin.

1	DISEASES OF THE CHEST WALL Conditions that stiffen the chest wall, such as kyphoscoliosis, or that weaken ventilatory muscles, such as myasthenia gravis or the Guillain-Barré syndrome, are also associated with PART 2 Cardinal Manifestations and Presentation of Diseases an increased effort to breathe. Large pleural effusions may contribute to dyspnea, both by increasing the work of breathing and by stimulating pulmonary receptors if there is associated atelectasis.

1	DISEASES OF THE LUNG PARENCHYMA Interstitial lung diseases, which may arise from infections, occupational exposures, or autoimmune disorders, are associated with increased stiffness (decreased compliance) of the lungs and increased work of breathing. In addition, V/Q mismatch and the destruction and/or thickening of the alveolar-capillary interface may lead to hypoxemia and an increased drive to breathe. Stimulation of pulmonary receptors may further enhance the hyperventilation characteristic of mild to moderate interstitial disease.

1	Cardiovascular System Dyspnea • DISEASES OF THE LEFT HEART Diseases of the myocardium resulting from coronary artery disease and nonischemic cardiomyopathies cause a greater left-ventricular end-diastolic volume and an elevation of the left-ventricular end-diastolic as well as pulmonary capillary pressures. These elevated pressures lead to interstitial edema and stimulation of pulmonary receptors, thereby causing dyspnea; hypoxemia due to V/Q mismatch may also contribute to breathlessness. Diastolic dysfunction, characterized by a very stiff left ventricle, may lead to severe dyspnea with relatively mild degrees of physical activity, particularly if it is associated with mitral regurgitation.

1	DISEASES OF THE PULMONARY VASCULATURE Pulmonary thromboembolic disease and primary diseases of the pulmonary circulation (primary pulmonary hypertension, pulmonary vasculitis) cause dyspnea via increased pulmonary-artery pressure and stimulation of pulmonary receptors. Hyperventilation is common, and hypoxemia may be present. However, in most cases, use of supplemental oxygen has only a minimal impact on the severity of dyspnea and hyperventilation. DISEASES OF THE PERICARDIUM Constrictive pericarditis and cardiac tamponade are both associated with increased intracardiac and pulmonary vascular pressures, which are the likely cause of dyspnea in these conditions. To the extent that cardiac output is limited (at rest or with exercise) metaboreceptors may be stimulated if cardiac output is compromised to the degree that lactic acidosis develops; chemoreceptors will also be activated.

1	Dyspnea with Normal Respiratory and Cardiovascular Systems Mild to moderate anemia is associated with breathing discomfort during exercise. This symptom is thought to be related to stimulation of metaboreceptors; oxygen saturation is normal in patients with anemia. The breathlessness associated with obesity is probably due to multiple mechanisms, including high cardiac output and impaired ventilatory pump function (decreased compliance of the chest wall). Cardiovascular deconditioning (poor fitness) is characterized by the early development of anaerobic metabolism and the stimulation of chemoreceptors and metaboreceptors. Dyspnea that is medically unexplained has been associated with increased sensitivity to the unpleasantness of acute hypercapnia. aHypoxemia and hypercapnia are not always present in these conditions. When hypoxemia is present, dyspnea usually persists, albeit at a reduced intensity, with correction of hypoxemia by the administration of supplemental oxygen.

1	Abbreviations: COPD, chronic obstructive pulmonary disease; CPE, cardiogenic pulmonary edema; ILD, interstitial lung disease; NCPE, noncardiogenic pulmonary edema; PVD, pulmonary vascular disease. APPROACH TO THE PATIENT: Dyspnea (see Fig. 47e-2)

1	APPROACH TO THE PATIENT: Dyspnea (see Fig. 47e-2) The patient should be asked to describe in his/her own words what the discomfort feels like as well as the effect of position, infections, and environmental stimuli on the dyspnea. Orthopnea is a common indicator of congestive heart failure (CHF), mechanical impairment of the diaphragm associated with obesity, or asthma triggered by esophageal reflux. Nocturnal dyspnea suggests CHF or asthma. Acute, intermittent episodes of dyspnea are more likely to reflect episodes of myocardial ischemia, bronchospasm, or pulmonary embolism, while chronic persistent dyspnea is typical of COPD, interstitial lung disease, and chronic thromboembolic disease. Information on risk factors for occupational lung disease and for coronary artery disease should be elicited. Left atrial myxoma or hepatopulmonary syndrome should be considered when the patient complains of platypnea—i.e., dyspnea in the upright position with relief in the supine position.

1	The physical examination should begin during the interview of the patient. Inability of the patient to speak in full sentences before stopping to get a deep breath suggests a condition that leads to stimulation of the controller or impairment of the ventilatory pump with reduced vital capacity. Evidence of increased work of breathing (supraclavicular retractions; use of accessory muscles of ventilation; and the tripod position, characterized by sitting with the hands braced on the knees) is indicative of increased airway resistance or stiffness of the lungs and the chest wall. When measuring the vital signs, the physician should accurately assess the respiratory rate and measure the pulsus paradoxus (Chap. 288); if the systolic pressure decreases by >10 mmHg, the presence of COPD, acute asthma, or pericardial disease should be considered. During the general examination, signs of anemia (pale conjunctivae), cyanosis, and cirrhosis (spider angiomata, gynecomastia) should be sought.

1	asthma, or pericardial disease should be considered. During the general examination, signs of anemia (pale conjunctivae), cyanosis, and cirrhosis (spider angiomata, gynecomastia) should be sought. Examination of the chest should focus on symmetry of movement; percussion (dullness is indicative of pleural effusion; hyperresonance is a sign of emphysema); and auscultation (wheezes, rhonchi, prolonged expiratory phase, and diminished breath sounds are clues to disorders of the airways; rales suggest interstitial edema or fibrosis). The cardiac examination should focus on signs of elevated right heart pressures (jugular venous distention, edema, accentuated pulmonic component to the second heart sound); left ventricular dysfunction (S3 and S4 gallops); and valvular disease (murmurs). When examining the abdomen with the patient in the supine position, the physician should note whether there is paradoxical movement of the abdomen: inward motion during inspiration is a sign of diaphragmatic

1	the abdomen with the patient in the supine position, the physician should note whether there is paradoxical movement of the abdomen: inward motion during inspiration is a sign of diaphragmatic weakness, and rounding of the abdomen during exhalation is suggestive of pulmonary edema. Clubbing of the digits may be an indication of interstitial pulmonary fibrosis, and joint swelling or deformation as well as changes consistent with Raynaud’s disease may be indicative of a collagen-vascular process that can be associated with pulmonary disease.

1	History Quality of sensation, timing, positional disposition Persistent vs intermittent Physical Exam General appearance: Speak in full sentences? Accessory muscles? Color? Vital Signs: Tachypnea? Pulsus paradoxus? Oximetry-evidence of desaturation? Chest: Wheezes, rales, rhonchi, diminished breath sounds? Hyperinflated? Cardiac exam: JVP elevated? Precordial impulse? Gallop? Murmur? Extremities: Edema? Cyanosis? At this point, diagnosis may be evident—if not, proceed to further evaluation Chest radiograph Assess cardiac size, evidence of CHF Assess for hyperinflation Assess for pneumonia, interstitial lung disease, pleural effusions If diagnosis still uncertain, obtain cardiopulmonary exercise test

1	If diagnosis still uncertain, obtain cardiopulmonary exercise test FIGuRE 47e-2 Algorithm for the evaluation of the patient with dyspnea. JVP, jugular venous pulse; CHF, congestive heart failure; ECG, electrocardiogram; CT, computed tomography. (Adapted from RM Schwartzstein, D Feller-Kopman, in E Braunwald, L Goldman [eds]. Primary Cardiology, 2nd ed. Philadelphia, WB Saunders, 2003.) Patients with exertional dyspnea should be asked to walk under observation in order to reproduce the symptoms. The patient should be examined during and at the end of exercise for new findings that were not present at rest and for changes in oxygen saturation.

1	After the history elicitation and the physical examination, a chest radiograph should be obtained. The lung volumes should be assessed: hyperinflation indicates obstructive lung disease, whereas low lung volumes suggest interstitial edema or fibrosis, diaphragmatic dysfunction, or impaired chest wall motion. The pulmonary parenchyma should be examined for evidence of interstitial disease and emphysema. Prominent pulmonary vasculature in the upper zones indicates pulmonary venous hypertension, while enlarged central pulmonary arteries suggest pulmonary arterial hypertension. An enlarged cardiac silhouette suggests dilated cardiomyopathy or valvular disease. Bilateral pleural effusions are typical of CHF and some forms of collagen-vascular disease. Unilateral effusions raise the specter of carcinoma and pulmonary embolism but may also occur in heart failure. CT of the chest is generally reserved for further evaluation of the lung parenchyma (interstitial lung disease) and possible

1	of carcinoma and pulmonary embolism but may also occur in heart failure. CT of the chest is generally reserved for further evaluation of the lung parenchyma (interstitial lung disease) and possible pulmonary embolism.

1	Laboratory studies should include electrocardiography to seek evidence of ventricular hypertrophy and prior myocardial infarction. Echocardiography is indicated when systolic dysfunction, pulmonary hypertension, or valvular heart disease is suspected. Bronchoprovocation testing is useful in patients with intermittent symptoms suggestive of asthma but normal physical examination and lung function; up to one-third of patients with the clinical diagnosis of asthma do not have reactive airways disease when formally tested. Measurement of brain natriuretic peptide levels in serum is increasingly used to assess for CHF in patients presenting with acute dyspnea but may be elevated in the presence of right ventricular strain as well.

1	If a patient has evidence of both pulmonary and cardiac disease, a cardiopulmonary exercise test should be carried out to determine which system is responsible for the exercise limitation. If, at peak exercise, the patient achieves predicted maximal ventilation, demonstrates an increase in dead space or hypoxemia, or develops bronchospasm, the respiratory system is probably the cause of the problem. Alternatively, if the heart rate is >85% of the predicted maximum, if the anaerobic threshold occurs early, if the blood pressure becomes excessively high or decreases during exercise, if the O2 pulse (O2 consumption/heart rate, an indicator of stroke volume) falls, or if there are ischemic changes on the electrocardiogram, an abnormality of the cardiovascular system is likely the explanation for the breathing discomfort. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases The first goal is to correct the underlying problem responsible for the symptom. If this is not possible, an effort is made to lessen the intensity of the symptom and its effect on the patient’s quality of life. Supplemental O2 should be administered if the resting O2 saturation is ≤89% or if the patient’s saturation drops to these levels with activity. For patients with COPD, pulmonary rehabilitation programs have demonstrated positive effects on dyspnea, exercise capacity, and rates of hospitalization. Studies of anxiolytics and antidepressants have not documented consistent benefit. Experimental interventions—e.g., cold air on the face, chest wall vibration, and inhaled furosemide—aimed at modulating the afferent information from receptors throughout the respiratory system are being studied. Morphine has been shown to reduce dyspnea out of proportion to the change in ventilation in laboratory models.

1	The extent to which fluid accumulates in the interstitium of the lung depends on the balance of hydrostatic and oncotic forces within the pulmonary capillaries and in the surrounding tissue. Hydrostatic pressure favors movement of fluid from the capillary into the interstitium. The oncotic pressure, which is determined by the protein concentration in the blood, favors movement of fluid into the vessel. Levels of albumin, the primary protein in the plasma, may be low in conditions such as cirrhosis and nephrotic syndrome. While hypoalbuminemia favors movement of fluid into the tissue for any given hydrostatic pressure in the capillary, it is usually not sufficient by itself to cause interstitial edema. In a healthy individual, the tight junctions of the capillary endothelium are impermeable to proteins, and the lymphatics in the tissue carry away the small amounts of protein that may leak out; together, these factors result in an oncotic force that maintains fluid in the capillary.

1	to proteins, and the lymphatics in the tissue carry away the small amounts of protein that may leak out; together, these factors result in an oncotic force that maintains fluid in the capillary. Disruption of the endothelial barrier, however, allows protein to escape the capillary bed and enhances the movement of fluid into the tissue of the lung.

1	(See also Chap. 326) Cardiac abnormalities that lead to an increase in pulmonary venous pressure shift the balance of forces between the capillary and the interstitium. Hydrostatic pressure is increased and fluid exits the capillary at an increased rate, resulting in interstitial and, in more severe cases, alveolar edema. The development of pleural effusions may further compromise respiratory system function and contribute to breathing discomfort. Early signs of pulmonary edema include exertional dyspnea and orthopnea. Chest radiographs show peribronchial thickening, prominent vascular markings in the upper lung zones, and Kerley B lines. As the pulmonary edema worsens, alveoli fill with fluid; the chest radio-graph shows patchy alveolar filling, typically in a perihilar distribution, which then progresses to diffuse alveolar infiltrates. Increasing airway edema is associated with rhonchi and wheezes.

1	In noncardiogenic pulmonary edema, lung water increases due to damage of the pulmonary capillary lining with consequent leakage of proteins and other macromolecules into the tissue; fluid follows the protein as oncotic forces are shifted from the vessel to the surrounding lung tissue. This process is associated with dysfunction of the surfactant lining the alveoli, increased surface forces, and a propensity for the alveoli to collapse at low lung volumes. Physiologically, noncardiogenic pulmonary edema is characterized by intrapulmonary shunt with hypoxemia and decreased pulmonary compliance leading to lower functional residual capacity. On pathologic examination, hyaline membranes are evident in the alveoli, and inflammation leading to pulmonary fibrosis may be seen. Clinically, the picture ranges from mild dyspnea to respiratory failure. Auscultation of the lungs may be relatively normal despite chest radiographs that show diffuse alveolar infiltrates. CT scans demonstrate that the

1	ranges from mild dyspnea to respiratory failure. Auscultation of the lungs may be relatively normal despite chest radiographs that show diffuse alveolar infiltrates. CT scans demonstrate that the distribution of alveolar edema is more heterogeneous than was once thought. Although normal intra-cardiac pressures are considered by many to be part of the definition of noncardiogenic pulmonary edema, the pathology of the process, as described above, is distinctly different, and a combination of cardiogenic and noncardiogenic pulmonary edema is observed in some patients.

1	It is useful to categorize the causes of noncardiogenic pulmonary edema in terms of whether the injury to the lung is likely to result from direct, indirect, or pulmonary vascular causes (Table 47e-3). Direct injuries are mediated via the airways (e.g., aspiration) or as the consequence of blunt chest trauma. Indirect injury is the consequence of mediators that reach the lung via the bloodstream. The third category includes conditions that may result from acute changes in pulmonary vascular pressures, possibly due to sudden autonomic discharge (in the case of neurogenic and high-altitude pulmonary edema) or sudden swings of pleural pressure as well as transient damage to the pulmonary capillaries (in the case of reexpansion pulmonary edema). Direct Injury to Lung Chest trauma, pulmonary contusion Aspiration Smoke inhalation Pneumonia Oxygen toxicity Pulmonary embolism, reperfusion Hematogenous Injury to Lung

1	Direct Injury to Lung Chest trauma, pulmonary contusion Aspiration Smoke inhalation Pneumonia Oxygen toxicity Pulmonary embolism, reperfusion Hematogenous Injury to Lung Sepsis Pancreatitis Nonthoracic trauma Leukoagglutination reactions Multiple transfusions Intravenous drug use (e.g., heroin) Cardiopulmonary bypass

1	The history is essential for assessing the likelihood of underlying cardiac disease as well as for identification of one of the conditions associated with noncardiogenic pulmonary edema. The physical examination in cardiogenic pulmonary edema is notable for evidence of increased intracardiac pressures (S3 gallop, elevated jugular venous pulse, peripheral edema) and rales and/or wheezes on auscultation of the chest. In contrast, the physical examination in noncardiogenic pulmonary edema is dominated by the findings of the precipitating condition; pulmonary findings may be relatively normal in the early stages. The chest radiograph in cardiogenic pulmonary edema typically shows an enlarged cardiac silhouette, vascular redistribution, interstitial thickening, and perihilar alveolar infiltrates; pleural effusions are common. In noncardiogenic pulmonary edema, heart size is normal, alveolar infiltrates are distributed more uniformly throughout the lungs, and pleural effusions are uncommon.

1	pleural effusions are common. In noncardiogenic pulmonary edema, heart size is normal, alveolar infiltrates are distributed more uniformly throughout the lungs, and pleural effusions are uncommon. Finally, the hypoxemia of cardiogenic pulmonary edema is due largely to V/Q to mismatch and responds to the administration of supplemental oxygen. In contrast, hypoxemia in noncardiogenic pulmonary edema is due primarily to intrapulmonary shunting and typically persists despite high concentrations of inhaled oxygen.

1	Cough and Hemoptysis Patricia A. Kritek, Christopher H. Fanta COugH Cough performs an essential protective function for human airways and lungs. Without an effective cough reflex, we are at risk for retained 48 airway secretions and aspirated material predisposing to infection, atelectasis, and respiratory compromise. At the other extreme, excessive coughing can be exhausting; can be complicated by emesis, syncope, muscular pain, or rib fractures; and can aggravate abdominal or inguinal hernias and urinary incontinence. Cough is often a clue to the presence of respiratory disease. In many instances, cough is an expected and accepted manifestation of disease, as in acute respiratory tract infection. However, persistent cough in the absence of other respiratory symptoms commonly causes patients to seek medical attention.

1	Spontaneous cough is triggered by stimulation of sensory nerve endings that are thought to be primarily rapidly adapting receptors and C fibers. Both chemical (e.g., capsaicin) and mechanical (e.g., particulates in air pollution) stimuli may initiate the cough reflex. A cationic ion channel—the type 1 vanilloid receptor—found on rapidly adapting receptors and C fibers is the receptor for capsaicin, and its expression is increased in patients with chronic cough. Afferent nerve endings richly innervate the pharynx, larynx, and airways to the level of the terminal bronchioles and extend into the lung parenchyma. They may also be located in the external auditory meatus (the auricular branch of the vagus nerve, or the Arnold nerve) and in the esophagus. Sensory signals travel via the vagus and superior laryngeal nerves to a region of the brainstem in the nucleus tractus solitarius vaguely identified as the “cough center.” The cough reflex involves a highly orchestrated series of

1	vagus and superior laryngeal nerves to a region of the brainstem in the nucleus tractus solitarius vaguely identified as the “cough center.” The cough reflex involves a highly orchestrated series of involuntary muscular actions, with the potential for input from cortical pathways as well. The vocal cords adduct, leading to transient upper-airway occlusion. Expiratory muscles contract, generating positive intrathoracic pressures as high as 300 mmHg. With sudden release of the laryngeal contraction, rapid expiratory flows are generated,

1	Coughs Volume (L) Flow (L/sec)FEV3 = 6.224 2 0 –2 –4 –6 –8 –10 12345678 91011 FIguRE 48-1 Flow-volume curve shows spikes of high expiratory flow achieved with cough. FEV1, forced expiratory volume in 1 s. FEV1 = 5.376 exceeding the normal “envelope” of maximal expiratory flow seen on the flow-volume curve (Fig. 48-1). Bronchial smooth-muscle contraction together with dynamic compression of airways narrows airway lumens and maximizes the velocity of exhalation. The kinetic energy available to dislodge mucus from the inside of airway walls is directly proportional to the square of the velocity of expiratory airflow. A deep breath preceding a cough optimizes the function of the expiratory muscles; a series of repetitive coughs at successively lower lung volumes sweeps the point of maximal expiratory velocity progressively further into the lung periphery.

1	Weak or ineffective cough compromises the ability to clear lower respiratory tract infections, predisposing to more serious infections and their sequelae. Weakness, paralysis, or pain of the expiratory (abdominal and intercostal) muscles is foremost on the list of causes of impaired cough (Table 48-1). Cough strength is generally assessed qualitatively; peak expiratory flow or maximal expiratory pressure at the mouth can be used as a surrogate marker for cough strength. A variety of assistive devices and techniques have been developed to improve cough strength, running the gamut from simple (splinting of the abdominal muscles with a tightly-held pillow to reduce postoperative pain while coughing) to complex (a mechanical cough-assist device supplied via face mask or tracheal tube that applies a cycle of positive pressure followed rapidly by negative pressure). Cough may fail to clear secretions despite a preserved ability to generate normal expiratory velocities; such failure may be

1	a cycle of positive pressure followed rapidly by negative pressure). Cough may fail to clear secretions despite a preserved ability to generate normal expiratory velocities; such failure may be due to either abnormal airway secretions (e.g., bronchiectasis due to cystic fibrosis) or structural abnormalities of the airways (e.g., tracheomalacia with expiratory collapse during cough).

1	The cough of chronic bronchitis in long-term cigarette smokers rarely leads the patient to seek medical advice. It lasts for only seconds to a CAuSES of iMPAiRED CougH

1	Central respiratory depression (e.g., anesthesia, sedation, or coma) 244 few minutes, is productive of benign-appearing mucoid sputum, and generally does not cause discomfort. Cough may occur in the context of other respiratory symptoms that together point to a diagnosis; for example, cough accompanied by wheezing, shortness of breath, and chest tightness after exposure to a cat or other sources of allergens suggests asthma. At times, however, cough is the dominant or sole symptom of disease, and it may be of sufficient duration and severity that relief is sought. The duration of cough is a clue to its etiology. Acute cough (<3 weeks) is most commonly due to a respiratory tract infection, aspiration, or inhalation of noxious chemicals or smoke. Subacute cough (3–8 weeks in duration) is a common residuum of tracheobronchitis, as in pertussis or “postviral tussive syndrome.” Chronic cough (>8 weeks) may be caused by a wide variety of cardiopulmonary diseases, including those of

1	is a common residuum of tracheobronchitis, as in pertussis or “postviral tussive syndrome.” Chronic cough (>8 weeks) may be caused by a wide variety of cardiopulmonary diseases, including those of inflammatory, infectious, neoplastic, and cardiovascular etiologies. When initial assessment with chest examination and radiography is normal, cough-variant asthma, gastroesophageal reflux, nasopharyngeal drainage, and medications (angiotensin-converting enzyme [ACE] inhibitors) are the most common causes of chronic cough.

1	Details as to the sound, the time of occurrence during the day, and the pattern of coughing infrequently provide useful etiologic clues. Regardless of cause, cough often worsens upon first lying down at night, with talking, or with the hyperpnea of exercise; it frequently improves with sleep. An exception may involve the cough that occurs only with certain allergic exposures or exercise in cold air, as in asthma. Useful historical questions include what circumstances surround the onset of cough, what makes the cough better or worse, and whether or not the cough produces sputum.

1	The physical examination seeks clues suggesting the presence of cardiopulmonary disease, including findings such as wheezing or crackles on chest examination. Examination of the auditory canals and tympanic membranes (for irritation of the latter resulting in stimulation of Arnold’s nerve), the nasal passageways (for rhinitis or polyps), and the nails (for clubbing) may also provide etiologic clues. Because cough can be a manifestation of a systemic disease such as sarcoidosis or vasculitis, a thorough general examination is equally important.

1	In virtually all instances, evaluation of chronic cough merits a chest radiograph. The list of diseases that can cause persistent cough without other symptoms and without detectable abnormalities on physical examination is long. It includes serious illnesses such as sarcoidosis or Hodgkin’s disease in young adults, lung cancer in older patients, and (worldwide) pulmonary tuberculosis. An abnormal chest film prompts an evaluation aimed at explaining the cough. In a patient with chronic productive cough, examination of expectorated sputum is warranted. Purulent-appearing sputum should be sent for routine bacterial culture and, in certain circumstances, mycobacterial culture as well. Cytologic examination of mucoid sputum may be useful to assess for malignancy and to distinguish neutrophilic from eosinophilic bronchitis. Expectoration of blood—whether streaks of blood, blood mixed with airway secretions, or pure blood—deserves a special approach to assessment and management (see

1	from eosinophilic bronchitis. Expectoration of blood—whether streaks of blood, blood mixed with airway secretions, or pure blood—deserves a special approach to assessment and management (see “Hemoptysis,” below).

1	It is commonly held that (alone or in combination) the use of an ACE inhibitor; postnasal drainage; gastroesophageal reflux; and asthma account for more than 90% of cases of chronic cough with a normal or noncontributory chest radiograph. However, clinical experience does not support this contention, and strict adherence to this concept discourages the search for alternative explanations by both clinicians and researchers. ACE inhibitor–induced cough occurs in 5–30% of patients taking these agents and is not dose dependent. ACE metabolizes bradykinin and other tachykinins, such as substance P. The mechanism of ACE inhibitor–associated cough may involve sensitization of sensory nerve endings due to accumulation of bradykinin. In support of this hypothesis, polymorphisms in the neurokinin-2 receptor gene are associated with ACE inhibitor–induced cough. Any patient with

1	PART 2 Cardinal Manifestations and Presentation of Diseases chronic unexplained cough who is taking an ACE inhibitor should have a trial period off the medication, regardless of the timing of the onset of cough relative to the initiation of ACE inhibitor therapy. In most instances, a safe alternative is available; angiotensin-receptor blockers do not cause cough. Failure to observe a decrease in cough after 1 month off medication argues strongly against this etiology. Postnasal drainage of any etiology can cause cough as a response to stimulation of sensory receptors of the cough-reflex pathway in the hypopharynx or aspiration of draining secretions into the trachea. Clues suggesting this etiology include postnasal drip, frequent throat clearing, and sneezing and rhinorrhea. On speculum examination of the nose, excess mucoid or purulent secretions, inflamed and edematous nasal mucosa, and/or polyps may be seen; in addition, secretions or a cobblestoned appearance of the mucosa along

1	examination of the nose, excess mucoid or purulent secretions, inflamed and edematous nasal mucosa, and/or polyps may be seen; in addition, secretions or a cobblestoned appearance of the mucosa along the posterior pharyngeal wall may be noted. Unfortunately, there is no means by which to quantitate postnasal drainage. In many instances, this diagnosis must rely on subjective information provided by the patient. This assessment must also be counterbalanced by the fact that many people who have chronic postnasal drainage do not experience cough.

1	Linking gastroesophageal reflux to chronic cough poses similar challenges. It is thought that reflux of gastric contents into the lower esophagus may trigger cough via reflex pathways initiated in the esophageal mucosa. Reflux to the level of the pharynx (laryngopharyngeal reflux), with consequent aspiration of gastric contents, causes a chemical bronchitis and possibly pneumonitis that can elicit cough for days afterward. Retrosternal burning after meals or on recumbency, frequent eructation, hoarseness, and throat pain may be indicative of gastroesophageal reflux. Nevertheless, reflux may also elicit minimal or no symptoms. Glottic inflammation detected on laryngoscopy may be a manifestation of recurrent reflux to the level of the throat, but it is a nonspecific finding. Quantification of the frequency and level of reflux requires a somewhat invasive procedure to measure esophageal pH directly (either nasopharyngeal placement of a catheter with a pH probe into the esophagus for 24 h

1	the frequency and level of reflux requires a somewhat invasive procedure to measure esophageal pH directly (either nasopharyngeal placement of a catheter with a pH probe into the esophagus for 24 h or endoscopic placement of a radio-transmitter capsule into the esophagus). The precise interpretation of test results that permits an etiologic linking of reflux events and cough remains debated. Again, assigning the cause of cough to gastroesophageal reflux must be weighed against the observation that many people with symptomatic reflux do not experience chronic cough.

1	Cough alone as a manifestation of asthma is common among children but not among adults. Cough due to asthma in the absence of wheezing, shortness of breath, and chest tightness is referred to as “cough-variant asthma.” A history suggestive of cough-variant asthma ties the onset of cough to exposure to typical triggers for asthma and the resolution of cough to discontinuation of exposure. Objective testing can establish the diagnosis of asthma (airflow obstruction on spirometry that varies over time or reverses in response to a bronchodilator) or exclude it with certainty (a negative response to a bronchoprovocation challenge—e.g., with methacholine). In a patient capable of taking reliable measurements, home expiratory peak flow monitoring can be a cost-effective method to support or discount a diagnosis of asthma.

1	Chronic eosinophilic bronchitis causes chronic cough with a normal chest radiograph. This condition is characterized by sputum eosinophilia in excess of 3% without airflow obstruction or bronchial hyperresponsiveness and is successfully treated with inhaled glucocorticoids.

1	Treatment of chronic cough in a patient with a normal chest radio-graph is often empirical and is targeted at the most likely cause(s) of cough as determined by history, physical examination, and possibly pulmonary-function testing. Therapy for postnasal drainage depends on the presumed etiology (infection, allergy, or vasomotor rhinitis) and may include systemic antihistamines; antibiotics; nasal saline irrigation; and nasal pump sprays with glucocorticoids, antihistamines, or anticholinergics. Antacids, histamine type 2 (H2) receptor antagonists, and proton-pump inhibitors are used to neutralize or decrease the production of gastric acid in gastroesophageal reflux disease; dietary changes, elevation of the head and torso during sleep, and medications to improve gastric emptying are additional therapeutic measures. Cough-variant asthma typically responds well to inhaled glucocorticoids and intermittent use of inhaled β-agonist bronchodilators.

1	Cough-variant asthma typically responds well to inhaled glucocorticoids and intermittent use of inhaled β-agonist bronchodilators. Patients who fail to respond to treatment targeting the common causes of chronic cough or who have had these causes excluded by appropriate diagnostic testing should undergo chest CT. Diseases causing cough that may be missed on chest x-ray include tumors, early interstitial lung disease, bronchiectasis, and atypical mycobacterial pulmonary infection. On the other hand, patients with chronic cough who have normal findings on chest examination, lung function testing, oxygenation assessment, and chest CT can be reassured as to the absence of serious pulmonary pathology.

1	Chronic idiopathic cough, also called cough hypersensitivity syndrome, is distressingly common. It is often experienced as a tickle or sensitivity in the throat, occurs more often in women, and is typically “dry” or at most productive of scant amounts of mucoid sputum. It can be exhausting, interfere with work, and cause social embarrassment. Once serious underlying cardiopulmonary pathology has been excluded, an attempt at cough suppression is appropriate. Most effective are narcotic cough suppressants, such as codeine or hydrocodone, which are thought to act in the “cough center” in the brainstem. The tendency of narcotic cough suppressants to cause drowsiness and constipation and their potential for addictive dependence limit their appeal for longterm use. Dextromethorphan is an over-the-counter, centrally acting cough suppressant with fewer side effects and less efficacy than the narcotic cough suppressants. Dextromethorphan is thought to have a different site of action than

1	centrally acting cough suppressant with fewer side effects and less efficacy than the narcotic cough suppressants. Dextromethorphan is thought to have a different site of action than narcotic cough suppressants and can be used in combination with them if necessary. Benzonatate is thought to inhibit neural activity of sensory nerves in the cough-reflex pathway. It is generally free of side effects; however, its effectiveness in suppressing cough is variable and unpredictable. Case series have reported benefit from off-label use of gabapentin or amitriptyline for chronic idiopathic cough. Novel cough suppressants without the limitations of currently available agents are greatly needed. Approaches that are being explored include the development of neurokinin receptor antagonists, type 1 vanilloid receptor antagonists, and novel opioid and opioid-like receptor agonists.

1	Hemoptysis, the expectoration of blood from the respiratory tract, can arise at any location from the alveoli to the glottis. It is important to distinguish hemoptysis from epistaxis (bleeding from the nasopharynx) and hematemesis (bleeding from the upper gastrointestinal tract). Hemoptysis can range from the expectoration of blood-tinged sputum to that of life-threatening large volumes of bright red blood. For most patients, any degree of hemoptysis can cause anxiety and often prompts medical evaluation.

1	While precise epidemiologic data are lacking, the most common etiology of hemoptysis is infection of the medium-sized airways. In the United States, the cause is usually viral or bacterial bronchitis. Hemoptysis can arise in the setting of acute bronchitis or during an exacerbation of chronic bronchitis. Worldwide, the most common cause of hemoptysis is infection with Mycobacterium tuberculosis, presumably because of the high prevalence of tuberculosis and its predilection for cavity formation. While these are the most common causes, the differential diagnosis for hemoptysis is extensive, and a step-wise approach to evaluation is appropriate.

1	One way to approach the source of hemoptysis is to search systematically for potential sites of bleeding from the alveolus to the mouth. Diffuse bleeding in the alveolar space, often referred to as diffuse alveolar hemorrhage (DAH), may present as hemoptysis. Causes of DAH can be inflammatory or noninflammatory. Inflammatory DAH is due to small-vessel vasculitis/capillaritis from a variety of diseases, including granulomatosis with polyangiitis and microscopic polyangiitis. Similarly, systemic autoimmune diseases such as systemic lupus erythematosus 245 can manifest as pulmonary capillaritis. Antibodies to the alveolar basement membrane, as are seen in Goodpasture’s disease, can also result in alveolar hemorrhage. In the early period after bone marrow transplantation, patients can develop a form of inflammatory DAH that can be catastrophic and life-threatening. The exact pathophysiology of this process is not well understood, but DAH should be suspected in patients with sudden-onset

1	a form of inflammatory DAH that can be catastrophic and life-threatening. The exact pathophysiology of this process is not well understood, but DAH should be suspected in patients with sudden-onset dyspnea and hypoxemia in the first 100 days after bone marrow transplantation.

1	Alveoli can also bleed due to direct inhalational injury, including thermal injury from fires, inhalation of illicit substances (e.g., cocaine), and inhalation of toxic chemicals. If alveoli are irritated from any process, patients with thrombocytopenia, coagulopathy, or antiplatelet or anticoagulant use will be at increased risk of hemoptysis.

1	Bleeding in hemoptysis most commonly arises from the smallto medium-sized airways. Irritation and injury of the bronchial mucosa can lead to small-volume bleeding. More significant hemoptysis can result from the proximity of the bronchial artery and vein to the airway, with these vessels and the bronchus running together in what is often referred to as the bronchovascular bundle. In the smaller airways, these blood vessels are close to the airspace, and lesser degrees of inflammation or injury can therefore result in their rupture into the airways. While alveolar hemorrhage arises from capillaries that are part of the low-pressure pulmonary circulation, bronchial bleeding generally originates from bronchial arteries, which are under systemic pressure and thus are predisposed to larger-volume bleeding.

1	Any infection of the airways can result in hemoptysis, although acute bronchitis is most commonly caused by viral infection. In patients with a history of chronic bronchitis, bacterial superinfection with organisms such as Streptococcus pneumoniae, Haemophilus influenzae, or Moraxella catarrhalis can also result in hemoptysis. Patients with bronchiectasis (a permanent dilation of the airways with loss of mucosal integrity) are particularly prone to hemoptysis due to chronic inflammation and anatomic abnormalities that bring the bronchial arteries closer to the mucosal surface. One common presentation of patients with advanced cystic fibrosis—the prototypical bronchiectatic lung disease—is hemoptysis, which can be life-threatening.

1	Pneumonias of any sort can cause hemoptysis. Tuberculous infection, which can lead to bronchiectasis or cavitary pneumonia, is a very common cause of hemoptysis worldwide. Patients may present with a chronic cough productive of blood-streaked sputum or with larger-volume bleeding. Rasmussen’s aneurysm (the dilation of a pulmonary artery in a cavity formed by previous tuberculous infection) remains a source of massive, life-threatening hemoptysis in the developing world. Community-acquired pneumonia and lung abscess can also result in bleeding. Once again, if the infection results in cavitation, there is a greater likelihood of bleeding due to erosion into blood vessels. Infections with Staphylococcus aureus and gram-negative rods (e.g., Klebsiella pneumoniae) are especially likely to cause necrotizing lung infections and thus to be associated with hemoptysis.

1	While not common in North America, pulmonary paragoni miasis (i.e., infection with the lung fluke Paragonimus wester mani) often presents as fever, cough, and hemoptysis. This infection is a public health issue in Southeast Asia and China and is frequently confused with active tuberculosis, in which the clinical picture can be similar. Paragonimiasis should be considered in recent immigrants from endemic areas who have new or recurrent hemoptysis. In addition, pulmonary paragonimiasis has been reported secondary to ingestion of crayfish or small crabs in the United States. Other causes of airway irritation resulting in hemoptysis include inhalation of toxic chemicals, thermal injury, and direct trauma from suctioning of the airways (particularly in intubated patients). All of these etiologies should be considered in light of the individual patient’s history and exposures.

1	Perhaps the most feared cause of hemoptysis is bronchogenic lung cancer, although hemoptysis is a presenting symptom in only ∼10% of patients. Cancers arising in the proximal airways are much more likely to cause hemoptysis, but any malignancy in the chest can do so. Because both squamous cell carcinomas and small-cell carcinomas are 246 more commonly in or adjacent to the proximal airways, and large at presentation, they are more often a cause of hemoptysis. These cancers can present with large-volume and life-threatening hemoptysis because of erosion into the hilar vessels. Carcinoid tumors, which are found almost exclusively as endobronchial lesions with friable mucosa, can also present with hemoptysis. In addition to cancers arising in the lung, metastatic disease in the pulmonary parenchyma can bleed. Malignancies that commonly metastasize to the lungs include renal cell, breast, colon, testicular, and thyroid cancers as well as melanoma. While hemoptysis is not a common

1	parenchyma can bleed. Malignancies that commonly metastasize to the lungs include renal cell, breast, colon, testicular, and thyroid cancers as well as melanoma. While hemoptysis is not a common manifestation of pulmonary metastases, the combination of multiple pulmonary nodules and hemoptysis should raise suspicion of this etiology. Finally, disease of the pulmonary vasculature can cause hemoptysis. Perhaps most frequently, congestive heart failure with transmission of elevated left atrial pressures can lead to rupture of small alveolar capillaries. These patients rarely present with bright red blood but more commonly have pink, frothy sputum or blood-tinged secretions. Patients with a focal jet of mitral regurgitation can present with an upper-lobe opacity on chest radiography together with hemoptysis. This finding is thought to be due to focal increases in pulmonary capillary pressure due to the regurgitant jet. Pulmonary arteriovenous malformations are prone to bleeding. Pulmonary

1	hemoptysis. This finding is thought to be due to focal increases in pulmonary capillary pressure due to the regurgitant jet. Pulmonary arteriovenous malformations are prone to bleeding. Pulmonary embolism can also lead to the development of hemoptysis, which is generally associated with pulmonary infarction. Pulmonary arterial hypertension from other causes rarely results in hemoptysis.

1	As with most signs of possible illness, the initial step in the evaluation of hemoptysis is a thorough history and physical examination (Fig. 48-2). As already mentioned, initial questioning should focus on ascertaining whether the bleeding is truly from the respiratory tract and not the nasopharynx or gastrointestinal tract; bleeding from the latter sources requires different approaches to evaluation and treatment. History and Physical Examination The specific characteristics of hemoptysis may be helpful in determining an etiology, such as whether the expectorated material consists of blood-tinged, purulent secretions; pink, frothy sputum; or pure blood. Information on specific triggers

1	PART 2 Cardinal Manifestations and Presentation of Diseases of the bleeding (e.g., recent inhalation exposures) as well as any previous episodes of hemoptysis should be elicited during history-taking. Monthly hemoptysis in a woman suggests catamenial hemoptysis from pulmonary endometriosis. Moreover, the volume of blood expectorated is important not only in determining the cause but also in gauging the urgency for further diagnostic and therapeutic maneuvers. Patients rarely exsanguinate from hemoptysis but can effectively “drown” in aspirated blood. Large-volume hemoptysis, referred to as massive hemoptysis, is variably defined as hemoptysis of >200–600 mL in 24 h. Massive hemoptysis should be considered a medical emergency. All patients should be asked about current or former cigarette smoking; this behavior predisposes to chronic bronchitis and increases the likelihood of bronchogenic cancer. Practitioners should inquire about symptoms and signs suggestive of respiratory tract

1	smoking; this behavior predisposes to chronic bronchitis and increases the likelihood of bronchogenic cancer. Practitioners should inquire about symptoms and signs suggestive of respiratory tract infection (including fever, chills, and dyspnea), recent inhalation exposures, recent use of illicit substances, and risk factors for venous thromboembolism.

1	A medical history of malignancy or treatment thereof, rheumatologic disease, vascular disease, or underlying lung disease (e.g., bronchiectasis) may be relevant to the cause of hemoptysis. Because many causes of DAH can be part of a pulmonary-renal syndrome, specific inquiry into a history of renal insufficiency is important.

1	The physical examination begins with an assessment of vital signs and oxygen saturation to gauge whether there is evidence of life-threatening bleeding. Tachycardia, hypotension, and decreased oxygen saturation mandate a more expedited evaluation of hemoptysis. A specific focus on respiratory and cardiac examinations is important; these examinations should include inspection of the nares, auscultation of the lungs and heart, assessment of the lower extremities for symmetric or asymmetric edema, and evaluation for jugular venous distention. Clubbing of the digits may suggest underlying lung diseases such as bronchogenic carcinoma or bronchiectasis, which predispose to hemoptysis. Similarly, mucocutaneous telangiectasias should raise the specter of pulmonary arterial-venous malformations.

1	Diagnostic Evaluation For most patients, the next step in evaluation of hemoptysis should be a standard chest radiograph. If a source of bleeding is not identified on plain film, CT of the chest should be performed. CT allows better delineation of bronchiectasis, alveolar filling, cavitary infiltrates, and masses than does chest radiograph. The practitioner should consider a CT protocol to assess for pulmonary embolism if the history or examination suggests venous thromboembolism as a cause of bleeding.

1	Quantify amount of bleeding History and physical exam Patient with hemoptysis Mild Moderate Massive Rule out other sources: • Oropharynx • Gastrointestinal tract No risk factors* Risk factors* or recurrent bleeding Treat underlying disease (usually infection) CT scan if unrevealing, bronchoscopy Bleeding continues Treat underlying disease CT scan Bronchoscopy CXR, CBC, coagulation studies, UA, creatinine Secure airway Treat underlying disease Persistent bleeding *Risk Factors: smoking, age >40 Bleeding stops Embolization or resection FIguRE 48-2 Decision tree for evaluation of hemoptysis. CBC, complete blood count; CT, computed tomography; CXR, chest x-ray; UA, urinalysis.

1	Laboratory studies should include a complete blood count to assess both the hematocrit and the platelet count as well as coagulation studies. Renal function should be evaluated and urinalysis conducted because of the possibility of pulmonary-renal syndromes presenting with hemoptysis. The documentation of acute renal insufficiency or the detection of red blood cells or their casts on urinalysis should elevate suspicion of small-vessel vasculitis, and studies such as antineutrophil cytoplasmic antibody, antiglomerular basement membrane antibody, and antinuclear antibody should be considered. If a patient is producing sputum, Gram’s and acid-fast staining as well as culture should be undertaken. If all of these studies are unrevealing, bronchoscopy should be considered. In any patient with a history of cigarette smoking, airway inspection should be part of the evaluation of new-onset hemoptysis as endobronchial lesions are not reliably visualized on CT.

1	For the most part, the treatment of hemoptysis varies with its etiology. However, large-volume, life-threatening hemoptysis generally requires immediate intervention regardless of the cause. The first step is to establish a patent airway, usually by endotracheal intubation and subsequent mechanical ventilation. As large-volume hemoptysis usually arises from an airway lesion, it is ideal to identify the site of bleeding by either chest imaging or bronchoscopy (more commonly rigid rather than flexible). The goals are then to isolate the bleeding to one lung and not to allow the preserved airspaces in the other lung to be filled with blood so that gas exchange is further impaired. Patients should be placed with the bleeding lung in a dependent position (i.e., bleeding-side down), and, if possible, dual-lumen endotracheal tubes or an airway blocker should be placed in the proximal airway of the bleeding lung. These interventions generally require the assistance of anesthesiologists,

1	possible, dual-lumen endotracheal tubes or an airway blocker should be placed in the proximal airway of the bleeding lung. These interventions generally require the assistance of anesthesiologists, interventional pulmonologists, or thoracic surgeons.

1	If the bleeding does not stop with treatment of the underlying cause and the passage of time, severe hemoptysis from bronchial arteries can be treated with angiographic embolization of the responsible bronchial artery. This intervention should be entertained only in the most severe and life-threatening cases of hemoptysis because of the risk of unintentional spinal-artery embolization and consequent paraplegia. Endobronchial lesions can be treated with a variety of bronchoscopically directed interventions, including cauterization and laser therapy. In extreme circumstances, surgical resection of the affected region of the lung is considered. Most cases of hemoptysis resolve with treatment of the infection or inflammatory process or with removal of the offending stimulus.

1	products from them. Proper maintenance of this function depends not only on intact cardiovascular and respiratory systems, but also on an adequate number of red blood cells and hemoglobin and a supply of inspired gas containing adequate O2. Decreased O2 availability to cells results in an inhibition of oxidative phosphorylation and increased anaerobic glycolysis. This switch from aerobic to anaerobic metabolism, the Pasteur effect, maintains some, albeit reduced, adenosine 5′-triphosphate (ATP) production. In severe hypoxia, when ATP production is inadequate to meet the energy requirements of ionic and osmotic equilibrium, cell membrane depolarization leads to uncontrolled Ca2+ influx and activation of Ca2+-dependent phospholipases and proteases. These events, in turn, cause cell swelling, activation of apoptotic pathways, and, ultimately, cell death.

1	The adaptations to hypoxia are mediated, in part, by the upregulation of genes encoding a variety of proteins, including glycolytic enzymes, such as phosphoglycerate kinase and phosphofructokinase, as well as the glucose transporters Glut-1 and Glut-2; and by growth factors, such as vascular endothelial growth factor (VEGF) and erythropoietin, which enhance erythrocyte production. The hypoxia-induced increase in expression of these key proteins is governed by the hypoxia-sensitive transcription factor, hypoxia-inducible factor-1 (HIF-1).

1	During hypoxia, systemic arterioles dilate, at least in part, by opening of KATP channels in vascular smooth-muscle cells due to the hypoxia-induced reduction in ATP concentration. By contrast, in pulmonary vascular smooth-muscle cells, inhibition of K+ channels causes depolarization which, in turn, activates voltage-gated Ca2+ channels raising the cytosolic [Ca2+] and causing smooth-muscle cell contraction. Hypoxia-induced pulmonary arterial constriction shunts blood away from poorly ventilated portions toward better ventilated portions of the lung; however, it also increases pulmonary vascular resistance and right ventricular afterload.

1	Effects on the Central Nervous System Changes in the central nervous system (CNS), particularly the higher centers, are especially important consequences of hypoxia. Acute hypoxia causes impaired judgment, motor incoordination, and a clinical picture resembling acute alcohol intoxication. High-altitude illness is characterized by headache secondary to cerebral vasodilation, gastrointestinal symptoms, dizziness, insomnia, fatigue, or somnolence. Pulmonary arterial and sometimes venous constriction causes capillary leakage and high-altitude pulmonary edema (HAPE) (Chap. 47e), which intensifies hypoxia, further promoting vasoconstriction. Rarely, high-altitude cerebral edema (HACE) develops, which is manifest by severe headache and papilledema and can cause coma. As hypoxia becomes more severe, the regulatory centers of the brainstem are affected, and death usually results from respiratory failure.

1	Effects on the Cardiovascular System Acute hypoxia stimulates the chemoreceptor reflex arc to induce venoconstriction and systemic arterial vasodilation. These acute changes are accompanied by transiently increased myocardial contractility, which is followed by depressed myocardial contractility with prolonged hypoxia.

1	CAuSES OF HYPOXIA Respiratory Hypoxia When hypoxia occurs from respiratory failure, Pao2 declines, and when respiratory failure is persistent, the hemoglobin-oxygen (Hb-O2) dissociation curve (see Fig. 127-2) is displaced to the right, with greater quantities of O2 released at any level of tissue Po2. Arterial hypoxemia, i.e., a reduction of O2 saturation of arterial blood (Sao2), and consequent cyanosis are likely to be more marked when such depression of Pao2 results from pulmonary disease than when the depression occurs as the result of a decline in the fraction of oxygen in inspired air (Fio2). In this latter situation, Paco2 falls secondary to anoxia-induced hyperventilation and the Hb-O2 dissociation curve is displaced to the left, limiting the decline in Sao2 at any level of Pao2.

1	The most common cause of respiratory hypoxia is ventilation-perfusion mismatch resulting from perfusion of poorly ventilated alveoli. Respiratory hypoxemia may also be caused by hypoventilation, in which case it is associated with an elevation of Paco2 (Chap. 306e). These two forms of respiratory hypoxia are usually correctable by 248 inspiring 100% O2 for several minutes. A third cause of respiratory hypoxia is shunting of blood across the lung from the pulmonary arterial to the venous bed (intrapulmonary right-to-left shunting) by perfusion of nonventilated portions of the lung, as in pulmonary atelectasis or through pulmonary arteriovenous connections. The low Pao2 in this situation is only partially corrected by an Fio2 of 100%.

1	Hypoxia Secondary to High Altitude As one ascends rapidly to 3000 m (~10,000 ft), the reduction of the O2 content of inspired air (Fio2) leads to a decrease in alveolar Po2 to approximately 60 mmHg, and a condition termed high-altitude illness develops (see above). At higher altitudes, arterial saturation declines rapidly and symptoms become more serious; and at 5000 m, unacclimated individuals usually cease to be able to function normally owing to the changes in CNS function described above. Hypoxia Secondary to Right-to-Left Extrapulmonary Shunting From a physiologic viewpoint, this cause of hypoxia resembles intrapulmonary right-to-left shunting but is caused by congenital cardiac malformations, such as tetralogy of Fallot, transposition of the great arteries, and Eisenmenger’s syndrome (Chap. 282). As in pulmonary right-toleft shunting, the Pao2 cannot be restored to normal with inspiration of 100% O2.

1	Anemic Hypoxia A reduction in hemoglobin concentration of the blood is accompanied by a corresponding decline in the O2-carrying capacity of the blood. Although the Pao2 is normal in anemic hypoxia, the absolute quantity of O2 transported per unit volume of blood is diminished. As the anemic blood passes through the capillaries and the usual quantity of O2 is removed from it, the Po2 and saturation in the venous blood decline to a greater extent than normal. Carbon Monoxide (CO) Intoxication (See also Chap. 472e) Hemoglobin that binds with CO (carboxy-hemoglobin, COHb) is unavailable for O2 transport. In addition, the presence of COHb shifts the Hb-O2 dissociation curve to the left (see Fig. 127-2) so that O2 is unloaded only at lower tensions, further contributing to tissue hypoxia.

1	Circulatory Hypoxia As in anemic hypoxia, the Pao2 is usually normal, but venous and tissue Po2 values are reduced as a consequence of reduced tissue perfusion and greater tissue O2 extraction. This pathophysiology leads to an increased arterial-mixed venous O2 difference (a-v-O2 difference), or gradient. Generalized circulatory hypoxia occurs in heart failure (Chap. 279) and in most forms of shock (Chap. 324).

1	Specific Organ Hypoxia Localized circulatory hypoxia may occur as a result of decreased perfusion secondary to arterial obstruction, as in localized atherosclerosis in any vascular bed, or as a consequence of vasoconstriction, as observed in Raynaud’s phenomenon (Chap. 302). Localized hypoxia may also result from venous obstruction and the resultant expansion of interstitial fluid causing arteriolar compression and, thereby, reduction of arterial inflow. Edema, which increases the distance through which O2 must diffuse before it reaches cells, can also cause localized hypoxia. In an attempt to maintain adequate perfusion to more vital organs in patients with reduced cardiac output secondary to heart failure or hypovolemic shock, vasoconstriction may reduce perfusion in the limbs and skin, causing hypoxia of these regions.

1	Increased O2 Requirements If the O2 consumption of tissues is elevated without a corresponding increase in perfusion, tissue hypoxia ensues and the Po2 in venous blood declines. Ordinarily, the clinical picture of patients with hypoxia due to an elevated metabolic rate, as in fever or thyrotoxicosis, is quite different from that in other types of hypoxia: the skin is warm and flushed owing to increased cutaneous blood flow that dissipates the excessive heat produced, and cyanosis is usually absent. Exercise is a classic example of increased tissue O2 requirements. These increased demands are normally met by several mechanisms operating simultaneously: (1) increase in the cardiac output and ventilation and, thus, O2 delivery to the tissues; (2) a preferential shift in blood flow to the exercising muscles by changing vascular resistances

1	PART 2 Cardinal Manifestations and Presentation of Diseases in the circulatory beds of exercising tissues, directly and/or reflexly; (3) an increase in O2 extraction from the delivered blood and a widening of the arteriovenous O2 difference; and (4) a reduction in the pH of the tissues and capillary blood, shifting the Hb-O2 curve to the right (see Fig. 127-2), and unloading more O2 from hemoglobin. If the capacity of these mechanisms is exceeded, then hypoxia, especially of the exercising muscles, will result. Improper Oxygen utilization Cyanide (Chap. 473e) and several other similarly acting poisons cause cellular hypoxia. The tissues are unable to use O2, and, as a consequence, the venous blood tends to have a high O2 tension. This condition has been termed histotoxic hypoxia.

1	An important component of the respiratory response to hypoxia originates in special chemosensitive cells in the carotid and aortic bodies and in the respiratory center in the brainstem. The stimulation of these cells by hypoxia increases ventilation, with a loss of CO2, and can lead to respiratory alkalosis. When combined with the metabolic acidosis resulting from the production of lactic acid, the serum bicarbonate level declines (Chap. 66). With the reduction of Pao2, cerebrovascular resistance decreases and cerebral blood flow increases in an attempt to maintain O2 delivery to the brain. However, when the reduction of Pao2 is accompanied by hyperventilation and a reduction of Paco2, cerebrovascular resistance rises, cerebral blood flow falls, and tissue hypoxia intensifies.

1	The diffuse, systemic vasodilation that occurs in generalized hypoxia increases the cardiac output. In patients with underlying heart disease, the requirements of peripheral tissues for an increase of cardiac output with hypoxia may precipitate congestive heart failure. In patients with ischemic heart disease, a reduced Pao2 may intensify myocardial ischemia and further impair left ventricular function.

1	One of the important compensatory mechanisms for chronic hypoxia is an increase in the hemoglobin concentration and in the number of red blood cells in the circulating blood, i.e., the development of polycythemia secondary to erythropoietin production (Chap. 131). In persons with chronic hypoxemia secondary to prolonged residence at a high altitude (>13,000 ft, 4200 m), a condition termed chronic mountain sickness develops. This disorder is characterized by a blunted respiratory drive, reduced ventilation, erythrocytosis, cyanosis, weakness, right ventricular enlargement secondary to pulmonary hypertension, and even stupor.

1	Cyanosis refers to a bluish color of the skin and mucous membranes resulting from an increased quantity of reduced hemoglobin (i.e., deoxygenated hemoglobin) or of hemoglobin derivatives (e.g., met-hemoglobin or sulfhemoglobin) in the small blood vessels of those tissues. It is usually most marked in the lips, nail beds, ears, and malar eminences. Cyanosis, especially if developed recently, is more commonly detected by a family member than the patient. The florid skin characteristic of polycythemia vera (Chap. 131) must be distinguished from the true cyanosis discussed here. A cherry-colored flush, rather than cyanosis, is caused by COHb (Chap. 473e).

1	The degree of cyanosis is modified by the color of the cutaneous pigment and the thickness of the skin, as well as by the state of the cutaneous capillaries. The accurate clinical detection of the presence and degree of cyanosis is difficult, as proved by oximetric studies. In some instances, central cyanosis can be detected reliably when the Sao2 has fallen to 85%; in others, particularly in dark-skinned persons, it may not be detected until it has declined to 75%. In the latter case, examination of the mucous membranes in the oral cavity and the conjunctivae rather than examination of the skin is more helpful in the detection of cyanosis.

1	The increase in the quantity of reduced hemoglobin in the mucocutaneous vessels that produces cyanosis may be brought about either by an increase in the quantity of venous blood as a result of dilation of the venules (including precapillary venules) or by a reduction in the Sao2 in the capillary blood. In general, cyanosis becomes apparent when the concentration of reduced hemoglobin in capillary blood exceeds 40 g/L (4 g/dL).

1	It is the absolute, rather than the relative, quantity of reduced hemoglobin that is important in producing cyanosis. Thus, in a patient with severe anemia, the relative quantity of reduced hemoglobin in the venous blood may be very large when considered in relation to the total quantity of hemoglobin in the blood. However, since the concentration of the latter is markedly reduced, the absolute quantity of reduced hemoglobin may still be low, and, therefore, patients with severe anemia and even marked arterial desaturation may not display cyanosis. Conversely, the higher the total hemoglobin content, the greater the tendency toward cyanosis; thus, patients with marked polycythemia tend to be cyanotic at higher levels of Sao2 than patients with normal hematocrit values. Likewise, local passive congestion, which causes an increase in the total quantity of reduced hemoglobin in the vessels in a given area, may cause cyanosis. Cyanosis is also observed when nonfunctional hemoglobin, such

1	congestion, which causes an increase in the total quantity of reduced hemoglobin in the vessels in a given area, may cause cyanosis. Cyanosis is also observed when nonfunctional hemoglobin, such as methemoglobin (consequential or acquired) or sulfhemoglobin (Chap. 127), is present in blood.

1	Cyanosis may be subdivided into central and peripheral types. In central cyanosis, the Sao2 is reduced or an abnormal hemoglobin derivative is present, and the mucous membranes and skin are both affected. Peripheral cyanosis is due to a slowing of blood flow and abnormally great extraction of O2 from normally saturated arterial blood; it results from vasoconstriction and diminished peripheral blood flow, such as occurs in cold exposure, shock, congestive failure, and peripheral vascular disease. Often in these conditions, the mucous membranes of the oral cavity or those beneath the tongue may be spared. Clinical differentiation between central and peripheral cyanosis may not always be simple, and in conditions such as cardiogenic shock with pulmonary edema, there may be a mixture of both types.

1	DIFFERENTIAL DIAgNOSIS Central Cyanosis (Table 49-1) Decreased Sao2 results from a marked reduction in the Pao2. This reduction may be brought about by a decline in the Fio2 without sufficient compensatory alveolar hyperventilation to maintain alveolar Po2. Cyanosis usually becomes manifest in an ascent to an altitude of 4000 m (13,000 ft). Seriously impaired pulmonary function, through perfusion of unventilated or poorly ventilated areas of the lung or alveolar hypoventilation, is a common cause of central cyanosis (Chap. 306e). Inhomogeneity in pulmonary ventilation and perfusion (perfusion of hypoventilated alveoli) Impaired oxygen diffusion Anatomic shunts Certain types of congenital heart disease Pulmonary arteriovenous fistulas Multiple small intrapulmonary shunts Hemoglobin with low affinity for oxygen Hemoglobin abnormalities Methemoglobinemia—hereditary, acquired Sulfhemoglobinemia—acquired Carboxyhemoglobinemia (not true cyanosis)

1	Hemoglobin with low affinity for oxygen Hemoglobin abnormalities Methemoglobinemia—hereditary, acquired Sulfhemoglobinemia—acquired Carboxyhemoglobinemia (not true cyanosis) Reduced cardiac output Cold exposure Redistribution of blood flow from extremities Arterial obstruction Venous obstruction This condition may occur acutely, as in extensive pneumonia or 249 pulmonary edema, or chronically, with chronic pulmonary diseases (e.g., emphysema). In the latter situation, secondary polycythemia is generally present and clubbing of the fingers (see below) may occur. Another cause of reduced Sao2 is shunting of systemic venous blood into the arterial circuit. Certain forms of congenital heart disease are associated with cyanosis on this basis (see above and Chap. 282).

1	Pulmonary arteriovenous fistulae may be congenital or acquired, solitary or multiple, microscopic or massive. The severity of cyanosis produced by these fistulae depends on their size and number. They occur with some frequency in hereditary hemorrhagic telangiectasia. Sao2 reduction and cyanosis may also occur in some patients with cirrhosis, presumably as a consequence of pulmonary arteriovenous fistulae or portal vein–pulmonary vein anastomoses. In patients with cardiac or pulmonary right-to-left shunts, the presence and severity of cyanosis depend on the size of the shunt relative to the systemic flow as well as on the Hb-O2 saturation of the venous blood. With increased extraction of O2 from the blood by the exercising muscles, the venous blood returning to the right side of the heart is more unsaturated than at rest, and shunting of this blood intensifies the cyanosis. Secondary polycythemia occurs frequently in patients in this setting and contributes to the cyanosis.

1	Cyanosis can be caused by small quantities of circulating methemoglobin (Hb Fe3+) and by even smaller quantities of sulfhemoglobin (Chap. 127); both of these hemoglobin derivatives impair oxygen delivery to the tissues. Although they are uncommon causes of cyanosis, these abnormal hemoglobin species should be sought by spectroscopy when cyanosis is not readily explained by malfunction of the circulatory or respiratory systems. Generally, digital clubbing does not occur with them. Peripheral Cyanosis Probably the most common cause of peripheral cyanosis is the normal vasoconstriction resulting from exposure to cold air or water. When cardiac output is reduced, cutaneous vasoconstriction occurs as a compensatory mechanism so that blood is diverted from the skin to more vital areas such as the CNS and heart, and cyanosis of the extremities may result even though the arterial blood is normally saturated.

1	Arterial obstruction to an extremity, as with an embolus, or arteriolar constriction, as in cold-induced vasospasm (Raynaud’s phenomenon) (Chap. 302), generally results in pallor and coldness, and there may be associated cyanosis. Venous obstruction, as in thrombophlebitis or deep venous thrombosis, dilates the subpapillary venous plexuses and thereby intensifies cyanosis. APPROACH TO THE PATIENT: Certain features are important in arriving at the cause of cyanosis: 1. It is important to ascertain the time of onset of cyanosis. Cyanosis present since birth or infancy is usually due to congenital heart disease. 2. Central and peripheral cyanosis must be differentiated. Evidence of disorders of the respiratory or cardiovascular systems is helpful. Massage or gentle warming of a cyanotic extremity will increase peripheral blood flow and abolish peripheral, but not central, cyanosis. 3.

1	3. The presence or absence of clubbing of the digits (see below) should be ascertained. The combination of cyanosis and clubbing is frequent in patients with congenital heart disease and right-to-left shunting and is seen occasionally in patients with pulmonary disease, such as lung abscess or pulmonary arteriovenous fistulae. In contrast, peripheral cyanosis or acutely developing central cyanosis is not associated with clubbed digits. 4. Pao2 and Sao2 should be determined, and, in patients with cyanosis in whom the mechanism is obscure, spectroscopic examination of the blood should be performed to look for abnormal types of hemoglobin (critical in the differential diagnosis of cyanosis).

1	The selective bulbous enlargement of the distal segments of the fingers and toes due to proliferation of connective tissue, particularly on the dorsal surface, is termed clubbing; there is also increased sponginess of the soft tissue at the base of the clubbed nail. Clubbing may be hereditary, idiopathic, or acquired and associated with a variety of disorders, including cyanotic congenital heart disease (see above), infective endocarditis, and a variety of pulmonary conditions (among them primary and metastatic lung cancer, bronchiectasis, asbestosis, sarcoidosis, lung abscess, cystic fibrosis, tuberculosis, and mesothelioma), as well as with some gastrointestinal diseases (including inflammatory bowel disease and hepatic cirrhosis). In some instances, it is occupational, e.g., in jackhammer operators.

1	Clubbing in patients with primary and metastatic lung cancer, mesothelioma, bronchiectasis, or hepatic cirrhosis may be associated with hypertrophic osteoarthropathy. In this condition, the subperiosteal formation of new bone in the distal diaphyses of the long bones of the extremities causes pain and symmetric arthritis-like changes in the shoulders, knees, ankles, wrists, and elbows. The diagnosis of hypertrophic osteoarthropathy may be confirmed by bone radiograph or magnetic resonance imaging (MRI). Although the mechanism of clubbing is unclear, it appears to be secondary to humoral substances that cause dilation of the vessels of the distal digits as well as growth factors released from platelet precursors in the digital circulation. In certain circumstances, clubbing is reversible, such as following lung transplantation for cystic fibrosis. Eugene Braunwald, Joseph Loscalzo PART 2 Cardinal Manifestations and Presentation of Diseases

1	About one-third of total-body water is confined to the extracellular space. Approximately 75% of the latter is interstitial fluid, and the remainder is the plasma. The forces that regulate the disposition of fluid between these two components of the extracellular compartment frequently are referred to as the Starling forces. The hydrostatic pressure within the capillaries and the colloid oncotic pressure in the interstitial fluid tend to promote movement of fluid from the vascular to the extravascular space. By contrast, the colloid oncotic pressure contributed by plasma proteins and the hydrostatic pressure within the interstitial fluid promote the movement of fluid into the vascular compartment. As a consequence, there is movement of water and diffusible solutes from the vascular space at the arteriolar end of the capillaries. Fluid is returned from the interstitial space into the vascular system at the venous end of the capillaries and by way of the lymphatics. These movements are

1	at the arteriolar end of the capillaries. Fluid is returned from the interstitial space into the vascular system at the venous end of the capillaries and by way of the lymphatics. These movements are usually balanced so that there is a steady state in the sizes of the intravascular and interstitial compartments, yet a large exchange between them occurs. However, if either the capillary hydrostatic pressure is increased and/or the oncotic pressure is reduced, a further net movement of fluid from intravascular to the interstitial spaces will take place.

1	Edema is defined as a clinically apparent increase in the interstitial fluid volume, which develops when Starling forces are altered so that there is increased flow of fluid from the vascular system into the interstitium. Edema due to an increase in capillary pressure may result from an elevation of venous pressure caused by obstruction to venous and/ or lymphatic drainage. An increase in capillary pressure may be generalized, as occurs in heart failure, or it may be localized to one extremity when venous pressure is elevated due to unilateral thrombophlebitis (see below). The Starling forces also may be imbalanced when the colloid oncotic pressure of the plasma is reduced owing to any factor that may induce hypoalbuminemia, as when large quantities of protein are lost in the urine such as in the nephrotic syndrome (see below), or when synthesis is reduced in a severe catabolic state.

1	Edema may also result from damage to the capillary endothelium, which increases its permeability and permits the transfer of proteins into the interstitial compartment. Injury to the capillary wall can result from drugs (see below), viral or bacterial agents, and thermal or mechanical trauma. Increased capillary permeability also may be a consequence of a hypersensitivity reaction and of immune injury. Damage to the capillary endothelium is presumably responsible for inflammatory edema, which is usually nonpitting, localized, and accompanied by other signs of inflammation—i.e., erythema, heat, and tenderness.

1	In many forms of edema, despite the increase in extracellular fluid volume, the effective arterial blood volume, a parameter that represents the filling of the arterial tree and that effectively perfuses the tissues, is reduced. Underfilling of the arterial tree may be caused by a reduction of cardiac output and/or systemic vascular resistance, by pooling of blood in the splanchnic veins (as in cirrhosis), and by hypoalbuminemia (Fig. 50-1A). As a consequence of underfilling, a series of physiologic responses designed to restore the effective arterial volume to normal are set into motion. A key element of these responses is the renal retention of sodium and, therefore, water, thereby restoring effective arterial volume, but sometimes also leading to or intensifying edema.

1	The diminished renal blood flow characteristic of states in which the effective arterial blood volume is reduced is translated by the renal juxtaglomerular cells (specialized myoepithelial cells surrounding the afferent arteriole) into a signal for increased renin release. Renin is an enzyme with a molecular mass of about 40,000 Da that acts on its substrate, angiotensinogen, an α2-globulin synthesized by the liver, to release angiotensin I, a decapeptide, which in turn is converted to angiotensin II (AII), an octapeptide. AII has generalized vasoconstrictor properties, particularly on the renal efferent arterioles. This action reduces the hydrostatic pressure in the peritubular capillaries, whereas the increased filtration fraction raises the colloid osmotic pressure in these vessels, thereby enhancing salt and water reabsorption in the proximal tubule as well as in the ascending limb of the loop of Henle.

1	The renin-angiotensin-aldosterone system (RAAS) operates as both a hormonal and paracrine system. Its activation causes sodium and water retention and thereby contributes to edema formation. Blockade of the conversion of angiotensin I to AII and blockade of the AII receptor enhance sodium and water excretion and reduce many forms of edema. AII that enters the systemic circulation stimulates the production of aldosterone by the zona glomerulosa of the adrenal cortex. Aldosterone in turn enhances sodium reabsorption (and potassium excretion) by the collecting tubule, further favoring edema formation. In patients with heart failure, not only is aldosterone secretion elevated but the biologic half-life of the hormone is prolonged secondary to the depression of hepatic blood flow, which reduces its hepatic catabolism and increases further the plasma level of the hormone. Blockade of the action of aldosterone by spironolactone or eplerenone (aldosterone antagonists) or by amiloride (a

1	its hepatic catabolism and increases further the plasma level of the hormone. Blockade of the action of aldosterone by spironolactone or eplerenone (aldosterone antagonists) or by amiloride (a blocker of epithelial sodium channels) often induces a moderate diuresis in edematous states.

1	(See also Chap. 404) The secretion of arginine vasopressin (AVP) occurs in response to increased intracellular osmolar concentration, and, by stimulating V2 receptors, AVP increases the reabsorption of free water in the distal tubules and collecting ducts of the kidneys, thereby increasing total-body water. Circulating AVP is elevated in many patients with heart failure secondary to a nonosmotic stimulus associated with decreased effective arterial volume and reduced compliance of the left atrium. Such patients fail to show the normal reduction of AVP with a reduction of osmolality, contributing to edema formation and hyponatremia.

1	Nonosmotic vasopressin stimulation Activation of RAAS ˜Extracellular fluid volume ˜Cardiac output Effective arterial volume Activation of ventricular and arterial receptors Restoration of effective arterial volume SNS stimulation Renal H2O retention Renal Na+ retention Low output heart failure, Pericardial tamponade Constructive pericarditis ˜Oncotic pressure and/or °capillary permeability °Systemic and renal arterial vascular resistance Sepsis Cirrhosis Activation of RAAS High-output cardiac failure Arteriovenous fistula Arterial vasodilators ˜Systemic vascular resistance Effective arterial volume Maintenance of arterial circulatory integrity Activation of arterial baroreceptors Nonosmotic AVP stimulation °Cardiac output Renal H2O retention SNS stimulation Pregnancy Renal Na+ retention °Systemic arterial, vascular, and renal resistance

1	FIguRE 50-1 Clinical conditions in which a decrease in cardiac output (A) and systemic vascular resistance (B) cause arterial underfilling with resulting neurohumoral activation and renal sodium and water retention. In addition to activating the neurohumoral axis, adrenergic stimulation causes renal vasoconstriction and enhances sodium and fluid transport by the proximal tubule epithelium. RAAS, renin-angiotensin aldosterone system; SNS, sympathetic nervous system. (Modified from RW Schrier: Ann Intern Med 113:155, 1990.) This potent peptide vasoconstrictor is released by endothelial cells. Its concentration in the plasma is elevated in patients with severe heart failure and contributes to renal vasoconstriction, sodium retention, and edema.

1	Atrial distention causes release into the circulation of atrial natriuretic peptide (ANP), a polypeptide; a high-molecular-weight precursor of ANP is stored in secretory granules within atrial myocytes. The closely related brain natriuretic peptide (pre-prohor-251 mone BNP) is stored primarily in ventricular myocytes and is released when ventricular diastolic pressure rises. Released ANP and BNP (which is derived from its precursor) bind to the natriuretic receptor-A, which causes: (1) excretion of sodium and water by augmenting glomerular filtration rate, inhibiting sodium reabsorption in the proximal tubule, and inhibiting release of renin and aldosterone; and (2) dilation of arterioles and venules by antagonizing the vasoconstrictor actions of AII, AVP, and sympathetic stimulation. Thus, elevated levels of natriuretic peptides have the capacity to oppose sodium retention in hypervolemic and edematous states.

1	Although circulating levels of ANP and BNP are elevated in heart failure and in cirrhosis with ascites, the natriuretic peptides are not sufficiently potent to prevent edema formation. Indeed, in edematous states, resistance to the actions of natriuretic peptides may be increased, further reducing their effectiveness. Further discussion of the control of sodium and water balance is found in Chap. 64e. A weight gain of several kilograms usually precedes overt manifestations of generalized edema, and a similar weight loss from diuresis can be induced in a slightly edematous patient before “dry weight” is achieved. Anasarca refers to gross, generalized edema. Ascites (Chap. 59) and hydrothorax refer to accumulation of excess fluid in the peritoneal and pleural cavities, respectively, and are considered special forms of edema.

1	Edema is recognized by the persistence of an indentation of the skin after pressure; this is known as “pitting” edema. In its more subtle form, edema may be detected by noting that after the stethoscope is removed from the chest wall, the rim of the bell leaves an indentation on the skin of the chest for a few minutes. Edema may be present when the ring on a finger fits more snugly than in the past or when a patient complains of difficulty putting on shoes, particularly in the evening. Edema may also be recognized by puffiness of the face, which is most readily apparent in the periorbital areas. The differences among the major causes of generalized edema are shown in Table 50-1. Cardiac, renal, hepatic, or nutritional disorders are responsible for a majority of patients with generalized edema. Consequently, the differential diagnosis of generalized edema should be directed toward identifying or excluding these several conditions.

1	Heart Failure (See also Chap. 279) In heart failure, the impaired systolic emptying of the ventricle(s) and/or the impairment of ventricular relaxation promotes an accumulation of blood in the venous circulation at the expense of the effective arterial volume. In addition, the heightened tone of the sympathetic nervous system causes renal vasoconstriction and reduction of glomerular filtration. In mild heart failure, a small increment of total blood volume may repair the deficit of 252 TABLE 50-1 PRinCiPAL CAuSES of gEnERALizED EDEMA: HiSToRy, PHySiCAL ExAMinATion, AnD LABoRAToRy finDingS PART 2 Cardinal Manifestations and Presentation of Diseases Renal (CRF) Usually chronic: may be associated with uremic signs and symptoms, including decreased appetite, altered (metallic or fishy) taste, altered sleep pattern, difficulty concentrating, restless legs, or myoclonus; dyspnea can be present, but generally less prominent than in heart failure

1	Abbreviations: CRF, chronic renal failure; NS, nephrotic syndrome. Elevated jugular venous pressure, ventricular (S3) gallop; occasionally with displaced or dyskinetic apical pulse; peripheral cyanosis, cool extremities, small pulse pressure when severe Frequently associated with ascites; jugular venous pressure normal or low; blood pressure lower than in renal or cardiac disease; one or more additional signs of chronic liver disease (jaundice, palmar erythema, Dupuytren’s contracture, spider angiomata, male gynecomastia; asterixis and other signs of encephalopathy) may be present Elevated blood pressure; hypertensive retinopathy; nitrogenous fetor; pericardial friction rub in advanced cases with uremia If severe, reductions in serum albumin, cholesterol, other hepatic proteins (transferrin, fibrinogen); liver enzymes elevated, depending on the cause and acuity of liver injury; tendency toward hypokalemia, respiratory alkalosis; macrocytosis from folate deficiency

1	Elevation of serum creatinine and cystatin C; albuminuria; hyperkalemia, metabolic acidosis, hyperphosphatemia, hypocalcemia, anemia (usually normocytic) Proteinuria (≥3.5 g/d); hypoalbuminemia; hypercholesterolemia; microscopic hematuria Source: Modified from GM Chertow: Approach to the patient with edema, in Primary Cardiology, 2nd ed, E Braunwald, L Goldman (eds). Philadelphia, Saunders, 2003, pp 117–128. effective arterial volume through the operation of Starling’s law of the heart, in which an increase in ventricular diastolic volume promotes a more forceful contraction and may thereby maintain the cardiac output. However, if the cardiac disorder is more severe, sodium and water retention continue, and the increment in blood volume accumulates in the venous circulation, raising venous pressure and causing edema (Fig. 50-1).

1	The presence of heart disease, as manifested by cardiac enlargement and/or ventricular hypertrophy, together with evidence of cardiac failure, such as dyspnea, basilar rales, venous distention, and hepatomegaly, usually indicates that edema results from heart failure. Noninvasive tests such as echocardiography may be helpful in establishing the diagnosis of heart disease. The edema of heart failure typically occurs in the dependent portions of the body.

1	Edema of Renal Disease (See also Chap. 338) The edema that occurs during the acute phase of glomerulonephritis is characteristically associated with hematuria, proteinuria, and hypertension. Although some evidence supports the view that the fluid retention is due to increased capillary permeability, in most instances, the edema results from primary retention of sodium and water by the kidneys owing to renal insufficiency. This state differs from most forms of heart failure in that it is characterized by a normal (or sometimes even increased) cardiac output. Patients with edema due to acute renal failure commonly have arterial hypertension as well as pulmonary congestion on chest roentgenogram, often without considerable cardiac enlargement, but they may not develop orthopnea. Patients with chronic renal failure may also develop edema due to primary renal retention of sodium and water.

1	Nephrotic Syndrome and other Hypoalbuminemic States The primary alteration in the nephrotic syndrome is a diminished colloid oncotic pressure due to losses of large quantities (≥3.5 g/d) of protein into the urine. With severe hypoalbuminemia (<35 g/L) and the consequent reduced colloid osmotic pressure, the sodium and water that are retained cannot be restrained within the vascular compartment, and total and effective arterial blood volumes decline. This process initiates the edema-forming sequence of events described above, including activation of the RAAS. The nephrotic syndrome may occur during the course of a variety of kidney diseases, which include glomerulonephritis, diabetic glomerulosclerosis, and hypersensitivity reactions. The edema is diffuse, symmetric, and most prominent in the dependent areas; as a consequence, periorbital edema is most prominent in the morning.

1	Hepatic Cirrhosis (See also Chap. 365) This condition is characterized in part by hepatic venous outflow blockade, which in turn expands the splanchnic blood volume and increases hepatic lymph formation. Intrahepatic hypertension acts as a stimulus for renal sodium retention and causes a reduction of effective arterial blood volume. These alterations are frequently complicated by hypoalbuminemia secondary to reduced hepatic synthesis of albumin, as well as peripheral arterial vasodilation. These effects reduce the effective arterial blood volume further, leading to activation of the RAAS and renal sympathetic nerves and to release of AVP, endothelin, and other sodium-and water-retaining mechanisms (Fig. 50-1B). The concentration of circulating aldosterone often is elevated by the failure of the liver to metabolize this hormone. Initially, the excess interstitial fluid is localized preferentially proximal (upstream) to the congested portal venous system and obstructed hepatic

1	failure of the liver to metabolize this hormone. Initially, the excess interstitial fluid is localized preferentially proximal (upstream) to the congested portal venous system and obstructed hepatic lymphatics, i.e., in the peritoneal cavity (causing ascites, Chap. 59). In later stages, particularly when there is severe hypoalbuminemia, peripheral edema may develop. A sizable accumulation of ascitic fluid may increase intraabdominal pressure and impede venous return from the lower extremities and contribute to the accumulation of edema of the lower extremities.

1	The excess production of prostaglandins (PGE2 and PGI2) in cirrhosis attenuates renal sodium retention. When the synthesis of these substances is inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs), renal function may deteriorate, and this may increase sodium retention further. Drug-Induced Edema A large number of widely used drugs can cause edema (Table 50-2). Mechanisms include renal vasoconstriction (NSAIDs and cyclosporine), arteriolar dilation (vasodilators), augmented renal sodium reabsorption (steroid hormones), and capillary damage.

1	Edema of Nutritional Origin A diet grossly deficient in protein over a prolonged period may produce hypoproteinemia and edema. The latter may be intensified by the development of beriberi heart disease, which also is of nutritional origin, in which multiple peripheral arteriovenous fistulae result in reduced effective systemic perfusion and effective arterial blood volume, thereby enhancing edema formation (Chap. 96e) (Fig. 50-1B). Edema may actually become intensified OKT3 monoclonal antibody Source: Modified from Chertow GM: Approach to the patient with edema, in Primary Cardiology, 2nd ed, E Braunwald, L Goldman (eds). Philadelphia, Saunders, 2003, pp 117–128.

1	OKT3 monoclonal antibody Source: Modified from Chertow GM: Approach to the patient with edema, in Primary Cardiology, 2nd ed, E Braunwald, L Goldman (eds). Philadelphia, Saunders, 2003, pp 117–128. when famished subjects are first provided with an adequate diet. The ingestion of more food may increase the quantity of sodium ingested, which is then retained along with water. So-called refeeding edema also may be linked to increased release of insulin, which directly increases tubular sodium reabsorption. In addition to hypoalbuminemia, hypokalemia and caloric deficits may be involved in the edema of starvation.

1	In this condition, the hydrostatic pressure in the capillary bed upstream (proximal) of the obstruction increases so that an abnormal quantity of fluid is transferred from the vascular to the interstitial space. Since the alternative route (i.e., the lymphatic channels) also may be obstructed or maximally filled, an increased volume of interstitial fluid in the limb develops (i.e., there is trapping of fluid in the interstitium of the extremity). The displacement of large quantities of fluid into a limb may occur at the expense of the blood volume in the remainder of the body, thereby reducing effective arterial blood volume and leading to the retention of NaCl and H2O until the deficit in plasma volume has been corrected.

1	Localized edema due to venous or lymphatic obstruction may be caused by thrombophlebitis, chronic lymphangitis, resection of regional lymph nodes, and filariasis, among other causes. Lymphedema is particularly intractable because restriction of lymphatic flow results in increased protein concentration in the interstitial fluid, a circumstance that aggravates fluid retention. Other Causes of Edema These causes include hypothyroidism (myxedema) and hyperthyroidism (pretibial myxedema secondary to Graves’ disease), the edema in which is typically nonpitting and due to deposition of hyaluronic acid and, in Graves’ disease, lymphocytic infiltration and inflammation; exogenous hyperadrenocortism; pregnancy; and administration of estrogens and vasodilators, particularly dihydropyridines such as nifedipine.

1	The distribution of edema is an important guide to its cause. Edema associated with heart failure tends to be more extensive in the legs and to be accentuated in the evening, a feature also determined largely by posture. When patients with heart failure are confined to bed, edema 253 may be most prominent in the presacral region. Severe heart failure may cause ascites that may be distinguished from the ascites caused by hepatic cirrhosis by the jugular venous pressure, which is usually elevated in heart failure and normal in cirrhosis.

1	Edema resulting from hypoproteinemia, as occurs in the nephrotic syndrome, characteristically is generalized, but it is especially evident in the very soft tissues of the eyelids and face and tends to be most pronounced in the morning owing to the recumbent posture assumed during the night. Less common causes of facial edema include trichinosis, allergic reactions, and myxedema. Edema limited to one leg or to one or both arms is usually the result of venous and/or lymphatic obstruction. Unilateral paralysis reduces lymphatic and venous drainage on the affected side and may also be responsible for unilateral edema. In patients with obstruction of the superior vena cava, edema is confined to the face, neck, and upper extremities in which the venous pressure is elevated compared with that in the lower extremities. APPROACH TO THE PATIENT: CHAPTER 51e Approach to the Patient with a Heart Murmur

1	APPROACH TO THE PATIENT: CHAPTER 51e Approach to the Patient with a Heart Murmur An important first question is whether the edema is localized or generalized. If it is localized, the local phenomena that may be responsible should be considered. If the edema is generalized, one should first determine if there is serious hypoalbuminemia, e.g., serum albumin <25 g/L. If so, the history, physical examination, urinalysis, and other laboratory data will help evaluate the question of cirrhosis, severe malnutrition, or the nephrotic syndrome as the underlying disorder. If hypoalbuminemia is not present, it should be determined if there is evidence of heart failure severe enough to promote generalized edema. Finally, it should be ascertained as to whether or not the patient has an adequate urine output or if there is significant oliguria or anuria. These abnormalities are discussed in Chaps. 61, 334, and 335. Approach to the Patient with a Patrick T. O’Gara, Joseph Loscalzo

1	Approach to the Patient with a Patrick T. O’Gara, Joseph Loscalzo This is a digital-only chapter. it is available on the DvD that accompanies this book, as well as on Access Medicine/Harrison’s online, and the eBook and “app” editions of HPiM 19e. The differential diagnosis of a heart murmur begins with a careful assessment of its major attributes and response to bedside maneuvers. The history, clinical context, and associated physical examination findings provide additional clues by which the significance of a heart murmur can be established. Accurate bedside identification of a heart murmur can inform decisions regarding the indications for noninvasive testing and the need for referral to a cardiovascular specialist. Preliminary discussions can be held with the patient regarding antibiotic or rheumatic fever prophylaxis, the need to restrict various forms of physical activity, and the potential role for family screening.

1	Approach to the Patient with a Heart Murmur Patrick T. O’Gara, Joseph Loscalzo The differential diagnosis of a heart murmur begins with a careful assessment of its major attributes and response to bedside maneuvers. 51e The history, clinical context, and associated physical examination findings provide additional clues by which the significance of a heart murmur can be established. Accurate bedside identification of a heart murmur can inform decisions regarding the indications for noninvasive testing and the need for referral to a cardiovascular specialist. Preliminary discussions can be held with the patient regarding antibiotic or rheumatic fever prophylaxis, the need to restrict various forms of physical activity, and the potential role for family screening.

1	Heart murmurs are caused by audible vibrations that are due to increased turbulence from accelerated blood flow through normal or abnormal orifices, flow through a narrowed or irregular orifice into a dilated vessel or chamber, or backward flow through an incompetent valve, ventricular septal defect, or patent ductus arteriosus. They traditionally are defined in terms of their timing within the cardiac cycle (Fig. 51e-1). Systolic murmurs begin with or after the first heart sound (S1) and terminate at or before the component (A2 or P2) of the second heart sound (S2) that corresponds to their site of origin (left or right, respectively). Diastolic murmurs begin with or after the associated component of S2 and end at or before the subsequent S1. Continuous murmurs are not confined to either phase of the cardiac cycle but instead begin in early systole and proceed through S2 into all or part of diastole. The accurate timing of heart murmurs is the first step in their identification. The

1	phase of the cardiac cycle but instead begin in early systole and proceed through S2 into all or part of diastole. The accurate timing of heart murmurs is the first step in their identification. The distinction between S1 and S2 and, therefore, systole and diastole is usually a straightforward process but can be difficult in the setting of a tachyarrhythmia, in which case the heart sounds can be distinguished by simultaneous palpation of the carotid upstroke, which should closely follow S1.

1	Duration and Character The duration of a heart murmur depends on the length of time over which a pressure difference exists between two cardiac chambers, the left ventricle and the aorta, the right ventricle and the pulmonary artery, or the great vessels. The magnitude and variability of this pressure difference, coupled with the geometry and compliance of the involved chambers or vessels, dictate the velocity of flow; the degree of turbulence; and the resulting frequency, configuration, and intensity of the murmur. The diastolic murmur of chronic aortic regurgitation (AR) is a blowing, high-frequency event, whereas the murmur of mitral stenosis (MS), indicative of the left atrial–left ventricular diastolic pressure gradient, is a low-frequency event, heard as a rumbling sound with the bell of the stethoscope. The frequency components of a heart murmur may vary at different sites of auscultation. The coarse systolic murmur of aortic stenosis (AS) may sound higher pitched and more

1	bell of the stethoscope. The frequency components of a heart murmur may vary at different sites of auscultation. The coarse systolic murmur of aortic stenosis (AS) may sound higher pitched and more acoustically pure at the apex, a phenomenon eponymously referred to as the Gallavardin effect. Some murmurs may have a distinct or unusual quality, such as the “honking” sound appreciated in some patients with mitral regurgitation (MR) due to mitral valve prolapse (MVP).

1	The configuration of a heart murmur may be described as crescendo, decrescendo, crescendo-decrescendo, or plateau. The decrescendo configuration of the murmur of chronic AR (Fig. 51e-1E) can be understood in terms of the progressive decline in the diastolic pressure gradient between the aorta and the left ventricle. The crescendo-decrescendo configuration of the murmur of AS reflects the changes in the systolic pressure gradient between the left ventricle and the aorta as ejection occurs, whereas the plateau configuration of the murmur of chronic MR (Fig. 51e-1B) is consistent with the large and nearly constant pressure difference between the left ventricle and the left atrium. Intensity The intensity of a heart murmur is graded on a scale of 1–6 (or I–VI). A grade 1 murmur is very soft and is heard only with great

1	Intensity The intensity of a heart murmur is graded on a scale of 1–6 (or I–VI). A grade 1 murmur is very soft and is heard only with great FIguRe 51e-1 Diagram depicting principal heart murmurs. A. Presystolic murmur of mitral or tricuspid stenosis. B. Holosystolic (pan-systolic) murmur of mitral or tricuspid regurgitation or of ventricular septal defect. C. Aortic ejection murmur beginning with an ejection click and fading before the second heart sound. D. Systolic murmur in pulmonic stenosis spilling through the aortic second sound, pulmonic valve closure being delayed. E. Aortic or pulmonary diastolic murmur. F. Long diastolic murmur of mitral stenosis after the opening snap (OS). G. Short mid-diastolic inflow murmur after a third heart sound.

1	H. Continuous murmur of patent ductus arteriosus. (Adapted from P Wood: Diseases of the Heart and Circulation, London, Eyre & Spottiswood, 1968. Permission granted courtesy of Antony and Julie Wood.) effort. A grade 2 murmur is easily heard but not particularly loud. A grade 3 murmur is loud but is not accompanied by a palpable thrill over the site of maximal intensity. A grade 4 murmur is very loud and is accompanied by a thrill. A grade 5 murmur is loud enough to be heard with only the edge of the stethoscope touching the chest, whereas a grade 6 murmur is loud enough to be heard with the stethoscope slightly off the chest. Murmurs of grade 3 or greater intensity usually signify important structural heart disease and indicate high blood flow velocity at the site of murmur production. Small ventricular septal defects (VSDs), for example, are accompanied by loud, usually grade 4 or greater, systolic murmurs as blood is ejected at high velocity from the left ventricle to the right

1	Small ventricular septal defects (VSDs), for example, are accompanied by loud, usually grade 4 or greater, systolic murmurs as blood is ejected at high velocity from the left ventricle to the right ventricle. Low-velocity events, such as left-to-right shunting across an atrial septal defect (ASD), are usually silent. The intensity of a heart murmur may be diminished by any process that increases the distance between the intracardiac source and the stethoscope on the chest wall, such as obesity, obstructive lung disease, and a large pericardial effusion. The intensity of a murmur also may be misleadingly soft when cardiac output is reduced significantly or when the pressure gradient between the involved cardiac structures is low.

1	Location and Radiation Recognition of the location and radiation of the murmur helps facilitate its accurate identification (Fig. 51e-2). Adventitious sounds, such as a systolic click or diastolic snap, or abnormalities of S1 or S2 may provide additional clues. Careful attention to the characteristics of the murmur and other heart sounds CHAPTER 51e Approach to the Patient with a Heart Murmur PART 2 Cardinal Manifestations and Presentation of Diseases

1	CHAPTER 51e Approach to the Patient with a Heart Murmur PART 2 Cardinal Manifestations and Presentation of Diseases FIguRe 51e-2 Maximal intensity and radiation of six isolated systolic murmurs. HOCM, hypertrophic obstructive cardiomyopathy; MR, mitral regurgitation; Pulm, pulmonary stenosis; Aortic, aortic stenosis; VSD, ventricular septal defect. (From JB Barlow: Perspectives on the Mitral Valve. Philadelphia, FA Davis, 1987, p 140.) during the respiratory cycle and the performance of simple bedside maneuvers complete the auscultatory examination. These features, along with recommendations for further testing, are discussed below in the context of specific systolic, diastolic, and continuous heart murmurs (Table 51e-1).

1	SYSTOLIC HeART MuRMuRS early Systolic Murmurs Early systolic murmurs begin with S1 and extend for a variable period, ending well before S2. Their causes are relatively few in number. Acute, severe MR into a normal-sized, relatively noncompliant left atrium results in an early, decrescendo systolic murmur best heard at or just medial to the apical impulse. These characteristics reflect the progressive attenuation of the pressure gradient between the left ventricle and the left atrium during systole owing to the rapid rise in left atrial pressure caused by the sudden volume load into an unprepared, noncompliant chamber and contrast sharply with the auscultatory features of chronic MR. Clinical settings in which acute, severe MR occur include (1) papillary muscle rupture complicating acute myocardial infarction (MI) (Chap. 295), (2) rupture of chordae tendineae in the setting of myxomatous mitral valve disease (MVP, Chap. 283), (3) infective endocarditis (Chap. 155), and (4) blunt chest

1	infarction (MI) (Chap. 295), (2) rupture of chordae tendineae in the setting of myxomatous mitral valve disease (MVP, Chap. 283), (3) infective endocarditis (Chap. 155), and (4) blunt chest wall trauma.

1	Acute, severe MR from papillary muscle rupture usually accompanies an inferior, posterior, or lateral MI and occurs 2–7 days after presentation. It often is signaled by chest pain, hypotension, and pulmonary edema, but a murmur may be absent in up to 50% of cases. The posteromedial papillary muscle is involved 6 to 10 times more frequently than the anterolateral papillary muscle. The murmur is to be distinguished from that associated with post-MI ventricular septal rupture, which is accompanied by a systolic thrill at the left sternal border in nearly all patients and is holosystolic in duration. A new heart murmur after an MI is an indication for transthoracic echocardiography (TTE) (Chap. 270e), which allows bedside delineation of its etiology and pathophysiologic significance. The distinction between acute MR and ventricular septal rupture also can be achieved with right heart catheterization, sequential determination of oxygen saturations, and analysis of the pressure waveforms

1	between acute MR and ventricular septal rupture also can be achieved with right heart catheterization, sequential determination of oxygen saturations, and analysis of the pressure waveforms (tall v wave in the pulmonary artery wedge pressure in MR). Post-MI mechanical complications of this nature mandate aggressive medical stabilization and prompt referral for surgical repair.

1	Spontaneous chordal rupture can complicate the course of myxomatous mitral valve disease (MVP) and result in new-onset or “acute on chronic” severe MR. MVP may occur as an isolated phenomenon, or the lesion may be part of a more generalized connective tissue disorder as seen, for example, in patients with Marfan syndrome. Acute, severe MR as a consequence of infective endocarditis results from destruction of leaflet tissue, chordal rupture, or both. Blunt chest wall trauma is usually self-evident but may be disarmingly trivial; it VSD Muscular Nonrestrictive with pulmonary hypertension Tricuspid TR with normal pulmonary artery pressure Mid-systolic Aortic Obstructive Supravalvular–supravalvular aortic stenosis, coarctation of the aorta Valvular–AS and aortic sclerosis Subvalvular–discrete, tunnel or HOCM Increased flow, hyperkinetic states, AR, complete heart block Dilation of ascending aorta, atheroma, aortitis Pulmonary

1	Increased flow, hyperkinetic states, AR, complete heart block Dilation of ascending aorta, atheroma, aortitis Pulmonary Increased flow, hyperkinetic states, left-to-right shunt (e.g., ASD) Dilation of pulmonary artery Late systolic Mitral MVP, acute myocardial ischemia Tricuspid TVP Holosystolic Atrioventricular valve regurgitation (MR, TR) Left-to-right shunt at ventricular level (VSD) Valvular: congenital (bicuspid valve), rheumatic deformity, endocarditis, prolapse, trauma, post-valvulotomy Dilation of valve ring: aorta dissection, annuloaortic ectasia, cystic medial degeneration, hypertension, ankylosing spondylitis Widening of commissures: syphilis

1	Pulmonic regurgitation Valvular: post-valvulotomy, endocarditis, rheumatic fever, carcinoid Dilation of valve ring: pulmonary hypertension; Marfan syndrome Congenital: isolated or associated with tetralogy of Fallot, VSD, pulmonic stenosis fever) Increased flow across nonstenotic mitral valve (e.g., MR, VSD, PDA, high-output states, and complete heart block) Tricuspid Tricuspid stenosis Increased flow across nonstenotic tricuspid valve (e.g., TR, ASD, and anomalous pulmonary venous return) Coronary AV fistula Mammary souffle of pregnancy Ruptured sinus of Valsalva aneurysm Pulmonary artery branch stenosis Cervical venous hum Small (restrictive) ASD with MS

1	Coronary AV fistula Mammary souffle of pregnancy Ruptured sinus of Valsalva aneurysm Pulmonary artery branch stenosis Cervical venous hum Small (restrictive) ASD with MS Anomalous left coronary artery Intercostal AV fistula Abbreviations: AR, aortic regurgitation; AS, aortic stenosis; ASD, atrial septal defect; AV, arteriovenous; HOCM, hypertrophic obstructive cardiomyopathy; MR, mitral regurgitation; MS, mitral stenosis; MVP, mitral valve prolapse; PDA, patent ductus arteriosus; TR, tricuspid regurgitation; TVP, tricuspid valve prolapse; VSD, ventricular septal defect. Source: E Braunwald, JK Perloff, in D Zipes et al (eds): Braunwald’s Heart Disease, 7th ed. Philadelphia, Elsevier, 2005; PJ Norton, RA O’Rourke, in E Braunwald, L Goldman (eds): Primary Cardiology, 2nd ed. Philadelphia, Elsevier, 2003.

1	can result in papillary muscle contusion and rupture, chordal detachment, or leaflet avulsion. TTE is indicated in all cases of suspected acute, severe MR to define its mechanism and severity, delineate left ventricular size and systolic function, and provide an assessment of suitability for primary valve repair.

1	A congenital, small muscular VSD (Chap. 282) may be associated with an early systolic murmur. The defect closes progressively during septal contraction, and thus, the murmur is confined to early systole. It is localized to the left sternal border (Fig. 51e-2) and is usually of grade 4 or 5 intensity. Signs of pulmonary hypertension or left ventricular volume overload are absent. Anatomically large and uncorrected VSDs, which usually involve the membranous portion of the septum, may lead to pulmonary hypertension. The murmur associated with the left-to-right shunt, which earlier may have been holosystolic, becomes limited to the first portion of systole as the elevated pulmonary vascular resistance leads to an abrupt rise in right ventricular pressure and an attenuation of the interventricular pressure gradient during the remainder of the cardiac cycle. In such instances, signs of pulmonary hypertension (right ventricular lift, loud and single or closely split S2) may predominate. The

1	pressure gradient during the remainder of the cardiac cycle. In such instances, signs of pulmonary hypertension (right ventricular lift, loud and single or closely split S2) may predominate. The murmur is best heard along the left sternal border but is softer. Suspicion of a VSD is an indication for TTE.

1	Tricuspid regurgitation (TR) with normal pulmonary artery pressures, as may occur with infective endocarditis, may produce an early systolic murmur. The murmur is soft (grade 1 or 2), is best heard at the lower left sternal border, and may increase in intensity with inspiration (Carvallo’s sign). Regurgitant “c-v” waves may be visible in the jugular venous pulse. TR in this setting is not associated with signs of right heart failure. Mid-Systolic Murmurs Mid-systolic murmurs begin at a short interval after , end before S (Fig. 51e-1C), and are usually crescendo-decrescendo in configuration. Aortic stenosis is the most common cause of a mid-systolic murmur in an adult. The murmur of AS is usually loudest to the right of the sternum in the second intercostal space (aortic area, Fig. 51e-2) and radiates into the carotids. Transmission of the mid-systolic murmur to the apex, where it becomes higher-pitched, is common (Gallavardin effect; see above).

1	Differentiation of this apical systolic murmur from MR can be difficult. The murmur of AS will increase in intensity, or become louder, in the beat after a premature beat, whereas the murmur of MR will have constant intensity from beat to beat. The intensity of the AS murmur also varies directly with the cardiac output. With a normal cardiac output, a systolic thrill and a grade 4 or higher murmur suggest severe AS. The murmur is softer in the setting of heart failure and low cardiac output. Other auscultatory findings of severe AS include a soft or absent A2, paradoxical splitting of S2, an apical S4, and a late-peaking systolic murmur. In children, adolescents, and young adults with congenital valvular AS, an early ejection sound (click) is usually audible, more often along the left sternal border than at the base. Its presence signifies a flexible, noncalcified bicuspid valve (or one of its variants) and localizes the left ventricular outflow obstruction to the valvular (rather

1	sternal border than at the base. Its presence signifies a flexible, noncalcified bicuspid valve (or one of its variants) and localizes the left ventricular outflow obstruction to the valvular (rather than subor supravalvular) level.

1	Assessment of the volume and rate of rise of the carotid pulse can provide additional information. A small and delayed upstroke (parvus et tardus) is consistent with severe AS. The carotid pulse examination is less discriminatory, however, in older patients with stiffened arteries. The electrocardiogram (ECG) shows signs of left ventricular hypertrophy (LVH) as the severity of the stenosis increases. TTE is indicated to assess the anatomic features of the aortic valve, the severity of the stenosis, left ventricular size, wall thickness and function, and the size and contour of the aortic root and proximal ascending aorta.

1	The obstructive form of hypertrophic cardiomyopathy (HOCM) is associated with a mid-systolic murmur that is usually loudest along the left sternal border or between the left lower sternal border and the apex (Chap. 287, Fig. 51e-2). The murmur is produced by both dynamic left ventricular outflow tract obstruction and MR, and thus, its configuration is a hybrid between ejection and regurgitant phenomena. The intensity of the murmur may vary from beat to beat and after provocative maneuvers but usually does not exceed grade 3. The murmur classically will increase in intensity with maneuvers that result 51e-3 in increasing degrees of outflow tract obstruction, such as a reduction in preload or afterload (Valsalva, standing, vasodilators), or with an augmentation of contractility (inotropic stimulation). Maneuvers that increase preload (squatting, passive leg raising, volume administration) or afterload (squatting, vasopressors) or that reduce contractility (β-adrenoreceptor blockers)

1	stimulation). Maneuvers that increase preload (squatting, passive leg raising, volume administration) or afterload (squatting, vasopressors) or that reduce contractility (β-adrenoreceptor blockers) decrease the intensity of the murmur. In rare patients, there may be reversed splitting of S2. A sustained left ventricular apical impulse and an S4 may be appreciated. In contrast to AS, the carotid upstroke is rapid and of normal volume. Rarely, it is bisferiens or bifid in contour (see Fig. 267-2D) due to mid-systolic closure of the aortic valve. LVH is present on the ECG, and the diagnosis is confirmed by TTE. Although the systolic murmur associated with MVP behaves similarly to that due to HOCM in response to the Valsalva maneuver and to standing/squatting (Fig. 51e-3), these two lesions can be distinguished on the basis of their associated findings, such as the presence of LVH in HOCM or a nonejection click in MVP.

1	The mid-systolic, crescendo-decrescendo murmur of congenital pulmonic stenosis (PS, Chap. 282) is best appreciated in the second and third left intercostal spaces (pulmonic area) (Figs. 51e-2 and 51e-4). The duration of the murmur lengthens and the intensity of P2 diminishes with increasing degrees of valvular stenosis (Fig. 51e1D). An early ejection sound, the intensity of which decreases with inspiration, is heard in younger patients. A parasternal lift and ECG evidence of right ventricular hypertrophy indicate severe pressure overload. If obtained, the chest x-ray may show poststenotic dilation of the main pulmonary artery. TTE is recommended for complete characterization. Significant left-to-right intracardiac shunting due to an ASD (Chap. 282) leads to an increase in pulmonary blood flow and a grade 2–3 mid-systolic murmur at the middle to upper left sternal border CHAPTER 51e Approach to the Patient with a Heart Murmur

1	CHAPTER 51e Approach to the Patient with a Heart Murmur FIguRe 51e-3 A mid-systolic nonejection sound (C) occurs in mitral valve prolapse and is followed by a late systolic murmur that crescendos to the second heart sound (S2). Standing decreases venous return; the heart becomes smaller; C moves closer to the first heart sound (S1), and the mitral regurgitant murmur has an earlier onset. With prompt squatting, venous return and afterload increase; the heart becomes larger; C moves toward S2; and the duration of the murmur shortens. (From JA Shaver, JJ Leonard, DF Leon: Examination of the Heart, Part IV, Auscultation of the Heart. Dallas, American Heart Association, 1990, p 13. Copyright, American Heart Association.) 51e-4 Pulmonic stenosis Tetralogy of Fallot P.Ej S1 S2S1 S2 PART 2 Cardinal Manifestations and Presentation of Diseases P.Ej A2 P2 A.Ej A2P.Ej P.Ej = Pulmonary ejection (valvular) A.Ej = Aortic ejection (root)

1	FIguRe 51e-4 Left. In valvular pulmonic stenosis with intact ventricular septum, right ventricular systolic ejection becomes progressively longer, with increasing obstruction to flow. As a result, the murmur becomes longer and louder, enveloping the aortic component of the second heart sound (A2). The pulmonic component (P2) occurs later, and splitting becomes wider but more difficult to hear because A2 is lost in the murmur and P2 becomes progressively fainter and lower pitched. As the pulmonic gradient increases, the isometric contraction phase shortens until the pulmonic valve ejection sound fuses with the first heart sound (S1). In severe pulmonic stenosis with concentric hypertrophy and decreasing right ventricular compliance, a fourth heart sound appears. Right. In tetralogy of Fallot with increasing obstruction at the pulmonic infundibular area, an increasing amount of right ventricular blood is shunted across the silent ventricular septal defect and flow across the obstructed

1	with increasing obstruction at the pulmonic infundibular area, an increasing amount of right ventricular blood is shunted across the silent ventricular septal defect and flow across the obstructed outflow tract decreases. Therefore, with increasing obstruction the murmur becomes shorter, earlier, and fainter. P2 is absent in severe tetralogy of Fallot. A large aortic root receives almost all cardiac output from both ventricular chambers, and the aorta dilates and is accompanied by a root ejection sound that does not vary with respiration. (From JA Shaver, JJ Leonard, DF Leon: Examination of the Heart, Part IV, Auscultation of the Heart. Dallas, American Heart Association, 1990, p 45. Copyright, American Heart Association.) attributed to increased flow rates across the pulmonic valve with fixed splitting of S2. Ostium secundum ASDs are the most common cause of these shunts in adults. Features suggestive of a primum ASD include the coexistence of MR due to a cleft anterior mitral valve

1	fixed splitting of S2. Ostium secundum ASDs are the most common cause of these shunts in adults. Features suggestive of a primum ASD include the coexistence of MR due to a cleft anterior mitral valve leaflet and left axis deviation of the QRS complex on the ECG. With sinus venosus ASDs, the left-to-right shunt is usually not large enough to result in a systolic murmur, although the ECG may show abnormalities of sinus node function. A grade 2 or 3 mid-systolic murmur may also be heard best at the upper left sternal border in patients with idiopathic dilation of the pulmonary artery; a pulmonary ejection sound is also present in these patients. TTE is indicated to evaluate a grade 2 or 3 mid-systolic murmur when there are other signs of cardiac disease.

1	An isolated grade 1 or 2 mid-systolic murmur, heard in the absence of symptoms or signs of heart disease, is most often a benign finding for which no further evaluation, including TTE, is necessary. The most common example of a murmur of this type in an older adult patient is the crescendo-decrescendo murmur of aortic valve sclerosis, heard at the second right interspace (Fig. 51e-2). Aortic sclerosis is defined as focal thickening and calcification of the aortic valve to a degree that does not interfere with leaflet opening. The carotid upstrokes are normal, and electrocardiographic LVH is not present. A grade 1 or 2 mid-systolic murmur often can be heard at the left sternal border with pregnancy, hyperthyroidism, or anemia, physiologic states that are associated with accelerated blood flow. Still’s murmur refers to a benign grade 2, vibratory or musical mid-systolic murmur at the mid or lower left sternal border in normal children and adolescents, best heard in the supine position

1	flow. Still’s murmur refers to a benign grade 2, vibratory or musical mid-systolic murmur at the mid or lower left sternal border in normal children and adolescents, best heard in the supine position (Fig. 51e-2).

1	Late Systolic Murmurs A late systolic murmur that is best heard at the left ventricular apex is usually due to MVP (Chap. 283). Often, this murmur is introduced by one or more nonejection clicks. The radiation of the murmur can help identify the specific mitral leaflet involved in the process of prolapse or flail. The term flail refers to the movement made by an unsupported portion of the leaflet after loss of its chordal attachment(s). With posterior leaflet prolapse or flail, the resultant jet of MR is directed anteriorly and medially, as a result of which the murmur radiates to the base of the heart and masquerades as AS. Anterior leaflet prolapse or flail results in a posteriorly directed MR jet that radiates to the axilla or left infrascapular region. Leaflet flail is associated with a murmur of grade 3 or 4 intensity that can be heard throughout the precordium in thin-chested patients. The presence of an S3 or a short, rumbling mid-diastolic murmur due to enhanced flow signifies

1	a murmur of grade 3 or 4 intensity that can be heard throughout the precordium in thin-chested patients. The presence of an S3 or a short, rumbling mid-diastolic murmur due to enhanced flow signifies severe MR.

1	Bedside maneuvers that decrease left ventricular preload, such as standing, will cause the click and murmur of MVP to move closer to the first heart sound, as leaflet prolapse occurs earlier in systole. Standing also causes the murmur to become louder and longer. With squatting, left ventricular preload and afterload are increased abruptly, leading to an increase in left ventricular volume, and the click and murmur move away from the first heart sound as leaflet prolapse is delayed; the murmur becomes softer and shorter in duration (Fig. 51e–3). As noted above, these responses to standing and squatting are directionally similar to those observed in patients with HOCM.

1	A late, apical systolic murmur indicative of MR may be heard transiently in the setting of acute myocardial ischemia; it is due to apical tethering and malcoaptation of the leaflets in response to structural and functional changes of the ventricle and mitral annulus. The intensity of the murmur varies as a function of left ventricular afterload and will increase in the setting of hypertension. TTE is recommended for assessment of late systolic murmurs.

1	Holosystolic Murmurs (Figs. 51e-1B and 51e-5) Holosystolic murmurs begin with S1 and continue through systole to S2. They are usually indicative of chronic mitral or tricuspid valve regurgitation or a VSD and warrant TTE for further characterization. The holosystolic murmur of chronic MR is best heard at the left ventricular apex and radiates to the axilla (Fig. 51e-2); it is usually high-pitched and plateau in configuration because of the wide difference between left ventricular and left atrial pressure throughout systole. In contrast to acute MR, left atrial compliance is normal or even increased in chronic MR. As a result, there is only a small increase in left atrial pressure for any increase in regurgitant volume.

1	Several conditions are associated with chronic MR and an apical holosystolic murmur, including rheumatic scarring of the leaflets, mitral annular calcification, postinfarction left ventricular remodeling, and severe left ventricular chamber enlargement. The circumference of the mitral annulus increases as the left ventricle enlarges and leads to failure of leaflet coaptation with central MR in patients with dilated cardiomyopathy (Chap. 287). The severity of the MR is worsened by any contribution from apical displacement of the papillary muscles and leaflet tethering (remodeling). Because the mitral annulus is contiguous with the left atrial endocardium, gradual enlargement of the left atrium from chronic MR will result in further stretching of the annulus and more MR; thus, “MR begets MR.” Chronic severe MR results in enlargement and leftward displacement of the left ventricular apex beat and, in some patients, a diastolic filling complex, as described previously.

1	The holosystolic murmur of chronic TR is generally softer than that of MR, is loudest at the left lower sternal border, and usually increases in intensity with inspiration (Carvallo’s sign). Associated signs include c-v waves in the jugular venous pulse, an enlarged and pulsatile liver, ascites, and peripheral edema. The abnormal jugular venous waveforms are the predominant finding and are seen very often in the HOLOSYSTOLIC MURMUR: DIFFERENTIAL DIAGNOSIS Maximum intensity over left sternal border Radiation to epigastrium and right sternal border Prominent c-v wave with sharp y descent in jugular venous pulse Maximum intensity over lower left third and fourth interspace Widespread radiation, palpable thrill Decreased intensity with amyl nitrate Wide splitting of S

1	Widespread radiation, palpable thrill Decreased intensity with amyl nitrate Wide splitting of S Hyperdynamic left ventricular impulse Wide splitting of S2 Sustained left ventricular impulse Single S2 or narrow splitting of S2 Prominent left parasternal diastolic impulse Normal brief left paraster-nal systolic impulse Normal P2 Rarely paradoxical S2 Sustained systolic left parasternal impulse Narrow splitting of S2 with marked increase in intensity of P2 Primary mitral regurgitation (e.g., rheumatic, ruptured chordae) Secondary mitral regurgitation (dilated cardiomyopathy; papillary muscle dysfunction, or late stage of primary mitral regurgitation) Primary Secondary to pulmonary hypertension Favors ventricular septal defect; often difficult to differentiate from mitral regurgitant murmur FIguRe 51e-5 Differential diagnosis of a holosystolic murmur.

1	FIguRe 51e-5 Differential diagnosis of a holosystolic murmur. absence of an audible murmur despite Doppler echocardiographic verification of TR. Causes of primary TR include myxomatous disease (prolapse), endocarditis, rheumatic disease, radiation, carcinoid, Ebstein’s anomaly, and chordal detachment as a complication of right ventricular endomyocardial biopsy. TR is more commonly a passive process that results secondarily from annular enlargement due to right ventricular dilatation in the face of volume or pressure overload.

1	The holosystolic murmur of a VSD is loudest at the midto lower left sternal border (Fig. 51e-2) and radiates widely. A thrill is present at the site of maximal intensity in the majority of patients. There is no change in the intensity of the murmur with inspiration. The intensity of the murmur varies as a function of the anatomic size of the defect. Small, restrictive VSDs, as exemplified by the maladie de Roger, create a very loud murmur due to the significant and sustained systolic pressure gradient between the left and right ventricles. With large defects, the ventricular pressures tend to equalize, shunt flow is balanced, and a murmur is not appreciated. The distinction between post-MI ventricular septal rupture and MR has been reviewed previously.

1	DIASTOLIC HeART MuRMuRS early Diastolic Murmurs (Fig. 51e-1E) Chronic AR results in a high-pitched, blowing, decrescendo, early to mid-diastolic murmur that begins after the aortic component of S2 (A2) and is best heard at the second right interspace (Fig. 51e-6). The murmur may be soft and difficult to hear unless auscultation is performed with the patient leaning forward at end expiration. This maneuver brings the aortic root closer to the anterior chest wall. Radiation of the murmur may provide a clue to the cause of the AR. With primary valve disease, such as that due to congenital bicuspid disease, prolapse, or endocarditis, the diastolic murmur tends to radiate along the left sternal border, where it is often louder than appreciated in the second right interspace. When AR is caused by aortic root disease, the diastolic murmur may radiate along the right sternal border. Diseases of the aortic root cause dilation or distortion of the aortic annulus and failure of leaflet

1	caused by aortic root disease, the diastolic murmur may radiate along the right sternal border. Diseases of the aortic root cause dilation or distortion of the aortic annulus and failure of leaflet coaptation. Causes include Marfan syndrome with aneurysm formation, annuloaortic ectasia, ankylosing spondylitis, and aortic dissection.

1	Chronic, severe AR also may produce a lower-pitched mid to late, grade 1 or 2 diastolic murmur at the apex (Austin Flint murmur), which is thought to reflect turbulence at the mitral inflow area from the admixture of regurgitant (aortic) and forward (mitral) blood flow (Fig. 51e-1G). This lower-pitched, apical diastolic murmur can be distinguished from that due to MS by the absence of an opening snap and the response of the murmur to a vasodilator challenge. Lowering after-load with an agent such as amyl nitrite will decrease the duration and magnitude of the aortic–left ventricular diastolic pressure gradient, and thus, the Austin Flint murmur of severe AR will become shorter and softer. The intensity of the diastolic murmur of mitral stenosis (Fig. 51e-6) may either remain constant or increase with afterload reduction because of the reflex increase in cardiac output and mitral valve flow.

1	Although AS and AR may coexist, a grade 2 or 3 crescendo-decrescendo mid-systolic murmur frequently is heard at the base of the heart in patients with isolated, severe AR and is due to an increased volume and rate of systolic flow. Accurate bedside identification of coexistent AS can be difficult unless the carotid pulse examination is abnormal or the mid-systolic murmur is of grade 4 or greater intensity. In the absence of heart failure, chronic severe AR is accompanied by several peripheral signs of significant diastolic run-off, including a wide pulse pressure, a “water-hammer” carotid upstroke (Corrigan’s pulse), and Quincke’s pulsations of the nail beds. The diastolic murmur of acute, severe AR is notably shorter in duration and lower pitched than the murmur of chronic AR. It can be very difficult to appreciate in the presence of a rapid heart rate. These attributes reflect the abrupt rate of rise of diastolic pressure within the unprepared and noncompliant left ventricle and the

1	difficult to appreciate in the presence of a rapid heart rate. These attributes reflect the abrupt rate of rise of diastolic pressure within the unprepared and noncompliant left ventricle and the correspondingly rapid decline in the aortic–left ventricular diastolic pressure gradient. Left ventricular diastolic pressure may increase sufficiently to result in premature closure of the mitral valve and a soft first heart sound. Peripheral signs of significant diastolic run-off are not present.

1	Pulmonic regurgitation (PR) results in a decrescendo, early to mid-diastolic murmur (Graham Steell murmur) that begins after the pulmonic component of S2 (P2), is best heard at the second left interspace, and radiates along the left sternal border. The intensity of the murmur may increase with inspiration. PR is most commonly due to dilation of the valve annulus from chronic elevation of the pulmonary artery pressure. Signs of pulmonary hypertension, including a right ventricular CHAPTER 51e Approach to the Patient with a Heart Murmur PART 2 Cardinal Manifestations and Presentation of Diseases O.S. O.S. FIguRe 51e-6 Diastolic filling murmur (rumble) in mitral stenosis.

1	In mild mitral stenosis, the diastolic gradient across the valve is limited to the phases of rapid ventricular filling in early diastole and presystole. The rumble may occur during either or both periods. As the stenotic process becomes severe, a large pressure gradient exists across the valve during the entire diastolic filling period, and the rumble persists throughout diastole. As the left atrial pressure becomes greater, the interval between A2 (or P2) and the opening snap (O.S.) shortens. In severe mitral stenosis, secondary pulmonary hypertension develops and results in a loud P2 and the splitting interval usually narrows. ECG, electrocardiogram. (From JA Shaver, JJ Leonard, DF Leon: Examination of the Heart, Part IV, Auscultation of the Heart. Dallas, American Heart Association, 1990, p 55. Copyright, American Heart Association.) lift and a loud, single or narrowly split S2, are present. These features also help distinguish PR from AR as the cause of a decrescendo diastolic

1	1990, p 55. Copyright, American Heart Association.) lift and a loud, single or narrowly split S2, are present. These features also help distinguish PR from AR as the cause of a decrescendo diastolic murmur heard along the left sternal border. PR in the absence of pulmonary hypertension can occur with endocarditis or a congenitally deformed valve. It is usually present after repair of tetralogy of Fallot in childhood. When pulmonary hypertension is not present, the diastolic murmur is softer and lower pitched than the classic Graham Steell murmur, and the severity of the PR can be difficult to appreciate.

1	TTE is indicated for the further evaluation of a patient with an early to mid-diastolic murmur. Longitudinal assessment of the severity of the valve lesion and ventricular size and systolic function help guide a potential decision for surgical management. TTE also can provide anatomic information regarding the root and proximal ascending aorta, although computed tomographic or magnetic resonance angiography may be indicated for more precise characterization (Chap. 270e).

1	Mid-Diastolic Murmurs (Figs. 51e-1G and 51e-1H) Mid-diastolic murmurs result from obstruction and/or augmented flow at the level of the mitral or tricuspid valve. Rheumatic fever is the most common cause of MS (Fig. 51e-6). In younger patients with pliable valves, S1 is loud and the murmur begins after an opening snap, which is a high-pitched sound that occurs shortly after S2. The interval between the pulmonic component of the second heart sound (P2) and the opening snap is inversely related to the magnitude of the left atrial–left ventricular pressure gradient. The murmur of MS is low-pitched and thus is best heard with the bell of the stethoscope. It is loudest at the left ventricular apex and often is appreciated only when the patient is turned in the left lateral decubitus position. It is usually of grade 1 or 2 intensity but may be absent when the cardiac output is severely reduced despite significant obstruction. The intensity of the murmur increases during maneuvers that

1	It is usually of grade 1 or 2 intensity but may be absent when the cardiac output is severely reduced despite significant obstruction. The intensity of the murmur increases during maneuvers that increase cardiac output and mitral valve flow, such as exercise. The duration of the murmur reflects the length of time over which left atrial pressure exceeds left ventricular diastolic pressure. An increase in the intensity of the murmur just before S1, a phenomenon known as presystolic accentuation (Figs. 51e-1A and 51e-6), occurs in patients in sinus rhythm and is due to a late increase in transmitral flow with atrial contraction. Presystolic accentuation does not occur in patients with atrial fibrillation.

1	The mid-diastolic murmur associated with tricuspid stenosis is best heard at the lower left sternal border and increases in intensity with inspiration. A prolonged y descent may be visible in the jugular venous waveform. This murmur is very difficult to hear and often is obscured by left-sided acoustical events.

1	There are several other causes of mid-diastolic murmurs. Large left atrial myxomas may prolapse across the mitral valve and cause variable degrees of obstruction to left ventricular inflow (Chap. 289e). The murmur associated with an atrial myxoma may change in duration and intensity with changes in body position. An opening snap is not present, and there is no presystolic accentuation. Augmented mitral diastolic flow can occur with isolated severe MR or with a large left-to-right shunt at the ventricular or great vessel level and produce a soft, rapid filling sound (S3) followed by a short, low-pitched mid-diastolic apical murmur. The Austin Flint murmur of severe, chronic AR has already been described.

1	A short, mid-diastolic murmur is rarely heard during an episode of acute rheumatic fever (Carey-Coombs murmur) and probably is due to flow through an edematous mitral valve. An opening snap is not present in the acute phase, and the murmur dissipates with resolution of the acute attack. Complete heart block with dyssynchronous atrial and ventricular activation may be associated with intermittent midto late diastolic murmurs if atrial contraction occurs when the mitral valve is partially closed. Mid-diastolic murmurs indicative of increased tricuspid valve flow can occur with severe, isolated TR and with large ASDs and significant left-to-right shunting. Other signs of an ASD are present (Chap. 282), including fixed splitting of S2 and a mid-systolic murmur at the midto upper left sternal border. TTE is indicated for evaluation of a patient with a midto late diastolic murmur. Findings specific to the diseases discussed above will help guide management.

1	(Figs. 51e-1H and 51e-7) Continuous murmurs begin in systole, peak near the second heart sound, and continue into all or part of diastole. Their presence throughout the cardiac cycle implies a pressure gradient between two chambers or vessels during both systole and diastole. The continuous murmur associated with a patent ductus arteriosus is best heard at the upper left sternal border. Large, uncorrected shunts may lead to pulmonary hypertension, attenuation or obliteration of the diastolic component of the murmur, reversal of shunt flow, and differential cyanosis of the lower extremities. A ruptured sinus of Valsalva aneurysm creates a continuous murmur of abrupt onset at the upper right sternal border. Rupture typically occurs into a right heart chamber, and the murmur is indicative of a continuous pressure difference between the aorta and either the right ventricle or the right atrium. A continuous murmur also may be audible along the left sternal border with a coronary

1	of a continuous pressure difference between the aorta and either the right ventricle or the right atrium. A continuous murmur also may be audible along the left sternal border with a coronary arteriovenous fistula and at the site of an arteriovenous fistula used for hemodialysis access. Enhanced flow through enlarged intercostal collateral arteries in patients with aortic coarctation may produce a continuous murmur along the course of one or more ribs. A cervical bruit with both systolic and diastolic components (a to-fro murmur, Fig. 51e-7) usually indicates a high-grade carotid artery stenosis.

1	Not all continuous murmurs are pathologic. A continuous venous hum can be heard in healthy children and young adults, especially during pregnancy; it is best appreciated in the right supraclavicular fossa and can be obliterated by pressure over the right internal jugular vein or by having the patient turn his or her head toward the examiner. The continuous mammary souffle of pregnancy is created by enhanced arterial flow through engorged breasts and usually appears during the late third trimester or early puerperium. The murmur is louder in systole. Firm pressure with the diaphragm of the stethoscope can eliminate the diastolic portion of the murmur. (Table 51e-2; see Table 267-1) Careful attention to the behavior of heart murmurs during simple maneuvers that alter cardiac hemodynamics can provide important clues to their cause and significance. Respiration Auscultation should be performed during quiet respiration or with a modest increase in inspiratory effort, as more forceful

1	Respiration Auscultation should be performed during quiet respiration or with a modest increase in inspiratory effort, as more forceful Continuous Murmur vs. To-Fro Murmur

1	FIguRe 51e-7 Comparison of the continuous murmur and the to-fro murmur. During abnormal communication between high-pressure and low-pressure systems, a large pressure gradient exists throughout the cardiac cycle, producing a continuous murmur. A classic example is patent ductus arteriosus. At times, this type of murmur can be confused with a to-fro murmur, which is a combination of systolic ejection murmur and a murmur of semilunar valve incompetence. A classic example of a to-fro murmur is aortic stenosis and regurgitation. A continuous murmur crescendos to near the second heart sound (S2), whereas a to-fro murmur has two components. The mid-systolic ejection component decrescendos and disappears as it approaches S2. (From JA Shaver, JJ Leonard, DF Leon: Examination of the Heart, Part IV, Auscultation of the Heart. Dallas, American Heart Association, 1990, p 55. Copyright, American Heart Association.) movement of the chest tends to obscure the heart sounds. Left-sided murmurs may be

1	Auscultation of the Heart. Dallas, American Heart Association, 1990, p 55. Copyright, American Heart Association.) movement of the chest tends to obscure the heart sounds. Left-sided murmurs may be best heard at end expiration, when lung volumes are minimized and the heart and great vessels are brought closer to the chest wall. This phenomenon is characteristic of the murmur of AR. Murmurs of right-sided origin, such as tricuspid or pulmonic regurgitation, increase in intensity during inspiration. The intensity of left-sided murmurs either remains constant or decreases with inspiration.

1	Bedside assessment also should evaluate the behavior of S2 with respiration and the dynamic relationship between the aortic and pulmonic components (Fig. 51e-8). Reversed splitting can be a feature of severe AS, HOCM, left bundle branch block, right ventricular pacing, or acute myocardial ischemia. Fixed splitting of S2 in the presence of a grade 2 or 3 mid-systolic murmur at the midor upper left sternal border indicates an ASD. Physiologic but wide splitting during the respiratory cycle implies either premature aortic valve closure, as can occur with severe MR, or delayed pulmonic valve closure due to PS or right bundle branch block.

1	Alterations of Systemic Vascular Resistance Murmurs can change characteristics after maneuvers that alter systemic vascular resistance and left ventricular afterload. The systolic murmurs of MR and VSD become louder during sustained handgrip, simultaneous inflation of blood pressure cuffs on both upper extremities to pressures 20–40 mmHg above systolic pressure for 20 s, or infusion of a vasopressor agent. The murmurs associated with AS or HOCM will become softer or remain unchanged with these maneuvers. The diastolic murmur of AR becomes louder in response to interventions that raise systemic vascular resistance. Opposite changes in systolic and diastolic murmurs may occur with the use of pharmacologic agents that lower systemic vascular resistance. DynAMiC AusCuLTATion: BEDsiDE MAnEuvERs THAT CAn BE usED To CHAngE THE inTEnsiTy of CARDiAC MuRMuRs (sEE TExT) 1. 2. 3. 4. Pharmacologic manipulation of preload and/or afterload 5. 6. 7. 8.

1	6. 7. 8. FIguRe 51e-8 Top. Normal physiologic splitting. During expiration, the aortic (A2) and pulmonic (P2) components of the second heart sound are separated by <30 ms and are appreciated as a single sound. During inspiration, the splitting interval widens, and A2 and P2 are clearly separated into two distinct sounds. Bottom. Audible expiratory splitting. Wide physiologic splitting is caused by a delay in P2. Reversed splitting is caused by a delay in A2, resulting in paradoxical movement; i.e., with inspiration P2 moves toward A2, and the splitting interval narrows. Narrow physiologic splitting occurs in pulmonary hypertension, and both A2 and P2 are heard during expiration at a narrow splitting interval because of the increased intensity and high-frequency composition of P2. (From JA Shaver, JJ Leonard, DF Leon: Examination of the Heart, Part IV, Auscultation of the Heart. Dallas, American Heart Association, 1990, p 17. Copyright, American Heart Association.)

1	Inhaled amyl nitrite is now rarely used for this purpose but can help distinguish the murmur of AS or HOCM from that of either MR or VSD, if necessary. The former two murmurs increase in intensity, whereas the latter two become softer after exposure to amyl nitrite. As noted previously, the Austin Flint murmur of severe AR becomes softer, but the mid-diastolic rumble of MS becomes louder, in response to the abrupt lowering of systemic vascular resistance with amyl nitrite.

1	Changes in Venous Return The Valsalva maneuver results in an increase in intrathoracic pressure, followed by a decrease in venous return, ventricular filling, and cardiac output. The majority of murmurs decrease in intensity during the strain phase of the maneuver. Two notable exceptions are the murmurs associated with MVP and obstructive HOCM, both of which become louder during the Valsalva maneuver. The murmur of MVP may also become longer as leaflet prolapse occurs earlier in systole at smaller ventricular volumes. These murmurs behave in a similar and parallel fashion with standing. Both the click and the murmur of MVP move closer in timing to S1 on rapid standing from a squatting position (Fig. 51e-3). The increase in the intensity of the murmur of HOCM is predicated on the augmentation of the dynamic left ventricular outflow tract gradient that occurs with reduced ventricular filling. Squatting results in abrupt increases in both venous return (preload) and left ventricular

1	of the dynamic left ventricular outflow tract gradient that occurs with reduced ventricular filling. Squatting results in abrupt increases in both venous return (preload) and left ventricular afterload that increase ventricular volume, changes that predictably cause a decrease in the intensity and duration of the murmurs associated with MVP and HOCM; the click and murmur of MVP move away from S1 with squatting. Passive leg raising can be used to increase venous return in patients who are unable to squat and stand. This maneuver may lead to a decrease in the intensity of the murmur associated with HOCM but has less effect in patients with MVP.

1	CHAPTER 51e Approach to the Patient with a Heart Murmur 51e-8 Post-Premature Ventricular Contraction A change in the intensity of a pulmonic, and mitral valves. Such signals are not likely to generate systolic murmur in the first beat after a premature beat, or in the beat after a long cycle length in patients with atrial fibrillation, can help distinguish AS from MR, particularly in an older patient in whom the murmur of AS is well transmitted to the apex. Systolic murmurs due to left ventricular outflow obstruction, including that due to AS, increase in intensity in the beat after a premature beat because of the combined effects of enhanced left ventricular filling and post-extrasystolic potentiation of contractile function. Forward flow accelerates, causing an increase in the gradient and a louder murmur. The intensity of the murmur of MR does not change in the post-premature beat as there is relatively little further increase in mitral valve flow or change in the left

1	gradient and a louder murmur. The intensity of the murmur of MR does not change in the post-premature beat as there is relatively little further increase in mitral valve flow or change in the left ventricular–left atrial gradient.

1	Additional clues to the etiology and importance of a heart murmur can be gleaned from the history and other physical examination findings. Symptoms suggestive of cardiovascular, neurologic, or pulmonary disease help focus the differential diagnosis, as do findings relevant to the jugular venous pressure and waveforms, the arterial pulses, other heart sounds, the lungs, the abdomen, the skin, and the extremities. In many instances, laboratory studies, an ECG, and/or a chest x-ray may have been obtained earlier and may contain valuable information. A patient with suspected infective endocarditis, for example, may have a murmur in the setting of fever, chills, anorexia, fatigue, dyspnea, splenomegaly, petechiae, and positive blood cultures. A new systolic murmur in a patient with a marked fall in blood pressure after a recent MI suggests myocardial rupture. By contrast, an isolated grade 1 or 2 mid-systolic murmur at the left sternal border in a healthy, active, and asymptomatic young

1	fall in blood pressure after a recent MI suggests myocardial rupture. By contrast, an isolated grade 1 or 2 mid-systolic murmur at the left sternal border in a healthy, active, and asymptomatic young adult is most likely a benign finding for which no further evaluation is indicated. The context in which the murmur is appreciated often dictates the need for further testing.

1	(Fig. 51e–9; Chaps. 267 and 270e) Echocardiography with color flow and spectral Doppler is a valuable tool for the assessment of cardiac murmurs. Information regarding valve structure and function, chamber size, wall thickness, ventricular function, estimated pulmonary artery pressures, intracardiac shunt flow, pulmonary and hepatic vein flow, and aortic flow can be ascertained readily. It is important to note that Doppler signals of trace or mild valvular regurgitation of no clinical consequence can be detected with structurally normal tricuspid, PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Cardiac murmur Systolic murmur Diastolic murmur Continuous murmur Mid-systolic, grade 2 or less • Early systolic • Mid-systolic, grade 3 or more • Late systolic • Holosystolic Asymptomatic and no associated findings Symptomatic or other signs of cardiac disease* TEE, cardiac MR, catheterization if appropriate • Venous hum • Mammary souffle TTE No further workup enough turbulence to create an audible murmur.

1	Echocardiography is indicated for the evaluation of patients with early, late, or holosystolic murmurs and patients with grade 3 or louder mid-systolic murmurs. Patients with grade 1 or 2 mid-systolic murmurs but other symptoms or signs of cardiovascular disease, including those from ECG or chest x-ray, should also undergo echocardiography. Echocardiography is also indicated for the evaluation of any patient with a diastolic murmur and for patients with continuous murmurs not due to a venous hum or mammary souffle. Echocardiography also should be considered when there is a clinical need to verify normal cardiac structure and function in a patient whose symptoms and signs are probably noncardiac in origin. The performance of serial echocardiography to follow the course of asymptomatic individuals with valvular heart disease is a central feature of their longitudinal assessment and provides valuable information that may have an impact on decisions regarding the timing of surgery.

1	individuals with valvular heart disease is a central feature of their longitudinal assessment and provides valuable information that may have an impact on decisions regarding the timing of surgery. Routine echocardiography is not recommended for asymptomatic patients with a grade 1 or 2 mid-systolic murmur without other signs of heart disease. For this category of patients, referral to a cardiovascular specialist should be considered if there is doubt about the significance of the murmur after the initial examination.

1	The selective use of echocardiography outlined above has not been subjected to rigorous analysis of its cost-effectiveness. For some clinicians, handheld or miniaturized cardiac ultrasound devices have replaced the stethoscope. Although several reports attest to the improved sensitivity of such devices for the detection of valvular heart disease, accuracy is highly operator-dependent, and incremental cost considerations and outcomes have not been addressed adequately. The use of electronic or digital stethoscopes with spectral display capabilities has also been proposed as a method to improve the characterization of heart murmurs and the mentored teaching of cardiac auscultation.

1	(Chap. 270e, Fig. 51e-9) In relatively few patients, clinical assessment and TTE do not adequately characterize the origin and significance of a heart murmur. Transesophageal echocardiography (TEE) can be considered for further evaluation, especially when the TTE windows are limited by body size, chest configuration, or intrathoracic pathology. TEE offers enhanced sensitivity for the detection of a wide range of structural cardiac disorders. Electrocardiographically gated cardiac magnetic resonance (CMR) imaging, although limited in its ability to display valvular morphology, can provide quantitative information regarding valvular function, stenosis severity, regurgitant fraction, regurgitant volume, shunt flow, chamber and great vessel size, ventricular function, and myocardial perfusion. CMR has largely supplanted the need for cardiac catheterization and invasive hemodynamic assessment when there is a discrepancy between the clinical and echocardiographic findings. Invasive coronary

1	CMR has largely supplanted the need for cardiac catheterization and invasive hemodynamic assessment when there is a discrepancy between the clinical and echocardiographic findings. Invasive coronary angiography is performed routinely in most adult patients before valve surgery, especially when there is a suspicion of coronary artery disease predicated on symptoms, risk factors, and/or age. The use of computed tomography coronary angiography (CCTA) to exclude coronary artery disease in patients with a low pretest probability of disease before valve surgery is gaining wider acceptance.

1	The accurate identification of a heart murmur begins with a systematic approach to cardiac auscultation. Characterization of its major attributes, FIguRe 51e-9 Strategy for evaluating heart murmurs. *If an electrocardiogram or as reviewed above, allows the examiner to construct chest x-ray has been obtained and is abnormal, echocardiography is indicated. TTE, a preliminary differential diagnosis, which is then transthoracic echocardiography; TEE, transesophageal echocardiography; MR, magnetic refined by integration of information available resonance. (Adapted from RO Bonow et al: J Am Coll Cardiol 32:1486, 1998.) from the history, associated cardiac findings, the CHAPTER 51e Approach to the Patient with a Heart Murmur

1	CHAPTER 51e Approach to the Patient with a Heart Murmur Palpitations Joseph Loscalzo Palpitations are extremely common among patients who present to their internists and can best be defined as an intermittent “thumping,” “pounding,” or “fluttering” sensation in the chest. This sensation can be either intermittent or sustained and either regular or irregular. Most 52254 patients interpret palpitations as an unusual awareness of the heartbeat and become especially concerned when they sense that they have had “skipped” or “missing” heartbeats. Palpitations are often noted when the patient is quietly resting, during which time other stimuli are minimal. Palpitations that are positional generally reflect a structural process within (e.g., atrial myxoma) or adjacent to (e.g., mediastinal mass) the heart.

1	Palpitations are brought about by cardiac (43%), psychiatric (31%), miscellaneous (10%), and unknown (16%) causes, according to one large series. Among the cardiovascular causes are premature atrial and ventricular contractions, supraventricular and ventricular arrhythmias, mitral valve prolapse (with or without associated arrhythmias), aortic insufficiency, atrial myxoma, and pulmonary embolism. Intermittent palpitations are commonly caused by premature atrial or ventricular contractions: the post-extrasystolic beat is sensed by the patient owing to the increase in ventricular end-diastolic dimension following the pause in the cardiac cycle and the increased strength of contraction (post-extrasystolic potentiation) of that beat. Regular, sustained palpitations can be caused by regular supraventricular and ventricular tachycardias. Irregular, sustained palpitations can be caused by atrial fibrillation. It is important to note that most arrhythmias are not associated with palpitations.

1	and ventricular tachycardias. Irregular, sustained palpitations can be caused by atrial fibrillation. It is important to note that most arrhythmias are not associated with palpitations. In those that are, it is often useful either to ask the patient to “tap out” the rhythm of the palpitations or to take his/her pulse during palpitations. In general, hyperdynamic cardiovascular states caused by catecholaminergic stimulation from exercise, stress, or pheochromocytoma can lead to palpitations. Palpitations are common among athletes, especially older endurance athletes. In addition, the enlarged ventricle of aortic regurgitation and accompanying hyperdynamic precordium frequently lead to the sensation of palpitations. Other factors that enhance the strength of myocardial contraction, including tobacco, caffeine, aminophylline, atropine, thyroxine, cocaine, and amphetamines, can cause palpitations.

1	Psychiatric causes of palpitations include panic attacks or disorders, anxiety states, and somatization, alone or in combination. Patients with psychiatric causes for palpitations more commonly report a longer duration of the sensation (>15 min) and other accompanying symptoms than do patients with other causes. Among the miscellaneous causes of palpitations are thyrotoxicosis, drugs (see above) and ethanol, spontaneous skeletal muscle contractions of the chest wall, pheochromocytoma, and systemic mastocytosis. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: The principal goal in assessing patients with palpitations is to determine whether the symptom is caused by a life-threatening arrhythmia. Patients with preexisting coronary artery disease (CAD) or risk factors for CAD are at greatest risk for ventricular arrhythmias (Chap. 276) as a cause for palpitations. In addition, the association of palpitations with other symptoms suggesting hemodynamic compromise, including syncope or lightheadedness, supports this diagnosis. Palpitations caused by sustained tachyarrhythmias in patients with CAD can be accompanied by angina pectoris or dyspnea, and, in patients with ventricular dysfunction (systolic or diastolic), aortic stenosis, hypertrophic cardiomyopathy, or mitral stenosis (with or without CAD), can be accompanied by dyspnea from increased left atrial and pulmonary venous pressure.

1	Key features of the physical examination that will help confirm or refute the presence of an arrhythmia as a cause for palpitations (as well as its adverse hemodynamic consequences) include measurement of the vital signs, assessment of the jugular venous pressure and pulse, and auscultation of the chest and precordium. A resting electrocardiogram can be used to document the arrhythmia. If exertion is known to induce the arrhythmia and accompanying palpitations, exercise electrocardiography can be used to make the diagnosis. If the arrhythmia is sufficiently infrequent, other methods must be used, including continuous electrocardiographic (Holter) monitoring; telephonic monitoring, through which the patient can transmit an electrocardiographic tracing during a sensed episode; loop recordings (external or implantable), which can capture the electrocardiographic event for later review; and mobile cardiac outpatient telemetry. Data suggest that Holter monitoring is of limited clinical

1	(external or implantable), which can capture the electrocardiographic event for later review; and mobile cardiac outpatient telemetry. Data suggest that Holter monitoring is of limited clinical utility, while the implantable loop recorder and mobile cardiac outpatient telemetry are safe and possibly more cost-effective in the assessment of patients with (infrequent) recurrent, unexplained palpitations.

1	Most patients with palpitations do not have serious arrhythmias or underlying structural heart disease. If sufficiently troubling to the patient, occasional benign atrial or ventricular premature contractions can often be managed with beta-blocker therapy. Palpitations incited by alcohol, tobacco, or illicit drugs need to be managed by abstention, while those caused by pharmacologic agents should be addressed by considering alternative therapies when appropriate or possible. Psychiatric causes of palpitations may benefit from cognitive therapy or pharmacotherapy. The physician should note that palpitations are at the very least bothersome and, on occasion, frightening to the patient. Once serious causes for the symptom have been excluded, the patient should be reassured that the palpitations will not adversely affect prognosis. PART 2 Cardinal Manifestations and Presentation of Diseases Ikuo Hirano, Peter J. Kahrilas

1	Dysphagia—difficulty with swallowing—refers to problems with the transit of food or liquid from the mouth to the hypopharynx or through the esophagus. Severe dysphagia can compromise nutrition, cause aspiration, and reduce quality of life. Additional terminology pertaining to swallowing dysfunction is as follows. Aphagia (inability to swallow) typically denotes complete esophageal obstruction, most commonly encountered in the acute setting of a food bolus or foreign body impaction. Odynophagia refers to painful swallowing, typically resulting from mucosal ulceration within the oropharynx or esophagus. It commonly is accompanied by dysphagia, but the converse is not true. Globus pharyngeus is a foreign body sensation localized in the neck that does not interfere with swallowing and sometimes is relieved by swallowing. Transfer dysphagia frequently results in nasal regurgitation and pulmonary aspiration during swallowing and is characteristic of oropharyngeal dysphagia. Phagophobia

1	is relieved by swallowing. Transfer dysphagia frequently results in nasal regurgitation and pulmonary aspiration during swallowing and is characteristic of oropharyngeal dysphagia. Phagophobia (fear of swallowing) and refusal to swallow may be psychogenic or related to anticipatory anxiety about food bolus obstruction, odynophagia, or aspiration.

1	Swallowing begins with a voluntary (oral) phase that includes preparation during which food is masticated and mixed with saliva. This is followed by a transfer phase during which the bolus is pushed into the pharynx by the tongue. Bolus entry into the hypopharynx initiates the pharyngeal swallow response, which is centrally mediated and involves a complex series of actions, the net result of which is to propel food through the pharynx into the esophagus while preventing its entry into the airway. To accomplish this, the larynx is elevated and pulled forward, actions that also facilitate upper esophageal sphincter (UES) opening. Tongue pulsion then propels the bolus through the UES, followed by a peristaltic contraction that clears residue from the pharynx and through the esophagus. The lower esophageal sphincter (LES) relaxes as the food enters the esophagus and remains relaxed until the peristaltic contraction has delivered the bolus into the stomach. Peristaltic contractions

1	The lower esophageal sphincter (LES) relaxes as the food enters the esophagus and remains relaxed until the peristaltic contraction has delivered the bolus into the stomach. Peristaltic contractions elicited in response to a swallow are called primary peristalsis and involve sequenced inhibition followed by contraction of the musculature along the entire length of the esophagus. The inhibition that precedes the peristaltic contraction is called deglutitive inhibition. Local distention of the esophagus anywhere along its length, as may occur with gastroesophageal reflux, activates secondary peristalsis that begins at the point of distention and proceeds distally. Tertiary esophageal contractions are nonperistaltic, disordered esophageal contractions that may be observed to occur spontaneously during fluoroscopic observation.

1	The musculature of the oral cavity, pharynx, UES, and cervical esophagus is striated and directly innervated by lower motor neurons carried in cranial nerves (Fig. 53-1). Oral cavity muscles are innervated by the fifth (trigeminal) and seventh (facial) cranial nerves; the tongue, by the twelfth (hypoglossal) cranial nerve. Pharyngeal muscles are innervated by the ninth (glossopharyngeal) and tenth (vagus) cranial nerves.

1	Physiologically, the UES consists of the cricopharyngeus muscle, the adjacent inferior pharyngeal constrictor, and the proximal portion of the cervical esophagus. UES innervation is derived from the vagus nerve, whereas the innervation to the musculature acting on the UES to facilitate its opening during swallowing comes from the fifth, seventh, and twelfth cranial nerves. The UES remains closed at rest owing to both its inherent elastic properties and neurogenically mediated contraction of the cricopharyngeus muscle. UES opening during swallowing involves both cessation of vagal excitation to the cricopha-255 ryngeus and simultaneous contraction of the suprahyoid and geniohyoid muscles that pull open the UES in conjunction with the upward and forward displacement of the larynx.

1	The neuromuscular apparatus for peristalsis is distinct in proximal and distal parts of the esophagus. The cervical esophagus, like the pharyngeal musculature, consists of striated muscle and is directly innervated by lower motor neurons of the vagus nerve. Peristalsis in the proximal esophagus is governed by the sequential activation of the vagal motor neurons in the nucleus ambiguus. In contrast, the distal esophagus and LES are composed of smooth muscle and are controlled by excitatory and inhibitory neurons within the esophageal myenteric plexus. Medullary preganglionic neurons from the dorsal motor nucleus of the vagus trigger peristalsis via these ganglionic neurons during primary peristalsis. Neurotransmitters of the excitatory ganglionic neurons are acetylcholine and substance P; those of the inhibitory neurons are vasoactive intestinal peptide and nitric oxide. Peristalsis results from the patterned activation of inhibitory followed by excitatory ganglionic neurons, with

1	P; those of the inhibitory neurons are vasoactive intestinal peptide and nitric oxide. Peristalsis results from the patterned activation of inhibitory followed by excitatory ganglionic neurons, with progressive dominance of the inhibitory neurons distally. Similarly, LES relaxation occurs with the onset of deglutitive inhibition and persists until the peristaltic sequence is complete. At rest, the LES is contracted because of excitatory ganglionic stimulation and its intrinsic myogenic tone, a property that distinguishes it from the adjacent esophagus. The function of the LES is supplemented by the surrounding muscle of the right diaphragmatic crus, which acts as an external sphincter during inspiration, cough, or abdominal straining.

1	Dysphagia can be subclassified both by location and by the circumstances in which it occurs. With respect to location, distinct considerations apply to oral, pharyngeal, or esophageal dysphagia. Normal transport of an ingested bolus depends on the consistency and size of the bolus, the caliber of the lumen, the integrity of peristaltic contraction, and deglutitive inhibition of both the UES and the LES. Dysphagia caused by an oversized bolus or a narrow lumen is called structural dysphagia, whereas dysphagia due to abnormalities of peristalsis or impaired sphincter relaxation after swallowing is called propulsive or Sagittal view of the pharynx Soft palate Thyrohyoid membrane Cricothyroid membrane Hyoid bone Vocal cord Transverse arytenoid ms. Hard palate Epiglottis Digastric (post. belly) Oral cavity Mylohoid ms. (hypopharynx) (ant. belly) Middle constrictor

1	FIguRE 53-1 Sagittal and diagrammatic views of the musculature involved in enacting oropharyngeal swallowing. Note the dominance of the tongue in the sagittal view and the intimate relationship between the entrance to the larynx (airway) and the esophagus. In the resting configuration illustrated, the esophageal inlet is closed. This is transiently reconfigured such that the esophageal inlet is open and the laryngeal inlet closed during swallowing. (Adapted from PJ Kahrilas, in DW Gelfand and JE Richter [eds]: Dysphagia: Diagnosis and Treatment. New York: Igaku-Shoin Medical Publishers, 1989, pp. 11–28.) 256 motor dysphagia. More than one mechanism may be operative in a patient with dysphagia. Scleroderma commonly presents with absent peristalsis as well as a weakened LES that predisposes patients to peptic stricture formation. Likewise, radiation therapy for head and neck cancer may compound the functional deficits in the oropharyngeal swallow attributable to the tumor and cause

1	patients to peptic stricture formation. Likewise, radiation therapy for head and neck cancer may compound the functional deficits in the oropharyngeal swallow attributable to the tumor and cause cervical esophageal stenosis.

1	Oral and Pharyngeal (Oropharyngeal) Dysphagia Oral-phase dysphagia is associated with poor bolus formation and control so that food has prolonged retention within the oral cavity and may seep out of the mouth. Drooling and difficulty in initiating swallowing are other characteristic signs. Poor bolus control also may lead to premature spillage of food into the hypopharynx with resultant aspiration into the trachea or regurgitation into the nasal cavity. Pharyngeal-phase dysphagia is associated with retention of food in the pharynx due to poor tongue or pharyngeal propulsion or obstruction at the UES. Signs and symptoms of concomitant hoarseness or cranial nerve dysfunction may be associated with oropharyngeal dysphagia.

1	Oropharyngeal dysphagia may be due to neurologic, muscular, structural, iatrogenic, infectious, and metabolic causes. Iatrogenic, neurologic, and structural pathologies are most common. Iatrogenic causes include surgery and radiation, often in the setting of head and neck cancer. Neurogenic dysphagia resulting from cerebrovascular accidents, Parkinson’s disease, and amyotrophic lateral sclerosis is a major source of morbidity related to aspiration and malnutrition. Medullary nuclei directly innervate the oropharynx. Lateralization of pharyngeal dysphagia implies either a structural pharyngeal lesion or a neurologic process that selectively targeted the ipsilateral brainstem nuclei or cranial nerve. Advances in functional brain imaging have elucidated an important role of the cerebral cortex in swallow function and dysphagia. Asymmetry in the cortical representation of the pharynx provides an explanation for the dysphagia that occurs as a consequence of unilateral cortical

1	cortex in swallow function and dysphagia. Asymmetry in the cortical representation of the pharynx provides an explanation for the dysphagia that occurs as a consequence of unilateral cortical cerebrovascular accidents.

1	Oropharyngeal structural lesions causing dysphagia include Zenker’s diverticulum, cricopharyngeal bar, and neoplasia. Zenker’s diverticulum typically is encountered in elderly patients, with an estimated prevalence between 1:1000 and 1:10,000. In addition to dysphagia, patients may present with regurgitation of particulate food debris, aspiration, and halitosis. The pathogenesis is related to stenosis of the cricopharyngeus that causes diminished opening of the UES and results in increased hypopharyngeal pressure during swallowing with development of a pulsion diverticulum immediately above the cricopharyngeus in a region of potential weakness known as Killian’s dehiscence. A cricopharyngeal bar, appearing as a prominent indentation behind the lower third of the cricoid cartilage, is related to Zenker’s diverticulum in that it involves limited distensibility of the cricopharyngeus and can lead to the formation of a Zenker’s diverticulum. However, a cricopharyngeal bar is a common

1	related to Zenker’s diverticulum in that it involves limited distensibility of the cricopharyngeus and can lead to the formation of a Zenker’s diverticulum. However, a cricopharyngeal bar is a common radiographic finding, and most patients with transient cricopharyngeal bars are asymptomatic, making it important to rule out alternative etiologies of dysphagia before treatment. Furthermore, cricopharyngeal bars may be secondary to other neuromuscular disorders.

1	Since the pharyngeal phase of swallowing occurs in less than a second, rapid-sequence fluoroscopy is necessary to evaluate for functional abnormalities. Adequate fluoroscopic examination requires that the patient be conscious and cooperative. The study incorporates recordings of swallow sequences during ingestion of food and liquids of varying consistencies. The pharynx is examined to detect bolus retention, regurgitation into the nose, or aspiration into the trachea. Timing and integrity of pharyngeal contraction and opening of the UES with a swallow are analyzed to assess both aspiration risk and the potential for swallow therapy. Structural abnormalities of the oropharynx, especially those which may require biopsies, also should be assessed by direct laryngoscopic examination.

1	Esophageal Dysphagia The adult esophagus measures 18–26 cm in length and is anatomically divided into the cervical esophagus, extending from the pharyngoesophageal junction to the suprasternal notch, and the thoracic esophagus, which continues to the diaphragmatic hiatus. When distended, the esophageal lumen has internal dimensions of

1	PART 2 Cardinal Manifestations and Presentation of Diseases about 2 cm in the anteroposterior plane and 3 cm in the lateral plane. Solid food dysphagia becomes common when the lumen is narrowed to <13 mm but also can occur with larger diameters in the setting of poorly masticated food or motor dysfunction. Circumferential lesions are more likely to cause dysphagia than are lesions that involve only a partial circumference of the esophageal wall. The most common structural causes of dysphagia are Schatzki’s rings, eosinophilic esophagitis, and peptic strictures. Dysphagia also occurs in the setting of gastroesophageal reflux disease without a stricture, perhaps on the basis of altered esophageal sensation, distensibility, or motor function.

1	Propulsive disorders leading to esophageal dysphagia result from abnormalities of peristalsis and/or deglutitive inhibition, potentially affecting the cervical or thoracic esophagus. Since striated muscle pathology usually involves both the oropharynx and the cervical esophagus, the clinical manifestations usually are dominated by oropharyngeal dysphagia. Diseases affecting smooth muscle involve both the thoracic esophagus and the LES. A dominant manifestation of this, absent peristalsis, refers to either the complete absence of swallow-induced contraction or the presence of nonperistaltic, disordered contractions. Absent peristalsis and failure of deglutitive LES relaxation are the defining features of achalasia. In diffuse esophageal spasm (DES), LES function is normal, with the disordered motility restricted to the esophageal body. Absent peristalsis combined with severe weakness of the LES is a nonspecific pattern commonly found in patients with scleroderma.

1	APPROACH TO THE PATIENT: Figure 53-2 shows an algorithm for the approach to a patient with dysphagia.

1	The patient history is extremely valuable in making a presumptive diagnosis or at least substantially restricting the differential diagnoses in most patients. Key elements of the history are the localization of dysphagia, the circumstances in which dysphagia is experienced, other symptoms associated with dysphagia, and progression. Dysphagia that localizes to the suprasternal notch may indicate either an oropharyngeal or an esophageal etiology as distal dysphagia is referred proximally about 30% of the time. Dysphagia that localizes to the chest is esophageal in origin. Nasal regurgitation and tracheobronchial aspiration manifest by coughing with swallowing are hallmarks of oropharyngeal dysphagia. Severe cough with swallowing may also be a sign of a tracheoesophageal fistula. The presence of hoarseness may be another important diagnostic clue. When hoarseness precedes dysphagia, the primary lesion is usually laryngeal; hoarseness that occurs after the development of dysphagia may

1	of hoarseness may be another important diagnostic clue. When hoarseness precedes dysphagia, the primary lesion is usually laryngeal; hoarseness that occurs after the development of dysphagia may result from compromise of the recurrent laryngeal nerve by a malignancy. The type of food causing dysphagia is a crucial detail. Intermittent dysphagia that occurs only with solid food implies structural dysphagia, whereas constant dysphagia with both liquids and solids strongly suggests a motor abnormality. Two caveats to this pattern are that despite having a motor abnormality, patients with scleroderma generally develop mild dysphagia for solids only and, somewhat paradoxically, that patients with oropharyngeal dysphagia often have greater difficulty managing liquids than solids. Dysphagia that is progressive over the course of weeks to months raises concern for neoplasia. Episodic dysphagia to solids that is unchanged over years indicates a benign disease process such as a Schatzki’s ring

1	is progressive over the course of weeks to months raises concern for neoplasia. Episodic dysphagia to solids that is unchanged over years indicates a benign disease process such as a Schatzki’s ring or eosinophilic esophagitis. Food impaction with a prolonged inability to pass an ingested bolus even with ingestion of liquid is typical of a structural dysphagia. Chest pain frequently accompanies dysphagia whether it is related to motor disorders, structural disorders, or reflux disease. A prolonged history of heartburn preceding the onset of dysphagia is suggestive of peptic stricture and, infrequently, esophageal adenocarcinoma. A history of prolonged nasogastric intubation, esophageal or head and neck surgery, ingestion of caustic to neck, nasal regurgitation, aspiration, neck, food impaction

1	FIguRE 53-2 Approach to the patient with dysphagia. Etiologies in bold print are the most common. ENT, ear, nose, and throat; GERD, gastroesophageal reflux disease. agents or pills, previous radiation or chemotherapy, or associated mucocutaneous diseases may help isolate the cause of dysphagia. With accompanying odynophagia, which usually is indicative of ulceration, infectious or pill-induced esophagitis should be suspected. In patients with AIDS or other immunocompromised states, esophagitis due to opportunistic infections such as Candida, herpes simplex virus, or cytomegalovirus and to tumors such as Kaposi’s sarcoma and lymphoma should be considered. A strong history of atopy increases concerns for eosinophilic esophagitis.

1	Physical examination is important in the evaluation of oral and pharyngeal dysphagia because dysphagia is usually only one of many manifestations of a more global disease process. Signs of bulbar or pseudobulbar palsy, including dysarthria, dysphonia, ptosis, tongue atrophy, and hyperactive jaw jerk, in addition to evidence of generalized neuromuscular disease, should be elicited. The neck should be examined for thyromegaly. A careful inspection of the mouth and pharynx should disclose lesions that may interfere with passage of food. Missing dentition can interfere with mastication and exacerbate an existing cause of dysphagia. Physical examination is less helpful in the evaluation of esophageal dysphagia as most relevant pathology is restricted to the esophagus. The notable exception is skin disease. Changes in the skin may suggest a diagnosis of scleroderma or mucocutaneous diseases such as pemphigoid, lichen planus and epidermolysis bullosa, all of which can involve the esophagus.

1	Although most instances of dysphagia are attributable to benign disease processes, dysphagia is also a cardinal symptom of several malignancies, making it an important symptom to evaluate. Cancer may result in dysphagia due to intraluminal obstruction (esophageal or proximal gastric cancer, metastatic deposits), extrinsic compression (lymphoma, lung cancer), or paraneoplastic syndromes. Even when not attributable to malignancy, dysphagia is usually a manifestation of an identifiable and treatable disease entity, making its evaluation beneficial to the patient and gratifying to the practitioner. The specific diagnostic algorithm to pursue is guided by the details of the history (Fig. 53-2). If oral or pharyngeal dysphagia is suspected, a fluoroscopic swallow study, usually done by a swallow therapist, is the procedure of choice. Otolaryngoscopic and neurologic evaluation also can be important, depending on the circumstances. For suspected esophageal dysphagia, upper endoscopy is the

1	therapist, is the procedure of choice. Otolaryngoscopic and neurologic evaluation also can be important, depending on the circumstances. For suspected esophageal dysphagia, upper endoscopy is the single most useful test. Endoscopy allows better visualization of mucosal lesions than does barium radiography and also allows one to obtain mucosal biopsies. Endoscopic or histologic abnormalities are evident in the leading causes of esophageal dysphagia: Schatzki ring, gastroesophageal reflux disease and eosinophilic esophagitis. Furthermore, therapeutic intervention with esophageal dilation can be done as part of the procedure if it is deemed necessary. The emergence of eosinophilic esophagitis as a leading cause of dysphagia in both children and adults has led to the recommendation that esophageal mucosal biopsies be obtained routinely in the evaluation of unexplained dysphagia even if endoscopically identified esophageal mucosal lesions are absent. For cases of suspected esophageal

1	esophageal mucosal biopsies be obtained routinely in the evaluation of unexplained dysphagia even if endoscopically identified esophageal mucosal lesions are absent. For cases of suspected esophageal motility disorders, endoscopy is still the appropriate initial evaluation as neoplastic and inflammatory conditions can secondarily produce patterns of either achalasia or esophageal spasm. Esophageal manometry is done if dysphagia is not adequately explained by endoscopy or to confirm the diagnosis of a suspected esophageal motor disorder. Barium radiography can provide useful adjunctive information in cases of subtle or complex esophageal strictures, prior esophageal surgery, esophageal diverticula, or paraesophageal herniation. In specific cases, computed tomography (CT) examination and endoscopic ultrasonography may be useful.

1	Treatment of dysphagia depends on both the locus and the specific etiology. Oropharyngeal dysphagia most commonly results from functional deficits caused by neurologic disorders. In such circumstances, the treatment focuses on utilizing postures or maneuvers devised to reduce pharyngeal residue and enhance airway protection learned under the direction of a trained swallow therapist. Aspiration risk may be reduced by altering the consistency of ingested food and liquid. Dysphagia resulting from a cerebrovascular accident usually, but not always, spontaneously improves within the first few weeks after the event. More severe and persistent cases may require gastrostomy and enteral feeding. Patients with myasthenia gravis (Chap. 461) and polymyositis (Chap. 388) may respond to medical treatment of the primary neuromuscular disease. Surgical intervention with cricopharyngeal myotomy is usually not helpful, with the exception of specific disorders such as the idiopathic cricopharyngeal bar,

1	of the primary neuromuscular disease. Surgical intervention with cricopharyngeal myotomy is usually not helpful, with the exception of specific disorders such as the idiopathic cricopharyngeal bar, Zenker’s diverticulum, and oculopharyngeal muscular dystrophy. Chronic neurologic disorders such as Parkinson’s disease and amyotrophic lateral sclerosis may manifest with severe oropharyngeal dysphagia. Feeding by a nasogastric tube or an endoscopically placed gastrostomy tube may be considered for nutritional support; however, these maneuvers do not provide protection against aspiration of salivary secretions or refluxed gastric contents.

1	Treatment of esophageal dysphagia is covered in detail in Chap. 347. The majority of causes of esophageal dysphagia are effectively managed by means of esophageal dilatation using bougie or balloon dilators. Cancer and achalasia are often managed surgically, although endoscopic techniques are available for both palliation and primary therapy, respectively. Infectious etiologies respond to antimicrobial medications or treatment of the underlying immunosuppressive state. Finally, eosinophilic esophagitis has emerged as an important cause of dysphagia that is amenable to treatment by elimination of dietary allergens or administration of swallowed, topically acting glucocorticoids. PART 2 Cardinal Manifestations and Presentation of Diseases nausea, vomiting, and 54 indigestion William L. Hasler

1	William L. Hasler Nausea is the subjective feeling of a need to vomit. Vomiting (emesis) is the oral expulsion of gastrointestinal contents due to contractions of gut and thoracoabdominal wall musculature. Vomiting is contrasted with regurgitation, the effortless passage of gastric contents into the mouth. Rumination is the repeated regurgitation of food residue, which may be rechewed and reswallowed. In contrast to emesis, these phenomena may exhibit volitional control. Indigestion is a term encompassing a range of complaints including nausea, vomiting, heartburn, regurgitation, and dyspepsia (the presence of symptoms thought to originate in the gastroduodenal region). Some individuals with dyspepsia report predominantly epigastric burning, gnawing, or pain. Others experience postprandial fullness, early satiety (an inability to complete a meal due to premature fullness), bloating, eructation (belching), and anorexia.

1	Vomiting is coordinated by the brainstem and is effected by responses in the gut, pharynx, and somatic musculature. Mechanisms underlying nausea are poorly understood but likely involve the cerebral cortex, as nausea requires conscious perception. This is supported by functional brain imaging studies showing activation of a range of cerebral cortical regions during nausea. Coordination of Emesis Brainstem nuclei—including the nucleus tractus solitarius; dorsal vagal and phrenic nuclei; medullary nuclei regulating respiration; and nuclei that control pharyngeal, facial, and tongue movements—coordinate initiation of emesis. Neurokinin NK1, serotonin 5-HT3, and vasopressin pathways participate in this coordination.

1	Somatic and visceral muscles respond stereotypically during emesis. Inspiratory thoracic and abdominal wall muscles contract, producing high intrathoracic and intraabdominal pressures that evacuate the stomach. The gastric cardia herniates above the diaphragm, and the larynx moves upward to propel the vomitus. Distally migrating gut contractions are normally regulated by an electrical phenomenon, the slow wave, which cycles at 3 cycles/min in the stomach and 11 cycles/ min in the duodenum. During emesis, the slow wave is abolished and is replaced by orally propagating spikes that evoke retrograde contractions that assist in expulsion of gut contents.

1	Activators of Emesis Emetic stimuli act at several sites. Emesis evoked by unpleasant thoughts or smells originates in the brain, whereas cranial nerves mediate vomiting after gag reflex activation. Motion sickness and inner ear disorders act on the labyrinthine system. Gastric irritants and cytotoxic agents like cisplatin stimulate gastroduodenal vagal afferent nerves. Nongastric afferents are activated by intestinal and colonic obstruction and mesenteric ischemia. The area postrema, in the medulla, responds to bloodborne stimuli (emetogenic drugs, bacterial toxins, uremia, hypoxia, ketoacidosis) and is termed the chemoreceptor trigger zone.

1	Neurotransmitters mediating vomiting are selective for different sites. Labyrinthine disorders stimulate vestibular muscarinic M1 and histaminergic H1 receptors. Vagal afferent stimuli activate serotonin 5-HT3 receptors. The area postrema is served by nerves acting on 5-HT , M , H , and dopamine D subtypes. Cannabinoid CB pathways may participate in the cerebral cortex. Optimal pharmacologic therapy of vomiting requires understanding of these pathways. Nausea and vomiting are caused by conditions within and outside the gut as well as by drugs and circulating toxins (Table 54-1).

1	Intraperitoneal Disorders Visceral obstruction and inflammation of hollow and solid viscera may elicit vomiting. Gastric obstruction results from ulcers and malignancy, whereas small-bowel and colon blockage occur because of adhesions, benign or malignant tumors, volvulus, intussusception, or inflammatory diseases like Crohn’s disease. The superior mesenteric artery syndrome, occurring after weight loss or prolonged bed rest, results when the duodenum is compressed by the overlying superior mesenteric artery. Abdominal irradiation impairs intestinal motor function and induces strictures. Biliary colic causes nausea by acting on local afferent nerves. Vomiting with pancreatitis, cholecystitis, and appendicitis is due to visceral irritation and induction of ileus. Enteric infections with viruses like norovirus or rotavirus or bacteria such as Staphylococcus aureus and Bacillus cereus often cause vomiting, especially in children. Opportunistic infections like cytomegalovirus or herpes

1	viruses like norovirus or rotavirus or bacteria such as Staphylococcus aureus and Bacillus cereus often cause vomiting, especially in children. Opportunistic infections like cytomegalovirus or herpes simplex virus induce emesis in immunocompromised individuals.

1	Gut sensorimotor dysfunction often causes nausea and vomiting. Gastroparesis presents with symptoms of gastric retention with evidence of delayed gastric emptying and occurs after vagotomy or with pancreatic carcinoma, mesenteric vascular insufficiency, or organic diseases like diabetes, scleroderma, and amyloidosis. Idiopathic gastroparesis is the most common etiology. It occurs in the absence of systemic illness and may follow a viral illness, suggesting an infectious trigger. Intestinal pseudoobstruction is characterized by disrupted intestinal and colonic motor activity with retention of food residue and secretions; bacterial overgrowth; nutrient malabsorption; and symptoms of nausea, vomiting, bloating, pain, and altered defecation. Intestinal pseudoobstruction may be idiopathic, inherited as a familial visceral myopathy or neuropathy, result from systemic disease, or occur as a paraneoplastic consequence of malignancy (e.g., small-cell lung carcinoma). Patients with

1	inherited as a familial visceral myopathy or neuropathy, result from systemic disease, or occur as a paraneoplastic consequence of malignancy (e.g., small-cell lung carcinoma). Patients with gastroesophageal reflux may report nausea and vomiting, as do some with irritable bowel syndrome (IBS) or chronic constipation.

1	Other functional gastroduodenal disorders without organic abnormalities have been characterized in adults. Chronic idiopathic nausea is defined as nausea without vomiting occurring several times a week. Functional vomiting is defined as one or more vomiting episodes weekly in the absence of an eating disorder or psychiatric disease. Cyclic vomiting syndrome presents with periodic discrete episodes of relentless nausea and vomiting in children and adults and shows an association with migraine headaches, suggesting that some cases may be migraine variants. Some adult cases have been described in association with rapid gastric emptying. A related condition, cannabinoid hyperemesis syndrome, presents with cyclical vomiting with intervening well periods in individuals (mostly men) who use large quantities of cannabis over many years and resolves with its discontinuation. Pathologic behaviors such as taking prolonged hot baths or showers are associated with the syndrome. Rumination

1	use large quantities of cannabis over many years and resolves with its discontinuation. Pathologic behaviors such as taking prolonged hot baths or showers are associated with the syndrome. Rumination syndrome, characterized by repetitive regurgitation of recently ingested food, is often misdiagnosed as refractory vomiting.

1	Extraperitoneal Disorders Myocardial infarction and congestive heart failure may cause nausea and vomiting. Postoperative emesis occurs after 25% of surgeries, most commonly laparotomy and orthopedic surgery. Increased intracranial pressure from tumors, bleeding, abscess, or blockage of cerebrospinal fluid outflow produces vomiting with or without nausea. Patients with psychiatric illnesses including anorexia nervosa, bulimia nervosa, anxiety, and depression often report significant nausea that may be associated with delayed gastric emptying.

1	Medications and Metabolic Disorders Drugs evoke vomiting by action on the stomach (analgesics, erythromycin) or area postrema (opiates, anti-parkinsonian drugs). Other emetogenic agents include antibiotics, cardiac antiarrhythmics, antihypertensives, oral hypoglycemics, antide-259 pressants (selective serotonin and serotonin norepinephrine reuptake inhibitors), smoking cessation drugs (varenicline, nicotine), and contraceptives. Cancer chemotherapy causes vomiting that is acute (within hours of administration), delayed (after 1 or more days), or anticipatory. Acute emesis from highly emetogenic agents (e.g., cisplatin) is mediated by 5-HT3 pathways, whereas delayed emesis is less dependent on 5-HT3 mechanisms. Anticipatory nausea may respond to anxiolytic therapy rather than antiemetics.

1	Metabolic disorders elicit nausea and vomiting. Pregnancy is the most prevalent endocrinologic cause, and nausea affects 70% of women in the first trimester. Hyperemesis gravidarum is a severe form of nausea of pregnancy that produces significant fluid loss and electrolyte disturbances. Uremia, ketoacidosis, adrenal insufficiency, and parathyroid and thyroid disease are other metabolic etiologies. Circulating toxins evoke emesis via effects on the area postrema. Endogenous toxins are generated in fulminant liver failure, whereas exogenous enterotoxins may be produced by enteric bacterial infection. Ethanol intoxication is a common toxic etiology of nausea and vomiting. APPROACH TO THE PATIENT:

1	The history helps define the etiology of nausea and vomiting. Drugs, toxins, and infections often cause acute symptoms, whereas established illnesses evoke chronic complaints. Gastroparesis and pyloric obstruction elicit vomiting within an hour of eating. Emesis from intestinal blockage occurs later. Vomiting occurring within minutes of meal consumption prompts consideration of rumination syndrome. With severe gastric emptying delays, the vomitus may contain food residue ingested hours or days before. Hematemesis raises suspicion of an ulcer, malignancy, or Mallory-Weiss tear. Feculent emesis is noted with distal intestinal or colonic obstruction. Bilious vomiting excludes gastric obstruction, whereas emesis of undigested food is consistent with a Zenker’s diverticulum or achalasia. Vomiting can relieve abdominal pain from a bowel obstruction, but has no effect in pancreatitis or cholecystitis. Profound weight loss raises concern about malignancy or obstruction. Fevers suggest

1	Vomiting can relieve abdominal pain from a bowel obstruction, but has no effect in pancreatitis or cholecystitis. Profound weight loss raises concern about malignancy or obstruction. Fevers suggest inflammation. An intracranial source is considered if there are headaches or visual field changes. Vertigo or tinnitus indicates labyrinthine disease.

1	The physical examination complements the history. Orthostatic hypotension and reduced skin turgor indicate intravascular fluid loss. Pulmonary abnormalities raise concern for aspiration of vomitus. Abdominal auscultation may reveal absent bowel sounds with ileus. High-pitched rushes suggest bowel obstruction, whereas a succussion splash upon abrupt lateral movement of the patient is found with gastroparesis or pyloric obstruction. Tenderness or involuntary guarding raises suspicion of inflammation, whereas fecal blood suggests mucosal injury from ulcer, ischemia, or tumor. Neurologic disease presents with papilledema, visual field loss, or focal neural abnormalities. Neoplasm is suggested by palpation of masses or adenopathy.

1	For intractable symptoms or an elusive diagnosis, selected screening tests can direct clinical care. Electrolyte replacement is indicated for hypokalemia or metabolic alkalosis. Iron-deficiency anemia mandates a search for mucosal injury. Pancreaticobiliary disease is indicated by abnormal pancreatic or liver biochemistries, whereas endocrinologic, rheumatologic, or paraneoplastic etiologies are suggested by hormone or serologic abnormalities. If bowel obstruction is suspected, supine and upright abdominal radiographs may show intestinal air-fluid levels with reduced colonic air. Ileus is characterized by diffusely dilated air-filled bowel loops. CHAPTER 54 Nausea, Vomiting, and Indigestion

1	CHAPTER 54 Nausea, Vomiting, and Indigestion Anatomic studies may be indicated if initial testing is nondiagnostic. Upper endoscopy detects ulcers, malignancy, and retained gastric food residue in gastroparesis. Small-bowel barium radiography or computed tomography (CT) diagnoses partial bowel obstruction. Colonoscopy or contrast enema radiography detects colonic obstruction. Ultrasound or CT defines intraperitoneal inflammation; CT and magnetic resonance imaging (MRI) enterography provide superior definition of inflammation in Crohn’s disease. CT or MRI of the head can delineate intracranial disease. Mesenteric angiography, CT, or MRI is useful for suspected ischemia.

1	Gastrointestinal motility testing may detect an underlying motor disorder when anatomic abnormalities are absent. Gastroparesis commonly is diagnosed by gastric scintigraphy, by which emptying of a radiolabeled meal is measured. Isotopic breath tests and wireless motility capsule methods are alternatives tests to define gastroparesis in different regions of the world. Intestinal pseudoobstruction often is suggested by abnormal barium transit and luminal dilation on small-bowel contrast radiography. Delayed small-bowel transit also may be detected by wireless capsule techniques. Small-intestinal manometry can confirm the diagnosis and further characterize the motor abnormality as neuropathic or myopathic based on contractile patterns. Such investigation can obviate the need for surgical intestinal biopsy to evaluate for smooth muscle or neuronal degeneration. Combined ambulatory esophageal pH/impedance testing and high-resolution manometry can facilitate diagnosis of rumination

1	intestinal biopsy to evaluate for smooth muscle or neuronal degeneration. Combined ambulatory esophageal pH/impedance testing and high-resolution manometry can facilitate diagnosis of rumination syndrome.

1	PART 2 Cardinal Manifestations and Presentation of Diseases Therapy of vomiting is tailored to correcting remediable abnormalities if possible. Hospitalization is considered for severe dehydration, especially if oral fluid replenishment cannot be sustained. Once oral intake is tolerated, nutrients are restarted with low-fat liquids, because lipids delay gastric emptying. Foods high in indigestible residue are avoided because these prolong gastric retention. Controlling blood glucose in poorly controlled diabetics can reduce hospitalizations in gastroparesis.

1	The most commonly used antiemetic agents act on central nervous system sites (Table 54-2). Antihistamines like dimenhydrinate and meclizine and anticholinergics like scopolamine act on labyrinthine pathways to treat motion sickness and inner ear disorders. Dopamine D2 antagonists treat emesis evoked by area postrema stimuli and are used for medication, toxic, and metabolic etiologies. Dopamine antagonists cross the blood-brain barrier and cause anxiety, movement disorders, and hyperprolactinemic effects (galactorrhea, sexual dysfunction).

1	Other classes exhibit antiemetic properties. 5-HT3 antagonists such as ondansetron and granisetron can prevent postoperative vomiting, radiation therapy–induced symptoms, and cancer chemotherapy–induced emesis, but also are used for other causes of emesis with limited evidence for efficacy. Tricyclic antidepressant agents provide symptomatic benefit in patients with chronic idiopathic nausea and functional vomiting as well as in long-standing diabetic patients with nausea and vomiting. Other antidepressants such as mirtazapine and olanzapine also may exhibit antiemetic effects.

1	Drugs that stimulate gastric emptying are used for gastroparesis (Table 54-2). Metoclopramide, a combined 5-HT4 agonist and D2 antagonist, is effective in gastroparesis, but antidopaminergic side effects, such as dystonias and mood and sleep disturbances, limit use in ∼25% of cases. Erythromycin increases gastroduodenal motility by action on receptors for motilin, an endogenous stimulant of fasting motor activity. Intravenous erythromycin is useful for inpatients with refractory gastroparesis, but oral forms have some utility. Domperidone, a D2 antagonist not available in the United States, exhibits prokinetic and antiemetic effects but does not cross into most brain regions; thus, anxiety and dystonic reactions are rare. The main side effects of domperidone relate to induction of hyperprolactinemia via effects on pituitary regions served by a porous blood-brain barrier.

1	Refractory motility disorders pose significant challenges. Intestinal pseudoobstruction may respond to the somatostatin analogue octreotide, which induces propagative small-intestinal motor complexes. Acetylcholinesterase inhibitors such as pyridostigmine are also observed to benefit some patients with small-bowel dysmotility. Pyloric injections of botulinum toxin are reported in uncontrolled studies to reduce gastroparesis symptoms, but small controlled trials observe benefits no greater than sham treatments. Surgical pyloroplasty has improved symptoms in case series. Placing a feeding jejunostomy reduces hospitalizations and improves overall health in some patients with drug-refractory gastroparesis. Postvagotomy gastroparesis may improve with near-total gastric resection; similar operations are now being tried for other gastroparesis etiologies. Implanted gastric electrical stimulators may reduce symptoms, enhance nutrition, improve quality of life, and decrease health care

1	operations are now being tried for other gastroparesis etiologies. Implanted gastric electrical stimulators may reduce symptoms, enhance nutrition, improve quality of life, and decrease health care expenditures in medication-refractory gastroparesis, but small controlled trials do not report convincing benefits.

1	Safety concerns about selected antiemetics have been emphasized. Centrally acting antidopaminergics, especially metoclopramide, can cause irreversible movement disorders such as tardive dyskinesia, particularly in older patients. This complication should be carefully explained and documented in the medical record. Some agents with antiemetic properties including domperidone, erythromycin, tricyclics, and 5-HT3 antagonists can induce dangerous cardiac rhythm disturbances, especially in those with QTc interval prolongation on electrocardiography (ECG). Surveillance ECG testing has been advocated for some of these agents.

1	Some cancer chemotherapies are intensely emetogenic (Chap. 103e). Combining a 5-HT3 antagonist, an NK1 antagonist, and a glucocorticoid provides significant control of both acute and delayed vomiting after highly emetogenic chemotherapy. Unlike other drugs in the same class, the 5-HT3 antagonist palonosetron exhibits efficacy at preventing delayed chemotherapy-induced vomiting. Benzodiazepines such as lorazepam can reduce anticipatory nausea and vomiting. Miscellaneous therapies with benefit in chemotherapy-induced emesis include cannabinoids, olanzapine, and alternative therapies like ginger. Most antiemetic regimens produce greater reductions in vomiting than in nausea.

1	Clinicians should exercise caution in managing pregnant patients with nausea. Studies of the teratogenic effects of antiemetic agents provide conflicting results. Few controlled trials have been performed in nausea of pregnancy. Antihistamines such as meclizine and doxylamine, antidopaminergics such as prochlorperazine, and antiserotonergics such as ondansetron demonstrate limited efficacy. Some obstetricians offer alternative therapies such as pyridoxine, acupressure, or ginger. Managing cyclic vomiting syndrome is a challenge. Prophylaxis with tricyclic antidepressants, cyproheptadine, or β-adrenoceptor antagonists can reduce the severity and frequency of attacks. Intravenous 5-HT3 antagonists combined with the sedating effects of a benzodiazepine like lorazepam are a mainstay of treatment of acute flares. Small studies report benefits with antimigraine agents, including the 5-HT1 agonist sumatriptan, as well as selected anticonvulsants such as zonisamide and levetiracetam.

1	The most common causes of indigestion are gastroesophageal reflux and functional dyspepsia. Other cases are a consequence of organic illness. gastroesophageal Reflux Gastroesophageal reflux results from many 261 physiologic defects. Reduced lower esophageal sphincter (LES) tone contributes to reflux in scleroderma and pregnancy and may be a factor in some patients without systemic illness. Others exhibit frequent transient LES relaxations (TLESRs) that cause bathing of the esophagus by acid or nonacidic fluid. Overeating and aerophagia override the barrier function of the LES, whereas reductions in esophageal body motility or salivary secretion prolong fluid exposure. Increased intragastric pressure promotes gastroesophageal reflux in obesity. The role of hiatal hernias is controversial—most reflux patients have hiatal hernias, but most with hiatal hernias do not have excess heartburn.

1	gastric Motor Dysfunction Disturbed gastric motility may contribute to gastroesophageal reflux in up to one-third of cases. Delayed gastric emptying is also found in ∼30% of functional dyspeptics. Conversely, some dyspeptics exhibit rapid gastric emptying. The relation of these defects to symptom induction is uncertain; studies show poor correlation between symptom severity and degrees of motor dysfunction. Impaired gastric fundus relaxation after eating (i.e., accommodation) may underlie selected dyspeptic symptoms like bloating, nausea, and early satiety in ∼40% of patients.

1	Visceral Afferent Hypersensitivity Disturbed gastric sensation is another pathogenic factor in functional dyspepsia. Visceral hypersensitivity was first reported in IBS with demonstration of heightened perception of rectal balloon inflation without changes in compliance. Similarly, ∼35% of dyspeptic patients note discomfort with fundic distention to lower pressures than healthy controls. Others with dyspepsia exhibit hypersensitivity to chemical stimulation with capsaicin or with acid or lipid exposure in the duodenum. Some individuals with functional heartburn without increased acid or nonacid reflux are believed to have heightened perception of normal esophageal pH and volume.

1	Other Factors Helicobacter pylori has a clear etiologic role in peptic ulcer disease, but ulcers cause a minority of dyspepsia cases. H. pylori is a minor factor in the genesis of functional dyspepsia. Functional dyspepsia is associated with chronic fatigue, produces reduced physical and mental well-being, and is exacerbated by stress. Anxiety, depression, and somatization may have contributing roles in some cases. Functional MRI studies show increased activation of several brain regions, emphasizing contributions from central nervous system factors. Analgesics cause dyspepsia, whereas nitrates, calcium channel blockers, theophylline, and progesterone promote gastroesophageal reflux. Other stimuli that induce reflux include ethanol, tobacco, and caffeine via LES relaxation. Genetic factors may promote development of reflux and dyspepsia.

1	DIFFERENTIAL DIAgNOSIS gastroesophageal Reflux Disease Gastroesophageal reflux disease (GERD) is prevalent. Heartburn is reported once monthly by 40% of Americans and daily by 7–10%. Most cases of heartburn occur because of excess acid reflux, but reflux of nonacidic fluid produces similar symptoms. Alkaline reflux esophagitis produces GERD-like symptoms most often in patients who have had surgery for peptic ulcer disease. Ten percent of patients with heartburn exhibit normal esophageal acid exposure and no increase in nonacidic reflux (functional heartburn).

1	Functional Dyspepsia Nearly 25% of the populace has dyspepsia at least six times yearly, but only 10–20% present to clinicians. Functional dyspepsia, the cause of symptoms in >60% of dyspeptic patients, is defined as ≥3 months of bothersome postprandial fullness, early satiety, or epigastric pain or burning with symptom onset at least 6 months before diagnosis in the absence of organic cause. Functional dyspepsia is subdivided into postprandial distress syndrome, characterized by meal-induced fullness, early satiety, and discomfort, and epigastric pain syndrome, which presents with epigastric burning pain unrelated to meals. Most cases follow a benign course, but some with H. pylori infection or on nonsteroidal anti-inflammatory drugs CHAPTER 54 Nausea, Vomiting, and Indigestion 262 (NSAIDs) develop ulcers. As with idiopathic gastroparesis, some cases of functional dyspepsia result from prior infection.

1	CHAPTER 54 Nausea, Vomiting, and Indigestion 262 (NSAIDs) develop ulcers. As with idiopathic gastroparesis, some cases of functional dyspepsia result from prior infection. ulcer Disease In most GERD patients, there is no destruction of the esophagus. However, 5% develop esophageal ulcers, and some form strictures. Symptoms cannot distinguish nonerosive from erosive or ulcerative esophagitis. A minority of cases of dyspepsia stem from gastric or duodenal ulcers. The most common causes of ulcer disease are H. pylori infection and use of NSAIDs. Other rare causes of gastroduodenal ulcers include Crohn’s disease (Chap. 351) and Zollinger-Ellison syndrome (Chap. 348), resulting from gastrin overproduction by an endocrine tumor.

1	Malignancy Dyspeptic patients often seek care because of fear of cancer, but few cases result from malignancy. Esophageal squamous cell carcinoma occurs most often with long-standing tobacco or ethanol intake. Other risks include prior caustic ingestion, achalasia, and the hereditary disorder tylosis. Esophageal adenocarcinoma usually complicates prolonged acid reflux. Eight to 20% of GERD patients exhibit esophageal intestinal metaplasia, termed Barrett’s metaplasia, a condition that predisposes to esophageal adenocarcinoma (Chap. 109). Gastric malignancies include adenocarcinoma, which is prevalent in certain Asian societies, and lymphoma.

1	Other Causes Opportunistic fungal or viral esophageal infections may produce heartburn but more often cause odynophagia. Other causes of esophageal inflammation include eosinophilic esophagitis and pill esophagitis. Biliary colic is in the differential diagnosis of unexplained upper abdominal pain, but most patients with biliary colic report discrete acute episodes of right upper quadrant or epigastric pain rather than the chronic burning, discomfort, and fullness of dyspepsia. Twenty percent of patients with gastroparesis report a predominance of pain or discomfort rather than nausea and vomiting. Intestinal lactase deficiency as a cause of gas, bloating, and discomfort occurs in 15–25% of whites of northern European descent but is more common in blacks and Asians. Intolerance of other carbohydrates (e.g., fructose, sorbitol) produces similar symptoms. Small-intestinal bacterial overgrowth may cause dyspepsia, often associated with bowel dysfunction, distention, and malabsorption.

1	carbohydrates (e.g., fructose, sorbitol) produces similar symptoms. Small-intestinal bacterial overgrowth may cause dyspepsia, often associated with bowel dysfunction, distention, and malabsorption. Eosinophilic infiltration of the duodenal mucosa is described in some dyspeptics, particularly with postprandial distress syndrome. Celiac disease, pancreatic disease (chronic pancreatitis, malignancy), hepatocellular carcinoma, Ménétrier’s disease, infiltrative diseases (sarcoidosis, eosinophilic gastroenteritis), mesenteric ischemia, thyroid and parathyroid disease, and abdominal wall strain cause dyspepsia. Gluten sensitivity in the absence of celiac disease is reported to evoke unexplained upper abdominal symptoms. Extraperitoneal etiologies of indigestion include congestive heart failure and tuberculosis.

1	APPROACH TO THE PATIENT: PART 2 Cardinal Manifestations and Presentation of Diseases Care of the indigestion patient requires a thorough interview. GERD classically produces heartburn, a substernal warmth that moves toward the neck. Heartburn often is exacerbated by meals and may awaken the patient. Associated symptoms include regurgitation of acid or nonacidic fluid and water brash, the reflex release of salty salivary secretions into the mouth. Atypical symptoms include pharyngitis, asthma, cough, bronchitis, hoarseness, and chest pain that mimics angina. Some patients with acid reflux on esophageal pH testing do not report heartburn, but note abdominal pain or other symptoms.

1	Dyspeptic patients typically report symptoms referable to the upper abdomen that may be meal-related, as with postprandial distress syndrome, or independent of food ingestion, as in epigastric pain syndrome. Functional dyspepsia overlaps with other disorders including GERD, IBS, and idiopathic gastroparesis. Family history of gastrointestinal malignancy The physical exam with GERD and functional dyspepsia usually is normal. In atypical GERD, pharyngeal erythema and wheezing may be noted. Recurrent acid regurgitation may cause poor denti tion. Dyspeptics may exhibit epigastric tenderness or distention.

1	Discriminating functional and organic causes of indigestion man dates excluding certain historic and exam features. Odynophagia suggests esophageal infection. Dysphagia is concerning for a benign or malignant esophageal blockage. Other alarm features include unexplained weight loss, recurrent vomiting, occult or gross bleed ing, jaundice, palpable mass or adenopathy, and a family history of gastrointestinal neoplasm. Because indigestion is prevalent and most cases result from GERD or functional dyspepsia, a general principle is to perform only lim ited and directed diagnostic testing of selected individuals.

1	Once alarm factors are excluded (Table 54-3), patients with typi cal GERD do not need further evaluation and are treated empiri cally. Upper endoscopy is indicated to exclude mucosal injury in cases with atypical symptoms, symptoms unresponsive to acid sup pression, or alarm factors. For heartburn >5 years in duration, espe cially in patients >50 years old, endoscopy is advocated to screen for Barrett’s metaplasia. The benefits and cost-effectiveness of this approach have not been validated in controlled studies. Ambulatory endoscopically attached to the esophageal wall is considered for chest pain. High-resolution esophageal manometry is ordered when surgical treatment of GERD is considered. A low LES pressure predicts failure of drug therapy and provides a rationale to proceed to surgery. Poor esophageal body peristalsis raises concern about postoperative dysphagia and directs the choice of surgical tech nique. Nonacidic reflux may be detected by combined esophageal impedance-pH

1	Poor esophageal body peristalsis raises concern about postoperative dysphagia and directs the choice of surgical tech nique. Nonacidic reflux may be detected by combined esophageal impedance-pH testing in medication-unresponsive patients.

1	Upper endoscopy is recommended as the initial test in patients with unexplained dyspepsia who are >55 years old or who have alarm factors because of the purported elevated risks of malig nancy and ulcer in these groups. However, endoscopic findings in unexplained dyspepsia include erosive esophagitis in 13%, peptic ulcer in 8%, and gastric or esophageal malignancy in only 0.3%. Management of patients <55 years old without alarm factors depends on the local prevalence of H. pylori infection. In regions with low H. pylori prevalence (<10%), a 4-week trial of an acid- suppressing medication such as a proton pump inhibitor (PPI) is recommended. If this fails, a “test and treat” approach is most commonly applied. H. pylori status is determined with urea breath testing, stool antigen measurement, or blood serology testing.

1	Those who are H. pylori positive are given therapy to eradicate the infection. If symptoms resolve on either regimen, no further inter vention is required. For patients in areas with high H. pylori preva lence (>10%), an initial test and treat approach is advocated, with a subsequent trial of an acid-suppressing regimen offered for those in whom H. pylori treatment fails or for those who are negative for the infection. In each of these patient subsets, upper endoscopy is reserved for those whose symptoms fail to respond to therapy.

1	Further testing is indicated in some settings. If bleeding is noted, a blood count can exclude anemia. Thyroid chemistries or calcium levels screen for metabolic disease, whereas specific serologies may suggest celiac disease. Pancreatic and liver chemistries are obtained for possible pancreaticobiliary causes. Ultrasound, CT, or MRI is performed if abnormalities are found. Gastric emptying testing is considered to exclude gastroparesis for dyspeptic symptoms that resemble postprandial distress when drug therapy fails and in some GERD patients, especially if surgical intervention is an option. Breath testing after carbohydrate ingestion detects lactase deficiency, intolerance to other carbohydrates, or small-intestinal bacterial overgrowth.

1	For mild indigestion, reassurance that a careful evaluation revealed no serious organic disease may be the only intervention needed. Drugs that cause gastroesophageal reflux or dyspepsia should be stopped, if possible. Patients with GERD should limit ethanol, caffeine, chocolate, and tobacco use due to their effects on the LES. Other measures in GERD include ingesting a low-fat diet, avoiding snacks before bedtime, and elevating the head of the bed. Patients with functional dyspepsia also may be advised to reduce intake of fat, spicy foods, caffeine, and alcohol. Specific therapies for organic disease should be offered when possible. Surgery is appropriate for biliary colic, whereas diet changes are indicated for lactase deficiency or celiac disease. Peptic ulcers may be cured by specific medical regimens. However, because most indigestion is caused by GERD or functional dyspepsia, medications that reduce gastric acid, modulate motility, or blunt gastric sensitivity are used.

1	Drugs that reduce or neutralize gastric acid are often prescribed for GERD. Histamine H2 antagonists like cimetidine, ranitidine, famotidine, and nizatidine are useful in mild to moderate GERD. For severe symptoms or for many cases of erosive or ulcerative esophagitis, PPIs such as omeprazole, lansoprazole, rabeprazole, pantoprazole, esomeprazole, or dexlansoprazole are needed. These drugs inhibit gastric H+, K+-ATPase and are more potent than H2 antagonists. Up to one-third of GERD patients do not respond to standard PPI doses; one-third of these patients have nonacidic reflux, whereas 10% have persistent acid-related disease. Furthermore, heartburn typically responds better to PPI therapy than regurgitation or atypical GERD symptoms. Some individuals respond to doubling of the PPI dose or adding an H2 antagonist at bedtime. Infrequent complications of long-term PPI therapy include infection, diarrhea (from Clostridium difficile infection or microscopic colitis), small-intestinal

1	or adding an H2 antagonist at bedtime. Infrequent complications of long-term PPI therapy include infection, diarrhea (from Clostridium difficile infection or microscopic colitis), small-intestinal bacterial overgrowth, nutrient deficiency (vitamin B12, iron, calcium), hypomagnesemia, bone demineralization, interstitial nephritis, and impaired medication absorption (e.g., clopidogrel). Many patients started on a PPI can be stepped down to an H2 antagonist or be switched to an on-demand schedule.

1	Acid-suppressing drugs are also effective in selected patients with functional dyspepsia. A meta-analysis of eight controlled trials calculated a risk ratio of 0.86, with a 95% confidence interval of 0.78–0.95, favoring PPI therapy over placebo. H2 antagonists also reportedly improve symptoms in functional dyspepsia; however, findings of trials of this drug class likely are influenced by inclusion of large numbers of GERD patients. Antacids are useful for short-term control of mild GERD but have less benefit in severe cases unless given at high doses that cause side effects (diarrhea and constipation with magnesiumand aluminum-containing agents, respectively). Alginic acid combined with antacids forms a floating barrier to reflux in patients with upright symptoms. Sucralfate, a salt of aluminum hydroxide and sucrose octasulfate that buffers acid and binds pepsin and bile salts, shows efficacy in GERD similar to H2 antagonists.

1	H. pylori eradication is definitively indicated only for peptic ulcer and mucosa-associated lymphoid tissue gastric lymphoma. The utility of eradication therapy in functional dyspepsia is limited, although some cases (particularly with the epigastric pain syndrome subtype) relate to this infection. A meta-analysis of 18 controlled trials calculated a relative risk reduction of 10%, with a 95% confidence interval of 6–14%, favoring H. pylori eradication over placebo. Most drug combinations (Chaps. 188 and 348) include 10–14 days of a PPI or bismuth subsalicylate in concert with two antibiotics. H. pylori infection is associated with reduced prevalence of GERD, especially in the elderly. However, eradication of the infection does not worsen GERD symptoms. No consensus recommendations regarding H. pylori eradication in GERD patients have been offered.

1	Prokinetics like metoclopramide, erythromycin, and domperidone have limited utility in GERD. The γ-aminobutyric acid B (GABA-B) agonist baclofen reduces esophageal exposure to acid and non-acidic fluids by reducing TLESRs by 40%; this drug is proposed for refractory acid and nonacid reflux. Several studies have promoted the efficacy of motor-stimulating drugs in functional dyspepsia, but publication bias and small sample sizes raise questions about reported benefits of these agents. Some clinicians suggest that patients with the postprandial distress subtype may respond preferentially to prokinetic drugs. The 5-HT1 agonist buspirone may improve some functional dyspepsia symptoms by enhancing meal-induced gastric accommodation. Acotiamide promotes gastric emptying and augments accommodation by enhancing gastric acetylcholine release via muscarinic receptor antagonism and acetylcholinesterase inhibition. This agent is approved for functional dyspepsia in Japan and is in testing

1	by enhancing gastric acetylcholine release via muscarinic receptor antagonism and acetylcholinesterase inhibition. This agent is approved for functional dyspepsia in Japan and is in testing elsewhere.

1	Antireflux surgery (fundoplication) to increase LES pressure may be offered to GERD patients who are young and require lifelong therapy, have typical heartburn and regurgitation, are responsive to PPIs, and show evidence of acid reflux on pH monitoring. Surgery also is effective for some cases of nonacidic reflux. Individuals who respond less well to fundoplication include those with atypical symptoms or who have esophageal body motor disturbances. Dysphagia, gas-bloat syndrome, and gastroparesis are long-term complications of these procedures; ∼60% develop recurrent GERD symptoms over time. The utility and safety of endoscopic therapies (radiofrequency ablation, transoral incisionless fundoplication) to enhance gastroesophageal barrier function have unproved durable benefits for refractory GERD.

1	Some patients with functional heartburn and functional dyspepsia refractory to standard therapies may respond to antidepressants in tricyclic and selective serotonin reuptake inhibitor classes, although studies are limited. Their mechanism of action may involve blunting of visceral pain processing in the brain. Gas and bloating are among the most troubling symptoms in some patients with indigestion and can be difficult to treat. Dietary exclusion of gas-producing foods such as legumes and use of simethicone or activated charcoal provide benefits in some cases. Low FODMAP (fermentable oligosaccharide, disaccharide, monosaccharide, and polyol) diets and therapies to modify gut flora (nonabsorbable antibiotics, probiotics) reduce gaseous symptoms in some IBS patients. The utility of low-FODMAP diets, antibiotics, and probiotics in functional dyspepsia is unproven. Herbal remedies such as STW 5 (Iberogast, a mixture of nine herbal agents) are useful in some dyspeptic patients.

1	of low-FODMAP diets, antibiotics, and probiotics in functional dyspepsia is unproven. Herbal remedies such as STW 5 (Iberogast, a mixture of nine herbal agents) are useful in some dyspeptic patients. Psychological treatments (e.g., behavioral therapy, psychotherapy, hypnotherapy) may be offered for refractory functional dyspepsia, but no convincing data confirm their efficacy.

1	CHAPTER 54 Nausea, Vomiting, and Indigestion

1	Diarrhea and Constipation Michael Camilleri, Joseph A. Murray Diarrhea and constipation are exceedingly common and, together, exact an enormous toll in terms of mortality, morbidity, social inconvenience, loss of work productivity, and consumption of medi-cal resources. Worldwide, >1 billion individuals suffer one or more 55264 episodes of acute diarrhea each year. Among the 100 million persons affected annually by acute diarrhea in the United States, nearly half must restrict activities, 10% consult physicians, ∼250,000 require hospitalization, and ∼5000 die (primarily the elderly). The annual economic burden to society may exceed $20 billion. Acute infectious diarrhea remains one of the most common causes of mortality in developing countries, particularly among impoverished infants, accounting for 1.8 million deaths per year. Recurrent, acute diarrhea in children in tropical countries results in environmental enteropathy with longterm impacts on physical and intellectual

1	accounting for 1.8 million deaths per year. Recurrent, acute diarrhea in children in tropical countries results in environmental enteropathy with longterm impacts on physical and intellectual development.

1	Constipation, by contrast, is rarely associated with mortality and is exceedingly common in developed countries, leading to frequent self-medication and, in a third of those, to medical consultation. Population statistics on chronic diarrhea and constipation are more uncertain, perhaps due to variable definitions and reporting, but the frequency of these conditions is also high. United States population surveys put prevalence rates for chronic diarrhea at 2–7% and for chronic constipation at 12–19%, with women being affected twice as often as men. Diarrhea and constipation are among the most common patient complaints presenting to internists and primary care physicians, and they account for nearly 50% of referrals to gastroenterologists.

1	Although diarrhea and constipation may present as mere nuisance symptoms at one extreme, they can be severe or life-threatening at the other. Even mild symptoms may signal a serious underlying gastrointestinal lesion, such as colorectal cancer, or systemic disorder, such as thyroid disease. Given the heterogeneous causes and potential severity of these common complaints, it is imperative for clinicians to appreciate the pathophysiology, etiologic classification, diagnostic strategies, and principles of management of diarrhea and constipation, so that rational and cost-effective care can be delivered.

1	While the primary function of the small intestine is the digestion and assimilation of nutrients from food, the small intestine and colon together perform important functions that regulate the secretion and absorption of water and electrolytes, the storage and subsequent transport of intraluminal contents aborally, and the salvage of some nutrients that are not absorbed in the small intestine after bacterial metabolism of carbohydrate allows salvage of short-chain fatty acids. The main motor functions are summarized in Table 55-1. Alterations in fluid and electrolyte handling contribute significantly to diarrhea. Alterations in motor and sensory functions of the colon result in Accommodation, trituration, mixing, transit Stomach ∼3 h Small bowel ∼3 h Colon: Irregular Mixing, Fermentation, Absorption, Transit Ascending, transverse: reservoirs Descending: conduit Sigmoid/rectum: volitional reservoir Abbreviation: MMC, migrating motor complex.

1	Colon: Irregular Mixing, Fermentation, Absorption, Transit Ascending, transverse: reservoirs Descending: conduit Sigmoid/rectum: volitional reservoir Abbreviation: MMC, migrating motor complex. PART 2 Cardinal Manifestations and Presentation of Diseases highly prevalent syndromes such as irritable bowel syndrome (IBS), chronic diarrhea, and chronic constipation.

1	PART 2 Cardinal Manifestations and Presentation of Diseases highly prevalent syndromes such as irritable bowel syndrome (IBS), chronic diarrhea, and chronic constipation. The small intestine and colon have intrinsic and extrinsic innervation. The intrinsic innervation, also called the enteric nervous system, comprises myenteric, submucosal, and mucosal neuronal layers. The function of these layers is modulated by interneurons through the actions of neurotransmitter amines or peptides, including acetylcholine, vasoactive intestinal peptide (VIP), opioids, norepinephrine, serotonin, adenosine triphosphate (ATP), and nitric oxide (NO). The myenteric plexus regulates smooth-muscle function through intermediary pacemaker-like cells called the interstitial cells of Cajal, and the submucosal plexus affects secretion, absorption, and mucosal blood flow. The enteric nervous system receives input from the extrinsic nerves, but it is capable of independent control of these functions.

1	The extrinsic innervations of the small intestine and colon are part of the autonomic nervous system and also modulate motor and secretory functions. The parasympathetic nerves convey visceral sensory pathways from and excitatory pathways to the small intestine and colon. Parasympathetic fibers via the vagus nerve reach the small intestine and proximal colon along the branches of the superior mesenteric artery. The distal colon is supplied by sacral parasympathetic nerves ) via the pelvic plexus; these fibers course through the wall of the (S2–4 colon as ascending intracolonic fibers as far as, and in some instances including, the proximal colon. The chief excitatory neurotransmitters controlling motor function are acetylcholine and the tachykinins, such as substance P. The sympathetic nerve supply modulates motor functions and reaches the small intestine and colon alongside their arterial vessels. Sympathetic input to the gut is generally excitatory to sphincters and inhibitory to

1	nerve supply modulates motor functions and reaches the small intestine and colon alongside their arterial vessels. Sympathetic input to the gut is generally excitatory to sphincters and inhibitory to non-sphincteric muscle. Visceral afferents convey sensation from the gut to the central nervous system (CNS); initially, they course along sympathetic fibers, but as they approach the spinal cord they separate, have cell bodies in the dorsal root ganglion, and enter the dorsal horn of the spinal cord. Afferent signals are conveyed to the brain along the lateral spinothalamic tract and the nociceptive dorsal column pathway and are then projected beyond the thalamus and brainstem to the insula and cerebral cortex to be perceived. Other afferent fibers synapse in the prevertebral ganglia and reflexly modulate intestinal motility, blood flow, and secretion.

1	On an average day, 9 L of fluid enter the gastrointestinal (GI) tract, ∼1 L of residual fluid reaches the colon, and the stool excretion of fluid constitutes about 0.2 L/d. The colon has a large capacitance and functional reserve and may recover up to four times its usual volume of 0.8 L/d, provided the rate of flow permits reabsorption to occur. Thus, the colon can partially compensate for excess fluid delivery to the colon that may result from intestinal absorptive or secretory disorders.

1	In the small intestine and colon, sodium absorption is predominantly electrogenic (i.e., it can be measured as an ionic current across the membrane because there is not an equivalent loss of a cation from the cell), and uptake takes place at the apical membrane; it is compensated for by the export functions of the basolateral sodium pump. There are several active transport proteins at the apical membrane, especially in the small intestine, whereby sodium ion entry is coupled to monosaccharides (e.g., glucose through the transporter SGLT1, or fructose through GLUT-5). Glucose then exits the basal membrane through a specific transport protein, GLUT-5, creating a glucose concentration gradient between the lumen and the intercellular space, drawing water and electrolytes passively from the lumen. A variety of neural and nonneural mediators regulate colonic fluid and electrolyte balance, including cholinergic, adrenergic, and serotonergic mediators. Angiotensin and aldosterone also

1	the lumen. A variety of neural and nonneural mediators regulate colonic fluid and electrolyte balance, including cholinergic, adrenergic, and serotonergic mediators. Angiotensin and aldosterone also influence colonic absorption, reflecting the common embryologic development of the distal colonic epithelium and the renal tubules.

1	During the fasting period, the motility of the small intestine is characterized by a cyclical event called the migrating motor complex (MMC), which serves to clear nondigestible residue from the small intestine (the intestinal “housekeeper”). This organized, propagated series of contractions lasts, on average, 4 min, occurs every 60–90 min, and usually involves the entire small intestine. After food ingestion, the small intestine produces irregular, mixing contractions of relatively low amplitude, except in the distal ileum where more powerful contractions occur intermittently and empty the ileum by bolus transfers.

1	The distal ileum acts as a reservoir, emptying intermittently by bolus movements. This action allows time for salvage of fluids, electrolytes, and nutrients. Segmentation by haustra compartmentalizes the colon and facilitates mixing, retention of residue, and formation of solid stools. There is increased appreciation of the intimate interaction between the colonic function and the luminal ecology. The resident microorganisms, predominantly anaerobic bacteria, in the colon are necessary for the digestion of unabsorbed carbohydrates that reach the colon even in health, thereby providing a vital source of nutrients to the mucosa. Normal colonic flora also keeps pathogens at bay by a variety of mechanisms. In health, the ascending and transverse regions of colon function as reservoirs (average transit time, 15 h), and the descending colon acts as a conduit (average transit time, 3 h). The colon is efficient at conserving sodium and water, a function that is particularly important in

1	transit time, 15 h), and the descending colon acts as a conduit (average transit time, 3 h). The colon is efficient at conserving sodium and water, a function that is particularly important in sodium-depleted patients in whom the small intestine alone is unable to maintain sodium balance. Diarrhea or constipation may result from alteration in the reservoir function of the proximal colon or the propulsive function of the left colon. Constipation may also result from disturbances of the rectal or sigmoid reservoir, typically as a result of dysfunction of the pelvic floor, the anal sphincters, the coordination of defecation, or dehydration.

1	The small intestinal MMC only rarely continues into the colon. However, short duration or phasic contractions mix colonic contents, and high-amplitude (>75 mmHg) propagated contractions (HAPCs) are sometimes associated with mass movements through the colon and normally occur approximately five times per day, usually on awakening in the morning and postprandially. Increased frequency of HAPCs may result in diarrhea or urgency. The predominant phasic contractions in the colon are irregular and non-propagated and serve a “mixing” function. Colonic tone refers to the background contractility upon which phasic contractile activity (typically contractions lasting <15 s) is superimposed. It is an important cofactor in the colon’s capacitance (volume accommodation) and sensation.

1	After meal ingestion, colonic phasic and tonic contractility increase for a period of ∼2 h. The initial phase (∼10 min) is mediated by the vagus nerve in response to mechanical distention of the stomach. The subsequent response of the colon requires caloric stimulation (e.g., intake of at least 500 kcal) and is mediated, at least in part, by hormones (e.g., gastrin and serotonin). Tonic contraction of the puborectalis reflex sympathetic innervation. As sigmoid and rectal contractions, as 265 well as straining (Valsalva maneuver), which increases intraabdominal pressure, increase the pressure within the rectum, the rectosigmoid angle opens by >15°. Voluntary relaxation of the external anal sphincter (striated muscle innervated by the pudendal nerve) in response to the sensation produced by distention permits the evacuation of feces. Defecation can also be delayed voluntarily by contraction of the external anal sphincter.

1	Diarrhea is loosely defined as passage of abnormally liquid or unformed stools at an increased frequency. For adults on a typical Western diet, stool weight >200 g/d can generally be considered diarrheal. Diarrhea may be further defined as acute if <2 weeks, persistent if 2–4 weeks, and chronic if >4 weeks in duration.

1	Two common conditions, usually associated with the passage of stool totaling <200 g/d, must be distinguished from diarrhea, because diagnostic and therapeutic algorithms differ. Pseudodiarrhea, or the frequent passage of small volumes of stool, is often associated with rectal urgency, tenesmus, or a feeling of incomplete evacuation, and accompanies IBS or proctitis. Fecal incontinence is the involuntary discharge of rectal contents and is most often caused by neuromuscular disorders or structural anorectal problems. Diarrhea and urgency, especially if severe, may aggravate or cause incontinence. Pseudodiarrhea and fecal incontinence occur at prevalence rates comparable to or higher than that of chronic diarrhea and should always be considered in patients complaining of “diarrhea.” Overflow diarrhea may occur in nursing home patients due to fecal impaction that is readily detectable by rectal examination. A careful history and physical examination generally allow these conditions to be

1	diarrhea may occur in nursing home patients due to fecal impaction that is readily detectable by rectal examination. A careful history and physical examination generally allow these conditions to be discriminated from true diarrhea.

1	More than 90% of cases of acute diarrhea are caused by infectious agents; these cases are often accompanied by vomiting, fever, and abdominal pain. The remaining 10% or so are caused by medications, toxic ingestions, ischemia, food indiscretions, and other conditions.

1	Infectious Agents Most infectious diarrheas are acquired by fecal-oral transmission or, more commonly, via ingestion of food or water contaminated with pathogens from human or animal feces. In the immunocompetent person, the resident fecal microflora, containing >500 taxonomically distinct species, are rarely the source of diarrhea muscle, which forms a sling around the rectoanal junction, is important AB Descent of the pelvic floor to maintain continence; during def-FIguRE 55-1 Sagittal view of the anorectum (A) at rest and (B) during straining to defecate. ecation, sacral parasympathetic nerves Continence is maintained by normal rectal sensation and tonic contraction of the internal anal relax this muscle, facilitating the sphincter and the puborectalis muscle, which wraps around the anorectum, maintaining an anorectal straightening of the rectoanal angle angle between 80° and 110°. During defecation, the pelvic floor muscles (including the puborectalis) (Fig. 55-1). Distention of

1	maintaining an anorectal straightening of the rectoanal angle angle between 80° and 110°. During defecation, the pelvic floor muscles (including the puborectalis) (Fig. 55-1). Distention of the rectum relax, allowing the anorectal angle to straighten by at least 15°, and the perineum descends by 1–3.5 cm. results in transient relaxation of the The external anal sphincter also relaxes and reduces pressure on the anal canal. (Reproduced with internal anal sphincter via intrinsic and permission from A Lembo, M Camilleri: N Engl J Med 349:1360, 2003.) 266 and may actually play a role in suppressing the growth of ingested pathogens. Disturbances of flora by antibiotics can lead to diarrhea by reducing the digestive function or by allowing the overgrowth of pathogens, such as Clostridium difficile (Chap. 161). Acute infection or injury occurs when the ingested agent overwhelms or bypasses the host’s mucosal immune and nonimmune (gastric acid, digestive enzymes, mucus secretion, peristalsis,

1	(Chap. 161). Acute infection or injury occurs when the ingested agent overwhelms or bypasses the host’s mucosal immune and nonimmune (gastric acid, digestive enzymes, mucus secretion, peristalsis, and suppressive resident flora) defenses. Established clinical associations with specific enteropathogens may offer diagnostic clues. In the United States, five high-risk groups are recognized: 1.

1	Travelers. Nearly 40% of tourists to endemic regions of Latin America, Africa, and Asia develop so-called traveler’s diarrhea, most commonly due to enterotoxigenic or enteroaggregative Escherichia coli as well as to Campylobacter, Shigella, Aeromonas, norovirus, Coronavirus, and Salmonella. Visitors to Russia (especially St. Petersburg) may have increased risk of Giardia-associated diarrhea; visitors to Nepal may acquire Cyclospora. Campers, backpackers, and swimmers in wilderness areas may become infected with Giardia. Cruise ships may be affected by outbreaks of gastroenteritis caused by agents such as norovirus. 2.

1	2. Consumers of certain foods. Diarrhea closely following food consumption at a picnic, banquet, or restaurant may suggest infection with Salmonella, Campylobacter, or Shigella from chicken; enterohemorrhagic E. coli (O157:H7) from undercooked hamburger; Bacillus cereus from fried rice or other reheated food; Staphylococcus aureus or Salmonella from mayonnaise or creams; Salmonella from eggs; Listeria from uncooked foods or soft cheeses; and Vibrio species, Salmonella, or acute hepatitis A from seafood, especially if raw. State departments of public health issue communications regarding food-related illnesses, which may have originated domestically or been imported, but ultimately cause epidemics in the United States (e.g., the Cyclospora epidemic of 2013 in midwestern states that resulted from bagged salads). 3.

1	3. Immunodeficient persons. Individuals at risk for diarrhea include those with either primary immunodeficiency (e.g., IgA deficiency, common variable hypogammaglobulinemia, chronic granulomatous disease) or the much more common secondary immunodeficiency states

1	PART 2 Cardinal Manifestations and Presentation of Diseases (e.g., AIDS, senescence, pharmacologic suppression). Common enteric pathogens often cause a more severe and protracted diarrheal illness, and, particularly in persons with AIDS, opportunistic infections, such as by Mycobacterium species, certain viruses (cytomegalovirus, adenovirus, and herpes simplex), and protozoa (Cryptosporidium, Isospora belli, Microsporida, and Blastocystis hominis) may also play a role (Chap. 226). In patients with AIDS, agents transmitted venereally per rectum (e.g., Neisseria gonorrhoeae, Treponema pallidum, Chlamydia) may contribute to proctocolitis. Persons with hemochromatosis are especially prone to invasive, even fatal, enteric infections with Vibrio species and Yersinia infections and should avoid raw fish. 4. Daycare attendees and their family members. Infections with Shigella, Giardia, Cryptosporidium, rotavirus, and other agents are very common and should be considered. 5.

1	4. Daycare attendees and their family members. Infections with Shigella, Giardia, Cryptosporidium, rotavirus, and other agents are very common and should be considered. 5. Institutionalized persons. Infectious diarrhea is one of the most frequent categories of nosocomial infections in many hospitals and long-term care facilities; the causes are a variety of microorganisms but most commonly C. difficile. C. difficile can affect those with no history of antibiotic use and may be acquired in the community.

1	The pathophysiology underlying acute diarrhea by infectious agents produces specific clinical features that may also be helpful in diagnosis (Table 55-2). Profuse, watery diarrhea secondary to small-bowel hypersecretion occurs with ingestion of preformed bacterial toxins, enterotoxin-producing bacteria, and enteroadherent pathogens. Diarrhea associated with marked vomiting and minimal or no fever may occur abruptly within a few hours after ingestion of the former two types; vomiting is usually less, abdominal cramping or bloating is greater, and fever is higher with the latter. Cytotoxin-producing and invasive microorganisms all cause high fever and abdominal pain. Invasive bacteria and Entamoeba histolytica often cause bloody diarrhea (referred to as dysentery). Yersinia invades the terminal ileal and proximal colon mucosa and may cause especially severe abdominal pain with tenderness mimicking acute appendicitis.

1	Finally, infectious diarrhea may be associated with systemic manifestations. Reactive arthritis (formerly known as Reiter’s syndrome), arthritis, urethritis, and conjunctivitis may accompany or follow 1–2+, watery, mushy 1–3+, usually watery, occasionally 1–3+, initially watery, quickly bloody 1–4+, watery or bloody Source: Adapted from DW Powell, in T Yamada (ed): Textbook of Gastroenterology and Hepatology, 4th ed. Philadelphia, Lippincott Williams & Wilkins, 2003. infections by Salmonella, Campylobacter, Shigella, and Yersinia.

1	infections by Salmonella, Campylobacter, Shigella, and Yersinia. Yersiniosis may also lead to an autoimmune-type thyroiditis, pericarditis, and glomerulonephritis. Both enterohemorrhagic E. coli (O157:H7) and Shigella can lead to the hemolytic-uremic syndrome with an attendant high mortality rate. The syndrome of postinfectious IBS has now been recognized as a complication of infectious diarrhea. Similarly, acute gastroenteritis may precede the diagnosis of celiac disease or Crohn’s disease. Acute diarrhea can also be a major symptom of several systemic infections including viral hepatitis, listeriosis, legionellosis, and toxic shock syndrome.

1	Other Causes Side effects from medications are probably the most common noninfectious causes of acute diarrhea, and etiology may be suggested by a temporal association between use and symptom onset. Although innumerable medications may produce diarrhea, some of the more frequently incriminated include antibiotics, cardiac antidysrhythmics, antihypertensives, nonsteroidal anti-inflammatory drugs (NSAIDs), certain antidepressants, chemotherapeutic agents, bronchodilators, antacids, and laxatives. Occlusive or nonocclusive ischemic colitis typically occurs in persons >50 years; often presents as acute lower abdominal pain preceding watery, then bloody diarrhea; and generally results in acute inflammatory changes in the sigmoid or left colon while sparing the rectum. Acute diarrhea may accompany colonic diverticulitis and graft-versus-host disease. Acute diarrhea, often associated with systemic compromise, can follow ingestion of toxins including organophosphate insecticides; amanita and

1	colonic diverticulitis and graft-versus-host disease. Acute diarrhea, often associated with systemic compromise, can follow ingestion of toxins including organophosphate insecticides; amanita and other mushrooms; arsenic; and preformed environmental toxins in seafood, such as ciguatera and scombroid. Acute anaphylaxis to food ingestion can have a similar presentation. Conditions causing chronic diarrhea can also be confused with acute diarrhea early in their course. This confusion may occur with inflammatory bowel disease (IBD) and some of the other inflammatory chronic diarrheas that may have an abrupt rather than insidious onset and exhibit features that mimic infection.

1	APPROACH TO THE PATIENT: The decision to evaluate acute diarrhea depends on its severity and duration and on various host factors (Fig. 55-2). Most episodes of acute diarrhea are mild and self-limited and do not justify the cost and potential morbidity rate of diagnostic or pharmacologic interventions. Indications for evaluation include profuse diarrhea with dehydration, grossly bloody stools, fever ≥38.5°C (≥101°F), duration >48 h without improvement, recent antibiotic use, new community outbreaks, associated severe abdominal pain in patients >50 years, and elderly (≥70 years) or immunocompromised patients. In some cases of moderately severe febrile diarrhea associated with fecal leukocytes (or increased fecal levels of the leukocyte proteins, such as calprotectin) or with gross blood, a diagnostic evaluation might be avoided in favor of an empirical antibiotic trial (see below).

1	The cornerstone of diagnosis in those suspected of severe acute infectious diarrhea is microbiologic analysis of the stool. Workup includes cultures for bacterial and viral pathogens, direct inspection for ova and parasites, and immunoassays for certain bacterial toxins (C. difficile), viral antigens (rotavirus), and protozoal antigens (Giardia, E. histolytica). The aforementioned clinical and epidemiologic associations may assist in focusing the evaluation. If a particular pathogen or set of possible pathogens is so implicated, then either the whole panel of routine studies may not be necessary or, in some instances, special cultures may be appropriate as for enterohemorrhagic and other types of E. coli, Vibrio species, and Yersinia. Molecular diagnosis of pathogens in stool can be made by identification of unique DNA sequences; and evolving microarray technologies have led to more rapid, sensitive, specific, and cost-effective diagnosis.

1	CHAPTER 55 Diarrhea and Constipation History and physical exam Moderate (activities altered) Mild (unrestricted) Observe Resolves Persists* Severe (incapacitated) Institute fluid and electrolyte replacement Antidiarrheal agents Resolves Persists* Stool microbiology studies Pathogen found Fever ˜38.5°C, bloody stools, fecal WBCs, immunocompromised or elderly host Evaluate and treat accordingly Acute Diarrhea Likely noninfectious Likely infectious Yes†No Yes†No Select specific treatment Empirical treatment + further evaluation FIguRE 55-2 Algorithm for the management of acute diarrhea. Consider empirical treatment before evaluation with (*) metronida-zole and with (†) quinolone. WBCs, white blood cells. Persistent diarrhea is commonly due to Giardia (Chap. 247), but additional causative organisms that should be considered include

1	Persistent diarrhea is commonly due to Giardia (Chap. 247), but additional causative organisms that should be considered include C. difficile (especially if antibiotics had been administered), E. histolytica, Cryptosporidium, Campylobacter, and others. If stool studies are unrevealing, flexible sigmoidoscopy with biopsies and upper endoscopy with duodenal aspirates and biopsies may be indicated. Brainerd diarrhea is an increasingly recognized entity characterized by an abrupt-onset diarrhea that persists for at least 4 weeks, but may last 1–3 years, and is thought to be of infectious origin. It may be associated with subtle inflammation of the distal small intestine or proximal colon.

1	Structural examination by sigmoidoscopy, colonoscopy, or abdominal computed tomography (CT) scanning (or other imaging approaches) may be appropriate in patients with uncharacterized persistent diarrhea to exclude IBD or as an initial approach in patients with suspected noninfectious acute diarrhea such as might be caused by ischemic colitis, diverticulitis, or partial bowel obstruction. Fluid and electrolyte replacement are of central importance to all forms of acute diarrhea. Fluid replacement alone may suffice for mild cases. Oral sugar-electrolyte solutions (iso-osmolar sport drinks or designed formulations) should be instituted promptly with severe diarrhea to limit dehydration, which is the major cause of death. Profoundly dehydrated patients, especially infants and the elderly, require IV rehydration.

1	In moderately severe nonfebrile and nonbloody diarrhea, anti-motility and antisecretory agents such as loperamide can be useful adjuncts to control symptoms. Such agents should be avoided with 268 febrile dysentery, which may be exacerbated or prolonged by them. Bismuth subsalicylate may reduce symptoms of vomiting and diarrhea but should not be used to treat immunocompromised patients or those with renal impairment because of the risk of bismuth encephalopathy. Judicious use of antibiotics is appropriate in selected instances of acute diarrhea and may reduce its severity and duration (Fig. 55-2). Many physicians treat moderately to severely ill patients with febrile dysentery empirically without diagnostic evaluation using a quinolone, such as ciprofloxacin (500 mg bid for 3–5 d). Empirical treatment can also be considered for suspected giardiasis with metronidazole (250 mg qid for 7 d). Selection of antibiotics and dosage regimens are otherwise dictated by specific pathogens,

1	Empirical treatment can also be considered for suspected giardiasis with metronidazole (250 mg qid for 7 d). Selection of antibiotics and dosage regimens are otherwise dictated by specific pathogens, geographic patterns of resistance, and conditions found (Chaps. 160, 186, and 190–196). Antibiotic coverage is indicated, whether or not a causative organism is discovered, in patients who are immunocompromised, have mechanical heart valves or recent vascular grafts, or are elderly. Bismuth subsalicylate may reduce the frequency of traveler’s diarrhea. Antibiotic prophylaxis is only indicated for certain patients traveling to high-risk countries in whom the likelihood or seriousness of acquired diarrhea would be especially high, including those with immunocompromise, IBD, hemochromatosis, or gastric achlorhydria. Use of ciprofloxacin, azithromycin, or rifaximin may reduce bacterial diarrhea in such travelers by 90%, though rifaximin is not suitable for invasive disease, but rather as

1	or gastric achlorhydria. Use of ciprofloxacin, azithromycin, or rifaximin may reduce bacterial diarrhea in such travelers by 90%, though rifaximin is not suitable for invasive disease, but rather as treatment for uncomplicated traveler’s diarrhea. Finally, physicians should be vigilant to identify if an outbreak of diarrheal illness is occurring and to alert the public health authorities promptly. This may reduce the ultimate size of the affected population.

1	PART 2 Cardinal Manifestations and Presentation of Diseases Diarrhea lasting >4 weeks warrants evaluation to exclude serious underlying pathology. In contrast to acute diarrhea, most of the causes of chronic diarrhea are noninfectious. The classification of chronic diarrhea by pathophysiologic mechanism facilitates a rational approach to management, although many diseases cause diarrhea by more than one mechanism (Table 55-3). Secretory Causes Secretory diarrheas are due to derangements in fluid and electrolyte transport across the enterocolonic mucosa. They are characterized clinically by watery, large-volume fecal outputs that are typically painless and persist with fasting. Because there is no malabsorbed solute, stool osmolality is accounted for by normal endogenous electrolytes with no fecal osmotic gap.

1	MEdICATIONS Side effects from regular ingestion of drugs and toxins are the most common secretory causes of chronic diarrhea. Hundreds of prescription and over-the-counter medications (see earlier section, “Acute Diarrhea, Other Causes”) may produce diarrhea. Surreptitious or habitual use of stimulant laxatives (e.g., senna, cascara, bisacodyl, ricinoleic acid [castor oil]) must also be considered. Chronic ethanol consumption may cause a secretory-type diarrhea due to enterocyte injury with impaired sodium and water absorption as well as rapid transit and other alterations. Inadvertent ingestion of certain environmental toxins (e.g., arsenic) may lead to chronic rather than acute forms of diarrhea. Certain bacterial infections may occasionally persist and be associated with a secretory-type diarrhea.

1	BOwEL RESECTION, MUCOSAL dISEASE, OR ENTEROCOLIC FISTULA These conditions may result in a secretory-type diarrhea because of inadequate surface for reabsorption of secreted fluids and electrolytes. Unlike other secretory diarrheas, this subset of conditions tends to worsen with eating. With disease (e.g., Crohn’s ileitis) or resection of <100 cm of terminal ileum, dihydroxy bile acids may escape absorption and stimulate colonic secretion (cholerheic diarrhea). This mechanism may contribute to so-called idiopathic secretory diarrhea or bile acid diarrhea (BAD), in which bile acids are functionally malabsorbed from a normal-appearing terminal ileum. This idiopathic bile acid malabsorption (BAM) may account for an average of 40% of unexplained chronic diarrhea. Reduced negative feedback regulation of bile acid

1	Exogenous stimulant laxatives Chronic ethanol ingestion Other drugs and toxins Endogenous laxatives (dihydroxy bile acids) Idiopathic secretory diarrhea or bile acid diarrhea Certain bacterial infections Bowel resection, disease, or fistula (↓ absorption) Partial bowel obstruction or fecal impaction Hormone-producing tumors (carcinoid, VIPoma, medullary cancer of thyroid, mastocytosis, gastrinoma, colorectal villous adenoma) Addison’s disease Congenital electrolyte absorption defects Osmotic laxatives (Mg2+, PO4–3, SO4 Lactase and other disaccharide deficiencies Nonabsorbable carbohydrates (sorbitol, lactulose, polyethylene glycol) Gluten and FODMAP intolerance Intraluminal maldigestion (pancreatic exocrine insufficiency, bacterial overgrowth, bariatric surgery, liver disease) Mucosal malabsorption (celiac sprue, Whipple’s disease, infections, abetalipoproteinemia, ischemia, drug-induced enteropathy)

1	Mucosal malabsorption (celiac sprue, Whipple’s disease, infections, abetalipoproteinemia, ischemia, drug-induced enteropathy) Idiopathic inflammatory bowel disease (Crohn’s, chronic ulcerative colitis) Lymphocytic and collagenous colitis Immune-related mucosal disease (1° or 2° immunodeficiencies, food allergy, eosinophilic gastroenteritis, graft-versus-host disease) Infections (invasive bacteria, viruses, and parasites, Brainerd diarrhea) Radiation injury Gastrointestinal malignancies Vagotomy, fundoplication Abbreviation: FODMAP, fermentable oligosaccharides, disaccharides, monosaccharides, and polyols.

1	Vagotomy, fundoplication Abbreviation: FODMAP, fermentable oligosaccharides, disaccharides, monosaccharides, and polyols. synthesis in hepatocytes by fibroblast growth factor 19 (FGF-19) produced by ileal enterocytes results in a degree of bile-acid synthesis that exceeds the normal capacity for ileal reabsorption, producing BAD. An alternative cause of BAD is a genetic variation in the receptor proteins (β-klotho and fibroblast growth factor 4) on the hepatocyte that normally mediate the effect of FGF-19. Dysfunction of these proteins prevents FGF-19 inhibition of hepatocyte bile acid synthesis. Partial bowel obstruction, ostomy stricture, or fecal impaction may paradoxically lead to increased fecal output due to fluid hypersecretion.

1	HORMONES Although uncommon, the classic examples of secretory diarrhea are those mediated by hormones. Metastatic gastrointestinal carcinoid tumors or, rarely, primary bronchial carcinoids may produce watery diarrhea alone or as part of the carcinoid syndrome that comprises episodic flushing, wheezing, dyspnea, and right-sided valvular heart disease. Diarrhea is due to the release into the circulation of potent intestinal secretagogues including serotonin, histamine, prostaglandins, and various kinins. Pellagra-like skin lesions may rarely occur as the result of serotonin overproduction with niacin depletion. Gastrinoma, one of the most common neuroendocrine tumors, most typically presents with refractory peptic ulcers, but diarrhea occurs in up to one-third of cases and may be the only clinical manifestation in 10%. While other secretagogues released with gastrin may play a role, the diarrhea most often results from fat maldigestion owing to pancreatic enzyme inactivation by low

1	clinical manifestation in 10%. While other secretagogues released with gastrin may play a role, the diarrhea most often results from fat maldigestion owing to pancreatic enzyme inactivation by low intraduodenal pH. The watery diarrhea hypokalemia achlorhydria syndrome, also called pancreatic cholera, is due to a non-β cell pancreatic adenoma, referred to as a VIPoma, that secretes VIP and a host of other peptide hormones including pancreatic polypeptide, secretin, gastrin, gastrin-inhibitory polypeptide (also called glucose-dependent insulinotropic peptide), neurotensin, calcitonin, and prostaglandins. The secretory diarrhea is often massive with stool volumes >3 L/d; daily volumes as high as 20 L have been reported. Life-threatening dehydration; neuromuscular dysfunction from associated hypokalemia, hypomagnesemia, or hypercalcemia; flushing; and hyperglycemia may accompany a VIPoma. Medullary carcinoma of the thyroid may present with watery diarrhea caused by calcitonin, other

1	hypokalemia, hypomagnesemia, or hypercalcemia; flushing; and hyperglycemia may accompany a VIPoma. Medullary carcinoma of the thyroid may present with watery diarrhea caused by calcitonin, other secretory peptides, or prostaglandins. Prominent diarrhea is often associated with metastatic disease and poor prognosis. Systemic mastocytosis, which may be associated with the skin lesion urticaria pigmentosa, may cause diarrhea that is either secretory and mediated by histamine or inflammatory due to intestinal infiltration by mast cells. Large colorectal villous adenomas may rarely be associated with a secretory diarrhea that may cause hypokalemia, can be inhibited by NSAIDs, and are apparently mediated by prostaglandins.

1	CONgENITAL dEFECTS IN ION ABSORPTION Rarely, defects in specific carriers associated with ion absorption cause watery diarrhea from birth. These disorders include defective Cl–/HCO3 exchange (congenital chloridorrhea) with alkalosis (which results from a mutated DRA [down-regulated in adenoma] gene) and defective Na+/H+ exchange (congenital sodium diarrhea), which results from a mutation in the NHE3 (sodium-hydrogen exchanger) gene and results in acidosis. Some hormone deficiencies may be associated with watery diarrhea, such as occurs with adrenocortical insufficiency (Addison’s disease) that may be accompanied by skin hyperpigmentation.

1	Some hormone deficiencies may be associated with watery diarrhea, such as occurs with adrenocortical insufficiency (Addison’s disease) that may be accompanied by skin hyperpigmentation. Osmotic Causes Osmotic diarrhea occurs when ingested, poorly absorbable, osmotically active solutes draw enough fluid into the lumen to exceed the reabsorptive capacity of the colon. Fecal water output increases in proportion to such a solute load. Osmotic diarrhea characteristically ceases with fasting or with discontinuation of the causative agent.

1	OSMOTIC LAXATIVES Ingestion of magnesium-containing antacids, health supplements, or laxatives may induce osmotic diarrhea typified by a stool osmotic gap (>50 mosmol/L): serum osmolarity (typically 290 mosmol/kg) – (2 × [fecal sodium + potassium concentration]). Measurement of fecal osmolarity is no longer recommended because, even when measured immediately after evacuation, it may be erroneous because carbohydrates are metabolized by colonic bacteria, causing an increase in osmolarity.

1	CARBOHYdRATE MALABSORPTION Carbohydrate malabsorption due to acquired or congenital defects in brush-border disaccharidases and other enzymes leads to osmotic diarrhea with a low pH. One of the most common causes of chronic diarrhea in adults is lactase deficiency, which affects three-fourths of nonwhites worldwide and 5–30% of persons in the United States; the total lactose load at any one time influences the symptoms experienced. Most patients learn to avoid milk products without requiring treatment with enzyme supplements. Some sugars, such as sorbitol, lactulose, or fructose, are frequently malabsorbed, and diarrhea ensues with ingestion of medications, gum, or candies sweetened with these poorly or incompletely absorbed sugars.

1	wHEAT ANd FOdMAP INTOLERANCE Chronic diarrhea, bloating, and 269 abdominal pain are recognized as symptoms of nonceliac gluten intolerance (which is associated with impaired intestinal or colonic barrier function) and intolerance of fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs). The latter’s effects represent the interaction between the GI microbiome and the nutrients.

1	Steatorrheal Causes Fat malabsorption may lead to greasy, foul-smelling, difficult-to-flush diarrhea often associated with weight loss and nutritional deficiencies due to concomitant malabsorption of amino acids and vitamins. Increased fecal output is caused by the osmotic effects of fatty acids, especially after bacterial hydroxylation, and, to a lesser extent, by the neutral fat. Quantitatively, steatorrhea is defined as stool fat exceeding the normal 7 g/d; rapid-transit diarrhea may result in fecal fat up to 14 g/d; daily fecal fat averages 15–25 g with small-intestinal diseases and is often >32 g with pancreatic exocrine insufficiency. Intraluminal maldigestion, mucosal malabsorption, or lymphatic obstruction may produce steatorrhea.

1	INTRALUMINAL MALdIgESTION This condition most commonly results from pancreatic exocrine insufficiency, which occurs when >90% of pancreatic secretory function is lost. Chronic pancreatitis, usually a sequel of ethanol abuse, most frequently causes pancreatic insufficiency. Other causes include cystic fibrosis; pancreatic duct obstruction; and, rarely, somatostatinoma. Bacterial overgrowth in the small intestine may deconjugate bile acids and alter micelle formation, impairing fat digestion; it occurs with stasis from a blind-loop, small-bowel diverticulum or dysmotility and is especially likely in the elderly. Finally, cirrhosis or biliary obstruction may lead to mild steatorrhea due to deficient intraluminal bile acid concentration.

1	MUCOSAL MALABSORPTION Mucosal malabsorption occurs from a variety of enteropathies, but it most commonly occurs from celiac disease. This gluten-sensitive enteropathy affects all ages and is characterized by villous atrophy and crypt hyperplasia in the proximal small bowel and can present with fatty diarrhea associated with multiple nutritional deficiencies of varying severity. Celiac disease is much more frequent than previously thought; it affects ∼1% of the population, frequently presents without steatorrhea, can mimic IBS, and has many other GI and extraintestinal manifestations. Tropical sprue may produce a similar histologic and clinical syndrome but occurs in residents of or travelers to tropical climates; abrupt onset and response to antibiotics suggest an infectious etiology. Whipple’s disease, due to the bacillus Tropheryma whipplei and histiocytic infiltration of the small-bowel mucosa, is a less common cause of steatorrhea that most typically occurs in young or middle-aged

1	disease, due to the bacillus Tropheryma whipplei and histiocytic infiltration of the small-bowel mucosa, is a less common cause of steatorrhea that most typically occurs in young or middle-aged men; it is frequently associated with arthralgias, fever, lymphadenopathy, and extreme fatigue, and it may affect the CNS and endocardium. A similar clinical and histologic picture results from Mycobacterium avium-intracellulare infection in patients with AIDS. Abetalipoproteinemia is a rare defect of chylomicron formation and fat malabsorption in children, associated with acanthocytic erythrocytes, ataxia, and retinitis pigmentosa. Several other conditions may cause mucosal malabsorption including infections, especially with protozoa such as Giardia; numerous medications (e.g., olmesartan, mycophenolate mofetil, colchicine, cholestyramine, neomycin); amyloidosis; and chronic ischemia.

1	POSTMUCOSAL LYMPHATIC OBSTRUCTION The pathophysiology of this condition, which is due to the rare congenital intestinal lymphangiectasia or to acquired lymphatic obstruction secondary to trauma, tumor, cardiac disease or infection, leads to the unique constellation of fat malabsorption with enteric losses of protein (often causing edema) and lymphocytopenia. Carbohydrate and amino acid absorption are preserved.

1	Inflammatory Causes Inflammatory diarrheas are generally accompanied by pain, fever, bleeding, or other manifestations of inflammation. The mechanism of diarrhea may not only be exudation but, depending on lesion site, may include fat malabsorption, disrupted fluid/ electrolyte absorption, and hypersecretion or hypermotility from 270 release of cytokines and other inflammatory mediators. The unifying feature on stool analysis is the presence of leukocytes or leukocyte-derived proteins such as calprotectin. With severe inflammation, exudative protein loss can lead to anasarca (generalized edema). Any middle-aged or older person with chronic inflammatory-type diarrhea, especially with blood, should be carefully evaluated to exclude a colorectal tumor.

1	IdIOPATHIC INFLAMMATORY BOwEL dISEASE The illnesses in this category, which include Crohn’s disease and chronic ulcerative colitis, are among the most common organic causes of chronic diarrhea in adults and range in severity from mild to fulminant and life-threatening. They may be associated with uveitis, polyarthralgias, cholestatic liver disease (primary sclerosing cholangitis), and skin lesions (erythema nodosum, pyoderma gangrenosum). Microscopic colitis, including both lymphocytic and collagenous colitis, is an increasingly recognized cause of chronic watery diarrhea, especially in middle-aged women and those on NSAIDs, statins, proton pump inhibitors (PPIs), and selective serotonin reuptake inhibitors (SSRIs); biopsy of a normal-appearing colon is required for histologic diagnosis. It may coexist with symptoms suggesting IBS or with celiac sprue or drug-induced enteropathy. It typically responds well to anti-inflammatory drugs (e.g., bismuth), to the opioid agonist loperamide,

1	It may coexist with symptoms suggesting IBS or with celiac sprue or drug-induced enteropathy. It typically responds well to anti-inflammatory drugs (e.g., bismuth), to the opioid agonist loperamide, or to budesonide.

1	PRIMARY OR SECONdARY FORMS OF IMMUNOdEFICIENCY Immunodeficiency may lead to prolonged infectious diarrhea. With selective IgA deficiency or common variable hypogammaglobulinemia, diarrhea is particularly prevalent and often the result of giardiasis, bacterial overgrowth, or sprue. EOSINOPHILIC gASTROENTERITIS Eosinophil infiltration of the mucosa, muscularis, or serosa at any level of the GI tract may cause diarrhea, pain, vomiting, or ascites. Affected patients often have an atopic history, Charcot-Leyden crystals due to extruded eosinophil contents may be seen on microscopic inspection of stool, and peripheral eosinophilia is present in 50–75% of patients. While hypersensitivity to certain foods occurs in adults, true food allergy causing chronic diarrhea is rare. OTHER CAUSES Chronic inflammatory diarrhea may be caused by radiation enterocolitis, chronic graft-versus-host disease, Behçet’s syndrome, and Cronkhite-Canada syndrome, among others.

1	OTHER CAUSES Chronic inflammatory diarrhea may be caused by radiation enterocolitis, chronic graft-versus-host disease, Behçet’s syndrome, and Cronkhite-Canada syndrome, among others. Dysmotility Causes Rapid transit may accompany many diarrheas as a secondary or contributing phenomenon, but primary dysmotility is an unusual etiology of true diarrhea. Stool features often suggest a secretory diarrhea, but mild steatorrhea of up to 14 g of fat per day can be produced by maldigestion from rapid transit alone. Hyperthyroidism, carcinoid syndrome, and certain drugs (e.g., prostaglandins, prokinetic agents) may produce hypermotility with resultant diarrhea. Primary visceral neuromyopathies or idiopathic acquired intestinal pseudoobstruction may lead to stasis with secondary bacterial overgrowth causing diarrhea. Diabetic diarrhea, often accompanied by peripheral and generalized autonomic neuropathies, may occur in part because of intestinal dysmotility.

1	The exceedingly common IBS (10% point prevalence, 1–2% per year incidence) is characterized by disturbed intestinal and colonic motor and sensory responses to various stimuli. Symptoms of stool frequency typically cease at night, alternate with periods of constipation, are accompanied by abdominal pain relieved with defecation, and rarely result in weight loss.

1	Factitial Causes Factitial diarrhea accounts for up to 15% of unexplained diarrheas referred to tertiary care centers. Either as a form of Munchausen syndrome (deception or self-injury for secondary gain) or eating disorders, some patients covertly self-administer laxatives alone or in combination with other medications (e.g., diuretics) or surreptitiously add water or urine to stool sent for analysis. Such patients are typically women, often with histories of psychiatric illness, and disproportionately from careers in health care. Hypotension and hypokalemia are common co-presenting features. The evaluation of such patients may be difficult: contamination of the stool with water or urine is suggested by very low or high stool osmolarity, respectively. PART 2 Cardinal Manifestations and Presentation of Diseases Such patients often deny this possibility when confronted, but they do benefit from psychiatric counseling when they acknowledge their behavior. APPROACH TO THE PATIENT:

1	The laboratory tools available to evaluate the very common problem of chronic diarrhea are extensive, and many are costly and invasive. As such, the diagnostic evaluation must be rationally directed by a careful history, including medications, and physical examination (Fig. 55-3A). When this strategy is unrevealing, simple triage tests are often warranted to direct the choice of more complex investigations (Fig. 55-3B). The history, physical examination (Table 55-4), and routine blood studies should attempt to characterize the mechanism of diarrhea, identify diagnostically helpful associations, and assess the patient’s fluid/electrolyte and nutritional status. Patients should be questioned about the onset, duration, pattern, aggravating (especially diet) and relieving factors, and stool characteristics of their diarrhea. The presence or absence of fecal incontinence, fever, weight loss, pain, certain exposures (travel, medications, contacts with diarrhea), and common extraintestinal

1	characteristics of their diarrhea. The presence or absence of fecal incontinence, fever, weight loss, pain, certain exposures (travel, medications, contacts with diarrhea), and common extraintestinal manifestations (skin changes, arthralgias, oral aphthous ulcers) should be noted. A family history of IBD or sprue may indicate those possibilities. Physical findings may offer clues such as a thyroid mass, wheezing, heart murmurs, edema, hepatomegaly, abdominal masses, lymphadenopathy, mucocutaneous abnormalities, perianal fistulas, or anal sphincter laxity. Peripheral blood leukocytosis, elevated sedimentation rate, or C-reactive protein suggests inflammation; anemia reflects blood loss or nutritional deficiencies; or eosinophilia may occur with parasitoses, neoplasia, collagen-vascular disease, allergy, or eosinophilic gastroenteritis. Blood chemistries may demonstrate electrolyte, hepatic, or other metabolic disturbances. Measuring IgA tissue transglutaminase antibodies may help

1	disease, allergy, or eosinophilic gastroenteritis. Blood chemistries may demonstrate electrolyte, hepatic, or other metabolic disturbances. Measuring IgA tissue transglutaminase antibodies may help detect celiac disease. Bile acid diarrhea is confirmed by a scintigraphic radiolabeled bile acid retention test; however, this is not available in many countries. Alternative approaches are a screening blood test (serum C4 or FGF-19), measurement of fecal bile acids, or a therapeutic trial with a bile acid sequestrant (e.g., cholestyramine or colesevelam).

1	A therapeutic trial is often appropriate, definitive, and highly cost-effective when a specific diagnosis is suggested on the initial physician encounter. For example, chronic watery diarrhea, which ceases with fasting in an otherwise healthy young adult, may justify a trial of a lactose-restricted diet; bloating and diarrhea persisting since a mountain backpacking trip may warrant a trial of metronidazole for likely giardiasis; and postprandial diarrhea persisting following resection of terminal ileum might be due to bile acid malabsorption and be treated with cholestyramine or colesevelam before further evaluation. Persistent symptoms require additional investigation.

1	Certain diagnoses may be suggested on the initial encounter (e.g., idiopathic IBD); however, additional focused evaluations may be necessary to confirm the diagnosis and characterize the severity or extent of disease so that treatment can be best guided. Patients suspected of having IBS should be initially evaluated with flexible sigmoidoscopy with colorectal biopsies to exclude IBD, or particularly microscopic colitis, which is clinically indistinguishable from IBS with diarrhea; those with normal findings might be reassured and, as indicated, treated empirically with antispasmodics, antidiarrheals, or antidepressants (e.g., tricyclic agents). Any patient who presents with chronic diarrhea and hematochezia should be evaluated with stool microbiologic studies and colonoscopy. In an estimated two-thirds of cases, the cause for chronic diarrhea remains unclear after the initial encounter, and further testing is required. Quantitative stool collection and analyses can yield

1	In an estimated two-thirds of cases, the cause for chronic diarrhea remains unclear after the initial encounter, and further testing is required. Quantitative stool collection and analyses can yield Exclude iatrogenic problem: medication, surgery Blood pr Colonoscopy + biopsy Features, e.g., stool, suggest malabsorption Small bowel: Imaging, biopsy, aspirate Pain aggravated before bm, relieved with bm, sense incomplete evacuation Suspect IBS Limited screen for organic disease No blood, features of malabsorption Consider functional diarrhea Dietary exclusion, e.g., lactose, sorbitol Chronic diarrhea

1	Limited screen for organic disease Chronic diarrhea Stool vol, OSM, pH; Laxative screen; Hormonal screen Persistent chronic diarrhea Stool fat >20 g/day Pancreatic function Titrate Rx to speed of transit Opioid Rx + follow-up Low K+ Screening tests all normal Colonoscopy + biopsy Normal and stool fat <14 g/day Small bowel: X-ray, biopsy, aspirate; stool 48-h fat Full gut transit Low Hb, Alb; abnormal MCV, MCH; excess fat in stool FIguRE 55-3 Chronic diarrhea. A. Initial management based on accompanying symptoms or features. B. Evaluation based on findings from a limited age-appropriate screen for organic disease. Alb, albumin; bm, bowel movement; Hb, hemoglobin; IBS, irritable bowel syndrome; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; OSM, osmolality; pr, per rectum. (Reprinted from M Camilleri: Clin Gastroenterol

1	Hepatol. 2:198, 2004.) important objective data that may establish a diagnosis or characterize the type of diarrhea as a triage for focused additional studies (Fig. 55-3B). If stool weight is >200 g/d, additional stool analyses should be performed that might include electrolyte concentration, 1. Are there general features to suggest malabsorption or inflammatory bowel disease (IBD) such as anemia, dermatitis herpetiformis, edema, or clubbing? 2. Are there features to suggest underlying autonomic neuropathy or collagen-vascular disease in the pupils, orthostasis, skin, hands, or joints? 3. Is there an abdominal mass or tenderness? 4. Are there any abnormalities of rectal mucosa, rectal defects, or altered anal sphincter functions? 5. Are there any mucocutaneous manifestations of systemic disease such as dermatitis herpetiformis (celiac disease), erythema nodosum (ulcerative colitis), flushing (carcinoid), or oral ulcers for IBD or celiac disease?

1	pH, occult blood testing, leukocyte inspection (or leukocyte protein assay), fat quantitation, and laxative screens.

1	For secretory diarrheas (watery, normal osmotic gap), possible medication-related side effects or surreptitious laxative use should be reconsidered. Microbiologic studies should be done including fecal bacterial cultures (including media for Aeromonas and Plesiomonas), inspection for ova and parasites, and Giardia antigen assay (the most sensitive test for giardiasis). Small-bowel bacterial overgrowth can be excluded by intestinal aspirates with quantitative cultures or with glucose or lactulose breath tests involving measurement of breath hydrogen, methane, or other metabolite. However, interpretation of these breath tests may be confounded by disturbances of intestinal transit. Upper endoscopy and colonoscopy with biopsies and small-bowel x-rays (formerly barium, but increasingly CT with enterography or magnetic resonance with enteroclysis) are helpful to rule out structural or occult inflammatory disease. When suggested by history or other findings, screens for peptide hormones

1	with enterography or magnetic resonance with enteroclysis) are helpful to rule out structural or occult inflammatory disease. When suggested by history or other findings, screens for peptide hormones should be pursued (e.g., serum gastrin, VIP, calcitonin, and thyroid hormone/thyroid-stimulating hormone, urinary 5-hydroxyindolacetic acid, histamine).

1	Further evaluation of osmotic diarrhea should include tests for lactose intolerance and magnesium ingestion, the two most common causes. Low fecal pH suggests carbohydrate malabsorption; lactose malabsorption can be confirmed by lactose breath testing or by a therapeutic trial with lactose exclusion and observation of the effect of lactose challenge (e.g., a liter of milk). Lactase determination on small-bowel biopsy is not generally available. If fecal magnesium or laxative levels are elevated, inadvertent or surreptitious ingestion should be considered and psychiatric help should be sought.

1	For those with proven fatty diarrhea, endoscopy with small-bowel biopsy (including aspiration for Giardia and quantitative cultures) should be performed; if this procedure is unrevealing, a small-bowel radiograph is often an appropriate next step. If small-bowel studies are negative or if pancreatic disease is suspected, pancreatic exocrine insufficiency should be excluded with direct tests, such as the secretin-cholecystokinin stimulation test or a variation that could be performed endoscopically. In general, indirect tests such as assay of fecal elastase or chymotrypsin activity or a bentiromide test have fallen out of favor because of low sensitivity and specificity. Chronic inflammatory-type diarrheas should be suspected by the presence of blood or leukocytes in the stool. Such findings warrant stool cultures; inspection for ova and parasites; C. difficile toxin assay; colonoscopy with biopsies; and, if indicated, small-bowel contrast studies.

1	Treatment of chronic diarrhea depends on the specific etiology and may be curative, suppressive, or empirical. If the cause can be eradicated, treatment is curative as with resection of a colorectal cancer, antibiotic administration for Whipple’s disease or tropical sprue, or discontinuation of a drug. For many chronic conditions, diarrhea can be controlled by suppression of the underlying mechanism. Examples include elimination of dietary lactose for lactase deficiency or gluten for celiac sprue, use of glucocorticoids or other anti-inflammatory agents for idiopathic IBDs, bile acid sequestrants for bile acid malabsorption, PPIs for the gastric hypersecretion of gastrinomas, somatostatin analogues such as octreotide for malignant carcinoid syndrome, prostaglandin inhibitors such as indomethacin for medullary carcinoma of the thyroid, and pancreatic enzyme replacement for pancreatic insufficiency. When the specific cause or mechanism of chronic diarrhea evades diagnosis, empirical

1	for medullary carcinoma of the thyroid, and pancreatic enzyme replacement for pancreatic insufficiency. When the specific cause or mechanism of chronic diarrhea evades diagnosis, empirical therapy may be beneficial. Mild opiates, such as diphenoxylate or loperamide, are often helpful in mild or moderate watery diarrhea. For those with more severe diarrhea, codeine or tincture of opium may be beneficial. Such antimotility agents should be avoided with severe IBD, because toxic megacolon may be precipitated. Clonidine, an α2-adrenergic agonist, may allow control of diabetic diarrhea, although the medication may be poorly tolerated because it causes postural hypotension. The 5-HT3 receptor antagonists (e.g., alosetron) may relieve diarrhea and urgency in patients with IBS diarrhea. For all patients with chronic diarrhea, fluid and electrolyte repletion is an important component of management (see “Acute Diarrhea,” earlier). Replacement of fat-soluble vitamins may also be necessary in

1	patients with chronic diarrhea, fluid and electrolyte repletion is an important component of management (see “Acute Diarrhea,” earlier). Replacement of fat-soluble vitamins may also be necessary in patients with chronic steatorrhea.

1	PART 2 Cardinal Manifestations and Presentation of Diseases Constipation is a common complaint in clinical practice and usually refers to persistent, difficult, infrequent, or seemingly incomplete defecation. Because of the wide range of normal bowel habits, constipation is difficult to define precisely. Most persons have at least three bowel movements per week; however, low stool frequency alone is not the sole criterion for the diagnosis of constipation. Many constipated patients have a normal frequency of defecation but complain of excessive straining, hard stools, lower abdominal fullness, or a sense of incomplete evacuation. The individual patient’s symptoms must be analyzed in detail to ascertain what is meant by “constipation” or “difficulty” with defecation.

1	Stool form and consistency are well correlated with the time elapsed from the preceding defecation. Hard, pellety stools occur with slow transit, whereas loose, watery stools are associated with rapid transit. Both small pellety or very large stools are more difficult to expel than normal stools. The perception of hard stools or excessive straining is more difficult to assess objectively, and the need for enemas or digital disimpaction is a clinically useful way to corroborate the patient’s perceptions of difficult defecation. Psychosocial or cultural factors may also be important. A person whose parents attached great importance to daily defecation will become greatly concerned when he or she misses a daily bowel movement; some children withhold stool to gain attention or because of fear of pain from anal irritation; and some adults habitually ignore or delay the call to have a bowel movement.

1	Pathophysiologically, chronic constipation generally results from inadequate fiber or fluid intake or from disordered colonic transit or anorectal function. These result from neurogastroenterologic disturbance, certain drugs, advancing age, or in association with a large number of systemic diseases that affect the GI tract (Table 55-5). Constipation of recent onset may be a symptom of significant organic disease such as tumor or stricture. In idiopathic constipation, a subset of patients exhibit delayed emptying of the ascending and transverse colon with prolongation of transit (often in the proximal colon) and a reduced frequency of propulsive HAPCs. Outlet obstruction to defecation (also called evacuation disorders) accounts for about a quarter of cases presenting with constipation in tertiary care and may cause delayed colonic transit, which is usually corrected by biofeedback retraining of the disordered defecation. Constipation of any cause may be exacerbated by hospitalization

1	tertiary care and may cause delayed colonic transit, which is usually corrected by biofeedback retraining of the disordered defecation. Constipation of any cause may be exacerbated by hospitalization or chronic illnesses that lead to physical or mental impairment and result in inactivity or physical immobility.

1	Types of Constipation and Causes Examples Colonic obstruction Neoplasm; stricture: ischemic, diverticular, inflammatory Anal sphincter spasm Anal fissure, painful hemorrhoids Medications Disorders of rectal evacuation Constipation-predominant, alternating Ca2+ blockers, antidepressants Slow-transit constipation, megacolon (rare Hirschsprung’s, Chagas’ diseases) Pelvic floor dysfunction; anismus; descending perineum syndrome; rectal mucosal prolapse; rectocele Hypothyroidism, hypercalcemia, pregnancy Depression, eating disorders, drugs Parkinsonism, multiple sclerosis, spinal APPROACH TO THE PATIENT:

1	A careful history should explore the patient’s symptoms and confirm whether he or she is indeed constipated based on frequency (e.g., fewer than three bowel movements per week), consistency (lumpy/hard), excessive straining, prolonged defecation time, or need to support the perineum or digitate the anorectum to facilitate stool evacuation. In the vast majority of cases (probably >90%), there is no underlying cause (e.g., cancer, depression, or hypothyroidism), and constipation responds to ample hydration, exercise, and supplementation of dietary fiber (15–25 g/d). A good diet and medication history and attention to psychosocial issues are key. Physical examination and, particularly, a rectal examination should exclude fecal impaction and most of the important diseases that present with constipation and possibly indicate features suggesting an evacuation disorder (e.g., high anal sphincter tone, failure of perineal descent, or paradoxical puborectalis contraction during straining to

1	constipation and possibly indicate features suggesting an evacuation disorder (e.g., high anal sphincter tone, failure of perineal descent, or paradoxical puborectalis contraction during straining to simulate stool evacuation).

1	The presence of weight loss, rectal bleeding, or anemia with constipation mandates either flexible sigmoidoscopy plus barium enema or colonoscopy alone, particularly in patients >40 years, to exclude structural diseases such as cancer or strictures. Colonoscopy alone is most cost-effective in this setting because it provides an opportunity to biopsy mucosal lesions, perform polypectomy, or dilate strictures. Barium enema has advantages over colonoscopy in the patient with isolated constipation because it is less costly and identifies colonic dilation and all significant mucosal lesions or strictures that are likely to present with constipation. Melanosis coli, or pigmentation of the colon mucosa, indicates the use of anthraquinone laxatives such as cascara or senna; however, this is usually apparent from a careful history. An unexpected disorder such as megacolon or cathartic colon may also be detected by colonic radiographs. Measurement of serum calcium, potassium, and

1	this is usually apparent from a careful history. An unexpected disorder such as megacolon or cathartic colon may also be detected by colonic radiographs. Measurement of serum calcium, potassium, and thyroid-stimulating hormone levels will identify rare patients with metabolic disorders.

1	Patients with more troublesome constipation may not respond to fiber alone and may be helped by a bowel-training regimen, which involves taking an osmotic laxative (e.g., magnesium salts, lactulose, sorbitol, polyethylene glycol) and evacuating with enema or suppository (e.g., glycerine or bisacodyl) as needed. After breakfast, a distraction-free 15–20 min on the toilet without straining is encouraged. Excessive straining may lead to development of hemorrhoids, and, if there is weakness of the pelvic floor or injury to the pudendal nerve, may result in obstructed defecation from descending perineum syndrome several years later. Those few who do not benefit from the simple measures delineated above or require long-term treatment or fail to respond to potent laxatives should undergo further investigation (Fig. 55-4). Novel agents that induce secretion (e.g., lubiprostone, a chloride channel activator, or linaclotide, a guanylate cyclase C agonist that activates chloride secretion) are

1	investigation (Fig. 55-4). Novel agents that induce secretion (e.g., lubiprostone, a chloride channel activator, or linaclotide, a guanylate cyclase C agonist that activates chloride secretion) are also available.

1	A small minority (probably <5%) of patients have severe or “intractable” constipation; about 25% have evacuation disorders. These are the patients most likely to require evaluation by gastroenterologists or in referral centers. Further observation of the patient may occasionally reveal a previously unrecognized cause, such as an evacuation disorder, laxative abuse, malingering, or psychological disorder. In these patients, evaluations of the physiologic function of the colon and pelvic floor and of psychological status aid in the rational choice of treatment. Even among these highly selected patients with severe constipation, a cause can be identified in only about one-third of tertiary referral patients, with the others being diagnosed with normal transit constipation.

1	Clinical and basic laboratory tests Bloods, chest and abd x-ray Exclude mechanical obstruction, e.g., colonoscopy Colonic transit Consider functional bowel disease Known disorder Rx No known underlying disorder Anorectal manometry and balloon expulsion Slow colonic transit Normal Rectoanal angle measurement, defecation proctography? Appropriate Rx: Rehabilitation program, surgery, or other Chronic Constipation Normal Abnormal FIguRE 55-4 Algorithm for the management of constipation. abd, abdominal.

1	FIguRE 55-4 Algorithm for the management of constipation. abd, abdominal. Measurement of Colonic Transit Radiopaque marker transit tests are easy, repeatable, generally safe, inexpensive, reliable, and highly applicable in evaluating constipated patients in clinical practice. Several validated methods are very simple. For example, radiopaque markers are ingested; an abdominal flat film taken 5 days later should indicate passage of 80% of the markers out of the colon without the use of laxatives or enemas. This test does not provide useful information about the transit profile of the stomach and small bowel.

1	Radioscintigraphy with a delayed-release capsule containing radio-labeled particles has been used to noninvasively characterize normal, accelerated, or delayed colonic function over 24–48 h with low radiation exposure. This approach simultaneously assesses gastric, small bowel (which may be important in ∼20% of patients with delayed colonic transit because they reflect a more generalized GI motility disorder), and colonic transit. The disadvantages are the greater cost and the need for specific materials prepared in a nuclear medicine laboratory.

1	Anorectal and Pelvic Floor Tests Pelvic floor dysfunction is suggested by the inability to evacuate the rectum, a feeling of persistent rectal fullness, rectal pain, the need to extract stool from the rectum digitally, application of pressure on the posterior wall of the vagina, support of the perineum during straining, and excessive straining. These significant symptoms should be contrasted with the simple sense of incomplete rectal evacuation, which is common in IBS. Formal psychological evaluation may identify eating disorders, “control issues,” depression, or post-traumatic stress disorders that may respond to cognitive or other intervention and may be important in restoring quality of life to patients who might present with chronic constipation.

1	A simple clinical test in the office to document a non-relaxing puborectalis muscle is to have the patient strain to expel the index finger during a digital rectal examination. Motion of the puborectalis posteriorly during straining indicates proper coordination of the pelvic floor muscles. Motion anteriorly with paradoxical contraction during simulated evacuation indicates pelvic floor dysfunction.

1	Measurement of perineal descent is relatively easy to gauge clinically by placing the patient in the left decubitus position and watching 274 the perineum to detect inadequate descent (<1.5 cm, a sign of pelvic floor dysfunction) or perineal ballooning during straining relative to bony landmarks (>4 cm, suggesting excessive perineal descent). A useful overall test of evacuation is the balloon expulsion test. A balloon-tipped urinary catheter is placed and inflated with 50 mL of water. Normally, a patient can expel it while seated on a toilet or in the left lateral decubitus position. In the lateral position, the weight needed to facilitate expulsion of the balloon is determined; normally, expulsion occurs with <200 g added or unaided within 2 min. Anorectal manometry, when used in the evaluation of patients with severe constipation, may find an excessively high resting (>80 mmHg) or squeeze anal sphincter tone, suggesting anismus (anal sphincter spasm). This test also identifies rare

1	of patients with severe constipation, may find an excessively high resting (>80 mmHg) or squeeze anal sphincter tone, suggesting anismus (anal sphincter spasm). This test also identifies rare syndromes, such as adult Hirschsprung’s disease, by the absence of the rectoanal inhibitory reflex. Defecography (a dynamic barium enema including lateral views obtained during barium expulsion or a magnetic resonance defecogram) reveals “soft abnormalities” in many patients; the most relevant findings are the measured changes in rectoanal angle, anatomic defects of the rectum such as internal mucosal prolapse, and enteroceles or rectoceles. Surgically remediable conditions are identified in only a few patients. These include severe, whole-thickness intussusception with complete outlet obstruction due to funnel-shaped plugging at the anal canal or an extremely large rectocele that fills preferentially during attempts at defecation instead of expulsion of the barium through the anus. In summary,

1	to funnel-shaped plugging at the anal canal or an extremely large rectocele that fills preferentially during attempts at defecation instead of expulsion of the barium through the anus. In summary, defecography requires an interested and experienced radiologist, and abnormalities are not pathognomonic for pelvic floor dysfunction. The most common cause of outlet obstruction is failure of the puborectalis muscle to relax; this is not identified by barium defecography, but can be demonstrated by magnetic resonance defecography, which provides more information about the structure and function of the pelvic floor, distal colorectum, and anal sphincters. Neurologic testing (electromyography) is more helpful in the evaluation of patients with incontinence than of those with symptoms suggesting obstructed defecation. The absence of neurologic signs in the lower extremities suggests that any documented denervation of the puborectalis results from pelvic (e.g., obstetric) injury or from

1	obstructed defecation. The absence of neurologic signs in the lower extremities suggests that any documented denervation of the puborectalis results from pelvic (e.g., obstetric) injury or from stretching of the pudendal nerve by chronic, long-standing straining. Constipation is common among patients with spinal cord injuries, neurologic diseases such as Parkinson’s disease, multiple sclerosis, and diabetic neuropathy. Spinal-evoked responses during electrical rectal stimulation or stimulation of external anal sphincter contraction by applying magnetic stimulation over the lumbosacral cord identify patients with limited sacral neuropathies with sufficient residual nerve conduction to attempt biofeedback training. In summary, a balloon expulsion test is an important screening test for anorectal dysfunction. Rarely, an anatomic evaluation of the rectum or anal sphincters and an assessment of pelvic floor relaxation are the tools for evaluating patients in whom obstructed defecation is

1	dysfunction. Rarely, an anatomic evaluation of the rectum or anal sphincters and an assessment of pelvic floor relaxation are the tools for evaluating patients in whom obstructed defecation is suspected and is associated with symptoms of rectal mucosal prolapse, pressure of the posterior wall of the vagina to facilitate defecation (suggestive of anterior rectocele), or prior pelvic surgery that may be complicated by enterocele.

1	After the cause of constipation is characterized, a treatment decision can be made. Slow-transit constipation requires aggressive medical or surgical treatment; anismus or pelvic floor dysfunction usually responds to biofeedback management (Fig. 40-4). The remaining ∼60% of patients with constipation has normal colonic transit and can be treated symptomatically. Patients with spinal cord injuries or other neurologic disorders require a dedicated bowel regimen that often includes rectal stimulation, enema therapy, and carefully timed laxative therapy. Patients with constipation are treated with bulk, osmotic, pro- kinetic, secretory, and stimulant laxatives including fiber, psyllium, milk of magnesia, lactulose, polyethylene glycol (colonic lavage

1	PART 2 Cardinal Manifestations and Presentation of Diseases solution), lubiprostone, linaclotide, and bisacodyl, or, in some countries, prucalopride, a 5-HT4 agonist. If a 3-to 6-month trial of medical therapy fails, unassociated with obstructed defecation, the patients should be considered for laparoscopic colectomy with ileorectostomy; however, this should not be undertaken if there is continued evidence of an evacuation disorder or a generalized GI dysmotility. Referral to a specialized center for further tests of colonic motor function is warranted. The decision to resort to surgery is facilitated in the presence of megacolon and megarectum. The complications after surgery include small-bowel obstruction (11%) and fecal soiling, particularly at night during the first postoperative year. Frequency of defecation is 3–8 per day during the first year, dropping to 1–3 per day from the second year after surgery.

1	Patients who have a combined (evacuation and transit/motility) disorder should pursue pelvic floor retraining (biofeedback and muscle relaxation), psychological counseling, and dietetic advice first. If symptoms are intractable despite biofeedback and optimized medical therapy, colectomy and ileorectostomy could be considered as long as the evacuation disorder is resolved and optimized medical therapy is unsuccessful. In patients with pelvic floor dysfunction alone, biofeedback training has a 70–80% success rate, measured by the acquisition of comfortable stool habits. Attempts to manage pelvic floor dysfunction with operations (internal anal sphincter or puborectalis muscle division) or injections with botulinum toxin have achieved only mediocre success and have been largely abandoned. Russell G. Robertson, J. Larry Jameson

1	Involuntary weight loss (IWL) is frequently insidious and can have important implications, often serving as a harbinger of serious underlying disease. Clinically important weight loss is defined as the loss of 10 pounds (4.5 kg) or >5% of one’s body weight over a period of 6–12 months. IWL is encountered in up to 8% of all adult outpatients and 27% of frail persons age 65 years and older. There is no identifiable cause in up to one-quarter of patients despite extensive investigation. Conversely, up to half of people who claim to have lost weight have no documented evidence of weight loss. People with no known cause of weight loss generally have a better prognosis than do those with known causes, particularly when the source is neoplastic. Weight loss in older persons is associated with a variety of deleterious effects, including hip fracture, pressure ulcers, impaired immune function, and decreased functional status. Not surprisingly, significant weight loss is associated with

1	with a variety of deleterious effects, including hip fracture, pressure ulcers, impaired immune function, and decreased functional status. Not surprisingly, significant weight loss is associated with increased mortality, which can range from 9% to as high as 38% within 1 to 2.5 years in the absence of clinical awareness and attention.

1	(See also Chaps. 94e and 415e) Among healthy aging people, total body weight peaks in the sixth decade of life and generally remains stable until the ninth decade, after which it gradually falls. In contrast, lean body mass (fat-free mass) begins to decline at a rate of 0.3 kg per year in the third decade, and the rate of decline increases further beginning at age 60 in men and age 65 in women. These changes in lean body mass largely reflect the age-dependent decline in growth hormone secretion and, consequently, circulating levels of insulin-like growth factor type I (IGF-I) that occur with normal aging. Loss of sex steroids, at menopause in women and more gradually with aging in men, also contributes to these changes in body composition. In the healthy elderly, an increase in fat tissue balances the loss in lean body mass until very old age, when loss of both fat and skeletal muscle occurs. Age-dependent changes also occur at the cellular level. Telomeres shorten, and body cell

1	balances the loss in lean body mass until very old age, when loss of both fat and skeletal muscle occurs. Age-dependent changes also occur at the cellular level. Telomeres shorten, and body cell mass—the fat-free portion of cells— declines steadily with aging.

1	Between ages 20 and 80, mean energy intake is reduced by up to 1200 kcal/d in men and 800 kcal/d in women. Decreased hunger is a reflection of reduced physical activity and loss of lean body mass, producing lower demand for calories and food intake. Several important age-associated physiologic changes also predispose elderly persons to weight loss, such as declining chemosensory function (smell and taste), reduced efficiency of chewing, slowed gastric emptying, and alterations in the neuroendocrine axis, including changes in levels of leptin, cholecystokinin, neuropeptide Y, and other hormones and peptides. These changes are associated with early satiety and a decline in both appetite and the hedonistic appreciation of food. Collectively, they contribute to the “anorexia of aging.”

1	Most causes of IWL belong to one of four categories: (1) malignant neoplasms, (2) chronic inflammatory or infectious diseases, (3) metabolic disorders (e.g., hyperthyroidism and diabetes), or (4) psychiatric disorders (Table 56-1). Not infrequently, more than one of these causes can be responsible for IWL. In most series, IWL is caused by malignant disease in a quarter of patients and by organic disease in one-third, with the remainder due to psychiatric disease, medications, or uncertain causes. The most common malignant causes of IWL are gastrointestinal, hepatobiliary, hematologic, lung, breast, genitourinary, ovarian, and prostate. Half of all patients with cancer lose some body weight; CAuSES of invoLunTARy wEigHT LoSS

1	CAuSES of invoLunTARy wEigHT LoSS Disorders of the mouth and teeth one-third lose more than 5% of their original body weight, and up to 20% 275 of all cancer deaths are caused directly by cachexia (through immobility and/or cardiac/respiratory failure). The greatest incidence of weight loss is seen among patients with solid tumors. Malignancy that reveals itself through significant weight loss usually has a very poor prognosis. In addition to malignancies, gastrointestinal causes are among the most prominent causes of IWL. Peptic ulcer disease, inflammatory bowel disease, dysmotility syndromes, chronic pancreatitis, celiac disease, constipation, and atrophic gastritis are some of the more common entities. Oral and dental problems are easily overlooked and may manifest with halitosis, poor oral hygiene, xerostomia, inability to chew, reduced masticatory force, nonocclusion, temporomandibular joint syndrome, edentulousness, and pain due to caries or abscesses.

1	Tuberculosis, fungal diseases, parasites, subacute bacterial endocarditis, and HIV are well-documented causes of IWL. Cardiovascular and pulmonary diseases cause unintentional weight loss through increased metabolic demand and decreased appetite and caloric intake. Uremia produces nausea, anorexia, and vomiting. Connective tissue diseases may increase metabolic demand and disrupt nutritional balance. As the incidence of diabetes mellitus increases with aging, the associated glucosuria can contribute to weight loss. Hyperthyroidism in the elderly may have less prominent sympathomimetic features and may present as “apathetic hyperthyroidism” or T3 toxicosis (Chap. 405).

1	Neurologic injuries such as stroke, quadriplegia, and multiple sclerosis may lead to visceral and autonomic dysfunction that can impair caloric intake. Dysphagia from these neurologic insults is a common mechanism. Functional disability that compromises activities of daily living (ADLs) is a common cause of undernutrition in the elderly. Visual impairment from ophthalmic or central nervous system disorders such as a tremor can limit the ability of people to prepare and eat meals. IWL may be one of the earliest manifestations of Alzheimer’s dementia.

1	Isolation and depression are significant causes of IWL that may manifest as an inability to care for oneself, including nutritional needs. A cytokine-mediated inflammatory metabolic cascade can be both a cause of and a manifestation of depression. Bereavement can be a cause of IWL and, when present, is more pronounced in men. More intense forms of mental illness such as paranoid disorders may lead to delusions about food and cause weight loss. Alcoholism can be a significant source of weight loss and malnutrition. Elderly persons living in poverty may have to choose whether to purchase food or use the money for other expenses, including medications. Institutionalization is an independent risk factor, as up to 30–50% of nursing home patients have inadequate food intake. Medications can cause anorexia, nausea, vomiting, gastrointestinal distress, diarrhea, dry mouth, and changes in taste. This is particularly an issue in the elderly, many of whom take five or more medications.

1	The four major manifestations of IWL are (1) anorexia (loss of appetite), (2) sarcopenia (loss of muscle mass), (3) cachexia (a syndrome that combines weight loss, loss of muscle and adipose tissue, anorexia, and weakness), and (4) dehydration. The current obesity epidemic adds complexity, as excess adipose tissue can mask the development of sarcopenia and delay awareness of the development of cachexia. If it is not possible to measure weight directly, a change in clothing size, corroboration of weight loss by a relative or friend, and a numeric estimate of weight loss provided by the patient are suggestive of true weight loss.

1	Initial assessment includes a comprehensive history and physical, a complete blood count, tests of liver enzyme levels, C-reactive protein, erythrocyte sedimentation rate, renal function studies, thyroid function tests, chest radiography, and an abdominal ultrasound (Table 56-2). Age, sex, and risk factor–specific cancer screening tests, such as mammography and colonoscopy, should be performed (Chap. 100). Patients at risk should have HIV testing. All elderly patients with weight loss should undergo screening for dementia and depression by using instruments such as the PART 2 Cardinal Manifestations and Presentation of Diseases 10% weight loss in 180 d Comprehensive electrolyte and metabolic panel, including liver and renal function tests

1	PART 2 Cardinal Manifestations and Presentation of Diseases 10% weight loss in 180 d Comprehensive electrolyte and metabolic panel, including liver and renal function tests Body mass index <21 Thyroid function tests 25% of food left uneaten after 7 d Erythrocyte sedimentation rate Change in fit of clothing C-reactive protein Change in appetite, smell, or taste Ferritin Abdominal pain, nausea, vomiting, HIV testing, if indicated diarrhea, constipation, dysphagia aMay be more specific to assess weight loss in the elderly.

1	Mini-Mental State Examination and the Geriatric Depression Scale, respectively (Chap. 11). The Mini Nutritional Assessment (www .mna-elderly.com) and the Nutrition Screening Initiative (http:// www.ncbi.nlm.nih.gov/pmc/articles/PMC1694757/) are also available for the nutritional assessment of elderly patients. Almost all patients with a malignancy and >90% of those with other organic diseases have at least one laboratory abnormality. In patients presenting with substantial IWL, major organic and malignant diseases are unlikely when a baseline evaluation is completely normal. Careful follow-up rather than undirected testing is advised since the prognosis of weight loss of undetermined cause is generally favorable.

1	The first priority in managing weight loss is to identify and treat the underlying causes systematically. Treatment of underlying metabolic, psychiatric, infectious, or other systemic disorders may be sufficient to restore weight and functional status gradually. Medications that cause nausea or anorexia should be withdrawn or changed, if possible. For those with unexplained IWL, oral nutritional supplements such as high-energy drinks sometimes reverse weight loss. Advising patients to consume supplements between meals rather than with a meal may help minimize appetite suppression and facilitate increased overall intake. Orexigenic, anabolic, and anticytokine agents are under investigation. In selected patients, the antidepressant mirtazapine results in a significant increase in body weight, body fat mass, and leptin concentration. Patients with wasting conditions who can comply with an appropriate exercise program gain muscle protein mass, strength, and endurance and may be more

1	weight, body fat mass, and leptin concentration. Patients with wasting conditions who can comply with an appropriate exercise program gain muscle protein mass, strength, and endurance and may be more capable of performing ADLs.

1	Gastrointestinal bleeding (GIB) accounts for ~150 hospitalizations per 100,000 population annually in the United States, with upper GIB (UGIB) ~1.5–2 times more common than lower GIB (LGIB) The incidence of GIB has decreased in recent decades, primarily due to a reduction in UGIB, and the mortality has also decreased to <5%. Patients today rarely die from exsanguination, but rather die due to decompensation of other underlying illnesses.

1	GIB presents as either overt or occult bleeding. Overt GIB is manifested by hematemesis, vomitus of red blood or “coffee-grounds” material; melena, black, tarry, foul-smelling stool; and/or hematochezia, passage of bright red or maroon blood from the rectum. Occult GIB may be identified in the absence of overt bleeding when patients present with symptoms of blood loss or anemia such as lightheadedness, syncope, angina, or dyspnea; or when routine diagnostic evaluation reveals iron deficiency anemia or a positive fecal occult blood test. GIB is also categorized by the site of bleeding as UGIB, LGIB, or obscure GIB if the source is unclear.

1	SOuRCES OF gASTROINTESTINAL BLEEDINg upper gastrointestinal Sources of Bleeding (Table 57-1) Peptic ulcers are the most common cause of UGIB, accounting for ∼50% of cases. Mallory-Weiss tears account for ~5–10% of cases. The proportion of patients bleeding from varices varies widely from ~5–40%, depending on the population. Hemorrhagic or erosive gastropathy (e.g., due to nonsteroidal anti-inflammatory drugs [NSAIDs] or alcohol) and erosive esophagitis often cause mild UGIB, but major bleeding is rare.

1	PEPTIC ULCERS Characteristics of an ulcer at endoscopy provide important prognostic information. One-third of patients with active bleeding or a nonbleeding visible vessel have further bleeding that requires urgent surgery if they are treated conservatively. These patients benefit from endoscopic therapy with bipolar electrocoagulation, heater probe, injection therapy (e.g., absolute alcohol, 1:10,000 epinephrine), and/or clips with reductions in bleeding, hospital stay, mortality, and costs. In contrast, patients with clean-based ulcers have rates of recurrent bleeding approaching zero. If stable with no other reason for hospitalization, such patients may be discharged home after endoscopy. Patients without clean-based ulcers usually remain in the hospital for 3 days because most episodes of recurrent bleeding occur within 3 days.

1	Randomized controlled trials document that high-dose, constant-infusion IV proton pump inhibitor (PPI) (80-mg bolus and 8-mg/h infusion), designed to sustain intragastric pH >6 and enhance clot stability, decreases further bleeding and mortality in patients with high-risk ulcers (active bleeding, nonbleeding visible vessel, adherent clot) when given after endoscopic therapy. Patients with lower-risk findings (flat pigmented spot or clean base) do not require endoscopic Sources of Bleeding Proportion of Patients, % Source: Data on hospitalizations from year 2000 onward from Am J Gastroenterol 98:1494, 2003; Gastrointest Endosc 57:AB147, 2003; 60;875, 2004; Eur J Gastroenterol Hepatol 16:177, 2004; 17:641, 2005; J Clin Gastroenterol 42:128, 2008; World J Gastroenterol 14:5046, 2008; Dig Dis Sci 54:333, 2009; Gut 60:1327, 2011; Endoscopy 44:998, 2012; J Clin Gastroenterol 48:113, 2014.

1	therapy and receive standard doses of oral PPI. Approximately one-third of patients with bleeding ulcers will rebleed within the next 1–2 years if no preventive strategies are employed. Prevention of recurrent bleeding focuses on the three main factors in ulcer pathogenesis, Helicobacter pylori, NSAIDs, and acid. Eradication of H. pylori in patients with bleeding ulcers decreases rates of rebleeding to <5%. If a bleeding ulcer develops in a patient taking NSAIDs, the NSAIDs should be discontinued. If NSAIDs must be given, a cyclooxygenase 2 (COX-2) selective inhibitor (coxib) plus a PPI should be used. PPI co-therapy alone or a coxib alone is associated with an annual rebleeding rate of ~10% in patients with a recent bleeding ulcer, whereas the combination of a coxib and PPI provides a further significant decrease in recurrent ulcer bleeding. Patients with established cardiovascular disease who develop bleeding ulcers while taking low-dose aspirin should restart aspirin as soon as

1	further significant decrease in recurrent ulcer bleeding. Patients with established cardiovascular disease who develop bleeding ulcers while taking low-dose aspirin should restart aspirin as soon as possible after their bleeding episode (1–7 days). A randomized trial showed that failure to restart aspirin was associated with no significant difference in rebleeding (5% vs. 10% at 30 days) but a significant increase in mortality at 30 days (9% vs. 1%) and 8 weeks (13% vs. 1%) compared with immediate reinstitution of aspirin. Patients with bleeding ulcers unrelated to H. pylori or NSAIDs should remain on PPI therapy indefinitely. Peptic ulcers are discussed in Chap. 348.

1	MALLORY-wEISS TEARS The classic history is vomiting, retching, or coughing preceding hematemesis, especially in an alcoholic patient. Bleeding from these tears, which are usually on the gastric side of the gastroesophageal junction, stops spontaneously in 80–90% of patients and recurs in only 0–10%. Endoscopic therapy is indicated for actively bleeding Mallory-Weiss tears. Angiographic therapy with embolization and operative therapy with oversewing of the tear are rarely required. Mallory-Weiss tears are discussed in Chap. 347.

1	ESOPHAgEAL VARICES Patients with variceal hemorrhage have poorer outcomes than patients with other sources of UGIB. Urgent endoscopy within 12 h is recommended in cirrhotics with UGIB, and if esophageal varices are present, endoscopic ligation is performed and an IV vasoactive medication (e.g., octreotide 50 μg bolus and 50 μg/h infusion) is given for 2–5 days. Combination endoscopic and medical therapy appears to be superior to either therapy alone in decreasing rebleeding. In patients with advanced liver disease (e.g., Child-Pugh class C with score 10–13), a transjugular intrahepatic portosystemic shunt (TIPS) should be strongly considered within the first 1–2 days of hospitalization because randomized trials show significant decreases in rebleeding and mortality compared with standard endoscopic and medical therapy. Over the long term, treatment with nonselective beta blockers plus endoscopic ligation is recommended because the combination of endoscopic and medical therapy is more

1	endoscopic and medical therapy. Over the long term, treatment with nonselective beta blockers plus endoscopic ligation is recommended because the combination of endoscopic and medical therapy is more effective than either alone in reduction of recurrent esophageal variceal bleeding.

1	In patients who have persistent or recurrent bleeding despite endoscopic and medical therapy, TIPS is recommended. Decompressive surgery (e.g., distal splenorenal shunt) may be considered instead of TIPS in patients with well-compensated cirrhosis. Portal hypertension is also responsible for bleeding from gastric varices, varices in the small and large intestine, and portal hypertensive gastropathy and enterocolopathy. Bleeding gastric varices due to cirrhosis are treated with endoscopic injection of tissue adhesive (e.g., n-butyl cyanoacrylate), if available; if not, TIPS is performed.

1	HEMORRHAgIC ANd EROSIVE gASTROPATHY (“gASTRITIS”) Hemorrhagic and erosive gastropathy, often labeled gastritis, refers to endoscopically visualized subepithelial hemorrhages and erosions. These are mucosal lesions and do not cause major bleeding due to the absence of arteries and veins in the mucosa. Erosions develop in various clinical settings, the most important of which are NSAID use, alcohol intake, and stress. Half of patients who chronically ingest NSAIDs have erosions, whereas up to 20% of actively drinking alcoholic patients with symptoms of UGIB have evidence of subepithelial hemorrhages or erosions.

1	Stress-related gastric mucosal injury occurs only in extremely sick patients, such as those who have experienced serious trauma, major surgery, burns covering more than one-third of the body surface area, 277 major intracranial disease, or severe medical illness (i.e., ventilator dependence, coagulopathy). Severe bleeding should not develop unless ulceration occurs. The mortality rate in these patients is quite high because of their serious underlying illnesses. The incidence of bleeding from stress-related gastric mucosal injury has decreased dramatically in recent years, most likely due to better care of critically ill patients. Pharmacologic prophylaxis for bleeding may be considered in the high-risk patients mentioned above. Meta-analyses of randomized trials indicate that PPIs are more effective than H2 receptor antagonists in reduction of overt and clinically important UGIB without differences in mortality or nosocomial pneumonia.

1	OTHER CAUSES Other less frequent causes of UGIB include erosive duodenitis, neoplasms, aortoenteric fistulas, vascular lesions (including hereditary hemorrhagic telangiectasias [Osler-Weber-Rendu] and gastric antral vascular ectasia [“watermelon stomach”]), Dieulafoy’s lesion (in which an aberrant vessel in the mucosa bleeds from a pinpoint mucosal defect), prolapse gastropathy (prolapse of proximal stomach into esophagus with retching, especially in alcoholics), and hemobilia or hemosuccus pancreaticus (bleeding from the bile duct or pancreatic duct).

1	Small-Intestinal Sources of Bleeding Small-intestinal sources of bleeding (bleeding from sites beyond the reach of the standard upper endoscope) are often difficult to diagnose and are responsible for the majority of cases of obscure GIB. Fortunately, small-intestinal bleeding is uncommon. The most common causes in adults are vascular ectasias, tumors (e.g., GI stromal tumor, carcinoid, adenocarcinoma, lymphoma, metastases), and NSAID-induced erosions and ulcers. Other less common causes in adults include Crohn’s disease, infection, ischemia, vasculitis, small-bowel varices, diverticula, Meckel’s diverticulum, duplication cysts, and intussusception. Meckel’s diverticulum is the most common cause of significant LGIB in children, decreasing in frequency as a cause of bleeding with age. In adults <40–50 years, small-bowel tumors often account for obscure GIB; in patients >50–60 years, vascular ectasias and NSAID-induced lesions are more commonly responsible.

1	Vascular ectasias should be treated with endoscopic therapy if possible. Although estrogen/progesterone compounds have been used for vascular ectasias, a large double-blind trial found no benefit in prevention of recurrent bleeding. Octreotide is also used, based on case series but no randomized trials. A randomized trial reported significant benefit of thalidomide and awaits further confirmation. Other isolated lesions, such as tumors, are generally treated with surgical resection.

1	Colonic Sources of Bleeding Hemorrhoids are probably the most common cause of LGIB; anal fissures also cause minor bleeding and pain. If these local anal processes, which rarely require hospitalization, are excluded, the most common causes of LGIB in adults are diverticula, vascular ectasias (especially in the proximal colon of patients >70 years), neoplasms (primarily adenocarcinoma), colitis (ischemic, infectious, idiopathic inflammatory bowel disease), and postpolypectomy bleeding. Less common causes include NSAID-induced ulcers or colitis, radiation proctopathy, solitary rectal ulcer syndrome, trauma, varices (most commonly rectal), lymphoid nodular hyperplasia, vasculitis, and aortocolic fistulas. In children and adolescents, the most common colonic causes of significant GIB are inflammatory bowel disease and juvenile polyps.

1	Diverticular bleeding is abrupt in onset, usually painless, sometimes massive, and often from the right colon; chronic or occult bleeding is not characteristic. Clinical reports suggest that bleeding colonic diverticula stop bleeding spontaneously in ~80% of patients and, on long-term follow-up, rebleed in ~15–25% of patients. Case series suggest endoscopic therapy may decrease recurrent bleeding in the uncommon case when colonoscopy identifies the specific bleeding diverticulum. When diverticular bleeding is found at angiography, transcatheter arterial embolization by superselective technique stops bleeding in a majority of patients. If bleeding persists or recurs, segmental surgical resection is indicated.

1	Bleeding from right colonic vascular ectasias in the elderly may be overt or occult; it tends to be chronic and only occasionally is hemodynamically significant. Endoscopic hemostatic therapy may be useful in the treatment of vascular ectasias, as well as discrete bleeding ulcers and postpolypectomy bleeding. Surgical therapy is generally required for major, persistent, or recurrent bleeding from the wide variety of colonic sources of GIB that cannot be treated medically, angiographically, or endoscopically. APPROACH TO THE PATIENT:

1	Measurement of the heart rate and blood pressure is the best way to initially assess a patient with GIB. Clinically significant bleeding leads to postural changes in heart rate or blood pressure, tachycardia, and, finally, recumbent hypotension. In contrast, the hemoglobin does not fall immediately with acute GIB, due to proportionate reductions in plasma and red cell volumes (i.e., “people bleed whole blood”). Thus, hemoglobin may be normal or only minimally decreased at the initial presentation of a severe bleeding episode. As extravascular fluid enters the vascular space to restore volume, the hemoglobin falls, but this process may take up to 72 h. Transfusion is recommended when the hemoglobin drops below 7 g/dL, based on a large randomized trial showing this restrictive transfusion strategy decreases rebleeding and death in acute UGIB compared with a transfusion threshold of 9 g/dL. Patients with slow, chronic GIB may have very low hemoglobin values despite normal blood pressure

1	decreases rebleeding and death in acute UGIB compared with a transfusion threshold of 9 g/dL. Patients with slow, chronic GIB may have very low hemoglobin values despite normal blood pressure and heart rate. With the development of iron-deficiency anemia, the mean corpuscular volume will be low and red blood cell distribution width will increase.

1	Hematemesis indicates an upper GI source of bleeding (above the ligament of Treitz). Melena indicates blood has been present in the GI tract for at least 14 h, and as long as 3–5 days. The more proximal the bleeding site, the more likely melena will occur. Hematochezia usually represents a lower GI source of bleeding, although an upper GI lesion may bleed so briskly that blood transits the bowel before melena develops. When hematochezia is the presenting symptom of UGIB, it is associated with hemodynamic instability and dropping hemoglobin. Bleeding lesions of the small bowel may present as melena or hematochezia. Other clues to UGIB include hyperactive bowel sounds and an elevated blood urea nitrogen (due to volume depletion and blood proteins absorbed in the small intestine). PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases A nonbloody nasogastric aspirate may be seen in up to ~18% of patients with UGIB, usually from a duodenal source. Even a bile-stained appearance does not exclude a bleeding postpyloric lesion because reports of bile in the aspirate are incorrect in ~50% of cases. Testing of aspirates that are not grossly bloody for occult blood is not useful. EVALuATION AND MANAgEMENT OF ugIB (FIg. 57-1)

1	At presentation, patients are generally stratified as higher or lower risk for further bleeding and death. Baseline characteristics predictive of rebleeding and death include hemodynamic compromise (tachycardia or hypotension), increasing age, and comorbidities. PPI infusion may be considered at presentation: it decreases high-risk ulcer stigmata (e.g., active bleeding) and need for endoscopic therapy but does not improve clinical outcomes such as further bleeding, surgery, or death. Treatment to improve endoscopic visualization with the promotility agent erythromycin, 250 mg intravenously ~30 min before endoscopy, also may be considered: it provides a small but significant increase in diagnostic yield and decrease in second endoscopies but is not documented to decrease further bleeding or death. Cirrhotic patients presenting with UGIB should be placed on antibiotics (e.g., quinolone, ceftriaxone) and started on a vasoactive medication (octreotide, terlipressin, somatostatin,

1	or death. Cirrhotic patients presenting with UGIB should be placed on antibiotics (e.g., quinolone, ceftriaxone) and started on a vasoactive medication (octreotide, terlipressin, somatostatin, vapreotide) upon presentation, even before endoscopy. Antibiotics decrease bacterial infections, rebleeding, and mortality in this population, and vasoactive medications appear to improve control of bleeding in the first 12 h after presentation.

1	Upper endoscopy should be performed within 24 h in most patients with UGIB. Patients at higher risk (e.g., hemodynamic instability, cirrhosis) may benefit from more urgent endoscopy within 12 h. Early endoscopy is also beneficial in low-risk patients for management decisions. Patients with major bleeding and high-risk endoscopic findings (e.g., varices, ulcers with active bleeding or a visible vessel) benefit from endoscopic hemostatic therapy, whereas patients with low-risk lesions (e.g., clean-based ulcers, nonbleeding Mallory-Weiss tears, erosive or hemorrhagic gastropathy) who have stable vital signs and hemoglobin and no other medical problems can be discharged home. EVALuATION AND MANAgEMENT OF LgIB (FIg. 57-2) Patients with hematochezia and hemodynamic instability should have upper endoscopy to rule out an upper GI source before evaluation of the lower GI tract.

1	Colonoscopy after an oral lavage solution is the procedure of choice in most patients admitted with LGIB unless bleeding is too massive, in which case angiography is recommended. Sigmoidoscopy is used primarily in patients <40 years old with minor bleeding. In patients with no source identified on colonoscopy, imaging studies may be employed. 99mTc-labeled red cell scan allows repeated imaging for up to 24 h and may identify the general location of bleeding. However, radionuclide scans should be interpreted with caution because results, especially from later images, are highly variable. Multidector computed tomography (CT) “angiography” is an increasingly used technique that is likely superior to nuclear scintigraphy. In active LGIB, angiography can detect the site of bleeding (extravasation of contrast into the gut) and permits treatment with embolization. Even after bleeding has stopped, angiography may identify lesions with abnormal vasculature, such as vascular ectasias or tumors.

1	Acute upper GI bleeding ICU for 1–2 days; ward for 2–3 days Ligation + IV vasoactive drug (e.g., octreotide) Esophageal varicesUlcer Mallory-Weiss tear Clean base Discharge No IV PPI or endoscopic therapy Active bleeding or visible vessel ICU for 1 day; ward for 2 days IV PPI therapy + endoscopic therapy Adherent clot Ward for 2–3 days IV PPI therapy +/– endoscopic therapy Flat, pigmented spot No IV PPI or endoscopic therapy Ward for 3 days Active bleeding No active bleeding No endoscopic therapy Discharge Endoscopic therapy Ward for 1–2 days FIguRE 57-1 Suggested algorithm for patients with acute upper gastrointestinal (GI) bleeding. Recommendations on level of care and time of discharge assume patient is stabilized without further bleeding or other concomitant medical problems. ICU, intensive care unit; PPI, proton pump inhibitor.

1	Hemodynamic instability Site identified; bleeding stops Angiography Obscure bleeding work-up Flexible sigmoidoscopy (colonoscopy if iron-deficiency anemia, familial colon cancer, or copious bleeding)* Bleeding persists Surgery Acute lower GI bleeding No hemodynamic instability Age ˜40 yrs Upper endoscopy^Age <40 yrs Colonoscopy Colonoscopy† Site identified; bleeding persists Site not identified; bleeding persists FIguRE 57-2 Suggested algorithm for patients with acute lower gastrointestinal (GI) bleeding. *Some suggest colonoscopy for any degree of rectal bleeding in patients <40 years as well. ^If upper GI endoscopy reveals definite source, no further evaluation is needed. †If massive bleeding does not allow time for colonic lavage, proceed to angiography.

1	Obscure GIB is defined as persistent or recurrent bleeding for which no source has been identified by routine endoscopic and contrast x-ray studies; it may be overt (melena, hematochezia) or occult (iron-deficiency anemia). Current guidelines suggest angiography as the initial test for massive obscure bleeding, and video capsule endoscopy, which allows examination of the entire small intestine, for all others. Push enteroscopy, usually performed with a pediatric colonoscope, to inspect the entire duodenum and proximal jejunum also may be considered as an initial evaluation. A systematic review of 14 trials comparing push enteroscopy to capsule revealed “clinically significant findings” in 26% and 56% of patients, respectively. However, in contrast to enteroscopy, lack of control of the capsule prevents its manipulation and full visualization of the intestine; in addition, tissue cannot be sampled and therapy cannot be applied.

1	If capsule endoscopy is positive, management is dictated by the finding. If capsule endoscopy is negative, current recommendations suggest patients may either be observed or, if their clinical course mandates (e.g., recurrent bleeding, need for transfusions or hospitalization), undergo further testing. “Deep” enteroscopy (e.g., double-balloon, single-balloon, and spiral enteroscopy) is commonly the next test undertaken in patients with clinically important obscure GIB because it allows the endoscopist to examine, obtain specimens from, and provide therapy to much or all of the small intestine. CT and magnetic resonance enterography also are used to examine the small intestine. Other imaging techniques sometimes used in evaluation of obscure GIB include 99mTc-labeled red blood cell scintigraphy, multidetector CT “angiography,” angiography, and 99mTc-pertechnetate scintigraphy for Meckel’s diverticulum (especially in young patients). If all tests are unrevealing, intraoperative

1	scintigraphy, multidetector CT “angiography,” angiography, and 99mTc-pertechnetate scintigraphy for Meckel’s diverticulum (especially in young patients). If all tests are unrevealing, intraoperative endoscopy is indicated in patients with severe recurrent or persistent bleeding requiring repeated transfusions.

1	Fecal occult blood testing is recommended only for colorectal cancer screening and may be used beginning at age 50 in average-risk adults and beginning at age 40 in adults with a first-degree relative with colorectal neoplasm at ≥60 years or two second-degree relatives with colorectal cancer. A positive test necessitates colonoscopy. If evaluation of the colon is negative, further workup is not recommended unless iron-deficiency anemia or GI symptoms are present. Savio John, Daniel S. Pratt

1	Jaundice, or icterus, is a yellowish discoloration of tissue resulting from the deposition of bilirubin. Tissue deposition of bilirubin occurs only in the presence of serum hyperbilirubinemia and is a sign of either liver disease or, less often, a hemolytic disorder. The degree of serum bilirubin elevation can be estimated by physical examination. Slight increases in serum bilirubin level are best detected by examining the sclerae, which have a particular affinity for bilirubin due to their high elastin content. The presence of scleral icterus indicates a serum bilirubin level of at least 51 μmol/L (3 mg/dL). The ability to detect scleral icterus is made more difficult if the examining room has fluorescent lighting. If the examiner suspects scleral icterus, a second site to examine is underneath the tongue. As serum bilirubin levels rise, the skin will eventually become yellow in light-skinned patients and even green if the process is long-standing; the green color is produced by

1	underneath the tongue. As serum bilirubin levels rise, the skin will eventually become yellow in light-skinned patients and even green if the process is long-standing; the green color is produced by oxidation of bilirubin to biliverdin.

1	The differential diagnosis for yellowing of the skin is limited. In addition to jaundice, it includes carotenoderma, the use of the drug quinacrine, and excessive exposure to phenols. Carotenoderma is the yellow color imparted to the skin of healthy individuals who ingest excessive amounts of vegetables and fruits that contain carotene, such as carrots, leafy vegetables, squash, peaches, and oranges. In jaundice the yellow coloration of the skin is uniformly distributed over the body, whereas in carotenoderma the pigment is concentrated on the palms, soles, forehead, and nasolabial folds. Carotenoderma can be distinguished from jaundice by the sparing of the sclerae. Quinacrine causes a yellow discoloration of the skin in 4–37% of patients treated with it.

1	Another sensitive indicator of increased serum bilirubin is darkening of the urine, which is due to the renal excretion of conjugated bilirubin. Patients often describe their urine as teaor cola-colored. Bilirubinuria indicates an elevation of the direct serum bilirubin fraction and, therefore, the presence of liver disease. Serum bilirubin levels increase when an imbalance exists between bilirubin production and clearance. A logical evaluation of the patient who is jaundiced requires an understanding of bilirubin production and metabolism. (See also Chap. 359) Bilirubin, a tetrapyrrole pigment, is a breakdown product of heme (ferroprotoporphyrin IX). About 70–80% of the 250– 300 mg of bilirubin produced each day is derived from the breakdown of hemoglobin in senescent red blood cells. The remainder comes from prematurely destroyed erythroid cells in bone marrow and from the turnover of hemoproteins such as myoglobin and cytochromes found in tissues throughout the body.

1	The formation of bilirubin occurs in reticuloendothelial cells, primarily in the spleen and liver. The first reaction, catalyzed by the microsomal enzyme heme oxygenase, oxidatively cleaves the α bridge of the porphyrin group and opens the heme ring. The end products of this reaction are biliverdin, carbon monoxide, and iron. The second reaction, catalyzed by the cytosolic enzyme biliverdin reductase, reduces the central methylene bridge of biliverdin and converts it to bilirubin. Bilirubin formed in the reticuloendothelial cells is virtually insoluble in water due to tight internal hydrogen bonding between the water-soluble moieties of bilirubin—i.e., the bonding of the proprionic acid carboxyl groups of one dipyrrolic half of the molecule with the imino and lactam groups of the opposite half. This configuration blocks solvent access to the polar residues of bilirubin and places the hydrophobic residues on the outside. To be transported in blood, bilirubin must be solubilized.

1	half. This configuration blocks solvent access to the polar residues of bilirubin and places the hydrophobic residues on the outside. To be transported in blood, bilirubin must be solubilized. Solubilization is accomplished by the reversible, noncovalent binding of bilirubin to albumin. Unconjugated bilirubin bound to albumin is transported to the liver. There, the bilirubin—but not the albumin—is taken up by hepatocytes via a process that at least partly involves carrier-mediated membrane transport. No specific bilirubin transporter has yet been identified (Chap. 359, Fig. 359-1).

1	After entering the hepatocyte, unconjugated bilirubin is bound in the cytosol to a number of proteins including proteins in the glutathione-S-transferase superfamily. These proteins serve both to reduce efflux of bilirubin back into the serum and to present the bilirubin for conjugation. In the endoplasmic reticulum, bilirubin is solubilized by conjugation to glucuronic acid, a process that disrupts the internal hydrogen bonds and yields bilirubin monoglucuronide and diglucuronide. The conjugation of glucuronic acid to bilirubin is catalyzed by bilirubin uridine diphosphate-glucuronosyl transferase (UDPGT). The now-hydrophilic bilirubin conjugates diffuse from the endoplasmic reticulum to the canalicular membrane, where bilirubin monoglucuronide and diglucuronide are actively transported into canalicular bile by an energy-dependent mechanism involving the multidrug resistance–associated protein 2 (MRP2).

1	The conjugated bilirubin excreted into bile drains into the duodenum and passes unchanged through the proximal small bowel. Conjugated bilirubin is not taken up by the intestinal mucosa. When the conjugated bilirubin reaches the distal ileum and colon, it is hydrolyzed to unconjugated bilirubin by bacterial β-glucuronidases. The unconjugated bilirubin is reduced by normal gut bacteria to form a group of colorless tetrapyrroles called urobilinogens. About 80–90% of these products are excreted in feces, either unchanged or oxidized to orange derivatives called urobilins. The remaining 10–20% of the urobilinogens are passively absorbed, enter the portal venous blood, and are re-excreted by the liver. A small fraction (usually <3 mg/dL) escapes hepatic uptake, filters across the renal glomerulus, and is excreted in urine.

1	The terms direct and indirect bilirubin—i.e., conjugated and unconjugated bilirubin, respectively—are based on the original van den Bergh reaction. This assay, or a variation of it, is still used in most clinical chemistry laboratories to determine the serum bilirubin level. In this assay, bilirubin is exposed to diazotized sulfanilic acid and splits into two relatively stable dipyrrylmethene azopigments that absorb PART 2 Cardinal Manifestations and Presentation of Diseases maximally at 540 nm, allowing photometric analysis. The direct fraction is that which reacts with diazotized sulfanilic acid in the absence of an accelerator substance such as alcohol. The direct fraction provides an approximation of the conjugated bilirubin level in serum. The total serum bilirubin is the amount that reacts after the addition of alcohol. The indirect fraction is the difference between the total and the direct bilirubin levels and provides an estimate of the unconjugated bilirubin in serum.

1	With the van den Bergh method, the normal serum bilirubin concentration usually is 17 μmol/L (<1 mg/dL). Up to 30%, or 5.1 μmol/L (0.3 mg/dL), of the total may be direct-reacting (conjugated) bilirubin. Total serum bilirubin concentrations are between 3.4 and 15.4 μmol/L (0.2 and 0.9 mg/dL) in 95% of a normal population.

1	Several new techniques, although less convenient to perform, have added considerably to our understanding of bilirubin metabolism. First, studies using these methods demonstrate that, in normal persons or those with Gilbert’s syndrome, almost 100% of the serum bilirubin is unconjugated; <3% is monoconjugated bilirubin. Second, in jaundiced patients with hepatobiliary disease, the total serum bilirubin concentration measured by these new, more accurate methods is lower than the values found with diazo methods. This finding suggests that there are diazo-positive compounds distinct from bilirubin in the serum of patients with hepatobiliary disease. Third, these studies indicate that, in jaundiced patients with hepatobiliary disease, monoglucuronides of bilirubin predominate over diglucuronides. Fourth, part of the direct-reacting bilirubin fraction includes conjugated bilirubin that is covalently linked to albumin. This albumin-linked bilirubin fraction (delta fraction, or biliprotein)

1	Fourth, part of the direct-reacting bilirubin fraction includes conjugated bilirubin that is covalently linked to albumin. This albumin-linked bilirubin fraction (delta fraction, or biliprotein) represents an important fraction of total serum bilirubin in patients with cholestasis and hepatobiliary disorders. The delta fraction (delta bilirubin) is formed in serum when hepatic excretion of bilirubin glucuronides is impaired and the glucuronides accumulate in serum. By virtue of its tight binding to albumin, the clearance rate of delta bilirubin from serum approximates the half-life of albumin (12–14 days) rather than the short half-life of bilirubin (about 4 h).

1	The prolonged half-life of albumin-bound conjugated bilirubin accounts for two previously unexplained enigmas in jaundiced patients with liver disease: (1) that some patients with conjugated hyperbilirubinemia do not exhibit bilirubinuria during the recovery phase of their disease because the bilirubin is covalently bound to albumin and therefore not filtered by the renal glomeruli, and (2) that the elevated serum bilirubin level declines more slowly than expected in some patients who otherwise appear to be recovering satisfactorily. Late in the recovery phase of hepatobiliary disorders, all the conjugated bilirubin may be in the albumin-linked form.

1	Unconjugated bilirubin is always bound to albumin in the serum, is not filtered by the kidney, and is not found in the urine. Conjugated bilirubin is filtered at the glomerulus, and the majority is reabsorbed by the proximal tubules; a small fraction is excreted in the urine. Any bilirubin found in the urine is conjugated bilirubin. The presence of bilirubinuria implies the presence of liver disease. A urine dipstick test (Ictotest) gives the same information as fractionation of the serum bilirubin and is very accurate. A false-negative result is possible in patients with prolonged cholestasis due to the predominance of delta bilirubin, which is covalently bound to albumin and therefore not filtered by the renal glomeruli. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: The goal of this chapter is not to provide an encyclopedic review of all of the conditions that can cause jaundice. Rather, the chapter is intended to offer a framework that helps a physician to evaluate the patient with jaundice in a logical way (Fig. 58-1). History (focus on medication/drug exposure) Physical examination Lab tests: Bilirubin with fractionation, ALT, AST, alkaline phosphatase, prothrombin time, and albumin

1	Isolated elevation of the bilirubin Indirect hyperbilirubinemia (direct < 15%) See Table 58-1 Direct hyperbilirubinemia (direct > 15%) See Table 58-1 Drugs Rifampicin Probenecid Inherited disorders Dubin-Johnson syndrome Rotor syndrome 1. Viral serologies Hepatitis A IgM Hepatitis B surface antigen and core antibody (IgM) Hepatitis C RNA 2. Toxicology screen Acetaminophen level 3. Ceruloplasmin (if patient < 40 years of age) 4. ANA, SMA, SPEP Inherited disorders Gilbert's syndrome Crigler-Najjar syndromes Hemolytic disorders Ineffective erythropoiesis Bilirubin and other liver tests elevated Hepatocellular pattern: ALT/AST elevated out of proportion to alkaline phosphatase See Table 58-2 Cholestatic pattern: Alkaline phosphatase out of proportion ALT/AST See Table 58-3 Dilated ducts Extrahepatic cholestasis CT/MRCP/ERCP Liver biopsy Liver biopsy MRCP/Liver biopsy Results negativeResults negative Additional virologic testing CMV DNA, EBV capsid antigen Hepatitis D antibody (if

1	Extrahepatic cholestasis CT/MRCP/ERCP Liver biopsy Liver biopsy MRCP/Liver biopsy Results negativeResults negative Additional virologic testing CMV DNA, EBV capsid antigen Hepatitis D antibody (if indicated) Hepatitis E IgM (if indicated) Results negative AMA positive Serologic testing AMA Hepatitis serologies Hepatitis A, CMV, EBV Review drugs (see Table 58-3) Ultrasound Ducts not dilated Intrahepatic cholestasis

1	FIguRE 58-1 Evaluation of the patient with jaundice. ALT, alanine aminotransferase; AMA, antimitochondrial antibody; ANA, antinuclear antibody; AST, aspartate aminotransferase; CMV, cytomegalovirus; EBV, Epstein-Barr virus; LKM, liver-kidney microsomal antibody; MRCP, magnetic resonance cholangiopancreatography; SMA, smooth-muscle antibody; SPEP, serum protein electrophoresis. Simply stated, the initial step is to perform appropriate blood tests in order to determine whether the patient has an isolated elevation of serum bilirubin. If so, is the bilirubin elevation due to an increased unconjugated or conjugated fraction? If the hyperbilirubinemia is accompanied by other liver test abnormalities, is the disorder hepatocellular or cholestatic? If cholestatic, is it intraor extrahepatic? All of these questions can be answered with a thoughtful history, physical examination, and interpretation of laboratory and radiologic tests and procedures.

1	The bilirubin present in serum represents a balance between input from the production of bilirubin and hepatic/biliary removal of the pigment. Hyperbilirubinemia may result from (1) overproduction of bilirubin; (2) impaired uptake, conjugation, or excretion of bilirubin; or (3) regurgitation of unconjugated or conjugated bilirubin from damaged hepatocytes or bile ducts. An increase in unconjugated bilirubin in serum results from overproduction, impaired uptake, or conjugation of bilirubin. An increase in conjugated bilirubin is due to decreased excretion into the bile ductules or backward leakage of the pigment. The initial steps in evaluating the patient with jaundice are to determine (1) whether the hyperbilirubinemia is predominantly conjugated or unconjugated in nature and (2) whether other biochemical liver tests are abnormal.

1	The thoughtful interpretation of limited data permits a rational evaluation of the patient (Fig. 58-1). The following discussion will focus solely on the evaluation of the adult patient with jaundice. ISOLATED ELEVATION OF SERuM BILIRuBIN unconjugated Hyperbilirubinemia The differential diagnosis of isolated unconjugated hyperbilirubinemia is limited (Table 58-1). The critical determination is whether the patient is suffering from a hemolytic process resulting in an overproduction of bilirubin (hemolytic disorders and ineffective erythropoiesis) or from impaired hepatic uptake/conjugation of bilirubin (drug effect or genetic disorders).

1	Hemolytic disorders that cause excessive heme production may be either inherited or acquired. Inherited disorders include spherocytosis, sickle cell anemia, thalassemia, and deficiency of red cell enzymes such as pyruvate kinase and glucose-6-phosphate dehydrogenase. In these conditions, the serum bilirubin level rarely exceeds 86 μmol/L (5 mg/dL). Higher levels may occur when there is coexistent renal or hepatocellular dysfunction or in acute hemolysis, such as a sickle cell crisis. In evaluating jaundice in patients with chronic hemolysis, it is important to remember the high incidence of pigmented (calcium bilirubinate) gallstones found in these CAuSES of iSoLATED HyPERBiLiRuBinEMiA PART 2 Cardinal Manifestations and Presentation of Diseases I. Indirect hyperbilirubinemia A. Hemolytic disorders 1. Inherited a. Spherocytosis, elliptocytosis, glucose-6-phosphate dehydrogenase and pyruvate kinase deficiencies b. 2. Acquired a. b. c. d. e. B. 1.

1	A. Hemolytic disorders 1. Inherited a. Spherocytosis, elliptocytosis, glucose-6-phosphate dehydrogenase and pyruvate kinase deficiencies b. 2. Acquired a. b. c. d. e. B. 1. Cobalamin, folate, and severe iron deficiencies 2. C. Increased bilirubin production 1. 2. Resorption of hematoma D. Drugs 1. 2. 3. E. Inherited conditions 1. 2. II. A. B. patients, which increases the likelihood of choledocholithiasis as an alternative explanation for hyperbilirubinemia. lytic anemia (e.g., hemolytic-uremic syndrome), paroxysmal noc turnal hemoglobinuria, spur cell anemia, immune hemolysis, and parasitic infections (e.g., malaria and babesiosis). Ineffective erythro poiesis occurs in cobalamin, folate, and iron deficiencies. Resorption increased hemoglobin release and overproduction of bilirubin. In the absence of hemolysis, the physician should consider a problem with the hepatic uptake or conjugation of bilirubin.

1	In the absence of hemolysis, the physician should consider a problem with the hepatic uptake or conjugation of bilirubin. Certain drugs, including rifampin and probenecid, may cause of bilirubin. Impaired bilirubin conjugation occurs in three genetic conditions: Crigler-Najjar syndrome types I and II and Gilbert’s syndrome. Crigler-Najjar type I is an exceptionally rare condition >342 μmol/L [>20 mg/dL]) and neurologic impairment due to kernicterus, frequently leading to death in infancy or childhood. These patients have a complete absence of bilirubin UDPGT activ ity, usually due to mutations in the critical 3′ domain of the UDPGT gene; are totally unable to conjugate bilirubin; and hence cannot excrete it. Crigler-Najjar type II is somewhat more common than type I. Patients live into adulthood with serum bilirubin levels of 103–428 μmol/L (6–25 mg/dL). In these patients, mutations in the bilirubin

1	Crigler-Najjar type II is somewhat more common than type I. Patients live into adulthood with serum bilirubin levels of 103–428 μmol/L (6–25 mg/dL). In these patients, mutations in the bilirubin UDPGT gene cause the reduction—but not the complete eradica tion—of the enzyme’s activity. Bilirubin UDPGT activity can be induced by the administration of phenobarbital, which can reduce serum bilirubin levels in these patients. Despite marked jaundice, these patients usually survive into adulthood, although they may be susceptible to kernicterus under the stress of intercurrent illness or surgery.

1	Gilbert’s syndrome is also marked by the impaired conjugation of bilirubin (to approximately one-third of normal) due to reduced bilirubin UDPGT activity. Patients with Gilbert’s syndrome have mild unconjugated hyperbilirubinemia, with serum levels almost always <103 μmol/L (6 mg/dL). The serum levels may fluctuate, and jaundice is often identified only during periods of fasting. The molecular defect in Gilbert’s syndrome is linked to a reduction in transcription of the bilirubin UDPGT gene due to mutations in the promoter and, rarely, in the coding region. Unlike both Crigler-Najjar syndromes, Gilbert’s syndrome is very common. The reported incidence is 3–7% of the population, with males predominating over females by a ratio of 2–7:1.

1	Conjugated Hyperbilirubinemia Elevated conjugated hyperbilirubinemia is found in two rare inherited conditions: Dubin-Johnson syndrome and Rotor syndrome (Table 58-1). Patients with either condition present with asymptomatic jaundice. The defect in Dubin-Johnson syndrome is the presence of mutations in the gene for MRP2. These patients have altered excretion of bilirubin into the bile ducts. Rotor syndrome may represent a deficiency of the major hepatic drug uptake transporters OATP1B1 and OATP1B3. Differentiating between these syndromes is possible but is clinically unnecessary due to their benign nature.

1	The remainder of this chapter will focus on the evaluation of patients with conjugated hyperbilirubinemia in the setting of other liver test abnormalities. This group of patients can be divided into those with a primary hepatocellular process and those with intraor extrahepatic cholestasis. This distinction, which is based on the history and physical examination as well as the pattern of liver test abnormalities, guides the clinician’s evaluation (Fig. 58-1).

1	History A complete medical history is perhaps the single most important part of the evaluation of the patient with unexplained jaundice. Important considerations include the use of or exposure to any chemical or medication, whether physician-prescribed, overthe-counter, complementary, or alternative medicines (e.g., herbal and vitamin preparations) or other drugs such as anabolic steroids. The patient should be carefully questioned about possible parenteral exposures, including transfusions, intravenous and intranasal drug use, tattooing, and sexual activity. Other important points include recent travel history; exposure to people with jaundice; exposure to possibly contaminated foods; occupational exposure to hepatotoxins; alcohol consumption; the duration of jaundice; and the presence of any accompanying signs and symptoms, such as arthralgias, myalgias, rash, anorexia, weight loss, abdominal pain, fever, pruritus, and changes in the urine and stool. While none of the latter

1	of any accompanying signs and symptoms, such as arthralgias, myalgias, rash, anorexia, weight loss, abdominal pain, fever, pruritus, and changes in the urine and stool. While none of the latter manifestations is specific for any one condition, any of them can suggest a particular diagnosis. A history of arthralgias and myalgias predating jaundice suggests hepatitis, either viral or drug-related. Jaundice associated with the sudden onset of severe right-upper-quadrant pain and shaking chills suggests choledocholithiasis and ascending cholangitis.

1	Physical Examination The general assessment should include evaluation of the patient’s nutritional status. Temporal and proximal muscle wasting suggests long-standing disease such as pancreatic cancer or cirrhosis. Stigmata of chronic liver disease, including spider nevi, palmar erythema, gynecomastia, caput medusae, Dupuytren’s contractures, parotid gland enlargement, and testicular atrophy, are commonly seen in advanced alcoholic (Laennec’s) cirrhosis and occasionally in other types of cirrhosis. An enlarged left supraclavicular node (Virchow’s node) or a periumbilical nodule (Sister Mary Joseph’s nodule) suggests an abdominal malignancy. Jugular venous distention, a sign of right-sided heart failure, suggests hepatic congestion. Right pleural effusion in the absence of clinically apparent ascites may be seen in advanced cirrhosis.

1	Jugular venous distention, a sign of right-sided heart failure, suggests hepatic congestion. Right pleural effusion in the absence of clinically apparent ascites may be seen in advanced cirrhosis. The abdominal examination should focus on the size and consistency of the liver, on whether the spleen is palpable and hence enlarged, and on whether ascites is present. Patients with cirrhosis may have an enlarged left lobe of the liver, which is felt below the xiphoid, and an enlarged spleen. A grossly enlarged nodular liver or an obvious abdominal mass suggests malignancy. An enlarged tender liver could signify viral or alcoholic hepatitis; an infiltrative process such as amyloidosis; or, less often, an acutely congested liver secondary to right-sided heart failure. Severe right-upper-quadrant tenderness with respiratory arrest on inspiration (Murphy’s sign) suggests cholecystitis. Ascites in the presence of jaundice suggests either cirrhosis or malignancy with peritoneal spread.

1	Laboratory Tests A battery of tests are helpful in the initial evaluation of a patient with unexplained jaundice. These include total and direct serum bilirubin measurement with fractionation; determination of serum aminotransferase, alkaline phosphatase, and albumin concentrations; and prothrombin time tests. Enzyme tests (alanine aminotransferase [ALT], aspartate aminotransferase [AST], and alkaline phosphatase [ALP]) are helpful in differentiating between a hepatocellular process and a cholestatic process (Table 358-1; Fig. 58-1)—a critical step in determining what additional workup is indicated. Patients with a hepatocellular process generally have a rise in the aminotransferases that is disproportionate to that in ALP, whereas patients with a cholestatic process have a rise in ALP that is disproportionate to that of the aminotransferases. The serum bilirubin can be prominently elevated in both hepatocellular and cholestatic conditions and therefore is not necessarily helpful in

1	is disproportionate to that of the aminotransferases. The serum bilirubin can be prominently elevated in both hepatocellular and cholestatic conditions and therefore is not necessarily helpful in differentiating between the two.

1	In addition to enzyme tests, all jaundiced patients should have additional blood tests—specifically, an albumin level and a prothrombin time—to assess liver function. A low albumin level suggests a chronic process such as cirrhosis or cancer. A normal albumin level is suggestive of a more acute process such as viral hepatitis or choledocholithiasis. An elevated prothrombin time indicates either vitamin K deficiency due to prolonged jaundice and malabsorption of vitamin K or significant hepatocellular dysfunction. The failure of the prothrombin time to correct with parenteral administration of vitamin K indicates severe hepatocellular injury. The results of the bilirubin, enzyme, albumin, and prothrombin time tests will usually indicate whether a jaundiced patient has a hepatocellular or a cholestatic disease and offer some indication of the duration and severity of the disease. The causes and evaluations of hepatocellular and cholestatic diseases are quite different.

1	Hepatocellular Conditions Hepatocellular diseases that can cause jaundice include viral hepatitis, drug or environmental toxicity, alcohol, and end-stage cirrhosis from any cause (Table 58-2). Wilson’s disease occurs primarily in young adults. Autoimmune hepatitis is typically seen in young to middle-aged women but may affect men and women of any age. Alcoholic hepatitis can be differentiated from viral and toxin-related hepatitis by the pattern of the aminotransferases: patients with alcoholic hepatitis typically have an AST-to-ALT ratio of at least 2:1, and the AST level rarely exceeds 300 U/L. Patients with acute viral hepatitis and toxin-related injury severe enough to produce jaundice typically have aminotransferase levels >500 U/L, with the ALT greater than or equal to the AST. While ALT and AST values <8 times normal may be seen in either hepatocellular or cholestatic liver disease, values 25 times normal or higher are seen primarily in acute hepatocellular diseases. Patients

1	ALT and AST values <8 times normal may be seen in either hepatocellular or cholestatic liver disease, values 25 times normal or higher are seen primarily in acute hepatocellular diseases. Patients with jaundice from cirrhosis can have normal or only slightly elevated aminotransferase levels.

1	When the clinician determines that a patient has a hepatocellular disease, appropriate testing for acute viral hepatitis includes Hepatitis A, B, C, D, and E Predictable, dose-dependent (e.g., acetaminophen) Unpredictable, idiosyncratic (e.g., isoniazid) Wild mushrooms—Amanita phalloides, A. verna a hepatitis A IgM antibody assay, a hepatitis B surface antigen and core IgM antibody assay, a hepatitis C viral RNA test, and, depend ing on the circumstances, a hepatitis E IgM antibody assay. Because it can take many weeks for hepatitis C antibody to become detect able, its assay is an unreliable test if acute hepatitis C is suspected. Studies for hepatitis D and E viruses, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) may also be indicated. Ceruloplasmin is the initial screening test for Wilson’s disease. Testing for autoim measurement of specific immunoglobulins.

1	Drug-induced hepatocellular injury can be classified as either predictable or unpredictable. Predictable drug reactions are dose- dependent and affect all patients who ingest a toxic dose of the drug in question. The classic example is acetaminophen hepatotoxicity. Unpredictable or idiosyncratic drug reactions are not dose-depen dent and occur in a minority of patients. A great number of drugs can cause idiosyncratic hepatic injury. Environmental toxins are also an important cause of hepatocellular injury. Examples include industrial chemicals such as vinyl chloride, herbal preparations containing pyrrolizidine alkaloids (Jamaica bush tea) or Kava Kava, and the mushrooms Amanita phalloides and A. verna, which con tain highly hepatotoxic amatoxins.

1	Cholestatic Conditions When the pattern of the liver tests suggests a cholestatic disorder, the next step is to determine whether it is intra or extrahepatic cholestasis (Fig. 58-1). Distinguishing intrahepatic from extrahepatic cholestasis may be difficult. History, physical examination, and laboratory tests often are not helpful. The next appropriate test is an ultrasound. The ultrasound is inexpensive, does not expose the patient to ionizing radiation, and can detect dilation of the intraand extrahepatic biliary tree with a high degree of sensitivity and specificity. The absence of biliary dilation suggests intrahepatic cholestasis, while its presence indicates extrahepatic cholestasis. False-negative results occur in patients with partial obstruction of the common bile duct or in patients with cirrhosis or primary sclerosing cholangitis (PSC), in which scarring prevents the intrahepatic ducts from dilating.

1	sis, it rarely identifies the site or cause of obstruction. The distal common bile duct is a particularly difficult area to visual ize by ultrasound because of overlying bowel gas. Appropriate next tests include CT, magnetic resonance cholangiopancreatog raphy (MRCP), endoscopic retrograde cholangiopancreatography (ERCP), and endoscopic ultrasound (EUS). CT scanning and

1	MRCP are better than ultrasonography for assessing the head of the pancreas and for identifying choledocholithiasis in the distal common bile duct, particularly when the ducts are not dilated. ERCP is the “gold standard” for identifying choledocholithiasis. Beyond its diagnostic capabilities, ERCP allows therapeutic interventions, including the removal of common bile duct stones and the placement of stents. MRCP has replaced ERCP as the initial diagnostic test in cases where the need for intervention is thought to be small. EUS displays sensitivity and specificity comparable to that of MRCP in the detection of bile duct obstruction. EUS also allows biopsy of suspected malignant lesions, but is invasive and requires sedation.

1	In patients with apparent intrahepatic cholestasis, the diagnosis is often made by serologic testing in combination with percutaneous liver biopsy. The list of possible causes of intrahepatic cholestasis is long and varied (Table 58-3). A number of conditions that typically cause a hepatocellular pattern of injury can also present as a cholestatic variant. Both hepatitis B and C viruses can cause cholestatic hepatitis (fibrosing cholestatic hepatitis). This disease variant has been reported in patients who have undergone solid organ transplantation. Hepatitis A and E, alcoholic hepatitis, and EBV or CMV infections may also present as cholestatic liver disease.

1	Drugs may cause intrahepatic cholestasis that is usually reversible after discontinuation of the offending agent, although it may take many months for cholestasis to resolve. Drugs most commonly associated with cholestasis are the anabolic and contraceptive steroids. Cholestatic hepatitis has been reported with chlorpromazine, imipramine, tolbutamide, sulindac, cimetidine, and erythromycin estolate. It also occurs in patients taking trimethoprim; sulfamethoxazole; and penicillin-based antibiotics such as ampicillin, dicloxacillin, and clavulanic acid. Rarely, cholestasis may be chronic and associated with progressive fibrosis despite early discontinuation of the offending drug. Chronic cholestasis has been associated with chlorpromazine and prochlorperazine.

1	Primary biliary cirrhosis is an autoimmune disease predominantly affecting middle-aged women and characterized by progressive destruction of interlobular bile ducts. The diagnosis is made by the detection of antimitochondrial antibody, which is found in 95% of patients. Primary sclerosing cholangitis is characterized by the destruction and fibrosis of larger bile ducts. The diagnosis of PSC is made with cholangiography (either MRCP or ERCP), which demonstrates the pathognomonic segmental strictures. Approximately 75% of patients with PSC have inflammatory bowel disease.

1	The vanishing bile duct syndrome and adult bile ductopenia are rare conditions in which a decreased number of bile ducts are seen in liver biopsy specimens. The histologic picture is similar to that in primary biliary cirrhosis. This picture is seen in patients who develop chronic rejection after liver transplantation and in those who develop graft-versus-host disease after bone marrow transplantation. Vanishing bile duct syndrome also occurs in rare cases of sarcoidosis, in patients taking certain drugs (including chlorpromazine), and idiopathically.

1	There are also familial forms of intrahepatic cholestasis. The familial intrahepatic cholestatic syndromes include progressive familial intrahepatic cholestasis (PFIC) types 1–3 and benign recurrent cholestasis (BRC). PFIC1 and BRC are autosomal recessive diseases that result from mutations in the ATP8B1 gene that encodes a protein belonging to the subfamily of P-type ATPases; the exact function of this protein remains poorly defined. While PFIC1 is a progressive condition that manifests in childhood, BRC presents later and is marked by recurrent episodes of jaundice and pruritus; the episodes are self-limited but can be debilitating. PFIC2 is caused by mutations in the ABCB11 gene, which encodes the bile salt export pump, and PFIC3 is caused by mutations in the multidrugresistant P-glycoprotein 3. Cholestasis of pregnancy occurs in the second and third trimesters and resolves after delivery. Its cause is unknown, but the condition is probably inherited, and cholestasis can be

1	P-glycoprotein 3. Cholestasis of pregnancy occurs in the second and third trimesters and resolves after delivery. Its cause is unknown, but the condition is probably inherited, and cholestasis can be triggered by estrogen administration.

1	PART 2 Cardinal Manifestations and Presentation of Diseases I. Intrahepatic A. Viral hepatitis 1. 2. Hepatitis A, Epstein-Barr virus infection, cytomegalovirus infection B. Alcoholic hepatitis C. Drug toxicity 1. 2. Cholestatic hepatitis—chlorpromazine, erythromycin estolate 3. D. Primary biliary cirrhosis E. Primary sclerosing cholangitis F. Vanishing bile duct syndrome 1. Chronic rejection of liver transplants 2. 3. G. Congestive hepatopathy and ischemic hepatitis H. Inherited conditions 1. 2. I. Cholestasis of pregnancy J. Total parenteral nutrition K. Nonhepatobiliary sepsis L. Benign postoperative cholestasis M. Paraneoplastic syndrome N. Veno-occlusive disease O. Graft-versus-host disease P. Infiltrative disease 1. 2. 3. Q. Infections 1. 2. II. A. 1. 2. 3. 4. 5. Malignant involvement of the porta hepatis lymph nodes B. Benign 1. 2. 3. 4. 5. 6. 7.

1	Other causes of intrahepatic cholestasis include total parenteral nutrition (TPN); nonhepatobiliary sepsis; benign postoperative cholestasis; and a paraneoplastic syndrome associated with a number of different malignancies, including Hodgkin’s disease, medullary thyroid cancer, renal cell cancer, renal sarcoma, T cell lymphoma, prostate cancer, and several gastrointestinal malignancies. The term Stauffer’s syndrome has been used for intrahepatic cholestasis specifically associated with renal cell cancer. In patients developing cholestasis in the intensive care unit, the major considerations should be sepsis, ischemic hepatitis (“shock liver”), and TPN jaundice. Jaundice occurring after bone marrow transplantation is most likely due to veno-occlusive disease or graft-versus-host disease. In addition to hemolysis, sickle cell disease may cause intrahepatic and extrahepatic cholestasis. Jaundice is a late finding in heart failure caused by hepatic congestion and hepatocellular hypoxia.

1	In addition to hemolysis, sickle cell disease may cause intrahepatic and extrahepatic cholestasis. Jaundice is a late finding in heart failure caused by hepatic congestion and hepatocellular hypoxia. Ischemic hepatitis is a distinct entity of acute hypoperfusion characterized by an acute and dramatic elevation in the serum aminotransferases followed by a gradual peak in serum bilirubin.

1	Jaundice with associated liver dysfunction can be seen in severe cases of Plasmodium falciparum malaria. The jaundice in these cases is due to a combination of indirect hyperbilirubinemia from hemolysis and both cholestatic and hepatocellular jaundice. Weil’s disease, a severe presentation of leptospirosis, is marked by jaundice with renal failure, fever, headache, and muscle pain.

1	Causes of extrahepatic cholestasis can be split into malignant and benign (Table 58-3). Malignant causes include pancreatic, gallbladder, and ampullary cancers as well as cholangiocarcinoma. This last malignancy is most commonly associated with PSC and is exceptionally difficult to diagnose because its appearance is often identical to that of PSC. Pancreatic and gallbladder tumors as well as cholangiocarcinoma are rarely resectable and have poor prognoses. Ampullary carcinoma has the highest surgical cure rate of all the tumors that present as painless jaundice. Hilar lymphadenopathy due to metastases from other cancers may cause obstruction of the extrahepatic biliary tree.

1	Choledocholithiasis is the most common cause of extrahepatic cholestasis. The clinical presentation can range from mild right-upper-quadrant discomfort with only minimal elevations of enzyme test values to ascending cholangitis with jaundice, sepsis, and circulatory collapse. PSC may occur with clinically important strictures limited to the extrahepatic biliary tree. IgG4-associated cholangitis is marked by stricturing of the biliary tree. It is critical that the clinician differentiate this condition from PSC as it is responsive to glucocorticoid therapy. In rare instances, chronic pancreatitis causes strictures of the distal common bile duct, where it passes through the head of the pancreas. AIDS cholangiopathy is a condition that is usually due to infection of the bile duct epithelium with CMV or cryptosporidia and has a cholangiographic appearance similar to that of PSC. The affected patients usually present with greatly elevated serum alkaline phosphatase levels (mean, 800 IU/L),

1	CMV or cryptosporidia and has a cholangiographic appearance similar to that of PSC. The affected patients usually present with greatly elevated serum alkaline phosphatase levels (mean, 800 IU/L), but the bilirubin level is often near normal. These patients do not typically present with jaundice.

1	gLOBAL CONSIDERATIONS While extrahepatic biliary obstruction and drugs are common causes of new-onset jaundice in developed countries, infections remain the leading cause in developing countries. Liver involvement and jaundice are observed with numerous infections, particularly malaria, babesiosis, severe leptospirosis, infections due to Mycobacterium tuberculosis and the Mycobacterium avium complex, typhoid fever, viral hepatitis secondary to infection with hepatitis viruses A–E, EBV and CMV infections, late phases of yellow fever, dengue hemorrhagic fever, schistosomiasis, fascioliasis, clonorchiasis, opisthorchiasis, ascariasis, echinococcosis, hepatosplenic candidiasis, disseminated histoplasmosis, cryptococcosis, coccidioimycosis, ehrlichiosis, chronic Q fever, yersiniosis, brucellosis, syphilis, and lep rosy. Bacterial infections that do not necessarily involve the liver and bile ducts may also lead to jaundice, as in cholestasis of sepsis.

1	This chapter is a revised version of chapters that have appeared in prior editions of Harrison’s in which Marshall M. Kaplan was a co-author together with Daniel Pratt. Kathleen E. Corey, Lawrence S. Friedman Abdominal swelling is a manifestation of numerous diseases. Patients may complain of bloating or abdominal fullness and may note increasing abdominal girth on the basis of increased clothing or belt size. Abdominal discomfort is often reported, but pain is less frequent. When abdominal pain does accompany swelling, it is frequently the result of an intraabdominal infection, peritonitis, or pancreatitis. Patients with abdominal distention from ascites (fluid in the abdomen) may report the new onset of an inguinal or umbilical hernia. Dyspnea may result from pressure against the diaphragm and the inability to expand the lungs fully.

1	The causes of abdominal swelling can be remembered conveniently as the six Fs: flatus, fat, fluid, fetus, feces, or a “fatal growth” (often a neoplasm).

1	Flatus Abdominal swelling may be the result of increased intestinal gas. The normal small intestine contains approximately 200 mL of gas made up of nitrogen, oxygen, carbon dioxide, hydrogen, and methane. Nitrogen and oxygen are consumed (swallowed), whereas carbon dioxide, hydrogen, and methane are produced intraluminally by bacterial fermentation. Increased intestinal gas can occur in a number of conditions. Aerophagia, the swallowing of air, can result in increased amounts of oxygen and nitrogen in the small intestine and lead to abdominal swelling. Aerophagia typically results from gulping food; chewing gum; smoking; or as a response to anxiety, which can lead to repetitive belching. In some cases, increased intestinal gas is the consequence of bacterial metabolism of excess fermentable substances such as lactose and other oligosaccharides, which can lead to production of hydrogen, carbon dioxide, or methane. In many cases, the precise cause of abdominal distention cannot be

1	substances such as lactose and other oligosaccharides, which can lead to production of hydrogen, carbon dioxide, or methane. In many cases, the precise cause of abdominal distention cannot be determined. In some persons, particularly those with irritable bowel syndrome and bloating, the subjective sense of abdominal pressure is attributable to impaired intestinal transit of gas rather than increased gas volume. Abdominal distention—an objective increase in girth—is the result of a lack of coordination between diaphragmatic contraction and anterior abdominal wall relaxation, a response in some cases to an increase in intraabdominal volume loads. Occasionally, increased lumbar lordosis accounts for apparent abdominal distention.

1	Fat Weight gain with an increase in abdominal fat can result in an increase in abdominal girth and can be perceived as abdominal swelling. Abdominal fat may be caused by an imbalance between caloric intake and energy expenditure associated with a poor diet and sedentary lifestyle; it also can be a manifestation of certain diseases, such as Cushing’s syndrome. Excess abdominal fat has been associated with an increased risk of insulin resistance and cardiovascular disease. Fluid The accumulation of fluid within the abdominal cavity (ascites) often results in abdominal distention and is discussed in detail below. Fetus Pregnancy results in increased abdominal girth. Typically, an increase in abdominal size is first noted at 12–14 weeks of gestation, when the uterus moves from the pelvis into the abdomen. Abdominal distention may be seen before this point as a result of fluid retention and relaxation of the abdominal muscles.

1	Feces In the setting of severe constipation or intestinal obstruction, increased stool in the colon leads to increased abdominal girth. These conditions are often accompanied by abdominal discomfort or pain, nausea, and vomiting and can be diagnosed by imaging studies. Fatal growth An abdominal mass can result in abdominal swelling. Enlargement of the intraabdominal organs, specifically the liver (hepatomegaly) or spleen (splenomegaly), or an abdominal aortic aneurysm 286 can result in abdominal distention. Bladder distention also may result in abdominal swelling. In addition, malignancies, abscesses, or cysts can grow to sizes that lead to increased abdominal girth. APPROACH TO THE PATIENT: PART 2 Cardinal Manifestations and Presentation of Diseases

1	APPROACH TO THE PATIENT: PART 2 Cardinal Manifestations and Presentation of Diseases Determining the etiology of abdominal swelling begins with history-taking and a physical examination. Patients should be questioned regarding symptoms suggestive of malignancy, including weight loss, night sweats, and anorexia. Inability to pass stool or flatus together with nausea or vomiting suggests bowel obstruction, severe constipation, or an ileus (lack of peristalsis). Increased eructation and flatus may point toward aerophagia or increased intestinal production of gas. Patients should be questioned about risk factors for or symptoms of chronic liver disease, including excessive alcohol use and jaundice, which suggest ascites. Patients should also be asked about other symptoms of medical conditions, including heart failure and tuberculosis, which may cause ascites.

1	Physical examination should include an assessment for signs of systemic disease. The presence of lymphadenopathy, especially supraclavicular lymphadenopathy (Virchow’s node), suggests metastatic abdominal malignancy. Care should be taken during the cardiac examination to evaluate for elevation of jugular venous pressure (JVP); Kussmaul’s sign (elevation of the JVP during inspiration); a pericardial knock, which may be seen in heart failure or constrictive pericarditis; or a murmur of tricuspid regurgitation. Spider angiomas, palmar erythema, dilated superficial veins around the umbilicus (caput medusae), and gynecomastia suggest chronic liver disease.

1	The abdominal examination should begin with inspection for the presence of uneven distention or an obvious mass. Auscultation should follow. The absence of bowel sounds or the presence of high-pitched localized bowel sounds points toward an ileus or intestinal obstruction. An umbilical venous hum may suggest the presence of portal hypertension, and a harsh bruit over the liver is heard rarely in patients with hepatocellular carcinoma or alcoholic hepatitis. Abdominal swelling caused by intestinal gas can be differentiated from swelling caused by fluid or a solid mass by percussion; an abdomen filled with gas is tympanic, whereas an abdomen containing a mass or fluid is dull to percussion. The absence of abdominal dullness, however, does not exclude ascites, because a minimum of 1500 mL of ascitic fluid is required for detection on physical examination. Finally, the abdomen should be palpated to assess for tenderness, a mass, enlargement of the spleen or liver, or presence of a nodular

1	ascitic fluid is required for detection on physical examination. Finally, the abdomen should be palpated to assess for tenderness, a mass, enlargement of the spleen or liver, or presence of a nodular liver suggesting cirrhosis or tumor. Light palpation of the liver may detect pulsations suggesting retrograde vascular flow from the heart in patients with right-sided heart failure, particularly tricuspid regurgitation.

1	Abdominal x-rays can be used to detect dilated loops of bowel sug gesting intestinal obstruction or ileus. Abdominal ultrasonography can detect as little as 100 mL of ascitic fluid, hepatosplenomegaly, a nodular liver, or a mass. Ultrasonography is often inadequate to detect retroperitoneal lymphadenopathy or a pancreatic lesion because of overlying bowel gas. If malignancy or pancreatic disease is suspected,

1	CT can be performed. CT may also detect changes associated with advanced cirrhosis and portal hypertension (Fig. 59-1). Laboratory evaluation should include liver biochemical testing, serum albumin level measurement, and prothrombin time determina tion (international normalized ratio) to assess hepatic function as well as a complete blood count to evaluate for the presence of cytopenias that may result from portal hypertension or of leukocytosis, anemia, and thrombocytosis that may result from systemic infection. Serum amylase and lipase levels should be checked to evaluate the patient for acute pancreatitis. Urinary protein quantitation is indicated when nephrotic syndrome, which may cause ascites, is suspected. FIguRE 59-1 CT of a patient with a cirrhotic, nodular liver (white arrow), splenomegaly (yellow arrow), and ascites (arrowheads).

1	FIguRE 59-1 CT of a patient with a cirrhotic, nodular liver (white arrow), splenomegaly (yellow arrow), and ascites (arrowheads). In selected cases, the hepatic venous pressure gradient (pressure across the liver between the portal and hepatic veins) can be measured via cannulation of the hepatic vein to confirm that ascites is caused by cirrhosis (Chap. 365). In some cases, a liver biopsy may be necessary to confirm cirrhosis.

1	Ascites in patients with cirrhosis is the result of portal hypertension and renal salt and water retention. Similar mechanisms contribute to ascites formation in heart failure. Portal hypertension signifies elevation of the pressure within the portal vein. According to Ohm’s law, pressure is the product of resistance and flow. Increased hepatic resistance occurs by several mechanisms. First, the development of hepatic fibrosis, which defines cirrhosis, disrupts the normal architecture of the hepatic sinusoids and impedes normal blood flow through the liver. Second, activation of hepatic stellate cells, which mediate fibrogenesis, leads to smooth-muscle contraction and fibrosis. Finally, cirrhosis is associated with a decrease in endothelial nitric oxide synthetase (eNOS) production, which results in decreased nitric oxide production and increased intrahepatic vasoconstriction.

1	The development of cirrhosis is also associated with increased systemic circulating levels of nitric oxide (contrary to the decrease seen intrahepatically) as well as increased levels of vascular endothelial growth factor and tumor necrosis factor that result in splanchnic arterial vasodilation. Vasodilation of the splanchnic circulation results in pooling of blood and a decrease in the effective circulating volume, which is perceived by the kidneys as hypovolemia. Compensatory vasoconstriction via release of antidiuretic hormone ensues; the consequences are free water retention and activation of the sympathetic nervous system and the renin angiotensin aldosterone system, which lead in turn to renal sodium and water retention.

1	Ascites in the absence of cirrhosis generally results from peritoneal carcinomatosis, peritoneal infection, or pancreatic disease. Peritoneal carcinomatosis can result from primary peritoneal malignancies such as mesothelioma or sarcoma, abdominal malignancies such as gastric or colonic adenocarcinoma, or metastatic disease from breast or lung carcinoma or melanoma (Fig. 59-2). The tumor cells lining the peritoneum produce a protein-rich fluid that contributes to the development of ascites. Fluid from the extracellular space is drawn into the peritoneum, further contributing to the development of ascites. Tuberculous peritonitis causes ascites via a similar mechanism; tubercles deposited on the peritoneum exude a proteinaceous fluid. Pancreatic ascites results from leakage of pancreatic enzymes into the peritoneum. FIguRE 59-2 CT of a patient with peritoneal carcinomatosis (white arrow) and ascites (yellow arrow).

1	FIguRE 59-2 CT of a patient with peritoneal carcinomatosis (white arrow) and ascites (yellow arrow). Cirrhosis accounts for 84% of cases of ascites. Cardiac ascites, peritoneal carcinomatosis, and “mixed” ascites resulting from cirrhosis and a second disease account for 10–15% of cases. Less common causes of ascites include massive hepatic metastasis, infection (tuberculosis, Chlamydia infection), pancreatitis, and renal disease (nephrotic syndrome). Rare causes of ascites include hypothyroidism and familial Mediterranean fever. Once the presence of ascites has been confirmed, the etiology of the ascites is best determined by paracentesis, a bedside procedure in which a needle or small catheter is passed transcutaneously to extract ascitic fluid from the peritoneum. The lower quadrants are the most frequent sites for paracentesis. The left lower quadrant is preferred because of the greater depth of ascites and the thinner abdominal wall.

1	Paracentesis is a safe procedure even in patients with coagulopathy; 287 complications, including abdominal wall hematomas, hypotension, hepatorenal syndrome, and infection, are infrequent. Once ascitic fluid has been extracted, its gross appearance should be examined. Turbid fluid can result from the presence of infection or tumor cells. White, milky fluid indicates a triglyceride level >200 mg/ dL (and often >1000 mg/dL), which is the hallmark of chylous ascites. Chylous ascites results from lymphatic disruption that may occur with trauma, cirrhosis, tumor, tuberculosis, or certain congenital abnormalities. Dark brown fluid can reflect a high bilirubin concentration and indicates biliary tract perforation. Black fluid may indicate the presence of pancreatic necrosis or metastatic melanoma.

1	The ascitic fluid should be sent for measurement of albumin and total protein levels, cell and differential counts, and, if infection is suspected, Gram’s stain and culture, with inoculation into blood culture bottles at the patient’s bedside to maximize the yield. A serum albumin level should be measured simultaneously to permit calculation of the serum-ascites albumin gradient (SAAG).

1	The SAAG is useful for distinguishing ascites caused by portal hypertension from nonportal hypertensive ascites (Fig. 59-3). The SAAG reflects the pressure within the hepatic sinusoids and correlates with the hepatic venous pressure gradient. The SAAG is calculated by subtracting the ascitic albumin concentration from the serum albumin level and does not change with diuresis. A SAAG ≥1.1 g/dL reflects the presence of portal hypertension and indicates that the ascites is due to increased pressure in the hepatic sinusoids. According to Starling’s law, a high SAAG reflects the oncotic pressure that counterbalances the portal pressure. Possible causes include cirrhosis, cardiac ascites, hepatic vein thrombosis (Budd-Chiari syndrome), sinusoidal obstruction syndrome (veno-occlusive disease), or massive liver metastases. A SAAG <1.1 g/dL indicates that the ascites is not related to portal hypertension, as in tuberculous peritonitis, peritoneal carcinomatosis, or pancreatic ascites.

1	For high-SAAG (≥1.1) ascites, the ascitic protein level can provide further clues to the etiology (Fig. 59-3). An ascitic protein level of ≥2.5 g/dL indicates that the hepatic sinusoids are normal and are allowing passage of protein into the ascites, as occurs in cardiac ascites, early Budd-Chiari syndrome, or sinusoidal obstruction syndrome. An ascitic protein level <2.5 g/dL indicates that the hepatic sinusoids have been damaged and scarred and no longer allow passage of protein, as occurs with cirrhosis, late Budd-Chiari syndrome, or massive liver metastases. Pro-brain-type natriuretic peptide (BNP) is a natriuretic hormone released by the heart as a result of increased volume and ventricular wall stretch. High levels of BNP in serum occur in heart failure and may be useful in identifying heart failure as the cause of high-SAAG ascites.

1	Further tests are indicated only in specific clinical circumstances. When secondary peritonitis resulting from a perforated hollow viscus is suspected, ascitic glucose and lactate dehydrogenase (LDH) levels can < 1.1 g/dL Ascitic protein ˜ 2.5 g/dL Cirrhosis Late Budd-Chiari syndrome Massive liver metastases ˜ 1.1 g/dL Heart failure/constrictive pericarditis Early Budd-Chiari syndrome IVC obstruction Sinusoidal obstruction syndrome Biliary leak Nephrotic syndrome Pancreatitis Peritoneal carcinomatosis Tuberculosis Ascitic protein < 2.5 g/dL SAAG FIguRE 59-3 Algorithm for the diagnosis of ascites according to the serum-ascites albumin gradient (SAAG). IVC, inferior vena cava.

1	288 be measured. In contrast to “spontaneous” bacterial peritonitis, which may complicate cirrhotic ascites (see “Complications,” below), secondary peritonitis is suggested by an ascitic glucose level <50 mg/dL, an ascitic LDH level higher than the serum LDH level, and the detection of multiple pathogens on ascitic fluid culture. When pancreatic ascites is suspected, the ascitic amylase level should be measured and is typically >1000 mg/dL. Cytology can be useful in the diagnosis of peritoneal carcinomatosis. At least 50 mL of fluid should be obtained and sent for immediate processing. Tuberculous peritonitis is typically associated with ascitic fluid lymphocytosis but can be difficult to diagnose by paracentesis. A smear for acid-fast bacilli has a diagnostic sensitivity of only 0 to 3%; a culture increases the sensitivity to 35–50%. In patients without cirrhosis, an elevated ascitic adenosine deaminase level has a sensitivity of >90% when a cut-off value of 30–45 U/L is used. When

1	a culture increases the sensitivity to 35–50%. In patients without cirrhosis, an elevated ascitic adenosine deaminase level has a sensitivity of >90% when a cut-off value of 30–45 U/L is used. When the cause of ascites remains uncertain, laparotomy or laparoscopy with peritoneal biopsies for histology and culture remains the gold standard.

1	The initial treatment for cirrhotic ascites is restriction of sodium intake to 2 g/d. When sodium restriction alone is inadequate to control ascites, oral diuretics—typically the combination of spironolactone and furosemide—are used. Spironolactone is an aldosterone antagonist that inhibits sodium resorption in the distal convoluted tubule of the kidney. Use of spironolactone may be limited by hyponatremia, hyperkalemia, and painful gynecomastia. If the gynecomastia is distressing, amiloride (5–40 mg/d) may be substituted for spironolactone. Furosemide is a loop diuretic that is generally combined with spironolactone in a ratio of 40:100; maximal daily doses of spironolactone and furosemide are 400 mg and 160 mg, respectively.

1	Refractory cirrhotic ascites is defined by the persistence of ascites despite sodium restriction and maximal (or maximally tolerated) diuretic use. Pharmacologic therapy for refractory ascites includes the addition of midodrine, an α1-adrenergic antagonist, or clonidine, an α2-adrenergic antagonist, to diuretic therapy. These agents act as vasoconstrictors, counteracting splanchnic vasodilation. Midodrine alone or in combination with clonidine improves systemic hemodynamics and control of ascites over that obtained with diuretics alone. Although β-adrenergic blocking agents (beta blockers) are often prescribed to prevent variceal hemorrhage in patients with cirrhosis, the use of beta blockers in patients with refractory ascites is associated with decreased survival rates.

1	When medical therapy alone is insufficient, refractory ascites can be managed by repeated large-volume paracentesis (LVP) or a transjugular intrahepatic peritoneal shunt (TIPS)—a radiologically placed portosystemic shunt that decompresses the hepatic sinusoids. Intravenous infusion of albumin accompanying LVP decreases the risk of “post-paracentesis circulatory dysfunction” and death. Patients undergoing LVP should receive IV albumin infusions of Part 2 Cardinal Manifestations and Presentation of Diseases 6–8 g/L of ascitic fluid removed. TIPS placement is superior to LVP in reducing the reaccumulation of ascites but is associated with an increased frequency of hepatic encephalopathy, with no difference in mortality rates. Malignant ascites does not respond to sodium restriction or diuretics. Patients must undergo serial LVPs, transcutaneous drainage catheter placement, or, rarely, creation of a peritoneovenous shunt (a shunt from the abdominal cavity to the vena cava).

1	Ascites caused by tuberculous peritonitis is treated with standard antituberculosis therapy. Noncirrhotic ascites of other causes is treated by correction of the precipitating condition. Spontaneous bacterial peritonitis (SBP; Chap. 159) is a common and potentially lethal complication of cirrhotic ascites. Occasionally, SBP also complicates ascites caused by nephrotic syndrome, heart failure, acute hepatitis, and acute liver failure but is rare in malignant ascites. Patients with SBP generally note an increase in abdominal girth; however, abdominal tenderness is found in only 40% of patients, and rebound tenderness is uncommon. Patients may present with fever, nausea, vomiting, or the new onset of or exacerbation of preexisting hepatic encephalopathy.

1	SBP is defined by a polymorphonuclear neutrophil (PMN) count of ffi250/ L in the ascitic fluid. Cultures of ascitic fluid typically reveal one bacterial pathogen. The presence of multiple pathogens in the setting of an elevated ascitic PMN count suggests secondary peritonitis from a ruptured viscus or abscess (Chap. 159). The presence of multiple pathogens without an elevated PMN count suggests bowel perforation from the paracentesis needle. SBP is generally the result of enteric bacteria that have translocated across an edematous bowel wall. The most common pathogens are gram-negative rods, including Escherichia coli and Klebsiella, as well as streptococci and enterococci.

1	Treatment of SBP with an antibiotic such as IV cefotaxime is effective against gram-negative and gram-positive aerobes. A 5-day course of treatment is sufficient if the patient improves clinically. Nosocomial or health care–acquired SBP is frequently caused by multidrug-resistant bacteria, and initial antibiotic therapy should be guided by the local bacterial epidemiology. Cirrhotic patients with a history of SBP, an ascitic fluid total protein concentration <1 g/dL, or active gastrointestinal bleeding should receive prophylactic antibiotics to prevent SBP; oral daily norfloxacin is commonly used. Diuresis increases the activity of ascitic fluid protein opsonins and may decrease the risk of SBP.

1	Hepatic hydrothorax occurs when ascites, often caused by cirrhosis, migrates via fenestrae in the diaphragm into the pleural space. This condition can result in shortness of breath, hypoxia, and infection. Treatment is similar to that for cirrhotic ascites and includes sodium restriction, diuretics, and, if needed, thoracentesis or TIPS placement. Chest tube placement should be avoided. Dysuria, Bladder Pain, and the Interstitial Cystitis/Bladder Pain Syndrome John W. Warren Dysuria and bladder pain are two symptoms that commonly call atten-60e SEC TION 7 tion to the lower urinary tract. Dysuria, or pain that occurs during urination, is commonly perceived as burning or stinging in the urethra and is a symptom of several syndromes. The presence or absence of other symptoms is often helpful in distinguishing among these conditions. Some of these syndromes differ in men and women.

1	Approximately 50% of women experience dysuria at some time in their lives; ∼20% report having had dysuria within the past year. Most dysuria syndromes in women can be categorized into two broad groups: bacterial cystitis and lower genital tract infections. Bacterial cystitis is usually caused by Escherichia coli; a few other gram-negative rods and Staphylococcus saprophyticus can also be responsible. Bacterial cystitis is acute in onset and manifests not only as dysuria but also as urinary frequency, urinary urgency, suprapubic pain, and/or hematuria.

1	The lower genital tract infections include vaginitis, urethritis, and ulcerative lesions; many of these infections are caused by sexually transmitted organisms and should be considered particularly in young women who have new or multiple sexual partners or whose partner(s) do not use condoms. The onset of dysuria associated with these syndromes is more gradual than in bacterial cystitis and is thought (but not proven) to result from the flow of urine over damaged epithelium. Frequency, urgency, suprapubic pain, and hematuria are reported less frequently than in bacterial cystitis. Vaginitis, caused by Candida albicans or Trichomonas vaginalis, presents as vaginal discharge or irritation. Urethritis is a consequence of infection by Chlamydia trachomatis or Neisseria gonorrhoeae. Ulcerative genital lesions may be caused by herpes simplex virus and several other specific organisms.

1	Among women presenting with dysuria, the probability of bacterial cystitis is ∼50%. This figure rises to >90% if four criteria are fulfilled: dysuria and frequency without vaginal discharge or irritation. Present standards suggest that women meeting these four criteria, if they are otherwise healthy, are not pregnant, and have an apparently normal urinary tract, can be diagnosed with uncomplicated bacterial cystitis and treated empirically with appropriate antibiotics. Other women with dysuria should be further evaluated by urine dipstick, urine culture, and a pelvic examination. Dysuria is less common among men. The syndromes presenting as dysuria are similar to those in women but with some important distinctions.

1	In the majority of men with dysuria, frequency, urgency, and/or suprapubic, penile, and/or perineal pain, the prostate is involved, either as the source of infection or as an obstruction to urine flow. Bacterial prostatitis is usually caused by E. coli or another gram-negative rod, with one of two presentations. Acute bacterial prostatitis presents with fever and chills; prostate examination should be gentle or not performed at all, as massage may result in a wave of bacteremia. Chronic bacterial prostatitis presents as recurrent episodes of bacterial cystitis; prostate examination with massage demonstrates prostatic bacteria and leukocytes. Benign prostatic hyperplasia (BPH) can obstruct urine flow, with consequent symptoms of weak stream, hesitancy, and dribbling. If a bacterial infection develops behind the obstructing prostate, dysuria and other symptoms of cystitis will occur. Men whose symptoms are consistent with bacterial cystitis should be evaluated with urinalysis and urine

1	develops behind the obstructing prostate, dysuria and other symptoms of cystitis will occur. Men whose symptoms are consistent with bacterial cystitis should be evaluated with urinalysis and urine culture.

1	Several sexually transmitted infections can manifest as dysuria. Urethritis (usually without urinary frequency) presents as a urethral discharge and can be caused by C. trachomatis, N. gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, or T. vaginalis. Herpes simplex, chancroid, and other ulcerous lesions may present as dysuria, again without urinary frequency. For further discussion, see Chaps. 162 and 163. Other causes of dysuria may be found in patients of either sex. Some cases are acute and include lower urinary tract stones, trauma, and urethral exposure to topical chemicals. Others may be relatively chronic and attributable to lower urinary tract cancers, certain medications, Behçet’s syndrome, reactive arthritis, a poorly understood entity known as chronic urethral syndrome, and interstitial cystitis/bladder pain syndrome (see below).

1	Studies indicate that patients perceive pain as coming from the urinary bladder if it is suprapubic in location, alters with bladder filling or emptying, and/or is associated with urinary symptoms such as urgency and frequency. Bladder pain occurring acutely (i.e., over hours or a day or two) is helpful in distinguishing bacterial cystitis from urethritis, vaginitis, and other genital infections. Chronic or recurrent bladder pain may accompany lower urinary tract stones; bladder, uterine, cervical, vaginal, urethral, or prostate cancer; urethral diverticulum; cystitis induced by radiation or certain medications; tuberculous cystitis; bladder neck obstruction; neurogenic bladder; urogenital prolapse; or BPH. In the absence of these conditions, the diagnosis of interstitial cystitis/bladder pain syndrome (IC/BPS) should be considered.

1	Most clinicians with outpatient practices see undiagnosed cases of IC/BPS. This chronic condition is characterized by pain perceived to be from the urinary bladder, urinary urgency and frequency, and nocturia. The majority of cases are diagnosed in women. Symptoms wax and wane for months or years or possibly even for the rest of the patient’s life. The spectrum of symptom intensity is broad. The pain can be excruciating, urgency can be distressing, frequency can be up to 60 times per 24 h, and nocturia can cause sleep deprivation. These symptoms can disrupt daily activities, work schedules, and personal relationships; patients with IC/BPS report less life satisfaction than do those with end-stage renal disease.

1	IC/BPS is not a new disease, having first been described in the late nineteenth century in a patient with the symptoms mentioned above and a single ulcer visible on cystoscopy (now called a Hunner’s lesion after the urologist who first reported it). Over the ensuing decades, it became clear that many patients with similar symptoms had no ulcer. It is now appreciated that only up to 10% of patients with IC/BPS have a Hunner’s lesion. The definition of IC/BPS, its diagnostic features, and even its name continue to evolve. The American Urological Association has defined IC/BPS as “an unpleasant sensation (pain, pressure, discomfort) perceived to be related to the urinary bladder, associated with lower urinary tract symptoms of more than six weeks’ duration, in the absence of infection or other identifiable causes.”

1	CHAPTER 60e Dysuria, Bladder Pain, and the Interstitial Cystitis/Bladder Pain Syndrome 60e-2 Many patients with IC/BPS also have other syndromes, such as fibromyalgia, chronic fatigue syndrome, irritable bowel syndrome, and migraine. These syndromes collectively are known as functional somatic syndromes (FSSs): chronic conditions in which pain and fatigue are prominent features but laboratory tests and histologic findings are normal. Like IC/BPS, the FSSs often are associated with depression and anxiety. The majority of FSSs affect more women than men, and more than one FSS can affect a single patient. Because of its similar features and comorbidity, IC/BPS sometimes is considered an FSS.

1	Contemporary population studies of IC/BPS in the United States indicate a prevalence of 3–6% among women and 2–4% among men. For decades, it was thought that IC/BPS occurred mostly in women. These prevalence findings, however, have generated research aimed at determining the proportion of men who have symptoms usually diagnosed as chronic prostatitis (now known as chronic prostatitis/chronic pelvic pain syndrome) but who actually have IC/BPS. Among women, the average age at onset of IC/BPS symptoms is the early forties, but the range is from childhood through the early sixties. Risk factors (antecedent features that distinguish cases from controls) primarily have been FSSs. Indeed, the odds of IC/BPS increase with the number of such syndromes present. Surgery was long thought to be a risk factor for IC/BPS, but analyses adjusting for FSSs refuted that association. About one-third of patients appear to have bacterial cystitis at the onset of IC/BPS.

1	The natural history of IC/BPS is not known. Although studies from urology and urogynecology practices have been interpreted as showing that IC/BPS lasts for the lifetime of the patient, population studies suggest that some individuals with IC/BPS do not consult specialists and may not seek medical care at all, and most prevalence studies do not show an upward trend with age—a pattern that would be expected with incident cases throughout adulthood followed by lifetime persistence of a nonfatal disease. It may be reasonable to conclude that patients in a urology practice represent those with the most severe and recalcitrant IC/BPS.

1	For the ≤10% of IC/BPS patients who have a Hunner’s lesion, the term interstitial cystitis may indeed describe the histopathologic picture. Most of these patients have substantive inflammation, mast cells, and granulation tissue. However, in the 90% of patients without such lesions, the bladder mucosa and interstitium are relatively normal, with scant inflammation.

1	Numerous hypotheses about the pathogenesis of IC/BPS have been put forward. It is not surprising that most early theories focused on the bladder. For instance, IC/BPS has been investigated as a chronic bladder infection. Sophisticated technologies have not identified a causative organism in urine or in bladder tissue; however, the patients studied by these methods had IC/BPS of long duration, and the results do not preclude the possibility that infection may trigger the syndrome or may be a feature of early IC/BPS. Other inflammatory factors, including a role for mast cells, have been postulated, but (as noted above) the 90% of patients without a Hunner’s ulcer have little bladder inflammation and do not have a prominence of mast cells in bladder tissue. Autoimmunity has been considered, but autoantibodies are low in titer, nonspecific, and thought to be a result rather than a cause of IC/BPS. Increased permeability of the bladder mucosa due to defective epithelium or

1	considered, but autoantibodies are low in titer, nonspecific, and thought to be a result rather than a cause of IC/BPS. Increased permeability of the bladder mucosa due to defective epithelium or glycosaminoglycan (the bladder’s mucous coating) has been studied frequently, but the findings have been inconclusive.

1	Investigations of causes outside the bladder have been prompted by the presence of comorbid FSSs. Many patients with FSSs have abnormal pain sensitivity as evidenced by (1) low pain thresholds in body areas unrelated to the diagnosed syndrome, (2) dysfunctional descending neurologic control of tactile signals, and (3) enhanced brain responses to touch in functional neuroimaging studies. Moreover, in patients with IC/BPS, body surfaces remote from the bladder are more sensitive to pain than is the case in individuals without IC/BPS. All PART 2 Cardinal Manifestations and Presentation of Diseases these findings are consistent with upregulation of sensory processing in the brain. Indeed, a prevailing theory is that these concomitantly occurring syndromes have in common an abnormality of brain processing of sensory input. However, antecedence is a critical criterion for causality, and no study has demonstrated that abnormal pain sensitivity precedes either IC/BPS or the FSSs.

1	In some patients, IC/BPS has a gradual onset, and/or the cardinal symptoms of pain, urgency, frequency, and nocturia appear sequentially in no consistent order. Other patients can identify the exact date of onset of IC/BPS symptoms. More than half of the latter patients describe dysuria beginning on that date. As stated, only a minority of IC/BPS patients who obtain medical care soon after symptom onset have uropathogenic bacteria or leukocytes in the urine. These patients—and many others with new-onset IC/BPS—are treated with antibiotics for presumptive bacterial cystitis or, if male, chronic bacterial prostatitis. Persistent or recurring symptoms without bacteriuria eventually prompt a differential diagnosis, and IC/BPS is considered. Traditionally, the diagnosis of IC/BPS has been delayed for years, but recent interest in the disease has shortened this interval.

1	The pain of IC/BPS includes suprapubic prominence and changes with the voiding cycle. Two-thirds of women with IC/BPS report two or more sites of pain. The most common site (involved in 80% of women) and generally the one with the most severe pain is the supra-pubic area. About 35% of female patients have pain in the urethra, 25% in other parts of the vulva, and 30% in nonurogenital areas, mostly the low back and also the anterior or posterior thighs or the buttocks. The pain of IC/BPS is most commonly described as aching, pressing, throbbing, tender, and/or piercing. What may distinguish IC/BPS from other pelvic pain is that, in 95% of patients, bladder filling exacerbates the pain and/or bladder emptying relieves it. Almost as many patients report a puzzling pattern in which certain dietary substances worsen the pain of IC/BPS. Smaller proportions—but still the majority—of patients report that their IC/BPS pain is worsened by menstruation, stress, tight clothing, exercise, and

1	dietary substances worsen the pain of IC/BPS. Smaller proportions—but still the majority—of patients report that their IC/BPS pain is worsened by menstruation, stress, tight clothing, exercise, and riding in a car as well as during or after vaginal intercourse.

1	The urethral and vulvar pains of IC/BPS merit special mention. In addition to the descriptive adjectives for IC/BPS mentioned above, these pains commonly are described as burning, stinging, and sharp and as being worsened by touch, tampons, and vaginal intercourse. Patients report that urethral pain increases during urination and generally lessens afterward. These characteristics have commonly resulted in diagnosis of the urethral pain of IC/BPS as chronic urethral syndrome and the vulvar pain as vulvodynia.

1	In many patients with IC/BPS, there is a link between pain and urinary urgency; that is, two-thirds of patients describe the urge to urinate as a desire to relieve their bladder pain. Only 20% report that the urge stems from a desire to prevent incontinence; indeed, very few patients with IC/BPS are incontinent. As mentioned above, urinary frequency can be severe, with ∼85% of patients voiding more than 10 times per 24 h and some as often as 60 times. Voiding continues through the night, and nocturia is common, frequent, and often associated with sleep deprivation.

1	Beyond these common symptoms of IC/BPS, additional urinary and other symptoms may be present. Among the urinary symptoms are difficulty in starting urine flow, perceptions of difficulty in emptying the bladder, and bladder spasms. Other symptoms include the manifestations of comorbid FSSs as well as symptoms that do not constitute recognized syndromes, such as numbness, muscle spasms, dizziness, ringing in the ears, and blurred vision. The pain, urgency, and frequency of IC/BPS can be debilitating. Proximity to a bathroom is a continual focus, and patients report difficulties in the workplace, leisure activities, travel, and simply leaving home. Familial and sexual relationships can be strained.

1	Traditionally, IC/BPS has been considered a rare condition that is diagnosed by urologists at cystoscopy. However, this disorder is much more common than once was thought; it is now being considered earlier in its course and is being diagnosed and managed more often by primary care clinicians. Results of physical examination, urinalysis, and urologic procedures are insensitive and/or nonspecific. Thus, diagnosis is based on the presence of appropriate symptoms and the exclusion of diseases with a similar presentation.

1	Three categories of disorders can be considered in the differential diagnosis of IC/BPS. The first comprises diseases that manifest as bladder pain (see above) or urinary symptoms. Among the latter diseases is overactive bladder, a chronic condition of women and men that presents as urgency and frequency and that can be distinguished from IC/BPS by the patient’s history: pain is not a feature of overactive bladder, and its urgency arises from the need to avoid incontinence. Endometriosis is a special case: it can be asymptomatic or can cause pelvic pain, dysmenorrhea, and dyspareunia—i.e., types of pain that mimic IC/BPS. Endometrial implants on the bladder (although uncommon) can cause urinary symptoms, and the resulting syndrome can mimic IC/BPS. Even if endometriosis is identified, it is difficult in the absence of bladder implants to determine whether it is causative of or incidental to the symptoms of IC/BPS in a specific woman.

1	The second category of disorders encompasses the FSSs that can accompany IC/BPS. IC/BPS can be misdiagnosed as gynecologic chronic pelvic pain, irritable bowel syndrome, or fibromyalgia. The correct diagnosis may be entertained only when either changes of pain with altered bladder volume or urinary symptoms become more prominent.

1	The third category involves syndromes that IC/BPS mimics by way of its referred pain, such as vulvodynia and chronic urethral syndrome. Therefore, IC/BPS should be considered in the differential diagnosis of persistent or recurrent “urinary tract infection” (UTI) with sterile urine cultures; overactive bladder with pain; chronic pelvic pain, endometriosis, vulvodynia, or FSSs with urinary symptoms; and “chronic prostatitis.” As mentioned above, important clues to the diagnosis of IC/BPS are pain that changes with bladder volume or with certain foods or drinks. Common among these are chilies, chocolate, citrus fruits, tomatoes, alcohol, caffeinated drinks, and carbonated beverages; full lists of common trigger foods are available at the websites cited in the treatment section below.

1	Cystoscopy under anesthesia formerly was thought to be necessary for the diagnosis of IC/BPS because of its capacity to reveal a Hunner’s lesion or—in the 90% of patients without an ulcer—petechial hemorrhages after bladder distention. However, because Hunner’s lesions are uncommon in IC/BPS and petechiae are nonspecific, cystoscopy is no longer necessary for diagnosis. Accordingly, the indications for urologic referral have evolved toward the need to rule out other diseases or to administer more advanced treatment.

1	A typical patient presents to the primary clinician after days, weeks, or months of pain, urgency, frequency, and/or nocturia. The presence of urinary nitrites, leukocytes, or uropathogenic bacteria should prompt treatment for UTI in women and chronic bacterial prostatitis in men. Persistence or recurrence of symptoms in the absence of bacteriuria should prompt a pelvic examination for women, an assay for serum prostate-specific antigen for men, and urine cytology and inclusion of IC/BPS in the differential diagnosis for both sexes.

1	In the diagnosis of IC/BPS, inquiries about pain, pressure, and discomfort are useful; IC/BPS should be considered if any of these sensations are noted in one or more anterior or posterior sites between the umbilicus and the upper thighs. Nondirective questions about the effect of bladder volume changes include “As your next urination approaches, does this pain get better, get worse, or stay the same?” and “After you urinate, does this pain get better, get worse, or stay the same?” Establishing that the pain is exacerbated by the consumption of certain foods and drinks not only supports the diagnosis of IC/BPS but also serves as the basis for one of the first steps in managing this syndrome. A nondirective way to ask about urgency is to describe it to the patient as a compelling urge to urinate that is difficult to postpone; follow-up questions can determine whether this urge is intended to relieve pain or prevent incontinence. To assess severity and provide quantitative baseline

1	to urinate that is difficult to postpone; follow-up questions can determine whether this urge is intended to relieve pain or prevent incontinence. To assess severity and provide quantitative baseline measures, pain and urgency should be estimated by the patient on a scale of 0–10, with 0 being none and 10 the worst 60e-3 imaginable. Frequency per 24-h period should be determined and nocturia assessed as the number of times per night the patient is awakened by the need to urinate.

1	About half of patients with IC/BPS have intermittent or persistent microscopic hematuria; this manifestation and the need to exclude bladder stones or cancer require urologic or urogynecologic referral. Initiation of therapy for IC/BPS does not hamper subsequent urologic evaluation. The goal of therapy is to relieve the symptoms of IC/BPS; the challenge lies in the fact that no treatment is uniformly successful. However, most patients eventually obtain relief, generally with a multifaceted approach. The American Urological Association’s guidelines for management of IC/BPS are an excellent resource. The correct strategy is to begin with conservative therapies and proceed to riskier measures only if necessary and under the supervision of a urologist or urogynecologist. Conservative tactics include education, stress reduction, dietary changes, medications, pelvic-floor physical therapy, and treatment of associated FSSs.

1	Months or even years may have passed since the onset of symptoms, and the patient’s life may have been disrupted continually, with repeated medical visits provoking frustration and dismay in both patient and physician. In this circumstance, simply giving a name to the syndrome is beneficial. The physician should discuss the disease, the diagnostic and therapeutic strategies, and the prognosis with the patient and with the spouse and/or other pertinent family members, who may need to be made aware that although IC/BPS has no visible manifestations, the patient is undergoing substantial pain and suffering. This information is particularly important for sexual partners, as exacerbation of pain during and after intercourse is a common feature of IC/BPS. Because stress can worsen IC/ BPS symptoms, stress reduction and active measures such as yoga or meditation exercises may be suggested. The Interstitial Cystitis Association (http://www.ichelp.com) and the Interstitial Cystitis Network

1	stress reduction and active measures such as yoga or meditation exercises may be suggested. The Interstitial Cystitis Association (http://www.ichelp.com) and the Interstitial Cystitis Network (http://www.ic-network.com) can be useful in this educational process.

1	In constructing a benign diet, some of the many patients who identify particular foods and drinks that exacerbate their symptoms find it useful to exclude all possible offenders and add items back into the diet one at a time to confirm which ones worsen their symptoms. Patients also should experiment with fluid volumes; some find relief with less fluid, others with more. The pelvic floor is often tender in IC/BPS patients. Two randomized controlled trials showed that weekly physical therapy directed at relaxation of the pelvic muscles yielded significantly more relief than a similar schedule of general body massage. This intervention can be initiated under the direction of a knowledgeable physical therapist who recognizes that the objective is to relax the pelvic floor, not to strengthen it.

1	Among oral medications, nonsteroidal anti-inflammatory drugs are commonly used but are controversial and often unsuccessful. Two randomized controlled trials showed that amitriptyline can diminish IC/BPS symptoms if an adequate dose (≥50 mg per night) can be given. This drug is used not for its antidepressant activity but because of its proven effects on neuropathic pain; however, it is not approved by the U.S. Food and Drug Administration for treatment of IC/BPS. An initial dose of 10 mg at bedtime is increased weekly up to 75 mg (or less if a lower dose adequately relieves symptoms). Side effects can be expected and include dry mouth, weight gain, sedation, and constipation. If this regimen does not control symptoms adequately, pentosan polysulfate, a semisynthetic polysaccharide, can be added at a dose of 100 mg three times a day. Its theoretical effect is to replenish a possibly defective glycosaminoglycan layer over the bladder mucosa; randomized controlled trials suggest only

1	CHAPTER 60e Dysuria, Bladder Pain, and the Interstitial Cystitis/Bladder Pain Syndrome 60e-4 a modest benefit over placebo. Adverse reactions are uncommon and include gastrointestinal symptoms, headache, and alopecia. Pentosan polysulfate has weak anticoagulant effects and perhaps should be avoided by patients with coagulation abnormalities. Anecdotal reports suggest that successful therapy for one FSS is accompanied by diminished symptoms of other FSSs. As has been noted here, IC/BPS often is associated with one or several FSSs. Thus, it seems reasonable to hope that, to the extent that accompanying FSSs are treated successfully, the symptoms of IC/BPS will be relieved as well. If several months of these therapies in combination do not relieve symptoms adequately, the patient should be referred to a urologist or urogynecologist who has access to additional modalities. Cystoscopy under anesthesia allows distention of the bladder with water, a procedure that provides ∼40% of patients

1	referred to a urologist or urogynecologist who has access to additional modalities. Cystoscopy under anesthesia allows distention of the bladder with water, a procedure that provides ∼40% of patients with several months of relief and can be repeated. For those few patients with a Hunner’s lesion, fulguration may offer relief. Bladder instillation of solutions containing lidocaine or dimethyl sulfoxide can be administered. Physicians experienced in the care of IC/BPS patients have used anticonvulsants, narcotics, and cyclosporine as components of therapy. Pain specialists can be of assistance. Sacral neuromodulation with a temporary percutaneous electrode can be tested and, if effective, can then be performed with an implanted device. In a very small number of patients with recalcitrant symptoms, surgeries, including cystoplasty, partial or total cystectomy, and urinary diversion, may provide relief.

1	PART 2 Cardinal Manifestations and Presentation of Diseases

1	Azotemia and urinary Abnormalities Julie Lin, Bradley M. Denker Normal kidney functions occur through numerous cellular pro-cesses to maintain body homeostasis. Disturbances in any of these 61 functions can lead to abnormalities that may be detrimental to survival. Clinical manifestations of these disorders depend on the pathophysiology of renal injury and often are identified as a complex of symptoms, abnormal physical findings, and laboratory changes that constitute specific syndromes. These renal syndromes (Table 61-1) may arise from systemic illness or as primary renal disease. Nephrologic syndromes usually consist of several elements that reflect the underlying pathologic processes, typically including one or more of the following: (1) reduction in glomerular filtration 289 rate (GFR) (azotemia), (2) abnormalities of urine sediment (red blood cells [RBCs], white blood cells [WBCs], casts, and crystals), abnormal excretion of serum proteins (proteinuria), (4) disturbances in urine

1	(azotemia), (2) abnormalities of urine sediment (red blood cells [RBCs], white blood cells [WBCs], casts, and crystals), abnormal excretion of serum proteins (proteinuria), (4) disturbances in urine volume (oliguria, anuria, polyuria), (5) presence of hypertension and/or expanded total body fluid volume (edema), (6) electrolyte abnormalities, and (7) in some syndromes, fever/pain. The specific combination of these findings should permit identification of one of the major nephrologic syndromes (Table 61-1) and allow differential diagnoses to be narrowed so that the appropriate diagnostic and therapeutic course can be determined. All these syndromes and their associated diseases are discussed in more detail in subsequent chapters. This chapter focuses on several aspects of renal abnormalities that are critically important for distinguishing among those processes: (1) reduction in GFR leading to azotemia, alterations of the urinary sediment and/or protein excretion, and abnormalities of

1	that are critically important for distinguishing among those processes: (1) reduction in GFR leading to azotemia, alterations of the urinary sediment and/or protein excretion, and abnormalities of urinary volume.

1	Monitoring the GFR is important in both hospital and outpatient settings, and several different methodologies are available. GFR is the primary metric for kidney “function,” and its direct measurement involves administration of a radioactive isotope (such as inulin or iothalamate) that is filtered at the glomerulus into the urinary space but is neither reabsorbed nor secreted throughout the tubule. GFR—i.e., the clearance of inulin or iothalamate in milliliters per minute—is calculated from the rate of appearance of the isotope in the urine over several hours. In most clinical circumstances, direct GFR measurement is not feasible, and the plasma creatinine level is used as a surrogate to estimate GFR. Plasma creatinine (PCr) is the most widely used marker for GFR, which is related directly to urine creatinine (UCr) excretion and inversely to PCr. On the basis of this relationship (with some important caveats, as discussed below), GFR will fall in roughly inverse proportion to the rise

1	to urine creatinine (UCr) excretion and inversely to PCr. On the basis of this relationship (with some important caveats, as discussed below), GFR will fall in roughly inverse proportion to the rise in PCr. Failure to account for GFR reductions in drug dosing can lead to significant morbidity and death from drug toxicities (e.g., digoxin, aminoglycosides). In the outpatient setting, PCr serves as an estimate for GFR (although much less accurate; see below). In patients with chronic progressive renal disease, there is an approximately linear relationship between 1/PCr (y axis) and time (x axis). The slope of that line will remain constant for an individual;

1	PART 2 Cardinal Manifestations and Presentation of Diseases when values deviate, an investigation for a superimposed acute process (e.g., volume depletion, drug reaction) should be initiated. Signs and symptoms of uremia develop at significantly different levels of PCr, depending on the patient (size, age, and sex), underlying renal disease, existence of concurrent diseases, and true GFR. Generally, patients do not develop symptomatic uremia until renal insufficiency is severe (GFR <15 mL/min).

1	A significantly reduced GFR (either acute or chronic) is usually reflected in a rise in PCr, leading to retention of nitrogenous waste products (defined as azotemia) such as urea. Azotemia may result from reduced renal perfusion, intrinsic renal disease, or postrenal processes (ureteral obstruction; see below and Fig. 61-1). Precise determination of GFR is problematic, as both commonly measured indices (urea and creatinine) have characteristics that affect their accuracy as markers of clearance. Urea clearance may underestimate GFR significantly because of urea reabsorption by the tubule. In contrast, creatinine is derived from muscle metabolism of creatine, and its generation varies little from day to day.

1	Creatinine clearance (CrCl), an approximation of GFR, is measured from plasma and urinary creatinine excretion rates for a defined period (usually 24 h) and is expressed in milliliters per minute: CrCl = (Uvol × UCr)/(PCr × Tmin). Creatinine is useful for estimating GFR because it is a small, freely filtered solute that is not reabsorbed by the tubules. PCr levels can increase acutely from

1	AZOTEMIA Urinalysis and Renal ultrasound HydronephrosisRenal size parenchyma Urinalysis Urologic evaluation Relieve obstruction Small kidneys, thin cortex, bland sediment, isosthenuria <3.5 g protein/24 h Normal size kidneys Intact parenchyma Bacteria Pyelonephritis Chronic Renal Failure Symptomatic treatment delay progression If end-stage, prepare for dialysis Normal urinalysis with oliguria Abnormal urinalysis WBC, casts eosinophils Interstitial nephritis Red blood cells Renal artery or vein occlusion Urine electrolytes Muddy brown casts, amorphous sediment + protein RBC casts Proteinuria Angiogram FeNa <1% U osmolality > 500 mosmol FeNa >1% U osmolality < 350 mosmol Renal biopsy Prerenal Azotemia Volume contraction, cardiac failure, vasodilatation, drugs, sepsis, renal vasoconstriction, impaired autoregulation Acute Tubular Necrosis Glomerulonephritis or vasculitis Immune complex, anti-GBM disease Acute Renal Failure

1	FIguRE 61-1 Approach to the patient with azotemia. FeNa, fractional excretion of sodium; GBM, glomerular basement membrane; RBC, red blood cell; WBC, white blood cell. dietary ingestion of cooked meat, however, and creatinine can be secreted into the proximal tubule through an organic cation pathway (especially in advanced progressive chronic kidney disease), leading to overestimation of GFR. When a timed collection for CrCl is not available, decisions about drug dosing must be based on PCr alone. Two formulas are used widely to estimate kidney function from PCr: (1) Cockcroft-Gault and (2) four-variable MDRD (Modification of Diet in Renal Disease). Cockcroft-Gault: CrCl (mL/min) = (140 − age (years) × weight (kg) ×[0.85 if female])/(72 × PCr (mg/dL). MDRD: eGFR (mL/min per 1.73 m2) = 186.3 × PCr (e−1.154) × age (e−0.203) ×(0.742 if female)×(1.21 if black).

1	Numerous websites are available to assist with these calculations (www.kidney.org/professionals/kdoqi/gfr_calculator.cfm). A newer CKDEPI eGFR, which was developed by pooling several cohorts with and without kidney disease who had data on directly measured GFR, appears to be more accurate: CKD-EPI: eGFR = 141 × min (PCr/k, 1)a × max (PCr/k, 1)−1.209 × 0.993Age × 1.018 [if female] × 1.159 [if black], where PCr is plasma creatinine, k is 0.7 for females and 0.9 for males, a is −0.329 for females and −0.411 for males, min indicates the minimum of PCr/k or 1, and max indicates the maximum of PCr/k or 1 (http:// www.qxmd.com/renal/Calculate-CKD-EPI-GFR.php).

1	There are limitations to all creatinine-based estimates of GFR. Each equation, along with 24-h urine collection for measurement of creatinine clearance, is based on the assumption that the patient is in steady state, without daily increases or decreases in PCr as a result of rapidly changing GFR. The MDRD equation is better correlated with true GFR when the GFR is <60 mL/min per 1.73 m2. The gradual loss of muscle from chronic illness, chronic use of glucocorticoids, or malnutrition can mask significant changes in GFR with small or imperceptible changes in PCr. Cystatin C, a member of the cystatin superfamily of cysteine protease inhibitors, is produced at a relatively constant rate from all nucleated cells. Serum cystatin C has been proposed to be a more sensitive marker of early GFR decline than is PCr; however, like serum creatinine, cystatin C is influenced by the patient’s age, race, and sex and also is associated with diabetes, smoking, and markers of inflammation.

1	APPROACH TO THE PATIENT:

1	Once GFR reduction has been established, the physician must decide if it represents acute or chronic renal injury. The clinical situation, history, and laboratory data often make this an easy distinction. However, the laboratory abnormalities characteristic of chronic renal failure, including anemia, hypocalcemia, and hyperphosphatemia, often are present as well in patients presenting with acute renal failure. Radiographic evidence of renal osteodystrophy (Chap. 335) can be seen only in chronic renal failure but is a very late finding, and these patients are usually undergoing dialysis. The urinalysis and renal ultrasound can facilitate distinguishing acute from chronic renal failure. An approach to the evaluation of azotemic patients is shown in Fig. 61-1. Patients with advanced chronic renal insufficiency often have some proteinuria, nonconcentrated urine (isosthenuria; isosmotic with plasma), and small kidneys on ultrasound, characterized by increased echogenicity and cortical

1	renal insufficiency often have some proteinuria, nonconcentrated urine (isosthenuria; isosmotic with plasma), and small kidneys on ultrasound, characterized by increased echogenicity and cortical thinning. Treatment should be directed toward slowing the progression of renal disease and providing symptomatic relief for edema, acidosis, anemia, and hyperphosphatemia, as discussed in Chap. 335. Acute renal failure (Chap. 334) can result from processes that affect renal blood flow (prerenal azotemia), intrinsic renal diseases (affecting small vessels, glomeruli, or tubules), or postrenal processes (obstruction of urine flow in ureters, bladder, or urethra) (Chap. 343).

1	Decreased renal perfusion accounts for 40–80% of cases of acute renal failure and, if appropriately treated, is readily reversible. The etiologies of prerenal azotemia include any cause of decreased circulating blood volume (gastrointestinal hemorrhage, burns, diarrhea, diuretics), volume sequestration (pancreatitis, peritonitis, rhabdomyolysis), or decreased effective arterial volume (cardiogenic shock, sepsis). Renal perfusion also can be affected by reductions in cardiac output from peripheral vasodilation (sepsis, drugs) or profound renal vasoconstriction (severe heart failure, hepatorenal syndrome, agents such as nonsteroidal anti-inflammatory drugs [NSAIDs]). True or “effective” arterial hypovolemia leads to a fall in mean arterial pressure, which in turn triggers a series of neural and humoral responses, including activation of the sympathetic nervous and renin-angiotensin-aldosterone systems and antidiuretic hormone (ADH) release. GFR is maintained by prostaglandin-mediated

1	and humoral responses, including activation of the sympathetic nervous and renin-angiotensin-aldosterone systems and antidiuretic hormone (ADH) release. GFR is maintained by prostaglandin-mediated relaxation of afferent arterioles and angiotensin II–mediated constriction of efferent arterioles. Once the mean arterial pressure falls below 80 mmHg, GFR declines steeply.

1	Blockade of prostaglandin production by NSAIDs can result in severe vasoconstriction and acute renal failure. Blocking angiotensin action with angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs) decreases efferent arteriolar tone and in turn decreases glomerular capillary perfusion pressure. Patients taking NSAIDs and/or ACE inhibitors/ARBs are most susceptible to hemodynamically mediated acute renal failure when blood volume is reduced for any reason. Patients with bilateral renal artery stenosis (or stenosis in a solitary kidney) are dependent on efferent arteriolar vasoconstriction for maintenance of glomerular filtration pressure and are particularly susceptible to a precipitous decline in GFR when given ACE inhibitors or ARBs.

1	Prolonged renal hypoperfusion may lead to acute tubular necrosis (ATN), an intrinsic renal disease that is discussed below. The urinalysis and urinary electrolyte measurements can be useful in distinguishing prerenal azotemia from ATN (Table 61-2). The urine Na and osmolality of patients with prerenal azotemia can be predicted from the stimulatory actions of norepinephrine, angiotensin II, ADH, and low tubule fluid flow rate. In prerenal conditions, the tubules are intact, leading to a concentrated urine (>500 mosmol), avid Na retention (urine Na concentration, <20 mmol/L; fractional excretion of Na, <1%), and UCr/PCr >40 (Table 61-2). The prerenal urine sediment is usually normal or has hyaline and granular casts, whereas the sediment of ATN usually is filled with cellular debris, tubular epithelial casts, and dark (muddy brown) granular casts.

1	Urinary tract obstruction accounts for <5% of cases of acute renal failure but is usually reversible and must be ruled out early in the evaluation (Fig. 61-1). Since a single kidney is capable of adequate BUN/PCr ratio Urine sodium UNa, meq/L Urine osmolality, mosmol/L H2O Fractional excretion of sodiuma Urine/plasma creatinine UCr/PCr Urinalysis (casts) FENa = . PNa × >20:1 granular 10–15:1 >40 <350 >2% <20 Muddy brown Abbreviations: BUN, blood urea nitrogen; PCr, plasma creatinine concentration; PNa, plasma sodium concentration; UCr, urine creatinine concentration; UNa, urine sodium concentration.

1	Abbreviations: BUN, blood urea nitrogen; PCr, plasma creatinine concentration; PNa, plasma sodium concentration; UCr, urine creatinine concentration; UNa, urine sodium concentration. clearance, obstructive acute renal failure requires obstruction at the urethra or bladder outlet, bilateral ureteral obstruction, or unilateral obstruction in a patient with a single functioning kidney. Obstruction is usually diagnosed by the presence of ureteral and renal pelvic dilation on renal ultrasound. However, early in the course of obstruction or if the ureters are unable to dilate (e.g., encasement by pelvic or periureteral tumors), the ultrasound examination may be negative. The specific urologic conditions that cause obstruction are discussed in Chap. 343.

1	When prerenal and postrenal azotemia have been excluded as etiologies of renal failure, an intrinsic parenchymal renal disease is present. Intrinsic renal disease can arise from processes involving large renal vessels, intrarenal microvasculature and glomeruli, or the tubulointerstitium. Ischemic and toxic ATN account for ~90% of cases of acute intrinsic renal failure. As outlined in Fig. 61-1, the clinical setting and urinalysis are helpful in separating the possible etiologies. Prerenal azotemia and ATN are part of a spectrum of renal hypoperfusion; evidence of structural tubule injury is present in ATN, whereas prompt reversibility occurs with prerenal azotemia upon restoration of adequate renal perfusion. Thus, ATN often can be distinguished from prerenal azotemia by urinalysis and urine electrolyte composition (Table 61-2 and Fig. 61-1). Ischemic ATN is observed most frequently in patients who have undergone major surgery, trauma, severe hypovolemia, overwhelming sepsis, or

1	urine electrolyte composition (Table 61-2 and Fig. 61-1). Ischemic ATN is observed most frequently in patients who have undergone major surgery, trauma, severe hypovolemia, overwhelming sepsis, or extensive burns. Nephrotoxic ATN complicates the administration of many common medications, usually by inducing a combination of intrarenal vasoconstriction, direct tubule toxicity, and/ or tubule obstruction. The kidney is vulnerable to toxic injury by virtue of its rich blood supply (25% of cardiac output) and its ability to concentrate and metabolize toxins. A diligent search for hypo-tension and nephrotoxins usually uncovers the specific etiology of ATN. Discontinuation of nephrotoxins and stabilization of blood pressure often suffice without the need for dialysis while the tubules recover. An extensive list of potential drugs and toxins implicated in ATN is found in Chap. 334.

1	Processes involving the tubules and interstitium can lead to acute kidney injury (AKI), a subtype of acute renal failure. These processes include drug-induced interstitial nephritis (especially by antibiotics, NSAIDs, and diuretics), severe infections (both bacterial and viral), systemic diseases (e.g., systemic lupus erythematosus), and infiltrative disorders (e.g., sarcoidosis, lymphoma, or leukemia). A list of drugs associated with allergic interstitial nephritis is found in Chap. 340. Urinalysis usually shows mild to moderate proteinuria, hematuria, and pyuria (~75% of cases) and occasionally WBC casts. The finding of RBC casts in interstitial nephritis has been reported but should prompt a search for glomerular diseases (Fig. 61-1). Occasionally, renal biopsy will be needed to distinguish among these possibilities. The finding of eosinophils in the urine is suggestive of allergic interstitial nephritis or atheroembolic renal disease and is optimally observed with Hansel staining.

1	among these possibilities. The finding of eosinophils in the urine is suggestive of allergic interstitial nephritis or atheroembolic renal disease and is optimally observed with Hansel staining. The absence of eosinophiluria, however, does not exclude these etiologies.

1	Occlusion of large renal vessels, including arteries and veins, is an uncommon cause of acute renal failure. A significant reduction in GFR by this mechanism suggests bilateral processes or, in a patient with a single functioning kidney, a unilateral process. Renal arteries can be occluded with atheroemboli, thromboemboli, in situ thrombosis, aortic dissection, or vasculitis. Atheroembolic renal failure can occur spontaneously but most often is associated with recent aortic instrumentation. The emboli are cholesterol-rich and lodge in medium and small renal arteries, with a consequent eosinophil-rich inflammatory reaction. Patients with atheroembolic acute renal failure often have a normal urinalysis, but the urine may contain eosinophils and casts. The diagnosis can be confirmed by renal biopsy, but this procedure is often unnecessary when other stigmata of atheroemboli are present (livedo reticularis, distal peripheral infarcts, eosinophilia). Renal artery thrombosis may lead

1	PART 2 Cardinal Manifestations and Presentation of Diseases HEMATURIA Proteinuria (>500 mg/24 h), Dysmorphic RBCs or RBC casts Pyuria, WBC casts Urine culture Urine eosinophils Hemoglobin electrophoresis Urine cytology UA of family members 24 h urinary calcium/uric acid IVP +/Renal ultrasound As indicated: retrograde pyelography or arteriogram, or cyst aspiration Cystoscopy Urogenital biopsy and evaluation Renal CT scan Renal biopsy of mass/lesion Follow periodic urinalysis Renal biopsy FIguRE 61-2 Approach to the patient with hematuria. ANCA, antineutrophil cytoplasmic antibody; ASLO, antistreptolysin O; CT, computed tomography; GBM, glomerular basement membrane; IVP, intravenous pyelography; RBC, red blood cell; UA, urinalysis; VDRL, Venereal Disease Research Laboratory; WBC, white blood cell.

1	Serologic and hematologic evaluation: blood cultures, anti-GBM antibody, ANCA, complement levels, cryoglobulins, hepatitis B and C serologies, VDRL, HIV, ASLO to mild proteinuria and hematuria, whereas renal vein thrombosis typically induces heavy proteinuria and hematuria. These vascular complications often require angiography for confirmation and are discussed in Chap. 341.

1	Diseases of the glomeruli (glomerulonephritis and vasculitis) and the renal microvasculature (hemolytic-uremic syndromes, thrombotic thrombocytopenic purpura, and malignant hypertension) usually present with various combinations of glomerular injury: proteinuria, hematuria, reduced GFR, and alterations of sodium excretion that lead to hypertension, edema, and circulatory congestion (acute nephritic syndrome). These findings may occur as primary renal diseases or as renal manifestations of systemic diseases. The clinical setting and other laboratory data help distinguish primary renal diseases from systemic diseases. The finding of RBC casts in the urine is an indication for early renal biopsy (Fig. 61-1), as the pathologic pattern has important implications for diagnosis, prognosis, and treatment. Hematuria without RBC casts can also be an indication of glomerular disease; this evaluation is summarized in Fig. 61-2. A detailed discussion of glomerulonephritis and diseases of the

1	and treatment. Hematuria without RBC casts can also be an indication of glomerular disease; this evaluation is summarized in Fig. 61-2. A detailed discussion of glomerulonephritis and diseases of the microvasculature is found in Chap. 340.

1	Oliguria refers to a 24-h urine output <400 mL, and anuria is the complete absence of urine formation (<100 mL). Anuria can be caused by total urinary tract obstruction, total renal artery or vein occlusion, and shock (manifested by severe hypotension and intense renal vasoconstriction). Cortical necrosis, ATN, and rapidly progressive glomerulonephritis occasionally cause anuria. Oliguria can accompany acute renal failure of any etiology and carries a more serious prognosis for renal recovery in all conditions except prerenal azotemia. Nonoliguria refers to urine output >400 mL/d in patients with acute or chronic azotemia. With nonoliguric ATN, disturbances of potassium and hydrogen balance are less severe than in oliguric patients, and recovery to normal renal function is usually more rapid.

1	The evaluation of proteinuria is shown schematically in Fig. 61-3 and typically is initiated after detection of proteinuria by dipstick examination. The dipstick measurement detects only albumin and gives false-positive results at pH >7.0 or when the urine is very concentrated or contaminated with blood. Because the dipstick relies on urinary albumin concentration, a very dilute urine may obscure significant proteinuria on dipstick examination. Quantification of urinary albumin on a spot urine sample (ideally from a first morning void) by measurement of an albumin-to-creatinine ratio (ACR) is helpful in approximating a 24-h albumin excretion rate (AER), where ACR (mg/g) ≈AER (mg/24 h). Furthermore, proteinuria that is not predominantly due to albumin will be missed by dipstick screening. This information is particularly important for the detection of Bence-Jones proteins in the urine of patients with multiple myeloma. Tests to measure total urine protein concentration accurately rely

1	This information is particularly important for the detection of Bence-Jones proteins in the urine of patients with multiple myeloma. Tests to measure total urine protein concentration accurately rely on precipitation with sulfosalicylic or trichloracetic acid (Fig. 61-3).

1	The magnitude of proteinuria and its composition in the urine depend on the mechanism of renal injury that leads to protein losses. Both charge and size selectivity normally prevent virtually all plasma albumin, globulins, and other high-molecular-weight proteins from crossing the glomerular wall; however, if this barrier is disrupted, plasma proteins may leak into the urine (glomerular proteinuria; Fig. 61-3). Smaller proteins (<20 kDa) are freely filtered but are readily reabsorbed by the proximal tubule. Traditionally, healthy individuals excrete <150 mg/d of total protein and <30 mg/d of albumin. However, even at albuminuria levels <30 mg/d, risk for progression to overt nephropathy or subsequent cardiovascular disease is increased. The remainder of the protein in the urine is secreted by 293 the tubules (Tamm-Horsfall, IgA, and urokinase) or represents small amounts of filtered β2-microglobulin, apoproteins, enzymes, and peptide hormones. Another mechanism of proteinuria entails

1	by 293 the tubules (Tamm-Horsfall, IgA, and urokinase) or represents small amounts of filtered β2-microglobulin, apoproteins, enzymes, and peptide hormones. Another mechanism of proteinuria entails excessive production of an abnormal protein that exceeds the capacity of the tubule for reabsorption. This situation most commonly occurs with plasma cell dyscrasias, such as multiple myeloma, amyloidosis, and lymphomas, that are associated with monoclonal production of immunoglobulin light chains.

1	The normal glomerular endothelial cell forms a barrier composed of pores of ~100 nm that retain blood cells but offer little impediment to passage of most proteins. The glomerular basement membrane traps most large proteins (>100 kDa), and the foot processes of epithelial cells (podocytes) cover the urinary side of the glomerular basement membrane and produce a series of narrow channels (slit diaphragms) to allow molecular passage of small solutes and water but not proteins. Some glomerular diseases, such as minimal change disease, cause fusion of glomerular epithelial cell foot processes, resulting in predominantly “selective” (Fig. 61-3) loss of albumin. Other glomerular diseases can present with disruption of the basement membrane and slit diaphragms (e.g., by immune complex deposition), resulting in losses of albumin and other plasma proteins. The fusion of foot processes causes increased pressure across the capillary basement membrane, resulting in areas with larger pore sizes

1	resulting in losses of albumin and other plasma proteins. The fusion of foot processes causes increased pressure across the capillary basement membrane, resulting in areas with larger pore sizes (and more severe “nonselective” proteinuria (Fig. 61-3).

1	When the total daily urinary excretion of protein is >3.5 g, hypoalbuminemia, hyperlipidemia, and edema (nephrotic syndrome; Fig. 61-3) are often present as well. However, total daily urinary protein excretion >3.5 g can occur without the other features of the nephrotic syndrome in a variety of other renal diseases, including diabetes (Fig. 61-3). Plasma cell dyscrasias (multiple myeloma) can be associated with large amounts of excreted light chains in the urine, which may not be detected by dipstick. The light chains are filtered by the glomerulus and overwhelm the reabsorptive capacity of the proximal tubule. Renal failure from these disorders occurs through a variety of mechanisms, including proximal tubule injury, tubule obstruction (cast nephropathy), and light chain deposition (Chap. 340). However, not all excreted light chains are nephrotoxic.

1	PROTEINURIA ON URINE DIPSTICK Quantify by 24-h urinary excretion of protein and albumin or first morning spot albumin-to-creatinine ratio RBCs or RBC casts on urinalysis In addition to disorders listed under microalbuminuria consider Myeloma-associated kidney disease (check UPEP) Intermittent proteinuria Postural proteinuria Congestive heart failure Fever Exercise Go to Fig. 61-2 Macroalbuminuria 300-3500 mg/d or 300-3500 mg/g Microalbuminuria 30-300 mg/d or 30-300 mg/g Nephrotic range > 3500 mg/d or > 3500 mg/g + Consider Early diabetes Essential hypertension Early stages of glomerulonephritis (especially with RBCs, RBC casts)Consider Early diabetes Essential hypertension Early stages of glomerulonephritis (especially with RBCs, RBC casts) Nephrotic syndrome Diabetes Amyloidosis Minimal change disease FSGS Membranous glomerulopathy IgA nephropathy

1	FIguRE 61-3 Approach to the patient with proteinuria. Investigation of proteinuria is often initiated by a positive dipstick on routine urinalysis. Conventional dipsticks detect predominantly albumin and provide a semiquantitative assessment (trace, 1+, 2+, or 3+), which is influenced by urinary concentration as reflected by urine specific gravity (minimum, <1.005; maximum, 1.030). However, more exact determination of proteinuria should employ a spot morning protein/creatinine ratio (mg/g) or a 24-h urine collection (mg/24 h). FSGS, focal segmental glomerulosclerosis; RBC, red blood cell; UPEP, urine protein electrophoresis.

1	Hypoalbuminemia in nephrotic syndrome occurs through excessive urinary losses and increased proximal tubule catabolism of filtered albumin. Edema forms from renal sodium retention and reduced plasma oncotic pressure, which favors fluid movement from capillaries to interstitium. To compensate for the perceived decrease in effective intravascular volume, activation of the renin-angiotensin system, stimulation of ADH, and activation of the sympathetic nervous system take place, promoting continued renal salt and water reabsorption and progressive edema. Despite these changes, hypertension is uncommon in primary kidney diseases resulting in the nephrotic syndrome (Fig. 61-3 and Chap. 338). The urinary loss of regulatory proteins and changes in hepatic synthesis contribute to the other manifestations of the nephrotic syndrome. A hypercoagulable state may arise from urinary losses of antithrombin III, reduced serum levels of proteins S and C, hyperfibrinogenemia, and enhanced platelet

1	of the nephrotic syndrome. A hypercoagulable state may arise from urinary losses of antithrombin III, reduced serum levels of proteins S and C, hyperfibrinogenemia, and enhanced platelet aggregation. Hypercholesterolemia may be severe and results from increased hepatic lipoprotein synthesis. Loss of immunoglobulins contributes to an increased risk of infection. Many diseases (some listed in Fig. 61-3) and drugs can cause the nephrotic syndrome; a complete list is found in Chap. 338.

1	HEMATuRIA, PYuRIA, AND CASTS

1	Isolated hematuria without proteinuria, other cells, or casts is often indicative of bleeding from the urinary tract. Hematuria is defined as two to five RBCs per high-power field (HPF) and can be detected by dipstick. A false-positive dipstick for hematuria (where no RBCs are seen on urine microscopy) may occur when myoglobinuria is present, often in the setting of rhabdomyolysis. Common causes of isolated hematuria include stones, neoplasms, tuberculosis, trauma, and prostatitis. Gross hematuria with blood clots usually is not an intrinsic renal process; rather, it suggests a postrenal source in the urinary collecting system. Evaluation of patients presenting with microscopic hematuria is outlined in Fig. 61-2. A single urinalysis with hematuria is common and can result from menstruation, viral illness, allergy, exercise, or mild trauma. Persistent or significant hematuria (>3 RBCs/ HPF on three urinalyses, a single urinalysis with >100 RBCs, or gross hematuria) is associated with

1	viral illness, allergy, exercise, or mild trauma. Persistent or significant hematuria (>3 RBCs/ HPF on three urinalyses, a single urinalysis with >100 RBCs, or gross hematuria) is associated with significant renal or urologic lesions in 9.1% of cases. The level of suspicion for urogenital neoplasms in patients with isolated painless hematuria and nondysmorphic RBCs increases with age. Neoplasms are rare in the pediatric population, and isolated hematuria is more likely to be “idiopathic” or associated with a congenital anomaly. Hematuria with pyuria and bacteriuria is typical of infection and should be treated with antibiotics after appropriate cultures. Acute cystitis or urethritis in women can cause gross hematuria. Hypercalciuria and hyperuricosuria are also risk factors for unexplained isolated hematuria in both children and adults. In some of these patients (50–60%), reducing calcium and uric acid excretion through dietary interventions can eliminate the microscopic hematuria.

1	Isolated microscopic hematuria can be a manifestation of glomerular diseases. The RBCs of glomerular origin are often dysmorphic when examined by phase-contrast microscopy. Irregular shapes of RBCs may also result from pH and osmolarity changes produced along the distal nephron. Observer variability in detecting dysmorphic RBCs is common. The most common etiologies of isolated glomerular hematuria are IgA nephropathy, hereditary nephritis, and thin basement membrane disease. IgA nephropathy and hereditary nephritis can lead to episodic gross hematuria. A family history of renal failure is often present in hereditary nephritis, and patients with thin basement membrane disease often have family members with microscopic hematuria. A renal biopsy is needed for the definitive diagnosis of these disorders, which are discussed in more detail in Chap. 338. Hematuria with dysmorphic RBCs, RBC casts, and protein excretion >500 mg/d is virtually diagnostic of glomerulonephritis. RBC casts form

1	disorders, which are discussed in more detail in Chap. 338. Hematuria with dysmorphic RBCs, RBC casts, and protein excretion >500 mg/d is virtually diagnostic of glomerulonephritis. RBC casts form as RBCs that enter the tubule fluid and become trapped in a cylindrical mold of gelled Tamm-Horsfall protein. Even in the absence of azotemia, these patients should undergo serologic evaluation and renal biopsy as outlined in Fig. 61-2.

1	PART 2 Cardinal Manifestations and Presentation of Diseases Isolated pyuria is unusual since inflammatory reactions in the kidney or collecting system also are associated with hematuria. The presence of bacteria suggests infection, and WBC casts with bacteria are indicative of pyelonephritis. WBCs and/or WBC casts also may be seen in acute glomerulonephritis as well as in tubulointerstitial processes such as interstitial nephritis and transplant rejection. Casts can be seen in chronic renal diseases. Degenerated cellular casts called waxy casts or broad casts (arising in the dilated tubules that have undergone compensatory hypertrophy in response to reduced renal mass) may be seen in the urine.

1	By history, it is often difficult for patients to distinguish urinary frequency (often of small volumes) from true polyuria (>3 L/d), and a quantification of volume by 24-h urine collection may be needed (Fig. 61-4). Polyuria results from two potential mechanisms: excretion of nonabsorbable solutes (such as glucose) or excretion of water (usually from a defect in ADH production or renal responsiveness). To distinguish a solute diuresis from a water diuresis and to determine whether the diuresis is appropriate for the clinical circumstances, urine osmolality is measured. The average person excretes between 600 and 800 mosmol of solutes per day, primarily as urea and electrolytes. If the urine output is >3 L/d and the urine is dilute (<250 mosmol/L), total mosmol excretion is normal and a water diuresis is present. This circumstance could arise from polydipsia, inadequate secretion of vasopressin (central diabetes insipidus), or failure of renal tubules to respond to vasopressin

1	and a water diuresis is present. This circumstance could arise from polydipsia, inadequate secretion of vasopressin (central diabetes insipidus), or failure of renal tubules to respond to vasopressin (nephrogenic diabetes insipidus). If the urine volume is >3 L/d and urine

1	POLYURIA (>3 L/24 h) Urine osmolality < 250 mosmol History, low serum sodium Water deprivation test or ADH level Primary polydipsia Psychogenic Hypothalamic disease Drugs (thioridazine, chlorpromazine, anticholinergic agents) > 300 mosmol Diabetes insipidus (DI) Solute diuresis Glucose, mannitol, radiocontrast, urea (from high protein feeding), medullary cystic diseases, resolving ATN, or obstruction, diuretics posthypophysectomy, trauma, histiocystosis or granuloma, encroachment by aneurysm, Sheehan's syndrome, infection, Guillain-Barré, fat embolus, Acquired tubular diseases: pyelonephritis, analgesic nephropathy, multiple myeloma, amyloidosis, obstruction, sarcoidosis, hypercalcemia, hypokalemia, Sjren’s syndrome, sickle cell anemia Drugs or toxins: lithium, demeclocycline, methoxyflurane, ethanol, diphenylhydantoin, propoxyphene, amphotericin Congenital: hereditary, polycystic or medullary cystic disease

1	Drugs or toxins: lithium, demeclocycline, methoxyflurane, ethanol, diphenylhydantoin, propoxyphene, amphotericin Congenital: hereditary, polycystic or medullary cystic disease FIguRE 61-4 Approach to the patient with polyuria. ADH, antidiuretic hormone; ATN, acute tubular necrosis. osmolality is >300 mosmol/L, a solute diuresis is clearly present and a search for the responsible solute(s) is mandatory.

1	Excessive filtration of a poorly reabsorbed solute such as glucose or mannitol can depress reabsorption of NaCl and water in the proximal tubule and lead to enhanced excretion in the urine. Poorly controlled diabetes mellitus with glucosuria is the most common cause of a solute diuresis, leading to volume depletion and serum hypertonicity. Since the urine sodium concentration is less than that of blood, more water than sodium is lost, causing hypernatremia and hypertonicity. Common iatrogenic solute diuresis occurs in association with mannitol administration, radiocontrast media, and high-protein feedings (enteral or parenteral), leading to increased urea production and excretion. Less commonly, excessive sodium loss may result from cystic renal diseases or Bartter’s syndrome or may develop during a tubulointerstitial process (such as resolving ATN). In these so-called salt-wasting disorders, the tubule damage results in direct impairment of sodium reabsorption and indirectly reduces

1	during a tubulointerstitial process (such as resolving ATN). In these so-called salt-wasting disorders, the tubule damage results in direct impairment of sodium reabsorption and indirectly reduces the responsiveness of the tubule to aldosterone. Usually, the sodium losses are mild, and the obligatory urine output is <2 L/d; resolving ATN and postobstructive diuresis are exceptions and may be associated with significant natriuresis and polyuria.

1	Formation of large volumes of dilute urine is usually due to polydipsic states or diabetes insipidus. Primary polydipsia can result from habit, psychiatric disorders, neurologic lesions, or medications. During deliberate polydipsia, extracellular fluid volume is normal or expanded and plasma vasopressin levels are reduced because serum osmolality tends to be near the lower limits of normal. Urine osmolality is also maximally dilute at 50 mosmol/L.

1	Central diabetes insipidus may be idiopathic in origin or secondary to a variety of conditions, including hypophysectomy, trauma, neoplastic, inflammatory, vascular, or infectious hypothalamic diseases. Idiopathic central diabetes insipidus is associated with selective destruction of the vasopressin-secreting neurons in the supraoptic and paraventricular nuclei and can either be inherited as an autosomal dominant trait or occur spontaneously. Nephrogenic diabetes insipidus can occur in a variety of clinical situations, as summarized in Fig. 61-4. A plasma vasopressin level is recommended as the best method for distinguishing between central and nephrogenic diabetes insipidus. Alternatively, a water deprivation test plus exogenous vasopressin may distinguish primary polydipsia from central and nephrogenic diabetes insipidus. For a detailed discussion, see Chap. 404.

1	Atlas of Urinary Sediments and Renal Biopsies Agnes B. Fogo, Eric G. Neilson Key diagnostic features of selected diseases in renal biopsy are illus-trated, with light, immunofluorescence, and electron microscopic 62e images. Common urinalysis findings are also documented. Figure 62e-1 Minimal-change disease. In minimal-change disease, light microscopy is unremarkable (A), whereas electron microscopy (B) reveals podocyte injury evidenced by complete foot process effacement. (ABF/Vanderbilt Collection.) CHAPTER 62e Atlas of Urinary Sediments and Renal Biopsies Figure 62e-2 Focal segmental glomerulosclerosis (FSGS). There is a well-defined segmental increase in matrix and obliteration of capil-lary loops (arrow), the sine qua non of segmental sclerosis not other-wise specified (NOS) type. (EGN/UPenn Collection.)

1	Figure 62e-3 Collapsing glomerulopathy. There is segmental col-lapse (arrow) of the glomerular capillary loops and overlying podocyte hyperplasia. This lesion may be idiopathic or associated with HIV infec-tion and has a particularly poor prognosis. (ABF/Vanderbilt Collection.) Figure 62e-4 Hilar variant of FSGS. There is segmental sclerosis of the glomerular tuft at the vascular pole with associated hyalinosis, also present in the afferent arteriole (arrows). This lesion often occurs as a secondary response when nephron mass is lost due to, e.g., scar-ring from other conditions. Patients usually have less proteinuria and less steroid response than FSGS, NOS type. (ABF/Vanderbilt Collection.) Figure 62e-5 Tip lesion variant of FSGS. There is segmental scle-rosis of the glomerular capillary loops at the proximal tubular outlet (arrow). This lesion has a better prognosis than other types of FSGS. (ABF/Vanderbilt Collection.)

1	PART 2 Cardinal Manifestations and Presentation of Diseases Figure 62e-6 Postinfectious (poststreptococcal) glomerulonephritis. The glomerular tuft shows proliferative changes with numerous poly-morphonuclear leukocytes (PMNs), with a crescentic reaction (arrow) in severe cases (A). These deposits localize in the mesangium and along the capillary wall in a subepithelial pattern and stain dominantly for C3 and to a lesser extent for IgG (B). Subepithelial hump-shaped deposits are seen by electron microscopy (arrow) (C). (ABF/Vanderbilt Collection.)

1	Figure 62e-7 Membranous glomerulopathy. Membranous glomerulopathy is due to subepithelial deposits, with resulting basement membrane reaction, resulting in the appearance of spike-like projections on silver stain (A). The deposits are directly visualized by fluorescent anti-IgG, revealing diffuse granular capillary loop staining (B). By electron microscopy, the subepithelial location of the deposits and early surrounding basement membrane reaction is evident, with overlying foot process effacement (C). (ABF/Vanderbilt Collection.) Figure 62e-8 IgA nephropathy. There is variable mesangial expansion due to mesangial deposits, with some cases also showing endocapillary proliferation or segmental sclerosis (A). By immunofluorescence, mesangial IgA deposits are evident (B). (ABF/Vanderbilt Collection.) CHAPTER 62e Atlas of Urinary Sediments and Renal Biopsies

1	CHAPTER 62e Atlas of Urinary Sediments and Renal Biopsies Figure 62e-9 Membranoproliferative glomerulonephritis. There is mesangial expansion and endocapillary proliferation with cellular interposition in response to subendothelial deposits, resulting in the “tram-track” of duplication of glomerular basement membrane. (EGN/ UPenn Collection.) Figure 62e-10 Dense deposit disease (membranoproliferative glomerulonephritis type II). By light microscopy, there is a membranoproliferative pattern. By electron microscopy, there is a dense transformation of the glomerular basement membrane with round, globular deposits within the mesangium. By immunofluorescence, only C3 staining is usually present. Dense deposit disease is part of the group of renal diseases called C3 glomerulopathy, related to underlying complement dysregulation. (ABF/Vanderbilt Collection.)

1	Figure 62e-12 C3 glomerulonephritis. By immunofluorescence, only C3 staining is usually present, with occasional minimal immu-noglobulin, in an irregular capillary wall and mesangial distribution. (ABF/Vanderbilt Collection.) PART 2 Cardinal Manifestations and Presentation of Diseases Figure 62e-11 C3 glomerulonephritis. By light microscopy, there is a membranoproliferative pattern. C3 glomerulonephritis is part of the group of renal diseases called C3 glomerulopathy, related to underlying complement dysregulation. (ABF/ Vanderbilt Collection.) Figure 62e-13 C3 glomerulonephritis. By electron microscopy, usual density deposits are present (arrows), including mesangial, subendothelial, and occasional hump-type subepithelial deposits. (ABF/ Vanderbilt Collection.)

1	(ABF/ Vanderbilt Collection.) Figure 62e-14 Mixed proliferative and membranous glomerulonephritis. This specimen shows pink subepithelial deposits with spike reaction, and the “tram-track” sign of reduplication of glomerular basement membrane, resulting from subendothelial deposits, as may be seen in mixed membranous and proliferative lupus nephritis (International Society of Nephrology [ISN]/Renal Pathology Society [RPS] class V and IV). (EGN/UPenn Collection.) Figure 62e-16 Granulomatosis with polyangiitis (Wegener’s). This pauci-immune necrotizing crescentic glomerulonephritis shows numerous breaks in the glomerular basement membrane with associated segmental fibrinoid necrosis and a crescent formed by proliferation of the parietal epithelium. Note that the uninvolved segment of the glomerulus (at ∼5 o’clock) shows no evidence of proliferation or immune complexes. (ABF/Vanderbilt Collection.) CHAPTER 62e Atlas of Urinary Sediments and Renal Biopsies

1	CHAPTER 62e Atlas of Urinary Sediments and Renal Biopsies Figure 62e-15 Lupus nephritis. Proliferative lupus nephritis, ISN/RPS class III (focal) or IV (diffuse), manifests as endocapillary proliferation, which may result in segmental necrosis due to deposits, particularly in the subendothelial area (A). By immunofluorescence, chunky irregular mesangial and capillary loop deposits are evident, with some of the peripheral loop deposits having a smooth, molded outer contour due to their subendothelial location. These deposits typically stain for all three immunoglobulins, IgG, IgA, IgM, and both C3 and C1q (B). By electron microscopy, subendothelial (arrow), mesangial (white rim arrowhead), and rare subepithelial (black arrowhead) dense immune complex deposits are evident, along with extensive foot process effacement (C). (ABF/Vanderbilt Collection.) PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Figure 62e-17 Anti–glomerular basement membrane antibody-mediated glomerulonephritis. There is segmental necrosis with a break of the glomerular basement membrane (arrow) and a cellular crescent (A), and immunofluorescence for IgG shows linear staining of the glomerular basement membrane with a small crescent at ∼1 o’clock (B). (ABF/Vanderbilt Collection.) Figure 62e-18 Amyloidosis. Amyloidosis shows amorphous, acellular expansion of the mesangium, with material often also infiltrating glomerular basement membranes, vessels, and the interstitium, with apple-green birefringence by polarized Congo red stain (A). The deposits are composed of randomly organized 9to 11-nm fibrils by electron microscopy (B). (ABF/Vanderbilt Collection.)

1	Figure 62e-19 Light chain deposition disease. There is mesangial expansion, often nodular by light microscopy (A), with immunofluorescence showing monoclonal staining, more commonly with kappa than lambda light chain, of tubules (B) and glomerular tufts. By electron microscopy (C), the deposits show an amorphous granular appearance and line the inside of the glomerular basement membrane (arrows) and are also found along the tubular basement membranes. (ABF/Vanderbilt Collection.) Figure 62e-20 Light chain cast nephropathy (myeloma kidney). Monoclonal light chains precipitate in tubules and result in a syncytial giant cell reaction (arrow) surrounding the casts and a surround-ing chronic interstitial nephritis with tubulointerstitial fibrosis. (ABF/ Vanderbilt Collection.) CHAPTER 62e Atlas of Urinary Sediments and Renal Biopsies

1	CHAPTER 62e Atlas of Urinary Sediments and Renal Biopsies Figure 62e-21 Fabry’s disease. Due to deficiency of α-galactosidase, there is abnormal accumulation of glycolipids, resulting in foamy podocytes by light microscopy (A). These deposits can be directly visualized by electron microscopy (B), where the glycosphingolipid appears as whorled so-called myeloid bodies, particularly in the podocytes. (ABF/Vanderbilt Collection.) Cardinal Manifestations and Presentation of Diseases Figure 62e-22 Alport’s syndrome and thin glomerular basement membrane lesion. In Alport’s syndrome, there is irregular thinning alternating with thickened so-called basket-weaving abnormal organization of the glomerular basement membrane (A). In benign familial hematuria, or in early cases of Alport’s syndrome or female carriers, only extensive thinning of the glomerular basement membrane is seen by electron microscopy (B). (ABF/Vanderbilt Collection.)

1	Figure 62e-23 Diabetic nephropathy. In the earliest stage of diabetic nephropathy, only mild mesangial increase and prominent glomerular basement membranes (confirmed to be thickened by electron microscopy) are present (A). In slightly more advanced stages, more marked mesangial expansion with early nodule formation develops, with evident arteriolar hyaline (B). In established diabetic nephropathy, there is nodular mesangial expansion, so-called Kimmelstiel-Wilson nodules, with increased mesangial matrix and cellularity, microaneurysm formation in the glomerulus on the left, and prominent glomerular basement membranes without evidence of immune deposits and arteriolar hyalinosis of both afferent and efferent arterioles (C). (ABF/Vanderbilt Collection.)

1	Figure 62e-24 Arterionephrosclerosis. Hypertension-associated injury often manifests extensive global sclerosis of glomeruli, with accompanying and proportional tubulointerstitial fibrosis and pericapsular fibrosis, and there may be segmental sclerosis (A). The vessels show disproportionately severe changes of intimal fibrosis, medial hypertrophy, and arteriolar hyaline deposits (B). (ABF/Vanderbilt Collection.) Figure 62e-26 Hemolytic-uremic syndrome. There are character-istic intraglomerular fibrin thrombi, with a chunky pink appearance (thrombotic microangiopathy) (arrow). The remaining portion of the capillary tuft shows corrugation of the glomerular basement mem-brane due to ischemia. (ABF/Vanderbilt Collection.)

1	B Figure 62e-27 Progressive systemic sclerosis. Acutely, there is fibrinoid necrosis of interlobular and larger vessels, with intervening normal vessels and ischemic change in the glomeruli (A). Chronically, this injury leads to intimal proliferation, the so-called onion-skinning appearance (B). (ABF/Vanderbilt Collection.) CHAPTER 62e Atlas of Urinary Sediments and Renal Biopsies Figure 62e-25 Cholesterol emboli. Cholesterol emboli cause cleft-like spaces (arrow) where the lipid has been extracted during processing, with smooth outer contours and surrounding fibrotic and mononuclear cell reaction in these arterioles. (ABF/Vanderbilt Collection.) Figure 62e-28 Acute pyelonephritis. There are characteristic intra-tubular plugs and casts of PMNs (arrow) with inflammation extending into the surrounding interstitium and accompanying tubular injury. (ABF/ Vanderbilt Collection.)

1	(ABF/ Vanderbilt Collection.) Figure 62e-29 Acute tubular injury. There is extensive flattening of the tubular epithelium and loss of the brush border, with mild intersti-tial edema, characteristic of acute tubular injury due to ischemia. (ABF/ Vanderbilt Collection.) Figure 62e-30 Acute interstitial nephritis. There is extensive interstitial lymphoplasmocytic infiltrate with mild edema and associated tubular injury (A), which is frequently associated with interstitial eosinophils (B) when caused by a drug hypersensitivity reaction. (ABF/Vanderbilt Collection.) B Figure 62e-31 Oxalosis. Calcium oxalate crystals have caused extensive tubular injury, with flattening and regeneration of tubular epithelium (A). Crystals are well visualized as sheaves when viewed under polarized light (B). (ABF/Vanderbilt Collection.)

1	Figure 62e-32 Acute phosphate nephropathy. There is extensive acute tubular injury with intratubular nonpolarizable calcium phos-phate crystals. (ABF/Vanderbilt Collection.) Figure 62e-35 Coarse granular cast. (ABF/Vanderbilt Collection.) Figure 62e-33 Sarcoidosis. There is chronic interstitial nephritis with numerous, confluent, nonnecrotizing granulomas. The glomeruli are unremarkable, but there is moderate tubular atrophy and interstitial fibrosis. (ABF/Vanderbilt Collection.) Figure 62e-36 Fine granular casts. (ABF/Vanderbilt Collection.) CHAPTER 62e Atlas of Urinary Sediments and Renal Biopsies Figure 62e-34 Hyaline cast. (ABF/Vanderbilt Collection.) Figure 62e-37 Red blood cell cast. (ABF/Vanderbilt Collection.) Figure 62e-38 White blood cell cast. (ABF/Vanderbilt Collection.) Figure 62e-40 “Maltese cross” formation in an oval fat body. (ABF/Vanderbilt Collection.)

1	Figure 62e-39 Triple phosphate crystals. (ABF/Vanderbilt Collection.) Figure 62e-41 Uric acid crystals. (ABF/Vanderbilt Collection.) 295 fluid and Electrolyte Disturbances David B. Mount SODIuM AND WATER COMPOSITION OF BODY FLuIDS Water is the most abundant constituent in the body, comprising approximately 50% of body weight in women and 60% in men. Total-63 body water is distributed in two major compartments: 55–75% is intracellular (intracellular fluid [ICF]), and 25–45% is extracellular (extracellular fluid [ECF]). The ECF is further subdivided into intravascular (plasma water) and extravascular (interstitial) spaces in a ratio of 1:3. Fluid movement between the intravascular and interstitial spaces occurs across the capillary wall and is determined by Starling forces, i.e., capillary hydraulic pressure and colloid osmotic pressure. The transcapillary hydraulic pressure gradient exceeds the corresponding oncotic pressure gradient, thereby favoring the movement of plasma

1	capillary hydraulic pressure and colloid osmotic pressure. The transcapillary hydraulic pressure gradient exceeds the corresponding oncotic pressure gradient, thereby favoring the movement of plasma ultrafiltrate into the extravascular space. The return of fluid into the intravascular compartment occurs via lymphatic flow.

1	The solute or particle concentration of a fluid is known as its osmolality, expressed as milliosmoles per kilogram of water (mOsm/kg). Water easily diffuses across most cell membranes to achieve osmotic equilibrium (ECF osmolality = ICF osmolality). Notably, the extracellular and intracellular solute compositions differ considerably owing to the activity of various transporters, channels, and ATP-driven membrane pumps. The major ECF particles are Na+ and its accompanying anions Cl– and HCO3–, whereas K+ and organic phosphate esters (ATP, creatine phosphate, and phospholipids) are the predominant ICF osmoles. Solutes that are restricted to the ECF or the ICF determine the “tonicity” or effective osmolality of that compartment. Certain solutes, particularly urea, do not contribute to water shifts across most membranes and are thus known as ineffective osmoles.

1	Water Balance Vasopressin secretion, water ingestion, and renal water transport collaborate to maintain human body fluid osmolality between 280 and 295 mOsm/kg. Vasopressin (AVP) is synthesized in magnocellular neurons within the hypothalamus; the distal axons of these neurons project to the posterior pituitary or neurohypophysis, from which AVP is released into the circulation. A network of central “osmoreceptor” neurons, which includes the AVP-expressing magnocellular neurons themselves, sense circulating osmolality via nonselective, stretch-activated cation channels. These osmoreceptor neurons are activated or inhibited by modest increases and decreases in circulating osmolality, respectively; activation leads to AVP release and thirst.

1	AVP secretion is stimulated as systemic osmolality increases above a threshold level of ~285 mOsm/kg, above which there is a linear relationship between osmolality and circulating AVP (Fig. 63-1). Thirst and thus water ingestion are also activated at ~285 mOsm/kg, beyond which there is an equivalent linear increase in the perceived intensity of thirst as a function of circulating osmolality. Changes in blood volume and blood pressure are also direct stimuli for AVP release and thirst, albeit with a less sensitive response profile. Of perhaps greater clinical relevance to the pathophysiology of water homeostasis, ECF volume strongly modulates the relationship between circulating osmolality and AVP release, such that hypovolemia reduces the osmotic threshold and increases the slope of the response curve to osmolality; hypervolemia has an opposite effect, increasing the osmotic threshold and reducing the slope of the response curve (Fig. 63-1). Notably, AVP has a half-life in the

1	the response curve to osmolality; hypervolemia has an opposite effect, increasing the osmotic threshold and reducing the slope of the response curve (Fig. 63-1). Notably, AVP has a half-life in the circulation of only 10–20 minutes; thus, changes in ECF volume and/or circulating osmolality can rapidly affect water homeostasis. In addition to volume status, a number of other “nonosmotic” stimuli have potent activating effects on osmosensitive neurons and AVP release, including nausea, intracerebral angiotensin II, serotonin, and multiple drugs.

1	The excretion or retention of electrolyte-free water by the kidney is modulated by circulating AVP. AVP acts on renal, V2-type receptors in FIguRE 63-1 Circulating levels of vasopressin (AVP) in response to changes in osmolality. Plasma AVP becomes detectable in euvolemic, healthy individuals at a threshold of ~285 mOsm/kg, above which there is a linear relationship between osmolality and circulating AVP. The vasopressin response to osmolality is modulated strongly by volume status. The osmotic threshold is thus slightly lower in hypovolemia, with a steeper response curve; hypervolemia reduces the sensitivity of circulating AVP levels to osmolality.

1	the thick ascending limb of Henle and principal cells of the collecting duct (CD), increasing intracellular levels of cyclic AMP and activating protein kinase A (PKA)–dependent phosphorylation of multiple transport proteins. The AVPand PKA-dependent activation of Na+-Cl– and K+ transport by the thick ascending limb of the loop of Henle (TALH) is a key participant in the countercurrent mechanism (Fig. 63-2). The countercurrent mechanism ultimately increases the interstitial osmolality in the inner medulla of the kidney, driving water absorption across the renal CD. However, water, salt, and solute transport by both proximal and distal nephron segments participates in the renal concentrating mechanism (Fig. 63-2). Water transport across apical and basolateral aquaporin-1 water channels in the descending thin limb of the loop of Henle is thus involved, as is passive absorption of Na+-Cl– PART 2 Cardinal Manifestations and Presentation of Diseases AQP2,3

1	PART 2 Cardinal Manifestations and Presentation of Diseases AQP2,3 Cortex excretion. The thiazide-sensitive apical Na+-Cl– cotransporter (NCC) reabsorbs 5–10% of filtered Na+-Cl– in the AQP2,3 DCT. Principal cells in the CNT and CD reabsorb Na+ via electrogenic, amiloride-sensitive epithelial Na+ channels (ENaC); Cl– ions are primarily reabsorbed by adjacent intercalated cells, via apical Cl– exchange (Cl–-OH– and Vasa Recta: – Cl–-HCO exchange, mediated by the SLC26A4 anion AQP1, UT-B 3 H2O exchanger) (Fig. 63-4). AQP2–4 Renal tubular reabsorption of filtered Na+-Cl– is regulated by multiple circulating and paracrine hormones, in addition to the activity of renal nerves. Angiotensin II activates proximal Na+-Cl– reabsorption, as do adrenergic receptors under the influence of renal sympathetic innervation; locally generated dopamine, in contrast, has a natriuretic effect. Aldosterone primarily activates

1	Na+-Cl– reabsorption within the aldosterone-sensitive FIguRE 63-2 The renal concentrating mechanism. Water, salt, and solute trans-distal nephron. In particular, aldosterone activates the port by both proximal and distal nephron segments participates in the renal con-ENaC channel in principal cells, inducing Na+ absorption centrating mechanism (see text for details). Diagram showing the location of the and promoting K+ excretion (Fig. 63-4). major transport proteins involved; a loop of Henle is depicted on the left, collect-Circulatory integrity is critical for the perfusion and ing duct on the right. AQP, aquaporin; CLC-K1, chloride channel; NKCC2, Na-K-2Cl function of vital organs. “Underfilling” of the artecotransporter; ROMK, renal outer medullary K+ channel; UT, urea transporter. (Used rial circulation is sensed by ventricular and vascular with permission from JM Sands: Molecular approaches to urea transporters. J Am Soc pressure receptors, resulting in a neurohumoral

1	(Used rial circulation is sensed by ventricular and vascular with permission from JM Sands: Molecular approaches to urea transporters. J Am Soc pressure receptors, resulting in a neurohumoral acti-Nephrol 13:2795, 2002.) vation (increased sympathetic tone, activation of the by the thin ascending limb, via apical and basolateral CLC-K1 chloride channels and paracellular Na+ transport. Renal urea transport in turn plays important roles in the generation of the medullary osmotic gradient and the ability to excrete solute-free water under conditions of both high and low protein intake (Fig. 63-2).

1	AVP-induced, PKA-dependent phosphorylation of the aquaporin-2 water channel in principal cells stimulates the insertion of active water channels into the lumen of the CD, resulting in transepithelial water absorption down the medullary osmotic gradient (Fig. 63-3). Under “antidiuretic” conditions, with increased circulating AVP, the kidney reabsorbs water filtered by the glomerulus, equilibrating the osmolality across the CD epithelium to excrete a hypertonic, “concentrated” urine (osmolality of up to 1200 mOsm/kg). In the absence of circulating AVP, insertion of aquaporin-2 channels and water absorption across the CD is essentially abolished, resulting in secretion of a hypotonic, dilute urine (osmolality as low as 30–50 mOsm/kg). Abnormalities in this “final common pathway” are involved in most disorders of water homeostasis, e.g., a reduced or absent insertion of active aquaporin-2 water channels into the membrane of principal cells in diabetes insipidus.

1	Maintenance of Arterial Circulatory Integrity Sodium is actively pumped out of cells by the Na+/K+-ATPase membrane pump. In consequence, 85–90% of body Na+ is extracellular, and the ECF volume (ECFV) is a function of total-body Na+ content. Arterial perfusion and circulatory integrity are, in turn, determined by renal Na+ retention or excretion, in addition to the modulation of systemic arterial resistance. Within the kidney, Na+ is filtered by the glomeruli and then sequentially reabsorbed by the renal tubules. The Na+ cation is typically reabsorbed with the chloride anion (Cl–), and, thus, chloride homeostasis also affects the ECFV. On a quantitative level, at a glomerular filtration rate (GFR) of 180 L/d and serum Na+ of ~140 mM, the kidney filters some 25,200 mmol/d of Na+. This is equivalent to ~1.5 kg of salt, which would occupy roughly 10 times the extracellular space; 99.6% of filtered Na+-Cl– must be reabsorbed to excrete 100 mM per day. Minute changes in renal Na+-Cl–

1	is equivalent to ~1.5 kg of salt, which would occupy roughly 10 times the extracellular space; 99.6% of filtered Na+-Cl– must be reabsorbed to excrete 100 mM per day. Minute changes in renal Na+-Cl– excretion will thus have significant effects on the ECFV, leading to edema syndromes or hypovolemia.

1	Approximately two-thirds of filtered Na+-Cl– is reabsorbed by the renal proximal tubule, via both paracellular and transcel lular mechanisms. The TALH subsequently reabsorbs another 25–30% of filtered Na+-Cl– via the apical, furose mide-sensitive Na+-K+-2Cl– cotransporter. The adjacent aldosterone-sensitive distal nephron, comprising the dis tal convoluted tubule (DCT), connecting tubule (CNT), and CD, accomplishes the “fine-tuning” of renal Na+-Cl– Vasopressin, also called antidiuretic hormone (ADH)

1	FIguRE 63-3 Vasopressin and the regulation of water permeability in the renal collecting duct. Vasopressin binds to the type 2 vasopressin receptor (V2R) on the basolateral membrane of principal cells, activates adenylyl cyclase (AC), increases intracellular cyclic adenosine monophosphatase (cAMP), and stimulates protein kinase A (PKA) activity. Cytoplasmic vesicles carrying aquaporin-2 (AQP) water channel proteins are inserted into the luminal membrane in response to vasopressin, thereby increasing the water permeability of this membrane. When vasopressin stimulation ends, water channels are retrieved by an endocytic process and water permeability returns to its low basal rate. The AQP3 and AQP4 water channels are expressed on the basolateral membrane and complete the transcellular pathway for water reabsorption. pAQP2, phosphorylated aquaporin-2. (From JM Sands, DG Bichet: Nephrogenic diabetes insipidus. Ann Intern Med 144:186, 2006, with permission.) renin-angiotensin-aldosterone

1	for water reabsorption. pAQP2, phosphorylated aquaporin-2. (From JM Sands, DG Bichet: Nephrogenic diabetes insipidus. Ann Intern Med 144:186, 2006, with permission.) renin-angiotensin-aldosterone axis, and increased circulating AVP) that synergistically increases renal Na+-Cl– reabsorption, vascular resistance, and renal water reabsorption. This occurs in the context of decreased cardiac output, as occurs in hypovolemic states, low-output cardiac failure, decreased oncotic pressure, and/or increased capillary permeability. Alternatively, excessive arterial vasodilation results in relative arterial underfilling, leading to neurohumoral activation in the defense of tissue perfusion. These physiologic responses play important roles in many of the disorders discussed in this chapter. In particular, it is important to appreciate that AVP functions in the defense of circulatory integrity, inducing vasoconstriction, increasing sympathetic nervous system tone, increasing renal retention of

1	it is important to appreciate that AVP functions in the defense of circulatory integrity, inducing vasoconstriction, increasing sympathetic nervous system tone, increasing renal retention of both water and Na+-Cl–, and modulating the arterial baroreceptor reflex. Most of these responses involve activation of systemic V1A AVP receptors, but concomitant activation of V2 receptors in the kidney can result in renal water retention and hyponatremia.

1	HYPOVOLEMIA Etiology True volume depletion, or hypovolemia, generally refers to a state of combined salt and water loss, leading to contraction of the ECFV. The loss of salt and water may be renal or nonrenal in origin. RENAL CAUSES Excessive urinary Na+-Cl– and water loss is a feature of several conditions. A high filtered load of endogenous solutes, such as glucose and urea, can impair tubular reabsorption of Na+-Cl– and water, leading to an osmotic diuresis. Exogenous mannitol, often used to decrease intracerebral pressure, is filtered by glomeruli but not reabsorbed by the proximal tubule, thus causing an osmotic diuresis. Pharmacologic diuretics selectively impair Na+-Cl– reabsorption at specific sites along the nephron, leading to increased urinary Na+-Cl– excretion. Other drugs can induce natriuresis as a side effect. For example, acetazolamide can inhibit proximal tubular Na+-Cl– absorption via its inhibition of carbonic anhydrase; other drugs, such as the

1	H2O AQP-2 AQP-3,4 H2O FIguRE 63-4 Sodium, water, and potassium transport in principal cells (PC) and adjacent β-intercalated cells (B-IC). The absorption of Na+ via the amiloride-sensitive epithelial sodium channel (ENaC) generates a lumen-negative potential difference, which drives K+ excretion through the apical secretory K+ channel ROMK (renal outer medullary K+ channel) and/or the flow-dependent BK channel. Transepithelial Cl– transport occurs in adjacent β-intercalated cells, via apical Cl–-HCO3 and Cl–-OH– exchange (SLC26A4 anion exchanger, also known as pendrin) basolateral CLC chloride channels. Water is absorbed down the osmotic gradient by principal cells, through the apical aquaporin-2 (AQP-2) and basolateral aquaporin-3 and aquaporin-4 (Fig. 63-3).

1	antibiotics trimethoprim and pentamidine, inhibit distal tubular Na+ reabsorption through the amiloride-sensitive ENaC channel, leading to urinary Na+-Cl– loss. Hereditary defects in renal transport proteins are also associated with reduced reabsorption of filtered Na+-Cl– and/ or water. Alternatively, mineralocorticoid deficiency, mineralocorticoid resistance, or inhibition of the mineralocorticoid receptor (MLR) can reduce Na+-Cl– reabsorption by the aldosterone-sensitive distal nephron. Finally, tubulointerstitial injury, as occurs in interstitial nephritis, acute tubular injury, or obstructive uropathy, can reduce distal tubular Na+-Cl– and/or water absorption.

1	Excessive excretion of free water, i.e., water without electrolytes, can also lead to hypovolemia. However, the effect on ECFV is usually less marked, given that two-thirds of the water volume is lost from the ICF. Excessive renal water excretion occurs in the setting of decreased circulating AVP or renal resistance to AVP (central and nephrogenic diabetes insipidus, respectively). EXTRARENAL CAUSES Nonrenal causes of hypovolemia include fluid loss from the gastrointestinal tract, skin, and respiratory system. Accumulations of fluid within specific tissue compartments, typically the interstitium, peritoneum, or gastrointestinal tract, can also cause hypovolemia.

1	Approximately 9 L of fluid enter the gastrointestinal tract daily, 2 L by ingestion and 7 L by secretion; almost 98% of this volume is absorbed, such that daily fecal fluid loss is only 100–200 mL. Impaired gastrointestinal reabsorption or enhanced secretion of fluid can cause hypovolemia. Because gastric secretions have a low pH (high H+ 298 concentration), whereas biliary, pancreatic, and intestinal secretions of hypovolemia, such as acute tubular necrosis; similarly, patients with are alkaline (high HCO3 concentration), vomiting and diarrhea are diabetes insipidus will have an inappropriately dilute urine. often accompanied by metabolic alkalosis and acidosis, respectively. Evaporation of water from the skin and respiratory tract (so-called “insensible losses”) constitutes the major route for loss of solute-free

1	PART 2 Cardinal Manifestations and Presentation of Diseases water, which is typically 500–650 mL/d in healthy adults. This evaporative loss can increase during febrile illness or prolonged heat exposure. Hyperventilation can also increase insensible losses via the respiratory tract, particularly in ventilated patients; the humidity of inspired air is another determining factor. In addition, increased exertion and/or ambient temperature will increase insensible losses via sweat, which is hypotonic to plasma. Profuse sweating without adequate repletion of water and Na+-Cl– can thus lead to both hypovolemia and hypertonicity. Alternatively, replacement of these insensible losses with a surfeit of free water, without adequate replacement of electrolytes, may lead to hypovolemic hyponatremia.

1	Excessive fluid accumulation in interstitial and/or peritoneal spaces can also cause intravascular hypovolemia. Increases in vascular permeability and/or a reduction in oncotic pressure (hypoalbuminemia) alter Starling forces, resulting in excessive “third spacing” of the ECFV. This occurs in sepsis syndrome, burns, pancreatitis, nutritional hypoalbuminemia, and peritonitis. Alternatively, distributive hypovolemia can occur due to accumulation of fluid within specific compartments, for example within the bowel lumen in gastrointestinal obstruction or ileus. Hypovolemia can also occur after extracorporeal hemorrhage or after significant hemorrhage into an expandable space, for example, the retroperitoneum.

1	Diagnostic Evaluation A careful history will usually determine the etiologic cause of hypovolemia. Symptoms of hypovolemia are nonspecific and include fatigue, weakness, thirst, and postural dizziness; more severe symptoms and signs include oliguria, cyanosis, abdominal and chest pain, and confusion or obtundation. Associated electrolyte disorders may cause additional symptoms, for example, muscle weakness in patients with hypokalemia. On examination, diminished skin turgor and dry oral mucous membranes are less than ideal markers of a decreased ECFV in adult patients; more reliable signs of hypovolemia include a decreased jugular venous pressure (JVP), orthostatic tachycardia (an increase of >15–20 beats/min upon standing), and orthostatic hypotension (a >10–20 mmHg drop in blood pressure on standing). More severe fluid loss leads to hypovolemic shock, with hypotension, tachycardia, peripheral vasoconstriction, and peripheral hypoperfusion; these patients may exhibit peripheral

1	on standing). More severe fluid loss leads to hypovolemic shock, with hypotension, tachycardia, peripheral vasoconstriction, and peripheral hypoperfusion; these patients may exhibit peripheral cyanosis, cold extremities, oliguria, and altered mental status.

1	Routine chemistries may reveal an increase in blood urea nitrogen (BUN) and creatinine, reflective of a decrease in GFR. Creatinine is the more dependable measure of GFR, because BUN levels may be influenced by an increase in tubular reabsorption (“prerenal azotemia”), an increase in urea generation in catabolic states, hyperalimentation, or gastrointestinal bleeding, and/or a decreased urea generation in decreased protein intake. In hypovolemic shock, liver function tests and cardiac biomarkers may show evidence of hepatic and cardiac ischemia, respectively. Routine chemistries and/or blood gases may reveal evidence of acid-base disorders. For example, bicarbonate loss due to diarrheal illness is a very common cause of metabolic acidosis; alternatively, patients with severe hypovolemic shock may develop lactic acidosis with an elevated anion gap.

1	The neurohumoral response to hypovolemia stimulates an increase in renal tubular Na+ and water reabsorption. Therefore, the urine Na+ concentration is typically <20 mM in nonrenal causes of hypovolemia, with a urine osmolality of >450 mOsm/kg. The reduction in both GFR and distal tubular Na+ delivery may cause a defect in renal potassium excretion, with an increase in plasma K+ concentration. Of note, patients with hypovolemia and a hypochloremic alkalosis due to vomiting, diarrhea, or diuretics will typically have a urine Na+ concentration >20 mM and urine pH of >7.0, due to the increase in filtered HCO3–; the urine Cl– concentration in this setting is a more accurate indicator of volume status, with a level <25 mM suggestive of hypovolemia. The urine Na+ concentration is often >20 mM in patients with renal causes

1	The therapeutic goals in hypovolemia are to restore normovolemia and replace ongoing fluid losses. Mild hypovolemia can usually be treated with oral hydration and resumption of a normal maintenance diet. More severe hypovolemia requires intravenous hydration, tailoring the choice of solution to the underlying pathophysiology. Isotonic, “normal” saline (0.9% NaCl, 154 mM Na+) is the most appropriate resuscitation fluid for normonatremic or hyponatremic patients with severe hypovolemia; colloid solutions such as intravenous albumin are not demonstrably superior for this purpose. Hypernatremic patients should receive a hypotonic solution, 5% dextrose if there has only been water loss (as in diabetes insipidus), or hypotonic saline (1/2 or 1/4 normal saline) if there has been water and Na+-Cl– loss. Patients with bicarbonate loss and metabolic acidosis, as occur frequently in diarrhea, should receive intravenous bicar bonate, either an isotonic solution (150 meq of Na+-HCO3 in 5%

1	Na+-Cl– loss. Patients with bicarbonate loss and metabolic acidosis, as occur frequently in diarrhea, should receive intravenous bicar bonate, either an isotonic solution (150 meq of Na+-HCO3 in 5% dextrose) or a more hypotonic bicarbonate solution in dextrose or dilute saline. Patients with severe hemorrhage or anemia should receive red cell transfusions, without increasing the hematocrit beyond 35%.

1	Disorders of serum Na+ concentration are caused by abnormalities in water homeostasis, leading to changes in the relative ratio of Na+ to body water. Water intake and circulating AVP constitute the two key effectors in the defense of serum osmolality; defects in one or both of these two defense mechanisms cause most cases of hyponatremia and hypernatremia. In contrast, abnormalities in sodium homeostasis per se lead to a deficit or surplus of whole-body Na+-Cl– content, a key determinant of the ECFV and circulatory integrity. Notably, volume status also modulates the release of AVP by the posterior pituitary, such that hypovolemia is associated with higher circulating levels of the hormone at each level of serum osmolality. Similarly, in “hypervolemic” causes of arterial underfilling, e.g., heart failure and cirrhosis, the associated neurohumoral activation is associated with an increase in circulating AVP, leading to water retention and hyponatremia. Therefore, a key concept in

1	e.g., heart failure and cirrhosis, the associated neurohumoral activation is associated with an increase in circulating AVP, leading to water retention and hyponatremia. Therefore, a key concept in sodium disorders is that the absolute plasma Na+ concentration tells one nothing about the volume status of a given patient, which furthermore must be taken into account in the diagnostic and therapeutic approach.

1	Hyponatremia, which is defined as a plasma Na+ concentration <135 mM, is a very common disorder, occurring in up to 22% of hospitalized patients. This disorder is almost always the result of an increase in circulating AVP and/or increased renal sensitivity to AVP, combined with an intake of free water; a notable exception is hyponatremia due to low solute intake (see below). The underlying pathophysiology for the exaggerated or “inappropriate” AVP response differs in patients with hyponatremia as a function of their ECFV. Hyponatremia is thus subdivided diagnostically into three groups, depending on clinical history and volume status, i.e., “hypovolemic,” “euvolemic,” and “hypervolemic” (Fig. 63-5).

1	Hypovolemic Hyponatremia Hypovolemia causes a marked neurohumoral activation, increasing circulating levels of AVP. The increase in circulating AVP helps preserve blood pressure via vascular and baroreceptor V1A receptors and increases water reabsorption via renal receptors; activation of V receptors can lead to hyponatremia in the setting of increased free water intake. Nonrenal causes of hypovolemic hyponatremia include GI loss (e.g., vomiting, diarrhea, tube drainage) and insensible loss (sweating, burns) of Na+-Cl– and water, in the absence of adequate oral replacement; urine Na+ concentration is typically <20 mM. Notably, these patients may be clinically classified as euvolemic, with only the reduced urinary Na+ concentration to FIguRE 63-5 The diagnostic approach to hyponatremia. (From S Kumar, T Berl: Diseases of water metabolism, in Atlas of Diseases of the Kidney,

1	FIguRE 63-5 The diagnostic approach to hyponatremia. (From S Kumar, T Berl: Diseases of water metabolism, in Atlas of Diseases of the Kidney, RW Schrier [ed]. Philadelphia, Current Medicine, Inc, 1999; with permission.) indicate the cause of their hyponatremia. Indeed, a urine Na+ concentration <20 mM, in the absence of a cause of hypervolemic hyponatremia, predicts a rapid increase in plasma Na+ concentration in response to intravenous normal saline; saline therapy thus induces a water diuresis in this setting, as circulating AVP levels plummet.

1	The renal causes of hypovolemic hyponatremia share an inappropriate loss of Na+-Cl– in the urine, leading to volume depletion and an increase in circulating AVP; urine Na+ concentration is typically >20 mM (Fig. 63-5). A deficiency in circulating aldosterone and/or its renal effects can lead to hyponatremia in primary adrenal insufficiency and other causes of hypoaldosteronism; hyperkalemia and hyponatremia in a hypotensive and/or hypovolemic patient with high urine Na+ concentration (much greater than 20 mM) should strongly suggest this diagnosis. Salt-losing nephropathies may lead to hyponatremia when sodium intake is reduced, due to impaired renal tubular function; typical causes include reflux nephropathy, interstitial nephropathies, postobstructive uropathy, medullary cystic disease, and the recovery phase of acute tubular necrosis. Thiazide diuretics cause hyponatremia via a number of mechanisms, including polydipsia and diuretic-induced volume depletion. Notably, thiazides do

1	and the recovery phase of acute tubular necrosis. Thiazide diuretics cause hyponatremia via a number of mechanisms, including polydipsia and diuretic-induced volume depletion. Notably, thiazides do not inhibit the renal concentrating mechanism, such that circulating AVP retains a full effect on renal water retention. In contrast, loop diuretics, which are less frequently associated with hyponatremia, inhibit Na+-Cl– and K+ absorption by the TALH, blunting the countercurrent mechanism and reducing the ability to concentrate the urine. Increased excretion of an osmotically active nonreabsorbable or poorly reabsorbable solute can also lead to volume depletion and hyponatremia; important causes include glycosuria, ketonuria (e.g., in starvation or in diabetic or alcoholic ketoacidosis), and bicarbonaturia (e.g., in renal tubular acidosis or metabolic alkalosis, where the associated bicarbonaturia leads to loss of Na+).

1	Finally, the syndrome of “cerebral salt wasting” is a rare cause of hypovolemic hyponatremia, encompassing hyponatremia with clinical hypovolemia and inappropriate natriuresis in association with intracranial disease; associated disorders include subarachnoid hemorrhage, traumatic brain injury, craniotomy, encephalitis, and meningitis. Distinction from the more common syndrome of inappropriate antidiuresis is critical because cerebral salt wasting will typically respond to aggressive Na+-Cl– repletion.

1	Hypervolemic Hyponatremia Patients with hypervolemic hyponatremia develop an increase in total-body Na+-Cl– that is accompanied by a proportionately greater increase in total-body water, leading to a reduced plasma Na+ concentration. As in hypovolemic hyponatremia, the causative disorders can be separated by the effect on urine Na+ concentration, with acute or chronic renal failure uniquely associated with an increase in urine Na+ concentration (Fig. 63-5). The pathophysiology of hyponatremia in the sodium-avid edematous disorders (congestive heart failure [CHF], cirrhosis, and nephrotic syndrome) is similar to that in hypovolemic hyponatremia, except that arterial filling and circulatory integrity is decreased due to the specific etiologic factors (e.g., cardiac dysfunction in CHF, peripheral vasodilation in cirrhosis). Urine Na+ concentration is typically very low, i.e., <10 mM, even after hydration with normal saline; this Na+-avid state may be obscured by diuretic therapy. The

1	vasodilation in cirrhosis). Urine Na+ concentration is typically very low, i.e., <10 mM, even after hydration with normal saline; this Na+-avid state may be obscured by diuretic therapy. The degree of hyponatremia provides an indirect index of the associated neurohumoral activation and is an important prognostic indicator in hypervolemic hyponatremia.

1	Euvolemic Hyponatremia Euvolemic hyponatremia can occur in moderate to severe hypothyroidism, with correction after achieving a euthyroid state. Severe hyponatremia can also be a consequence of secondary adrenal insufficiency due to pituitary disease; whereas the deficit in circulating aldosterone in primary adrenal insufficiency causes hypovolemic hyponatremia, the predominant glucocorticoid deficiency in secondary adrenal failure is associated with euvolemic hyponatremia. Glucocorticoids exert a negative feedback on AVP release by the posterior pituitary such that hydrocortisone replacement in these patients will rapidly normalize the AVP response to osmolality, reducing circulating AVP.

1	The syndrome of inappropriate antidiuresis (SIAD) is the most frequent cause of euvolemic hyponatremia (Table 63-1). The generation of hyponatremia in SIAD requires an intake of free water, with persistent intake at serum osmolalities that are lower than the usual threshold for thirst; as one would expect, the osmotic threshold and osmotic response curves for the sensation of thirst are shifted downward in patients with SIAD. Four distinct patterns of AVP secretion have been recognized in patients with SIAD, independent for the most part of the underlying cause. Unregulated, erratic AVP secretion is seen in about a third of patients, with no obvious correlation between serum osmolality and circulating AVP levels. Other patients fail to suppress AVP secretion at lower serum osmolalities, with a normal response curve to hyperosmolar conditions; others have a “reset osmostat,” with a lower threshold osmolality and a left-shifted osmotic response curve. Finally, the fourth subset of

1	with a normal response curve to hyperosmolar conditions; others have a “reset osmostat,” with a lower threshold osmolality and a left-shifted osmotic response curve. Finally, the fourth subset of patients have essentially no detectable

1	Disorders of the Central Malignant Diseases Pulmonary Disorders Nervous System Drugs Other Causes PART 2 Cardinal Manifestations and Presentation of Diseases ated with positive-pressure breathing porphyria Drugs that stimulate release of AVP or enhance its action Chlorpropamide SSRIs Tricyclic antidepressants Clofibrate Carbamazepine Vincristine Nicotine Narcotics Antipsychotic drugs Ifosfamide MDMA (“ecstasy”) AVP analogues Desmopressin Oxytocin Vasopressin Hereditary (gain-of-function mutations in the vasopressin V2 receptor) Idiopathic Transient Endurance exercise General anesthesia Nausea Pain Stress Abbreviations: AVP, vasopressin; MDMA; 3,4-methylenedioxymethamphetamine; SSRI, selective serotonin reuptake inhibitor. Source: From DH Ellison, T Berl: Syndrome of inappropriate antidiuresis. N Engl J Med 356:2064, 2007.

1	circulating AVP, suggesting either a gain in function in renal water reabsorption or a circulating antidiuretic substance that is distinct from AVP. Gain-in-function mutations of a single specific residue in the V2 AVP receptor have been described in some of these patients, leading to constitutive activation of the receptor in the absence of AVP and “nephrogenic” SIAD.

1	Strictly speaking, patients with SIAD are not euvolemic but are sub-clinically volume-expanded, due to AVP-induced water and Na+-Cl– retention; “AVP escape” mechanisms invoked by sustained increases in AVP serve to limit distal renal tubular transport, preserving a modestly hypervolemic steady state. Serum uric acid is often low (<4 mg/dL) in patients with SIAD, consistent with suppressed proximal tubular transport in the setting of increased distal tubular Na+-Cl– and water transport; in contrast, patients with hypovolemic hyponatremia will often be hyperuricemic, due to a shared activation of proximal tubular Na+-Cl– and urate transport.

1	Common causes of SIAD include pulmonary disease (e.g., pneumonia, tuberculosis, pleural effusion) and central nervous system (CNS) diseases (e.g., tumor, subarachnoid hemorrhage, meningitis). SIAD also occurs with malignancies, most commonly with small-cell lung carcinoma (75% of malignancy-associated SIAD); ~10% of patients with this tumor will have a plasma Na+ concentration of <130 mM at presentation. SIAD is also a frequent complication of certain drugs, most commonly the selective serotonin reuptake inhibitors (SSRIs). Other drugs can potentiate the renal effect of AVP, without exerting direct effects on circulating AVP levels (Table 63-1).

1	Low Solute Intake and Hyponatremia Hyponatremia can occasionally occur in patients with a very low intake of dietary solutes. Classically, this occurs in alcoholics whose sole nutrient is beer, hence the diagnostic label of beer potomania; beer is very low in protein and salt content, containing only 1–2 mM of Na+. The syndrome has also been described in nonalcoholic patients with highly restricted solute intake due to nutrient-restricted diets, e.g., extreme vegetarian diets. Patients with hyponatremia due to low solute intake typically present with a very low urine osmolality (<100–200 mOsm/kg) with a urine Na+ concentration that is <10–20 mM. The fundamental abnormality is the inadequate dietary intake of solutes; the reduced urinary solute excretion limits water excretion such that hyponatremia ensues after relatively modest polydipsia. AVP levels have not been reported in patients with beer potomania but are expected to be suppressed or rapidly suppressible with saline hydration;

1	ensues after relatively modest polydipsia. AVP levels have not been reported in patients with beer potomania but are expected to be suppressed or rapidly suppressible with saline hydration; this fits with the overly rapid correction in plasma Na+ concentration that can be seen with saline hydration. Resumption of a normal diet and/or saline hydration will also correct the causative deficit in urinary solute excretion, such that patients with beer potomania typically correct their plasma Na+ concentration promptly after admission to the hospital.

1	Clinical Features of Hyponatremia Hyponatremia induces generalized cellular swelling, a consequence of water movement down the osmotic gradient from the hypotonic ECF to the ICF. The symptoms of hyponatremia are primarily neurologic, reflecting the development of cerebral edema within a rigid skull. The initial CNS response to acute hyponatremia is an increase in interstitial pressure, leading to shunting of ECF and solutes from the interstitial space into the cerebrospinal fluid and then on into the systemic circulation. This is accompanied by an efflux of the major intracellular ions, Na+, K+, and Cl–, from brain cells. Acute hyponatremic encephalopathy ensues when these volume regulatory mechanisms are overwhelmed by a rapid decrease in tonicity, resulting in acute cerebral edema. Early symptoms can include nausea, headache, and vomiting. However, severe complications can rapidly evolve, including seizure activity, brainstem herniation, coma, and death. A key complication of acute

1	symptoms can include nausea, headache, and vomiting. However, severe complications can rapidly evolve, including seizure activity, brainstem herniation, coma, and death. A key complication of acute hyponatremia is normocapneic or hypercapneic respiratory failure; the associated hypoxia may amplify the neurologic injury. Normocapneic respiratory failure in this setting is typically due to noncardiogenic, “neurogenic” pulmonary edema, with a normal pulmonary capillary wedge pressure.

1	Acute symptomatic hyponatremia is a medical emergency, occurring in a number of specific settings (Table 63-2). Women, particularly before menopause, are much more likely than men to develop encephalopathy and severe neurologic sequelae. Acute hyponatremia often has an iatrogenic component, e.g., when hypotonic intravenous fluids are given to postoperative patients with an increase in circulating AVP. Exercise-associated hyponatremia, an important clinical issue at CAuSES of ACuTE HyPonATREMiA Postoperative: premenopausal women Hypotonic fluids with cause of ↑ vasopressin Glycine irrigation: TURP, uterine surgery Recent institution of thiazides MDMA (“ecstasy,”“Molly”) ingestion Multifactorial, e.g., thiazide and polydipsia Abbreviations: MDMA, 3,4-methylenedioxymethamphetamine; TURP, transurethral resection of the prostate.

1	MDMA (“ecstasy,”“Molly”) ingestion Multifactorial, e.g., thiazide and polydipsia Abbreviations: MDMA, 3,4-methylenedioxymethamphetamine; TURP, transurethral resection of the prostate. marathons and other endurance events, has similarly been linked to both a “nonosmotic” increase in circulating AVP and excessive free water intake. The recreational drugs Molly and ecstasy, which share an active ingredient (MDMA, 3,4-methylenedioxymethamphetamine), cause a rapid and potent induction of both thirst and AVP, leading to severe acute hyponatremia.

1	Persistent, chronic hyponatremia results in an efflux of organic osmolytes (creatine, betaine, glutamate, myoinositol, and taurine) from brain cells; this response reduces intracellular osmolality and the osmotic gradient favoring water entry. This reduction in intracellular osmolytes is largely complete within 48 h, the time period that clinically defines chronic hyponatremia; this temporal definition has considerable relevance for the treatment of hyponatremia (see below). The cellular response to chronic hyponatremia does not fully protect patients from symptoms, which can include vomiting, nausea, confusion, and seizures, usually at plasma Na+ concentration <125 mM. Even patients who are judged “asymptomatic” can manifest subtle gait and cognitive defects that reverse with correction of hyponatremia; notably, chronic “asymptomatic” hyponatremia increases the risk of falls. Chronic hyponatremia also increases the risk of bony fractures owing to the associated neurologic dysfunction

1	hyponatremia; notably, chronic “asymptomatic” hyponatremia increases the risk of falls. Chronic hyponatremia also increases the risk of bony fractures owing to the associated neurologic dysfunction and to a hyponatremia-associated reduction in bone density. Therefore, every attempt should be made to correct safely the plasma Na+ concentration in patients with chronic hyponatremia, even in the absence of overt symptoms (see the section on treatment of hyponatremia below).

1	The management of chronic hyponatremia is complicated significantly by the asymmetry of the cellular response to correction of plasma Na+ concentration. Specifically, the reaccumulation of organic osmolytes by brain cells is attenuated and delayed as osmolality increases after correction of hyponatremia, sometimes resulting in degenerative loss of oligodendrocytes and an osmotic demyelination syndrome (ODS). Overly rapid correction of hyponatremia (>8–10 mM in 24 h or 18 mM in 48 h) is also associated with a disruption in integrity of the blood-brain barrier, allowing the entry of immune mediators that may contribute to demyelination. The lesions of ODS classically affect the pons, a structure wherein the delay in the reaccumulation of osmotic osmolytes is particularly pronounced; clinically, patients with central pontine myelinolysis can present 1 or more days after overcorrection of hyponatremia with paraparesis or quadriparesis, dysphagia, dysarthria, diplopia, a “locked-in

1	clinically, patients with central pontine myelinolysis can present 1 or more days after overcorrection of hyponatremia with paraparesis or quadriparesis, dysphagia, dysarthria, diplopia, a “locked-in syndrome,” and/or loss of consciousness. Other regions of the brain can also be involved in ODS, most commonly in association with lesions of the pons but occasionally in isolation; in order of frequency, the lesions of extrapontine myelinolysis can occur in the cerebellum, lateral geniculate body, thalamus, putamen, and cerebral cortex or subcortex. Clinical presentation of ODS can, therefore, vary as a function of the extent and localization of extrapontine myelinolysis, with the reported development of ataxia, mutism, parkinsonism, dystonia, and catatonia. Relowering of plasma Na+ concentration after overly rapid correction can prevent or attenuate ODS (see the section on treatment of hyponatremia below). However, even appropriately slow correction can be associated with ODS,

1	concentration after overly rapid correction can prevent or attenuate ODS (see the section on treatment of hyponatremia below). However, even appropriately slow correction can be associated with ODS, particularly in patients with additional risk factors; these include alcoholism, malnutrition, hypokalemia, and liver 301 transplantation. Diagnostic Evaluation of Hyponatremia Clinical assessment of hyponatremic patients should focus on the underlying cause; a detailed drug history is particularly crucial (Table 63-1). A careful clinical assessment of volume status is obligatory for the classical diagnostic approach to hyponatremia (Fig. 63-5). Hyponatremia is frequently multifactorial, particularly when severe; clinical evaluation should consider all the possible causes for excessive circulating AVP, including volume status, drugs, and the presence of nausea and/or pain. Radiologic imaging may also be appropriate to assess whether patients have a pulmonary or CNS cause for hyponatremia.

1	AVP, including volume status, drugs, and the presence of nausea and/or pain. Radiologic imaging may also be appropriate to assess whether patients have a pulmonary or CNS cause for hyponatremia. A screening chest x-ray may fail to detect a small-cell carcinoma of the lung; computed tomography (CT) scanning of the thorax should be considered in patients at high risk for this tumor (e.g., patients with a smoking history).

1	Laboratory investigation should include a measurement of serum osmolality to exclude pseudohyponatremia, which is defined as the coexistence of hyponatremia with a normal or increased plasma tonicity. Most clinical laboratories measure plasma Na+ concentration by testing diluted samples with automated ion-sensitive electrodes, correcting for this dilution by assuming that plasma is 93% water. This correction factor can be inaccurate in patients with pseudohyponatremia due to extreme hyperlipidemia and/or hyperproteinemia, in whom serum lipid or protein makes up a greater percentage of plasma volume. The measured osmolality should also be converted to the effective osmolality (tonicity) by subtracting the measured concentration of urea (divided by 2.8, if in mg/dL); patients with hyponatremia have an effective osmolality of <275 mOsm/kg.

1	Elevated BUN and creatinine in routine chemistries can also indicate renal dysfunction as a potential cause of hyponatremia, whereas hyperkalemia may suggest adrenal insufficiency or hypoaldosteronism. Serum glucose should also be measured; plasma Na+ concentration falls by ~1.6–2.4 mM for every 100-mg/dL increase in glucose, due to glucose-induced water efflux from cells; this “true” hyponatremia resolves after correction of hyperglycemia. Measurement of serum uric acid should also be performed; whereas patients with SIAD-type physiology will typically be hypouricemic (serum uric acid <4 mg/dL), volume-depleted patients will often be hyperuricemic. In the appropriate clinical setting, thyroid, adrenal, and pituitary function should also be tested; hypothyroidism and secondary adrenal failure due to pituitary insufficiency are important causes of euvolemic hyponatremia, whereas primary adrenal failure causes hypovolemic hyponatremia. A cosyntropin stimulation test is necessary to

1	failure due to pituitary insufficiency are important causes of euvolemic hyponatremia, whereas primary adrenal failure causes hypovolemic hyponatremia. A cosyntropin stimulation test is necessary to assess for primary adrenal insufficiency.

1	Urine electrolytes and osmolality are crucial tests in the initial evaluation of hyponatremia. A urine Na+ concentration <20–30 mM is consistent with hypovolemic hyponatremia, in the clinical absence of a hypervolemic, Na+-avid syndrome such as CHF (Fig. 63-5). In contrast, patients with SIAD will typically excrete urine with an Na+ concentration that is >30 mM. However, there can be substantial overlap in urine Na+ concentration values in patients with SIAD and hypovolemic hyponatremia, particularly in the elderly; the ultimate “gold standard” for the diagnosis of hypovolemic hyponatremia is the demonstration that plasma Na+ concentration corrects after hydration with normal saline. Patients with thiazide-associated hyponatremia may also present with higher than expected urine Na+ concentration and other findings suggestive of SIAD; one should defer making a diagnosis of SIAD in these patients until 1–2 weeks after discontinuing the thiazide. A urine osmolality <100 mOsm/kg is

1	concentration and other findings suggestive of SIAD; one should defer making a diagnosis of SIAD in these patients until 1–2 weeks after discontinuing the thiazide. A urine osmolality <100 mOsm/kg is suggestive of polydipsia; urine osmolality >400 mOsm/kg indicates that AVP excess is playing a more dominant role, whereas intermediate values are more consistent with multifactorial pathophysiology (e.g., AVP excess with a significant component of polydipsia). Patients with hyponatremia due to decreased solute intake (beer potomania) typically have urine Na+ concentration <20 mM and urine osmolality in the range of <100 to the low 200s. Finally, the measurement of urine K+ concentration is required to calculate the urine-to-plasma electrolyte ratio, which is useful to predict the response to fluid restriction (see the section on treatment of hyponatremia below).

1	Three major considerations guide the therapy of hyponatremia. First, the presence and/or severity of symptoms determine the urgency and goals of therapy. Patients with acute hyponatremia (Table 63-2) present with symptoms that can range from headache, nausea, and/or vomiting, to seizures, obtundation, and central herniation; patients with chronic hyponatremia, present for >48 h, are less likely to have severe symptoms. Second, patients with chronic hyponatremia are at risk for ODS if plasma Na+ concentration is corrected by >8–10 mM within the first 24 h and/or by >18 mM within the first 48 h. Third, the response to interventions such as hypertonic saline, isotonic saline, or AVP antagonists can be highly unpredictable, such that frequent monitoring of plasma Na+ concentration during corrective therapy is imperative.

1	Once the urgency in correcting the plasma Na+ concentration has been established and appropriate therapy instituted, the focus should be on treatment or withdrawal of the underlying cause. Patients with euvolemic hyponatremia due to SIAD, hypothyroidism, or secondary adrenal failure will respond to successful treatment of the underlying cause, with an increase in plasma Na+ concentration. However, not all causes of SIAD are immediately reversible, necessitating pharmacologic therapy to increase the plasma Na+ concentration (see below). Hypovolemic hyponatremia will respond to intravenous hydration with isotonic normal saline, with a rapid reduction in circulating AVP and a brisk water diuresis; it may be necessary to reduce the rate of correction if the history suggests that hyponatremia has been chronic, i.e., present for more than 48 h (see below). Hypervolemic hyponatremia due to CHF will often respond to improved therapy of the underlying cardiomyopathy, e.g., following the

1	has been chronic, i.e., present for more than 48 h (see below). Hypervolemic hyponatremia due to CHF will often respond to improved therapy of the underlying cardiomyopathy, e.g., following the institution or intensification of angiotensin-converting enzyme (ACE) inhibition. Finally, patients with hyponatremia due to beer potomania and low solute intake will respond very rapidly to intravenous saline and the resumption of a normal diet. Notably, patients with beer potomania have a very high risk of developing ODS, due to the associated hypokalemia, alcoholism, malnutrition, and high risk of overcorrecting the plasma Na+ concentration.

1	Water deprivation has long been a cornerstone of the therapy of chronic hyponatremia. However, patients who are excreting minimal electrolyte-free water will require aggressive fluid restriction; this can be very difficult for patients with SIAD to tolerate, given that their thirst is also inappropriately stimulated. The urine-to-plasma electrolyte ratio (urinary [Na+] + [K+]/plasma [Na+]) can be exploited as a quick indicator of electrolyte-free water excretion (Table 63-3); patients with a ratio of >1 should be more aggressively restricted PART 2 Cardinal Manifestations and Presentation of Diseases 1. Estimate total-body water (TBW): 50% of body weight in women and 60% in men 2. Calculate free-water deficit: [(Na+ − 140)/140] × TBW 3.

1	Estimate total-body water (TBW): 50% of body weight in women and 60% in men 2. Calculate free-water deficit: [(Na+ − 140)/140] × TBW 3. Administer deficit over 48–72 h, without decrease in plasma Na+ concentration by >10 mM/24 h 4. Calculate free-water clearance, CeH2O: where V is urinary volume, U is urinary [Na+], U is urinary [K+], and P is 5. ~10 mL/kg per day: less if ventilated, more if febrile 6. Add components to determine water deficit and ongoing water loss; correct the water deficit over 48–72 h and replace daily water loss. Avoid correction of plasma [Na+] by >10 mM/d.

1	(<500 mL/d), those with a ratio of ~1 should be restricted to 500– 700 mL/d, and those with a ratio <1 should be restricted to <1 L/d. In hypokalemic patients, potassium replacement will serve to increase plasma Na+ concentration, given that the plasma Na+ concentration is a functional of both exchangeable Na+ and exchangeable K+ divided by total-body water; a corollary is that aggressive repletion of K+ has the potential to overcorrect the plasma Na+ concentration even in the absence of hypertonic saline. Plasma Na+ concentration will also tend to respond to an increase in dietary solute intake, which increases the ability to excrete free water; however, the use of oral urea and/or salt tablets for this purpose is generally not practical or well tolerated.

1	Patients in whom therapy with fluid restriction, potassium replacement, and/or increased solute intake fails may merit pharmacologic therapy to increase their plasma Na+ concentration. Many patients with SIAD respond to combined therapy with oral furosemide, 20 mg twice a day (higher doses may be necessary in renal insufficiency), and oral salt tablets; furosemide serves to inhibit the renal countercurrent mechanism and blunt urinary concentrating ability, whereas the salt tablets counteract diuretic-associated natriuresis. Demeclocycline is a potent inhibitor of principal cells and can be used in patients whose Na levels do not increase in response to furosemide and salt tablets. However, this agent can be associated with a reduction in GFR, due to excessive natriuresis and/or direct renal toxicity; it should be avoided in cirrhotic patients in particular, who are at higher risk of nephrotoxicity due to drug accumulation.

1	AVP antagonists (vaptans) are highly effective in SIAD and in hypervolemic hyponatremia due to heart failure or cirrhosis, reliably increasing plasma Na+ concentration due to their “aquaretic” effects (augmentation of free water clearance). Most of these agents specifically antagonize the V2 AVP receptor; tolvaptan is currently the only oral V2 antagonist to be approved by the U.S. Food and Drug Administration. Conivaptan, the only available intravenous vaptan, is a mixed V1A/V2 antagonist, with a modest risk of hypotension due to V1A receptor inhibition. Therapy with vaptans must be initiated in a hospital setting, with a liberalization of fluid restriction (>2 L/d) and close monitoring of plasma Na+ concentration. Although approved for the management of all but hypovolemic hyponatremia and acute hyponatremia, the clinical indications for these agents are not completely clear. Oral tolvaptan is perhaps most appropriate for the management of significant and persistent SIAD (e.g., in

1	and acute hyponatremia, the clinical indications for these agents are not completely clear. Oral tolvaptan is perhaps most appropriate for the management of significant and persistent SIAD (e.g., in small-cell lung carcinoma) that has not responded to water restriction and/or oral furosemide and salt tablets. Abnormalities in liver function tests have been reported with chronic tolvaptan therapy; hence, the use of this agent should be restricted to <1–2 months.

1	Treatment of acute symptomatic hyponatremia should include hypertonic 3% saline (513 mM) to acutely increase plasma Na+ concentration by 1–2 mM/h to a total of 4–6 mM; this modest increase is typically sufficient to alleviate severe acute symptoms, after which corrective guidelines for chronic hyponatremia are appropriate (see below). A number of equations have been developed to estimate the required rate of hypertonic saline, which has an Na+-Cl– concentration of 513 mM. The traditional approach is to calculate an Na+ deficit, where the Na+ deficit = 0.6 × body weight × (target plasma Na+ concentration – starting plasma Na+ concentration), followed by a calculation of the required rate. Regardless of the method used to determine the rate of administration, the increase in plasma Na+ concentration can be highly unpredictable during treatment with hypertonic saline, due to rapid changes in the underlying physiology; plasma Na+ concentration should be monitored every 2–4 h during

1	concentration can be highly unpredictable during treatment with hypertonic saline, due to rapid changes in the underlying physiology; plasma Na+ concentration should be monitored every 2–4 h during treatment, with appropriate changes in therapy based on the observed rate of change. The administration of supplemental oxygen and ventilatory support is also critical in acute hyponatremia, in the event that patients develop acute pulmonary edema or hypercapneic respiratory failure. Intravenous loop diuretics will help treat acute pulmonary edema and will also increase free water excretion, by interfering with the renal countercurrent multiplication system. AVP antagonists do not have an approved role in the management of acute hyponatremia.

1	The rate of correction should be comparatively slow in chronic hyponatremia (<8–10 mM in the first 24 h and <18 mM in the first 48 h), so as to avoid ODS; lower target rates are appropriate in patients at particular risk for ODS, such as alcoholics or hypokalemic patients. Overcorrection of the plasma Na+ concentration can occur when AVP levels rapidly normalize, for example following the treatment of patients with chronic hypovolemic hyponatremia with intravenous saline or following glucocorticoid replacement of patients with hypopituitarism and secondary adrenal failure. Approximately 10% of patients treated with vaptans will overcorrect; the risk is increased if water intake is not liberalized. In the event that the plasma Na+ concentration overcorrects following therapy, be it with hypertonic saline, isotonic saline, or a vaptan, hyponatremia can be safely reinduced or stabilized by the administration of the AVP agonist desmopressin acetate (DDAVP) and/or the administration of

1	hypertonic saline, isotonic saline, or a vaptan, hyponatremia can be safely reinduced or stabilized by the administration of the AVP agonist desmopressin acetate (DDAVP) and/or the administration of free water, typically intravenous D5W; the goal is to prevent or reverse the development of ODS. Alternatively, the treatment of patients with marked hyponatremia can be initiated with the twice-daily administration of DDAVP to maintain constant AVP bioactivity, combined with the administration of hypertonic saline to slowly correct the serum sodium in a more controlled fashion, thus reducing upfront the risk of overcorrection.

1	HYPERNATREMIA Etiology Hypernatremia is defined as an increase in the plasma Na+ concentration to >145 mM. Considerably less common than hyponatremia, hypernatremia is nonetheless associated with mortality rates of as high as 40–60%, mostly due to the severity of the associated underlying disease processes. Hypernatremia is usually the result of a combined water and electrolyte deficit, with losses of H2O in excess of Na+. Less frequently, the ingestion or iatrogenic administration of excess Na+ can be causative, for example after IV administration of excessive hypertonic Na+-Cl– or Na+-HCO3 (Fig. 63-6).

1	Elderly individuals with reduced thirst and/or diminished access to fluids are at the highest risk of developing hypernatremia. Patients with hypernatremia may rarely have a central defect in hypothalamic osmoreceptor function, with a mixture of both decreased thirst and reduced AVP secretion. Causes of this adipsic diabetes insipidus include primary or metastatic tumor, occlusion or ligation of the anterior communicating artery, trauma, hydrocephalus, and inflammation. FIguRE 63-6 The diagnostic approach to hypernatremia. ECF, extracellular fluid.

1	FIguRE 63-6 The diagnostic approach to hypernatremia. ECF, extracellular fluid. Hypernatremia can develop following the loss of water via both 303 renal and nonrenal routes. Insensible losses of water may increase in the setting of fever, exercise, heat exposure, severe burns, or mechanical ventilation. Diarrhea is, in turn, the most common gastrointestinal cause of hypernatremia. Notably, osmotic diarrhea and viral gastroenteritides typically generate stools with Na+ and K+ <100 mM, thus leading to water loss and hypernatremia; in contrast, secretory diarrhea typically results in isotonic stool and thus hypovolemia with or without hypovolemic hyponatremia.

1	Common causes of renal water loss include osmotic diuresis secondary to hyperglycemia, excess urea, postobstructive diuresis, or mannitol; these disorders share an increase in urinary solute excretion and urinary osmolality (see “Diagnostic Approach,” below). Hypernatremia due to a water diuresis occurs in central or nephrogenic diabetes insipidus (DI).

1	Nephrogenic DI (NDI) is characterized by renal resistance to AVP, which can be partial or complete (see “Diagnostic Approach,” below). Genetic causes include loss-of-function mutations in the X-linked V2 receptor; mutations in the AVP-responsive aquaporin-2 water channel can cause autosomal recessive and autosomal dominant NDI, whereas recessive deficiency of the aquaporin-1 water channel causes a more modest concentrating defect (Fig. 63-2). Hypercalcemia can also cause polyuria and NDI; calcium signals directly through the calcium-sensing receptor to downregulate Na+, K+, and Cl– transport by the TALH and water transport in principal cells, thus reducing renal concentrating ability in hypercalcemia. Another common acquired cause of NDI is hypokalemia, which inhibits the renal response to AVP and downregulates aquaporin-2 expression. Several drugs can cause acquired NDI, in particular lithium, ifosfamide, and several antiviral agents. Lithium causes NDI by multiple mechanisms,

1	to AVP and downregulates aquaporin-2 expression. Several drugs can cause acquired NDI, in particular lithium, ifosfamide, and several antiviral agents. Lithium causes NDI by multiple mechanisms, including direct inhibition of renal glycogen synthase kinase-3 (GSK3), a kinase thought to be the pharmacologic target of lithium in bipolar disease; GSK3 is required for the response of principal cells to AVP. The entry of lithium through the amiloride-sensitive Na+ channel ENaC (Fig. 63-4) is required for the effect of the drug on principal cells, such that combined therapy within lithium and amiloride can mitigate lithium-associated NDI. However, lithium causes chronic tubulointerstitial scarring and chronic kidney disease after prolonged therapy, such that patients may have a persistent NDI long after stopping the drug, with a reduced therapeutic benefit from amiloride.

1	Finally, gestational DI is a rare complication of late-term pregnancy wherein increased activity of a circulating placental protease with “vasopressinase” activity leads to reduced circulating AVP and polyuria, often accompanied by hypernatremia. DDAVP is an effective therapy for this syndrome, given its resistance to the vasopressinase enzyme.

1	Clinical Features Hypernatremia increases osmolality of the ECF, generating an osmotic gradient between the ECF and ICF, an efflux of intracellular water, and cellular shrinkage. As in hyponatremia, the symptoms of hypernatremia are predominantly neurologic. Altered mental status is the most frequent manifestation, ranging from mild confusion and lethargy to deep coma. The sudden shrinkage of brain cells in acute hypernatremia may lead to parenchymal or subarachnoid hemorrhages and/or subdural hematomas; however, these vascular complications are primarily encountered in pediatric and neonatal patients. Osmotic damage to muscle membranes can also lead to hypernatremic rhabdomyolysis. Brain cells accommodate to a chronic increase in ECF osmolality (>48 h) by activating membrane transporters that mediate influx and intracellular accumulation of organic osmolytes (creatine, betaine, glutamate, myoinositol, and taurine); this results in an increase in ICF water and normalization of brain

1	that mediate influx and intracellular accumulation of organic osmolytes (creatine, betaine, glutamate, myoinositol, and taurine); this results in an increase in ICF water and normalization of brain parenchymal volume. In consequence, patients with chronic hypernatremia are less likely to develop severe neurologic compromise. However, the cellular response to chronic hypernatremia predisposes these patients to the development of cerebral edema and seizures during overly rapid hydration (overcorrection of plasma Na+ concentration by >10 mM/d).

1	Diagnostic Approach The history should focus on the presence or absence of thirst, polyuria, and/or an extrarenal source for water loss, 304 such as diarrhea. The physical examination should include a detailed neurologic exam and an assessment of the ECFV; patients with a particularly large water deficit and/or a combined deficit in electrolytes and water may be hypovolemic, with reduced JVP and orthostasis. Accurate documentation of daily fluid intake and daily urine output is also critical for the diagnosis and management of hypernatremia. Laboratory investigation should include a measurement of serum and urine osmolality, in addition to urine electrolytes. The appropriate response to hypernatremia and a serum osmolality >295 mOsm/kg is an increase in circulating AVP and the excretion of low volumes (<500 mL/d) of maximally concentrated urine, i.e., urine with osmolality >800 mOsm/kg; should this be the case, then an extrarenal source of water loss is primarily responsible for the

1	of low volumes (<500 mL/d) of maximally concentrated urine, i.e., urine with osmolality >800 mOsm/kg; should this be the case, then an extrarenal source of water loss is primarily responsible for the generation of hypernatremia. Many patients with hypernatremia are polyuric; should an osmotic diuresis be responsible, with excessive excretion of Na+-Cl–, glucose, and/or urea, then daily solute excretion will be >750–1000 mOsm/d (>15 mOsm/kg body water per day) (Fig. 63-6). More commonly, patients with hypernatremia and polyuria will have a predominant water diuresis, with excessive excretion of hypotonic, dilute urine. Adequate differentiation between nephrogenic and central causes of DI requires the measurement of the response in urinary osmolality to DDAVP, combined with measurement of circulating AVP in the setting of hypertonicity. By definition, patients with baseline hypernatremia are hypertonic, with an adequate stimulus for AVP by the posterior pituitary. Therefore, in contrast

1	AVP in the setting of hypertonicity. By definition, patients with baseline hypernatremia are hypertonic, with an adequate stimulus for AVP by the posterior pituitary. Therefore, in contrast to polyuric patients with a normal or reduced baseline plasma Na+ concentration and osmolality, a water deprivation test (Chap. 61) is unnecessary in hypernatremia; indeed, water deprivation is absolutely contraindicated in this setting, given the risk for worsening the hypernatremia. Patients with NDI will fail to respond to DDAVP, with a urine osmolality that increases by <50% or <150 mOsm/kg from baseline, in combination with a normal or high circulating AVP level; patients with central DI will respond to DDAVP, with a reduced circulating AVP. Patients may exhibit a partial response to DDAVP, with a >50% rise in urine osmolality that nonetheless fails to reach 800 mOsm/kg; the level of circulating AVP will help differentiate the underlying cause, i.e., NDI versus central DI. In pregnant

1	with a >50% rise in urine osmolality that nonetheless fails to reach 800 mOsm/kg; the level of circulating AVP will help differentiate the underlying cause, i.e., NDI versus central DI. In pregnant patients, AVP assays should be drawn in tubes containing the protease inhibitor 1,10-phenanthroline, to prevent in vitro degradation of AVP by placental vasopressinase. For patients with hypernatremia due to renal loss of water, it is critical to quantify ongoing daily losses using the calculated electrolyte-free water clearance, in addition to calculation of the baseline water deficit (the relevant formulas are discussed in Table 63-3). This requires daily measurement of urine electrolytes, combined with accurate measurement of daily urine volume.

1	PART 2 Cardinal Manifestations and Presentation of Diseases The underlying cause of hypernatremia should be withdrawn or corrected, be it drugs, hyperglycemia, hypercalcemia, hypokalemia, or diarrhea. The approach to the correction of hypernatremia is outlined in Table 63-3. It is imperative to correct hypernatremia slowly to avoid cerebral edema, typically replacing the calculated free water deficit over 48 h. Notably, the plasma Na+ concentration should be corrected by no more than 10 mM/d, which may take longer than 48 h in patients with severe hypernatremia (>160 mM). A rare exception is patients with acute hypernatremia (<48 h) due to sodium loading, who can safely be corrected rapidly at a rate of 1 mM/h.

1	Water should ideally be administered by mouth or by nasogastric tube, as the most direct way to provide free water, i.e., water without electrolytes. Alternatively, patients can receive free water in dextrose-containing IV solutions, such as 5% dextrose (D5W); blood glucose should be monitored in case hyperglycemia occurs. Depending on the history, blood pressure, or clinical volume status, it may be appropriate to initially treat with hypotonic saline solutions (1/4 or 1/2 normal saline); normal saline is usually inappropriate in the absence of very severe hypernatremia, where normal saline is proportionally more hypotonic relative to plasma, or frank hypotension. Calculation of urinary electrolyte-free water clearance (Table 63-3) is required to estimate daily, ongoing loss of free water in patients with NDI or central DI, which should be replenished daily.

1	Additional therapy may be feasible in specific cases. Patients with central DI should respond to the administration of intravenous, intranasal, or oral DDAVP. Patients with NDI due to lithium may reduce their polyuria with amiloride (2.5–10 mg/d), which decreases entry of lithium into principal cells by inhibiting ENaC (see above); in practice, however, most patients with lithium-associated DI are able to compensate for their polyuria by simply increasing their daily water intake. Thiazides may reduce polyuria due to NDI, ostensibly by inducing hypovolemia and increasing proximal tubular water reabsorption. Occasionally, nonsteroidal anti-inflammatory drugs (NSAIDs) have been used to treat polyuria associated with NDI, reducing the negative effect of intrarenal prostaglandins on urinary concentrating mechanisms; however, this assumes the risks of NSAID-associated gastric and/or renal toxicity. Furthermore, it must be emphasized that thiazides, amiloride, and NSAIDs are only

1	urinary concentrating mechanisms; however, this assumes the risks of NSAID-associated gastric and/or renal toxicity. Furthermore, it must be emphasized that thiazides, amiloride, and NSAIDs are only appropriate for chronic management of polyuria from NDI and have no role in the acute management of associated hypernatremia, where the focus is on replacing free water deficits and ongoing free water loss.

1	3.5 and 5.0 mM, despite marked variation in dietary K+ intake. In a healthy individual at steady state, the entire daily intake of potassium is excreted, approximately 90% in the urine and 10% in the stool; thus, the kidney plays a dominant role in potassium homeostasis. However, more than 98% of total-body potassium is intracellular, chiefly in muscle; buffering of extracellular K+ by this large intracellular pool plays a crucial role in the regulation of plasma K+ concentration. Changes in the exchange and distribution of intraand extracellular K+ can thus lead to marked hypoor hyperkalemia. A corollary is that massive necrosis and the attendant release of tissue K+ can cause severe hyperkalemia, particularly in the setting of acute kidney injury and reduced excretion of K+.

1	Changes in whole-body K+ content are primarily mediated by the kidney, which reabsorbs filtered K+ in hypokalemic, K+-deficient states and secretes K+ in hyperkalemic, K+-replete states. Although K+ is transported along the entire nephron, it is the principal cells of the connecting segment (CNT) and cortical CD that play a dominant role in renal K+ secretion, whereas alpha-intercalated cells of the outer medullary CD function in renal tubular reabsorption of filtered K+ in K+-deficient states. In principal cells, apical Na+ entry via the amiloride-sensitive ENaC generates a lumen-negative potential difference, which drives passive K+ exit through apical K+ channels (Fig. 63-4). Two major K+ channels mediate distal tubular K+ secretion: the secretory K+ channel ROMK (renal outer medullary K+ channel; also known as Kir1.1 or KcnJ1) and the flow-sensitive big potassium (BK) or maxi-K K+ channel. ROMK is thought to mediate the bulk of constitutive K+ secretion, whereas increases in

1	K+ channel; also known as Kir1.1 or KcnJ1) and the flow-sensitive big potassium (BK) or maxi-K K+ channel. ROMK is thought to mediate the bulk of constitutive K+ secretion, whereas increases in distal flow rate and/or genetic absence of ROMK activate K+ secretion via the BK channel.

1	An appreciation of the relationship between ENaC-dependent Na+ entry and distal K+ secretion (Fig. 63-4) is required for the bedside interpretation of potassium disorders. For example, decreased distal delivery of Na+, as occurs in hypovolemic, prerenal states, tends to blunt the ability to excrete K+, leading to hyperkalemia; on the other hand, an increase in distal delivery of Na+ and distal flow rate, as occurs after treatment with thiazide and loop diuretics, can enhance K+ secretion and lead to hypokalemia. Hyperkalemia is also a predictable consequence of drugs that directly inhibit ENaC, due to the role of this Na+ channel in generating a lumen-negative potential difference. Aldosterone in turn has a major influence on potassium excretion, increasing the activity of ENaC channels and thus amplifying the driving force for K+ secretion across the luminal membrane of principal cells. Abnormalities in the renin-angiotensin-aldosterone system can thus cause both hypokalemia and

1	and thus amplifying the driving force for K+ secretion across the luminal membrane of principal cells. Abnormalities in the renin-angiotensin-aldosterone system can thus cause both hypokalemia and hyperkalemia. Notably, however, potassium excess and potassium restriction have opposing, aldosterone-independent effects on the density and activity of apical K+ channels in the distal nephron, i.e., factors other than aldosterone modulate the renal capacity to secrete K+. In addition, potassium restriction and hypokalemia activates aldosterone-independent distal reabsorption of filtered K+, activating apical H+/K+-ATPase activity in intercalated cells within the outer medullary CD. Reflective perhaps of this physiology, changes in plasma K+ concentration are not universal in disorders associated with changes in aldosterone activity.

1	Hypokalemia, defined as a plasma K+ concentration of <3.5 mM, occurs in up to 20% of hospitalized patients. Hypokalemia is associated with a 10-fold increase in in-hospital mortality, due to adverse effects on cardiac rhythm, blood pressure, and cardiovascular morbidity. Mechanistically, hypokalemia can be caused by redistribution of K+ between tissues and the ECF or by renal and nonrenal loss of K+ (Table 63-4). Systemic hypomagnesemia can also cause treatment-resistant hypokalemia, due to a combination of reduced cellular uptake of K+ and exaggerated renal secretion. Spurious hypokalemia or “pseudohypokalemia” can occasionally result from in vitro cellular uptake of K+ after venipuncture, for example, due to profound leukocytosis in acute leukemia.

1	Redistribution and Hypokalemia Insulin, β2-adrenergic activity, thyroid hormone, and alkalosis promote Na+/K+-ATPase-mediated cellular uptake of K+, leading to hypokalemia. Inhibition of the passive efflux of K+ can also cause hypokalemia, albeit rarely; this typically occurs in the setting of systemic inhibition of K+ channels by toxic barium ions. Exogenous insulin can cause iatrogenic hypokalemia, particularly during the management of K+-deficient states such as diabetic ketoacidosis. Alternatively, the stimulation of endogenous insulin can provoke hypokalemia, hypomagnesemia, and/or hypophosphatemia in malnourished patients given a carbohydrate load. Alterations in the activity of the endogenous sympathetic nervous system can cause hypokalemia in several settings, including alcohol withdrawal, hyperthyroidism, acute myocardial infarction, and severe head injury. β2 agonists, including both bronchodilators and tocolytics (ritodrine), are powerful activators of cellular K+ uptake;

1	hyperthyroidism, acute myocardial infarction, and severe head injury. β2 agonists, including both bronchodilators and tocolytics (ritodrine), are powerful activators of cellular K+ uptake; “hidden” sympathomimetics, such as pseudoephedrine and ephedrine in cough syrup or dieting agents, may also cause unexpected hypokalemia. Finally, xanthinedependent activation of cAMP-dependent signaling, downstream of the β2 receptor, can lead to hypokalemia, usually in the setting of overdose (theophylline) or marked overingestion (dietary caffeine).

1	Redistributive hypokalemia can also occur in the setting of hyperthyroidism, with periodic attacks of hypokalemic paralysis (thyrotoxic periodic paralysis [TPP]). Similar episodes of hypokalemic weakness in the absence of thyroid abnormalities occur in familial hypokalemic periodic paralysis, usually caused by missense mutations of voltage sensor domains within the α1 subunit of L-type calcium channels or the skeletal Na+ channel; these mutations generate an abnormal gating pore current activated by hyperpolarization. TPP develops more frequently in patients of Asian or Hispanic origin; this shared predisposition has been linked to genetic variation in Kir2.6, a muscle-specific, thyroid hormone–responsive K+ channel. Patients with TPP typically present with weakness of the extremities and limb girdles, with paralytic episodes that occur most frequently between 1 and 6 am. Signs and symptoms of hyperthyroidism are not invariably present. Hypokalemia is usually profound and almost

1	and limb girdles, with paralytic episodes that occur most frequently between 1 and 6 am. Signs and symptoms of hyperthyroidism are not invariably present. Hypokalemia is usually profound and almost invariably accompanied by hypophosphatemia and hypomagnesemia. The hypokalemia in TPP is attributed to both direct and indirect activation of the Na+/ K+-ATPase, resulting in increased uptake of K+ by muscle and other tissues. Increases in β-adrenergic activity play an important role in that high-dose propranolol (3 mg/kg) rapidly reverses the associated hypokalemia, hypophosphatemia, and paralysis.

1	Nonrenal Loss of Potassium The loss of K+ in sweat is typically low, except under extremes of physical exertion. Direct gastric losses of K+ due to vomiting or nasogastric suctioning are also minimal; however, the ensuing hypochloremic alkalosis results in persistent kaliuresis due to secondary hyperaldosteronism and bicarbonaturia, i.e., a renal loss CAuSES of HyPoKALEMiA I. Decreased intake A. Starvation B. Clay ingestion II. A. 1. Metabolic alkalosis B. Hormonal 1. 2. Increased β2-adrenergic sympathetic activity: post–myocardial infarction, head injury 3. β2-Adrenergic agonists – bronchodilators, tocolytics 4. 5. 6. Downstream stimulation of Na+/K+-ATPase: theophylline, caffeine C. Anabolic state 1. 2. 3. D. Other 1. 2. 3. 4. Barium toxicity: systemic inhibition of “leak” K+ channels III. A. 1. 2. B. Renal 1. Increased distal flow and distal Na+ delivery: diuretics, osmotic diuresis, salt-wasting nephropathies 2. Increased secretion of potassium a.

1	III. A. 1. 2. B. Renal 1. Increased distal flow and distal Na+ delivery: diuretics, osmotic diuresis, salt-wasting nephropathies 2. Increased secretion of potassium a. Mineralocorticoid excess: primary hyperaldosteronism (aldosterone-producing adenomas, primary or unilateral adrenal hyperplasia, idiopathic hyperaldosteronism due to bilateral adrenal hyperplasia, and adrenal carcinoma), genetic hyperaldosteronism (familial hyperaldosteronism types I/II/III, congenital adrenal hyperplasias), secondary hyperaldosteronism (malignant hypertension, renin-secreting tumors, renal artery stenosis, hypovolemia), Cushing’s syndrome, Bartter’s syndrome, Gitelman’s syndrome b.

1	Apparent mineralocorticoid excess: genetic deficiency of 11β-dehydrogenase-2 (syndrome of apparent mineralocorticoid excess), inhibition of 11β-dehydrogenase-2 (glycyrrhetinic/ glycyrrhizinic acid and/or carbenoxolone; licorice, food products, drugs), Liddle’s syndrome (genetic activation of epithelial Na+ channels) c.

1	Distal delivery of nonreabsorbed anions: vomiting, nasogastric suction, proximal renal tubular acidosis, diabetic ketoacidosis, glue-sniffing (toluene abuse), penicillin derivatives (penicillin, nafcillin, dicloxacillin, ticarcillin, oxacillin, and carbenicillin) 3. Magnesium deficiency of K+. Diarrhea is a globally important cause of hypokalemia, given the worldwide prevalence of infectious diarrheal disease. Noninfectious gastrointestinal processes such as celiac disease, ileostomy, villous adenomas, inflammatory bowel disease, colonic pseudo-obstruction (Ogilvie’s syndrome), VIPomas, and chronic laxative abuse can also cause significant hypokalemia; an exaggerated intestinal secretion of potassium by upregulated colonic BK channels has been directly implicated in the pathogenesis of hypokalemia in many of these disorders.

1	Renal Loss of Potassium Drugs can increase renal K+ excretion by a variety of different mechanisms. Diuretics are a particularly common cause, due to associated increases in distal tubular Na+ delivery and 306 distal tubular flow rate, in addition to secondary hyperaldosteronism. Thiazides have a greater effect on plasma K+ concentration than loop diuretics, despite their lesser natriuretic effect. The diuretic effect of thiazides is largely due to inhibition of the Na+-Cl– cotransporter NCC in DCT cells. This leads to a direct increase in the delivery of luminal Na+ to the principal cells immediately downstream in the CNT and cortical CD, which augments Na+ entry via ENaC, increases the lumen-negative potential difference, and amplifies K+ secretion. The higher propensity of thiazides to cause hypokalemia may also be secondary to thiazide-associated hypocalciuria, versus the hypercalciuria seen with loop diuretics; the increases in downstream luminal calcium in response to loop

1	to cause hypokalemia may also be secondary to thiazide-associated hypocalciuria, versus the hypercalciuria seen with loop diuretics; the increases in downstream luminal calcium in response to loop diuretics inhibit ENaC in principal cells, thus reducing the lumen-negative potential difference and attenuating distal K+ excretion. High doses of penicillin-related antibiotics (nafcillin, dicloxacillin, ticarcillin, oxacillin, and carbenicillin) can increase obligatory K+ excretion by acting as nonreabsorbable anions in the distal nephron. Finally, several renal tubular toxins cause renal K+ and magnesium wasting, leading to hypokalemia and hypomagnesemia; these drugs include aminoglycosides, amphotericin, foscarnet, cisplatin, and ifosfamide (see also “Magnesium Deficiency and Hypokalemia,” below). Aldosterone activates the ENaC channel in principal cells via multiple synergistic mechanisms, thus increasing the driving force for K+ excretion. In consequence, increases in aldosterone

1	below). Aldosterone activates the ENaC channel in principal cells via multiple synergistic mechanisms, thus increasing the driving force for K+ excretion. In consequence, increases in aldosterone bioactivity and/ or gains in function of aldosterone-dependent signaling pathways are associated with hypokalemia. Increases in circulating aldosterone (hyperaldosteronism) may be primary or secondary. Increased levels of circulating renin in secondary forms of hyperaldosteronism lead to increased angiotensin II and thus aldosterone; renal artery stenosis is perhaps the most frequent cause (Table 63-4). Primary hyperaldosteronism may be genetic or acquired. Hypertension and hypokalemia, due to increases in circulating 11-deoxycorticosterone, occur in patients with congenital adrenal hyperplasia caused by defects in either steroid 11β-hydroxylase or steroid 17α-hydroxylase; deficient 11β-hydroxylase results in associated virilization and other signs of androgen excess, whereas reduced sex

1	caused by defects in either steroid 11β-hydroxylase or steroid 17α-hydroxylase; deficient 11β-hydroxylase results in associated virilization and other signs of androgen excess, whereas reduced sex steroids in 17α-hydroxylase deficiency lead to hypogonadism. The major forms of isolated primary genetic hyperaldosteronism are familial hyperaldosteronism type I (FH-I, also known as glucocorticoid-remediable hyperaldosteronism [GRA]) and familial hyperaldosteronism types II and III (FH-II and FH-III), in which aldosterone production is not repressible by exogenous glucocorticoids. FH-I is caused by a chimeric gene duplication between the homologous 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes, fusing the adrenocorticotropic hormone (ACTH)– responsive 11β-hydroxylase promoter to the coding region of aldosterone synthase; this chimeric gene is under the control of ACTH and thus repressible by glucocorticoids. FH-III is caused by mutations in the KCNJ5 gene, which

1	promoter to the coding region of aldosterone synthase; this chimeric gene is under the control of ACTH and thus repressible by glucocorticoids. FH-III is caused by mutations in the KCNJ5 gene, which encodes the G-protein-activated inward rectifier K+ channel 4 (GIRK4); these mutations lead to the acquisition of sodium permeability in the mutant GIRK4 channels, causing an exaggerated membrane depolarization in adrenal glomerulosa cells and the activation of voltage-gated calcium channels. The resulting calcium influx is sufficient to produce aldosterone secretion and cell proliferation, leading to adrenal adenomas and hyperaldosteronism. Acquired causes of primary hyperaldosteronism include aldosteroneproducing adenomas (APAs), primary or unilateral adrenal hyperplasia (PAH), idiopathic hyperaldosteronism (IHA) due to bilateral adrenal hyperplasia, and adrenal carcinoma; APA and IHA account for close to 60% and 40%, respectively, of diagnosed hyperaldosteronism. Acquired somatic

1	hyperaldosteronism (IHA) due to bilateral adrenal hyperplasia, and adrenal carcinoma; APA and IHA account for close to 60% and 40%, respectively, of diagnosed hyperaldosteronism. Acquired somatic mutations in KCNJ5 or less frequently in the ATP1A1 (an Na+/K+ ATPase α subunit) and ATP2B3 (a Ca2+ ATPase) genes can be detected in APAs; as in FH-III (see above), the exaggerated depolarization of adrenal glomerulosa cells caused by these mutations is implicated in the excessive adrenal proliferation and the exaggerated release of aldosterone. Random testing of plasma renin activity (PRA) and aldosterone is a helpful screening tool in hypokalemic and/or hypertensive patients, with an aldosterone:PRA ratio of >50 suggestive of primary

1	PART 2 Cardinal Manifestations and Presentation of Diseases hyperaldosteronism. Hypokalemia and multiple antihypertensive drugs may alter the aldosterone:PRA ratio by suppressing aldosterone or increasing PRA, leading to a ratio of <50 in patients who do in fact have primary hyperaldosteronism; therefore, the clinical context should always be considered when interpreting these results.

1	The glucocorticoid cortisol has equal affinity for the mineralocorticoid receptor (MLR) to that of aldosterone, with resultant “mineralocorticoid-like” activity. However, cells in the aldosteronesensitive distal nephron are protected from this “illicit” activation by the enzyme 11β-hydroxysteroid dehydrogenase-2 (11βHSD-2), which converts cortisol to cortisone; cortisone has minimal affinity for the MLR. Recessive loss-of-function mutations in the 11βHSD-2 gene are thus associated with cortisol-dependent activation of the MLR and the syndrome of apparent mineralocorticoid excess (SAME), encompassing hypertension, hypokalemia, hypercalciuria, and metabolic alkalosis, with suppressed PRA and suppressed aldosterone. A similar syndrome is caused by biochemical inhibition of 11βHSD-2 by glycyrrhetinic/glycyrrhizinic acid and/or carbenoxolone. Glycyrrhizinic acid is a natural sweetener found in licorice root, typically encountered in licorice and its many guises or as a flavoring agent in

1	acid and/or carbenoxolone. Glycyrrhizinic acid is a natural sweetener found in licorice root, typically encountered in licorice and its many guises or as a flavoring agent in tobacco and food products.

1	Finally, hypokalemia may also occur with systemic increases in glucocorticoids. In Cushing’s syndrome caused by increases in pituitary ACTH (Chap. 406), the incidence of hypokalemia is only 10%, whereas it is 60–100% in patients with ectopic secretion of ACTH, despite a similar incidence of hypertension. Indirect evidence suggests that the activity of renal 11βHSD-2 is reduced in patients with ectopic ACTH compared with Cushing’s syndrome, resulting in SAME.

1	Finally, defects in multiple renal tubular transport pathways are associated with hypokalemia. For example, loss-of-function mutations in subunits of the acidifying H+-ATPase in alpha-intercalated cells cause hypokalemic distal renal tubular acidosis, as do many acquired disorders of the distal nephron. Liddle’s syndrome is caused by autosomal dominant gain-in-function mutations of ENaC subunits. Disease-associated mutations either activate the channel directly or abrogate aldosterone-inhibited retrieval of ENaC subunits from the plasma membrane; the end result is increased expression of activated ENaC channels at the plasma membrane of principal cells. Patients with Liddle’s syndrome classically manifest severe hypertension with hypokalemia, unresponsive to spironolactone yet sensitive to amiloride. Hypertension and hypokalemia are, however, variable aspects of the Liddle’s phenotype; more consistent features include a blunted aldosterone response to ACTH and reduced urinary

1	to amiloride. Hypertension and hypokalemia are, however, variable aspects of the Liddle’s phenotype; more consistent features include a blunted aldosterone response to ACTH and reduced urinary aldosterone excretion.

1	Loss of the transport functions of the TALH and DCT nephron segments causes hereditary hypokalemic alkalosis, Bartter’s syndrome (BS) and Gitelman’s syndrome (GS), respectively. Patients with classic BS typically suffer from polyuria and polydipsia, due to the reduction in renal concentrating ability. They may have an increase in urinary calcium excretion, and 20% are hypomagnesemic. Other features include marked activation of the renin-angiotensin-aldosterone axis. Patients with antenatal BS suffer from a severe systemic disorder characterized by marked electrolyte wasting, polyhydramnios, and hypercalciuria with nephrocalcinosis; renal prostaglandin synthesis and excretion are significantly increased, accounting for much of the systemic symptoms. There are five disease genes for BS, all of them functioning in some aspect of regulated Na+, K+, and Cl– transport by the TALH. In contrast, GS is genetically homogeneous, caused almost exclusively by loss-of-function mutations in the

1	all of them functioning in some aspect of regulated Na+, K+, and Cl– transport by the TALH. In contrast, GS is genetically homogeneous, caused almost exclusively by loss-of-function mutations in the thiazide-sensitive Na+-Cl– cotransporter of the DCT. Patients with GS are uniformly hypomagnesemic and exhibit marked hypocalciuria, rather than the hypercalciuria typically seen in BS; urinary calcium excretion is thus a critical diagnostic test in GS. GS is a milder phenotype than BS; however, patients with GS may suffer from chondrocalcinosis, an abnormal deposition of calcium pyrophosphate dihydrate (CPPD) in joint cartilage (Chap. 339).

1	Magnesium Deficiency and Hypokalemia Magnesium depletion has inhibitory effects on muscle Na+/K+-ATPase activity, reducing influx into muscle cells and causing a secondary kaliuresis. In addition, magnesium depletion causes exaggerated K+ secretion by the distal nephron; this effect is attributed to a reduction in the magnesium-dependent, intracellular block of K+ efflux through the secretory K+ channel of principal cells (ROMK; Fig. 63-4). In consequence, hypomagnesemic patients are clinically refractory to K+ replacement in the absence of Mg2+ repletion. Notably, magnesium deficiency is also a common concomitant of hypokalemia because many disorders of the distal nephron may cause both potassium and magnesium wasting (Chap. 339).

1	Clinical Features Hypokalemia has prominent effects on cardiac, skeletal, and intestinal muscle cells. In particular, hypokalemia is a major risk factor for both ventricular and atrial arrhythmias. Hypokalemia predisposes to digoxin toxicity by a number of mechanisms, including reduced competition between K+ and digoxin for shared binding sites on cardiac Na+/K+-ATPase subunits. Electrocardiographic changes in hypokalemia include broad flat T waves, ST depression, and QT prolongation; these are most marked when serum K+ is <2.7 mmol/L. Hypokalemia can thus be an important precipitant of arrhythmia in patients with additional genetic or acquired causes of QT prolongation. Hypokalemia also results in hyperpolarization of skeletal muscle, thus impairing the capacity to depolarize and contract; weakness and even paralysis may ensue. It also causes a skeletal myopathy and predisposes to rhabdomyolysis. Finally, the paralytic effects of hypokalemia on intestinal smooth muscle may cause

1	weakness and even paralysis may ensue. It also causes a skeletal myopathy and predisposes to rhabdomyolysis. Finally, the paralytic effects of hypokalemia on intestinal smooth muscle may cause intestinal ileus.

1	The functional effects of hypokalemia on the kidney can include Na+-Cl– and HCO3 retention, polyuria, phosphaturia, hypocitraturia, and an activation of renal ammoniagenesis. Bicarbonate retention and other acid-base effects of hypokalemia can contribute to the generation of metabolic alkalosis. Hypokalemic polyuria is due to a combination of central polydipsia and an AVP-resistant renal concentrating defect. Structural changes in the kidney due to hypokalemia include a relatively specific vacuolizing injury to proximal tubular cells, interstitial nephritis, and renal cysts. Hypokalemia also predisposes to acute kidney injury and can lead to end-stage renal disease in patients with longstanding hypokalemia due to eating disorders and/or laxative abuse.

1	Hypokalemia and/or reduced dietary K+ are implicated in the pathophysiology and progression of hypertension, heart failure, and stroke. For example, short-term K+ restriction in healthy humans and patients with essential hypertension induces Na+-Cl– retention and hypertension. Correction of hypokalemia is particularly important in hypertensive patients treated with diuretics, in whom blood pressure improves with the establishment of normokalemia.

1	Diagnostic Approach The cause of hypokalemia is usually evident from history, physical examination, and/or basic laboratory tests. The history should focus on medications (e.g., laxatives, diuretics, antibiotics), diet and dietary habits (e.g., licorice), and/or symptoms that suggest a particular cause (e.g., periodic weakness, diarrhea). The physical examination should pay particular attention to blood pressure, volume status, and signs suggestive of specific hypokalemic disorders, e.g., hyperthyroidism and Cushing’s syndrome. Initial laboratory evaluation should include electrolytes, BUN, creatinine, serum osmolality, Mg2+, Ca2+, a complete blood count, and urinary pH, osmolality, creatinine, and electrolytes (Fig. 63-7). The presence of a non–anion gap acidosis suggests a distal, hypokalemic renal tubular acidosis or diarrhea; calculation of the urinary anion gap can help differentiate these two diagnoses. Renal K+ excretion can be assessed with a 24-h urine collection; a 24-h K+

1	renal tubular acidosis or diarrhea; calculation of the urinary anion gap can help differentiate these two diagnoses. Renal K+ excretion can be assessed with a 24-h urine collection; a 24-h K+ excretion of <15 mmol is indicative of an extrarenal cause of hypokalemia (Fig. 63-7). If only a random, spot urine sample is available, serum and urine osmolality can be used to calculate the transtubular K+ gradient (TTKG), which should be <3 in the presence of hypokalemia (see also “Hyperkalemia”). Alternatively, a urinary K+-to-creatinine ratio of >13 mmol/g creatinine (>1.5 mmol/ mmol creatinine) is compatible with excessive renal K+ excretion. Urine Cl– is usually decreased in patients with hypokalemia from a nonreabsorbable anion, such as antibiotics or HCO3–. The most common causes of chronic hypokalemic alkalosis are surreptitious vomiting, diuretic abuse, and GS; these can be distinguished by the pattern of urinary electrolytes. Hypokalemic patients with vomiting due to bulimia will

1	hypokalemic alkalosis are surreptitious vomiting, diuretic abuse, and GS; these can be distinguished by the pattern of urinary electrolytes. Hypokalemic patients with vomiting due to bulimia will thus have a urinary Cl– <10 mmol/L; urine Na+, K+, and 307 Cl– are persistently elevated in GS, due to loss of function in the thiazide-sensitive Na+-Cl– cotransporter, but less elevated in diuretic abuse and with greater variability. Urine diuretic screens for loop diuretics and thiazides may be necessary to further exclude diuretic abuse.

1	Other tests, such as urinary Ca2+, thyroid function tests, and/or PRA and aldosterone levels, may also be appropriate in specific cases. A plasma aldosterone:PRA ratio of >50, due to suppression of circulating renin and an elevation of circulating aldosterone, is suggestive of hyperaldosteronism. Patients with hyperaldosteronism or apparent mineralocorticoid excess may require further testing, for example adrenal vein sampling (Chap. 406) or the clinically available testing for specific genetic causes (e.g., FH-I, SAME, Liddle’s syndrome). Patients with primary aldosteronism should thus be tested for the chimeric FH-I/GRA gene (see above) if they are younger than 20 years of age or have a family history of primary aldosteronism or stroke at a young age (<40 years). Preliminary differentiation of Liddle’s syndrome due to mutant ENaC channels from SAME due to mutant 11βHSD-2 (see above), both of which cause hypokalemia and hypertension with aldosterone suppression, can be made on a

1	of Liddle’s syndrome due to mutant ENaC channels from SAME due to mutant 11βHSD-2 (see above), both of which cause hypokalemia and hypertension with aldosterone suppression, can be made on a clinical basis and then confirmed by genetic analysis; patients with Liddle’s syndrome should respond to amiloride (ENaC inhibition) but not spironolactone, whereas patients with SAME will respond to spironolactone.

1	The goals of therapy in hypokalemia are to prevent life-threatening and/or serious chronic consequences, to replace the associated K+ deficit, and to correct the underlying cause and/or mitigate future hypokalemia. The urgency of therapy depends on the severity of hypokalemia, associated clinical factors (e.g., cardiac disease, digoxin therapy), and the rate of decline in serum K+. Patients with a prolonged QT interval and/or other risk factors for arrhythmia should be monitored by continuous cardiac telemetry during repletion. Urgent but cautious K+ replacement should be considered in patients with severe redistributive hypokalemia (plasma K+ concentration <2.5 mM) and/or when serious complications ensue; however, this approach has a risk of rebound hyperkalemia following acute resolution of the underlying cause. When excessive activity of the sympathetic nervous system is thought to play a dominant role in redistributive hypokalemia, as in TPP, theophylline overdose, and acute head

1	of the underlying cause. When excessive activity of the sympathetic nervous system is thought to play a dominant role in redistributive hypokalemia, as in TPP, theophylline overdose, and acute head injury, high-dose propranolol (3 mg/kg) should be considered; this nonspecific b-adrenergic blocker will correct hypokalemia without the risk of rebound hyperkalemia.

1	Oral replacement with K+-Cl– is the mainstay of therapy in hypokalemia. Potassium phosphate, oral or IV, may be appropriate in patients with combined hypokalemia and hypophosphatemia. Potassium bicarbonate or potassium citrate should be considered in patients with concomitant metabolic acidosis. Notably, hypomagnesemic patients are refractory to K+ replacement alone, such that concomitant Mg2+ deficiency should always be corrected with oral or intravenous repletion. The deficit of K+ and the rate of correction should be estimated as accurately as possible; renal function, medications, and comorbid conditions such as diabetes should also be considered, so as to gauge the risk of overcorrection. In the absence of abnormal K+ redistribution, the total deficit correlates with serum K+, such that serum K+ drops by approximately 0.27 mM for every 100-mmol reduction in total-body stores; loss of 400–800 mmol of total-body K+ results in a reduction in serum K+ by approximately 2.0 mM.

1	such that serum K+ drops by approximately 0.27 mM for every 100-mmol reduction in total-body stores; loss of 400–800 mmol of total-body K+ results in a reduction in serum K+ by approximately 2.0 mM. Notably, given the delay in redistributing potassium into intracellular compartments, this deficit must be replaced gradually over 24-48 h, with frequent monitoring of plasma K+ concentration to avoid transient overrepletion and transient hyperkalemia.

1	The use of intravenous administration should be limited to patients unable to use the enteral route or in the setting of severe complications (e.g., paralysis, arrhythmia). Intravenous K+-Cl– should always be administered in saline solutions, rather than dextrose, because the dextrose-induced increase in insulin can acutely

1	PART 2 Cardinal Manifestations and Presentation of Diseases Hypokalemia (Serum K+<3.5 mmol/l) <15 mmol/day OR <15 mmol/g Cr >15 mmol/g Cr OR >15 mmol/day Renal loss TTKG ˜ Distal K+ secretion ˜ Tubular flow -Osmotic diuresis BP and/or Volume Extrarenal loss/remote renal loss Metabolic acidosis -GI K+ loss Normal -Profuse sweating Metabolic alkalosis -Remote diuretic use -Remote vomiting or stomach drainage -Profuse sweating Non-reabsorbable anions other than HCO3 – -Hippurate -Penicillins Metabolic acidosis -Proximal RTA -Distal RTA -DKA -Amphotericin B -Acetazolamide Acid-base status Low OR normal Acid-base status Variable Aldosterone High Low High High Low High Normal Cortisol Renin Urine K+ Emergency? Pseudohypokalemia? Move to therapy History, physical examination & basic laboratory tests Clear evidence of transcellular shift No further workup Treat accordingly Clear evidence of low intake Treat accordingly and re-evaluate Yes Yes Yes Yes No No No No -Insulin excess -˜2-adrenergic

1	Clear evidence of transcellular shift No further workup Treat accordingly Clear evidence of low intake Treat accordingly and re-evaluate Yes Yes Yes Yes No No No No -Insulin excess -˜2-adrenergic agonists -FHPP -Hyperthyroidism -Barium intoxication -Theophylline -Chloroquine >4 >20 >0.20 <0.15 <10 <2 Metabolic alkalosis Urine Ca/Cr (molar ratio) -Vomiting -Chloride diarrhea Urine Cl– (mmol/l) -Loop diuretic -Bartter’s syndrome -Thiazide diuretic -Gitelman’s syndrome -RAS -RST -Malignant HTN -PA -FH-I -Cushing’s syndrome -Liddle’s syndrome -Licorice -SAME

1	FIguRE 63-7 The diagnostic approach to hypokalemia. See text for details. AME, apparent mineralocorticoid excess; BP, blood pressure; CCD, cortical collecting duct; DKA, diabetic ketoacidosis; FH-I, familial hyperaldosteronism type I; FHPP, familial hypokalemic periodic paralysis; GI, gastrointestinal; GRA, glucocorticoid remediable aldosteronism; HTN, hypertension; PA, primary aldosteronism; RAS, renal artery stenosis; RST, renin-secreting tumor; RTA, renal tubular acidosis; SAME, syndrome of apparent mineralocorticoid excess; TTKG, transtubular potassium gradient. (Used with permission from DB Mount, K Zandi-Nejad K: Disorders of potassium balance, in Brenner and Rector’s The Kidney, 8th ed, BM Brenner [ed]. Philadelphia, W.B. Saunders & Company, 2008, pp 547-587.) exacerbate hypokalemia. The peripheral intravenous dose is usually 20–40 mmol of K+-Cl– per liter; higher concentrations can cause localized pain from chemical phlebitis, irritation, and sclerosis. If hypokalemia is

1	The peripheral intravenous dose is usually 20–40 mmol of K+-Cl– per liter; higher concentrations can cause localized pain from chemical phlebitis, irritation, and sclerosis. If hypokalemia is severe (<2.5 mmol/L) and/or critically symptomatic, intravenous K+-Cl– can be administered through a central vein with cardiac monitoring in an intensive care setting, at rates of 10–20 mmol/h; higher rates should be reserved for acutely life-threatening complications. The absolute amount of administered K+ should be restricted (e.g., 20 mmol in 100 mL of saline solution) to prevent inadvertent infusion of a large dose. Femoral veins are preferable, because infusion through internal jugular or subclavian central lines can acutely increase the local concentration of K+ and affect cardiac conduction.

1	Strategies to minimize K+ losses should also be considered. These measures may include minimizing the dose of non-K+-sparing diuretics, restricting Na+ intake, and using clinically appropriate combinations of non-K+-sparing and K+-sparing medications (e.g., loop diuretics with angiotensin-converting enzyme inhibitors).

1	Hyperkalemia is defined as a plasma potassium level of 5.5 mM, occurring in up to 10% of hospitalized patients; severe hyperkalemia (>6.0 mM) occurs in approximately 1%, with a significantly increased risk of mortality. Although redistribution and reduced tissue uptake can acutely cause hyperkalemia, a decrease in renal K+ excretion is the most frequent underlying cause (Table 63-5). Excessive intake of K+ is a rare cause, given the adaptive capacity to increase renal secretion; however, dietary intake can have a major effect in susceptible patients, e.g., diabetics with hyporeninemic hypoaldosteronism and chronic kidney disease. Drugs that impact on the renin-angiotensin-aldosterone axis are also a major cause of hyperkalemia. CAuSES of HyPERKALEMiA I. Pseudohyperkalemia A. Cellular efflux; thrombocytosis, erythrocytosis, leukocytosis, in vitro hemolysis B. Hereditary defects in red cell membrane transport II. A. B.

1	CAuSES of HyPERKALEMiA I. Pseudohyperkalemia A. Cellular efflux; thrombocytosis, erythrocytosis, leukocytosis, in vitro hemolysis B. Hereditary defects in red cell membrane transport II. A. B. Hyperosmolality; radiocontrast, hypertonic dextrose, mannitol C. D. Digoxin and related glycosides (yellow oleander, foxglove, bufadienolide) E. F. Lysine, arginine, and ε-aminocaproic acid (structurally similar, positively charged) G. Succinylcholine; thermal trauma, neuromuscular injury, disuse atrophy, mucositis, or prolonged immobilization H. III. A. Inhibition of the renin-angiotensin-aldosterone axis; ↑ risk of hyperkalemia when used in combination 1. 2. Renin inhibitors; aliskiren (in combination with ACE inhibitors or angiotensin receptor blockers [ARBs]) 3. 4. Blockade of the mineralocorticoid receptor: spironolactone, eplerenone, drospirenone 5. Blockade of the epithelial sodium channel (ENaC): amiloride, triamterene, trimethoprim, pentamidine, nafamostat B.

1	Blockade of the epithelial sodium channel (ENaC): amiloride, triamterene, trimethoprim, pentamidine, nafamostat B. 1. 2. C. 1. Tubulointerstitial diseases: systemic lupus erythematosus (SLE), sickle cell anemia, obstructive uropathy 2. Diabetes, diabetic nephropathy 3. Drugs: nonsteroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase 2 (COX2) inhibitors, β-blockers, cyclosporine, tacrolimus 4. Chronic kidney disease, advanced age 5. Pseudohypoaldosteronism type II: defects in WNK1 or WNK4 kinases, Kelch-like 3 (KLHL3), or Cullin 3 (CUL3) D. Renal resistance to mineralocorticoid 1. Tubulointerstitial diseases: SLE, amyloidosis, sickle cell anemia, obstructive uropathy, post–acute tubular necrosis 2. Hereditary: pseudohypoaldosteronism type I; defects in the mineralocorticoid receptor or the epithelial sodium channel (ENaC) E. 1. 2. 3. F. 1. Autoimmune: Addison’s disease, polyglandular endocrinopathy 2.

1	E. 1. 2. 3. F. 1. Autoimmune: Addison’s disease, polyglandular endocrinopathy 2. Infectious: HIV, cytomegalovirus, tuberculosis, disseminated fungal infection 3. Infiltrative: amyloidosis, malignancy, metastatic cancer 4. Drug-associated: heparin, low-molecular-weight heparin 5. Hereditary: adrenal hypoplasia congenita, congenital lipoid adrenal hyperplasia, aldosterone synthase deficiency 6. Adrenal hemorrhage or infarction, including in antiphospholipid syndrome

1	Pseudohyperkalemia Hyperkalemia should be distinguished from factitious hyperkalemia or “pseudohyperkalemia,” an artifactual increase in serum K+ due to the release of K+ during or after venipuncture. Pseudohyperkalemia can occur in the setting of excessive muscle activity during venipuncture (e.g., fist clenching), a marked increase in cellular elements (thrombocytosis, leukocytosis, and/or erythrocytosis) with in vitro efflux of K+, and acute anxiety during venipuncture with respiratory alkalosis and redistributive hyperkalemia. Cooling of blood following venipuncture is another cause, due to reduced cellular uptake; the converse is the increased uptake of K+ by cells at high ambient temperatures, leading to normal values for hyperkalemic patients and/or to spurious hypokalemia in normokalemic patients. Finally, there are multiple genetic subtypes of hereditary pseudohyperkalemia, caused by increases in the passive K+ permeability of erythrocytes. For example, causative mutations

1	patients. Finally, there are multiple genetic subtypes of hereditary pseudohyperkalemia, caused by increases in the passive K+ permeability of erythrocytes. For example, causative mutations have been described in the red cell anion exchanger (AE1, encoded by the SLC4A1 gene), leading to reduced red cell anion transport, hemolytic anemia, the acquisition of a novel AE1mediated K+ leak, and pseudohyperkalemia.

1	Redistribution and Hyperkalemia Several different mechanisms can induce an efflux of intracellular K+ and hyperkalemia. Acidemia is associated with cellular uptake of H+ and an associated efflux of K+; it is thought that this effective K+-H+ exchange serves to help maintain extracellular pH. Notably, this effect of acidosis is limited to non– anion gap causes of metabolic acidosis and, to a lesser extent, respiratory causes of acidosis; hyperkalemia due to an acidosis-induced shift of potassium from the cells into the ECF does not occur in the anion gap acidoses lactic acidosis and ketoacidosis. Hyperkalemia due to hypertonic mannitol, hypertonic saline, and intravenous immune globulin is generally attributed to a “solvent drag” effect, as water moves out of cells along the osmotic gradient. Diabetics are also prone to osmotic hyperkalemia in response to intravenous hypertonic glucose, when given without adequate insulin. Cationic amino acids, specifically lysine, arginine, and the

1	Diabetics are also prone to osmotic hyperkalemia in response to intravenous hypertonic glucose, when given without adequate insulin. Cationic amino acids, specifically lysine, arginine, and the structurally related drug epsilonaminocaproic acid, cause efflux of K+ and hyperkalemia, through an effective cation-K+ exchange of unknown identity and mechanism. Digoxin inhibits Na+/K+-ATPase and impairs the uptake of K+ by 310 skeletal muscle, such that digoxin overdose predictably results in hyperkalemia. Structurally related glycosides are found in specific plants (e.g., yellow oleander, foxglove) and in the cane toad, Bufo marinus (bufadienolide); ingestion of these substances and extracts thereof can also cause hyperkalemia. Finally, fluoride ions also inhibit Na+/K+-ATPase, such that fluoride poisoning is typically associated with hyperkalemia. Succinylcholine depolarizes muscle cells, causing an efflux of K+ through acetylcholine receptors (AChRs). The use of this agent is

1	that fluoride poisoning is typically associated with hyperkalemia. Succinylcholine depolarizes muscle cells, causing an efflux of K+ through acetylcholine receptors (AChRs). The use of this agent is contraindicated in patients who have sustained thermal trauma, neuromuscular injury, disuse atrophy, mucositis, or prolonged immobilization. These disorders share a marked increase and redistribution of AChRs at the plasma membrane of muscle cells; depolarization of these upregulated AChRs by succinylcholine leads to an exaggerated efflux of K+ through the receptor-associated cation channels, resulting in acute hyperkalemia.

1	Hyperkalemia Caused by Excess Intake or Tissue Necrosis Increased intake of even small amounts of K+ may provoke severe hyperkalemia in patients with predisposing factors; hence, an assessment of dietary intake is crucial. Foods rich in potassium include tomatoes, bananas, and citrus fruits; occult sources of K+, particularly K+-containing salt substitutes, may also contribute significantly. Iatrogenic causes include simple overreplacement with K+-Cl– or the administration of a potassium-containing medication (e.g., K+-penicillin) to a susceptible patient. Red cell transfusion is a well-described cause of hyperkalemia, typically in the setting of massive transfusions. Finally, severe tissue necrosis, as in acute tumor lysis syndrome and rhabdomyolysis, will predictably cause hyperkalemia from the release of intracellular K+.

1	Hypoaldosteronism and Hyperkalemia Aldosterone release from the adrenal gland may be reduced by hyporeninemic hypoaldosteronism, medications, primary hypoaldosteronism, or isolated deficiency of ACTH (secondary hypoaldosteronism). Primary hypoaldosteronism may be genetic or acquired (Chap. 406) but is commonly caused by autoimmunity, either in Addison’s disease or in the context of a polyglandular endocrinopathy. HIV has surpassed tuberculosis as the most important infectious cause of adrenal insufficiency. The adrenal involvement in HIV disease is usually subclinical; however, adrenal insufficiency may be precipitated by stress, drugs such as ketoconazole that inhibit steroidogenesis, or the acute withdrawal of steroid agents such as megestrol.

1	Hyporeninemic hypoaldosteronism is a very common predisposing factor in several overlapping subsets of hyperkalemic patients: diabetics, the elderly, and patients with renal insufficiency. Classically, patients should have suppressed PRA and aldosterone; approximately 50% have an associated acidosis, with a reduced renal excretion of NH4+, a positive urinary anion gap, and urine pH <5.5. Most patients are volume expanded, with secondary increases in circulating atrial natriuretic peptide (ANP) that inhibit both renal renin release and adrenal aldosterone release.

1	Renal Disease and Hyperkalemia Chronic kidney disease and end-stage kidney disease are very common causes of hyperkalemia, due to the associated deficit or absence of functioning nephrons. Hyperkalemia is more common in oliguric acute kidney injury; distal tubular flow rate and Na+ delivery are less limiting factors in nonoliguric patients. Hyperkalemia out of proportion to GFR can also be seen in the context of tubulointerstitial disease that affects the distal nephron, such as amyloidosis, sickle cell anemia, interstitial nephritis, and obstructive uropathy.

1	Hereditary renal causes of hyperkalemia have overlapping clinical features with hypoaldosteronism, hence the diagnostic label pseudohypoaldosteronism (PHA). PHA type I (PHA-I) has both an autosomal recessive and an autosomal dominant form. The autosomal dominant form is due to loss-of-function mutations in the MLR; the recessive form is caused by various combinations of mutations in the three subunits of ENaC, resulting in impaired Na+ channel activity in principal cells and other tissues. Patients with recessive PHA-I suffer from lifelong salt wasting, hypotension, and hyperkalemia, whereas the phenotype of autosomal dominant PHA-I due to MLR dysfunction improves in adulthood. PHA type II (PHA-II; also known as hereditary hypertension with hyperkalemia) is in every respect the mirror image of PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases GS caused by loss of function in NCC, the thiazide-sensitive Na+-Cl– cotransporter (see above); the clinical phenotype includes hypertension, hyperkalemia, hyperchloremic metabolic acidosis, suppressed PRA and aldosterone, hypercalciuria, and reduced bone density. PHA-II thus behaves like a gain of function in NCC, and treatment with thiazides results in resolution of the entire clinical phenotype. However, the NCC gene is not directly involved in PHA-II, which is caused by mutations in the WNK1 and WNK4 serine-threonine kinases or the upstream Kelch-like 3 (KLHL3) and Cullin 3 (CUL3), two components of an E3 ubiquitin ligase complex that regulates these kinases; these proteins collectively regulate NCC activity, with PHA-II-associated activation of the transporter.

1	Medication-Associated Hyperkalemia Most medications associated with hyperkalemia cause inhibition of some component of the reninangiotensin-aldosterone axis. ACE inhibitors, angiotensin receptor blockers, renin inhibitors, and MLRs are predictable and common causes of hyperkalemia, particularly when prescribed in combination. The oral contraceptive agent Yasmin-28 contains the progestin drospirenone, which inhibits the MLR and can cause hyperkalemia in susceptible patients. Cyclosporine, tacrolimus, NSAIDs, and cyclooxygenase 2 (COX2) inhibitors cause hyperkalemia by multiple mechanisms, but share the ability to cause hyporeninemic hypoaldosteronism. Notably, most drugs that affect the renin-angiotensin-aldosterone axis also block the local adrenal response to hyperkalemia, thus attenuating the direct stimulation of aldosterone release by increased plasma K+ concentration.

1	Inhibition of apical ENaC activity in the distal nephron by amiloride and other K+-sparing diuretics results in hyperkalemia, often with a voltage-dependent hyperchloremic acidosis and/or hypovolemic hyponatremia. Amiloride is structurally similar to the antibiotics trimethoprim (TMP) and pentamidine, which also block ENaC; risk factors for TMP-associated hyperkalemia include the administered dose, renal insufficiency, and hyporeninemic hypoaldosteronism. Indirect inhibition of ENaC at the plasma membrane is also a cause of drug-associated hyperkalemia; nafamostat, a protease inhibitor used in some countries for the management of pancreatitis, inhibits aldosteroneinduced renal proteases that activate ENaC by proteolytic cleavage.

1	Clinical Features Hyperkalemia is a medical emergency due to its effects on the heart. Cardiac arrhythmias associated with hyperkalemia include sinus bradycardia, sinus arrest, slow idioventricular rhythms, ventricular tachycardia, ventricular fibrillation, and asystole. Mild increases in extracellular K+ affect the repolarization phase of the cardiac action potential, resulting in changes in T-wave morphology; further increase in plasma K+ concentration depresses intracardiac conduction, with progressive prolongation of the PR and QRS intervals. Severe hyperkalemia results in loss of the P wave and a progressive widening of the QRS complex; development of a sine-wave sinoventricular rhythm suggests impending ventricular fibrillation or asystole. Hyperkalemia can also cause a type I Brugada pattern in the electrocardiogram (ECG), with a pseudo–right bundle branch block and persistent coved ST segment elevation in at least two precordial leads. This hyperkalemic Brugada’s sign occurs

1	pattern in the electrocardiogram (ECG), with a pseudo–right bundle branch block and persistent coved ST segment elevation in at least two precordial leads. This hyperkalemic Brugada’s sign occurs in critically ill patients with severe hyperkalemia and can be differentiated from genetic Brugada’s syndrome by an absence of P waves, marked QRS widening, and an abnormal QRS axis. Classically, the electrocardiographic manifestations in hyperkalemia progress from tall peaked T waves (5.5–6.5 mM), to a loss of P waves (6.5–7.5 mM) to a widened QRS complex (7.0–8.0 mM), and, ultimately, a to a sine wave pattern (>8.0 mM). However, these changes are notoriously insensitive, particularly in patients with chronic kidney disease or end-stage renal disease.

1	Hyperkalemia from a variety of causes can also present with ascending paralysis, denoted secondary hyperkalemic paralysis to differentiate it from familial hyperkalemic periodic paralysis (HYPP). The presentation may include diaphragmatic paralysis and respiratory failure. Patients with familial HYPP develop myopathic weakness during hyperkalemia induced by increased K+ intake or rest after heavy exercise. Depolarization of skeletal muscle by hyperkalemia unmasks an inactivation defect in skeletal Na+ channel; autosomal dominant mutations in the SCN4A gene encoding this channel are the predominant cause.

1	Within the kidney, hyperkalemia has negative effects on the ability to excrete an acid load, such that hyperkalemia per se can contribute to metabolic acidosis. This defect appears to be due in part to competition between K+ and NH4+ for reabsorption by the TALH and subsequent countercurrent multiplication, ultimately reducing the medullary gradient for NH3/NH4 excretion by the distal nephron. Regardless of the underlying mechanism, restoration of normokalemia can, in many instances, correct hyperkalemic metabolic acidosis.

1	Diagnostic Approach The first priority in the management of hyperkalemia is to assess the need for emergency treatment, followed by a comprehensive workup to determine the cause (Fig. 63-8). History and physical examination should focus on medications, diet and dietary supplements, risk factors for kidney failure, reduction in urine output, blood pressure, and volume status. Initial laboratory 311 tests should include electrolytes, BUN, creatinine, serum osmolality, Mg2+ and Ca2+, a complete blood count, and urinary pH, osmolality, creatinine, and electrolytes. A urine Na+ concentration of <20 mM indicates that distal Na+ delivery is a limiting factor in K+ excretion; volume repletion with 0.9% saline or treatment with furosemide may be effective in reducing plasma K+ concentration. Serum and urine osmolality are required for calculation of the transtubular K+ gradient (TTKG) (Fig. 63-8). The expected values of the TTKG are largely based on historical data, and are <3 in the presence

1	and urine osmolality are required for calculation of the transtubular K+ gradient (TTKG) (Fig. 63-8). The expected values of the TTKG are largely based on historical data, and are <3 in the presence of hypokalemia and >7–8 in the presence of hyperkalemia.

1	CHAPTER 63 Fluid and Electrolyte Disturbances Hyperkalemia(Serum K+ ˜5.5 mmol/l) History, physical examination & basic laboratory tests Decreased urinary K+ excretion (<40 mmol/day) Urine electrolytes TTKG Evidence of increased potassium load Urine Na+ <25 mmol/L Reduced tubular flow Reduced distal K+ secretion (GFR >20 ml/min) Advanced kidney failure (GFR ˜20 ml/min) Reduced ECV TTKG <8 (Tubular resistance) TTKG ˜8 Low aldosterone Renin 9°-Fludrocortisone Treat accordingly and re-evaluate Pseudohyperkalemia? Evidence of transcellular shift No further actionK+ ˜6.0 or ECG changes Emergency therapy Yes Yes Decreased distal Na+ delivery Yes Treat accordingly and re-evaluate -Hypertonicity (e.g., mannitol) -Hyperglycemia -Succinylcholine -˛-aminocaproic acid -Digoxin -˝-blockers -Metabolic acidosis (non-organic) -Arginine or lysine infusion -Hyperkalemic periodic paralysis -˙Insulin -Exercise Yes No No No No >8 <5 High Low Drugs -Amiloride -Spironolactone -Triamterene -Trimethoprim

1	acidosis (non-organic) -Arginine or lysine infusion -Hyperkalemic periodic paralysis -˙Insulin -Exercise Yes No No No No >8 <5 High Low Drugs -Amiloride -Spironolactone -Triamterene -Trimethoprim -Pentamidine -Eplerenone -Drospirenone -Calcineurin inhibitors Other causes -Tubulointerstitial diseases -Urinary tract obstruction -PHA type I -PHA type II -Sickle cell disease -Renal transplant -SLE -Primary adrenal insufficiency -Isolated aldosterone deficiency -Heparin/ LMW heparin -ACE-I / ARB -Ketoconazole -Diabetes mellitus -Acute GN -Tubulointerstitial diseases -PHA type II -NSAIDs -˝-Blockers FIguRE 63-8 The diagnostic approach to hyperkalemia. See text for details. ACE-I, angiotensin-converting enzyme inhibitor; ARB, angioten-sin II receptor blocker; CCD, cortical collecting duct; ECG, electrocardiogram; ECV, effective circulatory volume; GFR, glomerular filtration rate; GN, glomerulonephritis; HIV, human immunodeficiency virus; LMW heparin, low-molecular-weight heparin; NSAIDs,

1	electrocardiogram; ECV, effective circulatory volume; GFR, glomerular filtration rate; GN, glomerulonephritis; HIV, human immunodeficiency virus; LMW heparin, low-molecular-weight heparin; NSAIDs, nonsteroidal anti-inflammatory drugs; PHA, pseudohypoaldosteronism; SLE, systemic lupus erythematosus; TTKG, transtubular potassium gradient. (Used with permission from DB Mount, K Zandi-Nejad K: Disorders of potassium balance, in Brenner and Rector’s The Kidney, 8th ed, BM Brenner [ed]. Philadelphia, W.B. Saunders & Company, 2008, pp 547-587.)

1	Electrocardiographic manifestations of hyperkalemia should be considered a medical emergency and treated urgently. However, patients with significant hyperkalemia (plasma K+ concentration ≥6.5 mM) in the absence of ECG changes should also be aggressively managed, given the limitations of ECG changes as a predictor of cardiac toxicity. Urgent management of hyperkalemia includes admission to the hospital, continuous cardiac monitoring, and immediate treatment. The treatment of hyperkalemia is divided into three stages: 1.

1	Immediate antagonism of the cardiac effects of hyperkalemia. Intravenous calcium serves to protect the heart, whereas other measures are taken to correct hyperkalemia. Calcium raises the action potential threshold and reduces excitability, without changing the resting membrane potential. By restoring the difference between resting and threshold potentials, calcium reverses the depolarization blockade due to hyperkalemia. The recommended dose is 10 mL of 10% calcium gluconate (3–4 mL of calcium chloride), infused intravenously over 2–3 min with cardiac monitoring. The effect of the infusion starts in 1–3 min and lasts 30–60 min; the dose should be repeated if there is no change in ECG findings or if they recur after initial improvement. Hypercalcemia potentiates the cardiac toxicity of digoxin; hence, intravenous calcium should be used with extreme caution in patients taking this medication; if judged necessary, 10 mL of 10% calcium gluconate can be added to 100 mL of 5% dextrose in

1	hence, intravenous calcium should be used with extreme caution in patients taking this medication; if judged necessary, 10 mL of 10% calcium gluconate can be added to 100 mL of 5% dextrose in water and infused over 20–30 min to avoid acute hypercalcemia.

1	2. Rapid reduction in plasma K+ concentration by redistribution into cells. Insulin lowers plasma K+ concentration by shifting K+ into cells. The recommended dose is 10 units of intravenous regular insulin followed immediately by 50 mL of 50% dextrose (D50W, 25 g of glucose total); the effect begins in 10–20 min, peaks at 30–60 min, and lasts for 4–6 h. Bolus D50W without insulin is never appropriate, given the risk of acutely worsening hyperkalemia due to the osmotic effect of hypertonic glucose. Hypoglycemia is common with insulin plus glucose; hence, this should be followed by an infusion of 10% dextrose at 50–75 mL/h, with close monitoring of plasma glucose concentration. In hyperkalemic patients with glucose concentrations of ≥200–250 mg/dL, insulin should be administered without glucose, again with close monitoring of glucose concentrations.

1	β2-agonists, most commonly albuterol, are effective but underused agents for the acute management of hyperkalemia. Albuterol and insulin with glucose have an additive effect on plasma K+ concentration; however, ~20% of patients with end-stage renal disease (ESRD) are resistant to the effect of β2-agonists; hence, these drugs should not be used without insulin. The recommended dose for inhaled albuterol is 10–20 mg of nebulized albuterol in 4 mL of normal saline, inhaled over 10 min; the effect starts at about 30 min, reaches its peak at about 90 min, and lasts for 2–6 h. Hyperglycemia is a side effect, along with tachycardia. β2-Agonists should be used with caution in hyperkalemic patients with known cardiac disease.

1	Intravenous bicarbonate has no role in the acute treatment of hyperkalemia, but may slowly attenuate hyperkalemia with sustained administration over several hours. It should not be given repeatedly as a hypertonic intravenous bolus of undiluted ampules, given the risk of associated hypernatremia, but should instead be infused in an isotonic or hypotonic fluid (e.g., 150 mEqu in 1 L of D5W). In patients with metabolic acidosis, a delayed drop in plasma K+ concentration can be seen after 4–6 h of isotonic bicarbonate infusion. 3. Removal of potassium. This is typically accomplished using cation exchange resins, diuretics, and/or dialysis. The cation exchange resin sodium polystyrene sulfonate (SPS) exchanges Na+ for K+ in the gastrointestinal tract and increases the fecal excretion of K+; alternative calcium-based resins, when available, may

1	Part 2 Cardinal Manifestations and Presentation of Diseases be more appropriate in patients with an increased ECFV. The recommended dose of SPS is 15–30 g of powder, almost always given in a premade suspension with 33% sorbitol. The effect of SPS on plasma K+ concentration is slow; the full effect may take up to 24 h and usually requires repeated doses every 4–6 h. Intestinal necrosis, typically of the colon or ileum, is a rare but usually fatal complication of SPS. Intestinal necrosis is more common in patients administered SPS via enema and/or in patients with reduced intestinal motility (e.g., in the postoperative state or after treatment with opioids). The coadministration of SPS with sorbitol appears to increase the risk of intestinal necrosis; however, this complication can also occur with SPS alone. If SPS without sorbitol is not available, clinicians must consider whether treatment with SPS in sorbitol is absolutely necessary. The low but real risk of intestinal necrosis with

1	with SPS alone. If SPS without sorbitol is not available, clinicians must consider whether treatment with SPS in sorbitol is absolutely necessary. The low but real risk of intestinal necrosis with SPS, which can sometimes be the only available or appropriate therapy for the removal of potassium, must be weighed against the delayed onset of efficacy. Whenever possible, alternative therapies for the acute management of hyperkalemia (i.e., aggressive redistributive therapy, isotonic bicarbonate infusion, diuretics, and/or hemodialysis) should be used instead of SPS.

1	Therapy with intravenous saline may be beneficial in hypovolemic patients with oliguria and decreased distal delivery of Na+, with the associated reductions in renal K+ excretion. Loop and thiazide diuretics can be used to reduce plasma K+ concentration in volume-replete or hypervolemic patients with sufficient renal function for a diuretic response; this may need to be combined with intravenous saline or isotonic bicarbonate to achieve or maintain euvolemia.

1	Hemodialysis is the most effective and reliable method to reduce plasma K+ concentration; peritoneal dialysis is considerably less effective. Patients with acute kidney injury require temporary, urgent venous access for hemodialysis, with the attendant risks; in contrast, patients with ESRD or advanced chronic kidney disease may have a preexisting venous access. The amount of K+ removed during hemodialysis depends on the relative distribution of K+ between ICF and ECF (potentially affected by prior therapy for hyperkalemia), the type and surface area of the dialyzer used, dialysate and blood flow rates, dialysate flow rate, dialysis duration, and the plasma-to-dialysate K+ gradient. Fluid and Electrolyte Imbalances and Acid-Base Disturbances: Case Examples David B. Mount, Thomas D. DuBose, Jr. CASE 1 64e

1	Fluid and Electrolyte Imbalances and Acid-Base Disturbances: Case Examples David B. Mount, Thomas D. DuBose, Jr. CASE 1 64e A 23-year-old woman was admitted with a 3-day history of fever, cough productive of blood-tinged sputum, confusion, and orthostasis. Past medical history included type 1 diabetes mellitus. A physical examination in the emergency department indicated postural hypo-tension, tachycardia, and Kussmaul respiration. The breath was noted to smell of “acetone.” Examination of the thorax suggested consolidation in the right lower lobe. Sodium 130 meq/L Potassium 5.0 meq/L Chloride 96 meq/L CO2 14 meq/L Blood urea nitrogen (BUN) 20 mg/dL Creatinine 1.3 mg/dL Glucose 450 mg/dL Pneumonic infiltrate, right lower lobe The diagnosis of the acid-base disorder should proceed in a stepwise fashion: 1. The normal anion gap (AG) is 8–10 meq/L, but in this case, the AG is elevated (20 meq/L). Therefore, the change in AG (ΔAG) = ~10 meq/L. 2.

1	The normal anion gap (AG) is 8–10 meq/L, but in this case, the AG is elevated (20 meq/L). Therefore, the change in AG (ΔAG) = ~10 meq/L. 2. Compare the ΔAG and the Δ[HCO3−]. In this case, the ΔAG, as noted above, is 10, and the Δ[HCO3−] (25 – 14) is 11. Therefore, the increment in the AG is approximately equal to the decrement in bicarbonate. 3. Estimate the respiratory compensatory response. In this case, the predicted Paco2 for an [HCO3−] of 14 should be approximately 29 mmHg. This value is obtained by adding 15 to the measured [HCO3−] (15 + 14 = 29) or by calculating the predicted Paco2 from the Winter equation: 1.5 × [HCO3−] + 8. In either case, the predicted value for Paco2 of 29 is significantly higher than the measured value of 24. Therefore, the prevailing Paco2 exceeds the range for compensation alone and is too low, indicating a superimposed respiratory alkalosis. 4.

1	4. Therefore, this patient has a mixed acid-base disturbance with two components: (a) high AG acidosis secondary to ketoacidosis and (b) respiratory alkalosis (which was secondary to community-acquired pneumonia in this case). The latter resulted in an additional compo-64e-1 nent of hyperventilation that exceeded the compensatory response driven by metabolic acidosis, explaining the normal pH. The finding of respiratory alkalosis in the setting of a high AG acidosis suggests another cause of the respiratory component. Respiratory alkalosis frequently accompanies community-acquired pneumonia. The clinical features in this case include hyperglycemia, hypovolemia, ketoacidosis, central nervous system (CNS) signs of confusion, and superimposed pneumonia. This clinical scenario is consistent with diabetic ketoacidosis (DKA) developing in a patient with known type 1 diabetes mellitus. Infections in DKA are common and may be a precipitating feature in the development of ketoacidosis.

1	The diagnosis of DKA is usually not challenging but should be considered in all patients with an elevated AG and metabolic acidosis. Hyperglycemia and ketonemia (positive acetoacetate at a dilution of 1:8 or greater) are sufficient criteria for diagnosis in patients with type 1 diabetes mellitus. The Δ[HCO3−] should approximate the increase in the plasma AG (ΔAG), but this equality can be modified by several factors. For example, the ΔAG will often decrease with IV hydration, as glomerular filtration increases and ketones are excreted into the urine. The decrement in plasma sodium is the result of hyperglycemia, which induces the movement of water into the extracellular compartment from the intracellular compartment of cells that require insulin for the transport of glucose. Additionally, a natriuresis occurs in response to an osmotic diuresis associated with hyperglycemia. Moreover, in patients with DKA, thirst is very common and water ingestion often continues. The plasma potassium

1	a natriuresis occurs in response to an osmotic diuresis associated with hyperglycemia. Moreover, in patients with DKA, thirst is very common and water ingestion often continues. The plasma potassium concentration is usually mildly elevated, but in the face of acidosis, and as a result of the ongoing osmotic diuresis, a significant total-body deficit of potassium is almost always present. Recognition of the total-body deficit of potassium is critically important. The inclusion of potassium replacement in the therapeutic regimen at the appropriate time and with the appropriate indications (see below) is essential. Volume depletion is a very common finding in DKA and is a pivotal component in the pathogenesis of the disorder.

1	Patients with DKA often have a sustained and significant deficit of sodium, potassium, water, bicarbonate, and phosphate. The general approach to treatment requires attention to all of these abnormalities. Successful treatment of DKA involves a stepwise approach, as follows: 1. Replace extracellular fluid (ECF) volume deficits. Because most patients present with actual or relative hypotension and, at times, impending shock, the initial fluid administered should be 0.9% NaCl infused rapidly until the systolic blood pressure is >100 mmHg or until 2–3 L cumulatively have been administered. During the initial 2–3 h of infusion of saline, the decline in blood glucose can be accounted for by dilution and increased renal excretion. Glucose should be added to the infusion as D5 normal saline (NS) or D5 0.45% NS once the plasma glucose declines to 230 mg/dL or below. 2.

1	2. Abate the production of ketoacids. Regular insulin is required during DKA as an initial bolus of 0.1 U/kg body weight (BW) IV, followed immediately by a continuous infusion of 0.1 U/kg BW per hour in NS. The effectiveness of IV insulin (not subcutaneous) can be tracked by observing the decline in plasma ketones. Because the increment in the AG above the normal value of 10 meq/L represents accumulated ketoacids in DKA, the disappearance of ketoacid anions is reflected by the narrowing and eventual correction of the AG. Typically, the plasma AG returns to normal within 8–12 h. 3. Replace potassium deficits. Although patients with DKA often have hyperkalemia due to insulin deficiency, they are usually severely K+ depleted. KCl (20 meq/L) should be added to each liter of IV fluids when urine output is established and insulin has been administered. 4.

1	4. Correct the metabolic acidosis. The plasma bicarbonate concentration will usually not increase for several hours because of dilution from administered IV NaCl. The plasma [HCO−] approaches 18 meq/L once ketoacidosis disappears. Sodium bicarbonate therapy is often not recommended or necessary and is contraindicated CHAPTER 64e Fluid and Electrolyte Imbalances and Acid-Base Disturbances: Case Examples 64e-2 for children. Bicarbonate is administered to adults with DKA for extreme acidemia (pH <7.1); for elderly patients (>70 years old), a threshold pH of 7.20 is recommended. Sodium bicarbonate, if administered, should only be given in small amounts. Because ketoacids are metabolized in response to insulin therapy, bicarbonate will be added to the ECF as ketoacids are converted. Overshoot alkalosis may occur from the combination of exogenously administered sodium bicarbonate plus metabolic production of bicarbonate. 5.

1	5. Phosphate. In the first 6–8 h of therapy, it may be necessary to infuse potassium with phosphate because of the unmasking of phosphate depletion during combined insulin and glucose therapy. The latter drives phosphate into the cell. Therefore, in patients with DKA, the plasma phosphate level should be followed closely, but phosphate should never be replaced empirically. Phosphate should be administered to patients with a declining plasma phosphate once the phosphate level declines into the low-normal level. Therapy is advisable in the form of potassium phosphate at a rate of 6 mmol/h. 6. Always seek underlying factors, such as infection, myocardial infarction, pancreatitis, cessation of insulin therapy, or other events, responsible for initiating DKA. The case presented here is illustrative of this common scenario. 7.

1	7. Volume overexpansion with IV fluid administration is not uncommon and contributes to the development of hyperchloremic acidosis during the later stages of treatment of DKA. Volume overexpansion should be avoided. A 25-year-old man with a 6-year history of HIV-AIDS complicated recently by Pneumocystis jiroveci pneumonia (PCP) was treated with intravenous trimethoprim-sulfamethoxazole (20 mg trimethoprim/kg per day). On day 4 of treatment, the following laboratory data were PART 2 Cardinal Manifestations and Presentation of Diseases H2O AQP-2AQP-3, 4 H2O

1	FIGuRE 64e-1 Water, sodium, potassium, ammonia, and proton transport in principal cells (PC) and adjacent type A intercalated cells (A-IC). Water is absorbed down the osmotic gradient by principal cells, through the apical aquaporin-2 (AQP-2) and basolateral aquaporin-3 (AQP-3) and aquaporin-4 (AQP-4) channels. The absorption of Na+ via the amiloride-sensitive epithelial sodium channel (ENaC) generates a lumen-negative potential difference, which drives K+ excretion through the apical secretory K+ channel, ROMK (renal outer medullary K+ channel), and/or the flow-dependent maxi-K channel. Transepithelial ammonia (NH3) transport and proton transport occur in adjacent type A intercalated cells, via apical and basolateral ammonia channels and apical H+-ATPase pumps, respectively; NH4+ is ultimately excreted in the urine, in the defense of systemic pH. Electrogenic proton secretion by type A intercalated cells is also affected by the lumen-negative potential difference generated by the

1	excreted in the urine, in the defense of systemic pH. Electrogenic proton secretion by type A intercalated cells is also affected by the lumen-negative potential difference generated by the adjacent principal cells, such that reduction of this lumen-negative electrical gradient can reduce H+ excretion. Type A intercalated cells also reabsorb filtered K+ in potassium-deficient states, via apical H+/K+-ATPase.

1	hyperkalemia and metabolic acidosis is not uncommon in this setting. H+ secretion via apical H+-ATPase pumps in adjacent type A intercalated cells (Fig. 64e-1) is also electrogenic, such that the reduction in the lumen-negative potential difference due to trimethoprim inhibits distal H+ secretion; this is often referred to as a “voltage defect” form of dRTA. Systemic hyperkalemia also suppresses renal ammoniagenesis, ammonium excretion, and, thus, acid excretion; i.e., hyperkalemia per se has multiple effects on urinary acidification.

1	The inhibitory effect of trimethoprim on K+ and H+ secretion in the cortical collecting tubule follows a dose-response relationship, and therefore, the higher doses of this agent used in HIV/AIDS patients with PCP or in deep tissue infections with methicillin-resistant Staphylococcus aureus (MRSA) result in a higher prevalence of hyperkalemia and acidosis. Conventional does of trimethoprim can also induce hyperkalemia and/or acidosis in predisposed patients, in particular the elderly, patients with renal insufficiency, and/or those with baseline hyporeninemic hypoaldosteronism. One means by which to assess the role of the kidney in the development of hyperkalemia is to calculate, from a spot urine and coincident plasma sample, the transtubular potassium gradient (TTKG). The TTKG is calculated as (Posmol ). The expected values of the TTKG are <3 in the presence of hypokalemia (see also Case 7 and Case 8) and >7–8 in the presence of hyperkalemia.

1	obtained: 135 60 6.5 15 110 43 15 0 7.30 5.5 14 — 0.9 — 268 270 What caused the hyperkalemia and metabolic acidosis in this patient? What other medications may be associated with a similar presentation? How does one use the urine electrolyte data to determine if the hyperkalemia is of renal origin or due to a shift from the cell to the extracellular compartment?

1	Hyperkalemia occurs in 15–20% of hospitalized patients with HIV/ AIDS. The usual causes are either adrenal insufficiency, the syndrome of hyporeninemic hypoaldosteronism, or one of several drugs, including trimethoprim, pentamidine, nonsteroidal anti-inflammatory drugs, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers, spironolactone, and eplerenone. Trimethoprim is usually given in combination with sulfamethoxazole or dapsone for PCP and, on average, increases the plasma K+ concentration by about 1 meq/L; however, the hyperkalemia may be severe. Trimethoprim is structurally and chemically related to amiloride and triamterene and, in this way, may function as a potassium-sparing diuretic. This effect results in inhibition of the epithelial sodium channel (ENaC) in the principal cell of the collecting duct. By blocking the Na+ channel, K+ secretion is also inhibited; K+ secretion is dependent on the lumen-negative potential difference generated by Na+

1	(ENaC) in the principal cell of the collecting duct. By blocking the Na+ channel, K+ secretion is also inhibited; K+ secretion is dependent on the lumen-negative potential difference generated by Na+ entry through the ENaC (Fig. 64e-1).

1	Trimethoprim is associated with a non-AG acidosis that parallels development of hyperkalemia such that the co-occurrence of In this case, the value for the TTKG of approximately 2 indicates that renal excretion of potassium is abnormally low for the prevailing hyperkalemia. Therefore, the inappropriately low TTKG indicates that the hyperkalemia is of renal tubular origin.

1	Knowledge of the factors controlling potassium secretion by the cortical collecting tubule principal cell can be helpful in understanding the basis for treatment of the hyperkalemia, especially if discontinuing the offending agent is not a reasonable clinical option. Potassium secretion is encouraged by a higher urine flow rate, increased distal delivery of sodium, distal delivery of a poorly reabsorbed anion (such as bicarbonate), and/or administration of a loop diuretic. Therefore, the approach to treatment in this patient should include intravenous 0.9% NaCl to expand the ECF and deliver more Na+ and Cl− to the cortical collecting tubule. In addition, because the trimethoprim molecule must be protonated to inhibit ENaC, alkalinization of the renal tubule fluid enhances distal tubular K+ secretion. As an alternative to inducing bicarbonaturia to assist in potassium secretion, a carbonic anhydrase inhibitor may be administered to induce a kaliuresis. However, in the case presented

1	K+ secretion. As an alternative to inducing bicarbonaturia to assist in potassium secretion, a carbonic anhydrase inhibitor may be administered to induce a kaliuresis. However, in the case presented here, for acetazolamide to be effective, the non-AG metabolic acidosis in this patient would first need to be corrected; Acetazolamide would, thus, require the coadministration of intravenous sodium bicarbonate for maximal benefit. Finally, systemic hyperkalemia directly suppresses renal ammoniagenesis, ammonium excretion, and, thus, acid excretion. Correcting the hyperkalemia with a potassium-binding resin (Kayexalate) is sometimes appropriate in these patients; the subsequent decline in the plasma K+ concentration will also increase urinary ammonium excretion, helping correct the acidosis.

1	A 63-year-old man was admitted to the intensive care unit (ICU) with a severe aspiration pneumonia. Past medical history included schizophrenia, for which he required institutional care; treatment had included neuroleptics and intermittent lithium, the latter restarted 6 months before admission. The patient was treated with antibiotics and intubated for several days, with the development of polyuria (3–5 L/d), hypernatremia, and acute renal insufficiency; the peak plasma Na+ concentration was 156 meq/L, and peak creatinine was 2.6 mg/dL. Urine osmolality was measured once and reported as 157 mOsm/kg, with a coincident plasma osmolality of 318 mOsm/kg. Lithium was stopped on admission to the ICU. On physical examination, the patient was alert, extubated, and thirsty. Weight was 97.5 kg. Urine output for the previous 24 h had been 3.4 L, with an IV intake of 2 L/d of D5W.

1	On physical examination, the patient was alert, extubated, and thirsty. Weight was 97.5 kg. Urine output for the previous 24 h had been 3.4 L, with an IV intake of 2 L/d of D5W. After 3 days of intravenous hydration, a water deprivation test was performed. A single dose of 2 μg IV desmopressin (DDAVP) was given at 9 h (+9): Why did the patient develop hypernatremia, polyuria, and acute renal insufficiency? What does the water deprivation test demonstrate? What is the underlying pathophysiology of this patient’s hypernatremic syndrome?

1	This patient became polyuric after admission to the ICU with severe pneumonia, developing significant hypernatremia and acute renal insufficiency. Polyuria can result from either an osmotic diuresis or a water diuresis. An osmotic diuresis can be caused by excessive excretion of Na+-Cl−, mannitol, glucose, and/or urea, with a daily solute excretion of >750–1000 mOsm/d (>15 mOsm/kg body water per day). In this case, however, the patient was excreting large volumes of very hypotonic urine, with a urine osmolality that was substantially lower than that of plasma; this, by definition, was a water diuresis, resulting in inappropriate excretion of free water and hypernatremia. The appropriate response to hypernatremia and a plasma osmolality >295 mOsm/kg is an increase in circulating vasopressin (AVP) and the excretion of low volumes (<500 mL/d) of maximally concentrated urine, i.e., urine with osmolality >800 mOsm/kg. This patient’s response to hypernatremia was clearly inappropriate, due

1	(AVP) and the excretion of low volumes (<500 mL/d) of maximally concentrated urine, i.e., urine with osmolality >800 mOsm/kg. This patient’s response to hypernatremia was clearly inappropriate, due to either a loss of circulating AVP (central diabetes insipidus [CDI]) or renal resistance to AVP (nephrogenic diabetes insipidus [NDI]). Ongoing loss of free water was sufficiently severe in this patient that absolute hypovolemia ensued, despite the fact that approximately two-thirds of the excreted water was derived from the intracellular fluid compartment rather than the ECF compartment. Hypovolemia led to an acute decrease in the glomerular filtration rate (GFR), i.e., acute renal insufficiency, with gradual improvement following hydration (see below).

1	Following the correction of hypernatremia and acute renal insufficiency with appropriate hydration (see below), the patient was subjected to a water deprivation test followed by administration of DDAVP. This test helps determine whether an inappropriate water diuresis is caused by CDI or NDI. The patient was water restricted beginning in the early morning, with careful monitoring of vital signs and urine output; overnight water deprivation of patients with diabetes insipidus is unsafe and clinically inappropriate, given the potential for severe hypernatremia. The plasma Na+ concentration, which is more accurate and more immediately available than plasma osmolality, was monitored hourly during water deprivation. A baseline AVP sample was drawn at the beginning of the test, with a second sample drawn once the plasma Na+ reached 148–150 meq/L. At this point, a single 2-μg dose of the V2 AVP receptor agonist DDAVP was administered. An alternative approach would have been to measure AVP

1	sample drawn once the plasma Na+ reached 148–150 meq/L. At this point, a single 2-μg dose of the V2 AVP receptor agonist DDAVP was administered. An alternative approach would have been to measure AVP and administer DDAVP when the patient was initially hypernatremic; however, it would have been less safe to administer DDAVP in the setting of renal impairment because clearance of DDAVP is renal dependent.

1	The patient’s water deprivation test was consistent with NDI, with an AVP level within the normal range in the setting of hypernatremia (i.e., no evidence of CDI) and an inappropriately low urine osmolality that failed to increase by >50% or >150 mOsm/kg after both water deprivation and the administration of DDAVP. This defect would be considered compatible with complete NDI; patients with partial NDI can achieve urine osmolalities of 500–600 mOsm/kg after DDAVP treatment but will not maximally concentrate their urine to 800 mOsm/kg or higher.

1	NDI has a number of genetic and acquired causes, which all share interference with some aspect of the renal concentrating mechanism. For example, loss-of-function mutations in the V2 AVP receptor cause X-linked NDI. This patient suffered from NDI due to lithium therapy, perhaps the most common cause of NDI in adult medicine. Lithium causes NDI via direct inhibition of renal glycogen synthase kinase-3 (GSK3), a kinase thought to be the pharmacologic target of lithium in psychiatric disease; renal GSK3 is required for the response of principal cells to AVP. Lithium also induces the expression of cyclooxygenase-2 (COX2) in the renal medulla; COX2-derived prostaglandins inhibit AVP-stimulated salt transport by the thick ascending limb and AVP-stimulated water transport by the collecting duct, thereby

1	CHAPTER 64e Fluid and Electrolyte Imbalances and Acid-Base Disturbances: Case Examples 64e-4 exacerbating lithium-associated polyuria. The entry of lithium through the amiloride-sensitive Na+ channel ENaC (Fig. 64e-1) is required for the effect of the drug on principal cells, such that combined therapy with lithium and amiloride can mitigate lithium-associated NDI. However, lithium causes chronic tubulointerstitial scarring and chronic kidney disease after prolonged therapy, such that patients may have a persistent NDI long after stopping the drug, with a reduced therapeutic benefit from amiloride. Notably, this particular patient had been treated intermittently for several years with lithium, with the development of chronic kidney disease (baseline creatinine of 1.3–1.4) and NDI that persisted after stopping the drug. How should this patient be treated? What are the major pitfalls of therapy?

1	How should this patient be treated? What are the major pitfalls of therapy? This patient developed severe hypernatremia due to a water diuresis from lithium-associated NDI. Treatment of hypernatremia must include both replacement of the existing free water deficit and daily replacement of ongoing free water loss. The first step is to estimate total-body water (TBW), typically estimated as 50% of the body weight in women and 60% in men. The free water deficit is then calculated as ([Na+ − 140]/140) × TBW. In this patient, the free water deficit was 4.2 L at a weight of 97.5 kg and plasma Na+ concentration of 150 meq/L. This free water deficit should be replaced slowly over 48–72 h to avoid increasing the plasma Na+ concentration by >10 meq/L per 24 h. A common mistake is to replace this deficit while neglecting to replace ongoing losses of free water, such that plasma Na+ concentration either fails to correct or, in fact, increases.

1	Ongoing losses of free water can be estimated using the equation for electrolyte-free water clearance: PART 2 Cardinal Manifestations and Presentation of Diseases where V is urinary volume, UNa is urinary [Na+], UK is urinary [K+], and PNa is plasma [Na+].

1	For this patient, the CeH2O was 2.5 L/d when initially evaluated, i.e., with urine Na+ and K+ concentrations of 34 and 5.2 meq/L, plasma Na+ concentration of 150 meq/L, and a urinary volume of 3.4 L. Therefore, the patient was given 2.5 L of D5W over the first 24 h to replace ongoing free water losses, along with 2.1 L of D5W to replace half his free water deficit. Daily random urine electrolytes and urinary volume measurement can be used to monitor CeH2O and adjust daily fluid administration in this manner, while following plasma Na+ concentration. Physicians often calculate the free water deficit to guide therapy of hypernatremia, providing half the deficit in the first 24 h. This approach can be adequate in patients who do not have significant ongoing losses of free water, e.g., with hypernatremia due to decreased free water intake. This case illustrates how free water requirements can be grossly underestimated in hypernatremic patients if ongoing, daily free water losses are not

1	hypernatremia due to decreased free water intake. This case illustrates how free water requirements can be grossly underestimated in hypernatremic patients if ongoing, daily free water losses are not taken into account.

1	A 78-year-old man was admitted with pneumonia and hyponatremia. Plasma Na+ concentration was initially 129 meq/L, decreasing within 3 days to 118–120 meq/L despite fluid restriction to 1 L/d. A chest computed tomography (CT) revealed a right 2.8 × 1.6 cm infrahilar mass and postobstructive pneumonia. The patient was an active smoker. Past medical history was notable for laryngeal carcinoma treated 15 years prior with radiation therapy, renal cell carcinoma, peripheral vascular disease, and hypothyroidism. On review of systems, he denied headache, nausea, and vomiting. He had chronic hip pain, managed with acetaminophen with codeine. Other medications included cilostazol, amoxicillin/clavulanate, digoxin, diltiazem, and thyroxine. He was euvolemic on examination, with no lymphadenopathy and a normal chest examination. Na+ 120 K+ 4.3 Cl− 89 HCO3− 23 BUN 8 Creat 1.0 Glu 93 Alb 3.1 Ca 8.9 Phos 2.8 Mg 2.0 Plasma osm 248 mOsm/kg Cortisol 25 μg/dL TSH 2.6 Uric acid 2.7 mg/dL

1	Na+ 120 K+ 4.3 Cl− 89 HCO3− 23 BUN 8 Creat 1.0 Glu 93 Alb 3.1 Ca 8.9 Phos 2.8 Mg 2.0 Plasma osm 248 mOsm/kg Cortisol 25 μg/dL TSH 2.6 Uric acid 2.7 mg/dL Urine: Na+ 97 K+ 22 Cl− 86 Osm 597 The patient was treated with furosemide, 20 mg PO bid, and salt tablets. The plasma Na+ concentration increased to 129 meq/L with this therapy; however, the patient developed orthostatic hypotension and dizziness. He was started on demeclocycline, 600 mg PO in the morning and 300 mg in the evening, just before discharge from hospital. Plasma Na+ concentration increased to 140 meq/L with a BUN of 23 and creatinine of 1.4, at which point demeclocycline was reduced to 300 mg PO bid. Bronchoscopic biopsy eventually showed small-cell lung cancer; the patient declined chemotherapy and was admitted to hospice. What factors contributed to this patient’s hyponatremia? What are the therapeutic options?

1	This patient developed hyponatremia in the context of a central lung mass and postobstructive pneumonia. He was clinically euvolemic, with a generous urine Na+ concentration and low plasma uric acid concentration. He was euthyroid, with no evidence of pituitary dysfunction or secondary adrenal insufficiency. The clinical presentation is consistent with the syndrome of inappropriate antidiuresis (SIAD). Although pneumonia was a potential contributor to the SIAD, it was notable that the plasma Na+ concentration decreased despite a clinical response to antibiotics. It was suspected that this patient had SIAD due to small-cell lung cancer, with a central lung mass on chest CT and a significant smoking history. There was a history of laryngeal cancer and renal cancer but with no evidence of recurrent disease; these malignancies were not considered contributory to his SIAD. Biopsy of the lung mass ultimately confirmed the diagnosis of small-cell lung cancer, which is responsible for ~75% of

1	disease; these malignancies were not considered contributory to his SIAD. Biopsy of the lung mass ultimately confirmed the diagnosis of small-cell lung cancer, which is responsible for ~75% of malignancy-associated SIAD; ~10% of patients with this neuroendocrine tumor will have a plasma Na+ concentration of <130 meq/L at presentation. The patient had no other “nonosmotic” stimuli for an increase in AVP, with no medications associated with SIAD and minimal pain or nausea.

1	The patient had no symptoms attributable to hyponatremia but was judged at risk for worsening hyponatremia from severe SIAD. Persistent, chronic hyponatremia (duration >48 h) results in an efflux of organic osmolytes (creatine, betaine, glutamate, myoinositol, and taurine) from brain cells; this response reduces intracellular osmolality and the osmotic gradient favoring water entry. This cellular response does not fully protect patients from symptoms, which can include vomiting, nausea, confusion, and seizures, usually at plasma Na+ concentration <125 meq/L. Even patients who are judged “asymptomatic” can manifest subtle gait and cognitive defects that reverse with correction of hyponatremia. Chronic hyponatremia also increases the risk of bony fractures due to an increased risk of falls and to a hyponatremia-associated reduction in bone density. Therefore, every attempt should be made to correct plasma Na+ concentration safely in patients with chronic hyponatremia. This is

1	of falls and to a hyponatremia-associated reduction in bone density. Therefore, every attempt should be made to correct plasma Na+ concentration safely in patients with chronic hyponatremia. This is particularly true in malignancy-associated SIAD, where it can take weeks to months for a tissue diagnosis and the subsequent reduction in AVP following initiation of chemotherapy, radiotherapy, and/or surgery.

1	What are the therapeutic options in SIAD? Water deprivation, a cornerstone of therapy for SIAD, had little effect on the plasma Na+ concentration in this patient. The urine:plasma electrolyte ratio (urinary [Na+] + [K+]/plasma [Na+]) can be used to estimate electrolyte-free water excretion and the required degree of water restriction; patients with a ratio of >1 should be more aggressively restricted (<500 mL/d), those with a ratio of ~1 should be restricted to 500–700 mL/d, and those with a ratio <1 should be restricted to <1 L/d. This patient had a urine:plasma electrolyte ratio of 1 and predictably did not respond to a moderate water restriction of ~1 L/d. A more aggressive water restriction would have theoretically been successful; however, this can be very difficult for patients with SIAD to tolerate, given that their thirst is also inappropriately stimulated.

1	Combined therapy with furosemide and salt tablets can often increase the plasma Na+ concentration in SIAD; furosemide reduces maximal urinary concentrating ability by inhibiting the countercurrent mechanism, whereas the salt tablets mitigate diuretic-associated NaCl loss and amplify the ability to excrete free water by increasing urinary solute excretion. This regimen is not always successful and requires careful titration of salt tablets to avoid volume depletion; indeed, in this particular patient, the plasma Na+ concentration remained <130 meq/L and the patient became orthostatic. The principal cell toxin, demeclocycline, is an alternative oral agent in SIAD. Treatment with demeclocycline was very successful in this patient, with an increase in plasma Na+ concentration to 140 meq/L. However, demeclocycline can be natriuretic, leading to a prerenal decrease in GFR. Demeclocycline has also been implicated in nephrotoxic injury, particularly in patients with cirrhosis and chronic

1	However, demeclocycline can be natriuretic, leading to a prerenal decrease in GFR. Demeclocycline has also been implicated in nephrotoxic injury, particularly in patients with cirrhosis and chronic liver disease, in whom the drug accumulates. Notably, this particular patient developed a significant but stable decrease in GFR while on demeclocycline, necessitating a reduction in the administered dose.

1	A major advance in the management of hyponatremia was the clinical development of AVP antagonists (vaptans). These agents inhibit the effect of AVP on renal V2 receptors, resulting in the excretion of electrolyte-free water and correction of hyponatremia. The specific indications for these agents are not as yet clear, despite U.S. Food and Drug Administration (FDA) approval for the management of both euvolemic and hypervolemic hyponatremia. It is, however, anticipated that the vaptans will have an increasing role in the management of SIAD and other causes of hyponatremia. Indeed, if this particular patient had continued with active therapy for his cancer, substitution of demeclocycline with oral tolvaptan (a V2-specific oral vaptan) would have been the next appropriate step, given the development of renal insufficiency with demeclocycline. As with other measures to correct hyponatremia (e.g., hypertonic saline, demeclocycline), the vaptans have the potential to “overcorrect” plasma

1	of renal insufficiency with demeclocycline. As with other measures to correct hyponatremia (e.g., hypertonic saline, demeclocycline), the vaptans have the potential to “overcorrect” plasma Na+ concentration (a rise of >8–10 meq/L per 24 h or 18 meq/L per 18 h), thus increasing the risk for osmotic demyelination (see Case 5). Therefore, the plasma Na+ concentration should be monitored closely during the initiation of therapy with these agents. In addition, long-term use of tolvaptan has been associated with abnormalities in liver function tests; hence, use of this agent should be restricted to only 1–2 months.

1	A 76-year-old woman presented with a several-month history of diarrhea, with marked worsening over the 2–3 weeks before admission (up to 12 stools a day). Review of systems was negative for fever, orthostatic dizziness, nausea and vomiting, or headache. Past medical history included hypertension, kidney stones, and hypercholesterolemia; medications included atenolol, spironolactone, and lovastatin. She also reliably consumed >2 L of liquid per day in management of the nephrolithiasis. The patient received 1 L of saline over the first 5 h of her hospital admission. On examination at hour 6, the heart rate was 72 sitting and 90 standing, and blood pressure was 105/50 mmHg lying and standing. Her jugular venous pressure (JVP) was indistinct with no peripheral edema. On abdominal examination, the patient had a slight increase in bowel sounds but a nontender abdomen and no organomegaly.

1	The plasma Na+ concentration on admission was 113 meq/L, with a creatinine of 2.35 (Table 64e-1). At hospital hour 7, the plasma Na+ concentration was 120 meq/L, potassium 5.4 meq/L, chloride 90 64e-5 meq/L, bicarbonate 22 meq/L, BUN 32 mg/dL, creatinine 2.02 mg/dL, glucose 89 mg/dL, total protein 5.0, and albumin 1.9. The hematocrit was 33.9, white count 7.6, and platelets 405. A morning cortisol was 19.5, with thyroid-stimulating hormone (TSH) of 1.7. The patient was treated with 1 μg of intravenous DDAVP, along with 75 mL/h of intravenous half-normal saline. After the plasma Na+ concentration dropped to 116 meq/L, intravenous fluid was switched to normal saline at the same infusion rate. The subsequent results are shown in Table 64e-1.

1	This patient presented with hypovolemic hyponatremia and a “prerenal” reduction in GFR, with an increase in serum creatinine. She had experienced diarrhea for some time and manifested an orthostatic tachycardia after a liter of normal saline. As expected for hypovolemic hyponatremia, the urine Na+ concentration was <20 meq/L in the absence of congestive heart failure or other causes of hypervolemic hyponatremia, and she responded to saline hydration with an increase in plasma Na+ concentration and a decrease in creatinine.

1	The initial hypovolemia increased the sensitivity of this patient’s AVP response to osmolality, both decreasing the osmotic threshold for AVP release and increasing the slope of the osmolality response curve. AVP has a half-life of only 10–20 min; therefore, the acute increase in intravascular volume after a liter of intravenous saline led to a rapid reduction in circulating AVP. The ensuing water diuresis is the primary explanation for the rapid increase in plasma Na+ concentration in the first 7 h of her hospitalization.

1	The key concern in this case was the evident chronicity of the patient’s hyponatremia, with several weeks of diarrhea followed by 2–3 days of acute exacerbation. This patient was judged to have chronic hyponatremia, i.e., with a suspected duration of >48 h; as such, she would be predisposed to osmotic demyelination were she to undergo too rapid a correction in her plasma Na+ concentration, i.e., by >8–10 meq/L in 24 h or 18 meq/L in 48 h. At presentation, she had no symptoms that one would typically attribute to acute hyponatremia, and the plasma Na+ concentration had already increased by a sufficient amount to protect from cerebral edema; however, she had corrected by 1 meq/L per hour within the first 7 h of admission, consistent with impending overcorrection. To reduce or halt the increase in plasma Na+ concentration, the patient received 1 μg of intravenous DDAVP along with intravenous free water. Given the hypovolemia and resolving acute renal insufficiency, a decision was made to

1	in plasma Na+ concentration, the patient received 1 μg of intravenous DDAVP along with intravenous free water. Given the hypovolemia and resolving acute renal insufficiency, a decision was made to administer half-normal saline as a source of free water, rather than D5W; this was switched to normal saline when plasma Na+ concentration acutely dropped to 117 meq/L (Table 64e-1).

1	Overcorrection of chronic hyponatremia is a major risk factor for the development of osmotic demyelination syndrome (ODS). Animal studies show a neurologic and survival benefit in ODS of “re-lowering” plasma Na+ concentration with DDAVP and free water administration; this approach is demonstrably safe in patients with hyponatremia, with no evident risk of seizure or other sequelae. This combination can be used to prevent an overcorrection or to re-lower plasma Na+ concentration in patients who have already overcorrected. DDAVP is required because in most of these patients endogenous AVP levels have plummeted, resulting in a free water diuresis; the administration of free water alone has minimal effect in this setting, given the relative absence of circulating AVP. An alternative approach in patients who present with severe hyponatremia is to treat them

1	CHAPTER 64e Fluid and Electrolyte Imbalances and Acid-Base Disturbances: Case Examples ity, coadministering hypertonic saline to increase slowly the plasma Na+ con- centration in a more controlled fashion.

1	Creatinine (mg/dL) 1.2 2.35 2.10 2.02 1.97 1.79 1.53 1.20 1.13 days after DDAVP administration. It is 64e-6 conceivable that residual hypovolemic hyponatremia attenuated the recovery of the plasma Na+ concentration. Alternatively, attenuated recovery was due to persistent effects of the single dose of DDAVP. Of note, although the plasma half-life of DDAVP is only 1–2 h, pharmacodynamic studies indicate a much more prolonged effect on urine output and/or urine osmolality. One final consideration is the effect of the patient’s initial renal dysfunction on the pharmacokinetics and pharmacodynamics of the administered DDAVP, which is renally excreted; DDAVP should be administered with caution for the reinduction of hyponatremia in patients with chronic kidney disease or acute renal dysfunction. A 44-year-old woman was referred from a local hospital after presenting with flaccid paralysis. Severe hypokalemia was documented (2.0 meq/L), and an infusion containing KCl was initiated.

1	A 44-year-old woman was referred from a local hospital after presenting with flaccid paralysis. Severe hypokalemia was documented (2.0 meq/L), and an infusion containing KCl was initiated. PART 2 Cardinal Manifestations and Presentation of Diseases Sodium 140 meq/L Potassium 2.6 meq/L Chloride 115 meq/L Bicarbonate 15 meq/L Anion gap 10 meq/L BUN 22 mg/dL Creatinine 1.4 mg/dL pH 7.32 U PaCO2 30 mmHg HCO3− 15 meq/L Rheumatoid factor positive, anti-Ro/SS-A positive, and anti-La/SS-B positive pH = 6.0, normal sediment without white or red blood cell casts and no bacteria. The urine protein-to-creatinine ratio was 0.150 g/g. Urinary electrolyte values were: Na+ 35, K+ 40, Cl− 18 meq/L. Therefore, the urine anion gap was positive, indicating low urine NH4+ excretion.

1	The diagnosis in this case is classic hypokalemic dRTA from Sjögren’s syndrome. This patient presented with a non-AG metabolic acidosis. The urine AG was positive, indicating an abnormally low excretion of ammonium in the face of systemic acidosis. The urine pH was inappropriately alkaline, yet there was no evidence of hypercalciuria, nephrocalcinosis, or bone disease. The patient was subsequently shown to exhibit hyperglobulinemia. These findings, taken together, indicate that the cause of this patient’s hypokalemia and non-AG metabolic acidosis was a renal tubular abnormality. The hypokalemia and abnormally low excretion of ammonium, as estimated by the urine AG, in the absence of glycosuria, phosphaturia, or aminoaciduria (Fanconi’s syndrome), defines the entity of classic distal renal tubular acidosis (dRTA), also known as type 1 RTA. Because of the hyperglobulinemia, additional serology was obtained, providing evidence for the diagnosis of primary Sjögren’s syndrome. Furthermore,

1	tubular acidosis (dRTA), also known as type 1 RTA. Because of the hyperglobulinemia, additional serology was obtained, providing evidence for the diagnosis of primary Sjögren’s syndrome. Furthermore, additional history indicated a 5-year history of xerostomia and keratoconjunctivitis sicca but without synovitis, arthritis, or rash.

1	Classic dRTA occurs frequently in patients with Sjögren’s syndrome and is a result of an immunologic attack on the collecting tubule, causing failure of the H+-ATPase to be inserted into the apical membrane of type A intercalated cells. Sjögren’s syndrome is one of the best-documented acquired causes of classic dRTA. The loss of H+-ATPase function also occurs with certain inherited forms of classic dRTA. There was no family history in the present case, and other family members were not affected. A number of autoantibodies have been associated with Sjögren’s syndrome; it is likely that these autoantibodies prevent trafficking or function of the H+-ATPase in the type A intercalated cell of the collecting tubule. Although proximal RTA has also been reported in patients with Sjögren’s syndrome, it is much less frequent, and there were no features of proximal tubule dysfunction (Fanconi’s syndrome) in this patient. The hypokalemia is due to secondary hyperaldosteronism from volume

1	syndrome, it is much less frequent, and there were no features of proximal tubule dysfunction (Fanconi’s syndrome) in this patient. The hypokalemia is due to secondary hyperaldosteronism from volume depletion.

1	The long-term renal prognosis for patients with classic dRTA due to Sjögren’s syndrome has not been established. Nevertheless, the metabolic acidosis and the hypokalemia respond to alkali replacement with either sodium citrate solution (Shohl’s solution) or sodium bicarbonate tablets. Obviously, potassium deficits must be replaced initially, but potassium replacement is usually not required in dRTA patients long term because sodium bicarbonate (or citrate) therapy expands volume and corrects the secondary hyperaldosteronism. A consequence of the interstitial infiltrate seen in patients with Sjögren’s syndrome and classic dRTA is progression of chronic kidney disease. Cytotoxic therapy plus glucocorticoids has been the mainstay of therapy in Sjögren’s syndrome for many years, although B lymphocyte infiltration in salivary gland tissue subsides and urinary acidification improves after treatment with rituximab.

1	A 32-year-old man was admitted to the hospital with weakness and hypokalemia. The patient had been very healthy until 2 months previously when he developed intermittent leg weakness. His review of systems was otherwise negative. He denied drug or laxative abuse and was on no medications. Past medical history was unremarkable, with no history of neuromuscular disease. Family history was notable for a sister with thyroid disease. Physical examination was notable only for reduced deep tendon reflexes. Sodium 139 143 meq/L Potassium 2.0 3.8 meq/L Chloride 105 107 meq/L Bicarbonate 26 29 meq/L BUN 11 16 mg/dL Creatinine 0.6 1.0 mg/dL Calcium 8.8 8.8 mg/dL Phosphate 1.2 mg/dL Albumin 3.8 meq/L TSH 0.08 μIU/L (normal 0.2–5.39) Free T4 41 pmol/L (normal 10–27)

1	This patient developed hypokalemia due to a redistribution of potassium between the intracellular and extracellular compartments; this pathophysiology was readily apparent following calculation of the TTKG. The TTKG is calculated as (P × U )/(P × U ). The expected values for the TTKG are <3 in the presence of hypokalemia and >7–8 in the presence of hyperkalemia (see also Case 2 and Case 8). Alternatively, a urinary K+-to-creatinine ratio of >13 mmol/g creatinine (>1.5 mmol/mmol creatinine) is compatible with excessive renal K+ excretion. In this case, the calculated TTKG was 2.5, consistent with appropriate renal conservation of K+ and a nonrenal cause for hypokalemia. In the absence of significant gastrointestinal loss of K+, the patient was diagnosed with a “redistributive” subtype of hypokalemia.

1	More than 98% of total-body potassium is intracellular; regulated buffering of extracellular K+ by this large intracellular pool plays a crucial role in the maintenance of a stable plasma K+ concentration. Clinically, changes in the exchange and distribution of intraand extracellular K+ can cause significant hypoor hyperkalemia. Insulin, β2-adrenergic activity, thyroid hormone, and alkalosis promote cellular uptake of K+ by multiple interrelated mechanisms, leading to hypokalemia. In particular, alterations in the activity of the endogenous sympathetic nervous system can cause hypokalemia in several settings, including alcohol withdrawal, hyperthyroidism, acute myocardial infarction, and severe head injury.

1	Weakness is common in severe hypokalemia; hypokalemia causes hyperpolarization of muscle, thereby impairing the capacity to depolarize and contract. In this particular patient, Graves’ disease caused hyperthyroidism and hypokalemic paralysis (thyrotoxic periodic paralysis [TPP]). TPP develops more frequently in patients of Asian or Hispanic origin. This predisposition has been linked to genetic variation in Kir2.6, a muscle-specific, thyroid hormone–induced K+ channel; however, the pathophysiologic mechanisms that link dysfunction of this ion channel to TPP have yet to be elucidated. The hypokalemia in TPP is attributed to both direct and indirect activation of the Na+/ K+-ATPase by thyroid hormone, resulting in increased uptake of K+ by muscle and other tissues. Thyroid hormone induces expression of multiple subunits of the Na+/K+-ATPase in skeletal muscle, increasing the capacity for uptake of K+; hyperthyroid increases in β-adrenergic activity are also thought to play an important

1	of multiple subunits of the Na+/K+-ATPase in skeletal muscle, increasing the capacity for uptake of K+; hyperthyroid increases in β-adrenergic activity are also thought to play an important role in TPP.

1	Clinically, patients with TPP present with weakness of the extremities and limb girdle, with paralytic episodes that occur most frequently between 1 and 6 am. Precipitants of weakness include high carbohydrate loads and strenuous exercise. Signs and symptoms of hyperthyroidism are not always present, often leading to delays in diagnosis. Hypokalemia is often profound and usually accompanied by redistributive hypophosphatemia, as in this case. A TTKG of <2–3 separates patients with TPP from those with hypokalemia due to renal potassium wasting, who will have TTKG values that are >4. This distinction is of considerable importance for therapy; patients with large potassium deficits require aggressive repletion with K+-Cl−, which has a significant risk of rebound hyperkalemia in TPP and related disorders.

1	Ultimately, definitive therapy for TPP requires treatment of the associated hyperthyroidism. In the short term, however, potassium replacement is necessary to hasten muscle recovery and prevent cardiac arrhythmias. The average recovery time of an acute attack is reduced by ~50% in patients treated with intravenous K+-Cl− at a rate of 10 meq/h; however, this incurs a significant risk of rebound hyperkalemia, with up to 70% developing a plasma K+ concentration of >5.0 meq/L. This potential for rebound hyperkalemia is a general problem in the management of all causes of redistributive hypokalemia, resulting in the need to distinguish these patients accurately and rapidly from those with a large K+ deficit due to renal or extrarenal loss of K+. An attractive alternative to K+-Cl− replacement in TPP is treatment with high-dose propranolol (3 mg/kg), which rapidly reverses the associated hypokalemia, hypophosphatemia, and paralysis. Notably, rebound hyperkalemia is not associated with this

1	in TPP is treatment with high-dose propranolol (3 mg/kg), which rapidly reverses the associated hypokalemia, hypophosphatemia, and paralysis. Notably, rebound hyperkalemia is not associated with this treatment.

1	A 66-year-old man was admitted to hospital with a plasma K+ concentration of 1.7 meq/L and profound weakness. The patient had noted progressive weakness over several days, to the point that he was unable to rise from bed. Past medical history was notable for small-cell lung cancer with metastases to brain, liver, and adrenals. The patient had been treated with one cycle of cisplatin/etoposide 1 year before this admission, which was complicated by acute kidney injury (peak creatinine of 5, with residual chronic kidney disease), and three subsequent cycles of cyclophosphamide/doxorubicin/vincristine, in addition to 15 treatments with whole-brain radiation. On physical examination, the patient was jaundiced. Blood pressure was 130/70 mmHg, increasing to 160/98 mmHg after 1 L of saline, with a JVP at 8 cm. There was generalized muscle weakness. Potassium 3.7 1.7 3.5 meq/L 7.47 Creatinine 2.8 2.9 2.3 mg/dL Magnesium 1.3 1.6 2.4 mg/dL Albumin 3.4 2.8 2.3 Total bilirubin 0.65 5.19 5.5

1	Potassium 3.7 1.7 3.5 meq/L 7.47 Creatinine 2.8 2.9 2.3 mg/dL Magnesium 1.3 1.6 2.4 mg/dL Albumin 3.4 2.8 2.3 Total bilirubin 0.65 5.19 5.5 Abbreviations: ACTH, adrenocorticotropic hormone; HD2, hospital day 2; PTA, prior to admission. The patient’s hospital course was complicated by acute respiratory failure attributed to pulmonary embolism; he died 2 weeks after admission. Why was this patient hypokalemic? Why was he weak? Why did he have an alkalosis? This patient suffered from metastatic small-cell lung cancer, which was persistent despite several rounds of chemotherapy and radiotherapy. He presented with profound hypokalemia, alkalosis, hypertension, severe weakness, jaundice, and worsening liver function tests.

1	With respect to the hypokalemia, there was no evident cause of nonrenal potassium loss, e.g., diarrhea. The urinary TTKG was 11.7, at a plasma K+ concentration of 1.7 meq/L; this TTKG value is consistent with inappropriate renal K+ secretion, despite severe hypokalemia. The TTKG is calculated as (P × U )/(P × U ). The expected values for the TTKG are <3 in the presence of hypokalemia and >7–8 in the presence of hyperkalemia (see also Case 2 and Case 6).

1	The patient had several explanations for excessive renal loss of potassium. First, he had had a history of cisplatin-associated acute kidney injury, with residual chronic kidney disease. Cisplatin can cause persistent renal tubular defects, with prominent hypokalemia and hypomagnesemia; however, this patient had not previously required potassium or magnesium repletion, suggesting that cisplatin-associated renal tubular defects did not play a major role in this presentation with severe hypokalemia. Second, he was hypomagnesemic on presentation, suggesting total-body magnesium depletion. Magnesium depletion has inhibitory effects on muscle Na+/K+-ATPase activity, reducing influx into muscle cells and causing a secondary increase in K+ excretion. Magnesium depletion also increases K+ secretion by the distal nephron; this is attributed to a reduction in the magnesium-dependent, intracellular block of K+ efflux through the secretory K+ channel of principal cells (ROMK, Fig. 64e-1).

1	secretion by the distal nephron; this is attributed to a reduction in the magnesium-dependent, intracellular block of K+ efflux through the secretory K+ channel of principal cells (ROMK, Fig. 64e-1). Clinically, hypomagnesemic patients are

1	CHAPTER 64e Fluid and Electrolyte Imbalances and Acid-Base Disturbances: Case Examples 64e-8 refractory to K+ replacement in the absence of Mg2+ repletion. Again, however, this patient had not previously developed significant hypokalemia, despite periodic hypomagnesemia, such that other factors must have caused the severe hypokalemia. The associated hypertension in this case suggested an increase in mineralocorticoid activity, causing increased activity of ENaC channels in principal cells, NaCl retention, hypertension, and hypokalemia. The increase in ENaC-mediated Na+ transport in principal cells would have led to an increase in the lumen-negative potential difference in the connecting tubule and cortical collecting duct, driving an increase in K+ secretion through apical K+ channels (Fig. 64e-1). This explanation is compatible with the very high TTKG, i.e., an increase in K+ excretion that is inappropriate for the plasma K+ concentration. What caused an increase in mineralocorticoid

1	64e-1). This explanation is compatible with the very high TTKG, i.e., an increase in K+ excretion that is inappropriate for the plasma K+ concentration. What caused an increase in mineralocorticoid activity in this patient? The patient had bilateral adrenal metastases, indicating that primary hyperaldosteronism was unlikely. The clinical presentation (hypokalemia, hypertension, and alkalosis) and the history of small-cell lung cancer suggested Cushing’s syndrome, with a massive increase in circulating glucocorticoids, in response to ectopic adrenocorticotropic hormone (ACTH) secretion by his small-cell lung cancer tumor. Confirmation of this diagnosis was provided by a very high plasma cortisol level, high ACTH level, and increased urinary cortisol (see the laboratory data above). Why would an increase in circulating cortisol cause an apparent increase in mineralocorticoid activity? Cortisol and aldosterone have equal affinity for the mineralocorticoid receptor (MLR); thus, cortisol

1	an increase in circulating cortisol cause an apparent increase in mineralocorticoid activity? Cortisol and aldosterone have equal affinity for the mineralocorticoid receptor (MLR); thus, cortisol has mineralocorticoid-like activity; however, cells in the aldosteronesensitive distal nephron (the distal convoluted tubule [DCT]), connecting tubule (CNT), and collecting duct are protected from circulating cortisol by the enzyme 11β-hydroxysteroid dehydrogenase-2 (11βHSD-2), which converts cortisol to cortisone (Fig. 64e-2); cortisone has minimal affinity for the MLR. Activation of the MLR causes activation of the basolateral Na+/K+-ATPase, activation of the thiazide-sensitive Na+-Cl− cotransporter in the DCT, and activation of apical ENaC

1	PART 2 Cardinal Manifestations and Presentation of Diseases

1	FIGuRE 64e-2 11β-Hydroxysteroid dehydrogenase-2 (11βHSD-2) and syndromes of apparent mineralocorticoid excess. The enzyme 11βHSD-2 protects cells in the aldosterone-sensitive distal nephron (the distal convoluted tubule [DCT ], connecting tubule [CNT], and collecting duct) from the illicit activation of mineralocorticoid receptors (MLR) by cortisol. Binding of aldosterone to the MLR leads to activation of the thiazide-sensitive Na+-Cl− cotransporter in DCT cells and the amiloride-sensitive epithelial sodium channel (ENaC) in principal cells (CNT and collecting duct). Aldosterone also activates basolateral Na+/K+-ATPase and, to a lesser extent, the apical secretory K+ channel ROMK (renal outer medullary K+ channel). Cortisol has equivalent affinity for the MLR to that of aldosterone; metabolism of cortisol to cortisone, which has no affinity for the MLR, prevents these cells from activation by circulating cortisol. Genetic deficiency of 11βHSD-2 or inhibition of its activity causes the

1	of cortisol to cortisone, which has no affinity for the MLR, prevents these cells from activation by circulating cortisol. Genetic deficiency of 11βHSD-2 or inhibition of its activity causes the syndromes of apparent mineralocorticoid excess (see Case 8).

1	channels in principal cells of the CNT and collecting duct (Fig. 64e-2). Recessive loss-of-function mutations in the 11βHSD-2 gene lead to cortisol-dependent activation of the MLR and the syndrome of apparent mineralocorticoid excess (SAME), comprising hypertension, hypokalemia, hypercalciuria, and metabolic alkalosis, with suppressed plasma renin activity (PRA) and suppressed aldosterone. A similar syndrome is caused by biochemical inhibition of 11βHSD-2 by glycyrrhetinic/ glycyrrhizinic acid (found in licorice, for example) and/or carbenoxolone.

1	In Cushing’s syndrome caused by increases in pituitary ACTH, the incidence of hypokalemia is only 10%, whereas it is ~70% in patients with ectopic secretion of ACTH, despite a similar incidence of hypertension. The activity of renal 11βHSD-2 is reduced in patients with ectopic ACTH compared with Cushing’s syndrome, resulting in SAME; the prevailing theory is that the much greater cortisol production in ectopic ACTH syndromes overwhelms the renal 11βHSD-2 enzyme, resulting in activation of renal MLRs by unmetabolized cortisol (Fig. 64e-2).

1	Why was the patient so weak? The patient was profoundly weak due to the combined effect of hypokalemia and increased cortisol. Hypokalemia causes hyperpolarization of muscle, thereby impairing the capacity to depolarize and contract. Weakness and even ascending paralysis can frequently complicate severe hypokalemia. Hypokalemia also causes a myopathy and predisposes to rhabdomyolysis; notably, however, the patient had a normal creatine phosphokinase (CPK) level. Cushing’s syndrome is often accompanied by a proximal myopathy, due to the protein-wasting effects of cortisol excess.

1	The patient presented with a mixed acid-base disorder, with a significant metabolic alkalosis and a bicarbonate concentration of 44 meq/L. A venous blood gas was drawn soon after his presentation; venous and arterial blood gases demonstrate a high level of agreement in hemodynamically stable patients, allowing for the interpretation of acid-base disorders with venous blood gas results. In response to his metabolic alkalosis, the Pco2 should have increased by 0.75 mmHg for each 1-meq/L increase in bicarbonate; the expected Pco2 should have been ~55 mmHg. Given the Pco2 of 62 mmHg, he had an additional respiratory acidosis, likely caused by respiratory muscle weakness from his acute hypokalemia and subacute hypercortisolism.

1	The patient’s albumin-adjusted AG was 21 + ([4 – 2.8] × 2.5) = 24; this suggests a third acid-base disorder, AG acidosis. Notably, the measured AG can increase in alkalosis, due to both increases in plasma protein concentrations (in hypovolemic alkalosis) and to the alkalemiaassociated increase in net negative charge of plasma proteins, both causing an increase in unmeasured anions; however, this patient was neither volume-depleted nor particularly alkalemic, suggesting that these effects played a minimal role in his increased AG. Alkalosis also stimulates an increase in lactic acid production, due to activation of phosphofructokinase and accelerated glycolysis; unfortunately, however, a lactic acid level was not measured in this patient. It should be noted in this regard that alkalosis typically increases lactic acid levels by a mere 1.5–3 meq/L and that the patient was not significantly alkalemic. Regardless of the underlying pathophysiology, the increased AG was likely related to

1	typically increases lactic acid levels by a mere 1.5–3 meq/L and that the patient was not significantly alkalemic. Regardless of the underlying pathophysiology, the increased AG was likely related to the metabolic alkalosis, given that the AG had decreased to 18 by hospital day 2, coincident with a reduction in plasma bicarbonate.

1	Why did the patient have a metabolic alkalosis? The activation of MLRs in the distal nephron increases distal nephron acidification and net acid secretion. In consequence, mineralocorticoid excess causes a saline-resistant metabolic alkalosis, which is exacerbated significantly by the development of hypokalemia. Hypokalemia plays a key role in the generation of most forms of metabolic alkalosis, stimulating proximal tubular ammonium production, proximal tubular bicarbonate reabsorption, and distal tubular H+/K+-ATPase activity.

1	The first priority in the management of this patient was to increase his plasma K+ and magnesium concentrations rapidly; hypomagnesemic patients are refractory to K+ replacement alone, resulting in the need to correct hypomagnesemia immediately. This was accomplished via the administration of both oral and intravenous K+-Cl−, giving a total of 240 meq over the first 18 h; 5 g of intravenous magnesium sulfate was also administered. Multiple 100-mL “minibags” of saline containing 20 meq each were infused, with cardiac monitoring and frequent measurement of plasma electrolytes. Of note, intravenous K+-Cl− should always be given in saline solutions because dextrose-containing solutions can increase insulin levels and exacerbate hypokalemia.

1	This case illustrates the difficulty in predicting the whole-body deficit of K+ in hypokalemic patients. In the absence of abnormal K+ redistribution, the total deficit correlates with plasma K+ concentration, which drops by approximately 0.27 mM for every 100-mmol reduction in total-body stores; this would suggest a deficit of ~650 meq of K+ in this patient, at the admission plasma K+ concentration of 1.7 meq/L. Notably, however, alkalemia induces a modest intracellular shift of circulating K+ such that this patient’s initial plasma K+ concentration was not an ideal indicator of the total potassium deficit. Regardless of the underlying pathophysiology in this case, close monitoring of plasma K+ concentration is always essential during the correction of severe hypokalemia in order to gauge the adequacy of repletion and to avoid overcorrection.

1	Subsequent management of this patient’s Cushing’s syndrome and ectopic ACTH secretion was complicated by the respiratory issues. The prognosis in patients with ectopic ACTH secretion depends on the tumor histology and the presence or absence of distant metastases. This patient had an exceptionally poor prognosis, with widely metastatic small-cell lung cancer that had failed treatment; other patients with ectopic ACTH secretion caused by more benign, isolated tumors, most commonly bronchial carcinoid tumors, have a much better prognosis. In the absence of successful surgical resection of the causative tumor, management of this syndrome can include surgical adrenalectomy or medical therapy to block adrenal steroid production.

1	A stuporous 22-year-old man was admitted with a history of behaving strangely. His friends indicated he experienced recent emotional problems stemming from a failed relationship and had threatened suicide. There was a history of alcohol abuse, but his friends were unaware of recent alcohol consumption. The patient was obtunded on admission, with no evident focal neurologic deficits. The remainder of the physical examination was unremarkable. Na+ 140 meq/L K+ 5 meq/L Cl− 95 meq/L HCO3− 10 meq/L Glucose 125 mg/dL BUN 15 mg/dL Creatinine 0.9 mg/dL Ionized calcium 4.0 mg/dL Plasma osmolality 325 mOsm kg/H2O Urinalysis revealed crystalluria, with a mixture of envelope-shaped and needle-shaped crystals.

1	This patient presented with CNS manifestations and a history of suspicious behavior, suggesting ingestion of a toxin. The AG was strikingly elevated at 35 meq/L. The ΔAG of 25 significantly exceeded the ΔHCO3− of 15. The fact that the Δ values were significantly disparate indicates that the most likely acid-base diagnosis in this patient is a mixed high-AG metabolic acidosis and a metabolic alkalosis. The metabolic alkalosis in this case may have been the result of vomiting. Nevertheless, the most useful finding is that the osmolar gap is elevated. The osmolar gap of 33 (difference in measured and calculated osmolality or 325 – 292) in the face of a high-AG metabolic acidosis is diagnostic of an osmotically active metabolite in plasma; a difference of >10 mOsm/kg indicates a significant concentration of an unmeasured osmolyte. Examples of toxic osmolytes include ethylene glycol, diethylene glycol, methanol, and propylene glycol.

1	Several caveats apply to the interpretation of the osmolar gap and 64e-9 AG in the differential diagnosis of toxic alcohol ingestions. First, unmeasured, neutral osmolytes can also accumulate in lactic acidosis and alcoholic ketoacidosis; i.e., an elevated osmolar gap is not specific to AG acidoses associated with toxic alcohol ingestions. Second, patients can present having extensively metabolized the ingested toxin, with an insignificant osmolar gap but a large AG; i.e., the absence of an elevated osmolar gap does not rule out toxic alcohol ingestion. Third, the converse can also be seen in patients who present earlier after ingestion of the toxin, i.e., a large osmolar gap with minimal elevation of the AG. Finally, clinicians should be aware of the effect of co-ingested ethanol, which can itself elevate the osmolar gap and can reduce metabolism of the toxic alcohols via competitive inhibition of alcohol dehydrogenase (see below), thus attenuating the expected increase in the AG.

1	Ethylene glycol is commonly available as antifreeze or solvents and may be ingested accidently or as a suicide attempt. The metabolism of ethylene glycol by alcohol dehydrogenase generates acids such as glycoaldehyde, glycolic acid, and oxalic acid. The initial effects of intoxication are on the CNS and, in the earliest stages, mimic inebriation, but may quickly progress to full-blown coma. Delay in treatment is one of the most common causes of mortality with toxic alcohol poisoning. The kidney shows evidence of acute tubular injury with widespread deposition of calcium oxalate crystals within tubular epithelial cells. Cerebral edema is common, as is crystal deposition in the brain; the latter is irreversible.

1	The co-occurrent crystalluria is typical of ethylene glycol intoxication; both needle-shaped monohydrate and envelope-shaped dihydrate calcium oxalate crystals can be seen in the urine as the process evolves. Circulating oxalate can also complex with plasma calcium, reducing the ionized calcium as in this case. Although ethylene glycol intoxication should be verified by measuring ethylene glycol levels, therapy must be initiated immediately in this life-threatening situation. Although therapy can be initiated with confidence in cases with known or witnessed ingestions, such histories are rarely available. Therapy should thus be initiated in patients with severe metabolic acidosis and elevated anion and osmolar gaps. Other diagnostic features, such as hypocalcemia or acute renal failure with crystalluria, can provide important confirmation for urgent, empiric therapy.

1	Because all four osmotically active toxic alcohols—ethylene glycol, diethylene glycol, methanol, and propylene glycol—are metabolized by alcohol dehydrogenase to generate toxic products, competitive inhibition of this key enzyme is common to the treatment of all four intoxications. The most potent inhibitor of alcohol dehydrogenase, and the drug of choice in this circumstance, is fomepizole (4-methyl pyrazole). Fomepizole should be administered intravenously as a loading dose (15 mg/kg) followed by doses of 10 mg/kg every 12 h for four doses, and then 15 mg/kg every 12 h thereafter until ethylene glycol levels have been reduced to <20 mg/dL and the patient is asymptomatic with a normal pH. Additional important components of the treatment of toxic alcohol ingestion include fluid resuscitation, thiamine, pyridoxine, folate, sodium bicarbonate, and hemodialysis. Hemodialysis is used to remove both the parent compound and toxic metabolites, but it also removes administered fomepizole,

1	thiamine, pyridoxine, folate, sodium bicarbonate, and hemodialysis. Hemodialysis is used to remove both the parent compound and toxic metabolites, but it also removes administered fomepizole, necessitating adjustment of dosage frequency. Gastric aspiration, induced emesis, or the use of activated charcoal is only effective if initiated within 30–60 min after ingestion of the toxin. When fomepizole is not available, ethanol, which has more than 10-fold affinity for alcohol dehydrogenase compared to other alcohols, may be substituted and is quite effective. Ethanol must be administered IV to achieve a blood level of 22 meq/L (100 mg/dL). A disadvantage of ethanol is the obtundation that follows its administration, which is additive to the CNS effects of ethylene glycol. Furthermore, if hemodialysis is used, the infusion rate of ethanol must be increased because it is rapidly dialyzed. In general, hemodialysis is indicated for all patients with ethylene glycol intoxication when the

1	hemodialysis is used, the infusion rate of ethanol must be increased because it is rapidly dialyzed. In general, hemodialysis is indicated for all patients with ethylene glycol intoxication when the arterial pH is <7.3 or the osmolar gap exceeds 20 mOsm/kg H2O.

1	CHAPTER 64e Fluid and Electrolyte Imbalances and Acid-Base Disturbances: Case Examples TABLE 65-1 CAuSES of HyPERCALCEMiA Sundeep Khosla Primary hyperparathyroidism (adenoma, hyperplasia, rarely carcinoma)

1	Sundeep Khosla Primary hyperparathyroidism (adenoma, hyperplasia, rarely carcinoma) The calcium ion plays a critical role in normal cellular function and signaling, regulating diverse physiologic processes such as neuromuscular signaling, cardiac contractility, hormone secretion, and blood coagulation. Thus, extracellular calcium concentrations are maintained within an exquisitely narrow range through a series of feedback mechanisms that involve parathyroid hormone (PTH) and the active vitamin D metabolite 1,25-dihydroxyvitmin D [1,25(OH)2D]. These feedback mechanisms are orchestrated by integrating signals between the parathyroid glands, kidney, intestine, and bone (Fig. 65-1; Chap. 423). Disorders of serum calcium concentration are relatively common and often serve as a harbinger of underlying disease. This chapter provides a brief summary of the approach to patients with altered serum calcium levels. See Chap. 424 for a detailed discussion of this topic.

1	The causes of hypercalcemia can be understood and classified based on derangements in the normal feedback mechanisms that regulate serum calcium (Table 65-1). Excess PTH production, which is not appropriately suppressed by increased serum calcium concentrations, occurs in primary neoplastic disorders of the parathyroid glands (parathyroid adenomas; hyperplasia; or, rarely, carcinoma) that are associated with 1,25 (OH)2D

1	FIguRE 65-1 Feedback mechanisms maintaining extracellular calcium concentrations within a narrow, physiologic range (8.9–10.1 mg/dL [2.2–2.5 mM]). A decrease in extracellular (ECF) calcium (Ca2+) triggers an increase in parathyroid hormone (PTH) secretion (1) via the calcium sensor receptor on parathyroid cells. PTH, in turn, results in increased tubular reabsorption of calcium by the kidney (2) and resorption of calcium from bone (2) and also stimulates renal 1,25(OH)2D production (3). 1,25(OH)2D, in turn, acts principally on the intestine to increase calcium absorption (4). Collectively, these homeostatic mechanisms serve to restore serum calcium levels to normal.

1	Tertiary hyperparathyroidism (long-term stimulation of PTH secretion in renal insufficiency) Ectopic PTH secretion (very rare) Inactivating mutations in the CaSR or in G proteins (FHH) Alterations in CaSR function (lithium therapy) Hypercalcemia of malignancy Overproduction of PTHrP (many solid tumors) Lytic skeletal metastases (breast, myeloma) Excessive 1,25(OH)2D production Granulomatous diseases (sarcoidosis, tuberculosis, silicosis) Lymphomas Vitamin D intoxication Primary increase in bone resorption Hyperthyroidism Immobilization Excessive calcium intake Milk-alkali syndrome Total parenteral nutrition Other causes Endocrine disorders (adrenal insufficiency, pheochromocytoma, VIPoma) Medications (thiazides, vitamin A, antiestrogens) Abbreviations: CaSR, calcium sensor receptor; FHH, familial hypocalciuric hypercalcemia; PTH, parathyroid hormone; PTHrP, PTH-related peptide.

1	increased parathyroid cell mass and impaired feedback inhibition by calcium. Inappropriate PTH secretion for the ambient level of serum calcium also occurs with heterozygous inactivating calcium sensor receptor (CaSR) or G protein mutations, which impair extracellular calcium sensing by the parathyroid glands and the kidneys, resulting in familial hypocalciuric hypercalcemia (FHH). Although PTH secretion by tumors is extremely rare, many solid tumors produce PTH-related peptide (PTHrP), which shares homology with PTH in the first 13 amino acids and binds the PTH receptor, thus mimicking effects of PTH on bone and the kidney. In PTHrP-mediated hypercalcemia of malignancy, PTH levels are suppressed by the high serum calcium levels. Hypercalcemia associated with granulomatous disease (e.g., sarcoidosis) or lymphomas is caused by enhanced conversion of 25(OH) D to the potent 1,25(OH)2D. In these disorders, 1,25(OH)2D enhances intestinal calcium absorption, resulting in hypercalcemia and

1	sarcoidosis) or lymphomas is caused by enhanced conversion of 25(OH) D to the potent 1,25(OH)2D. In these disorders, 1,25(OH)2D enhances intestinal calcium absorption, resulting in hypercalcemia and suppressed PTH. Disorders that directly increase calcium mobilization from bone, such as hyperthyroidism or osteolytic metastases, also lead to hypercalcemia with suppressed PTH secretion as does exogenous calcium overload, as in milk-alkali syndrome, or total parenteral nutrition with excessive calcium supplementation.

1	Mild hypercalcemia (up to 11–11.5 mg/dL) is usually asymptomatic and recognized only on routine calcium measurements. Some patients may complain of vague neuropsychiatric symptoms, including trouble concentrating, personality changes, or depression. Other presenting symptoms may include peptic ulcer disease or nephrolithiasis, and fracture risk may be increased. More severe hypercalcemia (>12–13 mg/dL), particularly if it develops acutely, may result in lethargy, stupor, or coma, as well as gastrointestinal symptoms (nausea, anorexia, constipation, or pancreatitis). Hypercalcemia decreases renal concentrating ability, which may cause polyuria and polydipsia. With longstanding hyperparathyroidism, patients may present with bone pain or pathologic fractures. Finally, hypercalcemia can result in significant electrocardiographic changes, including bradycardia, AV block, and short QT interval; changes in serum calcium can be monitored by following the QT interval.

1	The first step in the diagnostic evaluation of hyperor hypocalcemia is to ensure that the alteration in serum calcium levels is not due to abnormal albumin concentrations. About 50% of total calcium is ionized, and the rest is bound principally to albumin. Although direct measurements of ionized calcium are possible, they are easily influenced by collection methods and other artifacts; thus, it is generally preferable to measure total calcium and albumin to “correct” the serum calcium. When serum albumin concentrations are reduced, a corrected calcium concentration is calculated by adding 0.2 mM (0.8 mg/dL) to the total calcium level for every decrement in serum albumin of 1.0 g/dL below the reference value of 4.1 g/dL for albumin, and, conversely, for elevations in serum albumin.

1	A detailed history may provide important clues regarding the etiology of the hypercalcemia (Table 65-1). Chronic hypercalcemia is most commonly caused by primary hyperparathyroidism, as opposed to the second most common etiology of hypercalcemia, an underlying malignancy. The history should include medication use, previous neck surgery, and systemic symptoms suggestive of sarcoidosis or lymphoma.

1	Once true hypercalcemia is established, the second most important laboratory test in the diagnostic evaluation is a PTH level using a two-site assay for the intact hormone. Increases in PTH are often accompanied by hypophosphatemia. In addition, serum creatinine should be measured to assess renal function; hypercalcemia may impair renal function, and renal clearance of PTH may be altered depending on the fragments detected by the assay. If the PTH level is increased (or “inappropriately normal”) in the setting of elevated calcium and low phosphorus, the diagnosis is almost always primary hyperparathyroidism. Because individuals with FHH may also present with mildly elevated PTH levels and hypercalcemia, this diagnosis should be considered and excluded because parathyroid surgery is ineffective in this condition. A calcium/creatinine clearance ratio (calculated as urine calcium/ serum calcium divided by urine creatinine/serum creatinine) of <0.01 is suggestive of FHH, particularly when

1	in this condition. A calcium/creatinine clearance ratio (calculated as urine calcium/ serum calcium divided by urine creatinine/serum creatinine) of <0.01 is suggestive of FHH, particularly when there is a family history of mild, asymptomatic hypercalcemia. In addition, sequence analysis of the CaSR gene is now commonly performed for the definitive diagnosis of FHH, although in some families, FHH may be caused by mutations in G proteins that mediate signaling by the CaSR. Ectopic PTH secretion is extremely rare.

1	A suppressed PTH level in the face of hypercalcemia is consistent with non-parathyroid-mediated hypercalcemia, most often due to underlying malignancy. Although a tumor that causes hypercalcemia is generally overt, a PTHrP level may be needed to establish the diagnosis of hypercalcemia of malignancy. Serum 1,25(OH)2D levels are increased in granulomatous disorders, and clinical evaluation in combination with laboratory testing will generally provide a diagnosis for the various disorders listed in Table 65-1. PART 2 Cardinal Manifestations and Presentation of Diseases

1	Mild, asymptomatic hypercalcemia does not require immediate therapy, and management should be dictated by the underlying diagnosis. By contrast, significant, symptomatic hypercalcemia usually requires therapeutic intervention independent of the etiology of hypercalcemia. Initial therapy of significant hypercalcemia begins with volume expansion because hypercalcemia invariably leads to dehydration; 4–6 L of intravenous saline may be required over the first 24 h, keeping in mind that underlying comorbidities (e.g., congestive heart failure) may require the use of loop diuretics to enhance sodium and calcium excretion. However, loop diuretics should not be initiated until the volume status has been restored to normal. If there is increased calcium mobilization from bone (as in malignancy or severe hyperparathyroidism), drugs that inhibit bone resorption should be considered. Zoledronic acid (e.g., 4 mg intravenously over ∼30 min), pamidronate (e.g., 60–90 mg intravenously over 2–4 h),

1	severe hyperparathyroidism), drugs that inhibit bone resorption should be considered. Zoledronic acid (e.g., 4 mg intravenously over ∼30 min), pamidronate (e.g., 60–90 mg intravenously over 2–4 h), and ibandronate (2 mg intravenously over 2 h) are bisphosphonates that are commonly used for the treatment of hypercalcemia of malignancy in adults. Onset of action is within 1–3 days, with normalization of serum calcium levels occurring in 60–90% of patients. Bisphosphonate infusions may need to be repeated if hypercalcemia relapses. An alternative to the bisphosphonates is gallium nitrate (200 mg/m2 intravenously daily for 5 days), which is also effective, but has potential nephrotoxicity. In rare instances, dialysis may be necessary. Finally, although intravenous phosphate chelates calcium and decreases serum calcium levels, this therapy can be toxic because calcium-phosphate complexes may deposit in tissues and cause extensive organ damage.

1	In patients with 1,25(OH)2D-mediated hypercalcemia, glucocorticoids are the preferred therapy, as they decrease 1,25(OH)2D production. Intravenous hydrocortisone (100–300 mg daily) or oral prednisone (40–60 mg daily) for 3–7 days is used most often. Other drugs, such as ketoconazole, chloroquine, and hydroxychloroquine, may also decrease 1,25(OH)2D production and are used occasionally.

1	The causes of hypocalcemia can be differentiated according to whether serum PTH levels are low (hypoparathyroidism) or high (secondary hyperparathyroidism). Although there are many potential causes of hypocalcemia, impaired PTH production and impaired vitamin D production are the most common etiologies (Table 65-2) (Chap. 424). Because PTH is the main defense against hypocalcemia, disorders associated with deficient PTH production or secretion may be associated with profound, life-threatening hypocalcemia. In adults, hypoparathyroidism most commonly results from inadvertent damage to all four glands during thyroid or parathyroid gland surgery. Hypoparathyroidism is a cardinal feature of autoimmune endocrinopathies (Chap. 408); rarely, it Vitamin D deficiency or impaired 1,25(OH)2D production/action Nutritional vitamin D deficiency (poor intake or absorption) Renal insufficiency with impaired 1,25(OH)2D production Vitamin D resistance, including receptor defects

1	Drugs Calcium chelators Inhibitors of bone resorption (bisphosphonates, plicamycin) Altered vitamin D metabolism (phenytoin, ketoconazole) Miscellaneous causes Acute pancreatitis Acute rhabdomyolysis Hungry bone syndrome after parathyroidectomy Osteoblastic metastases with marked stimulation of bone formation (prostate cancer) Abbreviations: CaSR, calcium sensor receptor; PTH, parathyroid hormone. may be associated with infiltrative diseases such as sarcoidosis. Impaired PTH secretion may be secondary to magnesium deficiency or to activating mutations in the CaSR or in the G proteins that mediate CaSR signaling, which suppress PTH, leading to effects that are opposite to those that occur in FHH.

1	Vitamin D deficiency, impaired 1,25(OH)2D production (primarily secondary to renal insufficiency), or vitamin D resistance also cause hypocalcemia. However, the degree of hypocalcemia in these disorders is generally not as severe as that seen with hypoparathyroidism because the parathyroids are capable of mounting a compensatory increase in PTH secretion. Hypocalcemia may also occur in conditions associated with severe tissue injury such as burns, rhabdomyolysis, tumor lysis, or pancreatitis. The cause of hypocalcemia in these settings may include a combination of low albumin, hyperphosphatemia, tissue deposition of calcium, and impaired PTH secretion.

1	Patients with hypocalcemia may be asymptomatic if the decreases in serum calcium are relatively mild and chronic, or they may present with life-threatening complications. Moderate to severe hypocalcemia is associated with paresthesias, usually of the fingers, toes, and circumoral regions, and is caused by increased neuromuscular irritability. On physical examination, a Chvostek’s sign (twitching of the circumoral muscles in response to gentle tapping of the facial nerve just anterior to the ear) may be elicited, although it is also present in ∼10% of normal individuals. Carpal spasm may be induced by inflation of a blood pressure cuff to 20 mmHg above the patient’s systolic blood pressure for 3 min (Trousseau’s sign). Severe hypocalcemia can induce seizures, carpopedal spasm, bronchospasm, laryngospasm, and prolongation of the QT interval.

1	In addition to measuring serum calcium, it is useful to determine albumin, phosphorus, and magnesium levels. As for the evaluation of hypercalcemia, determining the PTH level is central to the evaluation of hypocalcemia. A suppressed (or “inappropriately low”) PTH level in the setting of hypocalcemia establishes absent or reduced PTH secretion (hypoparathyroidism) as the cause of the hypocalcemia. Further history will often elicit the underlying cause (i.e., parathyroid agenesis vs. destruction). By contrast, an elevated PTH level (secondary hyperparathyroidism) should direct attention to the vitamin D axis as the cause of the hypocalcemia. Nutritional vitamin D deficiency is best assessed by obtaining serum 25-hydroxyvitamin D levels, which reflect vitamin D stores. In the setting of renal insufficiency or suspected vitamin D resistance, serum 1,25(OH)2D levels are informative.

1	The approach to treatment depends on the severity of the hypocalcemia, the rapidity with which it develops, and the accompanying complications (e.g., seizures, laryngospasm). Acute, symptomatic hypocalcemia is initially managed with calcium gluconate, 10 mL 10% wt/vol (90 mg or 2.2 mmol) intravenously, diluted in 50 mL of 5% dextrose or 0.9% sodium chloride, given intravenously over 5 min. Continuing hypocalcemia often requires a constant intravenous infusion (typically 10 ampules of calcium gluconate or 900 mg of calcium in 1 L of 5% dextrose or 0.9% sodium chloride administered over 24 h). Accompanying hypomagnesemia, if present, should be treated with appropriate magnesium supplementation.

1	Chronic hypocalcemia due to hypoparathyroidism is treated with calcium supplements (1000–1500 mg/d elemental calcium in divided doses) and either vitamin D2 or D3 (25,000–100,000 U daily) or calcitriol [1,25(OH)2D, 0.25–2 μg/d]. Other vitamin D metabolites (dihydrotachysterol, alfacalcidiol) are now used less frequently. Vitamin D deficiency, however, is best treated using vitamin D supplementation, with the dose depending on the severity of the deficit and the underlying cause. Thus, nutritional vitamin D deficiency generally responds to relatively low doses of vitamin D (50,000 U, 2–3 times per week for several months), whereas vitamin D deficiency due to malabsorption may require much higher doses 315 (100,000 U/d or more). The treatment goal is to bring serum calcium into the low normal range and to avoid hypercalciuria, which may lead to nephrolithiasis.

1	In countries with more limited access to health care or screening laboratory testing of serum calcium levels, primary hyperparathyroidism often presents in its severe form with skeletal complications (osteitis fibrosa cystica) in contrast to the asymptomatic form that is common in developed countries. In addition, vitamin D deficiency is paradoxically common in some countries despite extensive sunlight (e.g., India) due to avoidance of sun exposure and poor dietary vitamin D intake. Thomas D. DuBose, Jr.

1	Thomas D. DuBose, Jr. Systemic arterial pH is maintained between 7.35 and 7.45 by extracellular and intracellular chemical buffering together with respiratory and renal regulatory mechanisms. The control of arterial CO2 tension (Paco2) by the central nervous system (CNS) and respiratory system and the control of plasma bicarbonate by the kidneys stabilize the arterial pH by excretion or retention of acid or alkali. The metabolic and respiratory components that regulate systemic pH are described by the Henderson-Hasselbalch equation: P = 6.1+ logPaco2 × 0.0301

1	P = 6.1+ logPaco2 × 0.0301 Under most circumstances, CO2 production and excretion are matched, and the usual steady-state Paco2 is maintained at 40 mmHg. Underexcretion of CO2 produces hypercapnia, and overexcretion causes hypocapnia. Nevertheless, production and excretion are again matched at a new steady-state Paco2. Therefore, the Paco2 is regulated primarily by neural respiratory factors and is not subject to regulation by the rate of CO2 production. Hypercapnia is usually the result of hypoventilation rather than of increased CO2 production. Increases or decreases in Paco2 represent derangements of neural respiratory control or are due to compensatory changes in response to a primary alteration in the plasma [HCO3 -]. The most common clinical disturbances are simple acid-base disorders; i.e., metabolic acidosis or alkalosis or respiratory acidosis or alkalosis.

1	The most common clinical disturbances are simple acid-base disorders; i.e., metabolic acidosis or alkalosis or respiratory acidosis or alkalosis. Primary respiratory disturbances (primary changes in Paco2) invoke compensatory metabolic responses (secondary changes in [HCO3 -]), and primary metabolic disturbances elicit predictable compensatory respiratory responses (secondary changes in Paco2). Physiologic compensation can be predicted from the relationships displayed in Table 66-1. In general, with one exception, compensatory responses return the pH toward, but not to, the normal value. Chronic respiratory alkalosis when prolonged is an exception to this rule and often returns the pH to a normal value. Metabolic acidosis due to an increase in endogenous acids (e.g., ketoacidosis) lowers extracellular fluid [HCO3 -] and decreases extracellular pH. This stimulates the medullary chemoreceptors to increase ventilation and to return

1	PART 2 Cardinal Manifestations and Presentation of Diseases Range of Values

1	Prediction of the ratio of [HCO3 -] to Paco2, and thus pH, toward, but not to, normal. The degree of respiratory compensation expected in a simple form of metabolic acidosis can be predicted from the relationship: Paco2 = (1.5 × [HCO3 -]) + 8 ± 2. Thus, a patient with metabolic acidosis and [HCO3 -] of 12 mmol/L would be expected to have a Paco2 between 24 and 28 mmHg. Values for Paco2 <24 or >28 mmHg define a mixed disturbance (metabolic acidosis and respiratory alkalosis or metabolic alkalosis and respiratory acidosis, respectively). Compensatory responses for primary metabolic disorders move the Paco2 in the same direction as the change in [HCO3 -], whereas, conversely, compensation for primary respiratory disorders moves the [HCO3 -] in the same direction as the primary change in Paco2 (Table 66-1). Therefore, changes in Paco2 and [HCO3 -] in opposite directions (i.e., Paco2 or [HCO3 -] is increased, whereas the other value is decreased) indicate a mixed disturbance. Another way

1	(Table 66-1). Therefore, changes in Paco2 and [HCO3 -] in opposite directions (i.e., Paco2 or [HCO3 -] is increased, whereas the other value is decreased) indicate a mixed disturbance. Another way to judge the appropriateness of the response in [HCO3 -] or Paco2 is to use an acid-base nomogram (Fig. 66-1). While the shaded areas of the nomogram show the 95% confidence limits for normal compensation in simple disturbances, finding acid-base values within the shaded area does not necessarily rule out a mixed disturbance. Imposition of one disorder over another may result in values lying within the area of a third. Thus, the nomogram, while convenient, is not a substitute for the equations in Table 66-1.

1	Mixed acid-base disorders—defined as independently coexisting disorders, not merely compensatory responses—are often seen in patients in critical care units and can lead to dangerous extremes of pH (Table 66-2). A patient with diabetic ketoacidosis (metabolic acidosis) may develop an independent respiratory problem (e.g., limits (range of values) of the normal respiratory and metabolic compensations for primary acid-base disturbances. (From TD DuBose Jr: Acid-base disorders, in Brenner and Rector’s The Kidney, 8th ed, BM Brenner [ed]. Philadelphia, Saunders, 2008, pp 505–546, with permission.) Key: Highor normal-AG metabolic acidosis; prevailing Paco2 below predicted value (Table 66-1) Example: Na+, 140; K+, 4.0; Cl-, 106; HCO3 -, 14; AG, 20; Paco2, 24; pH, 7.39 (lactic acidosis, sepsis in ICU) Metabolic acidosis—respiratory acidosis

1	Key: Highor normal-AG metabolic acidosis; prevailing Paco2 above predicted value (Table 66-1) Example: Na+, 140; K+, 4.0; Cl-, 102; HCO3 -, 18; AG, 20; Paco2, 38; pH, 7.30 (severe pneumonia, pulmonary edema) Metabolic alkalosis—respiratory alkalosis Key: Paco2 does not increase as predicted; pH higher than expected Example: Na+, 140; K+, 4.0; Cl-, 91; HCO3 -, 33; AG, 16; Paco2, 38; pH, 7.55 (liver Metabolic alkalosis—respiratory acidosis Key: Paco2 higher than predicted; pH normal Example: Na+, 140; K+, 3.5; Cl-, 88; HCO3 -, 42; AG, 10; Paco2, 67; pH, 7.42 Key: Only detectable with high-AG acidosis; ∆AG >> ∆HCO3 Example: Na+, 140; K+, 3.0; Cl-, 95; HCO3 -, 25; AG, 20; Paco2, 40; pH, 7.42 (uremia with vomiting) Key: Mixed high-AG—normal-AG acidosis; ∆HCO3 accounted for by combined change in ∆AG and ∆Cl- Example: Na+, 135; K+, 3.0; Cl-, 110; HCO3 -, 10; AG, 15; Paco2, 25; pH, 7.20 (diarrhea and lactic acidosis, toluene toxicity, treatment of diabetic ketoacidosis)

1	Example: Na+, 135; K+, 3.0; Cl-, 110; HCO3 -, 10; AG, 15; Paco2, 25; pH, 7.20 (diarrhea and lactic acidosis, toluene toxicity, treatment of diabetic ketoacidosis) Abbreviations: AG, anion gap; COPD, chronic obstructive pulmonary disease; ICU, intensive care unit.

1	pneumonia) leading to respiratory acidosis or alkalosis. Patients with underlying pulmonary disease (e.g., chronic obstructive pulmonary disease) may not respond to metabolic acidosis with an appropriate ventilatory response because of insufficient respiratory reserve. Such imposition of respiratory acidosis on metabolic acidosis can lead to severe acidemia. When metabolic acidosis and metabolic alkalosis coexist in the same patient, the pH may be normal or near normal. When the pH is normal, an elevated anion gap (AG; see below) reliably denotes the presence of an AG metabolic acidosis at a normal serum albumin of 4.5 g/dL. Assuming a normal AG of 10 mmol/L, a discrepancy in the ∆AG (prevailing minus normal AG) and the ∆HCO3 (normal value of 25 mmol/L minus abnormal HCO3 in the patient) indicates the presence of a mixed high-gap acidosis— metabolic alkalosis (see example below). A diabetic patient with ketoacidosis may have renal dysfunction resulting in simultaneous metabolic

1	indicates the presence of a mixed high-gap acidosis— metabolic alkalosis (see example below). A diabetic patient with ketoacidosis may have renal dysfunction resulting in simultaneous metabolic acidosis. Patients who have ingested an overdose of drug combinations such as sedatives and salicylates may have mixed disturbances as a result of the acid-base response to the individual drugs (metabolic acidosis mixed with respiratory acidosis or respiratory alkalosis, respectively). Triple acid-base disturbances are more complex. For example, patients with metabolic acidosis due to alcoholic ketoacidosis may develop metabolic alkalosis due to vomiting and superimposed respiratory alkalosis due to the hyperventilation of hepatic dysfunction or alcohol withdrawal.

1	APPROACH TO THE PATIENT:

1	A stepwise approach to the diagnosis of acid-base disorders follows (Table 66-3). Care should be taken when measuring blood gases to obtain the arterial blood sample without using excessive heparin. Blood for electrolytes and arterial blood gases should be drawn simultaneously prior to therapy, because an increase in [HCO3 -] occurs with metabolic alkalosis and respiratory acidosis. Conversely, a decrease in [HCO3 -] occurs in metabolic acidosis and respiratory alkalosis. In the determination of arterial blood gases by the clinical laboratory, both pH and Paco2 are measured, and the [HCO3 -] is calculated from the Henderson-Hasselbalch equation. This calculated value should be compared with the measured [HCO3 -] (total CO2) on the electrolyte panel. These two values should agree within 2 mmol/L. If they do not, the values may not have been drawn simultaneously, a laboratory error may be present, or an error could have been made in calculating the [HCO3 -]. After verifying the blood

1	2 mmol/L. If they do not, the values may not have been drawn simultaneously, a laboratory error may be present, or an error could have been made in calculating the [HCO3 -]. After verifying the blood acid-base values, the precise acid-base disorder can then be identified.

1	All evaluations of acid-base disorders should include a simple calculation of the AG; it represents those unmeasured anions in plasma (normally 8-10 mmol/L) and is calculated as follows: AG = Na+ – (Cl-+ HCO3 -). The unmeasured anions include anionic proteins (e.g., albumin), phosphate, sulfate, and organic anions. When acid anions, such as acetoacetate and lactate, accumulate in extracellular fluid, the AG increases, causing a high-AG 1. Obtain arterial blood gas (ABG) and electrolytes simultaneously. 2. Compare [HCO3 -] on ABG and electrolytes to verify accuracy. 3. Calculate anion gap (AG). 4. Know four causes of high-AG acidosis (ketoacidosis, lactic acid acidosis, renal failure, and toxins). 5. Know two causes of hyperchloremic or nongap acidosis (bicarbonate loss from gastrointestinal tract, renal tubular acidosis). 6. Estimate compensatory response (Table 66-1). 7. Compare ∆AG and ∆HCO3 . 8. Compare change in [Cl-] with change in [Na+].

1	acidosis. An increase in the AG is most often due to an increase in unmeasured anions and, less commonly, is due to a decrease in unmeasured cations (calcium, magnesium, potassium). In addition, the AG may increase with an increase in anionic albumin, because of either increased albumin concentration or alkalosis, which alters albumin charge. A decrease in the AG can be due to (1) an increase in unmeasured cations; (2) the addition to the blood of abnormal cations, such as lithium (lithium intoxication) or cationic immunoglobulins (plasma cell dyscrasias); (3) a reduction in the major plasma anion albumin concentration (nephrotic syndrome); (4) a decrease in the effective anionic charge on albumin by acidosis; or (5) hyperviscosity and severe hyperlipidemia, which can lead to an underestimation of sodium and chloride concentrations. A fall in serum albumin by 1 g/dL from the normal value (4.5 g/dL) decreases the AG by 2.5 meq/L. Know the common causes of a high-AG acidosis (Table

1	of sodium and chloride concentrations. A fall in serum albumin by 1 g/dL from the normal value (4.5 g/dL) decreases the AG by 2.5 meq/L. Know the common causes of a high-AG acidosis (Table 66-3).

1	In the face of a normal serum albumin, a high AG is usually due to non–chloride-containing acids that contain inorganic (phosphate, sulfate), organic (ketoacids, lactate, uremic organic anions), exogenous (salicylate or ingested toxins with organic acid production), or unidentified anions. The high AG is significant even if an additional acid-base disorder is superimposed to modify the [HCO3 -] independently. Simultaneous metabolic acidosis of the high-AG variety plus either chronic respiratory acidosis or metabolic alkalosis represents such a situation in which [HCO3 -] may be normal or even high (Table 66-3). Compare the change in [HCO3 -] (∆HCO3 -) and the change in the AG (∆AG).

1	Similarly, normal values for [HCO3 -], Paco2, and pH do not ensure the absence of an acid-base disturbance. For instance, an alcoholic who has been vomiting may develop a metabolic alkalosis with a pH of 7.55, Paco2 of 47 mmHg, [HCO3 -] of 40 mmol/L, [Na+] of 135, [Cl-] of 80, and [K+] of 2.8. If such a patient were then to develop a superimposed alcoholic ketoacidosis with a β-hydroxybutyrate concentration of 15 mM, arterial pH would fall to 7.40, [HCO3 -] to 25 mmol/L, and the Paco2 to 40 mmHg. Although these blood gases are normal, the AG is elevated at 30 mmol/L, indicating a mixed metabolic alkalosis and metabolic acidosis. A mixture of high-gap acidosis and metabolic alkalosis is recognized easily by comparing the differences (∆ values) in the normal to prevailing patient values. In this example, the ∆HCO3 is 0 (25 25 mmol/L), but the ∆AG is 20 (30 – 10 mmol/L). Therefore, 20 mmol/L is unaccounted for in the ∆/∆ value (∆AG to ∆HCO3 -).

1	Metabolic acidosis can occur because of an increase in endogenous acid production (such as lactate and ketoacids), loss of bicarbonate (as in diarrhea), or accumulation of endogenous acids (as in renal failure). Metabolic acidosis has profound effects on the respiratory, cardiac, and nervous systems. The fall in blood pH is accompanied by a char acteristic increase in ventilation, especially the tidal volume (Kussmaul respiration). Intrinsic cardiac contractility may be depressed, but ino tropic function can be normal because of catecholamine release. Both present; the decrease in central and pulmonary vascular compliance predisposes to pulmonary edema with even minimal volume overload. CNS function is depressed, with headache, lethargy, stupor, and, in some cases, even coma. Glucose intolerance may also occur. There are two major categories of clinical metabolic acidosis: high-AG and non-AG, or hyperchloremic, acidosis (Table 66-3 and Table 66-4).

1	Table 66-4). Treatment of metabolic acidosis with alkali should be reserved for severe acidemia except when the patient has no “potential HCO3 -” in plasma. Potential [HCO3 -] can be estimated from the increment (∆) in the AG (∆AG = patient’s AG – 10). It must be determined if

1	CAuSES of HigH-Anion gAP METABoLiC ACiDoSiS the acid anion in plasma is metabolizable (i.e., β-hydroxybutyrate, acetoacetate, and lactate) or nonmetabolizable (anions that accumulate in chronic renal failure and after toxin ingestion). The latter requires return of renal function to replenish the [HCO3 -] deficit, a slow and often unpredictable process. Consequently, patients with a normal AG acidosis (hyperchloremic acidosis), a slightly elevated AG (mixed hyperchloremic and AG acidosis), or an AG attributable to a nonmetabolizable anion in the face of renal failure should receive alkali therapy, either PO (NaHCO3 or Shohl’s solution) or IV (NaHCO3), in an amount necessary to slowly increase the plasma [HCO3 -] into the 20–22 mmol/L range. Overcorrection must be avoided.

1	Controversy exists, however, in regard to the use of alkali in patients with a pure AG acidosis owing to accumulation of a metabolizable organic acid anion (ketoacidosis or lactic acidosis). In general, severe acidosis (pH <7.10) warrants the IV administration of 50–100 meq of NaHCO3, over 30–45 min, during the initial 1–2 h of therapy. Provision of such modest quantities of alkali in this situation seems to provide an added measure of safety, but it is essential to monitor plasma electrolytes during the course of therapy, because the [K+] may decline as pH rises. The goal is to increase the [HCO3 -] to 10 meq/L and the pH to approximately 7.20, not to increase these values to normal. PART 2 Cardinal Manifestations and Presentation of Diseases APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: There are four principal causes of a high-AG acidosis: (1) lactic acidosis, (2) ketoacidosis, (3) ingested toxins, and (4) acute and chronic renal failure (Table 66-4). Initial screening to differentiate the high-AG acidoses should include (1) a probe of the history for evidence of drug and toxin ingestion and measurement of arterial blood gas to detect coexistent respiratory alkalosis (salicylates); (2) determination of whether diabetes mellitus is present (diabetic ketoacidosis); (3) a search for evidence of alcoholism or increased levels of β-hydroxybutyrate (alcoholic ketoacidosis); (4) observation for clinical signs of uremia and determination of the blood urea nitrogen (BUN) and creatinine (uremic acidosis); (5) inspection of the urine for oxalate crystals (ethylene glycol); and (6) recognition of the numerous clinical settings in which lactate levels may be increased (hypotension, shock, cardiac failure, leukemia, cancer, and drug or toxin ingestion).

1	Lactic Acidosis An increase in plasma l-lactate may be secondary to poor tissue perfusion (type A)—circulatory insufficiency (shock, cardiac failure), severe anemia, mitochondrial enzyme defects, and inhibitors (carbon monoxide, cyanide)—or to aerobic disorders (type B)— malignancies, nucleoside analogue reverse transcriptase inhibitors in HIV, diabetes mellitus, renal or hepatic failure, thiamine deficiency, severe infections (cholera, malaria), seizures, or drugs/toxins (biguanides, ethanol, methanol, propylene glycol, isoniazid, and fructose). Unrecognized bowel ischemia or infarction in a patient with severe atherosclerosis or cardiac decompensation receiving vasopressors is a common cause of lactic acidosis. Pyroglutamic acidemia has been reported in critically ill patients receiving acetaminophen, which is associated with depletion of glutathione. d-Lactic acid acidosis, which may be associated with jejunoileal bypass, short bowel syndrome, or intestinal obstruction, is due to

1	acetaminophen, which is associated with depletion of glutathione. d-Lactic acid acidosis, which may be associated with jejunoileal bypass, short bowel syndrome, or intestinal obstruction, is due to formation of d-lactate by gut bacteria.

1	APPROACH TO THE PATIENT: The underlying condition that disrupts lactate metabolism must first be corrected; tissue perfusion must be restored when inadequate. Vasoconstrictors should be avoided, if possible, because they may worsen tissue perfusion. Alkali therapy is generally advocated for acute, severe acidemia (pH <7.15) to improve cardiac function and lactate use. However, NaHCO3 therapy may paradoxically depress cardiac performance and exacerbate acidosis by enhanc ing lactate production (HCO3 stimulates phosphofructokinase). While the use of alkali in moderate lactic acidosis is controversial, it is generally agreed that attempts to return the pH or [HCO3 -] to normal by administration of exogenous NaHCO3 are deleterious. A reasonable approach is to infuse sufficient NaHCO3 to raise the arterial pH to no more than 7.2 over 30–40 min.

1	NaHCO3 therapy can cause fluid overload and hypertension because the amount required can be massive when accumulation of lactic acid is relentless. Fluid administration is poorly tolerated because of central venoconstriction, especially in the oliguric patient. When the underlying cause of the lactic acidosis can be remedied, blood lactate will be converted to HCO3 and may result in an overshoot alkalosis.

1	Ketoacidosis • dIABETIC kETOACIdOSIS (dkA) This condition is caused by increased fatty acid metabolism and the accumulation of ketoacids (acetoacetate and β-hydroxybutyrate). DKA usually occurs in insulin-dependent diabetes mellitus in association with cessation of insulin or an intercurrent illness such as an infection, gastroenteritis, pancreatitis, or myocardial infarction, which increases insulin requirements temporarily and acutely. The accumulation of ketoacids accounts for the increment in the AG and is accompanied most often by hyperglycemia (glucose >17 mmol/L [300 mg/dL]). The relationship between the ∆AG and ∆HCO3 is typically ∼1:1 in DKA. It should be noted that, because insulin prevents production of ketones, bicarbonate therapy is rarely needed except with extreme acidemia (pH < 7.1), and then in only limited amounts. Patients with DKA are typically volume depleted and require fluid resuscitation with isotonic saline. Volume overexpansion with IV fluid administration is

1	< 7.1), and then in only limited amounts. Patients with DKA are typically volume depleted and require fluid resuscitation with isotonic saline. Volume overexpansion with IV fluid administration is not uncommon, however, and contributes to the development of a hyperchloremic acidosis during treatment of DKA. The mainstay for treatment of this condition is IV regular insulin and is described in Chap. 417 in more detail.

1	ALCOHOLIC kETOACIdOSIS (AkA) Chronic alcoholics can develop ketoacidosis when alcohol consumption is abruptly curtailed and nutrition is poor. AKA is usually associated with binge drinking, vomiting, abdominal pain, starvation, and volume depletion. The glucose concentration is variable, and acidosis may be severe because of elevated ketones, predominantly β-hydroxybutyrate. Hypoperfusion may enhance lactic acid production, chronic respiratory alkalosis may accompany liver disease, and metabolic alkalosis can result from vomiting (refer to the relationship between ∆AG and ∆HCO3 -). Thus, mixed acid-base disorders are common in AKA. As the circulation is restored by administration of isotonic saline, the preferential accumulation of β-hydroxybutyrate is then shifted to acetoacetate. This explains the common clinical observation of an increasingly positive nitroprusside reaction as the patient improves. The nitroprusside ketone reaction (Acetest) can detect acetoacetic acid but not

1	explains the common clinical observation of an increasingly positive nitroprusside reaction as the patient improves. The nitroprusside ketone reaction (Acetest) can detect acetoacetic acid but not β-hydroxybutyrate, so that the degree of ketosis and ketonuria can not only change with therapy, but can be underestimated initially. Patients with AKA usually present with relatively normal renal function, as opposed to DKA, where renal function is often compromised because of volume depletion (osmotic diuresis) or diabetic nephropathy. The AKA patient with normal renal function may excrete relatively large quantities of ketoacids in the urine and, therefore, may have a relatively normal

1	AG and a discrepancy in the ∆AG/∆HCO3 relationship. Extracellular fluid deficits almost always accompany AKA and should be repleted by IV administration of saline and glucose (5% dextrose in 0.9% NaCl). Hypophosphatemia, hypokalemia, and hypomagnesemia may coexist and should be corrected. Hypophosphatemia usually emerges 12–24 h after admission, may be exacerbated by glucose infusion, and, if severe, may induce rhabdomyolysis or even respiratory arrest. Upper gastrointestinal hemorrhage, pancreatitis, and pneumonia may accompany this disorder. Drugand Toxin-Induced Acidosis • SALICYLATES (See also Chap. 472e) Salicylate intoxication in adults usually causes respiratory alkalosis or a mixture of high-AG metabolic acidosis and respiratory alkalosis. Only a portion of the AG is due to salicylates. Lactic acid production is also often increased.

1	Vigorous gastric lavage with isotonic saline (not NaHCO3) should be initiated immediately, followed by administration of activated charcoal per nasogastric tube. In the acidotic patient, to facilitate removal of salicylate, IV NaHCO3 is administered in amounts adequate to alkalinize the urine and to maintain urine output (urine pH >7.5). While this form of therapy is straightforward in acidotic patients, a coexisting respiratory alkalosis may make this approach hazardous. Alkalemic patients should not receive NaHCO3. Acetazolamide may be administered in the face of alkalemia, when an alkaline diuresis cannot be achieved, or to ameliorate volume overload associated with NaHCO3 administration, but this drug can cause systemic metabolic acidosis if HCO3 is not replaced. Hypokalemia should be anticipated with an alkaline diuresis and should be treated promptly and aggressively. Glucose-containing fluids should be administered because of the danger of hypoglycemia. Excessive insensible

1	be anticipated with an alkaline diuresis and should be treated promptly and aggressively. Glucose-containing fluids should be administered because of the danger of hypoglycemia. Excessive insensible fluid losses may cause severe volume depletion and hypernatremia. If renal failure prevents rapid clearance of salicylate, hemodialysis can be performed against a bicarbonate dialysate.

1	ALCOHOLS Under most physiologic conditions, sodium, urea, and glucose generate the osmotic pressure of blood. Plasma osmolality is calculated according to the following expression: Posm = 2Na+ + Glu + BUN (all in mmol/L), or, using conventional laboratory values in which glucose and BUN are expressed in milligrams per deciliter: Posm = 2Na+ + Glu/18 + BUN/2.8. The calculated and determined osmolality should agree within 10–15 mmol/kg H2O. When the measured osmolality exceeds the calculated osmolality by >10–15 mmol/kg H2O, one of two circumstances prevails. Either the serum sodium is spuriously low, as with hyperlipidemia or hyperproteinemia (pseudohyponatremia), or osmolytes other than sodium salts, glucose, or urea have accumulated in plasma. Examples of such osmolytes include mannitol, radiocontrast media, ethanol, isopropyl alcohol, ethylene glycol, propylene glycol, methanol, and acetone. In this situation, the difference between the calculated osmolality and the measured

1	radiocontrast media, ethanol, isopropyl alcohol, ethylene glycol, propylene glycol, methanol, and acetone. In this situation, the difference between the calculated osmolality and the measured osmolality (osmolar gap) is proportional to the concentration of the unmeasured solute. With an appropriate clinical history and index of suspicion, identification of an osmolar gap is helpful in identifying the presence of poison-associated AG acidosis. Three alcohols may cause fatal intoxications: ethylene glycol, methanol, and isopropyl alcohol. All cause an elevated osmolal gap, but only the first two cause a high-AG acidosis.

1	ETHYLENE gLYCOL (See also Chap. 472e) Ingestion of ethylene glycol (commonly used in antifreeze) leads to a metabolic acidosis and severe damage to the CNS, heart, lungs, and kidneys. The increased AG and osmolar gap are attributable to ethylene glycol and its metabolites, oxalic acid, glycolic acid, and other organic acids. Lactic acid production increases secondary to inhibition of the tricarboxylic acid cycle and altered intracellular redox state. Diagnosis is facilitated by recognizing oxalate crystals in the urine, the presence of an osmolar gap in serum, and a high-AG acidosis. Although use of a Wood’s lamp to 319 visualize the fluorescent additive to commercial antifreeze in the urine of patients with ethylene glycol ingestion, this is rarely reproducible. The combination of a high AG and high osmolar gap in a patient suspected of ethylene glycol ingestion should be taken as evidence of ethylene glycol toxicity. Treatment should not be delayed while awaiting measurement of

1	high AG and high osmolar gap in a patient suspected of ethylene glycol ingestion should be taken as evidence of ethylene glycol toxicity. Treatment should not be delayed while awaiting measurement of ethylene glycol levels in this setting.

1	This includes the prompt institution of a saline or osmotic diuresis, thiamine and pyridoxine supplements, fomepizole, and usually, hemodialysis. The IV administration of the alcohol dehydrogenase inhibitor fomepizole (4-methylpyrazole; 15 mg/kg as a loading dose) is the agent of choice and offers the advantages of a predictable decline in ethylene glycol levels without excessive obtundation as seen during ethyl alcohol infusion. If used, ethanol IV should be infused to achieve a blood level of 22 mmol/L (100 mg/dL). Both fomepizole and ethanol reduce toxicity because they compete with ethylene glycol for metabolism by alcohol dehydrogenase. Hemodialysis is indicated when the arterial pH is <7.3 or the osmolar gap exceeds 20 mOsm/kg.

1	METHANOL (See also Chap. 472e) The ingestion of methanol (wood alcohol) causes metabolic acidosis, and its metabolites formaldehyde and formic acid cause severe optic nerve and CNS damage. Lactic acid, ketoacids, and other unidentified organic acids may contribute to the acidosis. Due to its low molecular mass (32 Da), an osmolar gap is usually present. This is similar to that for ethylene glycol intoxication, including general supportive measures, fomepizole, and hemodialysis (as above).

1	This is similar to that for ethylene glycol intoxication, including general supportive measures, fomepizole, and hemodialysis (as above). PROPYLENE gLYCOL Propylene glycol is the vehicle used in IV administration of diazepam, lorazepam, phenobarbital, nitroglycerine, etomidate, enoximone, and phenytoin. Propylene glycol is generally safe for limited use in these IV preparations, but toxicity has been reported, most often in the setting of the intensive care unit in patients receiving frequent or continuous therapy. This form of high-gap acidosis should be considered in patients with unexplained high-gap acidosis, hyperosmolality, and clinical deterioration. Propylene glycol, like ethylene glycol and methanol, is metabolized by alcohol dehydrogenase. With intoxication by propylene glycol, the first response is to stop the offending infusion. Additionally, fomepizole should also be administered in acidotic patients.

1	ISOPROPYL ALCOHOL Ingested isopropanol is absorbed rapidly and may be fatal when as little as 150 mL of rubbing alcohol, solvent, or deicer is consumed. A plasma level >400 mg/dL is life-threatening. Isopropyl alcohol is metabolized by alcohol dehydrogenase to acetone. The characteristic features differ from ethylene glycol and methanol in that the parent compound, not the metabolites, causes toxicity, and an AG acidosis is not present because acetone is rapidly excreted. Both isopropyl alcohol and acetone increase the osmolal gap, and hypoglycemia is common. Alternative diagnoses should be considered if the patient does not improve significantly within a few hours. Patients with hemodynamic instability with plasma levels above 400 mg/dL should be considered for hemodialysis.

1	Isopropanol alcohol toxicity is treated by watchful waiting and supportive therapy, IV fluids, pressors, ventilatory support if needed, and occasionally hemodialysis for prolonged coma, hemodynamic instability, or levels >400 mg/dL. 320 PYROgLUTAMIC ACId Acetaminophen-induced high-AG metabolic acidosis is uncommon but is being recognized more often in either patients with acetaminophen overdose or malnourished or critically ill patients receiving acetaminophen in typical dosage. 5-Oxoproline accumulation after acetaminophen should be suspected in the setting of an unexplained high-AG acidosis without elevation of the osmolar gap in patients receiving acetaminophen. The first step in treatment is to immediately discontinue the drug. Additionally, sodium bicarbonate IV should be given. Although N-acetylcysteine has been suggested, it is not known if it hastens the metabolism of 5-oxoproline by increasing intracellular glutathione concentrations in this setting.

1	Renal Failure (See also Chap. 335) The hyperchloremic acidosis of moderate renal insufficiency is eventually converted to the high-AG acidosis of advanced renal failure. Poor filtration and reabsorption of organic anions contribute to the pathogenesis. As renal disease progresses, the number of functioning nephrons eventually becomes insufficient to keep pace with net acid production. Uremic acidosis is characterized, therefore, by a reduced rate of NH4+ production and excretion. The acid retained in chronic renal disease is buffered by alkaline salts from bone. Despite significant retention of acid (up to 20 mmol/d), the serum [HCO3 -] does not decrease further, indicating participation of buffers outside the extracellular compartment. Chronic metabolic acidosis results in significant loss of bone mass due to reduction in bone calcium carbonate. Chronic acidosis also increases urinary calcium excretion, proportional to cumulative acid retention.

1	Because of the association of renal failure acidosis with muscle catabolism and bone disease, both uremic acidosis and the hyperchloremic acidosis of renal failure require oral alkali replacement to maintain the [HCO3 -] >22 mmol/L. This can be accomplished with relatively modest amounts of alkali (1.0–1.5 mmol/kg body weight per day). Sodium citrate (Shohl’s solution) or NaHCO3 tablets (650-mg tablets contain 7.8 meq) are equally effective alkalinizing salts. Citrate enhances the absorption of aluminum from the gastrointestinal tract and should never be given together with aluminum-containing antacids because of the risk of aluminum intoxication. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Alkali can be lost from the gastrointestinal tract from diarrhea or from the kidneys (renal tubular acidosis, RTA). In these disorders (Table 66-5), reciprocal changes in [Cl-] and [HCO3 -] result in a normal AG. In pure non–AG acidosis, therefore, the increase in [Cl-] above the normal value approximates the decrease in [HCO3 -]. The absence of such a relationship suggests a mixed disturbance.

1	In diarrhea, stools contain a higher [HCO3 -] and decomposed HCO3 than plasma so that metabolic acidosis develops along with volume depletion. Instead of an acid urine pH (as anticipated with systemic acidosis), urine pH is usually >6 because metabolic acidosis and hypokalemia increase renal synthesis and excretion of NH4+, thus providing a urinary buffer that increases urine pH. Metabolic acidosis due to gastrointestinal losses with a high urine pH can be differentiated from RTA because urinary NH4+ excretion is typically low in RTA and high with diarrhea. Urinary NH4+ levels can be estimated by calculating the urine anion gap (UAG): UAG = [Na+ + K+]u – [Cl-]u. When [Cl-]u > [Na+ + K+]u, the UAG is negative by definition. This indicates that the urine ammonium level is appropriately increased, suggesting an extrarenal cause of the acidosis. Conversely, when the UAG is positive, the urine ammonium level is low, suggesting a renal cause of the acidosis.

1	CAuSES of non–Anion gAP ACiDoSiS 1. Gastrointestinal bicarbonate loss A. Diarrhea B. External pancreatic or small-bowel drainage C. Ureterosigmoidostomy, jejunal loop, ileal loop D. Drugs 1. 2. 3. II. A. 1. Proximal RTA (type 2) Drug-induced: acetazolamide, topiramate 2. Distal (classic) RTA (type 1) Drug induced: amphotericin B, ifosfamide B. Hyperkalemia 1. Generalized distal nephron dysfunction (type 4 RTA) a. b. Mineralocorticoid resistance (PHA I, autosomal dominant) c. Voltage defect (PHA I, autosomal recessive, and PHA II) d. C. 1. Chronic progressive kidney disease III. A. Potassium-sparing diuretics (amiloride, triamterene, spironolactone, eplerenone) B. C. D. E. F. IV. A. Acid loads (ammonium chloride, hyperalimentation) B. Loss of potential bicarbonate: ketosis with ketone excretion C. D. E.

1	B. C. D. E. F. IV. A. Acid loads (ammonium chloride, hyperalimentation) B. Loss of potential bicarbonate: ketosis with ketone excretion C. D. E. Abbreviations: ACE-I, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; PHA, pseudohypoaldosteronism; RTA, renal tubular acidosis. Proximal RTA (type 2 RTA) (Chap. 339) is most often due to generalized proximal tubular dysfunction manifested by glycosuria, generalized aminoaciduria, and phosphaturia (Fanconi syndrome). With a low plasma [HCO3 -], the urine pH is acid (pH <5.5). The fractional excretion of [HCO3 -] may exceed 10–15% when the serum HCO3 - >20 mmol/L. Because HCO3 is not reabsorbed normally in the proximal tubule, therapy with NaHCO3 will enhance renal potassium wasting and hypokalemia.

1	The typical findings in acquired or inherited forms of classic distal RTA (type 1 RTA) include hypokalemia, non-AG metabolic acidosis, low urinary NH4+ excretion (positive UAG, low urine [NH4+]), and inappropriately high urine pH (pH > 5.5). Most patients have hypocitraturia and hypercalciuria, so nephrolithiasis, nephrocalcinosis, and bone disease are common. In generalized distal RTA (type 4 RTA), hyperkalemia is disproportionate to the reduction in glomerular filtration rate (GFR) because of coexisting dysfunction of potassium and acid secretion. Urinary ammonium excretion is invariably depressed, and renal function may be compromised, for example, due to diabetic nephropathy, obstructive uropathy, or chronic tubulointerstitial disease.

1	Hyporeninemic hypoaldosteronism typically causes non-AG metabolic acidosis, most commonly in older adults with diabetes mellitus or tubulointerstitial disease and renal insufficiency. Patients usually have mild to moderate CKD (GFR, 20–50 mL/min) and acidosis, with elevation in serum [K+] (5.2–6.0 mmol/L), concurrent hypertension, and congestive heart failure. Both the metabolic acidosis and the hyperkalemia are out of proportion to impairment in GFR. Nonsteroidal anti-inflammatory drugs, trimethoprim, pentamidine, and angiotensin-converting enzyme (ACE) inhibitors can also cause non-AG metabolic acidosis in patients with renal insufficiency (Table 66-5).

1	Metabolic alkalosis is manifested by an elevated arterial pH, an increase in the serum [HCO3 -], and an increase in Paco2 as a result of compensatory alveolar hypoventilation (Table 66-1). It is often accompanied by hypochloremia and hypokalemia. The arterial pH establishes the diagnosis, because it is increased in metabolic alkalosis and decreased or normal in respiratory acidosis. Metabolic alkalosis frequently occurs in association with other disorders such as respiratory acidosis or alkalosis or metabolic acidosis. Metabolic alkalosis occurs as a result of net gain of [HCO3 -] or loss of nonvolatile acid (usually HCl by vomiting) from the extracellular fluid.

1	For HCO3 to be added to the extracellular fluid, it must be administered exogenously or synthesized endogenously, in part or entirely by the kidneys. Because it is unusual for alkali to be added to the body, the disorder involves a generative stage, in which the loss of acid usually causes alkalosis, and a maintenance stage, in which the kidneys fail to compensate by excreting HCO3 . Maintenance of metabolic alkalosis represents a failure of the kid neys to eliminate HCO3 in the usual manner. The kidneys will retain, rather than excrete, the excess alkali and maintain the alkalosis if (1) volume deficiency, chloride deficiency, and K+ deficiency exist in combination with a reduced GFR; or (2) hypokalemia exists because of autonomous hyperaldosteronism. In the first example, alkalosis is corrected by administration of NaCl and KCl, whereas, in the latter, it may be necessary to repair the alkalosis by pharmacologic or surgical intervention, not with saline administration.

1	To establish the cause of metabolic alkalosis (Table 66-6), it is necessary to assess the status of the extracellular fluid volume (ECFV), the recumbent and upright blood pressure, the serum [K+], and the renin-aldosterone system. For example, the presence of chronic hypertension and chronic hypokalemia in an alkalotic patient suggests either mineralocorticoid excess or that the hypertensive patient is receiving diuretics. Low plasma renin activity and normal urine [Na+] and [Cl-] in a patient who is not taking diuretics indicate a primary mineralocorticoid excess syndrome. The combination of hypokalemia and alkalosis in a normotensive, nonedematous patient can be due to Bartter’s or Gitelman’s syndrome, magnesium deficiency, vomiting, exogenous alkali, or diuretic ingestion. Determination of urine electrolytes (especially the urine [Cl-]) and screening of the urine for diuretics may be helpful. If the urine is alkaline, with an elevated [Na+] and [K+] but low [Cl-], the diagnosis is

1	of urine electrolytes (especially the urine [Cl-]) and screening of the urine for diuretics may be helpful. If the urine is alkaline, with an elevated [Na+] and [K+] but low [Cl-], the diagnosis is usually either vomiting (overt or surreptitious) or alkali ingestion. If the urine is relatively acid and has low concentrations of Na+, K+, and Cl-, the most likely possibilities are prior vomiting, the posthypercapnic state, or prior diuretic ingestion. If, on the other hand, neither the urine sodium, potassium, nor chloride concentrations are depressed, magnesium deficiency, Bartter’s or Gitelman’s syndrome, or current diuretic ingestion should be considered. Bartter’s syndrome is distinguished from Gitelman’s syndrome because of hypocalciuria and hypomagnesemia in the latter disorder.

1	Alkali Administration Chronic administration of alkali to individuals with normal renal function rarely causes alkalosis. However, in patients with coexistent hemodynamic disturbances, alkalosis can develop because the normal capacity to excrete HCO3 may be exceeded or there may be enhanced reabsorption of HCO3 -. Such - patients include those who receive HCO3 (PO or IV), acetate loads CAuSES of METABoLiC ALKALoSiS I. Exogenous HCO3 -loads A. Acute alkali administration B. Milk-alkali syndrome II. Effective ECFV contraction, normotension, K+ deficiency, and secondary hyperreninemic hyperaldosteronism A. 1. 2. 3. 4. B. Renal origin 1. 2. 3. 4. 5. Nonreabsorbable anions including penicillin, carbenicillin 6. Mg2+ deficiency 7. 8. Bartter’s syndrome (loss of function mutations of transporters and ion channels in TALH) 9. Gitelman’s syndrome (loss of function mutation in Na+-Clcotransporter in DCT) III.

1	8. Bartter’s syndrome (loss of function mutations of transporters and ion channels in TALH) 9. Gitelman’s syndrome (loss of function mutation in Na+-Clcotransporter in DCT) III. ECFV expansion, hypertension, K+ deficiency, and mineralocorticoid excess A. 1. 2. 3. 4. B. Low renin 1. Primary aldosteronism a. b. c. 2. Adrenal enzyme defects a. 11β-Hydroxylase deficiency b. 17α-Hydroxylase deficiency 3. 4. a. b. c. IV. Gain-of-function mutation of renal sodium channel with ECFV expansion, hypertension, K+ deficiency, and hyporeninemic-hypoaldosteronism A. Abbreviations: DCT, distal convoluted tubule; ECFV, extracellular fluid volume; TALH, thick ascending limb of Henle’s loop.

1	A. Abbreviations: DCT, distal convoluted tubule; ECFV, extracellular fluid volume; TALH, thick ascending limb of Henle’s loop. (parenteral hyperalimentation solutions), citrate loads (transfusions), or antacids plus cation-exchange resins (aluminum hydroxide and sodium polystyrene sulfonate). Nursing home patients receiving tube feedings have a higher incidence of metabolic alkalosis than nursing home patients receiving oral feedings. METABOLIC ALKALOSIS ASSOCIATED WITH ECFV CONTRACTION, K+ DEPLETION,

1	METABOLIC ALKALOSIS ASSOCIATED WITH ECFV CONTRACTION, K+ DEPLETION, AND SECONDARY HYPERRENINEMIC HYPERALDOSTERONISM gastrointestinal Origin Gastrointestinal loss of H+ from vomiting or gastric aspiration results in retention of HCO3 -. During active vomiting, the filtered load of bicarbonate is acutely increased to exceed the reabsorptive capacity of the proximal tubule for HCO3 so that the urine becomes alkaline and high in potassium. When vomiting ceases, the persistence of volume, potassium, and chloride depletion causes 322 maintenance of the alkalosis because of an enhanced capacity of the nephron to reabsorb HCO3 -. Correction of the contracted ECFV with NaCl and repair of K+ deficits corrects the acid-base disorder by restoring the ability of the kidney to excrete the excess bicarbonate.

1	Renal Origin • dIURETICS (See also Chap. 279) Drugs that induce chloruresis, such as thiazides and loop diuretics (furosemide, bumetanide, torsemide, and ethacrynic acid), acutely diminish the ECFV without altering the total body bicarbonate content. The serum [HCO3 -] increases because the reduced ECFV “contracts” the [HCO3 -] in the plasma (contraction alkalosis). The chronic administration of diuretics tends to generate an alkalosis by increasing distal salt delivery, so that K+ and H+ secretion are stimulated. The alkalosis is maintained by persistence of the contraction of the ECFV, secondary hyperaldosteronism, K+ deficiency, and the direct effect of the diuretic (as long as diuretic administration continues). Repair of the alkalosis is achieved by providing isotonic saline to correct the ECFV deficit. SOLUTE LOSINg dISORdERS: BARTTER’S SYNdROME ANd gITELMAN’S SYNdROME See Chap. 339.

1	SOLUTE LOSINg dISORdERS: BARTTER’S SYNdROME ANd gITELMAN’S SYNdROME See Chap. 339. NONREABSORBABLE ANIONS ANd MAgNESIUM dEFICIENCY Administration of large quantities of nonreabsorbable anions, such as penicillin or carbenicillin, can enhance distal acidification and K+ secretion by increasing the transepithelial potential difference. Mg2+ deficiency results in hypokalemic alkalosis by enhancing distal acidification through stimulation of renin and hence aldosterone secretion. POTASSIUM dEPLETION Chronic K+ depletion may cause metabolic alkalosis by increasing urinary acid excretion. Both NH4+ production and absorption are enhanced and HCO3 reabsorption is stimulated. Chronic K+ deficiency upregulates the renal H+, K+-ATPase to increase K+ absorption at the expense of enhanced H+ secretion. Alkalosis associated with severe K+ depletion is resistant to salt administration, but repair of the K+ deficiency corrects the alkalosis.

1	AFTER TREATMENT OF LACTIC ACIdOSIS OR kETOACIdOSIS When an underlying stimulus for the generation of lactic acid or ketoacid is removed rapidly, as with repair of circulatory insufficiency or with insulin therapy, the lactate or ketones are metabolized to yield an equivalent amount of HCO3 -. Other sources of new HCO3 are additive with the original amount generated by organic anion metabolism to create a surfeit of HCO3 -. Such sources include (1) new HCO3 -added to the blood by the kidneys as a result of enhanced acid excretion during the preexisting period of acidosis, and (2) alkali therapy during the treatment phase of the acidosis. Acidosis-induced contraction of the ECFV and K+ deficiency act to sustain the alkalosis. POSTHYPERCAPNIA Prolonged CO2 retention with chronic respiratory acidosis enhances renal HCO3 absorption and the generation of new

1	POSTHYPERCAPNIA Prolonged CO2 retention with chronic respiratory acidosis enhances renal HCO3 absorption and the generation of new HCO3 (increased net acid excretion). Metabolic alkalosis results from the effect of the persistently elevated [HCO3 -] when the elevated Paco2 is abruptly returned toward normal. METABOLIC ALKALOSIS ASSOCIATED WITH ECFV EXPANSION, HYPERTENSION, AND HYPERALDOSTERONISM Increased aldosterone levels may be the result of autonomous primary adrenal overproduction or of secondary aldosterone release due to renal overproduction of renin. Mineralocorticoid excess increases net acid excretion and may result in metabolic alkalosis, which may be worsened by associated K+ deficiency. ECFV expansion from salt retention causes hypertension. The kaliuresis persists because of mineralocorticoid excess and distal Na+ absorption causing enhanced K+ excretion, continued K+ depletion with polydipsia, inability to concentrate the urine, and polyuria.

1	Liddle’s syndrome (Chap. 339) results from increased activity of the collecting duct Na+ channel (ENaC) and is a rare monogenic form of hypertension due to volume expansion manifested as hypokalemic alkalosis and normal aldosterone levels. Symptoms With metabolic alkalosis, changes in CNS and peripheral nervous system function are similar to those of hypocalcemia (Chap. 423); symptoms include mental confusion; obtundation; and PART 2 Cardinal Manifestations and Presentation of Diseases a predisposition to seizures, paresthesia, muscular cramping, tetany, aggravation of arrhythmias, and hypoxemia in chronic obstructive pulmonary disease. Related electrolyte abnormalities include hypokalemia and hypophosphatemia. This is primarily directed at correcting the underlying stimulus for

1	This is primarily directed at correcting the underlying stimulus for HCO3 generation. If primary aldosteronism, renal artery stenosis, or Cushing’s syndrome is present, correction of the underlying cause will reverse the alkalosis. [H+] loss by the stomach or kidneys can be mitigated by the use of proton pump inhibitors or the discontinuation of diuretics. The second aspect of treatment is to remove the factors that sustain the inappropriate increase in HCO3 reabsorption, such as ECFV contraction or K+ deficiency. K+ deficits should always be repaired. Isotonic saline is usually sufficient to reverse the alkalosis if ECFV contraction is present.

1	If associated conditions preclude infusion of saline, renal HCO3 loss can be accelerated by administration of acetazolamide, a carbonic anhydrase inhibitor, which is usually effective in patients with adequate renal function but can worsen K+ losses. Dilute hydrochloric acid (0.1 N HCl) is also effective but can cause hemolysis, and must be delivered slowly in a central vein. Respiratory acidosis can be due to severe pulmonary disease, respiratory muscle fatigue, or abnormalities in ventilatory control and is recognized by an increase in Paco2 and decrease in pH (Table 66-7). In acute respiratory acidosis, there is an immediate compensatory elevation (due to cellular buffering mechanisms) in HCO3 -, which increases 1 mmol/L for every 10-mmHg increase in Paco2. In chronic respiratory acidosis (>24 h), renal adaptation increases the [HCO3 -] by 4 mmol/L for every 10-mmHg increase in Paco2. The serum HCO3 usually does not increase above 38 mmol/L.

1	The clinical features vary according to the severity and duration of the respiratory acidosis, the underlying disease, and whether there is accompanying hypoxemia. A rapid increase in Paco2 may cause anxiety, dyspnea, confusion, psychosis, and hallucinations and may progress to coma. Lesser degrees of dysfunction in chronic hypercapnia include sleep disturbances; loss of memory; daytime somnolence; personality changes; impairment of coordination; and motor disturbances such as tremor, myoclonic jerks, and asterixis. Headaches and other signs that mimic raised intracranial pressure, such as papilledema, abnormal reflexes, and focal muscle weakness, are due to vasoconstriction secondary to loss of the vasodilator effects of CO2.

1	Depression of the respiratory center by a variety of drugs, injury, or disease can produce respiratory acidosis. This may occur acutely with general anesthetics, sedatives, and head trauma or chronically with sedatives, alcohol, intracranial tumors, and the syndromes of sleep-disordered breathing including the primary alveolar and obesityhypoventilation syndromes (Chaps. 318 and 319). Abnormalities or disease in the motor neurons, neuromuscular junction, and skeletal muscle can cause hypoventilation via respiratory muscle fatigue. Mechanical ventilation, when not properly adjusted and supervised, may result in respiratory acidosis, particularly if CO2 production suddenly rises (because of fever, agitation, sepsis, or overfeeding) or alveolar ventilation falls because of worsening pulmonary function. High levels of positive end-expiratory pressure in the presence of reduced cardiac output may cause hypercapnia as a result of large increases in alveolar dead space (Chap. 306e).

1	pulmonary function. High levels of positive end-expiratory pressure in the presence of reduced cardiac output may cause hypercapnia as a result of large increases in alveolar dead space (Chap. 306e). Permissive hypercapnia is being used with increasing frequency because of studies suggesting lower mortality rates than with conventional mechanical ventilation, especially with severe CNS or heart disease. The respiratory acidosis associated with permissive hypercapnia may require administration of NaHCO3 to increase the arterial pH to 7.25, but overcorrection of the acidemia may be deleterious.

1	Acute hypercapnia follows sudden occlusion of the upper airway or generalized bronchospasm as in severe asthma, anaphylaxis, I. Alkalosis A. Central nervous system stimulation 1. 2. Anxiety, psychosis 3. 4. 5. Meningitis, encephalitis 6. 7. B. Hypoxemia or tissue hypoxia 1. 2. Pneumonia, pulmonary edema 3. 4. C. Drugs or hormones 1. Pregnancy, progesterone 2. 3. D. Stimulation of chest receptors 1. 2. 3. 4. E. Miscellaneous 1. 2. 3. 4. 5. II. A. 1. Drugs (anesthetics, morphine, sedatives) 2. 3. B. Airway 1. 2. C. Parenchyma 1. 2. 3. 4. 5. D. Neuromuscular 1. 2. 3. 4. E. Miscellaneous 1. 2. 3.

1	2. 3. 4. 5. II. A. 1. Drugs (anesthetics, morphine, sedatives) 2. 3. B. Airway 1. 2. C. Parenchyma 1. 2. 3. 4. 5. D. Neuromuscular 1. 2. 3. 4. E. Miscellaneous 1. 2. 3. inhalational burn, or toxin injury. Chronic hypercapnia and respiratory acidosis occur in end-stage obstructive lung disease. Restrictive disorders involving both the chest wall and the lungs can cause respiratory acidosis because the high metabolic cost of respiration causes ventilatory muscle fatigue. Advanced stages of intrapulmonary and extra-pulmonary restrictive defects present as chronic respiratory acidosis.

1	The diagnosis of respiratory acidosis requires the measurement of 323 Paco2 and arterial pH. A detailed history and physical examination often indicate the cause. Pulmonary function studies (Chap. 306e), including spirometry, diffusion capacity for carbon monoxide, lung volumes, and arterial Paco2 and O2 saturation, usually make it possible to determine if respiratory acidosis is secondary to lung disease. The workup for nonpulmonary causes should include a detailed drug history, measurement of hematocrit, and assessment of upper airway, chest wall, pleura, and neuromuscular function.

1	The management of respiratory acidosis depends on its severity and rate of onset. Acute respiratory acidosis can be life-threatening, and measures to reverse the underlying cause should be undertaken simultaneously with restoration of adequate alveolar ventilation. This may necessitate tracheal intubation and assisted mechanical ventilation. Oxygen administration should be titrated carefully in patients with severe obstructive pulmonary disease and chronic CO2 retention who are breathing spontaneously (Chap. 314). When oxygen is used injudiciously, these patients may experience progression of the respiratory acidosis. Aggressive and rapid correction of hypercapnia should be avoided, because the falling PaCO2 may provoke the same complications noted with acute respiratory alkalosis (i.e., cardiac arrhythmias, reduced cerebral perfusion, and seizures). The PaCO2 should be lowered gradually in chronic respiratory acidosis, aiming to restore the PaCO2 to baseline levels and to provide

1	cardiac arrhythmias, reduced cerebral perfusion, and seizures). The PaCO2 should be lowered gradually in chronic respiratory acidosis, aiming to restore the PaCO2 to baseline levels and to provide sufficient Cland K+ to enhance the renal excretion of HCO3 .

1	Chronic respiratory acidosis is frequently difficult to correct, but measures aimed at improving lung function (Chap. 314) can help some patients and forestall further deterioration in most.

1	Alveolar hyperventilation decreases Paco2 and increases the HCO3 -/ Paco2 ratio, thus increasing pH (Table 66-7). Nonbicarbonate cellular buffers respond by consuming HCO3 -. Hypocapnia develops when a sufficiently strong ventilatory stimulus causes CO2 output in the lungs to exceed its metabolic production by tissues. Plasma pH and [HCO3 -] appear to vary proportionately with Paco2 over a range from 40–15 mmHg. The relationship between arterial [H+] concentration and Paco2 is ∼0.7 mmol/L per mmHg (or 0.01 pH unit/mmHg), and that for plasma [HCO3 -] is 0.2 mmol/L per mmHg. Hypocapnia sustained for >2–6 h is further compensated by a decrease in renal ammonium and titratable acid excretion and a reduction in filtered HCO3 reabsorption. Full renal adaptation to respiratory alkalosis may take several days and requires normal volume status and renal function. The kidneys appear to respond directly to the lowered Paco2 rather than to alkalosis per se. In chronic respiratory alkalosis a

1	take several days and requires normal volume status and renal function. The kidneys appear to respond directly to the lowered Paco2 rather than to alkalosis per se. In chronic respiratory alkalosis a 1-mmHg decrease in Paco2 causes a 0.4-to 0.5-mmol/L drop in [HCO3 -] and a 0.3-mmol/L decrease (or 0.003 increase in pH) in [H+].

1	The effects of respiratory alkalosis vary according to duration and severity but are primarily those of the underlying disease. Reduced cerebral blood flow as a consequence of a rapid decline in Paco2 may cause dizziness, mental confusion, and seizures, even in the absence of hypoxemia. The cardiovascular effects of acute hypocapnia in the conscious human are generally minimal, but in the anesthetized or mechanically ventilated patient, cardiac output and blood pressure may fall because of the depressant effects of anesthesia and positive-pressure ventilation on heart rate, systemic resistance, and venous return. Cardiac arrhythmias may occur in patients with heart disease as a result of changes in oxygen unloading by blood from a left shift in the hemoglobin-oxygen dissociation curve (Bohr effect). Acute respiratory alkalosis causes intracellular shifts of Na+, K+, and PO42and reduces free [Ca2+] by increasing the protein-bound fraction. Hypocapnia-induced hypokalemia is usually

1	(Bohr effect). Acute respiratory alkalosis causes intracellular shifts of Na+, K+, and PO42and reduces free [Ca2+] by increasing the protein-bound fraction. Hypocapnia-induced hypokalemia is usually minor.

1	Chronic respiratory alkalosis is the most common acid-base disturbance in critically ill patients and, when severe, portends a poor prognosis. Many cardiopulmonary disorders manifest respiratory alkalosis 324 in their early to intermediate stages, and the finding of normocapnia and hypoxemia in a patient with hyperventilation may herald the onset of rapid respiratory failure and should prompt an assessment to determine if the patient is becoming fatigued. Respiratory alkalosis is common during mechanical ventilation. The hyperventilation syndrome may be disabling. Paresthesia; circumoral numbness; chest wall tightness or pain; dizziness; inability to take an adequate breath; and, rarely, tetany may be sufficiently stressful to perpetuate the disorder. Arterial blood-gas analysis demonstrates an acute or chronic respiratory alkalosis, often with hypocapnia in the range of 15–30 mmHg and no hypoxemia. CNS diseases or injury can produce several patterns of hyperventilation and sustained

1	an acute or chronic respiratory alkalosis, often with hypocapnia in the range of 15–30 mmHg and no hypoxemia. CNS diseases or injury can produce several patterns of hyperventilation and sustained Paco2 levels of 20–30 mmHg. Hyperthyroidism, high caloric loads, and exercise raise the basal metabolic rate, but ventilation usually rises in proportion so that arterial blood gases are unchanged and respiratory alkalosis does not develop. Salicylates are the most common cause of drug-induced respiratory alkalosis as a result of direct stimulation of the medullary chemoreceptor (Chap. 472e). The methylxanthines, theophylline, and aminophylline stimulate ventilation and increase the ventilatory response to CO2. Progesterone increases ventilation and lowers arterial Paco2 by as much as 5–10 mmHg. Therefore, chronic respiratory alkalosis is a common feature of pregnancy. Respiratory alkalosis is also prominent in liver failure, and the severity correlates with the degree of hepatic

1	5–10 mmHg. Therefore, chronic respiratory alkalosis is a common feature of pregnancy. Respiratory alkalosis is also prominent in liver failure, and the severity correlates with the degree of hepatic insufficiency. Respiratory alkalosis is often an early finding in gram-negative septicemia, before fever, hypoxemia, or hypotension develops.

1	The diagnosis of respiratory alkalosis depends on measurement of arterial pH and Paco2. The plasma [K+] is often reduced and the [Cl-] increased. In the acute phase, respiratory alkalosis is not associated with increased renal HCO3 excretion, but within hours net acid excretion is reduced. In general, the HCO3 concentration falls by 2.0 mmol/L for each 10-mmHg decrease in Paco2. Chronic hypocapnia reduces the serum [HCO3 -] by 4.0 mmol/L for each 10-mmHg decrease in Paco2. It is unusual to observe a plasma HCO3 <12 mmol/L as a result of a pure respiratory alkalosis. When a diagnosis of respiratory alkalosis is made, its cause should be investigated. The diagnosis of hyperventilation syndrome is made by exclusion. In difficult cases, it may be important to rule out other conditions such as pulmonary embolism, coronary artery disease, and hyperthyroidism.

1	The management of respiratory alkalosis is directed toward alleviation of the underlying disorder. If respiratory alkalosis complicates ventilator management, changes in dead space, tidal volume, and frequency can minimize the hypocapnia. Patients with the hyperventilation syndrome may benefit from reassurance, rebreathing from a paper bag during symptomatic attacks, and attention to underlying psychological stress. Antidepressants and sedatives are not recommended. β-Adrenergic blockers may ameliorate peripheral manifestations of the hyperadrenergic state. Kevin T. McVary

1	Kevin T. McVary Male sexual dysfunction affects 10–25% of middle-aged and elderly men, and female sexual dysfunction occurs with similar frequency. Demographic changes, the popularity of newer treatments, and greater awareness of sexual dysfunction by patients and society have led to increased diagnosis and associated health care expenditures for the management of this common disorder. Because many patients are reluctant to initiate discussion of their sex lives, physicians should address this topic directly to elicit a history of sexual dysfunction. Normal male sexual function requires (1) an intact libido, (2) the ability to achieve and maintain penile erection, (3) ejaculation, and (4) detumescence. Libido refers to sexual desire and is influenced by a variety of visual, olfactory, tactile, auditory, imaginative, and hormonal stimuli. Sex steroids, particularly testosterone, act to increase libido. Libido can be diminished by hormonal or psychiatric disorders and by medications.

1	Penile tumescence leading to erection depends on an increased flow of blood into the lacunar network accompanied by complete relaxation of the arteries and corporal smooth muscle. The microarchitecture of the corpora is composed of a mass of smooth muscle (trabecula) that contains a network of endothelial-lined vessels (lacunar spaces). Subsequent compression of the trabecular smooth muscle against the fibroelastic tunica albuginea causes a passive closure of the emissary veins and accumulation of blood in the corpora. In the presence of a full erection and a competent valve mechanism, the corpora become noncompressible cylinders from which blood does not escape.

1	The central nervous system (CNS) exerts an important influence by either stimulating or antagonizing spinal pathways that mediate erectile function and ejaculation. The erectile response is mediated by a combination of central (psychogenic) innervation and peripheral (reflexogenic) innervation. Sensory nerves that originate from receptors in the penile skin and glans converge to form the dorsal nerve of the penis, which travels to the S2-S4 dorsal root ganglia via the pudendal nerve. Parasympathetic nerve fibers to the penis arise from neurons in the intermediolateral columns of the S2-S4 sacral spinal segments. Sympathetic innervation originates from the T11 to the L2 spinal segments and descends through the hypogastric plexus.

1	Neural input to smooth-muscle tone is crucial to the initiation and maintenance of an erection. There is also an intricate interaction between the corporal smooth-muscle cell and its overlying endothelial cell lining (Fig. 67-1). Nitric oxide, which induces vascular relaxation, promotes erection and is opposed by endothelin 1 (ET-1) and Rho kinase, which mediate vascular contraction. Nitric oxide is synthesized from l-arginine by nitric oxide synthase and is released from the nonadrenergic, noncholinergic (NANC) autonomic nerve supply to act postjunctionally on smooth-muscle cells. Nitric oxide increases the production of cyclic 3′,5′-guanosine monophosphate (cyclic GMP), which induces relaxation of smooth muscle (Fig. 67-2). Cyclic GMP is gradually broken down by phosphodiesterase type 5 (PDE-5). Inhibitors of PDE-5, such as the oral medications sildenafil, vardenafil, and tadalafil, maintain erections by reducing the breakdown of cyclic GMP. However, if nitric oxide is not produced

1	(PDE-5). Inhibitors of PDE-5, such as the oral medications sildenafil, vardenafil, and tadalafil, maintain erections by reducing the breakdown of cyclic GMP. However, if nitric oxide is not produced at some level, PDE-5 inhibitors are ineffective, as these drugs facilitate, but do not initiate, the initial enzyme cascade. In addition to nitric oxide, vasoactive prostaglandins (PGE1, PGF2α) are synthesized within the cavernosal tissue and increase cyclic AMP levels, also leading to relaxation of cavernosal smooth-muscle cells.

1	Decreased Ca2+ PDE2, 3, 4

1	FIguRE 67-1 Pathways that regulate penile smooth-muscle relaxation and erection. A. Outflow from the parasympathetic nervous system leads to relaxation of the cavernous sinusoids in two ways, both of which increase the concentration of nitric oxide (NO) in smooth-muscle cells. First, NO is the neurotransmitter in nonadrenergic, noncholinergic (NANC) fibers; second, stimulation of endothelial nitric oxide synthase (eNOS) through cholinergic output causes increased production of NO. The NO produced in the endothelium then diffuses into the smooth-muscle cells and decreases its intracellular calcium concentration through a pathway mediated by cyclic guanosine monophosphate (cGMP), leading to relaxation. A separate mechanism that decreases the intracellular calcium level is mediated by cyclic adenosine monophosphate (cAMP). With increased cavernosal blood flow, as well as increased levels of vascular endothelial growth factor (VEGF), the endothelial release of NO is further sustained

1	adenosine monophosphate (cAMP). With increased cavernosal blood flow, as well as increased levels of vascular endothelial growth factor (VEGF), the endothelial release of NO is further sustained through the phosphatidylinositol 3 (PI3) kinase pathway. Active treatments (red boxes) include drugs that affect the cGMP pathway (phosphodiesterase [PDE] type 5 inhibitors and guanylyl cyclase agonists), the cAMP pathway (alprostadil), or both pathways (papaverine), along with neural-tone mediators (phentolamine and Rho kinase inhibitors). Agents that are being developed include guanylyl cyclase agonists (to bypass the need for endogenous NO) and Rho kinase inhibitors (to inhibit tonic contraction of smooth-muscle cells mediated through endothelin). α1, α-adrenergic receptor; GPCR, G-protein–coupled receptor, GTP, guanosine triphosphate; PGE, prostaglandin E; PGF, prostaglandin F. B. Biochemical pathways of NO synthesis and action. Sildenafil, vardenafil, and tadalafil enhance erectile

1	receptor, GTP, guanosine triphosphate; PGE, prostaglandin E; PGF, prostaglandin F. B. Biochemical pathways of NO synthesis and action. Sildenafil, vardenafil, and tadalafil enhance erectile function by inhibiting phosphodiesterase type 5 (PDE-5), thereby maintaining high levels of cyclic 3′,5′-guanosine monophosphate (cyclic GMP). iCa2+, intracellular calcium; NOS, nitric oxide synthase. (Part A from K McVary: N Engl J Med 357:2472, 2007; with permission.)

1	Ejaculation is stimulated by the sympathetic nervous system; this results in contraction of the epididymis, vas deferens, seminal vesicles, and prostate, causing seminal fluid to enter the urethra. Seminal fluid emission is followed by rhythmic contractions of the bulbocavernosus and ischiocavernosus muscles, leading to ejaculation. Premature ejaculation usually is related to anxiety or a learned behavior and is amenable to behavioral therapy or treatment with medications such as selective serotonin reuptake inhibitors (SSRIs). Retrograde ejaculation results when the internal urethral sphincter does not close; it may occur in men with diabetes or after surgery involving the bladder neck.

1	Detumescence is mediated by norepinephrine from the sympathetic nerves, endothelin from the vascular surface, and smooth-muscle contraction induced by postsynaptic α-adrenergic receptors and activation of Rho kinase. These events increase venous outflow and restore the flaccid state. Venous leak can cause premature detumescence and is caused by insufficient relaxation of the corporal smooth muscle rather than a specific anatomic defect. Priapism refers to a persistent and painful erection and may be associated with sickle cell anemia, hyper-coagulable states, spinal cord injury, or injection of vasodilator agents into the penis. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 67-2 Biochemical pathways modified by phosphodiesterase type 5 (PDE-5) inhibitors. Sildenafil, vardenafil, tadalafil and avanafil enhance erectile function by inhibiting PDE-5, thereby maintaining high levels of cyclic 3′,5′-guanosine monophosphate (cyclic GMP). iCa2+, intracellular calcium; NO, nitric oxide; NOS, nitric oxide synthase.

1	ERECTILE DYSFuNCTION Epidemiology Erectile dysfunction (ED) is not considered a normal part of the aging process. Nonetheless, it is associated with certain physiologic and psychological changes related to age. In the Massachusetts Male Aging Study (MMAS), a community-based survey of men age 40–70, 52% of responders reported some degree of ED. Complete ED occurred in 10% of respondents, moderate ED in 25%, and minimal ED in 17%. The incidence of moderate or severe ED more than doubled between the ages of 40 and 70. In the National Health and Social Life Survey (NHSLS), which included a sample of men and women age 18–59, 10% of men reported being unable to maintain an erection (corresponding to the proportion of men in the MMAS reporting severe ED). Incidence was highest among men in the age group 50–59 (21%) and men who were poor (14%), divorced (14%), and less educated (13%).

1	The incidence of ED is also higher among men with certain medical disorders, such as diabetes mellitus, obesity, lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH), heart disease, hypertension, decreased high-density lipoprotein (HDL) levels, and diseases associated with general systemic inflammation (e.g., rheumatoid arthritis). Cardiovascular disease and ED share etiologies as well as pathophysiology (e.g., endothelial dysfunction), and the degree of ED appears to correlate with the severity of cardiovascular disease. Consequently, ED represents a “sentinel symptom” in patients with occult cardiovascular and peripheral vascular disease.

1	Smoking is also a significant risk factor in the development of ED. Medications used in treating diabetes or cardiovascular disease are additional risk factors (see below). There is a higher incidence of ED among men who have undergone radiation or surgery for prostate cancer and in those with a lower spinal cord injury. Psychological causes of ED include depression, anger, stress from unemployment, and other stress-related causes. Pathophysiology ED may result from three basic mechanisms: (1) failure to initiate (psychogenic, endocrinologic, or neurogenic), (2) failure to fill (arteriogenic), and (3) failure to store adequate blood volume within the lacunar network (venoocclusive dysfunction). These categories are not mutually exclusive, and multiple factors contribute to ED in many patients. For example, diminished filling pressure can lead secondarily to venous leak. Psychogenic factors frequently coexist with other etiologic factors and should be considered in all cases.

1	Diabetic, atherosclerotic, and drug-related causes account for >80% of cases of ED in older men. VASCULOgENIC The most common organic cause of ED is a disturbance of blood flow to and from the penis. Atherosclerotic or traumatic arterial disease can decrease flow to the lacunar spaces, resulting in decreased rigidity and an increased time to full erection. Excessive outflow through the veins despite adequate inflow also may contribute to ED. Structural alterations to the fibroelastic components of the corpora may cause a loss of compliance and inability to compress the tunical veins. This condition may result from aging, increased cross-linking of collagen fibers induced by nonenzymatic glycosylation, hypoxemia, or altered synthesis of collagen associated with hypercholesterolemia.

1	NEUROgENIC Disorders that affect the sacral spinal cord or the autonomic fibers to the penis preclude nervous system relaxation of penile smooth muscle, thus leading to ED. In patients with spinal cord injury, the degree of ED depends on the completeness and level of the lesion. Patients with incomplete lesions or injuries to the upper part of the spinal cord are more likely to retain erectile capabilities than are those with complete lesions or injuries to the lower part. Although 75% of patients with spinal cord injuries have some erectile capability, only 25% have erections sufficient for penetration. Other neurologic disorders commonly associated with ED include multiple sclerosis and peripheral neuropathy. The latter is often due to either diabetes or alcoholism. Pelvic surgery may cause ED through disruption of the autonomic nerve supply.

1	ENdOCRINOLOgIC Androgens increase libido, but their exact role in erectile function is unclear. Individuals with castrate levels of testosterone can achieve erections from visual or sexual stimuli. Nonetheless, normal levels of testosterone appear to be important for erectile function, particularly in older males. Androgen replacement therapy can improve depressed erectile function when it is secondary to hypogonadism; however, it is not useful for ED when endogenous testosterone levels are normal. Increased prolactin may decrease libido by suppressing gonadotropin-releasing hormone (GnRH), and it also leads to decreased testosterone levels. Treatment of hyperprolactinemia with dopamine agonists can restore libido and testosterone.

1	dIABETIC ED occurs in 35–75% of men with diabetes mellitus. Pathologic mechanisms are related primarily to diabetes-associated vascular and neurologic complications. Diabetic macrovascular complications are related mainly to age, whereas microvascular complications correlate with the duration of diabetes and the degree of glycemic control (Chap. 417). Individuals with diabetes also have reduced amounts of nitric oxide synthase in both endothelial and neural tissues.

1	PSYCHOgENIC Two mechanisms contribute to the inhibition of erections in psychogenic ED. First, psychogenic stimuli to the sacral cord may inhibit reflexogenic responses, thereby blocking activation of vasodilator outflow to the penis. Second, excess sympathetic stimulation in an anxious man may increase penile smooth-muscle tone. The most common causes of psychogenic ED are performance anxiety, depression, relationship conflict, loss of attraction, sexual inhibition, conflicts over sexual preference, sexual abuse in childhood, and fear of pregnancy or sexually transmitted disease. Almost all patients with ED, even when it has a clear-cut organic basis, develop a psychogenic component as a reaction to ED.

1	MEdICATION-RELATEd Medication-induced ED (Table 67-1) is estimated to occur in 25% of men seen in general medical outpatient clinics. The adverse effects related to drug therapy are additive, especially in older men. In addition to the drug itself, the disease being treated is likely to contribute to sexual dysfunction. Among the antihypertensive agents, the thiazide diuretics and beta blockers have been implicated most frequently. Calcium channel blockers and angiotensin converting-enzyme inhibitors are cited less frequently. These drugs may act directly at the corporal level (e.g., calcium channel blockers) or indirectly by reducing pelvic blood pressure, which is important in the development of penile rigidity. α-Adrenergic blockers are less likely to Abbreviation: GnRH, gonadotropin-releasing hormone.

1	Abbreviation: GnRH, gonadotropin-releasing hormone. cause ED. Estrogens, GnRH agonists, H2 antagonists, and spironolactone cause ED by suppressing gonadotropin production or by blocking androgen action. Antidepressant and antipsychotic agents—particularly neuroleptics, tricyclics, and SSRIs—are associated with erectile, ejaculatory, orgasmic, and sexual desire difficulties. If there is a strong association between the institution of a drug and the onset of ED, alternative medications should be considered. Otherwise, it is often practical to treat the ED without attempting multiple changes in medications, as it may be difficult to establish a causal role for a drug. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: A good physician-patient relationship helps unravel the possible causes of ED, many of which require discussion of personal and sometimes embarrassing topics. For this reason, a primary care provider is often ideally suited to initiate the evaluation. However, a significant percentage of men experience ED and remain undiagnosed unless specifically questioned about this issue. By far the two most common reasons for underreporting of ED are patient embarrassment and perceptions of physicians’ inattention to the disease. Once the topic is initiated by the physician, patients are more willing to discuss their potency issues. A complete medical and sexual History: Medical, sexual, and psychosocial Physical examination Serum: Testosterone and prolactin levels Lifestyle risk management Medication review FIguRE 67-3 Algorithm for the evaluation and management of patients with erectile dysfunction. PDE, phosphodiesterase.

1	FIguRE 67-3 Algorithm for the evaluation and management of patients with erectile dysfunction. PDE, phosphodiesterase. history should be taken in an effort to assess whether the cause of ED is organic, psychogenic, or multifactorial (Fig. 67-3). Both the patient and his sexual partner should be interviewed regarding sexual history. ED should be distinguished from other sexual problems, such as premature ejaculation. Lifestyle factors such as sexual orientation, the patient’s distress from ED, performance anxiety, and details of sexual techniques should be addressed. Standardized questionnaires are available to assess ED, including the International Index of Erectile Function (IIEF) and the more easily administered Sexual Health Inventory for Men (SHIM), a validated abridged version of the IIEF.

1	The initial evaluation of ED begins with a review of the patient’s medical, surgical, sexual, and psychosocial histories. The history should note whether the patient has experienced pelvic trauma, surgery, or radiation. In light of the increasing recognition of the relationship between lower urinary tract symptoms and ED, it is advisable to evaluate for the presence of symptoms of bladder outlet obstruction. Questions should focus on the onset of symptoms, the presence and duration of partial erections, and the progression of ED. A history of nocturnal or early morning erections is useful for distinguishing physiologic ED from psychogenic ED. Nocturnal erections occur during rapid eye movement (REM) sleep and require intact neurologic and circulatory systems. Organic causes of ED generally are characterized by a gradual and persistent change in rigidity or the inability to sustain nocturnal, coital, or self-stimulated erections. The patient should be questioned about the presence of

1	are characterized by a gradual and persistent change in rigidity or the inability to sustain nocturnal, coital, or self-stimulated erections. The patient should be questioned about the presence of penile curvature or pain with coitus. It is also important to address libido, as decreased sexual drive and ED are sometimes the earliest signs of endocrine abnormalities (e.g., increased prolactin, decreased testosterone levels). It is useful to ask whether the problem is confined to coitus with one partner or also involves other partners; ED not uncommonly arises in association with new or extramarital sexual relationships. Situational ED, as opposed to consistent ED, suggests psychogenic causes. Ejaculation is much less commonly affected than erection, but questions should be asked about whether ejaculation is normal, premature, delayed, or absent. Relevant risk factors should be identified, such as diabetes mellitus, coronary artery disease (CAD), and neurologic disorders. The patient’s

1	ejaculation is normal, premature, delayed, or absent. Relevant risk factors should be identified, such as diabetes mellitus, coronary artery disease (CAD), and neurologic disorders. The patient’s surgical history should be explored with an emphasis on bowel, bladder, prostate, and vascular procedures. A complete drug history is also important. Social changes that may precipitate ED are also crucial to the evaluation, including health worries, spousal death, divorce, relationship difficulties, and financial concerns.

1	Because ED commonly involves a host of endothelial cell risk factors, men with ED report higher rates of overt and silent myocardial infarction. Therefore, ED in an otherwise asymptomatic male warrants consideration of other vascular disorders, including CAD. The physical examination is an essential element in the assessment of ED. Signs of hypertension as well as evidence of thyroid, hepatic, hematologic, cardiovascular, or renal diseases should be sought. An assessment should be made of the endocrine and vascular systems, the external genitalia, and the prostate gland. The penis should be palpated carefully along the corpora to detect fibrotic plaques. Reduced testicular size and loss of secondary sexual characteristics are suggestive of hypogonadism. Neurologic examination should include assessment of anal sphincter tone, investigation of the bulbocavernosus reflex, and testing for peripheral neuropathy.

1	Although hyperprolactinemia is uncommon, a serum prolactin level should be measured, as decreased libido and/or ED may be the presenting symptoms of a prolactinoma or another mass lesion of the sella (Chap. 403). The serum testosterone level should be measured, and if it is low, gonadotropins should be measured to determine whether hypogonadism is primary (testicular) or secondary (hypothalamic-pituitary) in origin (Chap. 411). If not performed recently, serum chemistries, complete blood count (CBC), and lipid profiles may be of value, as they can yield evidence of anemia, diabetes, hyperlipidemia, or other systemic diseases associated with ED. Determination of serum prostate-specific antigen (PSA) should be conducted according to recommended clinical guidelines (Chap. 115).

1	Additional diagnostic testing is rarely necessary in the evaluation of ED. However, in selected patients, specialized testing may provide insight into pathologic mechanisms of ED and aid in the selection of treatment options. Optional specialized testing includes studies of nocturnal penile tumescence and rigidity, (2) vascular testing (in-office injection of vasoactive substances, penile Doppler ultrasound, penile angiography, dynamic infusion cavernosography/ cavernosometry), (3) neurologic testing (biothesiometry-graded vibratory perception, somatosensory evoked potentials), and psychological diagnostic tests. The information potentially gained from these procedures must be balanced against their invasiveness and cost. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Patient and partner education is essential in the treatment of ED. In goal-directed therapy, education facilitates understanding of the disease, the results of the tests, and the selection of treatment. Discussion of treatment options helps clarify how treatment is best offered and stratify firstand second-line therapies. Patients with high-risk lifestyle issues such as obesity, smoking, alcohol abuse, and recreational drug use should be counseled on the role those factors play in the development of ED.

1	Therapies currently employed for the treatment of ED include oral PDE-5 inhibitor therapy (most commonly used), injection therapies, testosterone therapy, penile devices, and psychological therapy. In addition, limited data suggest that treatments for underlying risk factors and comorbidities—for example, weight loss, exercise, stress reduction, and smoking cessation—may improve erectile function. Decisions regarding therapy should take into account the preferences and expectations of patients and their partners.

1	Sildenafil, tadalafil, vardenafil, and avanafil are the only approved and effective oral agents for the treatment of ED. These four medications have markedly improved the management of ED because they are effective for the treatment of a broad range of causes, including psychogenic, diabetic, vasculogenic, post-radical prostatectomy (nerve-sparing procedures), and spinal cord injury. They belong to a class of medications that are selective and potent inhibitors of PDE-5, the predominant phosphodiesterase isoform found in the penis. They are administered in graduated doses and enhance erections after sexual stimulation. The onset of action is approximately 30–120 min, depending on the medication used and other factors, such as recent food intake. Reduced initial doses should be considered for patients who are elderly, are taking concomitant alpha blockers, have renal insufficiency, or are taking medications that inhibit the CYP3A4 metabolic pathway in the liver (e.g., erythromycin,

1	for patients who are elderly, are taking concomitant alpha blockers, have renal insufficiency, or are taking medications that inhibit the CYP3A4 metabolic pathway in the liver (e.g., erythromycin, cimetidine, ketoconazole, and possibly itraconazole and mibefradil), as they may increase the serum concentration of the PDE-5 inhibitors (PDE-5i) or promote hypotension.

1	Initially, there were concerns about the cardiovascular safety of PDE-5i drugs. These agents can act as a mild vasodilator, and warnings exist about orthostatic hypotension with concomitant use of alpha blockers. The use of PDE-5i is not contraindicated in men who are also receiving alpha blockers, but they must be stabilized on this blood pressure medication prior to initiating therapy. Concerns also existed that use of PDE-5i would increase cardiovascular events. However, the safety of these drugs has been confirmed in several controlled trials with no increase in myocardial ischemic events or overall mortality compared to the general population. Several randomized trials have demonstrated the efficacy of this class of medications. There are no compelling data to support the superiority of one PDE-5i over another. Subtle differences between agents have variable clinical relevance (Table 67-2).

1	Patients may fail to respond to a PDE-5i for several reasons (Table 67-3). Some patients may not tolerate PDE-5i secondary to adverse events from vasodilation in nonpenile tissues expressing PDE-5 or from the inhibition of homologous nonpenile isozymes (i.e., PDE-6 found in the retina). Abnormal vision attributed to the effects of PDE-5i on retinal PDE-6 is of short duration, reported only with sildenafil and not thought to be clinically significant. A more serious concern is the possibility that PDE-5i may cause nonarteritic anterior ischemic optic neuropathy; although data to support that association are limited, it is prudent to avoid the use of these agents in men with a prior history of nonarteritic anterior ischemic optic neuropathy.

1	Testosterone supplementation combined with a PDE-5i may be beneficial in improving erectile function in hypogonadal men with ED who are unresponsive to PDE-5i alone. These drugs do not affect ejaculation, orgasm, or sexual drive. Side effects associated with PDE-5i include headaches (19%), facial flushing (9%), dyspepsia (6%), and nasal congestion (4%). Approximately 7% of men using sildenafil may experience transient altered color vision (blue halo effect), and 6% of men taking tadalafil may experience loin pain. PDE-5i is contraindicated in men receiving nitrate therapy for cardiovascular disease, including agents delivered by the oral, sub-lingual, transnasal, and topical routes. These agents can potentiate its hypotensive effect and may result in profound shock. Likewise, amyl/butyl nitrate “poppers” may have a fatal synergistic effect on blood pressure. PDE-5i also should be avoided in patients with congestive heart failure and cardiomyopathy because of the risk of vascular

1	nitrate “poppers” may have a fatal synergistic effect on blood pressure. PDE-5i also should be avoided in patients with congestive heart failure and cardiomyopathy because of the risk of vascular collapse. Because sexual activity leads to an increase in physiologic expenditure (5–6 metabolic equivalents [METS]), physicians have been advised to exercise caution in prescribing any drug for sexual activity to those with active coronary disease, heart failure, borderline hypotension, or hypovolemia and to those on complex antihypertensive regimens.

1	Although the various forms of PDE-5i have a common mechanism of action, there are a few differences among the four agents (Table 67-2). Tadalafil is unique in its longer half-life, whereas avanafil appears to have the most rapid onset of action. All four drugs are effective for patients with ED of all ages, severities, and etiologies. Although there are pharmacokinetic and pharmacodynamic differences among these agents, clinically relevant differences are not clear. Testosterone replacement is used to treat both primary and secondary causes of hypogonadism (Chap. 411). Androgen supplementation Drug Onset of Action Half-Life Dose Adverse Effects Contraindications Sildenafil Tmax, 30-120 min 2–5 h 25–100 mg Headache, flushing, dyspepsia, nasal congestion, Duration, 4 h Starting dose, 50 mg altered vision Duration, 2 h Abbreviations: ETOH, alcohol; Tmax, time to maximum plasma concentration.

1	Duration, 4 h Starting dose, 50 mg altered vision Duration, 2 h Abbreviations: ETOH, alcohol; Tmax, time to maximum plasma concentration. in the setting of normal testosterone is rarely efficacious in the treatment of ED and is discouraged. Methods of androgen replacement include transdermal patches and gels, parenteral administration of long-acting testosterone esters (enanthate and cypionate), and oral preparations (17 α-alkylated derivatives) (Chap. 411). Oral androgen preparations have the potential for hepatotoxicity and should be avoided.

1	Men who receive testosterone should be reevaluated after 1–3 months and at least annually thereafter for testosterone levels, erectile function, and adverse effects, which may include gynecomastia, sleep apnea, development or exacerbation of lower urinary tract symptoms or BPH, prostate cancer, lowering of HDL, erythrocytosis, elevations of liver function tests, and reduced fertility. Periodic reevaluation should include measurement of CBC and PSA and digital rectal exam. Therapy should be discontinued in patients who do not respond within 3 months.

1	Vacuum constriction devices (VCDs) are a well-established noninvasive therapy. They are a reasonable treatment alternative for select patients who cannot take sildenafil or do not desire other interventions. VCDs draw venous blood into the penis and use a constriction ring to restrict venous return and maintain tumescence. Adverse events with VCD include pain, numbness, bruising, and altered ejaculation. Additionally, many patients complain that the devices are cumbersome and that the induced erections have a nonphysiologic appearance and feel. If a patient fails to respond to oral agents, a reasonable next choice is intraurethral or self-injection of vasoactive substances. iSSuES To ConSiDER if PATiEnTS REPoRT fAiLuRE of PDE-5i To iMPRovE ERECTiLE DySfunCTion A trial of medication on at least 6 different days at the maximal dose should be made before declaring patient nonresponsive to PDE-5i use

1	A trial of medication on at least 6 different days at the maximal dose should be made before declaring patient nonresponsive to PDE-5i use Failure to include physical and psychic stimulation at the time of foreplay to induce endogenous NO Unrecognized hypogonadism Abbreviations: NO, nitric oxide; PDE-5i, phosphodiesterase type 5 inhibitor. congestion, nasopharyngitis, back pain Nitrates Hypotension Cardiovascular risk factors Retinitis pigmentosa Change dose with some Should be on stable dose of alpha blockers Same as sildenafil May have minor prolongation Concomitant use of Class I antiarrhythmic Same as sildenafil Same as sildenafil

1	Should be on stable dose of alpha blockers Same as sildenafil May have minor prolongation Concomitant use of Class I antiarrhythmic Same as sildenafil Same as sildenafil Intraurethral prostaglandin E1 (alprostadil), in the form of a semisolid pellet (doses of 125–1000 μg), is delivered with an applicator. Approximately 65% of men receiving intraurethral alprostadil respond with an erection when tested in the office, but only 50% achieve successful coitus at home. Intraurethral insertion is associated with a markedly reduced incidence of priapism in comparison to intracavernosal injection.

1	Injection of synthetic formulations of alprostadil is effective in 70–80% of patients with ED, but discontinuation rates are high because of the invasive nature of administration. Doses range between 1 and 40 μg. Injection therapy is contraindicated in men with a history of hypersensitivity to the drug and men at risk for priapism (hypercoagulable states, sickle cell disease). Side effects include local adverse events, prolonged erections, pain, and fibrosis with chronic use. Various combinations of alprostadil, phentolamine, and/or papaverine sometimes are used.

1	A less frequently used form of therapy for ED involves the surgical implantation of a semirigid or inflatable penile prosthesis. The choice of prosthesis is dependent on patient preference and should take into account body habitus and manual dexterity, which may affect the ability of the patient to manipulate the device. Because of the permanence of prosthetic devices, patients should be advised to first consider less invasive options for treatment. These surgical treatments are invasive, are associated with potential complications, and generally are reserved for treatment of refractory ED. Despite their high cost and invasiveness, penile prostheses are associated with high rates of patient and partner satisfaction.

1	A course of sex therapy may be useful for addressing specific interpersonal factors that may affect sexual functioning. Sex therapy generally consists of in-session discussion and at-home exercises specific to the person and the relationship. Psychosexual therapy involves techniques such as sensate focus (nongenital massage), sensory awareness exercises, correction of misconceptions about sexuality, and interpersonal difficulties therapy (e.g., open communication about sexual issues, physical intimacy scheduling, and 330 behavioral interventions). These approaches may be useful in patients who have psychogenic or social components to their ED, although data from randomized trials are scanty and inconsistent. It is preferable if therapy includes both partners if the patient is involved in an ongoing relationship. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Female sexual dysfunction (FSD) has traditionally included disorders of desire, arousal, pain, and muted orgasm. The associated risk factors for FSD are similar to those in males: cardiovascular disease, endocrine disorders, hypertension, neurologic disorders, and smoking (Table 67-4). Epidemiologic data are limited, but the available estimates suggest that as many as 43% of women complain of at least one sexual problem. Despite the recent interest in organic causes of FSD, desire and arousal phase disorders (including lubrication complaints) remain the most common presenting problems when surveyed in a community-based population.

1	The female sexual response requires the presence of estrogens. A role for androgens is also likely but less well established. In the CNS, estrogens and androgens work synergistically to enhance sexual arousal and response. A number of studies report enhanced libido in women during preovulatory phases of the menstrual cycle, suggesting that hormones involved in the ovulatory surge (e.g., estrogens) increase desire. Sexual motivation is heavily influenced by context, including the environment and partner factors. Once sufficient sexual desire is reached, sexual arousal is mediated by the central and autonomic nervous systems. Cerebral sympathetic outflow is thought to increase desire, and peripheral parasympathetic activity results in clitoral vasocongestion and vaginal secretion (lubrication).

1	The neurotransmitters for clitoral corporal engorgement are similar to those in the male, with a prominent role for neural, smooth-muscle, and endothelial released nitric oxide (NO). A fine network of vaginal nerves and arterioles promotes a vaginal transudate. The major transmitters of this complex vaginal response are not certain, but roles for NO and vasointestinal polypeptide (VIP) are suspected. Investigators studying the normal female sexual response have challenged the long-held construct of a linear and unmitigated relationship between initial desire, arousal, vasocongestion, lubrication, and eventual orgasm. Caregivers Neurologic disease: stroke, spinal cord injury, parkinsonism Trauma, genital surgery, radiation Endocrinopathies: diabetes, hyperprolactinemia Psychological factors and interpersonal relationship disorders: sexual abuse, life stressors Antiandrogens: cimetidine, spironolactone Antidepressants, alcohol, hypnotics, sedatives

1	Psychological factors and interpersonal relationship disorders: sexual abuse, life stressors Antiandrogens: cimetidine, spironolactone Antidepressants, alcohol, hypnotics, sedatives Antihistamines, sympathomimetic amines Antihypertensives: diuretics, calcium channel blockers Abbreviation: GnRH, gonadotropin-releasing hormone. should consider a paradigm of a positive emotional and physical outcome with one, many, or no orgasmic peak and release.

1	Although there are anatomic differences as well as variation in the density of vascular and neural beds in males and females, the primary effectors of sexual response are strikingly similar. Intact sensation is important for arousal. Thus, reduced levels of sexual functioning are more common in women with peripheral neuropathies (e.g., diabetes). Vaginal lubrication is a transudate of serum that results from the increased pelvic blood flow associated with arousal. Vascular insufficiency from a variety of causes may compromise adequate lubrication and result in dyspareunia. Cavernosal and arteriole smooth-muscle relaxation occurs via increased nitric oxide synthase (NOS) activity and produces engorgement in the clitoris and the surrounding vestibule. Orgasm requires an intact sympathetic outflow tract; hence, orgasmic disorders are common in female patients with spinal cord injuries. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: Many women do not volunteer information about their sexual response. Open-ended questions in a supportive atmosphere are helpful in initiating a discussion of sexual fitness in women who are reluctant to discuss such issues. Once a complaint has been voiced, a comprehensive evaluation should be performed, including a medical history, a psychosocial history, a physical examination, and limited laboratory testing.

1	The history should include the usual medical, surgical, obstetric, psychological, gynecologic, sexual, and social information. Past experiences, intimacy, knowledge, and partner availability should also be ascertained. Medical disorders that may affect sexual health should be delineated. They include diabetes, cardiovascular disease, gynecologic conditions, obstetric history, depression, anxiety disorders, and neurologic disease. Medications should be reviewed as they may affect arousal, libido, and orgasm. The need for counseling and recognizing life stresses should be identified. The physical examination should assess the genitalia, including the clitoris. Pelvic floor examination may identify prolapse or other disorders. Laboratory studies are needed, especially if menopausal status is uncertain. Estradiol, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) are usually obtained, and dehydroepiandrosterone (DHEA) should be considered as it reflects adrenal androgen

1	is uncertain. Estradiol, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) are usually obtained, and dehydroepiandrosterone (DHEA) should be considered as it reflects adrenal androgen secretion. A CBC, liver function assessment, and lipid studies may be useful, if not otherwise obtained. Complicated diagnostic evaluations such as clitoral Doppler ultrasonography and biothesiometry require expensive equipment and are of uncertain utility. It is important for the patient to identify which symptoms are most distressing.

1	The evaluation of FSD previously occurred mainly in a psychosocial context. However, inconsistencies between diagnostic categories based only on psychosocial considerations and the emerging recognition of organic etiologies have led to a new classification of FSD. This diagnostic scheme is based on four components that are not mutually exclusive: (1) hypoactive sexual desire—the persistent or recurrent lack of sexual thoughts and/or receptivity to sexual activity, which causes personal distress; hypoactive sexual desire may result from endocrine failure or may be associated with psychological or emotional disorders; (2) sexual arousal disorder—the persistent or recurrent inability to attain or maintain sexual excitement, which causes personal distress; (3) orgasmic disorder—the persistent or recurrent loss of orgasmic potential after sufficient sexual stimulation and arousal, which causes personal distress; and (4) sexual pain disorder—persistent or recurrent genital pain associated

1	or recurrent loss of orgasmic potential after sufficient sexual stimulation and arousal, which causes personal distress; and (4) sexual pain disorder—persistent or recurrent genital pain associated with noncoital sexual stimulation, which causes personal distress. This newer classification emphasizes “personal distress” as a requirement for dysfunction and provides clinicians with an organized framework for evaluation before or in conjunction with more traditional counseling methods.

1	CAuSES of HiRSuTiSM An open discussion with the patient is important as couples may need to be educated about normal anatomy and physiologic responses, including the role of orgasm, in sexual encounters. Physiologic changes associated with aging and/or disease should be explained. Couples may need to be reminded that clitoral stimulation rather than coital intromission may be more beneficial.

1	Behavioral modification and nonpharmacologic therapies should be a first step. Patient and partner counseling may improve communication and relationship strains. Lifestyle changes involving known risk factors can be an important part of the treatment process. Emphasis on maximizing physical health and avoiding lifestyles (e.g., smoking, alcohol abuse) and medications likely to produce FSD is important (Table 67-4). The use of topical lubricants may address complaints of dyspareunia and dryness. Contributing medications such as antidepressants may need to be altered, including the use of medications with less impact on sexual function, dose reduction, medication switching, or drug holidays.

1	In postmenopausal women, estrogen replacement therapy may be helpful in treating vaginal atrophy, decreasing coital pain, and improving clitoral sensitivity (Chap. 413). Estrogen replacement in the form of local cream is the preferred method, as it avoids systemic side effects. Androgen levels in women decline substantially before menopause. However, low levels of testosterone or DHEA are not effective predictors of a positive therapeutic outcome with androgen therapy. The widespread use of exogenous androgens is not supported by the literature except in select circumstances (premature ovarian failure or menopausal states) and in secondary arousal disorders. The efficacy of PDE-5i in FDS has been a marked disappointment in light of the proposed role of nitric oxide–dependent physiology in the normal female sexual response. The use of PDE-5i for FSD should be discouraged pending proof that it is effective.

1	In patients with arousal and orgasmic difficulties, the option of using a clitoral vacuum device may be explored. This handheld battery-operated device has a small soft plastic cup that applies a vacuum over the stimulated clitoris. This causes increased cavernosal blood flow, engorgement, and vaginal lubrication.

1	Hirsutism David A. Ehrmann Hirsutism, which is defined as androgen-dependent excessive male-pattern hair growth, affects approximately 10% of women. Hirsutism is most often idiopathic or the consequence of androgen excess associ-ated with the polycystic ovarian syndrome (PCOS). Less frequently, it 68 may result from adrenal androgen overproduction as occurs in non-classic congenital adrenal hyperplasia (CAH) (Table 68-1). Rarely, it is a sign of a serious underlying condition. Cutaneous manifestations commonly associated with hirsutism include acne and male-pattern balding (androgenic alopecia). Virilization refers to a condition in which androgen levels are sufficiently high to cause additional signs and symptoms, such as deepening of the voice, breast atrophy, Syndromes of extreme insulin resistance (e.g., lipodystrophy)

1	Syndromes of extreme insulin resistance (e.g., lipodystrophy) Thecoma of pregnancy increased muscle bulk, clitoromegaly, and increased libido; virilization is an ominous sign that suggests the possibility of an ovarian or adrenal neoplasm. Hair can be categorized as either vellus (fine, soft, and not pigmented) or terminal (long, coarse, and pigmented). The number of hair follicles does not change over an individual’s lifetime, but the follicle size and type of hair can change in response to numerous factors, particularly androgens. Androgens are necessary for terminal hair and sebaceous gland development and mediate differentiation of pilosebaceous units (PSUs) into either a terminal hair follicle or a sebaceous gland. In the former case, androgens transform the vellus hair into a terminal hair; in the latter case, the sebaceous component proliferates and the hair remains vellus.

1	There are three phases in the cycle of hair growth: (1) anagen (growth phase), (2) catagen (involution phase), and (3) telogen (rest phase). Depending on the body site, hormonal regulation may play an important role in the hair growth cycle. For example, the eyebrows, eyelashes, and vellus hairs are androgen-insensitive, whereas the axillary and pubic areas are sensitive to low levels of androgens. Hair growth on the face, chest, upper abdomen, and back requires higher levels of androgens and is therefore more characteristic of the pattern typically seen in men. Androgen excess in women leads to increased hair growth in most androgen-sensitive sites except in the scalp region, where hair loss occurs because androgens cause scalp hairs to spend less time in the anagen phase.

1	Although androgen excess underlies most cases of hirsutism, there is only a modest correlation between androgen levels and the quantity of hair growth. This is due to the fact that hair growth from the follicle also 332 depends on local growth factors, and there is variability in end organ (PSU) sensitivity. Genetic factors and ethnic background also influence hair growth. In general, dark-haired individuals tend to be more hirsute than blond or fair individuals. Asians and Native Americans have relatively sparse hair in regions sensitive to high androgen levels, whereas people of Mediterranean descent are more hirsute.

1	Historic elements relevant to the assessment of hirsutism include the age at onset and rate of progression of hair growth and associated symptoms or signs (e.g., acne). Depending on the cause, excess hair growth typically is first noted during the second and third decades of life. The growth is usually slow but progressive. Sudden development and rapid progression of hirsutism suggest the possibility of an androgen-secreting neoplasm, in which case virilization also may be present.

1	The age at onset of menstrual cycles (menarche) and the pattern of the menstrual cycle should be ascertained; irregular cycles from the time of menarche onward are more likely to result from ovarian rather than adrenal androgen excess. Associated symptoms such as galactorrhea should prompt evaluation for hyperprolactinemia (Chap. 403) and possibly hypothyroidism (Chap. 405). Hypertension, striae, easy bruising, centripetal weight gain, and weakness suggest hypercortisolism (Cushing’s syndrome; Chap. 406). Rarely, patients with growth hormone excess (i.e., acromegaly) present with hirsutism. Use of medications such as phenytoin, minoxidil, and cyclosporine may be associated with androgen-independent excess hair growth (i.e., hypertrichosis). A family history of infertility and/or hirsutism may indicate disorders such as nonclassic CAH (Chap. 406). Lipodystrophy is often associated with increased ovarian androgen production that occurs as a consequence of insulin resistance. Patients

1	may indicate disorders such as nonclassic CAH (Chap. 406). Lipodystrophy is often associated with increased ovarian androgen production that occurs as a consequence of insulin resistance. Patients with lipodystrophy have a preponderance of central fat distribution together with scant subcutaneous adipose tissue in the upper and lower extremities.

1	Physical examination should include measurement of height and weight and calculation of body mass index (BMI). A BMI >25 kg/m2 is indicative of excess weight for height, and values >30 kg/m2 are often seen in association with hirsutism, probably the result of increased conversion of androgen precursors to testosterone. Notation should be made of blood pressure, as adrenal causes may be associated with hypertension. Cutaneous signs sometimes associated with androgen excess and insulin resistance include acanthosis nigricans and skin tags. Body fat distribution should also be noted.

1	An objective clinical assessment of hair distribution and quantity is central to the evaluation in any woman presenting with hirsutism. This assessment permits the distinction between hirsutism and hypertrichosis and provides a baseline reference point to gauge the response to treatment. A simple and commonly used method to grade hair growth is the modified scale of Ferriman and Gallwey (Fig. 68-1), in which each of nine androgen-sensitive sites is graded from 0 to 4. Approximately 95% of white women have a score below 8 on this scale; thus, it is normal for most women to have some hair growth in androgen-sensitive sites. Scores above 8 suggest excess androgen-mediated hair growth, a finding that should be assessed further by means of hormonal evaluation (see below). In racial/ethnic groups that are less likely to manifest hirsutism (e.g., Asian women), additional cutaneous evidence of androgen excess should be sought, including pustular acne and thinning scalp hair.

1	Androgens are secreted by the ovaries and adrenal glands in response to their respective tropic hormones: luteinizing hormone (LH) and adrenocorticotropic hormone (ACTH). The principal circulating steroids involved in the etiology of hirsutism are testosterone, androstenedione, and dehydroepiandrosterone (DHEA) and its sulfated form (DHEAS). The ovaries and adrenal glands normally contribute about equally to testosterone production. Approximately half of the total testosterone originates from direct glandular secretion, and the remainder is derived from the peripheral conversion of androstenedione and DHEA (Chap. 411). PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Although it is the most important circulating androgen, testosterone is in effect the penultimate androgen in mediating hirsutism; it is converted to the more potent dihydrotestosterone (DHT) by the enzyme 5α-reductase, which is located in the PSU. DHT has a higher affinity for, and slower dissociation from, the androgen receptor. The local production of DHT allows it to serve as the primary mediator of androgen action at the level of the pilosebaceous unit. There are two isoenzymes of 5α-reductase: Type 2 is found in the prostate gland and in hair follicles, and type 1 is found primarily in sebaceous glands.

1	One approach to the evaluation of hirsutism is depicted in Fig. 68-2. In addition to measuring blood levels of testosterone and DHEAS, it is important to measure the level of free (or unbound) testosterone. The fraction of testosterone that is not bound to its carrier protein, sex hormone–binding globulin (SHBG), is biologically available for conversion to DHT and binding to androgen receptors. Hyperinsulinemia and/or androgen excess decrease hepatic production of SHBG, resulting in levels of total testosterone within the high-normal range, whereas the unbound hormone is elevated more substantially. Although there is a decline in ovarian testosterone production after menopause, ovarian estrogen production decreases to an even greater extent, and the concentration of SHBG is reduced. Consequently, there is an increase in the relative proportion of unbound testosterone, and it may exacerbate hirsutism after menopause.

1	A baseline plasma total testosterone level >12 nmol/L (>3.5 ng/mL) usually indicates a virilizing tumor, whereas a level >7 nmol/L (>2 ng/ mL) is suggestive. A basal DHEAS level >18.5 μmol/L (>7000 μg/L) suggests an adrenal tumor. Although DHEAS has been proposed as a “marker” of predominant adrenal androgen excess, it is not unusual to find modest elevations in DHEAS among women with PCOS. Computed tomography (CT) or magnetic resonance imaging (MRI) should be used to localize an adrenal mass, and transvaginal ultrasound usually suffices to identify an ovarian mass if clinical evaluation and hormonal levels suggest these possibilities.

1	PCOS is the most common cause of ovarian androgen excess (Chap. 412). An increased ratio of LH to follicle-stimulating hormone (FSH) is characteristic in carefully studied patients with PCOS. However, because of the pulsatile nature of gonadotropin secretion, this finding may be absent in up to half of women with PCOS. Therefore, measurement of plasma LH and FSH is not needed to make a diagnosis of PCOS. Transvaginal ultrasound classically shows enlarged ovaries and increased stroma in women with PCOS. However, cystic ovaries also may be found in women without clinical or laboratory features of PCOS.

1	It has been suggested that the measurement of circulating levels of antimüllerian hormone (AMH) may help in making the diagnosis of PCOS; however, this remains controversial. AMH levels reflect ovarian reserve and correlate with follicular number. Measurement of AMH can be useful when considering premature ovarian insufficiency in a patient who presents with oligomenorrhea, in which case a subnormal level of AMH will be present.

1	Because adrenal androgens are readily suppressed by low doses of glucocorticoids, the dexamethasone androgen-suppression test may broadly distinguish ovarian from adrenal androgen overproduction. A blood sample is obtained before and after the administration of dexamethasone (0.5 mg orally every 6 h for 4 days). An adrenal source is suggested by suppression of unbound testosterone into the normal range; incomplete suppression suggests ovarian androgen excess. An overnight 1-mg dexamethasone suppression test, with measurement of 8:00 a.m. serum cortisol, is useful when there is clinical suspicion of Cushing’s syndrome (Chap. 406).

1	Nonclassic CAH is most commonly due to 21-hydroxylase deficiency but also can be caused by autosomal recessive defects in other steroidogenic enzymes necessary for adrenal corticosteroid synthesis (Chap. 406). Because of the enzyme defect, the adrenal gland cannot secrete glucocorticoids (especially cortisol) efficiently. This results in diminished negative feedback inhibition of ACTH, leading to compensatory adrenal hyperplasia and the accumulation of steroid precursors that subsequently are converted to androgen. Deficiency

1	FIguRE 68-1 Hirsutism scoring scale of Ferriman and Gallwey. The nine body areas that have androgen-sensitive areas are graded from 0 (no terminal hair) to 4 (frankly virile) to obtain a total score. A normal hirsutism score is <8. (Modified from DA Ehrmann et al: Hyperandrogenism, hirsutism, and polycystic ovary syndrome, in LJ DeGroot and JL Jameson [eds], Endocrinology, 5th ed. Philadelphia, Saunders, 2006; with permission.) of 21-hydroxylase can be reliably excluded by determining a morning 17-hydroxyprogesterone level <6 nmol/L (<2 μg/L) (drawn in the follicular phase). Alternatively, 21-hydroxylase deficiency can be diagnosed by measurement of 17-hydroxyprogesterone 1 h after the administration of 250 μg of synthetic ACTH (cosyntropin) intravenously. Treatment of hirsutism may be accomplished pharmacologically or by mechanical means of hair removal. Nonpharmacologic treatments should be considered in all patients either as the only treatment or as an adjunct to drug therapy.

1	PART 2 Cardinal Manifestations and Presentation of Diseases Laboratory Evaluation• Total, free testosterone• DHEAS ReassuranceNonpharmacologic approaches Rule out ovarian oradrenal neoplasmNormalIncreased Treat empirically or Consider further testing• Dexamethasone suppression ˜ adrenal vsovarian causes; R/O Cushing’s • ACTH stimulation ˜ assess nonclassic CAH Marked elevationTotal testosterone >7 nmol/L(>2 ng/mL)DHEAS >18.5 °mol/L (>7000 °g/L)Yes FIguRE 68-2 Algorithm for the evaluation and differential diagnosis of hirsutism. ACTH, adrenocorticotropic hormone; CAH, congenital adrenal hyperplasia; DHEAS, sulfated form of dehydroepiandrosterone; PCOS, polycystic ovarian syndrome.

1	Nonpharmacologic treatments include (1) bleaching; (2) depilatory (removal from the skin surface), such as shaving and chemical treatments; and (3) epilatory (removal of the hair including the root), such as plucking, waxing, electrolysis, and laser therapy. Despite perceptions to the contrary, shaving does not increase the rate or density of hair growth. Chemical depilatory treatments may be useful for mild hirsutism that affects only limited skin areas, though they can cause skin irritation. Wax treatment removes hair temporarily but is uncomfortable. Electrolysis is effective for more permanent hair removal, particularly in the hands of a skilled electrologist. Laser phototherapy appears to be efficacious for hair removal. It delays hair regrowth and causes permanent hair removal in most patients. The long-term effects and complications associated with laser treatment are being evaluated.

1	Pharmacologic therapy is directed at interrupting one or more of the steps in the pathway of androgen synthesis and action: suppression of adrenal and/or ovarian androgen production; enhancement of androgen-binding to plasma-binding proteins, particularly SHBG; (3) impairment of the peripheral conversion of androgen precursors to active androgen; and (4) inhibition of androgen action at the target tissue level. Attenuation of hair growth is typically not evident until 4–6 months after initiation of medical treatment and in most cases leads to only a modest reduction in hair growth.

1	Combination estrogen-progestin therapy in the form of an oral contraceptive is usually the first-line endocrine treatment for hirsutism and acne, after cosmetic and dermatologic management. The estrogenic component of most oral contraceptives currently in use is either ethinyl estradiol or mestranol. The suppression of LH leads to reduced production of ovarian androgens. The reduced androgen levels also result in a dose-related increase in SHBG, thus lowering the fraction of unbound plasma testosterone. Combination therapy also has been demonstrated to decrease DHEAS, perhaps by reducing ACTH levels. Estrogens also have a direct, dose-dependent suppressive effect on sebaceous cell function.

1	The choice of a specific oral contraceptive should be predicated on the progestational component, as progestins vary in their suppressive effect on SHBG levels and in their androgenic potential. Ethynodiol diacetate has relatively low androgenic potential, whereas progestins such as norgestrel and levonorgestrel are particularly androgenic, as judged from their attenuation of the estrogen-induced increase in SHBG. Norgestimate exemplifies the newer generation of progestins that are virtually nonandrogenic. Drospirenone, an analogue of spironolactone that has both antimineralocorticoid and antiandrogenic activities, has been approved for use as a progestational agent in combination with ethinyl estradiol.

1	Oral contraceptives are contraindicated in women with a history of thromboembolic disease and women with increased risk of breast or other estrogen-dependent cancers (Chap. 413). There is a relative contraindication to the use of oral contraceptives in smokers and those with hypertension or a history of migraine headaches. In most trials, estrogen-progestin therapy alone improves the extent of acne by a maximum of 50–70%. The effect on hair growth may not be evident for 6 months, and the maximum effect may require 9–12 months owing to the length of the hair growth cycle. Improvements in hirsutism are typically in the range of 20%, but there may be an arrest of further progression of hair growth.

1	Adrenal androgens are more sensitive than cortisol to the suppressive effects of glucocorticoids. Therefore, glucocorticoids are the mainstay of treatment in patients with CAH. Although glucocorticoids have been reported to restore ovulatory function in some women with PCOS, this effect is highly variable. Because of side effects from excessive glucocorticoids, low doses should be used. Dexamethasone (0.2– 0.5 mg) or prednisone (5–10 mg) should be taken at bedtime to achieve maximal suppression by inhibiting the nocturnal surge of ACTH.

1	Cyproterone acetate is the prototypic antiandrogen. It acts mainly by competitive inhibition of the binding of testosterone and DHT to the androgen receptor. In addition, it may enhance the metabolic clearance of testosterone by inducing hepatic enzymes. Although not available for use in the United States, cyproterone acetate is widely used in Canada, Mexico, and Europe. Cyproterone (50–100 mg) is given on days 1–15 and ethinyl estradiol (50 μg) is given on days 5–26 of the menstrual cycle. Side effects include irregular uterine bleeding, nausea, headache, fatigue, weight gain, and decreased libido. Menstrual Disorders and Pelvic Pain Janet E. Hall Menstrual dysfunction can signal an underlying abnormality that may have long-term health consequences. Although frequent or prolonged 69

1	Menstrual Disorders and Pelvic Pain Janet E. Hall Menstrual dysfunction can signal an underlying abnormality that may have long-term health consequences. Although frequent or prolonged 69 Spironolactone, which usually is used as a mineralocorticoid antagonist, is also a weak antiandrogen. It is almost as effective as cyproterone acetate when used at high enough doses (100–200 mg daily). Patients should be monitored intermittently for hyperkalemia or hypotension, although these side effects are uncommon. Pregnancy should be avoided because of the risk of feminization of a male fetus. Spironolactone can also cause menstrual irregularity. It often is used in combination with an oral contraceptive, which suppresses ovarian androgen production and helps prevent pregnancy.

1	Flutamide is a potent nonsteroidal antiandrogen that is effective in treating hirsutism, but concerns about the induction of hepatocellular dysfunction have limited its use. Finasteride is a competitive inhibitor of 5α-reductase type 2. Beneficial effects on hirsutism have been reported, but the predominance of 5α-reductase type 1 in the PSU appears to account for its limited efficacy. Finasteride would also be expected to impair sexual differentiation in a male fetus, and it should not be used in women who may become pregnant. Eflornithine cream (Vaniqa) has been approved as a novel treatment for unwanted facial hair in women, but long-term efficacy remains to be established. It can cause skin irritation under exaggerated conditions of use. Ultimately, the choice of any specific agent(s) must be tailored to the unique needs of the patient being treated. As noted previously, pharmacologic treatments for hirsutism should be used in conjunction with nonpharmacologic approaches. It is

1	must be tailored to the unique needs of the patient being treated. As noted previously, pharmacologic treatments for hirsutism should be used in conjunction with nonpharmacologic approaches. It is also helpful to review the pattern of female hair distribution in the normal population to dispel unrealistic expectations.

1	bleeding usually prompts a woman to seek medical attention, infrequent or absent bleeding may seem less troubling and the patient may not bring it to the attention of the physician. Thus, a focused menstrual history is a critical part of every encounter with a female patient. Pelvic pain is a common complaint that may relate to an abnormality of the reproductive organs but also may be of gastrointestinal, urinary tract, or musculoskeletal origin. Depending on its cause, pelvic pain may require urgent surgical attention.

1	Amenorrhea refers to the absence of menstrual periods. Amenorrhea is classified as primary if menstrual bleeding has never occurred in the absence of hormonal treatment or secondary if menstrual periods cease for 3–6 months. Primary amenorrhea is a rare disorder that occurs in <1% of the female population. However, between 3 and 5% of women experience at least 3 months of secondary amenorrhea in any specific year. There is no evidence that race or ethnicity influences the prevalence of amenorrhea. However, because of the importance of adequate nutrition for normal reproductive function, both the age at menarche and the prevalence of secondary amenorrhea vary significantly in different parts of the world.

1	Oligomenorrhea is defined as a cycle length >35 days or <10 menses per year. Both the frequency and the amount of vaginal bleeding are irregular in oligomenorrhea, and moliminal symptoms (premenstrual breast tenderness, food cravings, mood lability), suggestive of ovulation, are variably present. Anovulation can also present with intermenstrual 335 intervals <24 days or vaginal bleeding for >7 days. Frequent or heavy irregular bleeding is termed dysfunctional uterine bleeding if anatomic uterine and outflow tract lesions or a bleeding diathesis has been excluded.

1	Primary Amenorrhea The absence of menses by age 16 has been used traditionally to define primary amenorrhea. However, other factors, such as growth, secondary sexual characteristics, the presence of cyclic pelvic pain, and the secular trend toward an earlier age of menarche, particularly in African-American girls, also influence the age at which primary amenorrhea should be investigated. Thus, an evaluation for amenorrhea should be initiated by age 15 or 16 in the presence of normal growth and secondary sexual characteristics; age 13 in the absence of secondary sexual characteristics or if height is less than the third percentile; age 12 or 13 in the presence of breast development and cyclic pelvic pain; or within 2 years of breast development if menarche, defined by the first menstrual period, has not occurred.

1	Secondary Amenorrhea or Oligomenorrhea Anovulation and irregular cycles are relatively common for up to 2 years after menarche and for 1–2 years before the final menstrual period. In the intervening years, menstrual cycle length is ~28 days, with an intermenstrual interval normally ranging between 25 and 35 days. Cycle-to-cycle variability in an individual woman who is ovulating consistently is generally +/− 2 days. Pregnancy is the most common cause of amenorrhea and should be excluded early in any evaluation of menstrual irregularity. However, many women occasionally miss a single period. Three or more months of secondary amenorrhea should prompt an evaluation, as should a history of intermenstrual intervals >35 or <21 days or bleeding that persists for >7 days.

1	Evaluation of menstrual dysfunction depends on understanding the interrelationships between the four critical components of the reproductive tract: (1) the hypothalamus, (2) the pituitary, (3) the ovaries, and (4) the uterus and outflow tract (Fig. 69-1; Chap. 412). This system is maintained by complex negative and positive feedback loops involving the ovarian steroids (estradiol and progesterone) and peptides (inhibin B and inhibin A) and the hypothalamic (gonadotropin-releasing hormone [GnRH]) and pituitary (follicle-stimulating hormone [FSH] and luteinizing hormone [LH]) components of this system (Fig. 69-1).

1	Disorders of menstrual function can be thought of in two main categories: disorders of the uterus and outflow tract and disorders of ovulation. Many of the conditions that cause primary amenorrhea are congenital but go unrecognized until the time of normal puberty (e.g., genetic, chromosomal, and anatomic abnormalities). All causes of secondary amenorrhea also can cause primary amenorrhea.

1	Disorders of the uterus or Outflow Tract Abnormalities of the uterus and outflow tract typically present as primary amenorrhea. In patients with normal pubertal development and a blind vagina, the differential diagnosis includes obstruction by a transverse vaginal septum or imperforate hymen; müllerian agenesis (Mayer-Rokitansky-Kuster-Hauser syndrome), which has been associated with mutations in the WNT4 gene; and androgen insensitivity syndrome (AIS), which is an X-linked recessive disorder that accounts for ~10% of all cases of primary amenorrhea (Chap. 411). Patients with AIS have a 46,XY karyotype, but because of the lack of androgen receptor responsiveness, those with complete AIS have severe underandrogenization and female external genitalia. The absence of pubic and axillary hair distinguishes them clinically from patients with müllerian agenesis, as does an elevated testosterone level. Asherman’s syndrome presents as secondary amenorrhea or hypomenorrhea and results from

1	distinguishes them clinically from patients with müllerian agenesis, as does an elevated testosterone level. Asherman’s syndrome presents as secondary amenorrhea or hypomenorrhea and results from partial or complete obliteration of the uterine cavity by adhesions that prevent normal growth and shedding of the endometrium. Curettage performed for pregnancy complications accounts for >90% of cases; genital tuberculosis is an important cause in regions where it is endemic.

1	or secondary amenorrhea. They may occur in association with other features suggestive of hypothalamic or pituitary dysfunction, such as short stature, diabetes insipidus, galactorrhea, and headache. Hypogonadotropic hypogonadism also may be seen after cranial irradiation. In the postpartum period, it may be caused by pituitary necrosis (Sheehan’s syndrome) or lymphocytic hypophysitis. Because reproductive dysfunction is commonly associated with hyperprolactinemia from neuroanatomic lesions or medications, prolactin should be measured in all patients with hypogonadotropic hypogonadism (Chap. 403).

1	Isolated hypogonadotropic hypogonadism (IHH) occurs in women, although it is three times more common in men. IHH generally presents with primary amenorrhea, although 50% have some degree of breast development, and one to two menses have been described in ~10%. IHH is associated with anosmia in about 50% of women (termed Kallmann’s syndrome). Genetic causes of IHH have been identified in ~60% of patients (Chaps. 411 and 412). Functional hypothalamic amenorrhea (HA) is caused by a mismatch between energy expen-

1	FIguRE 69-1 Role of the hypothalamic-pituitary-gonadal axis in the etiology of diture and energy intake. Recent studies suggest amenorrhea. Gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus that variants in genes associated with IHH may increase susceptibility to these environmental the pituitary to induce ovarian folliculogenesis and steroidogenesis. Ovarian secretion of inputs, accounting in part for the clinical vari estradiol and progesterone controls the shedding of the endometrium, resulting in menses, ability in this disorder. Leptin secretion may and, in combination with the inhibins, provides feedback regulation of the hypothalamus play a key role in transducing the signals from and pituitary to control secretion of FSH and LH. The prevalence of amenorrhea resulting the periphery to the hypothalamus in HA. The from abnormalities at each level of the reproductive system (hypothalamus, pituitary, ovary, uterus, and outflow tract) varies depending on whether

1	the periphery to the hypothalamus in HA. The from abnormalities at each level of the reproductive system (hypothalamus, pituitary, ovary, uterus, and outflow tract) varies depending on whether amenorrhea is primary or secondary.

1	PART 2 Cardinal Manifestations and Presentation of Diseases PCOS, polycystic ovarian syndrome. Obstruction of the outflow tract requires surgical correction. The risk of endometriosis is increased with this condition, perhaps because of retrograde menstrual flow. Mlerian agenesis also may require surgical intervention to allow sexual intercourse, although vaginal dilatation is adequate in some patients. Because ovarian function is normal, assisted reproductive techniques can be used with a surrogate carrier. Androgen resistance syndrome requires gonadectomy because there is risk of gonadoblastoma in the dysgenetic gonads. Whether this should be performed in early childhood or after completion of breast development is controversial. Estrogen replacement is indicated after gonadectomy, and vaginal dilatation may be required to allow sexual intercourse.

1	Disorders of Ovulation Once uterus and outflow tract abnormalities have been excluded, other causes of amenorrhea involve disorders of ovulation. The differential diagnosis is based on the results of initial tests, including a pregnancy test, an FSH level (to determine whether the cause is likely to be ovarian or central), and assessment of hyperandrogenism (Fig. 69-2).

1	HYPOgONAdOTROPIC HYPOgONAdISM Low estrogen levels in combination with normal or low levels of LH and FSH are seen with anatomic, genetic, or functional abnormalities that interfere with hypothalamic GnRH secretion or normal pituitary responsiveness to GnRH. Although relatively uncommon, tumors and infiltrative diseases should be considered in the differential diagnosis of hypogonadotropic hypogonadism (Chap. 403). These disorders may present with primary play a role. The diagnosis of HA generally can be made on the basis of a careful history, a physical examination, and the demonstration of low levels of gonadotropins and normal prolactin levels. Eating disorders and chronic disease must be specifically excluded. An atypical history, headache, signs of other hypothalamic dysfunction, or hyperprolactinemia, even if mild, necessitates cranial imaging with computed tomography (CT) or magnetic resonance imaging (MRI) to exclude a neuroanatomic cause.

1	HYPERgONAdOTROPIC HYPOgONAdISM Ovarian failure is considered premature when it occurs in women <40 years old and accounts for ~10% of secondary amenorrhea. Primary ovarian insufficiency (POI) has generally replaced the terms premature menopause and premature ovarian failure in recognition that this disorder represents a continuum of impaired ovarian function. Ovarian insufficiency is associated with the loss of negative-feedback restraint on the hypothalamus and pituitary, resulting in increased FSH and LH levels. FSH is a better marker of ovarian failure as its levels are less variable than those of LH. Antimüllerian hormone (AMH) levels will also be low in patients with POI, but are more frequently used in management of infertility. As with natural menopause, POI may wax and wane, and serial measurements may be necessary to establish the diagnosis.

1	Once the diagnosis of POI has been established, further evaluation is indicated because of other health problems that may be associated with POI. For example, POI occurs in association with a variety of chromosomal abnormalities, including Turner’s syndrome, autoimmune polyglandular failure syndromes, radioand chemotherapy, and galactosemia. The recognition that early ovarian failure occurs in premutation carriers of the fragile X syndrome is important because of the increased risk of severe mental retardation in male children with FMR1 mutations. In the majority of cases, a cause for POI is not determined. Although there are increasing reports of genetic mutations in individuals and families with POI, testing for other than chromosomal abnormalities and FMR1 mutations is not recommended.

1	Neuroanatomic abnormality or idiopathic hypogonadotropic hypogonadism Hypothalamic amenorrhea 2° amenorrheaR/O drugs, °TSH 1° amenorrhea, short stature or clinical suspicion Hyperandrogenism ° testosterone, hirsutism, acne R/O tumor R/O 21 hydroxylase deficiency Polycystic ovarian syndrome Increased MRI Normal PRL Ovarian insufficiency Normal or low Increased (x2) GYN referral GYN referral Asherman’s syndrome FSH ˛-hCGPregnancy Normal Abnormal Normal PRL, FSH Negative trial of estrogen/ progesterone Mlerian agenesis, cervical stenosis, vaginal septum, imperfo-rate hymen Uterine instrumentation Androgen insensitivity syndrome – + R/O eating disorder, chronic disease FIguRE 69-2 Algorithm for evaluation of amenorrhea. β-hCG, human chorionic gonadotropin; FSH, follicle-stimulating hormone; GYN, gynecologist; MRI, magnetic resonance imaging; PRL, prolactin; R/O, rule out; TSH, thyroid-stimulating hormone.

1	Hypergonadotropic hypogonadism occurs rarely in other disorders, such as mutations in the FSH or LH receptors. Aromatase deficiency and 17α-hydroxylase deficiency are associated with decreased estrogen and elevated gonadotropins and with hyperandrogenism and hypertension, respectively. Gonadotropin-secreting tumors in women of reproductive age generally present with high, rather than low, estrogen levels and cause ovarian hyperstimulation or dysfunctional bleeding.

1	Amenorrhea almost always is associated with chronically low levels of estrogen, whether it is caused by hypogonadotropic hypogonadism or ovarian insufficiency. Development of secondary sexual characteristics requires gradual titration of estradiol replacement with eventual addition of progestin. Hormone replacement with either low-dose estrogen/progesterone regimens or oral contraceptive pills is recommended until the usual age of menopause for bone and cardiovascular protection. Patients with hypogonadotropic hypogonadism who are interested in fertility require treatment with exogenous FSH combined with LH or pulsatile GnRH. Patients with ovarian failure can consider oocyte donation, which has a high rate of success in this population, although its use in women with Turner’s syndrome is limited by significant maternal cardiovascular risk.

1	POLYCYSTIC OVARIAN SYNdROME (PCOS) PCOS is diagnosed based on a combination of clinical or biochemical evidence of hyperandrogenism, amenorrhea or oligomenorrhea, and the ultrasound appearance of polycystic ovaries. Approximately half of patients with PCOS are obese, and abnormalities in insulin dynamics are common, as is metabolic syndrome. Symptoms generally begin shortly after menarche and are slowly progressive. Lean oligo-ovulatory patients with PCOS generally have high LH levels in the presence of normal to low levels of FSH and estradiol. The LH/FSH ratio is less pronounced in obese patients in whom insulin resistance is a more prominent feature.

1	A major abnormality in patients with PCOS is the failure of regular, predictable ovulation. Thus, these patients are at risk for the development of dysfunctional bleeding and endometrial hyperplasia associated with unopposed estrogen exposure. Endometrial protection can be achieved with the use of oral contraceptives or progestins (medroxyprogesterone acetate, 5–10 mg, or prometrium, 200 mg daily for 10–14 days of each month). Oral contraceptives are also useful for management of hyperandrogenic symptoms, as are spironolactone and cyproterone acetate (not available in the United States), which function as weak androgen receptor blockers. Management of the associated metabolic syndrome may be appropriate for some patients (Chap. 422). For patients interested in fertility, weight control is a critical first step. Clomiphene citrate is highly effective as a first-line treatment, and there is increasing evidence that the aromatase inhibitor letrozole may also be effective. Exogenous

1	is a critical first step. Clomiphene citrate is highly effective as a first-line treatment, and there is increasing evidence that the aromatase inhibitor letrozole may also be effective. Exogenous gonadotropins can be used by experienced practitioners; a diagnosis of polycystic ovaries in the presence or absence of cycle abnormalities increases the risk of hyperstimulation.

1	The mechanisms that cause pelvic pain are similar to those that cause abdominal pain (Chap. 20) and include inflammation of the parietal peritoneum, obstruction of hollow viscera, vascular disturbances, and pain originating in the abdominal wall. Pelvic pain may reflect pelvic disease per se but also may reflect extrapelvic disorders that refer pain to the pelvis. In up to 60% of cases, pelvic pain can be attributed to 338 gastrointestinal problems, including appendicitis, cholecystitis, infections, intestinal obstruction, diverticulitis, and inflammatory bowel disease. Urinary tract and musculoskeletal disorders are also common causes of pelvic pain. APPROACH TO THE PATIENT: PART 2 Cardinal Manifestations and Presentation of Diseases

1	APPROACH TO THE PATIENT: PART 2 Cardinal Manifestations and Presentation of Diseases As with all types of abdominal pain, the first priority is to identify life-threatening conditions (shock, peritoneal signs) that may require emergent surgical management. The possibility of pregnancy should be identified as soon as possible by menstrual history and/or testing. A thorough history that includes the type, location, radiation, and status with respect to increasing or decreasing severity can help identify the cause of acute pelvic pain. Specific associations with vaginal bleeding, sexual activity, defecation, urination, movement, or eating should be specifically sought. Determination of whether the pain is acute versus chronic and cyclic versus noncyclic will direct further investigation (Table 69-1). However, disorders that cause cyclic pain occasionally may cause noncyclic pain, and the converse is also true.

1	Pelvic inflammatory disease most commonly presents with bilateral lower abdominal pain. It is generally of recent onset and is exacerbated by intercourse or jarring movements. Fever is present in about half of these patients; abnormal uterine bleeding occurs in about one-third. New vaginal discharge, urethritis, and chills may be present but are less specific signs. Adnexal pathology can present acutely and may be due to rupture, bleeding or torsion of cysts, or, much less commonly, neoplasms of the ovary, fallopian tubes, or paraovarian areas. Fever may be present with ovarian torsion. Ectopic pregnancy is associated with rightor left-sided lower abdominal pain, with clinical signs generally appearing 6–8 weeks after the last normal menstrual period. Amenorrhea is present in ~75% of cases and vaginal bleeding in ~50% of cases. Orthostatic signs and fever may be present. Risk factors include the presence of known tubal disease, previous ectopic pregnancies, a history of infertility,

1	and vaginal bleeding in ~50% of cases. Orthostatic signs and fever may be present. Risk factors include the presence of known tubal disease, previous ectopic pregnancies, a history of infertility, diethylstilbestrol (DES) exposure of the mother in utero, or a history of pelvic infections. Threatened abortion may also present with amenorrhea, abdominal pain, and vaginal bleeding. Although more common than ectopic pregnancy, it is rarely associated with systemic signs. Uterine pathology includes endometritis and, less frequently, degenerating leiomyomas (fibroids). Endometritis often is associated with vaginal bleeding and systemic signs of infection. It occurs in the setting of sexually transmitted infections, uterine instrumentation, or postpartum infection.

1	A sensitive pregnancy test, complete blood count with differential, urinalysis, tests for chlamydial and gonococcal infections, and abdominal ultrasound aid in making the diagnosis and directing further management. Treatment of acute pelvic pain depends on the suspected etiology but may require surgical or gynecologic intervention. Conservative management is an important consideration for ovarian cysts, if torsion is not suspected, to avoid unnecessary pelvic surgery and the subsequent risk of infertility due to adhesions. Surgical treatment may be required for ectopic pregnancies; however, approximately 35% of ectopic pregnancies are unruptured and may be appropriate for treatment with methotrexate, which is effective in ~90% of cases.

1	Some women experience discomfort at the time of ovulation (mittelschmerz). The pain can be quite intense but is generally of short duration. The mechanism is thought to involve rapid expansion of the dominant follicle, although it also may be caused by peritoneal irritation by follicular fluid released at the time of ovulation. Many women experience premenstrual symptoms such as breast discomfort, food cravings, and abdominal bloating or discomfort. These moliminal symptoms are a good marker of prior ovulation, although their absence is less helpful. Dysmenorrhea Dysmenorrhea refers to the crampy lower abdominal midline discomfort that begins with the onset of menstrual bleeding and gradually decreases over the next 12–72 h. It may be associated with nausea, diarrhea, fatigue, and headache and occurs in 60–93% of adolescents, beginning with the establishment of regular ovulatory cycles. Its prevalence decreases after pregnancy and with the use of oral contraceptives.

1	Primary dysmenorrhea results from increased stores of prostaglandin precursors, which are generated by sequential stimulation of the uterus by estrogen and progesterone. During menstruation, these precursors are converted to prostaglandins, which cause intense uterine contractions, decreased blood flow, and increased peripheral nerve hypersensitivity, resulting in pain.

1	Secondary dysmenorrhea is caused by underlying pelvic pathology. Endometriosis results from the presence of endometrial glands and stroma outside the uterus. These deposits of ectopic endometrium respond to hormonal stimulation and cause dysmenorrhea, which begins several days before menses. Endometriosis also may be associated with painful intercourse, painful bowel movements, and tender nodules in the uterosacral ligament. Fibrosis and adhesions can produce lateral displacement of the cervix. Transvaginal pelvic ultrasound is part of the initial workup and may detect an endometrioma within the ovary, rectovaginal or bladder nodules, or ureteral involvement. The CA125 level may be increased, but it has low negative predictive value. Definitive diagnosis requires laparoscopy. Symptomatology does not always predict the extent of endometriosis. The prevalence is lower in black and Hispanic women than in Caucasians and Asians. Other secondary causes of dysmenorrhea include adenomyosis, a

1	does not always predict the extent of endometriosis. The prevalence is lower in black and Hispanic women than in Caucasians and Asians. Other secondary causes of dysmenorrhea include adenomyosis, a condition caused by the presence of ectopic endometrial glands and stroma within the myometrium. Cervical stenosis may result from trauma, infection, or surgery.

1	Local application of heat; dietary dairy intake; use of vitamins B1, B6, and E and fish oil; acupuncture; yoga; and exercise are of some benefit for the treatment of dysmenorrhea. Studies of vitamin D3 are not yet adequate to provide a recommendation. However, nonsteroidal anti-inflammatory drugs (NSAIDs) are the most effective treatment and provide >80% sustained response rates. Ibuprofen, naproxen, ketoprofen, mefanamic acid, and nimesulide are all superior to placebo. Treatment should be started a day before expected menses and generally is continued for 2–3 days. Oral contraceptives also reduce symptoms of dysmenorrhea. The use of tocolytics, antiphosphodiesterase inhibitors, and magnesium has been suggested, but Approach to the Patient with a Skin Disorder Thomas J. Lawley, Kim B. Yancey The challenge of examining the skin lies in distinguishing normal from abnormal findings, distinguishing significant findings from 70 SECTion 9 ALTERATionS in THE SKin

1	CHAPTER 70 Approach to the Patient with a Skin Disorder trivial ones, and integrating pertinent signs and symptoms into an appropriate differential diagnosis. The fact that the largest organ in the body is visible is both an advantage and a disadvantage to those who examine it. It is advantageous because no special instrumentation is necessary and because the skin can be biopsied with little morbidity. However, the casual observer can be misled by a variety of stimuli and overlook important, subtle signs of skin or systemic disease. For instance, the sometimes minor differences in color and shape that distinguish a melanoma (Fig. 70-1) from a benign nevomelanocytic nevus (Fig. 70-2) can be difficult to recognize. A variety of descriptive terms have been developed that characterize cutaneous lesions (Tables 70-1, 70-2, and Tables 70-3; Fig. 70-3), thereby aiding in their interpretation and in the formulation of a differential diagnosis (Table 70-4). For example, the finding of scaling

1	lesions (Tables 70-1, 70-2, and Tables 70-3; Fig. 70-3), thereby aiding in their interpretation and in the formulation of a differential diagnosis (Table 70-4). For example, the finding of scaling papules, which are present in psoriasis or atopic dermatitis, places the patient in a different diagnostic category than would hemorrhagic papules, which may indicate vasculitis or sepsis (Figs. 70-4 and 70-5, respectively). It is also important to differentiate primary from secondary skin lesions. If the examiner focuses on linear erosions overlying an area of erythema and scaling, he or she may incorrectly assume that the erosion is the primary lesion and that the redness and scale are secondary, whereas the correct interpretation would be that the patient has a pruritic eczematous dermatitis with erosions caused by scratching.

1	FIguRE 70-1 Superficial spreading melanoma. This is the most common type of melanoma. Such lesions usually demonstrate asymmetry, border irregularity, color variegation (black, blue, brown, pink, and white), a diameter >6 mm, and a history of change (e.g., an increase in size or development of associated symptoms such as pruritus or pain). there are insufficient data to recommend them. Failure of response 339 to NSAIDs and/or oral contraceptives is suggestive of a pelvic disorder such as endometriosis, and diagnostic laparoscopy should be considered to guide further treatment. FIguRE 70-2 Nevomelanocytic nevus. Nevi are benign proliferations of nevomelanocytes characterized by regularly shaped hyperpigment-ed macules or papules of a uniform color. Macule: A flat, colored lesion, <2 cm in diameter, not raised above the surface of the surrounding skin. A “freckle,” or ephelid, is a prototypical pigmented macule.

1	Macule: A flat, colored lesion, <2 cm in diameter, not raised above the surface of the surrounding skin. A “freckle,” or ephelid, is a prototypical pigmented macule. Patch: A large (>2-cm) flat lesion with a color different from the surrounding skin. This differs from a macule only in size. Papule: A small, solid lesion, <0.5 cm in diameter, raised above the surface of the surrounding skin and thus palpable (e.g., a closed comedone, or whitehead, in acne). Nodule: A larger (0.5to 5.0-cm), firm lesion raised above the surface of the surrounding skin. This differs from a papule only in size (e.g., a large dermal nevomelanocytic nevus). Tumor: A solid, raised growth >5 cm in diameter. Plaque: A large (>1-cm), flat-topped, raised lesion; edges may either be distinct (e.g., in psoriasis) or gradually blend with surrounding skin (e.g., in eczematous dermatitis).

1	Plaque: A large (>1-cm), flat-topped, raised lesion; edges may either be distinct (e.g., in psoriasis) or gradually blend with surrounding skin (e.g., in eczematous dermatitis). Vesicle: A small, fluid-filled lesion, <0.5 cm in diameter, raised above the plane of surrounding skin. Fluid is often visible, and the lesions are translucent (e.g., vesicles in allergic contact dermatitis caused by Toxicodendron [poison ivy]). Pustule: A vesicle filled with leukocytes. Note: The presence of pustules does not necessarily signify the existence of an infection. Bulla: A fluid-filled, raised, often translucent lesion >0.5 cm in diameter. Wheal: A raised, erythematous, edematous papule or plaque, usually representing short-lived vasodilation and vasopermeability. Telangiectasia: A dilated, superficial blood vessel. Lichenification: A distinctive thickening of the skin that is characterized by accentuated skin-fold markings. Scale: Excessive accumulation of stratum corneum.

1	Lichenification: A distinctive thickening of the skin that is characterized by accentuated skin-fold markings. Scale: Excessive accumulation of stratum corneum. Crust: Dried exudate of body fluids that may be either yellow (i.e., serous crust) or red (i.e., hemorrhagic crust). Erosion: Loss of epidermis without an associated loss of dermis. Ulcer: Loss of epidermis and at least a portion of the underlying dermis. Excoriation: Linear, angular erosions that may be covered by crust and are caused by scratching. Atrophy: An acquired loss of substance. In the skin, this may appear as a depression with intact epidermis (i.e., loss of dermal or subcutaneous tissue) or as sites of shiny, delicate, wrinkled lesions (i.e., epidermal atrophy). Scar: A change in the skin secondary to trauma or inflammation. Sites may be erythematous, hypopigmented, or hyperpigmented depending on their age or character. Sites on hair-bearing areas may be characterized by destruction of hair follicles.

1	Alopecia: Hair loss, partial or complete. Annular: Ring-shaped. Cyst: A soft, raised, encapsulated lesion filled with semisolid or liquid contents. Herpetiform: In a grouped configuration. Lichenoid eruption: Violaceous to purple, polygonal lesions that resemble those seen in lichen planus. Milia: Small, firm, white papules filled with keratin. Morbilliform rash: Generalized, small erythematous macules and/or papules that resemble lesions seen in measles. Nummular: Coin-shaped. Poikiloderma: Skin that displays variegated pigmentation, atrophy, and telangiectases. Polycyclic lesions: A configuration of skin lesions formed from coalescing rings or incomplete rings.

1	Poikiloderma: Skin that displays variegated pigmentation, atrophy, and telangiectases. Polycyclic lesions: A configuration of skin lesions formed from coalescing rings or incomplete rings. Pruritus: A sensation that elicits the desire to scratch. Pruritus is often the predominant symptom of inflammatory skin diseases (e.g., atopic dermatitis, allergic contact dermatitis); it is also commonly associated with xerosis and aged skin. Systemic conditions that can be associated with pruritus include chronic renal disease, cholestasis, pregnancy, malignancy, thyroid disease, polycythemia vera, and delusions of parasitosis. PART 2 Cardinal Manifestations and Presentation of Diseases APPROACH TO THE PATIENT:

1	In examining the skin it is usually advisable to assess the patient before taking an extensive history. This approach ensures that the entire cutaneous surface will be evaluated, and objective findings can be integrated with relevant historical data. Four basic features of a skin lesion must be noted and considered during a physical examination: the distribution of the eruption, the types of primary and secondary lesions, the shape of individual lesions, and the arrangement of the lesions. An ideal skin examination includes evaluation of the skin, hair, and nails as well as the mucous membranes of the mouth, eyes, nose, nasopharynx, and anogenital region. In the initial examination, it is important that the patient be disrobed as completely as possible to minimize chances of missing important individual skin lesions and permit accurate assessment of the distribution of the eruption. The patient should first be viewed from a distance of about 1.5–2 m (4–6 ft) so that the general

1	important individual skin lesions and permit accurate assessment of the distribution of the eruption. The patient should first be viewed from a distance of about 1.5–2 m (4–6 ft) so that the general character of the skin and the distribution of lesions can be evaluated. Indeed, the distribution of lesions often correlates highly with diagnosis (Fig. 70-6). For example, a hospitalized patient with a generalized erythematous exanthem is more likely to have a drug eruption than is a patient with a similar rash limited to the sun-exposed portions of the face. Once the distribution of the lesions has been established, the nature of the primary lesion

1	FIguRE 70-3 A schematic representation of several common primary skin lesions (see Table 70-1).

1	must be determined. Thus, when lesions are distributed on elbows, knees, and scalp, the most likely possibility based solely on distribution is psoriasis or dermatitis herpetiformis (Figs. 70-7 and 70-8, respectively). The primary lesion in psoriasis is a scaly papule that soon forms erythematous plaques covered with a white scale, whereas that of dermatitis herpetiformis is an urticarial papule that quickly becomes a small vesicle. In this manner, identification of the primary lesion directs the examiner toward the proper diagnosis. Secondary changes in skin can also be quite helpful. For example, scale represents excessive epidermis, while crust is the result of a discontinuous epithelial cell layer. Palpation of skin lesions can yield insight into the character of an eruption. Thus, red papules on the lower extremities that blanch with pressure can be a manifestation of many different diseases, but hemorrhagic red papules that do not blanch with pressure indicate palpable purpura

1	papules on the lower extremities that blanch with pressure can be a manifestation of many different diseases, but hemorrhagic red papules that do not blanch with pressure indicate palpable purpura characteristic of necrotizing vasculitis (Fig. 70-4).

1	The shape of lesions is also an important feature. Flat, round, erythematous papules and plaques are common in many cutaneous diseases. However, target-shaped lesions that consist in part of erythematous plaques are specific for erythema multiforme (Fig. 70-9). Likewise, the arrangement of individual lesions is important. Erythematous papules and vesicles can occur in many conditions, but their arrangement in a specific linear array suggests an external etiology such as allergic contact dermatitis (Fig. 70-10) or primary irritant dermatitis. In contrast, lesions with a generalized arrangement are common and suggest a systemic etiology. As in other branches of medicine, a complete history should be obtained to emphasize the following features: 1. Evolution of lesions a. Site of onset b. Manner in which the eruption progressed or spread c. d. Periods of resolution or improvement in chronic eruptions 2. Symptoms associated with the eruption a. Itching, burning, pain, numbness b.

1	Manner in which the eruption progressed or spread c. d. Periods of resolution or improvement in chronic eruptions 2. Symptoms associated with the eruption a. Itching, burning, pain, numbness b. What, if anything, has relieved symptoms c. Time of day when symptoms are most severe 3. Current or recent medications (prescribed as well as over-thecounter) 4. Associated systemic symptoms (e.g., malaise, fever, arthralgias) 5. 6. History of allergies 7. Presence of photosensitivity 8. Review of systems 9. Family history (particularly relevant for patients with melanoma, atopy, psoriasis, or acne) 10. Social, sexual, or travel history Many skin diseases can be diagnosed on the basis of gross clinical appearance, but sometimes relatively simple diagnostic procedures can yield valuable information. In most instances, they can be performed at the bedside with a minimum of equipment.

1	Skin Biopsy A skin biopsy is a straightforward minor surgical procedure; however, it is important to biopsy a lesion that is most likely to yield diagnostic findings. This decision may require expertise in skin diseases and knowledge of superficial anatomic structures in selected areas of the body. In this procedure, a small area of skin is anesthetized with 1% lidocaine with or without epinephrine. The skin lesion in question can be excised or saucerized with a scalpel or removed by punch biopsy. In the latter technique, a punch is pressed against the surface of the skin and rotated with downward pressure until it penetrates to the subcutaneous tissue. The circular biopsy is then lifted with forceps, and the bottom is cut with iris scissors. Biopsy sites may or may not need suture closure, depending on size and location.

1	KOH Preparation A potassium hydroxide (KOH) preparation is per-hyphae in dermatophyte infections, pseudohyphae and budding formed on scaling skin lesions where a fungal infection is suspected. yeasts in Candida infections, and “spaghetti and meatballs” yeast The edge of such a lesion is scraped gently with a no. 15 scalpel forms in tinea versicolor. The same sampling technique can be used blade. The removed scale is collected on a glass microscope slide to obtain scale for culture of selected pathogenic organisms. and then treated with 1 or 2 drops of a solution of 10–20% KOH. KOH dissolves keratin and allows easier visualization of fungal ele-Tzanck Smear A Tzanck smear is a cytologic technique most often ments. Brief heating of the slide accelerates dissolution of keratin. used in the diagnosis of herpesvirus infections (herpes simplex When the preparation is viewed under the microscope, the refractile virus [HSV] or varicella zoster virus [VZV]) (see Figs. 217-1 hyphae are seen

1	diagnosis of herpesvirus infections (herpes simplex When the preparation is viewed under the microscope, the refractile virus [HSV] or varicella zoster virus [VZV]) (see Figs. 217-1 hyphae are seen more easily when the light intensity is reduced and and 217-3). An early vesicle, not a pustule or crusted lesion, is the condenser is lowered. This technique can be used to identify unroofed, and the base of the lesion is scraped gently with a scalpel

1	PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 70-4 Necrotizing vasculitis. Palpable purpuric papules on the lower legs are seen in this patient with cutaneous small-vessel vasculitis. (Courtesy of Robert Swerlick, MD; with permission.) blade. The material is placed on a glass slide, air-dried, and stained with Giemsa or Wright’s stain. Multinucleated epithelial giant cells suggest the presence of HSV or VZV; culture, immunofluorescence microscopy, or genetic testing must be performed to identify the specific virus. FIguRE 70-5 Meningococcemia. An example of fulminant meningococcemia with extensive angular purpuric patches. (Courtesy of Stephen E. Gellis, MD; with permission.)

1	FIguRE 70-5 Meningococcemia. An example of fulminant meningococcemia with extensive angular purpuric patches. (Courtesy of Stephen E. Gellis, MD; with permission.) Diascopy Diascopy is designed to assess whether a skin lesion will blanch with pressure as, for example, in determining whether a red lesion is hemorrhagic or simply blood-filled. Urticaria (Fig. 70-11) will blanch with pressure, whereas a purpuric lesion caused by necrotizing vasculitis (Fig. 70-4) will not. Diascopy is performed by pressing a microscope slide or magnifying lens against a lesion and noting the amount of blanching that occurs. Granulomas often have an opaque to transparent, brown-pink “apple jelly” appearance on diascopy. Wood’s Light A Wood’s lamp generates 360-nm ultraviolet (“black”) light that can be used to aid the evaluation of certain skin disorders. FIguRE 70-6 Distribution of some common dermatologic diseases and lesions.

1	FIguRE 70-6 Distribution of some common dermatologic diseases and lesions. FIguRE 70-7 Psoriasis. This papulosquamous skin disease is charac-terized by small and large erythematous papules and plaques with overlying adherent silvery scale. FIguRE 70-10 Allergic contact dermatitis (ACD). A. An example of ACD in its acute phase, with sharply demarcated, weeping, eczema-tous plaques in a perioral distribution. B. ACD in its chronic phase, with an erythematous, lichenified, weeping plaque on skin chronically exposed to nickel in a metal snap. (B, Courtesy of Robert Swerlick, MD; with permission.) CHAPTER 70 Approach to the Patient with a Skin Disorder FIguRE 70-8 Dermatitis herpetiformis. This disorder typically displays pruritic, grouped papulovesicles on elbows, knees, buttocks, and posterior scalp. Vesicles are often excoriated due to associated pruritus.

1	For example, a Wood’s lamp will cause erythrasma (a superficial, intertriginous infection caused by Corynebacterium minutissimum) to show a characteristic coral pink color, and wounds colonized by Pseudomonas will appear pale blue. Tinea capitis caused by certain dermatophytes (e.g., Microsporum canis or M. audouinii) exhibits a yellow fluorescence. Pigmented lesions of the epidermis such as freckles are accentuated, while dermal pigment such as postinflammatory hyper-pigmentation fades under a Wood’s light. Vitiligo (Fig. 70-12) appears FIguRE 70-9 Erythema multiforme. This eruption is characterized by multiple erythematous plaques with a target or iris morphology. It usually represents a hypersensitivity reaction to drugs (e.g., sulfon-amides) or infections (e.g., HSV). (Courtesy of the Yale Resident’s Slide Collection; with permission.) FIguRE 70-11 Urticaria. Discrete and confluent, edematous, erythematous papules and plaques are characteristic of this whealing eruption.

1	FIguRE 70-11 Urticaria. Discrete and confluent, edematous, erythematous papules and plaques are characteristic of this whealing eruption. CLiniCAL fEATuRES of AToPiC DERMATiTiS FIguRE 70-12 Vitiligo. Characteristic lesions display an acral distribu-tion and striking depigmentation as a result of loss of melanocytes. totally white under a Wood’s lamp, and previously unsuspected areas of involvement often become apparent. A Wood’s lamp may also aid in the demonstration of tinea versicolor and in recognition of ash leaf spots in patients with tuberous sclerosis.

1	Patch Tests Patch testing is designed to document sensitivity to a specific antigen. In this procedure, a battery of suspected allergens is applied to the patient’s back under occlusive dressings and allowed to remain in contact with the skin for 48 h. The dressings are removed, and the area is examined for evidence of delayed hypersensitivity reactions (e.g., erythema, edema, or papulovesicles). This test is best performed by physicians with special expertise in patch testing and is often helpful in the evaluation of patients with chronic dermatitis. Eczema, Psoriasis, Cutaneous infections, Acne, and other Common Skin Disorders Leslie P. Lawley, Calvin O. McCall, Thomas J. Lawley ECZEMA AND DERMATITIS 71

1	Eczema, Psoriasis, Cutaneous infections, Acne, and other Common Skin Disorders Leslie P. Lawley, Calvin O. McCall, Thomas J. Lawley ECZEMA AND DERMATITIS 71 Eczema is a type of dermatitis, and these terms are often used synonymously (e.g., atopic eczema or atopic dermatitis [AD]). Eczema is a reaction pattern that presents with variable clinical findings and the common histologic finding of spongiosis (intercellular edema of the epidermis). Eczema is the final common expression for a number of disorders, including those discussed in the following sections. Primary lesions may include erythematous macules, papules, and vesicles, which can coalesce to form patches and plaques. In severe eczema, secondary lesions from infection or excoriation, marked by weeping and crusting, may predominate. In chronic eczematous conditions, lichenification (cutaneous hypertrophy and accentuation of normal skin markings) may alter the characteristic appearance of eczema.

1	AD is the cutaneous expression of the atopic state, characterized by a family history of asthma, allergic rhinitis, or eczema. The prevalence of AD is increasing worldwide. Some of its features are shown in Table 71-1. The etiology of AD is only partially defined, but there is a clear genetic predisposition. When both parents are affected by AD, >80% of their children manifest the disease. When only one parent is affected, the prevalence drops to slightly over 50%. A characteristic defect in AD that contributes to the pathophysiology is an PART 2 Cardinal Manifestations and Presentation of Diseases 1. 2. 3. Lesions typical of eczematous dermatitis 4. Personal or family history of atopy (asthma, allergic rhinitis, food allergies, or eczema) 5. 6.

1	2. 3. Lesions typical of eczematous dermatitis 4. Personal or family history of atopy (asthma, allergic rhinitis, food allergies, or eczema) 5. 6. Lichenification of skin impaired epidermal barrier. In many patients, a mutation in the gene encoding filaggrin, a structural protein in the stratum corneum, is responsible. Patients with AD may display a variety of immunoregulatory abnormalities, including increased IgE synthesis; increased serum IgE levels; and impaired, delayed-type hypersensitivity reactions.

1	The clinical presentation often varies with age. Half of patients with AD present within the first year of life, and 80% present by 5 years of age. About 80% ultimately coexpress allergic rhinitis or asthma. The infantile pattern is characterized by weeping inflammatory patches and crusted plaques on the face, neck, and extensor surfaces. The childhood and adolescent pattern is typified by dermatitis of flexural skin, particularly in the antecubital and popliteal fossae (Fig. 71-1). AD may resolve spontaneously, but approximately 40% of all individuals affected as children will have dermatitis in adult life. The distribution of lesions in adults may be similar to those seen in childhood; however, adults frequently have localized disease manifesting as lichen simplex chronicus or hand eczema (see below). In patients with localized disease, AD may be suspected because of a typical personal or family history or the presence of cutaneous stigmata of AD such as perioral pallor, an extra

1	(see below). In patients with localized disease, AD may be suspected because of a typical personal or family history or the presence of cutaneous stigmata of AD such as perioral pallor, an extra fold of skin beneath the lower eyelid (Dennie-Morgan folds), increased palmar skin markings, and an increased incidence of cutaneous infections, particularly with Staphylococcus aureus. Regardless of other manifestations, pruritus is a prominent characteristic of AD in all age groups and is exacerbated by dry skin. Many of the cutaneous findings in affected patients, such as lichenification, are secondary to rubbing and scratching.

1	Therapy for AD should include avoidance of cutaneous irritants, adequate moisturizing through the application of emollients, judicious use of topical anti-inflammatory agents, and prompt treatment of secondary infection. Patients should be instructed to bathe no more often than daily, using warm or cool water, and to use only mild bath soap. Immediately after bathing, while the skin is still moist, a topical anti-inflammatory agent in a cream or ointment base should be applied to areas of dermatitis, and all other skin areas should be lubricated with a moisturizer. Approximately 30 g of a topical agent is required to cover the entire body surface of an average adult. FIguRE 71-1 Atopic dermatitis. Hyperpigmentation, lichenification, and scaling in the antecubital fossae are seen in this patient with atopic dermatitis. (Courtesy of Robert Swerlick, MD; with permission.)

1	Lowto mid-potency topical glucocorticoids are employed in most treatment regimens for AD. Skin atrophy and the potential for systemic absorption are constant concerns, especially with more potent agents. Low-potency topical glucocorticoids or nonglucocorticoid anti-inflammatory agents should be selected for use on the face and in intertriginous areas to minimize the risk of skin atrophy. Two nonglucocorticoid anti-inflammatory agents are available: tacrolimus ointment and pimecrolimus cream. These agents are macrolide immunosuppressants that are approved by the U.S. Food and Drug Administration (FDA) for topical use in AD. Reports of broader effectiveness appear in the literature. These agents do not cause skin atrophy, nor do they suppress the hypothalamic-pituitary-adrenal axis. However, concerns have emerged regarding the potential for lymphomas in patients treated with these agents. Thus, caution should be exercised when these agents are considered. Currently, they are also more

1	concerns have emerged regarding the potential for lymphomas in patients treated with these agents. Thus, caution should be exercised when these agents are considered. Currently, they are also more costly than topical glucocorticoids. Barrier-repair products that attempt to restore the impaired epidermal barrier are also nonglucocorticoid agents and are gaining popularity in the treatment of AD.

1	Secondary infection of eczematous skin may lead to exacerbation of AD. Crusted and weeping skin lesions may be infected with S. aureus. When secondary infection is suspected, eczematous lesions should be cultured and patients treated with systemic antibiotics active against S. aureus. The initial use of penicillinase-resistant penicillins or cephalosporins is preferable. Dicloxacillin or cephalexin (250 mg qid for 7–10 days) is generally adequate for adults; however, antibiotic selection must be directed by culture results and clinical response. More than 50% of S. aureus isolates are now methicillin resistant in some communities. Current recommendations for the treatment of infection with these community-acquired methicillinresistant S. aureus (CA-MRSA) strains in adults include trimethoprimsulfamethoxazole (1 double-strength tablet bid), minocycline (100 mg bid), doxycycline (100 mg bid), or clindamycin (300–450 mg qid). Duration of therapy should be 7–10 days. Inducible resistance

1	(1 double-strength tablet bid), minocycline (100 mg bid), doxycycline (100 mg bid), or clindamycin (300–450 mg qid). Duration of therapy should be 7–10 days. Inducible resistance may limit clindamycin’s usefulness. Such resistance can be detected by the double-disk diffusion test, which should be ordered if the isolate is erythromycin resistant and clindamycin sensitive. As an adjunct, antibacterial washes or dilute sodium hypochlorite baths (0.005% bleach) and intermittent nasal mupirocin may be useful.

1	Control of pruritus is essential for treatment, because AD often represents “an itch that rashes.” Antihistamines are most often used to control pruritus. Diphenhydramine (25 mg every 4–6 h), hydroxyzine (10–25mg every 6 h), or doxepin (10–25 mg at bedtime) are useful primarily due to their sedating action. Higher doses of these agents may be required, but sedation can become bothersome. Patients need to be counseled about driving or operating heavy equipment after taking these medications. When used at bedtime, sedating antihistamines may improve the patient’s sleep. Although they are effective in urticaria, non-sedating antihistamines and selective H2 blockers are of little use in controlling the pruritus of AD.

1	Treatment with systemic glucocorticoids should be limited to severe exacerbations unresponsive to topical therapy. In the patient with chronic AD, therapy with systemic glucocorticoids will generally clear the skin only briefly, and cessation of the systemic therapy will invariably be accompanied by a return, if not a worsening, of the dermatitis. Patients who do not respond to conventional therapies should be considered for patch testing to rule out allergic contact dermatitis (ACD). The role of dietary allergens in AD is controversial, and there is little evidence that they play any role outside of infancy, during which a small percentage of patients with AD may be affected by food allergens.

1	Lichen simplex chronicus may represent the end stage of a variety of pruritic and eczematous disorders, including AD. It consists of a circumscribed plaque or plaques of lichenified skin due to chronic 345 scratching or rubbing. Common areas involved include the posterior nuchal region, dorsum of the feet, and ankles. Treatment of lichen simplex chronicus centers on breaking the cycle of chronic itching and scratching. High-potency topical glucocorticoids are helpful in most cases, but, in recalcitrant cases, application of topical glucocorticoids under occlusion, or intralesional injection of glucocorticoids may be required.

1	Contact dermatitis is an inflammatory skin process caused by an exogenous agent or agents that directly or indirectly injure the skin. In irritant contact dermatitis (ICD), this injury is caused by an inherent characteristic of a compound—for example, a concentrated acid or base. Agents that cause ACD induce an antigen-specific immune response (e.g., poison ivy dermatitis). The clinical lesions of contact dermatitis may be acute (wet and edematous) or chronic (dry, thickened, and scaly), depending on the persistence of the insult (see Fig. 70-10).

1	Irritant Contact Dermatitis ICD is generally well demarcated and often localized to areas of thin skin (eyelids, intertriginous areas) or to areas where the irritant was occluded. Lesions may range from minimal skin erythema to areas of marked edema, vesicles, and ulcers. Prior exposure to the offending agent is not necessary, and the reaction develops in minutes to a few hours. Chronic low-grade irritant dermatitis is the most common type of ICD, and the most common area of involvement is the hands (see below). The most common irritants encountered are chronic wet work, soaps, and detergents. Treatment should be directed toward the avoidance of irritants and the use of protective gloves or clothing.

1	Allergic Contact Dermatitis ACD is a manifestation of delayed-type hypersensitivity mediated by memory T lymphocytes in the skin. Prior exposure to the offending agent is necessary to develop the hypersensitivity reaction, which may take as little as 12 h or as much as 72 h to develop. The most common cause of ACD is exposure to plants, especially to members of the family Anacardiaceae, including the genus Toxicodendron. Poison ivy, poison oak, and poison sumac are members of this genus and cause an allergic reaction marked by erythema, vesiculation, and severe pruritus. The eruption is often linear or angular, corresponding to areas where plants have touched the skin. The sensitizing antigen common to these plants is urushiol, an oleoresin containing the active ingredient pentadecylcatechol. The oleoresin may adhere to skin, clothing, tools, and pets, and contaminated articles may cause dermatitis even after prolonged storage. Blister fluid does not contain urushiol and is not

1	The oleoresin may adhere to skin, clothing, tools, and pets, and contaminated articles may cause dermatitis even after prolonged storage. Blister fluid does not contain urushiol and is not capable of inducing skin eruption in exposed subjects.

1	If contact dermatitis is suspected and an offending agent is identified and removed, the eruption will resolve. Usually, treatment with high-potency topical glucocorticoids is enough to relieve symptoms while the dermatitis runs its course. For those patients who require systemic therapy, daily oral prednisone—beginning at 1 mg/kg, but usually ≤60 mg/d—is sufficient. The dose should be tapered over 2–3 weeks, and each daily dose should be taken in the morning with food.

1	Identification of a contact allergen can be a difficult and time-consuming task. Allergic contact dermatitis should be suspected in patients with dermatitis unresponsive to conventional therapy or with an unusual and patterned distribution. Patients should be questioned carefully regarding occupational exposures and topical medications. Common sensitizers include preservatives in topical preparations, nickel sulfate, potassium dichromate, thimerosal, neomycin sulfate, fragrances, formaldehyde, and rubber-curing agents. Patch testing is helpful in identifying these agents but should not be attempted when patients have widespread active dermatitis or are taking systemic glucocorticoids. CHAPTER 71 Eczema, Psoriasis, Cutaneous Infections, Acne, and Other Common Skin Disorders PART 2 Cardinal Manifestations and Presentation of Diseases

1	CHAPTER 71 Eczema, Psoriasis, Cutaneous Infections, Acne, and Other Common Skin Disorders PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 71-2 Dyshidrotic eczema. This example is characterized by deep-seated vesicles and scaling on palms and lateral fingers, and the disease is often associated with an atopic diathesis.

1	Hand eczema is a very common, chronic skin disorder in which both exogenous and endogenous factors play important roles. It may be associated with other cutaneous disorders such as AD, and contact with various agents may be involved. Hand eczema represents a large proportion of cases of occupation-associated skin disease. Chronic, excessive exposure to water and detergents, harsh chemicals, or allergens may initiate or aggravate this disorder. It may present with dryness and cracking of the skin of the hands as well as with variable amounts of erythema and edema. Often, the dermatitis will begin under rings, where water and irritants are trapped. Dyshidrotic eczema, a variant of hand eczema, presents with multiple, intensely pruritic, small papules and vesicles on the thenar and hypothenar eminences and the sides of the fingers (Fig. 71-2). Lesions tend to occur in crops that slowly form crusts and then heal.

1	The evaluation of a patient with hand eczema should include an assessment of potential occupation-associated exposures. The history should be directed to identifying possible irritant or allergen exposures. Therapy for hand eczema is directed toward avoidance of irritants, identification of possible contact allergens, treatment of coexistent infection, and application of topical glucocorticoids. Whenever possible, the hands should be protected by gloves, preferably vinyl. The use of rubber gloves (latex) to protect dermatitic skin is sometimes associated with the development of hypersensitivity reactions to components of the gloves. Patients can be treated with cool moist compresses followed by application of a midto high-potency topical glucocorticoid in a cream or ointment base. As in AD, treatment of secondary infection is essential for good control. In addition, patients with hand eczema should be examined for dermatophyte infection by KOH preparation and culture (see below).

1	Nummular eczema is characterized by circular or oval “coinlike” lesions, beginning as small edematous papules that become crusted and scaly. The etiology of nummular eczema is unknown, but dry skin is a contributing factor. Common locations are the trunk or the extensor surfaces of the extremities, particularly on the pretibial areas or dorsum of the hands. Nummular eczema occurs more frequently in men and is most common in middle age. The treatment of nummular eczema is similar to that for AD.

1	Asteatotic eczema, also known as xerotic eczema or “winter itch,” is a mildly inflammatory dermatitis that develops in areas of extremely dry skin, especially during the dry winter months. Clinically, there may be considerable overlap with nummular eczema. This form of eczema accounts for a large number of physician visits because of the associated pruritus. Fine cracks and scale, with or without erythema, characteristically develop in areas of dry skin, especially on the anterior surfaces of the lower extremities in elderly patients. Asteatotic eczema responds well to topical moisturizers and the avoidance of cutaneous irritants. Overbathing and the use of harsh soaps exacerbate asteatotic eczema.

1	Stasis dermatitis develops on the lower extremities secondary to venous incompetence and chronic edema. Patients may give a history of deep venous thrombosis and may have evidence of vein removal or varicose veins. Early findings in stasis dermatitis consist of mild erythema and scaling associated with pruritus. The typical initial site of involvement is the medial aspect of the ankle, often over a distended vein (Fig. 71-3).

1	Stasis dermatitis may become acutely inflamed, with crusting and exudate. In this state, it is easily confused with cellulitis. Chronic stasis dermatitis is often associated with dermal fibrosis that is recognized clinically as brawny edema of the skin. As the disorder progresses, the dermatitis becomes progressively pigmented due to chronic erythrocyte extravasation leading to cutaneous hemosiderin deposition. Stasis dermatitis may be complicated by secondary infection and contact dermatitis. Severe stasis dermatitis may precede the development of stasis ulcers.

1	Patients with stasis dermatitis and stasis ulceration benefit greatly from leg elevation and the routine use of compression stockings with a gradient of at least 30–40 mmHg. Stockings providing less compression, such as antiembolism hose, are poor substitutes. Use of emollients and/or mid-potency topical glucocorticoids and avoidance of irritants are also helpful in treating stasis dermatitis. Protection of the legs from injury, including scratching, and control of chronic edema are essential to prevent ulcers. Diuretics may be required to adequately control chronic edema.

1	Stasis ulcers are difficult to treat, and resolution is slow. It is extremely important to elevate the affected limb as much as possible. The ulcer should be kept clear of necrotic material by gentle debridement and covered with a semipermeable dressing and a compression dressing or compression stocking. Glucocorticoids should not be applied to ulcers, because they may retard healing; however, they may be applied to the surrounding skin to control itching, scratching, and additional trauma. Secondarily infected lesions should be treated with appropriate oral antibiotics, but it should be noted that all ulcers will become colonized with bacteria, and the purpose of antibiotic therapy should not be to clear all bacterial growth. Care must be taken to exclude treatable causes of leg ulcers (hypercoagulation, vasculitis) before beginning the chronic management outlined above.

1	FIguRE 71-3 Stasis dermatitis. An example of stasis dermatitis showing erythematous, scaly, and oozing patches over the lower leg. Several stasis ulcers are also seen in this patient. FIguRE 71-4 Seborrheic dermatitis. Central facial erythema with overlying greasy, yellowish scale is seen in this patient. (Courtesy of Jean Bolognia, MD; with permission.)

1	FIguRE 71-4 Seborrheic dermatitis. Central facial erythema with overlying greasy, yellowish scale is seen in this patient. (Courtesy of Jean Bolognia, MD; with permission.) Seborrheic dermatitis is a common, chronic disorder characterized by greasy scales overlying erythematous patches or plaques. Induration and scale are generally less prominent than in psoriasis, but clinical overlap exists between these diseases (“sebopsoriasis”). The most common location is in the scalp, where it may be recognized as severe dandruff. On the face, seborrheic dermatitis affects the eyebrows, eyelids, glabella, and nasolabial folds (Fig. 71-4). Scaling of the external auditory canal is common in seborrheic dermatitis. In addition, the postauricular areas often become macerated and tender. Seborrheic dermatitis may also develop in the central chest, axilla, groin, submammary folds, and gluteal cleft. Rarely, it may cause widespread generalized dermatitis. Pruritus is variable.

1	Seborrheic dermatitis may be evident within the first few weeks of life, and within this context it typically occurs in the scalp (“cradle cap”), face, or groin. It is rarely seen in children beyond infancy but becomes evident again during adult life. Although it is frequently seen in patients with Parkinson’s disease, in those who have had cerebrovascular accidents, and in those with HIV infection, the overwhelming majority of individuals with seborrheic dermatitis have no underlying disorder.

1	Treatment with low-potency topical glucocorticoids in conjunction with a topical antifungal agent, such as ketoconazole cream or ciclopirox cream, is often effective. The scalp and beard areas may benefit from antidandruff shampoos, which should be left in place 3–5 min before rinsing. High-potency topical glucocorticoid solutions (betamethasone or clobetasol) are effective for control of severe scalp involvement. High-potency glucocorticoids should not be used on the face because this treatment is often associated with steroid-induced rosacea or atrophy.

1	Psoriasis is one of the most common dermatologic diseases, affecting up to 2% of the world’s population. It is an immune-mediated disease clinically characterized by erythematous, sharply demarcated papules and rounded plaques covered by silvery micaceous scale. The skin lesions of psoriasis are variably pruritic. Traumatized areas often develop lesions of psoriasis (the Koebner or isomorphic phenomenon). In addition, other external factors may exacerbate psoriasis, including infections, stress, and medications (lithium, beta blockers, and antimalarial drugs).

1	The most common variety of psoriasis is called plaque-type. Patients with plaque-type psoriasis have stable, slowly enlarging plaques, which remain basically unchanged for long periods of time. The most commonly involved areas are the elbows, knees, gluteal cleft, and scalp. Involvement tends to be symmetric. Plaque psoriasis generally develops slowly and runs an indolent course. It rarely remits spontaneously. Inverse psoriasis affects the intertriginous regions, including the axilla, groin, submammary region, and navel; it also tends to affect the scalp, palms, and soles. The individual lesions are sharply demarcated plaques (see Fig. 70-7), but they may be moist and without scale due to their locations.

1	Guttate psoriasis (eruptive psoriasis) is most common in children and young adults. It develops acutely in individuals without psoriasis or in those with chronic plaque psoriasis. Patients present with many small erythematous, scaling papules, frequently after upper respiratory tract infection with β-hemolytic streptococci. The differential diagnosis should include pityriasis rosea and secondary syphilis.

1	In pustular psoriasis, patients may have disease localized to the palms and soles, or the disease may be generalized. Regardless of the extent of disease, the skin is erythematous, with pustules and variable scale. Localized to the palms and soles, it is easily confused with eczema. When it is generalized, episodes are characterized by fever (39°–40°C [102.2°–104.0°F]) lasting several days, an accompanying generalized eruption of sterile pustules, and a background of intense erythema; patients may become erythrodermic. Episodes of fever and pustules are recurrent. Local irritants, pregnancy, medications, infections, and systemic glucocorticoid withdrawal can precipitate this form of psoriasis. Oral retinoids are the treatment of choice in nonpregnant patients. CHAPTER 71 Eczema, Psoriasis, Cutaneous Infections, Acne, and Other Common Skin Disorders

1	CHAPTER 71 Eczema, Psoriasis, Cutaneous Infections, Acne, and Other Common Skin Disorders Psoriasis Sharply demarcated, erythematous plaques with mica-like scale; predominantly on elbows, knees, and scalp; atypical forms may localize to intertriginous areas; eruptive forms may be associated with infection Lichen planus Purple polygonal papules marked by severe pruritus; lacy white markings, especially associated with mucous membrane lesions Pityriasis rosea Rash often preceded by herald patch; oval to round plaques with trailing scale; most often affects trunk; eruption lines up in skinfolds giving a “fir tree–like” appearance; generally spares palms and soles Dermatophytosis Polymorphous appearance depending on dermatophyte, body site, and host response; sharply defined to ill-demarcated scaly plaques with or without inflammation; may be associated with hair loss May be aggravated by certain drugs, infection; severe forms seen in association with HIV

1	Certain drugs may induce: thiazides, antimalarial drugs Variable pruritus; self-limited, resolving in 2–8 weeks; may be imitated by secondary syphilis KOH preparation may show branching hyphae; culture helpful Acanthosis, vascular proliferation PART 2 Cardinal Manifestations and Presentation of Diseases Fingernail involvement, appearing as punctate pitting, onycholysis, nail thickening, or subungual hyperkeratosis, may be a clue to the diagnosis of psoriasis when the clinical presentation is not classic.

1	According to the National Psoriasis Foundation, up to 30% of patients with psoriasis have psoriatic arthritis (PsA). There are five subtypes of PsA: symmetric, asymmetric, distal interphalangeal predominant (DIP), spondylitis, and arthritis mutilans. Symmetric arthritis resembles rheumatoid arthritis, but is usually milder. Asymmetric arthritis can involve any joint and may present as “sausage digits.” DIP is the classic form, but occurs in only about 5% of patients with PsA. It may involve fingers and toes. Spondylitis also occurs in about 5% of patients with PsA. Arthritis mutilans is severe and deforming. It affects primarily the small joints of the hands and feet. It accounts for fewer than 5% of PsA cases.

1	An increased risk of metabolic syndrome, including increased morbidity and mortality from cardiovascular events, has been demonstrated in psoriasis patients. Appropriate screening tests should be performed. The etiology of psoriasis is still poorly understood, but there is clearly a genetic component to the disease. In various studies, 30–50% of patients with psoriasis report a positive family history. Psoriatic lesions contain infiltrates of activated T cells that are thought to elaborate cytokines responsible for keratinocyte hyperproliferation, which results in the characteristic clinical findings. Agents inhibiting T cell activation, clonal expansion, or release of proinflammatory cytokines are often effective for the treatment of severe psoriasis (see below).

1	Treatment of psoriasis depends on the type, location, and extent of disease. All patients should be instructed to avoid excess drying or irritation of their skin and to maintain adequate cutaneous hydration. Most cases of localized, plaque-type psoriasis can be managed with mid-potency topical glucocorticoids, although their long-term use is often accompanied by loss of effectiveness (tachyphylaxis) and atrophy of the skin. A topical vitamin D analogue (calcipotriene) and a retinoid (tazarotene) are also efficacious in the treatment of limited psoriasis and have largely replaced other topical agents such as coal tar, salicylic acid, and anthralin.

1	Ultraviolet (UV) light, natural or artificial, is an effective therapy for many patients with widespread psoriasis. Ultraviolet B (UVB), narrowband UVB, and ultraviolet A (UVA) light with either oral or topical psoralens (PUVA) is used clinically. UV light’s immunosuppressive properties are thought to be responsible for its therapeutic activity in psoriasis. It is also mutagenic, potentially leading to an increased incidence of nonmelanoma and melanoma skin cancer. UV-light therapy is contraindicated in patients receiving cyclosporine and should be used with great care in all immunocompromised patients due to the increased risk of skin cancer.

1	Various systemic agents can be used for severe, widespread psoriatic disease (Table 71-3). Oral glucocorticoids should not be used for the treatment of psoriasis due to the potential for development of life-threatening pustular psoriasis when therapy is discontinued. Methotrexate is an effective agent, especially in patients with psoriatic arthritis. The synthetic retinoid acitretin is useful, especially when immunosuppression must be avoided; however, teratogenicity limits its use.

1	The evidence implicating psoriasis as a T cell–mediated disorder has directed therapeutic efforts to immunoregulation. Cyclosporine and other immunosuppressive agents can be very effective in the treatment of psoriasis, and much attention is currently directed toward the development of biologic agents with more selective immunosuppressive properties and better safety profiles (Table 71-4). Experience with these biologic agents is limited, and information regarding combination therapy and adverse events continues to emerge. Use of tumor necrosis factor (TNF-α) inhibitors may worsen congestive heart failure (CHF), and they should be used with caution in patients at risk for or known to have CHF. Further, none of the immunosuppressive agents used in the treatment of psoriasis should be initiated if the patient has a severe infection; patients on such therapy should be routinely screened for tuberculosis. There have been reports of progressive multifocal leukoencephalopathy in association

1	if the patient has a severe infection; patients on such therapy should be routinely screened for tuberculosis. There have been reports of progressive multifocal leukoencephalopathy in association with treatment with the TNF-α inhibitors. Malignancies, including a risk or history of certain malignancies, may limit the use of these systemic agents.

1	Administration Agent Mechanism of Action Indication Route Frequency Warnings Abbreviations: CHF, congestive heart failure; IL, interleukin; IM, intramuscular; Ps, psoriasis; PsA, psoriatic arthritis; SC, subcutaneous; TNF, tumor necrosis factor. FIguRE 71-5 Lichen planus. An example of lichen planus showing multiple flat-topped, violaceous papules and plaques. Nail dystrophy, as seen in this patient’s thumbnail, may also be a feature. (Courtesy of Robert Swerlick, MD; with permission.)

1	Lichen planus (LP) is a papulosquamous disorder that may affect the skin, scalp, nails, and mucous membranes. The primary cutaneous lesions are pruritic, polygonal, flat-topped, violaceous papules. Close examination of the surface of these papules often reveals a network of gray lines (Wickham’s striae). The skin lesions may occur anywhere but have a predilection for the wrists, shins, lower back, and genitalia (Fig. 71-5). Involvement of the scalp (lichen planopilaris) may lead to scarring alopecia, and nail involvement may lead to permanent deformity or loss of fingernails and toenails. LP commonly involves mucous membranes, particularly the buccal mucosa, where it can present on a spectrum ranging from a mild, white, reticulate eruption of the mucosa to a severe, erosive stomatitis. Erosive stomatitis may persist for years and may be linked to an increased risk of oral squamous cell carcinoma. Cutaneous eruptions clinically resembling LP have been observed after administration of

1	stomatitis may persist for years and may be linked to an increased risk of oral squamous cell carcinoma. Cutaneous eruptions clinically resembling LP have been observed after administration of numerous drugs, including thiazide diuretics, gold, antimalarial agents, penicillamine, and phenothiazines, and in patients with skin lesions of chronic graft-versus-host disease. In addition, LP may be associated with hepatitis C infection. The course of LP is variable, but most patients have spontaneous remissions 6 months to 2 years after the onset of disease. Topical glucocorticoids are the mainstay of therapy.

1	Pityriasis rosea (PR) is a papulosquamous eruption of unknown etiology occurring more commonly in the spring and fall. Its first manifestation is the development of a 2to 6-cm annular lesion (the herald patch). This is followed in a few days to a few weeks by the appearance of many smaller annular or papular lesions with a predilection to occur on the trunk (Fig. 71-6). The lesions are generally oval, with their long axis parallel to the skinfold lines. Individual lesions may range in color from red to brown and have a trailing scale. PR shares many clinical features with the eruption of secondary syphilis, but palm and sole lesions are extremely rare in PR and common in secondary syphilis. The eruption tends to be moderately pruritic and lasts 3–8 weeks. Treatment is directed at alleviating pruritus and consists of oral antihistamines; mid-potency topical glucocorticoids; and, in some cases, UVB phototherapy. IMPETIgO, ECTHYMA, AND FuRuNCuLOSIS

1	IMPETIgO, ECTHYMA, AND FuRuNCuLOSIS Impetigo is a common superficial bacterial infection of skin caused most often by S. aureus (Chap. 172) and in some cases by group A β-hemolytic streptococci (Chap. 173). The primary lesion is a superficial pustule that ruptures and forms a characteristic yellow-brown FIguRE 71-6 Pityriasis rosea. In this patient with pityriasis rosea, multiple round to oval erythematous patches with fine central scale are distributed along the skin tension lines on the trunk.

1	honey-colored crust (see Fig. 173-3). Lesions may occur on normal skin (primary infection) or in areas already affected by another skin disease (secondary infection). Lesions caused by staphylococci may be tense, clear bullae, and this less common form of the disease is called bullous impetigo. Blisters are caused by the production of exfoliative toxin by S. aureus phage type II. This is the same toxin responsible for staphylococcal scalded-skin syndrome, often resulting in dramatic loss of the superficial epidermis due to blistering. The latter syndrome is much more common in children than in adults; however, it should be considered along with toxic epidermal necrolysis and severe drug eruptions in patients with widespread blistering of the skin. Ecthyma is a deep non-bullous variant of impetigo that causes punched-out ulcerative lesions. It is more often caused by a primary or secondary infection with Streptococcus pyogenes. Ecthyma is a deeper infection than typical impetigo and

1	impetigo that causes punched-out ulcerative lesions. It is more often caused by a primary or secondary infection with Streptococcus pyogenes. Ecthyma is a deeper infection than typical impetigo and resolves with scars. Treatment of both ecthyma and impetigo involves gentle debridement of adherent crusts, which is facilitated by the use of soaks and topical antibiotics in conjunction with appropriate oral antibiotics.

1	Furunculosis is also caused by S. aureus, and this disorder has gained prominence in the last decade because of CA-MRSA. A furuncle, or boil, is a painful, erythematous nodule that can occur on any cutaneous surface. The lesions may be solitary but are most often multiple. Patients frequently believe they have been bitten by spiders or insects. Family members or close contacts may also be affected. Furuncles can rupture and drain spontaneously or may need incision and drainage, which may be adequate therapy for small solitary furuncles without cellulitis or systemic symptoms. Whenever possible, lesional material should be sent for culture. Current recommendations for methicillinsensitive infections are β-lactam antibiotics. Therapy for CA-MRSA was discussed previously (see “Atopic Dermatitis”). Warm compresses and nasal mupirocin are helpful therapeutic additions. Severe infections may require IV antibiotics. See Chap. 156.

1	See Chap. 156. Dermatophytes are fungi that infect skin, hair, and nails and include members of the genera Trichophyton, Microsporum, and Epidermophyton (Chap. 243). Tinea corporis, or infection of the relatively hairless skin of the body (glabrous skin), may have a variable appearance depending on the extent of the associated inflammatory reaction. Typical infections consist of erythematous, scaly plaques, with an annular appearance that accounts for the common name “ringworm.” Deep inflammatory nodules or granulomas occur in CHAPTER 71 Eczema, Psoriasis, Cutaneous Infections, Acne, and Other Common Skin Disorders

1	PART 2 Cardinal Manifestations and Presentation of Diseases some infections, most often those inappropriately treated with midto high-potency topical glucocorticoids. Involvement of the groin (tinea cruris) is more common in males than in females. It presents as a scaling, erythematous eruption sparing the scrotum. Infection of the foot (tinea pedis) is the most common dermatophyte infection and is often chronic; it is characterized by variable erythema, edema, scaling, pruritus, and occasionally vesiculation. The infection may be widespread or localized but generally involves the web space between the fourth and fifth toes. Infection of the nails (tinea unguium or onychomycosis) occurs in many patients with tinea pedis and is characterized by opacified, thickened nails and subungual debris. The distal-lateral variant is most common. Proximal subungual onychomycosis may be a marker for HIV infection or other immunocompromised states. Dermatophyte infection of the scalp (tinea capitis)

1	The distal-lateral variant is most common. Proximal subungual onychomycosis may be a marker for HIV infection or other immunocompromised states. Dermatophyte infection of the scalp (tinea capitis) continues to be common, particularly affecting inner-city children but also affecting adults. The predominant organism is Trichophyton tonsurans, which can produce a relatively noninflammatory infection with mild scale and hair loss that is diffuse or localized. T. tonsurans can also cause a markedly inflammatory dermatosis with edema and nodules. This latter presentation is a kerion.

1	The diagnosis of tinea can be made from skin scrapings, nail scrapings, or hair by culture or direct microscopic examination with potassium hydroxide (KOH). Nail clippings may be sent for histologic examination with periodic acid–Schiff (PAS) stain.

1	Both topical and systemic therapies may be used in dermatophyte infections. Treatment depends on the site involved and the type of infection. Topical therapy is generally effective for uncomplicated tinea corporis, tinea cruris, and limited tinea pedis. Topical agents are not effective as monotherapy for tinea capitis or onychomycosis (see below). Topical imidazoles, triazoles, and allylamines may be effective therapies for dermatophyte infections, but nystatin is not active against dermatophytes. Topicals are generally applied twice daily, and treatment should continue for 1 week beyond clinical resolution of the infection. Tinea pedis often requires longer treatment courses and frequently relapses. Oral antifungal agents may be required for recalcitrant tinea pedis or tinea corporis.

1	Oral antifungal agents are required for dermatophyte infections involving the hair and nails and for other infections unresponsive to topical therapy. A fungal etiology should be confirmed by direct microscopic examination or by culture before oral antifungal agents are prescribed. All of the oral agents may cause hepatotoxicity. They should not be used in women who are pregnant or breast-feeding.

1	Griseofulvin is approved in the United States for dermatophyte infections involving the skin, hair, or nails. When griseofulvin is used, a daily dose of 500 mg microsized or 375 mg ultramicrosized, administered with a fatty meal, is adequate for most dermatophyte infections. Higher doses are required for some cases of tinea pedis and tinea capitis. Markedly inflammatory tinea capitis may result in scarring and hair loss, and systemic or topical glucocorticoids may be helpful in preventing these sequelae. The duration of griseofulvin therapy may be 2 weeks for uncomplicated tinea corporis, 8–12 weeks for tinea capitis, or as long as 6–18 months for nail infections. Due to high relapse rates, griseofulvin is seldom used for nail infections. Common side effects of griseofulvin include gastrointestinal distress, headache, and urticaria.

1	Oral itraconazole is approved for onychomycosis. Itraconazole is given with food as either continuous daily therapy (200 mg/d) or pulses (200 mg bid for 1 week per month). Fingernails require 2 months of continuous therapy or two pulses. Toenails require 3 months of continuous therapy or three pulses. Itraconazole has the potential for serious interactions with other drugs requiring the P450 enzyme system for metabolism. Itraconazole should not be administered to patients with evidence of ventricular dysfunction or patients with known CHF. Terbinafine (250 mg/d) is also effective for onychomycosis, and the granule version is approved for treatment of tinea capitis. Therapy with terbinafine is continued for 6 weeks for fingernail and scalp infections and 12 weeks for toenail infections. Terbinafine has fewer interactions with other drugs than itraconazole, but caution should be used with patients who are on multiple medications. The risk/benefit ratio should be considered when an

1	Terbinafine has fewer interactions with other drugs than itraconazole, but caution should be used with patients who are on multiple medications. The risk/benefit ratio should be considered when an asymptomatic toenail infection is treated with systemic agents.

1	Tinea versicolor is caused by a nondermatophytic, dimorphic fungus, Malassezia furfur, a normal inhabitant of the skin. The expression of infection is promoted by heat and humidity. The typical lesions consist of oval scaly macules, papules, and patches concentrated on the chest, shoulders, and back but only rarely on the face or distal extremities. On dark skin the lesions often appear as hypopigmented areas, while on light skin they are slightly erythematous or hyperpigmented. A KOH preparation from scaling lesions will demonstrate a confluence of short hyphae and round spores (“spaghetti and meatballs”). Lotions or shampoos containing sulfur, salicylic acid, or selenium sulfide will clear the infection if used daily for 1–2 weeks and then weekly thereafter. These preparations are irritating if left on the skin for >10 min; thus, they should be washed off completely. Treatment with some oral antifungal agents is also effective, but they do not provide lasting results and are not FDA

1	if left on the skin for >10 min; thus, they should be washed off completely. Treatment with some oral antifungal agents is also effective, but they do not provide lasting results and are not FDA approved for this indication. A very short course of ketoconazole has been used, as have itraconazole and fluconazole. The patient must sweat after taking the medication if it is to be effective. Griseofulvin is not effective and terbinafine is not reliably effective for tinea versicolor.

1	Candidiasis is a fungal infection caused by a related group of yeasts whose manifestations may be localized to the skin and mucous membranes or, rarely, may be systemic and life-threatening (Chap. 240). The causative organism is usually Candida albicans. These organisms are normal saprophytic inhabitants of the gastrointestinal tract but may overgrow due to broad-spectrum antibiotic therapy, diabetes mellitus, or immunosuppression and cause disease. Candidiasis is a oral cavity is commonly involved. Lesions may occur on the tongue or very common infection in HIV-infected individuals (Chap. 226). The buccal mucosa (thrush) and appear as white plaques. Fissured, macerated lesions at the corners of the mouth (perléche) are often seen in individuals with poorly fitting dentures and may also be associated with candidal infection. In addition, candidal infections have an affinity for sites that are chronically wet and macerated, including the skin around nails (onycholysis and paronychia),

1	associated with candidal infection. In addition, candidal infections have an affinity for sites that are chronically wet and macerated, including the skin around nails (onycholysis and paronychia), and in intertriginous areas. Intertriginous lesions are characteristically edematous, erythematous, and scaly, with scattered “satellite pustules.” In males, there is often involvement of the penis and scrotum as well as the inner aspect of the thighs. In contrast to dermatophyte infections, candidal infections are frequently painful and accompanied by a marked inflammatory response. Diagnosis of candidal infection is based upon the clinical pattern and demonstration of yeast on KOH preparation or culture.

1	Treatment involves removal of any predisposing factors such as antibiotic therapy or chronic wetness and the use of appropriate topical or systemic antifungal agents. Effective topicals include nystatin or azoles (miconazole, clotrimazole, econazole, or ketoconazole). The associated inflammatory response accompanying candidal infection on glabrous skin can be treated with a mild glucocorticoid lotion or cream (2.5% hydrocortisone). Systemic therapy is usually reserved for immunosuppressed patients or individuals with chronic or recurrent disease who fail to respond to appropriate topical therapy. Oral agents approved for the treatment of candidiasis include itraconazole and fluconazole. Oral nystatin is effective only for candidiasis of the gastrointestinal tract. Griseofulvin and terbinafine are not effective.

1	Warts are cutaneous neoplasms caused by papillomaviruses. More than 100 different human papillomaviruses (HPVs) have been described. A typical wart, verruca vulgaris, is sessile, dome-shaped, and usually about a centimeter in diameter. Its surface is hyperkeratotic, consisting of many small filamentous projections. The HPV types that cause typical verruca vulgaris also cause typical plantar warts, flat warts (verruca plana), and filiform warts. Plantar warts are endophytic and are covered by thick keratin. Paring of the wart will generally reveal a central core of keratinized debris and punctate bleeding points. Filiform warts are most commonly seen on the face, neck, and skinfolds and present as papillomatous lesions on a narrow base. Flat warts are only slightly elevated and have a velvety, nonverrucous surface. They have a propensity for the face, arms, and legs and are often spread by shaving.

1	Genital warts begin as small papillomas that may grow to form large, fungating lesions. In women, they may involve the labia, perineum, or perianal skin. In addition, the mucosa of the vagina, urethra, and anus can be involved as well as the cervical epithelium. In men, the lesions often occur initially in the coronal sulcus but may be seen on the shaft of the penis, the scrotum, or the perianal skin or in the urethra.

1	Appreciable evidence has accumulated indicating that HPV plays a role in the development of neoplasia of the uterine cervix and anogenital skin (Chap. 117). HPV types 16 and 18 have been most intensely studied and are the major risk factors for intraepithelial neoplasia and squamous cell carcinoma of the cervix, anus, vulva, and penis. The risk is higher among patients immunosuppressed after solid organ transplantation and among those infected with HIV. Recent evidence also implicates other HPV types. Histologic examination of biopsied samples from affected sites may reveal changes associated with typical warts and/or features typical of intraepidermal carcinoma (Bowen’s disease). Squamous cell carcinomas associated with HPV infections have also been observed in extragenital skin (Chap. 105), most commonly in patients immunosuppressed after organ transplantation. Patients on long-term immunosuppression should be monitored for the development of squamous cell carcinoma and other

1	105), most commonly in patients immunosuppressed after organ transplantation. Patients on long-term immunosuppression should be monitored for the development of squamous cell carcinoma and other cutaneous malignancies.

1	Treatment of warts, other than anogenital warts, should be tempered by the observation that a majority of warts in normal individuals resolve spontaneously within 1–2 years. There are many modalities available to treat warts, but no single therapy is universally effective. Factors that influence the choice of therapy include the location of the wart, the extent of disease, the age and immunologic status of the patient, and the patient’s desire for therapy. Perhaps the most useful and convenient method for treating warts in almost any location is cryotherapy with liquid nitrogen. Equally effective for nongenital warts, but requiring much more patient compliance, is the use of keratolytic agents such as salicylic acid plasters or solutions. For genital warts, in-office application of a podophyllin solution is moderately effective but may be associated with marked local reactions. Prescription preparations of dilute, purified podophyllin are available for home use. Topical imiquimod, a

1	solution is moderately effective but may be associated with marked local reactions. Prescription preparations of dilute, purified podophyllin are available for home use. Topical imiquimod, a potent inducer of local cytokine release, has been approved for treatment of genital warts. A new topical compound composed of green tea extracts (sinecatechins) is also available. Conventional and laser surgical procedures may be required for recalcitrant warts. Recurrence of warts appears to be common to all these modalities. A highly effective vaccine for selected types of HPV has been approved by the FDA, and its use appears to reduce the incidence of anogenital and cervical carcinoma.

1	See Chap. 216. See Chap. 217. Acne vulgaris is a self-limited disorder primarily of teenagers and young adults, although perhaps 10–20% of adults may continue to experience some form of the disorder. The permissive factor for the expression of the disease in adolescence is the increase in sebum production by sebaceous glands after puberty. Small cysts, called comedones, form in hair follicles due to blockage of the follicular orifice by retention of keratinous material and sebum. The activity of bacteria (Propionibacterium acnes) within the comedones releases free fatty acids from sebum, causes inflammation within the cyst, and results in rupture of the cyst wall. An inflammatory foreign-body reaction develops as result of extrusion of oily and keratinous debris from the cyst.

1	The clinical hallmark of acne vulgaris is the comedone, which may be closed (whitehead) or open (blackhead). Closed comedones appear as 1to 2-mm pebbly white papules, which are accentuated when the skin is stretched. They are the precursors of inflammatory lesions of acne vulgaris. The contents of closed comedones are not easily expressed. Open comedones, which rarely result in inflammatory acne lesions, have a large dilated follicular orifice and are filled with easily expressible oxidized, darkened, oily debris. Comedones are usually accompanied by inflammatory lesions: papules, pustules, or nodules.

1	The earliest lesions seen in adolescence are generally mildly inflamed or noninflammatory comedones on the forehead. Subsequently, more typical inflammatory lesions develop on the cheeks, nose, and chin (Fig. 71-7). The most common location for acne is the face, but involvement of the chest and back is common. Most disease remains mild and does not lead to scarring. A small number of patients develop large inflammatory cysts and nodules, which may drain and result in significant scarring. Regardless of the severity, acne may affect a patient’s quality of life. With adequate treatment, this effect may be transient. In the case of severe, scarring acne, the effects can be permanent and profound. Early therapeutic intervention in severe acne is essential. Exogenous and endogenous factors can alter the expression of acne vulgaris. Friction and trauma (from headbands or chin straps of athletic helmets), application of comedogenic topical agents (cosmetics

1	CHAPTER 71 Eczema, Psoriasis, Cutaneous Infections, Acne, and Other Common Skin Disorders PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 71-7 Acne vulgaris. An example of acne vulgaris with inflammatory papules, pustules, and comedones. (Courtesy of Kalman Watsky, MD; with permission.) or hair preparations), or chronic topical exposure to certain industrial compounds may elicit or aggravate acne. Glucocorticoids, topical or systemic, may also elicit acne. Other systemic medications such as oral contraceptive pills, lithium, isoniazid, androgenic steroids, halogens, phenytoin, and phenobarbital may produce acneiform eruptions or aggravate preexisting acne. Genetic factors and polycystic ovary disease may also play a role.

1	Treatment of acne vulgaris is directed toward elimination of comedones by normalizing follicular keratinization, decreasing sebaceous gland activity, decreasing the population of P. acnes, and decreasing inflammation. Minimal to moderate pauci-inflammatory disease may respond adequately to local therapy alone. Although areas affected with acne should be kept clean, overly vigorous scrubbing may aggravate acne due to mechanical rupture of comedones. Topical agents such as retinoic acid, benzoyl peroxide, or salicylic acid may alter the pattern of epidermal desquamation, preventing the formation of comedones and aiding in the resolution of preexisting cysts. Topical antibacterial agents (such as azelaic acid, erythromycin, clindamycin, or dapsone) are also useful adjuncts to therapy.

1	Patients with moderate to severe acne with a prominent inflammatory component will benefit from the addition of systemic therapy, such as tetracycline in doses of 250–500 mg bid or doxycycline in doses of 100 mg bid. Minocycline is also useful. Such antibiotics appear to have anti-inflammatory effects independent of their antibacterial effects. Female patients who do not respond to oral antibiotics may benefit from hormonal therapy. Several oral contraceptives are now approved by the FDA for use in the treatment of acne vulgaris.

1	Patients with severe nodulocystic acne unresponsive to the therapies discussed above may benefit from treatment with the synthetic retinoid isotretinoin. Its dose is based on the patient’s weight, and it is given once daily for 5 months. Results are excellent in appropriately selected patients. Its use is highly regulated due to its potential for severe adverse events, primarily teratogenicity and depression. In addition, patients receiving this medication develop extremely dry skin and cheilitis and must be followed for development of hypertriglyceridemia.

1	At present, prescribers must enroll in a program designed to prevent pregnancy and adverse events while patients are taking isotretinoin. These measures are imposed to ensure that all prescribers are familiar with the risks of isotretinoin; that all female patients have two negative pregnancy tests prior to initiation of therapy and a negative pregnancy test prior to each refill; and that all patients have been warned about the risks associated with isotretinoin. FIguRE 71-8 Acne rosacea. Prominent facial erythema, telangiecta-sia, scattered papules, and small pustules are seen in this patient with acne rosacea. (Courtesy of Robert Swerlick, MD; with permission.)

1	Acne rosacea, commonly referred to simply as rosacea, is an inflammatory disorder predominantly affecting the central face. Persons most often affected are Caucasians of northern European background, but rosacea also occurs in patients with dark skin. Rosacea is seen almost exclusively in adults, only rarely affecting patients <30 years old. Rosacea is more common in women, but those most severely affected are men. It is characterized by the presence of erythema, telangiectases, and superficial pustules (Fig. 71-8) but is not associated with the presence of comedones. Rosacea rarely involves the chest or back.

1	There is a relationship between the tendency for facial flushing and the subsequent development of acne rosacea. Often, individuals with rosacea initially demonstrate a pronounced flushing reaction. This may be in response to heat, emotional stimuli, alcohol, hot drinks, or spicy foods. As the disease progresses, the flush persists longer and longer and may eventually become permanent. Papules, pustules, and telangiectases can become superimposed on the persistent flush. Rosacea of very long standing may lead to connective tissue overgrowth, particularly of the nose (rhinophyma). Rosacea may also be complicated by various inflammatory disorders of the eye, including keratitis, blepharitis, iritis, and recurrent chalazion. These ocular problems are potentially sight-threatening and warrant ophthalmologic evaluation.

1	Acne rosacea can be treated topically or systemically. Mild disease often responds to topical metronidazole, sodium sulfacetamide, or azaleic acid. More severe disease requires oral tetracyclines: tetracycline, 250–500 mg bid; doxycycline, 100 mg bid; or minocycline, 50–100 mg bid. Residual telangiectasia may respond to laser therapy. Topical glucocorticoids, especially potent agents, should be avoided because chronic use of these preparations may elicit rosacea. Application of topical agents to the skin is not effective treatment for ocular disease.

1	Although smallpox vaccinations were discontinued several decades ago for the general population, they are still required for certain military personnel and first responders. In the absence of a bioterrorism attack and a real or potential exposure to smallpox, such vaccination is contraindicated in persons with a history of skin diseases such as AD, eczema, and psoriasis, who have a higher incidence of adverse events associated with smallpox vaccination. In the case of such exposure, the risk of smallpox infection outweighs the risk of adverse events from the vaccine (Chap. 261e).

1	Skin Manifestations of internal Disease Jean L. Bolognia, Irwin M. Braverman It is a generally accepted concept in medicine that the skin can develop signs of internal disease. Therefore, in textbooks of medicine, one finds a chapter describing in detail the major systemic disorders that 72 can be identified by cutaneous signs. The underlying assumption of such a chapter is that the clinician has been able to identify the specific disorder in the patient and needs only to read about it in the textbook. In reality, concise differential diagnoses and the identification of these disorders are actually difficult for the nondermatologist because he or she is not well-versed in the recognition of cutaneous lesions or their spectrum of presentations. Therefore, this chapter covers this particular topic of cutaneous medicine not by simply focusing on individual diseases, but by describing the various presenting clinical signs and symptoms that point to specific disorders. Concise differential

1	topic of cutaneous medicine not by simply focusing on individual diseases, but by describing the various presenting clinical signs and symptoms that point to specific disorders. Concise differential diagnoses will be generated in which the significant diseases will be distinguished from the more common cutaneous disorders that have minimal or no significance with regard to associated internal disease. The latter disorders are reviewed in table form and always need to be excluded when considering the former. For a detailed description of individual diseases, the reader should consult a dermatologic text.

1	(Table 72-1) When an eruption is characterized by elevated lesions, either papules (<1 cm) or plaques (>1 cm), in association with scale, it is referred to as papulosquamous. The most common papulosquamous diseases—tinea, psoriasis, pityriasis rosea, and lichen planus—are primary cutaneous disorders (Chap. 71). When psoriatic lesions are accompanied by arthritis, the possibility of psoriatic arthritis or reactive arthritis (formerly known as Reiter’s syndrome) should be considered. A history of oral ulcers, conjunctivitis, uveitis, and/or urethritis points to the latter diagnosis. Lithium, beta blockers, HIV or streptococcal infections, and a rapid taper of systemic glucocorticoids are known to exacerbate psoriasis. Comorbidities in patients with psoriasis include cardiovascular disease and metabolic syndrome.

1	Whenever the diagnosis of pityriasis rosea or lichen planus is made, it is important to review the patient’s medications because the eruption may resolve by simply discontinuing the offending agent. Pityriasis rosea–like drug eruptions are seen most commonly with beta blockers, angiotensin-converting enzyme (ACE) inhibitors, and metronidazole, whereas the drugs that can produce a lichenoid eruption include thiazides, antimalarials, quinidine, beta blockers, and ACE inhibitors. In some populations, there is a higher prevalence of hepatitis C viral infection in patients with lichen planus. Lichen planus–like lesions are also observed in chronic graft-versus-host disease.

1	In its early stages, the mycosis fungoides (MF) form of cutaneous T cell lymphoma (CTCL) may be confused with eczema or psoriasis, but it often fails to respond to the appropriate therapy for those inflammatory diseases. MF can develop within lesions of large-plaque parapsoriasis and is suggested by an increase in the thickness of the lesions. The diagnosis of MF is established by skin biopsy in which collections of atypical T lymphocytes are found in the epidermis and dermis. As the disease progresses, cutaneous tumors and lymph node involvement may appear.

1	In secondary syphilis, there are scattered red-brown papules with thin scale. The eruption often involves the palms and soles and can resemble pityriasis rosea. Associated findings are helpful in making the diagnosis and include annular plaques on the face, nonscarring alopecia, condyloma lata (broad-based and moist), and mucous patches as well as lymphadenopathy, malaise, fever, headache, and myalgias. The interval between the primary chancre and the secondary stage is usually 4–8 weeks, and spontaneous resolution without appropriate therapy occurs. SELECTED CAuSES of PAPuLoSquAMouS SKin LESionS 1. Primary cutaneous disorders a. b. c. d. e. Parapsoriasis, small plaque and large plaque f. 2. 3. a. Lupus erythematosus, primarily subacute or chronic (discoid) lesionsc b. Cutaneous T cell lymphoma, in particular, mycosis fungoidesd c. d. Reactive arthritis (formerly known as Reiter's syndrome) e.

1	aDiscussed in detail in Chap. 71; cardiovascular disease and the metabolic syndrome are comorbidities in psoriasis; primarily in Europe, hepatitis C virus is associated with oral lichen planus. bAssociated with chronic sun exposure more often than exposure to arsenic; usually one or a few lesions. cSee also Red Lesions in “Papulonodular Skin Lesions.” dAlso cutaneous lesions of HTLV-1-associated adult T cell leukemia/lymphoma. eSee also Red-Brown Lesions in “Papulonodular Skin Lesions.” (Table 72-2) Erythroderma is the term used when the majority of the skin surface is erythematous (red in color). There may be associated scale, erosions, or pustules as well as shedding of the hair and nails. Potential systemic manifestations include fever, chills, hypothermia, reactive lymphadenopathy, peripheral edema, hypoalbuminemia, and high-output cardiac failure. The major etiologies of erythroderma are (1) cutaneous diseases such as psoriasis and dermatitis (Table 72-3); (2) drugs; (3) systemic

1	edema, hypoalbuminemia, and high-output cardiac failure. The major etiologies of erythroderma are (1) cutaneous diseases such as psoriasis and dermatitis (Table 72-3); (2) drugs; (3) systemic diseases, most commonly CTCL; and (4) idiopathic. In the first three groups, the location and description of the initial lesions, prior to the development of the erythroderma, aid in the diagnosis. For example, a history of red scaly plaques on the elbows and knees would point to psoriasis. It is also important to examine the skin carefully for a migration of the erythema and associated secondary changes such as pustules or erosions. Migratory waves of erythema studded with superficial pustules are seen in pustular psoriasis.

1	Drug-induced erythroderma (exfoliative dermatitis) may begin as an exanthematous (morbilliform) eruption (Chap. 74) or may arise as diffuse erythema. A number of drugs can produce an erythroderma, including penicillins, sulfonamides, carbamazepine, phenytoin, and allopurinol. Fever and peripheral eosinophilia often accompany the eruption, and there may also be facial swelling, hepatitis, myocarditis, thyroiditis, and allergic interstitial nephritis; this constellation is frequently referred to as drug reaction with eosinophilia and systemic symptoms (DRESS) or drug-induced hypersensitivity reaction (DIHS). In addition, these reactions, especially to aromatic anticonvulsants, can lead to a pseudolymphoma syndrome (with adenopathy and CAuSES of ERyTHRoDERMA 1. Primary cutaneous disorders a. b. c. 2. 3. a. Cutaneous T cell lymphoma (Sézary syndrome, erythrodermic mycosis fungoides) b. 4. Idiopathic (usually older men) a Discussed in detail in Chap. 71.

1	b. c. 2. 3. a. Cutaneous T cell lymphoma (Sézary syndrome, erythrodermic mycosis fungoides) b. 4. Idiopathic (usually older men) a Discussed in detail in Chap. 71. CHAPTER 72 Skin Manifestations of Internal Disease PART 2 Cardinal Manifestations and Presentation of Diseases Abbreviations: Ab, antibody; HSV, herpes simplex virus; IL, interleukin; IM, intramuscular; IV, intravenous; MTX, methotrexate; PUVA, psoralens + ultraviolet A irradiation; SAPHO, synovitis, acne, pustulosis, hyperostosis, and osteitis (also referred to as chronic recurrent multifocal osteomyelitis); TNF, tumor necrosis factor; UV-A, ultraviolet A irradiation; UV-B, ultraviolet B irradiation. circulating atypical lymphocytes), while reactions to allopurinol may The most common causes of nonscarring alopecia include androgebe accompanied by gastrointestinal bleeding. netic alopecia, telogen effluvium, alopecia areata, tinea capitis, and the

1	The most common malignancy that is associated with erythroderma early phase of traumatic alopecia (Table 72-5). In women with androis CTCL; in some series, up to 25% of the cases of erythroderma were genetic alopecia, an elevation in circulating levels of androgens may be due to CTCL. The patient may progress from isolated plaques and seen as a result of ovarian or adrenal gland dysfunction or neoplasm. tumors, but more commonly, the erythroderma is present throughout When there are signs of virilization, such as a deepened voice and the course of the disease (Sézary syndrome). In the Sézary syndrome, enlarged clitoris, the possibility of an ovarian or adrenal gland tumor there are circulating clonal atypical T lymphocytes, pruritus, and lymph-should be considered. adenopathy. In cases of erythroderma where there is no apparent cause Exposure to various drugs can also cause diffuse hair loss, (idiopathic), longitudinal evaluation is mandatory to monitor for the usually by inducing a

1	erythroderma where there is no apparent cause Exposure to various drugs can also cause diffuse hair loss, (idiopathic), longitudinal evaluation is mandatory to monitor for the usually by inducing a telogen effluvium. An exception is the anagen possible development of CTCL. There have been isolated case reports effluvium observed with antimitotic agents such as daunorubicin. of erythroderma secondary to some solid tumors—lung, liver, prostate, Alopecia is a side effect of the following drugs: warfarin, heparin, thyroid, and colon—but it is primarily during a late stage of the disease. propylthiouracil, carbimazole, isotretinoin, acitretin, lithium, beta blockers, interferons, colchicine, and amphetamines. Fortunately, spontaneous regrowth usually follows discontinuation of the offend- ing agent. (Table 72-4) The two major forms of alopecia are scarring and non-Less commonly, nonscarring alopecia is associated with lupus eryscarring. Scarring alopecia is associated with fibrosis,

1	ing agent. (Table 72-4) The two major forms of alopecia are scarring and non-Less commonly, nonscarring alopecia is associated with lupus eryscarring. Scarring alopecia is associated with fibrosis, inflammation, thematosus and secondary syphilis. In systemic lupus there are two and loss of hair follicles. A smooth scalp with a decreased number of forms of alopecia—one is scarring secondary to discoid lesions (see follicular openings is usually observed clinically, but in some patients, below), and the other is nonscarring. The latter form coincides with the changes are seen only in biopsy specimens from affected areas. In flares of systemic disease and may involve the entire scalp or just nonscarring alopecia, the hair shafts are absent or miniaturized, but the the frontal scalp, with the appearance of multiple short hairs (“lupus hair follicles are preserved, explaining the reversible nature of nonscar-hairs”) as a sign of initial regrowth. Scattered, poorly circumscribed ring

1	the appearance of multiple short hairs (“lupus hair follicles are preserved, explaining the reversible nature of nonscar-hairs”) as a sign of initial regrowth. Scattered, poorly circumscribed ring alopecia. patches of alopecia with a “moth-eaten” appearance are a manifestation

1	CAuSES of ALoPECiA I. Nonscarring alopecia A. Primary cutaneous disorders 1. 2. 3. 4. 5. B. Drugs C. Systemic diseases 1. 2. 3. 4. 5. 6. Deficiencies of protein, biotin, zinc, and perhaps iron II. A. 1. 2. 3. 4. 5. B. Systemic diseases 1. Discoid lesions in the setting of systemic lupus erythematosusb 2. 3. a Most patients with trichotillomania, pressure-induced alopecia, or early stages of traction alopecia. b While the majority of patients with discoid lesions have only cutaneous disease, these lesions do represent one of the 11 American College of Rheumatology criteria (1982) for systemic lupus erythematosus. c Can involve underlying muscles and osseous structures. of the secondary stage of syphilis. Diffuse thinning of the hair is also 355 associated with hypothyroidism and hyperthyroidism (Table 72-4).

1	of the secondary stage of syphilis. Diffuse thinning of the hair is also 355 associated with hypothyroidism and hyperthyroidism (Table 72-4). Scarring alopecia is more frequently the result of a primary cutaneous disorder such as lichen planus, folliculitis decalvans, chronic cutaneous (discoid) lupus, or linear scleroderma (morphea) than it is a sign of systemic disease. Although the scarring lesions of discoid lupus can be seen in patients with systemic lupus, in the majority of patients, the disease process is limited to the skin. Less common causes of scarring alopecia include sarcoidosis (see “Papulonodular Skin Lesions,” below) and cutaneous metastases.

1	In the early phases of discoid lupus, lichen planus, and folliculitis decalvans, there are circumscribed areas of alopecia. Fibrosis and subsequent loss of hair follicles are observed primarily in the center of these alopecic patches, whereas the inflammatory process is most prominent at the periphery. The areas of active inflammation in discoid lupus are erythematous with scale, whereas the areas of previous inflammation are often hypopigmented with a rim of hyperpigmentation. In lichen planus, perifollicular macules at the periphery are usually violet-colored. A complete examination of the skin and oral mucosa combined with a biopsy and direct immunofluorescence microscopy of inflamed skin will aid in distinguishing these two entities. The peripheral active lesions in folliculitis decalvans are follicular pustules; these patients can develop a reactive arthritis.

1	(Table 72-6) In figurate eruptions, the lesions form rings and arcs that are usually erythematous but can be skin-colored to brown. Most commonly, they are due to primary cutaneous diseases such as tinea, urticaria, granuloma annulare, and erythema annulare centrifugum (Chaps. 71 and 73). An underlying systemic illness is found in a second, less common group of migratory annular erythemas. It includes erythema migrans, erythema gyratum repens, erythema marginatum, and necrolytic migratory erythema. In erythema gyratum repens, one sees numerous mobile concentric arcs and wavefronts that resemble the grain in wood. A search for an CHAPTER 72 Skin Manifestations of Internal Disease Diffuse shedding of normal hairs Follows major stress (high fever, severe infection) or change in hormone levels (postpartum) Miniaturization of hairs along the midline of the scalp Recession of the anterior scalp line in men and some women

1	Miniaturization of hairs along the midline of the scalp Recession of the anterior scalp line in men and some women Well-circumscribed, circular areas of hair loss, 2–5 cm in diameter In extensive cases, coalescence of lesions and/or involvement of other hair-bearing surfaces of the body Pitting or sandpapered appearance of the nails Varies from scaling with minimal hair loss to discrete patches with “black dots” (broken infected hairs) to boggy plaque with pustules (kerion)b Broken hairs, often of varying lengths Irregular outline Stress causes more of the asynchronous growth cycles of individual hairs to become synchronous; therefore, larger numbers of growing (anagen) hairs simultaneously enter the dying (telogen) phase Increased sensitivity of affected hairs to the effects of androgens Increased levels of circulating androgens (ovarian or adrenal source in women) The germinative zones of the hair follicles are surrounded by T lymphocytes

1	Increased levels of circulating androgens (ovarian or adrenal source in women) The germinative zones of the hair follicles are surrounded by T lymphocytes Occasional associated diseases: hyperthyroidism, hypothyroidism, vitiligo, Down syndrome Invasion of hairs by dermatophytes, most commonly Trichophyton tonsurans Traction with curlers, rubber bands, braiding Exposure to heat or chemicals (e.g., hair straighteners) Mechanical pulling (trichotillomania) Observation; discontinue any drugs that have alopecia as a side effect; must exclude underlying metabolic causes, e.g., hypothyroidism, hyperthyroidism If no evidence of hyperandrogenemia, then topical minoxidil; finasteridea; spironolactone (women); hair transplant Oral griseofulvin or terbinafine plus 2.5% selenium sulfide or ketoconazole shampoo; examine family members

1	Oral griseofulvin or terbinafine plus 2.5% selenium sulfide or ketoconazole shampoo; examine family members Discontinuation of offending hair style or chemical treatments; diagnosis of trichotillomania may require observation of shaved hairs (for growth) or biopsy, possibly followed by psychotherapy aTo date, Food and Drug Administration–approved for men. bScarring alopecia can occur at sites of kerions. cMay also be scarring, especially late-stage traction alopecia. CAuSES of figuRATE SKin LESionS I. Primary cutaneous disorders A. Tinea B. Urticaria (primary in ≥90% of patients) C. Granuloma annulare D. Erythema annulare centrifugum E. Psoriasis II. A. 1. Erythema migrans (CDC case definition is ≥5 cm in diameter) 2. Urticaria (≤10% of patients) 3. 4. 5. 6. B. Nonmigratory 1. 2. 3. 4. Cutaneous T cell lymphoma (especially mycosis fungoides) aMigratory erythema with erosions; favors lower extremities and girdle area.

1	4. 5. 6. B. Nonmigratory 1. 2. 3. 4. Cutaneous T cell lymphoma (especially mycosis fungoides) aMigratory erythema with erosions; favors lower extremities and girdle area. Abbreviation: CDC, Centers for Disease Control and Prevention. underlying malignancy is mandatory in a patient with this eruption. Erythema migrans is the cutaneous manifestation of Lyme disease, which is caused by the spirochete Borrelia burgdorferi. In the initial stage (3–30 days after tick bite), a single annular lesion is usually seen, which can expand to ≥10 cm in diameter. Within several days, up to half of the patients develop multiple smaller erythematous lesions at sites distant from the bite. Associated symptoms include fever, headache, photophobia, myalgias, arthralgias, and malar rash. Erythema marginatum is seen in patients with rheumatic fever, primarily on the trunk. Lesions are pink-red in color, flat to minimally elevated, and transient.

1	There are additional cutaneous diseases that present as annular eruptions but lack an obvious migratory component. Examples include CTCL, subacute cutaneous lupus, secondary syphilis, and sarcoidosis (see “Papulonodular Skin Lesions,” below). (Table 72-7) In addition to acne vulgaris and acne rosacea, the two major forms of acne (Chap. 71), there are drugs and systemic diseases that can lead to acneiform eruptions. Patients with the carcinoid syndrome have episodes of flushing of the head, neck, and sometimes the trunk. Resultant skin changes of the face, in particular telangiectasias, may mimic the clinical appearance of acne rosacea. CAuSES of ACnEifoRM ERuPTionS I. Primary cutaneous disorders A. Acne vulgaris B. Acne rosacea II. Drugs, e.g., anabolic steroids, glucocorticoids, lithium, EGFRa inhibitors, iodides III. A. 1. Adrenal origin, e.g., Cushing’s disease, 21-hydroxylase deficiency 2. Ovarian origin, e.g., polycystic ovary syndrome, ovarian hyperthecosis

1	III. A. 1. Adrenal origin, e.g., Cushing’s disease, 21-hydroxylase deficiency 2. Ovarian origin, e.g., polycystic ovary syndrome, ovarian hyperthecosis B. Cryptococcosis, disseminated C. Dimorphic fungal infections D. Behçet’s disease aEGFR, epidermal growth factor receptor. PART 2 Cardinal Manifestations and Presentation of Diseases

1	Acneiform eruptions (see “Acne,” above) and folliculitis represent the most common pustular dermatoses. An important consideration in the evaluation of follicular pustules is a determination of the associated pathogen, e.g., normal flora, Staphylococcus aureus, Pseudomonas aeruginosa (“hot tub” folliculitis), Malassezia, dermatophytes (Majocchi’s granuloma), and Demodex spp. Noninfectious forms of folliculitis include HIVor immunosuppression-associated eosinophilic folliculitis and folliculitis secondary to drugs such as glucocorticoids, lithium, and epidermal growth factor receptor (EGFR) inhibitors. Administration of high-dose systemic glucocorticoids can result in a widespread eruption of follicular pustules on the trunk, characterized by lesions in the same stage of development. With regard to underlying systemic diseases, nonfollicular-based pustules are a characteristic component of pustular psoriasis (sterile) and can be seen in septic emboli of bacterial or fungal origin (see

1	regard to underlying systemic diseases, nonfollicular-based pustules are a characteristic component of pustular psoriasis (sterile) and can be seen in septic emboli of bacterial or fungal origin (see “Purpura,” below). In patients with acute generalized exanthematous pustulosis (AGEP) due primarily to medications (e.g., cephalosporins), there are large areas of erythema studded with multiple sterile pustules in addition to neutrophilia.

1	(Table 72-8) To distinguish the various types of telangiectasias, it is important to examine the shape and configuration of the dilated blood vessels. Linear telangiectasias are seen on the face of patients CAuSES of TELAngiECTASiAS I. Primary cutaneous disorders A. Linear/branching 1. 2. 3. 4. 5. 6. B. Poikiloderma 1. 2. Parapsoriasis, large plaque C. Spider angioma 1. 2. II. A. 1. 2. 3. B. Poikiloderma 1. 2. 3. C. Mat 1. Systemic sclerosis (scleroderma) D. Periungual/cuticular 1. 2. 3. 4. E. Papular 1. Hereditary hemorrhagic telangiectasia F. Spider angioma 1. Cirrhosis aBecoming less common.

1	2. 3. C. Mat 1. Systemic sclerosis (scleroderma) D. Periungual/cuticular 1. 2. 3. 4. E. Papular 1. Hereditary hemorrhagic telangiectasia F. Spider angioma 1. Cirrhosis aBecoming less common. with actinically damaged skin and acne rosacea, and they are found on the legs of patients with venous hypertension and generalized essential telangiectasia. Patients with an unusual form of mastocytosis (telangiectasia macularis eruptiva perstans) and the carcinoid syndrome (see “Acne,” above) also have linear telangiectasias. Lastly, linear telangiectasias are found in areas of cutaneous inflammation. For example, lesions of discoid lupus frequently have telangiectasias within them.

1	Poikiloderma is a term used to describe a patch of skin with: (1) reticulated hypoand hyperpigmentation, (2) wrinkling secondary to epidermal atrophy, and (3) telangiectasias. Poikiloderma does not imply a single disease entity—although it is becoming less common, it is seen in skin damaged by ionizing radiation as well as in patients with autoimmune connective tissue diseases, primarily dermatomyositis (DM), and rare genodermatoses (e.g., Kindler syndrome).

1	In systemic sclerosis (scleroderma) the dilated blood vessels have a unique configuration and are known as mat telangiectasias. The lesions are broad macules that usually measure 2–7 mm in diameter but occasionally are larger. Mats have a polygonal or oval shape, and their erythematous color may appear uniform, but, upon closer inspection, the erythema is the result of delicate telangiectasias. The most common locations for mat telangiectasias are the face, oral mucosa, and hands—peripheral sites that are prone to intermittent ischemia. The limited form of systemic sclerosis, often referred to as the CREST (calcinosis cutis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) variant (Chap. 382), is associated with a chronic course and anticentromere antibodies. Mat telangiectasias are an important clue to the diagnosis of this variant as well as the diffuse form of systemic sclerosis because they may be the only cutaneous finding.

1	Periungual telangiectasias are pathognomonic signs of the three major autoimmune connective tissue diseases: lupus erythematosus, systemic sclerosis, and DM. They are easily visualized by the naked eye and occur in at least two-thirds of these patients. In both DM and lupus, there is associated nailfold erythema, and in DM, the erythema is often accompanied by “ragged” cuticles and fingertip tenderness. Under 10× magnification, the blood vessels in the nailfolds of lupus patients are tortuous and resemble “glomeruli,” whereas in systemic sclerosis and DM, there is a loss of capillary loops and those that remain are markedly dilated.

1	In hereditary hemorrhagic telangiectasia (Osler-Rendu-Weber disease), the lesions usually appear during adolescence (mucosal) and adulthood (cutaneous) and are most commonly seen on the mucous membranes (nasal, orolabial), face, and distal extremities, including under the nails. They represent arteriovenous (AV) malformations of the dermal microvasculature, are dark red in color, and are usually slightly elevated. When the skin is stretched over an individual lesion, an eccentric punctum with radiating legs is seen. Although the degree of systemic involvement varies in this autosomal dominant disease (due primarily to mutations in either the endoglin or activin receptor– like kinase gene), the major symptoms are recurrent epistaxis and gastrointestinal bleeding. The fact that these mucosal telangiectasias are actually AV communications helps to explain their tendency to bleed.

1	(Table 72-9) Disorders of hypopigmentation are often classified as either diffuse or localized. The classic example of diffuse hypopigmentation is oculocutaneous albinism (OCA). The most common forms are due to mutations in the tyrosinase gene (type I) or the P gene (type II); patients with type IA OCA have a total lack of enzyme activity. At birth, different forms of OCA can appear similar—white hair, gray-blue eyes, and pink-white skin. However, the patients with no tyrosinase activity maintain this phenotype, whereas those with decreased activity will acquire some pigmentation of the eyes, hair, and skin as they age. The degree of pigment formation is also a function of racial background, and the pigmentary dilution is more readily apparent when patients are compared to their first-degree relatives. The ocular findings in OCA correlate with the degree of hypopigmentation and CAuSES of HyPoPigMEnTATion I. Primary cutaneous disorders A. Diffuse 1. Generalized vitiligoa

1	CAuSES of HyPoPigMEnTATion I. Primary cutaneous disorders A. Diffuse 1. Generalized vitiligoa B. Localized 1. 2. 3. 4. 5. 6. 7. II. A. 1. 2. Hermansky-Pudlak syndromeb,c 3. Chédiak-Higashi syndromeb,d 4. B. Localized 1. 2. Melanoma-associated leukoderma, spontaneous or immunotherapy-induced 3. 4. 5. 6. 7. 8. Linear nevoid hypopigmentation (hypomelanosis of Ito)e 9. 10. 11. aAbsence of melanocytes in areas of leukoderma. bNormal number of melanocytes. cPlatelet storage defect and restrictive lung disease secondary to deposits of ceroid-like material or immunodeficiency; due to mutations in β subunit of adaptor protein 3 as well as subunits of biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. dGiant lysosomal granules and recurrent infections. eMinority of patients in a nonreferral setting have systemic abnormalities (musculoskeletal, central nervous system, ocular).

1	include decreased visual acuity, nystagmus, photophobia, strabismus, and a lack of normal binocular vision.

1	The differential diagnosis of localized hypomelanosis includes the following primary cutaneous disorders: idiopathic guttate hypomelanosis, postinflammatory hypopigmentation, tinea (pityriasis) versicolor, vitiligo, chemicalor drug-induced leukoderma, nevus depigmentosus (see below), and piebaldism (Table 72-10). In this group of diseases, the areas of involvement are macules or patches with a decrease or absence of pigmentation. Patients with vitiligo also have an increased incidence of several autoimmune disorders, including Hashimoto’s thyroiditis, Graves’ disease, pernicious anemia, Addison’s disease, uveitis, alopecia areata, chronic mucocutaneous candidiasis, and the autoimmune polyendocrine syndromes (types I and II). Diseases of the thyroid gland are the most frequently associated disorders, occurring in up to 30% of patients with vitiligo. Circulating autoantibodies are often found, and the most common ones are antithyroglobulin, antimicrosomal, and antithyroid-stimulating

1	disorders, occurring in up to 30% of patients with vitiligo. Circulating autoantibodies are often found, and the most common ones are antithyroglobulin, antimicrosomal, and antithyroid-stimulating hormone receptor antibodies.

1	There are four systemic diseases that should be considered in a patient with skin findings suggestive of vitiligo—Vogt-Koyanagi-Harada syndrome, systemic sclerosis, onchocerciasis, and melanoma-associated leukoderma. A history of aseptic meningitis, nontraumatic uveitis, tinnitus, hearing loss, and/or dysacousia points to the diagnosis of the Vogt-Koyanagi-Harada syndrome. In these patients, the face and scalp are the most common locations of pigment loss. The CHAPTER 72 Skin Manifestations of Internal Disease 358 TABLE 72-10 HyPoPigMEnTATion (PRiMARy CuTAnEouS DiSoRDERS, LoCALizED) PART 2 Cardinal Manifestations and Presentation of Diseases Can develop within active lesions, as in subacute cutaneous lupus, or after the lesion fades, as in atopic dermatitis Upper trunk and neck (shawl-like distribution), groin Symmetric areas of complete pigment loss Periorificial—around mouth, nose, eyes, nipples, umbilicus, anus Other areas—flexor wrists, extensor distal extremities

1	Symmetric areas of complete pigment loss Periorificial—around mouth, nose, eyes, nipples, umbilicus, anus Other areas—flexor wrists, extensor distal extremities Segmental form is less common—unilateral, dermatomal-like Similar appearance to vitiligo Often begins on hands when associated with chemical exposure Satellite lesions in areas not exposed to chemicals Congenital, stable Areas of amelanosis contain normally pigmented and hyper-pigmented macules of various sizes Symmetric involvement of central forehead, ventral trunk, and mid regions of upper and lower extremities Less enhancement than vitiligo Enhancement of leukoderma and hyperpigmented macules Abrupt decrease in epidermal melanin content Type of inflammatory infiltrate depends on specific disease Absence of melanocytes Decreased number or absence of melanocytes Amelanotic areas—few to no melanocytes Possible somatic mutations as a reflection of aging or UV exposure

1	Absence of melanocytes Decreased number or absence of melanocytes Amelanotic areas—few to no melanocytes Possible somatic mutations as a reflection of aging or UV exposure Block in transfer of melanin from melanocytes to keratinocytes could be secondary to edema or decrease in contact time Destruction of melanocytes if inflammatory cells attack basal layer of epidermis Invasion of stratum corneum by the yeast Yeast is lipophilic and produces C9 and C11 dicarboxylic acids, which in vitro inhibit tyrosinase Autoimmune phenomenon that results in destruction of melanocytes—primarily cellular (circulating skin-homing autoreactive T cells) Exposure to chemicals that selectively destroy melanocytes, in particular phenols and catechols (germicides; adhesives) or ingestion of drugs such as imatinib Release of cellular antigens and activation of circulating lymphocytes may explain satellite phenomenon Possible inhibition of KIT receptor

1	Release of cellular antigens and activation of circulating lymphocytes may explain satellite phenomenon Possible inhibition of KIT receptor Defect in migration of melanoblasts from neural crest to involved skin or failure of melanoblasts to survive or differentiate in these areas Mutations within the c-kit protooncogene that encodes the tyrosine kinase receptor for stem cell growth factor (kit ligand) None Selenium sulfide 2.5%; topical imidazoles; oral triazoles Topical glucocorticoids; topical calcineurin inhibitors; NBUV-B; PUVA; transplants, if stable; depigmentation (topical MBEH), if widespread Avoid exposure to offending agent, then treat as vitiligo Drug-induced variant may undergo repigmentation when medication is discontinued Abbreviations: MBEH, monobenzylether of hydroquinone; NBUV-B, narrow band ultraviolet B; PUVA, psoralens + ultraviolet A irradiation.

1	vitiligo-like leukoderma seen in patients with systemic sclerosis has a clinical resemblance to idiopathic vitiligo that has begun to repigment as a result of treatment; that is, perifollicular macules of normal pigmentation are seen within areas of depigmentation. The basis of this leukoderma is unknown; there is no evidence of inflammation in areas of involvement, but it can resolve if the underlying connective tissue disease becomes inactive. In contrast to idiopathic vitiligo, melanoma-associated leukoderma often begins on the trunk, and its appearance, if spontaneous, should prompt a search for metastatic disease. It is also seen in patients undergoing immunotherapy for melanoma, including ipilimumab, with cytotoxic T lymphocytes presumably recognizing cell surface antigens common to melanoma cells and melanocytes, and is associated with a greater likelihood of a clinical response.

1	There are two systemic disorders (neurocristopathies) that may have the cutaneous findings of piebaldism (Table 72-9). They are Shah-Waardenburg syndrome and Waardenburg syndrome. A possible explanation for both disorders is an abnormal embryonic migration or survival of two neural crest–derived elements, one of them being melanocytes and the other myenteric ganglion cells (leading to Hirschsprung disease in Shah-Waardenburg syndrome) or auditory nerve cells (Waardenburg syndrome). The latter syndrome is characterized by congenital sensorineural hearing loss, dystopia canthorum (lateral displacement of the inner canthi but normal interpupillary distance), heterochromic irises, and a broad nasal root, in addition to the piebaldism. The facial dysmorphism can be explained by the neural crest origin of the connective tissues of the head and neck. Patients with Waardenburg syndrome have been shown to have mutations in four genes, including PAX-3 and MITF, all of which encode DNA-binding

1	origin of the connective tissues of the head and neck. Patients with Waardenburg syndrome have been shown to have mutations in four genes, including PAX-3 and MITF, all of which encode DNA-binding proteins, whereas patients with Hirschsprung disease plus white spotting have mutations in one of three genes—endothelin 3, endothelin B receptor, and SOX-10.

1	In tuberous sclerosis, the earliest cutaneous sign is macular hypomelanosis, referred to as an ash leaf spot. These lesions are often present at birth and are usually multiple; however, detection may require Wood’s lamp examination, especially in fair-skinned individuals. The pigment within them is reduced, but not absent. The average size is 1–3 cm, and the common shapes are polygonal and lance-ovate. Examination of the patient for additional cutaneous signs such as multiple angiofibromas of the face (adenoma sebaceum), ungual and gingival fibromas, fibrous plaques of the forehead, and connective tissue nevi (shagreen patches) is recommended. It is important to remember that an ash leaf spot on the scalp will result in a circumscribed patch of lightly pigmented hair. Internal manifestations include seizures, mental retardation, central nervous system (CNS) and retinal hamartomas, pulmonary lymphangioleiomyomatosis (women), renal angiomyolipomas, and cardiac rhabdomyomas. The latter

1	include seizures, mental retardation, central nervous system (CNS) and retinal hamartomas, pulmonary lymphangioleiomyomatosis (women), renal angiomyolipomas, and cardiac rhabdomyomas. The latter can be detected in up to 60% of children (<18 years) with tuberous sclerosis by echocardiography.

1	Nevus depigmentosus is a stable, well-circumscribed hypomelanosis that is present at birth. There is usually a single oval or rectangular lesion, but when there are multiple lesions, the possibility of tuberous sclerosis needs to be considered. In linear nevoid hypopigmentation, a term that is replacing hypomelanosis of Ito and segmental or systematized nevus depigmentosus, streaks and swirls of hypopigmentation are observed. Up to a third of patients in a tertiary care setting had associated abnormalities involving the musculoskeletal system (asymmetry), the CNS (seizures and mental retardation), and the eyes (strabismus and hypertelorism). Chromosomal mosaicism has been detected in these patients, lending support to the hypothesis that the cutaneous pattern is the result of the migration of two clones of primordial melanocytes, each with a different pigment potential.

1	Localized areas of decreased pigmentation are commonly seen as a result of cutaneous inflammation (Table 72-10) and have been observed in the skin overlying active lesions of sarcoidosis (see “Papulonodular Skin Lesions,” below) as well as in CTCL. Cutaneous infections also present as disorders of hypopigmentation, and in tuberculoid leprosy, there are a few asymmetric patches of hypomelanosis that have associated anesthesia, anhidrosis, and alopecia. Biopsy specimens of the palpable border show dermal granulomas that contain rare, if any, Mycobacterium leprae organisms.

1	(Table 72-11) Disorders of hyperpigmentation are also divided into two groups—localized and diffuse. The localized forms are due to an epidermal alteration, a proliferation of melanocytes, or an increase in pigment production. Both seborrheic keratoses and acanthosis nigricans belong to the first group. Seborrheic keratoses are common lesions, but in one rare clinical setting, they are a sign of systemic disease, and that setting is the sudden appearance of multiple lesions, often with an inflammatory base and in association with acrochordons (skin tags) and acanthosis nigricans. This is termed the sign of Leser-Trélat and alerts the clinician to search for an internal malignancy. Acanthosis nigricans can also be a reflection of an internal malignancy, most commonly of the gastrointestinal tract, and it appears as velvety hyperpigmentation, primarily in flexural areas. However, in the majority of patients, acanthosis nigricans is associated with obesity and insulin resistance,

1	tract, and it appears as velvety hyperpigmentation, primarily in flexural areas. However, in the majority of patients, acanthosis nigricans is associated with obesity and insulin resistance, although it may be a reflection of an endocrinopathy such as acromegaly, Cushing’s syndrome, polycystic ovary syndrome, or insulin-resistant diabetes mellitus (type A, type B, and lipodystrophic forms).

1	A proliferation of melanocytes results in the following pigmented lesions: lentigo, melanocytic nevus, and melanoma (Chap. 105). In an adult, the majority of lentigines are related to sun exposure, which explains their distribution. However, in the Peutz-Jeghers and LEOPARD (lentigines; ECG abnormalities, primarily conduction defects; ocular hypertelorism; pulmonary stenosis and subaortic valvular stenosis; abnormal genitalia [cryptorchidism, hypospadias]; retardation of growth; and deafness [sensorineural]) syndromes, lentigines do serve as a clue to systemic disease. In LEOPARD syndrome, hundreds of lentigines develop during childhood and are scattered over the entire surface of the body. The lentigines in patients with Peutz-Jeghers syndrome are located primarily around the nose and mouth, on the hands and feet, and within the oral cavity. While the pigmented macules on the face may fade with age, the oral lesions persist. However, similar intraoral lesions are also seen in

1	and mouth, on the hands and feet, and within the oral cavity. While the pigmented macules on the face may fade with age, the oral lesions persist. However, similar intraoral lesions are also seen in Addison’s disease, in Laugier-Hunziker syndrome (no internal manifestations), and as a normal finding in darkly pigmented individuals. Patients with this autosomal dominant syndrome (due to mutations in a novel serine threonine kinase gene) have multiple benign polyps of the gastrointestinal tract, testicular or ovarian tumors, and an increased risk of developing gastrointestinal (primarily colon) and pancreatic cancers.

1	In the Carney complex, numerous lentigines are also seen, but they are in association with cardiac myxomas. This autosomal dominant disorder is also known as the LAMB (lentigines, atrial myxomas, mucocutaneous myxomas, and blue nevi) syndrome or NAME (nevi, atrial myxoma, myxoid neurofibroma, and ephelides [freckles]) syndrome. These patients can also have evidence of endocrine overactivity in the form of Cushing’s syndrome (pigmented nodular adrenocortical disease) and acromegaly.

1	The third type of localized hyperpigmentation is due to a local increase in pigment production, and it includes ephelides and café au lait macules (CALMs). While a single CALM can be seen in up to 10% of the normal population, the presence of multiple or large-sized CALMs raises the possibility of an associated genodermatosis, e.g., neurofibromatosis (NF) or McCune-Albright syndrome. CALMs are flat, uniformly brown in color (usually two shades darker than uninvolved skin), and can vary in size from 0.5–12 cm. Approximately 80–90% of adult patients with type I NF will have six or more CALMs measuring ≥1.5 cm in diameter. Additional findings are discussed in the section on neurofibromas (see “Papulonodular Skin Lesions,” below). In comparison with NF, the CALMs in patients with McCune-Albright syndrome (polyostotic fibrous dysplasia with precocious puberty in females due to mosaicism for an activating mutation in a G protein [Gsα] gene) are usually larger, are more irregular in outline,

1	syndrome (polyostotic fibrous dysplasia with precocious puberty in females due to mosaicism for an activating mutation in a G protein [Gsα] gene) are usually larger, are more irregular in outline, and tend to respect the midline.

1	In incontinentia pigmenti, dyskeratosis congenita, and bleomycin pigmentation, the areas of localized hyperpigmentation form a pattern—swirled in the first, reticulated in the second, and flagellate in the third. In dyskeratosis congenita, atrophic reticulated CHAPTER 72 Skin Manifestations of Internal Disease CAuSES of HyPERPigMEnTATion I. Primary cutaneous disorders A. Localized 1. Epidermal alteration a. b. 2. Proliferation of melanocytes a. b. c. 3. Increased pigment production a. b. c. B. 1. Drugs (e.g., minocycline, hydroxychloroquine, bleomycin) II. A. 1. Epidermal alteration a. Seborrheic keratoses (sign of Leser-Trélat) b. Acanthosis nigricans (insulin resistance, other endocrine disorders, paraneoplastic) 2. Proliferation of melanocytes a. b. 3. Increased pigment production a. Café au lait macules (neurofibromatosis, McCune-Albright syndromeb) b. 4. Dermal pigmentation a. b. B. 1. Endocrinopathies a. b. c. d. 2. Metabolic a. b. c.

1	Café au lait macules (neurofibromatosis, McCune-Albright syndromeb) b. 4. Dermal pigmentation a. b. B. 1. Endocrinopathies a. b. c. d. 2. Metabolic a. b. c. Vitamin B12, folate deficiency d. e. Malabsorption, including Whipple’s disease 3. Melanosis secondary to metastatic melanoma 4. a. b. c. 5. Drugs and metals (e.g., arsenic) aAlso lentigines. bPolyostotic fibrous dysplasia. cSee also “Papulonodular Skin Lesions.” dLate 1980s. Abbreviations: LAMB, lentigines, atrial myxomas, mucocutaneous myxomas, and blue nevi; LEOPARD, lentigines, ECG abnormalities, ocular hypertelorism, pulmonary stenosis and subaortic valvular stenosis, abnormal genitalia, retardation of growth, and deafness (sensorineural); NAME, nevi, atrial myxoma, myxoid neurofibroma, and ephelides (freckles); POEMS, polyneuropathy, organomegaly, endocrinopathies, M-protein, and skin changes.

1	PART 2 Cardinal Manifestations and Presentation of Diseases hyperpigmentation is seen on the neck, trunk, and thighs and is accompanied by nail dystrophy, pancytopenia, and leukoplakia of the oral and anal mucosae. The latter often develops into squamous cell carcinoma. In addition to the flagellate pigmentation (linear streaks) on the trunk, patients receiving bleomycin often have hyperpigmentation overlying the elbows, knees, and small joints of the hand.

1	Localized hyperpigmentation is seen as a side effect of several other systemic medications, including those that produce fixed drug reactions (nonsteroidal anti-inflammatory drugs [NSAIDs], sulfonamides, barbiturates, and tetracyclines) and those that can complex with melanin (antimalarials) or iron (minocycline). Fixed drug eruptions recur in the exact same location as circular areas of erythema that can become bullous and then resolve as brown macules. The eruption usually appears within hours of administration of the offending agent, and common locations include the genitalia, distal extremities, and perioral region. Chloroquine and hydroxychloroquine produce gray-brown to blue-black discoloration of the shins, hard palate, and face, while blue macules (often misdiagnosed as bruises) can be seen on the lower extremities and in sites of inflammation with prolonged minocycline administration. Estrogen in oral contraceptives can induce melasma—symmetric brown patches on the face,

1	can be seen on the lower extremities and in sites of inflammation with prolonged minocycline administration. Estrogen in oral contraceptives can induce melasma—symmetric brown patches on the face, especially the cheeks, upper lip, and forehead. Similar changes are seen in pregnancy and in patients receiving phenytoin.

1	In the diffuse forms of hyperpigmentation, the darkening of the skin may be of equal intensity over the entire body or may be accentuated in sun-exposed areas. The causes of diffuse hyperpigmentation can be divided into four major groups—endocrine, metabolic, autoimmune, and drugs. The endocrinopathies that frequently have associated hyperpigmentation include Addison’s disease, Nelson syndrome, and ectopic ACTH syndrome. In these diseases, the increased pigmentation is diffuse but is accentuated in sun-exposed areas, the palmar creases, sites of friction, and scars. An overproduction of the pituitary hormones α-MSH (melanocyte-stimulating hormone) and ACTH can lead to an increase in melanocyte activity. These peptides are products of the proopiomelanocortin gene and exhibit homology; e.g., α-MSH and ACTH share 13 amino acids. A minority of patients with Cushing’s disease or hyperthyroidism have generalized hyperpigmentation.

1	The metabolic causes of hyperpigmentation include porphyria cutanea tarda (PCT), hemochromatosis, vitamin B12 deficiency, folic acid deficiency, pellagra, and malabsorption, including Whipple’s disease. In patients with PCT (see “Vesicles/Bullae,” below), the skin darkening is seen in sun-exposed areas and is a reflection of the photoreactive properties of porphyrins. The increased level of iron in the skin of patients with type 1 hemochromatosis stimulates melanin pigment production and leads to the classic bronze color. Patients with pellagra have a brown discoloration of the skin, especially in sun-exposed areas, as a result of nicotinic acid (niacin) deficiency. In the areas of increased pigmentation, there is a thin, varnish-like scale. These changes are also seen in patients who are vitamin B6 deficient, have functioning carcinoid tumors (increased consumption of niacin), or take isoniazid. Approximately 50% of the patients with Whipple’s disease have an associated generalized

1	vitamin B6 deficient, have functioning carcinoid tumors (increased consumption of niacin), or take isoniazid. Approximately 50% of the patients with Whipple’s disease have an associated generalized hyperpigmentation in association with diarrhea, weight loss, arthritis, and lymphadenopathy. A diffuse, slate-blue to gray-brown color is seen in patients with melanosis secondary to metastatic melanoma. The color reflects widespread deposition of melanin within the dermis as a result of the high concentration of circulating melanin precursors.

1	Of the autoimmune diseases associated with diffuse hyperpigmentation, biliary cirrhosis and systemic sclerosis are the most common, and occasionally, both disorders are seen in the same patient. The skin is dark brown in color, especially in sun-exposed areas. In biliary cirrhosis, the hyperpigmentation is accompanied by pruritus, jaundice, and xanthomas, whereas in systemic sclerosis, it is accompanied by sclerosis of the extremities, face, and, less commonly, the trunk. Additional clues to the diagnosis of systemic sclerosis are mat and periungual telangiectasias, calcinosis cutis, Raynaud’s phenomenon, and distal ulcerations (see “Telangiectasias,” above). The differential diagnosis of cutaneous sclerosis with hyperpigmentation includes the POEMS (polyneuropathy; organomegaly [liver, spleen, lymph nodes]; endocrinopathies [impotence, gynecomastia]; M-protein; and skin changes) syndrome. The skin changes include hyperpigmentation, induration, hypertrichosis, angiomas, clubbing, and

1	lymph nodes]; endocrinopathies [impotence, gynecomastia]; M-protein; and skin changes) syndrome. The skin changes include hyperpigmentation, induration, hypertrichosis, angiomas, clubbing, and facial lipoatrophy.

1	Diffuse hyperpigmentation that is due to drugs or metals can result from one of several mechanisms—induction of melanin pigment formation, complexing of the drug or its metabolites to melanin, and deposits of the drug in the dermis. Busulfan, cyclophosphamide, 5-fluorouracil, and inorganic arsenic induce pigment production. Complexes containing melanin or iron plus the drug or its metabolites are seen in patients receiving minocycline, and a diffuse, blue-gray, muddy appearance within sun-exposed areas may develop, in addition to pigmentation of the mucous membranes, teeth, nails, bones, and thyroid. Administration of amiodarone can result in both a phototoxic eruption (exaggerated sunburn) and/or a slate-gray to violaceous discoloration of sun-exposed skin. Biopsy specimens of the latter show yellow-brown granules in dermal macrophages, which represent intralysosomal accumulations of lipids, amiodarone, and its metabolites. Actual deposits of a particular drug or metal in the skin

1	show yellow-brown granules in dermal macrophages, which represent intralysosomal accumulations of lipids, amiodarone, and its metabolites. Actual deposits of a particular drug or metal in the skin are seen with silver (argyria), where the skin appears blue-gray in color; gold (chrysiasis), where the skin has a brown to blue-gray color; and clofazimine, where the skin appears reddish brown. The associated pigmentation is accentuated in sun-exposed areas, and discoloration of the eye is seen with gold (sclerae) and clofazimine (conjunctivae).

1	(Table 72-12) Depending on their size, cutaneous blisters are referred to as vesicles (<1 cm) or bullae (>1 cm). The primary autoimmune blistering disorders include pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, bullous pemphigoid, gestational pemphigoid, cicatricial pemphigoid, epidermolysis bullosa acquisita, linear IgA bullous dermatosis (LABD), and dermatitis herpetiformis (Chap. 73).

1	Vesicles and bullae are also seen in contact dermatitis, both allergic and irritant forms (Chap. 71). When there is a linear arrangement of vesicular lesions, an exogenous cause or herpes zoster should be suspected. Bullous disease secondary to the ingestion of drugs can take one of several forms, including phototoxic eruptions, isolated bullae, Stevens-Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN) (Chap. 74). Clinically, phototoxic eruptions resemble an exaggerated sunburn with diffuse erythema and bullae in sun-exposed areas. The most commonly associated drugs are doxycycline, quinolones, thiazides, NSAIDs, voriconazole, and psoralens. The development of a phototoxic eruption is dependent on the doses of both the drug and ultraviolet (UV)-A irradiation.

1	Toxic epidermal necrolysis is characterized by bullae that arise on widespread areas of tender erythema and then slough. This results in large areas of denuded skin. The associated morbidity, such as sepsis, and mortality rates are relatively high and are a function of the extent of epidermal necrosis. In addition, these patients may also have involvement of the mucous membranes and respiratory and intestinal tracts. Drugs are the primary cause of TEN, and the most common offenders are aromatic anticonvulsants (phenytoin, barbiturates, carbamazepine), sulfonamides, aminopenicillins, allopurinol, and NSAIDs. Severe acute graft-versus-host disease (grade 4), van-comycin-induced LABD, and the acute syndrome of apoptotic panepidermolysis (ASAP) in patients with lupus can also resemble TEN.

1	In erythema multiforme (EM), the primary lesions are pink-red macules and edematous papules, the centers of which may become vesicular. In contrast to a morbilliform exanthem, the clue to the diagnosis of EM, and especially SJS, is the development of a “dusky” violet color in the center of the lesions. Target lesions are also characteristic of EM and arise as a result of active centers and borders in combination with centrifugal spread. However, target lesions need not be present to make the diagnosis of EM. EM has been subdivided into two major groups: (1) EM minor due to herpes simplex virus (HSV) and (2) EM major due to HSV; Mycoplasma pneumoniae; or, occasionally, drugs. Involvement of the CAuSES of vESiCLES/BuLLAE I. Primary mucocutaneous diseases A. Primary blistering diseases (autoimmune) 1. Pemphigus, foliaceus and vulgarisa 2. 3. 4. 5. Dermatitis herpetiformisb,c 6. 7. Epidermolysis bullosa acquisitab,d B. Secondary blistering diseases 1.

1	Pemphigus, foliaceus and vulgarisa 2. 3. 4. 5. Dermatitis herpetiformisb,c 6. 7. Epidermolysis bullosa acquisitab,d B. Secondary blistering diseases 1. Contact dermatitisa,b 2. 3. 4. C. Infections 1. Varicella-zoster virusa,f 2. Herpes simplex virusa,f 3. Enteroviruses, e.g., hand-foot-and-mouth diseasef 4. Staphylococcal scalded-skin syndromea,g 5. II. A. 1. Paraneoplastic pemphigusa B. Infections 1. Cutaneous embolib C. Metabolic 1. Diabetic bullaea,b 2. 3. 4. 5. Bullous dermatosis of hemodialysisb D. Ischemia 1. Coma bullae aIntraepidermal. bSubepidermal. cAssociated with gluten enteropathy. dAssociated with inflammatory bowel disease. eDegeneration of cells within the basal layer of the epidermis can give impression split is subepidermal. fAlso systemic. gIn adults, associated with renal failure and immunocompromised state.

1	mucous membranes (ocular, nasal, oral, and genital) is seen more commonly in the latter form. Hemorrhagic crusts of the lips are characteristic of EM major and SJS as well as herpes simplex, pemphigus vulgaris, and paraneoplastic pemphigus. Fever, malaise, myalgias, sore throat, and cough may precede or accompany the eruption. The lesions of EM usually resolve over 2–4 weeks but may be recurrent, especially when due to HSV. In addition to HSV (in which lesions usually appear 7–12 days after the viral eruption), EM can also follow vaccinations, radiation therapy, and exposure to environmental toxins, including the oleoresin in poison ivy.

1	Induction of SJS is most often due to drugs, especially sulfonamides, phenytoin, barbiturates, lamotrigine, aminopenicillins, nonnucleoside reverse transcriptase inhibitors (e.g., nevirapine), and carbamazepine. Widespread dusky macules and significant mucosal involvement are characteristic of SJS, and the cutaneous lesions may or may not develop epidermal detachment. If the latter occurs, by definition, it is limited to <10% of the body surface area (BSA). Greater involvement leads to the diagnosis of SJS/TEN overlap (10–30% BSA) or TEN (>30% BSA). In addition to primary blistering disorders and hypersensitivity reactions, bacterial and viral infections can lead to vesicles and bullae. The most common infectious agents are HSV (Chap. 216), varicellazoster virus (Chap. 217), and S. aureus (Chap. 172). CHAPTER 72 Skin Manifestations of Internal Disease

1	Staphylococcal scalded-skin syndrome (SSSS) and bullous impetigo are two blistering disorders associated with staphylococcal (phage group II) infection. In SSSS, the initial findings are redness and tenderness of the central face, neck, trunk, and intertriginous zones. This is followed by short-lived flaccid bullae and a slough or exfoliation of the superficial epidermis. Crusted areas then develop, characteristically around the mouth in a radial pattern. SSSS is distinguished from TEN by the following features: younger age group (primarily infants), more superficial site of blister formation, no oral lesions, shorter course, lower morbidity and mortality rates, and an association with staphylococcal exfoliative toxin (“exfoliatin”), not drugs. A rapid diagnosis of SSSS versus TEN can be made by a frozen section of the blister roof or exfoliative cytology of the blister contents. In SSSS, the site of staphylococcal infection is usually extracutaneous (conjunctivitis, rhinorrhea,

1	be made by a frozen section of the blister roof or exfoliative cytology of the blister contents. In SSSS, the site of staphylococcal infection is usually extracutaneous (conjunctivitis, rhinorrhea, otitis media, pharyngitis, tonsillitis), and the cutaneous lesions are sterile, whereas in bullous impetigo, the skin lesions are the site of infection. Impetigo is more localized than SSSS and usually presents with honey-colored crusts. Occasionally, superficial purulent blisters also form. Cutaneous emboli from gram-negative infections may present as isolated bullae, but the base of the lesion is purpuric or necrotic, and it may develop into an ulcer (see “Purpura,” below).

1	Several metabolic disorders are associated with blister formation, including diabetes mellitus, renal failure, and porphyria. Local hypoxemia secondary to decreased cutaneous blood flow can also produce blisters, which explains the presence of bullae over pressure points in comatose patients (coma bullae). In diabetes mellitus, tense bullae with clear sterile viscous fluid arise on normal skin. The lesions can be as large as 6 cm in diameter and are located on the distal extremities. There are several types of porphyria, but the most common form with cutaneous findings is porphyria cutanea tarda (PCT). In sun-exposed areas (primarily the face and hands), the skin is very fragile, with trauma leading to erosions mixed with tense vesicles. These lesions then heal with scarring and formation of milia; the latter are firm, 1to 2-mm white or yellow papules that represent epidermoid inclusion cysts. Associated findings can include hypertrichosis of the lateral malar region (men) or face

1	of milia; the latter are firm, 1to 2-mm white or yellow papules that represent epidermoid inclusion cysts. Associated findings can include hypertrichosis of the lateral malar region (men) or face (women) and, in sun-exposed areas, hyperpigmentation and firm sclerotic plaques. An elevated level of urinary uroporphyrins confirms the diagnosis and is due to a decrease in uroporphyrinogen decarboxylase activity. PCT can be exacerbated by alcohol, hemochromatosis and other forms of iron overload, chlorinated hydrocarbons, hepatitis C and HIV infections, and hepatomas.

1	The differential diagnosis of PCT includes (1) porphyria variegata— the skin signs of PCT plus the systemic findings of acute intermittent porphyria; it has a diagnostic plasma porphyrin fluorescence emission at 626 nm; (2) drug-induced pseudoporphyria—the clinical and histologic findings are similar to PCT, but porphyrins are normal; etiologic agents include naproxen and other NSAIDs, furosemide, tetracycline, and voriconazole; (3) bullous dermatosis of hemodialysis—the same appearance as PCT, but porphyrins are usually normal or occasionally borderline elevated; patients have chronic renal failure and are on hemodialysis; (4) PCT associated with hepatomas and hemodialysis; and (5) epidermolysis bullosa acquisita (Chap. 73).

1	(Table 72-13) Exanthems are characterized by an acute generalized eruption. The most common presentation is erythematous macules and papules (morbilliform) and less often confluent blanching erythema (scarlatiniform). Morbilliform eruptions are usually due to either drugs or viral infections. For example, up to 5% of patients receiving penicillins, sulfonamides, phenytoin, or nevirapine will develop a maculopapular eruption. Accompanying signs may include pruritus, fever, eosinophilia, and transient lymphadenopathy. Similar maculopapular eruptions are seen in the classic childhood viral exanthems, including (1) rubeola (measles)—a prodrome of coryza, cough, and conjunctivitis followed by Koplik’s spots on the buccal mucosa; the eruption begins behind the ears, at the hairline, and on the forehead and then spreads down the body, often becoming confluent; (2) rubella—the eruption begins on the forehead and face PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases CAuSES of ExAnTHEMS I. Morbilliform A. Drugs B. Viral 1. 2. 3. Erythema infectiosum (erythema of cheeks; reticulated on extremities) 4. Epstein-Barr virus, echovirus, coxsackievirus, CMV, adenovirus, HHV-6/ HHV-7a, dengue virus, and West Nile virus infections 5. C. Bacterial 1. 2. 3. 4. 5. D. Acute graft-versus-host disease E. Kawasaki disease II. A. B. C. D. Early staphylococcal scalded-skin syndrome aPrimary infection in infants and reactivation in the setting of immunosuppression. Abbreviations: CMV, cytomegalovirus; HHV, human herpesvirus; HIV, human immunodeficiency virus.

1	Abbreviations: CMV, cytomegalovirus; HHV, human herpesvirus; HIV, human immunodeficiency virus. and then spreads down the body; it resolves in the same order and is associated with retroauricular and suboccipital lymphadenopathy; and (3) erythema infectiosum (fifth disease)—erythema of the cheeks is followed by a reticulated pattern on the extremities; it is secondary to a parvovirus B19 infection, and an associated arthritis is seen in adults.

1	Both measles and rubella can occur in unvaccinated adults, and an atypical form of measles is seen in adults immunized with either killed measles vaccine or killed vaccine followed in time by live vaccine. In contrast to classic measles, the eruption of atypical measles begins on the palms, soles, wrists, and ankles, and the lesions may become purpuric. The patient with atypical measles can have pulmonary involvement and be quite ill. Rubelliform and roseoliform eruptions are also associated with Epstein-Barr virus (5–15% of patients), echovirus, coxsackievirus, cytomegalovirus, adenovirus, dengue virus, and West Nile virus infections. Detection of specific IgM antibodies or fourfold elevations in IgG antibodies allow the proper diagnosis, but polymerase chain reaction (PCR) is gradually replacing serologic assays. Occasionally, a maculopapular drug eruption is a reflection of an underlying viral infection. For example, ~95% of the patients with infectious mononucleosis who are given

1	replacing serologic assays. Occasionally, a maculopapular drug eruption is a reflection of an underlying viral infection. For example, ~95% of the patients with infectious mononucleosis who are given ampicillin will develop a rash.

1	Of note, early in the course of infections with Rickettsia and meningococcus, prior to the development of petechiae and purpura, the lesions may be erythematous macules and papules. This is also the case in chickenpox prior to the development of vesicles. Maculopapular eruptions are associated with early HIV infection, early secondary syphilis, typhoid fever, and acute graft-versus-host disease. In the last, lesions frequently begin on the dorsal hands and forearms; the macular rose spots of typhoid fever involve primarily the anterior trunk.

1	The prototypic scarlatiniform eruption is seen in scarlet fever and is due to an erythrogenic toxin produced by bacteriophage-containing group A β-hemolytic streptococci, most commonly in the setting of pharyngitis. This eruption is characterized by diffuse erythema, which begins on the neck and upper trunk, and red follicular puncta. Additional findings include a white strawberry tongue (white coating with red papillae) followed by a red strawberry tongue (red tongue with red papillae); petechiae of the palate; a facial flush with circumoral pallor; linear petechiae in the antecubital fossae; and desquamation of the involved skin, palms, and soles 5–20 days after onset of the eruption. A similar desquamation of the palms and soles is seen in toxic shock syndrome (TSS), in Kawasaki disease, and after severe febrile illnesses. Certain strains of staphylococci also produce an erythrotoxin that leads to the same clinical findings as in streptococcal scarlet fever, except that the

1	disease, and after severe febrile illnesses. Certain strains of staphylococci also produce an erythrotoxin that leads to the same clinical findings as in streptococcal scarlet fever, except that the anti-streptolysin O or -DNase B titers are not elevated.

1	In toxic shock syndrome, staphylococcal (phage group I) infections produce an exotoxin (TSST-1) that causes the fever and rash as well as enterotoxins. Initially, the majority of cases were reported in menstruating women who were using tampons. However, other sites of infection, including wounds and nasal packing, can lead to TSS. The diagnosis of TSS is based on clinical criteria (Chap. 172), and three of these involve mucocutaneous sites (diffuse erythema of the skin, desquamation of the palms and soles 1–2 weeks after onset of illness, and involvement of the mucous membranes). The latter is characterized as hyperemia of the vagina, oropharynx, or conjunctivae. Similar systemic findings have been described in streptococcal toxic shock syndrome (Chap. 173), and although an exanthem is seen less often than in TSS due to a staphylococcal infection, the underlying infection is often in the soft tissue (e.g., cellulitis).

1	The cutaneous eruption in Kawasaki disease (Chap. 385) is polymorphous, but the two most common forms are morbilliform and scarlatiniform. Additional mucocutaneous findings include bilateral conjunctival injection; erythema and edema of the hands and feet followed by desquamation; and diffuse erythema of the oropharynx, red strawberry tongue, and dry fissured lips. This clinical picture can resemble TSS and scarlet fever, but clues to the diagnosis of Kawasaki disease are cervical lymphadenopathy, cheilitis, and thrombocytosis. The most serious associated systemic finding in this disease is coronary aneurysms secondary to arteritis. Scarlatiniform eruptions are also seen in the early phase of SSSS (see “Vesicles/Bullae,” above), in young adults with Arcanobacterium haemolyticum infection, and as reactions to drugs.

1	(Table 72-14) Urticaria (hives) are transient lesions that are composed of a central wheal surrounded by an erythematous halo or flare. Individual lesions are round, oval, or figurate and are often pruritic. Acute and chronic urticaria have a wide variety of allergic etiologies and reflect edema in the dermis. Urticarial lesions can also be seen in patients with mastocytosis (urticaria pigmentosa), hypoor hyperthyroidism, and systemic-onset juvenile idiopathic arthritis (Still’s disease). In both juvenileand adult-onset Still’s disease, the lesions CAuSES of uRTiCARiA AnD AngioEDEMA I. Primary cutaneous disorders A. Acute and chronic urticariaa B. Physical urticaria 1. 2. 3. 4. C. Angioedema (hereditary and acquired)b,c II. A. B. C. D. aA small minority develop anaphylaxis. bAlso systemic. cAcquired angioedema can be idiopathic, associated with a lymphoproliferative disorder, or due to a drug, e.g., angiotensin-converting enzyme (ACE) inhibitors.

1	coincide with the fever spike, are transient, and are due to dermal 363 infiltrates of neutrophils.

1	The common physical urticarias include dermatographism, solar urticaria, cold urticaria, and cholinergic urticaria. Patients with dermatographism exhibit linear wheals following minor pressure or scratching of the skin. It is a common disorder, affecting ~5% of the population. Solar urticaria characteristically occurs within minutes of sun exposure and is a skin sign of one systemic disease—erythropoietic protoporphyria. In addition to the urticaria, these patients have subtle pitted scarring of the nose and hands. Cold urticaria is precipitated by exposure to the cold, and therefore exposed areas are usually affected. In occasional patients, the disease is associated with abnormal circulating proteins—more commonly cryoglobulins and less commonly cryofibrinogens. Additional systemic symptoms include wheezing and syncope, thus explaining the need for these patients to avoid swimming in cold water. Autosomal dominantly inherited cold urticaria is associated with dysfunction of

1	symptoms include wheezing and syncope, thus explaining the need for these patients to avoid swimming in cold water. Autosomal dominantly inherited cold urticaria is associated with dysfunction of cryopyrin. Cholinergic urticaria is precipitated by heat, exercise, or emotion and is characterized by small wheals with relatively large flares. It is occasionally associated with wheezing.

1	Whereas urticarias are the result of dermal edema, subcutaneous edema leads to the clinical picture of angioedema. Sites of involvement include the eyelids, lips, tongue, larynx, and gastrointestinal tract as well as the subcutaneous tissue. Angioedema occurs alone or in combination with urticaria, including urticarial vasculitis and the physical urticarias. Both acquired and hereditary (autosomal dominant) forms of angioedema occur (Chap. 376), and in the latter, urticaria is rarely, if ever, seen.

1	Urticarial vasculitis is an immune complex disease that may be confused with simple urticaria. In contrast to simple urticaria, individual lesions tend to last longer than 24 h and usually develop central petechiae that can be observed even after the urticarial phase has resolved. The patient may also complain of burning rather than pruritus. On biopsy, there is a leukocytoclastic vasculitis of the small dermal blood vessels. Although many cases of urticarial vasculitis are idiopathic in origin, it can be a reflection of an underlying systemic illness such as lupus erythematosus, Sjögren’s syndrome, or hereditary complement deficiency. There is a spectrum of urticarial vasculitis that ranges from purely cutaneous to multisystem involvement. The most common systemic signs and symptoms are arthralgias and/or arthritis, nephritis, and crampy abdominal pain, with asthma and chronic obstructive lung disease seen less often. Hypocomplementemia occurs in oneto two-thirds of patients, even in

1	and/or arthritis, nephritis, and crampy abdominal pain, with asthma and chronic obstructive lung disease seen less often. Hypocomplementemia occurs in oneto two-thirds of patients, even in the idiopathic cases. Urticarial vasculitis can also be seen in patients with hepatitis B and hepatitis C infections, serum sickness, and serum sickness–like illnesses (e.g., due to cefaclor, minocycline).

1	(Table 72-15) In the papulonodular diseases, the lesions are elevated above the surface of the skin and may coalesce to form larger plaques. The location, consistency, and color of the lesions are the keys to their diagnosis; this section is organized on the basis of color.

1	In calcinosis cutis, there are firm white to white-yellow papules with an irregular surface. When the contents are expressed, a chalky white material is seen. Dystrophic calcification is seen at sites of previous inflammation or damage to the skin. It develops in acne scars as well as on the distal extremities of patients with systemic sclerosis and in the subcutaneous tissue and intermuscular fascial planes in DM. The latter is more extensive and is more commonly seen in children. An elevated calcium phosphate product, most commonly due to secondary hyperparathyroidism in the setting of renal failure, can lead to nodules of metastatic calcinosis cutis, which tend to be subcutaneous and periarticular. These patients can also develop calcification of muscular arteries and subsequent ischemic necrosis (calciphylaxis). Osteoma cutis, in the form of small papules, most commonly occurs on the face CHAPTER 72 Skin Manifestations of Internal Disease

1	CHAPTER 72 Skin Manifestations of Internal Disease PART 2 Cardinal Manifestations and Presentation of Diseases I. White A. Calcinosis cutis B. Osteoma cutis (also skin-colored or blue) II. A. B. C. Angiofibromas (tuberous sclerosis, MEN syndrome, type 1) D. Neuromas (MEN syndrome, type 2b) E. 1. 2. F. Osteomas (arise in skull and jaw in Gardner syndrome) G. Primary cutaneous disorders 1. 2. III. A. Amyloidosis, primary systemic B. C. IV. A. B. C. D. E. V. A. 1. 2. B. Papules/plaques 1. 2. 3. 4. C. Nodules 1. 2. Medium-sized vessel vasculitis (e.g., cutaneous polyarteritis nodosa) D. Primary cutaneous disorders 1. 2. 3. Infections, e.g., streptococcal cellulitis, sporotrichosis 4. 5. VI. A. B. C. D. VII. A. Venous malformations (e.g., blue rubber bleb syndrome) B. 1. 2. VIII. A. B. C. IX. A. B. C. X. XI. A.

1	5. VI. A. B. C. D. VII. A. Venous malformations (e.g., blue rubber bleb syndrome) B. 1. 2. VIII. A. B. C. IX. A. B. C. X. XI. A. aIf multiple with childhood onset, consider Gardner syndrome. bMay have darker hue in more darkly pigmented individuals. cSee also “Hyperpigmentation.” Abbreviation: MEN, multiple endocrine neoplasia. of individuals with a history of acne vulgaris, whereas plate-like lesions occur in rare genetic syndromes (Chap. 82).

1	There are several types of skin-colored lesions, including epidermoid inclusion cysts, lipomas, rheumatoid nodules, neurofibromas, angiofibromas, neuromas, and adnexal tumors such as tricholemmomas. Both epidermoid inclusion cysts and lipomas are very common mobile subcutaneous nodules—the former are rubbery and drain cheese-like material (sebum and keratin) if incised. Lipomas are firm and somewhat lobulated on palpation. When extensive facial epidermoid inclusion cysts develop during childhood or there is a family history of such lesions, the patient should be examined for other signs of Gardner syndrome, including osteomas and desmoid tumors. Rheumatoid nodules are firm 0.5to 4-cm nodules that favor the extensor aspect of joints, especially the elbows. They are seen in ~20% of patients with rheumatoid arthritis and 6% of patients with Still’s disease. Biopsies of the nodules show palisading granulomas. Similar lesions that are smaller and shorter-lived are seen in rheumatic fever.

1	Neurofibromas (benign Schwann cell tumors) are soft papules or nodules that exhibit the “button-hole” sign; that is, they invaginate into the skin with pressure in a manner similar to a hernia. Single lesions are seen in normal individuals, but multiple neurofibromas, usually in combination with six or more CALMs measuring >1.5 cm (see “Hyperpigmentation,” above), axillary freckling, and multiple Lisch nodules, are seen in von Recklinghausen’s disease (NF type I) (Chap. 118). In some patients, the neurofibromas are localized and unilateral due to somatic mosaicism.

1	Angiofibromas are firm pink to skin-colored papules that measure from 3 mm to a few centimeters in diameter. When multiple lesions are located on the central cheeks (adenoma sebaceum), the patient has tuberous sclerosis or multiple endocrine neoplasia (MEN) syndrome, type 1. The former is an autosomal disorder due to mutations in two different genes, and the associated findings are discussed in the section on ash leaf spots as well as in Chap. 118. Neuromas (benign proliferations of nerve fibers) are also firm, skin-colored papules. They are more commonly found at sites of amputation and as rudimentary supernumerary digits. However, when there are multiple neuromas on the eyelids, lips, distal tongue, and/or oral mucosa, the patient should be investigated for other signs of the MEN syndrome, type 2b. Associated findings include marfanoid habitus, protuberant lips, intestinal ganglioneuromas, and medullary thyroid carcinoma (>75% of patients; Chap. 408).

1	Adnexal tumors are derived from pluripotent cells of the epidermis that can differentiate toward hair, sebaceous, apocrine, or eccrine glands or remain undifferentiated. Basal cell carcinomas (BCCs) are examples of adnexal tumors that have little or no evidence of differentiation. Clinically, they are translucent papules with rolled borders, telangiectasias, and central erosion. BCCs commonly arise in sun-damaged skin of the head and neck as well as the upper trunk. When a patient has multiple BCCs, especially prior to age 30, the possibility of the nevoid basal cell carcinoma syndrome should be raised. It is inherited as an autosomal dominant trait and is associated with jaw cysts, palmar and plantar pits, frontal bossing, medulloblastomas, and calcification of the falx cerebri and diaphragma sellae. Tricholemmomas are also skin-colored adnexal tumors but differentiate toward hair follicles and can have a wartlike appearance. The presence of multiple tricholemmomas on the face and

1	sellae. Tricholemmomas are also skin-colored adnexal tumors but differentiate toward hair follicles and can have a wartlike appearance. The presence of multiple tricholemmomas on the face and cobblestoning of the oral mucosa points to the diagnosis of Cowden disease (multiple hamartoma syndrome) due to mutations in the phosphatase and tensin homolog (PTEN) gene. Internal organ involvement (in decreasing order of frequency) includes fibrocystic disease and carcinoma of the breast, adenomas and carcinomas of the thyroid, and gastrointestinal polyposis. Keratoses of the palms, soles, and dorsal aspect of the hands are also seen.

1	The cutaneous lesions associated with primary systemic amyloidosis are often pink in color and translucent. Common locations are the face, especially the periorbital and perioral regions, and flexural areas. On biopsy, homogeneous deposits of amyloid are seen in the dermis and in the walls of blood vessels; the latter lead to an increase in vessel wall fragility. As a result, petechiae and purpura develop in clinically normal skin as well as in lesional skin following minor trauma, hence the term pinch purpura. Amyloid deposits are also seen in the striated muscle of the tongue and result in macroglossia.

1	Even though specific mucocutaneous lesions are present in only ~30% of the patients with primary systemic (AL) amyloidosis, the diagnosis can be made via histologic examination of abdominal subcutaneous fat, in conjunction with a serum free light chain assay. By special staining, amyloid deposits are seen around blood vessels or individual fat cells in 40–50% of patients. There are also three forms of amyloidosis that are limited to the skin and that should not be construed as cutaneous lesions of systemic amyloidosis. They are macular amyloidosis (upper back), lichen amyloidosis (usually lower extremities), and nodular amyloidosis. In macular and lichen amyloidosis, the deposits are composed of altered epidermal keratin. Early-onset macular and lichen amyloidosis have been associated with MEN syndrome, type 2a.

1	Patients with multicentric reticulohistiocytosis also have pink-colored papules and nodules on the face and mucous membranes as well as on the extensor surface of the hands and forearms. They have a polyarthritis that can mimic rheumatoid arthritis clinically. On histologic examination, the papules have characteristic giant cells that are not seen in biopsies of rheumatoid nodules. Pink to skin-colored papules that are firm, 2–5 mm in diameter, and often in a linear arrangement are seen in patients with papular mucinosis. This disease is also referred to as generalized lichen myxedematosus or scleromyxedema. The latter name comes from the induration of the face and extremities that may accompany the papular eruption. Biopsy specimens of the papules show localized mucin deposition, and serum protein electrophoresis plus immunofixation electrophoresis demonstrates a monoclonal spike of IgG, usually with a λ light chain.

1	Several systemic disorders are characterized by yellow-colored cutaneous papules or plaques—hyperlipidemia (xanthomas), gout (tophi), diabetes (necrobiosis lipoidica), pseudoxanthoma elasticum, and Muir-Torre syndrome (sebaceous tumors). Eruptive xanthomas are the most common form of xanthomas and are associated with hypertriglyceridemia (primarily hyperlipoproteinemia types I, IV, and V). Crops of yellow papules with erythematous halos occur primarily on the extensor surfaces of the extremities and the buttocks, and they spontaneously involute with a fall in serum triglycerides. Types II and III result in one or more of the following types of xanthoma: xanthelasma, tendon xanthomas, and plane xanthomas. Xanthelasma are found on the eyelids, whereas tendon xanthomas are frequently associated with the Achilles and extensor finger tendons; plane xanthomas are flat and favor the palmar creases, neck, upper trunk, and flexural folds. Tuberous xanthomas are frequently associated with

1	with the Achilles and extensor finger tendons; plane xanthomas are flat and favor the palmar creases, neck, upper trunk, and flexural folds. Tuberous xanthomas are frequently associated with hypertriglyceridemia, but they are also seen in patients with hypercholesterolemia and are found most 365 frequently over the large joints or hand. Biopsy specimens of xanthomas show collections of lipid-containing macrophages (foam cells).

1	Patients with several disorders, including biliary cirrhosis, can have a secondary form of hyperlipidemia with associated tuberous and plane xanthomas. However, patients with plasma cell dyscrasias have normolipemic plane xanthomas. This latter form of xanthoma may be ≥12 cm in diameter and is most frequently seen on the upper trunk or side of the neck. It is important to note that the most common setting for eruptive xanthomas is uncontrolled diabetes mellitus. The least specific sign for hyperlipidemia is xanthelasma, because at least 50% of the patients with this finding have normal lipid profiles.

1	In tophaceous gout, there are deposits of monosodium urate in the skin around the joints, particularly those of the hands and feet. Additional sites of tophi formation include the helix of the ear and the olecranon and prepatellar bursae. The lesions are firm, yellow in color, and occasionally discharge a chalky material. Their size varies from 1 mm to 7 cm, and the diagnosis can be established by polarized light microscopy of the aspirated contents of a lesion. Lesions of necrobiosis lipoidica are found primarily on the shins (90%), and patients can have diabetes mellitus or develop it subsequently. Characteristic findings include a central yellow color, atrophy (transparency), telangiectasias, and a red to red-brown border. Ulcerations can also develop within the plaques. Biopsy specimens show necrobiosis of collagen and granulomatous inflammation.

1	In pseudoxanthoma elasticum (PXE), due to mutations in the gene ABCC6, there is an abnormal deposition of calcium on the elastic fibers of the skin, eye, and blood vessels. In the skin, the flexural areas such as the neck, axillae, antecubital fossae, and inguinal area are the primary sites of involvement. Yellow papules coalesce to form reticulated plaques that have an appearance similar to that of plucked chicken skin. In severely affected skin, hanging, redundant folds develop. Biopsy specimens of involved skin show swollen and irregularly clumped elastic fibers with deposits of calcium. In the eye, the calcium deposits in Bruch’s membrane lead to angioid streaks and choroiditis; in the arteries of the heart, kidney, gastrointestinal tract, and extremities, the deposits lead to angina, hypertension, gastrointestinal bleeding, and claudication, respectively.

1	Adnexal tumors that have differentiated toward sebaceous glands include sebaceous adenoma, sebaceous carcinoma, and sebaceous hyperplasia. Except for sebaceous hyperplasia, which is commonly seen on the face, these tumors are fairly rare. Patients with Muir-Torre syndrome have one or more sebaceous adenoma(s), and they can also have sebaceous carcinomas and sebaceous hyperplasia as well as keratoacanthomas. The internal manifestations of Muir-Torre syndrome include multiple carcinomas of the gastrointestinal tract (primarily colon) as well as cancers of the larynx, genitourinary tract, and breast.

1	Cutaneous lesions that are red in color have a wide variety of etiologies; in an attempt to simplify their identification, they will be subdivided into papules, papules/plaques, and subcutaneous nodules. Common red papules include arthropod bites and cherry hemangiomas; the latter are small, bright-red, dome-shaped papules that represent a benign proliferation of capillaries. In patients with AIDS (Chap. 226), the development of multiple red hemangioma-like lesions points to bacillary angiomatosis, and biopsy specimens show clusters of bacilli that stain positive with the Warthin-Starry stain; the pathogens have been identified as Bartonella henselae and Bartonella quintana. Disseminated visceral disease is seen primarily in immunocompromised hosts but can occur in immunocompetent individuals.

1	Multiple angiokeratomas are seen in Fabry disease, an X-linked recessive lysosomal storage disease that is due to a deficiency of α-galactosidase A. The lesions are red to red-blue in color and can be quite small in size (1–3 mm), with the most common location being the lower trunk. Associated findings include chronic renal disease, peripheral neuropathy, and corneal opacities (cornea verticillata). Electron photomicrographs of angiokeratomas and clinically normal skin demonstrate lamellar lipid deposits in fibroblasts, pericytes, and endothelial

1	CHAPTER 72 Skin Manifestations of Internal Disease 366 cells that are diagnostic of this disease. Widespread acute eruptions of erythematous papules are discussed in the section on exanthems. There are several infectious diseases that present as erythematous papules or nodules in a lymphocutaneous or sporotrichoid pattern, i.e., in a linear arrangement along the lymphatic channels. The two most common etiologies are Sporothrix schenckii (sporotrichosis) and the atypical mycobacterium Mycobacterium marinum. The organisms are introduced as a result of trauma, and a primary inoculation site is often seen in addition to the lymphatic nodules. Additional causes include Nocardia, Leishmania, and other atypical mycobacteria and dimorphic fungi; culture of lesional tissue will aid in the diagnosis. The diseases that are characterized by erythematous plaques with scale are reviewed in the papulosquamous section, and the various forms of dermatitis are discussed in the section on erythroderma.

1	The diseases that are characterized by erythematous plaques with scale are reviewed in the papulosquamous section, and the various forms of dermatitis are discussed in the section on erythroderma. Additional disorders in the differential diagnosis of red papules/ plaques include cellulitis, polymorphous light eruption (PMLE), cutaneous lymphoid hyperplasia (lymphocytoma cutis), cutaneous lupus, lymphoma cutis, and leukemia cutis. The first three diseases represent primary cutaneous disorders, although cellulitis may be accompanied by a bacteremia. PMLE is characterized by erythematous papules and plaques in a primarily sun-exposed distribution—dorsum of the hand, extensor forearm, and upper trunk. Lesions follow exposure to UV-B and/or UV-A, and in higher latitudes, PMLE is most severe in the late spring and early summer. A process referred to as “hardening” occurs with continued UV exposure, and the eruption fades, but in temperate climates, it will recur in the spring. PMLE must be

1	the late spring and early summer. A process referred to as “hardening” occurs with continued UV exposure, and the eruption fades, but in temperate climates, it will recur in the spring. PMLE must be differentiated from cutaneous lupus, and this is accomplished by observation of the natural history, histologic examination, and direct immunofluorescence of the lesions. Cutaneous lymphoid hyperplasia (pseudolymphoma) is a benign polyclonal proliferation of lymphocytes in the skin that presents as infiltrated pink-red to red-purple papules and plaques; it must be distinguished from lymphoma cutis. Several types of red plaques are seen in patients with systemic lupus, including (1) erythematous urticarial plaques across the cheeks and nose in the classic butterfly rash; (2) erythematous discoid lesions with fine or “carpet-tack” scale, telangiectasias, central hypopigmentation, peripheral hyperpigmentation, follicular plugging, and atrophy located on the face, scalp, external ears, arms,

1	lesions with fine or “carpet-tack” scale, telangiectasias, central hypopigmentation, peripheral hyperpigmentation, follicular plugging, and atrophy located on the face, scalp, external ears, arms, and upper trunk; and (3) psoriasiform or annular lesions of subacute cutaneous lupus with hypopigmented centers located primarily on the extensor arms and upper trunk. Additional mucocutaneous findings include (1) a violaceous flush on the face and V of the neck; (2) photosensitivity; (3) urticarial vasculitis (see “Urticaria,” above); (4) lupus panniculitis (see below); (5) diffuse alopecia; (6) alopecia secondary to discoid lesions; (7) periungual telangiectasias and erythema; (8) EM-like lesions that may become bullous; (9) oral ulcers; and (10) distal ulcerations secondary to Raynaud’s phenomenon, vasculitis, or livedoid vasculopathy. Patients with only discoid lesions usually have the form of lupus that is limited to the skin. However, up to 10% of these patients eventually develop

1	vasculitis, or livedoid vasculopathy. Patients with only discoid lesions usually have the form of lupus that is limited to the skin. However, up to 10% of these patients eventually develop systemic lupus. Direct immunofluorescence of involved skin, in particular discoid lesions, shows deposits of IgG or IgM and C3 in a granular distribution along the dermal-epidermal junction.

1	In lymphoma cutis, there is a proliferation of malignant lymphocytes in the skin, and the clinical appearance resembles that of cutaneous lymphoid hyperplasia—infiltrated pink-red to red-purple papules and plaques. Lymphoma cutis can occur anywhere on the surface of the skin, whereas the sites of predilection for lymphocytomas include the malar ridge, tip of the nose, and earlobes. Patients with nonHodgkin’s lymphomas have specific cutaneous lesions more often than those with Hodgkin’s disease, and, occasionally, the skin nodules precede the development of extracutaneous non-Hodgkin’s lymphoma or represent the only site of involvement (e.g., primary cutaneous B cell lymphoma). Arcuate lesions are sometimes seen in lymphoma and lymphocytoma cutis as well as in CTCL. Adult T cell leukemia/ lymphoma that develops in association with HTLV-1 infection is characterized by cutaneous plaques, hypercalcemia, and circulating CD25+ lymphocytes. Leukemia cutis has the same appearance as lymphoma

1	lymphoma that develops in association with HTLV-1 infection is characterized by cutaneous plaques, hypercalcemia, and circulating CD25+ lymphocytes. Leukemia cutis has the same appearance as lymphoma cutis, and specific lesions are seen more commonly in monocytic leukemias than in lymphocytic or granulocytic leukemias. Cutaneous

1	PART 2 Cardinal Manifestations and Presentation of Diseases chloromas (granulocytic sarcomas) may precede the appearance of circulating blasts in acute myelogenous leukemia and, as such, represent a form of aleukemic leukemia cutis.

1	Sweet syndrome is characterized by pink-red to red-brown edematous plaques that are frequently painful and occur primarily on the head, neck, and upper (and, less often, lower) extremities. The patients also have fever, neutrophilia, and a dense dermal infiltrate of neutrophils in the lesions. In ~10% of the patients, there is an associated malignancy, most commonly acute myelogenous leukemia. Sweet syndrome has also been reported with inflammatory bowel disease, systemic lupus erythematosus, and solid tumors (primarily of the genitourinary tract) as well as drugs (e.g., all-trans-retinoic acid, granulocyte colony-stimulating factor [G-CSF]). The differential diagnosis includes neutrophilic eccrine hidradenitis; bullous forms of pyoderma gangrenosum; and, occasionally, cellulitis. Extracutaneous sites of involvement include joints, muscles, eye, kidney (proteinuria, occasionally glomerulonephritis), and lung (neutrophilic infiltrates). The idiopathic form of Sweet syndrome is seen

1	sites of involvement include joints, muscles, eye, kidney (proteinuria, occasionally glomerulonephritis), and lung (neutrophilic infiltrates). The idiopathic form of Sweet syndrome is seen more often in women, following a respiratory tract infection.

1	Common causes of erythematous subcutaneous nodules include inflamed epidermoid inclusion cysts, acne cysts, and furuncles. Panniculitis, an inflammation of the fat, also presents as subcutaneous nodules and is frequently a sign of systemic disease. There are several forms of panniculitis, including erythema nodosum, erythema induratum/nodular vasculitis, lupus panniculitis, lipodermatosclerosis, α1-antitrypsin deficiency, factitial, and fat necrosis secondary to pancreatic disease. Except for erythema nodosum, these lesions may break down and ulcerate or heal with a scar. The shin is the most common location for the nodules of erythema nodosum, whereas the calf is the most common location for lesions of erythema induratum. In erythema nodosum, the nodules are initially red but then develop a blue color as they resolve. Patients with erythema nodosum but no underlying systemic illness can still have fever, malaise, leukocytosis, arthralgias, and/or arthritis. However, the possibility

1	a blue color as they resolve. Patients with erythema nodosum but no underlying systemic illness can still have fever, malaise, leukocytosis, arthralgias, and/or arthritis. However, the possibility of an underlying illness should be excluded, and the most common associations are streptococcal infections, upper respiratory viral infections, sarcoidosis, and inflammatory bowel disease, in addition to drugs (oral contraceptives, sulfonamides, penicillins, bromides, iodides). Less common associations include bacterial gastroenteritis (Yersinia, Salmonella) and coccidioidomycosis followed by tuberculosis, histoplasmosis, brucellosis, and infections with Chlamydophila pneumoniae or Chlamydia trachomatis, Mycoplasma pneumoniae, or hepatitis B virus.

1	Erythema induratum and nodular vasculitis have overlapping features clinically and histologically, and whether they represent two separate entities or the ends of a single disease spectrum is a point of debate; in general, the latter is usually idiopathic and the former is associated with the presence of Mycobacterium tuberculosis DNA by PCR within skin lesions. The lesions of lupus panniculitis are found primarily on the cheeks, upper arms, and buttocks (sites of abundant fat) and are seen in both the cutaneous and systemic forms of lupus. The overlying skin may be normal, erythematous, or have the changes of discoid lupus. The subcutaneous fat necrosis that is associated with pancreatic disease is presumably secondary to circulating lipases and is seen in patients with pancreatic carcinoma as well as in patients with acute and chronic pancreatitis. In this disorder, there may be an associated arthritis, fever, and inflammation of visceral fat. Histologic examination of deep

1	carcinoma as well as in patients with acute and chronic pancreatitis. In this disorder, there may be an associated arthritis, fever, and inflammation of visceral fat. Histologic examination of deep incisional biopsy specimens will aid in the diagnosis of the particular type of panniculitis.

1	Subcutaneous erythematous nodules are also seen in cutaneous polyarteritis nodosa and as a manifestation of systemic vasculitis when there is involvement of medium-sized vessels, e.g., systemic polyarteritis nodosa, allergic granulomatosis, or granulomatosis with polyangiitis (Wegener’s) (Chap. 385). Cutaneous polyarteritis nodosa presents with painful subcutaneous nodules and ulcers within a red-purple, netlike pattern of livedo reticularis. The latter is due to slowed blood flow through the superficial horizontal venous plexus. The majority of lesions are found on the lower extremities, and while arthralgias and myalgias may accompany cutaneous polyarteritis nodosa, there is no evidence of systemic involvement. In both the cutaneous and systemic forms of vasculitis, skin biopsy specimens of the associated nodules will show the changes characteristic of a necrotizing vasculitis and/or granulomatous inflammation.

1	The cutaneous lesions in sarcoidosis (Chap. 390) are classically red to red-brown in color, and with diascopy (pressure with a glass slide), a yellow-brown residual color is observed that is secondary to the granulomatous infiltrate. The waxy papules and plaques may be found anywhere on the skin, but the face is the most common location. Usually there are no surface changes, but occasionally the lesions will have scale. Biopsy specimens of the papules show “naked” granulomas in the dermis, i.e., granulomas surrounded by a minimal number of lymphocytes. Other cutaneous findings in sarcoidosis include annular lesions with an atrophic or scaly center, papules within scars, hypopigmented papules and patches, alopecia, acquired ichthyosis, erythema nodosum, and lupus pernio (see below).

1	The differential diagnosis of sarcoidosis includes foreign-body granulomas produced by chemicals such as beryllium and zirconium, late secondary syphilis, and lupus vulgaris. Lupus vulgaris is a form of cutaneous tuberculosis that is seen in previously infected and sensitized individuals. There is often underlying active tuberculosis elsewhere, usually in the lungs or lymph nodes. Lesions occur primarily in the head and neck region and are red-brown plaques with a yellow-brown color on diascopy. Secondary scarring and squamous cell carcinomas can develop within the plaques. Cultures or PCR analysis of the lesions should be performed, along with an interferon γ release assay of peripheral blood, because it is rare for the acid-fast stain to show bacilli within the dermal granulomas.

1	A generalized distribution of red-brown macules and papules is seen in the form of mastocytosis known as urticaria pigmentosa (Chap. 376). Each lesion represents a collection of mast cells in the dermis, with hyperpigmentation of the overlying epidermis. Stimuli such as rubbing cause these mast cells to degranulate, and this leads to the formation of localized urticaria (Darier’s sign). Additional symptoms can result from mast cell degranulation and include headache, flushing, diarrhea, and pruritus. Mast cells also infiltrate various organs such as the liver, spleen, and gastrointestinal tract, and accumulations of mast cells in the bones may produce either osteosclerotic or osteolytic lesions on radiographs. In the majority of these patients, however, the internal involvement remains indolent. A subtype of chronic cutaneous small-vessel vasculitis, erythema elevatum diutinum (EED), also presents with papules that are red-brown in color. The papules coalesce into plaques on the

1	indolent. A subtype of chronic cutaneous small-vessel vasculitis, erythema elevatum diutinum (EED), also presents with papules that are red-brown in color. The papules coalesce into plaques on the extensor surfaces of knees, elbows, and the small joints of the hand. Flares of EED have been associated with streptococcal infections.

1	Lesions that are blue in color are the result of vascular ectasias, hyperplasias and tumors or melanin pigment within the dermis. Venous lakes (ectasias) are compressible dark-blue lesions that are found commonly in the head and neck region. Venous malformations are also compressible blue papulonodules and plaques that can occur anywhere on the body, including the oral mucosa. When there are multiple rather than single congenital lesions, the patient may have the blue rubber bleb syndrome or Maffucci’s syndrome. Patients with the blue rubber bleb syndrome also have vascular anomalies of the gastrointestinal tract that may bleed, whereas patients with Maffucci’s syndrome have associated osteochondromas. Blue nevi (moles) are seen when there are collections of pigment-producing nevus cells in the dermis. These benign papular lesions are dome-shaped and occur most commonly on the dorsum of the hand or foot or in the head and neck region.

1	Violaceous papules and plaques are seen in lupus pernio, lymphoma cutis, and cutaneous lupus. Lupus pernio is a particular type of sarcoidosis that involves the tip and alar rim of the nose as well as the earlobes, with lesions that are violaceous in color rather than red-brown. This 367 form of sarcoidosis is associated with involvement of the upper respiratory tract. The plaques of lymphoma cutis and cutaneous lupus may be red or violaceous in color and were discussed above.

1	Purple-colored papules and plaques are seen in vascular tumors, such as Kaposi’s sarcoma (Chap. 226) and angiosarcoma, and when there is extravasation of red blood cells into the skin in association with inflammation, as in palpable purpura (see “Purpura,” below). Patients with congenital or acquired AV fistulas and venous hypertension can develop purple papules on the lower extremities that can resemble Kaposi’s sarcoma clinically and histologically; this condition is referred to as pseudo-Kaposi’s sarcoma (acral angiodermatitis). Angiosarcoma is found most commonly on the scalp and face of elderly patients or within areas of chronic lymphedema and presents as purple papules and plaques. In the head and neck region, the tumor often extends beyond the clinically defined borders and may be accompanied by facial edema. Brownand black-colored papules are reviewed in “Hyperpigmentation,” above.

1	Brownand black-colored papules are reviewed in “Hyperpigmentation,” above. These are discussed last because they can have a wide range of colors. Most commonly, they present as either firm, skin-colored subcutaneous nodules or firm, red to red-brown papulonodules. The lesions of lymphoma cutis range from pink-red to plum in color, whereas metastatic melanoma can be pink, blue, or black in color. Cutaneous metastases develop from hematogenous or lymphatic spread and are most often due to the following primary carcinomas: in men, melanoma, oropharynx, lung, and colon; and in women, breast, melanoma, and ovary. These metastatic lesions may be the initial presentation of the carcinoma, especially when the primary site is the lung.

1	(Table 72-16) Purpura are seen when there is an extravasation of red blood cells into the dermis and, as a result, the lesions do not blanch with pressure. This is in contrast to those erythematous or violet-colored lesions that are due to localized vasodilatation—they do blanch with pressure. Purpura (≥3 mm) and petechiae (≤2 mm) are divided into two major groups: palpable and nonpalpable. The most frequent causes of nonpalpable petechiae and purpura are primary cutaneous disorders such as trauma, solar (actinic) purpura, and capillaritis. Less common causes are steroid purpura and livedoid vasculopathy (see “Ulcers,” below). Solar purpura are seen primarily on the extensor forearms, whereas steroid purpura secondary to potent topical glucocorticoids or endogenous or exogenous Cushing’s syndrome can be more widespread. In both cases, there is alteration of the supporting connective tissue that surrounds the dermal blood vessels. In contrast, the petechiae that result from

1	Cushing’s syndrome can be more widespread. In both cases, there is alteration of the supporting connective tissue that surrounds the dermal blood vessels. In contrast, the petechiae that result from capillaritis are found primarily on the lower extremities. In capillaritis, there is an extravasation of erythrocytes as a result of perivascular lymphocytic inflammation. The petechiae are bright red, 1–2 mm in size, and scattered within yellow-brown patches. The yellow-brown color is caused by hemosiderin deposits within the dermis.

1	Systemic causes of nonpalpable purpura fall into several categories, and those secondary to clotting disturbances and vascular fragility will be discussed first. The former group includes thrombocytopenia (Chap. 140), abnormal platelet function as is seen in uremia, and clotting factor defects. The initial site of presentation for thrombocytopenia-induced petechiae is the distal lower extremity. Capillary fragility leads to nonpalpable purpura in patients with systemic amyloidosis (see “Papulonodular Skin Lesions,” above), disorders of collagen production such as Ehlers-Danlos syndrome, and scurvy. In scurvy, there are flattened corkscrew hairs with surrounding hemorrhage on the lower extremities, in addition to gingivitis. Vitamin C is a cofactor for lysyl hydroxylase, an enzyme involved in the posttranslational modification of procollagen that is necessary for cross-link formation. CHAPTER 72 Skin Manifestations of Internal Disease CAuSES of PuRPuRA

1	CHAPTER 72 Skin Manifestations of Internal Disease CAuSES of PuRPuRA PART 2 Cardinal Manifestations and Presentation of Diseases I. Primary cutaneous disorders A. Nonpalpable 1. 2. Solar (actinic, senile) purpura 3. 4. 5. Livedoid vasculopathy in the setting of venous hypertensiona II. A. 1. Clotting disturbances a. b. c. 2. Vascular fragility a. b. c. 3. Thrombi a. b. c. d. e. f. g. h. 4. Emboli a. b. 5. Possible immune complex a. b. B. 1. Vasculitis a. Cutaneous small-vessel vasculitis, including in the setting of systemic vasculitides b. 2. Embolib a. b. c. d. aAlso associated with underlying disorders that lead to hypercoagulability, e.g., factor V Leiden, protein C dysfunction/deficiency. bBacterial (including rickettsial), fungal, or parasitic. Abbreviation: ITP, idiopathic thrombocytopenic purpura.

1	In contrast to the previous group of disorders, the purpura (noninflammatory with a retiform outline) seen in the following group of diseases are associated with thrombi formation within vessels. It is important to note that these thrombi are demonstrable in skin biopsy specimens. This group of disorders includes disseminated intravascular coagulation (DIC), monoclonal cryoglobulinemia, thrombocytosis, thrombotic thrombocytopenic purpura, antiphospholipid antibody syndrome, and reactions to warfarin and heparin (heparin-induced thrombocytopenia and thrombosis). DIC is triggered by several types of infection (gramnegative, gram-positive, viral, and rickettsial) as well as by tissue injury and neoplasms. Widespread purpura and hemorrhagic infarcts of the distal extremities are seen. Similar lesions are found in purpura fulminans, which is a form of DIC associated with fever and hypotension that occurs more commonly in children following an infectious illness such as varicella, scarlet

1	lesions are found in purpura fulminans, which is a form of DIC associated with fever and hypotension that occurs more commonly in children following an infectious illness such as varicella, scarlet fever, or an upper respiratory tract infection. In both disorders, hemorrhagic bullae can develop in involved skin.

1	Monoclonal cryoglobulinemia is associated with plasma cell dyscrasias, chronic lymphocytic leukemia, and lymphoma. Purpura, primarily of the lower extremities, and hemorrhagic infarcts of the fingers, toes, and ears are seen in these patients. Exacerbations of disease activity can follow cold exposure or an increase in serum viscosity. Biopsy specimens show precipitates of the cryoglobulin within dermal vessels. Similar deposits have been found in the lung, brain, and renal glomeruli. Patients with thrombotic thrombocytopenic purpura can also have hemorrhagic infarcts as a result of intravascular thromboses. Additional signs include microangiopathic hemolytic anemia and fluctuating neurologic abnormalities, especially headaches and confusion.

1	Administration of warfarin can result in painful areas of erythema that become purpuric and then necrotic with an adherent black eschar; the condition is referred to as warfarin-induced necrosis. This reaction is seen more often in women and in areas with abundant subcutaneous fat—breasts, abdomen, buttocks, thighs, and calves. The erythema and purpura develop between the third and tenth day of therapy, most likely as a result of a transient imbalance in the levels of anticoagulant and procoagulant vitamin K–dependent factors. Continued therapy does not exacerbate preexisting lesions, and patients with an inherited or acquired deficiency of protein C are at increased risk for this particular reaction as well as for purpura fulminans and calciphylaxis.

1	Purpura secondary to cholesterol emboli are usually seen on the lower extremities of patients with atherosclerotic vascular disease. They often follow anticoagulant therapy or an invasive vascular procedure such as an arteriogram but also occur spontaneously from disintegration of atheromatous plaques. Associated findings include livedo reticularis, gangrene, cyanosis, and ischemic ulcerations. Multiple step sections of the biopsy specimen may be necessary to demonstrate the cholesterol clefts within the vessels. Petechiae are also an important sign of fat embolism and occur primarily on the upper body 2–3 days after a major injury. By using special fixatives, the emboli can be demonstrated in biopsy specimens of the petechiae. Emboli of tumor or thrombus are seen in patients with atrial myxomas and marantic endocarditis.

1	In the Gardner-Diamond syndrome (autoerythrocyte sensitivity), female patients develop large ecchymoses within areas of painful, warm erythema. Intradermal injections of autologous erythrocytes or phosphatidyl serine derived from the red cell membrane can reproduce the lesions in some patients; however, there are instances where a reaction is seen at an injection site of the forearm but not in the midback region. The latter has led some observers to view Gardner-Diamond syndrome as a cutaneous manifestation of severe emotional stress. More recently, the possibility of platelet dysfunction (as assessed via aggregation studies) has been raised. Waldenström’s hypergammaglobulinemic purpura is a chronic disorder characterized by petechiae on the lower extremities. There are circulating complexes of IgG–anti-IgG molecules, and exacerbations are associated with prolonged standing or walking.

1	Palpable purpura are further subdivided into vasculitic and embolic. In the group of vasculitic disorders, cutaneous small-vessel vasculitis, also known as leukocytoclastic vasculitis (LCV), is the one most commonly associated with palpable purpura (Chap. 385). Underlying etiologies include drugs (e.g., antibiotics), infections (e.g., hepatitis C virus), and autoimmune connective tissue diseases (e.g., rheumatoid arthritis, Sjögren’s syndrome, lupus). Henoch-Schönlein purpura (HSP) is a subtype of acute LCV that is seen more commonly in children and adolescents following an upper respiratory infection. The majority of lesions are found on the lower extremities and buttocks. Systemic manifestations include fever, arthralgias (primarily of the knees and ankles), abdominal pain, gastrointestinal bleeding, and nephritis. Direct immunofluorescence examination shows deposits of IgA within dermal blood vessel walls. Renal disease is of particular concern in adults with HSP. In polyarteritis

1	bleeding, and nephritis. Direct immunofluorescence examination shows deposits of IgA within dermal blood vessel walls. Renal disease is of particular concern in adults with HSP. In polyarteritis nodosa, specific cutaneous lesions result from a vasculitis of arterial vessels (arteritis), or there may be an associated LCV. Arteritis leads to an infarct of the skin, and

1	CAuSES of MuCoCuTAnEouS uLCERS this explains the irregular outline of the purpura (see below). Several types of infectious emboli can give rise to palpable purpura. These embolic lesions are usually irregular in outline as opposed to the lesions of LCV, which are circular in outline. The irregular outline is indicative of a cutaneous infarct, and the size corresponds to the area of skin that received its blood supply from that particular arteriole or artery. The palpable purpura in LCV are circular because the erythrocytes simply diffuse out evenly from the postcapillary venules as a result of inflammation. Infectious emboli are most commonly due to gram-negative cocci (meningococcus, gonococcus), gram-negative rods (Enterobacteriaceae), and gram-positive cocci (Staphylococcus). Additional causes include Rickettsia and, in immunocompromised patients, Aspergillus and other opportunistic fungi.

1	The embolic lesions in acute meningococcemia are found primarily on the trunk, lower extremities, and sites of pressure, and a gunmetal-gray color often develops within them. Their size varies from a few millimeters to several centimeters, and the organisms can be cultured from the lesions. Associated findings include a preceding upper respiratory tract infection; fever; meningitis; DIC; and, in some patients, a deficiency of the terminal components of complement. In disseminated gonococcal infection (arthritis-dermatitis syndrome), a small number of inflammatory papules and vesicopustules, often with central purpura or hemorrhagic necrosis, are found on the distal extremities. Additional symptoms include arthralgias, tenosynovitis, and fever. To establish the diagnosis, a Gram stain of these lesions should be performed. Rocky Mountain spotted fever is a tick-borne disease that is caused by Rickettsia rickettsii. A several-day history of fever, chills, severe headache, and photophobia

1	lesions should be performed. Rocky Mountain spotted fever is a tick-borne disease that is caused by Rickettsia rickettsii. A several-day history of fever, chills, severe headache, and photophobia precedes the onset of the cutaneous eruption. The initial lesions are erythematous macules and papules on the wrists, ankles, palms, and soles. With time, the lesions spread centripetally and become purpuric.

1	Lesions of ecthyma gangrenosum begin as edematous, erythematous papules or plaques and then develop central purpura and necrosis. Bullae formation also occurs in these lesions, and they are frequently found in the girdle region. The organism that is classically associated with ecthyma gangrenosum is Pseudomonas aeruginosa, but other gram-negative rods such as Klebsiella, Escherichia coli, and Serratia can produce similar lesions. In immunocompromised hosts, the list of potential pathogens is expanded to include Candida and other opportunistic fungi (e.g., Aspergillus, Fusarium). The approach to the patient with a cutaneous ulcer is outlined in Table 72-17. Peripheral vascular diseases of the extremities are reviewed in Chap. 302, as is Raynaud’s phenomenon.

1	The approach to the patient with a cutaneous ulcer is outlined in Table 72-17. Peripheral vascular diseases of the extremities are reviewed in Chap. 302, as is Raynaud’s phenomenon. Livedoid vasculopathy (livedoid vasculitis; atrophie blanche) represents a combination of a vasculopathy plus intravascular thrombosis. Purpuric lesions and livedo reticularis are found in association with painful ulcerations of the lower extremities. These ulcers are often slow to heal, but when they do, irregularly shaped white scars form. The majority of cases are secondary to venous hypertension, but possible underlying illnesses include cryofibrinogenemia and disorders of hypercoagulability, e.g., the antiphospholipid syndrome (Chaps. 142 and 379).

1	In pyoderma gangrenosum, the border of untreated active ulcers has a characteristic appearance consisting of an undermined necrotic violaceous edge and a peripheral erythematous halo. The ulcers often begin as pustules that then expand rather rapidly to a size as large as 20 cm. Although these lesions are most commonly found on the lower extremities, they can arise anywhere on the surface of the body, including sites of trauma (pathergy). An estimated 30–50% of cases are idiopathic, and the most common associated disorders are ulcerative colitis and Crohn’s disease. Less commonly, pyoderma gangrenosum is associated with seropositive rheumatoid arthritis, acute and chronic myelogenous leukemia, hairy cell leukemia, myelofibrosis, or a monoclonal gammopathy, usually IgA. Because the histology of pyoderma gangrenosum may be nonspecific I. Primary cutaneous disorders A. Peripheral vascular disease (Chap. 302) 1. 2. B. Livedoid vasculopathy in the setting of venous hypertensionb

1	I. Primary cutaneous disorders A. Peripheral vascular disease (Chap. 302) 1. 2. B. Livedoid vasculopathy in the setting of venous hypertensionb C. Squamous cell carcinoma, e.g., within scars, basal cell carcinomas D. Infections, e.g., ecthyma caused by Streptococcus (Chap. 173) E. Physical, e.g., trauma, pressure F. Drugs, e.g., hydroxyurea II. A. 1. 2. Hemoglobinopathies (Chap. 127) 3. Cryoglobulinemia,c cryofibrinogenemia 4. 5. 6. Antiphospholipid syndrome (Chap. 141) 7. Neuropathice (Chap. 417) 8. 9. Kaposi's sarcoma, acral angiodermatitis 10. B. Hands and feet 1. Raynaud's phenomenon (Chap. 302) 2. C. Generalized 1. Pyoderma gangrenosum, but most commonly legs 2. Calciphylaxis (Chap. 424) 3. Infections, e.g., dimorphic fungi, leishmaniasis 4. D. Face, especially perioral, and anogenital 1. Chronic herpes simplexf III. A. Behçet's syndrome (Chap. 387) B. Erythema multiforme major, Stevens-Johnson syndrome, TEN C.

1	D. Face, especially perioral, and anogenital 1. Chronic herpes simplexf III. A. Behçet's syndrome (Chap. 387) B. Erythema multiforme major, Stevens-Johnson syndrome, TEN C. Primary blistering disorders (Chap. 73) D. Lupus erythematosus, lichen planus E. F. G. Reactive arthritis (formerly known as Reiter's syndrome) aUnderlying atherosclerosis. bAlso associated with underlying disorders that lead to hypercoagulability, e.g., factor V Leiden, protein C dysfunction/deficiency, antiphospholipid antibodies. cReviewed in section on Purpura. dReviewed in section on Papulonodular Skin Lesions. eFavors plantar surface of the foot. fSign of immunosuppression. Abbreviation: TEN, toxic epidermal necrolysis.

1	Abbreviation: TEN, toxic epidermal necrolysis. (dermal infiltrate of neutrophils when in untreated state), the diagnosis requires clinicopathologic correlation, in particular, the exclusion of similar-appearing ulcers such as necrotizing vasculitis, Meleney’s ulcer (synergistic infection at a site of trauma or surgery), dimorphic fungi, cutaneous amebiasis, spider bites, and factitial. In the myeloproliferative disorders, the ulcers may be more superficial with a pustulobullous border, and these lesions provide a connection between classic pyoderma gangrenosum and acute febrile neutrophilic dermatosis (Sweet syndrome). The major considerations in a patient with a fever and a rash are inflammatory diseases versus infectious diseases. In the hospital setting, the most common scenario is a patient who has a drug rash plus a fever secondary to an underlying infection. However, it should be

1	CHAPTER 72 Skin Manifestations of Internal Disease lesions. immunologically Mediated Skin Diseases Kim B. Yancey, Thomas J. Lawley A number of immunologically mediated skin diseases and immuno-logically mediated systemic disorders with cutaneous manifestations 73 PART 2 Cardinal Manifestations and Presentation of Diseases emphasized that a drug reaction can lead to both a cutaneous eruption and a fever (“drug fever”), especially in the setting of DRESS, AGEP, or serum sickness–like reaction. Additional inflammatory diseases that are often associated with a fever include pustular psoriasis, erythroderma, and Sweet syndrome. Lyme disease, secondary syphilis, and viral and bacterial exanthems (see “Exanthems,” above) are examples of infectious diseases that produce a rash and a fever. Lastly, it is important to determine whether or not the cutaneous lesions represent septic emboli (see “Purpura,” above). Such lesions usually have evidence of ischemia in the form of purpura, necrosis, or

1	it is important to determine whether or not the cutaneous lesions represent septic emboli (see “Purpura,” above). Such lesions usually have evidence of ischemia in the form of purpura, necrosis, or impending necrosis (gunmetal-gray color). In the patient with thrombocytopenia, however, purpura can be seen in inflammatory reactions such as morbilliform drug eruptions and infectious are now recognized as distinct entities with consistent clinical, histologic, and immunopathologic findings. Clinically, these disorders are characterized by morbidity (pain, pruritus, disfigurement) and, in some instances, result in death (largely due to loss of epidermal barrier function and/or secondary infection). The major features of the more common immunologically mediated skin diseases are summarized in this chapter (Table 73-1), as are the autoimmune systemic disorders with cutaneous manifestations.

1	aAutoantigens bound by these patients’ autoantibodies are defined as follows: Dsg1, desmoglein 1; Dsg3, desmoglein 3; BPAG1, bullous pemphigoid antigen 1; BPAG2, bullous pemphigoid antigen 2. Abbreviation: BMZ, basement membrane zone. Pemphigus refers to a group of autoantibody-mediated intraepidermal blistering diseases characterized by loss of cohesion between epidermal cells (a process termed acantholysis). Manual pressure to the skin of these patients may elicit the separation of the epidermis (Nikolsky’s sign). This finding, while characteristic of pemphigus, is not specific to this group of disorders and is also seen in toxic epidermal necrolysis, Stevens-Johnson syndrome, and a few other skin diseases.

1	Pemphigus vulgaris (PV) is a mucocutaneous blistering disease that predominantly occurs in patients >40 years of age. PV typically begins on mucosal surfaces and often progresses to involve the skin. This disease is characterized by fragile, flaccid blisters that rupture to produce extensive denudation of mucous membranes and skin (Fig. 73-1). The mouth, scalp, face, neck, axilla, groin, and trunk are typically involved. PV may be associated with severe skin pain; some patients experience pruritus as well. Lesions usually heal without scarring except at sites complicated by secondary infection or mechanically induced dermal wounds. Postinflammatory hyperpigmentation is usually present for some time at sites of healed lesions.

1	Biopsies of early lesions demonstrate intraepidermal vesicle formation secondary to loss of cohesion between epidermal cells (i.e., acantholytic blisters). Blister cavities contain acantholytic epidermal cells, which appear as round homogeneous cells containing hyperchromatic nuclei. Basal keratinocytes remain attached to the epidermal basement membrane; hence, blister formation takes place within the suprabasal portion of the epidermis. Lesional skin may contain focal collections of intraepidermal eosinophils within blister cavities; dermal alterations are slight, often limited to an eosinophil-predominant leukocytic infiltrate. Direct immunofluorescence microscopy of lesional or intact patient skin shows deposits of IgG on the surface of keratinocytes; deposits of complement components are typically found in lesional but not in uninvolved skin. Deposits of IgG on keratinocytes are derived from circulating autoantibodies to cell-surface autoantigens. Such circulating autoantibodies

1	FIguRE 73-1 Pemphigus vulgaris. A. Flaccid bullae are easily ruptured, resulting in multiple erosions and crusted plaques.

1	B. Involvement of the oral mucosa, which is almost invariable, may present with erosions on the gingiva, buccal mucosa, palate, posterior pharynx, or tongue. (B, Courtesy of Robert Swerlick, MD; with permission.) can be demonstrated in 80–90% of PV patients by indirect immunofluorescence microscopy; monkey esophagus is the optimal substrate for these studies. Patients with PV have IgG autoantibodies to desmogleins (Dsgs), transmembrane desmosomal glycoproteins that belong to the cadherin family of calcium-dependent adhesion molecules. Such autoantibodies can be precisely quantitated by enzyme-linked immunosorbent assay (ELISA). Patients with early PV (i.e., mucosal disease) have IgG autoantibodies to Dsg3; patients with advanced PV (i.e., mucocutaneous disease) have IgG autoantibodies to both Dsg3 and Dsg1. Experimental studies have shown that autoantibodies from patients with PV are pathogenic (i.e., responsible for blister formation) and that their titer correlates with disease

1	to both Dsg3 and Dsg1. Experimental studies have shown that autoantibodies from patients with PV are pathogenic (i.e., responsible for blister formation) and that their titer correlates with disease activity. Recent studies have shown that the anti-Dsg autoantibody profile in these patients’ sera as well as the tissue distribution of Dsg3 and Dsg1 determine the site of blister formation in patients with PV. Coexpression of Dsg3 and Dsg1 by epidermal cells protects against pathogenic IgG antibodies to either of these cadherins but not against pathogenic autoantibodies to both.

1	PV can be life-threatening. Prior to the availability of glucocorticoids, mortality rates ranged from 60% to 90%; the current figure is ~5%. Common causes of morbidity and death are infection and complications of treatment with glucocorticoids. Bad prognostic 371 factors include advanced age, widespread involvement, and the requirement for high doses of glucocorticoids (with or without other immunosuppressive agents) for control of disease. The course of PV in individual patients is variable and difficult to predict. Some patients experience remission, while others may require long-term treatment or succumb to complications of their disease or its treatment. The mainstay of treatment is systemic glucocorticoids. Patients with moderate to severe PV are usually started on prednisone at 1 mg/kg per day. If new lesions continue to appear after 1–2 weeks of treatment, the dose may need to be increased and/or prednisone may need to be combined with other immunosuppressive agents such as

1	mg/kg per day. If new lesions continue to appear after 1–2 weeks of treatment, the dose may need to be increased and/or prednisone may need to be combined with other immunosuppressive agents such as azathioprine (2–2.5 mg/kg per day), mycophenolate mofetil (20–35 mg/kg per day), or cyclophosphamide (1–2 mg/kg per day). Patients with severe, treatment-resistant disease may derive benefit from plasmapheresis (six high-volume exchanges [i.e., 2–3 L per exchange] over ~2 weeks), IV immunoglobulin (IVIg) (2 g/kg over 3–5 days every 6–8 weeks), or rituximab (375 mg/m2 per week × 4, or 1000 mg on days 1 and 15). It is important to bring severe or progressive disease under control quickly in order to lessen the severity and/or duration of this disorder. Accordingly, some have suggested that rituximab and daily glucocorticoids should be used early in PV patients to avert the development of treatment-resistant disease.

1	Pemphigus foliaceus (PF) is distinguished from PV by several features. In PF, acantholytic blisters are located high within the epidermis, usually just beneath the stratum corneum. Hence, PF is a more superficial blistering disease than PV. The distribution of lesions in the two disorders is much the same, except that in PF mucous membranes are almost always spared. Patients with PF rarely have intact blisters but rather exhibit shallow erosions associated with erythema, scale, and crust formation. Mild cases of PF resemble severe seborrheic dermatitis; severe PF may cause extensive exfoliation. Sun exposure (ultraviolet irradiation) may be an aggravating factor.

1	PF has immunopathologic features in common with PV. Specifically, direct immunofluorescence microscopy of perilesional skin demonstrates IgG on the surface of keratinocytes. Similarly, patients with PF have circulating IgG autoantibodies directed against the surface of keratinocytes. In PF, autoantibodies are directed against Dsg1, a 160kDa desmosomal cadherin. These autoantibodies can be quantitated by ELISA. As noted for PV, the autoantibody profile in patients with PF (i.e., anti-Dsg1 IgG) and the tissue distribution of this autoantigen (i.e., expression in oral mucosa that is compensated by coexpression of Dsg3) are thought to account for the distribution of lesions in this disease.

1	Endemic forms of PF are found in south-central rural Brazil, where the disease is known as fogo salvagem (FS), as well as in selected sites in Latin America and Tunisia. Endemic PF, like other forms of this disease, is mediated by IgG autoantibodies to Dsg1. Clusters of FS overlap with those of leishmaniasis, a disease transmitted by bites of the sand fly Lutzomyia longipalis. Recent studies have shown that sand-fly salivary antigens (specifically, the LJM11 salivary protein) are recognized by IgG autoantibodies from FS patients (as well as by monoclonal antibodies to Dsg1 derived from these patients). Moreover, mice immunized with LJM11 produce antibodies to Dsg1. Thus, these findings suggest that insect bites may deliver salivary antigens that initiate a cross-reactive humoral immune response, which may lead to FS in genetically susceptible individuals.

1	Although pemphigus has been associated with several autoimmune diseases, its association with thymoma and/or myasthenia gravis is particularly notable. To date, >30 cases of thymoma and/or myasthenia gravis have been reported in association with pemphigus, usually with PF. Patients may also develop pemphigus as a consequence of drug exposure; drug-induced pemphigus usually resembles PF rather than PV. Drugs containing a thiol group in their chemical structure (e.g., penicillamine, captopril, enalapril) are most commonly associated with drug-induced pemphigus. Nonthiol drugs linked to pemphigus include penicillins, cephalosporins, and piroxicam. It has 372 been suggested that thiol-containing and non-thiol-containing drugs induce pemphigus via biochemical and immunologic mechanisms, respectively—hence, the better prognosis upon drug withdrawal in cases of pemphigus induced by thiol-containing medications. Some cases of drug-induced pemphigus are durable and require treatment with

1	the better prognosis upon drug withdrawal in cases of pemphigus induced by thiol-containing medications. Some cases of drug-induced pemphigus are durable and require treatment with systemic glucocorticoids and/or immunosuppressive agents. PF is generally a less severe disease than PV and carries a better prognosis. Localized disease can sometimes be treated with topical or intralesional glucocorticoids; more active cases can usually be controlled with systemic glucocorticoids. Patients with severe, treatment-resistant disease may require more aggressive interventions, as described above for patients with PV.

1	Paraneoplastic pemphigus (PNP) is an autoimmune acantholytic mucocutaneous disease associated with an occult or confirmed neoplasm. Patients with PNP typically have painful mucosal erosive lesions in association with papulosquamous and/or lichenoid eruptions that often progress to blisters. Palm and sole involvement are common in these patients and raise the possibility that prior reports of neoplasiaassociated erythema multiforme actually may have represented unrecognized cases of PNP. Biopsies of lesional skin from these patients show varying combinations of acantholysis, keratinocyte necrosis, and vacuolar-interface dermatitis. Direct immunofluorescence microscopy of a patient’s skin shows deposits of IgG and complement on the surface of keratinocytes and (variably) similar immunoreactants in the epidermal basement membrane zone. Patients with PNP have IgG autoantibodies to cytoplasmic proteins that are members of the plakin family (e.g., desmoplakins I and II, bullous pemphigoid

1	in the epidermal basement membrane zone. Patients with PNP have IgG autoantibodies to cytoplasmic proteins that are members of the plakin family (e.g., desmoplakins I and II, bullous pemphigoid antigen [BPAG]1, envoplakin, periplakin, and plectin) and to cell-surface proteins that are members of the cadherin family (e.g., Dsg1 and Dsg3). Passive transfer studies have shown that autoantibodies from patients with PNP are pathogenic in animal models.

1	The predominant neoplasms associated with PNP are non-Hodgkin’s lymphoma, chronic lymphocytic leukemia, thymoma, spindle cell tumors, Waldenström’s macroglobulinemia, and Castleman’s disease; the last-mentioned neoplasm is particularly common among children with PNP. Rare cases of seronegative PNP have been reported in patients with B cell malignancies previously treated with rituximab. In addition to severe skin lesions, many patients with PNP develop life-threatening bronchiolitis obliterans. PNP is generally resistant to conventional therapies (i.e., those used to treat PV); rarely, a patient’s disease may ameliorate or even remit following ablation or removal of underlying neoplasms.

1	Bullous pemphigoid (BP) is a polymorphic autoimmune subepidermal blistering disease usually seen in the elderly. Initial lesions may consist of urticarial plaques; most patients eventually display tense blisters on either normal-appearing or erythematous skin (Fig. 73-2). The lesions are usually distributed over the lower abdomen, groin, and flexor surface of the extremities; oral mucosal lesions are found in some patients. Pruritus may be nonexistent or severe. As lesions evolve, tense blisters tend to rupture and be replaced by erosions with or without surmounting crust. Nontraumatized blisters heal without scarring. The major histocompatibility complex class II allele HLA-DQβ1*0301 is prevalent in patients with BP. Despite isolated reports, several studies have shown that patients with BP do not have a higher incidence of malignancy than appropriately ageand gender-matched controls.

1	Biopsies of early lesional skin demonstrate subepidermal blisters and histologic features that roughly correlate with the clinical character of the particular lesion under study. Lesions on normal-appearing skin generally contain a sparse perivascular leukocytic infiltrate with some eosinophils; conversely, biopsies of inflammatory lesions typically show an eosinophil-rich infiltrate at sites of vesicle formation and in perivascular areas. In addition to eosinophils, cell-rich lesions also contain mononuclear cells and neutrophils. It is not possible to distinguish BP from other subepidermal blistering diseases by routine histologic studies alone. PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 73-2 Bullous pemphigoid with tense vesicles and bullae on erythematous, urticarial bases. (Courtesy of the Yale Resident’s Slide Collection; with permission.)

1	Direct immunofluorescence microscopy of normal-appearing perilesional skin from patients with BP shows linear deposits of IgG and/ or C3 in the epidermal basement membrane. The sera of ~70% of these patients contain circulating IgG autoantibodies that bind the epidermal basement membrane of normal human skin in indirect immunofluorescence microscopy. IgG from an even higher percentage of patients reacts with the epidermal side of 1 M NaCl split skin (an alternative immunofluorescence microscopy test substrate used to distinguish circulating IgG autoantibodies to the basement membrane in patients with BP from those in patients with similar, yet different, subepidermal blistering diseases; see below). In BP, circulating autoantibodies recognize 230and 180-kDa hemidesmosome-associated proteins in basal keratinocytes (i.e., BPAG1 and BPAG2, respectively). Autoantibodies to BPAG2 are thought to deposit in situ, activate complement, produce dermal mast-cell degranulation, and generate

1	in basal keratinocytes (i.e., BPAG1 and BPAG2, respectively). Autoantibodies to BPAG2 are thought to deposit in situ, activate complement, produce dermal mast-cell degranulation, and generate granulocyte-rich infiltrates that cause tissue damage and blister formation.

1	BP may persist for months to years, with exacerbations or remissions. Extensive involvement may result in widespread erosions and compromise cutaneous integrity; elderly and/or debilitated patients may die. The mainstay of treatment is systemic glucocorticoids. Local or minimal disease can sometimes be controlled with topical glucocorticoids alone; more extensive lesions generally respond to systemic glucocorticoids either alone or in combination with immunosuppressive agents. Patients usually respond to prednisone (0.75–1 mg/kg per day). In some instances, azathioprine (2–2.5 mg/kg per day), mycophenolate mofetil (20–35 mg/kg per day), or cyclophosphamide (1–2 mg/kg per day) are necessary adjuncts.

1	Pemphigoid gestationis (PG), also known as herpes gestationis, is a rare, nonviral, subepidermal blistering disease of pregnancy and the puerperium. PG may begin during any trimester of pregnancy or present shortly after delivery. Lesions are usually distributed over the abdomen, trunk, and extremities; mucous membrane lesions are rare. Skin lesions in these patients may be quite polymorphic and consist of erythematous urticarial papules and plaques, vesiculopapules, and/or frank bullae. Lesions are almost always extremely pruritic. Severe exacerbations of PG frequently follow delivery, typically within 24–48 h. PG tends to recur in subsequent pregnancies, often beginning earlier during such gestations. Brief flare-ups of disease may occur with resumption of menses and may develop in patients later exposed to oral contraceptives. Occasionally, infants of affected mothers have transient skin lesions.

1	Biopsies of early lesional skin show teardrop-shaped subepidermal vesicles forming in dermal papillae in association with an eosinophilrich leukocytic infiltrate. Differentiation of PG from other subepidermal bullous diseases by light microscopy is difficult. However, direct immunofluorescence microscopy of perilesional skin from PG patients reveals the immunopathologic hallmark of this disorder: linear deposits of C3 in the epidermal basement membrane. These deposits develop as a consequence of complement activation produced by low-titer IgG anti–basement membrane autoantibodies directed against BPAG2, the same hemidesmosome-associated protein that is targeted by auto-antibodies in patients with BP—a subepidermal bullous disease that resembles PG clinically, histologically, and immunopathologically.

1	The goals of therapy in patients with PG are to prevent the development of new lesions, relieve intense pruritus, and care for erosions at sites of blister formation. Many patients require treatment with moderate doses of daily glucocorticoids (i.e., 20–40 mg of prednisone) at some point in their course. Mild cases (or brief flare-ups) may be controlled by vigorous use of potent topical glucocorticoids. Infants born of mothers with PG appear to be at increased risk of being born slightly premature or “small for dates.” Current evidence suggests that there is no difference in the incidence of uncomplicated live births between PG patients treated with systemic glucocorticoids and those managed more conservatively. If systemic glucocorticoids are administered, newborns are at risk for development of reversible adrenal insufficiency.

1	Dermatitis herpetiformis (DH) is an intensely pruritic, papulovesicular skin disease characterized by lesions symmetrically distributed over extensor surfaces (i.e., elbows, knees, buttocks, back, scalp, and posterior neck) (see Fig. 70-8). Primary lesions in this disorder consist of papules, papulovesicles, or urticarial plaques. Because pruritus is prominent, patients may present with excoriations and crusted papules but no observable primary lesions. Patients sometimes report that their pruritus has a distinctive burning or stinging component; the onset of such local symptoms reliably heralds the development of distinct clinical lesions 12–24 h later. Almost all DH patients have associated, usually subclinical, gluten-sensitive enteropathy (Chap. 349), and >90% express the HLA-B8/DRw3 and HLA-DQw2 haplotypes. DH may present at any age, including in childhood; onset in the second to fourth decades is most common. The disease is typically chronic.

1	Biopsy of early lesional skin reveals neutrophil-rich infiltrates within dermal papillae. Neutrophils, fibrin, edema, and microvesicle formation at these sites are characteristic of early disease. Older lesions may demonstrate nonspecific features of a subepidermal bulla or an excoriated papule. Because the clinical and histologic features of this disease can be variable and resemble those of other subepidermal blistering disorders, the diagnosis is confirmed by direct immunofluorescence microscopy of normal-appearing perilesional skin. Such studies demonstrate granular deposits of IgA (with or without complement components) in the papillary dermis and along the epidermal basement membrane zone. IgA deposits in the skin are unaffected by control of disease with medication; however, these immunoreactants diminish in intensity or disappear in patients maintained for long periods on a strict gluten-free diet (see below). Patients with DH have granular deposits of IgA in their epidermal

1	immunoreactants diminish in intensity or disappear in patients maintained for long periods on a strict gluten-free diet (see below). Patients with DH have granular deposits of IgA in their epidermal basement membrane zone and should be distinguished from individuals with linear IgA deposits at this site (see below).

1	Although most DH patients do not report overt gastrointestinal symptoms or have laboratory evidence of malabsorption, biopsies of the small bowel usually reveal blunting of intestinal villi and a lymphocytic infiltrate in the lamina propria. As is true for patients with celiac disease, this gastrointestinal abnormality can be reversed by a gluten-free diet. Moreover, if maintained, this diet alone may control the skin disease and eventuate in clearance of IgA deposits from these patients’ epidermal basement membrane zones. Subsequent gluten exposure in such patients alters the morphology of their small bowel, elicits a flareup of their skin disease, and is associated with the reappearance of IgA in their epidermal basement membrane zones. As in patients with celiac disease, dietary gluten sensitivity in patients with DH is associ-373 ated with IgA endomysial autoantibodies that target tissue transglutaminase. Studies indicate that patients with DH also have high-avidity IgA

1	gluten sensitivity in patients with DH is associ-373 ated with IgA endomysial autoantibodies that target tissue transglutaminase. Studies indicate that patients with DH also have high-avidity IgA autoantibodies to epidermal transglutaminase 3 and that the latter is co-localized with granular deposits of IgA in the papillary dermis of DH patients. Patients with DH also have an increased incidence of thyroid abnormalities, achlorhydria, atrophic gastritis, and autoantibodies to gastric parietal cells. These associations likely relate to the high frequency of the HLA-B8/DRw3 haplotype in these patients, because this marker is commonly linked to autoimmune disorders. The mainstay of treatment of DH is dapsone, a sulfone. Patients respond rapidly (24–48 h) to dapsone (50–200 mg/d), but require careful pretreatment evaluation and close follow-up to ensure that complications are avoided or controlled. All patients taking dapsone at >100 mg/d will have some hemolysis and methemoglobinemia,

1	careful pretreatment evaluation and close follow-up to ensure that complications are avoided or controlled. All patients taking dapsone at >100 mg/d will have some hemolysis and methemoglobinemia, which are expected pharmacologic side effects of this agent. Gluten restriction can control DH and lessen dapsone requirements; this diet must rigidly exclude gluten to be of maximal benefit. Many months of dietary restriction may be necessary before a beneficial result is achieved. Good dietary counseling by a trained dietitian is essential.

1	Linear IgA disease, once considered a variant form of DH, is actually a separate and distinct entity. Clinically, patients with linear IgA disease may resemble individuals with DH, BP, or other subepidermal blistering diseases. Lesions typically consist of papulovesicles, bullae, and/or urticarial plaques that develop predominantly on central or flexural sites. Oral mucosal involvement occurs in some patients. Severe pruritus resembles that seen in patients with DH. Patients with linear IgA disease do not have an increased frequency of the HLA-B8/DRw3 haplotype or an associated enteropathy and therefore are not candidates for treatment with a gluten-free diet.

1	Histologic alterations in early lesions may be virtually indistinguishable from those in DH. However, direct immunofluorescence microscopy of normal-appearing perilesional skin reveals a linear band of IgA (and often C3) in the epidermal basement membrane zone. Most patients with linear IgA disease have circulating IgA basement membrane autoantibodies directed against neoepitopes in the proteolytically processed extracellular domain of BPAG2. These patients generally respond to treatment with dapsone (50–200 mg/d).

1	Epidermolysis bullosa acquisita (EBA) is a rare, noninherited, polymorphic, chronic, subepidermal blistering disease. (The inherited form is discussed in Chap. 427.) Patients with classic or noninflammatory EBA have blisters on noninflamed skin, atrophic scars, milia, nail dystrophy, and oral lesions. Because lesions generally occur at sites exposed to minor trauma, classic EBA is considered a mechanobullous disease. Other patients with EBA have widespread inflammatory scarring and bullous lesions that resemble severe BP. Inflammatory EBA may evolve into the classic, noninflammatory form of this disease. Rarely, patients present with lesions that predominate on mucous membranes. The HLA-DR2 haplotype is found with increased frequency in EBA patients. Studies suggest that EBA is sometimes associated with inflammatory bowel disease (especially Crohn’s disease).

1	The histology of lesional skin varies with the character of the lesion being studied. Noninflammatory bullae are subepidermal, feature a sparse leukocytic infiltrate, and resemble the lesions in patients with porphyria cutanea tarda. Inflammatory lesions consist of neutrophilrich subepidermal blisters. EBA patients have continuous deposits of IgG (and frequently C3) in a linear pattern within the epidermal basement membrane zone. Ultrastructurally, these immunoreactants are found in the sublamina densa region in association with anchoring fibrils. Approximately 50% of EBA patients have demonstrable circulating IgG basement membrane autoantibodies directed against type VII collagen—the collagen species that makes up anchoring fibrils. Such IgG autoantibodies bind the dermal side of 1 M NaCl split skin (in contrast to IgG autoantibodies in patients with BP). Studies have shown that passive transfer of experimental or clinical IgG against type 374 VII collagen can produce lesions in mice

1	split skin (in contrast to IgG autoantibodies in patients with BP). Studies have shown that passive transfer of experimental or clinical IgG against type 374 VII collagen can produce lesions in mice that clinically, histologically, and immunopathologically resemble those in patients with inflammatory EBA. Treatment of EBA is generally unsatisfactory. Some patients with inflammatory EBA may respond to systemic glucocorticoids, either alone or in combination with immunosuppressive agents. Other patients (especially those with neutrophil-rich inflammatory lesions) may respond to dapsone. The chronic, noninflammatory form of EBA is largely resistant to treatment, although some patients may respond to cyclosporine, azathioprine, or IVIg.

1	Mucous membrane pemphigoid (MMP) is a rare, acquired, subepithelial immunobullous disease characterized by erosive lesions of mucous membranes and skin that result in scarring of at least some sites of involvement. Common sites include the oral mucosa (especially the gingiva) and conjunctiva; other sites that may be affected include the nasopharyngeal, laryngeal, esophageal, and anogenital mucosa. Skin lesions (present in about one-third of patients) tend to predominate on the scalp, face, and upper trunk and generally consist of a few scattered erosions or tense blisters on an erythematous or urticarial base. MMP is typically a chronic and progressive disorder. Serious complications may arise as a consequence of ocular, laryngeal, esophageal, or anogenital lesions. Erosive conjunctivitis may result in shortened fornices, symblepharon, ankyloblepharon, entropion, corneal opacities, and (in severe cases) blindness. Similarly, erosive lesions of the larynx may cause hoarseness, pain,

1	may result in shortened fornices, symblepharon, ankyloblepharon, entropion, corneal opacities, and (in severe cases) blindness. Similarly, erosive lesions of the larynx may cause hoarseness, pain, and tissue loss that, if unrecognized and untreated, may eventuate in complete destruction of the airway. Esophageal lesions may result in stenosis and/or strictures that could place patients at risk for aspiration. Strictures may also complicate anogenital involvement.

1	Biopsies of lesional tissue generally show subepithelial vesiculobullae and a mononuclear leukocytic infiltrate. Neutrophils and eosinophils may be seen in biopsies of early lesions; older lesions may demonstrate a scant leukocytic infiltrate and fibrosis. Direct immunofluorescence microscopy of perilesional tissue typically reveals deposits of IgG, IgA, and/or C3 in the epidermal basement membrane. Because many patients with MMP exhibit no evidence of circulating basement membrane autoantibodies, testing of perilesional skin is important diagnostically. Although MMP was once thought to be a single nosologic entity, it is now largely regarded as a disease phenotype that may develop as a consequence of an autoimmune reaction to a variety of molecules in the epidermal basement membrane (e.g., BPAG2, laminin-332, type VII collagen, and other antigens yet to be completely defined). Studies suggest that MMP patients with autoantibodies to laminin-332 have an increased relative risk for

1	BPAG2, laminin-332, type VII collagen, and other antigens yet to be completely defined). Studies suggest that MMP patients with autoantibodies to laminin-332 have an increased relative risk for cancer. Treatment of MMP is largely dependent upon the sites of involvement. Due to potentially severe complications, patients with ocular, laryngeal, esophageal, and/or anogenital involvement require aggressive systemic treatment with dapsone, prednisone, or the latter in combination with another immunosuppressive agent (e.g., azathioprine, mycophenolate mofetil, cyclophosphamide, or rituximab) or IVIg. Less threatening forms of the disease may be managed with topical or intralesional glucocorticoids.

1	PART 2 Cardinal Manifestations and Presentation of Diseases The cutaneous manifestations of dermatomyositis (Chap. 388) are often distinctive but at times may resemble those of systemic lupus erythematosus (SLE) (Chap. 378), scleroderma (Chap. 382), or other overlapping connective tissue diseases (Chap. 382). The extent and severity of cutaneous disease may or may not correlate with the extent and severity of the myositis. The cutaneous manifestations of dermatomyositis are similar, whether the disease appears in children or in the elderly, except that calcification of subcutaneous tissue is a common late sequela in childhood dermatomyositis. The cutaneous signs of dermatomyositis may precede or follow the development of myositis by weeks to years. Cases lacking muscle

1	FIguRE 73-3 Dermatomyositis. Periorbital violaceous erythema characterizes the classic heliotrope rash. (Courtesy of James Krell, MD; with permission.) involvement (i.e., dermatomyositis sine myositis) have also been reported. The most common manifestation is a purple-red discoloration of the upper eyelids, sometimes associated with scaling (“heliotrope” erythema; Fig. 73-3) and periorbital edema. Erythema on the cheeks and nose in a “butterfly” distribution may resemble the malar eruption of SLE. Erythematous or violaceous scaling patches are common on the upper anterior chest, posterior neck, scalp, and the extensor surfaces of the arms, legs, and hands. Erythema and scaling may be particularly prominent over the elbows, knees, and dorsal interphalangeal joints. Approximately one-third of patients have violaceous, flat-topped papules over the dorsal interphalangeal joints that are pathognomonic of dermatomyositis (Gottron’s papules). Thin violaceous papules and plaques on the elbows

1	patients have violaceous, flat-topped papules over the dorsal interphalangeal joints that are pathognomonic of dermatomyositis (Gottron’s papules). Thin violaceous papules and plaques on the elbows and knees of patients with dermatomyositis are referred to as Gottron’s sign (Fig. 73-4). These lesions can be contrasted with the erythema and scaling on the dorsum of the fingers that spares the skin over the interphalangeal joints of some SLE patients. Periungual telangiectasia may be prominent in patients with dermatomyositis. Lacy or reticulated erythema may be associated with fine scaling on the extensor and lateral surfaces of the thighs and upper arms. Other patients, particularly those with long-standing disease, develop areas of hypopigmentation, hyperpigmentation, mild atrophy, and telangiectasia known as poikiloderma. Poikiloderma is rare in both SLE and scleroderma and thus can serve as a clinical sign that distinguishes dermatomyositis from these two diseases. Cutaneous

1	and telangiectasia known as poikiloderma. Poikiloderma is rare in both SLE and scleroderma and thus can serve as a clinical sign that distinguishes dermatomyositis from these two diseases. Cutaneous changes may be similar in dermatomyositis and various overlap syndromes where thickening and binding down of the skin of the hands (sclerodactyly) as well as Raynaud’s phenomenon can be seen. However, the presence of severe muscle disease, Gottron’s papules, heliotrope erythema, and poikiloderma serves to distinguish patients with dermatomyositis. Skin biopsy of the erythematous, scaling lesions of dermatomyositis may reveal only mild nonspecific inflammation but sometimes may show changes indistinguishable from those found in SLE, including epidermal atrophy, hydropic degeneration of basal keratinocytes, edema of the upper dermis, and a mild mononuclear cell infiltrate. Direct immunofluorescence microscopy of lesional skin is usually negative, although granular deposits of

1	of basal keratinocytes, edema of the upper dermis, and a mild mononuclear cell infiltrate. Direct immunofluorescence microscopy of lesional skin is usually negative, although granular deposits of immunoglobulin(s) and complement in the epidermal basement membrane zone have been described in some patients. Treatment should be directed at the systemic disease. Topical glucocorticoids are sometimes useful; patients should avoid exposure to ultraviolet irradiation and aggressively use photoprotective measures, including broad-spectrum sunscreens.

1	FIguRE 73-4 Gottron’s papules. Dermatomyositis often involves the hands as erythematous flat-topped papules over the knuckles. Periungual telangiectases are also evident. The cutaneous manifestations of lupus erythematosus (LE) (Chap. 378) can be divided into acute, subacute, and chronic or discoid types. Acute cutaneous LE is characterized by erythema of the nose and malar eminences in a “butterfly” distribution (Fig. 73-5A). The erythema is often sudden in onset, accompanied by edema and fine scale, and

1	FIguRE 73-5 Acute cutaneous lupus erythematosus (LE). A. Acute cutaneous LE on the face, showing prominent, scaly, malar erythema. Involvement of other sun-exposed sites is also common. B. Acute cutaneous LE on the upper chest, demonstrating brightly erythematous and slightly edematous papules and plaques. (B, Courtesy of Robert Swerlick, MD; with permission.) correlated with systemic involvement. Patients may have widespread 375 involvement of the face as well as erythema and scaling of the extensor surfaces of the extremities and upper chest (Fig. 73-5B). These acute lesions, while sometimes evanescent, usually last for days and are often associated with exacerbations of systemic disease. Skin biopsy of acute lesions may show only a sparse dermal infiltrate of mononuclear cells and dermal edema. In some instances, cellular infiltrates around blood vessels and hair follicles are notable, as is hydropic degeneration of basal cells of the epidermis. Direct immunofluorescence microscopy

1	edema. In some instances, cellular infiltrates around blood vessels and hair follicles are notable, as is hydropic degeneration of basal cells of the epidermis. Direct immunofluorescence microscopy of lesional skin frequently reveals deposits of immunoglobulin(s) and complement in the epidermal basement membrane zone. Treatment is aimed at control of systemic disease. Photoprotection is very important in this as well as in other forms of LE.

1	Subacute cutaneous lupus erythematosus (SCLE) is characterized by a widespread photosensitive, nonscarring eruption. In most patients, renal and central nervous system involvement is mild or absent. SCLE may present as a papulosquamous eruption that resembles psoriasis or as annular polycyclic lesions that resemble those seen in erythema multiforme. In the papulosquamous form, discrete erythematous papules arise on the back, chest, shoulders, extensor surfaces of the arms, and dorsum of the hands; lesions are uncommon on the central face and the flexor surfaces of the arms as well as below the waist. These slightly scaling papules tend to merge into large plaques, some with a reticulate appearance. The annular form involves the same areas and presents with erythematous papules that evolve into oval, circular, or polycyclic lesions. The lesions of SCLE are more widespread but have less tendency for scarring than lesions of discoid LE. Skin biopsy reveals a dense mononuclear cell

1	evolve into oval, circular, or polycyclic lesions. The lesions of SCLE are more widespread but have less tendency for scarring than lesions of discoid LE. Skin biopsy reveals a dense mononuclear cell infiltrate around hair follicles and blood vessels in the superficial dermis, combined with hydropic degeneration of basal cells in the epidermis. Direct immunofluorescence microscopy of lesional skin reveals deposits of immunoglobulin(s) in the epidermal basement membrane zone in about one-half of these cases. A particulate pattern of IgG deposition throughout the epidermis has been associated with SCLE. Most SCLE patients have anti-Ro autoantibodies. Local therapy alone is usually unsuccessful. Most patients require treatment with aminoquinoline antimalarial drugs. Low-dose therapy with oral glucocorticoids is sometimes necessary. Photoprotective measures against both ultraviolet B and ultraviolet A wavelengths are very important.

1	Discoid lupus erythematosus (DLE, also called chronic cutaneous LE) is characterized by discrete lesions, most often found on the face, scalp, and/or external ears. The lesions are erythematous papules or plaques with a thick, adherent scale that occludes hair follicles (follicular plugging). When the scale is removed, its underside shows small excrescences that correlate with the openings of hair follicles (so-called “carpet tacking”), a finding relatively specific for DLE. Longstanding lesions develop central atrophy, scarring, and hypopigmentation but frequently have erythematous, sometimes raised borders (Fig. 73-6). These lesions persist for years and tend to expand slowly. Up to 15% of patients with DLE eventually meet the American College of Rheumatology criteria for SLE. However, typical discoid lesions are frequently seen in patients with SLE. Biopsy of DLE lesions shows hyperkeratosis, follicular plugging, atrophy of the epidermis, hydropic degeneration of basal

1	However, typical discoid lesions are frequently seen in patients with SLE. Biopsy of DLE lesions shows hyperkeratosis, follicular plugging, atrophy of the epidermis, hydropic degeneration of basal keratinocytes, and a mononuclear cell infiltrate adjacent to epidermal, adnexal, and microvascular basement membranes. Direct immunofluorescence microscopy demonstrates immunoglobulin(s) and complement deposits at the basement membrane zone in ~90% of cases. Treatment is focused on control of local cutaneous disease and consists mainly of photoprotection and topical or intralesional glucocorticoids. If local therapy is ineffective, use of aminoquinoline antimalarial agents may be indicated.

1	The skin changes of scleroderma (Chap. 382) usually begin on the hands, feet, and face, with episodes of recurrent nonpitting edema. Sclerosis of the skin commences distally on the fingers (sclerodactyly) and spreads proximally, usually accompanied by resorption of bone of the fingertips, which may have punched out ulcers, stellate scars, or areas of hemorrhage (Fig. 73-7). The fingers may actually shrink and PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 73-6 Discoid (chronic cutaneous) lupus erythematosus. Violaceous, hyperpigmented, atrophic plaques, often with evidence of follicular plugging that may result in scarring, are typical.

1	become sausage-shaped, and, because the fingernails are usually unaffected, they may curve over the end of the fingertips. Periungual telangiectases are usually present, but periungual erythema is rare. In advanced cases, the extremities show contractures and calcinosis cutis. Facial involvement includes a smooth, unwrinkled brow, taut skin over the nose, shrinkage of tissue around the mouth, and perioral radial furrowing (Fig. 73-8). Matlike telangiectases are often present, particularly on the face and hands. Involved skin feels indurated, smooth, and bound to underlying structures; hyperand hypopigmentation are common as well. Raynaud’s phenomenon (i.e., cold-induced blanching, cyanosis, and reactive hyperemia) is documented in almost all patients and can precede development of scleroderma by many years. Linear scleroderma is a limited form of disease that presents in a linear, bandlike distribution and tends to involve deep as well as superficial layers of skin. The combination of

1	by many years. Linear scleroderma is a limited form of disease that presents in a linear, bandlike distribution and tends to involve deep as well as superficial layers of skin. The combination of calcinosis cutis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia has been termed the CREST syndrome. Centromere antibodies have been reported in a very high percentage of patients with the CREST syndrome but in only a small minority of patients with scleroderma. Skin biopsy reveals thickening of the dermis and homogenization of collagen bundles. Direct immunofluorescence microscopy of lesional skin is usually negative.

1	Morphea is characterized by localized thickening and sclerosis of skin; it dominates on the trunk. This disorder may affect children or adults. Morphea begins as erythematous or flesh-colored plaques that become sclerotic, develop central hypopigmentation, and have an FIguRE 73-7 Scleroderma showing acral sclerosis and focal digi-tal ulcers. FIguRE 73-8 Scleroderma often eventuates in development of an expressionless, masklike facies.

1	FIguRE 73-7 Scleroderma showing acral sclerosis and focal digi-tal ulcers. FIguRE 73-8 Scleroderma often eventuates in development of an expressionless, masklike facies. erythematous border. In most cases, patients have one or a few lesions, and the disease is termed localized morphea. In some patients, widespread cutaneous lesions may occur without systemic involvement (generalized morphea). Many adults with generalized morphea have concomitant rheumatic or other autoimmune disorders. Skin biopsy of morphea is indistinguishable from that of scleroderma. Scleroderma and morphea are usually quite resistant to therapy. For this reason, physical therapy to prevent joint contractures and to maintain function is employed and is often helpful. Treatment options for early, rapidly progressive disease include phototherapy (UVA1 or PUVA) or methotrexate (15–20 mg/week) alone or in combination with daily glucocorticoids.

1	Diffuse fasciitis with eosinophilia is a clinical entity that can sometimes be confused with scleroderma. There is usually a sudden onset of swelling, induration, and erythema of the extremities, frequently following significant physical exertion. The proximal portions of the extremities (upper arms, forearms, thighs, calves) are more often involved than are the hands and feet. While the skin is indurated, it usually displays a woody, dimpled, or “pseudocellulite” appearance rather than being bound down as in scleroderma; contractures may occur early secondary to fascial involvement. The latter may also cause muscle groups to be separated and veins to appear depressed (i.e., the “groove sign”). These skin findings are accompanied by peripheral-blood eosinophilia, increased erythrocyte sedimentation rate, and sometimes hypergammaglobulinemia. Deep biopsy of affected areas of skin reveals inflammation and thickening of the deep fascia overlying muscle. An inflammatory infiltrate

1	sedimentation rate, and sometimes hypergammaglobulinemia. Deep biopsy of affected areas of skin reveals inflammation and thickening of the deep fascia overlying muscle. An inflammatory infiltrate composed of eosinophils and mononuclear cells is usually found. Patients with eosinophilic fasciitis appear to be at increased risk for developing bone marrow failure or other hematologic abnormalities. While the ultimate course of eosinophilic fasciitis is uncertain, many patients respond favorably to treatment with prednisone in doses of 40–60 mg/d.

1	The eosinophilia-myalgia syndrome, a disorder with epidemic numbers of cases reported in 1989 and linked to ingestion of l-tryptophan manufactured by a single company in Japan, is a multisystem disorder characterized by debilitating myalgias and absolute eosinophilia in association with varying combinations of arthralgias, pulmonary symptoms, and peripheral edema. In a later phase (3–6 months after initial symptoms), these patients often develop localized sclerodermatous skin changes, weight loss, and/or neuropathy (Chap. 382). The precise cause of this syndrome, which may resemble other sclerotic skin conditions, is unknown. However, the implicated lots of l-tryptophan contained the contaminant 1,1-ethylidene bis[tryptophan]. This contaminant may be pathogenic or may be a marker for another substance that provokes the disorder. Cutaneous Drug Reactions Evidence suggests an immunologic basis for most acute drug eruptions. Drug reactions may result from immediate release of preformed

1	Cutaneous Drug Reactions Evidence suggests an immunologic basis for most acute drug eruptions. Drug reactions may result from immediate release of preformed Kanade Shinkai, Robert S. Stern, Bruce U. Wintroub mediators (e.g., urticaria, anaphylaxis), antibody-mediated reactions, Cutaneous reactions are among the most frequent adverse reactions to drugs. Most are benign, but a few can be life threatening. Prompt recognition of severe reactions, drug withdrawal, and appropriate therapeutic interventions can minimize toxicity. This chapter focuses on adverse cutaneous reactions to systemic medications; it covers their incidence, patterns, and pathogenesis and provides some practical guidelines on treatment, assessment of causality, and future use of drugs.

1	In the United States, more than 3 billion prescriptions for over 60,000 drug products, which include more than 2000 different active agents, are dispensed annually. Hospital inpatients alone annually receive about 120 million courses of drug therapy, and half of adult Americans receive prescription drugs on a regular outpatient basis. Many patients use over-the-counter medicines that may cause adverse cutaneous reactions. Several large cohort studies established that acute cutaneous reaction to drugs affected about 3% of hospital inpatients. Reactions usually occur a few days to 4 weeks after initiation of therapy. Many drugs of common use are associated with a 1–2% rate of rashes during premarketing clinical trials. The risk is often higher when medications are used in general, unselected populations. The rate may reach 3–7% for amoxicillin, sulfamethoxazole, many anticonvulsants, and anti-HIV agents.

1	In addition to acute eruptions, a variety of skin diseases can be induced or exacerbated by prolonged use of drugs (e.g., pruritus, pigmentation, nail or hair disorders, psoriasis, bullous pemphigoid, photosensitivity, and even cutaneous neoplasms). These drug reactions are not frequent, but neither their incidence nor their impact on public health has been evaluated.

1	In a series of 48,005 inpatients over a 20-year period, morbilliform rash (91%) and urticaria (6%) were the most frequent skin reactions. Severe reactions are actually too rare to be detected in such cohorts. Although rare, severe cutaneous reactions to drugs have an important impact on health because of significant sequelae, including mortality. Adverse drug rashes are responsible for hospitalization, increase the duration of hospital stay, and are life threatening. Some populations are at increased risk of drug reactions, including patients with collagen vascular diseases, bone marrow graft recipients, and those with acute Epstein-Barr virus infection. The pathophysiology underlying this association is unknown, but may be related to immunocompromise or immune dysregulation. Risk of drug allergy, including severe hypersensitivity reactions, is increased with HIV infection; individuals with advanced HIV disease (e.g., CD4 T lymphocyte count <200 cells/μL) have a fortyto fiftyfold

1	drug allergy, including severe hypersensitivity reactions, is increased with HIV infection; individuals with advanced HIV disease (e.g., CD4 T lymphocyte count <200 cells/μL) have a fortyto fiftyfold increased risk of adverse reactions to sulfamethoxazole (Chap. 226).

1	Adverse cutaneous responses to drugs can arise as a result of immunologic or nonimmunologic mechanisms. Examples of responses that arise from nonimmunologic mechanisms are pigmentary changes related to dermal accumulation of medications or their metabolites; alteration of hair follicles by antimetabolites and signaling inhibitors; and lipodystrophy associated with metabolic effects of anti-HIV medications. These side effects are mostly toxic, predictable, and sometimes can be avoided in part by simple preventive measures.

1	immune complex deposition, and antigen-specific responses. Drug-specific T cell clones can be derived from the blood or from skin lesions of patients with a variety of drug allergies, strongly suggesting that these T cells play a role in drug allergy in an antigen-specific manner. Specific clones were obtained with penicillin G, amoxicillin, cephalosporins, sulfamethoxazole, phenobarbital, carbamazepine, and lamotrigine (medications that are frequently a cause of drug eruptions). Both CD4 and CD8 clones have been obtained; however, their specific roles in the manifestations of allergy have not been elucidated. Drug presentation to T cells was major histocompatibility complex (MHC)-restricted and may involve drug-peptide complex recognition by specific T cell receptors (TCRs).

1	Once a drug has induced an immune response, the final phenotype of the reaction probably depends on the nature of effectors: cytotoxic (CD8+) T cells in blistering and certain hypersensitivity reactions, chemokines for reactions mediated by neutrophils or eosinophils, and collaboration with B cells for production of specific antibodies for urticarial reaction. Immunologic reactions have recently been classified into further subtypes that provide a useful framework for designating adverse drug reactions based on involvement of specific immune pathways (Table 74-1).

1	Immediate Reactions Immediate reactions depend on the release of mediators of inflammation by tissue mast cells or circulating basophils. These mediators include histamine, leukotrienes, prostaglandins, bradykinins, platelet-activating factor, enzymes, and proteoglycans. Drugs can trigger mediator release either directly (“anaphylactoid” reaction) or through IgE-specific antibodies. These reactions usually manifest in the skin and gastrointestinal, respiratory, and cardiovascular systems (Chap. 376). Primary symptoms and signs include pruritus, urticaria, nausea, vomiting, abdominal cramps, bronchospasm, laryngeal edema, and, occasionally, anaphylactic shock with hypotension and death. They occur within minutes of drug exposure. Nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin, and radiocontrast media are frequent causes of direct mast cell degranulation or anaphylactoid reactions, which can occur on first exposure. Penicillins and muscle relaxants used in general

1	aspirin, and radiocontrast media are frequent causes of direct mast cell degranulation or anaphylactoid reactions, which can occur on first exposure. Penicillins and muscle relaxants used in general anesthesia are the most frequent causes of IgE-dependent reactions to drugs, which require prior sensitization. Release of mediators is triggered when polyvalent drug protein conjugates cross-link IgE molecules fixed to sensitized cells. Certain routes of administration favor different clinical patterns (e.g., gastrointestinal effects from oral route, circulatory effects from intravenous route).

1	Immune Complex–Dependent Reactions Serum sickness is produced by tissue deposition of circulating immune complexes with consumption of complement. It is characterized by fever, arthritis, nephritis, neuritis, edema, and a urticarial, papular, or purpuric rash (Chap. 385). First described following administration of nonhuman sera, it currently occurs in the setting of monoclonal antibodies and other similar medications. In classic serum sickness, symptoms develop 6 days or more after exposure to a drug, the latent period representing the time needed to synthesize antibody. Cutaneous or systemic vasculitis, a relatively rare complication of drugs, may also be a result of immune complex deposition (Chap. 385). Cephalosporin and other medications, including monoclonal antibodies such as infliximab, rituximab, and omalizumab, may be associated with clinically similar “serum sickness–like” reactions. The mechanism of this reaction is unknown but is unrelated to complement activation and

1	rituximab, and omalizumab, may be associated with clinically similar “serum sickness–like” reactions. The mechanism of this reaction is unknown but is unrelated to complement activation and immune complex formation.

1	Delayed Hypersensitivity While not completely understood, delayed hypersensitivity directed by drug-specific T cells is an important mechanism underlying the most common drug eruptions, i.e., morbilliform eruptions, and also rare and severe forms such as drug-induced hypersensitivity syndrome (DIHS) (also known PART 2 Cardinal Manifestations and Presentation of Diseases Abbreviations: GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

1	PART 2 Cardinal Manifestations and Presentation of Diseases Abbreviations: GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor. as drug rash with eosinophilia and systemic symptoms [DRESS]), acute generalized exanthematous pustulosis (AGEP), Stevens-Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN) (Table 74-1). Drug-specific T cells have been detected in these types of drug eruptions. For example, drug-specific cytotoxic T cells have been detected in the skin lesions of fixed drug eruptions and of TEN. In TEN, skin lesions contain T lymphocytes reactive to autologous lymphocytes and keratinocytes in a drug-specific, HLA-restricted, and perforin/ granzyme-mediated pathway.

1	The mechanism(s) by which medications result in T cell activation is unknown. Two hypotheses prevail: first, that the antigens driving these reactions may be the native drug itself or components of the drug covalently complexed with endogenous proteins, presented in association with HLA molecules to T cells through the classical antigen presentation pathway, or alternatively, through direct interaction of the drug/metabolite with the T cell receptor or peptide-loaded HLA (e.g., the pharmacologic interaction of drugs with immune receptors, or p-i hypothesis). Recent x-ray crystallography data characterizing binding between specific HLA molecules to particular drugs known to cause hypersensitivity reactions demonstrates unique alterations to the MHC peptide-binding groove, suggesting a molecular basis for T cell activation and the development of hypersensitivity reactions.

1	Genetic determinants may predispose individuals to severe responses to drugs. Polymorphisms in cytochrome P450 enzymes, drug acetylation, methylation (such as thiopurine methyltransferase activity and azathioprine), and other forms of metabolism (such as glucose-6-phosphate dehydrogenase) may increase susceptibility to drug toxicity or underdosing, highlighting a role for differential pharmacokinetic or pharmacodynamic effects.

1	Associations between drug hypersensitivities and HLA haplotypes also suggest a key role for immune mechanisms. Hypersensitivity to the anti-HIV medication abacavir is strongly associated with HLAB*57:01 (Chap. 226). In Taiwan, within a homogeneous Han Chinese population, a 100% association was observed between SJS/TEN (but not DIHS) related to carbamazepine and HLA-B*15:02. In the same population, another 100% association was found between SJS, TEN, or DIHS related to allopurinol and HLA-B*58:01. These associations are drug and phenotype specific; that is, HLA-specific T cell stimulation by medications leads to distinct reactions and may explain why the reaction patterns are so clinically diverse. However, the strong associations found in Taiwan have not been observed in other countries with more heterogeneous populations.

1	Recognition of the HLA associations with drug hypersensitiv ity has been acknowledged by recommendations to screen high-risk populations. Genetic screening for HLA-B*57:01 to prevent abacavir hypersensitivity, which carries a 100% negative predictive value when patch test confirmed and 55% positive predictive value generalizable across races, is becoming the clinical standard of care worldwide (number needed to treat = 13). The U.S. Food and Drug Administration recently mandated new labeling of carbamazepine recommending HLA-B*15:02 screening of Asian individuals prior to receiving a new prescription of the medication. The American College of Rheumatology has recommended HLA-B*58:01 screening of Han Chinese patients prescribed allopurinol. To date, screening for a single HLA (but not multiple HLA haplotypes) in specific populations has been determined to be cost-effective.

1	Several investigators have proposed that specific HLA haplotypes associated with drug hypersensitivity indeed play a pathogenic role; stimulation of carbamazepine-specific cytotoxic T lymphocytes (CTL) in the context of HLA-B*15:02 results in production of a putative mediator of keratinocyte necrosis in TEN. Other studies have identified CTLs reactive to carbamazepine that use highly restricted V-alpha and V-beta TCR repertoires in patients with carbamazepine hypersensitivity that are not found in carbamazepine-tolerant individuals. Although not yet clinically available, some investigators have suggested combined genetic testing for specific HLA haplotypes and functional screening for TCR repertoire to best identify patients at risk.

1	NONIMMuNE CuTANEOuS REACTIONS Exacerbation or Induction of Dermatologic Diseases A variety of drugs can exacerbate preexisting diseases or sometimes induce a disease that may or may not disappear after withdrawal of the inducing medication. For example, NSAIDs, lithium, beta blockers, tumor necrosis factor (TNF) α cytokine antagonists, interferon (IFN) α, and angiotensinconverting enzyme (ACE) inhibitors can exacerbate plaque psoriasis, whereas antimalarials and withdrawal of systemic glucocorticoids can worsen pustular psoriasis. The situation of TNF-α inhibitors is unusual, as this class of medications is used to treat psoriasis; however, in other cases, they may induce psoriasis (especially palmar-plantar) in patients being treated for other conditions. Acne may be induced by glucocorticoids, androgens, lithium, and antidepressants. Follicular papular or pustular eruptions of the face and trunk, sometimes mimicking acne, frequently occur with epidermal growth factor (EGF) receptor

1	androgens, lithium, and antidepressants. Follicular papular or pustular eruptions of the face and trunk, sometimes mimicking acne, frequently occur with epidermal growth factor (EGF) receptor antagonists. In the case of EGF-receptor antagonists, the severity of the eruption correlates with a better anticancer effect. It may be secondarily impetiginized and often spares areas of prior or active radiation. Tetracycline antibiotics, topical corticosteroids, and topical anti-acne treatments (such as benzoyl peroxide and clindamycin lotion) are helpful.

1	Several medications induce or exacerbate autoimmune disease. Interleukin (IL) 2, IFN-α, and anti-TNF-α are associated with new-onset systemic lupus erythematosus (SLE). Drug-induced lupus is classically marked by antinuclear and antihistone antibodies and, in some cases, anti-double-stranded DNA (D-penicillamine, anti-TNF-α) or p-ANCA (minocycline) antibodies. Minocycline and thiazide diuretics may exacerbate subacute SLE; pemphigus can be induced by D-penicillamine and ACE inhibitors. Furosemide is associated with drug-induced bullous pemphigoid. Vancomycin is associated with linear IgA bullous dermatitis, a transient blistering disorder.

1	Other medications may cause highly selective cutaneous reactions. Gadolinium contrast has been associated with nephrogenic systemic fibrosis, a condition of sclerosing skin with rare internal organ involvement; advanced renal compromise may be an important risk factor. Granulocyte colony-stimulating factor may induce various neutrophilic dermatoses, including Sweet syndrome and pyoderma gangrenosum. Both systemic and topical glucocorticoids cause a variety of atrophic skin changes, including atrophy and striae, and, in sufficiently high doses, can impede wound healing. The hypothesis that a drug may be responsible should always be considered, especially in cases with atypical clinical presentation. Resolution of the cutaneous reaction may be delayed upon discontinuation of the medication (e.g., lichenoid drug eruptions may take years to resolve).

1	Photosensitivity Eruptions Photosensitivity eruptions are usually most marked in sun-exposed areas but may extend to sun-protected areas. The mechanism is almost always phototoxicity. Phototoxic reactions resemble sunburn and can occur with first exposure to a drug. Blistering may occur in drug-related pseudoporphyria, most commonly with NSAIDs (Fig. 74-1). The severity of the reactions depends on the tissue level of the drug, its efficiency as a photosensitizer, and the extent of exposure to the activating wavelengths of ultraviolet (UV) light (Chap. 75). Common orally administered photosensitizing drugs include fluoroquinolones and tetracycline antibiotics. Other drugs less frequently encountered are chlorpromazine, thiazides, and NSAIDs. Voriconazole may result in severe photosensitivity, accelerated photo-induced aging, and cutaneous carcinogenesis in organ transplant recipients.

1	Because UV-A and visible light, which trigger these reactions, are not easily absorbed by nonopaque sunscreens and are transmitted through window glass, photosensitivity reactions may be difficult to block. Photosensitivity reactions abate with removal of either the drug or UV radiation, use of sunscreens that block UV-A light, and treating the reaction as one would a sunburn. Rarely, individuals develop persistent reactivity to light, necessitating long-term avoidance of sun 379 exposure.

1	Pigmentation Changes Drugs, either systemic or topical, may cause a variety of pigmentary changes in the skin. Oral contraceptives may induce melasma. Long-term minocycline, pefloxacin, and amiodarone may cause blue-gray pigmentation. Phenothiazine, gold, and bismuth result in gray-brown pigmentation of sun-exposed areas. Numerous cancer chemotherapeutic agents may be associated with characteristic patterns of pigmentation (e.g., bleomycin, busulfan, daunorubicin, cyclophosphamide, hydroxyurea, and methotrexate). Clofazimine causes a drug-induced lipofuscinosis with characteristic red-brown coloration. Hyperpigmentation of the face, mucous membranes, and pretibial and subungual areas occurs with antimalarials. Quinacrine causes generalized, cutaneous yellow discoloration. Pigmentation changes may also occur in mucous membranes (busulfan, bismuth), conjunctiva (chlorpromazine, thioridazine, imipramine, clomipramine), nails (zidovudine, doxorubicin, cyclophosphamide, bleomycin,

1	changes may also occur in mucous membranes (busulfan, bismuth), conjunctiva (chlorpromazine, thioridazine, imipramine, clomipramine), nails (zidovudine, doxorubicin, cyclophosphamide, bleomycin, fluorouracil, hydroxyurea), hair, and teeth (tetracyclines).

1	Warfarin Necrosis of Skin This rare reaction (0.01–0.1%) usually occurs between the third and tenth days of therapy with warfarin, usually in women. Common sites are breasts, thighs, and buttocks (Fig. 74-2). Lesions are sharply demarcated, indurated, and erythematous or purpuric and may progress to form large, hemorrhagic bullae with eventual necrosis and slow-healing eschar formation. These lesions can be life threatening.

1	Development of the syndrome is unrelated to drug dose, and the course is not altered by discontinuation of the drug after onset of the eruption. Warfarin anticoagulation in heterozygous protein C deficiency causes a precipitous fall in circulating levels of protein C, permitting hypercoagulability and thrombosis in the cutaneous microvasculature, with consequent areas of necrosis. Heparin-induced necrosis may have clinically similar features but is probably due to heparin-induced platelet aggregation with subsequent occlusion of blood vessels; it can affect areas adjacent to the injection site or more distant sites if infused. Warfarin-induced cutaneous necrosis is treated with vitamin K, heparin, surgical debridement, and intensive wound care. Treatment with protein C concentrates may also be helpful. Newer agents such as dabigatran etexilate may avoid warfarin necrosis in high-risk patients.

1	Drug-Induced Hair Disorders • dRUg-INdUCEd HAIR LOSS Medications may affect hair follicles at two different phases of their growth cycle: anagen (growth) or telogen (resting). Anagen effluvium occurs within days of drug administration, especially with antimetabolite or other chemotherapeutic drugs. In contrast, in telogen effluvium, the delay is 2 to 4 months following initiation of a new medication. Both present as diffuse nonscarring alopecia most often reversible after discontinuation FIguRE 74-1 Pseudoporphyria due to nonsteroidal anti-inflammatory drugs. FIguRE 74-2 Warfarin necrosis.

1	FIguRE 74-1 Pseudoporphyria due to nonsteroidal anti-inflammatory drugs. FIguRE 74-2 Warfarin necrosis. 380 of the responsible agent. The prevalence and severity of alopecia depend on the drug as well as on an individual’s predisposition. A considerable number of drugs have been reported to induce hair loss. These include antineoplastic agents (alkylating agents, bleomycin, vinca alkaloids, platinum compounds), anticonvulsants (carbamazepine, valproate), antihypertensive drugs (beta blockers), antidepressants, antithyroid drugs, IFNs (especially IFN-α), oral contraceptives, and cholesterol-lowering agents.

1	dRUg-INdUCEd HAIR gROwTH Medications may also cause hair growth. Hirsutism is an excessive growth of terminal hair with masculine hair growth pattern in a female, most often on the face and trunk, due to androgenic stimulation of hormone-sensitive hair follicles (anabolic steroids, oral contraceptives, testosterone, corticotropin). Hypertrichosis is a distinct pattern of hair growth, not in a masculine pattern, typically located on the forehead and temporal regions of the face. Drugs responsible for hypertrichosis include anti-inflammatory drugs, glucocorticoids, vasodilators (diazoxide, minoxidil), diuretics (acetazolamide), anticonvulsants (phenytoin), immunosuppressive agents (cyclosporine A), psoralens, and zidovudine.

1	Changes in hair color or structure are uncommon adverse effects from medications. Hair discoloration may occur with chloroquine, IFN-α, chemotherapeutic agents, and tyrosine kinase inhibitors. Changes in hair structure have been observed in patients given epidermal growth factor receptor (EGFR) inhibitors, tyrosine kinase inhibitors (Fig. 74-3), and acitretin. Drug-Induced Nail Disorders Drug-related nail disorders usually involve all 20 nails and need months to resolve after withdrawal of the offending agent. The pathogenesis is most often toxic. Drug-induced nail changes include Beau’s line (transverse depression of the nail plate), onycholysis (detachment of the distal part of the nail plate), onychomadesis (detachment of the proximal part of the nail plate), pigmentation, and paronychia (inflammation of periungual skin).

1	ONYCHOLYSIS Onycholysis occurs with tetracyclines, fluoroquinolones, phenothiazines, and psoralens, as well as in persons taking NSAIDs, captopril, retinoids, sodium valproate, and many chemotherapeutic agents such as anthracyclines or taxanes including paclitaxel and docetaxel. The risk of onycholysis in patients receiving cytotoxic drugs, tetracyclines, quinolones, phenothiazines, and psoralens can be increased by exposure to sunlight. ONYCHOMAdESIS Onychomadesis is caused by temporary arrest of nail matrix mitotic activity. Common drugs reported to induce onychomadesis include carbamazepine, lithium, retinoids, and chemotherapeutic agents such as cyclophosphamide and vincristine. PARONYCHIA Paronychia and multiple pyogenic granuloma (Fig. 74-4) with progressive and painful periungual abscess of fingers and toes PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 74-3 Dysmorphic eyelashes in association with erlotinib.

1	PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 74-3 Dysmorphic eyelashes in association with erlotinib. FIguRE 74-4 Pyogenic granuloma in association with isotretinoin. are side effects of systemic retinoids, lamivudine, indinavir, and anti-EGFR monoclonal antibodies (cetuximab, gefitinib). NAIL dISCOLORATION Some drugs—including anthracyclines, taxanes, fluorouracil, psoralens, and zidovudine—may induce nail bed hyper-pigmentation through melanocyte stimulation. It appears to be reversible and dose-dependent. Toxic Erythema of Chemotherapy and Other Chemotherapy Reactions

1	Toxic Erythema of Chemotherapy and Other Chemotherapy Reactions Because many agents used in cancer chemotherapy inhibit cell division, rapidly proliferating elements of the skin, including hair, mucous membranes, and appendages, are sensitive to their effects. A broad spectrum of chemotherapy-related skin toxicities have been reported, including neutrophilic eccrine hidradenitis, sterile cellulitis, exfoliative dermatitis, and flexural erythema; although previously designated as distinct skin eruptions, recent nomenclature classifies these under the unifying diagnosis of toxic erythema of chemotherapy (TEC). Acral erythema, marked by dysesthesia and an erythematous, edematous eruption of the palms and soles, is caused by cytarabine, doxorubicin, methotrexate, hydroxyurea, and fluorouracil and may be alleviated by pyridoxine supplementation.

1	The recent introduction of many new monoclonal antibody and small molecular signaling inhibitors for the treatment of cancer has been accompanied by numerous reports of skin and hair toxicity; only the most common of these are mentioned here. Cetuximab and other EGFR antagonists induce follicular eruptions and nail toxicity after a mean interval of 10 days in a majority of patients. Xerosis, eczematous eruptions, acneiform eruptions, and pruritus are common. Erlotinib is associated with marked hair textural changes (Fig. 74-3). Sorafenib, a tyrosine kinase inhibitor, may result in follicular eruptions and bullous palmoplantar eruptions with dysesthesia (Fig. 74-5). BRAF inhibitors are associated with photosensitivity, dyskeratotic (Grover’s-like) rash, hyperkeratotic benign cutaneous neoplasms, and keratoacanthoma-like squamous cell carcinomas. Rash, pruritus, and vitiliginous depigmentation have been reported in association with ipilimumab (anti-CTLA4) treatment.

1	IMMuNE CuTANEOuS REACTIONS: COMMON Maculopapular Eruptions Morbilliform or maculopapular eruptions (Fig. 74-6) are the most common of all drug-induced reactions, often start on the trunk or intertriginous areas, and consist of erythematous macules and papules that are symmetric and confluent. Involvement of mucous membranes is unusual; the eruption may be associated with moderate to severe pruritus and fever. Diagnosis is rarely assisted by laboratory testing. Skin biopsy often shows nonspecific inflammatory changes. A viral exanthem is the principal differential diagnostic consideration, especially in children, and graft-versus-host disease is also a consideration in the proper clinical setting. Absence of enanthems; absence of ear, nose, throat, and upper respiratory tract symptoms; and polymorphism of the skin lesions support a drug rather than a viral eruption. Certain medications carry very high rates of morbilliform eruption, including nevirapine and lamotrigine, even in the

1	and polymorphism of the skin lesions support a drug rather than a viral eruption. Certain medications carry very high rates of morbilliform eruption, including nevirapine and lamotrigine, even in the absence of hypersensitivity reactions. Lamotrigine morbilliform rash is associated with higher starting doses, rapid dose escalation, concomitant use of valproate (which increases lamotrigine levels and half-life) and use in children, especially with seizure disorder.

1	FIguRE 74-5 Sorafenib-associated hand-foot syndrome. Maculopapular reactions usually develop within 1 week of initiation of therapy and last less than 2 weeks. Occasionally, these eruptions resolve despite continued use of the responsible drug. Because the eruption may also worsen, the suspect drug should be discontinued unless it is essential; it is important to note that the rash may continue to progress for a few days to up to one week following medication discontinuation. Oral antihistamines and emollients may help relieve pruritus. Short courses of potent topical glucocorticoids can reduce inflammation and symptoms. Systemic glucocorticoid treatment is rarely indicated.

1	Pruritus Pruritus is associated with almost all drug eruptions and, in some cases, may represent the only symptom of the adverse cutaneous reaction. It is usually alleviated by antihistamines such as hydroxyzine or diphenhydramine. Pruritus stemming from specific medications may require distinct treatment; opiate-related pruritus may require selective opiate antagonists to gain relief. Pruritus is a common complication of antimalarial therapy, occurring in up to 50% of black patients receiving chloroquine, and may be severe enough to lead to discontinuation of treatment. It is much rarer in Caucasians taking chloroquine. Intense pruritus, sometimes accompanied by an eczematous rash, may occur in 20% of patients receiving IFN and ribavirin for hepatitis C; addition of the protease inhibitor telaprevir may increase 381 this occurrence to 50% of treated patients. FIguRE 74-6 Morbilliform drug eruption.

1	FIguRE 74-6 Morbilliform drug eruption. urticaria/Angioedema/Anaphylaxis Urticaria, the second most frequent type of cutaneous reaction to drugs, is characterized by pruritic, red wheals of varying size rarely lasting more than 24 h. It has been observed in association with nearly all drugs, most frequently ACE inhibitors, aspirin, NSAIDs, penicillin, and blood products. However, medications account for no more than 10–20% of acute urticaria cases. Deep edema within dermal and subcutaneous tissues is known as angioedema and may involve respiratory and gastrointestinal mucous membranes as well. Urticaria and angioedema may be part of a life-threatening anaphylactic reaction.

1	Drug-induced urticaria may be caused by three mechanisms: an IgE-dependent mechanism, circulating immune complexes (serum sickness), and nonimmunologic activation of effector pathways. IgE-dependent urticarial reactions usually occur within 36 h of drug exposure but can occur within minutes. Immune complex–induced urticaria associated with serum sickness–like reactions usually occurs 6–12 days after first exposure. In this syndrome, the urticarial eruption (typically polycyclic plaques) may be accompanied by fever, hematuria, arthralgias, hepatic dysfunction, and neurologic symptoms. Certain drugs, such as NSAIDs, ACE inhibitors, angiotensin II antagonists, radiographic dye, and opiates, may induce urticarial reactions, angioedema, and anaphylaxis in the absence of drug-specific antibody through direct mast-cell degranulation.

1	Radiocontrast agents are a common cause of urticaria and, in rare cases, can cause anaphylaxis. High-osmolality radiocontrast media were about five times more likely to induce urticaria (1%) or anaphylaxis than were newer low-osmolality media. About one-third of those with mild reactions to previous exposure react on reexposure. Pretreatment with prednisone and diphenhydramine reduces reaction rates. Persons with a reaction to a high-osmolality contrast media may be given low-osmolality media if later contrast studies are required.

1	The treatment of urticaria or angioedema depends on the severity of the reaction. In severe cases with respiratory or cardiovascular compromise, epinephrine is the mainstay of therapy, but its effect is reduced in patients using beta blockers. Treatment with intravenous systemic glucocorticoids is helpful. For patients with urticaria without symptoms of angioedema or anaphylaxis, drug withdrawal and oral antihistamines are usually sufficient. Future drug avoidance is recommended; rechallenge, especially in individuals with severe reactions, should only occur in an intensive care setting. Anaphylactoid Reactions Vancomycin is associated with red man syndrome, a histamine-related anaphylactoid reaction characterized by flushing, diffuse maculopapular eruption, and hypotension. In rare cases, cardiac arrest may be associated with rapid IV infusion of the medication.

1	Irritant/Allergic Contact Dermatitis Patients using topical medications may develop an irritant or allergic contact dermatitis to the medication itself or to a preservative or other component of the formulation. Reactions to chlorhexidine, neomycin sulfate, and polymyxin B are common. Allergic contact dermatitis to topical glucocorticoids may also occur and is paradoxically partially masked by the anti-inflammatory nature of the medication itself; typically this allergy is selective for one of the four classes of glucocorticoid types, as subdivided by allergenic properties. Patch testing can be useful to determine whether a patient is steroid allergic. Desoximetasone is rarely allergenic.

1	Fixed Drug Eruptions These less common reactions are characterized by one or more sharply demarcated, dull red to brown lesions, sometimes with central bulla (Fig. 74-7). Hyperpigmentation often results after resolution of the acute inflammation. With rechallenge, the lesion recurs in the same (e.g., fixed) location. Lesions often involve the lips, hands, legs, face, genitalia, and oral mucosa and cause a burning sensation. Most patients have multiple lesions. Fixed drug eruptions have been associated with pseudoephedrine (frequently a nonpigmented reaction), phenolphthalein (in laxatives), sulfonamides, tetracyclines, NSAIDs, and barbiturates. FIguRE 74-7 Fixed drug eruption. PART 2 Cardinal Manifestations and Presentation of Diseases

1	FIguRE 74-7 Fixed drug eruption. PART 2 Cardinal Manifestations and Presentation of Diseases IMMuNE CuTANEOuS REACTIONS: RARE AND SEVERE Vasculitis Cutaneous small-vessel vasculitis often presents as palpable purpuric lesions that may be generalized or limited to the lower extremities or other dependent areas (Chap. 385). Pustular lesions and hemorrhagic blisters also occur. Vasculitis may involve other organs, including the liver, kidney, brain, and joints. Drugs are implicated as a cause of 10–15% of all cases of small-vessel vasculitides. Infection, malignancy, and collagen vascular disease are responsible for the majority of non-drug-related cases.

1	Propylthiouracil induces a cutaneous vasculitis that is accompanied by leukopenia and splenomegaly. Direct immunofluorescent changes in these lesions suggest immune-complex deposition. Common drugs implicated in vasculitis include allopurinol, thiazides, sulfonamides, antimicrobials, and NSAIDs. The presence of eosinophils in the perivascular infiltrate of skin biopsy suggests a drug etiology.

1	Pustular Eruptions AGEP is a rare reaction pattern (3–5 cases per million per year) that is often associated with exposure to drugs (Fig. 74-8). Usually beginning on the face or intertriginous areas, small nonfollicular pustules overlying erythematous and edematous skin may coalesce and lead to superficial erosion. Differentiating this eruption from TEN in its initial stages may be difficult. A skin biopsy is important and shows neutrophil collections and sparse necrotic keratinocytes in the upper part of the epidermis instead of the full-thickness epidermal necrosis that characterizes TEN. Fever and leukocytosis are common, and eosinophilia occurs in one-third of cases. Acute pustular psoriasis is the principal differential diagnostic consideration. DIHS with pustular features must also be clinically considered, although the timing for the onset of DIHS is distinct (much later onset). AGEP often begins within a few days of initiating drug treatment, most notably antibiotics, but may

1	be clinically considered, although the timing for the onset of DIHS is distinct (much later onset). AGEP often begins within a few days of initiating drug treatment, most notably antibiotics, but may occur as late as 7–14 days after initiation of treatment. A broad range of drug classes (anticonvulsants, mercury, radiocontrast dye) and infections (viral, Mycoplasma) are also associated with AGEP. Patch testing with the responsible drug results in a localized pustular eruption.

1	FIguRE 74-8 Acute generalized exanthematous pustulosis.

1	Drug-induced Hypersensitivity Syndrome Drug-induced hypersensitivity syndrome (DIHS) is a multiorgan drug reaction previously known as DRESS (drug reaction with eosinophilia and systemic symptoms); since eosinophilia is not always present, the term DIHS is now preferred. Allopurinol is the most common cause. Although less frequently prescribed, abacavir has been reported to cause DIHS with an incidence as high as 4–8%. It presents as a widespread erythematous eruption that may become purpuric, pustular, or lichenoid and is accompanied by many of the following features: fever, facial edema, lymphadenopathy, leukocytosis (often with atypical lymphocytes and eosinophilia), hepatitis, myositis (including myocarditis), and sometimes nephritis (with proteinuria) or pneumonitis. Distinct patterns of timing of onset and organ involvement may exist; e.g., allopurinol classically induces DIHS with renal involvement. Cardiac and lung involvement is more common with minocycline; gastrointestinal

1	of timing of onset and organ involvement may exist; e.g., allopurinol classically induces DIHS with renal involvement. Cardiac and lung involvement is more common with minocycline; gastrointestinal involvement is almost exclusively seen with abacavir, and some medications typically lack eosinophilia (abacavir, dapsone, lamotrigine). The cutaneous reaction usually begins 2–8 weeks after the drug is started and lasts longer than mild eruptions after drug cessation. Signs and symptoms may persist for several weeks, especially those associated with hepatitis. The eruption recurs with rechallenge, and cross-reactions among aromatic anticonvulsants, including phenytoin, carbamazepine, and barbiturates, are frequent. Other drugs causing this syndrome include sulfonamides and other antibiotics. Hypersensitivity to reactive drug metabolites, hydroxylamine for sulfamethoxazole, and arene oxide for aromatic anticonvulsants may be involved in the pathogenesis of DIHS. Reactivation of herpes

1	Hypersensitivity to reactive drug metabolites, hydroxylamine for sulfamethoxazole, and arene oxide for aromatic anticonvulsants may be involved in the pathogenesis of DIHS. Reactivation of herpes viruses, especially herpesvirus 6 and Epstein-Barr virus (EBV), has been frequently reported in this syndrome, although the causal role of viral infection has been debated. Recent research suggests that inciting drugs may reactivate quiescent herpes viruses, resulting in expansion of viral-specific CD8+ T lymphocytes with subsequent end-organ damage. Viral reactivation may be associated with a worse clinical prognosis. Mortality rates as high as 10% have been reported; mortality is highest in association with hepatitis. Systemic glucocorticoids (prednisone, 1–2 mg/kg daily) should be started with slow taper over 8–12 weeks. A steroid-sparing agent, such as mycophenolate mofetil, may be indicated in cases of rapid recurrence upon steroid taper. In all cases, rapid withdrawal of the suspected

1	slow taper over 8–12 weeks. A steroid-sparing agent, such as mycophenolate mofetil, may be indicated in cases of rapid recurrence upon steroid taper. In all cases, rapid withdrawal of the suspected drug is required. Given the severe long-term complications of myocarditis, patients should undergo cardiac evaluation if heart involvement is suspected by hypotension or arrhythmia. Patients should be closely monitored for resolution of organ dysfunction and for development of late-onset autoimmune thyroiditis (up to 6 months).

1	Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis SJS and TEN are characterized by blisters and mucosal/epidermal detachment resulting from full-thickness epidermal necrosis in the absence of substantial dermal inflammation (Fig. 74-9). The term Stevens-Johnson syndrome describes cases with blisters developing on target lesions, dusky or purpuric macules in which mucosal involvement is significant, and total body surface area blistering and eventual detachment in <10% of cases. The term Stevens-Johnson syndrome/toxic epidermal necrolysis overlap is used to describe cases with 10–30% detachment, and TEN is used to describe cases with >30% detachment.

1	Other blistering eruptions with mucositis associated with infections may be confused with SJS/TEN. Erythema multiforme (EM) associated with herpes simplex virus is characterized by mucosal involvement and target lesions often more acrally distributed and with limited skin detachment. Mycoplasma infection in children causes a clinically distinct presentation with prominent mucositis and limited blistering lesions; some believe that this clinical entity is the syndrome originally described by Stevens and Johnson. FIguRE 74-9 Toxic epidermal necrolysis. (Photo credit: Lindy Peta Fox, MD, and Jubin Ryu, MD, PhD.)

1	Patients with SJS, SJS/TEN, or TEN initially present with acute onset of painful skin lesions, fever >39°C (102.2°F), sore throat, and conjunctivitis resulting from mucosal lesions. Intestinal and pulmonary involvement is associated with a poor prognosis, as are a greater extent of epidermal detachment and older age. About 10% and 30% of SJSand TEN-affected persons die from their disease, respectively. Drugs that most commonly cause SJS or TEN are sulfonamides, nevirapine (1 in 1000 risk of SJS or TEN), allopurinol, lamotrigine, aromatic anticonvulsants, and NSAIDs, specifically oxicam. Frozen-section skin biopsy may aid in rapid diagnosis. At this time, SJS and TEN have no proven effective treatment. The best results come from early diagnosis, immediate discontinuation of any suspected drug, supportive therapy, and paying close attention to ocular complications and infection. Systemic glucocorticoid therapy (prednisone 1–2 mg/kg) may be useful early in the evolution of the disease,

1	drug, supportive therapy, and paying close attention to ocular complications and infection. Systemic glucocorticoid therapy (prednisone 1–2 mg/kg) may be useful early in the evolution of the disease, but long-term systemic glucocorticoid use has been associated with higher mortality. Cyclosporine may be a possible therapy for SJS/TEN. After initial enthusiasm for the use of intravenous immunoglobulin (IVIG) in the treatment of SJS/TEN, some recent data questions whether IVIG benefits these patients. Randomized studies to more definitively assess the potential benefit of systemic glucocorticoids and IVIG are lacking and difficult to perform but are necessary.

1	Overlap Hypersensitivity Syndromes An important emerging concept in the clinical approach to severe drug eruptions is the presence of overlap syndromes, most notably DIHS with TEN-like features, DIHS with pustular eruption (AGEP-like), and AGEP with TEN-like features. In several case series of AGEP, 50% of cases had TEN-like or DRESS-like features, and 20% of cases had mucosal involvement resembling SJS/ TEN. In one study, up to 20% of all severe drug eruptions had overlap features, suggesting that AGEP, DIHS, and SJS/TEN represent a clinical spectrum with common pathophysiologic mechanisms. Designation of a single diagnosis based on cutaneous and extracutaneous involvement may not always be possible in cases of hypersensitivity. There are four main questions to answer regarding an eruption: 1. Is it a drug reaction? 2. Is it a severe eruption or the onset of a form that may become severe? 3. Which drug(s) is (are) suspected, and which drug(s) should be withdrawn? 4.

1	Is it a drug reaction? 2. Is it a severe eruption or the onset of a form that may become severe? 3. Which drug(s) is (are) suspected, and which drug(s) should be withdrawn? 4. What is recommended for future use of drugs? Generalized erythema Facial edema Skin pain Palpable purpura Target lesions Skin necrosis Blisters or epidermal detachment Positive Nikolsky's sign Mucous membrane erosions Urticaria Swelling of tongue High fever (temperature >40°C [>104°F]) Enlarged lymph nodes Arthralgias or arthritis Shortness of breath, wheezing, hypotension Lymphocytosis with atypical lymphocytes Source: Adapted from JC Roujeau, RS Stern: N Engl J Med 331:1272, 1994.

1	Lymphocytosis with atypical lymphocytes Source: Adapted from JC Roujeau, RS Stern: N Engl J Med 331:1272, 1994. Rapid recognition of adverse drug reactions that may become serious or life threatening is paramount. Table 74-2 lists clinical and laboratory features that, if present, suggest that the reaction may be serious. Table 74-3 provides key features of the most serious adverse cutaneous reactions. Intensity of symptoms and rapid progression of signs should raise the suspicion of a severe eruption. Any doubt should lead to prompt consultation with a dermatologist and/or referral of the patient to a specialized center.

1	The probability of drug etiology varies with the pattern of the reaction. Only fixed drug eruptions are always drug-induced. Morbilliform eruptions are usually viral in children and drug-induced in adults. Among severe reactions, drugs account for 10–20% of anaphylaxis and vasculitis and between 70–90% of AGEP, DIHS, SJS, or TEN. Skin biopsy helps in characterizing the reaction but does not indicate drug causality. Blood counts and liver and renal function tests are important for evaluating organ involvement. The association of mild elevation of liver enzymes and high eosinophil count is frequent but not specific for a drug reaction. Blood tests that could identify an alternative cause, antihistone antibody tests (to rule out drug-induced lupus), and serology or polymerase chain reaction for infections may be of great importance to determine a cause.

1	Most cases of drug eruptions occur during the first course of treatment with a new medication. A notable exception is IgE-mediated urticaria and anaphylaxis that need presensitization and develop a few minutes to a few hours after rechallenge. Characteristic times of onset to drug reaction are as follows: 4–14 days for morbilliform eruptions, 2–4 days for AGEP, 5–28 days for SJS/TEN, and 14–48 days for DIHS. A drug chart, compiling information of all current and past medications/ supplements and the timing of administration relative to the rash, is a key diagnostic tool to identifying the inciting drug. Medications introduced for the first time in the relevant time frame are prime suspects. Two other important elements to suspect causality at this stage are (1) previous experience with the drug in the population and (2) alternative etiologic candidates.

1	PART 2 Cardinal Manifestations and Presentation of Diseases aOverlap of Stevens-Johnson syndrome and toxic epidermal necrolysis have features of both and attachment of 10–30% of body surface area may occur. Source: Adapted from JC Roujeau, RS Stern: N Engl J Med 331:1272, 1994.

1	The decision to continue or discontinue any medication will depend The usefulness of laboratory tests to determine causality is still on the severity of the reaction, the severity of the primary disease, the debated. Many in vitro immunologic assays have been developed, but degree of suspicion of causality, and the feasibility of an alternative the predictive value of these tests has not been validated in any large safer treatment. In any potentially fatal drug reaction, elimination series of affected patients; these tests exist primarily for research and of all possible suspect drugs or unnecessary medications should be not clinical purposes. attempted. Some rashes may resolve when “treating through” a benign In some cases, diagnostic rechallenge may be appropriate, even for drug-related eruption. The decision to treat through an eruption drugs with high rates of adverse reactions. Desensitization is often should, however, remain the exception and withdrawal of every sus-successful

1	eruption. The decision to treat through an eruption drugs with high rates of adverse reactions. Desensitization is often should, however, remain the exception and withdrawal of every sus-successful in HIV-infected patients with morbilliform eruptions to pect drug the general rule. On the other hand, drugs that are not sus-sulfonamides but is not recommended in HIV-infected patients who pected and are important for the patient (e.g., antihypertensive agents) manifested erythroderma or a bullous reaction in response to their generally should not be quickly withdrawn. This approach prevents earlier sulfonamide exposure. reluctance to future use of these agents. In patients with history suggesting immediate IgE-mediated reac tions to penicillin, skin-prick testing with penicillins or cephalosporins has proved useful for identifying patients at risk of anaphylactic reac-The aims are (1) to prevent the recurrence of the drug eruption and (2) tions to these agents. However, skin tests

1	has proved useful for identifying patients at risk of anaphylactic reac-The aims are (1) to prevent the recurrence of the drug eruption and (2) tions to these agents. However, skin tests themselves carry a small risknot to compromise future treatments by contraindicating otherwise of anaphylaxis. Negative skin tests do not totally rule out IgE-mediateduseful medications.

1	reactivity, but the risk of anaphylaxis in response to penicillin admin-Begin with thorough assessment of drug causality. Drug causality istration in patients with negative skin tests is about 1%. In contrast, is evaluated based on timing of the reaction, evaluation of other postwo-thirds of patients with a positive skin test experience an allergicsible causes, effect of drug withdrawal or continuation, and knowledge response upon rechallenge. of medications that have been associated with the observed reaction.

1	of medications that have been associated with the observed reaction. For patients with delayed-type hypersensitivity, the clinical utilityCombination of these criteria leads to considering the causality as of skin tests is more questionable. At least one of a combination ofdefinite, probable, possible, or unlikely. The RegiSCAR group has several tests (prick, patch, and intradermal) is positive in 50–70% ofproposed a useful algorithm called Algorithm of Drug Causality for patients with a reaction “definitely” attributed to a single medication. Epidermal Necrolysis (ALDEN) to determine drug causality in SJS/ This low sensitivity corresponds to the observation that readministra-TEN. A drug with a “definite” or “probable” causality should be tion of drugs with negative skin testing resulted in eruptions in 17%contraindicated, a warning card or medical alert tag (e.g., wristband) of cases.

1	should be given to the patient, and the drugs should be listed in the patient’s medical chart as an allergy. A drug with a “possible” causality may be submitted to further CROSS-SENSITIVITY investigations depending on the expected need for future treatment. Because of the possibility of cross-sensitivity among chemically related A drug with “unlikely” causality or that has been continued when drugs, many physicians recommend avoidance of not only the medicathe reaction improved or was reintroduced without a reaction can be tion that induced the reaction but also all drugs of the same pharmaadministered safely. cologic class.

1	There are two types of cross-sensitivity. Reactions that depend on a pharmacologic interaction may occur with all drugs that target the same pathway, whether they are structurally similar or not. This is the case with angioedema caused by NSAIDs and ACE inhibitors. In this situation, the risk of recurrence varies from drug to drug in a particular class; however, avoidance of all drugs in the class is usually recommended. Immune recognition of structurally related drugs is the second mechanism by which cross-sensitivity occurs. A classic example is hypersensitivity to aromatic antiepileptics (barbiturates, phenytoin, carbamazepine) with up to 50% reaction to a second drug in patients who reacted to one. For other drugs, in vitro as well as in vivo data have suggested that cross-reactivity existed only between compounds with very similar chemical structures. Sulfamethoxazolespecific lymphocytes may be activated by other antibacterial sulfonamides but not diuretics, antidiabetic drugs,

1	existed only between compounds with very similar chemical structures. Sulfamethoxazolespecific lymphocytes may be activated by other antibacterial sulfonamides but not diuretics, antidiabetic drugs, or anti-COX2 NSAIDs with a sulfonamide group. Approximately 10% of patients with penicillin allergies will also develop allergic reactions to cephalosporin class antibiotics.

1	Recent data suggest that although the risk of a drug eruption to another drug was increased in persons with a prior reaction, “crosssensitivity” was probably not the explanation. As an example, persons with a history of an allergic-like reaction to penicillin were at higher risk to develop a reaction to antibacterial sulfonamides than to cephalosporins. These data suggest that the list of drugs to avoid after a drug reaction should be limited to the causative one(s) and to a few very similar medications. Because of growing evidence that some severe cutaneous reactions to drugs are associated with HLA genes, it is recommended that first-degree family members of patients with severe cutaneous reactions also should avoid these causative medications. This may be most relevant to sulfonamides and antiseizure medications.

1	Desensitization can be considered in those with a history of reaction to a medication that must be used again. Efficacy of such procedures has been demonstrated in cases of immediate reaction to penicillin and positive skin tests, anaphylactic reactions to platinum chemotherapy, and delayed reactions to sulfonamides in patients with AIDS. Various protocols are available, including oral and parenteral approaches. Oral desensitization appears to have a lower risk of serious anaphylactic reactions. However, desensitization carries the risk of anaphylaxis regardless of how it is performed and should be performed in monitored clinical settings such as an intensive care unit. After desensitization, many patients experience non-life-threatening reactions during therapy with the culprit drug.

1	Any severe reaction to drugs should be reported to a regulatory agency or to pharmaceutical companies (e.g., MedWatch, http://www.fda.gov/ Safety/MedWatch/default.htm). Because severe reactions are too rare to be detected in premarketing clinical trials, spontaneous reports are of critical importance for early detection of unexpected life-threatening events. To be useful, the report should contain enough details to permit ascertainment of severity and drug causality. This enables recognition of similar cases that may be reported from several different sources. We acknowledge the contribution of Dr. Jean-Claude Roujeau to this chapter in the 17th edition. Photosensitivity and other 75 Reactions to Light Alexander G. Marneros, David R. Bickers

1	We acknowledge the contribution of Dr. Jean-Claude Roujeau to this chapter in the 17th edition. Photosensitivity and other 75 Reactions to Light Alexander G. Marneros, David R. Bickers Sunlight is the most visible and obvious source of comfort in the environment. The sun provides the beneficial effects of warmth and vitamin D synthesis. However, acute and chronic sun exposure also has pathologic consequences. Few effects of sun exposure beyond those affecting the skin have been identified, but cutaneous exposure to sunlight is the major cause of human skin cancer and can have immunosuppressive effects as well.

1	The sun’s energy reaching the earth’s surface is limited to components of the ultraviolet (UV) spectrum, the visible spectrum, and portions of the infrared spectrum. The cutoff at the short end of the UV spectrum at ~290 nm is due primarily to stratospheric ozone—formed by highly energetic ionizing radiation—that prevents penetration to the earth’s surface of the shorter, more energetic, potentially more harmful wavelengths of solar radiation. Indeed, concern about destruction of the ozone layer by chlorofluorocarbons released into the atmosphere has led to international agreements to reduce production of those chemicals.

1	Measurements of solar flux showed a twentyfold regional variation in the amount of energy at 300 nm that reaches the earth’s surface. This variability relates to seasonal effects, the path that sunlight traverses through ozone and air, the altitude (a 4% increase for each 300 m of elevation), the latitude (increasing intensity with decreasing latitude), and the amount of cloud cover, fog, and pollution. The major components of the photobiologic action spectrum that are capable of affecting human skin include the UV and visible wavelengths between 290 and 700 nm. In addition, the wavelengths beyond 700 nm in the infrared spectrum primarily emit heat and in certain circumstances may exacerbate the pathologic effects of energy in the UV and visible spectra.

1	The UV spectrum reaching the earth represents <10% of total incident solar energy and is arbitrarily divided into two major segments, UV-B and UV-A, which constitute the wavelengths from 290 to 400 nm. UV-B consists of wavelengths between 290 and 320 nm. This portion of the photobiologic action spectrum is the most efficient in producing redness or erythema in human skin and thus is sometimes known as the “sunburn spectrum.” UV-A includes wavelengths between 320 and 400 nm and is ~1000-fold less efficient in producing skin redness than is UV-B.

1	The wavelengths between 400 and 700 nm are visible to the human eye. The photon energy in the visible spectrum is not capable of damaging human skin in the absence of a photosensitizing chemical. Without the absorption of energy by a molecule, there can be no photosensitivity. Thus, the absorption spectrum of a molecule is defined as the range of wavelengths it absorbs, whereas the action spectrum for an effect of incident radiation is defined as the range of wavelengths that evoke the response. Photosensitivity occurs when a photon-absorbing chemical (chromophore) present in the skin absorbs incident energy, becomes excited, and transfers the absorbed energy to various structures or to molecular oxygen.

1	Human skin consists of two major compartments: the outer epidermis, which is a stratified squamous epithelium, and the underlying dermis, which is rich in matrix proteins such as collagens and elastin. Both compartments are susceptible to damage from sun exposure. The epidermis and the dermis contain several chromophores capable of absorbing incident solar energy, including nucleic acids, proteins, and lipids. The outermost epidermal layer, the stratum corneum, is a major absorber of UV-B, and <10% of incident UV-B wavelengths penetrate through the epidermis to the dermis. Approximately 3% of radiation

1	CHAPTER 75 Photosensitivity and Other Reactions to Light 386 below 300 nm, 20% of radiation below 360 nm, and 33% of short visible radiation reach the basal cell layer in untanned human skin. In contrast, UV-A readily penetrates to the dermis and is capable of altering structural and matrix proteins that contribute to photoaging of chronically sun-exposed skin, particularly in individuals of light complexion. Thus, longer wavelengths can penetrate more deeply into the skin.

1	Molecular Targets for uVR-Induced Skin Effects Epidermal DNA— predominantly in keratinocytes and in Langerhans cells, which are dendritic antigen-presenting cells—absorbs UV-B and undergoes structural changes between adjacent pyrimidine bases (thymine or cytosine), including the formation of cyclobutane dimers and 6,4-photoproducts. These structural changes are potentially mutagenic and are found in most basal cell and squamous cell carcinomas (BCCs and SCCs, respectively). They can be repaired by cellular mechanisms that result in their recognition and excision and the restoration of normal base sequences. The efficient repair of these structural aberrations is crucial, since individuals with defective DNA repair are at high risk for the development of cutaneous cancer. For example, patients with xeroderma pigmentosum, an autosomal recessive disorder, have a variably deficient repair of UV-induced photoproducts. The skin of these patients often shows the dry, leathery appearance of

1	with xeroderma pigmentosum, an autosomal recessive disorder, have a variably deficient repair of UV-induced photoproducts. The skin of these patients often shows the dry, leathery appearance of prematurely photoaged skin, and these patients have an increased frequency of skin cancer already in the first two decades of life. Studies in transgenic mice have verified the importance of functional genes that regulate these repair pathways in preventing the development of UV-induced skin cancer. DNA damage in Langerhans cells may also contribute to the known immunosuppressive effects of UV-B (see “Photoimmunology,” below).

1	In addition to DNA, molecular oxygen is a target for incident solar UVR, leading to the generation of reactive oxygen species (ROS). These ROS can damage skin components, such as epidermal lipids— either free lipids in the stratum corneum or cell membrane lipids. UVR also can target proteins, leading to increased cross-linking and degradation of matrix proteins in the dermis and accumulation of abnormal dermal elastin leading to photoaging changes known as solar elastosis.

1	Cutaneous Optics and Chromophores Chromophores are endogenous or exogenous chemical components that can absorb physical energy. Endogenous chromophores are of two types: (1) normal components of skin, including nucleic acids, proteins, lipids, and 7-dehydrocholesterol (the precursor of vitamin D); and (2) components that are synthesized elsewhere in the body and that circulate in the bloodstream and diffuse into the skin, such as porphyrins. Normally, only trace amounts of porphyrins are present in the skin, but, in selected diseases known as the porphyrias (Chap. 430), porphyrins are released into the circulation in increased amounts from the bone marrow and the liver and are transported to the skin, where they absorb incident energy both in the Soret band (around 400 nm; short visible) and, to a lesser extent, in the red portion of the visible spectrum (580–660 nm). This energy absorption results in the generation of ROS that can mediate structural damage to the skin, manifested as

1	and, to a lesser extent, in the red portion of the visible spectrum (580–660 nm). This energy absorption results in the generation of ROS that can mediate structural damage to the skin, manifested as erythema, edema, urticaria, or blister formation. It is of interest that photoexcited porphyrins are currently used in the treatment of nonmelanoma skin cancers and their precursor lesions, actinic keratoses. Known as photodynamic therapy, this modality generates ROS in the skin, leading to cell death. Topical photosensitizers used in photodynamic therapy are the porphyrin precursors 5-aminolevulinic acid and methyl aminolevulinate, which are converted to porphyrins in the skin. It is believed that photodynamic therapy targets tumor cells for destruction more selectively than it targets adjacent nonneoplastic cells. The efficacy of such therapy requires appropriate timing of the application of methyl aminolevulinate or 5-aminolevulinic acid to the affected skin followed by exposure to

1	nonneoplastic cells. The efficacy of such therapy requires appropriate timing of the application of methyl aminolevulinate or 5-aminolevulinic acid to the affected skin followed by exposure to artificial sources of visible light. High-intensity blue light has been used successfully for the treatment of thin actinic keratoses. Red light has a longer wavelength, penetrates more deeply into the skin, and is more beneficial in the treatment of superficial BCCs.

1	Acute Effects of Sun Exposure The acute effects of skin exposure to sunlight include sunburn and vitamin D synthesis. PART 2 Cardinal Manifestations and Presentation of Diseases

1	SUNBURN This painful skin condition is an acute inflammatory response of the skin, predominantly to UV-B. Generally, an individual’s ability to tolerate sunlight is inversely proportional to that individual’s degree of melanin pigmentation. Melanin, a complex polymer of tyrosine derivatives, is synthesized in specialized epidermal dendritic cells known as melanocytes and is packaged into melanosomes that are transferred via dendritic processes into keratinocytes, thereby providing photoprotection and simultaneously darkening the skin. Sun-induced melanogenesis is a consequence of increased tyrosinase activity in melanocytes. Central to the suntan response is the melanocortin-1 receptor (MC1R), and mutations in this gene contribute to the wide variation in human skin and hair color; individuals with red hair and fair skin typically have low MC1R activity. Genetic studies have revealed additional genes that influence skin color variation in humans, such as the gene for tyrosinase (TYR)

1	with red hair and fair skin typically have low MC1R activity. Genetic studies have revealed additional genes that influence skin color variation in humans, such as the gene for tyrosinase (TYR) and the genes APBA2[OCA2], SLC45A2, and SLC24A5. The human MC1R gene encodes a G protein–coupled receptor that binds α-melanocyte-stimulating hormone, which is secreted in the skin mainly by keratinocytes in response to UVR. The UV-induced expression of this hormone is controlled by the tumor suppressor p53, and absence of functional p53 attenuates the tanning response. Activation of the melanocortin receptor leads to increased intracellular cyclic adenosine 5′-monophosphate (cAMP) and protein kinase A activation, resulting in an increased transcription of the microphthalmia-associated transcription factor (MITF), which stimulates melanogenesis. Since the precursor of α-melanocytestimulating hormone, proopiomelanocortin, is also the precursor of β-endorphins, UVR may result in not only

1	factor (MITF), which stimulates melanogenesis. Since the precursor of α-melanocytestimulating hormone, proopiomelanocortin, is also the precursor of β-endorphins, UVR may result in not only increased pigmentation but also in increased β-endorphin production, an effect that has been hypothesized to promote sun-seeking behaviors.

1	The Fitzpatrick classification of human skin phototypes is based on the efficiency of the epidermal-melanin unit, which usually can be ascertained by asking an individual two questions: (1) Do you burn after sun exposure? (2) Do you tan after sun exposure? The answers to these questions permit division of the population into six skin types, varying from type I (always burn, never tan) to type VI (never burn, always tan) (Table 75-1).

1	Sunburn erythema is due to vasodilation of dermal blood vessels. There is a lag time (usually 4–12 h) between skin exposure to sunlight and the development of visible redness. The action spectrum for sunburn erythema includes UV-B and UV-A, although UV-B is much more efficient than UV-A in evoking the response. However, UV-A may contribute to sunburn erythema at midday, when much more UV-A than UV-B is present in the solar spectrum. The erythema that accompanies the inflammatory response induced by UVR results from the orchestrated release of cytokines along with growth factors and the generation of ROS. Furthermore, UV-induced activation of nuclear factor κB–dependent gene transcription can augment release of several pro-inflammatory cytokines and vasoactive mediators. These cytokines and mediators accumulate locally in sunburned skin, providing chemotactic factors that attract neutrophils, macrophages, and T lymphocytes, which promote the inflammatory response. UVR also stimulates

1	mediators accumulate locally in sunburned skin, providing chemotactic factors that attract neutrophils, macrophages, and T lymphocytes, which promote the inflammatory response. UVR also stimulates infiltration of inflammatory cells through induced expression of adhesion molecules such as E-selectin and intercellular adhesion molecule 1 on endothelial cells and keratinocytes. UVR also has been shown to activate phospholipase A2, resulting in increases in eicosanoids such as prostaglandin E2, which is known to be a potent inducer of sunburn erythema. The role of eicosanoids in this reaction has been verified by studies showing that nonsteroidal anti-inflammatory drugs (NSAIDs) can reduce it.

1	Epidermal changes in sunburn include the induction of “sunburn cells,” which are keratinocytes undergoing p53-dependent apoptosis as a defense, with elimination of cells that harbor UV-B-induced structural DNA damage.

1	VITAMIN d SYNTHESIS ANd PHOTOCHEMISTRY Cutaneous exposure to UV-B causes photolysis of epidermal 7-dehydrocholesterol, converting it to pre–vitamin D3, which then undergoes temperature-dependent isomerization to form the stable hormone vitamin D3. This compound diffuses to the dermal vasculature and circulates to the liver and kidney, where it is converted to the dihydroxylated functional hormone 1,25-dihydroxyvitamin D3. Vitamin D metabolites from the circulation and those produced in the skin itself can augment epidermal differentiation signaling and inhibit keratinocyte proliferation. These effects are exploited therapeutically in psoriasis with the topical application of synthetic vitamin D analogues. In addition, vitamin D is increasingly thought to have beneficial effects in several other inflammatory conditions, and some evidence suggests that—besides its classic physiologic effects on calcium metabolism and bone homeostasis—it is associated with a reduced risk of various

1	other inflammatory conditions, and some evidence suggests that—besides its classic physiologic effects on calcium metabolism and bone homeostasis—it is associated with a reduced risk of various internal malignancies. There is controversy regarding the risk-to-benefit ratio of sun exposure in vitamin D homeostasis. At present, it is important to emphasize that no clear-cut evidence suggests that the use of sunscreens substantially diminishes vitamin D levels. Since aging also substantially decreases the ability of human skin to photocatalytically produce vitamin D3, the widespread use of sunscreens that filter out UV-B has led to concerns that the elderly might be unduly susceptible to vitamin D deficiency. However, the amount of sunlight needed to produce sufficient vitamin D is small and does not justify the risks of skin cancer and other types of photodamage linked to increased sun exposure or tanning behavior. Nutritional supplementation of vitamin D is a preferable strategy for

1	does not justify the risks of skin cancer and other types of photodamage linked to increased sun exposure or tanning behavior. Nutritional supplementation of vitamin D is a preferable strategy for patients with vitamin D deficiency.

1	Chronic Effects of Sun Exposure: Nonmalignant The clinical features of photoaging (dermatoheliosis) consist of wrinkling, blotchiness, and telangiectasia as well as a roughened, irregular, “weather-beaten” leathery appearance.

1	UVR is important in the pathogenesis of photoaging in human skin, and ROS are likely involved. The dermis and its connective tissue matrix are major targets for sun-associated chronic damage that manifests as solar elastosis, a massive increase in thickened irregular masses of abnormal-appearing elastic fibers. Collagen fibers are also abnormally clumped in the deeper dermis of sun-damaged skin. The chromophore(s), the action spectra, and the specific biochemical events orchestrating these changes are only partially understood, although more deeply penetrating UV-A seems to be primarily involved. Chronologically aged sun-protected skin and photoaged skin share important molecular features, including connective tissue damage and elevated levels of matrix metalloproteinases (MMPs). MMPs are enzymes involved in the degradation of the extracellular matrix. UV-A induces expression of some MMPs, including MMP-1 and MMP-3, leading to increased collagen breakdown. In addition, UV-A reduces

1	enzymes involved in the degradation of the extracellular matrix. UV-A induces expression of some MMPs, including MMP-1 and MMP-3, leading to increased collagen breakdown. In addition, UV-A reduces type I procollagen mRNA expression. Thus, chronic UVR alters the structure and function of dermal collagen. On the basis of these observations, it is not surprising that high-dose UV-A phototherapy may have beneficial effects in some patients with localized fibrotic diseases of the skin, such as localized scleroderma.

1	Chronic Effects of Sun Exposure: Malignant One of the major known consequences of chronic excessive skin exposure to sunlight is non-melanoma skin cancer. The two most common types of nonmelanoma skin cancer are BCC and SCC (Chap. 105). A model for skin cancer induction involves three major steps: initiation, promotion, and progression. Exposure of human skin to sunlight results in initiation, a step by which structural (mutagenic) changes in DNA evoke an irreversible alteration in the target cell (keratinocyte) that begins the tumorigenic process. Exposure to a tumor initiator such as UV-B is believed to be a necessary but not a sufficient step in the malignant process, since initiated skin cells not exposed to tumor promoters 387 generally do not develop tumors. The second stage in tumor development is promotion, a multistep process by which chronic exposure to sunlight evokes further changes that culminate in the clonal expansion of initiated cells and cause the development, over

1	development is promotion, a multistep process by which chronic exposure to sunlight evokes further changes that culminate in the clonal expansion of initiated cells and cause the development, over many years, of premalignant growths known as actinic keratoses, a minority of which may progress to form SCCs. As a result of extensive studies, it seems clear that UV-B is a complete carcinogen, meaning that it can act as both a tumor initiator and a tumor promoter. The third and final step in the malignant process is malignant conversion of benign precursors into malignant lesions, a process thought to require additional genetic alterations.

1	On a molecular level, skin carcinogenesis results from the accumulation of gene mutations that cause inactivation of tumor suppressors, activation of oncogenes, or reactivation of cellular signaling pathways that normally are expressed only during epidermal embryologic development. Accumulation of mutations in the tumor-suppressor gene p53 secondary to UV-induced DNA damage occurs in both SCCs and BCCs and is important in promoting skin carcinogenesis. Indeed, the majority of both human and murine UV-induced skin cancers have characteristic p53 mutations (C → T and CC → TT transitions). Studies in mice have shown that sunscreens can substantially reduce the frequency of these signature mutations in p53 and inhibit the induction of tumors.

1	BCCs also harbor inactivating mutations in the tumor-suppressor gene patched, which result in activation of the sonic hedgehog signaling pathway and increased cell proliferation. Thus, these tumors can manifest mutations in tumor suppressors (p53 and patched) or oncogenes (smoothened). New evidence links alterations in the Wnt/βcatenin signaling pathway, which is known to be critical for hair follicle development, to skin cancer as well. Thus interactions between this pathway and the hedgehog signaling pathway appear to be involved in both skin carcinogenesis and embryologic development of the skin and hair follicles.

1	Clonal analysis in mouse models of BCC revealed that tumor cells arise from long-term resident progenitor cells of the interfollicular epidermis and the upper infundibulum of the hair follicle. These BCC-initiating cells are reprogrammed to resemble embryonic hair follicle progenitors, whose tumor-initiating ability depends on activation of the Wnt/β-catenin signaling pathway. SCC initiation occurs both in the interfollicular epidermis and in the hair follicle bulge stem cell populations. In mouse models, the combination of mutant K-Ras and p53 is sufficient to induce invasive SCCs from these cell populations. The transcription factor Myc is important for stem cell maintenance in the skin, and oncogenic activation of Myc has been implicated in the development of BCCs and SCCs. Thus, nonmelanoma skin cancer involves mutations and alterations in multiple genes and pathways that occur as a result of their chronic accumulation driven by exposure to environmental factors such as UVR.

1	Epidemiologic studies have linked excessive sun exposure to an increased risk of nonmelanoma cancers and melanoma of the skin; the evidence is far more direct for nonmelanoma skin cancers (BCCs and SCCs) than for melanoma. Approximately 80% of nonmelanoma skin cancers develop on sun-exposed body areas, including the face, neck, and hands. Major risk factors include male sex, childhood sun exposures, older age, fair skin, and residence at latitudes relatively close to the equator. Individuals with darker-pigmented skin have a lower risk of skin cancer than do fair-skinned individuals. More than 2 million individuals in the United States develop nonmelanoma skin cancer annually, and the lifetime risk that a fair-skinned individual will develop such a neoplasm is estimated at ~15%. The incidence of non-melanoma skin cancer in the population is increasing at a rate of 2–3% per year. One potential explanation is the widespread use of indoor tanning. It is estimated that 30 million people

1	of non-melanoma skin cancer in the population is increasing at a rate of 2–3% per year. One potential explanation is the widespread use of indoor tanning. It is estimated that 30 million people tan indoors in the United States annually, including >2 million adolescents.

1	The relationship of sun exposure to melanoma development is less direct, but strong evidence supports an association. Clear-cut risk

1	CHAPTER 75 Photosensitivity and Other Reactions to Light 388 factors include a positive family or personal history of melanoma and multiple dysplastic nevi. Melanomas can occur during adolescence; the implication is that the latent period for tumor growth is shorter than that for nonmelanoma skin cancer. For reasons that are only partially understood, melanomas are among the most rapidly increasing human malignancies (Chap. 105). Epidemiologic studies indicate that indoor tanning is a risk factor for melanoma, which may contribute to the increasing incidence of melanoma formation. Furthermore, epidemiologic studies suggest that life in a sunny climate from birth or early childhood may increase the risk of melanoma development. In general, risk does not correlate with cumulative sun exposure but may be related to the duration and extent of exposure in childhood. However, in contrast to nonmelanoma skin cancers, melanoma frequently develops in sun-protected skin, and oncogenic mutations

1	but may be related to the duration and extent of exposure in childhood. However, in contrast to nonmelanoma skin cancers, melanoma frequently develops in sun-protected skin, and oncogenic mutations in melanoma may also not be UVR-signature mutations; these observations suggest that UVR-independent factors contribute to melanomagenesis. Low MC1R activity leads to production of the red/yellow pheomelanin pigment in individuals with red hair and fair skin, while high MC1R activity results in increased production of the black/brown eumelanin. Experiments in mice suggest that high pheomelanin content in skin (as in individuals with red hair and fair skin) leads to a UVR-independent increase in the risk of melanoma through a mechanism that involves oxidative damage. Thus, both UVR-dependent and UVR-independent factors are likely to contribute to melanoma formation.

1	Photoimmunology Exposure to solar radiation causes both local immunosuppression (through inhibition of immune responses to antigens applied at the irradiated site) and systemic immunosuppression (through inhibition of immune responses to antigens applied at remote, unirradiated sites). For example, human skin exposure to modest doses of UV-B can deplete the epidermal antigen-presenting cells known as Langerhans cells, thereby reducing the degree of allergic sensitization to application of the potent contact allergen dinitrochlorobenzene at the irradiated skin site.

1	An example of the systemic immunosuppressive effects of higher doses of UVR is the diminished immunologic response to antigens introduced either epicutaneously or intracutaneously at sites distant from the irradiated site. Various immunomodulatory factors and immune cells have been implicated in UVR-induced systemic immunosuppression, including tumor necrosis factor α, interleukin 4, interleukin 10, cis-urocanic acid, and eicosanoids. Experimental evidence suggests that prostaglandin E2 signaling through prostaglandin E receptor subtype 4 mediates UVR-induced systemic immunosuppression by elevating the number of regulatory T cells, and this effect can be inhibited with NSAIDs.

1	The major chromophores in the upper epidermis that are known to initiate UV-mediated immunosuppression include DNA, transurocanic acid, and membrane components. The action spectrum for UV-induced immunosuppression closely mimics the absorption spectrum of DNA. Pyrimidine dimers in Langerhans cells may inhibit antigen presentation. The absorption spectrum of epidermal urocanic acid closely mimics the action spectrum for UV-B-induced immunosuppression. Urocanic acid is a metabolic product of the essential amino acid histidine and accumulates in the upper epidermis through breakdown of the histidine-rich protein filaggrin due to the absence of its catabolizing enzyme in keratinocytes. Urocanic acid is synthesized as a trans-isomer, and UV-induced trans-cis isomerization of urocanic acid in the stratum corneum drives immunosuppression. Cis-urocanic acid may exert its immunosuppressive effects through a variety of mechanisms, including inhibition of antigen presentation by Langerhans

1	in the stratum corneum drives immunosuppression. Cis-urocanic acid may exert its immunosuppressive effects through a variety of mechanisms, including inhibition of antigen presentation by Langerhans cells.

1	One important consequence of chronic sun exposure and associated immunosuppression is an enhanced risk of skin cancer. In part, UV-B activates regulatory T cells that suppress antitumor immune responses via interleukin 10 expression, whereas, in the absence of high UV-B exposure, epidermal Langerhans cells present tumor-associated antigens and induce protective immunity, thereby inhibiting skin tumorigenesis. UV-induced DNA damage is a major molecular trigger of this immunosuppressive effect. PART 2 Cardinal Manifestations and Presentation of Diseases

1	Perhaps the most graphic demonstration of the role of immunosuppression in enhancing the risk of nonmelanoma skin cancer comes from studies of organ transplant recipients who require lifelong immunosuppressive/antirejection drug regimens. More than 50% of organ transplant recipients develop BCCs and SCCs, and these cancers are the most common types of malignancies arising in these patients. Rates of BCC and SCC increase with the duration and degree of immunosuppression. These patients ideally should be screened prior to organ transplantation, be monitored closely thereafter, and adhere to rigorous photoprotection measures, including the use of sunscreens and protective clothing as well as sun avoidance. Notably, immunosuppressive drugs that target the mTOR pathway, such as sirolimus and everolimus, may reduce the risk of nonmelanoma skin cancer in organ transplant recipients from that associated with the use of calcineurin inhibitors (cyclosporine and tacrolimus), which may contribute

1	may reduce the risk of nonmelanoma skin cancer in organ transplant recipients from that associated with the use of calcineurin inhibitors (cyclosporine and tacrolimus), which may contribute to nonmelanoma skin cancer formation not only through their immunosuppressive effects but also through suppression of p53-dependent cancer cell senescence pathways independent of host immunity.

1	The diagnosis of photosensitivity requires elicitation of a careful history in order to define the duration of signs and symptoms, the length of time between exposure to sunlight and the development of subjective symptoms, and visible changes in the skin. The age of onset can also be a helpful diagnostic clue; for example, the acute photosensitivity of erythropoietic protoporphyria almost always begins in childhood, whereas the chronic photosensitivity of porphyria cutanea tarda (PCT) typically begins in the fourth and fifth decades of life. A patient’s history of exposure to topical and systemic drugs and chemicals may provide important diagnostic clues. Many classes of drugs can cause photosensitivity on the basis of either phototoxicity or photoallergy. Fragrances such as musk ambrette that were previously present in numerous cosmetic products are also potent photosensitizers.

1	Examination of the skin may offer important clues. Anatomic areas that are naturally protected from direct sunlight, such as the hairy scalp, the upper eyelids, the retroauricular areas, and the infranasal and submental regions, may be spared, whereas exposed areas show characteristic features of the pathologic process. These anatomic localization patterns are often helpful, but not infallible, in making the diagnosis. For example, airborne contact sensitizers that are blown onto the skin may produce dermatitis that can be difficult to distinguish from photosensitivity despite the fact that such material may trigger skin reactivity in areas shielded from direct sunlight.

1	Many dermatologic conditions may be caused or aggravated by sunlight (Table 75-2). The role of light in evoking these responses may be dependent on genetic abnormalities ranging from well-described defects in DNA repair that occur in xeroderma pigmentosum to the inherited abnormalities in heme synthesis that characterize the porphyrias. The chromophore has been identified in certain photosensitivity diseases, but the energy-absorbing agent remains unknown in the majority.

1	Polymorphous Light Eruption A common type of photosensitivity disease is polymorphous light eruption (PMLE). Many affected individuals never seek medical attention because the condition is often transient, becoming manifest in the spring with initial sun exposure but then subsiding spontaneously with continuing exposure, a phenomenon known as “hardening.” The major manifestations of PMLE include (often intensely) pruritic erythematous papules that may coalesce into plaques in a patchy distribution on exposed areas of the trunk and forearms. The face is usually less seriously involved. Whereas the morphologic skin findings remain similar for each patient with subsequent recurrences, significant interindividual variations in skin findings are characteristic (hence the term “polymorphous”).

1	A skin biopsy and phototest procedures in which skin is exposed to multiple erythemal doses of UV-A and UV-B may aid in the diagnosis. The action spectrum for PMLE is usually within these portions of the solar spectrum. Whereas the treatment of an acute flare of PMLE may require topical or systemic glucocorticoids, approaches to preventing PMLE are External Drugs, plants, food important and include the use of high-SPF and high UVA-protection broad-spectrum sunscreens as well as the induction of “hardening” by the cautious administration of artificial UV-B (broad-band or narrow-band) and/or UV-A radiation or the use of psoralen plus UV-A (PUVA) photochemotherapy for 2–4 weeks before initial sun exposure. Such prophylactic phototherapy or photochemotherapy at the beginning of spring may prevent the occurrence of PMLE throughout the summer.

1	Phototoxicity and Photoallergy These photosensitivity disorders are related to the topical or systemic administration of drugs and other chemicals. Both reactions require the absorption of energy by a drug or chemical with consequent production of an excited-state photosensitizer that can transfer its absorbed energy to a bystander molecule or to molecular oxygen, thereby generating tissue-destructive chemical species, including ROS. Phototoxicity is a nonimmunologic reaction that can be caused by drugs and chemicals, a few of which are listed in Table 75-3. The usual clinical manifestations include erythema resembling a sunburn reaction that quickly desquamates, or “peels,” within several days. In addition, edema, vesicles, and bullae may occur.

1	Photoallergy is much less common and is distinct in that it is an immunopathologic process. The excited-state photosensitizer may create highly unstable haptenic free radicals that bind covalently to macromolecules to form a functional antigen capable of evoking a delayed-type hypersensitivity response. Some drugs and chemicals that can produce photoallergy are listed in Table 75-4. The clinical manifestations typically differ from those of phototoxicity in that an intensely pruritic eczematous dermatitis tends to predominate and evolves into lichenified, thickened, “leathery” changes in sun-exposed areas. A small subset (perhaps 5–10%) of patients with photoallergy may develop a persistent exquisite hypersensitivity to light even when the offending drug or chemical is identified and eliminated, a condition known as persistent light reaction.

1	A very uncommon type of persistent photosensitivity is known as chronic actinic dermatitis. The affected patients are typically elderly men with a long history of preexisting allergic contact dermatitis or photosensitivity. These individuals are usually exquisitely sensitive to UV-B, UV-A, and visible wavelengths.

1	Phototoxicity and photoallergy often can be diagnostically confirmed by phototest procedures. In patients with suspected photo-toxicity, determining the minimal erythemal dose (MED) while the patient is exposed to a suspected agent and then repeating the MED after discontinuation of the agent may provide a clue to the causative drug or chemical. Photopatch testing can be performed to confirm the diagnosis of photoallergy. In this simple variant of ordinary patch testing, a series of known photoallergens is applied to the skin in duplicate, and one set is irradiated with a suberythemal dose of UV-A. The development of eczematous changes at sites exposed to sensitizer and light is a positive result. The characteristic abnormality in patients with persistent light reaction is a diminished threshold to erythema evoked + Halogenated salicylanilides + Hypericin (St. John’s wort) + + Musk ambrette + Piroxicam

1	CHAPTER 75 Photosensitivity and Other Reactions to Light 390 by UV-B. Patients with chronic actinic dermatitis usually manifest a broad spectrum of UV hyperresponsiveness and require meticulous photoprotection, including avoidance of sun exposure, use of high-SPF (>30) sunscreens, and, in severe cases, systemic immunosuppression, such as with azathioprine. The management of drug photosensitivity involves first and foremost the elimination of exposure to the chemical agents responsible for the reaction and the minimization of sun exposure. The acute symptoms of phototoxicity may be ameliorated by cool moist compresses, topical glucocorticoids, and systemically administered NSAIDs. In severely affected individuals, a rapidly tapered course of systemic glucocorticoids may be useful. Judicious use of analgesics may be necessary. Photoallergic reactions require a similar management approach. Furthermore, patients with persistent light reaction and chronic actinic dermatitis must be

1	use of analgesics may be necessary. Photoallergic reactions require a similar management approach. Furthermore, patients with persistent light reaction and chronic actinic dermatitis must be meticulously protected against light exposure. In selected patients to whom chronic systemic high-dose glucocorticoids pose unacceptable risks, it may be necessary to employ an immunosuppressive drug such as azathioprine, cyclophosphamide, cyclosporine, or mycophenolate mofetil.

1	Porphyria The porphyrias (Chap. 430) are a group of diseases that have in common inherited or acquired derangements in the synthesis of heme. Heme is an iron-chelated tetrapyrrole or porphyrin, and the nonmetal chelated porphyrins are potent photosensitizers that absorb light intensely in both the short (400–410 nm) and the long (580–650 nm) portions of the visible spectrum.

1	Heme cannot be reutilized and must be synthesized continuously. The two body compartments with the largest capacity for its production are the bone marrow and the liver. Accordingly, the porphyrias originate in one or the other of these organs, with an end result of excessive endogenous production of potent photosensitizing porphyrins. The porphyrins circulate in the bloodstream and diffuse into the skin, where they absorb solar energy, become photoexcited, generate ROS, and evoke cutaneous photosensitivity. The mechanism of porphyrin photosensitization is known to be photodynamic, or oxygen-dependent, and is mediated by ROS such as singlet oxygen and superoxide anions.

1	Porphyria cutanea tarda is the most common type of porphyria and is associated with decreased activity of the enzyme uroporphyrinogen decarboxylase. There are two basic types of PCT: (1) the sporadic or acquired type, generally seen in individuals ingesting ethanol or receiving estrogens; and (2) the inherited type, in which there is autosomal dominant transmission of deficient enzyme activity. Both forms are associated with increased hepatic iron stores.

1	In both types of PCT, the predominant feature is chronic photo-sensitivity characterized by increased fragility of sun-exposed skin, particularly areas subject to repeated trauma such as the dorsa of the hands, the forearms, the face, and the ears. The predominant skin lesions are vesicles and bullae that rupture, producing moist erosions (often with a hemorrhagic base) that heal slowly, with crusting and purplish discoloration of the affected skin. Hypertrichosis, mottled pigmentary change, and scleroderma-like induration are associated features. The diagnosis can be confirmed biochemically by measurement of urinary porphyrin excretion, plasma porphyrin assay, and assay of erythrocyte and/or hepatic uroporphyrinogen decarboxylase. Multiple mutations of the uroporphyrinogen decarboxylase gene have been identified in human populations. Some patients with PCT have associated mutations in the HFE gene, which is linked to hemochromatosis; these mutations could contribute to the iron

1	gene have been identified in human populations. Some patients with PCT have associated mutations in the HFE gene, which is linked to hemochromatosis; these mutations could contribute to the iron overload seen in PCT, although iron status as measured by serum ferritin, iron levels, and transferrin saturation is no different from that in PCT patients without HFE mutations. Prior hepatitis C virus infection appears to be an independent risk factor for PCT.

1	Treatment of PCT consists of repeated phlebotomies to diminish the excessive hepatic iron stores and/or intermittent low doses of chloroquine and hydroxychloroquine. Long-term remission of the disease can be achieved if the patient eliminates exposure to porphyrinogenic agents and prolonged exposure to sunlight. PART 2 Cardinal Manifestations and Presentation of Diseases Erythropoietic protoporphyria originates in the bone marrow and is due to a decrease in the mitochondrial enzyme ferrochelatase secondary to numerous gene mutations. The major clinical features include acute photosensitivity characterized by subjective burning and stinging of exposed skin that often develops during or just after sun exposure. There may be associated skin swelling and, after repeated episodes, a waxlike scarring.

1	The diagnosis is confirmed by demonstration of elevated levels of free erythrocyte protoporphyrin. Detection of increased plasma protoporphyrin helps distinguish erythropoietic protoporphyria from lead poisoning and iron-deficiency anemia, in both of which erythrocyte protoporphyrin levels are elevated in the absence of cutaneous photo-sensitivity and elevated plasma protoporphyrin levels. Treatment includes reduction of sun exposure and oral administration of the carotenoid β-carotene, which is an effective scavenger of free radicals. This drug increases tolerance to sun exposure in some affected individuals, although it has no effect on deficient ferrochelatase. An algorithm for managing patients with photosensitivity is presented in Fig. 75-1.

1	An algorithm for managing patients with photosensitivity is presented in Fig. 75-1. Laboratory screen Delayed Photosensitivity Phototesting Photo Patch Test Discontinue drug Rash persists History of exposure to photosensitizing drug History of association of rash to exposure UV-A Immediate Drug photosensitivity Unrelated Drug photosensitivity Photoallergic contact dermatitis Plasma porphyrin ANA Ro/La Rash disappears Solar urticaria UV-B (± UV-A) Lupus erythematosus dermatomyositis Porphyria Polymorphous light eruption Lupus erythematosus Atopic dermatitis with photoaggravation Chronic actinic dermatitis + – Phototest with UV-B, UV-A, and visible; read MED at 30 min Phototest with UV-B, UV-A, and visible; read MED at 24 h + + – – + – + + + – – FIguRE 75-1 Algorithm for the diagnosis of a patient with photo-sensitivity. ANA, antinuclear antibody; MED, minimal erythemal dose; UV-A and UV-B, ultraviolet spectrum segments including wavelengths of 320–400 nm and 290–320 nm, respectively.

1	Since photosensitivity of the skin results from exposure to sunlight, it follows that absolute avoidance of sunlight will eliminate these disorders. However, contemporary lifestyles make this approach impractical for most individuals. Thus better approaches to photoprotection have been sought. Natural photoprotection is provided by structural proteins in the epidermis, particularly keratins and melanin. The amount of melanin and its distribution in cells are genetically regulated, and individuals of darker complexion (skin types IV–VI) are at decreased risk for the development of acute sunburn and cutaneous malignancy. Other forms of photoprotection include clothing and sunscreens. Clothing constructed of tightly woven sun-protective fabrics, irrespective of color, affords substantial protection. Wide-brimmed hats, long sleeves, and trousers all reduce direct exposure. Sunscreens are now considered over-the-counter drugs, and a monograph from the

1	U.S. Food and Drug Administration (FDA) has recognized category I ingredients as safe and effective. Those ingredients are listed in Table 75-5. Sunscreens are rated for their photoprotective effect by their sun protection factor (SPF). The SPF is simply a ratio of the time required to produce sunburn erythema with and without sunscreen application. The SPF of most sunscreens reflects protection from UV-B but not from UV-A. The FDA monograph stipulates that sunscreens must be rated on a scale ranging from minimal (SPF ffi2 and <12) to moderate (SPF ffi12 and <30) to high (SPF ffi30, labeled as 30+). Broad-spectrum sunscreens contain both UV-B-absorbing and UV-A-absorbing chemicals, the latter including avobenzone and ecamsule (terephthalylidene dicamphor sulfonic acid). These chemicals absorb UVR and transfer the absorbed energy to surrounding cells. In contrast, physical UV blockers (zinc oxide and titanium dioxide) scatter or reflect UVR.

1	In addition to light absorption, a critical determinant of the sustained photoprotective effect of sunscreens is their water resistance. The FDA monograph has defined strict testing criteria for sunscreens that claim to possess a high degree of water resistance. Some degree of photoprotection can be achieved by limiting the time of sun exposure during the day. Since a large part of an individual’s total lifetime sun exposure may occur by age 18, it is important to educate parents and young children about the hazards of sunlight. Simply eliminating exposure at midday will substantially reduce lifetime UVR exposure.

1	UVR can be used therapeutically. The administration of UV-B alone or in combination with topically applied agents can induce remissions of many dermatologic diseases, including psoriasis and atopic dermatitis. In particular, narrow-band UV-B treatments (with fluorescent bulbs emitting radiation at ~311 nm) have enhanced efficacy over that obtained with broad-band UV-B in the treatment of psoriasis.

1	Photochemotherapy in which topically applied or systemically administered psoralens are combined with UV-A (PUVA) is effective in treating psoriasis and the early stages of cutaneous T cell lymphoma and vitiligo. Psoralens are tricyclic furocoumarins that, when intercalated into DNA and exposed to UV-A, form adducts with pyrimidine bases and eventually form DNA cross-links. These structural changes are thought to decrease DNA synthesis and to be related to the amelioration of psoriasis. Why PUVA photochemotherapy is effective in cutaneous T cell lymphoma is only partially understood, but it has been shown to induce apoptosis of atypical T lymphocyte populations in the skin. Consequently, direct treatment of circulating atypical lymphocytes by extracorporeal photochemotherapy (photopheresis) has been used in Sézary syndrome as well as in other severe systemic diseases with circulating atypical lymphocytes, such as graft-versus-host disease.

1	In addition to its effects on DNA, PUVA photochemotherapy stimulates epidermal thickening and melanin synthesis; the latter property, together with its anti-inflammatory effects, provides the rationale for use of PUVA in the depigmenting disease vitiligo. Oral 8-methoxypsoralen and UV-A appear to be most effective in this regard, but as many as 100 treatments extending over 12–18 months may be required for satisfactory repigmentation.

1	Not surprisingly, the major side effects of long-term UV-B photo-therapy and PUVA photochemotherapy mimic those seen in individuals with chronic sun exposure and include skin dryness, actinic keratoses, and an increased risk of skin cancer. Despite these risks, the therapeutic index of these modalities continues to be excellent. It is important to choose the most appropriate phototherapeutic approach for a specific dermatologic disease. For example, narrow-band UV-B has been reported in several studies to be as effective as PUVA photo-chemotherapy in the treatment of psoriasis but to pose a lower risk of skin cancer development than PUVA. CHAPTER 75 Photosensitivity and Other Reactions to Light

1	Atlas of Skin Manifestations of Internal Disease Thomas J. Lawley, Calvin McCall, Robert A. Swerlick In the practice of medicine, virtually every clinician encounters patients with skin disease. Physicians of all specialties face the daily 76e task of determining the nature and clinical implication of dermatologic disease. In patients with skin disease, the physician must confront the question of whether the cutaneous process is confined to the skin, representing a purely dermatologic event, or whether it is a manifestation of internal disease related to the patient’s overall medical condition. Evaluation and accurate diagnosis of skin lesions are particularly critical given the marked rise in both melanoma and nonmelanoma skin cancer. Dermatologic conditions can be classified and categorized in many ways. In this atlas, a selected group of inflammatory skin eruptions and neoplastic conditions are grouped in the following manner: common skin diseases and lesions, (2) nonmelanoma skin

1	in many ways. In this atlas, a selected group of inflammatory skin eruptions and neoplastic conditions are grouped in the following manner: common skin diseases and lesions, (2) nonmelanoma skin cancer, melanoma and benign pigmented lesions, (4) infectious disease and the skin, (5) immunologically mediated skin disease, and (6) skin manifestations of internal disease.

1	(Figs. 76e-1 to 76e-19) While most of these common inflammatory skin diseases and benign neoplastic and reactive lesions usually present as a predominantly dermatologic process, underlying systemic associations may be found in some settings. Atopic dermatitis is often present in patients with an atopic diathesis, including asthma or sinusitis. Psoriasis ranges from limited patches on the elbows and knees to severe erythrodermic and pustular involvement and associated psoriatic arthritis. Some patients with alopecia areata may have an underlying thyroid abnormality requiring screening. Finally, even acne vulgaris, one of the most common inflammatory dermatoses, can be associated with a systemic process such as polycystic ovarian syndrome.

1	(Figs. 76e-20 to 76e-27) In fair-skinned ethnic populations, rates of nonmelanoma skin cancer are increasing at an alarming rate. Basal cell carcinoma is the most common cancer in humans and is strongly linked to ultraviolet radiation. Squamous cell carcinoma, including keratoacanthoma, is the second most common skin cancer in most ethnic groups and is also most commonly linked to ultraviolet radiation. Less common cutaneous malignancies include cutaneous T cell lymphoma (mycosis fungoides) and carcinoma and lymphoma metastatic to skin. FIguRE 76e-1 Acne vulgaris, with inflammatory papules, pustules, and comedones. (Courtesy of Kalman Watsky, MD; with permission.) FIguRE 76e-2 Acne rosacea, with prominent facial erythema, telangiectasias, scattered papules, and small pustules. (Courtesy of Robert Swerlick, MD; with permission.)

1	FIguRE 76e-2 Acne rosacea, with prominent facial erythema, telangiectasias, scattered papules, and small pustules. (Courtesy of Robert Swerlick, MD; with permission.) FIguRE 76e-3 Psoriasis. A. Typical psoriasis is characterized by small and large erythematous plaques with adherent silvery scale. B. Acute inflammatory variants of psoriasis may present with widespread superficial pustules. CHAPTER 76e Atlas of Skin Manifestations of Internal Disease PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 76e-4 Atopic dermatitis, with hyperpigmentation, licheni-fication, and scaling in the antecubital fossae. (Courtesy of Robert Swerlick, MD; with permission.) FIguRE 76e-5 Dyshidrotic eczema, characterized by deep-seated vesicles and scaling on palms and lateral fingers, is often associated with an atopic diathesis. FIguRE 76e-6 Seborrheic dermatitis, with erythema and scale in the nasolabial fold. (Courtesy of Robert A. Swerlick, MD; with permission.)

1	FIguRE 76e-6 Seborrheic dermatitis, with erythema and scale in the nasolabial fold. (Courtesy of Robert A. Swerlick, MD; with permission.) FIguRE 76e-7 Stasis dermatitis, with erythematous, scaly, and oozing patches over the lower leg. Several stasis ulcers are also seen in this patient. FIguRE 76e-8 Allergic contact dermatitis. A. Acute phase, with sharply demarcated, weeping, eczematous plaques in a perioral distribution. B. Allergic contact reaction to nickel, chronic phase, with an erythematous, lichenified, weeping plaque on skin chronically exposed to a metal snap. (B: Courtesy of Robert Swerlick, MD; with permission.) FIguRE 76e-9 Lichen planus, with multiple flat-topped, violaceous papules and plaques. Nail dystrophy, as seen in this patient’s thumb-nail, may also be a feature. (Courtesy of Robert Swerlick, MD; with per-mission.) FIguRE 76e-11 Vitiligo in a typical acral distribution, with striking cutaneous depigmentation as a result of melanocyte loss.

1	FIguRE 76e-11 Vitiligo in a typical acral distribution, with striking cutaneous depigmentation as a result of melanocyte loss. CHAPTER 76e Atlas of Skin Manifestations of Internal Disease FIguRE 76e-10 Seborrheic keratoses are “stuck on,” waxy, verrucous papules and plaques with a variety of colors ranging from light tan to black. FIguRE 76e-12 Alopecia areata, characterized by a sharply demar-cated circular patch of scalp completely devoid of hairs. Preservation of follicular orifices is indicative of nonscarring alopecia. (Courtesy of Robert Swerlick, MD; with permission.) FIguRE 76e-13 Pityriasis rosea. Multiple round or oval erythematous patches with fine central scale are distributed along the skin tension lines on the trunk. FIguRE 76e-16 Keloids resulting from ear piercing, with firm exo-phytic flesh-colored to erythematous nodules of scar tissue. PART 2 Cardinal Manifestations and Presentation of Diseases

1	FIguRE 76e-16 Keloids resulting from ear piercing, with firm exo-phytic flesh-colored to erythematous nodules of scar tissue. PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 76e-14 A. Urticaria, with characteristic discrete and confluent, edematous, erythematous papules and plaques. B. Dermatographism. Erythema and whealing developed after firm stroking of the skin. (B: Courtesy of Robert Swerlick, MD; with permission.) FIguRE 76e-15 Epidermoid cysts. Several inflamed and noninflamed firm cystic nodules are seen in this patient. Often a patulous follicular punctum is observed on the overlying epidermal surface. FIguRE 76e-17 Cherry hemangiomas—multiple erythematous to dark-purple papules, usually located on the trunk—are very common and arise in middle-aged to older adults. FIguRE 76e-18 Frostbite of the hand, with vesiculation surrounded by edema and erythema. (Courtesy of Daniel F. Danzl, MD; with permission.)

1	FIguRE 76e-18 Frostbite of the hand, with vesiculation surrounded by edema and erythema. (Courtesy of Daniel F. Danzl, MD; with permission.) FIguRE 76e-22 Basal cell carcinoma, with central ulceration and a pearly, rolled, telangiectatic tumor border. FIguRE 76e-21 Non-Hodgkin’s lymphoma involving the skin, with typical violaceous, “plum-colored” nodules. (Courtesy of Jean Bolognia, MD; with permission.) FIguRE 76e-19 Frostbite of the foot, with vesiculation surrounded by edema and erythema. (Courtesy of Daniel F. Danzl, MD; with permission.) FIguRE 76e-20 Kaposi’s sarcoma in a patient with AIDS. Patch, plaque, and tumor stages are shown. CHAPTER 76e Atlas of Skin Manifestations of Internal Disease FIguRE 76e-23 Mycosis fungoides is a cutaneous T cell lymphoma. Plaque-stage lesions are seen in this patient.

1	(Figs. 76e-28 to 76e-33) As the prognosis of melanoma is related primarily to the microscopic depth of invasion, and as early detection with surgical treatment can be curative in a high percentage of patients, it is essential that all clinicians acquire some facility in evaluating pigmented lesions. Three clinicopathologic subtypes of melanoma—superficial spreading, lentigo maligna, and acral lentiginous melanoma—typically display features noted in the “ABCD rule”: asymmetry (one half of the lesion varies from the other half); border irregularity (the circumferential border exhibits an irregular, sometimes jagged appearance); color (there is uneven coloration and tone to the pigmented lesion, with various shades of brown, black, red, and white in different areas); and diameter (the diameter is typically >6 mm). The more uncommon subtype, nodular melanoma, may not manifest all these features but rather may present as a more symmetric, evenly pigmented, or amelanotic lesion. Dysplastic

1	is typically >6 mm). The more uncommon subtype, nodular melanoma, may not manifest all these features but rather may present as a more symmetric, evenly pigmented, or amelanotic lesion. Dysplastic (atypical) melanocytic nevi may occur as solitary or multiple lesions as well as in the setting of familial melanoma. These nevi display some degree of asymmetry, border irregularity, and color variation. Ordinary nevi may be acquired or congenital and are quite common.

1	FIguRE 76e-24 Metastatic carcinoma to the skin is characterized by inflammatory, often ulcerated dermal nodules. FIguRE 76e-27 Actinic keratoses consist of hyperkeratotic erythema-tous papules and patches on sun-exposed skin. They arise in middle-aged to older adults and have some potential for malignant transfor-mation. (Courtesy of Robert Swerlick, MD; with permission.) PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 76e-25 Keratoacanthoma is a low-grade squamous cell carci-noma that presents as an exophytic nodule with central keratinous debris. FIguRE 76e-26 Squamous cell carcinoma is seen here as a hyper-keratotic, crusted, and somewhat eroded plaque on the lower lip. Sun-exposed skin of the head, neck, hands, and arms are other typical sites of involvement. FIguRE 76e-28 Nevi are benign proliferations of nevomelanocytes characterized by regularly shaped hyperpigmented macules or pap-ules of a uniform color.

1	FIguRE 76e-28 Nevi are benign proliferations of nevomelanocytes characterized by regularly shaped hyperpigmented macules or pap-ules of a uniform color. FIguRE 76e-29 Dysplastic nevi are irregularly pigmented and shaped nevomelanocytic lesions that may be associated with familial melanoma. FIguRE 76e-32 Nodular melanoma most commonly manifests as a rapidly growing, often ulcerated or crusted black nodule. (Courtesy of S. Wright Caughman, MD; with permission.) CHAPTER 76e Atlas of Skin Manifestations of Internal Disease FIguRE 76e-30 Superficial spreading melanoma, the most com-mon type of malignant melanoma, is characterized by color variega-tion (black, blue, brown, pink, and white) and irregular borders.

1	FIguRE 76e-30 Superficial spreading melanoma, the most com-mon type of malignant melanoma, is characterized by color variega-tion (black, blue, brown, pink, and white) and irregular borders. (Figs. 76e-34 to 76e-58) One of the roles of the skin is to function as a barrier from the outside world. In this capacity, exposure to infectious agents occurs, and bacterial, viral, fungal, and parasitic infections may result. In addition, the skin may be secondarily involved and provides diagnostic clues to systemic infections such as meningococcemia, Rocky Mountain spotted fever, Lyme disease, and septic emboli. Most sexually transmitted bacterial and viral diseases exhibit cutaneous involvement; examples include primary and secondary syphilis, chancroid, genital herpes simplex, and condyloma acuminatum.

1	(Figs. 76e-59 to 76e-70) Immunologically mediated skin disease may be largely localized to skin and mucous membranes and manifest with blisters and erosions such as pemphigus, pemphigoid, and dermatitis herpetiformis. In diseases such as systemic lupus erythematosus, dermatomyositis, and vasculitis, skin manifestations are often only one element of a widespread process. FIguRE 76e-31 Lentigo maligna melanoma occurs on sun-exposed skin as a large, hyperpigmented macule or plaque with irregular bor-ders and variable pigmentation. (Courtesy of Alvin Solomon, MD; with permission.) FIguRE 76e-33 Acral lentiginous melanoma is more common among blacks, Asians, and Hispanics and occurs as an enlarging hyperpigmented macule or plaque on the palms or soles. Lateral pigment diffusion is present.

1	FIguRE 76e-37 Impetigo contagiosa is a superficial streptococcal or Staphylococcus aureus infection consisting of honey-colored crusts and erythematous weeping erosions. Bullous lesions are occasionally seen. PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 76e-34 Erysipelas is a streptococcal infection of the superficial dermis and consists of well-demarcated, erythematous, edematous, warm plaques. FIguRE 76e-38 Tender vesicles and erosions in the mouth of a patient with hand-foot-and-mouth disease. (Courtesy of Stephen D. Gellis, MD; with permission.) FIguRE 76e-35 Varicella, with numerous lesions in various stages of evolution: vesicles on an erythematous base, umbilicated vesicles, and crusts. (Courtesy of Robert Hartman, MD; with permission.) FIguRE 76e-39 Lacy reticular rash of erythema infectiosum (fifth disease).

1	FIguRE 76e-39 Lacy reticular rash of erythema infectiosum (fifth disease). FIguRE 76e-36 Herpes zoster is seen in this HIV-infected patient as hemorrhagic vesicles and pustules on an erythematous base in a dermatomal distribution. (Courtesy of Robert Swerlick, MD; with permission.) FIguRE 76e-40 Molluscum contagiosum is a cutaneous poxvirus infection characterized by multiple umbilicated flesh-colored or hypopigmented papules. (Courtesy of Yale Resident’s Slide Collection; with permission.) FIguRE 76e-43 Rocky Mountain spotted fever, with pinpoint pete-chial lesions on the palm and volar aspect of the wrist. (Courtesy of Robert Swerlick, MD; with permission.) CHAPTER 76e Atlas of Skin Manifestations of Internal Disease FIguRE 76e-44 Erythema migrans, the early cutaneous manifes-tation of Lyme disease, is characterized by erythematous annular patches, often with a central erythematous papule at the tick-bite site. (Courtesy of Yale Resident’s Slide Collection; with permission.)

1	(Courtesy of Yale Resident’s Slide Collection; with permission.) FIguRE 76e-41 Oral hairy leukoplakia often presents as white plaques on the lateral tongue and is associated with Epstein-Barr virus infection. (From K Wolff et al: Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005. www .accessmedicine.com.) FIguRE 76e-42 Fulminant meningococcemia, with extensive angu-lar purpuric patches. (Courtesy of Stephen D. Gellis, MD; with permission.) FIguRE 76e-45 Primary syphilis, with a firm, nontender chancre. (Courtesy of Gregory Cox, MD; with permission.) PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 76e-46 Secondary syphilis commonly affects the palms and soles, with scaling, firm, red-brown papules. (Courtesy of Alvin Solomon, MD; with permission.)

1	FIguRE 76e-46 Secondary syphilis commonly affects the palms and soles, with scaling, firm, red-brown papules. (Courtesy of Alvin Solomon, MD; with permission.) FIguRE 76e-47 Condylomata lata are moist, somewhat verrucous inter-triginous plaques seen in secondary syphilis. (Courtesy of Yale Resident’s Slide Collection; with permission.) FIguRE 76e-49 A. Tinea corporis is a superficial fungal infection, seen here as an erythematous annular scaly plaque with central clearing. B. A common presentation of chronic dermatophyte infection involves the feet (tinea pedis), hands (tinea manum), and nails (tinea unguium). FIguRE 76e-48 Secondary syphilis, with the characteristic papulo-squamous truncal eruption. FIguRE 76e-50 Scabies, with typical scaling erythematous papules and few linear burrows. FIguRE 76e-51 Skin lesions caused by Chironex fleckeri sting. (Courtesy of V. Pranava Murthy, MD; with permission.)

1	FIguRE 76e-51 Skin lesions caused by Chironex fleckeri sting. (Courtesy of V. Pranava Murthy, MD; with permission.) FIguRE 76e-53 Condylomata acuminata are lesions induced by human papillomavirus and in this patient are seen as multiple verrucous papules coalescing into plaques. (Courtesy of S. Wright Caughman, MD; with permission.) CHAPTER 76e Atlas of Skin Manifestations of Internal Disease FIguRE 76e-54 A patient with features of polar lepromatous FIguRE 76e-52 Chancroid, with characteristic penile ulcers and leprosy: multiple nodular skin lesions, particularly of the forehead, associated left inguinal adenitis (bubo). and loss of eyebrows. (Courtesy of Robert Gelber, MD; with permission.) PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 76e-55 Skin lesions of neutropenic patients. A. Hemorrhagic papules on the foot of a patient undergoing treatment for multiple myeloma. Biopsy and culture demonstrated Aspergillosis species. B. Eroded nodule on the hard palate of a patient undergoing chemotherapy. Biopsy and culture demonstrated Mucor species. C. Ecthyma gangrenosum in a neutropenic patient with Pseudomonas aeruginosa bacteremia. FIguRE 76e-56 Septic emboli, with hemorrhage and infarction due to acute Staphylococcus aureus endocarditis. (Courtesy of L. Baden, MD; with permission.) FIguRE 76e-57 Vegetations (arrows) due to viridans streptococcal endocarditis involving the mitral valve. (Courtesy of AW Karchmer, MD; with permission.) FIguRE 76e-58 Disseminated gonococcemia in the skin is seen as hemorrhagic papules and pustules with purpuric centers in an acral distribution. (Courtesy of Daniel M. Musher, MD; with permission.)

1	FIguRE 76e-58 Disseminated gonococcemia in the skin is seen as hemorrhagic papules and pustules with purpuric centers in an acral distribution. (Courtesy of Daniel M. Musher, MD; with permission.) CHAPTER 76e Atlas of Skin Manifestations of Internal Disease FIguRE 76e-60 Discoid lupus erythematosus. Atrophic, depigmented plaques and patches surrounded by hyperpigmentation and erythema in association with scarring and alopecia are characteristic of this cutaneous form of lupus. FIguRE 76e-59 Lupus erythematosus. A. Systemic lupus erythematosus, with prominent, scaly malar erythema. Involvement of other sun-exposed sites is also common. B. Acute lupus erythematosus on the upper chest, with brightly erythematous and slightly edematous coalescence of papules and plaques. (B: Courtesy of Robert Swerlick, MD; with permission.) FIguRE 76e-61 Dermatomyositis. Periorbital violaceous erythema characterizes the classic heliotrope rash. (Courtesy of James Krell, MD; with permission.)

1	FIguRE 76e-61 Dermatomyositis. Periorbital violaceous erythema characterizes the classic heliotrope rash. (Courtesy of James Krell, MD; with permission.) FIguRE 76e-62 Scleroderma characterized by typical expressionless, mask-like facies. PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 76e-65 Erythema multiforme is characterized by multiple erythematous plaques with a target or iris morphology and usually represents a hypersensitivity reaction to drugs or infections (especially herpes simplex virus). (Courtesy of Yale Resident’s Slide Collection; with permission.) FIguRE 76e-63 Scleroderma, with acral sclerosis and focal digital ulcers. FIguRE 76e-64 Dermatomyositis often involves the hands as ery-thematous flat-topped papules over the knuckles (Gottron’s sign) and periungual telangiectasias.

1	FIguRE 76e-64 Dermatomyositis often involves the hands as ery-thematous flat-topped papules over the knuckles (Gottron’s sign) and periungual telangiectasias. FIguRE 76e-66 Dermatitis herpetiformis, manifested by pruritic, grouped vesicles in a typical location. The vesicles are often excori-ated and may also occur on the knees, buttocks, elbows, and poste-rior scalp. FIguRE 76e-67 Pemphigus vulgaris. A. Eroded bullae on the back. B. The oral mucosa is almost invariably involved, sometimes with erosions on the gingiva, buccal mucosa, palate, posterior pharynx, or tongue. (B: Courtesy of Robert Swerlick, MD; with permission.) FIguRE 76e-68 Erythema nodosum is a panniculitis characterized by tender deep-seated nodules and plaques, usually located on the lower extremities. (Courtesy of Robert Swerlick, MD; with permission.) FIguRe 76e-69 Vasculitis. Palpable purpuric papules on the lower legs are seen in this patient with cutaneous small-vessel vasculitis.

1	FIguRe 76e-69 Vasculitis. Palpable purpuric papules on the lower legs are seen in this patient with cutaneous small-vessel vasculitis. (Courtesy of Robert Swerlick, MD; with permission.) CHAPTER 76e Atlas of Skin Manifestations of Internal Disease

1	FIguRE 76e-70 Bullous pemphigoid, with tense vesicles and bullae on an erythematous, urticarial base. (Courtesy of Yale Resident’s Slide Collection; with permission.) (Figs. 76e-71 to 76e-78) While many systemic diseases also have cutaneous manifestations, there are well-recognized dermatologic markers of internal disease, some of which are shown in this section. Many of these dermatologic markers may precede, accompany, or follow diagnosis of systemic disease. Acanthosis nigricans is a prototypical dermatologic process that often occurs in association with underlying systemic abnormalities, most commonly obesity and insulin resistance. It may also be associated with other endocrine disorders and several rare genetic syndromes. Malignant acanthosis nigricans may occur in association with several malignancies, especially adenocarcinoma of the gastrointestinal tract, lung, and breast. Other markers of internal disease in this section include pretibial myxedema, which is associated with

1	several malignancies, especially adenocarcinoma of the gastrointestinal tract, lung, and breast. Other markers of internal disease in this section include pretibial myxedema, which is associated with thyroid disease, and Sweet syndrome, which may be associated with hematologic malignancies, solid tumors, infections, or inflammatory bowel disease. The skin is also involved in many systemic inflammatory diseases such as sarcoidosis, rheumatoid arthritis, and lupus erythematosus.

1	FIguRE 76e-71 Acanthosis nigricans, with typical hyperpigmented plaques on a velvet-like, verrucous surface on the neck. FIguRE 76e-74 Bilateral rheumatoid nodules of the upper extremi-ties. (Courtesy of Robert Swerlick, MD; with permission.) PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 76e-75 Neurofibromatosis, with numerous flesh-colored cutaneous neurofibromas. FIguRE 76e-72 Pretibial myxedema manifesting as waxy, infiltrated plaques in a patient with Graves’ disease. FIguRE 76e-73 Erythematous, indurated plaque of Sweet syn-drome, with a pseudovesicular border. (Courtesy of Robert Swerlick, MD, with permission.) FIguRE 76e-76 Coumarin necrosis. Shown is cutaneous and subcutaneous necrosis of a breast. Other fatty areas, such as buttocks and thighs, are also common sites of involvement. (Courtesy of Kim Yancey, MD; with permission.)

1	FIguRE 76e-77 Sarcoid. A. Infiltrated papules and plaques of variable color are seen in a typical paranasal and periorbital location. B. Infiltrated, hyperpigmented, and slightly erythematous coalescent papules and plaques on the upper arm. (B: Courtesy of Robert Swerlick, MD; with permission.) FIguRE 76e-78 Pyoderma gangrenosum on the dorsal aspect of both hands. Multiple necrotic ulcers are surrounded by a violaceous and undermined border. (Courtesy of Robert Swerlick, MD; with permission.) CHAPTER 76e Atlas of Skin Manifestations of Internal Disease

1	PART 2 Anemia and Polycythemia John W. Adamson, Dan L. Longo HEMATOPOIESIS AND THE PHYSIOLOgIC BASIS OF RED CELL PRODuCTION Hematopoiesis is the process by which the formed elements of blood are produced. The process is regulated through a series of steps begin-ning with the hematopoietic stem cell. Stem cells are capable of pro-red blood cells. The size of the red cell mass reflects the balance of red cell production and destruction. The physiologic basis of red cell pro-duction and destruction provides an understanding of the mechanisms that can lead to anemia. The physiologic regulator of red cell production, the glycoprotein hormone EPO, is produced and released by peritubular capillary lining cells within the kidney. These cells are highly specialized epithelial-like cells. A small amount of EPO is produced by hepatocytes. The funda-mental stimulus for EPO production is the availability of O2 for tissue metabolic needs. Key to EPO gene regulation is hypoxia-inducible factor

1	amount of EPO is produced by hepatocytes. The funda-mental stimulus for EPO production is the availability of O2 for tissue metabolic needs. Key to EPO gene regulation is hypoxia-inducible factor (HIF)-1α. In the presence of O2, HIF-1α is hydroxylated at a key 77 SECTion 10 HEMAToLogiC ALTERATionS proline, allowing HIF-1α to be ubiquitinated and degraded via the pro ducing red cells, all classes of granulocytes, monocytes, platelets, and the cells of the immune system. The precise molecular mechanism— either intrinsic to the stem cell itself or through the action of extrinsic factors—by which the stem cell becomes committed to a given lineage is not fully defined. However, experiments in mice suggest that ery tor that does not develop in the absence of expression of the GATA-1 teasome pathway. If O2 becomes limiting, this critical hydroxylation step does not occur, allowing HIF-1α to partner with other proteins, translocate to the nucleus, and upregulate the expression of the EPO

1	pathway. If O2 becomes limiting, this critical hydroxylation step does not occur, allowing HIF-1α to partner with other proteins, translocate to the nucleus, and upregulate the expression of the EPO gene, among others.

1	Impaired O2 delivery to the kidney can result from a decreased red cell mass (anemia), impaired O2 loading of the hemoglobin molecule or a high O2 affinity mutant hemoglobin (hypoxemia), or, rarely, impaired blood flow to the kidney (renal artery stenosis). EPO governs the day to-day production of red cells, and ambient levels of the hormone can be measured in the plasma by sensitive immunoassays—the normal level being 10–25 U/L. When the hemoglobin concentration falls below 100–120 g/L (10–12 g/dL), plasma EPO levels increase in proportion to the severity of the anemia (Fig. 77-2). In circulation, EPO has a half-clearance time of 6–9 h. EPO acts by binding to specific receptors on the surface of marrow erythroid precursors, inducing them to pro liferate and to mature. With EPO stimulation, red cell production can increase fourto fivefold within a 1to 2-week period, but only in the presence of adequate nutrients, especially iron. The functional capacity of the erythron, therefore,

1	red cell production can increase fourto fivefold within a 1to 2-week period, but only in the presence of adequate nutrients, especially iron. The functional capacity of the erythron, therefore, requires normal renal production of EPO, a functioning erythroid marrow, and an adequate supply of substrates for hemoglobin synthesis. A defect in any of these key components can lead to anemia. Generally, anemia is recognized in the laboratory when a patient’s hemoglobin level or hematocrit is reduced below an expected value (the normal range). The likelihood and severity of anemia are defined based on the deviation of the patient’s hemoglobin/hematocrit from values expected for ageand sex-matched normal subjects. The hemoglobin concentration in adults has a Gaussian distribution. The and FOG-1 (friend of GATA-1) transcription factors (Chap. 89e).

1	Following lineage commitment, hematopoietic progenitor and precur sor cells come increasingly under the regulatory influence of growth factors and hormones. For red cell production, erythropoietin (EPO) is the primary regulatory hormone. EPO is required for the maintenance of committed erythroid progenitor cells that, in the absence of the hormone, undergo programmed cell death (apoptosis). The regulated process of red cell production is erythropoiesis, and its key elements are illustrated in Fig. 77-1. In the bone marrow, the first morphologically recognizable ery throid precursor is the pronormoblast. This cell can undergo four to five cell divisions, which result in the production of 16–32 mature red cells. With increased EPO production, or the administration of EPO as a drug, early progenitor cell numbers are amplified and, in turn, give rise to increased numbers of erythrocytes. The regulation of EPO production itself is linked to tissue oxygenation.

1	In mammals, O2 is transported to tissues bound to the hemoglobin contained within circulating red cells. The mature red cell is 8 μm in diameter, anucleate, discoid in shape, and extremely pliable in order to traverse the microcirculation successfully; its membrane integrity is maintained by the intracellular generation of ATP. Normal red cell production results in the daily replacement of 0.8–1% of all circulating red cells in the body, since the average red cell lives 100–120 days. The organ responsible for red cell production is called the erythron. The erythron is a dynamic organ made up of a rapidly proliferating pool of marrow erythroid precursor cells and a large mass of mature circulating FIguRE 77-2 Erythropoietin (EPO) levels in response to anemia. When the hemoglobin level falls to 120 g/L (12 g/dL), plasma EPO levels increase logarithmically. In the presence of chronic kidney

1	FIguRE 77-2 Erythropoietin (EPO) levels in response to anemia. When the hemoglobin level falls to 120 g/L (12 g/dL), plasma EPO levels increase logarithmically. In the presence of chronic kidney Cardinal Manifestations and Presentation of Diseases disease or chronic inflammation, EPO levels are typically lower than expected for the degree of anemia. As individuals age, the level of EPO needed to sustain normal hemoglobin levels appears to increase.

1	EPO needed to sustain normal hemoglobin levels appears to increase. FIguRE 77-1 The physiologic regulation of red cell production by (From RS Hillman et al: Hematology in Clinical Practice, 5th ed. New York, tissue oxygen tension. Hb, hemoglobin. McGraw-Hill, 2010.) mean hematocrit value for adult males is 47% (standard deviation, ±7%) and that for adult females is 42% (±5%). Any single hematocrit or hemoglobin value carries with it a likelihood of associated anemia. Thus, a hematocrit of <39% in an adult male or <35% in an adult female has only about a 25% chance of being normal. Hematocrit levels are less useful than hemoglobin levels in assessing anemia because they are calculated rather than measured directly. Suspected low hemoglobin or hematocrit values are more easily interpreted if previous values for the same patient are known for comparison. The World Health Organization (WHO) defines anemia as a hemoglobin level <130 g/L (13 g/dL) in men and <120 g/L (12 g/dL) in women.

1	The critical elements of erythropoiesis—EPO production, iron availability, the proliferative capacity of the bone marrow, and effective maturation of red cell precursors—are used for the initial classification of anemia (see below).

1	CLINICAL PRESENTATION OF ANEMIA Signs and Symptoms Anemia is most often recognized by abnormal screening laboratory tests. Patients less commonly present with advanced anemia and its attendant signs and symptoms. Acute anemia is due to blood loss or hemolysis. If blood loss is mild, enhanced O2 delivery is achieved through changes in the O2–hemoglobin dissociation curve mediated by a decreased pH or increased CO2 (Bohr effect). With acute blood loss, hypovolemia dominates the clinical picture, and the hematocrit and hemoglobin levels do not reflect the volume of blood lost. Signs of vascular instability appear with acute losses of 10–15% of the total blood volume. In such patients, the issue is not anemia but hypotension and decreased organ perfusion. When >30% of the blood volume is lost suddenly, patients are unable to compensate with the usual mechanisms of vascular contraction and changes in regional blood flow. The patient prefers to remain supine and will show postural

1	is lost suddenly, patients are unable to compensate with the usual mechanisms of vascular contraction and changes in regional blood flow. The patient prefers to remain supine and will show postural hypotension and tachycardia. If the volume of blood lost is >40% (i.e., >2 L in the average-sized adult), signs of hypovolemic shock including confusion, dyspnea, diaphoresis, hypotension, and tachycardia appear (Chap. 129). Such patients have significant deficits in vital organ perfusion and require immediate volume replacement.

1	With acute hemolysis, the signs and symptoms depend on the mechanism that leads to red cell destruction. Intravascular hemolysis with release of free hemoglobin may be associated with acute back pain, free hemoglobin in the plasma and urine, and renal failure. Symptoms associated with more chronic or progressive anemia depend on the age of the patient and the adequacy of blood supply to critical organs. Symptoms associated with moderate anemia include fatigue, loss of stamina, breathlessness, and tachycardia (particularly with physical exertion). However, because of the intrinsic compensatory mechanisms that govern the O2–hemoglobin dissociation curve, the gradual onset of anemia—particularly in young patients—may not be associated with signs or symptoms until the anemia is severe (hemoglobin <70–80 g/L [7–8 g/dL]). When anemia develops over a period of days or weeks, the total blood volume is normal to slightly increased, and changes in cardiac output and regional blood flow help

1	<70–80 g/L [7–8 g/dL]). When anemia develops over a period of days or weeks, the total blood volume is normal to slightly increased, and changes in cardiac output and regional blood flow help compensate for the overall loss in O2-carrying capacity. Changes in the position of the O2–hemoglobin dissociation curve account for some of the compensatory response to anemia. With chronic anemia, intracellular levels of 2,3-bisphosphoglycerate rise, shifting the dissociation curve to the right and facilitating O2 unloading. This compensatory mechanism can only maintain normal tissue O2 delivery in the face of a 20–30 g/L (2–3 g/dL) deficit in hemoglobin concentration. Finally, further protection of O2 delivery to vital organs is achieved by the shunting of blood away from organs that are relatively rich in blood supply, particularly the kidney, gut, and skin.

1	Certain disorders are commonly associated with anemia. Chronic inflammatory states (e.g., infection, rheumatoid arthritis, cancer) are associated with mild to moderate anemia, whereas lymphoproliferative disorders, such as chronic lymphocytic leukemia and certain other B cell neoplasms, may be associated with autoimmune hemolysis. APPROACH TO THE PATIENT:

1	The evaluation of the patient with anemia requires a careful history and physical examination. Nutritional history related to drugs or alcohol intake and family history of anemia should always be assessed. Certain geographic backgrounds and ethnic origins are associated with an increased likelihood of an inherited disorder of the hemoglobin molecule or intermediary metabolism. Glucose-6phosphate dehydrogenase (G6PD) deficiency and certain hemoglobinopathies are seen more commonly in those of Middle Eastern or African origin, including African Americans who have a high frequency of G6PD deficiency. Other information that may be useful includes exposure to certain toxic agents or drugs and symptoms related to other disorders commonly associated with anemia. These include symptoms and signs such as bleeding, fatigue, malaise, fever, weight loss, night sweats, and other systemic symptoms. Clues to the mechanisms of anemia may be provided on physical examination by findings of infection,

1	such as bleeding, fatigue, malaise, fever, weight loss, night sweats, and other systemic symptoms. Clues to the mechanisms of anemia may be provided on physical examination by findings of infection, blood in the stool, lymphadenopathy, splenomegaly, or petechiae. Splenomegaly and lymphadenopathy suggest an underlying lymphoproliferative disease, whereas petechiae suggest platelet dysfunction. Past laboratory measurements are helpful to determine a time of onset.

1	In the anemic patient, physical examination may demonstrate a forceful heartbeat, strong peripheral pulses, and a systolic “flow” murmur. The skin and mucous membranes may be pale if the hemoglobin is <80–100 g/L (8–10 g/dL). This part of the physical examination should focus on areas where vessels are close to the surface such as the mucous membranes, nail beds, and palmar creases. If the palmar creases are lighter in color than the surrounding skin when the hand is hyperextended, the hemoglobin level is usually <80 g/L (8 g/dL).

1	Table 77-1 lists the tests used in the initial workup of anemia. A routine complete blood count (CBC) is required as part of the evaluation and includes the hemoglobin, hematocrit, and red cell indices: the mean cell volume (MCV) in femtoliters, mean cell hemoglobin (MCH) in picograms per cell, and mean concentration of hemoglobin per volume of red cells (MCHC) in grams per liter (non-SI: grams per deciliter). The red cell indices are calculated as shown in Table 77-2, and the normal variations in the hemoglobin and hematocrit with age are shown in Table 77-3. A number of physiologic factors affect the CBC, including age, sex, pregnancy, smoking, and altitude. High-normal hemoglobin values may be seen in men and women who live at altitude or smoke heavily. Hemoglobin elevations due to smoking reflect normal compensation due to the displacement of O2 by CO in hemoglobin binding. Other important information is provided by the reticulocyte count and measurements of iron supply including

1	reflect normal compensation due to the displacement of O2 by CO in hemoglobin binding. Other important information is provided by the reticulocyte count and measurements of iron supply including serum iron, total iron-binding capacity (TIBC; an indirect measure of serum transferrin), and serum ferritin. Marked alterations in the red cell indices usually reflect disorders of maturation or iron deficiency. A careful evaluation of the peripheral blood smear is important, and clinical laboratories often provide a description of both the red and white cells, a white cell differential count, and the platelet count. In patients with severe anemia and abnormalities in red blood cell morphology and/or low reticulocyte counts, a bone marrow aspirate or biopsy can assist in the diagnosis. Other tests of value in the diagnosis of specific anemias are discussed in chapters on specific disease states.

1	The components of the CBC also help in the classification of anemia. Microcytosis is reflected by a lower than normal MCV (<80), whereas high values (>100) reflect macrocytosis. The MCH and MCHC reflect defects in hemoglobin synthesis (hypochromia). Automated cell counters describe the red cell volume distribution width (RDW). The MCV (representing the peak of the distribution curve) is insensitive to the appearance of small populations of macrocytes or microcytes. An experienced laboratory technician I. Complete blood count (CBC) A. Red blood cell count 1. 2. 3. B. Red blood cell indices 1. 2. 3. 4. C. White blood cell count 1. 2. Nuclear segmentation of neutrophils D. Platelet count E. Cell morphology 1. 2. 3. 4. 5. II. A. B. C. III. A. 1. 2. 3. B. Biopsy 1. 2. Morphology aM/E ratio, ratio of myeloid to erythroid precursors. PART 2 Cardinal Manifestations and Presentation of Diseases

1	2. 3. 4. 5. II. A. B. C. III. A. 1. 2. 3. B. Biopsy 1. 2. Morphology aM/E ratio, ratio of myeloid to erythroid precursors. PART 2 Cardinal Manifestations and Presentation of Diseases Mean cell hemoglobin concentration = (hemoglobin × 33 ± 2% 10)/hematocrit, or MCH/MCV TABLE 77-3 CHAngES in noRMAL HEMogLoBin/HEMAToCRiT vALuES wiTH AgE, SEx, AnD PREgnAnCy Age/Sex Hemoglobin, g/dL Hematocrit, % Source: From RS Hillman et al: Hematology in Clinical Practice, 5th ed. New York, McGraw-Hill, 2010. FIguRE 77-3 Normal blood smear (Wright stain). High-power field showing normal red cells, a neutrophil, and a few platelets. (From RS Hillman et al: Hematology in Clinical Practice, 5th ed. New York, McGraw-Hill, 2010.) will be able to identify minor populations of large or small cells or hypochromic cells before the red cell indices change.

1	Peripheral Blood Smear The peripheral blood smear provides important information about defects in red cell production (Chap. 81e). As a complement to the red cell indices, the blood smear also reveals variations in cell size (anisocytosis) and shape (poikilocytosis). The degree of anisocytosis usually correlates with increases in the RDW or the range of cell sizes. Poikilocytosis suggests a defect in the maturation of red cell precursors in the bone marrow or fragmentation of circulating red cells. The blood smear may also reveal polychromasia—red cells that are slightly larger than normal and grayish blue in color on the Wright-Giemsa stain. These cells are reticulocytes that have been prematurely released from the bone marrow, and their color represents residual amounts of ribosomal RNA. These cells appear in circulation in response to EPO stimulation or to architectural damage of the bone marrow (fibrosis, infiltration of the marrow by malignant cells, etc.) that results in their

1	These cells appear in circulation in response to EPO stimulation or to architectural damage of the bone marrow (fibrosis, infiltration of the marrow by malignant cells, etc.) that results in their disordered release from the marrow. The appearance of nucleated red cells, Howell-Jolly bodies, target cells, sickle cells, and others may provide clues to specific disorders (Figs. 77-3 to 77-11).

1	Reticulocyte Count An accurate reticulocyte count is key to the initial classification of anemia. Reticulocytes are red cells that FIguRE 77-4 Severe iron-deficiency anemia. Microcytic and hypo-chromic red cells smaller than the nucleus of a lymphocyte associated with marked variation in size (anisocytosis) and shape (poikilocytosis). (From RS Hillman et al: Hematology in Clinical Practice, 5th ed. New York, McGraw-Hill, 2010.) FIguRE 77-5 Macrocytosis. Red cells are larger than a small lympho-cyte and well hemoglobinized. Often macrocytes are oval shaped (macro-ovalocytes). FIguRE 77-8 Target cells. Target cells have a bull’s-eye appearance and are seen in thalassemia and in liver disease. (From RS Hillman et al: Hematology in Clinical Practice, 5th ed. New York, McGraw-Hill, 2010.) FIguRE 77-6 Howell-Jolly bodies. In the absence of a functional spleen, nuclear remnants are not culled from the red cells and remain as small homogeneously staining blue inclusions on Wright stain.

1	FIguRE 77-6 Howell-Jolly bodies. In the absence of a functional spleen, nuclear remnants are not culled from the red cells and remain as small homogeneously staining blue inclusions on Wright stain. (From RS Hillman et al: Hematology in Clinical Practice, 5th ed. New York, McGraw-Hill, 2010.) FIguRE 77-9 Red cell fragmentation. Red cells may become frag-mented in the presence of foreign bodies in the circulation, such as mechanical heart valves, or in the setting of thermal injury. (From RS Hillman et al: Hematology in Clinical Practice, 5th ed. New York, McGraw-Hill, 2010.) FIguRE 77-7 Red cell changes in myelofibrosis. The left panel shows a teardrop-shaped cell. The right panel shows a nucleated red cell. These forms can be seen in myelofibrosis.

1	FIguRE 77-7 Red cell changes in myelofibrosis. The left panel shows a teardrop-shaped cell. The right panel shows a nucleated red cell. These forms can be seen in myelofibrosis. FIguRE 77-10 Uremia. The red cells in uremia may acquire numerous regularly spaced, small, spiny projections. Such cells, called burr cells or echinocytes, are readily distinguishable from irregularly spiculated acanthocytes shown in Fig. 77-11. PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 77-11 Spur cells. Spur cells are recognized as distorted red cells containing several irregularly distributed thornlike projections. Cells with this morphologic abnormality are also called acanthocytes.

1	(From RS Hillman et al: Hematology in Clinical Practice, 5th ed. New York, McGraw-Hill, 2010.) have been recently released from the bone marrow. They are identified by staining with a supravital dye that precipitates the ribosomal RNA (Fig. 77-12). These precipitates appear as blue or black punctate spots and can be counted manually or, currently, by fluorescent emission of dyes that bind to RNA. This residual RNA is metabolized over the first 24–36 h of the reticulocyte’s life span in circulation. Normally, the reticulocyte count ranges from 1 to 2% and reflects the daily replacement of 0.8–1.0% of the circulating red cell population. A corrected reticulocyte count provides a reliable measure of effective red cell production.

1	In the initial classification of anemia, the patient’s reticulocyte count is compared with the expected reticulocyte response. In general, if the EPO and erythroid marrow responses to moderate anemia [hemoglobin <100 g/L (10 g/dL)] are intact, the red cell production rate increases to two to three times normal within 10 days following the onset of anemia. In the face of established anemia, a reticulocyte response less than two to three times normal indicates an inadequate marrow response. To use the reticulocyte count to estimate marrow response, two corrections are necessary. The first correction adjusts the reticulocyte count based on the reduced number of circulating red cells. With anemia, the percentage of reticulocytes may be increased while the absolute number is unchanged. To correct for this effect, the reticulocyte percentage is multiplied by the ratio of the patient’s hemoglobin or hematocrit to the expected hemoglobin/hematocrit

1	FIguRE 77-12 Reticulocytes. Methylene blue stain demonstrates residual RNA in newly made red cells. (From RS Hillman et al: Hematology in Clinical Practice, 5th ed. New York, McGraw-Hill, 2010.) Correction #1 for Anemia: This correction produces the corrected reticulocyte count. In a person whose reticulocyte count is 9%, hemoglobin 7.5 g/dL, and hematocrit 23%, the absolute reticulocyte count = 9 × (7.5/15) [or × (23/45)] = 4.5% Note. This correction is not done if the reticulocyte count is reported in absolute numbers (e.g., 50,000/μL of blood) Correction #2 for Longer Life of Prematurely Released Reticulocytes in the Blood: This correction produces the reticulocyte production index.

1	Correction #2 for Longer Life of Prematurely Released Reticulocytes in the Blood: This correction produces the reticulocyte production index. In a person whose reticulocyte count is 9%, hemoglobin 7.5 gm/dL, and hematocrit 23%, the reticulocyte production index (7.5/15)(hemoglobincorrection) =×9 =2.25 for the age and sex of the patient (Table 77-4). This provides an estimate of the reticulocyte count corrected for anemia. To convert the corrected reticulocyte count to an index of marrow production, a further correction is required, depending on whether some of the reticulocytes in circulation have been released from the marrow pre maturely. For this second correction, the peripheral blood smear is examined to see if there are polychromatophilic macrocytes present. These cells, representing prematurely released reticulocytes, are referred to as “shift” cells, and the relationship between the degree of shift and the necessary shift correction factor is shown in

1	These cells, representing prematurely released reticulocytes, are referred to as “shift” cells, and the relationship between the degree of shift and the necessary shift correction factor is shown in Fig. 77-13. The correction is necessary because these prematurely released cells survive as reticulocytes in circulation for >1 day, thereby providing a falsely high estimate of daily red cell produc tion. If polychromasia is increased, the reticulocyte count, already corrected for anemia, should be divided again by 2 to account for the prolonged reticulocyte maturation time. The second correction fac tor varies from 1 to 3 depending on the severity of anemia. In gen eral, a correction of 2 is simply used. An appropriate correction is shown in Table 77-4. If polychromatophilic cells are not seen on the blood smear, the second correction is not required. The now doubly 1.5 2.5

1	FIguRE 77-13 Correction of the reticulocyte count. To use the reticulocyte count as an indicator of effective red cell production, the reticulocyte percentage must be corrected based on the level of anemia and the circulating life span of the reticulocytes. Erythroid cells take ∼4.5 days to mature. At a normal hemoglobin, reticulocytes are released to the circulation with ∼1 day left as reticulocytes. However, with different levels of anemia, reticulocytes (and even earlier erythroid cells) may be released from the marrow prematurely. Most patients come to clinical attention with hematocrits in the mid20s, and thus a correction factor of 2 is commonly used because the observed reticulocytes will live for 2 days in the circulation before losing their RNA.

1	corrected reticulocyte count is the reticulocyte production index, and it provides an estimate of marrow production relative to normal. In many hospital laboratories, the reticulocyte count is reported not only as a percentage but also in absolute numbers. If so, no correction for dilution is required. A summary of the appropriate marrow response to varying degrees of anemia is shown in Table 77-5.

1	Premature release of reticulocytes is normally due to increased EPO stimulation. However, if the integrity of the bone marrow release process is lost through tumor infiltration, fibrosis, or other disorders, the appearance of nucleated red cells or polychromatophilic macrocytes should still invoke the second reticulocyte correction. The shift correction should always be applied to a patient with anemia and a very high reticulocyte count to provide a true index of effective red cell production. Patients with severe chronic hemolytic anemia may increase red cell production as much as sixto sevenfold. This measure alone confirms the fact that the patient has an appropriate EPO response, a normally functioning bone marrow, and sufficient iron available to meet the demands for new red cell formation. If the reticulocyte production index is <2 in the face of established anemia, a defect in erythroid marrow proliferation or maturation must be present.

1	Tests of Iron Supply and Storage The laboratory measurements that reflect the availability of iron for hemoglobin synthesis include the serum iron, the TIBC, and the percent transferrin saturation. The percent transferrin saturation is derived by dividing the serum iron level (× 100) by the TIBC. The normal serum iron ranges from 9 to 27 μmol/L (50–150 μg/dL), whereas the normal TIBC is 54–64 μmol/L (300–360 μg/dL); the normal transferrin saturation ranges from 25 to 50%. A diurnal variation in the serum iron leads to a variation in the percent transferrin saturation. The serum ferritin is used to evaluate total body iron stores. Adult males have serum ferritin levels that average ∼100 μg/L, corresponding to iron stores of ∼1 g. Adult females have lower serum ferritin levels averaging 30 μg/L, reflecting lower iron stores (∼300 mg). A serum ferritin level of 10–15 μg/L indicates depletion of body iron stores. However, ferritin is also an acute-phase reactant and, in the presence of

1	μg/L, reflecting lower iron stores (∼300 mg). A serum ferritin level of 10–15 μg/L indicates depletion of body iron stores. However, ferritin is also an acute-phase reactant and, in the presence of acute or chronic inflammation, may rise several-fold above baseline levels. As a rule, a serum ferritin >200 μg/L means there is at least some iron in tissue stores.

1	Bone Marrow Examination A bone marrow aspirate and smear or a needle biopsy can be useful in the evaluation of some patients with anemia. In patients with hypoproliferative anemia and normal iron status, a bone marrow is indicated. Marrow examination can diagnose primary marrow disorders such as myelofibrosis, a red cell maturation defect, or an infiltrative disease (Figs. 77-14 to 77-16). The increase or decrease of one cell lineage (myeloid vs erythroid) compared to another is obtained by a differential count of nucleated cells in a bone marrow smear (the myeloid/erythroid [M/E] ratio). A patient with a hypoproliferative anemia (see below) and a reticulocyte production index <2 will demonstrate an M/E ratio of 2 or 3:1. In contrast, patients with hemolytic disease and a production index >3 will have an M/E ratio of at least 1:1. Maturation disorders are identified from the discrepancy between the M/E ratio and the reticulocyte production index (see below). Either the marrow smear or

1	>3 will have an M/E ratio of at least 1:1. Maturation disorders are identified from the discrepancy between the M/E ratio and the reticulocyte production index (see below). Either the marrow smear or biopsy can be stained for the presence of iron stores or iron in developing red cells. The storage iron is in the form of ferritin or hemosiderin. On carefully prepared bone marrow smears, small ferritin granules can normally be seen under oil immersion in 20–40% of developing erythroblasts. Such cells are called sideroblasts.

1	FIguRE 77-14 Normal bone marrow. This is a low-power view of a section of a normal bone marrow biopsy stained with hematoxylin and eosin (H&E). Note that the nucleated cellular elements account for ∼40–50% and the fat (clear areas) accounts for ∼50–60% of the area. (From RS Hillman et al: Hematology in Clinical Practice, 5th ed. New York, McGraw-Hill, 2010.) FIguRE 77-15 Erythroid hyperplasia. This marrow shows an increase in the fraction of cells in the erythroid lineage as might be seen when a normal marrow compensates for acute blood loss or hemolysis. The myeloid/erythroid (M/E) ratio is about 1:1. (From RS Hillman et al: Hematology in Clinical Practice, 5th ed. New York, McGraw-Hill, 2010.)

1	FIguRE 77-16 Myeloid hyperplasia. This marrow shows an increase in the fraction of cells in the myeloid or granulocytic lineage as might be seen in a normal marrow responding to infection. The myeloid/ erythroid (M/E) ratio is >3:1. (From RS Hillman et al: Hematology in Clinical Practice, 5th ed. New York, McGraw-Hill, 2010.) Additional laboratory tests may be of value in confirming specific diagnoses. For details of these tests and how they are applied in individual disorders, see Chaps. 126 to 130. PART 2 Cardinal Manifestations and Presentation of Diseases

1	DEFINITION AND CLASSIFICATION OF ANEMIA Initial Classification of Anemia The functional classification of anemia has three major categories. These are (1) marrow production defects (hypoproliferation), (2) red cell maturation defects (ineffective erythropoiesis), and (3) decreased red cell survival (blood loss/hemolysis). The classification is shown in Fig. 77-17. A hypoproliferative anemia is typically seen with a low reticulocyte production index together with little or no change in red cell morphology (a normocytic, normochromic anemia) (Chap. 126). Maturation disorders typically have a slight to moderately elevated reticulocyte production index that is accompanied by either macrocytic (Chap. 128) or microcytic (Chaps. 126, 127) red cell indices. Increased red blood cell destruction secondary to hemolysis results in an increase in the reticulocyte production index to at least three times normal (Chap. 129), provided sufficient iron is available. Hemorrhagic anemia does not

1	secondary to hemolysis results in an increase in the reticulocyte production index to at least three times normal (Chap. 129), provided sufficient iron is available. Hemorrhagic anemia does not typically result in production indices of more than 2.0–2.5 times normal because of the limitations placed on expansion of the erythroid marrow by iron availability.

1	In the first branch point of the classification of anemia, a reticulocyte production index >2.5 indicates that hemolysis is most likely. A reticulocyte production index <2 indicates either a hypoproliferative anemia or maturation disorder. The latter two possibilities can often be distinguished by the red cell indices, by examination of the peripheral blood smear, or by a marrow examination. If the red cell indices are normal, the anemia is almost certainly hypoproliferative in nature. Maturation disorders are characterized by ineffective red cell production and a low reticulocyte production index. Bizarre red cell shapes—macrocytes or hypochromic microcytes—are seen on the peripheral blood smear. With a hypoproliferative anemia, no erythroid

1	Hemolysis/ hemorrhage Blood loss Intravascular hemolysis Metabolic defect Membrane abnormality Hemoglobinopathy Immune destruction Fragmentation Hypoproliferative Marrow damage • Infiltration/fibrosis • Aplasia Iron deficiency Stimulation • Inflammation Maturation disorder Cytoplasmic defects • Iron deficiency • Thalassemia • Sideroblastic anemia Nuclear defects Normocytic normochromic Micro or macrocytic Red cell morphology Index < 2.5 Index ˜ 2.5 Anemia CBC, reticulocyte count FIguRE 77-17 The physiologic classification of anemia. CBC, complete blood count. hyperplasia is noted in the marrow, whereas patients with ineffective red cell production have erythroid hyperplasia and an M/E ratio <1:1.

1	Hypoproliferative Anemias At least 75% of all cases of anemia are hypoproliferative in nature. A hypoproliferative anemia reflects absolute or relative marrow failure in which the erythroid marrow has not proliferated appropriately for the degree of anemia. The majority of hypoproliferative anemias are due to mild to moderate iron deficiency or inflammation. A hypoproliferative anemia can result from marrow damage, iron deficiency, or inadequate EPO stimulation. The last may reflect impaired renal function, suppression of EPO production by inflammatory cytokines such as interleukin 1, or reduced tissue needs for O2 from metabolic disease such as hypothyroidism. Only occasionally is the marrow unable to produce red cells at a normal rate, and this is most prevalent in patients with renal failure. With diabetes mellitus or myeloma, the EPO deficiency may be more marked than would be predicted by the degree of renal insufficiency. In general, hypoproliferative anemias are characterized

1	failure. With diabetes mellitus or myeloma, the EPO deficiency may be more marked than would be predicted by the degree of renal insufficiency. In general, hypoproliferative anemias are characterized by normocytic, normochromic red cells, although microcytic, hypochromic cells may be observed with mild iron deficiency or long-standing chronic inflammatory disease. The key laboratory tests in distinguishing between the various forms of hypoproliferative anemia include the serum iron and iron-binding capacity, evaluation of renal and thyroid function, a marrow biopsy or aspirate to detect marrow damage or infiltrative disease, and serum ferritin to assess iron stores. An iron stain of the marrow will determine the pattern of iron distribution. Patients with the anemia of acute or chronic inflammation show a distinctive pattern of serum iron (low), TIBC (normal or low), percent transferrin saturation (low), and serum ferritin (normal or high). These changes in iron values are brought

1	inflammation show a distinctive pattern of serum iron (low), TIBC (normal or low), percent transferrin saturation (low), and serum ferritin (normal or high). These changes in iron values are brought about by hepcidin, the iron regulatory hormone that is produced by the liver and is increased in inflammation (Chap. 126). A distinct pattern of results is noted in mild to moderate iron deficiency (low serum iron, high TIBC, low percent transferrin saturation, low serum ferritin) (Chap. 126). Marrow damage by drugs, infiltrative disease such as leukemia or lymphoma, or marrow aplasia is diagnosed from the peripheral blood and bone marrow morphology. With infiltrative disease or fibrosis, a marrow biopsy is required.

1	Maturation Disorders The presence of anemia with an inappropriately low reticulocyte production index, macroor microcytosis on smear, and abnormal red cell indices suggests a maturation disorder. Maturation disorders are divided into two categories: nuclear maturation defects, associated with macrocytosis, and cytoplasmic maturation defects, associated with microcytosis and hypochromia usually from defects in hemoglobin synthesis. The inappropriately low reticulocyte production index is a reflection of the ineffective erythropoiesis that results from the destruction within the marrow of developing erythroblasts. Bone marrow examination shows erythroid hyperplasia.

1	Nuclear maturation defects result from vitamin B12 or folic acid deficiency, drug damage, or myelodysplasia. Drugs that interfere with cellular DNA synthesis, such as methotrexate or alkylating agents, can produce a nuclear maturation defect. Alcohol, alone, is also capable of producing macrocytosis and a variable degree of anemia, but this is usually associated with folic acid deficiency. Measurements of folic acid and vitamin B12 are critical not only in identifying the specific vitamin deficiency but also because they reflect different pathogenetic mechanisms (Chap. 128).

1	Cytoplasmic maturation defects result from severe iron deficiency or abnormalities in globin or heme synthesis. Iron deficiency occupies an unusual position in the classification of anemia. If the iron-deficiency anemia is mild to moderate, erythroid marrow proliferation is blunted and the anemia is classified as hypoproliferative. However, if the anemia is severe and prolonged, the erythroid marrow will become hyperplastic despite the inadequate iron supply, and the anemia will be classified as ineffective erythropoiesis with a cytoplasmic maturation defect. In either case, an inappropriately low reticulocyte production index, microcytosis, and a classic pattern of iron values make the diagnosis clear and easily distinguish iron deficiency from other cytoplasmic maturation defects such as the thalassemias. Defects in heme synthesis, in contrast to globin synthesis, are less common and may be acquired or inherited (Chap. 430). Acquired abnormalities are usually associated with

1	such as the thalassemias. Defects in heme synthesis, in contrast to globin synthesis, are less common and may be acquired or inherited (Chap. 430). Acquired abnormalities are usually associated with myelodysplasia, may lead to either a macroor microcytic anemia, and are frequently associated with mitochondrial iron loading. In these cases, iron is taken up by the mitochondria of the developing erythroid cell but not incorporated into heme. The iron-encrusted mitochondria surround the nucleus of the erythroid cell, forming a ring. Based on the distinctive finding of so-called ringed sideroblasts on the marrow iron stain, patients are diagnosed as having a sideroblastic anemia—almost always reflecting myelodysplasia. Again, studies of iron parameters are helpful in the differential diagnosis of these patients.

1	Blood Loss/Hemolytic Anemia In contrast to anemias associated with an inappropriately low reticulocyte production index, hemolysis is associated with red cell production indices ≥2.5 times normal. The stimulated erythropoiesis is reflected in the blood smear by the appearance of increased numbers of polychromatophilic macrocytes. A marrow examination is rarely indicated if the reticulocyte production index is increased appropriately. The red cell indices are typically normocytic or slightly macrocytic, reflecting the increased number of reticulocytes. Acute blood loss is not associated with an increased reticulocyte production index because of the time required to increase EPO production and, subsequently, marrow proliferation. Subacute blood loss may be associated with modest reticulocytosis. Anemia from chronic blood loss presents more often as iron deficiency than with the picture of increased red cell production.

1	The evaluation of blood loss anemia is usually not difficult. Most problems arise when a patient presents with an increased red cell production index from an episode of acute blood loss that went unrecognized. The cause of the anemia and increased red cell production may not be obvious. The confirmation of a recovering state may require observations over a period of 2–3 weeks, during which the hemoglobin concentration will rise and the reticulocyte production index fall (Chap. 129).

1	Hemolytic disease, while dramatic, is among the least common forms of anemia. The ability to sustain a high reticulocyte production index reflects the ability of the erythroid marrow to compensate for hemolysis and, in the case of extravascular hemolysis, the efficient recycling of iron from the destroyed red cells to support red cell production. With intravascular hemolysis, such as paroxysmal nocturnal hemoglobinuria, the loss of iron may limit the marrow response. The level of response depends on the severity of the anemia and the nature of the underlying disease process. Hemoglobinopathies, such as sickle cell disease and the thalassemias, present a mixed picture. The reticulocyte index may be high but is inappropriately low for the degree of marrow erythroid hyperplasia (Chap. 127).

1	Hemolytic anemias present in different ways. Some appear suddenly as an acute, self-limited episode of intravascular or extravascular hemolysis, a presentation pattern often seen in patients with autoimmune hemolysis or with inherited defects of the Embden-Meyerhof pathway or the glutathione reductase pathway. Patients with inherited disorders of the hemoglobin molecule or red cell membrane generally have a lifelong clinical history typical of the disease process. Those with chronic hemolytic disease, such as hereditary spherocytosis, may actually present not with anemia but with a complication stemming from the prolonged increase in red cell destruction such as symptomatic bilirubin gallstones or splenomegaly. Patients with chronic hemolysis are also susceptible to aplastic crises if an infectious process interrupts red cell production.

1	The differential diagnosis of an acute or chronic hemolytic event requires the careful integration of family history, the pattern of clinical presentation, and—whether the disease is congenital or acquired— careful examination of the peripheral blood smear. Precise diagnosis may require more specialized laboratory tests, such as hemoglobin electrophoresis or a screen for red cell enzymes. Acquired defects in red cell survival are often immunologically mediated and require a direct or indirect antiglobulin test or a cold agglutinin titer to detect the presence of hemolytic antibodies or complement-mediated red cell destruction (Chap. 129).

1	An overriding principle is to initiate treatment of mild to moderate anemia only when a specific diagnosis is made. Rarely, in the acute setting, anemia may be so severe that red cell transfusions are required before a specific diagnosis is available. Whether the anemia is of acute or gradual onset, the selection of the appropriate treatment is determined by the documented cause(s) of the anemia. Often, the cause of the anemia is multifactorial. For example, a patient with severe rheumatoid arthritis who has been taking anti-inflammatory drugs may have a hypoproliferative anemia associated with chronic inflammation as well as chronic blood loss associated with intermittent gastrointestinal bleeding. In every circumstance, it is important to evaluate the patient’s iron status fully before and during the treatment of any anemia. Transfusion is discussed in Chap. 138e; iron therapy is discussed in Chap. 126; treatment of megaloblastic anemia is discussed in Chap. 128; treatment of other

1	and during the treatment of any anemia. Transfusion is discussed in Chap. 138e; iron therapy is discussed in Chap. 126; treatment of megaloblastic anemia is discussed in Chap. 128; treatment of other entities is discussed in their respective chapters (sickle cell anemia, Chap. 127; hemolytic anemias, Chap. 129; aplastic anemia and myelodysplasia, Chap. 130).

1	Therapeutic options for the treatment of anemias have expanded dramatically during the past 30 years. Blood component therapy is available and safe. Recombinant EPO as an adjunct to anemia management has transformed the lives of patients with chronic renal failure on dialysis and reduced transfusion needs of anemic cancer patients receiving chemotherapy. Eventually, patients with inherited disorders of globin synthesis or mutations in the globin gene, such as sickle cell disease, may benefit from the successful introduction of targeted genetic therapy (Chap. 91e).

1	Polycythemia is defined as an increase in the hemoglobin above normal. This increase may be real or only apparent because of a decrease in plasma volume (spurious or relative polycythemia). The term erythrocytosis may be used interchangeably with polycythemia, but some draw a distinction between them: erythrocytosis implies documentation of increased red cell mass, whereas polycythemia refers to any increase in red cells. Often patients with polycythemia are detected through an incidental finding of elevated hemoglobin or hematocrit levels. Concern that the hemoglobin level may be abnormally high is usually triggered at 170 g/L (17 g/dL) for men and 150 g/L (15 g/dL) for women. Hematocrit levels >50% in men or >45% in women may be abnormal. Hematocrits >60% in men and >55% in women are almost invariably associated with an increased red cell mass. Given that the machine that quantitates red cell parameters actually measures hemoglobin concentrations and calculates hematocrits,

1	are almost invariably associated with an increased red cell mass. Given that the machine that quantitates red cell parameters actually measures hemoglobin concentrations and calculates hematocrits, hemoglobin levels may be a better index.

1	Features of the clinical history that are useful in the differential diagnosis include smoking history; current living at high altitude; or a history of congenital heart disease, sleep apnea, or chronic lung disease.

1	Patients with polycythemia may be asymptomatic or experience symptoms related to the increased red cell mass or the underlying disease process that leads to the increased red cell mass. The dominant symptoms from an increased red cell mass are related to hyperviscosity and thrombosis (both venous and arterial), because the blood viscosity increases logarithmically at hematocrits >55%. Manifestations range from digital ischemia to Budd-Chiari syndrome with hepatic vein thrombosis. Abdominal vessel thromboses are particularly common. Neurologic symptoms such as vertigo, tinnitus, headache, and visual disturbances may occur. Hypertension is often present. Patients with polycythemia vera may have aquagenic pruritus and symptoms related to hepatosplenomegaly. Patients may have easy bruising, epistaxis, or bleeding from the gastrointestinal tract. Peptic ulcer disease is common. Patients with hypoxemia may develop cyanosis on minimal exertion or have headache, impaired mental acuity, and

1	epistaxis, or bleeding from the gastrointestinal tract. Peptic ulcer disease is common. Patients with hypoxemia may develop cyanosis on minimal exertion or have headache, impaired mental acuity, and fatigue.

1	The physical examination usually reveals a ruddy complexion. Splenomegaly favors polycythemia vera as the diagnosis (Chap. 131).

1	400 The presence of cyanosis or evidence of a right-to-left shunt suggests congenital heart disease presenting in the adult, particularly tetralogy of Fallot or Eisenmenger’s syndrome (Chap. 236). Increased blood viscosity raises pulmonary artery pressure; hypoxemia can lead to increased pulmonary vascular resistance. Together, these factors can produce cor pulmonale. Polycythemia can be spurious (related to a decrease in plasma volume; Gaisbock’s syndrome), primary, or secondary in origin. The secondary causes are all associated with increases in EPO levels: either a physiologically adapted appropriate elevation based on tissue hypoxia (lung disease, high altitude, CO poisoning, high-affinity hemoglobinopathy) or an abnormal overproduction (renal cysts, renal artery stenosis, tumors with ectopic EPO production). A rare familial form of polycythemia is associated with normal EPO levels but hyper-responsive EPO receptors due to mutations. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: PART 2 Cardinal Manifestations and Presentation of Diseases As shown in Fig. 77-18, the first step is to document the presence of an increased red cell mass using the principle of isotope dilution by administering 51Cr-labeled autologous red blood cells to the patient and sampling blood radioactivity over a 2-h period. If the red cell mass is normal (<36 mL/kg in men, <32 mL/kg in women), the patient has spurious or relative polycythemia. If the red cell mass is increased (>36 mL/kg in men, >32 mL/kg in women), serum EPO levels should be measured. If EPO levels are low or unmeasurable, the patient most likely has polycythemia vera. A mutation in JAK2 (Val617Phe), a key member of the cytokine intracellular signaling pathway, can be found in 90–95% of patients with polycythemia vera. Many of those without this particular JAK2 mutation have mutations in exon 12. As a practical matter, few centers assess red

1	Dx: Relative erythrocytosis Measure RBC mass Measure serum EPO levels Measure arterial O2 saturation elevated elevated Dx: O2 affinity hemoglobinopathy increased elevated normal Dx: Polycythemia vera Confirm JAK2mutation smoker? normal normal Dx: Smoker’s polycythemia normal Increased hct or hgb low low Diagnostic evaluation for heart or lung disease, e.g., COPD, high altitude, AV or intracardiac shunt Measure hemoglobin O2 affinity Measure carboxyhemoglobin levels Search for tumor as source of EPO IVP/renal ultrasound (renal Ca or cyst) CT of head (cerebellar hemangioma) CT of pelvis (uterine leiomyoma) CT of abdomen (hepatoma) no yes FIguRE 77-18 An approach to the differential diagnosis of patients with an elevated hemoglobin (possible polycythemia). AV, atrioventricular; COPD, chronic obstructive pulmonary disease; CT, computed tomography; EPO, erythropoietin; hct, hematocrit; hgb, hemoglobin; IVP, intravenous pyelogram; RBC, red blood cell.

1	AV, atrioventricular; COPD, chronic obstructive pulmonary disease; CT, computed tomography; EPO, erythropoietin; hct, hematocrit; hgb, hemoglobin; IVP, intravenous pyelogram; RBC, red blood cell. cell mass in the setting of an increased hematocrit. The short workup is to measure EPO levels, check for JAK2 mutation, and perform an abdominal ultrasound to assess spleen size. Tests that support the diagnosis of polycythemia vera include elevated white blood cell count, increased absolute basophil count, and thrombocytosis.

1	If serum EPO levels are elevated, one needs to distinguish whether the elevation is a physiologic response to hypoxia or related to autonomous EPO production. Patients with low arterial O2 saturation (<92%) should be further evaluated for the presence of heart or lung disease, if they are not living at high altitude. Patients with normal O2 saturation who are smokers may have elevated EPO levels because of CO displacement of O2. If carboxyhemoglobin (COHb) levels are high, the diagnosis is “smoker’s polycythemia.” Such patients should be urged to stop smoking. Those who cannot stop smoking require phlebotomy to control their polycythemia. Patients with normal O2 saturation who do not smoke either have an abnormal hemoglobin that does not deliver O2 to the tissues (evaluated by finding elevated O2–hemoglobin affinity) or have a source of EPO production that is not responding to the normal feedback inhibition. Further workup is dictated by the differential diagnosis of EPO-producing

1	elevated O2–hemoglobin affinity) or have a source of EPO production that is not responding to the normal feedback inhibition. Further workup is dictated by the differential diagnosis of EPO-producing neoplasms. Hepatoma, uterine leiomyoma, and renal cancer or cysts are all detectable with abdominopelvic computed tomography scans. Cerebellar hemangiomas may produce EPO, but they present with localizing neurologic signs and symptoms rather than polycythemia-related symptoms.

1	Barbara A. Konkle The human hemostatic system provides a natural balance between procoagulant and anticoagulant forces. The procoagulant forces include platelet adhesion and aggregation and fibrin clot formation; anticoagulant forces include the natural inhibitors of coagulation and fibrinolysis. Under normal circumstances, hemostasis is regulated to promote blood flow; however, it is also prepared to clot blood rapidly to arrest blood flow and prevent exsanguination. After bleeding is successfully halted, the system remodels the damaged vessel to restore normal blood flow. The major components of the hemostatic system, which function in concert, are (1) platelets and other formed elements of blood, such as monocytes and red cells; (2) plasma proteins (the coagulation and fibrinolytic factors and inhibitors); and (3) the vessel wall.

1	On vascular injury, platelets adhere to the site of injury, usually the denuded vascular intimal surface. Platelet adhesion is mediated primarily by Von Willebrand factor (VWF), a large multimeric protein present in both plasma and the extracellular matrix of the subendothelial vessel wall, which serves as the primary “molecular glue,” providing sufficient strength to withstand the high levels of shear stress that would tend to detach them with the flow of blood. Platelet adhesion is also facilitated by direct binding to subendothelial collagen through specific platelet membrane collagen receptors.

1	Platelet adhesion results in subsequent platelet activation and aggregation. This process is enhanced and amplified by humoral mediators in plasma (e.g., epinephrine, thrombin); mediators released from activated platelets (e.g., adenosine diphosphate, serotonin); and vessel wall extracellular matrix constituents that come in contact with adherent platelets (e.g., collagen, VWF). Activated platelets undergo the release reaction, during which they secrete contents that further promote aggregation and inhibit the naturally anticoagulant endothelial cell factors. During platelet aggregation (platelet-platelet interaction), additional platelets are recruited from the circulation to the site of vascular injury, leading to the formation of an occlusive platelet thrombus. The platelet plug is anchored and stabilized by the developing fibrin mesh.

1	The platelet glycoprotein (Gp) IIb/IIIa (αIIbβ3) complex is the most abundant receptor on the platelet surface. Platelet activation converts the normally inactive Gp IIb/IIIa receptor into an active receptor, enabling binding to fibrinogen and VWF. Because the surface of each platelet has about 50,000 Gp IIb/IIIa–binding sites, numerous activated platelets recruited to the site of vascular injury can rapidly form an occlusive aggregate by means of a dense network of intercellular fibrinogen bridges. Because this receptor is the key mediator of platelet aggregation, it has become an effective target for antiplatelet therapy.

1	Plasma coagulation proteins (clotting factors) normally circulate in plasma in their inactive forms. The sequence of coagulation protein reactions that culminate in the formation of fibrin was originally described as a waterfall or a cascade. Two pathways of blood coagulation have been described in the past: the so-called extrinsic, or tissue factor, pathway and the so-called intrinsic, or contact activation, pathway. We now know that coagulation is normally initiated through tissue factor (TF) exposure and activation through the classic extrinsic pathway but with critically important amplification through elements of the classic intrinsic pathway, as illustrated in Fig. 78-1. These reactions take place on phospholipid surfaces, usually the activated platelet surface. Coagulation testing in the laboratory can reflect other influences due to the artificial nature of the in vitro systems used (see below).

1	The immediate trigger for coagulation is vascular damage that exposes blood to TF that is constitutively expressed on the surfaces of subendothelial cellular components of the vessel wall, such as smooth muscle cells and fibroblasts. TF is also present in circulating microparticles, presumably shed from cells including monocytes and platelets. TF binds the serine protease factor VIIa; the complex activates factor X to factor Xa. Alternatively, the complex can indirectly activate factor X by initially converting factor IX to factor IXa, which then activates factor X. The participation of factor XI in hemostasis is not dependent on its activation by factor XIIa but rather on its positive feedback acti-

1	FIguRE 78-2 Fibrin formation and dissolution. (A) Fibrinogen is a trinodular structure consisting of two D domains and one E domain. Thrombin activation results in an ordered lateral assembly of protofibrils (B) with noncovalent associations. Factor XIIIa cross-links the D domains on adjacent molecules (C). Fibrin and fibrinogen (not shown) lysis by plasmin occurs at discrete sites and results in intermediary fibrin(ogen) degradation products (not shown). D-Dimers are the product of complete lysis of fibrin (D), maintaining the cross-linked D domains. vation by thrombin. Thus, factor XIa functions in the propagation and amplification, rather than in the initiation, of the coagulation cascade. Factor Xa can be formed through the actions of either the TF/ factor VIIa complex or factor IXa (with factor VIIIa as a cofactor) and converts prothrombin to thrombin, the pivotal protease of the coagulation system. The essential cofactor for this reaction is factor

1	Va. Like the homologous factor VIIIa, factor Va is produced by thrombininduced limited proteolysis of factor V. Thrombin is a multifunctional enzyme that converts soluble plasma fibrinogen to an insoluble fibrin matrix. Fibrin polymerization involves an orderly process of intermolecular associations (Fig. 78-2). Thrombin also activates factor XIII (fibrin-stabilizing factor) to factor XIIIa, which covalently cross-links and thereby stabilizes the fibrin clot.

1	The assembly of the clotting factors on activated cell membrane surfaces greatly accelerates their reaction rates and also serves to localize blood clotting to sites of vascular injury. The critical cell membrane components, acidic phospholipids, are not normally exposed on resting cell membrane surfaces. However, when Vessel injury TFPI IX IX XI VIIIa IXa (Prothrombin) Thrombin (IIa) Fibrinogen Fibrin XIaX Va Xa II X VIIa platelets, monocytes, and endothelial cells are activated by vascular injury or FIguRE 78-1 Coagulation is initiated by tissue factor (TF) exposure, which, with factor (F) VIIa, inflammatory stimuli, the procoagulant activates FIX and FX, which in turn, with FVIII and FV as cofactors, respectively, results in thrombin head groups of the membrane anionic formation and subsequent conversion of fibrinogen to fibrin. Thrombin activates FXI, FVIII, and FV, phospholipids become translocated to amplifying the coagulation signal. Once the TF/FVIIa/FXa complex is formed,

1	subsequent conversion of fibrinogen to fibrin. Thrombin activates FXI, FVIII, and FV, phospholipids become translocated to amplifying the coagulation signal. Once the TF/FVIIa/FXa complex is formed, tissue factor pathway the surfaces of these cells or released inhibitor (TFPI) inhibits the TF/FVIIa pathway, making coagulation dependent on the amplification as part of microparticles, making them loop through FIX/FVIII. Coagulation requires calcium (not shown) and takes place on phospholipid available to support and promote the surfaces, usually the activated platelet membrane. plasma coagulation reactions.

1	Several physiologic antithrombotic mechanisms act in concert to prevent clotting under normal circumstances. These mechanisms operate to preserve blood fluidity and to limit blood clotting to specific focal sites of vascular injury. Endothelial cells have many antithrombotic effects. They produce prostacyclin, nitric oxide, and ectoADPase/ CD39, which act to inhibit platelet binding, secretion, and aggregation. Endothelial cells produce anticoagulant factors including heparan proteoglycans, antithrombin, TF pathway inhibitor, and thrombomodulin. They also activate fibrinolytic mechanisms through the production of tissue plasminogen activator 1, urokinase, plasminogen activator inhibitor, and annexin-2. The sites of action of the major physiologic antithrombotic pathways are shown in Fig. 78-3.

1	Antithrombin (or antithrombin III) is the major plasma protease inhibitor of thrombin and the other clotting factors in coagulation. Antithrombin neutralizes thrombin and other activated coagulation factors by forming a complex between the active site of the enzyme and the reactive center of antithrombin. The rate of formation of these inactivating complexes increases by a factor of several thousand in the presence of heparin. Antithrombin inactivation of thrombin and other activated clotting factors occurs physiologically on vascular surfaces, where glycosoaminoglycans, including heparan sulfates, are present to catalyze these reactions. Inherited quantitative or qualitative deficiencies of antithrombin lead to a lifelong predisposition to venous thromboembolism.

1	Protein C is a plasma glycoprotein that becomes an anticoagulant when it is activated by thrombin. The thrombin-induced activation of protein C occurs physiologically on thrombomodulin, a transmembrane proteoglycan-binding site for thrombin on endothelial cell surfaces. The binding of protein C to its receptor on endothelial cells places it in PART 2 Cardinal Manifestations and Presentation of Diseases FIguRE 78-3 Sites of action of the four major physiologic anti-thrombotic pathways: antithrombin (AT); protein C/S (PC/PS); tissue factor pathway inhibitor (TFPI); and the fibrinolytic system, consisting of plasminogen, plasminogen activator (PA), and plasmin. PT, prothrombin; Th, thrombin; FDP, fibrin(ogen) degradation products.

1	(Modified from BA Konkle, AI Schafer, in DP Zipes et al [eds]: Braunwald’s Heart Disease, 7th ed. Philadelphia, Saunders, 2005.) proximity to the thrombin-thrombomodulin complex, thereby enhancing its activation efficiency. Activated protein C acts as an anticoagulant by cleaving and inactivating activated factors V and VIII. This reaction is accelerated by a cofactor, protein S, which, like protein C, is a glycoprotein that undergoes vitamin K–dependent posttranslational modification. Quantitative or qualitative deficiencies of protein C or protein S, or resistance to the action of activated protein C by a specific mutation at its target cleavage site in factor Va (factor V Leiden), lead to hypercoagulable states.

1	Tissue factor pathway inhibitor (TFPI) is a plasma protease inhibitor that regulates the TF-induced extrinsic pathway of coagulation. TFPI inhibits the TF/factor VIIa/factor Xa complex, essentially turning off the TF/factor VIIa initiation of coagulation, which then becomes dependent on the “amplification loop” via factor XI and factor VIII activation by thrombin. TFPI is bound to lipoprotein and can also be released by heparin from endothelial cells, where it is bound to glycosaminoglycans, and from platelets. The heparin-mediated release of TFPI may play a role in the anticoagulant effects of unfractionated and low-molecular-weight heparins.

1	Any thrombin that escapes the inhibitory effects of the physiologic anticoagulant systems is available to convert fibrinogen to fibrin. In response, the endogenous fibrinolytic system is then activated to dispose of intravascular fibrin and thereby maintain or reestablish the patency of the circulation. Just as thrombin is the key protease enzyme of the coagulation system, plasmin is the major protease enzyme of the fibrinolytic system, acting to digest fibrin to fibrin degradation products. The general scheme of fibrinolysis and its control is shown in Fig. 78-4.

1	The plasminogen activators, tissue type plasminogen activator (tPA) and the urokinase-type plasminogen activator (uPA), cleave the Arg560-Val561 bond of plasminogen to generate the active enzyme plasmin. The lysine-binding sites of plasmin (and plasminogen) permit it to bind to fibrin, so that physiologic fibrinolysis is “fibrin specific.” Both plasminogen (through its lysine-binding sites) and tPA possess specific affinity for fibrin and thereby bind selectively to clots. The assembly of a ternary complex, consisting of fibrin, plasminogen, and tPA, promotes the localized interaction between plasminogen and tPA and greatly accelerates the rate of plasminogen activation to plasmin. Moreover, partial degradation of fibrin by plasmin exposes new plasminogen and tPA-binding sites in carboxy-terminus lysine FIguRE 78-4 A schematic diagram of the fibrinolytic system.

1	FIguRE 78-4 A schematic diagram of the fibrinolytic system. Tissue plasminogen activator (tPA) is released from endothelial cells, binds the fibrin clot, and activates plasminogen to plasmin. Excess fibrin is degraded by plasmin to distinct degradation products (FDPs). Any free plasmin is complexed with α2-antiplasmin (α2Pl). PAI, plasminogen activator inhibitor; UPA, urokinase-type plasminogen activator. residues of fibrin fragments to enhance these reactions further. This creates a highly efficient mechanism to generate plasmin focally on the fibrin clot, which then becomes plasmin’s substrate for digestion to fibrin degradation products.

1	Plasmin cleaves fibrin at distinct sites of the fibrin molecule, leading to the generation of characteristic fibrin fragments during the process of fibrinolysis (Fig. 78-2). The sites of plasmin cleavage of fibrin are the same as those in fibrinogen. However, when plasmin acts on covalently cross-linked fibrin, d-dimers are released; hence, d-dimers can be measured in plasma as a relatively specific test of fibrin (rather than fibrinogen) degradation. d-Dimer assays can be used as sensitive markers of blood clot formation and have been validated for clinical use to exclude the diagnosis of deep venous thrombosis (DVT) and pulmonary embolism in selected populations. In addition, d-dimer measurement can be used to stratify patients, particularly women, for risk of recurrent venous thromboembolism (VTE) when measured 1 month after discontinuation of anticoagulation given for treatment of an initial idiopathic event. d-Dimer levels may be elevated in the absence of VTE in elderly people.

1	Physiologic regulation of fibrinolysis occurs primarily at three levels: (1) plasminogen activator inhibitors (PAIs), specifically PAI-1 and PAI-2, inhibit the physiologic plasminogen activators; (2) the thrombin-activatable fibrinolysis inhibitor (TAFI) limits fibrinolysis; and (3) α2-antiplasmin inhibits plasmin. PAI-1 is the primary inhibitor of tPA and uPA in plasma. TAFI cleaves the N-terminal lysine residues of fibrin, which aid in localization of plasmin activity. α2-Antiplasmin is the main inhibitor of plasmin in human plasma, inactivating any nonfibrin clot-associated plasmin. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: Disorders of hemostasis may be either inherited or acquired. A detailed personal and family history is key in determining the chronicity of symptoms and the likelihood of the disorder being inherited, as well as providing clues to underlying conditions that have contributed to the bleeding or thrombotic state. In addition, the history can give clues as to the etiology by determining (1) the bleeding (mucosal and/or joint) or thrombosis (arterial and/ or venous) site and (2) whether an underlying bleeding or clotting tendency was enhanced by another medical condition or the introduction of medications or dietary supplements.

1	History of Bleeding A history of bleeding is the most important predictor of bleeding risk. In evaluating a patient for a bleeding disorder, a history of at-risk situations, including the response to past surgeries, should be assessed. Does the patient have a history of spontaneous or trauma/surgery-induced bleeding? Spontaneous hemarthroses are a hallmark of moderate and severe factor VIII and IX deficiency and, in rare circumstances, of other clotting factor deficiencies. Mucosal bleeding symptoms are more suggestive of underlying platelet disorders or Von Willebrand disease (VWD), termed disorders of primary hemostasis or platelet plug formation. Disorders affecting primary hemostasis are shown in Table 78-1.

1	A bleeding score has been validated as a tool to predict patients more likely to have type 1 VWD (International Society on Thrombosis and Haemostasis Bleeding Assessment Tool [www.isth .org/resource/resmgr/ssc/isth-ssc_bleeding_assessment.pdf ]). This is most useful tool in excluding the diagnosis of a bleeding disorder, and thus avoiding unnecessary testing. One study found that a low bleeding score (≤3) and a normal activated partial thromboplastin time (aPTT) had 99.6% negative predictive value for the diagnosis of VWD. Bleeding symptoms that appear to be more common in patients with bleeding disorders include prolonged bleeding with surgery, dental procedures and extractions, and/or trauma, menorrhagia or postpartum hemorrhage, and large bruises (often described with lumps). Defects of Platelet Aggregation Glanzmann’s thrombasthenia (absence or dysfunction of platelet glycoprotein [Gp] IIb/IIIa) Defects of Platelet Secretion

1	Defects of Platelet Aggregation Glanzmann’s thrombasthenia (absence or dysfunction of platelet glycoprotein [Gp] IIb/IIIa) Defects of Platelet Secretion Drug-induced (aspirin, nonsteroidal anti-inflammatory agents, thienopyridines) Inherited Nonspecific inherited secretory defects Nonspecific drug effects Uremia Platelet coating (e.g., paraprotein, penicillin) Defect of Platelet Coagulant Activity

1	Nonspecific inherited secretory defects Nonspecific drug effects Uremia Platelet coating (e.g., paraprotein, penicillin) Defect of Platelet Coagulant Activity Easy bruising and menorrhagia are common complaints in patients with and without bleeding disorders. Easy bruising can also be a sign of medical conditions in which there is no identifiable coagulopathy; instead, the conditions are caused by an abnormality of blood vessels or their supporting tissues. In Ehlers-Danlos syndrome, there may be posttraumatic bleeding and a history of joint hyperextensibility. Cushing’s syndrome, chronic steroid use, and aging result in changes in skin and subcutaneous tissue, and subcutaneous bleeding occurs in response to minor trauma. The latter has been termed senile purpura.

1	Epistaxis is a common symptom, particularly in children and in dry climates, and may not reflect an underlying bleeding disorder. However, it is the most common symptom in hereditary hemorrhagic telangiectasia and in boys with VWD. Clues that epistaxis is a symptom of an underlying bleeding disorder include lack of seasonal variation and bleeding that requires medical evaluation or treatment, including cauterization. Bleeding with eruption of primary teeth is seen in children with more severe bleeding disorders, such as moderate and severe hemophilia. It is uncommon in children with mild bleeding disorders. Patients with disorders of primary hemostasis (platelet adhesion) may have increased bleeding after dental cleanings and other procedures that involve gum manipulation.

1	Menorrhagia is defined quantitatively as a loss of >80 mL of blood per cycle, based on the quantity of blood loss required to produce iron-deficiency anemia. A complaint of heavy menses is subjective and has a poor correlation with excessive blood loss. Predictors of menorrhagia include bleeding resulting in iron-deficiency anemia or a need for blood transfusion, passage of clots >1 inch in diameter, and changing a pad or tampon more than hourly. Menorrhagia is a common symptom in women with underlying bleeding disorders and is reported in the majority of women with VWD, women with factor XI deficiency, and symptomatic carriers of hemophilia. Women with underlying bleeding disorders are more likely to have other bleeding symptoms, including bleeding after dental extractions, postoperative bleeding, and postpartum bleeding, and are much more likely to have menorrhagia beginning at menarche than women with menorrhagia due to other causes.

1	Postpartum hemorrhage (PPH) is a common symptom in women with underlying bleeding disorders. In women with type 1 VWD and symptomatic carriers of hemophilia A in whom levels of VWF and factor VIII usually normalize during pregnancy, PPH may be delayed. Women with a history of PPH have a high risk of recurrence with subsequent pregnancies. Rupture of ovarian cysts with intraabdominal hemorrhage has also been reported in women with underlying bleeding disorders.

1	Tonsillectomy is a major hemostatic challenge, because intact hemostatic mechanisms are essential to prevent excessive bleeding from the tonsillar bed. Bleeding may occur early after surgery or after approximately 7 days postoperatively, with loss of the eschar at the operative site. Similar delayed bleeding is seen after colonic polyp resection. Gastrointestinal (GI) bleeding and hematuria are usually due to underlying pathology, and procedures to identify and treat the bleeding site should be undertaken, even in patients with known bleeding disorders. VWD, particularly types 2 and 3, has been associated with angiodysplasia of the bowel and GI bleeding.

1	Hemarthroses and spontaneous muscle hematomas are characteristic of moderate or severe congenital factor VIII or IX deficiency. They can also be seen in moderate and severe deficiencies of fibrinogen, prothrombin, and factors V, VII, and X. Spontaneous hemarthroses occur rarely in other bleeding disorders except for severe VWD, with associated factor VIII levels <5%. Muscle and soft tissue bleeds are also common in acquired factor VIII deficiency. Bleeding into a joint results in severe pain and swelling, as well as loss of function, but is rarely associated with discoloration from bruising around the joint. Life-threatening sites of bleeding include bleeding into the oropharynx, where bleeding can obstruct the airway, into the central nervous system, and into the retroperitoneum. Central nervous system bleeding is the major cause of bleeding-related deaths in patients with severe congenital factor deficiencies. Prohemorrhagic Effects of Medications and Dietary Supplements

1	Prohemorrhagic Effects of Medications and Dietary Supplements Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit cyclooxygenase 1 impair primary hemostasis and may exacerbate bleeding from another cause or even unmask a previously occult mild bleeding disorder such as VWD. All NSAIDs, however, can precipitate GI bleeding, which may be more severe in patients with underlying bleeding disorders. The aspirin effect on platelet function as assessed by aggregometry can persist for up to 7 days, although it has frequently returned to normal by 3 days after the last dose. The effect of other NSAIDs is shorter, as the inhibitor effect is reversed when the drug is removed. Thienopyridines (clopidogrel and prasugrel) inhibit ADP-mediated platelet aggregation and, like NSAIDs, can precipitate or exacerbate bleeding symptoms.

1	Many herbal supplements can impair hemostatic function (Table 78-2). Some are more convincingly associated with a bleeding risk than others. Fish oil or concentrated omega-3 fatty acid supplements impair platelet function. They alter platelet biochemistry to produce more PGI3, a more potent platelet inhibitor than prostacyclin (PGI2), and more thromboxane A3, a less potent platelet activator than thromboxane A2. In fact, diets naturally rich in omega-3 fatty acids can result in a prolonged bleeding time and abnormal platelet aggregation studies, but the actual associated bleeding risk is unclear. Vitamin E appears to inhibit protein kinase C–mediated platelet aggregation and nitric oxide production. In patients with unexplained bruising or bleeding, it is prudent to review any new medications or supplements and discontinue those that may be associated with bleeding.

1	underlying Systemic Diseases That Cause or Exacerbate a Bleeding Tendency Acquired bleeding disorders are commonly secondary to, or associated with, systemic disease. The clinical evaluation of a patient with a bleeding tendency must therefore include a thorough assessment for evidence of underlying disease. Bruising or mucosal bleeding may be the presenting complaint in liver disease, severe renal impairment, hypothyroidism, paraproteinemias or amyloidosis, and conditions causing bone marrow failure. All coagulation factors are synthesized in the liver, and hepatic failure results in combined factor deficiencies. This is often compounded by thrombocytopenia from splenomegaly due to portal PART 2 Cardinal Manifestations and Presentation of Diseases Herbs with Potential Antiplatelet Activity

1	PART 2 Cardinal Manifestations and Presentation of Diseases Herbs with Potential Antiplatelet Activity Ginkgo (Ginkgo biloba L.) Garlic (Allium sativum) Bilberry (Vaccinium myrtillus) Ginger (Gingiber officinale) Dong quai (Angelica sinensis) Feverfew (Tanacetum parthenium) Asian ginseng (Panax ginseng) American ginseng (Panax quinquefolius) Siberian ginseng/eleuthero (Eleutherococcus senticosus) Turmeric (Circuma longa) Meadowsweet (Filipendula ulmaria) Willow (Salix spp.) Chamomile (Matricaria recutita, Chamaemelum mobile) hypertension. Coagulation factors II, VII, IX, and X and proteins C, S, and Z are dependent on vitamin K for posttranslational modi fication. Although vitamin K is required in both procoagulant and anticoagulant processes, the phenotype of vitamin K deficiency or the warfarin effect on coagulation is bleeding. The normal blood platelet count is 150,000–450,000/μL.

1	The normal blood platelet count is 150,000–450,000/μL. Thrombocytopenia results from decreased production, increased destruction, and/or sequestration. Although the bleeding risk varies somewhat by the reason for the thrombocytopenia, bleeding rarely occurs in isolated thrombocytopenia at counts <50,000/μL and usually not until <10,000–20,000/μL. Coexisting coagulopathies, as is seen in liver failure or disseminated coagulation; infection; all increase the risk of bleeding in the thrombocytopenic patient. Most procedures can be performed in patients with a platelet count of 50,000/μL. The level needed for major surgery will depend on the type of surgery and the patient’s underlying medical state, although a count of approximately 80,000/μL is likely sufficient.

1	The risk of thrombosis, like that of bleeding, is influenced by both genetic and environmental influences. The major risk factor for arterial thrombosis is atherosclerosis, whereas for venous throm bosis, the risk factors are immobility, surgery, underlying medical conditions such as malignancy, medications such as hormonal therapy, obesity, and genetic predispositions. Factors that increase risks for venous and for both venous and arterial thromboses are shown in Table 78-3.

1	The most important point in a history related to venous throm bosis is determining whether the thrombotic event was idiopathic tated event. In patients without underlying malignancy, having an idiopathic event is the strongest predictor of recurrence of VTE. In patients who have a vague history of thrombosis, a history of being treated with warfarin suggests a past DVT. Age is an important risk factor for venous thrombosis—the risk of DVT increases per decade, with an approximate incidence of 1/100,000 per year in early childhood to 1/200 per year among octogenarians. Family history is helpful in determining if there is a genetic predisposition and how strong that predisposition appears to be. A genetic thrombophilia that confers a relatively small increased risk, such as being a het erozygote for the prothrombin G20210A or factor V Leiden muta tion, may be a minor determinant of risk in an elderly individual

1	Age Previous thrombosis Immobilization Major surgery Pregnancy and puerperium Hospitalization Obesity Infection APC resistance, nongenetic Smoking Elevated factor II, IX, XI Elevated TAFI levels Low levels of TFPI aUnknown whether risk is inherited or acquired. Abbreviations: APC, activated protein C; TAFI, thrombin-activatable fibrinolysis inhibitor; TFPI, tissue factor pathway inhibitor. undergoing a high-risk surgical procedure. As illustrated in Fig. 78-5, a thrombotic event usually has more than one contributing factor. Predisposing factors must be carefully assessed to determine the risk of recurrent thrombosis and, with consideration of the patient’s bleeding risk, determine the length of anticoagulation.

1	FIguRE 78-5 Thrombotic risk over time. Shown schematically is an individual’s thrombotic risk over time. An underlying factor V Leiden mutation provides a “theoretically” constant increased risk. The thrombotic risk increases with age and, intermittently, with oral contraceptive (OCP) or hormone replacement therapy (HRT) use; other events may increase the risk further. At some point, the cumulative risk may increase to the threshold for thrombosis and result in deep venous thrombosis (DVT). Note: The magnitude and duration of risk portrayed in the figure are meant for example only and may not precisely reflect the relative risk determined by clinical study. (From BA Konkle, A Schafer, in DP Zipes et al [eds]: Braunwald’s Heart Disease, 7th ed. Philadelphia, Saunders, 2005; modified with permission from FR Rosendaal: Venous thrombosis: A multicausal disease. Lancet 353:1167, 1999.)

1	Similar consideration should be given in determining the need, if any, to test the patient and family members for thrombophilias. Careful history taking and clinical examination are essential components in the assessment of bleeding and thrombotic risk. The use of laboratory tests of coagulation complement, but cannot substitute for, clinical assessment. No test exists that provides a global assessment of hemostasis. The bleeding time has been used to assess bleeding risk; however, it does not predict bleeding risk with surgery and it is not recommended for this indication. The PFA-100, an instrument that measures platelet-dependent coagulation under flow conditions, is more sensitive and specific for VWD than the bleeding time; however, it is not sensitive enough to rule out mild bleeding disorders. PFA-100 closure times are prolonged in patients with some, but not all, inherited platelet disorders. Also, its utility in predicting bleeding risk has not been determined.

1	For routine preoperative and preprocedure testing, an abnormal prothrombin time (PT) may detect liver disease or vitamin K deficiency that had not been previously appreciated. Studies have not confirmed the usefulness of an aPTT in preoperative evaluations in patients with a negative bleeding history. The primary use of coagulation testing should be to confirm the presence and type of bleeding disorder in a patient with a suspicious clinical history.

1	Because of the nature of coagulation assays, proper sample acquisition and handling is critical to obtaining valid results. In patients with abnormal coagulation assays who have no bleeding history, repeat studies with attention to these factors frequently results in normal values. Most coagulation assays are performed in sodium citrate anticoagulated plasma that is recalcified for the assay. Because the anticoagulant is in liquid solution and needs to be added to blood in proportion to the plasma volume, incorrectly filled or inadequately mixed blood collection tubes will give erroneous results. Vacutainer tubes should be filled to >90% of the recommended fill, which is usually denoted by a line on the tube. An elevated hematocrit (>55%) can result in a false value due to a decreased plasma-to-anticoagulant ratio.

1	Screening Assays The most commonly used screening tests are the PT, aPTT, and platelet count. The PT assesses the factors I (fibrinogen), II (prothrombin), V, VII, and X (Fig. 78-6). The PT measures the time for clot formation of the citrated plasma after recalcification and addition of thromboplastin, a mixture of TF and phospholipids. The sensitivity of the assay varies by the source of thromboplastin. The relationship between defects in secondary hemostasis (fibrin formation) and coagulation test abnormalities is shown in Table 78-4. To adjust for this variability, the overall sensitivity of different thromboplastins to reduction of the vitamin K–dependent clotting factors II, VII, IX, and X in anticoagulation patients is now expressed as the International Sensitivity Index (ISI). An inverse relationship exists between ISI and thromboplastin sensitivity. The international normalized ratio (INR) is then determined based on the formula: INR = (PTpatient/PT )ISI.

1	The INR was developed to assess stable anticoagulation due to reduction of vitamin K–dependent coagulation factors; it is commonly used in the evaluation of patients with liver disease. Although it does allow comparison between laboratories, reagent sensitivity as used to determine the ISI is not the same in liver disease as with warfarin anticoagulation. In addition, progressive liver failure is associated with variable changes in coagulation factors; the degree of prolongation of either the PT or the INR only roughly predicts the bleeding risk. Thrombin generation has been shown to be normal in many patients with mild to moderate liver dysfunction. Because the PT only measures one aspect of hemostasis affected by liver dysfunction, we likely overestimate the bleeding risk of a mildly elevated INR in this setting. The aPTT assesses the intrinsic and common coagulation pathways; factors XI, IX, VIII, X, V, and II; fibrinogen; prekallikrein;

1	The aPTT assesses the intrinsic and common coagulation pathways; factors XI, IX, VIII, X, V, and II; fibrinogen; prekallikrein; HMWK PK FXII FXI FIX FVIII FVII FX FV Prothrombin (FII) Fibrinogen (FI) aPTTPTFIguRE 78–6 Coagulation factor activity tested in the activated partial thromboplastin time (aPTT) in red and prothrombin time (PT) in green, or both. F, factor; HMWK, high-molecular-weight kininogen; PK, prekallikrein. No clinical bleeding—↓ factor XII, high-molecular-weight kininogen, prekallikrein Variable, but usually mild, bleeding—↓ factor XI, mild ↓ factor VIII and factor IX

1	No clinical bleeding—↓ factor XII, high-molecular-weight kininogen, prekallikrein Variable, but usually mild, bleeding—↓ factor XI, mild ↓ factor VIII and factor IX Frequent, severe bleeding—severe deficiencies of factors VIII and IX Heparin and direct thrombin inhibitors high-molecular-weight kininogen; and factor XII (Fig. 78-6). The aPTT reagent contains phospholipids derived from either animal or vegetable sources that function as a platelet substitute in the coagulation pathways and includes an activator of the intrinsic coagulation system, such as nonparticulate ellagic acid or the particulate activators kaolin, celite, or micronized silica.

1	The phospholipid composition of aPTT reagents varies, which influences the sensitivity of individual reagents to clotting factor deficiencies and to inhibitors such as heparin and lupus anticoagulants. Thus, aPTT results will vary from one laboratory to another, and the normal range in the laboratory where the testing occurs should be used in the interpretation. Local laboratories can relate their aPTT values to the therapeutic heparin anticoagulation by correlating aPTT values with direct measurements of heparin activity (anti-Xa or protamine titration assays) in samples from heparinized patients, although correlation between these assays is often poor. The aPTT reagent will vary in sensitivity to individual factor deficiencies and usually becomes prolonged with individual factor deficiencies of 30–50%.

1	Mixing Studies Mixing studies are used to evaluate a prolonged aPTT or, less commonly PT, to distinguish between a factor deficiency and an inhibitor. In this assay, normal plasma and patient plasma are mixed in a 1:1 ratio, and the aPTT or PT is determined immediately and after incubation at 37°C for varying times, typically 30, 60, and/or 120 min. With isolated factor deficiencies, the aPTT will correct with mixing and stay corrected with incubation. With aPTT prolongation due to a lupus anticoagulant, the mixing and incubation will show no correction. In acquired neutralizing factor antibodies, notably an acquired factor VIII inhibitor, the initial assay may or may not correct immediately after mixing but will prolong or remain prolonged with incubation at 37°C. Failure to correct with mixing can also be due to the presence of other inhibitors or interfering substances such as heparin, fibrin split products, and paraproteins.

1	Specific Factor Assays Decisions to proceed with specific clotting factor assays will be influenced by the clinical situation and the results of coagulation screening tests. Precise diagnosis and effective management of inherited and acquired coagulation deficiencies necessitate quantitation of the relevant factors. When bleeding is severe, specific assays are urgently required to guide appropriate therapy. Individual factor assays are usually performed as modifications of the mixing study, where the patient’s plasma is mixed with plasma deficient in the factor being studied. This will correct all factor deficiencies to >50%, thus making prolongation of clot formation due to a factor deficiency dependent on the factor missing from the added plasma.

1	Testing for Antiphospholipid Antibodies Antibodies to phospholipids (cardiolipin) or phospholipid-binding proteins (β2-microglobulin and others) are detected by enzyme-linked immunosorbent assay (ELISA). When these antibodies interfere with phospholipid-dependent coagulation tests, they are termed lupus anticoagulants. The aPTT has variability sensitivity to lupus anticoagulants, depending in part on the aPTT reagents used. An assay using a sensitive reagent has been termed an LA-PTT. The dilute Russell viper venom test (dRVVT) and the tissue thromboplastin inhibition (TTI) test are modifications of standard tests with the phospholipid reagent decreased, thus increasing the sensitivity to antibodies that interfere with the phospholipid component. The tests, however, are not specific for lupus anticoagulants, because factor deficiencies or other inhibitors will also result in prolongation. Documentation of a lupus anticoagulant requires not only prolongation of a phospholipid-dependent

1	anticoagulants, because factor deficiencies or other inhibitors will also result in prolongation. Documentation of a lupus anticoagulant requires not only prolongation of a phospholipid-dependent coagulation test but also lack of correction when mixed with normal plasma and correction with the addition of activated platelet membranes or certain phospholipids (e.g., hexagonal phase).

1	PART 2 Cardinal Manifestations and Presentation of Diseases Factor VII deficiency Vitamin K deficiency—early Warfarin anticoagulation Direct Xa inhibitors (rivaroxaban, apixaban) Factor II, V, X, or fibrinogen deficiency Vitamin K deficiency—late Direct thrombin inhibitors Heparin or heparin-like inhibitors Direct thrombin inhibitors (e.g., dabigatran, argatroban, bivalirudin) Mild or no bleeding—dysfibrinogenemia Frequent, severe bleeding—afibrinogenemia Prolonged PT and/or aPTT Not Corrected with Mixing with Normal Plasma Bleeding—specific factor inhibitor No symptoms, or clotting and/or pregnancy loss—lupus anticoagulant Disseminated intravascular coagulation Heparin or direct thrombin inhibitor Deficiency of α2-antiplasmin or plasminogen activator inhibitor 1 Treatment with fibrinolytic therapy

1	Other Coagulation Tests The thrombin time and the reptilase time measure fibrinogen conversion to fibrin and are prolonged when the fibrinogen level is low (usually <80–100 mg/dL) or qualitatively abnormal, as seen in inherited or acquired dysfibrinogenemias, or when fibrin/fibrinogen degradation products interfere. The thrombin time, but not the reptilase time, is prolonged in the presence of heparin. The thrombin time is markedly prolonged in the presence of the direct thrombin inhibitor, dabigatran; a dilute thrombin time can be used to assess drug activity. Measurement of anti–factor Xa plasma inhibitory activity is a test frequently used to assess lowmolecular-weight heparin (LMWH) levels, as a direct measurement of unfractionated heparin (UFH) activity, or to assess activity of the new direct Xa inhibitors rivaroxaban or apixaban. Drug in the patient sample inhibits the enzymatic conversion of an Xa-specific chromogenic substrate to colored product by factor Xa. Standard curves

1	new direct Xa inhibitors rivaroxaban or apixaban. Drug in the patient sample inhibits the enzymatic conversion of an Xa-specific chromogenic substrate to colored product by factor Xa. Standard curves are created using multiple concentrations of drug and are used to calculate the concentration of anti-Xa activity in the patient plasma.

1	Laboratory Testing for Thrombophilia Laboratory assays to detect thrombophilic states include molecular diagnostics and immunologic and functional assays. These assays vary in their sensitivity and specificity for the condition being tested. Furthermore, acute thrombosis, acute illnesses, inflammatory conditions, pregnancy, and medications affect levels of many coagulation factors and their inhibitors. Antithrombin is decreased by heparin and in the setting of acute thrombosis. Protein C and S levels may be increased in the setting of acute thrombosis and are decreased by warfarin. Antiphospholipid antibodies are frequently transiently positive in acute illness. Testing for genetic thrombophilias should, in general, only be performed when there is a strong family history of thrombosis and results would affect clinical decision making.

1	Because thrombophilia evaluations are usually performed to assess the need to extend anticoagulation, testing should be performed in a steady state, remote from the acute event. In most instances, warfarin anticoagulation can be stopped after the initial 3–6 months of treatment, and testing can be performed at least 3 weeks later. As a sensitive marker of coagulation activation, the quantitative d-dimer assay, drawn 4 weeks after stopping anticoagulation, can be used to stratify risk of recurrent thrombosis in patients who have an idiopathic event.

1	Measures of Platelet Function The bleeding time has been used to assess bleeding risk; however, it has not been found to predict bleeding risk with surgery, and it is not recommended for use for this indication. The PFA-100 and similar instruments that measure platelet-dependent coagulation under flow conditions are generally more sensitive and specific for platelet disorders and VWD than the bleeding time; however, data are insufficient to support their use to predict bleeding risk or monitor response to therapy, and they will be normal in some patients with platelet disorders or mild VWD. When they are used in the evaluation of a patient with bleeding symptoms, abnormal results, as with the bleeding time, require specific testing, such as VWF assays and/or platelet aggregation studies. Because all of these “screening” assays may miss patients with mild bleeding disorders, further studies are needed to define their role in hemostasis testing.

1	For classic platelet aggregometry, various agonists are added to the patient’s platelet-rich plasma and platelet aggregation is measured. Tests of platelet secretion in response to agonists can also be measured. These tests are affected by many factors, including numerous medications, and the association between minor defects in aggregation or secretion in these assays and bleeding risk is not clearly established. Robert I. Handin, MD, contributed this chapter in the 16th edition, and some material from that chapter has been retained here. Enlargement of Lymph nodes Patrick H. Henry, Dan L. Longo This chapter is intended to serve as a guide to the evaluation of patients who present with enlargement of the lymph nodes (lymphadenopathy) or the spleen (splenomegaly). Lymphadenopathy is a rather common clinical finding in primary care settings, whereas palpable splenomegaly is less so.

1	Lymphadenopathy may be an incidental finding in patients being examined for various reasons, or it may be a presenting sign or symptom of the patient’s illness. The physician must eventually decide whether the lymphadenopathy is a normal finding or one that requires further study, up to and including biopsy. Soft, flat, submandibular nodes (<1 cm) are often palpable in healthy children and young adults; healthy adults may have palpable inguinal nodes of up to 2 cm, which are considered normal. Further evaluation of these normal nodes is not warranted. In contrast, if the physician believes the node(s) to be abnormal, then pursuit of a more precise diagnosis is needed. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: Lymphadenopathy may be a primary or secondary manifestation of numerous disorders, as shown in Table 79-1. Many of these disorders are infrequent causes of lymphadenopathy. In primary care practice, more than two-thirds of patients with lymphadenopathy have nonspecific causes or upper respiratory illnesses (viral or bacterial), and <1% have a malignancy. In one study, 84% of patients referred for evaluation of lymphadenopathy had a “benign” diagnosis. The remaining 16% had a malignancy (lymphoma or metastatic adenocarcinoma). Of the patients with benign lymphadenopathy, 63% had a nonspecific or reactive etiology (no causative agent found), and the remainder had a specific cause demonstrated, most commonly infectious mononucleosis, toxoplasmosis, or tuberculosis. Thus, the vast majority of patients with lymphadenopathy will have a nonspecific etiology requiring few diagnostic tests.

1	The physician will be aided in the pursuit of an explanation for the lymphadenopathy by a careful medical history, physical examination, selected laboratory tests, and perhaps an excisional lymph node biopsy. The medical history should reveal the setting in which lymphadenopathy is occurring. Symptoms such as sore throat, cough, fever, night sweats, fatigue, weight loss, or pain in the nodes should be sought. The patient’s age, sex, occupation, exposure to pets, sexual behavior, and use of drugs such as diphenylhydantoin are other important historic points. For example, children and young adults usually have benign (i.e., nonmalignant) disorders that account for the observed lymphadenopathy such as viral or bacterial upper respiratory infections; infectious mononucleosis; toxoplasmosis; and, in some countries, tuberculosis. In contrast, after age 50, the incidence of malignant disorders increases and that of benign disorders decreases.

1	The physical examination can provide useful clues such as the extent of lymphadenopathy (localized or generalized), size of nodes, texture, presence or absence of nodal tenderness, signs of inflammation over the node, skin lesions, and splenomegaly. A thorough ear, nose, and throat (ENT) examination is indicated in adult patients CHAPTER 79 Enlargement of Lymph Nodes and Spleen 1. Infectious diseases a. Viral—infectious mononucleosis syndromes (EBV, CMV), infectious hepatitis, herpes simplex, herpesvirus-6, varicella-zoster virus, rubella, measles, adenovirus, HIV, epidemic keratoconjunctivitis, vaccinia, herpesvirus-8 b. Bacterial—streptococci, staphylococci, cat-scratch disease, brucellosis, tularemia, plague, chancroid, melioidosis, glanders, tuberculosis, atypical mycobacterial infection, primary and secondary syphilis, diphtheria, leprosy, Bartonella c. Fungal—histoplasmosis, coccidioidomycosis, paracoccidioidomycosis d. Chlamydial—lymphogranuloma venereum, trachoma e.

1	Fungal—histoplasmosis, coccidioidomycosis, paracoccidioidomycosis d. Chlamydial—lymphogranuloma venereum, trachoma e. Parasitic—toxoplasmosis, leishmaniasis, trypanosomiasis, filariasis f. Rickettsial—scrub typhus, rickettsialpox, Q fever 2. Immunologic diseases a. b. c. d. e. f. g. h. Drug hypersensitivity—diphenylhydantoin, hydralazine, allopurinol, primidone, gold, carbamazepine, etc. i. j. k. l. m. Autoimmune lymphoproliferative syndrome n. IgG4-related disease o. 3. Malignant diseases a. Hematologic—Hodgkin’s disease, non-Hodgkin’s lymphomas, acute or chronic lymphocytic leukemia, hairy cell leukemia, malignant histiocytosis, amyloidosis b. 4. Lipid storage diseases—Gaucher’s, Niemann-Pick, Fabry, Tangier 5. 6. a. b. c. d. e. f. Sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease) g. h. i. g. k. Vascular transformation of sinuses l. Inflammatory pseudotumor of lymph node m.

1	a. b. c. d. e. f. Sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease) g. h. i. g. k. Vascular transformation of sinuses l. Inflammatory pseudotumor of lymph node m. Congestive heart failure Abbreviations: CMV, cytomegalovirus; EBV, Epstein-Barr virus. with cervical adenopathy and a history of tobacco use. Localized or regional adenopathy implies involvement of a single anatomic area. Generalized adenopathy has been defined as involvement of three or more noncontiguous lymph node areas. Many of the causes of lymphadenopathy (Table 79-1) can produce localized or generalized adenopathy, so this distinction is of limited utility in the differential diagnosis. Nevertheless, generalized lymphadenopathy is frequently associated with nonmalignant disorders such as infectious

1	PART 2 Cardinal Manifestations and Presentation of Diseases mononucleosis (Epstein-Barr virus [EBV] or cytomegalovirus [CMV]), toxoplasmosis, AIDS, other viral infections, systemic lupus erythematosus (SLE), and mixed connective tissue disease. Acute and chronic lymphocytic leukemias and malignant lymphomas also produce generalized adenopathy in adults.

1	The site of localized or regional adenopathy may provide a useful clue about the cause. Occipital adenopathy often reflects an infection of the scalp, and preauricular adenopathy accompanies conjunctival infections and cat-scratch disease. The most frequent site of regional adenopathy is the neck, and most of the causes are benign—upper respiratory infections, oral and dental lesions, infectious mononucleosis, or other viral illnesses. The chief malignant causes include metastatic cancer from head and neck, breast, lung, and thyroid primaries. Enlargement of supraclavicular and scalene nodes is always abnormal. Because these nodes drain regions of the lung and retroperitoneal space, they can reflect lymphomas, other cancers, or infectious processes arising in these areas. Virchow’s node is an enlarged left supraclavicular node infiltrated with metastatic cancer from a gastrointestinal primary. Metastases to supraclavicular nodes also occur from lung, breast, testis, or ovarian

1	node is an enlarged left supraclavicular node infiltrated with metastatic cancer from a gastrointestinal primary. Metastases to supraclavicular nodes also occur from lung, breast, testis, or ovarian cancers. Tuberculosis, sarcoidosis, and toxoplasmosis are nonneoplastic causes of supraclavicular adenopathy. Axillary adenopathy is usually due to injuries or localized infections of the ipsilateral upper extremity. Malignant causes include melanoma or lymphoma and, in women, breast cancer. Inguinal lymphadenopathy is usually secondary to infections or trauma of the lower extremities and may accompany sexually transmitted diseases such as lymphogranuloma venereum, primary syphilis, genital herpes, or chancroid. These nodes may also be involved by lymphomas and metastatic cancer from primary lesions of the rectum, genitalia, or lower extremities (melanoma).

1	The size and texture of the lymph node(s) and the presence of pain are useful parameters in evaluating a patient with lymphadenopathy. Nodes <1.0 cm2 in area (1.0 cm × 1.0 cm or less) are almost always secondary to benign, nonspecific reactive causes. In one retrospective analysis of younger patients (9–25 years) who had a lymph node biopsy, a maximum diameter of >2 cm served as one discriminant for predicting that the biopsy would reveal malignant or granulomatous disease. Another study showed that a lymph node size of 2.25 cm2 (1.5 cm × 1.5 cm) was the best size limit for distinguishing malignant or granulomatous lymphadenopathy from other causes of lymphadenopathy. Patients with node(s) ≤1.0 cm2 should be observed after excluding infectious mononucleosis and/or toxoplasmosis unless there are symptoms and signs of an underlying systemic illness.

1	The texture of lymph nodes may be described as soft, firm, rubbery, hard, discrete, matted, tender, movable, or fixed. Tenderness is found when the capsule is stretched during rapid enlargement, usually secondary to an inflammatory process. Some malignant diseases such as acute leukemia may produce rapid enlargement and pain in the nodes. Nodes involved by lymphoma tend to be large, discrete, symmetric, rubbery, firm, mobile, and nontender. Nodes containing metastatic cancer are often hard, nontender, and non-movable because of fixation to surrounding tissues. The coexistence of splenomegaly in the patient with lymphadenopathy implies a systemic illness such as infectious mononucleosis, lymphoma, acute or chronic leukemia, SLE, sarcoidosis, toxoplasmosis, cat-scratch disease, or other less common hematologic disorders. The patient’s story should provide helpful clues about the underlying systemic illness.

1	Nonsuperficial presentations (thoracic or abdominal) of adenopathy are usually detected as the result of a symptom-directed diagnostic workup. Thoracic adenopathy may be detected by routine chest radiography or during the workup for superficial adenopathy. It may also be found because the patient complains of a cough or wheezing from airway compression; hoarseness from recurrent laryngeal nerve involvement; dysphagia from esophageal compression; or swelling of the neck, face, or arms secondary to compression of the superior vena cava or subclavian vein. The differential diagnosis of mediastinal and hilar adenopathy includes primary lung disorders and systemic illnesses that characteristically involve mediastinal or hilar nodes. In the young, mediastinal adenopathy is associated with infectious mononucleosis and sarcoidosis. In endemic regions, histoplasmosis can cause unilateral paratracheal lymph node involvement that mimics lymphoma. Tuberculosis can also cause unilateral

1	with infectious mononucleosis and sarcoidosis. In endemic regions, histoplasmosis can cause unilateral paratracheal lymph node involvement that mimics lymphoma. Tuberculosis can also cause unilateral adenopathy. In older patients, the differential diagnosis includes primary lung cancer (especially among smokers), lymphomas, metastatic carcinoma (usually lung), tuberculosis, fungal infection, and sarcoidosis.

1	Enlarged intraabdominal or retroperitoneal nodes are usually malignant. Although tuberculosis may present as mesenteric lymphadenitis, these masses usually contain lymphomas or, in young men, germ cell tumors.

1	The laboratory investigation of patients with lymphadenopathy must be tailored to elucidate the etiology suspected from the patient’s history and physical findings. One study from a family practice clinic evaluated 249 younger patients with “enlarged lymph nodes, not infected” or “lymphadenitis.” No laboratory studies were obtained in 51%. When studies were performed, the most common were a complete blood count (CBC) (33%), throat culture (16%), chest x-ray (12%), or monospot test (10%). Only eight patients (3%) had a node biopsy, and half of those were normal or reactive. The CBC can provide useful data for the diagnosis of acute or chronic leukemias, EBV or CMV mononucleosis, lymphoma with a leukemic component, pyogenic infections, or immune cytopenias in illnesses such as SLE. Serologic studies may demonstrate antibodies specific to components of EBV, CMV, HIV, and other viruses; Toxoplasma gondii; Brucella; and so on. If SLE is suspected, antinuclear and anti-DNA antibody studies

1	studies may demonstrate antibodies specific to components of EBV, CMV, HIV, and other viruses; Toxoplasma gondii; Brucella; and so on. If SLE is suspected, antinuclear and anti-DNA antibody studies are warranted.

1	The chest x-ray is usually negative, but the presence of a pulmonary infiltrate or mediastinal lymphadenopathy would suggest tuberculosis, histoplasmosis, sarcoidosis, lymphoma, primary lung cancer, or metastatic cancer and demands further investigation.

1	A variety of imaging techniques (computed tomography [CT], magnetic resonance imaging [MRI], ultrasound, color Doppler ultrasonography) have been used to differentiate benign from malignant lymph nodes, especially in patients with head and neck cancer. CT and MRI are comparably accurate (65–90%) in the diagnosis of metastases to cervical lymph nodes. Ultrasonography has been used to determine the long axis, short axis, and a ratio of long to short (L/S) axis in cervical nodes. An L/S ratio of <2.0 has a sensitivity and a specificity of 95% for distinguishing benign and malignant nodes in patients with head and neck cancer. This ratio has greater specificity and sensitivity than palpation or measurement of either the long or the short axis alone.

1	The indications for lymph node biopsy are imprecise, yet it is a valuable diagnostic tool. The decision to biopsy may be made early in a patient’s evaluation or delayed for up to 2 weeks. Prompt biopsy should occur if the patient’s history and physical findings suggest a malignancy; examples include a solitary, hard, nontender cervical node in an older patient who is a chronic user of tobacco; supraclavicular adenopathy; and solitary or generalized adenopathy that is firm, movable, and suggestive of lymphoma. If a primary head and neck cancer is suspected as the basis of a solitary, hard cervical node, then a careful ENT examination should be performed. Any mucosal lesion that is suspicious for a primary neoplastic process should be biopsied first. If no mucosal lesion is detected, an excisional biopsy of the largest node should be performed. Fine-needle aspiration should not be performed as the first diagnostic procedure. Most diagnoses require more tissue than such aspiration can

1	biopsy of the largest node should be performed. Fine-needle aspiration should not be performed as the first diagnostic procedure. Most diagnoses require more tissue than such aspiration can provide, and it often delays a definitive diagnosis. Fine-needle aspiration should be reserved for thyroid nodules and for confirmation of relapse in patients whose primary diagnosis is known. If the primary physician is uncertain about whether to proceed to biopsy, consultation with a hematologist or medical oncologist should be helpful. In primary care practices, <5% of lymphadenopathy patients will require a biopsy. That percentage will be considerably larger in referral practices, i.e., hematology, oncology, or ENT.

1	Two groups have reported algorithms that they claim will identify more precisely those lymphadenopathy patients who should have a biopsy. Both reports were retrospective analyses in referral practices. The first study involved patients 9–25 years of age who had a node biopsy performed. Three variables were identified that predicted those young patients with peripheral lymphadenopathy who should undergo biopsy; lymph node size >2 cm in diameter and abnormal chest x-ray had positive predictive values, whereas recent ENT symptoms had negative predictive values. The second study evaluated 220 lymphadenopathy patients in a hematology unit and identified five variables (lymph node size, location [supraclavicular or nonsupraclavicular], age [>40 years or <40 years], texture [nonhard or hard], and tenderness) that were used in a mathematical model to identify patients requiring a biopsy. Positive predictive value was found for age >40 years, supraclavicular location, node size >2.25 cm2, hard

1	tenderness) that were used in a mathematical model to identify patients requiring a biopsy. Positive predictive value was found for age >40 years, supraclavicular location, node size >2.25 cm2, hard texture, and lack of pain or tenderness. Negative predictive value was evident for age <40 years, node size <1.0 cm2, nonhard texture, and tender or painful nodes. Ninety-one percent of those who required biopsy were correctly classified by this model. Because both of these studies were retrospective analyses and one was limited to young patients, it is not known how useful these models would be if applied prospectively in a primary care setting.

1	Most lymphadenopathy patients do not require a biopsy, and at least half require no laboratory studies. If the patient’s history and physical findings point to a benign cause for lymphadenopathy, careful follow-up at a 2to 4-week interval can be used. The patient should be instructed to return for reevaluation if there is an increase in the size of the nodes. Antibiotics are not indicated for lymphadenopathy unless strong evidence of a bacterial infection is present. Glucocorticoids should not be used to treat lymphadenopathy because their lympholytic effect obscures some diagnoses (lymphoma, leukemia, Castleman’s disease), and they contribute to delayed healing or activation of underlying infections. An exception to this statement is the life-threatening pharyngeal obstruction by enlarged lymphoid tissue in Waldeyer’s ring that is occasionally seen in infectious mononucleosis. CHAPTER 79 Enlargement of Lymph Nodes and Spleen

1	CHAPTER 79 Enlargement of Lymph Nodes and Spleen The spleen is a reticuloendothelial organ that has its embryologic origin in the dorsal mesogastrium at about 5 weeks of gestation. It arises in a series of hillocks, migrates to its normal adult location in the left upper quadrant (LUQ), and is attached to the stomach via the gastrolienal ligament and to the kidney via the lienorenal ligament. When the hillocks fail to unify into a single tissue mass, accessory spleens may develop in around 20% of persons. The function of the spleen has been elusive. Galen believed it was the source of “black bile” or melancholia, and the word hypochondria (literally, beneath the ribs) and the idiom “to vent one’s spleen” attest to the beliefs that the spleen had an important influence on the psyche and emotions. In humans, its normal physiologic roles seem to be the following: 1.

1	Maintenance of quality control over erythrocytes in the red pulp by removal of senescent and defective red blood cells. The spleen accomplishes this function through a unique organization of its parenchyma and vasculature (Fig. 79-1). 2. Synthesis of antibodies in the white pulp. 3. The removal of antibody-coated bacteria and antibody-coated blood cells from the circulation. An increase in these normal functions may result in splenomegaly. The spleen is composed of red pulp and white pulp, which are Malpighi’s terms for the red blood–filled sinuses and reticuloendothelial Secondary follicle with germinal center (B cell area) PART 2 Cardinal Manifestations and Presentation of Diseases

1	FIguRE 79-1 Schematic spleen structure. The spleen comprises many units of red and white pulp centered around small branches of the splenic artery, called central arteries. White pulp is lymphoid in nature and contains B cell follicles, a marginal zone around the follicles, and T cell–rich areas sheathing arterioles. The red pulp areas include pulp sinuses and pulp cords. The cords are dead ends. In order to regain access to the circulation, red blood cells must traverse tiny openings in the sinusoidal lining. Stiff, damaged, or old red cells cannot enter the sinuses. RE, reticuloendothelial. (Bottom portion of figure from RS Hillman, KA Ault: Hematology in Clinical Practice, 4th ed. New York, McGraw-Hill, 2005.) cell–lined cords and the white lymphoid follicles arrayed within the red pulp matrix. The spleen is in the portal circulation. The reason for this is unknown but may relate to the fact that lower blood pressure allows less rapid flow and minimizes damage to normal

1	the red pulp matrix. The spleen is in the portal circulation. The reason for this is unknown but may relate to the fact that lower blood pressure allows less rapid flow and minimizes damage to normal erythrocytes. Blood flows into the spleen at a rate of about 150 mL/min through the splenic artery, which ultimately ramifies into central arterioles. Some blood goes from the arterioles to capillaries and then to splenic veins and out of the spleen, but the majority of blood from central arterioles flows into the macrophage-lined sinuses and cords. The blood entering the sinuses reenters the circulation through the splenic venules, but the blood entering the cords is subjected to an inspection of sorts. To return to the circulation, the blood cells in the cords must squeeze through slits in the cord lining to enter the sinuses that lead to the venules. Old and damaged erythrocytes are less deformable and are retained in the cords, where they are destroyed and their components recycled.

1	the cord lining to enter the sinuses that lead to the venules. Old and damaged erythrocytes are less deformable and are retained in the cords, where they are destroyed and their components recycled. Red cell–inclusion bodies such as parasites (Chaps. 248 and 250e), nuclear residua (Howell-Jolly bodies, see Fig. 77-6), or denatured hemoglobin (Heinz bodies) are pinched off in the process of passing through the slits, a process called pitting. The culling of dead and damaged cells and the pitting of cells with inclusions appear to occur without significant delay because the blood transit time through the spleen is only slightly slower than in other organs.

1	The spleen is also capable of assisting the host in adapting to its hostile environment. It has at least three adaptive functions: (1) clearance of bacteria and particulates from the blood, (2) the generation of immune responses to certain pathogens, and (3) the generation of cellular components of the blood under circumstances in which the marrow is unable to meet the needs (i.e., extramedullary hematopoiesis). The latter adaptation is a recapitulation of the blood-forming function the spleen plays during gestation. In some animals, the spleen also serves a role in the vascular adaptation to stress because it stores red blood cells (often hemoconcentrated to higher hematocrits than normal) under normal circumstances and contracts under the influence of β-adrenergic stimulation to provide the animal with an autotransfusion and improved oxygen-carrying capacity. However, the normal human spleen does not sequester or store red blood cells and does not contract in response to sympathetic

1	the animal with an autotransfusion and improved oxygen-carrying capacity. However, the normal human spleen does not sequester or store red blood cells and does not contract in response to sympathetic stimuli. The normal human spleen contains approximately one-third of the total body platelets and a significant number of marginated neutrophils. These sequestered cells are available when needed to respond to bleeding or infection.

1	APPROACH TO THE PATIENT: The most common symptoms produced by diseases involving the spleen are pain and a heavy sensation in the LUQ. Massive splenomegaly may cause early satiety. Pain may result from acute swelling of the spleen with stretching of the capsule, infarction, or inflammation of the capsule. For many years, it was believed that splenic infarction was clinically silent, which, at times, is true. However, Soma Weiss, in his classic 1942 report of the self-observations by a Harvard medical student on the clinical course of subacute bacterial endocarditis, documented that severe LUQ and pleuritic chest pain may accompany thromboembolic occlusion of splenic blood flow. Vascular occlusion, with infarction and pain, is commonly seen in children with sickle cell crises. Rupture of the spleen, from either trauma or infiltrative disease that breaks the capsule, may result in intraperitoneal bleeding, shock, and death. The rupture itself may be painless.

1	A palpable spleen is the major physical sign produced by diseases affecting the spleen and suggests enlargement of the organ. The normal spleen weighs <250 g, decreases in size with age, normally lies entirely within the rib cage, has a maximum cephalocaudad diameter of 13 cm by ultrasonography or maximum length of 12 cm and/or width of 7 cm by radionuclide scan, and is usually not palpable. However, a palpable spleen was found in 3% of 2200 asymptomatic, male, freshman college students. Follow-up at 3 years revealed that 30% of those students still had a palpable spleen without any increase in disease prevalence. Ten-year follow-up found no evidence for lymphoid malignancies. Furthermore, in some tropical countries (e.g., New Guinea), the incidence of splenomegaly may reach 60%. Thus, the presence of a palpable spleen does not always equate with presence of disease. Even when disease is present, splenomegaly may not reflect the primary disease but rather a reaction to it. For

1	the presence of a palpable spleen does not always equate with presence of disease. Even when disease is present, splenomegaly may not reflect the primary disease but rather a reaction to it. For example, in patients with Hodgkin’s disease, only two-thirds of the palpable spleens show involvement by the cancer.

1	Physical examination of the spleen uses primarily the techniques of palpation and percussion. Inspection may reveal fullness in the LUQ that descends on inspiration, a finding associated with a massively enlarged spleen. Auscultation may reveal a venous hum or friction rub.

1	Palpation can be accomplished by bimanual palpation, ballotment, and palpation from above (Middleton maneuver). For bimanual palpation, which is at least as reliable as the other techniques, the patient is supine with flexed knees. The examiner’s left hand is placed on the lower rib cage and pulls the skin toward the costal margin, allowing the fingertips of the right hand to feel the tip of the spleen as it descends while the patient inspires slowly, smoothly, and deeply. Palpation is begun with the right hand in the left lower quadrant with gradual movement toward the left costal margin, thereby identifying the lower edge of a massively enlarged spleen. When the spleen tip is felt, the finding is recorded as centimeters below the left costal margin at some arbitrary point, i.e., 10–15 cm, from the midpoint of the umbilicus or the xiphisternal junction. This allows other examiners to compare findings or the initial examiner to determine changes in size over time. Bimanual palpation

1	cm, from the midpoint of the umbilicus or the xiphisternal junction. This allows other examiners to compare findings or the initial examiner to determine changes in size over time. Bimanual palpation in the right lateral decubitus position adds nothing to the supine examination.

1	Percussion for splenic dullness is accomplished with any of three techniques described by Nixon, Castell, or Barkun: 1. Nixon’s method: The patient is placed on the right side so that the spleen lies above the colon and stomach. Percussion begins at the lower level of pulmonary resonance in the posterior axillary line and proceeds diagonally along a perpendicular line toward the lower midanterior costal margin. The upper border of dullness is normally 6–8 cm above the costal margin. Dullness >8 cm in an adult is presumed to indicate splenic enlargement. 2. Castell’s method: With the patient supine, percussion in the lowest intercostal space in the anterior axillary line (eighth or ninth) produces a resonant note if the spleen is normal in size. This is true during expiration or full inspiration. A dull percussion note on full inspiration suggests splenomegaly. 3.

1	3. Percussion of Traube’s semilunar space: The borders of Traube’s space are the sixth rib superiorly, the left midaxillary line laterally, and the left costal margin inferiorly. The patient is supine with the left arm slightly abducted. During normal breathing, this space is percussed from medial to lateral margins, yielding a normal resonant sound. A dull percussion note suggests splenomegaly.

1	Studies comparing methods of percussion and palpation with a standard of ultrasonography or scintigraphy have revealed sensitivity of 56–71% for palpation and 59–82% for percussion. Reproducibility among examiners is better for palpation than percussion. Both techniques are less reliable in obese patients or patients who have just eaten. Thus, the physical examination techniques of palpation and percussion are imprecise at best. It has been suggested that the examiner perform percussion first and, if positive, proceed to palpation; if the spleen is palpable, then one can be reasonably confident that splenomegaly exists. However, not all LUQ masses are enlarged spleens; gastric or colon tumors and pancreatic or renal cysts or tumors can mimic splenomegaly.

1	The presence of an enlarged spleen can be more precisely determined, if necessary, by liver-spleen radionuclide scan, CT, MRI, or ultrasonography. The latter technique is the current procedure of choice for routine assessment of spleen size (normal = a maximum cephalocaudad diameter of 13 cm) because it has high sensitivity and specificity and is safe, noninvasive, quick, mobile, and less costly. Nuclear medicine scans are accurate, sensitive, and reliable but are costly, require greater time to generate data, and use immobile equipment. They have the advantage of demonstrating accessory splenic tissue. CT and MRI provide accurate determination of spleen size, but the equipment is immobile and the procedures are expensive. MRI appears to offer no advantage over CT. Changes in spleen structure such as mass lesions, infarcts, inhomogeneous infiltrates, and cysts are more readily assessed by CT, MRI, or ultrasonography. None of these techniques is very reliable in the detection of patchy

1	such as mass lesions, infarcts, inhomogeneous infiltrates, and cysts are more readily assessed by CT, MRI, or ultrasonography. None of these techniques is very reliable in the detection of patchy infiltration (e.g., Hodgkin’s disease).

1	Many of the diseases associated with splenomegaly are listed in Table 79-2. They are grouped according to the presumed basic mechanisms responsible for organ enlargement: 1. Hyperplasia or hypertrophy related to a particular splenic function such as reticuloendothelial hyperplasia (work hypertrophy) in diseases such as hereditary spherocytosis or thalassemia syndromes that require removal of large numbers of defective red blood cells; immune hyperplasia in response to systemic infection (infectious mononucleosis, subacute bacterial endocarditis) or to immunologic diseases (immune thrombocytopenia, SLE, Felty’s syndrome). 2. Passive congestion due to decreased blood flow from the spleen in conditions that produce portal hypertension (cirrhosis, Budd-Chiari syndrome, congestive heart failure). 3. Infiltrative diseases of the spleen (lymphomas, metastatic cancer, amyloidosis, Gaucher’s disease, myeloproliferative disorders with extramedullary hematopoiesis).

1	3. Infiltrative diseases of the spleen (lymphomas, metastatic cancer, amyloidosis, Gaucher’s disease, myeloproliferative disorders with extramedullary hematopoiesis). The differential diagnostic possibilities are much fewer when the spleen is “massively enlarged,” palpable more than 8 cm below the left costal margin or has a drained weight of ≥1000 g (Table 79-3). The vast majority of such patients will have nonHodgkin’s lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, chronic myeloid leukemia, myelofibrosis with myeloid metaplasia, or polycythemia vera.

1	The major laboratory abnormalities accompanying splenomegaly are determined by the underlying systemic illness. Erythrocyte counts may be normal, decreased (thalassemia major syndromes, SLE, cirrhosis with portal hypertension), or increased (polycythemia vera). Granulocyte counts may be normal, decreased (Felty’s syndrome, congestive splenomegaly, leukemias), or increased (infections or inflammatory disease, myeloproliferative disorders). Similarly, the platelet count may be normal, decreased when there is enhanced sequestration or destruction of platelets in an enlarged spleen (congestive splenomegaly, Gaucher’s disease, immune thrombocytopenia), or increased in the myeloproliferative disorders such as polycythemia vera.

1	The CBC may reveal cytopenia of one or more blood cell types, which should suggest hypersplenism. This condition is characterized by splenomegaly, cytopenia(s), normal or hyperplastic bone marrow, and a response to splenectomy. The latter characteristic is less precise because reversal of cytopenia, particularly granulocytopenia, is sometimes not sustained after splenectomy. The cytopenias result from increased destruction of the cellular elements secondary to reduced flow of blood through enlarged and congested cords (congestive splenomegaly) or to immune-mediated mechanisms. In hypersplenism, various cell types usually have normal morphology on the peripheral blood smear, although the red cells may be spherocytic due to loss of surface area during their longer transit through the enlarged spleen. The increased marrow production of red cells should be reflected as an increased reticulocyte production index, although the value may be less than expected due to increased sequestration

1	spleen. The increased marrow production of red cells should be reflected as an increased reticulocyte production index, although the value may be less than expected due to increased sequestration of reticulocytes in the spleen.

1	The need for additional laboratory studies is dictated by the differential diagnosis of the underlying illness of which splenomegaly is a manifestation. Splenectomy is infrequently performed for diagnostic purposes, especially in the absence of clinical illness or other diagnostic tests that suggest underlying disease. More often, splenectomy is performed for symptom control in patients with massive splenomegaly, for disease control in patients with traumatic splenic rupture, or for correction of cytopenias in patients with hypersplenism or immune-mediated destruction of one or more cellular blood elements. Splenectomy is CHAPTER 79 Enlargement of Lymph Nodes and Spleen Enlargement Due to Increased Demand for Splenic Function PART 2 Cardinal Manifestations and Presentation of Diseases Reticuloendothelial system hyperplasia (for removal of defective erythrocytes) Response to infection (viral, bacterial, fungal, parasitic)

1	Reticuloendothelial system hyperplasia (for removal of defective erythrocytes) Response to infection (viral, bacterial, fungal, parasitic) Malaria Enlargement Due to Abnormal Splenic or Portal Blood Flow Extramedullary hematopoiesis Myelofibrosis Marrow damage by toxins, radiation, strontium Marrow infiltration by tumors, leukemias, Gaucher’s disease Leukemias (acute, chronic, lymphoid, myeloid, monocytic) Myeloproliferative syndromes (e.g., polycythemia vera, essential Metastatic tumors (melanoma is most common)

1	Leukemias (acute, chronic, lymphoid, myeloid, monocytic) Myeloproliferative syndromes (e.g., polycythemia vera, essential Metastatic tumors (melanoma is most common) Hemangiomas, fibromas, lymphangiomas necessary for staging of patients with Hodgkin’s disease only in those with clinical stage I or II disease in whom radiation therapy alone is contemplated as the treatment. Noninvasive staging of the spleen in Hodgkin’s disease is not a sufficiently reliable basis for treatment decisions because one-third of normal-sized spleens will be involved with Hodgkin’s disease and one-third of enlarged spleens will be tumor-free. The widespread use of systemic therapy to test all stages of Hodgkin’s disease has made staging laparotomy with splenectomy aThe spleen extends >8 cm below the left costal margin and/or weighs >1000 g.

1	unnecessary. Although splenectomy in chronic myeloid leukemia (CML) does not affect the natural history of disease, removal of the massive spleen usually makes patients significantly more comfortable and simplifies their management by significantly reducing transfusion requirements. The improvements in therapy of CML have reduced the need for splenectomy for symptom control. Splenectomy is an effective secondary or tertiary treatment for two chronic B cell leukemias, hairy cell leukemia and prolymphocytic leukemia, and for the very rare splenic mantle cell or marginal zone lymphoma. Splenectomy in these diseases may be associated with significant tumor regression in bone marrow and other sites of disease. Similar regressions of systemic disease have been noted after splenic irradiation in some types of lymphoid tumors, especially chronic lymphocytic leukemia and prolymphocytic leukemia. This has been termed the abscopal effect. Such systemic tumor responses to local therapy directed

1	some types of lymphoid tumors, especially chronic lymphocytic leukemia and prolymphocytic leukemia. This has been termed the abscopal effect. Such systemic tumor responses to local therapy directed at the spleen suggest that some hormone or growth factor produced by the spleen may affect tumor cell proliferation, but this conjecture is not yet substantiated.

1	A common therapeutic indication for splenectomy is traumatic or iatrogenic splenic rupture. In a fraction of patients with splenic rupture, peritoneal seeding of splenic fragments can lead to splenosis—the presence of multiple rests of spleen tissue not connected to the portal circulation. This ectopic spleen tissue may cause pain or gastrointestinal obstruction, as in endometriosis. A large number of hematologic, immunologic, and congestive causes of splenomegaly can lead to destruction of one or more cellular blood elements. In the majority of such cases, splenectomy can correct the cytopenias, particularly anemia and thrombocytopenia. In a large series of patients seen in two tertiary care centers, the indication for splenectomy was diagnostic in 10% of patients, therapeutic in 44%, staging for Hodgkin’s disease in 20%, and incidental to another procedure in 26%. Perhaps the only contraindication to splenectomy is the presence of marrow failure, in which the enlarged spleen is the

1	for Hodgkin’s disease in 20%, and incidental to another procedure in 26%. Perhaps the only contraindication to splenectomy is the presence of marrow failure, in which the enlarged spleen is the only source of hematopoietic tissue.

1	The absence of the spleen has minimal long-term effects on the hematologic profile. In the immediate postsplenectomy period, leukocytosis (up to 25,000/μL) and thrombocytosis (up to 1 × 106/μL) may develop, but within 2–3 weeks, blood cell counts and survival of each cell lineage are usually normal. The chronic manifestations of splenectomy are marked variation in size and shape of erythrocytes (anisocytosis, poikilocytosis) and the presence of Howell-Jolly bodies (nuclear remnants), Heinz bodies (denatured hemoglobin), basophilic stippling, and an occasional nucleated erythrocyte in the peripheral blood. When such erythrocyte abnormalities appear in a patient whose spleen has not been removed, one should suspect splenic infiltration by tumor that has interfered with its normal culling and pitting function.

1	The most serious consequence of splenectomy is increased susceptibility to bacterial infections, particularly those with capsules such as Streptococcus pneumoniae, Haemophilus influenzae, and some gram-negative enteric organisms. Patients under age 20 years are particularly susceptible to overwhelming sepsis with S. pneumoniae, and the overall actuarial risk of sepsis in patients who have had their spleens removed is about 7% in 10 years. The case–fatality rate for pneumococcal sepsis in splenectomized patients is 50–80%. About 25% of patients without spleens will develop a serious infection at some time in their life. The frequency is highest within the first 3 years after splenectomy. About 15% of the infections are polymicrobial, and lung, skin, and blood are the most common sites. No increased risk of viral infection has been noted in patients who have no spleen. The susceptibility to bacterial infections relates to the inability to remove opsonized bacteria from the bloodstream

1	increased risk of viral infection has been noted in patients who have no spleen. The susceptibility to bacterial infections relates to the inability to remove opsonized bacteria from the bloodstream and a defect in making antibodies to T cell–independent antigens such as the polysaccharide components of bacterial capsules. Pneumococcal vaccine should be administered to all patients 2 weeks before elective splenectomy. The Advisory Committee on Immunization Practices recommends that these patients receive repeat vaccination 5 years after splenectomy. Efficacy has not been proven for this group, and the recommendation discounts the possibility that administration of the vaccine may actually lower the titer of specific pneumococcal antibodies. A more effective pneumococcal conjugate vaccine that involves T cells in the response is now available (Prevenar, 7-valent). The vaccine to Neisseria meningitidis should also be given to patients in whom elective splenectomy is planned. Although

1	that involves T cells in the response is now available (Prevenar, 7-valent). The vaccine to Neisseria meningitidis should also be given to patients in whom elective splenectomy is planned. Although efficacy data for H. influenzae type b vaccine are not available for older children or adults, it may be given to patients who have had a splenectomy.

1	Splenectomized patients should be educated to consider any unexplained fever as a medical emergency. Prompt medical attention with evaluation and treatment of suspected bacteremia may be life-saving. Routine chemoprophylaxis with oral penicillin can result in the emergence of drug-resistant strains and is not recommended. In addition to an increased susceptibility to bacterial infections, splenectomized patients are also more susceptible to the parasitic disease babesiosis. The splenectomized patient should avoid areas where the parasite Babesia is endemic (e.g., Cape Cod, MA).

1	Surgical removal of the spleen is an obvious cause of hyposplenism. Patients with sickle cell disease often suffer from autosplenectomy as a result of splenic destruction by the numerous infarcts associated with sickle cell crises during childhood. Indeed, the presence of a palpable spleen in a patient with sickle cell disease after age 5 suggests a coexisting hemoglobinopathy, e.g., thalassemia or hemoglobin C.

1	In addition, patients who receive splenic irradiation for a neoplastic 413 or autoimmune disease are also functionally hyposplenic. The term hyposplenism is preferred to asplenism in referring to the physiologic consequences of splenectomy because asplenia is a rare, specific, and fatal congenital abnormality in which there is a failure of the left side of the coelomic cavity (which includes the splenic anlagen) to develop normally. Infants with asplenia have no spleens, but that is the least of their problems. The right side of the developing embryo is duplicated on the left so there is liver where the spleen should be, there are two right lungs, and the heart comprises two right atria and two right ventricles. Disorders of granulocytes and Steven M. Holland, John I. Gallin

1	Disorders of granulocytes and Steven M. Holland, John I. Gallin Leukocytes, the major cells comprising inflammatory and immune responses, include neutrophils, T and B lymphocytes, natural killer (NK) cells, monocytes, eosinophils, and basophils. These cells have specific functions, such as antibody production by B lymphocytes or destruction of bacteria by neutrophils, but in no single infectious disease is the exact role of the cell types completely established. Thus, whereas neutrophils are classically thought to be critical to host defense against bacteria, they may also play important roles in defense against viral infections.

1	The blood delivers leukocytes to the various tissues from the bone marrow, where they are produced. Normal blood leukocyte counts are 4.3–10.8 × 109/L, with neutrophils representing 45–74% of the cells, bands 0–4%, lymphocytes 16–45%, monocytes 4–10%, eosinophils 0–7%, and basophils 0–2%. Variation among individuals and among different ethnic groups can be substantial, with lower leukocyte numbers for certain African-American ethnic groups. The various leukocytes are derived from a common stem cell in the bone marrow. Three-fourths of the nucleated cells of bone marrow are committed to the production of leukocytes. Leukocyte maturation in the marrow is under the regulatory control of a number of different factors, known as colony-stimulating factors (CSFs) and interleukins (ILs). Because an alteration in the number and type of leukocytes is often associated with disease processes, total white blood cell (WBC) count (cells per μL) and differential counts are informative. This chapter

1	an alteration in the number and type of leukocytes is often associated with disease processes, total white blood cell (WBC) count (cells per μL) and differential counts are informative. This chapter focuses on neutrophils, monocytes, and eosinophils. Lymphocytes and basophils are discussed in Chaps. 372e and 376, respectively.

1	CHAPTER 80 Disorders of Granulocytes and Monocytes Important events in neutrophil life are summarized in Fig. 80-1. In normal humans, neutrophils are produced only in the bone marrow. The minimum number of stem cells necessary to support hematopoiesis is estimated to be 400–500 at any one time. Human blood monocytes, tissue macrophages, and stromal cells produce CSFs, hormones required for the growth of monocytes and neutrophils in the bone marrow. The hematopoietic system not only produces enough neutrophils (~1.3 × 1011 cells per 80-kg person per day) to carry out physiologic functions but also has a large reserve stored in the marrow, which can be mobilized in response to inflammation or infection. An increase in the number of blood neutrophils is called neutrophilia, and the presence of immature cells is termed a shift to the left. A decrease in the number of blood neutrophils is called neutropenia.

1	Neutrophils and monocytes evolve from pluripotent stem cells under the influence of cytokines and CSFs (Fig. 80-2). The proliferation phase through the metamyelocyte takes about 1 week, while the maturation phase from metamyelocyte to mature neutrophil takes PART 2 Cardinal Manifestations and Presentation of Diseases Stem O2, .OH,C3acell Diapedesis Activation of other limbs of host defense Redness (Rubor) O2 –, H2 Edema (Tumor) Pain (Dolor) Warmth (Calor) Chemokines, other chemoattractants Fever HOCl (bleach) Macrophages IL-1, TNF-˜IL-8, TNF-˜, IL-12 Integrins Increased endothelial stickiness Selectins Vasodilation Fluid Leakage Recruitment Cytokine secretion

1	FIguRE 80-1 Schematic events in neutrophil production, recruitment, and inflammation. The four cardinal signs of inflammation (rubor, tumor, calor, dolor) are indicated, as are the interactions of neutrophils with other cells and cytokines. G-CSF, granulocyte colony-stimulating factor; IL, interleukin; PMN, polymorphonuclear leukocyte; TNF-α, tumor necrosis factor α. FIguRE 80-2 Stages of neutrophil development shown schematically. Granulocyte colony-stimulating factor (G-CSF) and granulocytemacrophage colony-stimulating factor (GM-CSF) are critical to this process. Identifying cellular characteristics and specific cell-surface markers are listed for each maturational stage. CD33, CD13, CD15 CD33, CD13, CD15 CD33, CD13, CD15, CD14, CD11b CD33, CD13, CD15, CD14, CD11b CD33, CD13, CD15, CD14, CD11b CD10, CD16 CD33, CD13, CD15, CD14, CD11b CD10, CD16 Condensed, band– shaped nucleus Condensed, multilobed nucleus Secondary granule.

1	another week. The myeloblast is the first recognizable precursor cell and is followed by the promyelocyte. The promyelocyte evolves when the classic lysosomal granules, called the primary, or azurophil, granules are produced. The primary granules contain hydrolases, elastase, myeloperoxidase, cathepsin G, cationic proteins, and bactericidal/ permeability-increasing protein, which is important for killing gram-negative bacteria. Azurophil granules also contain defensins, a family of cysteine-rich polypeptides with broad antimicrobial activity against bacteria, fungi, and certain enveloped viruses. The promyelocyte divides to produce the myelocyte, a cell responsible for the synthesis of the specific, or secondary, granules, which contain unique (specific) constituents such as lactoferrin, vitamin B12–binding protein, membrane components of the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase required for hydrogen peroxide production, histaminase, and receptors for

1	vitamin B12–binding protein, membrane components of the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase required for hydrogen peroxide production, histaminase, and receptors for certain chemoattractants and adherence-promoting factors (CR3) as well as receptors for the basement membrane component, laminin. The secondary granules do not contain acid hydrolases and therefore are not classic lysosomes. Packaging of secondary granule contents during myelopoiesis is controlled by CCAAT/enhancer binding protein-ε. Secondary granule contents are readily released extracellularly, and their mobilization is important in modulating inflammation. During the final stages of maturation, no cell division occurs, and the cell passes through the metamyelocyte stage and then to the band neutrophil with a sausage-shaped nucleus (Fig. 80-3). As the band cell matures, the nucleus assumes a lobulated configuration. The nucleus of neutrophils normally contains up to four segments (Fig.

1	neutrophil with a sausage-shaped nucleus (Fig. 80-3). As the band cell matures, the nucleus assumes a lobulated configuration. The nucleus of neutrophils normally contains up to four segments (Fig. 80-4). Excessive segmentation (more than five nuclear lobes) may be a manifestation of folate or vitamin B12 deficiency or the congenital neutropenia syndrome of warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) described below. The Pelger-Hüet anomaly (Fig. 80-5), an infrequent dominant benign inherited trait, results in neutrophils with distinctive bilobed nuclei that must be distinguished from band forms. Acquired bilobed nuclei, pseudo Pelger-Hüet anomaly, can occur with acute infections or in myelodysplastic syndromes. The physiologic role of the normal multilobed nucleus of neutrophils is unknown, but it may allow great deformation of neutrophils during migration into tissues at sites of inflammation.

1	In severe acute bacterial infection, prominent neutrophil cytoplasmic granules, called toxic granulations, are occasionally seen. Toxic granulations are immature or abnormally staining azurophil granules. Cytoplasmic inclusions, also called Döhle bodies (Fig. 80-3), can be seen during infection and are fragments of ribosome-rich endoplasmic reticulum. Large neutrophil vacuoles are often present in acute bacterial infection and probably represent pinocytosed (internalized) membrane. FIguRE 80-3 Neutrophil band with Döhle body. The neutrophil with a sausage-shaped nucleus in the center of the field is a band form. Döhle bodies are discrete, blue-staining, nongranular areas found in the periphery of the cytoplasm of the neutrophil in infec-tions and other toxic states. They represent aggregates of rough endoplasmic reticulum.

1	FIguRE 80-4 Normal granulocyte. The normal granulocyte has a segmented nucleus with heavy, clumped chromatin; fine neutrophilic granules are dispersed throughout the cytoplasm. Neutrophils are heterogeneous in function. Monoclonal antibodies have been developed that recognize only a subset of mature neutrophils. The meaning of neutrophil heterogeneity is not known. The morphology of eosinophils and basophils is shown in Fig. 80-6. Specific signals, including IL-1, tumor necrosis factor α (TNF-α), the CSFs, complement fragments, and chemokines, mobilize leukocytes from the bone marrow and deliver them to the blood in an unstimulated state. Under normal conditions, ~90% of the neutrophil pool is in the bone marrow, 2–3% in the circulation, and the remainder in the tissues (Fig. 80-7).

1	The circulating pool exists in two dynamic compartments: one freely flowing and one marginated. The freely flowing pool is about one-half the neutrophils in the basal state and is composed of those cells that are in the blood and not in contact with the endothelium. Marginated leukocytes are those that are in close physical contact with the CHAPTER 80 Disorders of Granulocytes and Monocytes FIguRE 80-5 Pelger-Hüet anomaly. In this benign disorder, the majority of granulocytes are bilobed. The nucleus frequently has a spectacle-like, or “pince-nez,” configuration. FIguRE 80-6 Normal eosinophil (left) and basophil (right). The eosinophil contains large, bright orange granules and usually a bilobed nucleus. The basophil contains large purple-black granules that fill the cell and obscure the nucleus.

1	PART 2 Cardinal Manifestations and Presentation of Diseases endothelium (Fig. 80-8). In the pulmonary circulation, where an extensive capillary bed (~1000 capillaries per alveolus) exists, margination occurs because the capillaries are about the same size as a mature neutrophil. Therefore, neutrophil fluidity and deformability are necessary to make the transit through the pulmonary bed. Increased neutrophil rigidity and decreased deformability lead to augmented neutrophil trapping and margination in the lung. In contrast, in the systemic postcapillary venules, margination is mediated by the interaction of specific cell-surface molecules called selectins. Selectins are glycoproteins expressed on neutrophils and endothelial cells, among others, that cause a low-affinity interaction, resulting in “rolling” of the neutrophil along the endothelial surface. On neutrophils, the molecule L-selectin (cluster determinant [CD] 62L) binds to glycosylated proteins on endothelial cells (e.g.,

1	in “rolling” of the neutrophil along the endothelial surface. On neutrophils, the molecule L-selectin (cluster determinant [CD] 62L) binds to glycosylated proteins on endothelial cells (e.g., glycosylation-dependent cell adhesion molecule [GlyCAM1] and CD34). Glycoproteins on neutrophils, most importantly sialyl-Lewisx (SLex, CD15s), are targets for binding of selectins expressed on endothelial cells (E-selectin [CD62E] and P-selectin [CD62P]) and

1	FIguRE 80-7 Schematic neutrophil distribution and kinetics between the different anatomic and functional pools. other leukocytes. In response to chemotactic stimuli from injured tissues (e.g., complement product C5a, leukotriene B4, IL-8) or bacterial products (e.g., N-formylmethionylleucylphenylalanine [f-metleu-phe]), neutrophil adhesiveness increases through mobilization of intracellular adhesion proteins stored in specific granules to the cell surface, and the cells “stick” to the endothelium through integrins. The integrins are leukocyte glycoproteins that exist as complexes of a common CD18 β chain with CD11a (LFA-1), CD11b (called Mac-1, CR3, or the C3bi receptor), and CD11c (called p150,95 or CR4). CD11a/CD18 and CD11b/CD18 bind to specific endothelial receptors (intercellular adhesion molecules [ICAM] 1 and 2).

1	On cell stimulation, L-selectin is shed from neutrophils, and E-selectin increases in the blood, presumably because it is shed from endothelial cells; receptors for chemoattractants and opsonins are mobilized; and the phagocytes orient toward the chemoattractant source in the extravascular space, increase their motile activity (chemokinesis), and migrate directionally (chemotaxis) into tissues. The process of migration into tissues is called diapedesis and involves the crawling of neutrophils between postcapillary endothelial cells that open junctions between adjacent cells to permit leukocyte passage. Diapedesis involves platelet/endothelial cell adhesion molecule (PECAM) 1 (CD31), which is expressed on both the emigrating leukocyte and the endothelial cells. The endothelial responses (increased blood flow from increased vasodilation and permeability) are mediated by anaphylatoxins (e.g., C3a and C5a) as well as vasodilators such as histamine, bradykinin, serotonin, nitric oxide,

1	(increased blood flow from increased vasodilation and permeability) are mediated by anaphylatoxins (e.g., C3a and C5a) as well as vasodilators such as histamine, bradykinin, serotonin, nitric oxide, vascular endothelial growth factor (VEGF), and prostaglandins E and I. Cytokines regulate some of these processes (e.g., TNF-α induction of VEGF, interferon [IFN] γ inhibition of prostaglandin E).

1	In the healthy adult, most neutrophils leave the body by migration through the mucous membrane of the gastrointestinal tract. Normally, neutrophils spend a short time in the circulation (half-life, 6–7 h). Senescent neutrophils are cleared from the circulation by macrophages in the lung and spleen. Once in the tissues, neutrophils release enzymes, such as collagenase and elastase, which may help establish abscess cavities. Neutrophils ingest pathogenic materials that have been opsonized by IgG and C3b. Fibronectin and the tetrapeptide tuftsin also facilitate phagocytosis.

1	With phagocytosis comes a burst of oxygen consumption and activation of the hexose-monophosphate shunt. A membrane-associated NADPH oxidase, consisting of membrane and cytosolic components, is assembled and catalyzes the univalent reduction of oxygen to superoxide anion, which is then converted by superoxide dismutase to hydrogen peroxide and other toxic oxygen products (e.g., hydroxyl radical). Hydrogen peroxide + chloride + neutrophil myeloperoxidase generate hypochlorous acid (bleach), hypochlorite, and chlorine. These products oxidize and halogenate microorganisms and tumor cells and, when uncontrolled, can damage host tissue. Strongly cationic proteins, defensins, elastase, cathepsins, and probably nitric oxide also participate in microbial killing. Lactoferrin chelates iron, an important growth factor for microorganisms, especially fungi. Other enzymes, such as lysozyme and acid proteases, help digest microbial debris. After 1–4 days in tissues, neutrophils die. The apoptosis of

1	growth factor for microorganisms, especially fungi. Other enzymes, such as lysozyme and acid proteases, help digest microbial debris. After 1–4 days in tissues, neutrophils die. The apoptosis of neutrophils is also cytokine-regulated; granulocyte colony-stimulating factor (G-CSF) and IFN-γ prolong their life span. Under certain conditions, such as in delayed-type hypersensitivity, monocyte accumulation occurs within 6–12 h of initiation of inflammation. Neutrophils, monocytes, microorganisms in various states of digestion, and altered local tissue cells make up the inflammatory exudate, pus. Myeloperoxidase confers the characteristic green color to pus and may participate in turning off the inflammatory process by inactivating chemoattractants and immobilizing phagocytic cells.

1	Neutrophils respond to certain cytokines (IFN-γ, granulocytemacrophage colony-stimulating factor [GM-CSF], IL-8) and produce cytokines and chemotactic signals (TNF-α, IL-8, macrophage inflammatory protein [MIP] 1) that modulate the inflammatory response. In the presence of fibrinogen, f-met-leu-phe or leukotriene B4 induces IL-8 production by neutrophils, providing autocrine amplification of inflammation. Chemokines (chemoattractant cytokines) are small proteins CHAPTER 80 Disorders of Granulocytes and Monocytes Diapedesis CD18 CD11a,b CD31

1	FIguRE 80-8 Neutrophil travel through the pulmonary capillaries is dependent on neutrophil deformability. Neutrophil rigidity (e.g., caused by C5a) enhances pulmonary trapping and response to pulmonary pathogens in a way that is not so dependent on cell-surface receptors. Intraalveolar chemotactic factors, such as those caused by certain bacteria (e.g., Streptococcus pneumoniae), lead to diapedesis of neutrophils from the pulmonary capillaries into the alveolar space. Neutrophil interaction with the endothelium of the systemic postcapillary venules is dependent on molecules of attachment. The neutrophil “rolls” along the endothelium using selectins: neutrophil CD15s (sialyl-Lewisx) binds to CD62E (E-selectin) and CD62P (P-selectin) on endothelial cells; CD62L (L-selectin) on neutrophils binds to CD34 and other molecules (e.g., GlyCAM-1) expressed on endothelium. Chemokines or other activation factors stimulate integrin-mediated “tight adhesion”: CD11a/CD18 (LFA 1) and CD11b/CD18

1	binds to CD34 and other molecules (e.g., GlyCAM-1) expressed on endothelium. Chemokines or other activation factors stimulate integrin-mediated “tight adhesion”: CD11a/CD18 (LFA 1) and CD11b/CD18 (Mac-1, CR3) bind to CD54 (ICAM-1) and CD102 (ICAM-2) on the endothelium. Diapedesis occurs between endothelial cells: CD31 (PECAM-1) expressed by the emigrating neutrophil interacts with CD31 expressed at the endothelial cell-cell junction. CD, cluster determinant; GlyCAM, glycosylation-dependent cell adhesion molecule; ICAM, intercellular adhesion molecule; PECAM, platelet/endothelial cell adhesion molecule.

1	produced by many different cell types, including endothelial cells, fibroblasts, epithelial cells, neutrophils, and monocytes, that regulate neutrophil, monocyte, eosinophil, and lymphocyte recruitment and activation. Chemokines transduce their signals through heterotrimeric G protein– linked receptors that have seven cell membrane–spanning domains, the same type of cell-surface receptor that mediates the response to the classic chemoattractants f-met-leu-phe and C5a. Four major groups of chemokines are recognized based on the cysteine structure near the N terminus: C, CC, CXC, and CXXXC. The CXC cytokines such as IL-8 mainly attract neutrophils; CC chemokines such as MIP-1 attract lymphocytes, monocytes, eosinophils, and basophils; the C chemokine lymphotactin is T cell tropic; the CXXXC chemokine fractalkine attracts neutrophils, monocytes, and T cells. These molecules and their receptors not only regulate the trafficking and activation of inflammatory cells, but specific chemokine

1	chemokine fractalkine attracts neutrophils, monocytes, and T cells. These molecules and their receptors not only regulate the trafficking and activation of inflammatory cells, but specific chemokine receptors also serve as co-receptors for HIV infection (Chap. 226) and have a role in other viral infections such as West Nile infection and atherogenesis.

1	Defects in the neutrophil life cycle can lead to dysfunction and compromised host defenses. Inflammation is often depressed, and the clinical result is often recurrent, severe bacterial and fungal infections. Aphthous ulcers of mucous membranes (gray ulcers without pus) and gingivitis and periodontal disease suggest a phagocytic cell disorder. Patients with congenital phagocyte defects can have infections within the first few days of life. Skin, ear, upper and lower respiratory tract, and bone infections are common. Sepsis and meningitis are rare. In some disorders, the frequency of infection is variable, and patients can go for months or even years without major infection. Aggressive management of these congenital diseases has extended the life span of patients well beyond 30 years.

1	Neutropenia The consequences of absent neutrophils are dramatic. Susceptibility to infectious diseases increases sharply when neutrophil counts fall below 1000 cells/μL. When the absolute neutrophil count (ANC; band forms and mature neutrophils combined) falls to <500 cells/μL, control of endogenous microbial flora (e.g., mouth, gut) is impaired; when the ANC is <200/μL, the local inflammatory process is absent. Neutropenia can be due to depressed production, increased peripheral destruction, or excessive peripheral pooling. A falling neutrophil count or a significant decrease in the number of neutrophils below steady-state levels, together with a failure to increase neutrophil counts in the setting of infection or other challenge, requires investigation. Acute neutropenia, such as that caused by cancer chemotherapy, is more likely to be associated with increased risk of infection than neutropenia of long duration (months to years) that reverses in response to infection or carefully

1	caused by cancer chemotherapy, is more likely to be associated with increased risk of infection than neutropenia of long duration (months to years) that reverses in response to infection or carefully controlled administration of endotoxin (see “Laboratory Diagnosis and Management,” below).

1	Some causes of inherited and acquired neutropenia are listed in Table 80-1. The most common neutropenias are iatrogenic, resulting from the use of cytotoxic or immunosuppressive therapies for malignancy or control of autoimmune disorders. These drugs cause neutropenia because they result in decreased production of rapidly growing progenitor (stem) cells of the marrow. Certain antibiotics such as chloramphenicol, trimethoprim-sulfamethoxazole, flucytosine, vidarabine, and the antiretroviral drug zidovudine may cause neutropenia by inhibiting proliferation of myeloid precursors. Azathioprine and 6-mercaptopurine are metabolized by the enzyme thiopurine methyltransferase (TMPT), hypofunctional polymorphisms in which are found in 11% of whites and can lead to accumulation of 6-thioguanine and profound marrow toxicity. The marrow suppression is generally dose-related and dependent on continued administration of the drug. Cessation of the offending agent and recombinant human G-CSF usually

1	profound marrow toxicity. The marrow suppression is generally dose-related and dependent on continued administration of the drug. Cessation of the offending agent and recombinant human G-CSF usually reverse these forms of neutropenia.

1	Another important mechanism for iatrogenic neutropenia is the effect of drugs that serve as immune haptens and sensitize neutrophils or neutrophil precursors to immune-mediated peripheral destruction. This form of drug-induced neutropenia can be seen within 7 days of exposure to the drug; with previous drug exposure, resulting in preexisting antibodies, neutropenia may occur a few hours after Drug-induced—alkylating agents (nitrogen mustard, busulfan, chlorambucil, cyclophosphamide); antimetabolites (methotrexate, 6-mercaptopurine, 5-flucytosine); noncytotoxic agents (antibiotics [chloramphenicol, penicillins, sulfonamides], phenothiazines, tranquilizers [meprobamate], anticonvulsants [carbamazepine], antipsychotics [clozapine], certain diuretics, anti-inflammatory agents, antithyroid drugs, many others) Hematologic diseases—idiopathic, cyclic neutropenia, Chédiak-Higashi syndrome, aplastic anemia, infantile genetic disorders (see text) Tumor invasion, myelofibrosis

1	Hematologic diseases—idiopathic, cyclic neutropenia, Chédiak-Higashi syndrome, aplastic anemia, infantile genetic disorders (see text) Tumor invasion, myelofibrosis Nutritional deficiency—vitamin B12, folate (especially alcoholics) Infection—tuberculosis, typhoid fever, brucellosis, tularemia, measles, infectious mononucleosis, malaria, viral hepatitis, leishmaniasis, AIDS

1	Autoimmune disorders—Felty’s syndrome, rheumatoid arthritis, lupus erythematosus Drugs as haptens—aminopyrine, α-methyldopa, phenylbutazone, mercurial diuretics, some phenothiazines Granulomatosis with polyangiitis (Wegener’s) administration of the drug. Although any drug can cause this form of neutropenia, the most frequent causes are commonly used antibiotics, such as sulfa-containing compounds, penicillins, and cephalosporins. Fever and eosinophilia may also be associated with drug reactions, but often these signs are not present. Drug-induced neutropenia can be severe, but discontinuation of the sensitizing drug is sufficient for recovery, which is usually seen within 5–7 days and is complete by 10 days. Readministration of the sensitizing drug should be avoided, because abrupt neutropenia will often result. For this reason, diagnostic challenge should be avoided.

1	Autoimmune neutropenias caused by circulating antineutrophil antibodies are another form of acquired neutropenia that results in increased destruction of neutrophils. Acquired neutropenia may also be seen with viral infections, including infection with HIV. Acquired neutropenia may be cyclic in nature, occurring at intervals of several weeks. Acquired cyclic or stable neutropenia may be associated with an expansion of large granular lymphocytes (LGLs), which may be T cells, NK cells, or NK-like cells. Patients with large granular lymphocytosis may have moderate blood and bone marrow lymphocytosis, neutropenia, polyclonal hypergammaglobulinemia, splenomegaly, rheumatoid arthritis, and absence of lymphadenopathy. Such patients may have a chronic and relatively stable course. Recurrent bacterial infections are frequent. Benign and malignant forms of this syndrome occur. In some patients, a spontaneous regression has occurred even after 11 years, suggesting an immunoregulatory defect as

1	infections are frequent. Benign and malignant forms of this syndrome occur. In some patients, a spontaneous regression has occurred even after 11 years, suggesting an immunoregulatory defect as the basis for at least one form of the disorder. Glucocorticoids, cyclosporine, and methotrexate are commonly used to manage these cytopenias.

1	Hereditary Neutropenias Hereditary neutropenias are rare and may manifest in early childhood as a profound constant neutropenia or agranulocytosis. Congenital forms of neutropenia include Kostmann’s syndrome (neutrophil count <100/μL), which is often fatal and due to mutations in the antiapoptosis gene HAX-1; severe chronic neutropenia (neutrophil count of 300–1500/μL) due to mutations in neutrophil elastase (ELANE); hereditary cyclic neutropenia, or, more appropriately, cyclic hematopoiesis, also due to mutations in neutrophil elastase (ELANE); the cartilage-hair hypoplasia syndrome due to mutations in the mitochondrial RNA-processing endoribonuclease RMRP; Shwachman-Diamond syndrome associated with pancreatic insufficiency due to mutations in the Shwachman-Bodian-Diamond syndrome gene SBDS; the WHIM (warts, hypogammaglobulinemia,

1	PART 2 Cardinal Manifestations and Presentation of Diseases infections, myelokathexis [retention of WBCs in the marrow]) syndrome, characterized by neutrophil hypersegmentation and bone marrow myeloid arrest due to mutations in the chemokine receptor CXCR4; and neutropenias associated with other immune defects, such as X-linked agammaglobulinemia, Wiskott-Aldrich syndrome, and CD40 ligand deficiency. Mutations in the G-CSF receptor can develop in severe congenital neutropenia and are linked to leukemia. Absence of both myeloid and lymphoid cells is seen in reticular dysgenesis, due to mutations in the nuclear genome-encoded mitochondrial enzyme adenylate kinase-2 (AK2).

1	Maternal factors can be associated with neutropenia in the newborn. Transplacental transfer of IgG directed against antigens on fetal neutrophils can result in peripheral destruction. Drugs (e.g., thiazides) ingested during pregnancy can cause neutropenia in the newborn by either depressed production or peripheral destruction. In Felty’s syndrome—the triad of rheumatoid arthritis, splenomegaly, and neutropenia (Chap. 380)—spleen-produced antibodies can shorten neutrophil life span, while large granular lymphocytes can attack marrow neutrophil precursors. Splenectomy may increase the neutrophil count in Felty’s syndrome and lower serum neutrophilbinding IgG. Some Felty’s syndrome patients also have neutropenia associated with an increased number of LGLs. Splenomegaly with peripheral trapping and destruction of neutrophils is also seen in lysosomal storage diseases and in portal hypertension.

1	Neutrophilia Neutrophilia results from increased neutrophil production, increased marrow release, or defective margination (Table 80-2). The most important acute cause of neutrophilia is infection. Neutrophilia from acute infection represents both increased production and increased marrow release. Increased production is also associated with chronic inflammation and certain myeloproliferative diseases. Increased marrow release and mobilization of the marginated leukocyte pool are induced by glucocorticoids. Release of epinephrine, as with vigorous exercise, excitement, or stress, will demarginate neutrophils in the spleen and lungs and double the neutrophil count in minutes. Cigarette smoking can elevate neutrophil counts above the normal range. Leukocytosis with cell counts of 10,000–25,000/μL occurs in response to infection and other forms of acute inflammation and results from both release of the marginated pool and mobilization of marrow reserves. Persistent neutrophilia with cell

1	occurs in response to infection and other forms of acute inflammation and results from both release of the marginated pool and mobilization of marrow reserves. Persistent neutrophilia with cell counts of ≥30,000–50,000/μL is called a leukemoid reaction, a term

1	Idiopathic Drug-induced—glucocorticoids, G-CSF Infection—bacterial, fungal, sometimes viral Inflammation—thermal injury, tissue necrosis, myocardial and pulmonary infarction, hypersensitivity states, collagen vascular diseases Myeloproliferative diseases—myelocytic leukemia, myeloid metaplasia, polycythemia vera Drugs—epinephrine, glucocorticoids, nonsteroidal anti-inflammatory agents Stress, excitement, vigorous exercise Leukocyte adhesion deficiency type 1 (CD18); leukocyte adhesion deficiency type 2 (selectin ligand, CD15s); leukocyte adhesion deficiency type 3 (FERMT3) Metabolic disorders—ketoacidosis, acute renal failure, eclampsia, acute poi Other—metastatic carcinoma, acute hemorrhage or hemolysis Abbreviation: G-CSF, granulocyte colony-stimulating factor. Cause of Indicated Dysfunction Adherence-aggregation Aspirin, colchicine, alcohol, glucocorti-Neonatal state, hemodialysis Leukocyte adhesion deficiency types 1, 2, coids, ibuprofen, piroxicam and 3

1	Adherence-aggregation Aspirin, colchicine, alcohol, glucocorti-Neonatal state, hemodialysis Leukocyte adhesion deficiency types 1, 2, coids, ibuprofen, piroxicam and 3 Deformability Leukemia, neonatal state, diabetes mellitus, immature neutrophils Chemokinesis-chemotaxis Glucocorticoids (high dose), auranofin, Thermal injury, malignancy, malnutrition, Chédiak-Higashi syndrome, neutrophilcolchicine (weak effect), phenylbu-periodontal disease, neonatal state, systemic specific granule deficiency, hyper IgE–recurtazone, naproxen, indomethacin, lupus erythematosus, rheumatoid arthritis, rent infection (Job’s) syndrome (in some interleukin 2 diabetes mellitus, sepsis, influenza virus patients), Down’s syndrome, α-mannosidase infection, herpes simplex virus infection, deficiency, leukocyte adhesion deficiencies, acrodermatitis enteropathica, AIDS Wiskott-Aldrich syndrome

1	Microbicidal activity Colchicine, cyclophosphamide, gluco-Leukemia, aplastic anemia, certain neutrope-Chédiak-Higashi syndrome, neutrophil-specorticoids (high dose), TNF-α-blocking nias, tuftsin deficiency, thermal injury, sepsis, cific granule deficiency, chronic granulomaantibodies neonatal state, diabetes mellitus, malnutri-tous disease, defects in IFNγ/IL-12 axis tion, AIDS Abbreviations: IFNγ, interferon γ; IL, interleukin; TNF-α, tumor necrosis factor alpha. often used to distinguish this degree of neutrophilia from leukemia. In a leukemoid reaction, the circulating neutrophils are usually mature and not clonally derived. Abnormal Neutrophil Function Inherited and acquired abnormalities of phagocyte function are listed in Table 80-3. The resulting diseases are best considered in terms of the functional defects of adherence, chemotaxis, and microbicidal activity. The distinguishing features of the important inherited disorders of phagocyte function are shown in Table 80-4.

1	dISORdERS OF AdHESION Three main types of leukocyte adhesion deficiency (LAD) have been described. All are autosomal recessive and result in the inability of neutrophils to exit the circulation to sites of infection, leading to leukocytosis and increased susceptibility to infection (Fig. 80-8). Patients with LAD 1 have mutations in CD18, the common component of the integrins LFA-1, Mac-1, and p150,95, leading to a defect in tight adhesion between neutrophils and the endothelium. The heterodimer formed by CD18/CD11b (Mac-1) is also the receptor for the complement-derived opsonin C3bi (CR3). The CD18 gene is located on distal chromosome 21q. The severity of the defect determines the severity of clinical disease. Complete lack of expression of the leukocyte integrins results in a severe phenotype in which inflammatory stimuli do not increase the expression of leukocyte integrins on neutrophils or activated T and B cells. Neutrophils (and monocytes) from patients with LAD 1 adhere poorly

1	in which inflammatory stimuli do not increase the expression of leukocyte integrins on neutrophils or activated T and B cells. Neutrophils (and monocytes) from patients with LAD 1 adhere poorly to endothelial cells and protein-coated surfaces and exhibit defective spreading, aggregation, and chemotaxis. Patients with LAD 1 have recurrent bacterial infections involving the skin, oral and genital mucosa, and respiratory and intestinal tracts; persistent leukocytosis (resting neutrophil counts of 15,000–20,000/μL) because cells do not marginate; and, in severe cases, a history of delayed separation of the umbilical stump. Infections, especially of the skin, may become necrotic with progressively enlarging borders, slow healing, and development of dysplastic scars. The most common bacteria are Staphylococcus aureus and enteric gram-negative bacteria. LAD 2 is caused by an abnormality of fucosylation of SLex (CD15s), the ligand on neutrophils that interacts with selectins on endothelial

1	are Staphylococcus aureus and enteric gram-negative bacteria. LAD 2 is caused by an abnormality of fucosylation of SLex (CD15s), the ligand on neutrophils that interacts with selectins on endothelial cells and is responsible for neutrophil rolling along the endothelium. Infection susceptibility in LAD 2 appears to be less severe than in LAD 1. LAD 2 is also known as congenital disorder of glycosylation IIc (CDGIIc) due to mutation in a GDP-fucose transporter (SLC35C1). LAD 3 is characterized by infection susceptibility, leukocytosis, and petechial hemorrhage due to impaired integrin activation caused by mutations in the gene FERMT3.

1	dISORdERS OF NEUTROPHIL gRANULES The most common neutrophil defect is myeloperoxidase deficiency, a primary granule defect inherited as an autosomal recessive trait; the incidence is ~1 in 2000 persons. Isolated myeloperoxidase deficiency is not associated with clinically compromised defenses, presumably because other defense systems such as hydrogen peroxide generation are amplified. Microbicidal activity of neutrophils is delayed but not absent. Myeloperoxidase deficiency may make other acquired host defense defects more serious, and patients with myeloperoxidase deficiency and diabetes are more susceptible to Candida infections. An acquired form of myeloperoxidase deficiency occurs in myelomonocytic leukemia and acute myeloid leukemia.

1	Chédiak-Higashi syndrome (CHS) is a rare disease with autosomal recessive inheritance due to defects in the lysosomal transport protein LYST, encoded by the gene CHS1 at 1q42. This protein is required for normal packaging and disbursement of granules. Neutrophils (and all cells containing lysosomes) from patients with CHS characteristically have large granules (Fig. 80-9), making it a systemic disease. Patients with CHS have nystagmus, partial oculocutaneous albinism, and an increased number of infections resulting from many bacterial agents. Some CHS patients develop an “accelerated phase” in childhood with a hemophagocytic syndrome and an aggressive lymphoma requiring bone marrow transplantation. CHS neutrophils and monocytes have impaired chemotaxis and abnormal rates of microbial killing due to slow rates of fusion of the lysosomal granules with phagosomes. NK cell function is also impaired. CHS patients may develop a severe disabling peripheral neuropathy in adulthood that can

1	due to slow rates of fusion of the lysosomal granules with phagosomes. NK cell function is also impaired. CHS patients may develop a severe disabling peripheral neuropathy in adulthood that can lead to bed confinement.

1	Specific granule deficiency is a rare autosomal recessive disease in which the production of secondary granules and their contents, as well as the primary granule component defensins, is defective. The defect in killing leads to severe bacterial infections. One type of specific granule deficiency is due to a mutation in the CCAAT/enhancer binding protein-ε, a regulator of expression of granule components. A dominant mutation in C/EBP-ε has also been described.

1	CHRONIC gRANULOMATOUS dISEASE Chronic granulomatous disease (CGD) is a group of disorders of granulocyte and monocyte oxidative metabolism. Although CGD is rare, with an incidence of ~1 in 200,000 individuals, it is an important model of defective neutrophil oxidative metabolism. In about two-thirds of patients, CGD is inherited as an X-linked recessive trait; 30% of patients inherit the disease in an autosomal recessive pattern. Mutations in the genes for the five proteins that assemble at the plasma membrane account for all patients with CGD. Two proteins (a 91-kDa protein, abnormal in X-linked CGD, and a 22-kDa protein, absent in one form of autosomal recessive CGD) form the heterodimer cytochrome b-558 in the plasma membrane. Three other proteins (40, 47, and 67 kDa, abnormal in the other autosomal recessive forms of CGD) are cytoplasmic in origin and interact with the cytochrome after cell activation to form NADPH oxidase, required for hydrogen peroxide production. Leukocytes

1	other autosomal recessive forms of CGD) are cytoplasmic in origin and interact with the cytochrome after cell activation to form NADPH oxidase, required for hydrogen peroxide production. Leukocytes from patients with CGD have severely diminished hydrogen peroxide production. The genes involved in each of the defects have been cloned and sequenced and the chromosome locations identified. Patients with CGD characteristically have increased numbers of infections

1	CHAPTER 80 Disorders of Granulocytes and Monocytes Chronic Granulomatous Diseases (70% X-Linked, 30% Autosomal Recessive) Severe infections of skin, ears, lungs, liver, and bone with catalase-positive microorganisms such as Staphylococcus aureus, Burkholderia cepacia complex, Aspergillus spp., Chromobacterium violaceum; often hard to culture organism; excessive inflammation with granulomas, frequent lymph node suppuration; granulomas can obstruct GI or GU tracts; gingivitis, aphthous ulcers, seborrheic dermatitis No respiratory burst due to the lack of one of five DHR or NBT test; no superoxide and H2O2 NADPH oxidase subunits in neutrophils, monocytes, production by neutrophils; immunoblot and eosinophils for NADPH oxidase components; genetic PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Recurrent infections of skin, ears, and sinopulmonary tract; Abnormal chemotaxis, impaired respiratory burst Lack of secondary (specific) granules in delayed wound healing; decreased inflammation; bleed-and bacterial killing, failure to upregulate chemotac-neutrophils (Wright’s stain), no neutrophiling diathesis tic and adhesion receptors with stimulation, defect specific granule contents (i.e., lactoferrin), no in transcription of granule proteins; defect in CEBPE defensins, platelet α granule abnormality; genetic detection Clinically normal except in patients with underlying dis-No myeloperoxidase due to preand posttransla-No peroxidase in neutrophils; genetic detecease such as diabetes mellitus; then candidiasis or other tional defects in myeloperoxidase deficiency tion fungal infections

1	Type 1: Delayed separation of umbilical cord, sustained Impaired phagocyte adherence, aggregation, neutrophilia, recurrent infections of skin and mucosa, gin-spreading, chemotaxis, phagocytosis of C3bi-coated givitis, periodontal disease particles; defective production of CD18 subunit common to leukocyte integrins Type 2: Mental retardation, short stature, Bombay (hh) Impaired phagocyte rolling along endothelium; due blood phenotype, recurrent infections, neutrophilia to defects in fucose transporter Type 3: Petechial hemorrhage, recurrent infections Impaired signaling for integrin activation resulting in impaired adhesion due to mutation in FERMT3 Reduced phagocyte surface expression of the CD18-containing integrins with monoclonal antibodies against LFA-1 (CD18/ CD11a), Mac-1 or CR3 (CD18/CD11b), p150,95 (CD18/CD11c); genetic detection Reduced phagocyte surface expression of Sialyl-Lewisx, with monoclonal antibodies against CD15s; genetic detection

1	Reduced phagocyte surface expression of Sialyl-Lewisx, with monoclonal antibodies against CD15s; genetic detection Abbreviations: C/EBPε, CCAAT/enhancer binding protein-ε; DHR, dihydrorhodamine (oxidation test); DOCK8, dedicator of cytokinesis 8; GI, gastrointestinal; GU, genitourinary; HPV, human papilloma virus; HSV, herpes simplex virus; IFN, interferon; IL, interleukin; IRAK4, IL-1 receptor–associated kinase 4; LFA-1, leukocyte function–associated antigen 1; MyD88, myeloid differentiation primary response gene 88; NADPH, nicotinamide–adenine dinueleotide phosphate; NBT, nitroblue tetrazolium (dye test); NEMO, NF-κB essential modulator; NF-κB, nuclear factor-κB; NK, natural killer; STAT1–3, signal transducer and activator of transcription 1–3; TLR, Toll-like receptor; TNF, tumor necrosis factor.

1	FIguRE 80-9 Chédiak-Higashi syndrome. The granulocytes contain huge cytoplasmic granules formed from aggregation and fusion of azurophilic and specific granules. Large abnormal granules are found in other granule-containing cells throughout the body.

1	due to catalase-positive microorganisms (organisms that destroy their own hydrogen peroxide) such as S. aureus, Burkholderia cepacia, and Aspergillus species. When patients with CGD become infected, they often have extensive inflammatory reactions, and lymph node suppuration is common despite the administration of appropriate antibiotics. Aphthous ulcers and chronic inflammation of the nares are often present. Granulomas are frequent and can obstruct the gastrointestinal or genitourinary tracts. The excessive inflammation is due to failure to downregulate inflammation, reflecting failure to inhibit the synthesis of, degradation of, or response to chemoattractants or residual antigens, leading to persistent neutrophil accumulation. Impaired killing of intracellular microorganisms by macrophages may lead to persistent cell-mediated immune activation and granuloma formation. Autoimmune complications such as immune thrombocytopenic purpura and juvenile rheumatoid arthritis are also

1	macrophages may lead to persistent cell-mediated immune activation and granuloma formation. Autoimmune complications such as immune thrombocytopenic purpura and juvenile rheumatoid arthritis are also increased in CGD. In addition, for unexplained reasons, discoid lupus is more common in X-linked carriers. Late complications, including nodular regenerative hyperplasia and portal hypertension, are increasingly recognized in long-term survivors of severe CGD.

1	dISORdERS OF PHAgOCYTE ACTIVATION Phagocytes depend on cell-surface stimulation to induce signals that evoke multiple levels of the inflammatory response, including cytokine synthesis, chemotaxis, and antigen presentation. Mutations affecting the major pathway that signals through NF-κB have been noted in patients with a variety of infection susceptibility syndromes. If the defects are at a very late stage of signal transduction, in the protein critical for NF-κB activation known as the NF-κB essential modulator (NEMO), then affected males develop ectodermal dysplasia and severe immune deficiency with susceptibility to bacteria, fungi, mycobacteria, and viruses. If the defects in NF-κB activation are closer to the cell-surface receptors, in the proteins transducing Toll-like receptor signals, IL-1 receptor–associated kinase 4 (IRAK4), and myeloid differentiation primary response gene 88 (MyD88), then children have a marked susceptibility to pyogenic infections early in life but

1	signals, IL-1 receptor–associated kinase 4 (IRAK4), and myeloid differentiation primary response gene 88 (MyD88), then children have a marked susceptibility to pyogenic infections early in life but develop resistance to infection later.

1	The mononuclear phagocyte system is composed of monoblasts, promonocytes, and monocytes, in addition to the structurally diverse tissue macrophages that make up what was previously referred to as the reticuloendothelial system. Macrophages are long-lived phagocytic 421 cells capable of many of the functions of neutrophils. They are also secretory cells that participate in many immunologic and inflammatory processes distinct from neutrophils. Monocytes leave the circulation by diapedesis more slowly than neutrophils and have a half-life in the blood of 12–24 h.

1	After blood monocytes arrive in the tissues, they differentiate into macrophages (“big eaters”) with specialized functions suited for specific anatomic locations. Macrophages are particularly abundant in capillary walls of the lung, spleen, liver, and bone marrow, where they function to remove microorganisms and other noxious elements from the blood. Alveolar macrophages, liver Kupffer cells, splenic macrophages, peritoneal macrophages, bone marrow macrophages, lymphatic macrophages, brain microglial cells, and dendritic macrophages all have specialized functions. Macrophage-secreted products include lysozyme, neutral proteases, acid hydrolases, arginase, complement components, enzyme inhibitors (plasmin, α2-macroglobulin), binding proteins (transferrin, fibronectin, transcobalamin II), nucleosides, and cytokines (TNF-α; IL-1, -8, -12, -18). IL-1 (Chaps. 23 and 372e) has many functions, including initiating fever in the hypothalamus, mobilizing leukocytes from the bone marrow, and

1	nucleosides, and cytokines (TNF-α; IL-1, -8, -12, -18). IL-1 (Chaps. 23 and 372e) has many functions, including initiating fever in the hypothalamus, mobilizing leukocytes from the bone marrow, and activating lymphocytes and neutrophils. TNF-α is a pyrogen that duplicates many of the actions of IL-1 and plays an important role in the pathogenesis of gram-negative shock (Chap. 325). TNF-α stimulates production of hydrogen peroxide and related toxic oxygen species by macrophages and neutrophils. In addition, TNF-α induces catabolic changes that contribute to the profound wasting (cachexia) associated with many chronic diseases.

1	Other macrophage-secreted products include reactive oxygen and nitrogen metabolites, bioactive lipids (arachidonic acid metabolites and platelet-activating factors), chemokines, CSFs, and factors stimulating fibroblast and vessel proliferation. Macrophages help regulate the replication of lymphocytes and participate in the killing of tumors, viruses, and certain bacteria (Mycobacterium tuberculosis and Listeria monocytogenes). Macrophages are key effector cells in the elimination of intracellular microorganisms. Their ability to fuse to form giant cells that coalesce into granulomas in response to some inflammatory stimuli is important in the elimination of intracellular microbes and is under the control of IFN-γ. Nitric oxide induced by IFN-γ is an important effector against intracellular parasites, including tuberculosis and Leishmania.

1	Macrophages play an important role in the immune response (Chap. 372e). They process and present antigen to lymphocytes and secrete cytokines that modulate and direct lymphocyte development and function. Macrophages participate in autoimmune phenomena by removing immune complexes and other substances from the circulation. Polymorphisms in macrophage receptors for immunoglobulin (FcγRII) determine susceptibility to some infections and autoimmune diseases. In wound healing, they dispose of senescent cells, and they contribute to atheroma development. Macrophage elastase mediates development of emphysema from cigarette smoking.

1	Many disorders of neutrophils extend to mononuclear phagocytes. Monocytosis is associated with tuberculosis, brucellosis, subacute bacterial endocarditis, Rocky Mountain spotted fever, malaria, and visceral leishmaniasis (kala azar). Monocytosis also occurs with malignancies, leukemias, myeloproliferative syndromes, hemolytic anemias, chronic idiopathic neutropenias, and granulomatous diseases such as sarcoidosis, regional enteritis, and some collagen vascular diseases. Patients with LAD, hyperimmunoglobulin E–recurrent infection (Job’s) syndrome, CHS, and CGD all have defects in the mononuclear phagocyte system. Monocyte cytokine production or response is impaired in some patients with disseminated nontuberculous mycobacterial infection who are not infected with HIV. Genetic defects in the pathways regulated by IFN-γ and IL-12 lead to impaired killing of intracellular bacteria, mycobacteria, salmonellae, and certain viruses (Fig. 80-10).

1	CHAPTER 80 Disorders of Granulocytes and Monocytes IL-2RCD14IL-1518?TLRLPSIL-12IL-2IFN˜TNF°IFN˜RTNF°R˛1˛2AFBSalm.T/NKM˝STAT1GATA2ISG1512NRAMP1NEMOIRF8 PART 2 Cardinal Manifestations and Presentation of Diseases

1	FIguRE 80-10 Lymphocyte-macrophage interactions underlying resistance to mycobacteria and other intracellular pathogens such as Salmonella, Histoplasma, and Coccidioides. Mycobacteria (and others) infect macrophages, leading to the production of IL-12, which activates T or NK cells through its receptor, leading to production of IL-2 and IFN-γ. IFN-γ acts through its receptor on macrophages to upregulate TNF-γ and IL-12 and kill intracellular pathogens. Other critical interacting molecules include signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor 8 (IRF8), GATA2, and ISG15. Mutant forms of the cytokines and receptors shown in bold type have been found in severe cases of nontuberculous mycobacterial infection, salmonellosis and other intracellular pathogens. AFB, acid-fast bacilli; IFN, interferon; IL, interleukin; NEMO, nuclear factorκB essential modulator; NK, natural killer; TLR, Toll-like receptor; TNF, tumor necrosis factor.

1	Certain viral infections impair mononuclear phagocyte function. For example, influenza virus infection causes abnormal monocyte chemotaxis. Mononuclear phagocytes can be infected by HIV using CCR5, the chemokine receptor that acts as a co-receptor with CD4 for HIV. T lymphocytes produce IFN-γ, which induces FcR expression and phagocytosis and stimulates hydrogen peroxide production by mononuclear phagocytes and neutrophils. In certain diseases, such as AIDS, IFN-γ production may be deficient, whereas in other diseases, such as T cell lymphomas, excessive release of IFN-γ may be associated with erythrophagocytosis by splenic macrophages.

1	Autoinflammatory diseases are characterized by abnormal cytokine regulation, leading to excess inflammation in the absence of infection. These diseases can mimic infectious or immunodeficient syndromes. Gain-of-function mutations in the TNF-α receptor cause TNF-α receptor–associated periodic syndrome (TRAPS), which is characterized by recurrent fever in the absence of infection, due to persistent stimulation of the TNF-α receptor (Chap. 392). Diseases with abnormal IL-1 regulation leading to fever include familial Mediterranean fever due to mutations in PYRIN. Mutations in cold-induced autoinflammatory syndrome 1 (CIAS1) lead to neonatal-onset multisystem autoinflammatory disease, familial cold urticaria, and Muckle-Wells syndrome. The syndrome of pyoderma gangrenosum, acne, and sterile pyogenic arthritis (PAPA syndrome) is caused by mutations in PSTPIP1. In contrast to these syndromes of overexpression of proinflammatory cytokines, blockade of TNF-α by the antagonists infliximab,

1	pyogenic arthritis (PAPA syndrome) is caused by mutations in PSTPIP1. In contrast to these syndromes of overexpression of proinflammatory cytokines, blockade of TNF-α by the antagonists infliximab, adalimumab, certolizumab, golimumab, or etanercept has been associated with severe infections due to tuberculosis, nontuberculous mycobacteria, and fungi (Chap. 392).

1	Monocytopenia occurs with acute infections, with stress, and after treatment with glucocorticoids. Drugs that suppress neutrophil production in the bone marrow can cause monocytopenia. Persistent severe circulating monocytopenia is seen in GATA2 deficiency, even though macrophages are found at the sites of inflammation. Monocytopenia also occurs in aplastic anemia, hairy cell leukemia, acute myeloid leukemia, and as a direct result of myelotoxic drugs.

1	Eosinophils and neutrophils share similar morphology, many lysosomal constituents, phagocytic capacity, and oxidative metabolism. Eosinophils express a specific chemoattractant receptor and respond to a specific chemokine, eotaxin, but little is known about their required role. Eosinophils are much longer lived than neutrophils, and unlike neutrophils, tissue eosinophils can recirculate. During most infections, eosinophils appear unimportant. However, in invasive helminthic infections, such as hookworm, schistosomiasis, strongyloidiasis, toxocariasis, trichinosis, filariasis, echinococcosis, and cysticercosis, the eosinophil plays a central role in host defense. Eosinophils are associated with bronchial asthma, cutaneous allergic reactions, and other hypersensitivity states.

1	The distinctive feature of the red-staining (Wright’s stain) eosinophil granule is its crystalline core consisting of an arginine-rich protein (major basic protein) with histaminase activity, important in host defense against parasites. Eosinophil granules also contain a unique eosinophil peroxidase that catalyzes the oxidation of many substances by hydrogen peroxide and may facilitate killing of microorganisms.

1	Eosinophil peroxidase, in the presence of hydrogen peroxide and halide, initiates mast cell secretion in vitro and thereby promotes inflammation. Eosinophils contain cationic proteins, some of which bind to heparin and reduce its anticoagulant activity. Eosinophilderived neurotoxin and eosinophil cationic protein are ribonucleases that can kill respiratory syncytial virus. Eosinophil cytoplasm contains Charcot-Leyden crystal protein, a hexagonal bipyramidal crystal first observed in a patient with leukemia and then in sputum of patients with asthma; this protein is lysophospholipase and may function to detoxify certain lysophospholipids.

1	Several factors enhance the eosinophil’s function in host defense. T cell–derived factors enhance the ability of eosinophils to kill parasites. Mast cell–derived eosinophil chemotactic factor of anaphylaxis (ECFa) increases the number of eosinophil complement receptors and enhances eosinophil killing of parasites. Eosinophil CSFs (e.g., IL-5) produced by macrophages increase eosinophil production in the bone marrow and activate eosinophils to kill parasites.

1	Eosinophilia is the presence of >500 eosinophils per μL of blood and is common in many settings besides parasite infection. Significant tissue eosinophilia can occur without an elevated blood count. A common cause of eosinophilia is allergic reaction to drugs (iodides, aspirin, sulfonamides, nitrofurantoin, penicillins, and cephalosporins). Allergies such as hay fever, asthma, eczema, serum sickness, allergic vasculitis, and pemphigus are associated with eosinophilia. Eosinophilia also occurs in collagen vascular diseases (e.g., rheumatoid arthritis, eosinophilic fasciitis, allergic angiitis, and periarteritis nodosa) and malignancies (e.g., Hodgkin’s disease; mycosis fungoides; chronic myeloid leukemia; and cancer of the lung, stomach, pancreas, ovary, or uterus), as well as in Job’s syndrome, DOCK8 deficiency (see below), and CGD. Eosinophilia is commonly present in helminthic infections. IL-5 is the dominant eosinophil growth factor. Therapeutic administration of the cytokines IL-2

1	DOCK8 deficiency (see below), and CGD. Eosinophilia is commonly present in helminthic infections. IL-5 is the dominant eosinophil growth factor. Therapeutic administration of the cytokines IL-2 or GM-CSF frequently leads to transient eosinophilia. The most dramatic hypereosinophilic syndromes are Loeffler’s syndrome, tropical pulmonary eosinophilia, Loeffler’s endocarditis, eosinophilic leukemia, and idiopathic hypereosinophilic syndrome (50,000–100,000/μL). IL-5 is the dominant eosinophil growth factor and can be specifically inhibited with the monoclonal antibody mepolizumab.

1	The idiopathic hypereosinophilic syndrome represents a heterogeneous group of disorders with the common feature of prolonged eosinophilia of unknown cause and organ system dysfunction, including the heart, central nervous system, kidneys, lungs, gastrointestinal tract, and skin. The bone marrow is involved in all affected individuals, but the most severe complications involve the heart and central nervous system. Clinical manifestations and organ dysfunction are highly variable. Eosinophils are found in the involved tissues and likely cause tissue damage by local deposition of toxic eosinophil proteins such as eosinophil cationic protein and major basic protein. In the heart, the pathologic changes lead to thrombosis, endocardial fibrosis, and restrictive endomyocardiopathy. The damage to tissues in other organ systems is similar. Some cases are due to mutations involving the platelet-derived growth factor receptor, and these are extremely sensitive to the tyrosine kinase inhibitor

1	to tissues in other organ systems is similar. Some cases are due to mutations involving the platelet-derived growth factor receptor, and these are extremely sensitive to the tyrosine kinase inhibitor imatinib. Glucocorticoids, hydroxyurea, and IFN-α each have been used successfully, as have therapeutic antibodies against IL-5. Cardiovascular complications are managed aggressively.

1	The eosinophilia-myalgia syndrome is a multisystem disease, with prominent cutaneous, hematologic, and visceral manifestations, that frequently evolves into a chronic course and can occasionally be fatal. The syndrome is characterized by eosinophilia (eosinophil count >1000/μL) and generalized disabling myalgias without other recognized causes. Eosinophilic fasciitis, pneumonitis, and myocarditis; neuropathy culminating in respiratory failure; and encephalopathy may occur. The disease is caused by ingesting contaminants in L-tryptophan–containing products. Eosinophils, lymphocytes, macrophages, and fibroblasts accumulate in the affected tissues, but their role in pathogenesis is unclear. Activation of eosinophils and fibroblasts and the deposition of eosinophil-derived toxic proteins in affected tissues may contribute. IL-5 and transforming growth factor β have been implicated as potential mediators. Treatment is withdrawal of products containing L-tryptophan and the administration of

1	tissues may contribute. IL-5 and transforming growth factor β have been implicated as potential mediators. Treatment is withdrawal of products containing L-tryptophan and the administration of glucocorticoids. Most patients recover fully, remain stable, or show slow recovery, but the disease can be fatal in up to 5% of patients.

1	Eosinophilic neoplasms are discussed in Chapter 135e. Eosinopenia occurs with stress, such as acute bacterial infection, and after treatment with glucocorticoids. The mechanism of eosinopenia of acute bacterial infection is unknown but is independent of endogenous glucocorticoids, because it occurs in animals after total adrenalectomy. There is no known adverse effect of eosinopenia.

1	The hyperimmunoglobulin E–recurrent infection syndrome, or Job’s syndrome, is a rare multisystem disease in which the immune and somatic systems are affected, including neutrophils, monocytes, T cells, B cells, and osteoclasts. Autosomal dominant mutations in signal transducer and activator of transcription 3 (STAT3) lead to inhibition of normal STAT signaling with broad and profound effects. Patients have characteristic facies with broad nose, kyphoscoliosis, and eczema. The primary teeth erupt normally but do not deciduate, often requiring extraction. Patients develop recurrent sinopulmonary and cutaneous infections that tend to be much less inflamed than appropriate for the degree of infection and have been referred to as “cold abscesses.” Characteristically, pneumonias cavitate, leading to pneumatoceles. Coronary artery aneurysms are common, as are cerebral demyelinated plaques that accumulate with age. Importantly, IL-17–producing T cells, which are thought responsible for

1	leading to pneumatoceles. Coronary artery aneurysms are common, as are cerebral demyelinated plaques that accumulate with age. Importantly, IL-17–producing T cells, which are thought responsible for protection against extracellular and mucosal infections, are profoundly reduced in Job’s syndrome. Despite very high IgE levels, these patients do not have elevated levels of allergy. An important syndrome with clinical overlap with STAT3 deficiency is due to autosomal recessive defects in dedicator of cytokinesis 8 (DOCK8). In DOCK8 deficiency, IgE elevation is joined to severe allergy, viral susceptibility, and increased rates of cancer.

1	Initial studies of WBC and differential and often a bone marrow examination may be followed by assessment of bone marrow reserves (steroid challenge test), marginated circulating pool of cells (epinephrine challenge test), and marginating ability (endotoxin challenge test) (Fig. 80-7). In vivo assessment of inflammation is possible with a Rebuck skin window test or an in vivo skin blister assay, which measures the 423 ability of leukocytes and inflammatory mediators to accumulate locally in the skin. In vitro tests of phagocyte aggregation, adherence, chemotaxis, phagocytosis, degranulation, and microbicidal activity (for

1	S. aureus) may help pinpoint cellular or humoral lesions. Deficiencies of oxidative metabolism are detected with either the nitroblue tetrazolium (NBT) dye test or the dihydrorhodamine (DHR) oxidation test. These tests are based on the ability of products of oxidative metabolism to alter the oxidation states of reporter molecules so that they can be detected microscopically (NBT) or by flow cytometry (DHR). Qualitative studies of superoxide and hydrogen peroxide production may further define neutrophil oxidative function.

1	Patients with leukopenias or leukocyte dysfunction often have delayed inflammatory responses. Therefore, clinical manifestations may be minimal despite overwhelming infection, and unusual infections must always be suspected. Early signs of infection demand prompt, aggressive culturing for microorganisms, use of antibiotics, and surgical drainage of abscesses. Prolonged courses of antibiotics are often required. In patients with CGD, prophylactic antibiotics (trimethoprim-sulfamethoxazole) and antifungals (itraconazole) markedly diminish the frequency of life-threatening infections. Glucocorticoids may relieve gastrointestinal or genitourinary tract obstruction by granulomas in patients with CGD. Although TNF-α-blocking agents may markedly relieve inflammatory bowel symptoms, extreme caution must be exercised in their use in CGD inflammatory bowel disease, because it profoundly increases these patients’ already heightened susceptibility to infection. Recombinant human IFN-γ, which

1	must be exercised in their use in CGD inflammatory bowel disease, because it profoundly increases these patients’ already heightened susceptibility to infection. Recombinant human IFN-γ, which nonspecifically stimulates phagocytic cell function, reduces the frequency of infections in patients with CGD by 70% and reduces the severity of infection. This effect of IFN-γ in CGD is additive to the effect of prophylactic antibiotics. The recommended dose is 50 μg/m2 subcutaneously three times weekly. IFN-γ has also been used successfully in the treatment of leprosy, nontuberculous mycobacteria, and visceral leishmaniasis.

1	Rigorous oral hygiene reduces but does not eliminate the discomfort of gingivitis, periodontal disease, and aphthous ulcers; chlorhexidine mouthwash and tooth brushing with a hydrogen peroxide–sodium bicarbonate paste help many patients. Oral antifungal agents (fluconazole, itraconazole, voriconazole, posaconazole) have reduced mucocutaneous candidiasis in patients with Job’s syndrome. Androgens, glucocorticoids, lithium, and immunosuppressive therapy have been used to restore myelopoiesis in patients with neutropenia due to impaired production. Recombinant G-CSF is useful in the management of certain forms of neutropenia due to depressed neutrophil production, including those related to cancer chemotherapy. Patients with chronic neutropenia with evidence of a good bone marrow reserve need not receive prophylactic antibiotics. Patients with chronic or cyclic neutrophil counts <500/μL may benefit from prophylactic antibiotics and G-CSF during periods of neutropenia. Oral

1	reserve need not receive prophylactic antibiotics. Patients with chronic or cyclic neutrophil counts <500/μL may benefit from prophylactic antibiotics and G-CSF during periods of neutropenia. Oral trimethoprim-sulfamethoxazole (160/800 mg) twice daily can prevent infection. Increased numbers of fungal infections are not seen in patients with CGD on this regimen. Oral quinolones such as levofloxacin and ciprofloxacin are alternatives.

1	In the setting of cytotoxic chemotherapy with severe, persistent lymphocyte dysfunction, trimethoprim-sulfamethoxazole prevents Pneumocystis jiroveci pneumonia. These patients, and patients with phagocytic cell dysfunction, should avoid heavy exposure to airborne soil, dust, or decaying matter (mulch, manure), which are often rich in Nocardia and the spores of Aspergillus and other fungi. Restriction of activities or social contact has no proven role in reducing risk of infection for phagocyte defects.

1	Although aggressive medical care for many patients with phagocytic disorders can allow them to go for years without a life-threatening infection, there may still be delayed effects of prolonged antimicrobials and other inflammatory complications. Cure of most congenital phagocyte defects is possible by bone marrow transplantation, and rates of success are improving (Chap. 139e). The identification of specific gene defects in patients with LAD 1, CGD, and other immunodeficiencies has led to gene therapy trials in a number of genetic white cell disorders. CHAPTER 80 Disorders of Granulocytes and Monocytes

1	CHAPTER 80 Disorders of Granulocytes and Monocytes Atlas of Hematology and Analysis of Peripheral Blood Smears Dan L. Longo Some of the relevant findings in peripheral blood, enlarged lymph nodes, and bone marrow are illustrated in this chapter. Systematic his-81e tologic examination of the bone marrow and lymph nodes is beyond the scope of a general medicine textbook. However, every internist should know how to examine a peripheral blood smear.

1	The examination of a peripheral blood smear is one of the most informative exercises a physician can perform. Although advances in automated technology have made the examination of a peripheral blood smear by a physician seem less important, the technology is not a completely satisfactory replacement for a blood smear interpretation by a trained medical professional who also knows the patient’s clinical history, family history, social history, and physical findings. It is useful to ask the laboratory to generate a Wright’s-stained peripheral blood smear and examine it. The best place to examine blood cell morphology is the feathered edge of the blood smear where red cells lie in a single layer, side by side, just barely touching one another but not overlapping. The author’s approach is to look at the smallest cellular elements, the platelets, first and work his way up in size to red cells and then white cells.

1	Using an oil immersion lens that magnifies the cells 100-fold, one counts the platelets in five to six fields, averages the number per field, and multiplies by 20,000 to get a rough estimate of the platelet count. The platelets are usually 1–2 μm in diameter and have a blue granulated appearance. There is usually 1 platelet for every 20 or so red cells. Of course, the automated counter is much more accurate, but gross disparities between the automated and manual counts should be assessed. Large platelets may be a sign of rapid platelet turnover, as young platelets are often larger than old ones; alternatively, certain rare inherited syndromes can produce large platelets. Platelet clumping visible on the smear can be associated with falsely low automated platelet counts. Similarly, neutrophil fragmentation can be a source of falsely elevated automated platelet counts.

1	Next one examines the red blood cells. One can gauge their size by comparing the red cell to the nucleus of a small lymphocyte. Both are normally about 8 μm wide. Red cells that are smaller than the small lymphocyte nucleus may be microcytic; those larger than the small lymphocyte nucleus may be macrocytic. Macrocytic cells also tend to be more oval than spherical in shape and are sometimes called macroovalocytes. The automated mean corpuscular volume (MCV) can assist in making a classification. However, some patients may have both iron and vitamin B12 deficiency, which will produce an MCV in the normal range but wide variation in red cell size. When the red cells vary greatly in size, anisocytosis is said to be present. When the red cells vary greatly in shape, poikilocytosis is said to be present. The electronic cell counter provides an independent assessment of variability in red cell size. It measures the range of red cell volumes and reports the results as “red cell distribution

1	be present. The electronic cell counter provides an independent assessment of variability in red cell size. It measures the range of red cell volumes and reports the results as “red cell distribution width” (RDW). This value is calculated from the MCV; thus, cell width is not being measured but cell volume is. The term is derived from the curve displaying the frequency of cells at each volume, also called the distribution. The width of the red cell volume distribution curve is what determines the RDW. The RDW is calculated as follows: RDW = (standard deviation of MCV ÷ mean MCV) × 100. In the presence of morphologic anisocytosis, RDW (normally 11–14%) increases to 15–18%. The RDW is useful in at least two clinical settings. In patients with microcytic anemia, the differential diagnosis is generally between iron deficiency and thalassemia. In thalassemia, the small red cells are generally of uniform size with a normal small RDW. In iron deficiency, the size variability and the RDW are

1	generally between iron deficiency and thalassemia. In thalassemia, the small red cells are generally of uniform size with a normal small RDW. In iron deficiency, the size variability and the RDW are large. In addition, a large RDW can suggest a dimorphic anemia when a chronic atrophic gastritis can produce both vitamin B12 malabsorption to produce macrocytic anemia and blood loss to produce iron deficiency. In such settings, RDW is also large. An elevated

1	RDW also has been reported as a risk factor for all-cause mortality in 81e-1 population-based studies (Patel KV et al: Arch Intern Med 169:515, 2009), a finding that is unexplained currently. After red cell size is assessed, one examines the hemoglobin content of the cells. They are either normal in color (normochromic) or pale in color (hypochromic). They are never “hyperchromic.” If more than the normal amount of hemoglobin is made, the cells get larger—they do not become darker. In addition to hemoglobin content, the red cells are examined for inclusions. Red cell inclusions are the following: 1. Basophilic stippling—diffuse fine or coarse blue dots in the red cell usually representing RNA residue—especially common in lead poisoning 2. 3. Nuclei—red cells may be released or pushed out of the marrow prematurely before nuclear extrusion—often implies a myelophthisic process or a vigorous narrow response to anemia, usually hemolytic anemia 4.

1	(Chap. 250e) 5. Polychromatophilia—the red cell cytoplasm has a bluish hue, reflecting the persistence of ribosomes still actively making hemoglobin in a young red cell Vital stains are necessary to see precipitated hemoglobin called Heinz bodies.

1	Red cells can take on a variety of different shapes. All abnormally shaped red cells are poikilocytes. Small red cells without the central pallor are spherocytes; they can be seen in hereditary spherocytosis, hemolytic anemias of other causes, and clostridial sepsis. Dacrocytes are teardrop-shaped cells that can be seen in hemolytic anemias, severe iron deficiency, thalassemias, myelofibrosis, and myelodysplastic syndromes. Schistocytes are helmet-shaped cells that reflect microangiopathic hemolytic anemia or fragmentation on an artificial heart valve. Echinocytes are spiculated red cells with the spikes evenly spaced; they can represent an artifact of abnormal drying of the blood smear or reflect changes in stored blood. They also can be seen in renal failure and malnutrition and are often reversible. Acanthocytes are spiculated red cells with the spikes irregularly distributed. This process tends to be irreversible and reflects underlying renal disease, abetalipoproteinemia, or

1	often reversible. Acanthocytes are spiculated red cells with the spikes irregularly distributed. This process tends to be irreversible and reflects underlying renal disease, abetalipoproteinemia, or splenectomy. Elliptocytes are elliptical-shaped red cells that can reflect an inherited defect in the red cell membrane, but they also are seen in iron deficiency, myelodysplastic syndromes, megaloblastic anemia, and thalassemias. Stomatocytes are red cells in which the area of central pallor takes on the morphology of a slit instead of the usual round shape. Stomatocytes can indicate an inherited red cell membrane defect and also can be seen in alcoholism. Target cells have an area of central pallor that contains a dense center, or bull’s-eye. These cells are seen classically in thalassemia, but they are also present in iron deficiency, cholestatic liver disease, and some hemoglobinopathies. They also can be generated artifactually by improper slide making.

1	One last feature of the red cells to assess before moving to the white blood cells is the distribution of the red cells on the smear. In most individuals, the cells lie side by side in a single layer. Some patients have red cell clumping (called agglutination) in which the red cells pile upon one another; it is seen in certain paraproteinemias and autoimmune hemolytic anemias. Another abnormal distribution involves red cells lying in single cell rows on top of one another like stacks of coins. This is called rouleaux formation and reflects abnormal serum protein levels.

1	Finally, one examines the white blood cells. Three types of granulocytes are usually present: neutrophils, eosinophils, and basophils, in decreasing frequency. Neutrophils are generally the most abundant white cell. They are round, are 10–14 μm wide, and contain a lobulated nucleus with two to five lobes connected by a thin chromatin thread. Bands are immature neutrophils that have not completed nuclear condensation and have a U-shaped nucleus. Bands reflect a left shift in neutrophil maturation in an effort to make more cells more rapidly.

1	CHAPTER 81e Atlas of Hematology and Analysis of Peripheral Blood Smears 81e-2 Neutrophils can provide clues to a variety of conditions. Vacuolated neutrophils may be a sign of bacterial sepsis. The presence of 1to 2-μm blue cytoplasmic inclusions, called Dle bodies, can reflect infections, burns, or other inflammatory states. If the neutrophil granules are larger than normal and stain a darker blue, “toxic granulations” are said to be present, and they also suggest a systemic inflammation. The presence of neutrophils with more than five nuclear lobes suggests megaloblastic anemia. Large misshapen granules may reflect the inherited Chédiak-Higashi syndrome. Eosinophils are slightly larger than neutrophils, have bilobed nuclei, and contain large red granules. Diseases of eosinophils are associated with too many of them rather than any morphologic or qualitative change. They normally total less than one-thirtieth the number of neutrophils. Basophils are even rarer than eosinophils in the

1	with too many of them rather than any morphologic or qualitative change. They normally total less than one-thirtieth the number of neutrophils. Basophils are even rarer than eosinophils in the blood. They have large dark blue granules and may be increased as part of chronic myeloid leukemia. Lymphocytes can be present in several morphologic forms. Most common in healthy individuals are small lymphocytes with a small dark nucleus and scarce cytoplasm. In the presence of viral infections, more of the lymphocytes are larger, about the size of neutrophils, with abundant cytoplasm and a less condensed nuclear chromatin. These cells are called reactive lymphocytes. About 1% of lymphocytes are larger and contain blue granules in a light blue cytoplasm; they are called large granular lymphocytes. In chronic lymphoid leukemia, the small lymphocytes are increased in number, and many of them are ruptured in making the blood smear, leaving a smudge of nuclear material without a surrounding

1	In chronic lymphoid leukemia, the small lymphocytes are increased in number, and many of them are ruptured in making the blood smear, leaving a smudge of nuclear material without a surrounding cytoplasm or cell membrane; they are called smudge cells and are rare in the absence of chronic lymphoid leukemia.

1	Monocytes are the largest white blood cells, ranging from 15 to 22 μm in diameter. The nucleus can take on a variety of shapes but usually appears to be folded; the cytoplasm is gray. Abnormal cells may appear in the blood. Most often the abnormal cells originate from neoplasms of bone marrow–derived cells, including lymphoid cells, myeloid cells, and occasionally red cells. More rarely, other types of tumors can get access to the bloodstream, and rare epithelial malignant cells may be identified. The chances of seeing such abnormal cells is increased by examining blood smears made from buffy coats, the layer of cells that is visible on top of sedimenting red cells when blood is left in the test tube for an hour. Smears made from finger sticks may include rare endothelial cells. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Figure 81e-1 Normal peripheral blood smear. Small lymphocyte in center of field. Note that the diameter of the red blood cell is similar to the diameter of the small lymphocyte nucleus. Figure 81e-3 Hypochromic microcytic anemia of iron deficiency. Small lymphocyte in field helps assess the red blood cell size. Figure 81e-2 Reticulocyte count preparation. This new methylene blue–stained blood smear shows large numbers of heavily stained reticulocytes (the cells containing the dark blue–staining RNA precipitates). Figure 81e-4 Iron deficiency anemia next to normal red blood cells. Microcytes (right panel) are smaller than normal red blood cells (cell diameter <7 μm) and may or may not be poorly hemoglobinized (hypochromic). Figure 81e-5 Polychromatophilia. Note large red cells with light purple coloring. Figure 81e-8 Spherocytosis. Note small hyperchromatic cells with-out the usual clear area in the center.

1	Figure 81e-5 Polychromatophilia. Note large red cells with light purple coloring. Figure 81e-8 Spherocytosis. Note small hyperchromatic cells with-out the usual clear area in the center. CHAPTER 81e Atlas of Hematology and Analysis of Peripheral Blood Smears Figure 81e-6 Macrocytosis. These cells are both larger than normal (mean corpuscular volume >100) and somewhat oval in shape. Some morphologists call these cells macroovalocytes. Figure 81e-9 Rouleaux formation. Small lymphocyte in center of field. These red cells align themselves in stacks and are related to increased serum protein levels. Figure 81e-7 Hypersegmented neutrophils. Hypersegmented neutrophils (multilobed polymorphonuclear leukocytes) are larger than normal neutrophils with five or more segmented nuclear lobes. They are commonly seen with folic acid or vitamin B12 deficiency.

1	Figure 81e-10 Red cell agglutination. Small lymphocyte and seg-mented neutrophil in upper left center. Note irregular collections of aggregated red cells. Figure 81e-11 Fragmented red cells. Heart valve hemolysis. Figure 81e-14 Elliptocytosis. Small lymphocyte in center of field. Elliptical shape of red cells is related to weakened membrane struc-ture, usually due to mutations in spectrin. PART 2 Cardinal Manifestations and Presentation of Diseases Figure 81e-12 Sickle cells. Homozygous sickle cell disease. A nucle-ated red cell and neutrophil are also in the field. Figure 81e-15 Stomatocytosis. Red cells characterized by a wide transverse slit or stoma. This often is seen as an artifact in a dehydrat-ed blood smear. These cells can be seen in hemolytic anemias and in conditions in which the red cell is overhydrated or dehydrated.

1	Figure 81e-13 Target cells. Target cells are recognized by the bull’s-eye appearance of the cell. Small numbers of target cells are seen with liver disease and thalassemia. Larger numbers are typical of hemoglobin C disease. Figure 81e-16 Acanthocytosis. Spiculated red cells are of two types: acanthocytes are contracted dense cells with irregular membrane projections that vary in length and width; echinocytes have small, uniform, and evenly spaced membrane projections. Acanthocytes are present in severe liver disease, in patients with abetalipoproteinemia, and in rare patients with McLeod blood group. Echinocytes are found in patients with severe uremia, in glycolytic red cell enzyme defects, and in microangiopathic hemolytic anemia. Figure 81e-17 Howell-Jolly bodies. Howell-Jolly bodies are tiny nuclear remnants that normally are removed by the spleen. They appear in the blood after splenectomy (defect in removal) and with maturation/dysplastic disorders (excess production).

1	Figure 81e-20 Reticulin stain of marrow myelofibrosis. Silver stain of a myelofibrotic marrow showing an increase in reticulin fibers (black-staining threads). Figure 81e-18 Teardrop cells and nucleated red blood cells char-acteristic of myelofibrosis. A teardrop-shaped red blood cell (left panel) and a nucleated red blood cell (right panel) as typically seen with myelofibrosis and extramedullary hematopoiesis. CHAPTER 81e Atlas of Hematology and Analysis of Peripheral Blood Smears Figure 81e-21 Stippled red cell in lead poisoning. Mild hypochro-mia. Coarsely stippled red cell. Figure 81e-19 Myelofibrosis of the bone marrow. Total replace-ment of marrow precursors and fat cells by a dense infiltrate of reticu-lin fibers and collagen (H&E stain). Figure 81e-22 Heinz bodies. Blood mixed with hypotonic solution of crystal violet. The stained material is precipitates of denatured hemoglobin within cells.

1	Figure 81e-22 Heinz bodies. Blood mixed with hypotonic solution of crystal violet. The stained material is precipitates of denatured hemoglobin within cells. Figure 81e-23 Giant platelets. Giant platelets, together with a marked increase in the platelet count, are seen in myeloproliferative disorders, especially primary thrombocythemia. Figure 81e-26 Normal eosinophils. The film was prepared from the buffy coat of the blood from a normal donor. E, eosinophil; L, lymphocyte; N, neutrophil. PART 2 Cardinal Manifestations and Presentation of Diseases Figure 81e-27 Normal basophil. The film was prepared from the buffy coat of the blood from a normal donor. B, basophil; L, lymphocyte. Figure 81e-24 Normal granulocytes. The normal granulocyte has a segmented nucleus with heavy, clumped chromatin; fine neutrophilic granules are dispersed throughout the cytoplasm.

1	Figure 81e-24 Normal granulocytes. The normal granulocyte has a segmented nucleus with heavy, clumped chromatin; fine neutrophilic granules are dispersed throughout the cytoplasm. Figure 81e-25 Normal monocytes. The film was prepared from the buffy coat of the blood from a normal donor. L, lymphocyte; M, monocyte; N, neutrophil. Figure 81e-28 Pelger-Ht anomaly. In this benign disorder, the majority of granulocytes are bilobed. The nucleus frequently has a spectacle-like, or “pince-nez,” configuration. Figure 81e-29 Dle body. Neutrophil band with Döhle body. The neutrophil with a sausage-shaped nucleus in the center of the field is a band form. Döhle bodies are discrete, blue-staining nongranular areas found in the periphery of the cytoplasm of the neutrophil in infections and other toxic states. They represent aggregates of rough endoplasmic reticulum.

1	Figure 81e-32 Aplastic anemia bone marrow. Normal hematopoi-etic precursor cells are virtually absent, leaving behind fat cells, reticu-loendothelial cells, and the underlying sinusoidal structure. CHAPTER 81e Atlas of Hematology and Analysis of Peripheral Blood Smears Figure 81e-30 Chédiak-Higashi disease. Note giant granules in neutrophil. Figure 81e-33 Metastatic cancer in the bone marrow. Marrow biopsy specimen infiltrated with metastatic breast cancer and reactive fibrosis (H&E stain).

1	Figure 81e-33 Metastatic cancer in the bone marrow. Marrow biopsy specimen infiltrated with metastatic breast cancer and reactive fibrosis (H&E stain). Figure 81e-31 Normal bone marrow. Low-power view of normal adult marrow (hematoxylin and eosin [H&E] stain), showing a mix of fat cells (clear areas) and hematopoietic cells. The percentage of the space that consists of hematopoietic cells is referred to as marrow cellularity. In adults, normal marrow cellularity is 35–40%. If demands for increased marrow production occur, cellularity may increase to meet the demand. As people age, the marrow cellularity decreases and the marrow fat increases. Patients >70 years old may have a 20–30% marrow cellularity. Figure 81e-34 Lymphoma in the bone marrow. Nodular (follicular) lymphoma infiltrate in a marrow biopsy specimen. Note the character-istic paratrabecular location of the lymphoma cells. PART 2 Cardinal Manifestations and Presentation of Diseases

1	PART 2 Cardinal Manifestations and Presentation of Diseases Figure 81e-35 Erythroid hyperplasia of the marrow. Marrow aspirate specimen with a myeloid/erythroid ratio (M/E ratio) of 1:1–2, typical for a patient with a hemolytic anemia or one recovering from blood loss. Figure 81e-36 Myeloid hyperplasia of the marrow. Marrow aspi-rate specimen showing a myeloid/erythroid ratio of ≥3:1, suggesting either a loss of red blood cell precursors or an expansion of myeloid elements. Figure 81e-37 Megaloblastic erythropoiesis. High-power view of megaloblastic red blood cell precursors from a patient with a macrocytic anemia. Maturation is delayed, with late normoblasts showing a more immature-appearing nucleus with a lattice-like pattern with normal cytoplasmic maturation.

1	Figure 81e-38 Prussian blue staining of marrow iron stores. Iron stores can be graded on a scale of 0 to 4+. A. A marrow with excess iron stores (>4+); B. normal stores (2–3+); C. minimal stores (1+); and D. absent iron stores (0). Figure 81e-39 Ringed sideroblast. An orthochromatic normoblast with a collar of blue granules (mitochondria encrusted with iron) sur-rounding the nucleus. Figure 81e-42 Acute erythroleukemia. Note giant dysmorphic erythroblasts; two are binucleate, and one is multinucleate. Figure 81e-40 Acute myeloid leukemia. Leukemic myeloblast with an Auer rod. Note two to four large, prominent nucleoli in each cell. Figure 81e-43 Acute lymphoblastic leukemia. CHAPTER 81e Atlas of Hematology and Analysis of Peripheral Blood Smears Figure 81e-41 Acute promyelocytic leukemia. Note prominent cytoplasmic granules in the leukemia cells. Figure 81e-44 Burkitt’s leukemia, acute lymphoblastic leukemia. Figure 81e-45 Chronic myeloid leukemia in the peripheral blood.

1	Figure 81e-44 Burkitt’s leukemia, acute lymphoblastic leukemia. Figure 81e-45 Chronic myeloid leukemia in the peripheral blood. Figure 81e-48 Adult T cell leukemia. Peripheral blood smear show-ing leukemia cells with typical “flower-shaped” nucleus. PART 2 Cardinal Manifestations and Presentation of Diseases Figure 81e-46 Chronic lymphoid leukemia in the peripheral blood. Figure 81e-49 Follicular lymphoma in a lymph node. The normal nodal architecture is effaced by nodular expansions of tumor cells. Nodules vary in size and contain predominantly small lymphocytes with cleaved nuclei along with variable numbers of larger cells with vesicular chromatin and prominent nucleoli. Figure 81e-47 Sézary’s syndrome. Lymphocytes with frequently convoluted nuclei (Sézary cells) in a patient with advanced mycosis fungoides. Figure 81e-50 Diffuse large B cell lymphoma in a lymph node.

1	Figure 81e-50 Diffuse large B cell lymphoma in a lymph node. The neoplastic cells are heterogeneous but predominantly large cells with vesicular chromatin and prominent nucleoli. Figure 81e-51 Burkitt’s lymphoma in a lymph node. Burkitt’s lym-phoma with starry-sky appearance. The lighter areas are macrophages attempting to clear dead cells. Figure 81e-54 Lacunar cell; Reed-Sternberg cell variant in nodular sclerosing Hodgkin’s disease. High-power view of single mononuclear lacunar cell with retracted cytoplasm in a patient with nodular scleros-ing Hodgkin’s disease. CHAPTER 81e Atlas of Hematology and Analysis of Peripheral Blood Smears Figure 81e-52 Erythrophagocytosis accompanying aggressive lymphoma. The central macrophage is ingesting red cells, neutro-phils, and platelets. (Courtesy of Dr. Kiyomi Tsukimori, Kyushu University, Fukuoka, Japan.) Figure 81e-55 Normal plasma cell.

1	Figure 81e-55 Normal plasma cell. Figure 81e-53 Hodgkin’s disease. A Reed-Sternberg cell is present near the center of the field; a large cell with a bilobed nucleus and prominent nucleoli giving an “owl’s eyes” appearance. The majority of the cells are normal lymphocytes, neutrophils, and eosinophils that form a pleiomorphic cellular infiltrate. Figure 81e-56 Multiple myeloma. PART 2 Cardinal Manifestations and Presentation of Diseases Figure 81e-57 Serum color in hemoglobinemia. The distinctive red coloration of plasma (hemoglobinemia) in a spun blood sample in a patient with intravascular hemolysis. AcknowledgmentFigures in this e-chapter were borrowed from Williams Hematology, 7th edition, M Lichtman et al (eds). New York, McGraw-Hill, 2005; Hematology in General Practice, 4th edition, RS Hillman, KA Ault, New York, McGraw-Hill, 2005.

1	425part 3: Genes, the Environment, and Disease of the genome (and epigenome) in various malignancies has led toprinciples of human Genetics fundamental new insights into cancer biology and reveals that the genomic profile of mutations is in many cases more important in J. Larry Jameson, Peter Kopp determining the appropriate chemotherapy than the organ in which

1	The prevalence of genetic diseases, combined with their potential severity and chronic nature, imposes great human, social, and financial burdens on society. Human genetics refers to the study of individual genes, their role and function in disease, and their mode of inheritance. Genomics refers to an organism’s entire genetic information, the genome, and the function and interaction of DNA within the genome, as well as with environmental or nongenetic factors, such as a person’s lifestyle. With the characterization of the human genome, genomics complements traditional genetics in our efforts to elucidate the etiology and pathogenesis of disease and to improve therapeutic interventions and outcomes. Following impressive advances in genetics, genomics, and health care information technology, the consequences of this wealth of knowledge for the practice of medicine are profound and play an increasingly prominent role in the diagnosis, prevention, and treatment of disease (Chap. 84).

1	Personalized medicine, the customization of medical decisions to an individual patient, relies heavily on genetic information. For example, a patient’s genetic characteristics (genotype) can be used to optimize drug therapy and predict efficacy, adverse events, and drug dosing of selected medications (pharmacogenetics) (Chap. 5). The mutational profile of a malignancy allows the selection of therapies that target mutated or overexpressed signaling molecules. Although still investigational, genomic risk prediction models for common diseases are beginning to emerge.

1	Genetics has traditionally been viewed through the window of relatively rare single-gene diseases. These disorders account for ~10% of pediatric admissions and childhood mortality. Historically, genetics has focused predominantly on chromosomal and metabolic disorders, reflecting the long-standing availability of techniques to diagnose these conditions. For example, conditions such as trisomy 21 (Down’s syndrome) or monosomy X (Turner’s syndrome) can be diagnosed using cytogenetics (Chap. 83e). Likewise, many metabolic disorders (e.g., phenylketonuria, familial hypercholesterolemia) are diagnosed using biochemical analyses. The advances in DNA diagnostics have extended the field of genetics to include virtually all medical specialties and have led to the elucidation of the pathogenesis of numerous monogenic disorders. In addition, it is apparent that virtually every medical condition has a genetic component. As is often evident from a patient’s family history, many common disorders

1	of numerous monogenic disorders. In addition, it is apparent that virtually every medical condition has a genetic component. As is often evident from a patient’s family history, many common disorders such as hypertension, heart disease, asthma, diabetes mellitus, and mental illnesses are significantly influenced by the genetic background. These polygenic or multifactorial (complex) disorders involve the contributions of many different genes, as well as environmental factors that can modify disease risk (Chap. 84). Genome-wide association studies (GWAS) have elucidated numerous disease-associated loci and are providing novel insights into the allelic architecture of complex traits. These studies have been facilitated by the availability of comprehensive catalogues of human single-nucleotide polymorphism (SNP) haplotypes generated through the HapMap Project. The sequencing of whole genomes or exomes (the exons within the genome) is increasingly used in the clinical realm in order to

1	polymorphism (SNP) haplotypes generated through the HapMap Project. The sequencing of whole genomes or exomes (the exons within the genome) is increasingly used in the clinical realm in order to characterize individuals with complex undiagnosed conditions or to characterize the mutational profile of advanced malignancies in order to select better targeted therapies.

1	Cancer has a genetic basis because it results from acquired somatic mutations in genes controlling growth, apoptosis, and cellular differentiation (Chap. 101e). In addition, the development of many cancers is associated with a hereditary predisposition. Characterization the tumor originates. Hence, comprehensive mutational profiling of malignancies has increasing impact on cancer taxonomy, the choice of targeted therapies, and improved outcomes.

1	Genetic and genomic approaches have proven invaluable for the detection of infectious pathogens and are used clinically to identify agents that are difficult to culture such as mycobacteria, viruses, and parasites, or to track infectious agents locally or globally. In many cases, molecular genetics has improved the feasibility and accuracy of diagnostic testing and is beginning to open new avenues for therapy, including gene and cellular therapy (Chaps. 90e and 91e). Molecular genetics has also provided the opportunity to characterize the microbiome, a new field that characterizes the population dynamics of bacteria, viruses, and parasites that coexist with humans and other animals (Chap. 86e). Emerging data indicate that the microbiome has significant effects on normal physiology as well as various disease states.

1	Molecular biology has significantly changed the treatment of human disease. Peptide hormones, growth factors, cytokines, and vaccines can now be produced in large amounts using recombinant DNA technology. Targeted modifications of these peptides provide the practitioner with improved therapeutic tools, as illustrated by genetically modified insulin analogues with more favorable kinetics. Lastly, there is reason to believe that a better understanding of the genetic basis of human disease will also have an increasing impact on disease prevention.

1	The astounding rate at which new genetic information is being generated creates a major challenge for physicians, health care providers, and basic investigators. Although many functional aspects of the genome remain unknown, there are many clinical situations where sufficient evidence exits for the use of genetic and genomic information to optimize patient care and treatment. Much genetic information resides in databases or is being published in basic science journals. Databases provide easy access to the expanding information about the human genome, genetic disease, and genetic testing (Table 82-1). For example, several thousand monogenic disorders are summarized in a large, continuously evolving compendium, referred to as the Online Mendelian Inheritance in Man (OMIM) catalogue (Table 82-1). The ongoing refinement of bioinformatics is simplifying the analysis and access to this daunting amount of new information.

1	THE HUMAN GENOME Structure of the Human Genome • Human Genome Project The Human Genome Project was initiated in the mid-1980s as an ambitious effort to characterize the entire human genome. Although the prospect of determining the complete sequence of the human genome seemed daunting several years ago, technical advances in DNA sequencing and bioinformatics led to the completion of a draft human sequence in 2000 and the completion of the DNA sequence for the last of the human chromosomes in May 2006. Currently, facilitated by rapidly decreasing costs for comprehensive sequence analyses and improvement of bioinformatics pipelines for data analysis, the sequencing of whole genomes and exomes is used with increasing frequency in the clinical setting. The scope of a whole genome sequence analysis can be illustrated by the following analogy. Human DNA consists of ~3 billion base pairs (bp) of DNA per haploid genome, which is nearly 1000-fold greater than that of the Escherichia coli

1	analysis can be illustrated by the following analogy. Human DNA consists of ~3 billion base pairs (bp) of DNA per haploid genome, which is nearly 1000-fold greater than that of the Escherichia coli genome. If the human DNA sequence were printed out, it would correspond to about 120 volumes of Harrison’s Principles of Internal Medicine.

1	In addition to the human genome, the genomes of numerous organisms have been sequenced completely (~4000) or partially (~10,000) (Genomes Online Database [GOLD]; Table 82-1). They include, among others, eukaryotes such as the mouse (Mus musculus), Chapter 82 Principles of Human Genetics Catalog of Published Genome-Wide Association Studies Office of Biotechnology Activities, National Institutes of Health American College of Medical Genetics and Genomics American Society of Human Genetics MITOMAP, a human mitochondrial genome database Dolan DNA Learning Center, Cold Spring Harbor Laboratories The Online Metabolic and Molecular Bases of Inherited Disease (OMMBID)

1	MITOMAP, a human mitochondrial genome database Dolan DNA Learning Center, Cold Spring Harbor Laboratories The Online Metabolic and Molecular Bases of Inherited Disease (OMMBID) The Jackson Laboratory http://www.ncbi.nlm.nih.gov/ http://www.genome.gov/ http://www.genome.gov/ GWAStudies/ http://www.ensembl.org http://www.ncbi.nlm.nih.gov/ omim http://oba.od.nih.gov/oba http://www.acmg.net/ http://www.ashg.org http://cgap.nci.nih.gov/ http://www.genetests.org/ http://www.genomesonline.org/ http://www.genenames.org/ http://www.mitomap.org/ http://www.hapmap.org/ http://www.genome.gov/10005107 http://www.dnalc.org/ http://www.ommbid.com/ http://omia.angis.org.au/ http://www.jax.org/ http://www.informatics.jax.org Broad access to biomedical and genomic information, literature (PubMed), sequence databases, software for analyses of nucleotides and proteins Extensive links to other databases, genome resources, and tutorials

1	Extensive links to other databases, genome resources, and tutorials An institute of the National Institutes of Health focused on genomic and genetic research; links providing information about the human genome sequence, genomes of other organisms, and genomic research Maps and sequence information of eukaryotic genomes Online compendium of Mendelian disorders and human genes causing genetic disorders Information about recombinant DNA and gene transfer; medical, ethical, legal, and social issues raised by genetic testing; medical, ethical, legal, and social issues raised by xenotransplantation Extensive links to other databases relevant for the diagnosis, treatment, and prevention of genetic disease Information about advances in genetic research, professional and public education, social and scientific policies Information about gene expression profiles of normal, precancer, and cancer cells

1	Information about advances in genetic research, professional and public education, social and scientific policies Information about gene expression profiles of normal, precancer, and cancer cells International directory of genetic testing laboratories and prenatal diagnosis clinics; reviews and educational materials Gene names and symbols A compendium of polymorphisms and mutations of the human mitochondrial DNA Catalogue of haplotypes in different ethnic groups relevant for association studies and pharmacogenomics Encyclopedia of DNA Elements; catalogue of all functional elements in the human genome Educational material about selected genetic disorders, DNA, eugenics, and genetic origin Online version of the comprehensive text on the metabolic and molecular bases of inherited disease Online compendium of Mendelian disorders in animals Information about murine models and the mouse genome Mouse genome informatics

1	Online compendium of Mendelian disorders in animals Information about murine models and the mouse genome Mouse genome informatics Note: Databases are evolving constantly. Pertinent information may be found by using links listed in the few selected databases.

1	Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster; bacteria (e.g., E. coli); and Archaea, viruses, organelles (mitochondria, chloroplasts), and plants (e.g., Arabidopsis thaliana). Genomic information of infectious agents has significant impact for the characterization of infectious outbreaks and epidemics. Other ramifications arising from the availability of genomic data include, among others, (1) the comparison of entire genomes (comparative genomics), (2) the study of large-scale expression of RNAs (functional genomics) and proteins (proteomics) to detect differences between various tissues in health and disease, (3) the characterization of the variation among individuals by establishing catalogues of sequence variations and SNPs (HapMap Project), and (4) the identification of genes that play critical roles in the development of polygenic and multifactorial disorders.

1	cHromosomes The human genome is divided into 23 different chromosomes, including 22 autosomes (numbered 1–22) and the X and Y sex chromosomes (Fig. 82-1). Adult cells are diploid, meaning they contain two homologous sets of 22 autosomes and a pair of sex chromosomes. Females have two X chromosomes (XX), whereas males have one X and one Y chromosome (XY). As a consequence of meiosis, germ cells (sperm or oocytes) are haploid and contain one set of 22 autosomes and one of the sex chromosomes. At the time of fertilization, the diploid genome is reconstituted by pairing of the homologous chromosomes from the mother and father. With each cell division (mitosis), chromosomes are replicated, paired, segregated, and divided into two daughter cells.

1	structure of Dna DNA is a double-stranded helix composed of four different bases: adenine (A), thymidine (T), guanine (G), and cytosine (C). Adenine is paired to thymidine, and guanine is paired to cytosine, by hydrogen bond interactions that span the double helix (Fig. 82-1). DNA has several remarkable features that make it ideal for the transmission of genetic information. It is relatively stable, and the double-stranded nature of DNA and its feature of strict base-pair complementarity permit faithful replication during cell division. Complementarity also allows the transmission of genetic information from DNA → RNA → protein (Fig. 82-2). mRNA is encoded by the so-called sense or coding strand of the DNA double helix and is translated into proteins by ribosomes.

1	The presence of four different bases provides surprising genetic diversity. In the protein-coding regions of genes, the DNA bases are arranged into codons, a triplet of bases that specifies a particular amino acid. It is possible to arrange the four bases into 64 different triplet codons (43). Each codon specifies 1 of the 20 different amino acids, or a regulatory signal such as initiation and stop of translation. Because there are more codons than amino acids, the genetic code is degenerate; that is, most amino acids can be specified by several different codons. By arranging the codons in different combinations and in Nucleosome core Histone H2A, H2B, H4 p, short arm Centromere Solenoid q, long arm

1	Nucleosome core Histone H2A, H2B, H4 p, short arm Centromere Solenoid q, long arm FIGURE 82-1 Structure of chromatin and chromosomes. Chromatin is composed of double-strand DNA that is wrapped around histone and nonhistone proteins forming nucleosomes. The nucleosomes are further organized into solenoid structures. Chromosomes assume their characteristic structure, with short (p) and long (q) arms at the metaphase stage of the cell cycle. various lengths, it is possible to generate the tremendous diversity of primary protein structure.

1	various lengths, it is possible to generate the tremendous diversity of primary protein structure. DNA length is normally measured in units of 1000 bp (kilobases, kb) or 1,000,000 bp (megabases, Mb). Not all DNA encodes genes. In fact, genes account for only ~10–15% of DNA. Much of the remaining DNA consists of sequences, often of highly repetitive nature, the function of which is poorly understood. These repetitive DNA regions, along with nonrepetitive sequences that do not encode genes, serve, in part, a structural role in the packaging of DNA into chromatin (i.e., DNA bound to histone proteins, and chromosomes) and exert regulatory functions (Fig. 82-1).

1	Genes A gene is a functional unit that is regulated by transcription (see below) and encodes an RNA product, which is most commonly, but not always, translated into a protein that exerts activity within or outside the cell (Fig. 82-3). Historically, genes were identified because they conferred specific traits that are transmitted from one generation to the next. Increasingly, they are characterized based on expression in various tissues (transcriptome). The size of genes is quite broad; some genes are only a few hundred base pairs, whereas others are extraordinarily large (2 Mb). The number of genes greatly underestimates the complexity of genetic expression, because single genes can generate multiple spliced messenger RNA (mRNA) products (isoforms), which are translated into proteins that are subject to complex posttranslational modification such as phosphorylation. Exons refer to the portion of genes that are eventually spliced together to form mRNA. Introns refer to the spacing

1	that are subject to complex posttranslational modification such as phosphorylation. Exons refer to the portion of genes that are eventually spliced together to form mRNA. Introns refer to the spacing regions between the exons that are spliced out of precursor RNAs during RNA processing. The gene locus also includes regions that are necessary to control its expression (Fig. 82-2). Current 427 estimates predict 20,687 protein-coding genes in the human genome with an average of about four different coding transcripts per gene. Remarkably, the exome only constitutes 1.14% of the genome. In addition, thousands of noncoding transcripts (RNAs of various length such as microRNAs and long noncoding RNAs), which function, at least in part, as transcriptional and posttranscriptional regulators of gene expression, have been identified. Aberrant expression of microRNAs has been found to play a pathogenic role in numerous diseases.

1	sinGle-nucleotiDe PolymorPHisms An SNP is a variation of a single base pair in the DNA. The identification of the ~10 million SNPs estimated to occur in the human genome has generated a catalogue of common genetic variants that occur in human beings from distinct ethnic backgrounds (Fig. 82-3). SNPs are the most common type of sequence variation and account for ~90% of all sequence variation. They occur on average every 100 to 300 bases and are the major source of genetic heterogeneity. Remarkably, however, the primary DNA sequence of humans has ~99.9% similarity compared to that of any other human. SNPs that are in close proximity are inherited together (e.g., they are linked) and are referred to as haplotypes(Fig. 82-4). The HapMap describes the nature and location of these SNP haplotypes and how they are distributed among individuals within and among populations. The haplotype map information, referred to as HapMap, is greatly facilitating GWAS designed to elucidate the complex

1	and how they are distributed among individuals within and among populations. The haplotype map information, referred to as HapMap, is greatly facilitating GWAS designed to elucidate the complex interactions among multiple genes and lifestyle factors in multifactorial disorders (see below). Moreover, haplotype analyses are useful to assess variations in responses to medications (pharmacogenomics) and environmental factors, as well as the prediction of disease predisposition.

1	coPy number variations Copy number variations (CNVs) are relatively large genomic regions (1 kb to several Mb) that have been duplicated or deleted on certain chromosomes (Fig. 82-5). It has been estimated that as many as 1500 CNVs, scattered throughout the genome, are present in an individual. When comparing the genomes of two individuals, approximately 0.4–0.8% of their genomes differ in terms of CNVs. Of note, de novo CNVs have been observed between monozygotic twins, who otherwise have identical genomes. Some CNVs have been associated with susceptibility or resistance to disease, and CNVs can be elevated in cancer cells.

1	Replication of DNA and Mitosis Genetic information in DNA is transmitted to daughter cells under two different circumstances: (1) somatic cells divide by mitosis, allowing the diploid (2n) genome to replicate itself completely in conjunction with cell division; and (2) germ cells (sperm and ova) undergo meiosis, a process that enables the reduction of the diploid (2n) set of chromosomes to the haploid state (1n).

1	Prior to mitosis, cells exit the resting, or G0 state, and enter the cell cycle (Chap. 101e). After traversing a critical checkpoint in G1, cells undergo DNA synthesis (S phase), during which the DNA in each chromosome is replicated, yielding two pairs of sister chromatids (2n → 4n). The process of DNA synthesis requires stringent fidelity in order to avoid transmitting errors to subsequent generations of cells. Genetic abnormalities of DNA mismatch/repair include xeroderma pigmentosum, Bloom’s syndrome, ataxia telangiectasia, and hereditary nonpolyposis colon cancer (HNPCC), among others. Many of these disorders strongly predispose to neoplasia because of the rapid acquisition of additional mutations (Chap. 101e). After completion of DNA synthesis, cells enter G2 and progress through a second checkpoint before entering mitosis. At this stage, the chromosomes condense and are aligned along the equatorial plate at metaphase. The two identical sister chromatids, held together at the

1	a second checkpoint before entering mitosis. At this stage, the chromosomes condense and are aligned along the equatorial plate at metaphase. The two identical sister chromatids, held together at the centromere, divide and migrate to opposite poles of the cell. After formation of a nuclear membrane around the two separated sets of chromatids, the cell divides and two daughter cells are formed, thus restoring the diploid (2n) state.

1	Assortment and Segregation of Genes During Meiosis Meiosis occurs only in germ cells of the gonads. It shares certain features with mitosis but involves two distinct steps of cell division that reduce the chromosome number to the haploid state. In addition, there is active recombination that generates genetic diversity. During the first cell division, two Chapter 82 Principles of Human Genetics Regulation of Gene Expression

1	Chapter 82 Principles of Human Genetics Regulation of Gene Expression FIGURE 82-2 Flow of genetic information. Multiple extracellular signals activate intracellular signal cascades that result in altered regulation of gene expression through the interaction of transcription factors with regulatory regions of genes. RNA polymerase transcribes DNA into RNA that is processed to mRNA by excision of intronic sequences. The mRNA is translated into a polypeptide chain to form the mature protein after undergoing posttranslational processing. CBP, CREB-binding protein; CoA, co-activator; COOH, carboxyterminus; CRE, cyclic AMP responsive element; CREB, cyclic AMP response element–binding protein; GTF, general transcription factors; HAT, histone acetyl transferase; NH2, aminoterminus; RE, response element; TAF, TBP-associated factors; TATA, TATA box; TBP, TATA-binding protein.

1	sister chromatids (2n → 4n) are formed for each chromosome pair and there is an exchange of DNA between homologous paternal and maternal chromosomes. This process involves the formation of chiasmata, structures that correspond to the DNA segments that cross over between the maternal and paternal homologues (Fig. 82-6). Usually there is at least one crossover on each chromosomal arm; recombination occurs more frequently in female meiosis than in male meiosis. Subsequently, the chromosomes segregate randomly. Because there are 23 chromosomes, there exist 223 (>8 million) possible combinations of chromosomes. Together with the genetic exchanges that occur during recombination, chromosomal segregation generates tremendous diversity, and each gamete is genetically unique. The process of recombination and the independent segregation of chromosomes provide the foundation for performing linkage analyses, whereby one attempts to correlate the inheritance of certain chromosomal regions (or

1	and the independent segregation of chromosomes provide the foundation for performing linkage analyses, whereby one attempts to correlate the inheritance of certain chromosomal regions (or linked genes) with the presence of a disease or genetic trait (see below).

1	After the first meiotic division, which results in two daughter cells (2n), the two chromatids of each chromosome separate during a second meiotic division to yield four gametes with a haploid state (1n). When the egg is fertilized by sperm, the two haploid sets are combined, thereby restoring the diploid state (2n) in the zygote.

1	REGULATION OF GENE EXPRESSION Regulation by Transcription Factors The expression of genes is regulated by DNA-binding proteins that activate or repress transcription. The number of DNA sequences and transcription factors that regulate transcription is much greater than originally anticipated. Most genes contain at least 15–20 discrete regulatory elements within 300 bp of the transcription start site. This densely packed promoter region often contains binding sites for ubiquitous transcription factors such as CAAT box/enhancer binding protein (C/EBP), cyclic AMP response element–binding (CREB) protein, selective promoter factor 1 (Sp-1), or activator protein 1 (AP-1). However, factors involved in cell-specific expression may also bind to these sequences. Key regulatory elements may also reside at a large distance from the proximal promoter. The globin and the immunoglobulin genes, for example, contain locus control regions that are several kilobases away from the structural sequences

1	at a large distance from the proximal promoter. The globin and the immunoglobulin genes, for example, contain locus control regions that are several kilobases away from the structural sequences of the gene. Specific groups of transcription factors that bind to these promoter and enhancer sequences provide a combinatorial code for regulating transcription. In this manner, relatively ubiquitous factors interact with more restricted factors to allow each gene to be expressed and regulated in a unique manner that is dependent on developmental state, cell type, and numerous extracellular stimuli. Regulatory factors also bind within the gene itself, particularly in the intronic regions. The transcription factors that bind to DNA actually represent only the first level of regulatory control. Other proteins—co-activators and co-repressors—interact with the DNA-binding transcription factors to generate large regulatory complexes. These complexes are subject to control by numerous

1	control. Other proteins—co-activators and co-repressors—interact with the DNA-binding transcription factors to generate large regulatory complexes. These complexes are subject to control by numerous cell-signaling pathways and enzymes, leading to phosphorylation, acetylation, sumoylation, and ubiquitination. Ultimately, the recruited transcription factors interact with, and stabilize, components of the basal transcription complex that assembles at the site of the TATA box and initiator region. This basal transcription factor complex consists of >30 different proteins. Gene transcription occurs when RNA polymerase begins to synthesize RNA from the DNA template. A large number of identified genetic diseases involve transcription factors (Table 82-2).

1	The field of functional genomics is based on the concept that understanding alterations of gene expression under various physiologic and pathologic conditions provides insight into the underlying functional role of the gene. By revealing specific gene expression profiles, this knowledge may be of diagnostic and therapeutic relevance. The large-scale study of expression profiles, which takes advantage of microarray and bead array technologies, is also referred to as transcriptomics 429 Known Genes (1260) SNPs (612,977) p22.3 p22.1p21.3 p21.1 p15.3 p15.1 p14.3 p14.1 p13 p12.3 p12.1 p11.2 q11.21 q11.22 q11.23 q21.11 p21.13 q21.3 q22.1q22.3q31.1 q31.2 q31.31 q31.33 q32.1 q36.1 q36.3 in 7q31.2 containing the CFTR gene is shown below. The CFTR gene contains 27 exons. More than 1900 mutations in this gene have been found in patients with cystic fibrosis. A 20-kb region encompassing exons 4–9 is shown further amplified to illustrate the SNPs in this region.

1	Chapter 82 Principles of Human Genetics FIGURE 82-4 The origin of haplotypes is due to repeated recombination events occurring in multiple generations. Over time, this leads to distinct haplotypes. These haplotype blocks can often be characterized by genotyping selected Tag single-nucleotide polymorphisms (SNPs), an approach that facilitates performing genome-wide association studies (GWAS). because the complement of mRNAs transcribed by the cellular genome is called the transcriptome. Most studies of gene expression have focused on the regulatory DNA elements of genes that control transcription. However, it should be emphasized that gene expression requires a series of steps, including mRNA processing, protein translation, and posttranslational modifications, all of which are actively regulated (Fig. 82-2).

1	Epigenetic Regulation of Gene Expression Epigenetics describes mechanisms and phenotypic changes that are not a result of variation in the primary DNA nucleotide sequence, but are caused by secondary modifications of DNA or histones. These modifications include heritable changes such as X-inactivation and imprinting, but they can also result from dynamic posttranslational protein modifications in response to environmental influences such as diet, age, or drugs. The epigenetic modifications result in altered expression of individual genes or chromosomal loci encompassing multiple genes. The term epigenome describes the constellation of covalent modifications of DNA and histones that impact chromatin structure, as well as non-coding transcripts that modulate the transcriptional activity of DNA. Although the primary DNA sequence is usually identical in all cells of an organism, tissue-specific changes in the epigenome contribute to determining the transcriptional signature of a cell

1	of DNA. Although the primary DNA sequence is usually identical in all cells of an organism, tissue-specific changes in the epigenome contribute to determining the transcriptional signature of a cell (transcriptome) and hence the protein expression profile (proteome).

1	Mechanistically, DNA and histone modifications can result in the activation or silencing of gene expression (Fig. 82-7). DNA methylation involves the addition of a methyl group to cytosine residues. This is FIGURE 82-5 Copy number variations (CNV) encompass relatively large regions of the genome that have been duplicated or deleted. Chromosome 8 is shown with CNV detected by genomic hybridization. An increase in the signal strength indicates a duplication, a decrease reflects a deletion of the covered chromosomal regions. of the two X chromosome copies present in females.

1	FIGURE 82-6 Crossing-over and genetic recombination. During chiasma formation, either of the two sister chromatids on one chromosome pairs with one of the chromatids of the homologous chromosome. Genetic recombination occurs through crossing-over and results in recombinant and nonrecombinant chromosome segments in the gametes. Together with the random segregation of the maternal and paternal chromosomes, recombination contributes to genetic diversity and forms the basis of the concept of linkage.

1	usually restricted to cytosines of CpG dinucleotides, which are abundant throughout the genome. Methylation of these dinucleotides is thought to represent a defense mechanism that minimizes the expression of sequences that have been incorporated into the genome such as retroviral sequences. CpG dinucleotides also exist in so-called CpG islands, stretches of DNA characterized by a high CG content, which are found in the majority of human gene promoters. CpG islands in promoter regions are typically unmethylated, and the lack of methylation facilitates transcription.

1	Histone methylation involves the addition of a methyl group to lysine residues in histone proteins (Fig. 82-7). Depending on the specific lysine residue being methylated, this alters chromatin configuration, either making it more open or tightly packed. Acetylation of histone proteins is another well-characterized mechanism that results in an open chromatin configuration, which favors active transcription. Acetylation is generally more dynamic than methylation, and many transcriptional activation complexes have histone acetylase activity, whereas repressor complexes often contain deacetylases and remove acetyl groups from histones. Other histone modifications, whose effects are incompletely characterized, include phosphorylation and sumoylation. Lastly, noncoding RNAs that bind to DNA can have a significant impact on transcriptional activity.

1	Physiologically, epigenetic mechanisms play an important role in several instances. For example, X-inactivation refers to the relative silencing of one The inactivation process is a form of dosage compensation such that females (XX) do not generally express twice as many X-chromosomal gene products as males (XY). In a given cell, the choice of which chromosome is inactivated occurs randomly in humans. But once the maternal or paternal X chromosome is inactivated, it will remain inactive, and this information is transmitted with each cell division. The X-inactive specific transcript (Xist) gene encodes a large noncoding RNA that mediates the silencing of the X chromosome from which it is transcribed by coating it with Xist RNA. The inactive X chromosome is highly methylated and has low levels of histone acetylation.

1	Epigenetic gene inactivation also occurs on selected chromosomal regions of autosomes, a phenomenon referred to as genomic imprinting. Through this mechanism, a small subset of genes is only expressed in a monoallelic fashion. Imprinting is heritable and leads to the preferential expression of one of the parental alleles, which deviates from the usual biallelic expression seen for the majority of genes. Remarkably, imprinting can be limited to a subset of tissues. Imprinting is mediated through DNA methylation of one of the alleles. The epigenetic marks on imprinted genes are maintained throughout life, but during zygote formation, they are activated or inactivated in a sex-specific manner (imprint reset) (Fig. 82-8), which allows a differential expression pattern in the fertilized egg and the subsequent mitotic divisions. Appropriate expression of imprinted genes is important for normal development and cellular functions. Imprinting defects and uniparental disomy, which is the

1	egg and the subsequent mitotic divisions. Appropriate expression of imprinted genes is important for normal development and cellular functions. Imprinting defects and uniparental disomy, which is the inheritance of two chromosomes or chromosomal regions from the same parent, are the cause of several developmental disorders such as Beckwith-Wiedemann syndrome, Silver-Russell syndrome, Angelman’s syndrome, and Prader-Willi syndrome (see below). Monoallelic loss-of-function mutations in the GNAS1 gene lead to Albright’s hereditary osteodystrophy (AHO). Paternal transmission of GNAS1 mutations leads to an isolated AHO phenotype (pseudopseudohypoparathyroidism), whereas maternal transmission leads to AHO in combination with hormone resistance to parathyroid hormone, thyrotropin, and gonadotropins (pseudohypoparathyroidism type IA). These phenotypic differences are explained by tissue-specific imprinting of the GNAS1 gene, which is expressed primarily from the maternal allele in the

1	(pseudohypoparathyroidism type IA). These phenotypic differences are explained by tissue-specific imprinting of the GNAS1 gene, which is expressed primarily from the maternal allele in the thyroid, gonadotropes, and the proximal renal tubule. In most other tissues, the GNAS1 gene is expressed biallelically.

1	Abbreviations: CREB, cAMP responsive element–binding protein; HNF, hepatocyte nuclear factor; PML, promyelocytic leukemia; RAR, retinoic acid receptor; SRY, sex-determining region Y; VHL, von Hippel–Lindau. In patients with isolated renal resistance to parathyroid hormone (pseu-It is caused by mutations in the MECP2 gene, which encodes a methyldohypoparathyroidism type IB), defective imprinting of the GNAS1 binding protein. The ensuing aberrant methylation results in abnormal gene results in decreased Gsα expression in the proximal renal tubules. gene expression in neurons, which are otherwise normally developed. Rett’s syndrome is an X-linked dominant disorder resulting in devel-Remarkably, epigenetic differences also occur among monozygotic opmental regression and stereotypic hand movements in affected girls. twins. Although twins are epigenetically indistinguishable during the

1	FIGURE 82-7 Epigenetic modifications of DNA and histones. Methylation of cytosine residues is associated with gene silencing. Methylation of certain genomic regions is inherited (imprinting), and it is involved in the silencing of one of the two X chromosomes in females (X-inactivation). Alterations in methylation can also be acquired, e.g., in cancer cells. Covalent posttranslational modifications of histones play an important role in altering DNA accessibility and chromatin structure and hence in regulating transcription. Histones can be reversibly modified in their amino-terminal tails, which protrude from the nucleosome core particle, by acetylation of lysine, phosphorylation of serine, methylation of lysine and arginine residues, and sumoylation. Acetylation of histones by histone acetylases (HATs), e.g., leads to unwinding of chromatin and accessibility to transcription factors. Conversely, deacetylation by histone deacetylases (HDACs) results in a compact chromatin structure

1	acetylases (HATs), e.g., leads to unwinding of chromatin and accessibility to transcription factors. Conversely, deacetylation by histone deacetylases (HDACs) results in a compact chromatin structure and silencing of transcription.

1	early years of life, older monozygotic twins exhibit differences in the overall content and genomic distribution of DNA methylation and histone acetylation, which would be expected to alter gene expression in various tissues.

1	In cancer, the epigenome is characterized by simultaneous losses and gains of DNA methylation in different genomic regions, as well as repressive histone modifications. Hyperand hypomethylation are associated with mutations in genes that control DNA methylation. Hypomethylation is thought to remove normal control mechanisms that prevent expression of repressed DNA regions. It is also associated with genomic instability. Hypermethylation, in contrast, results in the silencing of CpG islands in promoter regions of genes, including tumor-suppressor genes. Epigenetic alterations are considered to be more easily reversible compared to genetic changes, and modification of the epigenome with demethylating agents and histone deacetylases is being explored in clinical trials. Several organisms have been studied extensively as genetic models, including M. musculus (mouse), D. melanogaster (fruit fly),

1	Several organisms have been studied extensively as genetic models, including M. musculus (mouse), D. melanogaster (fruit fly), C. elegans (nematode), S. cerevisiae (baker’s yeast), and E. coli (colonic bacterium). The ability to use these evolutionarily distant organisms as genetic models that are relevant

1	Maternal somatic cell Paternal somatic cell its functional consequences. Some mutations may be lethal, others are less deleterious, and some may confer an evolutionary advantage. Mutations can occur in the germline (sperm or oocytes); these can be transmitted to progeny. Alternatively, mutations can occur during embryogenesis or in somatic tissues. Mutations that occur during development lead to mosaicism, a situation in which tissues are composed of cells with different genetic constitutions. If the germline is mosaic, a mutation can be transmitted to some progeny but not others, which sometimes leads to confusion in assessing the pattern of inheritance. Somatic mutations that do not affect cell survival can sometimes be detected because of variable phenotypic effects in tissues (e.g., pigmented lesions in McCune-Albright syndrome). Other somatic mutations are associated with neoplasia because they confer a growth advantage to cells. Epigenetic events may also influence gene

1	(e.g., pigmented lesions in McCune-Albright syndrome). Other somatic mutations are associated with neoplasia because they confer a growth advantage to cells. Epigenetic events may also influence gene expression or facilitate genetic damage. With the exception of triplet nucleotide repeats, which can expand (see below), mutations are usually stable.

1	Mutations are structurally diverse—they can involve the entire genome, as in triploidy (one extra set of chromosomes), or gross numerical or structural alterations in chromosomes or individual genes (Chap. 83e). Large deletions may affect a portion of a gene or an entire gene, or, if several genes are involved, they may lead to a contiguous gene

1	Zygote syndrome. Unequal crossing-over between homologous genes can result in fusion gene mutations, as illustrated by color blindness. Mutations involving single nucleotides are referred to as point mutations. Substitutions are called transitions if a purine is replaced by another purine base (A ↔ G) or if a pyrimidine is replaced by another pyrimidine (C ↔ T). Changes from a purine to a pyrimidine, or vice versa, are referred to as transversions. If the DNA sequence change occurs in a coding region and alters an amino acid, it is called a missense mutation. Depending on the functional consequences of such a missense mutation, amino acid substitutions in different regions of the protein can lead to distinct

1	Inactive Methylated pat mat Active Unmethylated FIGURE 82-8 A few genomic regions are imprinted in a parent-specific fashion. The unmethylated chromosomal regions are actively expressed, whereas the methylated regions are silenced. In the germline, the imprint is reset in a parent-specific fashion: both chromo-somes are unmethylated in the maternal (mat) germline and methylated in the paternal (pat) germline. In the zygote, the resulting imprinting pattern is identical with the pattern in the somatic cells of the parents.

1	to human physiology reflects a surprising conservation of genetic pathways and gene function. Transgenic mouse models have been particularly valuable, because many human and mouse genes exhibit similar structure and function and because manipulation of the mouse genome is relatively straightforward compared to that of other mammalian species. Transgenic strategies in mice can be divided into two main approaches: (1) expression of a gene by random insertion into the genome, and (2) deletion or targeted mutagenesis of a gene by homologous recombination with the native endogenous gene (knock-out, knock-in). Previous versions of this chapter provide more detail about the technical principles underlying the development of genetically modified animals. Several databases provide comprehensive information about natural and transgenic animal models, the associated phenotypes, and integrated genetic, genomic, and biologic data (Table 82-1).

1	TRANSMISSION OF GENETIC DISEASE Origins and Types of Mutations A mutation can be defined as any change in the primary nucleotide sequence of DNA regardless of phenotypes. Mutations can occur in all domains of a gene (Fig. 82-9). A point mutation occurring within the coding region leads to an amino acid substitution if the codon is altered (Fig. 82-10). Point mutations that introduce a premature stop codon result in a truncated protein. Large deletions may affect a portion of a gene or an entire gene, whereas small deletions and insertions alter the reading frame if they do not represent a multiple of three bases. These “frameshift” mutations lead to an entirely altered carboxy terminus. Mutations in intronic sequences or in exon junctions may destroy or create splice donor or splice acceptor sites. Mutations may also be found in the regulatory sequences of genes, resulting in reduced or enhanced gene transcription.

1	Certain DNA sequences are particularly susceptible to mutagenesis. Successive pyrimidine residues (e.g., T-T or C-C) are subject to the formation of ultraviolet light–induced photoadducts. If these pyrimidine dimers are not repaired by the nucleotide excision repair pathway, mutations will be introduced after DNA synthesis. The dinucleotide C-G, or CpG, is also a hot spot for a specific type of mutation. In this case, methylation of the cytosine is associated with

1	FIGURE 82-9 Point mutations causing β thalassemia as example of allelic heterogeneity. The β-globin gene is located in the globin gene cluster. Point mutations can be located in the promoter, the CAP site, the 5’-untranslated region, the initiation codon, each of the three exons, the introns, or the polyadenylation signal. Many mutations introduce missense or nonsense mutations, whereas others cause defective RNA splicing. Not shown here are deletion mutations of the β-globin gene or larger deletions of the globin locus that can also result in thalassemia. ▼, promoter mutations; *, CAP site; •, 5’UTR; 1 , initiation codon; ♦, defective RNA processing; ✦, missense and nonsense Chapter 82 Principles of Human Genetics A, Poly A signal.

1	Chapter 82 Principles of Human Genetics A, Poly A signal. an enhanced rate of deamination to uracil, which is then replaced with thymine. This C → T transition (or G → A on the opposite strand) accounts for at least one-third of point mutations associated with polymorphisms and mutations. In addition to the fact that certain types of mutations (C → T or G → A) are relatively common, the nature of the genetic code also results in overrepresentation of certain amino acid substitutions.

1	Polymorphisms are sequence variations that have a frequency of at least 1%. Usually, they do not result in a perceptible phenotype. Often they consist of single base-pair substitutions that do not alter the protein coding sequence because of the degenerate nature of the genetic code (synonymous polymorphism), although it is possible that some might alter mRNA stability, translation, or the amino acid sequence (nonsynonymous polymorphism) (Fig. 82-10). The detection of sequence variants poses a practical problem because mutations is much greater in the male germline than the female germline, in which rates of aneuploidy are increased (Chap. 83e). Thus, the incidence of new point mutations in spermatogonia increases with paternal age (e.g., achondrodysplasia, Marfan’s syndrome, neurofibromatosis). It is estimated that about 1 in 10 sperm carries a new deleterious mutation. The rates for new mutations are calculated most readily for autosomal dominant and X-linked disorders and are

1	It is estimated that about 1 in 10 sperm carries a new deleterious mutation. The rates for new mutations are calculated most readily for autosomal dominant and X-linked disorders and are ~10−5−10−6/locus per generation. Because most monogenic diseases are relatively rare, new mutations account for a significant fraction of cases. This is important in the context of genetic counseling, because a new mutation can be transmitted to the affected individual but does not necessarily imply that the parents are at risk to transmit the disease to other children. An exception to this is when the new mutation occurs early in germline development, leading to gonadal mosaicism.

1	it is often unclear whether it creates 433 a mutation with functional consequences or a benign polymorphism. In this situation, the sequence alteration is described as variant of unknown significance (VUS).

1	mutation rates Mutations represent an important cause of genetic diversity as well as disease. Mutation rates are difficult to determine in humans because many mutations are silent and because testing is often not adequate to detect the phenotypic consequences. Mutation rates vary in different genes but are estimated to occur at a rate of ~10−10/bp per cell division. Germline mutation rates (as opposed to somatic mutations) are relevant in the transmission of genetic disease. Because the population of oocytes is established very early in development, only ~20 cell divisions are required for completed oogenesis, whereas spermatogenesis involves ~30 divisions by the time of puberty and 20 cell divisions each year thereafter. Consequently, the probability of acquiring new point 1 bp Deletion with frameshift

1	FIGURE 82-10 A. Examples of mutations. The coding strand is shown with the encoded amino acid sequence. B. Chromatograms of sequence analyses after amplification of genomic DNA by polymerase chain reaction.

1	unequal crossinG-over Normally, DNA recombination in germ cells occurs with remarkable fidelity to maintain the precise junction sites for the exchanged DNA sequences (Fig. 82-6). However, mispairing of homologous sequences leads to unequal crossover, with gene duplication on one of the chromosomes and gene deletion on the other chromosome. A significant fraction of growth hormone (GH) gene deletions, for example, involve unequal crossing-over (Chap. 402). The GH gene is a member of a large gene cluster that includes a GH variant gene as well as several structurally related chorionic somatomammotropin genes and pseudogenes (highly homologous but functionally inactive relatives of a normal gene). Because such gene clusters contain multiple homologous DNA sequences arranged in tandem, they are particularly prone to undergo recombination and, consequently, gene duplication or deletion. On the other hand, duplication of the PMP22 gene because of unequal crossing-over results in increased

1	are particularly prone to undergo recombination and, consequently, gene duplication or deletion. On the other hand, duplication of the PMP22 gene because of unequal crossing-over results in increased gene dosage and type IA Charcot-Marie-Tooth disease. Unequal crossing-over resulting in deletion of PMP22 causes a distinct neuropathy called hereditary liability to pressure palsy (Chap. 459).

1	Glucocorticoid-remediable aldosteronism (GRA) is caused by a gene fusion or rearrangement involving the genes that encode aldosterone synthase (CYP11B2) and steroid 11β-hydroxylase (CYP11B1), normally arranged in tandem on chromosome 8q. These two genes are 95% identical, predisposing to gene duplication and deletion by unequal crossing-over. The rearranged gene product contains the regulatory regions of 11β-hydroxylase fused to the coding sequence of aldosterone synthetase. Consequently, the latter enzyme is expressed in the adrenocorticotropic hormone (ACTH)–dependent zona fasciculata of the adrenal gland, resulting in overproduction of mineralocorticoids and hypertension (Chap. 406).

1	Gene conversion refers to a nonreciprocal exchange of homologous genetic information. It has been used to explain how an internal portion of a gene is replaced by a homologous segment copied from another allele or locus; these genetic alterations may range from a few nucleotides to a few thousand nucleotides. As a result of gene conversion, it is possible for short DNA segments of two chromosomes to be identical, even though these sequences are distinct in the parents. A practical consequence of this phenomenon is that nucleotide substitutions can occur during gene conversion between related genes, often altering the function of the gene. In disease states, gene conversion often involves intergenic exchange of DNA between a gene and a related pseudogene. For example, the 21-hydroxylase gene (CYP21A2) is adjacent to a nonfunctional pseudogene (CYP21A1P). Many of the nucleotide substitutions that are found in the CYP21A2 gene in patients with congenital adrenal hyperplasia correspond to

1	(CYP21A2) is adjacent to a nonfunctional pseudogene (CYP21A1P). Many of the nucleotide substitutions that are found in the CYP21A2 gene in patients with congenital adrenal hyperplasia correspond to sequences that are present in the CYP21A1P pseudogene, suggesting gene conversion as one cause of mutagenesis. In addition, mitotic gene conversion has been suggested as a mechanism to explain revertant mosaicism in which an inherited mutation is “corrected” in certain cells. For example, patients with autosomal recessive generalized atrophic benign epidermolysis bullosa have acquired reverse mutations in one of the two mutated COL17A1 alleles, leading to clinically unaffected patches of skin.

1	insertions anD Deletions Although many instances of insertions and deletions occur as a consequence of unequal crossing-over, there is also evidence for internal duplication, inversion, or deletion of DNA sequences. The fact that certain deletions or insertions appear to occur repeatedly as independent events indicates that specific regions within the DNA sequence predispose to these errors. For example, certain regions of the DMD gene, which encodes dystrophin, appear to be hot spots for deletions and result in muscular dystrophy (Chap. 462e). Some regions within the human genome are rearrangement hot spots and lead to CNVs.

1	errors in Dna rePair Because mutations caused by defects in DNA repair accumulate as somatic cells divide, these types of mutations are particularly important in the context of neoplastic disorders (Chap. 102e). Several genetic disorders involving DNA repair enzymes underscore their importance. Patients with xeroderma pigmentosum have defects in DNA damage recognition or in the nucleotide excision and repair pathway (Chap. 105). Exposed skin is dry and pigmented and is extraordinarily sensitive to the mutagenic effects of ultraviolet irradiation. More than 10 different genes have been shown to cause the different forms of xeroderma pigmentosum. This finding is consistent with the earlier classification of this disease into different complementation groups in which normal function is rescued by the fusion of cells derived from two different forms of xeroderma pigmentosum.

1	Ataxia telangiectasia causes large telangiectatic lesions of the face, cerebellar ataxia, immunologic defects, and hypersensitivity to ionizing radiation (Chap. 450). The discovery of the ataxia telangiectasia mutated (ATM) gene reveals that it is homologous to genes involved in DNA repair and control of cell cycle checkpoints. Mutations in the ATM gene give rise to defects in meiosis as well as increasing susceptibility to damage from ionizing radiation. Fanconi’s anemia is also associated with an increased risk of multiple acquired genetic abnormalities. It is characterized by diverse congenital anomalies and a strong predisposition to develop aplastic anemia and acute myelogenous leukemia (Chap. 132). Cells from these patients are susceptible to chromosomal breaks caused by a defect in genetic recombination. At least 13 different complementation groups have been identified, and the loci and genes associated with Fanconi’s anemia have been cloned. HNPCC (Lynch’s syndrome) is

1	in genetic recombination. At least 13 different complementation groups have been identified, and the loci and genes associated with Fanconi’s anemia have been cloned. HNPCC (Lynch’s syndrome) is characterized by autosomal dominant transmission of colon cancer, young age (<50 years) of presentation, predisposition to lesions in the proximal large bowel, and associated malignancies such as uterine cancer and ovarian cancer. HNPCC is predominantly caused by mutations in one of several different mismatch repair (MMR) genes including MutS homologue 2 (MSH2), MutL homologue 1 and 6 (MLH1, MLH6), MSH6, PMS1, and PMS2 (Chap. 110). These proteins are involved in the detection of nucleotide mismatches and in the recognition of slipped-strand trinucleotide repeats. Germline mutations in these genes lead to microsatellite instability and a high mutation rate in colon cancer. Genetic screening tests for this disorder are now being used for families considered to be at risk (Chap. 84). Recognition

1	lead to microsatellite instability and a high mutation rate in colon cancer. Genetic screening tests for this disorder are now being used for families considered to be at risk (Chap. 84). Recognition of HNPCC allows early screening with colonoscopy and the implementation of prevention strategies using nonsteroidal anti-inflammatory drugs.

1	unstable Dna sequences Trinucleotide repeats may be unstable and expand beyond a critical number. Mechanistically, the expansion is thought to be caused by unequal recombination and slipped mispairing. A premutation represents a small increase in trinucleotide copy number. In subsequent generations, the expanded repeat may increase further in length and result in an increasingly severe phenotype, a process called dynamic mutation (see below for discussion of anticipation). Trinucleotide expansion was first recognized as a cause of the fragile X syndrome, one of the most common causes of intellectual disability. Other disorders arising from a similar mechanism include Huntington’s disease (Chap. 448), X-linked spinobulbar muscular atrophy (Chap. 452), and myotonic dystrophy (Chap. 462e). Malignant cells are also characterized by genetic instability, indicating a breakdown in mechanisms that regulate DNA repair and the cell cycle.

1	Functional Consequences of Mutations Functionally, mutations can be broadly classified as gain-of-function and loss-of-function mutations. Gain-of-function mutations are typically dominant (e.g., they result in phenotypic alterations when a single allele is affected). Inactivating mutations are usually recessive, and an affected individual is homozygous or compound heterozygous (e.g., carrying two different mutant alleles of the same gene) for the disease-causing mutations. Alternatively, mutation in a single allele can result in haploinsufficiency, a situation in which one normal allele is not sufficient to maintain a normal phenotype. Haploinsufficiency is a commonly observed mechanism in diseases associated with mutations in transcription factors (Table 82-2). Remarkably, the clinical features among patients with an identical mutation in a transcription factor often vary significantly. One mechanism underlying this variability consists in the influence of modifying genes.

1	clinical features among patients with an identical mutation in a transcription factor often vary significantly. One mechanism underlying this variability consists in the influence of modifying genes. Haploinsufficiency can also affect the expression of rate-limiting enzymes. For example, haploinsufficiency in enzymes involved in heme synthesis can cause porphyrias (Chap. 430).

1	An increase in dosage of a gene product may also result in disease, as illustrated by the duplication of the DAX1 gene in dosage-sensitive sex reversal (Chap. 410). Mutation in a single allele can also result in loss of function due to a dominant-negative effect. In this case, the mutated allele interferes with the function of the normal gene product by one of several different mechanisms: (1) a mutant protein may interfere with the function of a multimeric protein complex, as illustrated by mutations in type 1 collagen (COL1A1, COL1A2) genes in osteogenesis imperfecta (Chap. 427); (2) a mutant protein may occupy binding sites on proteins or promoter response elements, as illustrated by thyroid hormone resistance, a disorder in which inactivated thyroid hormone receptor β binds to target genes and functions as an antagonist of normal receptors (Chap. 405); or (3) a mutant protein can be cytotoxic as in α1 antitrypsin deficiency (Chap. 314) or autosomal dominant neurohypophyseal

1	genes and functions as an antagonist of normal receptors (Chap. 405); or (3) a mutant protein can be cytotoxic as in α1 antitrypsin deficiency (Chap. 314) or autosomal dominant neurohypophyseal diabetes insipidus (Chap. 404), in which the abnormally folded proteins are trapped within the endoplasmic reticulum and ultimately cause cellular damage.

1	Genotype and Phnotype • alleles, GenotyPes, anD HaPlotyPes An observed trait is referred to as a phenotype; the genetic information defining the phenotype is called the genotype. Alternative forms of a gene or a genetic marker are referred to as alleles. Alleles may be polymorphic variants of nucleic acids that have no apparent effect on gene expression or function. In other instances, these variants may have subtle effects on gene expression, thereby conferring adaptive advantages associated with genetic diversity. On the other hand, allelic variants may reflect mutations that clearly alter the function of a gene product. The common Glu6Val (E6V) sickle cell mutation in the β-globin gene and the ΔF508 deletion of phenylalanine (F) in the CFTR gene are examples of allelic variants of these genes that result in disease. Because each individual has two copies of each chromosome (one inherited from the mother and one inherited from the father), he or she can have only two alleles at a

1	genes that result in disease. Because each individual has two copies of each chromosome (one inherited from the mother and one inherited from the father), he or she can have only two alleles at a given locus. However, there can be many different alleles in the population. The normal or common allele is usually referred to as wild type. When alleles at a given locus are identical, the individual is homozygous. Inheriting identical copies of a mutant allele occurs in many autosomal recessive disorders, particularly in circumstances of consanguinity or isolated populations. If the alleles are different on the maternal and the paternal copy of the gene, the individual is heterozygous at this locus (Fig. 82-10). If two different mutant alleles are inherited at a given locus, the individual is said to be a compound heterozygote. Hemizygous is used to describe males with a mutation in an X chromosomal gene or a female with a loss of one X chromosomal locus.

1	Genotypes describe the specific alleles at a particular locus. For example, there are three common alleles (E2, E3, E4) of the apolipoprotein E (APOE) gene. The genotype of an individual can therefore be described as APOE3/4 or APOE4/4 or any other variant. These designations indicate which alleles are present on the two chromosomes in the APOE gene at locus 19q13.2. In other cases, the genotype might be assigned arbitrary numbers (e.g., 1/2) or letters (e.g., B/b) to distinguish different alleles.

1	A haplotype refers to a group of alleles that are closely linked together at a genomic locus (Fig. 82-4). Haplotypes are useful for tracking the transmission of genomic segments within families and for detecting evidence of genetic recombination, if the crossover event occurs between the alleles (Fig. 82-6). As an example, various alleles at the histocompatibility locus antigen (HLA) on chromosome 6p are used to establish haplotypes associated with certain disease states. For example, 21-hydroxylase deficiency, complement deficiency, and hemochromatosis are each associated with specific HLA haplotypes. It is now recognized that these genes lie in close proximity to the HLA locus, which explains why HLA associations were identified even before the disease genes were cloned and localized. In other cases, specific HLA associations with diseases such as ankylosing spondylitis (HLA-B27) or type 1 diabetes mellitus (HLA-DR4) reflect the role of specific HLA allelic variants in

1	localized. In other cases, specific HLA associations with diseases such as ankylosing spondylitis (HLA-B27) or type 1 diabetes mellitus (HLA-DR4) reflect the role of specific HLA allelic variants in susceptibility to these autoimmune diseases. The characterization of common SNP haplotypes in numerous populations from different parts of the world through the HapMap Project is providing a novel tool for association studies designed to detect genes involved in the pathogenesis of complex disorders 435 (Table 82-1). The presence or absence of certain haplotypes may also become relevant for the customized choice of medical therapies (pharmacogenomics) or for preventive strategies.

1	Genotype-phenotype correlation describes the association of a specific mutation and the resulting phenotype. The phenotype may differ depending on the location or type of the mutation in some genes. For example, in von Hippel–Lindau disease, an autosomal dominant multisystem disease that can include renal cell carcinoma, hemangioblastomas, and pheochromocytomas, among others, the phenotype varies greatly and the identification of the specific mutation can be clinically useful in order to predict the phenotypic spectrum.

1	allelic HeteroGeneity Allelic heterogeneity refers to the fact that different mutations in the same genetic locus can cause an identical or similar phenotype. For example, many different mutations of the β-globin locus can cause β thalassemia (Table 82-3) (Fig. 82-9). In essence, allelic heterogeneity reflects the fact that many different mutations are capable of altering protein structure and function. For this reason, maps of inactivating mutations in genes usually show a near-random distribution. Exceptions include (1) a founder effect, in which a particular mutation that does not affect reproductive capacity can be traced to a single individual; (2) “hot spots” for mutations, in which the nature of the DNA sequence predisposes to a recurring mutation; and (3) localization of mutations to certain domains that are particularly critical for protein function. Allelic heterogeneity creates a practical problem for genetic testing because one must often examine the entire genetic locus

1	to certain domains that are particularly critical for protein function. Allelic heterogeneity creates a practical problem for genetic testing because one must often examine the entire genetic locus for mutations, because these can differ in each patient. For example, there are currently 1963 reported mutations in the CFTR gene (Fig. 82-3). Mutational analysis may initially focus on a panel of mutations that are particularly frequent (often taking the ethnic background of the patient into account), but a negative result does not exclude the presence of a mutation elsewhere in the gene. One should also be aware that mutational analyses generally focus on the coding region of a gene without considering regulatory and intronic regions. Because disease-causing mutations may be located outside the coding regions, negative results need to be interpreted with caution. The advent of more comprehensive sequencing technologies greatly facilitates concomitant mutational analyses of several genes

1	the coding regions, negative results need to be interpreted with caution. The advent of more comprehensive sequencing technologies greatly facilitates concomitant mutational analyses of several genes after targeted enrichment, or even mutational analysis of the whole exome or genome. However, comprehensive sequencing can result in significant diagnostic challenges because the detection of a sequence alteration alone is not always sufficient to establish that it has a causal role.

1	PHenotyPic HeteroGeneity Phenotypic heterogeneity occurs when more than one phenotype is caused by allelic mutations (e.g., different mutations in the same gene) (Table 82-3). For example, laminopathies are monogenic multisystem disorders that result from mutations in the LMNA gene, which encodes the nuclear lamins A and C. Twelve autosomal dominant and four autosomal recessive disorders are caused by mutations in the LMNA gene. They include several forms of lipodystrophies, Emery-Dreifuss muscular dystrophy, progeria syndromes, a form of neuronal Charcot-Marie-Tooth disease (type 2B1), and a group of overlapping syndromes. Remarkably, hierarchical cluster analysis has revealed that the phenotypes vary depending on the position of the mutation ( genotype-phenotype correlation). Similarly, identical mutations in the FGFR2 gene can result in very distinct phenotypes: Crouzon’s syndrome (craniofacial synostosis) or Pfeiffer’s syndrome (acrocephalopolysyndactyly).

1	locus or nonallelic HeteroGeneity anD PHenocoPies Nonallelic or locus heterogeneity refers to the situation in which a similar disease phenotype results from mutations at different genetic loci (Table 82-3). This often occurs when more than one gene product produces different subunits of an interacting complex or when different genes are involved in the same genetic cascade or physiologic pathway. For example, osteogenesis imperfecta can arise from mutations in two different procollagen genes (COL1A1 or COL1A2) that are located on different chromosomes, and at least eight other genes (Chap. 427). The effects of inactivating mutations in these two genes are similar because the protein products comprise different subunits

1	Chapter 82 Principles of Human Genetics of the helical collagen fiber. Similarly, muscular dystrophy syndromes can be caused by mutations in various genes, consistent with the fact that it can be transmitted in an X-linked (Duchenne or Becker), autosomal dominant (limb-girdle muscular dystrophy type 1), or autosomal recessive (limb-girdle muscular dystrophy type 2) manner (Chap. 462e). Mutations in the X-linked DMD gene, which encodes dystrophin, are the most common cause of muscular dystrophy. This feature reflects the large size of the gene as well as the fact that the phenotype is expressed in hemizygous males because they have only a single copy of the X chromosome. Dystrophin is associated with a large protein complex linked to the membrane-associated cytoskeleton in muscle. Mutations in several different components of this protein complex can also cause muscular dystrophy syndromes. Although the phenotypic features of some of these disorders are distinct, the phenotypic spectrum

1	in several different components of this protein complex can also cause muscular dystrophy syndromes. Although the phenotypic features of some of these disorders are distinct, the phenotypic spectrum caused by mutations in different genes overlaps, thereby leading to nonallelic heterogeneity. It should be noted that mutations in dystrophin also cause allelic heterogeneity. For example, mutations in the DMD gene can cause either Duchenne’s or the less severe Becker’s muscular dystrophy, depending on the severity of the protein defect.

1	Recognition of nonallelic heterogeneity is important for several reasons: (1) the ability to identify disease loci in linkage studies is reduced by including patients with similar phenotypes but different genetic disorders; (2) genetic testing is more complex because several different genes need to be considered along with the possibility of different mutations in each of the candidate genes; and (3) novel information is gained about how genes or proteins interact, providing unique insights into molecular physiology.

1	Phenocopies refer to circumstances in which nongenetic conditions mimic a genetic disorder. For example, features of toxin-or drug-induced neurologic syndromes can resemble those seen in Huntington’s disease, and vascular causes of dementia share phenotypic features with familial forms of Alzheimer’s dementia (Chap. 448). As in nonallelic heterogeneity, the presence of phenocopies has the potential to confound linkage studies and genetic testing. Patient history and subtle differences in phenotype can often provide clues that distinguish these disorders from related genetic conditions.

1	variable exPressivity anD incomPlete Penetrance The same genetic mutation may be associated with a phenotypic spectrum in different affected individuals, thereby illustrating the phenomenon of variable expressivity. This may include different manifestations of a disorder variably involving different organs (e.g., multiple endocrine neoplasia [MEN]), the severity of the disorder (e.g., cystic fibrosis), or the age of disease onset (e.g., Alzheimer’s dementia). MEN 1 illustrates several of these features. In this autosomal dominant tumor syndrome, affected individuals carry an inactivating germline mutation that is inherited in an autosomal dominant fashion. After somatic inactivation of the alternate allele, they can develop tumors of the parathyroid gland, endocrine pancreas, and the pituitary gland (Chap. 408). However, the pattern of tumors in the different glands, the age at which tumors develop, and the types of hormones produced vary among affected individuals, even within a

1	pituitary gland (Chap. 408). However, the pattern of tumors in the different glands, the age at which tumors develop, and the types of hormones produced vary among affected individuals, even within a given family. In this example, the phenotypic variability arises, in part, because of the requirement for a second somatic mutation in the normal copy of the MEN1 gene, as well as the large array of different cell types that are susceptible to the effects of MEN1 gene mutations. In part, variable expression reflects the influence of modifier genes, or genetic background, on the effects of a particular mutation. Even in identical twins, in whom the genetic constitution is essentially the same, one can occasionally see variable expression of a genetic disease.

1	Interactions with the environment can also influence the course of a disease. For example, the manifestations and severity of hemochromatosis can be influenced by iron intake (Chap. 428), and the course of phenylketonuria is affected by exposure to phenylalanine in the diet (Chap. 434e). Other metabolic disorders, such as hyperlipidemias and porphyria, also fall into this category. Many mechanisms, including genetic effects and environmental influences, can therefore lead to variable expressivity. In genetic counseling, it is particularly important to recognize this variability, because one cannot always predict the course of disease, even when the mutation is known.

1	Penetrance refers to the proportion of individuals with a mutant genotype that express the phenotype. If all carriers of a mutant express the phenotype, penetrance is complete, whereas it is said to be incomplete or reduced if some individuals do not exhibit features of the phenotype. Dominant conditions with incomplete penetrance are characterized by skipping of generations with unaffected carriers transmitting the mutant gene. For example, hypertrophic obstructive cardiomyopathy (HCM) caused by mutations in the myosin-binding protein C gene is a dominant disorder with clinical features in only a subset of patients who carry the mutation (Chap. 283). Patients who have the mutation but no evidence of the disease can still transmit the disorder to subsequent generations. In many conditions with postnatal onset, the proportion of gene carriers who are affected varies with age. Thus, when describing penetrance, one has to specify age. For example, for disorders such as Huntington’s

1	with postnatal onset, the proportion of gene carriers who are affected varies with age. Thus, when describing penetrance, one has to specify age. For example, for disorders such as Huntington’s disease or familial amyotrophic lateral sclerosis, which present later in life, the rate of penetrance is influenced by the age at which the clinical assessment is performed. Imprinting can also modify the penetrance of a disease. For example, in patients with Albright’s hereditary osteodystrophy, mutations in the Gsα subunit (GNAS1 gene) are expressed clinically only in individuals who inherit the mutation from their mother (Chap. 424).

1	sex-influenceD PHenotyPes Certain mutations affect males and females quite differently. In some instances, this is because the gene resides on the X or Y sex chromosomes (X-linked disorders and Y-linked disorders). As a result, the phenotype of mutated X-linked genes will be expressed fully in males but variably in heterozygous females, depending on the degree of X-inactivation and the function of the gene. For example, most heterozygous female carriers of factor VIII deficiency (hemophilia A) are asymptomatic because sufficient factor VIII is produced to prevent a defect in coagulation (Chap. 141). On the other hand, some females heterozygous for the X-linked lipid storage defect caused by α-galactosidase A deficiency (Fabry’s disease) experience mild manifestations of painful neuropathy, as well as other features of the disease (Chap. 432e). Because only males have a Y chromosome, mutations in genes such as SRY, which causes male-to-female sex reversal, or DAZ (deleted in

1	neuropathy, as well as other features of the disease (Chap. 432e). Because only males have a Y chromosome, mutations in genes such as SRY, which causes male-to-female sex reversal, or DAZ (deleted in azoospermia), which causes abnormalities of spermatogenesis, are unique to males (Chap. 410).

1	Other diseases are expressed in a sex-limited manner because of the differential function of the gene product in males and females. Activating mutations in the luteinizing hormone receptor cause dominant male-limited precocious puberty in boys (Chap. 411). 437 The phenotype is unique to males because activation of the receptor induces testosterone production in the testis, whereas it is functionally silent in the immature ovary. Biallelic inactivating mutations of the follicle-stimulating hormone (FSH) receptor cause primary ovarian failure in females because the follicles do not develop in the absence of FSH action. In contrast, affected males have a more subtle phenotype, because testosterone production is preserved (allowing sexual maturation) and spermatogenesis is only partially impaired (Chap. 411). In congenital adrenal hyperplasia, most commonly caused by 21-hydroxylase deficiency, cortisol production is impaired and ACTH stimulation of the adrenal gland leads to increased

1	(Chap. 411). In congenital adrenal hyperplasia, most commonly caused by 21-hydroxylase deficiency, cortisol production is impaired and ACTH stimulation of the adrenal gland leads to increased production of androgenic precursors (Chap. 406). In females, the increased androgen level causes ambiguous genitalia, which can be recognized at the time of birth. In males, the diagnosis may be made on the basis of adrenal insufficiency at birth, because the increased adrenal androgen level does not alter sexual differentiation, or later in childhood, because of the development of precocious puberty. Hemochromatosis is more common in males than in females, presumably because of differences in dietary iron intake and losses associated with menstruation and pregnancy in females (Chap. 428).

1	Chromosomal Disorders Chromosomal or cytogenetic disorders are caused by numerical or structural aberrations in chromosomes. For a detailed discussion of disorders of chromosome number and structure, see Chap. 83e. Deviations in chromosome number are common causes of abortions, developmental disorders, and malformations. Contiguous gene syndromes (e.g., large deletions affecting several genes) have been useful for identifying the location of new disease-causing genes. Because of the variable size of gene deletions in different patients, a systematic comparison of phenotypes and locations of deletion breakpoints allows positions of particular genes to be mapped within the critical genomic region.

1	Monogenic Mendelian Disorders Monogenic human diseases are frequently referred to as Mendelian disorders because they obey the principles of genetic transmission originally set forth in Gregor Mendel’s classic work. The continuously updated OMIM catalogue lists several thousand of these disorders and provides information about the clinical phenotype, molecular basis, allelic variants, and pertinent animal models (Table 82-1). The mode of inheritance for a given phenotypic trait or disease is determined by pedigree analysis. All affected and unaffected individuals in the family are recorded in a pedigree using standard symbols (Fig. 82-11). The principles of allelic segregation, and the transmission of alleles from parents to children, are illustrated in Fig. 82-12. One dominant (A) allele and one recessive (a) allele can display three Mendelian modes of inheritance: autosomal dominant, autosomal recessive, and X-linked. About 65% of human monogenic disorders are autosomal dominant,

1	and one recessive (a) allele can display three Mendelian modes of inheritance: autosomal dominant, autosomal recessive, and X-linked. About 65% of human monogenic disorders are autosomal dominant, 25% are autosomal recessive, and 5% are X-linked. Genetic testing is now available for many of these disorders and plays an increasingly important role in clinical medicine (Chap. 84).

1	autosomal Dominant DisorDers These disorders assume particular relevance because mutations in a single allele are sufficient to cause the disease. In contrast to recessive disorders, in which disease pathogenesis is relatively straightforward because there is loss of gene function, dominant disorders can be caused by various disease mechanisms, many of which are unique to the function of the genetic pathway involved.

1	In autosomal dominant disorders, individuals are affected in successive generations; the disease does not occur in the offspring of unaffected individuals. Males and females are affected with equal frequency because the defective gene resides on one of the 22 autosomes (Fig. 82-13A). Autosomal dominant mutations alter one of the two alleles at a given locus. Because the alleles segregate randomly at meiosis, the probability that an offspring will be affected is 50%. Unless there is a new germline mutation, an affected individual has an affected parent. Children with a normal genotype do not transmit the disorder. Due to differences in penetrance or expressivity (see above), the Chapter 82 Principles of Human Genetics Heterozygous Heterozygous Female male female carrier of X-linked trait FIGURE 82-11 Standard pedigree symbols.

1	Chapter 82 Principles of Human Genetics Heterozygous Heterozygous Female male female carrier of X-linked trait FIGURE 82-11 Standard pedigree symbols. clinical manifestations of autosomal dominant disorders may be variable. Because of these variations, it is sometimes challenging to determine the pattern of inheritance. It should be recognized, however, that some individuals acquire a mutated gene from an unaffected parent. De novo germline mutations occur more frequently during later cell divisions in gametogenesis, which explains why siblings are rarely affected. As noted before, new germline mutations occur more frequently in fathers of advanced age. For example, the average age of fathers with new germline mutations that cause Marfan’s syndrome is ~37 years, whereas fathers who transmit the disease by inheritance have an average age of ~30 years.

1	autosomal recessive DisorDers In recessive disorders, the mutated alleles result in a complete or partial loss of function. They frequently involve enzymes in metabolic pathways, receptors, or proteins in signaling cascades. In an autosomal recessive disease, the affected individual, who can be of either sex, is a homozygote or compound heterozygote for a single-gene defect. With a few important exceptions, autosomal recessive diseases are rare and often occur in the context of parental consanguinity. The relatively high frequency of certain recessive disorders such as sickle cell anemia, cystic fibrosis, and thalassemia, is partially explained by a selective biologic advantage for the heterozygous state (see below). Although heterozygous carriers of a defective allele are usually clinically normal, they may display subtle differences in phenotype that only become apparent with more precise testing or in the context of certain environmental influences. In sickle the offspring of

1	normal, they may display subtle differences in phenotype that only become apparent with more precise testing or in the context of certain environmental influences. In sickle the offspring of parents with one dominant (A) and one recessive (a) allele. The distribution of the parental alleles to their offspring depends on the combination present in the parents. Filled symbols = affected individuals.

1	B Autosomal recessive Autosomal recessive with pseudodominance FIGURE 82-13 (A) Dominant, (B) recessive, (C) X-linked, and (D)mitochondrial (matrilinear) inheritance. cell anemia, for example, heterozygotes are normally asymptomatic. However, in situations of dehydration or diminished oxygen pressure, sickle cell crises can also occur in heterozygotes (Chap. 127).

1	In most instances, an affected individual is the offspring of heterozygous parents. In this situation, there is a 25% chance that the offspring will have a normal genotype, a 50% probability of a heterozygous state, and a 25% risk of homozygosity for the recessive alleles (Figs. 82-10, 82-13B). In the case of one unaffected heterozygous and one affected homozygous parent, the probability of disease increases to 50% for each child. In this instance, the pedigree analysis mimics an autosomal dominant mode of inheritance (pseudodominance). In contrast to autosomal dominant disorders, new mutations in recessive alleles are rarely manifest because they usually result in an asymptomatic carrier state.

1	x-linkeD DisorDers Males have only one X chromosome; consequently, a daughter always inherits her father’s X chromosome in addition to one of her mother’s two X chromosomes. A son inherits the Y chromosome from his father and one maternal X chromosome. Thus, the characteristic features of X-linked inheritance are (1) the absence of father-to-son transmission, and (2) the fact that all daughters of an affected male are obligate carriers of the mutant allele (Fig. 82-13C). The risk of developing disease due to a mutant X-chromosomal gene differs in the two sexes. Because males have only one X chromosome, they are hemizygous for the mutant allele; thus, they are more likely to develop the mutant phenotype, regardless of whether the mutation is dominant or recessive. A female may be either heterozygous or homozygous for the mutant allele, which may be dominant or recessive. The terms X-linked dominant or X-linked recessive are therefore only applicable to expression of the mutant

1	heterozygous or homozygous for the mutant allele, which may be dominant or recessive. The terms X-linked dominant or X-linked recessive are therefore only applicable to expression of the mutant phenotype in women. In addition, the expression of X-chromosomal genes is influenced by X chromosome inactivation.

1	y-linkeD DisorDers The Y chromosome has a relatively small number of genes. One such gene, the sex-region determining Y factor (SRY), which encodes the testis-determining factor (TDF), is crucial for normal male development. Normally there is infrequent exchange of sequences on the Y chromosome with the X chromosome. The SRY region is adjacent to the pseudoautosomal region, a chromosomal segment on the X and Y chromosomes with a high degree of homology. A crossing-over event occasionally involves the SRY region with the distal tip of the X chromosome during meiosis in the male. Translocations can result in XY females with the Y chromosome lacking the SRY gene or XX males harboring the SRY gene on one of the X chromosomes (Chap. 410). Point mutations in the SRY gene may also result in individuals with an XY genotype and an incomplete female phenotype. Most of these mutations occur de novo. Men with oligospermia/azoospermia frequently have microdeletions on the long arm of the Y

1	in individuals with an XY genotype and an incomplete female phenotype. Most of these mutations occur de novo. Men with oligospermia/azoospermia frequently have microdeletions on the long arm of the Y chromosome that involve one or more of the azoospermia factor (AZF) genes.

1	Exceptions to Simple Mendelian Inheritance Patterns • mitocHonDrial DisorDers Mendelian inheritance refers to the transmission of genes encoded by DNA contained in the nuclear chromosomes. In addition, each mitochondrion contains several copies of a small circular chromosome (Chap. 85e). The mitochondrial DNA (mtDNA) is ~16.5 kb and encodes transfer and ribosomal RNAs and 13 core proteins that are components of the respiratory chain involved in oxidative phosphorylation and ATP generation. The mitochondrial genome does not recombine and is inherited through the maternal line because sperm does not contribute significant cytoplasmic components to the zygote. A noncoding region of the mitochondrial chromosome, referred to as D-loop, is highly polymorphic. This property, together with the absence of mtDNA recombination, makes it a valuable tool for studies tracing human migration and evolution, and it is also used for specific forensic applications.

1	Inherited mitochondrial disorders are transmitted in a matrilineal fashion; all children from an affected mother will inherit the disease, but it will not be transmitted from an affected father to his children (Fig. 82-13D). Alterations in the mtDNA that involves enzymes required for oxidative phosphorylation lead to reduction of ATP supply, generation of free radicals, and induction of apoptosis. Several syndromic disorders arising from mutations in the mitochondrial genome are known in humans and they affect both protein-coding and tRNA genes (Chap. 85e). The broad clinical spectrum often involves (cardio) myopathies and encephalopathies because of the high dependence of these tissues on oxidative phosphorylation. The age of onset and the clinical course are highly variable because of the unusual mechanisms of mtDNA transmission, which replicates independently from nuclear DNA. During cell replication, the proportion of wild-type and mutant mitochondria can drift among different

1	the unusual mechanisms of mtDNA transmission, which replicates independently from nuclear DNA. During cell replication, the proportion of wild-type and mutant mitochondria can drift among different cells and tissues. The resulting heterogeneity in the proportion of mitochondria with and without a mutation is referred to as heteroplasmia and underlies the phenotypic variability that is characteristic of mitochondrial diseases.

1	Acquired somatic mutations in mitochondria are thought to be involved in several age-dependent degenerative disorders affecting predominantly muscle and the peripheral and central nervous system (e.g., Alzheimer’s and Parkinson’s diseases). Establishing that an mtDNA alteration is causal for a clinical phenotype is challenging because of the high degree of polymorphism in mtDNA and the phenotypic variability characteristic of these disorders. Certain pharmacologic treatments may have an impact on mitochondria and/or their function. For example, treatment with the antiretroviral compound azidothymidine (AZT) causes an acquired mitochondrial myopathy through depletion of muscular mtDNA.

1	mosaicism Mosaicism refers to the presence of two or more genetically 439 distinct cell lines in the tissues of an individual. It results from a mutation that occurs during embryonic, fetal, or extrauterine development. The developmental stage at which the mutation arises will determine whether germ cells and/or somatic cells are involved. Chromosomal mosaicism results from nondisjunction at an early embryonic mitotic division, leading to the persistence of more than one cell line, as exemplified by some patients with Turner’s syndrome (Chap. 410). Somatic mosaicism is characterized by a patchy distribution of genetically altered somatic cells. The McCune-Albright syndrome, for example, is caused by activating mutations in the stimulatory G protein α (Gsα) that occur early in development (Chap. 424). The clinical phenotype varies depending on the tissue distribution of the mutation; manifestations include ovarian cysts that secrete sex steroids and cause precocious puberty,

1	(Chap. 424). The clinical phenotype varies depending on the tissue distribution of the mutation; manifestations include ovarian cysts that secrete sex steroids and cause precocious puberty, polyostotic fibrous dysplasia, café-au-lait skin pigmentation, growth hormone–secreting pituitary adenomas, and hypersecreting autonomous thyroid nodules (Chap. 412).

1	x-inactivation, imPrintinG, anD uniParental Disomy According to traditional Mendelian principles, the parental origin of a mutant gene is irrelevant for the expression of the phenotype. There are, however, important exceptions to this rule. X-inactivation prevents the expression of most genes on one of the two X chromosomes in every cell of a female. Gene inactivation through genomic imprinting occurs on selected chromosomal regions of autosomes and leads to inheritable preferential expression of one of the parental alleles. It is of pathophysiologic importance in disorders where the transmission of disease is dependent on the sex of the transmitting parent and, thus, plays an important role in the expression of certain genetic disorders. Two classic examples are the Prader-Willi syndrome and Angelman’s syndrome (Chap. 83e). Prader-Willi syndrome is characterized by diminished fetal activity, obesity, hypotonia, mental retardation, short stature, and hypogonadotropic hypogonadism.

1	and Angelman’s syndrome (Chap. 83e). Prader-Willi syndrome is characterized by diminished fetal activity, obesity, hypotonia, mental retardation, short stature, and hypogonadotropic hypogonadism. Deletions of the paternal copy of the Prader-Willi locus located on the short arm of chromosome 15 result in a contiguous gene syndrome involving missing paternal copies of the necdin and SNRPN genes, among others. In contrast, patients with Angelman’s syndrome, characterized by mental retardation, seizures, ataxia, and hypotonia, have deletions involving the maternal copy of this region on chromosome 15. These two syndromes may also result from uniparental disomy. In this case, the syndromes are not caused by deletions on chromosome 15 but by the inheritance of either two maternal chromosomes (Prader-Willi syndrome) or two paternal chromosomes (Angelman’s syndrome). Lastly, the two distinct phenotypes can also be caused by an imprinting defect that impairs the resetting of the imprint during

1	syndrome) or two paternal chromosomes (Angelman’s syndrome). Lastly, the two distinct phenotypes can also be caused by an imprinting defect that impairs the resetting of the imprint during zygote development (defect in the father leads to Prader-Willi syndrome; defect in the mother leads to Angelman’s syndrome).

1	Imprinting and the related phenomenon of allelic exclusion may be more common than currently documented, because it is difficult to examine levels of mRNA expression from the maternal and paternal alleles in specific tissues or in individual cells. Genomic imprinting, or uniparental disomy, is involved in the pathogenesis of several other disorders and malignancies (Chap. 83e). For example, hydatidiform moles contain a normal number of diploid chromosomes, but they are all of paternal origin. The opposite situation occurs in ovarian teratomata, with 46 chromosomes of maternal origin. Expression of the imprinted gene for insulin-like growth factor II (IGF-II) is involved in the pathogenesis of the cancer-predisposing Beckwith-Wiedemann syndrome (BWS) (Chap. 101e). These children show somatic overgrowth with organomegalies and hemihypertrophy, and they have an increased risk of embryonal malignancies such as Wilms’ tumor. Normally, only the paternally derived copy of the IGF-II gene is

1	overgrowth with organomegalies and hemihypertrophy, and they have an increased risk of embryonal malignancies such as Wilms’ tumor. Normally, only the paternally derived copy of the IGF-II gene is active and the maternal copy is inactive. Imprinting of the IGF-II gene is regulated by H19, which encodes an RNA transcript that is not translated into protein. Disruption or lack of H19 methylation leads to a relaxation of IGF-II imprinting and expression of both alleles. Alterations of the epigenome through gain and loss of DNA methylation, as well as altered histone modifications, play an important role in the pathogenesis of malignancies.

1	Chapter 82 Principles of Human Genetics somatic mutations Cancer can be considered a genetic disease at the cellular level (Chap. 101e). Cancers are monoclonal in origin, indicating that they have arisen from a single precursor cell with one or several mutations in genes controlling growth (proliferation or apoptosis) and/or differentiation. These acquired somatic mutations are restricted to the tumor and its metastases and are not found in the surrounding normal tissue. The molecular alterations include dominant gain-of-function mutations in oncogenes, recessive loss-offunction mutations in tumor-suppressor genes and DNA repair genes, gene amplification, and chromosome rearrangements. Rarely, a single mutation in certain genes may be sufficient to transform a normal cell into a malignant cell. In most cancers, however, the development of a malignant phenotype requires several genetic alterations for the gradual progression from a normal cell to a cancerous cell, a phenomenon termed

1	cell. In most cancers, however, the development of a malignant phenotype requires several genetic alterations for the gradual progression from a normal cell to a cancerous cell, a phenomenon termed multistep carcinogenesis (Chaps. 101e and 102e). Genome-wide analyses of cancers using deep sequencing often reveal somatic rearrangements resulting in fusion genes and mutations in multiple genes. Comprehensive sequence analyses provide further insight into genetic heterogeneity within malignancies; these include intratumoral heterogeneity among the cells of the primary tumor, intermetastatic and intrametastatic heterogeneity, and interpatient differences. These analyses further support the notion of cancer as an ongoing process of clonal evolution, in which successive rounds of clonal selection within the primary tumor and metastatic lesions result in diverse genetic and epigenetic alterations that require targeted (personalized) therapies. The heterogeneity of mutations within a tumor

1	within the primary tumor and metastatic lesions result in diverse genetic and epigenetic alterations that require targeted (personalized) therapies. The heterogeneity of mutations within a tumor can also lead to resistance to target therapies because cells with mutations that are resistant to the therapy, even if they are a minor part of the tumor population, will be selected as the more sensitive cells are killed. Most human tumors express telomerase, an enzyme formed of a protein and an RNA component, which adds telomere repeats at the ends of chromosomes during replication. This mechanism impedes shortening of the telomeres, which is associated with senescence in normal cells and is associated with enhanced replicative capacity in cancer cells. Telomerase inhibitors provide a novel strategy for treating advanced human cancers.

1	In many cancer syndromes, there is an inherited predisposition to tumor formation. In these instances, a germline mutation is inherited in an autosomal dominant fashion inactivating one allele of an autosomal tumor-suppressor gene. If the second allele is inactivated by a somatic mutation or by epigenetic silencing in a given cell, this will lead to neoplastic growth (Knudson two-hit model). Thus, the defective allele in the germline is transmitted in a dominant mode, although tumorigenesis results from a biallelic loss of the tumor-suppressor gene in an affected tissue. The classic example to illustrate this phenomenon is retinoblastoma, which can occur as a sporadic or hereditary tumor. In sporadic retinoblastoma, both copies of the retinoblastoma (RB) gene are inactivated through two somatic events. In hereditary retinoblastoma, one mutated or deleted RB allele is inherited in an autosomal dominant manner and the second allele is inactivated by a subsequent somatic mutation. This

1	somatic events. In hereditary retinoblastoma, one mutated or deleted RB allele is inherited in an autosomal dominant manner and the second allele is inactivated by a subsequent somatic mutation. This two-hit model applies to other inherited cancer syndromes such as MEN 1 (Chap. 408) and neurofibromatosis type 2 (Chap. 118).

1	nucleotiDe rePeat exPansion DisorDers Several diseases are associated with an increase in the number of nucleotide repeats above a certain threshold (Table 82-4). The repeats are sometimes located within the coding region of the genes, as in Huntington’s disease or the X-linked form of spinal and bulbar muscular atrophy (SBMA; Kennedy’s syndrome). In other instances, the repeats probably alter gene regulatory sequences. If an expansion is present, the DNA fragment is unstable and tends to expand further during cell division. The length of the nucleotide repeat often correlates with the severity of the disease. When repeat length increases from one generation to the next, disease manifestations may worsen or be observed at an earlier age; this phenomenon is referred to as anticipation. In Huntington’s disease, for example, there is a correlation between age of onset and length of the triplet codon expansion (Chap. 444e). Anticipation has also been documented in other diseases caused by

1	disease, for example, there is a correlation between age of onset and length of the triplet codon expansion (Chap. 444e). Anticipation has also been documented in other diseases caused by dynamic mutations in trinucleotide repeats (Table 82-4). The repeat number may also vary in a tissue-specific manner. In myotonic dystrophy, the CTG repeat may be tenfold greater in muscle tissue than in lymphocytes (Chap. 462e).

1	Complex Genetic Disorders The expression of many common diseases such as cardiovascular disease, hypertension, diabetes, asthma, psychiatric disorders, and certain cancers is determined by a combination of genetic background, environmental factors, and lifestyle. A trait is called polygenic if multiple genes contribute to the phenotype or multifactorial if multiple genes are assumed to interact with environmental factors. Genetic models for these complex traits need to account for genetic heterogeneity and interactions with other genes and the environment. Complex genetic traits may be influenced by modifier genes that are not linked to the main gene involved in the pathogenesis of the trait. This type of gene-gene interaction, or epistasis, plays an important role in polygenic traits that require the simultaneous presence of variations in multiple genes to result in a pathologic phenotype.

1	Type 2 diabetes mellitus provides a paradigm for considering a multifactorial disorder, because genetic, nutritional, and lifestyle factors are intimately interrelated in disease pathogenesis (Table 82-5) (Chap. 417).

1	The identification of genetic variations and environmental factors that either predispose to or protect against disease is essential for predicting disease risk, designing preventive strategies, and developing novel therapeutic approaches. The study of rare monogenic diseases may provide insight into some of the genetic and molecular mechanisms important in the pathogenesis of complex diseases. For example, the identification of the genes causing monogenic forms of permanent neonatal diabetes mellitus or maturity-onset diabetes defined them as candidate genes in the pathogenesis of diabetes mellitus type 2 (Tables 82-2 and 82-5). Genome scans have identified numerous genes and loci that may be associated with susceptibility to development of diabetes mellitus in certain populations. Efforts to identify susceptibility genes require very large sample sizes, and positive results may depend on ethnicity, ascertainment criteria, and statistical analysis. Association studies analyzing the

1	to identify susceptibility genes require very large sample sizes, and positive results may depend on ethnicity, ascertainment criteria, and statistical analysis. Association studies analyzing the potential influence of (biologically functional) SNPs and SNP haplotypes on a particular phenotype are providing new insights into the genes involved in the pathogenesis of these common disorders. Large variants ([micro]deletions, duplications, and inversions) present in the human population also contribute to the pathogenesis of complex disorders, but their contributions remain poorly understood.

1	Linkage and Association Studies There are two primary strategies for mapping genes that cause or increase susceptibility to human disease: (1) classic linkage can be performed based on a known genetic model or, when the model is unknown, by studying pairs of affected relatives; or (2) disease genes can be mapped using allelic association studies (Table 82-6). Genetic linkaGe Genetic linkage refers to the fact that genes are physically connected, or linked, to one another along the chromosomes.

1	Genetic linkaGe Genetic linkage refers to the fact that genes are physically connected, or linked, to one another along the chromosomes. Two fundamental principles are essential for understanding the concept of linkage: (1) when two genes are close together on a chromosome, they are usually transmitted together, unless a recombination event separates them (Figs. 82-6); and (2) the odds of a crossover, or recombination event, between two linked genes is proportional to the distance that separates them. Thus, genes that are farther apart are more likely to undergo a recombination event than genes that are very close together. The detection of chromosomal loci that segregate with a disease by linkage can be used to identify the gene responsible for the disease (positional cloning) and to predict the odds of disease gene transmission in genetic counseling.

1	Polymorphisms are essential for linkage studies because they provide a means to distinguish the maternal and paternal chromosomes in an individual. On average, 1 out of every 1000 bp varies from one person to the next. Although this degree of variation seems low (99.9% identical), it means that >3 million sequence differences exist between any two unrelated individuals and the probability that the sequence at such loci will differ on the two homologous chromosomes is high (often >70–90%). These sequence variations include variable number of tandem repeats (VNTRs), short tandem repeats (STRs), and SNPs. Most STRs, also called polymorphic microsatellite markers, consist of di-, tri-, or tetranucleotide repeats that can be characterized readily using the polymerase chain reaction (PCR). Characterization of SNPs, using DNA chips or beads, permits comprehensive analyses of genetic variation, linkage, and association studies. Although these sequence variations often have no apparent

1	Characterization of SNPs, using DNA chips or beads, permits comprehensive analyses of genetic variation, linkage, and association studies. Although these sequence variations often have no apparent functional consequences, they provide much of the basis for variation in genetic traits.

1	In order to identify a chromosomal locus that segregates with a disease, it is necessary to characterize polymorphic DNA markers from affected and unaffected individuals of one or several pedigrees. One can Abbreviation: GWAS, genome-wide association study. Suitable for identification of susceptibility genes in Requires large sample size and matched control polygenic and multifactorial disorders population Suitable for testing specific allelic variants of known False-positive results in the absence of suitable candidate loci control population

1	Suitable for testing specific allelic variants of known False-positive results in the absence of suitable candidate loci control population Facilitated by HapMap data, making GWAS more feasible Candidate gene approach does not permit detection of novel genes and pathways then assess whether certain marker alleles cosegregate with the disease. Markers that are closest to the disease gene are less likely to undergo recombination events and therefore receive a higher linkage score. Linkage is expressed as a lod (logarithm of odds) score—the ratio of the probability that the disease and marker loci are linked rather than unlinked. Lod scores of +3 (1000:1) are generally accepted as supporting linkage, whereas a score of –2 is consistent with the absence of linkage.

1	allelic association, linkaGe Disequilibrium, anD HaPlotyPes Allelic association refers to a situation in which the frequency of an allele is significantly increased or decreased in individuals affected by a particular disease in comparison to controls. Linkage and association differ in several aspects. Genetic linkage is demonstrable in families or sibships. Association studies, on the other hand, compare a population of affected individuals with a control population. Association studies can be performed as case-control studies that include unrelated affected individuals and matched controls or as family-based studies that compare the frequencies of alleles transmitted or not transmitted to affected children.

1	Allelic association studies are particularly useful for identifying susceptibility genes in complex diseases. When alleles at two loci occur more frequently in combination than would be predicted (based on known allele frequencies and recombination fractions), they are said to be in linkage disequilibrium. Evidence for linkage disequilibrium can be helpful in mapping disease genes because it suggests that the two loci are tightly linked.

1	Detecting the genetic factors contributing to the pathogenesis of common complex disorders remains a great challenge. In many instances, these are low-penetrance alleles (e.g., variations that individually have a subtle effect on disease development, and they can only be identified by unbiased GWAS) (Catalog of Published Genome-Wide Association Studies; Table 82-1) (Fig. 82-14). Most variants occur in noncoding or regulatory sequences but do not alter protein structure. The analysis of complex disorders is further complicated by ethnic differences in disease prevalence, differences in allele frequencies in known susceptibility genes among different populations, locus and allelic heterogeneity, gene-gene and gene-environment interactions, and the possibility of phenocopies. The data generated by the HapMap Project are greatly facilitating GWAS for the characterization of complex disorders. Adjacent SNPs are inherited together as blocks, and these blocks can be identified by genotyping

1	by the HapMap Project are greatly facilitating GWAS for the characterization of complex disorders. Adjacent SNPs are inherited together as blocks, and these blocks can be identified by genotyping selected marker SNPs, so-called Tag SNPs, thereby reducing cost and workload (Fig. 82-4). The availability of this information permits the characterization of a limited number of SNPs to identify the set of haplotypes present in an individual (e.g., in cases and controls). This, in turn, permits performing GWAS by searching for associations of certain haplotypes with a disease phenotype of interest, an essential step for unraveling the genetic factors contributing to complex disorders.

1	PoPulation Genetics In population genetics, the focus changes from alterations in an individual’s genome to the distribution pattern of different genotypes in the population. In a case where there are only two alleles, A and a, the frequency of the genotypes will be p2 + 2pq + q2 = 1, with p2 corresponding to the frequency of AA, 2pq to the frequency of Aa, and q2 to aa. When the frequency of an allele is known, the frequency of the genotype can be calculated. Alternatively, one can determine an allele frequency if the genotype frequency has been determined.

1	Allele frequencies vary among ethnic groups and geographic regions. For example, heterozygous mutations in the CFTR gene are relatively common in populations of European origin but are rare in the African population. Allele frequencies may vary because certain allelic variants confer a selective advantage. For example, heterozygotes for the sickle cell mutation, which is particularly common in West Africa, are more resistant to malarial infection because the erythrocytes of heterozygotes provide a less favorable environment for Plasmodium parasites. Although homozygosity for the sickle cell mutation is associated with severe anemia and sickle crises (Chap. 127), heterozygotes have a higher probability of survival because of the reduced morbidity and mortality from malaria; this phenomenon has led to an increased frequency of the mutant allele. Recessive conditions are more prevalent in geographically isolated populations because of the more restricted gene pool.

1	APPROACH TO THE PATIENT: For the practicing clinician, the family history remains an essential step in recognizing the possibility of a hereditary predisposition to disease. When taking the history, it is useful to draw a detailed 443 Rare alleles Mendelian disease Low frequency variants with intermediate effect Typical: Common variants with low effect on complex disease Rare: Common variants with high effect on complex disease Rare variants with small effect: difficult to identify Effect size 50 3.0 1.5 1.1 High Intermediate Modest Low 0.001 0.005 0.05 FIGURE 82-14 Relationship between allele frequency and effect size in monogenic and polygenic disorders. In classic Mendelian disorders, the allele frequency is typically low but has a high impact (single gene disorder). This contrasts with polygenic disorders that require the combination of multiple low impact alleles that are frequently quite common in the general population.

1	pedigree of the first-degree relatives (e.g., parents, siblings, and children), because they share 50% of genes with the patient. Standard symbols for pedigrees are depicted in Fig. 82-11. The family history should include information about ethnic background, age, health status, and deaths, including infants. Next, the physician should explore whether there is a family history of the same or related illnesses to the current problem. An inquiry focused on commonly occurring disorders such as cancers, heart disease, and diabetes mellitus should follow. Because of the possibility of age-dependent expressivity and penetrance, the family history will need intermittent updating. If the findings suggest a genetic disorder, the clinician should assess whether some of the patient’s relatives may be at risk of carrying or transmitting the disease. In this circumstance, it is useful to confirm and extend the pedigree based on input from several family members. This information may form the basis

1	at risk of carrying or transmitting the disease. In this circumstance, it is useful to confirm and extend the pedigree based on input from several family members. This information may form the basis for genetic counseling, carrier detection, early intervention, and disease prevention in relatives of the index patient (Chap. 84).

1	In instances where a diagnosis at the molecular level may be relevant, it is important to identify an appropriate laboratory that can perform the appropriate test. Genetic testing is available for a rapidly growing number of monogenic disorders through commercial laboratories. For uncommon disorders, the test may only be performed in a specialized research laboratory. Approved laboratories offering testing for inherited disorders can be identified in continuously updated online resources (e.g., GeneTests; Table 82-1). If genetic testing is considered, the patient and the family should be counseled about the potential implications of positive results, including psychological distress and the possibility of discrimination. The patient or caretakers should be informed about the meaning of a negative result, technical limitations, and the possibility of false-negative and inconclusive results. For these reasons, genetic testing should only be performed after obtaining informed consent.

1	a negative result, technical limitations, and the possibility of false-negative and inconclusive results. For these reasons, genetic testing should only be performed after obtaining informed consent. Published ethical guidelines address the specific aspects that should be considered when testing children and adolescents. Genetic testing should usually be limited to situations in which the results may have an impact on medical management.

1	Genomic medicine aims to enhance the quality of medical care through the use of genotypic analysis (DNA testing) to identify genetic predisposition to disease, to select more specific pharmacotherapy, and to design individualized medical care based on genotype. Genotype can be deduced by analysis of protein (e.g., hemoglobin, apoprotein E), mRNA, or DNA. However, technologic advances have made DNA analysis particularly useful because it can be readily applied.

1	DNA testing is performed by mutational analysis or linkage studies in individuals at risk for a genetic disorder known to be present in a family. Mass screening programs require tests of high sensitivity and specificity to be cost-effective. Prerequisites for the success of genetic screening programs include the following: that the disorder is potentially serious; that it can be influenced at a presymptomatic stage by changes in behavior, diet, and/or pharmaceutical manipulations; and that the screening does not result in any harm or discrimination. Screening in Jewish populations for the autosomal recessive neurodegenerative storage disease Tay-Sachs has reduced the number of affected individuals. In contrast, screening for sickle cell trait/disease in African Americans has led to unanticipated problems of discrimination by health insurers and employers. Mass screening programs harbor additional potential problems. For example, screening for the most common genetic alteration in

1	problems of discrimination by health insurers and employers. Mass screening programs harbor additional potential problems. For example, screening for the most common genetic alteration in cystic fibrosis, the ΔF508 mutation with a frequency of ~70% in northern Europe, is feasible and seems to be effective. One has to keep in mind, however, that there is pronounced allelic heterogeneity and that the disease can be caused by about 2000 other mutations. The search for these less common mutations would substantially increase costs but not the effectiveness of the screening program as a whole. Next-generation genome sequencing permits comprehensive and cost-effective mutational analyses after selective enrichment of candidate genes. For example, tests that sequence all the common genes causing hereditary deafness are already commercially available. Occupational screening programs aim to detect individuals with increased risk for certain professional activities (e.g., α1 antitrypsin

1	causing hereditary deafness are already commercially available. Occupational screening programs aim to detect individuals with increased risk for certain professional activities (e.g., α1 antitrypsin deficiency and smoke or dust exposure). Integrating genomic data into electronic medical records is evolving and may provide significant decision support at the point of care, for example, by providing the clinician with genomic data and decision algorithms for the prescription of drugs that are subject to pharmacogenetic influences.

1	Mutational Analyses DNA sequence analysis is now widely used as a diagnostic tool and has significantly enhanced diagnostic accuracy. It is used for determining carrier status and for prenatal testing in monogenic disorders (Chap. 84). Numerous techniques, Chapter 82 Principles of Human Genetics discussed in previous versions of this chapter, are available for the detection of mutations. In a very broad sense, one can distinguish between techniques that allow for screening of known mutations (screening mode) or techniques that definitively characterize mutations. Analyses of large alterations in the genome are possible using classic methods such as cytogenetics, fluorescent in situ hybridization (FISH), and Southern blotting (Chap. 83e), as well as more sensitive novel techniques that search for multiple single exon deletions or duplications.

1	More discrete sequence alterations rely heavily on the use of PCR, which allows rapid gene amplification and analysis. Moreover, PCR makes it possible to perform genetic testing and mutational analysis with small amounts of DNA extracted from leukocytes or even from single cells, buccal cells, or hair roots. DNA sequencing can be performed directly on PCR products or on fragments cloned into plasmid vectors amplified in bacterial host cells. Sequencing of all exons of the genome or selected chromosomes, or sequencing of numerous candidate genes in a single run, is now possible with next-generation sequencing platforms.

1	The majority of traditional diagnostic methods were gel-based. Novel technologies for the analysis of mutations, genotyping, large-scale sequencing, and mRNA expression profiles are undergoing rapid evolution. DNA chip technologies allow hybridization of DNA or RNA to hundreds of thousands of probes simultaneously. Microarrays are being used clinically for mutational analysis of several human disease genes, as well as for the identification of viral or bacterial sequence variations. With advances in high-throughput DNA sequencing technology, complete sequencing of the genome or an exome has entered the clinical realm. Although comprehensive sequencing of large genomic regions or multiple genes is already a reality, the subsequent bioinformatics analysis, assembly of sequence fragments, and comparative alignments remains a significant and commonly underestimated challenge. The discovery of incidental (or secondary) findings that are unrelated to the indication for the sequencing

1	and comparative alignments remains a significant and commonly underestimated challenge. The discovery of incidental (or secondary) findings that are unrelated to the indication for the sequencing analysis but indicators of other disorders of potential relevance for patient care can pose a difficult ethical dilemma. It can lead to the detection of undiagnosed medically actionable genetic conditions, but can also reveal deleterious mutations that cannot be influenced, as numerous sequence variants are of unknown significance.

1	A general algorithm for the approach to mutational analysis is outlined in Fig. 82-15. The importance of a detailed clinical phenotype cannot be overemphasized. This is the step where one should also consider the possibility of genetic heterogeneity and phenocopies. If obvious candidate genes are suggested by the phenotype, they can be analyzed directly. After identification of a mutation, it is essential to demonstrate that it segregates with the phenotype. The functional characterization of novel mutations is labor intensive and may require analyses in vitro or in transgenic models in order to document the relevance of the genetic alteration.

1	Prenatal diagnosis of numerous genetic diseases in instances with a high risk for certain disorders is now possible by direct DNA analysis. Amniocentesis involves the removal of a small amount of amniotic fluid, usually at 16 weeks of gestation. Cells can be collected and submitted for karyotype analyses, FISH, and mutational analysis of selected genes. The main indications for amniocentesis include advanced maternal age (>35 years), an abnormal serum triple marker test (α-fetoprotein, β human chorionic gonadotropin, pregnancy-associated plasma protein A, or unconjugated estriol), a family history of chromosomal abnormalities, or a Mendelian disorder amenable to genetic testing. Prenatal diagnosis can also be performed by chorionic villus sampling (CVS), in which a small amount of the chorion is removed by a transcervical or transabdominal biopsy. Chromosomes and DNA obtained from these cells can be submitted for cytogenetic and mutational analyses. CVS can be performed earlier in

1	chorion is removed by a transcervical or transabdominal biopsy. Chromosomes and DNA obtained from these cells can be submitted for cytogenetic and mutational analyses. CVS can be performed earlier in gestation (weeks 9–12) than amniocentesis, an aspect that may be of relevance when termination of pregnancy is a consideration. Later in pregnancy, beginning at about 18 weeks of gestation, percutaneous umbilical blood sampling (PUBS) permits collection of fetal blood for lymphocyte culture and analysis. Recently, the entire fetal genome has been determined prenatally from cells taken from the mother’s plasma through deep sequencing and the counting of parental haplotypes, or by inferring it from DNA sequences obtained from blood samples from the mother, father, and umbilical cord. These approaches enable screening for clinically relevant and deleterious alleles inherited from the parents, as well as for de novo germline mutations, and they may have the potential to change the diagnosis

1	enable screening for clinically relevant and deleterious alleles inherited from the parents, as well as for de novo germline mutations, and they may have the potential to change the diagnosis of genetic disorders in the prenatal setting.

1	In combination with in vitro fertilization (IVF) techniques, it is even possible to perform genetic diagnoses in a single cell removed from the fourto eight-cell embryo or to analyze the first polar body from an oocyte. Preconceptual diagnosis thereby avoids therapeutic abortions but is costly and labor intensive. It should be emphasized that excluding a specific disorder by any of these approaches is never equivalent to the assurance of having a normal child. Mutations in certain cancer susceptibility genes such as BRCA1 and BRCA2 may identify individuals with an increased risk for the development of malignancies and result in risk-reducing interventions. The detection of mutations is an important diagnostic and FIGURE 82-15 Approach to genetic disease.

1	prognostic tool in leukemias and lymphomas. The demonstration of the presence or absence of mutations and polymorphisms is also relevant for the rapidly evolving field of pharmacogenomics, including the identification of differences in drug treatment response or metabolism as a function of genetic background. For example, the thiopurine drugs 6-mercaptopurine and azathioprine are commonly used cytotoxic and immunosuppressive agents. They are metabolized by thiopurine methyltransferase (TPMT), an enzyme with variable activity associated with genetic polymorphisms in 10% of whites and complete deficiency in about 1 in 300 individuals. Patients with intermediate or deficient TPMT activity are at risk for excessive toxicity, including fatal myelosuppression. Characterization of these polymorphisms allows mercaptopurine doses to be modified based on TPMT genotype. Pharmacogenomics may increasingly permit individualized drug therapy, improve drug effectiveness, reduce adverse side effects,

1	allows mercaptopurine doses to be modified based on TPMT genotype. Pharmacogenomics may increasingly permit individualized drug therapy, improve drug effectiveness, reduce adverse side effects, and provide cost-effective pharmaceutical care (Chap. 5).

1	Determination of the association of genetic defects with disease, comprehensive data of an individual’s genome, and studies of genetic variation raise many ethical and legal issues. Genetic information is generally regarded as sensitive information that should not be readily accessible without explicit consent (genetic privacy). The disclosure of genetic information may risk possible discrimination by insurers or employers. The scientific components of the Human Genome Project have been paralleled by efforts to examine ethical, social, and legal implications. An important milestone emerging from these endeavors consists in the Genetic Information Nondiscrimination Act (GINA), signed into law in 2008, which aims to protect asymptomatic individuals against the misuse of genetic information for health insurance and employment. It does not, however, protect the symptomatic individual. Provisions of the U.S. Patient Protection and Affordable Care Act, effective in 2014, will fill this gap

1	for health insurance and employment. It does not, however, protect the symptomatic individual. Provisions of the U.S. Patient Protection and Affordable Care Act, effective in 2014, will fill this gap and prohibit exclusion from, or termination of, health insurance based on personal health status. Potential threats to the maintenance of genetic privacy consist in the emerging integration of genomic data into electronic medical records, compelled disclosures of health records, and direct-to-consumer genetic testing.

1	It is widely accepted that identifying disease-causing genes can lead to improvements in diagnosis, treatment, and prevention. However, the information gleaned from genotypic results can have quite different impacts, depending on the availability of strategies to modify the course of disease (Chap. 84). For example, the identification of mutations that cause MEN 2 or hemochromatosis allows specific interventions for affected family members. On the other hand, at present, the identification of an Alzheimer’s or Huntington’s disease gene does not currently alter therapy and outcomes. Most genetic disorders are likely to fall into an intermediate category where the opportunity for prevention or treatment is significant but limited (Chap. 84). However, the progress in this area is unpredictable, as underscored by the finding that angiotensin II receptor blockers may slow disease progression in Marfan’s syndrome. Genetic test results can generate anxiety in affected individuals and family

1	as underscored by the finding that angiotensin II receptor blockers may slow disease progression in Marfan’s syndrome. Genetic test results can generate anxiety in affected individuals and family members. Comprehensive sequence analyses are particularly challenging because most individuals can be expected to harbor several serious recessive gene mutations.

1	The impact of genetic testing on health care costs is currently unclear. It is likely to vary among disorders and depend on the availability of effective therapeutic modalities. A significant problem arises from the marketing of genetic testing directly to consumers by commercial companies. The validity of these tests has not been defined, and there are numerous concerns about the lack of appropriate regulatory oversight, the accuracy and confidentiality of genetic information, the availability of counseling, and the handling of these results.

1	Many issues raised by the genome project are familiar, in principle, to medical practitioners. For example, an asymptomatic patient with increased low-density lipoprotein (LDL) cholesterol, high blood pressure, or a strong family history of early myocardial infarction is known to be at increased risk of coronary heart disease. In such cases, it is clear that the identification of risk factors and an appropriate intervention are beneficial. Likewise, patients with phenylketonuria, cystic fibrosis, or sickle cell anemia are often identified as having a genetic disease early in life. These precedents can be helpful for adapting policies that relate to genetic information. We can anticipate similar efforts, whether based on genotypes or other markers of genetic predisposition, to be applied to many disorders. One confounding aspect of the rapid expansion of information is that our ability to make clinical decisions often lags behind initial insights into genetic mechanisms of disease. For

1	many disorders. One confounding aspect of the rapid expansion of information is that our ability to make clinical decisions often lags behind initial insights into genetic mechanisms of disease. For example, when genes that predispose to breast cancer such as BRCA are described, they generate tremendous public interest in the potential to predict disease, but many years of clinical research are still required to rigorously establish genotype and phenotype correlations.

1	Genomics may contribute to improvements in global health by providing a better understanding of pathogens and diagnostics, and through contributions to drug development. There is, however, concern about the development of a “genomics divide” because of the costs associated with these developments and uncertainty as to whether these advances will be accessible to the populations of developing countries. The World Health Organization has summarized the current issues and inequities surrounding genomic medicine in a detailed report titled “Genomics and World Health.”

1	Whether related to informed consent, participation in research, or the management of a genetic disorder that affects an individual or his or her family, there is a great need for more information about fundamental principles of genetics. The pervasive nature of the role of genetics in medicine makes it important for physicians and other health care professionals to become more informed about genetics and to provide advice and counseling in conjunction with trained genetic counselors (Chap. 84). The application of screening and prevention strategies will therefore require intensive patient and physician education, changes in health care financing, and legislation to protect patient’s rights. Chapter 82 Principles of Human Genetics

1	Chromosome Disorders Nancy B. Spinner, Laura K. Conlin CHROMOSOME DISORDERS Alterations of the chromosomes (numerical and structural) occur in about 1% of the general population, in 8% of stillbirths, and in close to 50% of spontaneously aborted fetuses. The 3 × 109 base pairs that 83e encode the human genome are packaged into 23 pairs of chromosomes, which consist of discrete portions of DNA, bound to several classes of regulatory proteins. Technical advances that led to the ability to analyze human chromosomes immediately translated into the revelation that human disorders can be caused by an abnormality of chromosome number. In 1959, the clinically recognizable disorder, Down syndrome, was demonstrated to result from having three copies of chromosome 21 (trisomy 21). Very soon thereafter, in 1960, a small, structurally abnormal chromosome was recognized in the cells of some patients with chronic myelogenous leukemia (CML), and this abnormal chromosome is now known as the

1	thereafter, in 1960, a small, structurally abnormal chromosome was recognized in the cells of some patients with chronic myelogenous leukemia (CML), and this abnormal chromosome is now known as the Philadelphia chromosome.

1	Since these early discoveries, the techniques for analysis of human chromosomes, and DNA in general, have gone through several revolutions, and with each technical advancement, our understanding of the role of chromosomal abnormalities in human disease has expanded. While early studies in the 1950s and 1960s easily identified abnormalities of chromosome number (aneuploidy) and large structural alterations such as deletions (chromosomes with missing regions), duplications (extra copies of chromosome regions), or translocations (where portions of the chromosomes are rearranged), many other types of structural alterations could only be identified as techniques improved. The first important technical advance was the introduction of chromosome banding in the late 1960s, a technique that allowed for the staining of the chromosomes, so that each chromosome could be recognized by its pattern of alternating dark and light (or fluorescent and nonfluorescent) bands. Other technical innovations

1	for the staining of the chromosomes, so that each chromosome could be recognized by its pattern of alternating dark and light (or fluorescent and nonfluorescent) bands. Other technical innovations ranged from the introduction of fluorescence in situ hybridization in the 1980s to use of array-based and sequencing technologies in the early 2000s. Currently, we can appreciate that many types of chromosome abnormalities contribute to human disease including aneuploidy; structural alterations such as deletions and duplications, translocations, or inversions; uniparental disomy, where two copies of one chromosome (or a portion of a chromosome) are inherited from one parent; complex alterations such as isochromosomes, markers, and rings; and mosaicism for all of the aforementioned abnormalities. The first chromosome disorders identified had very striking and generally severe phenotypes, because the abnormalities involved large regions of the genome, but as methods have become more sensitive,

1	The first chromosome disorders identified had very striking and generally severe phenotypes, because the abnormalities involved large regions of the genome, but as methods have become more sensitive, it is now possible to recognize many more subtle phenotypes, often involving smaller genomic regions.

1	Standard cytogenetic analysis refers to the examination of banded human chromosomes. Banded chromosome analysis allows for both the determination of the number and identity of chromosomes in the cell and recognition of abnormal banding patterns associated with a structural rearrangement. A stained band is defined as the part of a chromosome that is clearly distinguishable from its adjacent segments by appearing darker or lighter with one or more banding techniques. Cytogenetic analysis is most commonly carried out on cells in mitosis, requiring dividing cells. Actively growing cells are most often obtained from peripheral blood; however, it is only a small subset of the blood cells that are actually used for cytogenetic analysis. Often, chemicals, like phytohemagglutinin (PHA), are used to specially stimulate growth of T cells in a blood sample. Other sources of dividing cells include skin-derived fibroblasts, amniotic fluid or placental tissue (for prenatal diagnosis), or tumor

1	to specially stimulate growth of T cells in a blood sample. Other sources of dividing cells include skin-derived fibroblasts, amniotic fluid or placental tissue (for prenatal diagnosis), or tumor tissue (for cancer diagnosis). After culturing, cells are treated with a mitotic spindle inhibitor, which prevents the separation of the chromatids during metaphase. Halting mitosis 83e-1 in metaphase is essential, because chromosomes are at their most condensed state during this stage of mitosis. The banding pattern of a metaphase chromosome is easily recognizable and is ideal for karyotyping. There are several different types of chromosome staining techniques, including R-banding, C-banding, and quinacrine staining, but the most commonly used is G-banding. G-banding is accomplished by treatment of the chromosomes with a proteolytic enzyme, such as trypsin, which digests some of the proteins holding DNA in a three-dimensional structure, followed by staining with a dye (Giemsa) that binds

1	of the chromosomes with a proteolytic enzyme, such as trypsin, which digests some of the proteins holding DNA in a three-dimensional structure, followed by staining with a dye (Giemsa) that binds DNA. The resulting patterns have both dark and light bands; in general, the light bands occur in regions on the chromosome in which genes are actively being transcribed, and dark bands are in regions of less active transcription.

1	The banded human karyotype has now been standardized based on an internationally agreed upon system for designating not only individual chromosomes but also chromosome regions, providing a way in which structural rearrangements and variants can be described in terms of their composition. The normal human female karyotype is referred to as 46,XX (46 chromosomes, with 22 pairs of autosomes and two of the same type of sex chromosomes [two Xs], indicating this is a female); and the normal human male karyotype is referred to as 46,XY (46 chromosomes, with 22 pairs of autosomes and one of each type of sex chromosome [one X and one Y], indicating this is a male). The anatomy of a chromosome includes the central constriction, known as the centromere, which is critical for movement of the chromosomes during mitosis and meiosis; the two chromosome arms (p for the smaller or petite arm, and q for the longer arm); and the chromosome ends, which contain the telomeres. The telomeres are made up of

1	during mitosis and meiosis; the two chromosome arms (p for the smaller or petite arm, and q for the longer arm); and the chromosome ends, which contain the telomeres. The telomeres are made up of a hexanucleotide repeat (TTAGGG)n, and unlike the centromere, they are not visible at the light microscope level. Telomeres are functionally important because they confer stability to the end of the chromosome. Broken chromosomes tend to fuse end to end, whereas a normal chromosome with an intact telomere structure is stable. To create the standard chromosome-banding map, each chromosome is divided into segments that are numbered, and then further subdivided. The precise band names are recorded in an international document so that each band has a distinct number. Figure 83e-1 shows an ideogram (chromosome map with bands) of the X chromosome and a G-banded X chromosome. This system provides a way for a chromosome abnormality to be written, with an indication of which band is deleted,

1	(chromosome map with bands) of the X chromosome and a G-banded X chromosome. This system provides a way for a chromosome abnormality to be written, with an indication of which band is deleted, duplicated, or rearranged.

1	p22.3 p2 p21.3 p arm p11.2 centromere q1 q21.1 FIGuRE 83e-1 Ideogram of the X chromosome and a G-banded X chromosome. The labeling of the X ideogram shows the positioning of the p and q arms, the centromere, and the telomeres. The numbering of the bands is also demonstrated, indicating the broadest subbands (p1, p2, q1, q2) and the further subdivisions to the right. Numbering begins at the centromere and moves out along each arm toward the telomeres.

1	Molecular cytogenetics provides a link between chromosome and molecular analysis and overcomes some of the limitations of standard cytogenetics. Deletions smaller than several million base pairs are not routinely detectable by standard G-banding techniques, and chromosomal abnormalities with indistinct or novel banding patterns can be difficult or impossible to interpret. To carry out cytogenetic analysis, cells must be dividing, which is not always possible to obtain (e.g., in autopsy or tumor material that has already been fixed). Finally, growth selection or bias may occasionally cause the results of cytogenetic studies to be misleading because cells that proliferate in vitro may not be representative of the original population, as is often the case with tumor specimens.

1	Fluorescence in situ hybridization (FISH) is a combined cytogenetic-molecular technique that solves many of the aforementioned problems. FISH permits determination of the number and location of specific DNA sequences in human cells. FISH can be performed on metaphase chromosomes, as with G-banding, but can also be performed on cells not actively progressing through mitosis. FISH performed on nondividing cells is referred to as interphase or nuclear FISH (Fig. 83e-2). The FISH procedure relies on the complementarity between the two strands of the DNA double helix and uses a molecular probe, which can be a pool of sequences across an entire chromosome, a DNA sequence for a repetitive part of the genome (e.g., centromeres or telomeres), or a specific DNA sequence found only once in the genome (e.g., a disease-associated gene). The choice of probes for FISH studies is important and will vary with the information needed for the diagnosis of a particular disorder. The most common type of

1	(e.g., a disease-associated gene). The choice of probes for FISH studies is important and will vary with the information needed for the diagnosis of a particular disorder. The most common type of probes are locus-specific probes, which are used to determine if a critical gene or region is absent (indicating a deletion), or present in the normal number of copies, or if an additional copy of the region is present. FISH on metaphase chromosomes will give the additional information of the location of the additional copy, which is necessary information to determine whether a structural rearrangement, such as a translocation, is present. FISH can also be performed with probes that bind to repeated sequences, such as DNA found in centromeres or telomeres, or with probes that bind to an entire chromosome (“painting” probes), to determine the chromosome composition of an abnormal chromosome. Interphase FISH studies can also help to identify structural alterations when probes are used that map

1	(“painting” probes), to determine the chromosome composition of an abnormal chromosome. Interphase FISH studies can also help to identify structural alterations when probes are used that map to both sides of a translocation breakpoint. Each side of the breakpoint is labeled in a different color, and when no translocation is present the two probes appear to be overlapping. When a translocation is present, the two probes appear separate from one another. These set of probes, called “breakapart” probes, are commonly used to detect recurrent translocations in cancer cells.

1	Array-based methods were introduced into the clinical lab beginning in 2003 and quickly revolutionized the field of cytogenetics. These techniques used arrays (collections of DNA segments from the entire genome) which could be interrogated with respect to copy number. With standard cytogenetics, the missing or extra pieces of DNA have to be big enough to see in the microscope on banded chromosomes (usually larger than 5 Mb). FISH requires a preselection of an informative molecular probe prior to analysis. In contrast, array-based techniques permit analysis of many regions of the genome in a single analysis, with greatly increased resolution over standard cytogenetics. Array-based techniques allow for scanning of the genome for small deletions or duplications quickly and accurately. The resolution of the p13 –1.00 –0.75 –0.50 –0.25 0.00 0.25Log R ratio 0.50 0.75 1.00 p12 p11.2 q11.2 q14 q21.1 q21.3 q22.2 q23 q26.1 q26.2 q26.3 15

1	FIGuRE 83e-2 G-banding, fluorescence in situ hybridization (FISH), and single nucleotide polymorphism (SNP) array demonstrate an abnormal chromosome 15. A. G-banding shows an abnormal chromosome 15, with unrecognizable material in place of the p arm in the chromosome on the right (top arrow). B. Metaphase FISH (only chromosome 15s are shown) using a probe from the 15q telomere region (red) and a control probe that maps outside of the duplicated region (green). C. Interphase FISH demonstrates three copies of the 15q tel probe in red, and two copies of the 15q control probe (green). D. Genome-wide SNP array demonstrates the increased copy number for a portion of 15q. Note that the G-banding alone indicates the abnormal chromosome 15, but the origin of the extra material can only be demonstrated by FISH or array. The FISH analysis requires additional information about possible genetic causes to select the correct probe. The array can exactly identify the origin of the extra material, but

1	by FISH or array. The FISH analysis requires additional information about possible genetic causes to select the correct probe. The array can exactly identify the origin of the extra material, but by itself would not provide positional information.

1	test is a function of the number of probes or DNA sequences present on the array. Arrays may use probes of different sizes (ranging from 50 to 200,000 base pairs of DNA) and different probe densities depending on the requirements of the application. Low-resolution platforms can have hundreds of probes, targeted to known disease regions, whereas high-resolution platforms can have millions of probes spread across the entire genome. Depending on the size of the probes and the probe placement across the genome, array-based testing may be able to detect single exon deletions or duplications.

1	Comparative Genomic Hybridization (CGH) and Single Nucleotide Polymorphism (SNP) Analysis CGH and SNP-based genotyping arrays can both be used for the analysis of genomic deletions and duplications. For both techniques, oligonucleotide probes are placed onto a slide or chip in a grid format. Each of these probes is specific for a particular genomic region. In array CGH, the amount of DNA from a patient is compared to that in a clinically normal control, or pool of controls, for each of the probes present on the array. DNA from a patient is fluorescently labeled with a dye of one color, and DNA from a control individual is labeled with another color. These DNA samples are then hybridized at the same time to the array. The resulting fluorescent signal will vary depending on whether both the control and patient DNA are present in equal amounts or if one has a different copy number than the other. SNP platforms use arrays targeting SNPs that are distributed across the genome. SNP arrays

1	control and patient DNA are present in equal amounts or if one has a different copy number than the other. SNP platforms use arrays targeting SNPs that are distributed across the genome. SNP arrays vary in density of markers and in the technology used for genotyping, depending on the manufacturer of the array. SNP arrays were initially designed to determine genotypes at a biallelic, polymorphic base (e.g., CC, CT, or TT) and have been increasingly used in genome-wide association studies to identify disease susceptibility genes. SNP arrays were subsequently adapted to identify genomic deletions and duplications (Fig. 83e-2). SNP arrays, in addition to identifying copy number changes, can also detect regions of the genome that have an excess of homozygous genotypes and absence of heterozygous genotypes (e.g., CC and TT genotypes only, with no CT genotypes). Absence of heterozygosity is sometimes associated with uniparental disomy (discussed later in this chapter) but is also observed

1	genotypes (e.g., CC and TT genotypes only, with no CT genotypes). Absence of heterozygosity is sometimes associated with uniparental disomy (discussed later in this chapter) but is also observed when an individual’s parents are related to one another (identity by descent). Regions of homozygosity have been used to help identify genes in which homozygous mutations result in disease phenotypes in families with known consanguinity.

1	Array-based techniques (which we will now refer to as cytogenomic analysis) have proven superior to chromosome analysis in the identification of clinically significant deletions or duplications. It is estimated that for a deletion or duplication to be visualized by standard cytogenetics it must be minimally between 5 and 10 million base pairs in size. In almost all cases, deletions and duplications of this size contain multiple genes, and these deletions and duplications are disease causing. However, utilization of array-based cytogenomic testing, which can routinely identify deletions and duplications smaller than 50,000 base pairs, reveals that clinically normal individuals all have some deletions and duplications. This presents a dilemma for the analyst to discern which smaller copy number variations (CNVs) are disease causing (pathogenic) and which are likely benign polymorphisms. Although initially burdensome, the cytogenomics community has been curating these CNVs for almost a

1	variations (CNVs) are disease causing (pathogenic) and which are likely benign polymorphisms. Although initially burdensome, the cytogenomics community has been curating these CNVs for almost a decade, and databases have been created reporting CNVs routinely seen in clinically normal individuals and those routinely seen in individuals with clinical abnormalities. Nevertheless, each copy number variant that is identified in an individual undergoing genomic testing must be evaluated for gene content and overlap with CNVs in other patients and in controls.

1	Array technologies are DNA based, unlike cytogenetic technologies, which are cell based. Although resolution of gains and losses are greatly increased with array technology, this technique cannot identify structural changes. When DNA is extracted for array studies, chromosomal structure is lost because the DNA is fragmented for better hybridization to the slides. As an example, the array may be able to detect a duplication of a small region of a chromosome, but no information on the location of this extra material can be determined from this test. The location of this extra copy in the genome may be critical, as the chromosomal material may be involved in a translocation, insertion, marker, or other complex rearrangement. Depending on the chromosomal position of this extra material, the patient may have different clinical outcomes, and recurrence risks for the family can be significantly different. Often, combinations of array-based and cytogenetic-based techniques are required to

1	patient may have different clinical outcomes, and recurrence risks for the family can be significantly different. Often, combinations of array-based and cytogenetic-based techniques are required to fully characterize chromosomal abnormalities (see Table 83e-1 for comparison of these technologies).

1	Recent advances in genomic sequencing, known as next-generation sequencing (NGS), have vastly increased the speed and throughput of DNA sequence analysis. NGS is rapidly finding its way into the diagnostic lab for detection of clinically relevant intragenic mutations, and new bioinformatic tools for analysis of genomic deletions and duplications are being developed. It is anticipated that NGS will soon allow the complete analysis of a patient’s genome, with identification of intragenic mutations as well as chromosome abnormalities resulting in gain or loss of genetic material. Identification of completely balanced translocations is the most challenging for NGS, but recent reports of successes in this area suggest that in a matter of time, sequencing will be used for all types of genomic analysis.

1	Cytogenetic analysis is most commonly used for (1) examination of the fetal chromosomes or genome during pregnancy (prenatal diagnosis) or in the event of a spontaneous miscarriage; (2) examination of chromosomes in the neonatal or pediatric population to look for an underlying diagnosis in the case of congenital or developmental anomalies, including short stature and abnormalities of sexual differentiation or progression; (3) chromosome analysis in adults who are facing fertility problems; or (4) examination of cancer cells to look for alterations that aid in establishing a diagnosis or contributing to the prognosis of a tumor (Table 83e-2).

1	Prenatal diagnosis is carried out by analysis of samples obtained by four techniques: amniocentesis, chorionic villous sampling, fetal blood sampling, and analysis of cell free DNA from maternal serum. Amniocentesis, which has been the most commonly used test to date, is usually performed between 15 and 17 weeks of gestational age and carries a small but significant risk for miscarriage. Amniocentesis can be performed as early as 12 weeks, but because there is a lower volume of fluid, the risks for fetal injury or miscarriage are greater. Chorionic villous sampling (CVS) or placental biopsy is routinely carried out earlier than amniocentesis, between 10 and 12 weeks, but a reported increase in limb defects when the procedure is carried out earlier than

1	Timing of Testing Indications for Testing 10 weeks has resulted in reduced use of this test in some centers. Fetal blood sampling (percutaneous umbilical blood sampling [PUBS]) is a riskier procedure that is carried out in the second or third trimester of pregnancy, usually to follow up on an unclear finding from an amniocentesis (such as mosaicism) or an ultrasound abnormality that was detected later in pregnancy. One of the far-reaching recent advances in prenatal diagnosis of chromosome and other genetic disorders is the utilization of cell free fetal DNA that can be identified in maternal serum. The obvious advantages of using fetal DNA obtained from maternal serum is that the DNA can be obtained at minimal risk to the pregnancy, because it requires a maternal blood sample, rather than amniotic fluid which is obtained by puncturing the uterine membranes and carries a risk of miscarriage or infection. Although cell free fetal DNA screening, also called noninvasive prenatal

1	than amniotic fluid which is obtained by puncturing the uterine membranes and carries a risk of miscarriage or infection. Although cell free fetal DNA screening, also called noninvasive prenatal screening, has started to be offered clinically, it requires further confirmation of fetal tissues when an abnormal result is identified. Furthermore, ethical concerns have been raised, because it is feared that the ease of doing this test may encourage testing for individuals who are not truly prepared to deal with the choices that accompany diagnosis of a genetic disease and this testing may change the ethical implications of prenatal testing. Nevertheless, this is an active of area of research, both in terms of the technology and the utilization and implications.

1	Common Indications Common indications for prenatal diagnosis by cytogenetic or cytogenomic analysis are (1) advanced maternal age, (2) presence of an abnormality of the fetus on ultrasound examination, and (3) abnormalities in maternal serum screening that reveal an increased risk for chromosome abnormality.

1	Maternal age is well known to be an important risk factor for having a fetus with trisomy. At a maternal age less than 25 years, 2% of all clinically recognized pregnancies are trisomic, but by a maternal age of 36 years, this figure increases to 10%, and by the maternal age of 42 years, the figure increases to >33%. Based on the risk of having a chromosomally abnormal fetus in comparison to the risk for an adverse event from amniocentesis or CVS, the recommendation is that women over the age of 35 consider prenatal testing if they want to know the chromosomal status of their fetus. The precise mechanism for the maternal age effect is not known, but it is believed that it involves a breakdown in the process of chromosome segregation. A similar effect is not seen for trisomy and paternal age. This difference may reflect the fact that oocytes are generated early in ovary development in the female, whereas spermatogonia are generated continuously after puberty in the male.

1	Abnormalities on prenatal ultrasound are the second most frequent indication for prenatal genetic screening. Ultrasound screening can reveal structural or functional anomalies in the fetus, which might be associated with chromosome or genomic disorders. Follow-up chromosome studies may therefore be recommended. Maternal serum screening results are the third most frequent indication for prenatal chromosome analysis. There have been several versions of maternal serum screening offered over the past few decades. Currently, the “quad” screen analyzes levels of α fetoprotein (AFP), human chorionic gonadotropin (hCG), estriol, and inhibin-A. The values of these analytes are used to adjust the maternal age–predicted risk of a trisomy 21 or trisomy 18 fetus.

1	Postnatal indications for cytogenetic or cytogenomic analysis in neonates or children are varied, and the list has been growing with the increasing ability to diagnose smaller genomic alterations via array-based techniques. Common indications include multiple congenital anomalies, suspicion of a known cytogenetic or cytogenomic syndrome, intellectual disability or developmental delay both with and without accompanying dysmorphic features, autism, failure to thrive in infancy or short stature during childhood, and disorders of sexual development. The ability to detect smaller genomic alterations with involvement of fewer genes, sometimes as few as a single gene, suggests that a wider range of phenotypes could be investigated by cytogenomic analysis. Reasons for chromosome testing in adults include recurrent miscarriages or infertility, where balanced chromosome rearrangements such as reciprocal translocations may occur. Additionally, some adults with anomalies who were not diagnosed

1	include recurrent miscarriages or infertility, where balanced chromosome rearrangements such as reciprocal translocations may occur. Additionally, some adults with anomalies who were not diagnosed when they were children are referred for cytogenetic analysis, often when other members of their family want to understand any potential genetic implications, as they plan their own families.

1	Aneuploidy (extra or missing chromosomes) is the most common type of abnormality, occurring in 3/1000 newborns and at much higher frequency (about 35%) in spontaneously aborted fetuses. The only autosomal trisomies that are compatible with being live born in humans are trisomies 13, 18, and 21, although there are several chromosomes that can be trisomic in mosaic form. Trisomy 21 is associated with the relatively common disorder Down syndrome. Down syndrome has characteristic features including recognizable facial features, along with intellectual disability and abnormalities of multiple other organ systems including the heart. Both trisomy 13 and trisomy 18 are much more severe disorders than Down syndrome, with low frequency of patients surviving past 1 year of age. Trisomy 13 is characterized by low birth weight, postaxial polydactyly, microcephaly, ocular malformations such as anophthalmia or microphthalmia, cleft lip and palate, cardiac defects, and renal malformations. Trisomy

1	by low birth weight, postaxial polydactyly, microcephaly, ocular malformations such as anophthalmia or microphthalmia, cleft lip and palate, cardiac defects, and renal malformations. Trisomy 18 neonates have distinct facial characteristics at birth accompanied by an abnormal neurologic exam, underdeveloped genitalia, general lack of responsiveness, and structural birth defects such as congenital heart disease, esophageal atresia, and omphalocele.

1	Mosaicism refers to the presence of two or more populations of cells with distinct chromosome constitutions: for example, an individual with a normal female karyotype in some cells (46,XX) and trisomy 21 in other cells (47,XX,+21). In general, individuals who are mosaic for a chromosomal abnormality have less severe phenotypes than individuals with that same finding in every cell. The severity and presentation of phenotypes are related to the mosaic levels and the tissue distribution of the abnormal cells. There are a number of trisomies that have been reported in mosaic form including mosaic trisomies for chromosomes 8, 9, 14, 17, and 22. A number of trisomies have also been reported in spontaneous abortions (SABs) that have not been seen in live-born individuals, including trisomy 16, which is the most common trisomy in SABs. Monosomy for human chromosomes is very rare, with the single exception being monosomy for the X chromosome, associated with Turner syndrome (45,X). Monosomy

1	which is the most common trisomy in SABs. Monosomy for human chromosomes is very rare, with the single exception being monosomy for the X chromosome, associated with Turner syndrome (45,X). Monosomy for the X chromosome occurs in 1% of all conceptions, yet 98% of these conceptions do not go to term and result in SABs. Trisomies for the sex chromosomes also occur, with 47,XXX (trisomy X or triple X syndrome), 47,XXY (Klinefelter syndrome), and 47,XYY all reported in individuals with relatively mild phenotypes (Chap. 410). Klinefelter syndrome is the most common clinically recognized sex chromosome abnormality, and clinical features include gynecomastia, azoospermia, small testes, and hypogonadism. The 47,XYY karyotype is most often found in boys with developmental delay and or behavioral difficulties, but population-based studies have shown that intelligence for individuals with this karyotype is generally within the normal range, although slightly lower than that found in siblings.

1	Structural chromosome abnormalities include deletions, duplications, translocations, inversions, as well as other types of abnormalities, each relatively rare, but nonetheless contributing to clinical disease resulting from chromosome anomalies. These rare alterations include isochromosomes, ring chromosomes, dicentric chromosomes, and marker chromosomes (structurally abnormal chromosomes that cannot be identified based on cytogenetics alone). Both translocations and inversions can be completely balanced in some cases, such that there is no disruption of coding regions of the genome, with a completely normal clinical phenotype; however, carriers are at risk for unbalanced forms of these rearrangements in their offspring.

1	Reciprocal translocations are found in approximately 1/500–1/600 individuals in the general population and result from the exchange of chromosomal segments between at least two chromosomes. These usually occur between nonhomologous chromosomes and can be identified based on an altered banding pattern on G-banding. Balanced translocation carriers are at risk for abnormal chromosome segregation during meiosis and therefore have a higher risk for infertility, SAB, and live-born offspring with multiple congenital malformations. These phenotypes are observed when only one of the pairs of chromosomes involved in a translocation is inherited from a parent, resulting in an unbalanced genotype (Fig. 83e-3). Sometimes the exchanged segments are so small that they cannot be appreciated by banding (cryptic translocation), and these are sometimes recognized 83e-5 when a phenotypically affected child with an unbalanced form is born. Parental chromosomes can then be studied by FISH to determine if

1	translocation), and these are sometimes recognized 83e-5 when a phenotypically affected child with an unbalanced form is born. Parental chromosomes can then be studied by FISH to determine if the rearrangement is inherited from a parent with a balanced form of the translocation. The majority of reciprocal, apparently balanced translocations occur in phenotypically normal individuals. The risk for a clinical abnormality when a new reciprocal translocation is identified (usually during prenatal diagnostic studies) is about 7%. Analysis of cytogenetically reciprocal translocations using arrays has demonstrated that translocations in clinically normal individuals are more likely to show no deletions or duplications at the breakpoint, whereas translocations in clinically affected individuals are more likely to have breakpoint-associated deletions or duplications. Most reciprocal translocations occur uniquely, at apparently random positions throughout the genome; however, there are a few

1	are more likely to have breakpoint-associated deletions or duplications. Most reciprocal translocations occur uniquely, at apparently random positions throughout the genome; however, there are a few exceptions with multiple cases of recurrent translocations occurring. These recurrent translocations include t(11;22), which results in Emanuel syndrome in the unbalanced form, and several translocations involving a region on 4p, 8p, and 12p. These recurrent translocations occur in regions of the genome that contain specific types of AT-rich repeats, or other repeat sequences, that are prone to rearrangement. A special category of translocations is the Robertsonian translocations, which involve the acrocentric chromosomes. An acrocentric chromosome has unique genetic material only on the long arm of the chromosomes, whereas the short arm contains repetitive DNA. The acrocentric chromosomes are 13, 14, 15, 21, and 22. Robertsonian translocations occur when an entire long arm of an

1	the long arm of the chromosomes, whereas the short arm contains repetitive DNA. The acrocentric chromosomes are 13, 14, 15, 21, and 22. Robertsonian translocations occur when an entire long arm of an acrocentric chromosome is translocated onto the short arm of another acrocentric chromosome. Balanced carriers of a Robertsonian translocation contain only 45 chromosomes, with one chromosome consisting of two long arms of an acrocentric chromosome. Technically, this is an unbalanced translocation, as two short arms of the acrocentric chromosomes are missing; however, because the short arms are repetitive, there is no phenotypic consequence. Unbalanced Robertsonian carriers have 46 chromosomes, but have three copies of the long arm of an acrocentric chromosome. The most

1	FIGuRE 83e-3 Segregation of a balanced translocation in a mother, with inheritance of an unbalanced form in her child. Note that the mother has two rearranged chromosomes, but her child only received one of these, resulting in extra copies of a region of the blue chromosome, with loss of some material from the red chromosome. 14. Unbalanced Robertsonian translocations involving chromosomes 13 and 21 result in trisomy 13 and Down syndrome, respectively. Approximately 4% of patients with Down syndrome have a translocation, and because recurrence risks are different for families of these individuals, all patients with clinically identified Down syndrome should have a karyotype to look for translocations.

1	Inversions are another type of chromosome abnormality involving rearranged segments, where there are two breaks within a chromosome, with the intervening chromosomal material inserted in an inverted orientation. As with reciprocal translocations, if a break occurs within a gene or control region for a gene, a clinical phenotype may result, but often there are no consequences for the inversion carrier; however, there is risk for abnormalities in the offspring of carriers, as recombinant chromosomes may result after crossing over between a normal chromosome and an inverted chromosome during meiosis.

1	Deletion refers to the loss of a chromosomal segment, which results in the presence of only a single copy of that region in an individual’s genome. A deletion can be at the end of a chromosome (terminal), or it can be within the chromosome (interstitial). Deletions that are visible at the microscopic level in standard cytogenetic analysis are generally greater than 5 Mb in size. Smaller deletions have been identified by FISH and by chromosomal microarray. The clinical consequences of a deletion depend on the number and function of genes in the deleted region. Genes that cause a phenotype when a single copy is deleted are known as haploinsufficient genes (one copy is not sufficient), and it is estimated that less than 10% of genes are haploinsufficient. Genes associated with disease that are not haploinsufficient include genes for known recessive disorders, such as cystic fibrosis or Tay-Sachs disease.

1	The first chromosome deletion syndromes were diagnosed clinically and were subsequently demonstrated to be caused by a chromosome deletion on cytogenetic analysis. Examples of these disorders include the Wolf-Hirschhorn syndrome, which is associated with deletions of a small region of the short arm of chromosome 4 (4p); the cri-du-chat syndrome, associated with deletion of a small region of the short arm of chromosome 5 (5p); Williams syndrome, which is associated with interstitial deletions of the long arm of chromosome 7 (7q11.23); and the DiGeorge/velocardiofacial syndromes, associated with interstitial deletions of the long arm of chromosome 22 (22q11.2). Initial cytogenetic studies were able to provide a rough localization of the deletions in different patients, but with the increased usage of arrays, precise mapping of the extent and gene content of these deletions has become much easier. In many cases, one or two genes that are critical for the phenotype associated with these

1	usage of arrays, precise mapping of the extent and gene content of these deletions has become much easier. In many cases, one or two genes that are critical for the phenotype associated with these deletions have been identified. In other cases, the phenotype stems from the deletion of multiple genes. The increased utilization of genomic testing by array, which can identify deletions that are much smaller than those detectable by standard cytogenetic analysis, has resulted in the discovery of several new cytogenomic disorders. These include the 1q21.1, 15q13.3, 16p11.2, and 17q21.31 microdeletion syndromes.

1	Duplication of genomic regions is better tolerated than deletion, as evidenced by the viability of several autosomal trisomies (whole chromosome duplications) but no autosomal monosomies (whole chromosome deletions). There are several duplication syndromes where the duplicated region of the genome is present as a supernumerary chromosome. Utilization of chromosome microarray analysis has made analysis of the origins of duplicated chromosome material straightforward (Fig. 83e-2). Recurrent syndromes associated with supernumerary chromosomes include the inverted duplication 15 (inv dup 15) syndrome, caused by the presence of a marker chromosome derived from chromosome 15, with two copies of proximal 15q resulting in tetrasomy (four copies) of this region. The inv dup 15 syndrome has a distinct phenotype and is associated with hypotonia, developmental delay, intellectual disability, epilepsy, and autistic behavior. Another syndrome is the cat eye syndrome, named for the “cat-eyelike”

1	phenotype and is associated with hypotonia, developmental delay, intellectual disability, epilepsy, and autistic behavior. Another syndrome is the cat eye syndrome, named for the “cat-eyelike” appearance of the pupil, resulting from a coloboma of the iris. This syndrome results from a supernumerary chromosome derived from a portion of chromosome 22, and the marker chromosomes can vary in size and are often mosaic. Consistent with expectations of a mosaic disorder, the phenotype of this syndrome is highly variable and includes renal malformations, urinary tract anomalies, congenital heart defects, anal atresia with fistula, imperforate anus, and mild to moderate intellectual disability. Another rare duplication syndrome is the Pallister-Killian syndrome (PKS), which illustrates the principle of tissue-specific mosaicism. Individuals with PKS have coarse facial features with pigmentary skin anomalies, localized alopecia, profound intellectual disability, and seizures. The disorder is

1	of tissue-specific mosaicism. Individuals with PKS have coarse facial features with pigmentary skin anomalies, localized alopecia, profound intellectual disability, and seizures. The disorder is caused by a supernumerary isochromosome for the short arm of chromosome 12 (isochromosome 12p). Isochromosomes consist of two copies of one chromosome arm (p or q), rather than one copy of each arm. This isochromosome is not generally seen in peripheral blood lymphocytes when they are analyzed by G-banding, but it is detected in fibroblasts. Array technology has been reported to detect the isochromosome in uncultured peripheral blood in some patients, and it has been hypothesized that a growth bias against cells with the isochromosome prevents their identification in cytogenetic studies.

1	Numerical abnormalities, translocations, and deletions are the most common chromosome alterations observed in the diagnostic laboratory, but in addition to inversions and duplications, several other types of abnormal chromosomes have been reported, including ring chromosomes, where the two ends of the chromosome fuse to form a circle, and insertions, where a piece of one chromosome is inserted into another chromosome or elsewhere into the same chromosome.

1	Uniparental disomy (UPD) is the inheritance of a pair of chromosomes (or part of a chromosome) from only one parent. This usually occurs as a result of nondisjunction during meiosis, with a gamete missing or having an extra copy of a chromosome. A resulting fertilized egg would then have only one parental contribution for a given chromosome pair, or a trisomy for a given chromosome. If the monosomy or trisomy is not compatible with life, the embryo may undergo a “rescue” to normal copy number. If a monosomy is rescued, the single chromosome may be duplicated, resulting in a cell with two identical chromosomes (monosomy rescue) (Fig. 83e-4). In the case of trisomies, a subsequent nondisjunction can result in cells where one of the extra chromosomes is lost (trisomy rescue) (Fig. 83e-4). For trisomy rescue, there is a one in three chance that the lost FIGuRE 83e-4 Mechanisms of formation of uniparental disomy.

1	FIGuRE 83e-4 Mechanisms of formation of uniparental disomy. Panel A demonstrates nondisjunction in one parent (mother, represented in red), with trisomy in the zygote. A subsequent nondisjunction, with loss of the paternal chromosome (represented in blue), restores the diploid karyotype but leaves two copies of the maternal chromosome (maternal uniparental disomy [UPD]). Panel B demonstrates nondisjunction in one parent (mother, indicated by red oval), resulting in only one copy of this chromosome in the zygote. Subsequent nondisjunction duplicates the single chromosome, rescuing the monosomy, but resulting in two copies of the paternal chromosome (represented in blue; paternal UPD).

1	chromosome will be the sole chromosome from one parent, resulting in a cell with two chromosomes from the same parent. UPD is sometimes associated with clinical abnormalities, and this can occur by two mechanisms. UPD can cause disease when there is an imprinted gene on the involved chromosome, resulting in altered gene expression. Imprinting is the chemical marking of the parental origin of a chromosome, and genes that are imprinted are only expressed from either the maternal or paternal chromosome (Chap. 82). Imprinting therefore results in the differential expression of affected genes, based on parent of origin. Imprinting usually occurs through differential modification of the chromosome from one of the parents, and methylation is one of several epigenetic mechanisms (others include histone acetylation, ubiquitylation, and phosphorylation). Imprinted chromosomes that are associated with phenotypes include paternal UPD6 (associated with neonatal diabetes), maternal UPD7 and UPD11

1	histone acetylation, ubiquitylation, and phosphorylation). Imprinted chromosomes that are associated with phenotypes include paternal UPD6 (associated with neonatal diabetes), maternal UPD7 and UPD11 (associated with Russell-Silver syndrome), paternal UPD11 (associated with Beckwith-Wiedemann syndrome), paternal UPD14, maternal UPD15 (Angelman syndrome), and paternal UPD15 (Prader-Willi syndrome). UPD can also result in disease if the two copies from the same parent are the same chromosome (uniparental isodisomy), and the chromosome contains an allele involving a pathogenic mutation associated with a recessive disorder. Two copies of the deleterious allele would result in the associated disease, even though only one parent is a disease carrier.

1	Chromosome changes can occur during meiosis or mitosis and can occur at any point across the lifespan. Mosaicism for a developmental disorder is one consequence of mitotic chromosome abnormalities, and another consequence is cancer, when the chromosome change 83e-7 confers a growth or proliferation advantage on the cell. The types of chromosome abnormalities seen in cancer are similar to those seen in the developmental disorders (e.g., aneuploidy, deletion, duplication, translocation, isochromosomes, rings, inversion). Tumor cells often have multiple chromosome changes, some of which happen early in the development of a tumor, and may contribute to its selective advantage, whereas others are secondary effects of the deregulation that characterizes many tumors. Chromosome changes in cancer have been studied extensively and have been shown to provide important diagnostic, classification, and prognostic information. The identification of cancer type–specific translocation breakpoints has

1	have been studied extensively and have been shown to provide important diagnostic, classification, and prognostic information. The identification of cancer type–specific translocation breakpoints has led to the identification of a number of cancer-associated genes.�For example, the small abnormal chromosome found to be associated with chronic myelogenous leukemia (CML) in 1960 was shown to be the result of translocation between chromosomes 9 and 22 once techniques for analysis of banded chromosomes were introduced, and subsequently, the translocation breakpoint was cloned to reveal the c-abl oncogene on chromosome 9. This translocation produces a fusion protein, which has been targeted for treatment of CML. For detailed discussion of cancer genetics, see Chap. 101e.

1	446 the practice of Genetics in Clinical medicine Susan M. Domchek, J. Larry Jameson, Susan Miesfeldt APPLICATIONS OF MOLECULAR GENETICS IN CLINICAL MEDICINE Genetic testing for inherited abnormalities associated with disease 84 risk is increasingly used in the practice of clinical medicine. Germline alterations include chromosomal abnormalities (Chap. 83e), specific gene mutations with autosomal dominant or recessive patterns of transmission (Chap. 82), and single nucleotide polymorphisms with small relative risks associated with disease. Germline alterations are responsible for disorders beyond classic Mendelian conditions with genetic susceptibility to common adult-onset diseases such as asthma, hypertension, diabetes mellitus, macular degeneration, and many forms of cancer. For many of these diseases, there is a complex interplay of genes (often multiple) and environmental factors that affect lifetime risk, age of onset, disease severity, and treatment options.

1	The expansion of knowledge related to genetics is changing our understanding of pathophysiology and influencing our classification of diseases. Awareness of genetic etiology can have an impact on clinical management, including prevention and screening for or treatment of a range of diseases. Primary care physicians are relied upon to help patients navigate testing and treatment options. Consequently, they must understand the genetic basis for a large number of genetically influenced diseases, incorporate personal and family history to determine the risk for a specific mutation, and be positioned to provide counseling. Even if patients are seen by genetic specialists who assess genetic risk and coordinate testing, primary care providers should provide information to their patients regarding the indications, limitations, risks, and benefits of genetic counseling and testing. They must also be prepared to offer risk-based management following genetic risk assessment. Given the pace of

1	the indications, limitations, risks, and benefits of genetic counseling and testing. They must also be prepared to offer risk-based management following genetic risk assessment. Given the pace of genetics, this is an increasingly difficult task. The field of clinical genetics is rapidly moving from single gene testing to multigene panel testing, with techniques such as wholeexome and -genome sequencing on the horizon, increasing the complexity of test selection and interpretation, as well as patient education and medical decision making.

1	Adult-onset hereditary diseases follow multiple patterns of inheritance. Some are autosomal dominant conditions. These include many common cancer susceptibility syndromes such as hereditary breast and ovarian cancer (due to germline BRCA1 and BRCA2 mutations) and Lynch syndrome (caused by germline mutations in the mismatch repair genes MLH1, MSH2, MSH6, and PMS2). In both of these examples, inherited mutations are associated with a high penetrance (lifetime risk) of cancer, although risk is not 100%. In other conditions, although there is autosomal dominant transmission, there is lower penetrance, thereby making the disorders more difficult to recognize. For example, germline mutations in CHEK2 increase the risk of breast cancer, but with a moderate lifetime risk in the range of 20–40%, as opposed to 50–70% for mutations in BRCA1 or BRCA2. Other adult-onset hereditary diseases are transmitted in an autosomal recessive fashion where two mutant alleles are necessary to cause disease.

1	as opposed to 50–70% for mutations in BRCA1 or BRCA2. Other adult-onset hereditary diseases are transmitted in an autosomal recessive fashion where two mutant alleles are necessary to cause disease. Examples include hemochromatosis and MYH-associated colon cancer. There are more pediatric-onset autosomal recessive disorders, such as lysosomal storage diseases and cystic fibrosis.

1	The genetic risk for many adult-onset disorders is multifactorial. Risk can be conferred by genetic factors at a number of loci, which individually have very small effects (usually with relative risks of <1.5). These risk loci (generally single nucleotide polymorphisms [SNPs]) combine with other genes and environmental factors in ways that are not well understood. SNP panels are available to assess risk of disease, but the optimal way of using this information in the clinical setting remains uncertain.

1	Many diseases have multiple patterns of inheritance, adding to the complexity of evaluating patients and families for these conditions. For example, colon cancer can be associated with a single germline mutation in a mismatch repair gene (Lynch syndrome, autosomal dominant), biallelic mutations in MYH (autosomal recessive), or multiple SNPs (polygenic). Many more individuals will have SNP risk alleles than germline mutations in high-penetrance genes, but cumulative lifetime risk of colon cancer related to the former is modest, whereas the risk related to the latter is significant. Personal and family histories provide important insights into the possible mode of inheritance.

1	When two or more first-degree relatives are affected with asthma, cardiovascular disease, type 2 diabetes, breast cancer, colon cancer, or melanoma, the relative risk for disease among close relatives ranges from twoto fivefold, underscoring the importance of family history for these prevalent disorders. In most situations, the key to assessing the inherited risk for common adult-onset diseases is the collection and interpretation of a detailed personal and family medical history in conjunction with a directed physical examination.

1	Family history should be recorded in the form of a pedigree. Pedigrees should convey health-related data on firstand second-degree relatives. When such pedigrees suggest inherited disease, they should be expanded to include additional family members. The determination of risk for an asymptomatic individual will vary depending on the size of the pedigree, the number of unaffected relatives, the types of diagnoses, and the ages of disease onset. For example, a woman with two first-degree relatives with breast cancer is at greater risk for a specific Mendelian disorder if she has a total of 3 female first-degree relatives (with only 1 unaffected) than if she has a total of 10 female first-degree relatives (with 7 unaffected). Factors such as adoption and limited family structure (few women in a family) should to be taken into consideration in the interpretation of a pedigree. Additional considerations include young age of disease onset (e.g., a 30-year nonsmoking woman with a myocardial

1	a family) should to be taken into consideration in the interpretation of a pedigree. Additional considerations include young age of disease onset (e.g., a 30-year nonsmoking woman with a myocardial infarction), unusual diseases (e.g., male breast cancer or medullary thyroid cancer), and the finding of multiple potentially related diseases in an individual (e.g., a woman with a history of both colon and endometrial cancer). Some adult-onset diseases are more prevalent in certain ethnic groups. For instance, 2.5% of individuals of Ashkenazi Jewish ancestry carry one of three founder mutation in BRCA1 and BRCA2. Factor V Leiden mutations are much more common in Caucasians than in Africans or Asians.

1	Additional variables that should be documented are nonhereditary risk factors among those with disease (such as cigarette smoking and myocardial infarction; asbestos exposure and lung disease; and mantle radiation and breast cancer). Significant associated environmental exposures or lifestyle factors decrease the likelihood of a specific genetic disorder. In contrast, the absence of nonhereditary risk factors typically associated with a disease raises concern about a genetic association. A personal or family history of deep vein thrombosis in the absence of known environmental or medical risk factors suggests a hereditary thrombotic disorder. The physical examination may also provide important clues about the risk for a specific inherited disorder. A patient presenting with xanthomas at a young age should prompt consideration of familial hypercholesterolemia. The presence of trichilemmomas in a woman with breast cancer raises concern for Cowden syndrome, associated with PTEN

1	at a young age should prompt consideration of familial hypercholesterolemia. The presence of trichilemmomas in a woman with breast cancer raises concern for Cowden syndrome, associated with PTEN mutations.

1	Recall of family history is often inaccurate. This is especially so when the history is remote and families lose contact or separate geographically. It can be helpful to ask patients to fill out family history forms before or after their visits, because this provides them with an opportunity to contact relatives. Ideally, this information should be embedded in electronic health records and updated intermittently. Attempts should be made to confirm the illnesses reported in the family history before making important and, in certain circumstances, irreversible management decisions. This process is often labor

1	FIGURE 84-1 A 36-year-old woman (arrow) seeks consultation because of her family history of cancer. The patient expresses concern that the multiple cancers in her relatives imply an inherited predisposition to develop cancer. The family history is recorded, and records of the patient’s relatives confirm the reported diagnoses. Symbol key Breast cancer 52 Breast ca 44 46 Ovarian ca 43 Ovarian cancer 2 40 Ovarian ca 38 42 Breast ca 38 24 Pneumonia 56 36 62 69 Breast ca 44 55 Ovarian ca 54 6210 Accident 6 40 5 2 2 intensive and ideally involves interviews of additional family members or reviewing medical records, autopsy reports, and death certificates.

1	Although many inherited disorders will be suggested by the clustering of relatives with the same or related conditions, it is important to note that disease penetrance is incomplete for most genetic disorders. As a result, the pedigree obtained in such families may not exhibit a clear Mendelian inheritance pattern, because not all family members carrying the disease-associated alleles will manifest clinical evidence of the condition. Furthermore, genes associated with some of these disorders often exhibit variable disease expression. For example, the breast cancer–associated gene BRCA2 can predispose to several different malignancies in the same family, including cancers of the breast, ovary, pancreas, skin, and prostate. For common diseases such as breast cancer, some family members without the susceptibility allele (or genotype) may develop breast cancer (or phenotype) sporadically. Such phenocopies represent another confounding variable in the pedigree analysis.

1	Some of the aforementioned features of the family history are illustrated in Fig. 84-1. In this example, the proband, a 36-year-old woman (IV-1), has a strong history of breast and ovarian cancer on the paternal side of her family. The early age of onset and the co-occurrence of breast and ovarian cancer in this family suggest the possibility of an inherited mutation in BRCA1 or BRCA2. It is unclear however, without genetic testing, whether her father harbors such a mutation and transmitted it to her. After appropriate genetic counseling of the pro-band and her family, the most informative and cost-effective approach to DNA analysis in this family is to test the cancer-affected 42-year-old living cousin for the presence of a BRCA1 or BRCA2 mutation. If a mutation is found, then it is possible to test for this particular alteration in other family members, if they so desire. In the example shown, if the proband’s father has a BRCA1 mutation, there is a 50:50 probability that the

1	possible to test for this particular alteration in other family members, if they so desire. In the example shown, if the proband’s father has a BRCA1 mutation, there is a 50:50 probability that the mutation was transmitted to her, and genetic testing can be used to establish the absence or presence of this alteration. In this same example, if a mutation is not detected in the cancer-affected cousin, testing would not be indicated for cancer-unaffected relatives.

1	A critical first step before initiating genetic testing is to ensure that the correct clinical diagnosis has been made, whether it is based on family history, characteristic physical findings, pathology, or biochemical testing. Such careful clinical assessment can define the phenotype. In the traditional model of genetic testing, testing is directed initially toward the most probable genes (determined by the phenotype), which 447 prevents unnecessary testing. Many disorders exhibit the feature of locus heterogeneity, which refers to the fact that mutations in different genes can cause phenotypically similar disorders. For example, osteogenesis imperfecta (Chap. 427), long QT syndrome (Chap. 277), muscular dystrophy (Chap. 462e), and hereditary predisposition to breast (Chap. 108) or colon (Chap. 110) cancer can each be caused by mutations in a number of distinct genes. The patterns of disease transmission, disease risk, clinical course, and treatment may differ significantly depending

1	(Chap. 110) cancer can each be caused by mutations in a number of distinct genes. The patterns of disease transmission, disease risk, clinical course, and treatment may differ significantly depending on the specific gene affected. Historically, the choice of which gene to test has been determined by unique clinical and family history features and the relative prevalence of candidate genetic disorders. However, rapid changes in genetic testing techniques, as discussed below, may impact this paradigm. It is now technically and financially feasible to sequence many genes (or even the whole exome) at one time. The incorporation of multiplex testing for germline mutations is rapidly evolving.

1	Genetic testing is regulated and performed in much the same way as other specialized laboratory tests. In the United States, genetic testing laboratories are Clinical Laboratory Improvement Amendments (CLIA) approved to ensure that they meet quality and proficiency standards. A useful information source for various genetic tests is www.genetests.org. It should be noted that many tests need to be ordered through specialized laboratories.

1	Genetic testing is performed largely by DNA sequence analysis for mutations, although genotype can also be deduced through the study of RNA or protein (e.g., apolipoprotein E, hemoglobin S, and immunohistochemistry). For example, universal screening for Lynch syndrome via immunohistochemical analysis of colorectal cancers for absence of expression of mismatch repair proteins is under way at multiple hospitals throughout the United States. The determination of DNA sequence alterations relies heavily on the use of polymerase chain reaction (PCR), which allows rapid amplification and analysis of the gene of interest. In addition, PCR enables genetic testing on minimal amounts of DNA extracted from a wide range of tissue sources including leukocytes, mucosal epithelial cells (obtained via saliva or buccal swabs), and archival tissues. Amplified DNA can be analyzed directly by DNA sequencing, or it can be hybridized to DNA chips or blots to detect the presence of normal and altered DNA

1	saliva or buccal swabs), and archival tissues. Amplified DNA can be analyzed directly by DNA sequencing, or it can be hybridized to DNA chips or blots to detect the presence of normal and altered DNA sequences. Direct DNA sequencing is frequently used for determination of hereditary disease susceptibility and prenatal diagnosis. Analyses of large alterations of the genome are possible using cytogenetics, fluorescent in situ hybridization (FISH), Southern blotting, or multiplex ligation-dependent probe amplification (MLPA) (Chap. 83e).

1	Massively parallel sequencing (also called next-generation sequencing) is significantly altering the approach to genetic testing for adult-onset hereditary susceptibility disorder. This technology encompasses several high-throughput approaches to DNA sequencing, all of which can reliably sequence many genes at one time. Technically, this involves the use of amplified DNA templates in a flow cell, a very different process than traditional Sanger sequencing which is time-consuming and expensive.

1	Multiplex panels for inherited susceptibility are commercially available and include testing of a number of genes that have been associated with the condition of interest. For example, panels are available for Brugada syndrome, hypertrophic cardiomyopathy, and Charcot-Marie-Tooth neuropathy. For many syndromes, this type of panel testing may make sense. However, in other situations, the utility of panel testing is less certain. Currently available breast cancer susceptibility panels contain six genes or more. Many of the genes included in the larger panels are associated with only a modest risk of breast cancer, and the clinical application is uncertain. An additional problem of sequencing many genes (rather than the genes for which there is most suspicion) is the identification of one or more variants of uncertain significance (VUS), discussed below. Whole-exome sequencing (WES) is also now commercially available, although largely used in individuals with syndromes unexplained by

1	Whole-exome sequencing (WES) is also now commercially available, although largely used in individuals with syndromes unexplained by Chapter 84 The Practice of Genetics in Clinical Medicine traditional genetic testing. As cost declines, WES may be more widely used. Whole-genome sequencing is also commercially available. Although it may be quite feasible to sequence the entire genome, there are many issues in doing so, including the daunting task of analyzing the vast amount of data generated. Other issues include: (1) the optimal way in which to obtain informed consent, (2) interpretation of frequent sequence variation of uncertain significance, (3) interpretation of alterations in genes with unclear relevance to specific human pathology, and (4) management of unexpected but clinically significant genetic findings.

1	Testing strategies are evolving as a result of these new genetic testing platforms. As the cost of multiple gene panels and WES continue to fall, and as interpretation of such test results improve, there may be a shift from sequential single-gene (or a few genes) testing to multigene testing. For example, presently, a 30-year-old woman with breast cancer but no family history of cancer and no syndromic features would undergo BRCA1/2 testing. If negative, she would subsequently be offered TP53 testing. Notably, a reasonable number of individuals offered TP53 testing for Li-Fraumeni syndrome decline because mutations are associated with extremely high cancer risks (including childhood cancers) in multiple organs and there are no proven interventions to mitigate risk. Without features consistent with Cowden syndrome, the woman would not be routinely offered PTEN testing or testing for CHEK2, ATM, BRIP, BARD, NBN, and PALB2. However, it is now possible to synchronously analyze all of the

1	with Cowden syndrome, the woman would not be routinely offered PTEN testing or testing for CHEK2, ATM, BRIP, BARD, NBN, and PALB2. However, it is now possible to synchronously analyze all of the aforementioned genes, for a nominally higher cost than BRCA1/2 testing alone. Concerns about such panels include appropriate consent strategies related to unexpected findings, VUS, and unclear clinical utility of testing moderate-penetrance genes. Thus, changes from the traditional model of single-gene genetic testing should be done with caution (Fig. 84-2).

1	Limitations to the accuracy and interpretation of genetic testing exist. In addition to technical errors, genetics tests are sometimes designed to detect only the most common mutations. In addition, genetic testing has evolved over time. For example, it was not possible to obtain commercially available comprehensive large genomic rearrangement testing for BRCA1 and BRCA2 until 2006. Therefore, a negative result must be qualified by the possibility that the individual may have a mutation that was not included in the test. In addition, a negative result does not mean that there is not a mutation in some other gene that causes a similar inherited disorder. A negative result, unless there is known mutation in the family, is typically classified as uninformative.

1	VUS are another limitation to genetic testing. A VUS (also termed unclassified variant) is a sequence variation in a gene where the effect of the alteration on the function of the protein is not known. Many of these variants are single nucleotide substitutions (also called missense mutations) that result in a single amino acid change. Although many VUSs will ultimately be reclassified as benign polymorphisms, some will prove to be functionally important. As more genes are sequenced (for example, in a multiplex panel or through WES), the percentage of individuals found to have a VUS increases significantly. The finding of a VUS is difficult for patients and providers alike and complicates decisions regarding medical management.

1	Clinical utility is an important consideration because genetic testing for susceptibility to chronic diseases is increasingly integrated into the practice of medicine. In some situations, there is clear clinical utility to genetic testing with significant evidence-based changes in medical management decisions based on results. However, in many cases, the discovery of disease-associated genes has outpaced studies that assess how such information should be used in the clinical management of the patient and family. This is particularly true for moderate-and low-penetrance gene mutations. Therefore, predictive genetic testing should be approached with caution and only offered to patients who have been adequately counseled and have provided informed consent. Predictive genetic testing falls into two distinct categories. Presymptomatic testing applies to diseases where a specific genetic alteration is associated with a near 100% likelihood of developing disease. In contrast, predisposition

1	two distinct categories. Presymptomatic testing applies to diseases where a specific genetic alteration is associated with a near 100% likelihood of developing disease. In contrast, predisposition testing predicts a risk for disease that is less than 100%. For example, presymptomatic testing is available for those at risk for Huntington’s disease; whereas, predisposition testing is considered for those at risk for hereditary colon cancer. It is important to note that for the majority of adult-onset disorders, testing is only predictive. Test results cannot reveal with confidence whether, when, or how the disease will manifest itself. For example, not everyone with the apolipoprotein

1	Traditional approach to genetic testing Genetic testing in the era of E4 allele will develop Alzheimer’s disease, and next-generation sequencing? individuals without this genetic marker can still develop the disorder. The optimal testing strategy for a family is to initiate testing in an affected family member first. Identification of a mutation can direct the testing of other at-risk family members (whether symptomatic or not). In the absence of additional familial or environmental risk factors, individuals who test negative for the mutation found in the affected family member can be informed that they are at general population risk for that particular disease. Furthermore, they can be reassured that they are not at risk for passing the mutation on to their children. On the other hand, asymptomatic family members who test positive for the known mutation must be informed that they are at increased risk for disease development and for transmitting the alteration to their children.

1	asymptomatic family members who test positive for the known mutation must be informed that they are at increased risk for disease development and for transmitting the alteration to their children. Pretest counseling and education are important, as is an assessment of the patient’s ability to understand and cope with test results. Genetic testing has implications for entire families, and thus individuals interested in pursuing genetic testing must consider how test results might impact their relationships with relatives, partners, spouses, and children. In families with a known genetic mutation, those who test posi-FIGURE 84-2 Approach to genetic testing. tive must consider the impact of their carrier status on their present and future lifestyles; those who test negative may manifest survivor guilt. Parents who are found to have a disease-associated mutation often express considerable anxiety and despair as they address the issue of risk to their children. In addition, some individuals

1	guilt. Parents who are found to have a disease-associated mutation often express considerable anxiety and despair as they address the issue of risk to their children. In addition, some individuals consider options such as preimplantation genetic diagnosis in their reproductive decision making.

1	When a condition does not manifest until adulthood, clinicians and parents are faced with the question of whether at-risk children should be offered genetic testing and, if so, at what age. Although the matter is debated, several professional organizations have cautioned that genetic testing for adult-onset disorders should not be offered to children. Many of these conditions have no known interventions in childhood to prevent disease; consequently, such information can pose significant psychosocial risk to the child. In addition, there is concern that testing during childhood violates a child’s right to make an informed decision regarding testing upon reaching adulthood. On the other hand, testing should be offered in childhood for disorders that may manifest early in life, especially when management options are available. For example, children with multiple endocrine neoplasia 2 (MEN 2) may develop medullary thyroid cancer early in life and should be considered for prophylactic

1	when management options are available. For example, children with multiple endocrine neoplasia 2 (MEN 2) may develop medullary thyroid cancer early in life and should be considered for prophylactic thyroidectomy (Chap. 408). Similarly, children with familial adenomatous polyposis (FAP) due to a mutation in APC may develop polyps in their teens with progression to invasive cancer in the twenties, and therefore, colonoscopy screening is started between the ages of 10 and 15 years (Chap. 110).

1	Informed consent for genetic testing begins with education and counseling. The patient should understand the risks, benefits, and limitations of genetic testing, as well as the potential implications of test results. Informed consent should include a written document, drafted clearly and concisely in a language and format that is understandable to the patient. Because molecular genetic testing of an asymptomatic individual often allows prediction of future risk, the patient should understand all potential long-term medical, psychological, and social implications of testing. There have long been concerns about the potential for genetic discrimination. The Genetic Information Nondiscrimination Act (GINA) was passed in 2008 and provides some protections related to job and health insurance discrimination. It is important to explore with patients the potential impact of genetic test results on future health as well as disability and life insurance coverage. Patients should understand that

1	It is important to explore with patients the potential impact of genetic test results on future health as well as disability and life insurance coverage. Patients should understand that alternatives remain available if they decide not to pursue genetic testing, including the option of delaying testing to a later date. The option of DNA banking should be presented so that samples are readily available for future use by family members, if needed.

1	Depending on the nature of the genetic disorder, posttest interventions may include: (1) cautious surveillance and awareness; (2) specific medical interventions such as enhanced screening, chemoprevention, or risk-reducing surgery; (3) risk avoidance; and (4) referral to support services. For example, patients with known deleterious mutations in BRCA1 or BRCA2 are strongly encouraged to undergo risk-reducing salpingo-oophorectomy and are offered intensive breast cancer screening as well as the option of risk-reducing mastectomy. In addition, such women may wish to take chemoprevention with tamoxifen, raloxifene, or exemestane. Those with more limited medical management and prevention options, such as patients with Huntington’s disease, should be offered continued follow-up and supportive services, including physical and occupational therapy and social services or support groups as indicated. Specific interventions will change as research continues to enhance our understanding of the

1	services, including physical and occupational therapy and social services or support groups as indicated. Specific interventions will change as research continues to enhance our understanding of the medical management of these genetic conditions and more is learned about the functions of the gene products involved.

1	Individuals who test negative for a mutation in a disease-associated gene identified in an affected family member must be reminded that they may still be at risk for the disease. This is of particular importance for common diseases such as diabetes mellitus, cancer, and coronary artery disease. For example, a woman who finds that she does not carry 449 the disease-associated mutation in BRCA2 previously discovered in the family should be reminded that she still requires the same breast cancer screening recommended for the general population.

1	Genetic counseling should be distinguished from genetic testing and screening, although genetic counselors are often involved in issues related to testing. Genetic counseling refers to a communication process that deals with human problems associated with the occurrence of risk of a genetic disorder in a family. Genetic risk assessment is complex and often involves elements of uncertainty. Counseling, therefore, includes genetic education as well as psychosocial counseling. Genetic counseling can be useful in a wide range of situations (Table 84-1). The role of the genetic counselor includes the following: 1. Gather and document a detailed family history. 2. Educate patients about general genetic principles related to disease risk, both for themselves and for others in the family. 3. Assess and enhance the patient’s ability to cope with the genetic information offered. 4. Discuss how nongenetic factors may relate to the ultimate expression of disease. 5.

1	3. Assess and enhance the patient’s ability to cope with the genetic information offered. 4. Discuss how nongenetic factors may relate to the ultimate expression of disease. 5. Address medical management issues. 6. Assist in determining the role of genetic testing for the individual and the family. 7. Ensure the patient is aware of the indications, process, risks, benefits, and limitations of the various genetic testing options. 8. Assist the patient, family, and referring physician in the interpretation of the test results. 9. Refer the patient and other at-risk family members for additional medical and support services, if necessary.

1	Genetic counseling is generally offered in a nondirective manner, wherein patients learn to understand how their values factor into a particular medical decision. Nondirective counseling is particularly appropriate when there are no data demonstrating a clear benefit associated with a particular intervention or when an intervention is considered experimental. For example, nondirective genetic counseling is used when a person is deciding whether to undergo genetic testing for Huntington’s disease. At this time, there is no clear benefit (in terms of medical outcome) to an at-risk individual undergoing genetic testing for this disease because its course cannot be altered by therapeutic interventions. However, testing can have an important impact on the individual’s perception of advanced care planning and his or her interpersonal relationships and plans for childbearing. Therefore, the decision to pursue testing rests on the individual’s belief system and values. On the other hand, a

1	care planning and his or her interpersonal relationships and plans for childbearing. Therefore, the decision to pursue testing rests on the individual’s belief system and values. On the other hand, a more directive approach is appropriate when a condition can be treated. In a family with FAP, colon cancer screening and prophylactic colectomy should be recommended for known APC mutation carriers. The counselor and clinician following this family must ensure that the at-risk family members have access to the resources necessary to adhere to these recommendations.

1	Genetic education is central to an individual’s ability to make an informed decision regarding testing options and treatment. An adequate knowledge of patterns of inheritance will allow patients to understand the probability of disease risk for themselves and other family members. It is also important to impart the concepts of disease penetrance and expression. For most complex adult-onset genetic Previous history of a child with birth defects or a genetic disorder Personal or family history suggestive of a genetic disorder

1	Previous history of a child with birth defects or a genetic disorder Personal or family history suggestive of a genetic disorder Chapter 84 The Practice of Genetics in Clinical Medicine disorders, asymptomatic patients should be advised that a positive test result does not always translate into future disease development. In addition, the role of nongenetic factors, such as environmental exposures and lifestyle, must be discussed in the context of multi-factorial disease risk and disease prevention. Finally, patients should understand the natural history of the disease as well as the potential options for intervention, including screening, prevention, and in certain circumstances, pharmacologic treatment or prophylactic surgery.

1	Specific treatments are available for a number of genetic disorders. Strategies for the development of therapeutic interventions have a long history in childhood metabolic diseases; however, these principles have been applied in the diagnosis and management of adult-onset diseases as well (Table 84-2). Hereditary hemochromatosis is usually caused by mutations in HFE (although other genes have been less commonly associated) and manifests as a syndrome of iron overload, which can lead to liver disease, skin pigmentation, diabetes mellitus, arthropathy, impotence in males, and cardiac issues (Chap. 428). When identified early, the disorder can be managed effectively with therapeutic phlebotomy. Therefore, when the diagnosis of hemochromatosis has been made in a proband, it is important to counsel and offer testing to other family members in order to minimize the impact of the disorder.

1	Preventative measures and therapeutic interventions are not restricted to metabolic disorders. Identification of familial forms of long QT syndrome, associated with ventricular arrhythmias, allows early electrocardiographic testing and the use of prophylactic antiarrhythmic therapy, overdrive pacemakers, or defibrillators. Individuals with familial hypertrophic cardiomyopathy can be screened by ultrasound, treated with beta blockers or other drugs, and counseled about the importance of avoiding strenuous exercise and dehydration. Those with Marfan’s syndrome can be treated with beta blockers or Abbreviations: AD, autosomal dominant; AR, autosomal recessive; HNPCC, hereditary nonpolyposis colorectal cancer; MRI, magnetic resonance imaging; XL, X-linked. angiotensin II receptor blockers and monitored for the development of aortic aneurysms.

1	angiotensin II receptor blockers and monitored for the development of aortic aneurysms. The field of pharmacogenetics identifies genes that alter drug metabolism or confer susceptibility to toxic drug reactions. Pharmacogenetics seeks to individualize drug therapy in an attempt to improve treatment outcomes and reduce toxicity. Examples include thiopurine methyltransferase (TPMT) deficiency, dihydropyrimidine dehydrogenase deficiency, malignant hyperthermia, and glucose-6-phosphate deficiency. Despite successes in this area, it is not always clear how to incorporate pharmacogenetics into clinical care. For example, although there is an association with CYP and VKORCgenotypes and warfarin dosing, there is no evidence that incorporating genotyping into clinical practice improves patient outcomes.

1	The identification of germline abnormalities that increase the risk of specific types of cancer is rapidly changing clinical management. Identifying family members with mutations that predispose to FAP or Lynch syndrome leads to recommendations of early cancer screening and prophylactic surgery, as well as consideration of chemoprevention and attention to healthy lifestyle habits. Similar principles apply to familial forms of melanoma as well as cancers of the breast, ovary, and thyroid. In addition to increased screening and prophylactic surgery, the identification of germline mutations associated with cancer may also lead to the development of targeted therapeutics, for example, the ongoing development of PARP inhibitors in those with BRCA-associated cancers.

1	Although the role of genetic testing in the clinical setting continues to evolve, such testing holds the promise of allowing early and more targeted interventions that can reduce morbidity and mortality. Rapid technologic advances are changing the ways in which genetic testing is performed. As genetic testing becomes less expensive and technically easier to perform, it is anticipated that there will be an expansion of its use. This will present challenges, but also opportunities. It is critical that physicians and other health care professionals keep current with advances in genetic medicine in order to facilitate appropriate referral for genetic counseling and judicious use of genetic testing, as well as to provide state-of-the-art, evidence-based care for affected or at-risk patients and their relatives. Chapter 84 The Practice of Genetics in Clinical Medicine

1	Mitochondrial DNa and heritable traits and Diseases Karl Skorecki, Doron Behar Mitochondria are cytoplasmic organelles whose major function is to generate ATP by the process of oxidative phosphorylation under aero-85e bic conditions. This process is mediated by the respiratory electron transport chain (ETC) multiprotein enzyme complexes I–V and the two electron carriers, coenzyme Q (CoQ) and cytochrome c. Other cellular processes to which mitochondria make a major contribution include apoptosis (programmed cell death) and additional cell type–specific functions (Table 85e-1). The efficiency of the mitochondrial ETC in ATP production is a major determinant of overall body energy balance and thermogenesis. In addition, mitochondria are the predominant source of reactive oxygen species (ROS), whose rate of production also relates to the coupling of ATP production to oxygen consumption. Given the centrality of oxidative phosphorylation to the normal activities of almost all cells, it is

1	whose rate of production also relates to the coupling of ATP production to oxygen consumption. Given the centrality of oxidative phosphorylation to the normal activities of almost all cells, it is not surprising that mitochondrial dysfunction can affect almost any organ system (Fig. 85e-1). Thus, physicians in many disciplines might encounter patients with mitochondrial diseases and should be aware of their existence and characteristics.

1	The integrated activity of an estimated 1500 gene products is required for normal mitochondrial biogenesis, function, and integrity. Almost all of these are encoded by nuclear genes and thus follow the rules and patterns of nuclear genomic inheritance (Chap. 84). These nuclear-encoded proteins are synthesized in the cell cytoplasm and imported to their location of activity within the mitochondria through a complex biochemical process. In addition, the mitochondria contain their own small genome consisting of numerous copies (polyploidy) per mitochondrion of a circular, double-strand mitochondrial DNA (mtDNA) molecule comprising 16,569 nucleotides. This mtDNA sequence (also known as the “mitogenome”) might represent the remnants of endosymbiotic prokaryotes from which mitochondria are thought to have originated. The mtDNA sequence contains a total of 37 genes, of which 13 encode mitochondrial protein components of the ETC (Fig. 85e-2). The remaining 22 tRNAand 2 rRNA-encoding genes are

1	to have originated. The mtDNA sequence contains a total of 37 genes, of which 13 encode mitochondrial protein components of the ETC (Fig. 85e-2). The remaining 22 tRNAand 2 rRNA-encoding genes are dedicated to the process of translating the 13 mtDNAencoded proteins. This dual nuclear and mitochondrial genetic control of mitochondrial function results in unique and diagnostically challenging patterns of inheritance. The current chapter focuses on heritable traits and diseases related to the mtDNA component of the dual genetic control of mitochondrial function. The reader is referred to Chaps. 84 and 462e for consideration of mitochondrial disease originating from mutations in the nuclear genome. The latter include disorders due to mutations in nuclear genes directly encoding structural components or assembly factors of the oxidative phosphorylation complexes, (2) disorders due to mutations in nuclear genes encoding proteins indirectly related to oxidative phosphorylation, and of mtDNA

1	or assembly factors of the oxidative phosphorylation complexes, (2) disorders due to mutations in nuclear genes encoding proteins indirectly related to oxidative phosphorylation, and of mtDNA copy number in affected tissues without mutations or rear-85e-1 rangements in the mtDNA.

1	As a result of its circular structure and extranuclear location, the replication and transcription mechanisms of mtDNA differ from the corresponding mechanisms in the nuclear genome, whose nucleosomal packaging and structure are more complex. Because each cell contains many copies of mtDNA, and because the number of mitochondria can vary during the lifetime of each cell, mtDNA copy number is not directly coordinated with the cell cycle. Thus, vast differences in mtDNA copy number are observed between different cell types and tissues and during the lifetime of a cell. Another important feature of the mtDNA replication process is a reduced stringency of proofreading and replication error correction, leading to a greater degree of sequence variation compared to the nuclear genome. Some of these sequence variants are silent polymorphisms that do not have the potential for a phenotypic or pathogenic effect, whereas others may be considered pathogenic mutations.

1	With respect to transcription, initiation can occur on both strands and proceeds through the production of an intronless polycistronic precursor RNA, which is then processed to produce the 13 individual mRNA and 24 individual tRNA and rRNA products. The 37 mtDNA genes comprise fully 93% of the 16,569 nucleotides of the mtDNA in what is known as the coding region. The control region consists of ~1.1 kilobases (kb) of noncoding DNA, which is thought to have an important role in replication and transcription initiation.

1	In contrast to homologous pair recombination that takes place in the nucleus, mtDNA molecules do not undergo recombination, such that mutational events represent the only source of mtDNA genetic diversification. Moreover, with very rare exceptions, it is only the maternal DNA that is transmitted to the offspring. The fertilized oocyte degrades mtDNA carried from the sperm in a complex process involving the ubiquitin proteasome system. Thus, although mothers transmit their mtDNA to both their sons and daughters, only the daughters are able to transmit the inherited mtDNA to future generations. Accordingly, mtDNA sequence variation and associated phenotypic traits and diseases are inherited exclusively along maternal lines.

1	As noted below, because of the complex relationship between mtDNA mutations and disease expression, sometimes this maternal inheritance is difficult to recognize at the clinical or pedigree level. However, evidence of paternal transmission can almost certainly rule out an mtDNA genetic origin of phenotypic variation or disease; conversely, a disease affecting both sexes without evidence of paternal transmission strongly suggests a heritable mtDNA disorder (Fig. 85e-2). MULTIPLE COPY NUMBER (POLYPLOIDY), HIGH MUTATION RATE, HETEROPLASMY, AND MITOTIC SEGREGATION

1	Each aerobic cell in the body has multiple mitochondria, often numbering many hundreds or more in cells with extensive energy production requirements. Furthermore, the number of copies of mtDNA within each mitochondrion varies from several to hundreds; this is true of both somatic as well as germ cells, including oocytes in females. In the case of somatic cells, this means that the impact of most newly acquired somatic mutations is likely to be very small in terms of total cellular or organ system function; however, because of the manyfold higher mutation rate during mtDNA replication, numerous different mutations may accumulate with aging of the organism. It has been proposed that the total cumulative burden of acquired somatic mtDNA mutations with age may result in an overall perturbation of mitochondrial function, contributing to age-related reduction in the efficiency of oxidative phosphorylation and increased production of damaging ROS. The accumulation of such acquired somatic

1	of mitochondrial function, contributing to age-related reduction in the efficiency of oxidative phosphorylation and increased production of damaging ROS. The accumulation of such acquired somatic mtDNA mutations with aging may contribute to age-related diseases, such as metabolic syndrome and diabetes, cancer, and neurodegenerative and cardiovascular disease in any given individual. However, somatic mutations are not carried forward to the next generation, and the individual, play a pivotal role in the manifestation and severity of disease and are crucial to understanding the complexity of inheritance of mtDNA disorders. At the level of the oocyte, the percentage of mtDNA molecules bearing each version of the polymorphic sequence variant or mutation depends

1	Liver on stochastic events related to partition- Hepatopathy ing of mtDNA molecules during the process of oogenesis itself. Thus, oocytes differ from each other in the degree of ATP heteroplasmy for that sequence variant or mutation. In turn, the heteroplasmic state is carried forward to the zygote and to the organism as a whole, to varying Nuclear Subunits Oxidative degrees, depending on mitotic segrega-DNA phosphorylation tion of mtDNA molecules during organ Fanconi's syndrome system development and maintenance. Glomerulopathy For this reason, in vitro fertilization, Brain followed by preimplantation genetic diagnosis (PGD), is not as predictive of the genetic health of the offspring in Mitochondrial the case of mtDNA mutations as in the DNA case of the nuclear genome. Similarly, Dementia Pancreas Migraine Diabetes mellitus the impact of somatic mtDNA mutations acquired during development and subsequently also shows an enormous spectrum of variability.

1	Dementia Pancreas Migraine Diabetes mellitus the impact of somatic mtDNA mutations acquired during development and subsequently also shows an enormous spectrum of variability. Blood Mitotic segregation refers to the Pearson's syndrome unequal distribution of wild-type and mutant versions of mtDNA molecules Inner ear during all cell divisions that occur dur-Sensorineural ing prenatal development and subsehearing loss Colon quently throughout the lifetime of an Pseudo-obstruction individual. The phenotypic effect or FIGURE 85e-1 Dual genetic control and multiple organ system manifestations of mitochondrial disease impact will, thus, be a funcdisease. (Reproduced with permission from DR Johns: Mitochondrial DNA and disease. N Engl J Med tion not only of the inherent disruptive 333:638, 1995.) hereditary impact of mtDNA mutagenesis requires separate consideration of events in the female germline.

1	The multiple mtDNA copy number within each cell, including the maternal germ cells, results in the phenomenon of heteroplasmy, in contrast to much greater uniformity (homoplasy) of somatic nuclear DNA sequence. Heteroplasmy for a given mtDNA sequence variant or mutation arises as a result of the coexistence within a cell, tissue, or individual of mtDNA molecules bearing more than one version of the sequence variant (Fig. 85e-3). The importance of the heteroplasmy phenomena to the understanding of mtDNA-related mitochondrial diseases is critical. The coexistence of mutant and nonmutant mtDNA and the variation of the mutant load among individuals from the same maternal sibship, and across organs and tissues within the same FIGURE 85e-2 Maternal inheritance of mitochondrial DNA (mtDNA) disorders and heritable traits. Affected women (filled circles) transmit the trait to their children. Affected men (filled squares) do not transmit the trait to any of their offspring.

1	effect (pathogenicity) on the mtDNA tions) or integrity of the mtDNA molecule (control region mutations), but also of its distribution among the multiple copies of mtDNA in the various mitochondria, cells, and tissues of the affected individual. Thus, one consequence can be the generation of a bottleneck due to the marked decline in given sets of mtDNA variants, consequent to such mitotic segregation. Heterogeneity arises from differences in the degree of heteroplasmy among oocytes of the affected female, together with subsequent mitotic segregation of the pathogenic mutation during tissue and organ development, and throughout the lifetime of the individual offspring. The actual expression of disease might then depend on a threshold percentage of mitochondria whose function is disrupted by mtDNA mutations. This in turn confounds hereditary transmission patterns and hence genetic diagnosis of pathogenic heteroplasmic mutations. Generally, if the proportion of mutant mtDNA is less than

1	mtDNA mutations. This in turn confounds hereditary transmission patterns and hence genetic diagnosis of pathogenic heteroplasmic mutations. Generally, if the proportion of mutant mtDNA is less than 60%, the individual is unlikely to be affected, whereas proportions exceeding 90% cause clinical disease.

1	In contrast to classic mtDNA diseases, most of which begin in childhood and are the result of heteroplasmic mutations as noted above, during the course of human evolution, certain mtDNA sequence variants have drifted to a state of homoplasmy, wherein all of the mtDNA molecules in the organism contain the new sequence variant. This arises due to a “bottleneck” effect followed by genetic drift during the very process of oogenesis itself (Fig. 85e-3). In other words, during certain stages of oogenesis, the mtDNA copy number becomes so substantially reduced that the particular mtDNA species bearing the novel or derived sequence variant may become the increasingly predominant, mate because of the phenotypic heterogeneity that occurs as a function of heteroplasmy, the challenge of detecting and assessing heteroplasmy in different affected tissues, and the other unique features of mtDNA function and FIGURE 85e-3 Heteroplasmy and the mitochondrial genetic bottleneck.

1	FIGURE 85e-3 Heteroplasmy and the mitochondrial genetic bottleneck. inheritance described above. It is estimated that at least During the production of primary oocytes, a selected number of mitochondrial DNA (mtDNA) molecules are transferred into each oocyte. Oocyte maturation is mutation with the potential to causes disease, but that associated with the rapid replication of this mtDNA population. This restriction amplification event can lead to a random shift of mtDNA mutational load actually affect up to approximately 1 in 8500 individuals. between generations and is responsible for the variable levels of mutated mtDNA The true disease burden relating to mtDNA sequence observed in affected offspring from mothers with pathogenic mtDNA mutations. variation will only be known when the following capa-

1	The true disease burden relating to mtDNA sequence observed in affected offspring from mothers with pathogenic mtDNA mutations. variation will only be known when the following capa- Mitochondria that contain mutated mtDNA are shown in red, and those with bilities become available: (1) ability to distinguish a normal mtDNA are shown in green. (Reproduced with permission from R Taylor, D Turnbull: Mitochondrial DNA mutations in human disease. Nat Rev Genetics 6:389, 2005.) and eventually exclusive, version of the mtDNA for that particular nucleotide site. All of the offspring of a woman bearing an mtDNA sequence variant or mutation that has become homoplasmic will also be homoplasmic for that variant and will transmit the sequence variant forward in subsequent generations.

1	Considerations of reproductive fitness limit the evolutionary or population emergence of homoplasmic mutations that are lethal or cause severe disease in infancy or childhood. Thus, with a number of notable exceptions (e.g., mtDNA mutations causing Leber’s hereditary optic neuropathy; see below), most homoplasmic mutations are considered to be neutral markers of human evolution, which are useful and interesting in the population genetics analysis of shared maternal ancestry but which have little significance in human phenotypic variation or disease predisposition.

1	More importantly is the understanding that this accumulation of homoplastic mutations occurs at a genetic locus that is transmitted only through the female germline and that lacks recombination. In turn, this enables reconstruction of the sequential topology and radiating phylogeny of mutations accumulated through the course of human evolution since the time of the most recent common mtDNA ancestor of all contemporary mtDNA sequences, some 200,000 years ago. The term haplogroup is usually used to define major branching points in the human mtDNA phylogeny, nested one within the other, which often demonstrate striking continental geographic ancestral partitioning. At the level of the complete mtDNA sequence, the term haplotype is usually used to describe the sum of mutations observed for a given mtDNA sequence and as compared to a reference sequence, such that all haplotypes falling within a given haplogroup share the total sum of mutations that have accumulated since the most recent

1	a given mtDNA sequence and as compared to a reference sequence, such that all haplotypes falling within a given haplogroup share the total sum of mutations that have accumulated since the most recent common ancestor and the bifurcation point they mark. The remaining observed variants are private to each haplotype. Consequentially, human mtDNA sequence is an almost perfect molecular prototype for a nonrecombining locus, and its variation has been extensively used in phylogenetic studies. Moreover, the mtDNA mutation rate is considerably higher than the rate observed for the nuclear genome, especially in the control region, which contains the displacement, or

1	Oocyte maturation D-loop, in turn comprising two adjacent hypervariable 85e-3 and mtDNA amplification Fertilization regions (HVR-I and HVR-II). Together with the absence of recombination, this amplifies drift to high frequencies of novel haplotypes. As a result, mtDNA haplotypes are more highly partitioned across geographically defined High level of mutation (affected offspring) populations than sequence variants in other parts of the genome. Despite extensive research, it has not been well established that such haplotype-based partitioning has a significant influence on human health conditions. However, mtDNA-based phylogenetic analysis can be used both as a quality assurance tool and as a filter of mutation (mildly affected offspring) in distinguishing neutral mtDNA variants comprising human mtDNA phylogeny from potentially deleterious mutations.

1	Low level of mutation (unaffected offspring) The true prevalence of mtDNA disease is difficult to esti notype-modifying or pathogenic mutation, (2) accurate assessment of heteroplasmy that can be determined with fidelity, and (3) a systems biology approach (Chap. 87e) to determine the network of epistatic interactions of mtDNA sequence variations with mutations in the nuclear genome.

1	Given the vital roles of mitochondria in all nucleated cells, it is not surprising that mtDNA mutations can affect numerous tissues with pleiotropic effects. More than 200 different disease-causing, mostly heteroplasmic mtDNA mutations have been described affecting ETC function. Figure 85e-4 provides a partial mtDNA map of some of the better characterized of these disorders. A number of clinical clues can increase the index of suspicion for a heteroplasmic mtDNA mutation as an etiology of a heritable trait or disease, including (1) familial clustering with absence of paternal transmission; (2) adherence to one of the classic syndromes (see below) or paradigmatic combinations of disease phenotypes involving several organ systems that normally do not fit together within a single nuclear genomic mutation category; (3) a complex of laboratory and pathologic abnormalities that reflect disruption in cellular energetics (e.g., lactic acidosis and neurodegenerative and myodegenerative

1	genomic mutation category; (3) a complex of laboratory and pathologic abnormalities that reflect disruption in cellular energetics (e.g., lactic acidosis and neurodegenerative and myodegenerative symptoms with the finding of ragged red fibers, reflecting the accumulation of abnormal mitochondria under the muscle sarcolemmal membrane); or (4) a mosaic pattern reflecting a heteroplasmic state.

1	Heteroplasmy can sometimes be elegantly demonstrated at the tissue level using histochemical staining for enzymes in the oxidative phosphorylation pathway, with a mosaic pattern indicating heterogeneity of the genotype for the coding region for the mtDNA-encoded enzyme. Complex II, CoQ, and cytochrome c are exclusively encoded by nuclear DNA. In contrast, complexes I, III, IV, and V contain at least some subunits encoded by mtDNA. Just 3 of the 13 subunits of the ETC complex IV enzyme, cytochrome c oxidase, are encoded by mtDNA, and, therefore, this enzyme has the lowest threshold for dysfunction when a threshold level of mutated mtDNA is reached. Histochemical staining for cytochrome c oxidase activity in tissues of patients affected with heteroplasmic inherited mtDNA mutations Parkinsonism, LS, MELAS, PEO, LHON, MELAS, myopathy, cardiomyopathy, diabetes and deafness Myopathy, LHON cardiomyopathy, PEO Myopathy, MELAS Myopathy, lymphoma

1	Parkinsonism, LS, MELAS, PEO, LHON, MELAS, myopathy, cardiomyopathy, diabetes and deafness Myopathy, LHON cardiomyopathy, PEO Myopathy, MELAS Myopathy, lymphoma LS, ataxia, chorea, myopathy PEO Myopathy, PEO Myoglobinuria, motor neuron disease, PPK, deafness, Myopathy, multisystem disease, NARP, MILS, Myopathy, PEO Cardiomyopathy ECM ECM, LHON, myopathy, cardiomyopathy, MELAS and parkinsonism LHON, MELAS, diabetes, LHON and dystonia ND5 LS, MELAS Cardiomyopathy, ECM PEO, myopathy, LHON, myopathy, LHON and dystonia Progressive myoclonus, epilepsy, and optic atrophy Cardiomyopathy, encephalomyopathy FBSN SIDS, ECM Cardiomyopathy, LS, ECM, PEO, MERRF, myoglobinuria MELAS, deafness FIGURE 85e-4 Mutations in the human mitochondrial genome known to cause disease.

1	Disorders that are frequently or prominently associated with mutations in a particular gene are shown in boldface. Diseases due to mutations that impair mitochondrial protein synthesis are shown in blue. Diseases due to mutations in protein-coding genes are shown in red. ECM, encephalomyopathy; FBSN, familial bilateral striatal necrosis; LHON, Leber’s hereditary optic neuropathy; LS, Leigh syndrome; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; MERRF, myoclonic epilepsy with ragged red fibers; MILS, maternally inherited Leigh syndrome; NARP, neuropathy, ataxia, and retinitis pigmentosa; PEO, progressive external ophthalmoplegia; PPK, palmoplantar keratoderma; SIDS, sudden infant death syndrome. (Reproduced with permission from S DiMauro, E Schon: Mitochondrial respiratory-chain diseases. N Engl J Med 348:2656, 2003.) (or with the somatic accumulation of mtDNA mutations, see below) can show a mosaic pattern of reduced histochemical staining in

1	Mitochondrial respiratory-chain diseases. N Engl J Med 348:2656, 2003.) (or with the somatic accumulation of mtDNA mutations, see below) can show a mosaic pattern of reduced histochemical staining in comparison with histochemical staining for the complex II enzyme, succinate dehydrogenase (Fig. 85e-5). Heteroplasmy can also be detected at the genetic level through direct Sanger-type mtDNA genotyping under special conditions, although clinically significant low levels of heteroplasmy can escape detection in genomic samples extracted from whole blood using conventional genotyping and sequencing techniques.

1	The emerging next-generation sequencing (NGS) techniques and their rapid penetration and recognition as useful clinical diagnostic tools are expected to also dramatically improve the clinical genetic diagnostic evaluation of mitochondrial diseases at the level of both the nuclear genome and mtDNA. In the context of the larger nuclear genome, the ability of NGS techniques to dramatically increase the speed at which DNA can be sequenced at a fraction of the cost of conventional Sanger-type sequencing technology is particularly beneficial. Low sequencing costs and short turnaround time expedite “first-tier” screening of panels of hundreds of previously known or suspected mitochondrial disease genes or screening for the entire exome or genome in an attempt to identify novel genes and mutations affecting different patients or families. In the context of the mtDNA, NGS approaches hold the particular promise for rapid and reliable detection of heteroplasmy in different affected tissues.

1	affecting different patients or families. In the context of the mtDNA, NGS approaches hold the particular promise for rapid and reliable detection of heteroplasmy in different affected tissues. Although Sanger sequencing allows for complete coverage of the mtDNA, it is limited by the lack of deep coverage and low sensitivity for heteroplasmy detection when it is much less than 50%. In contrast, NGS technology is an excellent tool for rapidly and accurately obtaining a patient’s predominant mtDNA sequence and also lower frequency heteroplasmic variants. This is enabled by deep coverage of the genome through multiple independent sequence reads. Accordingly, recent studies making use of NGS techniques have demonstrated sequence accuracy equivalent to Sanger-type sequencing, but also have uncovered heretofore unappreciated heteroplasmy rates ranging between 10 and 50% and detection of single-nucleotide heteroplasmy down to levels of <10%.

1	Clinically, the most striking overall characteristic of mitochondrial genetic disease is the phenotypic heterogeneity associated with mtDNA mutations. This extends to intrafamilial phenotypic heterogeneity for the same mtDNA pathogenic mutation and, conversely, to the overlap of phenotypic disease manifestations with distinct mutations. Thus, although fairly consistent and well-defined “classic” syndromes have been attributed to specific mutations, frequently “nonclassic” combinations of disease phenotypes ranging from isolated myopathy to extensive multi-system disease are often encountered, rendering genotype-phenotype correlation challenging. In both classical and nonclassical mtDNA disorders, there is often a clustering of some combination of abnormalities affecting the neurologic system (including optic nerve atrophy, pigment retinopathy, and sensorineural hearing loss), cardiac and skeletal muscle (including extraocular muscles), and endocrine and metabolic systems (including

1	(including optic nerve atrophy, pigment retinopathy, and sensorineural hearing loss), cardiac and skeletal muscle (including extraocular muscles), and endocrine and metabolic systems (including diabetes mellitus).

1	Additional organ systems that may be affected include the hematopoietic, renal, hepatic, and gastrointestinal systems, although these are more frequently involved in infants and children. Disease-causing mtDNA coding region mutations can affect either one of the 13 protein encoding genes or one of the 24 protein synthetic genes. Clinical manifestations do not readily distinguish these two categories, although lactic acidosis and muscle pathologic findings tend to be more prominent in the latter. In all cases, either defective ATP production due to disturbances in the ETC or enhanced generation of ROS has been invoked as the mediating biochemical mechanism between mtDNA mutation and disease manifestation.

1	The clinical presentation of adult patients with mtDNA disease can be divided into three categories: (1) clinical features suggestive of mitochondrial disease (Table 85e-2), but not a well-defined classic syndrome; (2) classic mtDNA syndromes; and (3) clinical presentation confined to one organ system (e.g., isolated sensorineural deafness, cardiomyopathy, or diabetes mellitus). Table 85e-3 provides a summary of eight illustrative classic mtDNA syndromes or disorders that affect adult patients and highlights some of the most interesting features of mtDNA disease in terms of molecular pathogenesis, inheritance, and clinical presentation. The first five of these syndromes result from heritable point mutations in either protein-encoding or protein synthetic mtDNA genes; the other three result from rearrangements or deletions that usually do not involve the germline.

1	are often homoplasmic for the disease-85e-5 causing mutation. The somewhat later onset in young adulthood and modifying effect of protective background nuclear genomic haplotypes may have enabled homoplasmic pathogenic mutations to have escaped evolutionary censoring.

1	Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a multisystem disorder with a typical onset between 2 to 10 years of age. Following normal early psychomotor development, the most common initial symptoms are seizures, recurrent headaches, anorexia, and recurrent vomiting. Exercise intolerance or proximal limb weakness can be the initial manifestation, followed by generalized tonic-clonic seizures. Short stature is common. Seizures are often associated with stroke-like episodes of transient FIGURE 85e-5 Cytochrome c oxidase (COX) deficiency in mitochondrial DNA (mtDNA)– hemiparesis or cortical blindness that associated disease. Transverse tissue sections that have been stained for COX and succinate dehy-may produce altered consciousness drogenase (SDH) activities sequentially, with COX-positive cells shown in brown and COX-deficient and may recur. The cumulative residcells shown in blue. A. Skeletal muscle from a patient with a heteroplasmic

1	(SDH) activities sequentially, with COX-positive cells shown in brown and COX-deficient and may recur. The cumulative residcells shown in blue. A. Skeletal muscle from a patient with a heteroplasmic mitochondrial tRNA ual effects of the stroke-like episodes point mutation. The section shows a typical “mosaic” pattern of COX activity, with many muscle gradually impair motor abilities, vision, fibers harboring levels of mutated mtDNA that are above the crucial threshold to produce a func-and cognition, often by adolescence tional enzyme complex. B. Cardiac tissue (left ventricle) from a patient with a homoplasmic tRNA or young adulthood. Sensorineural mutation that causes hypertrophic cardiomyopathy, which demonstrates an absence of COX in hearing loss adds to the progressive most cells. C. A section of cerebellum from a patient with mtDNA rearrangement that highlights decline of these individuals. A plethora the presence of COX-deficient neurons. D, E. Tissues that show COX deficiency

1	A section of cerebellum from a patient with mtDNA rearrangement that highlights decline of these individuals. A plethora the presence of COX-deficient neurons. D, E. Tissues that show COX deficiency due to clonal expan-of less common symptoms have been sion of somatic mtDNA mutations within single cells—a phenomenon that is seen in both post-described including myoclonus, ataxia, mitotic cells (D; extraocular muscles) and rapidly dividing cells (E; colonic crypt) in aging humans. episodic coma, optic atrophy, cardio(Reproduced with permission from R Taylor, D Turnbull: Mitochondrial DNA mutations in human disease. myopathy, pigmentary retinopathy,

1	Nat Rev Genetics 6:389, 2005.)

1	Leber’s hereditary optic neuropathy (LHON) is a common cause of maternally inherited visual failure. LHON typically presents during young adulthood with subacute painless loss of vision in one eye, with symptoms developing in the other eye 6–12 weeks after the initial onset. In some instances, cerebellar ataxia, peripheral neuropathy, and cardiac conduction defects are observed. In >95% of cases, LHON is due to one of three homoplasmic point mutations of mtDNA that affect genes encoding different subunits of complex I of the mitochondrial ETC; however, not all individuals who inherit a primary LHON mtDNA mutation develop optic neuropathy, and males are four to five times more likely than females to be affected, indicating that additional environmental (e.g., tobacco exposure) or genetic factors are important in the etiology of the disorder. Both the nuclear and mitochondrial genomic backgrounds modify disease penetrance. Indeed, a region of the X chromosome containing a high-risk

1	factors are important in the etiology of the disorder. Both the nuclear and mitochondrial genomic backgrounds modify disease penetrance. Indeed, a region of the X chromosome containing a high-risk haplotype for LHON was recently identified, supporting the formulation that nuclear genes act as modifiers and affording an explanation for the male prevalence of LHON. This haplotype can be used in predictive genomic testing and prenatal screening for this disease. In contrast to the other classic mtDNA disorders, it is of interest that patients with this syndrome

1	Neurologic: stroke, epilepsy, migraine headache, peripheral neuropathy, cranial neuropathy (optic atrophy, sensorineural deafness, dysphagia, dysphasia) Skeletal myopathy: ophthalmoplegia, exercise intolerance, myalgia Cardiac: conduction block, cardiomyopathy Respiratory: hypoventilation, aspiration pneumonitis Endocrine: diabetes mellitus, premature ovarian failure, hypothyroidism, hypoparathyroidism

1	Ophthalmologic: cataracts, pigment retinopathy, neurologic and myopathic (optic atrophy, ophthalmoplegia) ophthalmoplegia, diabetes mellitus, hir sutism, gastrointestinal dysmotility, and nephropathy. The typical age of death ranges from 10 to 35 years, but some individuals live into their sixth decade. Intercurrent infections or intestinal obstructions are often the terminal events. Laboratory investigation commonly demonstrates elevated lactate concentrations at rest with excessive increase after moderate exercise. Brain imaging during stroke-like episodes shows areas of increased T2 signal, typically involving the posterior cerebrum and not conforming to the distribution of major arteries. Electrocardiogram (ECG) may show evidence of cardiomyopathy, preexcitation, or incomplete heart block. Electromyography and nerve conduction studies are consistent with a myopathic process, but axonal and sensory neuropathy may coexist. Muscle biopsy typically shows ragged red fibers with the

1	block. Electromyography and nerve conduction studies are consistent with a myopathic process, but axonal and sensory neuropathy may coexist. Muscle biopsy typically shows ragged red fibers with the modified Gomori trichrome stain or “ragged blue fibers” resulting from the hyperintense reaction with the histochemical staining for succinate dehydrogenase. The diagnosis of MELAS is based on a combination of clinical findings and molecular genetic testing. Mutations in the mtDNA gene MT-TL1 encoding tRNAleu are causative. The most common mutation, present in approximately 80% of individuals with typical clinical findings, is an A-to-G transition at nucleotide 3243 (m.3243A>G). Mutations can usually be detected in mtDNA from leukocytes in individuals with typical MELAS; however, the occurrence of heteroplasmy can result in varying tissue distribution of mutated mtDNA. In the absence of specific treatment, various manifestations of MELAS are treated according to standard modalities for

1	of heteroplasmy can result in varying tissue distribution of mutated mtDNA. In the absence of specific treatment, various manifestations of MELAS are treated according to standard modalities for prevention, surveillance, and treatment.

1	Myoclonic epilepsy with ragged red fibers (MERRF) is a multisystem disorder characterized by myoclonus, seizures, ataxia, and myopathy with ragged red fibers. Hearing loss, exercise intolerance, neuropathy, and short stature are often present. Almost all MERRF patients have mutation in the mtDNA tRNAlys gene, and the m.8344A>G mutation in the mtDNA gene encoding the lysine amino acid tRNA is responsible for 80–90% of MERRF cases. Abbreviations: CSF, cerebrospinal fluid; NARP, neuropathy, ataxia, and retinitis pigmentosa.

1	Abbreviations: CSF, cerebrospinal fluid; NARP, neuropathy, ataxia, and retinitis pigmentosa. Neuropathy, ataxia, and retinitis pigmentosa (NARP) is characterized by moderate diffuse cerebral and cerebellar atrophy and symmetric lesions of the basal ganglia on magnetic resonance imaging (MRI). A heteroplasmic m.8993T>G mutation in the ATPase 6 subunit gene has been identified as causative. Ragged red fibers are not observed in muscle biopsy. When >95% of mtDNA molecules are mutant, a more severe clinical, neuroradiologic. and neuropathologic picture (Leigh syndrome) emerges. Point mutations in the mtDNA gene encoding the 12S rRNA result in heritable nonsyndromic hearing loss. One such mutation causes heritable ototoxic susceptibility to aminoglycoside antibiotics, which opens a pathway for a simple pharmacogenetic test in the appropriate clinical settings.

1	Kearns-Sayre syndrome (KSS), sporadic progressive external ophthalmoplegia (PEO), and Pearson syndrome are three disease phenotypes caused by large-scale mtDNA rearrangements including partial deletions or partial duplication. The majority of single large-scale rearrangements of mtDNA are thought to result from clonal amplification of a single sporadic mutational event, occurring in the maternal oocyte or during early embryonic development. Because germline involvement is rare, most cases are sporadic rather than inherited. KSS is characterized by the triad of onset before age 20, chronic progressive external ophthalmoplegia, and pigmentary retinopathy. Cerebellar syndrome, heart block, increased cerebrospinal fluid protein content, diabetes mellitus, and short stature are also part of the syndrome. Single deletions/duplication can also result in milder phenotypes such as PEO, characterized by late-onset progressive external ophthalmoplegia, proximal myopathy, and exercise

1	part of the syndrome. Single deletions/duplication can also result in milder phenotypes such as PEO, characterized by late-onset progressive external ophthalmoplegia, proximal myopathy, and exercise intolerance. In both KSS and PEO, diabetes mellitus and hearing loss are frequent accompaniments. Pearson syndrome is also characterized by diabetes mellitus from pancreatic insufficiency, together with pancytopenia and lactic acidosis, caused by the large-scale sporadic deletion of several mtDNA genes.

1	Two important dilemmas in classic mtDNA disease have benefited from recent important research insights. The first relates to the greater involvement of neuronal, muscular, renal, hepatic, and pancreatic manifestations in mtDNA disease in these syndromes. This observation has appropriately been mostly attributed to the high energy utilization of the involved tissues and organ systems and, hence, greater dependency on mitochondrial ETC integrity and health. However, because mutations are stochastic events, mitochondrial mutations should occur in any organ during embryogenesis and development. Recently, additional explanations have been suggested based on studies of the common m.3243A>G transition. The proportion of this mutation in peripheral blood cells was shown to decrease exponentially with age. m.1555A>G mutation in 12S Homoplasmic Maternal rRNA m.7445A>G mutation in 12S Homoplasmic Maternal rRNA Single deletions or duplications Heteroplasmic Mostly sporadic, somatic mutations

1	m.1555A>G mutation in 12S Homoplasmic Maternal rRNA m.7445A>G mutation in 12S Homoplasmic Maternal rRNA Single deletions or duplications Heteroplasmic Mostly sporadic, somatic mutations Large deletion Heteroplasmic Sporadic, somatic mutations The 5-kb “common deletion” Heteroplasmic Sporadic, somatic mutations A selective process acting at the stem cell level with a strong bias against the mutated form would have its greatest effect to reduce the mutant mtDNA only in highly proliferating cells, such as those derived from the hematopoietic system. Tissues and organs with lower cell turnover, such as those involved with mtDNA mutations, would not benefit from this effect and, thus, would be the most affected.

1	The other dilemma arises from the observation that only a subset of mtDNA mutations accounts for the majority of the familial mtDNA diseases. The random occurrence of mutations in the mtDNA sequence should yield a more uniform distribution of disease-causing mutations. However, recent studies using the introduction of one severe and one mild point mutation into the female germline of experimental animals demonstrated selective elimination during oogenesis of the severe mutation and selective retention of the milder mutation, with the emergence of mitochondrial disease in offspring after multiple generations. Thus, oogenesis itself can act as an “evolutionary” filter for mtDNA disease.

1	The clinical presentations of classic syndromes, groupings of disease manifestations in multiple organ systems, or unexplained isolated presentations of one of the disease features of a classic mtDNA syndrome should prompt a systematic clinical investigation as outlined in Fig. 85e-6. Indeed, mitochondrial disease should be considered in the differential diagnosis of any progressive multisystem disorder. Despite the centrality of disruptive oxidative phosphorylation, an elevated blood lactate level is neither specific nor sensitive because there are many causes of blood lactic acidosis, and many patients with mtDNA defects presenting in adulthood have normal blood lactate. An elevated cerebrospinal fluid lactate is a more specific test for mitochondrial disease if there is central nervous system involvement. The serum creatine kinase may be elevated but is often normal, even in the presence of a proximal myopathy. Urinary organic and amino acids may also be abnormal, reflecting

1	system involvement. The serum creatine kinase may be elevated but is often normal, even in the presence of a proximal myopathy. Urinary organic and amino acids may also be abnormal, reflecting metabolic and kidney proximal tubule dysfunction. Every patient with seizures or cognitive decline should have an electroencephalogram. A brain computed tomography (CT) scan may show calcified basal ganglia or bilateral hypodense regions with cortical atrophy. MRI is indicated in patients with brainstem signs or stroke-like episodes.

1	For some mitochondrial diseases, it is possible to obtain an accurate diagnosis with a simple molecular genetic screen. For examples, 95% of patients with LHON harbor one of three mtDNA point mutations (m.11778A>G, m.A3460A>G, or m.14484T>C). These patients have very high levels of mutated mtDNA in peripheral blood cells, and Blood: creatine kinase, liver functions, glucose, lactate Urine: organic and amino acids CSF: glucose, protein, lactate Cardiac x-ray, ECG, ECHO EEG, EMG, nerve conduction Brain CT/MRI PCR/RFLP analysis of blood for known mutations Specific point mutation syndrome: e.g., MELAS, MERRF, and LHON

1	PCR/RFLP analysis of blood for known mutations Specific point mutation syndrome: e.g., MELAS, MERRF, and LHON FIGURE 85e-6 Clinical and laboratory investigation of a suspected mitochondrial DNA (mtDNA) disorder. CSF, cerebrospinal fluid; CT, computed tomography; ECG, electrocardiogram; ECHO, echocardiography; EEG, electroencephalogram; EMG, electromyogram; LHON, Leber’s hereditary optic neuropathy; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stoke-like episodes; MERFF, myoclonic epilepsy with ragged red fibers; MRI, magnetic resonance imaging; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism.

1	therefore, it is appropriate to send a blood sample for molecular genetic analysis by polymerase chain reaction (PCR) or restriction fragment length polymorphism. The same is true for most MERRF patients who harbor a point mutation in the lysine tRNA gene at position 8344. In contrast, patients with the m.3243A>G MELAS mutation often have low levels of mutated mtDNA in blood. If clinical suspicion is strong enough to warrant peripheral blood testing, then patients with a negative result should be investigated further by performing a skeletal muscle biopsy.

1	Muscle biopsy histochemical analysis is the cornerstone for investigation of patients with suspected mitochondrial disease. Histochemical analysis may show subsarcolemmal accumulation of mitochondria with the appearance of ragged red fibers. Electron microscopy might show abnormal mitochondria with paracrystalline inclusions. Muscle histochemistry may show cytochrome c oxidase (COX)–deficient fibers, which indicate mitochondrial dysfunction (Fig. 85e-5). Respiratory chain complex assays may also show reduced enzyme function. Either of these two abnormalities confirms the presence of a mitochondrial disease, to be followed by an in-depth molecular genetic analysis.

1	Recent evidence has provided important insights into the importance of nuclear-mtDNA genomic cross-talk and has provided a descriptive framework for classifying and understanding disorders that emanate from perturbations in this cross-talk. Although not strictly considered as mtDNA genetic disorders, manifestations do overlap those highlighted above (Fig. 85e-7).

1	The relationship among the degree of heteroplasmy, tissue distribution of the mutant mtDNA, and disease phenotype simplifies inference of a clear causative relationship between heteroplasmic mutation and disease. With the exception of certain mutations (e.g., those causing most cases of LHON), drift to homoplasmy of such mutations would be precluded normally by the severity of impaired oxidative phosphorylation and the consequent reduction in reproductive fitness. Therefore, sequence variants that have reached homoplasmy should be neutral in terms of human evolution and, hence, useful only for tracing human evolution, demography, and migration, as described above. One important exception is in the case of one or more of the homoplasmic population-level variants, which designate the mtDNA haplogroup J, and the interaction with the mtDNA mutations causing LHON. Reduced disease predilection suggests that one or more of the ancient sequence variants designating mtDNA haplogroup J appears

1	haplogroup J, and the interaction with the mtDNA mutations causing LHON. Reduced disease predilection suggests that one or more of the ancient sequence variants designating mtDNA haplogroup J appears to attenuate predisposition to degenerative disease, in the face of other risk factors. Whether or not additional epistatic interactions between population-level mtDNA haplotypes and common health conditions will be found remains to be determined. If such influences do exist, then they are more likely to be relevant to health conditions in the postreproductive age groups, wherein evolutionary filters would not have had the opportunity to censor deleterious effects and interactions and wherein the effects of oxidative stress may play a role. Although much has been written about the possible associations of population-level common mtDNA variants and human health and disease phenotypes or adaptation to different environmental influences (e.g., climate), a word of caution is in order.

1	Many studies that purport to show such associations with phenotypes such as longevity, athletic performance, and metabolic and neurodegenerative disease are limited by small sample sizes, possible genotyping inaccuracies, and the possibility of population stratification or ethnic ancestry bias. Because mtDNA haplogroups are so prominently partitioned along phylogeographic lines, it is difficult to rule out the possibility that a haplogroup for which an association has been found is simply a marker for differences in Succinyl-CoA synthase (SUCLA2, SUCLG1)

1	FIGURE 85e-7 Disorders associated with perturbations in nuclearmitochondrial genomic cross-talk. Clinical features and genes associated with multiple mitochondrial DNA (mtDNA) deletions, mtDNA depletion, and mitochondrial neurogastrointestinal encephalomyopathy syndromes. ANT, adenine nucleotide translocators; adPEO, autosomal dominant progressive external ophthalmoplegia; arPEO, autosomal recessive progressive external ophthalmoplegia; IOSCA, infantile-onset spinocerebellar ataxia; SCAE, spinocerebellar ataxia and epilepsy. (Reproduced with permission from A Spinazzola, M Zeviani: Disorders from perturbations of nuclear-mitochondrial intergenomic cross-talk. J Intern Med 265:174, 2009.) populations with a societal or environmental difference or with different allele frequencies at other genomic loci, which are actually causally related to the heritable trait or disease of interest. The difficulty in generating cellular or animal models to test the functional influence of homoplasmic

1	genomic loci, which are actually causally related to the heritable trait or disease of interest. The difficulty in generating cellular or animal models to test the functional influence of homoplasmic sequence variants (as a result of mtDNA polyploidy) further compounds the challenge. The most likely formulation is that the risk conferred by different mtDNA haplogroup–defining homoplasmic mutations for common diseases depends on the concomitant nuclear genomic background, together with environmental influences. Progress in minimizing potentially misleading associations in mtDNA heritable trait and disease studies should include ensuring adequate sample size taken from a large sample recruitment base, using carefully matched controls and population structure determination, and performing analysis that takes into account epistatic interactions with other genomic loci and environmental factors.

1	Studies on aging humans and animals have shown a potentially important correlation of age with the accumulation of heterogeneous mtDNA mutations, especially in those organ systems that undergo the most prominent age-related degenerative tissue phenotype. Sequencing of PCR-amplified single mtDNA molecules has demonstrated an average of two to three point mutations per molecule in elderly subjects when compared with younger ones. Point mutations observed include those responsible for known heritable heteroplasmic mtDNA disorders, such as the m.3344A>G and m.3243A>G mutations responsible for the MERRF and MELAS syndromes, respectively. However, the cumulative burden of these acquired somatic point mutations with age was observed to remain well below the threshold expected for phenotypic expression (<2%). Point mutations at other sites not normally involved in inherited mtDNA disorders have also been shown to accumulate to much higher levels in some tissues of elderly individuals, with

1	(<2%). Point mutations at other sites not normally involved in inherited mtDNA disorders have also been shown to accumulate to much higher levels in some tissues of elderly individuals, with the description of tissue-specific “hot spots” for mtDNA point mutations. Along the same lines, an age-associated and tissue-specific accumulation of mtDNA deletions has been observed, including deletions involved in known heritable mtDNA disorders, as well as others. The accumulation of functional mtDNA deletions in a given tissue is expected to be associated with mitochondrial dysfunction, as reflected in an age-associated patchy and reduced COX activity on histochemical staining, especially in skeletal and cardiac muscle and brain. A particularly well-studied and potentially important example is the accumulation of mtDNA deletions and COX deficiency observed in neurons of the substantia nigra in Parkinson’s disease patients.

1	The progressive accumulation of ROS has been proposed as the key factor connecting mtDNA mutations with aging and age-related disease pathogenesis (Fig. 85e-8). As noted above, ROS are a by-product of oxidative phosphorylation and are removed by detoxifying antioxidants into less harmful moieties; however, exaggerated production of ROS or impaired removal results in their accumulation. One of the main targets for ROS-mediated injury is DNA, and mtDNA is particularly vulnerable because of its lack of protective histones and less efficient injury repair systems compared with nuclear DNA. In turn, accumulation of mtDNA mutations results in inefficient oxidative phosphorylation, with the potential for excessive production of ROS, generating a “vicious cycle” of cumulative mtDNA damage. Indeed, measurement of the oxidative stress biomarker 8-hydroxy-2-deoxyguanosine has been used to measure age-dependent increases in mtDNA oxidative damage at a rate exceeding that of nuclear DNA. It should

1	measurement of the oxidative stress biomarker 8-hydroxy-2-deoxyguanosine has been used to measure age-dependent increases in mtDNA oxidative damage at a rate exceeding that of nuclear DNA. It should be noted that mtDNA mutation can potentially occur in postmitotic cells as well, because mtDNA replication is not synchronized with the cell cycle. Two other proposed links between mtDNA mutation and aging, besides ROS-mediated tissue injury, are the perturbations in efficiency of oxidative phosphorylation with disturbed cellular aerobic function

1	FIGURE 85e-8 Multiple pathways of mitochondrial DNA (mtDNA) damage and aging. Multiple factors may impinge on the integrity of mitochondria that lead to loss of cell function, apoptosis, and aging. The classic pathway is indicated with blue arrows; the generation of reactive oxygen species (ROS; superoxide anion, hydrogen peroxide, and hydroxyl radicals), as a by-product of mitochondrial oxidative phosphorylation, results in damage to mitochondrial macromolecules, including the mtDNA, with the latter leading to deleterious mutations. When these factors damage the mitochondrial energy-generating apparatus beyond a functional threshold, proteins are released from the mitochondria that activate the caspase pathway, leading to apoptosis, cell death, and aging. (Reproduced with permission from L Loeb et al: The mitochondrial theory of aging and its relationship to reactive oxygen species damage and somatic mtDNA mutations. Proc Natl Acad Sci USA 102:18769, 2005.) and perturbations in

1	from L Loeb et al: The mitochondrial theory of aging and its relationship to reactive oxygen species damage and somatic mtDNA mutations. Proc Natl Acad Sci USA 102:18769, 2005.) and perturbations in apoptotic pathways, whose execution steps involve mitochondrial activity.

1	Genetic intervention studies in animal models have sought to clarify the potential causative relationship between acquired somatic mtDNA mutation and the aging phenotype, and the role of ROS in particular. Replication of the mitochondrial genome is mediated by the activity of the nuclear-encoded polymerase gamma gene. A transgenic homozygous mouse knock-in mutation of this gene renders the polymerase enzyme deficient in proofreading and results in a threeto fivefold increase in mtDNA mutation rate. Such mice develop a premature aging phenotype, which includes subcutaneous lipoatrophy, alopecia, kyphonia, and weight loss with premature death. Although the finding of increased mtDNA mutation and mitochondrial dysfunction with age has been solidly established, the causative role and specific contribution of mitochondrial ROS to aging and age-related disease in humans has yet to be proved. Similarly, although many tumors display higher levels of heterogeneous mtDNA mutations, a causal

1	contribution of mitochondrial ROS to aging and age-related disease in humans has yet to be proved. Similarly, although many tumors display higher levels of heterogeneous mtDNA mutations, a causal relationship to tumorigenesis has not been proved.

1	Besides the age-dependent acquired accumulation in somatic cells of heterogeneous point mutations and deletions, a quite different effect of nonheritable and acquired mtDNA mutation has been described affecting tissue stem cells. In particular, disease phenotypes attributed to acquired mtDNA mutation have been observed in sporadic and apparently nonfamilial cases involving a single individual or even tissue, usually skeletal muscle. The presentation consists of decreased exercise tolerance and myalgias, sometimes progressing to rhabdomyolysis. As in the case of the sporadic, heteroplasmic, large-scale deletion, classic syndromes of chronic PEO, Pearson syndrome, and KSS, the absence of a maternal inheritance pattern, together with the finding of limited tissue distribution, suggests a molecular pathogenic mechanism emanating from mutations arising de novo in muscle stem cells after germline differentiation (somatic mutations that are not sporadic and occur in tissue-specific stem

1	molecular pathogenic mechanism emanating from mutations arising de novo in muscle stem cells after germline differentiation (somatic mutations that are not sporadic and occur in tissue-specific stem cells during fetal development or in the postnatal maintenance or postinjury repair stage). Such mutations would be expected to be propagated only within the progeny of that stem cell and affect a particular tissue within a given individual, without evidence of heritability.

1	No specific curative treatment for mtDNA disorders is currently available; therefore, the management of mitochondrial disease is largely supportive. Management issues may include early diagnosis and treatment of diabetes mellitus, cardiac pacing, ptosis correction, and intraocular lens replacement for cataracts. Less specific interventions in the case of other disorders involve combined treatment strategies including dietary intervention and removal of toxic metabolites. Cofactors and vitamin supplements are widely used in the treatment of diseases of mitochondrial oxidative phosphorylation, although there is little evidence, apart from anecdotal reports, to support their use. This includes administration of artificial electron acceptors, including vitamin K3, vitamin C, and ubiquinone (coenzyme Q10); administration of cofactors (coenzymes) including riboflavin, carnitine, and creatine; and use of oxygen radical scavengers, such as vitamin E, copper, selenium, ubiquinone, and

1	(coenzyme Q10); administration of cofactors (coenzymes) including riboflavin, carnitine, and creatine; and use of oxygen radical scavengers, such as vitamin E, copper, selenium, ubiquinone, and idebenone. Drugs that could interfere with mitochondrial function, such as the anesthetic agent propofol, barbiturates, and high doses of valproate, should be avoided. Supplementation with the nitric oxide synthase substrate, L-arginine, has been advocated as a vasodilator treatment during stroke-like episodes. The physician should also be familiar with environmental interactions, such as the strong and consistent association between visual loss in LHON and smoking. A clinical penetrance of 93% was found in men who smoked. Asymptomatic carriers of an LHON mtDNA mutation should, therefore, be strongly advised not to smoke and to moderate their alcohol intake. Although not a cure, these interventions might stave off the devastating clinical manifestations of the LHON mutation. Another example is

1	advised not to smoke and to moderate their alcohol intake. Although not a cure, these interventions might stave off the devastating clinical manifestations of the LHON mutation. Another example is strict avodiance of aminoglycosides in the familial syndrome of ototoxic susceptibility to aminoglycosides in the presence of the mtDNA m.1555A>G mutation of the 12SrRNA encoding gene.

1	GENETIC COUNSELING, PRENATAL DIAGNOSIS, AND PREIMPLANTATION GENETIC DIAGNOSIS IN MTDNA DISORDERS

1	The provision of accurate genetic counseling and reproductive options to families with mtDNA mutations is challenging due to the unique genetic features of mtDNA inheritance that distinguish it from Mendelian genetics. mtDNA defects are transmitted by maternal inheritance. mtDNA de novo mutations are often large deletions, affect one family member, and usually represent no significant risk to other members of the family. In contrast, mtDNA point mutations or duplications can be transmitted down the maternal line. Accordingly, the father of an affected individual has no risk of harboring the disease-causing mutation, and a male cannot transmit the mtDNA mutation to his offspring. In contrast, the mother of an affected individual usually harbors the same mutation but might be completely asymptomatic. This wide phenotypic variability is primarily related to the phenomena of heteroplasmy and the mutation load carried by different members of the same family. Consequently, a symptomatic or

1	This wide phenotypic variability is primarily related to the phenomena of heteroplasmy and the mutation load carried by different members of the same family. Consequently, a symptomatic or asymptomatic female harboring a disease-causing mutation in a heteroplasmic state will transmit to her offspring variable amounts of the mutant mtDNA molecules. The offspring will be symptomatic or asymptomatic primarily according to the mutant load transmitted via the oocyte and, to some extent, subsequent mitotic segregation during development. Interactions with the mtDNA haplotype background or nuclear human genome (as in the case of LHON) serve as an additional important determinant of disease penetrance. Because the severity of the disease phenotype associated with the heteroplasmic mutation load is a function of the stochastic differential segregation and copy number 85e-9 of mutant mtDNA during the oogenesis bottleneck and, subsequently, following tissue and organ development in the

1	load is a function of the stochastic differential segregation and copy number 85e-9 of mutant mtDNA during the oogenesis bottleneck and, subsequently, following tissue and organ development in the offspring, it is rarely predictable with any degree of accuracy. For this reason, prenatal diagnosis (PND) and PGD techniques that have evolved into integral and well-accepted standards of practice are severely hampered in the case of mtDNA-related diseases.

1	The value of PND and PGD is limited, partly due to the absence of data on the rules that govern the segregation of wild-type and mutant mtDNA species (heteroplasmy) among tissue in the developing embryo. Three factors are required to ensure the reliability of PND and PGD: (1) a close correlation between the mutant load and the disease severity, (2) a uniform distribution of mutant load among tissues, and (3) no major change in mutant load with time. These criteria are suggested to be fulfilled for the NARP m.8993T>G mutation but do not seem to apply to other mtDNA disorders. In fact, the level of mutant mtDNA in a chorionic villous or amniotic fluid sample may be very different from the level in the fetus, and it would be difficult to deduce whether the mutational load in the prenatal samples provides clinically useful information regarding the postnatal and adult state.

1	Because the treatment options for patients with mitochondrial disease are rather limited, preventive interventions that eliminate the likelihood of transmission of affected mtDNA into offspring are desirable. The lack of utility of PND and PGD techniques to reliably diagnose and predict mitochondrial disorders at preimplantation-stage products of conception has resulted in the search for alternative preventive approaches for the same problem. One possible approach to “diluting” or even entirely eliminating the mutant mtDNA is applicable only in the earliest embryonic state and in effect represents a form of germline preventive therapy (Fig. 85e-9). This possibility has been explored by using alternative assisted reproduction techniques such as ooplasmic transfer (OT), metaphase chromosome transfer (CT), pronuclear transfer (PNT), and germinal vesicle transfer (GVT) in animal models and, to an extent, in humans. OT is a technique wherein a certain volume (5–15%) of healthy donor oocyte

1	transfer (CT), pronuclear transfer (PNT), and germinal vesicle transfer (GVT) in animal models and, to an extent, in humans. OT is a technique wherein a certain volume (5–15%) of healthy donor oocyte cytoplasm with normal mitochondria is injected into the patient oocyte containing mutated mitochondria. The reasoning behind OT is to supplement the patient’s oocyte with uncompromised cytoplasmic factors such as mtDNA, mRNA, proteins, and other molecules by injecting cytoplasm from healthy oocytes. In PNT, following fertilization, pronuclei of a patient’s zygote are removed with a cytoplasm (“karyoplast”). The karyoplast is transferred to the perivitelline space of a donated zygote, which has been already enucleated. The karyoplast is then fused with enucleated zygote by electric pulses or inactivated Sendai viruses (HVJ). The reconstructed zygote contains a nucleus from the patient (patient nuclear DNA) and cytoplasm from the donor. Thus, the majority of the patient mtDNA is replaced

1	inactivated Sendai viruses (HVJ). The reconstructed zygote contains a nucleus from the patient (patient nuclear DNA) and cytoplasm from the donor. Thus, the majority of the patient mtDNA is replaced with mtDNA from the donor oocyte. In CT, meiosis II stage of oocyte maturation provides an opportunity for the reconstruction of oocytes with different nuclear and cytoplasmic components before fertilization takes place. Reconstructed oocytes by metaphase chromosome transfer are then fertilized to produce embryos with desired mtDNA haplotypes. In GVT, replacement of compromised cytoplasm with healthy cytoplasm through germinal vesicle transfer before the start of chromosome segregation is carried out.

1	These approaches have not yet met with widely reported clinical success, yet there is room for optimism. As noted above, analysis of heteroplasmy and inheritance patterns indicates that even a small increase in copies of nonmutant mtDNA can exceed the threshold required to ameliorate serious clinical disease. All of the approaches described above show promise in achieving this goal and thus reducing the burden of clinical mtDNA disease in the future. Nuclear transfer into Preimplantation donated oocytes: genetic diagnosis a future possibility? Mother’s oocytes fertilized with FIGURE 85e-9 Possible approaches for prevention of mitochondrial DNA (mtDNA) disease. A. No intervention: offspring’s mutant mtDNA load will vary greatly. B. Oocyte donation: currently permitted in some constituencies but limited by the availability of oocyte donors.

1	C. Preimplantation genetic diagnosis: available for some mtDNA diseases (reliable in determining background nuclear genomic haplotype risk). D. Nuclear transfer: research stage, including initial studies in nonhuman primates. Red represents mutant mtDNA, pink and white represent successively higher proportions of normal mtDNA. Blue represents genetic material from an unrelated donor. (Adapted with permission from J Poulton et al: Preventing transmission of maternally inherited mitochondrial DNA diseases. Br Med J 338:b94, 2009.) and prevention. Key terms used in the discussion of the human micro-86e-1 biome are defined in Table 86e-1. the human Microbiome We are holobionts—collections of human and microbial cells that 86e Jeffrey I. Gordon, Rob Knight function together in an elaborate symbiosis. The aggregate number

1	The technologies that allowed us to decipher the human genome have revolutionized our ability to delineate the composition and functions of the microbial communities that colonize our bodies and make up our microbiota. Each body habitat, including the skin, nose, mouth, airways, gastrointestinal tract, and vagina, harbors a distinctive community of microbes. Efforts to understand our microbiota and its collection of microbial genes (our microbiome) are changing our views of “self” and deepening our understanding of many normal physiologic, metabolic, and immunologic features and their interpersonal and intrapersonal variations. In addition, this area of research is beginning to provide new insights into diseases not previously known to have microbial “contributors” and is suggesting new strategies for treatment of microbial cells in our microbiota exceeds the number of human cells in our adult bodies by up to 10-fold, and each healthy adult is estimated to harbor 105–106 microbial

1	strategies for treatment of microbial cells in our microbiota exceeds the number of human cells in our adult bodies by up to 10-fold, and each healthy adult is estimated to harbor 105–106 microbial genes, in contrast to ~20,000 Homo sapiens genes. Members of our microbiota can function as mutualists (i.e., both host and microbe benefit from each other’s presence), as commensals (one partner benefits; the other is seemingly unaffected), and as potential or overt pathogens (one partner benefits; the other is harmed). Many clinicians view pathogens as individual microbial species or strains that can elicit disease in susceptible hosts. An emerging, more ecologic view is that pathogens do not function in isolation; rather, their invasion, emergence, and effects on the host reflect interactions with other members of a microbiota. An even more expansive view is that multiple organisms in a community conspire to

1	Culture-independent A type of analysis in which the culture of microbes is not required but rather information is extracted directly from environmental analysis samples Diversity (alpha and Alpha diversity measures the effective number of species (kinds of organisms) at the level of individual habitats, sites, or samples. Beta beta) diversity measures differences in the number of kinds of organisms across habitats, sites, or samples. Domains of life The three major branches of life on Earth: the Eukarya (including humans), the Bacteria, and the Archaea Dysbiosis Any deleterious condition arising from a structural and/or functional aberration in one or more of the host organism’s microbial communities Gnotobiotics The rearing of animals under sterile (germ-free) conditions. These animals can subsequently be colonized at various stages of the life cycle with defined collections of microbes.

1	Gnotobiotics The rearing of animals under sterile (germ-free) conditions. These animals can subsequently be colonized at various stages of the life cycle with defined collections of microbes. Holobiont The biologic entity consisting of a host and all its internal and external symbionts, their gene repertoires, and their functions

1	Holobiont The biologic entity consisting of a host and all its internal and external symbionts, their gene repertoires, and their functions Human microbiome In ecology, biome refers to a habitat and the organisms in it. In this sense, the human microbiome would be defined as the collection of microorganisms associated with the human body. However, the term microbiome is also used to refer to the collective genomes and genes present in members of a given microbiota (see “Microbiota,” below), and the human metagenome is the sum of the human genome and microbial genes (microbiome). A core human microbiome is defined as everything shared in a given body habitat among all or the vast majority of human microbiomes. A core microbiome may include a common set of genomes and genes encoding various protein families and/or metabolic capabilities. Microbial genes that are variably represented in different humans may contribute to distinctive physiologic/metabolic phenotypes.

1	Metagenomics An emerging field encompassing culture-independent studies of the structures and functions of microbial communities as well as the interactions of these communities with the habitats they occupy. Metagenomics includes (1) shotgun sequencing of microbial DNA isolated directly from a given environment and (2) high-throughput screening of expression libraries constructed from cloned community DNA to identify specific functions such as antibiotic resistance (functional metagenomics). DNA-level analyses provide the foundation for profiling of mRNAs and proteins produced by a microbiome (metatranscriptomics and metaproteomics) and for identification of a community’s metabolic network (metametabolomics).

1	Microbial source tracking A collection of methods for assessing the environments of origin for microbes. One method, SourceTracker, uses a Bayesian approach to identify each bacterial taxon’s origins and estimates the proportions of each community made up by bacteria originating from different environments. Microbiota A microbial community—including Bacteria, Archaea, Eukarya, and viruses—that occupies a given habitat Pan-genome The group of genes found in genomes that make up a given microbial phylotype, including both core genes found in all genomes and variably represented genes found in a subset of genomes within the phylotype Phylogenetic analysis Characterization of the evolutionary relationships between organisms and their gene products

1	Phylogenetic analysis Characterization of the evolutionary relationships between organisms and their gene products Phylogenetic tree A “tree” in which organisms are shown according to their relationships to hypothetical common ancestors. When built from molecular sequences, the branch lengths are proportional to the amount of evolutionary change separating each ancestor–descendant pair. Phylotype A phylogenetic group of microbes, currently defined by a threshold percentage identity shared among their small-subunit rRNA genes (e.g., ≥97% for a species-level phylotype) Principal coordinates An ordination method for visualizing multivariate data based on the similarity/dissimilarity of the measured entities (e.g., visualization analysis of bacterial communities based on their UniFrac distances; see “UniFrac,” below)

1	Random Forests analysis/ Machine learning is a collection of approaches that allow a computer to learn without being explicitly programmed. Random Forests machine learning is a machine-learning method for classification and regression that uses multiple decision trees during a training step. Rarefaction A procedure in which subsampling is used to assess whether all the diversity present in a given sample or set of samples has been observed at a given sampling depth and to extrapolate how much additional sampling would be needed to observe all the diversity Resilience A community’s ability to return to its initial state after a perturbation Shotgun sequencing A method for sequencing large DNA regions or collections of regions by fragmenting DNA and sequencing the resulting smaller sections

1	Shotgun sequencing A method for sequencing large DNA regions or collections of regions by fragmenting DNA and sequencing the resulting smaller sections Succession (primary and Succession (in an ecologic context) refers to changes in the structure of a community through time. Primary succession describes the secondary) sequence of colonizations and extinctions that occur in a new habitat. Secondary succession refers to changes in community structure after a disturbance. UniFrac A measure of the phylogenetic dissimilarity between two communities, calculated as the unshared proportion of the phylogenetic tree containing all the organisms present in either community Chapter 86e The Human Microbiome produce pathogenic effects in certain host and environmental contexts (a pathologic community).

1	Chapter 86e The Human Microbiome produce pathogenic effects in certain host and environmental contexts (a pathologic community). The ability to characterize microbial communities without culturing their component members has spawned the field of metagenomics (Table 86e-1). Metagenomics reflects a confluence of experimental and computational advances in the genome sciences as well as a more ecologic understanding of medical microbiology, according to which the functions of a given microbe and its impact on human biology depend on the context of other microbes in the same community. Traditional microbiology relies on culturing individual microbes, but metagenomics skips this step, instead sequencing DNA isolated directly from a given microbial community. The resulting datasets facilitate follow-up functional studies, such as the profiling of RNA and protein products expressed from the microbiome or the characterization of a microbial community’s metabolic activities.

1	Metagenomics provides insight into how microbial communities vary in several situations critical to human health. One such situation is how microbial communities are assembled following birth and how they operate over time, including responses of established communities to various perturbations. Another is how microbial communities normally vary between different anatomic sites within an individual and between different groups of people representing different ages, physiologic states, lifestyles, geographies, and gender. Yet another is how microbial communities vary in disease; whether such variations are consistent among individuals grouped according to current criteria for a disease or its subtypes; whether the microbiota or microbiome provides new ways of classifying disease states; and, importantly, whether the structural and functional configurations of microbial communities are a cause or a consequence of disease.

1	Analysis of our microbiomes also addresses one of the most fundamental questions in genetics: How does environment select our genes and directly influence their function? Each human encounters a unique environment during the course of his or her lifetime. Part of this personally experienced environment is incorporated into the genes and capabilities of our microbial communities. The microbiome therefore expands our conceptualization of “human” genetic potential from a single set of genes “fixed” at birth to a microbiome with additional genes and capabilities acquired via a process influenced by our family and life experiences, including modifiable lifestyle choices such as diet. This view recognizes a previously underappreciated dimension of human evolution that occurs at the level of our microbiomes and inspires us to determine how—and how fast—this microbial evolution effects changes in our human biology. For example, Westernization is associated with loss of bacterial species

1	our microbiomes and inspires us to determine how—and how fast—this microbial evolution effects changes in our human biology. For example, Westernization is associated with loss of bacterial species diversity (richness) in the microbiota, and this loss may be associated with the suite of Western diseases. The study of our microbiomes also raises important questions about personal identity, how we define the origins of health disparities, and privacy. Further, it offers the possibility of entirely new approaches to disease prevention and treatment, including regenerative medicine, which involves administration of microbial species (probiotics) to individuals harboring communities that have not developed into a mature, fully functional state or that have been perturbed in ways that can be restored by the addition of species that fill unoccupied “jobs” (niches).

1	This chapter provides a general overview of how human microbial communities are analyzed; reviews ecologic principles that guide our understanding of microbial communities in health and disease; summarizes recent studies that establish correlations and, in some cases, causal relationships between our microbiota/microbiomes and various diseases; and discusses challenges faced in the translation of these findings to new therapeutic interventions.

1	Life on Earth has been classified into three domains: Bacteria, Archaea, and Eukarya. The habitats of the surface-exposed human body harbor members of each domain plus their viruses. In large part, microbial diversity has not been characterized by culture-based approaches, partly because we do not know how to re-create the metabolic milieu fashioned by these communities in their native habitats and partly because a few organisms tend to outgrow the others. Culture-independent methods readily identify which organisms are present in a microbiota and their relative abundance. The gene widely used to identify microbes and their evolutionary relationships encodes the major RNA component of the small subunit (SSU) of ribosomes. Within each domain of life, the SSU gene is highly conserved, allowing the SSU gene sequences present in different organisms in that domain to be accurately aligned and regions of nucleotide sequence variation to be identified. Pairwise comparisons of SSU ribosomal

1	the SSU gene sequences present in different organisms in that domain to be accurately aligned and regions of nucleotide sequence variation to be identified. Pairwise comparisons of SSU ribosomal RNA (rRNA) genes from different microbes allow construction of a phylogenetic tree that represents an evolutionary map on which previously unknown organisms can be assigned a position. This approach, known as molecular phylogenetics, permits characterization of each organism on the basis of its evolutionary distance from other organisms. Different phylogenetic types (phylotypes) can be viewed as comprising branches on an evolutionary tree.

1	Characterization of Bacteria Because members of the Bacteria dominate our microbiota, most studies defining our various body habitat– associated microbial communities have sequenced the bacterial SSU gene that encodes 16S rRNA. This gene has a mosaic structure, with highly conserved domains flanking more variable regions. The most straightforward way to identify bacterial taxonomic groups (taxa) in a given community is to sequence polymerase chain reaction (PCR) products (amplicons) generated from the 16S rRNA genes present in that community. PCR primers directed at the conserved regions of the gene yield PCR amplicons encompassing one or more of that gene’s nine variable regions. PCR primer design is critical: differential annealing with primer pairs designed to amplify different variable regions can lead to overor underrepresentation of specific taxa, and different regions within the 16S rRNA gene can have different patterns of evolution. Therefore, caution must be exercised in

1	regions can lead to overor underrepresentation of specific taxa, and different regions within the 16S rRNA gene can have different patterns of evolution. Therefore, caution must be exercised in comparisons of the relative abundance of taxa in samples characterized in different studies, as methodologic differences can lead to larger perceived differences in the inferred taxonomy than actually exist.

1	A key innovation is multiplex sequencing. Amplicons from each microbial-community DNA sample are tagged by incorporation of a unique oligonucleotide barcode into the PCR primer. Amplicons harboring these sample-specific barcodes can then be pooled together so that multiple samples representing multiple communities can be sequenced simultaneously (Fig. 86e-1). One important choice is the tradeoff between the number of samples that can be processed simultaneously and the number of sequences generated per sample. Interpersonal differences in the bacterial components of the microbiota are typically large, as are differences between communities occupying different body habitats in the same individual (see below); thus fewer than 1000 16S rRNA reads are characteristically required to discriminate community type. However, the identification of systematic differences in microbiota composition that correlate with physiologic status or disease state is confounded by the substantial

1	discriminate community type. However, the identification of systematic differences in microbiota composition that correlate with physiologic status or disease state is confounded by the substantial interpersonal variation that occurs normally.

1	Sequencing of bacterial 16S rRNA genes creates a challenge for medical microbiology: how to define the taxonomic groups present in a community in a systematic and informative manner, so that one community can be compared with and contrasted to another. Within each domain of life, microbes are classified in a hierarchy beginning with phylum (the broadest group) followed by class, order, family, genus, and species. To determine taxonomy, 16S rRNA sequences are aligned on the basis of their sequence similarity—a process known as picking operational taxonomic units (OTUs). Grouping of 16S rRNA sequences from a given variable region into “bins” that share ≥97% nucleotide sequence identity (97%ID OTUs) is a commonly accepted, albeit arbitrary, way to define a species.

1	Looking beyond the 16S rRNA gene, we find that different isolates (strains) of a given bacterial species have overlapping but not identical sets of genes in their genomes. The aggregate set of genes identified in all isolates (strains) of a given species-level phylotype represents its pan-genome. Most species are represented by multiple strains, sometimes with markedly different functions (for example, Variable region Community 1 2 3 Align sequences, bin into OTUs, and infer phylogeny by placing OTUs on a master reference phylogenetic tree UniFrac analysis Cluster samples based on UniFrac distances calculated from (iii),

1	Chapter 86e The Human Microbiome (i) (ii) (iii) DE3 machine learning, feature identification, etc. PC2 PC1 1 2 FIGURE 86e-1 Pipeline for culture-independent studies of a microbiota. (A) DNA is extracted directly from a sampled human body habitat– associated microbial community. The precise location of the community and relevant patient clinical data are collected. Polymerase chain reac-tion (PCR) is used to amplify portions of small-subunit (SSU) rRNA genes (e.g., the genes encoding bacterial 16S rRNA) containing one or more variable regions. Primers with sample-specific, error-correcting barcodes are designed to recognize the more conserved regions of the 16S rRNA gene that flank the targeted variable region(s). (B) Barcoded amplicons from multiple samples (communities 1–3) are pooled and sequenced in batch in a highly parallel next-generation DNA sequencer. (C) The resulting reads are then processed, with barcodes denoting which sample the sequence came from. After barcode sequences

1	sequenced in batch in a highly parallel next-generation DNA sequencer. (C) The resulting reads are then processed, with barcodes denoting which sample the sequence came from. After barcode sequences are removed in silico, reads are aligned and grouped according to a specified level of shared identity; e.g., sequences that share ≥97% nucleotide sequence identity are regarded as representing a species. Once reads are binned into operational taxonomic units (OTUs) in this fashion, they are placed on a phylogenetic tree of all known bacteria and their phylogeny is inferred.

1	(D) Communities can be compared to one another by either taxon-based methods, in which phylogeny is not considered and the number of shared taxa are simply scored, or phylogenetic methods, in which community similarity is considered in light of the evolutionary relationships of community members. The UniFrac metric is commonly used for phylogeny-based comparisons. In stylized examples (i), (ii), and (iii), communities with varying degrees of similarity are shown. Each circle represents an OTU colored on the basis of its community of origin and placed on a master phylogenetic tree that includes all lineages from all communities. Branches (horizontal lines) are colored with each community that contains members from that branch. The three examples vary in the amount of branch length shared between the OTUs from each community. In (i), there is no shared branch length, and thus the three communities have a similarity score of 0. In (ii), the communities are identical, and a similarity

1	between the OTUs from each community. In (i), there is no shared branch length, and thus the three communities have a similarity score of 0. In (ii), the communities are identical, and a similarity score of 1 is assigned. In (iii), there is an intermediate level of similarity: communities represented in red and green share more branch length and thus have a higher similarity score than red vs. blue or green vs. blue. The amount of shared branch length in each pairwise community comparison provides a distance matrix. (E) The results of taxonor phylogeny-based distance matrices can be displayed by principal coordinates analysis (PCoA), which plots each community spatially such that the largest component of variance is captured on the x-axis (PC1) and the second largest component of variance is displayed on the y-axis (PC2). In the example shown, the three communities in example (iii) from panel D are compared. Note that for shotgun sequencing of whole-community DNA (microbiome

1	variance is displayed on the y-axis (PC2). In the example shown, the three communities in example (iii) from panel D are compared. Note that for shotgun sequencing of whole-community DNA (microbiome analysis), reads are compared with genes that are present in the genomes of sequenced cultured microbes and/or with genes that have been annotated by hierarchical functional classification schemes in various databases, such as the Kyoto Encyclopedia of Genes and Genomes (KEGG). Communities can then be compared on the basis of the distribution of functional groups in their microbiomes—an approach analogous to taxon-based methods for 16S rRNA–based comparisons—and the results plotted with PCoA.

1	enteropathogenic versus commensal Escherichia coli). Differences in perfused ecosystem exposed to the complex and varying set of sub-genome content among strains of a given species reflect differences stances we ingest, to adapt to changing circumstances rather than in community membership as well as differences in the selective pres-depending on one strain to occupy a given niche important for proper sures these strains experience within and between habitats. Horizontal community functioning. In ecologic studies of different environments, gene transfer among members of a microbiota—mediated by phage, such as grasslands, forests, and reefs, increased diversity within a com-plasmids, and other mechanisms—is a major contributor to this munity increases its capacity to respond to disturbances and to restore strain-level variation. itself (i.e., its resilience); the same is likely true of microbial ecosystems.

1	Strain-level diversity can be important in any consideration of how When characterizing the mechanisms by which a given species pro-microbial communities differ between individuals and how these com-duces an effect or effects on humans, it is important to consider the munities accommodate perturbations. For example, the great bacte-strain being tested; strain-level diversity has an impact on discovery rial strain-level diversity that exists in the gut is thought to be one of and development efforts aimed at identifying next-generation probiotthe features that allows this microbiota, which occupies a constantly ics that can be used therapeutically to promote health or treat disease.

1	Identification of Archaeal and Eukaryotic Members Surveys based on SSU rRNA gene sequencing have largely focused on Bacteria, yet the census of “who’s there” in human body habitat–associated communities must also include the other two domains of life: Archaea and Eukarya. Differences in the sequences of archaeal and bacterial 16S rRNA genes, first recognized by Carl Woese in 1977, allowed these two domains of life to be distinguished. The representation of Archaea in human microbial communities is less well defined than that of Bacteria, in part due to the difficulty in optimizing the design of PCR primers that specifically target conserved regions of archaeal (versus bacterial) 16S rRNA genes. Identifying archaeal members is important to our understanding of the functional properties of the microbiota. For example, a major challenge faced by microbial communities when breaking down polysaccharides (the most abundant biologic polymers on Earth) is the maintenance of redox balance in

1	the microbiota. For example, a major challenge faced by microbial communities when breaking down polysaccharides (the most abundant biologic polymers on Earth) is the maintenance of redox balance in the setting of maximal energy production. Many microbial species have branched fermentation pathways that allow them to dispose of reducing equivalents (e.g., by the production of H2, which is energetically efficient). However, there is a caveat: the hydrogen must be removed or it will inhibit reoxidation of pyridine nucleotides. Therefore, hydrogen-consuming (hydrogenotrophic) species are key to maximizing the energy-extracting capacity of primary fermenters.

1	In the human gut, hydrogenotrophs include a phylogenetically diverse group of bacterial acetogens, a more limited group of sulfate-reducing bacteria that generate hydrogen sulfide, and methane-producing archaeal organisms (methanogens) that can represent up to 10% of the anaerobes present in the feces of some humans. However, the degree of archaeal diversity in the gut microbiota of healthy individuals appears to be low.

1	Culture-independent surveys of eukaryotic diversity are also confounded by challenges related to the design of PCR primers that target the eukaryotic SSU gene (18S rRNA) as well as the internal transcribed spacer regions of rRNA operons. Metagenomic studies of healthy human adults living in countries with distinct cultural traditions and disparate geographic features and locations have revealed that the degree of eukaryotic diversity is lower than that of bacterial diversity. In the gut, which contains far more microbes than any other body habitat, the representation of fungi is significantly lower in individuals living in Westernized societies than in those living in non-Western societies. The most abundant fungal sequences belong to the phylum-level taxa Ascomycota and Microsporidia. The phyla Ascomycota and Basidiomycota appear to be mutually exclusive, and the presence of Candida in particular correlates with recent consumption of carbohydrates.

1	Elucidation of Viral Dynamics Viruses are the most abundant biologic entity on Earth. Viral particles outnumber microbial cells by 10:1 in most environments. Humans are no exception in terms of viral colonization; our feces alone contain 108–109 viral particles per gram. Despite this abundance, many eukaryotic viral communities remain incompletely characterized, in part because the identification of viruses within metagenomic sequencing datasets is itself very challenging. Characterizing viral diversity requires different approaches: because no single gene is found in all viruses, no universal phylogenetic “barcode of life” equivalent to the SSU rRNA gene exists. One approach has been to selectively purify virus-like particles from community biospecimens, amplify the small amounts of DNA that are recovered, and randomly fragment the DNA and sequence the fragments (shotgun sequencing). The resulting sequences can be assembled into larger contigs whose function can be computationally

1	that are recovered, and randomly fragment the DNA and sequence the fragments (shotgun sequencing). The resulting sequences can be assembled into larger contigs whose function can be computationally predicted from homology to known genes, and the information obtained can be used to populate/expand nonredundant viral databases. These annotated nonredundant databases can then be used for more targeted mining of the rapidly expanding number of shotgun sequencing datasets generated from total-community DNA for known or putative DNA viruses.

1	Given the dominance of bacteria in the gut microbiota, it is not surprising that phages (viruses that infect bacteria) dominate the identifiable components of the gut’s DNA virome. Prophages are a manifestation of a so-called temperate viral–bacterial host dynamic, in which a phage is integrated into its host bacterium’s genome. This temperate dynamic provides a way to constantly refashion the genomes of bacterial species through horizontal gene transfer. Genes encoded by a pro-phage genome may expand the niche and fitness of their bacterial host, for example, by enabling the metabolism of previously inaccessible nutrient sources. Prophage integration can also protect the host strain from superinfection, “immunizing” the strain against infection by closely related phages. A temperate prophage life cycle allows the virus to expand in a 1:1 ratio with its bacterial host. If the integrated virus conveys increased fitness, the prevalence of the bacterial host and its phage will increase

1	life cycle allows the virus to expand in a 1:1 ratio with its bacterial host. If the integrated virus conveys increased fitness, the prevalence of the bacterial host and its phage will increase in the microbiota. Induction of a lytic cycle, where the prophage replicates and kills the host, may follow. Lytic cycles can cause high bacterial turnover. Lysis debris (e.g., components of capsules) can be used as nutrient sources by surviving bacteria; this change in the energy dynamic in a community is referred to as a phage shunt. A subpopulation of bacteria that undergoes lytic induction may sweep away other sensitive species present in the community, thus increasing the niche space available for survivors (i.e., those bacteria that already have an integrated prophage). Periodic induction of prophages leads to a “constant diversity dynamic” that helps maintain community structure and function.

1	Interest in viral communities has expanded in recent years, especially given a potentially therapeutic role for phages as an alternative or adjunct to antibiotics. Virome members have evolved elegant survival mechanisms that allow them to evade host defenses, diversify, and establish elaborate and mutually beneficial symbioses with their hosts. A number of recent studies have tried to adapt these mechanisms for therapeutic purposes (e.g., the use of synthetic phages to treat Pseudomonas aeruginosa infections in burn patients or in other settings). Phage therapy is not a new idea: Félix d’Herelle, co-discoverer of phages, recognized their potential medical applications nearly a century ago. However, only recently have our technologic capabilities and our knowledge of the human microbiota made phage therapy realistically attainable within our lifetimes.

1	At many levels, different people are very much alike: our genomes are >99% identical, and we have similar collections of human cells. However, our microbial communities differ drastically, both between people and between habitats within a single human body. The greatest variation (beta diversity, described below) is between body sites. For example, the difference between the microbial communities residing in a person’s mouth versus the same person’s gut is comparable to the difference in communities residing in soil versus seawater. Even within a body site, the differences among people are not subtle: gut, skin, and oral communities can all differ by 80–90%, even from the broad, bacterial species–level view. The English poet John Donne said that “no man is an island”; however, from a microbial perspective, each of us consists of not just one isolated island but rather a whole archipelago of distinct habitats that exchange microbes with one another and with the outside environment at

1	perspective, each of us consists of not just one isolated island but rather a whole archipelago of distinct habitats that exchange microbes with one another and with the outside environment at some as yet undetermined level. Before we can discuss these differences and understand their relevance to human disease, it is important to understand some basic terms and ecologic principles.

1	Alpha Diversity Alpha diversity is defined as the effective number of species present in a given sample. Communities that are compositionally more diverse (i.e., have more OTUs) or that are phylogenetically more diverse are defined as having greater alpha diversity. Alpha diversity can be measured by plotting the number of different types of SSU rRNA sequences identified at a given phylogenetic level (species, genera, etc.) in a sample as a function of the number of SSU rRNA gene reads collected. The most commonly used metrics of alpha diversity are Sobs (the number of species observed in a given number of sequences), Chao1 (a measure based on the number of species observed only once), the Shannon index (a measure of the number of bits of information gained by revealing the identity of a randomly chosen member of the community), and phylogenetic diversity (a measure of the total branch length of a phylogenetic tree encompassing a sample). Diversity estimators are particularly

1	of a randomly chosen member of the community), and phylogenetic diversity (a measure of the total branch length of a phylogenetic tree encompassing a sample). Diversity estimators are particularly sensitive to errors introduced during PCR and sequencing.

1	Beta Diversity Beta diversity refers to the differences between communities and can be defined with phylogenetic or nonphylogenetic distance measurements. UniFrac is a commonly used phylogenetic metric that compares the evolutionary history of different microbial communities, noting the degree to which any two communities share branch length on a tree of microbial life: the more similar communities are to each other, the more branch length they share (Fig. 86e-1). UniFrac-based measurements of distances between communities can be visually represented with principal coordinates analysis or other geometric techniques that project a high-dimensional dataset down onto a small number of dimensions for a more approachable analysis (Fig. 86e-1). Principal coordinates analysis can also be applied to nonphylogenetic methods for comparing communities, such as Euclidean distance, Jensen-Shannon divergence, or Bray-Curtis dissimilarity, which operate independent of evolutionary tree data but can

1	to nonphylogenetic methods for comparing communities, such as Euclidean distance, Jensen-Shannon divergence, or Bray-Curtis dissimilarity, which operate independent of evolutionary tree data but can make biologic patterns more difficult to identify. The taxonomic data or distance matrices can also be used as input into a range of machine-learning algorithms (such as Random Forests) that employ supervised classification to identify differences between labeled groups of samples. Supervised classification is useful for identifying differences between cases and controls but can obscure important patterns intrinsic to the data, including confounding variables such as different sequencing runs or patient populations.

1	As noted above, the greatest beta diversity is that among body sites. This fact underscores the need to specify body habitat in microbiota analyses of any type, including microbial surveillance studies examining the flow of normal and pathogenic organisms into and out of different body sites in patients and their health care providers. Several other key points have emerged from beta diversity studies of human-associated microbial communities—notably, that (1) there is a high level of interpersonal variability in every body habitat studied to date, (2) intrapersonal variation in a given body habitat is less pronounced, and (3) family members have more similar communities than unrelated individuals living in separate households. Thus, a person is his/ her own best control, and examination of an individual over time as a function of disease state or treatment intervention is desirable. Similarly, family members serve as logical reference controls, although age is a major covariate that

1	of an individual over time as a function of disease state or treatment intervention is desirable. Similarly, family members serve as logical reference controls, although age is a major covariate that affects microbiota structure.

1	Studies of fecal samples obtained from twins over time have shown that the overall degree of phylogenetic similarity of bacterial communities does not differ significantly between monozygotic and dizygotic twin pairs, although monozygotic twin pairs may be more similar in some populations at earlier ages. These results, together with intervention studies in mice and epidemiologic observations in humans, emphasize that early environmental exposures are a very important determinant of adult-gut microbial ecology. In humans, the initial exposures depend on delivery mode: babies sampled within 20 min of birth have relatively undifferentiated microbial communities in the mouth, the skin, and the gut. For vaginally delivered babies, these communities resemble the specific microbial communities found in the mother’s vagina. For babies delivered by cesarean section, the communities resemble skin communities. Although studies of older children and of adults stratified by delivery mode are

1	found in the mother’s vagina. For babies delivered by cesarean section, the communities resemble skin communities. Although studies of older children and of adults stratified by delivery mode are still rare in the literature, these differences have been shown to persist until at least 4 months of age and perhaps until age 7 years. The infant gut microbiota changes to resemble the adult gut community over the first 3 years of life; comparable studies have not been done in other body habitats to date.

1	Exposures to environmental microbial reservoirs can continue to influence community structure. For example, unrelated cohabiting adults have more similar microbiotas in all of their body habitats than do non-cohabiting adults, and humans resemble the dogs they live with, at least in terms of skin microbiota. Gender and sexual maturation may also affect the microbiota structure, although efforts to isolate these variables are complicated by many confounding factors; any gender effect must be small compared with the effects of other variables such as diet (except in the case of the female urinary tract, which is influenced 86e-5 by the vaginal microbiota).

1	The vaginal microbiota illustrates another intriguing aspect of the contributions made by various factors to interpersonal differences in microbial community structure within a given body habitat. Bacterial 16S rRNA–based studies of the midvaginal microbiota in sexually active women have documented significant differences in community configurations between four self-reported ethnic groups: Caucasian, black, Hispanic, and Asian. Unlike most other body habitats that have been surveyed, this ecosystem is dominated by a single genus, Lactobacillus. Four species of this genus together account for more than half of the bacteria in most vaginal communities. Five community categories have been defined: four are dominated by L. iners,

1	L. crispatus, L. gasseri, and L. jensenii, respectively, and the fifth has proportionally fewer lactobacilli and more anaerobes. The representation of these community categories is distinct within each of the four ethnic groups and correlates with vaginal pH and Nugent score (the latter being a biomarker for bacterial vaginosis). Longitudinal studies of individuals are being conducted to identify factors that determine the assembly of these distinct communities—both within and among ethnic groups—as well as their resistance to or resilience after various physiologic and pathologic disturbances. For example, the menstrual cycle and pregnancy turn out to be surprisingly significant factors (cause larger changes) compared with sexual activity.

1	Yet another factor affecting beta diversity is spatial location within a habitat. Several surveys show that the skin harbors bacterial communities with predictable, albeit complex, biogeographic features. To determine whether these differences are due to differences in local environmental factors, to the history of a given site’s exposure to microbes, or to a combination of the two, reciprocal microbiota transplantation has been performed. Microbial communities from one region of the skin were depleted by treatment with germicidal agents, and the region (plot) was inoculated with a “foreign” microbiota harvested from different regions of the skin or from different body habitats from the same or another individual. Community assembly at the site of transplantation was then tracked over time. Remarkably, assembly proceeded differently at different sites: forearm plots receiving a tongue microbiota remained more similar to tongue communities than to native forearm communities in terms of

1	Remarkably, assembly proceeded differently at different sites: forearm plots receiving a tongue microbiota remained more similar to tongue communities than to native forearm communities in terms of their composition and diversity, while forehead plots inoculated with tongue bacteria changed to become more similar to native forehead communities. Thus, in addition to the history of exposure to tongue bacteria, environmental factors operating at the forehead plot likely shape community assembly. Intriguingly, the factors that shape fungal skin communities appear to be entirely different from those that shape bacterial skin communities. The palm and forearm have high bacterial and low fungal diversity, whereas the feet have the opposite diversity pattern. Moreover, fungal communities are generally shaped by location (foot, torso, head), whereas bacterial communities are generally shaped by moisture phenotype (dry, moist, or sebaceous).

1	Co-Occurrence Analysis Co-occurrence analysis seeks to identify which phylotypes are co-distributed across individuals in a given body habitat and/or between habitats and to determine the factors that explain the observed patterns of co-distribution. Positive correlations tend to reflect shared preferences for certain environmental features, while negative correlations typically reflect divergent preferences or a competitive relationship. Syntrophic (cross-feeding) relationships reflect interdependent interactions based on nutrient-sharing strategies. For example, in food webs, the products of one organism’s metabolism can be used by the other for its own unique metabolic capabilities (e.g., the interactions between fermentative organisms and methanogens).

1	Enterotype Analysis Enterotype analysis seeks to classify individuals into discrete groups based on the configuration of their microbiotas, essentially drawing boundaries on a map defined by principal coordinates analysis or other ordination techniques. The first enterotype analysis used supervised clustering to define three major types of human-gut microbial configurations across three distinct human studies and provided a view that presupposed the existence of three

1	Chapter 86e The Human Microbiome clusters. Subsequent work has shown that the range of variability in the gut microbiota of children and of non-Western populations greatly exceeds the variability captured in the populations used to define the original enterotypes; in addition, even in Western populations, the variability follows more of a continuum dominated by a gradient in the abundance of the genera Bacteroides and Prevotella. Another consideration in enterotype analysis is whether location on a map defined by healthy human variation is relevant to predisposition to disease or whether instead rare species with particular functions are more important discriminants.

1	Functional Redundancy Functional redundancy arises when functions are performed by many bacterial taxa. Thus interpersonal differences in microbial bacterial diversity (i.e., which bacteria are present) are not necessarily accompanied by comparable degrees of difference in functional diversity (i.e., what these bacteria can do). Characterization of a microbiome by shotgun sequencing is important because, unlike SSU rRNA analyses, shotgun sequencing provides a direct readout of the genes (and, via comparative genomics, their functions) in a given community. One fundamental question is the degree to which variations in the species occupying a given body habitat correlate with variations in a community’s functional capabilities. For example, the neutral theory of community assembly developed by macro-ecologists suggests that species are added to the community without respect to function, automatically endowing the community with functional redundancy. If applicable to the microbial

1	by macro-ecologists suggests that species are added to the community without respect to function, automatically endowing the community with functional redundancy. If applicable to the microbial world, neutral community assembly would predict a high level of variation in the types of microbial lineages that occupy a given body habitat in different individuals, although the broad functions encoded in the microbiomes of these communities could be quite similar.

1	Shotgun sequencing of the fecal microbiome has revealed that different microbial communities converge on the same functional state: in other words, there is a group of microbial genes represented in the guts of unrelated as well as related individuals. The same principle holds true at other body sites (Fig. 86e-2). The “core” gut microbiome is enriched in functions related to microbial survival (e.g., translation; metabolism of nucleotides, carbohydrates, and amino acids) and in functions that benefit the host (nutrient and energy partitioning from the diet to microbes and host). The latter functions encompass the food webs mentioned above, in which products of one type of microbe become the substrates for other microbes. These webs, which can be incredibly elaborate, change as microbes adjust their patterns of gene expression and metabolism in response to alterations in nutrient availability. Thus the sum of all the activities of the members of a microbial community can be viewed as

1	their patterns of gene expression and metabolism in response to alterations in nutrient availability. Thus the sum of all the activities of the members of a microbial community can be viewed as an emergent rather than a fixed property.

1	It is important to note that pairwise comparisons have shown that family members have functionally more similar gut microbiomes than do unrelated individuals. Thus, intrafamilial transmission of a gut microbiome within a given generation and across multiple generations could shape the biologic features of humans belonging to a kinship and modulate/mediate risks for a variety of diseases. Stability Like other ecosystems, human body habitat– associated microbial communities vary over time, and an understanding of this variation is essential for a functional

1	FIGURE 86e-2 Interpersonal variation in organismal representation in body habitat–associated communities is more extensive than interpersonal variation in gene functional features. Bacterial taxonomy and metabolic function are compared in 107 oral microbiota and microbiome samples (top) and in 139 fecal microbiota and microbiome samples (bottom). Samples represent an arbitrarily chosen subset from 242 healthy young adults living in the United States, with equal numbers of men and women. The same DNA extracts from the same samples were used for both taxonomic and functional classifications; each sample was analyzed by bacterial 16S rRNA amplicon sequencing (mean, 5400 sequences per sample) and by shotgun sequencing of community DNA (mean, 2.9 billion bases per sample). Taxonomic groups vary dramatically in their representation among different samples, with different characteristic bacterial phyla in the oral versus the fecal microbiota; e.g., members of the Actinobacteria and

1	vary dramatically in their representation among different samples, with different characteristic bacterial phyla in the oral versus the fecal microbiota; e.g., members of the Actinobacteria and Fusobacteria are far more common in the mouth than in the gut, while members of Bacteroidetes are far more common in fecal samples. In contrast, metabolic pathways are far more consistently represented in different samples, even when the species that contribute to these pathways are completely different. These results suggest a high degree of functional redundancy in microbial ecosystems—similar to that observed in macroecosystems, in which many fundamentally different lineages of organisms can play the same ecologic roles (e.g., pollinator or top predator). (Adapted from Human Microbiome Project Consortium: Nature 486:207, 2012; and CA Lozupone et al: Nature 489:220, 2012.) understanding of our microbiota. Few high-resolution time series of way for defining stability at the strain level than

1	Consortium: Nature 486:207, 2012; and CA Lozupone et al: Nature 489:220, 2012.) understanding of our microbiota. Few high-resolution time series of way for defining stability at the strain level than was available in the individual healthy adults have been published to date, but one available past. Application of these methods to the guts of healthy individualsdaily time series suggests that individuals tend to resemble themselves sampled over time has disclosed that a healthy adult gut harbors a permicrobially day to day over a span of 6–15 months, retaining their sistent collection of ~100 bacterial species and several hundred strains.separate identities during cohabitation. The development of low-error The stability of the bacterial components follows a power law: bacterial amplicon sequencing methods has provided a much more reliable strains acquired early in life can persist in the gut for decades, although their proportional representation changes as a function of numerous

1	sequencing methods has provided a much more reliable strains acquired early in life can persist in the gut for decades, although their proportional representation changes as a function of numerous factors, including diet. Whole-genome sequencing of culturable components of the microbiota of study participants has confirmed that strains are retained in individuals for prolonged periods and are shared among family members.

1	Resilience The ability of a microbiota or microbiome to rebound from a short-term perturbation, such as antibiotic administration or an infection, is defined as its resilience. This capacity can be visualized as a ball rolling over a landscape of local minima; essentially, the community moves into a new state and, to recover, must move through another, unstable state. In some cases, recovery will lead to the original stable state; in others, it will lead to a new stable state, which may be either healthy or unhealthy. Changes in, for example, diet or host physiologic status may introduce alterations into the landscape itself, making it easier to move from the initial state to any one of a number of other states, potentially with different health consequences. Microbial communities in our body habitats differ widely in resilience. For example, hand washing leads to profound changes in the microbial community, greatly increasing diversity (presumably because of the preferential removal

1	body habitats differ widely in resilience. For example, hand washing leads to profound changes in the microbial community, greatly increasing diversity (presumably because of the preferential removal of high-abundance, dominant phylotypes such as Propionibacterium). Within 6 h, the hand microbiota rebounds to resemble the original hand communities. The effects of repeated hand washing still need to be defined; for example, the surface microbiota on the skin (as measured by scrape biopsies) consists of ~50,000 microbial cells/cm2, whereas the subsurface microbiota (as measured by punch biopsies) consists of ~1,000,000 microbial cells/cm2.

1	In a study of three healthy adult volunteers given a short course of ciprofloxacin (500 mg by mouth twice a day for 5 days—a regimen commonly used against uncomplicated urinary tract infections), overall gut-community configuration came to resemble baseline within 6 months after treatment cessation, although some taxa failed to recover. However, the effects of the antibiotic perturbation were highly individualized. Administration of a second course of treatment months later led to altered-community states, relative to baseline, in all three volunteers; again, the extent of the alteration differed with the individual. Crucially, as shown in this and other studies, a given bacterial taxon can respond differently to the same antibiotic in different individuals; this observation suggests that the rest of the microbial community plays an important role in determining the effects of antibiotics on a per-individual basis.

1	In any body habitat, the microbial-community state after disturbance may be degraded. However, this degraded state may itself be resilient, and it may therefore be difficult to restore a more functional state. For example, Clostridium difficile infection can persist for years. The development and resilience of a degraded state may be driven by positive feedback loops, such as reactive oxygen species cascades involving host macrophages that promote the further growth of proinflammatory Proteobacteria, as well as negative-feedback loops such as depletion of the butyrate needed for promotion of a healthy gut epithelial barrier and further establishment of beneficial members of the microbiota. Consequently, microbiota-based therapies may require either (1) the elimination of a feedback loop that prevents establishment of a new community or (2) identification of a direction for change and a stimulus of sufficient magnitude (e.g., invasion and establishment of microbes from a fecal

1	loop that prevents establishment of a new community or (2) identification of a direction for change and a stimulus of sufficient magnitude (e.g., invasion and establishment of microbes from a fecal transplant or from a defined consortium of cultured, sequenced members of the human gut microbiota; see below) to overcome the resilience mechanisms inherent in the degraded state. A critical unresolved question that especially affects infants, whose microbiota is changing rapidly, is whether intervention during periods of rapid change or during periods of relative stability is generally more effective.

1	ESTABLISHING CAUSAL RELATIONSHIPS BETWEEN THE GUT MICROBIOTA AND NORMAL PHYSIOLOGIC, METABOLIC, AND IMMUNOLOGIC PHENOTYPES AS WELL AS DISEASE STATES Gnotobiotic animals are raised in germ-free environments—with no exposure to microbes—and then colonized at specific stages of life with specified microbial communities. Gnotobiotic mice provide an excellent system for controlling host genotype, microbial community composition, diet, and housing conditions. Microbial communities harvested from donor mice with defined genotypes and phenotypes 86e-7 can be used to determine how the donors’ microbial communities affect the properties of formerly germ-free recipients. The recipients may also affect the transplanted microbiota and its microbiome. Thus gnotobiotic mice afford investigators an opportunity to marry comparative studies of donor communities to functional assays of community properties and to determine how (and for how long) these functions influence host biology.

1	The Cardiovascular System The gut microbiota affects the elaborate microvasculature underlying the small-intestinal epithelium: capillary network density is markedly reduced in adult germ-free animals but can be restored to normal levels within 2 weeks after gut microbiota transplantation. Mechanistic studies have shown that the microbiota promotes vascular remodeling in the gut through effects on a novel extravascular tissue factor–protease-activated receptor (PAR1) signaling pathway. Heart weight measured echocardiographically or as wet mass and normalized to tibial length or lean body weight is significantly reduced in germ-free mice; this difference is eliminated within 2 weeks after colonization with a gut microbiota. During fasting, a gut microbiota–dependent increase in hepatic ketogenesis (regulated by peroxisome proliferator–activated receptor α) occurs, and myocardial metabolism is directed to ketone body utilization. Analyses of isolated, perfused working hearts from

1	ketogenesis (regulated by peroxisome proliferator–activated receptor α) occurs, and myocardial metabolism is directed to ketone body utilization. Analyses of isolated, perfused working hearts from germ-free and colonized animals, together with in vivo assessments, have shown that myocardial performance in germ-free mice is maintained by increasing glucose utilization. However, heart weight is significantly reduced in both fasted and fed mice; this heart-mass phenotype is completely reversed in germ-free mice fed a ketogenic diet. These findings illustrate how the gut microbiota benefits the host during periods of nutrient deprivation and represent one link between gut microbes and cardiovascular metabolism and health.

1	Conventionally raised apoE-deficient mice develop a less severe form of atherosclerosis than their germ-free counterparts when fed a high-fiber diet. This protective effect of the microbiota is obviated when animals are fed a diet low in fiber and high in simple sugars and fat. A number of the beneficial effects attributed to diets with high proportional representation of whole grains, fruits, and vegetables are thought to be mediated by end products of microbial metabolism of dietary compounds, including short-chain fatty acids and metabolites derived from flavonoids. Conversely, microbes can convert otherwise harmless dietary compounds into metabolites that increase risk for cardiovascular disease. Studies of mice and human volunteers have revealed that gut microbiota metabolism of dietary L-carnitine, which is present in large amounts in red meat, yields trimethylamineN-oxide, which can accelerate atherosclerosis in mice by suppressing reverse cholesterol transport.

1	Yet another facet of microbial influence on cardiovascular physiology was revealed in a study of mice deficient in Olfr78 (a G protein– coupled receptor expressed in the juxtaglomerular apparatus, where it regulates renin secretion in response to short-chain fatty acids) or Gpr41 (another short-chain fatty acid receptor that, together with Olfr78, is expressed in the smooth muscle cells present in small resistance vessels). This study demonstrated that the microbiota can modulate host blood pressure via short-chain fatty acids produced by microbial fermentation.

1	Bone Adult germ-free mice have greater bone mass than their conventionally raised counterparts. This increase in bone mass is associated with reduced numbers of osteoclasts per unit bone surface area, reduced numbers of CD11b+/GR1 osteoclast precursors in bone marrow, decreased numbers of CD4+ T cells, and reduced levels of expression of the osteolytic cytokine tumor necrosis factor α. Colonization with a normal gut microbiota resolves these observed differences between germ-free and conventionally raised animals. Brain Adult germ-free and conventionally raised mice differ significantly in levels of 38 out of 196 identified cerebral metabolites, 10 of which have known roles in brain function; included in the latter group are N-acetylaspartic acid (a marker of neuronal health and attenuation),

1	Chapter 86e The Human Microbiome pipecolic acid (a presynaptic modulator of γ-aminobutyric acid levels), and serine (an obligatory co-agonist at the glycine site of the N-methyl-d-aspartate receptor). Propionate, a short-chain fatty acid product of gut microbial-community metabolism of dietary fiber, affects expression of genes involved in intestinal gluconeogenesis via a gut–brain neural circuit involving free fatty-acid receptor 3; this effect provides a mechanistic explanation for the documented beneficial impact of dietary fiber in enhancing insulin sensitivity and reducing body mass and adiposity.

1	Studies of a mouse model (maternal immune activation) with stereotyped/repetitive and anxiety-like behaviors indicate that treatment with a member of the human gut microbiota, Bacteroides fragilis, corrects gut barrier (permeability) defects; reduces elevated levels of 4-ethylphenylsulfate, a metabolite seen in the maternal immune activation model that has been causally associated with the animals’ behavioral phenotypes; and ameliorates some behavioral effects. These observations highlight the importance of further exploration of potentially co-evolved relationships between the microbiota and host behavior.

1	Immune Function Many foundational studies have shown that the gut microbiota plays a key role in the maturation of the innate as well as the adaptive components of the immune system. The intestinal epithelium, which is composed of four principal cell lineages (enterocytes plus goblet, Paneth, and enteroendocrine cells), acts as a physical and functional barrier to microbial penetration. Goblet cells produce mucus that overlies the epithelium, where it forms two layers: an outer (luminal-facing) looser layer that harbors microbes and a denser lower layer that normally excludes microbes. Members of the Paneth cell lineage reside at the base of crypts of Lieberkühn and secrete antimicrobial peptides. Studies in mice have demonstrated that Paneth cells directly sense the presence of a microbiota through expression of the signaling adaptor protein MyD88, which helps transduce signals to host cells upon recognition of microbial products through Toll-like receptors (TLRs). This recognition

1	through expression of the signaling adaptor protein MyD88, which helps transduce signals to host cells upon recognition of microbial products through Toll-like receptors (TLRs). This recognition drives expression of antibacterial products (e.g., the lectin RegIIIγ) that act to prevent microbial translocation across the gut mucosal barrier.

1	The intestine is enriched for B cells that produce IgA, which is secreted into the lumen; there it functions to exclude microbes from crossing the mucosal barrier and to restrict dissemination of food antigens. The microbiota plays a key role in development of an IgA response: germ-free mice display a marked reduction in IgA+ B cells. The absence of a normal IgA response can lead to a massive increase in bacterial load. B cell–derived IgA that targets specific members of the gut microbiota plays an important role in preventing activation of microbiota-specific T cells.

1	Gut bacterial species elicit development of protective TH17 and TH1 responses that help ward off pathogen attack. Members of the microbiota also promote the development of a specialized population of CD4+ T cells that prevent unwarranted inflammatory responses. These regulatory T cells (Tregs) are characterized by expression of the transcription factor forkhead box P3 (FOXP3) and by expression of other cell-surface markers. There is a paucity of Tregs in the colonic lamina propria of germ-free mice. Specific members of the microbiota—including a consortium of Clostridium strains isolated from the mouse and human gut as well as several human-gut Bacteroides species —expand the Treg compartment and enhance immunosuppressive functions.

1	The microbiota is a key trigger in the development of inflammatory bowel disease (IBD) in mice that harbor mutations in genes associated with IBD risk in humans. Moreover, components of the gut microbiota can modify the activity of the immune system to ameliorate or prevent IBD. Mice containing a mutant ATG16L1 allele linked to Crohn’s disease are particularly susceptible to IBD. Upon infection with mouse norovirus and treatment with dextran sodium sulfate, expression of a hypomorphic ATG16L1 allele leads to defects in small-intestinal Paneth cells and renders mice significantly more susceptible to ileitis than are wild-type control animals. This process is dependent on the gut microbiota and highlights how the intersection of host genetics, infectious agents, and the microbiota can lead to severe immune pathology; i.e., the pathogenic potential of a microbiota may be context-dependent, requiring a confluence of factors. An important observation is that members of the gut microbiota,

1	to severe immune pathology; i.e., the pathogenic potential of a microbiota may be context-dependent, requiring a confluence of factors. An important observation is that members of the gut microbiota, including B. fragilis or members of Clostridium, prevent the severe inflammation that develops in mouse models mimicking various aspects of human IBD.

1	The gut microbiota has been implicated in promoting immunopathology outside of the intestine. Multiple sclerosis develops in conventionally raised mice whose CD4+ T cell compartment is reactive to myelin oligodendrocyte protein; their germ-free counterparts are completely protected from development of multiple sclerosis–like symptoms. This protection is reversed by colonization with a gut microbiota from conventionally raised animals.

1	Inflammasomes are cytoplasmic multiprotein complexes that sense stress and damage-associated patterns. Mice deficient in NLRP6, a component of the inflammasome, are more susceptible to colitis induced by administration of dextran sodium sulfate. This enhanced susceptibility is associated with alterations in the gut microbiota of these animals relative to that of wild-type controls. Mice are coprophagic, and co-housing of NLRP6-deficient mice with wild-type mice is sufficient to transfer the enhanced susceptibility to colitis induced by dextran sodium sulfate. Similar findings have been reported for mice deficient in the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain). ASC-deficient mice are more susceptible to the development of a model of nonalcoholic steatohepatitis. This susceptibility is associated with alterations in gut microbiota structure and can be transferred to wild-type animals by co-housing.

1	Obesity and Diabetes Germ-free mice are resistant to diet-induced obesity. Genetically obese ob/ob mice have gut microbial-community structures that are profoundly altered from those in their lean wild-type (+/+) and heterozygous +/ob littermates. Transplantation of the ob/ob mouse microbiota into wild-type germ-free animals transmits an increased-adiposity phenotype not seen in mice receiving microbiota transplants from +/+ and +/ob littermates. These differences are not attributable to differences in food consumption but rather are associated with differences in microbial community metabolism. Roux-en-Y gastric bypass produces pronounced decreases in weight and adiposity as well as improved glucose metabolism—changes that are not ascribable simply to decreased caloric intake or reduced nutrient absorption. 16S rRNA analyses have documented that changes in the gut microbiota after this surgery are conserved among mice, rats, and humans; animal studies have demonstrated these changes

1	nutrient absorption. 16S rRNA analyses have documented that changes in the gut microbiota after this surgery are conserved among mice, rats, and humans; animal studies have demonstrated these changes along the length of the gut but most prominently downstream of the site of surgical manipulation of the bowel. Notably, transplantation of the gut microbiota from mice that have undergone Roux-en-Y gastric bypass to germ-free mice that have not had this surgery produces reductions in weight and adiposity not seen in recipients of microbiotas from mice that underwent sham surgery.

1	The gut microbiota confers protection against the development of type 1 diabetes mellitus in the non-obese diabetic (NOD) mouse model. Disease incidence is significantly lower in conventionally raised male NOD mice than in their female counterparts, while germ-free males are as susceptible as their female counterparts. Castration of males increases disease incidence, while androgen treatment of females provides protection. Transfer of the gut microbiota from adult male NOD mice to female NOD weanlings is sufficient to reduce the severity of disease relative to that among females receiving a microbiota from an adult female or an unmanipulated female. The blocking of protection by treatment with flutamide highlights a functional role for testosterone signaling in this microbiota-mediated protection against type 1 diabetes.

1	NOD mice deficient in MyD88, a key component of the TLR signaling pathway, do not develop diabetes and exhibit increased relative abundance of members of the family-level taxon Lactobacillaceae. Consistent with these findings, investigators have documented lower levels of representation of members of the genus Lactobacillus in children with type 1 diabetes than in healthy controls. Components of lactobacilli have been shown to promote gut barrier integrity. Studies in various animal models indicate that translocation of bacterial components, including bacterial lipopolysaccharides, across a leaky gut barrier triggers low-grade inflammation, which contributes to insulin resistance. Mice deficient in TLR5 exhibit alterations in the gut microbiota and hyperphagia, and they develop features of metabolic syndrome, including hypertension, hyperlipidemia, insulin resistance, and increased adiposity.

1	The gut microbiota regulates biosynthesis as well as metabolism of host-derived products; these products can signal through host receptors to shape host physiology. An example of this symbiosis is provided by bile acids, which direct metabolic effects that are largely mediated through the farnesoid X receptor (FXR, also known as NR1H4). In leptin-deficient mice, FXR deficiency protects against obesity and improves insulin sensitivity. In mice with diet-induced obesity that are subjected to vertical sleeve gastrectomy, the surgical procedure results in elevated levels of circulating bile acids, changes in the gut microbiota, weight loss, and improved glucose homeostasis. However, weight reduction and improved insulin sensitivity are mitigated in animals with engineered FXR-deficiency. Xenobiotic Metabolism Evidence is accumulating that pharmacogenomic studies need to consider the gene repertoire present in our

1	Xenobiotic Metabolism Evidence is accumulating that pharmacogenomic studies need to consider the gene repertoire present in our H. sapiens genome as well as that in our microbiomes. For example, digoxin is inactivated by the human gut bacterium Eggerthella lenta, but only by strains with a cytochrome-containing operon. Expression of this operon is induced by digoxin and inhibited by arginine. Studies in gnotobiotic mice established that dietary protein affects (reduces) microbial metabolism of digoxin, with corresponding alterations in levels of the drug in both serum and urine. These findings reinforce the need to consider strain-level diversity in the gut microbiota when examining interpersonal variations in the metabolism of orally administered drugs.

1	Characterizing the Effects of the Human Microbiota on Host Biology in Mice and Humans Questions about the relationship between human microbial communities and health status can be posed in the following general format: Is there a consistent configuration of the microbiota definable in the study population that is associated with a given disease state? How is the configuration affected by remission/relapse or by treatment? If a reconfiguration does occur with treatment, is it durable? How is host biology related to the configuration or reconfiguration? What is the effect size? Are correlations robust to individuals from different families and communities representing different ages, geographic locales, and lifestyles?

1	As in all studies involving human microbial ecology, the issue of what constitutes a suitable reference control is extremely important. Should we choose the person himself or herself, family members, or ageor gender-matched individuals living in the same locale and representing similar cultural traditions? Critically, are the relationships observed between microbial community structure and expressed functions a response to disease state (i.e., side effects of other processes), or are they a contributing cause? In this sense, we are challenged to evolve a set of Koch’s postulates that can be applied to whole microbial communities or components of communities rather than just to a single purified organism. As in other circumstances in which experiments to determine causality of human disease are difficult or unethical, Hill’s criteria, which examine the strength, consistency, and biologic plausibility of epidemiologic data, can be useful.

1	Sets of monoand dizygotic twins and their family members represent a valuable resource for initially teasing out relationships between environmental exposures, genotypes, and our own microbial ecology. Similarly, monozygotic twins discordant for various disease states enhance the ability to determine whether various diseases can be linked to a person’s microbiota and microbiome. A twin-pair sampling design rather than a conventional unrelated case–control design has advantages owing to the pronounced between-family variability in microbiota/microbiome composition and the potential for multiple states of a community associated with disease. Transplantation of a microbiota from suitable human donor controls representing different disease states and communities (e.g., twins discordant for a disease) to germ-free mice is helpful in establishing a causal role for the com-86e-9 munity in pathogenesis and for providing insights relevant to underlying mechanisms. In addition, transplantation

1	disease) to germ-free mice is helpful in establishing a causal role for the com-86e-9 munity in pathogenesis and for providing insights relevant to underlying mechanisms. In addition, transplantation provides a preclinical platform for identifying next-generation probiotics, prebiotics, or combinations of the two (synbiotics). Obesity and obesity-associated metabolic dysfunction illustrate these points.

1	The gut microbiotas (and microbiomes) of obese individuals are significantly less diverse than those of lean individuals; the implication is that there may be unfilled niches (unexpressed functions) that contribute to obesity and its associated metabolic abnormalities. Le Chatelier and colleagues observed a bimodal distribution of gene abundance in their analysis of 292 fecal microbiomes: low-gene-count (LGC) individuals averaged 380,000 microbial genes per gut microbiome, while high-gene-count (HGC) individuals averaged 640,000 genes. LGC individuals had an increased risk for type 2 diabetes and other metabolic abnormalities, whereas the HGC group was metabolically healthy. When gene content was used to identify taxa that discriminated HGC and LGC individuals, the results revealed associations between anti-inflammatory bacterial species such as Faecalibacterium prausnitzii and the HGC group and between proinflammatory species such as Ruminococcus gnavus and the LGC group. LGC

1	associations between anti-inflammatory bacterial species such as Faecalibacterium prausnitzii and the HGC group and between proinflammatory species such as Ruminococcus gnavus and the LGC group. LGC microbiomes had significantly greater representation of genes assigned to tricarboxylic acid cycle modules, peroxidases, and catalases—an observation suggesting a greater capacity to handle oxygen exposure and oxidative stress; HGC microbiomes were enriched in genes involved in the production of organic acids, including lactate, propionate, and butyrate— a result suggesting increased fermentative capacity.

1	Transplantation of an uncultured fecal microbiota from twins stably discordant for obesity or of bacterial culture collections generated from their microbiota transmits their discordant adiposity phenotypes as well as obesity-associated metabolic abnormalities to recipient germ-free mice. Co-housing of the recipient coprophagic gnotobiotic mice results in invasion of specific bacterial species from the transplanted lean twin’s culture collection into the guts of cage mates harboring the obese twin’s culture collection (but not vice versa), thereby preventing the latter animals from developing obesity and its associated metabolic abnormalities. It is noteworthy that invasion and prevention of obesity and metabolic phenotypes are dependent on the type of human diets fed to animals: prevention is associated with a diet low in saturated fats and high in fruit and vegetable content, but not with a diet high in saturated fats and low in fruit and vegetable content.

1	This approach provides evidence for a causal role for the microbiota in obesity and its attendant metabolic abnormalities. It also provides a method for defining unoccupied niches in disease-associated microbial communities, the role of dietary components in determining how these niches can be filled by human gut–derived bacterial taxa, and the effects of such occupancy on microbial and host metabolism. It also provides a way to identify health-promoting diets and next-generation probiotics representing naturally occurring members of our indigenous microbial communities that are well adapted to persist in a given body habitat.

1	A key to this approach is the ability to harvest a microbial community from a donor representing a physiology, disease state, lifestyle, or geography of interest; to preserve the donor’s community by freezing it; and then to resurrect and replicate it in multiple recipient gnotobiotic animals that can be reared under conditions where environmental and host variables can be controlled and manipulated to a degree not achievable in clinical studies. Since these mice can be followed as a function of time prior to and after transplantation, in essence, a snapshot of a donor’s community can be converted into a movie. Transplantation of intact uncultured human (fecal) microbiota samples from multiple donors representing the phenotype of interest, with administration of the donors’ diets (or derivatives of those diets) to different groups of mice, is one way to assess whether transmissible responses are shared features of the microbiota or are highly donor specific. A second step is to

1	derivatives of those diets) to different groups of mice, is one way to assess whether transmissible responses are shared features of the microbiota or are highly donor specific. A second step is to determine whether the culturable component of a representative microbiota sample can transmit the phenotype(s) observed with the intact uncultured sample. Possession of a collection of cultured organisms that have co-evolved in a given donor’s body habitat sets the stage

1	Chapter 86e The Human Microbiome for the selection of subsets of the collection for testing in gnotobiotic mice, the determination of which members are responsible for effecting the phenotype, and the elucidation of the mechanisms underlying these effects. The models used may inform the design and interpretation of clinical studies of the very individuals and populations whose microbiota are selected for creating these models. Human-to-human fecal microbiota transplantation (FMT) is currently the most direct way to establish proof-of-concept for a causal role for the microbiota in disease pathogenesis. A human donor’s feces are provided to a recipient via nasogastric tube or another technique. Numerous small trials have documented the effects of FMT from healthy donors to recipients with diseases ranging from C. difficile infection to Crohn’s disease, ulcerative colitis, and type 2 diabetes. Only a few of these studies have used a double-blind, placebo-controlled design.

1	In a double-blind, controlled trial involving men 21–65 years old with a body mass index of >30 kg/m2 and documented insulin resistance, FMT was performed using a microbiota from metabolically healthy lean donors or from the study participants themselves. A microbiota from lean donors significantly improved peripheral insulin sensitivity over that in controls. This change was associated with an increase in the relative abundance of the butyrate-producing bacteria related to Roseburia intestinalis (in the feces) and Eubacterium hallii (in the small intestine).

1	The efficacy of FMT for the treatment of recurrent C. difficile infection has been assessed in a number of small trials. One unblinded, placebo-controlled trial assessed the use of FMT in 42 patients with recurrent C. difficile infection (defined as at least one relapse after treatment with vancomycin or metronidazole for ≥10 d). Patients were pretreated with oral vancomycin. The experimental group then received FMT via nasoduodenal tube from healthy volunteer donors (<60 years of age) selected from the community. Controls underwent sterile lavage or received oral vancomycin alone. In 10 weeks of follow-up, infection was cured (with cure defined as three negative fecal tests for C. difficile toxin) in 81% of patients in the FMT group (13 of 16) but in only 23% (3 of 13) in the bowel-lavage control arm and 31% (4 of 13) in the vancomycin-only group. Metagenomic analysis of microbiota samples collected before and after treatment revealed an increased representation of Bacteroidetes and

1	control arm and 31% (4 of 13) in the vancomycin-only group. Metagenomic analysis of microbiota samples collected before and after treatment revealed an increased representation of Bacteroidetes and Clostridium clusters IV and XIVa, along with a 100-fold decrease in the relative abundance of Proteobacteria, in the FMT group.

1	A meta-analysis of FMT in C. difficile infection examined 20 case-series publications, 15 case reports, and the one unblinded study described above. All but one of these studies used fresh (not frozen) fecal samples. Donor selection varied, although most donors were family members or relatives and most studies excluded donors who had recently received antibiotics. It is noteworthy that the concentrations of infused donor feces varied widely (i.e., from 5 g to 200 g, resuspended in 10–500 mL); these fecal suspensions were introduced at different sites along the gastrointestinal tract, including the stomach and points throughout the small intestine and colon. Resolution of infection, which was frequently assessed on the basis of symptom resolution (with C. difficile toxin testing rarely performed), was documented in 87% (467) of 536 treated patients. The most common adverse events reported were diarrhea (94% of cases) and abdominal cramps (31%) on the day of infusion. The meta-analysis

1	was documented in 87% (467) of 536 treated patients. The most common adverse events reported were diarrhea (94% of cases) and abdominal cramps (31%) on the day of infusion. The meta-analysis was limited to clinical outcomes and did not specifically address the role of the microbiota in disease resolution (e.g., the extent of invasion of donor taxa; their persistence; or the long-term effects of transplantation on various facets of host biology, which generally have not been evaluated).

1	Sober and thoughtful consideration needs to be applied to the therapeutic use of FMT, which represents an early and rudimentary approach to microbiota manipulation that very likely will be replaced by administration of defined collections of sequenced, cultured members of the human microbiota (probiotic consortia). A number of published reports on FMT have garnered significant public attention. This attention, coupled with an increasing public appreciation of the beneficial nature of our interactions with microbes, demands that the precautionary principle be honored and that risks versus benefits of such interventions be carefully evaluated.

1	To date, most FMT trials have failed to define (or have differed in) significant confounders, including (1) the criteria used for donor sample selection; (2) the methods used for donor sample preparation and characterization as well as the decision about whether or not to create a repository for donor and recipient samples that will permit retrospective analyses (and meta-analyses for given disease states); the development of minimal standards for assessing the invasion of recipient gut communities by taxa from donor microbiota (using microbial source-tracking methods) as well as the timing, duration, nature, and breadth of sampling of the recipient as a function of transplantation; (4) the adoption of minimal standards for collection of patients’ clinical data (e.g., age, diet, antibiotic use) and the establishment of databases for entering these data (including use of a defined vocabulary for annotating the clinical data); and (5) the development of standards for informed consent in

1	use) and the establishment of databases for entering these data (including use of a defined vocabulary for annotating the clinical data); and (5) the development of standards for informed consent in lieu of knowledge of the longterm effects of the procedure. The regulatory landscape is evolving. The U. S. Food and Drug Administration recently issued an enforcement policy specifically addressing the use of FMT for the treatment of recurrent C. difficile infection; this policy indicates that the agency intends to “exercise enforcement discretion regarding the investigational new drug (IND) requirements for the use of FMT to treat

1	C. difficile infection not responding to standard therapies,” but it does not waive IND requirements for other FMT studies.

1	The design of human microbiome studies is rapidly evolving, in part because the data are highly multivariate, are compositional, and do not meet distributional assumptions of standard statistical tests such as analysis of variance. Consequently, the proper number of subjects to enroll and the proper populations to target remain to be established. One useful approach is to review published studies and ask whether the reported conclusion could be obtained with fewer subjects (sample rarefaction) and/or fewer sequencing reads from SSU rRNA genes, whole-community DNA (microbiomes), or expressed community mRNA (metatranscriptomes) per subject (sequence rarefaction). A common yet critical problem to avoid is under-sampling of the types of objects under study. For example, if the goal is to compare factors applying to individuals (e.g., individual diet), then dozens of individuals in each clinical category may be needed. If the goal is to compare factors applying to populations (e.g.,

1	compare factors applying to individuals (e.g., individual diet), then dozens of individuals in each clinical category may be needed. If the goal is to compare factors applying to populations (e.g., demographic properties), then many populations may be needed.

1	Another key issue is whether the effect size to be studied, especially in meta-analysis, is greater than or less than technical effects. As noted above, different PCR primers will lead to different readouts of the taxonomy of a microbial community; these differences are, for example, greater than the differences between lean and obese subjects’ fecal microbiota but less than the difference between fecal communities in newborns and adults.

1	A central challenge in human microbiome research is establishing the extent to which diagnostic tests and therapeutic approaches are generalizable. This challenge is illustrated by studies of the capacities of gut microbiomes to metabolize orally administered drugs. The results could be very informative for the pharmaceutical industry as it seeks new and more accurate ways to predict bioavailability and toxicity. However, these studies should prompt consideration of the fact that many clinical trials are outsourced to countries where trial participants have diets and microbial community structures that differ from those of the intended initial recipients of the (marketed) drug. Capture and preservation of the wide range of microbial diversity present in different human populations—and thus of the capacity of our microbial communities to catalyze elaborate and in many respects uncharacterized biotransformations—represent potentially fertile ground for the discovery of new drugs (and

1	of the capacity of our microbial communities to catalyze elaborate and in many respects uncharacterized biotransformations—represent potentially fertile ground for the discovery of new drugs (and new industrial processes of societal value). The chemical entities that our microbial communities have evolved to synthesize in order to support their mutually beneficial relationships and the human genes that these chemotypes influence may become new classes of drugs and new targets for drug discovery, respectively. Therefore, characterization of groups of individuals living in countries that are undergoing rapid transformations in cultural traditions and socioeconomic conditions and are witnessing the emergence of a variety of diseases associated with increasingly Western lifestyles (globalization) is a timely challenge. Birth cohort studies (including studies of twins) initiated every 10 years in these countries may be able to capture the impact of globalization, including changing diets,

1	is a timely challenge. Birth cohort studies (including studies of twins) initiated every 10 years in these countries may be able to capture the impact of globalization, including changing diets, on human microbial ecology.

1	Although microbiome-associated diagnostics and therapeutics provide new and exciting dimensions for personalized medicine, attention must be paid to the potentially broad societal impact of this work. For example, studies of the human gut microbiome are likely to have a disruptive effect on current views of human nutrition, enhancing appreciation of how food and the metabolic output of interactions of dietary components with the microbiota are intimately connected to myriad features of human biology. Underlying the efforts to elucidate the relations among food, the microbiome, and human nutrition is a need to proactively develop materials for educational outreach with a narrative and vocabulary that is understandable to broad and varied consumer populations representing different cultural traditions and widely ranging degrees of scientific literacy. The results have the potential to catalyze efforts to integrate agricultural policies and practice, food production, and nutritional

1	traditions and widely ranging degrees of scientific literacy. The results have the potential to catalyze efforts to integrate agricultural policies and practice, food production, and nutritional recommendations for consumer populations representing different ages, geographic locales, and states of health.

1	Defining our metagenome (the genes embedded in our H. sapiens genome plus those in our microbiome) will likely lead to an entirely new level of refinement in our description of self, our genetic evolution, 86e-11 our postnatal development, the microbial legacy of our connection to family, and the consequences of personal lifestyle choices. While this information can help us understand the origins of certain yet unexplained health disparities, care must be taken to avoid stigmatization of individuals or groups of individuals having different cultural norms, belief systems, or behaviors. In partnership with human microbiome researchers, anthropologists need to examine the impact of studies of the human microbiome on the participants, assessing how this field and participants’ cultural traditions interact to affect these individuals’ perceptions about the natural world, the forces that affect their lives, and their connections to one another within the context of family and community.

1	Studies of human microbial ecology are an important manifestation of progress in the genome sciences, represent a timely step in our quest to achieve a better understanding of our place in the natural world, and reflect the evolving focus of twenty-first-century medicine on disease prevention, new definitions of health, new ways to determine the origins of individual biologic differences, and new approaches to evaluating the impact of changes in our lifestyles and biosphere on our biology. As microbiome-directed diagnostics and therapeutics emerge, we must be sensitive to the societal impact of this work.

1	Chapter 86e The Human Microbiome that challenge the discipline. Biologists study the experimental response of a variable of interest in a cell or organism while holding all other variables constant. In this way, it is possible to dissect the individual components of a biologic system and assume that a thorough understanding of a specific component (e.g., an enzyme or a transcription factor) will provide sufficient insight to explain the global behavior of that system (e.g., a metabolic pathway or a gene network, respectively). Biologic systems are, however, much more complex than this approach assumes and manifest behaviors that frequently (if not invariably) cannot be predicted from knowledge of their component parts characterized in isolation. Growing recognition of this shortcoming of conventional biologic research has led to the development of a new discipline, systems biology, which is defined as the holistic study of living organisms or their cellular or molecular network

1	of conventional biologic research has led to the development of a new discipline, systems biology, which is defined as the holistic study of living organisms or their cellular or molecular network components to predict their response to perturbations. Concepts of systems biology can be applied readily to human disease and therapy and define the field of systems pathobiology, in which genetic or environmental perturbations produce disease and drug perturbations restore normal system behavior.

1	Systems biology evolved from the field of systems engineering in which a linked collection of component parts constitute a network whose output the engineer wishes to predict. The simple example of an electronic circuit can be used to illustrate some basic systems engineering concepts. All the individual elements of the circuit—resistors, capacitors, transistors—have well-defined properties that can be characterized precisely. However, they can be linked (wired or configured) in a variety of ways, each of which yields a circuit whose response to voltage applied across it is different from the response of every other configuration. To predict the circuit’s (i.e., system’s) behavior, the engineer must study its response to perturbation (e.g., voltage applied across it) holistically rather than its individual components’ responses to that perturbation. Viewed another way, the resulting behavior of the system is greater than (or different from) the simple sum of its parts, and systems

1	than its individual components’ responses to that perturbation. Viewed another way, the resulting behavior of the system is greater than (or different from) the simple sum of its parts, and systems engineering utilizes rigorous mathematical approaches to predict these complex, often nonlinear, responses. By analogy to biologic systems, one can reason that detailed knowledge of a single enzyme in a metabolic pathway or of a single transcription factor in a gene network will not provide sufficient detail to predict the output of that metabolic pathway or transcriptional network, respectively. Only a systems-based approach will suffice.

1	It has taken biologists a long time to appreciate the importance of systems approaches to biomedical problems. Reductionism has reigned supreme for many decades, largely because it is experimentally and analytically simpler than holism, and because it has provided insights into biologic mechanisms and disease pathogenesis that have led to successful therapies. However, reductionism cannot solve all biomedical problems. For example, the so-called off-target effects of new drugs that frequently limit their adoption likely reflect the failure of a drug to be studied in holistic context, i.e., the failure to explore all possible actions aside from the principal target action for which it was developed. Other approaches to understanding biology therefore are clearly needed. With the growing body of genomic, proteomic, and metabolomic data sets in which dynamic changes in the expression of many genes and many metabolites are recorded after a perturbation and with the growth of rigorous

1	body of genomic, proteomic, and metabolomic data sets in which dynamic changes in the expression of many genes and many metabolites are recorded after a perturbation and with the growth of rigorous mathematical approaches to analyzing those changes, the stage has been set for applying systems engineering principles to modern biology.

1	Physiologists historically have had more of a (bio)engineering perspective on the conduct of their studies and have been among the first systems biologists. Yet, with few exceptions, they, too, have focused on comparatively simple physiologic systems that are tractable using 87e-1 conventional reductionist approaches. Efforts at integrative modeling of human physiologic systems, as first attempted by Guyton for blood pressure regulation, represent one application of systems engineering to human biology. These dynamic physiologic models often focus on the acute response of a measurable physiologic parameter to a system perturbation, and do so from a classic analytic perspective in which all the conventional physiologic determinants of the output parameter are known and can be modeled quantitatively.

1	Until recently, molecular systems analysis has been limited owing to inadequate knowledge of the molecular determinants of a biologic system of interest. Although biochemists have approached metabolic pathways from a systems perspective for over 50 years, their efforts have been limited by the inadequacy of key information for each enzyme (KM, kcat, and concentration) and substrate (concentration) in the pathway. With increasingly rich molecular data sets available for systems-based analyses, including genomic, transcriptomic, proteomic, and metabolomic data, biochemists are now poised to use systems biology approaches to explore biologic and pathobiologic phenomena.

1	To understand how best to apply the principles of systems biology to human biomedicine, it is necessary to review briefly the building blocks of any biologic system and the determinants of system complexity. All systems can be analyzed by defining their static topology (architecture) and their dynamic (i.e., time-dependent) response to perturbation. In the discussion that follows, system properties are described that derive from the consequences of topology (form) or dynamic response (function). Any system of interacting elements can be represented schematically as a network in which the individual elements are depicted as nodes and their connections are depicted as links. The nature of the links among nodes reflects the degree of complexity of the system. Simple systems are those in which the nodes are linearly linked with occasional feedback or feedforward loops modulating system throughput in highly predictable ways. By contrast, complex systems are nodes that are linked in more

1	the nodes are linearly linked with occasional feedback or feedforward loops modulating system throughput in highly predictable ways. By contrast, complex systems are nodes that are linked in more complicated, nonlinear networks; the behavior of these systems by definition is inherently more difficult to predict owing to the nature of the interacting links, the dependence of the system’s behavior on its initial conditions, and the inability to measure the overall state of the system at any specific time with great precision. Complex systems can be depicted as a network of lower-complexity interacting components or modules, each of which can be reduced further to simpler analyzable canonical motifs (such as feedback and feedforward loops, or negative and positive autoregulation); however, a central property of complex systems is that simplifying their structures by identifying and characterizing the individual nodes and links or even simpler substructures does not necessarily yield a

1	a central property of complex systems is that simplifying their structures by identifying and characterizing the individual nodes and links or even simpler substructures does not necessarily yield a predictable understanding of a system’s behavior. Thus, the functioning system is greater than (or different from) the sum of its individual, tractable parts.

1	Defined in this way, most biologic systems are complex systems that can be represented as networks whose behaviors are not readily predictable from simple reductionist principles. The nodes, for example, can be metabolites that are linked by the enzymes that cause their transformations, transcription factors that are linked by the genes whose expression they influence, or proteins in an interaction network that are linked by cofactors that facilitate interactions or by thermodynamic forces that facilitate their physical association. Biologic systems typically are organized as scale-free, rather than stochastic, networks of nodes. Scale-free networks are those in which a few nodes have many links to other nodes (highly linked nodes, or hubs) but most nodes have only a few links (weakly linked nodes). The term scale-free refers to the fact that the connectivity of nodes in the network is invariant with respect to the size of the network. This is quite different from two other common

1	linked nodes). The term scale-free refers to the fact that the connectivity of nodes in the network is invariant with respect to the size of the network. This is quite different from two other common network architectures: random (Poisson) and exponential distributions. Scale-free networks can be mathematically described by a power law that defines the probability of the number of

1	Chapter 87e Network Medicine: Systems Biology in Health and Disease FIGuRE 87e-1 Network representations and their distributions. A random network is depicted on the left, and its Poisson distribution of the number of nodal connections (k) is shown in the graph below it. A scale-free network is depicted on the right, and its power law distribution of the number of nodal connections (k) is shown in the graph below it. Highly connected nodes (hubs) are lightly shaded. links per node (P[k] = k−[γ], where k is the number of links per node and γ is the slope of the log P[k] versus log[k] plot); this unique property of most biologic networks is a reflection of their self-similarity or fractal nature (Fig. 87e-1).

1	There are unique properties of scale-free biologic systems that reflect their evolution and promote their adaptability and survival. Biologic networks likely evolved one node at a time in a process in which new nodes are more likely to link to a highly connected node than to a sparsely connected node. Furthermore, scale-free networks can become sparsely linked to one another, yielding more complex, modular scale-free topologies. This evolutionary growth of biologic networks has three important properties that affect system function and survival. First, this scale-free addition of new nodes promotes system redundancy, which minimizes the consequences of errors and accommodates adverse perturbations to the system robustly with minimal effects on critical functions (unless the highly connected nodes are the focus of the perturbation). Second, this resulting network redundancy provides a survival advantage to the system. In complex gene networks, for example, mutations or polymorphisms in

1	nodes are the focus of the perturbation). Second, this resulting network redundancy provides a survival advantage to the system. In complex gene networks, for example, mutations or polymorphisms in weakly linked genes account for biodiversity and biologic variability without disrupting the critical functions of the system; only mutations in highly linked (essential) genes (hubs) can shut down the system and cause embryonic lethality. Third, scale-free biologic systems facilitate the flow of information (e.g., metabolite flux) across the system compared with randomly organized biologic systems; this so-called “small-world” property of the system (in which the clustered nature of the highly linked hubs defines a local neighborhood within the network that communicates through weaker, less frequent links to other clusters) minimizes the energy cost for the dynamic action of the system (e.g., minimizes the transition time between states in a metabolic network).

1	These basic organizing principles of complex biologic systems lead to three unique properties that require emphasis. First, biologic systems are robust, which means that they are quite stable in response to most changes in external conditions or internal modification. Second, a corollary to the property of robustness is that complex biologic systems are sloppy, which means that they are insensitive to changes in external conditions or internal modification except under certain uncommon conditions (i.e., when a hub is involved in the change). Third, complex biologic systems exhibit emergent properties, which means that they manifest behaviors that cannot be predicted from the reductionist principles used to characterize their component parts. Examples of emergent behavior in biologic systems include spontaneous, self-sustained oscillations in glycolysis; spiral and scroll waves of depolarization in cardiac tissue that cause reentrant arrhythmias; and self-organizing patterns in

1	systems include spontaneous, self-sustained oscillations in glycolysis; spiral and scroll waves of depolarization in cardiac tissue that cause reentrant arrhythmias; and self-organizing patterns in biochemical systems governed by diffusion and chemical reaction.

1	The principles of systems biology have been applied to complex pathologic processes with some early successes. The key to these applications is the identification of emergent properties of the system under study in order to define novel, otherwise unpredictable (i.e., from the reductionist perspective) methods for regulating the system’s response. Systems biology approaches have been used to characterize epidemics and ways to control them, taking advantage of the scale-free properties of the network of infected individuals that constitute the epidemic. Through the use of a systems analysis of a neural protein-protein interaction network, unique disease-modifying proteins have been identified that are common to a wide range of cerebellar neurodegenerative disorders causing inherited ataxias. Systems analysis and disease network construction of a pulmonary arterial hypertension network led to the identification of a unique disease module involving a pathway governed by microRNA21.

1	Systems analysis and disease network construction of a pulmonary arterial hypertension network led to the identification of a unique disease module involving a pathway governed by microRNA21. Systems biology models have been used to dissect the dynamics of the inflammatory response using oscillatory changes in the transcription factor nuclear factor (NF) κB as the system output. Systems biology principles also have been used to predict the development of an idiotypy–anti-idiotypy antibody network, describe the dynamics of species growth in microbial biofilms, and analyze the innate immune response. In each of these examples, a systems (patho)biology approach provided insights into the behavior of these complex systems that could not have been recognized with conventional scientific reductionism.

1	A unique application of systems biology to biomedicine is in the area of drug development. Conventional drug development involves identifying a potential target protein and then designing or screening compounds to identify those that inhibit the function of that target. This reductionist analysis has identified many potential drug targets and drugs, yet only when a drug is tested in animal models or humans are the systems consequences of the drug’s action revealed; not uncommonly, so-called off-target effects may become apparent and be sufficiently adverse for researchers to cease development of the agent. A good example of this problem is the unexpected outcomes of the vitamin B–based regimens for lowering homocysteine levels. In these trials, plasma homocysteine levels were reduced effectively; however, there was no effect of this reduction on clinical vascular endpoints. One explanation for this outcome is that one of the B vitamins in the regimen, folate, has a panoply of effects

1	however, there was no effect of this reduction on clinical vascular endpoints. One explanation for this outcome is that one of the B vitamins in the regimen, folate, has a panoply of effects on cell proliferation and metabolism that probably offset its homocysteine-lowering benefits, promoting progressive atherosclerotic plaque growth and its consequences for clinical events. In addition to these types of unexpected outcomes exerted through pathways that were not considered ab initio, conventional approaches to drug development typically do not take into consideration the possibility of emergent behaviors of the organism or the metabolic pathway or the transcriptional network of interest. Thus, a systems-based analysis of potential drugs (drug-target network analysis) can benefit the development paradigm both by enhancing the likelihood that a compound of interest will not manifest unforeseen adverse effects and by promoting novel analytic methods for identifying unique control points

1	paradigm both by enhancing the likelihood that a compound of interest will not manifest unforeseen adverse effects and by promoting novel analytic methods for identifying unique control points or pathways in metabolic or genetic networks that would benefit from drug-based modulation.

1	SYSTEMS PATHOBIOLOGY AND HuMAN DISEASE CLASSIFICATION: NETWORK MEDICINE Perhaps most important, systems pathobiology can be used to revise and refine the definition of human disease. The classification of human disease used in this and all medical textbooks derives from the correlation between pathologic analysis and clinical syndromes that began in the nineteenth century. Although this approach has been very successful, serving as the basis for the development of many effective therapies, it has major shortcomings. Those shortcomings include a lack of sensitivity in defining preclinical disease, a primary focus on overtly manifest disease, failure to recognize different and potentially differentiable causes of common late-stage pathophenotypes, and a limited ability to incorporate the growing body of molecular and genetic determinants of pathophenotype into the conventional classification scheme.

1	Two examples will illustrate the weakness of simple correlation analyses grounded in the reductionist principle of simplification (Occam’s razor) in defining human disease. Sickle cell anemia, the “classic” Mendelian disorder, is caused by a Val6Gln substitution in the β chain of hemoglobin. If conventional genetic teaching holds, this single mutation should lead to a single phenotype in patients who harbor it (genotype-phenotype correlation). This assumption is, however, false, as patients with sickle cell disease manifest a variety of pathophenotypes, including hemolytic anemia, stroke, acute chest syndrome, bony infarction, and painful crisis, as well as an overtly normal phenotype. The reasons for these different phenotypic presentations include the presence of disease-modifying genes or gene products (e.g., hemoglobin F, hemoglobin C, glucose-6-phosphate dehydrogenase), exposure to adverse environmental factors (e.g., hypoxia, dehydration), and the genetic and environmental

1	genes or gene products (e.g., hemoglobin F, hemoglobin C, glucose-6-phosphate dehydrogenase), exposure to adverse environmental factors (e.g., hypoxia, dehydration), and the genetic and environmental determinants of common intermediate pathophenotypes (i.e., variations in those generic pathologic mechanisms underlying all human disease—inflammation, thrombosis/hemorrhage, fibrosis, cell proliferation, apoptosis/necrosis, immune response).

1	A second example of note is familial pulmonary arterial hypertension. This disorder is associated with over 100 different mutations in three members of the transforming growth factor β (TGF-β) super-family: bone morphogenetic protein receptor-2 (BMPR-2), activin receptor-like kinase-1 (Alk-1), and endoglin. All these different genotypes are associated with a common pathophenotype, and each leads to that pathophenotype by molecular mechanisms that range from haploinsufficiency to dominant negative effects. As only approximately one-fourth of individuals in families that harbor these mutations manifest the pathophenotype, other disease-modifying genes (e.g., 87e-3 the serotonin receptor 5-HT2B, the serotonin transporter 5-HTT), genomic and environmental determinants of common intermediate pathophenotypes, and environmental exposures (e.g., hypoxia, infective agents [HIV], anorexigens) probably account for the incomplete penetrance of the disorder.

1	On the basis of these and many other related examples, one can approach human disease from a systems pathobiology perspective in which each “disease” can be depicted as a network that includes the following modules: the primary disease-determining elements of the genome (or proteome, if posttranslationally modified), the disease-modifying elements of the genome or proteome, environmental determinants, and genomic and environmental determinants of the generic intermediate pathophenotypes. Figure 87e-2 graphically depicts these genotype-phenotype relationships as modules for the six common disease types with specific examples for each type. Figure 87e-3 shows a network-based depiction of sickle cell disease using this kind of modular approach.

1	Goh and colleagues developed the concept of a human disease network (Fig. 87e-4) in which they used a systems approach to characterize the disease-gene associations listed in the Online Mendelian Inheritance in Man database. Their analysis showed that genes linked to similar disorders are more likely to have products that physically associate and greater similarity between their transcription profiles than do genes not associated with similar disorders. In addition, proteins associated with the same pathophenotype are significantly more likely to interact with one another than with other proteins not associated with the pathophenotype. Finally, these authors showed that the great majority of disease-associated genes are not highly connected genes (i.e., not hubs) and are typically weakly linked nodes within the functional periphery of the network in which they operate.

1	This type of analysis validates the potential importance of defining disease on the basis of its systems pathobiologic determinants. Clearly, doing this will require a more careful dissection of the molecular elements in the relevant pathways (i.e., more precise molecular pathophenotyping), less reliance on overt manifestations of disease for their classification, and an understanding of the dynamics (not just the static architecture) of the pathobiologic networks that underlie pathophenotypes defined in this way. Figure 87e-5 illustrates the elements of a molecular network within which a disease module is contained. This network is first identified by determining the interactions (physical or regulatory) among the proteins or genes that comprise it (the “interactome”). These interactions then define a topologic module within which exists functional modules (pathways) and disease modules. One approach to constructing this module is illustrated in Fig. 87e-6. Examples of the use of

1	then define a topologic module within which exists functional modules (pathways) and disease modules. One approach to constructing this module is illustrated in Fig. 87e-6. Examples of the use of this approach in defining novel determinants of disease are given in Table 87e-1.

1	Chapter 87e Network Medicine: Systems Biology in Health and Disease Hereditary ataxias Many ataxia-causing Lim et al: Cell 125:801proteins share interact-814, 2006 ing partners that affect neurodegeneration Diabetes mellitus Metabolite-protein Wang-Sattler et al: Mol network analysis links Syst Biol 8:615, 2012 three unique metabolite abnormalities in prediabetics to seven type 2 diabetes genes through four enzymes Ebstein-Barr virus infec-Viral proteome exerts its Gulbahce et al: PLoS tion effects through linking One 8:e1002531, 2012 to host interactome Pulmonary arterial Network analysis indi-Parikh et al: Circulation hypertension cates adaptive role for 125:1520-1532, 2012 microRNA 21 in suppressing rho kinase pathway 87e-4 Classsic mendelian disorder: Classic mendelian disorder: Classic mendelian disorder: Example:? Polygenic disorder: Single phenotype Example: Essential hypertension Polygenic disorder: Multiple phenotypes Example: Ischemic heart disease

1	Example:? Polygenic disorder: Single phenotype Example: Essential hypertension Polygenic disorder: Multiple phenotypes Example: Ischemic heart disease Example: Subacute bacterial endocarditis FIGuRE 87e-2 Examples of modular representations of human disease. D, secondary human disease genome or proteome; E, environmental determinants; G, primary human disease genome or proteome; I, intermediate phenotype; P, pathophenotype. (Reproduced with permission from J Loscalzo et al: Molec Syst Biol 3:124, 2007.)

1	As yet another potential consideration, one can argue that disease reflects the later-stage consequences of the predilection of an organ system to manifest a particular intermediate pathophenotype in response to injury. This paradigm reflects a reverse causality view in which a disease is defined as a tendency to heightened inflammation, thrombosis, or fibrosis after an injurious perturbation. Where the process is manifest (i.e., the organ in which it occurs) is less important than that it occurs (with the exception of the organ-specific pathophysiologic consequences that may require acute attention). For example, from this perspective, acute myocardial infarction (AMI) and its consequences are a reflection of thrombosis (in the coronary artery), inflammation (in the acutely injured myocardium), and fibrosis (at the site or sites of cardiomyocyte death). In effect, the major therapies for AMI address these intermediate pathophenotypes (e.g., antithrombotics, statins) rather than any

1	and fibrosis (at the site or sites of cardiomyocyte death). In effect, the major therapies for AMI address these intermediate pathophenotypes (e.g., antithrombotics, statins) rather than any organ-specific disease-determining process. This paradigm would argue for a systems-based analysis that would first identify the intermediate pathophenotypes to which a person is predisposed, then determine how and when to intervene to attenuate that adverse predisposition, and finally limit the likelihood that a major organ-specific event will occur. Evidence for the validity of this approach is found in the work of Rzhetsky and colleagues, who reviewed 1.5 million patient records and 161 diseases and found that these disease phenotypes form a network of strong pairwise correlations. This result is consistent with the notion that underlying genetic predispositions to intermediate pathophenotypes form the predicate basis for conventionally defined end organ diseases.

1	Regardless of the specific nature of the systems pathobiologic approach used, these analyses will lead to a drastic revision of the way human disease is defined and treated, establishing the discipline of network medicine. This will be a lengthy and complicated process but ultimately will lead to better disease prevention and therapy and probably do so from an increasingly personalized perspective. The analysis of pathobiology from a systems-based perspective is likely to help define specific subsets of patients more likely to respond to particular interventions based on shared disease mechanisms. Although it is unlikely that the extreme of “individualized medicine” will ever be practical (or even desirable), complex diseases can be mechanistically subclassified and interventions may be tailored to those settings in which they are more likely to work.

1	G5,...,GnG1 G3 G2 G4 D5,...,DnD1 D3 D2 D4 I5 ... InI1 I3 I2 I4 E5 ... EnE1 E3 E2 E4 PS5 ... PSnPS1 PS3 PS2 PS4 P5 ... PnP1 P3 P2 P4 Primary disease genome Intermediate pathophenotype Pathophenome

1	HbSTGF˜HbC˜-ThalHbFImmuneresponseInflammationHemolyticanemiaAplasticanemiaStrokePainfulcrisisDisease-modifyinggenesIntermediatephenotypesPathophenotypeG6PDApoptosis/necrosisEnvironmentaldeterminantsHypoxiaDehydrationAcutechestsyndromeBoneinfarctThrombosisInfectiveagentFIGuRE 87e-3 A. Theoretical human disease network illustrating the relationships among genetic and environmental determinants of the pathophenotypes. Key: D, secondary disease genome or proteome; E, environmental determinants; G, primary disease genome or proteome; I, intermediate phenotype; PS, pathophysiologic states leading to P, pathophenotype. B. Example of this theoretical construct applied to sickle cell disease. Key: Red, primary molecular abnormality; gray, disease-modifying genes; yellow, intermediate phenotypes; green, environmental determinants; blue, pathophenotypes. (Reproduced with permission from J Loscalzo et al: Molec Syst Biol 3:124, 2007.)

1	Chapter 87e Network Medicine: Systems Biology in Health and Disease GNAS Developmental Ear, nose, throat Endocrine FIGuRE 87e-4 A. Human disease network. Each node corresponds to a specific disorder colored by class (22 classes, shown in the key to B). The size of each node is proportional to the number of genes contributing to the disorder. Edges between disorders in the same disorder class are colored with the same (lighter) color, and edges connecting different disorder classes are colored gray, with the thickness of the edge proportional to the number of genes shared by the disorders connected by it. B. Disease gene network. Each node is a single gene, and any two genes are connected if implicated in the same disorder. In this network map, the size of each node is proportional to the number of specific disorders in which the gene is implicated. (Reproduced with permission from KI Goh et al: Proc Natl Acad Sci USA 104:8685, 2007.)

1	FIGuRE 87e-5. The elements of the interactome. The interactome includes topologic modules (genes or gene products that are closely associated with one another through direct interactions), functional modules (genes or gene products that work together to define a pathway), and disease modules (genes or gene products that interact to yield a pathophenotype). (Reproduced with permission from AL Barabasi et al: Nat Rev Genet 12:56, 2011.) i. Interactome reconstruction iii. Disease module identification iv. Pathway identification v. Validation/prediction Potential sources: (i) OMIM (ii) GWAS ii. Disease gene (seed) identification Disease1 protein Disease2 protein Overlappingprotein Known disease2 protein Predicted disease2 protein Chapter 87e Network Medicine: Systems Biology in Health and Disease

1	Chapter 87e Network Medicine: Systems Biology in Health and Disease FIGuRE 87e-6. Approaches to identifying disease modules in molecular networks. A strategy for defining disease modules involves (i) reconstructing the interactome; (ii) ascertaining potential seed (disease) genes from the curated literature, the Online Mendelian Inheritance in Man (OMIM) database, or genomic analyses (genome-wide association studies [GWAS] or transcriptional profiling); (iii) identifying the disease module using different modeling or statistical approaches; (iv) identifying pathways and the role of disease genes or modules in those pathways; and (v) disease module validation and prediction. (Reproduced with permission from AL Barabasi et al: Nat Rev Genet 12:56, 2011.) http://vip.persianss.ir

1	Stem Cell Biology Minoru S. H. Ko Stem cell biology is a rapidly expanding field that explores the charac-teristics and possible clinical applications of a variety of stem cells that serve as the progenitors of more differentiated cell types. In addition to potential therapeutic applications (Chap. 90e), patient-derived 88 PART 4: Regenerative Medicine stem cells can also be used as disease models and as a means of testing drug efficacy. Stem cells and their niche are a major focus of medical research because they play central roles in tissue and organ homeostasis and repair, which are important aspects of aging and disease.

1	IDENTIFICATION, ISOLATION, AND DERIVATION OF STEM CELLS Resident Stem Cells The definition of stem cells remains elusive. Stem cells were originally postulated as unspecified or undifferentiated cells that provide a source of renewal of skin, intestine, and blood cells throughout life. These resident stem cells have been identified in a variety of organs (e.g., epithelia of the skin and digestive system, bone marrow, blood vessels, brain, skeletal muscle, liver, testis, and pancreas) based on their specific locations, morphology, and biochemical markers.

1	Isolated Stem Cells Unequivocal identification of stem cells requires their separation and purification, usually based on a combination of specific cell-surface markers. These isolated stem cells (e.g., hematopoietic stem [HS] cells) can be studied in detail and used in clinical applications, such as bone marrow transplantation (Chap. 89e). However, the lack of specific cell-surface markers for other types of stem cells has made it difficult to isolate them in large quantities. This challenge has been partially addressed in animal models by genetically marking different cell types with green-fluorescent protein driven by cell-specific promoters. Alternatively, putative stem cells have been isolated from a variety of tissues as side population (SP) cells using fluorescence-activated cell sorting after staining with the Hoechst 33342 dye.

1	Cultured Stem Cells It is desirable to culture and expand stem cells in vitro to obtain a sufficient quantity for analysis and potential therapeutic use. Although the derivation of stem cells in vitro has been a major obstacle in stem cell biology, the number and types of cultured stem cells have increased progressively (Table 88-1). Cultured stem cells derived from resident stem cells are often called adult stem cells or somatic stem cells to distinguish them from embryonic stem (ES) and embryonic germ (EG) cells. However, considering the existence of embryo-derived, tissue-specific stem cells (e.g., trophoblast stem [TS] cells) and the possible derivation of similar cells from an embryo/fetus (e.g., neural stem [NS] cells), it is more appropriate to use the term, tissue stem cells.

1	Successful derivation of cultured stem cells (both embryonic and tissue stem cells) often requires the identification of necessary growth factors and culture conditions, mimicking the microenvironment or niche of the resident stem cells. Recently, long-term maintenance of tissue stem cells in vitro is increasingly possible by growing them as three-dimensional (3D) organoids, which contain both stem cells and niche cells (Chap. 92e). For example, intestinal stem cells can now be cultured as “epithelial mini-guts” in the presence of R-spondin, epidermal growth factor (EGF), and noggin on Matrigel. Similarly, lung stem cells can be cultured as self-renewing “alveolospheres.” A growing list of cultured stem cells, although not comprehensive, is shown in Table 88-1. Please note that the establishment of cultured stem cells is often under dispute due to the difficulties in assessing the characteristics of these cells.

1	SELF-RENEWAL AND PROLIFERATION OF STEM CELLS Symmetric and Asymmetric Cell Division The most widely accepted stem cell definition is a cell with a unique capacity to produce unaltered daughter cells (self-renewal) and to generate specialized cell types (potency). Self-renewal can be achieved in two ways. Asymmetric cell division produces one daughter cell that is identical to the parental cell and one daughter cell that is different from the parental cell and is a progenitor or differentiated cell. Asymmetric cell division does not increase the number of stem cells. Symmetric cell division produces two Embryonic stem cells (ES, ESC) Blastocysts or immunosurgically isolated inner cell mass (ICM) from blastocysts Embryonic germ cells (EG, EGC) Primordial germ cells (PGCs) from embryos at E8.5–E12.5 (m); gonadal tissues from 5–11 week postfertilization embryo/fetus (h)

1	Embryonic germ cells (EG, EGC) Primordial germ cells (PGCs) from embryos at E8.5–E12.5 (m); gonadal tissues from 5–11 week postfertilization embryo/fetus (h) Trophoblast stem cells (TS, TSC) Trophectoderm of E3.5 blastocysts, extraembryonic ectoderm of E6.5 embryos, and chorionic ectoderm of E7.5 embryos (m) Embryonal carcinoma cells (EC) Teratocarcinoma—a type of cancer that develops in the testes and ovaries (m, h) Mesenchymal stem cells (MS, MSC) Bone marrow, muscle, adipose tissue, peripheral blood, and umbilical cord blood (m, h) Multipotent adult stem cells (MAPC) Bone marrow mononuclear cells (m, h); postnatal muscle and brain (m) Spermatogonial stem cells (SS, SSC) Newborn testis (m) Germline stem cells (GS, GSC) Neonatal testis (m) Multipotent adult germline stem cells Adult testis (m) (maGSC) Neural stem cells (NS, NSC) Fetal and adult brain (subventricular zone, ventricular zone, and hippo-campus) (m, h)

1	Neural stem cells (NS, NSC) Fetal and adult brain (subventricular zone, ventricular zone, and hippo-campus) (m, h) Unrestricted somatic stem cells Mononuclear fraction of cord blood (USSC) (h) Epistem cells (EpiSC) Early postimplantation epiblast (m) Induced pluripotent stem cells (iPS, Variety of terminally differentiated iPSC) cells and tissue stem cells (m, h) Lung stem cells Lung (m, h) Amniotic fluid-derived stem (AFS) Amniotic fluid (m, h) cells Umbilical cord blood stem cells Umbilical cord (h) Adipose stem cells (AST) Fat (m, h) Cardiac stem cells Heart (m, h) Renal stem cells Renal papilla (m, h) Crypt stem cells Intestine (m, h) Colon stem cells (CoSC) Colon (m, h) Hepatic stem cells Liver (m, h) Dental pulp stem cells (DPSC) Dental pulp (m, h) Hair follicle stem cells Hair (m, h) Abbreviations: h, human; m, mouse. identical daughter cells. For stem cells to proliferate in vitro, they must divide symmetrically.

1	Abbreviations: h, human; m, mouse. identical daughter cells. For stem cells to proliferate in vitro, they must divide symmetrically. Unlimited Expansion In Vitro Resident stem cells are often quiescent and divide infrequently. However, once the stem cells are successfully cultured in vitro, they often acquire the capacity to divide continuously and the ability to proliferate beyond the normal passage limit typical of primary cultured cells (sometimes called immortality). These features are primarily seen in ES cells but have also been demonstrated for tissue stem cells, such as NS cells and mesenchymal stem (MS) cells, thereby enhancing the potential of these cells for therapeutic use (Table 88-1).

1	Stability of Genotype and Phenotype The capacity to actively proliferate is often associated with the accumulation of chromosomal abnormalities and mutations. Mouse ES cells appear to be an exception to this rule and tend to maintain their euploid karyotype and genome integrity. By contrast, human ES cells appear to be more susceptible to mutations after long-term culture. However, it is also important to note that even euploid mouse ES cells can form teratomas when injected into immunosuppressed animals, raising concerns about the possible formation of tumors after transplanting actively dividing stem cells.

1	POTENCY AND DIFFERENTIATION OF STEM CELLS Developmental Potency The term potency is used to indicate a cell’s ability to differentiate into multiple specialized cell types. The current lack of knowledge about the molecular nature of potency requires the experimental manipulation of stem cells to demonstrate their potency. For example, in vivo testing can be done by injecting stem cells into mouse blastocysts or immunosuppressed adult mice and determining how many different cell types are formed from the injected cells. However, these in vivo assays are not applicable to human stem cells. In vitro testing can be performed by differentiating cells in various culture conditions to determine how many different cell types are formed from the cells. The formal test of self-renewal and potency is performed by demonstrating that a single cell possesses such abilities in vitro (clonality). Cultured stem cells are tentatively grouped according to their potency (Fig. 88-1). Only some examples

1	is performed by demonstrating that a single cell possesses such abilities in vitro (clonality). Cultured stem cells are tentatively grouped according to their potency (Fig. 88-1). Only some examples are shown, because many cultured stem cells, especially human cells, lack definitive information about their developmental potency.

1	From Totipotency to Unipotency Totipotent cells can form an entire organism autonomously. Only a fertilized egg (zygote) possesses this feature. Pluripotent cells (e.g., ES cells) can form almost all of the body’s cell lineages (endoderm, mesoderm, and ectoderm), including germ cells. Multipotent cells (e.g., HS cells) can form multiple cell lineages FIGURE 88-1 Potency and source developmental stage of cultured stem cells. For abbreviations of stem cells, see Table 88-1. Note that stem cells are often abbreviated with or without “cells,” e.g., ES cells or ESCs for embryonic stem cells. h, human; m, mouse.

1	but cannot form all of the body’s cell lineages. Oligopotent cells (e.g., NS cells) can form more than one cell lineage but are more restricted than multipotent cells. Oligopotent cells are sometimes called progenitor cells or precursor cells; however, these terms are often more strictly used to define partially differentiated or lineage-committed cells (e.g., myeloid progenitor cells) that can divide into different cell types but lack self-renewing capacity. Unipotent cells or monopotent cells (e.g., spermatogonial stem [SS] cells) can form a single differentiated cell lineage.

1	Nuclear Reprogramming Development naturally progresses from totipotent fertilized eggs to pluripotent epiblast cells to multipotent cells and, finally, to terminally differentiated cells. According to Waddington’s epigenetic landscape, this is analogous to a ball moving down a slope. The reversal of the terminally differentiated cells to totipotent or pluripotent cells (called nuclear reprogramming) can thus be seen as an uphill gradient. Nuclear reprogramming has been achieved using nuclear transplantation, or nuclear transfer (NT), procedures (often called “cloning”), where the nucleus of a differentiated cell is transferred into an enucleated oocyte. Although this is an error-prone procedure with a very low success rate, live animals have been produced using adult somatic cells as donors in sheep, mice, and other mammals. In mice, it has been demonstrated that ES cells derived from blastocysts made by somatic cell NT are indistinguishable from normal ES cells. NT can potentially be

1	in sheep, mice, and other mammals. In mice, it has been demonstrated that ES cells derived from blastocysts made by somatic cell NT are indistinguishable from normal ES cells. NT can potentially be used to produce patient-specific ES cells carrying a genome identical to that of the patient, although such strategies have not been pursued due to ethical issues and technical challenges. Recent success in generating human ES cells by NT has rekindled an interest in this area; however, the limited supply of human oocytes will still be a major problem for clinical applications of NT.

1	An alternative approach that has become a method of choice is the direct conversion of terminally differentiated cells into ES-like cells (called induced pluripotent stem [iPS] cells) by overexpressing a combination of key transcription factors (TFs). The original method was to infect mouse embryonic fibroblast cells with retrovirus vectors carrying four TFs [Pou5f1 (Oct4), Sox2, Klf4, and Myc] and to identify rare ES-like cells in culture. This approach was soon adapted to human cells, followed by a more refined procedure (e.g., the use of fewer TFs, different cell types, and different gene-delivery methods). Because a clinical trial using iPS cells is imminent, the safety of iPS-based therapy is a major concern and a variety of measures are being taken to ensure the safety. For example, it has now become a standard to use footprint-free methods such as an episomal vector, Sendai virus vector, and synthetic mRNAs to deliver reprogramming factors into cells, resulting in the

1	example, it has now become a standard to use footprint-free methods such as an episomal vector, Sendai virus vector, and synthetic mRNAs to deliver reprogramming factors into cells, resulting in the production of patient-specific iPS cells with minimal alteration of their genetic makeup. In addition to cell replacement therapy, disease-specific iPS cells are expected to play a role in modeling human disease in vitro and in screening drugs for personalized medicine.

1	It has also become possible to convert one type of terminally differentiated cell (e.g., fibroblast cell) into another type of terminally differentiated cell (e.g., cardiac muscle, neuron, or hepatocyte) by overexpressing specific sets of TFs (called direct reprogramming). Direct reprogramming can bypass the step of making iPS cells, possibly providing the safer route to desired cell types for therapy; however, the technology is currently limited by its low efficiency.

1	Stem Cell Plasticity, Transdifferentiation, and Facultative Stem Cells The prevailing paradigm in developmental biology is that once cells are differentiated, their phenotypes are stable. However, more recent studies show that tissue stem cells, which have traditionally been thought to be lineage-committed multipotent cells, possess the capacity to differentiate into cell types outside their lineage restrictions (called transdifferentiation). For example, HS cells may be converted into neurons as well as germ cells. This feature may provide a means to use tissue stem cells derived directly from a patient for therapeutic purposes, thereby eliminating the need to use embryonic stem cells or elaborate procedures such as nuclear reprogramming of a patient’s somatic cells. However, more strict criteria and rigorous validation are required to establish tissue stem cell plasticity. For example, observations of transdifferentiation may reflect cell fusion, contamination with progenitor cells

1	criteria and rigorous validation are required to establish tissue stem cell plasticity. For example, observations of transdifferentiation may reflect cell fusion, contamination with progenitor cells from other cell lineages, or persistence of pluripotent embryonic cells in adult organs. Therefore, the assignment of potency to each cultured stem cell in Fig. 88-1 should be considered with caution. Whether transdifferentiation exists and can be used for therapeutic purposes remains to be determined conclusively. A similar, but distinct, concept is the facultative stem cell, which is defined as a unipotent cell or a terminally differentiated cell that can function as a stem cell upon tissue injury. The presence of such cells has been proposed for some organs such as liver, intestine, pancreas, and testis, but is still debated.

1	Directed Differentiation of Stem Cells Pluripotent stem cells (e.g., ES and iPS cells) can differentiate into multiple cell types, but in culture, they normally differentiate into heterogeneous cell populations in a stochastic manner. However, for therapeutic uses, it is desirable to direct stem cells into specific cell types (e.g., insulin-secreting beta cells). This is an active area of stem cell research, and protocols are being developed to achieve this goal. In any of these directed cell differentiation systems, the cell phenotype must be evaluated critically. Alternatively, the heterogeneity of the cell population derived from pluripotent stem cells can be actively exploited, as different types of cells interact with each other in culture and further enhance their own differentiation. In some instances, e.g., optic cup, self-organizing tissue morphogenesis has been demonstrated in 3D culture.

1	MOLECULAR CHARACTERIZATION OF STEM CELLS Genomics and Proteomics In addition to standard molecular biological approaches, high-throughput genomics and proteomics have been extensively applied to the analysis of stem cells. For example, DNA microarray analyses have revealed the expression levels of essentially all genes and identified specific markers for some stem cells. Chromatin immunoprecipitation coupled with next-generation sequencing technologies, capable of producing billions of sequence reads in a single run, has revealed chromatin modifications (“epigenetic marks”) relevant to stem cell properties. Similarly, the protein profiles of stem cells have been assessed by using mass spectrometry. These methods are beginning to provide a novel means to characterize and classify various stem cells and the molecular mechanisms that give them their unique characteristics.

1	ES Cell Regulation It is important to identify genes involved in the regulation of stem cell function and to examine the effects of altered gene expression on ES and other stem cells. For example, core networks of TFs such as Pou5f1 (Oct4), Nanog, and Sox2, govern key gene regulatory pathways/networks for the maintenance of self-renewal and pluripotency of mouse and human ES cells. These TF networks are modulated by specific external factors through signal transduction pathways, such as leukemia inhibitory factor (Lif)/Stat3, mitogenactivated protein kinase 1/3 (Mapk1/3), the transforming growth factor β (TGFβ) superfamily, and Wnt/glycogen synthase kinase 3 beta (Gsk3b). Inhibitors of Mapk1/3 and Gsk3b signaling enhance the derivation of ES cells and help maintain ES cells in full pluripotency (“ground” or “naive state”). Recent data also indicate that 20–25 nucleotide RNAs, called microRNAs (miRNAs), play an important role in regulating stem cell function by repressing the

1	pluripotency (“ground” or “naive state”). Recent data also indicate that 20–25 nucleotide RNAs, called microRNAs (miRNAs), play an important role in regulating stem cell function by repressing the translation of their target genes. For example, it has been shown that miR-21 regulates cell cycle progression in ES cells and miR-128 prevents the differentiation of hematopoietic progenitor cells. These types of analyses should provide molecular clues about the function of stem cells and lead to a more effective means to manipulate stem cells for future therapeutic use.

1	Hematopoietic Stem Cells would ensue. In the blood, mature cells have variable average life spans, ranging David T. Scadden, Dan L. Longo from 7 h for mature neutrophils to a few months for red blood cells to All of the cell types in the peripheral blood and some cells in every tissue of the body are derived from hematopoietic (hemo: blood; poiesis: creation) stem cells. If the hematopoietic stem cell is damaged and can no longer function (e.g., due to a nuclear accident), a person would survive 2–4 weeks in the absence of extraordinary support measures. With the clinical use of hematopoietic stem cells, tens of thousands of lives are saved each year (Chap. 139e). Stem cells produce hundreds of billions of blood cells daily from a stem cell pool that is estimated to be only in the tens of thousands. How stem cells do this, how they persist for many decades despite the production demands, and how they may be better used in clinical care are important issues in medicine.

1	The study of blood cell production has become a paradigm for how other tissues may be organized and regulated. Basic research in hematopoiesis includes defining stepwise molecular changes accompanying functional changes in maturing cells, aggregating cells into functional subgroups, and demonstrating hematopoietic stem cell regulation by a specialized microenvironment; these concepts are worked out in hematology, but they offer models for other tissues. Moreover, these concepts may not be restricted to normal tissue function but extend to malignancy. Stem cells are rare cells among a heterogeneous population of cell types, and their behavior is assessed mainly in experimental animal models involving reconstitution of hematopoiesis. Thus, much of what we know about stem cells is imprecise and based on inferences from genetically manipulated animals.

1	All stem cell types have two cardinal functions: self-renewal and differentiation (Fig. 89e-1). Stem cells exist to generate, maintain, and repair tissues. They function successfully if they can replace a wide variety of shorter-lived mature cells over prolonged periods. The process of self-renewal (see below) assures that a stem cell population can be sustained over time. Without self-renewal, the stem cell pool would become exhausted and tissue maintenance would not be possible. The process of differentiation leads to production of the effectors of tissue function: mature cells. Without proper differentiation, the integrity of

1	FIGURE 89e-1 Signature characteristics of the stem cell. Stem cells have two essential features: the capacity to differentiate into a variety of mature cell types and the capacity for self-renewal. Intrinsic factors associated with self-renewal include expression of Bmi-1, Gfi-1, PTEN, STAT5, Tel/Atv6, p21, p18, MCL-1, Mel-18, RAE28, and HoxB4. Extrinsic signals for self-renewal include Notch, Wnt, SHH, and Tie2/Ang-1. Based mainly on murine studies, hematopoietic stem cells express the following cell surface molecules: CD34, Thy-1 (CD90), c-Kit receptor (CD117), CD133, CD164, and c-Mpl (CD110, also known as the thrombopoietin receptor).

1	many years for memory lymphocytes. However, the stem cell pool is the central, durable source of all blood and immune cells, maintaining a capacity to produce a broad range of cells from a single cell source, yet keeping itself vigorous over decades of life. As an individual stem cell divides, it has the capacity to accomplish one of three division outcomes: two stem cells, two cells destined for differentiation, or one stem cell and one differentiating cell. The former two outcomes are the result of symmetric cell division, whereas the latter indicates a different outcome for the two daughter cells—an event termed asymmetric cell division. The relative balance for these types of outcomes may change during development and under particular kinds of demands on the stem cell pool.

1	During development, blood cells are produced at different sites. Initially, the yolk sac provides oxygen-carrying red blood cells, and then the placenta and several sites of intraembryonic blood cell production become involved. These intraembryonic sites engage in sequential order, moving from the genital ridge at a site where the aorta, gonadal tissue, and mesonephros are emerging to the fetal liver and then, in the second trimester, to the bone marrow and spleen. As the location of stem cells changes, the cells they produce also change. The yolk sac provides red cells expressing embryonic hemoglobins while intraembryonic sites of hematopoiesis generate red cells, platelets, and the cells of innate immunity. The production of the cells of adaptive immunity occurs when the bone marrow is colonized and the thymus forms. Stem cell proliferation remains high, even in the bone marrow, until shortly after birth, when it appears to dramatically decline. The cells in the bone marrow are

1	is colonized and the thymus forms. Stem cell proliferation remains high, even in the bone marrow, until shortly after birth, when it appears to dramatically decline. The cells in the bone marrow are thought to arrive by the bloodborne transit of cells from the fetal liver after calcification of the long bones has begun. The presence of stem cells in the circulation is not unique to a time window in development; however, hematopoietic stem cells appear to circulate throughout life. The time that cells spend freely circulating appears to be brief (measured in minutes in the mouse), but the cells that do circulate are functional and can be used for transplantation. The number of stem cells that circulate can be increased in a number of ways to facilitate harvest and transfer to the same or a different host.

1	Cells entering and exiting the bone marrow do so through a series of molecular interactions. Circulating stem cells (through CD162 and CD44) engage the lectins (carbohydrate binding proteins) Pand E-selectin on the endothelial surface to slow the movement of the cells to a rolling phenotype. Stem cell integrins are then activated and accomplish firm adhesion between the stem cell and vessel wall, with a particularly important role for stem cell VCAM-1 engaging endothelial VLA-4. The chemokine CXCL12 (SDF1) interacting with stem cell CXCR4 receptors and ionic calcium interacting with the calcium sensing receptor appear to be important in the process of stem cells getting from the circulation to where they engraft in the bone marrow. This is particularly true in the developmental move from fetal liver to bone marrow.

1	However, the role for CXCR4 in adults appears to be more related to retention of stem cells in the bone marrow rather than the process of getting them there. Interrupting that retention process through either specific molecular blockers of the CXCR4/CXCL12 interaction, cleavage of CXCL12, or downregulation of the CXCR4 receptor can all result in the release of stem cells into the circulation. This process is an increasingly important aspect of recovering stem cells for therapeutic use as it has permitted the harvesting process to be done by leukapheresis rather than bone marrow punctures in the operating room. Granulocyte colony-stimulating factor and plerixafor, a macrocyclic compound that can block CXCR4, are both used clinically to mobilize marrow hematopoietic stem cells for transplant. Refining our knowledge of how stem cells get into and out of the bone marrow may improve our ability to obtain stem cells and make them more efficient at finding their way to the specific sites for

1	Refining our knowledge of how stem cells get into and out of the bone marrow may improve our ability to obtain stem cells and make them more efficient at finding their way to the specific sites for blood cell production, the so-called stem cell niche.

1	The concept of a specialized microenvironment, or stem cell niche, was first proposed to explain why cells derived from the bone marrow of one animal could be used in transplantation and again be found in the bone marrow of the recipient. This niche is more than just a housing site for stem cells, however. It is an anatomic location where regulatory signals are provided that allow the stem cells to thrive, to expand if needed, and to provide varying amounts of descendant daughter cells. In addition, unregulated growth of stem cells may be problematic based on their undifferentiated state and self-renewal capacity. Thus, the niche must also regulate the number of stem cells produced. In this manner, the niche has the dual function of serving as a site of nurture but imposing limits for stem cells: in effect, acting as both a nutritive and constraining home.

1	The niche for blood stem cells changes with each of the sites of blood production during development, but for most of human life it is located in the bone marrow. Within the bone marrow, the perivascular space particularly in regions of trabecular bone serves as a niche. The mesenchymal and endothelial cells of the marrow microvessels produce kit ligand and CXCL12, both known to be important for hematopoietic stem cells. Other cell types, such as sympathetic neurons, nonmyelinating Schwann cells, macrophages, osteoclasts, and osteoblasts, have been shown to regulate stem cells, but it is unclear whether their effects are direct or indirect. Extracellular matrix proteins like osteopontin also affect stem cell function. The endosteal region is particularly important for transplanted cells, suggesting that there may be distinctive features of that region that are yet to be defined that are important mediators of stem cell engraftment. The functioning of the niche as a supportive context

1	suggesting that there may be distinctive features of that region that are yet to be defined that are important mediators of stem cell engraftment. The functioning of the niche as a supportive context for stem cells is of obvious importance for maintaining hematopoiesis and in transplantation. An active area of study involves determining whether the niche is altered in disease and whether drugs can modify niche function to improve transplantation or normal stem cell function in hematologic disease.

1	In the absence of disease, one never runs out of hematopoietic stem cells. Indeed, serial transplantation studies in mice suggest that sufficient stem cells are present to reconstitute several animals in succession, with each animal having normal blood cell production. The fact that allogeneic stem cell transplant recipients also never run out of blood cells in their life span, which can extend for decades, argues that even the limiting numbers of stem cells provided to them are sufficient. How stem cells respond to different conditions to increase or decrease their mature cell production remains poorly understood. Clearly, negative feedback mechanisms affect the level of production of most of the cells, leading to the normal tightly regulated blood cell counts. However, many of the regulatory mechanisms that govern production of more mature progenitor cells do not apply or apply differently to stem cells. Similarly, most of the molecules shown to be able to change the size of the

1	mechanisms that govern production of more mature progenitor cells do not apply or apply differently to stem cells. Similarly, most of the molecules shown to be able to change the size of the stem cell pool have little effect on more mature blood cells. For example, the growth factor erythropoietin, which stimulates red blood cell production from more mature precursor cells, has no effect on stem cells. Similarly, granulocyte colony-stimulating factor drives the rapid proliferation of granulocyte precursors but has little or no effect on the cell cycling of stem cells. Rather, it changes the location of stem cells by indirect means, altering molecules such as CXCL12 that tether stem cells to their niche. Molecules shown to be important for altering the proliferation, self-renewal, or survival of stem cells, such as cyclin-dependent kinase inhibitors, transcription factors like Bmi-1, or microRNA-processing enzymes like Dicer, have little or different effects on progenitor cells.

1	survival of stem cells, such as cyclin-dependent kinase inhibitors, transcription factors like Bmi-1, or microRNA-processing enzymes like Dicer, have little or different effects on progenitor cells. Hematopoietic stem cells have governing mechanisms that are distinct from the cells they generate.

1	Hematopoietic stem cells sit at the base of a branching hierarchy of cells culminating in the many mature cell types that compose the blood and immune system (Fig. 89e-2). The maturation steps leading to terminally differentiated and functional blood cells take place both as a consequence of intrinsic changes in gene expression and niche-directed and cytokine-directed changes in the cells. Our knowledge of the details remains incomplete. As stem cells mature to progenitors, precursors, and, finally, mature effector cells, they undergo a series of functional changes. These include the obvious acquisition of functions defining mature blood cells, such as phagocytic capacity or hemoglobin synthesis. They also include the progressive loss of plasticity (i.e., the ability to become other cell types). For example, the myeloid progenitor can make all cells in the myeloid series but none in the lymphoid series. As common myeloid progenitors mature, they become precursors for either monocytes

1	types). For example, the myeloid progenitor can make all cells in the myeloid series but none in the lymphoid series. As common myeloid progenitors mature, they become precursors for either monocytes and granulocytes or erythrocytes and megakaryocytes, but not both. Some amount of reversibility of this process may exist early in the differentiation cascade, but that is lost beyond a distinct stage in normal physiologic conditions. With genetic interventions, however, blood cells, like other somatic cells, can be reprogrammed to become a variety of cell types.

1	As cells differentiate, they may also lose proliferative capacity (Fig. 89e-3). Mature granulocytes are incapable of proliferation and only increase in number by increased production from precursors. The exceptions to the rule are some resident macrophages, which appear capable of proliferation, and lymphoid cells. Lymphoid cells retain the capacity to proliferate but have linked their proliferation to the recognition of particular proteins or peptides by specific antigen receptors on their surface. Like many tissues with short-lived mature cells such as the skin and intestine, blood cell proliferation is largely accomplished by a more immature progenitor population. In general, cells within the highly proliferative progenitor cell compartment are also relatively short-lived, making their way through the differentiation process in a defined molecular program involving the sequential activation of particular sets of genes. For any particular cell type, the differentiation program is

1	way through the differentiation process in a defined molecular program involving the sequential activation of particular sets of genes. For any particular cell type, the differentiation program is difficult to speed up. The time it takes for hematopoietic progenitors to become mature cells is ~10–14 days in humans, evident clinically by the interval between cytotoxic chemotherapy and blood count recovery in patients.

1	Although hematopoietic stem cells are generally thought to have the capacity to form all cells of the blood, it is becoming clear that individual stem cells may not be equal in their differentiation potential. That is, some stem cells are “biased” to become mature cells of a particular type. In addition, the general concept of cells having a binary choice of lymphoid or myeloid differentiation is not entirely accurate. A cell population with limited myeloid (monocyte and granulocyte) and lymphoid potential is now added to the commitment steps stem cells may undergo.

1	The hematopoietic stem cell must balance its three potential fates: apoptosis, self-renewal, and differentiation. The proliferation of cells is generally not associated with the ability to undergo a self-renewing division except among memory T and B cells and among stem cells. Self-renewal capacity gives way to differentiation as the only option after cell division when cells leave the stem cell compartment, until they have the opportunity to become memory lymphocytes. In addition to this self-renewing capacity, stem cells have an additional feature characterizing their proliferation machinery. Stem cells in many mature adult tissues may be heterogeneous with some being deeply quiescent, serving as a deep reserve, whereas others are more proliferative and replenish the short-lived progenitor population. In the hematopoietic system, stem cells are generally cytokine-resistant, remaining dormant even when cytokines drive bone marrow progenitors to proliferation rates measured in hours.

1	population. In the hematopoietic system, stem cells are generally cytokine-resistant, remaining dormant even when cytokines drive bone marrow progenitors to proliferation rates measured in hours. Stem cells, in contrast, are thought to divide at far longer intervals, measured in months to years, for the most quiescent cells. This quiescence is difficult to overcome in vitro, limiting the ability to effectively expand human hematopoietic stem

1	Multipotent Progenitor IKAROS PU1 IL7 IL7 IL7 IL7 IL7 IL4 IL2 IL15 IL5 FLT-3 Ligand FLT-3 Ligand M-CSF G-CSF IL3, SCF EPOEPO TPO IL3, SCF TPO GM-CSF SCF TPO TPO Hox, Pbx1, SCL, GATA2, NOTCH Common Lymphoid Progenitor B Cell Progenitor T Cell Progenitor NK Cell Progenitor Monocyte Progenitor Granulocyte Monocyte Progenitor Granulocyte Progenitor Erythrocyte Progenitor Megakaryocyte Progenitor Megakaryocyte Erythroid Progenitor Common Myeloid Progenitor T/NK Cell Progenitor LEF1, E2A, EBF, PAX-5 NOTCH1 NOTCH1 Aiolos, PAX-5, AML-1 E2A, NOTCH1, GATA3 IKAROS, NOTCH,CBF1 Id2, Ets-1 RelB, ICSBP, ld2 Egn1, Myb GATA1 C/EBP˜C/EBP°Fli-1 AML-1 GATA1, FOG NF-E2, SCL Rbtn2

1	FIGURE 89e-2 Hierarchy of hematopoietic differentiation. Stem cells are multipotent cells that are the source of all descendant cells and have the capacity to provide either long-term (measured in years) or short-term (measured in months) cell production. Progenitor cells have a more limited spectrum of cells they can produce and are generally a short-lived, highly proliferative population also known as transient amplifying cells. Precursor cells are cells committed to a single blood cell lineage but with a continued ability to proliferate; they do not have all the features of a fully mature cell. Mature cells are the terminally differentiated product of the differentiation process and are the effector cells of specific activities of the blood and immune system. Progress through the pathways is mediated by alterations in gene expression. The regulation of the differentiation by soluble factors and cell-cell communications within the bone marrow niche are still being defined. The

1	pathways is mediated by alterations in gene expression. The regulation of the differentiation by soluble factors and cell-cell communications within the bone marrow niche are still being defined. The transcription factors that characterize particular cell transitions are illustrated on the arrows; the soluble factors that contribute to the differentiation process are in blue. This picture is a simplification of the process. Active research is revealing multiple discrete cell types in the maturation of B cells and T cells and has identified cells that are biased toward one lineage or another (rather than uncommitted) in their differentiation. EPO, erythropoi- etin; RBC, red blood cell; SCF, stem cell factor; TPO, thrombopoietin.

1	cells. The process may be controlled by particularly high levels of cyclin-dependent kinase inhibitors like p57 or CDKN1c that restrict entry of stem cells into the cell cycle, blocking the G1-S transition. Exogenous signals from the niche also appear to enforce quiescence, including the activation of the tyrosine kinase receptor Tie2 on stem cells by angiopoietin 1 on niche cells.

1	The regulation of stem cell proliferation also appears to change with age. In mice, the cyclin-dependent kinase inhibitor p16INK4a accumulates in stem cells in older animals and is associated with a change in five different stem cell functions, including cell cycling. Lowering expression of p16INK4a in older animals improves stem cell cycling and capacity to reconstitute hematopoiesis in adoptive hosts, making them similar to younger animals. Mature cell numbers are unaffected. Therefore, molecular events governing the specific functions of stem cells are being gradually made clear and offer the potential of new approaches to changing stem cell function for therapy. One critical stem cell function that remains poorly defined is the molecular regulation of self-renewal.

1	For medicine, self-renewal is perhaps the most important function of stem cells because it is critical in regulating the number of stem cells. Stem cell number is a key limiting parameter for both autologous and allogeneic stem cell transplantation. Were we to have the ability to use fewer stem cells or expand limited numbers of stem cells ex vivo, it might be possible to reduce the morbidity and expense of stem cell harvests and enable use of other stem cell sources. Specifically, umbilical cord blood is a rich source of stem cells. However, the volume of cord blood units is extremely small, and therefore, the total number of hematopoietic stem cells that can be obtained in any single cord blood unit is generally only sufficient to transplant an individual of <40 kg. This limitation restricts what would otherwise be an extremely promising source of stem cells. Two features of cord blood stem cells are particularly important. (1) They are derived from a diversity of individuals that

1	what would otherwise be an extremely promising source of stem cells. Two features of cord blood stem cells are particularly important. (1) They are derived from a diversity of individuals that far exceeds the adult donor pool and therefore can overcome the majority of immunologic cross-matching obstacles. (2) Cord blood stem cells have a large number of T cells associated with them, but (paradoxically) they appear to be associated with a lower

1	FIGURE 89e-3 Relative function of cells in the hematopoietic hierarchy. The boxes represent distinct functional features of cells in the myeloid (upper box) versus lymphoid (lower box) lineages. incidence of graft-versus-host disease when compared with similarly mismatched stem cells from other sources. If stem cell expansion by self-renewal could be achieved, the number of cells available might be sufficient for use in larger adults. An alternative approach to this problem is to improve the efficiency of engraftment of donor stem cells. Graft engineering is exploring methods of adding cell components that may enhance engraftment. Furthermore, at least some data suggest that depletion of host NK (natural killer) cells may lower the number of stem cells necessary to reconstitute hematopoiesis.

1	Some limited understanding of self-renewal exists and, intriguingly, implicates gene products that are associated with the chromatin state, a high-order organization of chromosomal DNA that influences transcription. These include members of the polycomb family, a group of zinc finger–containing transcriptional regulators that interact with the chromatin structure, contributing to the accessibility of groups of genes for transcription. One member, Bmi-1, is important in enabling hematopoietic stem cell self-renewal through modification of cell cycle regulators such as the cyclin-dependent kinase inhibitors. In the absence of Bmi-1 or of the transcriptional regulator, Gfi-1, hematopoietic stem cells decline in number and function. In contrast, dysregulation of Bmi-1 has been associated with leukemia; it may promote leukemic stem cell self-renewal when it is overexpressed. Other transcription regulators have also been associated with self-renewal, particularly homeobox, or “hox,” genes.

1	leukemia; it may promote leukemic stem cell self-renewal when it is overexpressed. Other transcription regulators have also been associated with self-renewal, particularly homeobox, or “hox,” genes. These transcription factors are named for their ability to govern large numbers of genes, including those determining body patterning in invertebrates. HoxB4 is capable of inducing extensive self-renewal of stem cells through its DNA-binding motif. Other members of the hox family of genes have been noted to affect normal stem cells, but they are also associated with leukemia. External signals that may influence the relative self-renewal versus differentiation outcomes of stem cell cycling include specific Wnt ligands. Intracellular signal transducing intermediates are also implicated in regulating self-renewal. They include PTEN, an inhibitor of the AKT pathway, and STAT5, both of which are downstream of activated growth factor receptors and necessary for normal stem cell functions

1	regulating self-renewal. They include PTEN, an inhibitor of the AKT pathway, and STAT5, both of which are downstream of activated growth factor receptors and necessary for normal stem cell functions including self-renewal, at least in mouse models. The connections between these molecules remain to be defined, and their role in physiologic regulation of stem cell self-renewal is still poorly understood.

1	The relationship of stem cells to cancer is an important evolving dimension of adult stem cell biology. Cancer may share principles of organization with normal tissues. Cancer cells are heterogeneous even within a given patient and may have a hierarchical organization of cells with a base of stem-like cells capable of the signature stem cell features: self-renewal and differentiation. These stem-like cells might be the basis for perpetuation of the tumor and represent a slowly dividing, rare population with distinct regulatory mechanisms, including a relationship with a specialized microenvironment. A subpopulation of self-renewing cells has been defined for some, but not all, cancers. A more sophisticated understanding of the stem cell organization of cancers may lead to improved strategies for developing new therapies for the many common and difficult-to-treat types of malignancies that have been relatively refractory to interventions aimed at dividing cells.

1	Does the concept of cancer stem cells provide insight into the cellular origin of cancer? The fact that some cells within a cancer have stem cell–like properties does not necessarily mean that the cancer arose in the stem cell itself. Rather, more mature cells could have acquired the self-renewal characteristics of stem cells. Any single genetic event is unlikely to be sufficient to enable full transformation of a normal cell to a frankly malignant one. Rather, cancer is a multistep process, and for the multiple steps to accumulate, the cell of origin must be able to persist for prolonged periods. It must also be able to generate large numbers of daughter cells. The normal stem cell has these properties and, by virtue of its having intrinsic self-renewal capability, may be more readily converted to a malignant phenotype. This hypothesis has been tested experimentally in the hematopoietic system. Taking advantage of the cell-surface markers that distinguish hematopoietic cells of

1	converted to a malignant phenotype. This hypothesis has been tested experimentally in the hematopoietic system. Taking advantage of the cell-surface markers that distinguish hematopoietic cells of varying maturity, stem cells, progenitors, precursors, and mature cells can be isolated. Powerful transforming gene constructs were placed in these cells, and it was found that the cell with the greatest potential to produce a malignancy was dependent on the transforming gene. In some cases, it was the stem cell, but in others, the progenitor cell functioned to initiate and perpetuate the cancer. This shows that cells can acquire stem cell–like properties in malignancy.

1	WHAT ELSE CAN HEMATOPOIETIC STEM CELLS DO? Some experimental data have suggested that hematopoietic stem cells or other cells mobilized into the circulation by the same factors that mobilize hematopoietic stem cells are capable of playing a role in healing the vascular and tissue damage associated with stroke and myocardial infarction. These data are controversial, and the applicability of a stem cell approach to nonhematopoietic conditions remains experimental. However, reprogramming technology offers the potential for using the readily obtained hematopoietic stem cell as a source for cells with other capabilities.

1	The stem cell, therefore, represents a true dual-edged sword. It has tremendous healing capacity and is essential for life. Uncontrolled, it can threaten the life it maintains. Understanding how stem cells function, the signals that modify their behavior, and the tissue niches that modulate stem cell responses to injury and disease are critical for more effectively developing stem cell–based medicine. That aspect of medicine will include the use of the stem cells and the use of drugs to target stem cells to enhance repair of damaged tissues. It will also include the careful balance of interventions to control stem cells where they may be dysfunctional or malignant.

1	Applications of Stem Cell Biology in Clinical Medicine John A. Kessler Damage to an organ initiates a series of events that lead to the recon-struction of the damaged tissue, including proliferation, differentiation, 90e stem cells Undifferentiated stem cells Erythropoietin Dopaminergic neurons Erythrocytes Hematopoietic stem cells Into striatum Into heart Intravenous FIGURE 90e-1 Strategies for transplantation of stem cells. 1. Undifferentiated or partially differentiated stem cells may be injected directly into the target organ or intravenously. 2. Stem cells may be differentiated ex vivo before injection into the target organ. 3. Growth factors or other drugs may be injected to stimulate endogenous stem cell populations.

1	and migration of various cell types; release of cytokines and chemokines; and remodeling of the extracellular matrix. Endogenous stem and progenitor cells are among the cell populations that are involved in these injury responses. In normal steady-state conditions, an equilibrium is maintained in which endogenous stem cells intrinsic to the tissue replenish dying cells. After tissue injury, stem cells in organs such as the liver and skin have a remarkable ability to regenerate the organ, whereas other stem cell populations, such as those in the heart and brain, have a much more limited capability for self-repair. In rare circumstances, circulating stem cells may contribute to regenerative responses by migrating into a tissue and differentiating into organ-specific cell types. The goal of stem cell therapies is to promote cell replacement in organs that are damaged beyond their ability to self-repair.

1	At least three different therapeutic concepts for cell replacement can be envisaged (Fig. 90e-1). One therapeutic approach involves direct administration of stem cells. The cells may be injected directly into the damaged organ, where they can differentiate into the desired cell type. Alternatively, stem cells may be injected systemically since they have the capacity to home in on damaged tissues by following gradients of cytokines and chemokines released by the diseased organ. A second approach involves transplantation of differentiated cells derived from stem cells. For example, pancreatic islet cells can be generated from stem cells before transplantation into diabetic patients, 90e-1 and cardiomyocytes can be generated to treat ischemic heart disease. A third approach involves stimulation of endogenous stem cells to facilitate repair. This goal might be accomplished by administration of appropriate growth factors and drugs that amplify the number of endogenous stem/progenitor cells

1	of endogenous stem cells to facilitate repair. This goal might be accomplished by administration of appropriate growth factors and drugs that amplify the number of endogenous stem/progenitor cells and/or direct them to differentiate into the desired cell types. Therapeutic stimulation of precursor cells is already a clinical reality in the hematopoietic system, where factors such as erythropoietin, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor are used to increase production of specific blood elements. In addition to these strategies for cell replacement, a number of other approaches could involve stem cells for ex vivo or in situ generation of tissues, a process termed tissue engineering (Chap. 92e). Stem cells are also excellent candidates as vehicles for cellular gene therapy (Chap. 91e). Finally, transplanted stem cells may exert paracrine effects on damaged tissues without the differentiation and replacement of lost cells.

1	Stem cell transplantation is not a new concept but rather is already part of established medical practice. Hematopoietic stem cells (Chap. 89e) are responsible for the long-term repopulation of all blood elements in recipients of bone marrow transplants, and hematopoietic stem cell transplantation is the gold standard against which other stem cell transplantation therapies will be measured. Transplantation of differentiated cells is also a clinical reality, and donated organs and tissues are often used to replace damaged tissues. However, the need for transplantable tissues and organs far outweighs the available supply, and organ transplantation has limited potential for some tissues, such as the brain. Stem cells offer the possibility of a renewable source of replacement cells for virtually all organs.

1	A variety of different types of stem cells (Chap. 88) could be used in regenerative strategies, including embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, umbilical-cord blood stem cells (USCs), organ-specific somatic stem cells (e.g., neural stem cells for treatment of the brain), and somatic stem cells that generate cell types specific for the target organ rather than the donor organ (e.g., bone marrow mesenchymal stem cells or CD34+ hematopoietic stem cells for cardiac repair). Although each cell type has potential advantages and disadvantages, there are a number of generic problems in developing any of these cell types into a useful and reliable clinical tool.

1	Embryonic Stem Cells Embryonic stem cells have the potential to generate all the cell types in the body; thus, in theory, there are no restrictions on the organs that could be regenerated. ES cells can self-renew endlessly, so that a single cell line with carefully characterized traits potentially could generate almost limitless numbers of cells. In the absence of moral or ethical constraints (see “Ethical Issues,” below), unused human blastocysts from fertility clinics could be used to derive new ES cell lines that are matched immunologically with potential transplant recipients. Alternatively, somatic cell nuclear transfer (“therapeutic cloning”) could be used to create ES cell lines that are genetically identical to those of the patient, although this endeavor has been technically refractory for human cells. However, human ES cells are difficult to culture and grow slowly. Techniques for differentiating them into specific cell types are just beginning

1	CHAPTER 90e Applications of Stem Cell Biology in Clinical Medicine to be developed. Cells tend to develop abnormal karyotypes and other abnormalities with increased time in culture, and ES cells have the potential to form teratomas if all cells are not committed to the desired cell types before transplantation. Further, human ES cells are ethically controversial and, on these grounds, would be unacceptable to some patients and physicians despite their therapeutic potential. Nevertheless, there have been limited clinical trials of ES-derived cells in a number of disorders, including macular degeneration, myopia, and spinal cord injury.

1	Induced Pluripotent Stem Cells The field of stem cell biology was transformed by the discovery that adult somatic cells can be converted (“reprogrammed”) into pluripotent cells through the overexpression of four transcription factors normally expressed in pluripotent cells (Chap. 88). These iPS cells share most properties with ES cells, although there are distinct differences in gene expression between ES and iPS cells. The initial use of viruses to insert the transcription factors into somatic cells made the resulting cells unsuitable for clinical use. However, a number of strategies have since been developed to circumvent this problem, including the insertion of modified mRNAs, proteins, or microRNAs rather than cDNAs; the use of non-integrating viruses such as Sendai virus; the insertion of transposons with the programming factors, followed by their subsequent removal; and the use of floxed viral constructs, followed by treatment with Cre recombinase to excise those constructs. The

1	of transposons with the programming factors, followed by their subsequent removal; and the use of floxed viral constructs, followed by treatment with Cre recombinase to excise those constructs. The safety of iPS cells in humans remains to be demonstrated, but clinical trials in macular degeneration and other disorders are planned. Potential advantages of iPS cells are that somatic cells from patients would generate pluripotent cells genetically identical to those of the patient and that these cells are not subject to the same ethical constraints as ES cells. It is not clear whether the differences in gene expression between ES and iPS cells will have any impact on their potential clinical utility, and studies of both cell types will be essential to resolve this issue.

1	Umbilical-Cord Stem Cells Umbilical-cord blood stem/progenitor cells (USCs) are widely and readily available. These cells appear to be associated with less graft-versus-host disease than are some other cell types, such as marrow stem cells. They have less human leukocyte antigen restriction than adult marrow stem cells and are less likely to be contaminated with herpesvirus. However, it is unclear how many different cell types can be generated from USCs, and methods for differentiating these cells into nonhematopoietic phenotypes are largely lacking. Nevertheless, there are ongoing clinical trials of these cells in dozens of disorders, including cirrhosis, cardiopathies, multiple sclerosis, burns, stroke, autism, and critical limb ischemia.

1	Organ-Specific Multipotent Stem Cells Organ-specific multipotent stem cells have the advantage of already being somewhat specialized so that the inducement of desired cell types may be easier. Cells potentially could be obtained from the patient and amplified in cell culture, circumventing the problems associated with immune rejection. Stem cells are relatively easy to harvest from some tissues, such as bone marrow and blood, but are difficult to harvest from other tissues, such as heart and brain. Moreover, these populations of cells are more limited in potentiality than are pluripotent ES or iPS cells, and they may be difficult to obtain in large quantities from many organs. Therefore, substantial efforts have been devoted to developing techniques for using more easily obtainable stem cell populations, such as bone marrow mesenchymal stem cells (MSCs), CD34+ hematopoietic stem cells (HSCs), cardiac mesenchymal cells, and adipose-derived stem cells (ASCs), for use in regenerative

1	cell populations, such as bone marrow mesenchymal stem cells (MSCs), CD34+ hematopoietic stem cells (HSCs), cardiac mesenchymal cells, and adipose-derived stem cells (ASCs), for use in regenerative strategies. Tissue culture evidence suggests that these stem cell populations may be able to generate differentiated cell types unrelated to their organ source (including myocytes, chondrocytes, tendon cells, osteoblasts, cardiomyocytes, adipocytes, hepatocytes, and neurons) in a process known as transdifferentiation. However, it is still unclear whether these stem cells are capable of generating differentiated cell types that integrate into organs, survive, and function after transplantation in vivo. A number of early studies of MSCs transplanted into heart, liver, and other organs suggested that the cells had differentiated into organ-specific cell types with beneficial effects in animal models of disease. Unfortunately, subsequent studies revealed that the stem cells had simply fused

1	that the cells had differentiated into organ-specific cell types with beneficial effects in animal models of disease. Unfortunately, subsequent studies revealed that the stem cells had simply fused with cells resident in the organs and that the observed beneficial effects were due to paracrine release of trophic and anti-inflammatory cytokines. Further studies will be necessary to determine whether transdifferentiation of MSCs, ASCs, or other stem cell populations occurs at a high enough frequency to make these cells useful for stem cell replacement therapy. Despite the remaining issues, clinical trials of MSCs, autologous HSCs, USCs, and ASCs are being performed in many disorders, including ischemic cardiac disease, cardiomyopathy, diabetes, stroke, cirrhosis, and muscular dystrophy.

1	Regardless of the source of the stem cells used in regenerative strategies, a number of generic problems must be overcome for the development of successful clinical applications. These problems include the devising of methods to reliably generate large numbers of specific cell types, to minimize the risk of tumor formation or proliferation of inappropriate cell types, to ensure the viability and function of the engrafted cells, to overcome immune rejection when autografts are not used, and to facilitate revascularization of regenerated tissue. Each organ system will also pose tissue-specific problems for stem cell therapies.

1	DISEASE-SPECIFIC APPLICATIONS OF STEM CELLS Ischemic Heart Disease and Cardiomyocyte Regeneration Because of the high prevalence of ischemic heart disease, extensive efforts have been devoted to the development of strategies for stem cell replacement of cardiomyocytes. Historically, the adult heart has been viewed as a terminally differentiated organ without the capacity for regeneration. However, recent studies have demonstrated that the heart has the capacity for low levels of cardiomyocyte regeneration (Chap. 265e). This regeneration appears to be accomplished by cardiac stem cells resident in the heart and possibly also by cells originating in the bone marrow. The heart might be an ideal source of stem cells for therapeutic use, but techniques for isolating, characterizing, and amplifying large numbers of these cells have not yet been perfected. For successful myocardial repair, stem cell therapy must deliver cells either systemically or locally, and the cells must survive,

1	amplifying large numbers of these cells have not yet been perfected. For successful myocardial repair, stem cell therapy must deliver cells either systemically or locally, and the cells must survive, engraft, and differentiate into functional cardiomyocytes that couple mechanically and electrically with the recipient myocardium. The optimal method for cell delivery is not clear, and various experimental and clinical studies have successfully employed intramyocardial, transendocardial, intravenous, intracoronary, and retrograde coronary venous injections. In experimental myocardial infarction, functional improvements have been achieved after transplantation of a variety of different cell types, including ES cells, HSCs, MSCs, USCs, and ASCs. Early studies suggested that each of these cell types might have the potential to engraft and generate cardiomyocytes. However, most investigators have found that the generation of new cardiomyocytes by these cells is at best a rare event and that

1	types might have the potential to engraft and generate cardiomyocytes. However, most investigators have found that the generation of new cardiomyocytes by these cells is at best a rare event and that graft survival over long periods is poor. The preponderance of evidence suggests that the observed beneficial effects of most experimental therapies were not derived from direct stem cell generation of cardiomyocytes but rather from indirect effects of the stem cells on resident cells. It is not clear whether these effects reflect the release of soluble trophic factors, the induction of angiogenesis, the release of anti-inflammatory cytokines, or another mechanism. A wide variety of cell delivery methods, cell types, and cell doses have been used in a progressively enlarging series of clinical trials, but the fate of the cells and the mechanisms by which they alter cardiac function are still open questions. In aggregate, however, these studies have shown a small but measurable improvement

1	trials, but the fate of the cells and the mechanisms by which they alter cardiac function are still open questions. In aggregate, however, these studies have shown a small but measurable improvement in cardiac function and, in some cases, reduction in infarct size. In short, the available evidence suggests that the beneficial clinical impact reflects an indirect effect of the transplanted cells rather than genuine cell replacement.

1	Diabetes Successes with islet cell and pancreas transplantation have provided proof of concept for cell-based therapies for type 1 diabetes. However, the demand for donor pancreases far exceeds the number available, and maintenance of long-term graft survival is a problem. The search for a renewable source of stem cells capable of regenerating pancreatic islets has therefore been intensive. Pancreatic beta cell turnover occurs even in the normal pancreas, although the source of the new beta cells remains controversial. This persistent turnover suggests that, in principle, it should be possible to develop strategies for reconstituting the beta cell population in diabetics. Attempts to devise techniques for promoting endogenous regenerative processes by using combinations of growth factors, drugs, and gene therapy have failed thus far, but this remains a potentially viable approach. A number of different cell types are candidates for use in stem cell replacement strategies, including

1	drugs, and gene therapy have failed thus far, but this remains a potentially viable approach. A number of different cell types are candidates for use in stem cell replacement strategies, including iPS cells, ES cells, hepatic progenitor cells, pancreatic ductal progenitor cells, and MSCs. Successful therapy will depend on the development of a source of cells that can be amplified to produce large numbers of progeny with the ability to synthesize, store, and release insulin when it is required, primarily in response to changes in the ambient level of glucose. The proliferative capacity of the replacement cells must be tightly regulated to avoid excessive expansion of beta cell numbers and the consequent development of hyperinsulinemia/hypoglycemia; moreover, the cells must withstand immune rejection. Although it has been reported that ES and iPS cells can be differentiated into cells that produce insulin, these cells have a low content of insulin and a high rate of apoptosis and

1	immune rejection. Although it has been reported that ES and iPS cells can be differentiated into cells that produce insulin, these cells have a low content of insulin and a high rate of apoptosis and generally lack the capacity to normalize blood glucose levels in diabetic animals. Thus, ES and iPS cells have not yet been useful for the large-scale production of differentiated islet cells. During embryogenesis, the pancreas, liver, and gastrointestinal tract are all derived from the anterior endoderm, and transdifferentiation of pancreas to liver and vice versa has been observed in a number of pathologic conditions. There is also substantial evidence that multipotential stem cells reside within gastric glands and intestinal crypts. These observations suggest that hepatic, pancreatic, and/or gastrointestinal precursor cells may be reasonable candidates for cell-based therapy for diabetes, although it is unclear whether insulin-producing cells derived from pancreatic stem cells or liver

1	gastrointestinal precursor cells may be reasonable candidates for cell-based therapy for diabetes, although it is unclear whether insulin-producing cells derived from pancreatic stem cells or liver progenitors can be expanded in vitro to clinically useful numbers. MSCs and neural stem cells both reportedly have the capacity to generate insulin-producing cells, but there is no convincing evidence that either cell type will be clinically useful. Clinical trials of MSCs, USCs, HSCs, and ASCs in both type 1 and type 2 diabetes are ongoing.

1	Nervous System Substantial progress has been made in the development of methodologies for generating neural cells from different stem cell populations. Human ES or iPS cells can be induced to generate cells with the properties of neural stem cells, and these cells in turn give rise to neurons, oligodendroglia, and astrocytes. Reasonably large numbers of these cells can be transplanted into the rodent brain with formation of appropriate cell types and no tumor formation. Multipotent stem cells present in the adult brain also can be easily amplified in number and used to generate all the major neural cell types, but the need for invasive procedures to obtain autologous cells is a major limitation. Fetal neural stem cells derived from miscarriages or abortions are an alternative but raise ethical concerns. Nevertheless, clinical trials of fetal neural stem cells have commenced in amyotrophic lateral sclerosis (ALS), stroke, and several other disorders. Transdifferentiation of MSCs and

1	ethical concerns. Nevertheless, clinical trials of fetal neural stem cells have commenced in amyotrophic lateral sclerosis (ALS), stroke, and several other disorders. Transdifferentiation of MSCs and ASCs into neural stem cells, and vice versa, has been reported by numerous investigators, and clinical trials of such cells have begun for a number of neurologic diseases. Clinical trials of a conditionally immortalized human cell line and of USCs in stroke are also in progress. Because of the incapacitating nature of neural disorders and the limited endogenous repair capacity of the nervous system, clinical trials of stem cells in neurologic disorders have been particularly numerous, including trials in spinal cord injury, multiple sclerosis, epilepsy, Alzheimer’s disease, ALS, acute and chronic stroke, numerous genetic disorders, traumatic brain injury, Parkinson’s disease, and others. In diseases such as ALS, possible benefits are more likely to be due to indirect trophic effects than

1	stroke, numerous genetic disorders, traumatic brain injury, Parkinson’s disease, and others. In diseases such as ALS, possible benefits are more likely to be due to indirect trophic effects than to neuron replacement. In Parkinson’s disease, the major motor features of the disorder result from the loss of a single cell population: dopaminergic neurons within the substantia nigra; this circumstance suggests that cell replacement should be relatively straightforward. However, two clinical trials of fetal nigral transplantation failed to meet their primary endpoint and were complicated by the development of dyskinesia. Transplantation of stem cell–derived dopamine-producing cells offers a number of 90e-3 potential advantages over the fetal transplants, including the ability of stem cells to migrate and disperse within tissue, the potential for engineering regulatable release of dopamine, and the ability to engineer cells to produce factors that will enhance cell survival. Nevertheless,

1	migrate and disperse within tissue, the potential for engineering regulatable release of dopamine, and the ability to engineer cells to produce factors that will enhance cell survival. Nevertheless, the experience with fetal transplants points out the difficulties that may be encountered.

1	At least some of the neurologic dysfunction after spinal cord injury reflects demyelination, and both ES cells and MSCs can facilitate remyelination after experimental spinal cord injury (SCI). Clinical trials of MSCs in this disorder have commenced in a number of countries, and SCI was the first disorder targeted for the clinical use of ES cells. However, the ES cell trial in SCI was terminated early for nonmedical reasons. At present, no population of transplanted stem cells has been shown to have the capacity to generate neurons that extend axons over long distances to form synaptic connections (as would be necessary for replacement of upper motor neurons in ALS, stroke, or other disorders). For many injuries, including SCI, the balance between scar formation and tissue repair/regeneration may prove to be an important consideration. For example, it may ultimately prove necessary to limit scar formation so that axons can reestablish connections.

1	Liver Liver transplantation is currently the only successful treatment for end-stage liver diseases, but the shortage of liver grafts limits its application. Clinical trials of hepatocyte transplantation demonstrate its potential as a substitute for organ transplantation, but this approach is limited by the paucity of available cells. Potential sources of stem cells for regenerative strategies include endogenous liver stem cells (such as oval cells), ES cells, MSCs, and USCs. Although a series of studies in humans as well as animals suggested that transplanted MSCs and HSCs can generate hepatocytes, fusion of the transplanted cells with endogenous liver cells, giving the erroneous appearance of new hepatocytes, appears to be the underlying event in most circumstances. The available evidence suggests that transplanted HSCs and MSCs can generate hepatocyte-like cells in the liver only at a very low frequency, but there are beneficial consequences presumably related to indirect paracrine

1	suggests that transplanted HSCs and MSCs can generate hepatocyte-like cells in the liver only at a very low frequency, but there are beneficial consequences presumably related to indirect paracrine effects. ES cells can be differentiated into hepatocytes and transplanted in animal models of liver failure without the formation of teratomas. Clinical trials are in progress in cirrhosis with numerous cell types, including MSCs, USCs, HSCs, and ASCs.

1	Other Organ Systems and the Future The use of stem cells in regenerative strategies has been studied for many other organ systems and cell types, including skin, eye, cartilage, bone, kidney, lung, endometrium, vascular endothelium, smooth muscle, and striated muscle, and clinical trials in these and other organs are ongoing. In fact, the potential for stem cell regeneration of damaged organs and tissues is virtually limitless. However, numerous obstacles must be overcome before stem cell therapies can become a widespread clinical reality. Only HSCs have been adequately characterized by surface markers so that they can be unambiguously identified, a prerequisite for reliable clinical applications. The pathways for differentiating stem cells into specific cellular phenotypes are largely unknown, and the ability to control the migration of transplanted cells or predict the response of the cells to the environment of diseased organs is presently limited. Some strategies may employ the

1	unknown, and the ability to control the migration of transplanted cells or predict the response of the cells to the environment of diseased organs is presently limited. Some strategies may employ the coadministration of scaffolding, artificial extracellular matrix, and/or growth factors to orchestrate differentiation of stem cells and their organization into appropriate constituents of the organ. There is currently no way to image stem cells in vivo after transplantation into humans, and it will be necessary to develop techniques to do so. Fortunately, stem cells can be engineered before transplantation to contain a contrast agent that may make their in vivo imaging feasible. The potential for tumor formation and the problems associated with immune rejection are impediments, and it will also be necessary to develop techniques for ensuring vascularization of regenerated tissues. There already are many strategies for supporting cell replacement, including coadministration of vasoactive

1	be necessary to develop techniques for ensuring vascularization of regenerated tissues. There already are many strategies for supporting cell replacement, including coadministration of vasoactive endothelial growth factor to foster vascularization of the transplant. Some strategies also include the genetic engineering of stem cells with an inducible suicide gene so that the cells can be easily eradicated in

1	CHAPTER 90e Applications of Stem Cell Biology in Clinical Medicine the event of tumor formation or another complication. The potential for stem cell therapies to revolutionize medical care is extraordinary, and disorders such as myocardial infarction, diabetes, and Parkinson’s disease, among many others, are potentially curable by such therapies. However, stem cell–based therapies are still at a very early stage of development, and perfection of techniques for clinical transplantation of predictable, well-characterized cells is going to be a difficult and lengthy undertaking.

1	Stem cell therapies raise ethical and socially contentious issues that must be addressed in parallel with the scientific and medical opportunities. Society has great diversity with respect to religious beliefs, concepts of individual rights, tolerance for uncertainty and risk, and boundaries for how scientific interventions should be used to alter the outcome of disease. In the United States, the federal government has authorized research using existing human ES cell lines but still restricts the use of federal funds for developing new human ES cell lines. Ongoing studies of existing lines have indicated that they develop abnormalities with time in culture and that they may be contaminated with mouse proteins. These findings highlight the need to develop new human ES cell lines. The development of iPS cell technology may lessen the need for deriving new ES cell lines, but it is still not clear whether the differences in gene expression by ES and iPS cells are important for potential

1	of iPS cell technology may lessen the need for deriving new ES cell lines, but it is still not clear whether the differences in gene expression by ES and iPS cells are important for potential clinical use.

1	In considering ethical issues associated with the use of stem cells, it is helpful to draw from experience with other scientific advances, such as organ transplantation, recombinant DNA technology, implantation of mechanical devices, neuroscience and cognitive research, in vitro fertilization, and prenatal genetic testing. These and other precedents have pointed to the importance of understanding and testing fundamental biology in the laboratory setting and in animal models before applying new techniques in carefully controlled clinical trials. When these trials occur, they must include full informed consent and careful oversight by external review groups.

1	Ultimately, there will be medical interventions that are scientifically feasible but ethically or socially unacceptable to some members of a society. Stem cell research raises fundamentally difficult questions about the definition of human life, and it has raised deep fears about the ability to balance issues of justice and safety with the needs of critically ill patients. Health care providers and experts with backgrounds in ethics, law, and sociology must help guard against the premature or inappropriate application of stem cell therapies and the inappropriate involvement of vulnerable population groups. However, these therapies offer important new strategies for the treatment of otherwise irreversible disorders. An open dialogue among the scientific community, physicians, patients and their advocates, lawmakers, and the lay population is critically important to raise and address important ethical issues and balance the benefits and risks associated with stem cell transfer.

1	by many factors, including an intense desire to develop therapies for 91e-1 Gene Therapy in Clinical Medicine hitherto untreatable diseases, lack of understanding of risks, and, in some cases, undisclosed financial conflicts of interest. After a teenager Katherine A. High died of complications related to vector infusion, the field underwent a

1	Gene transfer is a novel area of therapeutics in which the active agent is a nucleic acid sequence rather than a protein or small molecule. Because delivery of naked DNA or RNA to a cell is an inefficient process, most gene transfer is carried out using a vector, or gene delivery vehicle. These vehicles have generally been engineered from viruses by deleting some or all of the viral genome and replacing it with the therapeutic gene of interest under the control of a suitable promoter (Table 91e-1). Gene transfer strategies can thus be described in terms of three essential elements: (1) a vector; (2) a gene to be delivered, sometimes called the transgene; and (3) a physiologically relevant target cell to which the DNA or RNA is delivered. The series of steps in which the vector and donated DNA enter the target cell and express the transgene is referred to as transduction. Gene delivery can take place in vivo, in which the vector is directly injected into the patient, or, in the case of

1	DNA enter the target cell and express the transgene is referred to as transduction. Gene delivery can take place in vivo, in which the vector is directly injected into the patient, or, in the case of hematopoietic and some other target cells, ex vivo, with removal of the target cells from the patient, followed by return of the gene-modified autologous cells to the patient after manipulation in the laboratory. The latter approach effectively combines gene transfer techniques with cellular therapies (Chap. 90e).

1	Gene transfer is one of the most powerful concepts in modern molecular medicine and has the potential to address a host of diseases for which there are currently no available treatments. Clinical trials of gene therapy have been under way since 1990; a recent landmark in the field was the licensing, in 2012, of the first gene therapy product approved in Europe or the United States (see below). Given that vector-mediated gene therapy is arguably one of the most complex therapeutics yet developed, consisting of both a nucleic acid and a protein component, this time course from first clinical trial to licensed product is noteworthy for being similar to that seen with other novel classes of therapeutics, including monoclonal antibodies or bone marrow transplantation. Over 5000 subjects have been enrolled in gene transfer studies, and serious adverse events have been rare. Some of the initial trials were characterized by an overabundance of optimism and a failure to be appropriately

1	have been enrolled in gene transfer studies, and serious adverse events have been rare. Some of the initial trials were characterized by an overabundance of optimism and a failure to be appropriately critical of preclinical studies in animals; in addition, it was in some contexts not fully appreciated that animal studies are only a partial guide to safety profiles of products in humans (e.g., insertional mutagenesis). Initial exuberance was driven retrenchment; continued efforts led to a more nuanced understanding of the risks and benefits of these new therapies and more sophisticated selection of disease targets. Currently, gene therapies are being developed for a wide variety of disease entities (Fig. 91e-1).

1	Gene transfer strategies for genetic disease generally involve gene addition therapy, an approach characterized by transfer of the missing gene to a physiologically relevant target cell. However, other strategies are possible, including supplying a gene that achieves a similar biologic effect through an alternative pathway (e.g., factor VIIa for hemophilia A); supplying an antisense oligonucleotide to splice out a mutant exon if the sequence is not critical to the function of the protein (as has been done with the dystrophin gene in Duchenne’s muscular dystrophy); or downregulating a harmful effect through a small interfering RNA (siRNA). Two distinct strategies are used to achieve long-term gene expression: one is to transduce stem cells with an integrating vector, so that all progeny cells will carry the donated gene; and the other is to transduce long-lived cells, such as skeletal muscle or neurons. In the case of long-lived cells, integration into the target cell genome is

1	cells will carry the donated gene; and the other is to transduce long-lived cells, such as skeletal muscle or neurons. In the case of long-lived cells, integration into the target cell genome is unnecessary. Instead, because the cells are nondividing, the donated DNA, if stabilized in an episomal form, will give rise to expression for the life of the cell. This approach thus avoids problems related to integration and insertional mutagenesis.

1	IMMUNODEFICIENCY DISORDERS: PROOF OF PRINCIPLE Early attempts to effect gene replacement into hematopoietic stem cells (HSCs) were stymied by the relatively low transduction efficiency of retroviral vectors, which require dividing target cells for integration. Because HSCs are normally quiescent, they are a formidable transduction target. However, identification of cytokines that induced cell division without promoting differentiation of stem cells, along with technical improvements in the isolation and transduction of HSCs, led to modest but real gains in transduction efficiency. The first convincing therapeutic effect from gene transfer occurred with X-linked severe combined immunodeficiency disease (SCID), which results from mutations in the gene (IL2RG) encoding the γc subunit of cytokine receptors required for normal development of Immune responses to vector Theoretical risk of insertional mutagenesis (occurred in multiple cases)

1	Immune responses to vector Theoretical risk of insertional mutagenesis (occurred in multiple cases) Elicits few inflammatory responses, nonpathogenic 8.5 kb In need of a stable packaging system DNA RNA Large packaging Limited immune capacity with responses against persistent gene the vector transfer Residual cytotox-Transduced gene icity with neuron expression is specificity transient Abbreviations: AAV, adeno-associated virus; HSV, herpes simplex virus; SV, sarcoma virus. vector, so that errant clones can be quickly ablated, or using “insulator” elements in the cassette, which can limit the activation of genes surrounding the insertion site. Lentiviral vectors, which can efficiently transduce nondividing target cells, are also likely to be safer than retroviral vectors, based on patterns of integration; the field is thus gradually moving toward these to replace retroviral vectors.

1	More clear-cut success has been achieved in a gene therapy trial for another form of SCID, adenosine deaminase (ADA) deficiency (Chap. 374). ADASCID is clinically similar to X-linked SCID, although it can be treated by enzyme replacement therapy with a pegylated form of the enzyme (PEG-ADA), which leads to immune reconstitution but not always to normal T cell counts. Enzyme replacement therapy is expensive (annual costs: $200,000–$300,000 in U.S. dollars). The initial trials of gene therapy for

1	U.S. dollars). The initial trials of gene therapy for Number of trials (year 2013) ADA-SCID were unsuccessful, but modifications of FIGURE 91e-1 Indications in gene therapy clinical trials. The bar graph classifies this protocol to include the use of HSCs rather than clinical gene transfer studies by disease. A majority of trials have addressed cancer, T cells as the target for transduction; discontinuation with monogenic disorders, infectious diseases, and cardiovascular diseases the next of PEG-ADA at the time of vector infusion, so that the largest categories. (Adapted from SL Ginn et al: J Gene Med 15:65-77, 2013. Published transduced cells have a proliferative advantage over online in Wiley Online.)

1	T and natural killer (NK) cells (Chap. 374). Affected infants present in the first few months of life with overwhelming infections and/or failure to thrive. In this disorder, it was recognized that the transduced cells, even if few in number, would have a proliferative advantage compared to the nontransduced cells, which lack receptors for the cytokines required for lymphocyte development and maturation. Complete reconstitution of the immune system, including documented responses to standard childhood vaccinations, clearing of infections, and remarkable gains in growth occurred in most of the treated children. However, among 20 children treated in two separate trials, five eventually developed a syndrome similar to T cell acute lymphocytic leukemia, with splenomegaly, rising white counts, and the emergence of a single clone of T cells. Molecular studies revealed that, in most of these children, the retroviral vector had integrated within a gene, LMO-2 (LIM only-2), which encodes a

1	and the emergence of a single clone of T cells. Molecular studies revealed that, in most of these children, the retroviral vector had integrated within a gene, LMO-2 (LIM only-2), which encodes a component of a transcription factor complex involved in hematopoietic development. The retroviral long terminal repeat increases the expression of LMO-2, resulting in T cell leukemia.

1	The X-linked SCID studies were a watershed event in the evolution of gene therapy. They demonstrated conclusively that gene therapy could cure disease; of the 20 children eventually treated in these trials, 18 achieved correction of the immunodeficiency disorder. Unfortunately, 5 of the 20 patients later developed a leukemia-like disorder, and one died of this complication; the rest are alive and free of complications at time periods ranging up to 14 years after initial treatment. These studies demonstrated that insertional mutagenesis leading to cancer was more than a theoretical possibility (Table 91e-2). As a result of the experience in these trials, all protocols using integrating vectors in hematopoietic cells must include a plan for monitoring sites of insertion and clonal proliferation. Strategies to overcome this complication have included using a “suicide” gene cassette in the Gene silencing – repression of promoter

1	Gene silencing – repression of promoter Phenotoxicity – complications arising from overexpression or ectopic expression of the transgene Immunotoxicity – harmful immune response to either the vector or transgene Risks of horizontal transmission – shedding of infectious vector into environment

1	Risks of horizontal transmission – shedding of infectious vector into environment Risks of vertical transmission – germline transmission of donated DNA the nontransduced; and the use of a mild conditioning regimen to facilitate engraftment of the transduced cells have led to success without the complications seen in the X-linked SCID trials. There have been no complications in the 10 children treated on the Milan protocol, with a median follow-up of >8 years. ADA-SCID, then, is an example where gene therapy has changed therapeutic options for patients. For those with a human leukocyte antigen (HLA)-identical sibling, bone marrow transplantation is still the best treatment option, but this includes only a minority of those affected. For those without an HLA-identical match, gene therapy has comparable efficacy to PEG-ADA, does not require repetitive injections, and does not run the risk of neutralizing antibodies to the bovine enzyme.

1	NEURODEGENERATIVE DISEASES: EXTENSION OF PRINCIPLE

1	The SCID trials gave support to the hypothesis that gene transfer into HSCs could be used to treat any disease for which allogeneic bone marrow transplantation was therapeutic. Moreover, the use of genetically modified autologous cells carried several advantages including no risk of graft-versus-host disease, guaranteed availability of a “donor” (unless the disease itself damages the stem cell population of the patient), and low likelihood of failure of engraftment. Cartier and Aubourg capitalized on this realization to conduct the first trial of lentiviral vector transduction of HSCs for a neurodegenerative disorder, X-linked adrenoleukodystrophy (ALD). X-linked ALD is a fatal demyelinating disease of the central nervous system caused by mutations in the gene encoding an adenosine triphosphate–binding cassette transporter. Deficiency of this protein leads to accumulation of verylong-chain fatty acids in oligodendrocytes and microglia, disrupting myelin maintenance by these cells.

1	cassette transporter. Deficiency of this protein leads to accumulation of verylong-chain fatty acids in oligodendrocytes and microglia, disrupting myelin maintenance by these cells. Affected boys present with clinical and neuroradiographic evidence of disease at age 6–8 and usually die before adolescence. Following lentiviral transduction of autologous HSCs in young boys with the disease, dramatic stabilization of disease occurred, demonstrating that stem cell transduction could work for neurodegenerative as well as immunologic disorders. Investigators in Milan carried this observation one step further to develop a treatment for another neurodegenerative disorder that has previously responded poorly to bone marrow transplantation. Metachromatic leukodystrophy is a lysosomal storage disorder caused by mutations in the gene encoding arylsulfatase A (ARSA). The late infantile form of the disease is characterized by progressive motor and cognitive impairment, and death within a few years

1	caused by mutations in the gene encoding arylsulfatase A (ARSA). The late infantile form of the disease is characterized by progressive motor and cognitive impairment, and death within a few years of onset, due to accumulation of the ARSA substrate sulfatide in oligodendrocytes, microglia, and some neurons. Recognizing that endogenous levels of production of ARSA were too low to provide cross-correction by allogeneic transplant, Naldini and colleagues engineered a lentiviral vector that directed supraphysiologic levels of ARSA expression in transduced cells. Transduction of autologous HSCs from children born with the disease, at a point when they were still presymptomatic, led to preservation and continued acquisition of motor and cognitive milestones at time periods as long as 32 months after affected siblings had begun to lose milestones. These results illustrate that the ability to engineer levels of expression can allow gene therapy approaches to succeed where allogeneic bone

1	after affected siblings had begun to lose milestones. These results illustrate that the ability to engineer levels of expression can allow gene therapy approaches to succeed where allogeneic bone marrow transplantation cannot. It is likely that a similar approach will be used in other neurodegenerative conditions.

1	Transduction of HSCs to treat the hemoglobinopathies is an obvious extension of studies already conducted but represents a higher hurdle in terms of the extent of transduction required to achieve a therapeutic effect. Trials are now under way for thalassemia and for a number of other hematologic disorders, including Wiskott-Aldrich syndrome, and chronic granulomatous disease. LONG-TERM EXPRESSION IN GENETIC DISEASE: IN VIVO GENE TRANSFER WITH RECOMBINANT ADENO-ASSOCIATED VIRAL VECTORS

1	LONG-TERM EXPRESSION IN GENETIC DISEASE: IN VIVO GENE TRANSFER WITH RECOMBINANT ADENO-ASSOCIATED VIRAL VECTORS Recombinant adeno-associated viral (AAV) vectors have emerged as attractive gene delivery vehicles for genetic disease. Engineered from a small replication-defective DNA virus, they are devoid of viral coding sequences and trigger very little immune response in experimental animals. They are capable of transducing nondividing target cells, and the donated DNA is stabilized primarily in an episomal form, thus minimizing risks arising from insertional mutagenesis. Because the vector has a tropism for certain long-lived cell types, such as skeletal muscle, the central nervous system (CNS), and hepatocytes, long-term expression can be achieved even in the absence of integration.

1	These features of AAV were used to develop the first licensed gene therapy product in Europe, an AAV vector for treatment of the autosomal recessive disorder lipoprotein lipase (LPL) deficiency. This rare disorder (1–2/million) is due to loss-of-function mutations in the gene encoding LPL, an enzyme normally produced in skeletal muscle and required for the catabolism of triglyceride-rich lipoproteins and chylomicrons. Affected individuals have lipemic serum and may have eruptive xanthomas, hepatosplenomegaly, and in some cases, recurrent bouts of acute pancreatitis. Clinical trials demonstrated the safety of intramuscular injection of AAV-LPL and its efficacy in reducing frequency of pancreatitis episodes in affected individuals, leading to drug approval in Europe. Additional clinical trials currently under way that use AAV vectors in the setting of genetic disease include those for muscular dystrophies, α1 antitrypsin deficiency, Parkinson’s disease, Batten’s disease, hemophilia B,

1	currently under way that use AAV vectors in the setting of genetic disease include those for muscular dystrophies, α1 antitrypsin deficiency, Parkinson’s disease, Batten’s disease, hemophilia B, and several forms of congenital blindness.

1	Hemophilia (Chap. 78) has long been considered a promising disease model for gene transfer, because the gene product does not require precise regulation of expression and biologically active clotting factors can be synthesized in a variety of tissue types, permitting latitude in the choice of target tissue. Moreover, raising circulating factor levels from <1% (levels seen in those severely affected) into the range of 5% greatly improves the phenotype of the disease. Preclinical studies with recombinant AAV vectors infused into skeletal muscle or liver have resulted in long-term (>5 years) expression of factor VIII or factor IX in the hemophilic dog model. Administration to skeletal muscle of an AAV vector expressing factor IX in patients with hemophilia B was safe and resulted in long-term expression as measured on muscle biopsy, but circulating levels never rose to >1% for sustained periods, and a large number of IM injections (>80–100) was required to access a large muscle mass.

1	expression as measured on muscle biopsy, but circulating levels never rose to >1% for sustained periods, and a large number of IM injections (>80–100) was required to access a large muscle mass. Intravascular vector delivery has been used to access large areas of skeletal muscle in animal models of hemophilia and will likely be tested for this and other disorders in upcoming trials.

1	The first trial of an AAV vector expressing factor IX delivered to the liver in humans with hemophilia B resulted in therapeutic circulating levels at the highest dose tested, but expression at these levels (>5%) lasted for only 6–10 weeks before declining to baseline (<1%). A memory T cell response to viral capsid, present in humans but not 91e-3 in other animal species (which are not natural hosts for the virus), likely led to the loss of expression (Table 91e-2). In response to these findings, a second trial included a short course of prednisolone, to be administered if factor IX levels began to decline. This approach resulted in long-term expression of factor IX, in the range of 2–5%, in men with severe hemophilia B. Current efforts are focused on expanding these trials, and extending the approach to hemophilia A.

1	A logical conclusion from the early experience with AAV in liver in the hemophilia trial was that avoidance of immune responses was key to long-term expression. Thus immunoprivileged sites such as the retina began to attract substantial interest as therapeutic targets. This inference has been elegantly confirmed in the setting of the retinal degenerative disease Leber’s congenital amaurosis (LCA). Characterized by early-onset blindness, LCA is not currently treatable and is caused by mutations in several different genes; ~15% of cases of LCA are due to a mutation in a gene, RPE65, encoding a retinal pigment epithelial-associated 65-kDa protein. In dogs with a null mutation in RPE65, sight was restored after subretinal injection of an AAV vector expressing RPE65. Transgene expression appears to be stable, with the first animals treated >10 years ago continuing to manifest electroretinal and behavioral evidence of visual function. As is the case for X-linked SCID, gene transfer must

1	to be stable, with the first animals treated >10 years ago continuing to manifest electroretinal and behavioral evidence of visual function. As is the case for X-linked SCID, gene transfer must occur relatively early in life to achieve optimal correction of the genetic disease, although the exact limitations imposed by age have not yet been defined. AAV-RPE65 trials carried out in both the United States and the United Kingdom have shown restoration of visual and retinal function in over 30 subjects, with the most marked improvement occurring in the younger subjects. Trials for other inherited retinal degenerative disorders such as choroideremia are under way, as are studies for certain complex acquired disorders such as age-related macular degeneration, which affects several million people worldwide. The neovascularization that occurs in age-related macular degeneration can be inhibited by expression of vascular endothelial growth factor (VEGF) inhibitors such as angiostatin or

1	people worldwide. The neovascularization that occurs in age-related macular degeneration can be inhibited by expression of vascular endothelial growth factor (VEGF) inhibitors such as angiostatin or through the use of RNA interference (RNAi)-mediated knockdown of VEGF. Early-phase trials of siRNAs that target VEGF RNA are under way, but these require repeated intravitreal injection of the siRNAs; an AAV vector–mediated approach, which would allow long-term inhibition of the biological effects of VEGF through a soluble VEGF receptor, is now in clinical testing.

1	The majority of clinical gene transfer experience has been in subjects with cancer (Fig. 91e-1). As a general rule, a feature that distinguishes gene therapies from conventional cancer therapeutics is that the former are less toxic, in some cases because they are delivered locally (e.g., intratumoral injections), and in other cases because they are targeted specifically to elements of the tumor (immunotherapies, antiangiogenic approaches). Because cancer is a disease of aging, and many elderly are frail, the development of therapeutics with milder side effects is an important goal.

1	Cancer gene therapies can be divided into local and systemic approaches (Table 91e-3). Some of the earliest cancer gene therapy trials focused on local delivery of a prodrug or a suicide gene that would increase sensitivity of tumor cells to cytotoxic drugs. A frequently used strategy has been intratumoral injection of an adenoviral vector expressing the thymidine kinase (TK) gene. Cells that take up and express the TK gene can be killed after the administration of ganciclovir, which is phosphorylated to a toxic nucleoside by TK. Because cell division is required for the toxic nucleoside to affect cell viability, this strategy was initially used in aggressive brain tumors (glioblastoma multiforme) where the cycling tumor cells were affected but the nondividing normal neurons were not. More recently, this approach has been explored for locally recurrent prostate, breast, and colon tumors, among others.

1	Another local approach uses adenoviral-mediated expression of the tumor suppressor p53, which is mutated in a wide variety of cancers. This strategy has resulted in complete and partial responses in squamous cell carcinoma of the head and neck, esophageal cancer, and non-small-cell lung cancer after direct intratumoral injection of the vector. Response rates (~15%) are comparable to those of other single agents. The use of oncolytic viruses that selectively replicate in tumor cells but not in normal cells has also shown promise in squamous cell carcinoma of the head and neck and in other solid tumors. This approach is based on the observation that deletion of certain viral genes abolishes their ability to replicate in normal cells but not in tumor cells. An advantage of this strategy is that the replicating vector can proliferate and spread within the tumor, facilitating eventual tumor clearance. However, physical limitations to viral spread, including fibrosis, intermixed normal

1	is that the replicating vector can proliferate and spread within the tumor, facilitating eventual tumor clearance. However, physical limitations to viral spread, including fibrosis, intermixed normal cells, basement membranes, and necrotic areas within the tumor, may limit clinical efficacy. Oncolytic viruses are licensed and available in some countries but not in the United States.

1	Because metastatic disease rather than uncontrolled growth of the primary tumor is the source of mortality for most cancers, there has been considerable interest in developing systemic gene therapy approaches. One strategy has been to promote more efficient recognition of tumor cells by the immune system. Approaches have included transduction of tumor cells with immune-enhancing genes encoding cytokines, chemokines, or co-stimulatory molecules; and ex vivo manipulation of dendritic cells to enhance the presentation of tumor antigens. Recently, considerable success has been achieved using lentiviral transduction of autologous lymphocytes with a cDNA encoding a chimeric antigen receptor (CAR). The CAR moiety consists of a tumor antigen-binding domain (e.g., an antibody to the B cell antigen CD19) fused to an intracellular signaling domain that allows T cell activation. The transduced lymphocytes can then recognize and destroy cells bearing the antigen. This CAR–T cell approach has

1	CD19) fused to an intracellular signaling domain that allows T cell activation. The transduced lymphocytes can then recognize and destroy cells bearing the antigen. This CAR–T cell approach has proven extraordinarily successful in the setting of refractory chronic lymphocytic leukemia and pre-B-cell acute lymphoblastic leukemia. Infusion of gene-modified T cells engineered to recognize the B cell antigen CD19 has resulted in >1000-fold expansion in vivo, trafficking of the T cells to the bone marrow, and complete remission in a subset of patients who had failed multiple chemotherapy regimens. The cells persist as memory CAR+ T cells, providing ongoing antitumor functionality. Some patients experience a delayed tumor lysis syndrome requiring intensive medical management. This approach also causes an on-target toxicity, leading to B cell aplasia that necessitates lifelong IgG infusions. Current results indicate that long-lasting remissions can be achieved and the strategy can

1	also causes an on-target toxicity, leading to B cell aplasia that necessitates lifelong IgG infusions. Current results indicate that long-lasting remissions can be achieved and the strategy can theoretically be extended to other tumor types if a tumor antigen can be identified.

1	Gene transfer strategies have also been developed for inhibiting tumor angiogenesis. These have included constitutive expression of angiogenesis inhibitors such as angiostatin and endostatin; use of siRNA to reduce levels of VEGF or VEGF receptor; and combined approaches in which autologous T cells are genetically modified to recognize antigens specific to tumor vasculature. These studies are still in early-phase testing.

1	Another novel systemic approach is the use of gene transfer to protect normal cells from the toxicities of chemotherapy. The most extensively studied of these approaches has been transduction of hematopoietic cells with genes encoding resistance to chemotherapeutic agents, including the multidrug resistance gene MDRI or the gene encoding O6-methylguanine DNA methyltransferase (MGMT). Ex vivo transduction of hematopoietic cells, followed by autologous transplantation, is being investigated as a strategy for allowing administration of higher doses of chemotherapy than would otherwise be tolerated.

1	The third major category addressed by gene transfer studies is cardiovascular disease. Initial experience was in trials designed to increase blood flow to either skeletal (critical limb ischemia) or cardiac muscle (angina/myocardial ischemia). First-line treatment for both of these groups includes mechanical revascularization or medical management, but a subset of patients are not candidates for or fail these approaches. These patients formed the first cohorts for evaluation of gene transfer to achieve therapeutic angiogenesis. The major transgene used has been VEGF, attractive because of its specificity for endothelial cells; other transgenes have included fibroblast growth factor (FGF) and hypoxia-inducible factor 1, α subunit (HIF-1α). The design of most of the trials has included direct IM (or myocardial) injection of either a plasmid or an adenoviral vector expressing the transgene. Both of these vectors are likely to result in only short-term expression of VEGF, which may be

1	IM (or myocardial) injection of either a plasmid or an adenoviral vector expressing the transgene. Both of these vectors are likely to result in only short-term expression of VEGF, which may be adequate because there is no need for continued transgene expression once the new vessels have formed. Direct injection favors local expression, which should help to avoid systemic effects such as retinal neovascularization or new vessel formation in a nascent tumor. Initial trials of adeno-VEGF or plasmid-VEGF injection resulted in improvement over baseline in angiographically detectable vasculature, but no change in amputation frequency or cardiovascular mortality. Studies using different routes of administration or different transgenes are currently under way.

1	More recent studies have used AAV vectors to develop a therapeutic approach for individuals with refractory congestive heart failure. In preclinical studies, a vector encoding sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) demonstrated positive left ventricular inotropic effects in a swine model of volume-overloaded heart failure. Results of a phase II study in which vector was infused via the coronary arteries in patients with congestive heart failure demonstrated safety and some indications of efficacy; larger studies are now planned.

1	This chapter has focused on gene addition therapy, in which a normal gene is transferred to a target tissue to drive expression of a gene product with therapeutic effects. Another powerful technique under development is genome editing, in which a mutation is corrected in situ, generating a wild-type copy under the control of the endogenous regulatory signals. This approach makes use of novel reagents including zinc finger nucleases, TALENs and CRISPR, which introduce double-stranded breaks into the DNA near the site of the mutation and then rely on a donated repair sequence and cellular mechanisms for repair of double-strand breaks to reconstitute a functioning gene. Another strategy recently introduced into clinical trials is the use of siRNAs or short hairpin RNAs as transgenes to knock down expression of deleterious genes (e.g., mutant huntingtin in Huntington’s disease or genes of the hepatitis C genome in infected individuals).

1	The power and versatility of gene transfer approaches are such that there are few serious disease entities for which gene transfer therapies are not under development. The development of new classes of therapeutics typically takes two to three decades; monoclonal antibodies and recombinant proteins are recent examples. Gene therapeutics, which entered clinical testing in the early 1990s, traversed the same time course. Examples of clinical success are now abundant, and gene therapy approaches are likely to become increasingly important as a Elements of History for Subjects Enrolled in Gene Transfer Trials 1. What vector was administered? Is it predominantly integrating (retroviral, lentiviral, herpesvirus [latency and reactivation]) or nonintegrating (plasmid, adenoviral, adeno-associated viral)? 2. What was the route of administration of the vector? 3. What was the target tissue? 4. What gene was transferred in? A disease-related gene? A marker? 5.

1	2. What was the route of administration of the vector? 3. What was the target tissue? 4. What gene was transferred in? A disease-related gene? A marker? 5. Were there any adverse events noted after gene transfer? 1. Has a new malignancy been diagnosed? 2. Has a new neurologic/ophthalmologic disorder, or exacerbation of a preexisting disorder, been diagnosed? 3. Has a new autoimmune or rheumatologic disorder been diagnosed? 4. Has a new hematologic disorder been diagnosed? aFactors influencing long-term risk include: integration of the vector into the genome, vector persistence without integration, and transgene-specific effects.

1	aFactors influencing long-term risk include: integration of the vector into the genome, vector persistence without integration, and transgene-specific effects. therapeutic modality in the twenty-first century. A central question to 91e-5 be addressed is the long-term safety of gene transfer, and regulatory agencies have mandated a 15-year follow-up for subjects enrolled in gene therapy trials (Table 91e-4). Realization of the therapeutic benefits of modern molecular medicine will depend on continued progress in gene transfer technology.

1	Tissue Engineering Anthony Atala Tissue engineering is a field that applies principles of regenerative medicine to restore the function of various organs by combining cells with biomaterials. It is multidisciplinary, often combining the skills of physicians, cell biologists, bioengineers, and material scientists, to 92e recapitulate the native three-dimensional architecture of an organ, the appropriate cell types, and the supportive nutrients and growth factors that allow normal cell growth, differentiation, and function. Tissue engineering is a relatively new field, originating in the late 1970s. Early studies focused on efforts to create skin substitutes using biomaterials and epithelial skin cells with a goal of providing barrier protection for patients with burns. The early strategies employed a tissue biopsy, followed by ex vivo expansion of cells seeded on scaffolds. The cell– scaffold composite was later implanted back into the same patient, where the new tissue would mature.

1	employed a tissue biopsy, followed by ex vivo expansion of cells seeded on scaffolds. The cell– scaffold composite was later implanted back into the same patient, where the new tissue would mature. However, there were many hurdles to overcome. The three major challenges in the field of tissue engineering involved: (1) the ability to grow and expand normal primary human cells in large quantities; (2) the identification of appropriate biomaterials; and (3) the requirement for adequate vascularization and innervation of the engineered constructs.

1	The original model for tissue engineering focused largely on the isolation of tissue from the organ of interest, the growth and expansion of the tissue-specific cells, and the seeding of these cells onto three-dimensional scaffolds. Just a few decades ago, most primary cultures of human cells could not be grown and expanded in large quantities, representing a major impediment to the engineering of human tissues. However, the identification of specific tissue progenitor cells in the 1990s allowed expansion of multiple cell types, and progress has occurred steadily since then. Some cell types are more amenable to expansion than others, reflecting in part their native regenerative capacity but also varying requirements for nutrients, growth factors, and cell–cell contacts. As an example of progress, after years of effort, protocols for the growth and expansion of human cardiomyocytes are now available. However, there are still many tissue-specific cell types that cannot be expanded from

1	after years of effort, protocols for the growth and expansion of human cardiomyocytes are now available. However, there are still many tissue-specific cell types that cannot be expanded from tissue sources, including the pancreas, liver, and nerves. The discovery of pluripotent or highly multipotent stem cells (Chap. 88) may ultimately allow most human cell types to be used for tissue engineering. The stem cell characteristics depend on their origin and their degree of plasticity, with cells from the earliest developmental stages, such as embryonic stem cells, having the greatest plasticity. Induced pluripotent stem cells have the advantage that they can be derived from individual patients, allowing autologous transplants. They can also be differentiated, in vitro, along cell-specific lineages, although these protocols are still at an early stage of development. Human embryonic and induced pluripotent stem cells have a very high replicative potential, but they also have the potential

1	although these protocols are still at an early stage of development. Human embryonic and induced pluripotent stem cells have a very high replicative potential, but they also have the potential for rejection and tumor formation (e.g., teratomas). The more recently described amniotic fluid and placental stem cells have a high replicative potential but without an apparent propensity for tumor formation. Moreover, they have the potential to be used in an autologous manner without rejection. Adult stem cells, such as those derived from bone marrow, also have less propensity for tumor formation and, if used in an autologous manner, will not be rejected, but their replicative potential is limited, especially for endoderm and ectoderm cells.

1	Stem cells can be derived from autologous or heterologous sources. Heterologous cells can be used when only temporary coverage is needed, such as replacing skin after a burn or wound. However, if a more permanent construct is required, autologous cells are preferred to avoid rejection. There are also practical issues related to tissue sources. For example, if a patient presents with end-stage heart disease, obtaining a cardiac tissue biopsy for cell expansion is unlikely to be feasible, and bone marrow–derived mesenchymal cells may provide an alternative.

1	The biomaterials used to create the scaffolds for tissue engineering require specific properties to enhance the long-term success of the implanted constructs. Ideally, the biomaterials should be biocompatible; elicit minimal inflammatory responses; have appropriate biomechanical properties; and promote cell attachment, viability, proliferation, and differentiated function. Ideally, the scaffolds should replicate the biomechanical and structural properties of the tissue being replaced. In addition, biodegradation should be controlled such that the scaffold retains its structural integrity until the cells deposit their own matrix. If the scaffolds degrade too quickly, the constructs may collapse. If the scaffolds degrade too slowly, fibrotic tissue may form. Also, the degradation of the scaffolds should not alter the local environment unfavorably, because this can impair the function of cells or newly formed tissue.

1	The first scaffolds designed for tissue regeneration were naturally derived materials, such as collagen. The first artificially derived material for tissue engineering used a biodegradable scaffold made of polyglycolic acid. Naturally derived scaffolds have properties very similar to the native matrix, but there is an inherent batch-to-batch variability, whereas the production of artificially derived biomaterials can be better controlled, allowing for more uniform results. More recently, combination scaffolds, made of both naturally and artificially derived biomaterials, have been used for tissue engineering.

1	An emerging area is the use of peptide nanostructures to facilitate tissue engineering. Some of these are self-assembling peptide amphiphiles that allow scaffolds to form in vivo, for example at sites of spinal cord injury where they have been used experimentally to prevent scar formation and facilitate nerve and blood vessel regeneration. Peptide nanostructures can be combined with other biomaterials, and they can be linked to growth factors, antibodies, and various signaling molecules that can modulate cell behavior during organ regeneration.

1	Implanted tissue-engineered constructs require adequate vascularity and innervation. Judah Folkman, a pioneer in the field of angiogenesis, made the observation that cells could survive in volumes up to 3 mm3 via nutrient diffusion alone, but larger cell volumes required vascularization for survival. Adequate vascularity was also essential for normal innervation to occur. This was a major challenge in the field of tissue engineering, which largely depended on the patient’s native angiogenesis and innervation. Even if sufficient cell quantities are available, there is a theoretical limit on the types of tissue constructs that could be created. In response to this challenge, material scientists designed scaffolds with much greater porosity and architecture. Scaffold designs included the creation of thin, porous sponges comprised of 95% air, markedly increasing the surface area for the resident cells. These properties promoted increased vascularity and innervation. The addition of growth

1	of thin, porous sponges comprised of 95% air, markedly increasing the surface area for the resident cells. These properties promoted increased vascularity and innervation. The addition of growth factors, such as vascular endothelial growth factor and nerve growth factor, has been used to enhance angiogenesis and innervation.

1	All human tissues are complex. However, from an architectural aspect, tissues can be categorized under four levels. Flat tissue structures, such as skin, are the least complex (level 1), comprised predominantly of a single epithelial cell type. Tubular structures, such as blood vessels and the trachea, are more complex architecturally (level 2) and must be constructed to ensure that the structure does not collapse over time. These tissues typically have two major cell types. They are designed to act as a conduit for air or fluid at a steady state within a defined physiologic range. Hollow nontubular organs, such as the stomach, bladder, or uterus, are more complex architecturally (level 3). The cells are functionally more complex, and these cell types often have a functional interdependence. By far, the most complex are the solid organs (level 4), because the amount of cells per cm2 are exponentially greater than any of the other tissue types.

1	For the first three tissue levels (1–3), when the constructs are initially implanted, the cell layering on the scaffolds is thin, not unlike that seen in tissue culture matrices. The cell layering continues to mature, in concert with the recipient’s native angiogenesis and neoinnervation. For level 4 (solid) organs, the vascularity requirements are substantial, and native tissue angiogenesis is not sufficient. The engineering strategies for tissues vary according to their complexity level. The basic principles of tissue engineering involve the use of the relevant cell populations, where the cell biology is well understood and the cells can be reproducibly retrieved and expanded, and the use of optimized biomaterials and scaffold designs. Cell seeding can be performed using various techniques, including static or flow-based systems that use bioreactors. Most techniques for the engineering of tissues fall under one of five strategies (Fig. 92e-1): 1.

1	Most techniques for the engineering of tissues fall under one of five strategies (Fig. 92e-1): 1. Scaffolds can be used alone, without cells, and implanted, where they depend on native cell migration onto the scaffold from the adjacent tissue for regeneration. The first use of decellularized scaffolds for tissue regeneration was for urethral reconstruction. These techniques are most optimal when the size of the defect is relatively small, usually <0.5 cm from each tissue edge. Larger defects tend to heal by scarring, due to the deposition of fibroblasts, and eventual fibrosis. Scaffolds alone have also been used for other applications, including for wound coverage, soft tissue coverage after joint surgery, urogynecologic applications for sling surgery, and as materials for hernia repair. 2. A more recent strategy in tissue engineering involves the use of proteins, cytokines, genes, or small molecules that induce in situ Level 4 Heart, kidney, liver, lung

1	2. A more recent strategy in tissue engineering involves the use of proteins, cytokines, genes, or small molecules that induce in situ Level 4 Heart, kidney, liver, lung Strategies for tissue and organ engineering. tissue regeneration, either alone or with the use of scaffolds. For example, gene transcription factors used in the mouse pancreas led to tissue regeneration. Surgically implanted decellularized heart valve scaffolds, coated with proteins that attract vascular stem cells, led to the creation of in situ cell-seeded functional heart valves in sheep. Drugs that induce muscle regeneration are being tested clinically. Small molecules that induce tissue regeneration are currently under investigation for multiple applications, including growth of skin and hair and for musculoskeletal applications. 3.

1	The most common strategy for the engineering of tissues uses scaffolds seeded with cells. The most direct and established type of tissue engineering uses flat scaffolds, either artificial or naturally derived, that are seeded with cells and used for the replacement or repair of flat tissue structures. The flat scaffolds can also be sized and molded at the time of surgical implantation, or they can be shaped prior to cell seeding, for example, for tubular organs such as blood vessels or nontubular hollow tissues such as bladders. Bioreactors are often used to expose the cell–scaffold construct to mechanical forces, such as, stress, strain, and pulsatile flow that aid in the normal development of the cells into tissues (Video 92e-1, engineered heart valve in a pulsatile bioreactor showing the valves opening and closing). This strategy is the most common method used for tissue regeneration to date, and tissues and organs, such as skin, blood vessels, urethras, tracheas, vaginas, and

1	the valves opening and closing). This strategy is the most common method used for tissue regeneration to date, and tissues and organs, such as skin, blood vessels, urethras, tracheas, vaginas, and bladders, have been engineered and implanted in patients using these techniques.

1	4. The fourth strategy in tissue engineering is applicable for solid organs, where discarded organs are exposed to mild detergents and are decellularized, leaving behind a three-dimensional scaffold that preserves its vascular tree. The scaffold can then be reseeded with the patient’s own expanded vascular and tissue-specific cells. This strategy was used initially to create solid phallic structures in rabbits that were functional and able to produce offspring. Similar strategies were also used to recellularize miniature heart, liver, and kidney structures, with limited functionality to date, but with an established proof of concept (Video 92e-2, a dye is injected through the portal artery of a decellularized liver showing an intact vascular tree). These techniques are currently under investigation and have not been used clinically to date.

1	5. The fifth strategy for tissue engineering involves the use of bioprinting. These technologies arose through the use of modified desktop inkjet printers over a decade ago. The inkjet cartridges were filled with a cell–hydrogel combination instead of ink. A rudimentary three-dimensional elevator was lowered each time the cartridge deposited the cells and hydrogel, thus building miniature solid structures, such as two-chambered heart organoids, one layer at a time. More sophisticated bioprinters have now been built that have additional computer-aided design (CAD) and three-dimensional printing technologies. The information to print the organ can be personalized using the patient’s own imaging studies that help to define the size and shape of the particular tissue (Video 92e-3, a modified inkjet printer shows the three-dimensional construction of a two-chambered heart and how the structure beats with the cardiomyocytes in synchrony). Bioprinting is a tool that allows a scale-up option

1	inkjet printer shows the three-dimensional construction of a two-chambered heart and how the structure beats with the cardiomyocytes in synchrony). Bioprinting is a tool that allows a scale-up option for the production of engineered tissues. Its use is still experimental and has not been applied clinically to date.

1	A number of engineered tissues, including architecturally flat, tubular, and hollow nontubular organs, have been implanted in patients dating back to the 1990s. These include 92e-3 bladders, blood vessels, urethras, vaginal organs, tracheas, and skin for permanent replacement (Table 92e-1). Various types of skin substitutes, which were used as temporary “living wound dressings” to cover burn areas until skin grafts could be obtained from the same patient, were implanted starting in the 1990s. However, the use of engineered skin as a permanent replacement occurred only recently. Many engineered tissues are still being used in patients under regulatory guidelines for clinical trials. To date, solid organs have not yet been engineered for clinical use.

1	Tissue engineering is a rapidly evolving field where new technologies are continuously being applied to achieve success. The field still has many challenges ahead, including the long regulatory timelines required for the approval of widespread use, the need for improved scale-up production technologies, and the cost of the technologies, which include multiple processes involving biologics. Nonetheless, the list of tissues and organs being implanted in patients keeps growing, and the ability of these technologies to improve health has been demonstrated. More patients should be able to benefit from these technologies in the coming years. showing the valves opening and closing. VIDEO 92e-2 A dye is injected through the portal artery of a decellularized liver showing an intact vascular tree. VIDEO 92e-3 A modified inkjet printer shows the three-dimensional construction of a two-chambered heart and how the structure beats with the cardiomyocytes in synchrony.

1	VIDEO 92e-3 A modified inkjet printer shows the three-dimensional construction of a two-chambered heart and how the structure beats with the cardiomyocytes in synchrony. increase from 7 to 14% of the total population, and the United StatesWorld Demography of aging will soon have completed this same increase in 69 years. But in countries that started the transition later, the process is occurring much Richard M. Suzman, John G. Haaga more rapidly: Japan took 26 years to go from 7 to 14% age 65 and older,

1	Population aging is transforming the world in dramatic and fundamental ways. The age distributions of populations have changed and will continue to change radically, due to long-term declines in fertility rates and improvements in mortality rates (Table 93e-1). This transformation, known as the Demographic Transition, is also accompanied by an epidemiologic transition, in which noncommunicable chronic diseases are becoming the major causes of death and contributors to the burden of disease and disability. A concomitant of population aging is the change in key ratios expressing “dependency” of one form or another— the ratio of adults in the workforce to those typically out of the workforce, such as infants, children, retired “young old” (those still active but in ways other than paid work), and the oldest old. Global aging will affect economic growth, migration, patterns of work and retirement, family structures, pension and health systems, and even trade and the relative standing of

1	and the oldest old. Global aging will affect economic growth, migration, patterns of work and retirement, family structures, pension and health systems, and even trade and the relative standing of nations. Both absolute numbers (the size of an age group) and ratios (the ratio of those in working ages to dependents such as the young or retired, or the ratio of children to older people) are important. The size of age groups might affect the number of hospital beds needed, whereas the ratio of children to older people could affect the relative demand for pediatricians and geriatricians.

1	Although the increase in life expectancy, resulting from a series of social, economic, public health, and medical victories over disease, might very well be considered the crowning achievement of the past century and a half, the increased length of life coupled with the shifts in dependency ratios present formidable long-term challenges. The pace of the change is accelerating. In countries where the Demographic Transition began earlier, the process was slower: it took France 115 years for the proportion of the age group 65 and older to while China and Brazil are projected to require just 24 years.

1	Sometime around the year 2020, for the first time ever, the number of people age 65 and older in the world is expected to exceed that of children under the age of 5. Around the middle of the twentieth century, the under-5 age group constituted almost 15% of the total population and the over-65 age group 5%. It took about 70 years for these two to reach equal proportions. But demographers predict it will take only another 25–30 years for the 65 and older age group to equal about 15% and be about double the number of children under age 5. By the middle of their careers, medical students in most countries should expect to be practicing in far older populations. Preparations for these changes need to begin decades in advance, and the costs and penalties for delay can be very high. Although some governments have started planning for the long term, many, if not most, have yet to begin.

1	Population aging around the world in recent decades has followed a broadly similar pattern, starting with a decline in infant and childhood mortality that precedes a decline in fertility; at later stages, mortality at older ages declines as well. Declining fertility began as early as the beginning of the nineteenth century in the United States and France and extended to the rest of Europe and North America and parts of East Asia by the middle of the twentieth century. Since World War II, fertility declines have started in all other world regions. In fact, more than half the world’s population now lives in countries or provinces with fertility rates below the replacement level of just over two live births per woman. Mortality rates also began to change, relatively slowly at first, in Western Europe and North America during the nineteenth century. At first, changes were most evident at the youngest ages.

1	Chapter 93e World Demography of Aging taBLe 93e-1 SeLeCteD InDICatorS of popuLatIon agIng, eStImateS for 2009, anD projeCtIonS to 2050; SeLeCteD regIonS anD CountrIeS aUN Population Division defines Old Age Support Ratio as the number of people age 15 to 64 years for every person age 65 or older. bThe UN includes all European regions in its overall statistics; life expectancy at birth for males ranges from 63.8 years in Eastern Europe to 77.4 years in Western Europe. For women it ranges from 74.8 to 83.1 years in Western Europe. Source: United Nations Population Division, World Population Ageing 2012.

1	Source: United Nations Population Division, World Population Ageing 2012. Improvements in water supply and sewage handling, as well as in nutrition and housing, accounted for most of the improvement before the 1940s, when antibiotics and vaccines and increasing education of mothers began to make a major impact. Since the middle of the twentieth century, the “Child Survival Revolution” has spread to all parts of the world. Children almost everywhere in the world are much more likely to reach late middle age now than in previous generations. Especially since around 1960, mortality at older ages has improved steadily. This improvement has been primarily due to advances in care of heart disease and stroke and in control of conditions like hypertension and hypercholesterolemia that lead to circulatory diseases. In some parts of the world, smoking rates have declined, and these declines have led to lower incidence of many cancers, heart disease, and stroke.

1	The initial decline in fertility resulted in older age groups becoming a larger fraction of the total population. Declines in adult and old age mortality contributed to population aging in the later stages of the process. Life expectancy at birth—the average age to which someone is expected to live, under prevailing mortality conditions—has been calculated at around 28 years in ancient Greece, perhaps 30 years in medieval Britain, and less than 25 years in the colony of Virginia in North America. In the United States, life expectancy climbed slowly during the nineteenth century, reaching 49 years for white women by 1900. White men had a life expectancy 2 years lower than that for white women, and black Americans had a life expectancy 14 years lower than did white Americans in 1900. By the early twenty-first century, life expectancy in the United States had improved dramatically for all, with the sex gap wider and the racial gaps narrower than at the beginning of the century: 76 years

1	early twenty-first century, life expectancy in the United States had improved dramatically for all, with the sex gap wider and the racial gaps narrower than at the beginning of the century: 76 years for white men in 2006; 81 years for white women; and 70 and 76 years for black men and women, respectively. However, although the United States had a relatively high life expectancy compared to other high-income countries around 1980, almost all such countries have in the interim exceeded the United States in life expectancy. Female life expectancy, especially for whites in the United States, has done particularly poorly, and this has been attributed to relatively high rates of lifetime smoking.

1	At later stages of the demographic transition, mortality declines at the oldest ages, leading to increases in the 65 and older population, and the oldest old, those older than age 85 years. Migration can also affect population aging. An influx of young migrants with high birth rates can slow (though not stop) the process, as it has in the United States and Canada; or the out-migration of the young leaving older people behind can accelerate aging at the population level, as it has in many rural areas of the world.

1	Regions of the world are at very different stages of the demographic transition (Fig. 93e-1). Of a world population of 6.8 billion in 2012, approximately 11% were older than age 60 years, with Japan (32%) and Europe (22%) being the oldest regions (Germany and Italy 27% each) and the United States having 19%. The percentage of the population older than age 60 years in the United States has remained lower than in Europe, due both to modestly higher fertility rates and to higher rates of immigration. Asia has about 10% older than age 60 years, with the population giants close to the average—China (12%), Indonesia (9%), and India (7%). Middle Eastern and African countries have the lowest proportions of older people (5% or lower). 30-35 Based on estimates from the United Nations 25-30

1	30-35 Based on estimates from the United Nations 25-30 Population Division, 809 million people were 20-25 15-20 age 60 years or older in 2012, of whom 279 mil 5-10 million in less developed countries (as classified < 5 by the United Nations). The countries with the largest populations of those age 60 and older

1	Population projections make use of expected fertility, mortality, and migration rates and should be regarded as uncertain when applied 40 or more years in the future. However, the population that will be age 60 and older in 2050 have all been born and survived childhood in 2014, so uncertainty about their numbers (as distinct from their proportion of the total population) is not great. Comparing the maps of the world in 2010 (Fig. 93e-1) and 2050 (Fig. 93e-2), it is apparent that the middleand low-income countries in Latin America, Asia, and much of Africa will soon be joining the “oldest” category. In less than four decades between 2012 and 2050, the United Nations Population Division projects that the world population age 60 and older will more than double to 2.03 billion, with the least developed regions more than quadrupling. China’s 60+ population is projected to reach 439 million, India’s 323 million, and the United States’s 107 million. Over the same period, the median age of

1	developed regions more than quadrupling. China’s 60+ population is projected to reach 439 million, India’s 323 million, and the United States’s 107 million. Over the same period, the median age of the world’s population is expected to increase by 10 years.

1	Current global life expectancy at birth is estimated to be 65.4 for men and 69.8 for women, with the comparable figures for the more developed region being 73.6 and 80.5 years. Life expectancy in the least developed countries averaged only 57.2 for women and 54.7 for men. Life expectancy at birth is heavily influenced by infant and child mortality, which is considerably higher in poor countries. At older ages, the gap between rich and poor nations is narrower; so while women who have reached age 60 in wealthy countries can expect 23.7 more years of life on average, women at age 60 in poor countries live 16.8 years on average—a significant difference but not so stark as the difference in life expectancy at birth. At the lowest levels of per capita gross national product (GNP), life expectancy shows a powerful positive association with this measure of economic development, but then the slope of the relationship flattens out; for countries with average incomes above about $20,000 per

1	shows a powerful positive association with this measure of economic development, but then the slope of the relationship flattens out; for countries with average incomes above about $20,000 per year, life expectancy is not closely related to income. At each level of economic development, there is significant variation in life expectancy, indicating that many other factors influence life expectancy.

1	Japan, France, Italy, and Australia currently have some of the highest life expectancies in the world, while the United States has lagged behind other high-income countries since about 1980, especially in the case of white women. The causes of this lag are being explored, but the cumulative number of years that people have smoked tobacco by the time they reach older ages and the prevalence of obesity appear to play important roles.

1	A modern feature of population aging has been the almost explosive growth of the age group known as the oldest old, variously defined as those over age 80 or age 85. This is the age group with the highest burden of noncommunicable degenerative disease and related disability. Thirty years ago, this group attracted little attention because they were hidden within the overall older population in most statistical reports; for example, the U.S. Census Bureau merged them into a 65+ category. The reduction of mortality at older ages coupled with larger birth were China (181 million), India (100 million), FIGURE 93e-1 Percentages of national populations age 60+, in 2010. (From the U.S. and the United States (60 million). Census Bureau, International Database. StatPlanet Mapping Software.) are especially likely to understand and take 93e-3 advantage of measures to reduce infection. The effects of continuing progress will likely be seen in coming decades as well, because educational attainment

1	likely to understand and take 93e-3 advantage of measures to reduce infection. The effects of continuing progress will likely be seen in coming decades as well, because educational attainment is associated with improved health and survival at older ages. Countries vary in 30-35 25-30 the extent to which the “future elderly” cohorts 20-25 will be more educated. China in particular will15-20 tion in 2050 (with more than two-thirds of the school) than it did in 2000 (when only 10% of older people had a secondary education). In

1	FIGURE 93e-2 Percentages of national populations age 60 +, in 2050 (projections). the United States and other rich nations, this (From the U.S. Census Bureau, International Database. StatPlanet Mapping Software.) Chapter 93e World Demography of Aging cohorts surviving into old age led to the rapid growth of the oldest old. This age group is predicted to grow at a significantly higher rate than the 60+ population, and one estimate has the current 102 million age 80+ increasing to almost 400 million by 2050 (Table 93e-2). Projected increases are astounding: China’s 80+ population might increase from 20 to 96 million, India from 8 to 43 million, the United States from 12 to 32 million, and Japan from 9 to 16 million. The numbers of centenarians are increasing at an even faster rate.

1	The members of the population who could potentially become age 80 and older in 2050 are already alive today. The actual numbers of people who will be age 80 and older in 2050 will therefore depend almost solely on adult and old age mortality rates over the next 35 years. The history of the decline of mortality suggests that improvements in the standard of living, including increased and improved education and improved nutrition, coupled with improvements in public health stemming from an understanding of the germ theory of disease initially led to the decline in mortality, with medical achievements such as antibiotics and improved understanding of risk factors for cardiovascular and circulatory diseases becoming factors only in the post–World War II period; the largest strides in cardiovascular disease came only in more recent decades. The improvements in educational attainment of succeeding generations have been credited in large part for improvements changes in educational

1	cardiovascular disease came only in more recent decades. The improvements in educational attainment of succeeding generations have been credited in large part for improvements changes in educational attainment of the elderly population will be less dramatic.

1	Holding aside the possibility of new infectious diseases ravaging populations as AIDS did in some African countries, debates about future life expectancy revolve around the balance and influence of risk factors such as obesity; the possibility of reducing the deaths from current killers such as cancer, heart disease, and diabetes; whether there is some natural limit to life expectancy; and the distant though nonzero possibility that science will find a way to slow the basic processes of aging.

1	While some have posited natural limits to human life expectancy, the limits have been surpassed with some regularity, and at the very oldest ages in the leading countries with the highest life expectancy, there appears to be little evidence of any approaching asymptote. Indeed a surprising discovery was that life expectancy in the leading country over the last century and a half, with different countries taking the lead in different epochs, could be represented almost perfectly by a straight line, with the increase for females showing a steady and astonishing increase of three months per year or 2.5 years per decade (Fig. 93e-3). No single country kept that pace of improvement the entire time, but this trend calls into question the notion that improvement must slow down, at least in the near future.

1	There remains a great deal of diversity in health conditions both among and within national populations. There is nothing inevitable in child mortality during the past century, because educated mothers eStImateS (2012) anD projeCtIonS (2050) for the popuLatIon ageD 80 YearS anD oLDer: SeLeCteD regIonS about the mortality transition—in several African countries, the preva lence of AIDS has been high enough to cause life expectancy to fall below the levels of 1980. Though none has so far reached a scale to rival the AIDS epidemic, periodic outbreaks of new influenza viruses or “emerging infectious” agents remind us that infectious diseases could again come to the fore. Progress against chronic disease is also reversible: In Russia and some other countries that formed part of the Soviet Union before 1992, life expectancy for men has been declining, now reaching levels below those of men in South Asia. Much of the gap between Russian and Western European men is explainable by much greater

1	before 1992, life expectancy for men has been declining, now reaching levels below those of men in South Asia. Much of the gap between Russian and Western European men is explainable by much greater heart disease and injuries among the former.

1	Ratios of different age groups provide useful though crude indicators of potential demands on resources and resource availability. One set of ratios, known variously as dependency or support ratios, compare the age groups who are most likely to be in the labor force with the age groups typically dependent on the productive capacity of those work-ing—the young and the old, or just the old. A commonly used ratio is the number of persons age 15–64 per persons age 65 and older. Even though many in some countries do not enter the labor force until significantly older than age 15, retire before age 65, or work past age 65, the ratios do summarize important facts, especially in countries where financial support for the retired comes partially or mainly from those currently in the labor force through either a formal pension system or through informal support from the family. While many countries still have very basic pension systems with incomplete coverage, in Europe public pensions are

1	through either a formal pension system or through informal support from the family. While many countries still have very basic pension systems with incomplete coverage, in Europe public pensions are quite generous, and these countries face dramatic changes in their ratios of working age to older populations. Over the next 40 years, Western Europe faces a drop in the ratio from 4 to 2. In other words, while in crude terms there are today 4 workers supporting the pensions and other costs of each older person, by 2050 there will only be 2. China faces an even steeper drop from 9 persons of working age to only 3, while Japan declines from 3 to just 1. Even in India, projected to become the most populous country, the decline is quite steep from 13 to 5.

1	The dramatically declining number of workers per older person (however determined) is at the crux of the economic challenge of population aging. The extra years of life that can be considered the crowning achievement in medicine and public health of the last 150 years have to be financed. The economic model of the life cycle assumes that people are economically productive for a limited number of years and that the proceeds of their work during those years have to be smoothed over to finance consumption during less economically productive ages, either within families or by institutions such as the state in order to provide for the young, the old, and the infirm. There are only so many ways to meet the challenge of an extended period of dependency, including increasing the productivity of those in the labor force, saving more, reducing consumption, increasing the number of years worked by increasing the age of retirement, increasing the voluntary nonmonetary productive contributions of

1	in the labor force, saving more, reducing consumption, increasing the number of years worked by increasing the age of retirement, increasing the voluntary nonmonetary productive contributions of the retired, and immigration of very large numbers of young workers into the “old” countries. Pressures to increase retirement ages in industrialized countries and to reduce benefits are increasing. But no single one of these measures can bear the full load of adaptation to population aging, since the changes would have to be so severe and disruptive as to be politically impossible. More likely, there will be some combination of these measures.

1	Population health and the ability to function at work and in everyday life interact with these population ratios in significant ways. The physical and cognitive capacity to continue to work at older ages is crucial if the age of retirement is raised. Similarly, caregiving often requires significant physical and emotional stamina. Further, healthier older populations require less caregiving and medical services. Just two decades ago, the prevalent view of aging was highly pessimistic. Epidemiologists held that while modern medicine could keep older people alive, nothing much could be done to prevent, delay, or significantly treat the degenerative chronic diseases of aging. The result would be that more and more older people with chronic diseases

1	FIGURE 93e-4 Disability prevalence, various years 1982–2005, by age group over 65, United States. (Adapted from KG Manton et al: Proc Natl Acad Sci U S A. 103:18374, 2006.) would be kept from dying, with the consequent piling up of the older people disabled by chronic disease. Surprisingly, between 1984 and about 2000, the prevalence of disability in the 65+ population in the United States declined by about 25%, suggesting that in this respect, aging was more plastic than had been previously believed (Fig. 93e-4). All the causes of this significant shift in disability are not yet understood, but rising levels of education, improved treatment of cardiovascular diseases and cataracts, greater availability of assistive devices, and less physically demanding occupations have been found to contribute. One calculation showed that if the rate of improvement could be maintained until 2050, that the numbers of disabled in the older population could be kept constant in the United States despite

1	One calculation showed that if the rate of improvement could be maintained until 2050, that the numbers of disabled in the older population could be kept constant in the United States despite the aging of the baby boomers and the older population itself growing older. Unfortunately, the rapid increase in obesity rates could slow and perhaps even reverse this most positive trend. Because of the absence of comparable data in other countries, it is less certain whether the same pattern of improvement in disability rates (with recent deceleration) is occurring outside of the United States. Using estimates and projections of disease prevalence from the Global Burden of Disease Study, the global population of those “dependent and in need of care” is projected to rise from about 350 million in 2010 to over 600 million in 2050. Worldwide, about half of the older persons in need of care (two-thirds of the dependent population age 90 and above) suffer from dementia or cognitive impairment. A

1	2010 to over 600 million in 2050. Worldwide, about half of the older persons in need of care (two-thirds of the dependent population age 90 and above) suffer from dementia or cognitive impairment. A global network of longitudinal studies on aging, health, and retirement is now providing comparable data that may allow more definitive projections on disease and disability trends in the future. One estimate (World Alzheimer’s Report 2010) projected that the 36 million people with dementia worldwide in 2010 would increase to 115 million by 2050. The largest increases would occur in lowand middle-income countries where about two-thirds already live. The estimated costs were $604 billion in 2010 with 70% occurring in North America and Western Europe. A 2013 study using a nationally representative U.S. sample found that annual dementia costs could be as high as $215 billion. Direct costs of dementia care exceeded the direct costs for either heart disease or cancer. Given the age-associated

1	U.S. sample found that annual dementia costs could be as high as $215 billion. Direct costs of dementia care exceeded the direct costs for either heart disease or cancer. Given the age-associated prevalence of dementia and the expected increase in the older population, coupled with the associated decline in family members able to provide care, countries need to plan for a pandemic of individuals requiring long-term care.

1	Population aging, and related demographic changes including changes in family structure, could affect the “supply side” of long-term care as well as the demand for care and health care. In every country, long-term care of the disabled and the chronically ill relies heavily on informal, typically unpaid caregivers—usually spouses or children; and increasingly in more developed countries, caregivers for the oldest old are in their 60s and early 70s. Although there are many men who provide care, on the population level, informal caregiving is still mainly done by women. Because women live longer than men, lack of a spousal caregiver is especially likely to be a problem for older women. Both men and women have fewer children on whom they can call for informal caregiving, because of the worldwide decline in fertility rates. An increasing proportion of older men in Europe and North America have spent much or all of their adult lives apart from their biological children. Lower fertility

1	decline in fertility rates. An increasing proportion of older men in Europe and North America have spent much or all of their adult lives apart from their biological children. Lower fertility rates, delayed marriage, and increasing divorce rates mean that people approaching old age may be less likely to have close ties with daughters and daughters-in-law—the adults who have in the past been the most common caregivers apart from spouses. Adult women who in the past have provided uncompensated care (and much other essential volunteer work) are now more likely than in the past to be working for pay and thus have fewer hours to devote to the unpaid roles.

1	These broad demographic and economic trends do not dictate particular social adaptations or policy responses, of course. One can imagine many different responses to the challenges of caring for the disabled—increased reliance on home health agencies and assisted living communities, “naturally occurring retirement communities” in which neighbors fulfill many of the roles once reserved for close kin; private or even publicly financed direct payments to compensate formerly unpaid family caregivers (a reform that has proved very popular in Germany). These and other responses to the challenge of long-term care are being tested in aging countries, and continued experimentation will no doubt be needed.

1	The secular improvements in ages at death have been accompanied by changes in causes of death. In the broadest terms, the proportion of deaths due to infectious disease and conditions associated with pregnancy and delivery has fallen, and the proportion due to chronic, noncommunicable diseases, such as heart and cerebrovascular diseases, diabetes, cancers, and age-related neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases, has increased steadily and is expected to continue to increase. Figure 93e-5 shows results from an international comparative project that drew on a wide variety Communicable, maternal, perinatal, and nutritional conditions Noncommunicable diseases Injuries FIGURE 93e-5 Leading causes of burden of illness in world regions 2002 and projected for 2030. (Adapted from CD Mathers, D Loncar: PLoS Med 3:e442, 2006.)

1	FIGURE 93e-5 Leading causes of burden of illness in world regions 2002 and projected for 2030. (Adapted from CD Mathers, D Loncar: PLoS Med 3:e442, 2006.) Note: “Burden of disease” takes into account years of life lost due to death from this cause and also a weighted estimate for years spent with disability, pain, or impairments due to the condition. These estimates were aggregated from many different national reporting systems and special surveys or surveillance systems, with adjustments for incomplete coverage and different reporting schemes as part of the Global Burden of Disease Study 2010 updating previous global estimates. Abbreviation: COPD, chronic obstructive pulmonary disease. Source: CJL Murray et al: Lancet 380:2197, 2013, Fig. 5.

1	of data sources to provide estimates of the global burden of disease at the beginning of this century, with projections to future years based on recent trends in disease prevalence and demographic rates. Burden of disease in these pie charts is a composite measure, one that takes into account both the number of deaths due to a particular disease or condition and the timing of such deaths—an infant death represents a loss of more potential life-years lived than does the death of a very old person. Nor is death the only outcome that matters; most diseases or conditions cause significant disability and suffering even when nonfatal, so this measure of burden captures nonfatal outcomes using statistical weighting. As Table 93e-3 shows, the “modern plagues” of chronic noncommunicable diseases are already among the leading causes of premature death and disability even in low-income countries. This is due to a mix of factors—lower fertility rates mean fewer infants and children at prime ages

1	already among the leading causes of premature death and disability even in low-income countries. This is due to a mix of factors—lower fertility rates mean fewer infants and children at prime ages of susceptibility to infections; more people reaching older ages when chronic disease incidence is high; and often changing incidence rates due to increased exposure to tobacco, Western diets, and inactivity. Noncommunicable diseases, once thought of as “diseases of affluence,” are projected to account for more than half of the disease burden even in lowand middle-income countries by the year 2030 (Fig. 93e-5).

1	Population aging is a global phenomenon with profound shortand long-term implications for health and long-term care needs, and indeed for the economic and social well-being of nations. The timing and context of aging vary across and within world regions and countries; the industrialized nations became wealthy before they aged significantly, while many of the low-resource regions will age before they reach high-income levels. The variation at both the population level and individual levels indicates that there is much flexibility in successful aging, but meeting the challenges will require advance planning and preparation. The extent to which research can find solutions that reduce physical and cognitive disability at older ages will determine how countries cope with this fundamental transformation. Chapter 93e World Demography of Aging 94e 5000 The Biology of Aging Rafael de Cabo, David G. Le Couteur Mortality rate per 100,000

1	Mortality rate per 100,000 Aging and old age are among the most significant challenges facing medicine this century. The aging process is the major risk factor underlying disease and disability in developed nations, and older people respond differently to therapies developed for younger adults (usually with less effectiveness and more adverse reactions). Modern medicine and healthier lifestyles have increased the likelihood that younger adults will now achieve old age. However, this has led to rapidly increasing numbers of older people, often encumbered with age-related disorders that are predicted to overwhelm health care sys-0 tems. Improved health in old age and further extension of the human healthspan are now likely to result primarily from increased understanding of the biology of aging, age-related susceptibility to disease, and modifiable factors that influence the aging process.

1	Definitions of Aging Aging is easy to recognize but difficult to define. Most definitions of aging indicate that it is a progressive process associated with declines in structure and function, impaired maintenance and repair systems, increased susceptibility to disease and death, and reduced reproductive capacity. There are both statistical and phenotypic components to aging. As recognized by Gompertz in the nineteenth century, aging in humans is associated with an exponential risk of mortality with time (Fig. 94e-1), although it is now realized that this plateaus in extreme old age because of healthy survivor bias. The phenotypic components of aging include structural and functional changes that are separated, somewhat artificially, into either primary aging changes (e.g., sarcopenia, gray hair, oxidative stress, increased peripheral vascular resistance) or age-related disease (e.g., dementia, osteoporosis, arthritis, insulin resistance, hypertension).

1	Definitions of aging rarely acknowledge the possibility that some of those biological and functional changes with aging might be adaptive or even reflect improvement and gain. Nor do they emphasize the effect of aging on responses to medical treatments. Old age is associated with increased vulnerability to many perturbations, including therapeutic interventions. This is a critical issue for clinicians; the problem with aging would be more limited if our disease-specific therapies retained their balance of risk to benefit into old age. Aging and Disease Susceptibility Old age is the major independent risk factor for chronic diseases (and associated mortality) that are most prevalent in developed countries such as cardiovascular disease, cancers, and neurodegenerative disorders (Fig. 94e-2). Consequently,

1	FIGURE 94e-2 The rates of most common chronic diseases and related mortality increase with old age. (Data from USA 2008–2010 CDC.) older people have multiple comorbidities, usually in the range of 5 to 10 illnesses per person.

1	Disease in older people is typically multifactorial with a strong component related to the underlying aging process. For example, in younger patients with dementia, Alzheimer’s disease is a single disorder confirmed by examining brain tissue for plaques and tangles containing amyloid and tau proteins. However, the vast majority of people with dementia are elderly, and here the association between typical Alzheimer’s neuropathology and dementia becomes less definitive. In the oldest-old, the prevalence of Alzheimer’s-type brain pathology is similar in people with and without clinical features of dementia. On the other hand, brains of older people with dementia usually show mixed pathology, with evidence of Alzheimer’s pathology along with features of other dementias such as vascular lesions, Lewy bodies, and non-Alzheimer’s tauopathy. Typical aging changes, such as microvascular dysfunction, oxidative injury, and mitochondrial impairment, underlie many of the pathologic changes.

1	The Longevity Dividend Compression of morbidity refers to the concept that the burden of lifetime illness might be compressed by medical interventions into a shorter period before death without necessarily increasing longevity. However, continuing development of successful therapeutic and preventative interventions focusing on individual diseases is less effective in older people because of multiple comorbidities, complications of overtreatment, and competing causes of death. Therefore, it has been proposed that further gains in healthspan and life expectancy will be achieved by a single intervention that delays aging and age-related disease susceptibility, rather than multiple treat ments each targeting different individual age-related illnesses. This is 10000 called the longevity dividend and is driving an explosion of research into aging biology and, more importantly, interventions (genetic, 8000 pharmaceutical, and nutritional) that influence the rate of aging and delay

1	dividend and is driving an explosion of research into aging biology and, more importantly, interventions (genetic, 8000 pharmaceutical, and nutritional) that influence the rate of aging and delay age-related disease.

1	4000 At the most basic level, living things have only two approaches to maintain their existence: immortality or reproduction. In a changing2000 environment, reproduction combined with a finite lifespan has proved to be the successful strategy. Of course, finite lifespan is not the same Deaths (rate per 100,000) as aging, although aging, by definition, contributes to a finite lifespan. Many evolutionary theories related to aging are linked by their attempts to explain this interaction between reproduction and longevity (Fig. 94e-3). Most mainstream aging theories stem from the fact that FIGURE 94e-1 The rates of death in the United States (2010) evolution is driven by early reproductive success, whereas there is mini-showing exponential increase in mortality risk with chronologic age. mal selection pressure for late-life reproduction or postreproductive Tissue changes that predispose to disease Immunosenescence, inflammaging Detoxification Endocrine system Vascular system

1	Tissue changes that predispose to disease Immunosenescence, inflammaging Detoxification Endocrine system Vascular system Adaptive aging? Grandmother effect Adaptive senectitude FIGURE 94e-3 Schema linking evolution and cellular and tissue changes with aging. The call-out blue boxes indicate factors that might delay the aging process including nutrient response pathways and, possibly, adaptive evolutionary effects.

1	survival. Aging is seen as the random degeneration resulting from the inability of evolution to prevent it, i.e., the nonadaptive consequence of evolutionary “neglect.” This conclusion is supported by studies that restricted reproduction to later life in the fruit fly, Drosophila melanogaster, thus permitting natural selection to operate on later life traits and leading to an increase in longevity. There are some species of plants and animals that do not appear to age, or at least they undergo an extremely slow aging process, termed “negligible senescence.” The mortality rates of these species are relatively constant with time, and they do not display any obvious phenotypic changes of aging. Conversely, there are some living things that undergo programmed death immediately after reproduction, such as annual plants and semelparous animals (Fig. 94e-4). However, many other living things from yeast to humans undergo a gradual aging process leading to death that is surprisingly similar at

1	such as annual plants and semelparous animals (Fig. 94e-4). However, many other living things from yeast to humans undergo a gradual aging process leading to death that is surprisingly similar at the cellular and biochemical level across taxa. Some of the major classical evolutionary theories of aging include the following:

1	Negligible senescence Rapid death postreproduction (semelparous animals, annual plants) Rougheye rockfish Bristlecone pine Pacific salmon Sunflower FIGURE 94e-4 The typical features of aging (aging phenotype and exponential increase in risk of death) are not universal findings in liv-ing things. Some living things (e.g., rockfish and the bristlecone pine, sometimes called the Methuselah tree) undergo negligible senes-cence, whereas others die almost immediately after reproduction is completed (e.g., semelparous animals and annual plants). Programmed death. The first evolutionary theory of aging was proposed by Weissman in 1882. This theory states that aging and death are programmed and have evolved to remove older animals from the population so that environmental resources such as food and water are freed up for younger members of the species.

1	Mutation accumulation. This theory was proposed by Medawar in 1952. Natural selection is most powerful for those traits that influence reproduction in early life, and therefore, the ability of evolution to shape our biology declines with age. Germline mutations that are deleterious in later life can accumulate simply because natural selection cannot act to prevent them. Antagonistic pleiotropy. George C. Williams extended Medawar’s theory when he proposed that evolution can allow for the selection of genes that are pleiotropic, i.e., beneficial for survival and reproduction in early life, but harmful in old age. For example, genes for sex hormones are necessary for reproduction in early life but contribute to the risk of cancer in old age.

1	Life history theory. Evolution is influenced by the way that limited resources are allocated to all aspects of life including development, sexual maturation, reproduction, number of offspring, and senescence and death. Therefore, “trade-offs” occur between these phases of life. For example, in a hostile environment, survival is highest for those species that have large numbers of offspring and short lifespan, whereas in a safe and abundant environment, survival is highest for those species that invest resources in a smaller number of offspring and a longer life.

1	Disposable soma theory. Kirkwood and Holliday in 1979 combined many of these ideas in the disposable soma theory of aging. There are finite resources available for the maintenance and repair of both germ and soma cells, so there must be a trade-off between germ cells (i.e., reproduction) and soma cells (i.e., longevity and aging). The soma cells are disposable from an evolutionary perspective, so they accumulate damage that causes aging while resources are preferentially diverted to the maintenance and repair of the germ cells. For example, the longevity of the nematode worm, Caenorhabditis elegans, is increased when its germ cells are ablated early in life. All of these theories assume that natural selection has negligible or negative influences on aging. Some postmodern ideas propose that aspects of aging might be adaptive and raise the possibility that evolution can act on the aging process in a positive way. These include the following:

1	Grandmother hypothesis. The grandmother hypothesis proposed by Hamilton in 1966 describes how evolution can enhance old age. In some animals, including humans, the survival of multiple, dependent offspring is beyond the capacity and resources of a single parent. In this situation, the presence of a long-lived grandmother who shares in the care of her grandchildren can have a major impact on their survival. These children share some of the genes of their grandmother including those that promoted their grandmother’s longevity. Mother’s curse. Mitochondrial dysfunction is a key component of the aging process. Mitochondria contain their own DNA and are only passed on from mother to child because sperm cells contain almost no mitochondria. Therefore, natural selection can only act on the evolution of mitochondrial DNA in females. The “mother’s curse” of the maternal inheritance of mitochondrial DNA might explain why females live longer and age more slowly than males.

1	Adaptive senectitude. Many traits that are harmful in younger humans such as obesity, hypertension, and oxidative stress paradoxically appear to be associated with greater survival and function in very old people. Perhaps driven by the grandmother effect, this might represent “adaptive senectitude” or “reverse antagonistic pleiotropy,” whereby some traits that are harmful in young people become beneficial in older people. There are many cellular processes that change with aging. These are generally considered to be degenerative and stochastic or random changes that reflect some sort of time-dependent damage (Fig. 94e-3). Whether any of these is the root cause of aging is unknown, but they all contribute to the aging phenotype and disease susceptibility.

1	Oxidative Stress and the Free Radical Theory of Aging Free radicals are chemical species that are highly reactive because they contain unpaired electrons. Oxidants are oxygen-based free radicals that include the hydroxyl free radical, superoxide, and hydrogen peroxide. Most cellular oxidants are waste products generated by mitochondria during the production of ATP from oxygen. More recently, the role of oxidants in cellular signaling and inflammatory responses has been recognized. Unchecked, oxidants can generate chain reactions leading to widespread damage to biological molecules. Cells contain numerous antioxidant defense mechanisms to prevent such oxidative stress including enzymes (superoxide dismutase, catalase, glutathione peroxidase) and chemicals (uric acid, ascorbate). In 1956, Harman proposed the “free radical theory of aging,” whereby oxidants generated by metabolism or irradiation are responsible for age-related damage. It is now well established that old age in most

1	Harman proposed the “free radical theory of aging,” whereby oxidants generated by metabolism or irradiation are responsible for age-related damage. It is now well established that old age in most species is associated with increased oxidative stress, for example to DNA (8-hydroxyguanosine derivatives), proteins (carbonyls), lipids (lipoperoxides, malondialdehydes), and prostaglandins (isoprostanes). Conversely, many of the cellular antioxidant defense mechanisms, including the antioxidant enzymes, decline in old age. The free radical theory of aging has spawned numerous studies of supplementation with antioxidants such as vitamin E to delay aging in animals and humans. Unfortunately, meta-analyses of human clinical trials performed to treat and prevent various diseases with antioxidant supplements indicate that they have no effect on, or may even increase, mortality.

1	Mitochondrial Dysfunction Aging is characterized by altered mitochondrial production of ATP and oxygen-derived free radicals. This leads to a vicious cycle mediated by accumulation of oxidative injury to mitochondrial proteins and DNA. With age, the number of mitochondria in cells decreases, and there is an increase in their size (megamitochondria) associated with other structural changes including vacuolization and disrupted cristae. These morphologic aging changes are linked with decreased activity of mitochondrial complexes I, II, and IV and decreased ATP production. Of all of the complexes involved in 94e-3 ATP production, the activity of complex IV (COX) is usually reported to be most impaired in old age. Reduced energy production is linked with generation of hydrogen peroxide and superoxide radicals leading to oxidative injury to mitochondrial DNA and accumulation of carbonylated mitochondrial proteins and mitochondrial lipoperoxides. As well as being implicated in the aging

1	superoxide radicals leading to oxidative injury to mitochondrial DNA and accumulation of carbonylated mitochondrial proteins and mitochondrial lipoperoxides. As well as being implicated in the aging process, common geriatric syndromes including sarcopenia, frailty, and cognitive impairment are associated with mitochondrial dysfunction.

1	Telomere Shortening and Replicative Senescence Cells that are isolated from animal tissue and grown in culture only divide for a certain number of times before entering a senescent phase. This number of divisions is known as the Hayflick limit and tends to be less in cells isolated from older animals compared to younger animals. It has been suggested that aging in vivo might in part be secondary to some cells ceasing to divide because they have reached their Hayflick limit. One mechanism for replicative senescence relates to telomeres. Telomeres are repeat sequences of DNA at the end of linear chromosomes that shorten by around 50–200 base pairs during each cell division by mitosis. Once telomeres become too short, cell division can no longer occur. This mechanism contributes to the Hayflick limit and has been called the cellular clock. There are some studies that suggest that the length of telomeres in circulating leukocytes (leukocyte telomere length [LTL]) decreases with age in

1	limit and has been called the cellular clock. There are some studies that suggest that the length of telomeres in circulating leukocytes (leukocyte telomere length [LTL]) decreases with age in humans. However, the aging process also occurs in tissues that do not undergo repeated cell division such as neurons.

1	Altered Gene Expression, Epigenetics, and microRNA There are changes in the expression of many genes and proteins during the aging process. These changes are complicated and vary between species and tissue. Such heterogeneity reflects increasing dysregulation of gene expression with age while appearing to exclude a programmed and/or uniform response. With old age, there are often reductions in the expression of genes and proteins associated with mitochondrial function and increased expression of those involved with inflammation, genome repair, and oxidative stress. There are several factors controlling the regulation of gene and protein expression that change with aging. These include the epigenetic state of the chromosomes (e.g., DNA methylation and histone acetylation) and microRNAs (miRNA). DNA methylation correlates with age, although the pattern of change is complex. Histone acetylation is regulated by many enzymes including SIRT1, a protein that has marked effects on aging and

1	DNA methylation correlates with age, although the pattern of change is complex. Histone acetylation is regulated by many enzymes including SIRT1, a protein that has marked effects on aging and the response to dietary restriction in many species. miRNAs are a very large group of noncoding lengths of RNA (18–25 nucleotides) that inhibit translation of multiple different mRNAs through binding their 3′ untranslated regions (UTRs). The expression of miRNAs usually decreases with aging and is altered in some age-related diseases. Specific miRNAs linked with aging pathways include miR-21 (associated with target of rapamycin pathway) and miR-1 (associated with insulin/insulin-like growth factor 1 pathway).

1	Impaired Autophagy There are a number of ways that cells can remove damaged macromolecules and organelles, often generating cellular energy as a byproduct. Intracellular degradation is undertaken by the lysosomal system and the ubiquitin proteasomal system. Both are impaired with aging, leading to the accumulation of waste products that alter cellular functions. Such waste products include lipofuscin, a brown autofluorescent pigment found within lysosomes of most cells in old age and often considered to be one of the most characteristic histologic features of aging cells. They also include aggregated proteins characteristic of age-related neurodegenerative diseases (e.g., tau, β-amyloid, α-synuclein). Lysosomes are organelles that contain proteases, lipases, glycases, and nucleotidases that degrade intracellular macromolecules, membrane components, organelles, and some pathogens through a process called autophagy. The lysosomal process most impaired with aging is macroautophagy, which

1	degrade intracellular macromolecules, membrane components, organelles, and some pathogens through a process called autophagy. The lysosomal process most impaired with aging is macroautophagy, which is regulated by numerous autophagy-related genes (ATGs). Old age is associated with some impairment in chaperone-mediated autophagy, whereas the effect of aging on the third lysosomal process, microautophagy, is unclear.

1	CHAPTER 94e The Biology of Aging Aging changes in some tissues increase susceptibility to age-related disease as a secondary or downstream phenomenon (Fig. 94e-3). In humans, this includes, but is not limited to, the immune system (leading to increased infections and autoimmunity), hepatic detoxification (leading to increased exposure to disease-inducing endobiotics and xenobiotics), the endocrine system (leading to hypogonadism and bone disease), and the vascular system (leading to segmental or global ischemic changes in many tissues).

1	Inflammaging and Immunosenescence Old age is associated with increased background levels of inflammation including blood measurements of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and cytokines such as interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α). This has been termed inflammaging. T cells (particularly naïve T cells) are less numerous because of age-related atrophy of the thymus, whereas B cells overproduce autoantibodies, leading to the age-related increase in autoimmune diseases and gammopathies. Thus older people are generally considered to be immunocompromised and have reduced responses to infection (fever, leukocytosis) with increased mortality.

1	Detoxification and the Liver Old age is associated with impaired detoxification of various disease-causing endobiotics (e.g., lipoproteins) and xenobiotics (e.g., neurotoxins, carcinogens), leading to increased systemic exposure. In humans, the liver is the major organ for the clearance of such toxins. Hepatic clearance of many substrates is reduced in old age as a consequence of reduced hepatic blood flow, impaired hepatic microcirculation, and in some cases, reduced expression of xenobiotic metabolizing enzymes. These changes in hepatic detoxification also increase the likelihood of increased blood levels of, and adverse reactions to, medications.

1	Endocrine System Hormonal changes with aging have been a focus of aging research for over a century, partly because of the erroneous belief that supplementation with sex hormones will delay aging and rejuvenate older people. There are age-related reductions in sex steroids secondary to hypogonadism and, in females, menopause. Age-related declines in growth hormone and dehydroepiandrosterone (DHEA) are well established, as is the increase in insulin levels and associated insulin resistance. These hormonal changes contribute to some features of aging such as sarcopenia and osteoporosis, which may be delayed by hormonal supplementation. However, adverse effects of long-term hormonal supplementation outweigh any potential beneficial effects on lifespan.

1	Vascular Changes There is a continuum from vascular aging through to atherosclerotic disease, present in many, but not all, older people. Vascular aging changes overlap with the early stages of hypertension and atherosclerosis, with increasing arterial stiffness and vascular resistance. This contributes to myocardial ischemia and strokes but also appears to be associated with geriatric conditions such as dementia, sarcopenia, and osteoporosis. In these conditions, impaired exchange between blood and tissues is a common pathogenic factor. For example, the risk of Alzheimer’s disease and dementia is increased in patients with risk factors for vascular disease, and there is pathologic evidence for microvascular changes in postmortem studies of brains of people with established Alzheimer’s disease. Similarly, strong epidemiologic links have been found between osteoporosis and standard vascular risk factors, whereas there are significant age-associated changes in the microcirculation of

1	disease. Similarly, strong epidemiologic links have been found between osteoporosis and standard vascular risk factors, whereas there are significant age-associated changes in the microcirculation of osteoporotic bone. Sarcopenia might also be related to the effects of age on the muscle vasculature, which is altered in old age. The sinusoidal microcirculation of the liver becomes markedly altered during aging (pseudocapillarization), which influences hepatic uptake of lipoproteins and other substrates. In fact, it has often been overlooked that in his original exposition of the free radical theory of aging, Harman proposed that the primary target of oxidative stress was the vasculature and that many aging changes were secondary to impaired exchange across the damaged blood vessels.

1	There is variability in aging and lifespan in populations of genetically identical species such as mice that are housed in the same environment. Moreover, the heritability of lifespan in human twin studies is estimated to be only 25% (although there is stronger hereditary contribution to extreme longevity). These two observations indicate that the cause of aging is unlikely to lie only within the DNA code. On the other hand, genetic studies initially undertaken in the nematode worm C. elegans and, more recently, in models from yeast to mice have shown that manipulating genes can have profound effects on the rate of aging. Perhaps surprisingly, this can often be generated by variability in single genes, and for some genetic mechanisms, there is very strong evolutionary conservation.

1	Genetic Progeroid Syndromes There are a few very rare, genetic premature aging conditions that are called progeroid syndromes. These conditions recapitulate some, but not all, age-related diseases and senescent phenotypes. They are mostly caused by impairment of genome and nuclear maintenance. These syndromes include the following: Werner’s syndrome. This is an autosomal recessive condition caused by a mutation in the WRN gene. This gene codes for a RecQ helicase, which unwinds DNA for both repair and replication. It is typically diagnosed in teen years, and there is premature onset of atherosclerosis, osteoporosis, cancers, and diabetes, with death by age of 50 years.

1	Hutchinson-Gilford progeria syndrome (HGPS). This usually occurs as a de novo, noninherited mutation in the lamin A gene (LMNA), leading to an abnormal protein called progerin. LMNA is required for the nuclear lamina, which provides structural support to the nucleus. There are marked development changes obvious in infancy with subsequent onset of atherosclerosis, kidney failure, and scleroderma-like features and death during the teen years. Cockayne syndrome. This includes a number of autosomal recessive disorders with features such as impaired neurologic growth, photosensitivity (xeroderma pigmentosa), and death during childhood years. These disorders are caused by mutations in the genes for DNA excision repair proteins, ERCC-6 and ERCC-8.

1	Gene Studies in Long-Lived Humans The main genes that have been consistently associated with increased longevity in human candidate gene studies are APOE and FOXO3A. ApoE is an apoprotein found in chylomicrons, whereas the ApoE4 isoform is a risk factor for Alzheimer’s disease and cardiovascular disease, which might explain its association with reduced lifespan. FOXO3A is a transcription factor involved in the insulin/IGF-I pathway, and its homolog in C. elegans, daf16, has a substantial impact on aging in these nematodes. Genome-wide association studies (GWAS) of centenarians have confirmed the association of longevity with APOE. GWAS have been used to identify a range of other single nucleotide polymorphisms (SNPs) that might be associated with longevity including SNPs in the sirtuin genes and the progeroid syndrome genes, LMNA and WRN. Gene set analysis of GWAS studies has shown that both the insulin/IGF-I signaling pathway and the telomere maintenance pathway are associated with

1	and the progeroid syndrome genes, LMNA and WRN. Gene set analysis of GWAS studies has shown that both the insulin/IGF-I signaling pathway and the telomere maintenance pathway are associated with longevity.

1	Of particular interest are people with Laron-type dwarfism. These people have mutations in the growth hormone receptor, which causes severe growth hormone resistance. In mice, similar knockout of the growth hormone receptor (GHRKO mice, Methuselah mice) is associated with extremely long life. Therefore, subjects with Laron’s syndrome have been carefully studied, and it was found that they have very low rates of cancer and diabetes mellitus and, possibly, longer lives.

1	Nutrient-Sensing Pathways Many living things have evolved to respond to periods of nutritional shortage and famine by increasing cellular resilience and delaying reproduction until food supply becomes abundant once again. This increases the chances of reproductive success and survival of offspring. Lifelong food shortage, often termed caloric restriction (or dietary restriction) increases lifespan and delays aging in many animals, probably as a nonadaptive side effect of this famine response. Many of the genes and pathways that regulate the way that cells respond to nutritional undersupply have been identified, initially in yeast and C. elegans. In general, manipulation of these pathways (through genetic knockout or overexpression or pharmacologic agonists and antagonists) alters the aging benefits of caloric restriction and, in some cases, the lifespan of animals on normal diets. These pathways are all very influential cellular “switches” that control a wide range of key functions

1	benefits of caloric restriction and, in some cases, the lifespan of animals on normal diets. These pathways are all very influential cellular “switches” that control a wide range of key functions including protein translation, autophagy, mitochondrial function and bioenergetics, and the cellular metabolism of fats, proteins, and carbohydrates. The discovery of these nutrient-sensing pathways has led to targets for pharmacologic extension of lifespan. The main nutrient-sensing pathways that influence aging and responses to caloric restriction include the following:

1	SIRT1. The sirtuins are a class of histone deacetylases that inhibit gene expression. The key nutrient-sensing member of this class in mammals is SIRT1. The activity of SIRT1 is regulated by levels of reduced nicotinamide adenine dinucleotide (NAD+), which are increased when cellular energy stores are depleted. Important downstream targets include PGC1a and NRF2, which act on mitochondrial biogenesis. Target of rapamycin (TOR, or mTOR in mammals). mTOR is activated by branched-chain amino acids, providing a link to dietary protein intake. It is a complex that orchestrates two pathways (TORC1 and TORC2). Key downstream targets of mTOR of relevance to aging include the tuberous sclerosis protein (TSC) and 4EBP1, which influence protein production. 5′ Adenosine monophosphate–activated protein kinase (AMPK). AMPK is activated by increased levels of AMP, which reflect cellular energy status.

1	5′ Adenosine monophosphate–activated protein kinase (AMPK). AMPK is activated by increased levels of AMP, which reflect cellular energy status. • Insulin signaling and IGF-I/growth hormone. These two pathways are usually considered together because they are the same in lower animals and have diverged only in higher animals. Insulin responds to carbohydrate intake. An important downstream target for this pathway is a transcription factor called daf16 in worms and FOXO in mammals and the fruit fly.

1	Mitochondrial Genes Mitochondrial function is influenced by genes located both in the mitochondria (mtDNA) and the nucleus. mtDNA is considered to have a prokaryotic origin and is highly conserved across taxa. It forms a circular loop of 16,569 nucleotides in humans. Aging is associated with increased frequency of mutations in mtDNA as a consequence of its high exposure to oxygen-derived free radicals and relatively inefficient DNA repair machinery. Nuclear DNA encodes approximately 1000–1500 genes for mitochondrial function, including genes involved with oxidative phosphorylation, mitochondrial metabolic pathways, and enzymes required for biogenesis. These genes are thought to have originated in mtDNA but subsequently translocated to the nucleus, and unlike mtDNA genes, their sequence is stable with aging.

1	Genetic manipulation of mitochondrial genes in animals influences aging and lifespan. In C. elegans, many mutants with defective electron transfer chain function have increased lifespan. The mtDNA “mutator” mice, which lack the mtDNA proofreading enzyme, have increased mtDNA mutations and premature aging, whereas overexpression of mitochondrial uncoupling proteins leads to longer lifespan. In humans, hereditary variability in mtDNA is associated with diseases (mitochondriopathies such as Leigh’s disease) and aging. For example, in Europeans, mitochondrial DNA haplogroup J (haplogroups are combinations of genetic variants that exist in specific populations) is associated with longevity, and haplogroup D is overrepresented in Asian centenarians.

1	Aging is an intrinsic feature of human life whose manipulation has fascinated humans ever since becoming conscious of their own existence. Recent reports and scientific literature are shaping a picture where different dietary restriction regimes and exercise interventions 94e-5 may improve healthy aging in laboratory animals. Several long-term experimental interventions (e.g., resveratrol, rapamycin, spermidine, metformin) may open doors for corresponding pharmacologic strategies. Surprisingly, most of the effective aging interventions proposed converge on only a few molecular pathways: nutrient signaling, mitochondrial proteostasis, and the autophagic machinery.

1	Lifespan is inevitably accompanied by functional decline, a steady increase in a plethora of chronic diseases, and ultimately death. For millennia, it has been a dream of mankind to prolong both lifespan and healthspan. Developed countries have profited from the medical improvements and their transfer to public health care systems, as well as from better living conditions derived from their socioeconomic power, to achieve remarkable increases in life expectancy during the last century. In the United States, the percentage of the population age 65 years or older is projected to increase from 13% in 2010 to 19.3% in 2030. However, old age remains the main risk factor for major life-threatening disorders, and the number of people suffering from age-related diseases is anticipated to almost double over the next two decades. The prevalence of age-related pathologies represents a major threat as well as an economic burden that urgently needs effective interventions.

1	Molecules, drugs, and other interventions that might decelerate aging processes continue to raise interest among both the general public and scientists of all biologic and medical fields. Over the past two decades, this interest has taken root in the fact that many of the molecular mechanisms underlying aging are interconnected and linked with pathways that cause disease, including cancer and cardiovascular and neurodegenerative disorders. Unfortunately, among the many proposed aging interventions, only a few have reached a certain age themselves. Results often lack reproducibility because of a simple inherent problem: interventions in aging research take a lifetime. Experiments lasting the lifetime of animal models are prone to develop artifacts, increasing the possibilities and time windows for experimental discrepancies. Some inconsistencies in the field arise from overinterpreting lifespan-shortening models and scenarios as being related to accelerated aging.

1	Many substances and interventions have been claimed to be antiaging throughout history and into the present. In the following sections, interventions will be restricted to those that meet the following highly selective criteria: (1) promotion of lifespan and/or healthspan, (2) validation in at least three model organisms, and (3) confirmation by at least three different laboratories. These interventions include (1) caloric restriction and fasting regimens, (2) some pharmacotherapies (resveratrol, rapamycin, spermidine, metformin), and (3) exercise.

1	Caloric Restriction One of the most important and robust interventions that delays aging is caloric restriction. This outcome has been recorded in rodents, dogs, worms, flies, yeasts, monkeys, and prokaryotes. Calorie restriction is defined as a reduction in the total caloric intake, usually of about 30% and without malnutrition. Caloric restriction reduces the release of growth factors such as growth hormone, insulin, and IGF-I, which are activated by nutrients and have been shown to accelerate aging and enhance the probability for mortality in many organisms. Yet the effects of caloric restriction on aging were first discovered by McCay in 1935 long before the effects of such hormones and growth factors on aging were recognized. The cellular pathways that mediate this remarkable response have been explored in many experimental models. These include the nutrient-sensing pathways (TOR, AMPK, insulin/IGF-I, sirtuins) and transcription factors (FOXO in D. melanogaster and daf16 in C.

1	have been explored in many experimental models. These include the nutrient-sensing pathways (TOR, AMPK, insulin/IGF-I, sirtuins) and transcription factors (FOXO in D. melanogaster and daf16 in C. elegans). The transcription factor Nrf2 appears to confer most of the anticancer properties of caloric restriction in mice, even though it is dispensable for lifespan extension.

1	Two studies have reported the effects of caloric restriction in monkeys with different outcomes: one study observed prolonged life, while the other did not. However, both studies confirmed that caloric restriction increases healthspan by reducing the risk for diabetes, cardiovascular

1	CHAPTER 94e The Biology of Aging disease, and cancer. In humans, caloric restriction is associated with increased lifespan and healthspan. This is most convincingly demonstrated in Okinawa, Japan, where one of the most long-lived human populations resides. In comparison to the rest of the Japanese population, Okinawan people usually combine an above-average amount of daily exercise with a below-average food intake. However, when Okinawan families move to Brazil, they adopt a Western lifestyle that affects both exercise and nutrition, causing a rise in weight and a reduction in life expectancy by nearly two decades. In the Biosphere II project, where volunteers lived together for 24 months undergoing an unforeseen severe caloric restriction, there were improvements in insulin, blood sugar, glycated hemoglobin, cholesterol levels, and blood pressure—all outcomes that would be expected to benefit lifespan. Caloric restriction changes many aspects of human aging that might influence

1	glycated hemoglobin, cholesterol levels, and blood pressure—all outcomes that would be expected to benefit lifespan. Caloric restriction changes many aspects of human aging that might influence lifespan such as the transcriptome, hormonal status (especially IGF-I and thyroid hormones), oxidative stress, inflammation, mitochondrial function, glucose homeostasis, and cardiometabolic risk factors. Epigenetic modifications are an emerging target for caloric restriction.

1	It must be noted that maintaining caloric restriction and avoiding malnutrition is not only arduous in humans but is also linked with substantial side effects. For instance, prolonged reduction of calorie intake may decrease fertility and libido, impair wound healing, reduce the potential to combat infections, and lead to amenorrhea and osteoporosis.

1	Although extreme obesity (body mass index [BMI] >35) leads to a 29% increased risk of dying, people with BMI in the overweight range seem to have reduced mortality, at least in population studies of middle-aged and older subjects. People with a BMI in the overweight range seem more able to counteract and respond to disease, trauma, and infection, whereas caloric restriction impairs healing and immune responses. On the other hand, BMI is an insufficient denominator of body and body fat composition. A well-trained athlete may have a similar BMI as an overweight person because of the higher muscle mass density. The waist-to-hip ratio is a much better indicator for body fat and an excellent and stringent predictor of the risk of dying from cardiovascular disease: the lower the waist-to-hip ratio, the lower is the risk.

1	PERIODIC FASTING How can caloric restriction be translated to humans in a socially and medically feasible way? A whole series of periodic fasting regimens are asserting themselves as suitable strategies, among them the alternate-day fasting diet, the “five:two” intermittent fasting diet, and a 48-h fast once or twice each month. Periodic fasting is psychologically more viable, lacks some of the negative side effects, and is only accompanied by minimal weight loss.

1	It is striking that many cultures implement periodic fasting rituals, for example Buddhists, Christians, Hindus, Jews, Muslims, and some African animistic religions. It could be speculated that a selective advantage of fasting versus nonfasting populations is conferred by health-promoting attributes of religious routines that periodically limit caloric intake. Indeed, several lines of evidence indicate that intermittent fasting regimens exert antiaging effects. For example improved morbidity and longevity were observed among Spanish home nursing residents who underwent alternate-day fasting. Even rats subjected to alternate-day fasting live up to 83% longer than normally fed control animals, and one 24-h fasting period every 4 days is sufficient to generate lifespan extension

1	Repeated fasting and eating cycles may circumvent the negative side effects of sustained caloric restriction. This strategy may even yield effects despite extreme overeating during the nonfasting periods. In a spectacular experiment, mice fed a high-fat diet in a time-restricted manner, i.e., with regular fasting breaks, showed reduced inflammation markers and no fatty liver and were slim in comparison to mice with equivalent total calorie consumption but ad libitum. From an evolutionary point of view, this kind of feeding pattern may reflect mammalian adaptation to food availability: overeating in times of nutrient availability (e.g., after a hunting success) and starvation in between. This is how some indigenous peoples who have avoided Western lifestyles live today; those who have been investigated show limited signs of age-induced diseases such as cancer, neurodegeneration, diabetes, cardiovascular disease, and hypertension.

1	Fasting exerts beneficial effects on healthspan by minimizing the risk of developing agerelated diseases including hypertension, neurodegeneration, cancer, and cardiovascular diseases. The most effective and rapid repercussion of fasting is reduction in hypertension. Two weeks of water-only fasting resulted in a blood pressure below 120/80 mmHg in 82% of subjects with borderline hypertension. Ten days of fasting cured all hypertensive patients who had been taking antihypertensive medication previously.

1	Periodic fasting dampens the consequences of many age-related neurodegenerative diseases (Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and frontotemporal dementia, but not amyotrophic lateral sclerosis in mouse models). Fasting cycles are as effective as chemotherapy against certain tumors in mice. In combination with chemotherapy, fasting protected mice against the negative side effects of chemotherapeutic drugs, while it enhanced their efficacy against tumors. Combining fasting and chemotherapy rendered 20–60% of mice cancer-free when inoculated with highly aggressive tumors like glioblastoma or pancreatic tumors, which have 100% mortality even with chemotherapy. This approach has been attempted in people with some indication that toxicities of chemotherapy are reduced. Pharmacologic Interventions to Delay Aging and Increase Lifespan

1	Virtually all obese people know that stable weight reduction will reduce their elevated risk of cardiometabolic disease and enhance their overall survival, yet only 20% of overweight individuals are able to lose 10% of their weight for a period of at least 1 year. Even in the most motivated people (such as the “Cronies” who deliberately attempt long-term caloric restriction in order to extend their lives), long-term caloric restriction is extremely difficult. Thus, focus has been directed at the possibility of developing medicines that replicate the beneficial effects of caloric restriction without the need for reducing food intake (“CR-mimetics,” Fig. 94e-5): • Resveratrol. Resveratrol, an agonist of SIRT1, is a polyphenol that is found in grapes and in red wine. The potential of resveratrol to promote lifespan was first identified in yeast, and it has gathered fame since, at least in part because it might be responsible for the so-called French paradox whereby wine reduces some of

1	to promote lifespan was first identified in yeast, and it has gathered fame since, at least in part because it might be responsible for the so-called French paradox whereby wine reduces some of the cardiometabolic risks of a high-fat diet. Resveratrol has been reported to increase lifespan in many lower order species such as yeast, fruit flies, worms, and mice on high-fat diets. In monkeys fed a diet high in sugar and fat, resveratrol had beneficial outcomes related to inflammation and cardiometabolic parameters. Some studies in humans have also shown improvements in cardiometabolic function, whereas others have been negative. Gene expression studies in animals and humans reveal that resveratrol mimics some of the metabolic and gene expression changes of caloric restriction.

1	ResveratrolRapamycinSpermidineHOOOOOOOOOHOOOHONMetforminNHNHNNH2NHHOOHOHH2NNH2HNFIGURE 94e-5 Chemical structures of four agents (resveratrol, rapamycin, spermidine, and metformin) that have been shown to delay aging in experimental animal models. Rapamycin. Rapamycin, an inhibitor of mTOR, was originally discovered on Easter Island (Rapa Nui; hence its name) as a bacterial secretion with antibiotic properties. Before its immersion in the antiaging field, rapamycin already had a longstanding career as an immunosuppressant and cancer chemotherapeutic in humans. Rapamycin extends lifespan in all organisms tested so far, including yeast, flies, worms, and mice. However, the potential utility of rapamycin for human lifespan extension is likely to be limited by adverse effects related to immunosuppression, wound healing, proteinuria, and hypercholesterolemia, among others. An alternative strategy may be intermittent rapamycin feeding, which was found to increase mouse lifespan.

1	Spermidine. Spermidine is a physiologic polyamine that induces autophagy-mediated lifespan extension in yeast, flies, and worms. Spermidine levels decrease during the life of virtually all organisms including humans, with the stunning exception of centenarians. Oral administration of spermidine and upregulation of bacterial polyamine production in the gut both lead to lifespan extension in short-lived mouse models. Spermidine has also been found to have beneficial effects on neurodegeneration probably by increasing transcription of genes involved in autophagy.

1	Metformin. Metformin, an activator of AMPK, is a biguanide first isolated from the French lilac that is widely used for the treatment of type 2 diabetes mellitus. Metformin decreases hepatic gluconeogenesis and increases insulin sensitivity. Metformin has other actions including inhibition of mTOR and mitochondrial complex I and activation of the transcription factor SKN-1/Nrf2. Metformin increases lifespan in different mouse strains including female mouse strains predisposed to high incidence of mammary tumors. At a biochemical level, metformin supplementation is associated with reduced oxidative damage and inflammation and mimics some of the gene expression changes seen with caloric restriction.

1	Exercise and Physical Activity In humans and animals, regular exercise reduces the risk of morbidity and mortality. Given that cardiovascular diseases are the dominant cause of aging in humans but not in mice, the effects on human health may be even stronger than those seen in mouse experiments. An increase in aerobic exercise capacity, which declines during aging, is associated with favorable effects on blood pressure, lipids, glucose tolerance, bone density, and depression in older people. Likewise, exercise training protects against aging disorders such as cardiovascular diseases, diabetes mellitus, and osteoporosis. Exercise is the only treatment that can prevent or even reverse sarcopenia (age-related muscle wasting). Even moderate or low levels 94e-7 of exercise (30 min walking per day) have significant protective effects in obese subjects. In older people, regular physical activity has been found to increase the duration of independent living.

1	While clearly promoting health and thus quality of life, regular exercise does not extend lifespan. Furthermore, the combination of exercise with caloric restriction has no additive effect on maximal lifespan in rodents. On the other hand, alternate-day fasting with exercise is more beneficial for the muscle mass than single treatments alone. In nonobese humans, exercise combined with caloric restriction has synergistic effects on insulin sensitivity and inflammation. From the evolutionary perspective, the responses to hunger and exercise are linked: when food is scarce, increased activity is required to hunt and gather.

1	Hormesis The term hormesis describes the, at first sight paradoxic, protective effects conferred by the exposure to low doses of stressors or toxins (or as Nietzsche stated, “What does not kill him makes him stronger”). Adaptive stress responses elicited by noxious agents (chemical, thermal, or radioactive) precondition an organism, rendering it resistant to subsequent higher and otherwise lethal doses of the same trigger. Hormetic stressors have been found to influence aging and lifespan, presumably by increasing cellular resilience to factors that might contribute to aging such as oxidative stress.

1	Yeast cells that have been exposed to low doses of oxidative stress exhibit a marked antistress response that inhibits death following exposure to lethal doses of oxidants. During ischemic preconditioning in humans, short periods of ischemia protect the brain and the heart against a more severe deprivation of oxygen and subsequent reperfusion-induced oxidative stress. Similarly, the lifelong and periodic exposure to various stressors can inhibit or retard the aging process. Consistent with this concept, heat or mild doses of oxidative stress can lead to lifespan extension in C. elegans. Caloric restriction can also be considered to be a type of hormetic stress that results in the activation of antistress transcription factors (Rim15, Gis1, and Msn2/Msn4 in yeast and FOXO in mammals) that enhance the expression of free radical–scavenging factors and heat shock proteins.

1	Clinicians need to understand aging biology in order to better manage people who are elderly now. Moreover there is an urgent need to develop strategies based on aging biology that delay aging, reduce or postpone the onset of age-related disorders, and increase functional life and healthspan for future generations. Interventions related to nutritional interventions and drugs that act on nutrient-sensing pathways are being developed and, in some cases, are already being studied in humans. Whether these interventions are universally effective or species/individual specific needs to be determined.

1	CHAPTER 94e The Biology of Aging 95e-1 CHAPTER 95e Nutrient Requirements and Dietary Assessment Johanna Dwyer Nutrients are substances that are not synthesized in sufficient amounts in the body and therefore must be supplied by the diet. Nutrient requirements for groups of healthy persons have been determined Combinations of plant proteins that complement one another in bio-logic value or combinations of animal and plant proteins can increase biologic value and lower total protein requirements. In healthy people with adequate diets, the timing of protein intake over the course of the day has little effect. Protein needs increase during growth, pregnancy, lactation, and rehabilitation after injury or malnutrition. Tolerance to dietary protein is decreased in renal insufficiency (with consequent uremia) and in liver failure. Normal protein intake can precipitate encephalopathy in patients with cirrhosis of the liver. 95e PART 6: Nutrition and Weight Loss experimentally. The absence of

1	uremia) and in liver failure. Normal protein intake can precipitate encephalopathy in patients with cirrhosis of the liver. 95e PART 6: Nutrition and Weight Loss experimentally. The absence of essential nutrients leads to growth impairment, organ dysfunction, and failure to maintain nitrogen balance or adequate status of other nutrients. For good health, we require energy-providing nutrients (protein, fat, and carbohydrate), vitamins, minerals, and water. Requirements for organic nutrients include 9 essential amino acids, several fatty acids, glucose, 4 fat-soluble vitamins, 10 water-soluble vitamins, dietary fiber, and choline. Several inorganic substances, including 4 minerals, 7 trace minerals, 3 electrolytes, and the ultratrace elements, must also be supplied by diet.

1	The amounts of the essential nutrients that are required by individuals differ by age and physiologic state. Conditionally essential nutrients are not required in the diet but must be supplied to individuals who do not synthesize them in adequate amounts, such as those with genetic defects, those with pathologic conditions such as infection or trauma with nutritional implications, and developmentally immature infants. For example, inositol, taurine, arginine, and glutamine may be needed by premature infants. Many other organic and inorganic compounds that are present in foods, such as pesticides and lead, also have health effects.

1	ESSENTIAL NUTRIENT REQUIREMENTS Energy For weight to remain stable, energy intake must match energy output. The major components of energy output are resting energy expenditure (REE) and physical activity; minor components include the energy cost of metabolizing food (thermic effect of food, or specific dynamic action) and shivering thermogenesis (e.g., cold-induced thermogenesis). The average energy intake is ~2600 kcal/d for American men and ~1800 kcal/d for American women, though these estimates vary with body size and activity level. Formulas for roughly estimating REE are useful in assessing the energy needs of an individual whose weight is stable. Thus, for males, REE = 900 + 10m, and for females, REE = 700 + 7m, where is m mass in kilograms. The calculated REE is then adjusted for physical activity level by multiplying by 1.2 for sedentary, 1.4 for moderately active, or 1.8 for very active individuals. The final figure, the estimated energy requirement (EER), provides an

1	for physical activity level by multiplying by 1.2 for sedentary, 1.4 for moderately active, or 1.8 for very active individuals. The final figure, the estimated energy requirement (EER), provides an approximation of total caloric needs in a state of energy balance for a person of a certain age, sex, weight, height, and physical activity level.

1	For further discussion of energy balance in health and disease, see Chap. 97. Protein Dietary protein consists of both essential and nonessential amino acids that are required for protein synthesis. The nine essential amino acids are histidine, isoleucine, leucine, lysine, methionine/ cystine, phenylalanine/tyrosine, threonine, tryptophan, and valine. Certain amino acids, such as alanine, can also be used for energy and gluconeogenesis. When energy intake is inadequate, protein intake must be increased, because ingested amino acids are diverted into pathways of glucose synthesis and oxidation. In extreme energy deprivation, protein-calorie malnutrition may ensue (Chap. 97).

1	For adults, the recommended dietary allowance (RDA) for protein is ~0.6 g/kg desirable body mass per day, assuming that energy needs are met and that the protein is of relatively high biologic value. Current recommendations for a healthy diet call for at least 10–14% of calories from protein. Most American diets provide at least those amounts. Biologic value tends to be highest for animal proteins, followed by proteins from legumes (beans), cereals (rice, wheat, corn), and roots.

1	Fat and Carbohydrate Fats are a concentrated source of energy and constitute, on average, 34% of calories in U.S. diets. However, for optimal health, fat intake should total no more than 30% of calories. Saturated fat and trans fat should be limited to <10% of calories and polyunsaturated fats to <10% of calories, with monounsaturated fats accounting for the remainder of fat intake. At least 45–55% of total calories should be derived from carbohydrates. The brain requires ~100 g of glucose per day for fuel; other tissues use about 50 g/d. Some tissues (e.g., brain and red blood cells) rely on glucose supplied either exogenously or from muscle proteolysis. Over time, adaptations in carbohydrate needs are possible during hypocaloric states. Like fat (9 kcal/g), carbohydrate (4 kcal/g), and protein (4 kcal/g), alcohol (ethanol) provides energy (7 kcal/g). However, it is not a nutrient.

1	Water For adults, 1–1.5 mL of water per kilocalorie of energy expenditure is sufficient under usual conditions to allow for normal variations in physical activity, sweating, and solute load of the diet. Water losses include 50–100 mL/d in the feces; 500–1000 mL/d by evaporation or exhalation; and, depending on the renal solute load, ≥1000 mL/d in the urine. If external losses increase, intakes must increase accordingly to avoid underhydration. Fever increases water losses by ~200 mL/d per °C; diarrheal losses vary but may be as great as 5 L/d in severe diarrhea. Heavy sweating, vigorous exercise, and vomiting also increase water losses. When renal function is normal and solute intakes are adequate, the kidneys can adjust to increased water intake by excreting up to 18 L of excess water per day (Chap. 404). However, obligatory urine outputs can compromise hydration status when there is inadequate water intake or when losses increase in disease or kidney damage.

1	Infants have high requirements for water because of their large ratio of surface area to volume, their inability to communicate their thirst, and the limited capacity of the immature kidney to handle high renal solute loads. Increased water needs during pregnancy are ~30 mL/d. During lactation, milk production increases daily water requirements so that ~1000 mL of additional water is needed, or 1 mL for each milliliter of milk produced. Special attention must be paid to the water needs of the elderly, who have reduced total body water and blunted thirst sensation and are more likely to be taking medications such as diuretics. Other Nutrients See Chap. 96e for detailed descriptions of vitamins and trace minerals.

1	Other Nutrients See Chap. 96e for detailed descriptions of vitamins and trace minerals. Fortunately, human life and well-being can be maintained within a fairly wide range for most nutrients. However, the capacity for adaptation is not infinite—too much, as well as too little, intake of a nutrient can have adverse effects or alter the health benefits conferred by another nutrient. Therefore, benchmark recommendations regarding nutrient intakes have been developed to guide clinical practice. These quantitative estimates of nutrient intakes are collectively referred to as the dietary reference intakes (DRIs). The DRIs have supplanted the RDAs—the single reference values used in the United States until the early 1990s. DRIs include the estimated average requirement (EAR) for nutrients as well as other reference values used for dietary planning for individuals: the RDA, the adequate intake (AI), and the tolerable upper

1	Life-Stage Vitamin A Vitamin C Vitamin D Vitamin E Vitamin Thiamin Riboflavin Niacin Vitamin B6 Folate Vitamin B12 Pantothenic Biotin Choline Group (μg/d)a (mg/d) (μg/d)b,c (mg/d)d K (μg/d) (mg/d) (mg/d) (mg/d)e (mg/d) (μg/d)f (μg/d) Acid (mg/d) (μg/d) (mg/d)g

1	Birth to 400* 40* 10 4* 2.0* 0.2* 0.3* 2* 0.1* 65* 0.4* 1.7* 5* 125* 6 mo 6–12 mo 500* 50* 10 5* 2.5* 0.3* 0.4* 4* 0.3* 80* 0.5* 1.8* 6* 150* 1–3 y 300 1515 6 30* 0.5 0.5 6 0.5 150 0.9 2* 8* 200* 4–8 y 400 2515 7 55* 0.6 0.6 8 0.6 200 1.2 3* 12* 250* Males 9–13 y 600 4515 11 60* 0.9 0.9 12 1.0 300 1.8 4* 20* 375* 14–18 y 900 7515 15 75* 1.2 1.3 16 1.3 400 2.4 5* 25* 550* 19–30 y 900 9015 15 120* 1.2 1.3 16 1.3 400 2.4 5* 30* 550* 31–50 y 900 9015 15 120* 1.2 1.3 16 1.3 400 2.4 5* 30* 550* 51–70 y 900 9015 15 120* 1.2 1.3 16 1.7 400 2.4h 5* 30* 550* >70 y 900 9020 15 120* 1.2 1.3 16 1.7 400 2.4h 5* 30* 550* Females 9–13 y 600 4515 11 60* 0.9 0.9 12 1.0 300 1.8 4* 20* 375* 14–18 y 700 6515 15 75* 1.0 1.0 14 1.2 400i 2.4 5* 25* 400* 19–30 y 700 7515 15 90* 1.1 1.1 14 1.3 400i 2.4 5* 30* 425* 31–50 y 700 7515 15 90* 1.1 1.1 14 1.3 400i 2.4 5* 30* 425* 51–70 y 700 7515 15 90* 1.1 1.1 14 1.5 400 2.4h 5* 30* 425* >70 y 700 7520 15 90* 1.1 1.1 14 1.5 400 2.4h 5* 30* 425* Pregnant women 14–18

1	31–50 y 700 7515 15 90* 1.1 1.1 14 1.3 400i 2.4 5* 30* 425* 51–70 y 700 7515 15 90* 1.1 1.1 14 1.5 400 2.4h 5* 30* 425* >70 y 700 7520 15 90* 1.1 1.1 14 1.5 400 2.4h 5* 30* 425* Pregnant women 14–18 y 750 8015 15 75* 1.4 1.4 18 1.9 600j 2.6 6* 30* 450* 19–30 y 770 8515 15 90* 1.4 1.4 18 1.9 600j 2.6 6* 30* 450* 31–50 y 770 8515 15 90* 1.4 1.4 18 1.9 600j 2.6 6* 30* 450* Lactating women 14–18 y 1200 115 15 19 75* 1.4 1.6 17 2.0 500 2.8 7* 35* 550* 19–30 y 1300 120 15 19 90* 1.4 1.6 17 2.0 500 2.8 7* 35* 550* 31–50 y 1300 120 15 19 90* 1.4 1.6 17 2.0 500 2.8 7* 35* 550*

1	Note: This table (taken from the DRI reports; see www.nap.edu) presents recommended dietary allowances (RDAs) in bold type and adequate intakes (AIs) in ordinary type followed by an asterisk (*). An RDA is the average daily dietary intake level sufficient to meet the nutrient requirements of nearly all healthy individuals (97–98%) in a group. The RDA is calculated from an estimated average requirement (EAR). If sufficient scientific evidence is not available to establish an EAR and thus to calculate an RDA, an AI is usually developed. For healthy breast-fed infants, an AI is the mean intake. The AI for other life-stage and sex-specific groups is believed to cover the needs of all healthy individuals in those groups, but lack of data or uncertainty in the data makes it impossible to specify with confidence the percentage of individuals covered by this intake.

1	aAs retinol activity equivalents (RAEs). 1 RAE = 1 μg retinol, 12 μg β-carotene, 24 μg α-carotene, or 24 μg β-cryptoxanthin. The RAE for dietary provitamin A carotenoids is twofold greater than the retinol equivalent (RE), whereas the RAE for preformed vitamin A is the same as the RE. bAs cholecalciferol. 1 μg cholecalciferol = 40 IU vitamin D. cUnder the assumption of minimal sunlight. dAs α-tocopherol. α-Tocopherol includes RRR-α-tocopherol, the only form of α-tocopherol that occurs naturally in foods, and the 2R-stereoisomeric forms of α-tocopherol (RRR-, RSR-, RRS-, and RSS-α-tocopherol) that occur in fortified foods and supplements. It does not include the 2S-stereoisomeric forms of α-tocopherol (SRR-, SSR-, SRS-, and SSS-α-tocopherol) also found in fortified foods and supplements. eAs niacin equivalents (NEs). 1 mg of niacin = 60 mg of tryptophan; 0–6 months = preformed niacin (not NE). fAs dietary folate equivalents (DFEs). 1 DFE = 1 μg food folate = 0.6 μg of folic acid from

1	eAs niacin equivalents (NEs). 1 mg of niacin = 60 mg of tryptophan; 0–6 months = preformed niacin (not NE). fAs dietary folate equivalents (DFEs). 1 DFE = 1 μg food folate = 0.6 μg of folic acid from fortified food or as a supplement consumed with food = 0.5 μg of a supplement taken on an empty stomach. gAlthough AIs have been set for choline, there are few data to assess whether a dietary supply of choline is needed at all stages of the life cycle, and it may be that the choline requirement can be met by endogenous synthesis at some of these stages. hBecause 10–30% of older people may malabsorb food-bound B12, it is advisable for those >50 years of age to meet their RDA mainly by consuming foods fortified with B12 or a supplement containing B12. iIn view of evidence linking inadequate folate intake with neural tube defects in the fetus, it is recommended that all women capable of becoming pregnant consume 400 μg of folate from supplements or fortified foods in addition to intake of

1	intake with neural tube defects in the fetus, it is recommended that all women capable of becoming pregnant consume 400 μg of folate from supplements or fortified foods in addition to intake of food folate from a varied diet. jIt is assumed that women will continue consuming 400 μg from supplements or fortified food until their pregnancy is confirmed and they enter prenatal care, which ordinarily occurs after the end of the periconceptional period—the critical time for formation of the neural tube.

1	Source: Food and Nutrition Board, Institute of Medicine, National Academies (http://www.iom.edu/Activities/Nutrition/SummaryDRIs/DRI-Tables.aspx).

1	level (UL). The DRIs also include acceptable macronutrient distribution maintenance of body stores of nutrients; or, if available, the amount ranges (AMDRs) for protein, fat, and carbohydrate. The current DRIs of a nutrient that minimizes the risk of chronic degenerative disease. for vitamins and elements are provided in Tables 95e-1 and 95e-2, Current efforts focus on this last variable, but relevant markers often respectively. Table 95e-3 provides DRIs for water and macronutrients. are not available. EERs are discussed in Chap. 97 on energy balance in health and The EAR is the amount of a nutrient estimated to be adequate for disease. half of the healthy individuals of a specific age and sex. The types of evidence and criteria used to establish nutrient requirements varyEstimated Average Requirement When florid manifestations of the by nutrient, age, and physiologic group. The EAR is not an effectiveclassic dietary-deficiency diseases such as rickets (deficiency of vita-estimate of

1	Requirement When florid manifestations of the by nutrient, age, and physiologic group. The EAR is not an effectiveclassic dietary-deficiency diseases such as rickets (deficiency of vita-estimate of nutrient adequacy in individuals because it is a medianmin D and calcium), scurvy (deficiency of vitamin C), xerophthalmia requirement for a group; 50% of individuals in a group fall below the(deficiency of vitamin A), and protein-calorie malnutrition were com-requirement and 50% fall above it. Thus, a person with a usual intake at mon, nutrient adequacy was inferred from the absence of their clinical the EAR has a 50% risk of inadequate intake. For these reasons, othersigns. Later, biochemical and other changes were found to be evident standards, described below, are more useful for clinical purposes.

1	long before the deficiency became clinically apparent. Consequently, criteria of adequacy are now based on biologic markers when they Recommended Dietary Allowances The RDA is the average daily dietary are available. Priority is given to sensitive biochemical, physiologic, intake level that meets the nutrient requirements of nearly all healthy or behavioral tests that reflect early changes in regulatory processes; persons of a specific sex, age, life stage, or physiologic condition

1	Birth to 6 200* 0.2* 200* 0.01* 110* 0.27* 30* 0.003* 2* 100* 15* 2* 0.4* 0.12* 0.18* mo 6–12 mo 260* 5.5* 220* 0.5* 130* 11 75* 0.6* 3* 275* 20* 3 0.7* 0.37* 0.57* Children 1–3 y 700 11* 340 0.7* 907 80 1.2* 17 460 203 3.0* 1.0* 1.5* 4–8 y 1000 15* 440 1* 90 10 130 1.5* 22 500 305 3.8* 1.2* 1.9* Males 9–13 y 1300 25* 700 2* 120 8 240 1.9* 34 1250 408 4.5* 1.5* 2.3* 14–18 y 1300 35* 890 3* 150 11 410 2.2* 43 1250 55 11 4.7* 1.5* 2.3* 19–30 y 1000 35* 900 4* 150 8 400 2.3* 45 700 5511 4.7* 1.5* 2.3* 31–50 y 1000 35* 900 4* 150 8 420 2.3* 45 700 5511 4.7* 1.5* 2.3* 51–70 y 1000 30* 900 4* 150 8 420 2.3* 45 700 5511 4.7* 1.3* 2.0* >70 y 1200 30* 900 4* 150 8 420 2.3* 45 700 5511 4.7* 1.2* 1.8* Females 9–13 y 1300 21* 700 2* 120 8 240 1.6* 34 1250 408 4.5* 1.5* 2.3* 14–18 y 1300 24* 890 3* 150 15 360 1.6* 43 1250 559 4.7* 1.5* 2.3* 19–30 y 1000 25* 900 3* 150 18 310 1.8* 45 700 558 4.7* 1.5* 2.3* 31–50 y 1000 25* 900 3* 150 18 320 1.8* 45 700 558 4.7* 1.5* 2.3* 51–70 y 1200 20* 900 3* 150

1	15 360 1.6* 43 1250 559 4.7* 1.5* 2.3* 19–30 y 1000 25* 900 3* 150 18 310 1.8* 45 700 558 4.7* 1.5* 2.3* 31–50 y 1000 25* 900 3* 150 18 320 1.8* 45 700 558 4.7* 1.5* 2.3* 51–70 y 1200 20* 900 3* 150 8 320 1.8* 45 700 558 4.7* 1.3* 2.0* >70 y 1200 20* 900 3* 150 8 320 1.8* 45 700 558 4.7* 1.2* 1.8* Pregnant women 14–18 y 1300 29* 1000 3* 220 27 400 2.0* 50 1250 60 12 4.7* 1.5* 2.3* 19–30 y 1000 30* 1000 3* 220 27 350 2.0* 50 700 6011 4.7* 1.5* 2.3* 31–50 y 1000 30* 1000 3* 220 27 360 2.0* 50 700 6011 4.7* 1.5* 2.3* Lactating women 14–18 y 1300 44* 1300 3* 290 10 360 2.6* 50 1250 70 13 5.1* 1.5* 2.3* 19–30 y 1000 45* 1300 3* 290 9 310 2.6* 50 700 7012 5.1* 1.5* 2.3* 31–50 y 1000 45* 1300 3* 290 9 320 2.6* 50 700 7012 5.1* 1.5* 2.3*

1	Note: This table (taken from the DRI reports; see www.nap.edu) presents recommended dietary allowances (RDAs) in bold type and adequate intakes (AIs) in ordinary type followed by an asterisk (*). An RDA is the average daily dietary intake level sufficient to meet the nutrient requirements of nearly all healthy individuals (97–98%) in a group. The RDA is calculated from an estimated average requirement (EAR). If sufficient scientific evidence is not available to establish an EAR and thus to calculate an RDA, an AI is usually developed. For healthy breast-fed infants, an AI is the mean intake. The AI for other life-stage and sex-specific groups is believed to cover the needs of all healthy individuals in those groups, but lack of data or uncertainty in the data makes it impossible to specify with confidence the percentage of individuals covered by this intake.

1	Sources: Food and Nutrition Board, Institute of Medicine, National Academies (http://www.iom.edu/Activities/Nutrition/SummaryDRIs/DRI-Tables.aspx), based on: Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride (1997); Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline (1998); Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids (2000); and Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc (2001); Dietary Reference Intakes for Water, Potassium, Sodium, Chloride, and Sulfate (2005); and Dietary Reference Intakes for Calcium and Vitamin D (2011). These reports can be accessed via www.nap.edu.

1	(e.g., pregnancy or lactation). The RDA, which is the nutrient-intake based on observed or experimentally determined approximations of goal for planning diets of individuals, is defined statistically as two nutrient intakes in healthy people. In the DRIs, AIs rather than RDAs standard deviations above the EAR to ensure that the needs of any are proposed for nutrients consumed by infants (up to age 1 year) as given individual are met. The online tool at http://fnic.nal.usda.gov/ well as for chromium, fluoride, manganese, sodium, potassium, pantointeractiveDRI/ allows health professionals to calculate individualized thenic acid, biotin, choline, and water consumed by persons of all ages. daily nutrient recommendations for dietary planning based on the Vitamin D and calcium recommendations were recently revised, and DRIs for persons of a given age, sex, and weight. The RDAs are used more precise estimates are now available. to formulate food guides such as the U.S. Department of

1	were recently revised, and DRIs for persons of a given age, sex, and weight. The RDAs are used more precise estimates are now available. to formulate food guides such as the U.S. Department of Agriculture

1	Tolerable Upper Levels of Nutrient Intake Healthy individuals derive no (USDA) MyPlate Food Guide for individuals (www.supertracker.usda established benefit from consuming nutrient levels above the RDA or.gov/default.aspx), to create food-exchange lists for therapeutic diet AI. In fact, excessive nutrient intake can disturb body functions andplanning, and as a standard for describing the nutritional content of cause acute, progressive, or permanent disabilities. The tolerable ULfoods and nutrient-containing dietary supplements.

1	is the highest level of chronic nutrient intake (usually daily) that is The risk of dietary inadequacy increases as intake falls below the unlikely to pose a risk of adverse health effects for most of the popula-RDA. However, the RDA is an overly generous criterion for evaluating tion. Data on the adverse effects of large amounts of many nutrientsnutrient adequacy. For example, by definition, the RDA exceeds the are unavailable or too limited to establish a UL. Therefore, the lack ofactual requirements of all but ~2–3% of the population. Therefore, a UL does not mean that the risk of adverse effects from high intakemany people whose intake falls below the RDA may still be getting is nonexistent. Nutrients in commonly eaten foods rarely exceed theenough of the nutrient. On food labels, the nutrient content in a food UL. However, highly fortified foods and dietary supplements provideis stated by weight or as a percent of the daily value (DV), a variant of more concentrated amounts of

1	nutrient content in a food UL. However, highly fortified foods and dietary supplements provideis stated by weight or as a percent of the daily value (DV), a variant of more concentrated amounts of nutrients per serving and thus pose athe RDA used on the nutrition facts panel that, for an adult, represents potential risk of toxicity. Nutrient supplements are labeled with sup-the highest RDA for an adult consuming 2000 kcal.

1	plement facts that express the amount of nutrient in absolute units or Adequate Intake It is not possible to set an RDA for some nutrients as the percentage of the DV provided per recommended serving size. that do not have an established EAR. In this circumstance, the AI is Total nutrient consumption, including that in foods, supplements, DiETARy REfERENCE iNTAkEs (DRis): RECommENDED DiETARy AllowANCEs AND ADEquATE iNTAkEs foR ToTAl wATER AND mACRoNuTRiENTs

1	DiETARy REfERENCE iNTAkEs (DRis): RECommENDED DiETARy AllowANCEs AND ADEquATE iNTAkEs foR ToTAl wATER AND mACRoNuTRiENTs Birth to 6 mo 0.7* 60* NDc 31* 4.4* 0.5* 9.1* 6–12 mo 0.8* 95* ND 30* 4.6* 0.5* 11.0 Children 1–3 y 1.3* 130 19* ND 7* 0.7* 13 4–8 y 1.7* 130 25* ND 10* 0.9* 9–13 y 2.4* 130 31* ND 12* 1.2* 34 14–18 y 3.3* 130 38* ND 16* 1.6* 52 19–30 y 3.7* 130 38* ND 17* 1.6* 56 31–50 y 3.7* 130 38* ND 17* 1.6* 56 51–70 y 3.7* 130 30* ND 14* 1.6* 56 >70 y 3.7* 130 30* ND 14* 1.6* 56 Females 9–13 y 2.1* 130 26* ND 10* 1.0* 34 14–18 y 2.3* 130 26* ND 11* 1.1* 46 19–30 y 2.7* 130 25* ND 12* 1.1* 46 31–50 y 2.7* 130 25* ND 12* 1.1* 46 51–70 y 2.7* 130 21* ND 11* 1.1* 46 >70 y 2.7* 130 21* ND 11* 1.1* 46 Pregnant women 14–18 y 3.0* 175 28* ND 13* 1.4* 71 19–30 y 3.0* 175 28* ND 13* 1.4* 71 31–50 y 3.0* 175 28* ND 13* 1.4* 71 Lactating women 14–18 3.8* 210 29* ND 13* 1.3* 71 19–30 y 3.8* 210 29* ND 13* 1.3* 71 31–50 y 3.8* 210 29* ND 13* 1.3* 71

1	Note: This table (taken from the DRI reports; see www.nap.edu) presents recommended dietary allowances (RDAs) in bold type and adequate intakes (AIs) in ordinary type followed by an asterisk (*). An RDA is the average daily dietary intake level sufficient to meet the nutrient requirements of nearly all healthy individuals (97–98%) in a group. The RDA is calculated from an estimated average requirement (EAR). If sufficient scientific evidence is not available to establish an EAR and thus to calculate an RDA, an AI is usually developed. For healthy breast-fed infants, an AI is the mean intake. The AI for other life-stage and sex-specific groups is believed to cover the needs of all healthy individuals in those groups, but lack of data or uncertainty in the data make it impossible to specify with confidence the percentage of individuals covered by this intake.

1	aTotal water includes all water contained in food, beverages, and drinking water. bBased on grams of protein per kilogram of body weight for the reference body weight (e.g., for adults: 0.8 g/kg body weight for the reference body weight). cNot determined. Source: Food and Nutrition Board, Institute of Medicine, National Academies (http://www.iom.edu/Activities/Nutrition/SummaryDRIs/DRI-Tables.aspx), based on: Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein, and Amino Acids (2002/2005) and Dietary Reference Intakes for Water, Potassium, Sodium, Chloride, and Sulfate (2005). These reports can be accessed via www.nap.edu. and over-the-counter medications (e.g., antacids), should not exceed RDA levels.

1	and over-the-counter medications (e.g., antacids), should not exceed RDA levels. Acceptable Macronutrient Distribution Ranges The AMDRs are not experimentally determined but are rough ranges for energy-providing macronutrient intakes (protein, carbohydrate, and fat) that the Institute of Medicine’s Food and Nutrition Board considers to be healthful. These ranges are 10–35% of calories for protein, 20–35% of calories for fat, and 45–65% of calories for carbohydrate. Alcohol, which also provides energy, is not a nutrient; therefore, no recommendations are not provided. The DRIs are affected by age, sex, rate of growth, pregnancy, lactation, physical activity level, concomitant diseases, drugs, and dietary composition. If requirements for nutrient sufficiency are close to levels indicating excess of a nutrient, dietary planning is difficult.

1	Physiologic Factors Growth, strenuous physical activity, pregnancy, and lactation all increase needs for energy and several essential nutrients. Energy needs rise during pregnancy due to the demands of fetal growth and during lactation because of the increased energy required for milk production. Energy needs decrease with loss of lean body mass, the major determinant of REE. Because lean tissue, physical activity, and health often decline with age, energy needs of older persons, especially those over 70, tend to be lower than those of younger persons.

1	Dietary Composition Dietary composition affects the biologic availability and use of nutrients. For example, the absorption of iron may be impaired by large amounts of calcium or lead; likewise, non-heme iron uptake may be impaired by a lack of ascorbic acid and amino acids in the meal. Protein use by the body may be decreased when essential amino acids are not present in sufficient amounts—a rare scenario in U.S. diets. Animal foods, such as milk, eggs, and meat, have high biologic values, with most of the needed amino acids present in adequate amounts. Plant proteins in corn (maize), soy, rice, and wheat have lower biologic values and must be combined with other plant or animal proteins or fortified with the amino acids that are deficient to achieve optimal use by the body.

1	Route of Intake The RDAs apply only to oral intakes. When nutrients are administered parenterally, similar values can sometimes be used for amino acids, glucose (carbohydrate), fats, sodium, chloride, potassium, and most vitamins because their intestinal absorption rate is nearly 100%. However, the oral bioavailability of most mineral elements may be only half that obtained by parenteral administration. For some nutrients that are not readily stored in the body or that cannot be stored in large amounts, timing of administration may also be important. For example, amino acids cannot be used for protein synthesis if they are not supplied together; instead, they will be used for energy production, although in healthy individuals eating adequate diets, the distribution of protein intake over the course of the day has little effect on health.

1	Disease Dietary deficiency diseases include protein-calorie malnutrition, iron-deficiency anemia, goiter (due to iodine deficiency), rickets and osteomalacia (vitamin D deficiency), and xeropthalmia (vitamin A deficiency), megaloblastic anemia (vitamin B12 or folic acid deficiency), scurvy (vitamin C/ascorbic acid deficiency), beriberi (thiamin deficiency), and pellagra (niacin and tryptophan deficiency) (Chaps. 96e and 97). Each deficiency disease is characterized by imbalances at the cellular level between the supply of nutrients or energy and the body’s nutritional needs for growth, maintenance, and other functions. Imbalances and excesses in nutrient intakes are recognized as risk factors for certain chronic degenerative diseases, such as saturated fat and cholesterol in coronary artery disease; sodium in hypertension; obesity in hormone-dependent endometrial and breast cancers; and ethanol in alcoholism. Because the etiology and pathogenesis of these disorders are multifactorial,

1	disease; sodium in hypertension; obesity in hormone-dependent endometrial and breast cancers; and ethanol in alcoholism. Because the etiology and pathogenesis of these disorders are multifactorial, diet is only one of many risk factors. Osteoporosis, for example, is associated with calcium deficiency, sometimes secondary to vitamin D deficiency, as well as with risk factors related to environment (e.g., smoking, sedentary lifestyle), physiology (e.g., estrogen deficiency), genetic determinants (e.g., defects in collagen metabolism), and drug use (chronic steroid and aromatase inhibitors) (Chap. 425).

1	In clinical situations, nutritional assessment is an iterative process that involves: (1) screening for malnutrition, (2) assessing the diet and other data to establish either the absence or the presence of malnutrition and its possible causes, (3) planning and implementing the most appropriate nutritional therapy, and (4) reassessing intakes to make sure that they have been consumed. Some disease states affect the bioavailability, requirements, use, or excretion of specific nutrients. In these circumstances, specific measurements of various nutrients or their biomarkers may be required to ensure adequate replacement (Chap. 96e).

1	Most health care facilities have nutrition-screening processes in place for identifying possible malnutrition after hospital admission. Nutritional screening is required by the Joint Commission, which accredits and certifies health care organizations in the United States. However, there are no universally recognized or validated standards. The factors that are usually assessed include abnormal weight for height or body mass index (e.g., BMI <19 or >25); reported weight change (involuntary loss or gain of >5 kg in the past 6 months) (Chap. 56); diagnoses with known nutritional implications (e.g., metabolic disease, any disease affecting the gastrointestinal tract, alcoholism); present therapeutic dietary prescription; chronic poor appetite; presence of chewing and swallowing problems or major food intolerances; need for assistance with preparing or shopping for food, eating, or other aspects of self-care; and social isolation. The nutritional status of hospitalized patients should be

1	food intolerances; need for assistance with preparing or shopping for food, eating, or other aspects of self-care; and social isolation. The nutritional status of hospitalized patients should be reassessed periodically—at least once every week.

1	A more complete dietary assessment is indicated for patients who exhibit a high risk of or frank malnutrition on nutritional screening. The type of assessment varies with the clinical setting, the severity of the patient’s illness, and the stability of the patient’s condition.

1	Acute-Care Settings In acute-care settings, anorexia, various other diseases, test procedures, and medications can compromise dietary intake. Under such circumstances, the goal is to identify and avoid inadequate intake and to assure appropriate alimentation. Dietary assessment focuses on what patients are currently eating, whether or not they are able and willing to eat, and whether or not they experience any problems with eating. Dietary intake assessment is based on information from observed intakes; medical records; history; clinical examination; and anthropometric, biochemical, and functional status evaluations. The objective is to gather enough information to establish 95e-5 the likelihood of malnutrition due to poor dietary intake or other causes in order to assess whether nutritional therapy is indicated (Chap. 98e).

1	Simple observations may suffice to suggest inadequate oral intake. These include dietitians’ and nurses’ notes; observation of a patient’s frequent refusal to eat or the amount of food eaten on trays; the frequent performance of tests and procedures that are likely to cause meals to be skipped; adherence to nutritionally inadequate diet orders (e.g., clear liquids or full liquids) for more than a few days; the occurrence of fever, gastrointestinal distress, vomiting, diarrhea, or a comatose state; and the presence of diseases or use of treatments that involve any part of the alimentary tract. Acutely ill patients with diet-related diseases such as diabetes need assessment because an inappropriate diet may exacerbate these conditions and adversely affect other therapies. Abnormal biochemical values (serum albumin levels <35 g/L [<3.5 mg/dL]; serum cholesterol levels <3.9 mmol/L [<150 mg/dL]) are nonspecific but may indicate a need for further nutritional assessment.

1	Most therapeutic diets offered in hospitals are calculated to meet individual nutrient requirements and the RDA if they are eaten. Exceptions include clear liquids, some full-liquid diets, and test diets (such as those adhered to in preparation for gastrointestinal procedures), which are inadequate for several nutrients and should not be used, if possible, for more than 24 h. However, because as much as half of the food served to hospitalized patients is not eaten, it cannot be assumed that the intakes of hospitalized patients are adequate. Dietary assessment should compare how much and what kinds of food the patient has consumed with the diet that has been provided. Major deviations in intakes of energy, protein, fluids, or other nutrients of special concern for the patient’s illness should be noted and corrected.

1	Nutritional monitoring is especially important for patients who are very ill and who have extended lengths of hospital stay. Patients who are fed by enteral and parenteral routes also require special nutritional assessment and monitoring by physicians and/or dietitians with certification in nutritional support (Chap. 98e).

1	Ambulatory Settings The aim of dietary assessment in the outpatient setting is to determine whether or not the patient’s usual diet is a health risk in itself or if it contributes to existing chronic disease-related problems. Dietary assessment also provides the basis for planning a diet that fulfills therapeutic goals while ensuring patient adherence. The outpatient dietary assessment should review the adequacy of present and usual food intakes, including vitamin and mineral supplements, oral nutritional supplements, medical foods, other dietary supplements, medications, and alcohol, because all of these may affect the patient’s nutritional status. The assessment should focus on the dietary constituents that are most likely to be involved or compromised by a specific diagnosis as well as on any comorbidities that are present. More than one day’s intake should be reviewed to provide a better representation of the usual diet.

1	There are many ways to assess the adequacy of a patient’s habitual diet. These include use of a food guide, a food-exchange list, a diet history, or a food-frequency questionnaire. A commonly used food guide for healthy persons is the USDA’s Choose My Plate, which is useful as a rough guide for avoiding inadequate intakes of essential nutrients as well as likely excesses in the amounts of fat (especially saturated and trans fats), sodium, sugar, and alcohol consumed (Table 95e-4). The Choose My Plate graphic emphasizes a balance between calories and nutritional needs, encouraging increased intake of fruits and vegetables, whole grains, and low-fat milk in conjunction with reduced intake of sodium and high-calorie sugary drinks. The Web version of the guide provides a calculator that tailors the number of servings suggested for healthy patients of different weights, sexes, ages, and life-cycle stages to help them to meet their needs while avoiding excess

1	a calculator that tailors the number of servings suggested for healthy patients of different weights, sexes, ages, and life-cycle stages to help them to meet their needs while avoiding excess (http://www.supertracker.usda.gov/default.aspx and www .ChooseMyPlate.gov). Patients who follow ethnic or unusual dietary patterns may need extra instruction on how foods should be categorized and on the appropriate portion sizes that constitute a serving. The process of reviewing the guide with patients helps them transition exchange lists for diabetes and the Academy of Nutrition and Dietetics food-exchange lists for renal disease.

1	Examples of Standard Portion Sizes at Indicated Energy Level Dietary Factor, Unit of Lower: Moderate: Higher: Measure (Advice) 1600 kcal 2200 kcal 2800 kcal Note: Oils (formerly listed with portions of 5, 6, and 8 teaspoons for the lower, moderate, and higher energy levels, respectively) are no longer singled out in Choose My Plate, but rather are included in the empty calories/added sugar category with SOFAS (calories from solid fats and added sugars). The limit is the remaining number of calories in each food pattern above after intake of the recommended amounts of the nutrient-dense foods.

1	aFor example, 1 serving equals 1 slice bread, 1 cup ready-to-eat cereal, or 0.5 cup cooked rice, pasta, or cooked cereal. bFor example, 1 serving equals 1 oz lean meat, poultry, or fish; 1 egg; 1 tablespoon peanut butter; 0.25 cup cooked dry beans; or 0.5 oz nuts or seeds. cFor example, 1 serving equals 1 cup milk or yogurt, 1.5 oz natural cheese, or 2 oz processed cheese. dFormerly called “discretionary calorie allowance.” Portions are calculated as the number of calories remaining after all of the above allotments are accounted for. Abbreviation: oz eq, ounce equivalent. Source: Data from U.S. Department of Agriculture (http://www.Choosemyplate.gov). to healthier dietary patterns and identifies food groups eaten in excess of recommendations or in insufficient quantities. For persons on therapeutic diets, assessment against food-exchange lists may be useful. These include, for example, American Diabetes Association food-

1	Full nutritional status assessment is reserved for seriously ill patients and those at very high nutritional risk when the cause of malnutrition is still uncertain after the initial clinical evaluation and dietary assessment. It involves multiple dimensions, including documentation of dietary intake, anthropometric measurements, biochemical measurements of blood and urine, clinical examination, health history elicitation, and functional status evaluation. Therapeutic dietary prescriptions and menu plans for most diseases are available from most hospitals and from the Academy of Nutrition and Dietetics. For further discussion of nutritional assessment, see Chap. 97.

1	The DRIs (e.g., the EAR, the UL, and energy needs) are esti mates of physiologic requirements based on experimental evidence. Assuming that appropriate adjustments are made for age, sex, body size, and physical activity level, these estimates should be applicable to individuals in most parts of the world. However, the AIs are based on customary and adequate intakes in U.S. and Canadian populations, which appear to be compatible with good health, rather than on a large body of direct experimental evidence. Similarly, the AMDRs represent expert opinion regarding the approximate intakes of energy-providing nutrients that are healthful in these North American populations. Thus these measures should be used with caution in other settings. Nutrient-based standards like the DRIs have also been developed by the World Health Organization/Food and Agricultural Organization of the United Nations and are available on the Web (http://www.who.int/nutrition/topics/nutrecomm/en/index .html). The

1	developed by the World Health Organization/Food and Agricultural Organization of the United Nations and are available on the Web (http://www.who.int/nutrition/topics/nutrecomm/en/index .html). The European Food Safety Authority (EFSA) Panel on Dietetic Products, Nutrition and Allergies periodically publishes its recommendations in the EFSA Journal. Other countries have promulgated similar recommendations. The different standards have many similarities in their basic concepts, definitions, and nutrient recommendation levels, but there are some differences from the DRIs as a result of the functional criteria chosen, environmental differences, the timeliness of the evidence reviewed, and expert judgment.

1	Vitamin and Trace Mineral Deficiency and Excess Robert M. Russell, Paolo M. Suter Vitamins are required constituents of the human diet since they are synthesized inadequately or not at all in the human body. Only small 96e

1	Dietary Level per Day Associated Nutrient Clinical Finding with Overt Deficiency in Adults Contributing Factors to Deficiency amounts of these substances are needed to carry out essential biochemical reactions (e.g., by acting as coenzymes or prosthetic groups). Overt vitamin or trace mineral deficiencies are rare in Western countries because of a plentiful, varied, and inexpensive food supply; food fortification; and use of supplements. However, multiple nutrient deficiencies may appear together in persons who are chronically ill or alcoholic. After gastric bypass surgery, patients are at high risk for multiple nutrient deficiencies. Moreover, subclinical vitamin and trace mineral deficiencies, as diagnosed by laboratory testing, are quite common in the normal population, especially in the geriatric age group. Conversely, because of the widespread use of nutrient supplements, nutrient toxicities are gaining pathophysiologic and clinical importance.

1	Victims of famine, emergency-affected and displaced popula tions, and refugees are at increased risk for protein-energy malnutrition and classic micronutrient deficiencies (vitamin A, iron, iodine) as well as for overt deficiencies in thiamine (beriberi), riboflavin, vitamin C (scurvy), and niacin (pellagra). Body stores of vitamins and minerals vary tremendously. For example, stores of vitamin B12 and vitamin A are large, and an adult may not become deficient until ≥1 year after beginning to eat a deficient diet. However, folate and thiamine may become depleted within weeks among those eating a deficient diet. Therapeutic modalities can deplete essential nutrients from the body; for example, hemodialysis removes water-soluble vitamins, which must be replaced by supplementation.

1	Vitamins and trace minerals play several roles in diseases: (1) Deficiencies of vitamins and minerals may be caused by disease states such as malabsorption. (2) Either deficiency or excess of vitamins and minerals can cause disease in and of itself (e.g., vitamin A intoxication and liver disease). (3) Vitamins and minerals in high doses may be used as drugs (e.g., niacin for hypercholesterolemia). Since they are 96e-1 covered elsewhere, the hematologic-related vitamins and minerals (Chaps. 126 and 128) either are not considered or are considered only briefly in this chapter, as are the bone-related vitamins and minerals (vitamin D, calcium, phosphorus, magnesium; Chap. 423). See also Table 96e-1 and Fig. 96e-1.

1	See also Table 96e-1 and Fig. 96e-1. Thiamine was the first B vitamin to be identified and therefore is referred to as vitamin B1. Thiamine functions in the decarboxylation of α-ketoacids (e.g., pyruvate α-ketoglutarate) and branched-chain amino acids and thus is essential for energy generation. In addition, thiamine pyrophosphate acts as a coenzyme for a transketolase reaction that mediates the conversion of hexose and pentose phosphates. It has been postulated that thiamine plays a role in peripheral nerve conduction, although the exact chemical reactions underlying this function are not known.

1	Food Sources The median intake of thiamine in the United States from food alone is 2 mg/d. Primary food sources for thiamine include yeast, organ meat, pork, legumes, beef, whole grains, and nuts. Milled rice and grains contain little thiamine. Thiamine deficiency is therefore more common in cultures that rely heavily on a rice-based diet. Tea, coffee (regular and decaffeinated), raw fish, and shellfish contain thiaminases, which can destroy the vitamin. Thus, drinking large amounts of tea or coffee can theoretically lower thiamine body stores.

1	Deficiency Most dietary deficiency of thiamine worldwide is the result of poor dietary intake. In Western countries, the primary causes of thiamine deficiency are alcoholism and chronic illnesses such as cancer. Alcohol interferes directly with the absorption of thiamine and with the synthesis of thiamine pyrophosphate, and it increases urinary excretion. Thiamine should always be replenished when a patient with alcoholism is being refed, as carbohydrate repletion without adequate thiamine can precipitate acute thiamine deficiency with lactic acidosis. Other at-risk populations are women with prolonged hyperemesis gravidarum and anorexia, patients with overall poor nutritional status who are receiving parenteral glucose, patients who have had Thiamine Beriberi: neuropathy, muscle weakness and wasting, cardiomegaly, edema, ophthalmoplegia, confabulation Riboflavin Magenta tongue, angular stomatitis, seborrhea, cheilosis

1	Thiamine Beriberi: neuropathy, muscle weakness and wasting, cardiomegaly, edema, ophthalmoplegia, confabulation Riboflavin Magenta tongue, angular stomatitis, seborrhea, cheilosis Niacin Pellagra: pigmented rash of sun-exposed areas, bright red tongue, diarrhea, apathy, memory loss, disorientation Vitamin B6 Seborrhea, glossitis, convulsions, neuropathy, depression, confusion, microcytic anemia Folate Megaloblastic anemia, atrophic glossitis, depression, Vitamin B12 Megaloblastic anemia, loss of vibratory and position sense, abnormal gait, dementia, impotence, loss of bladder and bowel control, ↑ homocysteine, ↑ methylmalonic acid Vitamin C Scurvy: petechiae, ecchymosis, coiled hairs, inflamed and bleeding gums, joint effusion, poor wound healing, fatigue Vitamin A Xerophthalmia, night blindness, Bitot’s spots, follicular hyperkeratosis, impaired embryonic development, immune dysfunction Vitamin D Rickets: skeletal deformation, rachitic rosary, bowed legs; osteomalacia

1	Vitamin D Rickets: skeletal deformation, rachitic rosary, bowed legs; osteomalacia Vitamin E Peripheral neuropathy, spinocerebellar ataxia, skeletal muscle atrophy, retinopathy Vitamin K Elevated prothrombin time, bleeding <0.3 mg/1000 kcal <0.6 mg <9.0 niacin equivalents <0.2 mg <100 μg/d <1.0 μg/d <2.0 μg/d Not described unless underlying contributing factor is present <10 μg/d Alcoholism, chronic diuretic use, hyperemesis, thiaminases in food Alcoholism, vitamin B6 deficiency, riboflavin deficiency, tryptophan deficiency Alcoholism, isoniazid Alcoholism, sulfasalazine, pyrimethamine, triamterene Gastric atrophy (pernicious anemia), terminal ileal disease, strict vegetarianism, acid-reducing drugs (e.g., H2 blockers), metformin Smoking, alcoholism Fat malabsorption, infection, measles, alcoholism, protein-energy malnutrition Aging, lack of sunlight exposure, fat malabsorption, deeply pigmented skin

1	Smoking, alcoholism Fat malabsorption, infection, measles, alcoholism, protein-energy malnutrition Aging, lack of sunlight exposure, fat malabsorption, deeply pigmented skin Occurs only with fat malabsorption or genetic abnormalities of vitamin E metabolism/transport Fat malabsorption, liver disease, antibiotic use FIgurE 96e-1 Structures and principal functions of vitamins associated with human disorders. and peripheral neuritis. Patients with dry beriberi present with a symmetric peripheral neuropathy of the motor and sensory systems, with diminished reflexes. The neuropathy affects the legs most markedly, and patients have difficulty rising from a squatting position.

1	Alcoholic patients with chronic thiamine deficiency also may have central nervous system (CNS) manifestations known as Wernicke’s encephalopathy, which consists of horizontal nystagmus, ophthalmoplegia (due to weakness of one or more extraocular muscles), cerebellar ataxia, and mental impairment (Chap. 467). When there is an additional loss of memory and a confabulatory psychosis, the syndrome is known as Wernicke-Korsakoff syndrome. Despite the typical clinical picture and history, Wernicke-Korsakoff syndrome is underdiagnosed.

1	The laboratory diagnosis of thiamine deficiency usually is made by a functional enzymatic assay of transketolase activity measured before and after the addition of thiamine pyrophosphate. A >25% stimulation in response to the addition of thiamine pyrophosphate (i.e., an activity coefficient of 1.25) is interpreted as abnormal. Thiamine or the phosphorylated esters of thiamine in serum or blood also can be measured by high-performance liquid chromatography to detect deficiency.

1	In acute thiamine deficiency with either cardiovascular or neurologic signs, 200 mg of thiamine three times daily should be given intravenously until there is no further improvement in acute symptoms; oral thiamine (10 mg/d) should subsequently be given until recovery is complete. Cardiovascular and ophthalmoplegic improvement occurs within 24 h. Other manifestations gradually clear, although psychosis in Wernicke-Korsakoff syndrome may be permanent or may persist for several months. Other nutrient deficiencies should be corrected concomitantly. Toxicity Although anaphylaxis has been reported after high intravenous doses of thiamine, no adverse effects have been recorded from either food or supplements at high doses. Thiamine supplements may be bought over the counter in doses of up to 50 mg/d.

1	Riboflavin is important for the metabolism of fat, carbohydrate, and protein, acting as a respiratory coenzyme and an electron donor. Enzymes that contain flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as prosthetic groups are known as flavoenzymes (e.g., succinic acid dehydrogenase, monoamine oxidase, glutathione reductase). FAD is a cofactor for methyltetrahydrofolate reductase and therefore modulates homocysteine metabolism. The vitamin also plays a role in drug and steroid metabolism, including detoxification reactions. Although much is known about the chemical and enzymatic reactions of riboflavin, the clinical manifestations of riboflavin deficiency are nonspecific and are similar to those of other deficiencies of B vitamins. Riboflavin deficiency is manifested principally by lesions of the mucocutaneous surfaces of the mouth and skin. In addition, corneal 96e-4 vascularization, anemia, and personality changes have been described with riboflavin deficiency.

1	Deficiency and Excess Riboflavin deficiency almost always is due to dietary deficiency. Milk, other dairy products, and enriched breads and cereals are the most important dietary sources of riboflavin in the United States, although lean meat, fish, eggs, broccoli, and legumes are also good sources. Riboflavin is extremely sensitive to light, and milk should be stored in containers that protect against photodegradation. Laboratory diagnosis of riboflavin deficiency can be made by determination of red blood cell or urinary riboflavin concentrations or by measurement of erythrocyte glutathione reductase activity, with and without added FAD. Because the capacity of the gastrointestinal tract to absorb riboflavin is limited (~20 mg after one oral dose), riboflavin toxicity has not been described.

1	The term niacin refers to nicotinic acid and nicotinamide and their biologically active derivatives. Nicotinic acid and nicotinamide serve as precursors of two coenzymes, nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP), which are important in numerous oxidation and reduction reactions in the body. In addition, NAD and NADP are active in adenine diphosphate–ribose transfer reactions involved in DNA repair and calcium mobilization. Metabolism and requirements Nicotinic acid and nicotinamide are absorbed well from the stomach and small intestine. The bioavailability of niacin from beans, milk, meat, and eggs is high; bioavailability from cereal grains is lower. Since flour is enriched with “free” niacin (i.e., the non-coenzyme form), bioavailability is excellent. Median intakes of niacin in the United States considerably exceed the recommended dietary allowance (RDA).

1	The amino acid tryptophan can be converted to niacin with an efficiency of 60:1 by weight. Thus, the RDA for niacin is expressed in niacin equivalents. A lower-level conversion of tryptophan to niacin occurs in vitamin B6 and/or riboflavin deficiencies and in the presence of isoniazid. The urinary excretion products of niacin include 2-pyridone and 2-methyl nicotinamide, measurements of which are used in the diagnosis of niacin deficiency.

1	Deficiency Niacin deficiency causes pellagra, which is found mostly among people eating corn-based diets in parts of China, Africa, and India. Pellagra in North America is found mainly among alcoholics; among patients with congenital defects of intestinal and kidney absorption of tryptophan (Hartnup disease; Chap. 434e); and among patients with carcinoid syndrome (Chap. 113), in which there is increased conversion of tryptophan to serotonin. The antituberculosis drug isoniazid is a structural analog of niacin and can precipitate pellagra. In the setting of famine or population displacement, pellagra results from the absolute lack of niacin but also from the deficiency of micronutrients required for the conversion of tryptophan to niacin (e.g., iron, riboflavin, and pyridoxine). The early symptoms of pellagra include loss of appetite, generalized weakness and irritability, abdominal pain, and vomiting. Bright red glossitis then ensues and is followed by a characteristic skin rash that

1	symptoms of pellagra include loss of appetite, generalized weakness and irritability, abdominal pain, and vomiting. Bright red glossitis then ensues and is followed by a characteristic skin rash that is pigmented and scaling, particularly in skin areas exposed to sunlight. This rash is known as Casal’s necklace because it forms a ring around the neck; it is seen in advanced cases. Vaginitis and esophagitis also may occur. Diarrhea (due in part to proctitis and in part to malabsorption), depression, seizures, and dementia are also part of the pellagra syndrome. The primary manifestations of this syndrome are sometimes referred to as “the four D’s”: dermatitis, diarrhea, and dementia leading to death.

1	Treatment of pellagra consists of oral supplementation with 100– 200 mg of nicotinamide or nicotinic acid three times daily for 5 days. High doses of nicotinic acid (2 g/d in a time-release form) are used for the treatment of elevated cholesterol and triglyceride levels and/ or low high-density lipoprotein cholesterol levels (Chap. 421).

1	Toxicity Prostaglandin-mediated flushing due to binding of the vitamin to a G protein–coupled receptor has been observed at daily nicotinic acid doses as low as 30 mg taken as a supplement or as therapy for dyslipidemia. There is no evidence of toxicity from niacin that is derived from food sources. Flushing always starts in the face and may be accompanied by skin dryness, itching, paresthesia, and headache. Pharmaceutical preparations of nicotinic acid combined with laropiprant, a selective prostaglandin D2 receptor 1 antagonist, or premedication with aspirin may alleviate these symptoms. Flushing is subject to tachyphylaxis and often improves with time. Nausea, vomiting, and abdominal pain also occur at similar doses of niacin. Hepatic toxicity is the most serious toxic reaction caused by sustained-release niacin and may present as jaundice with elevated aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. A few cases of fulminant hepatitis requiring liver

1	by sustained-release niacin and may present as jaundice with elevated aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. A few cases of fulminant hepatitis requiring liver transplantation have been reported at doses of 3–9 g/d. Other toxic reactions include glucose intolerance, hyperuricemia, macular edema, and macular cysts. The combination of nicotinic acid preparations for dyslipidemia with 3-hydroxy3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors may increase the risk of rhabdomyolysis. The upper limit for daily niacin intake has been set at 35 mg. However, this upper limit does not pertain to the therapeutic use of niacin.

1	Vitamin B6 refers to a family of compounds that includes pyridoxine, pyridoxal, pyridoxamine, and their 5′-phosphate derivatives. 5′-Pyridoxal phosphate (PLP) is a cofactor for more than 100 enzymes involved in amino acid metabolism. Vitamin B6 also is involved in heme and neurotransmitter synthesis and in the metabolism of glycogen, lipids, steroids, sphingoid bases, and several vitamins, including the conversion of tryptophan to niacin. Dietary Sources Plants contain vitamin B6 in the form of pyridoxine, whereas animal tissues contain PLP and pyridoxamine phosphate. The vitamin B6 contained in plants is less bioavailable than that in animal tissues. Rich food sources of vitamin B6 include legumes, nuts, wheat bran, and meat, although it is present in all food groups.

1	Deficiency Symptoms of vitamin B6 deficiency include epithelial changes, as seen frequently with other B vitamin deficiencies. In addition, severe vitamin B6 deficiency can lead to peripheral neuropathy, abnormal electroencephalograms, and personality changes that include depression and confusion. In infants, diarrhea, seizures, and anemia have been reported. Microcytic hypochromic anemia is due to diminished hemoglobin synthesis, since the first enzyme involved in heme biosynthesis (aminolevulinate synthase) requires PLP as a cofactor (Chap. 126). In some case reports, platelet dysfunction has been reported. Since vitamin B6 is necessary for the conversion of homocysteine to cystathionine, it is possible that chronic low-grade vitamin B6 deficiency may result in hyperhomocysteinemia and increased risk of cardiovascular disease (Chaps. 291e and 434e). Independent of homocysteine, low levels of circulating vitamin B6 have been associated with inflammation and elevated levels of

1	and increased risk of cardiovascular disease (Chaps. 291e and 434e). Independent of homocysteine, low levels of circulating vitamin B6 have been associated with inflammation and elevated levels of C-reactive protein.

1	Certain medications, such as isoniazid, L-dopa, penicillamine, and cycloserine, interact with PLP due to a reaction with carbonyl groups. Pyridoxine should be given concurrently with isoniazid to avoid neuropathy. The increased ratio of AST to ALT seen in alcoholic liver disease reflects the relative vitamin B6 dependence of ALT. Vitamin B6 dependency syndromes that require pharmacologic doses of vitamin B6 are rare; they include cystathionine β-synthase deficiency, pyridoxineresponsive (primarily sideroblastic) anemias, and gyrate atrophy with chorioretinal degeneration due to decreased activity of the mitochondrial enzyme ornithine aminotransferase. In these situations, 100–200 mg/d of oral vitamin B6 is required for treatment. High doses of vitamin B6 have been used to treat carpal tunnel syndrome, premenstrual syndrome, schizophrenia, autism, and diabetic neuropathy but have not been found to be effective.

1	High doses of vitamin B6 have been used to treat carpal tunnel syndrome, premenstrual syndrome, schizophrenia, autism, and diabetic neuropathy but have not been found to be effective. The laboratory diagnosis of vitamin B6 deficiency is generally based on low plasma PLP values (<20 nmol/L). Vitamin B6 deficiency is treated with 50 mg/d; higher doses of 100–200 mg/d are given if the deficiency is related to medication use. Vitamin B6 should not be given with l-dopa, since the vitamin interferes with the action of this drug. Toxicity The safe upper limit for vitamin B6 has been set at 100 mg/d, although no adverse effects have been associated with high intakes of vitamin B6 from food sources only. When toxicity occurs, it causes severe sensory neuropathy, leaving patients unable to walk. Some cases of photosensitivity and dermatitis have been reported. See Chap. 128.

1	See Chap. 128. Both ascorbic acid and its oxidized product dehydroascorbic acid are biologically active. Actions of vitamin C include antioxidant activity, promotion of nonheme iron absorption, carnitine biosynthesis, conversion of dopamine to norepinephrine, and synthesis of many peptide hormones. Vitamin C is also important for connective tissue metabolism and cross-linking (proline hydroxylation), and it is a component of many drug-metabolizing enzyme systems, particularly the mixed-function oxidase systems. Absorption and Dietary Sources Vitamin C is almost completely absorbed if <100 mg is administered in a single dose; however, only 50% or less is absorbed at doses >1 g. Enhanced degradation and fecal and urinary excretion of vitamin C occur at higher intake levels.

1	Good dietary sources of vitamin C include citrus fruits, green vegetables (especially broccoli), tomatoes, and potatoes. Consumption of five servings of fruits and vegetables a day provides vitamin C in excess of the RDA of 90 mg/d for men and 75 mg/d for women. In addition, ~40% of the U.S. population consumes vitamin C as a dietary supplement in which “natural forms” of the vitamin are no more bioavailable than synthetic forms. Smoking, hemodialysis, pregnancy, and stress (e.g., infection, trauma) appear to increase vitamin C requirements.

1	Deficiency Vitamin C deficiency causes scurvy. In the United States, this condition is seen primarily among the poor and the elderly, in alcoholics who consume <10 mg/d of vitamin C, and in individuals consuming macrobiotic diets. Vitamin C deficiency also can occur in young adults who eat severely unbalanced diets. In addition to generalized fatigue, symptoms of scurvy primarily reflect impaired formation of mature connective tissue and include bleeding into the skin (petechiae, ecchymoses, perifollicular hemorrhages); inflamed and bleeding gums; and manifestations of bleeding into joints, the peritoneal cavity, the pericardium, and the adrenal glands. In children, vitamin C deficiency may cause impaired bone growth. Laboratory diagnosis of vitamin C deficiency is based on low plasma or leukocyte levels.

1	Administration of vitamin C (200 mg/d) improves the symptoms of scurvy within several days. High-dose vitamin C supplementation (e.g., 1–2 g/d) may slightly decrease the symptoms and duration of upper respiratory tract infections. Vitamin C supplementation has also been reported to be useful in Chédiak-Higashi syndrome (Chap. 80) and osteogenesis imperfecta (Chap. 427). Diets high in vitamin C have been claimed to lower the incidence of certain cancers, particularly esophageal and gastric cancers. If proved, this effect may be due to the fact that vitamin C can prevent the conversion of nitrites and secondary amines to carcinogenic nitrosamines. However, an intervention study from China did not show vitamin C to be protective. A potential role for parenteral ascorbic acid in the treatment of advanced cancers has been suggested.

1	Toxicity Taking >2 g of vitamin C in a single dose may result in abdominal pain, diarrhea, and nausea. Since vitamin C may be metabolized to oxalate, it is feared that chronic high-dose vitamin C supplementation could result in an increased prevalence of kidney stones. However, except in patients with preexisting renal disease, this association has not been borne out in several trials. Nevertheless, it is reasonable to advise patients with a history of kidney stones not to take large doses of vitamin C. There is also an unproven but possible risk that chronic high doses of vitamin C could promote iron overload and iron toxicity. High doses of vitamin C can induce hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency, and doses >1 g/d 96e-5 can cause false-negative guaiac reactions and interfere with tests for urinary glucose. High doses may interfere with the activity of certain drugs (e.g., bortezomib in myeloma patients).

1	Biotin is a water-soluble vitamin that plays a role in gene expression, gluconeogenesis, and fatty acid synthesis and serves as a CO2 carrier on the surface of both cytosolic and mitochondrial carboxylase enzymes. The vitamin also functions in the catabolism of specific amino acids (e.g., leucine) and in gene regulation by histone biotinylation. Excellent food sources of biotin include organ meat such as liver or kidney, soy and other beans, yeast, and egg yolks; however, egg white contains the protein avidin, which strongly binds the vitamin and reduces its bioavailability.

1	Biotin deficiency due to low dietary intake is rare; rather, deficiency is due to inborn errors of metabolism. Biotin deficiency has been induced by experimental feeding of egg white diets and by biotin-free parenteral nutrition in patients with short bowels. In adults, biotin deficiency results in mental changes (depression, hallucinations), paresthesia, anorexia, and nausea. A scaling, seborrheic, and erythematous rash may occur around the eyes, nose, and mouth as well as on the extremities. In infants, biotin deficiency presents as hypotonia, lethargy, and apathy. In addition, infants may develop alopecia and a characteristic rash that includes the ears. The laboratory diagnosis of biotin deficiency can be established on the basis of a decreased concentration of urinary biotin (or its major metabolites), increased urinary excretion of 3-hydroxyisovaleric acid after a leucine challenge, or decreased activity of biotin-dependent enzymes in lymphocytes (e.g., propionyl-CoA

1	(or its major metabolites), increased urinary excretion of 3-hydroxyisovaleric acid after a leucine challenge, or decreased activity of biotin-dependent enzymes in lymphocytes (e.g., propionyl-CoA carboxylase). Treatment requires pharmacologic doses of biotin–i.e., up to 10 mg/d. No toxicity is known.

1	Pantothenic acid is a component of coenzyme A and phosphopantetheine, which are involved in fatty acid metabolism and the synthesis of cholesterol, steroid hormones, and all compounds formed from isoprenoid units. In addition, pantothenic acid is involved in the acetylation of proteins. The vitamin is excreted in the urine, and the laboratory diagnosis of deficiency is based on low urinary vitamin levels.

1	The vitamin is ubiquitous in the food supply. Liver, yeast, egg yolks, whole grains, and vegetables are particularly good sources. Human pantothenic acid deficiency has been demonstrated only by experimental feeding of diets low in pantothenic acid or by administration of a specific pantothenic acid antagonist. The symptoms of pantothenic acid deficiency are nonspecific and include gastrointestinal disturbance, depression, muscle cramps, paresthesia, ataxia, and hypoglycemia. Pantothenic acid deficiency is believed to have caused the “burning feet syndrome” seen in prisoners of war during World War II. No toxicity of this vitamin has been reported.

1	Choline is a precursor for acetylcholine, phospholipids, and betaine. Choline is necessary for the structural integrity of cell membranes, cholinergic neurotransmission, lipid and cholesterol metabolism, methyl-group metabolism, and transmembrane signaling. Recently, a recommended adequate intake was set at 550 mg/d for men and 425 mg/d for women, although certain genetic polymorphisms can increase an individual’s requirement. Choline is thought to be a “conditionally essential” nutrient in that its de novo synthesis occurs in the liver and results in lesser-than-used amounts only under certain stress conditions (e.g., alcoholic liver disease). The dietary requirement for choline depends on the status of other nutrients involved in methyl-group metabolism (folate, vitamin B12, vitamin B6, and methionine) and thus varies widely. Choline is widely distributed in food (e.g., egg yolks, wheat germ, organ meat, milk) in the form of lecithin (phosphatidylcholine). Choline deficiency has

1	B6, and methionine) and thus varies widely. Choline is widely distributed in food (e.g., egg yolks, wheat germ, organ meat, milk) in the form of lecithin (phosphatidylcholine). Choline deficiency has occurred in patients receiving parenteral nutrition devoid of choline. Deficiency results in fatty liver, elevated aminotransferase levels, and skeletal muscle damage with high creatine phosphokinase values. The diagnosis of choline deficiency is 96e-6 currently based on low plasma levels, although nonspecific conditions (e.g., heavy exercise) may also suppress plasma levels. Toxicity from choline results in hypotension, cholinergic sweating, diarrhea, salivation, and a fishy body odor. The upper limit for choline intake has been set at 3.5 g/d. Because of its ability to lower cholesterol and homocysteine levels, choline treatment has been suggested for patients with dementia and patients at high risk of cardiovascular disease. However, the benefits of such treatment have not been firmly

1	homocysteine levels, choline treatment has been suggested for patients with dementia and patients at high risk of cardiovascular disease. However, the benefits of such treatment have not been firmly documented. Cholineand betaine-restricted diets are of therapeutic value in trimethylaminuria (“fish odor syndrome”).

1	Flavonoids constitute a large family of polyphenols that contribute to the aroma, taste, and color of fruits and vegetables. Major groups of dietary flavonoids include anthocyanidins in berries; catechins in green tea and chocolate; flavonols (e.g., quercitin) in broccoli, kale, leeks, onions, and the skins of grapes and apples; and isoflavones (e.g., genistein) in legumes. Isoflavones have a low bioavailability and are partially metabolized by the intestinal flora. The dietary intake of flavonoids is estimated at 10–100 mg/d; this figure is almost certainly an underestimate attributable to a lack of information on their concentrations in many foods. Several flavonoids have antioxidant activity and affect cell signaling. From observational epidemiologic studies and limited clinical (human and animal) studies, flavonoids have been postulated to play a role in the prevention of several chronic diseases, including neurodegenerative disease, diabetes, and osteoporosis. The ultimate

1	(human and animal) studies, flavonoids have been postulated to play a role in the prevention of several chronic diseases, including neurodegenerative disease, diabetes, and osteoporosis. The ultimate importance and usefulness of these compounds against human disease have not been demonstrated.

1	Vitamin A, in the strictest sense, refers to retinol. However, the oxidized metabolites retinaldehyde and retinoic acid are also biologically active compounds. The term retinoids includes all molecules (including synthetic molecules) that are chemically related to retinol. Retinaldehyde (11-cis) is the essential form of vitamin A that is required for normal vision, whereas retinoic acid is necessary for normal morphogenesis, growth, and cell differentiation. Retinoic acid does not function in vision and, in contrast to retinol, is not involved in reproduction. Vitamin A also plays a role in iron utilization, humoral immunity, T cell–mediated immunity, natural killer cell activity, and phagocytosis. Vitamin A is commercially available in esterified forms (e.g., acetate, palmitate), which are more stable than other forms.

1	There are more than 600 carotenoids in nature, ~50 of which can be metabolized to vitamin A. β-Carotene is the most prevalent carotenoid with provitamin A activity in the food supply. In humans, significant fractions of carotenoids are absorbed intact and are stored in liver and fat. It is estimated that ≥12 μg (range, 4–27 μg) of dietary all-trans β-carotene is equivalent to 1 μg of retinol activity, whereas the figure is ≥24 μg for other dietary provitamin A carotenoids (e.g., cryptoxanthin, α-carotene). The vitamin A equivalency for a β-carotene supplement in an oily solution is 2:1.

1	Metabolism The liver contains ~90% of the vitamin A reserves and secretes vitamin A in the form of retinol, which is bound to retinolbinding protein. Once binding has occurred, the retinol-binding protein complex interacts with a second protein, transthyretin. This trimolecular complex functions to prevent vitamin A from being filtered by the kidney glomerulus, thus protecting the body against the toxicity of retinol and allowing retinol to be taken up by specific cell-surface receptors that recognize retinol-binding protein. A certain amount of vitamin A enters peripheral cells even if it is not bound to retinol-binding protein. After retinol is internalized by the cell, it becomes bound to a series of cellular retinol-binding proteins, which function as sequestering and transporting agents as well as co-ligands for enzymatic reactions. Certain cells also contain retinoic acid–binding proteins, which have sequestering functions but also shuttle retinoic acid to the nucleus and enable

1	as well as co-ligands for enzymatic reactions. Certain cells also contain retinoic acid–binding proteins, which have sequestering functions but also shuttle retinoic acid to the nucleus and enable its metabolism.

1	Retinoic acid is a ligand for certain nuclear receptors that act as transcription factors. Two families of receptors (retinoic acid receptors [RARs] and retinoid X receptors [RXRs]) are active in retinoid-mediated gene transcription. Retinoid receptors regulate transcription by binding as dimeric complexes to specific DNA sites—the retinoic acid response elements—in target genes (Chap. 400e). The receptors can either stimulate or repress gene expression in response to their ligands. RARs bind all-trans retinoic acid and 9-cis-retinoic acid, whereas RXRs bind only 9-cis-retinoic acid.

1	The retinoid receptors play an important role in controlling cell proliferation and differentiation. Retinoic acid is useful in the treatment of promyelocytic leukemia (Chap. 132) and also is used in the treatment of cystic acne because it inhibits keratinization, decreases sebum secretion, and possibly alters the inflammatory reaction (Chap. 71). RXRs dimerize with other nuclear receptors to function as coregulators of genes responsive to retinoids, thyroid hormone, and calcitriol. RXR agonists induce insulin sensitivity experimentally, perhaps because RXRs are cofactors for the peroxisome proliferatoractivated receptors, which are targets for thiazolidinedione drugs such as rosiglitazone and troglitazone (Chap. 418).

1	Dietary Sources The retinol activity equivalent (RAE) is used to express the vitamin A value of food: 1 RAE is defined as 1 μg of retinol (0.003491 mmol), 12 μg of β-carotene, and 24 μg of other provitamin A carotenoids. In older literature, vitamin A often was expressed in international units (IU), with 1 μg of retinol equal to 3.33 IU of retinol and 20 IU of β-carotene, but these units are no longer in scientific use.

1	Liver, fish, and eggs are excellent food sources for preformed vitamin A; vegetable sources of provitamin A carotenoids include dark green and deeply colored fruits and vegetables. Moderate cooking of vegetables enhances carotenoid release for uptake in the gut. Carotenoid absorption is also aided by some fat in a meal. Infants are particularly susceptible to vitamin A deficiency because neither breast nor cow’s milk supplies enough vitamin A to prevent deficiency. In developing countries, chronic dietary deficiency is the main cause of vitamin A deficiency and is exacerbated by infection. In early childhood, low vitamin A status results from inadequate intakes of animal food sources and edible oils, both of which are expensive, coupled with seasonal unavailability of vegetables and fruits and lack of marketed fortified food products. Concurrent zinc deficiency can interfere with the mobilization of vitamin A from liver stores. Alcohol interferes with the conversion of retinol to

1	and lack of marketed fortified food products. Concurrent zinc deficiency can interfere with the mobilization of vitamin A from liver stores. Alcohol interferes with the conversion of retinol to retinaldehyde in the eye by competing for alcohol (retinol) dehydrogenase. Drugs that interfere with the absorption of vitamin A include mineral oil, neomycin, and cholestyramine.

1	Deficiency Vitamin A deficiency is endemic in areas where diets are chronically poor, especially in southern Asia, sub-

1	Saharan Africa, some parts of Latin America, and the western Pacific, including parts of China. Vitamin A status is usually assessed by measuring serum retinol (normal range, 1.05–3.50 μmol/L [30–100 μg/dL]) or blood-spot retinol or by tests of dark adaptation. Stable isotopic or invasive liver biopsy methods are available to estimate total body stores of vitamin A. As judged by deficient serum retinol (<0.70 μmol/L [20 μg/dL]), vitamin A deficiency worldwide is present in >90 million preschool-age children, among whom >4 million have an ocular manifestation of deficiency termed xerophthalmia. This condition includes milder stages of night blindness and conjunctival xerosis (dryness) with Bitot’s spots (white patches of keratinized epithelium appearing on the sclera) as well as rare, potentially blinding corneal ulceration and necrosis. Keratomalacia (softening of the cornea) leads to corneal scarring that blinds at least a quarter of a million children each year and is associated

1	blinding corneal ulceration and necrosis. Keratomalacia (softening of the cornea) leads to corneal scarring that blinds at least a quarter of a million children each year and is associated with fatality rates of 4–25%. However, vitamin A deficiency at any stage poses an increased risk of death from diarrhea, dysentery, measles, malaria, or respiratory disease. Vitamin A deficiency can compromise barrier, innate, and acquired immune defenses to infection. In areas where deficiency is widely prevalent, vitamin A supplementation can markedly reduce the risk of childhood mortality (by 23–34%, on average). About 10% of pregnant women in undernourished settings also develop night blindness (assessed by history) during the latter half of pregnancy, and this moderate vitamin A deficiency is associated with an increased risk of maternal infection and death.

1	Any stage of xerophthalmia should be treated with 60 mg (or RAE) of vitamin A in oily solution, usually contained in a soft-gel capsule. The same dose is repeated 1 and 14 days later. Doses should be reduced by half for patients 6–11 months of age. Mothers with night blindness or Bitot’s spots should be given vitamin A orally–either 3 mg daily or 7.5 mg twice a week for 3 months. These regimens are efficacious, and they are less expensive and more widely available than injectable water-miscible vitamin A. A common approach to prevention is to provide vitamin A supplementation every 4–6 months to young children and infants (both HIV-positive and HIV-negative) in high-risk areas. Infants 6–11 months of age should receive 30 mg vitamin A; children 12–59 months of age, 60 mg. For reasons that are not clear, vitamin A supplementation has not proven useful in high-risk settings for preventing morbidity or death among infants 1–5 months of age.

1	Uncomplicated vitamin A deficiency is rare in industrialized countries. One high-risk group—extremely low-birth-weight (<1000-g) infants—is likely to be vitamin A–deficient and should receive a supplement of 1500 μg (or RAE) three times a week for 4 weeks. Severe measles in any society can lead to secondary vitamin A deficiency. Children hospitalized with measles should receive two 60-mg doses of vitamin A on two consecutive days. Vitamin A deficiency most often occurs in patients with malabsorptive diseases (e.g., celiac sprue, short-bowel syndrome) who have abnormal dark adaptation or symptoms of night blindness without other ocular changes. Typically, such patients are treated for 1 month with 15 mg/d of a water-miscible preparation of vitamin A. This treatment is followed by a lower maintenance dose, with the exact amount determined by monitoring serum retinol.

1	No specific signs or symptoms result from carotenoid deficiency. It was postulated that β-carotene would be an effective chemopreventive agent for cancer because numerous epidemiologic studies had shown that diets high in β-carotene were associated with lower incidences of cancers of the respiratory and digestive systems. However, intervention studies in smokers found that treatment with high doses of β-carotene actually resulted in more lung cancers than did treatment with placebo. Non–provitamin A carotenoids such as lutein and zeaxanthin have been suggested to confer protection against macular degeneration, and one large-scale intervention study did not show a beneficial effect except in those with a low lutein status. The use of the non–provitamin A carotenoid lycopene to protect against prostate cancer has been proposed. Again, however, the effectiveness of these agents has not been proved by intervention studies, and the mechanisms underlying these purported biologic actions are

1	prostate cancer has been proposed. Again, however, the effectiveness of these agents has not been proved by intervention studies, and the mechanisms underlying these purported biologic actions are unknown.

1	Selective plant-breeding techniques that lead to a higher provitamin A content in staple foods may decrease vitamin A malnutrition in low-income countries. Moreover, a recently developed genetically modified food (Golden Rice) has an improved β-carotene–to– vitamin A conversion ratio of ~3:1.

1	Toxicity The acute toxicity of vitamin A was first noted in Arctic explorers who ate polar bear liver and has also been seen after administration of 150 mg to adults or 100 mg to children. Acute toxicity is manifested by increased intracranial pressure, vertigo, diplopia, bulging fontanels (in children), seizures, and exfoliative dermatitis; it may result in death. Among children being treated for vitamin A deficiency according to the protocols outlined above, transient bulging of fontanels occurs in 2% of infants, and transient nausea, vomiting, and headache occur in 5% of preschoolers. Chronic vitamin A intoxication is largely a concern in industrialized countries and has been seen in otherwise healthy adults who ingest 15 mg/d and children who ingest 6 mg/d over a period of several months. Manifestations include dry skin, cheilosis, glossitis, vomiting, alopecia, bone demineralization and pain, hypercalcemia, lymph node enlargement, hyperlipidemia, amenorrhea, and features of

1	months. Manifestations include dry skin, cheilosis, glossitis, vomiting, alopecia, bone demineralization and pain, hypercalcemia, lymph node enlargement, hyperlipidemia, amenorrhea, and features of pseudotumor cerebri with increased intracranial pressure and papilledema. Liver fibrosis with portal hypertension and 96e-7 bone demineralization may result from chronic vitamin A intoxication. Provision of vitamin A in excess to pregnant women has resulted in spontaneous abortion and in congenital malformations, including craniofacial abnormalities and valvular heart disease. In pregnancy, the daily dose of vitamin A should not exceed 3 mg. Commercially available retinoid derivatives are also toxic, including 13-cis-retinoic acid, which has been associated with birth defects. Thus contraception should be continued for at least 1 year and possibly longer in women who have taken 13-cis-retinoic acid.

1	In malnourished children, vitamin A supplements (30–60 mg), in amounts calculated as a function of age and given in several rounds over 2 years, are considered to amplify nonspecific effects of vaccines. However, for unclear reasons, there may be a negative effect on mortality rates in incompletely vaccinated girls.

1	High doses of carotenoids do not result in toxic symptoms but should be avoided in smokers due to an increased risk of lung cancer. Very high doses of β-carotene (~200 mg/d) have been used to treat or prevent the skin rashes of erythropoietic protoporphyria. Carotenemia, which is characterized by a yellowing of the skin (in creases of the palms and soles) but not the sclerae, may follow ingestion of >30 mg of β-carotene daily. Hypothyroid patients are particularly susceptible to the development of carotenemia due to impaired breakdown of carotene to vitamin A. Reduction of carotenes in the diet results in the disappearance of skin yellowing and carotenemia over a period of 30–60 days.

1	The metabolism of the fat-soluble vitamin D is described in detail in Chap. 423. The biologic effects of this vitamin are mediated by vitamin D receptors, which are found in most tissues; binding with these receptors potentially expands vitamin D actions on nearly all cell systems and organs (e.g., immune cells, brain, breast, colon, and prostate) as well as exerting classic endocrine effects on calcium metabolism and bone health. Vitamin D is thought to be important for maintaining normal function of many nonskeletal tissues such as muscle (including heart muscle), for immune function, and for inflammation as well as for cell proliferation and differentiation. Studies have shown that vitamin D may be useful as adjunctive treatment for tuberculosis, psoriasis, and multiple sclerosis or for the prevention of certain cancers. Vitamin D insufficiency may increase the risk of type 1 diabetes mellitus, cardiovascular disease (insulin resistance, hypertension, or low-grade inflammation), or

1	the prevention of certain cancers. Vitamin D insufficiency may increase the risk of type 1 diabetes mellitus, cardiovascular disease (insulin resistance, hypertension, or low-grade inflammation), or brain dysfunction (e.g., depression). However, the exact physiologic roles of vitamin D in these nonskeletal diseases and the importance of these roles have not been clarified.

1	The skin is a major source of vitamin D, which is synthesized upon skin exposure to ultraviolet B radiation (UV-B; wavelength, 290–320 nm). Except for fish, food (unless fortified) contains only limited amounts of vitamin D. Vitamin D2 (ergocalciferol) is obtained from plant sources and is the chemical form found in some supplements.

1	Deficiency Vitamin D status has been assessed by measuring serum levels of 25-dihydroxyvitamin D (25[OH]2 vitamin D); however, there is no consensus on a uniform assay or on optimal serum levels. The optimal level might, in fact, differ according to the targeted disease entity. Epidemiologic and experimental data indicate that a 25(OH)2 vitamin D level of >20 ng/mL (≥50 nmol/L; to convert ng/mL to nmol/L, multiply by 2.496) is sufficient for good bone health. Some experts advocate higher serum levels (e.g., >30 ng/mL) for other desirable endpoints of vitamin D action. There is insufficient evidence to recommend combined vitamin D and calcium supplementation as a primary preventive strategy for reduction of the incidence of fractures in healthy men and premenopausal women.

1	Risk factors for vitamin D deficiency are old age, lack of sun exposure, dark skin (especially among residents of northern latitudes), fat malabsorption, and obesity. Rickets represents the classic disease of vitamin D deficiency. Signs of deficiency are muscle soreness, weakness, and bone pain. Some of these effects are independent of calcium intake.

1	96e-8 The U.S. National Academy of Sciences recently concluded that the majority of North Americans are receiving adequate amounts of vitamin D (RDA = 15 μg/d or 600 IU/d; Chap. 95e). However, for people older than 70 years, the RDA is set at 20 μg/d (800 IU/d). The consumption of fortified or enriched foods as well as suberythemal sun exposure should be encouraged for people at risk for vitamin D deficiency. If adequate intake is impossible, vitamin D supplements should be taken, especially during the winter months. Vitamin D deficiency can be treated by the oral administration of 50,000 IU/week for 6–8 weeks followed by a maintenance dose of 800 IU/d (100 μg/d) from food and supplements once normal plasma levels have been attained. The physiologic effects of vitamin D2 and vitamin D3 are identical when these vitamins are ingested over long periods.

1	Toxicity The upper limit of intake has been set at 4000 IU/d. Contrary to earlier beliefs, acute vitamin D intoxication is rare and usually is caused by the uncontrolled and excessive ingestion of supplements or by faulty food fortification practices. High plasma levels of 1,25(OH)2 vitamin D and calcium are central features of toxicity and mandate discontinuation of vitamin D and calcium supplements; in addition, treatment of hypercalcemia may be required.

1	Vitamin E is the collective designation for all stereoisomers of tocopherols and tocotrienols, although only the RR tocopherols meet human requirements. Vitamin E acts as a chain-breaking antioxidant and is an efficient pyroxyl radical scavenger that protects low-density lipoproteins and polyunsaturated fats in membranes from oxidation. A network of other antioxidants (e.g., vitamin C, glutathione) and enzymes maintains vitamin E in a reduced state. Vitamin E also inhibits prostaglandin synthesis and the activities of protein kinase C and phospholipase A2. Absorption and Metabolism After absorption, vitamin E is taken up from chylomicrons by the liver, and a hepatic α-tocopherol transport protein mediates intracellular vitamin E transport and incorporation into very low density lipoprotein. The transport protein has a particular affinity for the RRR isomeric form of α-tocopherol; thus, this natural isomer has the most biologic activity.

1	requirement Vitamin E is widely distributed in the food supply, with particularly high levels in sunflower oil, safflower oil, and wheat germ oil; γ-tocotrienols are notably present in soybean and corn oils. Vitamin E is also found in meats, nuts, and cereal grains, and small amounts are present in fruits and vegetables. Vitamin E pills containing doses of 50–1000 mg are ingested by ~10% of the U.S. population. The RDA for vitamin E is 15 mg/d (34.9 μmol or 22.5 IU) for all adults. Diets high in polyunsaturated fats may necessitate a slightly higher intake of vitamin E.

1	Dietary deficiency of vitamin E does not exist. Vitamin E deficiency is seen only in severe and prolonged malabsorptive diseases, such as celiac disease, or after small-intestinal resection or bariatric surgery. Children with cystic fibrosis or prolonged cholestasis may develop vitamin E deficiency characterized by areflexia and hemolytic anemia. Children with abetalipoproteinemia cannot absorb or transport vitamin E and become deficient quite rapidly. A familial form of isolated vitamin E deficiency also exists; it is due to a defect in the α-tocopherol transport protein. Vitamin E deficiency causes axonal degeneration of the large myelinated axons and results in posterior column and spinocerebellar symptoms. Peripheral neuropathy is initially characterized by areflexia, with progression to an ataxic gait, and by decreased vibration and position sensations. Ophthalmoplegia, skeletal myopathy, and pigmented retinopathy may also be features of vitamin E deficiency. A deficiency of

1	to an ataxic gait, and by decreased vibration and position sensations. Ophthalmoplegia, skeletal myopathy, and pigmented retinopathy may also be features of vitamin E deficiency. A deficiency of either vitamin E or selenium in the host has been shown to increase certain viral mutations and, therefore, virulence. The laboratory diagnosis of vitamin E deficiency is based on low blood levels of α-tocopherol (<5 μg/mL, or <0.8 mg of α-tocopherol per gram of total lipids).

1	Symptomatic vitamin E deficiency should be treated with 800–1200 mg of α-tocopherol per day. Patients with abetalipoproteinemia may need as much as 5000–7000 mg/d. Children with symptomatic vitamin E deficiency should be treated orally with water-miscible esters (400 mg/d); alternatively, 2 mg/kg per day may be administered intramuscularly. Vitamin E in high doses may protect against oxygen-induced retrolental fibroplasia and bronchopulmonary dysplasia as well as intraventricular hemorrhage of prematurity. Vitamin E has been suggested to increase sexual performance, treat intermittent claudication, and slow the aging process, but evidence for these properties is lacking. When given in combination with other antioxidants, vitamin E may help prevent macular degeneration. High doses (60–800 mg/d) of vitamin E have been shown in controlled trials to improve parameters of immune function and reduce colds in nursing home residents, but intervention studies using vitamin E to prevent

1	mg/d) of vitamin E have been shown in controlled trials to improve parameters of immune function and reduce colds in nursing home residents, but intervention studies using vitamin E to prevent cardiovascular disease or cancer have not shown efficacy, and, at doses >400 mg/d, vitamin E may even increase all-cause mortality rates.

1	Toxicity All forms of vitamin E are absorbed and could contribute to toxicity; however, the toxicity risk seems to be rather low as long as liver function is normal. High doses of vitamin E (>800 mg/d) may reduce platelet aggregation and interfere with vitamin K metabolism and are therefore contraindicated in patients taking warfarin and anti-platelet agents (such as aspirin or clopidogrel). Nausea, flatulence, and diarrhea have been reported at doses >1 g/d. There are two natural forms of vitamin K: vitamin K1, also known as phylloquinone, from vegetable and animal sources, and vitamin K2, or menaquinone, which is synthesized by bacterial flora and found in hepatic tissue. Phylloquinone can be converted to menaquinone in some organs.

1	Vitamin K is required for the posttranslational carboxylation of glutamic acid, which is necessary for calcium binding to γ-carboxylated proteins such as prothrombin (factor II); factors VII, IX, and X; protein C; protein S; and proteins found in bone (osteocalcin) and vascular smooth muscle (e.g., matrix Gla protein). However, the importance of vitamin K for bone mineralization and prevention of vascular calcification is not known. Warfarin-type drugs inhibit γ-carboxylation by preventing the conversion of vitamin K to its active hydroquinone form. Dietary Sources Vitamin K is found in green leafy vegetables such as kale and spinach, and appreciable amounts are also present in margarine and liver. Vitamin K is present in vegetable oils; olive, canola, and soybean oils are particularly rich sources. The average daily intake by Americans is estimated to be ~100 μg/d.

1	Deficiency The symptoms of vitamin K deficiency are due to hemorrhage; newborns are particularly susceptible because of low fat stores, low breast milk levels of vitamin K, relative sterility of the infantile intestinal tract, liver immaturity, and poor placental transport. Intracranial bleeding as well as gastrointestinal and skin bleeding can occur in vitamin K–deficient infants 1–7 days after birth. Thus, vitamin K (0.5–1 mg IM) is given prophylactically at delivery.

1	Vitamin K deficiency in adults may be seen in patients with chronic small-intestinal disease (e.g., celiac disease, Crohn’s disease), in those with obstructed biliary tracts, or after small-bowel resection. Broad-spectrum antibiotic treatment can precipitate vitamin K deficiency by reducing numbers of gut bacteria, which synthesize menaquinones, and by inhibiting the metabolism of vitamin K. In patients with warfarin therapy, the anti-obesity drug orlistat can lead to international normalized ratio changes due to vitamin K malabsorption. Vitamin K deficiency usually is diagnosed on the basis of an elevated prothrombin time or reduced clotting factors, although vitamin K may also be measured directly by high-pressure liquid chromatography. Vitamin K deficiency is treated with a parenteral dose of 10 mg. For patients with chronic malabsorption, 1–2 mg/d should be given orally or 1–2 mg per week can be taken parenterally. Patients with liver disease may have an elevated prothrombin time

1	dose of 10 mg. For patients with chronic malabsorption, 1–2 mg/d should be given orally or 1–2 mg per week can be taken parenterally. Patients with liver disease may have an elevated prothrombin time because of liver cell destruction as well as vitamin K deficiency. If an elevated prothrombin time does not improve during vitamin K therapy, it can be deduced that this abnormality is not the result of vitamin K deficiency.

1	Toxicity Toxicity from dietary phylloquinones and menaquinones has not been described. High doses of vitamin K can impair the actions of oral anticoagulants. See also Table 96e-2. See Chap. 423. Zinc is an integral component of many metalloenzymes in the body; it is involved in the synthesis and stabilization of proteins, DNA, and RNA and plays a structural role in ribosomes and membranes. Zinc is necessary for the binding of steroid hormone receptors and several other transcription factors to DNA. Zinc is absolutely required for normal spermatogenesis, fetal growth, and embryonic development. Absorption The absorption of zinc from the diet is inhibited by dietary phytate, fiber, oxalate, iron, and copper as well as by certain drugs, including penicillamine, sodium valproate, and ethambutol. Meat, shellfish, nuts, and legumes are good sources of bioavailable zinc, whereas zinc in grains and legumes is less available for absorption.

1	diseases, including diabetes mellitus, HIV/AIDS, cirrhosis, alcoholism, inflammatory bowel disease, malabsorption syndromes, and sickle cell disease. In these diseases, mild chronic zinc deficiency can cause stunted growth in children, decreased taste sensation (hypogeusia), and impaired immune function. Severe chronic zinc deficiency has been described as a cause of hypogonadism and dwarfism in several Middle Eastern countries. In these children, hypopigmented hair is also part of the syndrome. Acrodermatitis enteropathica is a rare autosomal recessive disorder characterized by abnormalities in zinc absorption. Clinical manifestations include diarrhea, alopecia, muscle wasting, depression, irritability, and a rash involving the extremities, face, and perineum. The rash is characterized by vesicular and pustular crusting with scaling and erythema. Occasional patients with Wilson’s disease have developed zinc deficiency as a consequence of penicillamine therapy (Chap. 429).

1	Zinc deficiency is prevalent in many developing countries and usually coexists with other micronutrient deficiencies (especially iron deficiency). Zinc (20 mg/d until recovery) may be an effective adjunctive therapeutic strategy for diarrheal disease and pneumonia in children ≥ 6 months of age. The diagnosis of zinc deficiency is usually based on a serum zinc level <12 μmol/L (<70 μg/dL). Pregnancy and birth control pills may cause a slight depression in serum zinc levels, and hypoalbuminemia from any cause can result in hypozincemia. In acute stress situations, zinc may be redistributed from serum into tissues. Zinc deficiency may be treated with 60 mg of elemental zinc taken by mouth twice a day. Zinc gluconate lozenges (13 mg of elemental zinc every 2 h while awake) have been reported to reduce the duration and symptoms of the common cold in adults, but study results are conflicting.

1	96e-10 Toxicity Acute zinc toxicity after oral ingestion causes nausea, vomiting, and fever. Zinc fumes from welding may also be toxic and cause fever, respiratory distress, excessive salivation, sweating, and headache. Chronic large doses of zinc may depress immune function and cause hypochromic anemia as a result of copper deficiency. Intranasal zinc preparations should be avoided because they may lead to irreversible damage of the nasal mucosa and anosmia.

1	Copper is an integral part of numerous enzyme systems, including amine oxidases, ferroxidase (ceruloplasmin), cytochrome c oxidase, superoxide dismutase, and dopamine hydroxylase. Copper is also a component of ferroprotein, a transport protein involved in the basolateral transfer of iron during absorption from the enterocyte. As such, copper plays a role in iron metabolism, melanin synthesis, energy production, neurotransmitter synthesis, and CNS function; the synthesis and cross-linking of elastin and collagen; and the scavenging of superoxide radicals. Dietary sources of copper include shellfish, liver, nuts, legumes, bran, and organ meats.

1	Deficiency Dietary copper deficiency is relatively rare, although it has been described in premature infants who are fed milk diets and in infants with malabsorption (Table 96e-2). Copper-deficiency anemia (refractory to therapeutic iron) has been reported in patients with malabsorptive diseases and nephrotic syndrome and in patients treated for Wilson’s disease with chronic high doses of oral zinc, which can interfere with copper absorption. Menkes kinky hair syndrome is an X-linked metabolic disturbance of copper metabolism characterized by mental retardation, hypocupremia, and decreased circulating ceruloplasmin (Chap. 427). This syndrome is caused by mutations in the copper-transporting ATP7A gene. Children with this disease often die within 5 years because of dissecting aneurysms or cardiac rupture. Aceruloplasminemia is a rare autosomal recessive disease characterized by tissue iron overload, mental deterioration, microcytic anemia, and low serum iron and copper concentrations.

1	The diagnosis of copper deficiency is usually based on low serum levels of copper (<65 μg/dL) and low ceruloplasmin levels (<20 mg/ dL). Serum levels of copper may be elevated in pregnancy or stress conditions since ceruloplasmin is an acute-phase reactant and 90% of circulating copper is bound to ceruloplasmin. Toxicity Copper toxicity is usually accidental (Table 96e-2). In severe cases, kidney failure, liver failure, and coma may ensue. In Wilson’s disease, mutations in the copper-transporting ATP7B gene lead to accumulation of copper in the liver and brain, with low blood levels due to decreased ceruloplasmin (Chap. 429).

1	Selenium, in the form of selenocysteine, is a component of the enzyme glutathione peroxidase, which serves to protect proteins, cell membranes, lipids, and nucleic acids from oxidant molecules. As such, selenium is being actively studied as a chemopreventive agent against certain cancers, such as prostate cancer. Selenocysteine is also found in the deiodinase enzymes, which mediate the deiodination of thyroxine to triiodothyronine (Chap. 405). Rich dietary sources of selenium include seafood, muscle meat, and cereals, although the selenium content of cereal is determined by the soil concentration. Countries with low soil concentrations include parts of Scandinavia, China, and New Zealand. Keshan disease is an endemic cardiomyopathy found in children and young women residing in regions of China where dietary intake of selenium is low (<20 μg/d). Concomitant deficiencies of iodine and selenium may worsen the clinical manifestations of cretinism. Chronic ingestion of large amounts of

1	of China where dietary intake of selenium is low (<20 μg/d). Concomitant deficiencies of iodine and selenium may worsen the clinical manifestations of cretinism. Chronic ingestion of large amounts of selenium leads to selenosis, characterized by hair and nail brittleness and loss, garlic breath odor, skin rash, myopathy, irritability, and other abnormalities of the nervous system.

1	Chromium potentiates the action of insulin in patients with impaired glucose tolerance, presumably by increasing insulin receptor– mediated signaling, although its usefulness in treating type 2 diabetes is uncertain. In addition, improvement in blood lipid profiles has been reported in some patients. The usefulness of chromium supplements in muscle building has not been substantiated. Rich food sources of chromium include yeast, meat, and grain products. Chromium in the trivalent state is found in supplements and is largely nontoxic; however, chromium-6 is a product of stainless steel welding and is a known pulmonary carcinogen as well as a cause of liver, kidney, and CNS damage. See Chap. 423. FLuOrIDE, MANgANESE, AND uLTrATrACE ELEMENTS

1	See Chap. 423. FLuOrIDE, MANgANESE, AND uLTrATrACE ELEMENTS An essential function for fluoride in humans has not been described, although it is useful for the maintenance of structure in teeth and bones. Adult fluorosis results in mottled and pitted defects in tooth enamel as well as brittle bone (skeletal fluorosis). Manganese and molybdenum deficiencies have been reported in patients with rare genetic abnormalities and in a few patients receiving prolonged total parenteral nutrition. Several manganese-specific enzymes have been identified (e.g., manganese superoxide dismutase). Deficiencies of manganese have been reported to result in bone demineralization, poor growth, ataxia, disturbances in carbohydrate and lipid metabolism, and convulsions.

1	Ultratrace elements are defined as those needed in amounts <1 mg/d. Essentiality has not been established for most ultratrace elements, although selenium, chromium, and iodine are clearly essential (Chap. 405). Molybdenum is necessary for the activity of sulfite and xanthine oxidase, and molybdenum deficiency may result in skeletal and brain lesions.

1	459 CHAPTER 97 Malnutrition and Nutritional Assessment Douglas C. Heimburger Malnutrition can arise from primary or secondary causes, result-ing in the former case from inadequate or poor-quality food intake and in the latter case from diseases that alter food intake or nutri-97 ent requirements, metabolism, or absorption. Primary malnutrition occurs mainly in developing countries and under conditions of political unrest, war, or famine. Secondary malnutrition, the main form encountered in industrialized countries, was largely unrecognized until the early 1970s, when it was appreciated that persons with adequate food supplies can become malnourished as a result of acute or chronic diseases that alter nutrient intake or metabolism, particularly diseases that cause acute or chronic inflammation. Various studies have shown that protein-energy malnutrition (PEM) affects one-third to one-half of patients on general medical and surgical wards in teaching hospitals. The consistent finding

1	Various studies have shown that protein-energy malnutrition (PEM) affects one-third to one-half of patients on general medical and surgical wards in teaching hospitals. The consistent finding that nutritional status influences patient prognosis underscores the importance of preventing, detecting, and treating malnutrition.

1	Definitions for forms of PEM are in flux. Traditionally, the two major types of PEM have been marasmus and kwashiorkor. These conditions are compared in Table 97-1. Marasmus is the end result of a long-term deficit of dietary energy, whereas kwashiorkor has been understood to result from a protein-poor diet. Although the former concept remains essentially correct, evidence is accumulating that PEM syndromes are distinguished by two main features: insufficient dietary intake and underlying inflammatory processes. Energy-poor diets with minimal inflammation cause gradual erosion of body mass, resulting in classic marasmus. By contrast, inflammation from acute illnesses such as injury or sepsis or from chronic illnesses such as cancer, lung or heart disease, or HIV infection can erode lean body mass even in the presence of relatively sufficient dietary intake, leading to a kwashiorkor-like state. Quite often, inflammatory illnesses impair appetite and dietary intake, producing

1	lean body mass even in the presence of relatively sufficient dietary intake, leading to a kwashiorkor-like state. Quite often, inflammatory illnesses impair appetite and dietary intake, producing combinations of the two conditions.

1	Consensus committees have proposed the following revised definitions. Starvation–related malnutrition is suggested for instances of chronic starvation without inflammation, chronic disease–related malnutrition when inflammation is chronic and of mild to moderate degree, and acute disease– or injury–related malnutrition when inflammation is acute and of a severe degree. However, because distinguishing diagnostic criteria for these conditions have not been universally adopted, this chapter integrates the older and newer terms.

1	Marasmus (starvation–related malnutrition) is a state in which virtually all available body fat stores have been exhausted due to starvation without systemic inflammation. Cachexia (chronic disease–related malnutrition) is a state that involves substantial loss of lean body mass in the presence of chronic systemic inflammation. Conditions that produce cachexia tend to be chronic and indolent, such as cancer and chronic pulmonary disease, whereas, in high-income countries, the classic setting for marasmus is in patients with anorexia nervosa. These conditions are relatively easy to detect because of the patient’s starved aThe findings used to diagnose kwashiorkor/acute malnutrition must be unexplained by other causes. bTested by firmly pulling a lock of hair from the top (not the sides or back), grasping with the thumb and forefinger. An average of three or more hairs removed easily and painlessly is considered abnormal hair pluckability.

1	appearance. The diagnosis is based on fat and muscle wastage resulting from prolonged calorie deficiency and/or inflammation. Diminished skinfold thickness reflects the loss of fat reserves; reduced arm muscle circumference with temporal and interosseous muscle wasting reflects the catabolism of protein throughout the body, including in vital organs such as the heart, liver, and kidneys. Routine laboratory findings in cachexia/marasmus are relatively unremarkable. The creatinine-height index (24-h urinary creatinine excretion compared with normal values based on height) is low, reflecting the loss of muscle mass. Occasionally, the serum albumin level is reduced, but it remains above 2.8 g/dL when systemic inflammation is absent. Despite a morbid appearance, immunocompetence, wound healing, and the ability to handle short-term stress are reasonably well preserved in most patients.

1	Pure starvation–related malnutrition is a chronic, fairly well adapted form of starvation rather than an acute illness; it should be treated cautiously in an attempt to reverse the downward trend gradually. Although nutritional support is necessary, overly aggressive repletion can result in severe, even life-threatening metabolic imbalances such as hypophosphatemia and cardiorespiratory failure (refeeding syndrome). When possible, oral or enteral nutritional support is preferred; treatment started slowly allows readaptation of metabolic and intestinal functions (Chap. 98e).

1	By contrast, kwashiorkor (acute disease– or injury–related malnutrition) in developed countries occurs mainly in connection with acute, life-threatening conditions such as trauma and sepsis. The physiologic stress produced by these illnesses increases protein and energy requirements at a time when intake is often limited. A classic scenario is an acutely stressed patient who receives only 5% dextrose solutions for periods as brief as 2 weeks. Although the etiologic mechanisms are not fully known, the protein-sparing response normally seen in starvation is blocked by the stressed state and by carbohydrate infusion.

1	In its early stages, the physical findings of kwashiorkor/acute malnutrition are few and subtle. Initially unaffected fat reserves and muscle mass give the deceptive appearance of adequate nutrition. Signs that support the diagnosis include easy hair pluckability, edema, skin breakdown, and poor wound healing. The major sine qua non is severe reduction of levels of serum proteins such as albumin (<2.8 g/dL) and transferrin (<150 mg/dL) or of iron-binding capacity (<200 μg/dL). Cellular immune function is depressed, as reflected by lymphopenia (<1500 lymphocytes/μL in adults and older children) and lack of response to skin test antigens (anergy).

1	The prognosis of adult patients with full-blown kwashiorkor/acute malnutrition is not good even with aggressive nutritional support. Surgical wounds often dehisce (fail to heal), pressure sores develop, gastroparesis and diarrhea can occur with enteral feeding, the risk of gastrointestinal bleeding from stress ulcers is increased, host defenses are compromised, and death from overwhelming infection may occur despite antibiotic therapy. Unlike treatment of marasmus, therapy for kwashiorkor entails aggressive nutritional support to restore better metabolic balance rapidly (Chap. 98e). The metabolic characteristics and nutritional needs of hypermetabolic patients who are stressed from injury, infection, or chronic inflammatory illness differ from those of hypometabolic patients who are unstressed but chronically starved. In both cases, nutritional support is important, but misjudgments in selecting the appropriate approach may have serious adverse consequences.

1	The hypometabolic patient is typified by the relatively less stressed but mildly catabolic and chronically starved individual who, with time, will develop cachexia/marasmus. The hypermetabolic patient stressed from injury or infection is catabolic (experiencing rapid breakdown of body mass) and is at high risk for developing acute malnutrition/ kwashiorkor if nutritional needs are not met and/or the illness does not resolve quickly. As summarized in Table 97-2, the two states are distinguished by differing perturbations of metabolic rate, rates of protein breakdown (proteolysis), and rates of gluconeogenesis. These differences are mediated by proinflammatory cytokines and counter-regulatory hormones—tumor necrosis factor, interleukins 1 and 6, C-reactive protein, catecholamines (epinephrine and norepinephrine), glucagon, and cortisol—whose levels are relatively reduced in hypo-metabolic patients and increased in hypermetabolic patients. Although insulin levels are also elevated in

1	and norepinephrine), glucagon, and cortisol—whose levels are relatively reduced in hypo-metabolic patients and increased in hypermetabolic patients. Although insulin levels are also elevated in stressed patients, insulin resistance in the target tissues blocks insulin-mediated anabolic effects. Physiologic

1	Cytokines, catecholamines, ↓↑ glucagon, cortisol, insulin Metabolic rate, O2 consumption ↓↑ Proteolysis, gluconeogenesis ↓↑ Ureagenesis, urea excretion ↓↑ Fat catabolism, fatty acid utilization Relative ↑ Absolute ↑ Adaptation to starvation Normal Abnormal characteristics of patients at risk for chronic disease–related malnutrition are less predictable and likely represent a mixture of the two extremes depicted in Table 97-2. Metabolic rate In starvation and semistarvation, the resting metabolic rate falls between 10% and 30% as an adaptive response to energy restriction, slowing the rate of weight loss. By contrast, the resting metabolic rate rises in the presence of physiologic stress in proportion to the degree of the insult. The rate may increase by ~10% after elective surgery, 20–30% after bone fractures, 30–60% with severe infections such as peritonitis or gram-negative septicemia, and as much as 110% after major burns.

1	If the metabolic rate (energy requirement) is not matched by energy intake, weight loss results—slowly in hypometabolism and quickly in hypermetabolism. Losses of up to 10% of body mass are unlikely to be detrimental; however, greater losses in acutely ill hypermetabolic patients may be associated with rapid deterioration in body functions.

1	Protein Catabolism The rate of endogenous protein breakdown (catabolism) to supply energy needs normally falls during uncomplicated energy deprivation. After ~10 days of total starvation, an unstressed individual loses about 12–18 g of protein per day (equivalent to ~60 g of muscle tissue or ~2–3 g of nitrogen). In contrast, in injury and sepsis, protein breakdown accelerates in proportion to the degree of stress, reaching 30–60 g/d after elective surgery, 60–90 g/d with infection, 100–130 g/d with severe sepsis or skeletal trauma, and >175 g/d with major burns or head injuries. These losses are reflected by proportional increases in the excretion of urea nitrogen, the major by-product of protein breakdown.

1	Gluconeogenesis The major aim of protein catabolism during a state of starvation is to provide the glucogenic amino acids (especially alanine and glutamine) that serve as substrates for endogenous glucose production (gluconeogenesis) in the liver. In the hypometabolic/starved state, protein breakdown for gluconeogenesis is minimized, especially as ketones derived from fatty acids become the substrate preferred by certain tissues. In the hypermetabolic/stress state, gluconeogenesis increases dramatically and in proportion to the degree of the insult to increase the supply of glucose (the major fuel of reparation). Glucose is the only fuel that can be utilized by hypoxemic tissues (anaerobic glycolysis), white blood cells, and newly generated fibroblasts. Infusions of glucose partially offset a negative energy balance but do not significantly suppress the high rates of gluconeogenesis in catabolic patients. Hence, adequate supplies of protein are needed to replace the amino acids used

1	a negative energy balance but do not significantly suppress the high rates of gluconeogenesis in catabolic patients. Hence, adequate supplies of protein are needed to replace the amino acids used for this metabolic response.

1	In summary, a hypometabolic patient is adapted to starvation and conserves body mass through reduction of the metabolic rate and use of fat as the primary fuel (rather than glucose and its precursor amino acids). A hypermetabolic patient also uses fat as a fuel but rapidly breaks down body protein to produce glucose, with consequent loss of muscle and organ tissue and danger to vital body functions. The same illnesses and reductions in nutrient intake that lead to PEM often produce deficiencies of vitamins and minerals as well (Chap. 96e). Deficiencies of nutrients that are stored in small amounts (such as the water-soluble vitamins) occur because of loss through external secretions, such as zinc in diarrhea fluid or burn exudate, and are probably more common than is generally recognized.

1	Deficiencies of vitamin C, folic acid, and zinc are relatively common in sick patients. Signs of scurvy, such as corkscrew hairs on the lower extremities, are found frequently in chronically ill and/or alcoholic patients. The diagnosis can be confirmed by determination of plasma vitamin C levels. Folic acid intakes and blood levels are often less than optimal, even among healthy persons; with illness, alcoholism, poverty, or poor dentition, these deficiencies are common. Low blood zinc levels are prevalent in patients with malabsorption syndromes such as inflammatory bowel disease. Patients with zinc deficiency often exhibit poor wound healing, pressure ulcer formation, and impaired immunity. Thiamine deficiency is a common complication of alcohol-461 ism but may be prevented by therapeutic doses of thiamine in patients treated for alcohol abuse.

1	Patients with low plasma vitamin C levels usually respond to the doses in multivitamin preparations, but patients with deficiencies should be supplemented with 250–500 mg/d. Folic acid is absent from some oral multivitamin preparations; patients with deficiencies should be supplemented with ~1 mg/d. Patients with zinc deficiencies resulting from large external losses sometimes require oral supplementation with 220 mg of zinc sulfate one to three times daily. For these reasons, laboratory assessments of the micronutrient status of patients at high risk are desirable. Hypophosphatemia develops in hospitalized patients with remarkable frequency and generally results from rapid intracellular shifts of phosphate in underweight or alcoholic patients receiving intravenous glucose (Chap. 63). The adverse clinical sequelae are numerous; some, such as acute cardiopulmonary failure, are collectively called refeeding syndrome and can be life-threatening.

1	Many developing countries are still faced with high preva lences of the classic forms of PEM: marasmus and kwashior kor. Food insecurity, which characterizes many poor countries, prevents consistent dietary sufficiency and/or quality and leads to endemic or cyclic malnutrition. Factors threatening food security include marked seasonal variations in agricultural productivity (rainy season–dry season cycles), periodic droughts, political unrest or injustice, and disease epidemics (especially of HIV/AIDS). The coexistence of malnutrition and disease epidemics exacerbates the latter and increases complications and mortality rates, creating vicious cycles of malnutrition and disease.

1	As economic prosperity improves, developing countries have been observed to undergo an epidemiologic transition, a component of which has been termed the nutrition transition. As improved economic resources make greater dietary diversity possible, middle-income populations (e.g., in southern Asia, China, and Latin America) typically begin to adopt lifestyle habits of industrialized nations, with increased consumption of energy and fat and decreased levels of physical activity. These changes lead to rising levels of obesity, metabolic syndrome, diabetes, cardiovascular disease, and cancer, sometimes coexisting in populations with persistent undernutrition.

1	Micronutrient deficiencies also remain prevalent in many countries of the world, impairing functional status and productivity and increasing mortality rates. Vitamin A deficiency impairs vision and increases morbidity and mortality rates from infections such as measles. Mild to moderate iron deficiency may be prevalent in up to 50% of the world, resulting from poor dietary diversity coupled with periodic blood loss and pregnancies. Iodine deficiency remains prevalent, causing goiter, hypothyroidism, and cretinism. Zinc deficiency is endemic in many populations, producing growth retardation, hypogonadism, and dermatoses and impairing wound healing. Fortunately, public health supplementation programs have substantially improved vitamin A and zinc status in developing countries during the past two decades, reducing mortality rates from measles, diarrheal diseases, and other manifestations. However, with the advancing nutrition transition and a shift toward nutritionally related chronic

1	two decades, reducing mortality rates from measles, diarrheal diseases, and other manifestations. However, with the advancing nutrition transition and a shift toward nutritionally related chronic noncommunicable conditions, it is estimated that nutrition remains one of the three greatest contributors of risk for morbidity and mortality worldwide.

1	Because interactions between illness and nutrition are complex, many physical and laboratory findings reflect both underlying disease and nutritional status. Therefore, the nutritional evaluation of a patient requires an integration of history, physical examination, anthropometrics, and laboratory studies. This approach helps both to detect nutritional problems and to prevent the conclusion that isolated findings indicate nutritional problems when they do not. For example, hypoalbuminemia caused by an inflammatory illness does not necessarily indicate malnutrition. NuTRITIONAl DEfICIENCy: THE HIgH-RIsK PATIENT Underweight (body mass index <18.5) and/or recent loss of ≥10% of usual body mass Poor intake: anorexia, food avoidance (e.g., psychiatric condition), or NPOa status for more than ~5 days Protracted nutrient losses: malabsorption, enteric fistulas, draining abscesses or wounds, renal dialysis Hypermetabolic states: sepsis, protracted fever, extensive trauma or burns

1	Protracted nutrient losses: malabsorption, enteric fistulas, draining abscesses or wounds, renal dialysis Hypermetabolic states: sepsis, protracted fever, extensive trauma or burns Alcohol abuse or use of drugs with antinutrient or catabolic properties: glucocorticoids, antimetabolites (e.g., methotrexate), immunosuppressants, antitumor agents Impoverishment, isolation, advanced age aNil per os (nothing by mouth). Nutritional History Elicitation of a nutritional history is directed toward the identification of underlying mechanisms that put patients at risk for nutritional depletion or excess. These mechanisms include inadequate intake, impaired absorption, decreased utilization, increased losses, and increased requirements for nutrients. Individuals with the characteristics listed in Table 97-3 are at particular risk for nutritional deficiencies.

1	Individuals with the characteristics listed in Table 97-3 are at particular risk for nutritional deficiencies. Physical Examination Physical findings that suggest vitamin, mineral, and protein-energy deficiencies and excesses are outlined in Table 97-4. Most of the physical findings are not specific for individual nutrient deficiencies and must be integrated with historic, anthropometric, and laboratory findings. For example, follicular hyperkeratosis on the back of the arms is a fairly common, normal finding. However, if it is widespread in a person who consumes few fruits and vegetables and smokes regularly (increasing ascorbic acid requirements), vitamin C deficiency is likely. Similarly, easily pluck-able hair may be a consequence of chemotherapy but suggests acute malnutrition/kwashiorkor in a hospitalized patient who has poorly healing surgical wounds and hypoalbuminemia.

1	anthropometric Measurements Anthropometric measurements provide information on body muscle mass and fat reserves. The most practical and commonly used measurements are body weight, height, triceps skinfold (TSF), and midarm muscle circumference (MAMC). Body weight is one of the most useful nutritional parameters to follow in patients who are acutely or chronically ill. Unintentional weight loss during illness often reflects loss of lean body mass (muscle and organ tissue), especially if it is rapid and is not caused by diuresis. Such weight loss can be an ominous sign since it indicates use of vital body protein stores for metabolic fuel. The reference standard for normal body weight, body mass index (BMI: weight in kilograms divided by height, in meters, squared), is discussed in Chap. 416. BMI values <18.5 are considered underweight; <17, significantly underweight; and <16, severely wasted. Values of 18.5–24.9 are normal; 25–29.9, overweight; and ≥30, obese.

1	Measurement of skinfold thickness is useful for estimating body fat stores, because ~50% of body fat is normally located in the subcutaneous region. This measurement can also permit discrimination of fat mass from muscle mass. The triceps is a convenient site that is generally representative of the body’s overall fat level. A thickness <3 mm suggests virtually complete exhaustion of fat stores. The MAMC can be used to estimate skeletal muscle mass, calculated as follows: MAMC (cm) = upper arm circumference (cm) − [0.314 × TSF (mm)]

1	MAMC (cm) = upper arm circumference (cm) − [0.314 × TSF (mm)] Laboratory Studies A number of laboratory tests used routinely in clinical medicine can yield valuable information about a patient’s nutritional status if a slightly different approach to their interpretation is used. For example, abnormally low serum albumin levels, low total iron-binding capacity, and anergy may have a distinct explanation, but collectively they may represent kwashiorkor. In the clinical setting of a hypermetabolic, acutely ill patient who is edematous and has easily pluckable hair and inadequate protein intake, the diagnosis of acute malnutrition/kwashiorkor is clear-cut. Commonly used laboratory tests for assessing nutritional status are outlined in Table 97-5. The table also provides tips to avoid the assignment of nutritional significance to tests that may be abnormal for nonnutritional reasons.

1	Assessment of circulAting (viscerAl) proteins The serum proteins most commonly used to assess nutritional status include albumin, total iron-binding capacity (or transferrin), thyroxine-binding prealbumin (or transthyretin), and retinol-binding protein. Because they have different synthesis rates and half-lives (the half-life of serum albumin is ~21 days, whereas those of prealbumin and retinol-binding protein are ~2 days and ~12 h, respectively), some of these proteins reflect changes in nutritional status more quickly than do others. However, rapid fluctuations can also make shorter-half-life proteins less reliable.

1	Levels of circulating proteins are influenced by their rates of synthesis and catabolism, “third spacing” (loss into interstitial spaces), and, in some cases, external loss. Although an adequate intake of calories and protein is necessary for optimal circulating protein levels, serum protein levels generally do not reflect protein intake. For example, a drop in the serum level of albumin or transferrin often accompanies significant physiologic stress (e.g., from infection or injury) and is not necessarily an indication of malnutrition or poor intake. A low serum albumin level in a burned patient with both hypermetabolism and increased dermal losses of protein may not indicate malnutrition. However, adequate nutritional support of the patient’s calorie and protein needs is critical for returning circulating proteins to normal levels as stress resolves. Thus low values by themselves do not define malnutrition, but they often point to increased risk of malnutrition because of the

1	for returning circulating proteins to normal levels as stress resolves. Thus low values by themselves do not define malnutrition, but they often point to increased risk of malnutrition because of the hypermetabolic stress state. As long as significant physiologic stress persists, serum protein levels remain low, even with aggressive nutritional support. However, if the levels do not rise after the underlying illness improves, the patient’s protein and calorie needs should be reassessed to ensure that intake is sufficient.

1	Assessment of vitAmin And minerAl stAtus The use of laboratory tests to confirm suspected micronutrient deficiencies is desirable because the physical findings for those deficiencies are often equivocal or nonspecific. Low blood micronutrient levels can predate more serious clinical manifestations and also may indicate drug-nutrient interactions. A patient’s basal energy expenditure (BEE, measured in kilocalories per day) can be estimated from height, weight, age, and sex with the Harris-Benedict equations: Men: BEE = 66.47 + 13.75W + 5.00H − 6.76A Women: BEE = 655.10 + 9.56W + 1.85H − 4.68A

1	Men: BEE = 66.47 + 13.75W + 5.00H − 6.76A Women: BEE = 655.10 + 9.56W + 1.85H − 4.68A In these equations, W is weight in kilograms, H is height in centimeters, and A is age in years. After these equations are solved, total energy requirements are estimated by multiplying BEE by a factor that accounts for the stress of illness. Multiplying by 1.1–1.4 yields a range 10–40% above basal that estimates the 24-h energy expenditure of the majority of patients. The lower value (1.1) is used for patients without evidence of significant physiologic stress; the higher value (1.4) is appropriate for patients with marked stress such as sepsis or trauma. The result is used as a 24-h energy goal for feeding.

1	When it is important to have a more accurate assessment, energy expenditure can be measured at the bedside by indirect calorimetry. This technique is useful in patients who are thought to be hypermetabolic from sepsis or trauma and whose body weight cannot be ascertained accurately. Indirect calorimetry can also be useful in patients who have difficulty weaning from a ventilator and whose energy needs therefore should not be exceeded to avoid excessive CO2 production. Patients at the extremes of weight (e.g., obese persons) and/or age are good candidates as well, because the Harris-Benedict equations were developed from measurements in adults with roughly normal body weights. Because urea is a major by-product of protein catabolism, the amount of urea nitrogen excreted each day can be used to estimate the rate of protein catabolism and determine whether protein intake is adequate to

1	Edema Heart failure Hepatomegaly Parotid enlargement Sudden heart failure, death offset it. Total protein loss and protein balance can be calculated from urinary urea nitrogen (UUN) as follows: Protein catabolic rate (g/d) = [24-h UUN (g) + 4] × 6.25 (g protein/g nitrogen) The value of 4 g added to the UUN represents a liberal estimate of the unmeasured nitrogen lost in the urine (e.g., creatinine and uric acid), sweat, hair, skin, and feces. When protein intake is low (e.g., less than

1	Thiamine; acute malnutrition Thiamine (“wet” beriberi), phosphorus Acute malnutrition Vitamin A Acute malnutrition (consider also bulimia) Vitamin C ~20 g/d), the equation indicates both the patient’s protein requirement and the severity of the catabolic state (Table 97-5). More substantial protein intakes can raise the UUN because some of the ingested (or intravenously infused) protein is catabolized and converted to UUN. Thus, at lower protein intakes, the equation is useful for estimating requirements, and at higher protein intakes it is useful for assessing protein balance. Causes of Normal Value Test (Normal Values) Nutritional Use Despite Malnutrition Other Causes of Abnormal Value Serum albumin (3.5–5.5 g/dL) Serum prealbumin, also called transthyretin (20–40 mg/dL; lower in prepubertal children) Prothrombin time (2.0–15.5 s)

1	Serum albumin (3.5–5.5 g/dL) Serum prealbumin, also called transthyretin (20–40 mg/dL; lower in prepubertal children) Prothrombin time (2.0–15.5 s) Serum creatinine (0.6–1.6 mg/dL) 24-h urinary creatinine (500–1200 mg/d, standardized for height and sex) 24-h urinary urea nitrogen (UUN; <5 g/d; depends on level of protein intake) 2.8–3.5 g/dL: Protein depletion or systemic inflammation <2.8 g/dL: Possible acute malnutrition or severe inflammation 10–15 mg/dL: Mild protein depletion or inflammation 5–10 mg/dL: Moderate protein depletion or inflammation <5 mg/dL: Severe protein depletion or inflammation Increasing value reflects positive protein balance <200 μg/dL: Protein depletion or inflammatory state; Prolongation: vitamin K deficiency <0.6 mg/dL: Muscle wasting due to prolonged energy deficit Reflects muscle mass Low value: muscle wasting due to prolonged energy deficit

1	Prolongation: vitamin K deficiency <0.6 mg/dL: Muscle wasting due to prolonged energy deficit Reflects muscle mass Low value: muscle wasting due to prolonged energy deficit Determine level of catabolism (as long as protein intake is ≥10 g below calculated protein loss or <20 g total, and as long as carbohydrate intake has been at least 100 g) 5–10 g/d: Mild catabolism or normal fed state 10–15 g/d: Moderate catabolism >15 g/d: Severe catabolism loss (protein catabolic rate) = [24-h UUN (g) + 4] × 6.25. Adjustments required in burn patients and others with large nonurinary nitrogen losses and in patients with fluctuating levels of blood urea nitrogen (e.g., in renal failure) <8 mg/dL: Possibly inadequate protein intake 12–23 mg/dL: Possibly adequate protein intake >23 mg/dL: Possibly excessive protein intake If serum creatinine is normal, use BUN. If serum creatinine is elevated, use BUN/creatinine ratio. (Normal range is essentially the same as for BUN.)

1	If serum creatinine is elevated, use BUN/creatinine ratio. (Normal range is essentially the same as for BUN.) Infusion of albumin, fresh-frozen plasma, or whole blood Common: Infection and other stress, especially with poor protein intake Burns, trauma Congestive heart failure Fluid overload Severe liver disease Uncommon: Nephrotic syndrome Zinc deficiency Bacterial stasis/overgrowth of small intestine Similar to serum albumin Similar to serum albumin Despite muscle wasting: Renal failure Severe dehydration Severe liver disease Anabolic state Syndrome of inappropriate Despite poor protein intake: Renal failure (Use BUN/creatinine ratio.) Congestive heart failure Gastrointestinal hemorrhage

1	Enteral and Parenteral Nutrition Therapy Bruce R. Bistrian, L. John Hoffer, David F. Driscoll When correctly implemented, specialized nutritional support (SNS) plays a major and often life-saving role in medicine. SNS is used for 98e two main purposes: (1) to provide an appropriate nutritional substrate in order to maintain or replenish the nutritional status of patients unable to voluntarily ingest or absorb sufficient amounts of food, and (2) to maintain the nutritional and metabolic status of adequately nourished patients who are experiencing systemic hypercatabolic effects of severe inflammation, injury, or infection in the course of persistent critical illness. Patients with permanent major loss of intestinal length or function often require lifelong SNS. Many patients who require treatment in chronic-care facilities receive enteral SNS, most often because their voluntary food intake is deemed insufficient or because impaired chewing and swallowing create a high risk of

1	treatment in chronic-care facilities receive enteral SNS, most often because their voluntary food intake is deemed insufficient or because impaired chewing and swallowing create a high risk of aspiration pneumonia.

1	Enteral SNS is the provision of liquid formula meals through a tube placed into the gut. Parenteral SNS is the direct infusion of complete mixtures of crystalline amino acids, dextrose, triglyceride emulsions, and micronutrients into the bloodstream through a central venous catheter or (rarely in adults) via a peripheral vein. The enteral route is almost always preferred because of its relative simplicity and safety, its low cost, and the benefits of maintaining digestive, absorptive, and immunologic barrier functions of the gastrointestinal tract. Pliable, small-bore feeding tubes make placement relatively easy and acceptable to patients. Constant-rate infusion pumps increase the reliability of nutrient delivery. The chief disadvantage of enteral SNS is that many days may be required to meet the patient’s nutrient requirements.

1	For short-term use, the feeding tube can be placed via the nose into the stomach, duodenum, or jejunum. For long-term use, these sites may be accessed through the abdominal wall by endoscopic or surgical procedures. The chief disadvantage of tube feeding in acute illness is intolerance due to gastric retention, risk of vomiting, or diarrhea. The presence of severe coagulopathy is a relative contraindication to the insertion of a feeding tube. In adults, parenteral nutrition (PN) almost always requires aseptic insertion of a central venous catheter with a dedicated port. Many circumstances can delay or slow the progression of enteral SNS, whereas parenteral SNS can provide a complete substrate mix easily and promptly. This practical advantage is mitigated by the need to infuse relatively large fluid volumes and the real risk of inadvertent toxic overfeeding. APPROACH TO THE PATIENT:

1	Approximately one-fifth to one-quarter of patients in acute-care hospitals suffer from at least moderate protein-energy malnutrition (PEM), the defining features of which are malnutrition-induced weight loss and skeletal muscle atrophy. Usually, but not always, other features further compromise clinical responses; these features include a subnormal adipose tissue mass, with the accompanying adverse consequences of weakness, skin thinning, and breakdown; reduced ventilatory drive; ineffective cough; immunodeficiency; and impaired thermoregulation. Commonly, PEM is already present at the time of hospital admission and remains unimproved or worsens during the ensuing hospital stay. Common reasons for PEM worsening during hospitalization are refusal of food (because of anorexia, nausea, pain, or delirium), communication barriers, an unmet need for hand-feeding of patients with physical or sensory impairment, disordered or ineffective chewing or swallowing, and prolonged periods of

1	pain, or delirium), communication barriers, an unmet need for hand-feeding of patients with physical or sensory impairment, disordered or ineffective chewing or swallowing, and prolonged periods of physician-ordered fasting—all potentially

1	TAblE 98e-1 body MASS INdEx (bMI), MuSClE MASS, ANd PRoTEIN ENERgy MAlNuTRITIoN (PEM) taking place in a context of caregiver unawareness and inattention. Most patients who are suffering from in-hospital PEM do not, or ought not, to require SNS. A large proportion of these patients can be expected to improve with appropriate management of their primary disease. Others have a terminal disease whose downward course will not be altered by SNS. In yet other cases, the PEM is sufficiently mild that the benefits of SNS are exceeded by its risks. For patients who fall into this last category, the correct approach is to intensify and/or modify the patient’s oral nutrition as directed by the unit dietitian.

1	PEM is often classified as minimal, moderate, or severe on the basis of weight for height (body mass index, BMI) and percentage of body weight recently lost. As shown in Table 98e-1, the BMI (when corrected for abnormal extracellular fluid accumulation) is a crude but useful indicator of PEM severity. Note, however, that obesity does not preclude moderate or severe PEM, especially in older or bedridden patients; indeed, obesity can mask the presence of PEM if the patient’s muscle mass is not specifically examined.

1	The decision to implement SNS must be based on the determinations (1) that intensified or modified oral nutrition has failed or is impossible, impractical, or undesirable; and (2) that SNS will increase the patient’s rate and likelihood of recovery, reduce the risk of infection, improve healing, or otherwise shorten the hospital stay. In chronic-care situations, the decision to institute SNS is based on the likelihood that the intervention will extend the duration or quality of the patient’s life. An algorithm for determining when to use SNS is depicted in Fig. 98e-1. The decision to enhance oral nutrition or—that attempt failing— to resort to SNS is based on the anticipated consequences of nonintervention. The mnemonic “in-in-in” (for inanition-inflammation-inactivity) can serve as a reminder of the three main factors that come into play when deciding whether or not it is acceptable to withhold SNS from a patient with PEM.

1	Inanition Key issues include whether normal food intake is likely to be impossible for a prolonged period and whether the patient can tolerate prolonged starvation. A previously well-nourished person can tolerate ~7 days of starvation without harm, even in the presence of a moderate systemic response to inflammation (SRI), whereas the degree of tolerance to prolonged starvation is much less in patients whose skeletal muscle mass is already reduced, whether from PEM, from the muscle atrophy of old age (sarcopenia), or from muscle atrophy due to neuromuscular disease. Excess body fat does not exclude the possibility of coexisting muscle atrophy from any of these causes. In general, unintentional weight loss of >10% during the previous 6 months or a weight-to-height ratio that is <90% of standard, when associated with physiologic impairment, crudely predicts that the patient has moderate PEM. Weight loss >20% of usual or <80% of standard makes severe PEM more likely.

1	Inflammation The anorexia that invariably accompanies the SRI reduces the likelihood that a patient’s nutritional goals will be achieved by intensifying or modifying the diet, by providing counseling, or by hand-feeding. Furthermore, the protein-catabolic

1	Is disease process likely to cause nutritional impairment? Does the patient have PCM or is at risk for PCM? Would preventing or treating the malnutrition with SNS improve the prognosis and quality of life? What are the fluid, energy, mineral, and vitamin requirements and can these be provided enterally? Does the patient require total parenteral nutrition? Can requirements be met through oral foods and liquid supplements? Keep under surveillance with frequent calorie counts and clinical assessment Request feeding tube Needed for several weeks Needed for months or years Nasally inserted tube Percutaneously inserted tube Request CVC, PICC or peripheral catheter plus enteral nutrition Request CVC or PICC Need for several weeks Need for months or years Tunneled external catheter or subcutaneous infusion port Subclavian catheter or PICC Risks and discomfort of SNS outweigh potential benefits. Explain issue to patient or legal surrogate. Support patient

1	catheter or subcutaneous infusion port Subclavian catheter or PICC Risks and discomfort of SNS outweigh potential benefits. Explain issue to patient or legal surrogate. Support patient with general comfort measures including oral food and liquid supplements if desired. Yes Yes Yes No Yes Yes Yes No No No

1	FIgURE 98e-1 Decision-making for the implementation of specialized nutritional support (SNS). CVC, central venous catheter; PICC, peripherally inserted central catheter. (Adapted from the chapter on this topic in Harrison’s Principles of Internal Medicine, 16e, by Lyn Howard, MD.) effects of the SRI accelerate skeletal muscle wasting and substantially block normal protein-sparing adaptation to protein and energy starvation. Inactivity A nutritional red flag should be raised over every acutely ill patient who remains bedridden or inactive for a prolonged period. Such patients commonly manifest muscle atrophy (due to nutritional deficiencies and disuse) and anorexia with inadequate voluntary food intake.

1	Once it has been determined that a patient has significant— and, in particular, progressive—PEM despite meaningful efforts to reverse it by modifying the diet or the way food is provided, the next step is to decide whether SNS will have a net positive effect on the patient’s clinical outcome. The pathway to the end stage of most severe chronic diseases leads through PEM. In most patients with end-stage untreatable cancer or certain end-organ diseases, SNS will neither reverse PEM nor improve the quality of life. Provision of food and water is commonly regarded as an aspect of basic humane care; in contrast, enteral and parenteral SNS is a therapeutic intervention that can cause discomfort and pose risks. As with other life-support interventions, the discontinuation of enteral or parenteral SNS can be psychologically difficult for patients, their families, and their caregivers. Indeed, the difficulty can be greater with SNS than with other life-support interventions because the

1	SNS can be psychologically difficult for patients, their families, and their caregivers. Indeed, the difficulty can be greater with SNS than with other life-support interventions because the provision of food and water is often considered equivalent to comfort care. In such difficult, near end-of-life situations, it is prudent to explicitly state the treatment goals at the outset of a course of SNS therapy. Such clarity can smooth the way for subsequent appropriate discontinuation in those patients whose prognosis has become hopeless.

1	After the decision has been made that SNS is indeed appropriate, the next determinations are the route of delivery (enteral versus parenteral), timing, and calculation of the patient’s nutritional goals. Although enteral SNS is the default option, the choice of optimal route depends on the degree of gut function as well as on available technical resources. Both the choice of route and the timing of SNS require an evaluation of the patient’s current nutritional status, the presence and extent of the SRI, and the anticipated clinical course. Severe SRI is identified on the basis of the standard clinical signs of leukocytosis, tachycardia, tachypnea, and temperature elevation or depression.

1	Serum albumin is a negative acute-phase protein and hence a marker of the SRI. More severe hypoalbuminemia is a crude indicator of greater SRI severity, but this condition is almost certainly worsened by concurrent dietary protein deficiency. Despite the importance of adequate protein provision to patients with the SRI, no amount of SNS will raise serum albumin levels into the normal range as long as the SRI persists. The SRI can be graded as mild, moderate, or severe. Examples of a severe SRI include (1) sepsis or other major inflammatory diseases (e.g., pancreatitis) that require care in the intensive care unit; (2) multiple trauma with an Injury Severity Score >20–25 or an Acute Physiology and Chronic Health Evaluation II (APACHE

1	II) score >25; (3) closed head injury with a Glasgow Coma Scale <8; and (4) major third-degree burns of >40% of the body surface area. A moderate SRI occurs with less severe infections, injuries, or inflammatory conditions like pneumonia, uncomplicated major surgery, acute hepatic or renal injury, and exacerbations of ulcerative colitis or regional enteritis requiring hospitalization.

1	Patients with a severe SRI require the initiation of SNS within the first several days of care, for they are highly unlikely to consume an adequate amount of food voluntarily over the next 7 days. On the other hand, a moderate SRI, as is common during the period following major uncomplicated surgery without oral intake, may be tolerated for 5–7 days as long as the patient is initially well nourished. Patients awaiting elective major surgery benefit from preoperative nutritional repletion for 5–10 days but only in the presence of significant PEM. When adequate preoperative nutrition or SNS is impractical, early postoperative SNS is usually indicated. Furthermore, patients with a combination of a moderate SRI and moderate PEM are likely to benefit from early postoperative SNS.

1	The risks of enteral SNS are determined primarily by the patient’s state of alertness and swallowing competence, the anatomy and function of the gastrointestinal tract, and the experience of the supervising clinical team. The safest and least costly approach is to avoid SNS by close attention to oral food intake; personal encouragement; dietary modifications; hand-feeding, when possible; and, often, the addition of an oral liquid supplement. For this reason, all patients at nutritional risk should be assessed and followed by a nutritionist. There is increasing interest in the use, under selected circumstances and when not contraindicated, of pharmacologic doses of anabolic steroids to stimulate appetite and promote muscle anabolism.

1	Nasogastric tube insertion is a bedside procedure, but many critically ill patients have impaired gastric emptying and a high risk of aspiration pneumonia. This risk can be reduced by placing the tip of the feeding tube in the jejunum beyond the ligament of Treitz, a procedure that usually requires fluoroscopic or endoscopic guidance. When a laparotomy is planned for a patient who has other surgical conditions likely to necessitate prolonged SNS, it is advantageous to place a jejunal feeding tube at the time of surgery. A major disadvantage of enteral SNS is that the amounts of protein and calories provided to critically ill patients commonly fail to reach target goals within the first 7–14 days after SNS is initiated. This problem is compounded by the lack of enteral products that allow the provision of the recommended protein target of 1.5–2.0 g/kg without simultaneously inducing potentially harmful caloric overfeeding.

1	Enteral SNS is often required in patients with anorexia, impaired swallowing, or small-intestinal disease. The bowel and its associated digestive organs derive 70% of their required nutrients directly from nutritional substrates absorbed from the intestinal lumen. Enteral feeding also supports gut function by stimulating splanchnic blood flow, neuronal activity, IgA antibody release, and secretion of gastrointestinal hormones that stimulate gut trophic activity. These factors support the gut as an immunologic barrier against enteric pathogens. For these reasons, current evidence indicates that some luminal nutrition should be provided, even when PN is required to provide most of the nutritional support. The nonessential amino acids arginine and glutamine, short-chain fatty acids, long-chain omega 3 fatty acids, and nucleotides are available in some specialty enteral formulas and appear to have an important role in maintaining immune function. The addition of supplemental PN to enteral

1	omega 3 fatty acids, and nucleotides are available in some specialty enteral formulas and appear to have an important role in maintaining immune function. The addition of supplemental PN to enteral feeding (either by mouth or as SNS by enteral tube) may hasten the transition to full enteral feeding, which is usually successful when >50% of requirements can be met enterally. As long as protein and other essential nutrient requirements are met, substantial nutritional benefit can be achieved by providing ~50% of energy needs for periods of up to 10 days. As a rule of thumb, dietary protein provision should be increased by ~25–50% when energy intake is reduced by this amount, since negative energy balance reduces the efficiency of dietary protein retention. For longer periods and in patients who have a normal or increased body fat content, it may be preferable to provide only 75–80% of energy needs (together with increased protein), as the mild energy deficit improves gastrointestinal

1	who have a normal or increased body fat content, it may be preferable to provide only 75–80% of energy needs (together with increased protein), as the mild energy deficit improves gastrointestinal tolerance, makes glycemic control far easier, and avoids excess fluid administration.

1	The main risks of PN are related to the placement of a central venous catheter, with its complications of thrombosis and infection, and the relatively large intravenous volumes infused. Less often appreciated are the risks associated with the ease of inadvertently infusing excessive carbohydrate and lipid directly into the bloodstream. These risks include hyperglycemia, inadequate lipid clearance from the circulation, hepatic steatosis and inflammation, and even respiratory failure in patients with borderline pulmonary function. On the other hand, renal dysfunction does not reduce a patient’s requirement for protein or amino acids. In cases in which renal function is a limiting factor, appropriate renal replacement therapy must be provided along with SNS.

1	In the past, bowel rest through PN was the cornerstone of treatment for many severe gastrointestinal disorders. However, the value of providing even minimal amounts of enteral nutrition (EN) is now widely accepted. Protocols to facilitate more widespread use of EN include initiation within 24 h of ICU admission; aggressive use of the head-upright position; use of postpyloric and nasojejunal feeding tubes; use of prokinetic agents; more rapid increases in feeding rates; tolerance of higher gastric residuals; and adherence to nurse-directed algorithms for feeding progression. Parenteral SNS alone is generally necessary only for severe gut dysfunction due to prolonged ileus, intestinal obstruction, or severe hemorrhagic pancreatitis.

1	In critically ill patients, parenteral SNS can be commenced within the first 24 h of care, with the anticipation of a better clinical outcome and a lower mortality risk than those following delayed or inadequate enteral SNS; however, this point remains controversial. Some evidence suggests that early SNS is associated with a reduced risk of death but also with an increased risk of serious infection. More recent data, obtained in studies of moderately critically ill patients, suggest that early hypocaloric parenteral SNS lessens morbidity and mitigates muscle atrophy without an increased risk of infection, but also without a detectable reduction in mortality risk. Unfortunately, the current clinical-trial evidence fails to address several important unknowns. It is important to note that the level of protein substrate provided in published clinical trials generally falls well below the current recommendation, even in trials of supplemental parenteral SNS. Much of the increase in

1	that the level of protein substrate provided in published clinical trials generally falls well below the current recommendation, even in trials of supplemental parenteral SNS. Much of the increase in morbidity associated with parenteral and enteral SNS can be ascribed to hyperglycemia, which can be prevented by appropriately intensive insulin therapy. The level of glycemia necessary to prevent complications, whether <110 mg/dL or <150 mg/dL, remains unclear. Adequately fed surgical patients may benefit from the lower glucose range, but studies of intensive insulin therapy alone, without full feeding, suggest improved morbidity and mortality outcomes with looser control of glucose at <180 mg/dL.

1	In the early years of its use, PN was relatively expensive, but its components now are often less costly than specialty enteral formulas. Percutaneous placement of a central venous catheter into the subclavian vein or (less desirably) the internal jugular vein with advancement into the superior vena cava can be accomplished at the bedside by trained personnel using sterile techniques. Peripherally inserted central catheters (PICCs) can also be used, although they are usually more appropriate for non-ICU patients. Subclavian or internal jugular catheters carry the risks of pneumothorax or serious vascular damage but are generally well tolerated and, rather than requiring reinsertion, can be exchanged over a wire when catheter infection is suspected.

1	Although most SNS is delivered in hospitals, some patients require it on a long-term basis. At-home SNS requires a safe home environment, a stable clinical condition, and the patient’s ability and willingness to learn appropriate self-care techniques. Other important considerations in determining the appropriateness of at-home parenteral or enteral SNS are that the patient’s prognosis indicates survival for longer than several months and that the therapy enhances the patient’s quality of life.

1	The purpose of SNS is to correct and prevent malnutrition. Certain conditions require special modification of the SNS regimen. Protein intake may need to be limited in many stable patients with renal insufficiency or borderline liver function. In renal disease, except for brief periods, protein intakes should approach the required level for normal adults of at least 0.8 g/kg and should aim for 1.2 g/kg as long as severe azotemia does not occur. Patients with severe renal failure who require SNS need concurrent renal replacement therapy. In hepatic failure, protein intakes of 1.2–1.4 g/kg (up to 1.5 g/kg) should be provided as long as encephalopathy due to protein intolerance does not occur. In the presence of protein intolerance, formulas containing 33–50% branched-chain amino acids are available and can be provided at the 1.2to 1.4-g/kg level. Cardiac patients and many other severely stressed patients often benefit from fluid and sodium restriction to 1000 mL of PN formula and 5–20

1	available and can be provided at the 1.2to 1.4-g/kg level. Cardiac patients and many other severely stressed patients often benefit from fluid and sodium restriction to 1000 mL of PN formula and 5–20 meq of sodium per day. In patients with severe chronic PEM characterized by severe weight loss, it is important to initiate PN gradually because of the profound antinatriuresis, antidiuresis, and intracellular accumulation of potassium, magnesium, and phosphorus that develop as a consequence of the resulting high insulin levels. This modification of parenteral SNS is usually accomplished by limiting daily fluid intake initially to ~1000 mL; limiting carbohydrate intake to 10–20% dextrose; limiting sodium intake; and providing ample potassium, magnesium, and phosphorus, with careful daily assessment of fluid and electrolyte status. Protein need not be restricted.

1	Normal adults require ~30 mL of fluid/kg of body weight from all sources each day as well as the replacement of abnormal losses such as those caused by diuretic therapy, nasogastric tube drainage, wound output, high rates of perspiration (which can be several liters per day during periods of extreme heat), and diarrhea/ostomy losses. Electrolyte and mineral losses can be estimated or measured and need to be replaced (Table 98e-2). Fluid restriction may be necessary in patients with fluid overload. Total fluid input can usually be limited to 1200 mL/d as long as urine is the only significant source of fluid output. In severe fluid overload, a 1-L central vein PN solution of 7% crystalline amino acids (70 g) and 21% dextrose (210 g) can temporarily provide an acceptable amount of glucose and protein substrate in the absence of significant catabolic stress.

1	Patients who require PN or EN in the acute-care setting generally have associated hormonal adaptations to their underlying disease (e.g., increased secretion of antidiuretic hormone, aldosterone, insulin, glucagon, or cortisol), and these signals promote fluid retention and hyperglycemia. In critical illness, body weight is invariably increased due to fluid resuscitation and fluid retention. Lean-tissue accretion is minimal in the acute phase of critical illness, no matter how much protein and or how many calories are provided. Because excess fluid removal can be difficult, limiting fluid intake to allow for balanced intake and output is more effective.

1	Total energy expenditure comprises resting energy expenditure, activity energy expenditure, and the thermal effect of feeding (Chap. 97). Resting energy expenditure accounts for two-thirds of total energy expenditure, activity energy expenditure for one-fourth to one-third, and the thermal effect of feeding for ~10%. For normally nourished, healthy individuals, the total energy expenditure is ~30–35 kcal/kg. Critical illness increases resting energy expenditure, but this increase is significant only in initially well-nourished individuals with a robust SRI who experience, for example, severe multiple trauma, extensive burns, sepsis, sustained high fever, or closed head injury. In these situations, total energy expenditure can reach 40–45 kcal/kg. The chronically starved patient with adapted PEM has a reduced energy expenditure and is inactive, with a usual total energy expenditure of ~20–25 kcal/kg. Very few patients with adapted PEM require as much as 30 kcal/kg for energy balance.

1	PEM has a reduced energy expenditure and is inactive, with a usual total energy expenditure of ~20–25 kcal/kg. Very few patients with adapted PEM require as much as 30 kcal/kg for energy balance. Because providing ~50% of measured energy expenditure as SNS is at least as effective as 100% for the first 10 days of critical illness, actual measurement of energy expenditure generally is not necessary in the early period of SNS. However, in patients who remain critically ill beyond several weeks, in patients with severe PEM for whom estimates of energy expenditure are unreliable, and in patients who are difficult to wean from ventilators, it is reasonable to measure energy expenditure directly when the technique is available, targeting an energy intake of 100–120% of the measured energy expenditure.

1	Insulin resistance due to the SRI is associated with increased gluconeogenesis and reduced peripheral glucose utilization, with resulting hyperglycemia. Hyperglycemia is aggravated by excessive exogenous carbohydrate administration from SNS. In critically ill patients receiving SNS, normalization of blood glucose levels by insulin infusion reduces morbidity and mortality risk. In mildly or moderately malnourished patients, it is reasonable to provide metabolic support in order to improve protein synthesis and maintain metabolic homeostasis. Hypocaloric nutrition, with provision of ~1000 kcal and 70 g protein per day for up to 10 days, requires less fluid and reduces the likelihood of poor glycemic control, although a higher protein intake would be optimal. During the second week of SNS, energy and protein provision can be advanced to 20–25 kcal/kg and 1.5 g/kg per day, respectively, as metabolic conditions permit. As mentioned above, patients with multiple trauma, closed head injury,

1	and protein provision can be advanced to 20–25 kcal/kg and 1.5 g/kg per day, respectively, as metabolic conditions permit. As mentioned above, patients with multiple trauma, closed head injury, and severe burns often have greatly elevated energy expenditures, but there is little evidence that providing >30 kcal/kg daily confers further benefit, and such high caloric intake may well be harmful as it substantially increases the risk of hyperglycemia.

1	As a rule, amino acids and glucose are provided in an increasing dose until energy provision matches estimated resting energy expenditure. At this point, it becomes beneficial to add fat. A surfeit of glucose merely stimulates de novo lipogenesis—an energy-inefficient process. Polyunsaturated long-chain triglycerides (e.g., in soybean oil) are the chief ingredient in most parenteral fat emulsions and provide the majority of the fat in enteral feeding formulas. These vegetable oil–based emulsions provide essential fatty acids. The fat content of enteral feeding formulas ranges from 3% to 50% of energy. Parenteral fat is provided in separate containers as 20% and 30% emulsions that can be infused separately or mixed in the sterile pharmacy as an all-in-one or total nutrient admixture of amino acids, glucose, lipid, electrolytes, vitamins, and minerals. Although parenteral fat needs to make up only ~3% of the energy requirement in order to meet essential fatty acid requirements, when

1	amino acids, glucose, lipid, electrolytes, vitamins, and minerals. Although parenteral fat needs to make up only ~3% of the energy requirement in order to meet essential fatty acid requirements, when provided daily as an all-in-one mixture of carbohydrate, fat, and protein, the complete admixture has a fat content of 2–3 g/dL and provides 20–30% of the total energy requirement—an acceptable level that offers the advantage of ensuring emulsion stability. When given as a separate infusion, parenteral fat should not be provided at rates exceeding 0.11 g/kg of body mass or 100 g over 12 h—equivalent to 500 mL of 20% parenteral fat.

1	Medium-chain triglycerides containing saturated fatty acids with chain lengths of 6, 8, 10, or 12 carbons (>95% of which are C8 and C10) are included in a number of enteral feeding formulas because they are absorbed preferentially. Fish oil contains polyunsaturated fatty acids of the omega 3 family, which improve immune function and reduce the inflammatory response. At this time, fish oil injectable emulsions are available in the United States as an investigational new drug.

1	PN formulations provide carbohydrate as hydrous glucose (3.4 kcal/g). In enteral formulas, glucose is the carbohydrate source for so-called monomeric diets. These diets provide protein as amino acids and fat in minimal amounts (3%) to meet essential fatty acid requirements. Monomeric formulas are designed to optimize absorption in the seriously compromised gut. These formulas, like immune-enhancing diets, are expensive. In polymeric diets, the carbohydrate source is usually an osmotically less active polysaccharide, the protein is usually soy or casein protein, and fat is present at concentrations of 25–50%. Such formulas are usually well tolerated by patients with normal intestinal length, and some are acceptable for oral consumption.

1	The daily protein recommendation for healthy adults is 0.8 g/kg, but body proteins are replenished faster with 1.5 g/kg in patients with PEM, and net protein catabolism is reduced in critically ill patients when 1.5–2.0 g/kg is provided. In patients who are not critically ill but who require SNS in the acute-care setting, at least 1 g of protein/ kg is recommended, and larger amounts up to 1.5 g/kg are appropriate when volume, renal, and hepatic tolerances allow. The standard parenteral and enteral formulas contain protein of high biologic value and meet the requirements for the eight essential amino acids when nitrogen needs are met. Parenteral amino acid mixtures and elemental enteral mixtures consist of hydrated individual amino acids. Because of their hydrated status, elemental amino acid solutions deliver 17% less protein substrate than intact proteins. In protein-intolerant conditions such as renal and hepatic failure, modified amino acid formulas may be considered. In hepatic

1	acid solutions deliver 17% less protein substrate than intact proteins. In protein-intolerant conditions such as renal and hepatic failure, modified amino acid formulas may be considered. In hepatic failure, higher branched-chain, amino acid–enriched formulas appear to improve outcomes. Conditionally essential amino acids like arginine and glutamine may also have some benefit in supplemental amounts.

1	Protein (nitrogen) balance provides a measure of the efficacy of parenteral or enteral SNS. This balance is calculated as protein intake/6.25 (because proteins are, on average, 16% nitrogen) minus the 24-h urine urea nitrogen plus 4 g of nitrogen (the latter reflecting other nitrogen losses). In critical illness, a mild negative nitrogen balance of 2–4 g/d is often achievable. A similarly mild positive nitrogen balance is observed in the nonstressed recuperating patient. Each gram of nitrogen lost or gained represents ~30 g of lean tissue.

1	Parenteral electrolyte, vitamin, and trace mineral requirements are summarized in Tables 98e-3, 98e-4, and 98e-5, respectively. Electrolyte modifications are necessary with substantial gastrointestinal losses from nasogastric drainage or intestinal losses from fistulas, diarrhea, or ostomy outputs. Such losses also imply extra calcium, magnesium, and zinc losses. Zinc losses are high in secretory diarrhea. Secretory diarrhea contains ~12 mg of zinc/L, and patients with intestinal fistulas or chronic diarrhea require an average of ~12 mg of Acetate Calcium Magnesium Phosphorus 10 meq 10 meq 30 mmol 1–2 meq/kg + replacement, but can be as low as 5–40 meq/d 40–100 meq/d + replacement of unusual losses

1	Acetate Calcium Magnesium Phosphorus 10 meq 10 meq 30 mmol 1–2 meq/kg + replacement, but can be as low as 5–40 meq/d 40–100 meq/d + replacement of unusual losses As needed for acid-base balance, but usually 2:1 to 1:1 with acetate As needed for acid-base balance 10–20 meq/d 8–16 meq/d 20–40 mmol parenteral zinc/d (equivalent to 30 mg of oral elemental zinc) to maintain zinc balance. Excessive urinary potassium losses with amphotericin or magnesium losses with cisplatin or in renal failure necessitate adjustments in sodium, potassium, magnesium, phosphorus, and acid-base balance. Vitamin and trace element requirements are met by the daily provision of a complete parenteral vitamin supplement and trace elements via PN and by the provision of adequate amounts of enteral feeding formulas that contain these micronutrients.

1	Iron is a highly reactive catalyst of oxidative reactions and thus is not included in PN mixtures. The parenteral iron requirement is normally only ~1 mg/d. Iron deficiency occurs with considerable frequency in acutely ill hospitalized patients, especially those with PEM and gastrointestinal tract disease, and in patients subjected to frequent blood withdrawals. Iron deficiency is sometimes inadequately considered in hospitalized patients because there are commoner causes: the inflammation-mediated anemia of chronic disease (with an associated increase in serum ferritin, an acute-phase protein) and redistribution of the intravascular fluid volume during prolonged bed rest. Iron deficiency should be considered in every patient receiving SNS. A falling mean red cell volume, even if still in the low-normal range, together with an intermediate serum ferritin concentration is suggestive of iron deficiency. Intravenous iron infusions follow standard guidelines, always with a termination

1	in the low-normal range, together with an intermediate serum ferritin concentration is suggestive of iron deficiency. Intravenous iron infusions follow standard guidelines, always with a termination order and never as a standing order because of the risk of inadvertent iron overdosing. Major iron replacement during critical illness is of some concern because of the possibility that a substantial rise in the serum iron concentration may increase susceptibility to some bacterial infections.

1	3.6 mg 40 mg 600 μg 15 mg 6 mg 5 μg 60 μg 200 mg 200 IUa 10 IU 150 μg aThe current vitamin D requirement—a minimum of 600 IU/day—cannot be met with available injectable vitamin formulations. Calcitriol is not equivalent to vitamin D and is not a suitable replacement for it, since it is not a substrate for 25-hydroxyvitamin D biosynthesis. bA product is available without vitamin K. Vitamin K supplementation is recommended at 2–4 mg/week in patients not receiving oral anticoagulation therapy when the vitamin K–free product is used. aCommercial products are available with the first four, the first five, and all seven of these metals in recommended amounts. bThe basal IV zinc requirement is approximately one-third of the oral requirement, because only approximately one-third of orally ingested zinc is absorbed. Abbreviation: PN, parenteral nutrition.

1	Parenteral feeding through a peripheral vein is limited by osmolarity and volume constraints. Solutions with an osmolarity >900 mOsm/L (e.g., those which contain >3% amino acids and 5% glucose [290 kcal/L]) are poorly tolerated peripherally. Parenteral lipid emulsions (20%) can be given to increase the calories delivered. The total volume required for a marginal amino acid provision rate of 60 g (equivalent to 50 g of protein) and a total of 1680 kcal is 2.5 L. Moreover, the risk of significant morbidity and mortality from incompatibilities of calcium and phosphate salts is greatest in these low-osmolarity, low-glucose regimens. For short-term infusions, calcium may be temporarily limited or even omitted from the mixture. Parenteral feeding via a peripheral vein is generally intended as a supplement to oral feeding; it is not suitable for the critically ill. Peripheral PN may be enhanced by small amounts of heparin (1000 U/L) and co-infusion with parenteral fat to reduce osmolarity,

1	a supplement to oral feeding; it is not suitable for the critically ill. Peripheral PN may be enhanced by small amounts of heparin (1000 U/L) and co-infusion with parenteral fat to reduce osmolarity, but volume constraints still limit the value of this therapy, especially in critical illness.

1	PICCs may be used to infuse solutions of 20–25% dextrose and 4–7% amino acids, thus avoiding the traumatic complications of percutaneous central vein catheter placement. With PICC lines, however, flow can be position-related, and the lines cannot be exchanged over a wire for infection monitoring. It is important to withdraw blood samples carefully and appropriately from a dual-port PICC because intermixing of the blood sample with even tiny volumes of nutrient infusate will falsely indicate hyperglycemia and hyperkalemia. For all these reasons, centrally placed catheters are preferred in critical illness. The subclavian approach is best tolerated by the patient and is the easiest to dress. The jugular approach is less likely to cause a pneumothorax. Femoral vein catheterization is strongly discouraged because of the risk of catheter infection. For long-term feeding at home, tunneled catheters and implanted ports are used to reduce infection risk and are more acceptable to patients.

1	discouraged because of the risk of catheter infection. For long-term feeding at home, tunneled catheters and implanted ports are used to reduce infection risk and are more acceptable to patients. Tunneled catheters require placement in the operating room.

1	Catheters are made of Silastic®, polyurethane, or polyvinyl chloride. Silastic catheters are less thrombogenic and are best for tunneled catheters. Polyurethane is best for temporary catheters. To avoid infection, dressing changes with dry gauze should be performed at regular intervals by nurses skilled in catheter care. Chlorhexidine solution is more effective than alcohol or iodine compounds. Appropriate monitoring for patients receiving PN is summarized in Table 98e-6. Even though premixed solutions of crystalline amino acids and dextrose are in common use, the future of evidence-based PN lies in computer-controlled sterile compounders that rapidly and inexpensively General sense of well-being

1	General sense of well-being Strength, as evidenced by getting out of bed, walking, and resistance exercise as appropriate Vital signs, including temperature, blood pressure, pulse, and respiratory rate Fluid balance: weight (recorded at least several times weekly); fluid intake (parenteral and enteral) vs. fluid output (urine, stool, gastric drainage, wound, ostomy) Parenteral nutrition delivery equipment: tubing, pump, filter, catheter, dressing Blood glucose, Na, K, Cl, HCO3, BUN Daily until stable and fully advanced; then twice weekly Serum creatinine, albumin, PO4, Ca, Baseline; then twice weekly Mg, Hb/Hct, WBC count aParameters are assessed daily unless otherwise specified. Abbreviations: BUN, blood urea nitrogen; Hb, hemoglobin; Hct, hematocrit; INR, international normalized ratio; WBC, white blood cell. Source: Adapted from the chapter on this topic in Harrison’s Principles of Internal Medicine, 16e, by Lyn Howard, MD.

1	generate personalized solutions that meet the specific protein and calorie goals for different patients in different clinical situations. For example, 1 L of a standard mixture of 5% amino acids/25% dextrose solution provides 50 g of amino acids (41.5 g of protein substrate) and 1000 kcal; the use of this solution to meet the 1.5–to 2.0-g/kg protein requirement of an acutely ill 70-kg patient requires the infusion of 2.5–3.4 L of fluid and a potentially excessively high energy dose of 2500-3300 kcal. When the body fat store is adequate, clinical evidence increasingly supports the greater safety and efficacy of high-protein, moderately hypocaloric SNS in such patients. A sterile compounder can accurately generate an appropriate recipe for such a patient. For example, 1 L of a solution including 600 mL of 15% amino acids, 300 mL of 50% dextrose, and 100 mL of electrolyte/micronutrient mix contains 75 g of protein substrate and 800 kcal; thus it is feasible to meet the patient’s protein

1	600 mL of 15% amino acids, 300 mL of 50% dextrose, and 100 mL of electrolyte/micronutrient mix contains 75 g of protein substrate and 800 kcal; thus it is feasible to meet the patient’s protein requirement with only 1.4–1.9 L of solution and a more appropriate 1100–1520 kcal; any mild gap in energy provision is easily filled by use of intravenous lipid.

1	COMPLICATIONS Mechanical The insertion of a central venous catheter should be performed by trained and experienced personnel using aseptic techniques to limit the major common complications of pneumothorax and inadvertent arterial puncture or injury. The catheter’s position should be radiographically confirmed to be in the superior vena cava distal to the junction with the jugular or subclavian vein and not directly against the vessel wall. Thrombosis related to the catheter may occur at the site of entry into the vein and extend to encase the catheter. Catheter infection predisposes to thrombosis, as does the SRI. The addition of 6000 U of heparin to the daily parenteral formula for hospitalized patients with temporary catheters reduces the risk of fibrin sheath formation and catheter infection. Temporary catheters that develop a thrombus should be removed and, according to clinical findings, treated with anticoagulants. Thrombolytic therapy can be considered for patients with

1	infection. Temporary catheters that develop a thrombus should be removed and, according to clinical findings, treated with anticoagulants. Thrombolytic therapy can be considered for patients with permanent catheters, depending on the ease of replacement and the presence of alternative, reasonably acceptable venous access sites. Low-dose warfarin therapy (1 mg/d) reduces the risk of thrombosis in permanent catheters used for at-home parenteral SNS, but full anticoagulation may be required for patients who have recurrent thrombosis related to permanent catheters. A recent U.S. Food and Drug Administration mandate to reformulate parenteral multivitamins to include vitamin K at a dose of 150 μg/d may affect the efficacy of low-dose warfarin therapy. A “no vitamin K” version is available for patients receiving this therapy. Catheters can become occluded due to mechanical factors; by fibrin at the tip; or by fat, minerals, or drugs intraluminally. These occlusions can be managed with

1	for patients receiving this therapy. Catheters can become occluded due to mechanical factors; by fibrin at the tip; or by fat, minerals, or drugs intraluminally. These occlusions can be managed with low-dose alteplase for fibrin, with indwelling 70% alcohol for fat, with 0.1 N hydrochloric acid for mineral precipitates, and with either 0.1 N hydrochloric acid or 0.1 N sodium hydroxide for drugs, depending on the pH of the drug.

1	Metabolic The most common problems caused by parenteral SNS are fluid overload and hyperglycemia (Table 98e-7). Hypertonic dextrose stimulates a much higher insulin level than meal feeding. Because insulin is a potent antinatriuretic and antidiuretic hormone, Corrective Action Disturbance Cause with PN longed periods. Abbreviation: PN, parenteral nutrition.

1	hyperinsulinemia leads to sodium and fluid retention. Consequently, 98e-7 in the absence of gastrointestinal losses or renal dysfunction, net fluid retention is likely when total fluid intake exceeds 2000 mL/d. Close monitoring of body mass as well as of fluid intake and output is necessary to prevent this complication. In the absence of significant renal impairment, the sodium content of the urine is likely to be <10 meq/L. Provision of sodium in limited amounts (40 meq/d) and the use of both glucose and fat in the PN mixture will reduce serum glucose levels and help reduce fluid retention. The elevated insulin level also increases the intracellular transport of potassium, magnesium, and phosphorus, which can precipitate a dangerous re-feeding syndrome if the total glucose content of the PN solution is advanced too quickly in severely malnourished patients. To assess glucose tolerance, it is generally best to start PN with <200 g of glucose/d. Regular insulin can be added to the PN

1	PN solution is advanced too quickly in severely malnourished patients. To assess glucose tolerance, it is generally best to start PN with <200 g of glucose/d. Regular insulin can be added to the PN formula to establish glycemic control, and the insulin doses can be increased proportionately as the glucose content is advanced. As a general rule, patients with insulin-dependent diabetes require about twice their usual at-home insulin dose when receiving PN at 20–25 kcal/kg, largely as a consequence of parenteral glucose administration and some loss of insulin to the formula’s container. As a rough estimate, the amount of insulin provided can be proportionately similar to the number of calories provided as total parenteral nutrition (TPN) relative to full feeding, and the insulin can be placed in the TPN formula. Subcutaneous regular insulin can be provided to improve glucose control as assessed by measurements of blood glucose every 6 h. About two-thirds of the total 24-h amount can be

1	in the TPN formula. Subcutaneous regular insulin can be provided to improve glucose control as assessed by measurements of blood glucose every 6 h. About two-thirds of the total 24-h amount can be added to the next day’s order, with SC insulin supplements as needed. Advances in the TPN glucose concentration should be made when reasonable glucose control is established, and the insulin dose can be adjusted proportionately to the calories added as glucose and amino acids. These are general rules, and they are conservative. Given the adverse clinical impact of hyperglycemia, it may be necessary to use intensive insulin therapy as a separate infusion with a standard protocol to initially establish control. Once control is established, this insulin dose can be added to the PN formula. Acid-base imbalance is also common during parenteral SNS. Amino acid formulas are buffered, but critically ill patients are prone to metabolic acidosis, often due to renal tubular impairment. The use of

1	imbalance is also common during parenteral SNS. Amino acid formulas are buffered, but critically ill patients are prone to metabolic acidosis, often due to renal tubular impairment. The use of sodium and potassium acetate salts in the PN formula may address this problem. Bicarbonate salts should not be used because they are incompatible with TPN formulations. Nasogastric drainage produces hypochloremic alkalosis that can be managed by attention to chloride balance. Occasionally, hydrochloric acid may be required for a more rapid response or when diuretic therapy limits the ability to provide substantial sodium chloride. Up to 100 meq/L and up to 150 meq of hydrochloric acid per day may be placed in a fat-free TPN formula.

1	Infectious Infections of the central access catheter rarely occur in the first 72 h. Fever during this period is usually attributable to infection elsewhere or another cause. Fever that develops during parenteral SNS can be addressed by checking the catheter site and, if the site looks clean, exchanging the catheter over a wire, with cultures taken through the catheter and at the catheter tip. If these cultures are negative, as they usually are, the new catheter can continue to be used. If a culture is positive for a relatively nonpathogenic bacterium like Staphylococcus epidermidis, a second exchange over a wire with repeat cultures or replacement of the catheter can be considered in light of the clinical circumstances. If cultures are positive for more pathogenic bacteria or for fungi like Candida albicans, it is generally best to replace the catheter at a new site. Whether antibiotic treatment is required is a clinical decision, but C. albicans grown from the blood culture in a

1	like Candida albicans, it is generally best to replace the catheter at a new site. Whether antibiotic treatment is required is a clinical decision, but C. albicans grown from the blood culture in a patient receiving PN should always be treated with an antifungal drug because the consequences of failure to treat can be dire.

1	Catheter infections can be minimized by dedicating the feeding catheter to TPN, without blood sampling or medication administration. Central catheter infections are a serious complication, with an attributed mortality rate of 12–25%. Fewer than three infections per 1000 catheter-days should occur in central venous catheters dedicated to feeding. At-home TPN catheter infections may be treated through 98e-8 the catheter without its removal, particularly if the offending organism is S. epidermidis. Clearing of the biofilm and fibrin sheath by local treatment of the catheter with indwelling alteplase may increase the likelihood of eradication. Antibiotic lock therapy with high concentrations of antibiotic, with or without heparin in addition to systemic therapy, may improve efficacy. Sepsis with hypotension should precipitate catheter removal in either the temporary or the permanent TPN setting.

1	The types of enteral feeding tubes, methods of insertion, their clinical uses, and potential complications are outlined in Table 98e-8. The different types of enteral formulas are listed in Table 98e-9. Patients receiving EN are at risk for many of the same metabolic complications as those who receive PN and should be monitored in the same manner. EN can be a source of similar problems, but not to the same degree, because the insulin response to EN is about half of that to PN. Enteral feeding formulas have fixed electrolyte compositions that are generally modest in sodium and somewhat higher in potassium. Acid-base disturbances can be addressed to a more limited extent with EN.

1	External measurement: Short-term clinical situ-Aspiration; ulceration nostril, ear, xiphister-ation (weeks) or longer of nasal and esophanum; tube stiffened by periods with intermit-geal tissues, leading to ice water or stylet; posi-tent insertion; bolus stricture tion verified by air injec-feeding is simpler, but tion and auscultation or continuous drip with by x-ray pump is better tolerated External measurement: Short-term clinical Spontaneous pulling nostril, ear, anterior situations where gastric back into stomach superior iliac spine; tube emptying is impaired (position verified by stiffened by stylet and or proximal leak is aspirating content, pH passed through pylorus suspected; requires con->6); diarrhea common, under fluoroscopy or tinuous drip with pump fiber-containing formuwith endoscopic loop

1	Percutaneous place-Long-term clinical Aspiration; irritation ment endoscopically, situations, swallowing around tube exit site; radiologically, or surgi-disorders, or impaired peritoneal leak; balloon cally; after track is estab-small-bowel absorption migration and obstruclished, can be converted requiring continuous tion of pylorus to a gastric “button” drip Percutaneous place-Long-term clinical Clogging or displacement endoscopically or situations where gastric ment of tube; jejunal radiologically via pylorus emptying is impaired; fistula if large-bore tube or endoscopically or requires continuous drip is used; diarrhea from surgically directly into with pump; direct endo-dumping; irritation the jejunum scopic placement (PEJ) of surgical anchoring Abbreviation: PEJ, percutaneous endoscopic jejunostomy.

1	Abbreviation: PEJ, percutaneous endoscopic jejunostomy. Note: All small tubes are at risk for clogging, especially if used for crushed medications. In long-term enteral nutrition patients, gastrostomy and jejunostomy tubes can be exchanged for a low-profile “button” once the track is established. Source: Adapted from the chapter on this topic in Harrison’s Principles of Internal Medicine, 16e, by Lyn Howard, MD. 1. Caloric density: 1 kcal/mL 2. Protein: ~14% cals (caseinates, soy, lactalbumin) 3. Carbohydrate: ~60% cals (hydrolyzed corn starch, maltodextrin, sucrose) 4. Fat: ~30% cals (corn, soy, safflower oils) 5. Recommended daily intake of all minerals and vitamins in >1500 kcal/d 6. Osmolality: ~300 mosmol/kg 1. Caloric density: 1.5–2 kcal/mL (+) Fluid-restricted patients 2. a. b. Hydrolyzed protein to small Impaired absorption peptides (+) c. ↑ Arginine, glutamine, Immune-enhancing diets nucleotides, ω3 fat (+++) d.

1	a. b. Hydrolyzed protein to small Impaired absorption peptides (+) c. ↑ Arginine, glutamine, Immune-enhancing diets nucleotides, ω3 fat (+++) d. ↑ Branched-chain amino acids, Liver failure patients intolerant of aromatic amino acids (+++) 0.8 g of protein/kg e. Low protein of high biologic Renal failure patients for brief periods value if critically ill 3. Fat a. b. ↑ Fat (>40% cals) (++) Pulmonary failure with CO2 retention on standard formula, limited utility c. d. 4. Fiber: provided as soy Improved laxation polysaccharide (+) aCost: +, inexpensive; ++, moderately expensive; +++, very expensive. Note: ARDS, acute respiratory distress syndrome; MCT, medium-chain triglyceride; MUFA, monounsaturated fatty acids; ω3 or ω6, polyunsaturated fat with first double bond at carbon 3 (fish oils) or carbon 6 (vegetable oils). Source: Adapted from the chapter on this topic in Harrison’s Principles of Internal Medicine, 16e, by Lyn Howard, MD.

1	Source: Adapted from the chapter on this topic in Harrison’s Principles of Internal Medicine, 16e, by Lyn Howard, MD. Acetate salts can be added to the formula to treat chronic metabolic acidosis. Calcium chloride can be added to treat mild chronic metabolic alkalosis. Medications and other additives to enteral feeding formulas can clog the tubes (e.g., calcium chloride may interact with casein-based formulas to form insoluble calcium caseinate products) and may reduce the efficacy of some drugs (e.g., phenytoin). Since small-bore tubes are easily displaced, tube position should be checked at intervals by aspirating and measuring the pH of the gut fluid (normal: <4 in the stomach, >6 in the jejunum).

1	COMPLICATIONS Aspiration The debilitated patient with poor gastric emptying and impairment of swallowing and cough is at risk for aspiration; this complication is particularly common among patients who are mechanically ventilated. Tracheal suctioning induces coughing and gastric regurgitation, and cuffs on endotracheal or tracheostomy tubes seldom protect against aspiration. Preventive measures include elevating the head of the bed to 30°, using nurse-directed algorithms for formula advancement, combining enteral with parenteral feeding, and using post–ligament of Treitz feeding. Tube feeding should not be discontinued for gastric residuals of <300 mL unless there are other signs of gastrointestinal intolerance, such as nausea, vomiting, or abdominal distention. Continuous feeding using pumps is better tolerated intragastrically than bolus feeding and is essential for feeding into the jejunum. For small-bowel feeding, residuals are not assessed, but abdominal pain and distention

1	pumps is better tolerated intragastrically than bolus feeding and is essential for feeding into the jejunum. For small-bowel feeding, residuals are not assessed, but abdominal pain and distention should be monitored.

1	Diarrhea Enteral feeding often leads to diarrhea, especially if bowel function is compromised by disease or drugs (most often, broad-spectrum antibiotics). Sorbitol used to flavor some medications can also cause diarrhea. Diarrhea may be controlled by the use of a continuous drip, with a fiber-containing formula, or by the addition of an antidiarrheal agent to the formula. However, Clostridium difficile, which is a common cause of diarrhea in patients being tube-fed, should be ruled out as the etiology before antidiarrheal agents are used. H2 blockers may help reduce the net volume of fluid presented to the colon. Diarrhea associated with enteral feeding does not necessarily imply inadequate absorption of nutrients other than water and electrolytes. Amino acids and glucose are particularly well absorbed in the upper small bowel except in the most diseased or shortest bowel. Since luminal nutrients exert trophic effects on the gut mucosa, it is often appropriate to persist with tube

1	well absorbed in the upper small bowel except in the most diseased or shortest bowel. Since luminal nutrients exert trophic effects on the gut mucosa, it is often appropriate to persist with tube feeding despite diarrhea, even when this course necessitates supplemental parenteral fluid support.

1	Apart from conditions with drastically diminished small-intestinal 98e-9 absorptive function, there are no established indications for short peptide–based or elemental formulas.

1	In the United States, the only parenteral lipid emulsion avail able is made with soybean oil, whose constituent fatty acids have been suggested to be immunosuppressive under certain circumstances. In Europe and Japan, a number of other lipid emulsions are available, including those containing fish oil only; mixtures of fish oil, medium-chain triglycerides, and long-chain triglycerides as olive oil and/or soybean oil; mixtures of medium-chain triglycerides and long-chain triglycerides as soybean oil; and long-chain triglyceride mixtures as olive oil and soybean oil, which may be more beneficial in terms of metabolism and hepatic and immune function. Furthermore, a glutamine-containing dipeptide for inclusion in TPN formulas is available in Europe and may be helpful in terms of immune function and resistance to infection, although a recent study using a largerthan-recommended dose was associated with net harm.

1	The authors acknowledge the contributions of Lyn Howard, MD, the author in earlier editions of HPIM, to material in this chapter.

1	approach to the patient with Cancer Dan L. Longo The application of current treatment techniques (surgery, radiation therapy, chemotherapy, and biologic therapy) results in the cure of nearly two of three patients diagnosed with 99 seCtion 1 cancer. Nevertheless, patients experience the diagnosis of cancer as one of the most traumatic and revolutionary events that has ever happened to them. Independent of prognosis, the diagnosis brings with it a change in a person’s self-image and in his or her role in the home and workplace. The prognosis of a person who has just been found to have pancreatic cancer is the same as the prognosis of the person with aortic stenosis who develops the first symptoms of congestive heart failure (median survival, ~8 months). However, the patient with heart disease may remain functional and maintain a self-image as a fully intact person with just a malfunctioning part, a diseased organ (“a bum ticker”). By contrast, the patient with pancreatic cancer has a

1	may remain functional and maintain a self-image as a fully intact person with just a malfunctioning part, a diseased organ (“a bum ticker”). By contrast, the patient with pancreatic cancer has a completely altered self-image and is viewed differently by family and anyone who knows the diagnosis. He or she is being attacked and invaded by a disease that could be anywhere in the body. Every ache or pain takes on desperate significance. Cancer is an exception to the coordinated interaction among cells and organs. In general, the cells of a multicellular organism are programmed for collaboration. Many diseases occur because the specialized cells fail to perform their assigned task. Cancer takes this malfunction one step further. Not only is there a failure of the cancer cell to maintain its specialized function, but it also strikes out on its own; the cancer cell competes to survive using natural mutability and natural selection to seek advantage over normal cells in a recapitulation of

1	specialized function, but it also strikes out on its own; the cancer cell competes to survive using natural mutability and natural selection to seek advantage over normal cells in a recapitulation of evolution. One consequence of the traitorous behavior of cancer cells is that the patient feels betrayed by his or her body. The cancer patient feels that he or she, and not just a body part, is diseased.

1	No nationwide cancer registry exists; therefore, the incidence of cancer is estimated on the basis of the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) database, which tabulates cancer incidence and death figures from 13 sites, accounting for about 10% of the U.S. population, and from population data from the U.S. Census Bureau. In 2014, 1.665 million new cases of invasive cancer (855,220 men, 810,320 women) were diagnosed, and 585,720 persons (310,010 men, 275,710 women) died from cancer. The percent distribution of new cancer cases and cancer deaths by site for men and women is shown in Table 99-1. Cancer incidence has been declining by about 2% each year since 1992. Cancer is the cause of one in four deaths in the United States.

1	The most significant risk factor for cancer overall is age; two-thirds of all cases were in those older than age 65 years. Cancer incidence increases as the third, fourth, or fifth power of age in different sites. For the interval between birth and age 49 years, 1 in 29 men and 1 in 19 women will develop cancer; for the interval between ages 50 and 59 years, 1 in 15 men and 1 in 17 women will develop cancer; for the interval between ages 60 and 69 years, 1 in 6 men and 1 in 10 women will develop cancer; and for people age 70 and older, 1 in 3 men and 1 in 4 women will develop cancer. Overall, men have a 44% risk of developing cancer at some time during their lives; women have a 38% lifetime risk. Source: From R Siegel et al: Cancer statistics, 2014. CA Cancer J Clin 64:9, 2014.

1	Cancer is the second leading cause of death behind heart disease. Deaths from heart disease have declined 45% in the United States since 1950 and continue to decline. Cancer has overtaken heart disease as the number one cause of death in persons younger than age 85 years. Incidence trends over time are shown in Fig. 99-1. After a 70-year period of increase, cancer deaths began to decline in 1990–1991 (Fig. 99-2). Between 1990 and 2010, cancer deaths decreased by 21% among men and 12.3% among women. The magnitude of the decline is illustrated in Fig. 99-3. The five leading causes of cancer deaths are shown for various populations in Table 99-2. The 5-year survival for white patients was 39% in 1960–1963 and 69% in 2003–2009. Cancers are more often deadly in blacks; the 5-year survival was 61% for the 2003–2009 interval; however, the racial differences are narrowing over time. Incidence and mortality vary among racial and ethnic groups (Table 99-3). The basis for these differences is

1	61% for the 2003–2009 interval; however, the racial differences are narrowing over time. Incidence and mortality vary among racial and ethnic groups (Table 99-3). The basis for these differences is unclear.

1	In 2008, 12.7 million new cancer cases and 7.6 million cancer deaths were estimated worldwide, according to estimates of GLOBOCAN 2008, developed by the International Agency for Research on Cancer (IARC). When broken down by region of the world, ~45% of cases were in Asia, 26% in Europe, 14.5% in North America, 7.1% in Central/South America, 6% in Africa, and 1% in Australia/New Zealand (Fig. 99-4). Lung cancer is the most common cancer and the most common cause of cancer death in the world. Its Approach to the Patient with Cancer Rate per 100,000 population Year of diagnosis Year of diagnosis

1	FIGURE 99-1 Incidence rates for particular types of cancer over the last 35 years in men (A) and women (B). (From R Siegel et al: CA Cancer J Clin 64:9, 2014.) incidence is highly variable, affecting only 2 per 100,000 African women but as many as 61 per 100,000 North American men. Breast cancer is the second most common cancer worldwide; however, it ranks fifth as a cause of death behind lung, stomach, liver, and colorectal cancer. Among the eight most common forms of cancer, lung (2-fold), breast (3-fold), prostate (2.5-fold), and colorectal (3-fold) cancers are more common in more developed countries than in less developed countries. By contrast, liver (2-fold), cervical (2-fold), and esophageal (2to 3-fold) cancers are more common in less developed countries. Stomach cancer incidence is similar in more and less developed countries but is much more common in Asia than North America or Africa. The most common cancers in Africa are cervical, breast, and liver cancers. It has been

1	is similar in more and less developed countries but is much more common in Asia than North America or Africa. The most common cancers in Africa are cervical, breast, and liver cancers. It has been estimated that nine modifiable risk factors are responsible for more than one-third of cancers worldwide. These include smoking, alcohol consumption, obesity, physical inactivity, low fruit and vegetable consumption, unsafe sex, air pollution, indoor smoke from household fuels, and contaminated injections.

1	Important information is obtained from every portion of the routine history and physical examination. The duration of symptoms may reveal the chronicity of disease. The past medical history may alert the physician to the presence of underlying diseases that may affect the choice of therapy or the side effects of treatment. The social history may reveal occupational exposure to carcinogens or habits, such as smoking or alcohol consumption, that may influence the course of disease and its treatment. The family history may suggest an underlying familial cancer predisposition and point out the need to begin surveillance or other preventive therapy for unaffected siblings of the patient. The review of systems may suggest early symptoms of metastatic disease or a paraneoplastic syndrome.

1	The diagnosis of cancer relies most heavily on invasive tissue biopsy and should never be made without obtaining tissue; no noninvasive diagnostic test is sufficient to define a disease process as cancer. Although in rare clinical settings (e.g., thyroid nodules), fine-needle aspiration is an acceptable diagnostic procedure, the diagnosis generally depends on obtaining adequate tissue to permit careful evaluation of the histology of the tumor, its grade, and its invasiveness and to yield further molecular diagnostic information, such as the expression of cell-surface markers or intracellular proteins that typify a particular cancer, or the presence of a molecular marker, such as the t(8;14) translocation of Burkitt’s lymphoma. Increasing evidence links the expression of certain genes with the prognosis and response to therapy (Chaps. 101e and 102e).

1	Occasionally a patient will present with a metastatic disease process that is defined as cancer on biopsy but has no apparent primary site of disease. Efforts should be made to define the primary site based on age, sex, sites of involvement, histology and tumor markers, and personal and family history. Particular attention should be focused on ruling out the most treatable causes (Chap. 120e). Once the diagnosis of cancer is made, the management of the patient is best undertaken as a multidisciplinary collaboration among the primary care physician, medical oncologists, surgical oncologists, radiation oncologists, oncology nurse specialists, pharmacists, social workers, rehabilitation medicine specialists, and a number of other consulting professionals working closely with each other and with the patient and family. Deaths per 100,000 women 0 20 40 60 C. Females, by site 80 100 Ovary Lung and bronchus Colorectum Uterus Breast Pancreas Stomach

1	Deaths per 100,000 women 0 20 40 60 C. Females, by site 80 100 Ovary Lung and bronchus Colorectum Uterus Breast Pancreas Stomach Deaths per 100,000 men Deaths per 100,000 people A. All sites combined B. Males, by site 1930 1940 1950 1960 1970 1980 1990 2000 2010 Year of death FIGURE 99-2 Eighty-year trend in cancer death rates for (A) women and (B) men by site in the United States, 1930–2010. Rates are per 100,000 age-adjusted to the 2000 U.S. standard population. All sites combined (A), individual sites in men (B) and individual sites in women (C) are shown. (From R Siegel et al: CA Cancer J Clin 64:9, 2014.)

1	Approach to the Patient with Cancer histologic examination of all tissues removed during the surgical procedure. Surgical procedures performed may include a simple lymph node biopsy or more extensive procedures such as thoracotomy, mediastinoscopy, or laparotomy. Surgical staging may occur in a separate procedure or may be done at the time of definitive surgical resection of the primary tumor. 60–69 Knowledge of the predilection of particular tumors for spreading to adjacent or distant organs helps direct the staging evaluation.

1	60–69 Knowledge of the predilection of particular tumors for spreading to adjacent or distant organs helps direct the staging evaluation. 50–59 Information obtained from staging is used to define the extent of disease as localized, as exhibiting spread outside of the organ of origin to regional 40–49 but not distant sites, or as metastatic to distant sites. The most widely used system of staging is the TNM (tumor, node, metastasis) system codified by the International Union Against Cancer and the American Joint Committee on Cancer. The TNM classification is an anatomically based system that categorizes the tumor on the basis of the size of the primary tumor lesion (T1–4, where a higher number indicates a tumor of larger size), the presence of nodal involvement (usually N0 and N1 for the absence and presence, respectively, of involved nodes, although some tumors have more elaborate systems of nodal grading), and the presence of metastatic disease (M0

1	FIGURE 99-3 The decline in death rates from cancer is shown for different age ranges and M1 for the absence and presence, respectively, by sex and race for the 20-year period between 1991 and 2010 expressed as a percent-of metastases). The various permutations of T, N, age of the 1991 rate. (From R Siegel et al: CA Cancer J Clin 64:9, 2014.) The first priority in patient management after the diagnosis of cancer is established and shared with the patient is to determine the extent of disease. The curability of a tumor usually is inversely proportional to the tumor burden. Ideally, the tumor will be diagnosed before symptoms develop or as a consequence of screening efforts (Chap. 100). A very high proportion of such patients can be cured. However, most patients with cancer present with symptoms related to the cancer, caused either by mass effects of the tumor or by alterations associated with the production of cytokines or hormones by the tumor.

1	For most cancers, the extent of disease is evaluated by a variety of noninvasive and invasive diagnostic tests and procedures. This process is called staging. There are two types. Clinical staging is based on physical examination, radiographs, isotopic scans, computed tomography (CT) scans, and other imaging procedures; pathologic staging takes into account information obtained during a surgical procedure, which might include intraoperative palpation, resection of regional lymph nodes and/or tissue adjacent to the tumor, and inspection and biopsy of organs commonly involved in disease spread. Pathologic staging includes and M scores (sometimes including tumor histologic grade [G]) are then broken into stages, usually desig nated by the roman numerals I through IV. Tumor burden increases and curability decreases with increasing stage. Other anatomic staging systems are used for some tumors, e.g., the Dukes classification for colorectal cancers, the International Federation of

1	and curability decreases with increasing stage. Other anatomic staging systems are used for some tumors, e.g., the Dukes classification for colorectal cancers, the International Federation of Gynecologists and Obstetricians classification for gynecologic cancers, and the Ann Arbor classification for Hodgkin’s disease.

1	Certain tumors cannot be grouped on the basis of anatomic considerations. For example, hematopoietic tumors such as leukemia, myeloma, and lymphoma are often disseminated at presentation and do not spread like solid tumors. For these tumors, other prognostic factors have been identified (Chaps. 132-136).

1	In addition to tumor burden, a second major determinant of treatment outcome is the physiologic reserve of the patient. Patients who are bedridden before developing cancer are likely to fare worse, stage for stage, than fully active patients. Physiologic reserve is a determinant of how a patient is likely to cope with the physiologic stresses imposed by the cancer and its treatment. This factor is difficult to assess directly. Instead, surrogate markers for physiologic reserve are used, such as the patient’s age or Karnofsky performance status (Table 99-4) or Eastern Cooperative Oncology Group (ECOG) performance status the five LeaDing primary tumor sites for patients Dying of CanCer BaseD on age anD sex in 2010 Age, years 471taBLe 99-3 CanCer inCiDenCe anD mortaLity in raCiaL anD ethniC groups, uniteD states, 2006–2010 All M 217.3 276.6 132.4 191.0 152.2 F 153.6 171.2 92.1 139.0 101.3 Breast 22.7 30.8 11.5 15.5 14.8 Colorectal M 19.2 28.7 13.1 18.7 16.1

1	All M 217.3 276.6 132.4 191.0 152.2 F 153.6 171.2 92.1 139.0 101.3 Breast 22.7 30.8 11.5 15.5 14.8 Colorectal M 19.2 28.7 13.1 18.7 16.1 F 13.6 19.0 9.7 15.4 10.2 Kidney M 5.9 5.7 3.0 9.5 5.1 F 2.6 2.6 1.2 4.4 2.3 Liver M 7.1 11.8 14.4 13.2 12.3 F 2.9 4.1 6.0 6.1 5.4 Lung M 65.7 78.5 35.5 49.6 31.3 F 42.7 37.2 18.4 33.1 14.1 Prostate 21.3 50.9 10.1 20.7 19.2 Cervix 2.1 4.2 1.9 3.5 2.9 aBased on Indian Health Service delivery areas. Abbreviations: F, female; M, male. Source: From R Siegel R et al: Cancer statistics, 2014. CA Cancer J Clin 64:9, 2014. (Table 99-5). Older patients and those with a Karnofsky performance treatment is of the utmost importance in treatment planning. For some status <70 or ECOG performance status ≥3 have a poor prognosis cancers, chemotherapy or chemotherapy plus radiation therapy delivunless the poor performance is a reversible consequence of the tumor. ered before the use of definitive surgical treatment (so-called neoadju-

1	Increasingly, biologic features of the tumor are being related to prog-vant therapy) may improve the outcome, as seems to be the case for nosis. The expression of particular oncogenes, drug-resistance genes, locally advanced breast cancer and head and neck cancers. In certain apoptosis-related genes, and genes involved in metastasis is being settings in which combined-modality therapy is intended, coordinafound to influence response to therapy and prognosis. The presence tion among the medical oncologist, radiation oncologist, and surgeon of selected cytogenetic abnormalities may influence survival. Tumors is crucial to achieving optimal results. Sometimes the chemotherapy with higher growth fractions, as assessed by expression of proliferation-and radiation therapy need to be delivered sequentially, and other related markers such as proliferating cell nuclear antigen, behave more times concurrently. Surgical procedures may precede or follow other aggressively than tumors with lower

1	sequentially, and other related markers such as proliferating cell nuclear antigen, behave more times concurrently. Surgical procedures may precede or follow other aggressively than tumors with lower growth fractions. Information treatment approaches. It is best for the treatment plan either to follow obtained from studying the tumor itself will increasingly be used to a standard protocol precisely or else to be part of an ongoing clinical influence treatment decisions. Host genes involved in drug metabolism research protocol evaluating new treatments. Ad hoc modifications of can influence the safety and efficacy of particular treatments. standard protocols are likely to compromise treatment results.

1	Enormous heterogeneity has been noted by studying tumors; we The choice of treatment approaches was formerly dominated by the have learned that morphology is not capable of discerning certain local culture in both the university and the practice settings. However, distinct subsets of patients whose tumors have different sets of abnor-it is now possible to gain access electronically to standard treatment malities. Tumors that look the same by light microscopy can be very protocols and to every approved clinical research study in North different. Similarly, tumors that look quite different from one another America through a personal computer interface with the Internet.1 histologically can share genetic lesions that predict responses to treatments. Furthermore, tumor cells vary enormously within a single 1The National Cancer Institute maintains a database called PDQ (Physician patient even though the cells share a common origin. Data Query) that is accessible on the Internet under the

1	within a single 1The National Cancer Institute maintains a database called PDQ (Physician patient even though the cells share a common origin. Data Query) that is accessible on the Internet under the name CancerNet at www.cancer.gov/cancertopics/pdq/cancerdatabase. Information can be obtained MAKING A TREATMENT PLAN through a facsimile machine using CancerFax by dialing 301-402-5874. Patient From information on the extent of disease and the prognosis and information is also provided by the National Cancer Institute in at least in conjunction with the patient’s wishes, it is determined whether three formats: on the Internet via CancerNet at www.cancer.gov, through the the treatment approach should be curative or palliative in intent. CancerFax number listed above, or by calling 1-800-4-CANCER. The quality Cooperation among the various professionals involved in cancer control for the information provided through these services is rigorous.

1	Approach to the Patient with Cancer

1	Because cancer therapies are toxic (Chap. 103e), patient manage ment involves addressing complications of both the disease and its treatment as well as the complex psychosocial problems asso ciated with cancer. In the short term during a course of curative therapy, the patient’s functional status may decline. Treatment induced toxicity is less acceptable if the goal of therapy is palliation. The most common side effects of treatment are nausea 453.3 270.1385.3 246.3 378.1 302.3 358.2 285.2 354.0 273.5265.9 348.0 288.0 332.9 240.7 332.0 239.7 331.8 266.3 325.2 204.8 299.0 251.1 298.5 187.2 293.8 246.4 290.6 246.7 288.0 270.7 283.0 274.3 281.9 162.7 276.2 220.9 272.2 221.3 265.6 224.2 260.3 212.1 259.6 190.7 256.2 227.9 251.7 189.5 224.0 211.2 223.9 174.1 221.5 189.7 209.8 205.0 202.6 194.9 200.9 114.7 162.3 187.9 156.1 119.3 153.6 171.4155.4148.9 114.6105.386.3 80.3 Females Males and vomiting (see below), febrile neutropenia (Chap. 104), and myelosuppression (Chap. 103e). Tools are now

1	200.9 114.7 162.3 187.9 156.1 119.3 153.6 171.4155.4148.9 114.6105.386.3 80.3 Females Males and vomiting (see below), febrile neutropenia (Chap. 104), and myelosuppression (Chap. 103e). Tools are now available to minimize the acute toxicity of cancer treatment.

1	New symptoms developing in the course of cancer treatment should always be assumed to be reversible until proven other wise. The fatalistic attribution of anorexia, weight loss, and jaun dice to recurrent or progressive tumor could result in a patient dying from a reversible intercurrent cholecystitis. Intestinal obstruction may be due to reversible adhesions rather than progressive tumor. Systemic infections, sometimes with unusual pathogens, may be a consequence of the immunosuppression associated with cancer therapy. Some drugs used to treat cancer or its complications (e.g., nausea) may produce central nervous Rate per 100,000 paraneoplastic syndromes such as the syndrome of inappropriate antidiuretic hormone. A definitive diagnosis should be pursued and may even require a repeat biopsy.

1	Rate per 100,000 paraneoplastic syndromes such as the syndrome of inappropriate antidiuretic hormone. A definitive diagnosis should be pursued and may even require a repeat biopsy. A critical component of cancer management is assessing the response to treatment. In addition to a careful physical examination in which all sites of disease are physically measured and recorded in a flow chart by date, response assessment usually requires periodic repeating of imaging tests that were abnormal

1	Algeria, SetifIndia, ChennaiEcuador, QuitoUganda, KyadondoEgypt, GharbiahPakistan, South KarachiTurkey, IzmirZimbabwe, Harare: AfricanPhilippines, ManilaSingaporeChina, ShanghaiColombia, CaliRussia, St PetersburgArgentina, Bahia BlancaChina, Hong KongBrazil, GoianiaPoland, CracowFinlandUSA, SEER 14 (Hispanic)KoreaDenmarkIsraelThe NetherlandsNorwayJapan, MiyagiUK, Merseyside and CheshireSpain, NavarraCanadaCzech RepublicGermany, SaarlandItaly, ParmaSwitzerland, GenevaAustralia, SouthNew ZealandUSA, SEER 14 (White)France, Bas-RhinUSA, SEER 14 (Black)

1	Incidence (n = 10,864,499) Mortality (n = 6,724,931) Prevalence (n = 24,576,453) at the time of staging. If imaging tests have become normal, repeat biopsy of previously involved tissue is performed to docu-FIGURE 99-4 Worldwide overall annual cancer incidence, mortality, and ment complete response by pathologic criteria. Biopsies are not5-year prevalence for the period of 1993–2001. (Adapted from A Jemal et al: usually required if there is macroscopic residual disease. A com- Cancer Epidemiol Biomarkers Prev 19:1893, 2010.)

1	Cancer Epidemiol Biomarkers Prev 19:1893, 2010.) The skilled physician also has much to offer the patient for whom curative therapy is no longer an option. Often a combination of guilt and frustration over the inability to cure the patient and the pressure of a busy schedule greatly limit the time a physician spends with a patient who is receiving only palliative care. Resist these forces. In addition to the medicines administered to alleviate symptoms (see below), it is important to remember the comfort that is provided by holding the patient’s hand, continuing regular examinations, and taking time to talk.

1	plete response is defined as disappearance of all evidence of disease, and a partial response as >50% reduction in the sum of the products of the perpendicular diameters of all measurable lesions. The determination of partial response may also be based on a 30% decrease in the sums of the longest diameters of lesions (Response Evaluation Criteria in Solid Tumors [RECIST]). Progressive disease is defined as the appearance of any new lesion or an increase of >25% in the sum of the products of the perpendicular diameters of all measurable lesions (or an increase of 20% in the sums of the longest diameters by RECIST). Tumor shrinkage or growth that does not meet any of these criteria is considered stable disease. Some sites of involvement (e.g., bone) or patterns of involvement (e.g., lymphangitic lung or diffuse pulmonary infiltrates) are considered unmeasurable. No response is complete without biopsy documentation of their resolution, but partial responses may exclude their assessment

1	lung or diffuse pulmonary infiltrates) are considered unmeasurable. No response is complete without biopsy documentation of their resolution, but partial responses may exclude their assessment unless clear objective progression has occurred.

1	Status Functional Capability of the Patient 100 Normal; no complaints; no evidence of disease 90 Able to carry on normal activity; minor signs or symptoms of disease 80 Normal activity with effort; some signs or symptoms of Cares for self; unable to carry on normal activity or do the eastern Cooperative onCoLogy group (eCog) ECOG Grade 0: Fully active, able to carry on all predisease performance with out restriction ECOG Grade 1: Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature, e.g., light housework, office work ECOG Grade 2: Ambulatory and capable of all self-care but unable to carry out any work activities. Up and about more than 50% of waking hours

1	ECOG Grade 3: Capable of only limited self-care, confined to bed or chair more than 50% of waking hours ECOG Grade 4: Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair ECOG Grade 5: Dead Source: From MM Oken et al: Am J Clin Oncol 5:649, 1982. Human chorionic Gestational trophoblas-Pregnancy gonadotropin tic disease, gonadal Calcitonin Medullary cancer of the thyroid α Fetoprotein Hepatocellular carci-Cirrhosis, hepatitis noma, gonadal germ cell tumor Carcinoembryonic Adenocarcinomas of the Pancreatitis, hepatitis, antigen colon, pancreas, lung, inflammatory bowel breast, ovary disease, smoking Prostatic acid phos-Prostate cancer Prostatitis, prostatic phatase Neuron-specific enolase Small-cell cancer of the lung, neuroblastoma Lactate dehydrogenase Lymphoma, Ewing’s Hepatitis, hemolytic sarcoma anemia, many others Abbreviation: MGUS, monoclonal gammopathy of uncertain significance.

1	Lactate dehydrogenase Lymphoma, Ewing’s Hepatitis, hemolytic sarcoma anemia, many others Abbreviation: MGUS, monoclonal gammopathy of uncertain significance. Tumor markers may be useful in patient management in certain tumors. Response to therapy may be difficult to gauge with certainty. However, some tumors produce or elicit the production of markers that can be measured in the serum or urine, and in a particular patient, rising and falling levels of the marker are usually associated with increasing or decreasing tumor burden, respectively. Some clinically useful tumor markers are shown in Table 99-6. Tumor markers are not in themselves specific enough to permit a diagnosis of malignancy to be made, but once a malignancy has been diagnosed and shown to be associated with elevated levels of a tumor marker, the marker can be used to assess response to treatment.

1	The recognition and treatment of depression are important components of management. The incidence of depression in cancer patients is ~25% overall and may be greater in patients with greater debility. This diagnosis is likely in a patient with a depressed mood (dysphoria) and/or a loss of interest in pleasure (anhedonia) for at least 2 weeks. In addition, three or more of the following symptoms are usually present: appetite change, sleep problems, psychomotor retardation or agitation, fatigue, feelings of guilt or worthlessness, inability to concentrate, and suicidal ideation. Patients with these symptoms should receive therapy. Medical therapy with a serotonin reuptake inhibitor such as fluoxetine (10–20 mg/d), sertraline (50–150 mg/d), or paroxetine (10–20 mg/d) or a tricyclic antidepressant such as amitriptyline (50–100 mg/d) or desipramine (75–150 mg/d) should be tried, allowing 4–6 weeks for response. Effective therapy should be continued at least 6 months after resolution of

1	such as amitriptyline (50–100 mg/d) or desipramine (75–150 mg/d) should be tried, allowing 4–6 weeks for response. Effective therapy should be continued at least 6 months after resolution of symptoms. If therapy is unsuccessful, other classes of antidepressants may be used. In addition to medication, psychosocial interventions such as support groups, psychotherapy, and guided 473 imagery may be of benefit.

1	Many patients opt for unproven or unsound approaches to treatment when it appears that conventional medicine is unlikely to be curative. Those seeking such alternatives are often well educated and may be early in the course of their disease. Unsound approaches are usually hawked on the basis of unsubstantiated anecdotes and not only cannot help the patient but may be harmful. Physicians should strive to keep communications open and nonjudgmental, so that patients are more likely to discuss with the physician what they are actually doing. The appearance of unexpected toxicity may be an indication that a supplemental therapy is being taken.2

1	At the completion of treatment, sites originally involved with tumor are reassessed, usually by radiography or imaging techniques, and any persistent abnormality is biopsied. If disease persists, the multidisciplinary team discusses a new salvage treatment plan. If the patient has been rendered disease-free by the original treatment, the patient is followed regularly for disease recurrence. The optimal guidelines for follow-up care are not known. For many years, a routine practice has been to follow the patient monthly for 6–12 months, then every other month for a year, every 3 months for a year, every 4 months for a year, every 6 months for a year, and then annually. At each visit, a battery of laboratory and radiographic and imaging tests were obtained on the assumption that it is best to detect recurrent disease before it becomes symptomatic. However, where follow-up procedures have been examined, this assumption has been found to be untrue. Studies of breast cancer, melanoma, lung

1	detect recurrent disease before it becomes symptomatic. However, where follow-up procedures have been examined, this assumption has been found to be untrue. Studies of breast cancer, melanoma, lung cancer, colon cancer, and lymphoma have all failed to support the notion that asymptomatic relapses are more readily cured by salvage therapy than symptomatic relapses. In view of the enormous cost of a full battery of diagnostic tests and their manifest lack of impact on survival, new guidelines are emerging for less frequent follow-up visits, during which the history and physical examination are the major investigations performed.

1	As time passes, the likelihood of recurrence of the primary cancer diminishes. For many types of cancer, survival for 5 years without recurrence is tantamount to cure. However, important medical problems can occur in patients treated for cancer and must be examined (Chap. 125). Some problems emerge as a consequence of the disease and some as a consequence of the treatment. An understanding of these diseaseand treatment-related problems may help in their detection and management. Despite these concerns, most patients who are cured of cancer return to normal lives.

1	Despite these concerns, most patients who are cured of cancer return to normal lives. In many ways, the success of cancer therapy depends on the success of the supportive care. Failure to control the symptoms of cancer and its treatment may lead patients to abandon curative therapy. Of equal importance, supportive care is a major determinant of quality of life. Even when life cannot be prolonged, the physician must strive to preserve its quality. Quality-of-life measurements have become common endpoints of clinical research studies. Furthermore, palliative care has been shown to be cost-effective when approached in an organized fashion. A credo for oncology could be to cure sometimes, to extend life often, and to comfort always.

1	Pain Pain occurs with variable frequency in the cancer patient: 25–50% of patients present with pain at diagnosis, 33% have pain associated with treatment, and 75% have pain with progressive disease. The pain may have several causes. In ~70% of cases, pain is caused by the tumor itself—by invasion of bone, nerves, blood vessels, or mucous 2Information about unsound methods may be obtained from the National Council Against Health Fraud, Box 1276, Loma Linda, CA 92354, or from the Center for Medical Consumers and Health Care Information, 237 Thompson Street, New York, NY 10012.

1	Approach to the Patient with Cancer 474 membranes or obstruction of a hollow viscus or duct. In ~20% of cases, pain is related to a surgical or invasive medical procedure, to radiation injury (mucositis, enteritis, or plexus or spinal cord injury), or to chemotherapy injury (mucositis, peripheral neuropathy, phlebitis, steroid-induced aseptic necrosis of the femoral head). In 10% of cases, pain is unrelated to cancer or its treatment. Assessment of pain requires the methodical investigation of the history of the pain, its location, character, temporal features, provocative and palliative factors, and intensity (Chap. 18); a review of the oncologic history and past medical history as well as personal and social history; and a thorough physical examination. The patient should be given a 10-division visual analogue scale on which to indicate the severity of the pain. The clinical condition is often dynamic, making it necessary to reassess the patient frequently. Pain therapy should not

1	visual analogue scale on which to indicate the severity of the pain. The clinical condition is often dynamic, making it necessary to reassess the patient frequently. Pain therapy should not be withheld while the cause of pain is being sought. A variety of tools are available with which to address cancer pain. About 85% of patients will have pain relief from pharmacologic intervention. However, other modalities, including antitumor therapy (such as surgical relief of obstruction, radiation therapy, and strontium-89 or samarium-153 treatment for bone pain), neurostimulatory techniques, regional analgesia, or neuroablative procedures, are effective in an additional 12% or so. Thus, very few patients will have inadequate pain relief if appropriate measures are taken. A specific approach to pain relief is detailed in Chap. 10.

1	Nausea Emesis in the cancer patient is usually caused by chemotherapy (Chap. 103e). Its severity can be predicted from the drugs used to treat the cancer. Three forms of emesis are recognized on the basis of their timing with regard to the noxious insult. Acute emesis, the most common variety, occurs within 24 h of treatment. Delayed emesis occurs 1–7 days after treatment; it is rare, but, when present, usually follows cisplatin administration. Anticipatory emesis occurs before administration of chemotherapy and represents a conditioned response to visual and olfactory stimuli previously associated with chemotherapy delivery.

1	Acute emesis is the best understood form. Stimuli that activate signals in the chemoreceptor trigger zone in the medulla, the cerebral cortex, and peripherally in the intestinal tract lead to stimulation of the vomiting center in the medulla, the motor center responsible for coordinating the secretory and muscle contraction activity that leads to emesis. Diverse receptor types participate in the process, including dopamine, serotonin, histamine, opioid, and acetylcholine receptors. The serotonin receptor antagonists ondansetron and granisetron are the most effective drugs against highly emetogenic agents, but they are expensive.

1	As with the analgesia ladder, emesis therapy should be tailored to the situation. For mildly and moderately emetogenic agents, prochlorperazine, 5–10 mg PO or 25 mg PR, is effective. Its efficacy may be enhanced by administering the drug before the chemotherapy is delivered. Dexamethasone, 10–20 mg IV, is also effective and may enhance the efficacy of prochlorperazine. For highly emetogenic agents such as cisplatin, mechlorethamine, dacarbazine, and streptozocin, combinations of agents work best and administration should begin 6–24 h before treatment. Ondansetron, 8 mg PO every 6 h the day before therapy and IV on the day of therapy, plus dexamethasone, 20 mg IV before treatment, is an effective regimen. Addition of oral aprepitant (a substance P/neurokinin 1 receptor antagonist) to this regimen (125 mg on day 1, 80 mg on days 2 and 3) further decreases the risk of both acute and delayed vomiting. Like pain, emesis is easier to prevent than to alleviate.

1	Delayed emesis may be related to bowel inflammation from the therapy and can be controlled with oral dexamethasone and oral metoclopramide, a dopamine receptor antagonist that also blocks serotonin receptors at high dosages. The best strategy for preventing anticipatory emesis is to control emesis in the early cycles of therapy to prevent the conditioning from taking place. If this is unsuccessful, prophylactic antiemetics the day before treatment may help. Experimental studies are evaluating behavior modification.

1	Effusions Fluid may accumulate abnormally in the pleural cavity, pericardium, or peritoneum. Asymptomatic malignant effusions may not require treatment. Symptomatic effusions occurring in tumors responsive to systemic therapy usually do not require local treatment but respond to the treatment for the underlying tumor. Symptomatic effusions occurring in tumors unresponsive to systemic therapy may require local treatment in patients with a life expectancy of at least 6 months.

1	Pleural effusions due to tumors may or may not contain malignant cells. Lung cancer, breast cancer, and lymphomas account for ~75% of malignant pleural effusions. Their exudative nature is usually gauged by an effusion/serum protein ratio of ≥0.5 or an effusion/serum lactate dehydrogenase ratio of ≥0.6. When the condition is symptomatic, thoracentesis is usually performed first. In most cases, symptomatic improvement occurs for <1 month. Chest tube drainage is required if symptoms recur within 2 weeks. Fluid is aspirated until the flow rate is <100 mL in 24 h. Then either 60 units of bleomycin or 1 g of doxycycline is infused into the chest tube in 50 mL of 5% dextrose in water; the tube is clamped; the patient is rotated on four sides, spending 15 min in each position; and, after 1–2 h, the tube is again attached to suction for another 24 h. The tube is then disconnected from suction and allowed to drain by gravity. If <100 mL drains over the next 24 h, the chest tube is pulled, and

1	the tube is again attached to suction for another 24 h. The tube is then disconnected from suction and allowed to drain by gravity. If <100 mL drains over the next 24 h, the chest tube is pulled, and a radiograph is taken 24 h later. If the chest tube continues to drain fluid at an unacceptably high rate, sclerosis can be repeated. Bleomycin may be somewhat more effective than doxycycline but is very expensive. Doxycycline is usually the drug of first choice. If neither doxycycline nor bleomycin is effective, talc can be used.

1	Symptomatic pericardial effusions are usually treated by creating a pericardial window or by stripping the pericardium. If the patient’s condition does not permit a surgical procedure, sclerosis can be attempted with doxycycline and/or bleomycin. Malignant ascites is usually treated with repeated paracentesis of small volumes of fluid. If the underlying malignancy is unresponsive to systemic therapy, peritoneovenous shunts may be inserted. Despite the fear of disseminating tumor cells into the circulation, widespread metastases are an unusual complication. The major complications are occlusion, leakage, and fluid overload. Patients with severe liver disease may develop disseminated intravascular coagulation.

1	Nutrition Cancer and its treatment may lead to a decrease in nutrient intake of sufficient magnitude to cause weight loss and alteration of intermediary metabolism. The prevalence of this problem is difficult to estimate because of variations in the definition of cancer cachexia, but most patients with advanced cancer experience weight loss and decreased appetite. A variety of both tumor-derived factors (e.g., bombesin, adrenocorticotropic hormone) and host-derived factors (e.g., tumor necrosis factor, interleukins 1 and 6, growth hormone) contribute to the altered metabolism, and a vicious cycle is established in which protein catabolism, glucose intolerance, and lipolysis cannot be reversed by the provision of calories.

1	It remains controversial how to assess nutritional status and when and how to intervene. Efforts to make the assessment objective have included the use of a prognostic nutritional index based on albumin levels, triceps skinfold thickness, transferrin levels, and delayed-type hypersensitivity skin testing. However, a simpler approach has been to define the threshold for nutritional intervention as <10% unexplained body weight loss, serum transferrin level <1500 mg/L (150 mg/dL), and serum albumin <34 g/L (3.4 g/dL).

1	The decision is important, because it appears that cancer therapy is substantially more toxic and less effective in the face of malnutrition. Nevertheless, it remains unclear whether nutritional intervention can alter the natural history. Unless some pathology is affecting the absorptive function of the gastrointestinal tract, enteral nutrition provided orally or by tube feeding is preferred over parenteral supplementation. However, the risks associated with the tube may outweigh the benefits. Megestrol acetate, a progestational agent, has been advocated as a pharmacologic intervention to improve nutritional status. Research in this area may provide more tools in the future as cytokinemediated mechanisms are further elucidated.

1	Psychosocial Support The psychosocial needs of patients vary with their situation. Patients undergoing treatment experience fear, anxiety, and depression. Self-image is often seriously compromised by deforming surgery and loss of hair. Women who receive cosmetic advice that enables them to look better also feel better. Loss of control over how one spends time can contribute to the sense of vulnerability. Juggling the demands of work and family with the demands of treatment may create enormous stresses. Sexual dysfunction is highly prevalent and needs to be discussed openly with the patient. An empathetic health care team is sensitive to the individual patient’s needs and permits negotiation where such flexibility will not adversely affect the course of treatment.

1	Cancer survivors have other sets of difficulties. Patients may have fears associated with the termination of a treatment they associate with their continued survival. Adjustments are required to physical losses and handicaps, real and perceived. Patients may be preoccupied with minor physical problems. They perceive a decline in their job mobility and view themselves as less desirable workers. They may be victims of job and/or insurance discrimination. Patients may experience difficulty reentering their normal past life. They may feel guilty for having survived and may carry a sense of vulnerability to colds and other illnesses. Perhaps the most pervasive and threatening concern is the ever-present fear of relapse (the Damocles syndrome). Patients in whom therapy has been unsuccessful have other problems related to the end of life.

1	Death and Dying The most common causes of death in patients with cancer are infection (leading to circulatory failure), respiratory failure, hepatic failure, and renal failure. Intestinal blockage may lead to inanition and starvation. Central nervous system disease may lead to seizures, coma, and central hypoventilation. About 70% of patients develop dyspnea preterminally. However, many months usually pass between the diagnosis of cancer and the occurrence of these complications, and during this period, the patient is severely affected by the possibility of death. The path of unsuccessful cancer treatment usually occurs in three phases. First, there is optimism at the hope of cure; when the tumor recurs, there is the acknowledgment of an incurable disease, and the goal of palliative therapy is embraced in the hope of being able to live with disease; finally, at the disclosure of imminent death, another adjustment in outlook takes place. The patient imagines the worst in preparation

1	is embraced in the hope of being able to live with disease; finally, at the disclosure of imminent death, another adjustment in outlook takes place. The patient imagines the worst in preparation for the end of life and may go through stages of adjustment to the diagnosis. These stages include denial, isolation, anger, bargaining, depression, acceptance, and hope. Of course, patients do not all progress through all the stages or proceed through them in the same order or at the same rate. Nevertheless, developing an understanding of how the patient has been affected by the diagnosis and is coping with it is an important goal of patient management.

1	It is best to speak frankly with the patient and the family regarding the likely course of disease. These discussions can be difficult for the physician as well as for the patient and family. The critical features of the interaction are to reassure the patient and family that everything that can be done to provide comfort will be done. They will not be abandoned. Many patients prefer to be cared for in their homes or in a hospice setting rather than a hospital. The American College of Physicians has published a book called Home Care Guide for Cancer: How to Care for Family and Friends at Home that teaches an approach to successful problem-solving in home care. With appropriate planning, it should be possible to provide the patient with the necessary medical care as well as the psychological and spiritual support that will prevent the isolation and depersonalization that can attend in-hospital death.

1	The care of dying patients may take a toll on the physician. A “burnout” syndrome has been described that is characterized by fatigue, disengagement from patients and colleagues, and a loss of self-fulfillment. Efforts at stress reduction, maintenance of a balanced life, and setting realistic goals may combat this disorder.

1	End-of-Life Decisions Unfortunately, a smooth transition in treatment goals from curative to palliative may not be possible in all cases because of the occurrence of serious treatment-related complications or rapid disease progression. Vigorous and invasive medical support for a reversible disease or treatment complication is assumed to be justified. However, if the reversibility of the condition is in doubt, 475 the patient’s wishes determine the level of medical care. These wishes should be elicited before the terminal phase of illness and reviewed periodically. Information about advance directives can be obtained from the American Association of Retired Persons, 601 E Street, NW, Washington, DC 20049, 202-434-2277, or Choice in Dying, 250 West 57th Street, New York, NY 10107, 212-366-5540. Some states allow physicians to assist patients who choose to end their lives. This subject is challenging from an ethical and a medical point of view. Discussions of end-of-life decisions should

1	Some states allow physicians to assist patients who choose to end their lives. This subject is challenging from an ethical and a medical point of view. Discussions of end-of-life decisions should be candid and involve clear informed consent, waiting periods, second opinions, and documentation. A full discussion of end-of-life management is in Chap. 10.

1	Jennifer M. Croswell, Otis W. Brawley, Barnett S. Kramer Improved understanding of carcinogenesis has allowed cancer prevention and early detection (also known as cancer control) to expand beyond the identification and avoidance of carcinogens. Specific interventions to prevent cancer in those at risk, and effective screening for early detection of cancer, are the goals. Carcinogenesis is not an event but a process, a continuum of discrete tissue and cellular changes over time resulting in aberrant physiologic processes. Prevention concerns the identification and manipulation of the biologic, environmental, social, and genetic factors in the causal pathway of cancer.

1	Public education on the avoidance of identified risk factors for cancer and encouraging healthy habits contributes to cancer prevention and control. The clinician is a powerful messenger in this process. The patient-provider encounter provides an opportunity to teach patients about the hazards of smoking, the features of a healthy lifestyle, use of proven cancer screening methods, and avoidance of excessive sun exposure. Tobacco smoking is a strong, modifiable risk factor for cardiovascular disease, pulmonary disease, and cancer. Smokers have an approximately 1 in 3 lifetime risk of dying prematurely from a tobacco-related cancer, cardiovascular, or pulmonary disease. Tobacco use causes more deaths from cardiovascular disease than from cancer. Lung cancer and cancers of the larynx, oropharynx, esophagus, kidney, bladder, pancreas, and stomach are all tobacco-related.

1	The number of cigarettes smoked per day and the level of inhalation of cigarette smoke are correlated with risk of lung cancer mortality. Lightand low-tar cigarettes are not safer, because smokers tend to inhale them more frequently and deeply. Those who stop smoking have a 30–50% lower 10-year lung cancer mortality rate compared to those who continue smoking, despite the fact that some carcinogen-induced gene mutations persist for years after smoking cessation. Smoking cessation and avoidance would save more lives than any other public health activity. The risk of tobacco smoke is not limited to the smoker. Environmental tobacco smoke, known as secondhand or passive smoke, causes lung cancer and other cardiopulmonary diseases in nonsmokers.

1	The risk of tobacco smoke is not limited to the smoker. Environmental tobacco smoke, known as secondhand or passive smoke, causes lung cancer and other cardiopulmonary diseases in nonsmokers. Tobacco use prevention is a pediatric issue. More than 80% of adult American smokers began smoking before the age of 18 years. Approximately 20% of Americans in grades 9 through 12 have smoked a cigarette in the past month. Counseling of adolescents and young adults is critical to prevent smoking. A clinician’s simple advice can be of benefit. Providers should query patients on tobacco use and offer smokers assistance in quitting.

1	Prevention and Early Detection of Cancer 476 Current approaches to smoking cessation recognize smoking as an addiction (Chap. 470). The smoker who is quitting goes through identifiable stages that include contemplation of quitting, an action phase in which the smoker quits, and a maintenance phase. Smokers who quit completely are more likely to be successful than those who gradually reduce the number of cigarettes smoked or change to lower-tar or lower-nicotine cigarettes. More than 90% of the Americans who have successfully quit smoking did so on their own, without participation in an organized cessation program, but cessation programs are helpful for some smokers. The Community Intervention Trial for Smoking Cessation (COMMIT) was a 4-year program showing that light smokers (<25 cigarettes per day) were more likely to benefit from simple cessation messages and cessation programs than those who did not receive an intervention. Quit rates were 30.6% in the intervention group and 27.5%

1	per day) were more likely to benefit from simple cessation messages and cessation programs than those who did not receive an intervention. Quit rates were 30.6% in the intervention group and 27.5% in the control group. The COMMIT interventions were unsuccessful in heavy smokers (<25 cigarettes per day). Heavy smokers may need an intensive broad-based cessation program that includes counseling, behavioral strategies, and pharmacologic adjuncts, such as nicotine replacement (gum, patches, sprays, lozenges, and inhalers), bupropion, and/or varenicline. The health risks of cigars are similar to those of cigarettes. Smoking one or two cigars daily doubles the risk for oral and esophageal cancers; smoking three or four cigars daily increases the risk of oral cancers more than eightfold and esophageal cancer fourfold. The risks of occasional use are unknown. Smokeless tobacco also represents a substantial health risk. Chewing tobacco is a carcinogen linked to dental caries, gingivitis, oral

1	cancer fourfold. The risks of occasional use are unknown. Smokeless tobacco also represents a substantial health risk. Chewing tobacco is a carcinogen linked to dental caries, gingivitis, oral leukoplakia, and oral cancer. The systemic effects of smokeless tobacco (including snuff) may increase risks for other cancers. Esophageal cancer is linked to carcinogens in tobacco dissolved in saliva and swallowed. The net effects of e-cigarettes on health are poorly studied. Whether they aid in smoking cessation or serve as a “gateway” for nonsmoking children to acquire a smoking habit is debated.

1	Physical activity is associated with a decreased risk of colon and breast cancer. A variety of mechanisms have been proposed. However, such studies are prone to confounding factors such as recall bias, association of exercise with other health-related practices, and effects of preclinical cancers on exercise habits (reverse causality). International epidemiologic studies suggest that diets high in fat are associated with increased risk for cancers of the breast, colon, prostate, and endometrium. These cancers have their highest incidence and mortalities in Western cultures, where fat composes an average of one-third of the total calories consumed.

1	Despite correlations, dietary fat has not been proven to cause cancer. Case-control and cohort epidemiologic studies give conflicting results. In addition, diet is a highly complex exposure to many nutrients and chemicals. Low-fat diets are associated with many dietary changes beyond simple subtraction of fat. Other lifestyle changes are also associated with adherence to a low-fat diet.

1	In observational studies, dietary fiber is associated with a reduced risk of colonic polyps and invasive cancer of the colon. However, cancer-protective effects of increasing fiber and lowering dietary fat have not been proven in the context of a prospective clinical trial. The putative protective mechanisms are complex and speculative. Fiber binds oxidized bile acids and generates soluble fiber products, such as butyrate, that may have differentiating properties. Fiber does not increase bowel transit times. Two large prospective cohort studies of >100,000 health professionals showed no association between fruit and vegetable intake and risk of cancer. The Polyp Prevention Trial randomly assigned 2000 elderly persons, who had polyps removed, to a low-fat, high-fiber diet versus routine diet for 4 years. No differences were noted in polyp formation.

1	The U.S. National Institutes of Health Women’s Health Initiative, launched in 1994, was a long-term clinical trial enrolling >100,000 women age 45–69 years. It placed women in 22 intervention groups. Participants received calcium/vitamin D supplementation; hormone replacement therapy; and counseling to increase exercise, eat a low-fat diet with increased consumption of fruits, vegetables, and fiber, and cease smoking. The study showed that although dietary fat intake was lower in the diet intervention group, invasive breast cancers were not reduced over an 8-year follow-up period compared to the control group. No reduction was seen in the incidence of colorectal cancer in the dietary intervention arm. The difference in dietary fat averaged ∼10% between the two groups. Evidence does not currently establish the anticarcinogenic value of vitamin, mineral, or nutritional supplements in amounts greater than those provided by a balanced diet.

1	Risk of cancer appears to increase as body mass index increases beyond 25 kg/m2. Obesity is associated with increased risk for cancers of the colon, breast (female postmenopausal), endometrium, kidney (renal cell), and esophagus, although causality has not been established. In observational studies, relative risks of colon cancer are increased in obesity by 1.5–2 for men and 1.2–1.5 for women. Obese postmenopausal women have a 30–50% increased relative risk of breast cancer. An unproven hypothesis for the association is that adipose tissue serves as a depot for aromatase that facilitates estrogen production.

1	Nonmelanoma skin cancers (basal cell and squamous cell) are induced by cumulative exposure to ultraviolet (UV) radiation. Intermittent acute sun exposure and sun damage have been linked to melanoma, but the evidence is inconsistent. Sunburns, especially in childhood and adolescence, may be associated with an increased risk of melanoma in adulthood. Reduction of sun exposure through use of protective clothing and changing patterns of outdoor activities can reduce skin cancer risk. Sunscreens decrease the risk of actinic keratoses, the precursor to squamous cell skin cancer, but melanoma risk may not be reduced. Sunscreens prevent burning, but they may encourage more prolonged exposure to the sun and may not filter out wavelengths of energy that cause melanoma.

1	Educational interventions to help individuals assess their risk of developing skin cancer have some impact. In particular, appearance-focused behavioral interventions in young women can decrease indoor tanning use and other UV exposures. Self-examination for skin pigment characteristics associated with skin cancer, such as freckling, may be useful in identifying people at high risk. Those who recognize themselves as being at risk tend to be more compliant with sun-avoidance recommendations. Risk factors for melanoma include a propensity to sunburn, a large number of benign melanocytic nevi, and atypical nevi. Chemoprevention involves the use of specific natural or synthetic chemical agents to reverse, suppress, or prevent carcinogenesis before the development of invasive malignancy.

1	Cancer develops through an accumulation of tissue abnormalities associated with genetic and epigenetic changes, and growth regulatory pathways that are potential points of intervention to prevent cancer. The initial changes are termed initiation. The alteration can be inherited or acquired through the action of physical, infectious, or chemical carcinogens. Like most human diseases, cancer arises from an interaction between genetics and environmental exposures (Table 100-1). Influences that cause the initiated cell and its surrounding tissue microenvironment to progress through the carcinogenic process and change phenotypically are termed promoters. Promoters include hormones such as androgens, linked to prostate cancer, and estrogen, linked to breast and endometrial cancer. The distinction between an initiator and promoter is indistinct; some components of cigarette smoke are “complete carcinogens,” acting as both initiators and promoters. Cancer can be prevented or controlled

1	between an initiator and promoter is indistinct; some components of cigarette smoke are “complete carcinogens,” acting as both initiators and promoters. Cancer can be prevented or controlled through interference with the factors that cause cancer initiation, promotion, or progression. Compounds of interest in chemoprevention often have antimutagenic, hormone modulation, anti-inflammatory, antiproliferative, or proapoptotic activity (or a combination).

1	Alkylating agents Acute myeloid leukemia, bladder cancer Androgens Prostate cancer Aromatic amines (dyes) Bladder cancer Arsenic Cancer of the lung, skin Asbestos Cancer of the lung, pleura, peritoneum Benzene Acute myelocytic leukemia Chromium Lung cancer Diethylstilbestrol (prenatal) Vaginal cancer (clear cell) Epstein-Barr virus Burkitt’s lymphoma, nasal T cell lymphoma Estrogens Cancer of the endometrium, liver, breast Ethyl alcohol Cancer of the breast, liver, esophagus, head and neck Helicobacter pylori Gastric cancer, gastric MALT lymphoma Hepatitis B or C virus Liver cancer Human immunodeficiency Non-Hodgkin’s lymphoma, Kaposi’s sarcoma, virus squamous cell carcinomas (especially of the urogenital tract) Human papilloma virus Cancers of the cervix, anus, oropharynx Human T cell lymphotropic Adult T cell leukemia/lymphoma virus type 1 (HTLV-1) Immunosuppressive agents Non-Hodgkin’s lymphoma (azathioprine, cyclosporine, glucocorticoids)

1	Immunosuppressive agents Non-Hodgkin’s lymphoma (azathioprine, cyclosporine, glucocorticoids) Ionizing radiation (thera-Breast, bladder, thyroid, soft tissue, bone, peutic or diagnostic) hematopoietic, and many more Nitrogen mustard gas Cancer of the lung, head and neck, nasal sinuses Nickel dust Cancer of the lung, nasal sinuses Diesel exhaust Lung cancer (miners) Phenacetin Cancer of the renal pelvis and bladder Polycyclic hydrocarbons Cancer of the lung, skin (especially squamous cell carcinoma of scrotal skin) Radon gas Lung cancer Schistosomiasis Bladder cancer (squamous cell) Sunlight (ultraviolet) Skin cancer (squamous cell and melanoma) Tobacco (including Cancer of the upper aerodigestive tract, smokeless) bladder Vinyl chloride Liver cancer (angiosarcoma) aAgents that are thought to act as cancer initiators and/or promoters.

1	Vinyl chloride Liver cancer (angiosarcoma) aAgents that are thought to act as cancer initiators and/or promoters. Smoking causes diffuse epithelial injury in the oral cavity, neck, esophagus, and lung. Patients cured of squamous cell cancers of the lung, esophagus, oral cavity, and neck are at risk (as high as 5% per year) of developing second cancers of the upper aerodigestive tract. Cessation of cigarette smoking does not markedly decrease the cured cancer patient’s risk of second malignancy, even though it does lower the cancer risk in those who have never developed a malignancy. Smoking cessation may halt the early stages of the carcinogenic process (such as metaplasia), but it may have no effect on late stages of carcinogenesis. This “field carcinogenesis” hypothesis for upper aerodigestive tract cancer has made “cured” patients an important population for chemoprevention of second malignancies.

1	Oral human papilloma virus (HPV) infection, particularly HPV-16, increases the risk for cancers of the oropharynx. This association exists even in the absence of other risk factors such as smoking or alcohol use (although the magnitude of increased risk appears greater than additive when HPV infection and smoking are both present). Oral HPV infection is believed to be largely sexually acquired. Although no direct evidence currently exists to confirm the hypothesis, the introduction of the HPV vaccine may eventually reduce oropharyngeal cancer rates.

1	Oral leukoplakia, a premalignant lesion commonly found in smokers, has been used as an intermediate marker of chemopreventive activity in smaller shorter-duration, randomized, placebo-controlled 477 trials. Response was associated with upregulation of retinoic acid receptor-β (RAR-β). Therapy with high, relatively toxic doses of isotretinoin (13-cis-retinoic acid) causes regression of oral leukoplakia. However, the lesions recur when the therapy is withdrawn, suggesting the need for long-term administration. More tolerable doses of isotretinoin have not shown benefit in the prevention of head and neck cancer. Isotretinoin also failed to prevent second malignancies in patients cured of early-stage non-small cell lung cancer; mortality rates were actually increased in current smokers.

1	Several large-scale trials have assessed agents in the chemoprevention of lung cancer in patients at high risk. In the α-tocopherol/βcarotene (ATBC) Lung Cancer Prevention Trial, participants were male smokers, age 50–69 years at entry. Participants had smoked an average of one pack of cigarettes per day for 35.9 years. Participants received α-tocopherol, β-carotene, and/or placebo in a randomized, two-by-two factorial design. After median follow-up of 6.1 years, lung cancer incidence and mortality were statistically significantly increased in those receiving β-carotene. α-Tocopherol had no effect on lung cancer mortality, and no evidence suggested interaction between the two drugs. Patients receiving α-tocopherol had a higher incidence of hemorrhagic stroke.

1	The β-Carotene and Retinol Efficacy Trial (CARET) involved 17,000 American smokers and workers with asbestos exposure. Entrants were randomly assigned to one of four arms and received β-carotene, retinol, and/or placebo in a two-by-two factorial design. This trial also demonstrated harm from β-carotene: a lung cancer rate of 5 per 1000 subjects per year for those taking placebo and of 6 per 1000 subjects per year for those taking β-carotene. The ATBC and CARET results demonstrate the importance of testing chemoprevention hypotheses thoroughly before their widespread implementation because the results contradict a number of observational studies. The Physicians’ Health Trial showed no change in the risk of lung cancer for those taking β-carotene; however, fewer of its participants were smokers than those in the ATBC and CARET studies.

1	Many colon cancer prevention trials are based on the premise that most colorectal cancers develop from adenomatous polyps. These trials use adenoma recurrence or disappearance as a surrogate endpoint (not yet validated) for colon cancer prevention. Early clinical trial results suggest that nonsteroidal anti-inflammatory drugs (NSAIDs), such as piroxicam, sulindac, and aspirin, may prevent adenoma formation or cause regression of adenomatous polyps. The mechanism of action of NSAIDs is unknown, but they are presumed to work through the cyclooxygenase pathway. Although two randomized controlled trials (the Physicians’ Health Study and the Women’s Health Study) did not show an effect of aspirin on colon cancer or adenoma incidence in persons with no previous history of colonic lesions after 10 years of therapy, these trials did show an approximately 18% relative risk reduction for colonic adenoma incidence in persons with a previous history of adenomas after 1 year. Pooled findings from

1	10 years of therapy, these trials did show an approximately 18% relative risk reduction for colonic adenoma incidence in persons with a previous history of adenomas after 1 year. Pooled findings from observational cohort studies do demonstrate a 22% and 28% relative reduction in colorectal cancer and adenoma incidence, respectively, with regular aspirin use, and a well-conducted meta-analysis of four randomized controlled trials (albeit primarily designed to examine aspirin’s effects on cardiovascular events) found that aspirin at doses of at least 75 mg resulted in a 24% relative reduction in colorectal cancer incidence after 20 years, with no clear increase in efficacy at higher doses. Cyclooxygenase-2 (COX-2) inhibitors have also been considered for colorectal cancer and polyp prevention. Trials with COX-2 inhibitors were initiated, but an increased risk of cardiovascular events in those taking the COX-2 inhibitors was noted, suggesting that these agents are not suitable for

1	Trials with COX-2 inhibitors were initiated, but an increased risk of cardiovascular events in those taking the COX-2 inhibitors was noted, suggesting that these agents are not suitable for chemoprevention in the general population.

1	Epidemiologic studies suggest that diets high in calcium lower colon cancer risk. Calcium binds bile and fatty acids, which cause proliferation of colonic epithelium. It is hypothesized that calcium reduces intraluminal exposure to these compounds. The randomized controlled Calcium Polyp Prevention Study found that calcium supplementation

1	Prevention and Early Detection of Cancer 478 decreased the absolute risk of adenomatous polyp recurrence by 7% at 4 years; extended observational follow-up demonstrated a 12% absolute risk reduction 5 years after cessation of treatment. However, in the Women’s Health Initiative, combined use of calcium carbonate and vitamin D twice daily did not reduce the incidence of invasive colorectal cancer compared with placebo after 7 years. The Women’s Health Initiative demonstrated that postmenopausal women taking estrogen plus progestin have a 44% lower relative risk of colorectal cancer compared to women taking placebo. Of >16,600 women randomized and followed for a median of 5.6 years, 43 invasive colorectal cancers occurred in the hormone group and 72 in the placebo group. The positive effect on colon cancer is mitigated by the modest increase in cardiovascular and breast cancer risks associated with combined estrogen plus progestin therapy. A case-control study suggested that statins

1	on colon cancer is mitigated by the modest increase in cardiovascular and breast cancer risks associated with combined estrogen plus progestin therapy. A case-control study suggested that statins decrease the incidence of colorectal cancer; however, several subsequent case-control and cohort studies have not demonstrated an association between regular statin use and a reduced risk of colorectal cancer. No randomized controlled trials have addressed this hypothesis. A meta-analysis of statin use showed no protective effect of statins on overall cancer incidence or death.

1	Tamoxifen is an antiestrogen with partial estrogen agonistic activity in some tissues, such as endometrium and bone. One of its actions is to upregulate transforming growth factor β, which decreases breast cell proliferation. In randomized placebo-controlled trials to assess tamoxifen as adjuvant therapy for breast cancer, tamoxifen reduced the number of new breast cancers in the opposite breast by more than a third. In a randomized placebo-controlled prevention trial involving >13,000 preand postmenopausal women at high risk, tamoxifen decreased the risk of developing breast cancer by 49% (from 43.4 to 22 per 1000 women) after a median follow-up of nearly 6 years. Tamoxifen also reduced bone fractures; a small increase in risk of endometrial cancer, stroke, pulmonary emboli, and deep vein thrombosis was noted. The International Breast Cancer Intervention Study (IBIS-I) and the Italian Randomized Tamoxifen Prevention Trial also demonstrated a reduction in breast cancer incidence with

1	thrombosis was noted. The International Breast Cancer Intervention Study (IBIS-I) and the Italian Randomized Tamoxifen Prevention Trial also demonstrated a reduction in breast cancer incidence with tamoxifen use. A trial comparing tamoxifen with another selective estrogen receptor modulator, raloxifene, in postmenopausal women showed that raloxifene is comparable to tamoxifen in cancer prevention. This trial only included postmenopausal women. Raloxifene was associated with more invasive breast cancers and a trend toward more noninvasive breast cancers, but fewer thromboembolic events than tamoxifen; the drugs are similar in risks of other cancers, fractures, ischemic heart disease, and stroke. Both tamoxifen and raloxifene (the latter for post-menopausal women only) have been approved by the U.S. Food and Drug Administration (FDA) for reduction of breast cancer in women at high risk for the disease (1.66% risk at 5 years based on the Gail risk model:

1	women only) have been approved by the U.S. Food and Drug Administration (FDA) for reduction of breast cancer in women at high risk for the disease (1.66% risk at 5 years based on the Gail risk model: http://www.cancer.gov/bcrisktool/).

1	Because the aromatase inhibitors are even more effective than tamoxifen in adjuvant breast cancer therapy, it has been hypothesized that they would be more effective in breast cancer prevention. A randomized, placebo-controlled trial of exemestane reported a 65% relative reduction (from 5.5 to 1.9 per 1000 women) in the incidence of invasive breast cancer in women at elevated risk after a median follow-up of about 3 years. Common adverse effects included arthralgias, hot flashes, fatigue, and insomnia. No trial has directly compared aromatase inhibitors with selective estrogen receptor modulators for breast cancer chemoprevention.

1	Finasteride and dutasteride are 5-α-reductase inhibitors. They inhibit conversion of testosterone to dihydrotestosterone (DHT), a potent stimulator of prostate cell proliferation. The Prostate Cancer Prevention Trial (PCPT) randomly assigned men age 55 years or older at average risk of prostate cancer to finasteride or placebo. All men in the trial were being regularly screened with prostate-specific antigen (PSA) levels and digital rectal examination. After 7 years of therapy, the incidence of prostate cancer was 18.4% in the finasteride arm, compared with 24.4% in the placebo arm, a statistically significant difference. However, the finasteride group had more patients with tumors of Gleason score 7 and higher compared with the placebo arm (6.4 vs 5.1%). Reassuringly, long-term (10–15 years) follow-up did not reveal any statistically significant differences in overall mortality between all men in the finasteride and placebo arms or in men diagnosed with prostate cancer; differences

1	follow-up did not reveal any statistically significant differences in overall mortality between all men in the finasteride and placebo arms or in men diagnosed with prostate cancer; differences in prostate cancer in favor of finasteride persisted.

1	Dutasteride has also been evaluated as a preventive agent for prostate cancer. The Reduction by Dutasteride of Prostate Cancer Events (REDUCE) trial was a randomized double-blind trial in which approximately 8200 men with an elevated PSA (2.5–10 ng/mL for men age 50–60 years and 3–10 ng/mL for men age 60 years or older) and negative prostate biopsy on enrollment received daily 0.5 mg of dutasteride or placebo. The trial found a statistically significant 23% relative risk reduction in the incidence of biopsy-detected prostate cancer in the dutasteride arm at 4 years of treatment (659 cases vs 858 cases, respectively). Overall, across years 1 through 4, there was no difference between the arms in the number of tumors with a Gleason score of 7 to 10; however, during years 3 and 4, there was a statistically significant difference in tumors with Gleason score of 8 to 10 in the dutasteride arm (12 tumors vs 1 tumor, respectively).

1	The clinical importance of the apparent increased incidence of higher-grade tumors in the 5-α-reductase inhibitor arms of these trials is controversial. It may likely represent an increased sensitivity of PSA and digital rectal exam for high-grade tumors in men receiving these agents. The FDA has analyzed both trials, and it determined that the use of a 5-α-reductase inhibitor for prostate cancer chemoprevention would result in one additional high-grade (Gleason score 8 to 10) prostate cancer for every three to four lower-grade (Gleason score <6) tumors averted. Although it acknowledged that detection bias may have accounted for the finding, it stated that it could not conclusively dismiss a causative role for 5-α-reductase inhibitors. These agents are therefore not FDA-approved for prostate cancer prevention.

1	Because all men in both the PCPT and REDUCE trials were being screened and because screening approximately doubles the rate of prostate cancer, it is not known if finasteride or dutasteride decreases the risk of prostate cancer in men who are not being screened. Several favorable laboratory and observational studies led to the formal evaluation of selenium and α-tocopherol (vitamin E) as potential prostate cancer preventives. The Selenium and Vitamin E Cancer Prevention Trial (SELECT) assigned 35,533 men to receive 200 μg/d selenium, 400 IU/d α-tocopherol, selenium plus vitamin E, or placebo. After a median follow-up of 7 years, a trend toward an increased risk of developing prostate cancer was observed for those men taking vitamin E alone as compared to the placebo arm (hazard ratio 1.17; 95% confidence interval, 1.004–1.36).

1	A number of infectious agents cause cancer. Hepatitis B and C are linked to liver cancer; some HPV strains are linked to cervical, anal, and head and neck cancer; and Helicobacter pylori is associated with gastric adenocarcinoma and gastric lymphoma. Vaccines to protect against these agents may reduce the risk of their associated cancers. The hepatitis B vaccine is effective in preventing hepatitis and hepatomas due to chronic hepatitis B infection.

1	A quadrivalent HPV vaccine (covering HPV strains 6, 11, 16, and 18) and a bivalent vaccine (covering HPV strains 16 and 18) are available for use in the United States. HPV types 16 and 18 cause cervical and anal cancer; reduction in these HPV types could prevent >70% of cervical cancers worldwide. HPV types 6 and 11 cause genital papillomas. For individuals not previously infected with these HPV strains, the vaccines demonstrate high efficacy in preventing persistent strain-specific HPV infections; however, the trials and substudies that evaluated the vaccines’ ability to prevent cervical and anal cancer relied on surrogate outcome measures (cervical or anal intraepithelial neoplasia [CIN/AIN] I, II, and III), and the degree of durability of the immune response beyond 5 years is not currently known. The vaccines do not appear to impact preexisting infections and the efficacy appears to

1	Prevention and Early Detection of Cancer be markedly lower for populations that had previously been exposed to vaccine-specific HPV strains. The vaccine is recommended in the United States for females and males age 9–26 years. Some organs in some individuals are at such high risk of developing cancer that surgical removal of the organ at risk may be considered. Women with severe cervical dysplasia are treated with laser or loop electrosurgical excision or conization and occasionally even hysterectomy. Colectomy is used to prevent colon cancer in patients with familial polyposis or ulcerative colitis.

1	Prophylactic bilateral mastectomy may be chosen for breast cancer prevention among women with genetic predisposition to breast cancer. In a prospective series of 139 women with BRCA1 and BRCA2 mutations, 76 chose to undergo prophylactic mastectomy and 63 chose close surveillance. At 3 years, no cases of breast cancer had been diagnosed in those opting for surgery, but eight patients in the surveillance group had developed breast cancer. A larger (n = 639) retrospective cohort study reported that three patients developed breast cancer after prophylactic mastectomy compared with an expected incidence of 30–53 cases: a 90–94% reduction in breast cancer risk. Postmastectomy breast cancer–related deaths were reduced by 81–94% for high-risk women compared with sister controls and by 100% for moderate-risk women when compared with expected rates.

1	Prophylactic oophorectomy may also be employed for the prevention of ovarian and breast cancers among high-risk women. A prospective cohort study evaluating the outcomes of BRCA mutation carriers demonstrated a statistically significant association between prophylactic oophorectomy and a reduced incidence of ovarian or primary peritoneal cancer (36% relative risk reduction, or a 4.5% absolute difference). Studies of prophylactic oophorectomy for prevention of breast cancer in women with genetic mutations have shown relative risk reductions of approximately 50%; the risk reduction may be greatest for women having the procedure at younger (i.e., <50 years) ages.

1	All of the evidence concerning the use of prophylactic mastectomy and oophorectomy for prevention of breast and ovarian cancer in high-risk women has been observational in nature; such studies are prone to a variety of biases, including case selection bias, family relationships between patients and controls, and inadequate information about hormone use. Thus, they may give an overestimate of the magnitude of benefit. Screening is a means of detecting disease early in asymptomatic individuals, with the goal of decreasing morbidity and mortality. While screening can potentially reduce disease-specific deaths and has been shown to do so in cervical, colon, lung, and breast cancer, it is also subject to a number of biases that can suggest a benefit when actually there is none. Biases can even mask net harm. Early detection does not in itself confer benefit. Cause-specific mortality, rather than survival after diagnosis, is the preferred endpoint (see below).

1	Because screening is done on asymptomatic, healthy persons, it should offer substantial likelihood of benefit that outweighs harm. Screening tests and their appropriate use should be carefully evaluated before their use is widely encouraged in screening programs, as a matter of public policy.

1	A large and increasing number of genetic mutations and nucleotide polymorphisms have been associated with an increased risk of cancer. Testing for these genetic mutations could in theory define a high-risk population. However, most of the identified mutations have very low penetrance and individually provide minimal predictive accuracy. The ability to predict the development of a particular cancer may some day present therapeutic options as well as ethical dilemmas. It may eventually allow for early intervention to prevent a cancer or limit its severity. People at high risk may be ideal candidates for chemoprevention and screening; however, efficacy of these interventions in the high-risk population should be investigated. Currently, persons at high risk for a particular cancer can engage in intensive screening. While this course is clinically reasonable, it is not known if it reduces mortality in these populations.

1	Sensitivity The proportion of persons with the condition who test positive: a /(a + c) Specificity The proportion of persons without the condition who test negative: d /(b + d) Positive predictive value The proportion of persons with a positive test who (PPV) have the condition: a /(a + b) Negative predictive The proportion of persons with a negative test value who do not have the condition: d /(c + d) Prevalence, sensitivity, and specificity determine PPV aFor diseases of low prevalence, such as cancer, poor specificity has a dramatic adverse effect on PPV such that only a small fraction of positive tests are true positives.

1	The Accuracy of Screening A screening test’s accuracy or ability to discriminate disease is described by four indices: sensitivity, specificity, positive predictive value, and negative predictive value (Table 100-2). Sensitivity, also called the true-positive rate, is the proportion of persons with the disease who test positive in the screen (i.e., the ability of the test to detect disease when it is present). Specificity, or 1 minus the false-positive rate, is the proportion of persons who do not have the disease that test negative in the screening test (i.e., the ability of a test to correctly identify that the disease is not present). The positive predictive value is the proportion of persons who test positive that actually have the disease. Similarly, negative predictive value is the proportion testing negative that do not have the disease. The sensitivity and specificity of a test are independent of the underlying prevalence (or risk) of the disease in the population screened,

1	proportion testing negative that do not have the disease. The sensitivity and specificity of a test are independent of the underlying prevalence (or risk) of the disease in the population screened, but the predictive values depend strongly on the prevalence of the disease.

1	Screening is most beneficial, efficient, and economical when the target disease is common in the population being screened. Specificity is at least as important to the ultimate feasibility and success of a screening test as sensitivity. Potential Biases of Screening Tests Common biases of screening are lead time, length-biased sampling, and selection. These biases can make a screening test seem beneficial when actually it is not (or even causes net harm). Whether beneficial or not, screening can create the false impression of an epidemic by increasing the number of cancers diagnosed. It can also produce a shift in the proportion of patients diagnosed at an early stage and inflate survival statistics without reducing mortality (i.e., the number of deaths from a given cancer relative to the number of those at risk for the cancer). In such a case, the apparent duration of survival (measured from date of diagnosis) increases without lives being saved or life expectancy changed.

1	Lead-time bias occurs whether or not a test influences the natural history of the disease; the patient is merely diagnosed at an earlier date. Survival appears increased even if life is not really prolonged. The screening test only prolongs the time the subject is aware of the disease and spends as a patient.

1	Length-biased sampling occurs because screening tests generally can more easily detect slow-growing, less aggressive cancers than fast-growing cancers. Cancers diagnosed due to the onset of symptoms between scheduled screenings are on average more aggressive, and treatment outcomes are not as favorable. An extreme form of length bias sampling is termed overdiagnosis, the detection of “pseudo disease.” The reservoir of some undetected slow-growing tumors is large. Many of these tumors fulfill the histologic criteria of cancer but will 480 never become clinically significant or cause death. This problem is compounded by the fact that the most common cancers appear most frequently at ages when competing causes of death are more frequent. Selection bias must be considered in assessing the results of any screening effort. The population most likely to seek screening may differ from the general population to which the screening test might be applied. In general, volunteers for studies are

1	of any screening effort. The population most likely to seek screening may differ from the general population to which the screening test might be applied. In general, volunteers for studies are more health conscious and likely to have a better prognosis or lower mortality rate, irrespective of the screening result. This is termed the healthy volunteer effect.

1	Potential Drawbacks of Screening Risks associated with screening include harm caused by the screening intervention itself, harm due to the further investigation of persons with positive tests (both true and false positives), and harm from the treatment of persons with a true-positive result, whether or not life is extended by treatment (e.g., even if a screening test reduces relative cause-specific mortality by 20–30%, 70–80% of those diagnosed still go on to die of the target cancer). The diagnosis and treatment of cancers that would never have caused medical problems can lead to the harm of unnecessary treatment and give patients the anxiety of a cancer diagnosis. The psychosocial impact of cancer screening can also be substantial when applied to the entire population.

1	Assessment of Screening Tests Good clinical trial design can offset some biases of screening and demonstrate the relative risks and benefits of a screening test. A randomized controlled screening trial with cause-specific mortality as the endpoint provides the strongest support for a screening intervention. Overall mortality should also be reported to detect an adverse effect of screening and treatment on other disease outcomes (e.g., cardiovascular disease). In a randomized trial, two like populations are randomly established. One is given the usual standard of care (which may be no screening at all) and the other receives the screening intervention being assessed. The two populations are compared over time. Efficacy for the population studied is established when the group receiving the screening test has a better cause-specific mortality rate than the control group. Studies showing a reduction in the incidence of advanced-stage disease, improved survival, or a stage shift are weaker

1	test has a better cause-specific mortality rate than the control group. Studies showing a reduction in the incidence of advanced-stage disease, improved survival, or a stage shift are weaker (and possibly misleading) evidence of benefit. These latter criteria are early indicators but not sufficient to establish the value of a screening test.

1	Although a randomized, controlled screening trial provides the strongest evidence to support a screening test, it is not perfect. Unless the trial is population-based, it does not remove the question of generalizability to the target population. Screening trials generally involve thousands of persons and last for years. Less definitive study designs are therefore often used to estimate the effectiveness of screening practices. However, every nonrandomized study design is subject to strong confounders. In descending order of strength, evidence may also be derived from the findings of internally controlled trials using intervention allocation methods other than randomization (e.g., allocation by birth date, date of clinic visit); the findings of analytic observational studies; or the results of multiple time series studies with or without the intervention.

1	Screening for Specific Cancers Screening for cervical, colon, and breast cancer is beneficial for certain age groups. Depending on age and smoking history, lung cancer screening can also be beneficial in specific settings. Special surveillance of those at high risk for a specific cancer because of a family history or a genetic risk factor may be prudent, but few studies have assessed the influence on mortality. A number of organizations have considered whether or not to endorse routine use of certain screening tests. Because these groups have not used the same criteria to judge whether a screening test should be endorsed, they have arrived at different recommendations. The American Cancer Society (ACS) and the U.S. Preventive Services Task Force (USPSTF) publish screening guidelines (Table 100-3); the American Academy of Family Practitioners (AAFP) generally follow/ endorse the USPSTF recommendations; and the American College of Physicians (ACP) develops recommendations based on

1	(Table 100-3); the American Academy of Family Practitioners (AAFP) generally follow/ endorse the USPSTF recommendations; and the American College of Physicians (ACP) develops recommendations based on structured reviews of other organizations’ guidelines.

1	Breast cancer Breast self-examination, clinical breast examination by a caregiver, mammography, and magnetic resonance imaging (MRI) have all been variably advocated as useful screening tools.

1	A number of trials have suggested that annual or biennial screening with mammography or mammography plus clinical breast examination in normal-risk women older than age 50 years decreases breast cancer mortality. Each trial has been criticized for design flaws. In most trials, breast cancer mortality rate is decreased by 15–30%. Experts disagree on whether average-risk women age 40–49 years should receive regular screening (Table 100-3). The U.K. Age Trial, the only randomized trial of breast cancer screening to specifically evaluate the impact of mammography in women age 40–49 years, found no statistically significant difference in breast cancer mortality for screened women versus controls after about 11 years of follow-up (relative risk 0.83; 95% confidence interval 0.66–1.04); however, <70% of women received screening in the intervention arm, potentially diluting the observed effect. A meta-analysis of eight large randomized trials showed a 15% relative reduction in mortality

1	<70% of women received screening in the intervention arm, potentially diluting the observed effect. A meta-analysis of eight large randomized trials showed a 15% relative reduction in mortality (relative risk 0.85; 95% confidence interval 0.75–0.96) from mammography screening for women age 39–49 years after 11–20 years of follow-up. This is equivalent to a number needed to invite to screening of 1904 over 10 years to prevent one breast cancer death. At the same time, nearly half of women age 40–49 years screened annually will have false-positive mammograms necessitating further evaluation, often including biopsy. Estimates of overdiagnosis range from 10 to 40% of diagnosed invasive cancers. In the United States, widespread screening over the last several decades has not been accompanied by a reduction in incidence of metastatic breast cancer despite a large increase in early-stage disease, suggesting a substantial amount of overdiagnosis at the population level.

1	No study of breast self-examination has shown it to decrease mortality. A randomized controlled trial of approximately 266,000 women in China demonstrated no difference in breast cancer mortality between a group that received intensive breast self-exam instruction and reinforcement/reminders and controls at 10 years of follow-up. However, more benign breast lesions were discovered and more breast biopsies were performed in the self-examination arm.

1	Genetic screening for BRCA1 and BRCA2 mutations and other markers of breast cancer risk has identified a group of women at high risk for breast cancer. Unfortunately, when to begin and the optimal frequency of screening have not been defined. Mammography is less sensitive at detecting breast cancers in women carrying BRCA1 and BRCA2 mutations, possibly because such cancers occur in younger women, in whom mammography is known to be less sensitive. MRI screening may be more sensitive than mammography in women at high risk due to genetic predisposition or in women with very dense breast tissue, but specificity may be lower. An increase in overdiagnosis may accompany the higher sensitivity. The impact of MRI on breast cancer mortality with or without concomitant use of mammography has not been evaluated in a randomized controlled trial.

1	cervical cancer Screening with Papanicolaou (Pap) smears decreases cervical cancer mortality. The cervical cancer mortality rate has fallen substantially since the widespread use of the Pap smear. With the onset of sexual activity comes the risk of sexual transmission of HPV, the fundamental etiologic factor for cervical cancer. Screening guidelines recommend regular Pap testing for all women who have reached the age of 21 (prior to this age, even in individuals that have begun sexual activity, screening may cause more harm than benefit). The recommended interval for Pap screening is 3 years. Screening more frequently adds little benefit but leads to important harms, including unnecessary procedures and overtreatment of transient lesions. Beginning at age 30, guidelines also offer the alternative of combined Pap smear and HPV testing for women. The screening interval for women who test normal using this approach may be lengthened to 5 years.

1	An upper age limit at which screening ceases to be effective is not known, but women age 65 years with no abnormal results in the previous 10 years may choose to stop screening. Screening should be Women ≥40 years: “I” (as a standalone without mammography) Women 40–49 years: The decision should be an individual one, and take patient context/values into account (“C”) Women 50–74 years: Every 2 years (“B”) Women ≥75 years: “I” Women 21–65 years: Screen every 3 years (“A”) Women <21 years: “D” Women >65 years, with adequate, normal prior Pap screenings: “D” Women after total hysterectomy for noncancerous causes: “D” Women 30–65 years: Screen in combination with cytology every 5 years if woman desires to lengthen the screening interval (see Pap test, above) (“A”) Women <30 years: “D” Women >65 years, with adequate, normal prior Pap screenings: “D” Women after total hysterectomy for noncancerous causes: “D”

1	Women <30 years: “D” Women >65 years, with adequate, normal prior Pap screenings: “D” Women after total hysterectomy for noncancerous causes: “D” Adults 50–75 years: every 5 years in combination with high-sensitivity FOBT every 3 years (“A”)b Adults 76–85 years: “C” Adults ≥85 years: “D” Adults 50–75 years: Annually, for high-sensitivity FOBT (“A”) Adults 76–85 years: “C” Adults ≥85 years: “D” Adults 50–75 years: every 10 years (“A”) Adults 76–85 years: “C” Adults ≥85 years: “D” Adults 55–80 years, with a ≥30 pack-year smoking history, still smoking or have quit within past 15 years. Discontinue once a person has not smoked for 15 years or develops a health problem that substantially limits life expectancy or the ability to have curative lung surgery: “B” “D” Women ≥20 years: Breast self-exam is an option Women 20–39 years: Perform every 3 years Women ≥40 years: Perform annually Women ≥40 years: Screen annually for as long as the woman is in good health

1	Women with >20% lifetime risk of breast cancer: Screen with MRI plus mammography annually Women with 15–20% lifetime risk of breast cancer: Discuss option of MRI plus mammography annually Women with <15% lifetime risk of breast cancer: Do not screen annually with MRI Women 21–29 years: Screen every 3 years Women 30–65 years: Acceptable approach to screen with cytology every 3 years (see HPV test below) Women <21 years: No screening Women >65 years: No screening following adequate negative prior screening Women after total hysterectomy for noncancerous causes: Do not screen Women 30–65 years: Preferred approach to screen with HPV and cytology co-testing every 5 years (see Pap test above) Women <30 years: Do not use HPV testing Women >65 years: No screening following adequate negative prior screening Women after total hysterectomy for noncancerous causes: Do not screen Adults ≥50 years: Screen every 5 years Adults ≥50 years: Screen every year

1	Women after total hysterectomy for noncancerous causes: Do not screen Adults ≥50 years: Screen every 5 years Adults ≥50 years: Screen every year Adults ≥50 years: Screen every 10 years Adults ≥50 years: Screen, but interval uncertain Adults ≥50 years: Screen every year Adults ≥50 years: Screen every 5 years Men and women, 55–74 years, with ≥30 pack-year smoking history, still smoking or have quit within past 15 years: Discuss benefits, limitations, and potential harms of screening; only perform screening in facilities with the right type of CT scanner and with high expertise/specialists There is no sufficiently accurate test proven effective in the early detection of ovarian cancer. For women at high risk of ovarian cancer and/or who have unexplained, persistent symptoms, the combination of CA-125 and transvaginal ultrasound with pelvic exam may be offered. Prevention and Early Detection of Cancer Skin Complete skin examination by clinician or patient Men, all ages: “D”

1	Prevention and Early Detection of Cancer Skin Complete skin examination by clinician or patient Men, all ages: “D” Starting at age 50, men should talk to a doctor about the pros and cons of testing so they can decide if testing is the right choice for them. If African American or have a father or brother who had prostate cancer before age 65, men should have this talk starting at age 45. How often they are tested will depend on their PSA level. As for PSA; if men decide to be tested, they should have the PSA blood test with or without a rectal exam

1	Self-examination monthly; clinical exam as part of routine cancer-related checkup aSummary of the screening procedures recommended for the general population by the USPSTF and the ACS. These recommendations refer to asymptomatic persons who are not known to have risk factors, other than age or gender, for the targeted condition. bUSPSTF lettered recommendations are defined as follows: “A”: The USPSTF recommends the service, because there is high certainty that the net benefit is substantial; “B”: The USPSTF recommends the service, because there is high certainty that the net benefit is moderate or moderate certainty that the net benefit is moderate to substantial; “C”: The USPSTF recommends selectively offering or providing this service to individual patients based on professional judgment and patient preferences; there is at least moderate certainty that the net benefit is small; “D”: The USPSTF recommends against the service because there is moderate or high certainty that the

1	and patient preferences; there is at least moderate certainty that the net benefit is small; “D”: The USPSTF recommends against the service because there is moderate or high certainty that the service has no net benefit or that the harms outweigh the benefits; “I”: The USPSTF concludes that the current evidence is insufficient to assess the balance of benefits and harms of the service.

1	Abbreviations: ACS, American Cancer Society; USPSTF, U.S. Preventive Services Task Force. discontinued in women who have undergone a hysterectomy for noncancerous reasons. Although the efficacy of the Pap smear in reducing cervical cancer mortality has never been directly confirmed in a randomized, controlled setting, a clustered randomized trial in India evaluated the impact of one-time cervical visual inspection and immediate colposcopy, biopsy, and/or cryotherapy (where indicated) versus counseling on cervical cancer deaths in women age 30–59 years. After 7 years of follow-up, the age-standardized rate of death due to cervical cancer was 39.6 per 100,000 person-years in the intervention group versus 56.7 per 100,000 person-years in controls.

1	colorectal cancer Fecal occult blood testing (FOBT), digital rectal examination (DRE), rigid and flexible sigmoidoscopy, colonoscopy, and computed tomography (CT) colonography have been considered for colorectal cancer screening. A meta-analysis of four randomized controlled trials demonstrated a 15% relative reduction in colorectal cancer mortality with FOBT. The sensitivity for FOBT is increased if specimens are rehydrated before testing, but at the cost of lower specificity. The false-positive rate for rehydrated FOBT is high; 1–5% of persons tested have a positive test. Only 2–10% of those with occult blood in the stool have cancer. The high false-positive rate of FOBT dramatically increases the number of colonoscopies performed.

1	Fecal immunochemical tests appear to have higher sensitivity for colorectal cancer than nonrehydrated FOBT tests. Fecal DNA testing is an emerging testing modality; it may have increased sensitivity and comparable specificity to FOBT and could potentially reduce harms associated with follow-up of false-positive tests. The body of evidence on the operating characteristics and effectiveness of fecal DNA tests in reducing colorectal cancer mortality is limited.

1	Two meta-analyses of five randomized controlled trials of sigmoidoscopy (i.e., the NORCCAP, SCORE, PLCO, Telemark, and U.K. trials) found an 18% relative reduction in colorectal cancer incidence and a 28% relative reduction in colorectal cancer mortality. Participant ages ranged from 50 to 74 years, with follow-up ranging from 6 to 13 years. Diagnosis of adenomatous polyps by sigmoidoscopy should lead to evaluation of the entire colon with colonoscopy. The most efficient interval for screening sigmoidoscopy is unknown, but an interval of 5 years is often recommended. Case-control studies suggest that intervals of up to 15 years may confer benefit; the U.K. trial demonstrated benefit with one-time screening.

1	One-time colonoscopy detects ∼25% more advanced lesions (polyps >10 mm, villous adenomas, adenomatous polyps with high-grade dysplasia, invasive cancer) than one-time FOBT with sigmoidoscopy; comparative programmatic performance of the two modalities over time is not known. Perforation rates are about 3/1000 for colonoscopy and 1/1000 for sigmoidoscopy. Debate continues on whether colonoscopy is too expensive and invasive and whether sufficient provider capacity exists to be recommended as the preferred screening tool in standard-risk populations. Some observational studies suggest that efficacy of colonoscopy to decrease colorectal cancer mortality is primarily limited to the left side of the colon.

1	CT colonography, if done at expert centers, appears to have a sensitivity for polyps ≥6 mm comparable to colonoscopy. However, the rate of extracolonic findings of abnormalities of uncertain significance that must nevertheless be worked up is high (∼15–30%); the long-term cumulative radiation risk of repeated colonography screenings is also a concern.

1	lung cancer Chest x-ray and sputum cytology have been evaluated in several randomized lung cancer screening trials. The most recent and largest (n = 154,901) of these, one substudy of the Prostate, Lung, Colorectal, and Ovarian (PLCO) cancer screening trial, found that, compared with usual care, annual chest x-ray did not reduce the risk of dying from lung cancer (relative risk 0.99; 95% confidence interval 0.87–1.22) after 13 years. Low-dose CT has also been evaluated in several randomized trials. The largest and longest of these, the National Lung Screening Trial (NLST), was a randomized controlled trial of screening for lung cancer in approximately 53,000 persons age 55–74 years with a 30+ pack-year smoking history. It demonstrated a statistically significant relative reduction of about 15–20% in lung cancer mortality in the CT arm compared to the chest x-ray arm (or about 3 fewer deaths per 1000 people screened with CT). However, the harms include the potential radiation risks

1	15–20% in lung cancer mortality in the CT arm compared to the chest x-ray arm (or about 3 fewer deaths per 1000 people screened with CT). However, the harms include the potential radiation risks associated with multiple scans, the discovery of incidental findings of unclear significance, and a high rate of false-positive test results. Both incidental findings and false-positive tests can lead to invasive diagnostic procedures associated with anxiety, expense, and complications (e.g., pneumoor hemothorax after lung biopsy). The NLST was performed at experienced screening centers, and the balance of benefits and harms may differ in the community setting at less experienced centers.

1	ovarian cancer Adnexal palpation, transvaginal ultrasound (TVUS), and serum CA-125 assay have been considered for ovarian cancer screening. A large randomized controlled trial has shown that an annual screening program of TVUS and CA-125 in average-risk women does not reduce deaths from ovarian cancer (relative risk 1.21; 95% confidence interval 0.99–1.48). Adnexal palpation was dropped early in the study because it did not detect any ovarian cancers that were not detected by either TVUS or CA-125. The risks and costs associated with the high number of false-positive results are impediments to routine use of these modalities for screening. In the PLCO trial, 10% of participants had a false-positive result from TVUS or CA-125, and one-third of these women underwent a major surgical procedure; the ratio of surgeries to screen-detected ovarian cancer was approximately 20:1.

1	Prostate cancer The most common prostate cancer screening modalities are DRE and serum PSA assay. An emphasis on PSA screening has caused prostate cancer to become the most common nonskin cancer diagnosed in American males. This disease is prone to lead-time bias, length bias, and overdiagnosis, and substantial debate continues among experts as to whether screening should be offered unless the patient specifically asks to be screened. Virtually all organizations stress the importance of informing men about the uncertainty regarding screening efficacy and the harms associated with screening. Prostate cancer screening clearly detects many asymptomatic cancers, but the ability to distinguish tumors that are lethal but still curable from those that pose little or no threat to health is limited, and randomized trials indicate that the effect of PSA screening on prostate cancer mortality across a population is, at best, small. Men older than age 50 years have a high prevalence of indolent,

1	and randomized trials indicate that the effect of PSA screening on prostate cancer mortality across a population is, at best, small. Men older than age 50 years have a high prevalence of indolent, clinically insignificant prostate cancers (about 30–50% of men, increasing further as men age).

1	Two major randomized controlled trials of the impact of PSA screening on prostate cancer mortality have been published. The PLCO Cancer Screening Trial was a multicenter U.S. trial that randomized almost 77,000 men age 55–74 years to receive either annual PSA testing for 6 years or usual care. At 13 years of follow-up, no statistically significant difference in the number of prostate cancer deaths were noted between the arms (rate ratio 1.09; 95% confidence interval 0.87–1.36). Approximately 50% of men in the control arm received at least one PSA test during the trial, which may have potentially diluted a small effect.

1	The European Randomized Study of Screening for Prostate Cancer (ERSPC) was a multinational study that randomized approximately 182,000 men between age 50 and 74 years (with a predefined “core” screening group of men age 55–69 years) to receive PSA testing or no screening. Recruitment and randomization procedures, as well as actual frequency of PSA testing, varied by country. After a median follow-up of 11 years, a 20% relative reduction in the risk of prostate cancer death in the screened arm was noted in the “core” screening group. The trial found that 1055 men would need to be invited to screening, and 37 cases of prostate cancer detected, to avert 1 death from prostate cancer. Of the seven countries included in the mortality analysis, two demonstrated statistically significant reductions in prostate cancer deaths, whereas five did not. There was also an imbalance in treatment between the two study arms, with a higher proportion of men with clinically localized cancer receiving

1	in prostate cancer deaths, whereas five did not. There was also an imbalance in treatment between the two study arms, with a higher proportion of men with clinically localized cancer receiving radical prostatectomy in the screening arm and receiving it at experienced referral centers.

1	Treatments for low-stage prostate cancer, such as surgery and radiation therapy, can cause significant morbidity, including impotence and urinary incontinence. In a trial conducted in the United States after the initiation of widespread PSA testing, random assignment to radical prostatectomy compared with “watchful waiting” did not result in a statistically significant decrease in prostate cancer deaths (absolute risk reduction 2.7%; 95% confidence interval–1.3 to 6.2%). skIn cancer Visual examination of all skin surfaces by the patient or by a health care provider is used in screening for basal and squamous cell cancers and melanoma. No prospective randomized study has been performed to look for a mortality decrease. Unfortunately, screening is associated with a substantial rate of overdiagnosis. Prevention and Early Detection of Cancer

1	Prevention and Early Detection of Cancer Cancer Genetics Pat J. Morin, Jeffrey M. Trent, Francis S. Collins, Bert Vogelstein CANCER IS A GENETIC DISEASE Cancer arises through a series of somatic alterations in DNA that 101e

1	APC inactivation or ˜-catenin activation Early adenoma Intermed adenoma Late adenoma Carcinoma Metastasis K-RAS or BRAFactivation SMAD4or TGF˜ II inactivation P53inactivation Other alterations Microsatellite Instability (MIN) or Chromosomal Instability (CIN) Normal epithelium FIGURE 101e-2 Progressive somatic mutational steps in the development of colon carcinoma. The accumulation of alterations in a num-ber of different genes results in the progression from normal epithelium through adenoma to full-blown carcinoma. Genetic instability (micro-satellite or chromosomal) accelerates the progression by increasing the likelihood of mutation at each step. Patients with familial polyposis are already one step into this pathway, because they inherit a germline alteration of the APC gene. TGF, transforming growth factor.

1	result in unrestrained cellular proliferation. Most of these alterations involve actual sequence changes in DNA (i.e., mutations). They may originate as a consequence of random replication errors, exposure to carcinogens (e.g., radiation), or faulty DNA repair processes. While most cancers arise sporadically, familial clustering of cancers occurs in certain families that carry a germline mutation in a cancer gene.

1	The idea that cancer progression is driven by sequential somatic mutations in specific genes has only gained general acceptance in the past 25 years. Before the advent of the microscope, cancer was believed to be composed of aggregates of mucus or other noncellular matter. By the middle of the nineteenth century, it became clear that tumors were masses of cells and that these cells arose from the normal cells of the tissue from which the cancer originated. However, the molecular basis for the uncontrolled proliferation of cancer cells was to remain a mystery for another century. During that time, a number of theories for the origin of cancer were postulated. The great biochemist Otto Warburg proposed the combustion theory of cancer, which stipulated that cancer was due to abnormal oxygen metabolism. In addition, some believed that all cancers were caused by viruses, and that cancer was in fact a contagious disease.

1	In the end, observations of cancer occurring in chimney sweeps, studies of x-rays, and the overwhelming data demonstrating cigarette smoke as a causative agent in lung cancer, together with Ames’s work on chemical mutagenesis, provided convincing evidence that cancer originated through changes in DNA. Although the viral theory of cancer did not prove to be generally accurate (with the exception of human papillomaviruses, which can cause cervical and other cancers in human), the study of retroviruses led to the discovery of the first human oncogenes in the late 1970s. Soon after, the study of families with genetic predisposition to cancer was instrumental in the discovery of tumor-suppressor genes. The field that studies the type of mutations, as well as the consequence of these mutations in tumor cells, is now known as cancer genetics. Nearly all cancers originate from a single cell; this clonal origin is a critical discriminating feature between neoplasia and hyperplasia.

1	Nearly all cancers originate from a single cell; this clonal origin is a critical discriminating feature between neoplasia and hyperplasia. FIGURE 101e-1 Multistep clonal development of malignancy. In this diagram a series of five cumulative mutations (T , T , T , T , T ), each with a modest growth advantage acting alone, eventually results in a malignant tumor. Note that not all such alterations result in progression; for example, the T3 clone is a dead end. The actual number of cumulative mutations necessary to transform from the normal to the malignant state is unknown in most tumors. (After P Nowell: Science 194:23, 1976, with permission.)

1	Multiple cumulative mutational events are invariably required for the progression of a tumor from normal to fully malignant phenotype. The process can be seen as Darwinian microevolution in which, at each successive step, the mutated cells gain a growth advantage resulting in an increased representation relative to their neighbors (Fig. 101e-1). Based on observations of cancer frequency increases during aging, as well as molecular genetics work, it is believed that 5 to 10 accumulated mutations are necessary for a cell to progress from the normal to the fully malignant phenotype.

1	We are beginning to understand the precise nature of the genetic alterations responsible for some malignancies and to get a sense of the order in which they occur. The best-studied example is colon cancer, in which analyses of DNA from tissues extending from normal colon epithelium through adenoma to carcinoma have identified some of the genes mutated in the process (Fig. 101e-2). Other malignancies are believed to progress in a similar stepwise fashion, although the order and identity of genes affected may be different. TWO TYPES OF CANCER GENES: ONCOGENES AND TUMORSUPPRESSOR GENES There are two major types of cancer genes. The first type comprises genes that positively influence tumor formation and are known as oncogenes. The second type of cancer genes negatively impact tumor growth and have been named tumor-suppressor genes. Both oncogenes and tumor-suppressor genes exert their effects on tumor growth Abbreviations: AML, acute myeloid leukemia; CML, chronic myeloid leukemia.

1	through their ability to control cell division (cell birth) or cell death (apoptosis), although the mechanisms can be extremely complex. While tightly regulated in normal cells, oncogenes acquire mutations in cancer cells, and the mutations typically relieve this control and lead to increased activity of the gene products. This mutational event typically occurs in a single allele of the oncogene and acts in a dominant fashion. In contrast, the normal function of tumor-suppressor genes is usually to restrain cell growth, and this function is lost in cancer. Because of the diploid nature of mammalian cells, both alleles must be inactivated for a cell to completely lose the function of a tumor-suppressor gene, leading to a recessive mechanism at the cellular level. From these ideas and studies on the inherited form of retinoblastoma, Knudson and others formulated the two-hit hypothesis, which in its modern version states that both copies of a tumor-suppressor gene must be inactivated in

1	on the inherited form of retinoblastoma, Knudson and others formulated the two-hit hypothesis, which in its modern version states that both copies of a tumor-suppressor gene must be inactivated in cancer.

1	There is a subset of tumor-suppressor genes, the caretaker genes, that do not affect cell growth directly, but rather control the ability of the cell to maintain the integrity of its genome. Cells with a deficiency in these genes have an increased rate of mutations throughout their genomes, including in oncogenes and tumor-suppressor genes. This “mutator” phenotype was first hypothesized by Loeb to explain how the multiple mutational events required for tumorigenesis can occur in the lifetime of an individual. A mutator phenotype has now been observed in some forms of cancer, such as those associated with deficiencies in DNA mismatch repair. The great majority of cancers, however, do not harbor repair deficiencies, and their rate of mutation is similar to that observed in normal cells. Many of these cancers, however, appear to harbor a different kind of genetic instability, affecting the loss or gains of whole chromosomes or large parts thereof (as explained in more detail below).

1	Work by Peyton Rous in the early 1900s revealed that a chicken sarcoma could be transmitted from animal to animal in cell-free extracts, suggesting that cancer could be induced by an agent acting positively to promote tumor formation. The agent responsible for the transmission of the cancer was a retrovirus (Rous sarcoma virus, RSV) and the oncogene responsible was identified 75 years later as v-src. Other oncogenes were also discovered through their presence in the genomes of retroviruses that are capable of causing cancers in chickens, mice, and rats. The cellular homologues of these viral genes are called protooncogenes and are often targets of mutation or aberrant regulation in human cancer. Whereas many oncogenes were discovered because of their presence in retroviruses, other oncogenes, particularly those involved in translocations characteristic of particular leukemias and lymphomas, were isolated through genomic approaches. Investigators cloned the sequences surrounding the

1	particularly those involved in translocations characteristic of particular leukemias and lymphomas, were isolated through genomic approaches. Investigators cloned the sequences surrounding the chromosomal translocations observed cytogenetically and then deduced the nature of the genes that were the targets of these translocations (see below). Some of these were oncogenes known from retroviruses (like ABL, involved in chronic myeloid leukemia [CML]), whereas others were new (like BCL2, involved in B cell lymphoma). In the normal cellular environment, protooncogenes have crucial roles in cell proliferation and differentiation. Table 101e-1 is a partial list of oncogenes known to be involved in human cancer.

1	The normal growth and differentiation of cells is controlled by growth factors that bind to receptors on the surface of the cell. The signals generated by the membrane receptors are transmitted inside the cells through signaling cascades involving kinases, G proteins, and other regulatory proteins. Ultimately, these signals affect the activity of transcription factors in the nucleus, which regulate the expression of genes crucial in cell proliferation, cell differentiation, and cell death. Oncogene products have been found to function at critical steps in these pathways (Chap. 102e), and inappropriate activation of these pathways can lead to tumorigenesis.

1	Point mutation is a common mechanism of oncogene activation. For example, mutations in one of the RAS genes (HRAS, KRAS, or NRAS) are present in up to 85% of pancreatic cancers and 45% of colon cancers but are less common in other cancer types, although they can occur at significant frequencies in leukemia, lung, and thyroid cancers. Remarkably—and in contrast to the diversity of mutations found in tumor-suppressor genes (see below)—most of the activated RAS genes contain point mutations in codons 12, 13, or 61 (these mutations reduce RAS GTPase activity, leading to constitutive activation of the mutant RAS protein). The restricted pattern of mutations observed in oncogenes compared to that of tumor-suppressor genes reflects the fact that gain-of-function mutations are less likely to occur than mutations that simply lead to loss of activity. Indeed, inactivation of a gene can in theory be accomplished through the introduction of a stop codon anywhere in the coding sequence, whereas

1	than mutations that simply lead to loss of activity. Indeed, inactivation of a gene can in theory be accomplished through the introduction of a stop codon anywhere in the coding sequence, whereas activations require precise substitutions at residues that can somehow lead to an increase in the activity of the encoded protein. Importantly, the specificity of oncogene mutations provides diagnostic opportunities, as tests that identify mutations at defined positions are easier to design than tests aimed at detecting random changes in a gene.

1	The second mechanism for activation of oncogenes is DNA sequence amplification, leading to overexpression of the gene product. This increase in DNA copy number may cause cytologically recognizable chromosome alterations referred to as homogeneous staining regions (HSRs) if integrated within chromosomes, or double minutes (dmins) if extrachromosomal. The recognition of DNA amplification is accomplished through various cytogenetic techniques such as comparative genomic hybridization (CGH) or fluorescence in situ hybridization (FISH), which allow the visualization of chromosomal aberrations using fluorescent dyes. In addition, noncytogenetic, microarray-based approaches are now available for identifying changes in copy number at high resolution. Newer short-tag–based sequencing approaches have been used to evaluate amplifications. When paired with next-generation sequencing, this approach offers the highest degree of resolution and quantification available. With both microarray and

1	have been used to evaluate amplifications. When paired with next-generation sequencing, this approach offers the highest degree of resolution and quantification available. With both microarray and sequencing technologies, the entire genome can be surveyed for gains and losses of DNA sequences, thus pinpointing chromosomal regions likely to contain genes important in the development or progression of cancer.

1	Numerous genes have been reported to be amplified in cancer. Several of these genes, including NMYC and LMYC, were identified through their presence within the amplified DNA sequences of a tumor and had homology to known oncogenes. Because the region amplified often includes hundreds of thousands of base pairs, multiple oncogenes may be amplified in a single amplicon in some cancers (particularly in sarcomas). Indeed, MDM2, GLI, CDK4, and SAS at chromosomal location 12q13-15 have been shown to be simultaneously amplified in several types of sarcomas and other tumors. Amplification of a cellular gene is often a predictor of poor prognosis; for example, ERBB2/HER2 and NMYC are often amplified in aggressive breast cancers and neuroblastoma, respectively.

1	Chromosomal alterations provide important clues to the genetic changes in cancer. The chromosomal alterations in human solid tumors such as carcinomas are heterogeneous and complex and occur as a result of the frequent chromosomal instability (CIN) observed in these tumors (see below). In contrast, the chromosome alterations in myeloid and lymphoid tumors are often simple translocations, i.e., reciprocal transfers of chromosome arms from one chromosome to another. Consequently, many detailed and informative chromosome analyses have been performed on hematopoietic cancers. The breakpoints of recurring chromosome abnormalities usually occur at the site of cellular oncogenes. Table 101e-2 lists representative examples of recurring chromosome alterations in malignancy and the associated gene(s) rearranged or deregulated by the chromosomal rearrangement. Translocations are particularly common in lymphoid tumors, probably because these cell types have the capability to rearrange their DNA

1	rearranged or deregulated by the chromosomal rearrangement. Translocations are particularly common in lymphoid tumors, probably because these cell types have the capability to rearrange their DNA to generate antigen receptors. Indeed, antigen receptor genes are commonly involved in the translocations, implying that an imperfect regulation of receptor gene rearrangement may be involved in the pathogenesis. An interesting example is Burkitt’s lymphoma, a B cell tumor characterized by a reciprocal translocation between chromosomes 8 and 14. Molecular analysis of Burkitt’s lymphomas demonstrated that the breakpoints occurred within or near the MYC locus on chromosome 8 and within the immunoglobulin heavy chain locus on chromosome 14, resulting in the transcriptional activation of MYC. Enhancer activation by translocation, although not universal, appears to play an important role in malignant progression. In addition to transcription factors and signal transduction molecules, translocation

1	activation by translocation, although not universal, appears to play an important role in malignant progression. In addition to transcription factors and signal transduction molecules, translocation may result in the overexpression of cell cycle

1	Source: From R Hesketh: The Oncogene and Tumour Suppressor Gene Facts Book, 2nd ed. San Diego, Academic Press, 1997; with permission. regulatory proteins or proteins such as cyclins and of proteins that regulate cell death.

1	The first reproducible chromosome abnormality detected in human malignancy was the Philadelphia chromosome detected in CML. This cytogenetic abnormality is generated by reciprocal translocation involving the ABL oncogene on chromosome 9, encoding a tyrosine kinase, being placed in proximity to the BCR (breakpoint cluster region) gene on chromosome 22. Figure 101e-3 illustrates the generation of the translocation and its protein product. The consequence of expression of the BCR-ABL gene product is the activation of signal transduction pathways leading to cell growth independent of normal external signals. Imatinib (marketed as Gleevec), a drug that specifically blocks the activity of Abl tyrosine kinase, has shown remarkable efficacy with little toxicity in patients with CML. It is hoped that knowledge of genetic alterations in other cancers will likewise lead to mechanism-based design and development of a new generation of chemotherapeutic agents.

1	Solid tumors are generally highly aneuploid, containing an abnormal number of chromosomes; these chromosomes also exhibit structural alterations such as translocations, deletions, and amplifications. These abnormalities are collectively referred to as chromosomal instability (CIN). Normal cells possess several cell cycle checkpoints, essentially quality-control requirements that have to be met before subsequent events are allowed to take place. The mitotic checkpoint, which ensures proper chromosome attachment to the mitotic spindle before allowing the sister chromatids to separate, is altered in certain cancers. The molecular basis of CIN remains unclear, although a number of mitotic checkpoint genes are found mutated or abnormally expressed in various tumors. The exact effects of these changes on the mitotic checkpoint are unknown, and both weakening and overactivation of the checkpoint have been proposed. The identification of the cause of CIN

1	FIGURE 101e-3 Specific translocation seen in chronic myeloid leukemia (CML). The Philadelphia chromosome (Ph) is derived from a reciprocal translocation between chromosomes 9 and 22 with the breakpoint joining the sequences of the ABL oncogene with the BCR gene. The fusion of these DNA sequences allows the generation of an entirely novel fusion protein with modified function. in tumors will likely be a formidable task, considering that several hundred genes are thought to control the mitotic checkpoint and other cellular processes ensuring proper chromosome segregation. Regardless of the mechanisms underlying CIN, the measurement of the number of chromosomal alterations present in tumors is now possible with both cytogenetic and molecular techniques, and several studies have shown that this information can be useful for prognostic purposes. In addition, because the mitotic checkpoint is essential for cellular viability, it may become a target for novel therapeutic approaches.

1	The first indication of the existence of tumor-suppressor genes came from experiments showing that fusion of mouse cancer cells with normal mouse fibroblasts led to a nonmalignant phenotype in the fused cells. The normal role of tumor-suppressor genes is to restrain cell growth, and the function of these genes is inactivated in cancer. The two major types of somatic lesions observed in tumor-suppressor genes during tumor development are point mutations and large deletions. Point mutations in the coding region of tumor-suppressor genes will frequently lead to truncated protein products or otherwise nonfunctional proteins. Similarly, deletions lead to the loss of a functional product and sometimes encompass the entire gene or even the entire chromosome arm, leading to loss of heterozygosity (LOH) in the tumor DNA compared to the corresponding normal tissue DNA (Fig. 101e-4). LOH in tumor DNA is considered a hallmark for the presence of a tumor-suppressor gene at a particular chromosomal

1	(LOH) in the tumor DNA compared to the corresponding normal tissue DNA (Fig. 101e-4). LOH in tumor DNA is considered a hallmark for the presence of a tumor-suppressor gene at a particular chromosomal location, and LOH studies have been useful in the positional cloning of many tumor-suppressor genes.

1	Gene silencing, an epigenetic change that leads to the loss of gene expression and occurs in conjunction with hypermethylation of the promoter and histone deacetylation, is another mechanism of tumor-suppressor gene inactivation. (An epigenetic modification refers to a change in the genome, heritable by cell progeny, that does not involve a change in the DNA sequence. The inactivation of the second X chromosome in female cells is an example of an epigenetic silencing that prevents gene expression from the inactivated chromosome.) During embryologic development, regions of chromosomes from one parent are silenced and gene expression proceeds from the chromosome of the other parent. For most genes, expression occurs from both alleles or randomly from one allele or the other. The preferential expression of a particular gene exclusively from the allele contributed by one parent is called parental imprinting and is thought to be regulated by covalent modifications of chromatin protein and

1	expression of a particular gene exclusively from the allele contributed by one parent is called parental imprinting and is thought to be regulated by covalent modifications of chromatin protein and DNA (often methylation) of the silenced allele.

1	The role of epigenetic control mechanisms in the development of human cancer is unclear. However, a general decrease in the level of DNA methylation has been noted as a common change in cancer. In addition, numerous genes, including some tumor-suppressor genes, appear to become hypermethylated and silenced during tumorigenesis. VHL and p16INK4 are well-studied examples of such tumor-suppressor genes. Overall, epigenetic mechanisms may be responsible for reprogramming the expression of a large number of genes in cancer and, together with the mutation of specific genes, are likely to be crucial in the development of human malignancies. The use of drugs that can reverse epigenetic changes in cancer cells may represent a novel therapeutic option in certain cancers or premalignant conditions. For example, demethylating agents (azacitidine or decitabine) are now approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with high-risk myelodysplastic syndrome

1	For example, demethylating agents (azacitidine or decitabine) are now approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with high-risk myelodysplastic syndrome (MDS).

1	A small fraction of cancers occur in patients with a genetic predisposition. In these families, the affected individuals have a predisposing loss-of-function mutation in one allele of a tumor-suppressor gene. The tumors in these patients show a loss of the remaining normal allele as a result of somatic events (point mutations or deletions), in agreement with the two-hit hypothesis (Fig. 101e-4). Thus, most cells of an individual with an inherited loss-of-function mutation in a tumor-suppressor gene are functionally normal, and only the rare cells that develop a mutation in the remaining normal allele will exhibit uncontrolled regulation.

1	Roughly 100 syndromes of familial cancer have been reported, although many are rare. The majority are inherited as autosomal dominant traits, although some of those associated with DNA repair abnormalities (xeroderma pigmentosum, Fanconi’s anemia, ataxia telangiectasia) are autosomal recessive. Table 101e-3 shows a number of cancer predisposition syndromes and the responsible genes. The current paradigm states that the genes mutated in familial syndromes can also be targets for somatic mutations in sporadic (noninherited) tumors. The study of cancer syndromes has thus provided invaluable insights into the mechanisms of progression for many tumor types. This section examines the case of inherited colon cancer in detail, but A1 Loss of normal chr 13 A3 B1 B3 Markers:

1	FIGURE 101e-4 Diagram of possible mechanisms for tumor formation in an individual with hereditary (familial) retinoblastoma. On the left is shown the pedigree of an affected individual who has inherited the abnormal (Rb) allele from her affected mother. The normal allele is shown as a (+). The four chromosomes of her two parents are drawn to indicate their origin. Flanking the retinoblastoma locus are microsatellite markers (A and B) also analyzed in this family. Markers A3 and B3 are on the chromosome carrying the retinoblastoma disease gene. Tumor formation results when the normal allele, which this patient inherited from her father, is inactivated. On the right are shown four possible ways in which this could occur. In each case, the resulting chromosome 13 arrangement is shown, as well as the results of PCR typing using the microsatellite markers comparing normal tissue (N) with tumor tissue (T). Note that in the first three situations, the normal allele (B1) has been lost in the

1	as the results of PCR typing using the microsatellite markers comparing normal tissue (N) with tumor tissue (T). Note that in the first three situations, the normal allele (B1) has been lost in the tumor tissue, which is referred to as loss of heterozygosity (LOH) at this locus.

1	similar lessons can be applied to many of the cancer syndromes listed in Table 101e-3. In particular, the study of inherited colon cancer will clearly illustrate the difference between two types of tumor-suppressor genes: the gatekeepers, which directly regulate the growth of tumors, and the caretakers, which, when mutated, lead to genetic instability and therefore act indirectly on tumor growth.

1	Familial adenomatous polyposis (FAP) is a dominantly inherited colon cancer syndrome due to germline mutations in the adenomatous polyposis coli (APC) tumor-suppressor gene on chromosome 5. Patients with this syndrome develop hundreds to thousands of adenomas in the colon. Each of these adenomas has lost the normal remaining allele of APC but has not yet accumulated the required additional mutations to generate fully malignant cells (Fig. 101e-2). The loss of the second functional APC allele in tumors from FAP families often occurs through loss of heterozygosity. However, out of these thousands of benign adenomas, several will invariably acquire further abnormalities and a subset will even develop into fully malignant cancers. APC is thus considered to be a gatekeeper for colon tumorigenesis: in the absence of mutation of this gatekeeper (or a gene acting within the same pathway), a colorectal tumor simply cannot form. Figure 101e-5 shows germline and somatic mutations found in the

1	in the absence of mutation of this gatekeeper (or a gene acting within the same pathway), a colorectal tumor simply cannot form. Figure 101e-5 shows germline and somatic mutations found in the APC gene. The function of the APC protein is still not completely understood, but it likely provides differentiation and apoptotic cues to colonic cells as they migrate up the crypts. Defects in this process may lead to abnormal accumulation of cells that should normally undergo apoptosis.

1	In contrast to patients with FAP, patients with hereditary nonpolyposis colon cancer (HNPCC, or Lynch’s syndrome) do not develop multiple polyposis, but instead develop only one or a small number of adenomas that rapidly progress to cancer. Most HNPCC cases are due to mutations in one of four DNA mismatch repair genes (Table 101e-3), which are components of a repair system that is normally responsible for correcting errors in freshly replicated DNA. Germline mutations in MSH2 and MLH1 account for more than 90% of HNPCC cases, whereas mutations in MSH6 and PMS2 are much less frequent. When a somatic mutation inactivates the remaining wild-type allele of a mismatch repair gene, the cell develops a hypermutable phenotype characterized by profound genomic instability, especially for the short repeated sequences called microsatellites. This microsatellite instability (MSI) favors the development of cancer by increasing the rate of mutations in many genes, including oncogenes and

1	the short repeated sequences called microsatellites. This microsatellite instability (MSI) favors the development of cancer by increasing the rate of mutations in many genes, including oncogenes and tumor-suppressor genes (Fig. 101e-2). These genes can thus be considered caretakers. Interestingly, CIN can also be found in colon cancer, but MSI and CIN appear to be mutually exclusive, suggesting that they represent alternative mechanisms for the generation of a mutator phenotype in this cancer (Fig. 101e-2). Other cancer types rarely exhibit MSI, but most exhibit CIN.

1	Although most autosomal dominant inherited cancer syndromes are due to mutations in tumor-suppressor genes (Table 101e-3), there are a few interesting exceptions. Multiple endocrine neoplasia type 2, a dominant disorder characterized by pituitary adenomas, medullary carcinoma of the thyroid, and (in some pedigrees) pheochromocytoma, is due to gain-of-function mutations in the protooncogene RET on chromosome 10. Similarly, gain-of-function mutations in the tyrosine kinase domain of the MET oncogene lead to hereditary papillary renal carcinoma. Interestingly, loss-of-function mutations in the RET gene cause a completely different disease, Hirschsprung’s disease (aganglionic megacolon [Chaps. 353 and 408]). Tuberous sclerosis TSC1 9q34 AD Angiofibroma, renal angiomyolipoma TSC2 16p13.3 von Hippel–Lindau VHL 3p25-26 AD Kidney, cerebellum, pheochromocytoma Abbreviations: AD, autosomal dominant; AR, autosomal recessive

1	Although the Mendelian forms of cancer have taught us much addition, the decision to test should depend on whether effective inter-about the mechanisms of growth control, most forms of cancer do not ventions exist for the particular type of cancer to be tested. Despite follow simple patterns of inheritance. In many instances (e.g., lung these caveats, genetic cancer testing for some cancer syndromes cancer), a strong environmental contribution is at work. Even in such already appears to have greater benefits than risks. Companies offer circumstances, however, some individuals may be more genetically genetic testing for many of the cancer syndromes listed in Table 83-3, susceptible to developing cancer, given the appropriate exposure, due including FAP (APC gene), hereditary breast and ovarian cancer to the presence of modifier alleles. syndrome (BRCA1 and BRCA2 genes), Lynch’s syndrome (mismatch repair genes), Li-Fraumeni syndrome (TP53 gene), Cowden syndrome (PTEN gene), hereditary

1	cancer to the presence of modifier alleles. syndrome (BRCA1 and BRCA2 genes), Lynch’s syndrome (mismatch repair genes), Li-Fraumeni syndrome (TP53 gene), Cowden syndrome (PTEN gene), hereditary retinoblastoma (RB1 gene), and others.

1	Because of the inherent problems of genetic testing such as cost, The discovery of cancer susceptibility genes raises the possibility of specificity, and sensitivity, it is not yet appropriate to offer these tests to DNA testing to predict the risk of cancer in individuals of affected fam-the general population. However, testing may be appropriate in some ilies. An algorithm for cancer risk assessment and decision making in subpopulations with a known increased risk, even without a defined high-risk families using genetic testing is shown in Fig. 101e-6. Once family history. For example, two mutations in the breast cancer susa mutation is discovered in a family, subsequent testing of asymptom-ceptibility gene BRCA1, 185delAG and 5382insC, exhibit a sufficiently atic family members can be crucial in patient management. A nega-high frequency in the Ashkenazi Jewish population that genetic testing tive gene test in these individuals can prevent years of anxiety in the of an individual of

1	in patient management. A nega-high frequency in the Ashkenazi Jewish population that genetic testing tive gene test in these individuals can prevent years of anxiety in the of an individual of this ethnic group may be warranted. knowledge that their cancer risk is no higher than that of the general As noted above, it is important that genetic test results be com-population. On the other hand, a positive test may lead to alteration of municated to families by trained genetic counselors, especially for clinical management, such as increased frequency of cancer screening high-risk high-penetrance conditions such as the hereditary breast and and, when feasible and appropriate, prophylactic surgery. Potential ovarian cancer syndrome (BRCA1/BRCA2). To ensure that the families negative consequences of a positive test result include psychologi-clearly understand its advantages and disadvantages and the impact it cal distress (anxiety, depression) and discrimination, although the may have on

1	of a positive test result include psychologi-clearly understand its advantages and disadvantages and the impact it cal distress (anxiety, depression) and discrimination, although the may have on disease management and psyche, genetic testing should Genetic Information Nondiscrimination Act (GINA) makes it illegal never be done before counseling. Significant expertise is needed to for predictive genetic information to be used to discriminate in health communicate the results of genetic testing to families. For example, insurance or employment. Testing should therefore not be conducted one common mistake is to misinterpret the result of negative genetic without counseling before and after disclosure of the test result. In tests. For many cancer predisposition genes, the sensitivity of genetic

1	Number of mutations oncogene, leading to its downregula-101e-7 tion in leukemic cells and apoptosis. As another example of miRNAs’ involvement in oncogenic pathways, the p53 tumor suppressor can transcriptionally induce miR-34 following genotoxic stress, and this induction is important in mediating p53 function. The expression of miRNAs is extremely specific, and there is evi- Number of mutations lineage and differentiation state, as well as cancer diagnosis and outcome prediction. Certain human malignancies are asso ciated with viruses. Examples include Burkitt’s lymphoma (Epstein-Barr virus; Chap. 218), hepatocellular car-Amino acid number cinoma (hepatitis viruses), cervical

1	FIGURE 101e-5 Germline and somatic mutations in the tumor-suppressor gene APC. APC cancer (human papillomavirus [HPV]; encodes a 2843-amino-acid protein with six major domains: an oligomerization region (O), armadillo Chap. 222), and T cell leukemia (retrepeats (ARM), 15-amino-acid repeats (15 aa), 20-amino-acid repeats (20 aa), a basic region, and a roviruses; Chap. 225e). The mechadomain involved in binding EB1 and the Drosophila discs large homologue (E/D). Shown are the nisms of action of these viruses are positions within the APC gene of a total of 650 somatic and 826 germline mutations (from the APC varied but always involve activation of database at http://www.umd.be/APC). The vast majority of these mutations result in the truncation of growth-promoting pathways or inhithe APC protein. Germline mutations are found to be relatively evenly distributed up to codon 1600 bition of tumor-suppressor products except for two mutation hotspots at amino acids 1061 and 1309, which together

1	Germline mutations are found to be relatively evenly distributed up to codon 1600 bition of tumor-suppressor products except for two mutation hotspots at amino acids 1061 and 1309, which together account for one-in the infected cells. For example, third of the mutations found in familial adenomatous polyposis (FAP) families. Somatic APC mutations HPV proteins E6 and E7 bind and in colon tumors cluster in an area of the gene known as the mutation cluster region (MCR). The loca-inactivate cellular tumor suppressors tion of the MCR suggests that the 20-amino-acid domain plays a crucial role in tumor suppression. p53 and pRB, respectively. There are testing is less than 70% (i.e., of 100 kindreds tested, disease-causing mutations can be identified in 70 at most). Therefore, such testing should in general begin with an affected member of the kindred (the youngest family member still alive who has had the cancer of interest). If a mutation is not identified in this individual, then the test

1	in general begin with an affected member of the kindred (the youngest family member still alive who has had the cancer of interest). If a mutation is not identified in this individual, then the test should be reported as noninformative (Fig. 101e-6) rather than negative (because it is possible that, for technical reasons, the mutation in this individual is not detectable by standard genetic assays). On the other hand, if a mutation can be identified in this individual, then testing of other family members can be performed, and the sensitivity of such subsequent tests will be 100% (because the mutation in the family is in this case known to be detectable by the method used).

1	MicroRNAs (miRNAs) are small noncoding RNAs 20–22 nucleotides in length that are involved in posttranscriptional gene regulation. Studies in chronic lymphocytic leukemia first suggested a link between miRNAs and cancer when miR-15 and miR-16 were found to be deleted or downregulated in the vast majority of tumors. Various miRNAs have since been found abnormally expressed in several human malignancies. Aberrant expression of miRNAs in cancer has been attributed to several mechanisms, such as chromosomal rearrangements, genomic copy number change, epigenetic modifications, defects in miRNA biogenesis pathway, and regulation by transcriptional factors. Somatic mutations of miRNAs have been identified in many cancers, but the exact functional consequences of these changes on cancer development remain to be determined. The SomaMir database (http://compbio.uthsc.edu/SomamiR) catalogs somatic and germ-line miRNA mutations that have been identified in cancer.

1	Functionally, miRNAs have been suggested to contribute to tumorigenesis through their ability to regulate oncogenic signaling pathways. For example, miR-15 and miR-16 have been shown to target the BCL2 several HPV types, and some of these types have been associated with the development of several malignancies, including cervical, vulvar, vaginal, penile, anal, and oropharyngeal cancer. Viruses are not sufficient for cancer development, but constitute one alteration in the multistep process of cancer progression.

1	The tumorigenesis process, driven by alterations in tumor suppressors, oncogenes, and epigenetic regulation, is accompanied by changes in gene expression. The advent of powerful techniques for high-throughput gene expression profiling, based on sequencing or microarrays, has allowed the comprehensive study of gene expression in neoplastic cells. It is indeed possible to identify the expression levels of thousands of genes expressed in normal and cancer tissues. Figure 101e-7 shows a typical microarray experiment examining gene expression in cancer. This global knowledge of gene expression allows the identification of differentially expressed genes and, in principle, the understanding of the complex molecular circuitry regulating normal and neoplastic behaviors. Such studies have led to molecular profiling of tumors, which has suggested general methods for distinguishing tumors of various biologic behaviors (molecular classification), elucidating pathways relevant to the development of

1	profiling of tumors, which has suggested general methods for distinguishing tumors of various biologic behaviors (molecular classification), elucidating pathways relevant to the development of tumors, and identifying molecular targets for the detection and therapy of cancer. The first practical applications of this technology have suggested that global gene expression profiling can provide prognostic information not evident from other clinical or laboratory tests. The Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) is a searchable online repository for expression profiling data.

1	With the completion of the Human Genome Project and advances in sequencing technologies, systematic mutational analysis of the cancer genome has become possible. In fact, whole genome sequencing of cancer cells is now possible, and this technology has the potential to Patients (1) from family with a known cancer syndrome, (2) from a family with a history of cancer, (3) with early onset cancer

1	Patients (1) from family with a known cancer syndrome, (2) from a family with a history of cancer, (3) with early onset cancer End of testing, noninformative Informed consent Testing of cancer patient Identification of disease-causing mutation Screening of asymptomatic family members Failure to identify mutations Review of family history to confirm/identify possible cancer syndromes and candidate genes Pretest counseling Positive test: family member requires increased screening or other interventions Negative test: family member has no increased risk of cancer FIGURE 101e-6 Algorithm for genetic testing in a family with can-cer predisposition. The key step is the identification of a mutation in a cancer patient, which allows testing of asymptomatic family mem-bers. Asymptomatic family members who test positive may require increased screening or surgery, whereas others are at no greater risk for cancer than the general population.

1	revolutionize our approach to cancer prevention, diagnosis, and treatment. The International Cancer Genome Consortium (http://icgc. org/) was developed by leading cancer agencies worldwide, genome and cancer scientists, and statisticians with the goal to launch and coordinate cancer genomics research projects worldwide and to disseminate the data. Hundreds of cancer genomes from at least 25 cancer types have been sequenced through various collaborative efforts. In addition, exome sequencing (sequencing all the coding regions of the genome) has also been performed on a large number of tumors. These sequencing data have been used to elucidate the mutational profile of cancer, including the identification of driver mutations that are functionally involved in tumor development. There are generally 40 to 100 genetic alterations that affect protein sequence in a typical cancer, although statistical analyses suggest that only 8–15 are functionally involved in tumorigenesis. The picture that

1	40 to 100 genetic alterations that affect protein sequence in a typical cancer, although statistical analyses suggest that only 8–15 are functionally involved in tumorigenesis. The picture that emerges from these studies is that most genes found mutated in tumors are actually mutated at relatively low frequencies (<5%), whereas a small number of genes (such as p53, KRAS) are mutated in a large proportion of tumors (Fig. 101e-8). In the past, the focus of research has been on the frequently mutated genes, but it appears that the large number of genes that are infrequently mutated in cancer are major contributors to the cancer phenotype. Understanding the signaling pathways altered by mutations in these genes, as well as the functional relevance of these different mutations, represents the next challenge in the field. Moreover, a detailed knowledge of the genes altered in a particular tumor may allow for a new era of personalized treatment in cancer medicine (see below). A major effort

1	next challenge in the field. Moreover, a detailed knowledge of the genes altered in a particular tumor may allow for a new era of personalized treatment in cancer medicine (see below). A major effort in the United States, The Cancer Genome Atlas (http://cancergenome.nih.gov) is a coordinated effort from the National Cancer Institute and the National Human Genome

1	Prepare cDNA, label FIGURE 101e-7 A microarray experiment. RNA is prepared from cells, reverse transcribed to cDNA, and labeled with fluorescent dyes (typically green for normal cells and red for cancer cells). The fluorescent probes are mixed and hybridized to a cDNA array. Each spot on the array is an oligonucleotide (or cDNA fragment) that represents a different gene. The image is then captured with a fluorescence camera; red spots indicate higher expression in tumor cells compared with reference, while green spots represent the lower expression in tumor cells. Yellow signals indicate equal expression levels in normal and tumor specimens. After clustering analysis of multiple arrays, the results are typically represented graphically using a visualization software, which shows, for each sample, a color-coded representation of gene expression for every gene on the array.

1	Research Institute to systematically characterize the entire spectrum of genomic changes involved in human cancers. Similarly, COSMIC (Catalogue of Somatic Mutations in Cancer) is an initiative from the Welcome Trust Sanger Institute to store and display somatic mutation information and related details regarding human cancers (http:// cancer.sanger.ac.uk/). PERSONALIZED CANCER TREATMENT BASED ON MOLECULAR PROFILES: PRECISION THERAPY

1	Gene expression profiling and genomewide sequencing approaches have allowed for an unprecedented understanding of cancer at the molecular level. It has been suggested that individualized knowledge of pathways or genes deregulated in a given tumor (personalized genomics) may provide a guide for therapeutic options on the tumor, thus leading to personalized therapy (also called precision medicine). Because tumor behavior is highly heterogeneous, even within a tumor type, personalized information-based medicine will likely supplement or perhaps one day supplant the current histology-based therapy, especially in the case of tumors resistant to conventional therapeutic approaches. Molecular nosology has revealed similarities in tumors of diverse histotype. The success of this approach will be dependent on the identification of sufficient actionable changes (mutations or pathways that can be targeted with a specific drug). Examples of currently actionable changes include mutations in BRAF

1	dependent on the identification of sufficient actionable changes (mutations or pathways that can be targeted with a specific drug). Examples of currently actionable changes include mutations in BRAF (targeted by the drug vemurafenib) and RET (targeted by sunitinib and sorafenib), and ALK rearrangements (targeted by crizotinib). Interestingly, studies have reported that 20% of triple-negative breast cancers and 60% of lung cancers have potentially actionable genetic changes. Gene expression also offers the potential to predict drug sensitivities as well as provide prognostic information. Commercial diagnostic tests, such as Mammaprint and Oncotype DX for breast cancer, are available

1	FIGURE 101e-8 A two-dimensional maps of genes mutated in colorectal cancer. The two-dimensional landscape represents the positions of the RefSeq genes along the chromosomes and the height of the peaks represents the mutation frequency. On the top map, the taller peaks represent the genes that are commonly mutated in colon cancer, while the large number of smaller hills indicates the genes that are mutated at lower frequency. On the lower map, the mutations of two individual tumors are indicated. Note that there is little overlap between the mutated genes of the two colorectal tumors shown. These differences may represent the basis for the heterogeneity in terms of behavior and responsiveness to therapy observed in human cancer. (From LD Wood et al: Science 318:1108, 2007, with permission.) to help the patients and their physicians make treatment decisions. 101e-9 Personalized medicine is an exciting new avenue for cancer treatment based on matching the unique features of a tumor to an

1	to help the patients and their physicians make treatment decisions. 101e-9 Personalized medicine is an exciting new avenue for cancer treatment based on matching the unique features of a tumor to an effective therapy, and this concept is in the process of changing our approach to cancer therapy in fundamental ways. On a cautionary note, gene expression can vary enormously within a single person’s cancer and at different anatomic sites in the patient. We have not yet determined whether such clonal variation within an individual tumor will interfere with the goal tailoring therapy to a particular patient’s tumor.

1	A revolution in cancer genetics has occurred in the past 25 years. Identification of cancer genes has led to a deep understanding of the tumorigenesis process and has had important repercussions on all fields of cancer biology. In particular, the advancement of powerful techniques for genomewide expression profiling and mutation analyses has provided a detailed picture of the molecular defects present in individual tumors. Individualized treatment based on the specific genetic alterations within a given tumor has already become possible. Although these advances have not yet translated into overall changes in cancer prevention, prognosis, or treatment, it is expected that breakthroughs in these areas will continue to emerge and be applicable to an ever-increasing number of cancers. phenotypiC CharaCteristiCs of MaLignant CeLLs

1	phenotypiC CharaCteristiCs of MaLignant CeLLs Deregulated cell proliferation: Loss of function of negative growth regulators (tumor-suppressor genes, i.e., Rb, p53), and increased action of positive 102e Jeffrey W. Clark, Dan L. Longo growth regulators (oncogenes, i.e., Ras, Myc). Leads to aberrant cell cycle con- Cancers are characterized by unregulated cell division, avoidance of cell death, tissue invasion, and the ability to metastasize. A neoplasm is benign when it grows in an unregulated fashion without tissue invasion. The presence of unregulated growth and tissue invasion is characteristic of malignant neoplasms. Cancers are named based on their origin: those derived from epithelial tissue are called carcinomas, those derived from mesenchymal tissues are sarcomas, and those derived from hematopoietic tissue are leukemias, lymphomas, and plasma cell dyscrasias (including multiple myeloma).

1	Cancers nearly always arise as a consequence of genetic alterations, the vast majority of which begin in a single cell and therefore are monoclonal in origin. However, because a wide variety of genetic and epigenetic changes can occur in different cells within malignant tumors over time, most cancers are characterized by marked heterogeneity in the populations of cells. This heterogeneity significantly complicates the treatment of most cancers because it is likely that there are subsets of cells that will be resistant to therapy and will therefore survive and proliferate even if the majority of cells are killed.

1	A few cancers appear to, at least initially, be primarily driven by an alteration in a dominant gene that produces uncontrolled cell proliferation. Examples include chronic myeloid leukemia (abl), about half of melanomas (braf ), Burkitt’s lymphoma (c-myc), and subsets of lung adenocarcinomas (egfr, alk, ros1, and ret). The genes that can promote cell growth when altered are often called oncogenes. They were first identified as critical elements of viruses that cause animal tumors; it was subsequently found that the viral genes had normal counterparts with important functions in the cell and had been captured and mutated by viruses as they passed from host to host.

1	However, the vast majority of human cancers are characterized by a multiple-step process involving many genetic abnormalities, each of which contributes to the loss of control of cell proliferation and differentiation and the acquisition of capabilities, such as tissue invasion, the ability to metastasize, and angiogenesis. These properties are not found in the normal adult cell from which the tumor is derived. Indeed, normal cells have a large number of safeguards against uncontrolled proliferation and invasion. Many cancers go through recognizable steps of progressively more abnormal phenotypes: hyperplasia, to adenoma, to dysplasia, to carcinoma in situ, to invasive cancer with the ability to metastasize (Table 102e-1). For most cancers, these changes occur over a prolonged period of time, usually many years.

1	In most organs, only primitive undifferentiated cells are capable of proliferating and the cells lose the capacity to proliferate as they differentiate and acquire functional capability. The expansion of the primitive cells is linked to some functional need in the host through receptors that receive signals from the local environment or through hormonal and other influences delivered by the vascular supply. In the absence of such signals, the cells are at rest. The signals that keep the primitive cells at rest remain incompletely understood. These signals must be environmental, based on the observations that a regenerating liver stops growing when it has replaced the portion that has been surgically removed after partial hepatectomy and regenerating bone marrow stops growing when the peripheral blood counts return to normal. Cancer cells clearly have lost responsiveness to such controls and do not recognize when they have overgrown the niche normally occupied by the organ from which

1	blood counts return to normal. Cancer cells clearly have lost responsiveness to such controls and do not recognize when they have overgrown the niche normally occupied by the organ from which they are derived. A better understanding of the mechanisms of growth regulation is evolving.

1	Normal cells have a number of control mechanisms that are targeted by specific genetic alterations in cancer. Critical proteins in these control processes that are frequently mutated or otherwise inactivated in cancers are called tumor-suppressor genes. Examples include p53 trol and includes loss of normal checkpoint responses. Failure to differentiate: Arrest at a stage before terminal differentiation. May retain stem cell properties. (Frequently observed in leukemias due to transcriptional repression of developmental programs by the gene products of chromosomal translocations.) Loss of normal apoptosis pathways: Inactivation of p53, increases in Bcl-2 family members. This defect enhances the survival of cells with oncogenic mutations and genetic instability and allows clonal expansion and diversification within the tumor without activation of physiologic cell death pathways.

1	Genetic instability: Defects in DNA repair pathways leading to either single-nucleotide or oligonucleotide mutations (as in microsatellite instability, MIN) or more commonly chromosomal instability (CIN) leading to aneuploidy. Caused by loss of function of p53, BRCA1/2, mismatch repair genes, DNA repair enzymes, and the spindle checkpoint. Leads to accumulation of a variety of mutations in different cells within the tumor and heterogeneity. Loss of replicative senescence: Normal cells stop dividing in vitro after 25–50 population doublings. Arrest is mediated by the Rb, p16INK4a, and p53 pathways. Further replication leads to telomere loss, with crisis. Surviving cells often harbor gross chromosomal abnormalities. Relevance to human in vivo cancer remains uncertain. Many human cancers express telomerase.

1	Nonresponsiveness to external growth-inhibiting signals: Cancer cells have lost responsiveness to signals normally present to stop proliferating when they have overgrown the niche normally occupied by the organ from which they are derived. We know very little about this mechanism of growth regulation. Increased angiogenesis: Due to increased gene expression of proangiogenic factors (VEGF, FGF, IL-8) by tumor or stromal cells, or loss of negative regulators (endostatin, tumstatin, thrombospondin). Invasion: Loss of cell-cell contacts (gap junctions, cadherins) and increased production of matrix metalloproteinases (MMPs). Often takes the form of epithelial-to-mesenchymal transition (EMT), with anchored epithelial cells becoming more like motile fibroblasts. Metastasis: Spread of tumor cells to lymph nodes or distant tissue sites. Limited by the ability of tumor cells to survive in a foreign environment.

1	Metastasis: Spread of tumor cells to lymph nodes or distant tissue sites. Limited by the ability of tumor cells to survive in a foreign environment. Evasion of the immune system: Downregulation of MHC class I and II molecules; induction of T cell tolerance; inhibition of normal dendritic cell and/ or T cell function; antigenic loss variants and clonal heterogeneity; increase in regulatory T cells. Shift in cell metabolism: Energy generation shifts to aerobic glycolysis. Abbreviations: FGF, fibroblast growth factor; IL, interleukin; MHC, major histocompatibility complex; VEGF, vascular endothelial growth factor.

1	and Rb (discussed below). The progression of a cell through the cell division cycle is regulated at a number of checkpoints by a wide array of genes. In the first phase, G1, preparations are made to replicate the genetic material. The cell stops before entering the DNA synthesis phase, or S phase, to take inventory. Are we ready to replicate our DNA? Is the DNA repair machinery in place to fix any mutations that are detected? Are the DNA replicating enzymes available? Is there an adequate supply of nucleotides? Is there sufficient energy? The main brake on the process is the retinoblastoma protein, Rb. When the cell determines that it is prepared to move ahead, sequential activation of cyclin-dependent kinases (CDKs) results in the inactivation of the brake, Rb, by phosphorylation. Phosphorylated Rb releases the S phase–regulating transcription factor, E2F/DP1, and genes required for S phase progression are expressed. If the cell determines that it is unready to move ahead with DNA

1	Rb releases the S phase–regulating transcription factor, E2F/DP1, and genes required for S phase progression are expressed. If the cell determines that it is unready to move ahead with DNA replication, a number of inhibitors are capable of blocking the action of the CDKs, including p21Cip2/Waf1, p16Ink4a, and p27Kip1. Nearly every cancer has one or more genetic lesions in the G1 checkpoint that permits progression to S phase.

1	At the end of S phase, when the cell has exactly duplicated its DNA content, a second inventory is taken at the S checkpoint. Have all of the chromosomes been fully duplicated? Were any segments of DNA copied more than once? Do we have the right number of chromosomes and the right amount of DNA? If so, the cell proceeds to G2, in which the cell prepares for division by synthesizing mitotic spindle and other proteins needed to produce two daughter cells. When DNA damage is 1.DNA DAMAGE CHECKPOINT 2. ONCOGENE CHECKPOINT myc, E2F, EIA Induction of P14ARF Transcriptional activation of p53-responsive genes ATM/ATR chk1/chk2 mdm2 P14ARF mdm2 mdm2 P P P P P p53

1	FIGuRE 102e-1 Induction of p53 by the DNA damage and oncogene checkpoints. In response to noxious stimuli, p53 and mdm2 are phosphorylated by the ataxia-telangiectasia mutated (ATM) and related (ATR) serine/threonine kinases, as well as the immediate downstream checkpoint kinases, Chk1 and Chk2. This causes dissociation of p53 from mdm2, leading to increased p53 protein levels and transcription of genes leading to cell cycle arrest (p21Cip1/Waf1) or apoptosis (e.g., the proapoptotic Bcl-2 family members Noxa and Puma). Inducers of p53 include hypoxemia, DNA damage (caused by ultraviolet radiation, gamma irradiation, or chemotherapy), ribonucleotide depletion, and telomere shortening. A second mechanism of p53 induction is activated by oncogenes such as Myc, which promote aberrant G1/S transition. This pathway is regulated by a second product of the Ink4a locus, p14ARF (p19 in mice), which is encoded by an alternative reading frame of the same stretch of DNA that codes for p16Ink4a.

1	This pathway is regulated by a second product of the Ink4a locus, p14ARF (p19 in mice), which is encoded by an alternative reading frame of the same stretch of DNA that codes for p16Ink4a. Levels of ARF are upregulated by Myc and E2F, and ARF binds to mdm2 and rescues p53 from its inhibitory effect. This oncogene checkpoint leads to the death or senescence (an irreversible arrest in G1 of the cell cycle) of renegade cells that attempt to enter S phase without appropriate physiologic signals. Senescent cells have been identified in patients whose premalignant lesions harbor activated oncogenes, for instance, dysplastic nevi that encode an activated form of BRAF (see below), demonstrating that induction of senescence is a protective mechanism that operates in humans to prevent the outgrowth of neoplastic cells.

1	detected, the p53 pathway is normally activated. Called the guardian of the genome, p53 is a transcription factor that is normally present in the cell in very low levels. Its level is generally regulated through its rapid turnover. Normally, p53 is bound to mdm2, a ubiquitin ligase, that both inhibits p53 transcriptional activation and also targets p53 for degradation in the proteasome. When damage is sensed, the ATM (ataxia-telangiectasia mutated) pathway is activated; ATM phosphorylates mdm2, which no longer binds to p53, and p53 then stops cell cycle progression, directs the synthesis of repair enzymes, or if the damage is too great, initiates apoptosis of the cell to prevent the propagation of a damaged cell (Fig. 102e-1).

1	A second method of activating p53 involves the induction of p14ARF by hyperproliferative signals from oncogenes. p14ARF competes with p53 for binding to mdm2, allowing p53 to escape the effects of mdm2 and accumulate in the cell. Then p53 stops cell cycle progression by activating CDK inhibitors such as p21 and/or initiating the apoptosis pathway. Not surprisingly given its critical role in controlling cell cycle progression, mutations in the gene for p53 on chromosome 17p are found in more than 50% of human cancers. Most commonly these mutations are acquired in the malignant tissue in one allele and the second allele is deleted, leaving the cell unprotected from DNA-damaging agents or oncogenes. Some environmental exposures produce signature mutations in p53; for example, aflatoxin exposure leads to mutation of arginine to serine at codon 249 and leads to hepatocellular carcinoma. In rare instances, p53 mutations are in the germline (Li-Fraumeni syndrome) and produce a familial

1	exposure leads to mutation of arginine to serine at codon 249 and leads to hepatocellular carcinoma. In rare instances, p53 mutations are in the germline (Li-Fraumeni syndrome) and produce a familial cancer syndrome.

1	The absence of p53 leads to chromosome instability and the accumulation of DNA damage including the acquisition of properties that give the abnormal cell a proliferative and survival advantage. Like Rb dysfunction, most cancers have mutations that disable the p53 pathway. Indeed, the importance of p53 and Rb in the development of cancer is underscored by the neoplastic transformation mechanism of human papillomavirus. This virus has two main oncogenes, E6 and E7. E6 acts to increase the rapid turnover of p53, and E7 acts to inhibit Rb function; inhibition of these two targets is required for transformation of epithelial cells.

1	Another cell cycle checkpoint exists when the cell is undergoing division, the spindle checkpoint. The details of this checkpoint are still being discovered; however, it appears that if the spindle apparatus does not properly align the chromosomes for division, if the chromosome number is abnormal (i.e., greater or less than 4n), or if the centromeres are not properly paired with their duplicated partners, then the cell initiates a cell death pathway to prevent the production of aneuploid progeny (having an altered number of chromosomes). Abnormalities in the spindle checkpoint facilitate the development of aneuploidy. In some tumors, aneuploidy is a predominant genetic feature. In others, a defect in the cells’ ability to repair errors in the DNA due to mutations in genes coding for the proteins critical for mismatched DNA repair is the primary genetic lesion. This is usually detected by finding alterations in repeat sequences of DNA (called microsatellites) or microsatellite

1	the proteins critical for mismatched DNA repair is the primary genetic lesion. This is usually detected by finding alterations in repeat sequences of DNA (called microsatellites) or microsatellite instability in malignant cells. In general, tumors either have defects in chromosome number or microsatellite instability, but not both. Defects that lead to cancer include abnormal cell cycle checkpoints, inadequate DNA repair, and failure to preserve genome integrity.

1	Efforts are under way to therapeutically restore the defects in cell cycle regulation that characterize cancer, although this remains a challenging problem because it is much more difficult to restore normal biologic function than to inhibit abnormal function of proteins driving cell proliferation, such as oncogenes.

1	The fundamental cellular defects that create a malignant neoplasm act at the cellular level. However, that is not the entire story. Cancers behave as organs that have lost their specialized function and stopped responding to signals that normally limit their growth. Human cancers usually become clinically detectable when a primary mass is at least 1 cm in diameter—such a mass consists of about 109 cells. More commonly, patients present with tumors that are 1010 cells or greater. A lethal tumor burden is about 1012 to 1013 cells. If all tumor cells were dividing at the time of diagnosis, patients would reach a lethal tumor burden in a very short time. However, human tumors grow by Gompertzian kinetics—this means that not every daughter cell produced by a cell division is itself capable of dividing. The growth fraction of a tumor declines exponentially with time. The growth fraction of the first malignant cell is 100%, and by the time a patient presents for medical care, the growth

1	of dividing. The growth fraction of a tumor declines exponentially with time. The growth fraction of the first malignant cell is 100%, and by the time a patient presents for medical care, the growth fraction is 2–3% or less. This fraction is similar to the growth fraction of normal bone marrow and normal intestinal epithelium, the most highly proliferative normal tissues in the human body, a fact that may explain the dose-limiting toxicities of agents that target dividing cells.

1	The implication of these data is that the tumor is slowing its own growth over time. How does it do this? The tumor cells have multiple genetic lesions that tend to promote proliferation, yet by the time the tumor is clinically detectable, its capacity for proliferation has declined. We need to better understand how a tumor slows its own growth. A number of factors are known to contribute to the failure of tumor cells to proliferate in vivo. Some cells are hypoxemic and have inadequate supply of nutrients and energy. Some have sustained too much genetic damage to complete the cell cycle but have lost the capacity to undergo apoptosis and therefore survive but do not proliferate. However, an important subset is not actively dividing but retains the capacity to divide and can start dividing again under certain conditions such as when the tumor mass is reduced by treatments. Just as the bone marrow increases its rate of proliferation in response to bone marrow–damaging agents, the tumor

1	again under certain conditions such as when the tumor mass is reduced by treatments. Just as the bone marrow increases its rate of proliferation in response to bone marrow–damaging agents, the tumor also seems to sense when tumor cell numbers have been reduced and can respond by increasing growth rate. However, the critical difference is that the marrow stops growing when it has reached its production goals, whereas tumors do not.

1	Additional tumor cell vulnerabilities are likely to be detected when we learn more about how normal cells respond to “stop” signals from their environment and why and how tumor cells fail to heed such signals. IS IN VITRO SENESCENCE RELEVANT TO CARCINOGENESIS? When normal cells are placed in culture in vitro, most are not capable of sustained growth. Fibroblasts are an exception to this rule. When they are cultured, fibroblasts may divide 30–50 times and then they undergo what has been termed a “crisis” during which the majority of cells stop dividing (usually due to an increase in p21 expression, a CDK inhibitor), many die, and a small fraction emerge that have acquired genetic changes that permit their uncontrolled growth. The cessation of growth of normal cells in culture has been termed “senescence,” and whether this phenomenon is relevant to any physiologic event in vivo is debated.

1	Among the cellular changes during in vitro propagation is telomere shortening. DNA polymerase is unable to replicate the tips of chromosomes, resulting in the loss of DNA at the specialized ends of chromosomes (called telomeres) with each replication cycle. At birth, human telomeres are 15to 20-kb pairs long and are composed of tandem repeats of a six-nucleotide sequence (TTAGGG) that associates with specialized telomere-binding proteins to form a T-loop structure that protects the ends of chromosomes from being mistakenly recognized as damaged. The loss of telomeric repeats with each cell division cycle causes gradual telomere shortening, leading to growth arrest (called senescence) when one or more critically short telomeres trigger a p53-regulated DNA-damage checkpoint response. Cells can bypass this growth arrest if pRb and p53 are nonfunctional, but cell death usually ensues when the unprotected ends of chromosomes lead to chromosome fusions or other catastrophic DNA

1	Cells can bypass this growth arrest if pRb and p53 are nonfunctional, but cell death usually ensues when the unprotected ends of chromosomes lead to chromosome fusions or other catastrophic DNA rearrangements. The ability to bypass telomere-based growth limitations is thought to be a critical step in the evolution of most malignancies. This occurs by the reactivation of telomerase expression in cancer cells. Telomerase is an enzyme that adds TTAGGG repeats onto the 3′ ends of chromosomes. It contains a catalytic subunit with reverse transcriptase activity (hTERT) and an RNA component that provides the template for telomere extension. Most normal somatic cells do not express sufficient telomerase to prevent telomere attrition with each cell division. Exceptions include stem cells (such as those found in hematopoietic tissues, gut and skin epithelium, and germ cells) that require extensive cell division to maintain tissue homeostasis. More than 90% of human cancers express high levels

1	those found in hematopoietic tissues, gut and skin epithelium, and germ cells) that require extensive cell division to maintain tissue homeostasis. More than 90% of human cancers express high levels of telomerase that prevent telomere shortening to critical levels and allow indefinite cell proliferation. In vitro experiments indicate that inhibition of telomerase activity leads to tumor cell apoptosis. Major efforts are under way to develop methods to inhibit telomerase activity in cancer cells. For example, the protein component of telomerase (hTERT) may act as one of the most widely expressed tumor-associated antigens and be targeted by vaccine approaches.

1	Although most of the functions of telomerase relate to cell division, it also has several other effects including interfering with the differentiated functions of at least certain stem cells, although the impact on differentiated function of normal non-stem cells is less clear. Nevertheless, a major growth industry in medical research has been discovering an association between short telomeres and human diseases ranging from diabetes and coronary artery disease to Alzheimer’s disease. The picture is further complicated by the fact that rare genetic defects in the telomerase enzyme seem to cause pulmonary fibrosis, aplastic anemia, or dyskeratosis congenita (characterized by abnormalities in skin, nails, and oral mucosa with increased risk for certain malignancies) but not defects in nutrient absorption in the gut, a site that might be presumed to be highly sensitive to defective cell proliferation. Much remains to be learned about how telomere shortening and telomere maintenance are

1	absorption in the gut, a site that might be presumed to be highly sensitive to defective cell proliferation. Much remains to be learned about how telomere shortening and telomere maintenance are related to human illness in general and cancer in particular.

1	Signals that affect cell behavior come from adjacent cells, the stroma in which the cells are located, hormonal signals that originate remotely, and from the cells themselves (autocrine signaling). These signals generally exert their influence on the receiving cell through activation of signal transduction pathways that have as their end result the induction of activated transcription factors that mediate a change in cell behavior or function or the acquisition of effector machinery to accomplish a new task. Although signal transduction pathways can lead to a wide variety of outcomes, many such pathways rely on cascades of signals that sequentially activate different proteins or glycoproteins and lipids or glycolipids, and the activation steps often involve the addition or removal of one or more phosphate groups on a downstream target. Other chemical changes can result from signal transduction pathways, but phosphorylation and dephosphorylation play a major role. The proteins that add

1	more phosphate groups on a downstream target. Other chemical changes can result from signal transduction pathways, but phosphorylation and dephosphorylation play a major role. The proteins that add phosphate groups to proteins are called kinases. There are two major distinct classes of kinases; one class acts on tyrosine residues, and the other acts on serine/threonine residues. The tyrosine kinases often play critical roles in signal transduction pathways; they may be receptor tyrosine kinases, or they may be linked to other cell-surface receptors through associated docking proteins (Fig. 102e-2).

1	Normally, tyrosine kinase activity is short-lived and reversed by protein tyrosine phosphatases (PTPs). However, in many human cancers, tyrosine kinases or components of their downstream pathways are activated by mutation, gene amplification, or chromosomal translocations. Because these pathways regulate proliferation, survival, migration, and angiogenesis, they have been identified as important targets for cancer therapeutics.

1	Inhibition of kinase activity is effective in the treatment of a number of neoplasms. Lung cancers with mutations in the epidermal growth factor receptor are highly responsive to erlotinib and gefitinib (Table 102e-2). Lung cancers with activation of anaplastic lymphoma kinase (ALK) or ROS1 by translocations respond to crizotinib, an ALK and ROS1 inhibitor. A BRAF inhibitor is highly effective in melanomas and thyroid cancers in which BRAF is mutated. Targeting a protein (MEK) downstream of BRAF also has activity against BRAF mutant melanomas. Janus kinase inhibitors are active in myeloproliferative syndromes in which JAK2 activation is a pathogenetic event. Imatinib (which targets a number of tyrosine kinases) is an effective agent in tumors that have translocations of the c-Abl and BCR gene (such as chronic myeloid leukemia), mutant c-Kit (gastrointestinal stromal cell tumors), or mutant platelet-derived growth factor receptor (PDGFR; chronic myelomonocytic leukemia);

1	c-Abl and BCR gene (such as chronic myeloid leukemia), mutant c-Kit (gastrointestinal stromal cell tumors), or mutant platelet-derived growth factor receptor (PDGFR; chronic myelomonocytic leukemia); second-generation inhibitors of BCR-Abl, dasatinib, and nilotinib are even more effective. The third-generation agent bosutinib has activity in some patients who have progressed on other inhibitors, whereas the third-generation agent ponatinib has activity against the T315I mutation, which is resistant to the other agents. Sorafenib and sunitinib, agents that inhibit a large number of kinases, have shown antitumor activity in a number of malignancies, including renal cell cancer (RCC) (both), hepatocellular carcinoma (sorafenib), thyroid cancer (sorafenib), gastrointestinal stromal tumor (GIST) (sunitinib), and pancreatic neuroendocrine tumors (sunitinib). Inhibitors of the mammalian target of rapamycin (mTOR) are active in RCC, pancreatic neuroendocrine tumors, and breast cancer. The

1	(sunitinib), and pancreatic neuroendocrine tumors (sunitinib). Inhibitors of the mammalian target of rapamycin (mTOR) are active in RCC, pancreatic neuroendocrine tumors, and breast cancer. The list of active agents and treatment indications is growing rapidly. These new agents have ushered in a new era of personalized therapy. It is becoming more routine for resected tumors to be assessed for specific molecular changes that predict response and to have clinical decision-making guided by those results.

1	However, none of these therapies has yet been curative by themselves for any malignancy, although prolonged periods of disease control lasting many years frequently occur in chronic myeloid leukemia. The reasons for the failure to cure are not completely defined, although resistance to the treatment ultimately develops in most patients. In some tumors, resistance to kinase inhibitors is related to an acquired mutation in the target kinase that inhibits drug binding. Many of these kinase inhibitors act as competitive inhibitors of the ATP-binding pocket. ATP is the phosphate donor in these phosphorylation

1	FIGuRE 102e-2 Therapeutic targeting of signal transduction pathways in cancer cells. Three major signal transduction pathways are activated by receptor tyrosine kinases (RTK). 1. The protooncogene Ras is activated by the Grb2/mSOS guanine nucleotide exchange factor, which induces an association with Raf and activation of downstream kinases (MEK and ERK1/2). 2. Activated PI3K phosphorylates the membrane lipid PIP2 to generate PIP3, which acts as a membrane-docking site for a number of cellular proteins including the serine/threonine kinases PDK1 and Akt. PDK1 has numerous cellular targets, including Akt and mTOR. Akt phosphorylates target proteins that promote resistance to apoptosis and enhance cell cycle progression, whereas mTOR and its target p70S6K upregulate protein synthesis to potentiate cell growth. 3. Activation of PLC-γ leads to the formation of diacylglycerol (DAG) and increased intracellular calcium, with activation of multiple isoforms of PKC and other enzymes regulated

1	cell growth. 3. Activation of PLC-γ leads to the formation of diacylglycerol (DAG) and increased intracellular calcium, with activation of multiple isoforms of PKC and other enzymes regulated by the calcium/calmodulin system. Other important signaling pathways involve non-RTKs that are activated by cytokine or integrin receptors. Janus kinases (JAK) phosphorylate STAT (signal transducer and activator of transcription) transcription factors, which translocate to the nucleus and activate target genes. Integrin receptors mediate cellular interactions with the extracellular matrix (ECM), inducing activation of FAK (focal adhesion kinase) and c-Src, which activate multiple downstream pathways, including modulation of the cell cytoskeleton. Many activated kinases and transcription factors migrate into the nucleus, where they regulate gene transcription, thus completing the path from extracellular signals, such as growth factors, to a change in cell phenotype, such as induction of

1	migrate into the nucleus, where they regulate gene transcription, thus completing the path from extracellular signals, such as growth factors, to a change in cell phenotype, such as induction of differentiation or cell proliferation. The nuclear targets of these processes include transcription factors (e.g., Myc, AP-1, and serum response factor) and the cell cycle machinery (CDKs and cyclins). Inhibitors of many of these pathways have been developed for the treatment of human cancers. Examples of inhibitors that are currently being evaluated in clinical trials are shown in purple type.

1	reactions. Mutation in the BCR-ABL kinase in the ATP-binding pocket (such as the threonine to isoleucine change at codon 315 [T315I]) can prevent imatinib binding. Other resistance mechanisms include altering other signal transduction pathways to bypass the inhibited pathway. As resistance mechanisms become better defined, rational strategies to overcome resistance will emerge. In addition, many kinase inhibitors are less specific for an oncogenic target than was hoped, and toxicities related to off-target inhibition of kinases limit the use of the agent at a dose that would optimally inhibit the cancer-relevant kinase.

1	Targeted agents can also be used to deliver highly toxic compounds. An important component of the technology for developing effective conjugates is the design of the linker between the two, which needs to be stable. Currently approved antibody drug conjugates include brentuximab vedotin, which links the microtubule toxin monomethyl auristatin E (MMAE) to an antibody targeting the cell surface antigen CD30, which is expressed on a number of malignant cells but especially in Hodgkin’s disease and anaplastic lymphoma. The linker in this case is cleavable, which allows diffusion of the drug out of the cell after delivery. The second approved conjugate is ado-trastuzumab emtansine, which links the microtubule formation inhibitor mertansine and the monoclonal antibody trastuzumab targeted against human epidermal growth factor receptor 2 (HER2) on breast cancer cells. In this case, the linker is noncleavable, thus trapping the chemotherapeutic agent within the cells. There are theoretical

1	human epidermal growth factor receptor 2 (HER2) on breast cancer cells. In this case, the linker is noncleavable, thus trapping the chemotherapeutic agent within the cells. There are theoretical pluses and minuses to having either cleavable or noncleavable linkers, and it is likely that both will be used in future developments of antibody-drug conjugates.

1	Another strategy to enhance the antitumor effects of targeted agents is to use them in rational combinations with each other and in empiric combinations with chemotherapy agents that kill cells in ways distinct from targeted agents. Combinations of trastuzumab (a monoclonal antibody that targets the HER2 receptor [member of the epidermal growth factor receptor (EGFR) family]) with chemotherapy have significant activity against breast and stomach cancers that have high levels of expression of the HER2 protein. The activity of trastuzumab

1	Abbreviations: AML, acute myeloid leukemia; CTCL, cutaneous T cell lymphoma; EGFR, epidermal growth factor receptor; FDA, Food and Drug Administration; Flt-3, fms-like tyrosine kinase-3; GIST, gastrointestinal stromal tumor; MTC, medullary thyroid cancer; mTOR, mammalian target of rapamycin; PDGFR, platelet-derived growth factor receptor; PLGF, placental growth factor; PML-RARα, promyelocytic leukemia-retinoic acid receptor-alpha; RCC, renal cell cancer; t(15;17), translocation between chromosomes 15 and 17; TC, thyroid cancer; TGF-α, transforming growth factor-alpha; VEGFR, vascular endothelial growth factor receptor. and chemotherapy can be enhanced further by combinations with another targeted monoclonal antibody (pertuzumab), which prevents dimerization of the HER2 receptor with other HER family members including HER3.

1	Although targeted therapies have not yet resulted in cures when used alone, their use in the adjuvant setting and when combined with other effective treatments has substantially increased the fraction of patients cured. For example, the addition of rituximab, an anti-CD20 antibody, to combination chemotherapy in patients with diffuse large B cell lymphoma improves cure rates by 15–20%. The addition of trastuzumab, antibody to HER2, to combination chemotherapy in the adjuvant treatment of HER2-positive breast cancer reduces relapse rates by 50%.

1	A major effort is under way to develop targeted therapies for mutations in the ras family of genes, which are the most common mutations in oncogenes in cancers (especially kras) but have proved to be very difficult targets for a number of reasons related to how RAS proteins are activated and inactivated. Targeted therapies against proteins downstream of RAS (including mitogen-activated protein [MAP] kinase and ERK) are currently being studied, both individually and in combination. A large number of inhibitors of phospholipid signaling pathways such as the phosphatidylinositol-3-kinase (PI3K) and phospholipase C-gamma pathways, which are involved in a large number of cellular processes that are important in cancer development and progression, are being evaluated. The targeting of a variety of other pathways that are activated in malignant cells, such as the MET pathway, hedgehog pathway, and various angiogenesis pathways, is also being explored.

1	One of the strategies for new drug development is to take advantage of so-called oncogene addiction. This situation (Fig. 102e-3) is created when a tumor cell develops an activating mutation in an oncogene that becomes a dominant pathway for survival and growth with reduced contributions from other pathways, even when there may be abnormalities in those pathways. This dependency on a single pathway creates a cell that is vulnerable to inhibitors of that oncogene pathway. For example, cells harboring mutations in BRAF are very sensitive to MEK inhibitors that inhibit downstream signaling in the BRAF pathway.

1	Targeting proteins critical for transcription of proteins vital for malignant cell survival or proliferation provides another potential target for treating cancers. The transcription factor nuclear factor-κB (NF-κB) is a heterodimer composed of p65 and p50 subunits that associate with an inhibitor, IκB, in the cell cytoplasm. In response to growth factor or cytokine signaling, a multi-subunit kinase called IKK (IκB kinase) phosphorylates IκB and directs its degradation by the ubiquitin/proteasome system. NF-κB, free of its inhibitor, translocates to the nucleus and activates target genes, many of which promote the survival of tumor cells. Novel drugs called proteasome inhibitors block the proteolysis of IκB, thereby preventing NF-κB activation. For unexplained reasons, this is selectively toxic to tumor cells. The antitumor effects of proteasome inhibitors are more complicated and involve the inhibition of the degradation of multiple cellular proteins. Proteasome inhibitors (e.g.,

1	toxic to tumor cells. The antitumor effects of proteasome inhibitors are more complicated and involve the inhibition of the degradation of multiple cellular proteins. Proteasome inhibitors (e.g., bortezomib [Velcade]) have activity in patients with multiple myeloma, including partial and complete remissions. Inhibitors of IKK are also in development, with the hope of more selectively blocking the degradation of IκB, thus “locking” NF-κB in an inhibitory complex and rendering the cancer cell more susceptible to apoptosis-inducing agents. Many other transcription factors are activated by phosphorylation, which can be prevented by tyrosine kinase inhibitors or serine/threonine kinase inhibitors, a number of which are currently in clinical trials.

1	FIGuRE 102e-3 Synthetic lethality. Genes are said to have a synthetic lethal relationship when mutation of either gene alone is tolerated by the cell but mutation of both genes leads to lethality, as originally noted by Bridges and later named by Dobzhansky. Thus, mutant gene a and gene b have a synthetic lethal relationship, implying that the loss of one gene makes the cell dependent on the function of the other gene. In cancer cells, loss of function of a DNA repair gene like BRCA1, which repairs double-strand breaks, makes the cell dependent on base excision repair mediated in part by PARP. If the PARP gene product is inhibited, the cell attempts to repair the break using the error-prone nonhomologous end-joining method, which results in tumor cell death. High-throughput screens can now be performed using isogenic cell line pairs in which one cell line has a defined defect in a DNA repair pathway. Compounds can be identified that selectively kill the mutant cell line; targets of

1	now be performed using isogenic cell line pairs in which one cell line has a defined defect in a DNA repair pathway. Compounds can be identified that selectively kill the mutant cell line; targets of these compounds have a synthetic lethal relationship to the repair pathway and are potentially important targets for future therapeutics.

1	Estrogen receptors (ERs) and androgen receptors (ARs), members of the steroid hormone family of nuclear receptors, are targets of inhibition by drugs used to treat breast and prostate cancers, respectively. Tamoxifen, a partial agonist and antagonist of ER function, can mediate tumor regression in metastatic breast cancer and can prevent disease recurrence in the adjuvant setting. Tamoxifen binds to the ER and modulates its transcriptional activity, inhibiting activity in the breast but promoting activity in bone and uterine epithelium. Selective ER modulators (SERMs) have been developed with the hope of a more beneficial modulation of ER activity, i.e., antiestrogenic activity in the breast, uterus, and ovary, but estrogenic for bone, brain, and cardiovascular tissues. Aromatase inhibitors, which block the conversion of androgens to estrogens in breast and subcutaneous fat tissues, have demonstrated improved clinical efficacy compared with tamoxifen and are often used as first-line

1	which block the conversion of androgens to estrogens in breast and subcutaneous fat tissues, have demonstrated improved clinical efficacy compared with tamoxifen and are often used as first-line therapy in patients with ER-positive disease. A number of approaches have been developed for blocking androgen stimulation of prostate cancer, including decreasing production (e.g., orchiectomy, luteinizing hormone–releasing hormone agonists or antagonists, estrogens, ketoconazole, and inhibitors of enzymes such as CYP17 involved in androgen production) and AR blockers (Chap. 108).

1	The concepts of oncogene addiction and synthetic lethality have spurred new drug development targeting oncogeneand tumor-suppressor pathways. As discussed earlier in this chapter and outlined in Fig. 102e-3, cancer cells can become dependent on signaling pathways containing activated oncogenes; this can effect proliferation (i.e., mutated Kras, Braf, overexpressed Myc, or activated tyrosine kinases), DNA repair (loss of BRCA1 or BRCA2 gene function), survival (overexpression of Bcl-2 or NF-κB), cell metabolism (as occurs when mutant Kras enhances glucose uptake and aerobic glycolysis), and perhaps angiogenesis (production of VEGF in response to HIF-2α in RCC). In such cases, targeted inhibition of the pathway can lead to specific killing of the cancer cells. However, targeting defects in tumor-suppressor genes has been much more difficult, both because the target of mutation is often deleted and because it is much more difficult to restore normal function than to inhibit abnormal

1	in tumor-suppressor genes has been much more difficult, both because the target of mutation is often deleted and because it is much more difficult to restore normal function than to inhibit abnormal function of a protein. Synthetic lethality occurs when loss of function in either of two genes alone has limited effects on cell survival but loss of function in both genes leads to cell death. Identifying genes that have a synthetic lethal relationship to tumor-suppressor pathways that have been mutated in tumor cells may allow targeting of proteins required uniquely by those cells (Fig. 102e-3). Several examples of this have been identified. For instance, cells with mutations in the BRCA1 or BRCA2 tumor-suppressor genes (e.g., a subset of breast and ovarian cancers) are unable to repair DNA damage by homologous recombination. PARP are a family of proteins important for single-strand break (SSB) DNA repair. PARP inhibition results in selective killing of cancer cells with BRCA1 or BRCA2

1	damage by homologous recombination. PARP are a family of proteins important for single-strand break (SSB) DNA repair. PARP inhibition results in selective killing of cancer cells with BRCA1 or BRCA2 loss. Preliminary trials have suggested some effectiveness of PARP inhibition, especially in combination with chemotherapy; clinical trials are ongoing. The concept of synthetic lethality provides a framework for genetic screens to identify other synthetic lethal combinations involving known tumor-suppressor genes and development of novel therapeutic agents to target dependent pathways.

1	Chromatin structure regulates the hierarchical order of sequential gene transcription that governs differentiation and tissue homeostasis. Disruption of chromatin remodeling (the process of modifying chromatin structure to control exposure of specific genes to transcriptional proteins, thereby controlling the expression of those genes) leads to aberrant gene expression and can induce proliferation of undifferentiated cells. Epigenetics is defined as changes that alter the pattern of gene expression that persist across at least one cell division but are not caused by changes in the DNA code. Epigenetic changes include alterations of chromatin structure mediated by methylation of cytosine residues in CpG dinucleotides, modification of histones by acetylation or methylation, or changes in higher-order chromosome structure (Fig. 102e-4). The transcriptional regulatory regions of active genes 102e-7 often contain a high frequency of CpG dinucleotides (referred to as CpG islands), which are

1	chromosome structure (Fig. 102e-4). The transcriptional regulatory regions of active genes 102e-7 often contain a high frequency of CpG dinucleotides (referred to as CpG islands), which are normally unmethylated. Expression of these genes is controlled by transient association with repressor or activator proteins that regulate transcriptional activation. However, hypermethylation of promoter regions is a common mechanism by which tumor-suppressor loci are epigenetically silenced in cancer cells. Thus one allele may be inactivated by mutation or deletion (as occurs in loss of heterozygosity), while expression of the other allele is epigenetically silenced, usually by methylation.

1	Acetylation of the amino terminus of the core histones H3 and H4 induces an open chromatin conformation that promotes transcription initiation. Histone acetylases are components of coactivator complexes recruited to promoter/enhancer regions by sequence-specific transcription factors during the activation of genes (Fig. 102e-4). Histone deacetylases (HDACs; at least 17 are encoded in the human genome) are recruited to genes by transcriptional repressors and prevent the initiation of gene transcription. Methylated cytosine residues in promoter regions become associated with methyl cytosine–binding proteins that recruit protein complexes with HDAC activity. The balance between permissive and inhibitory chromatin structure is therefore largely determined by the activity of transcription factors in modulating the “histone code” and the methylation status of the genetic regulatory elements of genes.

1	The pattern of gene transcription is aberrant in all human cancers, and in many cases, epigenetic events are responsible. Unlike genetic events that alter DNA primary structure (e.g., deletions), epigenetic changes are potentially reversible and appear amenable to therapeutic intervention. In certain human cancers, including pancreatic cancer and multiple myeloma, the p16Ink4a promoter is inactivated by methylation, thus permitting the unchecked activity of CDK4/cyclin D and rendering pRb nonfunctional. In sporadic forms of renal, breast, and colon cancer, the von Hippel–Lindau (VHL), breast cancer 1 (BRCA1), and serine/threonine kinase 11 (STK11) genes, respectively, are epigenetically silenced. Other targeted genes include the p15Ink4b CDK inhibitor, glutathione-S-transferase (which detoxifies reactive oxygen species), and the E-cadherin molecule (important for junction formation between epithelial cells). Epigenetic silencing can occur in premalignant lesions and can affect genes

1	reactive oxygen species), and the E-cadherin molecule (important for junction formation between epithelial cells). Epigenetic silencing can occur in premalignant lesions and can affect genes involved in DNA repair, thus predisposing to further genetic damage. Examples include MLH1 (mut L homologue) in hereditary nonpolyposis colon cancer (HNPCC, also called Lynch’s syndrome), which is critical for repair of mismatched bases that occur during DNA synthesis, and O6-methylguanine-DNA methyltransferase, which removes alkylated guanine adducts from DNA and is often silenced in colon, lung, and lymphoid tumors.

1	Human leukemias often have chromosomal translocations that code for novel fusion proteins with enzymatic activities that alter chromatin structure. The promyelocytic leukemia–retinoic acid receptor (PML-RAR) fusion protein, generated by the t(15;17) observed in most cases of acute promyelocytic leukemia (APL), binds to promoters containing retinoic acid response elements and recruits HDAC to these promoters, effectively inhibiting gene expression. This arrests differentiation at the promyelocyte stage and promotes tumor cell proliferation and survival. Treatment with pharmacologic doses of all-trans retinoic acid (ATRA), the ligand for RARα, results in the release of HDAC activity and the recruitment of coactivators, which overcome the differentiation block. This induced differentiation of APL cells has improved treatment of these patients but also has led to a novel treatment toxicity when newly differentiated tumor cells infiltrate the lungs. However, ATRA represents a treatment

1	of APL cells has improved treatment of these patients but also has led to a novel treatment toxicity when newly differentiated tumor cells infiltrate the lungs. However, ATRA represents a treatment paradigm for the reversal of epigenetic changes in cancer. For other leukemia-associated fusion proteins, such as acute myeloid leukemia (AML)-eight-twentyone (ETO) and the MLL fusion proteins seen in AML and acute lymphocytic leukemia, no ligand is known. Therefore, efforts are ongoing to determine the structural basis for interactions between translocation fusion proteins and chromatin-remodeling proteins and to use this information to rationally design small molecules that will disrupt specific protein-protein associations, although this has proven to be technically difficult. Drugs that block the enzymatic activity of HDAC are

1	Nucleosomes permits binding of multiple Nucleosomes gene expression.

1	FIGuRE 102e-4 Epigenetic regulation of gene expression in cancer cells. Tumor-suppressor genes are often epigenetically silenced in cancer cells. In the upper portion, a CpG island within the promoter and enhancer regions of the gene has been methylated, resulting in the recruitment of methyl-cytosine binding proteins (MeCP) and complexes with histone deacetylase (HDAC) activity. Chromatin is in a condensed, non-permissive conformation that inhibits transcription. Clinical trials are under way using the combination of demethylating agents such as 5-aza-2′deoxycytidine plus HDAC inhibitors, which together confer an open, permissive chromatin structure (lower portion). Transcription factors bind to specific DNA sequences in promoter regions and, through protein-protein interactions, recruit coactivator complexes containing histone acetyl transferase (HAT) activity. This enhances transcription initiation by RNA polymerase II and associated general transcription factors. The expression of

1	coactivator complexes containing histone acetyl transferase (HAT) activity. This enhances transcription initiation by RNA polymerase II and associated general transcription factors. The expression of the tumor-suppressor gene commences, with phenotypic changes that may include growth arrest, differentiation, or apoptosis.

1	being tested. HDAC inhibitors have demonstrated antitumor activity in clinical studies against cutaneous T cell lymphoma (e.g., vorinostat) and some solid tumors. HDAC inhibitors may target cancer cells via a number of mechanisms, including upregulation of death receptors (DR4/5, FAS, and their ligands) and p21Cip1/Waf1, as well as inhibition of cell cycle checkpoints.

1	Efforts are also under way to reverse the hypermethylation of CpG islands that characterizes many malignancies. Drugs that induce DNA demethylation, such as 5-aza-2′-deoxycytidine, can lead to reexpression of silenced genes in cancer cells with restoration of function, and 5-aza-2′-deoxycytidine is approved for use in myelodysplastic syndrome (MDS). However, 5-aza-2′-deoxycytidine has limited aqueous solubility and is myelosuppressive. Other inhibitors of DNA methyltransferases are in development. In ongoing clinical trials, inhibitors of DNA methylation are being combined with HDAC inhibitors. The hope is that by reversing coexisting epigenetic changes, the deregulated patterns of gene transcription in cancer cells will be at least partially reversed.

1	Epigenetic gene regulation can also occur via microRNAs or long non-coding RNAs (lncRNAs). MicroRNAs are short (average 22 nucleotides in length) RNA molecules that silence gene expression after transcription by binding and inhibiting the translation or promoting the degradation of mRNA transcripts. It is estimated that more than 1000 microRNAs are encoded in the human genome. Each tissue has a distinctive repertoire of microRNA expression, and this pattern is altered in specific ways in cancers. However, specific correlations between microRNA expression and tumor biology and clinical behavior are just now emerging. Therapies targeting microRNAs are not currently at hand but represent a novel area of treatment development. lncRNAs are longer than 200 nucleotides and compose the largest group of noncoding RNAs. Some of them have been shown to play important roles in gene regulation. The potential for altering these RNAs for therapeutic benefit is an area of active investigation,

1	group of noncoding RNAs. Some of them have been shown to play important roles in gene regulation. The potential for altering these RNAs for therapeutic benefit is an area of active investigation, although much more needs to be learned before this will be feasible.

1	Tissue homeostasis requires a balance between the death of aged, terminally differentiated cells or severely damaged cells and their renewal by proliferation of committed progenitors. Genetic damage to growth-regulating genes of stem cells could lead to catastrophic results for the host as a whole. Thus, genetic events causing activation of oncogenes or loss of tumor suppressors, which would be predicted to lead to unregulated cell proliferation unless corrected, usually activate signal transduction pathways that block aberrant cell proliferation. These pathways can lead to a form of programmed cell death (apoptosis) or irreversible growth arrest (senescence). Much as a panoply of intra-and extracellular signals impinge upon the core cell cycle machinery to regulate cell division, so too are these signals transmitted to a core enzymatic machinery that regulates cell death and survival.

1	Apoptosis is induced by two main pathways (Fig. 102e-5). The extrinsic pathway of apoptosis is activated by cross-linking members of the tumor necrosis factor (TNF) receptor superfamily, such as CD95 (Fas) and death receptors DR4 and DR5, by their ligands, Fas ligand or TRAIL (TNF-related apoptosis-inducing ligand), respectively. This induces the association of FADD (Fas-associated death domain) and procaspase-8 to death domain motifs of the receptors. Caspase-8 is activated and then cleaves and activates effector caspases-3 and -7, which then target cellular constituents (including caspase-activated DNAse, cytoskeletal proteins, and a number of regulatory proteins), inducing the morphologic appearance characteristic of apoptosis, which pathologists term “karyorrhexis.” The intrinsic pathway of apoptosis is initiated by the release of cytochrome c and SMAC (second

1	APAF-1 dATP Pro-caspase 9 Cyt c Bak BcI2 Bax SMAC IAP BH3-only proteins Matrix Inter-membrane space p65 p50 Proteasome NF-˜B genes activated Cytoskeletal disruption Substrate cleavage Effector caspases BAD Caspase 9 FKHR IKK DNA degradation chromatin condensation Lamin cleavage I˜B Nucleus Outer membrane Mitochondrion receptor Death-inducing signals • DNA damage• Oncogene-induced proliferation • Loss of attachment to ECM • Chemotherapy, radiation therapy 5 8 2 743

1	FIGuRE 102e-5 Therapeutic strategies to overcome aberrant survival pathways in cancer cells. 1. The extrinsic pathway of apoptosis can be selectively induced in cancer cells by TRAIL (the ligand for death receptors 4 and 5) or by agonistic monoclonal antibodies. 2. Inhibition of antiapoptotic Bcl-2 family members with antisense oligonucleotides or inhibitors of the BH3-binding pocket will promote formation of Bakor Bax-induced pores in the mitochondrial outer membrane. 3. Epigenetic silencing of APAF-1, caspase-8, and other proteins can be overcome using demethylating agents and inhibitors of histone deacetylases. 4. Inhibitor of apoptosis proteins (IAP) blocks activation of caspases; small-molecule inhibitors of IAP function (mimicking SMAC action) should lower the threshold for apoptosis. 5. Signal transduction pathways originating with activation of receptor tyrosine kinase receptors (RTKs) or cytokine receptors promote survival of cancer cells by a number of mechanisms. Inhibiting

1	5. Signal transduction pathways originating with activation of receptor tyrosine kinase receptors (RTKs) or cytokine receptors promote survival of cancer cells by a number of mechanisms. Inhibiting receptor function with monoclonal antibodies, such as trastuzumab or cetuximab, or inhibiting kinase activity with small-molecule inhibitors can block the pathway. 6. The Akt kinase phosphorylates many regulators of apoptosis to promote cell survival; inhibitors of Akt may render tumor cells more sensitive to apoptosis-inducing signals; however, the possibility of toxicity to normal cells may limit the therapeutic value of these agents. 7 and 8. Activation of the transcription factor NF-κB (composed of p65 and p50 subunits) occurs when its inhibitor, IκB, is phosphorylated by IκB kinase (IKK), with subsequent degradation of IκB by the proteasome. Inhibition of IKK activity should selectively block the activation of NF-κB target genes, many of which promote cell survival. Inhibitors of

1	with subsequent degradation of IκB by the proteasome. Inhibition of IKK activity should selectively block the activation of NF-κB target genes, many of which promote cell survival. Inhibitors of proteasome function are Food and Drug Administration approved and may work in part by preventing destruction of IκB, thus blocking NF-κB nuclear localization. NF-κB is unlikely to be the only target for proteasome inhibitors.

1	mitochondrial activator of caspases) from the mitochondrial inter-membrane space in response to a variety of noxious stimuli, including DNA damage, loss of adherence to the extracellular matrix (ECM), oncogene-induced proliferation, and growth factor deprivation. Upon release into the cytoplasm, cytochrome c associates with dATP, procaspase-9, and the adaptor protein APAF-1, leading to the sequential activation of caspase-9 and effector caspases. SMAC binds to and blocks the function of inhibitor of apoptosis proteins (IAP), negative regulators of caspase activation.

1	The release of apoptosis-inducing proteins from the mitochondria is regulated by proand antiapoptotic members of the Bcl-2 family. Antiapoptotic members (e.g., Bcl-2, Bcl-XL, and Mcl-1) associate with the mitochondrial outer membrane via their carboxyl termini, exposing to the cytoplasm a hydrophobic binding pocket composed of Bcl-2 homology (BH) domains 1, 2, and 3 that is crucial for their activity. Perturbations of normal physiologic processes in specific cellular compartments lead to the activation of BH3-only proapoptotic family members (such as Bad, Bim, Bid, Puma, Noxa, and others) that can alter the conformation of the outer-membrane proteins Bax and Bak, which then oligomerize to form pores in the mitochondrial outer membrane resulting in cytochrome c release. If proteins composed only of BH3 domains are sequestered by Bcl-2, Bcl-XL, or Mcl-1, pores do not form and apoptosis-inducing proteins are not released from the mitochondria. The ratio of levels of antiapoptotic Bcl-2

1	only of BH3 domains are sequestered by Bcl-2, Bcl-XL, or Mcl-1, pores do not form and apoptosis-inducing proteins are not released from the mitochondria. The ratio of levels of antiapoptotic Bcl-2 family members and the levels of proapoptotic BH3-only proteins at the mitochondrial membrane determines the activation state of the intrinsic pathway. The mitochondrion must therefore be recognized not only as an organelle with vital roles in intermediary metabolism and oxidative phosphorylation but also as a central regulatory structure of the apoptotic process.

1	The evolution of tumor cells to a more malignant phenotype requires the acquisition of genetic changes that subvert apoptosis pathways and promote cancer cell survival and resistance to anticancer therapies. However, cancer cells may be more vulnerable than normal cells to therapeutic interventions that target the apoptosis pathways that cancer cells depend on. For instance, overexpression of Bcl-2 as a result of the t(14;18) translocation contributes to follicular lymphoma. Upregulation of Bcl-2 expression is also observed in prostate, breast, and lung cancers and melanoma. Targeting of antiapoptotic Bcl-2 family members has been accomplished by the identification of several low-molecular-weight compounds that bind to the hydrophobic pockets of either Bcl-2 or Bcl-XL and block their ability to associate with death-inducing BH3-only proteins. These compounds inhibit the antiapoptotic activities of Bcl-2 and Bcl-XL at nanomolar concentrations in the laboratory and are entering clinical

1	to associate with death-inducing BH3-only proteins. These compounds inhibit the antiapoptotic activities of Bcl-2 and Bcl-XL at nanomolar concentrations in the laboratory and are entering clinical trials.

1	Preclinical studies targeting death receptors DR4 and DR5 have demonstrated that recombinant, soluble, human TRAIL or humanized monoclonal antibodies with agonist activity against DR4 or DR5 can induce apoptosis of tumor cells while sparing normal cells. The mechanisms for this selectivity may include expression of decoy receptors or elevated levels of intracellular inhibitors (such as FLIP, which competes with caspase-8 for FADD) by normal cells but not tumor cells. Synergy has been shown between TRAIL-induced apoptosis and chemotherapeutic agents. For instance, some colon cancers encode mutated Bax protein as a result of mismatch repair (MMR) defects and are resistant to TRAIL. However, upregulation of Bak by chemotherapy restores the ability of TRAIL to activate the mitochondrial pathway of apoptosis. However, clinical studies have not yet shown significant activity of approaches targeting the TRAIL pathway.

1	Many of the signal transduction pathways perturbed in cancer promote tumor cell survival (Fig. 102e-5). These include activation of the PI3K/Akt pathway, increased levels of the NF-κB transcription factor, and epigenetic silencing of genes such as APAF-1 and caspase-8. Each of these pathways is a target for therapeutic agents that, in addition to affecting cancer cell proliferation or gene expression, may render cancer cells more susceptible to apoptosis, thus promoting synergy when combined with other chemotherapeutic agents.

1	Some tumor cells resist drug-induced apoptosis by expression of one or more members of the ABC family of ATP-dependent efflux pumps that mediate the multidrug-resistance (MDR) phenotype. The prototype, P-glycoprotein (PGP), spans the plasma membrane 12 times and has two ATP-binding sites. Hydrophobic drugs (e.g., anthracyclines and vinca alkaloids) are recognized by PGP as they enter the cell and are pumped out. Numerous clinical studies have failed to demonstrate that drug resistance can be overcome using inhibitors of PGP. However, ABC transporters have different substrate specificities, and inhibition of a single family member may not be sufficient to overcome the MDR phenotype. Efforts to reverse PGP-mediated drug resistance continue.

1	Cells, including cancer cells, can also undergo other mechanisms of cell death including autophagy (degradation of proteins and organelles by lysosomal proteases) and necrosis (digestion of cellular components and rupturing of the cell membrane). Necrosis usually occurs in response to external forces resulting in release of cellular components, which leads to inflammation and damage to surrounding tissues. Although necrosis was thought to be unprogrammed, evidence now suggests that at least some aspects may be programmed. The exact role of necrosis in cancer cell death in various settings is still being determined. In addition to its role in cell death, autophagy can serve as a homeostatic mechanism to promote survival for the cell by recycling cellular components to provide necessary energy. The mechanisms that control the balance between enhancing survival versus leading to cell death are still not fully understood. Autophagy appears to play conflicting roles in the development and

1	The mechanisms that control the balance between enhancing survival versus leading to cell death are still not fully understood. Autophagy appears to play conflicting roles in the development and survival of cancer. Early in the carcinogenic process, it can act as a tumor suppressor by preventing the cell from accumulating abnormal proteins and organelles. However, in established tumors, it may serve as a mechanism of survival for cancer cells when they are stressed by damage such as from chemotherapy. Inhibition of this process can enhance the sensitivity of cancer cells to chemotherapy. Better understanding of the factors that control the survival-promoting versus death-inducing aspects of autophagy is required in order to know how to best manipulate it for therapeutic benefit.

1	The metastatic process accounts for the vast majority of deaths from solid tumors, and therefore, an understanding of this process is critical. The biology of metastasis is complex and requires multiple steps. The three major features of tissue invasion are cell adhesion to the basement membrane, local proteolysis of the membrane, and movement of the cell through the rent in the membrane and the ECM. Cells that lose contact with the ECM normally undergo programmed cell death (anoikis), and this process has to be suppressed in cells that metastasize. Another process important for metastasizing epithelial cancer cells is epithelial-mesenchymal transition (EMT). This is a process by which cells lose their epithelial properties and gain mesenchymal properties. This normally occurs during the developmental process in embryos, allowing cells to migrate to their appropriate destinations in the embryo. It also occurs in wound healing, tissue regeneration, and fibrotic reactions, but in all of

1	process in embryos, allowing cells to migrate to their appropriate destinations in the embryo. It also occurs in wound healing, tissue regeneration, and fibrotic reactions, but in all of these processes, cells stop proliferating when the process is complete. Malignant cells that metastasize undergo EMT as an important step in that process but retain the capacity for unregulated proliferation. Malignant cells that gain access to the circulation must then repeat those steps at a remote site, find a hospitable niche in a foreign tissue, avoid detection by host defenses, and induce the growth of new blood vessels. The rate-limiting step for metastasis is the ability for tumor cells to survive and expand in the novel microenvironment of the metastatic site, and multiple host-tumor interactions determine the ultimate outcome (Fig. 102e-6). Few drugs have been developed to attempt to directly target the process of metastasis, in part because the specifics of the critical steps in the process

1	determine the ultimate outcome (Fig. 102e-6). Few drugs have been developed to attempt to directly target the process of metastasis, in part because the specifics of the critical steps in the process that would be potentially good targets for drugs are still being identified. However, a number of potential targets are known. HER2 can enhance the metastatic potential of breast cancer cells, and as discussed above, the monoclonal antibody trastuzumab, which targets HER2, improves survival in the adjuvant setting for HER2-positive breast cancer patients. Other potential targets that increase metastatic potential of cells in preclinical studies include HIF-1 and -2, transcription factors induced by hypoxia within tumors; growth factors (e.g., cMET and VEGFR); oncogenes (e.g., SRC); adhesion molecules (e.g., focal adhesion kinase [FAK]); ECM proteins (e.g., matrix metalloproteinases-1 and -2); and inflammatory molecules (e.g., COX-2).

1	The metastatic phenotype is likely restricted to a small fraction of tumor cells (Fig. 102e-6). A number of genetic and epigenetic changes are required for tumor cells to be able to metastasize, including activation of metastasis-promoting genes and inhibition of genes that suppress the metastatic ability. Cells with metastatic capability frequently express chemokine receptors that are likely important in the metastatic process. A number of candidate metastasis-suppressor genes have been identified, including genes coding for proteins that enhance apoptosis, suppress cell division, are involved in the interactions of cells with each other or the ECM, or suppress cell migration. The loss of function of these genes enhances metastasis. Gene expression profiling is being used to study the metastatic process and other properties of tumor cells that may predict susceptibilities.

1	An example of the ability of malignant cells to survive and grow in a novel microenvironment is bone metastasis. Bone metastases are extremely painful, cause fractures of weight-bearing bones, can lead to hypercalcemia, and are a major cause of morbidity for cancer patients. Osteoclasts and their monocyte-derived precursors express the surface receptor RANK (receptor activator of NF-κB), which is required for terminal differentiation and activation of osteoclasts. Osteoblasts and other stromal cells express RANK ligand (RANKL), as both a membrane-bound and soluble cytokine. Osteoprotegerin (OPG), a soluble receptor for RANKL produced by stromal cells, acts as a decoy receptor to inhibit RANK activation. The relative balance of RANKL

1	FIGuRE 102e-6 Oncogene signaling pathways are activated during tumor progression and promote metastatic potential. This figure shows a cancer cell that has undergone epithelial to mesenchymal transition (EMT) under the influence of several environmental signals. Critical components include activated transforming growth factor β (TGF-β) and the hepatocyte growth factor (HGF)/c-Met pathways, as well as changes in the expression of adhesion molecules that mediate cell-cell and cell–extracellular matrix interactions. Important changes in gene expression are mediated by the Snail and Twist family of transcriptional repressors (whose expression is induced by the oncogenic pathways), leading to reduced expression of E-cadherin, a key component of adherens junctions between epithelial cells. This, in conjunction with upregulation of N-cadherin, a change in the pattern of expression of integrins (which mediate cell–extracellular matrix associations that are important for cell motility), and a

1	in conjunction with upregulation of N-cadherin, a change in the pattern of expression of integrins (which mediate cell–extracellular matrix associations that are important for cell motility), and a switch in intermediate filament expression from cytokeratin to vimentin, results in the phenotypic change from adherent highly organized epithelial cells to motile and invasive cells with a fibroblast or mesenchymal morphology. EMT is thought to be an important step leading to metastasis in some human cancers. Host stromal cells, including tumor-associated fibroblasts and macrophages, play an important role in modulating tumor cell behavior through secretion of growth factors and proangiogenic cytokines, and matrix metalloproteinases that degrade the basement membrane. VEGF-A, -C, and -D are produced by tumor cells and stromal cells in response to hypoxemia or oncogenic signals and induce production of new blood vessels and lymphatic channels through which tumor cells metastasize to lymph

1	by tumor cells and stromal cells in response to hypoxemia or oncogenic signals and induce production of new blood vessels and lymphatic channels through which tumor cells metastasize to lymph nodes or tissues.

1	and OPG determines the activation state of RANK on osteoclasts. Many tumors increase osteoclast activity by secretion of substances such as parathyroid hormone (PTH), PTH-related peptide, interleukin (IL)-1, or Mip1 that perturb the homeostatic balance of bone remodeling by increasing RANK signaling. One example is multiple myeloma, where tumor cell–stromal cell interactions activate osteoclasts and inhibit osteoblasts, leading to the development of multiple lytic bone lesions. Inhibition of RANKL by an antibody (denosumab) can prevent further bone destruction. Bisphosphonates are also effective inhibitors of osteoclast function that are used in the treatment of cancer patients with bone metastases.

1	Only a small proportion of the cells within a tumor are capable of initiating colonies in vitro or forming tumors at high efficiency when injected into immunocompromised NOD/SCID mice. Acute and chronic myeloid leukemias (AML and CML) have a small population of cells (<1%) that have properties of stem cells, such as unlimited self-renewal and the capacity to cause leukemia when serially transplanted in mice. These cells have an undifferentiated phenotype (Thy1− CD34+CD38– and do not express other differentiation markers) and resemble normal stem cells in many ways, but are no longer under homeostatic control (Fig. 102e-7). Solid tumors may also contain a population of stem cells. Cancer stem cells, like their normal counterparts, have unlimited proliferative capacity and paradoxically traverse the cell cycle at a very slow rate; cancer growth occurs largely due to expansion of the stem cell pool, the unregulated proliferation of an amplifying population, and failure of apoptosis

1	traverse the cell cycle at a very slow rate; cancer growth occurs largely due to expansion of the stem cell pool, the unregulated proliferation of an amplifying population, and failure of apoptosis pathways (Fig. 102e-7). Slow cell cycle progression and high levels of expression of antiapoptotic Bcl-2 family members and drug efflux pumps of the MDR family render cancer stem cells less vulnerable to cancer chemotherapy or radiation therapy. Implicit in the cancer stem cell hypothesis is the idea that failure to cure most human cancers is due to the fact that current therapeutic agents do not kill the stem cells. If cancer stem cells can be identified and isolated, then aberrant signaling pathways that distinguish these cells from normal tissue stem cells can be identified and targeted. Evidence that cells with stem cell properties can arise from other epithelial cells within the cancer by processes such as epithelial mesenchymal transition also implies that it is essential to treat all

1	that cells with stem cell properties can arise from other epithelial cells within the cancer by processes such as epithelial mesenchymal transition also implies that it is essential to treat all of the cancer cells, and not just those with current stem cell-like properties, in order to eliminate the self-renewing cancer cell population. The exact nature of cancer stem cells remains an area of investigation. One of the unanswered questions is the exact origin of cancer stem cells for the different cancers.

1	Regulated activation of differentiation program Loss of self-renewal capacity Multi-lineage differentiation Partial differentiation Growth arrest No growth arrest Maintenance of tissue Loss of tissue architecture FIGuRE 102e-7 Cancer stem cells play a critical role in the initiation, progression, and resistance to therapy of malignant neoplasms.

1	In normal tissues (left), homeostasis is maintained by asymmetric division of stem cells, leading to one progeny cell that will differentiate and one cell that will maintain the stem cell pool. This occurs within highly specific niches unique to each tissue, such as in close apposition to osteoblasts in bone marrow, or at the base of crypts in the colon. Here, paracrine signals from stromal cells, such as sonic hedgehog or Notch ligands, as well as upregulation of β-catenin and telomerase, help to maintain stem cell features of unlimited self-renewal while preventing differentiation or cell death. This occurs in part through upregulation of the transcriptional repressor Bmi-1 and inhibition of the p16Ink4a/Arf and p53 pathways. Daughter cells leave the stem cells niche and enter a proliferative phase (referred to as transit-amplifying) for a specified number of cell divisions, during which time a developmental program is activated, eventually giving rise to fully differentiated cells

1	phase (referred to as transit-amplifying) for a specified number of cell divisions, during which time a developmental program is activated, eventually giving rise to fully differentiated cells that have lost proliferative potential. Cell renewal equals cell death, and homeostasis is maintained. In this hierarchical system, only stem cells are long-lived. The hypothesis is that cancers harbor stem cells that make up a small fraction (i.e., 0.001–1%) of all cancer cells. These cells share several features with normal stem cells, including an undifferentiated phenotype, unlimited self-renewal potential, and a capacity for some degree of differentiation; however, due to initiating mutations (mutations are indicated by lightning bolts), they are no longer regulated by environmental cues. The cancer stem cell pool is expanded, and rapidly proliferating progeny, through additional mutations, may attain stem cell properties, although most of this population is thought to have a limited

1	The cancer stem cell pool is expanded, and rapidly proliferating progeny, through additional mutations, may attain stem cell properties, although most of this population is thought to have a limited proliferative capacity. Differentiation programs are dysfunctional due to reprogramming of the pattern of gene transcription by oncogenic signaling pathways. Within the cancer transit-amplifying population, genomic instability generates aneuploidy and clonal heterogeneity as cells attain a fully malignant phenotype with metastatic potential. The cancer stem cell hypothesis has led to the idea that current cancer therapies may be effective at killing the bulk of tumor cells but do not kill tumor stem cells, leading to a regrowth of tumors that is manifested as tumor recurrence or disease progression. Research is in progress to identify unique molecular features of cancer stem cells that can lead to their direct targeting by novel therapeutic agents.

1	Cancer cells, and especially stem cells, have the capacity for significant plasticity, allowing them to alter multiple aspects of cell biology in response to external factors (e.g., chemotherapy, inflammation, immune response). Thus, a major problem in cancer therapy is that malignancies have a wide spectrum of mechanisms for both initial and adaptive resistance to treatments. These include inhibiting drug delivery to the cancer cells, blocking drug uptake and retention, increasing drug metabolism, altering levels of target proteins, acquiring mutations in target proteins, modifying metabolism and cell signaling pathways, using alternate signaling pathways, adjusting the cell replication process including mechanisms by which the cell deals with DNA damage, inhibiting apoptosis, and evading the immune system. Thus, most metastatic cancers (except those curable with chemotherapy such as germ cell tumors) eventually become resistant to the therapy being used. Overcoming resistance is a

1	the immune system. Thus, most metastatic cancers (except those curable with chemotherapy such as germ cell tumors) eventually become resistant to the therapy being used. Overcoming resistance is a major area of research.

1	One of the distinguishing characteristics of cancer cells is that they have altered metabolism as compared with normal cells in supporting survival and their high rates of proliferation. These cells must focus a significant fraction of their energy resources on synthesis of proteins and other molecules while still maintaining sufficient ATP production to survive and grow. Although normal proliferating cells also have similar needs, there are differences in how cancer cells metabolize glucose and a number of other compounds, including glutamine, as compared to normal cells. Many cancer cells use aerobic glycolysis (the Warburg effect) (Fig. 102e-8) to metabolize glucose, leading to increased lactic acid production, whereas normal cells use oxidative phosphorylation in mitochondria under aerobic conditions, a much more efficient process. One consequence is increased glucose uptake by cancer cells, a fact used in fluorodeoxyglucose (FDG) positron emission tomography (PET) scanning to

1	aerobic conditions, a much more efficient process. One consequence is increased glucose uptake by cancer cells, a fact used in fluorodeoxyglucose (FDG) positron emission tomography (PET) scanning to detect tumors. A number of proteins in cancer cells, including CMYC, HIF1, RAS, p53, pRB, and AKT, are all involved in modulating glycolytic processes and controlling the Warburg effect. Although these pathways remain difficult to target therapeutically, both the PI3 kinase pathway with signaling through mTOR and the AMP-activated kinase (AMPK) pathway, which inhibits mTOR complex 1 (mTORC1; a protein complex that includes mTOR), are important in controlling the glycolytic process and thus provide potential targets for inhibiting this process. The inefficient utilization of glucose also leads to a need for alternative metabolic pathways for other compounds as well, one of which is glutamine. Similar to glucose, this provides both a source for structural molecules as well as energy

1	leads to a need for alternative metabolic pathways for other compounds as well, one of which is glutamine. Similar to glucose, this provides both a source for structural molecules as well as energy production. Glutamine is also inefficiently used by cancer cells.

1	continually evolving. Both the complexity and dynamic nature of the microenvironment enhance the difficulty of treating tumors. or There are also a number of mechanisms by +O which the microenvironment can contribute +/–O2 to resistance to anticancer therapies. One of the critical elements of tumor cell proliferation is delivery of oxygen, nutrients, and circulating factors important for growth and survival. The diffusion limit for oxy- O2 gen in tissues is ~100–200 μm, and thus, a 5% 85% critical aspect in the growth of tumors is the development of new blood vessels, or angio-

1	Lactate genesis. The growth of primary and metastatic tumors to larger than a few millimeters requires the recruitment of blood vessels and vascular endothelial cells to support their metabolic requirements. Thus, a critical ele-Oxidative Anaerobic Aerobic ment in growth of primary tumors and forphosphorylation glycolysis glycolysis mation of metastatic sites is the angiogenic switch: the ability of the tumor to promote the formation of new capillaries from pre existing host vessels. The angiogenic switch FIGuRE 102e-8 Warburg effect versus oxidative phosphorylation. In most normal tissues, is a phase in tumor development when the the vast majority of cells are differentiated and dedicated to a particular function within the dynamic balance of proand antiangiogenic organ in which they reside. The metabolic needs are mainly for energy and not for building factors is tipped in favor of vessel formation blocks for new cells. In these tissues, ATP is generated by oxidative phosphorylation

1	The metabolic needs are mainly for energy and not for building factors is tipped in favor of vessel formation blocks for new cells. In these tissues, ATP is generated by oxidative phosphorylation that effi-by the effects of the tumor on its immediate ciently generates about 36 molecules of ATP for each molecule of glucose metabolized. By con-environment. Stimuli for tumor angiogentrast, proliferative tumor tissues, especially in the setting of hypoxia, a typical condition within esis include hypoxemia, inflammation, and tumors, use aerobic glycolysis to generate energy for cell survival and generation of building genetic lesions in oncogenes or tumor sup- blocks for new cells.

1	Mutations in genes involved in the metastatic process occur in a number of cancers. Among the most frequently found to date are mutations in isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). These have been most commonly seen in gliomas, AML, and intrahepatic cholangiocarcinomas. These mutations lead to the production of an oncometabolite (2-hydroxyglutarate [2HG]) instead of the normal product α-ketoglutarate. Although the exact mechanisms of oncogenesis by 2HG are still being elucidated, α-ketoglutarate is a key cofactor for a number of dioxygenases involved in controlling DNA methylation. 2HG can act as a competitive inhibitor for α-ketoglutarate, leading to alterations in methylation status (primarily hypermethylation) of genes (epigenetic changes) that can have profound effects on a number of cellular processes including differentiation. Inhibitors of mutant IDH1 and IDH2 are being developed.

1	Much needs to be learned about the specific differences in metabolism between cancer cells and normal cells; however, modulators of metabolism are being tested clinically. The first of these is the antidiabetic agent metformin, both alone and in combination with chemotherapeutic agents. Metformin inhibits gluconeogenesis and may have direct effects on tumor cells by activating the 5′-adenosine monophosphate-activated kinase (AMPK), a serine/threonine protein kinase that is downstream of the LKB1 tumor suppressor, and thus inhibiting mTORC1. This leads to decreased protein synthesis and proliferation. A second approach being tested involves dichloracetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK). PDK inhibits pyruvate dehydrogenase in cancer cells, leading to a switch from mitochondrial oxidative phosphorylation of glucose to cytoplasmic glycolysis (the Warburg effect). By blocking PDK, DCA inhibits glycolysis. Additional approaches targeting tumor metabolism will

1	from mitochondrial oxidative phosphorylation of glucose to cytoplasmic glycolysis (the Warburg effect). By blocking PDK, DCA inhibits glycolysis. Additional approaches targeting tumor metabolism will likely emerge.

1	TuMOR MICROENVIRONMENT, ANGIOGENESIS, AND IMMuNE EVASION Tumors consist not only of malignant cells but also of a complex microenvironment including many other types of cells (e.g., inflammatory cells), ECM, secreted factors (e.g., growth factors), reactive oxygen and nitrogen species, mechanical factors, blood vessels, and lymphatics. This microenvironment is not static but rather is dynamic and pressors that alter tumor cell gene expres sion. Angiogenesis consists of several steps, including the stimulation of endothelial cells (ECs) by growth factors, degradation of the ECM by proteases, proliferation and migration of ECs into the tumor, and the eventual formation of new capillary tubes.

1	Tumor blood vessels are not normal; they have chaotic architecture and blood flow. Due to an imbalance of angiogenic regulators such as VEGF and angiopoietins (see below), tumor vessels are tortuous and dilated with an uneven diameter, excessive branching, and shunting. Tumor blood flow is variable, with areas of hypoxemia and acidosis leading to the selection of variants that are resistant to hypoxemiainduced apoptosis (often due to the loss of p53 expression). Tumor vessel walls have numerous openings, widened interendothelial junctions, and discontinuous or absent basement membrane; this contributes to the high vascular permeability of these vessels and, together with lack of functional intratumoral lymphatics, causes increased interstitial pressure within the tumor (which also interferes with the delivery of therapeutics to the tumor; Figs. 102e-9, 102e-10, and 102e-11). Tumor blood vessels lack perivascular cells such as pericytes and smooth-muscle cells that normally regulate

1	with the delivery of therapeutics to the tumor; Figs. 102e-9, 102e-10, and 102e-11). Tumor blood vessels lack perivascular cells such as pericytes and smooth-muscle cells that normally regulate flow in response to tissue metabolic needs.

1	Unlike normal blood vessels, the vascular lining of tumor vessels is not a homogeneous layer of ECs but often consists of a mosaic of ECs and tumor cells with upregulated genes seen in ECs and vessel formation that can occur in hypoxic conditions because of their plasticity; the concept of cancer cell–derived vascular channels, which may be lined by ECM secreted by the tumor cells, is referred to as vascular mimicry. During tumor angiogenesis, ECs are highly proliferative and express a number of plasma membrane proteins that are characteristic of activated endothelium, including growth factor receptors and adhesion molecules such as integrins.

1	Tumors use a number of mechanisms to promote vascularization, subverting normal angiogenic processes for this purpose (Fig. 102e-9). Primary or metastatic tumor cells sometimes arise in proximity to host blood vessels and grow around these vessels, parasitizing nutrients by co-opting the local blood supply. However, most tumor blood vessels arise by the process of sprouting, in which tumors secrete trophic Vascular mimicry— CEP contributes newly tumor cells as differentiated EC part of vessel wall vessels Region of 100 ˝m New sprout Follows VEGF gradient to tumor

1	FIGuRE 102e-9 Tumor angiogenesis is a complex process involving many different cell types that must proliferate, migrate, invade, and differentiate in response to signals from the tumor microenvironment. Endothelial cells (ECs) sprout from host vessels in response to VEGF, bFGF, Ang2, and other proangiogenic stimuli. Sprouting is stimulated by VEGF/VEGFR2, Ang2/Tie2, and integrin/extracellular matrix (ECM) interactions. Bone marrow–derived circulating endothelial precursors (CEPs) migrate to the tumor in response to VEGF and differentiate into ECs, while hematopoietic stem cells differentiate into leukocytes, including tumor-associated macrophages that secrete angiogenic growth factors and produce matrix metalloproteinases (MMPs) that remodel the ECM and release bound growth factors. Tumor cells themselves may directly form parts of vascular channels within tumors. The pattern of vessel formation is haphazard: vessels are tortuous, dilated, and leaky and branch in random ways. This

1	cells themselves may directly form parts of vascular channels within tumors. The pattern of vessel formation is haphazard: vessels are tortuous, dilated, and leaky and branch in random ways. This leads to uneven blood flow within the tumor, with areas of acidosis and hypoxemia (which stimulate release of angiogenic factors) and high intratumoral pressures that inhibit delivery of therapeutic agents.

1	angiogenic molecules, the most potent being vascular endothelial growth factors (VEGF), that induce the proliferation and migration of host ECs into the tumor. Sprouting in normal and pathogenic angiogenesis is regulated by three families of transmembrane receptor tyrosine kinases (RTKs) expressed on ECs and their ligands (VEGFs, angiopoietins, ephrins; Fig. 102e-10), which are produced by tumor cells, inflammatory cells, or stromal cells in the tumor microenvironment.

1	When tumor cells arise in or metastasize to an avascular area, they grow to a size limited by hypoxemia and nutrient deprivation. Hypoxemia, a key regulator of tumor angiogenesis, causes the transcriptional induction of the gene encoding VEGF. VEGF and its receptors are required for embryonic vasculogenesis (development of new blood vessels when none preexist) and normal (wound healing, corpus luteum formation) and pathologic angiogenesis (tumor angiogenesis, inflammatory conditions such as rheumatoid arthritis). VEGF-A is a heparin-binding glycoprotein with at least four isoforms (splice variants) that regulates blood vessel formation by binding to the RTKs VEGFR1 and VEGFR2, which are expressed on all ECs in addition to a subset of hematopoietic cells (Fig. 102e-9). VEGFR2 regulates EC proliferation, migration, and survival, whereas VEGFR1 may act as an antagonist of R2 in ECs but is probably also important for angioblast differentiation during embryogenesis. Tumor vessels may be

1	proliferation, migration, and survival, whereas VEGFR1 may act as an antagonist of R2 in ECs but is probably also important for angioblast differentiation during embryogenesis. Tumor vessels may be more dependent on VEGFR signaling for growth and survival than normal ECs. Although VEGF signaling is a critical initiator of angiogenesis, this is a complex process regulated by additional signaling pathways (Fig. 102e-10). The angiopoietin, Ang1, produced by stromal cells, binds to the EC RTK Tie2 and promotes the interaction of ECs with the ECM and perivascular cells, such as pericytes and smooth-muscle cells, to form tight, nonleaky vessels. Platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) help to recruit these perivascular cells. Ang1 is required for maintaining the quiescence and stability of mature blood vessels and prevents the vascular permeability normally induced by VEGF and inflammatory cytokines.

1	and differentiation, of smooth-remodeling) vessel Endothelial cell proliferation, migration, survival FIGuRE 102e-10 Critical molecular determinants of endothelial cell biology. Angiogenic endothelium expresses a number of receptors not found on resting endothelium. These include receptor tyrosine kinases (RTKs) and integrins that bind to the extracellular matrix and mediate endothelial cell (EC) adhesion, migration, and invasion. ECs also express RTK (i.e., the FGF and PDGF receptors) that are found on many other cell types. Critical functions mediated by activated RTK include proliferation, migration, and enhanced survival of endothelial cells, as well as regulation of the recruitment of perivascular cells and bloodborne circulating endothelial precursors and hematopoietic stem cells to the tumor. Intracellular signaling via EC-specific RTK uses molecular pathways that may be targets for future antiangiogenic therapies.

1	For tumor cell–derived VEGF to initiate sprouting from host vessels, the stability conferred by the Ang1/Tie2 pathway must be perturbed; this occurs by the secretion of Ang2 by ECs that are undergoing active remodeling. Ang2 binds to Tie2 and is a competitive inhibitor of Ang1 action: under the influence of Ang2, preexisting blood vessels become more responsive to remodeling signals, with less adherence of ECs to stroma and associated perivascular cells and more responsiveness to VEGF. Therefore, Ang2 is required at early stages of tumor angiogenesis for destabilizing the vasculature by making host ECs more sensitive to angiogenic signals. Because tumor ECs are blocked by Ang2, there is no stabilization by the Ang1/Tie2 interaction, and tumor blood vessels are leaky, hemorrhagic, and have poor association of ECs with underlying stroma. Sprouting tumor ECs express high levels of the transmembrane protein ephrin-B2 and its receptor, the RTK EPH, whose signaling appears to work with the

1	poor association of ECs with underlying stroma. Sprouting tumor ECs express high levels of the transmembrane protein ephrin-B2 and its receptor, the RTK EPH, whose signaling appears to work with the angiopoietins during vessel remodeling. During embryogenesis, EPH receptors are expressed on the endothelium of primordial venous vessels while the transmembrane ligand ephrin-B2 is expressed by cells of primordial arteries; the reciprocal expression may regulate differentiation and patterning of the vasculature.

1	A number of ubiquitously expressed host molecules play critical roles in normal and pathologic angiogenesis. Proangiogenic cytokines, chemokines, and growth factors secreted by stromal cells or inflammatory cells make important contributions to neovascularization, including bFGF, transforming growth factor α (TGF-α), TNF-α, and IL-8. In contrast to normal endothelium, angiogenic endothelium overexpresses specific members of the integrin family of ECM-binding proteins that mediate EC adhesion, migration, and survival. Specifically, expression of integrins α β , α β , and α β mediates spreading and migration of ECs and is required for angiogenesis induced by VEGF and bFGF, which in turn can upregulate EC integrin expression. The αvβ3 integrin physically associates with VEGFR2 in the plasma membrane and promotes signal transduction from each receptor to promote EC proliferation (via focal adhesion kinase, src, PI3K, and other pathways) and survival (by inhibition of p53 and increasing

1	membrane and promotes signal transduction from each receptor to promote EC proliferation (via focal adhesion kinase, src, PI3K, and other pathways) and survival (by inhibition of p53 and increasing the Bcl-2/Bax expression ratio). In addition, αvβ3 forms cell-surface complexes with matrix metalloproteinases (MMPs), zinc-requiring proteases that cleave ECM proteins, leading to enhanced EC migration and the release of heparin-binding growth factors, including VEGF and bFGF. EC adhesion molecules can be upregulated (i.e., by VEGF, TNF-α) or downregulated (by TGF-β); this, together with chaotic blood flow, explains poor leukocyte-endothelial interactions in tumor blood vessels and may help tumor cells avoid immune surveillance.

1	Lymphatic vessels also exist within tumors. Development of tumor lymphatics is associated with expression of VEGFR3 and its ligands VEGF-C and VEGF-D. The role of these vessels in tumor cell metastasis to regional lymph nodes remains to be determined. However, VEGF-C levels correlate significantly with metastasis to regional lymph nodes in lung, prostate, and colorectal cancers.

1	Angiogenesis inhibitors function by targeting the critical molecular pathways involved in EC proliferation, migration, and/or survival, many of which are unique to the activated endothelium in tumors. Inhibition of growth factor and adhesion-dependent signaling pathways can induce EC apoptosis with concomitant inhibition of tumor growth. Different types of tumors can use distinct combinations of molecular mechanisms to activate the angiogenic switch. Therefore, it is doubtful that a single antiangiogenic strategy will suffice for all human cancers; rather, a number of agents or combinations of agents will be needed, depending on distinct programs of angiogenesis used by different human cancers. Despite this, experimental data indicate that for some tumor types, blockade of a single growth factor (e.g., VEGF) may inhibit tumor-induced vascular growth. A. Normal blood vessel B. Tumor blood vessel

1	A. Normal blood vessel B. Tumor blood vessel Pericytes Tumor cells Loss of EC junction complexes Irregular or no BM Absent (or few) pericyte Increased permeability C. Treatment with bevacizumab (Early) D. Treatment with bevacizumab (Late) Pericytes Tumor cells Death of EC due to loss of VEGF survival signals (plus chemotherapy or radiotherapy) Apoptosis of tumor due to starvation and/or effects of chemotherapy.

1	FIGuRE 102e-11 Normalization of tumor blood vessels due to inhibition of VEGF signaling. A. Blood vessels in normal tissues exhibit a regular hierarchical branching pattern that delivers blood to tissues in a spatially and temporally efficient manner to meet the metabolic needs of the tissue (top). At the microscopic level, tight junctions are maintained between endothelial cells (ECs), which are adherent to a thick and evenly distributed basement membrane (BM). Pericytes form a surrounding layer that provides trophic signals to the EC and helps maintain proper vessel tone. Vascular permeability is regulated, interstitial fluid pressure is low, and oxygen tension and pH are physiologic. B. Tumors have abnormal vessels with tortuous branching and dilated, irregular interconnecting branches, causing uneven blood flow with areas of hypoxemia and acidosis. This harsh environment selects genetic events that result in resistant tumor variants, such as the loss of p53. High levels of VEGF

1	causing uneven blood flow with areas of hypoxemia and acidosis. This harsh environment selects genetic events that result in resistant tumor variants, such as the loss of p53. High levels of VEGF (secreted by tumor cells) disrupt gap junction communication, tight junctions, and adherens junctions between EC via src-mediated phosphorylation of proteins such as connexin 43, zonula occludens-1, VE-cadherin, and α/β-catenins. Tumor vessels have thin, irregular BM, and pericytes are sparse or absent. Together, these molecular abnormalities result in a vasculature that is permeable to serum macromolecules, leading to high tumor interstitial pressure, which can prevent the delivery of drugs to the tumor cells. This is made worse by the binding and activation of platelets at sites of exposed BM, with release of stored VEGF and microvessel clot formation, creating more abnormal blood flow and regions of hypoxemia. C. In experimental systems, treatment with bevacizumab or blocking antibodies to

1	release of stored VEGF and microvessel clot formation, creating more abnormal blood flow and regions of hypoxemia. C. In experimental systems, treatment with bevacizumab or blocking antibodies to VEGFR2 leads to changes in the tumor vasculature that has been termed vessel normalization. During the first week of treatment, abnormal vessels are eliminated or pruned (dotted lines), leaving a more normal branching pattern. ECs partially regain features such as cell-cell junctions, adherence to a more normal BM, and pericyte coverage. These changes lead to a decrease in vascular permeability, reduced interstitial pressure, and a transient increase in blood flow within the tumor. Note that in murine models, this normalization period lasts only for ~5–6 days. D. After continued anti-VEGF/VEGFR therapy (which is often combined with chemoor radiotherapy), ECs die, leading to tumor cell death (either due to direct effects of the chemotherapy or lack of blood flow).

1	Bevacizumab, an antibody that binds VEGF, appears to potentiate the effects of a number of different types of active chemotherapeutic regimens used to treat a variety of different tumor types including colon cancer, lung cancer, cervical cancer, and RCC. Bevacizumab is administered IV every 2–3 weeks (its half-life is nearly 20 days) and is generally well tolerated. Hypertension is the most common side effect of inhibitors of VEGF (or its receptors), but can be treated with antihypertensive agents and rarely requires discontinuation of therapy. Rare but serious potential risks include arterial thromboembolic events, including stroke and myocardial infarction, and hemorrhage. Another serious complication is bowel perforation, which has been observed in 1–3% of patients (mainly those with colon and ovarian cancers). Inhibition of wound healing is also seen.

1	Several small-molecule inhibitors (SMIs) that target VEGFR tyrosine kinase activity but are also inhibitory to other kinases have also been approved to treat certain cancers. Sunitinib (see above and Table 102e-2) has activity directed against mutant c-Kit receptors (approved for GIST), but also targets VEGFR and PDGFR, and has shown significant antitumor activity against metastatic RCC, presumably on the basis of its antiangiogenic activity. Similarly, sorafenib, originally developed as a Raf kinase inhibitor but with potent activity against VEGFR and PDGFR, has activity against RCC, thyroid cancer, and hepatocellular cancer. Other inhibitors of VEGFR approved for the treatment of RCC include axitinib and pazopanib. The success in targeting tumor angiogenesis has led to enhanced enthusiasm for the development of drugs that target other aspects of the angiogenic process; some of these therapeutic approaches are outlined in Fig. 102e-12.

1	Cancers have a number of mechanisms that allow them to evade detection and elimination by the immune system. These include down-regulation of cell surface proteins involved in immune recognition (including MHC proteins and tumor-specific antigens), expression of other cell surface proteins that inhibit immune function (including members of the B7 family of proteins such as PD-L1), secretion of proteins and other molecules that are immunosuppressive, recruitment and expansion of immunosuppressive cells such as regulatory T cells, and induction of T cell tolerance. In addition, the inflammatory effects of some of the immune mediator cells in the tumor microenvironment (especially tissue-associated macrophages and myeloid-derived suppressor cells) can suppress T cell responses to the tumor as well as stimulate inflammation that can enhance tumor growth.

1	Immunotherapy approaches to treat cancer aimed at activating the immune response against tumors using immunostimulatory molecules such as interferons, IL-2, and monoclonal antibodies have had some successes. Another approach that has shown particular clinical promise is the targeting of proteins or cells (such as regulatory T cells) involved in normal homeostatic control to prevent autoimmune damage to the host but that malignant cells and their stroma can also use to inhibit the immune response directed against them. The approach that is furthest along clinically has involved targeting CTLA-4, PD-1, and PDL-1, co-inhibitory molecules that are expressed on the surface of cancer cells, cells of the immune system, and/or stromal cells and are involved in inhibiting the immune response against cancer (Fig. 102e13). Monoclonal antibodies directed against CTLA-4 and PD-1 are approved for the treatment of melanoma, and additional antibodies

1	VEGFR2 VEGF Tie2 receptor EPH receptor Novel inhibitors Novel inhibitors Stromal cell Ang 1 Ang 2 Kinase domain Anti-integrin MoAb, RGD peptides Extracellular matrix (ECM) Specific kinase inhibitors Proliferation survival migration Enhanced binding to ECM, vessel stabilization Ephrin-B2 Nucleus Microtubules 2-Methoxy estradiol ˜v°3 ˜v°5 ˜5°1 MMPs (invasion, growth factor Anti-VEGF MoAb Endothelial cell

1	FIGuRE 102e-12 Knowledge of the molecular events governing tumor angiogenesis has led to a number of therapeutic strategies to block tumor blood vessel formation. The successful therapeutic targeting of VEGF is described in the text. Other endothelial cell– specific receptor tyrosine kinase pathways (e.g., angiopoietin/Tie2 and ephrin/EPH) are likely targets for the future. Ligation of the αvβ3 integrin is required for endothelial cell (EC) survival. Integrins are also required for EC migration and are important regulators of matrix metalloproteinase (MMP) activity, which modulates EC movement through the extracellular matrix (ECM) as well as release of bound growth factors. Targeting of integrins includes development of blocking antibodies, small peptide inhibitors of integrin signaling, and arg-gly-asp–containing peptides that prevent integrin:ECM binding. Peptides derived from normal proteins by proteolytic cleavage, including endostatin and tumstatin, inhibit angiogenesis by

1	and arg-gly-asp–containing peptides that prevent integrin:ECM binding. Peptides derived from normal proteins by proteolytic cleavage, including endostatin and tumstatin, inhibit angiogenesis by mechanisms that include interfering with integrin function. Signal transduction pathways that are dysregulated in tumor cells indirectly regulate EC function. Inhibition of EGF-family receptors, whose signaling activity is upregulated in a number of human cancers (e.g., breast, colon, and lung cancers), results in downregulation of VEGF and IL-8, while increasing expression of the antiangiogenic protein thrombospondin-1. The Ras/MAPK, PI3K/Akt, and Src kinase pathways constitute important antitumor targets that also regulate the proliferation and survival of tumor-derived EC. The discovery that ECs from normal tissues express tissue-specific “vascular addressins” on their cell surface suggests that targeting specific EC subsets may be possible.

1	Tumor cells Elaboration of immunosuppressive cytokines TGF-˜ Interleukin-4 Interleukin-6 Interleukin-10 Induction of CTLA-4 Induction of PD-1 T cell inactivation Cell signaling disruption Degradation of T cell receptor ° chain Class I MHC loss in tumor cells STAT-3 signaling loss in T cells Generation of indoleamine 2, 3-dioxygenase Immunosuppressive immune cells FIGuRE 102e-13 Tumor-host interactions that suppress the immune response to the tumor.

1	FIGuRE 102e-13 Tumor-host interactions that suppress the immune response to the tumor. targeting PD-1 or PDL-1 have shown activity against melanoma, RCC, and lung cancer and continue to be evaluated against other malignancies as well. Combination approaches targeting more than one protein or involving other anticancer approaches (targeted agents, chemotherapy, radiation therapy) are also being explored and have shown promise in early studies. An important aspect of these approaches is balancing sufficient release of the negative control of the immune response to allow immune-mediated attack on the tumors while not allowing too much release and inducing severe autoimmune effects (such as against skin, thyroid, pituitary gland, or the gastrointestinal tract).

1	The explosion of information on tumor cell biology, metastasis, and tumor-host interactions (including angiogenesis and immune evasion by tumors) has ushered in a new era of rational targeted therapy for cancer. Furthermore, it has become clear that specific molecular factors detected in individual tumors (specific gene mutations, gene-expression profiles, microRNA expression, overexpression of specific proteins) can be used to tailor therapy and maximize antitumor effects. Robert G. Fenton contributed to this chapter in prior editions, and important material from those prior chapters has been included here.

1	Robert G. Fenton contributed to this chapter in prior editions, and important material from those prior chapters has been included here. principles of Cancer treatment Edward A. Sausville, Dan L. Longo CANCER PRESENTATION Cancer in a localized or systemic state is a frequent item in the differ-ential diagnosis of a variety of common complaints. Although not all forms of cancer are curable at diagnosis, affording patients the greatest 103e opportunity for cure or meaningful prolongation of life is greatly aided by diagnosing cancer at the earliest point possible in its natural history and defining treatments that prevent or retard its systemic spread. Indeed, certain forms of cancer, notably breast, colon, and possibly lung cancers in certain patients, can be prevented by screening appropriately selected asymptomatic patients; screening is arguably the earliest point in the spectrum of possible cancer-related interventions where cure is possible (Table 103e-1).

1	The term cancer, as used here, is synonymous with the term tumor, whose original derivation from Latin simply meant “swelling,” not otherwise specified. We now understand that the swelling that is a common physical manifestation of a tumor derives from increased interstitial fluid pressure and increased cellular and stromal mass per volume, compared to normal tissue. Tumors historically were referred to as carcinomas, or “crab-like” infiltrating tumors, or sarcomas, or “fleshy tumors,” derived from the Greek terms for “crab” and “flesh,” respectively. Leukemias are a special case of a cancer of the blood-forming tissues presenting in a disseminated form frequently without definable tumor masses. In addition to localized swelling, tumors present by altered function of the organ they afflict, such as dyspnea on exertion from the anemia caused by leukemia replacing normal hematopoietic cells, cough from lung cancers, jaundice from tumors disrupting the hepatobiliary tree, or seizures and

1	such as dyspnea on exertion from the anemia caused by leukemia replacing normal hematopoietic cells, cough from lung cancers, jaundice from tumors disrupting the hepatobiliary tree, or seizures and neurologic signs from brain tumors. Hemorrhage is also a frequent presenting sign of tumors involving hollow viscera, as are decreases in the number of platelets and inappropriate inhibition of blood coagulation. Thus, although statistically the fraction of patients with cancer underlying a particular presenting sign or symptom may be low, the implications for a patient with cancer of missing an early-stage tumor call for vigilance; therefore, persistent signs or symptoms should be evaluated as possibly coming from an early-stage tumor.

1	Evidence of a tumor’s existence can objectively be established by careful physical examination, such as enlarged lymph nodes in lymphomas or a palpable mass in a breast or soft tissue site. A mass SpeCtrum of CanCer-related InterventIonS Consideration of cancer in a differential diagnosis Physical examination, imaging, or endoscopy to define a possible tumor Diagnosis of cancer by biopsy or removal: Specialized histology: immunohistochemistry Staging the cancer: Where has it spread? During treatment: related to tumor effects on patient During treatment to counteract side effects of treatment Palliative and end of life

1	Staging the cancer: Where has it spread? During treatment: related to tumor effects on patient During treatment to counteract side effects of treatment Palliative and end of life When useful treatments are not feasible or desired may also be detected or confirmed by an imaging modality, such as 103e-1 plain x-ray, computed tomography (CT) scan, ultrasound, positron emission tomography (PET) imaging, or nuclear magnetic resonance approaches. Sensitivity of these technologies varies considerably, and the index of suspicion for a tumor should match the technology chosen. For example, low-dose helical CT scans are superior to plain chest radiographs in detecting lung cancers. Another way of initially establishing the existence of a possible tumor is through direct visualization of an afflicted organ by endoscopy.

1	Once the existence of a likely tumor is defined, unequivocally establishing the diagnosis is the next step in the spectrum of correctly addressing a patient’s needs. This is usually accomplished by a biopsy procedure and the emergence after pathologic examination of an unequivocal statement that cancer is present. The underlying principle in cancer diagnosis is to obtain as much tissue as safely as possible. Due to tumor heterogeneity, pathologists are better able to make the diagnosis when they have more tissue to examine. In addition to light microscopic inspection of a tumor for pattern of growth, degree of cellular atypia, invasiveness, and morphologic features that aid in the differential diagnosis, sufficient tissue is of value in searching for genetic abnormalities and protein expression patterns, such as hormone receptor expression in breast cancers, that may aid in the differential diagnosis or provide information about prognosis or likely response to treatment. Efforts to

1	patterns, such as hormone receptor expression in breast cancers, that may aid in the differential diagnosis or provide information about prognosis or likely response to treatment. Efforts to define “personalized” information from the biology of each patient’s tumor and pertinent to each patient’s treatment plan are becoming increasingly important in selecting treatment options. The general internist should make sure that a patient’s cancer biopsy is appropriately referred from the surgical suite for important molecular studies that can advise the best treatment (Table 103e-2).

1	Similar-appearing tumors by microscopic morphology may have very different gene expression patterns when assessed by such techniques as microarray analysis for gene expression patterns using gene dIagnoStIC bIopSy: Standard of Care moleCular and SpeCIal StudIeS Breast cancer: primary and suspected metastatic Hormone receptors: estrogen, progesterone HER2/neu oncoprotein Lung cancer: primary and suspected metastatic If nonsquamous non-small cell: epidermal growth factor receptor mutation; alk oncoprotein gene fusion Colon cancer: suspected metastatic Ki-ras mutation Gastrointestinal stromal tumor c-kit oncoprotein mutation Bcr-Abl fusion protein t(15,17) inversion 16 t(8,21) Lymphoma Immunohistochemistry for CD20, CD30, T cell markers Treatment defining chromosomal translocations: t(14,18) t(8,14)

1	Chapter 103e Principles of Cancer Treatment chips, with important differences in biology and response to treatment. Such testing requires that the tissue be handled properly (e.g., immunologic detection of proteins is more effective in fresh-frozen tissue rather than in formalin-fixed tissue). Coordination among the surgeon, pathologist, and primary care physician is essential to ensure that the amount of information learned from the biopsy material is maximized. These goals are best met by an excisional biopsy in which the entire tumor mass is removed with a small margin of normal tissue surrounding it. If an excisional biopsy cannot be performed, incisional biopsy is the procedure of second choice. A wedge of tissue is removed, and an effort is made to include the majority of the cross-sectional diameter of the tumor in the biopsy to minimize sampling error. Biopsy techniques that involve cutting into tumor carry with them a risk of facilitating the spread of the tumor, and

1	cross-sectional diameter of the tumor in the biopsy to minimize sampling error. Biopsy techniques that involve cutting into tumor carry with them a risk of facilitating the spread of the tumor, and consideration of whether the biopsy might be the prelude to a curative surgery if certain diagnoses are established should inform the actual approach taken. Core-needle biopsy usually obtains considerably less tissue, but this procedure often provides enough information to plan a definitive surgical procedure. Fine-needle aspiration generally obtains only a suspension of cells from within a mass. This procedure is minimally invasive, and if positive for cancer, it may allow inception of systemic treatment when metastatic disease is evident, or it can provide a basis for planning a more meticulous and extensive surgical procedure. However, a negative fine-needle aspiration for a neoplastic diagnosis cannot be taken as definitive evidence that a tumor is absent or make a definitive diagnosis

1	and extensive surgical procedure. However, a negative fine-needle aspiration for a neoplastic diagnosis cannot be taken as definitive evidence that a tumor is absent or make a definitive diagnosis in someone not known to have a cancer.

1	An essential component of correct patient management in many cancer types is defining the extent of disease, because this information critically informs whether localized treatments, “combined-modality” approaches, or systemic treatments should initially be considered. Radiographic and other imaging tests can be helpful in defining the clinical stage; however, pathologic staging requires defining the extent of involvement by documenting the histologic presence of tumor in tissue biopsies obtained through a surgical procedure. Axillary lymph node sampling in breast cancer and lymph node sampling at laparotomy for testicular, colon, and other intraabdominal cancers may provide crucial information for treatment planning and may determine the extent and nature of primary cancer treatment.

1	For tumors associated with a potential “primary site,” staging systems have evolved to define a “T” component related to the size of the tumor or its invasion into local structures, an “N” component related to the number and nature of lymph node groups adjacent to the tumor with evidence of tumor spread, and an “M” component, based on the presence of local or distant metastatic sites. The various “TNM” components are then aggregated to stages, usually stage I to III or IV, depending on the anatomic site. The numerical stages reflect similar long-term survival outcomes of the aggregated TNM groupings in a numeric stage after treatment tailored to the stage. In general, stage I tumors are T1 (reflecting small size), N0 or N1 (reflecting no or minimal node spread), and M0 (no metastases). Such early-stage tumors are amenable to curative approaches with local treatments. On the other hand, stage IV tumors usually have metastasized to distant sites or locally invaded viscera in a

1	Such early-stage tumors are amenable to curative approaches with local treatments. On the other hand, stage IV tumors usually have metastasized to distant sites or locally invaded viscera in a nonresectable way and are dealt with using techniques that have palliative intent, except for those diseases with exceptional sensitivity to systemic treatments such as chemotherapy or immunotherapy. Also, the TNM staging system is not useful in diseases such as leukemia, where bone marrow infiltration is never really localized, or central nervous system tumors, where tumor histology and the extent of anatomically feasible resection are more important in driving prognosis.

1	The goal of cancer treatment is first to eradicate the cancer. If this primary goal cannot be accomplished, the goal of cancer treatment shifts to palliation, the amelioration of symptoms, and preservation of quality of life while striving to extend life. The dictum primum non nocere may not always be the guiding principle of cancer therapy. When cure of cancer is possible, cancer treatments may be considered despite the certainty of severe and perhaps life-threatening toxicities. Every cancer treatment has the potential to cause harm, and treatment may be given that produces toxicity with no benefit. The therapeutic index of many interventions may be quite narrow, with treatments given to the point of toxicity. Conversely, when the clinical goal is palliation, careful attention to minimizing the toxicity of potentially toxic treatments becomes a significant goal.

1	Cancer treatments are divided into two main types: local and systemic. Local treatments include surgery, radiation therapy (including photodynamic therapy), and ablative approaches, including radio-frequency and cryosurgical approaches. Systemic treatments include chemotherapy (including hormonal therapy and molecularly targeted therapy) and biologic therapy (including immunotherapy). The modalities are often used in combination, and agents in one category can act by several mechanisms. For example, cancer chemotherapy agents can induce differentiation, and antibodies (a form of immunotherapy) can be used to deliver radiation therapy. Oncology, the study of tumors including treatment approaches, is a multidisciplinary effort with surgical, radiation, and internal medicine–related areas of oncologic expertise. Treatments for patients with hematologic malignancies are often shared by hematologists and medical oncologists.

1	In many ways, cancer mimics an organ attempting to regulate its own growth. However, cancers have not set an appropriate limit on how much growth should be permitted. Normal organs and cancers share the property of having (1) a population of cells actively progressing through the cell cycle with their division providing a basis for tumor growth, and (2) a population of cells not in cycle. In cancers, cells that are not dividing are heterogeneous; some have sustained too much genetic damage to replicate but have defects in their death pathways that permit their survival, some are starving for nutrients and oxygen, and some are out of cycle but poised to be recruited back into cycle and expand if needed (i.e., reversibly growth-arrested). Severely damaged and starving cells are unlikely to kill the patient. The problem is that the cells that are reversibly not in cycle are capable of replenishing tumor cells physically removed or damaged by radiation and chemotherapy. These include

1	to kill the patient. The problem is that the cells that are reversibly not in cycle are capable of replenishing tumor cells physically removed or damaged by radiation and chemotherapy. These include cancer stem cells, whose properties are being elucidated, as they may serve as a basis for giving rise to tumor initiating or repopulating cells. The stem cell fraction may define new targets for therapies that will retard their ability to reenter the cell cycle.

1	Tumors follow a Gompertzian growth curve (Fig. 103e-1), with the apparent growth fraction of a neoplasm being high with small tumor burdens and declining until, at the time of diagnosis, with a tumor burden of 1–5 × 109 tumor cells, the growth fraction is usually 1–4% for many solid tumors. By this view, the most rapid growth rate occurs before the tumor is detectable. An alternative explanation for such growth properties may also emerge from the ability of tumors at metastatic sites to recruit circulating tumor cells from the primary tumor or other metastases. An additional key feature of a successful tumor is the ability to stimulate the development of a new supporting stroma through angiogenesis and production of proteases to allow invasion through basement membranes and normal tissue barriers (Chap. 102e). Specific cellular mechanisms promote entry or withdrawal of tumor cells from the cell cycle. For example, when a tumor recurs after surgery or chemotherapy, frequently its

1	barriers (Chap. 102e). Specific cellular mechanisms promote entry or withdrawal of tumor cells from the cell cycle. For example, when a tumor recurs after surgery or chemotherapy, frequently its growth is accelerated and the growth fraction of the tumor is increased. This pattern is similar to that seen in regenerating organs. Partial resection of the liver results in the recruitment of cells into the cell cycle, and the resected liver volume is replaced. Similarly, chemotherapy-damaged bone marrow increases its growth to replace cells killed by chemotherapy. However, cancers do not recognize a limit on their expansion. Monoclonal gammopathy of uncertain significance may be an example of a clonal neoplasm with intrinsic features that stop its growth before a lethal tumor burden is reached. A fraction of patients with this disorder go on to develop fatal multiple myeloma, but probably this occurs because of the accumulation of additional genetic lesions. Elucidation of the mechanisms

1	A fraction of patients with this disorder go on to develop fatal multiple myeloma, but probably this occurs because of the accumulation of additional genetic lesions. Elucidation of the mechanisms that regulate this “organ-like” behavior of tumors may provide additional clues to cancer control and treatment.

1	necessary to obtain the best outcomes. Thus, lumpectomy with 103e-3 radiation therapy is as effective as modified radical mastectomy for breast cancer, and limb-sparing surgery followed by adjuvant radia rhabdomyosarcomas and osteosarcomas. More limited surgery is also being used to spare organ function, as in larynx and bladder cancer. The magnitude of operations necessary to optimally control and cure 1012 Lethal cancer has also been diminished by technical advances; for example, the circular anastomotic stapler has allowed narrower (<2 cm) margins Tumor burden logs of cells in colon cancer without compromise of local control rates, and many patients who would have had colostomies are able to maintain normal anatomy.

1	Tumor burden logs of cells in colon cancer without compromise of local control rates, and many patients who would have had colostomies are able to maintain normal anatomy. In some settings (e.g., bulky testicular cancer or stage III breast can of a tumor declines exponentially over time (top). The growth rate of a tumor peaks before it is clinically detectable (middle). Tumor size increases slowly, goes through an exponential phase, and slows again as the tumor reaches the size at which limitation of nutrients or autoregulatory or host regulatory influences can occur. The maximum growth rate occurs at 1/e, the point at which the tumor is about 37% of its maximum size (marked with an X ). Tumor becomes detectable at a burden of about 109 (1 cm3) cells and kills the patient at a tumor cell burden of about 1012 (1 kg). Efforts to treat the tumor and reduce its size can result in an increase in the growth fraction and an increase in growth rate.

1	Surgery is unquestionably the most effective means of treating cancer. Today at least 40% of cancer patients are cured by surgery. Unfortunately, a large fraction of patients with solid tumors (perhaps 60%) have metastatic disease that is not accessible for removal. However, even when the disease is not curable by surgery alone, the removal of tumor can obtain important benefits, including local control of tumor, preservation of organ function, debulking that permits subsequent therapy to work better, and staging information on extent of involvement. Cancer surgery aiming for cure is usually planned to excise the tumor completely with an adequate margin of normal tissue (the margin varies with the tumor and the anatomy), touching the tumor as little as possible to prevent vascular and lymphatic spread, and minimizing operative risk. Such a resection is defined as an R0 resection. R1 and R2 resections, in contrast, are imprecisely defined pathologically as having microscopic or

1	and lymphatic spread, and minimizing operative risk. Such a resection is defined as an R0 resection. R1 and R2 resections, in contrast, are imprecisely defined pathologically as having microscopic or macroscopic tumor at resection margins. Such outcomes may be necessitated by proximity of the tumor to vital structures or recognition only in the resected specimen of the extent of tumor involvement, and may be the basis for reoperation to obtain optimal margins if feasible. Extending the procedure to resect draining lymph nodes obtains prognostic information and may, in some anatomic locations, improve survival.

1	Increasingly, laparoscopic approaches are being used to address primary abdominal and pelvic tumors. Lymph node spread may be assessed using the sentinel node approach, in which the first draining lymph node a spreading tumor would encounter is defined by injecting a dye or radioisotope into the tumor site at operation and then resecting the first node to turn blue or collect label. The sentinel node assessment is continuing to undergo clinical evaluation but appears to provide reliable information without the risks (lymphedema, lymphangiosarcoma) associated with resection of all the regional nodes. Advances in adjuvant chemotherapy (chemotherapy given systemically after removal of all disease by operation and without evidence of active metastatic disease) and radiation therapy following surgery have permitted a substantial decrease in the extent of primary surgery cer), surgery is not the first treatment modality used. After an initial diagnostic biopsy, chemotherapy and/or radiation

1	have permitted a substantial decrease in the extent of primary surgery cer), surgery is not the first treatment modality used. After an initial diagnostic biopsy, chemotherapy and/or radiation therapy is delivered to reduce the size of the tumor and clinically control undetected metastatic disease. Such therapy is followed by a surgical procedure to remove residual masses; this is called neoadjuvant therapy. Because the sequence of treatment is critical to success and is different from the standard surgery-first approach, coordination among the surgical oncologist, radiation oncologist, and medical oncologist is crucial.

1	Surgery may be curative in a subset of patients with metastatic disease. Patients with lung metastases from osteosarcoma may be cured by resection of the lung lesions. In patients with colon cancer who have fewer than five liver metastases restricted to one lobe and no extrahepatic metastases, hepatic lobectomy may produce long-term disease-free survival in 25% of selected patients. Surgery can also be associated with systemic antitumor effects. In the setting of hormonally responsive tumors, oophorectomy and/or adrenalectomy may eliminate estrogen production, and orchiectomy may reduce androgen production, hormones that drive certain breast and all prostate cancers, respectively; both procedures can have useful effects on metastatic tumor growth. If resection of the primary lesion takes place in the presence of metastases, acceleration of metastatic growth has also been described in certain cases, perhaps based on the removal of a source of angiogenesis inhibitors and mass-related

1	place in the presence of metastases, acceleration of metastatic growth has also been described in certain cases, perhaps based on the removal of a source of angiogenesis inhibitors and mass-related growth regulators in the tumor.

1	In selecting a surgeon or center for primary cancer treatment, consideration must be given to the volume of cancer surgeries undertaken by the site. Studies in a variety of cancers have shown that increased annual procedure volume appears to correlate with outcome. In addition, facilities with extensive support systems—e.g., for joint thoracic and abdominal surgical teams with cardiopulmonary bypass, if needed—may allow resection of certain tumors that would otherwise not be possible.

1	Surgery is used in a number of ways for palliative or supportive care of the cancer patient, not related to the goal of curing the cancer. These include insertion and care of central venous catheters, control of pleural and pericardial effusions and ascites, caval interruption for recurrent pulmonary emboli, stabilization of cancer-weakened weight-bearing bones, and control of hemorrhage, among others. Surgical bypass of gastrointestinal, urinary tract, or biliary tree obstruction can alleviate symptoms and prolong survival. Surgical procedures may provide relief of otherwise intractable pain or reverse neurologic dysfunction (cord decompression). Splenectomy may relieve symptoms and reverse hypersplenism. Intrathecal or intrahepatic therapy relies on surgical placement of appropriate infusion portals. Surgery may correct other treatment-related toxicities such as adhesions or strictures. Surgical procedures are also valuable in rehabilitative efforts to restore health or function.

1	portals. Surgery may correct other treatment-related toxicities such as adhesions or strictures. Surgical procedures are also valuable in rehabilitative efforts to restore health or function. Orthopedic procedures may be necessary to ensure proper ambulation. Breast reconstruction can make an enormous impact on the patient’s perception of successful therapy. Plastic and reconstructive surgery can correct the effects of disfiguring primary treatment.

1	Surgery is also a tool valuable in the prevention of cancers in high-risk populations. Prophylactic mastectomy, colectomy, oophorectomy, and thyroidectomy are mainstays of prevention of genetic cancer syndromes. Resection of premalignant skin and uterine cervix lesions and colonic polyps prevents progression to frank malignancy. Chapter 103e Principles of Cancer Treatment

1	RADIATION Radiation Biology and Medicine Therapeutic radiation is ionizing; it damages any tissue in its path. The selectivity of radiation for causing cancer cell death may be due to defects in a cancer cell’s ability to repair sublethal DNA and other damage. Ionizing radiation causes breaks in DNA and generates free radicals from cell water that may damage cell membranes, proteins, and organelles. Radiation damage is augmented by oxygen; hypoxic cells are more resistant. Augmentation of oxygen presence is one basis for radiation sensitization. Sulfhydryl compounds interfere with free radical generation and may act as radiation protectors. X-rays and gamma rays are the forms of ionizing radiation most commonly used to treat cancer. They are both electromagnetic, nonparticulate waves that cause the ejection of an orbital electron when absorbed. This orbital electron ejection is called ionization. X-rays are generated by linear accelerators; gamma rays are generated from decay of

1	that cause the ejection of an orbital electron when absorbed. This orbital electron ejection is called ionization. X-rays are generated by linear accelerators; gamma rays are generated from decay of atomic nuclei in radioisotopes such as cobalt and radium. These waves behave biologically as packets of energy, called photons. Particulate ionizing radiation using protons has also become available. Most radiation-induced cell damage is due to the formation of hydroxyl radicals from tissue water:

1	Radiation is quantitated based on the amount of radiation absorbed by the tumor in the patient; it is not based on the amount of radiation generated by the machine. The International System (SI) unit for radiation absorbed is the Gray (Gy): 1 Gy refers to 1 J/kg of tissue; 1 Gy equals 100 centigrays (cGy) of absorbed dose. A historically used unit appearing in the oncology literature, the rad (radiation absorbed dose), is defined as 100 ergs of energy absorbed per gram of tissue and is equivalent to 1 cGy. Radiation dosage is defined by the energy absorbed per mass of tissue. Radiation dose is measured by placing detectors at the body surface or based on radiating phantoms that resemble human form and substance, containing internal detectors. The features that make a particular cell more sensitive or more resistant to the biologic effects of radiation are not completely defined and critically involve DNA repair proteins that, in their physiologic role, protect against environmentally

1	sensitive or more resistant to the biologic effects of radiation are not completely defined and critically involve DNA repair proteins that, in their physiologic role, protect against environmentally related DNA damage.

1	Localized Radiation Therapy Radiation effect is influenced by three determinants: total absorbed dose, number of fractions, and time of treatment. A frequent error is to omit the number of fractions and the duration of treatment. This is analogous to saying that a runner completed a race in 20 s; without knowing how far he or she ran, the result is difficult to interpret. The time could be very good for a 200-m race or very poor for a 100-m race. Thus, a typical course of radiation therapy should be described as 4500 cGy delivered to a particular target (e.g., mediastinum) over 5 weeks in 180-cGy fractions. Most curative radiation treatment programs are delivered once a day, 5 days a week, in 150to 200-cGy fractions.

1	A number of parameters influence the damage done to tissue (normal and tumor) by radiation. Hypoxic cells are relatively resistant. Nondividing cells are more resistant than dividing cells, and this is one rationale for delivering radiation in repeated fractions, to ultimately expose a larger number of tumor cells that have entered the division cycle. In addition to these biologic parameters, physical parameters of the radiation are also crucial. The energy of the radiation determines its ability to penetrate tissue. Low-energy orthovoltage beams (150–400 kV) scatter when they strike the body, much like light diffuses when it strikes particles in the air. Such beams result in more damage to adjacent normal tissues and less radiation delivered to the tumor. Megavoltage radiation (>1 MeV) has very low lateral scatter; this produces a skin-sparing effect, more homogeneous distribution of the radiation energy, and greater deposit of the energy in the tumor, or target volume. The tissues

1	has very low lateral scatter; this produces a skin-sparing effect, more homogeneous distribution of the radiation energy, and greater deposit of the energy in the tumor, or target volume. The tissues that the beam passes through to get to the tumor are called the transit volume. The maximum dose in the target volume is often the cause of complications to tissues in the transit volume, and the minimum dose in the target volume influences the likelihood of tumor recurrence. Dose homogeneity in the target volume is the goal. Computational approaches and delivery of many beams to converge on a target lesion are the basis for “gamma knife” and related approaches to deliver high doses to small volumes of tumor, sparing normal tissue.

1	Therapeutic radiation is delivered in three ways: (1) teletherapy, with focused beams of radiation generated at a distance and aimed at the tumor within the patient; (2) brachytherapy, with encapsulated sources of radiation implanted directly into or adjacent to tumor tissues; and (3) systemic therapy, with radionuclides administered, for example, intravenously but targeted by some means to a tumor site. Teletherapy with x-ray or gamma-ray photons is the most commonly used form of radiation therapy. Particulate forms of radiation are also used in certain circumstances, such as the use of proton beams. The difference between photons and protons relates to the volume in which the greatest delivery of energy occurs. Typically protons have a much narrower range of energy deposition, theoretically resulting in more precise delivery of radiation with improvement in the degree to which adjacent structures may be affected, in comparison to photons. Electron beams are a particulate form of

1	resulting in more precise delivery of radiation with improvement in the degree to which adjacent structures may be affected, in comparison to photons. Electron beams are a particulate form of radiation that, in contrast to photons and protons, have a very low tissue penetrance and are used to treat cutaneous tumors. Apart from sparing adjacent structures, particulate forms of radiation are in most applications not superior to x-rays or gamma rays in clinical studies reported thus far, but this is an active area of investigation.

1	Certain drugs used in cancer treatment may also act as radiation sensitizers. For example, compounds that incorporate into DNA and alter its stereochemistry (e.g., halogenated pyrimidines, cisplatin) augment radiation effects at local sites, as does hydroxyurea, another DNA synthesis inhibitor. These are important adjuncts to the local treatment of certain tumors, such as squamous head and neck, uterine cervix, and rectal cancers.

1	Toxicity of Radiation Therapy Although radiation therapy is most often administered to a local region, systemic effects, including fatigue, anorexia, nausea, and vomiting, may develop that are related in part to the volume of tissue irradiated, dose fractionation, radiation fields, and individual susceptibility. Injured tissues release cytokines that act systemically to produce these effects. Bone is among the most radio-resistant organs, with radiation effects being manifested mainly in children through premature fusion of the epiphyseal growth plate. By contrast, the male testis, female ovary, and bone marrow are the most sensitive organs. Any bone marrow in a radiation field will be eradicated by therapeutic irradiation. Organs with less need for cell renewal, such as heart, skeletal muscle, and nerves, are more resistant to radiation effects. In radiation-resistant organs, the vascular endothelium is the most sensitive component. Organs with more self-renewal as a part of normal

1	muscle, and nerves, are more resistant to radiation effects. In radiation-resistant organs, the vascular endothelium is the most sensitive component. Organs with more self-renewal as a part of normal homeostasis, such as the hematopoietic system and mucosal lining of the intestinal tract, are more sensitive. Acute toxicities include mucositis, skin erythema (ulceration in severe cases), and bone marrow toxicity. Often these can be alleviated by interruption of treatment.

1	Chronic toxicities are more serious. Radiation of the head and neck region often produces thyroid failure. Cataracts and retinal damage can lead to blindness. Salivary glands stop making saliva, which leads to dental caries and poor dentition. Taste and smell can be affected. Mediastinal irradiation leads to a threefold increased risk of fatal myocardial infarction. Other late vascular effects include chronic constrictive pericarditis, lung fibrosis, viscus stricture, spinal cord transection, and radiation enteritis. A serious late toxicity is the development of second solid tumors in or adjacent to the radiation fields. Such tumors can develop in any organ or tissue and occur at a rate of about 1% per year beginning in the second decade after treatment. Some organs vary in susceptibility to radiation carcinogenesis. A woman who receives mantle field radiation therapy for Hodgkin’s disease at age 25 years has a 30% risk of developing breast cancer by age 55 years. This is comparable

1	to radiation carcinogenesis. A woman who receives mantle field radiation therapy for Hodgkin’s disease at age 25 years has a 30% risk of developing breast cancer by age 55 years. This is comparable in magnitude to genetic breast cancer syndromes. Women treated after age 30 years have little or no increased risk of breast cancer. No data suggest that a threshold dose of therapeutic radiation exists below which the incidence of second cancers is decreased. High rates of second tumors occur in people who receive as little as 1000 cGy.

1	Endoscopy techniques may allow the placement of stents to unblock viscera by mechanical means, palliating, for example, gastrointestinal or biliary obstructions. Radiofrequency ablation (RFA) refers to the use of focused microwave radiation to induce thermal injury within a volume of tissue. RFA can be useful in the control of metastatic lesions, particularly in liver, that may threaten biliary drainage (as one example) and threaten quality and duration of useful life in patients with otherwise unresectable disease. Cryosurgery uses extreme cold to sterilize lesions in certain sites, such as prostate and kidney, when at a very early stage, eliminating the need for modalities with more side effects such as surgery or radiation.

1	Some chemicals (porphyrins, phthalocyanines) are preferentially taken up by cancer cells by mechanisms not fully defined. When light, usually delivered by a laser, is shone on cells containing these compounds, free radicals are generated and the cells die. Hematoporphyrins and light (phototherapy) are being used with increasing frequency to treat skin cancer; ovarian cancer; and cancers of the lung, colon, rectum, and esophagus. Palliation of recurrent locally advanced disease can sometimes be dramatic and last many months. Infusion of chemotherapeutic or biologic agents or radiation-bearing delivery devices such as isotope-coated glass spheres into local sites through catheters inserted into specific vascular sites such as liver or an extremity have been used in an effort to control disease limited to that site; in selected cases, prolonged control of truly localized disease has been possible.

1	The concept that systemically administered agents may have a useful effect on cancers was historically derived from three sets of observations. Paul Ehrlich in the nineteenth century observed that different dyes reacted with different cell and tissue components. He hypothesized the existence of compounds that would be “magic bullets” that might bind to tumors, owing to the affinity of the agent for the tumor. A second observation was the toxic effects of certain mustard gas derivatives on the bone marrow during World War I, leading to the idea that smaller doses of these agents might be used to treat tumors of marrow-derived cells. Finally, the observation that certain tumors from hormone-responsive tissues, e.g., breast tumors, could shrink after oophorectomy led to the idea that endogenous substances promoting the growth of a tumor might be antagonized. Chemicals achieving each of the goals are actually or intellectually the forbearers of the currently used cancer chemotherapy

1	substances promoting the growth of a tumor might be antagonized. Chemicals achieving each of the goals are actually or intellectually the forbearers of the currently used cancer chemotherapy agents.

1	Systemic cancer treatments are of four broad types. Conventional “cytotoxic” chemotherapy agents were historically derived by the empirical observation that these “small molecules” (generally with molecular mass <1500 Da) could cause major regression of experimental tumors growing in animals. These agents mainly target DNA structure or segregation of DNA as chromosomes in mitosis. Targeted agents refer to small molecules or “biologics” (generally macromolecules such as antibodies or cytokines) designed and developed to interact with a defined molecular target important in maintaining the malignant state or expressed by the tumor cells. As described in Chap. 102e, successful tumors have activated biochemical pathways that lead to uncontrolled proliferation through the action of, e.g., oncogene products, loss of cell cycle inhibitors, or loss of cell death regulation, and have acquired the capacity to replicate chromosomes indefinitely, invade, metastasize, and evade the immune system.

1	products, loss of cell cycle inhibitors, or loss of cell death regulation, and have acquired the capacity to replicate chromosomes indefinitely, invade, metastasize, and evade the immune system. Targeted therapies seek to capitalize on the biology behind the aberrant cellular behavior as a basis for therapeutic effects. Hormonal therapies (the first form of targeted therapy) capitalize on the biochemical pathways underlying estrogen and androgen function and action as a therapeutic basis for approaching patients with tumors of breast, prostate, uterus, and ovarian origin. Biologic therapies are often macromolecules that have a particular target (e.g., antigrowth factor or cytokine antibodies) or may have the capacity to regulate growth 103e-5 of tumor cells or induce a host immune response to kill tumor cells. Thus, biologic therapies include not only antibodies but also cytokines and gene therapies.

1	CANCER CHEMOTHERAPY Principles The usefulness of any drug is governed by the extent to which a given dose causes a useful result (therapeutic effect; in the case of anticancer agents, toxicity to tumor cells) as opposed to a toxic effect to the host. The therapeutic index is the degree of separation between toxic and therapeutic doses. Really useful drugs have large therapeutic indices, and this usually occurs when the drug target is expressed in the disease-causing compartment as opposed to the normal compartment. Classically, selective toxicity of an agent for a tissue or cell type is governed by the differential expression of a drug’s target in the “sensitive” cell type or by differential drug accumulation into or elimination from compartments where greater or lesser toxicity is experienced, respectively. Currently used chemotherapeutic agents have the unfortunate property that their targets are present in both normal and tumor tissues. Therefore, they have relatively narrow

1	experienced, respectively. Currently used chemotherapeutic agents have the unfortunate property that their targets are present in both normal and tumor tissues. Therefore, they have relatively narrow therapeutic indices.

1	Figure 103e-2 illustrates steps in cancer drug development. Following demonstration of antitumor activity in animal models, potentially useful anticancer agents are further evaluated to define an optimal schedule of administration and arrive at a drug formulation designed for a given route of administration and schedule. Safety testing in two species on an analogous schedule of administration defines the starting dose for a phase 1 trial in humans, usually but not always in patients with cancer who have exhausted “standard” (already approved) treatments. The initial dose is usually one-sixth to one-tenth of the dose just causing easily reversible toxicity in the more sensitive animal species. Escalating doses of the drug are then given during the human phase 1 trial until reversible toxicity is observed. Dose-limiting toxicity (DLT) defines a dose that conveys greater toxicity than would be acceptable in routine practice, allowing definition of a lower maximum-tolerated dose (MTD).

1	is observed. Dose-limiting toxicity (DLT) defines a dose that conveys greater toxicity than would be acceptable in routine practice, allowing definition of a lower maximum-tolerated dose (MTD). The occurrence of toxicity is, if possible, correlated with plasma drug concentrations. The MTD or a dose just lower than the MTD is usually the dose suitable for phase 2 trials, where a fixed dose is administered to a relatively homogeneous set of patients with a particular tumor type in an effort to define whether the drug causes regression of tumors. In a phase 3 trial, evidence of improved overall survival or improvement in the time to progression of disease on the part of the new drug is sought in comparison to an appropriate control population, which is usually receiving an acceptable “standard of care” approach. A favorable outcome of a phase 3 trial is the basis for application to a regulatory agency for approval of the new agent for commercial marketing as safe and possessing a measure

1	of care” approach. A favorable outcome of a phase 3 trial is the basis for application to a regulatory agency for approval of the new agent for commercial marketing as safe and possessing a measure of clinical effectiveness.

1	Response, defined as tumor shrinkage, is the most immediate indicator of drug effect. To be clinically valuable, responses must translate into clinical benefit. This is conventionally established by a beneficial effect on overall survival, or at least an increased time to further progression of disease. Karnofsky was among the first to champion the evaluation of a chemotherapeutic agent’s benefit by carefully quantitating its effect on tumor size and using these measurements to objectively decide the basis for further treatment of a particular patient or further clinical evaluation of a drug’s potential. A partial response (PR) is defined conventionally as a decrease by at least 50% in a tumor’s bidimensional area; a complete response (CR) connotes disappearance of all tumor; progression of disease signifies an increase in size of existing lesions by >25% from baseline or best response or development of new lesions; and stable disease fits into none of the above categories. Newer

1	of disease signifies an increase in size of existing lesions by >25% from baseline or best response or development of new lesions; and stable disease fits into none of the above categories. Newer evaluation systems, such as Response Evaluation Criteria in Solid Tumors (RECIST), use unidimensional measurement, but the intent is similar in rigorously defining evidence for the activity of the agent in assessing its value to the patient. An active chemotherapy agent conventionally has PR rates of at least 20–25% with reversible non-life-threatening side effects, and it may then be suitable for study in phase 3 trials to assess efficacy in comparison to standard or no therapy. Active efforts are being made to quantitate effects of anticancer

1	Chapter 103e Principles of Cancer Treatment Preclinical Model (e.g., mouse or rat) Rx :Time ? Phase II FIGuRE 103e-2 Steps in cancer drug discovery and development. Preclinical activity (top) in animal models of cancers may be used as evidence to support the entry of the drug candidate into phase 1 trials in humans to define a correct dose and observe any clinical antitumor effect that may occur. The drug may then be advanced to phase 2 trials directed against specific cancer types, with rigorous quantitation of antitumor effects (middle). Phase 3 trials then may reveal activity superior to standard or no treatment (bottom).

1	agents on quality of life. Cancer drug clinical trials conventionally use a toxicity grading scale where grade 1 toxicities do not require treatment, grade 2 toxicities may require symptomatic treatment but are not life-threatening, grade 3 toxicities are potentially life-threatening if untreated, grade 4 toxicities are actually life-threatening, and grade 5 toxicities are those that result in the patient’s death.

1	Development of targeted agents may proceed quite differently. While phase 1–3 trials are still conducted, molecular analysis of human tumors may allow the precise definition of target expression in a patient’s tumor that is necessary for or relevant to the drug’s action. This information might then allow selection of patients expressing the drug target for participation in all trial phases. These patients may then have a greater chance of developing a useful response to the drug by virtue of expressing the target in the tumor. Clinical trials may be designed to incorporate an assessment of the behavior of the target in relation to the drug (pharmacodynamic studies). Ideally, the plasma concentration that affects the drug target is known, so escalation to MTD may not be necessary. Rather, the correlation of host toxicity while achieving an “optimal biologic dose” becomes a more relevant endpoint for phase 1 and early phase 2 trials with targeted agents.

1	Useful cancer drug treatment strategies using conventional chemotherapy agents, targeted agents, hormonal treatments, or biologics have one of two valuable outcomes. They can induce cancer cell death, resulting in tumor shrinkage with corresponding improvement in patient survival, or increase the time until the disease progresses. Another potential outcome is to induce cancer cell differentiation or dormancy with loss of tumor cell replicative potential and reacquisition of phenotypic properties resembling normal cells. A blocking in normal cellular differentiation may be a key feature in the pathogenesis of certain leukemias.

1	Cell death is a closely regulated process. Necrosis refers to cell death induced, for example, by physical damage with the hallmarks of cell swelling and membrane disruption. Apoptosis, or programmed cell death, refers to a highly ordered process whereby cells respond to defined stimuli by dying, and it recapitulates the necessary cell death observed during the ontogeny of the organism. Cancer chemotherapeutic agents can cause both necrosis and apoptosis. Apoptosis is characterized by chromatin condensation (giving rise to “apoptotic bodies”), cell shrinkage, and, in living animals, phagocytosis by surrounding stromal cells without evidence of inflammation. This process is regulated either by signal transduction systems that promote a cell’s demise after a certain level of insult is achieved or in response to specific cell-surface receptors that mediate physiologic cell death responses, such as occurs in the developing organism or in the normal function of immune cells. Influencing

1	or in response to specific cell-surface receptors that mediate physiologic cell death responses, such as occurs in the developing organism or in the normal function of immune cells. Influencing apoptosis by manipulation of signal transduction pathways has emerged as a basis for understanding the actions of drugs and designing new strategies to improve their use. Autophagy is a cellular response to injury where the cell does not initially die but catabolizes itself in a way that can lead to loss of replicative potential. A general view of how cancer treatments work is that the interaction of a chemotherapeutic drug with its target induces a “cascade” of further signaling steps. These signals ultimately lead to cell death by triggering an “execution phase” where proteases, nucleases, and endogenous regulators of the cell death pathway are activated (Fig. 103e-3).

1	Targeted agents differ from chemotherapy agents in that they do not indiscriminately cause macromolecular lesions but regulate the action of particular pathways. For example, the p210bcr-abl fusion protein tyrosine kinase drives chronic myeloid leukemia (CML), and HER2/neu stimulates the proliferation of certain breast cancers. The tumor has been described as “addicted” to the function of these molecules in the sense that without the pathway’s continued action, the tumor cell cannot survive. In this way, targeted agents directed at or HER2/neu may alter the “threshold” tumors driven by these molecules may have for undergoing apoptosis without actually creating any molecular lesions such as direct DNA strand breakage or altered membrane function.

1	While apoptotic mechanisms are important in regulating cellular proliferation and the behavior of tumor cells in vitro, in vivo it is unclear whether all of the actions of chemotherapeutic agents to cause cell death can be attributed to apoptotic mechanisms. However, changes in molecules that regulate apoptosis are correlated with clinical outcomes (e.g., bcl2 overexpression in certain lymphomas conveys poor prognosis; proapoptotic bax expression is associated with a better outcome after chemotherapy for ovarian carcinoma). A better understanding of the relationship of cell death and cell survival mechanisms is needed.

1	Chemotherapy agents may be used for the treatment of active, clinically apparent cancer. The goal of such treatment in some cases is cure of the cancer, that is, elimination of all clinical and pathologic evidence of cancer and return of the patient to an expected survival no different than the general population. Table 103e-3, A lists those tumors considered curable by conventionally available chemotherapeutic agents chemotherapy prior to any surgery or radia-103e-7 tion to a local tumor in an effort to enhance the effect of the local treatment. Chemotherapy is routinely used in

1	Chemotherapy is routinely used in FASR “conventional” dose regimens. In general, TRAIL-R effects, primarily consisting of transient therapy regimens are predicated on the myelosuppression with or without gastro- intestinal toxicity (usually nausea), which are readily managed. “High-dose” chemo- observation that the dose-response curve for many anticancer agents is rather steep, increased therapeutic effect, although at the cost of potentially life-threatening com- plications that require intensive support, usually in the form of hematopoietic stem Cytochrome C PIGs, etc cell support from the patient (autologous) or from donors matched for histocompatibility loci (allogeneic), or pharmacologic “rescue” strategies to repair the effect of Chapter 103e Principles of Cancer Treatment the high-dose chemotherapy on normal tissues. High-dose regimens have definite curative potential in defined clinical settings (Table 103e-3, D).

1	If cure is not possible, chemotherapyFIGuRE 103e-3 Integration of cell death responses. Cell death through an apoptotic mecha-may be undertaken with the goal of palliatnism requires active participation of the cell. In response to interruption of growth factor (GF) or ing some aspect of the tumor’s effect on thepropagation of certain cytokine death signals (e.g., tumor necrosis factor receptor [TNF-R]), there host. In this usage, value is perceived by the is activation of “upstream” cysteine aspartyl proteases (caspases), which then directly digest demonstration of improved symptom relief,cytoplasmic and nuclear proteins, resulting in activation of “downstream” caspases; these cause progression-free survival, or overall survivalactivation of nucleases, resulting in the characteristic DNA fragmentation that is a hallmark of at a certain time from the inception of treatapoptosis. Chemotherapy agents that create lesions in DNA or alter mitotic spindle function ment in the treated

1	DNA fragmentation that is a hallmark of at a certain time from the inception of treatapoptosis. Chemotherapy agents that create lesions in DNA or alter mitotic spindle function ment in the treated population, compared seem to activate aspects of this process by damage ultimately conveyed to the mitochondria, to a relevant control population established perhaps by activating the transcription of genes whose products can produce or modulate the as the result of clinical research protocol ortoxicity of free radicals. In addition, membrane damage with activation of sphingomyelinases other organized comparative study. Such results in the production of ceramides that can have a direct action at mitochondria. The anti-clinical research protocols are the basis forapoptotic protein bcl2 attenuates mitochondrial toxicity, while proapoptotic gene products such U.S. Food and Drug Administration (FDA)as bax antagonize the action of bcl2. Damaged mitochondria release cytochrome C and approval of a

1	mitochondrial toxicity, while proapoptotic gene products such U.S. Food and Drug Administration (FDA)as bax antagonize the action of bcl2. Damaged mitochondria release cytochrome C and approval of a particular cancer treatment as apoptosis-activating factor (APAF), which can directly activate caspase 9, resulting in propaga-safe and effective and are the benchmark for tion of a direct signal to other downstream caspases through protease activation. Apoptosis-an evidence-based approach to the use ofinducing factor (AIF) is also released from the mitochondrion and then can translocate to the chemotherapeutic agents. Common tumorsnucleus, bind to DNA, and generate free radicals to further damage DNA. An additional proapop-that may be meaningfully addressed by chetotic stimulus is the bad protein, which can heterodimerize with bcl2 gene family members to motherapy with palliative intent are listedantagonize apoptosis. Importantly, though, bad protein function can be retarded by its

1	protein, which can heterodimerize with bcl2 gene family members to motherapy with palliative intent are listedantagonize apoptosis. Importantly, though, bad protein function can be retarded by its seques-in Table 103e-3, E. tration as phospho-bad through the 14-3-3 adapter proteins. The phosphorylation of bad is Usually, tumor-related symptoms manimediated by the action of the AKT kinase in a way that defines how growth factors that activate fest as pain, weight loss, or some localthis kinase can retard apoptosis and promote cell survival. symptom related to the tumor’s effect on normal structures. Patients treated with palliative intent should be aware of their when used to address disseminated or metastatic cancers. If a tumor diagnosis and the limitations of the proposed treatments, have accessis localized to a single site, serious consideration of surgery or primary to supportive care, and have suitable “performance status,” accordingradiation therapy should be given, because

1	have accessis localized to a single site, serious consideration of surgery or primary to supportive care, and have suitable “performance status,” accordingradiation therapy should be given, because these treatment modalities to assessment algorithms such as the one developed by Karnofskymay be curative as local treatments. Chemotherapy may then be used (see Table 99-4) or by the Eastern Cooperative Oncology Groupafter the failure of these modalities to eradicate a local tumor or as part (ECOG) (see Table 99-5). ECOG performance status 0 (PS0) patientsof multimodality approaches to offer primary treatment to a clinically are without symptoms; PS1 patients are ambulatory but restricted inlocalized tumor. In this event, it can allow organ preservation when strenuous physical activity; PS2 patients are ambulatory but unablegiven with radiation, as in the larynx or other upper airway sites, or to work and are up and about 50% or more of the time; PS3 patientssensitize tumors to radiation

1	are ambulatory but unablegiven with radiation, as in the larynx or other upper airway sites, or to work and are up and about 50% or more of the time; PS3 patientssensitize tumors to radiation when given, e.g., to patients concur-are capable of limited self-care and are up <50% of the time; and PS4rently receiving radiation for lung or cervix cancer (Table 103e-3, B). patients are totally confined to bed or chair and incapable of self-care.Chemotherapy can be administered as an adjuvant, i.e., in addition to Only PS0, PS1, and PS2 patients are generally considered suitable forsurgery or radiation (Table 103e-3, C), even after all clinically appar-palliative (noncurative) treatment. If there is curative potential, evenent disease has been removed. This use of chemotherapy has curative poor–performance status patients may be treated, but their prognosispotential in breast and colorectal neoplasms, as it attempts to eliminate is usually inferior to that of good–performance status patients

1	status patients may be treated, but their prognosispotential in breast and colorectal neoplasms, as it attempts to eliminate is usually inferior to that of good–performance status patients treated clinically unapparent tumor that may have already disseminated. As with similar regimens.noted above, small tumors frequently have high growth fractions and An important perspective the primary care provider may bring totherefore may be intrinsically more susceptible to the action of antipro-patients and their families facing incurable cancer is that, given theliferative agents. Neoadjuvant chemotherapy refers to administration of limited value of chemotherapeutic approaches at some point in the

1	CurabIlIty of CanCerS wIth Chemotherapy A. Advanced Cancers with D. Cancers Possibly Cured with Possible Cure “High-Dose” Chemotherapy with Stem Cell Support Acute lymphoid and acute myeloid leukemia (pediatric/ Relapsed leukemias, lymphoid adult) and myeloid Hodgkin’s disease (pediatric/ Relapsed lymphomas, Hodgkin’s adult) and non-Hodgkin’s E. Cancers Responsive with Embryonal carcinoma Useful Palliation, But Not Cure, by Chemotherapy B. Advanced Cancers Possibly Islet cell neoplasms Cured by Chemotherapy and F. Tumors Poorly Responsive in Advanced Stages to Carcinoma of the uterine cervix Biliary tract neoplasms (stage III) Thyroid carcinoma Small-cell lung carcinoma Carcinoma of the vulva C. Cancers Possibly Cured with Chemotherapy as Adjuvant to Surgery Prostate carcinoma Breast carcinoma Melanoma (subsets) Colorectal carcinomaa Hepatocellular carcinoma Osteogenic sarcoma Salivary gland cancer Soft tissue sarcoma aRectum also receives radiation therapy.

1	natural history of most metastatic cancers, palliative care or hospice-based approaches, with meticulous and ongoing attention to symptom relief and with family, psychological, and spiritual support, should receive prominent attention as a valuable therapeutic plan (Chaps. 10 and 99). Optimizing the quality of life rather than attempting to extend it becomes a valued intervention. Patients facing the impending progression of disease in a life-threatening way frequently choose to undertake toxic treatments of little to no potential value, and support provided by the primary caregiver in accessing palliative and hospice-based options in contrast to receiving toxic and ineffective regimen can be critical in providing a basis for patients to make sensible choices.

1	Cytotoxic Chemotherapy Agents Table 103e-4 lists commonly used cytotoxic cancer chemotherapy agents and pertinent clinical aspects of their use, with particular reference to adverse effects that might be encountered by the generalist in the care of patients. The drugs listed may be usefully grouped into two general categories: those affecting DNA and those affecting microtubules.

1	Direct DNA-iNterActive AgeNts DNA replication occurs during the synthesis or S-phase of the cell cycle, with chromosome segregation of the replicated DNA occurring in the M, or mitosis, phase. The G1 and G2 “gap phases” precede S and M, respectively. Historically, chemotherapeutic agents have been divided into “phase-nonspecific” agents, which can act in any phase of the cell cycle, and “phase-specific” agents, which require the cell to be at a particular cell cycle phase to cause greatest effect. Once the agent has acted, cells may progress to “checkpoints” in the cell cycle where the drug-related damage may be assessed and either repaired or allowed to initiate apoptosis. An important function of certain tumor-suppressor genes such as p53 may be to modulate checkpoint function.

1	Alkylating agents as a class are cell cycle phase–nonspecific agents. They break down, either spontaneously or after normal organ or tumor cell metabolism, to reactive intermediates that covalently modify bases in DNA. This leads to cross-linkage of DNA strands or the appearance of breaks in DNA as a result of repair efforts. “Broken” or cross-linked DNA is intrinsically unable to complete normal replication or cell division; in addition, it is a potent activator of cell cycle checkpoints and further activates cell-signaling pathways that can precipitate apoptosis. As a class, alkylating agents share similar toxicities: myelosuppression, alopecia, gonadal dysfunction, mucositis, and pulmonary fibrosis. They differ greatly in a spectrum of normal organ toxicities. As a class, they share the capacity to cause “second” neoplasms, particularly leukemia, many years after use, particularly when used in low doses for protracted periods.

1	Cyclophosphamide is inactive unless metabolized by the liver to 4-hydroxy-cyclophosphamide, which decomposes into an alkylating species, as well as to chloroacetaldehyde and acrolein. The latter causes chemical cystitis; therefore, excellent hydration must be maintained while using cyclophosphamide. If severe, the cystitis may be prevented from progressing or prevented altogether (if expected from the dose of cyclophosphamide to be used) by mesna (2-mercaptoethanesulfonate). Liver disease impairs cyclophosphamide activation. Sporadic interstitial pneumonitis leading to pulmonary fibrosis can accompany the use of cyclophosphamide, and high doses used in conditioning regimens for bone marrow transplant can cause cardiac dysfunction. Ifosfamide is a cyclophosphamide analogue also activated in the liver, but more slowly, and it requires coadministration of mesna to prevent bladder injury. Central nervous system (CNS) effects, including somnolence, confusion, and psychosis, can follow

1	in the liver, but more slowly, and it requires coadministration of mesna to prevent bladder injury. Central nervous system (CNS) effects, including somnolence, confusion, and psychosis, can follow ifosfamide use; the incidence appears related to low body surface area or decreased creatinine clearance.

1	Several alkylating agents are less commonly used. Nitrogen mustard (mechlorethamine) is the prototypic agent of this class, decomposing rapidly in aqueous solution to potentially yield a bifunctional carbonium ion. It must be administered shortly after preparation into a rapidly flowing intravenous line. It is a powerful vesicant, and infiltration may be symptomatically ameliorated by infiltration of the affected site with 1/6 M thiosulfate. Even without infiltration, aseptic thrombophlebitis is frequent. It can be used topically as a dilute solution or ointment in cutaneous lymphomas, with a notable incidence of hypersensitivity reactions. It causes moderate nausea after intravenous administration. Bendamustine is a nitrogen mustard derivative with evidence of activity in chronic lymphocytic leukemia and certain lymphomas.

1	Chlorambucil causes predictable myelosuppression, azoospermia, nausea, and pulmonary side effects. Busulfan can cause profound myelosuppression, alopecia, and pulmonary toxicity but is relatively “lymphocyte sparing.” Its routine use in treatment of CML has been curtailed in favor of imatinib (Gleevec) or dasatinib, but it is still used in transplant preparation regimens. Melphalan shows variable oral bioavailability and undergoes extensive binding to albumin and α1-acidic glycoprotein. Mucositis appears more prominently; however, it has prominent activity in multiple myeloma.

1	Nitrosoureas break down to carbamylating species that not only cause a distinct pattern of DNA base pair–directed toxicity but also can covalently modify proteins. They share the feature of causing relatively delayed bone marrow toxicity, which can be cumulative and long-lasting. Procarbazine is metabolized in the liver and possibly in tumor cells to yield a variety of free radical and alkylating species. In addition to Drug Toxicity Interactions, Issues Liver metabolism required to activate to phosphoramide mustard + acrolein Mesna protects against “high-dose” bladder damage Analogue of cyclophosphamide Must use mesna Greater activity vs testicular neoplasms and sarcomas Liver and tissue metabolism required Disulfiram-like effect with ethanol Acts as MAOI HBP after tyrosinase-rich foods Metabolic activation Maintain high urine flow; osmotic diuresis, monitor intake/ output K+, Mg2+ Emetogenic—prophylaxis needed Full dose if CrCl >60 mL/min and tolerate fluid push

1	Maintain high urine flow; osmotic diuresis, monitor intake/ output K+, Mg2+ Emetogenic—prophylaxis needed Full dose if CrCl >60 mL/min and tolerate fluid push Reduce dose according to CrCl: to AUC of 5–7 mg/mL per min [AUC = dose/(CrCl + 25)] Acute reversible neurotoxicity; chronic sensory neurotoxicity cumulative with dose; reversible laryngopharyngeal spasm Chapter 103e Principles of Cancer Treatment Drug Toxicity Interactions, Issues Marrow (WBCs > platelet) Alopecia Hypotension Hypersensitivity (rapid IV) Nausea Mucositis (high dose) Marrow Mucositis Nausea Mild alopecia Diarrhea: “early onset” with cramping, flush ing, vomiting; “late onset” after several doses Marrow Alopecia Nausea Vomiting Pulmonary Marrow Mucositis Alopecia Cardiovascular acute/chronic Vesicant Marrow Cardiac (less than doxorubicin) Marrow Cardiac Marrow Cardiac (less than doxorubicin) Vesicant (mild) Blue urine, sclerae, nails

1	Hepatic metabolism—renal 30% Reduce doses with renal failure Schedule-dependent (5-day schedule better than 1-day) Late leukemogenic Accentuate antimetabolite action Reduce dose with renal failure No liver toxicity Prodrug requires enzymatic clearance to active drug “SN 38” Early diarrhea likely due to biliary excretion Late diarrhea, use “high-dose” loperamide (2 mg q2–4 h) Heparin aggregate; coadministration increases clearance Acetaminophen, BCNU increase liver toxicity Radiation recall Interacts with heparin Less alopecia, nausea than doxorubicin Radiation recall Less alopecia, nausea than doxorubicin Excretes in urine Reduce dose for renal failure Inhibits adenosine deaminase Reduce dose for renal failure Variable bioavailability Metabolize by xanthine oxidase Decrease dose with allopurinol Increased toxicity with thiopurine methyltransferase deficiency Variable bioavailability Increased toxicity with thiopurine methyltransferase deficiency

1	Metabolizes to 6-MP; therefore, reduce dose with allopurinol Increase toxicity with thiopurine methyltransferase deficiency Decrease dose with renal failure Augments antimetabolite effect Drug Toxicity Interactions, Issues Asparaginase Protein synthesis; indirect inhibition of DNA synthesis by decreased histone synthesis Clotting factors Glucose Albumin Hypersensitivity CNS Pancreatitis Hepatic Toxicity lessened by “rescue” with leucovorin Excreted in urine Decrease dose in renal failure; NSAIDs increase renal toxicity Toxicity enhanced by leucovorin by increasing “ternary complex” with thymidylate synthase; dihydropyrimidine dehydrogenase deficiency increases toxicity; metabolism in tissue Prodrug of 5FU due to intratumoral metabolism Enhances activity of alkylating agents Metabolizes in tissues by deamination but renal excretion prominent at doses >500 mg; therefore, dose reduce in “high-dose” regimens in patients with decreased CrCl

1	Metabolizes in tissues by deamination but renal excretion prominent at doses >500 mg; therefore, dose reduce in “high-dose” regimens in patients with decreased CrCl Use limited to leukemia Altered methylation of DNA alters gene expression Dose reduction with renal failure Metabolized to F-ara converted to F-ara ATP in cells by deoxycytidine kinase Chapter 103e Principles of Cancer Treatment Vincristine Vesicant Hepatic clearance Marrow Dose reduction for bilirubin >1.5 mg/dL Neurologic Prophylactic bowel regimen GI: ileus/constipation; bladder hypotoxicity; Drug Toxicity Interactions, Issues trum to other vincas) Hypertension Raynaud’s spectrum to other vincas) aCommon allkylator: alopecia, pulmonary, infertility, plus teratogenesis. Hepatic clearance Dose reduction as with vincristine Premedicate with steroids, H1 and H2 blockers Hepatic clearance Dose reduction as with vincas Premedicate with steroids, H1 and H2 blockers

1	Hepatic clearance Dose reduction as with vincristine Premedicate with steroids, H1 and H2 blockers Hepatic clearance Dose reduction as with vincas Premedicate with steroids, H1 and H2 blockers Abbreviations: ALL, acute lymphocytic leukemia; AUC, area under the curve; CHF, congestive heart failure; CNS, central nervous system; CrCl, creatinine clearance; CV, cardiovascular; GI, gastrointestinal; HBP, high blood pressure; MAOI, monoamine oxidase inhibitor; NSAID, nonsteroidal anti-inflammatory drug; SIADH, syndrome of inappropriate antidiuretic hormone secretion.

1	myelosuppression, it causes hypnotic and other CNS effects, including vivid nightmares. It can cause a disulfiram-like syndrome on ingestion of ethanol. Altretamine (formerly hexa-methylmelamine) and thiotepa can chemically give rise to alkylating species, although the nature of the DNA damage has not been well characterized in either case. Dacarbazine (DTIC) is activated in the liver to yield the highly reactive methyl diazonium cation. It causes only modest myelosuppression 21–25 days after a dose but causes prominent nausea on day 1. Temozolomide is structurally related to dacarbazine but was designed to be activated by nonenzymatic hydrolysis in tumors and is bioavailable orally.

1	Cisplatin was discovered fortuitously by observing that bacteria present in electrolysis solutions with platinum electrodes could not divide. Only the cis diamine configuration is active as an antitumor agent. It is hypothesized that in the intracellular environment, a chloride is lost from each position, being replaced by a water molecule. The resulting positively charged species is an efficient bifunctional interactor with DNA, forming Pt-based cross-links.

1	Cisplatin requires administration with adequate hydration, including forced diuresis with mannitol to prevent kidney damage; even with the use of hydration, gradual decrease in kidney function is common, along with noteworthy anemia. Hypomagnesemia frequently attends cisplatin use and can lead to hypocalcemia and tetany. Other common toxicities include neurotoxocity with stocking-and-glove sensorimotor neuropathy. Hearing loss occurs in 50% of patients treated with conventional doses. Cisplatin is intensely emetogenic, requiring prophylactic antiemetics. Myelosuppression is less evident than with other alkylating agents. Chronic vascular toxicity (Raynaud’s phenomenon, coronary artery disease) is a more unusual toxicity. Carboplatin displays less nephro-, oto-, and neurotoxicity. However, myelosuppression is more frequent, and because the drug is exclusively cleared through the kidney, adjustment of dose for creatinine clearance must be accomplished through use of various dosing

1	myelosuppression is more frequent, and because the drug is exclusively cleared through the kidney, adjustment of dose for creatinine clearance must be accomplished through use of various dosing nomograms. Oxaliplatin is a platinum analogue with noteworthy activity in colon cancers refractory to other treatments. It is prominently neurotoxic.

1	ANtitumor ANtibiotics AND topoisomerAse poisoNs Antitumor antibiotics are substances produced by bacteria that in nature appear to provide a chemical defense against other hostile microorganisms. As a class, they bind to DNA directly and can frequently undergo electron transfer reactions to generate free radicals in close proximity to DNA, leading to DNA damage in the form of single-strand breaks or cross-links. Topoisomerase poisons include natural products or semisynthetic species derived ultimately from plants, and they modify enzymes that regulate the capacity of DNA to unwind to allow normal replication or transcription. These include topoisomerase I, which creates single-strand breaks that then rejoin following the passage of the other DNA strand through the break. Topoisomerase II creates double-strand breaks through which another segment of DNA duplex passes before rejoining. DNA damage from these agents can occur in any cell cycle phase, but cells tend to arrest in S-phase or

1	double-strand breaks through which another segment of DNA duplex passes before rejoining. DNA damage from these agents can occur in any cell cycle phase, but cells tend to arrest in S-phase or G2 of the cell cycle in cells with p53 and Rb pathway lesions as the result of defective checkpoint mechanisms in cancer cells. Owing to the role of topoisomerase I in the procession of the replication fork, topoisomerase I poisons cause lethality if the topoisomerase I–induced lesions are made in S-phase.

1	Doxorubicin can intercalate into DNA, thereby altering DNA structure, replication, and topoisomerase II function. It can also undergo reduction reactions by accepting electrons into its quinone ring system, with the capacity to undergo reoxidation to form reactive oxygen radicals after reoxidation. It causes predictable myelosuppression, alopecia, nausea, and mucositis. In addition, it causes acute cardiotoxicity in the form of atrial and ventricular dysrhythmias, but these are rarely of clinical significance. In contrast, cumulative doses >550 mg/m2 are associated with a 10% incidence of chronic cardiomyopathy. The incidence of cardiomyopathy appears to be related to schedule (peak serum concentration), with low-dose, frequent treatment or continuous infusions better tolerated than intermittent higher-dose exposures. Cardiotoxicity has been related to iron-catalyzed oxidation and reduction of doxorubicin, and not to topoisomerase action. Cardiotoxicity is related to peak plasma dose;

1	higher-dose exposures. Cardiotoxicity has been related to iron-catalyzed oxidation and reduction of doxorubicin, and not to topoisomerase action. Cardiotoxicity is related to peak plasma dose; thus, lower doses and continuous infusions are less likely to cause heart damage. Doxorubicin’s cardiotoxicity is increased when given together with trastuzumab (Herceptin), the anti-HER2/neu antibody. Radiation recall or interaction with concomitantly administered radiation to cause local site complications is frequent. The drug is a powerful vesicant, with necrosis of tissue apparent 4–7 days after an extravasation; therefore, it should be administered into a rapidly flowing intravenous line. Dexrazoxane is an antidote to doxorubicin-induced extravasation. Doxorubicin is metabolized by the liver, so doses must be reduced by 50–75% in the presence of liver dysfunction. Daunorubicin is closely related to doxorubicin and was actually introduced first into leukemia treatment, where it remains part

1	doses must be reduced by 50–75% in the presence of liver dysfunction. Daunorubicin is closely related to doxorubicin and was actually introduced first into leukemia treatment, where it remains part of curative regimens and has been shown preferable to doxorubicin owing to less mucositis and colonic damage. Idarubicin is also used in acute myeloid leukemia treatment and may be preferable to daunorubicin in activity. Encapsulation of daunorubicin into a liposomal formulation has attenuated cardiac toxicity and antitumor activity in Kaposi’s sarcoma, other sarcomas, multiple myeloma, and ovarian cancer.

1	Bleomycin refers to a mixture of glycopeptides that have the unique feature of forming complexes with Fe2+ while also bound to DNA. It remains an important component of curative regimens for Hodgkin’s disease and germ cell neoplasms. Oxidation of Fe2+ gives rise to superoxide and hydroxyl radicals. The drug causes little, if any, myelosuppression. The drug is cleared rapidly, but augmented skin and pulmonary toxicity in the presence of renal failure has led to the recommendation that doses be reduced by 50–75% in the face of a creatinine clearance <25 mL/min. Bleomycin is not a vesicant and can be administered intravenously, intramuscularly, or subcutaneously. Common side effects include fever and chills, facial flush, and Raynaud’s phenomenon. Hypertension can follow rapid intravenous administration, and the incidence of anaphylaxis with early preparations of the drug has led to the practice of administering a test dose of 0.5–1 unit before the rest of the dose. The most feared

1	administration, and the incidence of anaphylaxis with early preparations of the drug has led to the practice of administering a test dose of 0.5–1 unit before the rest of the dose. The most feared complication of bleomycin treatment is pulmonary fibrosis, which increases in incidence at >300 cumulative units administered and is minimally responsive to treatment (e.g., glucocorticoids). The earliest indicator of an adverse effect is 103e-13 usually a decline in the carbon monoxide diffusing capacity (DLco) or coughing, although cessation of drug immediately upon documentation of a decrease in DLco may not prevent further decline in pulmonary function. Bleomycin is inactivated by a bleomycin hydrolase, whose concentration is diminished in skin and lung. Because bleomycindependent electron transport is dependent on O2, bleomycin toxicity may become apparent after exposure to transient very high fraction of inspired oxygen (Fio2). Thus, during surgical procedures, patients with prior

1	transport is dependent on O2, bleomycin toxicity may become apparent after exposure to transient very high fraction of inspired oxygen (Fio2). Thus, during surgical procedures, patients with prior exposure to bleomycin should be maintained on the lowest Fio2 consistent with maintaining adequate tissue oxygenation.

1	Mitoxantrone is a synthetic compound that was designed to recapitulate features of doxorubicin but with less cardiotoxicity. It is quantitatively less cardiotoxic (comparing the ratio of cardiotoxic to therapeutically effective doses) but is still associated with a 10% incidence of cardiotoxicity at cumulative doses of >150 mg/m2. It also causes alopecia. Cases of acute promyelocytic leukemia (APL) have arisen shortly after exposure of patients to mitoxantrone, particularly in the adjuvant treatment of breast cancer. Although chemotherapy-associated leukemia is generally of the acute myeloid type, APL arising in the setting of prior mitoxantrone treatment had the typical t(15;17) chromosome translocation associated with APL, but the breakpoints of the translocation appeared to be at topoisomerase II sites that would be preferred sites of mitoxantrone action, clearly linking the action of the drug to the generation of the leukemia.

1	Etoposide was synthetically derived from the plant product podophyllotoxin; it binds directly to topoisomerase II and DNA in a reversible ternary complex. It stabilizes the covalent intermediate in the enzyme’s action where the enzyme is covalently linked to DNA. This “alkali-labile” DNA bond was historically a first hint that an enzyme such as a topoisomerase might exist. The drug therefore causes a prominent G2 arrest, reflecting the action of a DNA damage checkpoint. Prominent clinical effects include myelosuppression, nausea, and transient hypotension related to the speed of administration of the agent. Etoposide is a mild vesicant but is relatively free from other large-organ toxicities. When given at high doses or very frequently, topoisomerase II inhibitors may cause acute leukemia associated with chromosome 11q23 abnormalities in up to 1% of exposed patients.

1	Camptothecin was isolated from extracts of a Chinese tree and had notable antileukemia activity in preclinical mouse models. Early human clinical studies with the sodium salt of the hydrolyzed camptothecin lactone showed evidence of toxicity with little antitumor activity. Identification of topoisomerase I as the target of camptothecins and the need to preserve lactone structure allowed additional efforts to identify active members of this series. Topoisomerase I is responsible for unwinding the DNA strand by introducing single-strand breaks and allowing rotation of one strand about the other. In S-phase, topoisomerase I–induced breaks that are not promptly resealed lead to progress of the replication fork off the end of a DNA strand. The DNA damage is a potent signal for induction of apoptosis. Camptothecins promote the stabilization of the DNA linked to the enzyme in a so-called cleavable complex, analogous to the action of etoposide with topoisomerase II. Topotecan is a

1	of apoptosis. Camptothecins promote the stabilization of the DNA linked to the enzyme in a so-called cleavable complex, analogous to the action of etoposide with topoisomerase II. Topotecan is a camptothecin derivative approved for use in gynecologic tumors and small-cell lung cancer. Toxicity is limited to myelosuppression and mucositis. CPT-11, or irinotecan, is a camptothecin with evidence of activity in colon carcinoma. In addition to myelosuppression, it causes a secretory diarrhea related to the toxicity of a metabolite called SN-38. Levels of SN-38 are particularly high in the setting of Gilbert’s disease, characterized by defective glucuronyl transferase and indirect hyperbilirubinemia, a condition that affects about 10% of the white population in the United States. The diarrhea can be treated effectively with loperamide or octreotide.

1	iNDirect moDulAtors of Nucleic AciD fuNctioN: ANtimetAbolites A broad definition of antimetabolites would include compounds with structural similarity to precursors of purines or pyrimidines, or compounds that interfere with purine or pyrimidine synthesis. Some antimetabolites can cause DNA damage indirectly, through misincorporation into DNA, abnormal timing or progression through DNA synthesis, or Chapter 103e Principles of Cancer Treatment altered function of pyrimidine and purine biosynthetic enzymes. They tend to convey greatest toxicity to cells in S-phase, and the degree of toxicity increases with duration of exposure. Common toxic manifestations include stomatitis, diarrhea, and myelosuppression. Second malignancies are not associated with their use.

1	Methotrexate inhibits dihydrofolate reductase, which regenerates reduced folates from the oxidized folates produced when thymidine monophosphate is formed from deoxyuridine mono-phosphate. Without reduced folates, cells die a “thymine-less” death. N5-Tetrahydrofolate or N5-formyltetrahydrofolate (leucovorin) can bypass this block and rescue cells from methotrexate, which is maintained in cells by polyglutamylation. The drug and other reduced folates are transported into cells by the folate carrier, and high concentrations of drug can bypass this carrier and allow diffusion of drug directly into cells. These properties have suggested the design of “high-dose” methotrexate regimens with leucovorin rescue of normal marrow and mucosa as part of curative approaches to osteosarcoma in the adjuvant setting and hematopoietic neoplasms of children and adults. Methotrexate is cleared by the kidney via both glomerular filtration and tubular secretion, and toxicity is augmented by renal

1	the adjuvant setting and hematopoietic neoplasms of children and adults. Methotrexate is cleared by the kidney via both glomerular filtration and tubular secretion, and toxicity is augmented by renal dysfunction and drugs such as salicylates, probenecid, and nonsteroidal anti-inflammatory agents that undergo tubular secretion. With normal renal function, 15 mg/m2 leucovorin will rescue 10−8 to 10−6 M methotrexate in three to four doses. However, with decreased creatinine clearance, doses of 50–100 mg/m2 are continued until methotrexate levels are <5 × 10−8 M. In addition to bone marrow suppression and mucosal irritation, methotrexate can cause renal failure itself at high doses owing to crystallization in renal tubules; therefore, high-dose regimens require alkalinization of urine with increased flow by hydration. Methotrexate can be sequestered in third-space collections and diffuse back into the general circulation, causing prolonged myelosuppression. Less frequent adverse effects

1	flow by hydration. Methotrexate can be sequestered in third-space collections and diffuse back into the general circulation, causing prolonged myelosuppression. Less frequent adverse effects include reversible increases in transaminases and hypersensitivity-like pulmonary syndrome. Chronic low-dose methotrexate can cause hepatic fibrosis. When administered to the intrathecal space, methotrexate can cause chemical arachnoiditis and CNS dysfunction.

1	Pemetrexed is a novel folate-directed antimetabolite. It is “multitargeted” in that it inhibits the activity of several enzymes, including thymidylate synthetase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase, thereby affecting the synthesis of both purine and pyrimidine nucleic acid precursors. To avoid significant toxicity to the normal tissues, patients receiving pemetrexed should also receive low-dose folate and vitamin B12 supplementation. Pemetrexed has notable activity against certain lung cancers and, in combination with cisplatin, also against mesotheliomas. Pralatrexate is an antifolate approved for use in T cell lymphoma that is very efficiently transported into cancer cells.

1	5-Fluorouracil (5FU) represents an early example of “rational” drug design in that it originated from the observation that tumor cells incorporate radiolabeled uracil more efficiently into DNA than normal cells, especially gut. 5FU is metabolized in cells to 5´FdUMP, which inhibits thymidylate synthetase (TS). In addition, misincorporation can lead to single-strand breaks, and RNA can aberrantly incorporate FUMP. 5FU is metabolized by dihydropyrimidine dehydrogenase, and deficiency of this enzyme can lead to excessive toxicity from 5FU. Oral bioavailability varies unreliably, but orally administered analogues of 5FU such as capecitabine have been developed that allow at least equivalent activity to many parenteral 5FU-based approaches. Intravenous administration of 5FU leads to bone marrow suppression after short infusions but to stomatitis after prolonged infusions. Leucovorin augments the activity of 5FU by promoting formation of the ternary covalent complex of 5FU, the reduced

1	suppression after short infusions but to stomatitis after prolonged infusions. Leucovorin augments the activity of 5FU by promoting formation of the ternary covalent complex of 5FU, the reduced folate, and TS. Less frequent toxicities include CNS dysfunction, with prominent cerebellar signs, and endothelial toxicity manifested by thrombosis, including pulmonary embolus and myocardial infarction.

1	Cytosine arabinoside (ara-C) is incorporated into DNA after formation of ara-CTP, resulting in S-phase–related toxicity. Continuous infusion schedules allow maximal efficiency, with uptake maximal at 5–7 μM. Ara-C can be administered intrathecally. Adverse effects include nausea, diarrhea, stomatitis, chemical conjunctivitis, and cerebellar ataxia. Gemcitabine is a cytosine derivative that is similar to ara-C in that it is incorporated into DNA after anabolism to the triphosphate, rendering DNA susceptible to breakage and repair synthesis, which differs from that in ara-C in that gemcitabine-induced lesions are very inefficiently removed. In contrast to ara-C, gemcitabine appears to have useful activity in a variety of solid tumors, with limited nonmyelosuppressive toxicities.

1	6-Thioguanine and 6-mercaptopurine (6MP) are used in the treatment of acute lymphoid leukemia. Although administered orally, they display variable bioavailability. 6MP is metabolized by xanthine oxidase and therefore requires dose reduction when used with allopurinol. 6MP is also metabolized by thiopurine methyltransferase; genetic deficiency of thiopurine methyltransferase results in excessive toxicity.

1	Fludarabine phosphate is a prodrug of F-adenine arabinoside (F-ara-A), which in turn was designed to diminish the susceptibility of ara-A to adenosine deaminase. F-ara-A is incorporated into DNA and can cause delayed cytotoxicity even in cells with low growth fraction, including chronic lymphocytic leukemia and follicular B cell lymphoma. CNS and peripheral nerve dysfunction and T cell depletion leading to opportunistic infections can occur in addition to myelosuppression. 2-Chlorodeoxyadenosine is a similar compound with activity in hairy cell leukemia. 2-Deoxycoformycin inhibits adenosine deaminase, with resulting increase in dATP levels. This causes inhibition of ribonucleotide reductase as well as augmented susceptibility to apoptosis, particularly in T cells. Renal failure and CNS dysfunction are notable toxicities in addition to immunosuppression. Hydroxyurea inhibits ribonucleotide reductase, resulting in S-phase block. It is orally bioavailable and useful for the acute

1	CNS dysfunction are notable toxicities in addition to immunosuppression. Hydroxyurea inhibits ribonucleotide reductase, resulting in S-phase block. It is orally bioavailable and useful for the acute management of myeloproliferative states.

1	Asparaginase is a bacterial enzyme that causes breakdown of extracellular asparagine required for protein synthesis in certain leukemic cells. This effectively stops tumor cell DNA synthesis, as DNA synthesis requires concurrent protein synthesis. The outcome of asparaginase action is therefore very similar to the result of the small-molecule antimetabolites. Because asparaginase is a foreign protein, hypersensitivity reactions are common, as are effects on organs such as pancreas and liver that normally require continuing protein synthesis. This may result in decreased insulin secretion with hyperglycemia, with or without hyperamylasemia and clotting function abnormalities. Close monitoring of clotting functions should accompany use of asparaginase. Paradoxically, owing to depletion of rapidly turning over anticoagulant factors, thromboses particularly affecting the CNS may also be seen with asparaginase.

1	mitotic spiNDle iNhibitors Microtubules are cellular structures that form the mitotic spindle, and in interphase cells, they are responsible for the cellular “scaffolding” along which various motile and secretory processes occur. Microtubules are composed of repeating noncovalent multimers of a heterodimer of α and β isoform of the protein tubulin. Vincristine binds to the tubulin dimer with the result that microtubules are disaggregated. This results in the block of growing cells in M-phase; however, toxic effects in G1 and S-phase are also evident, reflecting effects on normal cellular activities of microtubules. Vincristine is metabolized by the liver, and dose adjustment in the presence of hepatic dysfunction is required. It is a powerful vesicant, and infiltration can be treated by local heat and infiltration of hyaluronidase. At clinically used intravenous doses, neurotoxicity in the form of glove-and-stocking neuropathy is frequent. Acute neuropathic effects include jaw pain,

1	local heat and infiltration of hyaluronidase. At clinically used intravenous doses, neurotoxicity in the form of glove-and-stocking neuropathy is frequent. Acute neuropathic effects include jaw pain, paralytic ileus, urinary retention, and the syndrome of inappropriate antidiuretic hormone secretion. Myelosuppression is not seen. Vinblastine is similar to vincristine, except that it tends to be more myelotoxic, with more frequent thrombocytopenia and also mucositis and stomatitis. Vinorelbine is a vinca alkaloid that appears to have differences in resistance patterns in comparison to vincristine and vinblastine; it may be administered orally.

1	The taxanes include paclitaxel and docetaxel. These agents differ from the vinca alkaloids in that the taxanes stabilize micro-tubules against depolymerization. The “stabilized” microtubules function abnormally and are not able to undergo the normal dynamic changes of microtubule structure and function necessary for cell cycle completion. Taxanes are among the most broadly active anti-neoplastic agents for use in solid tumors, with evidence of activity in ovarian cancer, breast cancer, Kaposi’s sarcoma, and lung tumors. They are administered intravenously, and paclitaxel requires use of a Cremophor-containing vehicle that can cause hypersensitivity reactions. Premedication with dexamethasone (8–16 mg orally or intravenously 12 and 6 h before treatment) and diphenhydramine (50 mg) and cimetidine (300 mg), both 30 min before treatment, decreases but does not eliminate the risk of hypersensitivity reactions to the paclitaxel vehicle. Docetaxel uses a polysorbate 80 formulation, which can

1	(300 mg), both 30 min before treatment, decreases but does not eliminate the risk of hypersensitivity reactions to the paclitaxel vehicle. Docetaxel uses a polysorbate 80 formulation, which can cause fluid retention in addition to hypersensitivity reactions, and dexamethasone premedication with or without antihistamines is frequently used. A protein-bound formulation of paclitaxel (called nab-paclitaxel) has at least equivalent antineoplastic activity and decreased risk of hypersensitivity reactions. Paclitaxel may also cause hypersensitivity reactions, myelosuppression, neurotoxicity in the form of glove-and-stocking numbness, and paresthesia. Cardiac rhythm disturbances were observed in phase 1 and 2 trials, most commonly asymptomatic bradycardia but also, much more rarely, varying degrees of heart block. These have not emerged as clinically significant in the majority of patients. Docetaxel causes comparable degrees of myelosuppression and neuropathy. Hypersensitivity reactions,

1	of heart block. These have not emerged as clinically significant in the majority of patients. Docetaxel causes comparable degrees of myelosuppression and neuropathy. Hypersensitivity reactions, including bronchospasm, dyspnea, and hypotension, are less frequent but occur to some degree in up to 25% of patients. Fluid retention appears to result from a vascular leak syndrome that can aggravate preexisting effusions. Rash can complicate docetaxel administration, appearing prominently as a pruritic maculopapular rash affecting the forearms, but it has also been associated with fingernail ridging, breakdown, and skin discoloration. Stomatitis appears to be somewhat more frequent than with paclitaxel. Cabazitaxel is a taxane with somewhat better activity in prostate cancers than earlier generations of taxanes, perhaps due to superior delivery to sites of disease.

1	Resistance to taxanes has been related to the emergence of efficient efflux of taxanes from tumor cells through the p170 P-glycoprotein (mdr gene product) or the presence of variant or mutant forms of tubulin. Epothilones represent a class of novel microtubule-stabilizing agents that have been conscientiously optimized for activity in taxane-resistant tumors. Ixabepilone has clear evidence of activity in breast cancers resistant to taxanes and anthracyclines such as doxorubicin. It retains acceptable expected side effects, including myelosuppression, and can also cause peripheral sensory neuropathy. Eribulin is a microtubule-directed agent with activity in patients who have had progression of disease on taxanes and is more similar to vinca alkaloids in its action but has similar side effects as vinca alkaloids and taxanes.

1	Estramustine was originally synthesized as a mustard derivative that might be useful in neoplasms that possessed estrogen receptors. However, no evidence of interaction with DNA was observed. Surprisingly, the drug caused metaphase arrest, and subsequent study revealed that it binds to microtubule-associated proteins, resulting in abnormal microtubule function. Estramustine binds to estramustinebinding proteins (EMBPs), which are notably present in prostate tumor tissue, where the drug is used. Gastrointestinal and cardiovascular adverse effects related to the estrogen moiety occur in up to 10% of patients, including worsened heart failure and thromboembolic phenomena. Gynecomastia and nipple tenderness can also occur.

1	Targeted Chemotherapy • hormoNe receptor–DirecteD therApy Steroid hormone receptor–related molecules have emerged as prominent targets for small molecules useful in cancer treatment. When bound to their cognate ligands, these receptors can alter gene transcription and, in certain tissues, induce apoptosis. The pharmacologic effect is a mirror or parody of the normal effects of the agents acting on nontransformed normal tissues, although the effects on tumors are mediated by indirect effects in some cases. While in some cases, such as breast cancer, demonstration of the target hormone receptor is necessary, in other cases such prostate cancer (androgen receptor) and lymphoid neoplasms (glucocorticoid receptor), the relevant receptor is 103e-15 always present in the tumor.

1	Glucocorticoids are generally given in “pulsed” high doses in leukemias and lymphomas, where they induce apoptosis in tumor cells. Cushing’s syndrome and inadvertent adrenal suppression on withdrawal from high-dose glucocorticoids can be significant complications, along with infections common in immunosuppressed patients, in particular Pneumocystis pneumonia, which classically appears a few days after completing a course of high-dose glucocorticoids.

1	Tamoxifen is a partial estrogen receptor antagonist; it has a 10-fold greater antitumor activity in breast cancer patients whose tumors express estrogen receptors than in those who have low or no levels of expression. It might be considered the prototypic “molecularly targeted” agent. Owing to its agonistic activities in vascular and uterine tissue, side effects include a somewhat increased risk of cardiovascular complications, such as thromboembolic phenomena, and a small increased incidence of endometrial carcinoma, which appears after chronic use (usually >5 years). Progestational agents—including medroxyprogesterone acetate, androgens including fluoxymesterone (Halotestin), and, paradoxically, estrogens—have approximately the same degree of activity in primary hormonal treatment of breast cancers that have elevated expression of estrogen receptor protein. Estrogen itself is not used often owing to prominent cardiovascular and uterotropic activity.

1	Aromatase refers to a family of enzymes that catalyze the formation of estrogen in various tissues, including the ovary and peripheral adipose tissue and some tumor cells. Aromatase inhibitors are of two types, the irreversible steroid analogues such as exemestane and the reversible inhibitors such as anastrozole or letrozole. Anastrozole is superior to tamoxifen in the adjuvant treatment of breast cancer in postmenopausal patients with estrogen receptor–positive tumors. Letrozole treatment affords benefit following tamoxifen treatment. Adverse effects of aromatase inhibitors may include an increased risk of osteoporosis.

1	Prostate cancer is classically treated by androgen deprivation. Diethylstilbestrol (DES) acting as an estrogen at the level of the hypothalamus to downregulate hypothalamic luteinizing hormone (LH) production results in decreased elaboration of testosterone by the testicle. For this reason, orchiectomy is equally as effective as moderate-dose DES, inducing responses in 80% of previously untreated patients with prostate cancer but without the prominent cardiovascular side effects of DES, including thrombosis and exacerbation of coronary artery disease. In the event that orchiectomy is not accepted by the patient, testicular androgen suppression can also be effected by luteinizing hormone–releasing hormone (LHRH) agonists such as leuprolide and goserelin. These agents cause tonic stimulation of the LHRH receptor, with the loss of its normal pulsatile activation resulting in decreased output of LH by the anterior pituitary. Therefore, as primary hormonal manipulation in prostate cancer,

1	of the LHRH receptor, with the loss of its normal pulsatile activation resulting in decreased output of LH by the anterior pituitary. Therefore, as primary hormonal manipulation in prostate cancer, one can choose orchiectomy or leuprolide, but not both. The addition of androgen receptor blockers, including flutamide or bicalutamide, is of uncertain additional benefit in extending overall response duration; the combined use of orchiectomy or leuprolide plus flutamide is referred to as total androgen blockade. Enzalutamide also binds to the androgen receptor and antagonizes androgen action in a mechanistically distinct way. Somewhat analogous to inhibitors of aromatase, agents have been derived that inhibit testosterone and other androgen synthesis in the testis, adrenal gland, and prostate tissue. Abiraterone inhibits 17 α-hydroxylase/C17,20 lyase (CYP 17A1) and has been shown to be active in prostate cancer patients experiencing progression despite androgen blockade.

1	Tumors that respond to a primary hormonal manipulation may frequently respond to second and third hormonal manipulations. Thus, breast tumors that had previously responded to tamoxifen have, on relapse, notable response rates to withdrawal of tamoxifen itself or to subsequent addition of an aromatase inhibitor or progestin. Likewise, initial treatment of prostate cancers with leuprolide plus flutamide may be followed after disease progression by response to withdrawal of flutamide. These responses may result from the removal Chapter 103e Principles of Cancer Treatment of antagonists from mutant steroid hormone receptors that have come to depend on the presence of the antagonist as a growth-promoting influence.

1	Chapter 103e Principles of Cancer Treatment of antagonists from mutant steroid hormone receptors that have come to depend on the presence of the antagonist as a growth-promoting influence. Additional strategies to treat refractory breast and prostate cancers that possess steroid hormone receptors may also address adrenal capacity to produce androgens and estrogens, even after orchiectomy or oophorectomy, respectively. Thus, aminoglutethimide or ketocon azole can be used to block adrenal synthesis by interfering with the enzymes of steroid hormone metabolism. Administration of these agents requires concomitant hydrocortisone replacement and additional glucocorticoid doses administered in the event of physiologic stress.

1	Humoral mechanisms can also result in complications from an underlying malignancy producing the hormone. Adrenocortical carcinomas can cause Cushing’s syndrome as well as syndromes of androgen or estrogen excess. Mitotane can counteract these by decreasing synthesis of steroid hormones. Islet cell neoplasms can cause debilitating diarrhea, treated with the somatostatin analogue octreotide. Prolactin-secreting tumors can be effectively managed by the dopaminergic agonist bromocriptine.

1	DiAgNosticAlly guiDeD therApy The basis for discovery of drugs of this type was the prior knowledge of the importance of the drugs’ molecular target to drive tumors in different contexts. Figure 103e-4 summarizes how FDA-approved targeted agents act. In the case of diagnostically guided targeted chemotherapy, prior demonstration of a specific target is necessary to guide the rational use of the agent, while in the case of targeted agents directed at oncogenic pathways, specific diagnosis of pathway activation is not yet necessary or in some cases feasible, although this is an area of ongoing clinical research. Table 103e-5 lists currently approved targeted chemotherapy agents, with features of their use.

1	In hematologic tumors, the prototypic agent of this type is imatinib, which targets the ATP binding site of the p210bcr-abl protein tyrosine kinase that is formed as the result of the chromosome 9;22 translocation producing the Philadelphia chromosome in CML. Imatinib is superior to interferon plus chemotherapy in the initial treatment of the chronic phase of this disorder. It has lesser activity in the blast phase of CML, where the cells may have acquired additional mutations in p210bcr-abl itself or other genetic lesions. Its side effects are relatively tolerable in most patients and include hepatic dysfunction, diarrhea, and fluid retention. Rarely, patients receiving imatinib have decreased cardiac function, which may persist after discontinuation of the drug. The quality of response to imatinib enters into the decision about when to refer patients with CML for consideration of transplant approaches. Nilotinib is a tyrosine protein kinase inhibitor with a similar spectrum of

1	to imatinib enters into the decision about when to refer patients with CML for consideration of transplant approaches. Nilotinib is a tyrosine protein kinase inhibitor with a similar spectrum of activity to imatinib, but with increased potency and perhaps better tolerance by certain patients. Dasatinib, another inhibitor of the p210bcr-abl oncoproteins, is active in certain mutant variants of p210bcr-abl that are refractory to imatinib and arise during therapy with imatinib or are present de novo. Dasatinib also has inhibitory action against kinases belonging to the src tyrosine protein kinase family; this activity may contribute to its effects in hematopoietic tumors and suggest a role in solid tumors where src kinases are active. The T315I mutant of p210bcr-abl is resistant to imatinib, nilotinib, bosutinib, and dasatinib; ponatinib has activity in patients with this p210bcr-abl variant, but ponatinib has noteworthy associated thromboembolic toxicity. Use of this class of targeted

1	nilotinib, bosutinib, and dasatinib; ponatinib has activity in patients with this p210bcr-abl variant, but ponatinib has noteworthy associated thromboembolic toxicity. Use of this class of targeted agents is thus critically guided not only by the presence of the p210bcr-abl tyrosine kinase, but also by the presence of different mutations in the ATP binding site.

1	All-trans-retinoic acid (ATRA) targets the PML-retinoic acid receptor (RAR) α fusion protein, which is the result of the chromosome 15;17 translocation pathogenic for most forms of APL. Administered orally, it causes differentiation of the neoplastic promyelocytes to mature granulocytes and attenuates the rate of hemorrhagic complications. Adverse effects include headache with or without pseudotumor cerebri and gastrointestinal and cutaneous toxicities. In epithelial solid tumors, the small-molecule epidermal growth factor (EGF) antagonists act at the ATP binding site of the EGF receptor

1	FIGuRE 103e-4 Targeted chemotherapeutic agents act in most instances by interrupting cell growth factor-mediated signaling pathways. After a growth factor binds to is cognate receptor (1), in many cases there is activation of tyrsosine kinase activity particularly after dimerization of the receptors (2). This leads to autophosphorylation of the receptor and docking of “adaptor” proteins. One important pathway activated occurs after exchange of GDP for GTP in the RAS family of proto-oncogene products (3). GTP-RAS activates the RAF proto-oncogene kinase (4), leading to a phosphorylation cascade of kinases (5, 6) that ultimately impart signals to regulators of gene function to produce transcripts which activate cell cycle progression and increase protein synthesis. In parallel, tyrosine phosphorylated receptors can activate the phosphatidylinositol-3-kinase to produce the phosphorylated lipid phosphatidyl-inositol-3phosphate (7). This leads to the activation of the AKT kinase(8) which in

1	receptors can activate the phosphatidylinositol-3-kinase to produce the phosphorylated lipid phosphatidyl-inositol-3phosphate (7). This leads to the activation of the AKT kinase(8) which in turn stimulates the mammalian “Target of Rapamycin” kinase (mTOR), which directly increases the translation of key mRNAs for gene products regulating cell growth. Erlotinib and afatinib, are examples of Epidermal Growth Factor receptor tyrosine kinase inhibitors; imatinib can act on the nonreceptor tyrosine kinase bcr-abl or c-KIT membrane bound tyrosine kinase. Vemurafenib and Dabrafenib act on the B isoform of RAF uniquely in melanoma, and c-RAF is inhibited by sorafenib. Trametinib acts on MEK. Temsirolimus and everolimus inhibit mTOR kinase to downregulate translation of oncogenic mRNAs.

1	tyrosine kinase. In early clinical trials, gefitinib showed evidence of responses in a small fraction of patients with non-small-cell lung cancer (NSCLC). Side effects were generally acceptable, consisting mostly of rash and diarrhea. Subsequent analysis of responding patients revealed a high frequency of activating mutations in the EGF receptor. Patients with such activating mutations who initially responded to gefitinib but who then had progression of the disease then acquired additional mutations in the enzyme, analogous functionally to mutational variants responsible for imatinib resistance in CML. Erlotinib is another EGF receptor tyrosine kinase antagonist with a superior outcome in clinical Chapter 103e Principles of Cancer Treatment Temsirolimus Renal cell carcinoma, second line or poor prognosis Stomatitis Thrombocytopenia Nausea Anorexia, fatigue Metabolic (glucose, lipid)

1	Chapter 103e Principles of Cancer Treatment Temsirolimus Renal cell carcinoma, second line or poor prognosis Stomatitis Thrombocytopenia Nausea Anorexia, fatigue Metabolic (glucose, lipid) Abbreviations: APL, acute promyelocytic leukemia; ALL, acute lymphocytic leukemia; CHF, congestive heart failure; CML, chronic myeloid leukemia; EGFR, epidermal growth factor receptor; GI, gastrointestinal; mTOR, mammalian target of rapamycin kinase; NSCLC, non-small-cell lung cancer; PDGFR, platelet-derived growth factor receptor; Pgp, P-glycoprotein; VEGFR, vascular endothelial growth factor receptor.

1	VEGFR, vascular endothelial growth factor receptor. trials in NSCLC; an overall survival advantage was demonstrated in subsets of patients who were treated after demonstrating progression of disease and who also had not been preselected for the presence of activating mutations. Thus, although even patients with wild-type EGF receptors may benefit from erlotinib treatment, the presence of EGF receptor tyrosine kinase mutations has recently been shown to be a basis for recommending erlotinib and afatinib for first-line treatment of advanced NSCLC. Likewise, crizotinib targeting the alk protooncogene fusion protein has value in the initial treatment of alk-positive NSCLC. Lapatinib is a tyrosine kinase inhibitor with both EGF receptor and HER2/neu antagonist activity, which is important in the treatment of breast cancers expressing the HER2/neu oncoprotein.

1	In addition to the p210bcr-abl kinase, imatinib also has activity against the c-kit tyrosine kinase (the receptor for the steel growth factor, also called stem cell factor) and the platelet-derived growth factor receptor (PDGFR), both of which can be expressed in gastrointestinal stromal sarcoma (GIST). Imatinib has found clinical utility in GIST, a tumor previously notable for its refractoriness to chemotherapeutic approaches. Imatinib’s degree of activity varies with the specific mutational variant of kit or PDGFR present in a particular patient’s tumor.

1	The BRAF V600E mutation has been detected in a notable fraction of melanomas, thyroid tumors, and hairy cell leukemia, and preclinical models supported the concept that BRAF V600E drives oncogenic signaling in these tumors. Vemurafenib and dabrafenib, with selective capacity to inhibit the BRAF V600E serine kinase activity, were each shown to cause noteworthy responses in patients with BRAF V600E– mutated melanomas, although early relapse occurred in many patients treated with the drugs as single agents. Trametinib, acting downstream of BRAF V600E by directly inhibiting the MEK serine kinase by a non-ATP binding site mechanism, also displayed noteworthy responses in BRAF V600E–mutated melanomas, and the combination of trametinib and dabrafenib is even more active, by targeting the BRAF V600E– driven pathway at two points in the pathway leading to gene activation.

1	oNcogeNicAlly ActivAteD pAthwAys This group of agents also targets specific regulatory molecules in promoting the viability of tumor cells, but they do not require the diagnostically verified presence of a particular target or target variant at this time.

1	“Multitargeted” kinase antagonists are small-molecule ATP site-directed antagonists that inhibit more than one protein kinase and have value in the treatment of several solid tumors. Drugs of this type with prominent activity against the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase have activity in renal cell carcinoma. Sorafenib is a VEGFR antagonist with activity against the raf serine-threonine protein kinase, and regorafenib is a closely related drug with value in relapsed advanced colon cancer. Pazopanib also prominently targets VEGFR and has activity in renal carcinoma and soft tissue sarcomas. Sunitinib has anti-VEGFR, anti-PDGFR, and anti-c-kit activity. It causes prominent responses and stabilization of disease in renal cell cancers and GISTs. Side effects for agents with anti-VEGFR activity prominently include hypertension, proteinuria, and, more rarely, bleeding and clotting disorders and perforation of scarred gastrointestinal lesions. Also

1	effects for agents with anti-VEGFR activity prominently include hypertension, proteinuria, and, more rarely, bleeding and clotting disorders and perforation of scarred gastrointestinal lesions. Also encountered are fatigue, diarrhea, and the hand-foot syndrome, with erythema and desquamation of the distal extremities, in some cases requiring dose modification, particularly with sorafenib.

1	Temsirolimus and everolimus are mammalian target of rapamycin (mTOR) inhibitors with activity in renal cancers. They produce stomatitis, fatigue, and some hyperlipidemia (10%), myelosuppression (10%), and rare lung toxicity. Everolimus is also useful in patients with hormone receptor–positive breast cancers displaying resistance to hormonal inhibition and in certain neuroendocrine and brain tumors, the latter arising in patients with sporadic or inherited mutations in the pathway activating mTOR.

1	In hematologic neoplasms, bortezomib is an inhibitor of the proteasome, the multisubunit assembly of protease activities responsible for the selective degradation of proteins important in regulating activation of transcription factors, including nuclear factor-κB (NF-κB) and proteins regulating cell cycle progression. It has activity in multiple myeloma and certain lymphomas. Adverse effects include neuropathy, orthostatic hypotension with or without hyponatremia, and reversible thrombocytopenia. Carfilzomib is a proteasome inhibitor chemically unrelated to bortezomib without prominent neuropathy, but with evidence of a cytokine release syndrome, which can be a cardiopulmonary stress. Other agents active in multiple myeloma and certain other hematologic neoplasms include the immunomodulatory agents related to thalidomide, including lenalidomide and pomalidomide. All these agents collectively inhibit aberrant angiogenesis in the bone marrow microenvironment, as well as influence

1	agents related to thalidomide, including lenalidomide and pomalidomide. All these agents collectively inhibit aberrant angiogenesis in the bone marrow microenvironment, as well as influence stromal cell immune functions to alter the cytokine milieu supporting the growth of myeloma cells. Thalidomide, although clinically active, has prominent cytopenic, neuropathic, procoagulant, and CNS toxicities that have been somewhat attenuated in the other drugs of the class, although use of these agents frequently entails concomitant anticoagulant prophylaxis.

1	Ibrutinib is representative a novel class of inhibitors directed at Bruton’s tyrosine kinase, which is important in the function of B cells. Initially approved for use in mantle cell lymphoma, it is potentially applicable to a number of B cell neoplasms that depend on signals through the B cell antigen receptor. Janus kinases likewise function downstream of a variety of cytokine receptors to amplify cytokine signals, and Janus kinase inhibitors including ruxolitinib have approved activity in myelofibrosis to ameliorate splenomegaly and systemic symptoms. Vorinostat is an inhibitor of histone deacetylases, which are responsible for maintaining the proper orientation of histones on DNA, with resulting capacity for transcriptional readiness. Acetylated histones allow access of transcription factors to target genes and therefore

1	Chapter 103e Principles of Cancer Treatment increase expression of genes that are selectively repressed in tumors. The result can be differentiation with the emergence of a more normal cellular phenotype, or cell cycle arrest with expression of endogenous regulators of cell cycle progression. Vorinostat is approved for clinical use in cutaneous T cell lymphoma, with dramatic skin clearing and very few side effects. Romidepsin is a distinct molecular class of his-tone deacetylase inhibitor also active in cutaneous T cell lymphoma. An active retinoid in cutaneous T cell lymphoma is the synthetic retinoid X receptor ligand bexarotene.

1	DNA methyltransferase inhibitors, including 5-aza-cytidine and 2´-deoxy-5-azacytidine (decitabine), can also increase transcription of genes “silenced” during the pathogenesis of a tumor by causing demethylation of the methylated cytosines that are acquired as an “epigenetic” (i.e., after the DNA is replicated) modification of DNA. These drugs were originally considered antimetabolites but have clinical value in myelodysplastic syndromes and certain leukemias when administered at low doses.

1	CANCER BIOLOGIC THERAPY Principles The goal of biologic therapy is to manipulate the host– tumor interaction in favor of the host, potentially at an optimum biologic dose that might be different than the MTD. As a class, biologic therapies may be distinguished from molecularly targeted agents in that many biologic therapies require an active response (e.g., reexpression of silenced genes or antigen expression) on the part of the tumor cell or on the part of the host (e.g., immunologic effects) to allow therapeutic effect. This may be contrasted with the more narrowly defined antiproliferative or apoptotic response that is the ultimate goal of molecularly targeted agents discussed above. However, there is much commonality in the strategies to evaluate and use molecularly targeted and biologic therapies.

1	Immune Cell–Mediated Therapies Tumors have a variety of means of avoiding the immune system: (1) they are often only subtly different from their normal counterparts; (2) they are capable of downregulating their major histocompatibility complex antigens, effectively masking them from recognition by T cells; (3) they are inefficient at presenting antigens to the immune system; (4) they can cloak themselves in a protective shell of fibrin to minimize contact with surveillance mechanisms; and (5) they can produce a range of soluble molecules, including potential immune targets, that can distract the immune system from recognizing the tumor cell or can kill or inactivate the immune effector cells. Some of the cell products initially polarize the immune response away from cellular immunity (shifting from TH1 to TH2 responses; Chap. 372e) and ultimately lead to defects in T cells that prevent their activation and cytotoxic activity. Cancer treatment further suppresses host immunity. A

1	(shifting from TH1 to TH2 responses; Chap. 372e) and ultimately lead to defects in T cells that prevent their activation and cytotoxic activity. Cancer treatment further suppresses host immunity. A variety of strategies are being tested to overcome these barriers.

1	Cell-Mediated Immunity The strongest evidence that the immune system can exert clinically meaningful antitumor effects comes from allogeneic bone marrow transplantation. Adoptively transferred T cells from the donor expand in the tumor-bearing host, recognize the tumor as being foreign, and can mediate impressive antitumor effects (graft-versus-tumor effects). Three types of experimental interventions are being developed to take advantage of the ability of T cells to kill tumor cells.

1	1. Transfer of allogeneic T cells. This occurs in three major settings: in allogeneic bone marrow transplantation; as purified lymphocyte transfusions following bone marrow recovery after allogeneic bone marrow transplantation; and as pure lymphocyte transfusions following immunosuppressive (nonmyeloablative) therapy (also called minitransplants). In each of these settings, the effector cells are donor T cells that recognize the tumor as being foreign, probably through minor histocompatibility differences. The main risk of such therapy is the development of graft-versus-host disease because of the minimal difference between the cancer and the normal host cells. This approach has been highly effective in certain hematologic cancers. 2.

1	Transfer of autologous T cells. In this approach, the patient’s own T cells are removed from the tumor-bearing host, manipulated in several ways in vitro, and given back to the patient. There are three major classes of autologous T-cell manipulation. First, tumor antigen–specific T cells can be developed and expanded to large numbers over many weeks ex vivo before administration. Second, the patient’s T cells can be activated by exposure to polyclonal stimulators such as anti-CD3 and anti-CD28 after a short period ex vivo, and then amplified in the host after transfer by stimulation with IL-2, for example. Short periods removed from the patient permit the cells to overcome the tumor-induced T cell defects, and such cells traffic and home to sites of disease better than cells that have been in culture for many weeks. In a third approach, genes that encode for a T cell receptor specific for an antigen expressed by the tumor along with genes that facilitate T cell activation can be

1	been in culture for many weeks. In a third approach, genes that encode for a T cell receptor specific for an antigen expressed by the tumor along with genes that facilitate T cell activation can be introduced into subsets of a patient’s T cells, which, after transfer back into the patient, allow homing of cytotoxic T cells to tumor cells expressing the antigen.

1	3.

1	Tumor vaccines aimed at boosting T cell immunity. The finding that mutant oncogenes that are expressed only intracellularly can be recognized as targets of T cell killing greatly expanded the possibilities for tumor vaccine development. No longer is it difficult to find something different about tumor cells. However, major difficulties remain in getting the tumor-specific peptides presented in a fashion to prime the T cells. Tumors themselves are very poor at presenting their own antigens to T cells at the first antigen exposure (priming). Priming is best accomplished by professional antigen-presenting cells (dendritic cells). Thus, a number of experimental strategies are aimed at priming host T cells against tumor-associated peptides. Vaccine adjuvants such as granulocytemacrophage colony-stimulating factor (GM-CSF) appear capable of attracting antigen-presenting cells to a skin site containing a tumor antigen. Such an approach has been documented to eradicate microscopic residual

1	factor (GM-CSF) appear capable of attracting antigen-presenting cells to a skin site containing a tumor antigen. Such an approach has been documented to eradicate microscopic residual disease in follicular lymphoma and give rise to tumor-specific T cells. Purified antigen-presenting cells can be pulsed with tumor, its membranes, or particular tumor antigens and delivered as a vaccine. One such vaccine, Sipuleucel-T, is approved for use in patients with hormone-independent prostate cancer. In this approach, the patient undergoes leukapheresis, wherein mononuclear cells (that include antigen-presenting cells) are removed from the patient’s blood. The cells are pulsed in a laboratory with an antigenic fusion protein comprising a protein frequently expressed by prostate cancer cells, prostate acid phosphatase, fused to GM-CSF, and matured to increase their capacity to present the antigen to immune effector cells. The cells are then returned to the patient in a well-tolerated treatment.

1	acid phosphatase, fused to GM-CSF, and matured to increase their capacity to present the antigen to immune effector cells. The cells are then returned to the patient in a well-tolerated treatment. Although no objective tumor response was documented in clinical trials, median survival was increased by about 4 months. Tumor cells can also be transfected with genes that attract antigen-presenting cells.

1	Another important vaccine strategy is directed at infectious agents whose action ultimately is tied to the development of human cancer. Hepatitis B vaccine in an epidemiologic sense prevents hepatocellular carcinoma, and a tetravalent human papillomavirus vaccine prevents infection by virus types currently accounting for 70% of cervical cancer. Unfortunately, these vaccines are ineffective at treating patients who have developed a virus-induced cancer.

1	Antibody-Mediated Therapeutic Approaches In general, antibodies are not very effective at killing cancer cells. Because the tumor seems to influence the host toward making antibodies rather than generating cellular immunity, it is inferred that antibodies are easier for the tumor to fend off. Many patients can be shown to have serum antibodies directed at their tumors, but these do not appear to influence disease progression. However, the ability to grow very large quantities of high-affinity antibody directed at a tumor by the hybridoma technique has led to the application of antibodies in the treatment of cancer. In this approach, antibodies are derived where the antigen-combining regions are grafted onto human immunoglobulin gene products (chimerized or humanized) or derived de novo from mice bearing Drug Target Indications and Features of Use

1	Drug Target Indications and Features of Use Rituximab CD20 B cell neoplasms (also emerging role in autoimmune disease); chimeric antibody with frequent mouse-derived sequences; frequent infusion reactions, particularly on initial doses; reactivation of infections, Ofatumumab CD20 active in CLL; fully human antibody with distinct binding site compared to rituximab; decreased intensity infusion reactions; Trastuzumab HER2/neu Active in breast cancer and GI cancers expressing HER2/neu; cardiotoxicity, particularly in setting of prior anthracyclines, requires monitoring; infusion reactions Pertuzumab HER2/neu Breast cancer; targets distinct binding site from trastuzumab, inhibiting dimerization of HER2 family members; infusion Cetuximab EGFR Colorectal cancers with wild-type Ki-ras oncoprotein; head and neck cancers with radiation; rash, diarrhea, infusion reactions

1	Cetuximab EGFR Colorectal cancers with wild-type Ki-ras oncoprotein; head and neck cancers with radiation; rash, diarrhea, infusion reactions Panitumumab EGFR Colorectal cancers with wild-type Ki-ras oncoprotein; fully humanized; decreased infusion reactions; different IgG subtype Bevacizumab VEGF Metastatic colorectal cancer and non-small-cell lung cancer (nonsquamous) with chemotherapy; renal cancer and glioblastoma as single agents; prominent HBP, proteinuria, GI perforations, hemorrhage, thrombosis (venous and arterial) adverse events Abbreviations: CLL, chronic lymphocytic leukemia; EGFR, epidermal growth factor receptor; GI, gastrointestinal; HBP, high blood pressure; VEGF, vascular endothelial growth factor.

1	human immunoglobulin gene loci. Three general strategies have emerged using antibodies. Tumor-regulatory antibodies target tumor cells directly or indirectly to modulate intracellular functions or attract immune or stromal cells. Immunoregulatory antibodies target antigens expressed on the tumor cells or host immune cells to modulate primarily the host’s immune responsiveness to the tumor. Finally, antibody conjugates can be made with the antibody linked to drugs, toxins, or radioisotopes to target these “warheads” for delivery to the tumor. Table 103e-6 lists features of currently used or promising antibodies for cancer treatment.

1	tumor-regulAtory ANtiboDies Humanized antibodies against the CD20 molecule expressed on B cell lymphomas (rituximab and ofatumumab) are exemplary of antibodies that affect both signaling events driving lymphomagenesis as well as activating immune responses against B cell neoplasms. They are used as single agents and in combination with chemotherapy and radiation in the treatment of B cell neoplasms. Obinutuzumab is an antibody with an altered glycosylation that enhances its ability to fix complement; it is also directed against CD20 and is of value in chronic lymphocytic leukemia. It seems to be more effective in this setting than rituximab.

1	The HER2/neu receptor overexpressed on epithelial cancers, especially breast cancer, was initially targeted by trastuzumab, with noteworthy activity in potentiating the action of chemotherapy in breast cancer as well as some evidence of single-agent activity. Trastuzumab also appears to interrupt intracellular signals derived from HER2/ neu and to stimulate immune mechanisms. The anti-HER2 antibody pertuzumab, specifically targeting the domain of HER2/neu responsible for dimerization with other HER2 family members, is more specifically directed against HER2 signaling function and augments the action of trastuzumab.

1	EGF receptor (EGFR)-directed antibodies (such as cetuximab and panitumumab) have activity in colorectal cancer refractory to chemotherapy, particularly when used to augment the activity of an additional chemotherapy program, and in the primary treatment of head and neck cancers treated with radiation therapy. The mechanism of action is unclear. Direct effects on the tumor may mediate an antiproliferative effect as well as stimulate the participation of host mechanisms involving immune cell or complement-mediated response to tumor cell–bound antibody. Alternatively, the antibody may alter the release of paracrine factors promoting tumor cell survival.

1	The anti-VEGF antibody bevacizumab shows little evidence of antitumor effect when used alone, but when combined with chemotherapeutic agents, it improves the magnitude of tumor shrinkage and time to disease progression in colorectal and nonsquamous lung cancers. The mechanism for the effect is unclear and may relate to the capacity of the antibody to alter delivery and tumor uptake of the active chemotherapeutic agent. Ziv-aflibercept is not an antibody, but a solubilized VEGF receptor VEGF binding domain, and therefore may have a distinct mechanism of action with comparable side effects.

1	Unintended side effects of any antibody use include infusion-related hypersensitivity reactions, usually limited to the first infusion, which can be managed with glucocorticoid and/or antihistamine prophylaxis. In addition, distinct syndromes have emerged with different antibodies. Anti-EGFR antibodies produce an acneiform rash that poorly responds to glucocorticoid cream treatment. Trastuzumab (anti-HER2) can inhibit cardiac function, particularly in patients with prior exposure to anthracyclines. Bevacizumab has a number of side effects of medical significance, including hypertension, thrombosis, proteinuria, hemorrhage, and gastrointestinal perforations with or without prior surgeries; these adverse events also occur with small-molecule drugs modulating VEGFR function.

1	immuNoregulAtory ANtiboDies Purely immunoregulatory antibodies stimulate immune responses to mediate tumor-directed cytotoxicity. First-generation approaches sought to activate complement and are exemplified by antibodies to CD52; these are active in chronic lymphoid leukemia and T cell malignancies. A more refined understanding of the tumor–host interface has defined that cytotoxic tumor-directed T cells are frequently inhibited by ligands upregulated in the tumor cells. The programmed death ligand 1 (PD-L1; also known as B7-homolog 1) was initially recognized as an entity that induced T cell death through a receptor present on T cells, termed the PD receptor (Fig 103e-5), which physiologically exists to regulate the intensity of the immune response. The PD family of ligands and receptors also regulates macrophage function, present in tumor stroma. These actions raised the hypothesis that antibodies directed against the PD signaling axis (both anti-PD-L1 and anti-PD) might be useful

1	also regulates macrophage function, present in tumor stroma. These actions raised the hypothesis that antibodies directed against the PD signaling axis (both anti-PD-L1 and anti-PD) might be useful in cancer treatment by allowing reactivation of the immune response against tumors. Indeed, nivolumab and lambrolizumab, both anti-PD antibodies, have shown evidence of important immune-mediated actions against certain solid tumors, including melanoma and lung cancers.

1	Chapter 103e Principles of Cancer Treatment

1	FIGuRE 103e-5 Tumors possess a microenvironment (tumor stroma) with immune cells including both helper T cells, suppressor T cells (both “regulatory” of other immune cell function), macrophages, and cytotoxic T cells. Cytokines found in the stroma and deriving from macrophawsges and regulatory T cells modulate the activities of cytotoxic T cells, which have the potential to kill tumor cells. Antigens released by tumor cells are taken up by Antigen Presenting Cells (APCs), also in the stroma. Antigens are processed by the APCs to peptides presented by the Major Histocompatibility Complex to T-cell antigen receptors, thus providing an (+) activation signal for the cytotoxic tumor cells to kill tumor cells bearing that antigen. Negative (–) signals inhibiting cytoxic T cell action include the CTLA4 receptor (on T cells), interacting with the B7 family of negative regulatory signals from APCs, and the PD receptor (on T cells), interacting with the PD-L1 (–) signal coming from tumor cells

1	CTLA4 receptor (on T cells), interacting with the B7 family of negative regulatory signals from APCs, and the PD receptor (on T cells), interacting with the PD-L1 (–) signal coming from tumor cells expressing the PD-1 ligand (PD-l1). As both CTLA4 and PD1 signals attenuate the anti-tumor T cell response, strategies which inhibit CTLA4 and PD1 function are a means of stimulating cytotoxic T cell activity to kill tumor cells. Cytokines from other immune cells and macrophages can provide both (+) and (–) signals for T cell action, and are under investigation as novel immunoregulatory therapeutics.

1	Already approved for clinical use in melanoma is ipilimumab, an antibody directed against the anti-CTLA4 (cytotoxic T lymphocyte antigen 4), which is expressed on T cells (not tumor cells), responds to signals from antigen-presenting cells (Fig. 103e-5), and also down-regulates the intensity of the T cell proliferative response to antigens derived from tumor cells. Indeed, manipulation of the CTLA4 axis was the first demonstration that purely immunoregulatory antibody strategies directed at T cell physiology could be safe and effective in the treatment of cancer, although it acts at a very early stage in T cell activation and can be considered somewhat nonspecific in its basis for T cell stimulation. Pembrolizumab, an anti-PD ligand blocking agent was also approved for melanoma, with a similar spectrum of potential adverse events, but acting in the tumor microenvironment. Indeed, prominent activation of autoimmune hepatic, endocrine, cutaneous, neurologic, and gastrointestinal

1	a similar spectrum of potential adverse events, but acting in the tumor microenvironment. Indeed, prominent activation of autoimmune hepatic, endocrine, cutaneous, neurologic, and gastrointestinal responses is a basis for adverse events with the use of ipilimumab; the emergent use of glucocorticoids may be required to attenuate severe toxicities, which unfortunately can cause potential attenuation of antitumor effect. Importantly for the general internist, these events may occur late after exposure to ipilimumab while the patient may otherwise be enjoying sustained control of tumor growth owing to the beneficial actions of ipilimumab.

1	Another class of immunoregulatory antibody is the “bispecific” antibody blinatumomab, which was constructed to have an anti-CD19 antigen combining site as one valency of an antibody with anti-CD3 binding site as the other valency. This antibody thus can bring T cells (with its anti-CD3 activity) close to B cells bearing the CD19 determinant. Blinatumomab is active in B cell neoplasms such as acute lymphocytic leukemia, which may not have prominent expression of the CD20 targeted by rituximab.

1	ANtiboDy coNjugAtes Conjugates of antibodies with drugs and isotopes have also been shown to be effective in the treatment of cancer and have the intent of increasing the therapeutic index of the drug or isotope by delivering the toxic “warhead” directly to the tumor cell or tumor microenvironment. Ado-trastuzumab is a conjugate of the HER2/neu-directed trastuzumab and a highly toxic microtubule targeted drug (emtansine), which by itself is too toxic for human use; the antibody-drug conjugate shows valuable activity in patients with breast cancer who have developed resistance to the “naked” antibody. Brentuximab vedotin is an anti-CD30 antibody drug conjugate with a distinct microtubule poison with activity in neoplasms such as Hodgkin’s lymphoma where the tumor cells frequently express CD30. Radioconjugates targeting CD20 on lymphomas have been approved for use (ibritumomab tiuxetan [Zevalin], using yttrium-90 or 131I-tositumomab). Toxicity concerns have limited their use.

1	Cytokines There are >70 separate proteins and glycoproteins with biologic effects in humans: interferon (IFN) α, β, and γ; interleukin (IL) 1 through 29 (so far); the tumor necrosis factor (TNF) family (including lymphotoxin, TNF-related apoptosis-inducing ligand [TRAIL], CD40 ligand, and others); and the chemokine family. Only a fraction of these has been tested against cancer; only IFN-α and IL-2 are in routine clinical use.

1	About 20 different genes encode IFN-α, and their biologic effects are indistinguishable. IFN induces the expression of many genes, inhibits protein synthesis, and exerts a number of different effects on diverse cellular processes. The two recombinant forms that are commercially available are IFN-α2a and -α2b. Interferon is not curative for any tumor but can induce partial responses in follicular lymphoma, hairy cell leukemia, CML, melanoma, and Kaposi’s sarcoma. It has been used in the adjuvant setting in stage II melanoma, multiple myeloma, and follicular lymphoma, with uncertain effects on survival. It produces fever, fatigue, a flulike syndrome, malaise, myelosuppression, and depression and can induce clinically significant autoimmune disease. IFN-α is not generally the treatment of choice for any cancer.

1	IL-2 exerts its antitumor effects indirectly through augmentation of immune function. Its biologic activity is to promote the growth and activity of T cells and natural killer (NK) cells. High doses of IL-2 can produce tumor regression in certain patients with metastatic melanoma and renal cell cancer. About 2–5% of patients may experience complete remissions that are durable, unlike any other treatment for these tumors. IL-2 is associated with myriad clinical side effects, including intravascular volume depletion, capillary leak syndrome, adult respiratory distress syndrome, hypotension, fever, chills, skin rash, and impaired renal and liver function. Patients may require blood pressure support and intensive care to manage the toxicity. However, once the agent is stopped, most of the toxicities reverse completely within 3–6 days.

1	Ligand Receptor–Directed Constructs High-affinity receptors for cytokines have led to the design of cytokine-toxin recombinant fusion proteins, such as IL-2 expressed in frame with a fragment of diphtheria toxin. A commercially available construct has activity against certain T cell lymphomas. Likewise, the high-affinity folate receptor is the target for folate conjugated to chemotherapeutic agents. In both cases, the drug’s utility derives from the internalization of the targeted receptor and cleavage of the active drug or toxin moiety.

1	Although total-body irradiation has a role in preparing a patient to received allogeneic stem cells, and antibodies as described above can specifically target radioisotopes, systemically administered isotopes of iodide salts have an important role in the treatment of thyroid neoplasms, owing to the selective upregulation of the iodide transporter in the tumor cell compartment. Likewise, isotopes of samarium and radium have been found useful in the palliation of symptoms from advanced bony metastases of prostate cancer owing to their selective deposition at the tumor–bone matrix interface, thereby potentially affecting the function of both tumor and stromal cells in the progressive growth of the metastatic deposit.

1	Resistance mechanisms to the conventional cytotoxic agents were initially characterized in the late twentieth century as defects in drug uptake, metabolism, or export by tumor cells. The multidrug resistance (mdr) gene defined in vitro in cell lines exposed to increasing concentrations of drugs led to the definition of a family of transport proteins that, when overexpressed, result in the facile transport of a variety of hydrophobic drugs out of the cancer cell. Although efforts to manipulate this transporter to promote drug residence in tumor cells have been pursued, none are clinically useful at this time. Drug-metabolizing enzymes such as cytidine deaminase are upregulated in resistant tumor cells, and this is the basis for so-called “high-dose cytarabine” regimens in the treatment of leukemia. Another resistance mechanism defined during this era involved increased expression of a drug’s target, exemplified by amplification of the dihydrofolate reductase gene, in patients who had

1	leukemia. Another resistance mechanism defined during this era involved increased expression of a drug’s target, exemplified by amplification of the dihydrofolate reductase gene, in patients who had lost responsiveness to methotrexate, or mutation of topoisomerase II in tumors that relapsed after topoisomerase II modulator treatment.

1	A second class of resistance mechanisms involves loss of the cellular apoptotic mechanism activated after the engagement of a drug’s target by the drug. This occurs in a way that is heavily influenced by the biology of the particular tumor type. For example, decreased alkylguanine alkyltransferase defines a subset of glioblastoma patients with the prospect of greatest benefit from treatment with temozolomide, but has no predictive value for benefit from temozolomide in epithelial neoplasms. Likewise, ovarian cancers resistant to platinating agents have decreased expression of the proapoptotic gene bax. These types of findings have prompted the idea that responsive tumors to chemotherapeutic agents are populated by cells that express drug-related cell death controlling genes, creating in effect a state of “synthetic lethality” of the drug (Chap. 102e) with the genes expressed in responsive tumors, analogous to the existence in yeast of mutations that are well tolerated in the absence

1	effect a state of “synthetic lethality” of the drug (Chap. 102e) with the genes expressed in responsive tumors, analogous to the existence in yeast of mutations that are well tolerated in the absence of a physiologic stressor but become lethal in the presence of that stressor. In the case of tumors, the chemotherapy inducing the cell death response is the analogous physiologic stressor.

1	A third class of resistance mechanisms emerged from sequencing of the targets of agents directed at oncogenic kinases. Thus, patients with CML resistant to imatinib have acquired mutations in the ATP binding domain of p210bcr-abl in some cases, leading to the screening and design of agents with activity against the mutant proteins. Entirely analogous resistance mechanisms have emerged in patients with lung cancer treated with the EGFR antagonists gefitinib and erlotinib.

1	A final category of tumor resistance mechanisms to targeted agents includes the upregulation of alternate means of activating the pathway targeted by the agent. Thus melanomas initially responsive to BRAF V600E antagonists such as vemurafenib may reactivate raf signaling by upregulating isoforms that can bypass the variant blocked by the drug. Likewise, inhibition of HER2/neu signaling in breast cancer cells can lead to the emergence of variants with distinct oncogenic signaling pathways such as PI3 kinase. Analogously in NSCLC, EGFR inhibitor treatment leads to the emergence of cells with a predominance of c-met protooncogene–dependent signaling in the resistant tumors.

1	The susceptibility of a tumor to different treatments as a function of its expression of potential drug targets or their mutational profile has led to efforts to define the dominant pathways driving a patient’s tumor by genomic techniques including whole exome sequencing. The difficulty with applying such data to patient treatment is recognizing that these pathways may change during the natural history of a tumor and that different sites in a single patient may have tumors with different patterns of gene mutation.

1	The common cytotoxic chemotherapeutic agents almost invariably affect bone marrow function. Titration of this effect determines the MTD of the agent on a given schedule. The normal kinetics of blood cell turnover influences the sequence and sensitivity of each of the formed elements. Polymorphonuclear leukocytes (PMNs; t1/2 = 6–8 h), platelets (t = 5–7 days), and red blood cells (RBCs; t = 120 days) have most, less, and least susceptibility, respectively, to usually administered cytotoxic agents. The nadir count of each cell type in response to classes of agents is characteristic. Maximal neutropenia occurs 6–14 days after conventional doses of anthracyclines, antifolates, and antimetabolites. Alkylating agents differ from each other in the timing of cytopenias. Nitrosoureas, DTIC, and procarbazine can display delayed marrow toxicity, first appearing 6 weeks after dosing.

1	Complications of myelosuppression result from the predictable sequelae of the missing cells’ function. Febrile neutropenia refers to the clinical presentation of fever (one temperature ≥38.5°C or three readings ≥38°C but ≤38.5°C per 24 h) in a neutropenic patient with an uncontrolled neoplasm involving the bone marrow or, more usually, in a patient undergoing treatment with cytotoxic agents. Mortality from uncontrolled infection varies inversely with the neutrophil count. If the nadir neutrophil count is >1000/μL, there is little risk; if <500/μL, risk of death is markedly increased. Management of febrile neutropenia has conventionally included empirical coverage with antibiotics for the duration of neutropenia (Chap. 104). Selection of antibiotics is governed by the expected association of infections with certain underlying neoplasms; careful physical examination (with scrutiny of catheter sites, dentition, mucosal surfaces, and perirectal and genital orifices by gentle palpation);

1	of infections with certain underlying neoplasms; careful physical examination (with scrutiny of catheter sites, dentition, mucosal surfaces, and perirectal and genital orifices by gentle palpation); chest x-ray; and Gram stain and culture of blood, urine, and sputum (if any) to define a putative site of infection. In the absence of any originating site, a broadly acting β-lactam with anti-Pseudomonas activity, such as ceftazidime, is begun empirically. The addition of vancomycin to cover potential cutaneous sites of origin (until these are ruled out or shown to originate from methicillin-sensitive organisms) or metronidazole or imipenem for abdominal or other sites favoring anaerobes reflects modifications tailored to individual patient presentations. The coexistence of pulmonary compromise raises a distinct set of potential pathogens, including Legionella, Pneumocystis, and fungal agents that may require further diagnostic evaluations, such as bronchoscopy with bronchoalveolar

1	raises a distinct set of potential pathogens, including Legionella, Pneumocystis, and fungal agents that may require further diagnostic evaluations, such as bronchoscopy with bronchoalveolar lavage. Febrile neutropenic patients can be stratified broadly into two prognostic groups. The first, with expected short duration of neutropenia and no evidence of hypotension or abdominal or other localizing symptoms, may be expected to do well even with oral regimens, e.g., ciprofloxacin or moxifloxacin, or amoxicillin plus clavulanic acid. A less favorable prognostic group is patients with expected prolonged neutropenia, evidence of sepsis, and end organ compromise, particularly pneumonia. These patients require tailoring of their antibiotic regimen to their underlying presentation, with frequent empirical addition of antifungal agents if fever and neutropenia persists for 7 days without identification of an adequately treated organism or site.

1	Transfusion of granulocytes has no role in the management of febrile neutropenia, owing to their exceedingly short half-life, mechanical fragility, and clinical syndromes of pulmonary compromise with leukostasis after their use. Instead, colony-stimulating factors (CSFs) are used to augment bone marrow production of PMNs. Early-acting factors such as IL-1, IL-3, and stem cell factor have not been as useful clinically as late-acting, lineage-specific factors such as granulocyte colony-stimulating factor (G-CSF) or GM-CSF, erythropoietin (EPO), thrombopoietin, IL-6, and IL-11. CSFs may easily become overused in oncology practice. The settings in which their use has been proved effective are limited. G-CSF, GM-CSF, EPO, and IL-11 are currently approved for use. The American Society of Clinical Oncology has developed practice guidelines for the use of G-CSF and GM-CSF (Table 103e-7).

1	Primary prophylaxis (i.e., shortly after completing chemotherapy to reduce the nadir) administers G-CSF to patients receiving cytotoxic Chapter 103e Principles of Cancer Treatment With the first cycle of chemotherapy (so-called primary CSF administration) Use if the probability of febrile neutropenia is ≥20% Age >65 years treated for lymphoma with curative intent or other tumor Dose-dense regimens in a clinical trial or with strong evidence of benefit With subsequent cycles if febrile neutropenia has previously occurred (so-called secondary CSF administration) Afebrile neutropenic patients No evidence of benefit Febrile neutropenic patients No evidence of benefit May feel compelled to use in the face of clinical deterioration from sepsis, pneumonia, or fungal infection, but benefit unclear In bone marrow or peripheral blood stem cell transplantation Use to mobilize stem cells from marrow Use to hasten myeloid recovery In acute myeloid leukemia G-CSF of minor or no benefit

1	Use to mobilize stem cells from marrow Use to hasten myeloid recovery In acute myeloid leukemia G-CSF of minor or no benefit GM-CSF of no benefit and may be harmful In myelodysplastic syndromes Use intermittently in subset with neutropenia and recurrent infection What Dose and Schedule Should Be Used? G-CSF: 5 mg/kg per day subcutaneously GM-CSF: 250 mg/m2 per day subcutaneously Pegfilgrastim: one dose of 6 mg 24 h after chemotherapy When Should Therapy Begin and End? When indicated, start 24–72 h after chemotherapy Continue until absolute neutrophil count is 10,000/μL Do not use concurrently with chemotherapy or radiation therapy Abbreviations: CSF, cerebrospinal fluid; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor. Source: From the American Society of Clinical Oncology: J Clin Oncol 24:3187, 2006.

1	regimens associated with a 20% incidence of febrile neutropenia. “Dose-dense” regimens, where cycling of chemotherapy is intended to be completed without delay of administered doses, may also benefit, but such patients should be on a clinical trial. Administration of G-CSF in these circumstances has reduced the incidence of febrile neutropenia in several studies by about 50%. Most patients, however, receive regimens that do not have such a high risk of expected febrile neutropenia, and therefore most patients initially should not receive G-CSF or GM-CSF. Special circumstances—such as a documented history of febrile neutropenia with the regimen in a particular patient or categories of patients at increased risk, such as patients older than age 65 years with aggressive lymphoma treated with curative chemotherapy regimens; extensive compromise of marrow by prior radiation or chemotherapy; or active, open wounds or deep-seated infection—may support primary treatment with G-CSF or GM-CSF.

1	curative chemotherapy regimens; extensive compromise of marrow by prior radiation or chemotherapy; or active, open wounds or deep-seated infection—may support primary treatment with G-CSF or GM-CSF. Administration of G-CSF or GM-CSF to afebrile neutropenic patients or to patients with low-risk febrile neutropenia is not recommended, and patients receiving concomitant chemoradiation treatment, particularly those with thoracic neoplasms, likewise are not generally recommended for treatment. In contrast, administration of G-CSF to high-risk patients with febrile neutropenia and evidence of organ compromise including sepsis syndrome, invasive fungal infection, concurrent hospitalization at the time fever develops, pneumonia, profound neutropenia (<0.1 × 109/L), or age >65 years is reasonable.

1	Secondary prophylaxis refers to the administration of CSFs in patients who have experienced a neutropenic complication from a prior cycle of chemotherapy; dose reduction or delay may be a reasonably considered alternative. G-CSF or GM-CSF is conventionally started 24–72 h after completion of chemotherapy and continued until a PMN count of 10,000/μL is achieved, unless a “depot” preparation of G-CSF such as pegfilgrastim is used, where one dose is administered at least 14 days before the next scheduled administration of chemotherapy. Also, patients with myeloid leukemias undergoing induction therapy may have a slight reduction in the duration of neutropenia if G-CSF is commenced after completion of therapy and may be of particular value in elderly patients, but the influence on long-term outcome has not been defined. GM-CSF probably has a more restricted utility than G-CSF, with its use currently limited to patients after autologous bone marrow transplants, although proper head-to-head

1	has not been defined. GM-CSF probably has a more restricted utility than G-CSF, with its use currently limited to patients after autologous bone marrow transplants, although proper head-to-head comparisons with G-CSF have not been conducted in most instances. GM-CSF may be associated with more systemic side effects.

1	Dangerous degrees of thrombocytopenia do not frequently complicate the management of patients with solid tumors receiving cytotoxic chemotherapy (with the possible exception of certain carboplatincontaining regimens), but they are frequent in patients with certain hematologic neoplasms where marrow is infiltrated with tumor. Severe bleeding related to thrombocytopenia occurs with increased frequency at platelet counts <20,000/μL and is very prevalent at counts <5000/μL.

1	The precise “trigger” point at which to transfuse patients has been defined as a platelet count of 10,000/μL or less in patients without medical comorbidities that may increase the risk of bleeding. This issue is important not only because of the costs of frequent transfusion, but unnecessary platelet transfusions expose the patient to the risks of allosensitization and loss of value from subsequent transfusion owing to rapid platelet clearance, as well as the infectious and hypersensitivity risks inherent in any transfusion. Prophylactic transfusions to keep platelets >20,000/μL are reasonable in patients with leukemia who are stressed by fever or concomitant medical conditions (the threshold for transfusion is 10,000/μL in patients with solid tumors and no other bleeding diathesis or physiologic stressors such as fever or hypotension, a level that might also be reasonably considered for leukemia patients who are thrombocytopenic but not stressed or bleeding). In contrast, patients

1	stressors such as fever or hypotension, a level that might also be reasonably considered for leukemia patients who are thrombocytopenic but not stressed or bleeding). In contrast, patients with myeloproliferative states may have functionally altered platelets despite normal platelet counts, and transfusion with normal donor platelets should be considered for evidence of bleeding in these patients. Careful review of medication lists to prevent exposure to nonsteroidal anti-inflammatory agents and maintenance of clotting factor levels adequate to support near-normal prothrombin and partial thromboplastin time tests are important in minimizing the risk of bleeding in the thrombocytopenic patient.

1	Certain cytokines in clinical investigation have shown an ability to increase platelets (e.g., IL-6, IL-1, thrombopoietin), but clinical benefit and safety are not yet proven. IL-11 (oprelvekin) is approved for use in the setting of expected thrombocytopenia, but its effects on platelet counts are small, and it is associated with side effects such as headache, fever, malaise, syncope, cardiac arrhythmias, and fluid retention. Eltrombopag and romiplostim are thrombopoietin agonists with demonstrated efficacy in certain thrombocytopenic states, but they have not been systematically studied in chemotherapy-induced thrombocytopenia.

1	Anemia associated with chemotherapy can be managed by transfusion of packed RBCs. Transfusion is not undertaken until the hemoglobin falls to <80 g/L (8 g/dL), compromise of end organ function occurs, or an underlying condition (e.g., coronary artery disease) calls for maintenance of hemoglobin >90 g/L (9 g/dL). Patients who are to receive therapy for >2 months on a “stable” regimen and who are likely to require continuing transfusions are also candidates for erythropoietin (EPO). Randomized trials in certain tumors have raised the possibility that EPO use may promote tumor-related adverse events. This information should be considered in the care of individual patients. In the event EPO treatment is undertaken, maintenance of hemoglobin of 90–100 g/L (9–10 g/dL) should be the target. In the setting of adequate iron stores and serum EPO levels <100 ng/mL, EPO, 150 U three times a week, can produce a slow increase in hemoglobin over about 2 months of administration. Depot formulations

1	the setting of adequate iron stores and serum EPO levels <100 ng/mL, EPO, 150 U three times a week, can produce a slow increase in hemoglobin over about 2 months of administration. Depot formulations can be administered less frequently. It is unclear whether higher hemoglobin levels, up to 110–120 g/L (11–12 g/dL), are associated with improved quality of life to a degree that justifies the more intensive EPO use. Efforts to achieve levels at or above 120 g/L (12 g/dL) have been associated with increased thromboses and mortality rates. EPO may rescue hypoxemic cells from death and contribute to tumor radioresistance.

1	The most common side effect of chemotherapy administration is nausea, with or without vomiting. Nausea may be acute (within 24 h of chemotherapy), delayed (>24 h), or anticipatory of the receipt of chemotherapy. Patients may be likewise stratified for their risk of susceptibility to nausea and vomiting, with increased risk in young, female, heavily pretreated patients without a history of alcohol or drug use but with a history of motion or morning sickness. Antineoplastic agents vary in their capacity to cause nausea and vomiting. Highly emetogenic drugs (>90%) include mechlorethamine, streptozotocin, DTIC, cyclophosphamide at >1500 mg/m2, and cisplatin; moderately emetogenic drugs (30–90% risk) include carboplatin, cytosine arabinoside (>1 mg/m2), ifosfamide, conventional-dose cyclophosphamide, and anthracyclines; low-risk (10–30%) agents include 5FU, taxanes, etoposide, and bortezomib, with minimal risk (<10%) afforded by treatment with antibodies, bleomycin, busulfan, fludarabine,

1	and anthracyclines; low-risk (10–30%) agents include 5FU, taxanes, etoposide, and bortezomib, with minimal risk (<10%) afforded by treatment with antibodies, bleomycin, busulfan, fludarabine, and vinca alkaloids. Emesis is a reflex caused by stimulation of the vomiting center in the medulla. Input to the vomiting center comes from the chemoreceptor trigger zone (CTZ) and afferents from the peripheral gastrointestinal tract, cerebral cortex, and heart. The different emesis “syndromes” require distinct management approaches. In addition, a conditioned reflex may contribute to anticipatory nausea arising after repeated cycles of chemotherapy. Accordingly, antiemetic agents differ in their locus and timing of action. Combining agents from different classes or the sequential use of different classes of agent is the cornerstone of successful management of chemotherapy-induced nausea and vomiting. Of great importance are the prophylactic administration of agents and such psychological

1	classes of agent is the cornerstone of successful management of chemotherapy-induced nausea and vomiting. Of great importance are the prophylactic administration of agents and such psychological techniques as the maintenance of a supportive milieu, counseling, and relaxation to augment the action of antiemetic agents.

1	Serotonin antagonists (5-HT3) and neurokinin 1 (NK1) receptor antagonists are useful in “high-risk” chemotherapy regimens. The combination acts at both peripheral gastrointestinal and CNS sites that control nausea and vomiting. For example, the 5-HT3 blocker dolasetron, 100 mg intravenously or orally; dexamethasone, 12 mg; and the NK1 antagonist aprepitant, 125 mg orally, are combined on the day of administration of severely emetogenic regimens, with repetition of dexamethasone (8 mg) and aprepitant (80 mg) on days 2 and 3 for delayed nausea. Alternate 5-HT3 antagonists include ondansetron, given as 0.15 mg/kg intravenously for three doses just before and at 4 and 8 h after chemotherapy; palonosetron at 0.25 mg over 30 s, 30 min before chemotherapy; and granisetron, given as a single dose of 0.01 mg/kg just before chemotherapy. Emesis from moderately emetic chemotherapy regimens may be prevented with a 5-HT3 antagonist and dexamethasone alone for patients not receiving doxorubicin and

1	of 0.01 mg/kg just before chemotherapy. Emesis from moderately emetic chemotherapy regimens may be prevented with a 5-HT3 antagonist and dexamethasone alone for patients not receiving doxorubicin and cyclophosphamide combinations; the latter combination requires the 5-HT3/dexamethasone/aprepitant on day 1 but aprepitant alone on days 2 and 3. Emesis from low-emetic-risk regimens may be prevented with 8 mg of dexamethasone alone or with non-5-HT3, non-NK1 antagonist approaches including the following.

1	Antidopaminergic phenothiazines act directly at the CTZ and include prochlorperazine (Compazine), 10 mg intramuscularly or intravenously, 10–25 mg orally, or 25 mg per rectum every 4–6 h for up to four doses; and thiethylperazine, 10 mg by potentially all of the above routes every 6 h. Haloperidol is a butyrophenone dopamine antagonist given at 1 mg intramuscularly or orally every 8 103e-25 h. Antihistamines such as diphenhydramine have little intrinsic anti-emetic capacity but are frequently given to prevent or treat dystonic reactions that can complicate use of the antidopaminergic agents. Lorazepam is a short-acting benzodiazepine that provides an anxiolytic effect to augment the effectiveness of a variety of agents when used at 1–2 mg intramuscularly, intravenously, or orally every 4–6 h. Metoclopramide acts on peripheral dopamine receptors to augment gastric emptying and is used in high doses for highly emetogenic regimens (1–2 mg/kg intravenously 30 min before chemotherapy and

1	h. Metoclopramide acts on peripheral dopamine receptors to augment gastric emptying and is used in high doses for highly emetogenic regimens (1–2 mg/kg intravenously 30 min before chemotherapy and every 2 h for up to three additional doses as needed); intravenous doses of 10–20 mg every 4–6 h as needed or 50 mg orally 4 h before and 8 and 12 h after chemotherapy are used for moderately emetogenic regimens. 5-9-Tetrahydrocannabinol (Marinol) is a rather weak antiemetic compared to other available agents, but it may be useful for persisting nausea and is used orally at 10 mg every 3–4 h as needed.

1	Regimens that include 5FU infusions and/or irinotecan may produce severe diarrhea. Similar to the vomiting syndromes, chemotherapy-induced diarrhea may be immediate or can occur in a delayed fashion up to 48–72 h after the drugs. Careful attention to maintained hydration and electrolyte repletion, intravenously if necessary, along with antimotility treatments such as “high-dose” loperamide, commenced with 4 mg at the first occurrence of diarrhea, with 2 mg repeated every 2 h until 12 h without loose stools, not to exceed a total daily dose of 16 mg. Octreotide (100–150 μg), a somatostatin analogue, or opiate-based preparations may be considered for patients not responding to loperamide.

1	Irritation and inflammation of the mucous membranes particularly afflicting the oral and anal mucosa, but potentially involving the gastrointestinal tract, may accompany cytotoxic chemotherapy. Mucositis is due to damage to the proliferating cells at the base of the mucosal squamous epithelia or in the intestinal crypts. Topical therapies, including anesthetics and barrier-creating preparations, may provide symptomatic relief in mild cases. Palifermin or keratinocyte growth factor, a member of the fibroblast growth factor family, is effective in preventing severe mucositis in the setting of high-dose chemotherapy with stem cell transplantation for hematologic malignancies. It may also prevent or ameliorate mucositis from radiation.

1	Chemotherapeutic agents vary widely in causing alopecia, with anthracyclines, alkylating agents, and topoisomerase inhibitors reliably causing near-total alopecia when given at therapeutic doses. Antimetabolites are more variably associated with alopecia. Psychological support and the use of cosmetic resources are to be encouraged, and “chemo caps” that reduce scalp temperature to decrease the degree of alopecia should be discouraged, particularly during treatment with curative intent of neoplasms, such as leukemia or lymphoma, or in adjuvant breast cancer therapy. The richly vascularized scalp can certainly harbor micrometastatic or disseminated disease.

1	Cessation of ovulation and azoospermia reliably result from alkylating agent– and topoisomerase poison–containing regimens. The duration of these effects varies with age and sex. Males treated for Hodgkin’s disease with mechlorethamineand procarbazine-containing regimens are effectively sterile, whereas fertility usually returns after regimens that include cisplatin, vinblastine, or etoposide and after bleomycin for testicular cancer. Sperm banking before treatment may be considered to support patients likely to be sterilized by treatment. Females experience amenorrhea with anovulation after alkylating agent therapy; they are likely to recover normal menses if treatment is completed before age 30 but unlikely to recover menses after age 35. Even those who regain menses usually experience premature

1	Chapter 103e Principles of Cancer Treatment menopause. Because the magnitude and extent of decreased fertility can be difficult to predict, patients should be counseled to maintain effective contraception, preferably by barrier means, during and after therapy. Resumption of efforts to conceive should be considered in the context of the patient’s likely prognosis. Hormone replacement therapy should be undertaken in women who do not have a hormonally responsive tumor. For patients who have had a hormone-sensitive tumor primarily treated by a local modality, conventional practice would counsel against hormone replacement, but this issue is under investigation.

1	Chemotherapy agents have variable effects on the success of pregnancy. All agents tend to have increased risk of adverse outcomes when administered during the first trimester, and strategies to delay chemotherapy, if possible, until after this milestone should be considered if the pregnancy is to continue to term. Patients in their second or third trimester can be treated with most regimens for the common neoplasms afflicting women in their childbearing years, with the exception of antimetabolites, particularly antifolates, which have notable teratogenic or fetotoxic effects throughout pregnancy. The need for anticancer chemotherapy per se is infrequently a clear basis to recommend termination of a concurrent pregnancy, although each treatment strategy in this circumstance must be tailored to the individual needs of the patient.

1	Treatment with EGFR-directed small molecules (e.g., erlotinib, afatinib, lapatinib), antibodies (e.g., cetuximab, panitumumab), and mTOR antagonists (e.g., everolimus, temsirolimus) reliably produces an acneiform rash that can be a source of distress to patients and can be ameliorated with topically applied clindamycin gels and low-potency corticosteroid creams. Diarrhea frequently accompanies tyrosine kinase inhibitor administration and may respond to antimotility agents such as loperamide or stool-bulking agents.

1	Anti-VEGFR-directed treatments, including the specific antibody bevacizumab, and the “multikinase” inhibitors with anti VEGFR activity, such as sorafenib, sunitinib, and pazopanib, reliably produce hypertension in a significant fraction of patients that typically can be treated with lisinopril, amlodipine, or clonidine alone or in combination. More difficult to treat is proteinuria with resultant azotemia; this can be a basis for discontinuing treatment depending on the clinical context. Thyroid function is prominently affected by chronic exposure to this group of multikinase inhibitors including sorafenib and pazopanib, and periodic surveillance of thyroid-stimulating hormone and thyroxine (T4) levels during treatment is reasonable. Gastrointestinal perforations, arterial thromboses, and hemorrhage likewise have no specific treatments and may be a basis to avoid this class of agents. Palmar-plantar dysesthesia (“hand-foot syndrome”) can be seen after administration of these agents

1	hemorrhage likewise have no specific treatments and may be a basis to avoid this class of agents. Palmar-plantar dysesthesia (“hand-foot syndrome”) can be seen after administration of these agents (as well as some cytotoxic agents including gemcitabine and liposomal preparations of doxorubicin) and is a basis for considering dose reduction if not responsive to topical emollients and analgesics.

1	Protein kinase antagonists as a class have been associated with poorly predicted hepatic and cardiac toxicities (imatinib, dasatinib, sorafenib, pazopanib) or cardiac conduction deficits including prolonged QT interval (pazopanib). The occurrence of new cardiac or liver abnormalities in a patient receiving treatment with a protein kinase antagonist should lead to a consideration of the risk versus benefit and the possible relation of the agent to the new adverse event. The existence of prior cardiac dysfunction is a relative contraindication to the use of certain targeted therapies (e.g., trastuzumab), although each patient’s needs should be individualized. Chronic effects of cancer treatment are reviewed in Chap. 125. of the epidermis, which allows bacteria to gain access to subcutaneous infections in patients with Cancer tissue and permits the development of cellulitis. The artificial closing of a normally patent orifice can also predispose to infection; for

1	Robert W. Finberg example, obstruction of a ureter by a tumor can cause urinary tract Infections are a common cause of death and an even more common cause of morbidity in patients with a wide variety of neoplasms. Autopsy studies show that most deaths from acute leukemia and half of deaths from lymphoma are caused directly by infection. With more intensive chemotherapy, patients with solid tumors have also become more likely to die of infection. Fortunately, an evolving approach to prevention and treatment of infectious complications of cancer has decreased infection-associated mortality rates and will probably continue to do so. This accomplishment has resulted from three major steps: 1.

1	The practice of using “early empirical” antibiotics reduced mortality rates among patients with leukemia and bacteremia from 84% in 1965 to 44% in 1972. Recent studies suggest that the mortality rate due to infection in febrile neutropenic patients dropped to <10% by 2013. This dramatic improvement is attributed to early intervention with appropriate antimicrobial therapy. 2. “Empirical” antifungal therapy has also lowered the incidence of disseminated fungal infection, with dramatic decreases in mortality rates. An antifungal agent is administered—on the basis of likely fungal infection—to neutropenic patients who, after 4–7 days of antibiotic therapy, remain febrile but have no positive cultures. 3.

1	3. Use of antibiotics for afebrile neutropenic patients as broad-spectrum prophylaxis against infections has decreased both mortality and morbidity even further. The current approach to treatment of severely neutropenic patients (e.g., those receiving high-dose chemotherapy for leukemia or high-grade lymphoma) is based on initial prophylactic therapy at the onset of neutropenia, subsequent “empirical” antibacterial therapy targeting the organisms whose involvement is likely in light of physical findings (most often fever alone), and finally “empirical” antifungal therapy based on the known likelihood that fungal infection will become a serious issue after 4–7 days of broad-spectrum antibacterial therapy.

1	A physical predisposition to infection in patients with cancer (Table 104-1) can be a result of the neoplasm’s production of a break in the skin. For example, a squamous cell carcinoma may cause local invasion infection, and obstruction of the bile duct can cause cholangitis. Part of the host’s normal defense against infection depends on the continuous emptying of a viscus; without emptying, a few bacteria that are present as a result of bacteremia or local transit can multiply and cause disease.

1	A similar problem can affect patients whose lymph node integrity has been disrupted by radical surgery, particularly patients who have had radical node dissections. A common clinical problem following radical mastectomy is the development of cellulitis (usually caused by streptococci or staphylococci) because of lymphedema and/or inadequate lymph drainage. In most cases, this problem can be addressed by local measures designed to prevent fluid accumulation and breaks in the skin, but antibiotic prophylaxis has been necessary in refractory cases.

1	A life-threatening problem common to many cancer patients is the loss of the reticuloendothelial capacity to clear microorganisms after splenectomy, which may be performed as part of the management of hairy cell leukemia, chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML) and in Hodgkin’s disease. Even after curative therapy for the underlying disease, the lack of a spleen predisposes such patients to rapidly fatal infections. The loss of the spleen through trauma similarly predisposes the normal host to overwhelming infection throughout life. The splenectomized patient should be counseled about the risks of infection with certain organisms, such as the protozoan Babesia (Chap. 249) and Capnocytophaga canimorsus, a bacterium carried in the mouths of animals (Chaps. 167e and 183e). Because encapsulated bacteria (Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis) are the organisms most commonly associated with postsplenectomy sepsis,

1	167e and 183e). Because encapsulated bacteria (Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis) are the organisms most commonly associated with postsplenectomy sepsis, splenectomized persons should be vaccinated (and revaccinated; Table 104-2 and Chap. 148) against the capsular polysaccharides of these organisms. Many clinicians recommend giving splenectomized patients a small supply of antibiotics effective against S. pneumoniae,

1	N. meningitidis, and H. influenzae to avert rapid, overwhelming sepsis in the event that they cannot present for medical attention immediately after the onset of fever or other signs or symptoms of bacterial infection. A few tablets of amoxicillin/clavulanic acid (or levofloxacin if resistant strains of S. pneumoniae are prevalent locally) are a reasonable choice for this purpose. Type of Defense Specific Lesion Cells Involved Organism Cancer Association Disease Emptying of fluid Occlusion of orifices: collections ureters, bile duct, colon Lymphatic function Node dissection Splenic clearance of Splenectomy Phagocytosis Lack of granulocytes Humoral immunity Lack of antibody Cellular immunity Lack of T cells Skin epithelial cells Luminal epithelial cells T cells and macrophages Staphylococci, streptococci Staphylococci, streptococci Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Babesia, Capnocytophaga canimorsus

1	T cells and macrophages Staphylococci, streptococci Staphylococci, streptococci Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Babesia, Capnocytophaga canimorsus Staphylococci, streptococci, enteric organisms, fungi S. pneumoniae, H. influenzae, N. meningitidis Mycobacterium tuberculosis, Listeria, herpesviruses, fungi, intracellular parasites Head and neck, squamous cell carcinoma Renal, ovarian, biliary tree, metastatic diseases of many cancers Hodgkin’s disease, leukemia Acute myeloid and acute lymphocytic leukemias, hairy cell leukemia Chronic lymphocytic leukemia, multiple myeloma Hodgkin’s disease, leukemia, T cell lymphoma Cellulitis, extensive skin infection Rapid, overwhelming bacteremia; urinary tract infection Rapid, overwhelming sepsis Infections with encapsulated organisms, sinusitis, pneumonia

1	Infections with intracellular bacteria, fungi, parasites; virus reactivation a case-by-case basis following reevaluation.) aThe latest recommendations by the Advisory Committee on Immunization Practices and the CDC guidelines can be found at http://www.cdc.gov/vaccines. bA single dose of TDaP (tetanus–diphtheria–acellular pertussis), followed by a booster dose of Td (tetanus-diphtheria) every 10 years, is recommended for adults. cLive-virus vaccine is contraindicated; inactivated vaccine should be used. dTwo types of vaccine are used to prevent pneumococcal disease. A conjugate vaccine active against 13 serotypes (13-valent pneumococcal conjugate vaccine, or PCV13) is currently administered in three separate doses to all children. A polysaccharide vaccine active against 23 serotypes (23-valent pneumococcal polysaccharide vaccine, or PPSV23) elicits titers of antibody lower than those achieved with the conjugate vaccine, and immunity may wane more rapidly. Because the ablative

1	(23-valent pneumococcal polysaccharide vaccine, or PPSV23) elicits titers of antibody lower than those achieved with the conjugate vaccine, and immunity may wane more rapidly. Because the ablative chemotherapy given to recipients of hematopoietic stem cell transplants (HSCTs) eradicates immunologic memory, revaccination is recommended for all such patients. Vaccination is much more effective once immunologic reconstitution has occurred; however, because of the need to prevent serious disease, pneumococcal vaccine should be administered 6–12 months after transplantation in most cases. Because PPSV23 includes serotypes not present in PCV13, HSCT recipients should receive a dose of PPSV23 at least 8 weeks after the last dose of PCV13. Although antibody titers from PPSV23 clearly decay, experience with multiple doses of PPSV23 is limited, as are data on the safety, toxicity, or efficacy of such a regimen. For this reason, the CDC currently recommends the administration of one additional

1	with multiple doses of PPSV23 is limited, as are data on the safety, toxicity, or efficacy of such a regimen. For this reason, the CDC currently recommends the administration of one additional dose of PPSV23 at least 5 years after the last dose to immunocompromised patients, including transplant recipients, as well as patients with Hodgkin’s disease, multiple myeloma, lymphoma, or generalized malignancies. Beyond this single additional dose, further doses are not recommended at this time. eMeningococcal conjugate vaccine MenACWY is recommended for adults ≤55 years old, and meningococcal polysaccharide vaccine (MPSV4) is recommended for those ≥56 years old. fIncludes both varicella vaccine for children and zoster vaccine for adults. gContact the manufacturer for more information on use in children with acute lymphocytic leukemia.

1	Infections in Patients with Cancer

1	The level of suspicion of infections with certain organisms should depend on the type of cancer diagnosed (Table 104-3). Diagnosis of multiple myeloma or CLL should alert the clinician to the possibility of hypogammaglobulinemia. While immunoglobulin replacement therapy can be effective, in most cases prophylactic antibiotics are a cheaper, more convenient method of eliminating bacterial infections in CLL patients with hypogammaglobulinemia. Patients with acute lymphocytic leukemia (ALL), patients with non-Hodgkin’s lymphoma, and all cancer patients treated with high-dose glucocorticoids (or glucocorticoid-containing chemotherapy regimens) should receive antibiotic prophylaxis for Pneumocystis infection (Table 104-3) for the duration of their chemotherapy. In addition to exhibiting susceptibility to certain infectious organisms, patients with cancer are likely to manifest their infections in characteristic ways. For example, fever—generally a sign of infection in normal

1	susceptibility to certain infectious organisms, patients with cancer are likely to manifest their infections in characteristic ways. For example, fever—generally a sign of infection in normal hosts—continues to be a reliable indicator in neutropenic patients. In contrast, patients receiving glucocorticoids and agents that impair T cell function and cytokine secretion may have serious infections in the absence of fever. Similarly, neutropenic patients commonly present with cellulitis without purulence and with pneumonia without sputum or even x-ray findings (see below).

1	The use of monoclonal antibodies that target B and T cells as well as drugs that interfere with lymphocyte signal transduction events is associated with reactivation of latent infections. The use of rituximab, the antibody to CD20 (a B cell surface protein), is associated with the development of reactivation tuberculosis as well as other latent viral infections, including hepatitis B and cytomegalovirus (CMV) infection. Like organ transplant recipients (Chap. 169), patients with latent bacterial disease (like tuberculosis) and latent viral disease (like herpes simplex or zoster) should be carefully monitored for reactivation disease. Skin lesions are common in cancer patients, and the appearance of these lesions may permit the diagnosis of systemic bacterial or fungal infection. While cellulitis caused by skin organisms such as aThe reason for this association is not well defined.

1	Streptococcus or Staphylococcus is common, neutropenic patients—i.e., those with <500 functional polymorphonuclear leukocytes (PMNs)/ μL—and patients with impaired blood or lymphatic drainage may develop infections with unusual organisms. Innocent-looking macules or papules may be the first sign of bacterial or fungal sepsis in immunocompromised patients (Fig. 104-1). In the neutropenic host, a macule progresses rapidly to ecthyma gangrenosum (see Fig. 25e-35), a usually painless, round, necrotic lesion consisting of a central black or gray-black eschar with surrounding erythema. Ecthyma gangrenosum, which is located in nonpressure areas (as distinguished from necrotic lesions associated with lack of circulation), is often associated with Pseudomonas aeruginosa bacteremia (Chap. 189) but may be caused by other bacteria.

1	Candidemia (Chap. 240) is also associated with a variety of skin conditions (see Fig. 25e-38) and commonly presents as a maculopapular rash. Punch biopsy of the skin may be the best method for diagnosis. Escherichia coli Serratia spp. Klebsiella spp. Acinetobacter spp.a Pseudomonas aeruginosa Stenotrophomonas spp. Enterobacter spp. Citrobacter spp. Non-aeruginosa Pseudomonas spp.a Candida spp. Mucor/Rhizopus Aspergillus spp. aOften associated with intravenous catheters.

1	Cellulitis, an acute spreading inflammation of the skin, is most often caused by infection with group A Streptococcus or Staphylococcus aureus, virulent organisms normally found on the skin (Chap. 156). Although cellulitis tends to be circumscribed in normal hosts, it may spread rapidly in neutropenic patients. A tiny break in the skin may lead to spreading cellulitis, which is characterized by pain and erythema; in the affected patients, signs of infection (e.g., purulence) are often lacking. What might be a furuncle in a normal host may require amputation because of uncontrolled infection in a patient presenting with leukemia. A dramatic response to an infection that might be trivial in a normal host can mark the first sign of leukemia. Fortunately, granulocytopenic patients are likely to be infected with certain types of organisms (Table 104-4); thus the selection of an antibiotic regimen is somewhat easier than it might otherwise be (see “Antibacterial Therapy,” below). It is

1	to be infected with certain types of organisms (Table 104-4); thus the selection of an antibiotic regimen is somewhat easier than it might otherwise be (see “Antibacterial Therapy,” below). It is essential to recognize cellulitis early and to treat it aggressively. Patients who are neutropenic or who have previously received antibiotics for other reasons may develop cellulitis with unusual organisms (e.g., Escherichia coli, Pseudomonas, or fungi). Early treatment, even of innocent-looking lesions, is essential to prevent necrosis and loss of tissue. Debridement to prevent spread may sometimes be necessary early in the course of disease, but it can often be performed after chemotherapy, when the PMN count increases.

1	FIGURE 104-1 A. Papules related to Escherichia coli bacteremia in a patient with acute lymphocytic leukemia. B. The same lesions on the following day.

1	Sweet syndrome, or febrile neutrophilic dermatosis, was originally described in women with elevated white blood cell (WBC) counts. The disease is characterized by the presence of leukocytes in the lower dermis, with edema of the papillary body. Ironically, this disease now is usually seen in neutropenic patients with cancer, most often in association with acute myeloid leukemia (AML) but also in association with a variety of other malignancies. Sweet syndrome usually presents as red or bluish-red papules or nodules that may coalesce and form sharply bordered plaques (see Fig. 25e-41). The edema may suggest vesicles, but on palpation the lesions are solid, and vesicles probably never arise in this disease. The lesions are most common on the face, neck, and arms. On the legs, they may be confused with erythema nodosum (see Fig. 25e-40). The development of lesions is often accompanied by high fevers and an elevated erythrocyte sedimentation rate. Both the lesions and the temperature

1	confused with erythema nodosum (see Fig. 25e-40). The development of lesions is often accompanied by high fevers and an elevated erythrocyte sedimentation rate. Both the lesions and the temperature elevation respond dramatically to glucocorticoid administration. Treatment begins with high doses of glucocorticoids (prednisone, 60 mg/d) followed by tapered doses over the next 2–3 weeks.

1	Data indicate that erythema multiforme (see Fig. 25e-25) with mucous membrane involvement is often associated with herpes simplex virus (HSV) infection and is distinct from Stevens-Johnson syndrome, which is associated with drugs and tends to have a more widespread distribution. Because cancer patients are both immunosuppressed (and therefore susceptible to herpes infections) and heavily treated with drugs (and therefore subject to Stevens-Johnson syndrome [see Fig. 46e-4]), both of these conditions are common in this population. Cytokines, which are used as adjuvants or primary treatments for cancer, can themselves cause characteristic rashes, further complicating the differential diagnosis. This phenomenon is a particular problem in bone marrow transplant recipients (Chap. 169), who, in addition to having the usual chemotherapy-, antibiotic-, and cytokineinduced rashes, are plagued by graft-versus-host disease.

1	Because IV catheters are commonly used in cancer chemotherapy and are prone to cause infection (Chap. 168), they pose a major problem in the care of patients with cancer. Some catheter-associated infections can be treated with antibiotics, whereas in others the catheter must be removed (Table 104-5). If the patient has a “tunneled” catheter (which consists of an entrance site, a subcutaneous tunnel, and an exit site), a red streak over the subcutaneous part of the line (the tunnel) is grounds for immediate device removal. Failure to remove catheters under these 487 circumstances may result in extensive cellulitis and tissue necrosis.

1	More common than tunnel infections are exit-site infections, often with erythema around the area where the line penetrates the skin. Most authorities (Chap. 172) recommend treatment (usually with vancomycin) for an exit-site infection caused by coagulase-negative Staphylococcus. Treatment of coagulase-positive staphylococcal infection is associated with a poorer outcome, and it is advisable to remove the catheter if possible. Similarly, most clinicians remove catheters associated with infections due to P. aeruginosa and Candida species, because such infections are difficult to treat and bloodstream infections with these organisms are likely to be deadly. Catheter infections caused by Burkholderia cepacia, Stenotrophomonas species, Agrobacterium species, Acinetobacter baumannii, Pseudomonas species other than aeruginosa, and carbapenem-resistant Enterobacteriaceae are likely to be very difficult to eradicate with antibiotics alone. Similarly, isolation of Bacillus, Corynebacterium, and

1	species other than aeruginosa, and carbapenem-resistant Enterobacteriaceae are likely to be very difficult to eradicate with antibiotics alone. Similarly, isolation of Bacillus, Corynebacterium, and Mycobacterium species should prompt removal of the catheter.

1	infections of the mouth The oral cavity is rich in aerobic and anaerobic bacteria (Chap. 201) that normally live in a commensal relationship with the host. The antimetabolic effects of chemotherapy cause a breakdown of mucosal host defenses, leading to ulceration of the mouth and the potential for invasion by resident bacteria. Mouth ulcerations afflict most patients receiving cytotoxic chemotherapy and have been associated with viridans streptococcal bacteremia. Candida infections of the mouth are very common. Fluconazole is clearly effective in the treatment of both local infections (thrush) and systemic infections (esophagitis) due to Candida albicans. Other azoles (e.g., voriconazole) as well as echinocandins offer similar efficacy as well as activity against the fluconazole-resistant organisms that are associated with chronic fluconazole treatment (Chap. 240).

1	Noma (cancrum oris), commonly seen in malnourished children, is a penetrating disease of the soft and hard tissues of the mouth and adjacent sites, with resulting necrosis and gangrene. It has a counterpart in immunocompromised patients and is thought to be due to invasion of the tissues by Bacteroides, Fusobacterium, and other normal inhabitants of the mouth. Noma is associated with debility, poor oral hygiene, and immunosuppression. Infections in Patients with Cancer Evidence of Infection, Negative Blood Cultures 488 Viruses, particularly HSV, are a prominent cause of morbidity in immunocompromised patients, in whom they are associated with severe mucositis. The use of acyclovir, either prophylactically or therapeutically, is of value. esoPhageal infections The differential diagnosis of esophagitis (usually presenting as substernal chest pain upon swallowing) includes herpes simplex and candidiasis, both of which are readily treatable.

1	Lower Gastrointestinal Tract Disease Hepatic candidiasis (Chap. 240) results from seeding of the liver (usually from a gastrointestinal source) in neutropenic patients. It is most common among patients being treated for AML and usually presents symptomatically around the time the neutropenia resolves. The characteristic picture is that of persistent fever unresponsive to antibiotics, abdominal pain and tenderness or nausea, and elevated serum levels of alkaline phosphatase in a patient with hematologic malignancy who has recently recovered from neutropenia. The diagnosis of this disease (which may present in an indolent manner and persist for several months) is based on the finding of yeasts or pseudohyphae in granulomatous lesions. Hepatic ultrasound or CT may reveal bull’s-eye lesions. MRI scans reveal small lesions not visible by other imaging modalities. The pathology (a granulomatous response) and the timing (with resolution of neutropenia and an elevation in granulocyte count)

1	MRI scans reveal small lesions not visible by other imaging modalities. The pathology (a granulomatous response) and the timing (with resolution of neutropenia and an elevation in granulocyte count) suggest that the host response to Candida is an important component of the manifestations of disease. In many cases, although organisms are visible, cultures of biopsied material may be negative. The designation hepatosplenic candidiasis or hepatic candidiasis is a misnomer because the disease often involves the kidneys and other tissues; the term chronic disseminated candidiasis may be more appropriate. Because of the risk of bleeding with liver biopsy, diagnosis is often based on imaging studies (MRI, CT). Treatment should be directed to the causative agent (usually C. albicans but sometimes Candida tropicalis or other less common Candida species).

1	Typhlitis Typhlitis (also referred to as necrotizing colitis, neutropenic colitis, necrotizing enteropathy, ileocecal syndrome, and cecitis) is a clinical syndrome of fever and right-lower-quadrant (or generalized abdominal) tenderness in an immunosuppressed host. This syndrome is classically seen in neutropenic patients after chemotherapy with cytotoxic drugs. It may be more common among children than among adults and appears to be much more common among patients with AML or ALL than among those with other types of cancer. Physical examination reveals right-lower-quadrant tenderness, with or without rebound tenderness. Associated diarrhea (often bloody) is common, and the diagnosis can be confirmed by the finding of a thickened cecal wall on CT, MRI, or ultrasonography. Plain films may reveal a right-lower-quadrant mass, but CT with contrast or MRI is a much more sensitive means of diagnosis. Although surgery is sometimes attempted to avoid perforation from ischemia, most cases

1	may reveal a right-lower-quadrant mass, but CT with contrast or MRI is a much more sensitive means of diagnosis. Although surgery is sometimes attempted to avoid perforation from ischemia, most cases resolve with medical therapy alone. The disease is sometimes associated with positive blood cultures (which usually yield aerobic gram-negative bacilli), and therapy is recommended for a broad spectrum of bacteria (particularly gram-negative bacilli, which are likely to be found in the bowel flora). Surgery is indicated in the case of perforation.

1	Clostridium difficile–Induced Diarrhea Patients with cancer are predisposed to the development of C. difficile diarrhea (Chap. 161) as a consequence of chemotherapy alone. Thus, they may test positive for C. difficile even without receiving antibiotics. Obviously, such patients are also subject to C. difficile–induced diarrhea as a result of antibiotic pressure. C. difficile should always be considered as a possible cause of diarrhea in cancer patients who have received either chemotherapy or antibiotics. CENTRAL NERVOUS SYSTEM–SPECIFIC SYNDROMES Meningitis The presentation of meningitis in patients with lymphoma or CLL and in patients receiving chemotherapy (particularly with glucocorticoids) for solid tumors suggests a diagnosis of cryptococcal or listerial infection. As noted previously, splenectomized patients are susceptible to rapid, overwhelming infection with encapsulated bacteria (including S. pneumoniae, H. influenzae, and N. meningitidis).

1	Similarly, patients who are antibody-deficient (e.g., those with CLL, those who have received intensive chemotherapy, or those who have undergone bone marrow transplantation) are likely to have infections caused by these bacteria. Other cancer patients, however, because of their defective cellular immunity, are likely to be infected with other pathogens (Table 104-3). Central nervous system (CNS) tuberculosis should be considered, especially in patients from countries where tuberculosis is highly prevalent in the population.

1	Encephalitis The spectrum of disease resulting from viral encephalitis is expanded in immunocompromised patients. A predisposition to infections with intracellular organisms similar to those encountered in patients with AIDS (Chap. 226) is seen in cancer patients receiving (1) high-dose cytotoxic chemotherapy, (2) chemotherapy affecting T cell function (e.g., fludarabine), or (3) antibodies that eliminate T cells (e.g., anti-CD3, alemtuzumab, anti-CD52) or cytokine activity (anti– tumor necrosis factor agents or interleukin 1 receptor antagonists). Infection with varicella-zoster virus (VZV) has been associated with encephalitis that may be caused by VZV-related vasculitis. Chronic viral infections may also be associated with dementia and encephalitic presentations. A diagnosis of progressive multifocal leukoencephalopathy (Chap. 164) should be considered when a patient who has received chemotherapy (rituximab in particular) presents with dementia (Table 104-6). Other abnormalities of

1	multifocal leukoencephalopathy (Chap. 164) should be considered when a patient who has received chemotherapy (rituximab in particular) presents with dementia (Table 104-6). Other abnormalities of the CNS that may be confused with infection include normal-pressure hydrocephalus and vasculitis resulting from CNS irradiation. It may be possible to differentiate these conditions by MRI.

1	Brain Masses Mass lesions of the brain most often present as headache with or without fever or neurologic abnormalities. Infections associated with mass lesions may be caused by bacteria (particularly Nocardia), fungi (particularly Cryptococcus or Aspergillus), or parasites (Toxoplasma). Epstein-Barr virus (EBV)–associated lymphoma may also present as single—or sometimes multiple—mass lesions of the brain. A biopsy may be required for a definitive diagnosis. Pneumonia (Chap. 153) in immunocompromised patients may be difficult to diagnose because conventional methods of diagnosis depend on the presence of neutrophils. Bacterial pneumonia in neutropenic patients may present without purulent sputum—or, in fact, without any sputum at all—and may not produce physical findings suggestive of chest consolidation (rales or egophony).

1	In granulocytopenic patients with persistent or recurrent fever, the chest x-ray pattern may help to localize an infection and thus to determine which investigative tests and procedures should be undertaken and which therapeutic options should be considered (Table 104-7). In this setting, a simple chest x-ray is a screening tool; because the impaired host response results in less evidence of consolidation or infiltration, high-resolution CT is recommended for the diagnosis of pulmonary infections. The difficulties encountered in the management of pulmonary infiltrates relate in part to the difficulties of performing diagnostic procedures on the patients involved. When platelet counts can be increased to adequate levels by transfusion, aHigh-dose glucocorticoid therapy, cytotoxic chemotherapy.

1	Cause of Pneumonia microscopic and microbiologic evaluation of the fluid obtained by endoscopic bronchial lavage is often diagnostic. Lavage fluid should be cultured for Mycoplasma, Chlamydia, Legionella, Nocardia, more common bacterial pathogens, fungi, and viruses. In addition, the possibility of Pneumocystis pneumonia should be considered, especially in patients with ALL or lymphoma who have not received prophylactic trimethoprim-sulfamethoxazole (TMP-SMX). The characteristics of the infiltrate may be helpful in decisions about further diagnostic and therapeutic maneuvers. Nodular infiltrates suggest fungal pneumonia (e.g., that caused by Aspergillus or Mucor). Such lesions may best be approached by visualized biopsy procedures. It is worth noting that while bacterial pneumonias classically present as lobar infiltrates in normal hosts, bacterial pneumonias in granulocytopenic hosts present with a paucity of signs, symptoms, or radiographic abnormalities; thus, the diagnosis is

1	present as lobar infiltrates in normal hosts, bacterial pneumonias in granulocytopenic hosts present with a paucity of signs, symptoms, or radiographic abnormalities; thus, the diagnosis is difficult.

1	Aspergillus species (Chap. 241) can colonize the skin and respiratory tract or cause fatal systemic illness. Although this fungus may cause aspergillomas in a previously existing cavity or may produce allergic bronchopulmonary disease in some patients, the major problem posed by this genus in neutropenic patients is invasive disease, primarily due to Aspergillus fumigatus or Aspergillus flavus. The organisms enter the host following colonization of the respiratory tract, with subsequent invasion of blood vessels. The disease is likely to present as a thrombotic or embolic event because of this ability of the fungi to invade blood vessels. The risk of infection with Aspergillus correlates directly with the duration of neutropenia. In prolonged neutropenia, positive surveillance cultures for nasopharyngeal colonization with Aspergillus may predict the development of disease.

1	Patients with Aspergillus infection often present with pleuritic chest pain and fever, which are sometimes accompanied by cough. Hemoptysis may be an ominous sign. Chest x-rays may reveal new focal infiltrates or nodules. Chest CT may reveal a characteristic halo consisting of a mass-like infiltrate surrounded by an area of low attenuation. The presence of a “crescent sign” on chest x-ray or chest CT, in which the mass progresses to central cavitation, is characteristic of invasive Aspergillus infection but may develop as the lesions are resolving. In addition to causing pulmonary disease, Aspergillus may invade through the nose or palate, with deep sinus penetration. The appearance of a discolored area in the nasal passages or on the hard palate should prompt a search for invasive Aspergillus. This situation is likely to require surgical debridement. Catheter infections with Aspergillus usually require both removal of the catheter and antifungal therapy.

1	Diffuse interstitial infiltrates suggest viral, parasitic, or Pneumocystis pneumonia. If the patient has a diffuse interstitial pattern on chest x-ray, it may be reasonable, while considering invasive diagnostic procedures, to institute empirical treatment for Pneumocystis with TMP-SMX and for Chlamydia, Mycoplasma, and Legionella with a quinolone or azithromycin. Noninvasive procedures, such as staining of induced sputum smears for Pneumocystis, serum cryptococcal antigen tests, and urine testing for Legionella antigen, may be helpful. Serum galactomannan and β-d-glucan tests may be of value in diagnosing Aspergillus infection, but their utility is limited by their lack of sensitivity and specificity. The presence of an elevated level of β-d-glucan in the serum of a patient being treated for cancer who is not receiving prophylaxis against Pneumocystis suggests the diagnosis of Pneumocystis pneumonia. Infections with viruses that cause only 489 upper respiratory symptoms in

1	treated for cancer who is not receiving prophylaxis against Pneumocystis suggests the diagnosis of Pneumocystis pneumonia. Infections with viruses that cause only 489 upper respiratory symptoms in immunocompetent hosts, such as respiratory syncytial virus (RSV), influenza viruses, and parainfluenza viruses, may be associated with fatal pneumonitis in immunocompromised hosts. CMV reactivation occurs in cancer patients receiving chemotherapy, but CMV pneumonia is most common among HSCT recipients (Chap. 169). Polymerase chain reaction testing now allows rapid diagnosis of viral pneumonia, which can lead to treatment in some cases (e.g., influenza). Multiplex studies that can detect a wide array of viruses in the lung and upper respiratory tract are now available and will lead to specific diagnoses of viral pneumonias.

1	Bleomycin is the most common cause of chemotherapy-induced lung disease. Other causes include alkylating agents (such as cyclophosphamide, chlorambucil, and melphalan), nitrosoureas (carmustine [BCNU], lomustine [CCNU], and methyl-CCNU), busulfan, procarbazine, methotrexate, and hydroxyurea. Both infectious and noninfectious (drugand/or radiation-induced) pneumonitis can cause fever and abnormalities on chest x-ray; thus, the differential diagnosis of an infiltrate in a patient receiving chemotherapy encompasses a broad range of conditions (Table 104-7). The treatment of radiation pneumonitis (which may respond dramatically to glucocorticoids) or drug-induced pneumonitis is different from that of infectious pneumonia, and a biopsy may be important in the diagnosis. Unfortunately, no definitive diagnosis can be made in ∼30% of cases, even after bronchoscopy.

1	Open-lung biopsy is the gold standard of diagnostic techniques. Biopsy via a visualized thoracostomy can replace an open procedure in many cases. When a biopsy cannot be performed, empirical treatment can be undertaken; a quinolone or an erythromycin derivative (azithromycin) and TMP-SMX are used in the case of diffuse infiltrates, and an antifungal agent is administered in the case of nodular infiltrates. The risks should be weighed carefully in these cases. If inappropriate drugs are administered, empirical treatment may prove toxic or ineffective; either of these outcomes may be riskier than biopsy.

1	Patients with Hodgkin’s disease are prone to persistent infections by Salmonella, sometimes (and particularly often in elderly patients) affecting a vascular site. The use of IV catheters deliberately lodged in the right atrium is associated with a high incidence of bacterial endocarditis, presumably related to valve damage followed by bacteremia. Nonbacterial thrombotic endocarditis (marantic endocarditis) has been described in association with a variety of malignancies (most often solid tumors) and may follow bone marrow transplantation as well. The presentation of an embolic event with a new cardiac murmur suggests this diagnosis. Blood cultures are negative in this disease of unknown pathogenesis.

1	Infections of the endocrine system have been described in immunocompromised patients. Candida infection of the thyroid may be difficult to diagnose during the neutropenic period. It can be defined by indium-labeled WBC scans or gallium scans after neutrophil counts increase. CMV infection can cause adrenalitis with or without resulting adrenal insufficiency. The presentation of a sudden endocrine anomaly in an immunocompromised patient can be a sign of infection in the involved end organ. Infection that is a consequence of vascular compromise, resulting in gangrene, can occur when a tumor restricts the blood supply to muscles, bones, or joints. The process of diagnosis and treatment of such infection is similar to that in normal hosts, with the following caveats: 1.

1	In terms of diagnosis, a lack of physical findings resulting from a lack of granulocytes in the granulocytopenic patient should make the clinician more aggressive in obtaining tissue rather than more willing to rely on physical signs. 2. In terms of therapy, aggressive debridement of infected tissues may be required. However, it is usually difficult to operate on patients

1	2. In terms of therapy, aggressive debridement of infected tissues may be required. However, it is usually difficult to operate on patients Infections in Patients with Cancer 490 who have recently received chemotherapy, both because of a lack of platelets (which results in bleeding complications) and because of a lack of WBCs (which may lead to secondary infection). A blood culture positive for Clostridium perfringens—an organism commonly associated with gas gangrene—can have a number of meanings (Chap. 179). Clostridium septicum bacteremia is associated with the presence of an underlying malignancy. Bloodstream infections with intestinal organisms such as Streptococcus bovis biotype 1 and C. perfringens may arise spontaneously from lower gastrointestinal lesions (tumor or polyps); alternatively, these lesions may be harbingers of invasive disease. The clinical setting must be considered in order to define the appropriate treatment for each case.

1	Infections of the urinary tract are common among patients whose ureteral excretion is compromised (Table 104-1). Candida, which has a predilection for the kidney, can invade either from the bloodstream or in a retrograde manner (via the ureters or bladder) in immunocompromised patients. The presence of “fungus balls” or persistent candiduria suggests invasive disease. Persistent funguria (with Aspergillus as well as Candida) should prompt a search for a nidus of infection in the kidney. Certain viruses are typically seen only in immunosuppressed patients. BK virus (polyomavirus hominis 1) has been documented in the urine of bone marrow transplant recipients and, like adenovirus, may be associated with hemorrhagic cystitis.

1	It is beyond the scope of this chapter to detail how all the immunologic abnormalities that result from cancer or from chemotherapy for cancer lead to infections. Disorders of the immune system are discussed in other sections of this book. As has been noted, patients with antibody deficiency are predisposed to overwhelming infection with encapsulated bacteria (including S. pneumoniae, H. influenzae, and N. meningitidis). Infections that result from the lack of a functional cellular immune system are described in Chap. 226. It is worth mentioning, however, that patients undergoing intensive chemotherapy for any form of cancer will have not only defects due to granulocytopenia but also lymphocyte dysfunction, which may be profound. Thus, these patients—especially those receiving glucocorticoid-containing regimens or drugs that inhibit either T cell activation (calcineurin inhibitors or drugs like fludarabine, which affect lymphocyte function) or cytokine induction—should be given

1	regimens or drugs that inhibit either T cell activation (calcineurin inhibitors or drugs like fludarabine, which affect lymphocyte function) or cytokine induction—should be given prophylaxis for Pneumocystis pneumonia.

1	Patients receiving treatment that eliminates B cells (e.g., with anti-CD20 antibodies or rituximab) are especially vulnerable to intercurrent viral infections. The incidence of progressive multifocal leukoencephalopathy (caused by JC virus) is elevated in these patients.

1	Initial studies in the 1960s revealed a dramatic increase in the incidence of infections (fatal and nonfatal) among cancer patients with a granulocyte count of <500/μL. The use of prophylactic antibacterial agents has reduced the number of bacterial infections, but 35–78% of febrile neutropenic patients being treated for hematologic malignancies develop infections at some time during chemotherapy. Aerobic pathogens (both gram-positive and gram-negative) predominate in all series, but the exact organisms isolated vary from center to center. Infections with anaerobic organisms are uncommon. Geographic patterns affect the types of fungi isolated. Tuberculosis and malaria are common causes of fever in the developing world and may present in this setting as well. Neutropenic patients are unusually susceptible to infection with a wide variety of bacteria; thus, antibiotic therapy should be initiated

1	Neutropenic patients are unusually susceptible to infection with a wide variety of bacteria; thus, antibiotic therapy should be initiated Physical examination: skin lesions, mucous membranes, IV catheter sites, perirectal area Granulocyte count: absolute count < 500/˜L; expected duration of neutropenia Blood cultures; chest radiogram; other appropriate studies based on history (sputum, urine, skin biopsy)

1	Continue treatment until neutropenia resolves (granulocyte count > 500/˜L). Add a broad spectrum antifungal agent. Febrile Afebrile InitialtherapyFollow-upSubsequenttherapyTreat with antibiotic(s) effective against both gram-negative and gram-positive aerobes. Treat the infection with the best available antibiotics. Do not narrow the spectrum unnecessarily. Continue to treat for both gram-positive and gram-negative aerobes. Obvious infectious site found No obvious infectious site Continue regimen. FIGURE 104-2 Algorithm for the diagnosis and treatment of fever and neutropenia.

1	promptly to cover likely pathogens if infection is suspected. Indeed, early initiation of antibacterial agents is mandatory to prevent deaths. Like most immunocompromised patients, neutropenic patients are threatened by their own microbial flora, including gram-positive and gram-negative organisms found commonly on the skin and mucous membranes and in the bowel (Table 104-4). Because treatment with narrow-spectrum agents leads to infection with organisms not covered by the antibiotics used, the initial regimen should target all pathogens likely to be the initial causes of bacterial infection in neutropenic hosts. As noted in the algorithm shown in Fig. 104-2, administration of antimicrobial agents is routinely continued until neutropenia resolves—i.e., the granulocyte count is sustained above 500 μL for at least 2 days. In some cases, patients remain febrile after resolution of neutropenia. In these instances, the risk of sudden death from overwhelming bacteremia is greatly reduced,

1	above 500 μL for at least 2 days. In some cases, patients remain febrile after resolution of neutropenia. In these instances, the risk of sudden death from overwhelming bacteremia is greatly reduced, and the following diagnoses should be seriously considered: (1) fungal infection, (2) bacterial abscesses or undrained foci of infection, and (3) drug fever (including reactions to antimicrobial agents as well as to chemotherapy or cytokines). In the proper setting, viral infection or graft-versus-host disease should be considered. In clinical practice, antibacterial therapy is usually discontinued when the patient is no longer neutropenic and all evidence of bacterial disease has been eliminated. Antifungal agents are then discontinued if there is no evidence of fungal disease. If the patient remains febrile, a search for viral diseases or unusual pathogens is conducted while unnecessary cytokines and other drugs are systematically eliminated from the regimen.

1	Hundreds of antibacterial regimens have been tested for use in patients with cancer. The major risk of infection is related to the degree of neutropenia seen as a consequence of either the disease or the therapy. Many of the relevant studies have involved small populations in which the outcomes have generally been good, and most have lacked the statistical power to detect differences among the regimens studied. Each febrile neutropenic patient should be approached as a unique problem, with particular attention given to previous infections and recent antibiotic exposures. Several general guidelines are useful in the initial treatment of neutropenic patients with fever (Fig. 104-2): 1. In the initial regimen, it is necessary to use antibiotics active against both gram-negative and gram-positive bacteria (Table 104-4). 2.

1	In the initial regimen, it is necessary to use antibiotics active against both gram-negative and gram-positive bacteria (Table 104-4). 2. Monotherapy with an aminoglycoside or an antibiotic lacking good activity against gram-positive organisms (e.g., ciprofloxacin or aztreonam) is not adequate in this setting. 3. The agents used should reflect both the epidemiology and the antibiotic resistance pattern of the hospital. 4. If the pattern of resistance justifies its use, a single third-generation cephalosporin constitutes an appropriate initial regimen in many hospitals. 5. Most standard regimens are designed for patients who have not previously received prophylactic antibiotics. The development of fever in a patient who has received antibiotics affects the choice of subsequent therapy, which should target resistant organisms and organisms known to cause infections in patients being treated with the antibiotics already administered. 6.

1	6. Randomized trials have indicated the safety of oral antibiotic regimens in the treatment of “low-risk” patients with fever and neutropenia. Outpatients who are expected to remain neutropenic for <10 days and who have no concurrent medical problems (such as hypotension, pulmonary compromise, or abdominal pain) can be classified as low risk and treated with a broad-spectrum oral regimen. 7. Several large-scale studies indicate that prophylaxis with a fluoroquinolone (ciprofloxacin or levofloxacin) decreases morbidity and mortality rates among afebrile patients who are anticipated to have neutropenia of long duration. Commonly used antibiotic regimens for the treatment of febrile patients in whom prolonged neutropenia (>7 days) is anticipated include (1) ceftazidime or cefepime, (2) piperacillin/tazobactam, or (3) imipenem/cilastatin or meropenem. All three regimens have shown equal efficacy in large trials. All three are active against

1	P. aeruginosa and a broad spectrum of aerobic gram-positive and gram-negative organisms. Imipenem/cilastatin has been associated with an elevated rate of C. difficile diarrhea, and many centers reserve carbapenem antibiotics for treatment of gram-negative bacteria that produce extended-spectrum β-lactamases; these limitations make carbapenems less attractive as an initial regimen. Despite the frequent involvement of coagulase-negative staphylococci, the initial use of vancomycin or its automatic addition to the initial regimen has not resulted in improved outcomes, and the antibiotic does exert toxic effects. For these reasons, only judicious use of vancomycin is recommended—for example, when there is good reason to suspect the involvement of coagulase-negative staphylococci (e.g., the appearance of erythema at the exit site of a catheter or a positive culture for methicillin-resistant S. aureus or coagulase-negative staphylococci). Because the sensitivities of bacteria vary from

1	appearance of erythema at the exit site of a catheter or a positive culture for methicillin-resistant S. aureus or coagulase-negative staphylococci). Because the sensitivities of bacteria vary from hospital to hospital, clinicians are advised to check their local sensitivities and to be aware that resistance patterns can change quickly, necessitating a change in approach to patients with fever and neutropenia. Similarly, infection control services should monitor for basic antibiotic resistance and for fungal infections. The appearance of a large number of Aspergillus infections, in particular, suggests the possibility of an environmental source that requires further investigation and remediation.

1	The initial antibacterial regimen should be refined on the basis of culture results (Fig. 104-2). Blood cultures are the most relevant basis for selection of therapy; surface cultures of skin and mucous membranes may be misleading. In the case of gram-positive bacteremia or another gram-positive infection, it is important that the antibiotic be optimal for the organism isolated. Once treatment with broad-spectrum antibiotics has begun, it is not desirable to discontinue all antibiotics because of the risk of failing to treat a potentially fatal bacterial infection; the addition of more and more antibacterial agents to the regimen is not appropriate unless there 491 is a clinical or microbiologic reason to do so. Planned progressive therapy (the serial, empirical addition of one drug after another without culture data) is not efficacious in most settings and may have unfortunate consequences. Simply adding another antibiotic for fear that a gram-negative infection is present is a

1	after another without culture data) is not efficacious in most settings and may have unfortunate consequences. Simply adding another antibiotic for fear that a gram-negative infection is present is a dubious practice. The synergy exhibited by β-lactams and aminoglycosides against certain gram-negative organisms (especially P. aeruginosa) provides the rationale for using two antibiotics in this setting, but recent analyses suggest that efficacy is not enhanced by the addition of aminoglycosides, while toxicity may be increased. Mere “double coverage,” with the addition of a quinolone or another antibiotic that is not likely to exhibit synergy, has not been shown to be of benefit and may cause additional toxicities and side effects. Cephalosporins can cause bone marrow suppression, and vancomycin is associated with neutropenia in some healthy individuals. Furthermore, the addition of multiple cephalosporins may induce β-lactamase production by some organisms; cephalosporins and double

1	is associated with neutropenia in some healthy individuals. Furthermore, the addition of multiple cephalosporins may induce β-lactamase production by some organisms; cephalosporins and double β-lactam combinations should probably be avoided altogether in Enterobacter infections.

1	Fungal infections in cancer patients are most often associated with neutropenia. Neutropenic patients are predisposed to the development of invasive fungal infections, most commonly those due to Candida and Aspergillus species and occasionally those caused by Mucor, Rhizopus, Fusarium, Trichosporon, Bipolaris, and others. Cryptococcal infection, which is common among patients taking immunosuppressive agents, is uncommon among neutropenic patients receiving chemotherapy for AML. Invasive candidal disease is usually caused by C. albicans or C. tropicalis but can be caused by C. krusei, C. parapsilosis, and C. glabrata.

1	For decades, it has been common clinical practice to add amphotericin B to antibacterial regimens if a neutropenic patient remains febrile despite 4–7 days of treatment with antibacterial agents. The rationale for this empirical addition is that it is difficult to culture fungi before they cause disseminated disease and that mortality rates from disseminated fungal infections in granulocytopenic patients are high. Before the introduction of newer azoles into clinical practice, amphotericin B was the mainstay of antifungal therapy. The insolubility of amphotericin B has resulted in the marketing of several lipid formulations that are less toxic than the amphotericin B deoxycholate complex. Echinocandins (e.g., caspofungin) are useful in the treatment of infections caused by azole-resistant Candida strains as well as in therapy for aspergillosis and have been shown to be equivalent to liposomal amphotericin B for the empirical treatment of patients with prolonged fever and neutropenia.

1	Candida strains as well as in therapy for aspergillosis and have been shown to be equivalent to liposomal amphotericin B for the empirical treatment of patients with prolonged fever and neutropenia. Newer azoles have also been demonstrated to be effective in this setting. Although fluconazole is efficacious in the treatment of infections due to many Candida species, its use against serious fungal infections in immunocompromised patients is limited by its narrow spectrum: it has no activity against Aspergillus or against several non-albicans Candida species. The broad-spectrum azoles (e.g., voriconazole and posaconazole) provide another option for the treatment of Aspergillus infections (Chap. 241), including CNS infection. Clinicians should be aware that the spectrum of each azole is somewhat different and that no drug can be assumed to be efficacious against all fungi. Aspergillus terreus is resistant to amphotericin B. Although voriconazole is active against Pseudallescheria boydii,

1	different and that no drug can be assumed to be efficacious against all fungi. Aspergillus terreus is resistant to amphotericin B. Although voriconazole is active against Pseudallescheria boydii, amphotericin B is not; however, voriconazole has no activity against Mucor. Posaconazole, which is administered orally, is useful as a prophylactic agent in patients with prolonged neutropenia. Studies in progress are assessing the use of these agents in combinations. For a full discussion of antifungal therapy, see Chap. 235.

1	The availability of a variety of agents active against herpes-group viruses, including some new agents with a broader spectrum of activity, has heightened focus on the treatment of viral infections,

1	Infections in Patients with Cancer 492 which pose a major problem in cancer patients. Viral diseases caused by the herpes group are prominent. Serious (and sometimes fatal) infections due to HSV and VZV are well documented in patients receiving chemotherapy. CMV may also cause serious disease, but fatalities from CMV infection are more common in HSCT recipients. The roles of human herpesvirus (HHV)-6, HHV-7, and HHV-8 (Kaposi’s sarcoma–associated herpesvirus) in cancer patients are still being defined (Chap. 219). EBV lymphoproliferative disease (LPD) can occur in patients receiving chemotherapy but is much more common among transplant recipients (Chap. 169). While clinical experience is most extensive with acyclovir, which can be used therapeutically or prophylactically, a number of derivative drugs offer advantages over this agent (Chap. 215e). In addition to the herpes group, several respiratory viruses (especially RSV) may cause serious disease in cancer patients. Although

1	of derivative drugs offer advantages over this agent (Chap. 215e). In addition to the herpes group, several respiratory viruses (especially RSV) may cause serious disease in cancer patients. Although influenza vaccination is recommended (see below), it may be ineffective in this patient population. The availability of antiviral drugs with activity against influenza viruses gives the clinician additional options for the prophylaxis and treatment of these patients (Chaps. 215e and 224).

1	Another way to address the problems posed by the febrile neutropenic patient is to replenish the neutrophil population. Although granulocyte transfusions may be effective in the treatment of refractory gram-negative bacteremia, they do not have a documented role in prophylaxis. Because of the expense, the risk of leukoagglutinin reactions (which has probably been decreased by improved cell-separation procedures), and the risk of transmission of CMV from unscreened donors (which has been reduced by the use of filters), granulocyte transfusion is reserved for patients whose condition is unresponsive to antibiotics. This modality is efficacious for documented gram-negative bacteremia refractory to antibiotics, particularly in situations where granulocyte numbers will be depressed for only a short period. The demonstrated usefulness of granulocyte colony-stimulating factor in mobilizing neutrophils and advances in preservation techniques may make this option more useful than in the past.

1	A variety of cytokines, including granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, enhance granulocyte recovery after chemotherapy and consequently shorten the period of maximal vulnerability to fatal infections. The role of these cytokines in routine practice is still a matter of some debate. Most authorities recommend their use only when neutropenia is both severe and prolonged. The cytokines themselves may have adverse effects, including fever, hypoxemia, and pleural effusions or serositis in other areas (Chap. 372e).

1	Once neutropenia has resolved, the risk of infection decreases dramatically. However, depending on what drugs they receive, patients who continue on chemotherapeutic protocols remain at high risk for certain diseases. Any patient receiving more than a maintenance dose of glucocorticoids (e.g., in many treatment regimens for diffuse lymphoma) should also receive prophylactic TMPSMX because of the risk of Pneumocystis infection; those with ALL should receive such prophylaxis for the duration of chemotherapy.

1	Outbreaks of fatal Aspergillus infection have been associated with construction projects and materials in several hospitals. The association between spore counts and risk of infection suggests the need for a high-efficiency air-handling system in hospitals that care for large numbers of neutropenic patients. The use of laminar-flow rooms and prophylactic antibiotics has decreased the number of infectious episodes in severely neutropenic patients. However, because of the expense of such a program and the failure to show that it dramatically affects mortality rates, most centers do not routinely use laminar flow to care for neutropenic patients. Some centers use “reverse isolation,” in which health care providers and visitors to a patient who is neutropenic wear gowns and gloves. Since most of the infections these patients develop are due to organisms that colonize the patients’ own skin and bowel, the validity of such schemes is dubious, and limited clinical data do not support their

1	of the infections these patients develop are due to organisms that colonize the patients’ own skin and bowel, the validity of such schemes is dubious, and limited clinical data do not support their use. Hand washing by all staff caring for neutropenic patients should be required to prevent the spread of resistant organisms.

1	The presence of large numbers of bacteria (particularly P. aeruginosa) in certain foods, especially fresh vegetables, has led some authorities to recommend a special “low-bacteria” diet. A diet consisting of cooked and canned food is satisfactory to most neutropenic patients and does not involve elaborate disinfection or sterilization protocols. However, there are no studies to support even this type of dietary restriction. Counseling of patients to avoid leftovers, deli foods, undercooked meat, and unpasteurized dairy products is recommended. Although few studies address this issue, patients with cancer are predisposed to infections resulting from anatomic compromise (e.g., lymphedema resulting from node dissections after radical mastectomy). Surgeons who specialize in cancer surgery can provide specific guidelines for the care of such patients, and patients benefit from common-sense advice about how to prevent infections in vulnerable areas.

1	Many patients with multiple myeloma or CLL have immunoglobulin deficiencies as a result of their disease, and all allogeneic bone marrow transplant recipients are hypogammaglobulinemic for a period after transplantation. However, current recommendations reserve intravenous immunoglobulin replacement therapy for those patients with severe (<400 mg of total IgG/dL), prolonged hypogammaglobulinemia and a history of repeated infections. Antibiotic prophylaxis has been shown to be cheaper and is efficacious in preventing infections in most CLL patients with hypogammaglobulinemia. Routine use of immunoglobulin replacement is not recommended. The use of condoms is recommended for severely immunocompromised patients. Any sexual practice that results in oral exposure to feces is not recommended. Neutropenic patients should be advised to avoid any practice that results in trauma, as even microscopic cuts may result in bacterial invasion and fatal sepsis.

1	Several studies indicate that the use of oral fluoroquinolones prevents infection and decreases mortality rates among severely neutropenic patients. Prophylaxis for Pneumocystis is mandatory for patients with ALL and for all cancer patients receiving glucocorticoid-containing chemotherapy regimens.

1	In general, patients undergoing chemotherapy respond less well to vaccines than do normal hosts. Their greater need for vaccines thus leads to a dilemma in their management. Purified proteins and inactivated vaccines are almost never contraindicated and should be given to patients even during chemotherapy. For example, all adults should receive diphtheria–tetanus toxoid boosters at the indicated times as well as seasonal influenza vaccine. However, if possible, vaccination should not be undertaken concurrent with cytotoxic chemotherapy. If patients are expected to be receiving chemotherapy for several months and vaccination is indicated (e.g., influenza vaccination in the fall), the vaccine should be given midcycle—as far apart in time as possible from the antimetabolic agents that will prevent an immune response. The meningococcal and pneumococcal polysaccharide vaccines should be given to patients before splenectomy, if possible. The H. influenzae type b conjugate vaccine should be

1	an immune response. The meningococcal and pneumococcal polysaccharide vaccines should be given to patients before splenectomy, if possible. The H. influenzae type b conjugate vaccine should be administered to all splenectomized patients.

1	In general, live virus (or live bacterial) vaccines should not be given to patients during intensive chemotherapy because of the risk of disseminated infection. Recommendations on vaccination are summarized in Table 104-2 (see www.cdc.gov/vaccine for updated recommendations). Cancer of the skin Walter J. Urba, Brendan D. Curti MELANOMA Pigmented lesions are among the most common findings on skin examination. The challenge is to distinguish cutaneous melanomas, which account for the overwhelming majority of deaths resulting from 105 skin cancer, from the remainder, which are usually benign. Cutaneous melanoma can occur in adults of all ages, even young individuals, and people of all colors; its location on the skin and its distinct clinical features make it detectable at a time when complete surgical excision is possible. Examples of malignant and benign pigmented lesions are shown in Fig. 105-1.

1	Melanoma is an aggressive malignancy of melanocytes, pigment-producing cells that originate from the neural crest and migrate to FIGURE 105-1 Atypical and malignant pigmented lesions. The most common melanoma is superficial spreading melanoma (not pictured). A. Acral lentiginous melanoma is the most common melanoma in blacks, Asians, and Hispanics and occurs as an enlarging hyperpigmented macule or plaque on the palms and soles. Lateral pigment diffusion is present. B. Nodular melanoma most commonly manifests as a rapidly growing, often ulcerated or crusted black nodule. C. Lentigo maligna melanoma occurs on sun-exposed skin as a large, hyperpigmented macule or plaque with irregular borders and variable pigmentation. D. Dysplastic nevi are irregularly pigmented and shaped nevomelanocytic lesions that may be associated with familial melanoma.

1	the skin, meninges, mucous membranes, upper esophagus, and eyes. 493 Melanocytes in each of these locations have the potential for malignant transformation. Cutaneous melanoma is predominantly a malignancy of white-skinned people (98% of cases), and the incidence correlates with latitude of residence, providing strong evidence for the role of sun exposure. Men are affected slightly more than women (1.3:1), and the median age at diagnosis is the late fifties. Dark-skinned populations (such as those of India and Puerto Rico), blacks, and East Asians also develop melanoma, albeit at rates 10–20 times lower than those in whites. Cutaneous melanomas in these populations are diagnosed more often at a higher stage, and patients tend to have worse outcomes. Furthermore, in nonwhite populations, there is a much higher frequency of acral (subungual, plantar, palmar) and mucosal melanomas. In 2014, more than 76,000 individuals in the United States were expected to develop melanoma, and

1	there is a much higher frequency of acral (subungual, plantar, palmar) and mucosal melanomas. In 2014, more than 76,000 individuals in the United States were expected to develop melanoma, and approximately 9700 were expected to die. There will be nearly 50,000 annual deaths worldwide as a result of melanoma. Data from the Connecticut Tumor Registry support an unremitting increase in the incidence and mortality of melanoma. In the past 60 years, there have been 17-fold and 9-fold increases in incidence for men and women, respectively. In the same six decades, there has been a tripling of mortality rates for men and doubling for women. Mortality rates begin to rise at age 55, with the greatest increase in men age >65 years. Of particular concern is the increase in rates among women <40 years of age. Much of this increase is believed to be associated with a greater emphasis on tanned skin as a marker of beauty, the increased availability and use of indoor tanning beds, and exposure to

1	of age. Much of this increase is believed to be associated with a greater emphasis on tanned skin as a marker of beauty, the increased availability and use of indoor tanning beds, and exposure to intense ultraviolet (UV) light in childhood. These statistics highlight the need to promote prevention and early detection.

1	RISK FACTORS Presence of Nevi The risk of developing melanoma is related to genetic, environmental, and host factors (Table 105-1). The strongest risk factors for melanoma are the presence of multiple benign or atypical nevi and a family or personal history of melanoma. The presence of melanocytic nevi, common or dysplastic, is a marker for increased risk of melanoma. Nevi have been referred to as precursor lesions because they can transform into melanomas; however, the actual risk for any specific nevus is exceedingly low. About one-quarter of melanomas are histologically associated with nevi, but the majority arise de novo. The number of clinically atypical moles may vary from one to several hundred, and they usually differ from one another in appearance. The borders are often hazy and indistinct, and the pigment pattern is more highly varied than that in benign acquired nevi. Individuals with clinically atypical moles and a strong family history of melanoma have been reported to

1	indistinct, and the pigment pattern is more highly varied than that in benign acquired nevi. Individuals with clinically atypical moles and a strong family history of melanoma have been reported to have a >50% lifetime risk for developing melanoma and warrant close follow-up with a dermatologist. Of the 90% of patients whose disease is sporadic (i.e., who lack a family history of melanoma), ∼40% have clinically atypical moles, compared with an estimated 5–10% of the population at large.

1	Congenital melanocytic nevi, which are classified as small (≤1.5 cm), medium (1.5–20 cm), and giant (>20 cm), can be precursors for melanoma. The risk is highest for the giant melanocytic nevus, also called the bathing trunk nevus, a rare malformation that affects 1 in 30,000–100,000 individuals. Since the lifetime risk of melanoma development is estimated to be as high as 6%, prophylactic excision early in life is prudent. This usually requires staged removal with coverage faCtors assoCiateD With inCreaseD risK of meLanoma CDKN2A, CDK4, MITF mutations

1	CDKN2A, CDK4, MITF mutations Cancer of the Skin 494 by split-thickness skin grafts. Surgery cannot remove all at-risk nevus cells, as some may penetrate into the muscles or central nervous system (CNS) below the nevus. Smallto medium-size congenital melanocytic nevi affect approximately 1% of persons; the risk of melanoma developing in these lesions is not known but appears to be relatively low. The management of smallto medium-size congenital melanocytic nevi remains controversial.

1	Personal and Family History Once diagnosed, patients with melanoma require a lifetime of surveillance because their risk of developing another melanoma is 10 times that of the general population. First-degree relatives have a higher risk of developing melanoma than do individuals without a family history, but only 5–10% of all melanomas are truly familial. In familial melanoma, patients tend to be younger at first diagnosis, lesions are thinner, survival is improved, and multiple primary melanomas are common.

1	Genetic Susceptibility Approximately 20–40% of cases of hereditary melanoma (0.2–2% of all melanomas) are due to germline mutations in the cell cycle regulatory gene cyclindependent kinase inhibitor 2A (CDKN2A). In fact, 70% of all cutaneous melanomas have mutations or deletions affecting the CDKN2A locus on chromosome 9p21. This locus encodes two distinct tumor-suppressor proteins from alternate reading frames: p16 and ARF (p14ARF). The p16 protein inhibits CDK4/6-mediated phosphorylation and inactivation of the retinoblastoma (RB) protein, whereas ARF inhibits MDM2 ubiquitin-mediated degradation of p53. The end result of the loss of CDKN2A is inactivation of two critical tumor-suppressor pathways, RB and p53, which control entry of cells into the cell cycle. Several studies have shown an increased risk of pancreatic cancer among melanoma-prone families with CDKN2A mutations. A second high-risk locus for melanoma susceptibility, CDK4, is located on chromosome 12q13 and encodes the

1	an increased risk of pancreatic cancer among melanoma-prone families with CDKN2A mutations. A second high-risk locus for melanoma susceptibility, CDK4, is located on chromosome 12q13 and encodes the kinase inhibited by p16. CDK4 mutations, which also inactivate the RB pathway, are much rarer than CDKN2A mutations. Germline mutations in the melanoma lineage-specific oncogene microphthalmia-associated transcription factor (MITF) predispose to both familial and sporadic melanomas.

1	The melanocortin-1 receptor (MC1R) gene is a moderate-risk inherited melanoma susceptibility factor. Solar radiation stimulates the production of melanocortin (α-melanocyte-stimulating hormone [α-MSH]), the ligand for MC1R, which is a G-protein-coupled receptor that signals via cyclic AMP and regulates the amount and type of pigment produced. MC1R is highly polymorphic, and among its 80 variants are those that result in partial loss of signaling and lead to the production of red/yellow pheomelanins, which are not sun-protective and produce red hair, rather than brown/black eumelanins that are photoprotective. This red hair color (RHC) phenotype is associated with fair skin, red hair, freckles, increased sun sensitivity, and increased risk of melanoma. In addition to its weak UV shielding capacity relative to eumelanin, increased pheomelanin production in patients with inactivating polymorphisms of MC1R also provides a UV-independent carcinogenic contribution to melanomagenesis via

1	capacity relative to eumelanin, increased pheomelanin production in patients with inactivating polymorphisms of MC1R also provides a UV-independent carcinogenic contribution to melanomagenesis via oxidative damage.

1	A number of other more common, low-penetrance polymorphisms that have small effects on melanoma susceptibility include other genes related to pigmentation, nevus count, immune responses, DNA repair, metabolism, and the vitamin D receptor.

1	Primary prevention of melanoma and nonmelanoma skin cancer (NMSC) is based on protection from the sun. Public health initiatives, such as the SunSmart program that started in Australia and now is operative in Europe and the United States, have demonstrated that behavioral change can decrease the incidence of NMSC and melanoma. Preventive measures should start early in life because damage from UV light begins early despite the fact that cancers develop years later. Biological factors are increasingly being understood, such as tanning addiction, which is postulated to involve stimulation of reward centers in the brain involving dopamine pathways, and cutaneous secretion of β-endorphins after UV exposure, and may represent another area for preventive intervention. Regular use of broad-spectrum sunscreens that block UVA and UVB with a sun protection factor (SPF) of at least 30 and protective clothing should be encouraged. Avoidance of tanning beds and midday (10:00 a.m. to 2:00 p.m.) sun

1	sunscreens that block UVA and UVB with a sun protection factor (SPF) of at least 30 and protective clothing should be encouraged. Avoidance of tanning beds and midday (10:00 a.m. to 2:00 p.m.) sun exposure is recommended.

1	Secondary prevention comprises education, screening, and early detection. Patients should be educated in the clinical features of melanoma (ABCDEs; see following “Diagnosis” section) and advised to report any growth or other change in a pigmented lesion. Brochures are available from the American Cancer Society, the American Academy of Dermatology, the National Cancer Institute, and the Skin Cancer Foundation. Self-examination at 6to 8-week intervals may enhance the likelihood of detecting change. Although the U.S. Preventive Services Task Force states that evidence is insufficient to recommend for or against skin cancer screening, a full-body skin exam seems to be a simple, practical way to approach reducing the mortality rate for skin cancer. Depending on the presence or absence of risk factors, strategies for early detection can be individualized. This is particularly true for patients with clinically atypical moles (dysplastic nevi) and those with a personal history of melanoma.

1	factors, strategies for early detection can be individualized. This is particularly true for patients with clinically atypical moles (dysplastic nevi) and those with a personal history of melanoma. For these individuals, surveillance should be performed by the dermatologist and include total-body photography and dermoscopy where appropriate. Individuals with three or more primary melanomas and families with at least one invasive melanoma and two or more cases of melanoma and/or pancreatic cancer among firstor second-degree relatives on the same side of the family may benefit from genetic testing. Precancerous and in situ lesions should be treated early. Early detection of small tumors allows the use of simpler treatment modalities with higher cure rates and lower morbidity.

1	The main goal is to identify a melanoma before tumor invasion and life-threatening metastases have occurred. Early detection may be facilitated by applying the ABCDEs: asymmetry (benign lesions are usually symmetric); border irregularity (most nevi have clear-cut borders); color variegation (benign lesions usually have uniform light or dark pigment); diameter >6 mm (the size of a pencil eraser); and evolving (any change in size, shape, color, or elevation or new symptoms such as bleeding, itching, and crusting). Benign nevi usually appear on sun-exposed skin above the waist, rarely involving the scalp, breasts, or buttocks; atypical moles usually appear on sun-exposed skin, most often on the back, but can involve the scalp, breasts, or buttocks. Benign nevi are present in 85% of adults, with 10–40 moles scattered over the body; atypical nevi can be present in the hundreds.

1	The entire skin surface, including the scalp and mucous membranes, as well as the nails should be examined in each patient. Bright room illumination is important, and a hand lens is helpful for evaluating variation in pigment pattern. Any suspicious lesions should be biopsied, evaluated by a specialist, or recorded by chart and/or photography for follow-up. A focused method for examining individual lesions, dermoscopy, employs low-level magnification of the epidermis and may allow a more precise visualization of patterns of pigmentation than is possible with the naked eye. Complete physical examination with attention to the regional lymph nodes is part of the initial evaluation in a patient with suspected melanoma. The patient should be advised to have other family members screened if either melanoma or clinically atypical moles (dysplastic nevi) are present. Patients who fit into high-risk groups should be instructed to perform monthly self-examinations.

1	Biopsy Any pigmented cutaneous lesion that has changed in size or shape or has other features suggestive of malignant melanoma is a candidate for biopsy. An excisional biopsy with 1to 3-mm margins is suggested. This facilitates pathologic assessment of the lesion, permits accurate measurement of thickness if the lesion is melanoma, and constitutes definitive treatment if the lesion is benign. For lesions that are large or on anatomic sites where excisional biopsy may not be feasible (such as the face, hands, and feet), an incisional biopsy through the most nodular or darkest area of the lesion is acceptable; this should include the vertical growth phase of the primary tumor, if present. Incisional biopsy does not appear to facilitate the spread of melanoma. For suspicious lesions, every attempt should be made to preserve the ability to assess the deep and peripheral margins and to perform immunohistochemistry. Shave biopsies are an acceptable alternative, particularly if the suspicion

1	should be made to preserve the ability to assess the deep and peripheral margins and to perform immunohistochemistry. Shave biopsies are an acceptable alternative, particularly if the suspicion of malignancy is low, but they should be deep and include underlying fat; cauterization should be avoided. The biopsy should be read by a pathologist experienced in pigmented lesions, and the report should include Breslow thickness, mitoses per square millimeter for lesions ≤1 mm, presence or absence of ulceration, and peripheral and deep margin status. Breslow thickness is the greatest thickness of a primary cutaneous melanoma measured on the slide from the top of the epidermal granular layer, or from the ulcer base, to the bottom of the tumor. To distinguish melanomas from benign nevi in cases with challenging histology, fluorescence in situ hybridization (FISH) with multiple probes and comparative genome hybridization (CGH) can be helpful.

1	Four major types of cutaneous melanoma have been recognized (Table 105-2). In three of these types—superficial spreading melanoma, lentigo maligna melanoma, and acral lentiginous melanoma— the lesion has a period of superficial (so-called radial) growth during which it increases in size but does not penetrate deeply. It is during this period that the melanoma is most capable of being cured by surgical excision. The fourth type—nodular melanoma —does not have a recognizable radial growth phase and usually presents as a deeply invasive lesion that is capable of early metastasis. When tumors begin to penetrate deeply into the skin, they are in the so-called vertical growth phase. Melanomas with a radial growth phase are characterized by irregular and sometimes notched borders, variation in pigment pattern, and variation in color. An increase in size or change in color is noted by the patient in 70% of early lesions. Bleeding, ulceration, and pain are late signs and are of little help in

1	pattern, and variation in color. An increase in size or change in color is noted by the patient in 70% of early lesions. Bleeding, ulceration, and pain are late signs and are of little help in early recognition. Superficial spreading melanoma is the most common variant observed in the white population. The back is the most common site for melanoma in men. In women, the back and the lower leg (from knee to ankle) are common sites. Nodular melanomas are dark brown-black to blue-black nodules. Lentigo maligna melanoma usually is confined to chronically sun-damaged sites in older individuals. Acral lentiginous melanoma occurs on the palms, soles, nail beds, and mucous membranes. Although this type occurs in whites, it occurs most frequently (along with nodular melanoma) in blacks and East Asians. A fifth type of melanoma, desmoplastic melanoma, is associated with a fibrotic response, neural invasion, and a greater tendency for local recurrence. Occasionally, melanomas appear clinically to

1	A fifth type of melanoma, desmoplastic melanoma, is associated with a fibrotic response, neural invasion, and a greater tendency for local recurrence. Occasionally, melanomas appear clinically to be amelanotic, in which case the diagnosis is established microscopically after biopsy of a new or a changing skin nodule. Melanomas can also arise in the mucosa of the head and neck (nasal cavity, paranasal sinuses and oral cavity), the gastrointestinal tract, the CNS, the female genital tract (vulva, vagina), and the uveal tract of the eye.

1	Although cutaneous melanoma subtypes are clinically and histo-495 pathologically distinct, this classification does not have independent prognostic value. Histologic subtype is not part of American Joint Committee on Cancer (AJCC) staging, although the College of American Pathologists (CAP) recommends inclusion in the pathology report. Newer classifications will increasingly emphasize molecular features of each melanoma (see below). The molecular analysis of individual melanomas will provide a basis for distinguishing benign nevi from melanomas, and determination of the mutational status of the tumor will help elucidate the molecular mechanisms of tumorigenesis and be used to identify targets that will guide therapy.

1	Considerable evidence from epidemiologic and molecular studies suggests that cutaneous melanomas arise via multiple causal pathways. There are both environmental and genetic components. UV solar radiation causes genetic changes in the skin, impairs cutaneous immune function, increases the production of growth factors, and induces the formation of DNA-damaging reactive oxygen species that affect keratinocytes and melanocytes. A comprehensive catalog of somatic mutations from a human melanoma revealed more than 33,000 base mutations with damage to almost 300 protein-coding segments compared with normal cells from the same patient. The dominant mutational signature reflected DNA damage due to UV light exposure. The melanoma also contained previously described driver mutations (i.e., mutations that confer selective clonal growth advantage and are implicated in oncogenesis). These driver mutations affect pathways that promote cell proliferation and inhibit normal pathways of apoptosis in

1	that confer selective clonal growth advantage and are implicated in oncogenesis). These driver mutations affect pathways that promote cell proliferation and inhibit normal pathways of apoptosis in response to DNA repair (see below). The altered melanocytes accumulate DNA damage, and selection occurs for all the attributes that constitute the malignant phenotype: invasion, metastasis, and angiogenesis.

1	An understanding of the molecular changes that occur during the transformation of normal melanocytes into malignant melanoma would not only help classify patients but also would contribute to the understanding of etiology and aid the development of new therapeutic options. A genome-wide assessment of melanomas classified into four groups based on their location and degree of exposure to the sun has confirmed that there are distinct genetic pathways in the development of melanoma. The four groups were cutaneous melanomas on skin without chronic sun-induced damage, cutaneous melanomas with chronic sun-induced damage, mucosal melanomas, and acral melanomas. Distinct patterns of DNA alterations were noted that varied with the site of origin and were independent of the histologic subtype of the tumor. Thus, although the genetic changes are diverse, the overall pattern of mutation, amplification, and loss of cancer genes indicates they have convergent effects on key biochemical pathways

1	the tumor. Thus, although the genetic changes are diverse, the overall pattern of mutation, amplification, and loss of cancer genes indicates they have convergent effects on key biochemical pathways involved in proliferation, senescence, and apoptosis. The p16 mutation that affects cell cycle arrest and the ARF mutation that results in defective apoptotic responses to genotoxic damage were described earlier. The proliferative pathways affected were the mitogen-activated protein (MAP) kinase and phosphatidylinositol 3’ kinase/AKT pathways (Fig. 105-2).

1	Cancer of the Skin Average Age at Duration of Known Type Site Diagnosis, Years Existence, Years Color aDuring much of this time, the precursor stage, lentigo maligna, is confined to the epidermis. Source: Adapted from AJ Sober, in NA Soter, HP Baden (eds): Pathophysiology of Dermatologic Diseases. New York, McGraw-Hill, 1984. in a subset of tumors FIGURE 105-2 Major pathways involved in melanoma. The MAP kinase and PI3K/AKT pathways, which promote proliferation and inhibit apoptosis, respectively, are subject to mutations in melanoma. ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase kinase; NF-1; neurofibromatosis type 1 gene; PTEN, phosphatase and tensin homolog.

1	RAS and BRAF, members of the MAP kinase pathway, which classically mediates the transcription of genes involved in cell proliferation and survival, undergo somatic mutation in melanoma and thereby generate potential therapeutic targets. N-RAS is mutated in approximately 20% of melanomas, and somatic activating BRAF mutations are found in most benign nevi and 40–60% of melanomas. Neither mutation by itself appears to be sufficient to cause melanoma; thus, they often are accompanied by other mutations. The BRAF mutation is most commonly a point mutation (T→A nucleotide change) that results in a valine-to-glutamate amino acid substitution (V600E). V600E BRAF mutations do not have the standard UV signature mutation (pyrimidine dimer); they are more common in younger patients and are present in most melanomas that arise on sites with intermittent sun exposure and are less common in melanomas from chronically sun-damaged skin.

1	Melanomas also harbor mutations in AKT (primarily in AKT3) and PTEN (phosphatase and tensin homolog). AKT can be amplified, and PTEN may be deleted or undergo epigenetic silencing that leads to constitutive activation of the PI3K/AKT pathway and enhanced cell survival by antagonizing the intrinsic pathway of apoptosis. Loss of PTEN, which dysregulates AKT activity, and mutation of AKT3 both prolong cell survival through inactivation of BAD, Bc12-antagonist of cell death, and activation of the forkhead transcription factor FOXO1, which leads to synthesis of prosurvival genes. A loss-of-function mutation in NF1, which can affect both MAP kinase and PI3K/AKT pathways, has been described in 10–15% of melanomas. In melanoma, these two signaling pathways (MAP kinase and PI3K/AKT) enhance tumorigenesis, chemoresistance, migration, and cell cycle dysregulation. Targeted agents that inhibit these pathways have been developed, and some are available for clinical use (see below). Optimal

1	tumorigenesis, chemoresistance, migration, and cell cycle dysregulation. Targeted agents that inhibit these pathways have been developed, and some are available for clinical use (see below). Optimal treatment of patients with melanoma may require simultaneous inhibition of both MAPK and PI3K pathways as well as promotion of immune eradication of malignancy.

1	The prognostic factors of greatest importance to a newly diagnosed patient are included in the staging classification (Table 105-3). The best predictor of metastatic risk is the lesion’s Breslow thickness. The Clark level, which defines melanomas on the basis of the layer of skin to which a melanoma has invaded, does not add significant prognostic information and has minimal influence on treatment decisions. The anatomic site of the primary is also prognostic; favorable sites are the forearm and leg (excluding the feet), and unfavorable sites include the scalp, hands, feet, and mucous membranes. In general, women with stage I or II disease have better survival than men, perhaps in part because of earlier diagnosis; women frequently have melanomas on the lower leg, where self-recognition is more likely and the prognosis is better. The effect of age is not straightforward. Older individuals, especially men over 60, have worse prognoses, a finding that has been explained in part by a

1	is more likely and the prognosis is better. The effect of age is not straightforward. Older individuals, especially men over 60, have worse prognoses, a finding that has been explained in part by a tendency toward later diagnosis (and thus thicker tumors) and in part by a higher proportion of acral melanomas in men. However, there is a greater risk of lymph node metastasis in young patients. Other important adverse factors recognized via the staging classification include high mitotic rate, presence of ulceration, microsatellite lesions and/or in-transit metastases, evidence of nodal involvement, elevated serum lactate dehydrogenase (LDH), and presence and site of distant metastases.

1	Once the diagnosis of melanoma has been made, the tumor must be staged to determine the prognosis and treatment. Staging helps determine prognosis and aids in treatment selection. The current melanoma staging criteria and estimated 15-year survival by stage are depicted in Table 105-3. The clinical stage of the patient is determined after the pathologic evaluation of the melanoma skin lesion and clinical/radiologic assessment for metastatic disease. Pathologic staging also includes the microscopic evaluation of the regional lymph nodes obtained at sentinel lymph node biopsy or completion lymphadenectomy as indicated. All patients should have a complete history, with attention to symptoms that may represent metastatic disease such as malaise, weight loss, headaches, visual changes, and pain, and physical examination directed to the site of the primary melanoma, looking for persistent disease or for dermal or subcutaneous nodules that could represent satellite or in-transit metastases,

1	and physical examination directed to the site of the primary melanoma, looking for persistent disease or for dermal or subcutaneous nodules that could represent satellite or in-transit metastases, and to the regional draining lymph nodes, CNS, liver, and lungs. A complete blood count (CBC), complete metabolic panel, and LDH should be performed. Although these are low-yield tests for uncovering occult metastatic disease, a microcytic anemia would raise the possibility of bowel metastases, particularly in the small bowel, and an unexplained elevated LDH should prompt a more extensive evaluation, including computed tomography (CT) scan or possibly a positron emission tomography (PET) (or CT/ PET combined) scan. If signs or symptoms of metastatic disease are present, appropriate diagnostic imaging should be performed. At initial presentation, more than 80% of patients will have disease confined to the skin and a negative history and physical exam, in which case imaging is not indicated.

1	MANAGEMENT OF CLINICALLY LOCALIZED MELANOMA (STAGE I, II) For a newly diagnosed cutaneous melanoma, wide surgical excision of the lesion with a margin of normal skin is necessary to remove all malignant cells and minimize possible local recurrence. The following margins are recommended for a primary melanoma: in situ, 0.5–1.0 cm; invasive up to 1 mm thick, 1 cm; >1.01–2 mm, 1–2 cm; and >2 mm, 2 cm. For lesions on the face, hands, and feet, strict adherence to these margins must give way to individual considerations about the constraints of surgery and minimization of morbidity. In all instances, however, inclusion of subcutaneous fat in the surgical specimen facilitates adequate thickness measurement and assessment of surgical margins by the pathologist. Topical imiquimod also has been used, particularly for lentigo maligna, in cosmetically sensitive locations.

1	Sentinel lymph node biopsy (SLNB) is a valuable staging tool that has replaced elective regional nodal dissection for the evaluation of regional nodal status. SLNB provides prognostic information and Cancer of the Skin helps identify patients at high risk for relapse who may be candidates for adjuvant therapy. The initial (sentinel) draining node(s) from the primary site is (are) identified by injecting a blue dye and a radioisotope around the primary site. The sentinel node(s) then is (are) identified by inspection of the nodal basin for the blue-stained node and/or the node with high uptake of the radioisotope. The identified nodes are removed and subjected to careful histopathologic analysis with serial section using hematoxylin and eosin stains as well as immunohistochemical stains (e.g., S100, HMB45, and MelanA) to identify melanocytes.

1	Not every patient requires a SLNB. Patients whose melanomas are ≤0.75 mm thick have <5% risk of sentinel lymph node (SLN) disease and do not require a SLNB. Patients with tumors >1 mm thick generally undergo SLNB. For melanomas 0.76–1.0 mm thick, SLNB may be considered for lesions with high-risk features such as ulceration, high mitotic index, or lymphovascular invasion, but wide excision alone is the usual definitive therapy. Most other patients with clinically negative lymph nodes should undergo a SLNB. Patients whose SLNB is negative are spared a complete node dissection and its attendant morbidities, and can simply be followed or, based on the features of the primary melanoma, be considered for adjuvant therapy or a clinical trial. The current standard of care for all patients with a positive SLN is to perform a complete lymphadenectomy; however, ongoing clinical studies will determine whether patients with small-volume SLN metastases can be managed safely without additional

1	a positive SLN is to perform a complete lymphadenectomy; however, ongoing clinical studies will determine whether patients with small-volume SLN metastases can be managed safely without additional surgery. Patients with microscopically positive lymph nodes should be considered for adjuvant therapy with interferon or enrollment in a clinical trial.

1	Melanomas may recur at the edge of the scar or graft, as satellite metastases, which are separate from but within 2 cm of the scar; as in-transit metastases, which are recurrences >2 cm from the primary lesion but not beyond the regional nodal basin; or, most commonly, as metastasis to a draining lymph node basin. Each of these presentations is managed surgically, following which there 498 is the possibility of long-term disease-free survival. Isolated limb perfusion or infusion with melphalan and hyperthermia are options for patients with extensive cutaneous regional recurrences in an extremity. High complete response rates have been reported and significant palliation of symptoms can be achieved, but there is no change in overall survival. Patients rendered free of disease after surgery may be at high risk for a local or distant recurrence and should be considered for adjuvant therapy. Radiotherapy can reduce the risk of local recurrence after lymphadenectomy, but does not affect

1	may be at high risk for a local or distant recurrence and should be considered for adjuvant therapy. Radiotherapy can reduce the risk of local recurrence after lymphadenectomy, but does not affect overall survival. Patients with large nodes (>3–4 cm), four or more involved lymph nodes, or extranodal spread on microscopic examination should be considered for radiation. Systemic adjuvant therapy is indicated primarily for patients with stage III disease, but high-risk, node-negative patients (>4 mm thick or ulcerated lesions) and patients with completely resected stage IV disease also may benefit. Either interferon α2b (IFN-α2b), which is given at 20 million units/m2 IV 5 days a week for 4 weeks followed by 10 million units/m2 SC three times a week for 11 months (1 year total), or subcutaneous peginterferon α2b (6 μg/kg per week for 8 weeks followed by 3 μg/kg per week for a total of 5 years) is acceptable adjuvant therapy. Treatment is accompanied by significant toxicity, including a

1	peginterferon α2b (6 μg/kg per week for 8 weeks followed by 3 μg/kg per week for a total of 5 years) is acceptable adjuvant therapy. Treatment is accompanied by significant toxicity, including a flu-like illness, decline in performance status, and the development of depression. Side effects can be managed in most patients by appropriate treatment of symptoms, dose reduction, and treatment interruption. Sometimes IFN must be permanently discontinued before all of the planned doses are administered because of unacceptable toxicity. The high-dose regimen is significantly more toxic than peginterferon, but the latter requires 4 additional years of therapy. Adjuvant treatment with IFN improves disease-free survival, but its impact on overall survival remains controversial. Enrollment in a clinical trial is appropriate for these patients, many of whom will otherwise be observed without treatment either because they are poor candidates for IFN or because the patient (or their oncologist)

1	trial is appropriate for these patients, many of whom will otherwise be observed without treatment either because they are poor candidates for IFN or because the patient (or their oncologist) does not believe the beneficial effects of IFN outweigh the toxicity. The recently approved immunotherapy and targeted agents are being evaluated in the adjuvant setting.

1	At diagnosis, most patients with melanoma will have early-stage disease; however, some will present with metastases, and others will develop metastases after initial therapy. Patients with a history of melanoma who develop signs or symptoms suggesting recurrent disease should undergo restaging that includes physical examination, CBC, complete metabolic panel, LDH, and appropriate diagnostic imaging that may include a magnetic resonance image (MRI) of the brain and total-body PET/CT or CT scans of the chest, abdomen, and pelvis. Distant metastases (stage IV), which may involve any organ, commonly involve the skin and lymph nodes as well as viscera, bone, or the brain. Historically, metastatic melanoma was considered incurable; median survival ranges from 6 to 15 months, depending on the organs involved. The prognosis is better for patients with skin and subcutaneous metastases (M1a) than for lung (M1b) and worst for those with metastases to liver, bone, and brain (M1c). An elevated

1	organs involved. The prognosis is better for patients with skin and subcutaneous metastases (M1a) than for lung (M1b) and worst for those with metastases to liver, bone, and brain (M1c). An elevated serum LDH is a poor prognostic factor and places the patient in stage M1c regardless of the site of the metastases (Table 105-3). Although historical data suggest that the 15-year survival of patients with M1a, M1b, and M1c disease is less than 10%, there is optimism that newer therapies will increase the number of melanoma patients with long-term survival, especially patients with M1a and M1b disease.

1	The treatment for patients with stage IV melanoma has changed dramatically in the past 2 years. Two new classes of therapeutic agents for melanoma have been approved by the U.S. Food and Drug Administration (FDA). The immune T cell checkpoint inhibitor, ipilimumab, and three new oral agents that target the MAP kinase pathway: the BRAF inhibitors, vemurafenib and dabrafenib, and the Surgery: Metastasectomy for small number of lesions Immunotherapy: Anti-CTLA-4: ipilimumab Anti-PD-1: nivolumab, lambrolizumab Molecular targeted therapy: BRAF inhibitor: vemurafenib, dabrafenib MEK inhibitor: trametinib Chemotherapy: dacarbazine, temozolomide, paclitaxel, albumin-bound paclitaxel (Abraxane), carboplatin MEK inhibitor, trametinib, are now available, so patients with stage IV disease now have multiple therapeutic options (Table 105-4).

1	MEK inhibitor, trametinib, are now available, so patients with stage IV disease now have multiple therapeutic options (Table 105-4). Patients with oligometastatic disease should be referred to a surgical oncologist for consideration of metastasectomy, because they may experience long-term disease-free survival after surgery. Patients with solitary metastases are the best candidates, but surgery increasingly is being used even for patients with metastases at more than one site. Patients rendered free of disease can be considered for IFN therapy or a clinical trial because their risk of developing additional metastases is very high. Surgery can also be used as an adjunct to immunotherapy when only a few of many metastatic lesions prove resistant to systemic therapy.

1	The cytokine interleukin 2 (IL-2 or aldesleukin) has been approved to treat patients with melanoma since 1995. IL-2 is used to treat stage IV patients who have a good performance status and is administered at centers with experience managing IL-2-related toxicity. Patients require hospitalization in an intensive care unit– like setting to receive high-dose IL-2 600,000 or 720,000 IU every 8 h for up to 14 doses (one cycle). Patients continue treatment until they achieve maximal benefit, usually 4–6 cycles. Treatment is associated with long-term disease-free survival (probable cures) in 5% of treated patients. The mechanism by which IL-2 causes tumor regression has not been identified, but it is presumed that IL-2 induces melanoma-specific T cells that eliminate tumor cells by recognizing specific antigens. Rosenberg and his colleagues at the National Cancer Institute (NCI) have combined adoptive transfer of in vitro–expanded tumor-infiltrating lymphocytes with high-dose IL-2 in

1	specific antigens. Rosenberg and his colleagues at the National Cancer Institute (NCI) have combined adoptive transfer of in vitro–expanded tumor-infiltrating lymphocytes with high-dose IL-2 in patients who were preconditioned with nonmyeloablative chemotherapy (sometimes combined with total-body irradiation). Tumor regression was observed in more than 50% of patients with IL-2-refractory metastatic melanoma.

1	Immune checkpoint blockade with monoclonal antibodies to the inhibitory immune receptors CTLA-4 and PD-1 has shown promising clinical efficacy. An array of inhibitory receptors are upregulated during an immune response. An absolute requirement to ensure proper regulation of a normal immune response, the continued expression of inhibitory receptors during chronic infection (hepatitis, HIV) and in cancer patients denotes exhausted T cells with limited potential for proliferation, cytokine production, or cytotoxicity (Fig. 105-3). Checkpoint blockade with a monoclonal antibody results in improved T cell function with eradication of tumor cells in preclinical animal models. Ipilimumab, a fully human IgG antibody that binds CTLA-4 and blocks inhibitory signals, was the first treatment of any kind to improve survival in patients with metastatic melanoma. A full course of therapy is four IV outpatient infusions of ipilimumab 3 mg/kg every 3 weeks. Although response rates were low (∼10%) in

1	kind to improve survival in patients with metastatic melanoma. A full course of therapy is four IV outpatient infusions of ipilimumab 3 mg/kg every 3 weeks. Although response rates were low (∼10%) in randomized clinical trials, survival of both previously treated and untreated patients was improved, and ipilimumab was approved by the FDA in March 2011.

1	FIGURE 105-3 Inhibitory regulatory pathways that influence T cell function, memory, and lifespan after engagement of the T cell receptor by antigen presented by antigen-presenting cells in the context of MHC I/II. CTLA-4 and PD-1 are part of the CD28 family and have inhibitory effects that can be mitigated by antagonistic antibodies to the receptors or ligand, resulting in enhanced T cell function and antitumor effects. CTLA-4, cytotoxic T lymphocyte antigen-4; MHC, major histocompatibility complex; PD-1, programmed death-1; PD-L1, programmed death ligand-1; PD-L2, programmed death ligand-2; TCR, T cell receptor.

1	In addition to its antitumor effects, ipilimumab’s interference with normal regulatory mechanisms produced a novel spectrum of side effects that resembled autoimmunity. The most common immune-related adverse events were skin rash and diarrhea (sometimes severe, life-threatening colitis), but toxicity could involve most any organ (e.g., hypophysitis, hepatitis, nephritis, pneumonitis, myocarditis, neuritis). Vigilance and early treatment with steroids that do not appear to interfere with the antitumor effects are required to manage these patients safely. Widespread use of ipilimumab has not been completely embraced by the oncology community because of the low objective response rate, significant toxicity (including death), and high cost (drug cost alone for a course of therapy is approximately $120,000 in 2013). Despite these reservations, ipilimumab’s overall survival benefit (17% of patients alive at 7 years) indicates that treatment should be strongly considered for all eligible

1	$120,000 in 2013). Despite these reservations, ipilimumab’s overall survival benefit (17% of patients alive at 7 years) indicates that treatment should be strongly considered for all eligible patients.

1	Chronic T cell activation also leads to induction of PD-1 on the surface of T cells. Expression of one of its ligands, PD-L1, on tumor cells can protect them from immune destruction (Fig. 105-3). Early trials attempting to block the PD-1:PD-L1 axis by IV administration of anti-PD-1 or anti-PD-L1 have shown substantial clinical activity in patients with advanced melanoma (and lung cancer) with significantly less toxicity than ipilimumab. Anti-PD-1 therapy looks promising, but is not currently available except by participation in clinical trials. Intriguingly, preliminary results from a clinical trial indicate that blocking both inhibitory pathways with ipilimumab and anti-PD-1 leads to superior antitumor activity than treatment with either agent alone. The main benefit to patients from immune-based therapy (IL-2, ipilimumab, and anti-PD-1) is the durability of the responses achieved. Although the percentage of patients whose tumors regress following immunotherapy is lower than the

1	immune-based therapy (IL-2, ipilimumab, and anti-PD-1) is the durability of the responses achieved. Although the percentage of patients whose tumors regress following immunotherapy is lower than the response rate after targeted therapy (see below), the durability of immunotherapy-induced responses (>10 years in some cases) appears to be superior to responses after targeted therapy and suggests that many of these patients have been cured.

1	RAF and MEK inhibitors of the MAP kinase pathway are a new and exciting approach for patients whose melanomas harbor a BRAF mutation. The high frequency of oncogenic mutations in the RASRAF-MEK-ERK pathway, which delivers proliferation and survival signals from the cell surface to the cytoplasm and nucleus, has led to the development of inhibitors to BRAF and MEK. Two BRAF inhibitors, vemurafenib and dabrafenib, have been approved for the treatment of stage IV patients whose melanomas harbor a mutation at position 600 in the gene for BRAF. The oral BRAF inhibitors cause 499 tumor regression in approximately 50% of patients, and overall survival is improved compared to treatment with chemotherapy. Treatment is accompanied by manageable side effects that differ from those following immunotherapy or chemotherapy. A class-specific complication of BRAF inhibition is the development of numerous skin lesions, some of which are well-differentiated squamous cell skin cancers (seen in up to a

1	or chemotherapy. A class-specific complication of BRAF inhibition is the development of numerous skin lesions, some of which are well-differentiated squamous cell skin cancers (seen in up to a quarter of patients). Patients should be co-managed with a dermatologist as these skin cancers will need excision. Metastases have not been reported, and treatment can be continued safely following simple excision. Long-term results following treatment with BRAF inhibitors are not yet available, but the current concern is that over time the vast majority of patients will relapse and eventually die from drug-resistant disease. There are a number of mechanisms by which resistance develops, usually via maintenance of MAP kinase signaling; however, mutations in the BRAF gene that affect binding of the inhibitor are not among them. The MEK inhibitor trametinib has activity as a single agent, but appears to be less effective than either of the BRAF inhibitors. Combined therapy with the BRAF inhibitor

1	are not among them. The MEK inhibitor trametinib has activity as a single agent, but appears to be less effective than either of the BRAF inhibitors. Combined therapy with the BRAF inhibitor and MEK inhibitor showed improved progression-free survival compared to BRAF inhibitor therapy alone; and, interestingly, the neoplastic skin lesions that were so troubling with BRAF inhibition alone did not occur. Although the durability of responses following combined therapy remains to be determined, its use in metastatic melanoma is FDA approved. Activating mutations in the c-kit receptor tyrosine kinase are found in a minority of cutaneous melanomas with chronic sun damage, but more commonly in mucosal and acral lentiginous subtypes. Overall, the number of patients with c-kit mutations is exceedingly small, but when present, they are largely identical to mutations found in gastrointestinal stromal tumors (GISTs); melanomas with activating c-kit mutations can have clinically meaningful

1	exceedingly small, but when present, they are largely identical to mutations found in gastrointestinal stromal tumors (GISTs); melanomas with activating c-kit mutations can have clinically meaningful responses to imatinib.

1	No chemotherapy regimen has ever been shown to improve survival in metastatic melanoma, and the advances in immunotherapy and targeted therapy have relegated chemotherapy to the palliation of symptoms. Drugs with antitumor activity include dacarbazine (DTIC) or its orally administered analog temozolomide (TMZ), cisplatin and carboplatin, the taxanes (paclitaxel alone or albumin-bound and docetaxel), and carmustine (BCNU), which have reported response rates of 12–20%.

1	Upon diagnosis of stage IV disease, whether by biopsy or diagnostic imaging, a sample of the patient’s tumor needs to undergo molecular testing to determine whether a druggable mutation (e.g., BRAF) is present. Analysis of a metastatic lesion is preferred, but any biopsy will suffice because there is little discordance between primary and metastatic lesions. Treatment algorithms start with the tumor’s BRAF status. For BRAF “wild-type” tumors, immunotherapy is recommended. For patients whose tumors harbor a BRAF mutation, initial therapy with either a BRAF inhibitor or immunotherapy is acceptable. Molecular testing may also include N-RAS and c-kit in appropriate tumors.

1	The majority of patients still die from their melanoma, despite improvements in therapy. Therefore, enrollment in a clinical trial is always an important consideration, even for previously untreated patients. Most patients with stage IV disease will eventually progress despite advances in therapy, and many, because of disease burden, poor performance status, or concomitant illness, will be unsuitable for therapy. Therefore, a major focus of care should be the timely integration of palliative care and hospice. Skin examination and surveillance at least once a year are recommended for all patients with melanoma. The National Comprehensive Cancer Network (NCCN) guidelines for patients with stage IA–IIA

1	Cancer of the Skin 500 melanoma recommend a comprehensive history and physical examination every 6–12 months for 5 years, and then annually as clinically indicated. Particular attention should be paid to the draining lymph nodes in stage I–III patients as resection of lymph node recurrences may still be curative. A CBC, LDH, and chest x-ray are recommended at the physician’s discretion, but are ineffective tools for the detection of occult metastases. Routine imaging for metastatic disease is not recommended at this time. For patients with higher stage disease (IIB–IV), imaging (chest x-ray, CT, and/or PET/CT scans) every 4–12 months can be considered. Because no discernible survival benefit has been demonstrated for routine surveillance, it is reasonable to perform scans only if clinically indicated.

1	Nonmelanoma skin cancer (NMSC) is the most common cancer in the United States. Although tumor registries do not routinely gather data on the incidence of basal cell and squamous cell skin cancers, it is estimated that the annual incidence is 1.5–2 million cases in the United States. Basal cell carcinomas (BCCs) account for 70–80% of NMSCs. Squamous cell carcinomas (SCCs), which comprise ∼20% of NMSCs, are more significant because of their ability to metastasize and account for 2400 NMSC deaths annually. There has also been an increase in the incidence of nonepithelial skin cancer, especially Merkel cell carcinoma, with nearly 5000 new diagnoses and 3000 deaths annually.

1	The most significant cause of BCC and SCC is UV exposure, whether through direct exposure to sunlight or by artificial UV light sources (tanning beds). Both UVA and UVB can induce DNA damage through free radical formation (UVA) or induction of pyrimidine dimers (UVB). The sun emits energy across the UV spectrum, whereas tanning bed equipment typically emits 97% UVA and 3% UVB. DNA damage induced by UV irradiation can result in cell death or repair of damaged DNA by nucleotide excision repair (NER). Inherited disorders of NER, such as xeroderma pigmentosum, are associated with a greatly increased incidence of skin cancer and help to establish the link between UV-induced DNA damage, inadequate DNA repair, and skin cancer. The genes damaged most commonly by UV in BCC involve the Hedgehog pathway (Hh). In SCC, p53 and N-RAS are commonly affected. There is a dose-response relationship between tanning bed use and the incidence of skin cancer. As few as four tanning bed visits per year

1	pathway (Hh). In SCC, p53 and N-RAS are commonly affected. There is a dose-response relationship between tanning bed use and the incidence of skin cancer. As few as four tanning bed visits per year confers a 15% increase in BCC and an 11% increase in SCC and melanoma. Tanning bed use as a teenager or young adult confers greater risk than comparable exposure in older individuals. Other associations include blond or red hair, blue or green eyes, a tendency to sunburn easily, and an outdoor occupation. The incidence of NMSC increases with decreasing latitude. Most tumors develop on sun-exposed areas of the head and neck. The risk of lip or oral SCC is increased with cigarette smoking. Human papillomaviruses and UV radiation may act as cocarcinogens.

1	Solid organ transplant recipients on chronic immunosuppression have a 65-fold increase in SCC and a 10-fold increase in BCC. The frequency of skin cancer is proportional to the level and duration of immunosuppression and the extent of sun exposure before and after transplantation. SCCs in this population also demonstrate higher rates of local recurrence, metastasis, and mortality. There is increasing use of tumor necrosis factor (TNF) antagonists to treat inflammatory bowel disease and autoimmune disorders such as rheumatoid and psoriatic arthritis. TNF antagonists may also confer an increased risk of NMSC. BRAF-targeted therapy can induce SCCs including keratoacanthoma-type SCCs in keratinocytes, with preexisting H-RAS overexpression present in approximately 60% of patients.

1	Other risk factors include HIV infection, ionizing radiation, thermal burn scars, and chronic ulcerations. Albinism, xeroderma pigmentosum, Muir-Torre syndrome, Rombo’s syndrome, BazexDupré-Christol syndrome, dyskeratosis congenita, and basal cell nevus syndrome (Gorlin syndrome) also increase the incidence of NMSC. Mutations in Hh genes encoding the tumor-suppressor patched

1	FIGURE 105-4 Influence of vismodegib on the hedgehog (Hh) pathway. Normally, one of three Hh ligands (sonic [SHh], Indian, or desert) binds to patched homolog 1 (PTCH1), causing its degradation and release of smoothened homolog (SMO). The downstream events of SMO release are the activation of Gli1, Gli2, and Gli3 through the transcriptional regulator known as SUFU. Gli1 and Gli2 translocate to the nucleus and promote gene transcription. Vismodegib is an SMO antagonist that decreases the interaction between SMO and PTCH1, resulting in decreased Hh pathway signaling, gene transcription, and cell division. The downstream Hh pathway events inhibited by vismodegib are indicated in red.

1	homolog 1 (PTCH1) and smoothened homolog (SMO) occur in BCC. Aberrant PTCH1 signaling is propagated by the nuclear transcription factors Gli1 and Gli2, which are salient in the development of BCC and have led to the FDA approval of an oral SMO inhibitor, vismodegib, to treat advanced inoperable or metastatic BCC (Fig. 105-4). Vismodegib also reduces the incidence of BCC in patients with basal cell nevus syndrome who have PTCH1 mutations, affirming the importance of Hh in the onset of BCC.

1	CLINICAL PRESENTATION Basal Cell Carcinoma BCC arises from epidermal basal cells. The least invasive of BCC subtypes, superficial BCC, consists of often subtle, erythematous scaling plaques that slowly enlarge and are most commonly seen on the trunk and proximal extremities (Fig. 105-5). This BCC subtype may be confused with benign inflammatory dermatoses, especially nummular eczema and psoriasis. BCC also can present as a small, slowly growing pearly nodule, often with tortuous telangiectatic vessels on its surface, rolled borders, and a central crust (nodular BCC). The occasional presence of melanin in this variant of nodular BCC (pigmented BCC) may lead to confusion with melanoma. Morpheaform (fibrosing), infiltrative, and micronodular BCC, the most invasive and potentially aggressive subtypes, manifest as solitary, flat or slightly depressed, indurated whitish, yellowish, or pink scar-like plaques. Borders are typically indistinct, and lesions can be subtle; thus, delay in

1	subtypes, manifest as solitary, flat or slightly depressed, indurated whitish, yellowish, or pink scar-like plaques. Borders are typically indistinct, and lesions can be subtle; thus, delay in treatment is common, and tumors can be more extensive than expected clinically.

1	Squamous Cell Carcinoma Primary cutaneous SCC is a malignant neoplasm of keratinizing epidermal cells. SCC has a variable clinical course, ranging from indolent to rapid growth kinetics, with the potential for metastasis to regional and distant sites. Commonly, SCC appears as an ulcerated erythematous nodule or superficial erosion on sun-exposed skin of the head, neck, trunk, and extremities (Fig. 105-5). It may also appear as a banal, firm, dome-shaped papule or rough-textured plaque. It is commonly mistaken for a wart or callous when the inflammatory response to the lesion is minimal. Clinically visible overlying telangiectasias are uncommon, although dotted or coiled vessels are a hallmark of SCC when viewed through a dermatoscope. FIGURE 105-5 Cutaneous neoplasms. A. Non-Hodgkin’s lymphoma involves the skin with typical violaceous, “plum-colored” nodules.

1	FIGURE 105-5 Cutaneous neoplasms. A. Non-Hodgkin’s lymphoma involves the skin with typical violaceous, “plum-colored” nodules. B. Squamous cell carcinoma is seen here as a hyperkeratotic crusted and somewhat eroded plaque on the lower lip. Sun-exposed skin in areas such as the head, neck, hands, and arms represent other typical sites of involvement. C. Actinic keratoses consist of hyperkeratotic erythematous papules and patches on sun-exposed skin. They arise in middle-aged to older adults and have some potential for malignant transformation. D. Metastatic carcinoma to the skin is characterized by inflammatory, often ulcerated dermal nodules. E. Mycosis fungoides is a cutaneous T cell lymphoma, and plaque-stage lesions are seen in this patient. F. Keratoacanthoma is a low-grade squamous cell carcinoma that presents as an exophytic nodule with central keratinous debris. G. This basal cell carcinoma shows central ulceration and a pearly, rolled telangiectatic tumor border.

1	Cancer of the Skin The margins of this tumor may be ill defined, and fixation to underlying structures may occur (“tethering”). A very rapidly growing but low-grade form of SCC, called keratoacanthoma (KA), typically appears as a large dome-shaped papule with a central keratotic crater. Some KAs regress spontaneously without therapy, but because progression to metastatic SCC has been documented, KAs should be treated in the same manner as other types of cutaneous SCC. KAs are also associated with medications that target BRAF mutations and occur in 15–25% of patients receiving these medications.

1	Actinic keratoses and cheilitis (actinic keratoses occurring on the lip), both premalignant forms of SCC, present as hyperkeratotic papules on sun-exposed areas. The potential for malignant degeneration in untreated lesions ranges from 0.25 to 20%. SCC in situ, also called Bowen’s disease, is the intraepidermal form of SCC and usually presents as a scaling, erythematous plaque. As with invasive SCC, SCC in situ most commonly arises on sun-damaged skin, but can occur anywhere on the body. Bowen’s disease forming secondary to infection with human papillomavirus (HPV) can arise on skin with minimal or no prior sun exposure, such as the buttock or posterior thigh. Treatment of premalignant and in situ lesions reduces the subsequent risk of invasive disease.

1	NATURAL HISTORY Basal Cell Carcinoma The natural history of BCC is that of a slowly enlarging, locally invasive neoplasm. The degree of local destruction and risk of recurrence vary with the size, duration, location, and histologic subtype of the tumor. Location on the central face, ears, or scalp may portend a higher risk. Small nodular, pigmented, cystic, or superficial BCCs respond well to most treatments. Large lesions and micronodular, infiltrative, and morpheaform subtypes may be more aggressive. The metastatic potential of BCC is low (0.0028–0.1% in immunocompetent patients), but the risk of recurrence or a new primary NMSC is about 40% over 5 years.

1	Squamous Cell Carcinoma The natural history of SCC depends on tumor and host characteristics. Tumors arising on sun-damaged skin have a lower metastatic potential than do those on non-sunexposed areas. Cutaneous SCC metastasizes in 0.3–5.2% of individuals, most frequently to regional lymph nodes. Tumors occurring on the lower lip and ear develop regional metastases in 13 and 11% of patients, respectively, whereas the metastatic potential of SCC arising in scars, chronic ulcerations, and genital or mucosal surfaces is higher. Recurrent SCC has a much higher potential for metastatic disease, approaching 30%. Large, poorly differentiated, deep tumors with perineural or lymphatic invasion, multifocal tumors, and those arising in immunosuppressed patients often behave aggressively.

1	Treatments used for BCC include electrodesiccation and curettage (ED&C), excision, cryosurgery, radiation therapy (RT), laser therapy, Mohs micrographic surgery (MMS), topical 5-fluorouracil, photo-dynamic therapy (PDT), and topical immunomodulators such as imiquimod. The therapy chosen depends on tumor characteristics including depth and location, patient age, medical status, and patient preference. ED&C remains the most commonly employed method for superficial, minimally invasive nodular BCCs and low-risk tumors (e.g., a small tumor of a less aggressive subtype in a favorable location). Wide local excision with standard margins is usually selected for invasive, ill-defined, and more aggressive subtypes of tumors, or for cosmetic reasons. MMS, a specialized type of surgical excision that provides the best method for tumor removal while preserving uninvolved tissue, is associated with cure rates >98%. It is the preferred modality for lesions that are recurrent, in high-risk or

1	that provides the best method for tumor removal while preserving uninvolved tissue, is associated with cure rates >98%. It is the preferred modality for lesions that are recurrent, in high-risk or cosmetically sensitive locations (including recurrent tumors in these locations), and in which maximal tissue conservation is critical (e.g., the eyelids, lips, ears, nose, and digits). RT can cure patients not considered surgical candidates and can be used as a surgical adjunct in high-risk tumors. Younger patients may not be good candidates 502 for RT because of the risks of long-term carcinogenesis and radio-dermatitis. Imiquimod can be used to treat superficial and smaller nodular BCCs, although it is not FDA-approved for nodular BCC. Topical 5-fluorouracil therapy should be limited to superficial BCC. PDT, which uses selective activation of a photoactive drug by visible light, has been used in patients with numerous tumors. Intralesional chemotherapy (5-fluorouracil and IFN) for NMSC

1	BCC. PDT, which uses selective activation of a photoactive drug by visible light, has been used in patients with numerous tumors. Intralesional chemotherapy (5-fluorouracil and IFN) for NMSC has existed since the mid-twentieth century, but is used so infrequently that recent consensus guidelines for the treatment of BCC and SCC do not include it. Like RT, it remains an option for well-selected patients who cannot or will not undergo surgery.

1	Therapy for cutaneous SCC should be based on the size, location, histologic differentiation, patient age, and functional status. Surgical excision and MMS are standard treatments. Cryosurgery and ED&C have been used for premalignant lesions and small, superficial, in situ primary tumors. Lymph node metastases are treated with surgical resection, RT, or both. Systemic chemotherapy combinations that include cisplatin can palliate patients with advanced disease. SCC and keratoacanthomas that develop in patients receiving BRAF-targeted therapy should be excised, but their development should not deter the continued use of BRAF therapy. Retinoid prophylaxis can also be considered for patients receiving BRAF-targeted therapy, although no prospective studies have been completed thus far.

1	The general principles for prevention are those described for melanoma earlier. Unique strategies for NMSC include active surveillance for patients on immunosuppressive medications or BRAF-targeted therapy. Chemoprophylaxis using synthetic retinoids and immunosuppression reduction when possible may be useful in controlling new lesions and managing patients with multiple tumors. Neoplasms of cutaneous adnexae and sarcomas of fibrous, mesenchymal, fatty, and vascular tissues make up the remaining 1–2% of NMSCs.

1	Merkel cell carcinoma (MCC) is a neural crest–derived highly aggressive malignancy with mortality rates approaching 33% at 3 years. An oncogenic Merkel cell polyomavirus is present in 80% of tumors. Many patients have detectable cellular or humoral immune responses to polyoma viral proteins, although this immune response is insufficient to eradicate the malignancy. Survival depends on extent of disease: 90% survive with local disease, 52% with nodal involvement, and only 10% with distant disease at 3 years. MCC incidence tripled over the last 20 years with an estimated 1600 cases per year in the United States. Immunosuppression can increase incidence and diminish prognosis. MCC lesions typically present as an asymptomatic rapidly expanding bluish-red/violaceous tumor on sun-exposed skin of older white patients. Treatment is surgical excision with sentinel lymph node biopsy for accurate staging in patients with localized disease, often followed by adjuvant RT. Patients with extensive

1	of older white patients. Treatment is surgical excision with sentinel lymph node biopsy for accurate staging in patients with localized disease, often followed by adjuvant RT. Patients with extensive disease can be offered systemic chemotherapy; however, there is no convincing survival benefit. Whenever possible a clinical trial should be considered for this rare but aggressive NMSC, especially in light of the potential for new treatments directed at the oncogenic virus that causes this malignancy.

1	Extramammary Paget’s disease is an uncommon apocrine malignancy arising from stem cells of the epidermis that are characterized histologically by the presence of Paget cells. These tumors present as moist erythematous patches on anogenital or axillary skin of the elderly. Outcomes are generally good with site-directed surgery, and 5-year disease specific survival is approximately 95% with localized disease. Advanced age and extensive disease at presentation are factors that confer diminished prognosis. RT or topical imiquimod can be considered for more extensive disease. Local management may be challenging because these tumors often extend far beyond clinical margins; surgical excision with MMS has the highest cure rates. Similarly, MMS is the treatment of choice in other rare cutaneous tumors with extensive subclinical extension such as dermatofibromasarcoma protuberans.

1	Kaposi’s sarcoma (KS) is a soft tissue sarcoma of vascular origin that is induced by the human herpesvirus 8. The incidence of KS increased dramatically during the AIDS epidemic, but has now decreased tenfold with the institution of highly active antiretroviral therapy. Carl V. Washington, MD, and Hari Nadiminti, MD, contributed to this chapter in the 18th edition, and material from that chapter is included here. Claudia Taylor, MD, and Steven Kolker, MD, provided valued feedback and suggested many improvements to this chapter. Everett E. Vokes

1	Everett E. Vokes Epithelial carcinomas of the head and neck arise from the mucosal surfaces in the head and neck and typically are squamous cell in origin. This category includes tumors of the paranasal sinuses, the oral cavity, and the nasopharynx, oropharynx, hypopharynx, and larynx. Tumors of the salivary glands differ from the more common carcinomas of the head and neck in etiology, histopathology, clinical presentation, and therapy. They are rare and histologically highly heterogeneous. Thyroid malignancies are described in Chap. 405.

1	Thyroid malignancies are described in Chap. 405. The number of new cases of head and neck cancers (oral cav ity, pharynx, and larynx) in the United States was 53,640 in 2013, accounting for about 3% of adult malignancies; 11,520 people died from the disease. The worldwide incidence exceeds half a million cases annually. In North America and Europe, the tumors usually arise from the oral cavity, oropharynx, or larynx. The incidence of oropharyngeal cancers is increasing in recent years. Nasopharyngeal cancer is more commonly seen in the Mediterranean countries and in the Far East, where it is endemic in some areas. Alcohol and tobacco use are the most significant risk factors for head and neck cancer, and when used together, they act synergistically. Smokeless tobacco is an etiologic agent for oral cancers. Other potential carcinogens include marijuana and occupational exposures such as nickel refining, exposure to textile fibers, and woodworking.

1	Some head and neck cancers have a viral etiology. Epstein-Barr virus (EBV) infection is frequently associated with nasopharyngeal cancer, especially in endemic areas of the Mediterranean and Far East. EBV antibody titers can be measured to screen high-risk populations. Nasopharyngeal cancer has also been associated with consumption of salted fish and in-door pollution.

1	In Western countries, the human papilloma virus (HPV) is associated with a rising incidence of tumors arising from the oropharynx, i.e., the tonsillar bed and base of tongue. Over 50% of oropharyngeal tumors are caused by HPV in the United States. HPV 16 is the dominant viral subtype, although HPV 18 and other oncogenic subtypes are seen as well. Alcoholand tobacco-related cancers, on the other hand, have decreased in incidence. HPV-related oropharyngeal cancer occurs in a younger patient population and is associated with increased numbers of sexual partners and oral sexual practices. It is associated with a better prognosis, especially for nonsmokers.

1	Dietary factors may contribute. The incidence of head and neck cancer is higher in people with the lowest consumption of fruits and vegetables. Certain vitamins, including carotenoids, may be protective if included in a balanced diet. Supplements of retinoids, such as cisretinoic acid, have not been shown to prevent head and neck cancers (or lung cancer) and may increase the risk in active smokers. No specific risk factors or environmental carcinogens have been identified for salivary gland tumors. HISTOPATHOLOGY, CARCINOGENESIS, AND MOLECULAR BIOLOGY

1	HISTOPATHOLOGY, CARCINOGENESIS, AND MOLECULAR BIOLOGY Squamous cell head and neck cancers are divided into well-differentiated, moderately well-differentiated, and poorly differentiated categories. Poorly differentiated tumors have a worse prognosis than well-differentiated tumors. For nasopharyngeal cancers, the less common differentiated squamous cell carcinoma is distinguished from nonkeratinizing and undifferentiated carcinoma (lymphoepithelioma) that contains infiltrating lymphocytes and is commonly associated with EBV. Salivary gland tumors can arise from the major (parotid, submandibular, sublingual) or minor salivary glands (located in the submucosa of the upper aerodigestive tract). Most parotid tumors are benign, but half of submandibular and sublingual gland tumors and most minor salivary gland tumors are malignant. Malignant tumors include mucoepidermoid and adenoid cystic carcinomas and adenocarcinomas.

1	The mucosal surface of the entire pharynx is exposed to alcohol-and tobacco-related carcinogens and is at risk for the development of a premalignant or malignant lesion. Erythroplakia (a red patch) or leukoplakia (a white patch) can be histopathologically classified as hyperplasia, dysplasia, carcinoma in situ, or carcinoma. However, most head and neck cancer patients do not present with a history of premalignant lesions. Multiple synchronous or metachronous cancers can also be observed. In fact, over time, patients with early-stage head and neck cancer are at greater risk of dying from a second malignancy than from a recurrence of the primary disease.

1	Second head and neck malignancies are usually not therapy-induced; they reflect the exposure of the upper aerodigestive mucosa to the same carcinogens that caused the first cancer. These second primaries develop in the head and neck area, the lung, or the esophagus. Thus, computed tomography (CT) screening for lung cancer in heavy smokers who have already developed a head and neck cancer should be considered. Rarely, patients can develop a radiation therapy–induced sarcoma after having undergone prior radiotherapy for a head and neck cancer.

1	Much progress has been made in describing the molecular features of head and neck cancer. These features have allowed investigators to describe the genetic and epigenetic alterations and the mutational spectrum of these tumors. Early reports demonstrated frequent overexpression of the epidermal growth factor receptor (EGFR). Overexpression was shown to correlate with poor prognosis. However, it has not proved to be a good predictor of tumor response to EGFR inhibitors, which are successful in only about 10–15% of patients. p53 mutations are also found frequently with other major affected oncogenic driver pathways including the mitotic signaling and Notch pathways and cell cycle regulation. The PI3K pathway is frequently altered, especially in HPV-positive tumors, where it is the only mutated cancer gene identified to date. Overall, these alterations affect mitogenic signaling, genetic stability, cellular proliferation, and differentiation. HPV is known to act through inhibition of the

1	cancer gene identified to date. Overall, these alterations affect mitogenic signaling, genetic stability, cellular proliferation, and differentiation. HPV is known to act through inhibition of the p53 and RB tumor-suppressor genes, thereby initiating the carcinogenic process, and has a mutational spectrum distinct from alcoholand tobacco-related tumors.

1	Most tobacco-related head and neck cancers occur in patients older than age 60 years. HPV-related malignancies are frequently diagnosed in younger patients, usually in their forties or fifties, whereas EBV-related nasopharyngeal cancer can occur in all ages, including teenagers. The manifestations vary according to the stage and primary site of the tumor. Patients with nonspecific signs and symptoms in the head and neck area should be evaluated with a thorough otolaryngologic exam, particularly if symptoms persist longer than 2–4 weeks. Males are more frequently affected than women by head and neck cancers, including HPV-positive tumors. Cancer of the nasopharynx typically does not cause early symptoms. However, it may cause unilateral serous otitis media due to obstruction of the eustachian tube, unilateral or bilateral nasal obstruction, or 503 epistaxis. Advanced nasopharyngeal carcinoma causes neuropathies of the cranial nerves due to skull base involvement.

1	Carcinomas of the oral cavity present as nonhealing ulcers, changes in the fit of dentures, or painful lesions. Tumors of the tongue base or oropharynx can cause decreased tongue mobility and alterations in speech. Cancers of the oropharynx or hypopharynx rarely cause early symptoms, but they may cause sore throat and/or otalgia. HPV-related tumors frequently present with neck lymphadenopathy as the first sign. Hoarseness may be an early symptom of laryngeal cancer, and persistent hoarseness requires referral to a specialist for indirect laryngoscopy and/or radiographic studies. If a head and neck lesion treated initially with antibiotics does not resolve in a short period, further workup is indicated; to simply continue the antibiotic treatment may be to lose the chance of early diagnosis of a malignancy.

1	Advanced head and neck cancers in any location can cause severe pain, otalgia, airway obstruction, cranial neuropathies, trismus, odynophagia, dysphagia, decreased tongue mobility, fistulas, skin involve ment, and massive cervical lymphadenopathy, which may be unilateral or bilateral. Some patients have enlarged lymph nodes even though no primary lesion can be detected by endoscopy or biopsy; these patients are considered to have carcinoma of unknown primary (Fig. 106-1). If the enlarged nodes are located in the upper neck and the tumor cells are of squamous cell histology, the malignancy probably arose from a mucosal surface in the head or neck. Tumor cells in supraclavicular lymph nodes may also arise from a primary site in the chest or abdomen.

1	The physical examination should include inspection of all visible mucosal surfaces and palpation of the floor of the mouth and of the tongue and neck. In addition to tumors themselves, leukoplakia (a white mucosal patch) or erythroplakia (a red mucosal patch) may be observed; these “premalignant” lesions can represent hyperplasia, dysplasia, or carcinoma in situ and require biopsy. Further examination should be performed by a specialist. Additional staging procedures include CT of the head and neck to identify the extent of the disease. Patients with lymph node involvement should have CT scan of the chest and upper abdomen to screen for distant metastases. In heavy smokers, the CT scan of the chest can also serve as a screening tool to rule out a second lung primary tumor. A positron emission tomography (PET) scan may also be administered and can help to identify or exclude distant metastases. The definitive staging procedure is an

1	Physical Examination in Office If lymphoma, sarcoma, or salivary gland tumor Panendoscopy and directed biopsies. Search for occult primary with biopsies of tonsils, nasopharynx, base of tongue, and pyriform sinus. FNA or excision of lymph node Specific workup If squamous cell carcinoma FIGURE 106-1 Evaluation of a patient with cervical adenopathy without a primary mucosal lesion; a diagnostic workup. FNA, fine-needle aspiration.

1	504 endoscopic examination under anesthesia, which may include laryngoscopy, esophagoscopy, and bronchoscopy; during this procedure, multiple biopsy samples are obtained to establish a primary diagnosis, define the extent of primary disease, and identify any additional premalignant lesions or second primaries. Head and neck tumors are classified according to the tumornode-metastasis (TNM) system of the American Joint Committee on Cancer (Fig. 106-2). This classification varies according to the specific anatomic subsite. In general, primary tumors are classified as T1 to T3 by increasing size, whereas T4 usually represents invasion of another structure such as bone, muscle, or root of tongue. Lymph nodes are staged by size, number, and location (ipsilateral vs contralateral to the primary). Distant metastases are found in <10% of patients at initial diagnosis and are more common in patients with advanced lymph node stage; microscopic involvement of the lungs, bones, or liver is more

1	Distant metastases are found in <10% of patients at initial diagnosis and are more common in patients with advanced lymph node stage; microscopic involvement of the lungs, bones, or liver is more common, particularly in patients with advanced neck lymph node disease. Modern imaging techniques may increase the number of patients with clinically detectable distant metastases in the future.

1	In patients with lymph node involvement and no visible primary, the diagnosis should be made by lymph node excision (Fig. 106-1). If the results indicate squamous cell carcinoma, a panendoscopy should be performed, with biopsy of all suspicious-appearing areas and directed biopsies of common primary sites, such as the nasopharynx, tonsil, tongue base, and pyriform sinus. HPV-positive tumors especially can have small primary tumors that spread early to locoregional lymph nodes. Patients with head and neck cancer can be grossly categorized into three clinical groups: those with localized disease, those with locally or regionally advanced disease (lymph node positive), and those with recurrent and/or metastatic disease. Comorbidities associated with tobacco and alcohol abuse can affect treatment outcome and define long-term risks for patients who are cured of their disease.

1	Nearly one-third of patients have localized disease, that is, T1 or T2 (stage I or stage II) lesions without detectable lymph node involvement or distant metastases. These patients are treated with curative intent by either surgery or radiation therapy. The choice of modality differs according to anatomic location and institutional expertise. Radiation therapy is often preferred for laryngeal cancer to preserve voice function, and surgery is preferred for small lesions in the oral cavity to avoid the long-term complications of radiation, such as xerostomia and dental decay. Overall 5-year survival is 60–90%. Most recurrences occur within the first 2 years following diagnosis and are usually local.

1	Locally or regionally advanced disease—disease with a large primary tumor and/or lymph node metastases—is the stage of presentation for >50% of patients. Such patients can also be treated with curative intent, but not with surgery or radiation therapy alone. Combined-modality therapy including surgery, radiation therapy, and chemotherapy is most successful. It can be administered as induction chemotherapy (chemotherapy before surgery and/or radiotherapy) or as concomitant (simultaneous) chemotherapy and radiation therapy. The latter is currently most commonly used and supported by the best evidence. Five-year survival rates exceed 50% in many trials, but part of this increased survival may be due to an increasing fraction of study populations with HPV-related tumors who carry a better prognosis. HPV testing of newly diagnosed tumors is now performed for most patients at the time of diagnosis, and clinical trials for HPV-related tumors are focused on exploring reductions in treatment

1	HPV testing of newly diagnosed tumors is now performed for most patients at the time of diagnosis, and clinical trials for HPV-related tumors are focused on exploring reductions in treatment intensity, especially radiation dose, in order to ameliorate long-term toxicities (fibrosis, swallowing dysfunction).

1	In patients with intermediate-stage tumors (stage III and early stage IV), concomitant chemoradiotherapy can be administered either as a primary treatment for patients with unresectable disease, to pursue an organ-preserving approach, or in the postoperative setting for intermediate-stage resectable tumors. Induction Chemotherapy In this strategy, patients receive chemotherapy (current standard is a three-drug regimen of docetaxel, cisplatin, and fluorouracil [5-FU]) before surgery and radiation therapy. Most patients who receive three cycles show tumor reduction, and the response is clinically “complete” in up to half of patients. This “sequential” multimodality therapy allows for organ preservation (omission of surgery) in patients with laryngeal and hypopharyngeal cancer, and it has been shown to result in higher cure rates compared with radiotherapy alone.

1	Concomitant Chemoradiotherapy With the concomitant strategy, chemotherapy and radiation therapy are given simultaneously rather than in sequence. Tumor recurrences from head and neck cancer develop most commonly locoregionally (in the head and neck area of the primary and draining lymph nodes). The concomitant approach is aimed at enhancing tumor cell killing by radiation therapy in the presence of chemotherapy (radiation enhancement) and is a conceptually attractive approach for bulky tumors. Toxicity 505 (especially mucositis, grade 3 or 4 in 70–80%) is increased with concomitant chemoradiotherapy. However, meta-analyses of randomized trials document an improvement in 5-year survival of 8% with concomitant chemotherapy and radiation therapy. Results seem more favorable in recent trials as more active drugs or more intensive radiotherapy schedules are used. In addition, concomitant chemoradiotherapy produces better laryngectomy-free survival (organ preservation) than radiation

1	as more active drugs or more intensive radiotherapy schedules are used. In addition, concomitant chemoradiotherapy produces better laryngectomy-free survival (organ preservation) than radiation therapy alone in patients with advanced larynx cancer. The use of radiation therapy together with cisplatin has also produced improved survival in patients with advanced nasopharyngeal cancer. The outcome of HPV-related cancers seems to be especially favorable following cisplatin-based chemoradiotherapy.

1	The success of concomitant chemoradiotherapy in patients with unresectable disease has led to the testing of a similar approach in patients with resected intermediate-stage disease as a postoperative therapy. Concomitant chemoradiotherapy produces a signifi cant improvement over postoperative radiation therapy alone for patients whose tumors demonstrate higher risk features, such as extracapsular spread beyond involved lymph nodes, involvement of multiple lymph nodes, or positive margins at the primary site following surgery. A monoclonal antibody to EGFR (cetuximab) increases survival rates when administered during radiotherapy. EGFR blockade results in radiation sensitization and has milder systemic side effects than traditional chemotherapy agents, although an acneiform skin rash is commonly observed. Nevertheless, the integration of cetuximab into current standard chemoradiotherapy regimens has failed to show additional improvement in survival and is not recommended.

1	Five to ten percent of patients present with metastatic disease, and 30–50% of patients with locoregionally advanced disease experience recurrence, frequently outside the head and neck region. Patients with recurrent and/or metastatic disease are, with few exceptions, treated with palliative intent. Some patients may require local or regional radiation therapy for pain control, but most are given chemotherapy. Response rates to chemotherapy average only 30–50%; the durations of response are short, and the median survival time is 8–10 months. Therefore, chemotherapy provides transient symptomatic benefit. Drugs with single-agent activity in this setting include methotrexate, 5-FU, cisplatin, paclitaxel, and docetaxel. Combinations of cisplatin with 5-FU, carboplatin with 5-FU, and cisplatin or carboplatin with paclitaxel or docetaxel are frequently used.

1	EGFR-directed therapies, including monoclonal antibodies (e.g., cetuximab) and tyrosine kinase inhibitors (TKIs) of the EGFR signaling pathway (e.g., erlotinib or gefitinib), have single-agent activity of approximately 10%. Side effects are usually limited to an acneiform rash and diarrhea (for the TKIs). The addition of cetuximab to standard combination chemotherapy with cisplatin or carboplatin and 5-FU was shown to result in a significant increase in median survival. Drugs targeting specific mutations are under investigation, but no such strategy has yet been shown to be feasible in head and neck cancer.

1	Complications from treatment of head and neck cancer are usually correlated to the extent of surgery and exposure of normal tissue structures to radiation. Currently, the extent of surgery has been limited or completely replaced by chemotherapy and radiation therapy as the primary approach. Acute complications of radiation include mucositis and dysphagia. Long-term complications include xerostomia, loss of taste, decreased tongue mobility, second malignancies, dysphagia, and neck fibrosis. The complications of chemotherapy vary with the regimen used but usually include myelosuppression, mucositis, nausea and vomiting, and nephrotoxicity (with cisplatin).

1	506 The mucosal side effects of therapy can lead to malnutrition and dehydration. Many centers address issues of dentition before starting treatment, and some place feeding tubes to ensure control of hydration and nutrition intake. About 50% of patients develop hypothyroidism from the treatment; thus, thyroid function should be monitored. Most benign salivary gland tumors are treated with surgical excision, and patients with invasive salivary gland tumors are treated with surgery and radiation therapy. These tumors may recur regionally; adenoid cystic carcinoma has a tendency to recur along the nerve tracks. Distant metastases may occur as late as 10–20 years after the initial diagnosis. For metastatic disease, therapy is given with palliative intent, usually chemotherapy with doxorubicin and/or cisplatin. Identification of novel agents with activity in these tumors is a high priority. neoplasms of the Lung Leora Horn, Christine M. Lovly, David H. Johnson

1	Lung cancer, which was rare prior to 1900 with fewer than 400 cases described in the medical literature, is considered a disease of modern man. By the mid-twentieth century, lung cancer had become epidemic and firmly established as the leading cause of cancer-related death in North America and Europe, killing over three times as many men as prostate cancer and nearly twice as many women as breast cancer. This fact is particularly troubling because lung cancer is one of the most preventable of all of the major malignancies. Tobacco consumption is the primary cause of lung cancer, a reality firmly established in the mid-twentieth century and codified with the release of the U.S. Surgeon General’s 1964 report on the health effects of tobacco smoking. Following the report, cigarette use started to decline in North America and parts of Europe, and with it, so did the incidence of lung cancer. To date, the decline in lung cancer is seen most clearly in men; only recently has the decline

1	to decline in North America and parts of Europe, and with it, so did the incidence of lung cancer. To date, the decline in lung cancer is seen most clearly in men; only recently has the decline become apparent among women in the United States. Unfortunately, in many parts of the world, especially in countries with developing economies, cigarette use continues to increase, and along with it, the incidence of lung cancers is also rising. Although tobacco smoking remains the primary cause of lung cancer worldwide, approximately 60% of new lung cancers in the United States occur in former smokers (smoked ≥100 cigarettes per lifetime, quit ≥1 year), many of whom quit decades ago, or never smokers (smoked <100 cigarettes per lifetime). Moreover, one in five women and one in 12 men diagnosed with lung cancer have never smoked. Given the magnitude of the problem, it is incumbent that every internist has a general knowledge of lung cancer and its management.

1	Lung cancer is the most common cause of cancer death among American men and women. More than 225,000 individuals will be diagnosed with lung cancer in the United States in 2013, and over 150,000 individuals will die from the disease. The incidence of lung cancer peaked among men in the late 1980s and has plateaued in women. Lung cancer is rare below age 40, with rates increasing until age 80, after which the rate tapers off. The projected lifetime probability of developing lung cancer is estimated to be approximately 8% among males and approximately 6% among females. The incidence of lung cancer varies by racial and ethnic group, with the highest age-adjusted incidence rates among African Americans. The excess in age-adjusted rates among African Americans occurs only among men, but examinations of age-specific rates show that below age 50, mortality from lung cancer is more than 25% higher among African American than Caucasian women. Incidence and mortality rates among Hispanics and

1	of age-specific rates show that below age 50, mortality from lung cancer is more than 25% higher among African American than Caucasian women. Incidence and mortality rates among Hispanics and Native and Asian Americans are approximately 40–50% those of whites.

1	Cigarette smokers have a 10-fold or greater increased risk of developing lung cancer compared to those who have never smoked. A deep sequencing study suggested that one genetic mutation is induced for every 15 cigarettes smoked. The risk of lung cancer is lower among persons who quit smoking than among those who continue smoking; former smokers have a ninefold increased risk of developing lung cancer compared to men who have never smoked versus the 20-fold excess in those who continue to smoke. The size of the risk reduction increases with the length of time the person has quit smoking, although generally even long-term former smokers have higher risks of lung cancer than those who never smoked. Cigarette smoking has been shown to increase the risk of all the major lung cancer cell types. Environmental tobacco smoke (ETS) or second-hand smoke is also an established cause of lung cancer. The risk from ETS is less than from active smoking, with about a 20–30% increase in lung cancer

1	Environmental tobacco smoke (ETS) or second-hand smoke is also an established cause of lung cancer. The risk from ETS is less than from active smoking, with about a 20–30% increase in lung cancer observed among never smokers married for many years to smokers, in comparison to the 2000% increase among continuing active smokers.

1	Although cigarette smoking is the cause of the majority of lung cancers, several other risk factors have been identified, including occupational exposures to asbestos, arsenic, bischloromethyl ether, hexavalent chromium, mustard gas, nickel (as in certain nickel-refining processes), and polycyclic aromatic hydrocarbons. Occupational observations also have provided insight into possible mechanisms of lung cancer induction. For example, the risk of lung cancer among asbestos-exposed workers is increased primarily among those with underlying asbestosis, raising the possibility that the scarring and inflammation produced by this fibrotic nonmalignant lung disease may in many cases (although likely not in all) be the trigger for asbestos-induced lung cancer. Several other occupational exposures have been associated with increased rates of lung cancer, but the causal nature of the association is not as clear.

1	The risk of lung cancer appears to be higher among individuals with low fruit and vegetable intake during adulthood. This observation led to hypotheses that specific nutrients, in particular retinoids and carotenoids, might have chemopreventative effects for lung cancer. However, randomized trials failed to validate this hypothesis. In fact, studies found the incidence of lung cancer was increased among smokers with supplementation. Ionizing radiation is also an established lung carcinogen, most convincingly demonstrated from studies showing increased rates of lung cancer among survivors of the atom bombs dropped on Hiroshima and Nagasaki and large excesses among workers exposed to alpha irradiation from radon in underground uranium mining. Prolonged exposure to low-level radon in homes might impart a risk of lung cancer equal or greater than that of ETS. Prior lung diseases such as chronic bronchitis, emphysema, and tuberculosis have been linked to increased risks of lung cancer as

1	might impart a risk of lung cancer equal or greater than that of ETS. Prior lung diseases such as chronic bronchitis, emphysema, and tuberculosis have been linked to increased risks of lung cancer as well.

1	Smoking Cessation Given the undeniable link between cigarette smoking and lung cancer (not even addressing other tobacco-related illnesses), physicians must promote tobacco abstinence. Physicians also must help their patients who smoke to stop smoking. Smoking cessation, even well into middle age, can minimize an individual’s subsequent risk of lung cancer. Stopping tobacco use before middle age avoids more than 90% of the lung cancer risk attributable to tobacco. However, there is little health benefit derived from just “cutting back.” Importantly, smoking cessation can even be beneficial in individuals with an established diagnosis of lung cancer, as it is associated with improved survival, fewer side effects from therapy, and an overall improvement in quality of life. Moreover, smoking can alter the metabolism of many chemotherapy drugs, potentially adversely altering the toxicities and therapeutic benefits of the agents. Consequently, it is important to promote smoking cessation

1	can alter the metabolism of many chemotherapy drugs, potentially adversely altering the toxicities and therapeutic benefits of the agents. Consequently, it is important to promote smoking cessation even after the diagnosis of lung cancer is established.

1	Physicians need to understand the essential elements of smoking cessation therapy. The individual must want to stop smoking and must be willing to work hard to achieve the goal of smoking abstinence. Self-help strategies alone only marginally affect quit rates, whereas individual and combined pharmacotherapies in combination with counseling can significantly increase rates of cessation. Therapy with an antidepressant (e.g., bupropion) and nicotine replacement therapy (varenicline, a α4β2 nicotinic acetylcholine receptor partial agonist) are approved by the U.S. Food and Drug Administration (FDA) as first-line treatments for nicotine dependence. However, both drugs have been reported to increase suicidal ideation and must be used with caution. In a randomized trial, varenicline was shown to be more efficacious than bupropion or placebo. Prolonged use of varenicline beyond the initial induction phase proved useful in maintaining smoking abstinence. Clonidine and nortriptyline are

1	to be more efficacious than bupropion or placebo. Prolonged use of varenicline beyond the initial induction phase proved useful in maintaining smoking abstinence. Clonidine and nortriptyline are recommended as second-line treatments. Of note, reducing cigarettes smoked before quit day and quitting abruptly, with no prior reduction, yield comparable quit rates. Therefore, patients can be given the choice to quit in either of these ways (Chap. 470).

1	Inherited Predisposition to Lung Cancer Exposure to environmental carcinogens, such as those found in tobacco smoke, induce or facilitate the transformation from bronchoepithelial cells to the malignant phenotype. The contribution of carcinogens on transformation is modulated by polymorphic variations in genes that affect aspects of carcinogen metabolism. Certain genetic polymorphisms of the P450 enzyme system, specifically CYP1A1, and chromosome fragility are associated with the development of lung cancer. These genetic variations occur at relatively high frequency in the population, but their contribution to an individual’s lung cancer risk is generally low. However, because of their population frequency, the overall impact on lung cancer risk could be high. In addition, environmental factors, as modified by inherited modulators, likely affect specific genes by deregulating important pathways to enable the cancer phenotype.

1	First-degree relatives of lung cancer probands have a twoto threefold excess risk of lung cancer and other cancers, many of which are not smoking-related. These data suggest that specific genes and/or genetic variants may contribute to susceptibility to lung cancer. However, very few such genes have yet been identified. Individuals with inherited mutations in RB (patients with retinoblastoma living to adulthood) and p53 (Li-Fraumeni syndrome) genes may develop lung cancer. Common gene variants involved in lung cancer have been recently identified through large, collaborative, genome-wide association studies. These studies identified three separate loci that are associated with lung cancer (5p15, 6p21, and 15q25) and include genes that regulate acetylcholine nicotinic receptors and telomerase production. A rare germline mutation (T790M) involving the epidermal growth factor receptor (EGFR) maybe be linked to lung cancer susceptibility in never smokers. Likewise, a susceptibility locus

1	production. A rare germline mutation (T790M) involving the epidermal growth factor receptor (EGFR) maybe be linked to lung cancer susceptibility in never smokers. Likewise, a susceptibility locus on chromosome 6q greatly increases risk lung cancer risk among light and never smokers. Although progress has been made, there is a significant amount of work that remains to be done in identifying heritable risk factors for lung cancer. Currently no molecular criteria are suitable to select patients for more intense screening programs or for specific chemopreventative strategies.

1	The World Health Organization (WHO) defines lung cancer as tumors arising from the respiratory epithelium (bronchi, bronchioles, and alveoli). The WHO classification system divides epithelial lung cancers into four major cell types: small-cell lung cancer (SCLC), adenocarcinoma, squamous cell carcinoma, and large-cell carcinoma; the latter three types are collectively known as non-small-cell carcinomas (NSCLCs) (Fig. 107-1). Small-cell carcinomas consist of small cells with scant cytoplasm, ill-defined cell borders, finely granular nuclear chromatin, absent or inconspicuous nucleoli, and a high mitotic count. SCLC may be distinguished from NSCLC by the presence of neuroendocrine markers including CD56, neural cell adhesion molecule (NCAM), synaptophysin, and chromogranin. In North America, adenocarcinoma is the most common histologic type of lung cancer. Adenocarcinomas possess glandular differentiation or mucin production and may show acinar, papillary, lepidic, or solid features or

1	adenocarcinoma is the most common histologic type of lung cancer. Adenocarcinomas possess glandular differentiation or mucin production and may show acinar, papillary, lepidic, or solid features or a mixture of these patterns. Squamous cell carcinomas of the

1	Small Cell Lung Cancer (SCLC) Non-Small Cell Lung Cancer (NSCLC): FIGURE 107-1 Traditional histologic view of lung cancer. lung are morphologically identical to extrapulmonary squamous cell carcinomas and cannot be distinguished by immunohistochemistry alone. Squamous cell tumors show keratinization and/or intercellular bridges that arise from bronchial epithelium. The tumor tends to consists of sheets of cells rather than the three-dimensional groups of cells characteristic of adenocarcinomas. Large-cell carcinomas comprise less than 10% of lung carcinomas. These tumors lack the cytologic and architectural features of small-cell carcinoma and glandular or squamous differentiation. Together these four histologic types account for approximately 90% of all epithelial lung cancers.

1	All histologic types of lung cancer can develop in current and former smokers, although squamous and small-cell carcinomas are most commonly associated with heavy tobacco use. Through the first half of the twentieth century, squamous carcinoma was the most common subtype of NSCLC diagnosed in the United States. However, with the decline in cigarette consumption over the past four decades, adenocarcinoma has become the most frequent histologic subtype of lung cancer in the United States as both squamous carcinoma and small-cell carcinoma are on the decline. In lifetime never smokers or former light smokers (<10 pack-year history), women, and younger adults (<60 years), adenocarcinoma tends to be the most common form of lung cancer.

1	Historically, the major pathologic distinction was simply between SCLC and NSCLC, because these tumors have quite different natural histories and therapeutic approaches (see below). Likewise, until fairly recently, there was no apparent need to distinguish among the various subtypes of NSCLC because there were no clear differences in therapeutic outcome based on histology alone. However, this perspective radically changed in 2004 with the recognition that a small percentage of lung adenocarcinomas harbored mutation in EGFR that rendered those tumors exquisitely sensitive to inhibitors of the EGFR tyrosine kinases (e.g., gefitinib and erlotinib). This observation, coupled with the subsequent identification of other “actionable” molecular alterations (Table 107-1) and the recognition that some active

1	Neoplasms of the Lung 508 chemotherapy agents performed quite differently in squamous carcinomas versus adenocarcinomas, firmly established the need for modifications in the then-existing 2004 WHO lung cancer classification system. The revised 2011 classification system, developed jointly by the International Association for the Study of Lung Cancer, the American Thoracic Society, and the European Respiratory Society, provides an integrated approach to the classification of lung adenocarcinomas that includes clinical, molecular, radiographic, and pathologic information. It also recognizes that most lung cancers present in an advanced stage and are often diagnosed based on small biopsies or cytologic specimens, rendering clear histologic distinctions difficult if not impossible. Previously, in the 2004 classification system, tumors failing to show definite glandular or squamous morphology in a small biopsy or cytologic specimen were simply classified as non-small-cell carcinoma, not

1	in the 2004 classification system, tumors failing to show definite glandular or squamous morphology in a small biopsy or cytologic specimen were simply classified as non-small-cell carcinoma, not otherwise specified. However, because the distinction between adenocarcinoma and squamous carcinoma is now viewed as critical to optimal therapeutic decision making, the modified classification approach recommends these lesions be further characterized using a limited special stain workup. This distinction can be achieved using a single marker for adenocarcinoma (thyroid transcription factor-1 or napsin-A) plus a squamous marker (p40 or p63) and/or mucin stains. The modified classification system also recommends preservation of sufficient specimen material for appropriate molecular testing necessary to help guide therapeutic decision making (see below). Another significant modification to the WHO classification system is the discontinuation of the terms bronchioloalveolar carcinoma and

1	necessary to help guide therapeutic decision making (see below). Another significant modification to the WHO classification system is the discontinuation of the terms bronchioloalveolar carcinoma and mixed-subtype adenocarcinoma. The term bronchioloalveolar carcinoma was dropped due to its inconsistent use and because it caused confusion in routine clinical care and research. As formerly used, the term encompassed at least five different entities with diverse clinical and molecular properties. The terms adenocarcinoma in situ and minimally invasive adenocarcinoma are now recommended for small solitary adenocarcinomas (≤3 cm) with either pure lepidic growth (term used to describe single-layered growth of atypical cuboidal cells coating the alveolar walls) or predominant lepidic growth with ≤5 mm invasion. Individuals with these entities experience 100% or near 100% 5-year disease-free survival with complete tumor resection. Invasive adenocarcinomas, representing more than 70–90% of

1	≤5 mm invasion. Individuals with these entities experience 100% or near 100% 5-year disease-free survival with complete tumor resection. Invasive adenocarcinomas, representing more than 70–90% of surgically resected lung adenocarcinomas, are now classified by their predominant pattern: lepidic, acinar, papillary, and solid patterns. Lepidic-predominant subtype has a favorable prognosis, acinar and papillary have an intermediate prognosis, and solid-predominant has a poor prognosis. The terms signet ring and clear cell adenocarcinoma have been eliminated from the variants of invasive lung adenocarcinoma, whereas the term micropapillary, a subtype with a particularly poor prognosis, has been added. Although EGFR mutations are encountered most frequently in nonmucinous adenocarcinomas with a lepidicor papillary-predominant pattern, most adenocarcinoma subtypes can harbor EGFR or KRAS mutations. The same is true of ALK, RET, and ROS1 rearrangements. What was previously termed mucinous

1	a lepidicor papillary-predominant pattern, most adenocarcinoma subtypes can harbor EGFR or KRAS mutations. The same is true of ALK, RET, and ROS1 rearrangements. What was previously termed mucinous bronchioloalveolar carcinoma is now called invasive mucinous adenocarcinoma. These tumors generally lack EGFR mutations and show a strong correlation with KRAS mutations. Overall, the revised WHO reclassification of lung cancer addresses important advances in diagnosis and treatment, most importantly, the critical advances in understanding the specific genes and molecular pathways that initiate and sustain lung tumorigenesis resulting in new “targeted” therapies with improved specificity and better antitumor efficacy.

1	The diagnosis of lung cancer most often rests on the morphologic or cytologic features correlated with clinical and radiographic findings. Immunohistochemistry may be used to verify neuroendocrine differentiation within a tumor, with markers such as neuron-specific enolase (NSE), CD56 or NCAM, synaptophysin, chromogranin, and Leu7. Immunohistochemistry is also helpful in differentiating primary from metastatic adenocarcinomas; thyroid transcription factor-1 (TTF-1), identified in tumors of thyroid and pulmonary origin, is positive in over 70% of pulmonary adenocarcinomas and is a reliable indicator of primary lung cancer, provided a thyroid primary has been excluded. A negative TTF-1, however, does not exclude the possibility of a lung primary. TTF-1 is also positive in neuroendocrine tumors of pulmonary and extrapulmonary origin. Napsin-A (Nap-A) is an aspartic protease that plays an important role in maturation of surfactant B7 and is expressed in cytoplasm of type II pneumocytes.

1	of pulmonary and extrapulmonary origin. Napsin-A (Nap-A) is an aspartic protease that plays an important role in maturation of surfactant B7 and is expressed in cytoplasm of type II pneumocytes. In several studies, Nap-A has been reported in >90% of primary lung adenocarcinomas. Notably, a combination of Nap-A and TTF-1 is useful in distinguishing primary lung adenocarcinoma (Nap-A positive, TTF-1 positive) from primary lung squamous cell carcinoma (Nap-A negative, TTF-1 negative) and primary SCLC (Nap-A negative, TTF-1 positive). Cytokeratins 7 and 20 used in combination can help narrow the differential diagnosis; nonsquamous NSCLC, SCLC, and mesothelioma may stain positive for CK7 and negative for CK20, whereas squamous cell lung cancer often will be both CK7 and CK20 negative. p63 is a useful marker for the detection of NSCLCs with squamous differentiation when used in cytologic pulmonary samples. Mesothelioma can be easily identified ultrastructurally, but it has historically been

1	useful marker for the detection of NSCLCs with squamous differentiation when used in cytologic pulmonary samples. Mesothelioma can be easily identified ultrastructurally, but it has historically been difficult to differentiate from adenocarcinoma through morphology and immunohistochemical staining. Several markers in the last few years have proven to be more helpful including CK5/6, calretinin, and Wilms tumor gene-1 (WT-1), all of which show positivity in mesothelioma.

1	Cancer is a disease involving dynamic changes in the genome. As proposed by Hanahan and Weinberg, virtually all cancer cells acquire six hallmark capabilities: self-sufficiency in growth signals, insensitivity to antigrowth signals, evading apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. The order in which these hallmark capabilities are acquired appears quite variable and can differ from tumor to tumor. Events leading to acquisition of these hallmarks can vary widely, although broadly, cancers arise as a result from accumulations of gain-of-function mutations in oncogenes and loss-of-function mutations in tumor-suppressor genes. Further complicating the study of lung cancer, the sequence of events that lead to disease is clearly different for the various histopathologic entities.

1	The exact cell of origin for lung cancers is not clearly defined. Whether one cell of origin leads to all histologic forms of lung cancer is unclear. However, for lung adenocarcinoma, evidence suggests that type II epithelial cells (or alveolar epithelial cells) have the capacity to give rise to tumors. For SCLC, cells of neuroendocrine origin have been implicated as precursors.

1	For cancers in general, one theory holds that a small subset of the cells within a tumor (i.e., “stem cells”) are responsible for the full malignant behavior of the tumor. As part of this concept, the large bulk of the cells in a cancer are “offspring” of these cancer stem cells. While clonally related to the cancer stem cell subpopulation, most cells by themselves cannot regenerate the full malignant phenotype. The stem cell concept may explain the failure of standard medical therapies to eradicate lung cancers, even when there is a clinical complete response. Disease recurs because therapies do not eliminate the stem cell component, which may be more resistant to chemotherapy. Precise human lung cancer stem cells have yet to be identified.

1	Lung cancer cells harbor multiple chromosomal abnormalities, including mutations, amplifications, insertions, deletions, and translocations. One of the earliest sets of oncogenes found to be aberrant was the MYC family of transcription factors (MYC, MYCN, and MYCL). MYC is most frequently activated via gene amplification or transcriptional dysregulation in both SCLC and NSCLC. Currently, there are no MYC-specific drugs. Among lung cancer histologies, adenocarcinomas have been the most extensively catalogued for recurrent genomic gains and losses as well as for somatic mutations (Fig. 107-2). While multiple different kinds of aberrations have been found, a major class involves “driver mutations,” which are mutations that occur in genes encoding signaling proteins that when aberrant, drive initiation and maintenance of tumor cells. Importantly, driver mutations can serve as potential Achilles’ heels for tumors, if their gene products can be targeted

1	FIGURE 107-2 Driver mutations in adenocarcinomas.

1	appropriately. For example, one set of mutations involves EGFR, which belongs to the ERBB (HER) family of protooncogenes, including EGFR (ERBB1), HER2/neu (ERBB2), HER3 (ERBB3), and HER4 (ERBB4). These genes encode cell-surface receptors consisting of an extracellular ligand-binding domain, a transmembrane structure, and an intracellular tyrosine kinase (TK) domain. The binding of ligand to receptor activates receptor dimerization and TK autophosphorylation, initiating a cascade of intracellular events, and leading to increased cell proliferation, angiogenesis, metastasis, and a decrease in apoptosis. Lung adenocarcinomas can arise when tumors express mutant EGFR. These same tumors display high sensitivity to small-molecule EGFR TK inhibitors (TKIs). Additional examples of driver mutations in lung adenocarcinoma include the GTPase KRAS, the serine-threonine kinase BRAF, and the lipid kinase PIK3CA. More recently, more subsets of lung adenocarcinoma have been identifed as defined by

1	in lung adenocarcinoma include the GTPase KRAS, the serine-threonine kinase BRAF, and the lipid kinase PIK3CA. More recently, more subsets of lung adenocarcinoma have been identifed as defined by the presence of specific chromsomal rearrangements resulting in the abberant activation of the TKs ALK, ROS1, and RET. Notably, most driver mutations in lung cancer appear to be mutually exclusive, suggesting that acquisition of one of these mutations is sufficient to drive tumorigenesis. Although driver mutations have mostly been found in adenocarinomas, three potential molecular targets recently have been identified in squamous cell lung carcinomas: FGFR1 amplification, DDR2 mutations, and PIK3CA mutations/PTEN loss (Table 107-1). Together, these potentially “actionable” defects occur in up to 50% of squamous carcinomas.

1	A large number of tumor-suppressor genes have also been identified that are inactivated during the pathogenesis of lung cancer. These include TP53, RB1, RASSF1A, CDKN2A/B, LKB1 (STK11), and FHIT. Nearly 90% of SCLCs harbor mutations in TP53 and RB1. Several tumor-suppressor genes on chromosome 3p appear to be involved in nearly all lung cancers. Allelic loss for this region occurs very early in lung cancer pathogenesis, including in histologically normal smoking-damaged lung epithelium.

1	In lung cancer, clinical outcome is related to the stage at diagnosis, and hence, it is generally assumed that early detection of occult tumors will lead to improved survival. Early detection is a process that involves screening tests, surveillance, diagnosis, and early treatment. Screening refers to the use of simple tests across a healthy population in order to identify individuals who harbor asymptomatic disease. For a screening program to be successful, there must be a high burden of disease within the target population; the test must be sensitive, specific, accessible, and cost effective; and there must be effective treatment that can reduce mortality. With any screening procedure, it is important to consider the possible influence of lead-time bias (detecting the cancer earlier without an effect on survival), length-time bias (indolent cancers are detected on screening and may not affect survival, whereas aggressive cancers are likely to cause symptoms earlier in patients and

1	an effect on survival), length-time bias (indolent cancers are detected on screening and may not affect survival, whereas aggressive cancers are likely to cause symptoms earlier in patients and are less likely to be detected), and overdiagnosis (diagnosing cancers so slow growing that they are unlikely to cause the death of the patient) (Chap. 100).

1	Because a majority of lung cancer patients present with advanced 509 disease beyond the scope of surgical resection, there is understandable skepticism about the value of screening in this condition. Indeed, randomized controlled trials conducted in the 1960s to 1980s using screening chest x-rays (CXR), with or without sputum cytology, reported no impact on lung cancer–specific mortality in patients characterized as high risk (males age ≥45 years with a smoking history). These studies have been criticized for their design, statistical analyses, and outdated imaging modalities. The results of the more recently conducted Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO) are consistent with these earlier reports. Initiated in 1993, participants in the PLCO lung cancer screening trial received annual CXR screening for 4 years, whereas participants in the usual care group received no interventions other than their customary medical care. The diagnostic follow-up of

1	screening trial received annual CXR screening for 4 years, whereas participants in the usual care group received no interventions other than their customary medical care. The diagnostic follow-up of positive screening results was determined by participants and their physicians. The PLCO trial differed from previous lung cancer screening studies in that women and never smokers were eligible. The study was designed to detect a 10% reduction in lung cancer mortality in the interventional group. A total of 154,901 individuals between 55 and 74 years of age were enrolled (77,445 assigned to annual CXR screenings; 77,456 assigned to usual care). Participant demographics and tumor characteristics were well balanced between the two groups. Through 13 years of follow-up, cumulative lung cancer incidence rates (20.1 vs 19.2 per 10,000 person-years; rate ratio [RR], 1.05; 95% confidence interval [CI], 0.98–1.12) and lung cancer mortality (n = 1213 vs n = 1230) were identical between the two

1	incidence rates (20.1 vs 19.2 per 10,000 person-years; rate ratio [RR], 1.05; 95% confidence interval [CI], 0.98–1.12) and lung cancer mortality (n = 1213 vs n = 1230) were identical between the two groups. The stage and histology of detected cancers in the two groups also were similar. These data corroborate previous recommendations against CXR screening for lung cancer.

1	In contrast to CXR, low-dose, noncontrast, thin-slice spiral chest computed tomography (LDCT) has emerged as an effective tool to screen for lung cancer. In nonrandomized studies conducted in the 1990s, LDCT scans were shown to detect more lung nodules and cancers than standard CXR in selected high-risk populations (e.g., age ≥60 years and a smoking history of ≥10 pack-years). Notably, up to 85% of the lung cancers discovered in these trials were classified as stage I disease and therefore considered potentially curable with surgical resection.

1	These data prompted the National Cancer Institute (NCI) to initiate the National Lung Screening Trial (NLST), a randomized study designed to determine if LDCT screening could reduce mortality from lung cancer in high-risk populations as compared with standard posterior anterior CXR. High-risk patients were defined as individuals between 55 and 74 years of age, with a ≥30 pack-year history of cigarette smoking; former smokers must have quit within the previous 15 years. Excluded from the trial were individuals with a previous lung cancer diagnosis, a history of hemoptysis, an unexplained weight loss of >15 lb in the preceding year, or a chest CT within 18 months of enrollment. A total of 53,454 persons were enrolled and randomized to annual screening yearly for three years (LDCT screening, n = 26,722; CXR screening, n = 26,732). Any noncalcified nodule measuring ≥4 mm in any diameter found on LDCT and CXR images with any noncalcified nodule or mass were classified as “positive.”

1	n = 26,722; CXR screening, n = 26,732). Any noncalcified nodule measuring ≥4 mm in any diameter found on LDCT and CXR images with any noncalcified nodule or mass were classified as “positive.” Participating radiologists had the option of not calling a final screen positive if a noncalcified nodule had been stable on the three screening exams. Overall, 39.1% of participants in the LDCT group and 16% in the CXR group had at least one positive screening result. Of those who screened positive, the false-positive rate was 96.4% in the LDCT group and 94.5% in the CXR group. This was consistent across all three rounds. In the LDCT group, 1060 cancers were identified compared with 941 cancers in the CXR group (645 vs 572 per 100,000 person-years; RR, 1.13; 95% CI, 1.03 to 1.23). Nearly twice as many early-stage IA cancers were detected in the LDCT group compared with the CXR group (40% vs 21%). The overall rates of lung cancer death were 247 and 309 deaths per 100,000 participants in the LDCT

1	early-stage IA cancers were detected in the LDCT group compared with the CXR group (40% vs 21%). The overall rates of lung cancer death were 247 and 309 deaths per 100,000 participants in the LDCT and CXR groups, respectively, representing a 20% reduction in lung cancer mortality in the LDCT-screened population (95% CI, 6.8–26.7%; p = .004). Compared with the CXR group, the rate of death in the LDCT group from any cause was reduced by 6.7% (95% CI, 1.2–13.6; p = .02) (Table 107-2). The

1	Neoplasms of the Lung Rates of Events per 100,000 Relative Risk Event Number Person-Years (95% CI) Source: Modified from PB Bach et al: JAMA 307:2418, 2012. number needed to screen (NNTS) to prevent one lung cancer death was calculated to be 320.

1	LDCT screening for lung cancer comes with known risks including a high rate of false-positive results, false-negative results, potential for unnecessary follow-up testing, radiation exposure, overdiagnosis, changes in anxiety and quality of life, and substantial financial costs. By far the biggest challenge confronting the use of CT screening is the high false-positive rate. False positives can have a substantial impact on patients through the expense and risk of unneeded further evaluation and emotional stress. The management of these patients usually consists of serial CT scans over time to see if the nodules grow, attempted fine-needle aspirates, or surgical resection. At $300 per scan (NCI estimated cost), the outlay for initial LDCT alone could run into the billions of dollars annually, an expense that only further escalates when factoring in various downstream expenditures an individual might incur in the assessment of positive findings. A formal cost-effectiveness analysis of

1	an expense that only further escalates when factoring in various downstream expenditures an individual might incur in the assessment of positive findings. A formal cost-effectiveness analysis of the NLST is expected soon that should help resolve this crucial concern.

1	Despite the aforementioned caveats, screening of individuals who meet the NLST criteria for lung cancer risk (or in some cases, modified versions of these criteria) seems warranted, provided comprehensive multidisciplinary coordinated care and follow-up similar to those provided to NLST participants are available. Algorithms to improve candidate selection are under development. When discussing the option of LDCT screening, use of absolute risks rather than relative risks is helpful because studies indicate the public can process absolute terminology more effectively than relative risk projections. A useful guide has been developed by the NCI to help patients and physicians assess the benefits and harms of LDCT screening for lung cancer (Table 107-3). Finally, even a small negative effect of screening on smoking behavior (either lower quit rates or higher recidivism) could easily offset the potential gains in a population. Fortunately no such impact has been reported to date.

1	effect of screening on smoking behavior (either lower quit rates or higher recidivism) could easily offset the potential gains in a population. Fortunately no such impact has been reported to date. Nonetheless, smoking cessation must be included as an indispensable component of any screening program.

1	Benefits: How Did CT Scans Help Compared To CXR? Harms: What Problems Did CT Scans Cause Compared to CXR? Abbreviations: CXR, chest x-ray; LDCT, low-dose computed tomography; NLST, National Lung Screening Trial. Source: Modified from S Woloshin et al: N Engl J Med 367:1677, 2012.

1	Over half of all patients diagnosed with lung cancer present with locally advanced or metastatic disease at the time of diagnosis. The majority of patients present with signs, symptoms, or laboratory abnormalities that can be attributed to the primary lesion, local tumor growth, invasion or obstruction of adjacent structures, growth at distant metastatic sites, or a paraneoplastic syndrome (Tables 107-4 and 107-5). The prototypical lung cancer patient is a current or former smoker of either sex, usually in the seventh decade of life. A history of chronic cough with or without hemoptysis in a current or former smoker with chronic obstructive pulmonary disease (COPD) age 40 years or older should prompt a thorough investigation for lung cancer even in the face of a normal CXR. A persistent pneumonia without constitutional symptoms and unresponsive to repeated courses of antibiotics also should prompt an evaluation for the underlying cause. Lung cancer arising in a lifetime never smoker

1	pneumonia without constitutional symptoms and unresponsive to repeated courses of antibiotics also should prompt an evaluation for the underlying cause. Lung cancer arising in a lifetime never smoker is more common in women and East Asians. Such patients also tend to be younger than their smoking counterparts at the time of diagnosis. The clinical presentation of lung cancer in never smokers tends to mirror that of current and former smokers.

1	Patients with central or endobronchial growth of the primary tumor may present with cough, hemoptysis, wheeze, stridor, dyspnea, or postobstructive pneumonitis. Peripheral growth of the primary tumor may cause pain from pleural or chest wall involvement, dyspnea on a restrictive basis, and symptoms of a lung abscess resulting from tumor cavitation. Regional spread of tumor in the thorax (by contiguous growth or by metastasis to regional lymph nodes) may cause tracheal obstruction, esophageal compression with dysphagia, recurrent laryngeal paralysis with hoarseness, phrenic nerve palsy with elevation of the hemidiaphragm and dyspnea, and sympathetic nerve paralysis with Horner’s syndrome (enophthalmos, ptosis, miosis, and anhydrosis). Malignant pleural effusions can cause pain, dyspnea, or cough. Pancoast (or superior sulcus tumor) syndromes result from local extension of a tumor growing in the apex of the lung with involvement of the eighth cervical and first and second thoracic

1	or cough. Pancoast (or superior sulcus tumor) syndromes result from local extension of a tumor growing in the apex of the lung with involvement of the eighth cervical and first and second thoracic nerves, and present with shoulder pain that characteristically radiates in the ulnar

1	Symptom and Signs Range of Frequency Source: Reproduced with permission from MA Beckles: Chest 123:97-104, 2003. Symptoms elicited in • Constitutional: weight loss >10 lb history • Musculoskeletal: • Neurologic: headaches, syncope, seizures, extremity weakness, recent change in mental status • Hoarseness, neurologic signs, papilledema Routine laboratory tests • Hematocrit, <40% in men; <35% in women • Elevated alkaline phosphatase, GGT, SGOT, and Abbreviations: GGT, gamma-glutamyltransferase; SGOT, serum glutamic-oxaloacetic transaminase. Source: Reproduced with permission from GA Silvestri et al: Chest 123(1 Suppl):147S, 2003.

1	distribution of the arm, often with radiologic destruction of the first and second ribs. Often Horner’s syndrome and Pancoast syndrome coexist. Other problems of regional spread include superior vena cava syndrome from vascular obstruction; pericardial and cardiac extension with resultant tamponade, arrhythmia, or cardiac failure; lymphatic obstruction with resultant pleural effusion; and lymphangitic spread through the lungs with hypoxemia and dyspnea. In addition, lung cancer can spread transbronchially, producing tumor growth along multiple alveolar surfaces with impairment of gas exchange, respiratory insufficiency, dyspnea, hypoxemia, and sputum production. Constitutional symptoms may include anorexia, weight loss, weakness, fever, and night sweats. Apart from the brevity of symptom duration, these parameters fail to clearly distinguish SCLC from NSCLC or even from neoplasms metastatic to lungs.

1	Extrathoracic metastatic disease is found at autopsy in more than 50% of patients with squamous carcinoma, 80% of patients with adenocarcinoma and large-cell carcinoma, and greater than 95% of patients with SCLC. Approximately one-third of patients present with symptoms as a result of distant metastases. Lung cancer metastases may occur in virtually every organ system, and the site of metastatic involvement largely determines other symptoms. Patients with brain metastases may present with headache, nausea and vomiting, seizures, or neurologic deficits. Patients with bone metastases may present with pain, pathologic fractures, or cord compression. The latter may also occur with epidural metastases. Individuals with bone marrow invasion may present with cytopenias or leukoerythroblastosis. Those with liver metastases may present with hepatomegaly, right upper quadrant pain, fever, anorexia, and weight loss. Liver dysfunction and biliary obstructions are rare. Adrenal metastases are

1	Those with liver metastases may present with hepatomegaly, right upper quadrant pain, fever, anorexia, and weight loss. Liver dysfunction and biliary obstructions are rare. Adrenal metastases are common but rarely cause pain or adrenal insufficiency unless they are large.

1	Paraneoplastic syndromes are common in patients with lung cancer, especially those with SCLC, and may be the presenting finding or the first sign of recurrence. In addition, paraneoplastic syndromes may mimic metastatic disease and, unless detected, lead to inappropriate palliative rather than curative treatment. Often the paraneoplastic syndrome may be relieved with successful treatment of the tumor. In some cases, the pathophysiology of the paraneoplastic syndrome is known, particularly when a hormone with biological activity is secreted by a tumor. However, in many cases, the pathophysiology is unknown. Systemic symptoms of anorexia, cachexia, weight loss (seen in 30% of patients), fever, and suppressed immunity are paraneoplastic syndromes of unknown etiology or at least not well defined. Weight loss greater than 10% of total body weight is considered a bad prognostic sign. Endocrine syndromes are seen in 12% of patients; hypercalcemia resulting from ectopic production of

1	defined. Weight loss greater than 10% of total body weight is considered a bad prognostic sign. Endocrine syndromes are seen in 12% of patients; hypercalcemia resulting from ectopic production of parathyroid hormone (PTH), or more commonly, PTH-related peptide, is the most common life-threatening metabolic complication of malignancy, primarily occurring with squamous cell carcinomas of the lung. Clinical symptoms include nausea, vomiting, abdominal pain, constipation, polyuria, 511 thirst, and altered mental status.

1	Hyponatremia may be caused by the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) or possibly atrial natriuretic peptide (ANP). SIADH resolves within 1-4 weeks of initiating chemotherapy in the vast majority of cases. During this period, serum sodium can usually be managed and maintained above 128 mEq/L via fluid restriction. Demeclocycline can be a useful adjunctive measure when fluid restriction alone is insufficient. Vasopressin receptor antagonists like tolvaptan also have been used in the management of SIADH. However, there are significant limitations to the use of tolvaptan including liver injury and overly rapid correction of the hyponatremia, which can lead to irreversible neurologic injury. Likewise, the cost of tolvaptan may be prohibitive (as high as $300 per tablet in some areas). Of note, patients with ectopic ANP may have worsening hyponatremia if sodium intake is not concomitantly increased. Accordingly, if hyponatremia fails to improve or worsens

1	per tablet in some areas). Of note, patients with ectopic ANP may have worsening hyponatremia if sodium intake is not concomitantly increased. Accordingly, if hyponatremia fails to improve or worsens after 3–4 days of adequate fluid restriction, plasma levels of ANP should be measured to determine the causative syndrome.

1	Ectopic secretion of ACTH by SCLC and pulmonary carcinoids usually results in additional electrolyte disturbances, especially hypokalemia, rather than the changes in body habitus that occur in Cushing’s syndrome from a pituitary adenoma. Treatment with standard medications, such as metyrapone and ketoconazole, is largely ineffective due to extremely high cortisol levels. The most effective strategy for management of the Cushing’s syndrome is effective treatment of the underlying SCLC. Bilateral adrenalectomy may be consid ered in extreme cases.

1	Skeletal–connective tissue syndromes include clubbing in 30% of cases (usually NSCLCs) and hypertrophic primary osteoarthropathy in 1–10% of cases (usually adenocarcinomas). Patients may develop periostitis, causing pain, tenderness, and swelling over the affected bones and a positive bone scan. Neurologic-myopathic syndromes are seen in only 1% of patients but are dramatic and include the myasthenic Eaton-Lambert syndrome and retinal blindness with SCLC, whereas peripheral neuropathies, subacute cerebellar degeneration, cortical degeneration, and polymyositis are seen with all lung cancer types. Many of these are caused by autoimmune responses such as the development of anti-voltage-gated calcium channel antibodies in Eaton-Lambert syndrome. Patients with this disorder present with proximal muscle weakness, usually in the lower extremities, occasional autonomic dysfunction, and rarely, cranial nerve symptoms or involvement of the bulbar or respiratory muscles. Depressed deep tendon

1	muscle weakness, usually in the lower extremities, occasional autonomic dysfunction, and rarely, cranial nerve symptoms or involvement of the bulbar or respiratory muscles. Depressed deep tendon reflexes are frequently present. In contrast to patients with myasthenia gravis, strength improves with serial effort. Some patients who respond to chemotherapy will have resolution of the neurologic abnormalities. Thus, chemotherapy is the initial treatment of choice. Paraneoplastic encephalomyelitis and sensory neuropathies, cerebellar degeneration, limbic encephalitis, and brainstem encephalitis occur in SCLC in association with a variety of antineuronal antibodies such as anti-Hu, antiCRMP5, and ANNA-3. Paraneoplastic cerebellar degeneration may be associated with anti-Hu, anti-Yo, or P/Q calcium channel autoantibodies. Coagulation or thrombotic or other hematologic manifestations occur in 1–8% of patients and include migratory venous thrombophlebitis (Trousseau’s syndrome), nonbacterial

1	channel autoantibodies. Coagulation or thrombotic or other hematologic manifestations occur in 1–8% of patients and include migratory venous thrombophlebitis (Trousseau’s syndrome), nonbacterial thrombotic (marantic) endocarditis with arterial emboli, and disseminated intravascular coagulation with hemorrhage, anemia, granulocytosis, and leukoerythroblastosis. Thrombotic disease complicating cancer is usually a poor prognostic sign. Cutaneous manifestations such as dermatomyositis and acanthosis nigricans are uncommon (1%), as are the renal manifestations of nephrotic syndrome and glomerulonephritis (≤1%).

1	Tissue sampling is required to confirm a diagnosis in all patients with suspected lung cancer. In patients with suspected metastatic disease, a biopsy of the most distant site of disease is preferred for tissue confirmation. Given the greater emphasis placed on molecular testing for NSCLC patients, a core biopsy is preferred to ensure adequate

1	Neoplasms of the Lung 512 tissue for analysis. Tumor tissue may be obtained via minimally invasive techniques such as bronchial or transbronchial biopsy during fiberoptic bronchoscopy, by fine-needle aspiration or percutaneous biopsy using image guidance, or via endobronchial ultrasound (EBUS) guided biopsy. Depending on the location, lymph node sampling may occur via transesophageal endoscopic ultrasound-guided biopsy (EUS), EBUS, or blind biopsy. In patients with clinically palpable disease such as a lymph node or skin metastasis, a biopsy may be obtained. In patients with suspected metastatic disease, a diagnosis may be confirmed by percutaneous biopsy of a soft tissue mass, lytic bone lesion, bone marrow, pleural or liver lesion, or an adequate cell block obtained from a malignant pleural effusion. In patients with a suspected malignant pleural effusion, if the initial thoracentesis is negative, a repeat thoracentesis is warranted. Although the majority of pleural effusions are

1	effusion. In patients with a suspected malignant pleural effusion, if the initial thoracentesis is negative, a repeat thoracentesis is warranted. Although the majority of pleural effusions are due to malignant disease, particularly if they are exudative or bloody, some may be parapneumonic. In the absence of distant disease, such patients should be considered for possible curative treatment. The diagnostic yield of any biopsy depends on several factors including location (accessibility) of the tumor, tumor size, tumor type, and technical aspects of the diagnostic procedure including the experience level of the bronchoscopist and pathologist. In general, central lesions such as squamous cell carcinomas, small-cell carcinomas, or endobronchial lesions such as carcinoid tumors are more readily diagnosed by bronchoscopic examination, whereas peripheral lesions such as adenocarcinomas and large-cell carcinomas are more amenable to transthoracic biopsy. Diagnostic accuracy for SCLC versus

1	diagnosed by bronchoscopic examination, whereas peripheral lesions such as adenocarcinomas and large-cell carcinomas are more amenable to transthoracic biopsy. Diagnostic accuracy for SCLC versus NSCLC for most specimens is excellent, with lesser accuracy for subtypes of NSCLC. Bronchoscopic specimens include bronchial brush, bronchial wash, bronchioloalveolar lavage, transbronchial fine-needle aspiration (FNA), and core biopsy. For more accurate histologic classification, mutation analysis, or investigational purposes, reasonable efforts (e.g., a core needle biopsy) should be made to obtain more tissue than what is contained in a routine cytology specimen obtained by FNA. Overall sensitivity for combined use of bronchoscopic methods is approximately 80%, and together with tissue biopsy, the yield increases to 85–90%. Like transbronchial core biopsy specimens, transthoracic core biopsy specimens are also preferred. Sensitivity is highest for larger lesions and peripheral tumors. In

1	the yield increases to 85–90%. Like transbronchial core biopsy specimens, transthoracic core biopsy specimens are also preferred. Sensitivity is highest for larger lesions and peripheral tumors. In general, core biopsy specimens, whether transbronchial, transthoracic, or EUS-guided, are superior to other specimen types. This is primarily due to the higher percentage of tumor cells with fewer confounding factors such as obscuring inflammation and reactive nonneoplastic cells.

1	Sputum cytology is inexpensive and noninvasive but has a lower yield than other specimen types due to poor preservation of the cells and more variability in acquiring a good-quality specimen. The yield for sputum cytology is highest for larger and centrally located tumors such as squamous cell carcinoma and small-cell carcinoma histology. The specificity for sputum cytology averages close to 100%, although sensitivity is generally <70%. The accuracy of sputum cytology improves with increased numbers of specimens analyzed. Consequently, analysis of at least three sputum specimens is recommended.

1	Lung cancer staging consists of two parts: first, a determination of the location of the tumor and possible metastatic sites (anatomic staging), and second, an assessment of a patient’s ability to withstand various antitumor treatments (physiologic staging). All patients with lung cancer should have a complete history and physical examination, with evaluation of all other medical problems, determination of performance status, and history of weight loss. The most significant dividing line is between those patients who are candidates for surgical resection and those who are inoperable but will benefit from chemotherapy, radiation therapy, or both. Staging with regard to a patient’s potential for surgical resection is principally applicable to NSCLC.

1	The accurate staging of patients with NSCLC is essential for determining the appropriate treatment in patients with resectable disease and avoiding unnecessary surgical procedures in patients with advanced disease (Fig. 107-3). All patients with NSCLC should undergo initial Complete history and physical examination Determination of performance status and weight loss Complete blood count with platelet determination Measurement of serum electrolytes, glucose, and calcium; renal and liver function tests PET scan to evaluate mediastinum and detect metastatic disease MRI brain if clinically indicated

1	Positive for metastatic disease No signs, symptoms, or imaging to suggest metastatic disease Patient has no contraindication to surgery or radiation therapy combined with chemotherapy Refer to surgeon for evaluation of mediastinum and possible resection No surgery Treatment with combined chemoradiation therapy N0 or N1 nodes N2 or N3 nodes Single suspicious lesion detected on imaging Multiple lesions detected on imaging Biopsy lesion See Fig. 107-6 Pulmonary function tests and arterial blood-gas measurements Cardiopulmonary exercise testing if performance status or pulmonary function tests are borderline Coagulation tests Negative for metastatic disease Stage IB <4 cm surgery alone >4 cm surgery followed by adjuvant chemotherapy Stage II or III Surgery followed by adjuvant chemotherapy Stage IA Surgery alone FIGURE 107-3 Algorithm for management of non-small-cell lung cancer. MRI, magnetic resonance imaging; PET, positron emission tomography.

1	radiographic imaging with CT scan, positron emission tomography (PET), or preferably CT-PET. PET scanning attempts to identify sites of malignancy based on glucose metabolism by measuring the uptake of 18F-fluorodeoxyglucose (FDG). Rapidly dividing cells, presumably in the lung tumors, will preferentially take up 18F-FDG and appear as a “hot spot.” To date, PET has been mostly used for staging and detection of metastases in lung cancer and in the detection of nodules >15 mm in diameter. Combined 18F-FDG PET-CT imaging has been shown to improve the accuracy of staging in NSCLC compared to visual correlation of PET and CT or either study alone. CT-PET has been found to be superior in identifying pathologically enlarged mediastinal lymph nodes and extrathoracic metastases. A standardized uptake value (SUV) of >2.5 on PET is highly suspicious for malignancy. False negatives can be seen in diabetes, in lesions <8 mm, and in slow-growing tumors (e.g., carcinoid tumors or well-differentiated

1	value (SUV) of >2.5 on PET is highly suspicious for malignancy. False negatives can be seen in diabetes, in lesions <8 mm, and in slow-growing tumors (e.g., carcinoid tumors or well-differentiated adenocarcinoma). False positives can be seen in certain infections and granulomatous disease (e.g., tuberculosis). Thus, PET should never be used alone to diagnose lung cancer, mediastinal involvement, or metastases. Confirmation with tissue biopsy is required. For brain metastases, magnetic resonance imaging (MRI) is the most effective method. MRI can also be useful in selected circumstances, such as superior sulcus tumors to rule out brachial plexus involvement, but in general, MRI does not play a major role in NSCLC staging.

1	In patients with NSCLC, the following are contraindications to potential curative resection: extrathoracic metastases, superior vena cava syndrome, vocal cord and, in most cases, phrenic nerve paralysis, malignant pleural effusion, cardiac tamponade, tumor within 2 cm of the carina (potentially curable with combined chemoradiotherapy), metastasis to the contralateral lung, metastases to supraclavicular lymph nodes, contralateral mediastinal node metastases (potentially curable with combined chemoradiotherapy), and involvement of the main pulmonary artery. In situations where it will make a difference in treatment, abnormal scan findings require tissue confirmation of malignancy so that patients are not precluded from having potentially curative therapy.

1	The best predictor of metastatic disease remains a careful history and physical examination. If signs, symptoms, or findings from the physical examination suggest the presence of malignancy, then sequential imaging starting with the most appropriate study should be performed. If the findings from the clinical evaluation are negative, then imaging studies beyond CT-PET are unnecessary and the search for metastatic disease is complete. More controversial is how one should assess patients with known stage III disease. Because these patients are more likely to have asymptomatic occult metastatic disease, current guidelines recommend a more extensive imaging evaluation including imaging of the brain with either CT scan or MRI. In patients in whom distant metastatic disease has been ruled out, lymph node status needs to be assessed via a combination of radiographic imaging and/or minimally invasive techniques such as those mentioned above and/or invasive techniques such as mediastinoscopy,

1	lymph node status needs to be assessed via a combination of radiographic imaging and/or minimally invasive techniques such as those mentioned above and/or invasive techniques such as mediastinoscopy, mediastinotomy, thoracoscopy, or thoracotomy. Approximately one-quarter to one-half of patients diagnosed with NSCLC will have mediastinal lymph node metastases at the time of diagnosis. Lymph node sampling is recommended in all patients with enlarged nodes detected by CT or PET scan and in patients with large tumors or tumors occupying the inner third of the lung. The extent of mediastinal lymph node involvement is important in determining the appropriate treatment strategy: surgical resection followed by adjuvant chemotherapy versus combined chemoradiation alone (see below). A standard nomenclature for referring to the location of lymph nodes involved with lung cancer has evolved (Fig. 107-4).

1	In SCLC patients, current staging recommendations include a CT scan of the chest and abdomen (because of the high frequency of hepatic and adrenal involvement), MRI of the brain (positive in 10% of asymptomatic patients), and radionuclide bone scan if symptoms or signs suggest disease involvement in these areas (Fig. 107-5). Although there are less data on the use of CT-PET in SCLC, the most recent American College of Chest Physicians Evidence-Based Clinical Practice Guidelines recommend PET scans in patients with clinical stage I SCLC who are being considered for curative intent surgical 513 resection. In addition, invasive mediastinal staging and extrathoracic imaging (head MRI/CT and PET or abdominal CT plus bone scan) is also recommended for patients with clinical stage I SCLC if curative intent surgical resection is contemplated. Some practice guidelines also recommend the use of PET scanning in the staging of SCLC patients who are potential candidates for the addition of

1	if curative intent surgical resection is contemplated. Some practice guidelines also recommend the use of PET scanning in the staging of SCLC patients who are potential candidates for the addition of thoracic radiotherapy to chemotherapy. Bone marrow biopsies and aspirations are rarely performed now given the low incidence of isolated bone marrow metastases. Confirmation of metastatic disease, ipsilateral or contra-lateral lung nodules, or metastases beyond the mediastinum may be achieved by the same modalities recommended earlier for patients with NSCLC.

1	If a patient has signs or symptoms of spinal cord compression (pain, weakness, paralysis, urinary retention), a spinal CT or MRI scan and examination of the cerebrospinal fluid cytology should be performed. If metastases are evident on imaging, a neurosurgeon should be consulted for possible palliative surgical resection and/or a radiation oncologist should be consulted for palliative radiotherapy to the site of compression. If signs or symptoms of leptomeningitis develop at any time in a patient with lung cancer, an MRI of the brain and spinal cord should be performed, as well as a spinal tap, for detection of malignant cells. If the spinal tap is negative, a repeat spinal tap should be considered. There is currently no approved therapy for the treatment of leptomeningeal disease.

1	The tumor-node-metastasis (TNM) international staging system provides useful prognostic information and is used to stage all patients with NSCLC. The various T (tumor size), N (regional node involvement), and M (presence or absence of distant metastasis) are combined to form different stage groups (Tables 107-6 and 107-7). The previous edition of the TNM staging system for lung cancer was developed based on a relatively small database of patients from a single institution. The latest seventh edition of the TNM staging system went into effect in 2010 and developed using a much more robust database of more than 100,000 patients with lung cancer who were treated in multiple countries between 1990 and 2000. Data from 67,725 patients with NSCLC were then used to reevaluate the prognostic value of the TNM descriptors (Table 107-8). The major distinction between the sixth and seventh editions of the international staging systems is within the T classification; T1 tumors are divided into

1	of the TNM descriptors (Table 107-8). The major distinction between the sixth and seventh editions of the international staging systems is within the T classification; T1 tumors are divided into tumors ≤2 cm in size, as these patients were found to have a better prognosis compared to patients with tumors >2 cm but ≤3 cm. T2 tumors are divided into those that are >3 cm but ≤5 cm and those that are >5 cm but ≤7 cm. Tumors that are >7 cm are considered T3 tumors. T3 tumors also include tumors with invasion into local structures such as chest wall and diaphragm and additional nodules in the same lobe. T4 tumors include tumors of any size with invasion into mediastinum, heart, great vessels, trachea, or esophagus or multiple nodules in the ipsilateral lung. No changes have been made to the current classification of lymph node involvement (N). Patients with metastasis may be classified as M1a (malignant pleural or pericardial effusion, pleural nodules, or nodules in the contralateral lung)

1	classification of lymph node involvement (N). Patients with metastasis may be classified as M1a (malignant pleural or pericardial effusion, pleural nodules, or nodules in the contralateral lung) or M1b (distant metastasis; e.g., bone, liver, adrenal, or brain metastasis). Based on these data, approximately one-third of patients have localized disease that can be treated with curative attempt (surgery or radiotherapy), one-third have local or regional disease that may or may not be amenable to a curative attempt, and one-third have metastatic disease at the time of diagnosis.

1	In patients with SCLC, it is now recommended that both the Veterans Administration system and the American Joint Committee on Cancer/ International Union Against Cancer seventh edition system (TNM) be used to classify the tumor stage. The Veterans Administration system is a distinct two-stage system dividing patients into those with limitedor extensive-stage disease. Patients with limited-stage disease (LD) have cancer that is confined to the ipsilateral hemithorax and can be encompassed within a tolerable radiation port. Thus, contralateral Neoplasms of the Lung Brachiocephalic (innominate) a. Azygos v. 12, 13, 14R 11L 10L 9 8 12, 13, 14L 7 Inf.pulm. ligt. L.pulmonary a. 3 6 5 Ao PA Phrenic n. N2 = single digit, ipsilateral N3 = single digit, contralateral or supraclavicular

1	Azygos v. 12, 13, 14R 11L 10L 9 8 12, 13, 14L 7 Inf.pulm. ligt. L.pulmonary a. 3 6 5 Ao PA Phrenic n. N2 = single digit, ipsilateral N3 = single digit, contralateral or supraclavicular FIGURE 107-4 Lymph node stations in staging non-small-cell lung cancer. The International Association for the Study of Lung Cancer (IASLC) lymph node map, including the proposed grouping of lymph node stations into “zones” for the purposes of prognostic analyses. a., artery; Ao, aorta; Inf. pulm. ligt., inferior pulmonary ligament; n., nerve; PA, pulmonary artery; v., vein.

1	supraclavicular nodes, recurrent laryngeal nerve involvement, and superior vena caval obstruction can all be part of LD. Patients with extensive-stage disease (ED) have overt metastatic disease by imaging or physical examination. Cardiac tamponade, malignant pleural effusion, and bilateral pulmonary parenchymal involvement generally qualify disease as ED, because the involved organs cannot be encompassed safely or effectively within a single radiation therapy port. Sixty to 70% of patients are diagnosed with ED at presentation. The TNM staging system is preferred in the rare SCLC patient presenting with what appears to be clinical stage I disease (see above).

1	Patients with lung cancer often have other comorbid conditions related to smoking including cardiovascular disease and COPD. To improve their preoperative condition, correctable problems (e.g., anemia, electrolyte and fluid disorders, infections, cardiac disease, and arrhythmias) should be addressed, appropriate chest physical therapy should be instituted, and patients should be encouraged to stop smoking. Because it is not always possible to predict whether a lobectomy or pneumonectomy will be required until the time of operation, a conservative approach is to restrict surgical resection to patients who could potentially tolerate a pneumonectomy. Patients with a forced expiratory volume in 1 s (FEV1) of greater than 2 L or greater than 80% of predicted can tolerate a pneumonectomy, and those with an FEV1 greater than 1.5 L have adequate reserve for a lobectomy. In patients with borderline lung function but a resectable tumor, cardiopulmonary exercise testing could be performed as

1	those with an FEV1 greater than 1.5 L have adequate reserve for a lobectomy. In patients with borderline lung function but a resectable tumor, cardiopulmonary exercise testing could be performed as part of the physiologic evaluation. This test allows an estimate of the maximal oxygen consumption (Vo ). A Vo <15 mL/(kg⋅min) predicts for a higher risk of postoperative complications. Patients deemed unable to tolerate lobectomy or pneumonectomy from a pulmonary functional standpoint may be candidates for more limited resections, such as wedge or anatomic segmental resection, although such procedures are associated with significantly higher rates of local

1	Complete history and physical examination Determination of performance status and weight loss Complete blood count with platelet determination Measurement of serum electrolytes, glucose, and calcium; renal and liver function tests CT scan of chest abdomen and pelvis to evaluate for metastatic disease MRI of brain Bone scan if clinically indicated

1	No signs, symptoms, or imaging to suggest metastatic disease Single lesion detected on imaging (For clinical stage I SCLC see “Anatomic Staging of Patients with Lung Cancer”) Multiple lesions detected on imaging Chemotherapy alone and/or radiation therapy for palliation of symptoms Patient has no contraindication to combined chemotherapy and radiation therapy Combined modality treatment with platinum-based therapy and etoposide and radiation therapy Sequential treatment with chemotherapy and radiation therapy Patient has contraindication to combined chemotherapy and radiation therapy Negative for metastatic disease Positive for metastatic disease Biopsy lesion Note: Regardless of disease stage, patients who have a good response to initial therapy should be considered for prophylactic cranial irradiation after therapy is completed. FIGURE 107-5 Algorithm for management of small-cell lung cancer. CT, computed tomography; MRI, magnetic resonance imaging.

1	FIGURE 107-5 Algorithm for management of small-cell lung cancer. CT, computed tomography; MRI, magnetic resonance imaging. Neoplasms of the Lung recurrence and a trend toward decreased overall survival. All patients should be assessed for cardiovascular risk using American College of Cardiology and American Heart Association guidelines. A myocardial infarction within the past 3 months is a contraindication to thoracic surgery because 20% of patients will die of reinfarction. An infarction in the past 6 months is a relative contraindication. Other major contraindications include uncontrolled arrhythmias, an FEV1 of less than 1 L, CO2 retention (resting Pco2 >45 mmHg), DLco <40%, and severe pulmonary hypertension. The overall treatment approach to patients with NSCLC is shown in Fig. 107-3.

1	The overall treatment approach to patients with NSCLC is shown in Fig. 107-3. Patients with severe atypia on sputum cytology have an increased risk of developing lung cancer compared to those without atypia. In the uncommon circumstance where malignant cells are identified in a sputum or bronchial washing specimen but the chest imaging appears normal (TX tumor stage), the lesion must be localized. More than 90% of tumors can be localized by meticulous examination of the bronchial tree with a fiberoptic bronchoscope under general anesthesia and collection of a series of differential brushings and biopsies. Surgical resection following bronchoscopic localization has been shown to improve survival compared to no treatment. Close follow-up of these patients is indicated because of the high incidence of second primary lung cancers (5% per patient per year).

1	A solitary pulmonary nodule is defined as an x-ray density completely surrounded by normal aerated lung with circumscribed margins, of any shape, usually 1–6 cm in greatest diameter. The approach to a patient with a solitary pulmonary nodule is based on an estimate of the probability of cancer, determined according to the patient’s smoking history, age, and characteristics on imaging (Table 107-9). Prior CXRs and CT scans should be obtained if available for comparison. A PET scan may be useful if the lesion is greater than 7–8 mm in diameter. If no diagnosis is apparent, Mayo investigators reported that clinical characteristics (age, cigarette smoking status, and prior cancer diagnosis) and three radiologic characteristics (nodule diameter, spiculation, and upper lobe location) were independent predictors of malignancy. At present, only two radiographic criteria are thought to predict the benign nature of a solitary pulmonary nodule: lack of growth over a period >2 years and certain

1	predictors of malignancy. At present, only two radiographic criteria are thought to predict the benign nature of a solitary pulmonary nodule: lack of growth over a period >2 years and certain characteristic patterns of calcification. Calcification alone, however, does not exclude malignancy; a dense central nidus, multiple punctuate foci, and “bulls eye” (granuloma) and “popcorn ball” (hamartoma) calcifications are highly suggestive of a benign lesion. In contrast, a relatively large lesion, lack of or asymmetric calcification, chest symptoms, associated atelectasis, pneumonitis, or growth of the lesion revealed by comparison with an old x-ray or CT scan or a positive PET scan may be suggestive of a malignant process and warrant further attempts to establish a histologic diagnosis. An algorithm for assessing these lesions is shown in Fig. 107-6.

1	Since the advent of screening CTs, small “ground-glass” opacities (GGOs) have often been observed, particularly as the increased sensitivity of CTs enables detection of smaller lesions. Many of these GGOs, when biopsied, are found to be atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), or minimally invasive adenocarcinoma (MIA). AAH is usually a nodule of <5 mm and is minimally hazy, also called nonsolid or ground glass (i.e., hazy slightly increased attenuation, no solid component, and preservation of bronchial and vascular margins). On thin-section CT, AIS is T1 Tumor ≤3 cm diameter, surrounded by lung or visceral pleura, without invasion more proximal than lobar bronchus T1a Tumor ≤2 cm in diameter T1b Tumor >2 cm but ≤ 3 cm in diameter T2 Tumor >3 cm but ≤7 cm, or tumor with any of the following features: Involves main bronchus, ≥2 cm distal to carina

1	Invades visceral pleura Associated with atelectasis or obstructive pneumonitis that extends to the hilar region but does not involve the entire lung T2a Tumor >3 cm but ≤5 cm T2b Tumor >5 cm but ≤7 cm T3 Tumor >7 cm or any of the following: Directly invades any of the following: chest wall, diaphragm, phrenic nerve, mediastinal pleura, parietal pericardium, main bronchus <2 cm from carina (without involvement of carina) Atelectasis or obstructive pneumonitis of the entire lung Separate tumor nodules in the same lobe T4 Tumor of any size that invades the mediastinum, heart, great vessels, trachea, recurrent laryngeal nerve, esophagus, vertebral body, carina, or with separate tumor nodules in a different ipsilateral lobe M0 No distant metastasis M1 Distant metastasis M1a Separate tumor nodule(s) in a contralateral lobe; tumor with pleural nodules or malignant pleural or pericardial effusion M1b Distant metastasis (in extrathoracic organs) Abbreviation: TNM, tumor-node-metastasis.

1	Source: Reproduced with permission from P Goldstraw et al: J Thorac Oncol 2:706, 2007. Stage IIA T1a,T1b,T2a N1 M0 Stage IIIA T1a,T1b,T2a,T2b N2 M0 T3 N1,N2 M0 T4 N0,N1 M0 Stage IIIB T4 N2 M0 Any T N3 M0 Stage IV Any T Any N M1a or M1b Abbreviation: TNM, tumor-node-metastasis. Source: Reproduced with permission from P Goldstraw P et al: J Thorac Oncol 2:706, 2007. Abbreviation: TNM, tumor-node-metastasis. usually a nonsolid nodule and tends to be slightly more opaque than AAH. MIA is mainly solid, usually with a small (<5 mm) central solid component. However, overlap exists among the imaging features of the preinvasive and minimally invasive lesions in the lung adenocarcinoma spectrum. Lepidic adenocarcinomas are usually solid but may be nonsolid. Likewise, the small invasive adenocarcinomas also are usually solid but may exhibit a small nonsolid component.

1	MANAGEMENT OF STAGES I AND II NSCLC Surgical Resection of Stage I and II NSCLC Surgical resection, ideally by an experienced thoracic surgeon, is the treatment of choice for patients with clinical stage I and II NSCLC who are able to tolerate the procedure. Operative mortality rates for patients resected by thoracic or cardiothoracic surgeons are lower compared to general surgeons. Moreover, survival rates are higher in patients who undergo resection in facilities with a high surgical volume compared to those performing fewer than 70 procedures per year, even though the higher-volume facilities often serve older and less socioeconomic advantaged populations. The improvement in survival is most evident in the immediate postoperative period. The extent of resection is a matter of surgical judgment based on findings at exploration. In patients with stage IA NSCLC, lobectomy is superior to wedge resection with respect to rates of local recurrence. There is also a trend toward improvement

1	based on findings at exploration. In patients with stage IA NSCLC, lobectomy is superior to wedge resection with respect to rates of local recurrence. There is also a trend toward improvement in overall survival. In patients with comorbidities, compromised pulmonary reserve, and small peripheral lesions, a limited resection, wedge resection, and segmentectomy (potentially by video-assisted thoracoscopic surgery) may be reasonable surgical option. Pneumonectomy is reserved for patients with central tumors and should be performed only in margins Source: Reproduced with permission from D Ost et al: N Engl J Med 348:2535, 2003.

1	SPN detected on CXR Obtain old films. Assess stability over ˜2 years or benign pattern of calcification Step 1 Solid SPN Negative Positive PET or PET-CT Obtain tissue diagnosis. (TTNA, TBBx, surgical resection)*** Serial CT chest** Neoplasms of the Lung FIGURE 107-6 A. Algorithm for evaluation of solitary pulmonary nodule (SPN). B. Algorithm for evaluation of solid SPN. C. Algorithm for evaluation of semisolid SPN. CT, computed tomography; CXR, chest radiograph; GGN, ground-glass nodule; PET, positron emission tomography; TTBx, transbronchial biopsy; TTNA, transthoracic needle biopsy. (Adapted from VK Patel et al: Chest 143:840, 2013.) No follow-up required Follow-up thin-section CT in 3 months. If nodule is unchanged, consider yearly low-dose CT scans.* If there is change in size or nodule characteristics, surgical resection should be strongly considered *Frequency and duration of follow-up CT scans have yet to be definitively established.

1	If there is change in size or nodule characteristics, surgical resection should be strongly considered *Frequency and duration of follow-up CT scans have yet to be definitively established. patients with excellent pulmonary reserve. The 5-year survival rates are 60–80% for patients with stage I NSCLC and 40–50% for patients with stage II NSCLC.

1	Accurate pathologic staging requires adequate segmental, hilar, and mediastinal lymph node sampling. Ideally this includes a mediastinal lymph node dissection. On the right side, mediastinal stations 2R, 4R, 7, 8R, and 9R should be dissected; on the left side, stations 5, 6, 7, 8L, and 9L should be dissected. Hilar lymph nodes are typically resected and sent for pathologic review, although it is helpful to specifically dissect and label level 10 lymph nodes when possible. On the left side, level 2 and sometimes level 4 lymph nodes are generally obscured by the aorta. Although the therapeutic benefit of nodal dissection versus nodal sampling is controversial, a pooled analysis of three trials involving patients with stages I to IIIA NSCLC demonstrated a superior 4-year survival in patients undergoing resection and a complete mediastinal lymph node dissection compared with lymph node sampling. Moreover, complete mediastinal lymphadenectomy added little morbidity to a pulmonary resection

1	undergoing resection and a complete mediastinal lymph node dissection compared with lymph node sampling. Moreover, complete mediastinal lymphadenectomy added little morbidity to a pulmonary resection for lung cancer when carried out by an experienced thoracic surgeon.

1	Radiation Therapy in Stages I and II NSCLC There is currently no role for postoperative radiation therapy in patients following resection of stage I or II NSCLC. However, patients with stage I and II disease Follow-up thin-section CT in months. If nodule unchanged and solid component is >8 mm, consider PET-CT. Further recommendations may include surgical resection, nodule biopsy, or serial CT scans. If there is a change in size or nodule characteristics, surgical resection should be strongly considered *Fleischner society guidelines; modified from: H. MacMahon, et al: Radiology 2005; 237;395–400 **ACCP guidelines (see MK Gould et al: Chest 2007;132(suppl 3):108s-130S. ***Consider patient preference, severity of medical comorbidities, center specific expertise prior to tissue diagnosis.

1	who either refuse or are not suitable candidates for surgery should be considered for radiation therapy with curative intent. Stereotactic body radiation therapy (SBRT) is a relatively new technique used to treat patients with isolated pulmonary nodules (≤5 cm) who are not candidates for or refuse surgical resection. Treatment is typically administered in three to five fractions delivered over 1–2 weeks. In uncontrolled studies, disease control rates are >90%, and 5-year survival rates of up to 60% have been reported with SBRT. By comparison, survival rates typically range from 13 to 39% in patients with stage I or II NSCLC treated with standard external-beam radiotherapy. Cryoablation is another technique occasionally used to treat small, isolated tumors (i.e., ≤3 cm). However, very little data exist on long-term outcomes with this technique.

1	Chemotherapy in Stages I and II NSCLC Although a landmark meta-analysis of cisplatin-based adjuvant chemotherapy trials in patients with resected stages I to IIIA NSCLC (the Lung Adjuvant Cisplatin Evaluation [LACE] Study) demonstrated a 5.4% improvement in 5-year survival for adjuvant chemotherapy compared to surgery alone, the survival benefit was seemingly confined to patients with stage II or III disease (Table 107-10). By contrast, survival was actually worsened in stage IA patients with the application of adjuvant therapy. In stage IB, there was a modest improvement in survival of questionable clinical significance. Adjuvant chemotherapy was .03 240 54 407 60 .017 433 58 548 50 .49 540 45 192 60 .90 189 58 173 59 .10 171 57 Abbreviations: ALPI, Adjuvant Lung Cancer Project Italy; ANITA, Adjuvant Navelbine International Trialist Association; BLT, Big Lung Trial; CALGB, Cancer and Lung Cancer Group B; IALT,

1	Abbreviations: ALPI, Adjuvant Lung Cancer Project Italy; ANITA, Adjuvant Navelbine International Trialist Association; BLT, Big Lung Trial; CALGB, Cancer and Lung Cancer Group B; IALT, International Adjuvant Lung Cancer Trial; MVP, mitomycin, vindesine, and cisplatin. also detrimental in patients with poor performance status (Eastern Cooperative Oncology Group [ECOG] performance status = 2). These data suggest that adjuvant chemotherapy is best applied in patients with resected stage II or III NSCLC. There is no apparent role for adjuvant chemotherapy in patients with resected stage IA or IB NSCLC. A possible exception to the prohibition of adjuvant therapy in this setting is the stage IB patient with a resected lesion ≥4 cm.

1	As with any treatment recommendation, the risks and benefits of adjuvant chemotherapy should be considered on an individual patient basis. If a decision is made to proceed with adjuvant chemotherapy, in general, treatment should be initiated 6–12 weeks after surgery, assuming the patient has fully recovered, and should be administered for no more than four cycles. Although a cisplatinbased chemotherapy is the preferred treatment regimen, carboplatin can be substituted for cisplatin in patients who are unlikely to tolerate cisplatin for reasons such as reduced renal function, presence of neuropathy, or hearing impairment. No specific chemotherapy regimen is considered optimal in this setting, although platinum plus vinorelbine is most commonly used.

1	Neoadjuvant chemotherapy, which is the application of chemotherapy administered before an attempted surgical resection, has been advocated by some experts on the assumption that such an approach will more effectively extinguish occult micrometastases compared to postoperative chemotherapy. In addition, it is thought that preoperative chemotherapy might render an inoperable lesion resectable. With the exception of superior sulcus tumors, however, the role of neoadjuvant chemotherapy in stage I to III disease is not well defined. However, a meta-analysis of 15 randomized controlled trials involving more than 2300 patients with stage I to III NSCLC suggested there may be a modest 5-year survival benefit (i.e., ∼5%) that is virtually identical to the survival benefit achieved with postoperative chemotherapy. Accordingly, neoadjuvant therapy may prove useful in selected cases (see below). A decision to use neoadjuvant chemotherapy should always be made in consultation with an experienced

1	chemotherapy. Accordingly, neoadjuvant therapy may prove useful in selected cases (see below). A decision to use neoadjuvant chemotherapy should always be made in consultation with an experienced surgeon.

1	In should be noted that all patients with resected NSCLC are at high risk of recurrence, most of which occurs within 18–24 months of surgery, or developing a second primary lung cancer. Thus, it is reasonable to follow these patients with periodic imaging studies. Given the results of the NLST, periodic CT scans appear to be the most appropriate screening modality. Based on the timing of most recurrences, some guidelines recommend a contrasted chest CT scan every 6 months for the first 3 years after surgery, followed by yearly CT scans of the chest without contrast thereafter.

1	Management of patients with stage III NSCLC usually requires a combined-modality approach. Patients with stage IIIA disease commonly are stratified into those with “nonbulky” or “bulky” mediastinal lymph node (N2) disease. Although the definition of “bulky” N2 disease varies somewhat in the literature, the usual criteria include the size of a dominant lymph node (i.e., >2–3 cm in short-axis diameter as measured by CT), groupings of multiple smaller lymph nodes, evidence of extracapsular nodal involvement, or involvement of more than two lymph node stations. The distinction between nonbulky and bulky stage IIIA disease is mainly used to select potential candidates for upfront surgical resection or for resection after neoadjuvant therapy. Many aspects of therapy of patients with stage III NSCLC remain controversial, and the optimal treatment strategy has not been clearly defined. Moreover, although there are many potential treatment options, none yields a very high probability of cure.

1	remain controversial, and the optimal treatment strategy has not been clearly defined. Moreover, although there are many potential treatment options, none yields a very high probability of cure. Furthermore, because stage III disease is highly heterogeneous, no single treatment approach can be recommended for all patients. Key factors guiding treatment choices include the particular combination of tumor (T) and nodal (N) disease, the ability to achieve a complete surgical resection if indicated, and the patient’s overall physical condition and preferences. For example, in carefully selected patients with limited stage IIIA disease where involved mediastinal lymph nodes can be completed resected, initial surgery followed by postoperative chemotherapy (with or without radiation therapy) may be indicated. By contrast, for patients with clinically evident bulky mediastinal lymph node involvement, the standard approach to treatment is concurrent chemoradiotherapy. Nevertheless, in some

1	may be indicated. By contrast, for patients with clinically evident bulky mediastinal lymph node involvement, the standard approach to treatment is concurrent chemoradiotherapy. Nevertheless, in some cases, the latter group of patients may be candidates for surgery following chemoradiotherapy.

1	Absent and Nonbulky Mediastinal (N2, N3) Lymph Node Disease For the subset of stage IIIA patients initially thought to have clinical stage I or II disease (i.e., pathologic involvement of mediastinal [N2] lymph nodes is not detected preoperatively), surgical resection is often the treatment of choice. This is followed by adjuvant chemotherapy in patients with microscopic lymph node involvement in a resection specimen. Postoperative radiation therapy (PORT) may also have a role for those with close or positive surgical margins. Patients with tumors involving the chest wall or proximal airways within 2 cm of the carina with hilar lymph node involvement (but not N2 disease) are classified as having T3N1 stage IIIA disease. They too are best managed with surgical resection, if technically feasible, followed by adjuvant chemotherapy if completely resected. Patients with tumors exceeding 7 cm in size also are now classified as T3 and are consider stage IIIA if tumor has spread to N1 nodes.

1	followed by adjuvant chemotherapy if completely resected. Patients with tumors exceeding 7 cm in size also are now classified as T3 and are consider stage IIIA if tumor has spread to N1 nodes. The appropriate initial management of these patients involves surgical resection when feasible, provided the mediastinal staging is negative, followed by adjuvant chemotherapy for those who achieve complete tumor resection. Patients with T3N0 or T3N1 disease due to the presence of satellite nodules within the same lobe as the primary tumor also are candidates for surgery, as are patients with ipsilateral nodules in another lobe and negative mediastinal nodes (IIIA, T4N0 or T4N1). Although data regarding adjuvant chemotherapy in the latter subsets of patients are limited, it is often recommended.

1	Patients with T4N0-1 were reclassified as having stage IIIA tumors in the seventh edition of the TNM system. These patients may have involvement of the carina, superior vena cava, or a vertebral body and yet still be candidates for surgical resection in selected circumstances. The decision to proceed with an attempted resection must be made in consultation with an experienced thoracic surgeon often in association with a vascular or cardiac surgeon and an orthopedic surgeon depending on tumor location. However, if an incomplete resection is inevitable or if there is evidence of N2 involvement (stage IIIB), surgery for T4 disease is contraindicated. Most T4 lesions are best treated with chemoradiotherapy.

1	The role of PORT in patients with completely resected stage III NSCLC is controversial. To a large extent, the use of PORT is dictated by the presence or absence of N2 involvement and, to a lesser degree, by the biases of the treating physician. Using the Surveillance, Epidemiology, and End Results (SEER) database, a recent meta-analysis of PORT identified a significant increase in survival in patients with N2 disease but not in patients with N0 or N1 disease. An earlier analysis by the PORT Meta-analysis Trialist Group using an older database produced similar results.

1	Known Mediastinal (N2, N3) Lymph Node Disease When pathologic involvement of mediastinal lymph nodes is documented preoperatively, a combined-modality approach is recommended assuming the patient is a candidate for treatment with curative intent. These patients are at high risk for both local and distant recurrence if managed with resection alone. For patients with stage III disease who are not candidates for initial surgical resection, concurrent chemoradiotherapy is most commonly used as the initial treatment. Concurrent chemoradiotherapy has been shown to produce superior survival compared to sequential chemoradiotherapy; however, it also is associated with greater host toxicities (including fatigue, esophagitis, and neutropenia). Therefore, for patients with a good performance status, concurrent chemoradiotherapy is the preferred treatment approach, whereas sequential chemoradiotherapy may be more appropriate for patients with a performance status that is not as good. For patients

1	concurrent chemoradiotherapy is the preferred treatment approach, whereas sequential chemoradiotherapy may be more appropriate for patients with a performance status that is not as good. For patients who are not candidates for a combined-modality treatment approach, typically due to a poor performance status or a comorbidity that makes chemotherapy untenable, radiotherapy alone may provide a modest survival benefit in addition to symptom palliation.

1	For patients with potentially resectable N2 disease, it remains uncertain whether surgery after neoadjuvant chemoradiotherapy improves survival. In an NCI-sponsored Intergroup randomized trial comparing concurrent chemoradiotherapy alone to concurrent chemoradiotherapy followed by attempted surgical resection, no survival benefit was observed in the trimodality arm compared to the bimodality therapy. In fact, patients subjected to a pneumonectomy had a worse survival outcome. By contrast, those treated with a lobectomy appeared to have a survival advantage based on a retrospective subset analysis. Thus, in carefully selected, otherwise healthy patients with nonbulky mediastinal lymph node involvement, surgery may be a reasonable option if the primary tumor can be fully resected with a lobectomy. This is not the case if a pneumonectomy is required to achieve complete resection.

1	Superior Sulcus Tumors (Pancoast Tumors) Superior sulcus tumors represent a distinctive subset of stage III disease. These tumors arise in the apex of the lung and may invade the second and third ribs, the brachial plexus, the subclavian vessels, the stellate ganglion, and adjacent vertebral bodies. They also may be associated with Pancoast syndrome, characterized by pain that may arise in the shoulder or chest wall or radiate to the neck. Pain characteristically radiates to the ulnar surface of the hand. Horner’s syndrome (enophthalmos, ptosis, miosis, and anhydrosis) due to invasion of the paravertebral sympathetic chain may be present as well. Patients with these tumors should undergo the same staging procedures as all patients with stage II and III NSCLC. Neoadjuvant chemotherapy or 519 combined chemoradiotherapy followed by surgery is reserved for those without N2 involvement. This approach yields excellent survival outcomes (>50% 5-year survival in patients with an R0

1	or 519 combined chemoradiotherapy followed by surgery is reserved for those without N2 involvement. This approach yields excellent survival outcomes (>50% 5-year survival in patients with an R0 resection). Patients with N2 disease are less likely to benefit from surgery and can be managed with chemoradiotherapy alone. Patients presenting with metastatic disease can be treated with radiation therapy (with or without chemotherapy) for symptom palliation.

1	Approximately 40% of NSCLC patients present with advanced, stage IV disease at the time of diagnosis. These patients have a poor median survival (4–6 months) and a 1-year survival of 10% when managed with best supportive care alone. In addition, a significant number of patients who first presented with early-stage NSCLC will eventually relapse with distant disease. Patients who have recurrent disease have a better prognosis than those presenting with metastatic disease at the time of diagnosis. Standard medical manage ment, the judicious use of pain medications, and the appropriate use of radiotherapy and chemotherapy form the cornerstone of management. Chemotherapy palliates symptoms, improves the quality of life, and improves survival in patients with stage IV NSCLC, particularly in patients with good performance status. In addition, economic analysis has found chemotherapy to be cost-effective palliation for stage IV NSCLC. However, the use of chemotherapy for NSCLC requires

1	in patients with good performance status. In addition, economic analysis has found chemotherapy to be cost-effective palliation for stage IV NSCLC. However, the use of chemotherapy for NSCLC requires clinical experience and careful judgment to balance potential benefits and toxicities. Of note, the early application of palliative care in conjunction with chemotherapy is associated with improved survival and a better quality of life.

1	First-Line Chemotherapy for Metastatic or Recurrent NSCLC A landmark meta-analysis published in 1995 provided the earliest meaningful indication that chemotherapy could provide a survival benefit in metastatic NSCLC as opposed to supportive care alone. However, the survival benefit was seemingly confined to cisplatin-based chemotherapy regimens (hazard ratio 0.73; 27% reduction in the risk of death; 10% improvement in survival at 1 year). These data launched two decades of clinical research aimed at detecting the optimal chemotherapy regimen for advanced NSCLC. For the most part, however, these efforts proved unsuccessful because the overwhelming majority of randomized trials showed no major survival improvement with any one regimen versus another (Table 107-11). On the other hand, differences in progression-free survival, cost, side effects, and schedule were frequently observed. These first-line studies were later extended to elderly patients, where doublet chemotherapy was found to

1	in progression-free survival, cost, side effects, and schedule were frequently observed. These first-line studies were later extended to elderly patients, where doublet chemotherapy was found to improve overall survival compared to single agents in the “fit” elderly (e.g., elderly patients with no major comorbidities) and in patients with an ECOG performance status of 2. An ongoing debate in the treatment of patients with advanced NSCLC is the appropriate duration of platinum-based chemotherapy. Several large phase III randomized trials have failed to show a meaningful benefit for increasing the duration of platinum-based doublet chemotherapy beyond four to six cycles. In fact, longer duration of chemotherapy has been associated with increased toxicities and impaired quality of life. Therefore, prolonged front-line therapy (beyond four to six cycles) with platinum-based regimens is not recommended. Maintenance therapy following initial platinum-based therapy is discussed below.

1	Although specific tumor histology was once considered irrelevant to treatment choice in NSCLC, with the recent recognition that selected chemotherapy agents perform quite differently in squamous versus adenocarcinomas, accurate determination of histology has become essential. Specifically, in a landmark randomized phase III trial, patients with nonsquamous NSCLC were found to have an improved survival when treated with cisplatin and pemetrexed compared to cisplatin and gemcitabine. By contrast, patients with squamous carcinoma had an improved survival when treated with cisplatin and gemcitabine. This survival difference is thought to be related to the differential expression of thymidylate synthase (TS), Neoplasms of the Lung Median No. of

1	Cisplatin + paclitaxel Cisplatin + gemcitabine Cisplatin + docetaxel Carboplatin + paclitaxel Cisplatin + docetaxel Cisplatin + vinorelbine Carboplatin + docetaxel Cisplatin + paclitaxel Cisplatin + gemcitabine Paclitaxel + gemcitabine Cisplatin + gemcitabine Carboplatin + paclitaxel Cisplatin + vinorelbine Cisplatin + vinorelbine Carboplatin + paclitaxel Cisplatin + irinotecan Carboplatin + paclitaxel Cisplatin + gemcitabine Cisplatin + vinorelbine Cisplatin + gemcitabine Cisplatin + pemetrexed Carboplatin + paclitaxel Gefitinib 288 21 7.8 288 22 8.1 289 17 7.4 290 17 8.1 406 32 11.3 394 25 10.1 404 24 9.4 159 32 8.1 160 37 8.9 161 28 6.7 205 30 9.8 204 32 9.9 203 30 9.5 202 28 8.0 206 25 8.0 145 31 13.9 145 32 12.3 146 30 14.0 145 33 11.4 863 28 10.3 862 31 10.3 608 32 17.3 609 43% 18.6 aEnrolled selected patients: 18 years of age or older, had histologic or cytologically confirmed stage IIIB or IV non-small-cell lung cancer with histologic features of adenocarcinoma (including

1	18.6 aEnrolled selected patients: 18 years of age or older, had histologic or cytologically confirmed stage IIIB or IV non-small-cell lung cancer with histologic features of adenocarcinoma (including bronchioloalveolar carcinoma), were nonsmokers (defined as patients who had smoked <100 cigarettes in their lifetime) or former light smokers (those who had stopped smoking at least 15 years previously and had a total of ≤10 pack-years of smoking), and had had no previous chemotherapy or biologic or immunologic therapy.

1	Abbreviations: ECOG, Eastern Cooperative Oncology Group; EORTC, European Organization for Research and Treatment of Cancer; ILCP, Italian Lung Cancer Project; SWOG, Southwest Oncology Group; FACS, Follow-up After Colorectal Surgery; iPASS, Iressa Pan-Asian Study. one of the targets of pemetrexed, between tumor types. Squamous cancers have a much higher expression of TS compared to adenocarcinomas, accounting for their lower responsiveness to pemetrexed. By contrast, the activity of gemcitabine is not impacted by the levels of TS. Bevacizumab, a monoclonal antibody against VEGF, has been shown to improve response rate, progression-free survival, and overall survival in patients with advanced disease when combined with chemotherapy (see below). However, bevacizumab cannot be given to patients with squamous cell histology NSCLC because of their tendency to experience serious hemorrhagic effects.

1	Agents That Inhibit Angiogenesis Bevacizumab, a monoclonal antibody directed against VEGF, was the first antiangiogenic agent approved for the treatment of patients with advanced NSCLC in the United States. This drug primarily acts by blocking the growth of new blood vessels, which are required for tumor viability. Two randomized phase III trials of chemotherapy with or without bevacizumab had conflicting results. The first trial, conducted in North America, compared carboplatin plus paclitaxel with or without bevacizumab in patients with recurrent or advanced nonsquamous NSCLC and reported a significant improvement in response rate, progression-free survival, and overall survival in patients treated with chemotherapy plus bevacizumab versus chemotherapy alone. Bevacizumab-treated patients had a significantly higher incidence of toxicities. The second trial, conducted in Europe, compared cisplatin/gemcitabine with or without bevacizumab in patients with recurrent or advanced

1	patients had a significantly higher incidence of toxicities. The second trial, conducted in Europe, compared cisplatin/gemcitabine with or without bevacizumab in patients with recurrent or advanced nonsquamous NSCLC and reported a significant improvement in progression-free survival but no improvement in overall survival for bevacizumab-treated patients. A randomized phase III trial compared carboplatin/pemetrexed and bevacizumab to carboplatin/paclitaxel and bevacizumab as first-line therapy in patients with recurrent or advanced nonsquamous NSCLC and reported no significant difference in progression-free survival or overall survival between treatment groups. Therefore, currently carboplatin/paclitaxel and bevacizumab or carboplatin/pemetrexed and bevacizumab are appropriate regimens for first-line treatment for stage IV nonsquamous NSCLC patients. Of note, there are many small-molecule inhibitors of VEGFR; however, these VEGFR TKIs have not proven to be effective in the treatment of

1	first-line treatment for stage IV nonsquamous NSCLC patients. Of note, there are many small-molecule inhibitors of VEGFR; however, these VEGFR TKIs have not proven to be effective in the treatment of NSCLC.

1	Maintenance Therapy for Metastatic NSCLC Maintenance chemotherapy in nonprogressing patients (patients with a complete response, partial response, or stable disease) is a controversial topic in the treatment of NSCLC. Conceptually, there are two types of maintenance strategies: (1) switch maintenance therapy, where patients receive four to six cycles of platinum-based chemotherapy and are switched to an entirely different regimen; and (2) continuation maintenance therapy, where patients receive four to six cycles of platinum-based chemotherapy and then the platinum agent is discontinued but the agent it is paired with is continued (Table 107-12). Two studies investigated switch maintenance single-agent chemotherapy with docetaxel or pemetrexed in nonprogressing patients following treatment with first-line platinum-based chemotherapy. Both trials randomized patients to immediate single-agent therapy versus observation and reported improvements in progression-free and overall survival.

1	with first-line platinum-based chemotherapy. Both trials randomized patients to immediate single-agent therapy versus observation and reported improvements in progression-free and overall survival. In both trials, a significant portion of patients in the observation arm did not receive therapy with the agent under investigation upon disease progression; 37% of study patients never received docetaxel in the docetaxel study and 81% of patients never received pemetrexed in the pemetrexed study. In the trial of maintenance docetaxel versus observation, survival was identical to the treatment group in the subset of patients who received docetaxel on progression, indicating this is an active agent in NSCLC. These data are not available for the pemetrexed study. Two additional trials evaluated switch maintenance therapy with erlotinib after platinum-based chemotherapy in patients with advanced

1	Fidias Immediate docetaxel 153 12.3 5.7 Delayed docetaxel 156 9.7 2.7 Ciuleanu Pemetrexed 444 13.4 4.3 BSC 222 10.6 2.6 Paramount Pemetrexed 472 13.9 4.1 BSC 297 11.0 2.8 ATLAS Bev + erlotinib 384 15.9 4.8 Bev + placebo 384 13.9 3.8 SATURN Erlotinib 437 12.3 2.9 Placebo 447 11.1 2.6 ECOG4599 Bev 15 mg/kg 444 12.3 6.2 BSC 434 10.3 4.5 AVAiL Bev 15 mg/kg 351 13.4 6.5 Bev 7.5 mg/kg 345 13.6 6.7 Placebo 347 13.1 6.1 8.6 Bev 15 mg/kg Bev 15 mg/kg 6.9 Abbreviations: Bev, bevacizumab; BSC, best supportive care; CT, chemotherapy; OS, overall survival; PFS, progression-free survival. NSCLC and reported an improvement in progression-free survival and overall survival in the erlotinib treatment group. Currently, maintenance pemetrexed or erlotinib following platinum-based chemotherapy in patients with advanced NSCLC are approved by the U.S. FDA. However, maintenance therapy is not without toxicity and, at this time, should be considered on an individual patient basis.

1	Targeted Therapies for Select Molecular Cohorts of NSCLC As the efficacy of traditional cytotoxic chemotherapeutic agents plateaued in NSCLC, there was a critical need to define novel therapeutic treatment strategies. These novel strategies have largely been based on the identification of somatic driver mutations within the tumor. These driver mutations occur in genes encoding signaling proteins that, when aberrant, drive initiation and maintenance of tumor cells. Importantly, driver mutations can serve as Achilles’ heels for tumors, if their gene products can be targeted therapeutically with small-molecule inhibitors. For example, EGFR mutations have been detected in 10–15% of North American patients diagnosed with NSCLC. EGFR mutations are associated with younger age, light (<10 pack-year) and nonsmokers, and adenocarcinoma histology. Approximately 90% of these mutations are exon 19 deletions or exon 21 L858R point mutations within the EGFR TK domain, resulting in hyperactivation of

1	and nonsmokers, and adenocarcinoma histology. Approximately 90% of these mutations are exon 19 deletions or exon 21 L858R point mutations within the EGFR TK domain, resulting in hyperactivation of both EGFR kinase activity and downstream signaling. Lung tumors that harbor activating mutations within the EGFR kinase domain display high sensitivity to small-molecule EGFR TKIs. Erlotinib and afatinib are FDA-approved oral small-molecule TKIs that inhibit EGFR. Outside the United States, gefitinib also is available. Several large, international, phase III studies have demonstrated improved response rates, progression-free survival, and overall survival in patients with EGFR mutation–positive NSCLC patients treated with an EGFR TKI as compared with standard first-line chemotherapy regimens (Table 107-13).

1	Although response rates with EGFR TKI therapy are clearly superior in patients with lung tumors harboring activating EGFR kinase domain mutations, the EGFR TKI erlotinib is also FDA approved for secondand third-line therapy in patients with advanced NSCLC irrespective of tumor genotype. The reason for this apparent discrepancy is that erlotinib was initially evaluated in lung cancer before the discovery of EGFR activating mutations. In fact, EGFR mutations were initially identified in lung cancer by studying the tumors of patients who had dramatic responses to this agent. With the rapid pace of scientific discovery, additional driver mutations in lung cancer have been identified and targeted therapeutically with impressive clinical results. For example, chromosomal rearrangements involving the anaplastic lymphoma kinase (ALK) gene on chromosome 2 have been found in ∼3–7% of NSCLC. The result of these ALK rearrangements is hyperactivation of the ALK TK domain. Similar to

1	No. of Study Therapy Patients ORR (%) PFS (months) Abbreviations: CbP, carboplatin and paclitaxel; CD, cisplatin and docetaxel; CG, cisplatin and gemcitabine; CP, cisplatin and paclitaxel; ORR, overall response rate; PFS, progression-free survival. EGFR, ALK rearrangements are typically (but not exclusively) associ-521 ated with younger age, light (<10 pack-year) and nonsmokers, and adenocarcinoma histology. Remarkably, ALK rearrangements were initially described in lung cancer in 2007, and by 2011, the first ALK inhibitor, crizotinib, received FDA approval for patients with lung tumors harboring ALK rearrangements.

1	In addition to EGFR and ALK, other driver mutations have been discovered with varying frequencies in NSCLC, including KRAS, BRAF, PIK3CA, NRAS, AKT1, MET, MEK1 (MAP2K1), ROS1, and RET. Mutations within the KRAS GTPase are found in approximately 20% of lung adenocarcinomas. To date, however, no small-molecule inhibitors are available to specifically target mutant KRAS. Each of the other driver mutations occurs in less than 1–3% of lung adenocarcinomas. The great majority of the driver mutations are mutually exclusive, and there are ongoing clinical studies for their specific inhibitors. For example, the BRAF inhibitor vemurafenib and the RET inhibitor cabozantinib have already demonstrated efficacy in patients with lung cancer harboring BRAF mutations or RET gene fusions, respectively.

1	Most of these mutations are present in adenocarcinoma; however, mutations that may be linked to future targeted therapies in squamous cell carcinomas are emerging. In addition, there are active research efforts aimed at defining novel targetable mutations in lung cancer as well as defining mechanisms of acquired resistance to small-molecule inhibitors used in the treatment of patients with NSCLC. to supportive care alone. As first-line chemotherapy regimens improve, a substantial number of patients will maintain a good performance status and a desire for further therapy when they develop recurrent disease. Currently, several agents are FDA approved for second-line use in NSCLC including docetaxel, pemetrexed, erlotinib (approved for second-line therapy regardless of tumor genotype), and crizotinib (for patients with ALK -mutant lung cancer only). Most of the survival benefit for any of these agents is realized in patients who maintain a good performance status.

1	Immunotherapy For more than 30 years, the investigation of vaccines and immunotherapies in lung cancer has yielded little in the way of meaningful benefit. Recently, however, this perception has changed based on preliminary results of studies using monoclonal antibodies that activate antitumor immunity through blockade of immune checkpoints. For example, ipilimumab, a monoclonal antibody directed at cytotoxic T lymphocyte antigen-4 (CTLA-4), was studied in combination with paclitaxel plus carboplatin in patients with both SCLC and NSCLC. There appeared to be a small but not statistically significant advantage to the combination when ipilimumab was instituted after several cycles of chemotherapy. A randomized phase III trial in SCLC is under way to validate these data. Antibodies to the T cell programmed cell death receptor 1 (PD-1), nivolumab and pembrolizumab, have been shown to produce responses in lung cancer, renal cell cancer, and melanoma. Many of these responses have had very

1	cell programmed cell death receptor 1 (PD-1), nivolumab and pembrolizumab, have been shown to produce responses in lung cancer, renal cell cancer, and melanoma. Many of these responses have had very long duration (i.e., >1 year). Monoclonal antibodies to the PD-1 ligand (anti-PDL-1), which may be expressed on the tumor cell, have also been shown to produce responses in patients with melanoma and lung cancer. Preliminary studies in melanoma suggest that the combination of ipilimumab and nivolumab could produce higher response rates compared to either agent alone. A similar strategy is being investigated in SCLC patients. Further evaluation of these agents in both NSCLC and SCLC is ongoing in combination with already approved chemotherapy and targeted agents.

1	Supportive Care No discussion of the treatment strategies for patients with advanced lung cancer would be complete without a mention of supportive care. Coincident with advances in chemotherapy and targeted therapy was a pivotal study that demonstrated that the early integration of palliative care with standard treatment strategies improved both quality of life and mood for patients with advanced lung cancer. Aggressive pain and symptom control is an important component for optimal treatment of these patients. Neoplasms of the Lung only FDA-approved agent for second-line therapy in patients with SCLC. Topotecan has only modest activity and can be given either

1	SCLC is a highly aggressive disease characterized by its rapid doubling time, high growth fraction, early development of disseminated disease, and dramatic response to first-line chemotherapy and radiation. In general, surgical resection is not routinely recommended for patients because even patients with LD-SCLC still have occult micrometastases. However, the most recent American College of Chest Physicians Evidence-Based Clinical Practice Guidelines recommend surgical resection over nonsurgical treatment in SCLC patients with clinical stage I disease after a thorough evaluation for distant metastases and invasive mediastinal stage evaluation (grade 2C). After resection, these patients should receive platinum-based adjuvant chemotherapy (grade 1C). If the histologic diagnosis of SCLC is made in patients on review of a resected surgical specimen, such patients should receive standard SCLC chemotherapy as well.

1	Chemotherapy significantly prolongs survival in patients with SCLC. Four to six cycles of platinum-based chemotherapy with either cisplatin or carboplatin plus either etoposide or irinotecan has been the mainstay of treatment for nearly three decades and is recommended over other chemotherapy regimens irrespective of initial stage. Cyclophosphamide, doxorubicin (Adriamycin), and vincristine (CAV) may be an alternative for patients who are unable to tolerate a platinum-based regimen. Despite response rates to first-line therapy as high as 80%, the median survival ranges from 12 to 20 months for patients with LD and from 7 to 11 months for patients with ED. Regardless of disease extent, the majority of patients relapse and develop chemotherapy-resistant disease. Only 6–12% of patients with LD-SCLC and 2% of patients with ED-SCLC live beyond 5 years. The prognosis is especially poor for patients who relapse within the first 3 months of therapy; these patients are said to have

1	with LD-SCLC and 2% of patients with ED-SCLC live beyond 5 years. The prognosis is especially poor for patients who relapse within the first 3 months of therapy; these patients are said to have chemotherapy-resistant disease. Patients are said to have sensitive disease if they relapse more than 3 months after their initial therapy and are thought to have a somewhat better overall survival. These patients also are thought to have the greatest potential benefit from second-line chemotherapy (Fig. 107-7). Topotecan is the

1	FIGURE 107-7 Management of recurrent small-cell lung cancer (SCLC). CAV, cyclophosphamide, doxorubicin, and vincristine. (Adapted with permission from JP van Meerbeeck et al: Lancet 378:1741, 2011.) intravenously or orally. In one randomized trial, 141 patients who were not considered candidates for further IV chemotherapy were randomized to receive either oral topotecan or best supportive care. Although the response rate to oral topotecan was only 7%, overall survival was significantly better in patients receiving chemotherapy (median survival time, 26 weeks vs 14 weeks; p = .01). Moreover, patients given topotecan had a slower decline in quality of life than did those not receiving chemotherapy. Other agents with similar low levels of activity in the second-line setting include irinotecan, paclitaxel, docetaxel, vinorelbine, oral etoposide, and gemcitabine. Clearly novel treatments for this all too common disease are desperately needed.

1	Thoracic radiation therapy (TRT) is a standard component of induction therapy for good performance status and limited-stage SCLC patients. Meta-analyses indicate that chemotherapy combined with chest irradiation improves 3-year survival by approximately 5% as compared with chemotherapy alone. The 5-year survival rate, however, remains disappointingly low at ∼10–15%. Most commonly, TRT is combined with cisplatin and etoposide chemotherapy due to a superior toxicity profile as compared to anthracycline-containing chemotherapy regimens. As observed in locally advanced NSCLC, concurrent chemoradiotherapy is more effective than sequential chemoradiation but is associated with significantly more esophagitis and hematologic toxicity. Ideally TRT should be administered with the first two cycles of chemotherapy because later application appears slightly less effective. If for reasons of fitness or availability, this regimen cannot be offered, TRT should follow induction chemotherapy. With

1	of chemotherapy because later application appears slightly less effective. If for reasons of fitness or availability, this regimen cannot be offered, TRT should follow induction chemotherapy. With respect to fractionation of TRT, twice-daily 1.5-Gy fractioned radiation therapy has been shown to improve survival in LD-SCLC patients but is associated with higher rates of grade 3 esophagitis and pulmonary toxicity. Although it is feasible to deliver once-daily radiation therapy doses up to 70 Gy concurrently with cisplatin-based chemotherapy, there are no data to support equivalency of this approach compared with the 45-Gy twice-daily radiotherapy dose. Therefore, the current standard regimen of a 45-Gy dose administered in 1.5-Gy fractions twice daily for 30 days is being compared with higher-dose regimens in two phase III trials, one in the United States and one in Europe. Patients should be carefully selected for concurrent chemoradiation therapy based on good performance status and

1	regimens in two phase III trials, one in the United States and one in Europe. Patients should be carefully selected for concurrent chemoradiation therapy based on good performance status and adequate pulmonary reserve. The role of radiotherapy in ED-SCLC is largely restricted to palliation of tumor-related symptoms such as bone pain and bronchial obstruction.

1	Prophylactic cranial irradiation (PCI) should be considered in all patients with either LD-SCLC or ED-SCLC who have responded well to initial therapy. A meta-analysis including seven trials and 987 patients with LD-SCLC who had achieved a complete remission after upfront chemotherapy yielded a 5.4% improvement in overall survival for patients treated with PCI. In patients with ED-SCLC who have responded to first-line chemotherapy, a prospective randomized phase III trial showed that PCI reduced the occurrence of symptomatic brain metastases and prolonged disease-free and overall survival compared to no radiation therapy. Long-term toxicities, including deficits in cognition, have been reported after PCI but are difficult to sort out from the effects of chemotherapy or normal aging.

1	The management of NSCLC has undergone major change in the past decade. To a lesser extent, the same is true for SCLC. For patients with early-stage disease, advances in radiotherapy and surgical procedures as well as new systemic therapies have greatly improved prognosis in both diseases. For patients with advanced disease, major progress in understanding tumor genetics has led to the development of

1	Core biopsy of most distant site of disease Squamous carcinoma Adenocartcinoma Obtain tissue Determine histology Determine molecular status Treatment options EGFRmut Erlotinib or afatinib Crizotinib No mutation or mutation for which there is no FDA approved therapy Platinum-based chemothearpy ± bevacizumab Cisplatin or carboplatin + gemcitabine, doc-etaxel, paclitaxel, or nab-paclitaxel Platinum-based chemotherapy ALK (+) Large-cell neuroendocrine carcinoma FIGURE 107-8 Approach to first-line therapy in a patient with stage IV non-small-cell lung cancer (NSCLC). EGFRmut, EGFR mutation; FDA, Food and Drug Administration.

1	targeted inhibitors based specifically on the tumor’s molecular profile. Furthermore, increased understanding of how to activate the immune system to drive antitumor immunity is proving to be a promising therapeutic strategy for some patients with advanced lung cancer. In Fig. 107-8, we propose an algorithm of the treatment approach for patient with stage IV NSCLC. However, the reality is that the majority of patients treated with targeted therapies or chemotherapy eventually develop resistance, which provides strong motivation for further research and enrollment of patients onto clinical trials in this rapidly evolving area. Marc E. Lippman

1	Marc E. Lippman Breast cancer is a malignant proliferation of epithelial cells lining the ducts or lobules of the breast. In the year 2014, about 180,000 cases of invasive breast cancer and 40,000 deaths will occur in the United States. In addition, about 2000 men will be diagnosed with breast cancer. Epithelial malignancies of the breast are the most common cause of cancer in women (excluding skin cancer), accounting for about one-third of all cancer in women. As a result of improved treatment and earlier detection, the mortality rate from breast cancer has begun to decrease very substantially in the United States. This Chapter will not consider rare malignancies presenting in the breast, such as sarcomas and lymphomas, but will focus on the epithelial cancers. Human breast cancer is a clonal disease; a single transformed cell—the product of a series of somatic (acquired) or germline mutations—is eventually able to express full malignant potential.

1	Human breast cancer is a clonal disease; a single transformed cell—the product of a series of somatic (acquired) or germline mutations—is eventually able to express full malignant potential. Thus, breast cancer may exist for a long period as either a noninvasive disease or an invasive but nonmetastatic disease. These facts have significant clinical ramifications. Not more than 10% of human breast cancers can be linked directly to germline mutations. Several genes have been implicated in familial cases. The Li-Fraumeni syndrome is characterized by inherited mutations in the p53 tumor-suppressor gene, which lead to an increased incidence of breast cancer, osteogenic sarcomas, and other malignancies. Inherited mutations in PTEN have also been reported in breast cancer.

1	Another tumor-suppressor gene, BRCA1, has been identified at the chromosomal locus 17q21; this gene encodes a zinc finger protein, and the protein product functions as a transcription factor and is involved in gene repair. Women who inherit a mutated allele of this gene from either parent have at least a 60–80% lifetime chance of developing breast cancer and about a 33% chance of developing ovarian cancer. The risk is higher among women born after 1940, presumably due to promotional effects of hormonal factors. Men who carry a mutant allele of the gene have an increased incidence of prostate cancer and breast cancer. A fourth gene, termed BRCA2, which has been localized to chromosome 13q12, is also associated with an increased incidence of breast cancer in men and women.

1	Germline mutations in BRCA1 and BRCA2 can be readily detected; patients with these mutations should be counseled appropriately. All women with strong family histories for breast cancer should be referred to genetic screening programs, particularly women of Ashkenazi Jewish descent who have a high likelihood of a specific founder BRCA1 mutation (substitution of adenine for guanine at position 185).

1	Even more important than the role these genes play in inherited forms of breast cancer may be their role in sporadic breast cancer. A p53 mutation is present in nearly 40% of human breast cancers as an acquired defect. Acquired mutations in PTEN occur in about 10% of the cases. BRCA1 mutation in sporadic primary breast cancer has not been reported. However, decreased expression of BRCA1 mRNA (possibly via gene methylation) and abnormal cellular location of the BRCA1 protein have been found in some breast cancers. Loss of heterozygosity of BRCA1 and BRCA2 suggests that tumor-suppressor 524 activity may be inactivated in sporadic cases of human breast cancer. Finally, increased expression of a dominant oncogene plays a role in about a quarter of human breast cancer cases. The product of this gene, a member of the epidermal growth factor receptor superfamily, is called erbB2 (HER/2 neu) and is overexpressed in these breast cancers due to gene amplification; this overexpression can

1	of this gene, a member of the epidermal growth factor receptor superfamily, is called erbB2 (HER/2 neu) and is overexpressed in these breast cancers due to gene amplification; this overexpression can contribute to transformation of human breast epithelium and is the target of effective systemic therapy in adjuvant and metastatic disease settings. A series of acquired “driver” mutations have been identified in sporadic breast cancer by major sequencing consortia. Unfortunately, most occur in no more than 5% of cases and generally do not have effective agents to target them, so “personalized medicine” is for now more of a dream than a reality.

1	Breast cancer is a hormone-dependent disease. Women without functioning ovaries who never receive estrogen replacement therapy do not develop breast cancer. The female-to-male ratio is about 150:1. For most epithelial malignancies, a log-log plot of incidence versus age shows a single-component straight-line increase with every year of life. A similar plot for breast cancer shows two components: a straight-line increase with age but with a decrease in slope beginning at the age of menopause. The three dates in a woman’s life that have a major impact on breast cancer incidence are age at menarche, age at first full-term pregnancy, and age at menopause. Women who experience menarche at age 16 years have only 50–60% of the breast cancer risk of a woman having menarche at age 12 years; the lower risk persists throughout life. Similarly, menopause occurring 10 years before the median age of menopause (52 years), whether natural or surgically induced, reduces lifetime breast cancer risk by

1	risk persists throughout life. Similarly, menopause occurring 10 years before the median age of menopause (52 years), whether natural or surgically induced, reduces lifetime breast cancer risk by about 35%. Women who have a first full-term pregnancy by age 18 years have a 30–40% lower risk of breast cancer compared with nulliparous women. Thus, length of menstrual life—particularly the fraction occurring before first full-term pregnancy—is a substantial component of the total risk of breast cancer. These three factors (menarche, age of first full-term pregnancy, and menopause) can account for 70–80% of the variation in breast cancer frequency in different countries. Also, duration of maternal nursing correlates with substantial risk reduction independent of either parity or age at first full-term pregnancy.

1	International variation in incidence has provided some of the most important clues on hormonal carcinogenesis. A woman living to age 80 years in North America has one chance in nine of developing invasive breast cancer. Asian women have one-fifth to one-tenth the risk of breast cancer of women in North America or Western Europe. Asian women have substantially lower concentrations of estrogens and progesterone. These differences cannot be explained on a genetic basis because Asian women living in a Western environment have sex steroid hormone concentrations and risks identical to those of their Western counterparts. These migrant women, and more notably their daughters, also differ markedly in height and weight from Asian women in Asia; height and weight are critical regulators of age of menarche and have substantial effects on plasma concentrations of estrogens.

1	The role of diet in breast cancer etiology is controversial. While there are associative links between total caloric and fat intake and breast cancer risk, the exact role of fat in the diet is unproven. Increased caloric intake contributes to breast cancer risk in multiple ways: earlier menarche, later age at menopause, and increased post-menopausal estrogen concentrations reflecting enhanced aromatase activities in fatty tissues. On the other hand, central obesity is both a risk factor for occurrence and recurrence of breast cancer. Moderate alcohol intake also increases the risk by an unknown mechanism. Folic acid supplementation appears to modify risk in women who use alcohol but is not additionally protective in abstainers. Recommendations favoring abstinence from alcohol must be weighed against other social pressures and the possible cardioprotective effect of moderate alcohol intake. Chronic low-dose aspirin use is associated with a decreased incidence of breast cancer.

1	be weighed against other social pressures and the possible cardioprotective effect of moderate alcohol intake. Chronic low-dose aspirin use is associated with a decreased incidence of breast cancer. Depression is also associated with both occurrence and recurrence of breast cancer.

1	Understanding the potential role of exogenous hormones in breast cancer is of extraordinary importance because millions of American women regularly use oral contraceptives and postmenopausal hormone replacement therapy. The most credible meta-analyses of oral contraceptive use suggest that these agents cause a small increased risk of breast cancer. By contrast, oral contraceptives offer a substantial protective effect against ovarian epithelial tumors and endometrial cancers. Hormone replacement therapy (HRT) has a powerful effect on breast cancer risk. Data from the Women’s Health Initiative (WHI) trial showed that conjugated equine estrogens plus progestins increased the risk of breast cancer and adverse cardiovascular events but decreased the risk of bone fractures and colorectal cancer. On balance, there were more negative events with HRT; 6–7 years of HRT nearly doubled the risk of breast cancer. A parallel WHI trial with >12,000 women enrolled testing conjugated estrogens alone

1	On balance, there were more negative events with HRT; 6–7 years of HRT nearly doubled the risk of breast cancer. A parallel WHI trial with >12,000 women enrolled testing conjugated estrogens alone (estrogen replacement therapy in women who have had hysterectomies) showed no significant increase in breast cancer incidence. Thus, there are serious concerns about longterm HRT use in terms of cardiovascular disease and breast cancer. The WHI trial of conjugated equine estrogen alone demonstrated few adverse effects for women age <70; however, no comparable safety data are available for other more potent forms of estrogen replacement, and they should not be routinely used as substitutes. HRT in women previously diagnosed with breast cancer increases recurrence rates. Rapid decrease in the number of women on HRT has already led to a coincident decrease in breast cancer incidence.

1	In addition to the other factors, radiation is a risk factor in younger women. Women who have been exposed before age 30 years to radiation in the form of multiple fluoroscopies (200–300 cGy) or treatment for Hodgkin’s disease (>3600 cGy) have a substantial increase in risk of breast cancer, whereas radiation exposure after age 30 years appears to have a minimal carcinogenic effect on the breast.

1	Because the breasts are a common site of potentially fatal malignancy in women, examination of the breast is an essential part of the physical examination. Unfortunately, internists frequently do not examine breasts in men, and in women, they are apt to defer this evaluation to gynecologists. Because of the plausible association between early detection and improved outcome, it is the duty of every physician to identify breast abnormalities at the earliest possible stage and to institute a diagnostic workup. Women should be trained in breast self-examination (BSE). Although breast cancer in men is unusual, unilateral lesions should be evaluated in the same manner as in women, with the recognition that gynecomastia in men can sometimes begin unilaterally and is often asymmetric.

1	Virtually all breast cancer is diagnosed by biopsy of a nodule detected either on a mammogram or by palpation. Algorithms have been developed to enhance the likelihood of diagnosing breast cancer and reduce the frequency of unnecessary biopsy (Fig. 108-1).

1	Women should be strongly encouraged to examine their breasts monthly. A potentially flawed study from China has suggested that BSE does not alter survival, but given its safety, the procedure should still be encouraged. At worst, this practice increases the likelihood of detecting a mass at a smaller size when it can be treated with more limited surgery. Breast examination by the physician should be performed in good light so as to see retractions and other skin changes. The nipple and areolae should be inspected, and an attempt should be made to elicit nipple discharge. All regional lymph node groups should be examined, and any lesions should be measured. Physical examination alone cannot exclude malignancy. Lesions with certain features are more likely to be cancerous (hard, irregular, tethered or fixed, or painless lesions). A negative mammogram in the presence of a persistent lump in the breast does not exclude malignancy. Palpable lesions require additional diagnostic procedures,

1	or fixed, or painless lesions). A negative mammogram in the presence of a persistent lump in the breast does not exclude malignancy. Palpable lesions require additional diagnostic procedures, including biopsy.

1	In premenopausal women, lesions that are either equivocal or nonsuspicious on physical examination should be reexamined in 2–4 Questionable mass “thickening” Reexamine follicular phase menstrual cycle Biopsy Mammogram Solid mass Postmenopausal Patient (with dominant mass) Management by “triple diagnosis” or biopsy Premenopausal Patient Routine screening Mass gone Cyst (see Fig. 108-3) Mass persists Suspicious Aspiration Dominant mass “Benign” FIGURE 108-1 Approach to a palpable breast mass.

1	FIGURE 108-1 Approach to a palpable breast mass. weeks, during the follicular phase of the menstrual cycle. Days 5–7 of the cycle are the best time for breast examination. A dominant mass in a postmenopausal woman or a dominant mass that persists through a menstrual cycle in a premenopausal woman should be aspirated by fine-needle biopsy or referred to a surgeon. If nonbloody fluid is aspirated, the diagnosis (cyst) and therapy have been accomplished together. Solid lesions that are persistent, recurrent, complex, or bloody cysts require mammography and biopsy, although in selected patients the so-called triple diagnostic technique (palpation, mammography, aspiration) can be used to avoid biopsy (Figs. 108-1, 108-2, and 108-3). Ultrasound can be used in place of fine-needle aspiration to distinguish cysts from solid lesions. Not all solid masses are detected by ultrasound; thus, a palpable mass that is not visualized on ultrasound must be presumed to be solid.

1	Several points are essential in pursuing these management decision trees. First, risk-factor analysis is not part of the decision structure. No constellation of risk factors, by their presence or absence, can be used to exclude biopsy. Second, fine-needle aspiration should be used only in centers that have proven skill in obtaining such specimens and analyzing them. The likelihood of cancer is low in the setting of a “triple negative” (benign-feeling lump, negative mammogram, and negative fine-needle aspiration), but it is not zero. The patient and physician FIGURE 108-2 The “triple diagnosis” technique. FIGURE 108-3 Management of a breast cyst. must be aware of a 1% risk of false negatives. Third, additional technologies such as magnetic resonance imaging (MRI), ultrasound, and sestamibi imaging cannot be used to exclude the need for biopsy, although in unusual circumstances, they may provoke a biopsy.

1	Diagnostic mammography should not be confused with screening mammography, which is performed after a palpable abnormality has been detected. Diagnostic mammography is aimed at evaluating the rest of the breast before biopsy is performed or occasionally is part of the triple-test strategy to exclude immediate biopsy. Subtle abnormalities that are first detected by screening mammography should be evaluated carefully by compression or magnified views. These abnormalities include clustered microcalcifications, densities (especially if spiculated), and new or enlarging architectural distortion. For some nonpalpable lesions, ultrasound may be helpful either to identify cysts or to guide biopsy. If there is no palpable lesion and detailed mammographic studies are unequivocally benign, the patient should have routine follow-up appropriate to the patient’s age. It cannot be stressed too strongly that in the presence of a breast lump a negative mammogram does not rule out cancer.

1	If a nonpalpable mammographic lesion has a low index of suspicion, mammographic follow-up in 3–6 months is reasonable. Workup of indeterminate and suspicious lesions has been rendered more complex by the advent of stereotactic biopsies. Morrow and colleagues have suggested that these procedures are indicated for lesions that require biopsy but are likely to be benign—that is, for cases in which the procedure probably will eliminate additional surgery. When a lesion is more probably malignant, open biopsy should be performed with a needle localization technique. Others have proposed more widespread use of stereotactic core biopsies for nonpalpable lesions on economic grounds and because diagnosis leads to earlier treatment planning. However, stereotactic diagnosis of a malignant lesion does not eliminate the need for definitive surgical procedures, particularly if breast conservation is attempted. For example, after a breast biopsy with needle localization (i.e., local excision) of a

1	not eliminate the need for definitive surgical procedures, particularly if breast conservation is attempted. For example, after a breast biopsy with needle localization (i.e., local excision) of a stereotactically diagnosed malignancy, reexcision may still be necessary to achieve negative margins. To some extent, these issues are decided on the basis of referral pattern and the availability of the resources for stereotactic core biopsies. A reasonable approach is shown in Fig. 108-4.

1	During pregnancy, the breast grows under the influence of estrogen, progesterone, prolactin, and human placental lactogen. Lactation is suppressed by progesterone, which blocks the effects of prolactin. After delivery, lactation is promoted by the fall in progesterone levels, which leaves the effects of prolactin unopposed. The development of a dominant mass during pregnancy or lactation should never be attributed to hormonal changes. A dominant mass must be treated with the same concern in a pregnant woman as any other. Breast Mammographic Abnormality Additional studies including spot magnification, oblique views, aspiration, and ultrasound as indicated. FIGURE 108-4 Approaches to abnormalities detected by mammogram.

1	Mammographic Abnormality Additional studies including spot magnification, oblique views, aspiration, and ultrasound as indicated. FIGURE 108-4 Approaches to abnormalities detected by mammogram. cancer develops in 1 in every 3000–4000 pregnancies. Stage for stage, breast cancer in pregnant patients is no different from premenopausal breast cancer in nonpregnant patients. However, pregnant women often have more advanced disease because the significance of a breast mass was not fully considered and/or because of endogenous hormone stimulation. Persistent lumps in the breast of pregnant or lactating women cannot be attributed to benign changes based on physical findings; such patients should be promptly referred for diagnostic evaluation.

1	Only about 1 in every 5–10 breast biopsies leads to a diagnosis of cancer, although the rate of positive biopsies varies in different countries and clinical settings. (These differences may be related to interpretation, medico-legal considerations, and availability of mammograms.) The vast majority of benign breast masses are due to “fibrocystic” disease, a descriptive term for small fluid-filled cysts and modest epithelial cell and fibrous tissue hyperplasia. However, fibrocystic disease is a histologic, not a clinical, diagnosis, and women who have had a biopsy with benign findings are at greater risk of developing breast cancer than those who have not had a biopsy. The subset of women with ductal or lobular cell proliferation (about 30% of patients), particularly the small fraction (3%) with atypical hyperplasia, have a fourfold greater risk of developing breast cancer than those women who have not had a biopsy, and the increase in the risk is about ninefold for women in this

1	(3%) with atypical hyperplasia, have a fourfold greater risk of developing breast cancer than those women who have not had a biopsy, and the increase in the risk is about ninefold for women in this category who also have an affected first-degree relative. Thus, careful follow-up of these patients is required. By contrast, patients with a benign biopsy without atypical hyperplasia are at little risk and may be followed routinely.

1	Breast cancer is virtually unique among the epithelial tumors in adults in that screening (in the form of annual mammography) improves survival. Meta-analysis examining outcomes from every randomized trial of mammography conclusively shows a 25–30% reduction in the chance of dying from breast cancer with annual screening after age 50 years; the data for women between ages 40 and 50 years are almost as positive; however, since the incidence is much lower in younger women, there are more false positives. While controversy continues to surround the assessment of screening mammography, the preponderance of data strongly supports the benefits of screening mammography. New analyses of older randomized studies have occasionally suggested that screening may not work. While the design defects in some older studies cannot be retrospectively corrected, most experts, including panels of the American Society of Clinical Oncology and the American Cancer Society (ACS), continue to believe that

1	in some older studies cannot be retrospectively corrected, most experts, including panels of the American Society of Clinical Oncology and the American Cancer Society (ACS), continue to believe that screening conveys substantial benefit. Furthermore, the profound drop in breast cancer mortality rate seen over the past decade is unlikely to be solely attributable to improvements in therapy. It seems prudent to recommend annual or biannual mammography for women past the age of 40 years. Although no randomized study of BSE has ever shown any improvement in survival, its major benefit is identification of tumors appropriate for conservative local therapy. Better mammographic technology, including digitized mammography, routine use of magnified views, and greater skill in mammographic interpretation, combined with newer diagnostic techniques (MRI, magnetic resonance spectroscopy, positron emission tomography, etc.) may make it possible to identify breast cancers even more reliably and

1	combined with newer diagnostic techniques (MRI, magnetic resonance spectroscopy, positron emission tomography, etc.) may make it possible to identify breast cancers even more reliably and earlier. Screening by any technique other than mammography is not indicated. However, the ACS suggests that younger women who are BRCA1 or BRCA2 carriers or untested first-degree relatives of women with cancer; women with a history of radiation therapy to the chest between ages 10 and 30 years; women with a lifetime risk of breast cancer of at least 20%; and women with a history of Li-Fraumeni, Cowden, or Bannayan-Riley-Ruvalcaba syndromes may benefit from MRI screening, where the higher sensitivity may outweigh the loss of specificity.

1	Correct staging of breast cancer patients is of extraordinary importance. Not only does it permit an accurate prognosis, but in many cases, therapeutic decision-making is based largely on the TNM (primary tumor, regional nodes, metastasis) classification (Table 108-1). Comparison with historic series should be undertaken with caution, as the staging has changed several times in the past 20 years. The current staging is complex and results in significant changes in outcome by stage as compared with prior staging systems. One of the most exciting aspects of breast cancer biology has been its subdivision into at least five subtypes based on gene expression profiling. 1. Luminal A: The luminal tumors express cytokeratins 8 and 18, have the highest levels of estrogen receptor expression, tend to be low grade, are most likely to respond to endocrine therapy, and have a favorable prognosis. They tend to be less responsive to chemotherapy. 2.

1	2. Luminal B: Tumor cells are also of luminal epithelial origin, but with a gene expression pattern distinct from luminal A. Prognosis is somewhat worse that luminal A. 3. Normal breast–like: These tumors have a gene expression profile reminiscent of nonmalignant “normal” breast epithelium. Prognosis is similar to the luminal B group. This subtype is somewhat controversial and may represent contamination of the sample by normal mammary epithelium. 4. HER2 amplified: These tumors have amplification of the HER2 gene on chromosome 17q and frequently exhibit coamplification and overexpression of other genes adjacent to HER2. Historically the clinical prognosis of such tumors was poor. However, with the advent of trastuzumab and other targeted therapies, the clinical outcome of HER2 -positive patients is markedly improving. 5.

1	5. Basal: These estrogen receptor/progesterone receptor–negative and HER2-negative tumors (so-called triple negative) are characterized by markers of basal/myoepithelial cells. They tend to be high grade, and express cytokeratins 5/6 and 17 as well as vimentin, p63, CD10, α-smooth muscle actin, and epidermal growth factor receptor (EGFR). Patients with BRCA mutations also fall within this molecular subtype. They also have stem cell characteristics. Breast-conserving treatments, consisting of the removal of the primary tumor by some form of lumpectomy with or without irra T0 No evidence of primary tumor TIS Carcinoma in situ T1 Tumor ≤2 cm T1a Tumor >0.1 cm but ≤0.5 cm T1b Tumor >0.5 but ≤1 cm T1c Tumor >1 cm but ≤2 cm T2 Tumor >2 cm but ≤5 cm T3 Tumor >5 cm T4 Extension to chest wall, inflammation, satellite lesions, ulcerations

1	M0 No distant metastasis M1 Distant metastasis (includes spread to ipsilateral supraclavicular nodes) aClinically apparent is defined as detected by imaging studies (excluding lymphoscintigraphy) or by clinical examination. Abbreviations: IHC, immunohistochemistry; RT-PCR, reverse transcriptase polymerase chain reaction. Source: Used with permission of the American Joint Committee on Cancer (AJCC), Chicago, Illinois. The original source for this material is the AJCC Cancer Staging Manual, 7th ed. New York, Springer, 2010; www.springeronline.com.

1	diating the breast, result in a survival that is as good as (or slightly tumors (i.e., T2 in size, positive margins, positive nodes). At pres-superior to) that after extensive surgical procedures, such as mas-ent, nearly one-third of women in the United States are managed tectomy or modified radical mastectomy, with or without further by lumpectomy. Breast-conserving surgery is not suitable for all irradiation. Postlumpectomy breast irradiation greatly reduces the patients: it is not generally suitable for tumors >5 cm (or for smaller risk of recurrence in the breast. While breast conservation is associ-tumors if the breast is small), for tumors involving the nipple-areola ated with a possibility of recurrence in the breast, 10-year survival is complex, for tumors with extensive intraductal disease involvat least as good as that after more extensive surgery. Postoperative ing multiple quadrants of the breast, for women with a history of radiation to regional nodes following mastectomy

1	disease involvat least as good as that after more extensive surgery. Postoperative ing multiple quadrants of the breast, for women with a history of radiation to regional nodes following mastectomy is also associated collagen-vascular disease, and for women who either do not have with an improvement in survival. Because radiation therapy can also the motivation for breast conservation or do not have convenient reduce the rate of local or regional recurrence, it should be strongly access to radiation therapy. However, these groups probably do not considered following mastectomy for women with high-risk primary account for more than one-third of patients who are treated with 528 mastectomy. Thus, a great many women still undergo mastectomy who could safely avoid this procedure and probably would if appropriately counseled. Sentinel lymph node biopsy (SLNB) is generally the standard of care for women with localized breast cancer and clinically negative axilla. If SLNB is negative, more

1	would if appropriately counseled. Sentinel lymph node biopsy (SLNB) is generally the standard of care for women with localized breast cancer and clinically negative axilla. If SLNB is negative, more extensive axillary surgery is not required, avoiding much of the risk of lymphedema following more extensive axillary dissections. In the presence of minimal involvement of a sentinel lymph node, further axillary surgery is not required. An extensive intraductal component is a predictor of recurrence in the breast, and so are several clinical variables. Both axillary lymph node involvement and involvement of vascular or lymphatic channels by metastatic tumor in the breast are associated with a higher risk of relapse in the breast but are not contraindications to breast-conserving treatment. When these patients are excluded, and when lumpectomy with negative tumor margins is achieved, breast conservation is associated with a recurrence rate in the breast of 5% or less. The survival of

1	When these patients are excluded, and when lumpectomy with negative tumor margins is achieved, breast conservation is associated with a recurrence rate in the breast of 5% or less. The survival of patients who have recurrence in the breast is somewhat worse than that of women who do not. Thus, recurrence in the breast is a negative prognostic variable for longterm survival. However, recurrence in the breast is not the cause of distant metastasis. If recurrence in the breast caused metastatic disease, then women treated with lumpectomy, who have a higher rate of recurrence in the breast, should have poorer survival than women treated with mastectomy, and they do not. Most patients should consult with a radiation oncologist before making a final decision concerning local therapy. However, a multimodality clinic in which the surgeon, radiation oncologist, medical oncologist, and other caregivers cooperate to evaluate the patient and develop a treatment plan is usually considered a major

1	multimodality clinic in which the surgeon, radiation oncologist, medical oncologist, and other caregivers cooperate to evaluate the patient and develop a treatment plan is usually considered a major advantage by patients.

1	Adjuvant Therapy The use of systemic therapy after local management of breast cancer substantially improves survival. More than half of the women who would otherwise die of metastatic breast cancer remain disease-free when treated with the appropriate systemic regimen. These data have grown more and more impressive with longer follow-up and more effective regimens.

1	Prognostic variaBles The most important prognostic variables are provided by tumor staging. The size of the tumor and the status of the axillary lymph nodes provide reasonably accurate information on the likelihood of tumor relapse. The relation of pathologic stage to 5-year survival is shown in Table 108-2. For most women, the need for adjuvant therapy can be readily defined on this basis alone. In the absence of lymph node involvement, involvement of microvessels (either capillaries or lymphatic channels) in tumors is nearly equivalent to lymph node involvement. The greatest controversy concerns women with intermediate prognoses. There is rarely justification for adjuvant chemotherapy in most women with tumors <1 cm in size whose axillary lymph nodes are negative. HER2positive tumors are a potential exception. Detection of breast cancer cells either in the circulation or bone marrow is associated with an increased relapse rate. The most exciting development in this area is the use

1	a potential exception. Detection of breast cancer cells either in the circulation or bone marrow is associated with an increased relapse rate. The most exciting development in this area is the use of gene expression arrays to analyze patterns of tumor gene expression. Several groups have independently defined gene sets that reliably predict disease-free and overall survival far more accu

1	Stage 5-Year Survival, % Source: Modified from data of the National Cancer Institute: Surveillance, Epidemiology, and End Results (SEER). rately than any single prognostic variable including the Oncotype DX® analysis of 21 genes. Also, the use of such standardized risk assessment tools such as Adjuvant! Online (www.adjuvantonline .com) is very helpful. These tools are highly recommended in otherwise ambiguous circumstances. Estrogen receptor status and progesterone receptor status are of prognostic significance. Tumors that lack either or both of these receptors are more likely to recur than tumors that have them.

1	Estrogen receptor status and progesterone receptor status are of prognostic significance. Tumors that lack either or both of these receptors are more likely to recur than tumors that have them. Several measures of tumor growth rate correlate with early relapse. S-phase analysis using flow cytometry is the most accurate measure. Indirect S-phase assessments using antigens associated with the cell cycle, such as PCNA (Ki67), are also valuable. Tumors with a high proportion (more than the median) of cells in S-phase pose a greater risk of relapse; chemotherapy offers the greatest survival benefit for these tumors. Assessment of DNA content in the form of ploidy is of modest value, with nondiploid tumors having a somewhat worse prognosis.

1	Histologic classification of the tumor has also been used as a prognostic factor. Tumors with a poor nuclear grade have a higher risk of recurrence than tumors with a good nuclear grade. Semiquantitative measures such as the Elston score improve the reproducibility of this measurement. Molecular changes in the tumor are also useful. Tumors that over-express erbB2 (HER2/neu) or have a mutated p53 gene have a worse prognosis. Particular interest has centered on erbB2 overexpression as measured by immunohistochemistry or fluorescence in situ hybridization. Tumors that overexpress erbB2 are more likely to respond to doxorubicin-containing regimens; erbB2 overexpression also predicts those tumors that will respond to HER2/neu antibodies (trastuzumab) (Herceptin) and HER2/neu kinase inhibitors.

1	Other variables that have also been used to evaluate prognosis include proteins associated with invasiveness, such as type IV collagenase, cathepsin D, plasminogen activator, plasminogen activator receptor, and the metastasis-suppressor gene nm23. None of these has been widely accepted as a prognostic variable for therapeutic decision-making. One problem in interpreting these prognostic variables is that most of them have not been examined in a study using a large cohort of patients.

1	adjuvant regimens Adjuvant therapy is the use of systemic therapies in patients whose known disease has received local therapy but who are at risk of relapse. Selection of appropriate adjuvant chemotherapy or hormone therapy is highly controversial in some situations. Meta-analyses have helped to define broad limits for therapy but do not help in choosing optimal regimens or in choosing a regimen for certain subgroups of patients. A summary of recommendations is shown in Table 108-3. In general, premenopausal women for whom any form of adjuvant systemic therapy is indicated should receive multidrug chemotherapy. Antihormone therapy improves survival in premenopausal patients who are estrogen receptor positive and should be added following completion of chemotherapy. Prophylactic surgical or medically induced castration may also be associated with a substantial survival benefit (primarily in estrogen receptor–positive patients) but is not widely used in this country.

1	Data on postmenopausal women are also controversial. The impact of adjuvant chemotherapy is quantitatively less clear-cut than in premenopausal patients, particularly in estrogen receptor– positive cases, although survival advantages have been shown. The first decision is whether chemotherapy or endocrine therapy should be used. While adjuvant endocrine therapy (aromatase inhibitors and tamoxifen) improves survival regardless of axillary lymph node status, the improvement in survival is modest for patients in whom multiple lymph nodes are involved. For this reason, it has been usual to give chemotherapy to postmenopausal patients who have no medical contraindications and who have more than one positive lymph node; hormone therapy is commonly given subsequently. For postmenopausal women for whom systemic therapy is warranted but who have a more favorable prognosis (based more commonly on analysis such as the Oncotype DX methodology), hormone therapy may be used alone. Large clinical

1	for whom systemic therapy is warranted but who have a more favorable prognosis (based more commonly on analysis such as the Oncotype DX methodology), hormone therapy may be used alone. Large clinical trials have aAs determined by pathologic examination.

1	shown superiority for aromatase inhibitors over tamoxifen alone in the adjuvant setting, although tamoxifen appears essentially equivalent in women who are obese and therefore presumably have higher endogenous concentrations of estrogen. Unfortunately the optimal plan is unclear. Tamoxifen for 5 years followed by an aromatase inhibitor, the reverse strategy, or even switching to an aromatase inhibitor after 2–3 years of tamoxifen has been shown to be better than tamoxifen alone. Continuation of tamoxifen for 10 years yields further benefit and is a reasonable decision for women with less favorable prognoses. Unfortunately, multiple studies have revealed very suboptimal adherence to long-term adjuvant endocrine regimens, and every effort should be made to encourage their continuous use. No valid information currently permits selection among the three clinically approved aromatase inhibitors. Concomitant use of bisphosphonates is almost always warranted; however, it is not finally

1	No valid information currently permits selection among the three clinically approved aromatase inhibitors. Concomitant use of bisphosphonates is almost always warranted; however, it is not finally settled as to whether their prophylactic use increases survival in addition to just decreasing recurrences in bone.

1	Most comparisons of adjuvant chemotherapy regimens show little difference among them, although small advantages for doxorubicincontaining regimens and “dose-dense” regimens are usually seen.

1	One approach—so-called neoadjuvant chemotherapy—involves the administration of adjuvant therapy before definitive surgery and radiation therapy. Because the objective response rates of patients with breast cancer to systemic therapy in this setting exceed 75%, many patients will be “downstaged” and may become candidates for breast-conserving therapy. However, overall survival has not been improved using this approach as compared with the same drugs given postoperatively. Patients who achieve a pathologic complete remission after neoadjuvant chemotherapy not unexpectedly have a substantially improved survival. The neoadjuvant setting also provides a wonderful opportunity for the evaluation of new agents. For example, a second HER2 targeting antibody, pertuzumab, has been shown to provide additional benefit when combined with trastuzumab in the neoadjuvant setting.

1	Other adjuvant treatments under investigation include the use of taxanes, such as paclitaxel and docetaxel, and therapy based on alternative kinetic and biologic models. In such approaches, high doses of single agents are used separately in relatively dose-intensive cycling regimens. Node-positive patients treated with doxorubicincyclophosphamide for four cycles followed by four cycles of a taxane have a substantial improvement in survival compared with women receiving doxorubicin-cyclophosphamide alone, particularly in women with estrogen receptor–negative tumors. In addition, administration of the same drug combinations at the same dose but at more frequent intervals (every 2 weeks with cytokine support as compared with the standard every 3 weeks) is even more effective. Among the 25% of women whose tumors overexpress HER2/neu, addition of trastuzumab given concurrently with a taxane and then for a year after chemotherapy produces significant improvement in survival. Although longer

1	women whose tumors overexpress HER2/neu, addition of trastuzumab given concurrently with a taxane and then for a year after chemotherapy produces significant improvement in survival. Although longer follow-up will be important, this is now the standard care for most women with HER2/neu-positive breast cancers. Cardiotoxicity, immediate and long-term, remains a concern, and further efforts to exploit non-anthracycline-containing regimens are being pursued. Very-high-dose therapy with stem cell transplantation in the adjuvant setting has not proved superior to standard-dose therapy and should not be routinely used.

1	A variety of exciting approaches are close to adoption, and the literature needs to be followed attentively. Tyrosine kinase inhibitors such as lapatinib and additional HER2-targeting antibodies such as pertuzumab are very promising. Finally, as described in the next section, a novel class of agents targeting DNA repair—the so-called poly–ADP ribose polymerase (PARP) inhibitors—is likely to have a major effect on breast cancers either caused by BRCA1 or BRCA2 mutations or sharing similar defects in DNA repair in their etiology.

1	About one-third of patients treated for apparently localized breast cancer develop metastatic disease. Although a small number of these patients enjoy long remissions when treated with combinations of systemic and local therapy, most eventually succumb to metastatic disease. The median survival for all patients diagnosed with metastatic breast cancer is less than 3 years. Soft tissue, bony, and visceral (lung and liver) metastases each account for approximately one-third of sites of initial relapses. However, by the time of death, most patients will have bony involvement. Recurrences can appear at any time after primary therapy. A very cruel fact about breast cancer recurrences is that at least half of all breast cancer recurrences occur >5 years after initial therapy. It is now clear that a variety of host factors can influence recurrence rates, including depression and central obesity, and these diseases should be managed as aggressively as possible.

1	Because the diagnosis of metastatic disease alters the outlook for the patient so drastically, it should rarely be made without a confirmatory biopsy. Every oncologist has seen patients with tuberculosis, gallstones, sarcoidosis, or other nonmalignant diseases misdiagnosed and treated as though they had metastatic breast cancer or even second malignancies such as multiple myeloma thought to be recurrent breast cancer. This is a catastrophic mistake and justifies biopsy for virtually every patient at the time of initial suspicion of metastatic disease. Furthermore, there are well-documented changes in hormone receptor status that can occur and substantially alter treatment decisions.

1	The choice of therapy requires consideration of local therapy needs, the overall medical condition of the patient, and the hormone receptor status of the tumor, as well as clinical judgment. Because therapy of systemic disease is palliative, the potential toxicities of therapies should be balanced against the response rates. Several variables influence the response to systemic therapy. For example, the presence of estrogen and progesterone receptors is a strong indication for endocrine therapy. On the other hand, patients 530 with short disease-free intervals, rapidly progressive visceral disease, lymphangitic pulmonary disease, or intracranial disease are unlikely to respond to endocrine therapy. In many cases, systemic therapy can be withheld while the patient is managed with appropriate local therapy. Radiation therapy and occasionally surgery are effective at relieving the symptoms of metastatic disease, particularly when bony sites are involved. Many patients with bone-only or

1	local therapy. Radiation therapy and occasionally surgery are effective at relieving the symptoms of metastatic disease, particularly when bony sites are involved. Many patients with bone-only or bone-dominant disease have a relatively indolent course. Under such circumstances, systemic chemotherapy has a modest effect, whereas radiation therapy may be effective for long periods. Other systemic treatments, such as strontium-89 and/ or bisphosphonates, may provide a palliative benefit without inducing objective responses. Most patients with metastatic disease, and certainly all who have bone involvement, should receive concurrent bisphosphonates. Because the goal of therapy is to maintain well-being for as long as possible, emphasis should be placed on avoiding the most hazardous complications of metastatic disease, including pathologic fracture of the axial skeleton and spinal cord compression. New back pain in patients with cancer should be explored aggressively on an emergent basis;

1	of metastatic disease, including pathologic fracture of the axial skeleton and spinal cord compression. New back pain in patients with cancer should be explored aggressively on an emergent basis; to wait for neurologic symptoms is a potentially catastrophic error. Metastatic involvement of endocrine organs can cause profound dysfunction, including adrenal insufficiency and hypopituitarism. Similarly, obstruction of the biliary tree or other impaired organ function may be better managed with a local therapy than with a systemic approach. Many patients are inappropriately treated with toxic regimens into their last days of life. Often oncologists are unwilling to have the difficult conversations that are required with patients nearing the end of life, and not uncommonly, patients and families can pressure physicians into treatments with very little survival value. Palliative care consultation and realistic assessment of treatment expectations need to be reviewed with patients and

1	families can pressure physicians into treatments with very little survival value. Palliative care consultation and realistic assessment of treatment expectations need to be reviewed with patients and families. We urge consideration of palliative care consultations for patients who have received at least two lines of therapy for metastatic disease.

1	Endocrine Therapy Normal breast tissue is estrogen dependent. Both primary and metastatic breast cancer may retain this phenotype. The best means of ascertaining whether a breast cancer is hormone dependent is through analysis of estrogen and progesterone receptor levels on the tumor. Tumors that are positive for the estrogen receptor and negative for the progesterone receptor have a response rate of ∼30%. Tumors that are positive for both receptors have a response rate approaching 70%. If neither receptor is present, the objective response rates are <5%. Receptor analyses provide information as to the correct ordering of endocrine therapies as opposed to chemotherapy. Because of their lack of toxicity and because some patients whose receptor analyses are reported as negative respond to endocrine therapy, an endocrine treatment should be attempted in virtually every patient with metastatic breast cancer. Potential endocrine therapies are summarized in Table 108-4. The choice of

1	to endocrine therapy, an endocrine treatment should be attempted in virtually every patient with metastatic breast cancer. Potential endocrine therapies are summarized in Table 108-4. The choice of endocrine therapy is usually determined by toxicity profile and availability. In most postmenopausal patients, the initial endocrine therapy should be an aromatase inhibitor rather than tamoxifen. For the subset of postmenopausal women who are estrogen receptor–positive but also HER2/neu-positive, response rates to aromatase inhibitors are substantially higher than to tamoxifen. Aromatase inhibitors are not used in premenopausal women because their hypothalamus can respond to estrogen deprivation by producing gonadotropins that promote estrogen synthesis. Newer “pure” antiestrogens that are free of agonistic effects are also effective. Cases in which tumors shrink in response to tamoxifen withdrawal (as well as withdrawal of pharmacologic doses of estrogens) have been reported. A series of

1	of agonistic effects are also effective. Cases in which tumors shrink in response to tamoxifen withdrawal (as well as withdrawal of pharmacologic doses of estrogens) have been reported. A series of studies with aromatase inhibitors, tamoxifen, and fulvestrant have all shown that the addition of everolimus to the hormonal treatment can lead to significant benefit after progression on the endocrine agent alone. Everolimus (an mTOR inhibitor) in coordination with endocrine agents is now being explored as front-line therapy and in the adjuvant setting. Endogenous estrogen formation may be blocked by analogues of aromatase inhibitors, tamoxifen, and aConsider retreatment with Everolimus in combination for disease progression

1	Abbreviation: LHRH, luteinizing hormone–releasing hormone. luteinizing hormone–releasing hormone in premenopausal women. Additive endocrine therapies, including treatment with progestogens, estrogens, and androgens, may also be tried in patients who respond to initial endocrine therapy; the mechanism of action of these latter therapies is unknown. Patients who respond to one endocrine therapy have at least a 50% chance of responding to a second endocrine therapy. It is not uncommon for patients to respond to two or three sequential endocrine therapies; however, combination endocrine therapies do not appear to be superior to individual agents, and combinations of chemotherapy with endocrine therapy are not useful. The median survival of patients with metastatic disease is approximately 2 years, although many patients, particularly older persons and those with hormone-dependent disease, may respond to endocrine therapy for 3–5 years or longer.

1	Chemotherapy Unlike many other epithelial malignancies, breast cancer responds to multiple chemotherapeutic agents, including anthracyclines, alkylating agents, taxanes, and antimetabolites. Multiple combinations of these agents have been found to improve response rates somewhat, but they have had little effect on duration of response or survival. The choice among multidrug combinations frequently depends on whether adjuvant chemotherapy was administered and, if so, what type. Although patients treated with adjuvant regimens such as cyclophosphamide, methotrexate, and fluorouracil (CMF regimens) may subsequently respond to the same combination in the metastatic disease setting, most oncologists use drugs to which the patients have not been previously exposed. Once patients have progressed after combination drug therapy, it is most common to treat them with single agents. Given the significant toxicity of most drugs, the use of a single effective agent will minimize toxicity by sparing

1	after combination drug therapy, it is most common to treat them with single agents. Given the significant toxicity of most drugs, the use of a single effective agent will minimize toxicity by sparing the patient exposure to drugs that would be of little value. No method to select the drugs most efficacious for a given patient has been demonstrated to be useful.

1	Most oncologists use either an anthracycline or paclitaxel following failure with the initial regimen. However, the choice has to be balanced with individual needs. One randomized study has suggested that docetaxel may be superior to paclitaxel. A nanoparticle formulation of paclitaxel (Abraxane) is also effective.

1	The use of a humanized antibody to erbB2 (trastuzumab [Herceptin]) combined with paclitaxel can improve response rate and survival for women whose metastatic tumors overexpress erbB2. A novel antibody conjugate (ADC) that links trastuzumab to a cytotoxic agent has been approved for management of HER2-positive breast cancer. The magnitude of the survival extension is modest in patients with metastatic disease. Similarly, the use of bevacizumab (Avastin) has improved the response rate and response duration to paclitaxel. Objective responses in previously treated patients may also be seen with gemcitabine, vinca alkaloids, capecitabine, vinorelbine, and oral etoposide, as well as a new class of agents, epothilones. There are few comparative trials of one agent versus another in metastatic disease. It is a sad fact that choices are often influenced by aggressive marketing of new very expensive agents that have not been shown to be superior to other generic agents. Platinum-based agents

1	disease. It is a sad fact that choices are often influenced by aggressive marketing of new very expensive agents that have not been shown to be superior to other generic agents. Platinum-based agents have become far more widely used in both the adjuvant and advanced disease settings for some breast cancers, particularly those of the “triple-negative” subtype.

1	high-dose chemotheraPy including autologous Bone marrow transPlantation Autologous bone marrow transplantation combined with high doses of single agents can produce objective responses even in heavily pretreated patients. However, such responses are rarely durable and do not alter the clinical course for most patients with advanced metastatic disease.

1	Between 10 and 25% of patients present with so-called locally advanced, or stage III, breast cancer at diagnosis. Many of these cancers are technically operable, whereas others, particularly cancers with chest wall involvement, inflammatory breast cancers, or cancers with large matted axillary lymph nodes, cannot be managed with surgery initially. Although no randomized trials have shown any survival benefit for neoadjuvant regimens as compared to adjuvant therapy, this approach has gained widespread use. More than 90% of patients with locally advanced breast cancer show a partial or better response to multidrug chemotherapy regimens that include an anthracycline. Early administration of this treatment reduces the bulk of the disease and frequently makes the patient a suitable candidate for salvage surgery and/or radiation therapy. These patients should be managed in multimodality clinics to coordinate surgery, radiation therapy, and systemic chemotherapy. Such approaches produce

1	for salvage surgery and/or radiation therapy. These patients should be managed in multimodality clinics to coordinate surgery, radiation therapy, and systemic chemotherapy. Such approaches produce long-term disease-free survival in about 30–50% of patients. The neoadjuvant setting is also an ideal time to evaluate the efficacy of novel treatments because the effect on the tumor can be directly assessed.

1	Women who have one breast cancer are at risk of developing a contralateral breast cancer at a rate of approximately 0.5% per year. When adjuvant tamoxifen or an aromatase inhibitor is administered to these patients, the rate of development of contralateral breast cancers is reduced. In other tissues of the body, tamoxifen has estrogen-like effects that are beneficial, including preservation of bone mineral density and long-term lowering of cholesterol. However, tamoxifen has estrogen-like effects on the uterus, leading to an increased risk of uterine cancer (0.75% incidence after 5 years on tamoxifen). Tamoxifen also increases the risk of cataract formation. The Breast Cancer Prevention Trial (BCPT) revealed a >49% reduction in breast cancer among women with a risk of at least 1.66% taking the drug for 5 years. Raloxifene has shown similar breast cancer prevention potency but may have different effects on bone and heart. The two agents have been compared in a prospective randomized

1	the drug for 5 years. Raloxifene has shown similar breast cancer prevention potency but may have different effects on bone and heart. The two agents have been compared in a prospective randomized prevention trial (the Study of Tamoxifen and Raloxifene [STAR] trial). The agents are approximately equivalent in preventing breast cancer with fewer thromboembolic events and endometrial cancers with raloxifene; however, raloxifene did not reduce noninvasive cancers as effectively as tamoxifen, so no clear winner has emerged. A newer selective estrogen receptor modulator (SERM), lasofoxifene, has been shown to reduce cardiovascular events in addition to breast cancer and fractures, and further studies of this agent should be watched with interest. It should be recalled that prevention of contralateral breast cancers in women diagnosed with one cancer is a reasonable surrogate for breast cancer prevention because these are second primaries not recurrences. In this regard, the aromatase

1	breast cancers in women diagnosed with one cancer is a reasonable surrogate for breast cancer prevention because these are second primaries not recurrences. In this regard, the aromatase inhibitors are all considerably more effective than tamoxifen; however, they are not approved for primary breast cancer prevention. It remains puzzling that agents with the safety 531 profile of raloxifene, which can reduce breast cancer risk by 50% with additional benefits in preventing osteoporotic fracture, are still so infrequently prescribed. They should be far more commonly offered to women than they are.

1	Breast cancer develops as a series of molecular changes in the epithelial cells that lead to ever more malignant behavior. Increased use of mammography has led to more frequent diagnoses of noninvasive breast cancer. These lesions fall into two groups: ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (lobular neoplasia). The management of both entities is controversial.

1	Ductal Carcinoma In Situ Proliferation of cytologically malignant breast epithelial cells within the ducts is termed DCIS. Atypical hyperplasia may be difficult to differentiate from DCIS. At least one-third of patients with untreated DCIS develop invasive breast cancer within 5 years. However, many low-grade DCIS lesions do not appear to progress over many years; therefore, many patients are overtreated. Unfortunately there is no reliable means of distinguishing patients who require treatment from those who may be safely observed. For many years, the standard treatment for this disease was mastectomy. However, treatment of this condition by lumpectomy and radiation therapy gives survival that is as good as the survival for invasive breast cancer treated by mastectomy. In one randomized trial, the combination of wide excision plus irradiation for DCIS caused a substantial reduction in the local recurrence rate as compared with wide excision alone with negative margins, although

1	trial, the combination of wide excision plus irradiation for DCIS caused a substantial reduction in the local recurrence rate as compared with wide excision alone with negative margins, although survival was identical in the two arms. No studies have compared either of these regimens to mastectomy. Addition of tamoxifen to any DCIS surgical/radiation therapy regimen further improves local control. Data for aromatase inhibitors in this setting are not available.

1	Several prognostic features may help to identify patients at high risk for local recurrence after either lumpectomy alone or lumpectomy with radiation therapy. These include extensive disease; age <40; and cytologic features such as necrosis, poor nuclear grade, and comedo subtype with overexpression of erbB2. Some data suggest that adequate excision with careful determination of pathologically clear margins is associated with a low recurrence rate. When surgery is combined with radiation therapy, recurrence (which is usually in the same quadrant) occurs with a frequency of ≤10%. Given the fact that half of these recurrences will be invasive, about 5% of the initial cohort will eventually develop invasive breast cancer. A reasonable expectation of mortality for these patients is about 1%, a figure that approximates the mortality rate for DCIS managed by mastectomy. Although this train of reasoning has not formally been proved valid, it is reasonable to recommend that patients who

1	1%, a figure that approximates the mortality rate for DCIS managed by mastectomy. Although this train of reasoning has not formally been proved valid, it is reasonable to recommend that patients who desire breast preservation, and in whom DCIS appears to be reasonably localized, be managed by adequate surgery with meticulous pathologic evaluation, followed by breast irradiation and tamoxifen. For patients with localized DCIS, axillary lymph node dissection is unnecessary. More controversial is the question of what management is optimal when there is any degree of invasion. Because of a significant likelihood (10–15%) of axillary lymph node involvement even when the primary lesion shows only microscopic invasion, it is prudent to do at least a sentinel lymph node sampling for all patients with any degree of invasion. Further management is dictated by the presence of nodal spread.

1	Lobular Neoplasia Proliferation of cytologically malignant cells within the lobules is termed lobular neoplasia. Nearly 30% of patients who have had adequate local excision of the lesion develop breast cancer (usually infiltrating ductal carcinoma) over the next 15–20 years. Ipsilateral and contralateral cancers are equally common. Therefore, lobular neoplasia may be a premalignant lesion that suggests an elevated risk of subsequent breast cancer, rather than a form of malignancy itself, and aggressive local management seems unreasonable. Most patients should be treated with an SERM or an aromatase inhibitor (for postmenopausal women) for 5 years and 532 followed with careful annual mammography and semiannual physical examinations. Additional molecular analysis of these lesions may make it possible to discriminate between patients who are at risk of further progression and require additional therapy and those in whom simple follow-up is adequate.

1	Breast cancer is about 1/150th as frequent in men as in women; 1720 men developed breast cancer in 2006. It usually presents as a unilateral lump in the breast and is frequently not diagnosed promptly. Given the small amount of soft tissue and the unexpected nature of the problem, locally advanced presentations are somewhat more common. When male breast cancer is matched to female breast cancer by age and stage, its overall prognosis is identical. Although gynecomastia may initially be unilateral or asymmetric, any unilateral mass in a man older than age 40 years should receive a careful workup including biopsy. On the other hand, bilateral symmetric breast development rarely represents breast cancer and is almost invariably due to endocrine disease or a drug effect. It should be kept in mind, nevertheless, that the risk of cancer is much greater in men with gynecomastia; in such men, gross asymmetry of the breasts should arouse suspicion of cancer. Male breast cancer is best managed

1	mind, nevertheless, that the risk of cancer is much greater in men with gynecomastia; in such men, gross asymmetry of the breasts should arouse suspicion of cancer. Male breast cancer is best managed by mastectomy and axillary lymph node dissection or SLNB. Patients with locally advanced disease or positive nodes should also be treated with irradiation. Approximately 90% of male breast cancers contain estrogen receptors, and approximately 60% of cases with metastatic disease respond to endocrine therapy. No randomized studies have evaluated adjuvant therapy for male breast cancer. Two historic experiences suggest that the disease responds well to adjuvant systemic therapy, and, if not medically contraindicated, the same criteria for the use of adjuvant therapy in women should be applied to men.

1	The sites of relapse and spectrum of response to chemotherapeutic drugs are virtually identical for breast cancers in either sex. Despite the availability of sophisticated and expensive imaging techniques and a wide range of serum tumor marker tests, survival is not influenced by early diagnosis of relapse. Surveillance guidelines are given in Table 108-5. Despite pressure from patients and their families, routine computed tomography scans (or other imaging) are not recommended. SERMs) Patient education about symptoms Ongoing of recurrence Coordination of care Ongoing Complete blood count Serum chemistry studies Chest radiographs Bone scans Ultrasound examination of the liver Computed tomography of chest, abdomen, or pelvis Tumor markers CA 15-3, CA 27-29, CEA Abbreviations: CEA, carcinoembryonic antigen; SERM, selective estrogen receptor modulator. Source: Recommended Breast Cancer Surveillance Guidelines, ASCO Education Book, Fall, 1997. Robert J. Mayer

1	Source: Recommended Breast Cancer Surveillance Guidelines, ASCO Education Book, Fall, 1997. Robert J. Mayer Upper gastrointestinal cancers include malignancies arising in the esophagus, stomach, and small intestine. Cancer of the esophagus is an increasingly common and extremely lethal malignancy. The diagnosis was made in 18,170 Americans in 2014 and led to 15,450 deaths. Almost all esophageal cancers are either squamous cell carcinomas or adenocarcinomas; the two histologic subtypes have a similar clinical presentation but different causative factors.

1	Worldwide, squamous cell carcinoma is the more common cell type, having an incidence that rises strikingly in association with geographic location. It occurs frequently within a region extending from the southern shore of the Caspian Sea on the west to northern China on the east, encompassing parts of Iran, central Asia, Afghanistan, Siberia, and Mongolia. Familial increased risk has been observed in regions with high incidence, although gene associations are not yet defined. High-incidence “pockets” of the disease are also present in such disparate locations as Finland, Iceland, Curaçao, southeastern Africa, and northwestern France. In North America and western Europe, the disease is more common in blacks than whites and in males than females; it appears most often after age 50 and seems to be associated with a lower socioeconomic status. Such cancers generally arise in the cervical and thoracic portions of the esophagus.

1	A variety of causative factors have been implicated in the development of squamous cell cancers of the esophagus (Table 109-1). In the United States, the etiology of such cancers is primarily related to excess alcohol consumption and/or cigarette smoking. The relative risk increases with the amount of tobacco smoked or alcohol consumed, with these factors acting synergistically. The consumption of whiskey is linked to a higher incidence than the consumption of wine or beer. Squamous cell esophageal carcinoma has also been associated with the ingestion of nitrates, smoked opiates, and fungal toxins in pickled vegetables, as well as mucosal damage caused by such physical insults as long-term exposure to extremely hot tea, the ingestion of lye, radiation-induced strictures, and chronic achalasia. The presence of an esophageal web in association with glossitis and iron deficiency (i.e., Plummer-Vinson or Paterson-Kelly syndrome) and congenital some etioLogiC faCtors assoCiateD With

1	achalasia. The presence of an esophageal web in association with glossitis and iron deficiency (i.e., Plummer-Vinson or Paterson-Kelly syndrome) and congenital some etioLogiC faCtors assoCiateD With squamous CeLL CanCer of the esophagus

1	Nitrates (converted to nitrites) Smoked opiates Fungal toxins in pickled vegetables Esophageal web with glossitis and iron deficiency (i.e., Plummer-Vinson or Paterson-Kelly syndrome) Congenital hyperkeratosis and pitting of the palms and soles (i.e., tylosis palmaris et plantaris) ? Dietary deficiencies of selenium, molybdenum, zinc, and vitamin A some etioLogiC faCtors assoCiateD With aDenoCarCinoma of the esophagus hyperkeratosis and pitting of the palms and soles (i.e., tylosis palmaris et plantaris) have each been linked with squamous cell esophageal cancer, as have dietary deficiencies of molybdenum, zinc, selenium, and vitamin A. Patients with head and neck cancer are at increased risk of squamous cell cancer of the esophagus.

1	For unclear reasons, the incidence of squamous cell esophageal cancer has decreased somewhat in both the black and white populations in the United States over the past 40 years, whereas the rate of adenocarcinoma has risen sevenfold, particularly in white males (male-tofemale ratio of 6:1). Whereas squamous cell cancers comprised the vast majority of esophageal cancers in the United States as recently as 40–50 years ago, more than 75% of esophageal tumors are now adenocarcinomas, with the incidence of this histologic subtype continuing to increase rapidly. Understanding the cause for this increase is the focus of current investigation.

1	Several strong etiologic associations have been observed to account for the development of adenocarcinoma of the esophagus (Table 109-2). Such tumors arise in the distal esophagus in association with chronic gastric reflux, often in the presence of Barrett’s esophagus (replacement of the normal squamous epithelium of the distal esophagus by columnar mucosa), which occurs more commonly in obese individuals. Adenocarcinomas arise within dysplastic columnar epithelium in the distal esophagus. Even before frank neoplasia is detectable, aneuploidy and p53 mutations are found in the dysplastic epithelium. These adenocarcinomas behave clinically like gastric adenocarcinomas, although they are not associated with Helicobacter pylori infections. Approximately 15% of esophageal adenocarcinomas overexpress the HER2/neu gene.

1	About 5% of esophageal cancers occur in the upper third of the esophagus (cervical esophagus), 20% in the middle third, and 75% in the lower third. Squamous cell carcinomas and adenocarcinomas cannot be distinguished radiographically or endoscopically.

1	Progressive dysphagia and weight loss of short duration are the initial symptoms in the vast majority of patients. Dysphagia initially occurs with solid foods and gradually progresses to include semisolids and liquids. By the time these symptoms develop, the disease is already very advanced, because difficulty in swallowing does not occur until >60% of the esophageal circumference is infiltrated with cancer. Dysphagia may be associated with pain on swallowing (odynophagia), pain radiating to the chest and/or back, regurgitation or vomiting, and aspiration pneumonia. The disease most commonly spreads to adjacent and supraclavicular lymph nodes, liver, lungs, pleura, and bone. Tracheoesophageal fistulas may develop, primarily in patients with upper and mid-esophageal tumors. As with other squamous cell carcinomas, hypercalcemia may occur in the absence of osseous metastases, probably from parathormone-related peptide secreted by tumor cells (Chap. 121).

1	Attempts at endoscopic and cytologic screening for carcinoma in patients with Barrett’s esophagus, while effective as a means of detecting high-grade dysplasia, have not yet been shown to reduce the likelihood of death from esophageal adenocarcinoma. Esophagoscopy should be performed in all patients suspected of having an esophageal abnormality, to both visualize and identify a tumor and also to obtain histopathologic confirmation of the diagnosis. Because the population of persons at risk for squamous cell carcinoma of the esophagus (i.e., smokers and drinkers) also has a high rate of cancers of the lung and the head and neck region, endoscopic inspection of the larynx, trachea, 533 and bronchi should also be carried out. A thorough examination of the fundus of the stomach (by retroflexing the endoscope) is imperative as well. The extent of tumor spread to the mediastinum and para-aortic lymph nodes should be assessed by computed tomography (CT) scans of the chest and abdomen and by

1	the endoscope) is imperative as well. The extent of tumor spread to the mediastinum and para-aortic lymph nodes should be assessed by computed tomography (CT) scans of the chest and abdomen and by endoscopic ultrasound. Positron emission tomography scanning provides a useful assessment of the presence of distant metastatic disease, offering accurate information regarding spread to mediastinal lymph nodes, which can be helpful in defining radiation therapy fields. Such scans, when performed sequentially, appear to provide a means of making an early assessment of responsiveness to preoperative chemotherapy.

1	The prognosis for patients with esophageal carcinoma is poor. Approximately 10% of patients survive 5 years after the diagnosis; thus, management focuses on symptom control. Surgical resection of all gross tumor (i.e., total resection) is feasible in only 45% of cases, with residual tumor cells frequently present at the resection margins. Such esophagectomies have been associated with a postoperative mortality rate of approximately 5% due to anastomotic fistulas, subphrenic abscesses, and cardiopulmonary complications. Although debate regarding the comparative benefits of trans-thoracic versus transhiatal resections has continued, experienced thoracic surgeons are now favoring minimally invasive transthoracic esophagectomies. Endoscopic resections of superficial squamous cell cancers or adenocarcinomas are being examined but have not yet been shown to result in a similar likelihood of survival as observed with conventional surgical procedures. Similarly, the value of endoscopic

1	or adenocarcinomas are being examined but have not yet been shown to result in a similar likelihood of survival as observed with conventional surgical procedures. Similarly, the value of endoscopic ablation of dysplastic lesions in an area of Barrett’s esophagus on reducing subsequent mortality from esophageal carcinoma is uncertain. Some experts have advocated fundoplication surgery (i.e., the removal of the gastroesophageal junction) as a means of cancer prevention in patients with Barrett’s esophagus; again, objective data are not yet available to fully assess the risks versus benefits of this invasive procedure. About 20% of patients who survive a total surgical resection live for 5 years. The evaluation of chemotherapeutic agents in patients with esophageal carcinoma has been hampered by ambiguity in the definition of “response” and the debilitated physical condition of many treated individuals, particularly those with squamous cell cancers. Nonetheless, significant reductions in

1	by ambiguity in the definition of “response” and the debilitated physical condition of many treated individuals, particularly those with squamous cell cancers. Nonetheless, significant reductions in the size of measurable tumor masses have been reported in 15–25% of patients given single-agent treatment and in 30–60% of patients treated with drug combinations that include cisplatin. In the small subset of patients whose tumors overexpress the HER2/neu gene, the addition of the monoclonal antibody trastuzumab (Herceptin) appears to further enhance the likelihood of benefit, particularly in patients with gastroesophageal lesions. The use of the antiangiogenic agent bevacizumab (Avastin) seems to be of limited value in the setting of esophageal cancer. Combination chemotherapy and radiation therapy as the initial therapeutic approach, either alone or followed by an attempt at operative resection, seems to be beneficial. When administered along with radiation therapy, chemotherapy

1	therapy as the initial therapeutic approach, either alone or followed by an attempt at operative resection, seems to be beneficial. When administered along with radiation therapy, chemotherapy produces a better survival outcome than radiation therapy alone. The use of preoperative chemotherapy and radiation therapy followed by esophageal resection appears to prolong survival compared with surgery alone according to several randomized trials and a meta-analysis; some reports suggest that no additional benefit accrues when surgery is added if significant shrinkage of tumor has been achieved by the chemoradiation combination.

1	For the incurable, surgically unresectable patient with esophageal cancer, dysphagia, malnutrition, and the management of tracheoesophageal fistulas are major issues. Approaches to palliation include repeated endoscopic dilatation, the surgical placement of a gastrostomy or jejunostomy for hydration and feeding, endoscopic placement of an expansive metal stent to bypass the tumor, and radiation therapy. Incidence and Epidemiology For unclear reasons, the incidence and mortality rates for gastric cancer have decreased in the

1	Incidence and Epidemiology For unclear reasons, the incidence and mortality rates for gastric cancer have decreased in the United States during the past 80 years, although the disease remains the second most frequent cause of worldwide cancer-related death. The mortality rate from gastric cancer in the United States has dropped in men from 28 to 5.8 per 100,000 persons, whereas in women, the rate has decreased from 27 to 2.8 per 100,000. Nonetheless, in 2014, 22,220 new cases of stomach cancer were diagnosed in the United States, and 10,990 Americans died of the disease. Although the incidence of gastric cancer has decreased worldwide, it remains high in such disparate geographic regions as Japan, China, Chile, and Ireland.

1	The risk of gastric cancer is greater among lower socioeconomic classes. Migrants from highto low-incidence nations maintain their susceptibility to gastric cancer, whereas the risk for their offspring approximates that of the new homeland. These findings suggest that an environmental exposure, probably beginning early in life, is related to the development of gastric cancer, with dietary carcinogens considered the most likely factor(s).

1	Pathology About 85% of stomach cancers are adenocarcinomas, with 15% due to lymphomas, gastrointestinal stromal tumors (GISTs), and leiomyosarcomas. Gastric adenocarcinomas may be subdivided into two categories: a diffuse type, in which cell cohesion is absent, so that individual cells infiltrate and thicken the stomach wall without forming a discrete mass; and an intestinal type, characterized by cohesive neoplastic cells that form glandlike tubular structures. The diffuse carcinomas occur more often in younger patients, develop throughout the stomach (including the cardia), result in a loss of distensibility of the gastric wall (so-called linitis plastica, or “leather bottle” appearance), and carry a poorer prognosis. Diffuse cancers have defective intercellular adhesion, mainly as a consequence of loss of expression of E-cadherin. Intestinal-type lesions are frequently ulcerative, more commonly appear in the antrum and lesser curvature of the stomach, and are often preceded by a

1	consequence of loss of expression of E-cadherin. Intestinal-type lesions are frequently ulcerative, more commonly appear in the antrum and lesser curvature of the stomach, and are often preceded by a prolonged precancerous process, often initiated by H. pylori infection. Although the incidence of diffuse carcinomas is similar in most populations, the intestinal type tends to predominate in the high-risk geographic regions and is less likely to be found in areas where the frequency of gastric cancer is declining. Thus, different etiologic factor(s) are likely involved in these two subtypes. In the United States, ∼30% of gastric cancers originate in the distal stomach, ∼20% arise in the midportion of the stomach, and ∼40% originate in the proximal third of the stomach. The remaining 10% involve the entire stomach.

1	Etiology The long-term ingestion of high concentrations of nitrates found in dried, smoked, and salted foods appears to be associated with a higher risk. The nitrates are thought to be converted to carcinogenic nitrites by bacteria (Table 109-3). Such bacteria may be introduced exogenously through the ingestion of partially decayed nitrate-Converting BaCteria as a faCtor in the Causation of gastriC CarCinomaa Exogenous sources of nitrate-converting bacteria: Bacterially contaminated food (common in lower socioeconomic classes, who have a higher incidence of the disease; diminished by improved food preservation and refrigeration) Helicobacter pylori infection

1	Endogenous factors favoring growth of nitrate-converting bacteria in the stomach: Decreased gastric acidity Prior gastric surgery (antrectomy) (15to 20-year latency period) Atrophic gastritis and/or pernicious anemia ? Prolonged exposure to histamine H2-receptor antagonists aHypothesis: Dietary nitrates are converted to carcinogenic nitrites by bacteria.

1	foods, which are consumed in abundance worldwide by the lower socioeconomic classes. Bacteria such as H. pylori may also contribute to this effect by causing chronic inflammatory atrophic gastritis, loss of gastric acidity, and bacterial growth in the stomach. Although the risk for developing gastric cancer is thought to be sixfold higher in people infected with H. pylori, it remains uncertain whether eradicating the bacteria after infection has already occurred actually reduces this risk. Loss of acidity may occur when acid-producing cells of the gastric antrum have been removed surgically to control benign peptic ulcer disease or when achlorhydria, atrophic gastritis, and even pernicious anemia develop in the elderly. Serial endoscopic examinations of the stomach in patients with atrophic gastritis have documented replacement of the usual gastric mucosa by intestinal-type cells. This process of intestinal metaplasia may lead to cellular atypia and eventual neoplasia. Because the

1	gastritis have documented replacement of the usual gastric mucosa by intestinal-type cells. This process of intestinal metaplasia may lead to cellular atypia and eventual neoplasia. Because the declining incidence of gastric cancer in the United States primarily reflects a decline in distal, ulcerating, intestinal-type lesions, it is conceivable that better food preservation and the availability of refrigeration for all socioeconomic classes have decreased the dietary ingestion of exogenous bacteria. H. pylori has not been associated with the diffuse, more proximal form of gastric carcinoma or with cancers arising at the gastroesophageal junction or in the distal esophagus. Approximately 10–15% of adenocarcinomas appearing in the proximal stomach, the gastroesophageal junction, and the distal esophagus overexpress the HER2/neu gene; individuals whose tumors demonstrate this over-expression benefit from treatment directed against this target (i.e., trastuzumab [Herceptin]).

1	Several additional etiologic factors have been associated with gastric carcinoma. Gastric ulcers and adenomatous polyps have occasionally been linked, but data on a cause-and-effect relationship are unconvincing. The inadequate clinical distinction between benign gastric ulcers and small ulcerating carcinomas may, in part, account for this presumed association. The presence of extreme hypertrophy of gastric rugal folds (i.e., Ménétrier’s disease), giving the impression of polypoid lesions, has been associated with a striking frequency of malignant transformation; such hypertrophy, however, does not represent the presence of true adenomatous polyps. Individuals with blood group A have a higher incidence of gastric cancer than persons with blood group O; this observation may be related to differences in the mucous secretion, leading to altered mucosal protection from carcinogens. A germline mutation in the E-cadherin gene (CDH1), inherited in an autosomal dominant pattern and coding for

1	in the mucous secretion, leading to altered mucosal protection from carcinogens. A germline mutation in the E-cadherin gene (CDH1), inherited in an autosomal dominant pattern and coding for a cell adhesion protein, has been linked to a high incidence of occult diffuse-type gastric cancers in young asymptomatic carriers. Duodenal ulcers are not associated with gastric cancer.

1	Clinical Features Gastric cancers, when superficial and surgically curable, usually produce no symptoms. As the tumor becomes more extensive, patients may complain of an insidious upper abdominal discomfort varying in intensity from a vague, postprandial fullness to a severe, steady pain. Anorexia, often with slight nausea, is very common but is not the usual presenting complaint. Weight loss may eventually be observed, and nausea and vomiting are particularly prominent in patients whose tumors involve the pylorus; dysphagia and early satiety may be the major symptoms caused by diffuse lesions originating in the cardia. There may be no early physical signs. A palpable abdominal mass indicates long-standing growth and predicts regional extension.

1	Gastric carcinomas spread by direct extension through the gastric wall to the perigastric tissues, occasionally adhering to adjacent organs such as the pancreas, colon, or liver. The disease also spreads via lymphatics or by seeding of peritoneal surfaces. Metastases to intraabdominal and supraclavicular lymph nodes occur frequently, as do metastatic nodules to the ovary (Krukenberg’s tumor), periumbilical region (“Sister Mary Joseph node”), or peritoneal cul-de-sac (Blumer’s shelf palpable on rectal or vaginal examination); malignant ascites may also develop. The liver is the most common site for hematogenous spread of tumor.

1	The presence of iron-deficiency anemia in men and of occult blood in the stool in both sexes mandates a search for an occult gastrointestinal tract lesion. A careful assessment is of particular importance in patients with atrophic gastritis or pernicious anemia. Unusual clinical features associated with gastric adenocarcinomas include migratory thrombophlebitis, microangiopathic hemolytic anemia, diffuse seborrheic keratoses (so-called Leser-Trélat sign), and acanthosis nigricans. Diagnosis The use of double-contrast radiographic examinations has been supplanted by esophagogastroscopy and CT scanning for the evaluation of patients with epigastric complaints.

1	Diagnosis The use of double-contrast radiographic examinations has been supplanted by esophagogastroscopy and CT scanning for the evaluation of patients with epigastric complaints. Gastric ulcers identified at the time of such endoscopic procedure may appear benign but merit biopsy in order to exclude a malignancy. Malignant gastric ulcers must be recognized before they penetrate into surrounding tissues, because the rate of cure of early lesions limited to the mucosa or submucosa is >80%. Because gastric carcinomas are difficult to distinguish clinically or endoscopically from gastric lymphomas, endoscopic biopsies should be made as deeply as possible, due to the submucosal location of lymphoid tumors. The staging system for gastric carcinoma is shown in Table 109-4. Data from ACS in the United States No. of 5-Year Stage TNM Features Cases, % Survival, % Abbreviations: ACS, American Cancer Society; TNM, tumor, node, metastasis.

1	Complete surgical removal of the tumor with resection of adjacent lymph nodes offers the only chance for cure. However, this is possible in less than a third of patients. A subtotal gastrectomy is the treatment of choice for patients with distal carcinomas, whereas total or near-total gastrectomies are required for more proximal tumors. The inclusion of extended lymph node dissection in these procedures appears to confer an added risk for complications without providing a meaningful enhancement in survival. The prognosis following complete surgical resection depends on the degree of tumor penetration into the stomach wall and is adversely influenced by regional lymph node involvement and vascular invasion, characteristics found in the vast majority of American patients. As a result, the probability of survival after 5 years for the 25–30% of patients able to undergo complete resection is ∼20% for distal tumors and <10% for proximal tumors, with recurrences continuing for at least 8

1	probability of survival after 5 years for the 25–30% of patients able to undergo complete resection is ∼20% for distal tumors and <10% for proximal tumors, with recurrences continuing for at least 8 years after surgery. In the absence of ascites or extensive hepatic or peritoneal metastases, even patients whose disease is believed to be incurable by surgery should be offered resection of the primary lesion. Reduction of tumor bulk is the best form of palliation and may enhance the probability of benefit from subsequent therapy. In high-incidence regions such as Japan and Korea, where the use of endoscopic screening programs has identified patients with superficial tumors, the use of laparoscopic gastrectomy has gained popularity. In the United States and western Europe, the use of this less invasive surgical approach remains investigational.

1	Gastric adenocarcinoma is a relatively radioresistant tumor, and the adequate control of the primary tumor requires doses of external-beam irradiation that exceed the tolerance of surrounding structures, such as bowel mucosa and spinal cord. As a result, the major role of radiation therapy in patients has been palliation of pain. Radiation therapy alone after a complete resection does not prolong survival. In the setting of surgically unresectable disease limited to the epigastrium, patients treated with 3500–4000 cGy did not live longer than similar patients not receiving radiotherapy; however, survival was prolonged slightly when 5-fluorouracil (5-FU) plus leucovorin was given in combination with radiation therapy (3-year survival 50% vs 41% for radiation therapy alone). In this clinical setting, the 5-FU likely functions as a radiosensitizer.

1	The administration of combinations of cytotoxic drugs to patients with advanced gastric carcinoma has been associated with partial responses in 30–50% of cases; responders appear to benefit from treatment. Such drug combinations have generally included cisplatin combined with epirubicin or docetaxel and infusional 5-FU or capecitabine, or with irinotecan. Despite the encouraging response rates, complete remissions are uncommon, the partial responses are transient, and the overall impact of multidrug therapy on survival has been limited; the median survival time for patients treated in this manner remains less than 12 months. As with adenocarcinomas arising in the esophagus, the addition of bevacizumab (Avastin) to chemotherapy regimens in treating gastric cancer appears to provide limited benefit. However, preliminary results utilizing another antiangiogenic compound—ramucirumab (Cyranza)—in the treatment of gastric cancer are encouraging. The use of adjuvant chemotherapy alone

1	benefit. However, preliminary results utilizing another antiangiogenic compound—ramucirumab (Cyranza)—in the treatment of gastric cancer are encouraging. The use of adjuvant chemotherapy alone following the complete resection of a gastric cancer has only minimally improved survival. However, combination chemotherapy administered before and after surgery (perioperative treatment) as well as postoperative chemotherapy combined with radiation therapy reduces the recurrence rate and prolongs survival.

1	Primary lymphoma of the stomach is relatively uncommon, accounting for <15% of gastric malignancies and ∼2% of all lymphomas. The stomach is, however, the most frequent extranodal site for lymphoma, and gastric lymphoma has increased in frequency during the past 536 35 years. The disease is difficult to distinguish clinically from gastric adenocarcinoma; both tumors are most often detected during the sixth decade of life; present with epigastric pain, early satiety, and generalized fatigue; and are usually characterized by ulcerations with a ragged, thickened mucosal pattern demonstrated by contrast radiographs or endoscopic appearance. The diagnosis of lymphoma of the stomach may occasionally be made through cytologic brushings of the gastric mucosa but usually requires a biopsy at gastroscopy or laparotomy. Failure of gastroscopic biopsies to detect lymphoma in a given case should not be interpreted as being conclusive, because superficial biopsies may miss the deeper lymphoid

1	gastroscopy or laparotomy. Failure of gastroscopic biopsies to detect lymphoma in a given case should not be interpreted as being conclusive, because superficial biopsies may miss the deeper lymphoid infiltrate. The macroscopic pathology of gastric lymphoma may also mimic adenocarcinoma, consisting of either a bulky ulcerated lesion localized in the corpus or antrum or a diffuse process spreading throughout the entire gastric submucosa and even extending into the duodenum. Microscopically, the vast majority of gastric lymphoid tumors are lymphomas of B-cell origin. Histologically, these tumors may range from well-differentiated, superficial processes (mucosa-associated lymphoid tissue [MALT]) to high-grade, large-cell lymphomas. Like gastric adenocarcinoma, infection with H. pylori increases the risk for gastric lymphoma in general and MALT lymphomas in particular. Large-cell lymphomas of the stomach spread initially to regional lymph nodes (often to Waldeyer’s ring) and may then

1	the risk for gastric lymphoma in general and MALT lymphomas in particular. Large-cell lymphomas of the stomach spread initially to regional lymph nodes (often to Waldeyer’s ring) and may then disseminate.

1	Primary gastric lymphoma is a far more treatable disease than adenocarcinoma of the stomach, a fact that underscores the need for making the correct diagnosis. Antibiotic treatment to eradicate

1	H. pylori infection has led to regression of about 75% of gastric MALT lymphomas and should be considered before surgery, radiation therapy, or chemotherapy is undertaken in patients having such tumors. A lack of response to such antimicrobial treatment has been linked to a specific chromosomal abnormality, i.e., t(11;18). Responding patients should undergo periodic endoscopic surveillance because it remains unclear whether the neoplastic clone is eliminated or merely suppressed, although the response to antimicrobial treatment is quite durable. Subtotal gastrectomy, usually followed by combination chemotherapy, has led to 5-year survival rates of 40–60% in patients with localized high-grade lymphomas. The need for a major surgical procedure has been questioned, particularly in patients with preoperative radiographic evidence of nodal involvement, for whom chemotherapy (CHOP [cyclophosphamide, doxorubicin, vincristine, and prednisone]) plus rituximab is highly effective therapy. A

1	with preoperative radiographic evidence of nodal involvement, for whom chemotherapy (CHOP [cyclophosphamide, doxorubicin, vincristine, and prednisone]) plus rituximab is highly effective therapy. A role for radiation therapy is not defined because most recurrences develop at distant sites.

1	Leiomyosarcomas and GISTs make up 1–3% of gastric neoplasms. They most frequently involve the anterior and posterior walls of the gastric fundus and often ulcerate and bleed. Even those lesions that appear benign on histologic examination may behave in a malignant fashion. These tumors rarely invade adjacent viscera and characteristically do not metastasize to lymph nodes, but they may spread to the liver and lungs. The treatment of choice is surgical resection. Combination chemotherapy should be reserved for patients with metastatic disease. All such tumors should be analyzed for a mutation in the c-kit receptor. GISTs are unresponsive to conventional chemotherapy; yet ∼50% of patients experience objective response and prolonged survival when treated with imatinib mesylate (Gleevec) (400–800 mg PO daily), a selective inhibitor of the c-kit tyrosine kinase. Many patients with GIST whose tumors have become refractory to imatinib subsequently benefit from sunitinib (Sutent) or

1	(400–800 mg PO daily), a selective inhibitor of the c-kit tyrosine kinase. Many patients with GIST whose tumors have become refractory to imatinib subsequently benefit from sunitinib (Sutent) or regorafenib (Stivarga), other inhibitors of the c-kit tyrosine kinase.

1	Small-bowel tumors comprise <3% of gastrointestinal neoplasms. Because of their rarity and inaccessibility, a correct diagnosis is often delayed. Abdominal symptoms are usually vague and poorly defined, and conventional radiographic studies of the upper and lower intestinal tract often appear normal. Small-bowel tumors should be considered in the differential diagnosis in the following situations: (1) recurrent, unexplained episodes of crampy abdominal pain; (2) intermittent bouts of intestinal obstruction, especially in the absence of inflammatory bowel disease (IBD) or prior abdominal surgery; (3) intussusception in the adult; and (4) evidence of chronic intestinal bleeding in the presence of negative conventional and endoscopic examination. A careful small-bowel barium study should be considered in such a circumstance; the diagnostic accuracy may be improved by infusing barium through a nasogastric tube placed into the duodenum (enteroclysis). Alternatively, capsule endoscopic

1	be considered in such a circumstance; the diagnostic accuracy may be improved by infusing barium through a nasogastric tube placed into the duodenum (enteroclysis). Alternatively, capsule endoscopic procedures have been used.

1	The histology of benign small-bowel tumors is difficult to predict on clinical and radiologic grounds alone. The symptomatology of benign tumors is not distinctive, with pain, obstruction, and hemorrhage being the most frequent symptoms. These tumors are usually discovered during the fifth and sixth decades of life, more often in the distal rather than the proximal small intestine. The most common benign tumors are adenomas, leiomyomas, lipomas, and angiomas.

1	Adenomas These tumors include those of the islet cells and Brunner’s glands as well as polypoid adenomas. Islet cell adenomas are occasionally located outside the pancreas; the associated syndromes are discussed in Chap. 113. Brunner’s gland adenomas are not truly neoplastic but represent a hypertrophy or hyperplasia of submucosal duodenal glands. These appear as small nodules in the duodenal mucosa that secrete a highly viscous alkaline mucus. Most often, this is an incidental radiographic finding not associated with any specific clinical disorder.

1	Polypoid Adenomas About 25% of benign small-bowel tumors are polypoid adenomas (see Table 110-2). They may present as single polypoid lesions or, less commonly, as papillary villous adenomas. As in the colon, the sessile or papillary form of the tumor is sometimes associated with a coexisting carcinoma. Occasionally, patients with Gardner’s syndrome develop premalignant adenomas in the small bowel; such lesions are generally in the duodenum. Multiple polypoid tumors may occur throughout the small bowel (and occasionally the stomach and colorectum) in the Peutz-Jeghers syndrome. The polyps are usually hamartomas (juvenile polyps) having a low potential for malignant degeneration. Mucocutaneous melanin deposits as well as tumors of the ovary, breast, pancreas, and endometrium are also associated with this autosomal dominant condition.

1	Leiomyomas These neoplasms arise from smooth-muscle components of the intestine and are usually intramural, affecting the overlying mucosa. Ulceration of the mucosa may cause gastrointestinal hemorrhage of varying severity. Cramping or intermittent abdominal pain is frequently encountered. Lipomas These tumors occur with greatest frequency in the distal ileum and at the ileocecal valve. They have a characteristic radiolucent appearance and are usually intramural and asymptomatic, but on occasion cause bleeding.

1	Angiomas While not true neoplasms, these lesions are important because they frequently cause intestinal bleeding. They may take the form of telangiectasia or hemangiomas. Multiple intestinal telangiectasias occur in a nonhereditary form confined to the gastrointestinal tract or as part of the hereditary Osler-Rendu-Weber syndrome. Vascular tumors may also take the form of isolated hemangiomas, most commonly in the jejunum. Angiography, especially during bleeding, is the best procedure for evaluating these lesions.

1	While rare, small-bowel malignancies occur in patients with longstanding regional enteritis and celiac sprue as well as in individuals with AIDS. Malignant tumors of the small bowel are frequently associated with fever, weight loss, anorexia, bleeding, and a palpable abdominal mass. After ampullary carcinomas (many of which arise from biliary or pancreatic ducts), the most frequently occurring small-bowel malignancies are adenocarcinomas, lymphomas, carcinoid tumors, and leiomyosarcomas.

1	The most common primary cancers of the small bowel are adenocarcinomas, accounting for ∼50% of malignant tumors. These cancers occur most often in the distal duodenum and proximal jejunum, where they tend to ulcerate and cause hemorrhage or obstruction. Radiologically, they may be confused with chronic duodenal ulcer disease or with Crohn’s disease if the patient has long-standing regional enteritis. The diagnosis is best made by endoscopy and biopsy under direct vision. Surgical resection is the treatment of choice with suggested postoperative adjuvant chemotherapy options generally following treatment patterns used in the management of colon cancer.

1	Lymphoma in the small bowel may be primary or secondary. A diagnosis of a primary intestinal lymphoma requires histologic confirmation in a clinical setting in which palpable adenopathy and hepatosplenomegaly are absent and no evidence of lymphoma is seen on chest radiograph, CT scan, or peripheral blood smear or on bone marrow aspiration and biopsy. Symptoms referable to the small bowel are present, usually accompanied by an anatomically discernible lesion. Secondary lymphoma of the small bowel consists of involvement of the intestine by a lymphoid malignancy extending from involved retroperitoneal or mesenteric lymph nodes (Chap. 134).

1	Primary intestinal lymphoma accounts for ∼20% of malignancies of the small bowel. These neoplasms are non-Hodgkin’s lymphomas; they usually have a diffuse, large-cell histology and are of T cell origin. Intestinal lymphoma involves the ileum, jejunum, and duodenum, in decreasing frequency—a pattern that mirrors the relative amount of normal lymphoid cells in these anatomic areas. The risk of small-bowel lymphoma is increased in patients with a prior history of malabsorptive conditions (e.g., celiac sprue), regional enteritis, and depressed immune function due to congenital immunodeficiency syndromes, prior organ transplantation, autoimmune disorders, or AIDS.

1	The development of localized or nodular masses that narrow the lumen results in periumbilical pain (made worse by eating) as well as weight loss, vomiting, and occasional intestinal obstruction. The diagnosis of small-bowel lymphoma may be suspected from the appearance on contrast radiographs of patterns such as infiltration and thickening of mucosal folds, mucosal nodules, areas of irregular ulceration, or stasis of contrast material. The diagnosis can be confirmed by surgical exploration and resection of involved segments. Intestinal lymphoma can occasionally be diagnosed by peroral intestinal mucosal biopsy, but because the disease mainly involves the lamina propria, full-thickness surgical biopsies are usually required.

1	Resection of the tumor constitutes the initial treatment modality. While postoperative radiation therapy has been given to some patients following a total resection, most authorities favor short-term (three cycles) systemic treatment with combination chemotherapy. The frequent presence of widespread intraabdominal disease at the time of diagnosis and the occasional multicentricity of the tumor often make a total resection impossible. The probability of sustained remission or cure is ∼75% in patients with localized disease but is ∼25% in individuals with unresectable lymphoma. In patients whose tumors are not resected, chemotherapy may lead to bowel perforation.

1	A unique form of small-bowel lymphoma, diffusely involving the entire intestine, was first described in oriental Jews and Arabs and is referred to as immunoproliferative small intestinal disease (IPSID), Mediterranean lymphoma, or α heavy chain disease. This is a B cell tumor. The typical presentation includes chronic diarrhea and steatorrhea associated with vomiting and abdominal cramps; clubbing of the digits may be observed. A curious feature in many patients with IPSID is the presence in the blood and intestinal secretions of an abnormal IgA that contains a shortened α heavy chain and is devoid of light chains. It is suspected that the abnormal α chains are produced by plasma cells infiltrating the small bowel. The clinical course of 537 patients with IPSID is generally one of exacerbations and remissions, with death frequently resulting from either progressive malnutrition and wasting or the development of an aggressive lymphoma. The use of oral antibiotics such as tetracycline

1	and remissions, with death frequently resulting from either progressive malnutrition and wasting or the development of an aggressive lymphoma. The use of oral antibiotics such as tetracycline appears to be beneficial in the early phases of the disorder, suggesting a possible infectious etiology. Combination chemotherapy has been administered during later stages of the disease, with variable results. Results are better when antibiotics and chemotherapy are combined.

1	Carcinoid tumors arise from argentaffin cells of the crypts of Lieberkühn and are found from the distal duodenum to the ascending colon, areas embryologically derived from the midgut. More than 50% of intestinal carcinoids are found in the distal ileum, with most congregating close to the ileocecal valve. Most intestinal carcinoids are asymptomatic and of low malignant potential, but invasion and metastases may occur, leading to the carcinoid syndrome (Chap. 113). Leiomyosarcomas often are >5 cm in diameter and may be palpable on abdominal examination. Bleeding, obstruction, and perforation are common. Such tumors should be analyzed for the expression of mutant c-kit receptor (defining GIST), and in the presence of metastatic disease, justifying treatment with imatinib mesylate (Gleevec) or, in imatinib-refractory patients, sunitinib (Sutent) or regorafenib (Stivarga). Robert J. Mayer Lower gastrointestinal cancers include malignant tumors of the colon, rectum, and anus.

1	Robert J. Mayer Lower gastrointestinal cancers include malignant tumors of the colon, rectum, and anus. Cancer of the large bowel is second only to lung cancer as a cause of cancer death in the United States: 136,830 new cases occurred in 2014, and 50,310 deaths were due to colorectal cancer. The incidence rate has decreased significantly during the past 25 years, likely due to enhanced and more compliantly followed screening practices. Similarly, mortality rates in the United States have decreased by approximately 25%, resulting largely from earlier detection and improved treatment.

1	Most colorectal cancers, regardless of etiology, arise from adenomatous polyps. A polyp is a grossly visible protrusion from the mucosal surface and may be classified pathologically as a nonneoplastic hamartoma (e.g., juvenile polyp), a hyperplastic mucosal proliferation (hyperplastic polyp), or an adenomatous polyp. Only adenomas are clearly premalignant, and only a minority of adenomatous polyps evolve into cancer. Adenomatous polyps may be found in the colons of ∼30% of middle-aged and ∼50% of elderly people; however, <1% of polyps ever become malignant. Most polyps produce no symptoms and remain clinically undetected. Occult blood in the stool is found in <5% of patients with polyps.

1	A number of molecular changes are noted in adenomatous polyps and colorectal cancers that are thought to reflect a multistep process in the evolution of normal colonic mucosa to life-threatening invasive carcinoma. These developmental steps toward carcinogenesis include, but are not restricted to, point mutations in the K-ras protooncogene; hypomethylation of DNA, leading to gene activation; loss of DNA 538 (allelic loss) at the site of a tumor-suppressor gene (the adenomatous polyposis coli [APC] gene) on the long arm of chromosome 5 (5q21); allelic loss at the site of a tumor-suppressor gene located on chromosome 18q (the deleted in colorectal cancer [DCC] gene); and allelic loss at chromosome 17p, associated with mutations in the p53 tumor-suppressor gene (see Fig. 101e-2). Thus, the altered proliferative pattern of the colonic mucosa, which results in progression to a polyp and then to carcinoma, may involve the mutational activation of an oncogene followed by and coupled with the

1	proliferative pattern of the colonic mucosa, which results in progression to a polyp and then to carcinoma, may involve the mutational activation of an oncogene followed by and coupled with the loss of genes that normally suppress tumorigenesis. It remains uncertain whether the genetic aberrations always occur in a defined order. Based on this model, however, cancer is believed to develop only in those polyps in which most (if not all) of these mutational events take place. Clinically, the probability of an adenomatous polyp becoming a cancer depends on the gross appearance of the lesion, its histologic features, and its size. Adenomatous polyps may be pedunculated (stalked) or sessile (flat-based). Invasive cancers develop more frequently in sessile polyps. Histologically, adenomatous polyps may be tubular, villous (i.e., papillary), or tubulovillous. Villous adenomas, most of which are sessile, become malignant more than three times as often as tubular adenomas. The likelihood that

1	may be tubular, villous (i.e., papillary), or tubulovillous. Villous adenomas, most of which are sessile, become malignant more than three times as often as tubular adenomas. The likelihood that any polypoid lesion in the large bowel contains invasive cancer is related to the size of the polyp, being negligible (<2%) in lesions <1.5 cm, intermediate (2–10%) in lesions 1.5–2.5 cm, and substantial (10%) in lesions >2.5 cm in size. Following the detection of an adenomatous polyp, the entire large bowel should be visualized endoscopically because synchronous lesions are noted in about one-third of cases. Colonoscopy should then be repeated periodically, even in the absence of a previously documented malignancy, because such patients have a 30–50% probability of developing another adenoma and are at a higher-than-average risk for developing a colorectal carcinoma. Adenomatous polyps are thought to require >5 years of growth before becoming clinically significant; colonoscopy need not be

1	are at a higher-than-average risk for developing a colorectal carcinoma. Adenomatous polyps are thought to require >5 years of growth before becoming clinically significant; colonoscopy need not be carried out more frequently than every 3 years for the vast majority of patients.

1	Risk factors for the development of colorectal cancer are listed in Table 110-1. Diet The etiology for most cases of large-bowel cancer appears to be related to environmental factors. The disease occurs more often in upper socioeconomic populations who live in urban areas. Mortality from colorectal cancer is directly correlated with per capita consumption of calories, meat protein, and dietary fat and oil as well as elevations in the serum cholesterol concentration and mortality from coronary artery disease. Geographic variations in incidence largely are unrelated to genetic differences, since migrant groups tend to assume the large-bowel cancer incidence rates of their adopted countries. Furthermore, population groups such as Mormons and Seventh Day Adventists, whose lifestyle and dietary habits differ somewhat risK faCtors for the DeveLopment of CoLoreCtaL CanCer

1	Diet: Animal fat ? Tobacco use from those of their neighbors, have significantly lower-than-expected incidence and mortality rates for colorectal cancer. The incidence of colorectal cancer has increased in Japan since that nation has adopted a more “Western” diet. At least three hypotheses have been proposed to explain the relationship to diet, none of which is fully satisfactory. animal fats One hypothesis is that the ingestion of animal fats found in red meats and processed meat leads to an increased proportion of anaerobes in the gut microflora, resulting in the conversion of normal bile acids into carcinogens. This provocative hypothesis is supported by several reports of increased amounts of fecal anaerobes in the stools of patients with colorectal cancer. Diets high in animal (but not vegetable) fats are also associated with high serum cholesterol, which is also associated with enhanced risk for the development of colorectal adenomas and carcinomas.

1	insulin resistance The large number of calories in Western diets coupled with physical inactivity has been associated with a higher prevalence of obesity. Obese persons develop insulin resistance with increased circulating levels of insulin, leading to higher circulating concentrations of insulin-like growth factor type I (IGF-I). This growth factor appears to stimulate proliferation of the intestinal mucosa. fiBer Contrary to prior beliefs, the results of randomized trials and case-controlled studies have failed to show any value for dietary fiber or diets high in fruits and vegetables in preventing the recurrence of colorectal adenomas or the development of colorectal cancer. The weight of epidemiologic evidence, however, implicates diet as being the major etiologic factor for colorectal cancer, particularly diets high in animal fat and in calories.

1	The weight of epidemiologic evidence, however, implicates diet as being the major etiologic factor for colorectal cancer, particularly diets high in animal fat and in calories. Up to 25% of patients with colorectal cancer have a family history of the disease, suggesting a hereditary predisposition. Inherited large-bowel cancers can be divided into two main groups: the well-studied but uncommon polyposis syndromes and the more common nonpolyposis syndromes (Table 110-2).

1	Polyposis Coli Polyposis coli (familial polyposis of the colon) is a rare condition characterized by the appearance of thousands of adenomatous polyps throughout the large bowel. It is transmitted as an autosomal dominant trait; the occasional patient with no family history probably developed the condition due to a spontaneous mutation. Polyposis coli is associated with a deletion in the long arm of chromosome 5 (including the APC gene) in both neoplastic (somatic mutation) and normal (germline mutation) cells. The loss of this genetic material (i.e., allelic loss) results in the absence of tumor-suppressor genes whose protein products would normally inhibit neoplastic growth. The presence of soft tissue and bony tumors, congenital hypertrophy of the retinal pigment epithelium, mesenteric desmoid tumors, and ampullary cancers in addition to the colonic polyps characterizes a subset of polyposis coli known as Gardner’s syndrome. The appearance of malignant tumors of the central nervous

1	desmoid tumors, and ampullary cancers in addition to the colonic polyps characterizes a subset of polyposis coli known as Gardner’s syndrome. The appearance of malignant tumors of the central nervous system accompanying polyposis coli defines Turcot’s syndrome. The colonic polyps in all these conditions are rarely present before puberty but are generally evident in affected individuals by age 25. If the polyposis is not treated surgically, colorectal cancer will develop in almost all patients before age 40. Polyposis coli results from a defect in the colonic mucosa, leading to an abnormal proliferative pattern and impaired DNA repair mechanisms. Once the multiple polyps are detected, patients should undergo a total colectomy. Medical therapy with nonsteroidal anti-inflammatory drugs (NSAIDs) such as sulindac and selective cyclooxygenase-2 inhibitors such as celecoxib can decrease the number and size of polyps in patients with polyposis coli; however, this effect on polyps is only

1	such as sulindac and selective cyclooxygenase-2 inhibitors such as celecoxib can decrease the number and size of polyps in patients with polyposis coli; however, this effect on polyps is only temporary, and the use of NSAIDs has not been shown to reduce the risk of cancer. Colectomy remains the primary therapy/prevention. The offspring of patients with polyposis coli, who often are prepubertal when the diagnosis is made in the parent, have a 50% risk for developing this premalignant disorder and should be carefully screened by annual flexible sigmoidoscopy until age 35. Proctosigmoidoscopy is a sufficient screening procedure because polyps tend to be evenly distributed from cecum to anus, making more invasive and expensive techniques such as colonoscopy or barium enema unnecessary. Testing for occult blood in the stool is an inadequate screening maneuver. If a causative germ-line AP C mutation has been identified in an affected family member, an alternative method for identifying

1	for occult blood in the stool is an inadequate screening maneuver. If a causative germ-line AP C mutation has been identified in an affected family member, an alternative method for identifying carriers is testing DNA from peripheral blood mononuclear cells for the presence of the specific APC mutation. The detection of such a germline mutation can lead to a definitive diagnosis before the development of polyps.

1	MYH-Associated Polyposis MYH-associated polyposis (MAP) is a rare autosomal recessive syndrome caused by a biallelic mutation in the MUT4H gene. This hereditary condition may have a variable clinical presentation, resembling polyposis coli or colorectal cancer occurring in younger individuals without polyposis. Screening and colectomy guidelines for this syndrome are less clear than for polyposis coli, but annual to biennial colonoscopic surveillance is generally recommended starting at age 25–30.

1	Hereditary Nonpolyposis Colon Cancer Hereditary nonpolyposis colon cancer (HNPCC), also known as Lynch’s syndrome, is another autosomal dominant trait. It is characterized by the presence of three or more relatives with histologically documented colorectal cancer, one of whom is a first-degree relative of the other two; one or more cases of colorectal cancer diagnosed before age 50 in the family; and colorectal cancer involving at least two generations. In contrast to polyposis coli, HNPCC is associated with an unusually high frequency of cancer arising in the proximal large bowel. The median age for the appearance of an adenocarcinoma is <50 years, 10–15 years younger than the median age for the general population. Despite having a poorly differentiated, mucinous histologic appearance, the proximal colon tumors that characterize HNPCC have a better prognosis than sporadic tumors from patients of similar age. Families with HNPCC often include individuals with multiple primary cancers;

1	proximal colon tumors that characterize HNPCC have a better prognosis than sporadic tumors from patients of similar age. Families with HNPCC often include individuals with multiple primary cancers; the association of colorectal cancer with either ovarian or endometrial carcinomas is especially strong in women, and an increased appearance of gastric, small-bowel, genitourinary, pancreaticobiliary, and sebaceous skin tumors has been reported as well. It has been recommended that members of such families undergo annual or biennial colonoscopy beginning at age 25 years, with intermittent pelvic ultrasonography and endometrial biopsy for afflicted women; such a screening strategy has not yet been validated. HNPCC is associated with germline muta-539 tions of several genes, particularly hMSH2 on chromosome 2 and hMLH1 on chromosome 3. These mutations lead to errors in DNA replication and are thought to result in DNA instability because of defective repair of DNA mismatches resulting in

1	on chromosome 2 and hMLH1 on chromosome 3. These mutations lead to errors in DNA replication and are thought to result in DNA instability because of defective repair of DNA mismatches resulting in abnormal cell growth and tumor development. Testing tumor cells through molecular analysis of DNA or immunohistochemical staining of paraffin-fixed tissue for “microsatellite instability” (sequence changes reflecting defective mismatch repair) in patients with colorectal cancer and a positive family history for colorectal or endometrial cancer may identify probands with HNPCC.

1	(Chap. 351) Large-bowel cancer is increased in incidence in patients with long-standing inflammatory bowel disease (IBD). Cancers develop more commonly in patients with ulcerative colitis than in those with granulomatous (i.e., Crohn’s) colitis, but this impression may result in part from the occasional difficulty of differentiating these two conditions. The risk of colorectal cancer in a patient with IBD is relatively small during the initial 10 years of the disease, but then appears to increase at a rate of ∼0.5–1% per year. Cancer may develop in 8–30% of patients after 25 years. The risk is higher in younger patients with pancolitis. Cancer surveillance strategies in patients with IBD are unsatisfactory. Symptoms such as bloody diarrhea, abdominal cramping, and obstruction, which may signal the appearance of a tumor, are similar to the complaints caused by a flare-up of the underlying disease.

1	In patients with a history of IBD lasting ≥15 years who continue to experience exacerbations, the surgical removal of the colon can significantly reduce the risk for cancer and also eliminate the target organ for the underlying chronic gastrointestinal disorder. The value of such surveillance techniques as colonoscopy with mucosal biopsies and brushings for less symptomatic individuals with chronic IBD is uncertain. The lack of uniformity regarding the pathologic criteria that characterize dysplasia and the absence of data that such surveillance reduces the development of lethal cancers have made this costly practice an area of controversy. OTHER HIGH-RISK CONDITIONS Streptococcus bovis Bacteremia For unknown reasons, individuals who develop endocarditis or septicemia from this fecal bacterium have a high incidence of occult colorectal tumors and, possibly, upper gastrointestinal cancers as well. Endoscopic or radiographic screening appears advisable.

1	Tobacco Use Cigarette smoking is linked to the development of colorectal adenomas, particularly after >35 years of tobacco use. No biologic explanation for this association has yet been proposed.

1	Several orally administered compounds have been assessed as possible inhibitors of colon cancer. The most effective class of chemopreventive agents is aspirin and other NSAIDs, which are thought to suppress cell proliferation by inhibiting prostaglandin synthesis. Regular aspirin use reduces the risk of colon adenomas and carcinomas as well as death from large-bowel cancer; such use also appears to diminish the likelihood for developing additional premalignant adenomas following successful treatment for a prior colon carcinoma. This effect of aspirin on colon carcinogenesis increases with the duration and dosage of drug use. Oral folic acid supplements and oral calcium supplements appear to reduce the risk of adenomatous polyps and colorectal cancers in case-controlled studies. The value of vitamin D as a form of chemoprevention is under study. Antioxidant vitamins such as ascorbic acid, tocopherols, and β-carotene are ineffective at reducing the incidence of subsequent adenomas in

1	of vitamin D as a form of chemoprevention is under study. Antioxidant vitamins such as ascorbic acid, tocopherols, and β-carotene are ineffective at reducing the incidence of subsequent adenomas in patients who have undergone the removal of a colon adenoma. Estrogen replacement therapy has been associated with a reduction in the risk of colorectal cancer in women, conceivably by an effect on bile acid synthesis and composition or by decreasing synthesis of IGF-I.

1	540 SCREENING The rationale for colorectal cancer screening programs is that the removal of adenomatous polyps will prevent colorectal cancer, and that earlier detection of localized, superficial cancers in asymptomatic individuals will increase the surgical cure rate. Such screening programs are particularly important for individuals with a family history of the disease in first-degree relatives. The relative risk for developing colorectal cancer increases to 1.75 in such individuals and may be even higher if the relative was afflicted before age 60. The prior use of proctosigmoidoscopy as a screening tool was based on the observation that 60% of early lesions are located in the rectosigmoid. For unexplained reasons, however, the proportion of large-bowel cancers arising in the rectum has been decreasing during the past several decades, with a corresponding increase in the proportion of cancers in the more proximal descending colon. As such, the potential for proctosigmoidoscopy to

1	been decreasing during the past several decades, with a corresponding increase in the proportion of cancers in the more proximal descending colon. As such, the potential for proctosigmoidoscopy to detect a sufficient number of occult neoplasms to make the procedure cost-effective has been questioned. Screening strategies for colorectal cancer that have been examined during the past several decades are listed in Table 110-3. Many programs directed at the early detection of colorectal cancers have focused on digital rectal examinations and fecal occult blood (i.e., stool guaiac) testing. The digital examination should be part of any routine physical evaluation in adults older than age 40 years, serving as a screening test for prostate cancer in men, a component of the pelvic examination in women, and an inexpensive maneuver for the detection of masses in the rectum. However, because of the proximal migration of colorectal tumors, its value as an overall screening modality for colorectal

1	and an inexpensive maneuver for the detection of masses in the rectum. However, because of the proximal migration of colorectal tumors, its value as an overall screening modality for colorectal cancer has become limited. The development of the fecal occult blood test has greatly facilitated the detection of occult fecal blood. Unfortunately, even when performed optimally, the fecal occult blood test has major limitations as a screening technique. About 50% of patients with documented colorectal cancers have a negative fecal occult blood test, consistent with the intermittent bleeding pattern of these tumors. When random cohorts of asymptomatic persons have been tested, 2–4% have fecal occult blood-positive stools. Colorectal cancers have been found in <10% of these “test-positive” cases, with benign polyps being detected in an additional 20–30%. Thus, a colorectal neoplasm will not be found in most asymptomatic individuals with occult blood in their stool. Nonetheless, persons found

1	with benign polyps being detected in an additional 20–30%. Thus, a colorectal neoplasm will not be found in most asymptomatic individuals with occult blood in their stool. Nonetheless, persons found to have fecal occult blood-positive stool routinely undergo further medical evaluation, including sigmoidoscopy and/or colonoscopy—procedures that are not only uncomfortable and expensive but also associated with a small risk for significant complications. The added cost of these studies would appear justifiable if the small number of patients found to have occult neoplasms because of fecal occult blood screening could be shown to have an improved prognosis and prolonged survival. Prospectively controlled trials have shown a statistically significant reduction in mortality rate from colorectal cancer for individuals undergoing annual stool guaiac screening. However, this benefit only emerged after >13 years of follow-up and was extremely expensive to achieve, because all positive tests

1	cancer for individuals undergoing annual stool guaiac screening. However, this benefit only emerged after >13 years of follow-up and was extremely expensive to achieve, because all positive tests (most of which were falsely positive) were followed by colonoscopy. Moreover, these colonoscopic examinations quite likely provided the opportunity for cancer prevention through the removal of potentially premalignant adenomatous polyps (i.e., computed tomography colonography) because the eventual development of cancer was reduced by 20% in the cohort undergoing annual screening.

1	With the appreciation that the carcinogenic process leading to the progression of the normal bowel mucosa to an adenomatous polyp and then to a cancer is the result of a series of molecular changes, investigators have examined fecal DNA for evidence of mutations associated with such molecular changes as evidence of the occult presence of precancerous lesions or actual malignancies. Such a strategy has been tested in more than 4000 asymptomatic individuals whose stool was assessed for occult blood and for 21 possible mutations in fecal DNA; these study subjects also underwent colonoscopy. Although the fecal DNA strategy suggested the presence of more advanced adenomas and cancers than did the fecal occult blood testing approach, the overall sensitivity, using colonoscopic findings as the standard, was less than 50%, diminishing enthusiasm for further pursuit of the fecal DNA screening strategy.

1	The use of imaging studies to screen for colorectal cancers has also been explored. Air contrast barium enemas had been used to identify sources of occult blood in the stool prior to the advent of fiberoptic endoscopy; the cumbersome nature of the procedure and inconvenience to patients limited its widespread adoption. The introduction of computed tomography (CT) scanning led to the development of virtual (i.e., CT) colonography as an alternative to the growing use of endoscopic screening techniques. Virtual colonography was proposed as being equivalent in sensitivity to colonoscopy and being available in a more widespread manner because it did not require the same degree of operator expertise as fiberoptic endoscopy. However, virtual colonography requires the same cathartic preparation that has limited widespread acceptance of endoscopic colonoscopy, is diagnostic but not therapeutic (i.e., patients with suspicious findings must undergo a subsequent endoscopic procedure for

1	that has limited widespread acceptance of endoscopic colonoscopy, is diagnostic but not therapeutic (i.e., patients with suspicious findings must undergo a subsequent endoscopic procedure for polypectomy or biopsy), and, in the setting of general radiology practices, appears to be less sensitive as a screening technique when compared with endoscopic procedures.

1	With the appreciation of the inadequacy of fecal occult blood testing alone, concerns about the practicality of imaging approaches, and the wider adoption of endoscopic examinations by the primary care community, screening strategies in asymptomatic persons have changed. At present, both the American Cancer Society and the National Comprehensive Cancer Network suggest either fecal occult blood testing annually coupled with flexible sigmoidoscopy every 5 years or colonoscopy every 10 years beginning at age 50 in asymptomatic individuals with no personal or family history of polyps or colorectal cancer. The recommendation for the inclusion of flexible sigmoidoscopy is strongly supported by the recently published results of three randomized trials performed in the United States, the United Kingdom, and Italy, involving more than 350,000 individuals, which consistently showed that periodic (even single) sigmoidoscopic examinations, after more than a decade of median follow-up, lead to an

1	and Italy, involving more than 350,000 individuals, which consistently showed that periodic (even single) sigmoidoscopic examinations, after more than a decade of median follow-up, lead to an approximate 21% reduction in the development of colorectal cancer and a more than 25% reduction in mortality from the malignant disease. Less than 20% of participants in these studies underwent a subsequent colonoscopy. In contrast to the cathartic preparation required before colonoscopic procedures, which is only performed by highly trained specialists, flexible sigmoidoscopy requires only an enema as preparation and can be accurately performed by nonspecialty physicians or physician-extenders. The randomized screening studies using flexible sigmoidoscopy led to the estimate that approximately 650 individuals needed to be screened to prevent one colorectal cancer death; this contrasts with the data for mammography where the number of women needing to be screened to prevent one breast cancer

1	individuals needed to be screened to prevent one colorectal cancer death; this contrasts with the data for mammography where the number of women needing to be screened to prevent one breast cancer death is 2500, reinforcing the efficacy of endoscopic surveillance for colorectal cancer screening. Presumably the benefit from the sigmoidoscopic screening is the result of the identification and removal of adenomatous polyps; it is intriguing that this benefit has been achieved using a technique that leaves the proximal half of the large bowel unvisualized.

1	It remains to be seen whether surveillance colonoscopy, which has gained increasing popularity in the United States for colorectal cancer screening, will prove to be more effective than flexible sigmoidoscopy. Ongoing randomized trials being conducted in Europe are addressing this issue. Although flexible sigmoidoscopy only visualizes the distal half of the large bowel, leading to the assumption that colonoscopy represents a more informative approach, colonoscopy has been reported as being less accurate for screening the proximal rather than the distal colon, perhaps due to technical considerations but also possibly because of a greater frequency of serrated (i.e., “flat”) polyps in the right colon, which are more difficult to identify. At present, colonoscopy performed every 10 years has been offered as an alternative to annual fecal occult blood testing with periodic (every 5 years) flexible sigmoidoscopy. Colonoscopy has been shown to be superior to double-contract barium enema and

1	been offered as an alternative to annual fecal occult blood testing with periodic (every 5 years) flexible sigmoidoscopy. Colonoscopy has been shown to be superior to double-contract barium enema and also to have a higher sensitivity for detecting villous or dysplastic adenomas or cancers than the strategy using occult fecal blood testing and flexible sigmoidoscopy. Whether colonoscopy performed every 10 years beginning at age 50 is medically superior and economically equivalent to flexible sigmoidoscopy remains to be determined.

1	CLINICAL FEATURES Presenting Symptoms Symptoms vary with the anatomic location of the tumor. Because stool is relatively liquid as it passes through the ileocecal valve into the right colon, cancers arising in the cecum and ascending colon may become quite large without resulting in any obstructive symptoms or noticeable alterations in bowel habits. Lesions of the right colon commonly ulcerate, leading to chronic, insidious blood loss without a change in the appearance of the stool. Consequently, patients with tumors of the ascending colon often present with symptoms such as fatigue, palpitations, and even angina pectoris and are found to have a hypochromic, microcytic anemia indicative of iron deficiency. Because the cancers may bleed intermittently, a random fecal occult blood test may be negative. As a result, the unexplained presence of iron-deficiency anemia in any adult (with the possible exception of a premenopausal, multiparous woman) mandates a thorough endoscopic and/or

1	may be negative. As a result, the unexplained presence of iron-deficiency anemia in any adult (with the possible exception of a premenopausal, multiparous woman) mandates a thorough endoscopic and/or radiographic visualization of the entire large bowel (Fig. 110-1).

1	Because stool becomes more formed as it passes into the transverse and descending colon, tumors arising there tend to impede the passage of stool, resulting in the development of abdominal cramping, occasional obstruction, and even perforation. Radiographs of the abdomen often reveal characteristic annular, constricting lesions (“apple-core” or “napkin-ring”) (Fig. 110-2). Cancers arising in the rectosigmoid are often associated with hematochezia, tenesmus, and narrowing of the caliber of stool; anemia is an infrequent finding. While these symptoms may lead patients and their physicians to suspect the presence of hemorrhoids, the development of rectal bleeding and/or altered bowel habits demands a prompt digital rectal examination and proctosigmoidoscopy.

1	Staging, Prognostic Factors, and Patterns of Spread The prognosis for individuals having colorectal cancer is related to the depth of tumor penetration into the bowel wall and the presence of both regional lymph node involvement and distant metastases. These variables are incorporated into the staging system introduced by Dukes and subsequently applied to a TNM classification method, in which T represents the depth of tumor penetration, N the presence of lymph node involvement, and M the presence or absence of distant metastases (Fig. 110-3). Superficial lesions that do not involve regional lymph nodes and do not penetrate through the submucosa (T1) or the muscularis (T2) are designated as stage I (T1–2N0M0) disease; tumors that penetrate through the muscularis but have not spread to lymph nodes are stage II disease (T3-4N0M0); regional lymph node involvement defines stage III (TXN1-2M0) disease; and metastatic spread to sites such as liver, lung, or bone indicates stage IV (TXNXM1)

1	nodes are stage II disease (T3-4N0M0); regional lymph node involvement defines stage III (TXN1-2M0) disease; and metastatic spread to sites such as liver, lung, or bone indicates stage IV (TXNXM1) disease. Unless gross evidence of metastatic disease is present, disease stage cannot be determined accurately before surgical resection and pathologic analysis of the operative specimens. It is not clear whether the detection of nodal metastases by special immunohistochemical molecular techniques has the same prognostic implications as disease detected by routine light microscopy.

1	Most recurrences after a surgical resection of a large-bowel cancer occur within the first 4 years, making 5-year survival a fairly reliable indicator of cure. The likelihood for 5-year survival in patients with colorectal cancer is stage-related (Fig. 110-3). That likelihood has improved during the past several decades when similar surgical stages have been compared. The most plausible explanation for this improvement is more thorough intraoperative and pathologic staging. In particular, more exacting attention to pathologic detail has revealed that the prognosis following the resection of a colorectal cancer is not related merely to the presence or absence of regional lymph node involvement; rather, prognosis may be more precisely gauged by the number of involved lymph nodes (one to three lymph nodes [“N1”] vs four or more lymph nodes [“N2”]) and the number of nodes examined. A minimum of 12 sampled lymph nodes is thought necessary to

1	FIGURE 110-1 Double-contrast air-barium enema revealing a ses-sile tumor of the cecum in a patient with iron-deficiency anemia and guaiac-positive stool. The lesion at surgery was a stage II adenocarci-noma. FIGURE 110-2 Annular, constricting adenocarcinoma of the descending colon. This radiographic appearance is referred to as an “apple-core” lesion and is always highly suggestive of malignancy. Staging of colorectal cancer FIGURE 110-3 Staging and prognosis for patients with colorectal cancer.

1	accurately define tumor stage, and the more nodes examined, the better. Other predictors of a poor prognosis after a total surgical resection include tumor penetration through the bowel wall into pericolic fat, poorly differentiated histology, perforation and/or tumor adherence to adjacent organs (increasing the risk for an anatomically adjacent recurrence), and venous invasion by tumor (Table 110-4). Regardless of the clinicopathologic stage, a preoperative elevation of the plasma carcinoembryonic antigen (CEA) level predicts eventual tumor recurrence. The presence of aneuploidy and specific chromosomal deletions, such as a mutation in the b-raf gene in tumor cells, appears to predict for a higher risk for metastatic spread. Conversely, the detection of microsatellite instability in tumor tissue indicates a more favorable outcome. In contrast to most other cancers, the prognosis in colorectal cancer is not influenced by the size of the primary lesion when adjusted for nodal

1	in tumor tissue indicates a more favorable outcome. In contrast to most other cancers, the prognosis in colorectal cancer is not influenced by the size of the primary lesion when adjusted for nodal involvement and histologic differentiation.

1	Cancers of the large bowel generally spread to regional lymph nodes or to the liver via the portal venous circulation. The liver represents the most frequent visceral site of metastasis; it is the initial site of distant spread in one-third of recurring colorectal cancers and is involved in more than two-thirds of such patients at the time of death. In general, colorectal cancer rarely spreads to the lungs, supraclavicular lymph nodes, bone, or brain without prior spread to the liver. A major exception to this rule occurs in patients having primary tumors in the distal rectum, from which tumor cells may spread through the paravertebral preDiCtors of poor outCome foLLoWing totaL surgiCaL reseCtion of CoLoreCtaL CanCer

1	Tumor spread to regional lymph nodes Number of regional lymph nodes involved Tumor penetration through the bowel wall Poorly differentiated histology Perforation Tumor adherence to adjacent organs Venous invasion Preoperative elevation of CEA titer (>5 ng/mL) Aneuploidy Specific chromosomal deletion (e.g., mutation in the b-raf gene) Abbreviation: CEA, carcinoembryonic antigen. venous plexus, escaping the portal venous system and thereby reaching the lungs or supraclavicular lymph nodes without hepatic involvement. The median survival after the detection of distant metastases has ranged in the past from 6–9 months (hepatomegaly, abnormal liver chemistries) to 24–30 months (small liver nodule initially identified by elevated CEA level and subsequent CT scan), but effective systemic therapy is significantly improving this prognosis.

1	Efforts to use gene expression profiles to identify patients at risk of recurrence or those particularly likely to benefit from adjuvant therapy have not yet yielded practice-changing results. Despite a burgeoning literature examining a host of prognostic factors, pathologic stage at diagnosis remains the best predictor of long-term prognosis. Patients with lymphovascular invasion and high preoperative CEA levels are likely to have a more aggressive clinical course.

1	Total resection of tumor is the optimal treatment when a malignant lesion is detected in the large bowel. An evaluation for the presence of metastatic disease, including a thorough physical examination, biochemical assessment of liver function, measurement of the plasma CEA level, and a CT scan of the chest, abdomen, and pelvis, should be performed before surgery. When possible, a colonoscopy of the entire large bowel should be performed to identify synchronous neoplasms and/or polyps. The detection of metastases should not preclude surgery in patients with tumor-related symptoms such as gastrointestinal bleeding or obstruction, but it often prompts the use of a less radical operative procedure. The necessity for a primary tumor resection in asymptomatic individuals with metastatic disease is an area of controversy. At the time of laparotomy, the entire peritoneal cavity should be examined, with thorough inspection of the liver, pelvis, and hemidiaphragm and careful palpation of the

1	is an area of controversy. At the time of laparotomy, the entire peritoneal cavity should be examined, with thorough inspection of the liver, pelvis, and hemidiaphragm and careful palpation of the full length of the large bowel. Following recovery from a complete resection, patients should be observed carefully for 5 years by semiannual physical examinations and blood chemistry measurements. If a complete colonoscopy was not performed preoperatively, it should be carried out within the first several postoperative months. Some authorities favor measuring plasma CEA levels at 3-month intervals because of the sensitivity of this test as a marker for otherwise undetectable tumor recurrence. Subsequent endoscopic surveillance of the large bowel, probably at triennial intervals, is indicated, because patients who have been cured of one colorectal cancer have a 3–5% probability of developing an additional bowel cancer during their lifetime and a >15% risk for the development of adenomatous

1	because patients who have been cured of one colorectal cancer have a 3–5% probability of developing an additional bowel cancer during their lifetime and a >15% risk for the development of adenomatous polyps. Anastomotic (“suture-line”) recurrences are infrequent in colorectal cancer patients, provided the surgical resection margins are adequate and free of tumor. The value of periodic CT scans of the abdomen, assessing for an early, asymptomatic indication of tumor recurrence, is an area of uncertainty, with some experts recommending the test be performed annually for the first 3 postoperative years.

1	Radiation therapy to the pelvis is recommended for patients with rectal cancer because it reduces the 20–25% probability of regional recurrences following complete surgical resection of stage II or III tumors, especially if they have penetrated through the serosa. This alarmingly high rate of local disease recurrence is believed to be due to the fact that the contained anatomic space within the pelvis limits the extent of the resection and because the rich lymphatic network of the pelvic side wall immediately adjacent to the rectum facilitates the early spread of malignant cells into surgically inaccessible tissue. The use of sharp rather than blunt dissection of rectal cancers (total mesorectal excision) appears to reduce the likelihood of local disease recurrence to ∼10%. Radiation therapy, either preor postoperatively, further reduces the likelihood of pelvic recurrences but does not appear to prolong survival. Combining radiation therapy with 5-fluorouracil (5-FU)-based

1	therapy, either preor postoperatively, further reduces the likelihood of pelvic recurrences but does not appear to prolong survival. Combining radiation therapy with 5-fluorouracil (5-FU)-based chemotherapy, preferably prior to surgical resection, lowers local recurrence rates and improves overall survival. Preoperative radiotherapy is indicated for patients with large, potentially unresectable rectal cancers; such lesions may shrink enough to permit subsequent surgical removal. Radiation therapy is not effective as the primary treatment of colon cancer.

1	Systemic therapy for patients with colorectal cancer has become more effective. 5-FU remains the backbone of treatment for this disease. Partial responses are obtained in 15–20% of patients. The probability of tumor response appears to be somewhat greater for patients with liver metastases when chemotherapy is infused directly into the hepatic artery, but intraarterial treatment is costly and toxic and does not appear to appreciably prolong survival. The concomitant administration of folinic acid (leucovorin) improves the efficacy of 5-FU in patients with advanced colorectal cancer, presumably by enhancing the binding of 5-FU to its target enzyme, thymidylate synthase. A threefold improvement in the partial response rate is noted when folinic acid is combined with 5-FU; however, the effect on survival is marginal, and the optimal dose schedule remains to be defined. 5-FU is generally administered intravenously but may also be given orally in the form of capecitabine (Xeloda) with

1	on survival is marginal, and the optimal dose schedule remains to be defined. 5-FU is generally administered intravenously but may also be given orally in the form of capecitabine (Xeloda) with seemingly similar efficacy.

1	Irinotecan (CPT-11), a topoisomerase 1 inhibitor, prolongs survival when compared to supportive care in patients whose disease has progressed on 5-FU. Furthermore, the addition of irinotecan to 5-FU and leucovorin (LV) (e.g., FOLFIRI) improves response rates and survival of patients with metastatic disease. The FOLFIRI regimen is as follows: irinotecan, 180 mg/m2 as a 90-min infusion on day 1; LV, 400 mg/m2 as a 2-h infusion during irinotecan administration; immediately followed by 5-FU bolus, 400 mg/m2, and 46-h continuous infusion of 2.4–3 g/m2 every 2 weeks. Diarrhea is the major side effect from irinotecan. Oxaliplatin, a platinum analogue, also improves the response rate when added to 5-FU and LV (FOLFOX) as initial treatment of patients with metastatic disease. The FOLFOX regimen is as follows: 2-h infusion of LV (400 mg/m2 per day) followed by a 5-FU bolus (400 mg/m2 per day) and 22-h infusion (1200 mg/m2) every 2 weeks, together with oxaliplatin, 85 mg/m2 as a 2-h infusion on

1	as follows: 2-h infusion of LV (400 mg/m2 per day) followed by a 5-FU bolus (400 mg/m2 per day) and 22-h infusion (1200 mg/m2) every 2 weeks, together with oxaliplatin, 85 mg/m2 as a 2-h infusion on day 1. Oxaliplatin frequently causes a dose-dependent sensory neuropathy that often but not always resolves following the cessation of therapy. FOLFIRI and FOLFOX are equal in efficacy. In metastatic disease, these regimens may produce median survivals of 2 years.

1	Monoclonal antibodies are also effective in patients with advanced colorectal cancer. Cetuximab (Erbitux) and panitumumab (Vectibix) are directed against the epidermal growth factor recep-543 tor (EGFR), a transmembrane glycoprotein involved in signaling pathways affecting growth and proliferation of tumor cells. Both cetuximab and panitumumab, when given alone, have been shown to benefit a small proportion of previously treated patients, and cetuximab appears to have therapeutic synergy with such chemotherapeutic agents as irinotecan, even in patients previously resistant to this drug; this suggests that cetuximab can reverse cellular resistance to cytotoxic chemotherapy. The antibodies are not effective in the approximate 40% subset of colon tumors that contain mutated K-ras. The use of both cetuximab and panitumumab can lead to an acne-like rash, with the development and severity of the rash being correlated with the likelihood of antitumor efficacy. Inhibitors of the EGFR tyrosine

1	both cetuximab and panitumumab can lead to an acne-like rash, with the development and severity of the rash being correlated with the likelihood of antitumor efficacy. Inhibitors of the EGFR tyrosine kinase such as erlotinib (Tarceva) or sunitinib (Sutent) do not appear to be effective in colorectal cancer.

1	Bevacizumab (Avastin) is a monoclonal antibody directed against the vascular endothelial growth factor (VEGF) and is thought to act as an antiangiogenesis agent. The addition of bevacizumab to irinotecan-containing combinations and to FOLFOX initially appeared to significantly improve the outcome observed with chemotherapy alone, but subsequent studies have suggested a lesser degree of benefit. The use of bevacizumab can lead to hypertension, proteinuria, and an increased likelihood of thromboembolic events. Patients with solitary hepatic metastases without clinical or radiographic evidence of additional tumor involvement should be considered for partial liver resection, because such procedures are associated with 5-year survival rates of 25–30% when performed on selected individuals by experienced surgeons.

1	The administration of 5-FU and LV for 6 months after resection of tumor in patients with stage III disease leads to a 40% decrease in recurrence rates and 30% improvement in survival. The likelihood of recurrence has been further reduced when oxaliplatin has been combined with 5-FU and LV (e.g., FOLFOX); unexpectedly, the addition of irinotecan to 5-FU and LV as well as the addition of either bevacizumab or cetuximab to FOLFOX did not significantly enhance outcome. Patients with stage II tumors do not appear to benefit appreciably from adjuvant therapy, with the use of such treatment generally restricted to those patients having biologic characteristics (e.g., perforated tumors, T4 lesions, lymphovascular invasion) that place them at higher likelihood for recurrence. The addition of oxaliplatin to adjuvant treatment for patients older than age 70 and those with stage II disease does not appear to provide any therapeutic benefit.

1	In rectal cancer, the delivery of preoperative or postoperative combined-modality therapy (5-FU plus radiation therapy) reduces the risk of recurrence and increases the chance of cure for patients with stage II and III tumors, with the preoperative approach being better tolerated. The 5-FU acts as a radiosensitizer when delivered together with radiation therapy. Life-extending adjuvant therapy is used in only about half of patients older than age 65 years. This age bias is unfortunate because the benefits and likely the tolerance of adjuvant therapy in patients age ≥65 years appear similar to those seen in younger individuals.

1	Cancers of the anus account for 1–2% of the malignant tumors of the large bowel. Most such lesions arise in the anal canal, the anatomic area extending from the anorectal ring to a zone approximately halfway between the pectinate (or dentate) line and the anal verge. Carcinomas arising proximal to the pectinate line (i.e., in the transitional zone between the glandular mucosa of the rectum and the squamous epithelium of the distal anus) are known as basaloid, cuboidal, or cloacogenic tumors; about one-third of anal cancers have this histologic pattern. Malignancies arising distal to the pectinate line have squamous histology, ulcerate more frequently, and constitute ∼55% of anal cancers. The prognosis for patients with basaloid and squamous cell cancers of 544 the anus is identical when corrected for tumor size and the presence or absence of nodal spread. The development of anal cancer is associated with infection by human papillomavirus, the same organism etiologically linked to

1	corrected for tumor size and the presence or absence of nodal spread. The development of anal cancer is associated with infection by human papillomavirus, the same organism etiologically linked to cervical cancer. The virus is sexually transmitted. The infection may lead to anal warts (condyloma acuminata), which may progress to anal intraepithelial neoplasia and on to squamous cell carcinoma. The risk for anal cancer is increased among homosexual males, presumably related to anal intercourse. Anal cancer risk is increased in both men and women with AIDS, possibly because their immunosuppressed state permits more severe papillomavirus infection. Vaccination against human papilloma viruses may reduce the eventual risk for anal cancer. Anal cancers occur most commonly in middle-aged persons and are more frequent in women than men. At diagnosis, patients may experience bleeding, pain, sensation of a perianal mass, and pruritus. Radical surgery (abdominal-perineal resection with lymph

1	and are more frequent in women than men. At diagnosis, patients may experience bleeding, pain, sensation of a perianal mass, and pruritus. Radical surgery (abdominal-perineal resection with lymph node sampling and a permanent colostomy) was once the treatment of choice for this tumor type. The 5-year survival rate after such a procedure was 55–70% in the absence of spread to regional lymph nodes and <20% if nodal involvement was present. An alternative therapeutic approach combining external beam radiation therapy with concomitant chemotherapy (5-FU and mitomycin C) has resulted in biopsy-proven disappearance of all tumor in >80% of patients whose initial lesion was <3 cm in size. Tumor recurrences develop in <10% of these patients, meaning that ∼70% of patients with anal cancers can be cured with nonoperative treatment and without the need for a colostomy. Surgery should be reserved for the minority of individuals who are found to have residual tumor after being managed initially

1	cured with nonoperative treatment and without the need for a colostomy. Surgery should be reserved for the minority of individuals who are found to have residual tumor after being managed initially with radia tion therapy combined with chemotherapy. tumors of the Liver and Biliary tree Brian I. Carr HEPATOCELLULAR CARCINOMA INCIDENCE 111

1	Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. The annual global incidence is approx imately 1 million cases, with a male-to-female ratio of approximately 4:1 (1:1 without cirrhosis to 9:1 in many high-incidence countries). The incidence rate equals the death rate. In the United States, approximately 22,000 new cases are diagnosed annually, with 18,000 deaths. The death rates in males in low-incidence countries such as the United States are 1.9 per 100,000 per year; in intermediate areas such as Austria and South Africa, they range from 5.1–20; and in high-incidence areas such as in the Orient (China and Korea), they are as high as 23.1– 150 per 100,000 per year (Table 111-1). The incidence of HCC in the United States is approximately 3 per 100,000 persons, with significant gender, ethnic, and geographic variations. These numbers are rapidly increasing and may be an underestimate. Approximately 4 million chronic hepatitis C virus (HCV) carriers are in

1	with significant gender, ethnic, and geographic variations. These numbers are rapidly increasing and may be an underestimate. Approximately 4 million chronic hepatitis C virus (HCV) carriers are in the United States alone. Approximately 10% of them, or 400,000, are likely to develop cirrhosis. Approximately 5%, or 20,000, of these patients may develop HCC annually. Add to this the two other common predisposing factors—hepatitis B virus (HBV) and chronic alcohol consumption—and 60,000 new HCC cases annually seem possible. Future advances in HCC survival will likely depend in part on immunization strategies for HBV (and HCV) and earlier diagnosis by screening of patients at risk of HCC development.

1	Current Directions With the U.S. HCV epidemic, HCC is increasing in most states, and obesity-associated liver disease (nonalcoholic steatohepatitis [NASH]) is increasingly recognized as a cause. Persons per 100,000 per Year Argentina 6.0 2.5 Brazil, Recife 9.2 8.3 Brazil, Sao Paulo 3.8 2.6 Mozambique 112.9 30.8 South Africa, Cape: Black 26.3 8.4 South Africa, Cape: White 1.2 0.6 Senegal 25.6 9.0 Nigeria 15.4 3.2 Gambia 33.1 12.6 Burma 25.5 8.8 Japan 7.2 2.2 Korea 13.8 3.2 China, Shanghai 34.4 11.6 India, Bombay 4.9 2.5 India, Madras 2.1 0.7 Great Britain 1.6 0.8 France 6.9 1.2 Italy, Varese 7.1 2.7 Norway 1.8 1.1 Spain, Navarra 7.9 4.7 There are two general types of epidemiologic studies of

1	India, Madras 2.1 0.7 Great Britain 1.6 0.8 France 6.9 1.2 Italy, Varese 7.1 2.7 Norway 1.8 1.1 Spain, Navarra 7.9 4.7 There are two general types of epidemiologic studies of HCC—those of country-based incidence rates (Table 111-1) and those of migrants. Endemic hot spots occur in areas of China and sub-Saharan Africa, which are associated both with high endemic hepatitis B carrier rates as well as mycotoxin contamination of foodstuffs (aflatoxin B1), stored grains, drinking water, and soil. Environmental factors are important, for example, Japanese in Japan have a higher incidence than Japanese living in Hawaii, who in turn have a higher incidence than those living in California.

1	studied along two general lines. First are agents identified as carcinogenic in experimental animals (particularly rodents) that are thought to be present in the human environment (Table 111-2). Second is the association of HCC with various other clinical conditions. Probably the best-studied and most potent ubiquitous natural chemical carcinogen is a product of the Aspergillus fungus, called aflatoxin B1. This mold and aflatoxin product can be found in a variety of stored grains in hot, humid places, where peanuts and rice are stored in unrefrigerated conditions. Aflatoxin contamination of foodstuffs correlates well with incidence rates in Africa and to some extent in China. In endemic areas of China, even farm animals such as ducks have HCC. The most potent carcinogens appear to be natural products of plants, fungi, and bacteria, such as bush trees containing pyrrolizidine alkaloids as well as tannic acid and safrole. Pollutants such as pesticides and insecticides are known rodent

1	products of plants, fungi, and bacteria, such as bush trees containing pyrrolizidine alkaloids as well as tannic acid and safrole. Pollutants such as pesticides and insecticides are known rodent carcinogens.

1	Hepatitis Both case-control and cohort studies have shown a strong association between chronic hepatitis B carrier rates and increased incidence of HCC. In Taiwanese male postal carriers who were hepatitis B surface antigen (HBsAg)-positive, a 98-fold greater risk for HCC was found compared to HBsAg-negative individuals. The incidence of HCC in Alaskan natives is markedly increased related to a high prevalence of HBV infection. HBV-based HCC may involve rounds of hepatic destruction with subsequent proliferation and not necessarily frank cirrhosis. The increase in Japanese Abbreviations: NAFL, nonalcoholic fatty liver; NASH, nonalcoholic steatohepatitis.

1	HCC incidence rates in the last three decades is thought to be from hepatitis C. A large-scale World Health Organization (WHO)sponsored intervention study is currently under way in Asia involving HBV vaccination of the newborn. HCC in African blacks is not associated with severe cirrhosis but is poorly differentiated and very aggressive. Despite uniform HBV carrier rates among the South African Bantu, there is a ninefold difference in HCC incidence between Mozambicans living along the coast and inland. These differences are attributed to the additional exposure to dietary aflatoxin B1 and other carcinogenic mycotoxins. A typical interval between HCV-associated transfusion and subsequent HCC is approximately 30 years. HCV-associated HCC patients tend to have more frequent and advanced cirrhosis, but in HBV-associated HCC, only half the patients have cirrhosis, with the remainder having chronic active hepatitis (Chap. 362).

1	Other Etiologic Conditions The 75–85% association of HCC with underlying cirrhosis has long been recognized, more typically with macronodular cirrhosis in Southeast Asia, but also with micronodular cirrhosis (alcohol) in Europe and the United States (Chap. 365). It is still not clear whether cirrhosis itself is a predisposing factor to the development of HCC or whether the underlying causes of the cirrhosis are actually the carcinogenic factors. However, ∼20% of U.S. patients with HCC do not have underlying cirrhosis. Several underlying conditions are associated with an increased risk for cirrhosis-associated HCC (Table 111-2), including hepatitis, alcohol, autoimmune chronic active hepatitis, cryptogenic cirrhosis, and NASH. A less common association is with primary biliary cirrhosis and several metabolic diseases including hemochromatosis, Wilson’s disease, α1 antitrypsin deficiency, tyrosinemia, porphyria cutanea tarda, glycogenesis types 1 and 3, citrullinemia, and orotic

1	and several metabolic diseases including hemochromatosis, Wilson’s disease, α1 antitrypsin deficiency, tyrosinemia, porphyria cutanea tarda, glycogenesis types 1 and 3, citrullinemia, and orotic aciduria. The etiology of HCC in those 20% of patients who have no cirrhosis is currently unclear, and their HCC natural history is not well-defined.

1	Current Directions Many patients have multiple etiologies, and the interactions of HBV, HCV, alcohol, smoking, and aflatoxins are just beginning to be explored.

1	Symptoms These include abdominal pain, weight loss, weak ness, abdominal fullness and swelling, jaundice, and nausea (Table 111-3). Presenting signs and symptoms differ somewhat between highand low-incidence areas. In high-risk areas, especially in South African blacks, the most common symptom is abdominal pain; by contrast, only 40–50% of Chinese and Japanese patients present with abdominal pain. Abdominal swelling may occur as a consequence of ascites due to the underlying chronic liver disease or may be due to a rapidly expanding tumor. Occasionally, central necrosis or acute hemorrhage into the peritoneal cavity leads to death. In countries with an active surveillance program, HCC tends to be identified at an earlier stage, when symptoms may be due only to the underlying disease. Jaundice is usually due to obstruction of the intrahepatic ducts from underlying liver disease. Hematemesis may occur due to Symptom No. of Patients (%)

1	Symptom No. of Patients (%) No symptom 129 (24) Abdominal pain 219 (40) Other (workup of anemia and various 64 (12) diseases) Routine physical exam finding, elevated LFTs 129 (24) Weight loss 112 (20) Appetite loss 59 (11) Weakness/malaise 83 (15) Jaundice 30 (5) Routine CT scan screening of known cirrhosis 92 (17) Cirrhosis symptoms (ankle swelling, 98 (18) abdominal bloating, increased girth, pruritus, GI bleed) Diarrhea 7 (1) Tumor rupture 1 Mean age (yr) 56 ± 13 Male:Female 3:1 Ethnicity Abbreviations: CT, computed tomography; GI, gastrointestinal; LFT, liver function test. esophageal varices from the underlying portal hypertension. Bone pain is seen in 3–12% of patients, but necropsies show pathologic bone metastases in ∼20% of patients. However, 25% of patients may be asymptomatic.

1	Physical Signs Hepatomegaly is the most common physical sign, occurring in 50–90% of the patients. Abdominal bruits are noted in 6–25%, and ascites occurs in 30–60% of patients. Ascites should be examined by cytology. Splenomegaly is mainly due to portal hypertension. Weight loss and muscle wasting are common, particularly with rapidly growing or large tumors. Fever is found in 10–50% of patients, from unclear cause. The signs of chronic liver disease may often be present, including jaundice, dilated abdominal veins, palmar erythema, gynecomastia, testicular atrophy, and peripheral edema. Budd-Chiari syndrome can occur due to HCC invasion of the hepatic veins, with tense ascites and a large tender liver (Chap. 365).

1	Paraneoplastic Syndromes Most paraneoplastic syndromes in HCC are biochemical abnormalities without associated clinical consequences. They include hypoglycemia (also caused by end-stage liver failure), erythrocytosis, hypercalcemia, hypercholesterolemia, dysfibrinogenemia, carcinoid syndrome, increased thyroxin-binding globulin, changes in secondary sex characteristics (gynecomastia, testicular atrophy, and precocious puberty), and porphyria cutanea tarda. Mild hypoglycemia occurs in rapidly growing HCC as part of

1	Tumors of the Liver and Biliary Tree 546 terminal illness, and profound hypoglycemia may occur, although the cause is unclear. Erythrocytosis occurs in 3–12% of patients and hypercholesterolemia in 10–40%. A high percentage of patients have thrombocytopenia associated with their fibrosis or leukopenia, resulting from portal hypertension, and not from cancer infiltration of bone marrow, as in other tumor types. Furthermore, large HCCs have normal or high platelet levels (thrombocytosis), as in ovarian and other gastrointestinal cancers, probably related to elevated interleukin 6 (IL-6) levels.

1	Multiple clinical staging systems for HCC have been described. A widely used one has been the American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) classification. However, the Cancer of the Liver Italian Program (CLIP) system is now popular because it takes cirrhosis into account, based on the original Okuda system (Table 111-4). Patients with Okuda stage III disease have a dire prognosis because they usually cannot be curatively resected, and the condition of their liver typically precludes chemotherapy. Other staging systems have been proposed, and a consensus is needed. They are all based on combining the prognostic features of liver damage with those of tumor aggressiveness and include the Barcelona Clinic Liver Cancer (BCLC) system from Spain (Fig. 111-1), which is externally validated and incorporates baseline survival estimates; the Chinese University Prognostic Index (CUPI); the important and simple Japan Integrated Staging Score (JIS); and SLiDe, which stands

1	validated and incorporates baseline survival estimates; the Chinese University Prognostic Index (CUPI); the important and simple Japan Integrated Staging Score (JIS); and SLiDe, which stands for s tage, li ver damage, and de s-γ-carboxy prothrombin. CLIP and BCLC appear most popular in the West, whereas JIS is favored in Japan. Each system i. Tumor number Single Multiple – Hepatic replacement by tumor (%) <50 <50 >50 ii. Child-Pugh score A B C iii. α Fetoprotein level (ng/mL) <400 ≥400 – iv. Portal vein thrombosis (CT) No Yes –

1	CLIP stages (score = sum of points): CLIP 0, 0 points; CLIP 1, 1 point; CLIP 2, 2 points; CLIP 3, 3 points. Okuda stages: stage 1, all (−); stage 2, 1 or 2 (+); stage 3, 3 or 4 (+). aExtent of liver occupied by tumor. Abbreviation: CLIP, Cancer of the Liver Italian Program. has its champions. The best prognosis is for stage I, solitary tumors less than 2 cm in diameter without vascular invasion. Adverse prognostic features include ascites, jaundice, vascular invasion, and elevated α fetoprotein (AFP). Vascular invasion in particular has profound effects

1	Resection Liver transplantation (CLT/LDLT) Curative treatments (30%) 5-yr survival: 40–70% Randomized controlled trials (50%) Median survival 11–20 months Symptomatic treatment (20%) Survival <3 months PEI/RF TACE Sorafenib Advanced stage (C) Portal invasion, N1, M1, PST 1-2 Intermediate stage (B) Multinodular, PST 0 Very early stage (0) Single <2 cm carcinoma in situ Single Portal pressure/ bilirubin Normal Increased Associated diseases No Yes 3 nodules ˜3 cm Stage 0 PST 0, Child-Pugh A Early stage (A) Single or 3 nodules <3 cm, PST 0 PST End stage (D) Stage D PST >2, Child-Pugh C Stage A-C PST 0–2, Child-Pugh A–B HCC

1	FIGURE 111-1 Barcelona Clinic Liver Cancer (BCLC) staging classification and treatment schedule. Patients with very early hepatocellular carcinoma (HCC) (stage 0) are optimal candidates for resection. Patients with early HCC (stage A) are candidates for radical therapy (resection, liver transplantation [LT], or local ablation via percutaneous ethanol injection [PEI] or radiofrequency [RF] ablation). Patients with intermediate HCC (stage B) benefit from transcatheter arterial chemoembolization (TACE). Patients with advanced HCC, defined as presence of macroscopic vascular invasion, extrahepatic spread, or cancer-related symptoms (Eastern Cooperative Oncology Group performance status 1 or 2) (stage C), benefit from sorafenib. Patients with end-stage disease (stage D) will receive symptomatic treatment. Treatment strategy will transition from one stage to another on treatment failure or contraindications for the procedures. CLT, cadaveric liver transplantation; LDLT, living donor liver

1	treatment. Treatment strategy will transition from one stage to another on treatment failure or contraindications for the procedures. CLT, cadaveric liver transplantation; LDLT, living donor liver transplantation; PST, Performance Status Test. (Modified from JM Llovet et al: JNCI 100:698, 2008.) on prognosis and may be microscopic or macroscopic (visible on computed tomography [CT] scans). Most large tumors have microscopic vascular invasion, so full staging can usually be made only after surgical resection. Stage III disease contains a mixture of lymph node– positive and–negative tumors. Stage III patients with positive lymph node disease have a poor prognosis, and few patients survive 1 year. The prognosis of stage IV is poor after either resection or transplantation, and 1-year survival is rare.

1	New Directions Consensus is needed on staging. These systems will soon be refined or upended by proteomics. APPROACH TO THE PATIENT: The history is important in evaluating putative predisposing factors, including a history of hepatitis or jaundice, blood transfusion, or use of intravenous drugs. A family history of HCC or hepatitis should be sought and a detailed social history taken to include job descriptions for industrial exposure to possible carcinogenic drugs as well as contraceptive hormones. Physical examination should include assessing stigmata of underlying liver disease such as jaundice, ascites, peripheral edema, spider nevi, palmar erythema, and weight loss. Evaluation of the abdomen for hepatic size, masses or ascites, hepatic nodularity and tenderness, and splenomegaly is needed, as is assessment of overall performance status and psychosocial evaluation.

1	AFP is a serum tumor marker for HCC; however, it is only increased in approximately one-half of U.S. patients. The lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) assay is thought to be more specific. The other widely used assay is that for des-γ-carboxy prothrombin (DCP), a protein induced by vitamin K absence (PIVKA-2). This protein is increased in as many as 80% of HCC patients but may also be elevated in patients with vitamin K deficiency; it is always elevated after warfarin use. It may also predict for portal vein invasion. Both AFP-L3 and DCP are U.S. Food and Drug Administration (FDA) approved. Many other assays have been developed, such as glypican-3, but none have greater aggregate sensitivity and specificity. In a patient presenting with either a new hepatic mass or other indications of recent hepatic decompensation, carcinoembryonic antigen (CEA), vitamin B12, AFP, ferritin, PIVKA2, and antimitochondrial antibody should be measured, and standard liver function

1	other indications of recent hepatic decompensation, carcinoembryonic antigen (CEA), vitamin B12, AFP, ferritin, PIVKA2, and antimitochondrial antibody should be measured, and standard liver function tests should be performed, including prothrombin time (PT), partial thromboplastin time (PTT), albumin, transaminases, γ-glutamyl transpeptidase, and alkaline phosphatase. γ-Glutamyl transpeptidase and alkaline phosphatase may be particularly important in the 50% of HCC patients who have low AFP levels. Decreases in platelet count and white blood cell count may reflect portal hypertension and associated hypersplenism. Hepatitis A, B, and C serology should be measured. If HBV or HCV serology is positive, quantitative measurements of HBV DNA or HCV RNA are needed.

1	New Directions Newer biomarkers are being evaluated, especially tissueand serum-based genomics profiling. Newer plasma biomarkers include glypican-3, osteopontin, insulin-like growth factor I, and vascular endothelial growth factor. However, they are still in process of validation. Furthermore, the commercial availability of kits for isolating circulating tumor cells is permitting the molecular profiling of HCCs without the need for further tissue biopsy.

1	An ultrasound examination of the liver is an excellent screening tool. The two characteristic vascular abnormalities are hypervascularity of the tumor mass (neovascularization or abnormal tumor-feeding arterial vessels) and thrombosis by tumor invasion of otherwise normal portal veins. To determine tumor size and extent and the presence of portal vein invasion accurately, a helical/ triphasic CT scan of the abdomen and pelvis, with fast-contrast bolus technique, should be performed to detect the vascular lesions typical of HCC. Portal vein invasion is normally detected as an obstruction and expansion of the vessel. A chest CT is used to exclude metastases. Magnetic resonance imaging (MRI) can also provide detailed information, especially with the newer contrast agents. Ethiodol (Lipiodol) is an ethiodized oil emulsion retained by liver tumors that can be delivered by hepatic artery injection (5–15 mL) for CT imaging 1 week later. For small tumors, Ethiodol injection is very helpful

1	is an ethiodized oil emulsion retained by liver tumors that can be delivered by hepatic artery injection (5–15 mL) for CT imaging 1 week later. For small tumors, Ethiodol injection is very helpful before biopsy because the histologic presence of the dye constitutes proof that the needle biopsied the mass under suspicion. A prospective comparison of triphasic CT, gadolinium-enhanced MRI, ultrasound, and fluorodeoxyglucose positron emission tomography (FDG-PET) showed similar results for CT, MRI, and ultrasound; PET imaging appears to be positive in only a subset of HCC patients. Abdominal CT versus MRI/CT uses a faster single breath-hold, is less complex, and is less dependent on patient cooperation. MRI requires a longer examination, and ascites can cause artifacts, but MRI is better able to distinguish dysplastic or regenerative nodules from HCC. Imaging criteria have been developed for HCC that do not require biopsy proof, as they have >90% specificity. The criteria include nodules

1	to distinguish dysplastic or regenerative nodules from HCC. Imaging criteria have been developed for HCC that do not require biopsy proof, as they have >90% specificity. The criteria include nodules >1 cm with arterial enhancement and portal venous washout and, for small tumors, specified growth rates on two scans performed less than 6 months apart (Organ Procurement and Transplant Network). Nevertheless, explant pathology after liver transplant for HCC has shown that ∼20% of patients diagnosed without biopsy did not actually have a tumor.

1	New Directions The altered tumor vascularity that is a consequence of molecularly targeted therapies is the basis for newer imaging techniques including contrast-enhanced ultrasound (CEUS) and dynamic MRI.

1	Histologic proof of the presence of HCC is obtained through a core liver biopsy of the liver mass under ultrasound guidance, as well as random biopsy of the underlying liver. Bleeding risk is increased compared to other cancers because (1) the tumors are hypervascular and (2) patients often have thrombocytopenia and decreased liver-dependent clotting factors. Bleeding risk is further increased in the presence of ascites. Tracking of tumor has an uncommon problem. Fine-needle aspirates can provide sufficient material for diagnosis of cancer, but core biopsies are preferred. Tissue architecture allows the distinction between HCC and adenocarcinoma. Laparoscopic approaches can also be used. For patients suspected of having portal vein involvement, a core biopsy of the portal vein may be performed safely. If positive, this is regarded as an exclusion criterion for transplantation for HCC.

1	New Directions Immunohistochemistry has become mainstream. Prognostic subgroupings are being defined based on growth signaling pathway proteins and genotyping strategies, including a prognostically significant five-gene profile score. Furthermore, molecular profiling of the underlying liver has provided evidence for a “fieldeffect” of cirrhosis in generating recurrent or new HCCs after primary resection. In addition, characteristics of HCC stem cells have been identified and include EpCAM, CD44, and CD90 expression, which may form the basis of stem cell therapeutic targeting strategies.

1	There are two goals of screening, both in patients at increased risk for developing HCC, such as those with cirrhosis. The first goal is to detect smaller tumors that are potentially curable by ablation. The second goal is to enhance survival, compared with patients who were not diagnosed by surveillance. Evidence from Taiwan has shown a survival advantage to population screening in HBV-positive patients, and other evidence has shown its efficacy in diagnosis for HCV. Prospective studies in

1	Tumors of the Liver and Biliary Tree 548 high-risk populations showed that ultrasound was more sensitive than AFP elevations alone, although most practitioners request both tests at 6-month intervals for HBV and HCV carriers, especially in the presence of cirrhosis or worsening of liver function tests. However, an Italian study in patients with cirrhosis identified a yearly HCC incidence of 3% but showed no increase in the rate of detection of potentially curable tumors with aggressive screening. Prevention strategies including universal vaccination against hepatitis are more likely to be effective than screening efforts. Despite absence of formal guidelines, most practitioners obtain 6-month AFP and ultrasound (cheap and ubiquitous, even in poor countries) or CT (more sensitive, especially in overweight patients, but more costly) studies when following high-risk patients (HBV carriers, HCV cirrhosis, family history of HCC).

1	Current Directions Cost-benefit analysis is not yet convincing, even though screening is intuitively sound. However, studies from areas with high HBV carrier rates have shown a survival benefit for screening as a result of earlier stage at diagnosis. A definitive clinical trial on screening is unlikely, due to difficulties in obtaining informed consent for patients who are not to be screened. γ-Glutamyl transpeptidase appears useful for detecting small tumors.

1	Prevention strategies can only be planned when the causes of a cancer are known or strongly suspected. This is true of few human cancers, with significant exceptions being smoking and lung cancer, papilloma virus and cancer of the cervix uteri, and cirrhosis of any cause or dietary contamination by aflatoxin B1 for HCC. Aflatoxin B1is one of the most potent known chemical carcinogens and is a product of the Aspergillus mold that grows on peanuts and rice when stored in hot and humid climates. The obvious strategy is to refrigerate these foodstuffs when stored and to conduct surveillance programs for elevated aflatoxin B1 levels, as happens in the United States, but not usually in Asia. HBV is commonly transmitted from mother to fetus in Asia (except Japan), and neonatal HBV vaccination programs have resulted in a big decrease in adolescent HBV and, thus, in predicted HCC rates. There are millions of HBV and HCV carriers (4 million with HCV in the United States) who are already

1	programs have resulted in a big decrease in adolescent HBV and, thus, in predicted HCC rates. There are millions of HBV and HCV carriers (4 million with HCV in the United States) who are already infected. Nucleoside analogue–based chemoprevention (entecavir) of HBV-mediated HCC in Japan resulted in a fivefold decrease in HCC incidence over 5 years in cirrhotic but not in non-cirrhotic HBV patients. More powerful and effective HCV therapies promise the possibility of prevention of HCV-based HCC in the future.

1	Most HCC patients have two liver diseases, cirrhosis and HCC, each of which is an independent cause of death. The presence of cirrhosis usually places constraints on resection surgery, ablative therapies, and chemotherapy. Thus patient assessment and treatment planning have to take the severity of the nonmalignant liver disease into account. The clinical management choices for HCC can be complex (Fig. 111-2, Tables 111-5 and 111-6). The natural history of HCC is highly variable. Patients presenting with advanced tumors (vascular invasion, symptoms, extrahepatic spread) have a median survival of ∼4 months, with or without treatment. Treatment results from the literature are difficult to interpret. Survival is not always a measure of the efficacy of therapy because of the adverse effects on survival of the underlying liver disease. A multidisciplinary team, including a hepatologist, interventional radiologist, surgical oncologist, resection surgeon, transplant surgeon, and medical

1	on survival of the underlying liver disease. A multidisciplinary team, including a hepatologist, interventional radiologist, surgical oncologist, resection surgeon, transplant surgeon, and medical oncologist, is important for the comprehensive management of HCC patients.

1	Early-stage tumors are successfully treated using various techniques, including surgical resection, local ablation (thermal, radiofrequency [RFA], or microwave ablation (MWA]), and local injection therapies (Table 111-6). Because the majority of patients with HCC suffer from a field defect in the cirrhotic liver, they are at risk for subsequent multiple primary liver tumors. Many will also have significant underlying liver disease and may not tolerate major surgical loss of hepatic parenchyma, and they may be eligible for orthotopic liver transplant (OLTX). Living related donor transplants have increased in popularity, resulting in absence of waiting for a transplant. An important principle in treating early-stage HCC in the nontransplant

1	PEI/RFA/MWA Single lesion <5 cm Child’s A/B Tumor progression Transplant TACE/90Yttrium/ New agent trials Multifocal >5 cm Child’s A/B Sorafenib/Palliative care hormonal therapies Child’s C Bilirubin ˜2 Metastases Neoadjuvant bridge therapy RFA/TACE/90Yttrium Living donor transplant Suitable donor UNOS + criteria Ablation candidate Resection/RFA Non-cirrhotic Child’s A Single lesion No metastases Transplant candidate Transplant evaluation 1 lesion <5 cm 3 lesions all less than 3 cm Child’s A/B/C; AFP <1000 ng/mL No gross vascular invasion Not suitable for surgery Medical evaluation Comorbid factors 4 lesions Gross vascular invasion LN (+) or metastasis HCC diagnosed Cadaver donor wait list Yes Sorafenib/New agent clinical trials FIGURE 111-2 Hepatocellular carcinoma (HCC) treatment algorithm. The initial clinical evaluation is aimed at assessing the extent of the tumor and the underlying functional compromise of the liver by cirrhosis. Patients are classified as having resectable

1	The initial clinical evaluation is aimed at assessing the extent of the tumor and the underlying functional compromise of the liver by cirrhosis. Patients are classified as having resectable disease or unresectable disease or as being candidates for transplantation. AFP, α fetoprotein; LN, lymph node; MWA, microwave ablation; OLTX, orthotopic liver trans-plantation; PEI, percutaneous ethanol injection; RFA, radiofrequency ablation; TACE, transcatheter arterial chemoembolization; UNOS, United Network for Organ Sharing. Child’s A/B/C refers to the Child-Pugh classification of liver failure.

1	Regional Therapies: Hepatic Artery Transcatheter Treatments Transarterial chemotherapy Transarterial embolization Transarterial chemoembolization Transarterial drug-eluting beads Transarterial radiotherapies: Molecularly targeted therapies (sorafenib, etc.) setting is to use liver-sparing treatments and to focus on treatment of both the tumor and the cirrhosis.

1	Surgical Excision The risk of major hepatectomy is high (5–10% mortality rate) due to the underlying liver disease and the potential for liver failure, but acceptable in selected cases and highly dependent on surgical experience. The risk is lower in high-volume centers. Preoperative portal vein occlusion can sometimes be performed to cause atrophy of the HCC-involved lobe and compensatory hypertrophy of the noninvolved liver, permitting safer resection. Intraoperative ultrasound is useful for planning the surgical approach. The ultrasound can image the proximity of major vascular structures that may be encountered during the dissection. In cirrhotic patients, any major liver surgery can result in liver failure. The Child-Pugh classification of liver failure is still a reliable prognosticator for tolerance of hepatic surgery, and only Child A patients should be considered for surgical resection. Child B and C patients with stages I and II HCC should be referred for OLTX if 549

1	for tolerance of hepatic surgery, and only Child A patients should be considered for surgical resection. Child B and C patients with stages I and II HCC should be referred for OLTX if 549 appropriate, as well as patients with ascites or a recent history of variceal bleeding. Although open surgical excision is the most reliable, the patient may be better served with a laparoscopic approach to resection, using RFA, MWA, or percutaneous ethanol injection (PEI). No adequate comparisons of these different techniques have been undertaken, and the choice of treatment is usually based on physician skill. However, RFA has been shown to be superior to PEI in necrosis induction for tumors <3 cm in diameter and is thought to be equivalent to open resection and, thus, is the treatment of first choice for these small tumors. As tumors get larger than 3 cm, especially ≥5 cm, the effectiveness of RFA-induced necrosis diminishes. The combination of transcatheter arterial chemoembolization (TACE) with

1	for these small tumors. As tumors get larger than 3 cm, especially ≥5 cm, the effectiveness of RFA-induced necrosis diminishes. The combination of transcatheter arterial chemoembolization (TACE) with RFA has shown superior results to TACE alone in a prospective, randomized trial. Although vascular invasion is a preeminent negative prognostic factor, microvascular invasion in small tumors appears not to be a negative factor.

1	Local Ablation Strategies RFA uses heat to ablate tumors. The maxi mum size of the probe arrays allows for a 7-cm zone of necrosis, which would be adequate for a 3to 4-cm tumor. The heat reliably kills cells within the zone of necrosis. Treatment of tumors close to the main portal pedicles can lead to bile duct injury and obstruction. This limits the location of tumors that are anatomically suited for this technique. RFA can be performed percutaneously with CT or ultrasound guidance, or at the time of laparoscopy with ultrasound guidance.

1	Local Injection Therapy Numerous agents have been used for local injection into tumors, most commonly ethanol (PEI). The relatively soft HCC within the hard background cirrhotic liver allows for injection of large volumes of ethanol into the tumor without diffusion into the hepatic parenchyma or leakage out of the liver. PEI causes direct destruction of cancer cells, but it is not selective for cancer and will destroy normal cells in the vicinity. However, it usually requires multiple injections (average three), in contrast to one for RFA. The maximum size of tumor reliably treated is 3 cm, even with multiple injections.

1	current directions Resection and RFA each obtain similar results. However, a distinction has been made between the causes and prevention strategies needed to prevent early versus late tumor recurrences after resection. Early recurrence has been linked to tumor invasion factors, especially microvascular tumor invasion with elevated transaminases, whereas late recurrence has been associated with cirrhosis and virus hepatitis factors and, thus, the development of new tumors. See the section on virus-directed adjuvant therapy below. Liver Transplantation (OLTX) A viable option for stages I and II tumors in the setting of cirrhosis is OLTX, with survival approaching that for noncancer cases. OLTX for

1	Tumors of the Liver and Biliary Tree patients with a single lesion ≤5 cm or three or fewer nodules, each ≤3 cm (Milan criteria), resulted in excellent taBLe 111-6 some ranDomizeD CLiniCaL triaLs invoLving transhepatiC artery ChemoemBoLization (taCe) for hepatoCeLLuLar CarCinoma 550 growing in the months until a donor liver becomes available. What remains unclear, however, is whether this translates into prolonged survival after transplant. Further, it is not known whether patients who have had their tumor(s) treated preoperatively follow the recurrence pattern predicted by their tumor status at the time of transplant (i.e., post–local ablative therapy), or if they follow the course set by their tumor parameters present before such treatment. The United Network for Organ Sharing (UNOS) point system for priority scoring of OLTX recipients now includes additional points for patients with HCC. The success of living related donor liver transplantation programs has also led to patients

1	point system for priority scoring of OLTX recipients now includes additional points for patients with HCC. The success of living related donor liver transplantation programs has also led to patients receiving transplantation earlier for HCC and often with greater than minimal tumors.

1	current directions Expanded criteria for larger HCCs beyond the Milan criteria (one lesion <5 cm or three lesions, each <3 cm), such as the University of California, San Francisco (UCSF) criteria (single lesion ≤6.5 cm or two lesions ≤4.5 cm with a total diameter ≤8 cm; 1and 5-year survival rates of 90 and 75%, respectively), are being increasingly accepted by various UNOS areas for OLTX with satisfactory longer-term survival comparable to Milan criteria results. Furthermore, downstaging of HCCs that are too large for the Milan criteria by medical therapy (TACE) is increasingly recognized as acceptable treatment before OLTX with equivalent outcomes to patients who originally were within Milan criteria. Within-criteria patients with AFP levels >1000 ng/mL have exceptionally high post-OLTX recurrence rates. Also, the use of “salvage” OLTX after recurrent HCC after resection has produced conflicting outcomes. Shortages of organs combined with advances in resection safety have led to

1	recurrence rates. Also, the use of “salvage” OLTX after recurrent HCC after resection has produced conflicting outcomes. Shortages of organs combined with advances in resection safety have led to increasing use of resection for patients with good liver function.

1	Adjuvant Therapy The role of adjuvant chemotherapy for patients after resection or OLTX remains unclear. Both adjuvant and neoadjuvant approaches have been studied, but no clear advantage in disease-free or overall survival has been found. However, a meta-analysis of several trials revealed a significant improvement in disease-free and overall survival. Although analysis of postoperative adjuvant systemic chemotherapy trials demonstrated no disease-free or overall survival advantage, single studies of TACE and neoadjuvant 131I-Ethiodol showed enhanced survival after resection. Antiviral therapy, instead of anticancer therapy, has been successful in decreasing postresection tumor recurrences in the postresection adjuvant setting. Nucleoside analogues in HBV-based HCC and peg-interferon plus ribavirin for HCV-based HCC have both been effective in reducing recurrence rates.

1	current directions A large adjuvant trial examining resection and transplantation, with or without sorafenib (see below) is in progress. The success of viral therapies in decreasing HCC recurrence after resection is part of a broader focus on the tumor microenvironment (stroma, blood vessels, inflammatory cells, and cytokines) as mediators of HCC progression and as targets for new therapies.

1	Fewer surgical options exist for stage III tumors involving major vascular structures. In patients without cirrhosis, a major hepatectomy is feasible, although prognosis is poor. Patients with Child A cirrhosis may be resected, but a lobectomy is associated with significant morbidity and mortality rates, and long-term prognosis is poor. Nevertheless, a small percentage of patients will achieve long-term survival, justifying an attempt at resection when feasible. Because of the advanced nature of these tumors, even successful resection can be followed by rapid recurrence. These patients are not considered candidates for transplantation because of the high tumor recurrence rates, unless their tumors can first be downstaged with neoadjuvant therapy. Decreasing the size of the primary tumor allows for less surgery, and the delay in surgery allows for extrahepatic disease to manifest on imaging studies and avoid unhelpful OLTX. The prognosis is poor for stage IV tumors, and no surgical

1	allows for less surgery, and the delay in surgery allows for extrahepatic disease to manifest on imaging studies and avoid unhelpful OLTX. The prognosis is poor for stage IV tumors, and no surgical treatment is recommended.

1	Systemic Chemotherapy A large number of controlled and uncontrolled clinical studies have been performed with most of the major classes of cancer chemotherapy. No single agent or combination of agents given systemically reproducibly leads to even a 25% response rate or has any effect on survival.

1	Regional Chemotherapy In contrast to the dismal results of systemic chemotherapy, a variety of agents given via the hepatic artery have activity for HCC confined to the liver (Table 111-6). Two randomized controlled trials have shown a survival advantage for TACE in a selected subset of patients. One used doxorubicin, and the other used cisplatin. Despite the fact that increased hepatic extraction of chemotherapy has been shown for very few drugs, some drugs such as cisplatin, doxorubicin, mitomycin C, and possibly neocarzinostatin, produce substantial objective responses when administered regionally. Few data are available on continuous hepatic arterial infusion for HCC, although pilot studies with cisplatin have shown encouraging responses. Because the reports have not usually stratified responses or survival based on TNM staging, it is difficult to know long-term prognosis in relation to tumor extent. Most of the studies on regional hepatic arterial chemotherapy also use an

1	responses or survival based on TNM staging, it is difficult to know long-term prognosis in relation to tumor extent. Most of the studies on regional hepatic arterial chemotherapy also use an embolizing agent such as Ethiodol, gelatin sponge particles (Gelfoam), starch (Spherex), or microspheres. Two products are composed of microspheres of defined size ranges—Embospheres (Biospheres) and Contour SE—using particles of 40–120, 100–300, 300–500, and 500–1000 μm in size. The optimal diameter of the particles for TACE has yet to be defined. Consistently higher objective response rates are reported for arterial administration of drugs together with some form of hepatic artery occlusion compared with any form of systemic chemotherapy to date. The widespread use of some form of embolization in addition to chemotherapy has added to its toxicities. These include a frequent but transient fever, abdominal pain, and anorexia (all in >60% of patients). In addition, >20% of patients have increased

1	to chemotherapy has added to its toxicities. These include a frequent but transient fever, abdominal pain, and anorexia (all in >60% of patients). In addition, >20% of patients have increased ascites or transient elevation of transaminases. Cystic artery spasm and cholecystitis are also not uncommon. However, higher responses have also been obtained. The hepatic toxicities associated with embolization may be ameliorated by the use of degradable starch microspheres, with 50–60% response rates. Two randomized studies of TACE versus placebo showed a survival advantage for treatment (Table 111-6). In addition, it is not clear that formal oncologic CT response criteria are adequate for HCC. A loss of vascularity on CT without size change may be an index of loss of viability and thus of response to TACE. A major problem that TACE trials have had in showing a survival advantage is that many HCC patients die of their underlying cirrhosis, not the tumor. Nevertheless, two randomized controlled

1	to TACE. A major problem that TACE trials have had in showing a survival advantage is that many HCC patients die of their underlying cirrhosis, not the tumor. Nevertheless, two randomized controlled trials, one using doxorubicin and the other using cisplatin, showed a survival advantage for TACE versus placebo (Table 111-6). However, improving quality of life is a legitimate goal of regional therapy. Drug-eluting beads using doxorubicin (DEB-TACE) have been claimed to produce equivalent survival with less toxicity, but this strategy has not been tested in a randomized trial.

1	Kinase Inhibitors A survival advantage has been observed for the oral multikinase inhibitor, sorafenib (Nexavar), versus placebo in two randomized trials. It targets both the Raf mitogenic pathway and the vascular endothelial growth factor receptor (VEGFR) endothelial vasculogenesis pathway. However, tumor responses were negligible, and the survival in the treatment arm in Asians was less than the placebo arm in the Western trial (Table 111-7). Sorafenib has considerable toxicity, with 30–40% of patients requiring “drug holidays,” dose reductions, or cessation of therapy. The most common toxicities include fatigue, hypertension, diarrhea, mucositis, and skin changes, such as the painful hand-foot syndrome, hair loss, and itching, each in 20–40% of patients. Several “look-alike” new agents that also target angiogenesis have either proved to be inferior or more toxic. These include sunitinib, brivanib, linifanib, everolimus, and bevacizumab (Table 111-8). The idea of angiogenesis alone

1	that also target angiogenesis have either proved to be inferior or more toxic. These include sunitinib, brivanib, linifanib, everolimus, and bevacizumab (Table 111-8). The idea of angiogenesis alone as a major HCC therapeutic target may need revision.

1	New Therapies Although prolonged survival has been reported in phase II trials using newer agents, such as bevacizumab plus Sorafenib vs placebo Raf, VEGFR, PDGFR 10.7 vs 7.9 Sorafenib vs placebo (Asians) Raf, VEGFR, PDGFR 6.5 vs 4.2 Abbreviations: PDGFR, platelet-derived growth factor receptor; Raf, rapidly accelerated fibrosarcoma; VEGFR vascular endothelial growth factor receptor.

1	erlotinib, the data from a phase III trial were disappointing. Several forms of radiation therapy have been used in the treatment of HCC, including external-beam radiation and conformal radiation therapy. Radiation hepatitis remains a dose-limiting problem. The pure beta emitter 90Yttrium attached to either glass (TheraSphere) or resin (SIR-Spheres) microspheres injected into a major branch hepatic artery has been assessed in phase II trials of HCC and has encouraging tumor control and survival effects with minimal toxicities. Randomized phase III trials comparing it to TACE have yet to be completed. The main attractiveness of 90Yttrium therapy is its safety in the presence of major branch portal vein thrombosis, where TACE is dangerous or contraindicated. Furthermore, external-beam radiation has been reported to be safe and useful in the control of major branch portal or hepatic vein invasion (thrombosis) by tumors. The studies have all been small. Vitamin K has been assessed in

1	has been reported to be safe and useful in the control of major branch portal or hepatic vein invasion (thrombosis) by tumors. The studies have all been small. Vitamin K has been assessed in clinical trials at high dosage for its HCC-inhibitory actions. This idea is based on the characteristic biochemical defect in HCC of elevated plasma levels of immature prothrombin (DCP or PIVKA-2), due to a defect in the activity of prothrombin carboxylase, a vitamin K–dependent enzyme. Two vitamin K randomized controlled trials from Japan show decreased tumor occurrence, but a major phase III trial aimed at limiting postresection recurrence was not successful.

1	current directions A number of new kinase inhibitors are being evaluated for HCC (Tables 111-9 and 111-10). These include the biologicals, such as Raf kinase and vascular endothelial growth factor (VEGF) inhibitors, and agents that target various steps of the cell growth pathway. Current hopes focus particularly on the Met pathway inhibitors such as tivantinib and several IGF receptor antagonists. 90Yttrium looks promising and without chemotherapy toxicities. It is particularly attractive because, unlike TACE, it seems safe in the presence of portal vein thrombosis, a pathognomonic feature of HCC aggressiveness. The bottleneck of liver donors for OLTX is at last widening with increasing use of living donors, and criteria for OLTX for larger HCCs are slowly expanding. Patient participation in clinical trials assessing new therapies is encouraged (www.clinicaltrials.gov).

1	The main effort now is the evaluation of combinations of the compounds listed in Tables 111-7 to 111-9 that target different pathways, as well as the combination of any of these targeted therapies, but especially sorafenib, with TACE or 90Yttrium radioembolization. Combining TACE with sorafenib appears to be safe in phase II studies with promising survival data, but randomized studies are still in progress. The same is true for intra-arterial 90Yttrium plus sorafenib as therapy for HCC and as bridge to transplant therapy. Ubiquitin-proteasome Bortezomib Abbreviations: EGF, epidermal growth factor; FGF1, fibroblast growth factor 1; IGF-I, insulin-like growth factor I; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor.

1	Tumor growth or spread is considered a poor prognostic sign and evidence of treatment failure. By contrast, patients receiving chemotherapy are judged to have a response if there is shrinkage of tumor size. Lack of response/size decrease has been thought of as treatment failure. Three considerations in HCC management have completely changed the views concerning nonshrinkage after therapy. First, the correlation between response to chemotherapy and survival is poor in various tumors; in some tumors, such as ovarian cancer and small-cell lung cancer, substantial tumor shrinkage on chemotherapy is followed by rapid

1	EGF receptor antagonists: erlotinib, gefitinib, lapatinib, cetuximab, brivanib Multikinase antagonists: sorafenib, sunitinib VEGF antagonist: bevacizumab VEGFR antagonist: ABT-869 (linifanib) mTOR antagonists: sirolimus, temsirolimus, everolimus Proteasome inhibitors: bortezomib Vitamin K 131I–Ethiodol (lipiodol) 131I–Ferritin 90Yttrium microspheres (TheraSphere, SIR-Spheres) 166Holmium, 188Rhenium Three-dimensional conformal radiation Proton beam high-dose radiotherapy Gamma knife, CyberKnife New targets: inhibitors of cyclin dependent kinases (Cdk), TRAIL induction caspases, and stem cells Abbreviations: EGF, epidermal growth factor; mTOR, mammalian target of rapamycin; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

1	Tumors of the Liver and Biliary Tree 552 tumor regrowth. Second, the Sorafenib HCC Assessment Randomized Protocol (SHARP) phase III trial of sorafenib versus placebo for unresectable HCC showed that survival could be significantly enhanced in the treatment arm with only 2% of the patients having tumor response but 70% of patients having disease stabilization. This observation has led to a reconsideration of the usefulness of response and the significance of disease stability. Third, HCC is a typically highly vascular tumor, and the vascularity is considered to be a measure of tumor viability. As a result, the Response Evaluation Criteria in Solid Tumors (RECIST) have been modified to mRECIST, which requires measurement of vascular/ viable tumor on the CT or MRI scan. A partial response is defined as a 30% decrease in the sum of diameters of viable (arterially enhancing) target tumors. The need for semiquantitation of tumor vascularity on scans has led to the introduction of

1	is defined as a 30% decrease in the sum of diameters of viable (arterially enhancing) target tumors. The need for semiquantitation of tumor vascularity on scans has led to the introduction of diffusion-weighted MRI imaging. Tissue-specific imaging agents such as gadoxetic acid (Primovist or Eovist) and the move to functional and genetic imaging mark a shift in approaches. Furthermore, plasma AFP response may be a biologic marker of radiologic response.

1	Long-term survival is associated with resection or ablation or transplantation, all of which can yield >70% 5-year survival. Liver transplant is the only therapy that can treat the tumor and the underlying liver disease simultaneously and may be the most important advance in HCC therapy in 50 years. Unfortunately, it benefits only patients with limited size tumors without macrovascular portal vein invasion. Untreated patients with multinodular asymptomatic tumors without vascular invasion or extrahepatic spread have a median survival of approximately 16 months. Chemoembolization (TACE) improves their median survival to 19–20 months and is considered standard therapy for these patients, who represent the majority of HCC patients, although 90Yttrium therapy may provide similar results with less toxicity. Patients with advanced-stage disease, vascular invasion, or metastases have a median survival of around 6 months. Among this group, outcomes may vary according to their underlying liver

1	toxicity. Patients with advanced-stage disease, vascular invasion, or metastases have a median survival of around 6 months. Among this group, outcomes may vary according to their underlying liver disease. It is this group at which kinase inhibitors are directed.

1	The Most Common Modes of Patient Presentation 1. A patient with known history of hepatitis, jaundice, or cirrhosis, with an abnormality on ultrasound or CT scan, or rising AFP or DCP (PIVKA-2) 2. A patient with an abnormal liver function test as part of a routine examination 3. 4. Symptoms of HCC including cachexia, abdominal pain, or fever 1. Clinical jaundice, asthenia, itching (scratches), tremors, or disorientation 2. Hepatomegaly, splenomegaly, ascites, peripheral edema, skin signs of liver failure 1. Blood tests: full blood count (splenomegaly), liver function tests, ammonia levels, electrolytes, AFP and DCP (PIVKA-2), Ca2+ and Mg2+; hepatitis B, C, and D serology (and quantitative HBV DNA or HCV RNA, if either is positive); neurotensin (specific for fibrolamellar HCC) 2.

1	Triphasic dynamic helical (spiral) CT scan of liver (if inadequate, then follow with an MRI); chest CT scan; upper and lower gastrointestinal endoscopy (for varices, bleeding, ulcers); and brain scan (only if symptoms suggest) 3. Core biopsy: of the tumor and separate biopsy of the underlying liver 1. HCC <2 cm: RFA, PEI, or resection 2. HCC >2 cm, no vascular invasion: liver resection, RFA, or OLTX 3. Multiple unilobar tumors or tumor with vascular invasion: TACE or sorafenib 4. Bilobar tumors, no vascular invasion: TACE with OLTX for patients with tumor response 5. Extrahepatic HCC or elevated bilirubin: sorafenib or bevacizumab plus erlotinib (combination agent trials are in progress)

1	This rarer variant of HCC has a quite different biology than adult-type HCC. None of the known HCC causative factors seem important here. It is typically a disease of younger adults, often teenagers and predominantly females. It is AFP-negative, but patients typically have elevated blood neurotensin levels, normal liver function tests, and no cirrhosis. Radiology is similar for HCC, except that characteristic adult-type portal vein invasion is less common. Although it is often multifocal in the liver, and therefore not resectable, metastases are common, especially to lungs and locoregional lymph nodes, but survival is often much better than with adult-type HCC. Resectable tumors are associated with 5-year survival ≥50%. Patients often present with a huge liver or unexplained weight loss, fever, or elevated liver function tests on routine evaluations. These huge masses suggest quite slow growth for many tumors. Surgical resection is the best management option, even for metastases, as

1	fever, or elevated liver function tests on routine evaluations. These huge masses suggest quite slow growth for many tumors. Surgical resection is the best management option, even for metastases, as these tumors respond much less well to chemotherapy than adult-type HCC. Although several series of OLTX for FL-HCC have been reported, the patients seem to die from tumor recurrences, with a 2-to 5-year lag compared with OLTX for adult-type HCC. Anecdotal responses to gemcitabine plus cisplatin-TACE are reported.

1	Epithelioid Hemangioendothelioma (EHE) This rare vascular tumor of adults is also usually multifocal and can also be associated with prolonged survival, even in the presence of metastases, which are commonly in the lung. There is usually no underlying cirrhosis. Histologically, these tumors are usually of borderline malignancy and express factor VIII, confirming their endothelial origin. OLTX may produce prolonged survival.

1	Cholangiocarcinoma (CCC) CCC typically refers to mucin-producing adenocarcinomas (different from HCC) that arise from the biliary tract and have features of cholangiocyte differentiation. They are grouped by their anatomic site of origin, as intrahepatic (IHC), perihilar (central, ∼65% of CCCs), and peripheral (or distal, ∼30% of CCCs). IHC is the second most common primary liver tumor. Depending on the site of origin, they have different features and require different treatments. They arise on the basis of cirrhosis less frequently than HCC, but may complicate primary biliary cirrhosis. However, cirrhosis and both primary biliary cirrhosis and HCV predispose to IHC. Nodular tumors arising at the bifurcation of the common bile duct are called Klatskin tumors and are often associated with a collapsed gallbladder, a finding that mandates visualization of the entire biliary tree. The approach to management of central and peripheral CCC is quite different. Incidence is increasing.

1	with a collapsed gallbladder, a finding that mandates visualization of the entire biliary tree. The approach to management of central and peripheral CCC is quite different. Incidence is increasing. Although most CCCs have no obvious cause (etiology unknown), a number of predisposing factors have been identified. Predisposing diseases include primary sclerosing cholangitis (10–20% of primary sclerosing cholangitis [PSC] patients), an autoimmune disease, and liver fluke in Asians, especially Opisthorchis viverrini and Clonorchis sinensis. CCC seems also to be associated with any cause of chronic biliary inflammation and injury, with alcoholic liver disease, choledocholithiasis, choledochal cysts (10%), and Caroli’s disease (a rare inherited form of bile duct ectasia). CCC most typically presents as painless jaundice, often with pruritus or weight loss. Diagnosis is made by biopsy, percutaneously for peripheral liver lesions, or more commonly via endoscopic retrograde

1	most typically presents as painless jaundice, often with pruritus or weight loss. Diagnosis is made by biopsy, percutaneously for peripheral liver lesions, or more commonly via endoscopic retrograde cholangiopancreatography (ERCP) under direct vision for central lesions. The tumors often stain positively for cytokeratins 7, 8, and 19 and negatively for cytokeratin 20. However, histology alone cannot usually distinguish CCC from metastases from colon or pancreas primary tumors. Serologic tumor markers appear to be nonspecific, but CEA, CA 19-9, and CA-125 are often elevated in CCC patients and are useful for following response to therapy. Radiologic evaluation typically starts with ultrasound, which is very useful in visualizing dilated bile ducts, and then proceeds with either MRI or magnetic resonance cholangiopancreatography (MRCP) or helical CT scans. Invasive cholangiopancreatography (ERCP) is then needed to define the biliary tree and obtain a biopsy or is needed therapeutically

1	resonance cholangiopancreatography (MRCP) or helical CT scans. Invasive cholangiopancreatography (ERCP) is then needed to define the biliary tree and obtain a biopsy or is needed therapeutically to decompress an obstructed biliary tree with internal stent placement. If that fails, then percutaneous biliary drainage will be needed, with the biliary drainage flowing into an external bag. Central tumors often invade the porta hepatis, and locoregional lymph node involvement by tumor is frequent. Incidence has been increasing in recent decades; few patients survive 5 years. The usual treatment is surgical, but combination systemic chemotherapy may be effective. After complete surgical resection for IHC, 5-year survival is 25–30%. Combination radiation therapy with liver transplant has produced a 5-year recurrence-free survival rate of 65%.

1	Hilar CCC is resectable in ∼30% of patients and usually involves bile duct resection and lymphadenectomy for prognostication. Typical survival is approximately 24 months, with recurrences being mainly in the operative bed but with ∼30% in the lungs and liver. Distal CCC, which involves the main ducts, is normally treated by resection of the extrahepatic bile ducts, often with pancreaticoduodenectomy. Survival is similar. Due to the high rates of locoregional recurrences or positive surgical margins, many patients receive postoperative adjuvant radiotherapy. Its effect on survival has not been assessed. Intraluminal brachyradiotherapy has also shown some promise. However, photodynamic therapy enhanced survival in one study. In this technique, sodium porfimer is injected intravenously and then subjected to intraluminal red light laser photoactivation. OLTX has been assessed for treatment of unresectable CCC. Five-year survival was ∼20%, so enthusiasm waned. However, neoadjuvant

1	and then subjected to intraluminal red light laser photoactivation. OLTX has been assessed for treatment of unresectable CCC. Five-year survival was ∼20%, so enthusiasm waned. However, neoadjuvant radiotherapy with sensitizing chemotherapy has shown better survival rates for CCC treated by OLTX and is currently used by UNOS for perihilar CCC <3 cm with neither intrahepatic or extrahepatic metastases. A 12-center data collection study of 287 patients with perihilar CCC confirmed the benefit of this approach in a subset of patients, with a 53% 5-year survival rate but with 10% patient dropout before transplantation. The patients had neoadjuvant external radiation with radiosensitizing therapy. Patients with tumors >3 cm had significantly shorter survival. Multiple chemotherapeutic agents have been assessed for activity and survival in unresectable CCC. Most have been inactive. However, both systemic and hepatic arterial gemcitabine have shown promising results. The combination of

1	have been assessed for activity and survival in unresectable CCC. Most have been inactive. However, both systemic and hepatic arterial gemcitabine have shown promising results. The combination of cisplatin plus gemcitabine has produced a survival advantage compared with gemcitabine alone in a 410-patient randomized controlled phase III trial for patients with locally advanced or metastatic CCC and is now considered standard therapy for unresectable CCC. Median overall survival in the combination arm was 11.7 months versus 8.1 months for gemcitabine alone. Significant responses were seen mainly in patients with IHC and gallbladder cancer. However, neither surgery for lymph node–positive disease nor regional chemotherapy in nonsurgical patients has shown any survival advantage thus far. Several case series have shown safety and some responses for hepatic arterial chemotherapy with gemcitabine, drug-eluting beads, and 90Yttrium microspheres, but no convincing clinical trials are

1	far. Several case series have shown safety and some responses for hepatic arterial chemotherapy with gemcitabine, drug-eluting beads, and 90Yttrium microspheres, but no convincing clinical trials are available. Clinical trials are under way with targeted therapies. Bevacizumab plus erlotinib gave a 10% partial response rate with median overall survival of 9.9 months. A sorafenib trial yielded an overall survival of 4.4 months, but 50% of the patients had received previous chemotherapy. Patients with unresectable tumors should be treated in clinical trials.

1	Gallbladder (GB) cancer has an even worse prognosis than CCC, with a typical survival of ∼6 months or less. Women are affected much more commonly than men (4:1), unlike HCC or CCC, and GB cancer occurs more frequently than CCC. Most patients have a history of antecedent 553 gallstones, but very few patients with gallstones develop GB cancer (∼0.2%). GB cancer presents similarly to CCC and is often diagnosed unexpectedly during gallstone or cholecystitis surgery. Presentation is typically that of chronic cholecystitis, chronic right upper quadrant pain, and weight loss. Useful but nonspecific serum markers include CEA and CA 19-9. CT scans or MRCP typically reveal a GB mass. The mainstay of treatment is surgical, either simple or radical cholecystectomy for stage I or II disease, respectively. Survival rates are near 100% at 5 years for stage I, and range from 60–90% at 5 years for stage II. More advanced GB cancer has worse survival, and many patients are unresectable. Adjuvant

1	Survival rates are near 100% at 5 years for stage I, and range from 60–90% at 5 years for stage II. More advanced GB cancer has worse survival, and many patients are unresectable. Adjuvant radiotherapy, used in the presence of local lymph node disease, has not been shown to enhance survival. Chemotherapy is not useful in advanced or metastatic GB cancer.

1	This tumor arises within 2 cm of the distal end of the common bile duct and is mainly (90%) an adenocarcinoma. Locoregional lymph nodes are commonly involved (50%), and the liver is the most frequent site for metastases. The most common clinical presentation is jaundice, and many patients also have pruritus, weight loss, and epigastric pain. Initial evaluation is performed with an abdominal ultrasound to assess vascular involvement, biliary dilation, and liver lesions. This is followed by a CT scan or MRI and especially MRCP. The most effective therapy is resection by pylorus-sparing pancreaticoduodenectomy, an aggressive procedure resulting in better survival rates than with local resection. Survival rates are ∼25% at 5 years in operable patients with involved lymph nodes and ∼50% in patients without involved nodes. Unlike CCC, approximately 80% of patients are thought to be resectable at diagnosis. Adjuvant chemotherapy or radiotherapy has not been shown to enhance survival. For

1	patients without involved nodes. Unlike CCC, approximately 80% of patients are thought to be resectable at diagnosis. Adjuvant chemotherapy or radiotherapy has not been shown to enhance survival. For metastatic tumors, chemotherapy is currently experimental.

1	These are predominantly from colon, pancreas, and breast primary tumors but can originate from any organ primary. Ocular melanomas are prone to liver metastasis. Tumor spread to the liver normally carries a poor prognosis for that tumor type. Colorectal and breast hepatic metastases were previously treated with continuous hepatic arterial infusion chemotherapy. However, more effective systemic drugs for each of these two cancers, especially the addition of oxaliplatin to colorectal cancer regimens, have reduced the use of hepatic artery infusion therapy. In a large randomized study of systemic versus infusional plus systemic chemotherapy for resected colorectal metastases to the liver, the patients receiving infusional therapy had no survival advantage, mainly due to extrahepatic tumor spread. 90Yttrium resin beads are approved in the United States for treatment of colorectal hepatic metastases. The role of this modality, either alone or in combination with chemotherapy, is being

1	spread. 90Yttrium resin beads are approved in the United States for treatment of colorectal hepatic metastases. The role of this modality, either alone or in combination with chemotherapy, is being evaluated in many centers. Palliation may be obtained from chemoembolization, PEI, or RFA.

1	Three common benign tumors occur and all are found predominantly in women. They are hemangiomas, adenomas, and focal nodular hyperplasia (FNH). FNH is typically benign, and usually no treatment is needed. Hemangiomas are the most common and are entirely benign. Treatment is unnecessary unless their expansion causes symptoms. Adenomas are associated with contraceptive hormone use. They can cause pain and can bleed or rupture, causing acute problems. Their main interest for the physician is a low potential for malignant change and a 30% risk of bleeding. For this reason, considerable effort has gone into differentiating these three entities radiologically. On discovery of a liver mass, patients are usually advised to stop taking sex steroids, because adenoma regression may then occasionally occur. Adenomas can often be large masses ranging from 8–15 cm. Due to their size and definite, but low, malignant potential and potential for bleeding, adenomas are typically resected. The most

1	occur. Adenomas can often be large masses ranging from 8–15 cm. Due to their size and definite, but low, malignant potential and potential for bleeding, adenomas are typically resected. The most useful diagnostic differentiating tool is a triphasic CT scan performed with HCC fast bolus protocol for arterial-phase imaging, together with subsequent delayed venous-phase imaging. Adenomas usually do not appear on

1	Tumors of the Liver and Biliary Tree 554 the basis of cirrhosis, although both adenomas and HCCs are intensely vascular on the CT arterial phase and both can exhibit hemorrhage (40% of adenomas). However, adenomas have smooth, well-defined edges, and enhance homogeneously, especially in the portal venous phase on delayed images, when HCCs no longer enhance. FNHs exhibit a characteristic central scar that is hypovascular on the arterial-phase and hypervascular on the delayed-phase CT images. MRI is even more sensitive in depicting the characteristic central scar of FNH. Elizabeth Smyth, David Cunningham

1	Elizabeth Smyth, David Cunningham Pancreatic cancer is the fourth leading cause of cancer death in the United States and is associated with a poor prognosis. Endocrine tumors affecting the pancreas are discussed in Chap. 113. Infiltrating ductal adenocarcinomas, the subject of this Chapter, account for the vast majority of cases and arise most frequently in the head of pancreas. At the time of diagnosis, 85–90% of patients have inoperable or metastatic disease, which is reflected in the 5-year survival rate of only 6% for all stages combined. An improved 5-year survival of up to 24% may be achieved when the tumor is detected at an early stage and when complete surgical resection is accomplished.

1	Pancreatic cancer represents 3% of all newly diagnosed malignancies in the United States. The most common age group at diagnosis is 65–84 years for both sexes. Pancreatic cancer was estimated to have been diagnosed in approximately 45,220 patients and accounted for approximately 38,460 deaths in 2013. Although survival rates have almost doubled over the past 35 years for this disease, overall survival remains low.

1	An estimated 278,684 cases of pancreatic cancer occur annu ally worldwide (the thirteenth most common cancer globally), with up to 60% of these cases diagnosed in more developed countries. It remains the eighth most common cause of cancer death in men and the ninth most common in women. The incidence is highest in the United States and western Europe and lowest in parts of Africa and South Central Asia. However, increasing rates of obesity, diabetes, and tobacco use in addition to access to diagnostic radiology in the developing world are likely to increase incidence rates in these countries. In this situation, consideration of the cost implications of adoption of current treatment paradigms in resource-constrained environments will be necessary. Primary prevention such as limiting tobacco use and avoiding obesity may be more cost effective than improvements in treatment of preexisting disease.

1	Cigarette smoking may be the cause of up to 20–25% of all pancreatic cancers and is the most common environmental risk factor for this disease. A longstanding history of type 1 or type 2 diabetes also appears to be a risk factor; however, diabetes may also occur in association with pancreatic cancer, possibly confounding this interpretation. Other risk factors may include obesity, chronic pancreatitis, and ABO blood group status. Alcohol does not appear to be a risk factor unless excess consumption gives rise to chronic pancreatitis.

1	Pancreatic cancer is associated with a number of well-defined molecular hallmarks. The four genes most commonly mutated or inactivated in pancreatic cancer are KRAS (predominantly codon 12, in 60–75% of pancreatic cancers), the tumor-suppressor genes p16 (deleted in 95% of tumors), p53 (inactivated or mutated in 50–70% of tumors), and SMAD4 (deleted in 55% of tumors). The pancreatic cancer precursor lesion pancreatic intraepithelial neoplasia (PanIN) acquires these genetic abnormalities in a progressive manner associated with increasing dysplasia; initial KRAS mutations are followed by p16 loss and finally p53 and SMAD4 alterations. SMAD4 gene inactivation is associated with a pattern of widespread metastatic disease in advanced-stage patients and poorer survival in patients with surgically resected pancreatic adenocarcinoma.

1	Up to 16% of pancreatic cancers may be inherited. Germline mutations in the following genes are associated with a significantly increased risk of pancreatic cancer and other cancers: (1) STK11 gene (Peutz-Jeghers syndrome), which carries a 132-fold increased lifetime risk of pancreatic cancer above the general population; (2) BRCA2 (increased risk of breast, ovarian, and pancreatic cancer); (3) p16/CDKN2A (familial atypical multiple mole melanoma), which carries an increased risk of melanoma and pancreatic cancer; (4) PALB2, which confers an increased risk of breast and pancreatic cancer; (5) hMLH1 and MSH2 (Lynch syndrome), which carries an increased risk of colon and pancreatic cancer; and (6) ATM (ataxia-telangiectasia), which carries an increased risk of breast cancer, lymphoma, and pancreatic cancer. Familial pancreatitis and an increased risk of pancreatic cancer are associated with mutations of the PRSS1 (serine protease 1) gene. However, for most familial pancreatic syndromes,

1	cancer. Familial pancreatitis and an increased risk of pancreatic cancer are associated with mutations of the PRSS1 (serine protease 1) gene. However, for most familial pancreatic syndromes, the underlying genetic cause remains unexplained. The absolute number of affected first-degree relatives is also correlated with increased cancer risk, and patients with at least two first-degree relatives with pancreatic cancer should be considered to have familial pancreatic cancer until proven otherwise.

1	The desmoplastic stroma surrounding pancreatic adenocarcinoma functions as a mechanical barrier to chemotherapy and secretes compounds essential for tumor progression and metastasis. Key mediators of these functions include the activated pancreatic stellate cell and the glycoprotein SPARC (secreted protein acidic and rich in cysteine), which is expressed in 80% of pancreatic ductal adenocarcinomas. Targeting this extracellular environment has become increasingly important in the treatment of advanced disease.

1	Screening is not routinely recommended because the incidence of pancreatic cancer in the general population is low (lifetime risk 1.3%), putative tumor markers such as carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) have insufficient sensitivity, and computed tomography (CT) has inadequate resolution to detect pancreatic dysplasia. Endoscopic ultrasound (EUS) is a more promising screening tool, and preclinical efforts are focused on identifying biomarkers that may detect pancreatic cancer at an early stage. Consensus practice recommendations based largely on expert opinion have chosen a threshold of greater than fivefold increased risk for developing pancreatic cancer to select individuals who may benefit from screening. This includes people with two or more first-degree relatives with pancreatic cancer, patients with Peutz-Jeghers syndrome, and BRCA 2, p16, and hereditary nonpolyposis colorectal cancer (HNPCC) mutation carriers with one or more affected

1	first-degree relatives with pancreatic cancer, patients with Peutz-Jeghers syndrome, and BRCA 2, p16, and hereditary nonpolyposis colorectal cancer (HNPCC) mutation carriers with one or more affected first-degree relatives.

1	PanIN represents a spectrum of small (<5 mm) neoplastic but noninvasive precursor lesions of the pancreatic ductal epithelium demonstrating mild, moderate, or severe dysplasia (PanIN 1–3, respectively); however, not all PanIN lesions will progress to frank invasive malignancy. Cystic pancreatic tumors such as intraductal mucinous papillary neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs) are increasingly detected radiologically and are frequently asymptomatic. Main duct IPMNs are more likely to occur in older persons and have higher malignant potential than branched duct IPMNs (invasive cancer in 45% vs 18% of resected lesions, respectively). In contrast, MCNs are solitary lesions of the distal pancreas that do not communicate with the duct system. MCNs have an almost exclusive female distribution (95%). The rate of invasive cancer in resected MCNs is lower (<18%) with increased rates associated with larger tumors or the presence of nodules.

1	CLINICAL FEATURES Clinical Presentation Obstructive jaundice occurs frequently when the cancer is located in the head of the pancreas. This may be accompanied by symptoms of abdominal discomfort, pruritus, lethargy, and weight loss. Less common presenting features include epigastric pain, backache, new-onset diabetes mellitus, and acute pancreatitis caused by pressure effects on the pancreatic duct. Nausea and vomiting, resulting from gastroduodenal obstruction, may also be a symptom of this disease. Physical Signs Patients can present with jaundice and cachexia, and scratch marks may be present. Of patients with operable tumors, 25% have a palpable gallbladder (Courvoisier’s sign). Physical signs related to the development of distant metastases include hepatomegaly, ascites, left supraclavicular lymphadenopathy (Virchow’s node), and periumbilical nodules (Sister Mary Joseph’s nodes).

1	DIAGNOSIS Diagnostic Imaging Patients who present with clinical features suggestive of pancreatic cancer undergo imaging to confirm the presence of a tumor and to establish whether the mass is likely to be inflammatory or malignant in nature. Other imaging objectives include the local and distant staging of the tumor, which will determine resectability and provide prognostic information. Dual-phase, contrast-enhanced spiral CT is the imaging modality of choice (Fig. 112-1). It provides accurate visualization of surrounding viscera, vessels, and lymph nodes, thus determining tumor resectability. Intestinal infiltration and liver and lung metastases are also reliably depicted on CT. There is no advantage of magnetic resonance imaging (MRI) over CT in predicting tumor resectability, but selected cases may benefit from MRI to characterize the nature of small indeterminate liver lesions and to evaluate the cause of biliary dilatation when no obvious mass is seen on CT. Endoscopic

1	but selected cases may benefit from MRI to characterize the nature of small indeterminate liver lesions and to evaluate the cause of biliary dilatation when no obvious mass is seen on CT. Endoscopic retrograde cholangiopancreatography (ERCP) is useful for revealing small pancreatic lesions, identifying stricture or obstruction in pancreatic or common bile ducts, and facilitating stent placement; however, it is associated with a risk of pancreatitis (Fig. 112-2). Magnetic resonance cholangiopancreatography (MRCP) is a noninvasive method for accurately depicting the level and degree of bile and pancreatic duct dilatation. EUS is highly sensitive in detecting lesions less than 3 cm in size (more sensitive than CT for lesions <2 cm) and is useful as a local staging tool for assessing vascular invasion and lymph node involvement. Fluorodeoxyglucose positron emission tomography (FDG-PET) should be considered before surgery or radical chemoradiotherapy (CRT), because it is superior to

1	invasion and lymph node involvement. Fluorodeoxyglucose positron emission tomography (FDG-PET) should be considered before surgery or radical chemoradiotherapy (CRT), because it is superior to conventional imaging in detecting distant metastases.

1	Tissue Diagnosis and Cytology Preoperative confirmation of malignancy is not always necessary in patients with radiologic appearances consistent with operable pancreatic cancer. However, EUS-guided fine-needle aspiration is the technique of choice when there is any doubt, and also for use in patients who require neoadjuvant treatment. It has an accuracy of approximately 90% and has a smaller risk of intraperitoneal dissemination compared with the percutaneous route. FIGURE 112-1 Coronal computed tomography showing pancre-atic cancer and dilated intrahepatic and pancreatic ducts (arrows). showing contrast in dilated pancreatic duct (arrows). Percutaneous biopsy of the pancreatic primary or liver metastases is only acceptable in patients with inoperable or metastatic disease. ERCP is a useful method for obtaining ductal brushings, but the sensitivity of ERCP for diagnosis ranges from 35 to 70%.

1	Serum Markers Tumor-associated CA19-9 is elevated in approximately 70–80% of patients with pancreatic carcinoma but is not recommended as a routine diagnostic or screening test because its sensitivity and specificity are inadequate for accurate diagnosis. Preoperative CA19-9 levels correlate with tumor stage, and postresection CA19-9 level has prognostic value. It is an indicator of asymptomatic recurrence in patients with completely resected tumors and is used as a biomarker of response in patients with advanced disease undergoing chemotherapy. A number of studies have established a high pretreatment CA19-9 level as an independent prognostic factor.

1	The American Joint Committee on Cancer (AJCC) tumor-nodemetastasis (TNM) staging of pancreatic cancer takes into account the location and size of the tumor, the involvement of lymph nodes, and distant metastasis. This information is then combined to assign a stage (Fig. 112-3). From a practical standpoint, patients are grouped according to whether the cancer is resectable, locally advanced (unresectable, but without distant spread), or metastatic.

1	Approximately 10% of patients present with localized nonmetastatic disease that is potentially suitable for surgical resection. Approximately 30% of patients have R1 resection (microscopic residual disease) following surgery. Those who undergo R0 resection (no microscopic or macroscopic residual tumor) and who receive adjuvant treatment have the best chance of cure, with an estimated median survival of 20–23 months and a 5-year survival of approximately 20%. Outcomes are more favorable in patients with small (<3 cm), well-differentiated tumors and lymph node–negative disease.

1	Patients should have surgery in dedicated pancreatic centers that have lower postoperative morbidity and mortality rates. The standard surgical procedure for patients with tumors of the pancreatic head or uncinate process is a pylorus-preserving pancreaticoduodenectomy (modified Whipple’s procedure). The procedure of choice for tumors of the pancreatic body and tail is a distal pancreatectomy, which routinely includes splenectomy. Postoperative treatment improves long-term outcomes in this group of patients. Adjuvant chemotherapy comprising six cycles of gemcitabine is common practice worldwide based on data from three randomized controlled trials (Table 112-1). The Charité FIGURE 112-3 Staging of pancreatic cancer, and survival according to stage. AJCC, American Joint Committee on Cancer. (Illustration by Stephen Millward.) Survival Study Comparator Arm No. of Patients PFS/DFS (months) Median Survival (months)

1	Survival Study Comparator Arm No. of Patients PFS/DFS (months) Median Survival (months) ESPAC-1, Neoptolemos et al: N Engl J Chemotherapy (folinic acid 289 PFS 15.3 vs 9.4. (p = .02) 20.1 vs 15.5 (HR 0.71; 95% CI Med 350:1200, 2004 + bolus 5-FU) vs no 0.55–0.92; p = .009) chemotherapy CONKO 001, Oettle et al: JAMA Gemcitabine vs observation 368 Median DFS 13.4 vs 6.9 22.1 vs 20.2 (p = .06) 297:267, 2007 (p <.001) ESPAC-3, Neoptolemos et al: JAMA 5-FU/LV vs gemcitabine 1088 23 vs 23.6 (HR 0.94; 95% CI 0.81–1.08, 304:1073, 2010 p = .39) Abbreviations: CI, confidence interval; CONKO, Charité Onkologie; DFS, disease-free survival; ESPAC, European Study Group for Pancreatic Cancer; 5-FU, 5-fluorouracil; HR, hazard ratio; LV, leucovorin; PFS, progression-free survival. Survival Study Comparator Arm No. of Patients PFS (months) Median Survival (months) Moore et al: J Clin Oncol 26:1960, Gemcitabine vs gemcitabine 569 2007 + erlotinib

1	Survival Study Comparator Arm No. of Patients PFS (months) Median Survival (months) Moore et al: J Clin Oncol 26:1960, Gemcitabine vs gemcitabine 569 2007 + erlotinib Cunningham et al: J Clin Oncol Gemcitabine vs gemcitabine 533 27:5513, 2009 + capecitabine (GEM-CAP) Von Hoff et al: N Engl J Med Gemcitabine vs gemcitabine 861 369:1691, 2013 + nab-paclitaxel Conroy et al: N Engl J Med Gemcitabine vs FOLFIRINOX 342 364:1817, 2011

1	Conroy et al: N Engl J Med Gemcitabine vs FOLFIRINOX 342 364:1817, 2011 Onkologie trial (CONKO 001) found that the use of gemcitabine after complete resection significantly delayed the development of recurrent disease compared with surgery alone. The European Study Group for Pancreatic Cancer 3 (ESPAC-3) trial, which investigated the benefit of adjuvant 5-fluorouracil/folinic acid (5-FU/FA) versus gemcitabine, revealed no survival difference between the two drugs. However, the toxicity profile of adjuvant gemcitabine was superior to 5-FU/FA by virtue of its lower incidence of stomatitis and diarrhea. Adjuvant radiotherapy is not commonly used in Europe based on the negative results of the ESPAC-1 study. Adjuvant 5-FU-based CRT with gemcitabine before and after radiotherapy as used in the Radiation Therapy Oncology Group (RTOG) 97-04 trial is preferred in the United States. This approach may be most beneficial in patients with bulky tumors involving the pancreatic head.

1	Approximately 30% of patients present with locally advanced, unresectable, but nonmetastatic pancreatic carcinoma. The median survival with gemcitabine is 9 months. Patients who respond to chemotherapy or who achieve stable disease after 3–6 months of gemcitabine have frequently been offered consolidation radiotherapy. However, a large, phase III, randomized controlled trial, LAP-07, did not demonstrate any improvement in survival for patients treated with CRT after 4 months of disease control on either gemcitabine or a gemcitabine/erlotinib combination. Approximately 60% of patients with pancreatic cancer present with metastatic disease. Patients with poor performance status do not usually benefit from chemotherapy. Gemcitabine was the standard

1	Approximately 60% of patients with pancreatic cancer present with metastatic disease. Patients with poor performance status do not usually benefit from chemotherapy. Gemcitabine was the standard Gastrointestinal (GI) neuroendocrine tumors (NETs) are tumors derived from the diffuse neuroendocrine system of the GI tract; that system is composed of amineand acid-producing cells with different hormonal profiles, depending on the site of origin. The tumors historically are divided into GI-NETs (in the GI tract) (also frequently called carcinoid tumors) and pancreatic neuroendocrine tumors (pNETs), although newer pathologic classifications have proposed that they all be classified as GI-NETs. The term GI-NET has been proposed to replace the term carcinoid; however, the term carcinoid is widely used, and many are not familiar with this change.

1	3.55 vs 3.75 (HR 0.77; 95% CI 5.91 vs 6.24 (HR 0.82; 95% CI 0.69– 0.64–0.92; p = .004) 0.99; p = .038) 3.8 vs 5.3 (HR 0.78; 95% CI 6.2 vs 7.1 (HR 0.86; 95% CI 0.72–1.02; 0.66–0.93; p = .004) p = .08) 3.7 vs 5.5 (HR 0.69; 95% CI 6.7 vs 8.5 (HR 0.72; 95% CI 0.62–0.83; 0.58–0.82; p <.001) p <.001) 3.3 vs 6.4 (HR 0.47; 95% CI 6.8 vs 11.1 (HR 0.57; 95% CI 0.45–0.73; 0.37–0.59; p <.001) p <.001) treatment with a median survival of 6 months and a 1-year survival rate of only 20%. The addition of nab-paclitaxel (an albumin bound nanoparticle formulation of paclitaxel) to gemcitabine results in significantly improved 1-year survival compared to gemcitabine alone (35% vs 22%, p <.001). Capecitabine, an oral fluoropyrimidine, has also been combined with gemcitabine (GEM-CAP) in a phase III trial that showed an improvement in response rate and progression-free survival over single-agent gemcitabine, but no overall survival benefit. However, pooling of two other randomized controlled trials with

1	showed an improvement in response rate and progression-free survival over single-agent gemcitabine, but no overall survival benefit. However, pooling of two other randomized controlled trials with this trial in a meta-analysis resulted in a survival advantage with GEM-CAP. Addition of erlotinib, a small-molecule epidermal growth factor receptor inhibitor, produced a statistically significant but clinically marginal benefit when added to gemcitabine in the advanced disease setting. A phase III trial limited to good performance status patients with metastatic pancreatic cancer showed improved survival with the combination of 5-FU/FA, irinotecan, and oxaliplatin (FOLFIRINOX) compared with gemcitabine, but with increased toxicity (Table 112-2).

1	The early detection and future treatment of pancreatic cancer relies on an improved understanding of molecular pathways involved in the development of this disease. This will ultimately lead to the discovery of novel agents and the identification of patient groups who are likely to benefit most from targeted therapy. Dr. Irene Chong is acknowledged for her work on this chapter in the 18th edition.

1	Dr. Irene Chong is acknowledged for her work on this chapter in the 18th edition. Accordingly, this chapter will use the term GI-NETs (carcinoids). These tumors originally were classified as APUDomas (for amine precursor uptake and decarboxylation), as were pheochromocytomas, melanomas, and medullary thyroid carcinomas, because they share certain cytochemical features as well as various pathologic, biologic, and molecular features (Table 113-1). It was originally proposed that APUDomas had a similar embryonic origin from neural crest cells, but it is now known the peptide-secreting cells are not of neuroectodermal origin. Nevertheless, the concept of APUDomas is useful because these tumors have important similarities as well as some differences (Table 113-1). In this section, the areas of similarity between pNETs and GI-NETs (carcinoids) will be discussed together, and areas in which there are important differences will be discussed separately.

1	NETs generally are composed of monotonous sheets of small round cells with uniform nuclei, and mitoses are uncommon. They can be identified tentatively on routine histology; however, these tumors are now recognized principally by their histologic staining patterns due to shared cellular proteins. Historically, silver staining was used, and Endocrine Tumors of the Gastrointestinal Tract and Pancreas endocrine tumors of the gastrointestinal tract and pancreas Robert T. Jensen GENERAL FEATURES OF GASTROINTESTINAL 113 generaL CharaCteristiCs of gastrointestinaL neuroenDoCrine tumors (gi-nets [CarCinoiDs], panCreatiC neuroenDoCrine tumors [pnets]) A. Share general neuroendocrine cell markers (identification used for diagnosis) 1. Chromogranins (A, B, C) are acidic monomeric soluble proteins found in the large secretory granules. Chromogranin A is the most widely used. 2.

1	Chromogranins (A, B, C) are acidic monomeric soluble proteins found in the large secretory granules. Chromogranin A is the most widely used. 2. Neuron-specific enolase (NSE) is the γ-γ dimer of the enzyme enolase and is a cytosolic marker of neuroendocrine differentiation. 3. Synaptophysin is an integral membrane glycoprotein of 38,000 molecular weight found in small vesicles of neurons and neuroendocrine tumors. B. Pathologic similarities 1. All are APUDomas showing amine precursor uptake and decarboxylation. 2. Ultrastructurally, they have dense-core secretory granules (>80 nm). 3. Histologically, they generally appear similar with few mitoses and uniform nuclei. 4. Frequently synthesize multiple peptides/amines, which can be detected immunocytochemically but may not be secreted. 5. Presence or absence of clinical syndrome or type cannot be predicted by immunocytochemical studies. 6.

1	5. Presence or absence of clinical syndrome or type cannot be predicted by immunocytochemical studies. 6. Histologic classifications (grading, TNM classification) have prognostic significance. Only invasion or metastases establish malignancy. C. Similarities of biologic behavior 1. Generally slow growing, but some are aggressive. 2. Most are well-differentiated tumors having low proliferative indices. 3. Secrete biologically active peptides/amines, which can cause clinical symptoms. 4. Generally have high densities of somatostatin receptors, which are used for both localization and treatment. 5. Most (>70%) secrete chromogranin A, which is frequently used as a tumor marker. D. Similarities/differences in molecular abnormalities 1. Similarities a. Uncommon—mutations in common oncogenes (ras, jun, fos, etc). b. Uncommon—mutations in common tumor-suppressor genes (p53, retinoblastoma). c.

1	Uncommon—mutations in common oncogenes (ras, jun, fos, etc). b. Uncommon—mutations in common tumor-suppressor genes (p53, retinoblastoma). c. Alterations at MEN 1 locus (11q13) (frequently foregut, less commonly mid/hindgut NETs) and p16INK4a (9p21) occur in a proportion (10–45%). d. Methylation of various genes occurs in 40–87% (ras-associated domain family I, p14, p16, O6-methylguanine methyltransferases, retinoic acid receptor β). 2. Differences a. pNETs—loss of 1p (21%), 3p (8–47%), 3q (8–41%), 11q (21–62%), 6q (18–68%), Y (45%). Gains at 17q (10–55%), 7q (16–68%), 4q (33%), 18 (up to 45%). b. GI-NETs (carcinoids)—loss of 18q (38–88%), >18p (33–43%), >9p, 16q21 (21–23%). Gains at 17q, 19p (57%), 4q (33%), 14q (20%), 5 (up to 36%). c. pNETs: ATRX/DAXX mutations in 43%, MEN 1 mutations in 44%, mTor mutations (14%); uncommon in midgut GI-NETs (0–2%).

1	c. pNETs: ATRX/DAXX mutations in 43%, MEN 1 mutations in 44%, mTor mutations (14%); uncommon in midgut GI-NETs (0–2%). Abbreviations: ATRX, alpha-thalassemia X-lined mental retardation protein; DAXX, death domain associated protein; MEN 1, multiple endocrine neoplasia type 1; TNM, tumor, node, metastasis. tumors were classified as showing an argentaffin reaction if they took up and reduced silver or as being argyrophilic if they did not reduce it. Currently, immunocytochemical localization of chromogranins (A, B, C), neuron-specific enolase, and synaptophysin, which are all neuroendocrine cell markers, is used (Table 113-1). Chromogranin A is the most widely used.

1	Ultrastructurally, these tumors possess electron-dense neurosecretory granules and frequently contain small clear vesicles that correspond to synaptic vesicles of neurons. NETs synthesize numerous peptides, growth factors, and bioactive amines that may be ectopically secreted, giving rise to a specific clinical syndrome (Table 113-2). The diagnosis of the specific syndrome requires the clinical features of the disease (Table 113-2) and cannot be made from the immunocytochemistry results alone. The presence or absence of a specific clinical syndrome also cannot be predicted from the immunocytochemistry alone (Table 113-1). Furthermore, pathologists cannot distinguish between benign and malignant NETs unless metastasis or invasion is present.

1	GI-NETs (carcinoids) frequently are classified according to their anatomic area of origin (i.e., foregut, midgut, hindgut) because tumors with similar areas of origin share functional manifestations, histochemistry, and secretory products (Table 113-3). Foregut tumors generally have a low serotonin (5-HT) content; are argentaffin-negative but argyrophilic; occasionally secrete adrenocorticotropic hormone (ACTH) or 5-hydroxytryptophan (5-HTP), causing an atypical carcinoid syndrome (Fig. 113-1); are often multihormonal; and may metastasize to bone. They uncommonly produce a clinical syndrome due to the secreted products. Midgut carcinoids are argentaffin-positive, have a high serotonin content, most frequently cause the typical carcinoid syndrome when they metastasize (Table 113-3, Fig. 113-1), release serotonin and tachykinins (substance P, neuropeptide K, substance K), rarely secrete 5-HTP or ACTH, and less commonly metastasize to bone. Hindgut carcinoids (rectum, transverse and

1	113-1), release serotonin and tachykinins (substance P, neuropeptide K, substance K), rarely secrete 5-HTP or ACTH, and less commonly metastasize to bone. Hindgut carcinoids (rectum, transverse and descending colon) are argentaffin-negative, are often argyrophilic, rarely contain serotonin or cause the carcinoid syndrome (Fig. 113-1, Table 113-3), rarely secrete 5-HTP or ACTH, contain numerous peptides, and may metastasize to bone.

1	pNETs can be classified into nine well-established specific functional syndromes (Table 113-2), six additional very rare specific functional syndromes (less than five cases described), five possible specific functional syndromes (pNETs secreting calcitonin, neurotensin, pancreatic polypeptide, ghrelin) (Table 113-2), and nonfunctional pNETs. Other functional hormonal syndromes due to nonpancreatic tumors (usually intraabdominal in location) have been described only rarely and are not included in (Table 113-2). These include secretion by intestinal and ovarian tumors of peptide tyrosine tyrosine (PYY), which results in altered motility and constipation, and ovarian tumors secreting renin or aldosterone causing alterations in blood pressure or somatostatin causing diabetes or reactive hypoglycemia. Each of the functional syndromes listed in Table 113-2 is associated with symptoms due to the specific hormone released. In contrast, nonfunctional pNETs release no products that cause a

1	Each of the functional syndromes listed in Table 113-2 is associated with symptoms due to the specific hormone released. In contrast, nonfunctional pNETs release no products that cause a specific clinical syndrome. “Nonfunctional” is a misnomer in the strict sense because those tumors frequently ectopically secrete a number of peptides (pancreatic polypeptide [PP], chromogranin A, ghrelin, neurotensin, α subunits of human chorionic gonadotropin, and neuron-specific enolase); however, they cause no specific clinical syndrome. The symptoms caused by nonfunctional pNETs are entirely due to the tumor per se. pNETs frequently ectopically secrete PP (60–85%), neurotensin (30–67%), calcitonin (30–42%), and to a lesser degree, ghrelin (5–65%). Whereas a few studies have proposed their secretion can cause a specific functional syndrome, most studies support the conclusion that their ectopic secretion is not associated with a specific clinical syndrome, and thus they are listed in Table 113-2

1	can cause a specific functional syndrome, most studies support the conclusion that their ectopic secretion is not associated with a specific clinical syndrome, and thus they are listed in Table 113-2 as possible clinical syndromes.

1	Endocrine Tumors of the Gastrointestinal Tract and Pancreas Abbreviations: ACTH, adrenocorticotropic hormone; GRFoma, growth hormone–releasing factor secreting pancreatic endocrine tumor; IGF-II, insulin-like growth factor II; MEN, multiple endocrine neoplasia; pNET, pancreatic neuroendocrine tumor; PPoma, tumor secreting pancreatic polypeptide; PTHrP, parathyroid hormone–related peptide; VIPoma, tumor secreting vasoactive intestinal peptide; WDHA, watery diarrhea, hypokalemia, and achlorhydria syndrome. aPancreatic polypeptide–secreting tumors (PPomas) are listed in two places because most authorities classify these as not associated with a specific hormonal syndrome (nonfunctional); however, rare cases of watery diarrhea proposed to be due to PPomas have been reported. Because a large proportion of nonfunctional pNETs (60–90%) secrete PP, these tumors are often referred to as PPomas (Table 113-2).

1	GI-NETs (carcinoids) can occur in almost any GI tissue (Table 113-3); however, at present, most (70%) have their origin in one of three sites: bronchus, jejunoileum, or colon/rectum. In the past, GI-NET (carcinoids) most frequently were reported in the appendix (i.e., 40%); however, the bronchus/lung, rectum, and small intestine are now the most common sites. Overall, the GI tract is the most common site for these tumors, accounting for 64%, with the respiratory tract a distant second at 28%. Both race and sex can affect the frequency as well as the distribution of GI-NETs (carcinoids). African Americans have a higher incidence of carcinoids. Race is particularly important for rectal carcinoids, which are found in 41% of Asians/Pacific Islanders with NETs compared to 32% of American Indians/Alaskan natives, 26% of African Americans, and 12% of white Americans. Females have a lower incidence of small intestinal and pancreatic carcinoids.

1	The term pancreatic neuroendocrine or endocrine tumor, although widely used and therefore retained here, is also a misnomer, strictly speaking, because these tumors can occur either almost entirely in the pancreas (insulinomas, glucagonomas, nonfunctional pNETs, pNETs causing hypercalcemia) or at both pancreatic and extrapancreatic sites (gastrinomas, VIPomas [vasoactive intestinal peptide], somatostatinomas, GRFomas [growth hormone–releasing factor]). pNETs are also called islet cell tumors; however, the use of this term is discouraged because it is not established that they originate from the islets, and many can occur at extrapancreatic sites.

1	Whereas the classification of GI neuroendocrine tumors into fore-gut, midgut, or hindgut is widely used and generally useful because the NETs within these areas have many similarities, they also have marked differences, particularly in biologic behavior, and it has not proved useful for prognostic purposes. More general classifications have been developed that allow NETs with similar features in different locations to be compared, have proven prognostic value, and are widely used. New classification systems have been developed for both GI-NETs (carcinoids) and pNETs by the World Health Organization (WHO), European Neuroendocrine Tumor Society (ENETS), and the American Joint Committee on Cancer/International Union Against Cancer (AJCC/UICC). Although there are some differences between these different classification systems, each uses similar information, and it is now recommended that the basic data underlying the classification be included in all standard pathology reports. These

1	these different classification systems, each uses similar information, and it is now recommended that the basic data underlying the classification be included in all standard pathology reports. These classification systems divide NETs from all sites into those that are well differentiated (low grade [G1] or intermediate grade [G2]) and those that are poorly differentiated (high grade [G3] divided into either small-cell carcinoma or large-cell neuroendocrine carcinoma). In these classification systems, both pNETs and GI-NETs (carcinoids) are classified as neuroendocrine tumors, and the old term of carcinoid is equivalent to well-differentiated neuroendocrine tumors of the GI tract. These classification systems are based on not only the differentiation of the NET, but also a grading system assessing proliferative indices (Ki-67 and the mitotic count). NETs are considered low grade (ENETS G1) if the Ki-67 is <3% and the mitotic count is <2 mitoses/high-power field (HPF), intermediate

1	assessing proliferative indices (Ki-67 and the mitotic count). NETs are considered low grade (ENETS G1) if the Ki-67 is <3% and the mitotic count is <2 mitoses/high-power field (HPF), intermediate grade (ENETS G2) if the Ki-67 is 3–20% and the mitotic count is 2–20 mitoses/HPF, and high grade (ENETS G3) if the Ki-67 is >20% and the mitotic count is >20 mitoses/HPF. In addition to the grading system, a TNM classification has been proposed that is based on the level of tumor invasion, tumor size, and tumor extent (see Table 113-4 for an example with pNETs and appendiceal GI-NETs [carcinoids]). Because of the proven prognostic value of these classification and grading systems, as well as the fact that NETs with different classifications/grades respond differently to treatments, the systems are now essential for the management of all NETs.

1	In addition to these classification/grading systems, a number of other factors have been identified that provide important prognostic information that can guide treatment (Table 113-5). The exact incidence of GI-NETs (carcinoids) or pNETs varies according to whether only symptomatic tumors or all tumors are considered. The incidence of clinically significant carcinoids is 7–13 cases/million population per year, whereas any malignant carcinoids taBLe 113-3 gi-net (CarCinoiD) LoCation, frequenCy of metastases, anD assoCiation With the CarCinoiD synDrome Esophagus <0.1 — — Stomach 4.6 10 9.5 Duodenum 2.0 — 3.4 Pancreas 0.7 71.9 20 Gallbladder 0.3 17.8 5 Bronchus, lung, trachea 27.9 5.7 13 Jejunum 1.8 {58.4 Ileum 14.9 9 Meckel’s diverticulum 0.5 — 13 Appendix 4.8 38.8 <1 Colon 8.6 51 5 Liver 0.4 32. — Ovary 1.0 2 32 50 Testis <0.1 — 50 Rectum 13.6 3.9 — Abbreviation: GI-NET, gastrointestinal neuroendocrine tumor.

1	Rectum 13.6 3.9 — Abbreviation: GI-NET, gastrointestinal neuroendocrine tumor. Source: Location is from the PAN-SEER data (1973–1999), and incidence of metastases is from the SEER data (1992–1999), reported by IM Modlin et al: Cancer 97:934, 2003. Incidence of carcinoid syndrome is from 4349 cases studied from 1950–1971, reported by JD Godwin: Cancer 36:560, 1975. FIGURE 113-1 Synthesis, secretion, and metabolism of serotonin (5-HT) in patients with typical and atypical carcinoid syndromes. 5-HIAA, 5-hydroxyindolacetic acid. Comparison of the Criteria for the tumor Category in the enets anD seventh eDition ajCC tnm CLassifiCations of panCreatiC anD appenDiCeaL nets I. Both GI-NETs (carcinoids) and pNETs

1	Comparison of the Criteria for the tumor Category in the enets anD seventh eDition ajCC tnm CLassifiCations of panCreatiC anD appenDiCeaL nets I. Both GI-NETs (carcinoids) and pNETs Symptomatic presentation (p <.05) Presence of liver metastases (p <.001) Extent of liver metastases (p <.001) Presence of lymph node metastases (p <.001) Development of bone or extrahepatic metastases (p <.01) Depth of invasion (p <.001) Rapid rate of tumor growth Elevated serum alkaline phosphatase levels (p = .003) Primary tumor site (p <.001) Primary tumor size (p <.005) High serum chromogranin A level (p <.01) Presence of one or more circulating tumor cells (p <.001) Various histologic features

1	Tumor differentiation (p <.001) High growth indices (high Ki-67 index, PCNA expression) High mitotic counts (p <.001) Necrosis present Presence of cytokeratin 19 (p <.02) Vascular or perineural invasion Vessel density (low microvessel density, increased lymphatic density) High CD10 metalloproteinase expression (in series with all grades of NETs) Flow cytometric features (i.e., aneuploidy) High VEGF expression (in low-grade or well-differentiated NETs only) WHO, ENETS, AJCC/UICC, and grading classification Presence of a pNET rather than GI-NET associated with poorer prognosis (p = .0001) Older age (p <.01) II. GI-NETs (Carcinoids) Location of primary: appendix < lung, rectum < small intestine < pancreas Presence of carcinoid syndrome Laboratory results (urinary 5-HIAA levels [p <.01], plasma neuropeptide

1	Location of primary: appendix < lung, rectum < small intestine < pancreas Presence of carcinoid syndrome Laboratory results (urinary 5-HIAA levels [p <.01], plasma neuropeptide K [p <.05], serum chromogranin A [p <.01]) Presence of a second malignancy Male sex (p <.001) Molecular findings (TGF-α expression [p <.05], chr 16q LOH or gain chr 4p [p <.05]) WHO, ENETS, AJCC/UICC, and grading classification Molecular findings (gain in chr 14, loss of 3p13 [ileal carcinoid], upregulation III. pNETs Location of primary: duodenal (gastrinoma) better than pancreatic Ha-ras oncogene or p53 overexpression Female gender MEN 1 syndrome absent Presence of nonfunctional tumor (some studies, not all) WHO, ENETS, AJCC/UICC, and grading classification Various histologic features: IHC positivity for c-KIT, low cyclin B1 expression (p <.01), loss of PTEN or of tuberous sclerosis-2 IHC, expression of

1	Molecular findings (increased HER2/neu expression [p = .032], chr 1q, 3p, 3q, or 6q LOH [p = .0004], EGF receptor overexpression [p = .034], gains in chr 7q, 17q, 17p, 20q; alterations in the VHL gene [deletion, methylation]; presence of FGFR4-G388R single-nucleotide polymorphism)

1	Abbreviations: 5-HIAA, 5-hydroxyindoleacetic acid; AJCC, American Joint Committee on Cancer; chr, chromosome; EGF, epidermal growth factor; FGFR, fibroblast growth factor receptor; GI-NET, gastrointestinal neuroendocrine tumor; IHC, immunohistochemistry; Ki-67, proliferation-associated nuclear antigen recognized by Ki-67 monoclonal antibody; LOH, loss of heterozygosity; MEN, multiple endocrine neoplasia; NET, neuroendocrine tumors; PCNA, proliferating cell nuclear antigen; pNET, pancreatic neuroendocrine tumor; PTEN, phosphatase and tensin homologue deleted from chromosome 10; TGF-α, transforming growth factor α; TNM, tumor, node, metastasis; UICC, International Union Against Cancer; VEGF, vascular endothelial growth factor; WHO, World Health Organization. Confined to pancreas, <2 cm Confined to pancreas, >2 cm Peripancreatic spread, but without major vascular invasion (truncus coeliacus, superior mesenteric artery) T1 ≤1 cm; invasion of

1	Confined to pancreas, <2 cm Confined to pancreas, >2 cm Peripancreatic spread, but without major vascular invasion (truncus coeliacus, superior mesenteric artery) T1 ≤1 cm; invasion of T1a, ≤1 cm; T1b, >1–2 cm muscularis propria >2–4 cm or invasion of of subserosa/mesoappendix T3 >2 cm or >3 mm invasion of >4 cm or invasion of ileum subserosa/mesoappendix T4 Invasion of peritoneum/ Invasion of peritoneum/ other organs Abbreviations: AJCC, American Joint Committee on Cancer; ENETS, European Neuroendocrine Tumor Society; NET, neuroendocrine tumor; pNET, pancreatic neuroendocrine tumor; TNM, tumor, node, metastasis; UICC, International Union Against Cancer. Source: Modified from DS Klimstra: Semin Oncol 40:23, 2013 and G Kloppel et al: Virchow Arch 456:595, 2010.

1	at autopsy are reported in 21–84 cases/million population per year. The incidence of GI-NETs (carcinoids) is approximately 25–50 cases per million in the United States, which makes them less common than adenocarcinomas of the GI tract. However, their incidence has increased sixfold in the last 30 years. In an analysis of 35,825 GI-NETs (carcinoids) (2004) from the U.S. Surveillance, Epidemiology, and End Results (SEER) database, their incidence was 5.25/100,000 per year, and the 29-year prevalence was 35/100,000. Clinically significant pNETs have a prevalence of 10 cases/million population, with insulinomas, gastrinomas, and nonfunctional pNETs having an incidence of 0.5–2 cases/million population per year (Table 113-2). pNETs account for 1–10% of all tumors arising in the pancreas and 1.3% of tumors in the SEER database, which consists primarily of malignant tumors. VIPomas are 2–8 times less common, glucagonomas are 17–30 times less common, and somatostatinomas are the least common.

1	of tumors in the SEER database, which consists primarily of malignant tumors. VIPomas are 2–8 times less common, glucagonomas are 17–30 times less common, and somatostatinomas are the least common. In autopsy studies, 0.5–1.5% of all cases have a pNET; however, in less than 1 in 1000 cases was a functional tumor thought to occur.

1	Both GI-NETs (carcinoids) and pNETs commonly show malignant behavior (Tables 113-2 and 113-3). With pNETs, except for insulinomas in which <10% are malignant, 50–100% in different series are malignant. With GI-NETs (carcinoids), the percentage showing malignant behavior varies in different locations (Table 113-3). For the three most common sites of occurrence, the incidence of metastases varies greatly from the jejunoileum (58%), lung/bronchus (6%), and rectum (4%) (Table 113-3). With both GI-NETs (carcinoids) and pNETs, a number of factors (Table 113-5) are important prognostic factors in determining survival and the aggressiveness of the tumor. Patients with pNETs (excluding insulinomas) generally have a poorer prognosis than do patients with GI-NETs (carcinoids). The presence of liver metastases is the single most important prognostic factor in single and multivariate analyses for both GI-NETs (carcinoids) and pNETs. Particularly important in the development of liver metastases is

1	metastases is the single most important prognostic factor in single and multivariate analyses for both GI-NETs (carcinoids) and pNETs. Particularly important in the development of liver metastases is the size of the primary tumor. For example, with small intestinal carcinoids, which are the most common cause of the carcinoid syndrome due to metastatic disease in the liver (Table 113-2), metastases occur in 15–25% if the tumor is <1 cm in diameter, 58–80% if it is 1–2 cm in diameter, and >75% if it is >2 cm in diameter. Similar data exist for gastrinomas and other pNETs; the size of the primary tumor is an independent predictor of the development of liver metastases.

1	Endocrine Tumors of the Gastrointestinal Tract and Pancreas 562 The presence of lymph node metastases or extrahepatic metastases; the depth of invasion; the rapid rate of growth; various histologic features (differentiation, mitotic rates, growth indices, vessel density, vascular endothelial growth factor [VEGF], and CD10 metalloproteinase expression); necrosis; presence of cytokeratin; elevated serum alkaline phosphatase levels; older age; presence of circulating tumor cells; and flow cytometric results, such as the presence of aneuploidy, are all important prognostic factors for the development of metastatic disease (Table 113-5). For patients with GI-NETs (carcinoids), additional associations with a worse prognosis include the development of the carcinoid syndrome (especially the development of carcinoid heart disease), male sex, the presence of a symptomatic tumor or greater increases in a number of tumor markers (5-hydroxyindolacetic acid [5-HIAA], neuropeptide K, chromogranin

1	of carcinoid heart disease), male sex, the presence of a symptomatic tumor or greater increases in a number of tumor markers (5-hydroxyindolacetic acid [5-HIAA], neuropeptide K, chromogranin A), and the presence of various molecular features. With pNETs or gastrinomas, a worse prognosis is associated with female sex, overexpression of the Ha-ras oncogene or p53, the absence of multiple endocrine neoplasia type 1 (MEN 1), higher levels of various tumor markers (i.e., chromogranin A, gastrin), and presence of various histologic features (immunohistochemistry for c-KIT, low cyclin B1, loss of PTEN/TSC-2, expression of fibroblast growth factor-13) and various molecular features (Table 113-5). The TNM classification systems and the grading systems (G1–G3) have important prognostic value. A number of diseases due to various genetic disorders are associated with an increased incidence of NETs (Table 113-6). Each one is caused by a loss of a possible tumor-suppressor gene. The most important

1	of diseases due to various genetic disorders are associated with an increased incidence of NETs (Table 113-6). Each one is caused by a loss of a possible tumor-suppressor gene. The most important is MEN 1, which is an autosomal dominant disorder due to a defect in a 10-exon gene on 11q13, which encodes for a 610-amino-acid nuclear protein, menin (Chap. 408). Patients with MEN 1 develop hyperparathyroidism due to parathyroid hyperplasia in 95–100% of cases, pNETs in 80–100%, pituitary adenomas in 54–80%, adrenal adenomas in 27–36%, bronchial carcinoids in 8%, thymic carcinoids in 8%, gastric carcinoids in 13–30% of patients with Zollinger-Ellison syndrome, skin tumors (angiofibromas [88%], collagenomas [72%]), central nervous system (CNS) tumors (meningiomas [<8%]), and smooth-muscle tumors (leiomyomas, leiomyosarcomas [1–7%]). Among patients with MEN 1, 80–100% develop nonfunctional pNETs (most are microscopic with 0–13% large/symptomatic), and functional pNETs occur in 20–80% in

1	(leiomyomas, leiomyosarcomas [1–7%]). Among patients with MEN 1, 80–100% develop nonfunctional pNETs (most are microscopic with 0–13% large/symptomatic), and functional pNETs occur in 20–80% in different series, with a mean of 54% developing Zollinger-Ellison syndrome, 18% insulinomas, 3% glucagonomas, 3%

1	Location of Gene Mutation and Gene Syndrome Product NETs Seen/Frequency Multiple endocrine 11q13 (encodes neoplasia type 1 610-amino-acid protein, (MEN 1) menin) von Recklinghausen’s 17q11.2 (encodes disease (neurofibro-2485-amino-acid protein, matosis 1 [NF-1]) neurofibromin) Tuberous sclerosis 9q34 (TSCI) (encodes 1164-amino-acid protein, hamartin), 16p13 (TSC2) (encodes 1807-aminoacid protein, tuberin) 80–100% develop pNETS (microscopic), 20–80% (clinical): (nonfunctional > gastrinoma > insulinoma) GI-NETs (Carcinoids): gastric (13–30%), bronchial/ thymic (8%) 0–10% develop pNETs, primarily duodenal somatostatinomas (usually nonfunctional) Rarely insulinoma, gastrinoma Uncommonly develop pNETS (nonfunctional and functional [insulinoma, gastrinoma]) VIPomas, and <1% GRFomas or somatostatinomas. MEN 1 is present in 20–25% of all patients with Zollinger-Ellison syndrome, 4% of patients with insulinomas, and a low percentage (<5%) of patients with other pNETs.

1	Three phacomatoses associated with NETs are von Hippel–Lindau disease (VHL), von Recklinghausen’s disease (neurofibromatosis type 1 [NF-1]), and tuberous sclerosis (Bourneville’s disease) (Table 113-6). VHL is an autosomal dominant disorder due to defects on chromosome 3p25, which encodes for a 213-amino-acid protein that interacts with the elongin family of proteins as a transcriptional regulator (Chaps. 118, 339, 407, and 408). In addition to cerebellar hemangioblastomas, renal cancer, and pheochromocytomas, 10–17% develop a pNET. Most are nonfunctional, although insulinomas and VIPomas have been reported. Patients with NF-1 (von Recklinghausen’s disease) have defects in a gene on chromosome 17q11.2 that encodes for a 2845-amino-acid protein, neurofibromin, which functions in normal cells as a suppressor of the ras signaling cascade (Chap. 118). Up to 10% of these patients develop an upper GI-NET (carcinoid), characteristically in the periampullary region (54%). Many are classified

1	as a suppressor of the ras signaling cascade (Chap. 118). Up to 10% of these patients develop an upper GI-NET (carcinoid), characteristically in the periampullary region (54%). Many are classified as somatostatinomas because they contain somatostatin immunocytochemically; however, they uncommonly secrete somatostatin and rarely produce a clinical somatostatinoma syndrome. NF-1 has rarely been associated with insulinomas and Zollinger-Ellison syndrome. NF-1 accounts for 48% of all duodenal somatostastinomas and 23% of all ampullary GI-NETs (carcinoids). Tuberous sclerosis is caused by mutations that alter either the 1164-amino-acid protein hamartin (TSC1) or the 1807-amino-acid protein tuberin (TSC2) (Chap. 118). Both hamartin and tuberin interact in a pathway related to phosphatidylinositol 3-kinases and mammalian target of rapamycin (mTOR) signaling cascades. A few cases including nonfunctional and functional pNETs (insulinomas and gastrinomas) have been reported in these patients

1	3-kinases and mammalian target of rapamycin (mTOR) signaling cascades. A few cases including nonfunctional and functional pNETs (insulinomas and gastrinomas) have been reported in these patients (Table 113-6). Mahvash disease is associated with the development of α-cell hyperplasia, hyperglucagonemia, and the development of NF pNETs and is due to a homozygous P86S mutation of the human glucagon receptor.

1	Mutations in common oncogenes (ras, myc, fos, src, jun) or common tumor-suppressor genes (p53, retinoblastoma susceptibility gene) are not commonly found in either pNETs or GI-NETs (carcinoids) (Table 113-1). However, frequent (70%) gene amplifications in MDM2, MDM4, and WIPI inactivating the p53 pathway are noted in well-differentiated pNETs, and the retinoblastoma pathway is altered in the majority of pNETs. In addition to these genes, additional alterations that may be important in their pathogenesis include changes in the MEN1 gene, p16/MTS1 tumor-suppressor gene, and DPC4/ Smad4 gene; amplification of the HER-2/neu protooncogene; alterations in transcription factors (Hoxc6 [GI carcinoids]), growth factors, and their receptors; methylation of a number of genes that probably results in their inactivation; and deletions of unknown tumor-suppressor genes as well as gains in other unknown genes (Table 113-1). The clinical antitumor activity of everolimus, an mTOR inhibitor, and

1	in their inactivation; and deletions of unknown tumor-suppressor genes as well as gains in other unknown genes (Table 113-1). The clinical antitumor activity of everolimus, an mTOR inhibitor, and sunitinib, a tyrosine kinase inhibitor (PDGFR, VEGFR1, VEGFR2, c-KIT, FLT-3), support the importance of the mTOR-AKT pathway and tyrosine kinase receptors in mediating growth of malignant NETs (especially pNETs). The importance of the mTOR pathway in pNET growth is further supported by the finding that a single-nucleotide polymorphism (FGFR4-G388R, in fibroblast growth factor receptor 4) affects selectivity to the mTOR inhibitor and can result in significantly higher risk of advanced pNET stage and liver metastases (Table 113-5). Comparative genomic hybridization, genome-wide allelotyping studies, and genome-wide single-nucleotide polymorphism analyses have shown that chromosomal losses and gains are common in pNETs and GI-NETs (carcinoids), but they differ between these two NETs, and some

1	and genome-wide single-nucleotide polymorphism analyses have shown that chromosomal losses and gains are common in pNETs and GI-NETs (carcinoids), but they differ between these two NETs, and some have prognostic significance (Table 113-5). Mutations in the MEN1 gene are probably particularly important. Loss of heterozygosity at the MEN 1 locus on chromosome 11q13 is noted in 93% of sporadic pNETs (i.e., in patients without MEN 1) and in 26–75% of sporadic GI-NETs (carcinoids). Mutations in the MEN1 gene are reported in 31–34% of sporadic gastrinomas. Exomic sequencing of sporadic pNETs found that the most frequently altered gene was

1	Abbreviations: GI, gastrointestinal; PNETs, pancreatic neuroendocrine tumors. MEN1, occurring in 44% of patients, followed by mutations in 43% of patients in genes encoding for two subunits of a transcription/ chromatin remodeling complex consisting of DAXX (death-domainassociated protein) and ATRX (α-thalassemia/mental retardation syndrome X-linked) and in 15% of patients in the mTOR pathway. The presence of a number of these molecular alterations in pNETs or GI-NETs (carcinoids) correlates with tumor growth, tumor size, and disease extent or invasiveness and may have prognostic significance (Table 113-5).

1	CHARACTERISTICS OF THE MOST COMMON GI-NETs (CARCINOIDS) Appendiceal NETs (Carcinoids) Appendiceal NETs (carcinoids) occur in 1 in every 200–300 appendectomies, usually in the appendiceal tip, have an incidence of 0.15/100,000 per year, comprise 2–5% of all GI-NETs (carcinoids), and comprise 32–80% of all appendiceal tumors. Most (i.e., >90%) are <1 cm in diameter without metastases in older studies, but more recently, 2–35% have had metastases (Table 113-3). In the SEER data of 1570 appendiceal carcinoids, 62% were localized, 27% had regional metastases, and 8% had distant metastases. The risk of metastases increases with size, with those <1 cm having a 0 to <10% risk of metastases and those >2 cm having a 25–44% risk. Besides tumor size, other important prognostic factors for metastases include basal location, invasion of mesoappendix, poor differentiation, advanced stage or WHO/ENETS classification, older age, and positive resection margins. The 5-year survival is 88–100% for

1	include basal location, invasion of mesoappendix, poor differentiation, advanced stage or WHO/ENETS classification, older age, and positive resection margins. The 5-year survival is 88–100% for patients with localized disease, 78–100% for patients with regional involvement, and 12–28% for patients with distal metastases. In patients with tumors <1 cm in diameter, the 5-year survival is 95–100%, whereas it is 29% if tumors are >2 cm in diameter. Most tumors are well-differentiated G1 tumors (87%) (Table 113-4), with the remainder primarily well-differentiated G2 tumors (13%); poorly differentiated G3 tumors are uncommon (<1%). Their percentage of the total number of carcinoids decreased from 43.9% (1950–1969) to 2.4% (1992–1999). Appendiceal goblet cell (GC) NETs (carcinoids)/ carcinomas are a rare subtype (<5%) that are mixed adeno-neuroendocrine carcinomas. They are malignant and are thought to comprise a distinct entity; they frequently present with advanced disease and are

1	are a rare subtype (<5%) that are mixed adeno-neuroendocrine carcinomas. They are malignant and are thought to comprise a distinct entity; they frequently present with advanced disease and are recommended to be treated as adenocarcinomas, not carcinoid tumors.

1	Small intestinal (SI) NETs (carcinoids) have a reported incidence of 0.67/100,000 in the United States, 0.32/100,000 in England, and 1.12/100,000 in Sweden and comprise >50% of all SI tumors. There is a male predominance (1.5:1), and race affects frequency, with a lower frequency in Asians and greater frequency in African Americans. The mean age of presentation is 52–63 years, with a wide range (1–93 years). Familial SI carcinoid families exist but are very uncommon. These are frequently multiple; 9–18% occur in the jeunum, 70–80% are present in the ileum, and 70% occur within 6 cm (2.4 in.) of the ileocecal valve. Forty percent are <1 cm in diameter, 32% are 1–2 cm, and 29% are >2 cm. They are characteristically well differentiated; however, they are generally invasive, with 1.2% being intramucosal in location, 27% penetrating the submucosa, and 20% invading the muscularis propria. Metastases occur in a mean of 47–58% (range 20–100%). Liver metastases occur in 38%, to lymph nodes in

1	in location, 27% penetrating the submucosa, and 20% invading the muscularis propria. Metastases occur in a mean of 47–58% (range 20–100%). Liver metastases occur in 38%, to lymph nodes in 37% and more distant in 20–25%. They characteristically cause a marked fibrotic reaction, which can lead to intestinal obstruction. Tumor size is an important variable in the frequency of metastases. However, even small NETs (carcinoids) of the small intestine (<1 cm) have metastases in 15–25% of cases, whereas the proportion increases to 58–100% for tumors 1–2 cm in diameter. Carcinoids also occur in the duodenum, with 31% having metastases. Duodenal tumors <1 cm virtually never metastasize, whereas 33% of those >2 cm had metastases. SI NETs (carcinoids) are the most common cause (60–87%) of the carcinoid syndrome and are discussed in a later section (Table 113-7). Important prognostic factors are listed in (Table 113-5), and particularly important are the

1	During Course At Presentation of Disease Mean 57 yrs 59.2 yrs tumor extent, proliferative index by grading, and stage (Table 113-4). The overall survival at 5 years is 55–75%; however, it varies markedly with disease extent, being 65–90% with localized disease, 66–72% with regional involvement, and 36–43% with distant disease.

1	Rectal NETs (Carcinoids) Rectal NETs (carcinoids) comprise 27% of all GI-NETs (carcinoids) and 16% of all NETs and are increasing in frequency. In the U.S. SEER data, they currently have an incidence of 0.86/100,000 per year (up from 0.2/100,000 per year in 1973) and represent 1–2% of all rectal tumors. They are found in approximately 1 in every 1500/2500 proctoscopies/colonoscopies or 0.05–0.07% of individuals undergoing these procedures. Nearly all occur between 4 and 13 cm above the dentate line. Most are small, with 66–80% being <1 cm in diameter, and rarely metastasize (5%). Tumors between 1 and 2 cm can metastasize in 5–30%, and those >2 cm, which are uncommon, in >70%. Most invade only to the submucosa (75%), with 2.1% confined to the mucosa, 10% to the muscular layer, and 5% to adjacent structures. Histologically, most are well differentiated (98%) with 72% ENETS/WHO grade G1 and 28% grade G2 (Table 113-4). Overall survival is 88%; however, it is very much dependent of the

1	adjacent structures. Histologically, most are well differentiated (98%) with 72% ENETS/WHO grade G1 and 28% grade G2 (Table 113-4). Overall survival is 88%; however, it is very much dependent of the stage, with 5-year survival of 91% for localized disease, 36–49% for regional disease, and 20–32% for distant disease. Risk factors are listed in Table 113-5 and particularly include tumor size, depth of invasion, presence of metastases, differentiation, and recent TNM classification and grade.

1	Bronchial NETs (Carcinoids) Bronchial NETs (carcinoids) comprise 25–33% of all well-differentiated NETs and 90% of all the poorly differentiated NETs found, likely due to a strong association with smoking. Their incidence ranges from 0.2 to 2/100,000 per year in the United States and European countries and is increasing at a rate of 6% per year. They are slightly more frequent in females and in whites compared with those of Hispanic/Asian/African descent, and are most commonly seen in the sixth decade of life, with a younger age of presentation for typical carcinoids (45 years) compared to atypical carcinoids (55 years).

1	A number of different classifications of bronchial GI-NETs (carcinoids) have been proposed. In some studies, they are classified into four categories: typical carcinoid (also called bronchial carcinoid tumor, Kulchitsky cell carcinoma I [KCC-I]), atypical carcinoid (also called well-differentiated neuroendocrine carcinoma [KC-II]), intermediate small-cell neuroendocrine carcinoma, and small-cell neuroendocarcinoma

1	Endocrine Tumors of the Gastrointestinal Tract and Pancreas 564 (KC-III). Another proposed classification includes three categories of lung NETs: benign or low-grade malignant (typical carcinoid), low-grade malignant (atypical carcinoid), and high-grade malignant (poorly differentiated carcinoma of the large-cell or small-cell type). The WHO classification includes four general categories: typical carcinoid, atypical carcinoid, large-cell neuroendocrine carcinoma, and small-cell carcinoma. The ratio of typical to atypical carcinoids is 8–10:1, with the typical carcinoids comprising 1–2% of lung tumors, atypical 0.1–0.2%, large-cell neuroendocrine tumors 0.3%, and small-cell lung cancer 9.8% of all lung tumors. These different categories of lung NETs have different prognoses, varying from excellent for typical carcinoid to poor for small-cell neuroendocrine carcinomas. The occurrence of large-cell and small-cell lung carcinoids, but not typical or atypical lung carcinoids, is related

1	excellent for typical carcinoid to poor for small-cell neuroendocrine carcinomas. The occurrence of large-cell and small-cell lung carcinoids, but not typical or atypical lung carcinoids, is related to tobacco use. The 5-year survival is very much influenced by the classification of the tumor, with survival of 92–100% for patients with a typical carcinoid, 61–88% with an atypical carcinoid, 13–57% with a large-cell neuroendocrine tumor, and 5% with a small-cell lung cancer.

1	Gastric NET (Carcinoids) Gastric NETs (carcinoids) account for 3 of every 1000 gastric neoplasms and 1.3–2% of all carcinoids, and their relative frequency has increased threeto fourfold over the last five decades (2.2% in 1950 to 9.6% in 2000–2007, SEER data). At present, it is unclear whether this increase is due to better detection with the increased use of upper GI endoscopy or to a true increase in incidence. Gastric NETs (carcinoids) are classified into three different categories, and this has important implications for pathogenesis, prognosis, and treatment. Each originates from gastric enterochromaffin-like (ECL) cells, one of the six types of gastric neuroendocrine cells, in the gastric mucosa. Two subtypes are associated with hypergastrinemic states, either chronic atrophic gastritis (type I) (80% of all gastric NETs [carcinoids]) or Zollinger-Ellison syndrome, which is almost always a part of the MEN 1 syndrome (type

1	I) (80% of all gastric NETs [carcinoids]) or Zollinger-Ellison syndrome, which is almost always a part of the MEN 1 syndrome (type II) (6% of all cases). These tumors generally pursue a benign course, with type I uncommonly (<10%) associated with metastases, whereas type II tumors are slightly more aggressive, with 10–30% associated with metastases. They are usually multiple, small, and infiltrate only to the submucosa. The third subtype of gastric NETs (carcinoids) (type III) (sporadic) occurs without hypergastrinemia (14–25% of all gastric carcinoids) and has an aggressive course, with 54–66% developing metastases. Sporadic carcinoids are usually single, large tumors; 50% have atypical histology, and they can be a cause of the carcinoid syndrome. Five-year survival is 99–100% in patients with type I, 60–90% in patients with type II, and 50% in patients with type III gastric NETs (carcinoids).

1	CLINICAL PRESENTATION OF NETs (CARCINOIDS) GI/Lung NET (Carcinoid) Without the Carcinoid Syndrome The age of patients at diagnosis ranges from 10 to 93 years, with a mean age of 63 years for the small intestine and 66 years for the rectum. The presentation is diverse and is related to the site of origin and the extent of malignant spread. In the appendix, NETs (carcinoids) usually are found incidentally during surgery for suspected appendicitis. SI NETs (carcinoids) in the jejunoileum present with periodic abdominal pain (51%), intestinal obstruction with ileus/invagination (31%), an abdominal tumor (17%), or GI bleeding (11%). Because of the vagueness of the symptoms, the diagnosis usually is delayed approximately 2 years from onset of the symptoms, with a range up to 20 years. Duodenal, gastric, and rectal NETs (carcinoids) are most frequently found by chance at endoscopy. The most common symptoms of rectal carcinoids are melena/bleeding (39%), constipation (17%), and diarrhea

1	gastric, and rectal NETs (carcinoids) are most frequently found by chance at endoscopy. The most common symptoms of rectal carcinoids are melena/bleeding (39%), constipation (17%), and diarrhea (12%). Bronchial NETs (carcinoids) frequently are discovered as a lesion on a chest radiograph, and 31% of the patients are asymptomatic. Thymic NETs (carcinoids) present as anterior mediastinal masses, usually on chest radiograph or computed tomography (CT) scan. Ovarian and testicular NETs (carcinoids) usually present as masses discovered on physical examination or ultrasound. Metastatic NETs (carcinoids) in the liver frequently presents as hepatomegaly in a patient who may have minimal symptoms and nearly normal liver function test results.

1	GI/lung NETs (carcinoids) immunocytochemically can contain numerous GI peptides: gastrin, insulin, somatostatin, motilin, neurotensin, tachykinins (substance K, substance P, neuropeptide K), glucagon, gastrin-releasing peptide, vasoactive intestinal peptide (VIP), PP, ghrelin, other biologically active peptides (ACTH, calcitonin, growth hormone), prostaglandins, and bioactive amines (serotonin). These substances may or may not be released in sufficient amounts to cause symptoms. In various studies of patients with GI-NETs (carcinoids), elevated serum levels of PP were found in 43%, motilin in 14%, gastrin in 15%, and VIP in 6%. Foregut NETs (carcinoids) are more likely to produce various GI peptides than are midgut NETs (carcinoids). Ectopic ACTH production causing Cushing’s syndrome is seen increasingly with foregut carcinoids (respiratory tract primarily) and, in some series, has been the most common cause of the ectopic ACTH syndrome, accounting for 64% of all cases. Acromegaly due

1	increasingly with foregut carcinoids (respiratory tract primarily) and, in some series, has been the most common cause of the ectopic ACTH syndrome, accounting for 64% of all cases. Acromegaly due to growth hormone–releasing factor release occurs with foregut NETs (carcinoids), as does the somatostatinoma syndrome, but rarely occurs with duodenal NETs (carcinoids). The most common systemic syndrome with GI-NETs (carcinoids) is the carcinoid syndrome, which is discussed in detail in the next section.

1	CARCINOID SYNDROME Clinical Features The cardinal features from a number of series at presentation as well as during the disease course are shown in Table 113-7. Flushing and diarrhea are the two most common symptoms, occurring in a mean of 69–70% of patients initially and in up to 78% of patients during the course of the disease. The characteristic flush is of sudden onset; it is a deep red or violaceous erythema of the upper body, especially the neck and face, often associated with a feeling of warmth and occasionally associated with pruritus, lacrimation, diarrhea, or facial edema. Flushes may be precipitated by stress; alcohol; exercise; certain foods, such as cheese; or certain agents, such as catecholamines, pentagastrin, and serotonin reuptake inhibitors. Flushing episodes may be brief, lasting 2–5 min, especially initially, or may last hours, especially later in the disease course. Flushing usually is associated with metastatic midgut NETs (carcinoids) but can also occur with

1	lasting 2–5 min, especially initially, or may last hours, especially later in the disease course. Flushing usually is associated with metastatic midgut NETs (carcinoids) but can also occur with foregut NETs (carcinoids). With bronchial NETs (carcinoids), the flushes frequently are prolonged for hours to days, reddish in color, and associated with salivation, lacrimation, diaphoresis, diarrhea, and hypotension. The flush associated with gastric NETs (carcinoids) can also be reddish in color, but with a patchy distribution over the face and neck, although the classic flush seen with midgut NETs (carcinoids) can also be seen with gastric NETs (carcinoids). It may be provoked by food and have accompanying pruritus.

1	Diarrhea usually occurs with flushing (85% of cases). The diarrhea usually is described as watery, with 60% of patients having <1 L/d of diarrhea. Steatorrhea is present in 67%, and in 46%, it is >15 g/d (normal <7 g). Abdominal pain may be present with the diarrhea or independently in 10–34% of cases.

1	Cardiac manifestations occur initially in 11–40% (mean 26%) of patients with carcinoid syndrome and in 14–41% (mean 30%) at some time in the disease course. The cardiac disease is due to the formation of fibrotic plaques (composed of smooth-muscle cells, myofibroblasts, and elastic tissue) involving the endocardium, primarily on the right side, although lesions on the left side also occur occasionally, especially if a patent foramen ovale exists. The dense fibrous deposits are most commonly on the ventricular aspect of the tricuspid valve and less commonly on the pulmonary valve cusps. They can result in constriction of the valves, and pulmonic stenosis is usually predominant, whereas the tricuspid valve is often fixed open, resulting in regurgitation predominating. Overall, in patients with carcinoid heart disease, 90–100% have tricuspid insufficiency, 43–59% have tricuspid stenosis, 50–81% have pulmonary insufficiency, 25–59% have pulmonary stenosis, and 11% (0–25%) left-side

1	with carcinoid heart disease, 90–100% have tricuspid insufficiency, 43–59% have tricuspid stenosis, 50–81% have pulmonary insufficiency, 25–59% have pulmonary stenosis, and 11% (0–25%) left-side lesions. Up to 80% of patients with cardiac lesions develop heart failure. Lesions on the left side are much less extensive, occur in 30% at autopsy, and most frequently affect the mitral valve. Up to 80% of patients with cardiac lesions have evidence of heart failure. At diagnosis in various series, 27–43% of patients are in New York Heart Association class I, 30–40% are in class II, 13–31% are in class III, and 3–12% are in class IV. At present, carcinoid heart disease is reported to be decreasing in frequency and severity, with mean occurrence in 20% of patients and occurrence in as few as 3–4% in some reports. Whether this decrease is due to the widespread use of somatostatin analogues, which control the release of bioactive agents thought involved in mediating the heart disease, is

1	3–4% in some reports. Whether this decrease is due to the widespread use of somatostatin analogues, which control the release of bioactive agents thought involved in mediating the heart disease, is unclear.

1	Other clinical manifestations include wheezing or asthma-like symptoms (8–18%), pellagra-like skin lesions (2–25%), and impaired cognitive function. A variety of noncardiac problems due to increased fibrous tissue have been reported, including retroperitoneal fibrosis causing urethral obstruction, Peyronie’s disease of the penis, intraabdominal fibrosis, and occlusion of the mesenteric arteries or veins.

1	Pathobiology Carcinoid syndrome occurred in 8% of 8876 patients with GI-NETs (carcinoids), with a rate of 1.7–18.4% in different studies. It occurs only when sufficient concentrations of products secreted by the tumor reach the systemic circulation. In 91–100% of cases, this occurs after distant metastases to the liver. Rarely, primary GI-NETs (carcinoids) with nodal metastases with extensive retroperitoneal invasion, pNETs (carcinoids) with retroperitoneal lymph nodes, or NETs (carcinoids) of the lung or ovary with direct access to the systemic circulation can cause the carcinoid syndrome without hepatic metastases. All GI-NETs (carcinoids) do not have the same propensity to metastasize and cause the carcinoid syndrome (Table 113-3). Midgut NETs (carcinoids) account for 57–67% of cases of carcinoid syndrome, fore-gut NETs (carcinoids) for 0–33%, hindgut for 0–8%, and an unknown primary location for 2–26% (Tables 113-3 and 113-7).

1	One of the main secretory products of GI-NETs (carcinoids) involved in the carcinoid syndrome is serotonin (5-HT) (Fig. 113-1), which is synthesized from tryptophan. Up to 50% of dietary tryptophan can be used in this synthetic pathway by tumor cells, and this can result in inadequate supplies for conversion to niacin; hence, some patients (2.5%) develop pellagra-like lesions. Serotonin has numerous biologic effects, including stimulating intestinal secretion with inhibition of absorption, stimulating increases in intestinal motility, and stimulating fibrogenesis. In various studies, 56–88% of all GI-NETs (carcinoids) were associated with serotonin overproduction; however, 12–26% of the patients did not have the carcinoid syndrome. In one study, platelet serotonin was elevated in 96% of patients with midgut NETs (carcinoids), 43% with foregut tumors, and 0% with hindgut tumors. In 90–100% of patients with the carcinoid syndrome, there is evidence of serotonin overproduction. Serotonin

1	with midgut NETs (carcinoids), 43% with foregut tumors, and 0% with hindgut tumors. In 90–100% of patients with the carcinoid syndrome, there is evidence of serotonin overproduction. Serotonin is thought to be predominantly responsible for the diarrhea. Patients with the carcinoid syndrome have increased colonic motility with a shortened transit time and possibly a secretory/absorptive alteration that is compatible with the known actions of serotonin in the gut mediated primarily through 5-HT3 and, to a lesser degree, 5-HT4 receptors. Serotonin receptor antagonists (especially 5-HT3 antagonists) relieve the diarrhea in many, but not all, patients. A tryptophan 5-hydroxylase inhibitor, LX-1031, which inhibits serotonin synthesis in peripheral tissues, is reported to cause a 44% decrease in bowel movement frequency and a 20% improvement in stool form in patients with the carcinoid syndrome. Additional studies suggest that tachykinins may be important mediators of diarrhea in some

1	in bowel movement frequency and a 20% improvement in stool form in patients with the carcinoid syndrome. Additional studies suggest that tachykinins may be important mediators of diarrhea in some patients. In one study, plasma tachykinin levels correlated with symptoms of diarrhea. Serotonin does not appear to be involved in the flushing because serotonin receptor antagonists do not relieve flushing. In patients with gastric carcinoids, the characteristic red, patchy pruritic flush is thought due to histamine release because H1 and H2 receptor antagonists can prevent it. Numerous studies have shown that tachykinins (substance P, neuropeptide K) are stored in GI-NETs (carcinoids) and released during flushing. However, some studies have demonstrated that octreotide can relieve the flushing induced by pentagastrin in these patients without altering the stimulated increase in plasma substance P, suggesting that other mediators must be involved in the flushing. A correlation between plasma

1	by pentagastrin in these patients without altering the stimulated increase in plasma substance P, suggesting that other mediators must be involved in the flushing. A correlation between plasma tachykinin levels (but not substance P levels) and flushing has been reported. Prostaglandin release could be involved in mediating either the diarrhea or flush, but conflicting data exist.

1	Both histamine and serotonin may be responsible for the wheezing as 565 well as the fibrotic reactions involving the heart, causing Peyronie’s disease and intraabdominal fibrosis. The exact mechanism of the heart disease remains unclear, although increasing evidence supports a central role for serotonin. Patients with heart disease have higher plasma levels of neurokinin A, substance P, plasma atrial natriuretic peptide (ANP), pro-brain natriuretic peptide, chromogranin A, and activin A as well as higher urinary 5-HIAA excretion.

1	The valvular heart disease caused by the appetite-suppressant drug dexfenfluramine is histologically indistinguishable from that observed in carcinoid disease. Furthermore, ergot-containing dopamine receptor agonists used for Parkinson’s disease (pergolide, cabergoline) cause valvular heart disease that closely resembles that seen in the carcinoid syndrome. Furthermore, in animal studies, the formation of valvular plaques/fibrosis occurs after prolonged treatment with serotonin as well as in animals with a deficiency of the 5-HIAA transporter gene, which results in an inability to inactivate serotonin. Metabolites of fenfluramine, as well as the dopamine receptor agonists, have high affinity for serotonin receptor subtype 5-HT2B receptors, whose activation is known to cause fibroblast mitogenesis. Serotonin receptor subtypes normally are expressed in human heart valve intersti- 5-HT1B,1D,2A,2B tial cells. High levels of 5-HT2B receptors are known to occur in heart valves and occur in

1	Serotonin receptor subtypes normally are expressed in human heart valve intersti- 5-HT1B,1D,2A,2B tial cells. High levels of 5-HT2B receptors are known to occur in heart valves and occur in cardiac fibroblasts and cardiomyocytes. Studies of cultured interstitial cells from human cardiac valves have demonstrated that these valvulopathic drugs induce mitogenesis by activating 5-HT2B receptors and stimulating upregulation of transforming growth factor β and collagen biosynthesis. These observations support the conclu sion that serotonin overproduction by GI-NETs (carcinoids) is important in mediating the valvular changes, possibly by activating 5-HT2B receptors in the endocardium. Both the magnitude of serotonin overproduction and prior chemotherapy are important predictors of progression of the heart disease, whereas patients with high plasma levels of ANP have a worse prognosis. Plasma connective tissue growth factor levels are elevated in many fibrotic conditions; elevated levels

1	of the heart disease, whereas patients with high plasma levels of ANP have a worse prognosis. Plasma connective tissue growth factor levels are elevated in many fibrotic conditions; elevated levels occur in patients with carcinoid heart disease and correlate with the presence of right ventricular dysfunction and the extent of valvular regurgitation in patients with GI-NETs (carcinoids).

1	Patients may develop either a typical or, rarely, an atypical carcinoid syndrome (Fig. 113-1). In patients with the typical form, which characteristically is caused by midgut NETs (carcinoids), the conversion of tryptophan to 5-HTP is the rate-limiting step (Fig. 113-1). Once 5-HTP is formed, it is rapidly converted to 5-HT and stored in secretory granules of the tumor or in platelets. A small amount remains in plasma and is converted to 5-HIAA, which appears in large amounts in the urine. These patients have an expanded serotonin pool size, increased blood and platelet serotonin, and increased urinary 5-HIAA. Some GI-NETs (carcinoids) cause an atypical carcinoid syndrome that is thought to be due to a deficiency in the enzyme dopa decarboxylase; thus, 5-HTP cannot be converted to 5-HT (serotonin), and 5-HTP is secreted into the bloodstream (Fig. 113-1). In these patients, plasma serotonin levels are normal but urinary levels may be increased because some 5-HTP is converted to 5-HT in

1	and 5-HTP is secreted into the bloodstream (Fig. 113-1). In these patients, plasma serotonin levels are normal but urinary levels may be increased because some 5-HTP is converted to 5-HT in the kidney. Characteristically, urinary 5-HTP and 5-HT are increased, but urinary 5-HIAA levels are only slightly elevated. Foregut carcinoids are the most likely to cause an atypical carcinoid syndrome; however, they also can cause a typical carcinoid syndrome.

1	One of the most immediate life-threatening complications of the carcinoid syndrome is the development of a carcinoid crisis. This is more common in patients who have intense symptoms or have greatly increased urinary 5-HIAA levels (i.e., >200 mg/d). The crisis may occur spontaneously; however, it is usually provoked by procedures such as anesthesia, chemotherapy, surgery, biopsy, endoscopy, or radiologic examinations such as during biopsies, hepatic artery embolization, and vessel catheterization. It can be provoked by stress or procedures as mild as repeated palpation of the tumor during physical examination. Patients develop intense flushing, diarrhea, abdominal pain, cardiac abnormalities including tachycardia, hypertension, or hypotension, and confusion or stupor. If not adequately treated, this can be a terminal event.

1	Endocrine Tumors of the Gastrointestinal Tract and Pancreas 566 DIAGNOSIS OF THE CARCINOID SYNDROME AND GI-NETs (CARCINOIDS) The diagnosis of carcinoid syndrome relies on measurement of urinary or plasma serotonin or its metabolites in the urine. The measurement of 5-HIAA is used most frequently. False-positive elevations may occur if the patient is eating serotonin-rich foods such as bananas, pineapples, walnuts, pecans, avocados, or hickory nuts or is taking certain medications (cough syrup containing guaifenesin, acetaminophen, salicylates, serotonin reuptake inhibitors, or l-dopa). The normal range for daily urinary 5-HIAA excretion is 2–8 mg/d. Serotonin overproduction was noted in 92% of patients with carcinoid syndrome in one study, and in another study, 5-HIAA had 73% sensitivity and 100% specificity for carcinoid syndrome. Serotonin overproduction is not synonymous with the presence of clinical carcinoid syndrome because 12–26% of patients with serotonin overproduction do not

1	100% specificity for carcinoid syndrome. Serotonin overproduction is not synonymous with the presence of clinical carcinoid syndrome because 12–26% of patients with serotonin overproduction do not have clinical evidence of the carcinoid syndrome. Most physicians use only the urinary 5-HIAA excretion rate; however, plasma and platelet serotonin levels, if available, may provide additional information. Platelet serotonin levels are more sensitive than urinary 5-HIAA but are not generally available. A single plasma 5-HIAA determination was found to correlate with the 24-h urinary values, raising the possibility that this could replace the standard urinary collection because of its greater convenience and avoidance of incomplete or improper collections. Because patients with foregut NETs (carcinoids) may produce an atypical carcinoid syndrome, if this syndrome is suspected and the urinary 5-HIAA is minimally elevated or normal, other urinary metabolites of tryptophan, such as 5-HTP and

1	may produce an atypical carcinoid syndrome, if this syndrome is suspected and the urinary 5-HIAA is minimally elevated or normal, other urinary metabolites of tryptophan, such as 5-HTP and 5-HT, should be measured (Fig. 113-1). Flushing occurs in a number of other diseases, including systemic mastocytosis, chronic myeloid leukemia with increased histamine release, menopause, reactions to alcohol or glutamate, and side effects of chlorpropamide, calcium channel blockers, and nicotinic acid. None of these conditions cause increased urinary 5-HIAA. The diagnosis of carcinoid tumor can be suggested by the carcinoid syndrome, recurrent abdominal symptoms in a healthy-appearing individual, or the discovery of hepatomegaly or hepatic metastases associated with minimal symptoms. Ileal NETs (carcinoids), which make up 25% of all clinically detected carcinoids, should be suspected in patients with bowel obstruction, abdominal pain, flushing, or diarrhea. Serum chromogranin A levels are elevated

1	which make up 25% of all clinically detected carcinoids, should be suspected in patients with bowel obstruction, abdominal pain, flushing, or diarrhea. Serum chromogranin A levels are elevated in 56–100% of patients with GI-NETs (carcinoids), and the level correlates with tumor bulk. Serum chromogranin A levels are not specific for GI-NETs (carcinoids) because they are also elevated in patients with pNETs and other NETs. Furthermore, a major problem is caused by potent acid antisecretory drugs such as proton pump inhibitors (omeprazole and related drugs) because they almost invariably cause elevation of plasma chromogranin A levels; the elevation occurs rapidly (3–5 days) with continued use, and the elevated levels overlap with the levels seen in many patients with NETs. Plasma neuron-specific enolase levels are also used as a marker of GI-NETs (carcinoids) but are less sensitive than chromogranin A, being increased in only 17–47% of patients. Newer markers have been proposed

1	enolase levels are also used as a marker of GI-NETs (carcinoids) but are less sensitive than chromogranin A, being increased in only 17–47% of patients. Newer markers have been proposed including pancreastatin (a chromogranin A breakdown product) and activin A. The former is not affected by proton pump inhibitors; however, its sensitivity and specificity are not established. Plasma activin elevations are reported to correlate with the presence of cardiac disease with a sensitivity of 87% and specificity of 57%.

1	Treatment includes avoiding conditions that precipitate flushing, dietary supplementation with nicotinamide, treatment of heart failure with diuretics, treatment of wheezing with oral bronchodilators, and control of the diarrhea with antidiarrheal agents such as loperamide and diphenoxylate. If patients still have symptoms, serotonin receptor antagonists or somatostatin analogues (Fig. 113-2) are the drugs of choice.

1	There are 14 subclasses of serotonin receptors, and antagonists for many are not available. The 5-HT1 and 5-HT2 receptor antagonists methysergide, cyproheptadine, and ketanserin have all been used to control the diarrhea but usually do not decrease flushing. The use of methysergide is limited because it can cause or enhance retroperitoneal fibrosis. Ketanserin diminishes diarrhea in 30–100% of patients. 5-HT3 receptor antagonists (ondansetron, tropisetron, alosetron) can control diarrhea and nausea in up to 100% of patients and occasionally ameliorate the flushing. A combination of histamine H1 and H2 receptor antagonists (i.e., diphenhydramine and cimetidine or ranitidine) may control flushing in patients with foregut carcinoids. The tryptophan 5-hydoxylase inhibitor telotristat etiprate decreased bowel frequency in 44% and improved stool consistency in 20%.

1	111In-[DTPA-D-Phe1, Tyr3]-octreotide = 111In-pentetreotide; Octreoscan-111 90Y-[DOTA0-D-Phe1 , Tyr3]-octreotide177Lu-[DOTA0-D-Phe1,Tyr3]-octreotateTHRCYSCYSSSCYSCYSSSCYSCYSSSD-PHELYSTYRTHRD-TRPLYSTYRTHRD-TRPLYSTYRTHRD-TRPHOOC-CH2CH2-COCH2-COOHCOOHHOOCNHHOOCN–(CH2)2–N–(CH2)2–ND-PHED-PHETHR-olTHR-olNNONN90Y111InCOOHHOOCNHHOOCNNONN177Lu FIGURE 113-2 Structure of somatostatin and synthetic analogues used for diagnostic or therapeutic indications.

1	Synthetic analogues of somatostatin (octreotide, lanreotide) are now the most widely used agents to control the symptoms of patients with carcinoid syndrome (Fig. 113-2). These drugs are effective at relieving symptoms and decreasing urinary 5-HIAA levels in patients with this syndrome. Octreotide-LAR and lanreotideSR/autogel (Somatuline) (sustained-release formulations allowing monthly injections) control symptoms in 74% and 68% of patients, respectively, with carcinoid syndrome and show a biochemical response in 51% and 64%, respectively. Patients with mild to moderate symptoms usually are treated initially with octreotide 100 µg SC every 8 h and then begun on the long-acting monthly depot forms (octreotide-LAR or lanreotide-autogel). Forty percent of patients escape control after a median time of 4 months, and the depot dosage may have to be increased as well as supplemented with the shorter-acting formulation, SC octreotide. Pasireotide (SOM230) is a somatostatin analogue with

1	median time of 4 months, and the depot dosage may have to be increased as well as supplemented with the shorter-acting formulation, SC octreotide. Pasireotide (SOM230) is a somatostatin analogue with broader selectivity (high-affinity somatostatin receptors [sst1, sst2, sst3, sst5]) than octreotide/lanreotide (sst2, sst5). In a phase II study of patients with refractory carcinoid syndrome, pasireotide controlled symptoms in 27%.

1	Carcinoid heart disease is associated with a decreased mean survival (3.8 years), and therefore, it should be sought for and carefully assessed in all patients with carcinoid syndrome. Transthoracic echocardiography remains a key element in establishing the diagnosis of carcinoid heart disease and determining the extent and type of cardiac abnormalities. Treatment with diuretics and somatostatin analogues can reduce the negative hemodynamic effects and secondary heart failure. It remains unclear whether long-term treatment with these drugs will decrease the progression of carcinoid heart disease. Balloon valvuloplasty for stenotic valves or cardiac valve surgery may be required.

1	In patients with carcinoid crises, somatostatin analogues are effective at both treating the condition and preventing their development during known precipitating events such as surgery, anesthesia, chemotherapy, and stress. It is recommended that octreotide 150–250 µg SC every 6 to 8 h be used 24–48 h before anesthesia and then continued throughout the procedure. Currently, sustained-release preparations of both octreotide (octreotide-LAR [long-acting release], 10, 20, 30 mg) and lanreotide (lanreotide-PR [prolonged release, lanreotide-autogel], 60, 90, 120 mg) are available and widely used because their use greatly facilitates long-term treatment. Octreotide-LAR (30 mg/month) gives a plasma level ≥1 ng/mL for 25 days, whereas this requires three to six injections a day of the non-sustained-release form. Lanreotideautogel (Somatuline) is given every 4–6 weeks.

1	Short-term side effects occur in up to one-half of patients. Pain at the injection site and side effects related to the GI tract (59% discomfort, 15% nausea, diarrhea) are the most common. They are usually short-lived and do not interrupt treatment. Important long-term side effects include gallstone formation, steatorrhea, and deterioration in glucose tolerance. The overall incidence of gallstones/biliary sludge in one study was 52%, with 7% having symptomatic disease that required surgical treatment.

1	Interferon α is reported to be effective in controlling symptoms of the carcinoid syndrome either alone or combined with hepatic artery embolization. With interferon α alone, the clinical response rate is 30–70%, and with interferon α with hepatic artery embolization, diarrhea was controlled for 1 year in 43% and flushing was controlled in 86%. Side effects develop in almost all patients, with the most frequent being a flu-like syndrome (80–100%), followed by anorexia and fatigue, even though these frequently improve with continued treatment. Other more severe side effects include bone marrow toxicity, hepatotoxicity, autoimmune disorders, and rarely CNS side effects (depression, mental disorders, visual problems).

1	Hepatic artery embolization alone or with chemotherapy (chemoembolization) has been used to control the symptoms of carcinoid syndrome. Embolization alone is reported to control symptoms in up to 76% of patients, and chemoembolization (5-fluorouracil, doxorubicin, cisplatin, mitomycin) controls symptoms in 60–75% of patients. Hepatic artery embolization can have major side effects, 567 including nausea, vomiting, pain, and fever. In two studies, 5–7% of patients died from complications of hepatic artery occlusion. Other drugs have been used successfully in small numbers of patients to control the symptoms of carcinoid syndrome. Parachlorophenylanine can inhibit tryptophan hydroxylase and therefore the conversion of tryptophan to 5-HTP. However, its severe side effects, including psychiatric disturbances, make it intolerable for long-term use. α-Methyldopa inhibits the conversion of 5-HTP to 5-HT, but its effects are only partial.

1	Peptide radioreceptor therapy (using radiotherapy with radio-labeled somatostatin analogues), the use of radiolabeled micro-spheres, and other methods for treatment of advanced metastatic disease may facilitate control of the carcinoid syndrome and are discussed in a later section dealing with treatment of advanced disease. Surgery is the only potentially curative therapy. Because with most

1	GI-NETs (carcinoids), the probability of metastatic disease increases with increasing size, the extent of surgical resection is determined accordingly. With appendiceal NETs (carcinoids) <1 cm, simple appendectomy was curative in 103 patients followed for up to 35 years. With rectal NETs (carcinoids) <1 cm, local resection is curative. With SI NETs (carcinoids) <1 cm, there is not complete agreement. Because 15–69% of SI NETs (carcinoids) this size have metastases in different studies, some recommend a wide resection with en bloc resection of the adjacent lymph-bearing mesentery. If the tumor is >2 cm for rectal, appendiceal, or SI NETs (carcinoids), a full cancer operation should be done. This includes a right hemicolectomy for appendiceal NETs (carcinoids), an abdominoperineal resection or low anterior resection for rectal NETs (carcinoids), and an en bloc resection of adjacent lymph nodes for SI NETs (carcinoids). For appendiceal NETs (carcinoids) 1–2 cm in diameter, a simple

1	or low anterior resection for rectal NETs (carcinoids), and an en bloc resection of adjacent lymph nodes for SI NETs (carcinoids). For appendiceal NETs (carcinoids) 1–2 cm in diameter, a simple appendectomy is proposed by some, whereas others favor a formal right hemicolectomy. For 1–2 cm rectal NETs (carcinoids), it is recommended that a wide, local, full-thickness excision be performed.

1	With type I or II gastric NETs (carcinoids), which are usually <1 cm, endoscopic removal is recommended. In type I or II gastric carcinoids, if the tumor is >2 cm or if there is local invasion, some recommend total gastrectomy, whereas others recommend antrectomy in type I to reduce the hypergastrinemia, which has led to regression of the carcinoids in a number of studies. For types I and II gastric NETs (carcinoids) of 1–2 cm, there is no agreement, with some recommending endoscopic treatment followed by chronic somatostatin treatment and careful follow-up and others recommending surgical treatment. With type III gastric NETs (carcinoids) >2 cm, excision and regional lymph node clearance are recommended. Most tumors <1 cm are treated endoscopically. Resection of isolated or limited hepatic metastases may be beneficial and will be discussed in a later section on treatment of advanced disease.

1	Resection of isolated or limited hepatic metastases may be beneficial and will be discussed in a later section on treatment of advanced disease. Functional pNETs usually present clinically with symptoms due to the hormone-excess state (Table 113-2). Only late in the course of the disease does the tumor per se cause prominent symptoms such as abdominal pain. In contrast, all the symptoms due to nonfunctional pNETs are due to the tumor per se. The overall result of this is that some functional pNETs may present with severe symptoms with a small or undetectable primary tumor, whereas nonfunctional tumors usually present late in the disease course with large tumors, which are frequently metastatic. The mean delay between onset of continuous symptoms and diagnosis of a functional pNET syndrome is 4–7 years. Therefore, the diagnoses frequently are missed for extended periods. Endocrine Tumors of the Gastrointestinal Tract and Pancreas

1	Endocrine Tumors of the Gastrointestinal Tract and Pancreas Treatment of pNETs requires two different strategies. First, treatment must be directed at the hormone-excess state such as the gastric acid hypersecretion in gastrinomas or the hypoglycemia in insulinomas. Ectopic hormone secretion usually causes the presenting symptoms and can cause life-threatening complications. Second, with all the tumors except insulinomas, >50% are malignant (Table 113-2); therefore, treatment must also be directed against the tumor per se. Because in many patients these tumors are not surgically curable due to the presence of advanced disease at diagnosis, surgical resection for cure, which addresses both treatment aspects, is often not possible.

1	A gastrinoma is an NET that secretes gastrin; the resultant hypergastrinemia causes gastric acid hypersecretion (Zollinger-Ellison syndrome [ZES]). The chronic hypergastrinemia results in marked gastric acid hypersecretion and growth of the gastric mucosa with increased numbers of parietal cells and proliferation of gastric ECL cells. The gastric acid hypersecretion characteristically causes peptic ulcer disease (PUD), often refractory and severe, as well as diarrhea. The most common presenting symptoms are abdominal pain (70–100%), diarrhea (37–73%), and gastroesophageal reflux disease (GERD) (30–35%); 10–20% of patients have diarrhea only. Although peptic ulcers may occur in unusual locations, most patients have a typical duodenal ulcer. Important observations that should suggest this diagnosis include PUD with diarrhea; PUD in an unusual location or with multiple ulcers; PUD refractory to treatment or persistent; PUD associated with prominent gastric folds; PUD associated with

1	diagnosis include PUD with diarrhea; PUD in an unusual location or with multiple ulcers; PUD refractory to treatment or persistent; PUD associated with prominent gastric folds; PUD associated with findings suggestive of MEN 1 (endocrinopathy, family history of ulcer or endocrinopathy, nephrolithiases); and PUD without Helicobacter pylori present. H. pylori is present in >90% of idiopathic peptic ulcers but is present in <50% of patients with gastrinomas. Chronic unexplained diarrhea also should suggest ZES.

1	Approximately 20–25% of patients with ZES have MEN 1 (MEN1/ ZES), and in most cases, hyperparathyroidism is present before the ZES develops. These patients are treated differently from those without MEN 1 (sporadic ZES); therefore, MEN 1 should be sought in all patients with ZES by family history and by measuring plasma ionized calcium and prolactin levels and plasma hormone levels (parathormone, growth hormone).

1	Most gastrinomas (50–90%) in sporadic ZES are present in the duodenum, followed by the pancreas (10–40%) and other intraabdominal sites (mesentery, lymph nodes, biliary tract, liver, stomach, ovary). Rarely, the tumor may involve extraabdominal sites (heart, lung cancer). In MEN 1/ZES the gastrinomas are also usually in the duodenum (70–90%), followed by the pancreas (10–30%), and are almost always multiple. About 60–90% of gastrinomas are malignant (Table 113-2) with metastatic spread to lymph nodes and liver. Distant metastases to bone occur in 12–30% of patients with liver metastases.

1	Diagnosis The diagnosis of ZES requires the demonstration of inappropriate fasting hypergastrinemia, usually by demonstrating hypergastrinemia occurring with an increased basal gastric acid output (BAO) (hyperchlorhydria). More than 98% of patients with ZES have fasting hypergastrinemia, although in 40–60% the level may be elevated less than tenfold. Therefore, when the diagnosis is suspected, a fasting gastrin is usually the initial test performed. It is important to remember that potent gastric acid suppressant drugs such as proton pump inhibitors (PPIs) (omeprazole, esomeprazole, pantoprazole, lansoprazole, rabeprazole) can suppress acid secretion sufficiently to cause hypergastrinemia; because of their prolonged duration of action, these drugs have to be tapered or frequently discontinued for a week before the gastrin determination. Withdrawal of PPIs should be performed carefully because PUD complications can rapidly develop in some patients and is best done in consultation with

1	for a week before the gastrin determination. Withdrawal of PPIs should be performed carefully because PUD complications can rapidly develop in some patients and is best done in consultation with GI units with experience in this area. The widespread use of PPIs can confound the diagnosis of ZES by raising a false-positive diagnosis by causing hypergastrinemia in a patient being treated with idiopathic PUD (without ZES) and lead to a false-negative diagnosis because at routine doses used to treat patients with idiopathic PUD, PPIs control symptoms in most ZES patients and thus mask the diagnosis. If ZES is suspected and the gastrin level is elevated, it is important to show that it is increased when gastric pH is ≤2.0 because physiologically hypergastrinemia secondary to achlorhydria (atrophic gastritis, pernicious anemia) is one of the most common causes of hypergastrinemia. Nearly all ZES patients have a fasting pH ≤2 when off antisecretory drugs. If the fasting gastrin is >1000 pg/mL

1	gastritis, pernicious anemia) is one of the most common causes of hypergastrinemia. Nearly all ZES patients have a fasting pH ≤2 when off antisecretory drugs. If the fasting gastrin is >1000 pg/mL (increased tenfold) and the pH is ≤2.0, which occurs in 40–60% of patients with ZES, the diagnosis of ZES is established after the possibility of retained antrum syndrome has been ruled out by history. In patients with hypergastrinemia with fasting gastrins <1000 pg/mL (<10-fold increased) and gastric pH ≤2.0, other conditions, such as H. pylori infections, antral G-cell hyperplasia/hyperfunction, gastric outlet obstruction, and, rarely, renal failure, can masquerade as ZES. To establish the diagnosis in this group, a determination of BAO and a secretin provocative test should be done. In patients with ZES without previous gastric acid–reducing surgery, the BAO is usually (>90%) elevated (i.e., >15 mEq/h). The secretin provocative test is usually positive, with the criterion of a >120-pg/mL

1	with ZES without previous gastric acid–reducing surgery, the BAO is usually (>90%) elevated (i.e., >15 mEq/h). The secretin provocative test is usually positive, with the criterion of a >120-pg/mL increase over the basal level having the highest sensitivity (94%) and specificity (100%). Unfortunately the diagnosis of ZES is becoming increasing more difficult. This is due not only to the widespread use of PPIs (leading to false-positive results as well as masking ZES presentation), but also recent studies demonstrate than many of the commercial gastrin kits that are used by most laboratories to measure fasting serum gastrin levels are not reliable. In one study, 7 of the 12 tested commercial gastrin kits inaccurately assessed the true serum concentration of gastrin primarily because the antibodies used had inappropriate specificity for the different circulating forms of gastrin and were not adequately validated. Both underestimation and overestimation of fasting serum gastrin levels

1	antibodies used had inappropriate specificity for the different circulating forms of gastrin and were not adequately validated. Both underestimation and overestimation of fasting serum gastrin levels occurred using these commercial kits. To circumvent this problem, it is either necessary to use one of the five reliable kits identified or, alternatively, to refer the patient to a center with expertise in making the diagnosis in your area, or if this is not possible, to contact such a center and use the gastrin assay they recommend. An accurate gastrin assay is essential for accurate measurement of fasting serum gastrin level as well as for assessing gastrin levels during the secretin provocative test, and thus, the diagnosis of ZES cannot reliably be made without one.

1	Gastric acid hypersecretion in patients with ZES can be controlled in almost every case by oral gastric antisecretory drugs. Because of their long duration of action and potency, which allows dosing once or twice a day, the PPIs (H+, K+-ATPase inhibitors) are the drugs of choice. Histamine H2-receptor antagonists are also effective, although more frequent dosing (q 4–8 h) and high doses are required. In patients with MEN 1/ZES with hyperparathyroidism, correction of the hyperparathyroidism increases the sensitivity to gastric antisecretory drugs and decreases the basal acid output. Long-term treatment with PPIs (>15 years) has proved to be safe and effective, without development of tachyphylaxis. Although patients with ZES, especially those with MEN 1/ZES, more frequently develop gastric NETs (carcinoids), no data suggest that the long-term use of PPIs increases this risk in these patients. With long-term PPI use in ZES patients, vitamin B12 deficiency can develop; thus, vitamin B12

1	NETs (carcinoids), no data suggest that the long-term use of PPIs increases this risk in these patients. With long-term PPI use in ZES patients, vitamin B12 deficiency can develop; thus, vitamin B12 levels should be assessed during follow-up. Epidemiologic studies suggest that long-term PPI use may be associated with an increased incidence of bone fractures; however, at present, there is no such report in ZES patients.

1	With the increased ability to control acid hypersecretion, more than 50% of patients who are not cured (>60% of patients) will die from tumor-related causes. At presentation, careful imaging studies are essential to localize the extent of the tumor to determine the appropriate treatment. A third of patients present with hepatic metastases, and in <15% of those patients, the disease is limited, so that surgical resection may be possible. Surgical short-term cure is possible in 60% of all patients without MEN 1/ZES or liver metastases (40% of all patients) and in 30% of patients long term. In patients with MEN 1/ZES, long-term surgical cure is rare because the tumors are multiple, frequently with lymph node metastases. Surgical studies demonstrate that successful resection of the gastrinoma not only decreases the chances of developing liver metastases but also increases the disease-related survival rate. Therefore, all patients with gastrinomas without MEN 1/ZES or a medical condition

1	not only decreases the chances of developing liver metastases but also increases the disease-related survival rate. Therefore, all patients with gastrinomas without MEN 1/ZES or a medical condition that limits life expectancy should undergo surgery by a surgeon experienced in the treatment of these disorders.

1	An insulinoma is an NET of the pancreas that is thought to be derived from beta cells that ectopically secrete insulin, which results in hypoglycemia. The average age of occurrence is 40–50 years old. The most common clinical symptoms are due to the effect of the hypoglycemia on the CNS (neuroglycemic symptoms) and include confusion, headache, disorientation, visual difficulties, irrational behavior, and even coma. Also, most patients have symptoms due to excess catecholamine release secondary to the hypoglycemia, including sweating, tremor, and palpitations. Characteristically, these attacks are associated with fasting. Insulinomas are generally small (>90% are <2 cm) and usually not multiple (90%); only 5–15% are malignant, and they almost invariably occur only in the pancreas, distributed equally in the pancreatic head, body, and tail.

1	Insulinomas should be suspected in all patients with hypoglycemia, especially when there is a history suggesting that attacks are provoked by fasting, or with a family history of MEN 1. Insulin is synthesized as pro-insulin, which consists of a 21-amino-acid α chain and a 30-amino-acid β chain connected by a 33-amino-acid connecting peptide (C peptide). In insulinomas, in addition to elevated plasma insulin levels, elevated plasma proinsulin levels are found, and C-peptide levels are elevated.

1	Diagnosis The diagnosis of insulinoma requires the demonstration of an elevated plasma insulin level at the time of hypoglycemia. A number of other conditions may cause fasting hypoglycemia, such as the inadvertent or surreptitious use of insulin or oral hypoglycemic agents, severe liver disease, alcoholism, poor nutrition, and other extrapancreatic tumors. Furthermore, postprandial hypoglycemia can be caused by a number of conditions that confuse the diagnosis of insulinoma. Particularly important here is the increased occurrence of hypoglycemia after gastric bypass surgery for obesity, which is now widely performed. A new entity, insulinomatosis, was described that can cause hypoglycemia and mimic insulinomas. It occurs in 10% of patients with persistent hyperinsulinemic hypoglycemia and is characterized by the occurrence of multiple macro-/microadenomas expressing insulin, and it is not clear how to distinguish this entity from insulinoma preoperatively. The most reliable test to

1	is characterized by the occurrence of multiple macro-/microadenomas expressing insulin, and it is not clear how to distinguish this entity from insulinoma preoperatively. The most reliable test to diagnose insulinoma is a fast up to 72 h with serum glucose, C-peptide, proinsulin, and insulin measurements every 4–8 h. If at any point the patient becomes symptomatic or glucose levels are persistently below <2.2 mmol/L (40 mg/dL), the test should be terminated, and repeat samples for the above studies should be obtained before glucose is given. Some 70–80% of patients will develop hypoglycemia during the first 24 h, and 98% by 48 h. In nonobese normal subjects, serum insulin levels should decrease to <43 pmol/L (<6 µU/mL) when blood glucose decreases to <2.2 mmol/L (<40 mg/ dL) and the ratio of insulin to glucose is <0.3 (in mg/dL). In addition to having an insulin level >6 µU/mL when blood glucose is <40 mg/ dL, some investigators also require an elevated C-peptide and serum proinsulin

1	of insulin to glucose is <0.3 (in mg/dL). In addition to having an insulin level >6 µU/mL when blood glucose is <40 mg/ dL, some investigators also require an elevated C-peptide and serum proinsulin level, an insulin/glucose ratio >0.3, and a decreased plasma β-hydroxybutyrate level for the diagnosis of insulinomas. Surreptitious use of insulin or hypoglycemic agents may be difficult to distinguish from insulinomas. The combination of proinsulin levels (normal in exogenous insulin/hypoglycemic agent users), C-peptide levels (low in exogenous insulin users), antibodies to insulin (positive in exogenous insulin users), and measurement of sulfonylurea levels in serum or plasma will allow the correct diagnosis to be made. The diagnosis of insulinoma has been complicated by the introduction of specific insulin 569 assays that do not also interact with proinsulin, as do many of the older radioimmunoassays (RIAs), and therefore give lower plasma insulin levels. The increased use of these

1	of specific insulin 569 assays that do not also interact with proinsulin, as do many of the older radioimmunoassays (RIAs), and therefore give lower plasma insulin levels. The increased use of these specific insulin assays has resulted in increased numbers of patients with insulinomas having lower plasma insulin values (<6 µU/mL) than levels proposed to be characteristic of insulinomas by RIA. In these patients, the assessment of proinsulin and C-peptide levels at the time of hypoglycemia is particularly helpful for establishing the correct diagnosis. An elevated proinsulin level when the fasting glucose level is <45 mg/dL is sensitive and specific.

1	Only 5–15% of insulinomas are malignant; therefore, after appropriate imaging (see below), surgery should be performed. In different studies, 75–100% of patients are cured by surgery. Before surgery, the hypoglycemia can be controlled by frequent small meals and the use of diazoxide (150–800 mg/d). Diazoxide is a benzothiadiazide whose hyperglycemic effect is attributed to inhibition of insulin release. Its side effects are sodium retention and GI symptoms such as nausea. Approximately 50–60% of patients respond to diazoxide. Other agents effective in some patients to control the hypoglycemia include verapamil and diphenylhydantoin. Long-acting somatostatin analogues such as octreotide and lanreotide are acutely effective in 40% of patients. However, octreotide must be used with care because it inhibits growth hormone secretion and can alter plasma glucagon levels; therefore, in some patients, it can worsen the hypoglycemia.

1	For the 5–15% of patients with malignant insulinomas, these drugs or somatostatin analogues are used initially. In a small number of patients with insulinomas, some with malignant tumors, mammalian target of rapamycin (mTOR) inhibitors (everolimus, rapamycin) are reported to control the hypoglycemia. If they are not effective, various antitumor treatments such as hepatic arterial embolization, chemoembolization, chemotherapy, and peptide receptor radiotherapy have been used (see below). Insulinomas, which are usually benign (>90%) and intrapancreatic in location, are increasingly resected using a laparoscopic approach, which has lower morbidity rates. This approach requires that the insulinoma be localized on preoperative imaging studies.

1	A glucagonoma is NET of the pancreas that secretes excessive amounts of glucagon, which causes a distinct syndrome characterized by dermatitis, glucose intolerance or diabetes, and weight loss. Glucagonomas principally occur between 45 and 70 years of age. The tumor is clinically heralded by a characteristic dermatitis (migratory necrolytic erythema) (67–90%), accompanied by glucose intolerance (40–90%), weight loss (66–96%), anemia (33–85%), diarrhea (15–29%), and thromboembolism (11–24%). The characteristic rash usually starts as an annular erythema at intertriginous and periorificial sites, especially in the groin or buttock. It subsequently becomes raised, and bullae form; when the bullae rupture, eroded areas form. The lesions can wax and wane. The development of a similar rash in patients receiving glucagon therapy suggests that the rash is a direct effect of the hyperglucagonemia. A characteristic laboratory finding is hypoaminoacidemia, which occurs in 26–100% of patients.

1	Glucagonomas are generally large tumors at diagnosis (5–10 cm). Some 50–80% occur in the pancreatic tail. From 50 to 82% have evidence of metastatic spread at presentation, usually to the liver. Glucagonomas are rarely extrapancreatic and usually occur singly. Two new entities have been described that can also cause hyperglucagonemia and may mimic glucagonomas. Mahvah disease is due to a homozygous P86S mutation of the human glucagon receptor. It is associated with the development of α-cell hyperplasia, hyperglucagonemia, and the development of nonfunctioning pNETs. A second disease called glucagon cell adenomatosis can mimic glucagonoma syndrome clinically and is characterized by the presence of hyperplastic islets staining positive for glucagon instead of a single glucagonoma.

1	Endocrine Tumors of the Gastrointestinal Tract and Pancreas 570 Diagnosis The diagnosis is confirmed by demonstrating an increased plasma glucagon level. Characteristically, plasma glucagon levels exceed 1000 pg/mL (normal is <150 pg/mL) in 90%; 7% are between 500 and 1000 pg/mL, and 3% are <500 pg/mL. A trend toward lower levels at diagnosis has been noted in the last decade. A plasma glucagon level >1000 pg/mL is considered diagnostic of glucagonoma. Other diseases causing increased plasma glucagon levels include cirrhosis, diabetic ketoacidosis, celiac disease, renal insufficiency, acute pancreatitis, hypercorticism, hepatic insufficiency, severe stress, and prolonged fasting or familial hyperglucagonemia, as well as danazol treatment. With the exception of cirrhosis, these disorders do not increase plasma glucagon >500 pg/mL. Necrolytic migratory erythema is not pathognomonic for glucagonoma and occurs in myeloproliferative disorders, hepatitis B infection, malnutrition,

1	do not increase plasma glucagon >500 pg/mL. Necrolytic migratory erythema is not pathognomonic for glucagonoma and occurs in myeloproliferative disorders, hepatitis B infection, malnutrition, short-bowel syndrome, inflammatory bowel disease, zinc deficiency, and malabsorption disorders.

1	In 50–80% of patients, hepatic metastases are present, and so curative surgical resection is not possible. Surgical debulking in patients with advanced disease or other antitumor treatments may be beneficial (see below). Long-acting somatostatin analogues such as octreotide and lanreotide improve the skin rash in 75% of patients and may improve the weight loss, pain, and diarrhea, but usually do not improve the glucose intolerance.

1	The somatostatinoma syndrome is due to an NET that secretes excessive amounts of somatostatin, which causes a distinct syndrome characterized by diabetes mellitus, gallbladder disease, diarrhea, and steatorrhea. There is no general distinction in the literature between a tumor that contains somatostatin-like immunoreactivity (somatostatinoma) and does (11–45%) or does not (55–90%) produce a clinical syndrome (somatostatinoma syndrome) by secreting somatostatin. In a review of 173 cases of somatostatinomas, only 11% were associated with the somatostatinoma syndrome. The mean age is 51 years. Somatostatinomas occur primarily in the pancreas and small intestine, and the frequency of the symptoms and occurrence of the somatostatinoma syndrome differ in each. Each of the usual symptoms is more common in pancreatic than in intestinal somatostatinomas: diabetes mellitus (95% vs 21%), gallbladder disease (94% vs 43%), diarrhea (92% vs 38%), steatorrhea (83% vs 12%), hypochlorhydria (86% vs

1	common in pancreatic than in intestinal somatostatinomas: diabetes mellitus (95% vs 21%), gallbladder disease (94% vs 43%), diarrhea (92% vs 38%), steatorrhea (83% vs 12%), hypochlorhydria (86% vs 12%), and weight loss (90% vs 69%). The somatostatinoma syndrome occurs in 30–90% of pancreatic and 0–5% of SI somatostatinomas. In various series, 43% of all duodenal NETs contain somatostatin; however, the somatostatinoma syndrome is rarely present (<2%). Somatostatinomas occur in the pancreas in 56–74% of cases, with the primary location being the pancreatic head. The tumors are usually solitary (90%) and large (mean size 4.5 cm). Liver metastases are common, being present in 69–84% of patients. Somatostatinomas are rare in patients with MEN 1, occurring in only 0.65%.

1	Somatostatin is a tetradecapeptide that is widely distributed in the CNS and GI tract, where it functions as a neurotransmitter or has paracrine and autocrine actions. It is a potent inhibitor of many processes, including release of almost all hormones, acid secretion, intestinal and pancreatic secretion, and intestinal absorption. Most of the clinical manifestations are directly related to these inhibitory actions. Diagnosis In most cases, somatostatinomas have been found by accident either at the time of cholecystectomy or during endoscopy. The presence of psammoma bodies in a duodenal tumor should particularly raise suspicion. Duodenal somatostatin-containing tumors are increasingly associated with von Recklinghausen’s disease (NF-1) (Table 113-6). Most of these tumors (>98%) do not cause the somatostatinoma syndrome. The diagnosis of the somatostatinoma syndrome requires the demonstration of elevated plasma somatostatin levels.

1	Pancreatic tumors are frequently (70–92%) metastatic at presentation, whereas 30–69% of SI somatostatinomas have metastases. Surgery is the treatment of choice for those without widespread hepatic metastases. Symptoms in patients with the somatostatinoma syndrome are also improved by octreotide treatment. VIPomas are NETs that secrete excessive amounts of vasoactive intestinal peptide (VIP), which causes a distinct syndrome characterized by large-volume diarrhea, hypokalemia, and dehydration. This syndrome also is called Verner-Morrison syndrome, pancreatic cholera, and WDHA syndrome for watery diarrhea, hypokalemia, and achlorhydria, which some patients develop. The mean age of patients with this syndrome is 49 years; however, it can occur in children, and when it does, it is usually caused by a ganglioneuroma or ganglioneuroblastoma.

1	The principal symptoms are large-volume diarrhea (100%) severe enough to cause hypokalemia (80–100%), dehydration (83%), hypochlorhydria (54–76%), and flushing (20%). The diarrhea is secretory in nature, persisting during fasting, and is almost always >1 L/d and in 70% is >3 L/d. In a number of studies, the diarrhea was intermittent initially in up to half the patients. Most patients do not have accompanying steatorrhea (16%), and the increased stool volume is due to increased excretion of sodium and potassium, which, with the anions, accounts for the osmolality of the stool. Patients frequently have hyperglycemia (25–50%) and hypercalcemia (25–50%).

1	VIP is a 28-amino-acid peptide that is an important neurotransmitter, ubiquitously present in the CNS and GI tract. Its known actions include stimulation of SI chloride secretion as well as effects on smooth-muscle contractility, inhibition of acid secretion, and vasodilatory effects, which explain most features of the clinical syndrome. In adults, 80–90% of VIPomas are pancreatic in location, with the rest due to VIP-secreting pheochromocytomas, intestinal carcinoids, and rarely ganglioneuromas. These tumors are usually solitary, 50–75% are in the pancreatic tail, and 37–68% have hepatic metastases at diagnosis. In children <10 years old, the syndrome is usually due to ganglioneuromas or ganglioblastomas and is less often malignant (10%).

1	Diagnosis The diagnosis requires the demonstration of an elevated plasma VIP level and the presence of large-volume diarrhea. A stool volume <700 mL/d is proposed to exclude the diagnosis of VIPoma. When the patient fasts, a number of diseases can be excluded that can cause marked diarrhea because the high volume of diarrhea is not sustained during the fast. Other diseases that can produce a secretory large-volume diarrhea include gastrinomas, chronic laxative abuse, carcinoid syndrome, systemic mastocytosis, rarely medullary thyroid cancer, diabetic diarrhea, sprue, and AIDS. Among these conditions, only VIPomas caused a marked increase in plasma VIP. Chronic surreptitious use of laxatives/diuretics can be particularly difficult to detect clinically. Hence, in a patient with unexplained chronic diarrhea, screens for laxatives should be performed; they will detect many, but not all, laxative abusers. Elevated plasma levels of VIP should not be the only basis of the diagnosis of

1	chronic diarrhea, screens for laxatives should be performed; they will detect many, but not all, laxative abusers. Elevated plasma levels of VIP should not be the only basis of the diagnosis of VIPomas because they can occur with some diarrheal states including inflammatory bowel disease, post small bowel resection, and radiation enteritis. Furthermore, nesidioblastosis can mimic VIPomas by causing elevated plasma VIP levels, diarrhea, and even false-positive location in the pancreatic region on somatostatin receptor scintigraphy.

1	The most important initial treatment in these patients is to correct their dehydration, hypokalemia, and electrolyte losses with fluid and electrolyte replacement. These patients may require 5 L/d of fluid and >350 mEq/d of potassium. Because 37–68% of adults with VIPomas have metastatic disease in the liver at presentation, a significant number of patients cannot be cured surgically. In these patients, long-acting somatostatin analogues such as octreotide and lanreotide are the drugs of choice.

1	Octreotide/lanreotide will control the diarrhea shortand longterm in 75–100% of patients. In nonresponsive patients, the combination of glucocorticoids and octreotide/lanreotide has proved helpful in a small number of patients. Other drugs reported to be helpful in small numbers of patients include prednisone (60– 100 mg/d), clonidine, indomethacin, phenothiazines, loperamide, lidamidine, lithium, propranolol, and metoclopramide. Treatment of advanced disease with cytoreductive surgery, embolization, chemoembolization, chemotherapy, radiotherapy, radiofrequency ablation, and peptide receptor radiotherapy may be helpful (see below).

1	NF-pNETs are NETs that originate in the pancreas and either secrete no products or their products do not cause a specific clinical syndrome. Their symptoms are due entirely to the tumor per se. NF-pNETs secrete chromogranin A (90–100%), chromogranin B (90–100%), α-HCG (human chorionic gonadotropin) (40%), neuron-specific enolase (31%), and β-HCG (20%), and because 40–90% secrete PP, they are also often called PPomas. Because the symptoms are due to the tumor mass, patients with NF-pNETs usually present late in the disease course with invasive tumors and hepatic metastases (64–92%), and the tumors are usually large (72% >5 cm). NF-pNETs are usually solitary except in patients with MEN 1, in which case they are multiple. They occur primarily in the pancreatic head. Even though these tumors do not cause a functional syndrome, immunocytochemical studies show that they synthesize numerous peptides and cannot be distinguished from functional pNETs by immunocytochemistry. In MEN 1, 80–100%

1	do not cause a functional syndrome, immunocytochemical studies show that they synthesize numerous peptides and cannot be distinguished from functional pNETs by immunocytochemistry. In MEN 1, 80–100% of patients have microscopic NF-pNETs, but they become large or symptomatic in a minority (0–13%) of cases. In VHL, 12–17% develop NF-pNETs, and in 4%, they are ≥3 cm in diameter.

1	The most common symptoms are abdominal pain (30–80%), jaundice (20–35%), and weight loss, fatigue, or bleeding; 10–35% are found incidentally. The average time from the beginning of symptoms to diagnosis is 5 years.

1	Diagnosis The diagnosis is established by histologic confirmation in a patient without either the clinical symptoms or the elevated plasma hormone levels of one of the established syndromes. The principal difficulty in diagnosis is to distinguish an NF-pNET from a nonendocrine pancreatic tumor, which is more common, as well as from a functional pNET. Even though chromogranin A levels are elevated in almost every patient, this is not specific for this disease as it can be found in functional pNETs, GI-NETs (carcinoids), and other neuroendocrine disorders. Plasma PP elevations should strongly suggest the diagnosis in a patient with a pancreatic mass because it is usually normal in patients with pancreatic adenocarcinomas. Elevated plasma PP is not diagnostic of this tumor because it is elevated in a number of other conditions, such as chronic renal failure, old age, inflammatory conditions, alcohol abuse, pancreatitis, hypoglycemia, postprandially, and diabetes. A positive somatostatin

1	in a number of other conditions, such as chronic renal failure, old age, inflammatory conditions, alcohol abuse, pancreatitis, hypoglycemia, postprandially, and diabetes. A positive somatostatin receptor scan in a patient with a pancreatic mass should suggest the presence of pNET/NF-pNET rather than a nonendocrine tumor.

1	Overall survival in patients with sporadic NF-pNET is 30–63% at 5 years, with a median survival of 6 years. Unfortunately, surgical curative resection can be considered only in a minority of these patients because 64–92% present with diffuse metastatic disease. Treatment needs to be directed against the tumor per se using the various modalities discussed below for advanced disease. The treatment of NF-pNETs in either MEN 1 patients or patients with VHL is controversial. Most recommend surgical resection for any tumor >2–3 cm in diameter; however, there is no consensus on smaller 571 NF-pNETs in these inherited disorders, with most recommending careful surveillance of these patients. The treatment of small sporadic, asymptomatic NF-pNETs (≤2 cm) is also controversial. Most of these are low-or intermediate-grade lesions, and <7% are malignant. Some advocate a nonoperative approach with careful, regular follow-up, whereas other recommend an operative approach with specially consideration

1	intermediate-grade lesions, and <7% are malignant. Some advocate a nonoperative approach with careful, regular follow-up, whereas other recommend an operative approach with specially consideration for a laparoscopic surgical approach.

1	GRFomas are NETs that secrete excessive amounts of growth hormone– releasing factor (GRF) that cause acromegaly. GRF is a 44-amino-acid peptide, and 25–44% of pNETs have GRF immunoreactivity, although it is uncommonly secreted. GRFomas are lung tumors in 47–54% of cases, pNETs in 29–30%, and SI carcinoids in 8–10%; up to 12% occur at other sites. Patients have a mean age of 38 years, and the symptoms usually are due to either acromegaly or the tumor per se. The acromegaly caused by GRFomas is indistinguishable from classic acromegaly. The pancreatic tumors are usually large (>6 cm), and liver metastases are present in 39%. They should be suspected in any patient with acromegaly and an abdominal tumor, a patient with MEN 1 with acromegaly, or a patient without a pituitary adenoma with acromegaly or associated with hyperprolactinemia, which occurs in 70% of GRFomas. GRFomas are an uncommon cause of acromegaly. GRFomas occur in <1% of MEN 1 patients. The diagnosis is established by

1	acromegaly or associated with hyperprolactinemia, which occurs in 70% of GRFomas. GRFomas are an uncommon cause of acromegaly. GRFomas occur in <1% of MEN 1 patients. The diagnosis is established by performing plasma assays for GRF and growth hormone. Most GRFomas have a plasma GRF level >300 pg/mL (normal <5 pg/mL men, <10 pg/mL women). Patients with GRFomas also have increased plasma levels of insulin-like growth factor type I (IGF-I) similar to those in classic acromegaly. Surgery is the treatment of choice if diffuse metastases are not present. Long-acting somatostatin analogues such as octreotide and lanreotide are the agents of choice, with 75–100% of patients responding.

1	Cushing’s syndrome (ACTHoma) due to a pNET occurs in 4–16% of all ectopic Cushing’s syndrome cases. It occurs in 5% of cases of sporadic gastrinomas, almost invariably in patients with hepatic metastases, and is an independent poor prognostic factor. Paraneoplastic hypercalcemia due to pNETs releasing parathyroid hormone–related peptide (PTHrP), a PTH-like material, or unknown factor, is rarely reported. The tumors are usually large, and liver metastases are usually present. Most (88%) appear to be due to release of PTHrP. pNETs occasionally can cause the carcinoid syndrome. A number of very rare pNET syndromes involving a few cases (less than five) have been described; these include a reninproducing pNET in a patient presenting with hypertension; pNETs secreting luteinizing hormone, resulting in masculinization or decreased libido; a pNET secreting erythropoietin, resulting in polycythemia; pNETs secreting IGF-II, causing hypoglycemia; and pNETs secreting enteroglucagon, causing

1	resulting in masculinization or decreased libido; a pNET secreting erythropoietin, resulting in polycythemia; pNETs secreting IGF-II, causing hypoglycemia; and pNETs secreting enteroglucagon, causing small intestinal hypertrophy, colonic/SI stasis, and malabsorption (Table 113-2). A number of other possible functional pNETs have been proposed, but most authorities classify these as unclear or as a nonfunctional pNET because in each case numerous patients have been described with similar plasma hormone elevations that do not cause any symptoms. These include pNETs secreting calcitonin, neurotensin (neurotensinoma), PP (PPoma), and ghrelin (Table 113-2).

1	Localization of the primary tumor and knowledge of the extent of the disease are essential to the proper management of all GI-NETs (carcinoids) and pNETs. Without proper localization studies, it is not possible to determine whether the patient is a candidate for surgical resection (curative or cytoreductive) or requires antitumor treatment, to determine whether the patient is responding to antitumor therapies, or to appropriately classify/stage the patient’s disease to assess prognosis. Numerous tumor localization methods are used in both types of NETs, including cross-sectional imaging studies (CT, magnetic resonance imaging [MRI], transabdominal ultrasound), selective angiography, somatostatin receptor scintigraphy (SRS), and positron emission

1	Endocrine Tumors of the Gastrointestinal Tract and Pancreas 572 tomography. In pNETs, endoscopic ultrasound (EUS) and functional localization by measuring venous hormonal gradients are also reported to be useful. Bronchial carcinoids are usually detected by standard chest radiography and assessed by CT. Rectal, duodenal, colonic, and gastric carcinoids are usually detected by GI endoscopy. Because of their wide availability, CT and MRI are generally initially used to determine the location of the primary NETs and the extent of disease. NETs are hyper-vascular tumors, and with both MRI and CT, contrast enhancement is essential for maximal sensitivity, and it is recommended that generally triple-phase scanning be used. The ability of cross-sectional imaging and, to a lesser extent, SRS to detect NETs is a function of NET size. With CT and MRI, <10% of tumors <1 cm in diameter are detected, 30–40% of tumors 1–3 cm are detected, and >50% of tumors >3 cm are detected. Many primary GI-NETs

1	NETs is a function of NET size. With CT and MRI, <10% of tumors <1 cm in diameter are detected, 30–40% of tumors 1–3 cm are detected, and >50% of tumors >3 cm are detected. Many primary GI-NETs (carcinoids) are small, as are insulinomas and duodenal gastrinomas, and are frequently not detected by cross-sectional imaging, whereas most other pNETs present late in the course of their disease and are large (>4 cm). Selective angiography is more sensitive, localizing 60–90% of all NETs; however, it is now used infrequently. For detecting liver metastases, CT and MRI are more sensitive than ultrasound, and with recent improvements, 5–25% of patients with liver metastases will be missed by CT and/or MRI. pNETs, as well as GI-NETs (carcinoids), frequently (>80%) overexpress high-affinity somatostatin receptors in both the primary tumors and the metastases. Of the five types of somatostatin receptors (sst1–5), radiolabeled octreotide binds with high affinity to sst2 and sst5, has a lower

1	receptors in both the primary tumors and the metastases. Of the five types of somatostatin receptors (sst1–5), radiolabeled octreotide binds with high affinity to sst2 and sst5, has a lower affinity for sst3, and has a very low affinity for sst1 and sst4. Between 80 and 100% of GI-NETs (carcinoids) and pNETs possess sst2, and many also have the other four sst subtypes. Interaction with these receptors can be used to treat these tumors as well as to localize NETs by using radiolabeled somatostatin analogues (SRS). In the United States, [111In-DTPA-d-Phe1]octreotide (octreoscan) is generally used with gamma camera detection using single-photon emission computed tomography (SPECT) imaging. Numerous studies, primarily in Europe, using gallium-68-labeled somatostatin analogues and positron emission tomography (PET) detection, demonstrate even greater sensitivity than with SRS with 111In-labeled somatostatin analogues. Although not yet approved in the United States, there are a number of

1	tomography (PET) detection, demonstrate even greater sensitivity than with SRS with 111In-labeled somatostatin analogues. Although not yet approved in the United States, there are a number of centers starting to use this approach. Because of its sensitivity and ability to localize tumor throughout the body, SRS is the initial imaging modality of choice for localizing both the primary tumor and metastatic NETs. SRS localizes tumor in 73–95% of patients with GI-NETs (carcinoids) and in 56–100% of patients with pNETs, except insulinomas. Insulinomas are usually small and have low densities of sst receptors, resulting in SRS being positive in only 12–50% of patients with insulinomas. SRS identifies >90–95% of patients with liver metastases due to NETs. Figure 113-3 shows an example of the increased sensitivity of SRS in a patient with a GI-NET (carcinoid) tumor. The CT scan showed a single liver metastasis, whereas the SRS demonstrated three metastases in the liver in multiple locations.

1	sensitivity of SRS in a patient with a GI-NET (carcinoid) tumor. The CT scan showed a single liver metastasis, whereas the SRS demonstrated three metastases in the liver in multiple locations. Occasional false-positive responses with SRS can occur (12% in one study) because numerous other normal tissues as well as diseases can have high densities of sst receptors, including granulomas (sarcoid, tuberculosis, etc.), thyroid diseases (goiter, thyroiditis), and activated lymphocytes (lymphomas, wound infections). If liver metastases are identified by SRS, to plan the proper treatment, either a CT or an MRI (with contrast enhancement) is recommended to assess the size and exact location of the metastases because SRS does not provide information on tumor size. For pNETs in the pancreas, EUS is highly sensitive, localizing 77–100% of insulinomas, which occur almost exclusively within the pancreas. Endoscopic ultrasound is less sensitive for extrapancreatic tumors. It is increasingly used in

1	highly sensitive, localizing 77–100% of insulinomas, which occur almost exclusively within the pancreas. Endoscopic ultrasound is less sensitive for extrapancreatic tumors. It is increasingly used in patients with MEN 1, and to a lesser extent VHL, to detect small pNETs not seen with other modalities or for serial pNET assessments to determine size changes or rapid growth in patients in whom surgery is deferred. EUS with cytologic evaluation also is used frequently to distinguish an NF-pNET from a pancreatic adenocarcinoma or another nonendocrine pancreatic tumor. Not infrequently patients present with liver metastases due to an NET and the primary site is unclear. Occult small intestinal NETs (carcinoids) are increasingly detected by double-balloon enteroscopy or capsule endoscopy.

1	FIGURE 113-3 Ability of computed tomography (CT) scanning (top) or somatostatin receptor scintigraphy (SRS) (bottom) to localize metastatic carcinoid in the liver.

1	Insulinomas frequently overexpress receptors for glucagon-like peptide-1 (GLP-1), and radiolabeled GLP-1 analogues have been developed that can detect occult insulinomas not localized by other imaging modalities. Functional localization by measuring hormonal gradients is now uncommonly used with gastrinomas (after intra-arterial secretin injections) but is still frequently used in insulinoma patients in whom other imaging studies are negative (assessing hepatic vein insulin concentrations post-intra-arterial calcium injections). Functional localization measuring hormone gradients in insulinomas or gastrin gradients in gastrinoma is a sensitive method, being positive in 80–100% of patients. The intra-arterial calcium test may also allow differentiation of the cause of the hypoglycemia and indicate whether it is due to an insulinoma or a nesidioblastosis. The latter entity is becoming increasingly important because hypoglycemia after gastric bypass surgery for obesity is increasing in

1	indicate whether it is due to an insulinoma or a nesidioblastosis. The latter entity is becoming increasingly important because hypoglycemia after gastric bypass surgery for obesity is increasing in frequency, and it is primarily due to nesidioblastosis, although it can occasionally be due to an insulinoma.

1	PET and use of hybrid scanners such as CT and SRS may have increased sensitivity. PET scanning with 18F-fluoro-DOPA in patients with carcinoids or with 11C-5-HTP in patients with pNETs or GI-NETs (carcinoids) has greater sensitivity than cross-sectional imaging studies and may be used increasingly in the future. PET scanning for GI-NETs is not currently approved in the United States. The single most important prognostic factor for survival is the presence of liver metastases (Fig. 113-4). For patients with foregut carcinoids without hepatic metastases, the 5-year survival in one A.ENETS Stage D. Appendiceal NETs (Carcinoids) ---Stage 3–4 ENETS pT classification pT1–2 vs pT3–4: p = 0.0004 Probability of survival .75 .5 .25 B.UICC/AJCC/WHO2010 Stage E. Appendiceal NETs (Carcinoids) Endocrine Tumors of the Gastrointestinal Tract and Pancreas WHO/AJCC pT classification pT1–2 vs pT3–4: p <0.0001 C.ENETS/WHO Grade F.Midgut NETs (Carcinoids)

1	Endocrine Tumors of the Gastrointestinal Tract and Pancreas WHO/AJCC pT classification pT1–2 vs pT3–4: p <0.0001 C.ENETS/WHO Grade F.Midgut NETs (Carcinoids) Probability of survival .75 .5 .25 .75 .5 .25

1	FIGURE 113-4 Survival (Kaplan-Meier plots) of patients with pancreatic neuroendocrine tumors (pNETs; n = 1072) (A–C) or gastrointestinal neuroendocrine tumors (GI-NETs; carcinoids) (appendix, n = 138; midgut, n = 238) (D–F) stratified according to recent proposed classification and grading systems. (Panels A–C are drawn from data in G Rindi et al: J Natl Cancer Inst 104:764, 2012; panels D and E are drawn from data in M Volante et al: Am J Surg Pathol 37:606, 2013; and panel F is drawn from data in MS Khan: Br J Cancer 108:1838, 2013.) study was 95%, and with distant metastases, it was 20% (Fig. 113-4). With gastrinomas, the 5-year survival without liver metastases is 98%; with limited metastases in one hepatic lobe, it is 78%; and with diffuse metastases, 16% (Fig. 113-4). In a large study of 156 patients (67 pNETs, rest carcinoids), the overall 5-year survival rate was 77%; it was 96% without liver metastases, 73% with liver metastases, and 50% with distant disease. Another very

1	study of 156 patients (67 pNETs, rest carcinoids), the overall 5-year survival rate was 77%; it was 96% without liver metastases, 73% with liver metastases, and 50% with distant disease. Another very important prognostic factor is whether the NET is well-differentiated (G1/G2) or poorly differentiated (<1% of all NETs) (G3). Well-differentiated NETs have a 5-year survival of 50–80%, whereas poorly differentiated NETs have a 5-year survival of only 0–15%.

1	Therefore, treatment for advanced metastatic disease is an important challenge. A number of different modalities are reported to be effective, including cytoreductive surgery (surgically or by radiofrequency ablation [RFA]), treatment with chemotherapy, somatostatin analogues, interferon α, hepatic embolization alone or with chemotherapy (chemoembolization), molecular targeted therapy, radiotherapy with radiolabeled beads/microspheres, peptide radioreceptor therapy (PRRT), and liver transplantation.

1	Cytoreductive surgery is considered if either all of the visible metastatic disease or at last 90% is thought resectable; however, unfortunately, this is possible in only the 9–22% of patients who present with limited hepatic metastases. Although no randomized studies have proven that it extends life, results from a number of studies suggest that it may increase survival; therefore, it is recommended, if possible. RFA can be applied to NET liver metastases if they are 574 limited in number (usually less than five) and size (usually <3.5 cm in diameter). It can be used at the time of surgery (either general or laparoscopic) or using radiologic guidance. Response rates are >80%, the responses can last up to 3 years, the morbidity rate is low, and this procedure may be particularly helpful in patients with functional pNETs that are difficult to control medically. Although RFA has not been established in a controlled trial, both the European and North American Neuroendocrine Tumor Society

1	patients with functional pNETs that are difficult to control medically. Although RFA has not been established in a controlled trial, both the European and North American Neuroendocrine Tumor Society guidelines (ENETS, NANETS) state it can be an effective antitumor treatment for both refractory functional syndromes and for palliative treatment. Chemotherapy plays a different role in the treatment of patients with pNETs and GI-NETs (carcinoids). Chemotherapy continues to be widely used in the treatment of patients with advanced pNETs with moderate success (response rates 20–70%); however, in general, its results in patients with metastatic GI-NETs (carcinoids) has been disappointing, with response rates of 0–30% with various twoand three-drug combinations, and thus, it is infrequently used in these patients. An important distinction in patients with pNETs is whether the tumor is well differentiated (G1/G2) or poorly differentiated (G3). The chemotherapeutic approach is different for

1	in these patients. An important distinction in patients with pNETs is whether the tumor is well differentiated (G1/G2) or poorly differentiated (G3). The chemotherapeutic approach is different for these two groups. The current regimen of choice for patients with well-differentiated pNETs is the combination of streptozotocin and doxorubicin with or without 5-fluorouracil. Streptozotocin is a glucosamine nitrourea compound originally found to have cytotoxic effects on pancreatic islets, and later in studies with doxorubicin with or without 5-fluorouracil, it produced response rates of 20–45% in advanced pNETs. Streptozotocin causes considerable morbidity, with 70–100% of patients developing side effects (most prominent being nausea/vomiting in 60–100% or leukopenia/thrombocytopenia) and 15–40% of patients developing some degree of renal dysfunction (proteinuria in 40–50%, decreased creatine clearance). The combination of temozolomide (TMZ) with capecitabine produces partial response

1	15–40% of patients developing some degree of renal dysfunction (proteinuria in 40–50%, decreased creatine clearance). The combination of temozolomide (TMZ) with capecitabine produces partial response rates as high as 70% in patients with advance pNETs and a 2-year survival of 92%. The use of TMZ or another alkylating agent in advanced pNETs is supported by studies that show low levels of the DNA repair enzyme O6-methylguanine DNA methyltransferase in pNETs, but not in GI-NETs (carcinoids), which increases the sensitivity of pNETs to TMZ. In poorly differentiated NETs (G3), chemotherapy with a cisplatin-based regimen with etoposide or other agents (vincristine, paclitaxel) is the recommended treatment, with response rates of 40–70%; however, responses are generally short-lived (<12 months). This chemotherapy regimen can be associated with significant toxicity including GI toxicities (nausea, vomiting), myelosuppression, and renal toxicity. In addition to the effectiveness in

1	months). This chemotherapy regimen can be associated with significant toxicity including GI toxicities (nausea, vomiting), myelosuppression, and renal toxicity. In addition to the effectiveness in controlling the functional hormonal state, long-acting somatostatin analogues such as octreotide and lanreotide are increasingly used for their antiproliferative effects. Whereas somatostatin analogues rarely decrease tumor size (i.e., 0–17%), these drugs have tumoristatic effects, stopping additional growth in 26–95% of patients with NETs. In a randomized, double-blind study in patients with metastatic midgut carcinoids (PROMID study) octreotide-LAR demonstrated a marked lengthening of time to progression (14.3 vs 6 months, p = .000072). This improvement was seen in patients with limited liver involvement. This study did not assess whether such treatment will extend survival. A double-blind, randomized, placebo-controlled, phase III study in patients with well-differentiated, metastatic,

1	involvement. This study did not assess whether such treatment will extend survival. A double-blind, randomized, placebo-controlled, phase III study in patients with well-differentiated, metastatic, inoperable pNETs (45%) or GI-NETs (carcinoids) (55%) (CLARINET study) showed that monthly treatment with lanreotide-autogel reduced tumor progression or death by 53%. Somatostatin analogues can induce apoptosis in GI-NETs (carcinoids), which probably contributes to their tumoristatic effects. Treatment with somatostatin analogues is generally well-tolerated, with most side effects being mild and uncommonly leading to stopping the drug. Potential longterm side effects include diabetes/glucose intolerance, steatorrhea, and the development of gallbladder sludge/gallstones (10–80%), although only 1% of patients develop symptomatic gallbladder disease. Because of these phase III studies, somatostatin analogues are generally recommended as first-line treatment for patients with

1	although only 1% of patients develop symptomatic gallbladder disease. Because of these phase III studies, somatostatin analogues are generally recommended as first-line treatment for patients with well-differentiated metastatic NETs.

1	Interferon α, similar to somatostatin analogues, is effective at controlling the hormonal excess symptoms of NETs and has antiproliferative effects in NETs, which primarily result in disease stabilization (30–80%), with a decrease in tumor size in <15% of patients. Interferon can inhibit DNA synthesis, block cell cycle progression in the G1 phase, inhibit protein synthesis, inhibit angiogenesis, and induce apoptosis. Interferon α treatment results in side effects in the majority of patients, with the most frequent being a flu-like syndrome (80–100%), anorexia with weight loss, and fatigue. These side effects frequently decrease in severity with continued treatment. In addition, patients become accommodated to the symptoms. More serious side effects include hepatotoxicity (31%), hyperlipidemia (31%), bone marrow toxicity, thyroid disease (19%), and rarely CNS side effects (depression, mental/visual disorders). ENETS 2012 guidelines conclude that in patients with well-differentiated

1	(31%), bone marrow toxicity, thyroid disease (19%), and rarely CNS side effects (depression, mental/visual disorders). ENETS 2012 guidelines conclude that in patients with well-differentiated NETs that are slowly progressive, interferon α treatment should be considered if the tumor is somatostatin receptor negative or if somatostatin treatment fails.

1	Selective internal radiation therapy (SIRT) using yttrium-90 (90Y) glass or resin microspheres is a relatively newer approach being evaluated in patients with unresectable NET liver metastases, with approximately 500 NET patients treated. The treatment requires careful evaluation for vascular shunting before treatment and a pretreatment angiogram to evaluate placement of the catheter and is generally is reserved for patients without extrahepatic metastatic disease and with adequate hepatic reserve. One of two types of 90Y microspheres are used: either microspheres with a 20to 60-µm diameter and 50 Bq/sphere (SIR-Spheres) or glass microspheres (TheraSpheres) with a 20to 30-µm diameter and 2500 Bq/sphere. The 90Y-microspheres are delivered to the liver by intra-arterial injection from percutaneously placed catheters. In four studies involving metastatic NETs, the response rate varied from 50–61% (partial or complete), tumor stabilization occurred in 22–41%, 60–100% had symptomatic

1	percutaneously placed catheters. In four studies involving metastatic NETs, the response rate varied from 50–61% (partial or complete), tumor stabilization occurred in 22–41%, 60–100% had symptomatic improvement, and overall survival varied from 25–70 months. Side effects include postembolization syndrome (pain, fever, nausea/vomiting [frequent]), which is usually mild, although grade 2 (43%) or grade 3 (1%) symptoms can occur; radiation-induced liver disease (<1%); and radiation pneumonitis (<1%). Contraindications to use include excess shunting to the GI tract or lung, inability to isolate the liver arterial supply, and inadequate liver reserve. Because of the limited data available in the ENETS 2012 guidelines, treatment with SIRTs is considered experimental.

1	Molecular targeted medical treatment with either an mTOR inhibitor (everolimus) or a tyrosine kinase inhibitor (sunitinib) is now approved treatment in the United States and Europe for patients with metastatic unresectable pNET, each supported by a phase III, double-blind, prospective, placebo-controlled trial. mTOR is a serine-threonine kinase that plays an important role in proliferation, cell growth, and apoptosis in both normal and neoplastic cells. Activation of the mTOR cascade is important in mediating NET cell growth, especially in pNETs. A number of mTOR inhibitors have shown promising antitumor activity in NETs including everolimus and temsirolimus, with the former undergoing a phase III trial (RADIANT-3) involving 410 patients with advance progressive pNETs. Everolimus caused significant improvement in progression-free survival (11 vs 4.6 months, p <.001) and increased by a factor of 3.7 the proportion of patients progression-free at 18 months (37% vs 9%). Everolimus

1	significant improvement in progression-free survival (11 vs 4.6 months, p <.001) and increased by a factor of 3.7 the proportion of patients progression-free at 18 months (37% vs 9%). Everolimus treatment was associated with frequent side effects, causing a twofold increase in adverse events, with the most frequent being grade 1 or 2. Grade 3 or 4 side effects included hematologic, GI (diarrhea), stomatitis, or hypoglycemia occurring in 3–7% of patients. Most grade 3 or 4 side effects were controlled by dose reduction or drug interruption. The ENETS 2012 guidelines conclude that everolimus, similar to sunitinib (below), should be considered as a first-line treatment in selected cases of well-differentiated pNETs that are unresectable. NETs, like other normal and neoplastic cells, frequently possess multiple types of the 20 different tyrosine kinase (TK) receptors that are known and mediate the action of different growth factors. Numerous studies demonstrate that TK receptors in normal

1	possess multiple types of the 20 different tyrosine kinase (TK) receptors that are known and mediate the action of different growth factors. Numerous studies demonstrate that TK receptors in normal and neoplastic tissues as well as NETs are especially important in mediating cell growth, angiogenesis, differentiation, and apoptosis. Whereas a number of TK inhibitors show antiproliferative activity in NETs only sunitinib has undergone a phase III controlled trial. Sunitinib is an orally active small-molecule inhibitor of TK receptors (PDGFRs, VEGFR-1, VEGFR-2, c-KIT, FLT-3). In a phase III study in which 171 patients with progressive, metastatic, nonresectable pNETs were treated with sunitinib (37.5 mg/d) or placebo, sunitinib treatment caused a doubling of progression-free survival (11.4 vs 4.5 months, p <.001), an increase in objective tumor response rate (9% vs 0%, p = .007), and an increase in overall survival. Sunitinib treatment was associated with an overall threefold increase in

1	4.5 months, p <.001), an increase in objective tumor response rate (9% vs 0%, p = .007), and an increase in overall survival. Sunitinib treatment was associated with an overall threefold increase in side effects, although most were grade 1 or 2. The most frequent grade 3 or 4 side effects were neutropenia (12%) and hypertension (9.6%), which were controlled by dose reduction or temporary interruption. There is no consensus regarding the order of sunitinib or everolimus use in patients with advanced, well-differentiated, progressive pNETs.

1	PRRT for NETs involves treatment with radiolabeled somatostatin analogues. The success of this approach is based on the finding that somatostatin receptors (sst) are overexpressed or ectopically expressed by 60–100% of all NETs, which allows the targeting of cytotoxic, radiolabeled somatostatin receptor ligands.

1	Three different radionuclides are being used. High doses of [111In-DTPA-D-Phe1]octreotide, which emits γ-rays, internal conversion, and Auger electrons; 90yttrium, which emits high-energy β-particles coupled by a DOTA chelating group to octreotide or octreotate; and 177lutetium-coupled analogues, which emit both, are all in clinical studies. At present, the 177lutetium-coupled analogues are the most widely used. 111Indium-, 90yttrium-, and 177lutetiumlabeled compounds caused tumor stabilization in 41–81%, 44–88%, and 23–40%, respectively, and a decrease in tumor size in 8–30%, 6–37%, and 38%, respectively, of patients with advanced metastatic NETs. In one large study involving 504 patients with malignant NETs, 177lutetium-labeled analogues produced a reduction of tumor size of >50% in 30% of patients (2% complete) and tumor stabilization in 51% of patients. An effect on survival has not been established. At present, PRRT is not approved for use in either the United States or Europe,

1	of patients (2% complete) and tumor stabilization in 51% of patients. An effect on survival has not been established. At present, PRRT is not approved for use in either the United States or Europe, but because of the above promising results, a large phase III study is now being conducted in both the United States and Europe. The ENETS 2012, NANETS 2010, Nordic 2010, and European Society for Medical Oncology (ESMO) guidelines list PRRT as an experimental or investigational treatment at present.

1	The use of liver transplantation has been abandoned for treatment of most metastatic tumors to the liver. However, for metastatic NETs, it is still a consideration. Among 213 European patients with NETs (50% functional NETs) who had liver transplantation from 1982 to 2009, the overall 5-year survival was 52% and disease free-survival was 30%. In various studies, the postoperative mortality rate is 10–14%. These results are similar to the United Network for Organ Sharing data in the United States in which 150 NET patients had liver transplants and the 5-year survival was 49%. In various studies, important prognostic factors for a poor outcome include a major resection performed in addition at the time of the liver transplant; poor tumor differentiation; hepatomegaly; age >45 years; a primary NET in the duodenum or pancreas; the presence of extrahepatic metastatic disease or extensive liver involvement (>50%); Ki-67 proliferative index >10%; and abnormal E-cadherin staining. The ENETS

1	NET in the duodenum or pancreas; the presence of extrahepatic metastatic disease or extensive liver involvement (>50%); Ki-67 proliferative index >10%; and abnormal E-cadherin staining. The ENETS 2012 guidelines conclude that liver transplantation should be viewed as providing palliative care, with cure an exception, and recommend it be reserved for patients with life-threatening hormonal disturbances refractory to other treatments or for selected patients with a nonfunctional tumor with diffuse liver metastatic disease refractory to all other treatments.

1	Howard I. Scher, Jonathan E. Rosenberg, Robert J. Motzer Transitional cell epithelium lines the urinary tract from the renal pelvis to the ureter, urinary bladder, and the proximal two-thirds of the urethra. Cancers can occur at any point: 90% of malignancies develop in the bladder, 8% in the renal pelvis, and 2% in the ureter or urethra. Bladder cancer is the fourth most common cancer in men and the thirteenth in women, with an estimated 72,570 new cases and 15,210 deaths in the United States predicted for the year 2013. The almost 5:1 ratio of incidence to mortality reflects the higher frequency of the less lethal superficial variants compared to the more lethal invasive and metastatic variants. The incidence is roughly four times higher in men than in women and twofold higher in white men than in black men, with a median age of 65 years.

1	Once diagnosed, urothelial tumors exhibit polychronotropism, which is the tendency to recur over time in new locations in the urothelial tract. As long as urothelium is present, continuous monitoring is required. Cigarette smoking is believed to contribute to up to 50% of urothelial cancers in men and nearly 40% in women. The risk of developing a urothelial cancer in male smokers is increased twoto fourfold relative to nonsmokers and continues for 10 years or longer after cessation. Other implicated agents include aniline dyes, the drugs phenacetin and chlornaphazine, and external beam radiation. Chronic cyclophosphamide exposure also increases risk, whereas vitamin A supplements appear to be protective. Exposure to Schistosoma haematobium, a parasite found in many developing countries, is associated with an increase in both squamous and transitional cell carcinomas of the bladder.

1	Clinical subtypes are grouped into three categories: 75% are superficial, 20% invade muscle, and 5% are metastatic at presentation. Staging of the tumor within the bladder is based on the pattern of growth and depth of invasion. The revised tumor, node, metastasis (TNM) staging system is illustrated in Fig. 114-1. About half of invasive tumors presented originally as superficial lesions that later progressed. Tumors are also rated by grade. Low-grade (highly differentiated) tumors rarely progress to a higher stage, whereas high-grade tumors do.

1	More than 95% of urothelial tumors in the United States are transitional cell in origin. Pure squamous cancers with keratinization constitute 3%, adenocarcinomas 2%, and small cell tumors (often with paraneoplastic syndromes) <1%. Adenocarcinomas develop primarily in the urachal remnant in the dome of the bladder or in the periurethral tissues. Paragangliomas, lymphomas, and melanomas are rare. Of the transitional cell tumors, low-grade papillary lesions that grow on a central stalk are most common. These tumors are very friable, have a tendency to bleed, and have a high risk for recurrence, yet they rarely progress to the more lethal invasive variety. In contrast, carcinoma in situ (CIS) is a high-grade tumor that is considered a precursor of the more lethal muscle-invasive disease.

1	The multicentric nature of the disease and high recurrence suggests a field effect in the urothelium that results in a predisposition to develop cancer. Molecular genetic analyses suggest that the superficial and invasive lesions develop along distinct molecular pathways. Low-grade noninvasive papillary tumors harbor constitutive activation of the receptor tyrosine kinase-Ras signal transduction pathway and high frequencies of fibroblast growth factor receptor 3 and phosphoinositide-3 576 kinase α subunit mutations. In contrast, CIS and invasive tumors have a higher frequency of TP53 and RB gene alterations. Within all clinical stages, including Tis, T1, and T2 or greater lesions, tumors with alterations in p53, p21, and/or RB have a higher probability of recurrence, metastasis, and death from disease. CLINICAL PRESENTATION, DIAGNOSIS, AND STAGING

1	CLINICAL PRESENTATION, DIAGNOSIS, AND STAGING Hematuria occurs in 80–90% of patients and often reflects exophytic tumors. The bladder is the most common source of gross hematuria (40%), but benign cystitis (22%) is a more common cause than bladder cancer (15%) (Chap. 61). Microscopic hematuria is more commonly of prostate origin (25%); only 2% of bladder cancers produce microscopic hematuria. Once hematuria is documented, a urinary cytology, visualization of the urothelial tract by computed tomography (CT) or magnetic resonance urogram or intravenous pyelogram, and cystoscopy are recommended if no other etiology is found. Screening asymptomatic individuals for hematuria increases the diagnosis of tumors at an early stage but has not been shown to prolong life. After hematuria, irritative symptoms are the next most common presentation. Ureteral obstruction may cause flank pain. Symptoms of metastatic disease are rarely the first presenting sign.

1	The endoscopic evaluation includes an examination under anesthesia to determine whether a palpable mass is present. A flexible endoscope is inserted into the bladder, and bladder barbotage for cytology is performed. Visual inspection includes mapping the location, size, and number of lesions, as well as a description of the growth pattern (solid vs papillary). All visible tumors should be resected, and a sample of the muscle underlying the tumor should be obtained to assess the depth of invasion. Normal-appearing areas are biopsied at random to ensure no CIS is present. A notation is made as to whether a tumor was completely or incompletely resected. Selective catheterization and visualization of the upper tracts should be performed if the cytology is positive and no disease is visible in the bladder. Ultrasonography, CT, and/or magnetic resonance imaging (MRI) are used to determine whether a tumor extends to perivesical fat (T3) and to document nodal spread. Distant metastases are

1	in the bladder. Ultrasonography, CT, and/or magnetic resonance imaging (MRI) are used to determine whether a tumor extends to perivesical fat (T3) and to document nodal spread. Distant metastases are assessed by CT of the chest and abdomen, MRI, or radionuclide imaging of the skeleton.

1	Stage TNM 5-Year Survival Superficial Superficial Infiltrating Invasion of adjacent structures Lymph node invasion Distant extension IV IV IV I III III T1 Ta Tis T3a T4a T4b L. Nodes% 26 7–30 10–20% T3b 50 35–50% 90% 70 100 100 60 Ois Oa T2 70%II FIGURE 114-1 Bladder staging. TNM, tumor, node, metastasis. Management depends on whether the tumor invades muscle and whether it has spread to the regional lymph nodes and beyond. The probability of spread increases with increasing T stage.

1	Management depends on whether the tumor invades muscle and whether it has spread to the regional lymph nodes and beyond. The probability of spread increases with increasing T stage. At a minimum, the management is complete endoscopic resection with or without intravesical therapy. The decision to recommend intravesical therapy depends on the histologic subtype, number of lesions, depth of invasion, presence or absence of CIS, and antecedent history. Recurrences develop in upward of 50% of cases, of which 5–20% progress to a more advanced stage. In general, solitary papillary lesions are managed by transurethral surgery alone. CIS and recurrent disease are treated by transurethral surgery followed by intravesical therapy.

1	Intravesical therapies are used in two general contexts: as an adjuvant to a complete endoscopic resection to prevent recurrence or to eliminate disease that cannot be controlled by endoscopic resection alone. Intravesical treatments are advised for patients with diffuse CIS, recurrent disease, >40% involvement of the bladder surface by tumor, or T1 disease. The standard therapy, based on randomized comparisons, is Bacillus Calmette-Guérin (BCG) in six weekly instillations, often followed by maintenance administrations for ≥1 year. Other agents with activity include mitomycin C, interferon, and gemcitabine. The side effects of intravesical therapies include dysuria, urinary frequency, and, depending on the drug, myelosuppression or contact dermatitis. Rarely, intravesical BCG may produce a systemic illness associated with granulomatous infections in multiple sites requiring antituberculin therapy.

1	Following the endoscopic resection, patients are monitored for recurrence at 3-month intervals during the first year. Recurrence may develop anywhere along the urothelial tract, including the renal pelvis, ureter, or urethra. Persistent disease in the bladder and new tumors are treated with a second course of BCG or intravesical chemotherapy with valrubicin or gemcitabine. In some cases, cystectomy is recommended. Tumors in the ureter or renal pelvis are typically managed by resection during retrograde examination or, in some cases, by instillation through the renal pelvis. Prostatic urethral tumors may require cystoprostatectomy if the tumor cannot be resected completely.

1	The treatment of a tumor that has invaded muscle can be separated into control of the primary tumor and systemic chemotherapy to treat micrometastatic disease. Radical cystectomy is the standard treatment in the United States, although in selected cases, a bladder-sparing approach is used. This approach includes complete endoscopic resection; partial cystectomy; or a combination of resection, systemic chemotherapy, and external beam radiation therapy. In some countries, external beam radiation therapy is considered standard. In the United States, it is generally limited to those patients deemed unfit for cystectomy, those with unresectable local disease, or as part of an experimental bladder-sparing approach.

1	Indications for cystectomy include muscle-invading tumors not suitable for segmental resection; non–muscle-invasive tumors unsuitable for conservative management (e.g., due to multicentric and frequent recurrences resistant to intravesical instillations); high-grade T1 tumors especially if associated with CIS; and bladder symptoms (e.g., frequency or hemorrhage) that impair quality of life.

1	Radical cystectomy is major surgery that requires appropriate preoperative evaluation and management. It involves removal of the bladder and pelvic lymph nodes and creation of a conduit or reservoir for urinary flow. Grossly abnormal lymph nodes are evaluated by frozen section. If metastases are confirmed, the procedure is often aborted. In males, radical cystectomy includes the removal of the prostate, seminal vesicles, and proximal urethra. Impotence is universal unless the nerves responsible for erectile function are preserved. In females, the procedure includes removal of the bladder, urethra, uterus, fallopian tubes, ovaries, anterior vaginal wall, and surrounding fascia.

1	Several options are frequently used for urinary diversion. Ileal conduits bring urine directly from the ureter to the abdominal wall. Some patients receive either a continent cutaneous reservoir constructed from detubularized bowel or an orthotopic neobladder. Approximately 25% of men receive a neobladder, leading to 85–90% continence during the day. Cutaneous reservoirs are drained by intermittent catheterization. Contraindications to a neobladder include renal insufficiency, an inability to self-catheterize, or CIS or an exophytic tumor in the urethra. Diffuse CIS in the bladder is a relative contraindication based on the risk of a urethral recurrence. Concurrent ulcerative colitis or Crohn’s disease may hinder the use of bowel.

1	A partial cystectomy may be considered when the disease is limited to the dome of the bladder, a ≥2 cm margin can be achieved, there is no associated CIS, and the bladder capacity is adequate after resection. This occurs in 5–10% of cases. Carcinomas in the ureter or in the renal pelvis are treated with nephroureterectomy with a bladder cuff to remove the tumor. The probability of recurrence following surgery is based on pathologic stage, presence or absence of lymphatic or vascular invasion, and nodal spread. Among those whose cancers recur, the recurrence develops in a median of 1 year. Long-term outcomes vary by pathologic stage and histology (Table 114-1). The number of lymph nodes removed is also prognostic, whether or not the nodes contained tumor.

1	Chemotherapy (described below) has been shown to prolong the survival of patients with muscle-invasive disease when combined with definitive treatment of the bladder by radical cystectomy or radiation therapy. Presurgical (or neoadjuvant) chemotherapy has been the most thoroughly explored, and increases the cure rate by 5–15%, whereas postsurgical (adjuvant) chemotherapy has not been proven definitively beneficial. For the majority of patients, chemotherapy alone is inadequate to eradicate the disease. Use of neoadjuvant chemotherapy is increasing, although it still remains underused. Experimental studies are evaluating bladder preservation strategies by combining chemotherapy and radiation therapy in patients whose tumors were endoscopically removed.

1	The primary goal of metastatic disease treatment is to achieve complete remission with chemotherapy alone or with a combined-modality approach of chemotherapy followed by surgical resection of residual disease. One can define a goal in terms of cure or palliation on the basis of the probability of achieving a complete response 577 to chemotherapy using prognostic factors, such as Karnofsky performance status (KPS) (<80%) and whether the pattern of spread is nodal or visceral (liver, lung, or bone). For those with zero, one, or two risk factors, the probability of complete remission is 38, 25, and 5%, respectively, and median survival is 33, 13.4, and 9.3 months, respectively. Patients who have low KPS or who have visceral disease or bone metastases rarely achieve long-term survival. The toxicities also vary as a function of risk, and treatment-related mortality rates are as high as 3–4% using some combinations in these poor-risk patient groups. For most patients, treatment is

1	The toxicities also vary as a function of risk, and treatment-related mortality rates are as high as 3–4% using some combinations in these poor-risk patient groups. For most patients, treatment is palliative, aimed at delaying or relieving cancer-related symptoms, because few patients experience durable complete remissions.

1	A number of chemotherapeutic drugs have activity as single agents; cisplatin, paclitaxel, and gemcitabine are considered most active. Standard therapy consists of two-, three-, or four-drug combina tions. Overall response rates of >50% have been reported using combinations such as methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC); gemcitabine and cisplatin (GC); or gemcitabine, paclitaxel, and cisplatin (GPC). MVAC was considered standard, but the toxicities of neutropenia and fever, mucositis, diminished renal and auditory function, and peripheral neuropathy led to the development of alternative regimens. At present, GC is used more commonly than MVAC based on the results of a comparative trial of MVAC versus GC that showed less neutropenia and fever and less mucositis for the GC regimen with similar response rates and median overall survival. Anemia and thrombocytopenia were more common with GC. GPC is not more effective than GC.

1	Chemotherapy has also been tested in the neoadjuvant and adjuvant settings. In a randomized trial, patients receiving three cycles of neoadjuvant MVAC followed by cystectomy had a significantly better median (6.2 years) and 5-year survival (57%) compared to cystectomy alone (median survival 3.8 years; 5-year survival 42%). Similar results were obtained in an international study of three cycles of cisplatin, methotrexate, and vinblastine (CMV) followed by either radical cystectomy or radiation therapy. The decision to administer adjuvant therapy is based on recurrence risk after cystectomy. Studies of adjuvant chemotherapy have been underpowered, and most closed for lack of accrual. One underpowered study using the GPC regimen suggested that adjuvant treatment improved survival, although many patients never received chemotherapy for metastases. Another underpowered study did not show a benefit for GC chemotherapy. Therefore, preoperative chemotherapy is preferred when medically

1	many patients never received chemotherapy for metastases. Another underpowered study did not show a benefit for GC chemotherapy. Therefore, preoperative chemotherapy is preferred when medically appropriate. Indications for adjuvant chemotherapy in patients who did not receive neoadjuvant treatment include nodal disease, extravesical tumor extension, or vascular invasion in the resected specimen.

1	The management of bladder cancer is summarized in Table 114-2.

1	About 5000 cases of renal pelvis and ureter cancer occur each year; nearly all are transitional cell carcinomas similar to bladder cancer in biology and appearance. This tumor is associated with chronic phenacetin abuse and aristolochic acid consumption in Chinese herbal preparations; aristolochic acid also seems to be associated with Balkan nephropathy, a chronic interstitial nephritis endemic in Bulgaria, Greece, Bosnia-Herzegovina, taBLe 114-1 survivaL foLLoWing surgery for BLaDDer CanCer Pathologic Stage 5-Year Survival, % 10-Year Survival, % taBLe 114-2 management of BLaDDer CanCer Nature of Lesion Management Approach 578 and Romania. In addition, upper tract urothelial carcinoma is linked to hereditary nonpolyposis colorectal cancer. The most common symptom is painless gross hematuria, and the disease is usually detected on imaging during the workup for hematuria. Patterns of spread are like bladder cancer. For low-grade disease localized to the renal pelvis and ureter,

1	hematuria, and the disease is usually detected on imaging during the workup for hematuria. Patterns of spread are like bladder cancer. For low-grade disease localized to the renal pelvis and ureter, nephroureterectomy (including excision of the distal ureter with a portion of the bladder) is associated with 5-year survival of 80–90%. More invasive or poorly differentiated tumors are more likely to recur locally and to metastasize. Metastatic disease is treated with the chemotherapy used in bladder cancer, and the outcome is similar to that of metastatic bladder cancer.

1	Renal cell carcinomas account for 90–95% of malignant neoplasms arising from the kidney. Notable features include resistance to cytotoxic agents, infrequent responses to biologic response modifiers such as interleukin (IL) 2, robust activity to antiangiogenesis targeted agents, and a variable clinical course for patients with metastatic disease, including anecdotal reports of spontaneous regression.

1	The incidence of renal cell carcinoma continues to rise and is now nearly 65,000 cases annually in the United States, resulting in 13,700 deaths. The male-to-female ratio is 2:1. Incidence peaks between the ages of 50 and 70 years, although this malignancy may be diagnosed at any age. Many environmental factors have been investigated as possible contributing causes; the strongest association is with cigarette smoking. Risk is also increased for patients who have acquired cystic disease of the kidney associated with end-stage renal disease and for those with tuberous sclerosis. Most cases are sporadic, although familial forms have been reported. One is associated with von Hippel-Lindau (VHL) syndrome. VHL syndrome is an autosomal dominant disorder. Genetic studies identified the VHL gene on the short arm of chromosome 3. Approximately 35% of individuals with VHL disease develop clear cell renal cell carcinoma. Other associated neoplasms include retinal hemangioma, hemangioblastoma of

1	on the short arm of chromosome 3. Approximately 35% of individuals with VHL disease develop clear cell renal cell carcinoma. Other associated neoplasms include retinal hemangioma, hemangioblastoma of the spinal cord and cerebellum, pheochromocytoma, neuroendocrine tumors and cysts, and cysts in the epididymis of the testis in men and the broad ligament in women.

1	Renal cell neoplasia represents a heterogeneous group of tumors with distinct histopathologic, genetic, and clinical features ranging from benign to high-grade malignant (Table 114-3). They are classified on the basis of morphology and histology. Categories include clear cell carcinoma (60% of cases), papillary tumors (5–15%), chromophobe tumors (5–10%), oncocytomas (5–10%), and collecting or Bellini duct tumors (<1%). Papillary tumors tend to be bilateral and multifocal. Chromophobe tumors have a more indolent clinical course, and oncocytomas are considered benign neoplasms. In contrast, Bellini duct carcinomas, which are thought to arise from the collecting ducts within the renal medulla, are rare but often very aggressive. Clear cell tumors, the predominant histology, are found in >80% of patients who Carcinoma Growth Type Pattern Cell of Origin Cytogenetics Clear cell Acinar or Proximal tubule 3p-, 5q+, 14qsarcomatoid

1	Carcinoma Growth Type Pattern Cell of Origin Cytogenetics Clear cell Acinar or Proximal tubule 3p-, 5q+, 14qsarcomatoid Papillary Papillary or Proximal tubule +7, +17, -Y sarcomatoid

1	Chromophobe Solid, tubular, Distal tubules/corti-Whole arm losses or sarcomatoid cal collecting duct (1, 2, 6, 10, 13, 17, and 21) develop metastases. Clear cell tumors arise from the epithelial cells of the proximal tubules and usually show chromosome 3p deletions. Deletions of 3p21–26 (where the VHL gene maps) are identified in patients with familial as well as sporadic tumors. VHL encodes a tumor suppressor protein that is involved in regulating the transcription of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and a number of other hypoxia-inducible proteins. Inactivation of VHL leads to overexpression of these agonists of the VEGF and PDGF receptors, which promote tumor angiogenesis and tumor growth. Agents that inhibit proangiogenic growth factor activity show antitumor effects. Enormous genetic variability has been documented in tumors from individual patients. Although the tumors have a clear clonal origin and often contain VHL mutations in

1	activity show antitumor effects. Enormous genetic variability has been documented in tumors from individual patients. Although the tumors have a clear clonal origin and often contain VHL mutations in common, different portions of the primary tumor and different metastatic sites may have wide variation in genetic lesions they contain. This tumor heterogeneity may underlie the emergence of treatment resistance.

1	The presenting signs and symptoms include hematuria, abdominal pain, and a flank or abdominal mass. Other symptoms are fever, weight loss, anemia, and a varicocele. The tumor is most commonly detected as an incidental finding on a radiograph. Widespread use of radiologic cross-sectional imaging procedures (CT, ultrasound, MRI) contributes to earlier detection, including incidental renal masses detected during evaluation for other medical conditions. The increasing number of incidentally discovered low-stage tumors has contributed to an improved 5-year survival for patients with renal cell carcinoma and increased use of nephron-sparing surgery (partial nephrectomy). A spectrum of paraneoplastic syndromes has been associated with these malignancies, including erythrocytosis, hypercalcemia, nonmetastatic hepatic dysfunction (Stauffer’s syndrome), and acquired dysfibrinogenemia. Erythrocytosis is noted at presentation in only about 3% of patients. Anemia, a sign of advanced disease, is

1	nonmetastatic hepatic dysfunction (Stauffer’s syndrome), and acquired dysfibrinogenemia. Erythrocytosis is noted at presentation in only about 3% of patients. Anemia, a sign of advanced disease, is more common.

1	The standard evaluation of patients with suspected renal cell tumors includes a CT scan of the abdomen and pelvis, chest radio-graph, urine analysis, and urine cytology. If metastatic disease is suspected from the chest radiograph, a CT of the chest is warranted. MRI is useful in evaluating the inferior vena cava in cases of suspected tumor involvement or invasion by thrombus. In clinical practice, any solid renal masses should be considered malignant until proven otherwise; a definitive diagnosis is required. If no metastases are demonstrated, surgery is indicated, even if the renal vein is invaded. The differential diagnosis of a renal mass includes cysts, benign neoplasms (adenoma, angiomyolipoma, oncocytoma), inflammatory lesions (pyelonephritis or abscesses), and other primary or metastatic cancers. Other malignancies that may involve the kidney include transitional cell carcinoma of the renal pelvis, sarcoma, lymphoma, and Wilms’ tumor. All of these are less common causes of

1	metastatic cancers. Other malignancies that may involve the kidney include transitional cell carcinoma of the renal pelvis, sarcoma, lymphoma, and Wilms’ tumor. All of these are less common causes of renal masses than is renal cell cancer.

1	Staging is based on the American Joint Committee on Cancer (AJCC) staging system (Fig. 114-2). Stage I tumors are <7 cm in greatest diameter and confined to the kidney, stage II tumors are ≥7 cm and confined to the kidney, stage III tumors extend through the renal capsule but are confined to Gerota’s fascia (IIIa) or involve a single hilar lymph node (N1), and stage IV disease includes tumors that have invaded adjacent organs (excluding the adrenal gland) or involve multiple lymph nodes or distant metastases. The 5-year survival rate varies by stage: >90% for stage I, 85% for stage II, 60% for stage III, and 10% for stage IV. FIGURE 114-2 Renal cell carcinoma staging. TNM, tumor, node, metastasis.

1	FIGURE 114-2 Renal cell carcinoma staging. TNM, tumor, node, metastasis. its contents, including the kidney, the ipsilateral adrenal gland in some cases, and adjacent hilar lymph nodes. The role of a regional lymphadenectomy is controversial. Extension into the renal vein or inferior vena cava (stage III disease) does not preclude resection even if cardiopulmonary bypass is required. If the tumor is resected, half of these patients have prolonged survival. Nephron-sparing approaches via open or laparoscopic surgery may be appropriate for patients who have only one kidney, depending on the size and location of the lesion. A nephron-sparing approach can also be used for patients with bilateral tumors. Partial nephrectomy techniques are applied electively to resect small masses for patients with a normal contralateral kidney. Adjuvant therapy following this surgery does not improve outcome, even in cases with a poor prognosis.

1	Surgery has a limited role for patients with metastatic disease. Longterm survival may occur in patients who relapse after nephrectomy in a solitary site that is removed. One indication for nephrectomy with metastases at initial presentation is to alleviate pain or hemorrhage of a primary tumor. Also, a cytoreductive nephrectomy before systemic treatment improves survival for carefully selected patients with stage IV tumors. Metastatic renal cell carcinoma is refractory to chemotherapy. Cytokine therapy with IL-2 or interferon α (IFN-α) produces regression in 10–20% of patients. IL-2 produces durable complete remission in a small proportion of cases. In general, cytokine therapy is considered unsatisfactory for most patients.

1	The situation changed dramatically when two large-scale randomized trials established a role for antiangiogenic therapy, as predicted by the genetic studies. These trials separately evaluated two orally administered antiangiogenic agents, sorafenib and sunitinib, that inhibited receptor tyrosine kinase signaling through the VEGF and PDGF receptors. Both showed efficacy as second-line treatment following progression during cytokine treatment, resulting in approval by regulatory authorities for the treatment of advanced renal cell carcinoma. A randomized phase III trial comparing sunitinib to IFN-α showed superior efficacy for sunitinib with an acceptable safety profile. The trial resulted in a change in the standard first-line treatment from IFN to sunitinib. Sunitinib is usually given orally at a dose of 50 mg/d for 4 out of 6 weeks. Pazopanib and axitinib are newer agents of the same class. Pazopanib was compared to sunitinib in a randomized first-line phase III trial. Efficacy was

1	at a dose of 50 mg/d for 4 out of 6 weeks. Pazopanib and axitinib are newer agents of the same class. Pazopanib was compared to sunitinib in a randomized first-line phase III trial. Efficacy was similar, and there was less fatigue and skin toxicity, resulting in better quality of life scores for pazopanib compared with sunitinib. Temsirolimus and everolimus, inhibitors of the mammalian target of rapamycin (mTOR), show activity in patients with untreated poor-prognosis tumors and in sunitinib/sorafenib-refractory tumors. Patients benefit from the sequential use of axitinib and everolimus following progression to sunitinib or pazopanib first-line therapy.

1	The prognosis of metastatic renal cell carcinoma is variable. In one analysis, no prior nephrectomy, a KPS <80, low hemoglobin, high corrected calcium, and abnormal lactate dehydrogenase were poor prognostic factors. Patients with zero, one or two, and three or more factors had a median survival of 24, 12, and 5 months, respectively. These tumors may follow an unpredictable and protracted clinical course. It may be best to document progression before considering systemic treatment. 115 of the prostate Howard I. Scher, James A. Eastham

1	Benign and malignant changes in the prostate increase with age. Autopsies of men in the eighth decade of life show hyperplastic changes in >90% and malignant changes in >70% of individuals. The high prevalence of these diseases among the elderly, who often have competing causes of morbidity and mortality, mandates a risk-adapted approach to diagnosis and treatment. This can be achieved by considering these diseases as a series of states. Each state represents a distinct clinical milestone for which therapy(ies) may be recommended based on current symptoms, the risk of developing symptoms, or death from disease in relation to death from other causes within a given time frame. For benign proliferative disorders, symptoms of urinary frequency, infection, and potential for obstruction are weighed against the side effects and complications of medical or surgical intervention. For prostate malignancies, the risks of developing the disease, symptoms, or death from cancer are balanced against

1	against the side effects and complications of medical or surgical intervention. For prostate malignancies, the risks of developing the disease, symptoms, or death from cancer are balanced against the morbidities of the recommended treatments and preexisting comorbidities.

1	The prostate is located in the pelvis and is surrounded by the rectum, the bladder, the periprostatic and dorsal vein complexes and neurovascular bundles that are responsible for erectile function, and the urinary sphincter that is responsible for passive urinary control. The

1	Benign and Malignant Diseases of the Prostate 580 prostate is composed of branching tubuloalveolar glands arranged in lobules surrounded by fibromuscular stroma. The acinar unit includes an epithelial compartment made up of epithelial, basal, and neuroendocrine cells and separated by a basement membrane, and a stromal compartment that includes fibroblasts and smooth-muscle cells. Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) are produced in the epithelial cells. Both prostate epithelial cells and stromal cells express androgen receptors (ARs) and depend on androgens for growth. Testosterone, the major circulating androgen, is converted by the enzyme 5α-reductase to dihydrotestosterone in the gland. The periurethral portion of the gland increases in size during puberty and after the age of 55 years due to the growth of nonmalignant cells in the transition zone of the prostate that surrounds the urethra. Most cancers develop in the peripheral zone, and cancers in

1	and after the age of 55 years due to the growth of nonmalignant cells in the transition zone of the prostate that surrounds the urethra. Most cancers develop in the peripheral zone, and cancers in this location may be palpated during a digital rectal examination (DRE).

1	In 2013, approximately 238,590 prostate cancer cases were diagnosed, and 29,720 men died from prostate cancer in the United States. The absolute number of prostate cancer deaths has decreased in the past 5 years, which has been attributed by some to the widespread use of PSA-based detection strategies. However, the benefit of screening on survival is unclear. The paradox of management is that although 1 in 6 men will eventually be diagnosed with the disease, and the disease remains the second leading cause of cancer deaths in men, only 1 man in 30 with prostate cancer will die of his disease.

1	Epidemiologic studies show that the risk of being diagnosed with prostate cancer increases by a factor of two if one first-degree relative is affected and by four if two or more are affected. Current estimates are that 40% of early-onset and 5–10% of all prostate cancers are hereditary. Prostate cancer affects ethnic groups differently. Matched for age, African-American males have both a higher incidence of prostate cancer and larger tumors and more worrisome histologic features than white males. Polymorphic variants of the AR, the cytochrome P450 C17, and the steroid 5α-reductase type II (SRD5A2) genes have been implicated in the variations in incidence.

1	The prevalence of autopsy-detected cancers is similar around the world, while the incidence of clinical disease varies. Thus, environmental and dietary factors may play a role in prostate cancer growth and progression. High consumption of dietary fats, such as α-linoleic acid or the polycyclic aromatic hydrocarbons that form when red meats are cooked, is believed to increase risk. Similar to breast cancer in Asian women, the risk of prostate cancer in Asian men increases when they move to Western environments. Protective factors include consumption of the isoflavonoid genistein (which inhibits 5α-reductase) found in many legumes, cruciferous vegetables that contain the isothiocyanate sulforaphane, retinoids such as lycopene found in tomatoes, and inhibitors of cholesterol biosynthesis (e.g., statin drugs). The development of prostate cancer is a multistep process. One early change is hypermethylation of the GSTP1 gene promoter, which leads to loss of function of a gene that detoxifies

1	statin drugs). The development of prostate cancer is a multistep process. One early change is hypermethylation of the GSTP1 gene promoter, which leads to loss of function of a gene that detoxifies carcinogens. The finding that many prostate cancers develop adjacent to a lesion termed proliferative inflammatory atrophy (PIA) suggests a role for inflammation.

1	Currently no drugs or dietary supplements are approved by the U.S. Food and Drug Administration (FDA) for prevention of prostate cancer, nor are any recommended by the major clinical guidelines. Although statins may have some protective effect, the potential risks outweigh the benefits given the small number of men who die of prostate cancer. The results from several large, double-blind, randomized chemoprevention trials established 5α-reductase inhibitors (5ARI) as the most likely therapy to reduce the future risk of a prostate cancer diagnosis. The Prostate Cancer Prevention Trial (PCPT), in which men older than age 55 years received placebo or the 5ARI finasteride, which inhibits the type 1 isoform, showed a 25% (95% confidence interval 19–31%) reduction in the period prevalence of prostate cancer across all age groups in favor of finasteride (18.4%) over placebo (24.4%). In the Reduction by Dutasteride of Prostate Cancer Events (REDUCE) trial, a similar 23% reduction in the 4-year

1	cancer across all age groups in favor of finasteride (18.4%) over placebo (24.4%). In the Reduction by Dutasteride of Prostate Cancer Events (REDUCE) trial, a similar 23% reduction in the 4-year period prevalence was observed in favor of dutasteride (p = .001). Dutasteride inhibits both the type 1 and type 2 5ARI isoforms. While both studies met their endpoint, there was concern that most of the cancers that were prevented were low risk and that there was a slightly higher rate of clinically significant cancers (those with higher Gleason score) in the treatment arm. Neither drug was FDA-approved for prostate cancer prevention. In comparison, the Selenium and Vitamin E Cancer Prevention Trial (SELECT), which enrolled African-American men age ≥50 years and others age ≥55 years, showed no difference in cancer incidence in patients receiving vitamin E (4.6%) or selenium (4.9%) alone or in combination (4.6%) relative to placebo (4.4%). A similar lack of benefit for vitamin E, vitamin C,

1	difference in cancer incidence in patients receiving vitamin E (4.6%) or selenium (4.9%) alone or in combination (4.6%) relative to placebo (4.4%). A similar lack of benefit for vitamin E, vitamin C, and selenium was seen in the Physicians Health Study II.

1	The prostate cancer continuum—from the appearance of a preneoplastic and invasive lesion localized to the prostate, to a metastatic lesion that results in symptoms and, ultimately, mortality—can span decades. To facilitate disease management, competing risks are considered in the context of a series of clinical states (Fig. 115-1). The states are defined operationally on the basis of whether or not a cancer diagnosis has been established and, for those with a diagnosis, whether or not metastases are detectable on imaging studies and the measured level of testosterone in the blood. With this approach, an individual resides in only one state and remains in that state until he has progressed. At each assessment, the decision to offer treatment and the specific form of treatment are based on the risk posed by the cancer relative to competing causes of mortality that may be present in that individual. It follows that the more advanced the disease, the greater is the need for treatment.

1	For those without a cancer diagnosis, the decision to undergo testing to detect a cancer is based on the individual’s estimated life expectancy and, separately, the probability that a clinically significant cancer may be present. For those with a prostate cancer diagnosis, the clinical states model considers the probability of developing symptoms or dying from prostate cancer. Thus, a patient with localized prostate cancer who has had all cancer removed surgically remains in the state of localized disease as long as the PSA remains undetectable. The time within a state becomes a measure of the efficacy of an intervention, although the effect may not be assessable for years. Because many men with active cancer are not at risk for metastases, symptoms, or death, the clinical states model allows a distinction between cure—the elimination of all cancer cells, the primary therapeutic objective when treating most cancers—and cancer control, in which the tempo of the illness is altered and

1	a distinction between cure—the elimination of all cancer cells, the primary therapeutic objective when treating most cancers—and cancer control, in which the tempo of the illness is altered and symptoms are controlled until the patient dies of other causes. These can be equivalent therapeutically from a patient standpoint if the patient has not experienced symptoms of the disease or the treatment needed to control it. Even when a recurrence is documented, immediate therapy is not always necessary. Rather, as at the time of diagnosis, the need for intervention is based on the tempo of the illness as it unfolds in the individual, relative to the risk-to-benefit ratio of the therapy being considered.

1	SCREENING AND DIAGNOSIS Physical Examination The need to pursue a diagnosis of prostate cancer is based on symptoms, an abnormal DRE, or, more typically, a change in or an elevated serum PSA. The urologic history should focus on symptoms of outlet obstruction, continence, potency, or change in ejaculatory pattern. The DRE focuses on prostate size and consistency and abnormalities within or beyond the gland. Many cancers occur in the peripheral zone and may be palpated on DRE. Carcinomas are characteristically hard, nodular, and irregular, while induration may also be due to benign prostatic hypertrophy (BPH) or calculi. Overall, 20–25% of men with an abnormal DRE have cancer. No Cancer Diagnosis Clinically Localized Disease Rising PSA: No Visible Metastases: Non-Castrate Rising PSA: No Visible Metastases: Castrate Clinical Metastases: Non-Castrate Death from cancer exceeds death from other causes FIGURE 115-1 Clinical states of prostate cancer. PSA, prostate-specific antigen.

1	Prostate-Specific Antigen PSA (kallikrein-related peptidase 3; KLK3) is a kallikrein-related serine protease that causes liquefaction of seminal coagulum. It is produced by both nonmalignant and malignant epithelial cells and, as such, is prostate-specific, not prostate cancer–specific. Serum levels may also increase from prostatitis and BPH. Serum levels are not significantly affected by DRE, but the performance of a prostate biopsy can increase PSA levels up to tenfold for 8–10 weeks. PSA circulating in the blood is inactive and mainly occurs as a complex with the protease inhibitor α1-antichymotrypsin and as free (unbound) PSA forms. The formation of complexes between PSA, α2-macroglobulin, or other protease inhibitors is less significant. Free PSA is rapidly eliminated from the blood by glomerular filtration with an estimated half-life of 12–18 h. Elimination of PSA bound to α1-antichymotrypsin is slow (estimated half-life of 1–2 weeks) because it too is largely cleared by the

1	by glomerular filtration with an estimated half-life of 12–18 h. Elimination of PSA bound to α1-antichymotrypsin is slow (estimated half-life of 1–2 weeks) because it too is largely cleared by the kidneys. Levels should be undetectable after about 6 weeks if the prostate has been removed. Immunohistochemical staining for PSA can be used to establish a prostate cancer diagnosis.

1	Psa-Based screening and early detection PSA testing was approved by the U.S. FDA in 1994 for early detection of prostate cancer, and the widespread use of the test has played a significant role in the proportion of men diagnosed with early-stage cancers: more than 70–80% of newly diagnosed cancers are clinically organ-confined. The level of PSA in blood is strongly associated with the risk and outcome of prostate cancer. A single PSA measured at age 60 is associated (area under the curve [AUC] of 0.90) with lifetime risk of death from prostate cancer. Most prostate cancer deaths (90%) occur among men with PSA levels in the top quartile (>2 ng/mL), although only a minority of men with PSA >2 ng/mL will develop lethal prostate cancer. Despite this and mortality rate reductions reported from large randomized prostate cancer screening trials, routine use of the test remains controversial.

1	The U.S. Preventive Services Task Force (USPSTF) reviewed the evidence for screening for prostate cancer and made a clear recommendation against screening. By giving a grade of “D” in the recommendation statement that was based on this review, the USPSTF concluded that “there is moderate or high certainty that this service has no net benefit or that the harms outweigh the benefits.” Whether the harms of screening, overdiagnosis, and overtreatment are justified by the benefits in terms of reduced prostate cancer mortality is open to reasonable doubt. In response to the USPSTF, the American Urological Association (AUA) updated their consensus statement regarding prostate cancer screening.

1	They concluded that the quality of evidence for the benefits of screening was moderate, and evidence for harm was high for men age 55–69 years. For men outside this age range, evidence was lacking for benefit, but the harms of screening, including overdiagnosis and overtreatment, remained. The AUA recommends shared decision making considering PSA-based screening for men age 55–69, a target age group for whom benefits may outweigh harms. Outside this age range, PSA-based screening as a routine test was not recommended based on the available evidence. The entire guideline is available at www.AUAnet.org/education/guidelines/ prostate-cancer-detection.cfm.

1	The PSA criteria used to recommend a diagnostic prostate biopsy have evolved over time. However, based on the commonly used cut point for prostate biopsy (a total PSA ≥4 ng/mL), most men with a PSA elevation do not have histologic evidence of prostate cancer at biopsy. In addition, many men with PSA levels below this cut point harbor cancer cells in their prostate. Information from the PCPT demonstrates that there is no PSA below which the risk of prostate cancer is zero. Thus, the PSA level establishes the likelihood that a man will harbor cancer if he undergoes a prostate biopsy. The goal is to increase the sensitivity of the test for younger men more likely to die of the disease and to reduce the frequency of detecting cancers of low malignant potential in elderly men more likely to die of other causes. Patients with symptomatic prostatitis should have a course of antibiotics before biopsy. However, the routine use of antibiotics in an asymptomatic man with an elevated PSA level is

1	of other causes. Patients with symptomatic prostatitis should have a course of antibiotics before biopsy. However, the routine use of antibiotics in an asymptomatic man with an elevated PSA level is strongly discouraged.

1	Prostate Biopsy A diagnosis of cancer is established by an image-guided needle biopsy. Direct visualization by transrectal ultrasound (TRUS) or magnetic resonance imaging (MRI) assures that all areas of the gland are sampled. Contemporary schemas advise an extended-pattern 12-core biopsy that includes sampling from the peripheral zone as well as a lesion-directed palpable nodule or suspicious image-guided sampling. Men with an abnormal PSA and negative biopsy are advised to undergo a repeat biopsy. BioPsy Pathology Each core of the biopsy is examined for the presence of cancer, and the amount of cancer is quantified based on the length of the cancer within the core and the percentage of the core involved. Of the cancers identified, >95% are adenocarcinomas; the rest are squamous or transitional cell tumors or, rarely, carcinosarcomas. Metastases to the prostate are rare, but in some cases colon cancers or transitional cell tumors of the bladder invade the gland by direct extension.

1	Benign and Malignant Diseases of the Prostate Tx Primary tumor cannot be assessed T0 No evidence of primary tumor T1 Clinically inapparent tumor, neither palpable nor visible by imaging T1a Tumor incidental histologic finding in ≤5% of resected tissue; not palpable T1b Tumor incidental histologic finding in >5% of resected tissue T1c Tumor identified by needle biopsy (e.g., because of elevated PSA) T2 Tumor confined within prostateb T2a Tumor involves half of one lobe or less T2b Tumor involves more than one half of one lobe, not both lobes T2c Tumor involves both lobes T3 Tumor extends through the prostate capsulec T3a Extracapsular extension (unilateral or bilateral) T3b Tumor invades seminal vesicle(s) T4 Tumor is fixed or invades adjacent structures other than seminal vesicles such as external sphincter, rectum, bladder, levator muscles, and/or pelvic wall.

1	aRevised from SB Edge et al (eds): AJCC Cancer Staging Manual, 7th ed. New York, Springer, 2010. bTumor found in one or both lobes by needle biopsy, but not palpable or reliably visible by imaging, is classified as T1c. cInvasion into the prostatic apex or into (but not beyond) the prostatic capsule is classified not as T3 but as T2. Abbreviations: PSA, prostate-specific antigen; TNM, tumor, node, metastasis. When prostate cancer is diagnosed, a measure of histologic aggressiveness is assigned using the Gleason grading system, in which the dominant and secondary glandular histologic patterns are scored from 1 (well-differentiated) to 5 (undifferentiated) and summed to give a total score of 2–10 for each tumor. The most poorly differentiated area of tumor (i.e., the area with the highest histologic grade) often determines biologic behavior. The presence or absence of perineural invasion and extracapsular spread is also recorded.

1	Prostate Cancer Staging The tumor, node, metastasis (TNM) staging system includes categories for cancers identified solely on the basis of an abnormal PSA (T1c), those that are palpable but clinically confined to the gland (T2), and those that have extended outside the gland (T3 and T4) (Table 115-1, Fig. 115-2). DRE alone is inaccurate in determining the extent of disease within the gland, the presence or absence of capsular invasion, involvement of seminal vesicles, and extension of disease to lymph nodes. Because of the inadequacy of DRE for staging, the TNM staging system was modified to include the results of imaging. Unfortunately, no single test has proven to accurately indicate the stage or the presence of organ-confined disease, seminal vesicle involvement, or lymph node spread.

1	TRUS is the imaging technique most frequently used to assess the primary tumor, but its chief use is directing prostate biopsies, not staging. No TRUS finding consistently indicates cancer with certainty. Computed tomography (CT) lacks sensitivity and specificity to detect extraprostatic extension and is inferior to MRI in visualization of lymph nodes. In general, MRI performed with an endorectal coil is superior to CT to detect cancer in the prostate and to assess local disease extent. T1-weighted MRI produces a high signal in the periprostatic fat, periprostatic venous plexus, perivesicular tissues, lymph nodes, and bone marrow. T2-weighted MRI demonstrates the internal architecture of the prostate and seminal vesicles. Most cancers have a low signal, while the normal peripheral zone has a high signal, although the technique lacks sensitivity and specificity. MRI is also useful for the planning of surgery and radiation therapy.

1	Radionuclide bone scans (bone scintigraphy) are used to evaluate spread to osseous sites. This test is sensitive but relatively nonspecific because areas of increased uptake are not always related to metastatic disease. Healing fractures, arthritis, Paget’s disease, and other conditions will also cause abnormal uptake. True-positive bone scans are uncommon when the PSA is <10 ng/mL unless the tumor is high grade.

1	Clinically localized prostate cancers are those that appear to be nonmetastatic after staging studies are performed. Patients with clinically localized disease are managed by radical prostatectomy, radiation therapy, or active surveillance. Choice of therapy requires the consideration of several factors: the presence of symptoms, the probability that the untreated tumor will adversely affect the quality or duration of survival and thus require treatment, and the probability that the tumor can be cured by single-modality therapy directed at the prostate or that it will require both local and systemic therapy to achieve cure.

1	Data from the literature do not provide clear evidence for the superiority of any one treatment relative to another. Comparison of outcomes of various forms of therapy is limited by the lack of prospective trials, referral bias, the experience of the treating teams, and differences in endpoints and cancer control definitions. Often, PSA relapse– free survival is used because an effect on metastatic progression

1	FIGURE 115-2 T stages of prostate cancer. (A) T1—Clinically inapparent tumor, neither palpable nor visible by imaging; (B) T2—Tumor confined within prostate; (C ) T3—Tumor extends through prostate capsule and may invade the seminal vesicles; (D) T4—Tumor is fixed or invades adjacent structures. Eighty-one percent of patients present with local disease (T1 and T2), which is associated with a 5-year survival rate of 100%. An additional 12% of patients present with regional disease (T3 and T4 without metastases), which is also associated with a 100% survival rate after 5 years. Four percent of patients present with distant disease (T4 with metastases), which is associated with a 28% 5-year survival rate. (Three percent of patients are ungraded, and this group is associated with a 73% 5-year survival rate.) (Data from AJCC, http://seer .cancer.gov/statfacts/html/prost.html. Figure © 2014 Memorial Sloan-Kettering Cancer Center; used with permission.) or survival may not be apparent for

1	survival rate.) (Data from AJCC, http://seer .cancer.gov/statfacts/html/prost.html. Figure © 2014 Memorial Sloan-Kettering Cancer Center; used with permission.) or survival may not be apparent for years. After radical surgery to remove all prostate tissue, PSA should become undetectable in the blood within 6 weeks. If PSA remains or becomes detectable after radical prostatectomy, the patient is considered to have persistent disease. After radiation therapy, in contrast, PSA does not become undetectable because the remaining nonmalignant elements of the gland continue to produce PSA even if all cancer cells have been eliminated. Similarly, cancer control is not well defined for a patient managed by active surveillance because PSA levels will continue to rise in the absence of therapy. Other outcomes are time to objective progression (local or systemic), cancer-specific survival, and overall survival; however, these outcomes may take years to assess.

1	The more advanced the disease, the lower the probability of local control and the higher the probability of systemic relapse. More important is that within the categories of T1, T2, and T3 disease are cancers with a range of prognoses. Some T3 tumors are curable with therapy directed solely at the prostate, and some T1 lesions have a high probability of systemic relapse that requires the integration of local and systemic therapy to achieve cure. For T1c cancers in particular, stage alone is inadequate to predict outcome and select treatment; other factors must be considered.

1	Nomograms To better assess risk and guide treatment selection, many groups have developed prognostic models or nomograms that use a combination of the initial clinical T stage, biopsy Gleason score, and baseline PSA. Some use discrete cut points (PSA <10 or ≥10 ng/mL; Gleason score of ≤6, 7, or ≥8); others employ nomograms that use PSA and Gleason score as continuous variables. More than 100 nomograms have been reported to predict the probability that a clinically significant prostate cancer is present, disease extent (organ-confined vs non–organ-confined, node-negative or -positive), or the probability of success of treatment for specific local therapies using pretreatment variables. Considerable controversy exists over what constitutes “high risk” based on a predicted probability of success or failure. In these situations, nomograms and predictive models can only go so far. Exactly what probability of success or failure would lead a physician to recommend and a patient to seek

1	success or failure. In these situations, nomograms and predictive models can only go so far. Exactly what probability of success or failure would lead a physician to recommend and a patient to seek alternative approaches is controversial. As an example, it may be appropriate to recommend radical surgery for a younger patient with a low probability of cure. Nomograms are being refined continually to incorporate additional clinical parameters, biologic determinants, and year of treatment, which can also affect outcomes, making treatment decisions a dynamic process.

1	Treatment-Related Adverse Events The frequency of adverse events varies by treatment modality and the experience of the treating team. For example, following radical prostatectomy, incontinence rates range from 2–47% and impotence rates range from 25–89%. Part of the variability relates to how the complication is defined and whether the patient or physician is reporting the event. The time of the assessment is also important. After surgery, impotence is immediate but may reverse over time, while with radiation therapy impotence is not immediate but may develop over time. Of greatest concern to patients are the effects on continence, sexual potency, and bowel function.

1	Radical Prostatectomy The goal of radical prostatectomy is to excise the cancer completely with a clear margin, to maintain continence by preserving the external sphincter, and to preserve potency by sparing the autonomic nerves in the neurovascular bundle. The procedure is advised for patients with a life expectancy of 10 years or more and is performed via a retropubic or perineal approach or via a minimally invasive robotic-assisted or hand-held laparoscopic approach. Outcomes can be predicted using postoperative nomograms that consider pretreatment factors and the pathologic findings at surgery. PSA failure is usually defined as a value greater than 0.1 or 0.2 ng/mL. Specific criteria to guide the choice of one approach over another are lacking. Minimally invasive approaches offer the advantage of a shorter hospital stay and reduced blood loss. Rates of cancer control, recovery of continence, and recovery of erectile function are comparable between open and minimally invasive

1	the advantage of a shorter hospital stay and reduced blood loss. Rates of cancer control, recovery of continence, and recovery of erectile function are comparable between open and minimally invasive approaches. The individual surgeon rather than the surgi-583 cal approach used is most important in determining outcomes after surgery.

1	Neoadjuvant hormonal therapy has also been explored in an attempt to improve the outcomes of surgery for high-risk patients, using a variety of definitions. The results of several large trials testing 3 or 8 months of androgen depletion before surgery showed that serum PSA levels decreased by 96%, prostate volumes decreased by 34%, and margin positivity rates decreased from 41% to 17%. Unfortunately, hormones did not produce an improvement in PSA relapse–free survival. Thus, neoadjuvant hormonal therapy is not recommended.

1	Factors associated with incontinence following radical prostatectomy include older age and urethral length, which impacts the ability to preserve the urethra beyond the apex and the distal sphincter. The skill and experience of the surgeon are also factors. Recovery of erectile function is associated with younger age, quality of erections before surgery, and the absence of damage to the neurovas cular bundles. In general, erectile function begins to return about 6 months after surgery if both neurovascular bundles are preserved. Potency is reduced by half if at least one neurovascular bundle is sacrificed. Overall, with the availability of drugs such as phosphodiesterase-5 (PDE5) inhibitors, intraurethral inserts of alprostadil, and intracavernosal injections of vasodilators, many patients recover satisfactory sexual function. Radiation Therapy Radiation therapy is given by external beam, by radioactive sources implanted into the gland, or by a combination of the two techniques.

1	Radiation Therapy Radiation therapy is given by external beam, by radioactive sources implanted into the gland, or by a combination of the two techniques. external-Beam radiation theraPy Contemporary external-beam radiation therapy requires three-dimensional conformal treatment plans to maximize the dose to the prostate and to minimize the exposure of the surrounding normal tissue. Intensity-modulated radiation therapy (IMRT) permits shaping of the dose and allows the delivery of higher doses to the prostate and a further reduction in normal tissue exposure than three-dimensional conformal treatment alone. These advances have enabled the safe administration of doses >80 Gy and resulted in higher local control rates and fewer side effects.

1	Cancer control after radiation therapy has been defined by various criteria, including a decline in PSA to <0.5 or 1 ng/mL, “nonrising” PSA values, and a negative biopsy of the prostate 2 years after completion of treatment. The current standard definition of biochemical failure (the Phoenix definition) is a rise in PSA by ≥2 ng/mL higher than the lowest PSA achieved. The date of failure is “at call” and not backdated. Radiation dose is critical to the eradication of prostate cancer. In a representative study, a PSA nadir of <1.0 ng/mL was achieved in 90% of patients receiving 75.6 or 81.0 Gy versus 76% and 56% of those receiving 70.2 and 64.8 Gy, respectively. Positive biopsy rates at 2.5 years were 4% for those treated with 81 Gy versus 27% and 36% for those receiving 75.6 and 70.2 Gy, respectively.

1	Overall, radiation therapy is associated with a higher frequency of bowel complications (mainly diarrhea and proctitis) than surgery. The frequency relates directly to the volume of the anterior rectal wall receiving full-dose treatment. In one series, grade 3 rectal or urinary toxicities were seen in 2.1% of patients who received a median dose of 75.6 Gy, whereas grade 3 urethral strictures requiring dilatation developed in 1% of cases, all of whom had undergone a transurethral resection of the prostate (TURP). Pooled data show that the frequency of grade 3 and 4 toxicities is 6.9% and 3.5%, respectively, for patients who received >70 Gy. The frequency of erectile dysfunction is related to the age of the patient, the quality of erections pretreatment, the dose administered, and the time of assessment. Postradiation erectile dysfunction is related to a disruption of the vascular supply and not the nerve fibers.

1	Neoadjuvant hormone therapy before radiation therapy has the aim of decreasing the size of the prostate and, consequently, reducing the exposure of normal tissues to full-dose radiation, increasing local control rates, and decreasing the rate of systemic failure. Benign and Malignant Diseases of the Prostate 584 Short-term hormone therapy can reduce toxicities and improve local control rates, but long-term treatment (2–3 years) is needed to prolong the time to PSA failure and lower the risk of metastatic disease in men with high-risk cancers. The impact on survival has been less clear.

1	BrachytheraPy Brachytherapy is the direct implantation of radioactive sources (seeds) into the prostate. It is based on the principle that the deposition of radiation energy in tissues decreases as a function of the square of the distance from the source (Chap. 103e). The goal is to deliver intensive irradiation to the prostate, minimizing the exposure of the surrounding tissues. The current standard technique achieves a more homogeneous dose distribution by placing seeds according to a customized template based on imaging assessment of the cancer and computer-optimized dosimetry. The implantation is performed transperineally as an outpatient procedure with real-time imaging.

1	Improvements in brachytherapy techniques have resulted in fewer complications and a marked reduction in local failure rates. In a series of 197 patients followed for a median of 3 years, 5-year actuarial PSA relapse–free survival for patients with pretherapy PSA levels of 0–4, 4–10, and >10 ng/mL were 98%, 90%, and 89%, respectively. In a separate report of 201 patients who underwent posttreatment biopsies, 80% were negative, 17% were indeterminate, and 3% were positive. The results did not change with longer follow-up. Nevertheless, many physicians feel that implantation is best reserved for patients with good or intermediate prognostic features.

1	Brachytherapy is well tolerated, although most patients experience urinary frequency and urgency that can persist for several months. Incontinence has been seen in 2–4% of cases. Higher complication rates are observed in patients who have undergone a prior TURP, whereas those with obstructive symptoms at baseline are at a higher risk for retention and persistent voiding symptoms. Proctitis has been reported in <2% of patients.

1	Active Surveillance Although prostate cancer is the most common form of cancer affecting men in the United States, patients are being diagnosed earlier and more frequently present with early-stage disease. Active surveillance, described previously as watchful waiting or deferred therapy, is the policy of monitoring the illness at fixed intervals with DREs, PSA measurements, and repeat prostate biopsies as indicated until histopathologic or serologic changes correlative of progression warrant treatment with curative intent. It evolved from studies that evaluated predominantly elderly men with well-differentiated tumors who demonstrated no clinically significant progression for protracted periods, recognition of the contrast between incidence and disease-specific mortality, the high prevalence of autopsy cancers, and an effort to reduce overtreatment. A recent screening study estimated that between 50–100 men with low-risk disease would need to be treated to prevent one prostate

1	prevalence of autopsy cancers, and an effort to reduce overtreatment. A recent screening study estimated that between 50–100 men with low-risk disease would need to be treated to prevent one prostate cancer–specific death.

1	Arguing against active surveillance are the results of a Swedish randomized trial of radical prostatectomy versus active surveillance. With a median follow-up of 6.2 years, men treated by radical surgery had a lower risk of prostate cancer death relative to active surveillance patients (4.6% vs 8.9%) and a lower risk of metastatic progression (hazard ratio 0.63). Case selection is critical, and determining clinical parameters predictive of cancer aggressiveness that can be used to reliably select men most likely to benefit from active surveillance is an area of intense study. In one prostatectomy series, it was estimated that 10–15% of those treated had “insignificant” disease. One set of criteria includes men with clinical T1c tumors that are biopsy Gleason grade 6 or less involving three or fewer cores, each of them having less than 50% involvement by tumor and a PSA density of less than 0.15.

1	Concerns about active surveillance include the limited ability to predict pathologic findings by needle biopsy even when multiple cores are obtained, the recognized multifocality of the disease, and the possibility of a missed opportunity to cure the disease. Nomograms to help predict which patients can safely be managed by active surveillance continue to be refined, and as their predictive accuracy improves, it can be anticipated that more patients will be candidates. This term is applied to a group of patients in whom the sole manifestation of disease is a rising PSA after surgery and/or radiation therapy. By definition, there is no evidence of disease on an imaging study. For these patients, the central issue is whether the rise in PSA results from persistent disease in the primary site, systemic disease, or both. In theory, disease in the primary site may still be curable by additional local treatment.

1	The decision to recommend radiation therapy after prostatectomy is guided by the pathologic findings at surgery, because imaging studies such as CT and bone scan are typically uninformative. Some recommend a choline-11 positron emission tomography (PET) scan, but availability in the United States is limited. Others recommend that a biopsy of the urethrovesical anastomosis be obtained before considering radiation, whereas others treat empirically based on risk. Factors that predict for response to salvage radiation therapy are a positive surgical margin, lower Gleason score in the radical prostatectomy specimen, long interval from surgery to PSA failure, slow PSA doubling time, absence of disease in the lymph nodes, and a low (<0.5–1 ng/mL) PSA value at the time of radiation treatment. Radiation therapy is generally not recommended if the PSA was persistently elevated after surgery, which usually indicates that the disease has spread outside of the area of the prostate bed and is

1	Radiation therapy is generally not recommended if the PSA was persistently elevated after surgery, which usually indicates that the disease has spread outside of the area of the prostate bed and is unlikely to be controlled with radiation therapy. As is the case for other disease states, nomograms to predict the likelihood of success are available.

1	For patients with a rising PSA after radiation therapy, salvage local therapy can be considered if the disease was “curable” at the time of diagnosis, if persistent disease has been documented by a biopsy of the prostate, and if no metastatic disease is seen on imaging studies. Unfortunately, case selection is poorly defined in most series, and morbidities are significant. Options include salvage radical prostatectomy, salvage cryotherapy, salvage radiation therapy, and salvage irreversible electroporation.

1	The rise in PSA after surgery or radiation therapy may indicate subclinical or micrometastatic disease with or without local recurrence. In these cases, the need for treatment depends, in part, on the estimated probability that the patient will develop clinically detectable metastatic disease on a scan and in what time frame. That immediate therapy is not always required was shown in a series where patients who developed a biochemical recurrence after radical prostatectomy received no systemic therapy until metastatic disease was documented. Overall, the median time to metastatic progression by imaging was 8 years, and 63% of the patients with rising PSA values remained free of metastases at 5 years. Factors associated with progression included the Gleason score of the radical prostatectomy specimen, time to recurrence, and PSA doubling time. For those with Gleason grade ≥8, the probability of metastatic progression was 37%, 51%, and 71% at 3, 5, and 7 years, respectively. If the time

1	specimen, time to recurrence, and PSA doubling time. For those with Gleason grade ≥8, the probability of metastatic progression was 37%, 51%, and 71% at 3, 5, and 7 years, respectively. If the time to recurrence was <2 years and PSA doubling time was long (>10 months), the proportions with metastatic disease at the same time intervals were 23%, 32%, and 53%, versus 47%, 69%, and 79% if the doubling time was short (<10 months). PSA doubling times are also prognostic for survival. In one series, all patients who succumbed to disease had PSA doubling times of 3 months or less.

1	Most physicians advise treatment if the PSA doubling time is 12 months or less. A difficulty with predicting the risk of metastatic spread, symptoms, or death from disease in the rising PSA state is that most patients receive some form of therapy before the development of metastases. Nevertheless, predictive models continue to be refined. METASTATIC DISEASE: NONCASTRATE The state of noncastrate metastatic prostate cancer includes men with metastases visible on an imaging study and noncastrate levels of testosterone (>150 ng/dL). The patient may be newly diagnosed or have a recurrence after treatment for localized disease. Symptoms of metastatic disease include pain from osseous spread, although many patients are asymptomatic despite extensive spread. Less common are symptoms related to marrow compromise (myelophthisis), spinal cord compression, or a coagulopathy.

1	Standard treatment is to deplete/lower androgens by medical or surgical means and/or to block androgen binding to the AR with antiandrogens. More than 90% of male hormones originate in the testes; <10% are synthesized in the adrenal gland. Surgical orchiectomy is the “gold standard” but is rarely used due to the availability of effective medical therapies and the more widespread use of hormones on an intermittent basis by which patients are treated for defined periods of time, following which the treatments are intentionally discontinued (discussed further below) (Fig. 115-3).

1	Testosterone-Lowering Agents Medical therapies that lower testosterone levels include the gonadotropin-releasing hormone (GnRH) agonists/antagonists, 17,20-lyase inhibitors, CYP17 inhibitors, estrogens, and progestational agents. Of these, GnRH analogues such as leuprolide acetate and goserelin acetate initially produce a rise in luteinizing hormone and follicle-stimulating hormone, followed by a downregulation of receptors in the pituitary gland, which effects a chemical castration. They were approved on the basis of randomized comparisons showing an improved safety profile (specifically, reduced cardiovascular toxicities) relative to diethylstilbestrol (DES), with equivalent potency. The initial rise in testosterone may result in a clinical flare of the disease. As such, these agents are relatively contraindicated in men with significant obstructive symptoms, cancer-related pain, or spinal cord compromise. GnRH antagonists such as degarelix achieve castrate levels of testosterone

1	are relatively contraindicated in men with significant obstructive symptoms, cancer-related pain, or spinal cord compromise. GnRH antagonists such as degarelix achieve castrate levels of testosterone within 48 h without the initial rise in serum testosterone and do not cause a flare in the disease. Estrogens such as DES are rarely used due to the risk of vascular complications such as fluid retention, phlebitis, embolic events, and stroke. Progestational agents alone are less efficacious.

1	Agents that lower testosterone are associated with an androgen-depletion syndrome that includes hot flushes, weakness, fatigue, loss of libido, impotence, sarcopenia, anemia, change in personality, and depression. Changes in lipids, obesity, and insulin resistance, along with an increased risk of diabetes and cardiovascular disease, can also occur, mimicking the metabolic syndrome. A decrease in bone density may also result that worsens over time and results in an increased risk of clinical fractures. This is a particular concern, often underappreciated, for men with preexisting osteopenia secondary to hypogonadism or glucocorticoid or alcohol use. Baseline fracture risk can be assessed using the Fracture Risk Assessment Scale (FRAX), and to minimize fracture risk, patients are advised to take calcium and vitamin D supplementation, along with a bisphosphonate or the RANK ligand inhibitor, denosumab.

1	Antiandrogens First-generation nonsteroidal antiandrogens such as flutamide, bicalutamide, and nilutamide block ligand binding to the AR and were initially approved to block the disease flare that may occur with the rise in serum testosterone associated with GnRH agonist therapy. When antiandrogens are given alone, testosterone levels typically increase above baseline, but relative to testosterone-lowering therapies, they cause fewer hot flushes, less of an effect on libido, less muscle wasting, fewer personality changes, and less bone loss. Gynecomastia remains a significant problem but can be alleviated in part by tamoxifen.

1	Most reported randomized trials suggest that the cancer-specific outcomes are inferior when antiandrogens are used alone. Bicalutamide, even at 150 mg (three times the recommended dose), was associated with a shorter time to progression and inferior survival compared to surgical castration for patients with established metastatic disease. Nevertheless, some men may accept the trade-off of a potentially inferior cancer outcome for an improved quality of life. Combined androgen blockade, the administration of an antiandrogen plus a GnRH analogue or surgical orchiectomy, and triple androgen blockade, which includes the addition of a 5ARI, have not

1	Combined androgen blockade, the administration of an antiandrogen plus a GnRH analogue or surgical orchiectomy, and triple androgen blockade, which includes the addition of a 5ARI, have not Chapter 115 Benign and Malignant Diseases of the Prostate XXTestis HypothalmusPituitary ProstateProstate cell Prostate cell nucleus DNA Adrenal Glands CYP17 inhibitors abiraterone Testosterone androstenedione DHEA DHEA-S GnRH GnRH agonists GnRH antagonist Estrogens dutasteride prednisone ACTHLH CRH DHT DHT DHT AR AR AR AR AR Next generation anti-androgens ARE XXenzalutamide FIGURE 115-3 Sites of action of different hormone therapies. ACTH, adrenocorticotropic hormone; AR, androgen receptor; ARE, androgen-response element; CRH, corticotropin-releasing hormone; DHEA, dehydroepiandrosterone; DHEA-S, dehydroepi-androsterone sulphate; DHT, dihydrotestosterone; GnRH, gonado-tropin-releasing hormone; LH, luteinizing hormone.

1	586 been shown to be superior to androgen depletion monotherapies and are no longer recommended. In practice, most patients who are treated with a GnRH agonist receive an antiandrogen for the first 2–4 weeks of treatment to protect against the flare.

1	Intermittent Androgen Deprivation Therapy (IADT) The use of hormones in an “on-and-off” approach was initially proposed as a way to prevent the selection of cells that are resistant to androgen depletion and to reduce side effects. The hypothesis is that by allowing endogenous testosterone levels to rise, the cells that survive androgen depletion will induce a normal differentiation pathway. It is postulated that by allowing the surviving cells to proliferate in the presence of androgen, sensitivity to subsequent androgen depletion will be retained and the chance of developing a castration-resistant state will be reduced. Applied in the clinic, androgen depletion is continued for 2–6 months beyond the point of maximal response. Once treatment is stopped, endogenous testosterone levels increase, and the symptoms associated with hormone treatment abate. PSA levels also begin to rise, and at some level, treatment is restarted. With this approach, multiple cycles of regression and

1	increase, and the symptoms associated with hormone treatment abate. PSA levels also begin to rise, and at some level, treatment is restarted. With this approach, multiple cycles of regression and proliferation have been documented in individual patients. It is unknown whether the intermittent approach increases, decreases, or does not change the overall duration of sensitivity to androgen depletion. The approach is safe, but long-term data are needed to assess the course in men with low PSA levels. A randomized trial showed similar survival time between patients treated with intermittent versus continuous treatment, with a slightly higher risk of prostate cancer–specific mortality in the intermittent group, and higher cardiovascular mortality in patients on continuous therapy. The intermittent therapy was better tolerated.

1	Outcomes of Androgen Depletion The anti–prostate cancer effects of the various androgen depletion/blockade strategies are similar, and the outcomes predictable: an initial response, then a period of stability in which tumor cells are dormant and nonproliferative, followed after a variable period of time by a rise in PSA and tumor regrowth as a castration-resistant lesion that for most men is invariably lethal. Androgen depletion is not curative because cells that survive castration are present when the disease is first diagnosed. Considered by disease manifestation, PSA levels return to normal in 60–70% of cases, and measurable lesions regress in about 50%; improvements in bone scan occur in 25% of cases, but the majority of cases remain stable. The duration of response and survival is inversely proportional to disease extent at the time androgen depletion is first started, whereas the degree of PSA decline at 6 months has been shown to be prognostic. In a large-scale trial, PSA nadir

1	proportional to disease extent at the time androgen depletion is first started, whereas the degree of PSA decline at 6 months has been shown to be prognostic. In a large-scale trial, PSA nadir proved prognostic.

1	An active question is whether hormones should be given in the adjuvant setting after surgery or radiation treatment of the primary tumor or whether to wait until PSA recurrence, metastatic disease, or symptoms are documented. Trials in support of early therapy have often been underpowered relative to the reported benefit or have been criticized on methodologic grounds. One trial showing a survival benefit for patients treated with radiation therapy and 3 years of androgen depletion, relative to radiation alone, was criticized for the poor outcomes of the control group. Another showing a survival benefit for patients with positive lymph nodes who were randomized to immediate medical or surgical castration compared to observation (p = .02) was criticized because the confidence intervals around the 5and 8-year survival distributions for the two groups overlapped. A large randomized study comparing early to late hormone treatment (orchiectomy or GnRH analogue) in patients with locally

1	around the 5and 8-year survival distributions for the two groups overlapped. A large randomized study comparing early to late hormone treatment (orchiectomy or GnRH analogue) in patients with locally advanced or asymptomatic metastatic disease showed that patients treated early were less likely to progress from M0 to M1 disease, to develop pain, and to die of prostate cancer. This trial was criticized because therapy was delayed “too long” in the late-treatment group. Noteworthy is that the American Society of Clinical Oncology Guidelines recommend deferring treatment until the disease has recurred and the prognosis has been reassessed. These guidelines do not support immediate therapy.

1	METASTATIC DISEASE: CASTRATE

1	Castration-resistant prostate cancer (CRPC) is defined as disease that progresses despite androgen suppression by medical or surgical therapies where the measured levels of testosterone are 50 ng/ mL or lower. The rise in PSA indicates continued signaling through the AR signaling axis, the result of a series of oncogenic changes that include overexpression of androgen biosynthetic enzymes that can lead to increased intratumoral androgens, and overexpression of the receptor itself that enables signaling to occur even in the setting of low levels of androgen. The majority of CRPC cases are not “hormone-refractory,” and considering them as such can deny patients safe and effective treatment. CRPC can manifest in many ways. For some, it is a rise in PSA with no change in radiographs and no new symptoms. In others, it is a rising PSA and progression in bone with or without symptoms of disease. Still others will show soft tissue disease with or without osseous metastases, and others have

1	new symptoms. In others, it is a rising PSA and progression in bone with or without symptoms of disease. Still others will show soft tissue disease with or without osseous metastases, and others have visceral spread.

1	For the individual patient, it is first essential to ensure that a castrate status be documented. Patients receiving an antiandrogen alone, whose serum testosterone levels are elevated, should be treated first with a GnRH analogue or orchiectomy and observed for response. Patients on an antiandrogen in combination with a GnRH analogue should have the antiandrogen discontinued, because approximately 20% will respond to the selective discontinuation of the antiandrogen.

1	Chemotherapy and New Agents Through 2009, docetaxel was the only systemic therapy proven to prolong life. As a single agent, the drug produced PSA declines in 50% of patients, measurable disease regression in 25%, and improvement in both preexisting pain and prevention of future cancer-related pain. Since then, six agents with diverse mechanisms of action that target the tumor itself or other aspects of the metastatic process have been proven to prolong life and were FDA approved. The first was sipuleucel-T, the first biologic approach shown to prolong life in which antigen-presenting cells are activated ex vivo, pulsed with antigen, and reinfused. The second, cabazitaxel, a non–cross-resistant taxane, was shown to be superior to mitoxantrone in the post-docetaxel setting. This was followed by the CYP17 inhibitor abiraterone acetate, which lowers androgen levels in the tumor, adrenal glands, and testis, and the next-generation antiandrogen enzalutamide, which not only has a higher

1	by the CYP17 inhibitor abiraterone acetate, which lowers androgen levels in the tumor, adrenal glands, and testis, and the next-generation antiandrogen enzalutamide, which not only has a higher binding affinity to the AR relative to first-generation compounds, but uniquely inhibits nuclear location and DNA binding of the receptor complex. Both abiraterone acetate and enzalutamide were first approved for postchemotherapy treated patients on the basis of placebo-controlled phase III trials—a further indication that these tumors are not uniformly hormone-refractory. The indication for abiraterone acetate was later expanded to the prechemotherapy setting, based on a second trial using a co-primary endpoint of radiographic progression–free survival and overall survival. Similar results were seen with enzalutamide, for which an expanded indication is also anticipated. Alpharadin (radium-223 chloride), an alpha-emitting bone-seeking radioisotope, has been shown to prolong life in patients

1	seen with enzalutamide, for which an expanded indication is also anticipated. Alpharadin (radium-223 chloride), an alpha-emitting bone-seeking radioisotope, has been shown to prolong life in patients with symptoms related to osseous disease. The alpharadin result validated the bone microenvironment as a therapeutic target independent of direct effects on the tumor itself, as no declines in PSA were observed in the trial. Notable is that in addition to a survival benefit, the drug also reduced the development of significant skeletal events.

1	Other bone-targeted agents, such as the bisphosphonates and the RANK ligand inhibitor denosumab, protect against bone loss associated with androgen depletion and also reduce skeletal-related events by targeting bone osteoclasts. In one trial, denosumab was shown to be superior to zoledronic acid with respect to skeletal-related events, but had a slightly higher frequency of osteonecrosis of the jaw.

1	In clinical practice, most men seek to avoid chemotherapy and are first treated with a biologic agent and/or newer hormonal agent approved for this indication. It is crucial to the management of the individual patient to define therapeutic objectives before initiating treatment, as there are defined standards of care for different disease manifestations. For example, sipuleucel-T is not indicated for patients with symptoms or visceral disease because the effects on the disease occur late. Similarly, alpharadin is not indicated for patients with disease that is predominantly in soft tissue or who have osseous disease that is not causing symptoms.

1	Pain Management Management of pain secondary to osseous metastatic disease is a critical part of therapy. Optimal palliation requires assessing whether the symptoms are from metastases that threaten or that are already affecting the spinal cord, the cauda equina, or the base of the skull, which are best treated with external-beam radiation, as are single sites of pain. Neurologic symptoms require emergency evaluation because loss of function may be permanent if not addressed quickly. Because the disease is often diffuse, palliation at one site is often followed by the emergence of symptoms in a separate site that had not received radiation. In these cases, bone-seeking radioisotopes such as alpharadin or the beta emitter 153Sm-EDTMP (Quadramet) can be considered in addition to abiraterone acetate, docetaxel, and mitoxantrone, each of which is formally approved for the palliation of pain due to prostate cancer metastases.

1	BPH is a pathologic process that contributes to the development of lower urinary tract symptoms in men. Such symptoms, arising from lower urinary tract dysfunction, are further subdivided into obstructive symptoms (urinary hesitancy, straining, weak stream, terminal dribbling, prolonged voiding, incomplete emptying) and irritative symptoms (urinary frequency, urgency, nocturia, urge incontinence, small voided volumes). Lower urinary tract symptoms and other sequelae of BPH are not just due to a mass effect, but are also likely due to a combination of the prostatic enlargement and age-related detrusor dysfunction.

1	The symptoms are generally measured using a validated, reproducible index that is designed to determine disease severity and response to therapy—the AUA’s Symptom Index (AUASI), also adopted as the International Prostate Symptom Score (IPSS) (Table 115–2). Serial AUASI is particularly useful in following patients as they are treated with various forms of therapy. Asymptomatic patients do not require treatment regardless of the size of the gland, whereas patients with an inability to urinate, gross hematuria, recurrent infection, or bladder stones may require surgery. In patients with 587 symptoms, uroflowmetry can identify those with normal flow rates who are unlikely to benefit from treatment, and bladder ultrasound can identify those with high postvoid residuals who may need intervention. Pressure-flow (urodynamic) studies detect primary bladder dysfunction. Cystoscopy is recommended if hematuria is documented and to assess the urinary outflow tract before surgery. Imaging of the

1	Pressure-flow (urodynamic) studies detect primary bladder dysfunction. Cystoscopy is recommended if hematuria is documented and to assess the urinary outflow tract before surgery. Imaging of the upper tracts is advised for patients with hematuria, a history of calculi, or prior urinary tract problems.

1	Symptomatic relief is the most common reason men seek treatment for BPH, and therefore the goal of therapy for BPH is usually relief of these symptoms. Alpha-adrenergic receptor antagonists are thought to treat the dynamic aspect of BPH by reducing sympathetic tone of the bladder outlet, thereby decreasing resistance and improving urinary flow. 5ARIs are thought to treat the static aspect of BPH by reducing prostate volume and having a similar, albeit delayed effect. They have also proven to be beneficial in the prevention of BPH progression, as measured by prostate volume, the risk of developing acute urinary retention, and the risk of having BPH-related surgery. The use of an alpha-adrenergic receptor antagonist and a 5ARI as combination therapy seeks to provide symptomatic relief while preventing progression of BPH.

1	Another class of medications that has shown improvement in lower urinary tract symptoms secondary to BPH is PDE5 inhibitors, used currently in the treatment of erectile dysfunction. All three of the PDE5 inhibitors available in the United States, sildenafil, vardenafil, and tadalafil, appear to be effective in the treatment of symp toms secondary to BPH. The use of PDE5 inhibitors is not without controversy, however, given the fact that short-acting phosphodiesterase inhibitors such as sildenafil need to be dosed separately from alpha blockers such as tamsulosin because of potential hypotensive effects. Newer classes of pharmacologic agents have been used to treat symptoms secondary to BPH. Symptoms due to BPH often coexist with symptoms due to overactive bladder, and the most common pharmacologic agents for the treatment of overactive bladder symptoms are anticholinergics. This has led to multiple studies evaluating the efficacy of anticholinergics for the treatment of lower urinary

1	agents for the treatment of overactive bladder symptoms are anticholinergics. This has led to multiple studies evaluating the efficacy of anticholinergics for the treatment of lower urinary tract symptoms secondary to BPH. Surgical therapy is now considered second-line therapy and is usually reserved for patients after a trial of medical therapy. The goal of surgical therapy is to reduce the size of the prostate, effectively reducing resistance to urine flow.

1	Surgical approaches include TURP, transurethral incision, or removal of the gland via a retropubic, suprapubic, or perineal approach. Also used are transurethral ultrasound-guided laser-induced prostatectomy (TULIP), stents, and hyperthermia. Benign and Malignant Diseases of the Prostate Less Than Less Than About Half More Than Almost Questions to Be Answered Not at All 1 Time in 5 Half the Time the Time Half the Time Always Over the past month, how often have you had a sensation of not emptying your bladder completely after you finished urinating? Over the past month, how often have you had to urinate again less than 2 h after you finished urinating? Over the past month, how often have you found you stopped and started again several times when you urinated? Over the past month, how often have you found it difficult to postpone urination? Over the past month, how often have you had a weak urinary stream?

1	Over the past month, how often have you had a weak urinary stream? Over the past month, how often have you had to push or strain to begin urination? Over the past month, how many times did you most typically get up to urinate from the time you went to bed at night until the time you got up in the morning? Sum of 7 circled numbers (AUA Symptom Score): ____ Abbreviation: AUA, American Urological Association. Source: MJ Barry et al: J Urol 148:1549, 1992. Used with permission.

1	Source: MJ Barry et al: J Urol 148:1549, 1992. Used with permission. 588 testicular Cancer Robert J. Motzer, Darren R. Feldman, George J. Bosl Primary germ cell tumors (GCTs) of the testis arising by the malig-nant transformation of primordial germ cells constitute 95% of all testicular neoplasms. Infrequently, GCTs arise from an extragonadal site, including the mediastinum, retroperitoneum, and, very rarely, the 116 pineal gland. This disease is notable for the young age of the afflicted patients, the totipotent capacity for differentiation of the tumor cells, and its curability; approximately 95% of newly diagnosed patients are cured. Experience in the management of GCTs leads to improved outcome.

1	The incidence of testicular GCT is now approximately 8000 cases annually in the United States, resulting in nearly 400 deaths. The tumor occurs most frequently in men between the ages of 20 and 40 years. A testicular mass in a male ≥50 years should be regarded as a lymphoma until proved otherwise. GCT is at least four to five times more common in white than in African-American males, and a higher incidence has been observed in Scandinavia and New Zealand than in the United States.

1	Cryptorchidism is associated with a several-fold higher risk of GCT. Abdominal cryptorchid testes are at a higher risk than inguinal crypt-orchid testes. Orchiopexy should be performed before puberty, if possible. Early orchiopexy reduces the risk of GCT and improves the ability to save the testis. An abdominal cryptorchid testis that cannot be brought into the scrotum should be removed. Approximately 2% of men with GCTs of one testis will develop a primary tumor in the other testis. Testicular feminization syndromes and family history increase the risk of testicular GCT, and Klinefelter’s syndrome is associated with mediastinal GCT. An isochromosome of the short arm of chromosome 12 [i(12p)] is pathognomonic for GCT. Excess 12p copy number, either in the form of i(12p) or as increased 12p on aberrantly banded marker chromosomes, occurs in nearly all GCTs, but the gene(s) on 12p involved in the pathogenesis are not yet defined.

1	A painless testicular mass is pathognomonic for a testicular malignancy. More commonly, patients present with testicular discomfort or swelling suggestive of epididymitis and/or orchitis. In this circumstance, a trial of antibiotics is reasonable. However, if symptoms persist or a residual abnormality remains, then testicular ultrasound examination is indicated. Ultrasound of the testis is indicated whenever a testicular malignancy is considered and for persistent or painful testicular swelling. If a testicular mass is detected, a radical inguinal orchiectomy should be performed. Because the testis develops from the gonadal ridge, its blood supply and lymphatic drainage originate in the abdomen and descend with the testis into the scrotum. An inguinal approach is taken to avoid breaching anatomic barriers and permitting additional pathways of spread.

1	Back pain from retroperitoneal metastases is common and must be distinguished from musculoskeletal pain. Dyspnea from pulmonary metastases occurs infrequently. Patients with increased serum levels of human chorionic gonadotropin (hCG) may present with gynecomastia. A delay in diagnosis is associated with a more advanced stage and possibly worse survival.

1	The staging evaluation for GCT includes a determination of serum levels of α fetoprotein (AFP), hCG, and lactate dehydrogenase (LDH). After orchiectomy, a computed tomography (CT) scan of the chest, abdomen, and pelvis is generally performed. Stage I disease is limited to the testis, epididymis, or spermatic cord. Stage II disease is limited to retroperitoneal (regional) lymph nodes. Stage III disease is disease outside the retroperitoneum, involving supradiaphragmatic nodal sites or viscera. The staging may be “clinical”—defined solely by physical examination, blood marker evaluation, and radiographs—or “pathologic”—defined by an operative procedure.

1	The regional draining lymph nodes for the testis are in the retro-peritoneum, and the vascular supply originates from the great vessels (for the right testis) or the renal vessels (for the left testis). As a result, the lymph nodes that are involved first by a right testicular tumor are the interaortocaval lymph nodes just below the renal vessels. For a left testicular tumor, the first involved lymph nodes are lateral to the aorta (para-aortic) and below the left renal vessels. In both cases, further retroperitoneal nodal spread is inferior, contralateral, and, less commonly, above the renal hilum. Lymphatic involvement can extend cephalad to the retrocrural, posterior mediastinal, and supraclavicular lymph nodes. Treatment is determined by tumor histology (seminoma versus nonseminoma) and clinical stage (Fig. 116-1).

1	GCTs are divided into nonseminoma and seminoma subtypes. Nonseminomatous GCTs are most frequent in the third decade of life and can display the full spectrum of embryonic and adult cellular differentiation. This entity comprises four histologies: embryonal carcinoma, teratoma, choriocarcinoma, and endodermal sinus (yolk sac) tumor. Choriocarcinoma, consisting of both cytotrophoblasts and syncytiotrophoblasts, represents malignant trophoblastic differentiation and is invariably associated with secretion of hCG. Endodermal sinus tumor is the malignant counterpart of the fetal yolk sac and is associated with secretion of AFP. Pure embryonal carcinoma may secrete AFP or hCG, or both; this pattern is biochemical evidence of differentiation. Teratoma is composed of somatic cell types derived from two or more germ layers (ectoderm, mesoderm, or endoderm). Each of these histologies may be present alone or in combination with others. Nonseminomatous GCTs tend to metastasize early to sites such

1	two or more germ layers (ectoderm, mesoderm, or endoderm). Each of these histologies may be present alone or in combination with others. Nonseminomatous GCTs tend to metastasize early to sites such as the retroperitoneal lymph nodes and lung parenchyma. Sixty percent of patients present with disease limited to the testis (stage I), 20% with retroperitoneal metastases (stage II), and 20% with more extensive supradiaphragmatic nodal or visceral metastases (stage III).

1	Seminoma represents approximately 50% of all GCTs, has a median age in the fourth decade, and generally follows a more indolent clinical course. Eighty percent of patients present with stage I disease, approximately 10% with stage II disease, and 10% with stage III disease; lung or other visceral metastases are rare. When a tumor contains both seminoma and nonseminoma components, patient management is directed by the more aggressive nonseminoma component.

1	Careful monitoring of the serum tumor markers AFP and hCG is essential in the management of patients with GCT, because these markers are important for diagnosis, as prognostic indicators, in monitoring treatment response, and in the early detection of relapse. Approximately 70% of patients presenting with disseminated nonseminomatous GCT have increased serum concentrations of AFP and/or hCG. Although hCG concentrations may be increased in patients with either nonseminoma or seminoma histology, the AFP concentration is increased only in patients with nonseminoma. The presence of an increased AFP level in a patient whose tumor shows only seminoma indicates that an occult nonseminomatous component exists, and the patient should be treated for nonseminomatous GCT. LDH levels are less specific than AFP or hCG but are increased in 50–60% patients with metastatic nonseminoma and in up to 80% of patients with advanced seminoma.

1	AFP, hCG, and LDH levels should be determined before and after orchiectomy. Increased serum AFP and hCG concentrations decay according to first-order kinetics; the half-life is 24–36 h for hCG and 5–7 days for AFP. AFP and hCG should be assayed serially during and after treatment. The reappearance of hCG and/or AFP or the failure of these markers to decline according to the predicted half-life is an indicator of persistent or recurrent tumor. Stage Extent of Disease Stage Extent of Disease Testis only,

1	Stage Extent of Disease Stage Extent of Disease Testis only, IIA IIC IB IIB Observation Chemotherapy or RT Observation Chemotherapy or RT Observation Retroperitoneal Nodes 2-5 cm RT or Chemotherapy RTRetroperitoneal Nodes < 2 cm RPLND +/– adjuvant Chemotherapy or Chemotherapy, often followed by RPLND Retroperitoneal Nodes > 5 cm Chemotherapy Chemotherapy, often followed by RPLND Testis only, with vascular/lymphatic invasion (T2), or extension through tunica albuginea (T2), or involvement of spermatic cord (T3) or scrotum (T4) RPLND or Chemotherapy Treatment Option Distant Metastases Nonseminoma Seminoma Chemotherapy, often followed by RPLND FIGURE 116-1 Germ cell tumor staging and treatment. RPLND, retroperitoneal lymph node dissection; RT, radiotherapy.

1	Patients with radiographs and physical examination showing no evidence of disease and serum AFP and hCG concentrations that are either normal or declining to normal according to the known half-life have clinical stage I disease. Approximately 20–50% of such patients will have retroperitoneal lymph node metastases (pathologic stage II) but will still be cured in over 95% of cases. Depending on risk of relapse, which is determined by the pathology (see below), surveillance, a nerve-sparing retroperitoneal lymph node dissection (RPLND), or adjuvant chemotherapy (one to two cycles of bleomycin, etoposide, and cisplatin [BEP]) may be appropriate choices depending on the availability of surgical expertise and patient and physician preference. If the primary tumor shows no evidence for lymphatic or vascular invasion and is limited to the testis (T1, clinical stage IA), then the risk of relapse is only 10–20%. Because over 80% of patients with clinical stage IA nonseminoma are cured with

1	or vascular invasion and is limited to the testis (T1, clinical stage IA), then the risk of relapse is only 10–20%. Because over 80% of patients with clinical stage IA nonseminoma are cured with orchiectomy alone and there is no survival advantage to RPLND (or adjuvant chemotherapy), surveillance is the preferred treatment option. This avoids overtreatment with the potential for both acute and long-term toxicities (see below). Surveillance requires patients to be carefully followed with periodic chest radiography, physical examination, CT scan of the abdomen, and serum tumor marker determinations. The median time to relapse is approximately 7 months, and late relapses (>2 years) are rare. Noncompliant patients can be considered for RPLND or adjuvant BEP.

1	If lymphatic or vascular invasion is present or the tumor extends through the tunica, spermatic cord, or scrotum (T2 through T4, clinical stage IB), then the risk of relapse is approximately 50%, and RPLND and adjuvant chemotherapy can be considered. Relapse rates are reduced to 3–5% after one to two cycles of adjuvant BEP. All three approaches (surveillance, RPLND, and adjuvant BEP) should cure >95% of patients with clinical stage IB disease.

1	RPLND is the standard operation for removal of the regional lymph nodes of the testis (retroperitoneal nodes). The operation removes the lymph nodes draining the primary site and the nodal groups adjacent to the primary landing zone. The standard (modified bilateral) RPLND removes all node-bearing tissue down to the bifurcation of the great vessels, including the ipsilateral iliac nodes. The major long-term effect of this operation is retrograde ejaculation with resultant infertility. Nerve-sparing RPLND can preserve anterograde ejaculation in ~90% of patients. Patients with pathologic stage I disease are observed, and only the <10% who relapse require additional therapy. If nodes are found to be involved at RPLND, then a decision regarding adjuvant chemotherapy is made on the basis of the extent of retroperitoneal disease (see “Stage II Nonseminoma” below). Hence, because less than 20% of patients require chemotherapy, of the three approaches, RPLND results in the lowest number of

1	the extent of retroperitoneal disease (see “Stage II Nonseminoma” below). Hence, because less than 20% of patients require chemotherapy, of the three approaches, RPLND results in the lowest number of patients at risk for the late toxicities of chemotherapy.

1	Patients with limited, ipsilateral retroperitoneal adenopathy ≤2 cm in largest diameter and normal levels of AFP and hCG can be treated with either a modified bilateral nerve-sparing RPLND or chemotherapy. The local recurrence rate after a properly performed RPLND is very low. Depending on the extent of disease, the postoperative management options include either surveillance or two cycles of adjuvant chemotherapy. Surveillance is the preferred approach for patients with resected “low-volume” metastases (tumor nodes ≤2 cm in diameter and <6 nodes involved) because the probability of relapse is one-third or less. For those who relapse, risk-directed chemotherapy is indicated (see section on advanced GCT below). Because relapse occurs in ≥50% of patients with “highvolume” metastases (>6 nodes involved, or any involved node >2 cm in largest diameter, or extranodal tumor extension), two cycles of adjuvant chemotherapy should be considered, as it results in a cure in ≥98% of patients.

1	nodes involved, or any involved node >2 cm in largest diameter, or extranodal tumor extension), two cycles of adjuvant chemotherapy should be considered, as it results in a cure in ≥98% of patients. Regimens consisting of etoposide plus cisplatin (EP) with or without bleomycin every 3 weeks are effective and well tolerated.

1	Increased levels of either AFP or hCG imply metastatic disease outside the retroperitoneum; full-dose (not adjuvant) chemotherapy is used in this setting. Primary management with chemotherapy is also favored for patients with larger (>2 cm) or bilateral retroperitoneal nodes (see section on advanced GCT below).

1	Inguinal orchiectomy followed by immediate retroperitoneal radiation therapy or surveillance with treatment at relapse both result in cure in nearly 100% of patients with stage I seminoma. Historically, radiation was the mainstay of treatment, but the reported association between radiation and secondary malignancies and the absence of a survival advantage of radiation over surveillance has led many to favor surveillance for compliant patients. Approximately 15% of patients relapse, which is usually treated with chemotherapy. Longterm follow-up is essential, because approximately 30% of relapses occur after 2 years and 5% occur after 5 years. A single dose of carboplatin has also been investigated as an alternative to radiation therapy; the outcome was similar, but long-term safety data are lacking, and the retroperitoneum remained the most frequent site of relapse.

1	Generally, nonbulky retroperitoneal disease (stage IIA and small IIB) is treated with retroperitoneal radiation therapy. Approximately 90% of patients achieve relapse-free survival with retroperitoneal masses <3 cm in diameter. Due to higher relapse rates after radiation for bulkier disease, initial chemotherapy is preferred for all stage IIC and some stage IIB patients. Chemotherapy has been studied as an alternative to radiation for stage IIA and small stage IIB seminoma with lower recurrence rates compared with historical controls. These results, combined with studies demonstrating a threefold increase in the incidence of secondary malignancies and cardiovascular disease among patients who receive both radiation and chemotherapy (patients relapsing after radiation fall into this category), have led some experts to prefer chemotherapy for all stage II seminomas.

1	Regardless of histology, all patients with stage IIC and stage III and most with stage IIB GCT are treated with chemotherapy. Combination chemotherapy programs based on cisplatin at doses of 100 mg/m2 plus etoposide at doses of 500 mg/m2 per cycle cure 70–80% of such patients, with or without bleomycin, depending on risk stratification (see below). A complete response (the complete disappearance of all clinical evidence of tumor on physical examination and radiography plus normal serum levels of AFP and hCG for ≥1 month) occurs after chemotherapy alone in ~60% of patients, and another 10–20% become disease free with surgical resection of residual masses containing viable GCT. Lower doses of cisplatin result in inferior survival rates.

1	The toxicity of four cycles of the BEP is substantial. Nausea, vomiting, and hair loss occur in most patients, although nausea and vomiting have been markedly ameliorated by modern antiemetic regimens. Myelosuppression is frequent, and symptomatic bleomycin pulmonary toxicity occurs in ~5% of patients. Treatment-induced mortality due to neutropenia with septicemia or bleomycin-induced pulmonary failure occurs in 1–3% of patients. Dose reductions for myelosuppression are rarely indicated. Long-term permanent toxicities include nephrotoxicity (reduced glomerular filtration and persistent magnesium wasting), ototoxicity, peripheral neuropathy, and infertility. When bleomycin is administered by weekly bolus injection, Raynaud’s phenomenon appears in 5–10% of patients. Other evidence of small blood vessel damage, such as transient ischemic attacks and myocardial infarction, is seen less often.

1	Because not all patients are cured and treatment may cause significant toxicities, patients are stratified into “good-risk,” “intermediaterisk,” and “poor-risk” groups according to pretreatment clinical features established by the International Germ Cell Cancer Consensus Group (Table 116-1). For good-risk patients, the goal is to achieve necessary. If viable tumor is present but is completely excised, two 591 additional cycles of chemotherapy are given. If the initial histology is pure seminoma, mature teratoma is Abbreviations: AFP, α fetoprotein; hCG, human chorionic gonadotropin; LDH, lactate dehydrogenase. Source: From International Germ Cell Cancer Consensus Group. maximum efficacy with minimal toxicity. For intermediateand poor-risk patients, the goal is to identify more effective therapy with tolerable toxicity.

1	maximum efficacy with minimal toxicity. For intermediateand poor-risk patients, the goal is to identify more effective therapy with tolerable toxicity. The marker cut offs are included in the TNM (primary tumor, regional nodes, metastasis) staging of GCT. Hence, TNM stage groupings are based on both anatomy (site and extent of disease) and biology (marker status and histology). Seminoma is either goodor intermediate-risk, based on the absence or presence of nonpulmonary visceral metastases. No poor-risk category exists for seminoma. Marker levels and primary site play no role in defining risk for seminoma. Nonseminomas have good-, intermediate-, and poor-risk categories based on the primary site of the tumor, the presence or absence of nonpulmonary visceral metastases, and marker levels.

1	For ~90% of patients with good-risk GCTs, four cycles of EP or three cycles of BEP produce durable complete responses, with minimal acute and chronic toxicity, and a low relapse rate. Pulmonary toxicity is absent when bleomycin is not used and is rare when therapy is limited to 9 weeks; myelosuppression with neutropenic fever is less frequent; and the treatment mortality rate is negligible. Approximately 75% of intermediate-risk patients and 50% of poor-risk patients achieve durable complete remission with four cycles of BEP, and no regimen has proved superior.

1	POSTCHEMOTHERAPY SURGERY Resection of residual metastases after the completion of chemotherapy is an integral part of therapy. If the initial histology is nonseminoma and the marker values have normalized, all sites of residual disease should be resected. In general, residual retroperitoneal disease requires a modified bilateral RPLND. Thoracotomy (unilateral or bilateral) and neck dissection are less frequently required to remove residual mediastinal, pulmonary parenchymal, or cervical nodal disease. Viable tumor (seminoma, embryonal carcinoma, yolk sac tumor, or choriocarcinoma) will be present in 15%, mature teratoma in 40%, and necrotic debris and fibrosis in 45% of resected specimens. The frequency of teratoma or viable disease is highest in residual mediastinal tumors. If necrotic debris or mature teratoma is present, no further chemotherapy is rarely present, and the most frequent finding is necrotic debris. For residual retroperitoneal disease, a complete RPLND is technically

1	or mature teratoma is present, no further chemotherapy is rarely present, and the most frequent finding is necrotic debris. For residual retroperitoneal disease, a complete RPLND is technically difficult due to extensive postchemotherapy fibrosis. Observation is recommended when no radiographic abnormality exists on CT scan. Positive findings on a positron emission tomography (PET) scan correlate with viable seminoma in residua and mandate surgical excision or biopsy.

1	Of patients with advanced GCT, 20–30% fail to achieve a durable complete response to first-line chemotherapy. A combination of vinblastine, ifosfamide, and cisplatin (VeIP) will cure approximately 25% of patients as a second-line therapy. Patients are more likely to achieve a durable complete response if they had a testicular primary tumor and relapsed from a prior complete remission to first-line cisplatin-containing chemotherapy. Substitution of paclitaxel for vinblastine (TIP) in this setting was associated with durable remission in nearly two-thirds of patients. In contrast, for patients with a primary mediastinal nonseminoma or who did not achieve a complete response with first-line chemotherapy, then VeIP standard-dose salvage therapy is rarely beneficial. Such patients are usually managed with high-dose chemotherapy and/or surgical resection.

1	Chemotherapy consisting of dose-intensive, high-dose carboplatin plus high-dose etoposide, with peripheral blood stem cell support, induces a complete response in 25–40% of patients who have progressed after ifosfamide-containing salvage chemotherapy. Approximately one-half of the complete responses will be durable. High-dose therapy is standard of care for this patient population and has been suggested as the treatment of choice for all patients with relapsed or refractory disease. Paclitaxel is active when incorporated into high-dose combination programs. Cure is still possible in some relapsed patients.

1	The prognosis and management of patients with extragonadal GCT depends on the tumor histology and site of origin. All patients with a diagnosis of extragonadal GCT should have a testicular ultrasound examination. Nearly all patients with retroperitoneal or mediastinal seminoma achieve a durable complete response to BEP or EP. The clinical features of patients with primary retroperitoneal nonseminoma GCT are similar to those of patients with a primary tumor of testis origin, and careful evaluation will find evidence of a primary testicular GCT in about two-thirds of cases. In contrast, a primary mediastinal nonseminomatous GCT is associated with a poor prognosis; one-third of patients are cured with standard therapy (four cycles of BEP). Patients with newly diagnosed mediastinal nonseminoma are considered to have poor-risk disease and should be considered for clinical trials testing regimens of possibly greater efficacy. In addition, mediastinal nonseminoma is associated with

1	are considered to have poor-risk disease and should be considered for clinical trials testing regimens of possibly greater efficacy. In addition, mediastinal nonseminoma is associated with hematologic disorders, including acute myelogenous leukemia, myelodysplastic syndrome, and essential thrombocytosis unrelated to previous chemotherapy. These hematologic disorders are very refractory to treatment. Nonseminoma of any primary site may change into other malignant histologies such as embryonal rhabdomyosarcoma or adenocarcinoma. This is called malignant transformation. i(12p) has been identified in the transformed cell type, indicating GCT clonal origin.

1	A group of patients with poorly differentiated tumors of unknown histogenesis, midline in distribution, and not associated with secretion of AFP or hCG has been described; a few (10–20%) are cured by standard cisplatin-containing chemotherapy. An i(12p) is present in ~25% of such tumors (the fraction that are cisplatin-responsive), confirming their origin from primitive germ cells. This finding is also predictive of the response to cisplatin-based chemotherapy and resulting long-term survival. These tumors are heterogeneous; neuroepithelial tumors and lymphoma may also present in this fashion.

1	592 FERTILITY Infertility is an important consequence of the treatment of GCTs. Preexisting infertility or impaired fertility is often present. Azoospermia and/or oligospermia are present at diagnosis in at least 50% of patients with testicular GCTs. Ejaculatory dysfunction is associated with RPLND, and germ cell damage may result from cisplatin-containing chemotherapy. Nerve-sparing techniques to preserve the retroperitoneal sympathetic nerves have made retrograde ejaculation less likely in the subgroups of patients who are candidates for this operation. Spermatogenesis does recur in some patients after chemotherapy. However, because of the significant risk of impaired reproductive capacity, semen analysis and cryopreservation of sperm in a sperm bank should be recommended to all patients before treatment.

1	part 7 gynecologic malignancies Michael V. Seiden OVARIAN CANCER INCIDENCE AND PATHOLOGY Cancer arising in or near the ovary is actually a collection of diverse malignancies. This collection of malignancies, often referred to as 117 “ovary cancer,” is the most lethal gynecologic malignancy in the United States and other countries that routinely screen women for cervical neoplasia. In 2014, it was estimated that there were 21,980 cases of ovarian cancer with 14,270 deaths in the United States. The ovary is a complex and dynamic organ and, between the ages of approximately 11 and 50 years, is responsible for follicle maturation associated with egg maturation, ovulation, and cyclical sex steroid hormone production. These complex and linked biologic functions are coordinated through a variety of cells within the ovary, each of which possesses neoplastic potential. By far the most common and most lethal of the ovarian neoplasms arise from the ovarian epithelium or, alternatively, the

1	of cells within the ovary, each of which possesses neoplastic potential. By far the most common and most lethal of the ovarian neoplasms arise from the ovarian epithelium or, alternatively, the neighboring specialized epithelium of the fallopian tube, uterine corpus, or cervix. Epithelial tumors may be benign (50%), malignant (33%), or of borderline malignancy (16%). Age influences risk of malignancy; tumors in younger women are more likely benign. The most common of the ovarian epithelial malignancies are serous tumors (50%); tumors of mucinous (25%), endometrioid (15%), clear cell (5%), and transitional cell histology or Brenner tumor (1%) represent smaller proportions of epithelial ovarian tumors. In contrast, stromal tumors arise from the steroid hormone–producing cells and likewise have different phenotypes and clinical presentations largely dependent on the type and quantity of hormone production. Tumors arising in the germ cell are most similar in biology and behavior to

1	have different phenotypes and clinical presentations largely dependent on the type and quantity of hormone production. Tumors arising in the germ cell are most similar in biology and behavior to testicular tumors in males (Chap. 116).

1	Tumors may also metastasize to the ovary from breast, colon, appendiceal, gastric, and pancreatic primaries. Bilateral ovarian masses from metastatic mucin-secreting gastrointestinal cancers are termed Krukenberg tumors.

1	OVARIAN CANCER OF EPITHELIAL ORIGIN Epidemiology and Pathogenesis A female has approximately a 1 in 72 lifetime risk (1.6%) of developing ovarian cancer, with the majority of affected women developing epithelial tumors. Each of the histologic variants of epithelial tumors is distinct with unique molecular features. As a group of malignancies, epithelial tumors of the ovary have a peak incidence in women in their sixties, although age at presentation can range across the extremes of adult life, with cases being reported in women in their twenties to nineties. Each histologic subtype of ovarian cancer likely has its own associated risk factors. Serous cancer, the most common type of epithelial ovarian cancer, is seen with increased frequency in women who are nulliparous or have a history of use of talc agents applied to the perineum; other risk factors include obesity and probably hormone replacement therapy. Protective factors include the use of oral contraceptives, multiparity, and

1	of use of talc agents applied to the perineum; other risk factors include obesity and probably hormone replacement therapy. Protective factors include the use of oral contraceptives, multiparity, and breast-feeding. These protective factors are thought to work through suppression of ovulation and perhaps the associated reduction of ovulation associated inflammation of the ovarian epithelium or, alternatively, the serous epithelium located within the fimbriae of the fallopian tube. Other protective factors, such as fallopian tube ligation, are thought to protect the ovarian epithelium (or perhaps the distal fallopian tube fimbriae) from carcinogens that migrate from the vagina to the tubes and ovarian surface epithelium. Mucinous tumors are more frequent in women with a history of cigarette smoking, whereas endometrioid and clear cell tumors are more frequent in women with a history of endometriosis.

1	Considerable evidence now suggests that the precursor cell to serous carcinoma of the ovary might actually arise in the fimbria of the fallopian tube with extension or metastasis to the ovarian surface or capture of preneoplastic or neoplastic exfoliating tubal cells into an involuting ovarian follicle around the time of ovulation. Careful histologic and molecular analysis of tubal epithelium demonstrates molecular and histologic abnormalities, termed serous tubular intraepithelial carcinoma (STIC) lesions, in a high proportion of women undergoing risk-reducing salpingo-oophorectomies in the context of high-risk germline mutations in BRCA1 and BRCA2, as well as a modest proportion of women with ovarian cancer in the absence of such mutations. Genetic Risk Factors A variety of genetic syndromes substan tially increase a woman’s risk of developing ovarian cancer.

1	Approximately 10% of women with ovarian cancer have a germline mutation in one of two DNA repair genes: BRCA1 (chromosome 17q12-21) or BRCA2 (chromosome 13q12-13). Individuals inheriting a single copy of a mutant allele have a very high incidence of breast and ovarian cancer. Most of these women have a family history that is notable for multiple cases of breast and/or ovarian cancer, although inheritance through male members of the family can camouflage this genotype through several generations. The most common malignancy in these women is breast carcinoma, although women harboring germline BRCA1 mutations have a marked increased risk of developing ovarian malignancies in their forties and fifties with a 30–50% lifetime risk of developing ovarian cancer. Women harboring a mutation in BRCA2 have a lower penetrance of ovarian cancer with perhaps a 20–40% chance of developing this malignancy, with onset typically in their fifties or sixties. Women with a BRCA2 mutation also are at

1	BRCA2 have a lower penetrance of ovarian cancer with perhaps a 20–40% chance of developing this malignancy, with onset typically in their fifties or sixties. Women with a BRCA2 mutation also are at slightly increased risk of pancreatic cancer. Likewise women with mutations in the DNA mismatch repair genes associated with Lynch syndrome, type 2 (MSH2, MLH1, MLH6, PMS1, PMS2) may have a risk of ovarian cancer as high as 1% per year in their forties and fifties. Finally, a small group of women with familial ovarian cancer may have mutations in other BRCA-associated genes such as RAD51, CHK2, and others. Screening studies in this select population suggest that current screening techniques, including serial evaluation of the CA-125 tumor marker and ultrasound, are insufficient at detecting early-stage and curable disease, so women with these germline mutations are advised to undergo prophylactic removal of ovaries and fallopian tubes typically after completing childbearing and ideally

1	early-stage and curable disease, so women with these germline mutations are advised to undergo prophylactic removal of ovaries and fallopian tubes typically after completing childbearing and ideally before age 35–40 years. Early prophylactic oophorectomy also protects these women from subsequent breast cancer with a reduction of breast cancer risk of approximately 50%.

1	Presentation Neoplasms of the ovary tend to be painless unless they undergo torsion. Symptoms are therefore typically related to compression of local organs or due to symptoms from metastatic disease. Women with tumors localized to the ovary do have an increased incidence of symptoms including pelvic discomfort, bloating, and perhaps changes in a woman’s typical urinary or bowel pattern. Unfortunately, these symptoms are frequently dismissed by either the woman or her health care team. It is believed that high-grade tumors metastasize early in the neoplastic process. Unlike other epithelial malignancies, these tumors tend to exfoliate throughout the peritoneal cavity and thus present with symptoms associated with disseminated intraperitoneal tumors. The most common symptoms at presentation include a multimonth period of progressive complaints that typically include some combination of heartburn, nausea, early satiety, indigestion, constipation, and abdominal pain. Signs include the

1	include a multimonth period of progressive complaints that typically include some combination of heartburn, nausea, early satiety, indigestion, constipation, and abdominal pain. Signs include the rapid increase in abdominal girth due to the accumulation of ascites that typically alerts the patient and her physician that the concurrent gastrointestinal symptoms are likely associated with serious pathology. Radiologic evaluation typically demonstrates a complex adnexal mass and ascites. Laboratory evaluation usually demonstrates a markedly elevated CA-125, a shed mucin (Muc 16) associated with, but not specific for, ovarian cancer. Hematogenous and lymphatic spread are seen but are not the typical presentation. Ovarian cancers are divided into four stages, with stage I tumors confined to the ovary, stage II malignancies confined to the pelvis, and stage III tumors confined to the peritoneal cavity (Table 117-1). These three stages are subdivided, with the most common presentation, stage

1	ovary, stage II malignancies confined to the pelvis, and stage III tumors confined to the peritoneal cavity (Table 117-1). These three stages are subdivided, with the most common presentation, stage IIIC, defined as tumors with bulky intraperitoneal disease. About 60% of women present with stage IIIC disease. Stage IV disease includes women with parenchymal metastases (liver, lung, spleen) or, alternatively, abdominal wall or pleural disease. The 40% not presenting with stage IIIC disease are roughly evenly distributed among the other stages, although mucinous and clear cell tumors are overrepresented in stage I tumors.

1	Screening Ovarian cancer is the fifth most lethal malignancy in women in the United States. It is curable in early stages, but seldom curable in advanced stages; hence, the development of effective screening strategies is of considerable interest. Furthermore, the ovary is well visualized with a variety of imaging techniques, most notably trans-vaginal ultrasound. Early-stage tumors often produce proteins that can be measured in the blood such as CA-125 and HE-4. Nevertheless, the incidence of ovarian cancer in the middle-aged female population is low, with only approximately 1 in 2000 women between the ages of 50 and 60 carrying an asymptomatic and undetected tumor. Thus effective screening techniques must be sensitive but, more importantly, highly specific to minimize the number of false-positive results. Even a screening test with 98% specificity and 50% sensitivity would have a positive predictive value of only about 1%. A large randomized study of active screening versus usual

1	results. Even a screening test with 98% specificity and 50% sensitivity would have a positive predictive value of only about 1%. A large randomized study of active screening versus usual standard care demonstrated that a screening program consisting of six annual CA-125 measurements and four annual transvaginal ultrasounds in a population of women age 55–74 was not effective at reducing death from ovarian cancer and was associated with significant morbidity in the screened arm due to complications associated with diagnostic testing in the screened group. Although ongoing studies are evaluating the utility of alternative screening strategies, currently screening of normal-risk women is not recommended outside of a clinical trial.

1	In women presenting with a localized ovarian mass, the principal diagnostic and therapeutic maneuver is to determine if the tumor is benign or malignant and, in the event that the tumor is malignant, whether the tumor arises in the ovary or is a site of metastatic disease. Metastatic disease to the ovary can be seen from primary tumors of the colon, appendix, stomach (Krukenberg tumors), and breast. Typically women undergo a unilateral salpingo-oophorectomy, and if pathology reveals a primary ovarian malignancy, then the procedure is followed by a hysterectomy, removal of the remaining tube and ovary, omentectomy, and pelvic node sampling along with some random biopsies of the peritoneal cavity. This extensive surgical procedure is performed because approximately 30% of tumors that by visual inspection appear to be confined to the ovary have already disseminated to the peritoneal cavity and/or surrounding lymph nodes.

1	If there is evidence of bulky intraabdominal disease, a comprehensive attempt at maximal tumor cytoreduction is attempted even if it involves partial bowel resection, splenectomy, and in certain cases more extensive upper abdominal surgery. The ability to debulk metastatic ovarian cancer to minimal visible disease is associated with an improved prognosis compared with women left with visible disease. Patients without gross residual disease after resection have a median survival of 39 months, compared with 17 months for those left with macroscopic tumor. Once tumors have been surgically debulked, women receive therapy with a platinum agent, typically a taxane. Debate continues as to whether this therapy should be delivered intravenously or, alternatively, whether some of the therapy should be delivered directly into the peritoneal cavity via a catheter. Three randomized studies have demonstrated improved survival with intraperitoneal therapy, but this approach is still not widely

1	be delivered directly into the peritoneal cavity via a catheter. Three randomized studies have demonstrated improved survival with intraperitoneal therapy, but this approach is still not widely accepted due to technical challenges associated with this delivery route and increased toxicity. In women who present with bulky intraabdominal disease, an alternative approach is to treat with platinum plus a taxane for several cycles before attempting a surgical debulking procedure (neoadjuvant therapy). Subsequent surgical procedures are more effective at leaving the patient without gross residual tumor and appear to be less morbid. Two studies have demonstrated that the neoadjuvant approach is associated with an overall survival that is comparable to the traditional approach of primary surgery followed by chemotherapy.

1	With optimal debulking surgery and platinum-based chemotherapy (usually carboplatin dosed to an area under the curve [AUC] of 6 plus paclitaxel 175 mg/m2 by 3-h infusion in 21-day cycles), 70% of women who present with advanced-stage tumors respond, and 40–50% experience a complete remission with normalization of their CA-125, computed tomography (CT) scans, and physical examination. Unfortunately, a small proportion of women who obtain a complete response to therapy will remain in remission. Disease recurs within 1–4 years from the completion of their primary therapy in 75% of the complete responders. CA-125 levels often increase as a first sign of relapse; however, data are not clear that early intervention in relapsing patients influences survival. Recurrent disease is effectively managed, but not cured, with a variety of chemotherapeutic agents. Eventually all women with recurrent disease develop chemotherapy-refractory disease at which point refractory ascites, poor bowel

1	managed, but not cured, with a variety of chemotherapeutic agents. Eventually all women with recurrent disease develop chemotherapy-refractory disease at which point refractory ascites, poor bowel motility, and obstruction or pseudoobstruction due to a 594 tumor-infiltrated aperistaltic bowel are common. Limited surgery to relieve intestinal obstruction, localized radiation therapy to relieve pressure or pain from masses, or palliative chemotherapy may be helpful. Agents with >15% response rates include gemcitabine, topotecan, liposomal doxorubicin, pemetrexed, and bevacizumab. Approximately 10% of ovarian cancers are HER2/neu positive, and trastuzumab may induce responses in this subset. Five-year survival correlates with the stage of disease: stage I, 85–90%; stage II, 70–80%; stage III, 20–50%; and stage IV, 1–5% (Table 117-1). Low-grade serous tumors are molecularly distinct from high-grade serous tumors and are, in general, poorly responsive to chemotherapy. Targeted therapies

1	20–50%; and stage IV, 1–5% (Table 117-1). Low-grade serous tumors are molecularly distinct from high-grade serous tumors and are, in general, poorly responsive to chemotherapy. Targeted therapies focused on inhibiting kinases downstream of RAS and BRAF are being tested. Patients with tumors of low malignant potential are managed by surgery; chemotherapy and radiation therapy do not improve survival.

1	OVARIAN SEX CORD AND STROMAL TUMORS Epidemiology, Presentation, and Predisposing Syndromes Approximately 7% of ovarian neoplasms are stromal or sex cord tumors, with approximately 1800 cases expected each year in the United States. Ovarian stromal tumors or sex cord tumors are most common in women in their fifties or sixties, but tumors can present in the extremes of age, including the pediatric population. These tumors arise from the mesenchymal components of the ovary, including steroid-producing cells as well as fibroblasts. Essentially all of these tumors are of low malignant potential and present as unilateral solid masses. Three clinical presentations are common: the detection of an abdominal mass; abdominal pain due to ovarian torsion, intratumoral hemorrhage, or rupture; or signs and symptoms due to hormonal production by these tumors.

1	The most common hormone-producing tumors include thecomas, granulosa cell tumor, or juvenile granulosa tumors in children. These estrogen-producing tumors often present with breast tenderness as well as isosexual precocious pseudopuberty in children, menometrorrhagia, oligomenorrhea, or amenorrhea in premenopausal women, or alternatively as postmenopausal bleeding in older women. In some women, estrogen-associated secondary malignancies, such as endometrial or breast cancer, may present as synchronous malignancies. Alternatively, endometrial cancer may serve as the presenting malignancy with evaluation subsequently identifying a unilateral solid ovarian neoplasm that proves to be an occult granulosa cell tumor. Sertoli-Leydig tumors often present with hirsutism, virilization, and occasionally Cushing’s syndrome due to increased production of testosterone, androstenedione, or other 17-ketosteroids. Hormonally inert tumors include fibroma that presents as a solitary mass often in

1	Cushing’s syndrome due to increased production of testosterone, androstenedione, or other 17-ketosteroids. Hormonally inert tumors include fibroma that presents as a solitary mass often in association with ascites and occasionally hydrothorax also known as Meigs’ syndrome. A subset of these tumors present in individuals with a variety of inherited disorders that predispose them to mesenchymal neoplasia. Associations include juvenile granulosa cell tumors and perhaps Sertoli-Leydig tumors with Ollier’s disease (multiple enchondromatosis) or Maffucci’s syndrome, ovarian sex cord tumors with annular tubules with Peutz-Jeghers syndrome, and fibromas with Gorlin’s disease. Essentially all granulosa tumors and a minority of juvenile granulosa cell tumors and thecomas have a defined somatic point mutation in the FOXL2 gene at C134W generated by replacement of cysteine with a guanine at position 402. About 30% of Sertoli-Leydig tumors harbor a mutation in the RNA-processing gene DICER in the

1	mutation in the FOXL2 gene at C134W generated by replacement of cysteine with a guanine at position 402. About 30% of Sertoli-Leydig tumors harbor a mutation in the RNA-processing gene DICER in the RNAIIIb domain.

1	The mainstay of treatment for sex cord tumors is surgical resection. Most women present with tumors confined to the ovary. For the small subset of women who present with metastatic disease or develop evidence of tumor recurrence after primary resection, survival is still typically long, often in excess of a decade. Because these tumors are slow growing and relatively refractory to chemotherapy, women with metastatic disease are often debulked because disease is usually peritoneal-based (as with epithelial ovarian cancer). Definitive data that surgical debulking of metastatic or recurrent disease prolongs survival are lacking, but ample data document women who have survived years or, in some cases, decades after resection of recurrent disease. In addition, large peritoneal-based metastases also have a proclivity for hemorrhage, sometimes with catastrophic complications. Chemotherapy is occasionally effective, and women tend to receive regimens designed to treat epithelial or germ cell

1	also have a proclivity for hemorrhage, sometimes with catastrophic complications. Chemotherapy is occasionally effective, and women tend to receive regimens designed to treat epithelial or germ cell tumors. Bevacizumab has some activity in clinical trials but is not approved for this specific indication. These tumors often produce high levels of müllerian inhibiting substance (MIS), inhibin, and, in the case of Sertoli-Leydig tumors, α fetoprotein (AFP). These proteins are detectable in serum and can be used as tumor markers to monitor women for recurrent disease because the increase or decrease of these proteins in the serum tends to reflect the changing bulk of systemic tumor.

1	Germ cell tumors, like their counterparts in the testis, are cancers of germ cells. These totipotent cells contain the programming for differentiation to essentially all tissue types, and hence the germ cell tumors include a histologic menagerie of bizarre tumors, including benign teratomas and a variety of malignant tumors, such as immature teratomas, dysgerminomas, yolk sac malignancies, and choriocarcinomas. Benign teratoma (or dermoid cyst) is the most common germ cell neoplasm of the ovary and often presents in young woman. These tumors include a complex mixture of differentiated tissue including tissues from all three germ layers. In older women, these differentiated tumors can develop malignant transformation, most commonly squamous cell carcinomas. Malignant germ cell tumors include dysgerminomas, yolk sac tumors, immature teratomas, and embryonal carcinoma and choriocarcinomas. There are no known genetic abnormalities that unify these tumors. A subset of dysgerminomas harbor

1	dysgerminomas, yolk sac tumors, immature teratomas, and embryonal carcinoma and choriocarcinomas. There are no known genetic abnormalities that unify these tumors. A subset of dysgerminomas harbor mutations in c-Kit oncogenes (as seen in gastrointestinal stromal tumors [GIST]), whereas a subset of germ cell tumors have isochromosome 12 abnormalities, as seen in testicular malignancies. In addition, a subset of dysgerminomas is associated with dysgenetic ovaries. Identification of a dysgerminoma arising in genotypic XY gonads is important in that it highlights the need to identify and remove the contralateral gonad due to risk of gonadoblastoma.

1	Presentation Germ cell tumors can present at all ages, but the peak age of presentation tends to be in females in their late teens or early twenties. Typically these tumors will become large ovarian masses, which eventually present as palpable low abdominal or pelvic masses. Like sex cord tumors, torsion or hemorrhage may present urgently or emergently as acute abdominal pain. Some of these tumors produce elevated levels of human chorionic gonadotropin (hCG), which can lead to isosexual precocious puberty when tumors present in younger girls. Unlike epithelial ovarian cancer, these tumors have a higher proclivity for nodal or hematogenous metastases. As with testicular tumors, some of these tumors tend to produce AFP (yolk sac tumors) or hCG (embryonal carcinoma, choriocarcinomas, and some dysgerminomas) that are reliable tumor markers.

1	Germ cell tumors typically present in women who are still of childbearing age, and because bilateral tumors are uncommon (except in dysgerminoma, 10–15%), the typical treatment is unilateral oophorectomy or salpingo-oophorectomy. Because nodal metastases to pelvic and para-aortic nodes are common and may affect treatment choices, these nodes should be carefully inspected and, if enlarged, should be resected if possible. Women with malignant germ cell tumors typically receive bleomycin, etoposide, and cisplatin (BEP) chemotherapy. In the majority of women, even those with advanced-stage disease, cure is expected. Close follow-up without adjuvant therapy of women with stage I tumors is reasonable if there is high confidence that the patient and health care team are committed to compulsive and careful follow-up, as chemotherapy at the time of tumor recurrence is likely to be curative.

1	Dysgerminoma is the ovarian counterpart of testicular seminoma. The 5-year disease-free survival is 100% in early-stage patients and 61% in stage III disease. Although the tumor is highly radiation-sensitive, radiation produces infertility in many patients. BEP chemotherapy is as effective or more so without causing infertility. The use of BEP following incomplete resection is associated with a 2-year disease-free survival rate of 95%. This chemotherapy is now the treatment of choice for dysgerminoma.

1	Transport of the egg to the uterus occurs via transit through the fallopian tube, with the distal ends of these tubes composed of fimbriae that drape about the ovarian surface and capture the egg as it erupts from the ovarian cortex. Fallopian tube malignancies are typically serous tumors. Previous teaching was that these malignancies were rare, but more careful histologic examination suggests that many “ovarian malignancies” might actually arise in the distal fimbria of the fallopian tube (see above). These women often present with adnexal masses, and like ovarian cancer, these tumors spread relatively early throughout the peritoneal cavity and respond to platinum and taxane therapy and have a natural history that is essentially identical to ovarian cancer (Table 117-1).

1	Cervical cancer is the second most common and most lethal malignancy in women worldwide likely due to the widespread infection with high-risk strains of human papillomavirus (HPV) and limited utilization of or access to Pap smear screening in many nations throughout the world. Nearly 500,000 cases of cervical cancer are expected worldwide, with approximately 240,000 deaths annually. Cancer incidence is particularly high in women residing in Central and South America, the Caribbean, and southern and eastern Africa. Mortality rate is disproportionately high in Africa. In the United States, 12,360 women were diagnosed with cervical cancer and 4020 women died in 2014. Developed countries have looked at high-technology screening techniques for HPV involving automated polymerase chain reaction in thin preps that identify dysplastic cytology as well as high-risk HPV genetic material. Visual inspection of the cervix coated with acetic acid has demonstrated the ability to reduce mortality from

1	thin preps that identify dysplastic cytology as well as high-risk HPV genetic material. Visual inspection of the cervix coated with acetic acid has demonstrated the ability to reduce mortality from cervical cancer with potential broad applicability in low-resource environments. The development of effective vaccines for high-risk HPV types makes it imperative to determine economical, socially acceptable, and logistically feasible strategies to deliver and distribute this vaccine to girls and boys before their engagement in sexual activity.

1	HPV is the primary neoplastic-initiating event in the vast majority of women with invasive cervical cancer. This double-strand DNA virus infects epithelium near the transformation zone of the cervix. More than 60 types of HPV are known, with approximately 20 types having the ability to generate high-grade dysplasia and malignancy. HPV16 and -18 are the types most frequently associated with high-grade dysplasia and targeted by both U.S. Food and Drug Administration– approved vaccines. The large majority of sexually active adults are exposed to HPV, and most women clear the infection without specific intervention. The 8-kilobase HPV genome encodes seven early genes, most notably E6 and E7, which can bind to RB and p53, respectively. High-risk types of HPV encode E6 and E7 molecules that are particularly effective at inhibiting the normal cell cycle checkpoint functions of these regulatory proteins, leading to immortalization but not full transformation of cervical epithelium. A minority

1	particularly effective at inhibiting the normal cell cycle checkpoint functions of these regulatory proteins, leading to immortalization but not full transformation of cervical epithelium. A minority of woman will fail 595 to clear the infection with subsequent HPV integration into the host genome. Over the course of as short as months but more typically years, some of these women develop high-grade dysplasia. The time from dysplasia to carcinoma is likely years to more than a decade and almost certainly requires the acquisition of other poorly defined genetic mutations within the infected and immortalized epithelium.

1	Risk factors for HPV infection and, in particular, dysplasia include a high number of sexual partners, early age of first intercourse, and history of venereal disease. Smoking is a cofactor; heavy smokers have a higher risk of dysplasia with HPV infection. HIV infection, especially when associated with low CD4+ T cell counts, is associated with a higher rate of high-grade dysplasia and likely a shorter latency period between infection and invasive disease. The administration of highly active antiretroviral therapy reduces the risk of high-grade dysplasia associated with HPV infection.

1	Currently approved vaccines include the recombinant proteins to the late proteins, L1 and L2, of HPV-16 and -18. Vaccination of women before the initiation of sexual activity dramatically reduces the rate of HPV-16 and -18 infection and subsequent dysplasia. There is also partial protection against other HPV types, although vaccinated women are still at risk for HPV infection and still require standard Pap smear screening. Although no randomized trial data demonstrate the utility of Pap smears, the dramatic drop in cervical cancer incidence and death in developed countries employing wide-scale screening provides strong evidence for its effectiveness. In addition, even visual inspection of the cervix with preapplication of acetic acid using a “see and treat” strategy has demonstrated a 30% reduction in cervical cancer death. The incorporation of HPV testing by polymerase chain reaction or other molecular techniques increases the sensitivity of detecting cervical pathology but at the

1	reduction in cervical cancer death. The incorporation of HPV testing by polymerase chain reaction or other molecular techniques increases the sensitivity of detecting cervical pathology but at the cost of identifying many women with transient infections who require no specific medical intervention.

1	The majority of cervical malignancies are squamous cell carcinomas associated with HPV. Adenocarcinomas are also HPV-related and arise deep in the endocervical canal; they are typically not seen by visual inspection of the cervix and thus are often missed by Pap smear screening. A variety of rarer malignancies including atypical epithelial tumors, carcinoids, small cell carcinomas, sarcomas, and lymphomas have also been reported.

1	The principal role of Pap smear testing is the detection of asymptomatic preinvasive cervical dysplasia of squamous epithelial lining. Invasive carcinomas often have symptoms or signs including post-coital spotting or intermenstrual cycle bleeding or menometrorrhagia. Foul-smelling or persistent yellow discharge may also be seen. Presentations that include pelvic or sacral pain suggest lateral extension of the tumor into pelvic nerve plexus by either the primary tumor or a pelvic node and are signs of advanced-stage disease. Likewise, flank pain from hydronephrosis from ureteral compression or deep venous thrombosis from iliac vessel compression suggests either extensive nodal disease or direct extension of the primary tumor to the pelvic sidewall. The most common finding of physical exam is a visible tumor on the cervix.

1	Scans are not part of the formal clinical staging of cervical cancer yet are very useful in planning appropriate therapy. CT can detect hydronephrosis indicative of pelvic sidewall disease but is not accurate at evaluating other pelvic structures. Magnetic resonance imaging (MRI) is more accurate at estimating uterine extension and paracervical extension of disease into soft tissues typically bordered by broad and cardinal ligaments that support the uterus in the central pelvis. Positron emission tomography (PET) scan is the most accurate technique for evaluating the pelvis and more importantly nodal (pelvic, para-aortic, and scalene) sites for disease. Staging of cervix cancer Extent of Carcinoma Confined Disease Disease Invades bladder, tumor in-situ to cervix beyond cervix to pelvic rectum or but not to pelvic wall or metastasis wall or lower lower 1/3 1/3 of vagina vagina

1	FIGURE 117-1 Anatomic display of the stages of cervix cancer defined by location, extent of tumor, frequency of presentation, and 5-year survival. This technique seems more prognostic and accurate than CT, MRI, or lymphangiogram, especially in the para-aortic region.

1	Stage I cervical tumors are confined to the cervix, whereas stage II tumors extend into the upper vagina or paracervical soft tissue (Fig. 117-1). Stage III tumors extend to the lower vagina or the pelvic sidewalls, whereas stage IV tumors invade the bladder or rectum or have spread to distant sites. Very small stage I cervical tumors can be treated with a variety of surgical procedures. In young women desiring to maintain fertility, radical trachelectomy removes the cervix with subsequent anastomosis of the upper vagina to the uterine corpus. Larger cervical tumors confined to the cervix can be treated with either surgical resection or radiation therapy in combination with cisplatin-based chemotherapy with a high chance of cure. Larger tumors that extend regionally down the vagina or into the paracervical soft tissues or the pelvic sidewalls are treated with combination chemotherapy and radiation therapy. The treatment of recurrent or metastatic disease is unsatisfactory due to the

1	the paracervical soft tissues or the pelvic sidewalls are treated with combination chemotherapy and radiation therapy. The treatment of recurrent or metastatic disease is unsatisfactory due to the relative resistance of these tumors to chemotherapy and currently available biological agents, although bevacizumab, a monoclonal antibody that is said to inhibit tumor-associated angiogenesis, has demonstrated clinically meaningful activity in the management of metastatic disease.

1	Several different tumor types arise in uterine corpus. Most tumors arise in the glandular lining and are endometrial adenocarcinomas. Tumors can also arise in the smooth muscle; most are benign (uterine leiomyoma), with a small minority of tumors being sarcomas. The endometrioid histologic subtype of endometrial cancer is the most common gynecologic malignancy in the United States. In 2014, an estimated 52,630 women were diagnosed with cancer of the uterine corpus, with 8590 deaths from the disease. Development of these tumors is a multistep process, with estrogen playing an important early role in driving endometrial gland proliferation. Relative overexposure to this class of hormones is a risk factor for the subsequent development of endometrioid tumors. In contrast, progestins drive glandular maturation and are protective. Hence, women with high endogenous or pharmacologic exposure to estrogens, especially if unopposed by progesterone, are at high risk for endometrial cancer.

1	Obese women, women treated with unopposed estrogens, or women with estrogen-producing tumors (such as granulosa cell tumors of the ovary) are at higher risk for endometrial cancer. In addition, treatment with tamoxifen, which has antiestrogenic effects in breast tissue but estrogenic effects in uterine epithelium, is associated with an increased risk of endometrial cancer. Events such as the loss of the PTEN tumor suppressor gene with activation and often additional mutations in the PIK-3CA/AKT pathways likely serve as secondary events in carcinogenesis. The Cancer Genome Atlas Research Network has demonstrated that endometrioid tumors can be divided into four subgroups: ultramutated, microsatellite instability hypermutated, copy number low, and copy number high subgroups. These groups have different natural histories; therapy for these subgroups may eventually be individualized. Serous tumors of the uterine corpus represent approximately 5–10% of epithelial tumors of the uterine

1	have different natural histories; therapy for these subgroups may eventually be individualized. Serous tumors of the uterine corpus represent approximately 5–10% of epithelial tumors of the uterine corpus and possess distinct molecular characteristics that are most similar to those seen in serous tumors arising in the ovary or fallopian tube.

1	Women with a mutation in one of a series of DNA mismatch repair genes associated with the Lynch syndrome, also known as hereditary nonpolyposis colon cancer (HNPCC), are at increased risk for endometrioid endometrial carcinoma. These individuals have germline mutations in MSH2, MLH1, and in rare cases PMS1 and PMS2, with resulting microsatellite instability and hypermutation. Individuals who carry these mutations typically have a family history of cancer and are at markedly increased risk for colon cancer and modestly increased risk for ovarian cancer and a variety of other tumors. Middle-aged women with HNPCC carry a 4% annual risk of endometrial cancer and a relative overall risk of approximately 200-fold as compared to age-matched women without HNPCC.

1	The majority of women with tumors of the uterine corpus present with postmenopausal vaginal bleeding due to shedding of the malignant endometrial lining. Premenopausal women often will present with atypical bleeding between typical menstrual cycles. These signs typically bring a woman to the attention of a health care professional, and hence the majority of women present with early-stage disease with the tumor confined to the uterine corpus. Diagnosis is typically established by endometrial biopsy. Epithelial tumors may spread to pelvic or para-aortic lymph nodes. Pulmonary metastases can appear later in the natural history of this disease but are very uncommon at initial presentation. Serous tumors tend to have patterns of spread much more reminiscent of ovarian cancer with many patients presenting with disseminated peritoneal disease and sometimes ascites. Some women presenting with uterine sarcomas will present with pelvic pain. Nodal metastases are uncommon with sarcomas, which

1	presenting with disseminated peritoneal disease and sometimes ascites. Some women presenting with uterine sarcomas will present with pelvic pain. Nodal metastases are uncommon with sarcomas, which are more likely to present with either intraabdominal disease or pulmonary metastases.

1	Most women with endometrial cancer have disease that is localized to the uterus (75% are stage I, Table 117-1), and definitive treatment typically involves a hysterectomy with removal of the ovaries and fallopian tubes. The resection of lymph nodes does not improve outcome but does provide prognostic information. Node involvement defines stage III disease, which is present in 13% of patients. Tumor grade and depth of invasion are the two key prognostic variables in early-stage tumors, and women with low-grade and/ or minimally invasive tumors are typically observed after definitive surgical therapy. Patients with high-grade tumors or tumors that are deeply invasive (stage IB, 13%) are at higher risk for pelvic recurrence or recurrence at the vaginal cuff, which is typically prevented by vaginal vault brachytherapy.

1	Women with regional metastases or metastatic disease (3% of patients) with low-grade tumors can be treated with progesterone. Poorly differentiated tumors are typically resistant to hormonal manipulation and thus are treated with chemotherapy. The role of chemotherapy in the adjuvant setting is currently under investigation. Chemotherapy for metastatic disease is delivered with palliative intent. Drugs that effectively target and inhibit signaling of the AKT-mTOR pathway are currently under investigation. Five-year survival is 89% for stage I, 73% for stage II, 52% for stage III, and 17% for stage IV disease (Table 117-1).

1	Five-year survival is 89% for stage I, 73% for stage II, 52% for stage III, and 17% for stage IV disease (Table 117-1). Gestational trophoblastic diseases represent a spectrum of neo plasia from benign hydatidiform mole to choriocarcinoma due to persistent trophoblastic disease associated most commonly with molar pregnancy but occasionally seen after normal gestation. The most common presentations of trophoblastic tumors are partial and complete molar pregnancies. These represent approximately 1 in 1500 conceptions in developed Western countries. The incidence widely varies globally, with areas in Southeast Asia having a much higher incidence of molar pregnancy. Regions with high molar pregnancy rates are often associated with diets low in carotene and animal fats.

1	Trophoblastic tumors result from the outgrowth or persistence of placental tissue. They arise most commonly in the uterus but can also arise in other sites such as the fallopian tubes due to ectopic pregnancy. Risk factors include poorly defined dietary and environmental factors as well as conceptions at the extremes of reproductive age, with the incidence particularly high in females conceiving younger than age 16 or older than age 50. In older women, the incidence of molar pregnancy might be as high as one in three, likely due to increased risk of abnormal fertilization of the aged ova. Most trophoblastic neoplasms are associated with complete moles, diploid tumors with all genetic material from the paternal donor (known as parental disomy). This is thought to occur when a single sperm fertilizes an enucleate egg that subsequently duplicates the paternal DNA. Trophoblastic proliferation occurs with exuberant villous stroma. If pseudopregnancy extends out past the 12th week, fluid

1	fertilizes an enucleate egg that subsequently duplicates the paternal DNA. Trophoblastic proliferation occurs with exuberant villous stroma. If pseudopregnancy extends out past the 12th week, fluid progressively accumulates within the stroma, leading to “hydropic changes.” There is no fetal development in complete moles.

1	Partial moles arise from the fertilization of an egg with two sperm; 597 hence two-thirds of genetic material is paternal in these triploid tumors. Hydropic changes are less dramatic, and fetal development can often occur through late first trimester or early second trimester at which point spontaneous abortion is common. Laboratory findings will include excessively high hCG and high AFP. The risk of persistent gestational trophoblastic disease after partial mole is approximately 5%. Complete and partial moles can be noninvasive or invasive. Myometrial invasion occurs in no more than one in six complete moles and a lower portion of partial moles.

1	The clinical presentation of molar pregnancy is changing in developed countries due to the early detection of pregnancy with home pregnancy kits and the very early use of Doppler and ultrasound to evaluate the early fetus and uterine cavity for evidence of a viable fetus. Thus, in these countries, the majority of women presenting with trophoblastic disease have their moles detected early and have typical symptoms of early pregnancy including nausea, amenorrhea, and breast tenderness. With uterine evacuation of early complete and partial moles, most women experience spontaneous remission of their disease as monitored by serial hCG levels. These women require no chemotherapy. Patients with persistent elevation of hCG or rising hCG after evacuation have persistent or actively growing gestational trophoblastic disease and require therapy. Most series suggest that between 15 and 25% of women will have evidence of persistent gestational trophoblastic disease after molar evacuation.

1	In women who lack access to prenatal care, presenting symptoms can be life threatening including the development of preeclampsia or even eclampsia. Hyperthyroidism can also be seen. Evacuation of large moles can be associated with life-threatening complications including uterine perforation, volume loss, high-output cardiac failure, and adult respiratory distress syndrome (ARDS). For women with evidence of rising hCG or radiologic confirmation of metastatic or persistent regional disease, prognosis can be estimated through a variety of scoring algorithms that identify those women at low, intermediate, and high risk for requiring multiagent chemotherapy. In general, women with widely metastatic nonpulmonary disease, very elevated hCG, and prior normal antecedent term pregnancy are considered at high risk and typically require multiagent chemotherapy for cure.

1	The management for a persistent and rising hCG after evacuation of a molar conception is typically chemotherapy, although surgery can play an important role for disease that is persistently isolated in the uterus (especially if childbearing is complete) or to control hemorrhage. For women wishing to maintain fertility or with metastatic disease, the preferred treatment is chemotherapy. Chemotherapy is guided by the hCG level, which typically drops to undetectable levels with effective therapy. Single-agent treatment with methotrexate or dactinomycin cures 90% of women with low-risk disease. Patients with high-risk disease (high hCG levels, presentation 4 or more months after pregnancy, brain or liver metastases, failure of methotrexate therapy) are typically treated with multiagent chemotherapy (e.g., etoposide, methotrexate, and dactinomycin alternating with cyclophosphamide and vincristine [EMA-CO]), which is typically curative even in women with extensive metastatic disease.

1	chemotherapy (e.g., etoposide, methotrexate, and dactinomycin alternating with cyclophosphamide and vincristine [EMA-CO]), which is typically curative even in women with extensive metastatic disease. Cisplatin, bleomycin, and either etoposide or vinblastine are also active combinations. Survival in high-risk disease exceeds 80%. Cured women may get pregnant again without evidence of increased fetal or maternal complications.

1	598 primary and metastatic tumors of the nervous system Lisa M. DeAngelis, Patrick Y. Wen Primary brain tumors are diagnosed in approximately 52,000 people each year in the United States. At least one-half of these tumors are 118 malignant and associated with a high mortality. Glial tumors account for about 30% of all primary brain tumors, and 80% of those are malignant. Meningiomas account for 35%, vestibular schwannomas 10%, and central nervous system (CNS) lymphomas about 2%. Brain metastases are three times more common than all primary brain tumors combined and are diagnosed in approximately 150,000 people each year. Metastases to the leptomeninges and epidural space of the spinal cord each occur in approximately 3–5% of patients with systemic cancer and are also a major cause of neurologic disability. APPROACH TO THE PATIENT: primary and metastatic tumors of the nervous system

1	Brain tumors of any type can present with a variety of symptoms and signs that fall into two categories: general and focal; patients often have a combination of the two (Table 118-1). General or nonspecific symptoms include headache, with or without nausea or vomiting, cognitive difficulties, personality change, and gait disorder. Generalized symptoms arise when the enlarging tumor and its surrounding edema cause an increase in intracranial pressure or direct compression of cerebrospinal fluid (CSF) circulation leading to hydrocephalus. The classic headache associated with a brain tumor is most evident in the morning and improves during the day, but this particular pattern is actually seen in a minority of patients. Headaches are often holocephalic but can be ipsilateral to the side of a tumor. Occasionally, headaches have features of a typical migraine with unilateral throbbing pain associated with visual scotoma. Personality changes may include apathy and withdrawal from social

1	a tumor. Occasionally, headaches have features of a typical migraine with unilateral throbbing pain associated with visual scotoma. Personality changes may include apathy and withdrawal from social circumstances, mimicking depression. Focal or lateralizing findings include hemiparesis, aphasia, or visual field defect. Lateralizing symptoms are typically subacute and progressive. A visual field defect is often unnoticed by the patient; its presence may only be revealed after it leads to an injury such as an automobile accident occurring in the blind visual field. Language difficulties may be mistaken for confusion. Seizures are a common presentation of brain tumors, occurring in about 25% of patients with brain metastases or malignant gliomas but can be the presenting symptom in up to 90% of patients with a low-grade glioma. All seizures that arise from a brain tumor will have a focal onset whether or not it is apparent clinically.

1	Cranial MRI is the preferred diagnostic test for any patient suspected of having a brain tumor and should be performed with gadolinium contrast administration. Computed tomography (CT) scan should be reserved for those patients unable to undergo magnetic resonance imaging (MRI; e.g., pacemaker). Malignant brain tumors—whether primary or metastatic—typically enhance with gadolinium and may have central areas of necrosis; they are characteristically surrounded by edema of the neighboring white matter. Low-grade gliomas usually do not enhance with gadolinium and are best appreciated on fluid-attenuated inversion recovery (FLAIR) MRIs. Meningiomas have a characteristic appearance on MRI because they are duralbased with a dural tail and compress but do not invade the brain. Dural metastases or a dural lymphoma can have a similar appearance. Imaging is characteristic for many primary and metastatic tumors and sometimes will suffice to establish a diagnosis when the location precludes

1	or a dural lymphoma can have a similar appearance. Imaging is characteristic for many primary and metastatic tumors and sometimes will suffice to establish a diagnosis when the location precludes surgical intervention (e.g., brainstem glioma). Functional MRI is useful in presurgical planning to define eloquent sensory, motor, or language cortex. Positron emission tomography (PET) is useful in determining the metabolic activity of the lesions seen on MRI; MR perfusion and spectroscopy can provide information on blood flow or tissue composition. These techniques may help distinguish tumor progression from necrotic tissue as a consequence of treatment with radiation and chemotherapy or identify foci of high-grade tumor in an otherwise low-grade-appearing glioma.

1	Neuroimaging is the only test necessary to diagnose a brain tumor. Laboratory tests are rarely useful, although patients with metastatic disease may have elevation of a tumor marker in their serum that reflects the presence of brain metastases (e.g., β human chorionic gonadotropin [β-hCG] from testicular cancer). Additional testing such as cerebral angiogram, electroencephalogram (EEG), or lumbar puncture is rarely indicated or helpful.

1	Therapy of any intracranial malignancy requires both symptomatic and definitive treatments. Definitive treatment is based on the specific tumor type and includes surgery, radiotherapy, and chemotherapy. However, symptomatic treatments apply to brain tumors of any type. Most high-grade malignancies are accompanied by substantial surrounding edema, which contributes to neurologic disability and raised intracranial pressure. Glucocorticoids are highly effective at reducing perilesional edema and improving neurologic function, often within hours of administration. Dexamethasone has been the glucocorticoid of choice because of its relatively low mineralocorticoid activity. Initial doses are typically 12–16 mg/d in divided doses given orally or IV (both are equivalent). Although glucocorticoids rapidly ameliorate symptoms and signs, their long-term use causes substantial toxicity including insomnia, weight gain, diabetes mellitus, steroid myopathy, and personality changes. Consequently, a

1	rapidly ameliorate symptoms and signs, their long-term use causes substantial toxicity including insomnia, weight gain, diabetes mellitus, steroid myopathy, and personality changes. Consequently, a taper is indicated as definitive treatment is administered and the patient improves.

1	Patients with brain tumors who present with seizures require antiepileptic drug therapy. There is no role for prophylactic antiepileptic drugs in patients who have not had a seizure. The agents of choice are those drugs that do not induce the hepatic microsomal enzyme system. These include levetiracetam, topiramate, lamotrigine, valproic acid, and lacosamide (Chap. 445). Other drugs, such as phenytoin and carbamazepine, are used less frequently because they are potent enzyme inducers that can interfere with both glucocorticoid metabolism and the metabolism of chemotherapeutic agents needed to treat the underlying systemic malignancy or the primary brain tumor. Venous thromboembolic disease occurs in 20–30% of patients with high-grade gliomas and brain metastases. Therefore, prophylactic anticoagulants should be used during hospitalization and in nonambulatory patients. Those who have had either a deep vein thrombosis or pulmonary embolus can receive therapeutic doses of

1	prophylactic anticoagulants should be used during hospitalization and in nonambulatory patients. Those who have had either a deep vein thrombosis or pulmonary embolus can receive therapeutic doses of anticoagulation safely and without increasing the risk for hemorrhage into the tumor. Inferior vena cava filters are reserved for patients with absolute contraindications to anticoagulation such as recent craniotomy.

1	No underlying cause has been identified for the majority of primary brain tumors. The only established risk factors are exposure to ionizing radiation (meningiomas, gliomas, and schwannomas) and immunosuppression (primary CNS lymphoma). Evidence for an association with exposure to electromagnetic fields including cellular telephones, head injury, foods containing N-nitroso compounds, or occupational risk factors are unproven. A small minority of patients have a family history of brain tumors. Some of these familial cases are associated with genetic syndromes (Table 118-2).

1	As with other neoplasms, brain tumors arise as a result of a multi-step process driven by the sequential acquisition of genetic alterations. These include loss of tumor-suppressor genes (e.g., p53 and phosphatase and tensin homolog on chromosome 10 [PTEN]) and amplification and overexpression of protooncogenes such as the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptors (PDGFR). The accumulation of these genetic abnormalities results in uncontrolled cell growth and tumor formation.

1	Important progress has been made in understanding the molecular pathogenesis of several types of brain tumors, including glioblastoma and medulloblastoma. Morphologically indistinguishable glioblastomas can be separated into four subtypes defined by molecular profiling: (1) classical, characterized by overactivation of the EGFR pathway; (2) proneural, characterized by overexpression of PDGFRA, mutations of the isocitrate dehydrogenase (IDH) 1 and 2 genes, and expression 599 of neural markers; (3) mesenchymal, defined by expression of mesenchymal markers and loss of NF1; and (4) neural, characterized by overactivity of EGFR and expression of neural markers. The clinical implications of these subtypes are under study. Medulloblastoma is the other primary brain tumor that has been highly analyzed, and four molecular subtypes have also been identified: (1) the Wnt subtype is defined by a mutation in β-catenin and has an excellent prognosis; (2) the SHH subtype has mutations in PTCH1, SMO,

1	and four molecular subtypes have also been identified: (1) the Wnt subtype is defined by a mutation in β-catenin and has an excellent prognosis; (2) the SHH subtype has mutations in PTCH1, SMO, GLI2, or SUFU and has an intermediate prognosis; (3) group 3 has elevated MYC expression and has the worst prognosis; and (4) group 4 is characterized by isochromosome 17q. Targeted therapeutics are under development for some of the medulloblastoma subtypes, especially the SHH group.

1	These are infiltrative tumors with a presumptive glial cell of origin. The World Health Organization (WHO) classifies astrocytomas into four prognostic grades based on histologic features: grade I (pilocytic astrocytoma, subependymal giant cell astrocytoma); grade II (diffuse astrocytoma); grade III (anaplastic astrocytoma); and grade IV (glioblastoma). Grades I and II are considered low-grade astrocytomas, and grades III and IV are considered high-grade astrocytomas. most common tumor of childhood. They occur typically in the cerebellum but may also be found elsewhere in the neuraxis, including the optic nerves and brainstem. Frequently they appear as cystic lesions Primary and Metastatic Tumors of the Nervous System aVarious DNA mismatch repair gene mutations may cause a similar clinical phenotype, also referred to as Turcot syndrome, in which there is a predisposition to nonpolyposis colon cancer and brain tumors.

1	Abbreviations: AD, autosomal dominant; APC, adenomatous polyposis coli; AR, autosomal recessive; ch, chromosome; PTEN, phosphatase and tensin homologue; TSC, tuberous sclerosis complex. FIGURE 118-1 Fluid-attenuated inversion recovery (FLAIR) MRI of a left frontal low-grade astrocytoma. This lesion did not enhance. FIGURE 118-2 Postgadolinium T1 MRI of a large cystic left frontal glioblastoma. with an enhancing mural nodule. These are well-demarcated lesions that are potentially curable if they can be resected completely. Giant-cell subependymal astrocytomas are usually found in the ventricular wall of patients with tuberous sclerosis. They often do not require intervention but can be treated surgically or with inhibitors of the mammalian target of rapamycin (mTOR).

1	grade ii astrocytomas These are infiltrative tumors that usually present with seizures in young adults. They appear as nonenhancing tumors with increased T2/FLAIR signal (Fig. 118-1). If feasible, patients should undergo maximal surgical resection, although complete resection is rarely possible because of the invasive nature of the tumor. Radiation therapy (RT) is helpful, but there is no difference in overall survival between RT administered postoperatively or delayed until the time of tumor progression. There is increasing evidence that chemotherapeutic agents such as temozolomide, an oral alkylating agent, can be helpful in some patients. The tumor transforms to a malignant astrocytoma in the majority of patients, leading to variable survival with a median of about 5 years.

1	grade iii (anaPlastic) astrocytoma These account for approximately 15–20% of high-grade astrocytomas. They generally present in the fourth and fifth decades of life as variably enhancing tumors. Treatment is the same as for glioblastoma, consisting of maximal safe surgical resection followed by RT with concurrent and adjuvant temozolomide or by RT and adjuvant temozolomide alone.

1	grade iv astrocytoma (glioBlastoma) Glioblastoma accounts for the majority of high-grade astrocytomas. They are the most common malignant primary brain tumor, with over 10,000 cases diagnosed each year in the United States. Patients usually present in the sixth and seventh decades of life with headache, seizures, or focal neurologic deficits. The tumors appear as ring-enhancing masses with central necrosis and surrounding edema (Fig. 118-2). These are highly infiltrative tumors, and the areas of increased T2/FLAIR signal surrounding the main tumor mass contain invading tumor cells. Treatment involves maximal surgical resection followed by partial-field external-beam RT (6000 cGy in thirty 200-cGy fractions) with concomitant temozolomide, followed by 6–12 months of adjuvant temozolomide. With this regimen, median survival is increased to 14.6 months compared to only 12 months with RT alone, and 2-year survival is increased to 27%, compared to 10% with RT alone. Patients whose tumor

1	With this regimen, median survival is increased to 14.6 months compared to only 12 months with RT alone, and 2-year survival is increased to 27%, compared to 10% with RT alone. Patients whose tumor contains the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) are relatively resistant to temozolomide and have a worse prognosis compared to those whose tumors contain low levels of MGMT as a result of silencing of the MGMT gene by promoter hypermethylation. Implantation of biodegradable polymers containing the chemotherapeutic agent carmustine into the tumor bed after resection of the tumor also produces a modest improvement in survival.

1	Despite optimal therapy, glioblastomas invariably recur. Treatment options for recurrent disease may include reoperation, carmustine wafers, and alternate chemotherapeutic regimens. Reirradiation is rarely helpful. Bevacizumab, a humanized vascular endothelial growth factor (VEGF) monoclonal antibody, has activity in recurrent glioblastoma, increasing progression-free survival and reducing peritumoral edema and glucocorticoid use (Fig. 118-3). Treatment decisions for patients with recurrent glioblastoma must be made on an individual basis, taking into consideration such factors as previous therapy, time to relapse, performance status, and quality of life. Whenever feasible, patients with recurrent disease should be enrolled in clinical trials. Novel therapies undergoing evaluation in patients with glioblastoma include targeted molecular agents directed at receptor tyrosine kinases and signal transduction pathways; antiangiogenic agents, especially those directed at the VEGF receptors;

1	with glioblastoma include targeted molecular agents directed at receptor tyrosine kinases and signal transduction pathways; antiangiogenic agents, especially those directed at the VEGF receptors; chemotherapeutic agents that cross the blood-brain barrier more effectively than currently available drugs; gene therapy; immunotherapy; and infusion of radiolabeled drugs and targeted toxins into the tumor and surrounding brain by means of convection-enhanced delivery.

1	The most important adverse prognostic factors in patients with high-grade astrocytomas are older age, histologic features of glioblastoma, poor Karnofsky performance status, and unresectable tumor. Patients whose tumor contains an unmethylated MGMT promoter resulting in the presence of the repair enzyme in tumor cells and resistance to temozolomide also have a worse prognosis. Gliomatosis Cerebri Rarely, patients may present with a highly infiltrating, nonenhancing tumor of variable histologic grade involving more than two lobes of the brain. These tumors may be indolent initially, but will eventually behave aggressively and have a poor outcome. Treatment involves RT and temozolomide chemotherapy. Oligodendrogliomas account for approximately 15–20% of gliomas. They are classified by the WHO into well-differentiated oligodendrogliomas (grade II) or anaplastic oligodendrogliomas (AOs) (grade III). Tumors with oligodendroglial components have distinctive pathologic

1	FIGURE 118-3 Postgadolinium T1 MRI of a recurrent glioblastoma before (A) and after (B) administration of bevacizumab. Note the decreased enhancement and mass effect. features such as perinuclear clearing—giving rise to a “fried-egg” appearance—and a reticular pattern of blood vessel growth. Some tumors have both an oligodendroglial as well as an astrocytic component. These mixed tumors, or oligoastrocytomas (OAs), are also classified into well-differentiated OA (grade II) or anaplastic oligoastrocytomas (AOAs) (grade III). Grade II oligodendrogliomas and OAs are generally more responsive to therapy and have a better prognosis than pure astrocytic tumors. These tumors present similarly to grade II astrocytomas in young adults. The tumors are nonenhancing and often partially calcified. They should be treated with surgery and, if necessary, RT and chemotherapy. Patients with oligodendrogliomas have a median survival in excess of 10 years.

1	AOs and AOAs present in the fourth and fifth decades as variably enhancing tumors. They are more responsive to therapy than grade III astrocytomas. Co-deletion of chromosomes 1p and 19q, mediated by an unbalanced translocation of 19p to 1q, occurs in 61–89% of patients with AO and 14–20% of patients with AOA. Tumors with the 1p and 19q co-deletion are particularly sensitive to chemotherapy with procarbazine, lomustine (cyclohexylchloroethylnitrosourea [CCNU]), and vincristine (PCV) or temozolomide, as well as to RT. Median survival of patients with AO or AOA is approximately 3–6 years, but 601 those with co-deleted tumors can have a median survival of 10–14 years if treated with RT and chemotherapy.

1	Ependymomas are tumors derived from ependymal cells that line the ventricular surface. They account for approximately 5% of childhood tumors and frequently arise from the wall of the fourth ventricle in the posterior fossa. Although adults can have intracranial ependymomas, they occur more commonly in the spine, especially in the filum terminale of the spinal cord where they have a myxopapillary histology. Ependymomas that can be completely resected are potentially curable. Partially resected ependymomas will recur and require irradiation. The less common anaplastic ependymoma is more aggressive and is treated with resection and RT; chemotherapy has limited efficacy. Subependymomas are slow-growing benign lesions arising in the wall of ventricles that often do not require treatment.

1	Gangliogliomas and pleomorphic xanthoastrocytomas occur in young adults. They behave as more indolent forms of grade II gliomas and are treated in the same way. Brainstem gliomas usually occur in children or young adults. Despite treatment with RT and chemotherapy, the prognosis is poor, with a median survival of only 1 year. Gliosarcomas contain both an astrocytic as well as a sarcomatous component and are treated in the same way as glioblastomas. Primary central nervous system lymphoma (PCNSL) is a rare non-Hodgkin lymphoma accounting for less than 3% of primary brain tumors. For unclear reasons, its incidence is increasing, particularly in immunocompetent individuals.

1	PCNSL in immunocompetent patients usually consists of a diffuse large B cell lymphoma. PCNSL may also occur in immunocompromised patients, usually those infected with the human immunodeficiency virus (HIV) or organ transplant recipients on immunosuppressive therapy. PCNSL in immunocompromised patients is typically large cell with immunoblastic and more aggressive features. These patients are usually severely immunocompromised, with CD4 counts of less than 50/mL. The Epstein-Barr virus (EBV) frequently plays an important role in the pathogenesis of HIV-related PCNSL. Immunocompetent patients with PCNSL are older (median 60 years) compared to patients with HIV-related PCNSL (median 31 years). PCNSL usually presents as a mass lesion, with neuropsychiatric symptoms, symptoms of increased intracranial pressure, lateralizing signs, or seizures.

1	On contrast-enhanced MRI, PCNSL usually appears as a densely enhancing tumor (Fig. 118-4). Immunocompetent patients have solitary lesions more often than immunosuppressed patients. Frequently there is involvement of the basal ganglia, corpus callosum, or periventricular region. Although the imaging features are often characteristic, PCNSL can sometimes be difficult to differentiate from high-grade gliomas, infections, or demyelination. Stereotactic biopsy is necessary to obtain a histologic diagnosis. Whenever possible, glucocorticoids should be withheld until after the biopsy has been obtained because they have a cytolytic effect on lymphoma cells and may lead to nondiagnostic tissue. In addition, patients should be tested for HIV and the extent of disease should be assessed by performing PET or CT of the body, MRI of the spine, CSF analysis, and slit-lamp examination of the eye. Bone marrow biopsy and testicular ultrasound are occasionally performed.

1	PCNSL is more sensitive to glucocorticoids, chemotherapy, and RT than other primary brain tumors. Durable complete responses and long-term survival are possible with these treatments. High-dose methotrexate, a folate antagonist that interrupts DNA synthesis, produces response rates ranging from 35–80% and median Primary and Metastatic Tumors of the Nervous System FIGURE 118-4 Postgadolinium T1 MRI demonstrating a large bifrontal primary central nervous system lymphoma (PCNSL). The periventricular location and diffuse enhancement pattern are characteristic of lymphoma.

1	survival of up to 50 months. The combination of methotrexate with other chemotherapeutic agents such as cytarabine increases the response rate to 70–100%. The addition of whole-brain RT to methotrexate-based chemotherapy prolongs progression-free survival but not overall survival. Furthermore, RT is associated with delayed neurotoxicity, especially in patients over the age of 60 years. As a result, full-dose RT is frequently omitted, but there may be a role for reduced-dose RT. The anti-CD20 monoclonal antibody rituximab has activity in PCNSL and is often incorporated into the chemotherapy regimen. For some patients, high-dose chemotherapy with autologous stem cell rescue may offer the best chance of preventing relapse. At least 50% of patients will eventually develop recurrent disease. Treatment options include RT for patients who have not had prior irradiation, re-treatment with methotrexate, as well as other agents such as temozolomide, rituximab, procarbazine, topotecan, and

1	Treatment options include RT for patients who have not had prior irradiation, re-treatment with methotrexate, as well as other agents such as temozolomide, rituximab, procarbazine, topotecan, and pemetrexed. High-dose chemotherapy with autologous stem cell rescue may have a role in selected patients with relapsed disease.

1	PCNSL in immunocompromised patients often produces multiple ring-enhancing lesions that can be difficult to differentiate from metastases and infections such as toxoplasmosis. The diagnosis is usually established by examination of the CSF for cytology and EBV DNA, toxoplasmosis serologic testing, brain PET imaging for hypermetabolism of the lesions consistent with tumor instead of infection, and, if necessary, brain biopsy. Since the advent of highly active antiretroviral drugs, the incidence of HIV-related PCNSL has declined. These patients may be treated with whole-brain RT, high-dose methotrexate, and initiation of highly active antiretroviral therapy. In organ transplant recipients, reduction of immunosuppression may improve outcome.

1	Medulloblastomas are the most common malignant brain tumor of childhood, accounting for approximately 20% of all primary CNS tumors among children. They arise from granule cell progenitors or from multipotent progenitors from the ventricular zone. Approximately 5% of children have inherited disorders with germline mutations of genes that predispose to the development of medulloblastoma. Gorlin syndrome, the most common of these inherited disorders, is due to mutations in the patched-1 (PTCH-1) gene, a key component in the sonic hedgehog pathway. Turcot syndrome, caused by mutations in the adenomatous polyposis coli (APC) gene and familial adenomatous polyposis, has also been associated with an increased incidence of medulloblastoma. Histologically, medulloblastomas are highly cellular tumors with abundant dark staining, round nuclei, and rosette formation (Homer-Wright rosettes). They present with headache, ataxia, and signs of brainstem involvement. On MRI they appear as densely

1	tumors with abundant dark staining, round nuclei, and rosette formation (Homer-Wright rosettes). They present with headache, ataxia, and signs of brainstem involvement. On MRI they appear as densely enhancing tumors in the posterior fossa, sometimes associated with hydrocephalus. Seeding of the CSF is common. Treatment involves maximal surgical resection, craniospinal irradiation, and chemotherapy with agents such as cisplatin, lomustine, cyclophosphamide, and vincristine. Approximately 70% of patients have long-term survival but usually at the cost of significant neurocognitive impairment. A major goal of current research is to improve survival while minimizing long-term complications.

1	A large number of tumors can arise in the region of the pineal gland. These typically present with headache, visual symptoms, and hydrocephalus. Patients may have Parinaud syndrome characterized by impaired upgaze and accommodation. Some pineal tumors such as pineocytomas and benign teratomas can be treated simply by surgical resection. Germinomas respond to irradiation, whereas pineoblastomas and malignant germ cell tumors require craniospinal radiation and chemotherapy. Meningiomas are diagnosed with increasing frequency as more people undergo neuroimaging for various indications. They are now the most common primary brain tumor, accounting for approximately 35% of the total. Their incidence increases with age. They tend to be more common in women and in patients with neurofibromatosis type 2. They also occur more commonly in patients with a past history of cranial irradiation.

1	Meningiomas arise from the dura mater and are composed of neoplastic meningothelial (arachnoidal cap) cells. They are most commonly located over the cerebral convexities, especially adjacent to the sagittal sinus, but can also occur in the skull base and along the dorsum of the spinal cord. Meningiomas are classified by the WHO into three histologic grades of increasing aggressiveness: grade I (benign), grade II (atypical), and grade III (malignant). Many meningiomas are found incidentally following neuroimaging for unrelated reasons. They can also present with headaches, seizures, or focal neurologic deficits. On imaging studies they have a characteristic appearance usually consisting of a partially calcified, densely enhancing extraaxial tumor arising from the dura (Fig. 118-5). Occasionally they may have a dural tail, consisting of thickened, enhanced dura extending like a tail from the mass. The main differential diagnosis of meningioma is a dural metastasis.

1	If the meningioma is small and asymptomatic, no intervention is necessary and the lesion can be observed with serial MRI studies. Larger, symptomatic lesions should be resected. If complete resection is achieved, the patient is cured. Incompletely resected tumors tend to recur, although the rate of recurrence can be very slow with grade I tumors. Tumors that cannot be resected, or can only be partially removed, may benefit from treatment with external-beam RT or stereotactic radiosurgery (SRS). These treatments may also be helpful in patients whose tumor has recurred after surgery. Hormonal therapy and chemotherapy are currently unproven. Rarer tumors that resemble meningiomas include hemangiopericytomas and solitary fibrous tumors. These are treated with surgery and RT but have a higher propensity to recur locally or metastasize systemically.

1	These are generally benign tumors arising from the Schwann cells of cranial and spinal nerve roots. The most common schwannomas, termed vestibular schwannomas or acoustic neuromas, arise from the vestibular portion of the eighth cranial nerve and account for approximately 9% of primary brain tumors. Patients with neurofibromatosis type 2 have a high incidence of vestibular schwannomas that are frequently bilateral. Schwannomas arising from other cranial nerves, such as the trigeminal nerve (cranial nerve V), occur with much lower frequency. Neurofibromatosis type 1 is associated with an increased incidence of schwannomas of the spinal nerve roots. FIGURE 118-5 Postgadolinium T1 MRI demonstrating multiple meningiomas along the falx and left parietal cortex.

1	FIGURE 118-5 Postgadolinium T1 MRI demonstrating multiple meningiomas along the falx and left parietal cortex. Vestibular schwannomas may be found incidentally on neuroimaging or present with progressive unilateral hearing loss, dizziness, tinnitus, or less commonly, symptoms resulting from compression of the brainstem and cerebellum. On MRI they appear as densely enhancing lesions, enlarging the internal auditory canal and often extending into the cerebellopontine angle (Fig. 118-6). The differential diagnosis includes meningioma. Very small, asymptomatic lesions can be observed with serial MRIs. Larger lesions should be treated with surgery or SRS. The optimal treatment will depend on the size of the tumor, symptoms, and the patient’s preference. In patients with small vestibular schwannomas and relatively intact hearing, early surgical 603 intervention increases the chance of preserving hearing.

1	FIGURE 118-6 Postgadolinium MRI of a right vestibular schwan-noma. The tumor can be seen to involve the internal auditory canal. PITUITARY TUMORS (CHAP. 401e)

1	PITUITARY TUMORS (CHAP. 401e) These account for approximately 9% of primary brain tumors. They can be divided into functioning and nonfunctioning tumors. Functioning tumors are usually microadenomas (<1 cm in diameter) that secrete hormones and produce specific endocrine syndromes (e.g., acromegaly for growth hormone–secreting tumors, Cushing syndrome for adrenocorticotropic hormone [ACTH]-secreting tumors, and galactorrhea, amenorrhea, and infertility for prolactin-secreting tumors). Nonfunctioning pituitary tumors tend to be macroadenomas (>1 cm) that produce symptoms by mass effect, giving rise to headaches, visual impairment (such as bitemporal hemianopia), and hypopituitarism. Prolactin-secreting tumors respond well to dopamine agonists such as bromocriptine and cabergoline. Other pituitary tumors usually require treatment with surgery and sometimes RT or radiosurgery and hormonal therapy.

1	Craniopharyngiomas are rare, usually suprasellar, partially calcified, solid, or mixed solid-cystic benign tumors that arise from remnants of Rathke’s pouch. They have a bimodal distribution, occurring predominantly in children but also between the ages of 55 and 65 years. They present with headaches, visual impairment, and impaired growth in children and hypopituitarism in adults. Treatment involves surgery, RT, or a combination of the two. Dysembryoplastic Neuroepithelial Tumors (DNTs) These are benign, supratentorial tumors, usually in the temporal lobe. They typically occur in children and young adults with a long-standing history of seizures. Surgical resection is curative.

1	Epidermoid Cysts These consist of squamous epithelium surrounding a keratin-filled cyst. They are usually found in the cerebellopontine angle and the intrasellar and suprasellar regions. They may present with headaches, cranial nerve abnormalities, seizures, or hydrocephalus. Imaging studies demonstrate extraaxial lesions with characteristics that are similar to CSF but have restricted diffusion. Treatment involves surgical resection.

1	Dermoid Cysts Like epidermoid cysts, dermoid cysts arise from epithelial cells that are retained during closure of the neural tube. They contain both epidermal and dermal structures such as hair follicles, sweat glands, and sebaceous glands. Unlike epidermoid cysts, these tumors usually have a midline location. They occur most frequently in the posterior fossa, especially the vermis, fourth ventricle, and suprasellar cistern. Radiographically, dermoid cysts resemble lipomas, demonstrating T1 hyperintensity and variable signal on T2. Symptomatic dermoid cysts can be treated with surgery. Colloid Cysts These usually arise in the anterior third ventricle and may present with headaches, hydrocephalus, and, very rarely, sudden death. Surgical resection is curative, or a third ventriculostomy may relieve the obstructive hydrocephalus and be sufficient therapy.

1	A number of genetic disorders are characterized by cutaneous lesions and an increased risk of brain tumors. Most of these disorders have an autosomal dominant inheritance with variable penetrance. NF1 is an autosomal dominant disorder with an incidence of approximately 1 in 2600–3000. Approximately one-half the cases are familial; the remainder are caused by new mutations arising in patients with unaffected parents. The NF1 gene on chromosome 17q11.2 encodes a protein, neurofibromin, a guanosine triphosphatase (GTPase)-activating protein (GAP) that modulates signaling through the ras pathway. Mutations of NF1 result in a large number of nervous system tumors

1	Primary and Metastatic Tumors of the Nervous System 604 including neurofibromas, plexiform neurofibromas, optic nerve gliomas, astrocytomas, and meningiomas. In addition to neurofibromas, which appear as multiple, soft, rubbery cutaneous tumors, other cutaneous manifestations of NF1 include café-au-lait spots and axillary freckling. NF1 is also associated with hamartomas of the iris termed Lisch nodules, pheochromocytomas, pseudoarthrosis of the tibia, scoliosis, epilepsy, and mental retardation.

1	NF2 is less common than NF1, with an incidence of 1 in 25,000– 40,000. It is an autosomal dominant disorder with full penetrance. As with NF1, approximately one-half the cases arise from new mutations. The NF2 gene on 22q encodes a cytoskeletal protein, merlin (moesin, ezrin, radixin-like protein) that functions as a tumor suppressor. NF2 is characterized by bilateral vestibular schwannomas in over 90% of patients, multiple meningiomas, and spinal ependymomas and astrocytomas. Treatment of bilateral vestibular schwannomas can be challenging because the goal is to preserve hearing for as long as possible. These patients may also have diffuse schwannomatosis that may affect the cranial, spinal, or peripheral nerves; posterior subcapsular lens opacities; and retinal hamartomas.

1	This is an autosomal dominant disorder with an incidence of approximately 1 in 5000–10,000 live births. It is caused by mutations in either the TSC1 gene, which maps to chromosome 9q34 and encodes a protein termed hamartin, or the TSC2 gene, which maps to chromosome 16p13.3 and encodes the protein tuberin. Hamartin forms a complex with tuberin, which inhibits cellular signaling through the mTOR, and acts as a negative regulator of the cell cycle. Patients with tuberous sclerosis may have seizures, mental retardation, adenoma sebaceum (facial angiofibromas), shagreen patch, hypomelanotic macules, periungual fibromas, renal angiomyolipomas, and cardiac rhabdomyomas. These patients have an increased incidence of subependymal nodules, cortical tubers, and subependymal giant-cell astrocytomas (SEGA). Patients frequently require anticonvulsants for seizures. SEGAs do not always require therapeutic intervention, but the most effective therapy is with the mTOR inhibitors sirolimus or

1	(SEGA). Patients frequently require anticonvulsants for seizures. SEGAs do not always require therapeutic intervention, but the most effective therapy is with the mTOR inhibitors sirolimus or everolimus, which often decrease seizures as well as SEGA size.

1	Brain metastases arise from hematogenous spread and frequently either arise from a lung primary or are associated with pulmonary metastases. Most metastases develop at the gray matter–white matter junction in the watershed distribution of the brain where intravascular tumor cells lodge in terminal arterioles. The distribution of metastases in the brain approximates the proportion of blood flow such that about 85% of all metastases are supratentorial and 15% occur in the posterior fossa. The most common sources of brain metastases are lung and breast carcinomas; melanoma has the greatest propensity to metastasize to the brain, being found in 80% of patients at autopsy Table 118-3). Other tumor Abbreviations: ESCC, epidural spinal cord compression; GIT, gastrointestinal tract; LM, leptomeningeal metastases.

1	Abbreviations: ESCC, epidural spinal cord compression; GIT, gastrointestinal tract; LM, leptomeningeal metastases. types such as ovarian and esophageal carcinoma rarely metastasize to the brain. Prostate and breast cancer also have a propensity to metastasize to the dura and can mimic meningioma. Leptomeningeal metastases are common from hematologic malignancies and also breast and lung cancers. Spinal cord compression primarily arises in patients with prostate and breast cancer, tumors with a strong propensity to metastasize to the axial skeleton.

1	Brain metastases are best visualized on MRI, where they usually appear as well-circumscribed lesions (Fig. 118-7). The amount of perilesional edema can be highly variable, with large lesions causing minimal edema and sometimes very small lesions causing extensive edema. Enhancement may be in a ring pattern or diffuse. Occasionally, intracranial metastases will hemorrhage; although melanoma, thyroid, and kidney cancer have the greatest propensity to hemorrhage, the most common cause of a hemorrhagic metastasis is lung cancer because it accounts for the majority of brain metastases. The radiographic appearance of brain metastasis is nonspecific, and similar-appearing lesions can occur with infection including brain abscesses and also with demyelinating lesions, sarcoidosis, radiation necrosis in a previously treated patient, or a primary brain tumor that may be a second malignancy in a patient with systemic cancer. However, biopsy is rarely necessary for diagnosis in most patients

1	in a previously treated patient, or a primary brain tumor that may be a second malignancy in a patient with systemic cancer. However, biopsy is rarely necessary for diagnosis in most patients because imaging alone in the appropriate clinical situation usually suffices. This is straightforward for the majority of patients with brain metastases because they have a known systemic cancer. However, in approximately 10% of patients, a systemic cancer may present with a brain metastasis, and if there is not an easily accessible systemic site to biopsy, then a brain lesion must be removed for diagnostic purposes.

1	The number and location of brain metastases often determine the therapeutic options. The patient’s overall condition and the current or potential control of the systemic disease are also major determinants. Brain metastases are single in approximately one-half of patients and multiple in the other half.

1	The standard treatment for brain metastases has been whole-brain radiotherapy (WBRT) usually administered to a total dose of 3000 cGy in 10 fractions. This affords rapid palliation, and approximately 80% of patients improve with glucocorticoids and RT. However, it is not curative. Median survival is only 4–6 months. More recently, SRS delivered through a variety of techniques including the gamma knife, linear accelerator, proton beam, and CyberKnife all can deliver highly focused doses of RT, usually in a single fraction. SRS can effectively sterilize the visible lesions and afford local disease control in 80–90% of patients. In addition, there are some patients who have clearly been cured of their brain metastases using SRS, whereas this is distinctly rare with WBRT. However, SRS can be used only for lesions 3 cm or less in diameter and should be confined to patients with only one to three metastases. The addition of WBRT to SRS improves disease control in the nervous system but does

1	only for lesions 3 cm or less in diameter and should be confined to patients with only one to three metastases. The addition of WBRT to SRS improves disease control in the nervous system but does not prolong survival.

1	Randomized controlled trials have demonstrated that surgical extirpation of a single brain metastasis followed by WBRT is superior to WBRT alone. Removal of two lesions or a single symptomatic mass, particularly if compressing the ventricular system, can also be useful. This is particularly useful in patients who have highly radioresistant lesions such as renal carcinoma. Surgical resection can afford rapid symptomatic improvement and prolonged survival. WBRT administered after complete resection of a brain metastasis improves disease control but does not prolong survival.

1	Chemotherapy is rarely useful for brain metastases. Metastases from certain tumor types that are highly chemosensitive, such as germ cell tumors or small-cell lung cancer, may respond to chemotherapeutic regimens chosen according to the underlying malignancy. Increasingly, there are data demonstrating responsiveness of brain metastases to chemotherapy including small molecule–targeted therapy when the lesion possesses the target. This has been best illustrated in patients with lung cancer harboring EGFR mutations that sensitize them to EGFR inhibitors. Antiangiogenic agents such as bevacizumab may also prove efficacious in the treatment of CNS metastases.

1	Leptomeningeal metastases are also identified as carcinomatous meningitis, meningeal carcinomatosis, or in the case of specific tumors, leukemic or lymphomatous meningitis. Among the hematologic malignancies, acute leukemia is the most common to metastasize to the subarachnoid space, and in lymphomas the aggressive diffuse lymphomas can metastasize to the subarachnoid space frequently as well. Among solid tumors, breast and lung carcinomas and melanoma most frequently spread in this fashion. Tumor cells reach the subarachnoid space via the arterial circulation or occasionally through retrograde flow in venous systems that drain metastases along the bony spine or cranium. In addition, leptomeningeal metastases may develop as a direct consequence of prior brain metastases and can develop in almost 40% of patients who have a metastasis resected from the cerebellum.

1	Leptomeningeal metastases are characterized clinically by multilevel symptoms and signs along the neuraxis. Combinations of lumbar and cervical radiculopathies, cranial neuropathies, seizures, confusion, and encephalopathy from hydrocephalus or raised intracranial pressure can be present. Focal deficits such as hemiparesis or aphasia are rarely due to leptomeningeal metastases unless there is direct brain infiltration, and they are more often associated with coexisting brain lesions. New-onset limb pain in patients with breast cancer, lung cancer, or melanoma should prompt consideration of leptomeningeal spread.

1	Leptomeningeal metastases are particularly challenging to diagnose because identification of tumor cells in the subarachnoid compartment may be elusive. MRI can be definitive in patients when there are clear tumor nodules adherent to the cauda equina or spinal cord, enhancing cranial nerves, or subarachnoid enhancement on brain imaging (Fig. 118-8). Imaging is diagnostic in approximately 75% of patients and is more often positive in patients with solid tumors. Demonstration of tumor cells in the CSF is definitive and often considered the gold standard. However, CSF cytologic examination is positive in only 50% of patients on the first lumbar puncture and still misses 10% after three CSF samples. CSF cytologic examination is most useful in hematologic malignancies. Accompanying CSF abnormalities include an elevated protein concentration and an elevated white count. Hypoglycorrhachia is noted in less than 25% of patients but is useful when present. Identification of tumor markers or

1	include an elevated protein concentration and an elevated white count. Hypoglycorrhachia is noted in less than 25% of patients but is useful when present. Identification of tumor markers or molecular confirmation of clonal proliferation with techniques such as flow cytometry within the CSF can also be definitive when present. Tumor markers are usually specific to solid tumors, and chromosomal or molecular markers are most useful in patients with hematologic malignancies. New technologies, such as rare cell capture, may enhance identification of tumor cells in the CSF.

1	The treatment of leptomeningeal metastasis is palliative because there is no curative therapy. RT to the symptomatically involved areas, such as skull base for cranial neuropathy, can relieve pain and sometimes improve function. Whole-neuraxis RT has extensive toxicity with myelosuppression and gastrointestinal irritation as well as limited effectiveness. Systemic chemotherapy with agents that can penetrate the blood-CSF barrier may be helpful. Alternatively, intrathecal chemotherapy can be effective, particularly in hematologic malignancies. This is optimally delivered through an intraventricular cannula (Ommaya reservoir) rather than by lumbar puncture. Few drugs can be delivered safely into the subarachnoid space, and they have a limited spectrum of antitumor activity, perhaps accounting for the relatively poor response to this approach. In addition, impaired CSF flow dynamics can compromise intrathecal drug delivery. Surgery has a limited role in the treatment of leptomeningeal

1	for the relatively poor response to this approach. In addition, impaired CSF flow dynamics can compromise intrathecal drug delivery. Surgery has a limited role in the treatment of leptomeningeal metastasis, but placement of a ventriculoperitoneal shunt can relieve raised intracranial pressure. However, it compromises delivery of chemotherapy into the CSF.

1	Primary and Metastatic Tumors of the Nervous System FIGURE 118-8 Postgadolinium MRI images of extensive leptomeningeal metastases from breast cancer. Nodules along the dorsal surface of the spinal cord (A) and cauda equina (B) are seen. Epidural metastasis occurs in 3–5% of patients with a systemic malignancy and causes neurologic compromise by compressing the spinal cord or cauda equina. The most common cancers that metastasize to the epidural space are those malignancies that spread to bone, such as breast and prostate. Lymphoma can cause bone involvement and compression, but it can also invade the intervertebral foramens and cause spinal cord compression without bone destruction. The thoracic spine is affected most commonly, followed by the lumbar and then cervical spine.

1	Back pain is the presenting symptom of epidural metastasis in virtually all patients; the pain may precede neurologic findings by weeks or months. The pain is usually exacerbated by lying down; by contrast, arthritic pain is often relieved by recumbency. Leg weakness is seen in about 50% of patients, as is sensory dysfunction. Sphincter problems are present in about 25% of patients at diagnosis. FIGURE 118-9 Postgadolinium T1 MRI showing circumferential epidural tumor around the thoracic spinal cord from esophageal cancer.

1	FIGURE 118-9 Postgadolinium T1 MRI showing circumferential epidural tumor around the thoracic spinal cord from esophageal cancer. Diagnosis is established by imaging, with MRI of the complete spine being the best test (Fig. 118-9). Contrast is not needed to identify spinal or epidural lesions. Any patient with cancer who has severe back pain should undergo an MRI. Plain films, bone scans, or even CT scans may show bone metastases, but only MRI can reliably delineate epidural tumor. For patients unable to have an MRI, CT myelography should be performed to outline the epidural space. The differential diagnosis of epidural tumor includes epidural abscess, acute or chronic hematomas, and rarely, extramedullary hematopoiesis.

1	Epidural metastasis requires immediate treatment. A randomized controlled trial demonstrated the superiority of surgical resection followed by RT compared to RT alone. However, patients must be able to tolerate surgery, and the surgical procedure of choice is a complete removal of the mass, which is typically anterior to the spinal canal, necessitating an extensive approach and resection. Otherwise, RT is the mainstay of treatment and can be used for patients with radiosensitive tumors, such as lymphoma, or for those unable to undergo surgery. Chemotherapy is rarely used for epidural metastasis unless the patient has minimal to no neurologic deficit and a highly chemosensitive tumor such as lymphoma or germinoma. Patients generally fare well if treated before there is severe neurologic deficit. Recovery after paraparesis is better after surgery than with RT alone, but survival is often short due to widespread metastatic tumor.

1	RT can cause a variety of toxicities in the CNS. These are usually described based on their relationship in time to the administration of RT: acute (occurring within days of RT), early delayed (months), or late delayed (years). In general, the acute and early delayed syndromes resolve and do not result in persistent deficits, whereas the late delayed toxicities are usually permanent and sometimes progressive. Acute Toxicity Acute cerebral toxicity usually occurs during RT to the brain. RT can cause a transient disruption of the blood-brain barrier, resulting in increased edema and elevated intracranial pressure. This is usually manifest as headache, lethargy, nausea, and vomiting and can be both prevented and treated with the administration of glucocorticoids. There is no acute RT toxicity that affects the spinal cord.

1	Early Delayed Toxicity Early delayed toxicity is usually apparent weeks to months after completion of cranial irradiation and is likely due to focal demyelination. Clinically it may be asymptomatic or take the form of worsening or reappearance of a preexisting neurologic deficit. At times a contrast-enhancing lesion can be seen on MRI/ CT that can mimic the tumor for which the patient received the RT. For patients with a malignant glioma, this has been described as “pseudoprogression” because it mimics tumor recurrence on MRI but actually represents inflammation and necrotic debris engendered by effective therapy. This is seen with increased frequency when chemotherapy, particularly temozolomide, is given concurrently with RT. Pseudoprogression can resolve on its own or, if very symptomatic, may require resection. A rare form of early delayed toxicity is the somnolence syndrome that occurs primarily in children and is characterized by marked sleepiness.

1	In the spinal cord, early delayed RT toxicity is manifest as a Lhermitte symptom with paresthesias of the limbs or along the spine when the patient flexes the neck. Although frightening, it is benign, resolves on its own, and does not portend more serious problems.

1	Late Delayed Toxicity Late delayed toxicities are the most serious because they are often irreversible and cause severe neurologic deficits. In the brain, late toxicities can take several forms, the most common of which include radiation necrosis and leukoencephalopathy. Radiation necrosis is a focal mass of necrotic tissue that is contrast enhancing on CT/MRI and may be associated with significant edema. This may appear identical to pseudoprogression but is seen months to years after RT and is always symptomatic. Clinical symptoms and signs include seizure and lateralizing findings referable to the location of the necrotic mass. The necrosis is caused by the effect of RT on cerebral vasculature with resultant fibrinoid necrosis and occlusion of the blood vessels. It can mimic tumor radiographically, but unlike tumor, it is typically hypometabolic on a PET scan and has reduced perfusion on perfusion MR sequences. It may require resection for diagnosis and treatment unless it can be

1	but unlike tumor, it is typically hypometabolic on a PET scan and has reduced perfusion on perfusion MR sequences. It may require resection for diagnosis and treatment unless it can be managed with glucocorticoids. There are rare reports of improvement with hyperbaric oxygen or anticoagulation, but the usefulness of these approaches is questionable.

1	Leukoencephalopathy is seen most commonly after WBRT as opposed to focal RT. On T2 or FLAIR MR sequences, there is diffuse increased signal seen throughout the hemispheric white matter, often bilaterally and symmetrically. There tends to be a periventricular predominance that may be associated with atrophy and ventricular enlargement. Clinically, patients develop cognitive impairment, gait disorder, and later urinary incontinence, all of which can progress over time. These symptoms mimic those of normal pressure hydrocephalus, and placement of a ventriculoperitoneal shunt can improve function in some patients but does not reverse the deficits completely. Increased age is a risk factor for leukoencephalopathy but not for radiation necrosis. Necrosis appears to depend on an as yet unidentified predisposition.

1	Other late neurologic toxicities include endocrine dysfunction if the pituitary or hypothalamus was included in the RT port. An RT-induced neoplasm can occur many years after therapeutic RT for either a prior CNS tumor or a head and neck cancer; accurate diagnosis requires surgical resection or biopsy. In addition, RT causes accelerated atherosclerosis, which can cause stroke either from intracranial vascular disease or carotid plaque from neck irradiation. The peripheral nervous system is relatively resistant to RT toxicities. Peripheral nerves are rarely affected by RT, but the plexus is more Acute encephalopathy (delirium) Seizures Methotrexate (high-dose IV, IT) Methotrexate Cisplatin Etoposide (high-dose) Vincristine Cisplatin Asparaginase Vincristine Procarbazine Asparaginase 5-Fluorouracil (± levamisole) Nitrogen mustard Cytarabine (high-dose) Carmustine Nitrosoureas (high-dose or Dacarbazine (intraarterial or

1	Abbreviations: IT, intrathecal; IV, intravenous; PRES, posterior reversible encephalopathy syndrome. vulnerable. Plexopathy develops more commonly in the brachial distribution than in the lumbosacral distribution. It must be differentiated from tumor progression in the plexus, which is usually accomplished with CT/MR imaging of the area or PET scan demonstrating tumor infiltrating the region. Clinically, tumor progression is usually painful, whereas RT-induced plexopathy is painless. Radiation plexopathy is also more commonly associated with lymphedema of the affected limb. Sensory loss and weakness are seen in both.

1	Neurotoxicity is second to myelosuppression as the dose-limiting toxicity of chemotherapeutic agents (Table 118-4). Chemotherapy causes peripheral neuropathy from a number of commonly used agents, and the type of neuropathy can differ, depending on the drug. Vincristine causes paresthesias but little sensory loss and is associated with motor dysfunction, autonomic impairment (frequently ileus), and rarely cranial nerve compromise. Cisplatin causes large fiber sensory loss resulting in sensory ataxia but little cutaneous sensory loss and no weakness. The taxanes also cause a predominately sensory neuropathy. Agents such as bortezomib and thalidomide also cause neuropathy.

1	Encephalopathy and seizures are common toxicities from chemotherapeutic drugs. Ifosfamide can cause a severe encephalopathy, which is reversible with discontinuation of the drug and the use of methylene blue for severely affected patients. Fludarabine also causes a severe global encephalopathy that may be permanent. Bevacizumab and other anti-VEGF agents can cause posterior reversible encephalopathy syndrome. Cisplatin can cause hearing loss and less frequently vestibular dysfunction. Immunotherapy with anti-CTLA-4 monoclonal antibodies, such as ipilimumab, can cause an autoimmune hypophysitis. Primary and Metastatic Tumors of the Nervous System

1	Primary and Metastatic Tumors of the Nervous System Soft tissue and Bone Sarcomas and Bone Metastases Shreyaskumar R. Patel, Robert S. Benjamin Sarcomas are rare (<1% of all malignancies) mesenchymal neoplasms that arise in bone and soft tissues. These tumors are usually of meso-119e dermal origin, although a few are derived from neuroectoderm, and they are biologically distinct from the more common epithelial malignancies. Sarcomas affect all age groups; 15% are found in children <15 years of age, and 40% occur after age 55 years. Sarcomas are one of the most common solid tumors of childhood and are the fifth most common cause of cancer deaths in children. Sarcomas may be divided into two groups, those derived from bone and those derived from soft tissues.

1	Soft tissues include muscles, tendons, fat, fibrous tissue, synovial tissue, vessels, and nerves. Approximately 60% of soft tissue sarcomas arise in the extremities, with the lower extremities involved three times as often as the upper extremities. Thirty percent arise in the trunk, the retroperitoneum accounting for 40% of all trunk lesions. The remaining 10% arise in the head and neck. Approximately 11,410 new cases of soft tissue sarcomas occurred in the United States in 2013. The annual age-adjusted incidence is 3 per 100,000 population, but the incidence varies with age. Soft tissue sarcomas constitute 0.7% of all cancers in the general population and 6.5% of all cancers in children.

1	Malignant transformation of a benign soft tissue tumor is extremely rare, with the exception that malignant peripheral nerve sheath tumors (neurofibrosarcoma, malignant schwannoma) can arise from neurofibromas in patients with neurofibromatosis. Several etiologic factors have been implicated in soft tissue sarcomas. Environmental Factors Trauma or previous injury is rarely involved, but sarcomas can arise in scar tissue resulting from a prior operation, burn, fracture, or foreign body implantation. Chemical carcinogens such as polycyclic hydrocarbons, asbestos, and dioxin may be involved in the pathogenesis. Iatrogenic Factors Sarcomas in bone or soft tissues occur in patients who are treated with radiation therapy. The tumor nearly always arises in the irradiated field. The risk increases with time.

1	Iatrogenic Factors Sarcomas in bone or soft tissues occur in patients who are treated with radiation therapy. The tumor nearly always arises in the irradiated field. The risk increases with time. Viruses Kaposi’s sarcoma (KS) in patients with HIV type 1, classic KS, and KS in HIV-negative homosexual men is caused by human herpesvirus (HHV) 8 (Chap. 219). No other sarcomas are associated with viruses. Immunologic Factors Congenital or acquired immunodeficiency, including therapeutic immunosuppression, increases the risk of sarcoma.

1	Li-Fraumeni syndrome is a familial cancer syndrome in which affected individuals have germline abnormalities of the tumor- suppressor gene p53 and an increased incidence of soft tissue sarcomas and other malignancies, including breast cancer, osteosarcoma, brain tumor, leukemia, and adrenal carcinoma (Chap. 101e). Neurofibromatosis 1 (NF-1, peripheral form, von Recklinghausen’s disease) is characterized by multiple neurofibromas and café-au-lait spots. Neurofibromas occasionally undergo malignant degeneration to become malignant peripheral nerve sheath tumors. The gene for NF-1 is located in the pericentromeric region of chromosome 17 and encodes neurofibromin, a tumor-suppressor protein with guanosine 5′-triphosphate (GTP)ase-activating activity that inhibits Ras function 119e-1 (Chap. 118). Germline mutation of the Rb-1 locus (chromosome 13q14) in patients with inherited retinoblastoma is associated with the development of osteosarcoma in those who survive the retinoblastoma and of

1	Germline mutation of the Rb-1 locus (chromosome 13q14) in patients with inherited retinoblastoma is associated with the development of osteosarcoma in those who survive the retinoblastoma and of soft tissue sarcomas unrelated to radiation therapy. Other soft tissue tumors, including desmoid tumors, lipomas, leiomyomas, neuroblastomas, and paragangliomas, occasionally show a familial predisposition.

1	Ninety percent of synovial sarcomas contain a characteristic chromosomal translocation t(X;18)(p11;q11) involving a nuclear transcription factor on chromosome 18 called SYT and two breakpoints on X. Patients with translocations to the second X breakpoint (SSX2) may have longer survival than those with translocations involving SSX1. Insulin-like growth factor (IGF) type II is produced by some sarcomas and may act as an autocrine growth factor and as a motility factor that promotes metastatic spread. IGF-II stimulates growth through IGF-I receptors, but its effects on motility are through different receptors. If secreted in large amounts, IGF-II may produce hypoglycemia (Chaps. 121 and 420).

1	Approximately 20 different groups of sarcomas are recognized on the basis of the pattern of differentiation toward normal tissue. For example, rhabdomyosarcoma shows evidence of skeletal muscle fibers with cross-striations; leiomyosarcomas contain interlacing fascicles of spindle cells resembling smooth muscle; and liposarcomas contain adipocytes. When precise characterization of the group is not possible, the tumors are called unclassified sarcomas. All of the primary bone sarcomas can also arise from soft tissues (e.g., extraskeletal osteosarcoma). The entity malignant fibrous histiocytoma (MFH) includes many tumors previously classified as fibrosarcomas or as pleomorphic variants of other sarcomas and is characterized by a mixture of spindle (fibrous) cells and round (histiocytic) cells arranged in a storiform pattern with frequent giant cells and areas of pleomorphism. As immunohistochemical suggestion of differentiation, particularly myogenic differentiation, may be found in a

1	arranged in a storiform pattern with frequent giant cells and areas of pleomorphism. As immunohistochemical suggestion of differentiation, particularly myogenic differentiation, may be found in a significant fraction of these patients, many are now characterized as poorly differentiated leiomyosarcomas, and the terms undifferentiated pleomorphic sarcoma (UPS) and myxofibrosarcoma are replacing MFH and myxoid MFH.

1	For purposes of treatment, most soft tissue sarcomas can be considered together. However, some specific tumors have distinct features. For example, liposarcoma can have a spectrum of behaviors. Pleomorphic liposarcomas and dedifferentiated liposarcomas behave like other high-grade sarcomas; in contrast, well-differentiated liposarcomas (better termed atypical lipomatous tumors) lack metastatic potential, and myxoid liposarcomas metastasize infrequently, but, when they do, they have a predilection for unusual metastatic sites containing fat, such as the retroperitoneum, mediastinum, and subcutaneous tissue. Rhabdomyosarcomas, Ewing’s sarcoma, and other small-cell sarcomas tend to be more aggressive and are more responsive to chemotherapy than other soft tissue sarcomas.

1	Gastrointestinal stromal cell tumors (GISTs), previously classified as gastrointestinal leiomyosarcomas, are now recognized as a distinct entity within soft tissue sarcomas. Its cell of origin resembles the interstitial cell of Cajal, which controls peristalsis. The majority of malignant GISTs have activating mutations of the c-kit gene that result in ligand-independent phosphorylation and activation of the KIT receptor tyrosine kinase, leading to tumorigenesis. Approximately 5–10% of tumors will have a mutation in the platelet-derived growth factor receptor α (PDGFRA). GISTs that are wild type for both KIT and PDGFRA mutations may show mutations in SDH B, C, or D and may be driven by the IGF-I pathway.

1	The most common presentation is an asymptomatic mass. Mechanical symptoms referable to pressure, traction, or entrapment of nerves or muscles may be present. All new and persistent or growing masses should be biopsied, either by a cutting needle (core-needle biopsy) or by a small incision, placed so that it can be encompassed in the to that achieved by radical excisions and Moderately differentiated (G2) >5 cm (T2) Involved (N1) Present (M1) amputations. Limb-sparing approaches Poorly differentiated (G3) Superficial fascial involvement (Ta) are indicated except when negative margins are not obtainable, when the risks of radiation are prohibitive, or when neuro-

1	Poorly differentiated (G3) Superficial fascial involvement (Ta) are indicated except when negative margins are not obtainable, when the risks of radiation are prohibitive, or when neuro- Disease Stage 5-Year Survival, % subsequent excision without compromising a definitive resection. Lymph node metastases occur in 5%, except in synovial and epithelioid sarcomas, clear-cell sarcoma (melanoma of the soft parts), angiosarcoma, and rhabdomyosarcoma, where nodal spread may be seen in 17%. The pulmonary parenchyma is the most common site of metastases. Exceptions are GISTs, which metastasize to the liver; myxoid liposarcomas, which seek fatty tissue; and clear-cell sarcomas, which may metastasize to bones. Central nervous system metastases are rare, except in alveolar soft part sarcoma.

1	Radiographic Evaluation Imaging of the primary tumor is best with plain radiographs and magnetic resonance imaging (MRI) for tumors of the extremities or head and neck and by computed tomography (CT) for tumors of the chest, abdomen, or retroperitoneal cavity. A radiograph and CT scan of the chest are important for the detection of lung metastases. Other imaging studies may be indicated, depending on the symptoms, signs, or histology. The histologic grade, relationship to fascial planes, and size of the primary tumor are the most important prognostic factors. The current American Joint Committee on Cancer (AJCC) staging system is shown in Table 119e-1. Prognosis is related to the stage. Cure is common in the absence of metastatic disease, but a small number of patients with metastases can also be cured. Most patients with stage IV disease die within 12 months, but some patients may live with slowly progressive disease for many years.

1	AJCC stage I patients are adequately treated with surgery alone. Stage II patients are considered for adjuvant radiation therapy. Stage III patients may benefit from adjuvant chemotherapy. Stage IV patients are managed primarily with chemotherapy, with or without other modalities. Soft tissue sarcomas tend to grow along fascial planes, with the surrounding soft tissues compressed to form a pseudocapsule that gives the sarcoma the appearance of a well-encapsulated lesion. This is invariably deceptive because “shelling out,” or marginal excision, of such lesions results in a 50–90% probability of local recurrence. Wide excision with a negative margin, incorporating the biopsy site, is the standard surgical procedure for local disease. The adjuvant use of radiation therapy and/or chemotherapy vascular structures are involved so that resection will result in serious functional consequences to the limb.

1	External-beam radiation therapy is an adjuvant to limb-sparing surgery for improved local control. Preoperative radi ation therapy allows the use of smaller a higher rate of wound complications. given to larger fields, because the entire surgical bed must be encompassed, and in higher doses to compensate for hypoxia in the operated field. This results in a higher rate of late complications. Brachytherapy or interstitial therapy, in which the radiation source is inserted into the tumor bed, is comparable in efficacy (except in low-grade lesions), less time-consuming, and less expensive.

1	Chemotherapy is the mainstay of treatment for Ewing’s primitive neuroectodermal tumors (PNET) and rhabdomyosarcomas. Meta-analysis of 14 randomized trials revealed a significant improvement in local control and disease-free survival in favor of doxorubicinbased chemotherapy. Overall survival improvement was 4% for all sites and 7% for the extremity site. An updated meta-analysis including four additional trials with doxorubicin and ifosfamide combination has reported a statistically significant 6% survival advantage in favor of chemotherapy. A chemotherapy regimen including an anthracycline and ifosfamide with growth factor support improved overall survival by 19% for high-risk (high-grade, ≥5 cm primary, or locally recurrent) extremity soft tissue sarcomas.

1	Metastatic soft tissue sarcomas are largely incurable, but up to 20% of patients who achieve a complete response become long-term survivors. The therapeutic intent, therefore, is to produce a complete remission with chemotherapy (<10%) and/or surgery (30–40%). Surgical resection of metastases, whenever possible, is an integral part of the management. Some patients benefit from repeated surgical excision of metastases. The two most active chemotherapeutic agents are doxorubicin and ifosfamide. These drugs show a steep dose-response relationship in sarcomas. Gemcitabine with or without docetaxel has become an established second-line regimen and is particularly active in patients with undifferentiated pleomorphic sarcoma (UPS) and leiomyosarcomas. Dacarbazine also has some modest activity. Taxanes have selective activity in angiosarcomas, and vincristine, etoposide, and irinotecan are effective in rhabdomyosarcomas and Ewing’s sarcomas. Pazopanib, an inhibitor of the vascular endothelial

1	have selective activity in angiosarcomas, and vincristine, etoposide, and irinotecan are effective in rhabdomyosarcomas and Ewing’s sarcomas. Pazopanib, an inhibitor of the vascular endothelial growth factor, platelet-derived growth factor (PDGF), and c-kit is now approved for patients with advanced soft tissue sarcomas excluding liposarcomas after failure of chemotherapy. Imatinib targets the KIT and PDGF tyrosine kinase activity and is standard therapy for advanced/metastatic GISTs and dermatofibrosarcoma protuberans. Imatinib is now also indicated as adjuvant therapy for completely resected primary GISTs. Three years of adjuvant imatinib appears to be superior to 1 year of therapy for high-risk GISTs, although the optimal treatment duration remains unknown.

1	Bone sarcomas are rarer than soft tissue sarcomas; they accounted for only 0.2% of all new malignancies and 2890 new cases in the United States in 2013. Several benign bone lesions have the potential for malignant transformation. Enchondromas and osteochondromas can transform into chondrosarcoma; fibrous dysplasia, bone infarcts, and Paget’s disease of bone can transform into either UPS or osteosarcoma. CLASSIFICATION Benign Tumors The common benign bone tumors include enchondroma, osteochondroma, chondroblastoma, and chondromyxoid fibroma, of cartilage origin; osteoid osteoma and osteoblastoma, of bone origin; fibroma and desmoplastic fibroma, of fibrous tissue origin; hemangioma, of vascular origin; and giant-cell tumor, of unknown origin.

1	Malignant Tumors The most common malignant tumors of bone are plasma cell tumors (Chap. 136). The four most common malignant nonhematopoietic bone tumors are osteosarcoma, chondrosarcoma, Ewing’s sarcoma, and UPS. Rare malignant tumors include chordoma (of notochordal origin), malignant giant-cell tumor and adamantinoma (of unknown origin), and hemangioendothelioma (of vascular origin).

1	Musculoskeletal Tumor Society Staging System Sarcomas of bone are staged according to the Musculoskeletal Tumor Society staging system based on grade and compartmental localization. A Roman numeral reflects the tumor grade: stage I is low grade, stage II is high grade, and stage III includes tumors of any grade that have lymph node or distant metastases. In addition, the tumor is given a letter reflecting its compartmental localization. Tumors designated A are intracompartmental (i.e., confined to the same soft tissue compartment as the initial tumor), and tumors designated B are extracompartmental (i.e., extending into the adjacent soft tissue compartment or into bone). The tumor-nodemetastasis (TNM) staging system is shown in Table 119e-2.

1	Osteosarcoma, accounting for almost 45% of all bone sarcomas, is a spindle cell neoplasm that produces osteoid (unmineralized bone) or bone. Approximately 60% of all osteosarcomas occur in children and adolescents in the second decade of life, and approximately 10% occur in the third decade of life. Osteosarcomas in the fifth and sixth decades of life are frequently secondary to either radiation therapy or transformation in a preexisting benign condition, such as Paget’s disease. Males are affected 1.5–2 times as often as females. Osteosarcoma has a predilection for metaphyses of long bones; the most common sites of involvement are the distal femur, proximal tibia, and proximal humerus. The classification of osteosarcoma is complex, but 75% of osteosarcomas fall into the “classic” category, which include osteoblastic, chondroblastic, and fibroblastic osteosarcomas. The remaining 25% are classified as “variants” on the basis of (1) clinical characteristics, as in the case of

1	category, which include osteoblastic, chondroblastic, and fibroblastic osteosarcomas. The remaining 25% are classified as “variants” on the basis of (1) clinical characteristics, as in the case of osteosarcoma of the jaw, postradiation osteosarcoma, or Paget’s osteosarcoma; (2) morphologic characteristics, as in the case of telangiectatic osteosarcoma, small-cell osteosarcoma, or epithelioid osteosarcoma; or (3) location, as in parosteal or periosteal osteosarcoma. Diagnosis usually requires a synthesis of clinical, radiologic, and pathologic features. Patients typically present with pain and swelling of the affected area. A plain radiograph reveals a destructive lesion with a moth-eaten appearance, a spiculated periosteal reaction (sunburst appearance), and a cuff of periosteal new bone formation at the margin of the soft tissue mass (Codman’s triangle). A CT scan of the primary tumor is best for defining bone destruction and the pattern of calcification, whereas MRI is better for

1	formation at the margin of the soft tissue mass (Codman’s triangle). A CT scan of the primary tumor is best for defining bone destruction and the pattern of calcification, whereas MRI is better for defining intramedullary and soft tissue extension. A chest radiograph and CT scan are used to detect lung metastases. Metastases to the bony skeleton should be imaged by a bone scan or by fluorodeoxyglucose positron emission tomography (FDG-PET). Almost all osteosarcomas are hypervascular. Angiography is not helpful for diagnosis, but it is the most sensitive test for assessing the response to preoperative

1	Primary tumor (T) TX Primary tumor cannot be assessed T0 No evidence of primary tumor T1 Tumor ≤8 cm in greatest dimension T2 Tumor >8 cm in greatest dimension T3 Discontinuous tumors in the primary Histologic grade (G) GX Grade cannot be assessed G1 Well differentiated—low grade G2 Moderately differentiated—low grade G3 Poorly differentiated—high grade G4 Undifferentiated—high grade (Ewing’s is Stage IA T1 N0 M0 G1,2 low grade Stage IB T2 N0 M0 G1,2 low grade Stage IIA T1 N0 M0 G3,4 high grade

1	Stage IIB T2 N0 M0 G3,4 high grade chemotherapy. Pathologic diagnosis is established either with a core-needle biopsy, where feasible, or with an open biopsy with an appropriately placed incision that does not compromise future limb-sparing resection. Most osteosarcomas are high-grade. The most important prognostic factor for long-term survival is response to chemotherapy. Preoperative chemotherapy followed by limb-sparing surgery (which can be accomplished in >80% of patients) followed by postoperative chemotherapy is standard management. The effective drugs are doxorubicin, ifosfamide, cisplatin, and high-dose methotrexate with leucovorin rescue. The various combinations of these agents that have been used have all been about equally successful. Long-term survival rates in extremity osteosarcoma range from 60 to 80%. Osteosarcoma is radioresistant; radiation therapy has no role in the routine management. UPS is considered a part of the spectrum of osteosarcoma and is managed

1	osteosarcoma range from 60 to 80%. Osteosarcoma is radioresistant; radiation therapy has no role in the routine management. UPS is considered a part of the spectrum of osteosarcoma and is managed similarly.

1	Chondrosarcoma, which constitutes ~20–25% of all bone sarcomas, is a tumor of adulthood and old age with a peak incidence in the fourth to sixth decades of life. It has a predilection for the flat bones, especially the shoulder and pelvic girdles, but can also affect the diaphyseal portions of long bones. Chondrosarcomas can arise de novo or as a malignant transformation of an enchondroma or, rarely, of the cartilaginous cap of an osteochondroma. Chondrosarcomas have an indolent natural history and typically present as pain and swelling. Radiographically, the lesion may have a lobular appearance with mottled or punctate or annular calcification of the cartilaginous matrix. It is difficult to distinguish low-grade chondrosarcoma from benign lesions by x-ray or histologic examination. The diagnosis is therefore influenced by clinical history and physical examination. A new onset of pain, signs of inflammation, and progressive increase in the size of the mass suggest malignancy. The

1	diagnosis is therefore influenced by clinical history and physical examination. A new onset of pain, signs of inflammation, and progressive increase in the size of the mass suggest malignancy. The histologic classification is complex, but most tumors fall within the classic category. Like other bone sarcomas, high-grade chondrosarcomas spread to the lungs. Most chondrosarcomas are resistant to chemotherapy, and surgical resection of primary or recurrent tumors, including pulmonary metastases, is the mainstay of therapy. This rule does not hold for two histologic variants. Dedifferentiated chondrosarcoma has a high-grade osteosarcoma or a malignant fibrous histiocytoma component that responds to chemotherapy. Mesenchymal chondrosarcoma, a rare variant composed of a small-cell element, also is responsive to systemic chemotherapy and is treated like Ewing’s sarcoma.

1	Ewing’s sarcoma, which constitutes ~10–15% of all bone sarcomas, is common in adolescence and has a peak incidence in the second decade of life. It typically involves the diaphyseal region of long bones and also has an affinity for flat bones. The plain radiograph may show a characteristic “onion peel” periosteal reaction with a generous soft tissue mass, which is better demonstrated by CT or MRI. This mass is composed of sheets of monotonous, small, round, blue cells and can be confused with lymphoma, embryonal rhabdomyosarcoma, and small-cell carcinoma. The presence of p30/32, the product of the mic-2 gene (which maps to the pseudoautosomal region of the X and Y chromosomes), is a cell-surface marker for Ewing’s sarcoma (and other members of the Ewing’s family of tumors, sometimes called PNETs). Most PNETs arise in soft tissues; they include peripheral neuroepithelioma, Askin’s tumor (chest wall), and esthesioneuroblastoma. Glycogen-filled cytoplasm detected by staining with

1	called PNETs). Most PNETs arise in soft tissues; they include peripheral neuroepithelioma, Askin’s tumor (chest wall), and esthesioneuroblastoma. Glycogen-filled cytoplasm detected by staining with periodic acid–Schiff is also characteristic of Ewing’s sarcoma cells. The classic cytogenetic abnormality associated with this disease (and other PNETs) is a reciprocal translocation of the long arms of chromosomes 11 and 22, t(11;22), which creates a chimeric gene product of unknown function with components from the fli-1 gene on chromosome 11 and ews on 22. This disease is very aggressive, and it is therefore considered a systemic disease. Common sites of metastases are lung, bones, and bone marrow. Systemic chemotherapy is the mainstay of therapy, often being used before surgery. Doxorubicin, cyclophosphamide or ifosfamide, etoposide, vincristine, and dactinomycin are active drugs. Topotecan or irinotecan in combination with an alkylating agent is often used in relapsed patients.

1	cyclophosphamide or ifosfamide, etoposide, vincristine, and dactinomycin are active drugs. Topotecan or irinotecan in combination with an alkylating agent is often used in relapsed patients. Targeted therapy with an anti-IGF-I receptor antibody in combination with an inhibitor of mammalian target of rapamycin (mTOR) appears to have promising activity in refractory cases. Local treatment for the primary tumor includes surgical resection, usually with limb salvage or radiation therapy. Patients with lesions below the elbow and below the mid-calf have a 5-year survival rate of 80% with effective treatment. Ewing’s sarcoma at first presentation is a curable tumor, even in the presence of obvious metastatic disease, especially in children <11 years old.

1	Bone is a common site of metastasis for carcinomas of the prostate, breast, lung, kidney, bladder, and thyroid and for lymphomas and sarcomas. Prostate, breast, and lung primaries account for 80% of all bone metastases. Metastatic tumors of bone are more common than primary bone tumors. Tumors usually spread to bone hematogenously, but local invasion from soft tissue masses also occurs. In descending order of frequency, the sites most often involved are the vertebrae, proximal femur, pelvis, ribs, sternum, proximal humerus, and skull. Bone metastases may be asymptomatic or may produce pain, swelling, nerve root or spinal cord compression, pathologic fracture, or myelophthisis (replacement of the marrow). Symptoms of hypercalcemia may be noted in cases of bony destruction.

1	Pain is the most frequent symptom. It usually develops gradually over weeks, is usually localized, and often is more severe at night. When patients with back pain develop neurologic signs or symptoms, emergency evaluation for spinal cord compression is indicated (Chap. 331). Bone metastases exert a major adverse effect on quality of life in cancer patients.

1	Cancer in the bone may produce osteolysis, osteogenesis, or both. Osteolytic lesions result when the tumor produces substances that can directly elicit bone resorption (vitamin D–like steroids, prostaglandins, or parathyroid hormone–related peptide) or cytokines that can induce the formation of osteoclasts (interleukin 1 and tumor necrosis factor). Osteoblastic lesions result when the tumor produces cytokines that activate osteoblasts. In general, purely osteolytic lesions are best detected by plain radiography, but they may not be apparent until they are >1 cm. These lesions are more commonly associated with hypercalcemia and with the excretion of hydroxyproline-containing peptides indicative of matrix destruction. When osteoblastic activity is prominent, the lesions may be readily detected using radionuclide bone scanning (which is sensitive to new bone formation), and the radiographic appearance may show increased bone density or sclerosis. Osteoblastic lesions are associated with

1	using radionuclide bone scanning (which is sensitive to new bone formation), and the radiographic appearance may show increased bone density or sclerosis. Osteoblastic lesions are associated with higher serum levels of alkaline phosphatase and, if extensive, may produce hypocalcemia. Although some tumors may produce mainly osteolytic lesions (e.g., kidney cancer) and others mainly osteoblastic lesions (e.g., prostate cancer), most metastatic lesions produce both types of lesion and may go through stages where one or the other predominates.

1	In older patients, particularly women, it may be necessary to distinguish metastatic disease of the spine from osteoporosis. In osteoporosis, the cortical bone may be preserved, whereas cortical bone destruction is usually noted with metastatic cancer.

1	Treatment of metastatic bone disease depends on the underlying malignancy and the symptoms. Some metastatic bone tumors are curable (lymphoma, Hodgkin’s disease), and others are treated with palliative intent. Pain may be relieved by local radiation therapy. Hormonally responsive tumors are responsive to hormone inhibition (antiandrogens for prostate cancer, antiestrogens for breast cancer). Strontium-89, samarium-153, and radium-223 are bone-seeking radionuclides that can exert antitumor effects and relieve symptoms. Denosumab, a monoclonal antibody that binds to RANK ligand, inhibits osteoclastic activity and increases bone mineral density. Bisphosphonates such as pamidronate may relieve pain and inhibit bone resorption, thereby maintaining bone mineral density and reducing risk of fractures in patients with osteolytic metastases from breast cancer and multiple myeloma. Careful monitoring of serum electrolytes and creatinine is recommended. Monthly administration prevents

1	of fractures in patients with osteolytic metastases from breast cancer and multiple myeloma. Careful monitoring of serum electrolytes and creatinine is recommended. Monthly administration prevents bone-related clinical events and may reduce the incidence of bone metastases in women with breast cancer. When the integrity of a weight-bearing bone is threatened by an expanding metastatic lesion that is refractory to radiation therapy, prophylactic internal fixation is indicated. Overall survival is related to the prognosis of the underlying tumor. Bone pain at the end of life is particularly common; an adequate pain relief regimen including sufficient amounts of narcotic analgesics is required. The management of hypercalcemia is discussed in Chap. 424.

1	Carcinoma of Unknown primary Gauri R. Varadhachary, James L. Abbruzzese Carcinoma of unknown primary (CUP) is a biopsy-proven malig-nancy for which the anatomic site of origin remains unidentified after 120e an intensive search. CUP is one of the 10 most frequently diagnosed cancers worldwide, accounting for 3–5% of all cancers. Most investigators limit CUP to epithelial cancers and do not include lymphomas, metastatic melanomas, and metastatic sarcomas because these cancers have specific histologyand stage-based treatments that guide management.

1	The emergence of sophisticated imaging, robust immunohistochemistry (IHC), and genomic and proteomic tools has challenged the “unknown” designation. Additionally, effective targeted therapies in several cancers have moved the paradigm from empiricism to considering a personalized approach to CUP management. The reasons cancers present as CUP remain unclear. One hypothesis is that the primary tumor either regresses after seeding the metastasis or remains so small that it is not detected. It is possible that CUP falls on the continuum of cancer presentation where the primary has been contained or eliminated by the natural body defenses. Alternatively, CUP may represent a specific malignant event that results in an increase in metastatic spread or survival relative to the primary. Whether the CUP metastases truly define a clone that is genetically and phenotypically unique to this diagnosis remains to be determined.

1	Studies looking for unique signature abnormalities in CUP tumors have not been positive. Abnormalities in chromosomes 1 and 12 and other complex cytogenetic abnormalities have been reported. Aneuploidy has been described in 70% of CUP patients with metastatic adenocarcinoma or undifferentiated carcinoma. The overexpression of various genes, including Ras, bcl-2 (40%), her-2 (11%), and p53 (26–53%), has been studied in CUP samples, but they have no effect on response to therapy or survival. The extent of angiogenesis in CUP relative to that in metastases from known primaries has also been evaluated, but no consistent findings have emerged. Using the Sequenom (SQM) Massarray platform, a study in consecutive CUP patients showed that the overall mutational rate was surprisingly low (18%). No “new” low-frequency mutations were found using a panel of mutations involving the P13K/AKT pathway, MEK pathway, receptors, and downstream effectors. Nevertheless, it is possible that newer “deep

1	No “new” low-frequency mutations were found using a panel of mutations involving the P13K/AKT pathway, MEK pathway, receptors, and downstream effectors. Nevertheless, it is possible that newer “deep sequencing” techniques in select patients may yield consistent abnormalities.

1	Initial CUP evaluation has two goals: search for the primary tumor based on pathologic evaluation of the metastases and determine the extent of disease. Obtaining a thorough medical history from CUP patients is essential, including paying particular attention to previous surgeries, removed lesions, and family medical history to assess potential hereditary cancers. Adequate physical examination, including a digital rectal examination in men and breast and pelvic examinations in women, should be performed based on clinical presentation.

1	Role of Serum Tumor Markers and Cytogenetics Most tumor markers, including CEA, CA-125, CA 19-9, and CA 15-3, when elevated, are nonspecific and not helpful in determining the primary tumor site. Men who present with adenocarcinoma and osteoblastic metastasis should undergo a prostate-specific antigen (PSA) test. In patients with undifferentiated or poorly differentiated carcinoma (especially with a midline tumor), elevated β-human chorionic gonadotropin (β-hCG) and α fetoprotein (AFP) levels suggest the possibility of an extragonadal germ cell (testicular) tumor. With the availability of IHC, cytogenetic studies are rarely needed.

1	Role of Imaging Studies In the absence of contraindications, a baseline 120e-1 IV contrast computed tomography (CT) scan of the chest, abdomen, and pelvis is the standard of care. This helps to search for the primary tumor, evaluate the extent of disease, and select the most accessible biopsy site. Older studies suggested that the primary tumor site is detected in 20–35% of patients who undergo a CT scan of the abdomen and pelvis, although by current definition, these patients do not have CUP. These studies also suggest a latent primary tumor prevalence of 20%; with more sophisticated imaging, this has decreased to ≤5% today.

1	Mammography should be performed in all women who present with metastatic adenocarcinoma, especially in those with adenocarcinoma and isolated axillary lymphadenopathy. Magnetic resonance imaging (MRI) of the breast is a follow-up modality in patients with axillary adenopathy and suspected occult primary breast carcinoma following a negative mammography and ultrasound. The results of these imaging modalities can influence surgical management; a negative breast MRI result predicts a low tumor yield at mastectomy.

1	A conventional workup for a squamous cell carcinoma and cervical CUP (neck lymphadenopathy with no known primary tumor) includes a CT scan or MRI and invasive studies, including indirect and direct laryngoscopy, bronchoscopy, and upper endoscopy. Ipsilateral (or bilateral) staging tonsillectomy has been recommended for these patients. 18-Fluorodeoxyglucose positron emission tomography (18-FDG-PET) scans are useful in this patient population and may help guide the biopsy; determine the extent of disease; facilitate the appropriate treatment, including planning radiation fields; and help with disease surveillance. A smaller radiation field encompassing the primary (when found) and metastatic adenopathy decreases the risk of chronic xerostomia. Several studies have evaluated the utility of PET in patients with squamous cervical CUP, and head and neck primary tumors were identified in ~21–30%.

1	The diagnostic contribution of PET to the evaluation of other CUP (outside of the neck adenopathy indication) remains controversial and is not routinely recommended. PET-CT can be helpful for patients who are candidates for surgical intervention for solitary metastatic disease because the presence of disease outside the primary site may affect surgical planning. Invasive studies, including upper endoscopy, colonoscopy, and bronchoscopy, should be limited to symptomatic patients or those with laboratory, imaging, or pathologic abnormalities that suggest that these techniques will result in a high yield in finding a primary cancer. Role of Pathologic Studies A detailed pathologic examination of the most accessible biopsied tissue specimen is mandatory in CUP patients. Pathologic evaluation typically consists of hematoxylin and eosin stains and immunohistochemical tests.

1	LIGHT MICROSCOPY EVALUATION Adequate tissue obtained preferably by excisional biopsy or core-needle biopsy (instead of only a fine-needle aspiration) is stained with hematoxylin and eosin and subjected to light microscopic examination. On light microscopy, 60–65% of CUP is adenocarcinoma, and 5% is squamous cell carcinoma. The remaining 30–35% is poorly differentiated adenocarcinoma, poorly differentiated carcinoma, or poorly differentiated neoplasm. A small percentage of lesions are diagnosed as neuroendocrine cancers (2%), mixed tumors (adenosquamous or sarcomatoid carcinomas), or undifferentiated neoplasms (Table 120e-1). Histology Proportion, % Chapter 120e Carcinoma of Unknown Primary Well to moderately differentiated adenocarcinoma 60 Squamous cell cancer 5 Poorly differentiated adenocarcinoma, poorly FIGURE 120e-1 Approach to cytokeratin (CK7 and CK20) markers used in adenocarcinoma of unknown primary.

1	ROLE OF IMMUNOHISTOCHEMICAL ANALYSIS Immunohistochemical stains are peroxidase-labeled antibodies against specific tumor antigens that are used to define tumor lineage. The number of available immunohistochemical stains is ever-increasing. However, in CUP cases, more is not necessarily better, and immunohistochemical stains should be used in conjunction with the patient’s clinical presentation and imaging studies to select the best therapy. Communication between the clinician and pathologist is essential. No stain is 100% specific, and overinterpretation should be avoided. PSA and thyroglobulin tissue markers, which are positive in prostate and thyroid cancer, respectively, are the most specific of the current marker panel. However, these cancers rarely present as CUP, so the yield of these tests may be low. Fig. 120e-1 delineates a simple algorithm for immunohistochemical staining in CUP cases. Table 120e-2 lists additional tests that may be useful to further define the tumor

1	of these tests may be low. Fig. 120e-1 delineates a simple algorithm for immunohistochemical staining in CUP cases. Table 120e-2 lists additional tests that may be useful to further define the tumor lineage. A more comprehensive algorithm may improve the diagnostic accuracy but can make the process complex. With the use of immunohistochemical markers, electron microscopic analysis, which is time-consuming and expensive, is rarely needed.

1	There are >20 subtypes of cytokeratin (CK) intermediate filaments with different molecular weights and differential expression in various cell types and cancers. Monoclonal antibodies to specific CK subtypes have been used to help classify tumors according to their site of origin; commonly used CK stains in adenocarcinoma CUP are CK7 and CK20. CK7 is found in tumors of the lung, ovary, endometrium, breast, and upper gastrointestinal tract including pancreaticobiliary cancers, whereas CK20 is normally expressed in the gastrointestinal epithelium, urothelium, and Merkel cells. The nuclear CDX-2 transcription factor, which is the product of a homeobox gene necessary for intestinal organogenesis, is often used to aid in the diagnosis of gastrointestinal adenocarcinomas.

1	Thyroid transcription factor 1 (TTF-1) nuclear staining is typically positive in lung and thyroid cancers. Approximately 68% of adenocarcinomas and 25% of squamous cell lung cancers stain positive for TTF-1, which helps differentiate a lung primary tumor from metastatic adenocarcinoma in a pleural effusion, the mediastinum, or the lung parenchyma. Gross cystic disease fibrous protein-15, a 15-kDa monomer protein, is a marker of apocrine differentiation that is detected in 62–72% of breast carcinomas. UROIII, high-molecular-weight cytokeratin, thrombomodulin, and CK20 are the markers used to diagnose lesions of urothelial origin.

1	IHC performs the best when used in groups that give rise to patterns that are strongly indicative of certain profiles. For example, the TTF-1/CK7+ and CK20+/CDX-2+/CK7– phenotypes have been reported as very suggestive of lung and lower gastrointestinal cancer profiles, respectively, although these patterns have not been validated prospectively in the absence of a primary cancer. IHC is not without its limitations; several factors affect tissue antigenicity (antigen retrieval, specimen processing, and fixation), interpretation of stains in tumor (nuclear, cytoplasmic, membrane) versus normal tissue, inter-and intraobserver variability, and tissue heterogeneity and inadequacy (given small biopsy sizes). Communication with the pathologist is critical to determine if additional tissue will be beneficial in the pathologic evaluation.

1	ROLE OF TISSUE OF ORIGIN MOLECULAR PROFILING In the absence of a known primary, developing therapeutic strategies for CUP is challenging. The current diagnostic yield with imaging and immunochemistry is ~20– 30% for CUP patients. The use of gene expression studies holds the Commonly Considered IHC to Assist in Likely Primary Profile Differential Diagnosis of CUPa aPatterns emerging from coexpression of stains are better than individual stains to suggest putative primary site. Even with optimization, no IHC panel is 100% sensitive or specific (e.g., ovarian mucinous carcinoma can exhibit positivity with intestinal markers). Abbreviations: AFP, α fetoprotein; β-hCG, β human chorionic gonadotropin; CUP, carcinoma of unknown primary; IHC, immunohistochemistry; PSA, prostate-specific antigen. promise of substantially increasing this yield. Gene expression profiles are most commonly generated using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) or DNA microarray.

1	promise of substantially increasing this yield. Gene expression profiles are most commonly generated using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) or DNA microarray. Neural network programs have been used to develop predictive algorithms from the gene expression profiles. Typically, a training set of gene profiles from known cancers (preferably from metastatic sites) is used to train the software. The program can then be used to predict the putative origin of a test tumor and presumably of true CUP. Comprehensive gene expression databases that have become available for common malignancies may also be useful in CUP. These approaches have been effective in testing against known primary cancers and their metastases.

1	mRNA-or microRNA-based tissue of origin molecular profiling assays have been studied in prospective and retrospective CUP trials. Most of the CUP studies have evaluated assay performance, although the challenge with validating the accuracy of an assay for CUP is that, by definition, the primary cancer diagnosis cannot be verified. Thus, current estimates of tissue of origin test accuracy have relied on indirect metrics including comparison with IHC, clinical presentation, and appearance of latent primaries. Using these measures, the assays suggest a plausible primary in ~70% of patients studied. The only out-comes-based study is a single-arm study reporting a median survival of 12.5 months for patients who received assay-directed site-specific therapy. Firm conclusions of therapeutic impact cannot be drawn from this study given the nonrandomized design, statistical biases, confounding variables including use of subsequent lines of (empiric) therapy, and the heterogeneity of the CUP

1	cannot be drawn from this study given the nonrandomized design, statistical biases, confounding variables including use of subsequent lines of (empiric) therapy, and the heterogeneity of the CUP cancers. Additional studies are needed to better understand the clinical influence of tissue of origin profiling tools and how these assays complement IHC and help guide therapy.

1	The treatment of CUP continues to evolve, albeit slowly. The median survival duration of most patients with disseminated CUP is ~6–10 months. Systemic chemotherapy is the primary treatment modality in most patients with disseminated disease, but the careful integra-120e-3 tion of surgery, radiation therapy, and even periods of observation is important in the overall management of this condition (Figs. 120e-2 and 120e-3). Prognostic factors include performance status, site and number of metastases, response to chemotherapy, and serum lactate dehydrogenase (LDH) levels. Culine and colleagues developed a prognostic model using performance status and serum LDH levels, which allowed the assignment of patients into two subgroups with divergent outcomes. Future prospective trials using this prognostic model are warranted. Clinically, some CUP diagnoses fall into a favorable prognostic subset. Others, including those with disseminated CUP, do not and have a more unfavorable prognosis.

1	TREATMENT OF FAVORABLE CUP SUBSETS Women with Isolated Axillary Adenopathy Women with isolated axillary adenopathy with adenocarcinoma or carcinoma are usually treated for stage II or III breast cancer based on pathologic findings. These patients should undergo a breast MRI if mammogram and ultrasound are negative. Radiation therapy to the ipsilateral breast is indicated if the breast MRI is positive. Chemotherapy and/or hormonal therapy are indicated based on patient’s age (premenopausal or postmenopausal), nodal disease bulk, and hormone receptor status (Chap. 108). It is important to verify that the pathology suggests a breast cancer profile (morphology, immunohistochemical breast markers including estrogen receptor, mammoglobin, GCDFP15, HER-2 gene expression) before embarking on a breast cancer therapeutic program.

1	The term primary peritoneal papillary serous carcinoma (PPSC) has been used to describe CUP with carcinomatosis with the pathologic and laboratory (elevated CA-125 antigen) characteristics of ovarian cancer but no ovarian primary tumor identified on transvaginal sonography or laparotomy. Studies suggest that ovarian cancer and PPSC, which are both of müllerian origin, have similar gene expression profiles. Similar to patients with ovarian cancer, patients with PPSC are candidates for cytoreductive surgery, followed by adjuvant taxaneand platinum-based chemotherapy. In one Chapter 120e Carcinoma of Unknown Primary

1	Chapter 120e Carcinoma of Unknown Primary Isolated axillary nodes in women Bone only metastases in men (blastic) Solitary site of metastasis Peritoneal carcinoma Disseminated cancer, 2 or more sites involved Breast MRI if mammogram and ultrasound are negative If PSA not elevated, C or RT as indicated MRI (+). Breast surgery or radiation. C and/or hormonal therapy for breast cancer. MRI (–). No surgery, consider radiation. C for breast cancer. Check PSA (in tumor and serum). If elevated, Rx as prostate cancer. If resectable, resect with or without prior C or CRT. If unresectable, C, RT, or CRT depending on location of tumor If not suggestive of primary peritoneal, GI workup for primary. C, if good performance status. If suggestive of primary peritoneal cancer, treat as ovarian cancer C, if good performance status FIGURE 120e-2 Treatment algorithm for adenocarcinoma and poorly differentiated adenocarcinoma of unknown primary (CUP).

1	FIGURE 120e-2 Treatment algorithm for adenocarcinoma and poorly differentiated adenocarcinoma of unknown primary (CUP). C, chemotherapy; CRT, chemoradiation; GI, gastrointestinal; IHC, immunohistochemistry; MRI, magnetic resonance imaging; PSA, prostate-specific antigen; RT, radiation. Squamous cell CUP Disseminated, visceral metastases Metastatic inguinal adenopathy Metastatic cervical adenopathy Directed invasive tests as needed Perineal exam, anoscopy if needed. Pelvic examination in women. PET is optional. Triple endoscopy, consider tonsillectomy. CT neck and chest. PET is optional. C in good performance status patients. RT as indicated. If localized, lymph node dissection, followed by local RT in select patients If no extra-cervical disease—neck dissection followed by adjuvant RT vs. RT alone. C for bulky disease.

1	FIGURE 120e-3 Treatment algorithm for squamous cell carcinoma of unknown primary (CUP). C, chemotherapy; CT, computed tomography; PET, positron emission tomography; RT, radiation. retrospective study of 258 women with peritoneal carcinomatosis who had undergone cytoreductive surgery and chemotherapy, 22% of patients had a complete response to chemotherapy; the median survival duration was 18 months (range 11–24 months). However, not all peritoneal carcinomatosis in women is PPSC. Careful pathologic evaluation can help diagnose a colon cancer profile (CDX-2+, CK-20+, CK7−) or a pancreaticobiliary cancer or even a mislabeled peritoneal mesothelioma (calretinin positive).

1	Men with poorly differentiated or undifferentiated carcinoma that presents as a midline adenopathy should be evaluated for extragonadal germ cell malignancy. If diagnosed and treated as such, they often experience a good response to treatment with platinum-based combination chemotherapy. Response rates of >50% have been noted, and long-term survival rates of 10–15% long have been reported. Older patients (especially smokers) who present with mediastinal adenopathy are more likely to have a lung or head-andneck cancer profile.

1	Low-grade neuroendocrine carcinoma often has an indolent course, and treatment decisions are based on symptoms and tumor bulk. Urine 5-HIAA and serum chromogranin may be elevated and can be followed as markers. Often the patient is treated with somatostatin analogues alone for hormone-related symptoms (diarrhea, flushing, nausea). Specific local therapies or systemic therapy would only be indicated if the patient is symptomatic with local pain secondary to significant growth of the metastasis or the hormone-related symptoms are not controlled with endocrine therapy. Patients with high-grade neuroendocrine carcinoma are treated as having small-cell lung cancer and are responsive to chemotherapy; 20–25% show a complete response, and up to 10% patients survive more than 5 years.

1	Patients with early-stage squamous cell carcinoma involving the cervical lymph nodes are candidates for node dissection and radiation therapy, which can result in long-term survival. The role of chemotherapy in these patients is undefined, although chemoradiation therapy or induction chemotherapy is often used and is beneficial in bulky N2/N3 lymph node disease. Patients with solitary metastases can also experience good treatment outcomes. Some patients who present with locoregional disease are candidates for aggressive trimodality management; both prolonged disease-free interval and occasionally cure are possible. Blastic bone-only metastasis is a rare presentation, and elevated serum PSA or tumor staining with PSA may provide confirmatory evidence of prostate cancer in these patients. Those with elevated levels are candidates for hormonal therapy for prostate cancer, although it is important to rule out other primary tumors (lung most common).

1	Patients who present with liver, brain, and adrenal metastatic disease usually have a poor prognosis. Patients with nonserous papillary primary peritoneal carcinomatosis can have a large differential diagnosis, which is mainly of gastrointestinal profile and includes gastric, appendiceal, colon, and pancreaticobiliary profiles. Traditionally, platinum-based combination chemotherapy regimens have been used to treat CUP. Several broadly used regimens have been studied in the last two decades; these include paclitaxelcarboplatin, gemcitabine-cisplatin, gemcitabine-oxaliplatin, and irinotecan and fluoropyrimidine-based therapies. These chemotherapeutic agents used as empiric regimens have shown a response rate of 25–40%, and their use obtains median survival times of 6–13 months.

1	Outside of favorable subsets, there is a small group of patients with a “definitive” IHC. These patients usually have a single diagnosis based on their clinicopathologic presentation and are often treated for the putative primary tumor. This does not guarantee a response, although it increases the probability of response when select drugs are chosen from a class of drugs known to work in that cancer type. Patients who do not fall into those categories are candidates for broad-spectrum platinum-based regimens, clinical trials, and additional trial-based genomic and proteomic tests. Today, we do not have effective drugs for several CUP cancer profiles, and treatments overlap for some cancers. However, as novel therapies are developed for additional known cancers, tissue of origin and assessment of molecular features of the tumor will be important and might direct more selective treatment.

1	Patients with CUP should undergo a directed diagnostic search for the primary tumor on the basis of clinical and pathologic data. Subsets of patients have prognostically favorable disease, as defined by clinical or histologic criteria, and may substantially benefit from aggressive treatment and expect prolonged survival. However, for most patients who present with advanced CUP, the prognosis remains poor with early resistance to available cytotoxic therapy. The current focus has shifted away from empirical chemotherapeutic trials to understanding the metastatic phenotype, tissue of origin profiling, and evaluating molecular targets in CUP patients. paraneoplastic syndromes: endocrinologic/hematologic J. Larry Jameson, Dan L. Longo Neoplastic cells can produce a variety of products that can stimulate hormonal, hematologic, dermatologic, and neurologic responses. 121

1	Paraneoplastic syndromes is the term used to refer to the disorders that accompany benign or malignant tumors but are not directly related to mass effects or invasion. Tumors of neuroendocrine origin, such as small-cell lung carcinoma (SCLC) and carcinoids, produce a wide array of peptide hormones and are common causes of paraneoplastic syndromes. However, almost every type of tumor has the potential to produce hormones or to induce cytokine and immunologic responses. Careful studies of the prevalence of paraneoplastic syndromes indicate that they are more common than is generally appreciated. The signs, symptoms, and metabolic alterations associated with paraneoplastic disorders may be overlooked in the context of a malignancy and its treatment. Consequently, atypical clinical manifestations in a patient with cancer should prompt consideration of a paraneoplastic syndrome. The most common endocrinologic and hematologic syndromes associated with underlying neoplasia will be discussed

1	in a patient with cancer should prompt consideration of a paraneoplastic syndrome. The most common endocrinologic and hematologic syndromes associated with underlying neoplasia will be discussed here.

1	Etiology Hormones can be produced from eutopic or ectopic sources. Eutopic refers to the expression of a hormone from its normal tissue of origin, whereas ectopic refers to hormone production from an atypical tissue source. For example, adrenocorticotropic hormone (ACTH) is expressed eutopically by the corticotrope cells of the anterior pituitary, but it can be expressed ectopically in SCLC. Many hormones are produced at low levels from a wide array of tissues in addition to the classic endocrine source. Thus, ectopic expression is often a quantitative change rather than an absolute change in tissue expression. Nevertheless, the term ectopic expression is firmly entrenched and conveys the abnormal physiology associated with hormone production by neoplastic cells. In addition to high levels of hormones, ectopic expression typically is characterized by abnormal regulation of hormone production (e.g., defective feedback control) and peptide processing (resulting in large, unprocessed

1	of hormones, ectopic expression typically is characterized by abnormal regulation of hormone production (e.g., defective feedback control) and peptide processing (resulting in large, unprocessed precursors).

1	A diverse array of molecular mechanisms has been suggested to cause ectopic hormone production. In rare instances, genetic rearrangements explain aberrant hormone expression. For example, translocation of the parathyroid hormone (PTH) gene can result in high levels of PTH expression in tissues other than the parathyroid gland because the genetic rearrangement brings the PTH gene under the control of atypical regulatory elements. A related phenomenon is well documented in many forms of leukemia and lymphoma, in which somatic genetic rearrangements confer a growth advantage and alter cellular differentiation and function (Chap. 134). Although genetic rearrangements cause selected cases of ectopic hormone production, this mechanism is rare, as many tumors are associated with excessive production of numerous peptides. Cellular dedifferentiation probably underlies most cases of ectopic hormone production. Many cancers are poorly differentiated, and certain tumor products, such as human

1	of numerous peptides. Cellular dedifferentiation probably underlies most cases of ectopic hormone production. Many cancers are poorly differentiated, and certain tumor products, such as human chorionic gonadotropin (hCG), parathyroid hormone–related protein (PTHrP), and α fetoprotein, are characteristic of gene expression at earlier developmental stages. In contrast, the propensity of certain cancers to produce particular hormones (e.g., squamous cell carcinomas produce PTHrP) suggests that dedifferentiation is partial or that selective pathways are derepressed. These expression profiles probably reflect epigenetic modifications that alter transcriptional repression, microRNA expression, and other pathways that govern cell differentiation.

1	In SCLC, the pathway of differentiation has been relatively well defined. The neuroendocrine phenotype is dictated in part by the basic-helix-loop-helix (bHLH) transcription factor human achaetescute homologue 1 (hASH-1), which is expressed at abnormally high Hypercalcemia of Parathyroid hormone–related protein (PTHrP) Squamous cell (head and neck, lung, skin), breast, genitourinary, malignancy gastrointestinal 1,25-dihydroxyvitamin D Lymphomas Parathyroid hormone (PTH) (rare) Lung, ovary Prostaglandin E2 (PGE2) (rare) Renal, lung Syndrome of inappropriate Vasopressin Lung (squamous, small cell), gastrointestinal, genitourinary, ovary antidiuretic hormone secretion (SIADH) Cushing’s syndrome Adrenocorticotropic hormone (ACTH) Lung (small cell, bronchial carcinoid, adenocarcinoma, squamous), thymus, pancreatic islet, medullary thyroid carcinoma

1	Cushing’s syndrome Adrenocorticotropic hormone (ACTH) Lung (small cell, bronchial carcinoid, adenocarcinoma, squamous), thymus, pancreatic islet, medullary thyroid carcinoma Corticotropin-releasing hormone (CRH) (rare) Pancreatic islet, carcinoid, lung, prostate Ectopic expression of gastric inhibitory peptide (GIP), Macronodular adrenal hyperplasia luteinizing hormone (LH)/human chorionic gonadotropin (hCG), other G protein–coupled receptors (rare) aOnly the most common tumor types are listed. For most ectopic hormone syndromes, an extensive list of tumors has been reported to produce one or more hormones. bhCG is produced eutopically by trophoblastic tumors. Certain tumors produce disproportionate amounts of the hCG α or hCG β subunit. High levels of hCG rarely cause hyperthyroidism because of weak binding to the TSH receptor. cCalcitonin is produced eutopically by medullary thyroid carcinoma and is used as a tumor marker.

1	levels in SCLC associated with ectopic ACTH. The activity of hASH-1 is inhibited by hairy enhancer of split 1 (HES-1) and by Notch proteins, which also are capable of inducing growth arrest. Thus, abnormal expression of these developmental transcription factors appears to provide a link between cell proliferation and differentiation. Ectopic hormone production would be merely an epiphenomenon associated with cancer if it did not result in clinical manifestations. Excessive and unregulated production of hormones such as ACTH, PTHrP, and vasopressin can lead to substantial morbidity and complicate the cancer treatment plan. Moreover, the paraneoplastic endocrinopathies may be a presenting clinical feature of underlying malignancy and prompt the search for an unrecognized tumor.

1	A large number of paraneoplastic endocrine syndromes have been described, linking overproduction of particular hormones with specific types of tumors. However, certain recurring syndromes emerge from this group (Table 121-1). The most common paraneoplastic endocrine syndromes include hypercalcemia from overproduction of PTHrP and other factors, hyponatremia from excess vasopressin, and Cushing’s syndrome from ectopic ACTH. (See also Chap. 424)

1	(See also Chap. 424) Etiology Humoral hypercalcemia of malignancy (HHM) occurs in up to 20% of patients with cancer. HHM is most common in cancers of the lung, head and neck, skin, esophagus, breast, and genitourinary tract and in multiple myeloma and lymphomas. There are several distinct humoral causes of HHM, but it is caused most commonly by overproduction of PTHrP. In addition to acting as a circulating humoral factor, bone metastases (e.g., breast, multiple myeloma) may produce PTHrP, leading to local osteolysis and hypercalcemia. PTHrP may also affect the initiation and progression of tumors by acting through pro-survival and chemokine pathways.

1	PTHrP is structurally related to PTH and binds to the PTH receptor, explaining the similar biochemical features of HHM and hyperparathyroidism. PTHrP plays a key role in skeletal development and regulates cellular proliferation and differentiation in other tissues, including skin, bone marrow, breast, and hair follicles. The mechanism of PTHrP induction in malignancy is incompletely understood; however, tumor-bearing tissues commonly associated with HHM normally produce PTHrP during development or cell renewal. PTHrP expression is stimulated by hedgehog pathways and Gli transcription factors that are active in many malignancies. Transforming growth factor β (TGF-β), which is produced by many tumors, also stimulates PTHrP, in part by activating the Gli pathway. Mutations in certain oncogenes, such as Ras, also can activate PTHrP expression. In adult T cell lymphoma, the transactivating Tax protein produced by human T cell lymphotropic virus 1 (HTLV-1) stimulates PTHrP promoter

1	oncogenes, such as Ras, also can activate PTHrP expression. In adult T cell lymphoma, the transactivating Tax protein produced by human T cell lymphotropic virus 1 (HTLV-1) stimulates PTHrP promoter activity. Metastatic lesions to bone are more likely to produce PTHrP than are metastases in other tissues, suggesting that bone produces factors (e.g., TGF-β) that enhance PTHrP production or that PTHrP-producing metastases have a selective growth advantage in bone. PTHrP activates the pro-survival AKT pathway and the chemokine receptor CXCR4. Thus, PTHrP production can be stimulated by mutations in oncogenes, altered expression of viral or cellular transcription factors, and local growth factors. In addition to its role in HHM, the PTHrP pathway may also provide a potential target for therapeutic intervention to impede cancer growth.

1	Paraneoplastic Syndromes: Endocrinologic/Hematologic 610 Another relatively common cause of HHM is excess production of 1,25-dihydroxyvitamin D. Like granulomatous disorders associated with hypercalcemia, lymphomas can produce an enzyme that converts 25-hydroxyvitamin D to the more active 1,25-dihydroxyvitamin D, leading to enhanced gastrointestinal calcium absorption. Other causes of HHM include tumor-mediated production of osteolytic cytokines and inflammatory mediators. Clinical Manifestations The typical presentation of HHM is a patient with a known malignancy who is found to be hypercalcemic on routine laboratory tests. Less often, hypercalcemia is the initial presenting feature of malignancy. Particularly when calcium levels are markedly increased (>3.5 mmol/L [>14 mg/dL]), patients may experience fatigue, mental status changes, dehydration, or symptoms of nephrolithiasis.

1	Diagnosis Features that favor HHM, as opposed to primary hyperparathyroidism, include known malignancy, recent onset of hypercalcemia, and very high serum calcium levels. Like hyperparathyroidism, hypercalcemia caused by PTHrP is accompanied by hypercalciuria and hypophosphatemia. Patients with HHM typically have metabolic alkalosis rather than hyperchloremic acidosis, as is seen in hyperparathyroidism. Measurement of PTH is useful to exclude primary hyperparathyroidism; the PTH level should be suppressed in HHM. An elevated PTHrP level confirms the diagnosis, and it is increased in ~80% of hypercalcemic patients with cancer. 1,25-Dihydroxyvitamin D levels may be increased in patients with lymphoma.

1	The management of HHM begins with removal of excess calcium in the diet, medications, or IV solutions. Saline rehydration (typically 200–500 mL/h) is used to dilute serum calcium and promote calciuresis; exercise caution in patients with cardiac, hepatic, or renal insufficiency. Forced diuresis with furosemide (20–80 mg IV in escalating doses) or other loop diuretics can enhance calcium excretion but provides relatively little value except in life-threatening hypercalcemia. When used, loop diuretics should be administered only after complete rehydration and with careful monitoring of fluid balance. Oral phosphorus (e.g., 250 mg Neutra-Phos 3–4 times daily) should be given until serum phosphorus is >1 mmol/L (>3 mg/dL). Bisphosphonates such as pamidronate (60–90 mg IV), zoledronate (4–8 mg IV), and etidronate (7.5 mg/kg per day PO for 3–7 consecutive days) can reduce serum calcium within 1–2 days and suppress calcium release for several weeks. Bisphosphonate infusions can be repeated,

1	IV), and etidronate (7.5 mg/kg per day PO for 3–7 consecutive days) can reduce serum calcium within 1–2 days and suppress calcium release for several weeks. Bisphosphonate infusions can be repeated, or oral bisphosphonates can be used for chronic treatment. Dialysis should be considered in severe hypercalcemia when saline hydration and bisphosphonate treatments are not possible or are too slow in onset. Previously used agents such as calcitonin and mithramycin have little utility now that bisphosphonates are available. Calcitonin (2–8 U/kg SC every 6–12 h) should be considered when rapid correction of severe hypercalcemia is needed. Hypercalcemia associated with lymphomas, multiple myeloma, or leukemia may respond to glucocorticoid treatment (e.g., prednisone 40–100 mg PO in four divided doses).

1	ECTOPIC VASOPRESSIN: TUMOR-ASSOCIATED SIADH (See also Chap. 63) Etiology Vasopressin is an antidiuretic hormone normally produced by the posterior pituitary gland. Ectopic vasopressin production by tumors is a common cause of the syndrome of inappropriate antidiuretic hormone (SIADH), occurring in at least half of patients with SCLC. SIADH also can be caused by a number of nonneoplastic conditions, including central nervous system (CNS) trauma, infections, and medications (Chap. 404). Compensatory responses to SIADH, such as decreased thirst, may mitigate the development of hyponatremia. However, with prolonged production of excessive vasopressin, the osmostat controlling thirst and hypothalamic vasopressin secretion may become reset. In addition, intake of free water, orally or intravenously, can quickly worsen hyponatremia because of reduced renal diuresis.

1	Tumors with neuroendocrine features, such as SCLC and carcinoids, are the most common sources of ectopic vasopressin production, but it also occurs in other forms of lung cancer and with CNS lesions, head and neck cancer, and genitourinary, gastrointestinal, and ovarian cancers. The mechanism of activation of the vasopressin gene in these tumors is unknown but often involves concomitant expression of the adjacent oxytocin gene, suggesting derepression of this locus. Clinical Manifestations Most patients with ectopic vasopressin secretion are asymptomatic and are identified because of the presence of hyponatremia on routine chemistry testing. Symptoms may include weakness, lethargy, nausea, confusion, depressed mental status, and seizures. The severity of symptoms reflects the rapidity of onset as well as the severity of hyponatremia. Hyponatremia usually develops slowly but may be exacerbated by the administration of IV fluids or the institution of new medications.

1	Diagnosis The diagnostic features of ectopic vasopressin production are the same as those of other causes of SIADH (Chaps. 63 and 404). Hyponatremia and reduced serum osmolality occur in the setting of an inappropriately normal or increased urine osmolality. Urine sodium excretion is normal or increased unless volume depletion is present. Other causes of hyponatremia should be excluded, including renal, adrenal, or thyroid insufficiency. Physiologic sources of vasopressin stimulation (CNS lesions, pulmonary disease, nausea), adaptive circulatory mechanisms (hypotension, heart failure, hepatic cirrhosis), and medications, including many chemotherapeutic agents, also should be considered as possible causes of hyponatremia. Vasopressin measurements are not usually necessary to make the diagnosis.

1	Most patients with ectopic vasopressin production develop hyponatremia over several weeks or months. The disorder should be corrected gradually unless mental status is altered or there is risk of seizures. Treatment of the underlying malignancy may reduce ectopic vasopressin production, but this response is slow if it occurs at all. Fluid restriction to less than urine output, plus insensible losses, is often sufficient to correct hyponatremia partially. However, strict monitoring of the amount and types of liquids consumed or administered intravenously is required for fluid restriction to be effective. Salt tablets and saline are not helpful unless volume depletion is also present. Demeclocycline (150–300 mg orally three to four times daily) can be used to inhibit vasopressin action on the renal distal tubule, but its onset of action is relatively slow (1–2 weeks). Conivaptan, a nonpeptide V2-receptor antagonist, can be administered either PO (20–120 mg bid) or IV (10–40 mg) and is

1	the renal distal tubule, but its onset of action is relatively slow (1–2 weeks). Conivaptan, a nonpeptide V2-receptor antagonist, can be administered either PO (20–120 mg bid) or IV (10–40 mg) and is particularly effective when used in combination with fluid restriction in euvolemic hyponatremia. Tolvaptan (15 mg PO daily) is another vasopressin antagonist. The dose can be increased to 30–60 mg/d based on response. Severe hyponatremia (Na <115 meq/L) or mental status changes may require treatment with hypertonic (3%) or normal saline infusion together with furosemide to enhance free water clearance. The rate of sodium correction should be slow (0.5–1 meq/L per hour) to prevent rapid fluid shifts and the possible development of central pontine myelinolysis.

1	(See also Chap. 406)

1	Etiology Ectopic ACTH production accounts for 10–20% of cases of Cushing’s syndrome. The syndrome is particularly common in neuroendocrine tumors. SCLC is the most common cause of ectopic ACTH, followed by bronchial and thymic carcinoids, islet cell tumors, other carcinoids, and pheochromocytomas. Ectopic ACTH production is caused by increased expression of the proopiomelanocortin (POMC) gene, which encodes ACTH, along with melanocyte-stimulating hormone (MSH), β lipotropin, and several other peptides. In many tumors, there is abundant but aberrant expression of the POMC gene from an internal promoter, proximal to the third exon, which encodes ACTH. However, because this product lacks the signal sequence necessary for protein processing, it is not secreted. Increased production of ACTH arises instead from less abundant, but unregulated, POMC expression from the same promoter site used in the pituitary. However, because the tumors lack many of the enzymes needed to process the POMC

1	arises instead from less abundant, but unregulated, POMC expression from the same promoter site used in the pituitary. However, because the tumors lack many of the enzymes needed to process the POMC polypeptide, it is typically released as multiple large, biologically inactive fragments along with relatively small amounts of fully processed, active ACTH.

1	Rarely, corticotropin-releasing hormone (CRH) is produced by pancreatic islet cell tumors, SCLC, medullary thyroid cancer, carcinoids, or prostate cancer. When levels are high enough, CRH can cause pituitary corticotrope hyperplasia and Cushing’s syndrome. Tumors that produce CRH sometimes also produce ACTH, raising the possibility of a paracrine mechanism for ACTH production. A distinct mechanism for ACTH-independent Cushing’s syndrome involves ectopic expression of various G protein–coupled receptors in the adrenal nodules. Ectopic expression of the gastric inhibitory peptide (GIP) receptor is the best-characterized example of this mechanism. In this case, meals induce GIP secretion, which inappropriately stimulates adrenal growth and glucocorticoid production.

1	Clinical Manifestations The clinical features of hypercortisolemia are detected in only a small fraction of patients with documented ectopic ACTH production. Patients with ectopic ACTH syndrome generally exhibit less marked weight gain and centripetal fat redistribution, probably because the exposure to excess glucocorticoids is relatively brief and because cachexia reduces the propensity for weight gain and fat deposition. The ectopic ACTH syndrome is associated with several clinical features that distinguish it from other causes of Cushing’s syndrome (e.g., pituitary adenomas, adrenal adenomas, iatrogenic glucocorticoid excess). The metabolic manifestations of ectopic ACTH syndrome are dominated by fluid retention and hypertension, hypokalemia, metabolic alkalosis, glucose intolerance, and occasionally steroid psychosis. The very high ACTH levels often cause increased pigmentation, and melanotrope-stimulating hormone (MSH) activity derived from the POMC precursor peptide is also

1	and occasionally steroid psychosis. The very high ACTH levels often cause increased pigmentation, and melanotrope-stimulating hormone (MSH) activity derived from the POMC precursor peptide is also increased. The extraordinarily high glucocorticoid levels in patients with ectopic sources of ACTH can lead to marked skin fragility and easy bruising. In addition, the high cortisol levels often overwhelm the renal 11β-hydroxysteroid dehydrogenase type II enzyme, which normally inactivates cortisol and prevents it from binding to renal mineralocorticoid receptors. Consequently, in addition to the excess mineralocorticoids produced by ACTH stimulation of the adrenal gland, high levels of cortisol exert activity through the mineralocorticoid receptor, leading to severe hypokalemia.

1	Diagnosis The diagnosis of ectopic ACTH syndrome is usually not difficult in the setting of a known malignancy. Urine free cortisol levels fluctuate but are typically greater than two to four times normal, and the plasma ACTH level is usually >22 pmol/L (>100 pg/mL). A suppressed ACTH level excludes this diagnosis and indicates an ACTH-independent cause of Cushing’s syndrome (e.g., adrenal or exogenous glucocorticoid). In contrast to pituitary sources of ACTH, most ectopic sources of ACTH do not respond to glucocorticoid suppression. Therefore, high-dose dexamethasone (8 mg PO) suppresses 8:00 a.m. serum cortisol (50% decrease from baseline) in ~80% of pituitary ACTH-producing adenomas but fails to suppress ectopic ACTH in ~90% of cases. Bronchial and other carcinoids are well-documented exceptions to these general guidelines, as these ectopic sources of ACTH may exhibit feedback regulation indistinguishable from pituitary adenomas, including suppression by high-dose dexamethasone,

1	exceptions to these general guidelines, as these ectopic sources of ACTH may exhibit feedback regulation indistinguishable from pituitary adenomas, including suppression by high-dose dexamethasone, and ACTH responsiveness to adrenal blockade with metyrapone. If necessary, petrosal sinus catheterization can be used to evaluate a patient with ACTH-dependent Cushing’s syndrome when the source of ACTH is unclear. After CRH stimulation, a 3:1 petrosal sinus:peripheral ACTH ratio strongly suggests a pituitary ACTH source. Imaging studies (computed tomography or magnetic reso-611 nance imaging) are also useful in the evaluation of suspected carcinoid lesions, allowing biopsy and characterization of hormone production using special stains. If available, positron emission tomography or octreotide scanning may identify some sources of ACTH production.

1	The morbidity associated with the ectopic ACTH syndrome can be substantial. Patients may experience depression or personality changes because of extreme cortisol excess. Metabolic derangements, including diabetes mellitus and hypokalemia, can worsen fatigue. Poor wound healing and predisposition to infections can complicate the surgical management of tumors, and opportunistic infections caused by organisms such as Pneumocystis carinii and mycoses are often the cause of death in patients with ectopic ACTH production. These patients likely have increased risk of venous thromboembolism reflecting the combination of malignancy and altered coagulation factor profiles. Depending on prognosis and treatment plans for the underlying malignancy, measures to reduce cortisol levels are often indicated. Treatment of the underlying malignancy may reduce ACTH levels but is rarely sufficient to reduce cortisol levels to normal. Adrenalectomy is not practical for most of these patients but should be

1	Treatment of the underlying malignancy may reduce ACTH levels but is rarely sufficient to reduce cortisol levels to normal. Adrenalectomy is not practical for most of these patients but should be considered during surgery for the malignancy or if the underlying tumor is not resectable and the prognosis is otherwise favorable (e.g., carcinoid). Medical therapy with ketoconazole (300–600 mg PO bid), metyrapone (250–500 mg PO every 6 h), mitotane (3–6 g PO in four divided doses, tapered to maintain low cortisol production), or other agents that block steroid synthesis or action is often the most practical strategy for managing the hypercortisolism associated with ectopic ACTH production. Glucocorticoid replacement should be provided to prevent adrenal insufficiency (Chap. 406). Unfortunately, many patients eventually progress despite medical blockade.

1	(See also Chap. 420) Mesenchymal tumors, hemangiopericytomas, hepatocellular tumors, adrenal carcinomas, and a variety of other large tumors have been reported to produce excessive amounts of insulin-like growth factor type II (IGF-II) precursor, which binds weakly to insulin receptors and more strongly to IGF-I receptors, leading to insulin-like actions. The gene encoding IGF-II resides on a chromosome 11p15 locus that is normally imprinted (that is, expression is exclusively from a single parental allele). Biallelic expression of the IGF-II gene occurs in a subset of tumors, suggesting loss of methylation and loss of imprinting as a mechanism for gene induction. In addition to increased IGF-II production, IGF-II bioavailability is increased due to complex alterations in circulating binding proteins. Increased IGF-II suppresses growth hormone (GH) and insulin, resulting in reduced IGF binding protein 3 (IGFBP-3), IGF-I, and acid-labile subunit (ALS). The reduction in ALS and IGFBP-3,

1	proteins. Increased IGF-II suppresses growth hormone (GH) and insulin, resulting in reduced IGF binding protein 3 (IGFBP-3), IGF-I, and acid-labile subunit (ALS). The reduction in ALS and IGFBP-3, which normally sequester IGF-II, causes it to be displaced to a small circulating complex that has greater access to insulin target tissues. For this reason, circulating IGF-II levels may not be markedly increased despite causing hypoglycemia. In addition to IGF-II–mediated hypoglycemia, tumors may occupy enough of the liver to impair gluconeogenesis.

1	In most cases, a tumor causing hypoglycemia is clinically apparent (usually >10 cm in size) and hypoglycemia develops in association with fasting. The diagnosis is made by documenting low serum glucose and suppressed insulin levels in association with symptoms of hypoglycemia. Serum IGF-II levels may not be increased (IGF-II assays may not detect IGF-II precursors). Increased IGF-II mRNA expression is found in most of these tumors. Any medications associated with hypoglycemia should be eliminated. Treatment of the underlying malignancy, if possible, may reduce the predisposition to hypoglycemia. Frequent meals and IV glucose, especially during sleep or fasting, are often Paraneoplastic Syndromes: Endocrinologic/Hematologic 612 necessary to prevent hypoglycemia. Glucagon and glucocorticoids have also been used to enhance glucose production.

1	hCG is composed of α and β subunits and can be produced as intact hormone, which is biologically active, or as uncombined biologically inert subunits. Ectopic production of intact hCG occurs most often in association with testicular embryonal tumors, germ cell tumors, extragonadal germinomas, lung cancer, hepatoma, and pancreatic islet tumors. Eutopic production of hCG occurs with trophoblastic malignancies. hCG α subunit production is particularly common in lung cancer and pancreatic islet cancer. In men, high hCG levels stimulate steroidogenesis and aromatase activity in testicular Leydig cells, resulting in increased estrogen production and the development of gynecomastia. Precocious puberty in boys or gynecomastia in men should prompt measurement of hCG and consideration of a testicular tumor or another source of ectopic hCG production. Most women are asymptomatic. hCG is easily measured. Treatment should be directed at the underlying malignancy.

1	Hypophosphatemic oncogenic osteomalacia, also called tumor-induced osteomalacia (TIO), is characterized by markedly reduced serum phosphorus and renal phosphate wasting, leading to muscle weakness, bone pain, and osteomalacia. Serum calcium and PTH levels are normal, and 1,25-dihydroxyvitamin D is low. Oncogenic osteomalacia is usually caused by benign mesenchymal tumors, such as hemangiopericytomas, fibromas, and giant cell tumors, often of the skeletal extremities or head. It has also been described in sarcomas and in patients with prostate and lung cancer. Resection of the tumor reverses the disorder, confirming its humoral basis. The circulating phosphaturic factor is called phosphatonin—a factor that inhibits renal tubular reabsorption of phosphate and renal conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D. Phosphatonin has been identified as fibroblast growth factor 23 (FGF23). FGF23 levels are increased in some, but not all, patients with osteogenic osteomalacia.

1	D to 1,25-dihydroxyvitamin D. Phosphatonin has been identified as fibroblast growth factor 23 (FGF23). FGF23 levels are increased in some, but not all, patients with osteogenic osteomalacia. FGF23 forms a ternary complex with the klotho protein and renal FGF receptors to reduce renal phosphate reabsorption. Treatment involves removal of the tumor, if possible, and supplementation with phosphate and vitamin D. Octreotide treatment reduces phosphate wasting in some patients with tumors that express somatostatin receptor subtype 2. Octreotide scans may also be useful in detecting these tumors.

1	The elevation of granulocyte, platelet, and eosinophil counts in most patients with myeloproliferative disorders is caused by the proliferation of the myeloid elements due to the underlying disease rather than to a paraneoplastic syndrome. The paraneoplastic hematologic syndromes in patients with solid tumors are less well characterized than are the endocrine syndromes because the ectopic hormone(s) or cytokines responsible have not been identified in most of these tumors (Table 121-2). The extent of the paraneoplastic syndromes parallels the course of the cancer. Ectopic production of erythropoietin by cancer cells causes most paraneoplastic erythrocytosis. The ectopically produced erythropoietin stimulates the production of red blood cells (RBCs) in the bone marrow and raises the hematocrit. Other lymphokines and hormones produced by cancer cells may stimulate erythropoietin release but have not been proved to cause erythrocytosis.

1	Most patients with erythrocytosis have an elevated hematocrit (>52% in men, >48% in women) that is detected on a routine blood count. Approximately 3% of patients with renal cell cancer, 10% of patients with hepatoma, and 15% of patients with cerebellar hemangioblastomas have erythrocytosis. In most cases, the erythrocytosis is asymptomatic. Patients with erythrocytosis due to a renal cell cancer, hepatoma, or CNS cancer should have measurement of red cell mass. If the red cell Cancers Typically Associated Syndrome Proteins with Syndrome Abbreviations: G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin. mass is elevated, the serum erythropoietin level should be measured. Patients with an appropriate cancer, elevated erythropoietin levels, and no other explanation for erythrocytosis (e.g., hemoglobinopathy that causes increased O2 affinity; Chap. 77) have the paraneoplastic syndrome.

1	Successful resection of the cancer usually resolves the erythrocytosis. If the tumor cannot be resected or treated effectively with radiation therapy or chemotherapy, phlebotomy may control any symptoms related to erythrocytosis. Approximately 30% of patients with solid tumors have granulocytosis (granulocyte count >8000/μL). In about half of patients with granulocytosis and cancer, the granulocytosis has an identifiable nonparaneoplastic etiology (infection, tumor necrosis, glucocorticoid administration, etc.). The other patients have proteins in urine and serum that stimulate the growth of bone marrow cells. Tumors and tumor cell lines from patients with lung, ovarian, and bladder cancers have been documented to produce granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and/or interleukin 6 (IL-6). However, the etiology of granulocytosis has not been characterized in most patients.

1	Patients with granulocytosis are nearly all asymptomatic, and the differential white blood cell count does not have a shift to immature forms of neutrophils. Granulocytosis occurs in 40% of patients with lung and gastrointestinal cancers, 20% of patients with breast cancer, 30% of patients with brain tumors and ovarian cancers, 20% of patients with Hodgkin’s disease, and 10% of patients with renal cell carcinoma. Patients with advanced-stage disease are more likely to have granulocytosis than are those with early-stage disease. Paraneoplastic granulocytosis does not require treatment. The granulocytosis resolves when the underlying cancer is treated.

1	Paraneoplastic granulocytosis does not require treatment. The granulocytosis resolves when the underlying cancer is treated. Some 35% of patients with thrombocytosis (platelet count >400,000/μL) have an underlying diagnosis of cancer. IL-6, a candidate molecule for the etiology of paraneoplastic thrombocytosis, stimulates the production of platelets in vitro and in vivo. Some patients with cancer and thrombocytosis have elevated levels of IL-6 in plasma. Another candidate molecule is thrombopoietin, a peptide hormone that stimulates megakaryocyte proliferation and platelet production. The etiology of thrombocytosis has not been established in most cases.

1	Patients with thrombocytosis are nearly all asymptomatic. Thrombocytosis is not clearly linked to thrombosis in patients with cancer. Thrombocytosis is present in 40% of patients with lung and gastrointestinal cancers; 20% of patients with breast, endometrial, and ovarian cancers; and 10% of patients with lymphoma. Patients with thrombocytosis are more likely to have advanced-stage disease and have a poorer prognosis than do patients without thrombocytosis. In ovarian cancer, IL-6 has been shown to directly promote tumor growth. Paraneoplastic thrombocytosis does not require treatment other than treatment of the underlying tumor. Eosinophilia is present in ~1% of patients with cancer. Tumors and tumor cell lines from patients with lymphomas or leukemia may produce IL-5, which stimulates eosinophil growth. Activation of IL-5 transcription in lymphomas and leukemias may involve translocation of the long arm of chromosome 5, to which the genes for IL-5 and other cytokines map.

1	Patients with eosinophilia are typically asymptomatic. Eosinophilia is present in 10% of patients with lymphoma, 3% of patients with lung cancer, and occasional patients with cervical, gastrointestinal, renal, and breast cancer. Patients with markedly elevated eosinophil counts (>5000/μL) can develop shortness of breath and wheezing. A chest radiograph may reveal diffuse pulmonary infiltrates from eosinophil infiltration and activation in the lungs. Definitive treatment is directed at the underlying malignancy: Tumors should be resected or treated with radiation or chemotherapy. In most patients who develop shortness of breath related to eosinophilia, symptoms resolve with the use of oral or inhaled glucocorticoids. IL-5 antagonists exist but have not been evaluated in this clinical setting.

1	Deep venous thrombosis and pulmonary embolism are the most common thrombotic conditions in patients with cancer. Migratory or recurrent thrombophlebitis may be the initial manifestation of cancer. Nearly 15% of patients who develop deep venous thrombosis or pulmonary embolism have a diagnosis of cancer (Chap. 142). The coexistence of peripheral venous thrombosis with visceral carcinoma, particularly pancreatic cancer, is called Trousseau’s syndrome.

1	Pathogenesis Patients with cancer are predisposed to thromboembolism because they are often at bed rest or immobilized, and tumors may obstruct or slow blood flow. Postoperative deep venous thrombosis is twice as common in cancer patients who undergo surgery. Chronic IV catheters also predispose to clotting. In addition, clotting may be promoted by release of procoagulants or cytokines from tumor cells or associated inflammatory cells or by platelet adhesion or aggregation. The specific molecules that promote thromboembolism have not been identified. Chemotherapeutic agents, particularly those associated with endothelial damage, can induce venous thrombosis. The annual risk of venous thrombosis in patients with cancer receiving chemotherapy is about 11%, sixfold higher than the risk in the general population. Bleomycin, l-asparaginase, thalidomide analogues, cisplatin-based regimens, and high doses of busulfan and carmustine are all associated with an increased risk.

1	In addition to cancer and its treatment causing secondary thrombosis, primary thrombophilic diseases may be associated with cancer. For example, the antiphospholipid antibody syndrome is associated with a wide range of pathologic manifestations (Chap. 379). About 20% of patients with this syndrome have cancers. Among patients with cancer and antiphospholipid antibodies, 35–45% develop thrombosis.

1	Clinical Manifestations Patients with cancer who develop deep venous thrombosis usually develop swelling or pain in the leg, and physical examination reveals tenderness, warmth, and redness. Patients who present with pulmonary embolism develop dyspnea, chest pain, and syncope, and physical examination shows tachycardia, cyanosis, and 613 hypotension. Some 5% of patients with no history of cancer who have a diagnosis of deep venous thrombosis or pulmonary embolism will have a diagnosis of cancer within 1 year. The most common cancers associated with thromboembolic episodes include lung, pancreatic, gastrointestinal, breast, ovarian, and genitourinary cancers; lymphomas; and brain tumors. Patients with cancer who undergo surgical procedures requiring general anesthesia have a 20–30% risk of deep venous thrombosis.

1	Diagnosis The diagnosis of deep venous thrombosis in patients with cancer is made by impedance plethysmography or bilateral compression ultrasonography of the leg veins. Patients with a noncompressible venous segment have deep venous thrombosis. If compression ultrasonography is normal and there is a high clinical suspicion for deep venous thrombosis, venography should be done to look for a luminal filling defect. Elevation of D-dimer is not as predictive of deep venous thrombosis in patients with cancer as it is in patients without cancer; elevations are seen in people over age 65 years without concomitant evidence of thrombosis, probably as a consequence of increased thrombin deposition and turnover in aging.

1	Patients with symptoms and signs suggesting a pulmonary embolism should be evaluated with a chest radiograph, electrocardiogram, arterial blood gas analysis, and ventilation-perfusion scan. Patients with mismatched segmental perfusion defects have a pulmonary embolus. Patients with equivocal ventilation-perfusion findings should be evaluated as described above for deep venous thrombosis in their legs. If deep venous thrombosis is detected, they should be anticoagulated. If deep venous thrombosis is not detected, they should be considered for a pulmonary angiogram.

1	Patients without a diagnosis of cancer who present with an initial episode of thrombophlebitis or pulmonary embolus need no additional tests for cancer other than a careful history and physical examination. In light of the many possible primary sites, diagnostic testing in asymptomatic patients is wasteful. However, if the clot is refractory to standard treatment or is in an unusual site or if the thrombophlebitis is migratory or recurrent, efforts to find an underlying cancer are indicated.

1	Patients with cancer and a diagnosis of deep venous thrombosis or pulmonary embolism should be treated initially with IV unfractionated heparin or low-molecular-weight heparin for at least 5 days, and warfarin should be started within 1 or 2 days. The warfarin dose should be adjusted so that the international normalized ratio (INR) is 2–3. Patients with proximal deep venous thrombosis and a relative contraindication to heparin anticoagulation (hemorrhagic brain metastases or pericardial effusion) should be considered for placement of a filter in the inferior vena cava (Greenfield filter) to prevent pulmonary embolism. Warfarin should be administered for 3–6 months. An alternative approach is to use low-molecularweight heparin for 6 months. Patients with cancer who undergo a major surgical procedure should be considered for heparin prophylaxis or pneumatic boots. Breast cancer patients undergoing chemotherapy and patients with implanted catheters should be considered for prophylaxis.

1	procedure should be considered for heparin prophylaxis or pneumatic boots. Breast cancer patients undergoing chemotherapy and patients with implanted catheters should be considered for prophylaxis. Guidelines recommend that hospitalized patients with cancer and patients receiving a thalidomide analogue receive prophylaxis with low-molecular-weight heparin or low-dose aspirin. Use of prophylaxis routinely during chemotherapy is controversial and not recommended by the American Society of Clinical Oncology.

1	Cutaneous paraneoplastic syndromes are discussed in Chap. 72. Neurologic paraneoplastic syndromes are discussed in Chap. 122. The authors acknowledge the contributions of Bruce E. Johnson to prior versions of this chapter. Paraneoplastic Syndromes: Endocrinologic/Hematologic taBLe 122-2 antiBoDies to intraCeLLuLar antigens, synDromes, anD assoCiateD CanCers 614 paraneoplastic neurologic syndromes and autoimmune encephalitis Josep Dalmau, Myrna R. Rosenfeld Paraneoplastic neurologic disorders (PNDs) are cancer-related syn-122 Anti-Hu (ANNA1) Encephalomyelitis, subacute SCLC sensory neuronopathy Anti-Yo (PCA1) Cerebellar degeneration Ovary, breast Anti-Ri (ANNA2) Cerebellar degeneration, Breast, gynecologic, opsoclonus, brainstem SCLC encephalitis dromes that can affect any part of the nervous system (Table 122-1).

1	Anti-Ri (ANNA2) Cerebellar degeneration, Breast, gynecologic, opsoclonus, brainstem SCLC encephalitis dromes that can affect any part of the nervous system (Table 122-1). They are caused by mechanisms other than metastasis or by any of the complications of cancer such as coagulopathy, stroke, metabolic and Anti-CRMP5 (CV2) Encephalomyelitis, chorea, SCLC, thymoma, other optic neuritis, uveitis, nutritional conditions, infections, and side effects of cancer therapy. In 60% of patients, the neurologic symptoms precede the cancer diagnosis. Anti-Ma proteins Limbic, hypothalamic, Testicular (Ma2), Clinically disabling PNDs occur in 0.5–1% of all cancer patients, but they affect 2–3% of patients with neuroblastoma or small-cell lung can Anti-amphiphysin Stiff-person syndrome, Breast, SCLC cer (SCLC) and 30–50% of patients with thymoma or sclerotic myeloma.

1	Anti-amphiphysin Stiff-person syndrome, Breast, SCLC cer (SCLC) and 30–50% of patients with thymoma or sclerotic myeloma. Recoverin, bipolar Cancer-associated retinopathy SCLC (CAR), melanoma cell antibodies, (CAR) (MAR) Most PNDs are mediated by immune responses triggered by neuronal othersa proteins (onconeuronal antigens) expressed by tumors. In PNDs of the central nervous system (CNS), many antibody-associated immune Anti-GAD Stiff-person, cerebellar Infrequent tumor responses have been identified (Table 122-2). These antibodies react syndromes, limbic encephalitis association (thymoma) with the patient’s tumor, and their detection in serum or cerebrospinal aA variety of target antigens have been identified. fluid (CSF) usually predicts the presence of cancer. When the anti- Abbreviations: CRMP, collapsing response-mediator protein; SCLC, small-cell lung cancer.

1	fluid (CSF) usually predicts the presence of cancer. When the anti- Abbreviations: CRMP, collapsing response-mediator protein; SCLC, small-cell lung cancer. gens are intracellular, most syndromes are associated with extensive infiltrates of CD4+ and CD8+ T cells, microglial activation, gliosis, and variable neuronal loss. The infiltrating T cells are often in close contact with neurons undergoing degeneration, suggesting a primary pathogenic role. T cell–mediated cytotoxicity may contribute directly taBLe 122-3 antiBoDies to CeLL surfaCe or synaptiC antigens, synDromes, anD assoCiateD tumors to cell death in these PNDs. Thus both humoral and cellular immune mechanisms participate in the pathogenesis of many PNDs. This complex immunopathogenesis may underlie the resistance of many of Anti-AChR Myasthenia gravis Thymoma (muscle)a these conditions to therapy.

1	In contrast to the disorders associated with immune responses Anti-AChR Autonomic ganglionopathy SCLC (neuronal)a against intracellular antigens, those associated with antibodies to antigens expressed on the neuronal cell surface of the CNS or at the neu-Anti-VGCCb LEMS, cerebellar SCLC romuscular junction are more responsive to immunotherapy (Table 122-3, Fig. 122-1). These disorders occur with and without a cancer Anti-NMDARa Anti-NMDAR encephalitis Teratoma in young women association and may affect children and young adults, and there is increasing evidence that they are mediated by the antibodies. Anti-LGI1c Limbic encephalitis, hypona-Rarely thymoma Other PNDs are likely immune-mediated, although their antigens tremia, faciobrachial tonic or are unknown. These include several syndromes of inflammatory neuropathies and myopathies. In addition, many patients with typical Anti-Caspr2c Morvan’s syndrome, neuro-Thymoma, prostate cancer PND syndromes are antibody-negative.

1	Anti-Caspr2c Morvan’s syndrome, neuro-Thymoma, prostate cancer PND syndromes are antibody-negative. Anti-GABABRd Limbic encephalitis, seizures SCLC, neuroendocrine Anti-GABAARd Encephalitis with prominent Rarely thymoma Classic Syndromes: Usually Nonclassic Syndromes: May Occur Occur with Cancer Association with and Without Cancer Association Limbic encephalitis Stiff-person syndrome Anti-AMPARd Limbic encephalitis with SCLC, thymoma, breast relapses Cerebellar degeneration (adults) Necrotizing myelopathy Glycine Encephalomyelitis with rigid-Rarely, thymoma, lung receptord ity, stiff-person syndrome cancer Subacute sensory neuronopathy Guillain-Barré syndrome

1	Anti-DPPXd Agitation, myoclonus, tremor, No cancer, but frequent Gastrointestinal paresis or Subacute and chronic mixed seizures, hyperekplexia, diarrhea or cachexia sugpseudo-obstruction sensory-motor neuropathies encephalomyelitis with gesting paraneoplasia Dermatomyositis (adults) Neuropathy associated with plasma aA direct pathogenic role of these antibodies has been demonstrated. bAnti-VGCC anti-syndrome Vasculitis of nerve bodies are pathogenic for LEMS. cPreviously named voltage-gated potassium channel antibodies (VGKC); currently included under the term VGKC-complex proteins. Of note, the significance of antibodies to VGKC-complex proteins other than LGI1 and Caspr2 is uncerretinopathy Acute necrotizing myopathy tain (the antigens are unknown, and the response to immunotherapy is variable) dThese antibodies are strongly suspected to be pathogenic. Polymyositis Abbreviations: AChR, acetylcholine receptor; AMPAR, α-amino-3-hydroxy-5

1	Polymyositis Abbreviations: AChR, acetylcholine receptor; AMPAR, α-amino-3-hydroxy-5 Vasculitis of muscle methylisoxazole-4-propionic acid receptor; Caspr2, contactin-associated protein-like 2; Optic neuropathy DPPX, dipeptidyl-peptidase-like protein-6; GABABR, γ-aminobutyric acid B receptor; GAD, glutamic acid decarboxylase; LEMS, Lambert-Eaton myasthenic syndrome; LGI1, leucine- rich glioma-inactivated 1; NMDAR, N-methyl-D-aspartate receptor; SCLC, small-cell lung Abbreviation: BDUMP, bilateral diffuse uveal melanocytic proliferation. cancer; VGCC, voltage-gated calcium channel.

1	FIGURE 122-1 Antibodies to the GluN1 subunit of the N-methyl-D-aspartate (NMDA) receptor in a patient with anti-NMDA receptor encephalitis and ovarian teratoma. (A) Coronal section of rat brain immunolabeled (green fluorescence) with the patient’s antibodies. The reactivity predominates in the hippocampus, which is highly enriched in NMDA receptors. (B) This image shows the antibody reactivity with cultures of rat hippocampal neurons; the intense green immunolabeling is due to the antibodies against the GluN1 subunit of NMDA receptors. (C–E) Images of HEK cells (a human kidney cell line) transfected to express NMDA receptors, showing reactivity with patient’s antibodies (C) and with a commercial monoclonal antibody against NMDA receptors (E); the patient’s antibody reactivity co-labels only the cells that express NMDA receptors (D). (From J Dalmau et al: Lancet Neurol 7:1091, 2008; with permission.)

1	For still other PNDs, the cause remains quite obscure. These include, among others, several neuropathies that occur in the terminal stages of cancer and a number of neuropathies associated with plasma cell dyscrasias or lymphoma without evidence of inflammatory infiltrates or deposits of immunoglobulin, cryoglobulin, or amyloid. APPROACH TO THE PATIENT: Three key concepts are important for the diagnosis and management of PNDs. First, it is common for symptoms to appear before the presence of a tumor is known; second, the neurologic syndrome usually develops rapidly, producing severe deficits in a short period of time; and third, there is evidence that prompt tumor control improves the neurologic outcome. Therefore, the major concern of the physician is to recognize a disorder promptly as paraneoplastic and to identify and treat the tumor.

1	When symptoms involve brain, spinal cord, or dorsal root ganglia, the suspicion of PND is usually based on a combination of clinical, radiologic, and CSF findings. Presence of antineuronal antibodies (Tables 122-2 and 122-3) may help in the diagnosis, but only 60–70% of PNDs of the CNS and less than 20% of those involving the peripheral nervous system have neuronal or neuromuscular junction antibodies that can be used as diagnostic tests.

1	Magnetic resonance imaging (MRI) and CSF studies are important to rule out neurologic complications due to the direct spread of cancer, particularly metastatic and leptomeningeal disease. In most PNDs, the MRI findings are nonspecific. Paraneoplastic limbic encephalitis is usually associated with characteristic MRI abnormalities in the mesial temporal lobes (see below), but similar findings can occur with other disorders (e.g., nonparaneoplastic autoimmune limbic encephalitis and human herpesvirus type 6 [HHV-6] encephalitis) (Fig. 122-2). The CSF profile of patients with PND of the CNS or dorsal root ganglia typically consists of mild to moderate pleocytosis (<200 mononuclear cells, predominantly lymphocytes), an increase in the protein concentration, and a variable presence of oligoclonal bands. There are no specific electrophysiologic tests that are diagnostic of PND. Moreover, a biopsy of the affected tissue is often difficult to obtain, and although useful to rule out other

1	bands. There are no specific electrophysiologic tests that are diagnostic of PND. Moreover, a biopsy of the affected tissue is often difficult to obtain, and although useful to rule out other disorders (e.g., metastasis) the pathologic findings are not specific for PND.

1	If symptoms involve peripheral nerve, neuromuscular junction, or muscle, the diagnosis of a specific PND is usually established on clinical, electrophysiologic, and pathologic grounds. The clinical FIGURE 122-2 Fluid-attenuated inversion recovery sequence magnetic resonance imaging of a patient with limbic encephalitis and LGI1 antibodies. Note the abnormal hyperintensity involving the medial aspect of the temporal lobes.

1	history, accompanying symptoms (e.g., anorexia, weight loss), and type of syndrome dictate the studies and degree of effort needed to demonstrate a neoplasm. For example, the frequent association of Lambert-Eaton myasthenic syndrome (LEMS) with SCLC should lead to a chest and abdomen computed tomography (CT) or body positron emission tomography (PET) scan and, if negative, periodic tumor screening for at least 3 years after the neurologic diagnosis. In contrast, the weak association of polymyositis with cancer calls into question the need for repeated cancer screenings in this situation. Serum and urine immunofixation studies should be considered in patients with peripheral neuropathy of unknown cause; detection of a monoclonal gammopathy suggests the need for additional studies to uncover a B cell or plasma cell malignancy. In paraneoplastic neuropathies, diagnostically useful antineuronal antibodies are limited to anti-CV2/CRMP5 and anti-Hu.

1	For any type of PND, if antineuronal antibodies are negative, the diagnosis relies on the demonstration of cancer and the exclusion of other cancer-related or independent neurologic disorders. Combined CT and PET scans often uncover tumors undetected by other tests. For germ cell tumors of the testis and teratomas of the ovary, ultrasound and CT or MRI of the abdomen and pelvis may reveal tumors undetectable by PET.

1	The term encephalomyelitis describes an inflammatory process with multifocal involvement of the nervous system, including brain, brain-stem, cerebellum, and spinal cord. It is often associated with dorsal root ganglia and autonomic dysfunction. For any given patient, the clinical manifestations are determined by the areas predominantly involved, but pathologic studies almost always reveal abnormalities beyond the symptomatic regions. Several clinicopathologic syndromes may occur alone or in combination: (1) cortical encephalitis, which may present as “epilepsia partialis continua”; (2) limbic encephalitis, characterized by confusion, depression, agitation, anxiety, severe short-term memory deficits, partial complex seizures, and sometimes dementia (the MRI usually shows unilateral or bilateral medial temporal lobe abnormalities, best seen with T2 and fluid-attenuated inversion recovery sequences); (3) brainstem encephalitis, resulting in eye movement disorders (nystagmus, opsoclonus,

1	medial temporal lobe abnormalities, best seen with T2 and fluid-attenuated inversion recovery sequences); (3) brainstem encephalitis, resulting in eye movement disorders (nystagmus, opsoclonus, supranuclear or nuclear paresis), cranial nerve paresis, dysarthria, dysphagia, and central autonomic dysfunction; (4) cerebellar gait and limb ataxia; (5) myelitis, which may cause lower or upper motor neuron symptoms, myoclonus, muscle rigidity, and spasms; and (6) autonomic dysfunction as a result of involvement of the neuraxis at multiple levels, including hypothalamus, brainstem, and autonomic nerves (see Paraneoplastic Peripheral Neuropathies, below). Cardiac arrhythmias, postural hypotension, and central hypoventilation are frequent causes of death in patients with encephalomyelitis.

1	Paraneoplastic encephalomyelitis and focal encephalitis are usually associated with SCLC, but many other cancers have been implicated. Patients with SCLC and these syndromes usually have anti-Hu antibodies in serum and CSF. Anti-CRMP5 antibodies occur less frequently; some of these patients may develop chorea, uveitis, or optic neuritis. Antibodies to Ma proteins are associated with limbic, hypothalamic, and brainstem encephalitis and occasionally with cerebellar symptoms (Fig. 122-3); some patients develop hypersomnia, cataplexy, and severe hypokinesia. MRI abnormalities are frequent, including those described with limbic encephalitis and variable involvement of the hypothalamus, basal ganglia, or upper brainstem. The oncologic associations of these antibodies are shown in Table 122-2.

1	Most types of paraneoplastic encephalitis and encephalomyelitis respond poorly to treatment. Stabilization of symptoms or partial neurologic improvement may occasionally occur, particularly if there is a satisfactory response of the tumor to treatment. Controlled trials of therapy are lacking, but many experts recommend treatment initially with glucocorticoids. If there is FIGURE 122-3 Magnetic resonance imaging (MRI) and tumor of a patient with anti-Ma2-associated encephalitis. (A and B) Fluid-attenuated inversion recovery MRI sequences showing abnormal hyperintensities in the medial temporal lobes, hypothalamus, and upper brainstem. (C) This image corresponds to a section of the patient’s orchiectomy incubated with a specific marker (Oct4) of germ cell tumors. The positive (brown) cells correspond to an intratubular germ cell neoplasm.

1	no response within several days, one can advance to intravenous immunoglobulin (IVIg) or plasma exchange, and then to immunosuppression with either rituximab or cyclophosphamide. Approximately 30% of patients with anti-Ma2-associated encephalitis respond to treatment of the tumor (usually a germ cell neoplasm of the testis) and immunotherapy. These disorders are important for three reasons: (1) they can occur with and without tumor association, (2) some syndromes predominate in young individuals and children, and (3) despite the severity of the symptoms patients usually respond to treatment of the tumor, if found, and immunotherapy (e.g., glucocorticoids, IVIg, plasma exchange, rituximab, or cyclophosphamide).

1	Encephalitis with N-methyl-d-aspartate (NMDA) receptor antibodies (Fig. 122-1) usually occurs in young women and children, but men and older patients of both sexes can be affected. The disorder has a characteristic pattern of symptom progression that includes a prodrome resembling a viral process, followed in a few days by the onset of severe psychiatric symptoms, memory loss, seizures, decreased level of consciousness, abnormal movements (orofacial, limb, and trunk dyskinesias, dystonic postures), autonomic instability, and frequent hypoventilation. Monosymptomatic episodes, such as pure psychosis, occur in 4% of the patients. Clinical relapses occur in 12–24% of patients (12% during the first 2 years after initial presentation). Most patients have intrathecal synthesis of antibodies, likely by infiltrating plasma cells in brain and meninges (Fig. 122-4A). The syndrome is often misdiagnosed as a viral or idiopathic encephalitis, neuroleptic malignant syndrome, or encephalitis

1	likely by infiltrating plasma cells in brain and meninges (Fig. 122-4A). The syndrome is often misdiagnosed as a viral or idiopathic encephalitis, neuroleptic malignant syndrome, or encephalitis lethargica, and many patients are initially evaluated by psychiatrists with the suspicion of acute psychosis. The detection of an associated teratoma is dependent on age and gender: 46% of female patients 12 years or older have unior bilateral ovarian teratomas, whereas less than 7% of girls younger than 12 have a teratoma (Fig. 122-4B). In male patients, the detection of a tumor is rare. Patients older than 45 years are more frequently male; about 20% of these patients have tumors (e.g., cancer of the breast, ovary, or lung).

1	Encephalitis with leucine-rich glioma-inactivated 1 (LGI1) antibodies predominates in patients older than 50 years (65% male) and frequently presents with memory loss and seizures (limbic encephalopathy), along with hyponatremia and sleep dysfunction. In a small number of patients, the encephalitis is preceded by or occurs with myoclonic-like movements called faciobrachial dystonic or tonic seizures. Less than 10% of patients have thymoma. Encephalitis with contactin-associated protein-like 2 (Caspr2) antibodies predominates in patients older than 50 years and is associated with Morvan’s syndrome (encephalitis, insomnia, confusion, hal-617 lucinations, autonomic dysfunction, and neuromyotonia) and, less frequently, with limbic encephalitis, neuromyotonia, and neuropathic pain. About 30–40% of patients have thymoma.

1	Encephalitis with γ-aminobutyric acid type B (GABAB) receptor antibodies is usually associated with limbic encephalitis and seizures. In rare instances, patients develop cerebellar symptoms and opsoclonus. Fifty percent of patients have SCLC or a neuroendocrine tumor of the lung. Patients may have additional antibodies to glutamic acid decarboxylase (GAD), which are of unclear significance. Other antibodies to nonneuronal proteins are often found in these patients as well as in patients with α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antibodies, indicating a general tendency to autoimmunity.

1	Encephalitis with GABAA receptor antibodies may affect children and adults. When antibodies are present at high titer in serum and CSF, the disorder associates with prominent seizures and status epilepticus, often requiring pharmacologically induced coma. Low titer antibodies in serum are often associated with other autoimmune condi tions, and the spectrum of symptoms is wider, including encephalitis, seizures, opsoclonus, or stiff-person syndrome. Most patients do not have an underlying tumor, but some may have thymoma. Encephalitis with AMPA receptor antibodies affects middle-aged women, who develop acute limbic dysfunction or, less frequently, prominent psychiatric symptoms; 70% of the patients have an underlying tumor in the lung, breast, or thymus. Neurologic relapses may occur; these also respond to immunotherapy and are not necessarily associated with tumor recurrence.

1	Encephalitis with glycine receptor (GlyR) antibodies has been described in adults with progressive encephalomyelitis with rigidity and myoclonus (PERM) and stiff-person spectrum of symptoms (with or without GAD antibodies). The disorder usually occurs without tumor association, although some patients have lung cancer, thymoma, or Hodgkin’s lymphoma. Encephalitis with dipeptidyl-peptidase-like protein-6 (or DPPX) antibodies results in symptoms of CNS hyperexcitability including agitation, hallucinations, paranoid delusions, tremor, myoclonus, nystagmus, seizures, and sometimes hyperekplexia. Some patients develop progressive encephalomyelitis with rigidity and myoclonus. Diarrhea, other gastrointestinal symptoms, and substantial loss of weight often suggest the presence of an underlying tumor, but no tumor association has been identified. The disorder responds to immunotherapy.

1	This disorder is often preceded by a prodrome that may include dizziness, oscillopsia, blurry or double vision, nausea, and vomiting. A few

1	FIGURE 122-4 Pathologic findings in anti–N-methyl-D-aspartate (NMDA) receptor encephalitis. Infiltrates of plasma cells (brown cells; stained for CD138) in the meninges and brain of a patient (A); the inset is a magnification of some plasma cells. (B) Neurons and neuronal processes in the teratoma of a patient (brown cells; stained with MAP2); these neurons express NMDA receptors (not shown). (From E Martinez-Hernandez et al: Neurology 77:589, 2011, with permission.) 618 days or weeks later, patients develop dysarthria, gait and limb ataxia, and variable dysphagia. The examination usually shows downbeating nystagmus and, rarely, opsoclonus. Brainstem dysfunction, upgoing toes, or a mild neuropathy may occur. Early in the course, MRI studies are usually normal; later, the MRI reveals cerebellar atrophy. The disorder results from extensive degeneration of Purkinje cells, with variable involvement of other cerebellar cortical neurons, deep cerebellar nuclei, and spinocerebellar tracts.

1	atrophy. The disorder results from extensive degeneration of Purkinje cells, with variable involvement of other cerebellar cortical neurons, deep cerebellar nuclei, and spinocerebellar tracts. The tumors more frequently involved are SCLC, cancer of the breast and ovary, and Hodgkin’s lymphoma. Anti-Yo antibodies in patients with breast and gynecologic cancers and anti-Tr antibodies in patients with Hodgkin’s lymphoma are the two immune responses typically associated with prominent or pure cerebellar degeneration. Antibodies to P/Q-type voltage-gated calcium channels (VGCC) occur in some patients with SCLC and cerebellar dysfunction; only some of these patients develop LEMS. A variable degree of cerebellar dysfunction can be associated with virtually any of the antibodies and PND of the CNS shown in Table 122-2. A number of single case reports have described neurologic improvement after tumor removal, plasma exchange, IVIg, cyclophosphamide, rituximab, or glucocorticoids. However, most

1	shown in Table 122-2. A number of single case reports have described neurologic improvement after tumor removal, plasma exchange, IVIg, cyclophosphamide, rituximab, or glucocorticoids. However, most patients with paraneoplastic cerebellar degeneration do not improve with treatment.

1	Opsoclonus is a disorder of eye movement characterized by involuntary, chaotic saccades that occur in all directions of gaze; it is frequently associated with myoclonus and ataxia. Opsoclonus-myoclonus may be cancer-related or idiopathic. When the cause is paraneoplastic, the tumors involved are usually cancer of the lung and breast in adults, neuroblastoma in children, and ovarian teratoma in adolescents and young women. The pathologic substrate of opsoclonus-myoclonus is unclear, but studies suggest that disinhibition of the fastigial nucleus of the cerebellum is involved. Most patients do not have antineuronal antibodies. A small subset of patients with ataxia, opsoclonus, and other eye-movement disorders develop anti-Ri antibodies; in rare instances, muscle rigidity, laryngeal spasms, autonomic dysfunction, and dementia also occur. The tumors most frequently involved in anti-Ri-associated syndromes are breast and ovarian cancer. If the tumor is not successfully treated, the

1	autonomic dysfunction, and dementia also occur. The tumors most frequently involved in anti-Ri-associated syndromes are breast and ovarian cancer. If the tumor is not successfully treated, the syndrome in adults often progresses to encephalopathy, coma, and death. In addition to treating the tumor, symptoms may respond to immunotherapy (glucocorticoids, plasma exchange, and/or IVIg).

1	At least 50% of children with opsoclonus-myoclonus have an underlying neuroblastoma. Hypotonia, ataxia, behavioral changes, and irritability are frequent accompanying symptoms. Neurologic symptoms often improve with treatment of the tumor and glucocorticoids, adrenocorticotropic hormone (ACTH), plasma exchange, IVIg, and rituximab. Many patients are left with psychomotor retardation and behavioral and sleep problems.

1	The number of reports of paraneoplastic spinal cord syndromes, such as subacute motor neuronopathy and acute necrotizing myelopathy, has decreased in recent years. This may represent a true decrease in incidence, due to improved and prompt oncologic interventions, or the identification of nonparaneoplastic etiologies. Some patients with cancer develop upper or lower motor neuron dysfunction or both, resembling amyotrophic lateral sclerosis. It is unclear whether these disorders have a paraneoplastic etiology or simply coincide with the presence of cancer. There are isolated case reports of cancer patients with motor neuron dysfunction who had neurologic improvement after tumor treatment. A search for lymphoma should be undertaken in patients with a rapidly progressive motor neuron syndrome and a monoclonal protein in serum or CSF.

1	Paraneoplastic myelitis may present with upper or lower motor neuron symptoms, segmental myoclonus, and rigidity, and can be the first manifestation of encephalomyelitis. Neuromyelitis optica (NMO) with aquaporin 4 antibodies may occur in rare instances as a paraneoplastic manifestation of a cancer.

1	This disorder is characterized by progressive muscle rigidity, stiffness, and painful spasms triggered by auditory, sensory, or emotional stimuli. Rigidity mainly involves the lower trunk and legs, but it can affect the upper extremities and neck. Sometimes, only one extremity is affected (stiff-limb syndrome). Symptoms improve with sleep and general anesthetics. Electrophysiologic studies demonstrate continuous motor unit activity. The associated antibodies target proteins (GAD, amphiphysin) involved in the function of inhibitory synapses using γ-aminobutyric acid (GABA) or glycine as neurotransmitters. The presence of amphiphysin antibodies usually indicates a paraneoplastic etiology related to SCLC and breast cancer. By contrast, GAD antibodies may occur in some cancer patients but are much more frequently present in the nonparaneoplastic disorder. GlyR antibodies may occur in some patients with stiff-person syndrome; these antibodies are also detectable in patients with PERM.

1	Optimal treatment of stiff-person syndrome requires therapy of the underlying tumor, glucocorticoids, and symptomatic use of drugs that enhance GABA-ergic transmission (diazepam, baclofen, sodium valproate, tiagabine, vigabatrin). IVIg and plasma exchange are transiently effective in some patients.

1	This syndrome is characterized by sensory deficits that may be symmetric or asymmetric, painful dysesthesias, radicular pain, and decreased or absent reflexes. All modalities of sensation and any part of the body including face and trunk can be involved. Specialized sensations such as taste and hearing can also be affected. Electrophysiologic studies show decreased or absent sensory nerve potentials with normal or near-normal motor conduction velocities. Symptoms result from an inflammatory, likely immune-mediated, process that targets the dorsal root ganglia, causing neuronal loss and secondary degeneration of the posterior columns of the spinal cord. The dorsal and, less frequently, the anterior nerve roots and peripheral nerves may also be involved. This disorder often precedes or is associated with encephalomyelitis and autonomic dysfunction and has the same immunologic and oncologic associations (Hu antibodies, SCLC).

1	As with anti-Hu-associated encephalomyelitis, the therapeutic approach focuses on prompt treatment of the tumor. Glucocorticoids occasionally produce clinical stabilization or improvement. The benefit of IVIg and plasma exchange is not proven.

1	These disorders may develop any time during the course of the neoplastic disease. Neuropathies occurring at late stages of cancer or lymphoma usually cause mild to moderate sensorimotor deficits due to axonal degeneration of unclear etiology. These neuropathies are often masked by concurrent neurotoxicity from chemotherapy and other cancer therapies. In contrast, the neuropathies that develop in the early stages of cancer frequently show a rapid progression, sometimes with a relapsing and remitting course, and evidence of inflammatory infiltrates and axonal loss or demyelination. If demyelinating features predominate (Chaps. 459 and 460), IVIg, plasma exchange, or glucocorticoids may improve symptoms. Occasionally anti-CRMP5 antibodies are present; detection of anti-Hu suggests concurrent dorsal root ganglionitis.

1	Guillain-Barré syndrome and brachial plexitis have occasionally been reported in patients with lymphoma, but there is no clear evidence of a paraneoplastic association (Chap. 460).

1	Malignant monoclonal gammopathies include: (1) multiple myeloma and sclerotic myeloma associated with IgG or IgA monoclonal proteins; and (2) Waldenström’s macroglobulinemia, B cell lymphoma, and chronic B cell lymphocytic leukemia associated with IgM monoclonal proteins. These disorders may cause neuropathy by a variety of mechanisms, including compression of roots and plexuses by metastasis to vertebral bodies and pelvis, deposits of amyloid in peripheral nerves, and paraneoplastic mechanisms. The paraneoplastic variety has several distinctive features. Approximately half of patients with sclerotic myeloma develop a sensorimotor neuropathy with predominantly motor deficits, resembling a chronic inflammatory demyelinating neuropathy (Chap. 460); some patients develop elements of the POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, M protein, skin changes). Treatment of the plasmacytoma or sclerotic lesions usually improves the neuropathy. In contrast, the sensorimotor or

1	syndrome (polyneuropathy, organomegaly, endocrinopathy, M protein, skin changes). Treatment of the plasmacytoma or sclerotic lesions usually improves the neuropathy. In contrast, the sensorimotor or sensory neuropathy associated with multiple myeloma is more refractory to treatment. Between 5 and 10% of patients with Waldenstr’s macroglobulinemia develop a distal symmetric sensorimotor neuropathy with predominant involvement of large sensory fibers. These patients may have IgM antibodies in their serum against myelin-associated glycoprotein and various gangliosides (Chap. 460). In addition to treating the Waldenstr’s macroglobulinemia, other therapies may improve the neuropathy, including plasma exchange, IVIg, chlorambucil, cyclophosphamide, fludarabine, or rituximab.

1	Vasculitis of the nerve and muscle causes a painful symmetric or asymmetric distal axonal sensorimotor neuropathy with variable proximal weakness. It predominantly affects elderly men and is associated with an elevated erythrocyte sedimentation rate and increased CSF protein concentration. SCLC and lymphoma are the primary tumors involved. Glucocorticoids and cyclophosphamide often result in neurologic improvement.

1	Peripheral nerve hyperexcitability (neuromyotonia, or Isaacs’ syndrome) is characterized by spontaneous and continuous muscle fiber activity of peripheral nerve origin. Clinical features include cramps, muscle twitching (fasciculations or myokymia), stiffness, delayed muscle relaxation (pseudomyotonia), and spontaneous or evoked carpal or pedal spasms. The involved muscles may be hypertrophic, and some patients develop paresthesias and hyperhidrosis. CNS dysfunction, including mood changes, sleep disorder, hallucinations, and autonomic symptoms may occur. The electromyogram (EMG) shows fibrillations; fasciculations; and doublet, triplet, or multiplet single-unit (myokymic) discharges that have a high intraburst frequency. Some patients have Caspr2 antibodies in the context of Morvan’s syndrome, but most cases of isolated neuromyotonia are antibody negative. The disorder often occurs without cancer; if paraneoplastic, benign and malignant thymomas and SCLC are the usual tumors.

1	syndrome, but most cases of isolated neuromyotonia are antibody negative. The disorder often occurs without cancer; if paraneoplastic, benign and malignant thymomas and SCLC are the usual tumors. Phenytoin, carbamazepine, and plasma exchange improve symptoms.

1	Paraneoplastic autonomic neuropathy usually develops as a component of other disorders, such as LEMS and encephalomyelitis. It may rarely occur as a pure or predominantly autonomic neuropathy with cholinergic or adrenergic dysfunction at the preor postganglionic levels. Patients can develop several life-threatening complications, such as gastrointestinal paresis with pseudo-obstruction, cardiac dysrhythmias, and postural hypotension. Other clinical features include abnormal pupillary responses, dry mouth, anhidrosis, erectile dysfunction, and problems in sphincter control. The disorder occurs in association with several tumors, including SCLC, cancer of the pancreas or testis, carcinoid tumors, and lymphoma. Because autonomic symptoms can be the presenting feature of encephalomyelitis, serum anti-Hu and anti-CRMP5 antibodies should be sought. Antibodies to ganglionic (alpha3-type) neuronal acetylcholine receptors are the cause of autoimmune autonomic ganglionopathy, a disorder that

1	serum anti-Hu and anti-CRMP5 antibodies should be sought. Antibodies to ganglionic (alpha3-type) neuronal acetylcholine receptors are the cause of autoimmune autonomic ganglionopathy, a disorder that frequently occurs without cancer association (Chap. 454).

1	LEMS is discussed in Chap. 461. Myasthenia gravis is discussed in Chap. 461. Polymyositis and dermatomyositis are discussed in detail in Chap. 388. Patients with this syndrome develop myalgias and rapid progression of weakness involving the extremities and the pharyngeal and respiratory muscles, often resulting in death. Serum muscle enzymes are elevated, and muscle biopsy shows extensive necrosis with minimal or absent inflammation and sometimes deposits of complement. The disorder occurs as a paraneoplastic manifestation of a variety of cancers including SCLC and cancer of the gastrointestinal tract, breast, kidney, and prostate, among others. Glucocorticoids and treatment of the underly-619 ing tumor rarely control the disorder.

1	This group of disorders involves the retina and, less frequently, the uvea and optic nerves. The term cancer-associated retinopathy is used to describe paraneoplastic cone and rod dysfunction characterized by photosensitivity, progressive loss of vision and color perception, central or ring scotomas, night blindness, and attenuation of photopic and scotopic responses in the electroretinogram (ERG). The most commonly associated tumor is SCLC. Melanoma-associated retinopathy affects patients with metastatic cutaneous melanoma. Patients develop acute onset of night blindness and shimmering, flickering, or pulsating photopsias that often progress to visual loss. The ERG shows reduced b waves with normal dark adapted a waves. Paraneoplastic optic neuritis and uveitis are very uncommon and can develop in association with encephalomyelitis. Some patients with paraneoplastic uveitis and optic neuritis have anti-CRMP5 antibodies.

1	Some paraneoplastic retinopathies are associated with serum antibodies that specifically react with the subset of retinal cells undergoing degeneration, supporting an immune-mediated pathogenesis (Table 122-2). Paraneoplastic retinopathies usually fail to improve with treatment, although rare responses to glucocorticoids, plasma exchange, and IVIg have been reported. thymoma Once a mediastinal mass is detected, a surgical procedure is required Dan L. Longo for definitive diagnosis. An initial mediastinoscopy or limited thoracotomy can be undertaken to get sufficient tissue to make an accurate

1	The thymus is derived from the third and fourth pharyngeal pouches and is located in the anterior mediastinum. It is composed of epithelial and stromal cells derived from the pharyngeal pouch and lymphoid precursors derived from mesodermal cells. It is the site to which bone marrow precursors that are committed to differentiate into T cells migrate to complete their differentiation. Like many organs, it is organized into functional regions, in this case the cortex and the medulla. The cortex of the thymus contains ~85% of the lymphoid cells, and the medulla contains ~15%. It appears that the primitive bone marrow progenitors enter the thymus at the corticomedullary junction and migrate first through the cortex toward the periphery of the gland and then toward the medulla as they mature. Medullary thymocytes have a phenotype that cannot be distinguished readily from that of mature peripheral blood and lymph node T cells.

1	Several things can go wrong with the thymus, but thymic abnormalities are very rare. If the thymus does not develop properly, serious deficiencies in T-cell development ensue and severe immunodeficiency is seen (e.g., DiGeorge syndrome, Chap. 374). If a lymphoid cell within the thymus becomes neoplastic, the disease that develops is a lymphoma. The majority of lymphoid tumors that develop in the thymus are derived from the precursor T cells, and the tumor is a precursor T-cell lymphoblastic lymphoma (Chap. 134). Rare B cells exist in the thymus, and when they become neoplastic, the tumor is a mediastinal (thymic) B cell lymphoma (Chap. 134). Hodgkin’s disease, particularly the nodular sclerosing subtype, often involves the anterior mediastinum. Extranodal marginal zone (mucosa-associated lymphoid tissue [MALT]) lymphomas have been reported to involve the thymus in the setting of Sjren’s syndrome or other autoimmune disorders, and the lymphoma cells often express IgA instead of IgM on

1	lymphoid tissue [MALT]) lymphomas have been reported to involve the thymus in the setting of Sjren’s syndrome or other autoimmune disorders, and the lymphoma cells often express IgA instead of IgM on their surface. Castleman’s disease can involve the thymus. Germ cell tumors and carcinoid tumors occasionally may arise in the thymus. If the epithelial cells of the thymus become neoplastic, the tumor that develops is a thymoma.

1	Thymoma, although rare (0.1–0.15 cases per 100,000 person-years), is the most common cause of an anterior mediastinal mass in adults, accounting for ~40% of all mediastinal masses. The other major causes of anterior mediastinal masses are lymphomas, germ cell tumors, and substernal thyroid tumors. Carcinoid tumors, lipomas, and thymic cysts also may produce radiographic masses. After combination chemotherapy for another malignancy, teenagers and young adults may develop a rebound thymic hyperplasia in the first few months after treatment. Granulomatous inflammatory diseases (tuberculosis, sarcoidosis) can produce thymic enlargement. Thymomas are most common in the fifth and sixth decades, are uncommon in children, and are distributed evenly between men and women.

1	About 40–50% of patients are asymptomatic; masses are detected incidentally on routine chest radiographs. When symptomatic, patients may have cough, chest pain, dyspnea, fever, wheezing, fatigue, weight loss, night sweats, or anorexia. Occasionally, thymomas may obstruct the superior vena cava. Pericardial effusion may be present. About 40% of patients with thymoma have another systemic autoimmune illness related to the thymoma. About 30% of patients with thymoma have myasthenia gravis, 5–8% have pure red cell aplasia, and ~5% have hypogammaglobulinemia. Thymoma with hypogammaglobulinemia also is called Good’s syndrome. Among patients with myasthenia gravis, ~10–15% have a thymoma. Thymoma more rarely may be associated with polymyositis, systemic lupus erythematosus, thyroiditis, Sjren’s syndrome, ulcerative colitis, pernicious anemia, Addison’s disease, stiff person syndrome, scleroderma, and panhypopituitarism. In one series, 70% of patients with thymoma were found to have another

1	syndrome, ulcerative colitis, pernicious anemia, Addison’s disease, stiff person syndrome, scleroderma, and panhypopituitarism. In one series, 70% of patients with thymoma were found to have another systemic illness.

1	diagnosis. Fine-needle aspiration is poor at distinguishing between lymphomas and thymomas but is more reliable in diagnosing germ cell tumors and metastatic carcinoma. Thymomas and lymphomas require sufficient tissue to examine the tumor architecture to assure an accurate diagnosis and obtain prognostic information.

1	Once a diagnosis of thymoma is defined, subsequent staging generally occurs at surgery. However, chest computed tomography (CT) scans can assess local invasiveness in some instances. Magnetic resonance imaging (MRI) has a defined role in the staging of posterior mediastinal tumors, but it is not clear that it adds important information to the CT scan in anterior mediastinal tumors. Somatostatin receptor imaging with indium-labeled somatostatin analogues may be of value. If invasion is not distinguished by noninvasive testing, an effort to resect the entire tumor should be undertaken. If invasion is present, neoadjuvant chemotherapy may be warranted before surgery (see “Treatment” section below). Some 90% of thymomas are in the anterior mediastinum, but some may be in other mediastinal sites or even the neck, based on aberrant migration of the developing thymic enlage.

1	The staging system for thymoma was developed by Masaoka and colleagues (Table 123e-1). It is an anatomic system in which the stage is increased on the basis of the degree of invasiveness. The 5-year survival of patients in the various stages is as follows: stage I, 96%; stage II, 86%; stage III, 69%; and stage IV, 50%. The French Study Group on Thymic Tumors (GETT) has proposed modifications to the Masaoka scheme based on the degree of surgical removal because the extent of surgery has been noted to be a prognostic indicator. In their system, stage I tumors are divided into A and B on the basis of whether the surgeon suspects adhesions to adjacent structures; stage III tumors are divided into A and B based on whether disease was subtotally resected or only biopsied. The concurrence between the two systems is high.

1	Thymomas are epithelial tumors, and all of them have malignant potential. It is not worthwhile to try to divide them into benign and malignant forms; the key prognostic feature is whether they are noninvasive or invasive. About 65% of thymomas are encapsulated Source: From A Masaoka et al: Cancer 48:2485, 1981. Updated from S Tomaszek et al: Ann Thorac Surg 87:1973, 2009, and CB Falkson et al: J Thorac Oncol 4:911, 2009. Prognosis (10-year Type Distribution, % disease-free survival), % Source: From S Tomaszek et al: Ann Thorac Surg 87:1973, 2009.

1	Prognosis (10-year Type Distribution, % disease-free survival), % Source: From S Tomaszek et al: Ann Thorac Surg 87:1973, 2009. and noninvasive, and about 35% are invasive. They may have a variable percentage of lymphocytes within the tumor, but genetic studies suggest that the lymphocytes are benign polyclonal cells. The epithelial component of the tumor may consist primarily of round or oval cells derived mainly from the cortex or spindle-shaped cells derived mainly from the medulla or combinations of the two types (World Health Organization classification; Table 123e-2). Cytologic features are not reliable predictors of biologic behavior. In part, this unreliability may be related to the moderate reproducibility of the system. About 90% of A, AB, and B1 tumors are localized. A very small number of patients have aggressive histology features characteristic of carcinomas. Thymic carcinomas are invasive and have a poor prognosis.

1	Genetic lesions are common in thymomas. The most common abnormalities affect chromosome 6p21.3 (the MHC locus) and 6p25.2-25.3 (usually loss of heterozygosity). Abnormalities affecting a number of other genes altered in other types of tumors are also seen, including p53, RB, FHIT, and APC. Thymic carcinomas may overexpress c-kit, HER2, or growth factor receptor genes (epidermal growth factor receptor and insulin-like growth factor receptor). Some data suggest that Epstein-Barr virus may be associated with thymomas. Some tumors overexpress the p21 ras gene product. However, molecular pathogenesis remains undefined. A thymoma susceptibility locus has been defined on rat chromosome 7, but the relationship between this gene locus, termed Tsr1, and human thymoma has not been examined.

1	Patients with myasthenia gravis have a high incidence of thymic abnormalities (~80%), but overt thymoma is present in only ~10–15% of patients with myasthenia gravis. It is thought that the thymus plays a role in breaking self-tolerance and generating T cells that recognize the acetylcholine receptor as a foreign antigen. Although patients with thymoma and myasthenia gravis are less likely to have a remission in the myasthenia as a consequence of thymectomy than are patients with thymic abnormalities other than thymoma, the course of myasthenia gravis is not significantly different in patients with or without thymoma. Thymectomy produces at least some symptomatic improvement in ~65% of patients with myasthenia gravis. In one large series, thymoma patients with myasthenia gravis had a better long-term survival from thymoma resection than did those without myasthenia gravis.

1	About 30–50% of patients with pure red cell aplasia have a thymoma. Thymectomy results in the resolution of pure red cell aplasia in ~30% of patients. About 10% of patients with hypogammaglobulinemia have a thymoma, but hypogammaglobulinemia rarely responds to thymectomy.

1	Treatment is determined by the stage of disease. For patients with encapsulated tumors and stage I disease, complete resection is sufficient to cure 96% of patients. For patients with stage II disease, complete resection may be followed by 30–60 Gy of postoperative radiation therapy to the site of the primary tumor. However, the value of radiation therapy in this setting has not been established. The main predictors of long-term survival are Masaoka stage and completeness of resection. For patients with stage III and IV disease, the use of neoadjuvant chemotherapy followed by radical surgery, with or without additional radiation therapy, and additional consolidation chemotherapy has been associated with excellent survival. Chemotherapy regimens that are most effective generally include a platinum compound (either cisplatin or carboplatin) and an anthracycline. Addition of cyclophosphamide, vincristine, and prednisone seems to improve response rates. Response rates of 50–93% have been

1	platinum compound (either cisplatin or carboplatin) and an anthracycline. Addition of cyclophosphamide, vincristine, and prednisone seems to improve response rates. Response rates of 50–93% have been reported in series of patients each of which involved fewer than 40 patients. A single most effective regimen has not been defined. No randomized controlled phase III studies have been reported. If surgery after neoadjuvant chemotherapy fails to produce a complete resection of residual disease, radiation therapy (50–60 Gy) may help reduce recurrence rates.

1	This multimodality approach appears to be superior to the use of surgery followed by radiation therapy alone, which produces a 5-year survival of ≤50% in patients with advanced-stage disease. Some thymic carcinomas express c-kit, and one patient whose c-kit locus was mutated responded dramatically to imatinib. Many thymomas express epidermal growth factor receptors, but the antibodies to the receptor and the kinase inhibitors that block its action have not been evaluated systematically. Octreotide plus prednisone produces responses in about one-third of patients.

1	Neoplasia During pregnancy Michael F. Greene, Dan L. Longo Cancer complicates ~1 in every 1000 pregnancies. Of all the cancers that occur in women, less than 1% complicate pregnancies. The four cancers that most commonly complicate pregnancies are cervical cancer, breast cancer, melanoma, and lymphomas (particularly Hodgkin’s lymphoma); 124e however, virtually every form of cancer has been reported in pregnant women (Table 124e-1). In addition to cancers developing in other organs of the mother, gestational trophoblastic tumors can arise from the placenta. The problem of cancer in a pregnant woman is complex. One must take into account (1) the possible influence of the pregnancy on the natural history of the cancer, (2) effects on the mother and fetus of complications from the malignancy (e.g., anorexia, nausea, vomiting, malnutrition), (3) potential effects of diagnostic and staging procedures, and (4) potential effects of cancer treatments on both the mother and the developing

1	(e.g., anorexia, nausea, vomiting, malnutrition), (3) potential effects of diagnostic and staging procedures, and (4) potential effects of cancer treatments on both the mother and the developing fetus. Generally, the management that optimizes maternal physiology is also best for the fetus. However, the dilemma occasionally arises that what is best for the mother may be harmful to the fetus, and what is best for the fetus may compromise the ultimate prognosis for the mother. The best way to approach management of a pregnant woman with cancer is to ask, “What would we do for this woman in this clinical situation if she was not pregnant? Now, which, if any, of those plans need to be modified because she is pregnant?”

1	Pregnancy is associated with a number of physiologic changes that frequently result in symptoms that may make it difficult to recognize symptoms or physical findings suggestive of a neoplasm. Increased sensitivity of central chemoreceptors to Pco2 drives an increase in minute ventilation that many women perceive as dyspnea at rest or with minimal exertion. The combination of increased total body water, decreased colloid oncotic pressure, and some obstruction of venous return from the lower extremities causes demonstrable dependent edema in more than 50% of pregnant women. Decreased gastrointestinal motility due to high serum progesterone levels and mechanical compression from an enlarging uterus cause early satiety, gastroesophageal reflux, nausea, vomiting, and constipation. Hemorrhoids develop and often bleed. Breasts enlarge and increase in density and “lumpiness.” These changes may result in delayed recognition and more advanced disease at diagnosis.

1	Physiologic changes in the maternal immune system necessary to facilitate retention of the fetal semi-allograft raise concerns that the relationship of a cancer with its host may be altered to the detriment of the maternal host. One half of all the genes necessary to create a new individual by sexual reproduction come from each parent. This provides the opportunity for many antigenic differences between the conceptus and the mother. Mammalian placentation has been a very successful method of reproduction, but it has necessitated some combination of both fetal and maternal evolutionary immune adaptations. These mechanisms are incompletely understood and remain an area of active investigation. It does seem likely, however, that this has been accomplished without a general, nonspecific blunting of the maternal Incidence per 10,000 Tumor Type Pregnanciesa % of Casesb Cervical cancer 1.2–4.5 25% Thyroid cancer 1.2 15% Hodgkin’s disease 1.6 10% Melanoma 1–2.6 8% Ovarian cancer 0.8 2%

1	Incidence per 10,000 Tumor Type Pregnanciesa % of Casesb Cervical cancer 1.2–4.5 25% Thyroid cancer 1.2 15% Hodgkin’s disease 1.6 10% Melanoma 1–2.6 8% Ovarian cancer 0.8 2% All sites 10 100% aThese are estimates based on extrapolations from a review of more than 3 million pregnancies (LH Smith et al: Am J Obstet Gynecol 184:1504, 2001). bBased on accumulating case reports from the literature; the precision of these data is not high.

1	immune response, which would be maladaptive to the mother. The 124e-1 multiple mechanisms likely include some “masking” of fetal antigens from recognition by the maternal immune system, blunting the maternal inflammatory response locally at the placental–maternal interface and induction of fetal-specific maternal immune tolerance to avoid rejection. Attention has turned to a subset of CD4+ induced, peripherally produced regulatory T cells that express the X chromosome encoded transcription factor Foxp3 (so-called Tregs). When these Foxp3 cells develop centrally in the thymus, they are termed “Tregs.” When Foxp3-expressing cells develop peripherally, they are called “Pregs.” These regulatory cells suppress the immune response against “self” and foreign antigens. They seem to be capable of suppressing the maternal response to paternal antigens expressed by the fetus and creating memory cells that retain tolerance to the same paternal antigens in subsequent pregnancies. Unfortunately, in

1	the maternal response to paternal antigens expressed by the fetus and creating memory cells that retain tolerance to the same paternal antigens in subsequent pregnancies. Unfortunately, in a mouse model, the interleukin (IL) 10 produced by these cells enhanced susceptibility to infection by Listeria and Salmonella, while ironically not proving essential for retaining the fetal graft. Undoubtedly much remains to be learned about this critical immune balance.

1	Exposure of developing fetuses to ionizing radiation may cause adverse fetal effects; awareness among physicians of this potential toxicity has resulted in a disproportionate aversion to diagnostic imaging in pregnancy. First, it must be stated that there are very useful imaging modalities (i.e., ultrasound and magnetic resonance imaging [MRI]) that do not use any ionizing radiation and are not associated with any demonstrable adverse fetal effects. There are three potential adverse fetal effects of ionizing radiation: teratogenesis (induction of anatomic birth defects), mutagenesis, and carcinogenesis. The fetus is most sensitive to teratogenesis during organogenesis in the first trimester. The dose of ionizing radiation necessary to induce birth defects in human fetuses is derived from studies of the survivors of the atomic bomb explosions and by extrapolation from controlled experiments in nonhuman mammals. From these data sources, it is clear that a minimum of 5 rem and more

1	from studies of the survivors of the atomic bomb explosions and by extrapolation from controlled experiments in nonhuman mammals. From these data sources, it is clear that a minimum of 5 rem and more likely greater than 10 rem exposure is needed to induce birth defects in the first trimester. The fetal doses of radiation associated with some common diagnostic radiologic procedures are displayed in Table 124e-2. The data in Table 124e-2 show that no single procedure or selective combination of diagnostic procedures will exceed the very conservative 5 rem teratogenic threshold. Teratogenic effects later in pregnancy are largely limited to microcephaly and require exposures exceeding 25 rem. The reason for the disproportionate concern about radiation exposure and birth defects is that 2.5% of all fetuses are affected with birth defects without radiation exposure and, therefore, 2.5% of women undergoing any diagnostic imaging procedure will deliver malformed fetuses. Spontaneous mutations

1	all fetuses are affected with birth defects without radiation exposure and, therefore, 2.5% of women undergoing any diagnostic imaging procedure will deliver malformed fetuses. Spontaneous mutations occur relatively infrequently, and high doses of radiation (>150 rem) are required to cause a demonstrable increase in that rate. The magnitude of the risk of carcinogenesis in offspring exposed as fetuses to diagnostic doses of radiation has been very difficult to

1	Source: Data from FG Cunningham et al: General considerations and maternal evaluation. In Williams Obstetrics, 21st ed. New York: McGraw-Hill; 2001, pp. 1143–1158.

1	measure due to the relative rarity of cancer in children and the long duration of follow-up that might credibly be needed to see the effect. The inconsistent results and small effect sizes observed from diagnostic exposures make it likely that, if there is an effect, it is very small and, if there is not a significant effect, it will be impossible to prove that fact to everyone’s satisfaction. No imaging using ionizing radiation should be done without a compelling reason and due consideration to obtaining the necessary information by other imaging modalities. Exposure to diagnostic and therapeutic radionuclides, especially radioactive iodine, poses unique risks, but a full discussion of these is beyond the scope of this chapter. Radiation therapy uses radiation doses three orders of magnitude greater than diagnostic procedures, entails substantial risks if the fetus is in the radiation field, and is rarely appropriate in pregnancy. Finally, although difficult to prove, it is likely

1	greater than diagnostic procedures, entails substantial risks if the fetus is in the radiation field, and is rarely appropriate in pregnancy. Finally, although difficult to prove, it is likely that more harm has come to pregnant women from failing to perform appropriate diagnostic procedures than has been done to their offspring from performing appropriate diagnostic procedures.

1	There are a number of reasons why it is impossible to make many definitive statements regarding the safety and efficacy of chemotherapy in pregnancy. All of the available data in the literature are published as case reports or case series. The quality and completeness of the data are inconsistent and often poor. Reports may come from medical oncologists, obstetricians, pediatricians, or other treating physicians familiar with the information important to the report from their own perspective but missing information important for other specialty areas. Reports frequently lack critical details of drug administration, such as dose, duration, cumulative dose, and timing of exposure in gestation, and outcomes, including birth weight and gestational age at delivery, indication for or cause of premature delivery, and follow-up of offspring beyond the immediate neonatal period. There are a wide variety of agents available to treat cancer, and they are usually used in combinations. This

1	of premature delivery, and follow-up of offspring beyond the immediate neonatal period. There are a wide variety of agents available to treat cancer, and they are usually used in combinations. This results in the fact that every patient is almost unique (an experiment of one) in the combination of agents, doses, durations, and gestational ages of administration, making it very difficult to attribute what benefit or toxicity accrues to which agent. Fortunately, cancer in pregnant women is sufficiently rare that it takes quite a while to accumulate enough information for any one agent or combination of agents to be confident about what toxicities (including congenital malformations) are truly associated with which agents. There is such rapid progress in cancer chemotherapy that by the time there may seem to be enough information about the agents currently in use to use them intelligently and counsel patients meaningfully, the cancer community has moved on to newer, more efficacious, and

1	may seem to be enough information about the agents currently in use to use them intelligently and counsel patients meaningfully, the cancer community has moved on to newer, more efficacious, and hopefully less toxic agents for which there is little or no experience in pregnancy. Finally, for obvious reasons, there are no untreated controls for comparison. It may be very difficult to sort out the maternal consequences (nausea, vomiting, fever, weight loss, dehydration) that might result directly from the malignancy and cause adverse pregnancy outcomes from some of the toxicities of the chemotherapeutic agents used to treat the malignancy.

1	Generally, toxic chemotherapy should be avoided during pregnancy, if at all possible. It should virtually never be given in the first trimester. However, a variety of single agents and combinations have been given in the second and third trimesters, without a high frequency of toxic effects to the pregnancy or the fetus, but data on safety are sparse. Maternal factors that may influence the pharmacology of chemotherapeutic agents include the 50% increase in plasma volume, altered absorption and protein binding, increased glomerular filtration rate, increased hepatic mixed function oxidase activity, and third space created by amniotic fluid. The fetus is protected from some agents by placental expression of drug efflux pumps, but decreased fetal hepatic mixed function oxidase and glucuronidation activity may prolong the half-life of agents that do cross the placenta. A database on the risks associated with individual chemotherapy agents is available on the Internet

1	and glucuronidation activity may prolong the half-life of agents that do cross the placenta. A database on the risks associated with individual chemotherapy agents is available on the Internet (http://ntp.niehs.nih.gov/ntp/ohat/cancer_chemo_preg/ chemopregnancy_monofinal_508.pdf ).

1	Optimal management strategies have not been developed based on prospective clinical trials. Management of a malignancy complicating pregnancy will be critically determined by the gestational age when the malignancy is diagnosed and the anticipated natural history of the lesion, if left untreated. On one extreme, if the malignancy is slowly progressive, the patient is near her delivery date, and waiting until delivery to begin treatment would not be anticipated to compromise maternal prognosis, then treatment could be delayed until after delivery to avoid fetal exposure to chemotherapy. If there is a greater sense of urgency to begin definitive treatment to avoid compromising maternal prognosis, and the patient is beyond 24 weeks of gestation but remote from her delivery date, then treatment (surgical, medical, or both) might be initiated during pregnancy and plans made to deliver the fetus early to avoid exposure to more chemotherapy than absolutely necessary. Finally, if the patient

1	(surgical, medical, or both) might be initiated during pregnancy and plans made to deliver the fetus early to avoid exposure to more chemotherapy than absolutely necessary. Finally, if the patient is in her first trimester and toxic chemotherapy must be initiated promptly to avoid a very poor maternal outcome, then it may be necessary to consider therapeutic abortion to avoid maternal disaster and fetal survival with injury resulting in long-term morbid sequelae. No two cases are precisely alike, and inevitably, decision making must be individualized, preferably with consultation with a multidisciplinary team including medical oncology, surgical oncology if appropriate, maternal–fetal medicine, neonatology, and anesthesia. Pregnancy appears to have little or no impact on the natural history of malignancies, despite the hormonal influences. Spread of the mother’s cancer to the fetus (so-called vertical transmission) is exceedingly rare.

1	The incidence of cervical cancer in pregnant women is roughly comparable to that of age-matched controls who are not pregnant. Invasive cervical cancer complicates about 0.45 in 1000 live births, and carcinoma in situ is seen in 1 in 750 pregnancies. About 1% of women diagnosed with cervical cancer are pregnant at the time of diagnosis. Early signs of cervical cancer include vaginal spotting or discharge, pain, and postcoital bleeding, which are also common features of pregnancy. Early visual changes in the cervix related to invasive cancer can be mistaken for cervical decidualization or ectropion (columnar epithelium on the cervix) due to pregnancy. Women diagnosed with cervical cancer during pregnancy report having had symptoms for 4.5 months on average.

1	Approximately 95% of all cervical cancer is caused by human papillomavirus (HPV) infections, with types 16 and 18 accounting for about 70% of cervical cancer. The rate of carriage of these serotypes is highest among women in their early twenties and can be reduced with the use of vaccination before exposure. Women generally tend to clear the infection by age 30, with the risk of cervical cancer being highest among those who fail to clear the infection. Screening is recommended at the first prenatal visit and 6 weeks postpartum. The rate of cytologic abnormalities on Pap smear in pregnant women is about 5–8% and is not much different than the rate in nonpregnant women of the same age.

1	In 2012, several sets of recommendations were published for screening for cervical cancer: one by the American Cancer Society (ACS), the American Society for Colposcopy and Cervical Pathology (ASCCP), and the American Society for Clinical Pathology (ASCP); a second by the U.S. Preventive Services Task Force (USPSTF); and a third by the American College of Obstetricians and Gynecologists (ACOG). Although the details of the recommendations for screening and management of abnormal results differ slightly among the three sets of guidelines, there is general consensus that cytology screening should start at age 21 and continue every 3 years through age 29. After age 30, cytology screening frequency may be reduced to every 5 years if accompanied by co-testing for HPV. Recommendations for management of abnormal cytology findings are complex and determined by the degree of abnormality of the cytology finding (e.g., atypical squamous cells of undetermined significance; atypical squamous cells,

1	of abnormal cytology findings are complex and determined by the degree of abnormality of the cytology finding (e.g., atypical squamous cells of undetermined significance; atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion; low-grade squamous intraepithelial lesion; or high-grade squamous intraepithelial lesion), the HPV status of the patient, the age of the patient, and whether this is the first abnormal finding or a persistent abnormality. A full discussion of all the treatment recommendations based on these factors is beyond the scope of this chapter. Some of the diagnostic procedures recommended for evaluation of nonpregnant women are contraindicated in pregnancy, and the indications for some procedures are modified in the setting of pregnancy. Suffice it to say that the evaluation of women with abnormal cervical cytology in pregnancy should be referred to knowledgeable and experienced gynecologists or gynecologic oncologists.

1	Cervical intraepithelial neoplasia is a slowly progressive lesion and has a low risk of progression to invasive cancer during pregnancy (~0.4%), and many low-grade lesions (36–70%) regress spontaneously. Accordingly, some physicians defer definitive diagnostic procedures in pregnant women until 6 weeks postpartum unless they are at high risk for invasive disease. If invasive disease is suspected and the pregnancy is between 16 and 20 weeks, a cone biopsy may be performed to make the diagnosis and may be curative for some lesions; however, the procedure may cause heavy bleeding due to the increased vasculature in the gravid cervix and increases the risk of premature rupture of membranes and preterm labor twoto threefold. Cone biopsy should not be done within 4 weeks of delivery. The only indication for therapy of cervical neoplasia in pregnant women is the documentation of invasive cancer.

1	Management of invasive disease is guided by the stage of disease, the gestational age of the fetus, and the desire of the mother to have the baby. If the disease is in early stage and the pregnancy is desired, it is safe to delay treatment regardless of gestational age until fetal maturity allows for safe delivery. Abortion followed by definitive therapy is recommended for women with advanced, but potentially curable, cancer in the first or second trimester (Chap. 117). If the disease is in an advanced stage in early pregnancy and the patient declines pregnancy termination to permit prompt definitive therapy, she must be informed of the fact that the maternal safety of delaying therapy is unproven. In women in the third trimester with advanced disease, the mother should be treated with betamethasone to accelerate fetal lung maturation and the baby should be delivered at the earliest possible gestational age followed immediately by stage-appropriate therapy. Most women with invasive

1	betamethasone to accelerate fetal lung maturation and the baby should be delivered at the earliest possible gestational age followed immediately by stage-appropriate therapy. Most women with invasive cancer have early-stage disease. If the disease is microinvasive, vaginal delivery can take place and be followed by definitive treatment, usually conization. If a lesion is visible on the cervix, delivery is best done by caesarian section and followed by radical hysterectomy.

1	Breast cancer complicates approximately 1 in 3000 to 10,000 live births. About 5% of all breast cancers occur in women age 40 years or younger. Among all premenopausal women with breast cancer, 25–30% were pregnant at the time of diagnosis. It has been recognized for some time that breast cancer associated with pregnancy generally seems to have a poorer prognosis for both overall survival and progression-free survival. The definition of pregnancy-associated breast cancer (PABC) has differed in various publications, but a generally accepted definition is breast cancer diagnosed during pregnancy or within 1 year of delivery. There are likely several reasons for the observation of the poorer prognosis. Breast cancers diagnosed during pregnancy are often diagnosed at a later stage of disease and so have a poorer outcome. The late diagnosis is often due to the fact that early physical signs of the disease are missed or attributed to the changes that occur in the breast normally as a

1	and so have a poorer outcome. The late diagnosis is often due to the fact that early physical signs of the disease are missed or attributed to the changes that occur in the breast normally as a function of pregnancy. However, a discreet breast mass in a pregnant woman should never be assumed to be normal. Another reason is the more aggressive behavior of the cancer possibly related to the hormonal milieu (estrogen increases 100-fold; progesterone increases 1000-fold) of the pregnancy. However, about 70% of the breast cancers found in pregnancy are estrogen receptor– negative. About 28–58% of the tumors express HER2, a biologically more aggressive breast cancer subset. Another factor is that aggressive, definitive chemotherapy and radiation therapy are often delayed due aLower measured levels could be in part artifactual due to the increased levels of estrogen in the milieu.

1	to concerns about the consequences of those treatments for the fetus. Younger women with breast cancer have a higher likelihood of having mutations in BRCA1 or BRCA2.

1	Differences in presentation between PABC and breast cancers diagnosed in nonpregnant women are shown in Table 124e-3. About 20% of breast cancers are detected in the first trimester, 45% in the second trimester, and 35% in the third trimester. Some argue that stage for stage, the outcome is the same for breast cancer diagnosed in pregnant and nonpregnant women. Primary tumors in pregnant women are 3.5 cm on average, compared to <2 cm in nonpregnant women. A dominant mass and a nipple discharge are the most common presenting signs, and they should prompt ultrasonography and breast MRI exam (if available) followed by lumpectomy if the mass is solid and aspiration if the mass is cystic. Mammography is less reliable in pregnancy due to the increased breast density. Needle aspirates of breast masses in pregnant women are often nondiagnostic or falsely positive. Even in pregnancy, most breast masses are benign (~80% are adenoma, lobular hyperplasia, milk retention cyst, fibrocystic disease,

1	in pregnant women are often nondiagnostic or falsely positive. Even in pregnancy, most breast masses are benign (~80% are adenoma, lobular hyperplasia, milk retention cyst, fibrocystic disease, fibroadenoma, or other rarer entities).

1	Many studies comparing outcomes among women with PABC to those of nonpregnant women have small sample sizes, and there is considerable heterogeneity among the study results, but a formal meta-analysis including multiple adjustments and sensitivity analyses confirms the clinical impression of poorer outcomes for women with PABC. The hazard ratios were 1.44 for poorer overall survival and 1.60 for poorer disease-free survival.

1	Although having had a pregnancy is a protective factor against breast cancer in women in general, it is questionable as to whether it retains its protective effect in carriers of BRCA1 and BRCA2 mutations. Cullinane et al. (Int J Cancer 117:988, 2005) found a statistically insignificant difference (odds ratio 0.94) in breast cancer risk among BRCA1 carriers who had ever been pregnant versus those who never had a pregnancy. Stratifying the risk of breast cancer according to the number of prior pregnancies versus no pregnancies, no statistically significant protective trend was observed. For BRCA2 carriers, there was a marginally statistically significant increased risk of breast cancer among women with prior pregnancies. In an international study with more than 65,000 person-years of observation (Andrieu J: Natl Cancer Inst 98:535, 2006), there was no significant effect in either direction of pregnancy on breast cancer risk for carriers of either mutation. Staging the axillary lymph

1	(Andrieu J: Natl Cancer Inst 98:535, 2006), there was no significant effect in either direction of pregnancy on breast cancer risk for carriers of either mutation. Staging the axillary lymph nodes is currently somewhat controversial. Sentinel lymph node sampling is not straightforward in pregnant women. Blue dye has been carcinogenic in rats, and fetuses cannot be shielded from administered radionuclides. For this reason, many surgeons favor axillary node dissection to stage the nodes. Largely due to the typical delay in diagnosis, axillary nodes are more often positive in pregnant than in nonpregnant women.

1	As with other types of cancer in pregnant women, counseling following diagnosis in the first trimester should include a discussion of pregnancy termination to allow definitive therapeutic intervention at the earliest possible time without the potential for permanent injury to a surviving fetus. While definitive local surgery can safely be performed in the first trimester, radiation therapy and chemotherapy are considerably more risky. Delay in administration of systemic therapy can increase the risk of axillary spread. In the second and third trimesters, chemotherapy (particularly anthracycline-based combinations) is both safe and effective (Chap. 108). Lumpectomy followed by adjuvant chemotherapy is frequently used; fluorouracil and cyclophosphamide with either doxorubicin or epirubicin have been given without major risk to the fetus. Taxanes and gemcitabine are also beginning to be used; however, safety data are sparse. Methotrexate and other folate antagonists are to be avoided

1	have been given without major risk to the fetus. Taxanes and gemcitabine are also beginning to be used; however, safety data are sparse. Methotrexate and other folate antagonists are to be avoided because of effects on the fetal nervous system. Myelotoxic therapy is generally not administered after 33 or 34 weeks of gestation to allow 3 weeks off therapy before delivery for recovery of blood counts. Endocrine therapy and trastuzumab are unsafe during pregnancy. Experience with lapatinib is anecdotal, but no fetal malformations have been reported. Antiemetics and colony-stimulating factors are also considered safe. Women being treated into the postpartum period should not breast-feed their babies because of excretion of cancer chemotherapy agents, particularly alkylating agents, in milk.

1	Subsequent pregnancies following gestational breast cancer do not appear to influence relapse rate or overall survival. A meta-analysis found that pregnancy in breast cancer survivors was associated with a reduced the risk of dying from breast cancer by as much as 42%. This finding, however, is heavily confounded by the “healthy survivor effect”; women with more extensive or advanced disease are more likely to avoid pregnancy.

1	Speculation about melanoma occurring during pregnancy based largely on anecdotal evidence and small case series concluded that it occurred with increased frequency, was more aggressive in its natural history, and was caused in part by the hormonal changes that also produced hyperpigmentation (so-called melasma) during pregnancy. However, more complete epidemiologic data suggest that melanoma is no more frequent in pregnant women than in nonpregnant women in the same age group, that melanoma is not more aggressive during pregnancy, and that hormones seem to have little or nothing to do with the etiology. Pregnant and nonpregnant women do not differ in the location of primary tumor, depth of primary tumor, tumor ulceration, or vascular invasion.

1	Suspicious lesions should be looked for and managed definitively with excisional biopsy during pregnancy. Wide excision with sampling of regional lymph nodes is warranted. If lymph nodes are involved, the course of action is less clear. Several agents have demonstrated some activity in melanoma, but none have been used during pregnancy. Adjuvant interferon α is toxic, and its safety in pregnancy has not been documented. Agents active in advanced disease include dacarbazine, IL-2, ipilimumab (antibody to CTLA-4), and in those with BRAF mutation V600E, a BRAF kinase inhibitor. In the setting of metastatic disease, abortion may be indicated so that systemic therapy can be initiated as soon as possible (Chap. 105). Melanoma is one of the very few cancers that are well documented to metastasize transplacentally to the fetus, where it seems to have a predilection for the head and neck. It has a very grave prognosis in the offspring. Fortunately, transplacental spread is rare.

1	Pregnancy subsequent to the diagnosis and treatment of melanoma is not associated with an increased risk of melanoma recurrence.

1	(See Chap. 134) Hodgkin’s disease occurs mainly in the age range that coincides with child-bearing. However, Hodgkin’s disease is not more common in pregnant than nonpregnant women. Hodgkin’s disease is diagnosed in approximately 1 in 6000 pregnancies. It generally presents as a nontender lymph node swelling, most often in the left supraclavicular region. It may be accompanied by B symptoms (fever, night sweats, unexplained weight loss). Excisional biopsy is the preferred diagnostic procedure because fine-needle aspiration cannot reveal the architectural framework that is an essential component of Hodgkin’s disease diagnosis. The stage at presentation appears to be unaffected by pregnancy. Women diagnosed in the second and third trimester can be treated safely with combination chemotherapy, usually doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD). In general, the patient in the first trimester is asymptomatic, and a woman with a desired pregnancy can be followed until the

1	usually doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD). In general, the patient in the first trimester is asymptomatic, and a woman with a desired pregnancy can be followed until the second or third trimester when definitive multiagent chemotherapy can be safely given. Radiation therapy is not given during pregnancy and is not necessary for optimal management of the pregnant patient. If symptoms requiring treatment appear during the first trimester, anecdotal evidence suggests that Hodgkin’s disease symptoms can be controlled with weekly low-dose vinblastine. Such an approach has been safely used to avoid termination of pregnancy. Pregnancy does not have an adverse effect on treatment outcome.

1	Non-Hodgkin’s lymphomas are more unusual in pregnancy (approximately 0.8 per 100,000 pregnancies), but are usually tumors with an aggressive natural history, such as diffuse large B cell lymphoma, Burkitt’s lymphoma, or peripheral T cell lymphoma. Diagnosis relies on an excisional biopsy of a tumor mass, not fine-needle aspiration. Staging evaluation is generally limited to ultrasound or MRI examinations. Diagnosis in the first trimester should prompt termination of the pregnancy followed by definitive treatment with combination chemotherapy, because aggressive lymphomas are not likely to be held at bay with single-agent chemotherapy. Women diagnosed in the second or third trimesters can be treated with standard chemotherapy, such as with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). The experience with rituximab in this setting is anecdotal. However, infants born of mothers who have received rituximab may have transient delay in B cell development that typically

1	(CHOP). The experience with rituximab in this setting is anecdotal. However, infants born of mothers who have received rituximab may have transient delay in B cell development that typically normalizes by 6 months. The treatment outcome is similar in lymphomas diagnosed in pregnant and nonpregnant women of the same clinical stage.

1	(See Chap. 405) Thyroid cancer, along with melanomas, brain tumors, and lymphomas, are cancers that are increasing in incidence in the general population. Thyroid cancers are rising faster among women in North America than the other increasing tumor types. The Endocrine Society has developed practice guidelines to inform the management of patients with thyroid disease during pregnancy (http://www.endocrine .org/~/media/endosociety/Files/Publications/Clinical%20Practice%20 Guidelines/Thyroid-Exec-Summ.pdf ). Thyroid nodules 1 cm or larger are approached by fine-needle aspiration. If a malignancy is diagnosed, surgery is generally recommended in the second and third trimesters. However, surgical complications appear to be twice as common when the patient is pregnant. Because the growth of thyroid tumors is often indolent, surgery can safely be postponed until after the first trimester. Patients with follicular cancer or early papillary cancer can be observed until the postpartum period.

1	tumors is often indolent, surgery can safely be postponed until after the first trimester. Patients with follicular cancer or early papillary cancer can be observed until the postpartum period. The fetal thyroid begins trapping iodine by 12 weeks of gestation and does so with very high avidity. Even small doses of radioactive iodine given during pregnancy can completely ablate the fetal thyroid with serious consequences for the fetus and should be avoided throughout pregnancy. Radioactive iodine can be safely administered after delivery. Patients with a history of thyroid cancer who become pregnant should be maintained on thyroid hormone replacement during pregnancy because of the adverse impact of maternal hypothyroidism on the fetus. Women who are breast-feeding should not be treated with radioactive iodine, and women treated with radioactive iodine should not become pregnant for 6–12 months after treatment.

1	The assessment of thyroid function during pregnancy is challenging because of the physiologic changes that occur during pregnancy. Women who have previously been treated for thyroid cancer are at risk of hypothyroidism. The demand for thyroid hormone increases during pregnancy, and doses to maintain normal function may increase by 30–50%. Total T4 levels are higher during pregnancy, but target therapeutic levels also increase (Table 124e-4). It is recommended that the upper and lower limits of the laboratory range be multiplied by 1.5 in the second and third trimester to establish a pregnancy-specific normal range. The target thyroid-stimulating hormone (TSH) level is <2.5 mIU/L. Source: Based on the National Health and Nutrition Examination Survey III (NHANES III) (OP Soldin et al: Ther Drug Monit 17:303, 2007).

1	(See Chap. 117) Gestational trophoblastic disease encompasses hydatidiform mole, choriocarcinoma, placental site trophoblastic tumor, and assorted miscellaneous and unclassifiable trophoblastic tumors. Moles are the most common, occurring in 1 in 1500 pregnancies in the United States. The incidence is higher in Asia. In general, if the serum level of β-human chorionic gonadotropin (β-hCG) returns to normal after surgical removal (evacuation) of the mole, the illness is considered gestational trophoblastic disease. By contrast, if the β-hCG level remains elevated after mole evacuation, the patient is considered to have gestational trophoblastic neoplasia. Choriocarcinoma occurs in 1 in 25,000 pregnancies. Maternal age >45 years and prior history of molar pregnancy are risk factors. A previous molar pregnancy makes choriocarcinoma about 1000 times more likely to occur (incidence 1–2%).

1	Hydatidiform moles are characterized by clusters of villi with hydropic changes, trophoblastic hyperplasia, and absence of fetal blood vessels. Invasive moles are distinguished by invasion of the myometrium. Placental site trophoblastic tumors are composed mainly of cytotrophoblast cells arising at the site of placental implantation. Choriocarcinomas contain anaplastic trophoblastic tissue with both cytotrophoblast and syncytiotrophoblast features and no identifiable villi.

1	Moles can be partial, typically associated with fetal tissue, or complete, typically not associated with any fetal or embryonic tissue. Partial moles have a distinct molecular origin and usually are smaller tumors with less hydropic villi and considerably less potential for persistent or malignant disease. Partial moles result from fertilization of an egg by two sperm, resulting in diandric triploidy. Complete moles usually have a 46,XX genotype; 95% develop by a single male sperm fertilizing an empty egg and undergoing gene duplication (diandric diploidy); 5% develop from dispermic fertilization of an empty egg (diandric dispermy).

1	Women with molar gestations often present with first-trimester bleeding, disproportionately high serum β-hCG levels for menstrual age, unusually large uterine size for menstrual age, hyperemesis gravidarum, theca lutein cysts in the ovaries (due to β-hCG stimulation), and hyperthyroidism (due to cross-reactivity of β-hCG and TSH) and may develop preeclampsia before 20 weeks of menstrual age. Pelvic ultrasound imaging of complete moles shows absence of fetal parts, an enlarged echo-bright, hydropic placenta in an enlarged uterus, and enlarged multicystic ovaries. If the diagnosis is uncertain at the initial examination and the pregnancy is desired, then a serum β-hCG level should be obtained and the examination repeated in a week. If no embryo is seen within 7–10 days and the serum β-hCG is elevated, 124e-5 then this is a nonviable pregnancy that should be evacuated. Diagnosis of partial molar pregnancies can be more difficult because an embryo or fetus with visible heart motion is

1	is elevated, 124e-5 then this is a nonviable pregnancy that should be evacuated. Diagnosis of partial molar pregnancies can be more difficult because an embryo or fetus with visible heart motion is usually present, and the hydropic changes in the placenta, uterine enlargement, and elevations of β-hCG are not usually as dramatic. Although an embryo or fetus is present, it rarely grows normally with normal anatomy, and repeated ultrasound examinations usually make the diagnosis. Amniocentesis will also make the diagnosis by demonstration of triploidy.

1	Patients with molar pregnancies require prompt uterine evacuation with suction curettage, which may be complicated by very heavy bleeding. Following evacuation of complete moles, approximately 20% will result in persistent, invasive, or metastatic disease. Partial moles are considerably less likely (<5%) to result in persistent disease. Patients should be monitored with serial determinations of serum β-hCG until the values fall below the lower limit of the assay and remain low for at least 6 months. Patients should be advised not to become pregnant for at least 12 months. A variety of criteria have been used to make the diagnosis of post-molar gestational trophoblastic disease, but current consensus guidelines as adopted by the International Federation of Gynecology and Obstetrics are listed below: 1. A β-hCG level plateau of four values plus or minus 10% recorded over a 3-week duration (days 1, 7, 14, and 21) 2.

1	A β-hCG level plateau of four values plus or minus 10% recorded over a 3-week duration (days 1, 7, 14, and 21) 2. A β-hCG level increase of more than 10% in three values recorded over a 2-week duration (days 1, 7, and 14) 3. Persistence of detectable β-hCG for more than 6 months after molar evacuation About half of choriocarcinomas develop after a molar pregnancy, and half develop after ectopic pregnancy or, rarely, after a normal full-term pregnancy. Disease is classified as stage I if it is confined to the uterus, stage II if disease is limited to genital structures (~30% have vaginal involvement), stage III if disease has spread to the lungs but no other organs, and stage IV if disease has spread to liver, brain, or other organs.

1	Patients without widely metastatic disease are generally managed with single-agent methotrexate (either 30 mg/m2 IM weekly until β-hCG normalizes or 1 mg/kg IM every other day for four doses followed by leucovorin 0.1 mg/kg IV 24 h after methotrexate), which cures >90% of patients. Patients with very high β-hCG levels, presenting >4 months after a pregnancy, with brain or liver metastases, or failing to be cured by single-agent methotrexate are treated with combination chemotherapy. Etoposide, methotrexate, and dactinomycin alternating with cyclophosphamide and vincristine (EMA-CO) is the most commonly used regimen, producing long-term survival in >80% of patients. Brain metastases can usually be controlled with brain radiation therapy. The vast majority of choriocarcinomas can be cured with chemotherapy alone. Hysterectomy is reserved for women who have completed their child-bearing, women with chemotherapy-resistant disease in the uterus, and women with rare placental site

1	cured with chemotherapy alone. Hysterectomy is reserved for women who have completed their child-bearing, women with chemotherapy-resistant disease in the uterus, and women with rare placental site trophoblastic tumors confined to the uterus because these tumors are less reliably sensitive to chemotherapy. Women cured of trophoblastic disease who have not undergone hysterectomy do not appear to have increased risk of fetal abnormalities or maternal complications with subsequent pregnancies.

1	Oncology and Hematology Late Consequences of Cancer and Its Treatment Carl E. Freter, Dan L. Longo There are over 10 million American cancer survivors. The vast major-ity of these will bear some mark of their cancer and its treatment, and 125 a large proportion will experience long-term consequences including medical problems, psychosocial dysfunction, economic hardship, sexual dysfunction, and discrimination regarding employment and insurance. Many of these problems are directly related to cancer treatment. As patients survive longer from more types of malignancies, we are increasingly recognizing the biologic toll our very imperfect therapies take in terms of morbidity and mortality. The human face of these consequences of therapy confronts the cancer specialist who treats them every day. Although long-term survivors of childhood leukemias, Hodgkin’s lymphoma, and testicular cancer, as examples, have taught us much about the consequences of cancer treatment, we keep learning more as

1	Although long-term survivors of childhood leukemias, Hodgkin’s lymphoma, and testicular cancer, as examples, have taught us much about the consequences of cancer treatment, we keep learning more as patients survive longer with newer therapies. Newer “targeted” chemotherapy drugs have their own, often unique, long-term toxicities about which we remain in a learning process. Cancer “survivorship” clinics are increasing to expressly follow patients for long-term toxicities of cancer treatment.

1	The pace of developing therapies that mitigate treatment-related consequences has been slow, partly due to an understandable aversion to alter regimens that work and partly due to a lack of new, effective, less toxic therapeutic agents with less “collateral damage” to replace known agents with known toxicities. The types of damage from cancer treatment vary. Often, a final common pathway is irreparable damage to DNA. Surgery can create dysfunction, including blind gut loops with absorption problems and loss of function of removed body parts. Radiation may damage end-organ function, for example, loss of potency in prostate cancer patients, pulmonary fibrosis, and neurocognitive impairment, and may act as a direct carcinogen. Cancer chemotherapy can be a direct carcinogen and has a kaleidoscope of other toxicities discussed in this chapter. Table 125-1 lists the late effects of cancer treatment.

1	The first goal of therapy is to eradicate or control the malignancy. Late treatment consequences are, indeed, testimony to the increasing success of such treatment. Their occurrence sharply underlines the necessity to develop more effective therapies with less long-term morbidity and mortality. At the same time, a sense of perspective and relative risk is necessary; fear of long-term complications should not prevent the application of effective, particularly curative, cancer treatment.

1	Cardiovascular toxicity of cancer chemotherapeutic agents includes dysrhythmias, cardiac ischemia, cardiomyopathic congestive heart failure (CHF), pericardial disease, and peripheral vascular disease. Because these cardiac toxicities are difficult to distinguish from disease that is not associated with cancer treatment, clear etiologic implication of cancer chemotherapeutic agents may be difficult. Cardiovascular complications occurring in an unexpected clinical setting in patients who have undergone cancer therapy are often important in raising suspicion. Dose-dependent myocardial toxicity of anthracyclines with characteristic myofibrillar dropout is pathologically pathognomonic on endomyocardial biopsy. Anthracycline cardiotoxicity occurs through a root mechanism of chemical free radical damage. Fe3+doxorubicin complexes damage DNA, nuclear and cytoplasmic membranes, and mitochondria. About 5% of patients receiving >450–550 mg/m2 of doxorubicin will develop CHF. Cardiotoxicity in

1	damage. Fe3+doxorubicin complexes damage DNA, nuclear and cytoplasmic membranes, and mitochondria. About 5% of patients receiving >450–550 mg/m2 of doxorubicin will develop CHF. Cardiotoxicity in relation to the dose of anthracycline is clearly not a step function, but rather a continuous function, and occasional patients are seen with CHF at substantially lower doses. Advanced age, other concomitant cardiac disease, hypertension, diabetes, and thoracic radiation therapy are all important cofactors in promoting anthracycline-associated CHF. The risk of cardiac failure appears to be substantially lower when doxorubicin is administered by continuous infusion. Anthracycline-related CHF is difficult to reverse and has a mortality rate as high as 50%, making prevention crucial. Some anthracyclines such as mitoxantrone are associated with less cardiotoxicity, and continuous-infusion regimens and liposomally encapsulated doxorubicin are associated with less cardiotoxicity. Dexrazoxane, an

1	such as mitoxantrone are associated with less cardiotoxicity, and continuous-infusion regimens and liposomally encapsulated doxorubicin are associated with less cardiotoxicity. Dexrazoxane, an intracellular iron chelator, may limit anthracycline toxicity, but the concern of limiting chemotherapeutic efficacy has somewhat limited its use. Monitoring patients for cardiac toxicity typically involves periodic gated nuclear cardiac blood pool ejection fraction testing (multigated acquisition scan [MUGA]) or cardiac ultrasonography. More recently, cardiac magnetic resonance imaging (MRI) has been used, but MRI is not standard or widespread. Testing is performed more frequently at higher cumulative doses, with additional risk factors, and certainly for any newly developing CHF or other symptoms of cardiac dysfunction.

1	After anthracyclines, trastuzumab is the next most frequent cardiotoxic drug currently in use. Trastuzumab is frequently used as adjuvant breast cancer therapy, sometimes in conjunction with anthracyclines, which is believed to result in additive or possibly synergistic toxicity. In contrast to anthracyclines, cardiotoxicity is not dose-related, is usually reversible, is not associated with pathologic changes of anthracyclines on cardiac myofibrils, and has a different biochemical mechanism inhibiting intrinsic cardiac repair mechanisms. Toxicity is typically routinely monitored every three to four doses using functional cardiac testing as mentioned earlier for anthracyclines. Other cardiotoxic drugs include lapatinib, phosphoramide mustards (cyclophosphamide), ifosfamide, interleukin 2, ponatinib, imatinib, and sunitinib.

1	Radiation therapy that includes the heart can cause interstitial myocardial fibrosis, acute and chronic pericarditis, valvular disease, and accelerated premature atherosclerotic coronary artery disease. Repeated or high (>6000 cGy) radiation doses are associated with greater risk, as is concomitant or distant cardiotoxic cancer chemo-621 therapy exposure. Symptoms of acute pericarditis, which peaks about 9 months after treatment, include dyspnea, chest pain, and fever. Chronic constrictive pericarditis may develop 5–10 years following radiation therapy. Cardiac valvular disease includes aortic insufficiency from fibrosis or papillary muscle dysfunction resulting in mitral regurgitation. A threefold increased risk of fatal myocardial infarction is associated with mantle field radiation with accelerated coronary artery disease. Carotid radiation similarly increases the risk of embolic stroke.

1	Therapy for chemotherapeutic/radiation-induced cardiovascular disease is essentially the same as therapy for disease not associated with cancer treatment. Discontinuation of the offending agent is the first step. Diuretics, fluid and sodium restriction, and antiarrhythmic agents are often useful for acute symptoms. Afterload reduction with angiotensin-converting enzyme (ACE) inhibitors or, in some cases, β-adrenergic blockers (carvedilol) often is of significant benefit, and digitalis may be helpful as well. A hybrid discipline of “cardio-oncology” has been developing in clinics to expressly follow chemotherapy-treated patients for cardiotoxicity. The goals are early intervention using more sensitive techniques, management of cardiotoxicity before it becomes symptomatic, and using clinical trials to identify cardioprotective strategies.

1	Bleomycin generates activated free radical oxygen species and causes pneumonitis associated with a radiographic or interstitial ground-glass appearance diffusely throughout both lungs, often worse in the lower lobes. A nonproductive cough with or without fever may be an early sign. This toxicity is dose-related and dose-limiting. The diffusion capacity of the lungs for carbon dioxide (DLCO) is a sensitive measure of toxicity and recovery, and a baseline value is generally obtained for future comparison prior to bleomycin therapy. Additive or synergistic risk factors include age, prior lung disease, and concomitant use of other chemotherapy, lung irradiation, and high concentrations of inspired oxygen. Other chemotherapeutic agents notable for pulmonary toxicity include mitomycin, nitrosoureas, doxorubicin with radiation, gemcitabine combined with weekly docetaxel, methotrexate, and fludarabine. High-dose alkylating agents, cyclophosphamide, ifosfamide, and melphalan are frequently

1	doxorubicin with radiation, gemcitabine combined with weekly docetaxel, methotrexate, and fludarabine. High-dose alkylating agents, cyclophosphamide, ifosfamide, and melphalan are frequently used in the hematopoietic stem cell transplant setting, often with whole-body radiation. This therapy may result in severe pulmonary fibrosis and/or pulmonary venoocclusive disease.

1	Risk factors for radiation pneumonitis include advanced age, poor performance status, preexisting compromised pulmonary function, and radiation volume and dose. The dose “threshold” is thought to be in the range of 5 to 20 Gy. Hypoxemia and dyspnea on exertion are characteristic. Fine, high-pitched “Velcro rales” may be an accompanying physical finding, and fever, cough, and pleuritic chest pain are common symptoms. The DLCO is the most sensitive measure of pulmonary functional impairment, and ground-glass infiltrates often correspond with relatively sharp edges to the irradiated volume, although the pneumonitis may progress beyond the field and even occasionally involve the contralateral unirradiated lung. Late Consequences of Cancer and Its Treatment processing. There is no clear mechanistic explanation for its cause and no clearly effective therapy. This entity is justifiably attracting more

1	Chemotherapyand radiation-induced pneumonitis is generally very corticosteroid responsive, except in the case of nitrosoureas. Prednisone 1 mg/kg is often used to control acute symptoms and pulmonary dysfunction with a generally slow taper. Prolonged glucocorticoid therapy requires gastrointestinal protection with proton pump inhibitors, management of hyperglycemia, heightened infection management, and treatment of steroid-induced osteoporosis. Antibiotics, bronchodilators, oxygen in only necessary doses, and diuretics may all play an important role in management of pneumonitis, and consultation with a pulmonologist should be routinely undertaken. Amifostine has been studied as a pulmonary radioprotectant, with inconclusive results, and is associated with skin rash, fatigue, and nausea; hence, it is not considered standard therapy at this time. Transforming growth factor β (TGF-β) is believed to be a major inducer of radiation fibrosis and represents a therapeutic target for

1	nausea; hence, it is not considered standard therapy at this time. Transforming growth factor β (TGF-β) is believed to be a major inducer of radiation fibrosis and represents a therapeutic target for development of anti-TGF-β therapies.

1	Chemotherapyand radiation-induced neurologic dysfunction is unfortunately increasing in both incidence and severity as a result of improved supportive care leading to more aggressive regimens and longer cancer survivorship allowing the development of late toxicity. Direct effects on myelin, glial cells, and neurons have all been implicated, with alterations in cellular cytoskeleton, axonal transport, and cellular metabolism as mechanisms.

1	Vinca alkaloids produce a characteristic “stocking-glove” neuropathy with numbness and tingling advancing to loss of motor function, which is highly dose related. Distal sensorimotor polyneuropathy prominently involves loss of deep tendon reflexes with initially loss of pain and temperature sensation, followed by proprioceptive and vibratory loss. This requires careful patient history and physical examination by experienced oncologists to decide when the drug must be stopped due to toxicity. Milder toxicity often slowly completely resolves. Vinca alkaloids may sometimes be associated with jaw claudication, autonomic neuropathy, ileus, cranial nerve palsies, and, in severe cases, encephalopathy, seizures, and coma. Cisplatin is associated with sensorimotor neuropathy and hearing loss, especially at doses >400 mg/m2, requiring audiometry in patients with preexisting hearing compromise. Carboplatin is often substituted in such cases given its lesser effect on hearing.

1	Many of the agents that target kinase enzymes in tumor cells and 5-fluorouracil congeners produce dysesthesias and painful hands and feet known as hand-foot syndrome or palmar-plantar erythrodysesthesia. Symptoms usually abate when the agent is stopped.

1	Neurocognitive dysfunction has been well described in childhood survivors of acute lymphoblastic leukemia (ALL) treatment, including intrathecal methotrexate or cytosine arabinoside in conjunction with prophylactic cranial irradiation. Methotrexate alone may cause acute leukoencephalopathy characterized by somnolence and confusion that is often reversible. Acute toxicity is dose related, especially at doses >3 g/m2, with younger patients being at greater risk. Subacute methotrexate toxicity occurs weeks after therapy and is often ameliorated with glucocorticoid therapy. Chronic methotrexate toxicity (leukoencephalopathy) develops months or years after treatment and is characterized clinically as progressive loss of cognitive function and focal neurologic signs, which are irreversible, promoted by synchronous or metachronous radiation therapy, and more pronounced at a younger age.

1	Neurocognitive decline following chemotherapy alone occurs notably in breast cancer patients receiving adjuvant chemotherapy; this has been referred to as “chemo brain.” It is clinically associated with impaired memory, learning, attention, and speed of information attention and clearly needs to be studied to develop effective therapy or prophylaxis. Many cancer patients experience intrusive or debilitating concerns about cancer recurrence following successful therapy. In addition, these patients may experience job, insurance, stress, relationship, financial, and sexual difficulties. Oncologists need to ask about and address these issues explicitly with patients and provide appropriate counseling or support systems. Suicidal ideation and suicide have an increased incidence in cancer patients and survivors.

1	Acute radiation central nervous system (CNS) toxicity occurs within weeks; is characterized by nausea, drowsiness, hypersomnia, and ataxia; and is most often associated with recovery. Early delayed toxicity occurring weeks to 3 months following therapy is associated with similar symptoms as acute toxicity and is pathologically associated with reversible demyelination. Chronic, late radiation injury occurs 9 months to up to 10 years following therapy. Focal necrosis is a common pathologic finding, and glucocorticoid therapy may be helpful. Diffuse radiation injury is associated with global CNS neurologic dysfunction and diffuse white matter changes on computed tomography (CT) or MRI. Pathologically, small vessel changes are prominent. Glucocorticoids may be symptomatically useful but do not alter the course. Necrotizing encephalopathy is the most severe form of radiation injury and almost always is associated with chemotherapy, notably methotrexate.

1	Cranial radiation may also be associated with an array of endocrine abnormalities with disruption of normal pituitary/hypothalamic axis function, and a high index of suspicion needs to be maintained to identify and treat this toxicity. Radiation-associated spinal cord injury (myelopathy) is highly dose-dependent and rarely occurs with modern radiation therapy. An early, self-limited form involving electric sensations down the spine on neck flexion (Lhermitte’s sign) is seen 6–12 weeks after treatment and generally resolves over weeks. Peripheral nerve toxicity is quite rare owing to relative radiation resistance.

1	Long-term hepatic damage from standard chemotherapy regimens is rare. Long-term methotrexate or high-dose chemotherapy alone or with radiation therapy, for example, in preparative regimens for bone marrow transplantation, may result in venoocclusive disease of the liver. This potentially lethal complication classically presents with anicteric ascites, elevated alkaline phosphatase, and hepatosplenomegaly. Pathologically, there is venous congestion, epithelial cell proliferation, and hepatocyte atrophy progressing to frank fibrosis. Frequent monitoring of liver function tests during any chemotherapy is necessary to avoid both idiosyncratic and expected toxicities. Certain nucleoside drugs have been associated with hepatic dysfunction; however, this complication is rare in oncology.

1	Certain nucleoside drugs have been associated with hepatic dysfunction; however, this complication is rare in oncology. Hepatic radiation damage depends on dose, volume, fractionation, preexisting liver disease, and synchronous or metachronous chemotherapy. In general, radiation doses to the liver >1500 cGy can produce hepatic dysfunction with a steep dose-injury curve. Radiation-induced liver disease closely mimics hepatic venoocclusive disease.

1	Cisplatin produces reversible decrements in renal function, but may also produce severe irreversible toxicity in the presence of renal disease and may predispose to accentuated damage with subsequent renal insults. Cyclophosphamide and ifosfamide, as prodrugs primarily activated in the liver, have cleavage products (acrolein) that can produce hemorrhagic cystitis. This can be prevented with the free radical scavenger MESNA (mercaptoethane sulfonate), which is required for ifosfamide administration. Hemorrhagic cystitis caused by these agents may predispose to bladder cancer.

1	Alkylating agents are associated with the highest rates of male and female infertility, which is directly dependent on age, dose, and duration of treatment. The age at treatment is an important determinant of fertility outcome, with prepubertal patients having the highest tolerance. Ovarian failure is age related, and females who resume menses after treatment are still at increased risk for premature menopause. Males generally have reversible azoospermia during lower intensity alkylator chemotherapy, and long-term infertility is associated with doses of cyclophosphamide >9 g/m2 and with high-intensity therapy, such as that used in hematopoietic stem cell transplantation. Males undergoing potentially sterilizing chemotherapy should be offered sperm banking. Gonadotropin-releasing hormone (GnRH) analogs remain experimental to preserve ovarian function. Assisted reproductive technologies can be helpful to couples with chemotherapy-induced infertility.

1	Testicles and ovaries in prepubertal patients are less sensitive to radiation damage; spermatogenesis is affected by low doses of radiation, and complete azoospermia occurs at 600–700 cGy. Leydig cell dysfunction, in contrast, occurs at <2000 cGy, and hence, endocrine function is lost at much higher radiation doses than spermatogenesis. Erectile dysfunction occurs in up to 80% of men treated with external-beam radiation therapy for prostate cancer. Sildenafil may be useful in reversing erectile dysfunction. Ovarian function damage with radiation is age related and occurs at doses of 150–500 cGy. Premature induction of menopause can have serious medical and psychological sequelae. Hormone replacement therapy is often contraindicated (as in estrogen receptor–positive breast cancer). Attention must be paid to maintenance of bone mass with calcium and vitamin D supplements and oral bisphosphonates, and bone mass should be monitored using bone density determinations. Paroxetine, clonidine,

1	must be paid to maintenance of bone mass with calcium and vitamin D supplements and oral bisphosphonates, and bone mass should be monitored using bone density determinations. Paroxetine, clonidine, pregabalin, and other drugs may be useful in symptomatically controlling hot flashes.

1	Long-term survivors of childhood cancer (e.g., ALL) who have received cranial radiation may have altered leptin biology and growth hormone deficiency, leading to obesity and reduced strength, exercise tolerance, and bone density. Radiation therapy to the neck (e.g., in Hodgkin’s lymphoma) may lead to hypothyroidism, Graves’ disease, thyroiditis, and thyroid malignancies. Thyroid-stimulating hormone (TSH) is followed routinely in such patients to prevent hypothyroidism, and to suppress persistently elevated levels of TSH which may cause or drive thyroid cancer. Cataracts may be caused by glucocorticoids, depending on duration and dose; radiation therapy; and uncommonly tamoxifen. Orbital radiation therapy may cause blindness. Radiation therapy can produce xerostomia (dry mouth), with an attendant increase in caries and poor dentition. Taste and appetite may be suppressed. Bisphosphonate use may result in osteonecrosis of the jaw.

1	Up to 40% of patients treated with bleomycin may develop Raynaud’s phenomenon as a result of an unknown mechanism.

1	Second malignancies in patients cured of cancer are a major cause of death, and treated cancer patients must be monitored for their occurrence. The induction of second malignancies is governed by the complex interplay of a number of factors including age, gender, environmental exposures, genetic susceptibility, and cancer treatment itself. In a number of settings, the events leading to the primary cancer themselves increase the risk of second malignancies. Patients with lung cancer are at increased risk of esophageal and head and neck cancers, and vice versa, due to shared risk factors including alcohol and tobacco abuse. Indeed, the risk of developing a second primary head and neck, esophageal, or lung cancer is also increased in these patients. Patients with breast cancer are at increased risk of breast cancer in the opposite breast. Patients with Hodgkin’s lymphoma are at risk for nonHodgkin’s lymphomas. Genetic cancer syndromes (e.g., multiple endocrine neoplasia or Li-Fraumeni,

1	risk of breast cancer in the opposite breast. Patients with Hodgkin’s lymphoma are at risk for nonHodgkin’s lymphomas. Genetic cancer syndromes (e.g., multiple endocrine neoplasia or Li-Fraumeni, Lynch’s, Cowden’s, and Gardner’s syndromes) are examples of genetically based second malignancies of specific types. Cancer treatment itself does not appear to be responsible for the risk of these secondary malignancies. Deficient DNA repair can greatly increase the risk of cancers from DNA-damaging agents, as in ataxia-telangiectasia. Importantly, the risk of treatment-related second malignancies is at least additive and often synergistic with combined chemotherapy and radiation therapy, and hence for such combined-therapy treatment approaches, it is important to establish the necessity of each in the treatment program. All of these patients require special surveillance or, in some cases, prophylactic surgery as part of appropriate treatment and follow-up.

1	Chemotherapy is significantly associated with two fatal second malignancies, acute leukemia and myelodysplastic syndromes. Two types of leukemia have been described; in patients treated with alkylating agents, acute myeloid leukemia is associated with deletions in chromosome 5 or 7. The lifetime risk is about 1–5%, is increased by radiation therapy, and increases with age. The incidence of these leukemias peaks at 4–6 years, with risk returning close to baseline at 10 years. The other type of acute myeloid leukemia is related to therapy with topoisomerase inhibitors, is associated with chromosome 10q23 trans-locations, has an incidence of <1%, and generally occurs 1.5–3 years after treatment. Both of these acute myeloid leukemias are refractory to treatment and have a high mortality. The development of myelodysplastic syndromes is increased following chemotherapy, and these are often associated with leukemic progression and a dismal prognosis.

1	Patients receiving radiation have an increasing and lifelong risk of second malignancies that is 1–2% in the second decade following treatment but increases to >25% after 25 years. These malignancies include cancers of the thyroid and breast, sarcomas, and CNS cancers, which often tend to be aggressive and have a poor prognosis. An example of organ-, age-, and sex-dependent radiation-induced secondary malignancy is breast cancer, in which the risk is small with radiation in women under age 30 but increases about 20-fold over baseline in women over 30. A 25-year-old woman treated with mantle radiation for Hodgkin’s lymphoma has a 29% actuarial risk of developing breast cancer by age 55.

1	Treatment of breast cancer with tamoxifen for 5 years or longer is associated with a 1–2% risk of endometrial cancer. Surveillance is generally effective at finding these cancers at an early stage. The risk of mortality from tamoxifen-induced endometrial cancer is low compared to the benefit of tamoxifen as adjuvant therapy for breast cancer. Immunosuppressive therapy, as used in allogeneic bone marrow transplantation, particularly with T cell depletion using antithymocyte globulin or other means, increases the risk of Epstein Barr virus–associated Late Consequences of Cancer and Its Treatment Pediatric cancers Majority have at least one late effect 30% with moderate/severe problems Cardiovascular: radiation, anthracyclines Lungs: radiation Skeletal abnormalities: radiation Psychological, cognitive, and sexual problems Second neoplasms significant cause of death

1	Hodgkin’s lymphoma Thyroid dysfunction: radiation Premature coronary artery disease: radiation Gonadal dysfunction: chemotherapy Postsplenectomy sepsis Myelodysplasia Acute myeloid leukemia Non-Hodgkin’s lymphomas Breast cancer, lung cancer, and melanoma Fatigue, psychological and sexual problems Peripheral neuropathy Acute leukemia Second malignancies: hematologic, solid tumors Neuropsychiatric dysfunction Subnormal growth Thyroid abnormalities Infertility Graft-versus-host disease (allogeneic transplant) Psychosexual dysfunction. Head and neck cancer Poor dentition, dry mouth, poor nutrition: radiation Breast cancer Tamoxifen: endometrial cancer, blood clots Aromatase inhibitors: osteoporosis, arthritis Cardiomyopathy: anthracycline ± radiation, trastuzumab Acute leukemia Hormone deficiency symptoms: hot flashes, vaginal dryness, dyspareunia Psychosocial dysfunction “Chemo brain”

1	Testicular cancer Raynaud’s phenomenon Renal dysfunction Pulmonary dysfunction Retrograde ejaculation: surgery 15% sexual dysfunction Colon cancer Major risk is second colon cance. Quality of life high in survivors Prostate cancer Impotence Urinary incontinence (0–15%) Chronic proctitis, prostatitis/cystitis: radiation B cell lymphoproliferative disorder. The incidence at 10 years after T cell depletion is 9–12%. Discontinuing immunosuppressive therapy, if possible, is often associated with complete disease regression.

1	All former cancer patients should be followed indefinitely. This is most often done by oncologists, but demographic changes suggest that more primary care physicians will need to be trained in the follow-up of treated cancer patients in remission. Cancer patients need to be educated about signs and symptoms of recurrence and potentially adverse effects related to therapy. Localized pain or palpable abnormality in a previously radiated field should prompt radiographic evaluation. Screening tests, when available and validated, should be used on a routine and regular basis (e.g., mammography and Pap smear), particularly in patients receiving radiation to specific organs. Annual mammography should start no later than 10 years after breast radiation. Patients receiving radiation fields encompassing thyroid tissue should have regular thyroid exams and TSH testing. Patients treated with alkylating agents or topoisomerase inhibitors should have a complete blood count every 6–12 months, and

1	thyroid tissue should have regular thyroid exams and TSH testing. Patients treated with alkylating agents or topoisomerase inhibitors should have a complete blood count every 6–12 months, and cytopenias, abnormal cells on peripheral smear, or macrocytosis should be evaluated with bone marrow biopsy and aspirate, to include cytogenetics, flow cytometry, or fluorescence in situ hybridization (FISH) studies as appropriate.

1	As the population of cancer survivors lives longer and grows, cancer survivorship has become an increasingly recognized subject, and the Institute of Medicine and National Research Council have published a monograph entitled From Cancer Patient to Cancer Survivor: Lost in Transition. The monograph proposes a plan that would inform clinicians caring for cancer survivors in complete detail of their previous treatments, complications thereof, signs and symptoms of late effects, and recommended screening and follow-up procedures. Table 125-2 lists long-term treatment effects by cancer type.

1	Clearly, the challenge for the future is to combine chemotherapy, targeted agents, biologic therapies, radiation, and surgery to produce better outcomes with less toxicity, including late effects of therapy. This is easily said but less easily accomplished. As treatment becomes more effective in new patient populations (ovarian, bladder, anal, and laryngeal cancers, for example), we will expect to discover new populations at risk for late effects. These populations will need to be followed carefully, so that such effects are recognized and treated. Cancer survivors represent an underused resource for prevention studies. Childhood cancer survivors, especially, suffer multiple chronic health impairments. The incidence of these late treatment consequences appears to have no plateau with age, throwing in stark relief the necessity of close monitoring and therapies with fewer late consequences of treatment. John W. Adamson

1	John W. Adamson Anemias associated with normocytic and normochromic red cells and an inappropriately low reticulocyte response (reticulocyte index <2–2.5) are hypoproliferative anemias. This category includes early iron deficiency (before hypochromic microcytic red cells develop), acute and chronic inflammation (including many malignancies), renal disease, hypometabolic states such as protein malnutrition and endocrine deficiencies, and anemias from marrow damage. Marrow damage states are discussed in Chap. 130. Hypoproliferative anemias are the most common anemias, and in the clinic, iron deficiency anemia is the most common of these followed by the anemia of inflammation. The anemia of inflammation, similar to iron deficiency, is related in part to abnormal iron metabolism. The anemias associated with renal disease, inflammation, cancer, and hypometabolic states are characterized by a suboptimal erythro poietin response to the anemia.

1	Internal iron exchange. Normally 80% of iron passing through the plasma transferrin pool is recycled from senescent red Iron is a critical element in the function of all cells, although the amount of iron required by individual tissues varies during development. At the same time, the body must protect itself from free iron, which is highly toxic in that it participates in chemical reactions that generate free radicals such as singlet O2 or OH-. Consequently, elaborate mechanisms have evolved that allow iron to be made available for physiologic functions while at the same time conserving this element and handling it in such a way that toxicity is avoided.

1	The major role of iron in mammals is to carry O2 as part of hemoglobin. O2 is also bound by myoglobin in muscle. Iron is a critical element in iron-containing enzymes, including the cytochrome system in mitochondria. Iron distribution in the body is shown in Table 126-1. Without iron, cells lose their capacity for electron transport and energy metabolism. In erythroid cells, hemoglobin synthesis is impaired, resulting in anemia and reduced O2 delivery to tissue.

1	Figure 126-1 outlines the major pathways of internal iron exchange in humans. Iron absorbed from the diet or released from stores circulates in the plasma bound to transferrin, the iron transport protein. Transferrin is a bilobed glycoprotein with two iron binding sites. Transferrin that carries iron exists in two forms—monoferric (one iron atom) or diferric (two iron atoms). The turnover (half-clearance time) of transferrin-bound iron is very rapid—typically 60–90 min. Because almost all of the iron transported by transferrin is delivered to the erythroid marrow, the clearance time of transferrin-bound iron from the circulation is affected most by the plasma iron level and the erythroid marrow activity. When erythropoiesis is markedly stimulated, the pool of erythroid cells requiring iron increases and the clearance time of iron from the circulation decreases. The half-clearance time of iron in

1	Iron Content, mg cells. Absorption of approximately 1 mg/d is required from the diet in men, and 1.4 mg/d in women to maintain homeostasis. As long as transferrin saturation is maintained between 20 and 60% and erythropoiesis is not increased, use of iron stores is not required. However, in the event of blood loss, dietary iron deficiency, or inadequate iron absorption, up to 40 mg/d of iron can be mobilized from stores. RE, reticuloendothelial. the presence of iron deficiency is as short as 10–15 min. With suppression of erythropoiesis, the plasma iron level typically increases and the half-clearance time may be prolonged to several hours. Normally, the iron bound to transferrin turns over 6–8 times per day. Assuming a normal plasma iron level of 80–100 μg/dL, the amount of iron passing through the transferrin pool is 20–24 mg/d.

1	The iron-transferrin complex circulates in the plasma until it interacts with specific transferrin receptors on the surface of marrow erythroid cells. Diferric transferrin has the highest affinity for transferrin receptors; apotransferrin (not carrying iron) has very little affinity. Although transferrin receptors are found on cells in many tissues within the body—and all cells at some time during development will display transferrin receptors—the cell having the greatest number of receptors (300,000–400,000/cell) is the developing erythroblast.

1	Once the iron-bearing transferrin interacts with its receptor, the complex is internalized via clathrin-coated pits and transported to an acidic endosome, where the iron is released at the low pH. The iron is then made available for heme synthesis while the transferrin-receptor complex is recycled to the surface of the cell, where the bulk of the transferrin is released back into circulation and the transferrin receptor reanchors into the cell membrane. At this point a certain amount of the transferrin receptor protein may be released into circulation and can be measured as soluble transferrin receptor protein. Within the erythroid cell, iron in excess of the amount needed for hemoglobin synthesis binds to a storage protein, apoferritin, forming ferritin. This mechanism of iron exchange also takes place in other cells of the body expressing transferrin receptors, especially liver parenchymal cells where the iron can be incorporated into heme-containing enzymes or stored. The iron

1	also takes place in other cells of the body expressing transferrin receptors, especially liver parenchymal cells where the iron can be incorporated into heme-containing enzymes or stored. The iron incorporated into hemoglobin subsequently enters the circulation as new red cells are released from the bone marrow. The iron is then part of the red cell mass and will not become available for reutilization until the red cell dies.

1	626 In a normal individual, the average red cell life span is 120 days. Thus, 0.8–1% of red cells are replaced each day. At the end of its life span, the red cell is recognized as senescent by the cells of the reticuloendothelial (RE) system, and the red cell undergoes phagocytosis. Once within the RE cell, the ingested hemoglobin is broken down, the globin and other proteins are returned to the amino acid pool, and the iron is shuttled back to the surface of the RE cell, where it is presented to circulating transferrin. It is the efficient and highly conserved recycling of iron from senescent red cells that supports steady-state (and even mildly accelerated) erythropoiesis. Because each milliliter of red cells contains 1 mg of elemental iron, the amount of iron needed to replace those red cells lost through senescence amounts to 20 mg/d (assuming an adult with a red cell mass of 2 L). Any additional iron required for daily red cell production comes from the diet. Normally, an adult

1	cells lost through senescence amounts to 20 mg/d (assuming an adult with a red cell mass of 2 L). Any additional iron required for daily red cell production comes from the diet. Normally, an adult male will need to absorb at least 1 mg of elemental iron daily to meet needs, while females in the childbearing years will need to absorb an average of 1.4 mg/d. However, to achieve a maximum proliferative erythroid marrow response to anemia, additional iron must be available. With markedly stimulated erythropoiesis, demands for iron are increased by as much as sixto eightfold. With extravascular hemolytic anemia, the rate of red cell destruction is increased, but the iron recovered from the red cells is efficiently reutilized for hemoglobin synthesis. In contrast, with intra-vascular hemolysis or blood loss anemia, the rate of red cell production is limited by the amount of iron that can be mobilized from stores. Typically, the rate of mobilization under these circumstances will not support

1	blood loss anemia, the rate of red cell production is limited by the amount of iron that can be mobilized from stores. Typically, the rate of mobilization under these circumstances will not support red cell production more than 2.5 times normal. If the delivery of iron to the stimulated marrow is suboptimal, the marrow’s proliferative response is blunted, and hemoglobin synthesis is impaired. The result is a hypoproliferative marrow accompanied by microcytic, hypochromic anemia. Whereas blood loss or hemolysis places a demand on the iron supply, inflammatory conditions interfere with iron release from stores and can result in a rapid decrease in the serum iron (see below).

1	The balance of iron in humans is tightly controlled and designed to conserve iron for reutilization. There is no regulated excretory pathway for iron, and the only mechanisms by which iron is lost are blood loss (via gastrointestinal bleeding, menses, or other forms of bleeding) and the loss of epithelial cells from the skin, gut, and genitourinary tract. Normally, the only route by which iron comes into the body is via absorption from food or from medicinal iron taken orally. Iron may also enter the body through red cell transfusions or injection of iron complexes. The margin between the amount of iron available for absorption and the requirement for iron in growing infants and the adult female is narrow; this accounts for the great prevalence of iron deficiency worldwide—currently estimated at one-half billion people.

1	The amount of iron required from the diet to replace losses averages approximately 10% of body iron content a year in men and 15% in women of childbearing age. Dietary iron content is closely related to total caloric intake (approximately 6 mg of elemental iron per 1000 calories). Iron bioavailability is affected by the nature of the foodstuff, with heme iron (e.g., red meat) being most readily absorbed. In the United States, the average iron intake in an adult male is 15 mg/d with 6% absorption; for the average female, the daily intake is 11 mg/d with 12% absorption. An individual with iron deficiency can increase iron absorption to approximately 20% of the iron present in a meat-containing diet but only 5–10% of the iron in a vegetarian diet. As a result, one-third of the female population in the United States has virtually no iron stores. Vegetarians are at an additional disadvantage because certain foodstuffs that include phytates and phosphates reduce iron absorption by

1	population in the United States has virtually no iron stores. Vegetarians are at an additional disadvantage because certain foodstuffs that include phytates and phosphates reduce iron absorption by approximately 50%. When ionizable iron salts are given together with food, the amount of iron absorbed is reduced. When the percentage of iron absorbed from individual food items is compared with the percentage for an equivalent amount of ferrous salt, iron in vegetables is only about one-twentieth as available, egg iron one-eighth, liver iron one-half, and heme iron one-half to two-thirds.

1	Infants, children, and adolescents may be unable to maintain normal iron balance because of the demands of body growth and lower dietary intake of iron. During the last two trimesters of pregnancy, daily iron requirements increase to 5–6 mg, and iron supplements are strongly recommended for pregnant women in developed countries.

1	Iron absorption takes place largely in the proximal small intestine and is a carefully regulated process. For absorption, iron must be taken up by the luminal cell. That process is facilitated by the acidic contents of the stomach, which maintains the iron in solution. At the brush border of the absorptive cell, the ferric iron is converted to the ferrous form by a ferrireductase. Transport across the membrane is accomplished by divalent metal transporter type 1 (DMT-1, also known as natural resistance macrophage-associated protein type 2 [Nramp 2] or DCT-1). DMT-1 is a general cation transporter. Once inside the gut cell, iron may be stored as ferritin or transported through the cell to be released at the basolateral surface to plasma transferrin through the membrane-embedded iron exporter, ferroportin. The function of ferroportin is negatively regulated by hepcidin, the principal iron regulatory hormone. In the process of release, iron interacts with another ferroxidase, hephaestin,

1	ferroportin. The function of ferroportin is negatively regulated by hepcidin, the principal iron regulatory hormone. In the process of release, iron interacts with another ferroxidase, hephaestin, which oxidizes the iron to the ferric form for transferrin binding. Hephaestin is similar to ceruloplasmin, the copper-carrying protein.

1	Iron absorption is influenced by a number of physiologic states. Erythroid hyperplasia stimulates iron absorption even in the face of normal or increased iron stores, and hepcidin levels are inappropriately low. Thus, patients with anemias associated with high levels of ineffective erythropoiesis absorb excess amounts of dietary iron. The molecular mechanism underlying this relationship is not known. Over time, this may lead to iron overload and tissue damage. In iron deficiency, hepcidin levels are also low and iron is much more efficiently absorbed; the contrary is true in states of secondary iron overload. The normal individual can reduce iron absorption in situations of excessive intake or medicinal iron intake; however, while the percentage of iron absorbed goes down, the absolute amount goes up. This accounts for the acute iron toxicity occasionally seen when children ingest large numbers of iron tablets. Under these circumstances, the amount of iron absorbed exceeds the

1	amount goes up. This accounts for the acute iron toxicity occasionally seen when children ingest large numbers of iron tablets. Under these circumstances, the amount of iron absorbed exceeds the transferrin binding capacity of the plasma, resulting in free iron that affects critical organs such as cardiac muscle cells.

1	Iron deficiency is one of the most prevalent forms of malnutri tion. Globally, 50% of anemia is attributable to iron deficiency and accounts for approximately 841,000 deaths annually worldwide. Africa and parts of Asia bear 71% of the global mortality burden; North America represents only 1.4% of the total morbidity and mortality associated with iron deficiency.

1	The progression to iron deficiency can be divided into three stages (Fig. 126-2). The first stage is negative iron balance, in which the demands for (or losses of) iron exceed the body’s ability to absorb iron from the diet. This stage results from a number of physiologic mechanisms, including blood loss, pregnancy (in which the demands for red cell production by the fetus outstrip the mother’s ability to provide iron), rapid growth spurts in the adolescent, or inadequate dietary iron intake. Blood loss in excess of 10–20 mL of red cells per day is greater than the amount of iron that the gut can absorb from a normal diet. Under these circumstances, the iron deficit must be made up by mobilization of iron from RE storage sites. During this period, iron stores—reflected by the serum ferritin level or the appearance of stain-able iron on bone marrow aspirations—decrease. As long as iron stores are present and can be mobilized, the serum iron, total iron-binding capacity (TIBC), and red

1	level or the appearance of stain-able iron on bone marrow aspirations—decrease. As long as iron stores are present and can be mobilized, the serum iron, total iron-binding capacity (TIBC), and red cell protoporphyrin levels remain within normal limits. At this stage, red cell morphology and indices are normal.

1	When iron stores become depleted, the serum iron begins to fall. Gradually, the TIBC increases, as do red cell protoporphyrin levels. By definition, marrow iron stores are absent when the serum ferritin level is <15 μg/L. As long as the serum iron remains within the normal

1	FIGURE 126-2 Laboratory studies in the evolution of iron deficiency. Measurements of marrow iron stores, serum ferritin, and total iron-binding capacity (TIBC) are sensitive to early iron-store depletion. Iron-deficient erythropoiesis is recognized from additional abnormalities in the serum iron (SI), percent transferrin saturation, the pattern of marrow sideroblasts, and the red blood cell (RBC) protoporphyrin level. Patients with iron-deficiency anemia demonstrate all the same abnormalities plus hypochromic microcytic anemia. (From RS Hillman, CA Finch: The Red Cell Manual, 7th ed. Philadelphia, F.A.Davis and Co., 1996, with permission.) range, hemoglobin synthesis is unaffected despite the dwindling iron stores. Once the transferrin saturation falls to 15–20%, hemoglobin synthesis becomes impaired. This is a period of iron-deficient erythropoiesis. Careful evaluation of the peripheral blood smear reveals the first appearance of microcytic cells, and if the laboratory technology is

1	impaired. This is a period of iron-deficient erythropoiesis. Careful evaluation of the peripheral blood smear reveals the first appearance of microcytic cells, and if the laboratory technology is available, one finds hypochromic reticulocytes in circulation. Gradually, the hemoglobin and hematocrit begin to fall, reflecting iron-deficiency anemia. The transferrin saturation at this point is 10–15%.

1	When moderate anemia is present (hemoglobin 10–13 g/dL), the bone marrow remains hypoproliferative. With more severe anemia (hemoglobin 7–8 g/dL), hypochromia and microcytosis become more prominent, target cells and misshapen red cells (poikilocytes) appear on the blood smear as cigaror pencil-shaped forms, and the erythroid marrow becomes increasingly ineffective. Consequently, with severe prolonged iron-deficiency anemia, erythroid hyperplasia of the marrow develops, rather than hypoproliferation. Conditions that increase demand for iron, increase iron loss, or decrease iron intake or absorption can produce iron deficiency (Table 126-2). Chronic blood loss Menses Acute blood loss Blood donation Phlebotomy as treatment for polycythemia vera Malabsorption from disease (sprue, Crohn’s disease) Malabsorption from surgery (gastrectomy and some forms of bariatric surgery)

1	Malabsorption from disease (sprue, Crohn’s disease) Malabsorption from surgery (gastrectomy and some forms of bariatric surgery) LABORATORY IRON STUDIES Serum Iron and Total Iron-Binding Capacity The serum iron level represents the amount of circulating iron bound to transferrin. The TIBC is an indirect measure of the circulating transferrin. The normal range for the serum iron is 50–150 μg/dL; the normal range for TIBC is 300–360 μg/dL. Transferrin saturation, which is normally 25–50%, is obtained by the following formula: serum iron × 100 ÷ TIBC. Iron-deficiency states are associated with saturation levels below 20%. There is a diurnal variation in the serum iron. A transferrin saturation % >50% indicates that a disproportionate amount of the iron bound to transferrin is being delivered to nonerythroid tissues. If this persists for an extended time, tissue iron overload may occur.

1	Serum Ferritin Free iron is toxic to cells, and the body has established an elaborate set of protective mechanisms to bind iron in various tissue compartments. Within cells, iron is stored complexed to protein as ferritin or hemosiderin. Apoferritin binds to free ferrous iron and stores it in the ferric state. As ferritin accumulates within cells of the RE system, protein aggregates are formed as hemosiderin. Iron in ferritin or hemosiderin can be extracted for release by the RE cells, although hemosiderin is less readily available. Under steady-state conditions, the serum ferritin level correlates with total body iron stores; thus, the serum ferritin level is the most convenient laboratory test to estimate iron stores. The normal value for ferritin varies according to the age and gender of the individual (Fig. 126-3). Adult males have serum Serum ferritin, ˜g/L

1	Serum ferritin, ˜g/L Certain clinical conditions carry an increased likelihood of iron deficiency. Pregnancy, adolescence, periods of rapid growth, and an intermittent history of blood loss of any kind should alert the clini-0 cian to possible iron deficiency. A cardinal rule is that the appearance of iron deficiency in an adult male means gastrointestinal blood loss until proven otherwise. Signs related to iron deficiency depend on the severity and chronicity of the anemia in addition to the usual signs of anemia—fatigue, pallor, and reduced exercise capacity. Cheilosis (fissures at the corners of the mouth) and koilonychia (spooning of the fingernails) are signs of advanced tissue iron deficiency. The diagnosis of iron deficiency is typically based on laboratory results. 0 1020304050607080 Age, years FIGURE 126-3 Serum ferritin levels as a function of sex and age.

1	0 1020304050607080 Age, years FIGURE 126-3 Serum ferritin levels as a function of sex and age. Iron store depletion and iron deficiency are accompanied by a decrease in serum ferritin level below 20 μg/L. (From RS Hillman et al: Hematology in Clinical Practice, 5th ed. New York, McGraw-Hill, 2011, with permission.) Iron Stores Marrow Iron Stain, 0–4+ Serum Ferritin, μg/L 1–300 mg Trace to 1+ 15–30 ferritin values averaging 100 μg/L, while adult females have levels averaging 30 μg/L. As iron stores are depleted, the serum ferritin falls to <15 μg/L. Such levels are diagnostic of absent body iron stores.

1	Evaluation of Bone Marrow Iron Stores Although RE iron stores can be estimated from the iron stain of a bone marrow aspirate or biopsy, the measurement of serum ferritin has largely supplanted these procedures for determination of storage iron (Table 126-3). The serum ferritin level is a better indicator of iron overload than the marrow iron stain. However, in addition to storage iron, the marrow iron stain provides information about the effective delivery of iron to developing erythroblasts. Normally, when the marrow smear is stained for iron, 20–40% of developing erythroblasts—called sideroblasts—will have visible ferritin granules in their cytoplasm. This represents iron in excess of that needed for hemoglobin synthesis. In states in which release of iron from storage sites is blocked, RE iron will be detectable, and there will be few or no sideroblasts. In the myelodysplastic syndromes, mitochondrial dysfunction can occur, and accumulation of iron in mitochondria appears in a

1	RE iron will be detectable, and there will be few or no sideroblasts. In the myelodysplastic syndromes, mitochondrial dysfunction can occur, and accumulation of iron in mitochondria appears in a necklace fashion around the nucleus of the erythroblast. Such cells are referred to as ringed sideroblasts.

1	Red Cell Protoporphyrin Levels Protoporphyrin is an intermediate in the pathway to heme synthesis. Under conditions in which heme synthesis is impaired, protoporphyrin accumulates within the red cell. This reflects an inadequate iron supply to erythroid precursors to support hemoglobin synthesis. Normal values are <30 μg/dL of red cells. In iron deficiency, values in excess of 100 μg/dL are seen. The most common causes of increased red cell protoporphyrin levels are absolute or relative iron deficiency and lead poisoning.

1	Serum Levels of Transferrin Receptor Protein Because erythroid cells have the highest numbers of transferrin receptors of any cell in the body, and because transferrin receptor protein (TRP) is released by cells into the circulation, serum levels of TRP reflect the total erythroid marrow mass. Another condition in which TRP levels are elevated is absolute iron deficiency. Normal values are 4–9 μg/L determined by immunoassay. This laboratory test is becoming increasingly available and, along with the serum ferritin, has been proposed to distinguish between iron deficiency and the anemia of inflammation (see below).

1	Other than iron deficiency, only three conditions need to be considered in the differential diagnosis of a hypochromic microcytic anemia (Table 126-4). The first is an inherited defect in globin chain synthesis: the thalassemias. These are differentiated from iron deficiency most readily by serum iron values; normal or increased serum iron levels and transferrin saturation are characteristic of the thalassemias. In addition, the red blood cell distribution width (RDW) index is generally normal in thalassemia and elevated in iron deficiency.

1	The second condition is the anemia of inflammation (AI; also referred to as the anemia of chronic disease) with inadequate iron supply to the erythroid marrow. The distinction between true iron-deficiency anemia and AI is among the most common diagnostic problems encountered by clinicians (see below). Usually, AI is normocytic and normochromic. The iron values usually make the differential diagnosis clear, as the ferritin level is normal or increased and the percent transferrin saturation and TIBC are typically below normal. Finally, the myelodysplastic syndromes represent the third and least common condition. Occasionally, patients with myelodysplasia have impaired hemoglobin synthesis with mitochondrial dysfunction, resulting in impaired iron incorporation into heme. The iron values again reveal normal stores and more than an adequate supply to the marrow, despite the microcytosis and hypochromia.

1	The severity and cause of iron-deficiency anemia will determine the appropriate approach to treatment. As an example, symptomatic elderly patients with severe iron-deficiency anemia and cardiovascular instability may require red cell transfusions. Younger individuals who have compensated for their anemia can be treated more conservatively with iron replacement. The foremost issue for the latter patient is the precise identification of the cause of the iron deficiency. For the majority of cases of iron deficiency (pregnant women, growing children and adolescents, patients with infrequent episodes of bleeding, and those with inadequate dietary intake of iron), oral iron therapy will suffice. For patients with unusual blood loss or malabsorption, specific diagnostic tests and appropriate therapy take priority. Once the diagnosis of iron-deficiency anemia and its cause is made, there are three major therapeutic approaches.

1	Transfusion therapy is reserved for individuals who have symptoms of anemia, cardiovascular instability, and continued and excessive blood loss from whatever source and who require immediate intervention. The management of these patients is less related to the iron deficiency than it is to the consequences of the severe anemia. Not only do transfusions correct the anemia acutely, but the transfused red cells provide a source of iron for reutilization, assuming they are not lost through continued bleeding. Transfusion therapy will stabilize the patient while other options are reviewed. In the asymptomatic patient with established iron-deficiency anemia, treatment with oral iron is usually adequate. Multiple preparations are available, ranging from simple iron salts to complex iron compounds designed for sustained release throughout the small intestine (Table 126-5). Although the various preparations

1	Tablet (Iron Elixir (Iron Content), Generic Name Content), mg mg in 5 mL contain different amounts of iron, they are generally all absorbed well and are effective in treatment. Some come with other compounds designed to enhance iron absorption, such as ascorbic acid. It is not clear whether the benefits of such compounds justify their costs. Typically, for iron replacement therapy, up to 200 mg of elemental iron per day is given, usually as three or four iron tablets (each containing 50–65 mg elemental iron) given over the course of the day. Ideally, oral iron preparations should be taken on an empty stomach, since food may inhibit iron absorption. Some patients with gastric disease or prior gastric surgery require special treatment with iron solutions, because the retention capacity of the stomach may be reduced. The retention capacity is necessary for dissolving the shell of the iron tablet before the release of iron. A dose of 200 mg of elemental iron per day should result in the

1	the stomach may be reduced. The retention capacity is necessary for dissolving the shell of the iron tablet before the release of iron. A dose of 200 mg of elemental iron per day should result in the absorption of iron up to 50 mg/d. This supports a red cell production level of two to three times normal in an individual with a normally functioning marrow and appropriate erythropoietin stimulus. However, as the hemoglobin level rises, erythropoietin stimulation decreases, and the amount of iron absorbed is reduced. The goal of therapy in individuals with iron-deficiency anemia is not only to repair the anemia, but also to provide stores of at least 0.5–1 g of iron. Sustained treatment for a period of 6–12 months after correction of the anemia will be necessary to achieve this.

1	Of the complications of oral iron therapy, gastrointestinal distress is the most prominent and is seen in 15–20% of patients. Abdominal pain, nausea, vomiting, or constipation may lead to noncompliance. Although small doses of iron or iron preparations with delayed release may help somewhat, the gastrointestinal side effects are a major impediment to the effective treatment of a number of patients.

1	The response to iron therapy varies, depending on the erythropoietin stimulus and the rate of absorption. Typically, the reticulocyte count should begin to increase within 4–7 days after initiation of therapy and peak at 1–1½ weeks. The absence of a response may be due to poor absorption, noncompliance (which is common), or a confounding diagnosis. A useful test in the clinic to determine the patient’s ability to absorb iron is the iron tolerance test. Two iron tablets are given to the patient on an empty stomach, and the serum iron is measured serially over the subsequent 2 h. Normal absorption will result in an increase in the serum iron of at least 100 μg/ dL. If iron deficiency persists despite adequate treatment, it may be necessary to switch to parenteral iron therapy.

1	Intravenous iron can be given to patients who are unable to tolerate oral iron; whose needs are relatively acute; or who need iron on an ongoing basis, usually due to persistent gastrointestinal blood loss. Parenteral iron use has been increasing rapidly in the last several years with the recognition that recombinant erythropoietin (EPO) therapy induces a large demand for iron—a demand that frequently cannot be met through the physiologic release of iron from RE sources or oral iron absorption. The safety of parenteral iron— particularly iron dextran—has been a concern. The serious adverse reaction rate to intravenous high-molecular-weight iron dextran is 0.7%. Fortunately, newer iron complexes are available in the United States, such as ferumoxytol (Feraheme), sodium ferric gluconate (Ferrlecit), iron sucrose (Venofer), and ferric carboxy-629 maltose (Injectafer), that have much lower rates of adverse effects. Ferumoxytol delivers 510 mg of iron per injection; ferric gluconate 125 mg

1	iron sucrose (Venofer), and ferric carboxy-629 maltose (Injectafer), that have much lower rates of adverse effects. Ferumoxytol delivers 510 mg of iron per injection; ferric gluconate 125 mg per injection, ferric carboxymaltose 750 mg per injection, and iron sucrose 200 mg per injection.

1	Parenteral iron is used in two ways: one is to administer the total dose of iron required to correct the hemoglobin deficit and provide the patient with at least 500 mg of iron stores; the second is to give repeated small doses of parenteral iron over a protracted period. The latter approach is common in dialysis centers, where it is not unusual for 100 mg of elemental iron to be given weekly for 10 weeks to augment the response to recombinant EPO therapy. The amount of iron needed by an individual patient is calculated by the following formula: Body weight (kg) × 2.3 × (15 – patient’s hemoglobin, g/dL) + 500 or 1000 mg (for stores).

1	Body weight (kg) × 2.3 × (15 – patient’s hemoglobin, g/dL) + 500 or 1000 mg (for stores). In administering intravenous iron dextran, anaphylaxis is a con cern. Anaphylaxis is much rarer with the newer preparations. The factors that have correlated with an anaphylactic-like reaction include a history of multiple allergies or a prior allergic reaction to dextran (in the case of iron dextran). Generalized symptoms appearing several days after the infusion of a large dose of iron can include arthralgias, skin rash, and low-grade fever. These may be dose-related, but they do not preclude the further use of parenteral iron in the patient. To date, patients with sensitivity to iron dextran have been safely treated with other parenteral iron preparations.

1	If a large dose of iron dextran is to be given (>100 mg), the iron preparation should be diluted in 5% dextrose in water or 0.9% NaCl solution. The iron solution can then be infused over a 60to 90-min period (for larger doses) or at a rate convenient for the attending nurse or physician. Although a test dose (25 mg) of parenteral iron dextran is recommended, in reality a slow infusion of a larger dose of parenteral iron solution will afford the same kind of early warning as a separately injected test dose. Early in the infusion of iron, if chest pain, wheezing, a fall in blood pressure, or other systemic symptoms occur, the infusion of iron should be stopped immediately.

1	In addition to mild to moderate iron-deficiency anemia, the hypo-proliferative anemias can be divided into four categories: (1) chronic inflammation, (2) renal disease, (3) endocrine and nutritional deficiencies (hypometabolic states), and (4) marrow damage (Chap. 130). With chronic inflammation, renal disease, or hypometabolism, endogenous EPO production is inadequate for the degree of anemia observed. For the anemia of chronic inflammation, the erythroid marrow also responds inadequately to stimulation, due in part to defective iron reutilization. As a result of the lack of adequate EPO stimulation, an examination of the peripheral blood smear will disclose only an occasional polychromatophilic (“shift”) reticulocyte. In cases of iron deficiency or marrow damage, appropriate elevations in endogenous EPO levels are typically found, and shift reticulocytes will be present on the blood smear.

1	AI—which encompasses inflammation, infection, tissue injury, and conditions (such as cancer) associated with the release of proinflammatory cytokines—is one of the most common forms of anemia seen clinically. It is the most important anemia in the differential diagnosis of iron deficiency, because many of the features of the anemia are brought about by inadequate iron delivery to the marrow, despite the presence of normal or increased iron stores. This is reflected by a low serum iron, increased red cell protoporphyrin, a hypoproliferative marrow, transferrin saturation in the range of 15–20%, and a normal or increased serum ferritin. The serum ferritin values are often the most distinguishing features between true iron-deficiency anemia and the iron-restricted erythropoiesis associated with inflammation. Typically, serum ferritin values increase threefold over basal levels 630 Neoplasms with angina, exercise intolerance, and shortness of breath. The eryth-Bacterial infections

1	with inflammation. Typically, serum ferritin values increase threefold over basal levels 630 Neoplasms with angina, exercise intolerance, and shortness of breath. The eryth-Bacterial infections ropoietic profile that distinguishes the anemia of inflammation from the other causes of hypoproliferative anemias is shown in Table 126-6.

1	Progressive CKD is usually associated with a moderate to severe hypoproliferative anemia; the level of the anemia corre-EPO Marrow lates with the stage of CKD. Red cells are typically normocytic and normochromic, and reticulocytes are decreased. The anemiaIron is primarily due to a failure of EPO production by the diseased kidney and a reduction in red cell survival. In certain forms of acuteInterferon ° renal failure, the correlation between the anemia and renal function is weaker. Patients with the hemolytic-uremic syndrome increase erythropoiesis in response to the hemolysis, despite renal failure requiring Rheumatoid arthritis dialysis. Polycystic kidney disease also shows a smaller degree of EPO FIGURE 126-4 Suppression of erythropoiesis by inflammatory deficiency for a given level of renal failure. By contrast, patients with cytokines. Through the release of tumor necrosis factor (TNF) and diabetes or myeloma have more severe EPO deficiency for a given level interferon γ

1	level of renal failure. By contrast, patients with cytokines. Through the release of tumor necrosis factor (TNF) and diabetes or myeloma have more severe EPO deficiency for a given level interferon γ (IFN-γ), neoplasms and bacterial infections suppress of renal failure. erythropoietin (EPO) production and the proliferation of erythroid Assessment of iron status provides information to distinguish the progenitors (erythroid burst-forming units and erythroid colony-anemia of CKD from the other forms of hypoproliferative anemia forming units [BFU/CFU-E]). The mediators in patients with vasculitis (Table 126-6) and to guide management. Patients with the anemia of and rheumatoid arthritis include interleukin 1 (IL-1) and IFN-γ. The red CKD usually present with normal serum iron, TIBC, and ferritin levarrows indicate sites of inflammatory cytokine inhibitory effects. RBC, els. However, those maintained on chronic hemodialysis may develop red blood cell. iron deficiency from blood loss

1	ferritin levarrows indicate sites of inflammatory cytokine inhibitory effects. RBC, els. However, those maintained on chronic hemodialysis may develop red blood cell. iron deficiency from blood loss through the dialysis procedure. Iron must be replenished in these patients to ensure an adequate response to EPO therapy (see below).

1	in the face of inflammation. These changes are due to the effects of inflammatory cytokines and hepcidin, the key iron regulatory hor-ANEMIA IN HYPOMETABOLIC STATES mone, acting at several levels of erythropoiesis (Fig. 126-4). Patients who are starving, particularly for protein, and those with a

1	Interleukin 1 (IL-1) directly decreases EPO production in response variety of endocrine disorders that produce lower metabolic rates, may to anemia. IL-1, acting through accessory cell release of interferon γ develop a mild to moderate hypoproliferative anemia. The release of (IFN-γ), suppresses the response of the erythroid marrow to EPO—an EPO from the kidney is sensitive to the need for O2, not just O2 levels. effect that can be overcome by EPO administration in vitro and in vivo. Thus, EPO production is triggered at lower levels of blood O2 content In addition, tumor necrosis factor (TNF), acting through the release in disease states (such as hypothyroidism and starvation) where meta-of IFN-γ by marrow stromal cells, also suppresses the response to bolic activity, and thus O2 demand, is decreased. EPO. Hepcidin, made by the liver, is increased in inflammation via an

1	Endocrine Deficiency States The difference in the levels of hemoglobinIL-6 mediated pathway, and acts to suppress iron absorption and iron between men and women is related to the effects of androgen andrelease from storage sites. The overall result is a chronic hypoproliferaestrogen on erythropoiesis. Testosterone and anabolic steroids augtive anemia with classic changes in iron metabolism. The anemia is furment erythropoiesis; castration and estrogen administration to malesther compounded by a mild to moderate shortening in red cell survival.

1	decrease erythropoiesis. Patients who are hypothyroid or have deficitsWith chronic inflammation, the primary disease will determine in pituitary hormones also may develop a mild anemia. Pathogenesisthe severity and characteristics of the anemia. For example, many may be complicated by other nutritional deficiencies because iron andpatients with cancer also have anemia that is typically normocytic and folic acid absorption can be affected by these disorders. Usually, cornormochromic. In contrast, patients with long-standing active rheurection of the hormone deficiency reverses the anemia.

1	matoid arthritis or chronic infections such as tuberculosis will have a Anemia may be more severe in Addison’s disease, depending on themicrocytic, hypochromic anemia. In both cases, the bone marrow is level of thyroid and androgen hormone dysfunction; however, anemiahypoproliferative, but the differences in red cell indices reflect differ-may be masked by decreases in plasma volume. Once such patients areences in the availability of iron for hemoglobin synthesis. Occasionally, given cortisol and volume replacement, the hemoglobin level may fallconditions associated with chronic inflammation are also associated rapidly. Mild anemia complicating hyperparathyroidism may be duewith chronic blood loss. Under these circumstances, a bone marrow to decreased EPO production as a consequence of the renal effects ofaspirate stained for iron may be necessary to rule out absolute iron hypercalcemia or to impaired proliferation of erythroid progenitors.

1	deficiency. However, the administration of iron in this case will correct the iron deficiency component of the anemia and leave the inflamma-Protein Starvation Decreased dietary intake of protein may lead to tory component unaffected. mild to moderate hypoproliferative anemia; this form of anemia may

1	The anemia associated with acute infection or inflammation is typi-be prevalent in the elderly. The anemia can be more severe in patients cally mild but becomes more pronounced over time. Acute infection with a greater degree of starvation. In marasmus, where patients are can produce a decrease in hemoglobin span. The fever and cytokines released Anemia Mild to severe Mild Mild to severe Mild exert a selective pressure against cells MCV (fL) 60–90 80–90 90 90 with more limited capacity to maintain Morphology Normo-microcytic Normocytic Normocytic Normocytic the red cell membrane. In most individu- als, the mild anemia is reasonably well tolerated, and symptoms, if present, are associated with the underlying disease. Occasionally, in patients with preexisting cardiac disease, moderate anemia (hemo-Iron stores 0 2–4+ 1–4+ Normal globin 10–11 g/dL) may be associated Abbreviations: MCV, mean corpuscular volume; SI, serum iron; TIBC, total iron-binding capacity.

1	both protein and calorie deficient, the release of EPO is impaired in proportion to the reduction in metabolic rate; however, the degree of Disorders of Hemoglobin anemia may be masked by volume depletion and becomes apparent Edward J. Benz, Jr. after refeeding. Deficiencies in other nutrients (iron, folate) may also complicate the clinical picture but may not be apparent at diagnosis. Changes in the erythrocyte indices on refeeding should prompt evaluation of iron, folate, and B12 status.

1	Anemia in Liver Disease A mild hypoproliferative anemia may develop in patients with chronic liver disease from nearly any cause. The peripheral blood smear may show spur cells and stomatocytes from the accumulation of excess cholesterol in the membrane from a deficiency of lecithin-cholesterol acyltransferase. Red cell survival is shortened, and the production of EPO is inadequate to compensate. In alcoholic liver disease, nutritional deficiencies are common and complicate the management. Folate deficiency from inadequate intake, as well as iron deficiency from blood loss and inadequate intake, can alter the red cell indices.

1	Many patients with hypoproliferative anemias experience recovery of normal hemoglobin levels when the underlying disease is appropriately treated. For those in whom such reversals are not possible— such as patients with end-stage kidney disease, cancer, and chronic inflammatory diseases—symptomatic anemia requires treatment. The two major forms of treatment are transfusions and EPO.

1	Thresholds for transfusion should be determined based on the patient’s symptoms. In general, patients without serious underlying cardiovascular or pulmonary disease can tolerate hemoglobin levels above 7–8 g/dL and do not require intervention until the hemoglobin falls below that level. Patients with more physiologic compromise may need to have their hemoglobin levels kept above 11 g/dL. Usually, a unit of packed red cells increases the hemoglobin level by 1 g/dL. Transfusions are associated with certain infectious risks (Chap. 138e), and chronic transfusions can produce iron overload. Importantly, the liberal use of blood has been associated with increased morbidity and mortality, particularly in the intensive care setting. Therefore, in the absence of documented tissue hypoxia, a conservative approach to the use of red cell transfusions is preferable.

1	EPO is particularly useful in anemias in which endogenous EPO levels are inappropriately low, such as CKD or AI. Iron status must be evaluated and iron replaced to obtain optimal effects from EPO. In patients with CKD, the usual dose of EPO is 50–150 U/kg three times a week intravenously. Hemoglobin levels of 10–12 g/dL are usually reached within 4–6 weeks if iron levels are adequate; 90% of these patients respond. Once a target hemoglobin level is achieved, the EPO dose can be decreased. A decrease in hemoglobin level occurring in the face of EPO therapy usually signifies the development of an infection or iron depletion. Aluminum toxicity and hyperparathyroidism can also compromise the response to EPO. When an infection intervenes, it is best to interrupt the EPO therapy and rely on transfusions to correct the anemia until the infection is adequately treated. The dose of EPO needed to correct chemotherapy-induced anemia in patients with cancer is higher, up to 300 U/kg three times a

1	to correct the anemia until the infection is adequately treated. The dose of EPO needed to correct chemotherapy-induced anemia in patients with cancer is higher, up to 300 U/kg three times a week, and only approximately 60% of patients respond. Because of evidence that there is an increased risk of thromboembolic complications and tumor progression with EPO administration, the risks and benefits of using EPO in such patients must be weighed carefully, and the target hemoglobin should be that necessary to avoid transfusions.

1	Longer-acting preparations of EPO can reduce the frequency of injections. Darbepoetin alfa, a molecularly modified EPO with additional carbohydrate, has a half-life in the circulation that is three to four times longer than recombinant human EPO, permitting weekly or every other week dosing. Hemoglobin is critical for normal oxygen delivery to tissues; it is also present in erythrocytes in such high concentrations that it can alter red cell shape, deformability, and viscosity. Hemoglobinopathies are disorders affecting the structure, function, or production of hemoglobin. These conditions are usually inherited and range in severity from asymptomatic laboratory abnormalities to death in utero. Different forms may present as hemolytic anemia, erythrocytosis, cyanosis, or vasoocclusive stigmata.

1	Different hemoglobins are produced during embryonic, fetal, and adult life (Fig. 127-1). Each consists of a tetramer of globin polypeptide chains: a pair of α-like chains 141 amino acids long and a pair of β-like chains 146 amino acids long. The major adult hemoglobin, HbA, has the structure α2β2. HbF (α2γ2) predominates during most of gestation, and HbA2 (α2δ2) is minor adult hemoglobin. Embryonic hemoglobins need not be considered here. Each globin chain enfolds a single heme moiety, consisting of a protoporphyrin IX ring complexed with a single iron atom in the ferrous state (Fe2+). Each heme moiety can bind a single oxygen molecule; a molecule of hemoglobin can transport up to four oxygen molecules.

1	The amino acid sequences of the various globins are highly homologous to one another. Each has a highly helical secondary structure. Their globular tertiary structures cause the exterior surfaces to be rich in polar (hydrophilic) amino acids that enhance solubility, and the interior to be lined with nonpolar groups, forming a hydrophobic pocket into which heme is inserted. The tetrameric quaternary structure of HbA contains two αβ dimers. Numerous tight interactions (i.e., α1β1 contacts) hold the α and β chains together. The complete tetramer is held together by interfaces (i.e., α1β2 contacts) between the α-like chain of one dimer and the non-α chain of the other dimer.

1	The hemoglobin tetramer is highly soluble, but individual globin chains are insoluble. Unpaired globin precipitates, forming inclusions that damage the cell and can trigger apoptosis. Normal globin chain synthesis is balanced so that each newly synthesized α or non-α globin chain will have an available partner with which to pair. Solubility and reversible oxygen binding are the key properties deranged in hemoglobinopathies. Both depend most on the hydrophilic surface amino acids, the hydrophobic amino acids lining the heme pocket, a key histidine in the F helix, and the amino acids forming the α1β1 and α1β2 contact points. Mutations in these strategic regions tend to be the ones that alter oxygen affinity or solubility. FIGURE 127-1 The globin genes. The α-like genes (α, ζ) are encoded on chromosome 16; the β-like genes (β, γ, δ, ε) are encoded on chromosome 11. The ζ and ε genes encode embryonic globins.

1	Disorders of Hemoglobin 632 FUNCTION OF HEMOGLOBIN To support oxygen transport, hemoglobin must bind O2 efficiently at the partial pressure of oxygen (Po2) of the alveolus, retain it in the circulation, and release it to tissues at the Po2 of tissue capillary beds. Oxygen acquisition and delivery over a relatively narrow range of oxygen tensions depend on a property inherent in the tetrameric arrangement of heme and globin subunits within the hemoglobin molecule called cooperativity or heme-heme interaction. At low oxygen tensions, the hemoglobin tetramer is fully deoxygenated (Fig. 127-2). Oxygen binding begins slowly as O2 tension rises. However, as soon as some oxygen has been bound by the tetramer, an abrupt increase occurs in the slope of the curve. Thus, hemoglobin molecules that have bound some oxygen develop a higher oxygen affinity, greatly accelerating their ability to combine with more oxygen. This S-shaped oxygen equilibrium curve (Fig. 127-2), along which substantial

1	have bound some oxygen develop a higher oxygen affinity, greatly accelerating their ability to combine with more oxygen. This S-shaped oxygen equilibrium curve (Fig. 127-2), along which substantial amounts of oxygen loading and unloading can occur over a narrow range of oxygen tensions, is physiologically more useful than the high-affinity hyperbolic curve of individual monomers. Oxygen affinity is modulated by several factors. The Bohr effect is the ability of hemoglobin to deliver more oxygen to tissues at low pH. It arises from the stabilizing action of protons on deoxyhemoglobin, which binds protons more readily than oxyhemoglobin because the latter is a weaker acid (Fig. 127-2). Thus, hemoglobin has a lower oxygen affinity at low pH. The major small molecule that alters oxygen affinity in humans is 2,3-bisphosphoglycerate (2,3-BPG; formerly 2,3DPG), which lowers oxygen affinity when bound to hemoglobin. HbA has a reasonably high affinity for 2,3-BPG. HbF does not bind 2,3BPG, so

1	in humans is 2,3-bisphosphoglycerate (2,3-BPG; formerly 2,3DPG), which lowers oxygen affinity when bound to hemoglobin. HbA has a reasonably high affinity for 2,3-BPG. HbF does not bind 2,3BPG, so it tends to have a higher oxygen affinity in vivo. Hemoglobin also binds nitric oxide reversibly; this interaction influences vascular tone, but its clinical relevance remains incompletely understood. Proper oxygen transport depends on the tetrameric structure of the proteins, the proper arrangement of hydrophilic and hydrophobic amino acids, and interaction with protons or 2,3-BPG.

1	Red cells first appearing at about 6 weeks after conception contain the embryonic hemoglobins Hb Portland (ζ2γ2), Hb Gower I (ζ2ε2), and Hb Gower II (α2ε2). At 10–11 weeks, fetal hemoglobin (HbF; α2γ2) becomes predominant. The switch to nearly exclusive synthesis of adult hemoglobin (HbA; α2β2) occurs at about 38 weeks (Fig. 127-1).

1	Fetuses and newborns therefore require α-globin but not β-globin for normal gestation. A major advance in understanding the HbF to HbA transition has been the demonstration that the transcription factor Bcl11a plays a pivotal role in its regulation. Small amounts of HbF are produced during postnatal life. A few red cell clones called F cells are progeny of a small pool of immature committed erythroid precursors (BFU-e) that retain the ability to produce HbF. Profound erythroid stresses, such as severe hemolytic anemias, bone marrow transplantation, or cancer chemotherapy, cause more of the F-potent BFU-e to be recruited. HbF levels thus tend to rise in some patients with sickle cell anemia or thalassemia. This phenomenon probably explains the ability of hydroxyurea to increase levels of HbF in adults. Agents such as butyrate and histone deacetylase inhibitors can also activate fetal globin genes partially after birth.

1	The human hemoglobins are encoded in two tightly linked gene clusters; the α-like globin genes are clustered on chromosome 16 and the β-like genes on chromosome 11 (Fig. 127-1). The α-like cluster consists of two α-globin genes and a single copy of the ζ gene. The non-α gene cluster consists of a single ε gene, the Gγ and Aγ fetal globin genes, and the adult δ and β genes.

1	Important regulatory sequences flank each gene. Immediately upstream are typical promoter elements needed for the assembly of the transcription initiation complex. Sequences in the 5′ flanking region of the γ and the β genes appear to be crucial for the correct developmental regulation of these genes, whereas elements that function like classic enhancers and silencers are in the 3′ flanking regions. The locus control region (LCR) elements located far upstream appear to control the overall level of expression of each cluster. These elements achieve their regulatory effects by interacting with transacting transcription factors. Some of these factors are ubiquitous (e.g., Sp1 and YY1), while others are more or less limited to erythroid cells or hematopoietic cells (e.g., GATA-1, NFE-2, and EKLF). The LCR controlling the α-globin gene cluster is modulated by a SWI/SNF-like protein called ATRX; this protein appears to influence chromatin remodeling and DNA methylation. The association of α

1	The LCR controlling the α-globin gene cluster is modulated by a SWI/SNF-like protein called ATRX; this protein appears to influence chromatin remodeling and DNA methylation. The association of α thalassemia with mental retardation and myelodysplasia in some families appears to be related to mutations in the ATRX pathway. This pathway also modulates genes specifically expressed during erythropoiesis, such

1	Percent Saturation of Hemoglobin Tissue PO (mmHg) 0 25 50 75 100 Oxyhemoglobin Oxyhemoglobin Deoxyhemoglobin Deoxyhemoglobin Salt bridges Oxygen HEME P50 pH 2,3-BPG less O2 delivered ˜˜˜˜°°°°2,3-BPG T° pH more O2 delivered 2,3-BPG T°°

1	FIGURE 127-2 Hemoglobin-oxygen dissociation curve. The hemoglobin tetramer can bind up to four molecules of oxygen in the iron-containing sites of the heme molecules. As oxygen is bound, 2,3-bisphosphoglycerate (2,3-BPG) and carbon dioxide (CO2) are expelled. Salt bridges are broken, and each of the globin molecules changes its conformation to facilitate oxygen binding. Oxygen release to the tissues is the reverse process, with salt bridges being formed and 2,3-BPG and CO2 bound. Deoxyhemoglobin does not bind oxygen efficiently until the cell returns to conditions of higher pH, the most important modulator of O2 affinity (Bohr effect). When acid is produced in the tissues, the dissociation curve shifts to the right, facilitating oxygen release and CO2 binding. Alkalosis has the opposite effect, reducing oxygen delivery.

1	as those that encode the enzymes for heme biosynthesis. Normal red blood cell (RBC) differentiation requires the coordinated expression of the globin genes with the genes responsible for heme and iron metabolism. RBC precursors contain a protein, α-hemoglobinstabilizing protein (AHSP), that enhances the folding and solubility of α globin, which is otherwise easily denatured, leading to insoluble precipitates. These precipitates play an important role in the thalassemia syndromes and certain unstable hemoglobin disorders. Polymorphic variation in the amounts and/or functional capacity of AHSP might explain some of the clinical variability seen in patients inheriting identical thalassemia mutations.

1	There are five major classes of hemoglobinopathies (Table 127-1). Structural hemoglobinopathies occur when mutations alter the amino acid sequence of a globin chain, altering the physiologic properties of the variant hemoglobins and producing the characteristic clinical abnormalities. The most clinically relevant variant hemoglobins polymerize abnormally, as in sickle cell anemia, or exhibit altered solubility or oxygen-binding affinity. Thalassemia syndromes arise from mutations that impair production or translation of globin mRNA, leading to deficient globin chain biosynthesis. Clinical abnormalities are attributable to the inadequate supply of hemoglobin and the imbalances in the production of individual globin chains, leading to premature destruction of erythroblasts and RBC. Thalassemic hemoglobin variants combine features of thalassemia (e.g., abnormal globin biosynthesis) and of structural hemoglobinopathies (e.g., an abnormal amino acid sequence). Hereditary persistence of

1	hemoglobin variants combine features of thalassemia (e.g., abnormal globin biosynthesis) and of structural hemoglobinopathies (e.g., an abnormal amino acid sequence). Hereditary persistence of fetal hemoglobin (HPFH) is characterized by synthesis of high levels of fetal hemoglobin in adult life. Acquired hemoglobinopathies include modifications of the hemoglobin molecule by toxins (e.g., acquired methemoglobinemia) and clonal abnormalities of hemoglobin synthesis (e.g., high levels of HbF production in preleukemia and α thalassemia in myeloproliferative disorders).

1	I. Structural hemoglobinopathies—hemoglobins with altered amino acid sequences that result in deranged function or altered physical or chemical properties A. Abnormal hemoglobin polymerization—HbS, hemoglobin sickling B. Altered O2 affinity 1. 2. Low affinity—cyanosis, pseudoanemia C. Hemoglobins that oxidize readily 1. Unstable hemoglobins—hemolytic anemia, jaundice 2. M hemoglobins—methemoglobinemia, cyanosis II. Thalassemias—defective biosynthesis of globin chains A. B. C. δβ, γδβ, αβ Thalassemias III. Thalassemic hemoglobin variants—structurally abnormal Hb associated with coinherited thalassemic phenotype A. B. C. IV. Hereditary persistence of fetal hemoglobin—persistence of high levels of HbF into adult life V. A. Methemoglobin due to toxic exposures B. Sulfhemoglobin due to toxic exposures C. D. E. Elevated HbF in states of erythroid stress and bone marrow dysplasia

1	V. A. Methemoglobin due to toxic exposures B. Sulfhemoglobin due to toxic exposures C. D. E. Elevated HbF in states of erythroid stress and bone marrow dysplasia Hemoglobinopathies are especially common in areas in which malaria is endemic. This clustering of hemoglobinopathies is assumed to reflect a selective survival advantage for the abnormal RBC, which presumably provides a less hospitable environment during the obligate RBC stages of the parasitic life cycle. Very young children with α thalassemia are more susceptible to infection with the nonlethal Plasmodium vivax. Thalassemia might then favor a natural protection against infection with the more lethal Plasmodium falciparum.

1	Thalassemias are the most common genetic disorders in the world, affecting nearly 200 million people worldwide. About 15% of African Americans are silent carriers for α thalassemia; α thalassemia trait (minor) occurs in 3% of African American and in 1–15% of persons of Mediterranean origin. β Thalassemia has a 10–15% incidence in individuals from the Mediterranean and Southeast Asia and 0.8% in African Americans. The number of severe cases of thalassemia in the United States is about 1000. Sickle cell disease is the most common structural hemoglobinopathy, occurring in heterozygous form in ~8% of African Americans and in homozygous form in 1 in 400. Between 2 and 3% of African Americans carry a hemoglobin C allele.

1	Hemoglobinopathies are autosomal codominant traits—thus, compound heterozygotes who inherit a different abnormal mutant allele from each parent exhibit composite features of each. For example, patients inheriting sickle β thalassemia exhibit features of β thalassemia and sickle cell anemia. The α chain is present in HbA, HbA2, and HbF; α-chain mutations thus cause abnormalities in all three. The α-globin hemoglobinopathies are symptomatic in utero and after birth because normal function of the α-globin gene is required throughout gestation and adult life. In contrast, infants with β-globin hemoglobinopathies tend to be asymptomatic until 3–9 months of age, when HbA has largely replaced HbF. Prevention or partial reversion of the switch should thus be an effective therapeutic strategy for β-chain hemoglobinopathies.

1	Electrophoretic techniques are still widely used for hemoglobin analysis. Electrophoresis at pH 8.6 on cellulose acetate membranes is especially simple, inexpensive, and reliable for initial screening. Agar gel electrophoresis at pH 6.1 in citrate buffer is often used as a complementary method because each method detects different variants. Some important variants are electrophoretically silent. These mutant hemoglobins can usually be characterized by more specialized techniques such as mass spectroscopy, which is rapidly replacing electrophoresis for initial analysis.

1	Quantitation of the hemoglobin profile is often desirable. HbA2 is frequently elevated in β thalassemia trait and depressed in iron deficiency. HbF is elevated in HPFH, some β thalassemia syndromes, and occasional periods of erythroid stress or marrow dysplasia. For characterization of sickle cell trait, sickle thalassemia syndromes, or HbSC disease, and for monitoring the progress of exchange transfusion therapy to lower the percentage of circulating HbS, quantitation of individual hemoglobins is also required. In most laboratories, quantitation is performed only if the test is specifically ordered. Complete characterization, including amino acid sequencing or gene cloning and sequencing, is readily available from several reference laboratories.

1	Because some variants can comigrate with HbA or HbS (sickle hemoglobin), electrophoretic assessment should always be regarded as incomplete unless functional assays for hemoglobin sickling, solubility, or oxygen affinity are also performed, as dictated by the clinical presentation. The best sickling assays involve measurement of the degree to which the hemoglobin sample becomes insoluble, or gelated, as it is deoxygenated (i.e., sickle solubility test). Unstable hemoglobins are detected by their precipitation in isopropanol or after heating to 50°C. High-O2 affinity and low-O2 affinity variants are detected Disorders of Hemoglobin ing blood viscosity. However, natural history and drug therapy trials suggest that an increase in the hematocrit and feedback inhibition of reticulocytosis might be beneficial, even at the expense of increased blood viscosity. The role of adhesive reticulocytes in vasoocclusion might account for these paradoxical effects.

1	Granulocytosis is common. The white count can fluctuate substantially and unpredictably during and between painful crises, infectious episodes, and other intercurrent illnesses. Vasoocclusion causes protean manifestations. Intermittent episodes of vasoocclusion in connective and musculoskeletal structures produce ischemia manifested by acute pain and tenderness, fever, tachycardia, and anxiety. These recurrent episodes, called painful crises, are the most common clinical manifestation. Their frequency and severity vary greatly. Pain can develop almost anywhere in the body and may last from a few hours to 2 weeks. Repeated crises requiring hospitalization (>3 episodes per year) correlate with reduced survival in adult life, suggesting that these episodes are associated with accumulation of chronic end-organ damage. Provocative factors include infection, fever, excessive exercise, anxiety, abrupt changes in temperature, hypoxia, or hypertonic dyes.

1	FIGURE 127-3 Pathophysiology of sickle cell crisis. Repeated microinfarction can destroy tissues having microvascular beds prone to sickling. Thus, splenic function is frequently lost within the first 18–36 months of life, causing susceptibility to infection,by quantitating the P50, the partial pressure of oxygen at which the particularly by pneumococci. Acute venous obstruction of the spleenhemoglobin sample becomes 50% saturated with oxygen. Direct tests (splenic sequestration crisis), a rare occurrence in early childhood, mayfor the percent carboxyhemoglobin and methemoglobin, using spec-require emergency transfusion and/or splenectomy to prevent traptrophotometric techniques, can readily be obtained from most clinical ping of the entire arterial output in the obstructed spleen. Occlusionlaboratories on an urgent basis.

1	of retinal vessels can produce hemorrhage, neovascularization, andLaboratory evaluation remains an adjunct, rather than the sole eventual detachments. Renal papillary necrosis invariably producesdiagnostic aid. Diagnosis is best established by recognition of a characisosthenuria. More widespread renal necrosis leads to renal failure in teristic history, physical findings, peripheral blood smear morphology, adults, a common late cause of death. Bone and joint ischemia can leadand abnormalities of the complete blood cell count (e.g., profound to aseptic necrosis, especially of the femoral or humeral heads; chronic microcytosis with minimal anemia in thalassemia trait).

1	arthropathy; and unusual susceptibility to osteomyelitis, which may be caused by organisms, such as Salmonella, rarely encountered in other settings. The hand-foot syndrome is caused by painful infarcts of the SICKLE CELL SYNDROMES digits and dactylitis. Stroke is especially common in children; a small The sickle cell syndromes are caused by a mutation in the β-globin subset tends to suffer repeated episodes. Stroke is less common in gene that changes the sixth amino acid from glutamic acid to adults and is often hemorrhagic. A particularly painful complication valine. HbS (α2β26 Glu→Val) polymerizes reversibly when deoxygenated in males is priapism, due to infarction of the penile venous outflow to form a gelatinous network of fibrous polymers that stiffen the RBC tracts; permanent impotence is a frequent consequence. Chronic lower membrane, increase viscosity, and cause dehydration due to potas-leg ulcers probably arise from ischemia and superinfection in the distal sium leakage and

1	is a frequent consequence. Chronic lower membrane, increase viscosity, and cause dehydration due to potas-leg ulcers probably arise from ischemia and superinfection in the distal sium leakage and calcium influx (Fig. 127-3). These changes also circulation. produce the sickle shape. Sickled cells lose the pliability needed to Acute chest syndrome is a distinctive manifestation character-traverse small capillaries. They possess altered “sticky” membranes ized by chest pain, tachypnea, fever, cough, and arterial oxygen that are abnormally adherent to the endothelium of small venules. desaturation. It can mimic pneumonia, pulmonary emboli, bone These abnormalities provoke unpredictable episodes of microvascular marrow infarction and embolism, myocardial ischemia, or in situ vasoocclusion and premature RBC destruction (hemolytic anemia). lung infarction. Acute chest syndrome is thought to reflect in situ Hemolysis occurs because the spleen destroys the abnormal RBC. The sickling within the

1	RBC destruction (hemolytic anemia). lung infarction. Acute chest syndrome is thought to reflect in situ Hemolysis occurs because the spleen destroys the abnormal RBC. The sickling within the lung, producing pain and temporary pulmonary rigid adherent cells clog small capillaries and venules, causing tissue dysfunction. Often it is difficult or impossible to distinguish among ischemia, acute pain, and gradual end-organ damage. This venooc-other possibilities. Pulmonary infarction and pneumonia are the clusive component usually dominates the clinical course. Prominent most frequent underlying or concomitant conditions in patients manifestations include episodes of ischemic pain (i.e., painful crises) with this syndrome. Repeated episodes of acute chest pain correlate and ischemic malfunction or frank infarction in the spleen, central with reduced survival. Acutely, reduction in arterial oxygen saturanervous system, bones, joints, liver, kidneys, and lungs (Fig. 127-3). tion is

1	or frank infarction in the spleen, central with reduced survival. Acutely, reduction in arterial oxygen saturanervous system, bones, joints, liver, kidneys, and lungs (Fig. 127-3). tion is especially ominous because it promotes sickling on a massive scale. Chronic acute or subacute pulmonary crises lead to pulmonary

1	Arterial PO2 oxy Hbs (soluble) Capillary venous PO2 deoxy Hbs (polymerized) Stiff, viscous sickle cell Membrane changes Ca2+ influx, K leakage Capillary venule occlusion Shortened red cell survival (hemolytic anemia) Microinfarction Ischemic tissue pain Ischemic organ malfunction Autoinfarction of spleen Anemia, Jaundice, Gallstones, Leg ulcers hypertension and cor pulmonale, an increasingly common cause of death as patients survive longer. Considerable controversy exists about the possible role played by free plasma HbS in scavenging nitrogen dioxide (NO2), thus raising pulmonary vascular tone. Trials of sildenafil to restore NO2 levels were terminated because of adverse effects.

1	Chronic subacute central nervous system damage in the absence of an overt stroke is a distressingly common phenomenon beginning in early childhood. Modern functional imaging techniques have pinpointed circulatory dysfunction due to a likely CNS sickle vasculopathy; these changes correlate with an array of cognitive and behavioral abnormalities in children and young adults. It is important to be aware of these often subtle changes because they can complicate clinical management or be misinterpreted as “difficult patient” behaviors.

1	Sickle cell syndromes are remarkable for their clinical heterogeneity. Some patients remain virtually asymptomatic into or even through adult life, while others suffer repeated crises requiring hospitalization from early childhood. Patients with sickle thalassemia and sickle-HbE tend to have similar, slightly milder symptoms, perhaps because of the ameliorating effects of production of other hemoglobins within the RBC. Hemoglobin SC disease, one of the more common variants of sickle cell anemia, is frequently marked by lesser degrees of hemolytic anemia and a greater propensity for the development of retinopathy and aseptic necrosis of bones. In most respects, however, the clinical manifestations resemble sickle cell anemia. Some rare hemoglobin variants actually aggravate the sickling phenomenon.

1	The clinical variability in different patients inheriting the same disease-causing mutation (sickle hemoglobin) has made sickle cell disease the focus of efforts to identify modifying genetic polymorphisms in other genes that might account for the heterogeneity. The complexity of the data obtained thus far has dampened the expectation that genome-wide analysis will yield individualized profiles that predict a patient's clinical course. Nevertheless, a number of interesting patterns have emerged from these modifying gene analyses. For example, genes affecting the inflammatory response or cytokine expression appear to be modifying candidates. Genes that affect transcriptional regulation of lymphocytes may also be involved.

1	Clinical Manifestations of Sickle Cell Trait Sickle cell trait is often asymptomatic. Anemia and painful crises are rare. An uncommon but highly distinctive symptom is painless hematuria often occurring in adolescent males, probably due to papillary necrosis. Isosthenuria is a more common manifestation of the same process. Sloughing of papillae with urethral obstruction has been reported, as have isolated cases of massive sickling or sudden death due to exposure to high altitudes or extremes of exercise and dehydration. Avoidance of dehydration or extreme physical stress should be advised.

1	Diagnosis Sickle cell syndromes are suspected on the basis of hemolytic anemia, RBC morphology (Fig. 127-4), and intermittent episodes of ischemic pain. Diagnosis is confirmed by hemoglobin electrophoresis, mass spectroscopy, and the sickling tests already discussed. Thorough characterization of the exact hemoglobin profile of the patient is important, because sickle thalassemia and hemoglobin SC disease have distinct prognoses or clinical features. Diagnosis is usually established in childhood, but occasional patients, often with compound heterozygous states, do not develop symptoms until the onset of puberty, pregnancy, or early adult life. Genotyping of family members and potential parental partners is critical for genetic counseling. Details of the childhood history establish prognosis and need for aggressive or experimental therapies. Factors associated with increased morbidity and reduced survival include more than three crises requiring hospitalization per year, chronic

1	and need for aggressive or experimental therapies. Factors associated with increased morbidity and reduced survival include more than three crises requiring hospitalization per year, chronic neutrophilia, a history of splenic sequestration or hand-foot syndrome, and second episodes of acute chest syndrome. Patients with a history of cerebrovascular accidents are at higher risk for repeated episodes and require partial exchange transfusion and especially close monitoring using Doppler carotid flow measurements. Patients with severe or repeated episodes of acute chest syndrome may need lifelong transfusion support, using partial exchange transfusion, if possible.

1	FIGURE 127-4 Sickle cell anemia. The elongated and crescent-shaped red blood cells seen on this smear represent circulating irreversibly sickled cells. Target cells and a nucleated red blood cell are also seen. Patients with sickle cell syndromes require ongoing continuity of care. Familiarity with the pattern of symptoms provides the best safeguard against excessive use of the emergency room, hospitalization, and habituation to addictive narcotics. Additional preventive measures include regular slit-lamp examinations to monitor development of retinopathy; antibiotic prophylaxis appropriate for splenectomized patients during dental or other invasive procedures; and vigorous oral hydration during or in anticipation of periods of extreme exercise, exposure to heat or cold, emotional stress, or infection. Pneumococcal and Haemophilus influenzae vaccines are less effective in splenectomized individuals. Thus, patients with sickle cell anemia should be vaccinated early in life.

1	The management of an acute painful crisis includes vigorous hydration, thorough evaluation for underlying causes (such as infection), and aggressive analgesia administered by a standing order and/or patient-controlled analgesia (PCA) pump. Morphine (0.1–0.15 mg/kg every 3–4 h) should be used to control severe pain. Bone pain may respond as well to ketorolac (30–60 mg initial dose, then 15–30 mg every 6–8 h). Inhalation of nitrous oxide can provide short-term pain relief, but great care must be exercised to avoid hypoxia and respiratory depression. Nitrous oxide also elevates O2 affinity, reducing O2 delivery to tissues. Its use should be restricted to experts. Many crises can be managed at home with oral hydration and oral analgesia. Use of the emergency room should be reserved for especially severe symptoms or circumstances in which other processes, e.g., infection, are strongly suspected. Nasal oxygen should be used as appropriate to protect arterial saturation. Most crises resolve

1	severe symptoms or circumstances in which other processes, e.g., infection, are strongly suspected. Nasal oxygen should be used as appropriate to protect arterial saturation. Most crises resolve in 1–7 days. Use of blood transfusion should be reserved for extreme cases: transfusions do not shorten the duration of the crisis.

1	No tests are definitive to diagnose acute painful crisis. Critical to good management is an approach that recognizes that most patients reporting crisis symptoms do indeed have crisis or another significant medical problem. Diligent diagnostic evaluation for underlying causes is imperative, even though these are found infrequently. In adults, the possibility of aseptic necrosis or sickle arthropathy must be considered, especially if pain and immobility become repeated or chronic at a single site. Nonsteroidal anti-inflammatory agents are often effective for sickle cell arthropathy. Acute chest syndrome is a medical emergency that may require management in an intensive care unit. Hydration should be monitored carefully to avoid the development of pulmonary edema, and oxygen therapy should be especially vigorous for protection of

1	Disorders of Hemoglobin 636 arterial saturation. Diagnostic evaluation for pneumonia and pulmonary embolism should be especially thorough, since these may occur with atypical symptoms. Critical interventions are transfusion to maintain a hematocrit >30, and emergency exchange transfusion if arterial saturation drops to <90%. As patients with sickle cell syndrome increasingly survive into their fifth and sixth decades, end-stage renal failure and pulmonary hypertension are becoming increasingly prominent causes of end-stage morbidity. A sickle cell cardiomyopathy and/or premature coronary artery disease may compromise cardiac function in later years. Sickle cell patients have received kidney transplants, but they often experience an increase in the frequency and severity of crises, possibly due to increased infection as a consequence of immunosuppression. The most significant advance in the therapy of sickle cell anemia has been the introduction of hydroxyurea as a mainstay of therapy

1	due to increased infection as a consequence of immunosuppression. The most significant advance in the therapy of sickle cell anemia has been the introduction of hydroxyurea as a mainstay of therapy for patients with severe symptoms. Hydroxyurea (10–30 mg/kg per day) increases fetal hemoglobin and may also exert beneficial effects on RBC hydration, vascular wall adherence, and suppression of the granulocyte and reticulocyte counts; dosage is titrated to maintain a white cell count between 5000 and 8000/μL. White cells and reticulocytes may play a major role in the pathogenesis of sickle cell crisis, and their suppression may be an important side benefit of hydroxyurea therapy. Hydroxyurea should be considered in patients experiencing repeated episodes of acute chest syndrome or with more than three crises per year requiring hospitalization. The utility of this agent for reducing the incidence of other complications (priapism, retinopathy) is under evaluation, as are the long-term side

1	three crises per year requiring hospitalization. The utility of this agent for reducing the incidence of other complications (priapism, retinopathy) is under evaluation, as are the long-term side effects. To date, however, minimal risk of bone marrow dyscrasias or other neoplasms has been documented. Hydroxyurea offers broad benefits to most patients whose disease is severe enough to impair their functional status, and it may improve survival. HbF levels increase in most patients within a few months. The antitumor drug 5-azacytidine was the first agent found to elevate HbF. It never achieved widespread use because of concerns about acute toxicity and carcinogenesis. However, low doses of the related agent 5-deoxyazacytidine (decitabine) can elevate HbF with more acceptable toxicity. Bone marrow transplantation can provide definitive cures but is known to be effective and safe only in children. Clinical trials studying partially myeloablative conditioning regimens (“mini” transplants)

1	marrow transplantation can provide definitive cures but is known to be effective and safe only in children. Clinical trials studying partially myeloablative conditioning regimens (“mini” transplants) are likely to support more widespread use of this modality in older patients. Prognostic features justifying bone marrow transplant are the presence of repeated crises early in life, a high neutrophil count, or the development of hand-foot syndrome. Children at risk for stroke can now be identified through the use of Doppler ultrasound techniques. Prophylactic exchange transfusion appears to substantially reduce the risk of stroke in this population. Children who do suffer a cerebrovascular accident should be maintained for at least 3–5 years on a program of vigorous exchange transfusion, as the risk of second strokes is extremely high. Gene therapy for sickle cell anemia is being intensively pursued, but no safe measures are currently available. The development of newer methods of direct

1	risk of second strokes is extremely high. Gene therapy for sickle cell anemia is being intensively pursued, but no safe measures are currently available. The development of newer methods of direct gene correction in situ (e.g., zinc finger nucleases, or “CRISPR” [clustered regularly interspaced short palindromic repeats] technology) could well find clinical use in these patients. Experimental methods of derepressing HbF by interfering with Bcl11a are also being explored.

1	Amino acid substitutions that reduce solubility or increase susceptibility to oxidation result in unstable hemoglobins that precipitate, forming inclusion bodies injurious to the RBC membrane. Representative mutations are those that interfere with contact points between the α and β subunits (e.g., Hb Philly [β35Tyr→Phe]), alter the helical segments (e.g., Hb Genova [β28Leu→Pro]), or disrupt interactions of the hydrophobic pockets of the globin subunits with heme (e.g., Hb Köln [β98Val→Met]) (Table 127-3). The inclusions, called Heinz bodies, are clinically aSee text for details. detectable by staining with supravital dyes such as crystal violet. Removal of these inclusions by the spleen generates pitted, rigid cells that have shortened life spans, producing hemolytic anemia of variable severity, sometimes requiring chronic transfusion support. Splenectomy may be needed to correct the anemia. Leg ulcers and premature gallbladder disease due to bilirubin loading are frequent stigmata.

1	Unstable hemoglobins occur sporadically, often by spontaneous new mutations. Heterozygotes are often symptomatic because a significant Heinz body burden can develop even when the unstable variant accounts for only a portion of the total hemoglobin. Symptomatic unstable hemoglobins tend to be β-globin variants, because sporadic mutations affecting only one of the four α globins alleles would generate only 20–30% abnormal hemoglobin.

1	High-affinity hemoglobins (e.g., Hb Yakima [β99Asp→His]) bind oxygen more readily but deliver less O2 to tissues at normal capillary Po2 levels (Fig. 127-2). Mild tissue hypoxia ensues, stimulating RBC production and erythrocytosis (Table 127-3). In extreme cases, the hematocrits can rise to 60–65%, increasing blood viscosity and producing typical symptoms (headache, somnolence, or dizziness). Phlebotomy may be required. Typical mutations alter interactions within the heme pocket or disrupt the Bohr effect or salt-bond site. Mutations that impair the interaction of HbA with 2,3-BPG can increase O2 affinity because 2,3-BPG binding lowers O2 affinity.

1	Low-affinity hemoglobins (e.g., Hb Kansas [β102Asn→Lys]) bind sufficient oxygen in the lungs, despite their lower oxygen affinity, to achieve nearly full saturation. At capillary oxygen tensions, they lose sufficient amounts of oxygen to maintain homeostasis at a low hematocrit (Fig. 127-2) (pseudoanemia). Capillary hemoglobin desaturation can also be sufficient to produce clinically apparent cyanosis. Despite these findings, patients usually require no specific treatment. Methemoglobin is generated by oxidation of the heme iron moieties to the ferric state, causing a characteristic bluish-brown muddy color resembling cyanosis. Methemoglobin has such high oxygen affinity that virtually no oxygen is delivered. Levels >50–60% are often fatal.

1	Congenital methemoglobinemia arises from globin mutations that stabilize iron in the ferric state (e.g., HbM Iwata [α87His→Tyr], Table 127-3) or from mutations that impair the enzymes that reduce met-hemoglobin to hemoglobin (e.g., methemoglobin reductase, NADP diaphorase). Acquired methemoglobinemia is caused by toxins that oxidize heme iron, notably nitrate and nitrite-containing compounds, including drugs commonly used in cardiology and anesthesiology. DIAGNOSIS AND MANAGEMENT OF PATIENTS WITH UNSTABLE HEMOGLOBINS, HIGH-AFFINITY HEMOGLOBINS, AND METHEMOGLOBINEMIA

1	DIAGNOSIS AND MANAGEMENT OF PATIENTS WITH UNSTABLE HEMOGLOBINS, HIGH-AFFINITY HEMOGLOBINS, AND METHEMOGLOBINEMIA Unstable hemoglobin variants should be suspected in patients with nonimmune hemolytic anemia, jaundice, splenomegaly, or premature biliary tract disease. Severe hemolysis usually presents during infancy as neonatal jaundice or anemia. Milder cases may present in adult life with anemia or only as unexplained reticulocytosis, hepatosplenomegaly, premature biliary tract disease, or leg ulcers. Because spontaneous mutation is common, family history of anemia may be absent. The peripheral blood smear often shows anisocytosis, abundant cells with punctate inclusions, and irregular shapes (i.e., poikilocytosis).

1	The two best tests for diagnosing unstable hemoglobins are the Heinz body preparation and the isopropanol or heat stability test. Many unstable Hb variants are electrophoretically silent. A normal electrophoresis does not rule out the diagnosis. Mass spectroscopy or direct gene analysis will provide a definitive diagnosis. Severely affected patients may require transfusion support for the first 3 years of life, because splenectomy before age 3 is associated with a significantly higher immune deficit. Splenectomy is usually effective thereafter, but occasional patients may require lifelong transfusion support. After splenectomy, patients can develop cholelithiasis and leg ulcers, hypercoagulable states, and susceptibility to overwhelming sepsis. Splenectomy should thus be avoided or delayed unless it is the only alternative. Precipitation of unstable hemoglobins is aggravated by oxidative stress, e.g., infection and antimalarial drugs, which should be avoided where possible.

1	High-O2 affinity hemoglobin variants should be suspected in patients with erythrocytosis. The best test for confirmation is measurement of the P50. A high-O2 affinity hemoglobin causes a significant left shift (i.e., lower numeric value of the P50); confounding conditions, e.g., tobacco smoking or carbon monoxide exposure, can also lower the P 50.

1	High-affinity hemoglobins are often asymptomatic; rubor or plethora may be telltale signs. When the hematocrit approaches 60%, symptoms of high blood viscosity and sluggish flow (headache, lethargy, dizziness, etc.) may be present. These persons may benefit from judicious phlebotomy. Erythrocytosis represents an appropriate attempt to compensate for the impaired oxygen delivery by the abnormal variant. Overzealous phlebotomy may stimulate increased erythropoiesis or aggravate symptoms by thwarting this compensatory mechanism. The guiding principle of phlebotomy should be to improve oxygen delivery by reducing blood viscosity and increasing blood flow rather than restoration of a normal hematocrit. Phlebotomy-induced modest iron deficiency may aid in control.

1	Low-affinity hemoglobins should be considered in patients with cyanosis or a low hematocrit with no other reason apparent after thorough evaluation. The P50 test confirms the diagnosis. Counseling and reassurance are the interventions of choice. Methemoglobin should be suspected in patients with hypoxic symptoms who appear cyanotic but have a Pao2 sufficiently high that hemoglobin should be fully saturated with oxygen. A history of nitrite or other oxidant ingestions may not always be available; some exposures may be inapparent to the patient, and others may result from suicide attempts. The characteristic muddy appearance of freshly drawn blood can be a critical clue. The best diagnostic test is methemoglobin assay, which is usually available on an emergency basis.

1	Methemoglobinemia often causes symptoms of cerebral ischemia at levels >15%; levels >60% are usually lethal. Intravenous injection of 1 mg/kg of methylene blue is effective emergency therapy. Milder cases and follow-up of severe cases can be treated orally with methylene blue (60 mg three to four times each day) or ascorbic acid (300–600 mg/d). The thalassemia syndromes are inherited disorders of αor β-globin biosynthesis. The reduced supply of globin diminishes production of hemoglobin tetramers, causing hypochromia and microcytosis. Unbalanced accumulation of α and β subunits occurs because the synthesis of the unaffected globins proceeds at a normal rate. Unbalanced chain accumulation dominates the clinical phenotype. Clinical severity varies widely, depending on the degree to which the synthesis of the affected globin is impaired, altered synthesis of other globin chains, and coinheritance of other abnormal globin alleles.

1	Mutations causing thalassemia can affect any step in the pathway of globin gene expression: transcription, processing of the mRNA precursor, translation, and posttranslational metabolism of the β-globin polypeptide chain. The most common forms arise from mutations that derange splicing of the mRNA precursor or prematurely terminate translation of the mRNA. FIGURE 127-5 β Thalassemia intermedia. Microcytic and hypochromic red blood cells are seen that resemble the red blood cells of severe iron-deficiency anemia. Many elliptical and teardrop-shaped red blood cells are noted.

1	Hypochromia and microcytosis characterize all forms of β thalassemia because of the reduced amounts of hemoglobin tetramers (Fig. 127-5). In heterozygotes (β thalassemia trait), this is the only abnormality seen. Anemia is minimal. In more severe homozygous states, unbalanced αand β-globin accumulation causes accumulation of highly insoluble unpaired α chains. They form toxic inclusion bodies that kill developing erythroblasts in the marrow. Few of the proerythroblasts beginning erythroid maturation survive. The surviving RBCs bear a burden of inclusion bodies that are detected in the spleen, shortening the RBC life span and producing severe hemolytic anemia. The resulting profound anemia stimulates erythropoietin release and compensatory erythroid hyperplasia, but the marrow response is sabotaged by the ineffective erythropoiesis. Anemia persists. Erythroid hyperplasia can become exuberant and produce masses of extramedullary erythropoietic tissue in the liver and spleen.

1	Massive bone marrow expansion deranges growth and development. Children develop characteristic “chipmunk” facies due to maxillary marrow hyperplasia and frontal bossing. Thinning and pathologic fracture of long bones and vertebrae may occur due to cortical invasion by erythroid elements and profound growth retardation. Hemolytic anemia causes hepatosplenomegaly, leg ulcers, gallstones, and high-output congestive heart failure. The conscription of caloric resources to support erythropoiesis leads to inanition, susceptibility to infection, endocrine dysfunction, and in the most severe cases, death during the first decade of life. Chronic transfusions with RBCs improve oxygen delivery, suppress the excessive ineffective erythropoiesis, and prolong life, but the inevitable side effects, notably iron overload, often prove fatal by age 30 years.

1	Severity is highly variable. Known modulating factors are those that ameliorate the burden of unpaired α-globin inclusions. Alleles associated with milder synthetic defects and coinheritance of α thalassemia trait reduce clinical severity by reducing accumulation of excess α globin. HbF persists to various degrees in β thalassemias. γ-Globin gene chains can substitute for β chains, generating more hemoglobin and reducing the burden of α-globin inclusions. The terms β thalassemia major and β thalassemia intermedia are used to reflect the clinical heterogeneity. Patients with β thalassemia major require intensive transfusion support to survive. Patients with β thalassemia intermedia have a somewhat milder phenotype and can survive without transfusion. The terms β thalassemia minor and β thalassemia trait describe asymptomatic heterozygotes for β thalassemia.

1	The four classic α thalassemias, most common in Asians, are α thalassemia-2 trait, in which one of the four α-globin loci is deleted; Disorders of Hemoglobin Hemoglobin Hemoglobin H Hemoglobin Level, vive without chronic hypertransfusion. Condition A, % (β4), % g/L (g/dL) MCV, fL Management is particularly challenging

1	Normal Silent thalassemia: −α/αα Thalassemia trait: or −−/αα heterozygous α-thal-1a Hemoglobin H disease: 1/α-thal-2 Hydrops fetalis: −−/−− homozygous α-thal-1 90 because a number of factors can aggra 90 vate the anemia, including infection, 70–80 onset of puberty, and development of splenomegaly and hypersplenism. Some patients may eventually benefit from splenectomy. The expanded erythron 60–70 can cause absorption of excessive dietary iron and hemosiderosis, even without transfusion. Some patients eventually become transfusion dependent. β Thalassemia minor (i.e., thalas aWhen both α alleles on one chromosome are deleted, the locus is called α-thal-1; when only a single α allele on one chromo-semia trait) usually presents as profound some is deleted, the locus is called α-thal-2. b90–95% of the hemoglobin is hemoglobin Barts (tetramers of γ chains). microcytosis and hypochromia with tar α thalassemia-1 trait, with two deleted loci; HbH disease, with three loci deleted; and hydrops

1	of the hemoglobin is hemoglobin Barts (tetramers of γ chains). microcytosis and hypochromia with tar α thalassemia-1 trait, with two deleted loci; HbH disease, with three loci deleted; and hydrops fetalis with Hb Barts, with all four loci deleted (Table 127-4). Nondeletion forms of α thalassemia also exist.

1	α Thalassemia-2 trait is an asymptomatic, silent carrier state. α Thalassemia-1 trait resembles β thalassemia minor. Offspring doubly heterozygous for α thalassemia-2 and α thalassemia-1 exhibit a more severe phenotype called HbH disease. Heterozygosity for a deletion that removes both genes from the same chromosome (cis deletion) is common in Asians and in those from the Mediterranean region, as is homozygosity for α thalassemia-2 (trans deletion). Both produce asymptomatic hypochromia and microcytosis.

1	In HbH disease, HbA production is only 25–30% normal. Fetuses accumulate some unpaired γ chains (Hb Barts; γ-chain tetramers). In adults, unpaired β chains accumulate and are soluble enough to form β4 tetramers called HbH. HbH forms few inclusions in erythroblasts and precipitates in circulating RBC. Patients with HbH disease have thalassemia intermedia characterized by moderately severe hemolytic anemia but milder ineffective erythropoiesis. Survival into midadult life without transfusions is common.

1	The homozygous state for the α thalassemia-1 cis deletion (hydrops fetalis) causes total absence of α-globin synthesis. No physiologically useful hemoglobin is produced beyond the embryonic stage. Excess γ globin forms tetramers called Hb Barts (γ4), which has a very high oxygen affinity. It delivers almost no O2 to fetal tissues, causing tissue asphyxia, edema (hydrops fetalis), congestive heart failure, and death in utero. α Thalassemia-2 trait is common (15–20%) among people of African descent. The cis α thalassemia-1 deletion is almost never seen, however. Thus, α thalassemia-2 and the trans form of α thalassemia-1 are very common, but HbH disease and hydrops fetalis are rare. It has been known for some time that some patients with myelodysplasia or erythroleukemia produce RBC clones containing HbH. This phenomenon is due to mutations in the ATRX pathway that affect the LCR of the α-globin gene cluster.

1	The diagnosis of β-thalassemia major is readily made during childhood on the basis of severe anemia accompanied by the characteristic signs of massive ineffective erythropoiesis: hepatosplenomegaly, profound microcytosis, a characteristic blood smear (Fig. 127-5), and elevated levels of HbF, HbA2, or both. Many patients require chronic hypertransfusion therapy designed to maintain a hematocrit of at least 27–30% so that erythropoiesis is suppressed. Splenectomy is required if the annual transfusion requirement (volume of RBCs per kilogram of body weight per year) increases by >50%. Folic acid supplements may be useful. Vaccination with Pneumovax in anticipation of eventual splenectomy is advised, as is close monitoring for infection, leg ulcers, and biliary tract disease. Many patients develop endocrine deficiencies as a result of iron overload. Early endocrine evaluation is required for glucose intolerance, thyroid dysfunction, and delayed onset of puberty or secondary sexual

1	develop endocrine deficiencies as a result of iron overload. Early endocrine evaluation is required for glucose intolerance, thyroid dysfunction, and delayed onset of puberty or secondary sexual characteristics.

1	get cells, but only minimal or mild ane mia. The mean corpuscular volume is rarely >75 fL; the hematocrit is rarely <30–33%. Hemoglobin analysis classically reveals an elevated HbA2 (3.5–7.5%), but some forms are associated with normal HbA2 and/or elevated HbF. Genetic counseling and patient education are essential. Patients with β thalassemia trait should be warned that their blood picture resembles iron deficiency and can be misdiagnosed. They should eschew empirical use of iron, yet iron deficiency requiring replacement therapy can develop during pregnancy or from chronic bleeding.

1	Persons with α thalassemia trait may exhibit mild hypochromia and microcytosis usually without anemia. HbA2 and HbF levels are normal. Affected individuals usually require only genetic counseling. HbH disease resembles β thalassemia intermedia, with the added complication that the HbH molecule behaves like moderately unstable hemoglobin. Patients with HbH disease should undergo splenectomy if excessive anemia or a transfusion requirement develops. Oxidative drugs should be avoided. Iron overload leading to death can occur in more severely affected patients. Antenatal diagnosis of thalassemia syndromes is now widely available. DNA diagnosis is based on polymerase chain reaction (PCR) amplification of fetal DNA, obtained by amniocentesis or chorionic villus biopsy followed by hybridization to allele-specific oligonucleotide probes or direct DNA sequencing. Thalassemic structural variants are characterized by both defective synthesis and abnormal structure.

1	Thalassemic structural variants are characterized by both defective synthesis and abnormal structure. Hb Lepore [α2(δβ)2] arises by an unequal crossover and recombination event that fuses the proximal end of the δ-gene with the distal end of the closely linked β-gene. It is common in the Mediterranean basin. The resulting chromosome contains only the fused δβ gene. The Lepore (δβ) globin is synthesized poorly because the fused gene is under the control of the weak δ-globin promoter. Hb Lepore alleles have a phenotype like β thalassemia, except for the added presence of 2–20% Hb Lepore. Compound heterozygotes for Hb Lepore and a classic β thalassemia allele may also have severe thalassemia.

1	HbE (i.e., α2β226Glu→Lys) is extremely common in Cambodia, Thailand, and Vietnam. The gene has become far more prevalent in the United States as a result of immigration of Asian persons, especially in California, where HbE is the most common variant detected. HbE is mildly unstable but not enough to affect RBC life span significantly. Heterozygotes resemble individuals with a mild β-thalassemia trait. Homozygotes have somewhat more marked abnormalities but are asymptomatic. Compound heterozygotes for HbE and a β thalassemia gene can have β thalassemia intermedia or β thalassemia major, depending on the severity of the coinherited thalassemic gene.

1	HbE and a β thalassemia gene can have β thalassemia intermedia or β thalassemia major, depending on the severity of the coinherited thalassemic gene. The βE allele contains a single base change in codon 26 that causes the amino acid substitution. This mutation also activates a cryptic RNA splice site, generating a structurally abnormal globin mRNA that cannot be translated, from about 50% of the initial pre-mRNA molecules. The remaining 40–50% are normally spliced and generate functional mRNA that is translated into βE-globin because the mature mRNA carries the base change that alters codon 26. Genetic counseling of the persons at risk for HbE should focus especially on the interaction of HbE with β thalassemia, because HbE homozygosity is a condition associated with mildly asymptomatic microcytosis, hypochromia, and hemoglobin levels rarely <100 g/L (<10 g/dL).

1	HPFH is characterized by continued synthesis of high levels of HbF in adult life. No deleterious effects are apparent, even when all of the hemoglobin produced is HbF. These rare patients demonstrate convincingly that prevention or reversal of the fetal to adult hemoglobin switch would provide effective therapy for sickle cell anemia and β thalassemia. The two most important acquired hemoglobinopathies are carbon monoxide poisoning and methemoglobinemia (see above). Carbon monoxide has a higher affinity for hemoglobin than does oxygen; it can replace oxygen and diminish O2 delivery. Chronic elevation of carboxyhemoglobin levels to 10 or 15%, as occurs in smokers, can lead to secondary polycythemia. Carboxyhemoglobin is cherry red in color and masks the development of cyanosis usually associated with poor O2 delivery to tissues.

1	Abnormalities of hemoglobin biosynthesis have also been described in blood dyscrasias. In some patients with myelodysplasia, erythroleukemia, or myeloproliferative disorders, elevated HbF or a mild form of HbH disease may also be seen. The abnormalities are not severe enough to alter the course of the underlying disease.

1	Chronic blood transfusion can lead to bloodborne infection, alloimmunization, febrile reactions, and lethal iron overload (Chap. 138e). A unit of packed RBCs contains 250–300 mg iron (1 mg/mL). The iron assimilated by a single transfusion of 2 units of packed RBCs is thus equal to a 1to 2-year oral intake of iron. Iron accumulates in chronically transfused patients because no mechanisms exist for increasing iron excretion: an expanded erythron causes especially rapid development of iron overload because accelerated erythropoiesis promotes excessive absorption of dietary iron. Vitamin C should not be supplemented because it generates free radicals in iron excess states.

1	Patients who receive >100 units of packed RBCs usually develop hemosiderosis. The ferritin level rises, followed by early endocrine dysfunction (glucose intolerance and delayed puberty), cirrhosis, and cardiomyopathy. Liver biopsy shows both parenchymal and reticuloendothelial iron. The superconducting quantum-interference device (SQUID) is accurate at measuring hepatic iron but not widely available. Cardiac toxicity is often insidious. Early development of pericarditis is followed by dysrhythmia and pump failure. The onset of heart failure is ominous, often presaging death within a year (Chap. 428).

1	The decision to start long-term transfusion support should also prompt one to institute therapy with iron-chelating agents. Deferoxamine (Desferal) is for parenteral use. Its iron-binding kinetics require chronic slow infusion via a metering pump. The constant presence of the drug improves the efficiency of chelation and pro-639 tects tissues from occasional releases of the most toxic fraction of iron—low-molecular-weight iron—which may not be sequestered by protective proteins.

1	Deferoxamine is relatively nontoxic. Occasional cataracts, deafness, and local skin reactions, including urticaria, occur. Skin reactions can usually be managed with antihistamines. Negative iron balance can be achieved, even in the face of a high transfusion requirement, but this alone does not prevent long-term morbidity and mortality in chronically transfused patients. Irreversible end-organ deterioration develops at relatively modest levels of iron overload, even if symptoms do not appear for many years thereafter. To enjoy a significant survival advantage, chelation must begin before 5–8 years of age in β thalassemia major.

1	Deferasirox is an oral iron-chelating agent. Single daily doses of 20–30 mg/kg deferasirox produced reductions in liver iron concentration comparable to deferoxamine in long-term transfused adult and pediatric patients. Deferasirox produces some elevations in liver enzymes and slight but persistent increases in serum creatinine, without apparent clinical consequence. Other toxicities are similar to those of deferoxamine. Its toxicity profile is acceptable, although long-term effects are still being evaluated. BONE MARROW TRANSPLANTATION, GENE THERAPY, AND MANIPULATION OF HbF

1	BONE MARROW TRANSPLANTATION, GENE THERAPY, AND MANIPULATION OF HbF Bone marrow transplantation provides stem cells able to express normal hemoglobin; it has been used in a large number of patients with β thalassemia and a smaller number of patients with sickle cell anemia. Early in the course of disease, before end-organ damage occurs, transplantation is curative in 80–90% of patients. In highly experienced centers, the treatment-related mortality is <10%. Because survival into adult life is possible with conventional therapy, the decision to transplant is best made in consultation with specialized centers. Gene therapy of thalassemia and sickle cell disease has proved to be an elusive goal, but experimental advances are raising expectations.

1	Gene therapy of thalassemia and sickle cell disease has proved to be an elusive goal, but experimental advances are raising expectations. Reestablishing high levels of fetal hemoglobin synthesis should ameliorate the symptoms of β-chain hemoglobinopathies. Cytotoxic agents such as hydroxyurea and cytarabine promote high levels of HbF synthesis, probably by stimulating proliferation of the primitive HbF-producing progenitor cell population (i.e., F cell progenitors). Unfortunately, this regimen has not yet been effective in β thalassemia. Butyrates stimulate HbF production, but only transiently. Pulsed or intermittent administration has been found to sustain HbF induction in the majority of patients with sickle cell disease. It is unclear whether butyrates will have similar activity in patients with β thalassemia.

1	Patients with hemolytic anemias sometimes exhibit an alarming decline in hematocrit during and immediately after acute illnesses. Bone marrow suppression occurs in almost everyone during acute and chronic inflammatory illnesses. In patients with short RBC life spans, suppression can affect RBC counts more dramatically. These hypoplastic crises are usually transient and self-correcting before intervention is required.

1	Aplastic crisis refers to a profound cessation of erythroid activity in patients with chronic hemolytic anemias. It is associated with a rapidly falling hematocrit. Episodes are usually self-limited. Aplastic crises are caused by infection with a particular strain of parvovirus, B19A. Children infected with this virus usually develop permanent immunity. Aplastic crises do not often recur and are rarely seen in adults. Management requires close monitoring of the hematocrit and reticulocyte count. If anemia becomes symptomatic, transfusion support is indicated. Most crises resolve spontaneously within 1–2 weeks. Disorders of Hemoglobin A. Victor Hoffbrand

1	Disorders of Hemoglobin A. Victor Hoffbrand The megaloblastic anemias are a group of disorders characterized by the presence of distinctive morphologic appearances of the developing red cells in the bone marrow. The marrow is usually hypercellular and the anemia is based on ineffective erythropoiesis. The cause is usually a deficiency of either cobalamin (vitamin B12) or folate, but megaloblastic anemia may occur because of genetic or acquired abnormalities that affect the metabolism of these vitamins or because of defects in DNA synthesis not related to cobalamin or folate (Table 128-1). Cobalamin and folate absorption and metabolism are described next, followed by the biochemical basis, clinical and laboratory features, causes, and treatment of megaloblastic anemia.

1	Cobalamin (vitamin B12) exists in a number of different chemical forms. All have a cobalt atom at the center of a corrin ring. In nature, the vitamin is mainly in the 2-deoxyadenosyl (ado) form, which is located in mitochondria. It is the cofactor for the enzyme methylmalonyl coenzyme A (CoA) mutase. The other major natural cobalamin is methylcobalamin, the form in human plasma and in cell cytoplasm. It is the cofactor for methionine synthase. There are also minor amounts of hydroxocobalamin to which methyland adocobalamin are converted rapidly by exposure to light.

1	Cobalamin is synthesized solely by microorganisms. Ruminants obtain cobalamin from the foregut, but the only source for humans is food of animal origin, e.g., meat, fish, and dairy products. Vegetables, fruits, and other foods of nonanimal origin are free from cobalamin unless they are contaminated by bacteria. A normal Western diet contains 5–30 μg of cobalamin daily. Adult daily losses (mainly in the urine and feces) are 1–3 μg (~0.1% of body stores), and because the body does not have the ability to degrade cobalamin, daily requirements are also about 1–3 μg. Body stores are of the order of 2–3 mg, sufficient for 3–4 years if supplies are completely cut off.

1	Two mechanisms exist for cobalamin absorption. One is passive, occurring equally through buccal, duodenal, and ileal mucosa; it is rapid but extremely inefficient, with <1% of an oral dose being absorbed by this process. The normal physiologic mechanism is active; it occurs through the ileum and is efficient for small (a few micro-grams) oral doses of cobalamin, and it is mediated by gastric intrinsic factor (IF). Dietary cobalamin is released from protein complexes by enzymes in the stomach, duodenum, and jejunum; it combines rapidly with a salivary glycoprotein that belongs to the family of cobalaminbinding proteins known as haptocorrins (HCs). In the intestine, the haptocorrin is digested by pancreatic trypsin and the cobalamin is transferred to IF. Cobalamin deficiency or abnormalities of cobalamin metabolism (see Tables 128-3, 128-4) Folate deficiency or abnormalities of folate metabolism (see Table 128-5) Therapy with antifolate drugs (e.g., methotrexate)

1	Folate deficiency or abnormalities of folate metabolism (see Table 128-5) Therapy with antifolate drugs (e.g., methotrexate) Independent of either cobalamin or folate deficiency and refractory to cobalamin and folate therapy: Some cases of acute myeloid leukemia, myelodysplasia Therapy with drugs interfering with synthesis of DNA (e.g., cytosine arabinoside, hydroxyurea, 6-mercaptopurine, azidothymidine [AZT]) Orotic aciduria (responds to uridine)

1	Therapy with drugs interfering with synthesis of DNA (e.g., cytosine arabinoside, hydroxyurea, 6-mercaptopurine, azidothymidine [AZT]) Orotic aciduria (responds to uridine) IF (gene at chromosome 11q13) is produced in the gastric parietal cells of the fundus and body of the stomach, and its secretion parallels that of hydrochloric acid. Normally, there is a vast excess of IF. The IF-cobalamin complex passes to the ileum, where IF attaches to a specific receptor (cubilin) on the microvillus membrane of the enterocytes. Cubilin also is present in yolk sac and renal proximal tubular epithelium. Cubilin appears to traffic by means of amnionless (AMN), an endocytic receptor protein that directs sublocalization and endocytosis of cubilin with its ligand IF-cobalamin complex. The cobalamin-IF complex enters the ileal cell, where IF is destroyed. After a delay of about 6 h, the cobalamin appears in portal blood attached to transcobalamin (TC) II.

1	Between 0.5 and 5 μg of cobalamin enter the bile each day. This binds to IF, and a major portion of biliary cobalamin normally is reabsorbed together with cobalamin derived from sloughed intestinal cells. Because of the appreciable amount of cobalamin undergoing enterohepatic circulation, cobalamin deficiency develops more rapidly in individuals who malabsorb cobalamin than it does in vegans, in whom reabsorption of biliary cobalamin is intact.

1	Two main cobalamin transport proteins exist in human plasma; they both bind cobalamin—one molecule for one molecule. One HC, also known as TC I, is closely related to other cobalamin-binding HCs in milk, gastric juice, bile, saliva, and other fluids. The gene TCNL is at chromosome 11q11-q12.3. These HCs differ from each other only in the carbohydrate moiety of the molecule. TC I is derived primarily from the specific granules in neutrophils. Normally, it is about two-thirds saturated with cobalamin, which it binds tightly. TC I does not enhance cobalamin entry into tissues. Glycoprotein receptors on liver cells are involved in the removal of TC I from plasma, and TC I may play a role in the transport of cobalamin analogues (which it binds more effectively than IF) to the liver for excretion in bile.

1	The other major cobalamin transport protein in plasma is transcobalamin, also known as TC II. The gene is on chromosome 22q11-q13.1. As for IF and HC, there are nine exons. The three proteins are likely to have a common ancestral origin. TC II is synthesized by liver and by other tissues, including macrophages, ileum, and vascular endothelium. It normally carries only 20–60 ng of cobalamin per liter of plasma and readily gives up cobalamin to marrow, placenta, and other tissues, which it enters by receptor-mediated endocytosis involving the TC II receptor and megalin (encoded by the LRP-2 gene). The TC II cobalamin is internalized by endocytosis via clathrin-coated pits; the complex is degraded, but the receptor probably is recycled to the cell membrane as is the case for transferrin. Export of “free” cobalamin is via the ATP-binding cassette drug transporter alias multidrug resistance protein 1.

1	Folic (pteroylglutamic) acid is a yellow, crystalline, water-soluble substance. It is the parent compound of a large family of natural folate compounds, which differ from it in three respects: (1) they are partly or completely reduced to dior tetrahydrofolate (THF) derivatives, (2) they usually contain a single carbon unit (Table 128-2), and (3) 70–90% of natural folates are folate-polyglutamates.

1	Most foods contain some folate. The highest concentrations are found in liver, yeast, spinach, other greens, and nuts (>100 μg/100 g). The total folate content of an average Western diet is ~250 μg daily, but the amount varies widely according to the type of food eaten and the method of cooking. Folate is easily destroyed by heating, particularly in large volumes of water. Total body folate in the adult is ~10 mg, with the liver containing the largest store. Daily adult requirements are ~100 μg, and so stores are sufficient for only 3–4 months in normal adults and severe folate deficiency may develop rapidly.

1	Folates are absorbed rapidly from the upper small intestine. The absorption of folate polyglutamates is less efficient than that of monoglutamates; on average, ~50% of food folate is absorbed. Polyglutamate forms are hydrolyzed to the monoglutamate derivatives either in the and dG(guanine)TP (purines), dT(thymine)TP and dC(cytosine) lumen of the intestine or within the mucosa. All dietary folates are con-TP (pyrimidines). In deficiencies of either folate or cobalamin, verted to 5-methylTHF (5-MTHF) within the small intestinal mucosa there is failure to convert deoxyuridine monophosphate (dUMP) to before entering portal plasma. The monoglutamates are actively trans-deoxythymidine monophosphate (dTMP), the precursor of dTTP ported across the enterocyte by a proton-coupled folate transporter (Fig 128-1). This is the case because folate is needed as the coenzyme (PCFT, SCL46A1). This is situated at the apical brush border and is 5,10-methylene-THF polyglutamate for conversion of dUMP to

1	(Fig 128-1). This is the case because folate is needed as the coenzyme (PCFT, SCL46A1). This is situated at the apical brush border and is 5,10-methylene-THF polyglutamate for conversion of dUMP to most active at pH 5.5, which is about the pH of the duodenal and dTMP; the availability of 5,10-methylene-THF is reduced in either jejunal surface. Genetic mutations of this protein underlie hereditary cobalamin or folate deficiency. An alternative theory for megaloblastic malabsorption of folate (see below). Pteroylglutamic acid at doses >400 anemia in cobalamin or folate deficiency is misincorporation of uracil μg is absorbed largely unchanged and converted to natural folates in into DNA because of a buildup of deoxyuridine triphosphate (dUTP) the liver. Lower doses are converted to 5-MTHF during absorption at the DNA replication fork as a consequence of the block in converthrough the intestine. sion of dUMP to dTMP.

1	641glycine. Methionine, the other product of the methionine synthase reaction, is the precursor for S-adenosylmethionine (SAM), the universal methyl donor involved in >100 methyltransferase TABLE 128-2 BIOCHEMICAL rEACTIONs Of fOLATE COENzYMEs Reaction Coenzyme Form of Folate Involved Single Carbon Unit Transferred Importance About 60–90 μg of folate enters the bile each day and is excreted into the small intestine. Loss of this folate, together with the folate of COBALAMIN-FOLATE RELATIONS sloughed intestinal cells, accelerates the speed with which folate defi-Folate is required for many reactions in mammalian tissues. Only two ciency develops in malabsorption conditions. reactions in the body are known to require cobalamin. Methylmalonyl

1	CoA isomerization requires adocobalamin, and the methylation of homocysteine to methionine requires both methylcobalamin and Folate is transported in plasma; about one-third is loosely bound to 5-MTHF (Fig. 128-1). This reaction is the first step in the pathway byalbumin, and two-thirds is unbound. In all body fluids (plasma, cerewhich 5-MTHF, which enters bone marrow and other cells from plasma,brospinal fluid, milk, bile), folate is largely, if not entirely, 5-MTHF is converted into all the intracellular folate coenzymes. The coenzymes in the monoglutamate form. Three types of folate-binding protein are all polyglutamated (the larger size aiding retention in the cell), but are involved. A reduced folate transporter (RFC, SLC19A1) is the the enzyme folate polyglutamate synthase can use only THF, not MTHF,major route of delivery of plasma folate (5-MTHF) to cells. Two folate as substrate. In cobalamin deficiency, MTHF accumulates in plasma,receptors, FR2 and FR3 embedded in the cell

1	only THF, not MTHF,major route of delivery of plasma folate (5-MTHF) to cells. Two folate as substrate. In cobalamin deficiency, MTHF accumulates in plasma,receptors, FR2 and FR3 embedded in the cell membrane by a glycosyl and intracellular folate concentrations fall due to failure of formation phosphatidylinositol anchor, transport folate into the cell via receptor-of THF, the substrate on which folate polyglutamates are built. This hasmediated endocytosis. The third protein, PCFT, transports folate at been termed THF starvation, or the methylfolate trap.

1	low pH from the vesicle to the cell cytoplasm. The reduced folate This theory explains the abnormalities of folate metabolism thattransporter also mediates uptake of methotrexate by cells. occur in cobalamin deficiency (high serum folate, low cell folate, positive purine precursor aminoimidazole carboxamide ribonucleotide [AICAR] excretion; Table 128-2) and also why the anemia of cobala-Folates (as the intracellular polyglutamate derivatives) act as coenzymes min deficiency responds to folic acid in large doses.

1	in the transfer of single-carbon units (Fig. 128-1 and Table 128-2). Two of these reactions are involved in purine synthesis and one in pyrimi- dine synthesis necessary for DNA and RNA replication. Folate is also a coenzyme for methionine synthesis, in which methylcobalamin is also Many symptomless patients are detected through the finding of a raised involved and in which THF is regenerated. THF is the acceptor of single mean corpuscular volume (MCV) on a routine blood count. The main carbon units newly entering the active pool via conversion of serine to clinical features in more severe cases are those of anemia. Anorexia is

1	Methylated product Folic acid Folic acid Deoxythymidine monophosphate Purines Serine Glycine S-Adenosylhomocysteine (SAH) 5-Methyl tetrahydrofolate 5, 10-Methylene tetrahydrofolate 5-Methyl tetrahydrofolate (monoglutamate) Plasma Cell Tetrahydrofolate Dihydrofolate S-Adenosylmethionine (SAM) Homocysteine Methionine Cystathionine Pyruvate GSH Cysteine THE METHYLATION CYCLE Substrate ATP (e.g., methylated lipids, myelin basic protein, DOPA, DNA) Methyltransferases Methionine synthase methylcobalamin Cystathionine synthase vitamin B6 5,10-Methylene-tetrahydrofolate reductase DHF reductase Polyglutamate synthase + glutamates 10-Formyl tetrahydrofolate DNA CYCLE (CELL REPLICATION) Formate Deoxyuridine monophosphate FIGURE 128-1 The role of folates in DNA synthesis and in formation of S-adenosylmethionine (SAM), which is involved in numerous meth-ylation reactions. DHF, dihydrofolate; GSH, glutathione. (Reprinted from AV Hoffbrand et al [eds]: Postgraduate Haematology, 5th ed. Oxford, UK,

1	(SAM), which is involved in numerous meth-ylation reactions. DHF, dihydrofolate; GSH, glutathione. (Reprinted from AV Hoffbrand et al [eds]: Postgraduate Haematology, 5th ed. Oxford, UK, Blackwell Publishing, 2005; with permission.) usually marked, and there may be weight loss, diarrhea, or constipation. Glossitis, angular cheilosis, a mild fever in more severely anemic patients, jaundice (unconjugated), and reversible melanin skin hyper-pigmentation also may occur with a deficiency of either folate or cobalamin. Thrombocytopenia sometimes leads to bruising, and this may be aggravated by vitamin C deficiency or alcohol in malnourished patients. The anemia and low leukocyte count may predispose to infections, particularly of the respiratory and urinary tracts. Cobalamin deficiency has also been associated with impaired bactericidal function of phagocytes.

1	GENERAL TISSUE EFFECTS OF COBALAMIN AND FOLATE DEFICIENCIES Epithelial Surfaces After the marrow, the next most frequently affected tissues are the epithelial cell surfaces of the mouth, stomach, and small intestine and the respiratory, urinary, and female genital tracts. The cells show macrocytosis, with increased numbers of multinucleate and dying cells. The deficiencies may cause cervical smear abnormalities. Complications of Pregnancy The gonads are also affected, and infertility is common in both men and women with either deficiency. Maternal folate deficiency has been implicated as a cause of prematurity, and both folate deficiency and cobalamin deficiency have been implicated in recurrent fetal loss and neural tube defects, as discussed below.

1	Neural Tube Defects Folic acid supplements at the time of conception and in the first 12 weeks of pregnancy reduce by ~70% the incidence of neural tube defects (NTDs) (anencephaly, meningomyelocele, encephalocele, and spina bifida) in the fetus. Most of this protective effect can be achieved by taking folic acid, 0.4 mg daily, at the time of conception. The incidence of cleft palate and harelip also can be reduced by prophylactic folic acid. There is no clear simple relationship between maternal folate status and these fetal abnormalities, although overall the lower the maternal folate, the greater the risk to the fetus. NTDs also can be caused by antifolate and antiepileptic drugs.

1	An underlying maternal folate metabolic abnormality has also been postulated. One abnormality has been identified: reduced activity of the enzyme 5,10-methylene-THF reductase (MTHFR) (Fig. 128-1) caused by a common C677T polymorphism in the MTHFR gene. In one study, the prevalence of this polymorphism was found to be higher than in controls in the parents of NTD fetuses and in the fetuses themselves: homozygosity for the TT mutation was found in 13% of cases compared with 5% of control subjects. The polymorphism codes for a thermolabile form of MTHFR. The homozygous state results in a lower mean serum and red cell folate level compared with control subjects, as well as significantly higher serum homocysteine levels. Tests for mutations in other enzymes possibly associated with NTDs, e.g., methionine synthase and serine–glycine hydroxymethylase, have been negative. Serum vitamin B12 levels are also lower in the sera of mothers of NTD infants than in controls. In addition, maternal TC

1	methionine synthase and serine–glycine hydroxymethylase, have been negative. Serum vitamin B12 levels are also lower in the sera of mothers of NTD infants than in controls. In addition, maternal TC II receptor polymorphisms are associated with increased risk of NTD births. There are, however, no studies showing dietary fortification with vitamin B12 reduces the incidence of NTDs.

1	Cardiovascular Disease Children with severe homocystinuria (blood levels ≥100 μmol/L) due to deficiency of one of three enzymes, methionine synthase, MTHFR, or cystathionine synthase (Fig. 128-1), have vascular disease, e.g., ischemic heart disease, cerebrovascular disease, or pulmonary embolus, as teenagers or in young adulthood. Lesser degrees of raised serum homocysteine and low levels of serum folate and homozygous inherited mutations of MTHFR have been found to be associated with cerebrovascular, peripheral vascular, and coronary heart disease and with deep vein thrombosis. Prospective randomized trials of lowering homocysteine levels with supplements of folic acid, vitamin B12, and vitamin B6 against placebo over a 5-year period in patients with vascular disease or diabetes have not, however, shown a reduction of first event fatal or nonfatal myocardial infarction, nor have these supplements reduced the risk of recurrent cardiovascular disease after an acute myocardial infarct.

1	however, shown a reduction of first event fatal or nonfatal myocardial infarction, nor have these supplements reduced the risk of recurrent cardiovascular disease after an acute myocardial infarct. Meta-analysis showed an 18% reduction in strokes but no significant prevention of death from any cause. Venous thrombosis has been reported to be more frequent in vitamin B12–deficient subjects than in controls. This was ascribed to raised plasma homocysteine levels in vitamin B12 deficiency.

1	Malignancy Prophylactic folic acid in pregnancy has been found in some but not all studies to reduce the subsequent incidence of acute lymphoblastic leukemia (ALL) in childhood. A significant negative association has also been found with the MTHFR C677T polymorphism and leukemias with mixed lineage leukemia (MLL) translocations, but a positive association with hyperdiploidy in infants with ALL or acute myeloid leukemia or with childhood ALL. A second polymorphism in the MTHFR gene, A1298C, is also strongly associated with hyperdiploid leukemia. There are various positive and negative associations between polymorphisms in folate-dependent enzymes and the incidence of adult ALL. The C677T polymorphism is thought to lead to increased thymidine pools and “better quality” of DNA synthesis by shunting one-carbon groups toward thymidine and purine synthesis. This may explain its reported association with a lower risk for colorectal cancer. Most but not all studies suggest that prophylactic

1	shunting one-carbon groups toward thymidine and purine synthesis. This may explain its reported association with a lower risk for colorectal cancer. Most but not all studies suggest that prophylactic folic acid also protects against colon adenomas. Other tumors that have been associated with folate polymorphisms or status include follicular lymphoma, breast cancer, and gastric cancer. A meta-analysis of 50,000 individuals given folic acid or placebo in cardiovascular or colon adenoma prevention trials found that folic acid supplementation did not substantially increase or decrease the incidence of site-specific cancer during the first 5 years of treatment. Because folic acid may “feed” tumors, it probably should be avoided in those with established tumors unless there is severe megaloblastic anemia due to folate deficiency.

1	Neurologic Manifestations Cobalamin deficiency may cause a bilateral peripheral neuropathy or degeneration (demyelination) of the posterior and pyramidal tracts of the spinal cord and, less frequently, optic atrophy or cerebral symptoms. The patient, more frequently male, presents with paresthesias, muscle weakness, or difficulty in walking and sometimes dementia, psychotic disturbances, or visual impairment. Long-term nutritional cobalamin deficiency in infancy leads to poor brain development and impaired intellectual development. Folate deficiency has been suggested to cause organic nervous disease, but this is uncertain, although methotrexate injected into the cerebrospinal fluid may cause brain or spinal cord damage.

1	An important clinical problem is the nonanemic patient with neurologic or psychiatric abnormalities and a low or borderline serum cobalamin level. In such patients, it is necessary to try to establish whether there is significant cobalamin deficiency, e.g., by careful examination of the blood film, tests for serum gastrin level and for antibodies to IF or parietal cells, along with serum methylmalonic acid (MMA) measurement if available. A trial of cobalamin therapy for at 643 least 3 months will usually also be needed to determine whether the symptoms improve. The biochemical basis for cobalamin neuropathy remains obscure. Its occurrence in the absence of methylmalonic aciduria in TC II deficiency suggests that the neuropathy is related to the defect in homocysteine-methionine conversion. Accumulation of S-adenosylhomocysteine in the brain, resulting in inhibition of transmethylation reactions, has been suggested.

1	Psychiatric disturbance is common in both folate and cobalamin deficiencies. This, like the neuropathy, has been attributed to a failure of the synthesis of SAM, which is needed in methylation of biogenic amines (e.g., dopamine) as well as that of proteins, phospholipids, and neurotransmitters in the brain (Fig. 128-1). Associations between lower serum folate or cobalamin levels and higher homocysteine levels and the development of decreased cognitive function and dementia in Alzheimer’s disease have been reported. A meta-analysis of randomized, placebo-controlled trials of homocysteine-lowering B-vitamin supplementation of individuals with and without cognitive impairment, however, showed that supplementation with vitamin B12, vitamin B6, and folic acid alone or in combination did not improve cognitive function. It is unknown whether prolonged treatment with these B vitamins can reduce the risk of dementia in later life.

1	Oval macrocytes, usually with considerable anisocytosis and poikilo cytosis, are the main feature (Fig. 128-2A). The MCV is usually >100 fL unless a cause of microcytosis (e.g., iron deficiency or thalassemia trait) is present. Some of the neutrophils are hypersegmented (more than five nuclear lobes). There may be leukopenia due to a reduction in granulocytes and lymphocytes, but this is usually >1.5 × 109/L; the platelet count may be moderately reduced, rarely to <40 × 109/L. The severity of all these changes parallels the degree of anemia. In a non-anemic patient, the presence of a few macrocytes and hypersegmented neutrophils in the peripheral blood may be the only indication of the underlying disorder.

1	In a severely anemic patient, the marrow is hypercellular with an accumulation of primitive cells due to selective death by apoptosis of more mature forms. The erythroblast nucleus maintains a primitive appearance despite maturation and hemoglobinization of the cytoplasm. The cells are larger than normoblasts, and an increased number of cells with eccentric lobulated nuclei or nuclear fragments may be present (Fig. 128-2B). Giant and abnormally shaped metamyelocytes and enlarged hyperpolyploid megakaryocytes are characteristic. In severe cases, the accumulation of primitive cells may mimic acute myeloid leukemia, whereas in less anemic patients, the changes in the marrow may be difficult to recognize. The terms intermediate, mild, and early have been used. The term megaloblastoid does not mean mildly megaloblastic. It is used to describe cells with both immature-appearing nuclei and defective hemoglobinization and is usually seen in myelodysplasia.

1	Bone marrow cells, transformed lymphocytes, and other proliferating cells in the body show a variety of changes, including random breaks, reduced contraction, spreading of the centromere, and exaggeration of secondary chromosomal constrictions and overprominent satellites. Similar abnormalities may be produced by antimetabolite drugs (e.g., cytosine arabinoside, hydroxyurea, and methotrexate) that interfere with either DNA replication or folate metabolism and that also cause megaloblastic appearances. There is an accumulation of unconjugated bilirubin in plasma due to the death of nucleated red cells in the marrow (ineffective erythropoiesis). Other evidence for this includes raised urine urobilinogen, reduced haptoglobins and positive urine hemosiderin, and a raised

1	FIGURE 128-2 A. The peripheral blood in severe megaloblastic anemia. B. The bone marrow in severe megaloblastic anemia. (Reprinted from AV Hoffbrand et al [eds]: Postgraduate Haematology, 5th ed. Oxford, UK, Blackwell Publishing, 2005; with permission.) serum lactate dehydrogenase. A weakly positive direct antiglobulin test due to complement can lead to a false diagnosis of autoimmune hemolytic anemia. Cobalamin deficiency is usually due to malabsorption. The only other cause is inadequate dietary intake.

1	Cobalamin deficiency is usually due to malabsorption. The only other cause is inadequate dietary intake. INADEQUATE DIETARY INTAKE Adults Dietary cobalamin deficiency arises in vegans who omit meat, fish, eggs, cheese, and other animal products from their diet. The largest group in the world consists of Hindus, and it is likely that many millions of Indians are at risk of deficiency of cobalamin on a nutritional basis. Subnormal serum cobalamin levels are found in up to 50% of randomly selected, young, adult Indian vegans, but the deficiency usually does not progress to megaloblastic anemia since the diet of most vegans is not totally lacking in cobalamin and the enterohepatic circulation of cobalamin is intact. Dietary cobalamin deficiency may also arise rarely in nonvegetarian individuals who exist on grossly inadequate diets because of poverty or psychiatric disturbance.

1	Infants Cobalamin deficiency has been described in infants born to severely cobalamin-deficient mothers. These infants develop megaloblastic anemia at about 3–6 months of age, presumably because they are born with low stores of cobalamin and because they are fed breast milk with low cobalamin content. The babies have also shown growth retardation, impaired psychomotor development, and other neurologic sequelae. See Tables 128-3 and 128-4.

1	See Tables 128-3 and 128-4. Pernicious Anemia Pernicious anemia (PA) may be defined as a severe lack of IF due to gastric atrophy. It is a common disease in north Europeans but occurs in all countries and ethnic groups. The overall incidence is about 120 per 100,000 population in the United Kingdom (UK). The ratio of incidence in men and women among whites is ~1:1.6, and the peak age of onset is 60 years, with only 10% of patients being <40 years of age. However, in some ethnic groups, notably black individuals and Latin Americans, the age at onset of PA is generally lower. The disease occurs more commonly than by chance

1	Nutritional Vegans Malabsorption Pernicious anemia Gastric causes Congenital absence of intrinsic factor or functional abnormality Total or partial gastrectomy Intestinal causes Intestinal stagnant loop syndrome: jejunal diver ticulosis, ileocolic fistula, anatomic blind loop, intestinal stricture, etc. Ileal resection and Crohn’s disease Selective malabsorption with proteinuria Tropical sprue Transcobalamin II deficiency Fish tapeworm Gastric causes Simple atrophic gastritis (food cobalamin malabsorption) Zollinger-Ellison syndrome Gastric bypass surgery Use of proton pump inhibitors Deficiencies of cobalamin, folate, protein, ?riboflavin, ?nicotinic acid

1	Therapy with colchicine, para-aminosalicylate, neomycin, slow-release potassium chloride, anticonvulsant drugs, metformin, phenformin, cytotoxic drugs Alcohol in close relatives and in persons with other organ-specific autoimmune diseases, e.g., thyroid diseases, vitiligo, hypoparathyroidism, and Addison’s disease. It is also associated with hypogammaglobulinemia, with premature graying or blue eyes, and persons of blood group A. An association with human leukocyte antigen (HLA) 3 has been reported in some but not all series and, in those with endocrine disease, with HLA-B8, -B12, and -BW15. Life expectancy is normal in women once regular treatment has begun. Men have a slightly subnormal life expectancy as a result of a higher incidence of carcinoma of the stomach than in control subjects. Gastric output of hydrochloric acid, pepsin, and IF is severely reduced. The serum gastrin level is raised, and serum pepsinogen I levels are low.

1	Gastric Biopsy A single endoscopic examination is recommended if PA is diagnosed. Gastric biopsy usually shows atrophy of all layers of the body and fundus, with loss of glandular elements, an absence of parietal and chief cells and replacement by mucous cells, a mixed inflammatory cell infiltrate, and perhaps intestinal metaplasia. The infiltrate of plasma cells and lymphocytes contains an excess of CD4 cells. These are directed against gastric H/K-ATPase. The antral mucosa is usually well preserved. Helicobacter pylori infection occurs infrequently in PA, but it has been suggested that H. pylori gastritis occurs at an early phase of atrophic gastritis and presents in younger patients as iron-deficiency anemia but in older patients as PA. H. pylori is suggested to stimulate an autoimmune process directed against parietal cells, with the H. pylori infection then being gradually replaced, in some individuals, by an autoimmune process.

1	Serum Antibodies Two types of IF immunoglobulin G antibody may be found in the sera of patients with PA. One, the “blocking,” or type I, antibody, prevents the combination of IF and cobalamin, whereas the “binding,” or type II, antibody prevents attachment of IF to ileal mucosa. Type I occurs in the sera of ~55% of patients, and type II in 35%. IF antibodies cross the placenta and may cause temporary IF deficiency in a newborn infant. Patients with PA also show cell-mediated immunity to IF. Type I antibody has been detected rarely in the sera of patients without PA but with thyrotoxicosis, myxedema, Hashimoto’s disease, or diabetes mellitus and in relatives of PA patients. IF antibodies also have been detected in gastric juice in ~80% of PA patients. These gastric antibodies may reduce absorption of dietary cobalamin by combining with small amounts of remaining IF.

1	Parietal cell antibody is present in the sera of almost 90% of adult patients with PA but is frequently present in other subjects. Thus, it occurs in as many as 16% of randomly selected female subjects age >60 years. The parietal cell antibody is directed against the α and β subunits of the gastric proton pump (H+,K+-ATPase). This usually occurs in older children and resembles PA of adults. Gastric atrophy, achlorhydria, and serum IF antibodies are all present, although parietal cell antibodies are usually absent. About one-half of these patients show an associated endocrinopathy such as autoimmune thyroiditis, Addison’s disease, or hypoparathyroidism; in some, mucocutaneous candidiasis occurs.

1	An affected child usually presents with megaloblastic anemia in the first to third year of life; a few have presented as late as the second decade. The child usually has no demonstrable IF but has a normal gastric mucosa and normal secretion of acid. The inheritance is autosomal recessive. Parietal cell and IF antibodies are absent. Variants have been described in which the child is born with IF that can be detected immunologically but is unstable or functionally inactive, unable to bind cobalamin or to facilitate its uptake by ileal receptors. After total gastrectomy, cobalamin deficiency is inevitable, and prophylactic cobalamin therapy should be commenced immediately after the operation. After partial gastrectomy, 10–15% of patients also develop this deficiency. The exact incidence and time of onset are most influenced by the size of the resection and the preexisting size of 645 cobalamin body stores.

1	Failure of release of cobalamin from binding proteins in food is believed to be responsible for this condition, which is more common in the elderly. It is associated with low serum cobalamin levels, with or without raised serum levels of MMA and homocysteine. Typically, these patients have normal cobalamin absorption, as measured with crystalline cobalamin, but show malabsorption when a modified test using food-bound cobalamin is used. The frequency of progression to severe cobalamin deficiency and the reasons for this progression are not clear. INTESTINAL CAUSES OF COBALAMIN MALABSORPTION Intestinal Stagnant Loop Syndrome Malabsorption of cobalamin occurs in a variety of intestinal lesions in which there is colonization of the upper small intestine by fecal organisms. This may occur in patients with jejunal diverticulosis, enteroanastomosis, or an intestinal stricture or fistula or with an anatomic blind loop due to Crohn’s disease, tuberculosis, or an operative procedure.

1	Ileal Resection Removal of ≥1.2 m of terminal ileum causes malabsorption of cobalamin. In some patients after ileal resection, particularly if the ileocecal valve is incompetent, colonic bacteria may contribute further to the onset of cobalamin deficiency. Selective Malabsorption of Cobalamin with Proteinuria (Imerslund’s

1	Syndrome; Imerslund-Gräsbeck Syndrome; Congenital Cobalamin Malabsorption; Autosomal Recessive Megaloblastic Anemia; MGA1) This autosomally recessive disease is the most common cause of megaloblastic anemia due to cobalamin deficiency in infancy in Western countries. More than 200 cases have been reported, with familial clusters in Finland, Norway, the Middle East, and North Africa. The patients secrete normal amounts of IF and gastric acid but are unable to absorb cobalamin. In Finland, impaired synthesis, processing, or ligand binding of cubilin due to inherited mutations is found. In Norway, mutation of the gene for AMN has been reported. Other tests of intestinal absorption are normal. Over 90% of these patients show nonspecific proteinuria, but renal function is otherwise normal and renal biopsy has not shown any consistent renal defect. A few have shown aminoaciduria and congenital renal abnormalities, such as duplication of the renal pelvis.

1	Tropical Sprue Nearly all patients with acute and subacute tropical sprue show malabsorption of cobalamin; this may persist as the principal abnormality in the chronic form of the disease, when the patient may present with megaloblastic anemia or neuropathy due to cobalamin deficiency. Absorption of cobalamin usually improves after antibiotic therapy and, in the early stages, folic acid therapy. Fish Tapeworm Infestation The fish tapeworm (Diphyllobothrium latum) lives in the small intestine of humans and accumulates cobalamin from food, rendering the cobalamin unavailable for absorption. Individuals acquire the worm by eating raw or partly cooked fish. Infestation is common around the lakes of Scandinavia, Germany, Japan, North America, and Russia. Megaloblastic anemia or cobalamin neuropathy occurs only in those with a heavy infestation.

1	Gluten-Induced Enteropathy Malabsorption of cobalamin occurs in ~30% of untreated patients (presumably those in whom the disease extends to the ileum). Cobalamin deficiency is not severe in these patients and is corrected with a gluten-free diet. Severe Chronic Pancreatitis In this condition, lack of trypsin is thought to cause dietary cobalamin attached to gastric non-IF (R) binder to be unavailable for absorption. It also has been proposed that in pancreatitis, the concentration of calcium ions in the ileum falls below the level needed to maintain normal cobalamin absorption. HIV Infection Serum cobalamin levels tend to fall in patients with HIV infection and are subnormal in 10–35% of those with AIDS. 646 Malabsorption of cobalamin not corrected by IF has been shown in some, but not all, patients with subnormal serum cobalamin levels. Cobalamin deficiency sufficiently severe to cause megaloblastic anemia or neuropathy is rare.

1	Zollinger-Ellison Syndrome Malabsorption of cobalamin has been reported in the Zollinger-Ellison syndrome. It is thought that there is a failure to release cobalamin from R-binding protein due to inactivation of pancreatic trypsin by high acidity, as well as interference with IF binding of cobalamin. Radiotherapy Both total-body irradiation and local radiotherapy to the ileum (e.g., as a complication of radiotherapy for carcinoma of the cervix) may cause malabsorption of cobalamin. Graft-versus-Host Disease This commonly affects the small intestine. Malabsorption of cobalamin due to abnormal gut flora, as well as damage to ileal mucosa, is common. Drugs The drugs that have been reported to cause malabsorption of cobalamin are listed in Table 105-4. However, megaloblastic anemia due to these drugs is rare.

1	Drugs The drugs that have been reported to cause malabsorption of cobalamin are listed in Table 105-4. However, megaloblastic anemia due to these drugs is rare. ABNORMALITIES OF COBALAMIN METABOLISM Congenital Transcobalamin II Deficiency or Abnormality Infants with TC II deficiency usually present with megaloblastic anemia within a few weeks of birth. Serum cobalamin and folate levels are normal, but the anemia responds to massive (e.g., 1 mg three times weekly) injections of cobalamin. Some cases show neurologic complications. The protein may be present but functionally inert. Genetic abnormalities found include mutations of an intra-exonic cryptic splice site, extensive deletion, single nucleotide deletion, nonsense mutation, and an RNA editing defect. Malabsorption of cobalamin occurs in all cases, and serum immunoglobulins are usually reduced. Failure to institute adequate cobalamin therapy or treatment with folic acid may lead to neurologic damage.

1	Congenital Methylmalonic Acidemia and Aciduria Infants with this abnormality are ill from birth with vomiting, failure to thrive, severe metabolic acidosis, ketosis, and mental retardation. Anemia, if present, is normocytic and normoblastic. The condition may be due to a functional defect in either mitochondrial methylmalonyl CoA mutase or its cofactor adocobalamin. Mutations in the methylmalonyl CoA mutase are not responsive, or only poorly responsive, to treatment with cobalamin. A proportion of infants with failure of adocobalamin synthesis respond to cobalamin in large doses. Some children have combined methylmalonic aciduria and homocystinuria due to defective formation of both cobalamin coenzymes. This usually presents in the first year of life with feeding difficulties, developmental delay, microcephaly, seizures, hypotonia, and megaloblastic anemia. Acquired Abnormality of Cobalamin Metabolism: Nitrous Oxide Inhalation

1	Acquired Abnormality of Cobalamin Metabolism: Nitrous Oxide Inhalation Nitrous oxide (N2O) irreversibly oxidizes methylcobalamin to an inactive precursor; this inactivates methionine synthase. Megaloblastic anemia has occurred in patients undergoing prolonged N2O anesthesia (e.g., in intensive care units). A neuropathy resembling cobalamin neuropathy has been described in dentists and anesthetists who are exposed repeatedly to N2O. Methylmalonic aciduria does not occur as adocobalamin is not inactivated by N2O.

1	Dietary folate deficiency is common. Indeed, in most patients with folate deficiency a nutritional element is present. Certain individuals are particularly prone to have diets containing inadequate amounts of folate (Table 128-5). In the United States and other countries where fortification of the diet with folic acid has been adopted, the prevalence of folate deficiency has dropped dramatically and is now almost restricted to high-risk groups with increased folate needs. Nutritional Particularly in: old age, infancy, poverty, alcoholism, chronic invalids, and the psychiatrically disturbed; may be associated with scurvy or kwashiorkor Major causes of deficiency Tropical sprue, gluten-induced enteropathy in children and adults, and in association with dermatitis herpetiformis, specific malabsorption of folate, intestinal megaloblastosis caused by severe cobalamin or folate deficiency Minor causes of deficiency

1	Minor causes of deficiency Extensive jejunal resection, Crohn’s disease, partial gastrectomy, congestive heart failure, Whipple’s disease, scleroderma, amyloid, diabetic enteropathy, systemic bacterial infection, lymphoma, sulfasalazine (Salazopyrin) Pregnancy and lactation, prematurity Hematologic diseases: chronic hemolytic anemias, sickle cell anemia, thalassemia major, myelofibrosis Malignant diseases: carcinoma, lymphoma, leukemia, myeloma Inflammatory diseases: tuberculosis, Crohn’s disease, psoriasis, exfoliative dermatitis, malaria Metabolic disease: homocystinuria Excess urinary loss: congestive heart failure, active liver disease Hemodialysis, peritoneal dialysis Anticonvulsant drugs (phenytoin, primidone, barbiturates), sulfasalazine Nitrofurantoin, tetracycline, antituberculosis (less well documented)

1	Hemodialysis, peritoneal dialysis Anticonvulsant drugs (phenytoin, primidone, barbiturates), sulfasalazine Nitrofurantoin, tetracycline, antituberculosis (less well documented) Liver diseases, alcoholism, intensive care units aIn severely folate-deficient patients with causes other than those listed under Dietary, poor dietary intake is often present. bDrugs inhibiting dihydrofolate reductase are discussed in the text. folate deficiency occurs in kwashiorkor and scurvy and in infants with repeated infections or those who are fed solely on goats’ milk, which has a low folate content.

1	Malabsorption of dietary folate occurs in tropical sprue and in gluten-induced enteropathy. In the rare congenital recessive syndrome of selective malabsorption of folate due to mutation of the proton-coupled folate transporter (PCFT), there is an associated defect of folate transport into the cerebrospinal fluid, and these patients show megaloblastic anemia, which responds to physiologic doses of folic acid given parenterally but not orally. They also show mental retardation, convulsions, and other central nervous system abnormalities. Minor degrees of malabsorption may also occur after jejunal resection or partial gastrectomy, in Crohn’s disease, and in systemic infections, but in these conditions, if severe deficiency occurs, it is usually largely due to poor nutrition. Malabsorption of folate has been described in patients receiving sulfasalazine (Salazopyrin), cholestyramine, and triamterene.

1	EXCESS UTILIZATION OR LOSS Pregnancy Folate requirements are increased by 200–300 μg to ~400 μg daily in a normal pregnancy, partly because of transfer of the vitamin to the fetus but mainly because of increased folate catabolism due to cleavage of folate coenzymes in rapidly proliferating tissues. Megaloblastic anemia due to this deficiency is prevented by prophylactic folic acid therapy. It occurred in 0.5% of pregnancies in the UK and other Western countries before prophylaxis with folic acid, but the incidence is much higher in countries where the general nutritional status is poor.

1	Prematurity A newborn infant, whether full term or premature, has higher serum and red cell folate concentrations than does an adult. However, a newborn infant’s demand for folate has been estimated to be up to 10 times that of adults on a weight basis, and the neonatal folate level falls rapidly to the lowest values at about 6 weeks of age. The falls are steepest and are liable to reach subnormal levels in premature babies, a number of whom develop megaloblastic anemia responsive to folic acid at about 4–6 weeks of age. This occurs particularly in the smallest babies (<1500 g birth weight) and those who have feeding difficulties or infections or have undergone multiple exchange transfusions. In these babies, prophylactic folic acid should be given.

1	Hematologic Disorders Folate deficiency frequently occurs in chronic hemolytic anemia, particularly in sickle cell disease, autoimmune hemolytic anemia, and congenital spherocytosis. In these and other conditions of increased cell turnover (e.g., myelofibrosis, malignancies), folate deficiency arises because it is not completely reutilized after performing coenzyme functions. Inflammatory Conditions Chronic inflammatory diseases such as tuberculosis, rheumatoid arthritis, Crohn’s disease, psoriasis, exfoliative dermatitis, bacterial endocarditis, and chronic bacterial infections cause deficiency by reducing the appetite and increasing the demand for folate. Systemic infections also may cause malabsorption of folate. Severe deficiency is virtually confined to the patients with the most active disease and the poorest diet.

1	Homocystinuria This is a rare metabolic defect in the conversion of homocysteine to cystathionine. Folate deficiency occurring in most of these patients may be due to excessive utilization because of compensatory increased conversion of homocysteine to methionine. Long-Term Dialysis Because folate is only loosely bound to plasma proteins, it is easily removed from plasma by dialysis. In patients with anorexia, vomiting, infections, and hemolysis, folate stores are particularly likely to become depleted. Routine folate prophylaxis is now given. Congestive Heart Failure, Liver Disease Excess urinary folate losses of >100 μg per day may occur in some of these patients. The explanation appears to be release of folate from damaged liver cells.

1	Congestive Heart Failure, Liver Disease Excess urinary folate losses of >100 μg per day may occur in some of these patients. The explanation appears to be release of folate from damaged liver cells. A large number of epileptics who are receiving long-term therapy with phenytoin or primidone, with or without barbiturates, develop low serum and red cell folate levels. The exact mechanism is unclear. Alcohol may also be a folate antagonist, as patients who are drinking spirits may develop megaloblastic anemia that will respond to normal quantities of dietary folate or to physiologic doses of folic acid only if alcohol is withdrawn. Macrocytosis of red cells is associated with chronic alcohol intake even when folate levels are normal. Inadequate folate intake is the major factor in the development of deficiency in spirit-drinking alcoholics. Beer is relatively folate-rich in some countries, depending on the technique used for brewing.

1	The drugs that inhibit DHF reductase include methotrexate, pyrimethamine, and trimethoprim. Methotrexate has the most powerful action against the human enzyme, whereas trimethoprim is most active against the bacterial enzyme and is likely to cause megaloblastic anemia only when used in conjunction with sulfamethoxazole in patients with preexisting folate or cobalamin deficiency. The activity of pyrimethamine is intermediate. The antidote to these drugs is folinic acid (5-formyl-THF). Some infants with congenital defects of folate enzymes (e.g., cyclohydrolase or methionine synthase) have had megaloblastic anemia. The diagnosis of cobalamin or folate deficiency has traditionally depended on the recognition of the relevant abnormalities in the peripheral blood and analysis of the blood levels of the vitamins.

1	COBALAMIN DEFICIENCY 647 Serum Cobalamin This is measured by an automated enzyme-linked immunosorbent assay (ELISA) or competitive-binding luminescence assay (CBLA). Normal serum levels range from 118–148 pmol/L (160–200 ng/L) to ~738 pmol/L (1000 ng/L). In patients with megaloblastic anemia due to cobalamin deficiency, the level is usually <74 pmol/L (100 ng/L). In general, the more severe the deficiency, the lower is the serum cobalamin level. In patients with spinal cord damage due to the deficiency, levels are very low even in the absence of anemia. Values between 74 and 148 pmol/L (100 and 200 ng/L) are regarded as borderline. They may occur, for instance, in pregnancy, in patients with megaloblastic anemia due to folate deficiency. They may also be due to heterozygous, homozygous, or compound heterozygous mutations of the gene TCN1 that codes for haptocorrin (transcobalamin I). There is no clinical or hematologic abnormality. The serum cobalamin level is sufficiently robust,

1	or compound heterozygous mutations of the gene TCN1 that codes for haptocorrin (transcobalamin I). There is no clinical or hematologic abnormality. The serum cobalamin level is sufficiently robust, cost-effective, and most convenient to rule out cobalamin deficiency in the vast majority of patients suspected of having this problem. However, problems have arisen with commercial

1	CBLA assays involving intrinsic factor in PA patients with intrinsic antibodies in serum. These antibodies may cause false normal serum vitamin B12 levels in up to 50% of cases tested. Where clinical indications of PA are strong, a normal serum vitamin B12 does not rule out the diagnosis. Serum MMA levels will be elevated in PA (see below).

1	Serum Methylmalonate and Homocysteine In patients with cobalamin deficiency sufficient to cause anemia or neuropathy, the serum MMA level is raised. Sensitive methods for measuring MMA and homocysteine in serum have been introduced and recommended for the early diagnosis of cobalamin deficiency, even in the absence of hematologic abnormalities or subnormal levels of serum cobalamin. Serum MMA levels fluctuate, however, in patients with renal failure. Mildly elevated serum MMA and/or homocysteine levels occur in up to 30% of apparently healthy volunteers, with serum cobalamin levels up to 258 pmol/L (350 ng/L) and normal serum folate levels; 15% of elderly subjects, even with cobalamin levels >258 pmol/L (>350 ng/L), have this pattern of raised metabolite levels. These findings bring into question the exact cutoff points for normal MMA and homocysteine levels. It is also unclear at present whether these mildly raised metabolite levels have clinical consequences.

1	Serum homocysteine is raised in both early cobalamin and folate deficiency but may be raised in other conditions, e.g., chronic renal disease, alcoholism, smoking, pyridoxine deficiency, hypothyroidism, and therapy with steroids, cyclosporine, and other drugs. Levels are also higher in serum than in plasma, in men than in premenopausal women, in women taking hormone replacement therapy or in oral contraceptive users, and in elderly persons and patients with several inborn errors of metabolism affecting enzymes in trans-sulfuration pathways of homocysteine metabolism. Thus, homocysteine levels must be carefully interpreted for diagnosis of cobalamin or folate deficiency.

1	Tests for the Cause of Cobalamin Deficiency Only vegans, strict vegetarians, or people living on a totally inadequate diet will become vitamin deficient because of inadequate intake. Studies of cobalamin absorption once were widely used, but difficulty in obtaining radioactive cobalamin and ensuring that IF preparations are free of viruses has made these tests obsolete. Tests to diagnose PA include serum gastrin, which is raised; serum pepsinogen I, which is low in PA (90–92%) but also in other conditions; and gastric endoscopy. Tests for IF and parietal cell antibodies are also used, as well as tests for individual intestinal diseases.

1	FOLATE DEFICIENCY Serum Folate This is also measured by an ELISA technique. In most laboratories, the normal range is from 11 nmol/L (2 μg/L) to ~82 nmol/L (15 μg/L). The serum folate level is low in all folate-deficient patients. It also reflects recent diet. Because of this, serum folate may be low before there is hematologic or biochemical evidence of deficiency. Serum folate rises in severe cobalamin deficiency because of the block in conversion of MTHF to THF inside cells; raised levels have also 648 been reported in the intestinal stagnant loop syndrome due to absorption of bacterially synthesized folate.

1	Red Cell Folate The red cell folate assay is a valuable test of body folate stores. It is less affected than the serum assay by recent diet and traces of hemolysis. In normal adults, concentrations range from 880–3520 μmol/L (160–640 μg/L) of packed red cells. Subnormal levels occur in patients with megaloblastic anemia due to folate deficiency but also in nearly two-thirds of patients with severe cobalamin deficiency. False-normal results may occur if a folate-deficient patient has received a recent blood transfusion or if a patient has a raised reticulocyte count. Serum homocysteine assay is discussed earlier. Tests for the Cause of Folate Deficiency The diet history is important. Tests for transglutaminase antibodies are performed to confirm or exclude celiac disease. If positive, duodenal biopsy is needed. An underlying disease causing increased folate breakdown should also be excluded.

1	It is usually possible to establish which of the two deficiencies, folate or cobalamin, is the cause of the anemia and to treat only with the appropriate vitamin. In patients who enter the hospital severely ill, however, it may be necessary to treat with both vitamins in large doses once blood samples have been taken for cobalamin and folate assays and a bone marrow biopsy has been performed (if deemed necessary). Transfusion is usually unnecessary and inadvisable. If it is essential, packed red cells should be given slowly, one or two units only, with the usual treatment for heart failure if present. Potassium supplements have been recommended to obviate the danger of the hypokalemia but are not necessary. Occasionally, an excessive rise in platelets occurs after 1–2 weeks of therapy. Antiplatelet therapy, e.g., aspirin, should be considered if the platelet count rises to >800 × 109/L.

1	It is usually necessary to treat patients who have developed cobalamin deficiency with lifelong regular cobalamin injections. In the UK, the form used is hydroxocobalamin; in the United States, cyanocobalamin. In a few instances, the underlying cause of cobalamin deficiency can be permanently corrected, e.g., fish tapeworm, tropical sprue, or an intestinal stagnant loop that is amenable to surgery. The indications for starting cobalamin therapy are a well-documented megaloblastic anemia or other hematologic abnormalities and neuropathy due to the deficiency. Patients with borderline serum cobalamin levels but no hematologic or other abnormality may be followed to make sure that the cobalamin deficiency does not progress (see below). If malabsorption of cobalamin or rises in serum MMA levels have been demonstrated, however, these patients also should be given regular maintenance cobalamin therapy. Cobalamin should be given routinely to all patients who have had a total gastrectomy or

1	have been demonstrated, however, these patients also should be given regular maintenance cobalamin therapy. Cobalamin should be given routinely to all patients who have had a total gastrectomy or ileal resection. Patients who have undergone gastric reduction for control of obesity or who are receiving longterm treatment with proton pump inhibitors should be screened and, if necessary, given cobalamin replacement.

1	Replenishment of body stores should be complete with six 1000-μg IM injections of hydroxocobalamin given at 3to 7-day intervals. More frequent doses are usually used in patients with cobalamin neuropathy, but there is no evidence that they produce a better response. Allergic reactions are rare and may require desensitization or antihistamine or glucocorticoid cover. For maintenance therapy, 1000 μg hydroxocobalamin IM once every 3 months is satisfactory. Because of the poorer retention of cyanocobalamin, protocols generally use higher and more frequent doses, e.g., 1000 μg IM, monthly, for maintenance treatment.

1	Because a small fraction of cobalamin can be absorbed passively through mucous membranes even when there is complete failure of physiologic IF-dependent absorption, large daily oral doses (1000– 2000 μg) of cyanocobalamin have been used in PA for replacement and maintenance of normal cobalamin status in, e.g., food malabsorption of cobalamin. Sublingual therapy has also been proposed for those in whom injections are difficult because of a bleeding tendency and who may not tolerate oral therapy. If oral therapy is used, it is important to monitor compliance, particularly with elderly, forgetful patients. This author prefers parenteral therapy for initial treatment, particularly in severe anemia or if a neuropathy is present, and for maintenance.

1	For treatment of patients with subnormal serum vitamin B12 (B12) levels with a normal MCV and no hypersegmentation of neutrophils, a negative IF antibody test in the absence of tests of B12 absorption is problematic. Some (perhaps 15%) cases may be due to TC I (HC) deficiency. Homocysteine and/or MMA measurements may help, but in the absence of these tests and with otherwise normal gastrointestinal function, repeat serum B12 assay after 6–12 months may help one decide whether to start cobalamin therapy.

1	Vitamin B12 injections are used in a wide variety of diseases, often neurologic, despite normal serum B12 and folate levels and a normal blood count and in the absence of randomized, double-blind, controlled trials. These conditions include multiple sclerosis and chronic fatigue syndrome/myalgic encephalomyelitis (ME). It seems probable that any benefit is due to the placebo effect of a usually painless, pink injection. In ME, oral B12 therapy, despite providing equally large amounts of B12, has not been beneficial, supporting the view of the effect of the injections being placebo only.

1	Oral doses of 5–15 mg folic acid daily are satisfactory, as sufficient folate is absorbed from these extremely large doses even in patients with severe malabsorption. The length of time therapy must be continued depends on the underlying disease. It is customary to continue therapy for about 4 months, when all folate-deficient red cells will have been eliminated and replaced by new folate-replete populations. Before large doses of folic acid are given, cobalamin deficiency must be excluded and, if present, corrected; otherwise cobalamin neuropathy may develop despite a response of the anemia of cobalamin deficiency to folate therapy. Studies in the United States, however, suggest that there is no increase in the proportion of individuals with low serum cobalamin levels and no anemia since food fortification with folic acid, but it is unknown if there has been a change in incidence of cobalamin neuropathy.

1	Long-term folic acid therapy is required when the underlying cause of the deficiency cannot be corrected and the deficiency is likely to recur, e.g., in chronic dialysis or hemolytic anemias. It may also be necessary in gluten-induced enteropathy that does not respond to a gluten-free diet. Where mild but chronic folate deficiency occurs, it is preferable to encourage improvement in the diet after correcting the deficiency with a short course of folic acid. In any patient receiving long-term folic acid therapy, it is important to measure the serum cobalamin level at regular (e.g., once-yearly) intervals to exclude the coincidental development of cobalamin deficiency. Folinic Acid (5-Formyl-THF) This is a stable form of fully reduced folate. It is given orally or parenterally to overcome the toxic effects of methotrexate or other DHF reductase inhibitors, e.g., trimethoprim or cotrimoxazole.

1	Prophylactic folic acid is used in chronic dialysis patients and in parenteral feeds. Prophylactic folic acid has been used to reduce homocysteine levels to prevent cardiovascular disease and for cognitive function in the elderly, but there are no firm data to show any benefit.

1	Pregnancy In over 70 countries (but none in Europe), food is fortified with folic acid (in grain or flour) to reduce the risk of NTDs. Nevertheless, folic acid, 400 μg daily, should be given as a supplement before and throughout pregnancy to prevent megaloblastic anemia and reduce the incidence of NTDs, even in countries with fortification of the diet. The levels of fortification provide up to 400 μg daily on average in Chile, but in most countries, it is nearer to 200 μg, so periconceptual folic acid is still needed. Studies in early pregnancy show significant lack of compliance with the folic acid supplements, emphasizing the benefit of food fortification. Supplemental folic acid reduces the incidence of birth defects in babies born to diabetic mothers. In women who have had a previous fetus with an NTD, 5 mg daily is recommended when pregnancy is contemplated and throughout the subsequent pregnancy.

1	Infancy and Childhood The incidence of folate deficiency is so high in the smallest premature babies during the first 6 weeks of life that folic acid (e.g., 1 mg daily) should be given routinely to those weighing <1500 g at birth and to larger premature babies who require exchange transfusions or develop feeding difficulties, infections, or vomiting and diarrhea. The World Health Organization currently recommends routine supplementation with iron and folic acid in children in countries where iron deficiency is common and child mortality, largely due to infectious diseases, is high. However, some studies suggest that in areas where malaria rates are high, this approach may increase the incidence of severe illness and death. Even where malaria is rare, there appears to be no survival benefit.

1	This may occur with many antimetabolic drugs (e.g., hydroxyurea, cytosine arabinoside, 6-mercaptopurine) that inhibit DNA replication. Antiviral nucleoside analogues used in treatment of HIV infection may also cause macrocytosis and megaloblastic marrow changes. In the rare disease orotic aciduria, two consecutive enzymes in purine synthesis are defective. The condition responds to therapy with uridine, which bypasses the block. In thiamine-responsive megaloblastic anemia, there is a genetic defect in the high-affinity thiamine transport (SLC19A2) gene. This causes defective RNA ribose synthesis through impaired activity of transketolase, a thiamine-dependent enzyme in the pentose cycle. This leads to reduced nucleic acid production. It may be associated with diabetes mellitus and deafness and the presence of many ringed sideroblasts in the marrow. The explanation is unclear for megaloblastic changes in the marrow in some patients with acute myeloid leukemia and myelodysplasia. Lucio

1	and the presence of many ringed sideroblasts in the marrow. The explanation is unclear for megaloblastic changes in the marrow in some patients with acute myeloid leukemia and myelodysplasia. Lucio Luzzatto DEFINITIONS 129

1	Hemolytic Anemias and Anemia Due to Acute Blood Loss A finite life span is a distinct characteristic of red cells. Hence, a logical, time-honored classification of anemias is in three groups: (1) decreased production of red cells, (2) increased destruction of red cells, and (3) acute blood loss. Decreased production is covered in Chaps. 126, 128, and 130; increased destruction and acute blood loss are covered in this chapter. All patients who are anemic as a result of either increased destruc tion of red cells or acute blood loss have one important element in common: the anemia results from overconsumption of red cells from the peripheral blood, whereas the supply of cells from the bone marrow is normal (indeed, it is usually increased). On the other hand, these two groups differ in that physical loss of red cells from the bloodstream or from the body itself, as in acute hemorrhage, is fundamentally different from destruction of red cells within the body, as in hemolytic anemias.

1	aHereditary causes correlate with intracorpuscular defects, because these defects are due to inherited mutations; the one exception is PNH, because the defect is due to an acquired somatic mutation. Similarly, acquired causes correlate with extracorpuscular factors, because mostly these factors are exogenous; the one exception is familial hemolytic-uremic syndrome (HUS; often referred to as atypical HUS), because here an inherited abnormality allows complement activation to be excessive, with bouts of production of membrane attack complex capable of destroying normal red cells. Therefore, the clinical aspects and pathophysiology of anemia in these two groups of patients are quite different, and they will be considered separately.

1	Therefore, the clinical aspects and pathophysiology of anemia in these two groups of patients are quite different, and they will be considered separately. With respect to primary etiology, anemias due to increased destruction of red cells, which we know as hemolytic anemias (HAs), may be inherited or acquired; from a clinical point of view, they may be more acute or more chronic, and they may vary from mild to very severe; the site of hemolysis may be predominantly intravascular or extravascular. With respect to mechanisms, HAs may be due to intracorpuscular causes or to extracorpuscular causes (Table 129-1). But before reviewing the individual types of HA, it is appropriate to consider what they have in common.

1	The clinical presentation of a patient with anemia is greatly influenced in the first place by whether the onset is abrupt or gradual, and HAs are no exception. A patient with autoimmune HA or with favism may be a medical emergency, whereas a patient with mild hereditary spherocytosis or with cold agglutinin disease may be diagnosed after years. This is due in large measure to the remarkable ability of the body to adapt to anemia when it is slowly progressing (Chap. 77).

1	What differentiates HAs from other anemias is that the patient has signs and symptoms arising directly from hemolysis (Table 129-2). At the clinical level, the main sign is jaundice; in addition, the patient may report discoloration of the urine. In many cases of HA, the spleen is enlarged, because it is a preferential site of hemolysis; and in some cases, the liver may be enlarged as well. In all severe congenital forms of HA, there may also be skeletal changes due to overactivity of the bone marrow (although they are never as severe as they are in thalassemia). Abbreviations: LDH, lactate dehydrogenase; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume.

1	Hemolytic Anemias and Anemia Due to Acute Blood Loss 650 The laboratory features of HA are related to hemolysis per se and the erythropoietic response of the bone marrow. Hemolysis regularly produces an increase in unconjugated bilirubin and aspartate aminotransferase (AST) in the serum; urobilinogen will be increased in both urine and stool. If hemolysis is mainly intravascular, the telltale sign is hemoglobinuria (often associated with hemosiderinuria); in the serum, there is hemoglobin, lactate dehydrogenase (LDH) is increased, and haptoglobin is reduced. In contrast, the bilirubin level may be normal or only mildly elevated. The main sign of the erythropoietic response by the bone marrow is an increase in reticulocytes (a test all too often neglected in the initial workup of a patient with anemia). Usually the increase will be reflected in both the percentage of reticulocytes (the more commonly quoted figure) and the absolute reticulocyte count (the more definitive parameter). The

1	with anemia). Usually the increase will be reflected in both the percentage of reticulocytes (the more commonly quoted figure) and the absolute reticulocyte count (the more definitive parameter). The increased number of reticulocytes is associated with an increased mean corpuscular volume (MCV) in the blood count. On the blood smear, this is reflected in the presence of macrocytes; there is also polychromasia, and sometimes one sees nucleated red cells. In most cases, a bone marrow aspirate is not necessary in the diagnostic workup; if it is done, it will show erythroid hyperplasia. In practice, once an HA is suspected, specific tests will usually be required for a definitive diagnosis of a specific type of HA.

1	The mature red cell is the product of a developmental pathway that brings the phenomenon of differentiation to an extreme. An orderly sequence of events produces synchronous changes, whereby the gradual accumulation of a huge amount of hemoglobin in the cytoplasm (to a final level of 340 g/L, i.e., about 5 mM) goes hand in hand with the gradual loss of cellular organelles and of biosynthetic abilities. In the end, the erythroid cell undergoes a process that has features of apoptosis, including nuclear pyknosis and actual loss of the nucleus. However, the final result is more altruistic than suicidal; the cytoplasmic body, instead of disintegrating, is now able to provide oxygen to all cells in the human organism for some remaining 120 days of the red cell life span.

1	As a result of this unique process of differentiation and maturation, intermediary metabolism is drastically curtailed in mature red cells (Fig. 129-1); for instance, cytochrome-mediated oxidative phosphorylation has been lost with the loss of mitochondria (through a process of physiologic autophagy); therefore, there is no backup to anaerobic glycolysis, which in the red cell is the only provider of adenosine triphosphate (ATP). Also the capacity of making protein has been lost with the loss of ribosomes. This places the cell’s limited metabolic apparatus at risk, because if any protein component deteriorates, it cannot be replaced, as it would be in most other cells; and in fact the activity of most enzymes gradually decreases as red cells age. At the same time, during their long time in circulation, various red cell components inevitably accumulate damage; in senescent red cells, the membrane protein band 3 molecules (see below and Fig. 129-1), having bound hemichromes on their

1	in circulation, various red cell components inevitably accumulate damage; in senescent red cells, the membrane protein band 3 molecules (see below and Fig. 129-1), having bound hemichromes on their intracellular domains, tend to cluster. Now they bind anti–band 3 IgG antibodies (present in most people) and C3 complement fragments; thus they become opsonized and are eventually removed by phagocytosis in the reticuloendothelial system.

1	Another consequence of the relative simplicity of red cells is that they have a very limited range of ways to manifest distress under hardship; in essence, any sort of metabolic failure will eventually lead either to structural damage to the membrane or to failure of the cation pump. In either case, the life span of the red cell is reduced, which is the definition of a hemolytic disorder. If the rate of red cell destruction exceeds the capacity of the bone marrow to produce more red cells, the hemolytic disorder will manifest as HA.

1	Thus, the essential pathophysiologic process common to all HAs is an increased red cell turnover; and in many HAs, this is due at least in part to an acceleration of the senescence process described above. The gold standard for proving that the life span of red cells is reduced (compared to the normal value of about 120 days) is a red cell survival study, which can be carried out by labeling the red cells with 51Cr and measuring residual radioactivity over several days or weeks: however, this classic test is now available in very few centers, and it is rarely necessary.

1	glucose phosphate isomerase glucose hexokinase ATP ADP glucose-6-phosphate fructose-6-phosphate ATP ADP phosphofructokinase fructose-1, 6-diphosphate aldolase glyceraldehyde-3-phosphate NAD+ glyceraldehyde 3-phosphate dehydrogenase 1,3-bisphosphoglycerate ADP ATP phosphoglycerate kinase 3-phosphoglycerate 3-phosphoglycerate mutase 2-phosphoglycerate NADH GSH GSSG NADPHNADP+ 6-phosphogluconate 2,3-bisphosphoglycerate mutase 2,3-bisphosphoglycerate phosphatase reductase G6PD 2,3-bisphosphoglycerate

1	FIGURE 129-1 Red blood cell (RBC) metabolism. The Embden-Meyerhof pathway (glycolysis) generates ATP for energy and membrane maintenance. The generation of NADPH maintains hemoglobin in a reduced state. The hexose monophosphate shunt generates NADPH that is used to reduce glutathione, which protects the red cell against oxidant stress. Regulation of 2,3-bisphosphoglycerate levels is a critical determinant of oxygen affinity of hemoglobin. Enzyme deficiency states in order of prevalence: glucose 6-phosphate dehydrogenase (G6PD) > pyruvate kinase > glucose-6-phosphate isomerase > rare deficiencies of other enzymes in the pathway. The more common enzyme deficiencies are encircled.

1	If the hemolytic event is transient, it does not usually cause any longterm consequences, except for an increased requirement for erythropoietic factors, particularly folic acid. However, if hemolysis is recurrent or persistent, the increased bilirubin production favors the formation of gallstones. If a considerable proportion of hemolysis takes place in the spleen, as is often the case, splenomegaly may become increasingly a feature, and hypersplenism may develop, with consequent neutropenia and/or thrombocytopenia.

1	The increased red cell turnover also has metabolic consequences. In normal subjects, the iron from effete red cells is very efficiently recycled by the body; however, with chronic intravascular hemolysis, the persistent hemoglobinuria will cause considerable iron loss, needing replacement. With chronic extravascular hemolysis, the opposite problem, iron overload, is more common, especially if the patient needs frequent blood transfusions. Chronic iron overload will cause secondary hemochromatosis; this will cause damage particularly to the liver, eventually leading to cirrhosis, and to the heart muscle, eventually causing heart failure.

1	Compensated Hemolysis Versus Hemolytic Anemia Red cell destruction is a potent stimulus for erythropoiesis, which is mediated by erythropoietin (EPO) produced by the kidney. This mechanism is so effective that in many cases the increased output of red cells from the bone marrow can fully balance an increased destruction of red cells. In such cases, we say that hemolysis is compensated. The pathophysiology of compensated hemolysis is similar to what we have just described, except there is no anemia. This notion is important from the diagnostic point of view, because a patient with a hemolytic condition, even an inherited one, may present without anemia; and it is also important from the point of view of management, because compensated hemolysis may become “decompensated,” i.e., anemia may suddenly appear, in certain circumstances, for instance in pregnancy, folate deficiency, or renal failure interfering with adequate EPO production. Another general feature of chronic HAs is seen when

1	suddenly appear, in certain circumstances, for instance in pregnancy, folate deficiency, or renal failure interfering with adequate EPO production. Another general feature of chronic HAs is seen when any intercurrent condition, such as an acute infection, depresses erythropoiesis. When this happens, in view of the increased rate of red cell turnover, the effect will be predictably much more marked than in a person who does not have hemolysis. The most dramatic example is infection by parvovirus B19, which may cause a rather precipitous fall in hemoglobin—an occurrence sometimes referred to as aplastic crisis.

1	There are three essential components in the red cell: (1) hemoglobin, (2) the membrane-cytoskeleton complex, and (3) the metabolic machinery necessary to keep hemoglobin and the membrane-cytoskeleton complex in working order. Diseases caused by abnormalities of hemoglobin, or hemoglobinopathies, are covered in Chap. 127. Here we will deal with diseases of the other two components.

1	Hemolytic Anemias due to Abnormalities of the Membrane-Cytoskeleton Complex The detailed architecture of the red cell membrane is complex, but its basic design is relatively simple (Fig. 129-2). The lipid bilayer incorporates phospholipids and cholesterol, and it is spanned by a number of proteins that have their hydrophobic transmembrane domain(s) embedded in the membrane; most of these proteins also extend to both the outside (extracellular domains) and the inside of the cell (cytoplasmic domains). Other proteins are tethered to the membrane through a glycosylphosphatidylinositol (GPI) anchor; these have only an extracellular domain, and they include ion channels, receptors for complement components, and receptors for other ligands. The most abundant red cell membrane proteins are glycophorins and the so-called band 3, an anion transporter. The extracellular domains of many of these proteins are heavily glycosylated, and they carry antigenic 4.2 GPA determinants that correspond to

1	and the so-called band 3, an anion transporter. The extracellular domains of many of these proteins are heavily glycosylated, and they carry antigenic 4.2 GPA determinants that correspond to blood groups. Underneath the mem-651 brane, and tangential to it, is a network of other proteins that make up the cytoskeleton. The main cytoskeletal protein is spectrin, the basic unit of which is a dimer of α-spectrin and β-spectrin. The membrane is physically linked to the cytoskeleton by a third set of proteins (includ ing ankyrin and the so-called band 4.1 and band 4.2), which thus make these two structures intimately connected to each other.

1	The membrane-cytoskeleton complex is so integrated that, not surprisingly, an abnormality of almost any of its components will be disturbing or disruptive, causing structural failure, which results ultimately in hemolysis. These abnormalities are almost invariably inherited mutations; thus, diseases of the membrane-cytoskeleton complex belong to the category of inherited HAs. Before the red cells lyse, they often exhibit more or less specific morphologic changes that alter the normal biconcave disk shape. Thus, the majority of the dis eases in this group have been known for over a century as hereditary spherocytosis and hereditary elliptocytosis. Over the past 20 years, their tions can arise from mutations in several genes with considerable overlap (Fig. 129-3).

1	Hereditary spHerocytosis (Hs) This is a relatively common type of genetically determined HA, with an estimated frequency of at least 1 in 5000. Its identification is credited to Minkowksy and Chauffard, who, at the end of the nineteenth century, reported families who had the presence of numerous spherocytes in the peripheral blood (Fig 129-4A). In vitro studies revealed that the red cells were abnormally susceptible to lysis in hypotonic media; indeed, the presence of osmotic fragility became the main diagnostic test for HS. Today we know that HS, thus defined, is genetically heterogeneous; i.e., it can arise from a variety of mutations in one of several genes (Table 129-3). It has been also recognized that the inheritance of HS is not always autosomal dominant (with the patient being heterozygous); indeed, some of the most severe forms are instead autosomal recessive (with the patient being homozygous).

1	clinical presentation and diagnosis The spectrum of clinical severity of HS is broad. Severe cases may present in infancy with severe anemia, whereas mild cases may present in young adults or even later in life. The main clinical findings are jaundice, an enlarged spleen, and often gallstones; indeed, it may be the finding of gallstones in a young person that triggers diagnostic investigations. -Spectrin Dematin4.1R Hemolytic Anemias and Anemia Due to Acute Blood Loss

1	FIGURE 129-2 The red cell membrane. In this figure, one sees, within the lipid bilayer, several membrane proteins, of which band 3 (anion exchanger 1 [AE1]) is the most abundant; the α-β spectrin dimers that associate to form most of the cytoskeleton; and several proteins (e.g., ankyrin) that connect the membrane to the cytoskeleton. In addition, as examples of glycosylphosphatidylinositol (GPI)-linked proteins, one sees acetylcholinesterase (AChE) and the two complement-regulatory proteins CD59 and CD55. The (nonrealistic) shapes of the protein moieties of the GPI-linked proteins are meant to indicate that they can be very different from each other and that, unlike with the other membrane proteins shown, the entire polypeptide chain is extracellular. Branched lines symbolize carbohydrate moiety of proteins. The molecules are obviously not drawn to the same scale. Additional explanations can be found in the text. (From N Young et al: Clinical Hematology. Copyright Elsevier, 2006; with

1	moiety of proteins. The molecules are obviously not drawn to the same scale. Additional explanations can be found in the text. (From N Young et al: Clinical Hematology. Copyright Elsevier, 2006; with permission.)

1	FIGURE 129-3 Hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary stomatocytosis (HSt) are three morphologically distinct forms of congenital hemolytic anemia. It has emerged that each one can arise from mutation of one of several genes and that different mutations of the same gene can give one or another form. (See also Table 129-3.)

1	The variability in clinical manifestations that is observed among patients with HS is largely due to the different underlying molecular lesions (Table 129-3). Not only are mutations of several genes involved, but also individual mutations of the same gene can also give very different clinical manifestations. In milder cases, hemolysis is often compensated (see above), and this may cause variation in time even in the same patient, due to the fact that intercurrent conditions (e.g., pregnancy, infection) may cause decompensation. The anemia is usually normocytic, with the characteristic morphology that gives the disease its name. An increased mean corpuscular hemoglobin concentration (MCHC) on an ordinary blood count report should raise the suspicion of HS, because HS is almost the only condition in which this abnormality occurs. It has been apparent for a long time that the spleen plays a special role in HS through a dual mechanism. On one hand, like in many other HAs, the spleen

1	condition in which this abnormality occurs. It has been apparent for a long time that the spleen plays a special role in HS through a dual mechanism. On one hand, like in many other HAs, the spleen itself is a major site of destruction; on the other hand, transit through the splenic circulation makes the defective red cells more spherocytic and, therefore, accelerates their demise, even though that may take place elsewhere.

1	When there is a family history, it is usually easy to make a diagnosis based on features of HA and typical red cell morphology. However, there may be no family history for at least two reasons. First, the patient may have a de novo mutation, i.e., a mutation that has taken place in a germ cell of one of his parents or early after zygote formation. Second, the patient may have a recessive form of HS (Table 129-3). In such cases, more extensive laboratory investigations are required, including osmotic fragility, the acid glycerol lysis test, the eosin-5′-maleimide (EMA)–binding test, and SDS-gel electrophoresis of membrane proteins; these tests are usually carried out in laboratories with special expertise in this area. Sometimes a definitive diagnosis can be obtained only by molecular studies demonstrating a mutation in one of the genes underlying HS (Table 129-3).

1	We do not have a causal treatment for HS; i.e., no way has yet been found to correct the basic defect in the membrane-cytoskeleton structure. Given the special role of the spleen in HS (see above), it has long been thought that an almost obligatory therapeutic measure was splenectomy. Because this operation may have more than trivial consequences, today we have more articulate recommendations, based on disease severity (having found out, whenever possible, about the outcome of splenectomy in the patient’s relatives with HS), as follows. In mild cases, avoid splenectomy. Delay splenectomy until puberty in moderate cases or until 4–6 years of age in severe cases. Antipneumococcal vaccination before splenectomy FIGURE 129-4 Peripheral blood smear from patients with membrane-cytoskeleton abnormalities. A. Hereditary spherocytosis. B. Hereditary elliptocytosis, heterozygote. C. Elliptocytosis, with both alleles of the α-spectrin gene mutated.

1	is imperative, whereas penicillin prophylaxis after splenectomy is controversial. Along with splenectomy, cholecystectomy should not be regarded as automatic; it should be carried out, usually by the laparoscopic approach, when clinically indicated. Abbreviations: HE, hereditary elliptocytosis; HS, hereditary spherocytosis.

1	Hereditary elliptocytosis (He) HE is at least as heterogeneous as HS, both from the genetic point of view (Table 129-3, Fig. 129-3) and from the clinical point of view. Again, it is the shape of the red cells (Fig. 129-4B) that gives the name to the condition, but there is no direct correlation between the elliptocytic morphology and clinical severity. In fact, some mild or even asymptomatic cases may have nearly 100% elliptocytes, whereas in severe cases, all kinds of bizarre poikilocytes can predominate. Clinical features and recommended management are similar to those outlined above for HS. Although the spleen may not have the specific role it has in HS, in severe cases, splenectomy may be beneficial. The prevalence of HE causing clinical disease is similar to that of HS. However, an in-frame deletion of nine amino acids in the SLC4A1 gene encoding band 3, causing the so-called Southeast Asia ovalocytosis, has a frequency of up to 7% in certain populations, presumably as a result

1	in-frame deletion of nine amino acids in the SLC4A1 gene encoding band 3, causing the so-called Southeast Asia ovalocytosis, has a frequency of up to 7% in certain populations, presumably as a result of malaria selection; it is asymptomatic in heterozygotes and probably lethal in homozygotes.

1	Disorders of Cation Transport These rare conditions with autosomal dominant inheritance are characterized by increased intracellular sodium in red cells, with concomitant loss of potassium; indeed, they are sometimes discovered through the incidental finding, in a blood test, of a high serum K+ (pseudohyperkalemia). In patients from some families, the cation transport disturbance is associated with gain of water; as a result, the red cells are overhydrated (low MCHC), and on a blood smear, the normally round-shaped central pallor is replaced by a linear-shaped central pallor, which has earned this disorder the name stomatocytosis (Fig. 129-3). In patients from other families, instead, the red cells are dehydrated (high MCHC), and their consequent rigidity has earned this disorder the name xerocytosis. One would surmise that in these disorders the primary defect may be in a cation transporter; indeed, xerocytosis results from mutations in PIEZO1. In other patients with stomatocytosis,

1	xerocytosis. One would surmise that in these disorders the primary defect may be in a cation transporter; indeed, xerocytosis results from mutations in PIEZO1. In other patients with stomatocytosis, mutations are found in other genes also related to solute transport (Table 129-3), including SLC4A1 (encoding band 3), the Rhesus gene RHAG, and the glucose transporter gene SLC2A1 responsible for a special form called cryohydrocytosis. Hemolysis can vary from relatively mild to quite severe. From the practical point of view, it is important to know that in stomatocytosis, splenectomy is strongly contraindicated because it has been followed in a significant proportion of cases by severe thromboembolic complications.

1	channel. Enzyme Abnormalities When there is an important defect in the membrane or in the cytoskeleton, hemolysis is a direct consequence of the fact that the very structure of the red cell is abnormal. Instead, when one of the enzymes is defective, the consequences will depend on the precise role of that enzyme in the metabolic machinery of the red cell, which, in first approximation, has two important functions: (1) to provide energy in the form of ATP and (2) to prevent oxidative damage to hemoglobin and to other proteins by providing sufficient reductive potential; the key molecule for this is NADPH.

1	abnormalities of tHe glycolytic patHway Because red cells, in the course of their differentiation, have sacrificed not only their nucleus and their ribosomes, but also their mitochondria, they rely exclusively on the anaerobic portion of the glycolytic pathway for producing energy in the form of ATP. Most of the ATP is required by the red cell for cation transport against a concentration gradient across the membrane. If this fails, due to a defect of any of the enzymes of the glycolytic pathway (Table 129-4), the result will be hemolytic disease.

1	pyruvate kinase deficiency Abnormalities of the glycolytic pathway are all inherited and all rare. Among them, deficiency of pyruvate kinase (PK) is the least rare, with an estimated prevalence in most populations of the order of 1:10,000. However, very recently, a polymorphic PK mutation (E277K) was found in some African populations, with heterozygote frequencies of 1–7%, suggesting that this may be another malaria-related polymorphism. The clinical picture of homozygous (or compound biallelic) PK deficiency is that of an HA that often presents in the newborn with neonatal jaundice; the jaundice persists, and it is usually associated with a very high reticulocytosis. The anemia is of variable severity; sometimes it is so severe as to require regular blood transfusion treatment, whereas sometimes it is mild, bordering on a nearly compensated hemolytic disorder. As a result, the diagnosis may be delayed, and in some cases, it is made, for instance, in a young woman during her first

1	sometimes it is mild, bordering on a nearly compensated hemolytic disorder. As a result, the diagnosis may be delayed, and in some cases, it is made, for instance, in a young woman during her first pregnancy, when the anemia may get worse. The delay in diagnosis may be also helped by the fact that the anemia is remarkably well tolerated, because the metabolic block at the last step in glycolysis causes an increase in bisphosphoglycerate (or DPG; Fig. 129-1), a major effector of the hemoglobin-oxygen dissociation curve; thus, the oxygen delivery to the tissues is enhanced, a remarkable compensatory feat.

1	Hemolytic Anemias and Anemia Due to Acute Blood Loss aThe numbers from (1) to (4) indicate the ranking order of these enzymopathies in terms of frequency. Abbreviations: AHA, acquired hemolytic anemia; CNS, central nervous system; NM, neuromuscular.

1	The management of PK deficiency is mainly supportive. In view of the marked increase in red cell turnover, oral folic acid supplements should be given constantly. Blood transfusion should be used as necessary, and iron chelation may have to be added if the blood transfusion requirement is high enough to cause iron overload. In these patients, who have more severe disease, splenectomy may be beneficial. There is a single case report of curative treatment of PK deficiency by bone marrow transplantation from an HLA-identical PK-normal sibling. This seems a viable option for severe cases when a sibling donor is available. Rescue of inherited PK deficiency through lentiviral-mediated human PK gene transfer has been successful in mice. Prenatal diagnosis has been carried out in a mother who had already had an affected child.

1	other glycolytic enzyme abnormalities All of these defects are rare to very rare (Table 129-4), and all cause hemolytic anemia with varying degrees of severity. It is not unusual for the presentation to be in the guise of severe neonatal jaundice, which may require exchange transfusion; if the anemia is less severe, it may present later in life, or it may even remain asymptomatic and be detected incidentally when a blood count is done for unrelated reasons. The spleen is often enlarged. When other systemic manifestations occur, they can involve the central nervous system (sometimes entailing severe mental retardation, particularly in the case of triose phosphate isomerase deficiency), the neuromuscular system, or both. This is not altogether surprising, if we consider that these are housekeeping genes. The diagnosis of hemolytic anemia is usually not difficult, thanks to the triad of normomacrocytic anemia, reticulocytosis, and hyperbilirubinemia. Enzymopathies should be considered in

1	genes. The diagnosis of hemolytic anemia is usually not difficult, thanks to the triad of normomacrocytic anemia, reticulocytosis, and hyperbilirubinemia. Enzymopathies should be considered in the differential diagnosis of any chronic Coombs-negative hemolytic anemia. Unlike with membrane disorders where the red cells show characteristic morphologic abnormalities, in most cases of glycolytic enzymopathies, these are conspicuous by their absence. A definitive diagnosis can be made only by demonstrating the deficiency of an individual enzyme by quantitative assays; these are carried out in only a few specialized laboratories. If a particular molecular abnormality is already known in the family, then one could test directly for that defect at the DNA level, thus bypassing the need for enzyme assays. Of course the time may be getting nearer when a patient will present with her or his exome already sequenced, and we will need to concentrate on which genes to look up within the file. The

1	assays. Of course the time may be getting nearer when a patient will present with her or his exome already sequenced, and we will need to concentrate on which genes to look up within the file. The principles for the management of these conditions are similar as for PK deficiency. In one case of phosphoglycerate kinase deficiency, allogeneic bone marrow transplantation (BMT) effectively controlled the hematologic manifestations but did not reverse neurologic damage.

1	abnormalities of redox metabolism glucose 6-phosphate dehydrogenase (g6pd) deficiency G6PD is a housekeeping enzyme critical in the redox metabolism of all aerobic cells (Fig. 129-1). In red cells, its role is even more critical, because it is the only source of NADPH, which directly and via glutathione (GSH) defends these cells against oxidative stress (Fig. 129-5). G6PD deficiency is a prime example of an HA due to interaction between FIGURE 129-5 Diagram of redox metabolism in the red cell. 6PG, 6-phosphogluconate; G6P, glucose 6-phosphate; G6PD, glucose 6-phosphate dehydrogenase; GSH, reduced glutathione; GSSG, oxidized glutathione; Hb, hemoglobin; MetHb, methemoglobin; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate.

1	an intracorpuscular cause and an extracorpuscular cause, because in the majority of cases hemolysis is triggered by an exogenous agent. Although a decrease in G6PD activity is present in most tissues of G6PD-deficient subjects, in other cells, the decrease is much less marked than in red cells, and it does not seem to impact on clinical expression. The G6PD gene is X-linked, and this has important implications.

1	First, because males have only one G6PD gene (i.e., they are hemizy gous for this gene), they must be either normal or G6PD deficient. By contrast, females, who have two G6PD genes, can be either normal or deficient (homozygous) or intermediate (heterozygous). As a result of the phenomenon of X chromosome inactivation, heterozygous females are genetic mosaics, with a highly variable ratio of G6PDnormal to G6PD-deficient cells and an equally variable degree of clinical expression; some heterozygotes can be just as affected as hemizygous males. The enzymatically active form of G6PD is either a dimer or a tetramer of a single protein subunit of 514 amino acids. G6PDdeficient subjects have been found invariably to have mutations in the coding region of the G6PD gene (Fig. 129-5). Almost all of the approximately 180 different mutations known are single missense point mutations, entailing single amino acid replacements in the G6PD protein. In most cases, these mutations cause G6PD

1	all of the approximately 180 different mutations known are single missense point mutations, entailing single amino acid replacements in the G6PD protein. In most cases, these mutations cause G6PD deficiency by decreasing the in vivo stability of the protein; thus, the physiologic decrease in G6PD activity that takes place with red cell aging is greatly accelerated. In some cases, an amino acid replacement can also affect the catalytic function of the enzyme.

1	Among these mutations, those underlying chronic nonspherocytic hemolytic anemia (CNSHA; see below) are a discrete subset. This much more severe clinical phenotype can be ascribed in some cases to adverse qualitative changes (for instance, a decreased affinity for the substrate, glucose 6-phosphate) or simply to the fact that the enzyme deficit is more extreme, because of a more severe instability of the enzyme. For instance, a cluster of mutations map at or near the dimer interface, and clearly they compromise severely the formation of the dimer.

1	epidemiology G6PD deficiency is widely distributed in tropical and subtropical parts of the world (Africa, Southern Europe, the Middle East, Southeast Asia, and Oceania) (Fig. 129-6) and wherever people from those areas have migrated. A conservative estimate is that at least 400 million people have a G6PD deficiency gene. In several of these areas, the frequency of a G6PD deficiency gene may be as high as 20% or more. It would be quite extraordinary for a trait that causes significant pathology to spread widely and reach high frequencies in many populations without conferring some biologic advantage. Indeed, G6PD is one of the best-characterized examples of genetic polymorphisms in the human species. Clinical field studies and in vitro experiments strongly support the view that G6PD deficiency 655 has been selected by Plasmodium falciparum malaria, by virtue of the fact that it confers a relative resistance against this highly lethal infection. Different G6PD variants underlie G6PD

1	655 has been selected by Plasmodium falciparum malaria, by virtue of the fact that it confers a relative resistance against this highly lethal infection. Different G6PD variants underlie G6PD deficiency in different parts of the world. Some of the more widespread variants are G6PD Mediterranean on the shores of that sea, in the Middle East, and in India; G6PD A– in Africa and in Southern Europe; G6PD Vianchan and G6PD Mahidol in Southeast Asia; G6PD Canton in China; and G6PD Union worldwide. The heterogeneity of polymorphic G6PD variants is proof of their independent origin, and it supports the notion that they have been selected by a common environmental agent, in keeping with the concept of convergent evolution (Fig. 129-6).

1	clinical manifestations The vast majority of people with G6PD deficiency remain clinically asymptomatic throughout their lifetime; however, all of them have an increased risk of developing neonatal jaundice (NNJ) and a risk of developing acute HA (AHA) when challenged by a number of oxidative agents. NNJ related to G6PD deficiency is very rarely present at birth; the peak incidence of clinical onset is between day 2 and day 3, and in most cases, the anemia is not severe. However, NNJ can be very severe in some G6PD-deficient babies, especially in association with prematurity, infection, and/or environmental factors (such as naphthalene-camphor balls, which are used in babies’ bedding and clothing), and the risk of severe NNJ is also increased by the coexistence of a monoallelic or biallelic mutation in the uridyl transferase gene (UGT1A1; the same mutations are associated with Gilbert’s syndrome). If inadequately managed, NNJ associated with G6PD deficiency can produce kernicterus and

1	mutation in the uridyl transferase gene (UGT1A1; the same mutations are associated with Gilbert’s syndrome). If inadequately managed, NNJ associated with G6PD deficiency can produce kernicterus and permanent neurologic damage.

1	AHA can develop as a result of three types of triggers: (1) fava beans, (2) infections, and (3) drugs (Table 129-5). Typically, a hemolytic attack starts with malaise, weakness, and abdominal or lumbar pain. After an interval of several hours to 2–3 days, the patient develops jaundice and often dark urine. The onset can be extremely abrupt, especially with favism in children. The anemia is moderate to extremely severe, usually normocytic and normochromic, and due partly to intravascular hemolysis; hence, it is associated with hemoglobinemia, hemoglobinuria, high LDH, and low or absent plasma haptoglobin. The blood film shows anisocytosis, polychromasia, and spherocytes typical of hemolytic anemias. The most typical feature of G6PD deficiency is the presence of bizarre poikilocytes, with red cells that appear to have unevenly distributed hemoglobin (“hemighosts”) and red cells that appear to have had parts of them bitten away (“bite cells” or “blister cells”) (Fig. 129-7). A classical

1	cells that appear to have unevenly distributed hemoglobin (“hemighosts”) and red cells that appear to have had parts of them bitten away (“bite cells” or “blister cells”) (Fig. 129-7). A classical test, now rarely carried out, is supravital staining with methyl violet, which, if done promptly, reveals the presence of Heinz bodies (consisting of precipitates of denatured hemoglobin and hemichromes), which are regarded as a signature of oxidative damage to red cells (they are also seen with unstable hemoglobins). LDH is high, and so is the unconjugated

1	CHAPTEr129 Hemolytic Anemias and Anemia Due to Acute Blood Loss FIGURE 129-6 Epidemiology of glucose 6-phosphate dehydrogenase (G6PD) deficiency throughout the world. The different shadings indicate increasingly high levels of prevalence, up to about 20%; the different colored symbols indicate individual genetic variants of G6PD, each one having a different mutation. (From L Luzzatto et al, in C Scriver et al [eds]: The Metabolic & Molecular Bases of Inherited Disease, 8th ed. New York, McGraw-Hill, 2001.) bilirubin, indicating that there is also extravascular hemolysis. The most serious threat from AHA in adults is the development of acute renal failure (this is exceedingly rare in children). Once the threat of acute anemia is over and in the absence of comorbidity, full recovery from AHA associated with G6PD deficiency is the rule.

1	Although it was primaquine (PQ) that led to the discovery of G6PD deficiency, this drug has not been very prominent subsequently, because it is not necessary for the treatment of life-threatening P. falciparum malaria. Today there is a revival of interest in PQ because it is the only effective agent for eliminating the gametocytes of P. falciparum (thus preventing further transmission) and eliminating the hypnozoites of Plasmodium vivax (thus preventing endogenous relapse). In countries aiming to eliminate malaria, there may be a call for mass administration of PQ; this ought to be associated with G6PD testing. At the other end of the historic spectrum, the latest addition to the list of potentially hemolytic drugs (Table 129-5) is rasburicase; again G6PD testing ought to be made mandatory before giving this drug because fatal cases have been reported in newborns with kidney injury and in adults with tumor lysis syndrome.

1	FIGURE 129-7 Peripheral blood smear from a glucose 6-phosphate dehydrogenase (G6PD)-deficient boy experiencing hemolysis. Note the red cells that are misshapen and called “bite” cells. (From MA Lichtman et al: Lichtman's Atlas of Hematology: http://www .accessmedicine.com. Copyright © The McGraw-Hill Companies, Inc. All rights reserved.)

1	A very small minority of subjects with G6PD deficiency have chronic nonspherocytic hemolytic anemia (CNSHA) of variable severity. The patient is nearly always a male, usually with a history of NNJ, who may present with anemia, unexplained jaundice, or gallstones later in life. The spleen may be enlarged. The severity of anemia ranges in different patients from borderline to transfusion dependent. The anemia is usually normomacrocytic, with reticulocytosis. Bilirubin and LDH are increased. Although hemolysis is, by definition, chronic in these patients, they are also vulnerable to acute oxidative damage, and therefore the same agents that can cause AHA in people with the ordinary type of G6PD deficiency will cause severe exacerbations in people with CNSHA associated with G6PD deficiency. In some cases of CNSHA, the deficiency of G6PD is so severe in granulocytes that it becomes rate-limiting for their oxidative burst, with consequent increased susceptibility to some bacterial

1	In some cases of CNSHA, the deficiency of G6PD is so severe in granulocytes that it becomes rate-limiting for their oxidative burst, with consequent increased susceptibility to some bacterial infections.

1	laboratory diagnosis The suspicion of G6PD deficiency can be confirmed by semiquantitative methods often referred to as screening tests, which are suitable for population studies and can correctly classify male subjects, in the steady state, as G6PD normal or G6PD deficient. However, in clinical practice, a diagnostic test is usually needed when the patient has had a hemolytic attack; this implies that the oldest, most G6PD-deficient red cells have been selectively destroyed, and young red cells, having higher G6PD activity, are being released into the circulation. Under these conditions, only a quantitative test can give a definitive result. In males, this test will identify normal hemizygotes and G6PD-deficient hemizygotes; among females, some heterozygotes will be missed, but those who are at most risk of hemolysis will be identified. Of course, G6PD deficiency also can be diagnosed by DNA testing.

1	The AHA of G6PD deficiency is largely preventable by avoiding exposure to triggering factors of previously screened subjects. Of course, the practicability and cost-effectiveness of screening depend on the prevalence of G6PD deficiency in each individual community. Favism is entirely preventable in G6PD-deficient subjects by not eating fava beans. Drug-induced hemolysis can be prevented by testing for G6PD deficiency before prescribing; in most cases, one can use alternative drugs. When AHA develops and once its cause is recognized, in most cases, no specific treatment is needed. However, if the anemia is severe, it may be a medical emergency, especially in children, requiring immediate action, including blood transfusion. This has been the case with an antimalarial drug combination containing dapsone (called Lapdap, introduced in 2003) that has caused severe acute hemolytic episodes in children with malaria in several African countries; after a few years, the drug was taken off the

1	dapsone (called Lapdap, introduced in 2003) that has caused severe acute hemolytic episodes in children with malaria in several African countries; after a few years, the drug was taken off the market. If there is acute renal failure, hemodialysis may be necessary, but if there is no previous kidney disease, recovery is the rule. The management of NNJ associated with G6PD deficiency is no different from that of NNJ due to other causes.

1	In cases with CNSHA, if the anemia is not severe, regular folic acid supplements and regular hematologic surveillance will suffice. It will be important to avoid exposure to potentially hemolytic drugs, and blood transfusion may be indicated when exacerbations occur, mostly in concomitance with intercurrent infection. In rare patients, regular blood transfusions may be required, in which case appropriate iron chelation should be instituted. Unlike in HS, there is no evidence of selective red cell destruction in the spleen; however, in practice, splenectomy has proven beneficial in severe cases.

1	other abnormalities of the redox system As mentioned above, GSH is a key player in the defense against oxidative stress. Inherited defects of GSH metabolism are exceedingly rare, but each one can give rise to chronic HA (Table 129-4). A rare, peculiar, usually self-limited severe HA of the first month of life, called infantile poikilocytosis, may be associated with deficiency of glutathione peroxidase (GSHPX) due not to an inherited abnormality, but to transient nutritional deficiency of selenium, an element essential for the activity of GSHPX.

1	pyrimidine 5′-nucleotidase (p5n) deficiency P5N is a key enzyme in the catabolism of nucleotides arising from the degradation of nucleic acids that takes place in the final stages of erythroid cell maturation. How exactly its deficiency causes HA is not well understood, but a highly distinctive feature of this condition is a morphologic abnormality of the red cells known as basophilic stippling. The condition is rare, but it probably ranks third in frequency among red cell enzyme defects (after G6PD deficiency and PK deficiency). The anemia is lifelong, of variable severity, and may benefit from splenectomy.

1	Familial (Atypical) Hemolytic-Uremic Syndrome The term familial (atypical) hemolytic-uremic syndrome is used to designate a group of rare disorders, mostly affecting children, characterized by microangiopathic HA with presence of fragmented erythrocytes in the peripheral blood smear, thrombocytopenia (usually mild), and acute renal failure. (The word atypical is part of the phrase for historical reasons; it was hemolytic-uremic syndrome [HUS] caused by infection with Escherichia coli producing the Shiga toxin that was regarded as typical). The genetic basis of atypical HUS (aHUS) has been elucidated. Studies of 657 >100 families have revealed that those family members who developed HUS had mutations in any one of several genes encoding complement regulatory proteins: complement factor H (CFH), CD46 or membrane cofactor protein (MCP), complement factor I (CFI), complement component C3, complement factor B (CFB), and thrombomodulin. Thus, whereas all other inherited HAs are due to

1	H (CFH), CD46 or membrane cofactor protein (MCP), complement factor I (CFI), complement component C3, complement factor B (CFB), and thrombomodulin. Thus, whereas all other inherited HAs are due to intrinsic red cell abnormalities, this group is unique in that hemolysis results from an inherited defect external to red cells (Table 129-1). Because the regulation of the complement cascade has considerable redundancy, in the steady state, any of the above abnormalities can be tolerated. However, when an intercurrent infection or some other trigger activates complement through the alternative pathway, the deficiency of one of the complement regulators becomes critical. Endothelial cells get damaged, especially in the kidney; at the same time, and partly as a result of this, there will be brisk hemolysis (thus, the more common Shiga toxin–related HUS [Chap. 149] can be regarded as a phenocopy of aHUS). aHUS is a severe disease, with up to 15% mortality in the acute phase and up to 50% of

1	hemolysis (thus, the more common Shiga toxin–related HUS [Chap. 149] can be regarded as a phenocopy of aHUS). aHUS is a severe disease, with up to 15% mortality in the acute phase and up to 50% of cases progressing to end-stage renal disease. Not infrequently, aHUS undergoes spontaneous remission; but because its basis is an inherited abnormality, it is not surprising that, given renewed exposure to a trigger, the syndrome will tend to recur; when it does, the prognosis is always serious. The standard treatment has been plasma exchange, which will supply the deficient complement regulator. The anti-C5 complement inhibitor eculizumab (see below) was found to greatly ameliorate the microangiopathic picture, with improvement in platelet counts and in renal function, thus abrogating the need for plasma exchange. It remains to be seen for how long eculizumab treatment will have to be continued in individual patients and whether it will influence the controversial issue of kidney (and

1	need for plasma exchange. It remains to be seen for how long eculizumab treatment will have to be continued in individual patients and whether it will influence the controversial issue of kidney (and liver) transplantation.

1	ACQUIRED HEMOLYTIC ANEMIA Mechanical Destruction of Red Cells Although red cells are characterized by the remarkable deformability that enables them to squeeze through capillaries narrower than themselves for thousands of times in their lifetime, there are at least two situations in which they succumb to shear, if not to wear and tear; the result is intravascular hemolysis, resulting in hemoglobinuria (Table 129-6). One situation is acute and self-inflicted, march hemoglobinuria. Why sometimes a marathon runner may develop this complication, whereas on another occasion, this does not happen, we do not know (perhaps her or his footwear needs attention). A similar syndrome may develop after prolonged barefoot ritual dancing or intense playing of bongo drums. The other situation is chronic and iatrogenic (it has been called microangiopathic hemolytic anemia). It takes place in patients with prosthetic heart valves, especially when paraprosthetic regurgitation is present. If the hemolysis

1	iatrogenic (it has been called microangiopathic hemolytic anemia). It takes place in patients with prosthetic heart valves, especially when paraprosthetic regurgitation is present. If the hemolysis consequent on mechanical trauma to the red cells is mild, and if the supply of iron is adequate, the loss may be largely compensated; if more

1	Hemolytic Anemias and Anemia Due to Acute Blood Loss Paroxysmal nocturnal hemo-Chronic with acute Complement (C)-mediated destruc-Flow cytometry to display a Exacerbations due to C activation globinuria (PNH) exacerbations tion of CD59(−) red cells CD59(−) red cell population through any pathway 658 than mild anemia develops, reintervention to correct regurgitation may be required.

1	Infection By far the most frequent infectious cause of HA, in endemic areas, is malaria (Chap. 248). In other parts of the world, the most frequent direct cause is probably Shiga toxin–producing E. coli O157:H7, now recognized as the main etiologic agent of HUS, which is more common in children than in adults (Chap. 149). Life-threatening intravascular hemolysis, due to a toxin with lecithinase activity, occurs with Clostridium perfringens sepsis, particularly following open wounds, septic abortion, or as a disastrous accident due to a contaminated blood unit. Rarely, and if at all in children, HA is seen with sepsis or endocarditis from a variety of organisms. In addition, bacterial and viral infections can cause HA by indirect mechanisms (see above section on G6PD deficiency and Table 129-6).

1	Immune Hemolytic Anemias These can arise through at least two distinct mechanisms. (1) There is a true autoantibody directed against a red cell antigen, i.e., a molecule present on the surface of red cells. (2) When an antibody directed against a certain molecule (e.g., a drug) reacts with that molecule, red cells may get caught in the reaction, whereby they are damaged or destroyed. Because the antibodies involved differ in optimum reactivity temperatures, they are classified in the time-honored categories of “cold” and “warm” (Table 129-7). Autoantibody-mediated HAs may be seen in isolation (when they are called idiopathic) or as part of a systemic autoimmune disorder such as systemic lupus erythematosus. Here we discuss the most distinctive clinical pictures.

1	autoimmune Hemolytic anemia (aiHa) Once a red cell is coated by an autoantibody (see [1] above), it will be destroyed by one or more mechanisms. In most cases, the Fc portion of the antibody will be recognized by the Fc receptor of macrophages, and this will trigger erythrophagocytosis. Thus, destruction of red cells will take place wherever macrophages are abundant, i.e., in the spleen, liver, or bone marrow; this is called extravascular hemolysis (Fig. 129-8). Because of the special anatomy of the spleen, this organ is particularly efficient in Type of Antibody Drug-dependent: antibody destroys red cells only when drug present (e.g., rarely penicillin) Drug-independent: antibody can destroy red cells even when drug no longer present (e.g., methyldopa)

1	Drug-dependent: antibody destroys red cells only when drug present (e.g., rarely penicillin) Drug-independent: antibody can destroy red cells even when drug no longer present (e.g., methyldopa) Abbreviations: AIHA, autoimmune hemolytic anemia; CAD, cold agglutinin disease; CLL, chronic lymphocytic leukemia; CMV, cytomegalovirus; EBV, Epstein-Barr virus; HIV, human immunodeficiency virus; HSCT, hematopoietic stem cell transplantation; IBD, inflammatory bowel disease; SLE, systemic lupus erythematosus. trapping antibody-coated red cells, and often this is the predominant site of red cell destruction. In some cases, the nature of the antibody is such (usually an IgM antibody) that the antigen-antibody complex on the surface of red cells is able to activate complement (C); as a result, a large amount of membrane attack complex will form, and the red cells may be destroyed directly; this is known as intravascular hemolysis.

1	clinical features AIHA is a serious condition; without appropriate treatment, it may have a mortality of approximately 10%. The onset is often abrupt and can be dramatic. The hemoglobin level can drop, within days, to as low as 4 g/dL; the massive red cell removal will produce jaundice; and sometimes the spleen is enlarged. When this triad is present, the suspicion of AIHA must be high. When hemolysis is (in part) intravascular, the telltale sign will be hemoglobinuria, which the patient may report or about which we must enquire or test for. The diagnostic test for AIHA is the direct antiglobulin test developed in 1945 by R. R. A. Coombs and known since by this name. The beauty of this test is that it detects directly the pathogenetic mediator of the disease, i.e., the presence of antibody on the red cells themselves. When the test is positive, it clinches the diagnosis; when it is negative, the diagnosis is unlikely. However, the sensitivity of the Coombs test varies depending on the

1	on the red cells themselves. When the test is positive, it clinches the diagnosis; when it is negative, the diagnosis is unlikely. However, the sensitivity of the Coombs test varies depending on the technique that is used, and in doubtful cases, a repeat in a specialized lab is advisable; the term Coombs-negative AIHA is a last resort. In some cases, the autoantibody has a defined identity; it may be specific for an antigen belonging to the Rhesus system (it is often anti-e). In many cases, it is regarded as “nonspecific” because it reacts with virtually all types of red cells.

1	When AIHA develops in a person who is already known to have, for instance, systemic lupus or chronic lymphocytic leukemia (Table 129-7), we call it a complication; conversely, when AIHA presents on its own, it may be a pointer to an underlying condition that we ought to seek out. In both cases, what brings about AIHA remains, as in other autoimmune disorders, obscure. In some cases, AIHA can be associated, on first presentation or subsequently, with autoimmune thrombocytopenia (Evans’ syndrome).

1	Severe acute AIHA can be a medical emergency. The immediate treatment almost invariably includes transfusion of red cells. This may pose a special problem because, if the antibody involved is nonspecific, all of the blood units cross-matched will be incompatible. In these cases, it is often correct, paradoxically, to transfuse incompatible blood, with the rationale being that the transfused red cells will be destroyed no less but no more than the patient’s own red cells, but in the meantime, the patient stays alive. A situation like this requires close liaison and understanding between the clinical unit treating the patient and the blood transfusion/serology lab. Whenever the anemia is not immediately life-threatening, blood transfusion should be withheld (because compatibility problems may increase with each unit of blood transfused), and medical treatment started immediately with prednisone (1 mg/kg per day), which will produce a remission promptly in at least one-half of patients.

1	increase with each unit of blood transfused), and medical treatment started immediately with prednisone (1 mg/kg per day), which will produce a remission promptly in at least one-half of patients. Rituximab (anti-CD20) was regarded as second-line treatment, but it is increasingly likely that a relatively low dose (100 mg/wk × 4) of rituximab together with prednisone will become a first-line standard. It is especially encouraging that this approach seems to reduce the rate of relapse, a common occurrence in AIHA. For patients who do relapse or are refractory to medical treatment, one may have to consider splenectomy, which, although it does not cure the disease, can produce significant benefit by removing a major site of hemolysis, thus improving the anemia and/or reducing the need for other therapies (e.g., the dose of prednisone). Since the introduction of rituximab, azathioprine, cyclophosphamide, cyclosporine, and intravenous immunoglobulin have become secondor third-line agents.

1	therapies (e.g., the dose of prednisone). Since the introduction of rituximab, azathioprine, cyclophosphamide, cyclosporine, and intravenous immunoglobulin have become secondor third-line agents. In very rare severe refractory cases, either autologous or allogeneic hematopoietic stem cell transplantation may have to be considered.

1	Destroyed red cell with formation of membrane and membrane free hemoglobin Reticuloendothelial attack complex system

1	FIGURE 129-8 Mechanism of antibody-mediated immune destruction of red blood cells (RBCs). ADCC, antibody-dependent cell-mediated cytotoxicity. (From N Young et al: Clinical Hematology. Philadelphia, Elsevier, 2006; with permission.) paroxysmal cold Hemoglobinuria (pcH) PCH is a rather rare form of AIHA occurring mostly in children, usually triggered by a viral infection, usually self-limited, and characterized by the involvement of the so-called Donath-Landsteiner antibody. In vitro, this antibody has unique serologic features; it has anti-P specificity and binds to red cells only at a low temperature (optimally at 4°C), but when the temperature is shifted to 37°C, lysis of red cells takes place in the presence of complement. Consequently, in vivo there is intravascular hemolysis, resulting in hemoglobinuria. Clinically the differential diagnosis must include other causes of hemoglobinuria (Table 129-6), but the presence of the Donath-Landsteiner antibody will prove PCH. Active

1	in hemoglobinuria. Clinically the differential diagnosis must include other causes of hemoglobinuria (Table 129-6), but the presence of the Donath-Landsteiner antibody will prove PCH. Active supportive treatment, including blood transfusion, is needed to control the anemia; subsequently, recovery is the rule.

1	cold agglutinin disease (cad) This designation is used for a form of chronic AIHA that usually affects the elderly and has special clinical and pathologic features. First, the term cold refers to the fact that the autoantibody involved reacts with red cells poorly or not at all at 37°C, whereas it reacts strongly at lower temperatures. As a result, hemolysis is more prominent the more the body is exposed to the cold. The antibody is usually IgM; usually it has an anti-I specificity (the I antigen is present on the red cells of almost everybody), and it may have a very high titer (1:100,000 or more has been observed). Second, the antibody is produced by an expanded clone of B lymphocytes, and sometimes its concentration in the plasma is high enough to show up as a spike in plasma protein electrophoresis, i.e., as a monoclonal gammopathy. Third, because the antibody is IgM, CAD is related to Waldenström’s macroglobulinemia (WM) (Chap. 136), although in most cases, the other clinical

1	electrophoresis, i.e., as a monoclonal gammopathy. Third, because the antibody is IgM, CAD is related to Waldenström’s macroglobulinemia (WM) (Chap. 136), although in most cases, the other clinical features of this disease are not present. Thus, CAD must be regarded as a form of WM (i.e., as a low-grade mature B cell lymphoma) that manifests at an earlier stage precisely because the unique biologic properties of the IgM that it produces give the clinical picture of chronic HA.

1	In mild forms of CAD, avoidance of exposure to cold may be all that is needed to enable the patient to have a reasonably comfortable quality of life; but in more severe forms, the management of CAD is not easy. Blood transfusion is not very effective because donor red cells are I positive and will be rapidly removed. Immunosuppressive/cytotoxic treatment with azathioprine or cyclophosphamide can reduce the antibody titer, but clinical efficacy is limited, and in view of the chronic nature of the disease, the side effects may prove unacceptable. Unlike in AIHA, prednisone and splenectomy are ineffective. Plasma exchange will remove antibody and is, therefore, in theory, a rational approach, but it is laborious and must be carried out at frequent intervals if it is to be beneficial. The management of CAD has changed significantly with the advent of rituximab; although its impact on CAD is not as great as on AIHA, up to 60% of patients respond, and remissions may be more durable with a

1	of CAD has changed significantly with the advent of rituximab; although its impact on CAD is not as great as on AIHA, up to 60% of patients respond, and remissions may be more durable with a rituximab-fludarabine combination. Given the long clinical course of CAD, it remains to be seen with what schedule or periodicity these agents will need to be administered.

1	Toxic Agents and Drugs A number of chemicals with oxidative potential, whether medicinal or not, can cause hemolysis even in people who are not G6PD deficient (see above). Examples are hyperbaric oxygen (or 100% oxygen), nitrates, chlorates, methylene blue, dapsone, cisplatin, and numerous aromatic (cyclic) compounds. Other chemicals may be hemolytic through nonoxidative, largely unknown mechanisms; examples include arsine, stibine, copper, and lead. The HA caused by lead poisoning is characterized by basophilic stippling; it is in fact a phenocopy of that seen in P5N deficiency (see above), suggesting it is mediated at least in part by lead inhibiting this enzyme.

1	In these cases, hemolysis appears to be mediated by a direct chemical action on red cells. But drugs can cause hemolysis through at least two other mechanisms. (1) A drug can behave as a hapten and induce antibody production; in rare subjects, this happens, for instance, with penicillin. Upon a subsequent exposure, red cells are caught, as innocent bystanders, in the reaction between penicillin and antipenicillin antibodies. Hemolysis will subside as soon as penicillin administration is stopped. (2) A drug can trigger, perhaps through mimicry, the production of an antibody against a red cell antigen. The best known example is methyldopa, an antihypertensive agent no longer in use, which in a small fraction of patients stimulated the production of the

1	Hemolytic Anemias and Anemia Due to Acute Blood Loss 660 Rhesus antibody anti-e. In patients who have this antigen, the anti-e is a true autoantibody, which then causes an autoimmune HA (see below). Usually this will gradually subside once methyldopa is discontinued. Severe intravascular hemolysis can be caused by the venom of certain snakes (cobras and vipers), and HA can also follow spider bites. Paroxysmal Nocturnal Hemoglobinuria (PNH) PNH is an acquired chronic HA characterized by persistent intravascular hemolysis subject to recurrent exacerbations. In addition to hemolysis, there is often pancytopenia and a distinct tendency to venous thrombosis. This triad makes PNH a truly unique clinical condition; however, when not all of these three features are manifest on presentation, the diagnosis is often delayed, although it can always be made by appropriate laboratory investigations (see below).

1	PNH has about the same frequency in men and women and is encountered in all populations throughout the world, but it is a rare disease; its prevalence is estimated to be approximately 5 per million (it may be somewhat less rare in Southeast Asia and in the Far East). There is no evidence of inherited susceptibility. PNH has never been reported as a congenital disease, but it can present in small children or as late as in the seventies, although most patients are young adults.

1	clinical features The patient may seek medical attention because, one morning, she or he passed blood instead of urine (Fig. 129-9). This distressing or frightening event may be regarded as the classical presentation; however, more frequently, this symptom is not noticed or is suppressed. Indeed, the patient often presents simply as a problem in the differential diagnosis of anemia, whether symptomatic or discovered incidentally. Sometimes, the anemia is associated from the outset with neutropenia, thrombocytopenia, or both, thus signaling an element of bone marrow failure (see below). Some patients may present with recurrent attacks of severe abdominal pain defying a specific diagnosis and eventually found to be related to thrombosis. When thrombosis affects the hepatic veins, it may produce acute hepatomegaly and ascites, i.e., a full-fledged Budd-Chiari syndrome, which, in the absence of liver disease, ought to raise the suspicion of PNH.

1	The natural history of PNH can extend over decades. Without treatment, the median survival is estimated to be about 8–10 years; in the past, the most common cause of death has been venous thrombosis, followed by infection secondary to severe neutropenia and hemorrhage secondary to severe thrombocytopenia. Rarely (estimated 1–2% of all cases), PNH may terminate in acute myeloid leukemia. On the other hand, full spontaneous recovery from PNH has been documented, albeit rarely.

1	laboratory investigations and diagnosis The most consistent blood finding is anemia, which may range from mild to moderate to very severe. The anemia is usually normomacrocytic, with unremarkable red cell morphology. If the MCV is high, it is usually largely accounted for by reticulocytosis, which may be quite marked (up to 20%, or up to 400,000/μL). The anemia may become microcytic if the patient is allowed to become iron deficient as a result of chronic urinary blood loss through hemoglobinuria. Unconjugated bilirubin is mildly or moderately elevated; LDH is typically markedly elevated (values in the thousands are common); and haptoglobin is usually undetectable. All of these findings make the diagnosis of hemolytic anemia compelling. Hemoglobinuria may be overt in a random urine sample; if it is not, it may be helpful to obtain serial urine samples, because hemoglobinuria can vary dramatically from day to day and even from hour to hour. The bone marrow is usually cellular, with

1	if it is not, it may be helpful to obtain serial urine samples, because hemoglobinuria can vary dramatically from day to day and even from hour to hour. The bone marrow is usually cellular, with marked to massive erythroid hyperplasia, often with mild to moderate dyserythropoietic features (not to be confused with myelodysplastic syndrome). At some stage of the disease, the marrow may become hypocellular or even frankly aplastic (see below).

1	FIGURE 129-9 Consecutive urine samples from a patient with par-oxysmal nocturnal hemoglobinuria (PNH). The variation in the severity of hemoglobinuria within hours is probably unique to this condition. The definitive diagnosis of PNH must be based on the demonstration that a substantial proportion of the patient’s red cells have an increased susceptibility to complement (C), due to the deficiency on their surface of proteins (particularly CD59 and CD55) that normally protect the red cells from activated C. The sucrose hemolysis test is unreliable; in contrast, the acidified serum (Ham) test is highly reliable but is carried out only in a few labs. The gold standard today is flow cytometry, which can be carried out on granulocytes as well as on red cells. A bimodal distribution of cells, with a discrete population that is CD59 and CD55 negative, is diagnostic of PNH. In PNH patients, this population is at least 5% of the total red cells and at least 20% of the total granulocytes.

1	patHopHysiology Hemolysis in PNH is mainly intravascular and is due to an intrinsic abnormality of the red cell, which makes it exquisitely sensitive to activated C, whether it is activated through the alternative pathway or through an antigen-antibody reaction. The former mechanism is mainly responsible for chronic hemolysis in PNH; the latter explains why the hemolysis can be dramatically exacerbated in the course of a viral or bacterial infection. Hypersusceptibility to C is due to deficiency of several protective membrane proteins (Fig. 129-10), of which CD59 is the most important, because it hinders the insertion into the membrane of C9 polymers. The molecular basis for the deficiency of these proteins has been pinpointed not to a defect in any of the respective genes, but rather to the shortage of a unique glycolipid molecule, GPI (Fig. 129-2), which, through a peptide bond, anchors these proteins to the surface membrane of cells. The shortage of GPI is due in turn to a mutation

1	shortage of a unique glycolipid molecule, GPI (Fig. 129-2), which, through a peptide bond, anchors these proteins to the surface membrane of cells. The shortage of GPI is due in turn to a mutation in an X-linked gene, called PIG-A, required for an early step in GPI biosynthesis. In virtually each patient, the PIGA mutation is different. This is not surprising, because these mutations are not inherited; rather, each one takes place de novo in a hemopoietic stem cell (i.e., they are somatic mutations). As a result, the patient’s marrow is a mosaic of mutant and nonmutant cells, and the peripheral blood always contains both PNH cells and normal (non-PNH) cells. Thrombosis is one of the most immediately life-threatening complications of PNH and yet one of the least understood in its pathogenesis. It could be that deficiency of CD59 on the PNH platelet causes inappropriate platelet activation; however, other mechanisms are possible.

1	bone marrow failure (bmf) and relationsHip between pnH and aplastic anemia (aa) It is not unusual that patients with firmly established PNH have a previous history of well-documented AA; indeed, BMF preceding overt PNH is probably the rule rather than the exception. On the other hand, sometimes a patient with PNH becomes less hemolytic and more pancytopenic and ultimately has the clinical picture of AA. Because AA is probably an organ-specific autoimmune disease, in which T cells cause damage to hematopoietic stem cells, the same may be true of PNH, with the specific proviso that the damage spares PNH stem cells. PIG-A mutations can be demonstrated in normal people, and there is evidence from mouse models that PNH stem cells do not expand when the rest of the bone marrow is normal. Thus, we can visualize PNH as always having two components: failure of normal hematopoiesis and massive expansion of a PNH clone. Findings supporting this notion include skewing of the T cell repertoire and

1	can visualize PNH as always having two components: failure of normal hematopoiesis and massive expansion of a PNH clone. Findings supporting this notion include skewing of the T cell repertoire and the demonstration of GPI-reactive T cells in patients with PNH.

1	A Normal, steady state A normal (CD55+, CD59+) red cell can withstand the hazard of complement activation. Intact normal (CD55+/CD59+) B PNH, steady state RBCs CHAPTEr 129 An abnormal (CD55–, CD59–) red cell (PNH cell) will be lysed sooner or later by activated complement (intravascular hemolysis). Hemolytic Anemias and Anemia Due to Acute Blood Loss C PNH, on eculizumab C3 C3 C3 opsonization RES macrophages (liver, spleen) Amplification loop C5 convertase Eculizumab + Lectin pathway Alternative pathway Classical pathway C3 C5b C6 C7 C8 C9 C5C3b C3b EcuC5C5C5C555 C3convertase C3convertase C3convertase MAC With C5 blocked, a PNH red cell will be protected from undergoing intravascular hemolysis, but once opsonized by C3 it will become prey to macrophages.

1	FIGURE 129-10 The complement cascade and the fate of red cells. A. Normal red cells are protected from complement activation and subsequent hemolysis by CD55 and CD59. These two proteins, being GPI-linked, are missing from the surface of PNH red cells as a result of a somatic mutation of the X-linked PIG-A gene that encodes a protein required for an early step of the GPI molecule biosynthesis. B. In the steady state, PNH erythrocytes suffer from spontaneous (tick-over) complement activation, with consequent intravascular hemolysis through formation of the membrane attack complex (MAC); when extra complement is activated through the classical pathway, an exacerbation of hemolysis will result. C. On eculizumab, PNH erythrocytes are protected from hemolysis from the inhibition of C5 cleavage; however, upstream complement activation may lead to C3 opsonization and possible extravascular hemolysis. GPI, glycosylphosphatidylinositol; PNH, paroxysmal nocturnal hemoglobinuria. (From L

1	however, upstream complement activation may lead to C3 opsonization and possible extravascular hemolysis. GPI, glycosylphosphatidylinositol; PNH, paroxysmal nocturnal hemoglobinuria. (From L Luzzatto et al: Haematologica 95:523, 2010.) which, for some patients, means quite frequently. Folic acid supplements (at least 3 mg/d) are mandatory; the serum iron should be checked periodically, and iron supplements should be administered as appropriate. Long-term glucocorticoids are not indi-

1	Unlike other acquired hemolytic anemias, PNH may be a lifelong condition, and most patients receive supportive treatment only, including transfusion of filtered red cells1 whenever necessary, 1Now that filters with excellent retention of white cells are routinely used, the traditional cated because there is no evidence that they have any effect on chronic hemolysis; in fact, they are contraindicated because their side effects are considerable and potentially dangerous. A major advance in the management of PNH has been the development of a humanized monoclonal antibody, eculizumab, which binds to washing of red cells, aiming to avoid white cell reactions triggering hemolysis, is no the complement component C5 near the site that, when cleaved, longer necessary and is wasteful. will trigger the distal part of the complement cascade leading to 662 the formation of membrane attack complex (MAC). In an international, placebo-controlled, randomized trial of 87 patients (so far the only

1	the distal part of the complement cascade leading to 662 the formation of membrane attack complex (MAC). In an international, placebo-controlled, randomized trial of 87 patients (so far the only controlled therapeutic trial in PNH) who had been selected on grounds of having severe hemolysis making them transfusion-dependent, eculizumab proved effective and was licensed in 2007. Eculizumab, by abrogating complement-dependent intravascular hemolysis, significantly improves the quality of life of PNH patients. One would expect that the need for blood transfusion would also be abrogated; indeed, this is the case in about one-half of patients, in many of whom there is also a rise in hemoglobin levels. In the remaining patients, however, the anemia remains sufficiently severe to require blood transfusion. One reason for this is that, once the distal complement pathway is blocked, red cells no longer destroyed by the MAC become opsonized by complement (C3) fragments and undergo extravascular

1	One reason for this is that, once the distal complement pathway is blocked, red cells no longer destroyed by the MAC become opsonized by complement (C3) fragments and undergo extravascular hemolysis (Fig. 129-10). The extent to which this happens depends in part on a genetic polymorphism of the complement receptor CR1. Based on its half-life, eculizumab must be administered intravenously every 14 days. The only form of treatment that currently can provide a definitive cure for PNH is allogeneic BMT. When an HLA-identical sibling is available, BMT should be offered to any young patient with severe PNH; the availability of eculizumab has decreased significantly the proportion of patients receiving BMT. For patients with the PNH-AA syndrome, immunosuppressive treatment with antithymocyte globulin and cyclosporine A may be indicated, especially in order to relieve severe thrombocytopenia and/or neutropenia in patients in whom these were the main problem(s); of course, this treatment will

1	and cyclosporine A may be indicated, especially in order to relieve severe thrombocytopenia and/or neutropenia in patients in whom these were the main problem(s); of course, this treatment will have little or no effect on hemolysis. Any patient who has had venous thrombosis or who has a genetically determined thrombophilic state in addition to PNH complications that do not resolve otherwise, ment with tissue plasminogen activator may be indicated.

1	The latter type of anemia is covered in Chap. 126 cerned with the former type, i.e., posthemorrhagic anemia lows acute blood loss. This can be external ric hemorrhage) or internal tract, rupture of the spleen, rupture of an ectopic pregnancy, subarachnoid hemorrhage). In any of these cases, after the sudden loss of a large amount of blood, there are three clinical/pathophysiologic stages. (1) At first, the dominant feature is hypovolemia, which poses a threat particularly to organs that normally have a high blood supply, like the brain and the kidneys; therefore, loss of consciousness and acute renal failure are major threats. It is important to note that at this stage an ordinary blood count will not show anemia, because the hemoglobin concentration is not affected. (2) Next, as an emergency response, baroreceptors and stretch receptors will cause release of vasopressin and other peptides, and the body will shift fluid from the extravascular to the intravascular compartment,

1	an emergency response, baroreceptors and stretch receptors will cause release of vasopressin and other peptides, and the body will shift fluid from the extravascular to the intravascular compartment, producing hemodilution; thus, the hypovolemia gradually converts to anemia. The degree of anemia will reflect the amount of blood lost. If after 3 days the hemoglobin is, for example, 7 g/dL, it means that about half of the entire blood has been lost. (3) Provided bleeding does not continue, the bone marrow response will gradually ameliorate the anemia.

1	The diagnosis of acute posthemorrhagic anemia (APHA) is usually straightforward, although sometimes internal bleeding episodes (e.g., after a traumatic injury), even when large, may not be immediately obvious. Whenever an abrupt fall in hemoglobin has taken place, whatever history is given by the patient, APHA should be suspected. Supplementary history may have to be obtained by asking the appropriate questions, and appropriate investigations (e.g., a sonogram or an endoscopy) may have to be carried out. With respect to treatment, a two-pronged approach is imperative.

1	With respect to treatment, a two-pronged approach is imperative. (1) In many cases, the blood lost needs to be replaced promptly. Unlike with many chronic anemias, when finding and correcting the cause of the anemia is the first priority and blood transfusion may not be even necessary because the body is adapted to the anemia, with acute blood loss the reverse is true; because the body is not adapted to the anemia, blood transfusion takes priority. (1) While the emergency is being confronted, it is imperative to stop the hemorrhage and to eliminate its source.

1	A special type of APHA is blood loss during and immediately after surgery, which can be substantial (e.g., up to 2 L in the case of a radical prostatectomy). Of course with elective surgical procedures, the patient’s own stored blood may be available (through preoperative autologous blood donation), and in any case, blood loss ought to have been carefully monitored/measured. The fact that this blood loss is iatrogenic dictates that ever more effort should be invested in optimizing its management.

1	A Holy Grail of emergency medicine for a long time has been the idea of a blood substitute that would be universally available, suitable for all recipients, easy to store and to transport, safe, and as effective as blood itself. Two main paths have been pursued: (1) fluorocarbon synthetic chemicals that bind oxygen reversibly, and (2) artificially modified hemoglobins, known as hemoglobin-based oxygen carriers (HBOCs). Although there are numerous anecdotal reports of the use of both approaches in humans, and although HBOCs have reached the stage of phase 2–3 clinical trials, no “blood substitute” has yet become standard treatment. Neal S. Young

1	Neal S. Young The hypoproliferative anemias are normochromic, normocytic, or macrocytic and are characterized by a low reticulocyte count. Hypoproliferative anemia is also a prominent feature of hematologic diseases that are described as bone marrow failure states; these include aplastic anemia, myelodysplastic syndrome (MDS), pure red cell aplasia (PRCA), and myelophthisis. Anemia in these disorders is often not a solitary or even the major hematologic finding. More frequent in bone marrow failure is pancytopenia: anemia, leukopenia, and thrombocytopenia. Low blood counts in the marrow failure diseases result from deficient hematopoiesis, as distinguished from blood count depression due to peripheral destruction of red cells (hemolytic anemias), platelets (idiopathic thrombocytopenic purpura [ITP] or due to splenomegaly), and granulocytes (as in the immune leukopenias). Marrow damage and dysfunction also may be secondary to infection, inflammation, or cancer.

1	Hematopoietic failure syndromes are classified by dominant morphologic features of the bone marrow (Table 130-1). Although practical distinction among these syndromes usually is clear, some processes are so closely related that the diagnosis may be complex. Patients may seem to suffer from two or three related diseases simultaneously, or one diagnosis may appear to evolve into another. Many of these syndromes share an immune-mediated mechanism of marrow destruction and some element of genomic instability resulting in a higher rate of malignant transformation. It is important that the internist and general practitioner recognize the marrow failure syndromes, as their prognosis may be poor if the Pancytopenia with Hypocellular Bone Marrow Acquired aplastic anemia Constitutional aplastic anemia (Fanconi anemia, dyskeratosis congenita) Some myelodysplasia Rare aleukemic leukemia Some acute lymphoid leukemia Some lymphomas of bone marrow Pancytopenia with Cellular Bone Marrow

1	Pancytopenia with Cellular Bone Marrow Primary bone marrow diseases Secondary to systemic diseases Myelodysplasia Systemic lupus erythematosus Paroxysmal nocturnal Hypersplenism B12, folate deficiency Myelofibrosis Overwhelming infection Some aleukemic leukemia Alcohol Myelophthisis Brucellosis Bone marrow lymphoma Sarcoidosis Hairy cell leukemia Tuberculosis Q fever Legionnaires’ disease Anorexia nervosa, starvation patient is untreated; effective therapies are often available but sufficiently complicated in their choice and delivery so as to warrant the care of a hematologist or oncologist.

1	Aplastic anemia is pancytopenia with bone marrow hypocellularity. Acquired aplastic anemia is distinguished from iatrogenic aplasia, marrow hypocellularity after intensive cytotoxic chemotherapy for cancer. Aplastic anemia can also be constitutional: the genetic diseases Fanconi anemia and dyskeratosis congenita, although frequently associated with typical physical anomalies and the development of pancytopenia early in life, can also present as marrow failure in normal-appearing adults. Acquired aplastic anemia is often stereotypical in its manifestations, with the abrupt onset of low blood counts in a previously well young adult; seronegative hepatitis or a course of an incriminated medical drug may precede the onset. The diagnosis in these instances is uncomplicated. Sometimes blood count depression is moderate or incomplete, resulting in anemia, leukopenia, and thrombocytopenia in some combination. Aplastic anemia is related to both paroxysmal nocturnal hemoglobinuria (PNH; Chap.

1	depression is moderate or incomplete, resulting in anemia, leukopenia, and thrombocytopenia in some combination. Aplastic anemia is related to both paroxysmal nocturnal hemoglobinuria (PNH; Chap. 129) and to MDS, and in some cases, a clear distinction among these disorders may not be possible.

1	The incidence of acquired aplastic anemia in Europe and Israel is two cases per million persons annually. In Thailand and China, rates of five to seven per million have been established. In general, men and women are affected with equal frequency, but the age distribution is biphasic, with the major peak in the teens and twenties and a second rise in older adults. The origins of aplastic anemia have been inferred from several recurring clinical associations (Table 130-2); unfortunately, these relationships are not reliable in an individual patient and may not be etiologic. In addition, although most cases of aplastic anemia are idiopathic, little other than history separates these cases from those with a presumed etiology such as a drug exposure. Epstein-Barr virus (infectious Preleukemia (monosomy 7, etc.) mononucleosis) Hepatitis (non-A, non-B, non-C Nonhematologic syndrome (Down, hepatitis) Dubowitz, Seckel) Parvovirus B19 (transient aplastic crisis, PRCA) HIV-1 (AIDS)

1	Parvovirus B19 (transient aplastic crisis, PRCA) HIV-1 (AIDS) Abbreviation: PRCA, pure red cell aplasia. Radiation Marrow aplasia is a major acute sequela of radiation. Radiation damages DNA; tissues dependent on active mitosis are particularly susceptible. Nuclear accidents involve not only power plant workers but also employees of hospitals, laboratories, and industry (food sterilization, metal radiography, etc.), as well as innocents exposed to stolen, misplaced, or misused sources. Whereas the radiation dose can be approximated from the rate and degree of decline in blood counts, dosimetry by reconstruction of the exposure can help to estimate the patient’s prognosis and also to protect medical personnel from contact with radioactive tissue and excreta. MDS and leukemia, but probably not aplastic anemia, are late effects of radiation.

1	Chemicals Benzene is a notorious cause of bone marrow failure: epidemiologic, clinical, and laboratory data link benzene to aplastic anemia, acute leukemia, and blood and marrow abnormalities. For leukemia, incidence is correlated with cumulative exposure, but susceptibility must also be important, because only a minority of even heavily exposed workers develop myelotoxicity. The employment history is important, especially in industries where benzene is used for a secondary purpose, usually as a solvent. Benzene-related blood diseases have declined with regulation of industrial exposure. Although benzene is no longer generally available as a household solvent, exposure to its metabolites occurs in the normal diet and in the environment. The association between marrow failure and other chemicals is much less well substantiated. Agents that regularly produce marrow depression as major toxicity in com monly used doses or normal exposures:

1	Agents that regularly produce marrow depression as major toxicity in com monly used doses or normal exposures: Cytotoxic drugs used in cancer chemotherapy: alkylating agents, antime tabolites, antimitotics, some antibiotics Agents that frequently but not inevitably produce marrow aplasia: Agents associated with aplastic anemia but with a relatively low probability: Antiprotozoals: quinacrine and chloroquine, mepacrine Nonsteroidal anti-inflammatory drugs (including phenylbutazone, indomethacin, ibuprofen, sulindac, aspirin) Anticonvulsants (hydantoins, carbamazepine, phenacemide, felbamate) Heavy metals (gold, arsenic, bismuth, mercury) Sulfonamides: some antibiotics, antithyroid drugs (methimazole, methyl- thiouracil, propylthiouracil), antidiabetes drugs (tolbutamide, chlorprop amide), carbonic anhydrase inhibitors (acetazolamide and methazolamide) Antihistamines (cimetidine, chlorpheniramine) Agents whose association with aplastic anemia is more tenuous:

1	Antihistamines (cimetidine, chlorpheniramine) Agents whose association with aplastic anemia is more tenuous: Other antibiotics (streptomycin, tetracycline, methicillin, mebendazole, trimethoprim/sulfamethoxazole, flucytosine) Sedatives and tranquilizers (chlorpromazine, prochlorperazine, piperaceta zine, chlordiazepoxide, meprobamate, methyprylon) Carbimazole Note: Terms set in italics show the most consistent association with aplastic anemia.

1	Drugs (Table 130-3) Many chemotherapeutic drugs have marrow suppression as a major toxicity; effects are dose dependent and will occur in all recipients. In contrast, idiosyncratic reactions to a large and diverse group of drugs may lead to aplastic anemia without a clear dose-response relationship. These associations rest largely on accumulated case reports until a large international study in Europe in the 1980s quantitated drug relationships, especially for nonsteroidal analgesics, sulfonamides, thyrostatic drugs, some psychotropics, penicillamine, allopurinol, and gold. Association does not equal causation: a drug may have been used to treat the first symptoms of bone marrow failure (antibiotics for fever or the preceding viral illness) or provoked the first symptom of a preexisting disease (petechiae by nonsteroidal anti-inflammatory agents administered to the thrombocytopenic patient). In the context of total drug use, idiosyncratic reactions, although individually devastating,

1	(petechiae by nonsteroidal anti-inflammatory agents administered to the thrombocytopenic patient). In the context of total drug use, idiosyncratic reactions, although individually devastating, are rare events. Risk estimates are usually lower when determined in population-based studies. Furthermore, the low absolute risk is also made more obvious: even a 10or 20-fold increase in risk translates, in a rare disease, to just a handful of drug-induced aplastic anemia cases among hundreds of thousands of exposed persons.

1	Infections Hepatitis is the most common preceding infection, and posthepatitis marrow failure accounts for approximately 5% of etiologies in most series. Patients are usually young men who have recovered from a bout of liver inflammation 1 to 2 months earlier; the subsequent pancytopenia is very severe. The hepatitis is seronegative (non-A, non-B, non-C) and possibly due to an as yet undiscovered infectious agent. Fulminant liver failure in childhood also follows seronegative hepatitis, and marrow failure occurs at a high rate in these patients. Aplastic anemia can rarely follow infectious mononucleosis. Parvovirus B19, the cause of transient aplastic crisis in hemolytic anemias and of some PRCAs (see below), does not usually cause generalized bone marrow failure. Mild blood count depression is frequent in the course of many viral and bacterial infections but resolves with the infection.

1	Immunologic Diseases Aplasia is a major consequence and the inevitable cause of death in transfusion-associated graft-versus-host disease (GVHD) that can occur after infusion of nonirradiated blood products to an immunodeficient recipient. Aplastic anemia is strongly associated with the rare collagen vascular syndrome eosinophilic fasciitis that is characterized by painful induration of subcutaneous tissues (Chap. 382). Thymoma and hypoimmunoglobulinemia are occasional associations with aplastic anemia. Pancytopenia with marrow hypoplasia can also occur in systemic lupus erythematosus (SLE). Pregnancy Aplastic anemia very rarely may occur and recur during pregnancy and resolve with delivery or with spontaneous or induced abortion.

1	Paroxysmal Nocturnal Hemoglobinuria An acquired mutation in the PIG-A gene in a hematopoietic stem cell is required for the development of PNH, but PIG-A mutations probably occur commonly in normal individuals. If the PIG-A mutant stem cell proliferates, the result is a clone of progeny deficient in glycosylphosphatidylinositol-linked cell surface membrane proteins (Chap. 129). Small clones of deficient cells can be detected by sensitive flow cytometry tests in one-half or more of patients with aplastic anemia at the time of presentation. Functional studies of bone marrow from PNH patients, even those with mainly hemolytic manifestations, show evidence of defective hematopoiesis. Patients with an initial clinical diagnosis of PNH, especially younger individuals, may later develop frank marrow aplasia and pancytopenia; patients with an initial diagnosis of aplastic anemia may suffer from hemolytic PNH years after recovery of blood counts.

1	Constitutional Disorders Fanconi anemia, an autosomal recessive disorder, manifests as congenital developmental anomalies, progressive pancytopenia, and an increased risk of malignancy. Chromosomes in Fanconi anemia are peculiarly susceptible to DNA cross-linking agents, the basis for a diagnostic assay. Patients with Fanconi anemia typically have short stature, café au lait spots, and anomalies involving the thumb, radius, and genitourinary tract. At least 16 different genetic defects (all but one with an identified gene) have been defined; the most common, type A Fanconi anemia, is due to a mutation in FANCA. Most of the Fanconi anemia gene products form a protein complex that activates FANCD2 by monoubiquitination to play a role in the cellular response to DNA damage and especially interstrand cross-linking.

1	Dyskeratosis congenita is characterized by the triad of mucous membrane leukoplasia, dystrophic nails, reticular hyperpigmentation, and with the development of aplastic anemia in childhood. Dyskeratosis is due to mutations in genes of the telomere repair complex, which acts to maintain telomere length in replicating cells: the X-linked variety is due to mutations in the DKC1 (dyskerin) gene; the more unusual autosomal dominant type is due to mutation in TERC, which encodes an RNA template, and TERT, which encodes the catalytic reverse transcriptase, telomerase. Mutations in TNF2, a component of the shelterin complex, proteins that bind the telomere DNA, also occur. In Shwachman-Diamond syndrome, presentation is early in life with neutropenia with pancreatic insufficiency and malabsorption; most patients have compound heterozygous mutations in SBDS that may affect both ribosomal biogenesis (as in Diamond-Blackfan anemia; see below) and marrow stroma function.

1	While these constitutional syndromes can on occasion present in adults, genetic mutations are also risk factors for bone marrow failure. In the recently recognized telomeropathies, mutations in TERT and TERC have subtle effects on hematopoietic function. Typical presentations include not only severe but also moderate aplastic anemia, which can be chronic and not progressive, and isolated macrocytic anemia or thrombocytopenia. Physical anomalies are usually not found in the patient, although early hair gray ing is a clue to the diagnosis. A careful family history may disclose pulmonary fibrosis and hepatic cirrhosis. Specific involvement of the bone marrow, liver, and lung is highly variable, as is penetrance of clinical phenotype, both within families and among kindreds. Variable penetrance means that TERT and TERC mutations represent risk factors for marrow failure, as family members with the same mutations may have normal or only slight hematologic abnormalities but more subtle

1	means that TERT and TERC mutations represent risk factors for marrow failure, as family members with the same mutations may have normal or only slight hematologic abnormalities but more subtle evidence of (compensated) hematopoietic insufficiency.

1	Bone marrow failure results from severe damage to the hematopoietic cell compartment. In aplastic anemia, replacement of the bone marrow by fat is apparent in the morphology of the biopsy specimen (Fig. 130-1) and magnetic resonance imaging (MRI) of the spine. Cells bearing the CD34 antigen, a marker of early hematopoietic cells, are greatly diminished, and in functional studies, committed and primitive progenitor cells are virtually absent; in vitro assays BD have suggested that the stem cell pool FIGURE 130-1 Normal and aplastic bone marrow. A. Normal bone marrow biopsy. B. Normal is reduced to ≤1% of normal in severe bone marrow aspirate smear. The marrow is normally 30–70% cellular, and there is a heterogeneous disease at the time of presentation. mix of myeloid, erythroid, and lymphoid cells. C. Aplastic anemia biopsy. D. Marrow smear in aplas-

1	An intrinsic stem cell defect exists tic anemia. The marrow shows replacement of hematopoietic tissue by fat and only residual stromal for the constitutional aplastic anemias: and lymphoid cells. cells from patients with Fanconi anemia exhibit chromosome damage and death on exposure to certain chemical agents. Telomeres are short in some patients with aplastic anemia, due to heterozygous mutations in genes of the telomere repair complex. Telomeres may also shorten physiologically in acquired marrow failure due to replicative demands on a limited stem cell pool.

1	Drug Injury Extrinsic damage to the marrow follows massive physical or chemical insults such as high doses of radiation and toxic chemicals. For the more common idiosyncratic reaction to modest doses of medical drugs, altered drug metabolism has been invoked as a likely mechanism. The metabolic pathways of many drugs and chemicals, especially if they are polar and have limited water solubility, involve enzymatic degradation to highly reactive electrophilic compounds; these intermediates are toxic because of their propensity to bind to cellular macromolecules. For example, derivative hydroquinones and quinolones are responsible for benzene-induced tissue injury. Excessive generation of toxic intermediates or failure to detoxify the intermediates may be genetically determined and apparent only on specific drug challenge; the complexity and specificity of the pathways imply multiple susceptibility loci and would provide an explanation for the rarity of idiosyncratic drug reactions.

1	Immune-Mediated Injury The recovery of marrow function in some patients prepared for bone marrow transplantation with antilymphocyte globulin first suggested that aplastic anemia might be immune mediated. Consistent with this hypothesis was the frequent failure of simple bone marrow transplantation from a syngeneic twin, without conditioning cytotoxic chemotherapy, which also argued both against simple stem cell absence as the cause and for the presence of a host factor producing marrow failure. Laboratory data support an important role for the immune system in aplastic anemia. Blood and bone marrow cells of patients can suppress normal hematopoietic progenitor cell growth, and removal of T cells from aplastic anemia bone marrow improves colony formation in vitro. Increased numbers of activated cytotoxic T cell clones are observed in aplastic anemia patients and usually decline with successful immunosuppressive therapy; type 1 cytokines are implicated; and interferon γ (IFN-γ) induces

1	cytotoxic T cell clones are observed in aplastic anemia patients and usually decline with successful immunosuppressive therapy; type 1 cytokines are implicated; and interferon γ (IFN-γ) induces Fas expression on CD34 cells, leading to apoptotic cell death. The early immune system events in aplastic anemia are not well understood, but an oligoclonal, T cell response implies antigenic stimulus. The rarity of aplastic anemia despite common exposures (medicines, seronegative hepatitis) suggests that genetically determined features of the immune response can convert a normal physiologic response into a sustained abnormal autoimmune process, including polymorphisms in histocompatibility antigens, cytokine genes, and genes that regulate T cell polarization and effector function.

1	CLINICAL FEATURES History Aplastic anemia can appear abruptly or insidiously. Bleeding is the most common early symptom; a complaint of days to weeks of easy bruising, oozing from the gums, nose bleeds, heavy menstrual flow, and sometimes petechiae will have been noticed. With thrombocytopenia, massive hemorrhage is unusual, but small amounts of bleeding in the central nervous system can result in catastrophic intra-cranial or retinal hemorrhage. Symptoms of anemia are also frequent, including lassitude, weakness, shortness of breath, and a pounding sensation in the ears. Infection is an unusual first symptom in aplastic anemia (unlike in agranulocytosis, where pharyngitis, anorectal infection, or frank sepsis occurs early). A striking feature of aplastic anemia is the restriction of symptoms to the hematologic system, and patients often feel and look remarkably well despite drastically reduced blood counts. Systemic complaints and weight loss should point to other etiologies of

1	symptoms to the hematologic system, and patients often feel and look remarkably well despite drastically reduced blood counts. Systemic complaints and weight loss should point to other etiologies of pancytopenia. Prior drug use, chemical exposure, and preceding viral illnesses must often be elicited with repeated questioning. A family history of hematologic diseases or blood abnormalities, of pulmonary or liver fibrosis, or of early hair graying points to a telomeropathy.

1	666 Physical Examination Petechiae and ecchymoses are typical, and retinal hemorrhages may be present. Pelvic and rectal examinations can often be deferred but, when performed, should be undertaken with great gentleness to avoid trauma; these will often show bleeding from the cervical os and blood in the stool. Pallor of the skin and mucous membranes is common except in the most acute cases or those already transfused. Infection on presentation is unusual but may occur if the patient has been symptomatic for a few weeks. Lymphadenopathy and splenomegaly are highly atypical of aplastic anemia. Café au lait spots and short stature suggest Fanconi anemia; peculiar nails and leukoplakia suggest dyskeratosis congenita; early graying (and use of hair dyes to mask it!) suggests a telomerase defect.

1	LABORATORY STUDIES Blood The smear shows large erythrocytes and a paucity of platelets and granulocytes. Mean corpuscular volume (MCV) is commonly increased. Reticulocytes are absent or few, and lymphocyte numbers may be normal or reduced. The presence of immature myeloid forms suggests leukemia or MDS; nucleated red blood cells (RBCs) suggest marrow fibrosis or tumor invasion; abnormal platelets suggest either peripheral destruction or MDS.

1	Bone Marrow The bone marrow is usually readily aspirated but dilute on smear, and the fatty biopsy specimen may be grossly pale on withdrawal; a “dry tap” instead suggests fibrosis or myelophthisis. In severe aplasia, the smear of the aspirated specimen shows only red cells, residual lymphocytes, and stromal cells; the biopsy (which should be >1 cm in length) is superior for determination of cellularity and shows mainly fat under the microscope, with hematopoietic cells occupying <25% of the marrow space; in the most serious cases, the biopsy is virtually all fat. The correlation between marrow cellularity and disease severity is imperfect, in part because marrow cellularity declines physiologically with aging. Additionally, some patients with moderate disease by blood counts will have empty iliac crest biopsies, whereas “hot spots” of hematopoiesis may be seen in severe cases. If an iliac crest specimen is inadequate, cells may also be obtained by aspiration from the sternum.

1	have empty iliac crest biopsies, whereas “hot spots” of hematopoiesis may be seen in severe cases. If an iliac crest specimen is inadequate, cells may also be obtained by aspiration from the sternum. Residual hematopoietic cells should have normal morphology, except for mildly megaloblastic erythropoiesis; megakaryocytes are invariably greatly reduced and usually absent. Granulomas may indicate an infectious etiology of the marrow failure.

1	Ancillary Studies Chromosome breakage studies of peripheral blood using diepoxybutane or mitomycin C should be performed on children and younger adults to exclude Fanconi anemia. Very short telomere length (available commercially) strongly suggests the presence of a telomerase or shelterin mutation, which can be pursued by family studies and nucleotide sequencing. Chromosome studies of bone marrow cells are often revealing in MDS and should be negative in typical aplastic anemia. Flow cytometry offers a sensitive diagnostic test for PNH. Serologic studies may show evidence of viral infection, such as Epstein-Barr virus and HIV. Posthepatitis aplastic anemia is seronegative. The spleen size should be determined by computed tomography (CT) scanning or ultrasound if the physical examination of the abdomen is unsatisfactory. Occasionally MRI may be helpful to assess the fat content of vertebrae in order to distinguish aplasia from MDS.

1	The diagnosis of aplastic anemia is usually straightforward, based on the combination of pancytopenia with a fatty bone marrow. Aplastic anemia is a disease of the young and should be a leading diagnosis in the pancytopenic adolescent or young adult. When pancytopenia is secondary, the primary diagnosis is usually obvious from either history or physical examination: the massive spleen of alcoholic cirrhosis, the history of metastatic cancer or SLE, or miliary tuberculosis on chest radiograph (Table 130-1).

1	Diagnostic problems can occur with atypical presentations and among related hematologic diseases. Although pancytopenia is most common, some patients with bone marrow hypocellularity have depression of only one or two of three blood lines, with later progression to pancytopenia. The bone marrow in constitutional aplastic anemia is morphologically indistinguishable from the aspirate in acquired disease. The diagnosis can be suggested by family history, abnormal blood counts since childhood, or the presence of associated physical anomalies. Aplastic anemia may be difficult to distinguish from the hypocellular variety of MDS: MDS is favored by finding morphologic abnormalities, particularly of megakaryocytes and myeloid precursor cells, and typical cytogenetic abnormalities (see below).

1	The natural history of severe aplastic anemia is rapid deterioration and death. Historically, provision first of RBC and later of platelet transfusions and effective antibiotics were of some benefit, but few patients show spontaneous recovery. The major prognostic determinant is the blood count. Severe disease has been defined by the presence of two of three parameters: absolute neutrophil count <500/μL, platelet count <20,000/μL, and corrected reticulocyte count <1% (or absolute reticulocyte count <60,000/μL). In the era of effective immunosuppressive therapies, absolute numbers of reticulocytes (>25,000/μL) and lymphocytes (>1000/μL) may be better predictors of response to treatment and long-term outcome.

1	Severe acquired aplastic anemia can be cured by replacement of the absent hematopoietic cells (and the immune system) by stem cell transplant, or it can be ameliorated by suppression of the immune system to allow recovery of the patient’s residual bone marrow function. Glucocorticoids are not of value as primary therapy. Suspect exposures to drugs or chemicals should be discontinued; however, spontaneous recovery of severe blood count depression is rare, and a waiting period before beginning treatment may not be advisable unless the blood counts are only modestly depressed.

1	This is the best therapy for the younger patient with a fully histocompatible sibling donor (Chap. 139e). Human leukocyte antigen (HLA) typing should be ordered as soon as the diagnosis of aplastic anemia is established in a child or younger adult. In transplant candidates, transfusion of blood from family members should be avoided so as to prevent sensitization to histocompatibility antigens, but limited numbers of blood products probably do not greatly affect outcome. For allogeneic transplant from fully matched siblings, long-term survival rates for children are approximately 90%. Transplant morbidity and mortality are increased among adults, due to the higher risk of chronic GVHD and serious infections.

1	Most patients do not have a suitable sibling donor. Occasionally, a full phenotypic match can be found within the family and serve as well. Far more available are other alternative donors, either unrelated but histocompatible volunteers or closely but not perfectly matched family members. High-resolution matching at HLA and more effective conditioning regimens and GVHD prophylaxis have led to improved survival rates in patients who proceed to alternative donor transplant, in some series approximating results with conventional sibling donors. Patients will be at risk for late complications, especially a higher rate of cancer, if radiation is used as a component of conditioning.

1	The standard regimen of antithymocyte globulin (ATG) in combination with cyclosporine induces hematologic recovery (independence from transfusion and a leukocyte count adequate to prevent infection) in 60–70% of patients. Children do especially well, whereas older adult patients often suffer complications due to the presence of comorbidities. An early robust hematologic response correlates with long-term survival. Improvement in granulocyte number is generally apparent within 2 months of treatment. Most recovered patients continue to have some degree of blood count depression, the MCV remains elevated, and bone marrow cellularity returns toward normal very slowly if at all. Relapse (recurrent pancytopenia) is frequent, often occurring as cyclosporine is discontinued; most, but not all, patients respond to reinstitution of immunosuppression, but some responders become dependent on continued cyclosporine administration. Development of MDS, with typical marrow morphologic or cytogenetic

1	respond to reinstitution of immunosuppression, but some responders become dependent on continued cyclosporine administration. Development of MDS, with typical marrow morphologic or cytogenetic abnormalities, occurs in approximately 15% of treated patients, usually but not invariably associated with a return of pancytopenia, and some patients develop leukemia. A laboratory diagnosis of PNH can generally be made at the time of presentation of aplastic anemia by flow cytometry; recovered patients may have frank hemolysis if the PNH clone expands. Bone marrow examinations should be performed if there is an unfavorable change in blood counts.

1	Horse ATG is administered as intravenous infusions over 4 days. ATG binds to peripheral blood cells; therefore, platelet and granulocyte numbers may decrease further during active treatment. Serum sickness, a flulike illness with a characteristic cutaneous eruption and arthralgia, often develops approximately 10 days after initiating treatment. Methylprednisolone is administered with ATG to ameliorate the immune consequences of heterologous protein infusion. Excessive or extended glucocorticoid therapy is associated with avascular joint necrosis. Cyclosporine is administered orally at an initial high dose, with subsequent adjustment according to blood levels obtained every 2 weeks; rough levels should be between 150 and 200 ng/mL. The most important side effects are nephrotoxicity, hypertension, seizures, and opportunistic infections, especially Pneumocystis jiroveci (prophylactic treatment with monthly inhaled pentamidine is recommended).

1	Most patients with aplastic anemia lack a suitable marrow donor, and immunosuppression is the treatment of choice. Overall survival is equivalent with transplantation and immunosuppression. However, successful transplant cures marrow failure, whereas patients who recover adequate blood counts after immunosuppression remain at risk of relapse and malignant evolution. Because of excellent results in children and younger adults, allogeneic transplant should be performed if a suitable sibling donor is available. Increasing age and the severity of neutropenia are the most important factors weighing in the decision between transplant and immunosuppression in adults who have a matched family donor: older patients do better with ATG and cyclosporine, whereas transplant is preferred if granulocytopenia is profound.

1	Outcomes following both transplant and immunosuppression have improved with time. High doses of cyclophosphamide, without stem cell rescue, have been reported to produce durable hematologic recovery, without relapse or evolution to MDS, but this treatment can produce sustained severe fatal neutropenia, and response is often delayed. The effectiveness of androgens has not been verified in controlled trials, but occasional patients will respond or even demonstrate blood count dependence on continued therapy. Sex hormones upregulate telomerase gene activity in vitro, which is possibly also their mechanism of action in improving marrow function. For patients with moderate disease, especially if a telomere defect is present, or those with severe pancytopenia in whom immunosuppression has failed, a 3to 4-month trial is appropriate.

1	Hematopoietic growth factors (HGFs) such as erythropoietin and granulocyte colony-stimulating factor (G-CSF) are not definitive therapy for severe aplastic anemia, and even their roles as adjuncts to immunosuppression are not clear. In research protocols, thrombopoietin mimetics have shown surprising activity in patients with refractory aplastic anemia, with patterns of blood count recovery suggesting that they act as stem cell stimulants.

1	Meticulous medical attention is required so that the patient may survive to benefit from definitive therapy or, having failed treatment, to maintain a reasonable existence in the face of pancytopenia. First and most important, infection in the presence of severe neutropenia must be aggressively treated by prompt institution 667 of parenteral, broad-spectrum antibiotics, usually ceftazidime or a combination of an aminoglycoside, cephalosporin, and semisynthetic penicillin. Therapy is empirical and must not await results of culture, although specific foci of infection such as oropharyngeal or anorectal abscesses, pneumonia, sinusitis, and typhlitis (necrotizing colitis) should be sought on physical examination and with radiographic studies. When indwelling plastic catheters become contaminated, vancomycin should be added. Persistent or recrudescent fever implies fungal disease: Candida and Aspergillus are common, especially after several courses of antibacterial antibiotics. A major

1	vancomycin should be added. Persistent or recrudescent fever implies fungal disease: Candida and Aspergillus are common, especially after several courses of antibacterial antibiotics. A major reason for the improved prognosis in aplastic anemia has been the development of better antifungal drugs and the timely institution of such therapy when infection is suspected. Granulocyte transfusions using G-CSF–mobilized peripheral blood may be effective in the treatment of overwhelming or refractory infections. Hand washing, the single best method of preventing the spread of infection, remains a neglected practice. Nonabsorbed antibiotics for gut decontamination are poorly tolerated and not of proven value. Total reverse isolation does not reduce mortality from infections.

1	Both platelet and erythrocyte numbers can be maintained by transfusion. Alloimmunization historically limited the usefulness of platelet transfusions and is now minimized by several strategies, including use of single donors to reduce exposure and physical or chemical methods to diminish leukocytes in the product; HLA-matched platelets are often effective in patients refractory to random donor products. Inhibitors of fibrinolysis such as aminocaproic acid have not been shown to relieve mucosal oozing; the use of low-dose glucocorticoids to induce “vascular stability” is unproven and not recommended. Whether platelet transfusions are better used prophylactically or only as needed remains unclear. Any rational regimen of prophylaxis requires transfusions once or twice weekly to maintain the platelet count >10,000/μL (oozing from the gut increases precipitously at counts <5000/μL). Menstruation should be suppressed either by oral estrogens or nasal follicle-stimulating

1	to maintain the platelet count >10,000/μL (oozing from the gut increases precipitously at counts <5000/μL). Menstruation should be suppressed either by oral estrogens or nasal follicle-stimulating hormone/luteinizing hormone (FSH/LH) antagonists. Aspirin and other nonsteroidal anti-inflammatory agents inhibit platelet function and must be avoided.

1	RBCs should be transfused to maintain a normal level of activity, usually at a hemoglobin value of 70 g/L (90 g/L if there is underlying cardiac or pulmonary disease); a regimen of 2 units every 2 weeks will replace normal losses in a patient without a functioning bone marrow. In chronic anemia, the iron chelators, deferoxamine and deferasirox, should be added at approximately the fiftieth transfusion to avoid secondary hemochromatosis.

1	Other, more restricted forms of marrow failure occur, in which only a single circulating cell type is affected and the marrow shows corresponding absence or decreased numbers of specific precursor cells: aregenerative anemia as in PRCA (see below), thrombocytopenia with amegakaryocytosis (Chap. 140), and neutropenia without marrow myeloid cells in agranulocytosis (Chap. 80). In general, and in contrast to aplastic anemia and MDS, the unaffected lineages appear quantitatively and qualitatively normal. Agranulocytosis, the most frequent of these syndromes, is usually a complication of medical drug use (with agents similar to those related to aplastic anemia), either by a mechanism of direct chemical toxicity or by immune destruction. Agranulocytosis has an incidence similar to aplastic anemia but is especially frequent among older adults and in women. The syndrome should resolve with discontinuation of exposure, but significant mortality is attached to neutropenia in the older and often

1	but is especially frequent among older adults and in women. The syndrome should resolve with discontinuation of exposure, but significant mortality is attached to neutropenia in the older and often previously unwell patient. Both pure white cell aplasia (agranulocytosis without incriminating drug exposure) and amegakaryocytic thrombocytopenia are exceedingly rare and, like PRCA, appear to be due to destructive antibodies or lymphocytes and can respond to immunosuppressive therapies. In all of the single-lineage failure syndromes, progression to pancytopenia or leukemia is unusual.

1	is probably the more common immune mechanism. Cytotoxic lym- Transient erythroblastopenia of childhood killer cell activity inhibitory of erythropoiesis have been demonstrated Transient aplastic crisis of hemolysis (acute B19 parvovirus infection) in particularly well-studied individual cases.

1	Transient aplastic crisis of hemolysis (acute B19 parvovirus infection) in particularly well-studied individual cases. Fetal red blood cell aplasia Nonimmune hydrops fetalis (in utero B19 parvovirus infection) PERSISTENT PARVOVIRUS B19 INFECTION Hereditary pure red cell aplasia Chronic parvovirus infection is an important, treatable cause of PRCA. Congenital pure red cell aplasia (Diamond-Blackfan anemia) This common virus causes a benign exanthem of childhood (fifth disease) and a polyarthralgia/arthritis syndrome in adults. In patients with production), parvovirus infection can cause a transient aplastic crisis and an abrupt but temporary worsening of the anemia due to failed erythropoiesis. In normal individuals, acute infection is resolved by Paraneoplastic to solid tumors production of neutralizing antibodies to the virus, but in the setting of Connective tissue disorders with immunologic abnormalities congenital, acquired, or iatrogenic immunodeficiency, persistent viral

1	Systemic lupus erythematosus, juvenile rheumatoid arthritis, rheumatoid infection may occur. The bone marrow shows red cell aplasia and the arthritis presence of giant pronormoblasts (Fig. 130-2), which is the cytopathic Multiple endocrine gland insufficiency sign of B19 parvovirus infection. Viral tropism for human erythroid progenitor cells is due to its use of erythrocyte P antigen as a cellular receptor for entry. Direct cytotoxicity of virus causes anemia if demands Persistent B19 parvovirus, hepatitis, adult T cell leukemia virus, Epstein-Barr on erythrocyte production are high; in normal individuals, the tempo- rary cessation of red cell production is not clinically apparent, and skin and joint symptoms are mediated by immune complex deposition. Especially phenytoin, azathioprine, chloramphenicol, procainamide, isoniazid Antibodies to erythropoietin

1	Especially phenytoin, azathioprine, chloramphenicol, procainamide, isoniazid Antibodies to erythropoietin History, physical examination, and routine laboratory studies may disclose an underlying disease or a drug exposure. Thymoma should be sought by radiographic procedures. Tumor excision PRCA is characterized by anemia, reticulocytopenia, and absent or rare erythroid precursor cells in the bone marrow. The classification of PRCA is shown in Table 130-4. In adults, PRCA is acquired. An identical syndrome can occur constitutionally: Diamond-Blackfan anemia, or congenital PRCA, is diagnosed at birth or in early childhood and often responds to glucocorticoid treatment; mutations in ribosome protein genes are etiologic. Temporary red cell failure occurs in transient aplastic crisis of hemolytic anemias due to acute parvovirus infection (Chap. 221) and in transient erythroblastopenia of child- hood, which occurs in normal children.

1	PRCA has important associations with immune system diseases. A small minority of cases occur with a thymoma. More frequently, red cell aplasia can be the major manifestation of large granular lymphocytosis or complicate chronic lymphocytic leukemia. Some patients may be hypogammaglobulinemic. Infrequently (compared to agranulocytosis), PRCA can be due to an idiosyncratic drug reaction. Subcutaneous administration of erythropoietin (EPO) has provoked PRCA mediated by neu-CD tralizing antibodies. FIGURE 130-2 Pathognomonic cells in marrow failure syndromes. A. Giant pronormoblast, the

1	Like aplastic anemia, PRCA results cytopathic effect of B19 parvovirus infection of the erythroid progenitor cell. B. Uninuclear mega-from diverse mechanisms. Antibodies karyocyte and microblastic erythroid precursors typical of the 5q–myelodysplasia syndrome. to RBC precursors are frequently pres-C. Ringed sideroblast showing perinuclear iron granules. D. Tumor cells present on a touch preparaent in the blood, but T cell inhibition tion made from the marrow biopsy of a patient with metastatic carcinoma. is indicated, but anemia does not necessarily improve with surgery. The diagnosis of parvovirus infection requires detection of viral DNA sequences in the blood (IgG and IgM antibodies are commonly absent). The presence of erythroid colonies has been considered predictive of response to immunosuppressive therapy in idiopathic PRCA.

1	Red cell aplasia is compatible with long-term survival with supportive care alone: a combination of erythrocyte transfusions and iron chelation. For persistent B19 parvovirus infection, almost all patients respond to intravenous immunoglobulin therapy (e.g., 0.4 g/kg daily for 5 days), although relapse and retreatment may be expected, especially in patients with AIDS. The majority of patients with idiopathic PRCA respond favorably to immunosuppression. Most first receive a course of glucocorticoids. Also effective are cyclosporine, ATG, azathioprine, and cyclophosphamide.

1	The myelodysplastic syndromes (MDS) are a heterogeneous group of hematologic disorders broadly characterized by both (1) cytopenias due to bone marrow failure and (2) a high risk of development of acute myeloid leukemia (AML). Anemia, often with thrombocytopenia and 669 neutropenia, occurs with dysmorphic (abnormal appearing) and usually cellular bone marrow, which is evidence of ineffective blood cell production. In patients with “low-risk” MDS, marrow failure dominates the clinical course. In other patients, myeloblasts are present at diagnosis, chromosomes are abnormal, and the “high risk” is due to leukemic progression. MDS may be fatal due to the complications of pancytopenia or the incurability of leukemia, but a large proportion of patients will die of concurrent disease, the comorbidities typical in an elderly population. A clinically useful nosology of these often confusing entities was first developed by the French-American-British Cooperative Group in 1983. Five entities

1	typical in an elderly population. A clinically useful nosology of these often confusing entities was first developed by the French-American-British Cooperative Group in 1983. Five entities were defined: refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess blasts (RAEB), refractory anemia with excess blasts in transformation (RAEB-t), and chronic myelomonocytic leukemia (CMML). The World Health Organization (WHO) classification (2002) recognized that the distinction between RAEB-t and AML is arbitrary and grouped them together as acute leukemia and that CMML behaves as a myeloproliferative disease; the WHO classification also separated refractory anemias with dysmorphic change restricted to erythroid lineage from those with multilineage changes. In a 2008 revision, specific categories for unilineage dysplasias were added (Table 130-5).

1	Refractory cytopenias with unilineage dysplasia (RCUD): Refractory anemia (RA) 10–20% Anemia Unilineage erythroid dysplasia (in ≥10% of cells) <1% of blasts <5% blasts Refractory neutropenia (RN) <1% Neutropenia Unilineage granulocytic dysplasia <1% blasts <5% blasts Refractory thrombocytopenia (RT) <1% Thrombocytopenia Unilineage megakaryocytic dysplasia <1% blasts <5% blasts Refractory anemia with ringed 3–11% Anemia Unilineage erythroid dysplasia ≥15% of erythroid sideroblasts (RARS) precursors are ringed sideroblasts Refractory cytopenias with 30% Cytopenia(s) Multilineage dysplasia ± ringed sideroblasts multilineage dysplasia (RCMD) Refractory anemia with excess blasts, 40% Cytopenia(s) Unilineage or multilineage dysplasia Refractory anemia with excess blasts, MDS associated with isolated del(5q) Uncommon Anemia Isolated 5q31 chromosome deletion Childhood MDS, including refractory <1% Pancytopenia <5% marrow blasts for RCC cytopenia of childhood (provisional) (RCC)

1	Childhood MDS, including refractory <1% Pancytopenia <5% marrow blasts for RCC cytopenia of childhood (provisional) (RCC) MDS, unclassifiable (MDS-U) ? Cytopenia Does not fit other categories

1	MDS, unclassifiable (MDS-U) ? Cytopenia Does not fit other categories If no dysplasia, MDS-associated karyotype Note: If peripheral blood blasts are 2–4%, the diagnosis is RAEB-1 even if marrow blasts are <5%. If Auer rods are present, the WHO considers the diagnosis RAEB-2 if the blast proportion is <20% (even if <10%), or acute myeloid leukemia (AML) if at least 20% blasts. For all subtypes, peripheral blood monocytes are <1 × 109/L. Bicytopenia may be observed in RCUD subtypes, but pancytopenia with unilineage marrow dysplasia should be classified as MDS-U. Therapy-related MDS (t-MDS), whether due to alkylating agents or topoisomerase II inhibitors (t-MDS/t-AML) is now included in the WHO classification of myeloid neoplasms. The listing in this table excludes MDS/myeloproliferative neoplasm overlap categories, such as chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, and the provisional entity RARS with thrombocytosis.

1	Abbreviation: MDS, myelodysplastic syndrome. 670 The diagnosis of MDS may be a challenge, because sometimes subtle clinical and pathologic features must be distinguished and precise diagnostic categorization requires a hematopathologist knowledgeable in the latest classification scheme. Nonetheless, it is important that the internist and primary care physician be sufficiently familiar with MDS to expedite referral to a hematologist, both because many new therapies are now available to improve hematopoietic function and the judicious use of supportive care can improve the patient’s quality of life.

1	Idiopathic MDS is a disease of the elderly; the mean age at onset is older than 70 years. There is a slight male preponderance. MDS is a relatively common form of bone marrow failure, with reported incidence rates of 35 to >100 per million persons in the general population and 120 to >500 per million in the older adult. MDS is rare in children, but monocytic leukemia can be seen. Secondary or therapy-related MDS is not age related. Rates of MDS have increased over time, due to better recognition of the syndrome by physicians and an aging population.

1	MDS is associated with environmental exposures such as radiation and benzene; other risk factors have been reported inconsistently. Secondary MDS occurs as a late toxicity of cancer treatment, usually a combination of radiation and the radiomimetic alkylating agents such as busulfan, nitrosourea, or procarbazine (with a latent period of 5–7 years) or the DNA topoisomerase inhibitors (2-year latency). Acquired aplastic anemia, Fanconi anemia, and other constitutional marrow failure diseases can evolve into MDS. However, the typical MDS patient does not have a suggestive environmental exposure history or a preceding hematologic disease. MDS is a disease of aging, suggesting random cumulative intrinsic and environmental damage to marrow cells.

1	MDS is a clonal hematopoietic stem cell disorder characterized by disordered cell proliferation and impaired differentiation, resulting in cytopenias and risk of progression to leukemia. Both chromosomal and genetic instability have been implicated, and both are likely aging-related. Cytogenetic abnormalities are found in approximately one-half of patients, and some of the same specific lesions are also seen in frank leukemia; aneuploidy (chromosome loss or gain) is more frequent than translocations. More sensitive assays, such as comparative genomic hybridization and single nucleotide polymorphism arrays, reveal chromosomal abnormalities in a large proportion of patients with normal conventional cytogenetics. Accelerated telomere attrition may destabilize the genome in marrow failure and predispose to acquisition of chromosomal lesions. Cytogenetic abnormalities are not random (loss of all or part of 5, 7, and 20, trisomy of 8) and may be related to etiology (11q23 following

1	and predispose to acquisition of chromosomal lesions. Cytogenetic abnormalities are not random (loss of all or part of 5, 7, and 20, trisomy of 8) and may be related to etiology (11q23 following topoisomerase II inhibitors). The type and number of cytogenetic abnormalities strongly correlate with the probability of leukemic transformation and survival.

1	Genomics has illuminated the role of point mutations in the pathophysiology of MDS. Recurrent somatic mutations, acquired in the abnormal marrow cells and absent in the germline, have been identified in almost 100 genes. Many of the same genes are also mutated in AML without MDS, whereas others are distinctive in subtypes of MDS. A prominent example of the latter is the discovery of mutations in genes of the RNA splicing machinery, especially SF3B1, which strongly associate with sideroblastic anemia. Some mutations correlate with prognosis: spliceosome defects with favorable outcome, and mutations in EZH2, TP53, RUNX1, and ASXL1 with poor outcome. Mutations and cytogenetic abnormalities are not independent: TP53 mutations associate with complex cytogenetic abnormalities and TET2 mutations with normal cytogenetics. Correlation and exclusion in the pattern of mutations indicate a functional genomic architecture. Analysis of deep sequencing results in patients whose MDS evolved to AML

1	with normal cytogenetics. Correlation and exclusion in the pattern of mutations indicate a functional genomic architecture. Analysis of deep sequencing results in patients whose MDS evolved to AML has shown evidence of clonal succession, with founder clones acquiring further mutations that allow clonal dominance. Furthermore, the prevalence of abnormal cells by morphology underestimates bone marrow involvement by MDS clones, as cells normal in appearance are apparently derived from the abnormal clones. Both presenting and evolving hematologic manifestations result from the accumulation of multiple genetic lesions: loss of tumor-suppressor genes, activating oncogene mutations, epigenetic pathways that affect mRNA processing and methylation status, or other harmful alterations. Pathophysiology has been linked to mutations and chromosome abnormalities in some specific MDS syndromes. The 5q– deletion leads to heterozygous loss of a ribosomal protein gene that is also mutant in

1	has been linked to mutations and chromosome abnormalities in some specific MDS syndromes. The 5q– deletion leads to heterozygous loss of a ribosomal protein gene that is also mutant in Diamond-Blackfan anemia, and both are characterized by deficient erythropoiesis. An immune pathophysiology may underlie trisomy 8 MDS, in which patients often experience improved blood counts after immunosuppressive therapy; there is T cell activity directed to hematopoietic progenitors, which the cytogenetically aberrant clone resists. However, in general for MDS, the role of the immune system and its cells and cytokines; the role of the hematopoietic stem cell niche, the microenvironment, and cell-cell interactions; the fate of normal cells in the Darwinian competitive environment of the dysplastic marrow; and how mutant cells produce marrow failure in MDS are not well understood.

1	Anemia dominates the early course. Most symptomatic patients complain of the gradual onset of fatigue and weakness, dyspnea, and pallor, but at least one-half the patients are asymptomatic, and their MDS is discovered only incidentally on routine blood counts. Previous chemotherapy or radiation exposure is an important historic fact. Fever and weight loss should point to a myeloproliferative rather than myelodysplastic process. MDS in childhood is rare and, when diagnosed, increases the likelihood of an underlying genetic disease. Children with Down syndrome are susceptible to MDS, and a family history may indicate a hereditary form of sideroblastic anemia, Fanconi anemia, or a telomeropathy. Inherited GATA2 mutations, as in the MonoMAC syndrome (with increased susceptibility to viral, mycobacteria, and fungal infections, as well as deficient numbers of monocytes, natural killer cells, and B lymphocytes), also cause MDS in young patients.

1	The physical examination is remarkable for signs of anemia; approximately 20% of patients have splenomegaly. Some unusual skin lesions, including Sweet syndrome (febrile neutrophilic dermatosis), occur with MDS. Accompanying autoimmune syndromes are not infrequent. In the younger patient, stereotypical anomalies point to a constitutional syndrome (short stature, abnormal thumbs in Fanconi anemia; early graying in the telomeropathies; cutaneous warts in GATA2 deficiency).

1	LABORATORY STUDIES Blood Anemia is present in most cases, either alone or as part of bi-or pancytopenia; isolated neutropenia or thrombocytopenia is more unusual. Macrocytosis is common, and the smear may be dimorphic with a distinctive population of large red blood cells. Platelets are also large and lack granules. In functional studies, they may show marked abnormalities, and patients may have bleeding symptoms despite seemingly adequate numbers. Neutrophils are hypogranulated; have hyposegmented, ringed, or abnormally segmented nuclei; contain Döhle bodies; and may be functionally deficient. Circulating myeloblasts usually correlate with marrow blast numbers, and their quantity is important for classification and prognosis. The total white blood cell count (WBC) is usually normal or low, except in chronic myelomonocytic leukemia. As in aplastic anemia, MDS can be associated with a clonal population of PNH cells. Genetic testing is commercially available for constitutional

1	or low, except in chronic myelomonocytic leukemia. As in aplastic anemia, MDS can be associated with a clonal population of PNH cells. Genetic testing is commercially available for constitutional syndromes.

1	Bone Marrow The bone marrow is usually normal or hypercellular, but in about 20% of cases, it is sufficiently hypocellular to be confused with aplasia. No single characteristic feature of marrow morphology distinguishes MDS, but the following are commonly observed: dyserythropoietic changes (especially nuclear abnormalities) and ringed sideroblasts in the erythroid lineage; hypogranulation and hyposegmentation in granulocytic precursors, with an increase in myeloblasts; and megakaryocytes showing reduced numbers of or disorganized aGood, normal, –Y, del(5q), del (20q); poor, complex (≥3 abnormalities) or chromosome 7 abnormalities; intermediate, all other abnormalities. bCytopenias defined as hemoglobin <100 g/L, platelet count <100,000/μL, and absolute neutrophil count <1500/μL.

1	nuclei. Megaloblastic nuclei associated with defective hemoglobinization in the erythroid lineage are common. Prognosis strongly correlates with the proportion of marrow blasts. Cytogenetic analysis and fluorescent in situ hybridization can identify chromosomal abnormalities. Deficiencies of vitamin B12 or folate should be excluded by appropriate blood tests; vitamin B6 deficiency can be assessed by a therapeutic trial of pyridoxine if the bone marrow shows ringed sideroblasts. Marrow dysplasia can be observed in acute viral infections, drug reactions, or chemical toxicity but should be transient. More difficult are the distinctions between hypocellular MDS and aplasia or between refractory anemia with excess blasts and early acute leukemia. The WHO considers the presence of 20% blasts in the marrow as the criterion that separates AML from MDS. In young patients, underlying, predisposing genetic diseases should be considered (see above).

1	The median survival varies greatly from years for patients with 5q– or sideroblastic anemia to a few months in refractory anemia with excess blasts or severe pancytopenia associated with monosomy 7; an International Prognostic Scoring System (IPSS; Table 130-6) assists in making predictions. Even “low-risk” MDS has significant morbidity and mortality. Most patients die as a result of complications of pancytopenia and not due to leukemic transformation; perhaps one-third will succumb to other diseases unrelated to their MDS. Precipitous worsening of pancytopenia, acquisition of new chromosomal abnormalities on serial cytogenetic determination, increase in the number of blasts, and marrow fibrosis are all poor prognostic indicators. The outlook in therapy-related MDS, regardless of type, is extremely poor, and most patients will progress within a few months to refractory AML.

1	Historically, the therapy of MDS has been unsatisfactory, but new drugs recently have been approved for this disease. Several regimens appear to not only improve blood counts but to delay onset of leukemia and to improve survival. The choice of therapy for an individual patient, administration of treatment, and management of toxicities are complicated and require hematologic expertise.

1	Only hematopoietic stem cell transplantation offers cure of MDS. The current survival rate in selected patient cohorts is ~50% at 3 years and is improving. Results using unrelated matched donors are now similar to those obtained using siblings, and patients in their 50s and 60s have been successfully transplanted. Nevertheless, treatment-related mortality and morbidity increase with recipient age. Complicating the decision to undertake transplant is that the high-risk patient, for whom the procedure is most obviously indicated, has a high probability of a poor outcome from transplant-related mortality or disease relapse, whereas the low-risk patient, who is more likely to tolerate transplant, also may do well for years 671 with less aggressive therapies.

1	MDS has been regarded as particularly refractory to cytotoxic chemotherapy regimens, and as in AML in the older adult, drug toxicity is frequent and often fatal, and remissions if achieved are brief. Low doses of cytotoxic drugs have been administered for their “differentiation” potential, and from this experience, drug therapies have emerged based on pyrimidine analogues. These new drugs are classified as epigenetic modulators, believed to act through a demethylating mechanism to alter gene regulation and allow differentiation to mature blood cells from the abnormal MDS stem cell (although global methylation status has not correlated with clinical efficacy). Azacitidine and decitabine are two epigenetic modifiers frequently used in bone marrow failure clinics. Azacitidine improves blood counts and survival in MDS, compared to best supportive care. Azacitidine is usually administered subcutaneously, daily for 7 days, at 4-week intervals, for at least four cycles before assessing for

1	counts and survival in MDS, compared to best supportive care. Azacitidine is usually administered subcutaneously, daily for 7 days, at 4-week intervals, for at least four cycles before assessing for response. Overall, generally improved blood counts with a decrease in transfusion requirements occurred in ~50% of patients in published trials. Response is dependent on continued drug administration, and most patients eventually will no longer respond and experience recurrent cytopenias or progression to AML. Decitabine is closely related to azacitidine and more potent; 30–50% of patients show responses in blood counts, with a duration of response of almost a year. Decitabine is usually administered by continuous intravenous infusion in regimens of varying doses and durations of 3 to 10 days in repeating cycles. The major toxicity of azacitidine and decitabine is myelosuppression, leading to worsened blood counts. Other symptoms associated with cancer chemotherapy frequently occur.

1	days in repeating cycles. The major toxicity of azacitidine and decitabine is myelosuppression, leading to worsened blood counts. Other symptoms associated with cancer chemotherapy frequently occur. Demethylating agents are frequently used in the high-risk patient who is not a candidate for stem cell transplant. In the lower risk patient, they are also effective, but alternative therapies should be considered.

1	Lenalidomide, a thalidomide derivative with a more favorable toxicity profile, is particularly effective in reversing anemia in MDS patients with 5q– syndrome; not only do a high proportion of these patients become transfusion independent with normal or near-normal hemoglobin levels, but their cytogenetics also become normal. The drug has many biologic activities, and it is unclear which is critical for clinical efficacy. Lenalidomide is administered orally. Most patients will improve within 3 months of initiating therapy. Toxicities include myelosuppression (worsening thrombocytopenia and neutropenia, necessitating blood count monitoring) and an increased risk of deep vein thrombosis and pulmonary embolism.

1	Immunosuppression, as used in aplastic anemia, also may produce sustained independence from transfusion and improve survival. ATG, cyclosporine, and the anti-CD52 monoclonal antibody alemtuzumab are especially effective in younger MDS patients (<60 years old) with more favorable IPSS scores and who bear the histocompatibility antigen HLA-DR15. HGFs can improve blood counts but, as in most other marrow failure states, have been most beneficial to patients with the least severe pancytopenia. EPO alone or in combination with G-CSF can improve hemoglobin levels, but mainly in those with low serum EPO levels who have no or only a modest need for transfusions. Survival does not appear to be improved by G-CSF treatment alone but may be enhanced by erythropoietin and amelioration of anemia. G-CSF treatment alone failed to improve survival in a controlled trial.

1	The same principles of supportive care described for aplastic anemia apply to MDS. Despite improvements in drug therapy, many patients will be anemic for years. RBC transfusion support should be accompanied by iron chelation to prevent secondary hemochromatosis.

1	Fibrosis of the bone marrow (see Fig. 129-2), usually accompanied by a characteristic blood smear picture called leukoerythroblastosis, can occur as a primary hematologic disease, called myelofibrosis or myeloid metaplasia (Chap. 131), and as a secondary process, called 672 myelophthisis. Myelophthisis, or secondary myelofibrosis, is reactive. Fibrosis can be a response to invading tumor cells, usually an epithelial cancer of breast, lung, or prostate origin or neuroblastoma. Marrow fibrosis may occur with infection of mycobacteria (both Mycobacterium tuberculosis and Mycobacterium avium), fungi, or HIV and in sarcoidosis. Intracellular lipid deposition in Gaucher’s disease and obliteration of the marrow space related to absence of osteoclast remodeling in congenital osteopetrosis also can produce fibrosis. Secondary myelofibrosis is a late consequence of radiation therapy or treatment with radiomimetic drugs. Usually the infectious or malignant underlying processes are obvious.

1	can produce fibrosis. Secondary myelofibrosis is a late consequence of radiation therapy or treatment with radiomimetic drugs. Usually the infectious or malignant underlying processes are obvious. Marrow fibrosis can also be a feature of a variety of hematologic syndromes, especially chronic myeloid leukemia, multiple myeloma, lymphomas, myeloma, and hairy cell leukemia. The pathophysiology has three distinct features: proliferation of fibroblasts in the marrow space (myelofibrosis); the extension of hematopoiesis into the long bones and into extramedullary sites, usually the spleen, liver, and lymph nodes (myeloid metaplasia); and ineffective erythropoiesis. The etiology of the fibrosis is unknown but most likely involves dysregulated production of growth factors: platelet-derived growth factor and transforming growth factor β have been implicated. Abnormal regulation of other hematopoietins would lead to localization of blood-producing cells in nonhematopoietic tissues and

1	growth factor and transforming growth factor β have been implicated. Abnormal regulation of other hematopoietins would lead to localization of blood-producing cells in nonhematopoietic tissues and uncoupling of the usually balanced processes of stem cell proliferation and differentiation. Myelofibrosis is remarkable for pancytopenia despite very large numbers of circulating hematopoietic progenitor cells. Anemia is dominant in secondary myelofibrosis, usually normocytic and normochromic. The diagnosis is suggested by the characteristic leukoerythroblastic smear (see Fig. 129-1). Erythrocyte morphology is highly abnormal, with circulating nucleated RBCs, teardrops, and shape distortions. WBC numbers are often elevated, sometimes mimicking a leukemoid reaction, with circulating myelocytes, promyelocytes, and myeloblasts. Platelets may be abundant and are often of giant size. Inability to aspirate the bone marrow, the characteristic “dry tap,” can allow a presumptive diagnosis in the

1	promyelocytes, and myeloblasts. Platelets may be abundant and are often of giant size. Inability to aspirate the bone marrow, the characteristic “dry tap,” can allow a presumptive diagnosis in the appropriate setting before the biopsy is decalcified. The course of secondary myelofibrosis is determined by its etiology, usually a metastatic tumor or an advanced hematologic malignancy. Treatable causes must be excluded, especially tuberculosis and fungus. Transfusion support can relieve symptoms.

1	Polycythemia Vera and Other Myeloproliferative Neoplasms Jerry L. Spivak The World Health Organization (WHO) classification of the chronic myeloproliferative neoplasms (MPNs) includes eight disorders, some of which are rare or poorly characterized (Table 131-1) but all of 131 which share an origin in a multipotent hematopoietic progenitor cell; overproduction of one or more of the formed elements of the blood without significant dysplasia; and a predilection to extramedullary hematopoiesis, myelofibrosis, and transformation at varying rates to acute leukemia. Within this broad classification, however, significant phenotypic heterogeneity exists. Some diseases such as chronic myelogenous leukemia (CML), chronic neutrophilic leukemia (CNL), and chronic eosinophilic leukemia (CEL) express primarily a myeloid phenotype, whereas in other diseases, such as polycythemia vera (PV), primary myelofibrosis (PMF), and essential thrombocytosis (ET), erythroid or megakaryocytic hyperplasia

1	primarily a myeloid phenotype, whereas in other diseases, such as polycythemia vera (PV), primary myelofibrosis (PMF), and essential thrombocytosis (ET), erythroid or megakaryocytic hyperplasia predominates. The latter three disorders, in contrast to the former three, also appear capable of transforming into each other.

1	Chronic myeloid leukemia, bcr-abl–positive Chronic eosinophilic leukemia, not otherwise specified Myeloproliferative neoplasms, unclassifiable

1	Such phenotypic heterogeneity has a genetic basis; CML is the consequence of the balanced translocation between chromosomes 9 and 22 [t(9;22)(q34;11)]; CNL has been associated with a t(15;19) translocation; and CEL occurs with a deletion or balanced translocations involving the PDGFRα gene. By contrast, to a greater or lesser extent, PV, PMF, and ET are characterized by a mutation, V617F, that causes constitutive activation of JAK2, a tyrosine kinase essential for the function of the erythropoietin and thrombopoietin receptors but not the granulocyte colony-stimulating factor receptor. This important distinction is also reflected in the natural histories of CML, CNL, and CEL, which are usually measured in years, and their high rate of leukemic transformation. By contrast, the natural history of PV, PMF, and ET is usually measured in decades, and transformation to acute leukemia is uncommon in PV and ET in the absence of exposure to mutagenic drugs. This chapter, therefore, will focus

1	of PV, PMF, and ET is usually measured in decades, and transformation to acute leukemia is uncommon in PV and ET in the absence of exposure to mutagenic drugs. This chapter, therefore, will focus only on PV, PMF, and ET, because their clinical and genetic overlap is substantial even though their clinical courses are distinctly different.

1	The other chronic myeloproliferative neoplasms will be discussed in Chaps. 133 and 135e. PV is a clonal disorder involving a multipotent hematopoietic progenitor cell in which phenotypically normal red cells, granulocytes, and platelets accumulate in the absence of a recognizable physiologic stimulus. The most common of the chronic MPNs, PV occurs in 2.5 per 100,000 persons, sparing no adult age group and increasing with age to rates over 10/100,000. Familial transmission is infrequent, and women predominate among sporadic cases.

1	The etiology of PV is unknown. Although nonrandom chromo some abnormalities such as deletion 20q and trisomy 8 and 9 have been documented in up to 30% of untreated PV patients, unlike CML, no consistent cytogenetic abnormality has been associated with the disorder. However, a mutation in the autoinhibitory pseudokinase domain of the tyrosine kinase JAK2—that replaces valine with phenylalanine (V617F), causing constitutive kinase activation—appears to have a central role in the pathogenesis of PV.

1	JAK2 is a member of an evolutionarily well-conserved, nonreceptor tyrosine kinase family and serves as the cognate tyrosine kinase for the erythropoietin and thrombopoietin receptors. It also functions as an obligate chaperone for these receptors in the Golgi apparatus and is responsible for their cell-surface expression. The conformational change induced in the erythropoietin and thrombopoietin receptors following binding to their respective cognate ligands, erythropoietin or thrombopoietin, leads to JAK2 autophosphorylation, receptor phosphorylation, and phosphorylation of proteins involved in cell proliferation, differentiation, and resistance to apoptosis. Transgenic animals lacking JAK2 die as embryos from severe anemia. Constitutive activation of JAK2, on the other hand, explains the erythropoietin hypersensitivity, erythropoietin-independent erythroid colony formation, rapid terminal differentiation, increase in Bcl-XL expression, and apoptosis resistance in the absence of

1	the erythropoietin hypersensitivity, erythropoietin-independent erythroid colony formation, rapid terminal differentiation, increase in Bcl-XL expression, and apoptosis resistance in the absence of erythropoietin that characterize the in vitro behavior of PV erythroid progenitor cells.

1	Importantly, the JAK2 gene is located on the short arm of chromo some 9, and loss of heterozygosity on chromosome 9p due to mitotic recombination is the most common cytogenetic abnormality in PV. The segment of 9p involved contains the JAK2 locus, and loss of heterozygosity in this region leads to homozygosity for JAK2 V617F. More than 95% of PV patients express this mutation, as do approximately 50% of PMF and ET patients. Homozygosity for the mutation occurs in approximately 30% of PV patients and 60% of PMF patients but is rare in ET. Over time, a portion of PV JAK2 V617F heterozygotes become homozygotes due to mitotic recombination, but usually not after 10 years of the disease. Most PV patients who do not express JAK2 V617F express a mutation in exon 12 of the kinase and are not clinically different from those who do, nor do JAK2 V617F heterozygotes differ clinically from homozygotes. Interestingly, the predisposition to acquire mutations in JAK2 appears to be associated with a

1	different from those who do, nor do JAK2 V617F heterozygotes differ clinically from homozygotes. Interestingly, the predisposition to acquire mutations in JAK2 appears to be associated with a specific JAK2 gene haplotype, GGCC. JAK2 V617F is the basis for many of the phenotypic and biochemical characteristics of PV such as elevation of the leukocyte alkaline phosphatase (LAP) score; however, it cannot solely account for the entire PV phenotype and is probably not the initiating lesion in the three MPNs. First, PV patients with the same phenotype and documented clonal disease lack any mutation of JAK2. Second, ET and PMF patients have the same mutation but different clinical phenotypes. Third, familial PV can occur without the mutation, even when other members of the same family express it. Fourth, not all the cells of the malignant clone express JAK2 V617F. Fifth, JAK2 V617F has been observed in patients with long-standing idiopathic erythrocytosis. Sixth, in some patients, JAK2 V617F

1	Fourth, not all the cells of the malignant clone express JAK2 V617F. Fifth, JAK2 V617F has been observed in patients with long-standing idiopathic erythrocytosis. Sixth, in some patients, JAK2 V617F appears to be acquired after another mutation. Finally, in some JAK2 V617F–positive PV or ET patients, acute leukemia can occur in a JAK2 V617F–negative progenitor cell. However, although JAK2 V617F alone may not be sufficient to cause PV, it appears essential for the transformation of ET to PV, although not for its transformation to PMF.

1	Although isolated thrombocytosis, leukocytosis, or splenomegaly may be the initial presenting manifestation of PV, most often the disorder is first recognized by the incidental discovery of a high hemoglobin or hematocrit. With the exception of aquagenic pruritus, no symptoms distinguish PV from other causes of erythrocytosis.

1	Uncontrolled erythrocytosis causes hyperviscosity, leading to neurologic symptoms such as vertigo, tinnitus, headache, visual disturbances, and transient ischemic attacks (TIAs). Systolic hypertension is also a feature of the red cell mass elevation. In some patients, venous or arterial thrombosis may be the presenting manifestation of PV. Any vessel can be affected; but cerebral, cardiac, or mesenteric vessels are most commonly involved. Intraabdominal venous thrombosis is particularly common in young women and may be catastrophic if a sudden and complete obstruction of the hepatic vein occurs. Indeed, PV should be suspected in any patient who develops hepatic vein thrombosis. Digital ischemia, easy bruising, epistaxis, acid-peptic disease, or gastrointestinal hemorrhage may occur due to vascular stasis or thrombocytosis. Erythema, burning, and pain in the extremities, a symptom complex known as erythromelalgia, are other complications of the thrombocytosis of PV due to increased

1	to vascular stasis or thrombocytosis. Erythema, burning, and pain in the extremities, a symptom complex known as erythromelalgia, are other complications of the thrombocytosis of PV due to increased platelet stickiness. Given the large turnover of hematopoietic cells, hyperuricemia with secondary gout, uric acid stones, and symptoms due to hypermetabolism can also complicate the disorder.

1	When PV presents with erythrocytosis in combination with leukocytosis, thrombocytosis, or splenomegaly or a combination of these, the diagnosis is apparent. However, when patients present with an elevated hemoglobin or hematocrit alone, the diagnostic evaluation is more complex because of the many diagnostic possibilities (Table 131-2). Furthermore, unless the hemoglobin level is ≥20 g/dL (hematocrit ≥60%), it is not possible to distinguish true erythrocytosis from disorders causing plasma volume contraction. This is because uniquely in PV, in contrast to other causes of true erythrocytosis, there is expansion of the plasma volume, which can mask the elevated red Hemoconcentration secondary to dehydration, diuretics, ethanol abuse, androgens, or tobacco abuse Adrenal tumors Right to left cardiac or vascular glomerulonephritis Erythropoietin receptor mutation Postrenal transplantation VHL mutations (Chuvash polycythemia) Renal cysts 2,3-BPG mutationBartter’s syndrome

1	Abbreviations: 2,3-BPG, 2,3-bisphosphoglycerate; VHL, von Hippel-Lindau. cell mass; thus, red cell mass and plasma volume determinations are necessary to establish the presence of an absolute erythrocytosis and to distinguish this from relative erythrocytosis due to a reduction in plasma volume alone (also known as stress or spurious erythrocytosis or Gaisböck’s syndrome). Figure 77-18 illustrates a diagnostic algorithm for the evaluation of suspected erythrocytosis. Assay for JAK2 mutations in the presence of a normal arterial oxygen saturation provides an alternative diagnostic approach to erythrocytosis when red cell mass and plasma volume determinations are not available; a normal serum erythropoietin level does not exclude the presence of PV, but an elevated erythropoietin level is more consistent with a secondary cause for the erythrocytosis.

1	Other laboratory studies that may aid in diagnosis include the red cell count, mean corpuscular volume, and red cell distribution width (RDW), particularly when the hematocrit or hemoglobin levels are less than 60% or 20 g/dL, respectively. Only three situations cause microcytic erythrocytosis: β thalassemia trait, hypoxic erythrocytosis, and PV. With β thalassemia trait, the RDW is normal, whereas with hypoxic erythrocytosis and PV, the RDW may be elevated due to associated iron deficiency. Today, however, the assay for JAK2 V617F has superseded other tests for establishing the diagnosis of PV. Of course, in patients with associated acid-peptic disease, occult gastrointestinal bleeding may lead to a presentation with hypochromic, microcytic anemia, masking the presence of PV.

1	A bone marrow aspirate and biopsy provide no specific diagnostic information because these may be normal or indistinguishable from ET or PMF. Similarly, no specific cytogenetic abnormality is associated with the disease, and the absence of a cytogenetic marker does not exclude the diagnosis.

1	Many of the clinical complications of PV relate directly to the increase in blood viscosity associated with red cell mass elevation and indirectly to the increased turnover of red cells, leukocytes, and platelets with the attendant increase in uric acid and cytokine production. The latter appears to be responsible for constitutional symptoms. Peptic ulcer disease can also be due to Helicobacter pylori infection, the incidence of which is increased in PV, while the pruritus associated with this disorder may be a consequence of mast cell activation by JAK2 V617F. A sudden increase in spleen size can be associated with painful splenic infarction. Myelofibrosis appears to be part of the natural history of the disease but is a reactive, reversible process that does not itself 674 impede hematopoiesis and by itself has no prognostic significance. In approximately 15% of patients, however, myelofibrosis is accompanied by significant extramedullary hematopoiesis, hepatosplenomegaly, and

1	and by itself has no prognostic significance. In approximately 15% of patients, however, myelofibrosis is accompanied by significant extramedullary hematopoiesis, hepatosplenomegaly, and transfusion-dependent anemia, which are manifestations of stem cell failure. The organomegaly can cause significant mechanical discomfort, portal hypertension, and progressive cachexia. Although the incidence of acute nonlymphocytic leukemia is increased in PV, the incidence of acute leukemia in patients not exposed to chemotherapy or radiation therapy is low. Interestingly, chemotherapy, including hydroxyurea, has been associated with acute leukemia in JAK2 V617F– negative stem cells in some PV patients. Erythromelalgia is a curious syndrome of unknown etiology associated with thrombocytosis, primarily involving the lower extremities and usually manifested by erythema, warmth, and pain of the affected appendage and occasionally digital infarction. It occurs with a variable frequency and is usually

1	involving the lower extremities and usually manifested by erythema, warmth, and pain of the affected appendage and occasionally digital infarction. It occurs with a variable frequency and is usually responsive to salicylates. Some of the central nervous system symptoms observed in patients with PV, such as ocular migraine, appear to represent a variant of erythromelalgia. Left uncontrolled, erythrocytosis can lead to thrombosis involving vital organs such as the liver, heart, brain, or lungs. Patients with massive splenomegaly are particularly prone to thrombotic events because the associated increase in plasma volume masks the true extent of the red cell mass elevation measured by the hematocrit or hemoglobin level. A “normal” hematocrit or hemoglobin level in a PV patient with massive splenomegaly should be considered indicative of an elevated red cell mass until proven otherwise.

1	PV is generally an indolent disorder, the clinical course of which is measured in decades, and its management should reflect its tempo. Thrombosis due to erythrocytosis is the most significant complication and often the presenting manifestation, and maintenance of the hemoglobin level at ≤140 g/L (14 g/dL; hematocrit <45%) in men and ≤120 g/L (12 g/dL; hematocrit <42%) in women is mandatory to avoid thrombotic complications. Phlebotomy serves initially to reduce hyperviscosity by bringing the red cell mass into the normal range while further expanding the plasma volume. Periodic phlebotomies thereafter serve to maintain the red cell mass within the normal range and to induce a state of iron deficiency that prevents an accelerated reexpansion of the red cell mass. In most PV patients, once an iron-deficient state is achieved, phlebotomy is usually only required at 3-month intervals. Neither phlebotomy nor iron deficiency increases the platelet count relative to the effect of the

1	once an iron-deficient state is achieved, phlebotomy is usually only required at 3-month intervals. Neither phlebotomy nor iron deficiency increases the platelet count relative to the effect of the disease itself, and thrombocytosis is not correlated with thrombosis in PV, in contrast to the strong correlation between erythrocytosis and thrombosis in this disease. The use of salicylates as a tonic against thrombosis in PV patients is not only potentially harmful if the red cell mass is not controlled by phlebotomy, but is also an unproven remedy. Anticoagulants are only indicated when a thrombosis has occurred and can be difficult to monitor if the red cell mass is substantially elevated owing to the artifactual imbalance between the test tube anticoagulant and plasma that occurs when blood from these patients is assayed for prothrombin or partial thromboplastin activity. Asymptomatic hyperuricemia (<10 mg/dL) requires no therapy, but allopurinol should be administered to avoid

1	blood from these patients is assayed for prothrombin or partial thromboplastin activity. Asymptomatic hyperuricemia (<10 mg/dL) requires no therapy, but allopurinol should be administered to avoid further elevation of the uric acid when chemotherapy is used to reduce splenomegaly or leukocytosis or to treat pruritus. Generalized pruritus intractable to antihistamines or antidepressants such as doxepin can be a major problem in PV; interferon α (IFN-α), psoralens with ultraviolet light in the A range (PUVA) therapy, and hydroxyurea are other methods of palliation. Asymptomatic thrombocytosis requires no therapy unless the platelet count is sufficiently high to cause bleeding due an acquired form of von Willebrand’s disease in which there is adsorption and proteolysis of high-molecular-weight von Willebrand factor (VWF) multimers by the expanded platelet mass. Symptomatic splenomegaly can be treated with pegylated IFN-α. Pegylated IFN-α can also produce complete hematologic and

1	von Willebrand factor (VWF) multimers by the expanded platelet mass. Symptomatic splenomegaly can be treated with pegylated IFN-α. Pegylated IFN-α can also produce complete hematologic and molecular remissions in PV, and its role in this disorder is currently under investigation. Anagrelide, a phosphodiesterase inhibitor, can reduce the platelet count and, if tolerated, is preferable to hydroxyurea because it lacks marrow toxicity and is protective against venous thrombosis. A reduction in platelet number may be necessary for the treatment of erythromelalgia or ocular migraine if salicylates are not effective or if the platelet count is sufficiently high to increase the risk of hemorrhage but only to the degree that symptoms are alleviated. Alkylating agents and radioactive sodium phosphate (32P) are leukemogenic in PV, and their use should be avoided. If a cytotoxic agent must be used, hydroxyurea is preferred, but this drug does not prevent either thrombosis or myelofibrosis in PV,

1	(32P) are leukemogenic in PV, and their use should be avoided. If a cytotoxic agent must be used, hydroxyurea is preferred, but this drug does not prevent either thrombosis or myelofibrosis in PV, is itself leukemogenic, and should be used for as short a time as possible. Previously, PV patients with massive splenomegaly unresponsive to reduction by chemotherapy or interferon required splenectomy. However, with the introduction of the nonspecific JAK2 inhibitor ruxolitinib, it has been possible in the majority of patients with PV complicated by myelofibrosis and myeloid metaplasia to reduce spleen size while at the same time alleviating constitutional symptoms to due to cytokine release. This drug is currently undergoing clinical trials in PV patients intolerant of hydroxyurea. In some patients with end-stage disease, pulmonary hypertension may develop due to fibrosis or extramedullary hematopoiesis. A role for allogeneic bone marrow transplantation in PV has not been defined.

1	Most patients with PV can live long lives without functional impairment when their red cell mass is effectively managed with phlebotomy alone. Chemotherapy is never indicated to control the red cell mass unless venous access is inadequate.

1	Chronic PMF (other designations include idiopathic myelofibrosis, agnogenic myeloid metaplasia, or myelofibrosis with myeloid metaplasia) is a clonal disorder of a multipotent hematopoietic progenitor cell of unknown etiology characterized by marrow fibrosis, extramedullary hematopoiesis, and splenomegaly. PMF is the least common chronic MPN, and establishing this diagnosis in the absence of a specific clonal marker is difficult because myelofibrosis and splenomegaly are also features of both PV and CML. Furthermore, myelofibrosis and splenomegaly also occur in a variety of benign and malignant disorders (Table 131-3), many of which are amenable to specific therapies not effective in PMF. In contrast to the other chronic MPNs and so-called acute or malignant myelofibrosis, which can occur at any age, PMF primarily afflicts men in their sixth decade or later.

1	The etiology of PMF is unknown. Nonrandom chromosome abnormalities such as 9p, 20q−, 13q−, trisomy 8 or 9, or partial trisomy 1q are common, but no cytogenetic abnormality specific to the disease has been identified. JAK2 V617F is present in approximately 50% of PMF patients, and mutations in the thrombopoietin receptor Mpl occur in about 5%. Most of the rest have mutations in the

1	Acute leukemia (lymphocytic, HIV infection myelogenous, megakaryocytic) calreticulin gene (CALR) that alter the carboxy-terminal portion of the gene product. The degree of myelofibrosis and the extent of extramedullary hematopoiesis are also not related. Fibrosis in this disorder is associated with overproduction of transforming growth factor β and tissue inhibitors of metalloproteinases, whereas osteosclerosis is associated with overproduction of osteoprotegerin, an osteoclast inhibitor. Marrow angiogenesis occurs due to increased production of vascular endothelial growth factor. Importantly, fibroblasts in PMF are polyclonal and not part of the neoplastic clone.

1	No signs or symptoms are specific for PMF. Many patients are asymptomatic at presentation, and the disease is usually detected by the discovery of splenic enlargement and/or abnormal blood counts during a routine examination. However, in contrast to its companion MPN, night sweats, fatigue, and weight loss are common presenting complaints. A blood smear will show the characteristic features of extramedullary hematopoiesis: teardrop-shaped red cells, nucleated red cells, myelocytes, and promyelocytes; myeloblasts may also be present (Fig. 131-1). Anemia, usually mild initially, is the rule, whereas the leukocyte and platelet counts are either normal or increased, but either can be depressed. Mild hepatomegaly may accompany the splenomegaly but is unusual in the absence of splenic enlargement; isolated lymphadenopathy should suggest another diagnosis. Both serum lactate dehydrogenase and alkaline phosphatase levels can be elevated. The LAP score can be low, normal, or high. Marrow is

1	isolated lymphadenopathy should suggest another diagnosis. Both serum lactate dehydrogenase and alkaline phosphatase levels can be elevated. The LAP score can be low, normal, or high. Marrow is usually inaspirable due to the myelofibrosis (Fig. 131-2), and bone x-rays may reveal osteosclerosis. Exuberant extramedullary hematopoiesis can cause ascites; portal, pulmonary, or intracranial hypertension; intestinal or ureteral obstruction; pericardial tamponade; spinal cord compression; or skin nodules. Splenic enlargement can be sufficiently rapid to cause splenic infarction with fever and pleuritic chest pain. Hyperuricemia and secondary gout may ensue.

1	While the clinical picture described above is characteristic of PMF, all of the clinical features described can also be observed in PV or CML. Massive splenomegaly commonly masks erythrocytosis in PV, and reports of intraabdominal thrombosis in PMF most likely represent instances of unrecognized PV. In some patients with PMF, erythrocytosis has developed during the course of the disease. Furthermore, because many other disorders have features that overlap with PMF but respond to distinctly different therapies, the diagnosis of PMF is one of exclusion, which requires that the disorders listed in Table 131-3 be ruled out. FIGURE 131-1 Teardrop-shaped red blood cells indicative of mem-brane damage from passage through the spleen, a nucleated red blood cell, and immature myeloid cells indicative of extramedullary hematopoiesis are noted. This peripheral blood smear is related to any cause of extramedullary hematopoiesis.

1	FIGURE 131-2 This marrow section shows the marrow cavity replaced by fibrous tissue composed of reticulin fibers and collagen. When this fibrosis is due to a primary hematologic process, it is called myelofibrosis. When the fibrosis is secondary to a tumor or a granulomatous process, it is called myelophthisis.

1	The presence of teardrop-shaped red cells, nucleated red cells, myelocytes, and promyelocytes establishes the presence of extramedullary hematopoiesis, while the presence of leukocytosis, thrombocytosis with large and bizarre platelets, and circulating myelocytes suggests the presence of an MPN as opposed to a secondary form of myelofibrosis (Table 131-3). Marrow is usually inaspirable due to increased marrow reticulin, but marrow biopsy will reveal a hypercellular marrow with trilineage hyperplasia and, in particular, increased numbers of megakaryocytes in clusters and with large, dysplastic nuclei. However, there are no characteristic bone marrow morphologic abnormalities that distinguish PMF from the other chronic MPNs. Splenomegaly due to extramedullary hematopoiesis may be sufficiently massive to cause portal hypertension and variceal formation. In some patients, exuberant extramedullary hematopoiesis can dominate the clinical picture. An intriguing feature of PMF is the

1	massive to cause portal hypertension and variceal formation. In some patients, exuberant extramedullary hematopoiesis can dominate the clinical picture. An intriguing feature of PMF is the occurrence of autoimmune abnormalities such as immune complexes, antinuclear antibodies, rheumatoid factor, or a positive Coombs’ test. Whether these represent a host reaction to the disorder or are involved in its pathogenesis is unknown. Cytogenetic analysis of blood is useful both to exclude CML and for prognostic purposes, because complex karyotype abnormalities portend a poor prognosis in PMF. For unknown reasons, the number of circulating CD34+ cells is markedly increased in PMF (>15,000/μL) compared to the other chronic MPNs, unless they too develop myeloid metaplasia.

1	Importantly, approximately 50% of PMF patients, like patients with its companion myeloproliferative disorders PV and ET, express the JAK2 V617F mutation, often as homozygotes. Such patients are usually older and have higher hematocrits than the patients who are JAK2 V617F–negative, whereas PMF patients expressing an MPL mutation tend to be more anemic and have lower leukocyte counts. Somatic mutations in exon 9 of the calreticulin gene (CALR) have been found in a majority of patients with PMF and ET who lack mutations in either JAK2 or MPL, and their clinical course appears to be more indolent than patients expressing either a JAK2 or an MPL mutation.

1	Survival in PMF varies according to specific risk factors at diagnosis (Tables 131-4 and 131-5) but is shorter in most patients than in PV or ET patients. The natural history of PMF is one of increasing marrow failure with transfusion-dependent anemia and increasing organomegaly due to extramedullary hematopoiesis. As with CML, PMF can evolve from a chronic phase to an accelerated phase with constitutional symptoms and increasing marrow failure. About 10% of patients spontaneously transform to an aggressive form of acute leukemia for which therapy is usually ineffective. Additional important prognostic

1	Note: The Dynamic International Prognostic Scoring System (DIPSS) was developed to determine if the International Prognostic Scoring System (IPSS) risk factors identified as important for survival at the time of primary myelofibrosis (PMF) diagnosis could also be used for risk stratification following their acquisition during the course of the disease. One point is assigned to each risk factor for IPSS scoring. For DIPSS, the same is true, but age >65 years, anemia, blood blasts, and constitutional symptoms are assigned 2 points each. The DIPSS Plus scoring system represents recognition that the addition of unfavorable karyotype, thrombocytopenia, and transfusion dependence improved the DIPSS risk stratification system for which additional points are assigned (Table 131-5). More recent studies suggest that mutational analysis of the ASXL1, EZH2, SRSF2, and IDH1/2 genes further improves risk stratification for survival and leukemic transformation (Leukemia 27:1861, 2013).

1	factors for disease acceleration during the course of PMF include the presence of complex cytogenetic abnormalities, thrombocytopenia, and transfusion-dependent anemia. Most recently, mutations in the ASXL1, EZH2, SRSF2, and IDH1/2 genes have been identified as risk factors for early death or transformation to acute leukemia and may prove to be more useful for PMF risk assessment than any clinical scoring system.

1	No specific therapy exists for PMF. The causes for anemia are multifarious and include ineffective erythropoiesis uncompensated by splenic extramedullary hematopoiesis, hemodilution due to splenomegaly, splenic sequestration, blood loss secondary to thrombocytopenia or portal hypertension, folic acid deficiency, systemic inflammation, and autoimmune hemolysis. Neither recombinant erythropoietin nor androgens such as danazol have proven to be consistently effective as therapy for anemia. Erythropoietin may worsen splenomegaly and will be ineffective if the serum erythropoietin level is >125 mU/L. Given the inflammatory milieu that characterizes PMF, corticosteroids can ameliorate anemia as well as constitutional symptoms such as fever, chills, night sweats, anorexia, and weight loss, and low-dose thalidomide together with prednisone has proved effective as well. Thrombocytopenia can be due to impaired marrow function, splenic sequestration, or autoimmune destruction

1	Number of Risk Factors aThe corresponding survival curves for each risk category can be found in the references cited in the footnotes of Table 131-4. Abbreviations: DIPSS, Dynamic International Prognostic Scoring System; IPSS, International Prognostic Scoring System.

1	and may also respond to low-dose thalidomide together with prednisone. Splenomegaly is by far the most distressing and intractable problem for PMF patients, causing abdominal pain, portal hypertension, easy satiety, and cachexia, whereas surgical removal of a massive spleen is associated with significant postoperative complications including mesenteric venous thrombosis, hemorrhage, rebound leukocytosis and thrombocytosis, and hepatic extramedullary hematopoiesis with no amelioration of either anemia or thrombocytopenia when present. For unexplained reasons, splenectomy also increases the risk of blastic transformation. Splenic irradiation is, at best, temporarily palliative and associated with a significant risk of neutropenia, infection, and subsequent operative hemorrhage if splenectomy is attempted. Allopurinol can control significant hyperuricemia, and bone pain can be alleviated by local irradiation. The role of IFN-α is still undefined; its side effects are more pronounced in

1	is attempted. Allopurinol can control significant hyperuricemia, and bone pain can be alleviated by local irradiation. The role of IFN-α is still undefined; its side effects are more pronounced in the older individuals, and it may exacerbate the bone marrow failure. The JAK2 inhibitor, ruxolitinib, has proved effective in reducing splenomegaly and alleviating constitutional symptoms in a majority of advanced PMF patients while also prolonging survival, although it does not significantly influence the JAK2 V617F allele burden. Although anemia and thrombocytopenia are its major side effects, these are dose-dependent, and with time, anemia stabilizes and thrombocytopenia may improve. Allogeneic bone marrow transplantation is the only curative treatment for PMF and should be considered in younger patients; nonmyeloablative conditioning regimens may permit hematopoietic cell transplantation to be extended to older individuals, but this approach is currently under investigation.

1	Essential thrombocytosis (other designations include essential thrombocythemia, idiopathic thrombocytosis, primary thrombocytosis, and hemorrhagic thrombocythemia) is a clonal disorder of unknown etiology involving a multipotent hematopoietic progenitor cell manifested clinically by overproduction of platelets without a definable cause. ET is an uncommon disorder, with an incidence of 1–2/100,000 and a distinct female predominance. No clonal marker is available to consistently distinguish ET from the more common nonclonal, reactive forms of thrombocytosis (Table 131-6), making its diagnosis difficult. Once considered a disease of the elderly and responsible for significant morbidity due to hemorrhage or thrombosis, with the widespread use of electronic cell counters, it is now clear that ET can occur at any age in adults and often without symptoms or disturbances of hemostasis. There is an unexplained female predominance in contrast to PMF or the reactive forms of thrombocytosis where

1	can occur at any age in adults and often without symptoms or disturbances of hemostasis. There is an unexplained female predominance in contrast to PMF or the reactive forms of thrombocytosis where no sex difference exists. Because no specific clonal marker is available, clinical criteria have been proposed to distinguish ET from the other chronic MPNs, which may also present with thrombocytosis but have differing prognoses and therapies (Table 131-6). These criteria do not establish clonality; therefore, they are truly useful only in identifying disorders such as CML, PV, or myelodysplasia, which can masquerade as ET, as opposed to actually establishing the presence of ET. Furthermore, as with “idiopathic” erythrocytosis, nonclonal benign forms of thrombocytosis

1	Tissue inflammation: collagen vascular Hemorrhage disease, inflammatory bowel disease Myeloproliferative disorders: polycythe-Rebound: Correction of vitamin mia vera, primary myelofibrosis, essential B12 or folate deficiency, postthrombocytosis, chronic myelogenous ethanol abuse leukemia Myelodysplastic disorders: 5q–syndrome, Hemolysis idiopathic refractory sideroblastic anemia Postsplenectomy or hyposplenism Familial: Thrombopoietin overproduction, MPL mutations exist (such as hereditary overproduction of thrombopoietin) that are not widely recognized because we currently lack adequate diagnostic tools. Approximately 50% of ET patients carry the JAK2 V617F mutation, but its absence does not exclude the disorder.

1	Megakaryocytopoiesis and platelet production depend on thrombopoietin and its receptor Mpl. As in the case of early erythroid and myeloid progenitor cells, early megakaryocytic progenitors require the presence of interleukin 3 (IL-3) and stem cell factor for optimal proliferation in addition to thrombopoietin. Their subsequent development is also enhanced by the chemokine stromal cell-derived factor 1 (SDF-1). However, megakaryocyte maturation requires thrombopoietin.

1	Megakaryocytes are unique among hematopoietic progenitor cells because reduplication of their genome is endomitotic rather than mitotic. In the absence of thrombopoietin, endomitotic megakaryocytic reduplication and, by extension, the cytoplasmic development necessary for platelet production are impaired. Like erythropoietin, thrombopoietin is produced in both the liver and the kidneys, and an inverse correlation exists between the platelet count and plasma thrombopoietic activity. Unlike erythropoietin, thrombopoietin is only constitutively produced, and the plasma thrombopoietin level is controlled by the size of its progenitor cell pool. Also, in contrast to erythropoietin, but like its myeloid counterparts, granulocyte and granulocyte-macrophage colony-stimulating factors, thrombopoietin not only enhances the proliferation of its target cells but also enhances the reactivity of their end-stage product, the platelet. In addition to its role in thrombopoiesis, thrombopoietin also

1	not only enhances the proliferation of its target cells but also enhances the reactivity of their end-stage product, the platelet. In addition to its role in thrombopoiesis, thrombopoietin also enhances the survival of multipotent hematopoietic stem cells and their bone marrow residence.

1	The clonal nature of ET was established by analysis of glucose-6-phosphate dehydrogenase isoenzyme expression in patients hemizygous for this gene, by analysis of X-linked DNA polymorphisms in informative female patients, and by the expression in patients of nonrandom, though variable, cytogenetic abnormalities. Although thrombocytosis is its principal manifestation, like the other chronic MPNs, a multipotent hematopoietic progenitor cell is involved in ET. Furthermore, a number of families have been described in which ET was inherited, in one instance as an autosomal dominant trait. In addition to ET, PMF and PV have also been observed in some kindreds. Like PMF, most patients who do not have JAK2 mutations have CALR mutations.

1	Clinically, ET is most often identified incidentally when a platelet count is obtained during the course of a routine medical evaluation. Occasionally, review of previous blood counts will reveal that an elevated platelet count was present but overlooked for many years. No symptoms or signs are specific for ET, but these patients can have hemorrhagic and thrombotic tendencies expressed as easy bruising for the former and microvascular occlusive events for the latter such as erythromelalgia, ocular migraine, or a TIA. Physical examination is generally unremarkable except occasionally for mild splenomegaly. Splenomegaly is indicative of another MPN, in particular PV, PMF, or CML.

1	Anemia is unusual, but a mild neutrophilic leukocytosis is not. The blood smear is most remarkable for the number of platelets present, some of which may be very large. The large mass of circulating platelets may prevent the accurate measurement of serum potassium due to release of platelet potassium upon blood clotting. This type of hyperkalemia is a laboratory artifact and not associated with electrocardiographic abnormalities. Similarly, arterial oxygen measurements can be inaccurate unless thrombocythemic blood is collected on ice. The prothrombin and partial thromboplastin times are normal, whereas abnormalities of platelet function such as a prolonged bleeding time and impaired platelet aggregation can be present. However, despite much study, no platelet function abnormality is characteristic of ET, and no platelet function test predicts the risk of clinically significant bleeding or thrombosis.

1	The elevated platelet count may hinder marrow aspiration, but 677 marrow biopsy usually reveals megakaryocyte hypertrophy and hyperplasia, as well as an overall increase in marrow cellularity. If marrow reticulin is increased, another diagnosis should be considered. The absence of stainable iron demands an explanation because iron deficiency alone can cause thrombocytosis, and absent marrow iron in the presence of marrow hypercellularity is a feature of PV. Nonrandom cytogenetic abnormalities occur in ET but are uncommon, and no specific or consistent abnormality is notable, even those involving chromosomes 3 and 1, where the genes for thrombopoietin and its receptor Mpl, respectively, are located.

1	Thrombocytosis is encountered in a broad variety of clinical disorders (Table 131-6), in many of which production of cytokines is increased. The absolute level of the platelet count is not a useful diagnostic aid for distinguishing between benign and clonal causes of thrombocytosis. About 50% of ET patients express the JAK2 V617F mutation.

1	When JAK2 V617F is absent, cytogenetic evaluation is mandatory to determine if the thrombocytosis is due to CML or a myelodysplastic disorder such as the 5q− syndrome. Because the bcr-abl translocation can be present in the absence of the Ph chromosome, and because bcr-abl reverse transcriptase polymerase chain reaction is associated with false-positive results, fluorescence in situ hybridization (FISH) analysis for bcr-abl is the preferred assay in patients with thrombocytosis in whom a cytogenetic study for the Ph chromosome is negative. CALR mutations are present in most patients who do not have JAK2 mutations, but diagnostic tools to detect these mutations are not yet widespread. Anemia and ringed sideroblasts are not features of ET, but they are features of idiopathic refractory sideroblastic anemia, and in some of these patients, the thrombocytosis occurs in association with JAK2 V617F expression. Splenomegaly should suggest the presence of another MPN, and in this setting, a

1	anemia, and in some of these patients, the thrombocytosis occurs in association with JAK2 V617F expression. Splenomegaly should suggest the presence of another MPN, and in this setting, a red cell mass determination should be performed because splenomegaly can mask the presence of erythrocytosis. Importantly, what appears to be ET can evolve into PV or PMF after a period of many years, revealing the true nature of the underlying MPN. There is sufficient overlap of the JAK2 V617F neutrophil allele burden between ET and PV that this cannot be used as a distinguishing diagnostic feature; only a red cell mass and plasma volume determination can distinguish PV from ET, and importantly in this regard, 64% of JAK2 V617F–positive ET patients actually were found to have PV when red cell mass and plasma volume determinations were performed.

1	Perhaps no other condition in clinical medicine has caused otherwise astute physicians to intervene inappropriately more often than thrombocytosis, particularly if the platelet count is >1 × 106/μL. It is commonly believed that a high platelet count causes intravascular stasis and thrombosis; however, no controlled clinical study has ever established this association, and in patients younger than age 60 years, the incidence of thrombosis was not greater in patients with thrombocytosis than in age-matched controls, and tobacco use appears to be the most important risk factor for thrombosis in ET patients.

1	To the contrary, very high platelet counts are associated primarily with hemorrhage due to acquired von Willebrand’s disease. This is not meant to imply that an elevated platelet count cannot cause symptoms in an ET patient, but rather that the focus should be on the patient, not the platelet count. For example, some of the most dramatic neurologic problems in ET are migraine-related and respond only to lowering of the platelet count, whereas other symptoms such as erythromelalgia respond simply to platelet cyclooxygenase-1 inhibitors such as aspirin or ibuprofen, without a reduction in platelet number. Still others may represent an interaction between an atherosclerotic vascular system and a high platelet count, and others may have no relationship to the platelet count whatsoever. Recognition that PV can present with thrombocytosis alone as well as the discovery of previously unrecognized causes of hypercoagulability (Chap. 142) make the older literature on the complications of

1	that PV can present with thrombocytosis alone as well as the discovery of previously unrecognized causes of hypercoagulability (Chap. 142) make the older literature on the complications of thrombocytosis unreliable.

1	678 ET can also evolve into PMF, but whether this is a feature of ET or represents PMF presenting initially with isolated thrombocytosis is unknown.

1	Survival of patients with ET is not different than for the general population. An elevated platelet count in an asymptomatic patient without cardiovascular risk factors requires no therapy. Indeed, before any therapy is initiated in a patient with thrombocytosis, the cause of symptoms must be clearly identified as due to the elevated platelet count. When the platelet count rises above 1 × 106/μL, a substantial quantity of high-molecular-weight von Willebrand multimers are removed from the circulation and destroyed by the enlarged platelet mass, resulting in an acquired form of von Willebrand’s disease. This can be identified by a reduction in ristocetin cofactor activity. In this situation, aspirin could promote hemorrhage. Bleeding in this situation usually responds to ε-aminocaproic acid, which can be given prophylactically before and after elective surgery. Plateletpheresis is at best a temporary and inefficient remedy that is rarely required. Importantly, ET patients treated with

1	which can be given prophylactically before and after elective surgery. Plateletpheresis is at best a temporary and inefficient remedy that is rarely required. Importantly, ET patients treated with 32P or alkylating agents are at risk of developing acute leukemia without any proof of benefit; combining either therapy with hydroxyurea increases this risk. If platelet reduction is deemed necessary on the basis of symptoms refractory to salicylates alone, pegylated IFN-α, the quinazoline derivative, anagrelide, or hydroxyurea can be used to reduce the platelet count, but none of these is uniformly effective or without significant side effects. Hydroxyurea and aspirin are more effective than anagrelide and aspirin for prevention of TIAs but not more effective for the prevention of other types of arterial thrombosis and are actually less effective for venous thrombosis. The effectiveness of hydroxyurea in preventing TIAs is because it is an NO donor. Normalizing the platelet count also does

1	arterial thrombosis and are actually less effective for venous thrombosis. The effectiveness of hydroxyurea in preventing TIAs is because it is an NO donor. Normalizing the platelet count also does not prevent either arterial or venous thrombosis. The risk of gastrointestinal bleeding is also higher when aspirin is combined with anagrelide.

1	As more clinical experience is acquired, ET appears more benign than previously thought. Evolution to acute leukemia is more likely to be a consequence of therapy than of the disease itself. In managing patients with thrombocytosis, the physician’s first obligation is to do no harm.

1	Acute Myeloid Leukemia Guido Marcucci, Clara D. Bloomfield INCIDENCE Acute myeloid leukemia (AML) is a neoplastic disease characterized by infiltration of the blood, bone marrow, and other tissues by prolifera-tive, clonal undifferentiated cells of the hematopoietic system. These 132 leukemias comprise a spectrum of malignancies that, untreated, range from rapidly fatal to slowly growing. In 2013, the estimated number of new AML cases in the United States was 14,590. The incidence of AML is ~3.5 per 100,000 people per year, and the age-adjusted incidence is higher in men than in women (4.5 vs 3.1). AML incidence increases with age; it is 1.7 in individuals age <65 years and 15.9 in those age >65 years. The median age at diagnosis is 67 years.

1	Heredity Certain syndromes with somatic cell chromosome aneuploidy, such as trisomy 21 noted in Down syndrome, are associated with an increased incidence of AML. Inherited diseases with defective DNA repair, e.g., Fanconi anemia, Bloom syndrome, and ataxiatelangiectasia, are also associated with AML. Congenital neutropenia (Kostmann syndrome) is a disease with mutations in the genes encoding the granulocyte colony-stimulating factor (G-CSF) receptor and, often, neutrophil elastase that may evolve into AML. Germline mutations of CCAAT/enhancer-binding protein α (CEBPA), runt-related transcription factor 1 (RUNX1), and tumor protein p53 (TP53) have also been associated with a higher predisposition to AML in some series.

1	Radiation High-dose radiation, like that experienced by survivors of the atomic bombs in Japan or nuclear reactor accidents, increases the risk of myeloid leukemias that peaks 5–7 years after exposure. Therapeutic radiation alone seems to add little risk of AML but can increase the risk in people also exposed to alkylating agents. Chemical and Other Exposures Exposure to benzene, a solvent used in the chemical, plastic, rubber, and pharmaceutical industries, is associated with an increased incidence of AML. Smoking and exposure to petroleum products, paint, embalming fluids, ethylene oxide, herbicides, and pesticides have also been associated with an increased risk of AML.

1	Drugs Anticancer drugs are the leading cause of therapy-associated AML. Alkylating agent–associated leukemias occur on average 4–6 years after exposure, and affected individuals have aberrations in chromosomes 5 and 7. Topoisomerase II inhibitor–associated leukemias occur 1–3 years after exposure, and affected individuals often have aberrations involving chromosome 11q23. Newer agents for treatment of other hematopoietic malignancies and solid tumors are also under scrutiny for increased risk of AML. Chloramphenicol, phenylbutazone, and, less commonly, chloroquine and methoxypsoralen can result in bone marrow failure that may evolve into AML.

1	The current categorization of AML uses the World Health Organization (WHO) classification (Table 132-1), which includes different biologically distinct groups based on clinical features and cytogenetic and molecular abnormalities in addition to morphology. In contrast to the previously used French-American-British (FAB) schema, the WHO classification places limited reliance on cytochemistry. A major difference between the WHO and the FAB systems is the blast cutoff for a diagnosis of AML as opposed to myelodysplastic syndrome (MDS); it is 20% in the WHO classification and 30% in the FAB. However, within the WHO classification, specific chromosomal rearrangements, i.e., t(8;21)(q22;q22), inv(16)(p13.1q22), t(16;16)(p13.1;q22), and t(15;17) (q22;q12), define AML even with <20% blasts.

1	Immunophenotype and Relevance to the WHO Classification The immunophenotype of human leukemia cells can be studied by multiparameter flow cytometry after the cells are labeled with monoclonal antibodies to cell-surface antigens. This can be important for separating AML from acute lymphoblastic leukemia (ALL) and identifying some subtypes of AML. For example, AML with minimal differentiation that is characterized by immature morphology and no lineage-specific cytochemical reactions may be diagnosed by flow-cytometric demonstration of the myeloid-specific antigens cluster designation (CD) 13 and/ or 117. Similarly, acute megakaryoblastic leukemia can often be diagnosed only by expression of the platelet-specific antigens CD41 and/ or CD61. Although flow cytometry is useful, widely used, and in some cases essential for the diagnosis of AML, it is supportive only in establishing the different subtypes of AML through the WHO classification.

1	Clinical Features and Relevance to the WHO Classification The WHO ETIOLOGY classification also considers clinical features in subdividing AML. For Heredity, radiation, chemical and other occupational exposures, and example, it identifies therapy-related AML as a separate entity that drugs have been implicated in the development of AML. No direct develops following prior therapy (e.g., alkylating agents, topoisomevidence suggests a viral etiology. erase II inhibitors, ionizing radiation). It also identifies AML with AML with recurrent genetic abnormalities

1	AML with recurrent genetic abnormalities AML with t(8;21)(q22;q22); RUNX1-RUNX1T1b AML with inv(16)(pl3.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11b Acute promyelocytic leukemia with t(15;17)(q22;q12); PML-RARAb AML with t(9;11)(p22;q23); MLLT3-MLL AML with t(6;9)(p23;q34); DEK-NUP214 AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1 AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1 Provisional entity: AML with mutated NPM1 Provisional entity: AML with mutated CEBPA AML with myelodysplasia-related changes Therapy-related myeloid neoplasms AML, not otherwise specified AML with minimal differentiation AML with maturation Acute panmyelosis with myelofibrosis Myeloid sarcoma Myeloid proliferations related to Down syndrome

1	AML with minimal differentiation AML with maturation Acute panmyelosis with myelofibrosis Myeloid sarcoma Myeloid proliferations related to Down syndrome Transient abnormal myelopoiesis Myeloid leukemia associated with Down syndrome aFrom SH Swerdlow et al (eds): World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, IARC Press, 2008. bDiagnosis is AML regardless of blast count. myelodysplasia-related changes based in part on medical history of an antecedent MDS or myelodysplastic/myeloproliferative neoplasm. The clinical features likely contribute to the prognosis of AML and have therefore been included in the classification. Genetic Findings and Relevance to the WHO Classification The

1	WHO classification uses clinical, morphologic, and cytogenetic and/or molecular criteria to identify subtypes of AML and forces the clinician to take the appropriate steps to correctly identify the entity and thus tailor treatment(s) accordingly. The WHO classification is indeed the first AML classification that incorporates genetic (chromosomal and molecular) information. In this classification, subtypes of AML are recognized based on the presence or absence of specific recurrent genetic abnormalities. For example, the diagnosis of acute promyelocytic leukemia (APL) is based on the presence of either the t(15;17)(q22;q12) cytogenetic rearrangement or the PML-RARA fusion product of the translocation. A similar approach is taken with regard to core binding factor (CBF) AML that is now designated based on the presence of t(8;21)(q22;q22), inv(16)(p13.1q22), or t(16;16) (p13.1;q22) or the respective fusion products RUNX1-RUNX1T1 and CBFB-MYH11.

1	The WHO classification incorporates cytogenetics in the AML classification by recognizing a category of AML with recurrent genetic abnormalities and a category of AML with myelodysplasia-related changes (Table 132-1). The latter category is diagnosed not only by morphologic changes, but also in part by selected myelodysplasiarelated cytogenetic abnormalities (e.g., complex karyotypes and unbalanced and balanced changes involving, among others, chromosomes 5, 7, and 11). Only one cytogenetic abnormality has been invariably associated with specific morphologic features: t(15;17)(q22;q12) with APL. Other chromosomal abnormalities have been associated primarily with one morphologic/immunophenotypic group, including inv(16) (p13.1q22) with AML with abnormal bone marrow eosinophils; t(8;21) 679 (q22;q22) with slender Auer rods, expression of CD19, and increased normal eosinophils; and t(9;11)(p22;q23), and other translocations involving 11q23, with monocytic features. Recurring chromosomal

1	(q22;q22) with slender Auer rods, expression of CD19, and increased normal eosinophils; and t(9;11)(p22;q23), and other translocations involving 11q23, with monocytic features. Recurring chromosomal abnormalities in AML may also be associated with specific clinical characteristics. More commonly associated with younger age are t(8;21) and t(15;17), and with older age, del(5q) and del(7q). Myeloid sarcomas (see below) are associated with t(8;21), and disseminated intravascular coagulation (DIC) is associated with t(15;17).

1	The WHO classification also incorporates molecular abnormalities by recognizing fusion genes that are products of recurrent cytogenetic aberrations or have been found mutated and may be involved in leukemogenesis. For instance, t(15;17) results in the fusion gene PML-RARA that encodes a chimeric protein, promyelocytic leukemia (Pml)–retinoic acid receptor α (Rarα), which is formed by the fusion of the retinoic acid receptor α (RARA) gene from chromosome 17 and the promyelocytic leukemia (PML) gene from chromosome 15. The RARA gene encodes a member of the nuclear hormone receptor family of transcription factors. After binding retinoic acid, RARA can promote expression of a variety of genes. The 15;17 translocation juxtaposes PML with RARA in a head-to-tail configuration that is under the transcriptional control of PML. Three different breakpoints in the PML gene lead to various fusion protein isoforms. The Pml-Rarα fusion protein tends to suppress gene transcription and blocks

1	the transcriptional control of PML. Three different breakpoints in the PML gene lead to various fusion protein isoforms. The Pml-Rarα fusion protein tends to suppress gene transcription and blocks differentiation of the cells. Pharmacologic doses of the Rarα ligand, all-trans-retinoic acid (tretinoin), relieve the block and promote hematopoietic cell differentiation (see below). Similar examples of molecular subtypes of the disease included in the category of AML with recurrent genetic abnormalities are those characterized by the leukemogenic fusion genes RUNX1-RUNX1T1, CBFB-MYH11, MLLT3-MLL, and DEK-NUP214, resulting, respectively, from t(8;21), inv(16) or t(16;16), t(9;11), and t(6;9)(p23;q34).

1	Two new provisional entities defined by the presence of gene mutations, rather than microscopic chromosomal abnormalities, have been added to the category of AML with recurrent genetic abnormalities: AML with mutated nucleophosmin (nucleolar phosphoprotein B23, numatrin) (NPM1) and AML with mutated CEBPA. AML with fmsrelated tyrosine kinase 3 (FLT3) mutations is not considered a distinct entity, although determining the presence of such mutations is recommended by WHO in patients with cytogenetically normal AML (CN-AML) because the relatively frequent FLT3-internal tandem duplication (ITD) carries a negative prognostic significance and therefore is clinically relevant. FLT3 encodes a tyrosine kinase receptor important in the development of myeloid and lymphoid lineages. Activating mutations of FLT3 are present in ~30% of adult AML patients due to ITDs in the juxtamembrane domain or point mutations of the activating loop of the kinase (called tyrosine kinase domain mutations). Aberrant

1	of FLT3 are present in ~30% of adult AML patients due to ITDs in the juxtamembrane domain or point mutations of the activating loop of the kinase (called tyrosine kinase domain mutations). Aberrant activation of the FLT3-encoded protein provides increased proliferation and antiapoptotic signals to the myeloid progenitor cell. FLT3-ITD, the more common of the FLT3 mutations, occurs preferentially in patients with CN-AML. The importance of identifying FLT3-ITD at diagnosis relates to the fact that not only is it a useful prognosticator but it also may predict response to specific treatment such as the tyrosine kinase inhibitors that are in clinical investigation.

1	Several factors have been demonstrated to predict outcome of AML patients treated with chemotherapy, and they can be used for risk stratification and treatment guidance.

1	Chromosome findings at diagnosis are currently the most important independent prognostic factors. Several studies have categorized patients as having favorable, intermediate, or poor cytogenetic risk based on the presence of structural and/or numerical aberrations. Patients with t(15;17) have a very good prognosis (~85% cured), and those with t(8;21) and inv(16) have a good prognosis (~55% cured), whereas those with no cytogenetic abnormality have an intermediate outcome risk (~40% cured). Patients with a complex karyotype, t(6;9), inv(3), or –7 have a very poor prognosis. Another cytogenetic subgroup, the monosomal karyotype, has been suggested to adversely 680 impact the outcome of AML patients other than those with t(15;17), t(8;21), or inv(16) or t(16;16). The monosomal karyotype subgroup is defined by the presence of at least two autosomal monosomies (loss of chromosomes other than Y or X) or a single autosomal monosomy with additional structural abnormalities. For patients

1	subgroup is defined by the presence of at least two autosomal monosomies (loss of chromosomes other than Y or X) or a single autosomal monosomy with additional structural abnormalities. For patients lacking prognostic cytogenetic abnormalities, such as those with CN-AML, outcome prediction uses mutated or aberrantly expressed genes. NPM1 mutations without concurrent presence of FLT3-ITD, and CEBPA mutations, especially if concurrently present in two different alleles, have been shown to predict favorable outcome, whereas FLT3-ITD predicts poor outcome. Given the proven prognostic importance of NPM1 and CEBPA mutations and FLT3-ITD, molecular assessment of these genes at diagnosis has been incorporated in AML management guidelines by the National Comprehensive Cancer Network (NCCN) and the European LeukemiaNet (ELN). The same markers have also been incorporated in the definitions of the genetic groups of the ELN standardized reporting system, which are based on both cytogenetic and

1	the European LeukemiaNet (ELN). The same markers have also been incorporated in the definitions of the genetic groups of the ELN standardized reporting system, which are based on both cytogenetic and molecular abnormalities and used for comparing clinical features and treatment response among subsets of patients reported in different studies (Table 132-2). More recently, the prognostic impact of the genetic groups recognized by the ELN reporting system has been demonstrated. Thus, these genetic groups may also be used for risk stratification and treatment guidance. In addition to NPM1 and CEBPA mutations and FLT3-ITD, other molecular aberrations (Table 132-3) may in the future be routinely used for prognostication in AML and incorporated in the WHO classification and the ELN reporting system. Among these prognostic mutated genes are those encoding receptor tyrosine kinases (e.g., v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog [KIT]), transcription factors (i.e., RUNX1

1	Among these prognostic mutated genes are those encoding receptor tyrosine kinases (e.g., v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog [KIT]), transcription factors (i.e., RUNX1 and Wilms tumor 1 [WT1]), and epigenetic modifiers (i.e., additional sex combs like transcriptional regulator 1 [ASXL1], DNA (cytosine-5-)-methyltransferase 3 alpha [DNMT3A], isocitrate dehydrogenase 1 (NADP+), soluble [IDH1] and isocitrate dehydrogenase 2 (NADP+), mitochondrial [IDH2], lysine (K)-specific methyltransferase 2A [KMT2A, also known as MLL], and tet methylcytosine dioxygenase 2 [TET2]). Although KIT mutations are almost exclusively present in CBF AML and impact adversely the outcome, the remaining markers have been reported primarily in CN-AML. These gene mutations have been shown to be associated with outcome in multivariable analyses independently from other prognostic factors. However, for some of them, the prognostic impact (e.g., TET2 mutations) or the type (adverse vs

1	to be associated with outcome in multivariable analyses independently from other prognostic factors. However, for some of them, the prognostic impact (e.g., TET2 mutations) or the type (adverse vs favorable) of prognostic impact (e.g., IDH1, IDH2) has been found in the majority, but not in all, of the reported studies. An independent prognostic impact remains to be determined for mutated genes that are either associated primarily with unfavorable

1	Favorable t(8;21)(q22;q22); RUNX1-RUNX1T1 inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11 Mutated NPM1 without FLT3-ITD (normal karyotype) Mutated CEBPA (normal karyotype) Intermediate-II t(9;11)(p22;q23); MLLT3-MLL Cytogenetic abnormalities not classified as favorable or adverse Adverse inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1 t(6;9)(p23;q34); DEK-NUP214 t(v;11)(v;q23); MLL rearranged –5 or del(5q); –7; abn(17p); complex karyotype (≥3 abnormalities) aH Döhner et al: Blood 115:453, 2010. Abbreviation: ITD, internal tandem duplication. Genes Included in the WHO Classification and ELN Reporting System Abbreviations: AML, acute myeloid leukemia; ELN, European LeukemiaNet; ITD, internal tandem duplication; PTD, partial tandem duplication; TKD, tyrosine kinase domain; WHO, World Health Organization.

1	cytogenetic aberrations (e.g., TP53) or are found with a relatively lower frequency in AML patients like those encoding epigenetic modifiers (e.g., enhancer of zeste 2 polycomb repressive complex 2 subunit [EZH2]), phosphatases (e.g., protein tyrosine phosphatase, non-receptor type 11 (PTPN11]), putative transcription factors (e.g., PHD finger protein 6 [PHF6]), splicing factors (e.g., U2 small nuclear RNA auxiliary factor 1 [U2AF1]), and proteins involved in chromosome segregation and genome stability (e.g., structural maintenance of chromosomes 1A [SMC1A] or structural maintenance of chromosomes 3 [SMC3]). Finally, other mutated genes are recognized as predictors of treatment response to distinct therapies rather than prognosticators; for example, neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS) and Kirsten rat sarcoma viral oncogene homolog (KRAS) predict a better response to high-dose cytarabine in CBF AML.

1	In addition to gene mutations, deregulation of the expression levels of coding genes and of short noncoding RNAs (microRNAs) have been reported to provide prognostic information (Table 132-3). Overexpression of genes such as brain and acute leukemia, cytoplasmic (BAALC), v-ets avian erythroblastosis virus E26 oncogene homologue (avian) (ERG), meningioma (disrupted in balanced translocation) 1 (MN1), and MDS1 and EVI1 complex locus (MECOM, also known as EVI1) have been found to be predictive for poor outcome, especially in CN-AML. Similarly, deregulated expression levels of microRNAs, naturally occurring noncoding RNAs that have been shown to regulate the expression of proteins involved in hematopoietic differentiation and survival pathways by degradation or translation inhibition of target coding RNAs, have been associated with prognosis in AML. Overexpression of miR-155 and miR-3151 has been found to affect outcome adversely in CN-AML, whereas overexpression of miR-181a predicts a

1	coding RNAs, have been associated with prognosis in AML. Overexpression of miR-155 and miR-3151 has been found to affect outcome adversely in CN-AML, whereas overexpression of miR-181a predicts a favorable outcome both in CN-AML and cytogenetically abnormal AML.

1	Because prognostic molecular markers in AML are not mutually exclusive and often occur concurrently (>80% patients have at least two or more prognostic gene mutations), the likelihood that distinct marker combinations may be more informative than single markers is being recognized. Epigenetic changes (e.g., DNA methylation) and microRNAs are often involved in deregulation of genes involved in hematopoiesis, contribute to leukemogenesis, and are often associated with the previously discussed prognostic gene mutations. These changes not only have been shown to provide biologic insights into leukemogenic mechanisms, but also independent prognostic information. Indeed, it is anticipated that with the enormous progress made in DNA and RNA sequencing technology, additional genetic and epigenetic aberrations will soon be discovered and will contribute to classification and reporting systems and outcome risk determination in AML patients.

1	In addition to cytogenetics and/or molecular aberrations, several other factors are associated with outcome in AML. Age at diagnosis is one of the most important risk factors. Advancing age is associated with a poorer prognosis not only because of its influence on the ability to survive induction therapy due to coexisting comorbidities, but also because with each successive decade of age, a greater proportion of patients have an intrinsically more resistant disease. A prolonged symptomatic interval with cytopenias preceding diagnosis or a history of antecedent hematologic disorders including myeloproliferative neoplasms is often found in older patients and is a clinical feature associated with a lower complete remission (CR) rate and shorter survival time. The CR rate is lower in patients who have had anemia, leukopenia, and/or thrombocytopenia for >3 months before the diagnosis of AML when compared to those without such a history. Responsiveness to chemotherapy declines as the

1	who have had anemia, leukopenia, and/or thrombocytopenia for >3 months before the diagnosis of AML when compared to those without such a history. Responsiveness to chemotherapy declines as the duration of the antecedent disorder(s) increases. AML developing after treatment with cytotoxic agents for other malignancies is usually difficult to treat successfully. Finally, it is likely that AML in older patients is also associated with poor outcome because of the presence of distinct biologic features that may increase the aggressiveness of the disease and reduce the likelihood of treatment response. The leukemic cells in older patients more commonly express the multidrug resistance 1 (MDR1) efflux pump that conveys resistance to natural product–derived agents such as the anthracyclines that are frequently incorporated into the initial treatment. In addition, older patients less frequently harbor favorable cytogenetic abnormalities [i.e., t(8;21), inv(16), and t(16;16)] and more

1	that are frequently incorporated into the initial treatment. In addition, older patients less frequently harbor favorable cytogenetic abnormalities [i.e., t(8;21), inv(16), and t(16;16)] and more frequently harbor adverse cytogenetic (e.g., complex and monosomal karyotypes) and/or molecular (e.g., ASXL1, IDH2, RUNX1, TET2) abnormalities.

1	Other factors independently associated with worse outcome are a low performance status that influences ability to survive induction therapy and thus respond to treatment and a high presenting leukocyte count that in some series is an adverse prognostic factor for attaining a CR. Among patients with hyperleukocytosis (>100,000/μL), early central nervous system bleeding and pulmonary leukostasis contribute to poor outcome with initial therapy.

1	Achievement of CR is associated with better outcome and longer survival. CR is defined after examination of both blood and bone marrow. The blood neutrophil count must be ≥1000/μL and the platelet count ≥100,000/μL. Hemoglobin concentration is not considered in determining CR. Circulating blasts should be absent. Although rare blasts may be detected in the blood during marrow regeneration, they should disappear on successive studies. The bone marrow should contain <5% blasts, and Auer rods should be absent. Extramedullary leukemia should not be present. Patients who achieve CR after one induction cycle have longer CR durations than those requiring multiple cycles. CLINICAL PRESENTATION Symptoms Patients with AML most often present with nonspecific symptoms that begin gradually or abruptly and are the consequence of anemia, leukocytosis, leukopenia or leukocyte dysfunction, or thrombocytopenia. Nearly half have had symptoms for ≤3 months before the leukemia was diagnosed.

1	Half of patients mention fatigue as the first symptom, but most complain of fatigue or weakness at the time of diagnosis. Anorexia and weight loss are common. Fever with or without an identifiable infection is the initial symptom in approximately 10% of patients. 681 Signs of abnormal hemostasis (bleeding, easy bruising) are noted first in 5% of patients. On occasion, bone pain, lymphadenopathy, nonspecific cough, headache, or diaphoresis is the presenting symptom. Rarely patients may present with symptoms from a myeloid sarcoma that is a tumor mass consisting of myeloid blasts occurring at anatomic sites other than bone marrow. Sites involved are most commonly the skin, lymph node, gastrointestinal tract, soft tissue, and testis. This rare presentation, often characterized by chromosome aberrations [e.g., monosomy 7, trisomy 8, MLL rearrangement, inv(16), trisomy 4, t(8;21)], may precede or coincide with AML.

1	Physical Findings Fever, splenomegaly, hepatomegaly, lymphadenopathy, sternal tenderness, and evidence of infection and hemorrhage are often found at diagnosis. Significant gastrointestinal bleeding, intrapulmonary hemorrhage, or intracranial hemorrhage occurs most often in APL. Bleeding associated with coagulopathy may also occur in monocytic AML and with extreme degrees of leukocytosis or thrombocytopenia in other morphologic subtypes. Retinal hemorrhages are detected in 15% of patients. Infiltration of the gingivae, skin, soft tissues, or meninges with leukemic blasts at diagnosis is characteristic of the monocytic subtypes and those with 11q23 chromosomal abnormalities.

1	Hematologic Findings Anemia is usually present at diagnosis and can be severe. The degree varies considerably, irrespective of other hematologic findings, splenomegaly, or duration of symptoms. The anemia is usually normocytic normochromic. Decreased erythropoiesis often results in a reduced reticulocyte count, and red blood cell (RBC) sur vival is decreased by accelerated destruction. Active blood loss also contributes to the anemia.

1	The median presenting leukocyte count is about 15,000/μL. Between 25 and 40% of patients have counts <5000/μL, and 20% have counts >100,000/μL. Fewer than 5% have no detectable leukemic cells in the blood. The morphology of the malignant cell varies in different subsets. In AML, the cytoplasm often contains primary (nonspecific) granules, and the nucleus shows fine, lacy chromatin with one or more nucleoli characteristic of immature cells. Abnormal rod-shaped granules called Auer rods are not uniformly present, but when they are, myeloid lineage is virtually certain (Fig. 132-1). Poor neutrophil function may be noted functionally by impaired phagocytosis and migration and morphologically by abnormal lobulation and deficient granulation.

1	Platelet counts <100,000/μL are found at diagnosis in ~75% of patients, and about 25% have counts <25,000/μL. Both morphologic and functional platelet abnormalities can be observed, including large and bizarre shapes with abnormal granulation and inability of platelets to aggregate or adhere normally to one another.

1	Pretreatment Evaluation Once the diagnosis of AML is suspected, a rapid evaluation and initiation of appropriate therapy should follow. In addition to clarifying the subtype of leukemia, initial studies should evaluate the overall functional integrity of the major organ systems, including the cardiovascular, pulmonary, hepatic, and renal systems (Table 132-4). Factors that have prognostic significance, either for achieving CR or for predicting the duration of CR, should also be assessed before initiating treatment, including cytogenetics and molecular markers (see above). Leukemic cells should be obtained from all patients and cryopreserved for future use as new tests and therapeutics become available. All patients should be evaluated for infection.

1	Most patients are anemic and thrombocytopenic at presentation. Replacement of the appropriate blood components, if necessary, should begin promptly. Because qualitative platelet dysfunction or the presence of an infection may increase the likelihood of bleeding, evidence of hemorrhage justifies the immediate use of platelet transfusion, even if the platelet count is only moderately decreased. About 50% of patients have a mild to moderate elevation of serum uric acid at presentation. Only 10% have marked elevations, but renal precipitation of uric acid and the nephropathy that may result is a serious but uncommon complication. The initiation of chemotherapy may aggravate hyperuricemia, and patients are usually started immediately on allopurinol and hydration at diagnosis. Rasburicase (recombinant

1	FIGURE 132-1 Morphology of acute myeloid leukemia (AML) cells. A. Uniform population of primitive myeloblasts with immature chromatin, nucleoli in some cells, and primary cytoplasmic granules. B. Leukemic myeloblast containing an Auer rod. C. Promyelocytic leukemia cells with prominent cytoplasmic primary granules. D. Peroxidase stain shows dark blue color characteristic of peroxidase in granules in AML. uric oxidase) is also useful for treating uric acid nephropathy and often can normalize the serum uric acid level within hours with a single dose of treatment. The presence of high concentrations of lysozyme, a marker for monocytic differentiation, may be etiologic in renal tubular dysfunction, which could worsen other renal problems that arise during the initial phases of therapy.

1	Treatment of the newly diagnosed patient with AML is usually divided into two phases, induction and postremission management (Fig. 132-2). The initial goal is to induce CR. Once CR is obtained, further therapy must be used to prolong survival and achieve cure. The initial induction treatment and subsequent postremission therapy are often chosen based on the patient’s age. Intensifying therapy with traditional chemotherapy agents such as cytarabine and anthracyclines in younger patients (<60 years) appears to increase the cure rate of AML. In older patients, the benefit of intensive therapy is controversial; novel approaches for selecting patients predicted to be responsive to treatment and new therapies are being pursued.

1	The most commonly used CR induction regimens (for patients other than those with APL) consist of combination chemotherapy with cytarabine and an anthracycline (e.g., daunorubicin, idarubicin, mitoxantrone). Cytarabine is a cell cycle S-phase–specific antimetabolite that becomes phosphorylated intracellularly to an active triphosphate form that interferes with DNA synthesis. Anthracyclines are DNA intercalators. Their primary mode of action is thought to be inhibition of topoisomerase II, leading to DNA breaks.

1	In younger adults (age <60 years), cytarabine is used either at standard dose (100–200 mg/m2) administered as a continuous intravenous infusion for 7 days or higher dose (2 g/m2) administered intravenously every 12 h for 6 days. With standard-dose cytarabine, anthracycline therapy generally consists of daunorubicin (60–90 mg/m2) or idarubicin (12 mg/m2) intravenously on days 1, 2, and 3 (the 7 and 3 regimen). Other agents can be added (i.e., cladribine) when 60 mg/m2 of daunorubicin is used.

1	High-dose cytarabine-based regimens have also been shown to induce high CR rates. When given in high doses, higher intracellular levels of cytarabine may be achieved, thereby saturating the cytarabine-inactivating enzymes and increasing the intracellular levels of 1-β-D-arabinofuranylcytosine-triphosphate, the active metabolite incorporated into DNA. Thus, higher doses of cytarabine may increase the inhibition of DNA synthesis and thereby overcome resistance to standard-dose cytarabine. With high-dose cytarabine, daunorubicin 60 mg/m2 or idarubicin 12 mg/m2 is generally used. The hematologic toxicity of high-dose cytarabine-based induction regimens has typically been greater than that associated with 7 and 3 regimens. Toxicity with high-dose cytarabine also includes pulmonary toxicity and significant and occasionally irreversible

1	Increasing fatigue or decreased exercise tolerance (anemia) Excess bleeding or bleeding from unusual sites (DIC, thrombocytopenia) Fevers or recurrent infections (neutropenia) Headache, vision changes, nonfocal neurologic abnormalities (CNS leukemia or bleed) Early satiety (splenomegaly) Family history of AML (Fanconi, Bloom, or Kostmann syndromes or ataxia-telangiectasia) History of cancer (exposure to alkylating agents, radiation, topoisomerase II inhibitors) Occupational exposures (radiation, benzene, petroleum products, paint, smoking, pesticides)

1	Performance status (prognostic factor) Ecchymosis and oozing from IV sites (DIC, possible acute promyelocytic leukemia) Fever and tachycardia (signs of infection) Papilledema, retinal infiltrates, cranial nerve abnormalities (CNS leukemia) Poor dentition, dental abscesses Gum hypertrophy (leukemic infiltration, most common in monocytic leukemia) Skin infiltration or nodules (leukemia infiltration, most common in monocytic leukemia) Lymphadenopathy, splenomegaly, hepatomegaly Back pain, lower extremity weakness [spinal granulocytic sarcoma, most likely in t(8;21) patients]

1	CBC with manual differential cell count Chemistry tests (electrolytes, creatinine, BUN, calcium, phosphorus, uric acid, hepatic enzymes, bilirubin, LDH, amylase, lipase) Clotting studies (prothrombin time, partial thromboplastin time, fibrinogen, D-dimer) Viral serologies (CMV, HSV-1, varicella-zoster) RBC type and screen HLA typing for potential allogeneic HSCT Bone marrow aspirate and biopsy (morphology, cytogenetics, flow cytometry, molecular studies for NPM1 and CEBPA mutations and FLT3-ITD) Cryopreservation of viable leukemia cells Myocardial function (echocardiogram or MUGA scan) PA and lateral chest radiograph Placement of central venous access device Dental evaluation (for those with poor dentition) Lumbar puncture (for those with symptoms of CNS involvement) Screening spine MRI (for patients with back pain, lower extremity weakness, paresthesias) Social work referral for patient and family psychosocial support

1	Provide patients with information regarding their disease, financial counseling, and support group contacts Abbreviations: AML, acute myeloid leukemia; BUN, blood urea nitrogen; CBC, complete blood count; CMV, cytomegalovirus; CNS, central nervous system; DIC, disseminated intravascular coagulation; HLA, human leukocyte antigen; HSCT, hematopoietic stem cell transplantation; HSV, herpes simplex virus; IV, intravenous; LDH, lactate dehydrogenase; MRI, magnetic reso nance imaging; MUGA, multigated acquisition; PA, posteroanterior; RBC, red blood (cell) count.

1	cerebellar toxicity. All patients treated with high-dose cytarabine must be closely monitored for cerebellar toxicity. Full cerebellar testing should be performed before each dose, and further high-dose cytarabine should be withheld if evidence of cerebellar toxicity develops. This toxicity occurs more commonly in patients with renal impairment and in those older than age 60 years. The increased toxicity observed with high-dose cytarabine has limited the use of this therapy in older AML patients.

1	Incorporation of novel and molecular targeting agents into these regimens is currently under investigation. For patients with FLT3ITD AML, trials with tyrosine kinase inhibitors are ongoing. Patients with CBF AML may benefit from the combination of gemtuzumab ozogamicin, a monoclonal CD33 antibody linked to the cytotoxic agent calicheamicin, with induction and consolidation chemotherapies. This agent, initially approved for older patients with relapsed disease, has been withdrawn from the U.S. market at the request of the U.S. Food and Drug Administration due to concerns about the product’s toxicity, including myelosuppression, infusion toxicity, and venoocclusive disease and the clinical benefit of the initially recommended higher doses. However, the aforementioned recent results are encouraging and support the reintroduction of this agent into the therapeutic armamentarium for AML.

1	In older patients (age ≥60 years), the outcome is generally poor likely due to a higher induction treatment–related mortality rate and frequency of resistant disease, especially in patients with prior hematologic disorders (MDS or myeloproliferative syndromes) or who have received chemotherapy treatment for another malignancy or harbor cytogenetic and genetic abnormalities that adversely impact on clinical outcome. These patients should be considered for clinical trials. Alternatively, older patients can be also treated with the 7 and 3 regimen with standard-dose cytarabine and idarubicin (12 mg/m2), daunorubicin (45–90 mg/m2), or mitoxantrone (12 mg/ m2). For patients older than 65 years, higher dose daunorubicin (90 mg/m2) has not shown benefit due to the increased toxicity and is not recommended. The combination of gemtuzumab ozogamicin with chemotherapy reduces the risk of relapse for patients age 50–70 years with previously untreated AML. Finally, older patients may be considered

1	The combination of gemtuzumab ozogamicin with chemotherapy reduces the risk of relapse for patients age 50–70 years with previously untreated AML. Finally, older patients may be considered for single-agent therapies with clofarabine or hypomethylating agents (i.e., 5-azacitidine or decitabine). The latter are often used for patients unfit for more intensive therapies.

1	Diagnosis AML Previously untreated CBF AML: t(8;21) or inv(16) or t(16;16) Low risk normal cytogenetics (CEBPA double mutations or NPM1 mutations without FLT3-ITD) High risk normal cytogenetics (FLT3-ITD and/or NPM1 wild type) and High risk abnormal cytogenetics Salvage treatment Patient candidate for allogeneic HSCT and has suitable donor Yes: Allogeneic HSCT No: Investigational therapy If CR: investigational therapy If CR, consolidation therapy: Allogeneic HSCT If CR: Investigational molecular targeted therapy If CR, consolidation therapy: High-dose cytarabine or autologous HSCT If CR: Investigational molecular targeted therapy If CR, consolidation therapy: High-dose cytarabine Investigational therapy Induction therapy: Daunorubicin+ cytarabine-based regimen Induction therapy: Daunorubicin+ cytarabine-based regimen Induction therapy: Daunorubicin+ cytarabine-based regimen Investigational molecular targeted therapy Investigational molecular targeted therapy Either option acceptable

1	cytarabine-based regimen Induction therapy: Daunorubicin+ cytarabine-based regimen Investigational molecular targeted therapy Investigational molecular targeted therapy Either option acceptable Either option acceptable Either option acceptable Refractory or relapsed Refractory or relapsed

1	FIGURE 132-2 Flow chart for the therapy of newly diagnosed acute myeloid leukemia (AML). For all forms of AML except acute promyelocytic leukemia (APL), standard therapy includes a regimen based on a 7-day continuous infusion of cytarabine (100–200 mg/m2 per day) and a 3-day course of daunorubicin (60–90 mg/m2 per day) with or without additional drugs. Idarubicin (12–13 mg/m2 per day) could be used in place of daunorubicin (not shown). Patients who achieve complete remission (CR) undergo postremission consolidation therapy, including sequential courses of high-dose cytarabine, autologous hematopoietic stem cell transplantation (HSCT), allogeneic HSCT, or novel therapies, based on their predicted risk of relapse (i.e., risk-stratified therapy). Patients with APL (see text for treatment) usually receive tretinoin and arsenic trioxide–based regimens with or without anthracycline-based chemotherapy and possibly maintenance with tretinoin. CBF, core binding factor; ITD, internal tandem

1	usually receive tretinoin and arsenic trioxide–based regimens with or without anthracycline-based chemotherapy and possibly maintenance with tretinoin. CBF, core binding factor; ITD, internal tandem duplication.

1	After one cycle of the 7 and 3 chemotherapy induction regimen, if persistence of leukemia is documented, the patient is usually retreated with the same agents (cytarabine and the anthracycline) for 5 and 2 days, respectively. Our recommendation, however, is to consider changing therapy in this setting. Induction of a durable first CR is critical to long-term disease-free survival in AML. However, without further therapy, virtually all patients experience relapse. Thus, postremission therapy is designed to eradicate residual leukemic cells to prevent relapse and prolong survival. The type of postremission therapy in AML is often based on age and cytogenetic and molecular risk.

1	For younger patients, most studies include intensive chemotherapy and allogeneic or autologous hematopoietic stem cell transplantation (HSCT). In the postremission setting, high-dose cytarabine for three to four cycles is more effective than standard-dose cytarabine. The Cancer and Leukemia Group B (CALGB), for example, compared the duration of CR in patients randomly assigned after remission to four cycles of high (3 g/m2, every 12 h on days 1, 3, and 5), intermediate (400 mg/m2 for 5 days by continuous infusion), or standard (100 mg/m2 per day for 5 days by continuous infusion) doses of cytarabine. A dose-response effect for cytarabine in patients with AML who were age ≤60 years was demonstrated. High-dose cytarabine significantly prolonged CR and increased the fraction cured in patients with favorable [t(8;21) and inv(16)] and normal cytogenetics, but it had no significant effect on patients with other abnormal karyotypes. As discussed, high-dose cytarabine has increased toxicity

1	with favorable [t(8;21) and inv(16)] and normal cytogenetics, but it had no significant effect on patients with other abnormal karyotypes. As discussed, high-dose cytarabine has increased toxicity in older patients. Therefore, in this age group, for patients without CBF AML, exploration of attenuated chemotherapy regimens has been pursued. However, because the outcome of older patients is poor, allogeneic HSCT, when feasible, should be strongly considered. Postremission therapy is also a setting for introduction of new agents (Table 132-5).

1	Autologous HSCT preceded by one to two cycles of high-dose cytarabine is also an option for intensive consolidation therapy. Autologous HSCT has been generally applied to AML patients in the context of a clinical trial or when the risk of repetitive intensive chemotherapy represents a higher risk than the autologous HSCT (e.g., in patients with severe platelet alloimmunization) or when other factors including patient age, comorbid conditions, and fertility are considered.

1	Allogeneic HSCT is used in patients age <70–75 years with a human leukocyte antigen (HLA)-compatible donor who have high-risk cytogenetics. Selected high-risk patients are also considered for alternative donor transplants (e.g., mismatched unrelated, haploidentical related, and unrelated umbilical cord donors). In patients with CN-AML and high-risk molecular features such as FLT3-ITD, allogeneic HSCT is best applied in the context of clinical trials because the impact of aggressive therapy on outcome is unknown. For older patients, exploration of reduced-intensity allogeneic HSCT has been pursued.

1	Trials comparing intensive chemotherapy and autologous and allogeneic HSCT have shown improved duration of remission with allogeneic HSCT compared to autologous HSCT or chemotherapy alone. However, overall survival is generally not different; the improved disease control with allogeneic HSCT is erased by the increase in fatal toxicity. In fact, relapse following allogeneic HSCT occurs in only a small fraction of patients, but treatment-related toxicity is relatively high; complications include venoocclusive disease, graft-versus-host disease (GVHD), and infections. Autologous HSCT can be administered in young and older patients and uses the same preparative regimens. Patients subsequently receive their own stem cells collected while in remission. The toxicity is relatively low with Class of Drugs Examples of Agents in Class Inhibitors of Mutant proteins Tyrosine kinase inhibitors Dasatinib, midostaurin, quizartinib, sorafenib IDH2 mutation inhibitor AG-221

1	Class of Drugs Examples of Agents in Class Inhibitors of Mutant proteins Tyrosine kinase inhibitors Dasatinib, midostaurin, quizartinib, sorafenib IDH2 mutation inhibitor AG-221 Inhibitors of Cell Proliferation Inhibitors of Protein Synthesis and Degradation HSP-90 antagonists 17-Allylaminogeldanamycin (17-AAG), DMAG, or derivatives Nucleoside analogues Clofarabine, troxacitabine, elacytarabine, sapacitabine Compounds with Immuno-Mediated Mechanisms Antibodies CSL362 (anti-CD123), anti-CD33 (SGN33), anti-KIR Immunomodulatory Lenalidomide, interleukin 2, histamine dihydrochloride autologous HSCT (5% mortality rate), but the relapse rate is higher than with allogeneic HSCT, due to the absence of the graft-versusleukemia (GVL) effect seen with allogeneic HSCT and possible contamination of the autologous stem cells with residual tumor cells.

1	Prognostic factors may ultimately help to select the appropriate postremission therapy in patients in first CR. Our approach includes allogeneic HSCT in first CR for patients without favorable cytogenetics or genotype (e.g., patients who do not have CEBPA biallelic mutations or NPM1 mutations without FLT3-ITD) and/or with other poor risk factors (e.g., an antecedent hematologic disorder or failure to attain remission with a single induction course). If a suitable HLA donor does not exist, investigational therapeutic approaches are considered. Indeed, postremission therapy is also a setting for introduction of new agents (Table 132-5). Because FLT3-ITD can be targeted with emerging novel inhibitors, patients with this molecular abnormality should be considered for clinical trials with these agents whenever possible.

1	Patients with the favorable CBF AML [i.e., t(8;21), inv(16), or t(16;16)] are treated with repetitive doses of high-dose cytarabine, which offers a high frequency of cure without the morbidity of transplant. Among AML patients with t(8;21) and inv(16), those with KIT mutations, who have a worse prognosis, may be considered for novel investigational studies, including tyrosine kinase inhibitors. The inclusion of gemtuzumab ozogamicin in induction and consolidation chemotherapy-based treatment has been reported to be beneficial in this subset of patients.

1	For patients in morphologic CR, immunophenotyping to detect minute populations of blasts or sensitive molecular assays (e.g., reverse transcriptase polymerase chain reaction [RT-PCR]) to detect AML-associated molecular abnormalities (e.g., NPM1 mutation, the CBF AML RUNX1/RUNX1T1 and CBFB/MYH11 transcripts, the APL PML/RARA transcript), and the less sensitive metaphase cytogenetics or interphase cytogenetics by fluorescence in situ hybridization (FISH) to detect AML-associated cytogenetic aberrations, can be performed to assess whether clinically meaningful minimal residual disease (MRD) is present at sequential time points during 685 or after treatment. Detection of MRD may be a reliable discriminator between patients who will continue in CR and those who are destined to experience disease recurrence and therefore require early therapeutic intervention before clinical relapse occurs. Although assessment of MRD in bone marrow and/or blood during CR is routinely used in the clinic to

1	recurrence and therefore require early therapeutic intervention before clinical relapse occurs. Although assessment of MRD in bone marrow and/or blood during CR is routinely used in the clinic to anticipate clinical relapse and initiate timely salvage treatment for APL patients, for other cytogenetic and molecular subtypes of AML, this is an area of current investigation.

1	Measures geared to supporting patients through several weeks of neutropenia and thrombocytopenia are critical to the success of AML therapy. Patients with AML should be treated in centers expert in providing supportive measures. Multilumen right atrial catheters should be inserted as soon as patients with newly diagnosed AML have been stabilized. They should be used thereafter for administration of intravenous medications and transfusions, as well as for blood drawing.

1	Adequate and prompt blood bank support is critical to therapy of AML. Platelet transfusions should be given as needed to maintain a platelet count ≥10,000/μL. The platelet count should be kept at higher levels in febrile patients and during episodes of active bleeding or DIC. Patients with poor posttransfusion platelet count increments may benefit from administration of platelets from HLA-matched donors. RBC transfusions should be administered to keep the hemoglobin level >80 g/L (8 g/dL) in the absence of active bleed ing, DIC, or congestive heart failure, which require higher hemoglobin levels. Blood products leukodepleted by filtration should be used to avert or delay alloimmunization as well as febrile reactions. Blood products should also be irradiated to prevent transfusion-associated GVHD. Cytomegalovirus (CMV)-negative blood products should be used for CMV-seronegative patients who are potential candidates for allogeneic HSCT. Leukodepleted products are also effective for

1	GVHD. Cytomegalovirus (CMV)-negative blood products should be used for CMV-seronegative patients who are potential candidates for allogeneic HSCT. Leukodepleted products are also effective for these patients if CMV-negative products are not available.

1	Neutropenia (neutrophils <500/μL or <1000/μL and predicted to decline to <500/μL over the next 48 h) can be part of the initial presentation and/or a side effect of the chemotherapy treatment in AML patients. Thus, infectious complications remain the major cause of morbidity and death during induction and postremission chemotherapy for AML. Antibacterial (i.e., quinolones) and antifungal (i.e., posaconazole) prophylaxis in the absence of fever is likely to be beneficial. For patients who are herpes simplex virus or varicellazoster seropositive, antiviral prophylaxis should be initiated (e.g., acyclovir, valacyclovir).

1	Fever develops in most patients with AML, but infections are documented in only half of febrile patients. Early initiation of empirical broad-spectrum antibacterial and antifungal antibiotics has significantly reduced the number of patients dying of infectious complications (Chap. 104). An antibiotic regimen adequate to treat gram-negative organisms should be instituted at the onset of fever in a neutropenic patient after clinical evaluation, including a detailed physical examination with inspection of the indwelling catheter exit site and a perirectal examination, as well as procurement of cultures and radiographs aimed at documenting the source of fever. Specific antibiotic regimens should be based on antibiotic sensitivity data obtained from the institution at which the patient is being treated. Acceptable regimens for empiric antibiotic therapy include monotherapy with imipenem-cilastatin, meropenem, piperacillin/ tazobactam, or an extended-spectrum antipseudomonal cephalosporin

1	treated. Acceptable regimens for empiric antibiotic therapy include monotherapy with imipenem-cilastatin, meropenem, piperacillin/ tazobactam, or an extended-spectrum antipseudomonal cephalosporin (cefepime or ceftazidime). The combination of an aminoglycoside with an antipseudomonal penicillin (e.g., piperacillin) or an aminoglycoside in combination with an extended-spectrum antipseudomonal cephalosporin should be considered in complicated or resistant cases. Aminoglycosides should be avoided if possible in patients with renal insufficiency. Empirical vancomycin should be added in neutropenic patients with catheter-related infections, blood cultures positive for gram-positive bacteria before final identification and susceptibility testing, hypotension or shock, 686 or known colonization with penicillin/cephalosporin-resistant pneumococci or methicillin-resistant Staphylococcus aureus. In special situations where decreased susceptibility to vancomycin, vancomycin-resistant organisms,

1	with penicillin/cephalosporin-resistant pneumococci or methicillin-resistant Staphylococcus aureus. In special situations where decreased susceptibility to vancomycin, vancomycin-resistant organisms, or vancomycin toxicity is documented, other options including linezolid, daptomycin, and quinupristin/ dalfopristin need to be considered. Caspofungin (or a similar echinocandin), voriconazole, or liposomal amphotericin B should be considered for antifungal treatment if fever persists for 4–7 days following initiation of empiric antibiotic therapy. Amphotericin B has long been used for antifungal therapy. Although liposomal formulations have improved the toxicity profile of this agent, its use has been limited to situations with high risk of or documented mold infections. Caspofungin has been approved for empiric antifungal treatment. Voriconazole has also been shown to be equivalent in efficacy and less toxic than amphotericin B. Antibacterial and antifungal antibiotics should be

1	been approved for empiric antifungal treatment. Voriconazole has also been shown to be equivalent in efficacy and less toxic than amphotericin B. Antibacterial and antifungal antibiotics should be continued until patients are no longer neutropenic, regardless of whether a specific source has been found for the fever. Recombinant hematopoietic growth factors have been incorporated into clinical trials in AML. These trials have been designed to lower the infection rate after chemotherapy. Both G-CSF and granulocyte-macrophage colony-stimulating factor (GM-CSF) have reduced the median time to neutrophil recovery. This accelerated rate of neutrophil recovery, however, has not generally translated into significant reductions in infection rates or shortened hospitalizations. In most randomized studies, both G-CSF and GM-CSF have failed to improve the CR rate, disease-free survival, or overall survival. Although receptors for both G-CSF and GM-CSF are present on AML blasts, therapeutic

1	studies, both G-CSF and GM-CSF have failed to improve the CR rate, disease-free survival, or overall survival. Although receptors for both G-CSF and GM-CSF are present on AML blasts, therapeutic efficacy is neither enhanced nor inhibited by these agents. The use of growth factors as supportive care for AML patients is controversial. We favor their use in elderly patients with complicated courses, those receiving intensive postremission regimens, patients with uncontrolled infections, or those participating in clinical trials.

1	With the 7 and 3 regimen, 65–75% of younger and 50–60% of older patients with primary AML achieve CR. Two-thirds achieve CR after a single course of therapy, and one-third require two courses. Of patients who do not achieve CR, approximately 50% have a drug-resistant leukemia, and 50% do not achieve CR because of fatal complications of bone marrow aplasia or impaired recovery of normal stem cells. Patients with refractory disease after induction should be considered for salvage treatments, preferentially on clinical trials, before receiving allogeneic HSCT usually administered in patients who achieve a disease-free status. Because these patients are usually not cured even if they achieve second CR with salvage chemotherapy, allogeneic HSCT is a necessary therapeutic step.

1	In patients who relapse after achieving CR, the length of first CR is predictive of response to salvage chemotherapy treatment; patients with longer first CR (>12 months) generally relapse with drug-sensitive disease and have a higher chance of attaining a CR, even with the same chemotherapeutic agents used for first remission induction. Whether initial CR was achieved with one or two courses of chemotherapy and the type of postremission therapy may also predict achievement of second CR. Similar to patients with refractory disease, patients with relapsed disease are rarely cured by the salvage chemotherapy treatments. Therefore, patients who eventually achieve a second CR and are eligible for allogeneic HSCT should be transplanted.

1	Because achievement of a second CR with routine salvage therapies is relatively uncommon, especially in patients who relapse rapidly after achievement of first CR (<12 months), these patients and those lacking HLA-compatible donors or who are not candidates for allogeneic HSCT should be considered for innovative approaches on clinical trials (Table 132-5). The discovery of novel gene mutations and mechanisms of leukemogenesis that might represent actionable therapeutic targets has prompted the development of new targeting agents. In addition to kinase inhibitors for FLT3and KIT-mutated AML, other compounds targeting the aberrant activity of mutant proteins (e.g., IDH2 inhibitors) or biologic mechanisms deregulating epigenetics (e.g., histone deacetylase and DNA methyltransferase inhibitors), cell proliferation (e.g., farnesyl transferase inhibitors), protein synthesis (e.g., aminopeptide inhibitors) and folding (e.g., heat shock protein inhibitors), and ubiquitination, or with novel

1	cell proliferation (e.g., farnesyl transferase inhibitors), protein synthesis (e.g., aminopeptide inhibitors) and folding (e.g., heat shock protein inhibitors), and ubiquitination, or with novel cytotoxic mechanisms (e.g., clofarabine, sapacitabine), are being tested in clinical trials. Furthermore, approaches with antibodies targeting commonly expressed leukemia blasts (e.g., CD33) or leukemia initiating cells (e.g., CD123) and immunomodulatory agents (e.g., lenalidomide) are also under investigation. Once these compounds have demonstrated safety and activity as single agents, investigation of combinations with other molecular targeting compounds and/or chemotherapy should be pursued.

1	APL is a highly curable subtype of AML, and approximately 85% of these patients achieve long-term survival with current approaches. APL has long been shown to be responsive to cytarabine and daunorubicin, but previously patients treated with these drugs alone frequently died from DIC induced by the release of granule components by the chemotherapy-treated leukemia cells. However, the prognosis of APL patients has changed dramatically from adverse to favorable with the introduction of tretinoin, an oral drug that induces the differentiation of leukemic cells bearing the t(15;17), where disruption of the RARA gene encoding a retinoid acid receptor occurs. Tretinoin decreases the frequency of DIC but produces another complication called the APL differentiation syndrome. Occurring within the first 3 weeks of treatment, it is characterized by fever, fluid retention, dyspnea, chest pain, pulmonary infiltrates, pleural and pericardial effusions, and hypoxemia. The syndrome is related to

1	the first 3 weeks of treatment, it is characterized by fever, fluid retention, dyspnea, chest pain, pulmonary infiltrates, pleural and pericardial effusions, and hypoxemia. The syndrome is related to adhesion of differentiated neoplastic cells to the pulmonary vasculature endothelium. Glucocorticoids, chemotherapy, and/ or supportive measures can be effective for management of the APL differentiation syndrome. Temporary discontinuation of tretinoin is necessary in cases of severe APL differentiation syndrome (i.e., patients developing renal failure or requiring admission to the intensive care unit due to respiratory distress). The mortality rate of this syndrome is about 10%.

1	Tretinoin (45 mg/m2 per day orally until remission is documented) plus concurrent anthracycline-based (i.e., idarubicin or daunorubicin) chemotherapy appears to be among the most effective treatment for APL, leading to CR rates of 90–95%. The role of cytarabine in APL induction and consolidation is controversial. The addition of cytarabine, although not demonstrated to increase the CR rate, seemingly decreases the risk for relapse. Following achievement of CR, patients should receive at least two cycles of anthracyclinebased chemotherapy.

1	Arsenic trioxide has significant antileukemic activity and is being explored as part of initial treatment in clinical trials of APL. In a randomized trial, arsenic trioxide improved outcome if used after achievement of CR and before consolidation therapy with anthracycline-based chemotherapy. Patients receiving arsenic trioxide are at risk of APL differentiation syndrome, especially when it is administered during induction or salvage treatment after disease relapse. In addition, arsenic trioxide may prolong the QT interval, increasing the risk of cardiac arrhythmias.

1	Given the progress made in APL resulting in high cure rates, in recent years the goal has been to identify patients with low risk of relapse (i.e., those presenting with a leukocyte count ≤10,000/μL) where attempts are being made to decrease the amount of therapy administered and to identify patients at greatest risk of relapse (i.e., those presenting with a leukocyte count ≥10,000/μL) where new approaches can be developed to increase cure. A study compared the gold standard (tretinoin plus chemotherapy) in newly diagnosed non-high-risk APL with a chemotherapy-free combination of tretinoin and arsenic trioxide. An equivalent outcome was demonstrated between the two arms, and the chemotherapy-free regimen will likely become a new standard for non-high-risk APL patients. Combinations of tretinoin, arsenic trioxide, and/or chemotherapy and/or gemtuzumab ozogamicin have shown favorable responses in high-risk APL patients at diagnosis.

1	Combinations of tretinoin, arsenic trioxide, and/or chemotherapy and/or gemtuzumab ozogamicin have shown favorable responses in high-risk APL patients at diagnosis. Assessment of residual disease by RT-PCR amplification of the t(15;17) chimeric gene product PML-RARA following the final cycle of chemotherapy is an important step in the management of APL patients. Disappearance of the signal is associated with long-term disease-free survival; its persistence documented by two consecutive tests performed 2 weeks apart invariably predicts relapse. Sequential monitoring of RT-PCR for PML-RARA is now considered standard for postremission monitoring of APL, especially in high-risk patients.

1	The benefit from maintenance therapy with tretinoin has been documented in some studies and not in others. Thus, the use of tretinoin depends on which regimen has been used for induction and consolidation treatment and the risk category of the patients, with those with high-risk disease seemingly benefiting the most from maintenance therapy. Patients in molecular, cytogenetic, or clinical relapse should be salvaged with arsenic trioxide with or without tretinoin; it produces meaningful responses in up to 85% of patients and can be followed by autologous or, less frequently, especially if RT-PCR positive for PML-RARA, allogeneic HSCT.

1	Chronic Myeloid Leukemia Hagop Kantarjian, Jorge Cortes Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder. The disease is driven by the BCR-ABL1 chimeric gene prod-uct, a constitutively active tyrosine kinase, resulting from a reciprocal balanced translocation between the long arms of chromosomes 9 and 133 22, t(9;22) (q34;q11.2), cytogenetically detected as the Philadelphia chromosome (Ph) (Fig. 133-1). Untreated, the course of CML may be biphasic or triphasic, with an early indolent or chronic phase, followed often by an accelerated phase and a terminal blastic phase. Before the era of selective BCR-ABL1 tyrosine kinase inhibitors (TKIs), the median survival in CML was 3–7 years, and the 10-year survival rate was 30% or less. Introduced into CML therapy in 2000, TKIs have revolutionized the treatment, natural history, and prognosis of CML. Today, the estimated 10-year survival rate with imatinib mesylate, the first BCR-ABL1 TKI approved, is 85%. Allogeneic

1	TKIs have revolutionized the treatment, natural history, and prognosis of CML. Today, the estimated 10-year survival rate with imatinib mesylate, the first BCR-ABL1 TKI approved, is 85%. Allogeneic stem cell transplantation (SCT), a curative but risky treatment approach, is now offered as secondor third-line therapy after failure of TKIs.

1	CML accounts for 15% of all cases of leukemia. There is a slight male preponderance (male:female ratio 1.6:1). The median age at diagnosis is 55–65 years. It is uncommon in children; only 3% of patients with CML are younger than 20 years. CML incidence increases slowly with age, with a steeper increase after the age of 40–50 years. The annual incidence of CML is 1.5 cases per 100,000 individuals. In the United States, this translates into 4500–5000 new cases per year. The incidence of CML has not changed over several decades. By extrapolation, the worldwide annual incidence of CML is about 100,000 cases. With a median survival of 6 years before 2000, the disease prevalence in the United States was 20,000–30,000 cases. With TKI therapy, the annual mortality has been reduced from 10–20% to about 2%. Therefore, the prevalence of CML in the United States is expected to continue to increase (about 80,000 in 2013) and reach a plateau of approximately 180,000 cases around 2030. The worldwide

1	2%. Therefore, the prevalence of CML in the United States is expected to continue to increase (about 80,000 in 2013) and reach a plateau of approximately 180,000 cases around 2030. The worldwide prevalence will depend on the treatment penetration of TKIs and their effect on reduction of worldwide annual mortality. Ideally, with full TKI treatment penetration, the worldwide prevalence should plateau at 35 times the incidence, or around 3 million patients.

1	There are no familial associations in CML. The risk of developing CML is not increased in monozygotic twins or in relatives of patients. No etiologic agents are incriminated, and no associations exist with exposures to benzene or other toxins, fertilizers, insecticides, or viruses. CML is not a frequent secondary leukemia following therapy of other cancers with alkylating agents and/or radiation. Exposure to ionizing radiation (e.g., nuclear accidents, radiation treatment for ankylosing spondylitis or cervical cancer) has increased the risk of CML, which peaks at 5–10 years after exposure and is dose-related. The median time to development of CML among atomic bomb survivors was 6.3 years. Following the Chernobyl accident, the incidence of CML did not increase, suggesting that only large doses of radiation can cause CML. Because of adequate protection, the risk of CML is not increased in individuals working in the nuclear industry or among radiologists in recent times.

1	The t(9;22) (q34;q11.2) is present in more than 90% of classical CML cases. It results from a balanced reciprocal translocation between the long arms of chromosomes 9 and 22. It is present in hematopoietic cells (myeloid, erythroid, megakaryocytes, and monocytes; less often mature B lymphocytes; rarely mature T lymphocytes, but not stromal cells), but not in other cells in the human body. As a result of the translocation, DNA sequences from the cellular oncogene ABL1 are translocated next to the major breakpoint cluster region (BCR) gene on chromosome 22, generating a hybrid oncogene, BCR-ABL1. This fusion gene codes for a novel oncoprotein of molecular weight 210 kDa, referred to as p210BCR-ABL1 (Fig. 133-1B). This BCR-ABL1 oncoprotein exhibits constitutive kinase activity that leads to excessive proliferation and reduced apoptosis of CML cells, endowing them with a growth advantage over their normal counterparts. Over time, normal hematopoiesis is suppressed, but normal stem cells

1	excessive proliferation and reduced apoptosis of CML cells, endowing them with a growth advantage over their normal counterparts. Over time, normal hematopoiesis is suppressed, but normal stem cells can persist and may reemerge following effective therapy, for example with TKIs. In Ph-positive acute lymphocytic leukemia (ALL) and in rare cases of CML, the breakpoint in BCR is more centromeric, in a region called the minor BCR region (mBCR). As a result, a shorter sequence of BCR is fused to ABL1, with a consequent smaller BCR-ABL1 oncoprotein, p190BCR-ABL1. When occurring in Ph-positive CML, this translocation may predict for a worse outcome. A third rare breakpoint in BCR occurs telomeric to the major BCR region and is called micro-BCR (μ-BCR). It juxtaposes a larger fragment of the BCR gene to ABL1 and produces a larger p230BCR-ABL1 oncoprotein, which is associated with a more indolent CML course.

1	The constitutive activation of BCR-ABL1 results in autophosphorylation and activation of multiple downstream pathways that modify gene transcription, apoptosis, skeletal organization, and degradation of inhibitory proteins. These transduction pathways may involve RAS, mitogenactivated protein (MAP) kinases, signal transducers and activators of transcription (STAT), phosphatidylinositol-3-kinase (PI3k), MYC, and others. These interactions are mostly mediated through tyrosine phosphorylation and require binding of BCR-ABL1 to adapter proteins such as GRB-2, CRK, CRK-like (CRK-L) protein, and Src homology containing proteins (SHC). BCR-ABL1 TKIs bind to the BCR-ABL1 kinase domain (KD), preventing the activation of transformation pathways and inhibiting downstream signaling. As a result, proliferation of CML cells is inhibited and apoptosis induced, leading to the reemergence of normal hematopoiesis. A plethora of signaling pathways have been implicated in BCR-ABL1-mediated cellular

1	proliferation of CML cells is inhibited and apoptosis induced, leading to the reemergence of normal hematopoiesis. A plethora of signaling pathways have been implicated in BCR-ABL1-mediated cellular transformation. The emerging picture is a complex and redundant transformation network. An additional layer of complexity is related to differences in signal transduction between CML differentiated cells and early progenitors. Beta-catenin, Wnt1, Foxo3a, transforming growth factor β, interleukin-6, PP2A, SIRT1, and others have been implicated in CML stem cell survival.

1	Experimental models have established the causal relationship between the Ph-related BCR-ABL1 molecular events and the development of CML. In animal models, expression of BCR-ABL1 in normal hematopoietic cells produced CML-like disorders or lymphoid leukemia, demonstrating the leukemogenic potential of BCR-ABL1 as a single oncogenic abnormality. q34q11.2 t(9;22)(q34;q11.2) FIGURE 133-1 A. The Philadelphia (Ph) chromosome cytogenetic abnormality. B. Breakpoints in the long arms of chromosome 9 (ABL locus) and chromosome 22 (BCR regions) result in three different BCR-ABL oncoprotein messages, p210BCR-ABL1 (most common message in chronic myeloid leukemia [CML]), p190BCR-ABL1 (present in two-thirds of patients with Ph-positive acute lymphocytic leukemia; rare in CML), and p230BCR-ABL1 (rare in CML and associated with an indolent course). (© 2013 The University of Texas MD Anderson Cancer Center.)

1	The cause of the BCR-ABL1 molecular rearrangement is unknown. Molecular techniques that detect BCR-ABL1 at a level of 1 in 108 identify this molecular abnormality in the blood of up to 25% of normal adults and 5% of infants, but 0% of cord blood samples. This suggests that BCR-ABL1 is not sufficient to cause overt CML in the overwhelming majority of individuals in whom it occurs. Because CML develops in only 1.5 of 100,000 individuals annually, it is evident that additional molecular events or poor immune recognition of the rearranged cells are needed to cause overt CML.

1	CML is defined by the presence of BCR-ABL1 abnormality in a patient with a myeloproliferative neoplasm. In some patients with a typical morphologic picture of CML, the Ph abnormality is not detectable by standard cytogenetic analysis, but fluorescence in situ hybridization (FISH) and molecular studies (polymerase chain reaction [PCR]) detect BCR-ABL1. These patients have a course similar to Ph-positive CML and respond to TKI therapy. Many of the remaining patients have atypical morphologic or clinical features and belong to other diagnostic groups, such as atypical CML or chronic myelomonocytic leukemia. These individuals do not respond to TKI therapy and have a poor prognosis with a median survival of about 2–3 years. Detection of mutations in the granulocyte colony-stimulating factor receptor (CSF3R) in chronic neutrophilic leukemia and in some cases of atypical CML and of mutations in SETBP1 in atypical CML confirmed that they are distinct entities.

1	The mechanisms associated with the transition of CML from a chronic to accelerated-blastic phase are poorly understood. They are often associated with characteristic chromosomal abnormalities such as a double Ph, trisomy 8, isochromosome 17 or deletion of 17p (loss of TP53), 20q–, and others. Molecular events associated with transformation include mutations in TP53, retinoblastoma 1 (RB1), myeloid transcriptions factors like Runx1, and cell cycle regulators like p16. A plethora of other mutations or functional abnormalities have been implicated in blastic transformation, but no unifying theme has emerged other than that BCR-ABL1 itself induces genetic instability that leads to the acquisition of additional mutations and eventually to blastic transformation. In this frame of thinking, one critical effect of TKIs is their ability to stabilize the CML genome, leading to a much reduced transformation rate. In particular, the previously observed sudden blastic transformations (i.e., abrupt

1	effect of TKIs is their ability to stabilize the CML genome, leading to a much reduced transformation rate. In particular, the previously observed sudden blastic transformations (i.e., abrupt transformation to blastic phase in a patient who had been in cytogenetic response) have become uncommon, occurring rarely in younger patients in the first 1–2 years of TKI therapy (usually sudden lymphoid blastic transformations). Sudden transformations beyond the third year of TKI therapy are rare in patients who continue on TKI therapy. Moreover, initial experience suggests that the course of CML has become significantly more indolent, even without cytogenetic responses, in patients on TKI-based therapy compared to previous experience with hydroxyurea/busulfan.

1	Among patients developing resistance to TKIs, several resistance mechanisms have been observed. The most clinically relevant one is the development of different ABL1 kinase domain mutations that prevent the binding of TKIs to the catalytic site (ATP binding site) of the kinase. More than 100 BCR-ABL1 mutations have now been described, many of which confer relative or absolute resistance to imatinib. This has resulted in the development of second-generation TKIs (i.e., dasatinib, nilotinib, bosutinib) and of a third-generation TKI (ponatinib) with selective efficacy against T315I, a mutation of the gatekeeper residue of the kinase that causes resistance to all other TKIs.

1	The presenting signs and symptoms in CML depend on the availability of and access to health care procedures, including physical exams and screening tests. In the United States, because of the easy access to health care screening and physical exams, 50–60% of patients are diagnosed on routine blood tests and have minimal symptoms at presentation, such as fatigue. In geographic locations where access to health care is more limited, patients often present with high CML burden including splenomegaly, anemia, and related symptoms (abdominal pain, weight loss, fatigue), as well as a higher frequency of high-risk CML. Presenting findings in patients diagnosed in the United States are shown in Table 133-1.

1	Symptoms Most patients with CML (90%) present in the indolent or chronic phase. Depending on the timing of diagnosis, patients are often asymptomatic (if the diagnosis is discovered during health care screening tests). Common symptoms, when present, are manifestations of anemia and splenomegaly. These may include fatigue, malaise, weight loss (if high leukemia burden), or early satiety and left upper quadrant pain or masses (from splenomegaly). Less common presenting findings include thrombotic or vasoocclusive events (from severe leukocytosis or thrombocytosis). These include priapism, cardiovascular complications, myocardial infarction, venous thrombosis, visual disturbances, dyspnea and pulmonary insufficiency, drowsiness, loss of coordination, confusion, or cerebrovascular accidents. Bleeding diatheses findings include retinal hemorrhages, gastrointestinal bleeding,

1	Cytogenetic clonal evolution other than the Philadelphia chromosome Sokal risk Low Intermediate High and others. Patients who present with, or progress to, the accelerated 689 or blastic phases have additional symptoms including unexplained fever, significant weight loss, severe fatigue, bone and joint aches, bleeding and thrombotic events, and infections.

1	Physical Findings Splenomegaly is the most common physical finding, occurring in 20–70% of patients depending on health care screening frequency. Other less common findings include hepatomegaly (10–20%), lymphadenopathy (5–10%), and extramedullary disease (skin or subcutaneous lesions). The latter indicates CML transformation if a biopsy confirms the presence of sheets of blasts. Other physical findings are manifestations of complications of high tumor burden described earlier (e.g., cardiovascular, cerebrovascular, bleeding). High basophil counts may be associated with histamine overproduction causing pruritus, diarrhea, flushing, and even gastrointestinal ulcers.

1	Hematologic and Marrow Findings In untreated CML, leukocytosis ranging from 10–500 × 109/L is common. The peripheral blood differential shows left-shifted hematopoiesis with predominance of neutrophils and the presence of bands, myelocytes, metamyelocytes, promyelocytes, and blasts (usually ≤5%). Basophils and/or eosinophils are frequently increased. Thrombocytosis is common, but thrombocytopenia is rare and, when present, suggests a worse prognosis, disease acceleration, or an unrelated etiology. Anemia is present in one-third of patients. Cyclic oscillations of counts are noted in 25% of patients without treatment. Biochemical abnormalities include a low leukocyte alkaline phosphatase score and high levels of vitamin B12, uric acid, lactic dehydrogenase, and lysozyme. The presence of unexplained and sustained leukocytosis, with or without splenomegaly, should lead to a marrow examination and cytogenetic analysis.

1	The bone marrow is hypercellular with marked myeloid hyperplasia and a high myeloid-to-erythroid ratio of 15–20:1. Marrow blasts are 5% or less; when higher, they carry a worse prognosis or represent acceleration (if they are ≥15%). Increased reticulin fibrosis (by Snook’s silver stain) is common, with 30–40% of patients demonstrating grade 3–4 reticulin fibrosis. This was considered adverse in the pre-TKI era. With TKI therapy, reticulin fibrosis resolves in most patients and is not an indicator of poor prognosis. Collagen fibrosis (Wright-Giemsa stain) is rare at diagnosis. Disease progression with a “spent phase” of myelofibrosis (myelophthisis, or burnt-out marrow) was common with busulfan therapy (20–30%) but is rare with TKI therapy.

1	Cytogenetic and Molecular Findings The diagnosis of CML is straightforward and depends on documenting t(9;22)(q34;q11.2), which is found in 90% of cases. This is known as the Philadelphia-chromosome abnormality (discovered in Philadelphia) and was initially identified as a shortened chromosome, later identified to be chromosome 22 (22q–) (Fig. 133-1). Some patients may have complex translocations (variant Ph) involving three or more translocations that include chromosomes 9 and 22 and one or more other chromosomes. Others may have a “masked Ph,” involving translocations between chromosome 9 and a chromosome other than 22. The prognosis of these patients and their response to TKI therapy are similar to those in patients with Ph. About 5–10% of patients may have additional chromosomal abnormalities in the Ph-positive cells. These usually involve trisomy 8, a double Ph, isochromosome 17 or 17p deletion, 20q–, or others. This is referred to as clonal evolution and was historically a sign

1	in the Ph-positive cells. These usually involve trisomy 8, a double Ph, isochromosome 17 or 17p deletion, 20q–, or others. This is referred to as clonal evolution and was historically a sign of adverse prognosis, particularly when trisomy 8, double Ph, or chromosome 17 abnormalities were noted.

1	Techniques such as FISH and PCR are now used to aid in the diagnosis of CML. They are more sensitive approaches to estimate the CML burden in patients on TKI therapy. They can be done on peripheral samples, and thus are less painful and more convenient. Patients with CML at diagnosis should have a FISH analysis to quantify the percentage of Ph-positive cells, if FISH is used to replace marrow cytogenetic analysis in monitoring response to therapy. FISH may not detect additional chromosomal abnormalities (clonal evolution); thus, a cytogenetic analysis is usually recommended at the time of diagnosis. The BCR-ABL1 RNA message is usually one of two variants: e13a2 (formerly b2a2) and e14a2 (formerly b3a2). About 2–5% of patients 690 may have other RNA fusion types (e.g., e1a2, e13a3, or e14a3). In these patients, the routine PCR primers may not amplify the BCR-ABL1 transcripts, thus leading to false-negative results. Therefore, molecular studies at diagnosis are important to document the

1	In these patients, the routine PCR primers may not amplify the BCR-ABL1 transcripts, thus leading to false-negative results. Therefore, molecular studies at diagnosis are important to document the type and presence of BCR-ABL1 transcripts to avoid erroneously “undetectable” BCRABL1 transcripts on follow-up studies, with the misconception of a complete molecular response. Both FISH and PCR studies can be falsely positive at low levels or falsely negative because of technical issues. Therefore, a diagnosis of CML must always rely on a marrow analysis with routine cytogenetics. The diagnostic bone marrow confirms the presence of the Ph chromosome, detects clonal evolution, i.e., chromosomal abnormalities in the Ph-positive cells (which may be prognostic), and also quantifies the percentage of marrow blasts and basophils. In 10% of patients, the percentage of marrow blasts and basophils can be significantly higher than in the peripheral blood, suggesting poorer prognosis or even disease

1	of marrow blasts and basophils. In 10% of patients, the percentage of marrow blasts and basophils can be significantly higher than in the peripheral blood, suggesting poorer prognosis or even disease transformation. Monitoring patients on TKI therapy by cytogenetics, FISH, and molecular studies has become an important standard practice to assess response to therapy, emphasize compliance, evaluate possible treatment resistance, change TKI therapy, and order mutational analysis studies. It is thus important to recognize the comparability of these measures in monitoring response. A partial cytogenetic response is defined as the presence of 35% less Ph-positive metaphases by routine cytogenetic analysis. This is roughly equivalent to BCR-ABL1 transcripts by the International Scale (IS) of 10% or less. A complete cytogenetic response refers to the absence of Ph-positive metaphases (0% Ph positivity). This is approximately equivalent to BCR-ABL1 transcripts (IS) of 1% or less. A major

1	10% or less. A complete cytogenetic response refers to the absence of Ph-positive metaphases (0% Ph positivity). This is approximately equivalent to BCR-ABL1 transcripts (IS) of 1% or less. A major molecular response refers to BCR-ABL1 transcripts (IS) ≤0.1%, or roughly a 3-log or greater reduction of CML burden from baseline. A complete molecular response usually refers to BCR-ABL1 transcripts (IS) <0.0032% (undetectable by current techniques), roughly equivalent to a more than 4.5-log reduction of CML burden from baseline.

1	Findings in CML Transformation Progression of CML is usually associated with leukocytosis resistant to therapy, increasing anemia, fever and constitutional symptoms, and increased blasts and basophils in the peripheral blood or marrow. Criteria of accelerated-phase CML, historically associated with median survival of less than 1.5 years, include the presence of 15% or more peripheral blasts, 30% or more peripheral blasts plus promyelocytes, 20% or more peripheral basophils, cytogenetic clonal evolution (presence of chromosomal abnormalities in addition to Ph), and thrombocytopenia <100 × 109/L (unrelated to 1.0 therapy). About 5–10% of patients present with de novo accelerated phase or blastic phase. The prognosis of de novo accelerated phase with TKI therapy has improved significantly, with an estimated 8-year survival rate of 75%. The median survival of accelerated phase evolving from chronic phase has also improved from a historical median survival of 18 months to an estimated

1	with an estimated 8-year survival rate of 75%. The median survival of accelerated phase evolving from chronic phase has also improved from a historical median survival of 18 months to an estimated 4-year survival rate of 70% on TKI therapy. Therefore, the criteria for accelerated-phase CML should be revisited because most have lost much of their prognostic significance. Blastic-phase CML is defined by the presence of 30% or more peripheral or marrow blasts or the presence of sheets of blasts in extramedullary disease (usually skin, soft tissues, or lytic bone lesions). Blasticphase CML is commonly myeloid (60%) but can present uncommonly as erythroid, promyelocytic, monocytic, or megakaryocytic. Lymphoid blastic phase occurs in about 25% of patients. Lymphoblasts are terminal deoxynucleotide transferase positive and peroxidase negative (although occasionally with low positivity up to 3–5%) and express lymphoid markers (CD10, CD19, CD20, CD22). However, they also often express myeloid

1	transferase positive and peroxidase negative (although occasionally with low positivity up to 3–5%) and express lymphoid markers (CD10, CD19, CD20, CD22). However, they also often express myeloid markers (50–80%), resulting in diagnostic confusion. This is important because, unlike other morphologic blastic phases, lymphoid blastic-phase CML is quite responsive to antiALL-type chemotherapy (e.g., hyper-CVAD [cyclophosphamide, vincristine, doxorubicin, and dexamethasone]) in combination with TKIs.

1	Before the imatinib era, the annual mortality in CML was 10% in the first 2 years and 15–20% thereafter. The median survival time in CML was 3–7 years (with hydroxyurea-busulfan and interferon α). Without a curative option of allogeneic SCT, the course of CML was inexorable toward transformation to, and death from, accelerated or blastic phases. The disease stability was unpredictable, with some patients demonstrating sudden transformation to a blastic phase. With imatinib therapy, the annual mortality in CML has decreased to 2% in the first 12 years of observation. Half of the deaths are from factors other than CML, such as old age, accidents, suicides, other cancers, and other medical conditions (e.g., infections, surgical procedures). The estimated 8to 10-year survival rate is now 85%, or 93% if only CML-related deaths are considered (Fig. 133-2). The course of CML has also become quite predictable. In the first 2 years of TKI therapy, rare sudden transformations are still noted

1	or 93% if only CML-related deaths are considered (Fig. 133-2). The course of CML has also become quite predictable. In the first 2 years of TKI therapy, rare sudden transformations are still noted (1–2%), usually lymphoid blastic transformations that respond to combinations of chemotherapy and TKIs followed by allogeneic SCT. These may be explained by the intrinsic mechanisms of sudden transformation already existing in the CML clones before the start of therapy that were not amenable to TKI inhibition, in particular imatinib. Second-generation TKIs (nilotinib, 1.0 0.0 0.0 100 1 2 3 4 5 6 7 8 9 Years 41% 19% 5% 2% Accelerated 1980–2000 398 325 28 p = 0.015 Accelerated 2001–2013 258 86 88 Blastic 1980–2000 196 189 5 Blastic 2001–2013 175 133 7 p < 0.001 p < 0.001

1	FIGURE 133-2 A. Survival in newly diagnosed chronic-phase chronic myeloid leukemia (CML) by era of therapy (M.D. Anderson Cancer Center experience from 1965 to present). Causes of non-CML deaths in 22 patients were other cancers (n = 7), postsurgical complications (n = 3), car accident (n = 2), suicide (n = 1), neurologic events (n = 3), cardiac (n = 3), pneumonia (n = 1), and unknown (n = 2). B. Survival in patients with acceleratedand blastic-phase CML referred to M.D. Anderson Cancer Center by era of therapy, demonstrating the significant survival benefit in the tyrosine kinase inhibitor (TKI) era in accelerated-phase CML but the modest benefit in blastic-phase CML. Referred cases included de novo and post-chronic-phase transformations.

1	0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 dasatinib) used as frontline therapy have reduced the incidence of transformation in the first 2–3 years from 6–8% with imatinib to 2–4% with nilotinib or dasatinib. Disease transformation to accelerated or blastic phase is rare on continued TKI therapy, estimated at <1% annually in years 4–8 of follow-up on the original imatinib trials. Patients usually develop resistance in the form of cytogenetic relapse, followed by hematologic relapse and subsequent transformation, rather than the previously feared sudden transformations without the warning signals of cytogenetic-hematologic relapse.

1	Before the imatinib era, several pretreatment prognostic factors predicted for worse outcome in CML and have been incorporated into prognostic models and staging systems. These have included older age, significant splenomegaly, anemia, thrombocytopenia or thrombocytosis, high percentages of blasts and basophils (and/or eosinophils), marrow fibrosis, deletions in the long arm of chromosome 9, clonal evolution, and others. Different risk models and staging systems, derived from multivariate analyses, were proposed to define different risk groups. As with the introduction of cisplatin into testicular cancer therapy, the introduction of TKIs into CML therapy has nullified or lessened the prognostic impact of most of these prognostic factors and the significance of the CML models (e.g., Sokal, Hasford, European Treatment and Outcome Study [EUTOS]). Treatment-related prognostic factors have emerged as the most important prognostic factors in the era of imatinib therapy. Achievement of

1	Hasford, European Treatment and Outcome Study [EUTOS]). Treatment-related prognostic factors have emerged as the most important prognostic factors in the era of imatinib therapy. Achievement of complete cytogenetic response has become the major therapeutic endpoint and is the only endpoint associated with improvement in survival. Achievement of a major molecular response is associated with decreased risk of events (relapse) and CML progression, may predict for differences in event-free survival (depending on the definition of an event) and for small differences in transformation rates, but has not been associated with survival prolongation. Among patients in complete cytogenetic response, survival is similar independent of whether they achieve a major molecular response or not. This may be due to the efficacy of salvage TKI therapies, which are and should be implemented at the first evidence of cytogenetic relapse. Achievement of complete molecular response (undetectable BCR-ABL1

1	be due to the efficacy of salvage TKI therapies, which are and should be implemented at the first evidence of cytogenetic relapse. Achievement of complete molecular response (undetectable BCR-ABL1 transcripts), particularly when durable (>2 years), may offer the possibility of durable molecular response (molecular cure rather than functional cure) in the context of investigational trials and may allow temporary therapy interruption in women eager to have babies. The lack of achievement of major or complete molecular responses should not be considered as “failure” of a particular TKI therapy and/or an indication to change the TKI or to consider allogeneic SCT.

1	Pretreatment prognostic factors and prognostic models have lost much of their clinical relevance to define prognosis and to select different therapies. However TKI-associated therapeutic responses have gained major clinical relevance and dictate appropriate and careful monitoring of patients to optimize their treatment.

1	The introduction of TKI therapy, first in the form of imatinib mesylate in 2001, has revolutionized the treatment and prognosis in CML. Before 2000, allogeneic SCT was frontline therapy, when available, because of its potentially curative capacity. Otherwise, patients 691 were offered interferon α therapy (approved for the treatment of CML in 1986), which had modest benefits (improving survival from a median of 3–4 years with hydroxyurea-busulfan to a median of 6–7 years), but also significant side effects. Other alternatives included hydroxyurea, busulfan, and other nonspecific chemotherapies. With TKI therapy, the estimated 10-year survival in CML is 85%. Since 2001, six agents have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of CML. These include five oral BCR-ABL1-selective TKIs: imatinib (Gleevec), nilotinib (Tasigna), dasatinib (Sprycel), bosutinib (Bosulif), and ponatinib (Iclusig). Imatinib 400 mg orally daily, nilotinib 300 mg orally twice a

1	oral BCR-ABL1-selective TKIs: imatinib (Gleevec), nilotinib (Tasigna), dasatinib (Sprycel), bosutinib (Bosulif), and ponatinib (Iclusig). Imatinib 400 mg orally daily, nilotinib 300 mg orally twice a day (on an empty stomach), and dasatinib 100 mg orally daily are approved for frontline therapy of CML. All three are also approved for salvage therapy (nilotinib 400 mg twice daily), in addition to bosutinib (500 mg daily) and ponatinib (45 mg daily). Imatinib, dasatinib (140 mg daily), bosutinib, and ponatinib are also approved for the treatment of CML transformation (accelerated and blastic phase), whereas nilotinib is only approved for chronic and accelerated phase. Dasatinib, nilotinib, and bosutinib are referred to as sec-ond-generation TKIs; ponatinib is referred to as a third-generation TKI. The sixth approved agent is omacetaxine (Synribo), a protein synthesis inhibitor with presumed more selective inhibition of the synthesis of the BCR-ABL1 oncoprotein. It is approved for the

1	TKI. The sixth approved agent is omacetaxine (Synribo), a protein synthesis inhibitor with presumed more selective inhibition of the synthesis of the BCR-ABL1 oncoprotein. It is approved for the treatment of chronicand accelerated-phase CML after failure of two or more TKIs, at 1.25 mg/m2 subcutaneously twice a day for 14 days for induction and for 7 days for consolidation-maintenance. Nilotinib is similar in structure to imatinib but 30 times more potent. Dasatinib and bosutinib are dual SRC-ABL1 TKIs (dasatinib is reported to be 300 times more potent and bosutinib 30–50 times more potent than imatinib). Ponatinib is effective against wild-type and mutant BCRABL1 clones. It is unique in being the only currently available BCRABL1 TKI that is active against T315I, a gatekeeper mutant resistant to the other four TKIs (Table 133-2).

1	Imatinib, nilotinib, and dasatinib are all acceptable frontline therapies in CML. The long-term results of imatinib are very favorable. The 8-year follow-up results show a cumulative complete cytogenetic response rate (occurring at least once) of 83%, with 60–65% of patients being in complete cytogenetic response at 5-year follow-up. The estimated 8-year event-free survival rate is 81%, and the overall survival rate is 85%. Among patients continuing on imatinib, the annual rate of transformation to accelerated-blastic phase in years 4–8 is <1%. In two randomized studies, one comparing nilotinib 300 mg twice daily or 400 mg twice daily with imatinib (ENEST-nd) and the other comparing dasatinib 100 mg daily with imatinib (DASISION), the second-generation TKIs were associated with better outcomes in early surrogate endpoints, including higher rates of complete cytogenetic responses (85–87% vs 77–82%), major molecular responses (65–76% vs 46–63%), and undetectable BCR-ABL1 transcripts

1	outcomes in early surrogate endpoints, including higher rates of complete cytogenetic responses (85–87% vs 77–82%), major molecular responses (65–76% vs 46–63%), and undetectable BCR-ABL1 transcripts (IS) (32–37% vs 15–30%), and lower rates of transformation to accelerated-blastic phase (2–4% vs 6%). However, neither study showed a survival benefit with second-generation TKIs (median follow-up times of 4–5 years). This may be because salvage 692 therapy with other TKIs (following close observation and treatment change at progression) provides highly effective salvage therapy that rebalances the negative effect of the relapse. Salvage therapy in chronic phase with dasatinib, nilotinib, bosutinib, or ponatinib is associated with complete cytogenetic response rates of 30–60% of patients, depending on the salvage status (cytogenetic vs hematologic relapse), prior response to other TKIs, and the mutations at the time of relapse. Complete cytogenetic responses are generally durable,

1	depending on the salvage status (cytogenetic vs hematologic relapse), prior response to other TKIs, and the mutations at the time of relapse. Complete cytogenetic responses are generally durable, particularly in the absence of clonal evolution and mutations. Ponatinib is the only TKI active in the setting of T315I mutation, with complete cytogenetic response rates of 50–70%. The estimated 3to 5-year survival rates with new TKIs as salvage are 70–80% (compared with <50% before their availability). For example, with dasatinib salvage after imatinib failure in chronic-phase CML, the major molecular response rates were 40–43%, the estimated 6-year survival rates were 74–83%, and progression-free survival rates were 40–51%. Thus, TKIs in the salvage setting have already reduced the annual mortality from the historical rate of 10–15% to ≤5%. The goal of CML therapy is viewed differently in the context of research versus standard practice. In current practice, functional cure, defined as

1	from the historical rate of 10–15% to ≤5%. The goal of CML therapy is viewed differently in the context of research versus standard practice. In current practice, functional cure, defined as survival with CML similar to survival among normal individuals, is the current goal of therapy. CML is now considered an indolent disease, which, with appropriate TKI therapy, treatment compliance, careful monitoring, and early change to other TKIs as indicated, can be associated with close to normal survival. Therefore, in standard practice, achievement and maintenance of a complete cytogenetic response are the aims of therapy, because complete cytogenetic response is the only treatment-related factor associated with survival prolongation. Lack of achievement of a major molecular response (protects against events; associated with longer event-free survival) or of negative BCR-ABL1 transcripts (offers the potential of TKI interruption on investigational studies) should not be considered

1	against events; associated with longer event-free survival) or of negative BCR-ABL1 transcripts (offers the potential of TKI interruption on investigational studies) should not be considered indications to change TKI therapy or to consider allogeneic SCT. A general practice rule is to continue the particular TKI chosen at the most tolerable dose schedule not associated with grade 3–4 side effects or with bothersome chronic side effects, for as long as possible, until either cytogenetic relapse or the persistence of unacceptable side effects. These two factors (i.e., cytogenetic relapse and intolerable side effects as judged by the patient and treating physician) are the indicators of “failure” of a particular TKI therapy. Because of the increasing prevalence of CML (cost of TKI therapy) and the emerging long-term low rates of significant organ toxicities, the ultimate goal of CML therapy in the research setting is to achieve eradication of the disease (molecular cure) that is

1	and the emerging long-term low rates of significant organ toxicities, the ultimate goal of CML therapy in the research setting is to achieve eradication of the disease (molecular cure) that is prolonged and durable, with recovery of nonneoplastic, nonclonal hematopoiesis off TKI therapy. The first step toward this aim is to obtain the highest rates of undetectable BCR-ABL1 transcripts lasting for at least 2 or more years. Recommendations provided by the National Comprehensive Cancer Network (NCCN) and by the European LeukemiaNet (ELN) discuss optimal/expected, suboptimal/warning, and failure response scenarios at different time points of TKI treatment duration. Unfortunately, they may have been misinterpreted in current practice, because oncologists often report that their aim of treatment is the achievement of major molecular response and disease eradication. Significantly, a substantial proportion of oncologists consider a change of TKI therapy in a patient in complete cytogenetic

1	is the achievement of major molecular response and disease eradication. Significantly, a substantial proportion of oncologists consider a change of TKI therapy in a patient in complete cytogenetic response if they note “loss of major molecular response” (increase of BCR-ABL1 transcripts ([IS] from <0.1% to >0.1%). This perception may be the result of confusion regarding the NCCN and ELN guidelines, which have been updated often as a result of maturing data and have multiple treatment endpoint considerations. Although such endpoints have been suggested by these recommendations as possible criteria for failure, it is important to emphasize that no randomized study has yet shown that a change of TKI treatment in patients with complete cytogenetic response because of a loss of major molecular response, versus changing at the time of cytogenetic relapse, has been shown to improve survival. This is likely because of the high efficacy of salvage TKI therapy at the time of cytogenetic

1	response, versus changing at the time of cytogenetic relapse, has been shown to improve survival. This is likely because of the high efficacy of salvage TKI therapy at the time of cytogenetic relapse.

1	Side effects of TKIs are generally mild to moderate, although with long-term TKI therapy, they could affect the patient’s quality of life. Serious side effects occur in less than 5–10% of patients. With imatinib therapy, common mild to moderate side effects include fluid retention, weight gain, nausea, diarrhea, skin rashes, periorbital edema, bone or muscle aches, fatigue, and others (rates of 10–20%). In general, second-generation TKIs are associated with lower rates of these bothersome adverse events. However, dasatinib is associated with higher rates of myelosuppression (20–30%), particularly thrombocytopenia, and with pleural (10–25%) or pericardial effusions (≤5%). Nilotinib is associated with higher rates of hyperglycemia (10–20%), pruritus and skin rashes, and headaches. Nilotinib is also associated with rare events of pancreatitis (<5%). Bosutinib is associated with higher rates of early and self-limited gastrointestinal complications like diarrhea (50–70%). Ponatinib is

1	is also associated with rare events of pancreatitis (<5%). Bosutinib is associated with higher rates of early and self-limited gastrointestinal complications like diarrhea (50–70%). Ponatinib is associated with higher rates of skin rashes (10–15%), pancreatitis (5%), elevations of amylase/lipase (10%), and vasospastic/vasoocclusive events (10–20%). Nilotinib and dasatinib may cause prolongation of the QTc interval; therefore, they should be evaluated cautiously in patients with prolonged QTc interval on electrocardiogram (>470–480 ms), and drugs given for other medical conditions should have relatively smaller or no effects on QTc. These side effects can often be dose-dependent and are generally reversible with treatment interruptions and dose reductions. Dose reductions can be individualized. However, the lowest estimated effective doses of TKIs (from different studies and treatment practices) are imatinib 300 mg daily; nilotinib 200 mg twice daily; dasatinib 20 mg daily; bosutinib

1	However, the lowest estimated effective doses of TKIs (from different studies and treatment practices) are imatinib 300 mg daily; nilotinib 200 mg twice daily; dasatinib 20 mg daily; bosutinib 300 mg daily; and ponatinib 15 mg daily.

1	With long-term follow-up, rare but clinically relevant serious toxicities are emerging. Renal dysfunction and renal failure (creatinine elevations >2–3 mg/dL) are observed in 2–3% of patients and reverse with TKI discontinuation and empirical use of other TKIs. Pulmonary hypertension has been reported with dasatinib (<1–2%) and should be considered in a patient with shortness of breath and a normal chest x-ray (echocardiogram with emphasis on measurement of pulmonary artery pressure). This may be reversible with dasatinib discontinuation and occasionally the use of sildenafil citrate. Systemic hypertension has been observed more often with ponatinib therapy, as well as other TKIs. Hyperglycemia and diabetes have been noted more frequently with nilotinib. Finally, midand small-vessel vasoocclusive and vasospastic events have been reported at low but significant rates with nilotinib and ponatinib and should be considered possibly TKI-related and represent indications to interrupt or

1	and vasospastic events have been reported at low but significant rates with nilotinib and ponatinib and should be considered possibly TKI-related and represent indications to interrupt or reduce the dose of the TKI. These events include angina, coronary artery disease, myocardial infarction, peripheral arterial occlusive disease, transient ischemic attacks, cerebral vascular accidents, Raynaud’s phenomenon, and accelerated atherosclerosis. Although these events are uncommon (<5%), they are clinically significant for the patient’s long-term prognosis and occur at significantly higher rates than in the general population (5–20 times more often).

1	Allogeneic SCT, a curative modality in CML, is associated with longterm survival rates of 40–60% when implemented in the chronic phase. It is associated with early (1-year) mortality rates of 5–30%. Although the 5to 10-year survival rates were reported to be around 50–60% (and considered as cure rates), about 10–15% of patients die in the subsequent 1–2 decades from subtle long-term complications of the transplant (rather than from CML relapse). These are related to chronic graft-versus-host disease (GVHD), organ dysfunction, development of second cancers, and hazard ratios for mortality higher than in the normal population. Other significant morbidities include infertility, chronic immune-mediated complications, cataracts, hip necrosis, and other morbidities affecting quality of life. The cure and early mortality rates in chronic-phase CML are also associated with several factors: patient age, duration of chronic phase, whether the donor is related or unrelated, degree of matching,

1	cure and early mortality rates in chronic-phase CML are also associated with several factors: patient age, duration of chronic phase, whether the donor is related or unrelated, degree of matching, preparative regimen, and others. In accelerated-phase CML, the cure rates with allogeneic SCT are 20–40%, depending on the definition of acceleration. Patients with clonal evolution as the only criterion have cure rates of up to 40–50%. Patients undergoing allogeneic SCT in second chronic phase have cure rates of 40–50%. The cure rates with allogeneic SCT in blastic phase CML are ≤15%. Post–allogeneic SCT strategies are now implemented in the setting of molecular or cytogenetic relapse or in hematologic relapse/transformation. These include the use of TKIs for prevention or treatment of relapse, donor lymphocyte infusions, and second allogeneic SCTs, among others. TKIs appear to be highly successful at reinducing cytogenetic/molecular remissions in the setting of cytogenetic or molecular

1	donor lymphocyte infusions, and second allogeneic SCTs, among others. TKIs appear to be highly successful at reinducing cytogenetic/molecular remissions in the setting of cytogenetic or molecular relapse after allogeneic SCT.

1	Choice and Timing of Allogeneic SCT Allogeneic SCT was considered first-line CML therapy before 2000. The maturing positive experience with TKIs has now relegated its use to after first-line TKI failures. An important question is the optimal timing and sequence of TKIs and allogeneic SCT (whether allogeneic SCT should be used as secondor third-line therapy). Among patients who present with or evolve to blastic phase, combinations of chemotherapy and TKIs should be used to induce remission, followed by allogeneic SCT as soon as possible. The same applies to patients who evolve from chronic to accelerated phase. Patients with de novo accelerated-phase CML may do well with long-term TKI therapy (estimated 8-year survival rate 75%); the timing of allogeneic SCT depends on their optimal response to TKI (achievement of complete cytogenetic response). Among patients who relapse in chronic phase, the treatment sequence depends on several factors: (1) patient age and availability of

1	response to TKI (achievement of complete cytogenetic response). Among patients who relapse in chronic phase, the treatment sequence depends on several factors: (1) patient age and availability of appropriate donors; (2) risk of allogeneic SCT; (3) presence or absence of clonal evolution and mutations; (4) patient’s prior history and comorbidities; and (5) patient and physician preferences (Table 133-3). Patients with T315I mutations at relapse should be offered ponatinib and considered for allogeneic SCT (because of the short follow-up with ponatinib). Patients with mutations involving Y253H, E255K/V, and F359V/C/I respond better to dasatinib or bosutinib. Patients with mutations involving V299L, T315A, and F317L/F/I/C respond better to nilotinib. Comorbidities such as diabetes, hypertension, pulmonary hypertension, chronic lung disease, cardiac conditions, and pancreatitis may influence the choice for or against a particular TKI. Patients with clonal evolution, unfavorable mutations,

1	pulmonary hypertension, chronic lung disease, cardiac conditions, and pancreatitis may influence the choice for or against a particular TKI. Patients with clonal evolution, unfavorable mutations, or lack of major/complete cytogenetic

1	Consideration of CML Phase Use of TKI Allogeneic SCT Note: Mutations involving Y253H, E255K/V, or F359V/C/I: prefer dasatinib or bosutinib. Mutations involving V299L, T315A, or F317L/F/I/C: prefer nilotinib.

1	response within 1 year of salvage TKI therapy have short remission 693 durations and should consider allogeneic SCT as more urgent in the setting of salvage. Patients without clonal evolution or mutations at relapse and who achieve a complete cytogenetic response with TKI salvage, have long-lasting complete remissions and may delay the option of allogeneic SCT to third-line therapy. Finally, older patients (age 65–70 years or older) and those with high risk of mortality with allogeneic SCT may forgo this curative option for several years of disease control in chronic phase with or without cytogenetic response (Table 133-3). Historically, before the availability of TKIs, patients without cytogenetic response on interferon α or hydroxy-urea had expected short median survival times (2–3 years) with expected rapid disease transformation. The maturing experience with TKIs suggests a different course, whereby patients may remain in chronic phase on TKI-based therapies (combinations

1	years) with expected rapid disease transformation. The maturing experience with TKIs suggests a different course, whereby patients may remain in chronic phase on TKI-based therapies (combinations including hydroxyurea, cytarabine, decitabine, and others), with or without cytogenetic response, for many years. Table 133-3 summarizes a general guidance to the choice of TKIs versus allogeneic SCT.

1	Achievement of complete cytogenetic response by 12 months of imatinib therapy and its persistence later, the only consistent prognostic factor associated with survival, is now the main therapeutic endpoint in CML. Failure to achieve a complete cytogenetic response by 12 months or occurrence of later cytogenetic or hematologic relapse is considered as treatment failure and an indication to change therapy. Because salvage therapy with other TKIs reestab lishes good outcome, it is important to ensure patient compliance to continued TKI therapy and change therapy at the first sign of cytogenetic relapse. Patients on frontline imatinib therapy should be closely monitored until documentation of complete cytogenetic response, at which time they can be monitored every 6 months with peripheral blood FISH and PCR studies (to check for concordance of results), or more frequently if there are concerns about changes in BCR-ABL1 transcripts (e.g., every 3 months). Monitoring by molecular studies

1	FISH and PCR studies (to check for concordance of results), or more frequently if there are concerns about changes in BCR-ABL1 transcripts (e.g., every 3 months). Monitoring by molecular studies only is reasonable in patients who are in major molecular response. Cytogenetic relapse on imatinib is an indication of treatment failure and need to change TKI therapy. Mutational analysis in this instance helps in the selection of the next TKI and identifies mutations in 30–50% of patients. Mutational studies in patients in complete cytogenetic response (in whom there may be concerns of increasing BCR-ABL1 transcripts) identify mutations in ≤5% and are therefore not indicated. Earlier response has been identified as a prognostic factor for long-term outcome, including achievement of partial cytogenetic response (BCR-ABL1 transcripts ≤10%) by 3–6 months of therapy. Failure to achieve such a response on imatinib therapy has been associated with significantly worse survival in some studies

1	cytogenetic response (BCR-ABL1 transcripts ≤10%) by 3–6 months of therapy. Failure to achieve such a response on imatinib therapy has been associated with significantly worse survival in some studies (particularly when second-generation TKIs were not readily available as salvage therapy), but not in others (when they were).

1	The use of second-generation TKIs (nilotinib, dasatinib) as front-line therapy changed the monitoring approach slightly. Patients are expected to achieve complete cytogenetic response by 3–6 months of therapy. Failure to do so is associated with worse event-free survival, transformation rates, and survival. However, the 3to 5-year estimated survival among such patients is still high, around 80–90%, which is better than what would be anticipated if such patients were offered allogeneic SCT at that time. Thus, this adverse response to therapy is considered a warning signal, but it is not known whether changing therapy to other TKIs at that time would improve longer term outcome.

1	Patients in accelerated or blastic phase may receive therapy with TKIs, preferably secondor third-generation TKIs (dasatinib, nilotinib, bosutinib, ponatinib), alone or in combination with chemotherapy, to reduce the CML burden, before undergoing allogeneic SCT. Response rates with single-agent TKIs range from 30 to 50% in accelerated phase and from 20 to 30% in blastic phase. Cytogenetic 694 responses, particularly complete cytogenetic responses, are uncommon (10–30%) and transient in blastic phase. Studies of TKIs in combination with chemotherapy are ongoing; the general experience suggests that combined TKI-chemotherapy strategies increase the response rates and their durability and improve survival. In CML lymphoid blastic phase, the combination of anti-ALL chemotherapy with TKIs results in complete response rates of 60–70% and median survival times of 2–3 years (compared with historical response rates of 40–50% and median survival times of 12–18 months). This allows many patients

1	in complete response rates of 60–70% and median survival times of 2–3 years (compared with historical response rates of 40–50% and median survival times of 12–18 months). This allows many patients to undergo allogeneic SCT in a state of minimal CML burden or secondary chronic phase, which are associated with higher cure rates. In CML nonlymphoid blastic phase, anti-AML chemotherapy combined with TKIs results in CR rates of 30–50% and median survival times of 9–12 months (compared with historical response rates of 20–30% and median survival times of 3–5 months). In accelerated phase, response to single TKIs is significant in conditions where “softer” accelerated phase criteria are considered (e.g., clonal evolution alone, thrombocytosis alone, significant splenomegaly or resistance to hydroxyurea, but without evidence of high blast and basophil percentages). In accelerated phase, combinations usually include TKIs with low-intensity chemotherapy such as low-dose cytarabine, low-dose

1	but without evidence of high blast and basophil percentages). In accelerated phase, combinations usually include TKIs with low-intensity chemotherapy such as low-dose cytarabine, low-dose idarubicin, decitabine, interferon α, hydroxyurea, or others.

1	OTHER TREATMENTS AND SPECIAL THERAPEUTIC CONSIDERATIONS Interferon α Interferon α was a standard of care before 2000. Today, it is considered in combination with TKIs (an investigational approach), sometimes after CML failure on TKIs, occasionally in patients during pregnancy, or as part of investigational strategies with TKIs to eradicate residual molecular disease.

1	Chemotherapeutic Agents Hydroxyurea and busulfan were commonly used chemotherapeutic agents in the past. Hydroxyurea remains a safe and effective agent (at daily doses of 0.5–10 g) to reduce initial CML burden, as a temporary measure in between definitive therapies, or in combination with TKIs to sustain complete hematologic or cytogenetic responses. Busulfan is often used in allogeneic SCT preparative regimens. Because of its side effects (delayed myelosuppression, Addison-like disease, pulmonary and cardiac fibrosis, myelofibrosis), it is now only rarely used in the chronic management of CML. Low-dose cytarabine, decitabine, anthracyclines, 6-mercaptopurine, 6-thioguanine, thiotepa, anagrelide, and other agents are useful in different CML settings to control the disease burden.

1	Others Splenectomy is occasionally considered to alleviate symp toms of massive splenomegaly and/or hypersplenism. Splenic irradi ation is rarely used, if at all, because of the postirradiation adhesions and complications. Leukapheresis is rarely used in patients presenting with extreme leukocytosis and leukostatic complications. Single doses of high-dose cytarabine or high doses of hydroxyurea, with tumor lysis management, may be as effective and less cumbersome. and may be indicative of the genetic instability of the hematopoietic stem cells that predispose the patient to develop CML in the first place. Rarely, abnormalities involving chromosomes 5 or 7 may be truly clonal and evolve into myelodysplastic syndrome or acute myeloid leukemia. This is thought to be part of the natural course of patients in whom CML was suppressed and who live long enough to develop other hematologic malignancies.

1	Routine physical exams and blood tests in the United States and advanced countries result in early detection of CML in most patients. About 50–70% of patients with CML are diagnosed accidentally, and high-risk CML as defined by prognostic models (e.g., Sokal risk groups) is found in only 10–20% of patients. This is not the same situation in emerging nations (e.g., India, China, African countries, the Middle East), where most patients are diagnosed following evaluation for symptoms and many present with high tumor burdens, such as massive splenomegaly, and advanced phases of CML (high-risk CML documented in 30–50%). Therefore, the prognosis of such patients on TKI therapy may be worse than the published experience.

1	The high cost of TKI therapies (annual costs of $90,000–$140,000 in the United States; lower but variable in the rest of the world) makes the general affordability of such treatments difficult. Although TKI treatment penetration is high in nations where cost of therapy is not an issue (e.g., Sweden, European Union), it may be less so in other nations, even in advanced ones like the United States, where out-ofpocket expenses may be prohibitive to a subset of patients (perhaps 10–20%). Based on the sales of imatinib worldwide and charity free drug supplies, it is estimated that less than 30% of patients are treated with imatinib (or other TKIs) consistently. Although the estimated 10-year survival in CML is 85% in single-institution studies (e.g.,

1	M.D. Anderson Cancer Center), in national studies in countries with TKI affordability (Sweden) (Figs. 133-2 and 133-3) or in company-sponsored studies (where all patients have access to TKIs throughout their care), the estimated 10-year survival worldwide, even 12 years after the introduction of TKI therapies, is likely to be less than 50%. The Surveillance, Epidemiology, and End Results (SEER) data from 1.0 0.8 0.6 0.4

1	Special Considerations Women with CML who become pregnant should discontinue TKI therapy immediately. Among 125 babies delivered to women with CML who discontinued TKI therapy as soon as the pregnancy was known, three babies were born with ocular, skeletal, and renal malformations, suggesting the uncommon teratogenicity of imatinib. There are no or little data with other TKIs. Control of CML during pregnancy can be managed with leukapheresis for severe symptomatic leukocytosis in the first trimester and with hydroxyurea subsequently until delivery. There are case reports of successful pregnancies and deliveries of normal babies with interferon α therapy and registry studies in essential thrombocytosis of its safety, but interferon α can be antiangiogenic and may increase the risk of spontaneous abortions.

1	Patients on TKI therapy may develop chromosomal abnormalities in the Ph-negative cells. These may involve loss of chromosome Y, trisomy 8, 20q–, chromosome 5 or 7 abnormalities, and others. Most chromosomal abnormalities disappear spontaneously on follow-up 0.2 0.0 No. at risk FIGURE 133-3 Survival in chronic (CP), accelerated (AP), and blastic crisis (BC) phases of chronic myeloid leukemia (CML) in the population-based Swedish national registry study. The acceleratedand blastic-phase cases are de novo presentations. The favorable outcome with de novo blastic phase may be due to use of 20% blasts or more to define blastic phase. (With permission from Dr. Martin Hoglund, Swedish CML Registry, 2013.) the United States report an estimated 5-year survival rate of 60% in

1	Malignancies of Lymphoid Cells Dan L. Longo Malignancies of lymphoid cells range from the most indolent to the most aggressive human malignancies. These cancers arise from cells of the immune system at different stages of differentiation, resulting in a wide range of morphologic, immunologic, and clinical findings. 134 the era of TKIs.

1	The current high cost of TKI therapies poses two additional considerations. The first are the treatment pathways and guidelines in nations where TKIs may not be affordable by patients or the health care system. In these conditions, there are trends of pathways advocating frontline allogeneic SCT (a one-time cost of $30,000–$50,000) despite the associated mortality and morbidities. The second is the choice of frontline TKI therapy once imatinib becomes available in generic forms (hopefully at much lower annual prices, e.g., $2,000–$10,000). This will depend on the maturing data in randomized studies of second-generation TKIs versus imatinib in relation to important longterm outcome endpoints, particularly survival, but also event-free survival and transformation-free survival. Insights on the normal immune system have allowed a better understanding of these sometimes confusing disorders.

1	Insights on the normal immune system have allowed a better understanding of these sometimes confusing disorders. Some malignancies of lymphoid cells almost always present as leukemia (i.e., primary involvement of bone marrow and blood), while others almost always present as lymphomas (i.e., solid tumors of the immune system). However, other malignancies of lymphoid cells can present as either leukemia or lymphoma. In addition, the clinical pattern can change over the course of the illness. This change is more often seen in a patient who seems to have a lymphoma and then develops the manifestations of leukemia over the course of the illness. BIOLOGY OF LYMPHOID MALIGNANCIES: CONCEPTS OF THE WORLD HEALTH ORGANIZATION CLASSIFICATION OF LYMPHOID MALIGNANCIES

1	The classification of lymphoid cancers evolved steadily throughout the twentieth century. The distinction between leukemia and lymphoma was made early, and separate classification systems were developed for each. Leukemias were first divided into acute and chronic subtypes based on average survival. Chronic leukemias were easily subdivided into those of lymphoid or myeloid origin based on morphologic characteristics. However, a spectrum of diseases that were formerly all called chronic lymphoid leukemia has become apparent (Table 134-1). The acute leukemias were usually malignancies of blast cells with few identifying characteristics. When cytochemical stains became available, it was possible to divide these objectively into myeloid malignancies and acute leukemias of lymphoid cells. Acute leukemias of lymphoid cells have been subdivided based on morphologic characteristics by the French-American-British (FAB) group (Table 134-2). Using this system, lymphoid malignancies of small

1	Immunologic % of FAB Subtype Cases Subtype Cytogenetic Abnormalities Abbreviation: FAB, French-American-British classification. uniform blasts (e.g., typical childhood acute lymphoblastic leukemia) were called L1, lymphoid malignancies with larger and more variable size cells were called L2, and lymphoid malignancies of uniform cells with basophilic and sometimes vacuolated cytoplasm were called L3 (e.g., typical Burkitt’s lymphoma cells). Acute leukemias of lymphoid cells have also been subdivided based on immunologic (i.e., T cell vs B cell) and cytogenetic abnormalities (Table 134-2). Major cytogenetic subgroups include the t(9;22) (e.g., Philadelphia chromosome– positive acute lymphoblastic leukemia) and the t(8;14) found in the L3 or Burkitt’s leukemia.

1	Non-Hodgkin’s lymphomas were separated from Hodgkin’s lymphoma by recognition of the Sternberg-Reed cells early in the twentieth century. The histologic classification for non-Hodgkin’s lymphomas has been one of the most contentious issues in oncology. Imperfect morphologic systems were supplanted by imperfect immunologic systems, and poor reproducibility of diagnosis has hampered progress. In 1999, the World Health Organization (WHO) classification of lymphoid malignancies was devised through a process of consensus development among international leaders in hematopathology and clinical oncology. The WHO classification takes into account morphologic, clinical, immunologic, and genetic information and attempts to divide nonHodgkin’s lymphomas and other lymphoid malignancies into clinical/ pathologic entities that have clinical and therapeutic relevance. This system is presented in Table 134-3. This system is clinically relevant and has a higher degree of diagnostic accuracy than those

1	pathologic entities that have clinical and therapeutic relevance. This system is presented in Table 134-3. This system is clinically relevant and has a higher degree of diagnostic accuracy than those used previously. The possibilities for subdividing lymphoid malignancies are extensive. However, Table 134-3 presents in bold those malignancies that occur in at least 1% of patients. Specific lymphoma subtypes will be dealt with in more detail below.

1	Lymphomas occurring in fewer than 1% of patients with lymphoproliferative diseases are discussed in Chap. 135e, and lymphomas associated with HIV infection are discussed in Chap. 226. The relative frequency of the various lymphoid malignancies is shown in Fig. 134-1. Chronic lymphoid leukemia (CLL) is the most prevalent form of leukemia in Western countries. It occurs most frequently in older adults and is exceedingly rare in children. In 2014, 15,720 new cases were diagnosed in the United States, but because of the prolonged survival associated with this disorder, the total prevalence is many times higher. CLL is more common in men than in women and more common in whites than in blacks. This is an uncommon malignancy in Asia. The etiologic factors for typical CLL are unknown.

1	In contrast to CLL, acute lymphoid leukemias (ALLs) are predominantly cancers of children and young adults. The L3 or Burkitt’s leukemia occurring in children in developing countries seems to be associated with infection by the Epstein-Barr virus (EBV) in infancy. However, the explanation for the etiology of more common subtypes of ALL is much less certain. Childhood ALL occurs more often in higher socioeconomic subgroups. Children with trisomy 21 (Down’s syndrome) have an increased risk for childhood ALL as well as acute myeloid leukemia (AML). Exposure to high-energy radiation in early childhood increases the risk of developing T-cell ALL. The etiology of ALL in adults is also uncertain. ALL is unusual in middle-aged adults but increases in incidence in the elderly. However, AML is still much more common in older patients. Environmental exposures, including certain industrial exposures, exposure to agricultural chemicals, and smoking, might increase the risk of developing

1	Malignancies of Lymphoid Cells Abbreviations: HHV, human herpesvirus; HTLV, human T-cell lymphotropic virus; MALT, mucosa-associated lymphoid tissue; NK, natural killer; WHO, World Health Organization. Source: Adapted from SH Swerdlow et al: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th ed. World Health Organization, 2008. Plasma cell disorders 16% Non-Hodgkin’s lymphoma 62.4% CLL 9% ALL 3.8% Hodgkin’s disease 8.2% FIGURE 134-1 Relative frequency of lymphoid malignancies. ALL, acute lymphoid leukemia; CLL, chronic lymphoid leukemia; MALT, mucosa-associated lymphoid tissue. 7.6% MALT lymphoma 7.6% Mature T-cell lymphoma 6.7% Small lymphocytic lymphoma 2.4% Mediastinal large B-cell lymphoma 2.4% Anaplastic large cell lymphoma 2.4% Burkitt’s lymphoma 1.8% Nodal marginal zone lymphoma 1.7% Precursor T lymphoblastic lymphoma 1.2% Lymphoplasmacytic lymphoma 7.4% Others

1	ALL as an adult. ALL was diagnosed in 6020 persons and AML in 18,860 persons in the United States in 2014.

1	The preponderance of evidence suggests that Hodgkin’s lymphoma is of B-cell origin. The incidence of Hodgkin’s lymphoma appears fairly stable, with 9190 new cases diagnosed in 2014 in the United States. Hodgkin’s lymphoma is more common in whites than in blacks and more common in males than in females. A bimodal distribution of age at diagnosis has been observed, with one peak incidence occurring in patients in their twenties and the other in those in their eighties. Some of the late age peak may be attributed to confusion among entities with similar appearance such as anaplastic large cell lymphoma and T-cell–rich B-cell lymphoma. Patients in the younger age groups diagnosed in the United States largely have the nodular sclerosing subtype of Hodgkin’s lymphoma. Elderly patients, patients infected with HIV, and patients in Third World countries more commonly have mixed-cellularity Hodgkin’s lymphoma or lymphocyte-depleted Hodgkin’s lymphoma. Infection by HIV is a risk factor for

1	infected with HIV, and patients in Third World countries more commonly have mixed-cellularity Hodgkin’s lymphoma or lymphocyte-depleted Hodgkin’s lymphoma. Infection by HIV is a risk factor for developing Hodgkin’s lymphoma. In addition, an association between infection by EBV and Hodgkin’s lymphoma has been suggested. A monoclonal or oligoclonal proliferation of EBV-infected cells in 20–40% of the patients with Hodgkin’s lymphoma has led to proposals for this virus having an etiologic role in Hodgkin’s lymphoma. However, the matter is not settled definitively.

1	For unknown reasons, non-Hodgkin’s lymphomas increased in frequency in the United States at the rate of 4% per year and increased 2–8% per year globally between 1950 and the late 1990s. The rate of increase in the past few years seems to be decreasing. About 70,800 new cases of non-Hodgkin’s lymphoma were diagnosed in the United States in 2014 and nearly 360,000 cases worldwide. Non-Hodgkin’s lymphomas are more frequent in the elderly and more frequent in men. Patients with both primary and secondary immunodeficiency states are predisposed to developing non-Hodgkin’s lymphomas. These include patients with HIV infection; patients who have undergone organ transplantation; and patients with inherited immune deficiencies, the sicca syndrome, and rheumatoid arthritis.

1	The incidence of non-Hodgkin’s lymphomas and the patterns of expression of the various subtypes differ geographically. T-cell lymphomas are more common in Asia than in Western countries, while certain subtypes of B-cell lymphomas such as follicular lymphoma are more common in Western countries. A specific subtype of nonHodgkin’s lymphoma known as the angiocentric nasal T/natural killer (NK) cell lymphoma has a striking geographic occurrence, being most frequent in Southern Asia and parts of Latin America. Another subtype of non-Hodgkin’s lymphoma associated with infection by human T-cell lymphotropic virus (HTLV) 1 is seen particularly in southern Japan and the Caribbean (Chap. 225e).

1	A number of environmental factors have been implicated in the occurrence of non-Hodgkin’s lymphoma, including infectious agents, chemical exposures, and medical treatments. Several studies have demonstrated an association between exposure to agricultural chemicals and an increased incidence of non-Hodgkin’s lymphoma. Patients treated for Hodgkin’s lymphoma can develop non-Hodgkin’s lymphoma; it is unclear whether this is a consequence of the Hodgkin’s lymphoma or its treatment. However, a number of non-Hodgkin’s lymphomas are associated with infectious agents (Table 134-4). HTLV-1 infects T cells and leads directly to the development of adult T-cell lymphoma in a small percentage of infected patients. The cumulative lifetime risk of developing lymphoma in an infected patient is 2.5%. The virus is transmitted by infected lymphocytes ingested by nursing babies of infected mothers, bloodborne transmission, or sexually. The median age of patients with adult T-cell lymphoma is ~56 years,

1	virus is transmitted by infected lymphocytes ingested by nursing babies of infected mothers, bloodborne transmission, or sexually. The median age of patients with adult T-cell lymphoma is ~56 years, emphasizing the long latency. HTLV-1 is also the cause of tropical spastic paraparesis—a neurologic disorder that occurs somewhat more frequently than lymphoma and with shorter latency and usually from transfusion-transmitted virus (Chap. 225e).

1	EBV is associated with the development of Burkitt’s lymphoma in Central Africa and the occurrence of aggressive non-Hodgkin’s lymphomas in immunosuppressed patients in Western countries. The majority of primary central nervous system (CNS) lymphomas Abbreviations: CNS, central nervous system; HIV, human immunodeficiency virus; HTLV, human T-cell lymphotropic virus; MALT, mucosa-associated lymphoid tissue; NK, natural killer.

1	are associated with EBV. EBV infection is strongly associated with the occurrence of extranodal nasal T/NK cell lymphomas in Asia and South America. Infection with HIV predisposes to the development of aggressive, B-cell non-Hodgkin’s lymphoma. This may be through overexpression of interleukin 6 by infected macrophages. Infection of the stomach by the bacterium Helicobacter pylori induces the development of gastric MALT (mucosa-associated lymphoid tissue) lymphomas. This association is supported by evidence that patients treated with antibiotics to eradicate H. pylori have regression of their MALT lymphoma. The bacterium does not transform lymphocytes to produce the lymphoma; instead, a vigorous immune response is made to the bacterium, and the chronic antigenic stimulation leads to the neoplasia. MALT lymphomas of the skin may be related to Borrelia sp. infections, those of the eyes to Chlamydophila psittaci, and those of the small intestine to Campylobacter jejuni.

1	Chronic hepatitis C virus infection has been associated with the development of lymphoplasmacytic lymphoma. Human herpesvirus 8 is associated with primary effusion lymphoma in HIV-infected persons and multicentric Castleman’s disease, a diffuse lymphadenopathy associated with systemic symptoms of fever, malaise, and weight loss. In addition to infectious agents, a number of other diseases or exposures may predispose to developing lymphoma (Table 134-5).

1	In addition to infectious agents, a number of other diseases or exposures may predispose to developing lymphoma (Table 134-5). All lymphoid cells are derived from a common hematopoietic progenitor that gives rise to lymphoid, myeloid, erythroid, monocyte, and megakaryocyte lineages. Through the ordered and sequential activation of a series of transcription factors, the cell first becomes committed to the lymphoid lineage and then gives rise to B and T cells. About 75% of all lymphoid leukemias and 90% of all lymphomas are of B-cell origin. A cell becomes committed to B-cell development when it begins to rearrange its immunoglobulin genes. The sequence of cellular changes, Common variable immunodeficiency disease Phenytoin Acquired immunodeficiency diseases Dioxin, phenoxy herbicides Iatrogenic immunosuppression Radiation HIV-1 infection Prior chemotherapy and radiation therapy Malignancies of Lymphoid Cells

1	Malignancies of Lymphoid Cells FIGURE 134-2 Pathway of normal B-cell differentiation and relationship to B-cell lymphomas. HLA-DR, CD10, CD19, CD20, CD21, CD22, CD5, and CD38 are cell markers used to distinguish stages of development. Terminal transferase (TdT) is a cellular enzyme. Immunoglobulin heavy chain gene rearrangement (HCR) and light chain gene rearrangement or deletion (κR or D, λR or D) occur early in B-cell development. The approximate normal stage of differentiation associated with particular lymphomas is shown. ALL, acute lymphoid leukemia; CLL, chronic lymphoid leukemia; SL, small lymphocytic lymphoma. including changes in cell-surface phenotype, that characterizes normal B-cell development is shown in Fig. 134-2. A cell becomes committed to T-cell differentiation upon migration to the thymus and rearrangement of T-cell antigen receptor genes. The sequence of the events that characterize T-cell development is depicted in Fig. 134-3.

1	Although lymphoid malignancies often retain the cell-surface phenotype of lymphoid cells at particular stages of differentiation, this information is of little consequence. The so-called stage of differentiation of a malignant lymphoma does not predict its natural history. For example, the clinically most aggressive lymphoid leukemia is Burkitt’s leukemia, which has the phenotype of a mature follicle center IgM-bearing B cell. Leukemias bearing the immunologic cell-surface phenotype of more primitive cells (e.g., pre-B ALL, CD10+) are less aggressive and more amenable to curative therapy than the “more mature” appearing Burkitt’s leukemia cells. Furthermore, the apparent stage of differentiation of the malignant cell does not reflect the stage at which the genetic lesions that gave rise to the malignancy developed. For example, follicular lymphoma has the cell-surface phenotype of a follicle center cell, but its characteristic chromosomal translocation, the t(14;18), which involves

1	to the malignancy developed. For example, follicular lymphoma has the cell-surface phenotype of a follicle center cell, but its characteristic chromosomal translocation, the t(14;18), which involves juxtaposition of the antiapoptotic bcl-2 gene next to the immunoglobulin heavy chain gene (see below), had to develop early in ontogeny as an error in the process of immunoglobulin gene rearrangement. Why the subsequent steps that led to transformation became manifest in a cell of follicle center differentiation is not clear.

1	The major value of cell-surface phenotyping is to aid in the differential diagnosis of lymphoid tumors that appear similar by light microscopy. For example, benign follicular hyperplasia may resemble follicular lymphoma; however, the demonstration that all the cells bear the same immunoglobulin light chain isotype strongly suggests the mass is a clonal proliferation rather than a polyclonal response to an exogenous stimulus.

1	Malignancies of lymphoid cells are associated with recurring genetic abnormalities. While specific genetic abnormalities have not been identified for all subtypes of lymphoid malignancies, it is presumed that they exist. Genetic abnormalities can be identified at a variety of levels including gross chromosomal changes (i.e., translocations, additions, or deletions); rearrangement of specific genes that may or may not be apparent from cytogenetic studies; and overexpression, under-expression, or mutation of specific oncogenes. Altered expression or mutation of specific proteins is particularly important. Many lymphomas contain balanced chromosomal translocations involving the antigen receptor genes; immunoglobulin genes on chromosomes 2, 14, and 22 in B cells; and T-cell antigen receptor genes on chromosomes 7 and 14 in T cells. The rearrangement of chromosome segments to generate mature antigen receptors must create a site of vulnerability to aberrant recombination. B cells are even

1	genes on chromosomes 7 and 14 in T cells. The rearrangement of chromosome segments to generate mature antigen receptors must create a site of vulnerability to aberrant recombination. B cells are even more susceptible to acquiring mutations during their maturation in germinal centers; the generation of antibody of higher affinity requires the introduction of mutations into the variable region genes in the germinal centers. Other nonimmunoglobulin genes, e.g., bcl-6, may acquire mutations as well.

1	In the case of diffuse large B-cell lymphoma, the translocation t(14;18) occurs in ~30% of patients and leads to overexpression of the Majority of Minority of T-ALL Majority of T-LL Minority of T-LL Majority of Mature T Helper T-CLL, CTCL, Cell Sezary Cell, NHL CD: 2,3,4,5,6,7; TCR Minority of Suppressor Cell T-CLL, NHL CD: 2,3,4,5,6,7; TCR FIGURE 134-3 Pathway of normal T-cell differentiation and relationship to T-cell lymphomas. CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD38, and CD71 are cell markers used to distinguish stages of development. T-cell antigen receptors (TCR) rearrange in the thymus, and mature T cells emigrate to nodes and peripheral blood. ALL, acute lymphoid leukemia; T-ALL, T-cell ALL; T-LL, T-cell lymphoblastic lymphoma; T-CLL, T-cell chronic lymphoid leukemia; CTCL, cutaneous T-cell lymphoma; NHL, non-Hodgkin’s lymphoma.

1	bcl-2 gene found on chromosome 18. Some other patients without the translocation also overexpress the BCL-2 protein. This protein is involved in suppressing apoptosis—i.e., the mechanism of cell death most often induced by cytotoxic chemotherapeutic agents. A higher relapse rate has been observed in patients whose tumors overexpress the BCL-2 protein, but not in those patients whose lymphoma cells show only the translocation. Thus, particular genetic mechanisms have clinical ramifications.

1	Table 134-6 presents the most common translocations and associated oncogenes for various subtypes of lymphoid malignancies. In some cases, such as the association of the t(14;18) in follicular lymphoma, the t(2;5) in anaplastic large T/null cell lymphoma, the t(8;14) in Burkitt’s lymphoma, and the t(11;14) in mantle cell lymphoma, the great majority of tumors in patients with these diagnoses display these abnormalities. In other types of lymphoma where a minority of the patients have tumors expressing specific genetic abnormalities, the defects may have prognostic significance. No specific genetic abnormalities have been identified in Hodgkin’s lymphoma other than aneuploidy.

1	In typical B-cell CLL, trisomy 12 conveys a poorer prognosis. In ALL in both adults and children, genetic abnormalities have important prognostic significance. Patients whose tumor cells display the t(9;22) and translocations involving the MLL gene on chromosome 11q23 have a much poorer outlook than patients who do not have these translocations. Other genetic abnormalities that occur frequently in adults with ALL include the t(4;11) and the t(8;14). The t(4;11) is associated with younger age, female predominance, high white cell counts, and L1 morphology. The t(8;14) is associated with older age, male predominance, frequent CNS involvement, and L3 morphology. Both are associated with a poor prognosis. In childhood ALL, hyperdiploidy has been shown to have a favorable prognosis. Gene profiling using array technology allows the simultaneous assessment of the expression of thousands of genes. This technology aNumerous sites of translocation may be involved with these genes.

1	Gene profiling using array technology allows the simultaneous assessment of the expression of thousands of genes. This technology aNumerous sites of translocation may be involved with these genes. Abbreviations: CLL, chronic lymphoid leukemia; IgH, immunoglobulin heavy chain; MALT, mucosa-associated lymphoid tissue.

1	provides the possibility to identify new genes with pathologic importance in lymphomas, the identification of patterns of gene expression with diagnostic and/or prognostic significance, and the identification of new therapeutic targets. Recognition of patterns of gene expression is complicated and requires sophisticated mathematical techniques. Early successes using this technology in lymphoma include the identification of previously unrecognized subtypes of diffuse large B-cell lymphoma whose gene expression patterns resemble either those of follicular center B cells or activated peripheral blood B cells. Patients whose lymphomas have a germinal center B-cell pattern of gene expression have a considerably better prognosis than those whose lymphomas have a pattern resembling activated peripheral blood B cells. This improved prognosis is independent of other known prognostic factors. Similar information is being generated in follicular lymphoma and mantle cell lymphoma. The challenge

1	blood B cells. This improved prognosis is independent of other known prognostic factors. Similar information is being generated in follicular lymphoma and mantle cell lymphoma. The challenge remains to provide information from such techniques in a clinically useful time frame.

1	APPROACH TO THE PATIENT: Regardless of the type of lymphoid malignancy, the initial evaluation of the patient should include performance of a careful history and physical examination. These will help confirm the diagnosis, identify those manifestations of the disease that might require prompt attention, and aid in the selection of further studies to optimally characterize the patient’s status to allow the best choice of therapy. It is difficult to overemphasize the importance of a carefully done history and physical examination. They might provide observations that lead to reconsidering the diagnosis, provide hints at etiology, clarify the stage, and allow the physician to establish rapport with the patient that will make it possible to develop and carry out a therapeutic plan.

1	For patients with ALL, evaluation is usually completed after a complete blood count, chemistry studies reflecting major organ function, a bone marrow biopsy with genetic and immunologic studies, and a lumbar puncture. The latter is necessary to rule out occult CNS involvement. At this point, most patients would Malignancies of Lymphoid Cells be ready to begin therapy. In ALL, prognosis is dependent on the genetic characteristics of the tumor, the patient’s age, the white cell count, and the patient’s overall clinical status and major organ function.

1	In CLL, the patient evaluation should include a complete blood count, chemistry tests to measure major organ function, serum protein electrophoresis, and a bone marrow biopsy. However, some physicians believe that the diagnosis would not always require a bone marrow biopsy. Patients often have imaging studies of the chest and abdomen looking for pathologic lymphadenopathy. Patients with typical B-cell CLL can be subdivided into three major prognostic groups. Those patients with only blood and bone marrow involvement by leukemia but no lymphadenopathy, organomegaly, or signs of bone marrow failure have the best prognosis. Those with lymphadenopathy and organomegaly have an intermediate prognosis, and patients with bone marrow failure, defined as hemoglobin <100 g/L (10 g/dL) or platelet count <100,000/μL, have the worst prognosis. The pathogenesis of the anemia or thrombocytopenia is important to discern. The prognosis is adversely affected when either or both of these abnormalities

1	count <100,000/μL, have the worst prognosis. The pathogenesis of the anemia or thrombocytopenia is important to discern. The prognosis is adversely affected when either or both of these abnormalities are due to progressive marrow infiltration and loss of productive marrow. However, either or both may be due to autoimmune phenomena or to hypersplenism that can develop during the course of the disease. These destructive mechanisms are usually completely reversible (glucocorticoids for autoimmune disease; splenectomy for hypersplenism) and do not influence disease prognosis.

1	Two popular staging systems have been developed to reflect these prognostic groupings (Table 134-7). Patients with typical B-cell CLL can have their course complicated by immunologic abnormalities, including autoimmune hemolytic anemia, autoimmune thrombocytopenia, and hypogammaglobulinemia. Patients with hypogammaglobulinemia benefit from regular (monthly) γ globulin administration. Because of expense, γ globulin is often withheld until the patient experiences a significant infection. These abnormalities do not have a clear prognostic significance and should not be used to assign a higher stage.

1	Two other features may be used to assess prognosis in B-cell CLL, but neither has yet been incorporated into a staging classification. At least two subsets of CLL have been identified based on the cytoplasmic expression of ZAP-70; expression of this protein, which is usually expressed in T cells, identifies a subgroup with poorer prognosis. A less powerful subsetting tool is CD38 expression. CD38+ tumors tend to have a poorer prognosis than CD38– tumors. A less easily measured feature, the presence of immunoglobulin variable region gene mutations, is also able to separate prognostic groups; patients with mutated immunoglobulin variable region genes respond better to treatment and have better survival than those with unmutated immunoglobulin variable region genes.

1	The initial evaluation of a patient with Hodgkin’s lymphoma or non-Hodgkin’s lymphoma is similar. In both situations, the determination of an accurate anatomic stage is an important part of the evaluation. Staging is done using the Ann Arbor staging system originally developed for Hodgkin’s lymphoma (Table 134-8).

1	Evaluation of patients with Hodgkin’s lymphoma will typically include a complete blood count; erythrocyte sedimentation rate; chemistry studies reflecting major organ function; computed tomography (CT) scans of the chest, abdomen, and pelvis; and a bone marrow biopsy. Neither a positron emission tomography (PET) scan nor a gallium scan is absolutely necessary for primary staging, but one performed at the completion of therapy allows evaluation of persisting radiographic abnormalities, particularly the mediastinum. Knowing that the PET scan or gallium scan is abnormal before treatment can help in this assessment. In most cases, these studies will allow assignment of anatomic stage and the development of a therapeutic plan.

1	In patients with non-Hodgkin’s lymphoma, the same evaluation described for patients with Hodgkin’s lymphoma is usually carried out. In addition, serum levels of lactate dehydrogenase (LDH) and β2-microglobulin and serum protein electrophoresis are often included in the evaluation. Anatomic stage is assigned in the same manner as used for Hodgkin’s lymphoma. However, the prognosis of patients with non-Hodgkin’s lymphoma is best assigned using the International Prognostic Index (IPI) (Table 134-9). This is a powerful predictor of outcome in all subtypes of non-Hodgkin’s lymphoma. Patients are assigned an IPI score based on the presence or absence of five adverse prognostic factors and may have none or all five of these adverse prognostic factors. Figure 134-4 shows the prognostic significance of this score in 1300 patients with all types of non-Hodgkin’s lymphoma. With the addition of rituximab to CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), treatment outcomes have

1	of this score in 1300 patients with all types of non-Hodgkin’s lymphoma. With the addition of rituximab to CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), treatment outcomes have improved and the original IPI has lost some of its discrimination power. A revised IPI has been proposed that better predicts outcome of rituximab plus chemotherapy-based programs (Table 134-9). CT scans are routinely used in the evaluation of patients with all subtypes of non-Hodgkin’s lymphoma, but PET and

1	Five clinical risk factors: Age ≥60 years Serum lactate dehydrogenase levels elevated Performance status ≥2 (ECOG) or ≤70 (Karnofsky) Ann Arbor stage III or IV >1 site of extranodal involvement Patients are assigned a number for each risk factor they have Patients are grouped differently based on the type of lymphoma For diffuse large B-cell lymphoma: 0, 1 factor = low risk: 35% of cases; 5-year survival, 73% 2 factors = low-intermediate risk: 27% of cases; 5-year survival, 51% 3 factors = high-intermediate risk: 22% of cases; 5-year survival, 43% 4, 5 factors = high risk: 16% of cases; 5-year survival, 26% For diffuse large B-cell lymphoma treated with R-CHOP: 0 factor = very good: 10% of cases; 5-year survival, 94% 1, 2 factors = good: 45% of cases; 5-year survival, 79% 3, 4, 5 factors = poor: 45% of cases; 5-year survival, 55% Abbreviations: ECOG, Eastern Cooperative Oncology Group; R-CHOP, rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone.

1	Abbreviations: ECOG, Eastern Cooperative Oncology Group; R-CHOP, rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone. gallium scans are much more useful in aggressive subtypes such as diffuse large B-cell lymphoma than in more indolent subtypes such as follicular lymphoma or small lymphocytic lymphoma. Although the IPI does divide patients with follicular lymphoma into subsets with distinct prognoses, the distribution of such patients is skewed toward lower-risk categories. A follicular lymphoma–specific IPI (FLIPI) has been proposed that replaces performance status with hemoglobin level (<120 g/L [<12 g/dL]) and number of extranodal sites with number of nodal sites (more than four). Low risk (zero or one factor) was assigned to 36% of patients, intermediate risk (two factors) to 37%, and poor risk (more than two factors) to 27% of patients. CLINICAL FEATURES, TREATMENT, AND PROGNOSIS OF SPECIFIC LYMPHOID MALIGNANCIES

1	CLINICAL FEATURES, TREATMENT, AND PROGNOSIS OF SPECIFIC LYMPHOID MALIGNANCIES PRECURSOR CELL B-CELL NEOPLASMS Precursor B-Cell Lymphoblastic Leukemia/Lymphoma The most common cancer in childhood is B-cell ALL. Although this disorder can also present as a lymphoma in either adults or children, presentation as lymphoma is rare. The malignant cells in patients with precursor B-cell lymphoblastic leukemia are most commonly of pre–B cell origin. Patients typically 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Log rank test: p < .001 IPI: 4/5 (n = 171) 0.0 0 12345678910 FIGURE 134-4 Relationship of International Prognostic Index (IPI) to survival. Kaplan-Meier survival curves for 1300 patients with various kinds of lymphoma stratified according to the IPI. FIGURE 134-5 Acute lymphoblastic leukemia. The cells are hetero-geneous in size and have round or convoluted nuclei, high nuclear/ cytoplasmic ratio, and absence of cytoplasmic granules.

1	FIGURE 134-5 Acute lymphoblastic leukemia. The cells are hetero-geneous in size and have round or convoluted nuclei, high nuclear/ cytoplasmic ratio, and absence of cytoplasmic granules. present with signs of bone marrow failure such as pallor, fatigue, bleeding, fever, and infection related to peripheral blood cytopenias. Peripheral blood counts regularly show anemia and thrombocytopenia but might show leukopenia, a normal leukocyte count, or leukocytosis based largely on the number of circulating malignant cells (Fig. 134-5). Extramedullary sites of disease are frequently involved in patients who present with leukemia, including lymphadenopathy, hepatoor splenomegaly, CNS disease, testicular enlargement, and/or cutaneous infiltration.

1	The diagnosis is usually made by bone marrow biopsy, which shows infiltration by malignant lymphoblasts. Demonstration of a pre–B cell immunophenotype (Fig. 134-2) and, often, characteristic cytogenetic abnormalities (Table 134-6) confirm the diagnosis. An adverse prognosis in patients with precursor B-cell ALL is predicted by a very high white cell count, the presence of symptomatic CNS disease, and unfavorable cytogenetic abnormalities. For example, t(9;22), frequently found in adults with B-cell ALL, has been associated with a very poor outlook. The bcr/abl kinase inhibitors have improved the prognosis.

1	The treatment of patients with precursor B-cell ALL involves remission induction with combination chemotherapy, a consolidation phase that includes administration of high-dose systemic therapy and treatment to eliminate disease in the CNS, and a period of continuing therapy to prevent relapse and effect cure. The overall cure rate in children is 90%, whereas ~50% of adults are long-term disease-free survivors. This reflects the high proportion of adverse cytogenetic abnormalities seen in adults with precursor B-cell ALL. Precursor B-cell lymphoblastic lymphoma is a rare presentation of precursor B-cell lymphoblastic malignancy. These patients often have a rapid transformation to leukemia and should be treated as though they had presented with leukemia. The few patients who present with the disease confined to lymph nodes have a high cure rate.

1	MATURE (PERIPHERAL) B-CELL NEOPLASMS B-Cell Chronic Lymphoid Leukemia/Small Lymphocytic Lymphoma B-cell CLL/small lymphocytic lymphoma represents the most common lymphoid leukemia, and when presenting as a lymphoma, it accounts for ~7% of non-Hodgkin’s lymphomas. Presentation can be as either leukemia or lymphoma. The major clinical characteristics of B-cell CLL/ small lymphocytic lymphoma are presented in Table 134-10. The diagnosis of typical B-cell CLL is made when an increased number of circulating lymphocytes (i.e., >4 × 109/L and usually Malignancies of Lymphoid Cells Abbreviation: MALT, mucosa-associated lymphoid tissue.

1	Malignancies of Lymphoid Cells Abbreviation: MALT, mucosa-associated lymphoid tissue. >10 × 109/L) is found (Fig. 134-6) that are monoclonal B cells expressing the CD5 antigen. Finding bone marrow infiltration by the same cells confirms the diagnosis. The peripheral blood smear in such patients typically shows many “smudge” or “basket” cells, nuclear remnants of cells damaged by the physical shear stress of making the blood smear. If cytogenetic studies are performed, trisomy 12 is found in 25–30% of patients. Abnormalities in chromosome 13 are also seen. If the primary presentation is lymphadenopathy and a lymph node biopsy is performed, pathologists usually have little difficulty in making the diagnosis of small lymphocytic lymphoma based on morphologic findings and immunophenotype. However, even in these patients, 70–75% will be found to have bone marrow involvement and circulating monoclonal B lymphocytes are often present.

1	The differential diagnosis of typical B-cell CLL is extensive (Table 134-1). Immunophenotyping will eliminate the T-cell disorders and can often help sort out other B-cell malignancies. For example, only mantle cell lymphoma and typical B-cell CLL are usually CD5 positive. Typical B-cell small lymphocytic lymphoma can be confused with other B-cell disorders, including lymphoplasmacytic lymphoma (i.e., the tissue manifestation of Waldenström’s macroglobulinemia), nodal marginal zone B-cell lymphoma, and mantle cell lymphoma. In addition, some small lymphocytic lymphomas have areas of large cells that can lead to confusion with diffuse large B-cell lymphoma. An expert hematopathologist is vital for making this distinction.

1	FIGURE 134-6 Chronic lymphocytic leukemia. The peripheral white blood cell count is high due to increased numbers of small, well-differentiated, normal-appearing lymphocytes. The leukemia lympho-cytes are fragile, and substantial numbers of broken, smudged cells are usually also present on the blood smear.

1	Typical B-cell CLL is often found incidentally when a complete blood count is done for another reason. However, complaints that might lead to the diagnosis include fatigue, frequent infections, and new lymphadenopathy. The diagnosis of typical B-cell CLL should be considered in a patient presenting with an autoimmune hemolytic anemia or autoimmune thrombocytopenia. B-cell CLL has also been associated with red cell aplasia. When this disorder presents as lymphoma, the most common abnormality is asymptomatic lymphadenopathy, with or without splenomegaly. The staging systems predict prognosis in patients with typical B-cell CLL (Table 134-7). The evaluation of a new patient with typical B-cell CLL/small lymphocytic lymphoma will include many of the studies (Table 134-11) that are used in patients with other non-Hodgkin’s lymphomas. In addition, particular attention needs to be given to detecting immune abnormalities such as autoimmune hemolytic anemia, autoimmune thrombocytopenia,

1	in patients with other non-Hodgkin’s lymphomas. In addition, particular attention needs to be given to detecting immune abnormalities such as autoimmune hemolytic anemia, autoimmune thrombocytopenia, hypogammaglobulinemia, and red cell aplasia. Molecular analysis of immunoglobulin gene sequences in CLL has demonstrated that about half the patients have tumors expressing mutated immunoglobulin genes and half have tumors expressing unmutated or germline immunoglobulin sequences. Patients with unmutated immunoglobulins tend to have a more aggressive clinical course and are less responsive to therapy. Unfortunately, immunoglobulin gene sequencing is not routinely available. CD38

1	Physical examination Documentation of B symptoms Laboratory evaluation Serum β2-microglobulin Chest radiograph CT scan of abdomen, pelvis, and usually chest Bone marrow biopsy Lumbar puncture in lymphoblastic, Burkitt’s, and diffuse large B-cell lym phoma with positive marrow biopsy Gallium scan (SPECT) or PET scan in large cell lymphoma Abbreviations: CT, computed tomography; PET, positron emission tomography; SPECT, single-photon emission computed tomography. expression is said to be low in the better-prognosis patients expressing mutated immunoglobulin and high in poorer-prognosis patients expressing unmutated immunoglobulin, but this test has not been confirmed as a reliable means of distinguishing the two groups. ZAP-70 expression correlates with the presence of unmutated immunoglobulin genes, but the assay is not yet standardized and widely available.

1	Patients whose presentation is typical B-cell CLL with no manifestations of the disease other than bone marrow involvement and lymphocytosis (i.e., Rai stage 0 and Binet stage A; Table 134-7) can be followed without specific therapy for their malignancy. These patients have a median survival >10 years, and some will never require therapy for this disorder. If the patient has an adequate number of circulating normal blood cells and is asymptomatic, many physicians would not initiate therapy for patients in the intermediate stage of the disease manifested by lymphadenopathy and/or hepatosplenomegaly. However, the median survival for these patients is ~7 years, and most will require treatment in the first few years of follow-up. Patients who present with bone marrow failure (i.e., Rai stage III or IV or Binet stage C) will require initial therapy in almost all cases. These patients have a serious disorder with a median survival of only 1.5 years. It must be remembered that immune

1	stage III or IV or Binet stage C) will require initial therapy in almost all cases. These patients have a serious disorder with a median survival of only 1.5 years. It must be remembered that immune manifestations of typical B-cell CLL should be managed independently of specific antileukemia therapy. For example, glucocorticoid therapy for autoimmune cytopenias and γ globulin replacement for patients with hypogammaglobulinemia should be used whether or not antileukemia therapy is given.

1	Patients who present primarily with lymphoma and have a low IPI score have a 5-year survival of ~75%, but those with a high IPI score have a 5-year survival of <40% and are more likely to require early therapy.

1	The most common treatments for patients with typical B-cell CLL/small lymphocytic lymphoma have been chlorambucil or fludarabine, alone or in combination. Chlorambucil can be administered orally with few immediate side effects, while fludarabine is administered IV and is associated with significant immune suppression. However, fludarabine is by far the more active agent and is the only drug associated with a significant incidence of complete remission. The combination of rituximab (375–500 mg/m2 day 1), fludarabine (25 mg/m2 days 2–4 on cycle 1 and days 1–3 in subsequent cycles), and cyclophosphamide (250 mg/m2 with fludarabine) achieves complete responses in 69% of patients, and those responses are associated with molecular remissions in half of the cases. Half the patients experience grade III or IV neutropenia. For young patients presenting with leukemia requiring therapy, regimens containing fludarabine are the treatment of choice. Because fludarabine is an effective second-line

1	III or IV neutropenia. For young patients presenting with leukemia requiring therapy, regimens containing fludarabine are the treatment of choice. Because fludarabine is an effective second-line agent in patients with tumors unresponsive to chlorambucil, the latter agent is often chosen in elderly patients who require therapy. Bendamustine, an alkylating agent structurally related to nitrogen mustard, is highly effective and is vying with fludarabine as the primary treatment of choice. Patients who present with lymphoma (rather than leukemia) are also highly responsive to bendamustine, and some patients will receive a combination chemotherapy regimen used in other lymphomas such as CVP (cyclophosphamide, vincristine, and prednisone) or CHOP plus rituximab. Alemtuzumab (anti-CD52) is an antibody with activity in the disease, but it kills both B and T cells and is associated with more immune compromise than rituximab. Young patients with this disease can be candidates for bone marrow

1	antibody with activity in the disease, but it kills both B and T cells and is associated with more immune compromise than rituximab. Young patients with this disease can be candidates for bone marrow transplantation. Allogeneic bone marrow transplantation can be curative but is associated with a significant treatment-related mortality rate. Mini-transplants using immunosuppressive rather than myeloablative doses of preparative drugs are being studied (Chap. 139e). The use of autologous transplantation in patients with this disorder has been discouraging.

1	At least two newer anti-CD20 monoclonal antibodies have 703 become available, ofatumumab and obinutuzumab. Both have activity in previously treated patients. Agents targeting signaling pathways, such as ibrutinib, an irreversible inhibitor of Bruton’s tyrosine kinase, and idelalisib, an inhibitor of phosphoinositide3-kinase delta, also have antitumor effects. The ideal combination and sequence of these therapies have not been defined. Extranodal Marginal Zone B-Cell Lymphoma of MALT Type Extranodal marginal zone B-cell lymphoma of MALT type (MALT lymphoma) makes up ~8% of non-Hodgkin’s lymphomas. This small cell lymphoma presents in extranodal sites. It was previously considered a small lymphocytic lymphoma or sometimes a pseudolymphoma. The recognition that the gastric presentation of this lymphoma was associated with H. pylori infection was an important step in recognizing it as a separate entity. The clinical characteristics of MALT lymphoma are presented in Table 134-10.

1	The diagnosis of MALT lymphoma can be made accurately by an expert hematopathologist based on a characteristic pattern of infiltration of small lymphocytes that are monoclonal B cells and CD5 negative. In some cases, transformation to diffuse large B-cell lymphoma occurs, and both diagnoses may be made in the same biopsy. The differential diagnosis includes benign lymphocytic infiltration of extra-nodal organs and other small cell B-cell lymphomas.

1	MALT lymphoma may occur in the stomach, orbit, intestine, lung, thyroid, salivary gland, skin, soft tissues, bladder, kidney, and CNS. It may present as a new mass, be found on routine imaging studies, or be associated with local symptoms such as upper abdominal discomfort in gastric lymphoma. Most MALT lymphomas are gastric in origin. At least two genetic forms of gastric MALT exist: one (accounting for ~50% of cases) characterized by t(11;18)(q21;q21) that juxtaposes the amino terminal of the API2 gene with the carboxy terminal of the MALT1 gene creating an API2/MALT1 fusion product, and the other characterized by multiple sites of genetic instability including trisomies of chromosomes 3, 7, 12, and 18. About 95% of gastric MALT lymphomas are associated with H. pylori infection, and those that are do not usually express t(11;18). The t(11;18) usually results in activation of nuclear factor-κB (NF-κB), which acts as a survival factor for the cells. Lymphomas with t(11;18)

1	and those that are do not usually express t(11;18). The t(11;18) usually results in activation of nuclear factor-κB (NF-κB), which acts as a survival factor for the cells. Lymphomas with t(11;18) translocations are genetically stable and do not evolve to diffuse large B-cell lymphoma. By contrast, t(11;18)-negative MALT lymphomas often acquire BCL6 mutations and progress to aggressive histology lymphoma. MALT lymphomas are localized to the organ of origin in ~40% of cases and to the organ and regional lymph nodes in ~30% of patients. However, distant metastasis can occur—particularly with transformation to diffuse large B-cell lymphoma. Many patients who develop this lymphoma will have an autoimmune or inflammatory process such as Sjögren’s syndrome (salivary gland MALT), Hashimoto’s thyroiditis (thyroid MALT), Helicobacter gastritis (gastric MALT), C. psittaci conjunctivitis (ocular MALT), or Borrelia skin infections (cutaneous MALT).

1	Evaluation of patients with MALT lymphoma follows the pattern (Table 134-11) for staging a patient with non-Hodgkin’s lymphoma. In particular, patients with gastric lymphoma need to have studies performed to document the presence or absence of H. pylori infection. Endoscopic studies including ultrasound can help define the extent of gastric involvement. Most patients with MALT lymphoma have a good prognosis, with a 5-year survival of ~75%. In patients with a low IPI score, the 5-year survival is ~90%, whereas it drops to ~40% in patients with a high IPI score. MALT lymphoma is often localized. Patients with gastric MALT lymphomas who are infected with H. pylori can achieve remission in the 80% of cases with eradication of the infection. These remissions can be durable, but molecular evidence of persisting neoplasia is not

1	Malignancies of Lymphoid Cells 704 infrequent. After H. pylori eradication, symptoms generally improve quickly, but molecular evidence of persistent disease may be present for 12–18 months. Additional therapy is not indicated unless progressive disease is documented. Patients with more extensive disease or progressive disease are most often treated with single-agent chemotherapy such as chlorambucil. Combination regimens that include rituximab are also highly effective. Coexistent diffuse large B-cell lymphoma must be treated with combination chemotherapy (see below). The additional acquired mutations that mediate the histologic progression also convey Helicobacter independence to the growth.

1	Mantle Cell Lymphoma Mantle cell lymphoma makes up ~6% of all non-Hodgkin’s lymphomas. This lymphoma was previously placed in a number of other subtypes. Its existence was confirmed by the recognition that these lymphomas have a characteristic chromosomal trans-location, t(11;14), between the immunoglobulin heavy chain gene on chromosome 14 and the bcl-1 gene on chromosome 11, and regularly overexpress the BCL-1 protein, also known as cyclin D1. Table 134-10 shows the clinical characteristics of mantle cell lymphoma. The diagnosis of mantle cell lymphoma can be made accurately by an expert hematopathologist. As with all subtypes of lymphoma, an adequate biopsy is important. The differential diagnosis of mantle cell lymphoma includes other small cell B-cell lymphomas. In particular, mantle cell lymphoma and small lymphocytic lymphoma share a characteristic expression of CD5. Mantle cell lymphoma usually has a slightly indented nucleus.

1	The most common presentation of mantle cell lymphoma is with palpable lymphadenopathy, frequently accompanied by systemic symptoms. The median age is 63 years, and men are affected four times as commonly as women. Approximately 70% of patients will be stage IV at the time of diagnosis, with frequent bone marrow and peripheral blood involvement. Of the extranodal organs that can be involved, gastrointestinal involvement is particularly important to recognize. Patients who present with lymphomatosis polyposis in the large intestine usually have mantle cell lymphoma. Table 134-11 outlines the evaluation of patients with mantle cell lymphoma. Patients who present with gastrointestinal tract involvement often have Waldeyer’s ring involvement, and vice versa. The 5-year survival for all patients with mantle cell lymphoma is ~25%, with only occasional patients who present with a high IPI score surviving 5 years and ~50% of patients with a low IPI score surviving 5 years.

1	Current therapies for mantle cell lymphoma are evolving. Patients with localized disease might be treated with combination chemotherapy followed by radiotherapy; however, these patients are exceedingly rare. For the usual presentation with disseminated disease, standard lymphoma treatments have been unsatisfactory, with the minority of patients achieving complete remission. Aggressive combination chemotherapy regimens followed by autologous or allogeneic bone marrow transplantation are frequently offered to younger patients. For the occasional elderly, asymptomatic patient, observation followed by single-agent chemotherapy might be the most practical approach. An intensive combination chemotherapy regimen originally used in the treatment of acute leukemia, HyperC-VAD (cyclophosphamide, vincristine, doxorubicin, dexamethasone, cytarabine, and methotrexate), in combination with rituximab, seems to be associated with better response rates, particularly in younger patients. Alternating

1	vincristine, doxorubicin, dexamethasone, cytarabine, and methotrexate), in combination with rituximab, seems to be associated with better response rates, particularly in younger patients. Alternating two regimens, HyperC-VAD with rituximab added (R-HyperC-VAD) and rituximab plus high-dose methotrexate and cytarabine, can achieve complete responses in >80% of patients and an 8-year survival of 56%, comparable to regimens using high-dose therapy and autologous hematopoietic stem cell transplantation. Bendamustine plus rituximab has been found to induce complete responses in about 31% of patients, but the responses are generally not long lasting. Bortezomib and temsirolimus are single agents that induce transient partial responses in a minority of patients and are being added to primary combinations.

1	FIGURE 134-7 Follicular lymphoma. The normal nodal architecture is effaced by nodular expansions of tumor cells. Nodules vary in size and contain predominantly small lymphocytes with cleaved nuclei along with variable numbers of larger cells with vesicular chromatin and prominent nucleoli. Follicular Lymphoma Follicular lymphomas make up 22% of nonHodgkin’s lymphomas worldwide and at least 30% of non-Hodgkin’s lymphomas diagnosed in the United States. This type of lymphoma can be diagnosed accurately on morphologic findings alone and has been the diagnosis in the majority of patients in therapeutic trials for “low-grade” lymphoma in the past. The clinical characteristics of follicular lymphoma are presented in Table 134-10.

1	Evaluation of an adequate biopsy by an expert hematopathologist is sufficient to make a diagnosis of follicular lymphoma. The tumor is composed of small cleaved and large cells in varying proportions organized in a follicular pattern of growth (Fig. 134-7). Confirmation of B-cell immunophenotype and the existence of the t(14;18) and abnormal expression of BCL-2 protein are confirmatory. The major differential diagnosis is between lymphoma and reactive follicular hyperplasia. The coexistence of diffuse large B-cell lymphoma must be considered. Patients with follicular lymphoma are often subclassified into those with predominantly small cells, those with a mixture of small and large cells, and those with predominantly large cells. Although this distinction cannot be made simply or very accurately, these subdivisions do have prognostic significance. Patients with follicular lymphoma with predominantly large cells have a higher proliferative fraction, progress more rapidly, and have a

1	these subdivisions do have prognostic significance. Patients with follicular lymphoma with predominantly large cells have a higher proliferative fraction, progress more rapidly, and have a shorter overall survival with simple chemotherapy regimens.

1	The most common presentation for follicular lymphoma is with new, painless lymphadenopathy. Multiple sites of lymphoid involvement are typical, and unusual sites such as epitrochlear nodes are sometimes seen. However, essentially any organ can be involved, and extranodal presentations do occur. Most patients do not have fevers, sweats, or weight loss, and an IPI score of 0 or 1 is found in ~50% of patients. Fewer than 10% of patients have a high (i.e., 4 or 5) IPI score. The staging evaluation for patients with follicular lymphoma should include the studies shown in Table 134-11.

1	Follicular lymphoma is one of the malignancies most responsive to chemotherapy and radiotherapy. In addition, tumors in as many as 25% of the patients undergo spontaneous regression—usually transient—without therapy. In an asymptomatic patient, no initial treatment and watchful waiting can be an appropriate management strategy and is particularly likely to be adopted for older patients with advanced-stage disease. For patients who do require treat ment, single-agent chlorambucil or cyclophosphamide or combination chemotherapy with CVP or CHOP is most frequently used. With adequate treatment, 50–75% of patients will achieve a complete remission. Although most patients relapse (median response duration is ~2 years), at least 20% of complete responders will remain in remission for >10 years. For the rare patients (15%) with localized follicular lymphoma, involved-field radiotherapy produces longterm disease-free survival in the majority.

1	A number of therapies have been shown to be active in the treatment of patients with follicular lymphoma. These include cytotoxic agents such as fludarabine, biologic agents such as interferon α, monoclonal antibodies with or without radionuclides, and lymphoma vaccines. In patients treated with a doxorubicin-containing combination chemotherapy regimen, interferon α given to patients in complete remission seems to prolong survival, but interferon toxicities can affect quality of life. The monoclonal antibody rituximab can cause objective responses in 35–50% of patients with relapsed follicular lymphoma, and radiolabeled antibodies appear to have response rates well in excess of 50%. The addition of rituximab to CHOP and other effective combination chemotherapy programs achieves prolonged overall survival and a decreased risk of histologic progression. Complete remissions can be noted in 85% or more of patients treated with R-CHOP, and median remission durations can exceed 6 or 7

1	overall survival and a decreased risk of histologic progression. Complete remissions can be noted in 85% or more of patients treated with R-CHOP, and median remission durations can exceed 6 or 7 years. Maintenance intermittent rituximab therapy can prolong remissions even further, although it is not completely clear that overall survival is prolonged. Some trials with tumor vaccines have been encouraging. Both autologous and allogeneic hematopoietic stem cell transplantations yield high complete response rates in patients with relapsed follicular lymphoma, and long-term remissions can occur in 40% or more of patients.

1	Patients with follicular lymphoma with a predominance of large cells have a shorter survival when treated with single-agent chemotherapy but seem to benefit from receiving an anthracyclinecontaining combination chemotherapy regimen plus rituximab. When their disease is treated aggressively, the overall survival for such patients is no lower than for patients with other follicular lymphomas, and the failure-free survival is superior.

1	Patients with follicular lymphoma have a high rate of histologic transformation to diffuse large B-cell lymphoma (5–7% per year). This is recognized ~40% of the time during the course of the illness by repeat biopsy and is present in almost all patients at autopsy. This transformation is usually heralded by rapid growth of lymph nodes—often localized—and the development of systemic symptoms such as fevers, sweats, and weight loss. Although these patients have a poor prognosis, aggressive combination chemotherapy regimens can sometimes cause a complete remission in the diffuse large B-cell lymphoma, at times leaving the patient with persisting follicular lymphoma. With more frequent use of R-CHOP to treat follicular lymphoma at diagnosis, it appears that the rate of histologic progression is decreasing. R-CHOP or bendamustine plus rituximab with intermittent rituximab maintenance for 2 years are the most commonly used treatment approaches.

1	Diffuse Large B-Cell Lymphoma Diffuse large B-cell lymphoma is the most common type of non-Hodgkin’s lymphoma, representing approximately one-third of all cases. This lymphoma makes up the majority of cases in previous clinical trials of “aggressive” or “intermediate-grade” lymphoma. Table 134-10 shows the clinical characteristics of diffuse large B-cell lymphoma.

1	The diagnosis of diffuse large B-cell lymphoma can be made accurately by an expert hematopathologist (Fig. 134-8). Cytogenetic and molecular genetic studies are not necessary for diagnosis, but some evidence has accumulated that patients whose tumors overexpress the BCL-2 protein might be more likely to relapse than others. A subset of patients have tumors with mutations in BCL6 and translocations involving MYC; these are called “double-hit” lymphomas and typically have more aggressive growth and are more poorly responsive to treatment than other diffuse large B-cell lymphomas. Patients with prominent mediastinal involvement are sometimes diagnosed as a separate subgroup having primary mediastinal diffuse large B-cell lymphoma. This latter group of patients has a younger median age (i.e., 37 years) and a female predominance (66%). Subtypes of diffuse large B-cell lymphoma, including those with an immunoblastic subtype and tumors with extensive fibrosis, are recognized by pathologists

1	37 years) and a female predominance (66%). Subtypes of diffuse large B-cell lymphoma, including those with an immunoblastic subtype and tumors with extensive fibrosis, are recognized by pathologists but do not appear to have important independent prognostic significance.

1	FIGURE 134-8 Diffuse large B-cell lymphoma. The neoplastic cells are heterogeneous but predominantly large cells with vesicular chro-matin and prominent nucleoli. Diffuse large B-cell lymphoma can present as either primary lymph node disease or at extranodal sites. More than 50% of patients will have some site of extranodal involvement at diagnosis, with the most common sites being the gastrointestinal tract and bone marrow, each being involved in 15–20% of patients. Essentially any organ can be involved, making a diagnostic biopsy imperative. For example, diffuse large B-cell lymphoma of the pancreas has a much better prognosis than pancreatic carcinoma but would be missed without biopsy. Primary diffuse large B-cell lymphoma of the brain is being diagnosed with increasing frequency. Other unusual subtypes of diffuse large B-cell lymphoma such as pleural effusion lymphoma and intravascular lymphoma have been difficult to diagnose and associated with a very poor prognosis.

1	Table 134-11 shows the initial evaluation of patients with diffuse large B-cell lymphoma. After a careful staging evaluation, ~50% of patients will be found to have stage I or II disease, and ~50% will have widely disseminated lymphoma. Bone marrow biopsy shows involvement by lymphoma in ~15% of cases, with marrow involvement by small cells more frequent than by large cells.

1	The initial treatment of all patients with diffuse large B-cell lymphoma should be with a combination chemotherapy regimen. The most popular regimen in the United States is CHOP plus rituximab, although a variety of other anthracycline-containing combination chemotherapy regimens appear to be equally efficacious. Patients with stage I or nonbulky stage II disease can be effectively treated with three to four cycles of combination chemotherapy with or without subsequent involved-field radiotherapy. The need for radiation therapy is unclear. Cure rates of 70–80% in stage II disease and 85–90% in stage I disease can be expected.

1	For patients with bulky stage II, stage III, or stage IV disease, six to eight cycles of CHOP plus rituximab are usually administered. A large randomized trial showed the superiority of CHOP combined with rituximab over CHOP alone in elderly patients. A frequent approach would be to administer four cycles of therapy and then reevaluate. If the patient has achieved a complete remission after four cycles, two more cycles of treatment might be given and then therapy discontinued. Using this approach, 70–80% of patients can

1	Malignancies of Lymphoid Cells 706 be expected to achieve a complete remission, and 50–70% of complete responders will be cured. The chances for a favorable response to treatment are predicted by the IPI. In fact, the IPI was developed based on the outcome of patients with diffuse large B-cell lymphoma treated with CHOP-like regimens. For the 35% of patients with a low IPI score of 0–1, the 5-year survival is >70%, whereas for the 20% of patients with a high IPI score of 4–5, the 5-year survival is ~20%. The addition of rituximab to CHOP has improved each of those numbers by ~15%. A number of other factors, including molecular features of the tumor, levels of circulating cytokines and soluble receptors, and other surrogate markers, have been shown to influence prognosis. However, they have not been validated as rigorously as the IPI and have not been uniformly applied clinically. Because a number of patients with diffuse large B-cell lymphoma are either initially refractory to therapy

1	not been validated as rigorously as the IPI and have not been uniformly applied clinically. Because a number of patients with diffuse large B-cell lymphoma are either initially refractory to therapy or relapse after apparently effective chemotherapy, 30–40% of patients will be candidates for salvage treatment at some point. Alternative combination chemotherapy regimens can induce complete remission in as many as 50% of these patients, but long-term disease-free survival is seen in ≤10%. Autologous bone marrow transplantation is superior to salvage chemotherapy at usual doses and leads to longterm disease-free survival in ~40% of patients whose lymphomas remain chemotherapy-sensitive after relapse.

1	Burkitt’s Lymphoma/Leukemia Burkitt’s lymphoma/leukemia is a rare disease in adults in the United States, making up <1% of non-Hodgkin’s lymphomas, but it makes up ~30% of childhood non-Hodgkin’s lymphoma. Burkitt’s leukemia, or L3 ALL, makes up a small proportion of childhood and adult acute leukemias. Table 134-10 shows the clinical features of Burkitt’s lymphoma.

1	Burkitt’s lymphoma can be diagnosed morphologically by an expert hematopathologist with a high degree of accuracy. The cells are homogeneous in size and shape (Fig. 134-9). Demonstration of a very high proliferative fraction and the presence of the t(8;14) or one of its variants, t(2;8) (c-myc and the λ light chain gene) or t(8;22) (c-myc and the κ light chain gene), can be confirmatory. Burkitt’s cell leukemia is recognized by the typical monotonous mass of medium-sized cells with round nuclei, multiple nucleoli, and basophilic cytoplasm with cytoplasmic vacuoles. Demonstration of surface expression of immunoglobulin and one of the above-noted cytogenetic abnormalities is confirmatory. Three distinct clinical forms of Burkitt’s lymphoma are recognized: endemic, sporadic, and immunodeficiency-associated. Endemic and sporadic Burkitt’s lymphomas occur frequently in children in Africa, and the sporadic form occurs in Western countries.

1	FIGURE 134-9 Burkitt’s lymphoma. The neoplastic cells are homo-geneous, medium-sized B cells with frequent mitotic figures, a mor-phologic correlate of high growth fraction. Reactive macrophages are scattered through the tumor, and their pale cytoplasm in a back-ground of blue-staining tumor cells gives the tumor a so-called starry sky appearance. Immunodeficiency-associated Burkitt’s lymphoma is seen in patients with HIV infection.

1	Immunodeficiency-associated Burkitt’s lymphoma is seen in patients with HIV infection. Pathologists sometimes have difficulty distinguishing between Burkitt’s lymphoma and diffuse large B-cell lymphoma. In the past, a separate subgroup of non-Hodgkin’s lymphoma intermediate between the two was recognized. When tested, this subgroup could not be diagnosed accurately. Distinction between the two major types of B-cell aggressive non-Hodgkin’s lymphoma can sometimes be made based on the extremely high proliferative fraction seen in patients with Burkitt’s lymphoma (i.e., essentially 100% of tumor cells are in cycle) caused by c-myc deregulation.

1	Most patients in the United States with Burkitt’s lymphoma present with peripheral lymphadenopathy or an intraabdominal mass. The disease is rapidly progressive and has a propensity to metastasize to the CNS. Initial evaluation should always include an examination of cerebrospinal fluid to rule out metastasis in addition to the other staging evaluations noted in Table 134-11. Once the diagnosis of Burkitt’s lymphoma is suspected, a diagnosis must be made promptly, and staging evaluation must be accomplished expeditiously. This is the most rapidly progressive human tumor, and any delay in initiating therapy can adversely affect the patient’s prognosis.

1	Treatment of Burkitt’s lymphoma in both children and adults should begin within 48 h of diagnosis and involves the use of intensive combination chemotherapy regimens incorporating high doses of cyclophosphamide. Prophylactic therapy to the CNS is mandatory. Burkitt’s lymphoma was one of the first cancers shown to be curable by chemotherapy. Today, cure can be expected in 70–80% of both children and young adults when effective therapy is administered precisely. Salvage therapy has been generally ineffective in patients in whom the initial treatment fails, emphasizing the importance of the initial treatment approach. involves blood and marrow infiltration by large lymphocytes with prominent nucleoli. Patients typically have a high white cell count, splenomegaly, and minimal lymphadenopathy. The chances for a complete response to therapy are poor.

1	Hairy cell leukemia is a rare disease that presents predominantly in older males. Typical presentation involves pancytopenia, although occasional patients will have a leukemic presentation. Splenomegaly is usual. The malignant cells appear to have “hairy” projections on light and electron microscopy and show a characteristic staining pattern with tartrate-resistant acid phosphatase. Bone marrow is typically not able to be aspirated, and biopsy shows a pattern of fibrosis with diffuse infiltration by the malignant cells. Patients with this disorder have monocytopenia and are prone to unusual infections, including infection by Mycobacterium avium intracellulare, and to vasculitic syndromes. Hairy cell leukemia is responsive to chemotherapy with interferon α, pentostatin, or cladribine, with the latter being the usually preferred treatment. Clinical complete remissions with cladribine occur in the majority of patients, and long-term disease-free survival is frequent. Many of these tumors

1	the latter being the usually preferred treatment. Clinical complete remissions with cladribine occur in the majority of patients, and long-term disease-free survival is frequent. Many of these tumors have the V600E BRAF mutation and accordingly are responsive to BRAF inhibitors like vemurafenib.

1	Splenic marginal zone lymphoma involves infiltration of the splenic white pulp by small, monoclonal B cells. This is a rare disorder that can present as leukemia as well as lymphoma. Definitive diagnosis is often made at splenectomy, which is also an effective therapy. This is an extremely indolent disorder, but when chemotherapy is required, the most usual treatment has been chlorambucil.

1	Lymphoplasmacytic lymphoma is the tissue manifestation of Waldenström’s macroglobulinemia (Chap. 136). Many of these tumors harbor a specific mutation, L265P, in MYD88, a change that leads to NF-κB activation. This type of lymphoma has been associated with chronic hepatitis C virus infection, and an etiologic association has been proposed. Patients typically present with lymphadenopathy, splenomegaly, bone marrow involvement, and occasionally peripheral blood involvement. The tumor cells do not express CD5. Patients often have a monoclonal IgM protein, high levels of which can dominate the clinical picture with the symptoms of hyperviscosity. Treatment of lymphoplasmacytic lymphoma can be aimed primarily at reducing the abnormal protein, if present, but will usually also involve chemotherapy. Chlorambucil, fludarabine, and cladribine have been used. The median 5-year survival for patients with this disorder is ~60%.

1	Nodal marginal zone lymphoma, also known as monocytoid cell lymphoma, represents ~1% of non-Hodgkin’s lymphomas. This lymphoma has a slight female predominance and presents with disseminated disease (i.e., stage III or IV) in 75% of patients. Approximately one-third of patients have bone marrow involvement, and a leukemic presentation occasionally occurs. The staging evaluation and therapy should use the same approach as used for patients with follicular lymphoma. Approximately 60% of the patients with nodal marginal zone lymphoma will survive 5 years after diagnosis. Other more uncommon B-cell malignancies are discussed in Chap. 135e. PRECURSOR T-CELL MALIGNANCIES Precursor T-Cell Lymphoblastic Leukemia/Lymphoma Precursor T-cell malignancies can present either as ALL or as an aggressive lymphoma. These malignancies are more common in children and young adults, with males more frequently affected than females.

1	Precursor T-cell ALL can present with bone marrow failure, although the severity of anemia, neutropenia, and thrombocytopenia is often less than in precursor B-cell ALL. These patients sometimes have very high white cell counts, a mediastinal mass, lymphadenopathy, and hepatosplenomegaly. Precursor T-cell lymphoblastic lymphoma is most often found in young men presenting with a large mediastinal mass and pleural effusions. Both presentations have a propensity to metastasize to the CNS, and CNS involvement is often present at diagnosis.

1	Children with precursor T-cell ALL seem to benefit from very intensive remission induction and consolidation regimens. The majority of patients treated in this manner can be cured. Older children and young adults with precursor T-cell lymphoblastic lymphoma are also often treated with “leukemia-like” regimens. Patients who present with localized disease have an excellent prognosis. However, advanced age is an adverse prognostic factor. Adults with precursor T-cell lymphoblastic lymphoma who present with high LDH levels or bone marrow or CNS involvement are often offered bone marrow transplantation as part of their primary therapy. MATURE (PERIPHERAL) T-CELL DISORDERS Mycosis Fungoides Mycosis fungoides is also known as cutaneous T-cell lymphoma. This lymphoma is more often seen by dermatologists than internists. The median age of onset is in the mid-fifties, and the disease is more common in males and in blacks.

1	Mycosis fungoides is an indolent lymphoma with patients often having several years of eczematous or dermatitic skin lesions before the diagnosis is finally established. The skin lesions progress from patch stage to plaque stage to cutaneous tumors. Early in the disease, biopsies are often difficult to interpret, and the diagnosis may only become apparent by observing the patient over time. In advanced stages, the lymphoma can spread to lymph nodes and visceral organs. Patients with this lymphoma may develop generalized erythroderma and circulating tumor cells, called Sézary’s syndrome.

1	Rare patients with localized early-stage mycosis fungoides can be cured with radiotherapy, often total-skin electron beam irradiation. More advanced disease has been treated with topical glucocorticoids, topical nitrogen mustard, phototherapy, psoralen with ultraviolet A (PUVA), extracorporeal photopheresis, retinoids (bexarotene), elec-707 tron beam radiation, interferon, antibodies, fusion toxins, histone deacetylase inhibitors, and systemic cytotoxic therapy. Unfortunately, these treatments are palliative.

1	Adult T-Cell Lymphoma/Leukemia Adult T-cell lymphoma/leukemia is one manifestation of infection by the HTLV-1 retrovirus. Patients can be infected through transplacental transmission, mother’s milk, blood transfusion, and by sexual transmission of the virus. Patients who acquire the virus from their mother through breast milk are most likely to develop lymphoma, but the risk is still only 2.5% and the latency averages 55 years. Nationwide testing for HTLV-1 antibodies and the aggressive implementation of public health measures could theoretically lead to the disappearance of adult T-cell lymphoma/leukemia. Tropical spastic paraparesis, another manifestation of HTLV-1 infection (Chap. 225e), occurs after a shorter latency (1–3 years) and is most common in individuals who acquire the virus during adulthood from transfusion or sex.

1	The diagnosis of adult T-cell lymphoma/leukemia is made when an expert hematopathologist recognizes the typical morphologic picture, a T-cell immunophenotype (i.e., CD4 positive), and the presence in serum of antibodies to HTLV-1. Examination of the peripheral blood will usually reveal characteristic, pleomorphic abnormal CD4-positive cells with indented nuclei, which have been called “flower” cells (Fig. 134-10).

1	A subset of patients have a smoldering clinical course and long survival, but most patients present with an aggressive disease manifested by lymphadenopathy, hepatosplenomegaly, skin infiltration, pulmo nary infiltrates, hypercalcemia, lytic bone lesions, and elevated LDH levels. The skin lesions can be papules, plaques, tumors, and ulcerations. Lung lesions can be either tumor or opportunistic infection in light of the underlying immunodeficiency in the disease. Bone marrow involvement is not usually extensive, and anemia and thrombocytopenia are not usually prominent. Although treatment with combination chemotherapy regimens can result in objective responses, true complete remissions are unusual, and the median survival of patients is ~7 months. A small phase II study reported a high response rate with interferon plus zidovudine and arsenic trioxide.

1	Anaplastic Large T/Null-Cell Lymphoma Anaplastic large T/null-cell lymphoma was previously usually diagnosed as undifferentiated carcinoma or malignant histiocytosis. Discovery of the CD30 (Ki-1) antigen and the recognition that some patients with previously unclassified malignancies displayed this antigen led to the identification of a new type of lymphoma. Subsequently, discovery of the t(2;5) and the resultant frequent overexpression of the anaplastic lymphoma kinase (ALK) protein confirmed the existence of this entity. This lymphoma accounts for ~2% of all non-Hodgkin’s lymphomas. Table 134-10 shows the clinical characteristics of patients with anaplastic large T/null cell lymphoma. FIGURE 134-10 Adult T-cell leukemia/lymphoma. Peripheral blood smear showing leukemia cells with typical “flower-shaped” nucleus.

1	Malignancies of Lymphoid Cells 708 The diagnosis of anaplastic large T/null-cell lymphoma is made when an expert hematopathologist recognizes the typical morphologic picture and a T-cell or null-cell immunophenotype with CD30 positivity. Documentation of the t(2;5) and/or overexpression of ALK protein confirm the diagnosis. Some diffuse large B-cell lymphomas can also have an anaplastic appearance but have the same clinical course or response to therapy as other diffuse large B-cell lymphomas. A small percentage of anaplastic lymphomas are ALK negative. Patients with anaplastic large T/null-cell lymphoma are typically young (median age, 33 years) and male (~70%). Some 50% of patients present in stage I/II, and the remainder present with more extensive disease. Systemic symptoms and elevated LDH levels are seen in about one-half of patients. Bone marrow and the gastrointestinal tract are rarely involved, but skin involvement is frequent. Some patients with disease confined to the skin

1	LDH levels are seen in about one-half of patients. Bone marrow and the gastrointestinal tract are rarely involved, but skin involvement is frequent. Some patients with disease confined to the skin have a different and more indolent disorder that has been termed cutaneous anaplastic large T/null-cell lymphoma and might be related to lymphomatoid papulosis.

1	Treatment regimens appropriate for other aggressive lymphomas, such as diffuse large B-cell lymphoma, should be used in patients with anaplastic large T/null-cell lymphoma, with the exception that the B-cell–specific antibody, rituximab, is omitted. Surprisingly, given the anaplastic appearance, this disorder has the best survival rate of any aggressive lymphoma. The 5-year survival is >75%. While traditional prognostic factors such as the IPI predict treatment outcome, overexpression of the ALK protein is an important prognostic factor, with patients overexpressing this protein having a superior treatment outcome. The ALK inhibitor crizotinib appears highly active as well. In addition, the CD30 immunotoxin, brentuximab vedotin, is active in the disease.

1	Peripheral T-Cell Lymphoma The peripheral T-cell lymphomas make up a heterogeneous morphologic group of aggressive neoplasms that share a mature T-cell immunophenotype. They represent ~7% of all cases of non-Hodgkin’s lymphoma. A number of distinct clinical syndromes are included in this group of disorders. Table 134-10 shows the clinical characteristics of patients with peripheral T-cell lymphoma.

1	The diagnosis of peripheral T-cell lymphoma, or any of its specific subtypes, requires an expert hematopathologist, an adequate biopsy, and immunophenotyping. Most peripheral T-cell lymphomas are CD4+, but a few will be CD8+, both CD4+ and CD8+, or have an NK cell immunophenotype. No characteristic genetic abnormalities have yet been identified, but translocations involving the T-cell antigen receptor genes on chromosomes 7 or 14 may be detected. The differential diagnosis of patients suspected of having peripheral T-cell lymphoma includes reactive T-cell infiltrative processes. In some cases, demonstration of a monoclonal T-cell population using T-cell receptor gene rearrangement studies will be required to make a diagnosis.

1	The initial evaluation of a patient with a peripheral T-cell lymphoma should include the studies in Table 134-11 for staging patients with non-Hodgkin’s lymphoma. Unfortunately, patients with peripheral T-cell lymphoma usually present with adverse prognostic factors, with >80% of patients having an IPI score ≥2 and >30% having an IPI score ≥4. As this would predict, peripheral T-cell lymphomas are associated with a poor outcome, and only 25% of the patients survive 5 years after diagnosis. Treatment regimens are the same as those used for diffuse large B-cell lymphoma (omitting rituximab), but patients with peripheral T-cell lymphoma have a poorer response to treatment. Because of this poor treatment outcome, hematopoietic stem cell transplantation is often considered early in the care of young patients.

1	A number of specific clinical syndromes are seen in the peripheral T-cell lymphomas. Angioimmunoblastic T-cell lymphoma is one of the more common subtypes, making up ~20% of T-cell lymphomas. These patients typically present with generalized lymphadenopathy, fever, weight loss, skin rash, and polyclonal hypergammaglobulinemia. In some cases, it is difficult to separate patients with a reactive disorder from those with true lymphoma.

1	Extranodal T/NK-cell lymphoma of nasal type has also been called angiocentric lymphoma and was previously termed lethal midline granuloma. This disorder is more frequent in Asia and South America than in the United States and Europe. EBV is thought to play an etiologic role. Although most frequent in the upper airway, it can involve other organs. The course is aggressive, and patients frequently have the hemophagocytic syndrome. When marrow and blood involvement occur, distinction between this disease and leukemia might be difficult. Some patients will respond to aggressive combination chemotherapy regimens, but the overall outlook is poor.

1	Enteropathy-type intestinal T-cell lymphoma is a rare disorder that occurs in patients with untreated gluten-sensitive enteropathy. Patients are frequently wasted and sometimes present with intestinal perforation. The prognosis is poor. Hepatosplenic γδ T-cell lymphoma is a systemic illness that presents with sinusoidal infiltration of the liver, spleen, and bone marrow by malignant T cells. Tumor masses generally do not occur. The disease is associated with systemic symptoms and is often difficult to diagnose. Treatment outcome is poor. Subcutaneous panniculitis-like T-cell lymphoma is a rare disorder that is often confused with panniculitis. Patients present with multiple subcutaneous nodules, which progress and can ulcerate. Hemophagocytic syndrome is common. Response to therapy is poor. The development of the hemophagocytic syndrome (profound anemia, ingestion of erythrocytes by monocytes and macrophages, elevated ferritin levels) in the course of any peripheral T-cell lymphoma is

1	The development of the hemophagocytic syndrome (profound anemia, ingestion of erythrocytes by monocytes and macrophages, elevated ferritin levels) in the course of any peripheral T-cell lymphoma is generally associated with a fatal outcome.

1	HODGKIN’S LYMPHOMA Classical Hodgkin’s Lymphoma Hodgkin’s lymphoma occurs in 9000 patients in the United States each year, and the disease does not appear to be increasing in frequency. Most patients present with palpable lymphadenopathy that is nontender; in most patients, these lymph nodes are in the neck, supraclavicular area, and axilla. More than half the patients will have mediastinal adenopathy at diagnosis, and this is sometimes the initial manifestation. Subdiaphragmatic presentation of Hodgkin’s lymphoma is unusual and more common in older males. One-third of patients present with fevers, night sweats, and/or weight loss—B symptoms in the Ann Arbor staging classification (Table 134-8). Occasionally, Hodgkin’s lymphoma can present as a fever of unknown origin. This is more common in older patients who are found to have mixed-cellularity Hodgkin’s lymphoma in an abdominal site. Rarely, the fevers persist for days to weeks, followed by afebrile intervals and then recurrence of

1	in older patients who are found to have mixed-cellularity Hodgkin’s lymphoma in an abdominal site. Rarely, the fevers persist for days to weeks, followed by afebrile intervals and then recurrence of the fever. This pattern is known as Pel-Ebstein fever. Hodgkin’s lymphoma can occasionally present with unusual manifestations. These include severe and unexplained itching, cutaneous disorders such as erythema nodosum and ichthyosiform atrophy, paraneoplastic cerebellar degeneration and other distant effects on the CNS, nephrotic syndrome, immune hemolytic anemia and thrombocytopenia, hypercalcemia, and pain in lymph nodes on alcohol ingestion.

1	The diagnosis of Hodgkin’s lymphoma is established by review of an adequate biopsy specimen by an expert hematopathologist. In the United States, most patients have nodular sclerosing Hodgkin’s lymphoma, with a minority of patients having mixed-cellularity Hodgkin’s lymphoma. Lymphocyte-predominant and lymphocyte-depleted Hodgkin’s lymphoma are rare. Mixed-cellularity Hodgkin’s lymphoma or lymphocyte-depletion Hodgkin’s lymphoma are seen more frequently in patients infected by HIV (Fig. 134-11). Hodgkin’s lymphoma is a tumor characterized by rare neoplastic cells of B-cell origin (immunoglobulin genes are rearranged but not expressed) in a tumor mass that is largely polyclonal inflammatory infiltrate, probably a reaction to cytokines produced by the tumor cells. The differential diagnosis of a lymph node biopsy suspicious for Hodgkin’s lymphoma includes inflammatory processes, mononucleosis, non-Hodgkin’s lymphoma, phenytoin-induced adenopathy, and nonlymphomatous malignancies.

1	FIGURE 134-11 Mixed-cellularity Hodgkin’s lymphoma. A Reed-Sternberg cell is present near the center of the field; a large cell with a bilobed nucleus and prominent nucleoli giving an “owl’s eyes” appearance. The majority of the cells are normal lymphocytes, neutrophils, and eosinophils that form a pleomorphic cellular infiltrate.

1	The staging evaluation for a patient with Hodgkin’s lymphoma would typically include a careful history and physical examination; complete blood count; erythrocyte sedimentation rate; serum chemistry studies including LDH; chest radiograph; CT scan of the chest, abdomen, and pelvis; and bone marrow biopsy. Many patients would also have a PET scan or a gallium scan. Although rarely used, a bipedal lymphangiogram can be helpful. PET and gallium scans are most useful to document remission. Staging laparotomies were once popular for most patients with Hodgkin’s lymphoma but are now done rarely because of an increased reliance on systemic rather than local therapy.

1	Patients with localized Hodgkin’s lymphoma are cured >90% of the time. In patients with good prognostic factors, extended-field radiotherapy has a high cure rate. Increasingly, patients with all stages of Hodgkin’s lymphoma are treated initially with chemotherapy. Patients with localized or good-prognosis disease receive a brief course of chemotherapy followed by radiotherapy to sites of node involvement. Patients with more extensive disease or those with B symptoms receive a complete course of chemotherapy. The most popular chemotherapy regimen used in Hodgkin’s lymphoma is a combination of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD). Today, most patients in the United States receive ABVD, but a weekly chemotherapy regimen administered for 12 weeks called Stanford V is becoming increasingly popular, but it includes radiation therapy, which has been associated with life-threatening late toxicities such as premature coronary artery disease and second solid tumors. In

1	becoming increasingly popular, but it includes radiation therapy, which has been associated with life-threatening late toxicities such as premature coronary artery disease and second solid tumors. In Europe, a high-dose regimen called BEACOPP incorporating alkylating agents has become popular and might have a better response rate in very-high-risk patients. Long-term disease-free survival in patients with advanced disease can be achieved in >75% of patients who lack systemic symptoms and in 60–70% of patients with systemic symptoms.

1	Patients who relapse after primary therapy of Hodgkin’s lymphoma can frequently still be cured. Patients who relapse after initial treatment with only radiotherapy have an excellent outcome when treated with chemotherapy. Patients who relapse after an effective chemotherapy regimen are usually not curable with subsequent chemotherapy administered at standard doses. However, patients with a long initial remission can be an exception to this rule. Autologous bone marrow transplantation can cure half of patients in whom effective chemotherapy regimens fail to induce durable remissions. The immunotoxin, brentuximab vedotin, a CD30-directed chemotherapy that selectively targets cells expressing CD30, is active in the salvage setting and is being integrated into ABVD for initial 709 treatment.

1	Because of the very high cure rate in patients with Hodgkin’s lymphoma, long-term complications have become a major focus for clinical research. In fact, in some series of patients with early-stage disease, more patients died from late complications of therapy than from Hodgkin’s lymphoma itself. This is particularly true in patients with localized disease. The most serious late side effects include second malignancies and cardiac injury. Patients are at risk for the development of acute leukemia in the first 10 years after treatment with combination chemotherapy regimens that contain alkylating agents plus radiation therapy. The risk for development of acute leukemia appears to be greater after MOPP-like (mechlorethamine, vincristine, procarbazine, prednisone) regimens than with ABVD. The risk of development of acute leukemia after treatment for Hodgkin’s lymphoma is also related to the number of exposures to potentially leukemogenic agents (i.e., multiple treatments after relapse)

1	risk of development of acute leukemia after treatment for Hodgkin’s lymphoma is also related to the number of exposures to potentially leukemogenic agents (i.e., multiple treatments after relapse) and the age of the patient being treated, with those age >60 years at par ticularly high risk. The development of carcinomas as a complication of treatment for Hodgkin’s lymphoma has become a major problem. These tumors usually occur ≥10 years after treatment and are associated with use of radiotherapy. For this reason, young women treated with thoracic radiotherapy for Hodgkin’s lymphoma should institute screening mammograms 5–10 years after treatment, and all patients who receive thoracic radiotherapy for Hodgkin’s lymphoma should be discouraged from smoking. Thoracic radiation also accelerates coronary artery disease, and patients should be encouraged to minimize risk factors for coronary artery disease such as smoking and elevated cholesterol levels. Cervical radiation therapy increases

1	coronary artery disease, and patients should be encouraged to minimize risk factors for coronary artery disease such as smoking and elevated cholesterol levels. Cervical radiation therapy increases the risk of carotid atherosclerosis and stroke.

1	A number of other late side effects from the treatment of Hodgkin’s lymphoma are well known. Patients who receive thoracic radiotherapy are at very high risk for the eventual development of hypothyroidism and should be observed for this complication; intermittent measurement of thyrotropin should be made to identify the condition before it becomes symptomatic. Lhermitte’s syndrome occurs in ~15% of patients who receive thoracic radiotherapy. This syndrome is manifested by an “electric shock” sensation into the lower extremities on flexion of the neck. Infertility is a concern for all patients undergoing treatment for Hodgkin’s lymphoma. In both women and men, the risk of permanent infertility is age-related, with younger patients more likely to recover fertility. In addition, treatment with ABVD increases the chances to retain fertility.

1	Nodular Lymphocyte-Predominant Hodgkin’s Lymphoma Nodular lymphocyte-predominant Hodgkin’s lymphoma is now recognized as an entity distinct from classical Hodgkin’s lymphoma. Previous classification systems recognized that biopsies from a subset of patients diagnosed as having Hodgkin’s lymphoma contained a predominance of small lymphocytes and rare Reed-Sternberg cells (Fig. 134-11). A subset of these patients have tumors with nodular growth pattern and a clinical course that varied from that of patients with classical Hodgkin’s lymphoma. This is an unusual clinical entity and represents <5% of cases of Hodgkin’s lymphoma.

1	Nodular lymphocyte-predominant Hodgkin’s lymphoma has a number of characteristics that suggest its relationship to nonHodgkin’s lymphoma. These include a clonal proliferation of B cells and a distinctive immunophenotype; tumor cells express J chain and display CD45 and epithelial membrane antigen (EMA) and do not express two markers normally found on Reed-Sternberg cells, CD30 and CD15. This lymphoma tends to have a chronic, relapsing course and sometimes transforms to diffuse large B-cell lymphoma. The treatment of patients with nodular lymphocyte-predominant Hodgkin’s lymphoma is controversial. Some clinicians favor no treatment and merely close follow-up. In the United States, most physicians will treat localized disease with radiotherapy and disseminated disease with regimens used for patients with classical Hodgkin’s lymphoma. Regardless of the therapy used, most series report a long-term survival of >80%. Malignancies of Lymphoid Cells

1	Malignancies of Lymphoid Cells The most common condition that pathologists and clinicians might confuse with lymphoma is reactive, atypical lymphoid hyperplasia. Patients might have localized or disseminated lymphadenopathy and might have the systemic symptoms characteristic of lymphoma. Underlying causes include a drug reaction to phenytoin or carbamazepine. Immune disorders such as rheumatoid arthritis and lupus erythematosus, viral infections such as cytomegalovirus and EBV, and bacterial infections such as cat-scratch disease may cause adenopathy (Chap. 79). In the absence of a definitive diagnosis after initial biopsy, continued follow-up, further testing, and repeated biopsies, if necessary, constitute the appropriate approach, rather than instituting therapy.

1	Specific conditions that can be confused with lymphoma include Castleman’s disease, which can present with localized or disseminated lymphadenopathy; some patients have systemic symptoms. The disseminated form is often accompanied by anemia and polyclonal hypergammaglobulinemia, and the condition has been associated with overproduction of interleukin 6 (IL-6), in some cases produced by human herpesvirus 8 infection. Patients with localized disease can be treated effectively with local therapy, whereas the initial treatment for patients with disseminated disease is usually with systemic glucocorticoids. IL-6-directed therapy (tocilizumab) has produced short-term responses. Rituximab appears to produce longer remissions than tocilizumab.

1	Sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease) usually presents with bulky lymphadenopathy in children or young adults. The disease is usually nonprogressive and self-limited, but patients can manifest autoimmune hemolytic anemia. Lymphomatoid papulosis is a cutaneous lymphoproliferative disorder that is often confused with anaplastic large cell lymphoma involving the skin. The cells of lymphomatoid papulosis are similar to those seen in lymphoma and stain for CD30, and T-cell receptor gene rearrangements are sometimes seen. However, the condition is characterized by waxing and waning skin lesions that usually heal, leaving small scars. In the absence of effective communication between the clinician and the pathologist regarding the clinical course in the patient, this disease will be misdiagnosed. Since the clinical picture is usually benign, misdiagnosis is a serious mistake.

1	James Armitage was a coauthor of this chapter in prior editions, and substantial material from those editions has been included here. Less Common hematologic Malignancies Ayalew Tefferi, Dan L. Longo The most common lymphoid malignancies are discussed in Chap. 134, myeloid leukemias in Chaps. 132 and 133, myelodysplastic syndromes 135e in Chap. 130, and myeloproliferative syndromes in Chap. 131. This chapter will focus on the more unusual forms of hematologic malignancy. The diseases discussed here are listed in Table 135e-1. Each of these entities accounts for less than 1% of hematologic neoplasms. Precursor B-cell and precursor T-cell neoplasms are discussed in Chap. 134. All the lymphoid tumors discussed here are mature B cell or T cell, natural killer (NK) cell neoplasms.

1	Precursor B-cell and precursor T-cell neoplasms are discussed in Chap. 134. All the lymphoid tumors discussed here are mature B cell or T cell, natural killer (NK) cell neoplasms. MATURE B-CELL NEOPLASMS B-Cell Prolymphocytic Leukemia (B-PLL) This is a malignancy of medium-sized (about twice the size of a normal small lymphocyte), round lymphocytes with a prominent nucleolus and light blue cytoplasm on Wright’s stain. It dominantly affects the blood, bone marrow, and spleen and usually does not cause adenopathy. The median age of

1	Mature T-cell and natural killer (NK) cell neoplasms T-cell prolymphocytic leukemia T-cell large granular lymphocytic leukemia Aggressive NK cell leukemia Extranodal NK/T-cell lymphoma, nasal type Enteropathy-type T-cell lymphoma Hepatosplenic T-cell lymphoma Subcutaneous panniculitis-like T-cell lymphoma Blastic NK cell lymphoma Primary cutaneous CD30+ T-cell lymphoma Angioimmunoblastic T-cell lymphoma affected patients is 70 years, and men are more often affected than 135e-1 women (male-to-female ratio is 1.6). This entity is distinct from chronic lymphoid leukemia (CLL) and does not develop as a consequence of that disease.

1	Clinical presentation is generally from symptoms of splenomegaly or incidental detection of an elevated white blood cell (WBC) count. The clinical course can be rapid. The cells express surface IgM (with or without IgD) and typical B-cell markers (CD19, CD20, CD22). CD23 is absent, and about one-third of cases express CD5. The CD5 expression along with the presence of the t(11;14) translocation in 20% of cases leads to confusion in distinguishing B-PLL from the leukemic form of mantle cell lymphoma. No reliable criteria for the distinction have emerged. About half of patients have mutation or loss of p53, and deletions have been noted in 11q23 and 13q14. Nucleoside analogues like fludarabine and cladribine and combination chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP]) have produced responses. CHOP plus rituximab may be more effective than CHOP alone, but the disease is sufficiently rare that large series have not been reported. Splenectomy can produce

1	[CHOP]) have produced responses. CHOP plus rituximab may be more effective than CHOP alone, but the disease is sufficiently rare that large series have not been reported. Splenectomy can produce palliation of symptoms but appears to have little or no impact on the course of the disease.

1	Splenic Marginal Zone Lymphoma (SMZL) This tumor of mainly small lymphocytes originates in the marginal zone of the spleen white pulp, grows to efface the germinal centers and mantle, and invades the red pulp. Splenic hilar nodes, bone marrow, and peripheral blood may be involved. The circulating tumor cells have short surface villi and are called villous lymphocytes. Table 135e-2 shows differences in tumor cells of a number of neoplasms of small lymphocytes that aid in the differential diagnosis. SMZL cells express surface immunoglobulin and CD20, but are negative for CD5, CD10, CD43, and CD103. Lack of CD5 distinguishes SMZL from CLL, and lack of CD103 separates SMZL from hairy cell leukemia.

1	The median age of patients with SMZL is mid-fifties, and men and women are equally represented. Patients present with incidental or symptomatic splenomegaly or incidental detection of lymphocytosis in the peripheral blood with villous lymphocytes. Autoimmune anemia or thrombocytopenia may be present. The immunoglobulin produced by these cells contains somatic mutations that reflect transit through a germinal center, and ongoing mutations suggest that the mutation machinery has remained active. About 40% of patients have either deletions or translocations involving 7q21, the site of the FLNC gene (filamin Cγ, involved in cross-linking actin filaments in the cytoplasm). NOTCH2 mutations are seen in 25% of patients. Chromosome 8p deletions may also be noted. The genetic lesions typically found in extranodal marginal zone lymphomas [e.g., trisomy 3 and t(11;18)] are uncommon in SMZL.

1	The clinical course of disease is generally indolent with median survivals exceeding 10 years. Patients with elevated lactate dehydrogenase (LDH) levels, anemia, and hypoalbuminemia generally have a poorer prognosis. Long remissions can be seen after splenectomy. Rituximab is also active. A small fraction of patients undergo histologic progression to diffuse large B-cell lymphoma with a concomitant change to a more aggressive natural history. Experience with combination chemotherapy in SMZL is limited.

1	Hairy Cell Leukemia Hairy cell leukemia is a tumor of small lymphocytes with oval nuclei, abundant cytoplasm, and distinctive membrane projections (hairy cells). Patients have splenomegaly and diffuse bone marrow involvement. While some circulating cells are noted, the clinical picture is dominated by symptoms from the enlarged spleen and pancytopenia. The mechanism of the pancytopenia is not completely clear and may be mediated by both inhibitory cytokines and marrow replacement. The marrow has an increased level of reticulin fibers; indeed, hairy cell leukemia is a common cause of inability to aspirate bone marrow or so-called “dry tap” (Table 135e-3). Monocytopenia is profound and may explain a predisposition to atypical mycobacterial infection that is observed clinically. The tumor cells have strong expression of CD22, CD25, and CD103; soluble CD25 level in serum is an excellent tumor marker for disease activity. The cells also express tartrate-resistant acid phosphatase. The

1	cells have strong expression of CD22, CD25, and CD103; soluble CD25 level in serum is an excellent tumor marker for disease activity. The cells also express tartrate-resistant acid phosphatase. The immunoglobulin genes are

1	Abbreviations: neg, negative; pos, positive. rearranged and mutated, indicating the influence of a germinal center. No specific cytogenetic abnormality has been found, but most cases contain the activating BRAF mutation V600E. The median age of affected patients is mid-fifties, and the maleto-female ratio is 5:1. Treatment options are numerous. Splenectomy is often associated with prolonged remission. Nucleosides including cladribine and deoxycoformycin are highly active but are also associated with further immunosuppression and can increase the risk of certain opportunistic infections. However, after brief courses of these agents, patients usually obtain very durable remissions during which immune function spontaneously recovers. Interferon α is also an effective therapy but is not as effective as nucleosides. Chemotherapy-refractory patients have responded to vemurafenib, a BRAF inhibitor.

1	Nodal Marginal Zone B-Cell Lymphoma This rare node-based disease bears an uncertain relationship with extranodal marginal zone lymphomas, which are often mucosa-associated and are called mucosa-associated lymphoid tissue (MALT) lymphomas, and SMZLs. Patients may have localized or generalized adenopathy. The neoplastic cell is a marginal zone B cell with monocytoid features and has been called monocytoid B-cell lymphoma in the past. Up to one-third of the patients may have extranodal involvement, and involvement of the lymph nodes can be secondary to the spread of a mucosal primary lesion. In authentic nodal primaries, the cytogenetic abnormalities associated with MALT lymphomas [trisomy 3 and t(11;18)] are very rare. The clinical course is indolent. Patients often respond to combination chemotherapy, although remissions have not been durable. Few patients have received CHOP plus rituximab, which is likely to be an effective approach to management.

1	Mediastinal (Thymic) Large B-Cell Lymphoma This entity was originally considered a subset of diffuse large B-cell lymphoma; however, additional study has identified it as a distinct entity with its own characteristic clinical, genetic, and immunophenotypic features. This is a disease that can be bulky in size but usually remains confined to the mediastinum. It can be locally aggressive, including progressing to produce a superior vena cava obstruction syndrome or pericardial effusion. About one-third of patients develop pleural effusions, and 5–10% can disseminate widely to kidney, adrenal, liver, skin, and even brain. The disease affects women more often than men (male-to-female ratio is 1:2–3), and the median age is 35–40 years.

1	The tumor is composed of sheets of large cells with abundant cytoplasm accompanied by variable, but often abundant, fibrosis. It is distinguished from nodular sclerosing Hodgkin’s disease by the paucity of normal lymphoid cells and the absence of lacunar variants differentiaL diagnosis of “dry tap”—inaBiLity to aspirate Bone Marrow

1	Dry taps occur in about 4% of attempts and are associated with: of Reed-Sternberg cells. However, more than one-third of the genes that are expressed to a greater extent in primary mediastinal large B-cell lymphoma than in usual diffuse large B-cell lymphoma are also overexpressed in Hodgkin’s disease, suggesting a possible pathogenetic relationship between the two entities that affect the same anatomic site. Tumor cells may overexpress MAL. The genome of tumor cells is characterized by frequent chromosomal gains and losses. The tumor cells in mediastinal large B-cell lymphoma express CD20, but surface immunoglobulin and HLA class I and class II molecules may be absent or incompletely expressed. Expression of lower levels of class II HLA identifies a subset with poorer prognosis. The cells are CD5 and CD10 negative but may show light staining with anti-CD30. The cells are CD45 positive, unlike cells of classical Hodgkin’s disease.

1	Methotrexate, leucovorin, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin (MACOP-B) and rituximab plus CHOP are effective treatments, achieving 5-year survival of 75–87%. Dose-adjusted therapy with prednisone, etoposide, vincristine, cyclophosphamide, and doxorubicin (EPOCH) plus rituximab has produced 5-year survival of 97%. A role for mediastinal radiation therapy has not been definitively demonstrated, but it is frequently used, especially in patients whose mediastinal area remains positron emission tomography–avid after four to six cycles of chemotherapy.

1	Intravascular Large B-Cell Lymphoma This is an extremely rare form of diffuse large B-cell lymphoma characterized by the presence of lymphoma in the lumen of small vessels, particularly capillaries. It is also known as malignant angioendotheliomatosis or angiotropic large cell lymphoma. It is sufficiently rare that no consistent picture has emerged to define a clinical syndrome or its epidemiologic and genetic features. It is thought to remain inside vessels because of a defect in adhesion molecules and homing mechanisms, an idea supported by scant data suggesting absence of expression of β-1 integrin and ICAM-1. Patients commonly present with symptoms of small-vessel occlusion, skin lesions, or neurologic symptoms. The tumor cell clusters can promote thrombus formation. In general, the clinical course is aggressive and the disease is poorly responsive to therapy. Often a diagnosis is not made until very late in the course of the disease.

1	Primary Effusion Lymphoma This entity is another variant of diffuse large B-cell lymphoma that presents with pleural effusions, usually without apparent tumor mass lesions. It is most common in the setting of immune deficiency disease, especially AIDS, and is caused by human herpes virus 8 (HHV-8)/Kaposi’s sarcoma herpes virus (KSHV). It is also known as body cavity–based lymphoma. Some patients have been previously diagnosed with Kaposi’s sarcoma. It can also occur in the absence of immunodeficiency in elderly men of Mediterranean heritage, similar to Kaposi’s sarcoma but even less common.

1	The malignant effusions contain cells positive for HHV-8/KSHV, and many are also co-infected with Epstein-Barr virus. The cells are large with large nuclei and prominent nucleoli that can be confused with Reed-Sternberg cells. The cells express CD20 and CD79a (immunoglobulin-signaling molecule), although they often do not express immunoglobulin. Some cases aberrantly express T-cell markers such as CD3 or rearranged T-cell receptor genes. No characteristic genetic lesions have been reported, but gains in chromosome 12 and X material has been seen, similar to other HIV-associated lymphomas. The clinical course is generally characterized by rapid progression and death within 6 months.

1	Lymphomatoid Granulomatosis This is an angiocentric, angiodestructive lymphoproliferative disease comprised by neoplastic Epstein-Barr virus–infected monoclonal B cells accompanied and outnumbered by a polyclonal reactive T-cell infiltrate. The disease is graded based on histologic features such as cell number and atypia in the B cells. It is most often confused with extranodal NK–T cell lymphoma, nasal type, which can also be angiodestructive and is Epstein-Barr virus–related. The disease usually presents in adults (males > females) as a pulmonary infiltrate. Involvement is often entirely extranodal and can include kidney (32%), liver (29%), skin (25%), and brain (25%). The disease often but not always occurs in the setting of immune deficiency.

1	The disease can be remitting and relapsing in nature or can be rapidly progressive. The course is usually predicted by the histologic grade. The disease is highly responsive to combination chemotherapy and is curable in most cases. Some investigators have claimed that low-grade disease (grade I and II) can be treated with interferon α.

1	MATURE T-CELL AND NK CELL NEOPLASMS T-Cell Prolymphocytic Leukemia This is an aggressive leukemia of medium-sized prolymphocytes involving the blood, marrow, nodes, liver, spleen, and skin. It accounts for 1–2% of all small lymphocytic leukemias. Most patients present with elevated WBC count (often >100,000/μL), hepatosplenomegaly, and adenopathy. Skin involvement occurs in 20%. The diagnosis is made from peripheral blood smear, which shows cells about 25% larger than those in small lymphocytes, with cytoplasmic blebs and nuclei that may be indented. The cells express T-cell markers like CD2, CD3, and CD7; two-thirds of patients have cells that are CD4+ and CD8–, and 25% have cells that are CD4+ and CD8+. T-cell receptor β chains are clonally rearranged. In 80% of patients, inversion of chromosome 14 occurs between q11 and q32. Ten percent have t(14;14) translocations that bring the T-cell receptor alpha/beta gene locus into juxtaposition with oncogenes TCL1 and TCL1b at 14q32.1.

1	of chromosome 14 occurs between q11 and q32. Ten percent have t(14;14) translocations that bring the T-cell receptor alpha/beta gene locus into juxtaposition with oncogenes TCL1 and TCL1b at 14q32.1. Chromosome 8 abnormalities are also common. Deletions in the ATM gene are also noted. Activating JAK3 mutations have also been reported.

1	The course of the disease is generally rapid, with median survival of about 12 months. Responses have been seen with the anti-CD52 antibody, nucleoside analogs, and CHOP chemotherapy. Small numbers of patients with T-cell prolymphocytic leukemia have also been treated with high-dose therapy and allogeneic bone marrow transplantation after remission has been achieved with conventional-dose therapy.

1	T-Cell Large Granular Lymphocytic Leukemia T-cell large granular lymphocytic leukemia (LGL leukemia) is characterized by increases in the number of LGLs in the peripheral blood (2000–20,000/μL) often accompanied by severe neutropenia, with or without concomitant anemia. Patients may have splenomegaly and frequently have evidence of systemic autoimmune disease, including rheumatoid arthritis, hypergammaglobulinemia, autoantibodies, and circulating immune complexes. Bone marrow involvement is mainly interstitial in pattern, with fewer than 50% lymphocytes on differential count. Usually the cells express CD3, T-cell receptors, and CD8; NK-like variants may be CD3–. The leukemic cells often express Fas and Fas ligand.

1	The course of the disease is generally indolent and dominated by the neutropenia. Paradoxically, immunosuppressive therapy with cyclosporine, methotrexate, or cyclophosphamide plus glucocorticoids can produce an increase in granulocyte counts. Nucleosides have been used anecdotally. Occasionally the disease can accelerate to a more aggressive clinical course.

1	Aggressive NK Cell Leukemia NK neoplasms are very rare, and they may follow a range of clinical courses from very indolent to highly aggressive. They are more common in Asians than whites, and the cells frequently harbor a clonal Epstein-Barr virus episome. The peripheral blood white count is usually not greatly elevated, but abnormal large lymphoid cells with granular cytoplasm are noted. The aggressive form is characterized by symptoms of fever and laboratory abnormalities of pancytopenia. Hepatosplenomegaly is common; node involvement is less common. Patients may have hemophagocytosis, coagulopathy, or multiorgan failure. Serum levels of Fas ligand are elevated.

1	The cells express CD2 and CD56 and do not have rearranged T-cell 135e-3 receptor genes. Deletions involving chromosome 6 are common. The disease can be rapidly progressive. Some forms of NK neoplasms are more indolent. They tend to be discovered incidentally with LGL lymphocytosis and do not manifest the fever and hepatosplenomegaly characteristic of the aggressive leukemia. The cells are also CD2 and CD56 positive, but they do not contain clonal forms of Epstein-Barr virus and are not accompanied by pancytopenia or autoimmune disease.

1	Extranodal NK/T-Cell Lymphoma, Nasal Type Like lymphomatoid granulomatosis, extranodal NK/T-cell lymphoma tends to be an angiocentric and angiodestructive lesion, but the malignant cells are not B cells. In most cases, they are CD56+ Epstein-Barr virus–infected cells; occasionally they are CD56– Epstein-Barr virus–infected cytotoxic T cells. They are most commonly found in the nasal cavity. Historically, this illness was called lethal midline granuloma, polymorphic reticulosis, and angiocentric immunoproliferative lesion. This form of lymphoma is prevalent in Asia, Mexico, and Central and South America; it affects males more commonly than females. When it spreads beyond the nasal cavity, it may affect soft tissue, the gastrointestinal tract, or the testis. In some cases, hemophagocytic syndrome may influence the clinical picture. Patients may have B symptoms. Many of the systemic manifestations of disease are related to the production of cytokines by the tumor cells and the cells

1	syndrome may influence the clinical picture. Patients may have B symptoms. Many of the systemic manifestations of disease are related to the production of cytokines by the tumor cells and the cells responding to their signals. Deletions and inversions of chromosome 6 are common.

1	Many patients with extranodal NK/T-cell lymphoma, nasal type have excellent antitumor responses with combination chemotherapy regimens, particularly those with localized disease. Radiation therapy is often used after completion of chemotherapy. Four risk factors have been defined, including B symptoms, advanced stage, elevated LDH, and regional lymph node involvement. Patient survival is linked to the number of risk factors: 5-year survival is 81% for zero risk factors, 64% for one risk factor, 32% for two risk factors, and 7% for three or four risk factors. Combination regimens without anthracyclines have been touted as superior to CHOP, but data are sparse. High-dose therapy with stem cell transplantation has been used, but its role is unclear.

1	Enteropathy-Type T-Cell Lymphoma Enteropathy-type T-cell lymphoma is a rare complication of longstanding celiac disease. It most commonly occurs in the jejunum or the ileum. In adults, the lymphoma may be diagnosed at the same time as celiac disease, but the suspicion is that the celiac disease was a longstanding precursor to the development of lymphoma. The tumor usually presents as multiple ulcerating mucosal masses, but may also produce a dominant exophytic mass or multiple ulcerations. The tumor expresses CD3 and CD7 nearly always and may or may not express CD8. The normal-appearing lymphocytes in the adjacent mucosa often have a similar phenotype to the tumor. Most patients have the HLA genotype associated with celiac disease, HLA DQA1*0501 or DQB1*0201.

1	The prognosis of this form of lymphoma is typically (median survival is 7 months) poor, but some patients have a good response to CHOP chemotherapy. Patients who respond can develop bowel perforation from responding tumor. If the tumor responds to treatment, recurrence may develop elsewhere in the celiac disease–affected small bowel.

1	Hepatosplenic T-Cell Lymphoma Hepatosplenic T-cell lymphoma is a malignancy derived from T cells expressing the gamma/delta T-cell antigen receptor that affects mainly the liver and fills the sinusoids with medium-size lymphoid cells. When the spleen is involved, dominantly the red pulp is infiltrated. It is a disease of young people, especially young people with an underlying immunodeficiency or with an autoimmune disease that demands immunosuppressive therapy. The use of thiopurine and infliximab is particularly common in the history of patients with this disease. The cells are CD3+ and usually CD4– and CD8–. The cells may contain isochromosome 7q, often together with trisomy 8. The lymphoma has an aggressive natural history. Combination chemotherapy may induce remissions, but most patients relapse. Median survival is about 2 years. The tumor does not appear to respond to reversal of immunosuppressive therapy.

1	Subcutaneous Panniculitis-Like T-Cell Lymphoma Subcutaneous panniculitis-like T-cell lymphoma involves multiple subcutaneous collections of neoplastic T cells that are usually cytotoxic cells in phenotype (i.e., contain perforin and granzyme B and express CD3 and CD8). The rearranged T-cell receptor is usually alpha/beta-derived, but occasionally the gamma/delta receptors are involved, particularly in the setting of immunosuppression. The cells are negative for Epstein-Barr virus. Patients may have a hemophagocytic syndrome in addition to the skin infiltration; fever and hepatosplenomegaly may also be present. Nodes are generally not involved. Patients frequently respond to combination chemotherapy, including CHOP. When the disease is progressive, the hemophagocytic syndrome can be a component of a fulminant downhill course. Effective therapy can reverse the hemophagocytic syndrome.

1	Blastic NK Cell Lymphoma The neoplastic cells express NK cell markers, especially CD56, and are CD3 negative. They are large blasticappearing cells and may produce a leukemia picture, but the dominant site of involvement is the skin. Morphologically, the cells are similar to the neoplastic cells in acute lymphoid and myeloid leukemia. No characteristic chromosomal abnormalities have been described. The clinical course is rapid, and the disease is largely unresponsive to typical lymphoma treatments.

1	Primary Cutaneous CD30+ T-Cell Lymphoma This tumor involves the skin and is composed of cells that appear similar to the cells of anaplastic T-cell lymphoma. Among cutaneous T-cell tumors, about 25% are CD30+ anaplastic lymphomas. If dissemination to lymph nodes occurs, it is difficult to distinguish between the cutaneous and systemic forms of the disease. The tumor cells are often CD4+, and the cells contain granules that are positive for granzyme B and perforin in 70% of cases. The typical t(2;5) of anaplastic T-cell lymphoma is absent; indeed, its presence should prompt a closer look for systemic involvement and a switch to a diagnosis of anaplastic T-cell lymphoma. This form of lymphoma has sporadically been noted as a rare complication of silicone on saline breast implants. Cutaneous CD30+ T-cell lymphoma often responds to therapy. Radiation therapy can be effective, and surgery can also produce long-term disease control. Five-year survival exceeds 90%.

1	Angioimmunoblastic T-Cell Lymphoma Angioimmunoblastic T-cell lymphoma is a systemic disease that accounts for about 15% of all T-cell lymphomas. Patients frequently have fever, advanced stage, diffuse adenopathy, hepatosplenomegaly, skin rash, polyclonal hypergammaglobulinemia, and a wide range of autoantibodies including cold agglutinins, rheumatoid factor, and circulating immune complexes. Patients may have edema, arthritis, pleural effusions, and ascites. The nodes contain a polymorphous infiltrate of neoplastic T cells and nonneoplastic inflammatory cells together with proliferation of high endothelial venules and follicular dendritic cells. The most common chromosomal abnormalities are trisomy 3, trisomy 5, and an extra X chromosome. Aggressive combination chemotherapy can induce regressions. The underlying immune defects make conventional lymphoma treatments more likely to produce infectious complications.

1	The World Health Organization (WHO) system uses peripheral blood counts and smear analysis, bone marrow morphology, and cytogenetic and molecular genetic tests in order to classify myeloid malignancies into five major categories (Table 135e-4). In this chapter, we focus on chronic neutrophilic leukemia; atypical chronic myeloid leukemia, BCR-ABL1 negative; chronic myelomonocytic leukemia; juvenile myelomonocytic leukemia; chronic eosinophilic leukemia, not otherwise specified; mastocytosis; myeloproliferative neoplasm (MPN), unclassifiable (MPN-U); myelodysplastic syndrome (MDS)/ MPN, unclassifiable (MDS/MPN-U); refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T); and myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1. This chapter also includes histiocytic and dendritic cell neoplasms, transient myeloproliferative disorders, worLd heaLth organization CLassifiCation of MyeLoid MaLignanCies 1. 2.

1	2. 2.1. Chronic myelogenous leukemia, BCR-ABL1 positive (CML) 2.2. BCR-ABL1-negative MPN 2.2.1. Polycythemia vera 2.2.2. Primary myelofibrosis 2.2.3. Essential thrombocythemia 2.3. Chronic neutrophilic leukemia 2.4. Chronic eosinophilic leukemia, not otherwise specified (CEL-NOS) 2.5. Mastocytosis 2.6. Myeloproliferative neoplasm, unclassifiable (MPN-U) 3.

1	3.1. Refractory cytopeniab with unilineage dysplasia (RCUD) 3.1.1. Refractory anemia (ring sideroblasts <15% of erythroid precursors) 3.1.2. Refractory neutropenia 3.1.3. Refractory thrombocytopenia 3.2. Refractory anemia with ring sideroblasts (RARS; dysplasia limited to erythroid lineage and ring sideroblasts ≥15% of bone marrow erythroid precursors) 3.3. Refractory cytopenia with multilineage dysplasia (RCMD; ring sideroblast count does not matter) 3.4. Refractory anemia with excess blasts (RAEB) 3.4.1. RAEB-1 (2–4% circulating or 5–9% marrow blasts) 3.4.2. RAEB-2 (5–19% circulating or 10–19% marrow blasts or Auer rods present) 3.5. MDS associated with isolated del(5q) 3.6. MDS, unclassifiable (MDS-U) 4. MDS/MPN overlap 4.1. Chronic myelomonocytic leukemia (CMML) 4.2. Atypical chronic myeloid leukemia, BCR-ABL1 negative (aCML) 4.3. Juvenile myelomonocytic leukemia (JMML) 4.4. MDS/MPN, unclassifiable (MDS/MPN-U) 4.4.1. Provisional entity: Refractory anemia with ring sideroblasts

1	myeloid leukemia, BCR-ABL1 negative (aCML) 4.3. Juvenile myelomonocytic leukemia (JMML) 4.4. MDS/MPN, unclassifiable (MDS/MPN-U) 4.4.1. Provisional entity: Refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T) 5. Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1c 5.1. Myeloid and lymphoid neoplasms with PDGFRA rearrangement 5.2. Myeloid neoplasms with PDGFRB rearrangement 5.3. Myeloid and lymphoid neoplasms with FGFR1 abnormalities aAML-related precursor neoplasms include therapy-related MDS and myeloid sarcoma. bEither monocytopenia or bicytopenia: hemoglobin level <10 g/dL, absolute neutrophil count <1.8 × 109/L, or platelet count <100 × 109/L. However, higher blood counts do not exclude the diagnosis in the presence of unequivocal histologic/cytogenetic evidence for MDS. cGenetic rearrangements involving platelet-derived growth factor receptor α/β (PDGFRA/PDGFRB) or fibroblast growth factor receptor 1

1	presence of unequivocal histologic/cytogenetic evidence for MDS. cGenetic rearrangements involving platelet-derived growth factor receptor α/β (PDGFRA/PDGFRB) or fibroblast growth factor receptor 1 (FGFR1).

1	and a broader discussion on primary eosinophilic disorders including hypereosinophilic syndrome (HES). Chronic neutrophilic leukemia (CNL) is characterized by mature neutrophilic leukocytosis with few or no circulating immature granulocytes. CNL is associated with activating mutations of the gene (CSF3R) encoding for the receptor for granulocyte colony-stimulating factor (G-CSF), also known as colony-stimulating factor 3 (CSF3). Patients with CNL might be asymptomatic at presentation but also display constitutional symptoms, splenomegaly, anemia, and thrombocytopenia. Median survival is approximately 2 years, and causes of death include leukemic transformation, progressive disease associated with severe cytopenias, and marked treatment-refractory leukocytosis. CNL is rare, with less than 200 reported cases. Median age at diagnosis is approximately 67 years, and the disease is equally prevalent in both genders.

1	Pathogenesis CSF3 is the main growth factor for granulocyte proliferation and differentiation. Accordingly, recombinant CSF3 is used for the treatment of severe neutropenia, including severe congenital neutropenia (SCN). Some patients with SCN acquire CSF3R mutations, and the frequency of such mutations is significantly higher (~80%) in patients who experience leukemic transformation. SCN-associated CSF3R mutations occur in the region of the gene coding for the cytoplasmic domain of CSF3R and result in truncation of the C-terminal-negative regulatory domain. A different class of CSF3R mutations is noted in ~90% of patients with CNL; these are mostly membrane proximal, with the most frequent being a C-to-T substitution at nucleotide 1853 (T618I). About 40% of the T618I-mutated cases also harbored SETBP1 mutations. CSF3R T618I induces a lethal myeloproliferative disorder in a mouse model and is associated with in vitro sensitivity to JAK inhibition.

1	Diagnosis Diagnosis of CNL requires exclusion of the more common causes of neutrophilia including infections and inflammatory processes. In addition, one should be mindful of the association between some forms of metastatic cancer or plasma cell neoplasms with secondary neutrophilia. Neoplastic neutrophilia also occurs in other myeloid malignancies including atypical chronic myeloid leukemia and chronic myelomonocytic leukemia. Accordingly, the WHO diagnostic criteria for CNL are designed to exclude the possibilities of both secondary/reactive neutrophilia and leukocytosis associated with myeloid malignancies other than CNL (Table 135e-5): leukocytosis (≥25 × 109/L), >80% segmented/band neutrophils, <10% immature myeloid cells, <1% circulating blasts, and absence of dysgranulopoiesis or monocytosis. Bone marrow in CNL is hypercellular and displays increased number and percentage of neutrophils with a very high myeloid-to-erythroid ratio and minimal left shift, myeloid dysplasia, or

1	monocytosis. Bone marrow in CNL is hypercellular and displays increased number and percentage of neutrophils with a very high myeloid-to-erythroid ratio and minimal left shift, myeloid dysplasia, or reticulin fibrosis.

1	Treatment Current treatment in CNL is largely palliative and suboptimal in its efficacy. Several drugs alone or in combination have been tried, and none have shown remarkable efficacy. As such, allogeneic stem cell transplantation (ASCT) is reasonable to consider in the presence of symptomatic disease, especially in younger patients. Otherwise, cytoreductive therapy with hydroxyurea is probably as good as any treatment, and a more intensive combination chemother-135e-5 apy may not have additional value. However, response to hydroxyurea therapy is often transient, and some have successfully used interferon α as an alternative drug. Response to treatment with ruxolitinib (a JAK1 and JAK2 inhibitor) has been reported but has not been confirmed.

1	Atypical chronic myeloid leukemia, BCR-ABL1 negative (aCML) is formally classified under the MDS/MPN category of myeloid malignancies and is characterized by left shifted granulocytosis and dysgranulopoiesis. The differential diagnosis of aCML includes chronic myeloid leukemia (CML), which is distinguished by the presence of BCR-ABL1; CNL, which is distinguished by the absence of dysgranulopoiesis and presence of CSF3R mutations; and chronic myelomonocytic leukemia, which is distinguished by the presence of monocytosis (absolute monocyte count >1 × 109/L). The WHO diagnostic criteria for aCML are listed in Table 135e-5 and include granulocytosis (WBC ≥13 × 109/L), neutrophilia with dysgranulopoiesis, ≥10% immature granulocytes, <20% peripheral blood myeloblasts, <10% peripheral blood monocytes, <2% basophils, and absence of otherwise specific mutations such as BCR-ABL1. The bone marrow is hypercellular with granulocyte proliferation and dysplasia with or without erythroid or

1	monocytes, <2% basophils, and absence of otherwise specific mutations such as BCR-ABL1. The bone marrow is hypercellular with granulocyte proliferation and dysplasia with or without erythroid or megakaryocytic dysplasia.

1	The molecular pathogenesis of aCML is incompletely understood; about one-fourth of the patients express SETBP1 mutations, which are, however, also found in several other myeloid malignancies, including CNL and chronic myelomonocytic leukemia. SETBP1 mutations in aCML were prognostically detrimental and mostly located between codons 858 and 871; similar mutations are seen with Schinzel-Giedion syndrome (a congenital disease with severe developmental delay and various physical stigmata including midface retraction, large forehead, and macroglossia).

1	In a series of 55 patients with WHO-defined aCML, median age at diagnosis was 62 years with female preponderance (57%); splenomegaly was reported in 54% of the patients, red cell transfusion requirement in 65%, abnormal karyotype in 20% (20q– and trisomy 8 being the most frequent), and leukemic transformation in 40%. Median survival was 25 months. Outcome was worse in patients with marked worLd heaLth organization diagnostiC Criteria for ChroniC neUtrophiLiC LeUkeMia (CnL); atypiCaL ChroniC MyeLoid LeUkeMia, BCRABL1negative (aCML); and ChroniC MyeLoMonoCytiC LeUkeMia (CMML) <10% Bone marrow ↑Neutrophils, number and % ↑Granulocyte proliferation Dysplasia in ≥1 myeloid lineages <5% blasts Granulocytic dysplasia ± erythroid/ or megakaryocyte dysplasia

1	Megakaryocytes normal or left shifted BCR-ABL1 No No No PDGFRA, PDGFRB, or FGFR1 mutation No No No PB and BM blasts/promonocytes <20% <20% <20% Hepatosplenomegaly ± ± ± Evidence for other MDS/MPN No No No Evidence for other MPN No No No Evidence for reactive leukocytosisb or No No No monocytosis aImmature granulocytes include myeloblasts, promyelocytes, myelocytes, and metamyelocytes. bCauses of reactive neutrophilia include plasma cell neoplasms, solid tumor, infections, and inflammatory processes. Abbreviations: BM, bone marrow; MDS, myelodysplastic syndromes; MPN, myeloproliferative neoplasms; PB, peripheral blood. leukocytosis, transfusion requirement, and increased immature cells in the peripheral blood. Conventional chemotherapy is largely ineffective in the treatment of aCML. However, a favorable experience with ASCT was reported in nine patients; after a median follow-up of 55 months, the majority of the patients remained in complete remission.

1	Chronic myelomonocytic leukemia (CMML) is classified under the WHO category of MDS/MPN and is defined by an absolute monocyte count (AMC) of >1 × 109/L in the peripheral blood. Median age at diagnosis ranges between 65 and 75 years, and there is a 2:1 male predominance. Clinical presentation is variable and depends on whether the disease presents with MDS-like or MPN-like phenotype; the former is associated with cytopenias and the latter with splenomegaly and features of myeloproliferation such as fatigue, night sweats, weight loss, and cachexia. About 20% of patients with CMML experience serositis involving the joints (arthritis), pericardium (pericarditis and pericardial effusion), pleura (pleural effusion), or peritoneum (ascites).

1	Pathogenesis Clonal cytogenetic abnormalities are seen in about one-third of patients with CMML and include trisomy 8 and abnormalities of chromosome 7. Almost all patients with CMML harbor somatic mutations involving epigenetic regulator genes (e.g., ASXL1, TET2), spliceosome pathway genes (e.g., SRSF2), DNA damage response genes (e.g., TP53), and tyrosine kinases/transcription factors (e.g., KRAS, NRAS, CBL, and RUNX1). However, none of these mutations are specific to CMML, and their precise pathogenetic contribution is unclear.

1	Diagnosis Reactive monocytosis is uncommon but has been reported in association with certain infections and inflammatory conditions. Clonal (i.e., neoplastic) monocytosis defines CMML but is also seen with juvenile myelomonocytic leukemia and acute myeloid leukemia with monocytic differentiation. The WHO diagnostic criteria for CMML are listed in Table 135e-5 and include persistent AMC >1 × 109/L, absence of BCR-ABL1, absence of the PDGFRA or PDGFRB mutations, <20% blasts and promonocytes in the peripheral blood and bone marrow, and dysplasia involving one or more myeloid lineages.

1	The bone marrow in CMML is hypercellular with granulocytic and monocytic proliferation. Dysplasia is often present and may involve one, two, or all myeloid lineages. On immunophenotyping, the abnormal cells often express myelomonocytic antigens such as CD13 and CD33, with variable expression of CD14, CD68, CD64, and CD163. Monocytic-derived cells are almost always positive for the cytochemical nonspecific esterases (e.g., butyrate esterase), whereas normal granulocytic precursors are positive for lysozyme and chloroacetate esterase. In CMML, it is common to have a hybrid cytochemical staining pattern with cells expressing both chloroacetate and butyrate esterases simultaneously (dual esterase staining).

1	Prognosis A meta-analysis showed median survival of 1.5 years in CMML. Numerous prognostic systems have attempted to better define and stratify the natural history of CMML. One of these, the Mayo prognostic model, assigns one point each to the following four independent prognostic variables: AMC >10 × 109/L, presence of circulating immature cells, hemoglobin <10 g/dL, and platelet count <100,000/mL. This model stratified patients into three risk groups: low (0 points), intermediate (1 point), and high (≥2 points), translating to median survival times of 32, 18, and 10 months, respectively.

1	A French study incorporated ASXL1 mutational status in 312 CMML patients. In a multivariable model, independent predictors of poor survival were WBC >15 × 109/L (3 points), ASXL1 mutations (2 points), age >65 years (2 points), platelet count <100,000/mL (2 points), and hemoglobin <10 g/dL in females and <11 g/dL in males (2 points). This model stratified patients into three groups: low (0–4 points), intermediate (5–7 points), and high risk (8–12 points), with median survival times of not reached, 38.5 months, and 14.4 months, respectively.

1	Treatment Current treatment consists of hydroxyurea and supportive care, including red cell transfusions and use of erythropoiesisstimulating agents (ESAs). The value of hydroxyurea was reinforced by a randomized trial against oral etoposide. No other single or combination chemotherapy has been shown to be superior to hydroxyurea. ASCT is a viable treatment option for transplant-eligible patients with poor prognostic features. Given the MDS/MPN overlap phenotype and the presence of MDS-like genetic/methylation abnormalities in CMML, hypomethylating agents such as 5-azacitidine and decitabine have been used with limited efficacy.

1	Juvenile myelomonocytic leukemia (JMML) is primarily a disease of early childhood and is included, along with CMML, in the MDS/ MPN WHO category. Both CMML and JMML feature leukocytosis, monocytosis, and hepatosplenomegaly. Additional characteristic features in JMML include thrombocytopenia and elevated fetal hemoglobin. Myeloid progenitors in JMML display granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity that has been attributed to dysregulated RAS/MAPK signaling. The latter is believed to result from mutually exclusive mutations involving RAS, PTPN11, and NF1. A third of patients with JMML that is not associated with Noonan’s syndrome carry PTPN11 mutations, whereas the incidence of NF1 in patients without neurofibromatosis type 1 and RAS mutations is approximately 15% each. Drug therapy is relatively ineffective in JMML, and the treatment of choice is ASCT, which results in a 5-year survival of approximately 50%.

1	The WHO classifies patients with morphologic and laboratory features that resemble both MDS and MPN as MDS/MPN overlap. This category includes CMML, aCML, and JMML, which have been described above. In addition, MDS/MPN includes a fourth category referred to as MDS/MPN, unclassifiable (MDS/MPN-U). Diagnosis of MDS/ MPN-U requires the presence of both MDS and MPN features that are not adequate to classify patients as CMML, aCML, or JMML. MDS/ MPN includes the provisional category of RARS-T.

1	RARS-T is classified in the MDS/MPN category because it shares dysplastic features with RARS and myeloproliferative features with essential thrombocythemia (ET). In one study, 111 patients with RARS-T were compared with 33 patients with RARS. The frequency of SF3B1 mutations in RARS-T (87%) was similar to that in RARS (85%). JAK2 V617F mutation was detected in 49% of RARS-T patients (including 48% of those mutated for SF3B1) but none of those with RARS. In RARS-T, SF3B1 mutations were more frequent in females (95%) than in males (77%), and mean ring sideroblast counts were higher in SF3B1-mutated patients. Median overall survival was 6.9 years in SF3B1-mutated patients versus 3.3 years in unmutated patients. Six-year survival was 67% in JAK2-mutated patients versus 32% in unmutated patients. Multivariable analysis identified younger age and JAK2 and SF3B1 mutations as favorable factors.

1	In one series, 85 patients with non-RARS-T MDS/MPN, median age was 70 years, and 72% were males. Splenomegaly at presentation was present in 33%, thrombocytosis in 13%, leukocytosis in 18%, JAK2 mutations in 30%, and abnormal karyotype in 51%; the most frequent cytogenetic abnormality was trisomy 8. Median survival was 12.4 months and favorably affected by thrombocytosis. Treatment with hypomethylating agents, immunomodulators, or ASCT did not appear to favorably affect survival. MYELOPROLIFERATIVE NEOPLASM, UNCLASSIFIABLE (MPN-U)

1	MYELOPROLIFERATIVE NEOPLASM, UNCLASSIFIABLE (MPN-U) The category of MPN-U includes MPN-like neoplasms that cannot be clearly classified as one of the other seven subcategories of MPN (Table 135e-4). Examples include patients presenting with unusual thrombosis or unexplained organomegaly with normal blood counts but found to carry MPN-characteristic mutations such as JAK2 and CALR or display bone marrow morphology that is consistent with MPN. It is possible that some cases of MPN-U represent earlier disease stages in polycythemia vera (PV) or ET that fail to meet the threshold hemoglobin levels (18.5 g/dL in men or 16.5 g/dL in women) or platelet counts (450 × 109/L) that are required by the WHO diagnostic criteria. Specific treatment interventions might not be necessary in asymptomatic patients diagnosis of ChroniC eosinophiLiC LeUkeMia and hypereosinophiLiC syndroMe

1	Required: Persistent eosinophilia ≥1500/μL in blood, increased marrow eosinophils, and myeloblasts <20% in blood or marrow. 1. Exclude all causes of reactive eosinophilia: allergy, parasites, infection, pulmonary disease (e.g., hypersensitivity pneumonitis, Loeffler’s), and collagen vascular diseases 2. Exclude primary neoplasms associated with secondary eosinophilia: T-cell lymphomas, Hodgkin’s disease, acute lymphoid leukemia, mastocytosis 3. Exclude other primary myeloid neoplasms that may involve eosinophils: chronic myeloid leukemia, acute myeloid leukemia with inv(16) or t(16;16) (p13;q22), other myeloproliferative syndromes, and myelodysplasia 4. Exclude T-cell reaction with increased interleukin 5 or other cytokine production If these entities have been excluded and no evidence documents a clonal myeloid disorder, the diagnosis is hypereosinophilic syndrome.

1	If these entities have been excluded and no evidence documents a clonal myeloid disorder, the diagnosis is hypereosinophilic syndrome. If these entities have been excluded and the myeloid cells show a clonal chromosome abnormality or some other evidence of clonality and blast cells are present in the peripheral blood (>2%) or are increased in the marrow (but <20%), the diagnosis is chronic eosinophilic leukemia. with MPN-U, whereas patients with arterial thrombotic complications might require cytoreductive and aspirin therapy and those with venous thrombosis might require systemic anticoagulation.

1	TMD constitutes an often but not always transient phenomenon of abnormal megakaryoblast proliferation, which occurs in approximately 10% of infants with Down’s syndrome. TMD is usually recognized at birth and either undergoes spontaneous regression (75% of cases) or progresses into acute megakaryoblastic leukemia (AMKL) (25% of cases). Almost all patients with TMD and TMD-derived AMKL display somatic GATA1 mutations. TMD-associated GATA1 mutations constitute exon 2 insertions, deletions, or missense mutations, affecting the N-terminal transactivation domain of GATA-1, and result in loss of full-length (50-kDa) GATA-1 and its replacement with a shorter isoform (40-kDa) that retains friend of GATA-1 (FOG 1) binding. In contrast, inherited forms of exon 2 GATA1 mutations produce a phenotype with anemia, whereas exon 4 mutations that affect the N-terminal, FOG-1-interactive domain produce familial dyserythropoietic anemia with thrombocytopenia or X-linked macrothrombocytopenia.

1	Eosinophilia refers to a peripheral blood absolute eosinophil count (AEC) that is above the upper normal limit of the reference range. The term hypereosinophilia is used when the AEC is >1500 × 109/L. Eosinophilia is operationally classified as secondary (nonneoplastic proliferation of eosinophils) and primary (proliferation of eosinophils that is either neoplastic or otherwise unexplained) (Table 135e-6). Secondary eosinophilia is by far the most frequent cause of eosinophilia and is often associated with infections, especially those related to tissue-invasive helminths; allergic/vasculitic diseases; drugs; and metastatic cancer. Primary eosinophilia is the focus of this chapter and is considered when a cause for secondary eosinophilia is not readily 135e-7 apparent.

1	Primary Eosinophilia Primary eosinophilia is classified as clonal or idiopathic. Diagnosis of clonal eosinophilia requires morphologic, cytogenetic, or molecular evidence of a myeloid neoplasm. Idiopathic eosinophilia is considered when both secondary and clonal eosinophilias have been ruled out as a possibility. HES is a subcategory of idiopathic eosinophilia with persistent AEC of ≥1.5 × 109/L and associated with eosinophil-mediated organ damage (Table 135e-7). An HES-like disorder that is associated with clonal or phenotypically abnormal T cells is referred to as lymphocytic variant hypereosinophilia (Table 135e-7).

1	Clonal Eosinophilia Examples of clonal eosinophilia include eosinophilia associated with acute myeloid leukemia (AML), MDS, CML, mastocytosis, and MDS/MPN overlap. Myeloid neoplasm-associated eosinophilia also includes the WHO MPN subcategory of chronic eosinophilic leukemia, not otherwise specified (CEL-NOS) and the WHO myeloid malignancy subcategory referred to as myeloid/lymphoid neoplasms with eosinophilia and mutations involving platelet-derived growth factor receptor (PDGFR) α/β or fibroblast growth factor receptor 1 (FGFR1).

1	The diagnostic workup for clonal eosinophilia that is not associated with morphologically overt myeloid malignancy should start with peripheral blood mutation screening for FIP1L1-PDGFRA and PDGFRB mutations using fluorescence in situ hybridization (FISH) or reverse transcription polymerase chain reaction. This is crucial because such eosinophilia is easily treated with imatinib. If mutation screening is negative, a bone marrow examination with cytogenetic studies is indicated. In this regard, one must first pay attention to the presence or absence of 5q33, 4q12, or 8p11.2 translocations, which, if present, would suggest PDGFRB-, PDGFRA-, or FGFR1-rearranged clonal eosinophilia, respectively. The presence of 5q33 or 4q12 trans-locations predicts favorable response to treatment with imatinib mesylate, whereas 8p11.2 translocations are associated with aggressive myeloid malignancies that are refractory to current drug therapy.

1	CEL-NOS is considered in the presence of cytogenetic/morphologic evidence of a myeloid malignancy that is otherwise not classifiable. Specifically, CEL-NOS is distinguished from HES by the presence of either a cytogenetic abnormality or greater than 2% peripheral blood blasts or greater than 5% bone marrow blasts (Table 135e-7). HES or idiopathic eosinophilia is considered in the absence of both morphologic and molecular evidence of clonal eosinophilia. However, before making a working diagnosis of HES, one has to exclude lymphocytic variant hypereosinophilia by excluding the presence of phenotypically abnormal T lymphocytes (by flow cytometry) and clonal T-cell gene rearrangements.

1	Chronic Eosinophilic Leukemia, Not Otherwise Specified (CEL-NOS) CELNOS is a subset of clonal eosinophilia that is neither molecularly defined nor classified as an alternative clinicopathologically assigned myeloid malignancy. We prefer to use the term strictly in patients with an HES phenotype who also display either a clonal cytogenetic/molecular abnormality or excess blasts in the bone marrow or peripheral blood. The WHO defines CEL-NOS in the presence of an AEC ≥1.5 × 109/L that is accompanied by either the presence of myeloblast excess (either >2% in the peripheral blood or 5–19% in the bone marrow) or evidence of myeloid clonality. Cytogenetic abnormalities in CEL, other than those that are associated with molecularly defined eosinophilic disorders, include trisomy 8 (the most frequent), t(10;11)(p14;q21), and t(7;12)(q11;p11). CEL-NOS does not respond to imatinib, and treatment strategies are often not different from those used in other similar MPNs and include ASCT for

1	frequent), t(10;11)(p14;q21), and t(7;12)(q11;p11). CEL-NOS does not respond to imatinib, and treatment strategies are often not different from those used in other similar MPNs and include ASCT for transplant-eligible patients with poor risk factors and participation in experimental treatment protocols otherwise.

1	PDGFR-Mutated Eosinophilia Both platelet-derived growth factor receptors α (PDGFRA located on chromosome 4q12) and β (PDGFRB located on chromosome 5q31-q32) are involved in MPN-relevant activating mutations. Clinical phenotype in both instances includes prominent blood eosinophilia and excellent response to imatinib therapy. In regard to PDGFRA mutations, the most popular is FIP1L1-PDGFRA, a karyotypically occult del(4)(q12) that was described in 2003 as an imatinib-sensitive activating mutation. Functional studies have demonstrated transforming properties in cell lines and the induction of MPN in mice. Cloning of the FIP1L1-PDGFRA fusion gene identified a novel molecular mechanism for generating this constitutively active fusion tyrosine kinase, wherein a ~800-kb interstitial deletion within 4q12 fuses the 5′ portion of FIP1L1 to the 3′ portion of PDGFRA. FIP1L1-PDGFRA occurs in a very small subset of patients who present with the phenotypic features of either systemic mastocytosis

1	4q12 fuses the 5′ portion of FIP1L1 to the 3′ portion of PDGFRA. FIP1L1-PDGFRA occurs in a very small subset of patients who present with the phenotypic features of either systemic mastocytosis or HES, but the presence of the mutation reliably predicts complete hematologic and molecular response to imatinib therapy.

1	The association between eosinophilic myeloid malignancies and PDGFRB rearrangement was first characterized and published in 1994 when fusion of the tyrosine kinase–encoding region of PDGFRB to the ets-like gene, ETV6 [ETV6-PDGFRB, t(5;12)(q33;p13)] was demonstrated. The fusion protein was transforming to cell lines and resulted in constitutive activation of PDGFRB signaling. Since then, several other PDGFRB fusion transcripts with similar disease phenotypes have been described, cell line transformation and myeloproliferative disease (MPD) induction in mice has been demonstrated, and imatinib therapy was proven effective when used.

1	FGFR1-Mutated Eosinophilia The 8p11 myeloproliferative syndrome (EMS) (also known as human stem cell leukemic/lymphoma syndrome) constitutes a clinical phenotype with features of both lymphoma and eosinophilic MPN and characterized by a fusion mutation that involves the gene for fibroblast growth factor receptor 1 (FGFR1), which is located on chromosome 8p11. In EMS, both myeloid and lymphoid lineage cells exhibit the 8p11 translocation, thus demonstrating the stem cell origin of the disease. The disease features several 8p11-linked chromosome translocations, and some of the corresponding fusion FGFR1 mutants have been shown to transform cell lines and induce EMSor CML-like disease in mice depending on the specific FGFR1 partner gene (ZNF198 or BCR, respectively). Consistent with this laboratory observation, some patients with BCR-FGFR1 mutation manifest a more indolent CML-like disease. The mechanism of FGFR1 activation in EMS is similar to that seen with PDGFRB-associated MPD; the

1	observation, some patients with BCR-FGFR1 mutation manifest a more indolent CML-like disease. The mechanism of FGFR1 activation in EMS is similar to that seen with PDGFRB-associated MPD; the tyrosine kinase domain of FGFR1 is juxtaposed to a dimerization domain from the partner gene. EMS is aggressive and requires combination chemotherapy followed by ASCT.

1	Hypereosinophilic Syndrome (HES) Blood eosinophilia that is neither secondary nor clonal is operationally labeled as being idiopathic. HES is a subcategory of idiopathic eosinophilia with persistent increase of the AEC to ≥1.5 × 109/L and presence of eosinophil-mediated organ damage, including cardiomyopathy, gastroenteritis, cutaneous lesions, sinusitis, pneumonitis, neuritis, and vasculitis. In addition, some patients manifest thromboembolic complications, hepatosplenomegaly, and either cytopenia or cytosis.

1	Bone marrow histologic and cytogenetic/molecular studies should be examined before a working diagnosis of HES is made. Additional blood studies that are currently recommended during the evaluation of HES include serum tryptase (an increased level suggests systemic mastocytosis and warrants molecular studies to detect FIP1L1PDGFRA), T-cell immunophenotyping, and T-cell receptor antigen gene rearrangement analysis (a positive test suggests an underlying clonal or phenotypically abnormal T-cell disorder). In addition, initial evaluation in HES should include echocardiogram and measurement of serum troponin levels to screen for myocardial involvement by the disease.

1	Initial evaluation of the patient with eosinophilia should include tests that facilitate assessment of target organ damage, including complete blood count, chest x-ray, echocardiogram, and serum troponin level. An increased level of serum cardiac troponin has been shown to correlate with the presence of cardiomyopathy in HES. Typical echocardiographic findings in HES include ventricular apical thrombus, posterior mitral leaflet or tricuspid valve abnormality, endocardial thickening, dilated left ventricle, and pericardial effusion.

1	Glucocorticoids are the cornerstone of therapy in HES. Treatment with oral prednisone is usually started at 1 mg/kg per day and continued for 1–2 weeks before the dose is tapered slowly over the ensuing 2–3 months. If symptoms recur at a prednisone dose level of >10 mg/d, either hydroxyurea or interferon α is used as steroid-sparing agent. In patients who do not respond to usual therapy as outlined above, mepolizumab or alemtuzumab might be considered. Mepolizumab targets interleukin 5 (IL-5), a well-recognized survival factor for eosinophils. Alemtuzumab targets the CD52 antigen, which has been shown to be expressed by eosinophils but not by neutrophils.

1	Mast cell disease (MCD) is defined as tissue infiltration by morphologically and immunophenotypically abnormal mast cells. MCD is classified into two broad categories: cutaneous mastocytosis and systemic mastocytosis (SM). MCD in adults is usually systemic, and the clinical course can be either indolent or aggressive, depending on the respective absence or presence of impaired organ function. Symptoms and signs of MCD include urticaria pigmentosa, mast cell mediator release symptoms (e.g., headache, flushing, lightheadedness, syncope, anaphylaxis, pruritus, urticaria, angioedema, nausea, diarrhea, abdominal cramps), and organ damage (lytic bone lesions, osteoporosis, hepatosplenomegaly, cytopenia). Aggressive SM can be associated with another myeloid malignancy, including MPN, MDS, or MDS/ MPN overlap (e.g., CMML), or present as overt mast cell leukemia. In general, life expectancy is near normal in indolent SM but significantly shortened in aggressive SM.

1	Diagnosis of SM is based on bone marrow examination that shows clusters of morphologically abnormal, spindle-shaped mast cells that are best evaluated by the use of immunohistochemical stains that are specific to mast cells (tryptase, CD117). In addition, mast cell immunophenotyping reveals aberrant CD25 expression by neoplastic mast cells. Other laboratory findings in SM include increased levels of serum tryptase, histamine and urine histamine metabolites, and prostaglandins. SM is associated with KIT mutations, usually KIT D816V, in the majority of patients. Accordingly, mutation screening for KIT D816V is diagnostically useful. However, the ability to detect KIT D816V depends on assay sensitivity and mast cell content of the test sample.

1	Both indolent and aggressive SM patients might experience mast cell mediator release symptoms, which are usually managed by both H1 and H2 histamine receptor blockers as well as cromolyn sodium. In addition, patients with propensity to vasodilatory shock should wear a medical alert bracelet and carry an Epi-Pen self-injector for self-administration of subcutaneous epinephrine. Urticaria pigmentosa shows variable response to both topical and systemic glucocorticoid therapy. Cytoreductive therapy is not recommended for indolent SM. In aggressive SM, either interferon α or cladribine is considered first-line therapy and benefits the majority of patients. In contrast, imatinib is ineffective in the treatment of PDGFR-unmutated SM.

1	Dendritic cell (DC) and histiocyte/macrophage neoplasms are extremely rare. DCs are antigen-presenting cells, whereas histiocyte/ macrophages are antigen-processing cells. Bone marrow myeloid stem cells (CD34+) give rise to monocyte (CD14+, CD68+, CD11c+, CD1a–) and DC (CD14–, CD11c+/–, CD1a+/c) precursors. Monocyte precursors, in turn, give rise to macrophages (CD14+, CD68+, CD11c+, CD163+, lysozyme+) and interstitial DCs (CD68+, CD1a–). DC precursors give rise to Langerhans cell DCs (Birbeck granules, CD1a+, S100+, langerin+) and plasmacytoid DCs (CD68+, CD123+). Follicular DCs (CD21+, CD23+, CD35+) originate from mesenchymal stem cells. Dendritic and histiocytic neoplasms are operationally classified into macrophage/histiocyte-related and DC-related neoplasms. The former includes histiocytic sarcoma/malignant histiocytosis and the latter Langerhans cell histiocytosis, Langerhans cell sarcoma, interdigitating DC sarcoma, and follicular DC sarcoma.

1	Histiocytic Sarcoma/Malignant Histiocytosis Histiocytic sarcoma represents malignant proliferation of mature tissue histiocytes and is often localized. Median age at diagnosis is estimated at 46 years with slight male predilection. Some patients might have history of lymphoma, MDS, or germ cell tumors at time of disease presentation. The three typical disease sites are lymph nodes, skin, and the gastrointestinal system. Patients may or may not have systemic symptoms including fever and weight loss, and other symptoms include hepatosplenomegaly, lytic bone lesions, and pancytopenia. Immunophenotype includes presence of histiocytic markers (CD68, lysozyme, CD11c, CD14) and absence of myeloid or lymphoid markers. Prognosis is poor, and treatment is often ineffective. The term malignant histiocytosis refers to a disseminated disease and systemic symptoms. Lymphoma-like treatment induces complete remissions in some patients, and median survival is estimated at 2 years.

1	Langerhans Cell Histiocytosis Langerhans cells (LCs) are specialized DCs that reside in mucocutaneous tissue and upon activation become specialized for antigen presentation to T cells. LC histiocytosis (LCH; also known as histiocytosis X) represents neoplastic proliferation of LCs (S-100+, CD1a+, and Birbeck granules on electron microscopy). LCH incidence is estimated at 5 per million, and the disease typically affects children with a male predilection. Presentation can be either unifocal (eosinophilic granuloma) or multifocal. The former usually affects bones and less frequently lymph nodes, skin, and lung, whereas the latter is more disseminated. Unifocal disease often affects older children and adults, whereas multisystem disease affects infants. LCH of the lung in adults is characterized by bilateral nodules. Prognosis depends on organs involved. Only 10% of patients progress from 135e-9 unifocal to multiorgan disease. LCH of the lung might improve upon cessation of smoking.

1	Langerhans Cell Sarcoma Langerhans cell sarcoma (LCS) also represents neoplastic proliferation of LCs with overtly malignant morphology. The disease can present de novo or progress from antecedent LCH. There is a female predilection, and median age at diagnosis is estimated at 41 years. Immunophenotype is similar to that seen in LCH, and liver, spleen, lung, and bone are the usual sites of disease. Prognosis is poor, and treatment is generally ineffective. Interdigitating Dendritic Cell Sarcoma Interdigitating DC sarcoma (IDCS), also known as reticulum cell sarcoma, represents neoplastic proliferation of interdigitating DCs. The disease is extremely rare and affects elderly adults with no sex predilection. Typical presentation is asymptomatic solitary lymphadenopathy. Immunophenotype includes S-100+ and negative for vimentin and CD1a. Prognosis ranges from benign local disease to widespread lethal disease.

1	Follicular Dendritic Cell Neoplasm Follicular DCs (FDCs) reside in B-cell follicles and present antigen to B cells. FDC neoplasms (FDCNs) are usually localized and often affect adults. FDCN might be associated with Castleman’s disease in 10–20% of cases, and increased incidence in schizophrenia has been reported. Cervical lymph nodes are the most frequent site of involvement in FDCN, and other sites include maxillary, mediastinal, and retroperitoneal lymph nodes; oral cavity; gastrointestinal system; skin; and breast. Sites of metastasis include lung and liver. Immunophenotype includes CD21, CD35, and CD23. Clinical course is typically indolent, and treatment includes surgical excision followed by regional radiotherapy and sometimes systemic chemotherapy.

1	Hemophagocytic Syndromes Hemophagocytic syndrome (HPS) represents nonneoplastic proliferation and activation of macrophages that induce cytokine-mediated bone marrow suppression and features of intense phagocytosis in bone marrow and liver. HPS may result from genetic or acquired disorders of macrophages. The former entail genetically determined inability to regulate macrophage proliferation and activation. Acquired HPS is often precipitated by viral infections, most notably Epstein-Barr virus. HPS might also accompany certain malignancies such as T-cell lymphoma. Clinical course is often fulminant and fatal.

1	Plasma Cell Disorders Nikhil C. Munshi, Dan L. Longo, Kenneth C. Anderson The plasma cell disorders are monoclonal neoplasms related to each other by virtue of their development from common progenitors in the B-lymphocyte lineage. Multiple myeloma, Waldenström’s mac-roglobulinemia, primary amyloidosis (Chap. 137), and the heavy 136 chain diseases comprise this group and may be designated by a variety of synonyms such as monoclonal gammopathies, paraproteinemias, plasma cell dyscrasias, and dysproteinemias. Mature B lymphocytes destined to produce IgG bear surface immunoglobulin molecules of both M and G heavy chain isotypes with both isotypes having identical idiotypes (variable regions). Under normal circumstances, maturation to antibody-secreting plasma cells and their proliferation is stimulated by exposure to the antigen for which the surface immunoglobulin is specific; however, in the plasma cell disorders, the control over this process is lost. The clinical manifestations of all

1	by exposure to the antigen for which the surface immunoglobulin is specific; however, in the plasma cell disorders, the control over this process is lost. The clinical manifestations of all the plasma cell disorders relate to the expansion of the neoplastic cells, to the secretion of cell products (immunoglobulin molecules or subunits, lymphokines), and to some extent to the host’s response to the tumor. Normal development of B lymphocytes is discussed in Chap. 372e and depicted in Fig. 134-2.

1	There are three categories of structural variation among immunoglobulin molecules that form antigenic determinants, and these are used to classify immunoglobulins. Isotypes are those determinants that distinguish among the main classes of antibodies of a given species and are the same in all normal individuals of that species. Therefore, isotypic determinants are, by definition, recognized by antibodies from a distinct species (heterologous sera) but not by antibodies from the same species (homologous sera). There are five heavy chain isotypes (M, G, A, D, E) and two light chain isotypes (κ, λ). Allotypes are distinct determinants that reflect regular small differences between individuals of the same species in the amino acid sequences of otherwise similar immunoglobulins. These differences are determined by allelic genes; by definition, they are detected by antibodies made in the same species. Idiotypes are the third category of antigenic determinants. They are unique to the

1	are determined by allelic genes; by definition, they are detected by antibodies made in the same species. Idiotypes are the third category of antigenic determinants. They are unique to the molecules produced by a given clone of antibody-producing cells. Idiotypes are formed by the unique structure of the antigen-binding portion of the molecule.

1	Antibody molecules (Fig. 136-1) are composed of two heavy chains (~50,000 mol wt) and two light chains (~25,000 mol wt). Each chain has a constant portion (limited amino acid sequence variability) and a variable region (extensive sequence variability). The light and heavy chains are linked by disulfide bonds and are aligned so that their variable regions are adjacent to one another. This variable region forms the antigen recognition site of the antibody molecule; its unique structural features form idiotypes that are reliable markers for a particular clone of cells because each antibody is formed and secreted by a single clone. Because of the mechanics of the gene rearrangements necessary to specify the immunoglobulin variable regions (VDJ joining for the heavy chain, VJ joining for the light chain), a particular clone rearranges only one of the two chromosomes to produce an immunoglobulin molecule of only one light chain isotype and only one allotype (allelic exclusion) (Fig. 136-1).

1	chain), a particular clone rearranges only one of the two chromosomes to produce an immunoglobulin molecule of only one light chain isotype and only one allotype (allelic exclusion) (Fig. 136-1). After exposure to antigen, the variable region may become associated with a new heavy chain isotype (class switch). Each clone of cells performs these sequential gene arrangements in a unique way. This results in each clone producing a unique immunoglobulin molecule. In most plasma cells, light chains are synthesized in slight excess, secreted as free light chains, and cleared by the kidney, but <10 mg of such light chains is excreted per day.

1	Electrophoretic analysis permits separation of components of the serum proteins (Fig. 136-2). The immunoglobulins move heterogeneously in an electric field and form a broad peak in the gamma region, which is usually increased in the sera of patients with plasma cell tumors. There is a sharp spike in this region called an M component (M for monoclonal). Less commonly, the M component may appear in the

1	FIGURE 136-1 Immunoglobulin genetics and the relationship of gene segments to the antibody protein. The top portion of the figure is a schematic of the organization of the immunoglobulin genes, λ on chromosome 22, κ on chromosome 2, and the heavy chain locus on chromosome 14. The heavy chain locus is longer than 2 megabases, and some of the D region gene segments are only a few bases long, so the figure depicts the schematic relationship among the segments, not their actual size. The bottom portion of the figure outlines the steps in going from the noncontiguous germline gene segments to an intact antibody molecule. Two recombination events juxtapose the V-D-J (or V-J for light chains) segments. The rearranged gene is transcribed, and RNA splicing cuts out intervening sequences to produce an mRNA, which is then translated into an antibody light or heavy chain. The sites on the antibody that bind to antigen (the so called CDR3 regions) are encoded by D and J segments for heavy chains

1	an mRNA, which is then translated into an antibody light or heavy chain. The sites on the antibody that bind to antigen (the so called CDR3 regions) are encoded by D and J segments for heavy chains and the J segments for light chains. (From K Murphy: Janeway’s Immunobiology, 8th ed. Garland Science, 2011.) β2 or α2 globulin region. The monoclonal antibody must be present at and electrophoresis provide qualitative and quantitative assessment a concentration of at least 5 g/L (0.5 g/dL) to be accurately quantitated of the M component, respectively. Once the presence of an M compoby this method. This corresponds to ~109 cells producing the anti-nent has been confirmed, the amount of M component in the serum body. Confirmation of the type of immunoglobulin and that it is truly is a reliable measure of the tumor burden, making M component an monoclonal is determined by immunoelectrophoresis that reveals a excellent tumor marker to manage therapy, yet it is not specific enough single heavy

1	of the tumor burden, making M component an monoclonal is determined by immunoelectrophoresis that reveals a excellent tumor marker to manage therapy, yet it is not specific enough single heavy and/or light chain type. Hence immunoelectrophoresis to be used to screen asymptomatic patients. In addition to the plasma

1	FIGURE 136-2 Representative patterns of serum electrophoresis and immunofixation. The upper panels represent agarose gel, middle panels are the densitometric tracing of the gel, and lower panels are immunofixation patterns. Panel on the left illustrates the normal pattern of serum protein on electrophoresis. Because there are many different immunoglobulins in the serum, their differing mobilities in an electric field produce a broad peak. In conditions associated with increases in polyclonal immunoglobulin, the broad peak is more prominent (middle panel). In monoclonal gammopathies, the predominance of a product of a single cell produces a “church spire” sharp peak, usually in the γ globulin region (right panel). The immunofixation (lower panel) identifies the type of immunoglobulin. For example, normal and polyclonal increase in immunoglobulins produce no distinct bands; however, the right panel shows distinct bands in IgG and lambda protein lanes, confirming the presence of IgG

1	For example, normal and polyclonal increase in immunoglobulins produce no distinct bands; however, the right panel shows distinct bands in IgG and lambda protein lanes, confirming the presence of IgG lambda monoclonal protein. (Courtesy of Dr. Neal I. Lindeman; with permission.) cell disorders, M components may be detected in other lymphoid neoplasms such as chronic lymphocytic leukemia and lymphomas of Bor T-cell origin; nonlymphoid neoplasms such as chronic myeloid leukemia, breast cancer, and colon cancer; a variety of nonneoplastic conditions such as cirrhosis, sarcoidosis, parasitic diseases, Gaucher’s disease, and pyoderma gangrenosum; and a number of autoimmune conditions, including rheumatoid arthritis, myasthenia gravis, and cold agglutinin disease. Monoclonal proteins are also observed in immunosuppressed patients after organ transplant and, rarely, allogeneic transplant. At least two very rare skin diseases—lichen myxedematosus (also known as papular mucinosis) and

1	also observed in immunosuppressed patients after organ transplant and, rarely, allogeneic transplant. At least two very rare skin diseases—lichen myxedematosus (also known as papular mucinosis) and necrobiotic xanthogranuloma—are associated with a monoclonal gammopathy. In papular mucinosis, highly cationic IgG is deposited in the dermis of patients. This organ specificity may reflect the specificity of the antibody for some antigenic component of the dermis. Necrobiotic xanthogranuloma is a histiocytic infiltration of the skin, usually of the face, that produces red or yellow nodules that can enlarge to plaques. Approximately 10% progress to myeloma. Five percent of patients with sensory motor neuropathy also have a monoclonal paraprotein.

1	The nature of the M component is variable in plasma cell disorders. It may be an intact antibody molecule of any heavy chain subclass, or it may be an altered antibody or fragment. Isolated light or heavy chains may be produced. In some plasma cell tumors such as extramedullary or solitary bone plasmacytomas, less than one-third of patients will have an M component. In ~20% of myelomas, only light chains are produced and, in most cases, are secreted in the urine as Bence Jones proteins. The frequency of myelomas of a particular heavy chain class is roughly proportional to the serum concentration, and therefore, IgG myelomas are more common than IgA and IgD myelomas. In approximately 1% of patients with myeloma, biclonal or triclonal gammopathy is observed.

1	Multiple myeloma represents a malignant proliferation of plasma cells derived from a single clone. The tumor, its products, and the host response to it result in a number of organ dysfunctions and symptoms, including bone pain or fracture, renal failure, susceptibility to infection, anemia, hypercalcemia, and occasionally clotting abnormalities, neurologic symptoms, and manifestations of hyperviscosity.

1	The cause of myeloma is not known. Myeloma occurred with increased frequency in those exposed to the radiation of nuclear warheads in World War II after a 20-year latency. Myeloma has been seen more commonly than expected among farmers, wood workers, leather workers, and those exposed to petroleum products. A variety of chromosomal alterations have been found in patients with myeloma: hyperdiploidy, 13q14 deletions, translocations t(11;14)(q13;q32), t(4;14) (p16;q32), and t(14;16), and 17p13 deletions. Evidence is strong that errors in switch recombination—the genetic mechanism to change antibody heavy chain isotype—participate in the transformation process. However, no common molecular pathogenetic pathway has yet emerged. Genome sequencing studies have failed to identify any recurrent mutation with frequency >20%; N-ras, K-ras, and B-raf mutations are most common and combined occur in over 40% of patients. There is also evidence of complex clusters of subclonal variants at diagnosis

1	with frequency >20%; N-ras, K-ras, and B-raf mutations are most common and combined occur in over 40% of patients. There is also evidence of complex clusters of subclonal variants at diagnosis that acquire additional mutations over time, indicative of genomic evolution that may drive disease progression. The neoplastic event in myeloma may involve cells earlier in B-cell differentiation than the plasma cell. Interleukin (IL) 6 may play a role in driving myeloma cell proliferation. It remains difficult to distinguish benign from malignant plasma cells based on morphologic criteria in all but a few cases (Fig. 136-3).

1	An estimated 24,050 new cases of myeloma were diagnosed in 2014, and 11,090 people died from the disease in the United States. Myeloma increases in incidence with age. The median age at diagnosis is 70 years; it is uncommon under age 40. Males are more commonly affected than females, and blacks have nearly twice the incidence of whites. Myeloma accounts for 1.3% of all malignancies in whites and 2% in blacks, and 13% of all hematologic cancers in whites and 33% in blacks. FIGURE 136-3 Multiple myeloma (marrow). The cells bear characteristic morphologic features of plasma cells, round or oval cells with an eccentric nucleus composed of coarsely clumped chromatin, a densely basophilic cytoplasm, and a perinuclear clear zone containing the Golgi apparatus. Binucleate and multinucleate malignant plasma cells can be seen. The incidence of myeloma is highest in African Americans

1	The incidence of myeloma is highest in African Americans American whites; and lowest in people from developing countries including Asia. The higher incidence in more developed countries may result from the combination of a longer life expectancy and more frequent medical surveillance. Incidence of multiple myeloma in other ethnic groups including native Hawaiians, female Hispanics, American Indians from New Mexico, and Alaskan natives is higher relative to U.S. whites in the same geographic area. Chinese and Japanese populations have a lower incidence than whites. Immunoproliferative small-intestinal disease with alpha heavy chain disease is most prevalent in the Mediterranean area. Despite these differences in prevalence, the characteristics, response to therapy, and prognosis of myeloma are similar worldwide.

1	Multiple myeloma (MM) cells bind via cell-surface adhesion molecules to bone marrow stromal cells (BMSCs) and extracellular matrix (ECM), which triggers MM cell growth, survival, drug resistance, and migration in the bone marrow milieu (Fig. 136-4). These effects are due both to direct MM cell–BMSC binding and to induction of various cytokines, including IL-6, insulin-like growth factor type I (IGF-I), vascular endothelial growth factor (VEGF), and stromal cell–derived growth factor (SDF)-1α. Growth, drug resistance, and migration are mediated via Ras/Raf/mitogen-activated protein kinase, PI3K/Akt, and protein kinase C signaling cascades, respectively.

1	Bone pain is the most common symptom in myeloma, affecting nearly 70% of patients. Unlike the pain of metastatic carcinoma, which often is worse at night, the pain of myeloma is precipitated by movement. Persistent localized pain in a patient with myeloma usually signifies a pathologic fracture. The bone lesions of myeloma are caused by the proliferation of tumor cells, activation of osteoclasts that destroy bone, and suppression of osteoblasts that form new bone. The increased osteoclast activity is mediated by osteoclast activat ing factors (OAFs) made by the myeloma cells (OAF activity can be mediated by several cytokines, including IL-1, lymphotoxin, VEGF, receptor activator of NF-κB [RANK] ligand, macrophage inhibitory factor [MIP]-1α, and tumor necrosis factor [TNF]). The bone lesions are lytic in nature and are rarely associated with osteoblastic new bone formation due to their suppression by dickhoff-1 (DKK-1) produced by myeloma cells. Therefore, radioisotopic bone scanning

1	are lytic in nature and are rarely associated with osteoblastic new bone formation due to their suppression by dickhoff-1 (DKK-1) produced by myeloma cells. Therefore, radioisotopic bone scanning is less useful in diagnosis than is plain radiography. The bony lysis results in substantial mobilization of calcium from bone, and serious acute and chronic complications of hypercalcemia may dominate the clinical picture (see below). Localized bone lesions may expand to the point that mass lesions may be palpated, especially on the skull (Fig. 136-5), clavicles, and sternum; and the collapse of vertebrae may lead to spinal cord compression. The next most common clinical problem in patients with myeloma is susceptibility to bacterial infections. The most common infections are pneumonias and pyelonephritis, and the most frequent pathogens are Streptococcus pneumoniae, Staphylococcus aureus, and Klebsiella pneumoniae in the lungs and Escherichia coli and other gram-negative organisms in the

1	and the most frequent pathogens are Streptococcus pneumoniae, Staphylococcus aureus, and Klebsiella pneumoniae in the lungs and Escherichia coli and other gram-negative organisms in the urinary tract. In ~25% of patients, recurrent infections are the presenting features, and >75%

1	FIGURE 136-4 Pathogenesis of multiple myeloma. Multiple myeloma (MM) cells interact with bone marrow stromal cells (BMSCs) and extracellular matrix proteins via adhesion molecules, triggering adhesion-mediated signaling as well as cytokine production. This triggers cytokinemediated signaling that provides growth, survival, and antiapoptotic effects as well as development of drug resistance.

1	FIGURE 136-5 Bony lesions in multiple myeloma. The skull dem-onstrates the typical “punched out” lesions characteristic of multiple myeloma. The lesion represents a purely osteolytic lesion with little or no osteoblastic activity. (Courtesy of Dr. Geraldine Schechter; with permission.) of patients will have a serious infection at some time in their course. The susceptibility to infection has several contributing causes. First, patients with myeloma have diffuse hypogammaglobulinemia if the M component is excluded. The hypogammaglobulinemia is related to both decreased production and increased destruction of normal antibodies. Moreover, some patients generate a population of circulating regulatory cells in response to their myeloma that can suppress normal antibody synthesis. In the case of IgG myeloma, normal IgG antibodies are broken down more rapidly than normal because the catabolic rate for IgG antibodies varies directly with the serum concentration. The large M component results

1	IgG myeloma, normal IgG antibodies are broken down more rapidly than normal because the catabolic rate for IgG antibodies varies directly with the serum concentration. The large M component results in fractional catabolic rates of 8–16% instead of the normal 2%. These patients have very poor antibody responses, especially to polysaccharide antigens such as those on bacterial cell walls. Most measures of T-cell function in myeloma are normal, but a subset of CD4+ cells may be decreased. Granulocyte lysozyme content is low, and granulocyte migration is not as rapid as normal in patients with myeloma, probably the result of a tumor product. There are also a variety of abnormalities in complement functions in myeloma patients. All these factors contribute to the immune deficiency of these patients. Some commonly used therapeutic agents, e.g., dexamethasone, suppress immune responses and increase susceptibility to bacterial and fungal infection, and bortezomib predisposes to herpesvirus

1	Some commonly used therapeutic agents, e.g., dexamethasone, suppress immune responses and increase susceptibility to bacterial and fungal infection, and bortezomib predisposes to herpesvirus reactivation.

1	Renal failure occurs in nearly 25% of myeloma patients, and some renal pathology is noted in more than 50%. Many factors contribute to this. Hypercalcemia is the most common cause of renal failure. Glomerular deposits of amyloid, hyperuricemia, recurrent infections, frequent use of nonsteroidal anti-inflammatory agents for pain control, use of iodinated contrast dye for imaging, bisphosphonate use, and occasional infiltration of the kidney by myeloma cells all may contribute to renal dysfunction. However, tubular damage associated with the excretion of light chains is almost always present. Normally, light chains are filtered, reabsorbed in the tubules, and catabolized. With the increase in the amount of light chains presented to the tubule, the tubular cells become overloaded with these proteins, and tubular damage results either directly from light chain toxic effects or indirectly from the release of intracellular lysosomal enzymes. The earliest manifestation of this tubular damage

1	and tubular damage results either directly from light chain toxic effects or indirectly from the release of intracellular lysosomal enzymes. The earliest manifestation of this tubular damage is the adult Fanconi’s syndrome (a type 2 proximal renal tubular acidosis), with loss of glucose and amino acids, as well as defects in the ability of the kidney to acidify and concentrate the urine. The proteinuria is not accompanied by hypertension, and the protein is nearly all light chains. Generally, very little albumin is in the urine because glomerular function is usually normal. When the glomeruli are involved, nonselective proteinuria is also observed. Patients with myeloma also have a decreased anion gap [i.e., Na+ – (Cl− + HCO3−)] because the M component is cationic, resulting in retention of chloride. This is often accompanied by hyponatremia that is felt to be artificial (pseudohyponatremia) because each volume of serum has less water as a result of the increased protein. Renal

1	of chloride. This is often accompanied by hyponatremia that is felt to be artificial (pseudohyponatremia) because each volume of serum has less water as a result of the increased protein. Renal dysfunction due to light chain deposition disease, light chain cast nephropathy, and amyloidosis is partially reversible with effective therapy. Myeloma patients are susceptible to developing acute renal failure if they become dehydrated.

1	Normocytic and normochromic anemia occurs in ~80% of myeloma patients. It is usually related to the replacement of normal marrow by expanding tumor cells, to the inhibition of hematopoiesis by factors made by the tumor, to reduced production of erythropoietin by the kidney, and to the effects of long-term therapy. In addition, mild hemolysis may contribute to the anemia. A larger than expected fraction of patients may have megaloblastic anemia due to either folate or vitamin B12 deficiency. Granulocytopenia and thrombocytopenia are rare except when therapy-induced. Clotting abnormalities may be seen due to the failure of antibody-coated platelets to function properly; the interaction of the M component with clotting factors I, II, V, VII, or VIII; antibody to clotting factors; or amyloid damage of endothelium. Deep venous thrombosis is also observed with use of thalidomide, lenalidomide, or pomalidomide in combination with dexamethasone. Raynaud’s phenomenon and impaired circulation

1	damage of endothelium. Deep venous thrombosis is also observed with use of thalidomide, lenalidomide, or pomalidomide in combination with dexamethasone. Raynaud’s phenomenon and impaired circulation may result if the M component forms cryoglobulins, and hyperviscosity syndromes may develop depending on the physical properties of the M component (most common with IgM, IgG3, and IgA paraproteins). Hyperviscosity is defined based on the relative viscosity of serum as compared with water. Normal relative serum viscosity is 1.8 (i.e., serum is normally almost twice as viscous as water). Symptoms of hyperviscosity occur at a level greater than 4 centipoise (cP), which is usually reached at paraprotein concentrations of ~40 g/L (4 g/dL) for IgM, 50 g/L (5 g/dL) for IgG3, and 70 g/L (7 g/dL) for IgA; however, depending on chemical and physical properties of the paraprotein molecule, it can occasionally be observed at lower levels.

1	Although neurologic symptoms occur in a minority of patients, they may have many causes. Hypercalcemia may produce lethargy, weakness, depression, and confusion. Hyperviscosity may lead to headache, fatigue, shortness of breath, exacerbation or precipitation of heart failure, visual disturbances, ataxia, vertigo, retinopathy, somnolence, and coma. Bony damage and collapse may lead to cord compression, radicular pain, and loss of bowel and bladder control. Infiltration of peripheral nerves by amyloid can be a cause of carpal tunnel syndrome and other sensorimotor monoand polyneuropathies. Neuropathy associated with monoclonal gammopathy of undetermined significance (MGUS) and myeloma is more frequently sensory than motor neuropathy and is associated with IgM more than other isotypes. In >50% of patients with neuropathy, the IgM monoclonal protein is directed against myelin-associated globulin (MAG). Sensory neuropathy is also a side effect of thalidomide and bortezomib therapy.

1	Many of the clinical features of myeloma, e.g., cord compression, pathologic fractures, hyperviscosity, sepsis, and hypercalcemia, can present as medical emergencies. Despite the widespread distribution of plasma cells in the body, tumor expansion is dominantly within bone and bone marrow and, for reasons unknown, rarely causes enlargement of spleen, lymph nodes, or gut-associated lymphatic tissue.

1	The diagnosis of myeloma requires marrow plasmacytosis (>10%), a serum and/or urine M component, and end organ damage detailed in Table 136-1. Bone marrow plasma cells are CD138 and either monoclonal kappa or lambda light chain positive. The most important differential diagnosis in patients with myeloma involves their separation from individuals with MGUS or smoldering multiple myeloma (SMM). MGUS is vastly more common than myeloma, occurring in 1% of the population older than age 50 years and in up to 10% of individuals older than age 75 years. The diagnostic criteria for MGUS, SMM, and myeloma are described in Table 136-1. Although ~1% of patients per year with MGUS go on to develop myeloma, all TABLE 136-1 DIAgNOsTIC CrITErIA fOr MuLTIPLE MYELOMA, MYELOMA VArIANTs, AND MONOCLONAL gAMMOPATHY Of uNDETErMINED sIgNIfICANCE Monoclonal Gammopathy of Undetermined Significance (MGUS)

1	TABLE 136-1 DIAgNOsTIC CrITErIA fOr MuLTIPLE MYELOMA, MYELOMA VArIANTs, AND MONOCLONAL gAMMOPATHY Of uNDETErMINED sIgNIfICANCE Monoclonal Gammopathy of Undetermined Significance (MGUS) M protein in serum <30 g/L Bone marrow clonal plasma cells <10% No evidence of other B cell proliferative disorders No myeloma-related organ or tissue impairment (no end organ damage, M protein in serum ≥30 g/L and/or Bone marrow clonal plasma cells ≥10% No myeloma-related organ or tissue impairment (no end organ damage, M protein in serum and/or urine Bone marrow (clonal) plasma cellsb or plasmacytoma Myeloma-related organ or tissue impairment (end organ damage, including No M protein in serum and/or urine with immunofixation Bone marrow clonal plasmacytosis ≥10% or plasmacytoma Myeloma-related organ or tissue impairment (end organ damage, including Solitary Plasmacytoma of Bone Single area of bone destruction due to clonal plasma cells Bone marrow not consistent with multiple myeloma

1	Solitary Plasmacytoma of Bone Single area of bone destruction due to clonal plasma cells Bone marrow not consistent with multiple myeloma Normal skeletal survey (and magnetic resonance imaging of spine and pelvis if done) All of the following four criteria must be met: 1. 2. 3. Any one of the following: (a) sclerotic bone lesions; (b) Castleman’s disease; (c) elevated levels of vascular endothelial growth factor (VEGF) 4.

1	Any one of the following: (a) organomegaly (splenomegaly, hepatomegaly, or lymphadenopathy); (b) extravascular volume overload (edema, pleural effusion, or ascites); (c) endocrinopathy (adrenal, thyroid, pituitary, gonadal, parathyroid, and pancreatic); (d) skin changes (hyperpigmentation, hypertrichosis, glomeruloid hemangiomata, plethora, acrocyanosis, flushing, and white nails); (e) papilledema; (f ) thrombocytosis/polycythemiad aMyeloma-related organ or tissue impairment (end organ damage): calcium levels increased: serum calcium >0.25 mmol/L above the upper limit of normal or >2.75 mmol/L; renal insufficiency: creatinine >173 mmol/L; anemia: hemoglobin 2 g/dL below the lower limit of normal or hemoglobin <10 g/dL; bone lesions: lytic lesions or osteoporosis with compression fractures (magnetic resonance imaging or computed tomography may clarify); other: symptomatic hyperviscosity, amyloidosis, recurrent bacterial infections (>2 episodes in 12 months). bIf flow cytometry is

1	(magnetic resonance imaging or computed tomography may clarify); other: symptomatic hyperviscosity, amyloidosis, recurrent bacterial infections (>2 episodes in 12 months). bIf flow cytometry is performed, most plasma cells (>90%) will show a “neoplastic” phenotype. cA small M component may sometimes be present. dThese features should have no attributable other causes and have temporal relation with each other.

1	Abbreviation: POEMS, polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes.

1	myeloma is preceded by MGUS. Non-IgG subtype, abnormal kappa/ lambda free light chain ratio, and serum M protein >15 g/L (1.5 g/ dL) are associated with higher incidence of progression of MGUS to myeloma. Absence of all three features predicts a 5% chance of progression, whereas higher risk MGUS with the presence of all three features predicts a 60% chance of progression over 20 years. The features responsible for higher risk of progression from SMM to MM are bone marrow plasmacytosis >10%, abnormal kappa/lambda free light chain ratio, and serum M protein >30 g/L (3 g/dL). Patients with only one of these three features have a 25% chance of progression to MM in 5 years, whereas patients with high-risk SMM with all three features have a 76% chance of progression. There are two important variants 715 of myeloma—solitary bone plasmacytoma and solitary extramedullary plasmacytoma. These lesions are associated with an M component in <30% of the cases, they may affect younger individuals,

1	715 of myeloma—solitary bone plasmacytoma and solitary extramedullary plasmacytoma. These lesions are associated with an M component in <30% of the cases, they may affect younger individuals, and both are associated with median survivals of ≥10 years. Solitary bone plasmacytoma is a single lytic bone lesion without marrow plasmacytosis. Extramedullary plasmacytomas usually involve the submucosal lymphoid tissue of the nasopharynx or paranasal sinuses without marrow plasmacytosis. Both tumors are highly responsive to local radiation therapy. If an M component is present, it should disappear after treatment. Solitary bone plasmacytomas may recur in other bony sites or evolve into myeloma. Extramedullary plasmacytomas rarely recur or progress.

1	The clinical evaluation of patients with myeloma includes a careful physical examination searching for tender bones and masses. Chest and bone radiographs may reveal lytic lesions or diffuse osteopenia. Magnetic resonance imaging (MRI) offers a sensitive means to document extent of bone marrow infiltration and cord or root compression in patients with pain syndromes. A complete blood count with differential may reveal anemia. Erythrocyte sedimentation rate is elevated. Rare patients (~1%) may have plasma cell leukemia with >2000 plasma cells/μL. This may be seen in disproportionate frequency in IgD (12%) and IgE (25%) myelomas. Serum calcium, urea nitrogen, creatinine, and uric acid levels may be elevated. Protein electrophoresis and measurement of serum immunoglobulins and free light chains are useful for detecting and characterizing M spikes, supplemented by immunoelectrophoresis, which is especially sensitive for identifying low concentrations of M components not detectable by

1	chains are useful for detecting and characterizing M spikes, supplemented by immunoelectrophoresis, which is especially sensitive for identifying low concentrations of M components not detectable by protein electrophoresis. A 24-h urine specimen is necessary to quantitate Bence Jones protein excretion. Serum alkaline phosphatase is usually normal even with extensive bone involvement because of the absence of osteoblastic activity. It is also important to quantitate serum β2-microglobulin and albumin (see below).

1	The serum M component will be IgG in 53% of patients, IgA in 25%, and IgD in 1%; 20% of patients will have only light chains in serum and urine. Dipsticks for detecting proteinuria are not reliable at identifying light chains, and the heat test for detecting Bence Jones protein is falsely negative in ~50% of patients with light chain myeloma. Fewer than 1% of patients have no identifiable M component; these patients usually have light chain myeloma in which renal catabolism has made the light chains undetectable in the urine. In most of these patients, light chains can now be detected by serum free light chain assay. IgD myeloma may also present with light chain disease. About two-thirds of patients with serum M components also have urinary light chains. The light chain isotype may have an impact on survival. Patients secreting lambda light chains have a significantly shorter overall survival than those secreting kappa light chains. Whether this is due to some genetically important

1	an impact on survival. Patients secreting lambda light chains have a significantly shorter overall survival than those secreting kappa light chains. Whether this is due to some genetically important determinant of cell proliferation or because lambda light chains are more likely to cause renal damage and form amyloid than are kappa light chains is unclear. The heavy chain isotype may have an impact on patient management as well. About half of patients with IgM paraproteins develop hyperviscosity compared with only 2–4% of patients with IgA and IgG M components. Among IgG myelomas, it is the IgG3 subclass that has the highest tendency to form both concentrationand temperature-dependent aggregates, leading to hyperviscosity and cold agglutination at lower serum concentrations.

1	Serum β2-microglobulin is the single most powerful predictor of survival and can substitute for staging. β2-Microglobulin is a protein of 11,000 mol wt with homologies to the constant region of immunoglobulins that is the light chain of the class I major histocompatibility antigens (HLAA, -B, -C) on the surface of every cell. Patients with β2-microglobulin levels <0.004 g/L have a median survival of 43 months, and those with levels >0.004 g/L have a survival of only 12 months. Combination of serum β2-microglobulin and albumin levels forms the basis for a three-stage International Staging System (ISS) (Table 136-2) that predicts survival. With the use of high-dose therapy and the newer agents, the Median Survival, Stage Months β2M <3.5, alb ≥3.5 I (28%)a 62 β2M <3.5, alb <3.5 or II (39%) 44 β2M = 3.5–5.5 β2M >5.5 III (33%) 29 Other features suggesting high-risk disease: aPercentage of patients presenting at each stage.

1	Abbreviations: β2M, serum β2-microglobulin in mg/L; alb, serum albumin in g/dL; FISH, fluorescent in situ hybridization. Durie-Salmon staging system is unable to predict outcome and thus is no longer used. High labeling index, circulating plasma cells, performance status, and high levels of lactate dehydrogenase are also associated with poor prognosis. Other factors that may influence prognosis are the presence of cytogenetic abnormalities and hypodiploidy by karyotype, fluorescent in situ hybridization (FISH)–identified chromosome 17p deletion, and translocations t(4;14), (14;16), and t(14;20). Chromosome 13q deletion, previously thought to predict poor outcome, is not a predictor following the use of newer agents. Microarray profiling and comparative genomic hybridization have formed the basis for RNAand DNA-based prognostic staging systems, respectively. The ISS system, along with cytogenetic changes, is the most widely used method for assessing prognosis (Table 136-2).

1	No specific intervention is indicated for patients with MGUS. Follow-up once a year or less frequently is adequate except in higher risk MGUS, where serum protein electrophoresis, complete blood count, creatinine, and calcium should be repeated every 6 months. A patient with MGUS and severe polyneuropathy is considered for therapeutic intervention if a causal relationship can be assumed, especially in absence of any other potential causes for neuropathy. Therapy can include plasmapheresis and occasionally rituximab in patients with IgM MGUS or myeloma-like therapy in those with IgG or IgA disease. About 10% of patients with myeloma are asymptomatic (SMM) and will have an indolent course demonstrating only very slow progression of disease over many years. For these patients, no specific therapeutic intervention is indicated, although early intervention with lenalidomide and dexamethasone may prevent progression from high-risk SMM to active MM. At present, patients with SMM only require

1	intervention is indicated, although early intervention with lenalidomide and dexamethasone may prevent progression from high-risk SMM to active MM. At present, patients with SMM only require antitumor therapy when the disease becomes symptomatic with development of anemia, hypercalcemia, progressive lytic bone lesions, renal dysfunction, or recurrent infections. Patients with solitary bone plasmacytomas and extramedullary plasmacytomas may be expected to enjoy prolonged disease-free survival after local radiation therapy at a dose of around 40 Gy. There is a low incidence of occult marrow involvement in patients with solitary bone plasmacytoma. Such patients are usually identified because their serum M component falls slowly or disappears initially, only to return after a few months. These patients respond well to systemic therapy.

1	Patients with symptomatic and/or progressive myeloma require therapeutic intervention. In general, such therapy is of two sorts: (1) systemic therapy to control the progression of myeloma and (2) symptomatic supportive care to prevent serious morbidity from the complications of the disease. Therapy can significantly prolong survival and improve the quality of life for myeloma patients. The therapy of myeloma includes an initial induction regimen followed by consolidation and/or maintenance therapy and, on subsequent progression, management of relapsed disease. The therapy is partly dictated by the patient’s age and comorbidities, which may affect a patient’s ability to undergo high-dose therapy and transplantation.

1	Thalidomide (200 mg daily), when combined with dexamethasone, achieved responses in two-thirds of newly diagnosed MM patients. Subsequently, lenalidomide (25 mg/d on days 1–21 every 4 weeks), an immunomodulatory derivative of thalidomide, and bortezomib (1.3 mg/m2 on days 1, 4, 8, and 11 every 3 weeks), a proteasome inhibitor, have each been combined with dexamethasone (40 mg once every week) and obtained high response rates (>80%) in newly diagnosed patients with MM. Importantly, their superior toxicity profile with improved efficacy has made them the preferred agents for induction therapy. Efforts to improve the fraction of patients responding and the degree of response have involved adding agents to the treatment regimen. The combination of lenalidomide, bortezomib, and dexamethasone achieves close to a 100% response rate and 30% complete response rate, making it one of the preferred induction regimens in transplant-eligible patients. Other similar three-drug combinations

1	achieves close to a 100% response rate and 30% complete response rate, making it one of the preferred induction regimens in transplant-eligible patients. Other similar three-drug combinations (bortezomib, thalidomide, and dexamethasone or bortezomib, cyclophosphamide, and dexamethasone) also achieve >90% response rate. Herpes zoster prophylaxis is indicated if bortezomib is used, and neuropathy attendant to bortezomib can be decreased both by its subcutaneous administration and administration on a weekly schedule. Lenalidomide use requires prophylaxis for deep vein thrombosis (DVT) with either aspirin or warfarin or low-molecular-weight heparin if patients are at a greater risk of DVT. In patients receiving lenalidomide, stem cells should be collected within 6 months, because the continued use of lenalidomide may compromise the ability to collect adequate numbers of stem cells. Initial therapy is continued until maximal cytoreduction. In patients who are transplant candidates,

1	use of lenalidomide may compromise the ability to collect adequate numbers of stem cells. Initial therapy is continued until maximal cytoreduction. In patients who are transplant candidates, alkylating agents such as melphalan should be avoided because they damage stem cells, leading to decreased ability to collect stem cells for autologous transplant.

1	In patients who are not transplant candidates due to physiologic age >70 years, significant cardiopulmonary problems, or other comorbid illnesses, the same twoor three-drug combinations described above are considered standard of care as induction therapy. Previously, therapy consisting of intermittent pulses of melphalan, an alkylating agent, with prednisone (MP; melphalan, 0.25 mg/kg per day, and prednisone, 1 mg/kg per day for 4 days) every 4–6 weeks was used. However, a number of studies have combined novel agents with MP and reported superior response and survival outcomes. In patients >65 years old, combining thalidomide with MP (MPT) obtains higher response rates and overall survival compared with MP alone. Similarly, significantly improved response (71 vs 35%) and overall survival (3-year survival 72 vs 59%) were observed with the combination of bortezomib and MP compared with MP alone. Lenalidomide added to MP followed by lenalidomide maintenance also prolonged

1	survival (3-year survival 72 vs 59%) were observed with the combination of bortezomib and MP compared with MP alone. Lenalidomide added to MP followed by lenalidomide maintenance also prolonged progression-free survival compared with MP alone. These combinations of novel agents with MP also achieve high complete response rates (MPT, ~15%; MP plus bortezomib, ~30%; MP plus lenalidomide, ~20%; and MP, ~2–4%). Although combinations of MP with newer agents are an alternative in these patients, most studies favor continuous therapy with nonMP-containing regimens (e.g., lenalidomide plus dexamethasone) due to longer term safety profile and efficacy.

1	Improvement in the serum M component may lag behind the symptomatic improvement. The fall in M component depends on the rate of tumor kill and the fractional catabolic rate of immunoglobulin, which in turn depends on the serum concentration (for IgG). Light chain excretion, with a functional half-life of ~6 h, may fall within the first week of treatment. Because urine light chain levels may relate to renal tubular function, they are not a reliable measure of tumor cell kill, especially in patients with renal dysfunction; however, improvements in serum free light chain measurement are often seen sooner. Although patients may not achieve complete remission, clinical responses may last for long periods of time.

1	High-dose therapy and consolidation/maintenance are standard practice in the majority of eligible patients. Randomized studies comparing standard-dose therapy to high-dose melphalan therapy (HDT) with hematopoietic stem cell support have shown that HDT can achieve high overall response rates, with up to 25–40% additional complete responses and prolonged progression-free and overall survival; however, few, if any, patients are cured. Although two successive HDTs (tandem transplantations) are more effective than single HDT, the benefit is only observed in the subset of patients who do not achieve a complete or very good partial response to the first transplantation, which is rare. Moreover, a randomized study failed to show any significant difference in overall survival between early transplantation after induction therapy versus delayed transplantation at relapse. These data allow an option to delay transplantation, especially with the availability of more agents and combinations.

1	after induction therapy versus delayed transplantation at relapse. These data allow an option to delay transplantation, especially with the availability of more agents and combinations. Allogeneic transplantations may also produce high response rates, but treatment-related mortality may be as high as 40%. Nonmyeloablative allogeneic transplantation can reduce toxicity but is recommended only under the auspices of a clinical trial to exploit an immune graft-versus-myeloma effect while avoiding attendant toxicity.

1	Maintenance therapy prolongs remissions following standard-dose regimens as well as HDT. Two phase 3 studies have demonstrated improved progression-free survival, and one study showed prolonged overall survival in patients receiving lenalidomide compared to placebo as maintenance therapy after HDT. In non-transplant candidates, another phase 3 study showed prolonged progression-free survival with lenalidomide maintenance after MP plus lenalidomide induction therapy. Although there is concern regarding an increased incidence of second primary malignancies in patients receiving lenalidomide maintenance, its benefits far outweigh the risk of progressive disease and death from myeloma. In patients with high-risk cytogenetics, lenalidomide and bortezomib have been combined and show promise as maintenance therapy after transplantation.

1	Relapsed myeloma can be treated with a number of agents including lenalidomide and/or bortezomib. These agents in combination with dexamethasone can achieve a partial response rate of up to 60% and a 10–15% complete response rate in patients with relapsed disease. The combination of bortezomib and liposomal doxorubicin is active in relapsed myeloma. Thalidomide, if not used as initial therapy, can achieve responses in refractory cases. The second-generation proteasome inhibitor carfilzomib and immunomodulatory agent pomalidomide have shown efficacy in relapsed and refractory MM, even MM refractory to lenalidomide and bortezomib. High-dose melphalan and stem cell transplantation, if not used earlier, also have activity as salvage therapy in patients with refractory disease.

1	The median overall survival of patients with myeloma is 7–8+ years, with subsets of younger patients surviving more than 10 years. The major causes of death are progressive myeloma, renal failure, sepsis, or therapy-related myelodysplasia. Nearly a quarter of patients die of myocardial infarction, chronic lung disease, diabetes, or stroke—all intercurrent illnesses related more to the age of the patient group than to the tumor.

1	Supportive care directed at the anticipated complications of 717 the disease may be as important as primary antitumor therapy. Hypercalcemia generally responds well to bisphosphonates, glucocorticoid therapy, hydration, and natriuresis, and rarely requires calcitonin as well. Bisphosphonates (e.g., pamidronate 90 mg or zoledronate 4 mg once a month) reduce osteoclastic bone resorption and preserve performance status and quality of life, decrease bone-related complications, and may also have antitumor effects. Osteonecrosis of the jaw and renal dysfunction can occur in a minority of patients receiving aminobisphosphonate therapy. Treatments aimed at strengthening the skeleton such as fluorides, calcium, and vitamin D, with or without androgens, have been suggested, but are not of proven efficacy. Kyphoplasty or vertebroplasty should be considered in patients with painful collapsed vertebra. Iatrogenic worsening of renal function may be prevented by maintaining a high fluid intake to

1	efficacy. Kyphoplasty or vertebroplasty should be considered in patients with painful collapsed vertebra. Iatrogenic worsening of renal function may be prevented by maintaining a high fluid intake to prevent dehydration and enhance excretion of light chains and calcium. In the event of acute renal failure, plasmapheresis is ~10 times more effective at clearing light chains than peri toneal dialysis; however, its role in reversing renal failure remains controversial. Importantly, reducing the protein load by effective antitumor therapy with agents such as bortezomib may result in improvement in renal function in over half of the patients. Use of lenalidomide in renal failure is possible but requires dose modification, because it is renally excreted. Urinary tract infections should be watched for and treated early. Plasmapheresis may be the treatment of choice for hyperviscosity syndromes. Although the pneumococcus is a dreaded pathogen in myeloma patients, pneumococ cal polysaccharide

1	for and treated early. Plasmapheresis may be the treatment of choice for hyperviscosity syndromes. Although the pneumococcus is a dreaded pathogen in myeloma patients, pneumococ cal polysaccharide vaccines may not elicit an antibody response. Prophylactic administration of intravenous γ globulin preparations is used in the setting of recurrent serious infections. Chronic oral antibiotic prophylaxis is not warranted. Patients developing neurologic symptoms in the lower extremities, severe localized back pain, or problems with bowel and bladder control may need emergency MRI and local radiation therapy and glucocorticoids if cord compression is identified. In patients in whom neurologic deficit is increasing or substantial, emergent surgical decompression may be necessary. Most bone lesions respond to analgesics and systemic therapy, but certain painful lesions may respond most promptly to localized radiation. The anemia associated with myeloma may respond to erythropoietin along with

1	respond to analgesics and systemic therapy, but certain painful lesions may respond most promptly to localized radiation. The anemia associated with myeloma may respond to erythropoietin along with hematinics (iron, folate, cobalamin). The pathogenesis of the anemia should be established and specific therapy instituted, whenever possible.

1	In 1948, Waldenström described a malignancy of lymphoplasmacytoid cells that secreted IgM. In contrast to myeloma, the disease was associated with lymphadenopathy and hepatosplenomegaly, but the major clinical manifestation was hyperviscosity syndrome. The disease resembles the related diseases chronic lymphocytic leukemia, myeloma, and lymphocytic lymphoma. It originates from a post– germinal center B cell that has undergone somatic mutations and antigenic selection in the lymphoid follicle and has the characteristics of an IgM-bearing memory B cell. Waldenström’s macroglobulinemia (WM) and IgM myeloma follow a similar clinical course, but therapeutic options are different. The diagnosis of IgM myeloma is usually reserved for patients with lytic bone lesions and predominant infiltration with CD138+ plasma cells in the bone marrow. Such patients are at greater risk of pathologic fractures than patients with WM.

1	A familial occurrence is common in WM, but its molecular bases are yet unclear. A distinct MYD88 L265P somatic mutation has been reported in over 90% of patients with WM and the majority of IgM MGUS. Presence of this mutation is now used as a diagnostic test to discriminate WM from marginal zone lymphomas (MZLs), IgM-secreting myeloma, and chronic lymphocytic leukemia (CLL) with plasmacytic differentiation. This mutation also explains the molecular pathogenesis of the disease, with involvement of Toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) signaling leading to 718 activation of IL-1R–associated kinase (IRAK) 4 and IRAK1 followed by nuclear factor-κB (NF-κB) activation. The disease is similar to myeloma in being slightly more common in men and occurring with increased incidence with increasing age (median 64 years). There have been reports that the IgM in some patients with macroglobulinemia may have specificity for myelin-associated glycoprotein (MAG), a protein that

1	with increasing age (median 64 years). There have been reports that the IgM in some patients with macroglobulinemia may have specificity for myelin-associated glycoprotein (MAG), a protein that has been associated with demyelinating disease of the peripheral nervous system and may be lost earlier and to a greater extent than the better known myelin basic protein in patients with multiple sclerosis. Sometimes patients with macroglobulinemia develop a peripheral neuropathy, and half of these patients are positive for anti-MAG antibody. The neuropathy may precede the appearance of the neoplasm. There is speculation that the whole process begins with a viral infection that may elicit an antibody response that cross-reacts with a normal tissue component. Like myeloma, the disease involves the bone marrow, but unlike myeloma, it does not cause bone lesions or hypercalcemia. Bone marrow shows >10% infiltration with lymphoplasmacytic cells (surface IgM+, CD19+, CD20+, and CD22+, rarely CD5+,

1	bone marrow, but unlike myeloma, it does not cause bone lesions or hypercalcemia. Bone marrow shows >10% infiltration with lymphoplasmacytic cells (surface IgM+, CD19+, CD20+, and CD22+, rarely CD5+, but CD10− and CD23−) with an increase in number of mast cells. Like myeloma, an M component is present in the serum in excess of 30 g/L (3 g/dL), but unlike myeloma, the size of the IgM paraprotein results in little renal excretion, and only ~20% of patients excrete light chains. Therefore, renal disease is not common. The light chain isotype is kappa in 80% of the cases. Patients present with weakness, fatigue, and recurrent infections similar to myeloma patients, but epistaxis, visual disturbances, and neurologic symptoms such as peripheral neuropathy, dizziness, headache, and transient paresis are much more common in macroglobulinemia. Physical examination reveals adenopathy and hepatosplenomegaly, and ophthalmoscopic examination may reveal vascular segmentation and dilation of the

1	paresis are much more common in macroglobulinemia. Physical examination reveals adenopathy and hepatosplenomegaly, and ophthalmoscopic examination may reveal vascular segmentation and dilation of the retinal veins characteristic of hyperviscosity states. Patients may have a normocytic, normochromic anemia, but rouleaux formation and a positive Coombs’ test are much more common than in myeloma. Malignant lymphocytes are usually present in the peripheral blood. About 10% of macroglobulins are cryoglobulins. These are pure M components and are not the mixed cryoglobulins seen in rheumatoid arthritis and other autoimmune diseases. Mixed cryoglobulins are composed of IgM or IgA complexed with IgG, for which they are specific. In both cases, Raynaud’s phenomenon and serious vascular symptoms precipitated by the cold may occur, but mixed cryoglobulins are not commonly associated with malignancy. Patients suspected of having a cryoglobulin based on history and physical examination should have

1	by the cold may occur, but mixed cryoglobulins are not commonly associated with malignancy. Patients suspected of having a cryoglobulin based on history and physical examination should have their blood drawn into a warm syringe and delivered to the laboratory in a container of warm water to avoid errors in quantitating the cryoglobulin.

1	Control of serious hyperviscosity symptoms such as an altered state of consciousness or paresis can be achieved acutely by plasmapheresis because 80% of the IgM paraprotein is intravascular. The median survival of affected individuals is ~50 months, similar to that of MM. However, many patients with WM have indolent disease that does not require therapy. Pretreatment parameters including older age, male sex, general symptoms, and cytopenias define a high-risk population. Treatment is usually not initiated unless the disease is symptomatic or increasing anemia, hyperviscosity, lymphadenopathy, or hepatosplenomegaly is present. Bortezomib and bendamustine are two agents with significant efficacy in WM. Rituximab (anti-CD20) can produce responses, alone or combined with either of these two agents. Rituximab can produce IgM flare, so its use is initially withheld in patients with high IgM levels. Fludarabine (25 mg/m2 per day for 5 days every 4 weeks) and cladribine (0.1 mg/kg per day for

1	Rituximab can produce IgM flare, so its use is initially withheld in patients with high IgM levels. Fludarabine (25 mg/m2 per day for 5 days every 4 weeks) and cladribine (0.1 mg/kg per day for 7 days every 4 weeks) are also highly effective single agents. With identification of the MYD88 mutation, BTK and IRAK1/4 inhibitors are being evaluated and show significant responses. Although high-dose therapy plus autologous transplantation is an option, its use has declined due to the availability of other effective agents.

1	The features of this syndrome are polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes (POEMS). Diagnostic criteria are described in Table 136-1. Patients usually have a severe, progressive sensorimotor polyneuropathy associated with sclerotic bone lesions from myeloma. Polyneuropathy occurs in ~1.4% of myelomas, but the POEMS syndrome is only a rare subset of that group. Unlike typical myeloma, hepatomegaly and lymphadenopathy occur in about two-thirds of patients, and splenomegaly is seen in one-third. The lymphadenopathy frequently resembles Castleman’s disease histologically, a condition that has been linked to IL-6 overproduction. The endocrine manifestations include amenorrhea in women and impotence and gynecomastia in men. Hyperprolactinemia due to loss of normal inhibitory control by the hypothalamus may be associated with other central nervous system manifestations such as papilledema and elevated cerebrospinal fluid pressure and protein. Type 2 diabetes

1	inhibitory control by the hypothalamus may be associated with other central nervous system manifestations such as papilledema and elevated cerebrospinal fluid pressure and protein. Type 2 diabetes mellitus occurs in about one-third of patients. Hypothyroidism and adrenal insufficiency are occasionally noted. Skin changes are diverse: hyperpigmentation, hypertrichosis, skin thickening, and digital clubbing. Other manifestations include peripheral edema, ascites, pleural effusions, fever, and thrombocytosis. Not all the components of POEMS syndrome may be present initially.

1	The pathogenesis of the disease is unclear, but high circulating levels of the proinflammatory cytokines IL-1, IL-6, VEGF, and TNF have been documented, and levels of the inhibitory cytokine transforming growth factor β are lower than expected. Treatment of the myeloma may result in an improvement in the other disease manifestations. Patients are often treated similarly to those with myeloma. Plasmapheresis does not appear to be of benefit in POEMS syndrome. Patients presenting with isolated sclerotic lesions may have resolution of neuropathic symptoms after local therapy for plasmacytoma with radiotherapy. Similar to multiple myeloma, novel agents and high-dose therapy with autologous stem cell transplantation have been pursued in selected patients and have been associated with prolonged progression-free survival.

1	The heavy chain diseases are rare lymphoplasmacytic malignancies. Their clinical manifestations vary with the heavy chain isotype. Patients have absence of light chain and secrete a defective heavy chain that usually has an intact Fc fragment and a deletion in the Fd region. Gamma, alpha, and mu heavy chain diseases have been described, but no reports of delta or epsilon heavy chain diseases have appeared. Molecular biologic analysis of these tumors has revealed structural genetic defects that may account for the aberrant chain secreted.

1	This disease affects individuals of widely different age groups and countries of origin. It is characterized by lymphadenopathy, fever, anemia, malaise, hepatosplenomegaly, and weakness. It is frequently associated with autoimmune diseases, especially rheumatoid arthritis. Its most distinctive symptom is palatal edema, resulting from involvement of nodes in Waldeyer’s ring, and this may progress to produce respiratory compromise. The diagnosis depends on the demonstration of an anomalous serum M component (often <20 g/L [<2 g/dL]) that reacts with anti-IgG but not anti–light chain reagents. The M component is typically present in both serum and urine. Most of the paraproteins have been of the γ1 subclass, but other subclasses have been seen. The patients may have thrombocytopenia, eosinophilia, and nondiagnostic bone marrow that may show increased numbers of lymphocytes or plasma cells that do not stain for light chain. Patients usually have a rapid downhill course and die of

1	eosinophilia, and nondiagnostic bone marrow that may show increased numbers of lymphocytes or plasma cells that do not stain for light chain. Patients usually have a rapid downhill course and die of infection; however, some patients have survived 5 years with chemotherapy. Therapy is indicated when symptomatic and involves chemotherapeutic combinations used in low-grade lymphoma. Rituximab has also been reported to show efficacy.

1	This is the most common of the heavy chain diseases. It is closely related to a malignancy known as Mediterranean lymphoma, a disease that affects young persons in parts of the world where intestinal parasites are common, such as the Mediterranean, Asia, and South America. The disease is characterized by an infiltration of the lamina propria of the small intestine with lymphoplasmacytoid cells that secrete truncated alpha chains. Demonstrating alpha heavy chains is difficult because the alpha chains tend to polymerize and appear as a smear instead of a sharp peak on electrophoretic profiles. Despite the polymerization, hyperviscosity is not a common problem in alpha heavy chain disease. Without J chain–facilitated dimerization, viscosity does not increase dramatically. Light chains are absent from serum and urine. The patients present with chronic diarrhea, weight loss, and malabsorption and have extensive mesenteric and paraaortic adenopathy. Respiratory tract involvement occurs

1	absent from serum and urine. The patients present with chronic diarrhea, weight loss, and malabsorption and have extensive mesenteric and paraaortic adenopathy. Respiratory tract involvement occurs rarely. Patients may vary widely in their clinical course. Some may develop diffuse aggressive histologies of malignant lymphoma. Chemotherapy may produce long-term remissions. Rare patients appear to have responded to antibiotic therapy, raising the question of the etiologic role of antigenic stimulation, perhaps by some chronic intestinal infection. Chemotherapy plus antibiotics may be more effective than chemotherapy alone. Immunoproliferative small-intestinal disease (IPSID) is recognized as an infectious pathogen–associated human lymphoma that has association with Campylobacter jejuni. It involves mainly the proximal small intestine resulting in malabsorption, diarrhea, and abdominal pain. IPSID is associated with excessive plasma cell differentiation and produces truncated alpha heavy

1	mainly the proximal small intestine resulting in malabsorption, diarrhea, and abdominal pain. IPSID is associated with excessive plasma cell differentiation and produces truncated alpha heavy chain proteins lacking the light chains as well as the first constant domain. Early-stage IPSID responds to antibiotics (30–70% complete remission). Most untreated IPSID patients progress to lymphoplasmacytic and immunoblastic lymphoma. Patients not responding to antibiotic therapy are considered for treatment with combination chemotherapy used to treat low-grade lymphoma.

1	The secretion of isolated mu heavy chains into the serum appears to occur in a very rare subset of patients with CLL. The only features that may distinguish patients with mu heavy chain disease are the presence of vacuoles in the malignant lymphocytes and the excretion of kappa light chains in the urine. The diagnosis requires ultracentrifugation or gel filtration to confirm the nonreactivity of the paraprotein with the light chain reagents, because some intact macroglobulins fail to interact with these serums. The tumor cells seem to have a defect in the assembly of light and heavy chains, because they appear to contain both in their cytoplasm. There is no evidence that such patients should be treated differently from other patients with CLL (Chap. 134).

1	erone proteins during the process of synthesis and secretion, to ensure that they achieve correct tertiary conformation and function, and to eliminate proteins that misfold. However, genetic mutation, incorrect processing, and other factors may favor misfolding, with consequent loss of normal protein function and intracellular or extracellular aggregation. Many diseases, ranging from cystic fibrosis to Alzheimer’s disease, are now known to involve protein misfolding. In the amyloidoses, the aggregates are typically extracellular, and the misfolded protein subunits assume a common antiparallel, β-pleated sheet–rich structural conformation that leads to the formation of higher-order oligomers and then of fibrils with unique staining properties. The term amyloid was coined around 1854 by the pathologist Rudolf Virchow, who thought that these deposits resembled starch (Latin amylum) 719 under the microscope.

1	Amyloid diseases, defined by the biochemical nature of the protein composing the fibril deposits, are classified according to whether they are systemic or localized, whether they are acquired or inherited, and their clinical patterns (Table 137-1). The standard nomenclature is AX, where A indicates amyloidosis and X represents the protein present in the fibril. This chapter focuses primarily on the systemic forms. AL refers to amyloid composed of immunoglobulin light chains (LCs); this disorder, formerly termed primary systemic amyloidosis, arises from a clonal B cell or plasma cell disorder and can be associated with myeloma or lymphoma. AF refers to the familial amyloidoses, which are most commonly due to mutations in transthyretin (TTR), the transport protein for thyroid hormone and retinol-binding protein. AA amyloid is composed of the acute-phase reactant protein serum amyloid A (SAA) and occurs in the setting of chronic inflammatory or infectious diseases; for this reason, this

1	protein. AA amyloid is composed of the acute-phase reactant protein serum amyloid A (SAA) and occurs in the setting of chronic inflammatory or infectious diseases; for this reason, this type was formerly known as secondary amyloidosis. Aβ2M amyloid results from misfolded β2-microglobulin, occurring in individuals with long-standing renal disease who have undergone dialysis, typically for years. Aβ, the most common form of localized amyloidosis, is found in the brain of patients with Alzheimer’s disease after abnormal proteolytic processing and aggregation of polypeptides derived from the amyloid precursor protein.

1	Diagnosis and treatment of the amyloidoses rest upon the histopathologic identification of amyloid deposits and immunohistochemical, biochemical, or genetic determination of amyloid type (Fig. 137-1).

1	In the systemic amyloidoses, the clinically involved organs can be biopsied, but amyloid deposits may be found in any tissue of the body. Historically, blood vessels of the gingiva or rectal mucosa were often examined, but the most easily accessible tissue—positive in more than 80% of patients with systemic amyloidosis—is fat. After local anesthesia, fat is aspirated from the abdominal pannus with a 16-gauge needle. Fat globules expelled onto a glass slide can be stained, thus avoiding a surgical procedure. If this material is negative, more invasive biopsies of the kidney, heart, liver, or gastrointestinal tract can be considered in patients in whom amyloidosis is suspected. The regular β-sheet structure of amyloid deposits exhibits a unique “apple green” birefringence by polarized light microscopy when stained with Congo red dye; other regular protein structures (e.g., collagen) appear white under these conditions. The 10-nm-diameter fibrils can also be visualized by electron

1	light microscopy when stained with Congo red dye; other regular protein structures (e.g., collagen) appear white under these conditions. The 10-nm-diameter fibrils can also be visualized by electron microscopy of paraformaldehyde-fixed tissue. Once amyloid is found, the protein type must be determined by immunohistochemistry, immunoelectron microscopy, or extraction and biochemical analysis employing mass spectrometry; gene sequencing is used to identify mutants causing AF amyloid. The patient’s history, physical findings, and clinical presentation, including age and ethnic origin, organ system involvement, underlying diseases, and family history, may provide helpful clues as to the type of amyloid. However, there can be considerable overlap in clinical presentations, and accurate typing is essential to guide appropriate therapy.

1	The mechanisms of fibril formation and tissue toxicity remain controversial. The “amyloid hypothesis,” as it is currently understood, proposes that precursor proteins undergo a process of reversible unfolding or misfolding; misfolded proteins form oligomeric aggregates, higher-order polymers, and then fibrils that deposit in tissues. Accumulating evidence suggests that the oligomeric intermediates may constitute the most toxic species. Oligomers are more capable than large fibrils of interacting with cells and inducing formation of reactive oxygen species and stress signaling. Ultimately, the fibrillar tissue deposits are likely to interfere with normal organ function. A more sophisticated understanding of the mechanisms leading to amyloid formation and cell and tissue dysfunction will continue to provide new targets for therapies.

1	The clinical syndromes of the amyloidoses are associated with relatively nonspecific alterations in routine laboratory tests. Blood counts are usually normal, although the erythrocyte sedimentation rate is frequently elevated. Patients with glomerular kidney involvement generally have proteinuria, often in the nephrotic range, leading to Amyloidosis David C. Seldin, John L. Berk GENERAL PRINCIPLES Amyloidosis is the term for a group of protein folding disorders char-acterized by the extracellular deposition of insoluble polymeric protein fibrils in tissues and organs. A robust cellular machinery exists to chap-137 aLocalized AL deposits can occur in skin, conjunctiva, urinary bladder, and the tracheobronchial tree. bSecondary to chronic inflammation or infection or to a hereditary periodic fever syndrome such as familial Mediterranean fever. Wild-type transthyretin (usually males >65, cardiac)

1	Wild-type transthyretin (usually males >65, cardiac) Mutant ApoAI, ApoAII, fibrinogen, lysozyme, gelsolin No further work-up (Screen for cardiac, renal, hepatic, autonomic involvement, and factor X deficiency) (Screen for renal, (Screen for neuropathy, cardiomyopathy; screen relatives) Familial amyloidosis of rare type (Screen for renal, hepatic, GI involvement) FIGURE 137-1 Algorithm for the diagnosis of amyloidosis and determination of type. Clinical suspicion: unexplained nephropathy, cardiomyopathy, neuropathy, enteropathy, arthropathy, and macroglossia. ApoAI, apolipoprotein AI; ApoAII, apolipoprotein AII; GI, gastrointestinal.

1	hypoalbuminemia that may be severe; patients with serum albumin levels below 2 g/dL generally have pedal edema or anasarca. Amyloid cardiomyopathy is characterized by concentric ventricular hypertrophy and diastolic dysfunction associated with elevation of brain natriuretic peptide or N-terminal pro–brain natriuretic peptide as well as troponin. These cardiac biomarkers can be used for disease staging, prognostication, and disease activity monitoring in patients with AL amyloidosis. Notably, renal insufficiency can falsely elevate levels of these biomarkers. Recently, biomarkers of cardiac remodeling— i.e., matrix metalloproteinases and tissue inhibitors of metalloproteinases—have been found to be altered in the serum of patients with amyloid cardiomyopathy. Electrocardiographic and echocardiographic features of amyloid cardiomyopathy are described below. Patients with liver involvement, even when advanced, usually develop cholestasis with an elevated alkaline phosphatase

1	and echocardiographic features of amyloid cardiomyopathy are described below. Patients with liver involvement, even when advanced, usually develop cholestasis with an elevated alkaline phosphatase concentration but minimal alteration of the aminotransferases and preservation of synthetic function. In AL amyloidosis, endocrine organs may be infiltrated with fibrils, and hypothyroidism, hypoadrenalism, or even hypopituitarism can occur. Although none of these findings is specific for amyloidosis, the presence of abnormalities in multiple organ systems should raise suspicion regarding this diagnosis.

1	AL AMYLOIDOSIS Etiology and Incidence AL amyloidosis is most frequently caused by a clonal expansion of bone-marrow plasma cells that secrete a monoclonal immunoglobulin LC depositing as amyloid fibrils in tissues. Whether the clonal plasma cells produce an LC that misfolds and leads to AL amyloidosis or an LC that folds properly, allowing the cells to inexorably expand over time and develop into multiple myeloma (Chap. 136), may depend upon primary sequence or other genetic or epigenetic factors. AL amyloidosis can occur with multiple myeloma or other B lymphoproliferative diseases, including non-Hodgkin’s lymphoma (Chap. 134) and Waldenström’s macroglobulinemia (Chap. 136). AL amyloidosis is the most common type of systemic amyloidosis diagnosed in North America. Its incidence has been estimated at 4.5 cases/100,000 population; however, ascertainment continues to be inadequate, and the true incidence may be much higher. AL amyloidosis, like other plasma cell diseases, usually occurs

1	at 4.5 cases/100,000 population; however, ascertainment continues to be inadequate, and the true incidence may be much higher. AL amyloidosis, like other plasma cell diseases, usually occurs after age 40 and is often rapidly progressive and fatal if untreated.

1	Pathology and Clinical Features Amyloid deposits are usually widespread in AL amyloidosis and can be present in the interstitium of any organ outside the central nervous system. The amyloid fibril deposits are composed of full-length 23-kDa monoclonal immunoglobulin LCs as well as fragments. Accessory molecules co-deposited with LC fibrils (as well as with other amyloid fibrils) include serum amyloid P component, other proteins, glycosaminoglycans, and metal ions. Although all kappa and lambda LC subtypes have been identified in AL amyloid fibrils, lambda subtypes predominate. The lambda 6 subtype appears to have unique structural properties that predispose it to fibril formation, often in the kidney.

1	AL amyloidosis is often a rapidly progressive disease that presents as a pleiotropic set of clinical syndromes, recognition of which is key for initiation of the appropriate workup. Nonspecific symptoms of fatigue and weight loss are common; however, the diagnosis is rarely considered until symptoms referable to a specific organ develop. The kidneys are the most frequently involved organ and are affected in 70–80% of patients. Renal amyloidosis usually manifests as proteinuria, often in the nephrotic range and associated with hypoalbuminemia, secondary hypercholesterolemia and hypertriglyceridemia, and edema or anasarca. In some patients, interstitial rather than glomerular amyloid deposition can produce azotemia without proteinuria. The heart is the second most commonly affected organ (50–60% of patients), and cardiac involvement is the leading cause of death from AL amyloidosis. Early on, the electrocardiogram may show low voltage in the limb leads with a pseudo-infarct pattern.

1	(50–60% of patients), and cardiac involvement is the leading cause of death from AL amyloidosis. Early on, the electrocardiogram may show low voltage in the limb leads with a pseudo-infarct pattern. Echocardiographic features of disease include concentrically thickened ventricles and diastolic dysfunction with an abnormal strain pattern a “sparkly” appearance has been described but is often not seen with modern high-resolution echocardiographic techniques. Poor atrial contractility occurs even in sinus rhythm, and patients with cardiac amyloidosis are at risk for development of atrial thrombi and stroke. Cardiac MRI can show increased wall thickness, and characteristic enhancement of the subendocardium has been described following injection of gadolinium contrast. Nervous system symptoms include peripheral sensorimotor neuropathy and/or autonomic dysfunction manifesting as gastrointestinal motility disturbances (early satiety, diarrhea, constipation), impotence, orthostatic

1	symptoms include peripheral sensorimotor neuropathy and/or autonomic dysfunction manifesting as gastrointestinal motility disturbances (early satiety, diarrhea, constipation), impotence, orthostatic hypotension, and/or neurogenic bladder. Macroglossia (Fig. 137-2A), a pathognomonic sign of AL amyloidosis, is seen in only ~10% of patients. Liver involvement causes cholestasis and hepatomegaly. The spleen is frequently involved, and there may 721 be functional hyposplenism in the absence of significant splenomegaly. Many patients experience “easy bruising” due to amyloid deposits in capillaries or deficiency of clotting factor X, which can bind to amyloid fibrils; cutaneous ecchymoses appear, particularly around the eyes, producing the “raccoon-eye” sign Fig. 137-2B), another uncommon but pathognomonic finding. Other findings include nail dystrophy (Fig. 137-2C), alopecia, and amyloid arthropathy with thickening of synovial membranes in the wrists and shoulders. The presence of a

1	but pathognomonic finding. Other findings include nail dystrophy (Fig. 137-2C), alopecia, and amyloid arthropathy with thickening of synovial membranes in the wrists and shoulders. The presence of a multisystemic illness or general fatigue along with any of these clinical syndromes should prompt a workup for amyloidosis.

1	Diagnosis Identification of an underlying clonal plasma cell or B lymphoproliferative process and a clonal LC are key to the diagnosis of AL amyloidosis. Serum protein electrophoresis and urine protein electrophoresis, although of value in multiple myeloma, are not useful screening tests if AL amyloidosis is suspected because the clonal LC or whole immunoglobulin often is not present in sufficient amounts to produce a monoclonal “M-spike” in the serum or LC (Bence Jones) protein in the urine. However, more than 90% of patients with AL amyloidosis have serum or urine monoclonal LC or whole immunoglobulin detectable by immunofixation electrophoresis of serum (SIFE) or urine (UIFE) (Fig. 137-3A) or by nephelometric measurement of “free” LCs (i.e., LCs circulating in monomeric form rather than in an immunoglobulin tetramer with heavy chain). Examining the ratio as well as the absolute amount of free LCs is essential, as renal insufficiency reduces LC clearance, elevating both isotypes. In

1	in an immunoglobulin tetramer with heavy chain). Examining the ratio as well as the absolute amount of free LCs is essential, as renal insufficiency reduces LC clearance, elevating both isotypes. In addition, an increased percentage of plasma cells in the bone marrow—typically 5–30% of nucleated cells—is found in ~90% of patients. Kappa or lambda clonality should be demonstrated by flow cytometry, immunohistochemistry, or in situ hybridization for LC mRNA (Fig. 137-3B).

1	A monoclonal serum protein by itself is not diagnostic of amyloidosis, since monoclonal gammopathy of uncertain significance is common in older patients (Chap. 136). However, when monoclonal gammopathy of uncertain significance is found in patients with biopsy-proven amyloidosis, the AL type should be strongly suspected. Similarly, patients thought to have “smoldering myeloma” because of a modest elevation of bone-marrow plasma cells should be screened for AL amyloidosis if they have signs or symptoms of renal, cardiac, or neurologic disease. Accurate tissue amyloid typing is essential for appropriate treatment. Immunohistochemical staining of the amyloid deposits is useful if they bind one LC antibody in preference to the other; some AL deposits bind antibodies nonspecifically. Immunoelectron microscopy is more reliable, and mass spectrometry–based microsequencing of small amounts of protein extracted from fibril deposits can also be undertaken. In ambiguous cases, other forms of

1	microscopy is more reliable, and mass spectrometry–based microsequencing of small amounts of protein extracted from fibril deposits can also be undertaken. In ambiguous cases, other forms of amyloidosis should be thoroughly excluded with appropriate genetic and other testing.

1	Extensive multisystemic involvement typifies AL amyloidosis, and the median survival period without treatment is usually only ~1–2 years from the time of diagnosis. Current therapies target the FIGURE 137-2 Clinical signs of AL amyloidosis. A. Macroglossia. B. Periorbital ecchymoses. C. Fingernail dystrophy.

1	FIGURE 137-3 Laboratory features of AL amyloidosis. A. Serum immunofixation electrophoresis reveals an IgGκ monoclonal protein in this example; serum protein electrophoresis is often normal. B. Bone-marrow biopsy sections stained by immunohistochemistry with antibody to CD138 (syndecan, highly expressed on plasma cells) (left) or by in situ hybridization with fluorescein-tagged probes (Ventana Medical Systems) binding to κ mRNA (center) and λ mRNA (right) in plasma cells. (Photomicrograph courtesy of C. O’Hara; with permission.) clonal bone-marrow plasma cells, using approaches employed for multiple myeloma. Treatment with oral melphalan and prednisone can decrease the plasma cell burden but rarely leads to complete hematologic remission, meaningful organ responses, or improved survival and is no longer widely used. The substitution of dexamethasone for prednisone produces a higher response rate and more durable remissions, although dexamethasone is not always well tolerated by

1	and is no longer widely used. The substitution of dexamethasone for prednisone produces a higher response rate and more durable remissions, although dexamethasone is not always well tolerated by patients with significant edema or cardiac disease. High-dose IV melphalan followed by autologous stem cell transplantation (HDM/SCT) produces complete hematologic responses in ~40% of treated patients, as determined by loss of clonal plasma cells in the bone marrow and disappearance of the monoclonal LC, as determined by SIFE/UIFE and free LC quantitation. Hematologic responses can be followed in the subsequent 6–12 months as improvements in organ function and quality of life. Hematologic responses appear to be more durable after HDM/SCT than in multiple myeloma, with remissions continuing in some patients beyond 15 years without additional treatment. Unfortunately, only about half of AL amyloidosis patients are suitable for aggressive treatment, and, even at specialized treatment centers,

1	patients beyond 15 years without additional treatment. Unfortunately, only about half of AL amyloidosis patients are suitable for aggressive treatment, and, even at specialized treatment centers, transplantation-related mortality rates are higher than those for other hematologic diseases because of impaired organ function. Amyloid cardiomyopathy, poor nutritional and performance status, and multiorgan disease contribute to excess morbidity and mortality. A bleeding diathesis resulting from adsorption of clotting factor X to amyloid fibrils also increases mortality rates; however, this syndrome occurs in only 5–10% of patients. A randomized multicenter trial conducted in France compared oral melphalan and dexamethasone with HDM/ SCT and failed to show a benefit of dose-intensive treatment, although the transplantation-related mortality rate in this study was very high. It has become clear that careful selection of patients and expert peritransplantation management are essential in

1	although the transplantation-related mortality rate in this study was very high. It has become clear that careful selection of patients and expert peritransplantation management are essential in reducing transplantation-related mortality.

1	For patients with impaired cardiac function or arrhythmias due to amyloid involvement of the myocardium, the median survival period is only ~6 months without treatment. In these patients, cardiac transplantation can be performed and followed by HDM/SCT to eliminate the noxious clone and prevent amyloid deposition in the transplanted heart or other organs. Novel anti–plasma cell agents have been investigated for treatment of plasma cell diseases. The immunomodulators thalidomide, lenalidomide, and pomalidomide display activity; dosing may need to be adjusted compared to their usage for myeloma. The proteasome inhibitor bortezomib has also been found to be effective in single-center and multicenter trials. Anti-fibril small molecules and humanized monoclonal antibodies are also being tested. Clinical trials are essential in improving therapy for this rare disease.

1	Supportive care is important for patients with any type of amyloidosis. For nephrotic syndrome, diuretics and supportive stockings can ameliorate edema; angiotensin-converting enzyme inhibitors should be used with caution and have not been shown to slow renal disease progression. Effective diuresis can be facilitated with albumin infusions to raise intravascular oncotic pressure. Congestive heart failure due to amyloid cardiomyopathy is best treated with diuretics; it is important to note that digitalis, calcium channel blockers, and beta blockers are relatively contraindicated as they can interact with amyloid fibrils and produce heart block and worsening heart failure. Amiodarone has been used for atrial and ventricular arrhythmias. Automatic implantable defibrillators have reduced effectiveness due to the thickened myocardium, but they may benefit some patients. Atrial ablation is an effective approach for atrial fibrillation. For conduction abnormalities, ventricular pacing may be

1	due to the thickened myocardium, but they may benefit some patients. Atrial ablation is an effective approach for atrial fibrillation. For conduction abnormalities, ventricular pacing may be indicated. Atrial contractile dysfunction is common in amyloid cardiomyopathy and is an indication for anticoagulation even in the absence of atrial fibrillation. Autonomic neuropathy can be treated with α agonists such as midodrine to support the blood pressure; gastrointestinal dysfunction may respond to motility or bulk agents. Nutritional supplementation, either oral or parenteral, is also important.

1	In localized AL disease, amyloid deposits can be produced by clonal plasma cells infiltrating local sites in the airways, bladder, skin, or lymph nodes (Table 137-1). These deposits may respond to surgical intervention or low-dose radiation therapy (typically only 20 Gy); systemic treatment generally is not appropriate. Patients should be referred to a center familiar with management of these rare manifestations of amyloidosis.

1	AA AMYLOIDOSIS Etiology and Incidence AA amyloidosis can occur in association with almost any chronic inflammatory state (e.g., rheumatoid arthritis, inflammatory bowel disease, familial Mediterranean fever [Chap. 392], or other periodic fever syndromes) or chronic infections such as tuberculosis or subacute bacterial endocarditis. In the United States and Europe, AA amyloidosis has become less common, occurring in fewer than 2% of patients with these diseases, presumably because of advances in anti-inflammatory and antimicrobial therapies. It has also been described in association with Castleman’s disease, and patients with AA amyloidosis should undergo CT scanning to look for such tumors as well as serologic and microbiologic studies. AA amyloidosis can also be seen without any identifiable underlying disease. AA is the only type of systemic amyloidosis that occurs in children.

1	Pathology and Clinical Features Organ involvement in AA amyloidosis usually begins in the kidneys. Hepatomegaly, splenomegaly, and autonomic neuropathy can also occur as the disease progresses; cardiomyopathy occurs, albeit rarely. The symptoms and signs of AA disease cannot be reliably distinguished from those of AL amyloidosis. AA amyloid fibrils are usually composed of an 8-kDa, 76-amino-acid N-terminal portion of the 12-kDa precursor protein SAA. This acute-phase apoprotein is synthesized in the liver and transported by high-density lipoprotein (HDL3) in the plasma. Several years of an underlying inflammatory disease causing chronic elevation of SAA levels usually precede fibril formation, although infections can lead to AA deposition more rapidly.

1	Primary therapy for AA amyloidosis consists of treatment of the underlying inflammatory or infectious disease. Treatment that suppresses or eliminates the inflammation or infection also decreases the SAA concentration. For familial Mediterranean fever, colchicine at a dose of 1.2–1.8 mg/d is the standard treatment. However, 723 colchicine has not been helpful for AA amyloidosis of other causes or for other amyloidoses. Tumor necrosis factor and interleukin 1 antagonists can be effective in syndromes related to cytokine elevation. For this disease, there is also a fibril-specific agent: eprodisate was designed to interfere with the interaction of AA amyloid protein with glycosaminoglycans and to prevent or disrupt fibril formation. The drug is well tolerated and delays progression of AA renal disease. Randomized phase III clinical trials with eprodisate are ongoing; the drug is not otherwise available.

1	The familial amyloidoses are autosomal dominant diseases in which, beginning in midlife, a variant (FINE) plasma protein forms amyloid deposits. These diseases are rare, with an estimated incidence of <1 case/100,000 population in the United States, although founder effects in isolated areas of Portugal, Sweden, and Japan have led to a much higher incidence. The most common form of AF amyloidosis is ATTRm in the updated nomenclature, caused by mutation of the abundant plasma protein transthyretin (TTR, also known as prealbumin). More than 100 TTR mutations are known, and most are associated with ATTR amyloidosis. One variant, V122I, has a carrier frequency that may be as high as 4% in the African-American population and is associated with late-onset cardiac amyloidosis. The actual incidence and penetrance of disease in the African-American population is the subject of ongoing research, but ATTR amyloidosis warrants consideration in the differential diagnosis of African-American

1	and penetrance of disease in the African-American population is the subject of ongoing research, but ATTR amyloidosis warrants consideration in the differential diagnosis of African-American patients who present with concentric cardiac hypertrophy and evidence of diastolic dysfunction, particularly in the absence of a history of hypertension. Other familial amyloidoses, caused by variant apolipoproteins AI or AII, gelsolin, fibrinogen Aα, or lysozyme, are reported in only a few families worldwide. New amyloidogenic serum proteins continue to be identified periodically, including recently the leukocyte chemotactic factor LECT2, a cause of renal amyloidosis in Hispanic and Pakistani populations. To date, no mutation in the coding sequence for the LECT2 gene has been identified, so the heritability of ALECT2 is uncertain.

1	TTR deposits composed of unmutated fibrils occur with aging, and ATTRwt is being diagnosed with increasing frequency in Caucasian men >65 years of age with amyloid cardiomyopathy. Formerly termed senile systemic amyloidosis, ATTRwt has been found at autopsy in 25% of hearts from patients older than age 80 years. Why a wild type protein becomes amyloidogenic, and why patients bearing mutant TTR genes do not express disease until adulthood, remains a mystery.

1	Clinical Features and Diagnosis AF amyloidosis has a presentation that is variable but is usually consistent within kindreds affected by the same mutant protein. A family history makes AF disease more likely, but many patients present sporadically with new mutations. ATTR amyloidosis typically presents as a syndrome of familial amyloidotic polyneuropathy or familial amyloidotic cardiomyopathy. Peripheral neuropathy begins as a small-fiber lower-extremity sensory and motor neuropathy and progresses to the upper extremities. Autonomic neuropathy manifests as diarrhea with weight loss and orthostatic hypo-tension. Patients with TTR V30M, the most common mutation, have normal electrocardiograms but may develop conduction defects late in the disease. Patients with TTR T60A and several other mutations have myocardial thickening similar to that caused by AL amyloidosis, although heart failure is less common and long-term survival rates are usually better. Vitreous opacities caused by amyloid

1	have myocardial thickening similar to that caused by AL amyloidosis, although heart failure is less common and long-term survival rates are usually better. Vitreous opacities caused by amyloid deposits are pathognomonic for ATTR amyloidosis.

1	Typical syndromes associated with other forms of AF disease include renal amyloidosis with mutant fibrinogen, lysozyme, or apolipoproteins; hepatic amyloidosis with apolipoprotein AI; and amyloidosis of cranial nerves and cornea with gelsolin. Patients with AF amyloidosis can present with clinical syndromes that mimic those of patients with AL disease. Rarely, AF carriers can develop AL disease or AF patients may have monoclonal gammopathy without AL. 724 Thus, it is important to screen both for plasma cell disorders and for mutations in patients with amyloidosis. Variant TTRs can usually be detected by isoelectric focusing, but DNA sequencing is now standard for diagnosis of ATTR and other AF mutations.

1	Without intervention, the survival period after onset of ATTR disease is 5–15 years. Orthotopic liver transplantation replaces the major source of variant TTR production with a source of normal TTR. While liver transplantation can slow disease progression and improve chances of survival, it does not reverse sensorimotor neuropathy. Liver transplants are most successful in young patients with early peripheral neuropathy; older patients with familial amyloidotic cardiomyopathy or advanced polyneuropathy often experience end-organ disease progression despite successful liver transplantation. Progressive disease has been attributed to accumulation of wild-type TTR in fibrillar deposits initiated by the mutant.

1	The rate-limiting step in ATTR amyloidosis is dissociation of the TTR tetramer into monomer followed by misfolding and aggregation. TTR tetramers can be stabilized by thyroxine binding or by small molecules such as the non-steroidal anti-inflammatory drug diflunisal or the rationally designed small-molecule therapeutic tafamidis. A placebo-controlled randomized trial of diflunisal demonstrated a reduction in the progression of polyneuropathy and maintenance of quality of life in patients with a wide variety of ATTR mutations who received the “repurposed” diflunisal. Tafamidis tested in a similar fashion in patients with the V30M ATTR mutation failed to meet its primary endpoints, but tafamidis was approved by the European Medicines Agency since most secondary endpoints favored the drug. These agents are now being investigated for effects on cardiomyopathy, and in ATTRwt. In vitro data and serendipitous observations in patients suggest that ATTRm disease can be ameliorated by

1	drug. These agents are now being investigated for effects on cardiomyopathy, and in ATTRwt. In vitro data and serendipitous observations in patients suggest that ATTRm disease can be ameliorated by “trans-suppression,” in which a T119M TTR variant stabilizes tetramers that also contain amyloidogenic subunits. Interestingly, in a large population study in Denmark, 0.5% of participants were heterozygous for the T119M allele, and this small group had higher levels of TTR in their blood, a reduced incidence of cerebrovascular disease, and a 5to 10-year survival advantage compared with participants lacking this allele.

1	Aβ2M amyloid is composed of β2-microglobulin, the invariant chain of class I human leukocyte antigens, and produces rheumatologic manifestations in patients undergoing long-term hemodialysis. β2Microglobulin is excreted by the kidney, and levels become elevated in end-stage renal disease. The molecular mass of β2M is 11.8 kDa— above the cutoff of some dialysis membranes. The incidence of this disease appears to be declining with the use of newer membranes in high-flow dialysis techniques. Aβ2M amyloidosis usually presents as carpal tunnel syndrome, persistent joint effusions, spondyloarthropathy, or cystic bone lesions. Carpal tunnel syndrome is often the first symptom. In the past, persistent joint effusions accompanied by mild discomfort were found in up to 50% of patients who had undergone dialysis for >12 years. Involvement is bilateral, and large joints (shoulders, knees, wrists, and hips) are most frequently affected. The synovial fluid is noninflammatory, and β2M amyloid can be

1	dialysis for >12 years. Involvement is bilateral, and large joints (shoulders, knees, wrists, and hips) are most frequently affected. The synovial fluid is noninflammatory, and β2M amyloid can be found if the sediment is stained with Congo red. Although less common, visceral β2M amyloid deposits do occasionally occur in the gastrointestinal tract, heart, tendons, and subcutaneous tissues of the buttocks. There is no specific therapy for Aβ2M amyloidosis, but cessation of dialysis after renal allografting may lead to symptomatic improvement.

1	A diagnosis of amyloidosis should be considered in patients with unexplained nephropathy, cardiomyopathy (particularly with diastolic dysfunction), neuropathy (either peripheral or autonomic), enteropathy, or the pathognomonic soft tissue findings of macroglossia or periorbital ecchymoses. Pathologic identification of amyloid fibrils can be made with Congo red staining of aspirated abdominal fat or of an involved-organ biopsy specimen. Accurate typing by a combination of immunologic, biochemical, and genetic testing is essential in selecting appropriate therapy (Fig. 137-1). Systemic amyloidosis should not be considered an untreatable condition, as anti–plasma cell chemotherapy is highly effective in AL disease and targeted therapies are being developed for AA and ATTR disease. Tertiary referral centers can provide specialized diagnostic techniques and access to clinical trials for patients with these rare diseases.

1	This chapter represents a revised version of a chapter that was coauthored by Dr. Martha Skinner and Dr. David Seldin in previous editions of Harrison’s Principles of Internal Medicine. transfusion Biology and therapy Jeffery S. Dzieczkowski, Kenneth C. Anderson BLOOD GROUP ANTIGENS AND ANTIBODIES The study of red blood cell (RBC) antigens and antibodies forms the 138e foundation of transfusion medicine. Serologic studies initially characterized these antigens, but now the molecular composition and structure of many are known. Antigens, either carbohydrate or protein, are assigned to a blood group system based on the structure and similarity of the determinant epitopes. Other cellular blood elements and plasma proteins are also antigenic and can result in alloimmunization, the production of antibodies directed against the blood group antigens of another individual. These antibodies are called alloantibodies.

1	Antibodies directed against RBC antigens may result from “natural” exposure, particularly to carbohydrates that mimic some blood group antigens. Those antibodies that occur via natural stimuli are usually produced by a T cell–independent response (thus, generating no memory) and are IgM isotype. Autoantibodies (antibodies against autologous blood group antigens) arise spontaneously or as the result of infectious sequelae (e.g., from Mycoplasma pneumoniae) and are also often IgM. These antibodies are often clinically insignificant due to their low affinity for antigen at body temperature. However, IgM antibodies can activate the complement cascade and result in hemolysis. Antibodies that result from allogeneic exposure, such as transfusion or pregnancy, are usually IgG. IgG antibodies commonly bind to antigen at warmer temperatures and may hemolyze RBCs. Unlike IgM antibodies, IgG antibodies can cross the placenta and bind fetal erythrocytes bearing the corresponding antigen, resulting

1	bind to antigen at warmer temperatures and may hemolyze RBCs. Unlike IgM antibodies, IgG antibodies can cross the placenta and bind fetal erythrocytes bearing the corresponding antigen, resulting in hemolytic disease of the newborn, or hydrops fetalis.

1	Alloimmunization to leukocytes, platelets, and plasma proteins may also result in transfusion complications such as fevers and urticaria but generally does not cause hemolysis. Assay for these other alloantibodies is not routinely performed; however, they may be detected using special assays.

1	The first blood group antigen system, recognized in 1900, was ABO, the most important in transfusion medicine. The major blood groups of this system are A, B, AB, and O. O type RBCs lack A or B antigens. These antigens are carbohydrates attached to a precursor backbone, may be found on the cellular membrane either as glycosphingolipids or glycoproteins, and are secreted into plasma and body fluids as glycoproteins. H substance is the immediate precursor on which the A and B antigens are added. This H substance is formed by the addition of fucose to the glycolipid or glycoprotein backbone. The subsequent addition of N-acetylgalactosamine creates the A antigen, whereas the addition of galactose produces the B antigen.

1	The genes that determine the A and B phenotypes are found on chromosome 9p and are expressed in a Mendelian codominant manner. The gene products are glycosyl transferases, which confer the enzymatic capability of attaching the specific antigenic carbohydrate. Individuals who lack the “A” and “B” transferases are phenotypically type “O,” whereas those who inherit both transferases are type “AB.” Rare individuals lack the H gene, which codes for fucose transferase, and cannot form H substance. These individuals are homozygous for the silent h allele (hh) and have Bombay phenotype (Oh).

1	The ABO blood group system is important because essentially all individuals produce antibodies to the ABH carbohydrate antigen that they lack. The naturally occurring anti-A and anti-B antibodies are termed isoagglutinins. Thus, type A individuals produce anti-B, whereas type B individuals make anti-A. Neither isoagglutinin is found in type AB individuals, whereas type O individuals produce both anti-A and anti-B. Thus, persons with type AB are “universal recipients” because they do not have antibodies against any ABO phenotype, whereas persons with type O blood can donate to essentially all recipi-138e-1 ents because their cells are not recognized by any ABO isoagglutinins. The rare individuals with Bombay phenotype produce antibodies to H substance (which is present on all red cells except those of hh phenotype) as well as to both A and B antigens and are therefore compatible only with other hh donors.

1	In most people, A and B antigens are secreted by the cells and are present in the circulation. Nonsecretors are susceptible to a variety of infections (e.g., Candida albicans, Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae) because many organisms may bind to polysaccharides on cells. Soluble blood group antigens may block this binding.

1	The Rh system is the second most important blood group system in pretransfusion testing. The Rh antigens are found on a 30to 32-kDa RBC membrane protein that has no defined function. Although >40 different antigens in the Rh system have been described, five determinants account for the vast majority of phenotypes. The presence of the D antigen confers Rh “positivity,” whereas persons who lack the D antigen are Rh negative. Two allelic antigen pairs, E/e and C/c, are also found on the Rh protein. The three Rh genes, E/e, D, and C/c, are arranged in tandem on chromosome 1 and inherited as a haplotype, i.e., cDE or Cde. Two haplotypes can result in the phenotypic expression of two to five Rh antigens. The D antigen is a potent alloantigen. About 15% of individuals lack this antigen. Exposure of these Rh-negative people to even small amounts of Rh-positive cells, by either transfusion or pregnancy, can result in the production of anti-D alloantibody.

1	More than 100 blood group systems are recognized, composed of more than 500 antigens. The presence or absence of certain antigens has been associated with various diseases and anomalies; antigens also act as receptors for infectious agents. Alloantibodies of importance in routine clinical practice are listed in Table 138e-1. Antibodies to Lewis system carbohydrate antigens are the most common cause of incompatibility during pretransfusion screening. The Lewis gene product is a fucosyl transferase and maps to chromosome 19. The antigen is not an integral membrane structure but is adsorbed to the RBC membrane from the plasma. Antibodies to Lewis antigens are usually IgM and cannot cross the placenta. Lewis antigens may be adsorbed onto tumor cells and may be targets of therapy.

1	I system antigens are also oligosaccharides related to H, A, B, and Le. I and i are not allelic pairs but are carbohydrate antigens that differ only in the extent of branching. The i antigen is an unbranched chain that is converted by the I gene product, a glycosyltransferase, into a branched chain. The branching process affects all the ABH antigens, which become progressively more branched in the first 2 years of life. Some patients with cold agglutinin disease or lymphomas can produce anti-I autoantibodies that cause RBC destruction. Occasional patients with mononucleosis or Mycoplasma pneumonia may develop cold agglutinins of either anti-I or anti-i specificity. Most adults lack i Rh (D, C/c, E/e) RBC protein IgG HTR, HDN Lewis (Lea , Leb) Oligosaccharide IgM/IgG Rare HTR Kell (K/k) RBC protein IgG HTR, HDN Duffy (Fya/Fyb) RBC protein IgG HTR, HDN Kidd (Jka/Jkb) RBC protein IgG HTR (often delayed), MNSsU RBC protein IgM/IgG Anti-M rare HDN, anti- S, -s, and -U HDN, HTR

1	MNSsU RBC protein IgM/IgG Anti-M rare HDN, anti- S, -s, and -U HDN, HTR Abbreviations: HDN, hemolytic disease of the newborn; HTR, hemolytic transfusion reac tion; RBC, red blood cell. expression; thus, finding a donor for patients with anti-i is not difficult. Even though most adults express I antigen, binding is generally low at body temperature. Thus, administration of warm blood prevents isoagglutination. The P system is another group of carbohydrate antigens controlled by specific glycosyltransferases. Its clinical significance is in rare cases of syphilis and viral infection that lead to paroxysmal cold hemoglobinuria. In these cases, an unusual autoantibody to P is produced that binds to RBCs in the cold and fixes complement upon warming. Antibodies with these biphasic properties are called Donath-Landsteiner antibodies. The P antigen is the cellular receptor of parvovirus B19 and also may be a receptor for Escherichia coli binding to urothelial cells.

1	The MNSsU system is regulated by genes on chromosome 4. M and N are determinants on glycophorin A, an RBC membrane protein, and S and s are determinants on glycophorin B. Anti-S and anti-s IgG antibodies may develop after pregnancy or transfusion and lead to hemolysis. Anti-U antibodies are rare but problematic; virtually every donor is incompatible because nearly all persons express U.

1	The Kell protein is very large (720 amino acids), and its secondary structure contains many different antigenic epitopes. The immunogenicity of Kell is third behind the ABO and Rh systems. The absence of the Kell precursor protein (controlled by a gene on X) is associated with acanthocytosis, shortened RBC survival, and a progressive form of muscular dystrophy that includes cardiac defects. This rare condition is called the McLeod phenotype. The Kx gene is linked to the 91-kDa component of the NADPH-oxidase on the X chromosome, deletion or mutation of which accounts for about 60% of cases of chronic granulomatous disease. The Duffy antigens are codominant alleles, Fya and Fyb, that also serve as receptors for Plasmodium vivax. More than 70% of persons in malaria-endemic areas lack these antigens, probably from selective influences of the infection on the population. For unknown reasons, the lack of the Duffy antigen receptor for cytokines (DARC) is associated with mild neutropenia.

1	The Kidd antigens, Jka and Jkb, may elicit antibodies transiently. A delayed hemolytic transfusion reaction that occurs with blood tested as compatible is often related to delayed appearance of anti-Jka. Pretransfusion testing of a potential recipient consists of the “type and screen.” The “forward type” determines the ABO and Rh phenotype of the recipient’s RBC by using antisera directed against the A, B, and D antigens. The “reverse type” detects isoagglutinins in the patient’s serum and should correlate with the ABO phenotype, or forward type. The alloantibody screen identifies antibodies directed against other RBC antigens. The alloantibody screen is performed by mixing patient serum with type O RBCs that contain the major antigens of most blood group systems and whose extended phenotype is known. The specificity of the alloantibody is identified by correlating the presence or absence of antigen with the results of the agglutination.

1	Cross-matching is ordered when there is a high probability that the patient will require a packed RBC (PRBC) transfusion. Blood selected for cross-matching must be ABO compatible and lack antigens for which the patient has alloantibodies. Nonreactive cross-matching confirms the absence of any major incompatibility and reserves that unit for the patient. In the case of Rh-negative patients, every attempt must be made to provide Rh-negative blood components to prevent alloimmunization to the D antigen. In an emergency, Rh-positive blood can be safely transfused to an Rh-negative patient who lacks anti-D; however, the recipient is likely to become alloimmunized and produce anti-D. Rh-negative women of childbearing age who are transfused with products containing Rh-positive RBCs should receive passive immunization with anti-D (RhoGam or WinRho) to reduce or prevent sensitization.

1	Blood products intended for transfusion are routinely collected as whole blood (450 mL) in various anticoagulants. Most donated blood is processed into components: PRBCs, platelets, and fresh-frozen plasma (FFP) or cryoprecipitate (Table 138e-2). Whole blood is first separated into PRBCs and platelet-rich plasma by slow centrifugation. The platelet-rich plasma is then centrifuged at high speed to yield one unit of random donor (RD) platelets and one unit of FFP. Cryoprecipitate is produced by thawing FFP to precipitate the plasma proteins and then separated by centrifugation. Apheresis technology is used for the collection of multiple units of platelets from a single donor. These single-donor apheresis platelets (SDAP) contain the equivalent of at least six units of RD platelets and have fewer contaminating leukocytes than pooled RD platelets.

1	Plasma may also be collected by apheresis. Plasma derivatives such as albumin, intravenous immunoglobulin, antithrombin, and coagulation factor concentrates are prepared from pooled plasma from many donors and are treated to eliminate infectious agents. Whole blood provides both oxygen-carrying capacity and volume expansion. It is the ideal component for patients who have sustained acute hemorrhage of ≥25% total blood volume loss. Whole blood is stored at 4°C to maintain erythrocyte viability, but platelet dysfunction and degradation of some coagulation factors occurs. In addition, 2,3-bisphosphoglycerate levels fall over time, leading to an increase in the oxygen affinity of the hemoglobin and a decreased capacity to deliver oxygen to the tissues, a problem with all red cell storage. Fresh whole blood avoids these problems, but it is typically used only in emergency settings (i.e., military). Whole blood is not readily available, because it is routinely processed into components.

1	This product increases oxygen-carrying capacity in the anemic patient. Adequate oxygenation can be maintained with a hemoglobin content of 70 g/L in the normovolemic patient without cardiac disease; however, comorbid factors may necessitate transfusion at a higher threshold. The decision to transfuse should be guided by the clinical situation and not by an arbitrary laboratory value. In the critical care setting, liberal use of transfusions to maintain near-normal levels of hemoglobin has not proven advantageous. In most patients requiring transfusion, levels of hemoglobin of 100 g/L are sufficient to keep oxygen supply from being critically low.

1	PRBCs may be modified to prevent certain adverse reactions. The majority of cellular blood products are now leukocyte reduced, and universal prestorage leukocyte reduction has been recommended. Prestorage filtration appears superior to bedside filtration as smaller amounts of cytokines are generated in the stored product. These PRBC units contain <5 × 106 donor white blood cells (WBCs), and their use Component Volume, mL Content Clinical Response RBCs with variable leukocyte content and small amount of plasma 5.5 × 1010/RD unit Plasma proteins—coagulation factors, proteins C and S, antithrombin Cold-insoluble plasma proteins, fibrinogen, factor VIII, VWF Increase platelet count 5000–10,000/μL CCI ≥10 × 109/L within 1 h and ≥7.5 × 109/L within 24 h posttransfusion Topical fibrin glue, also 80 IU factor VIII

1	Increase platelet count 5000–10,000/μL CCI ≥10 × 109/L within 1 h and ≥7.5 × 109/L within 24 h posttransfusion Topical fibrin glue, also 80 IU factor VIII Abbreviations: CCI, corrected count increment; FFP, fresh-frozen plasma; PRBC, packed red blood cells; RBC, red blood cell; RD, random donor; SDAP, single-donor apheresis platelets; VWF, von Willebrand factor. lowers the incidence of posttransfusion fever, cytomegalovirus (CMV) infections, and alloimmunization. Other theoretical benefits include less immunosuppression in the recipient and lower risk of infections. Plasma, which may cause allergic reactions, can be removed from cellular blood components by washing.

1	Thrombocytopenia is a risk factor for hemorrhage, and platelet transfusion reduces the incidence of bleeding. The threshold for prophylactic platelet transfusion is 10,000/μL. In patients without fever or infections, a threshold of 5000/μL may be sufficient to prevent spontaneous hemorrhage. For invasive procedures, the usual target level is 50,000/μL platelets.

1	Platelets are given either as pools prepared from RDs or as SDAPs from a single donor. In an unsensitized patient without increased platelet consumption (splenomegaly, fever, disseminated intravascular coagulation [DIC]), two units of transfused RD per square-meter body surface area (BSA) is anticipated to increase the platelet count by approximately 10,000/μL. Patients who have received multiple transfusions may be alloimmunized to many HLAand platelet-specific antigens and have little or no increase in their posttransfusion platelet counts. Patients who may require multiple transfusions are best served by receiving SDAP and leukocyte-reduced components to lower the risk of alloimmunization.

1	Refractoriness to platelet transfusion may be evaluated using the corrected count increment (CCI): where BSA is body surface area measured in square meters. The platelet count performed 1 h after the transfusion is acceptable if the CCI is 10 × 109/mL, and after 18–24 h an increment of 7.5 × 109/mL is expected. Patients who have suboptimal responses are likely to have received multiple transfusions and have antibodies directed against class I HLA antigens. Refractoriness can be investigated by detecting anti-HLA antibodies in the recipient’s serum. Patients who are sensitized will often react with 100% of the lymphocytes used for the HLA-antibody screen, and HLA-matched SDAPs should be considered for patients who require transfusion. Although ABO-identical HLA-matched SDAPs provide the best chance for increasing the platelet count, locating these products is difficult. Platelet cross-matching is available in some centers. Additional clinical causes for a low platelet CCI include

1	best chance for increasing the platelet count, locating these products is difficult. Platelet cross-matching is available in some centers. Additional clinical causes for a low platelet CCI include fever, bleeding, splenomegaly, DIC, or medications in the recipient.

1	FFP contains stable coagulation factors and plasma proteins: fibrinogen, antithrombin, albumin, and proteins C and S. Indications for FFP include correction of coagulopathies, including the rapid reversal of warfarin; supplying deficient plasma proteins; and treatment of thrombotic thrombocytopenic purpura. FFP should not be routinely used to expand blood volume. FFP is an acellular component and does not transmit intracellular infections, e.g., CMV. Patients who are IgA-deficient and require plasma support should receive FFP from IgA-deficient donors to prevent anaphylaxis (see below).

1	Cryoprecipitate is a source of fibrinogen, factor VIII, and von Willebrand factor (VWF). It is ideal for supplying fibrinogen to the volume-sensitive patient. When factor VIII concentrates are not available, cryoprecipitate may be used because each unit contains approximately 80 units of factor VIII. Cryoprecipitate may also supply VWF to patients with dysfunctional (type II) or absent (type III) von Willebrand’s disease. Plasma from thousands of donors may be pooled to derive specific protein concentrates, including albumin, intravenous immunoglobulin, antithrombin, and coagulation factors. In addition, donors who have high-titer antibodies to specific agents or antigens provide hyper-138e-3 immune globulins, such as anti-D (RhoGam, WinRho), and antisera to hepatitis B virus (HBV), varicella-zoster virus, CMV, and other infectious agents.

1	Adverse reactions to transfused blood components occur despite multiple tests, inspections, and checks. Fortunately, the most common reactions are not life threatening, although serious reactions can present with mild symptoms and signs. Some reactions can be reduced or prevented by modified (filtered, washed, or irradiated) blood components. When an adverse reaction is suspected, the transfusion should be stopped and reported to the blood bank for investigation. Transfusion reactions may result from immune and nonimmune mechanisms. Immune-mediated reactions are often due to preformed donor or recipient antibody; however, cellular elements may also cause adverse effects. Nonimmune causes of reactions are due to the chemical and physical properties of the stored blood component and its additives.

1	Transfusion-transmitted viral infections are increasingly rare due to improved screening and testing. As the risk of viral infection is reduced, the relative risk of other reactions increases, such as hemolytic transfusion reactions and sepsis from bacterially contaminated components. Pretransfusion quality assurance improvements further increase the safety of transfusion therapy. Infections, like any adverse transfusion reaction, must be brought to the attention of the blood bank for appropriate studies (Table 138e-3). IMMUNE-MEDIATED REACTIONS Acute Hemolytic Transfusion Reactions Immune-mediated hemolysis occurs when the recipient has preformed antibodies that lyse donor erythrocytes. The ABO isoagglutinins are responsible for the majority of these reactions. However, alloantibodies directed against other RBC antigens, i.e., Rh, Kell, and Duffy, are responsible for more fatal hemolytic transfusion reactions.

1	Acute hemolytic reactions may present with hypotension, tachypnea, tachycardia, fever, chills, hemoglobinemia, hemoglobinuria, chest and/or flank pain, and discomfort at the infusion site. Monitoring Frequency, Episodes: Unit aInfectious agents rarely associated with transfusion, theoretically possible, or of unknown risk include West Nile virus, hepatitis A virus, parvovirus B19, Babesia microti and Babesia duncani (babesiosis), Borrelia burgdorferi (Lyme disease), Anaplasma phagocytophilum (human granulocytic ehrlichiosis), Trypanosoma cruzi (Chagas’ disease), Treponema pallidum, and human herpesvirus-8. Abbreviations: FNHTR, febrile nonhemolytic transfusion reaction; HTLV, human T lymphotropic virus; RBC, red blood cell; TRALI, transfusion-related acute lung injury.

1	Abbreviations: FNHTR, febrile nonhemolytic transfusion reaction; HTLV, human T lymphotropic virus; RBC, red blood cell; TRALI, transfusion-related acute lung injury. the patient’s vital signs before and during the transfusion is important to identify reactions promptly. When acute hemolysis is suspected, the transfusion must be stopped immediately, intravenous access maintained, and the reaction reported to the blood bank. A correctly labeled posttransfusion blood sample and any untransfused blood should be sent to the blood bank for analysis. The laboratory evaluation for hemolysis includes the measurement of serum haptoglobin, lactate dehydrogenase (LDH), and indirect bilirubin levels.

1	The immune complexes that result in RBC lysis can cause renal dysfunction and failure. Diuresis should be induced with intravenous fluids and furosemide or mannitol. Tissue factor released from the lysed erythrocytes may initiate DIC. Coagulation studies including prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, and platelet count should be monitored in patients with hemolytic reactions.

1	Errors at the patient’s bedside, such as mislabeling the sample or transfusing the wrong patient, are responsible for the majority of these reactions. The blood bank investigation of these reactions includes examination of the preand posttransfusion samples for hemolysis and repeat typing of the patient samples; direct antiglobulin test (DAT), sometimes called the direct Coombs test, of the posttransfusion sample; repeating the cross-matching of the blood component; and checking all clerical records for errors. DAT detects the presence of antibody or complement bound to RBCs in vivo (Fig. 138e-1). Delayed Hemolytic and Serologic Transfusion Reactions Delayed hemolytic transfusion reactions (DHTRs) are not completely preventable.

1	Delayed Hemolytic and Serologic Transfusion Reactions Delayed hemolytic transfusion reactions (DHTRs) are not completely preventable. These reactions occur in patients previously sensitized to RBC alloantigens who have a negative alloantibody screen due to low antibody levels. When the patient is transfused with antigen-positive blood, an anamnestic response results in the early production of alloantibody that binds donor RBCs. The alloantibody is detectable 1–2 weeks following the transfusion, and the posttransfusion DAT may become positive due to circulating donor RBCs coated with antibody or complement. The transfused, alloantibody-coated erythrocytes are cleared by the reticuloendothelial system. These reactions are detected most commonly in the blood bank when a subsequent patient sample reveals a positive alloantibody screen or a new alloantibody in a recently transfused recipient.

1	No specific therapy is usually required, although additional RBC transfusions may be necessary. Delayed serologic transfusion reactions are similar to DHTR, because the DAT is positive and alloantibody is detected; however, RBC clearance is not increased.

1	Febrile Nonhemolytic Transfusion Reaction The most frequent reaction associated with the transfusion of cellular blood components is a febrile nonhemolytic transfusion reaction (FNHTR). These reactions are characterized by chills and rigors and a ≥1°C rise in temperature. FNHTR is diagnosed when other causes of fever in the transfused patient are ruled out. Antibodies directed against donor leukocyte and HLA antigens may mediate these reactions; thus, multiply transfused patients and multiparous women are felt to be at increased risk. Although anti-HLA antibodies may be demonstrated in the recipient’s serum, investigation is not routinely done because of the mild nature test result Positive test result Indirect Coombs test/ indirect antiglobulin test (Coombs reagent) the reb blood cell surface anti-RBC antibodies Blood sample from a patient with immune mediated haemolytic anaemia: antibodies are shown attached to antigens on the RBC surface. Recipient’s serum is obtained, containing

1	anti-RBC antibodies Blood sample from a patient with immune mediated haemolytic anaemia: antibodies are shown attached to antigens on the RBC surface. Recipient’s serum is obtained, containing anitbodies (Ig’s). Donor’s blood sample is added to the tube with serum. Recipient’s Ig’s that target the donor’s red blood cells form antibody-antigen complexes. Anti-human Ig’s (Coombs antibodies) are added to the solution. Agglutination of red blood cells occurs, because human Ig’s are attached to red blood cells. The patient’s washed RBCs are incubated with antihuman antibodies (Coombs reagent). RBCs agglutinate: antihuman antibodies form links between RBCs by binding to the human antibodies on the RBCs. FIGURE 138E-1 Direct and indirect Coombs test. The direct Coombs (antiglobulin) test detects the presence of antibodies (or complement) on the surface of erythrocytes. The indirect Coombs (antiglobulin) test detects antibodies in the serum that may bind to donor erythrocytes. RBC, red blood

1	the presence of antibodies (or complement) on the surface of erythrocytes. The indirect Coombs (antiglobulin) test detects antibodies in the serum that may bind to donor erythrocytes. RBC, red blood cell. (Adapted from http://upload.wikimedia.org/wikipedia/commons/1/1c/coombs_test_schematic.png.) of most FNHTR. The use of leukocyte-reduced blood products may prevent or delay sensitization to leukocyte antigens and thereby reduce the incidence of these febrile episodes. Cytokines released from cells within stored blood components may mediate FNHTR; thus, leukoreduction before storage may prevent these reactions.

1	Allergic Reactions Urticarial reactions are related to plasma proteins found in transfused components. Mild reactions may be treated symptomatically by temporarily stopping the transfusion and administering antihistamines (diphenhydramine, 50 mg orally or intramuscularly). The transfusion may be completed after the signs and/or symptoms resolve. Patients with a history of allergic transfusion reaction should be premedicated with an antihistamine. Cellular components can be washed to remove residual plasma for the extremely sensitized patient.

1	Anaphylactic Reaction This severe reaction presents after transfusion of only a few milliliters of the blood component. Symptoms and signs include difficulty breathing, coughing, nausea and vomiting, hypotension, bronchospasm, loss of consciousness, respiratory arrest, and shock. Treatment includes stopping the transfusion, maintaining vascular access, and administering epinephrine (0.5–1 mL of 1:1000 dilution subcutaneously). Glucocorticoids may be required in severe cases. Patients who are IgA-deficient, <1% of the population, may be sensitized to this Ig class and are at risk for anaphylactic reactions associated with plasma transfusion. Individuals with severe IgA deficiency should therefore receive only IgA-deficient plasma and washed cellular blood components. Patients who have anaphylactic or repeated allergic reactions to blood components should be tested for IgA deficiency.

1	Graft-Versus-Host Disease Graft-versus-host disease (GVHD) is a frequent complication of allogeneic stem cell transplantation, in which lymphocytes from the donor attack and cannot be eliminated by an immunodeficient host. Transfusion-related GVHD is mediated by donor T lymphocytes that recognize host HLA antigens as foreign and mount an immune response, which is manifested clinically by the development of fever, a characteristic cutaneous eruption, diarrhea, and liver function abnormalities. GVHD can also occur when blood components that contain viable T lymphocytes are transfused to immunodeficient recipients or to immunocompetent recipients who share HLA antigens with the donor (e.g., a family donor). In addition to the aforementioned clinical features of GVHD, transfusion-associated GVHD (TA-GVHD) is characterized by marrow aplasia and pancytopenia. TA-GVHD is highly resistant to treatment with immunosuppressive therapies, including glucocorticoids, cyclosporine, antithymocyte

1	GVHD (TA-GVHD) is characterized by marrow aplasia and pancytopenia. TA-GVHD is highly resistant to treatment with immunosuppressive therapies, including glucocorticoids, cyclosporine, antithymocyte globulin, and ablative therapy followed by allogeneic bone marrow transplantation. Clinical manifestations appear at 8–10 days, and death occurs at 3–4 weeks after transfusion.

1	TA-GVHD can be prevented by irradiation of cellular components (minimum of 2500 cGy) before transfusion to patients at risk. Patients at risk for TA-GVHD include fetuses receiving intrauterine transfusions, selected immunocompetent (e.g., lymphoma patients) or immunocompromised recipients, recipients of donor units known to be from a blood relative, and recipients who have undergone marrow transplantation. Directed donations by family members should be discouraged (they are not less likely to transmit infection); lacking other options, the blood products from family members should always be irradiated.

1	Transfusion-Related Acute Lung Injury Transfusion-related acute lung injury (TRALI) is the most common cause of transfusion related fatalities. The recipient develops symptoms of hypoxia (Pao2/FIo2 <300 mmHg) and signs of noncardiogenic pulmonary edema, including bilateral interstitial infiltrates on chest x-ray, either during or within 6 h of transfusion. Treatment is supportive, and patients usually recover without sequelae. TRALI usually results from the transfusion of donor plasma that contains high-titer anti-HLA class II antibodies that bind recipient leukocytes. The leukocytes aggregate in the pulmonary vasculature and release mediators that increase capillary permeability. Testing the donor’s plasma for anti-HLA antibodies can support this diagnosis. The implicated donors are frequently multiparous women. The transfusion of plasma from male and nulliparous women donors reduces the risk of TRALI. Recipient factors that are associated 138e-5 with increased risk of TRALI include

1	multiparous women. The transfusion of plasma from male and nulliparous women donors reduces the risk of TRALI. Recipient factors that are associated 138e-5 with increased risk of TRALI include smoking, chronic alcohol use, shock, liver surgery (transplantation), mechanical ventilation with >30 cmH2O pressure support and positive fluid balance.

1	Posttransfusion Purpura This reaction presents as thrombocytopenia 7–10 days after platelet transfusion and occurs predominantly in women. Platelet-specific antibodies are found in the recipient’s serum, and the most frequently recognized antigen is HPA-1a found on the platelet glycoprotein IIIa receptor. The delayed thrombocytopenia is due to the production of antibodies that react to both donor and recipient platelets. Additional platelet transfusions can worsen the thrombocytopenia and should be avoided. Treatment with intravenous immunoglobulin may neutralize the effector antibodies, or plasmapheresis can be used to remove the antibodies.

1	Alloimmunization A recipient may become alloimmunized to a number of antigens on cellular blood elements and plasma proteins. Alloantibodies to RBC antigens are detected during pretransfusion testing, and their presence may delay finding antigen-negative crossmatch-compatible products for transfusion. Women of childbearing age who are sensitized to certain RBC antigens (i.e., D, c, E, Kell, or Duffy) are at risk for bearing a fetus with hemolytic disease of the newborn. Matching for D antigen is the only pretransfusion selection test to prevent RBC alloimmunization.

1	Alloimmunization to antigens on leukocytes and platelets can result in refractoriness to platelet transfusions. Once alloimmunization has developed, HLA-compatible platelets from donors who share similar antigens with the recipient may be difficult to find. Hence, prudent transfusion practice is directed at preventing sensitization through the use of leukocyte-reduced cellular components, as well as limiting antigenic exposure by the judicious use of transfusions and use of SDAPs. NONIMMUNOLOGIC REACTIONS Fluid Overload Blood components are excellent volume expanders, and transfusion may quickly lead to transfusion-associated circulatory overload (TACO). Dyspnea with PaO2 <90% on room air, bilateral infiltrates on chest x-ray, and systolic hypertension are found with TACO. Brain natriuretic peptide (BNP) is often elevated (>1.5) compared with pretransfusion levels. Monitoring the rate and volume of the transfusion and using a diuretic can minimize this problem.

1	Hypothermia Refrigerated (4°C) or frozen (−18°C or below) blood components can result in hypothermia when rapidly infused. Cardiac dysrhythmias can result from exposing the sinoatrial node to cold fluid. Use of an in-line warmer will prevent this complication. Electrolyte Toxicity RBC leakage during storage increases the concentration of potassium in the unit. Neonates and patients in renal failure are at risk for hyperkalemia. Preventive measures, such as using fresh or washed RBCs, are warranted for neonatal transfusions because this complication can be fatal.

1	Citrate, commonly used to anticoagulate blood components, chelates calcium and thereby inhibits the coagulation cascade. Hypocalcemia, manifested by circumoral numbness and/or tingling sensation of the fingers and toes, may result from multiple rapid transfusions. Because citrate is quickly metabolized to bicarbonate, calcium infusion is seldom required in this setting. If calcium or any other intravenous infusion is necessary, it must be given through a separate line. Iron Overload Each unit of RBCs contains 200–250 mg of iron. Symptoms and signs of iron overload affecting endocrine, hepatic, and cardiac function are common after 100 units of RBCs have been transfused (total-body iron load of 20 g). Preventing this complication by using alternative therapies (e.g., erythropoietin) and judicious transfusion is preferable and cost effective. Chelating agents, such as deferoxamine and deferasirox, are available, but the response is often suboptimal.

1	Hypotensive Reactions Transient hypotension may be noted among transfused patients who take angiotensin-converting enzyme (ACE) inhibitors. Because blood products contain bradykinin that is normally degraded by ACE, patients on ACE inhibitors may have increased bradykinin levels that cause hypotension in the recipient. The blood pressure typically returns to normal without intervention. Immunomodulation Transfusion of allogeneic blood is immunosuppressive. Multiply transfused renal transplant recipients are less likely to reject the graft, and transfusion may result in poorer outcomes in cancer patients and increase the risk of infections. Transfusion-related immunomodulation is thought to be mediated by transfused leukocytes. Leukocyte-depleted cellular products may cause less immunosuppression, although controlled data are unlikely to be obtained as the blood supply becomes universally leukocyte depleted.

1	The blood supply is initially screened by selecting healthy donors without high-risk lifestyles, medical conditions, or exposure to transmissible pathogens, such as intravenous drug use or visiting malaria endemic areas. Multiple tests performed on donated blood to detect the presence of infectious agents using nucleic acid amplification testing (NAAT) or evidence of prior infections by testing for antibodies to pathogens and sterility of platelet products further reduce the risk of transfusion-acquired infections. Viral Infections • Hepatitis C virus (HCv) Blood donations are tested for antibodies to HCV and HCV RNA. The risk of acquiring HCV through transfusion is now calculated to be approximately 1 in 2,000,000 units. Infection with HCV may be asymptomatic or lead to chronic active hepatitis, cirrhosis, and liver failure.

1	Human immunodefiCienCy virus type 1 (Hiv-1) Donated blood is tested for antibodies to HIV-1, HIV-1 p24 antigen, and HIV RNA using NAAT. Approximately a dozen seronegative donors have been shown to harbor HIV RNA. The risk of HIV-1 infection per transfusion episode is 1 in 2,000,000. Antibodies to HIV-2 are also measured in donated blood. No cases of HIV-2 infection have been reported in the United States since 1992. Hepatitis B virus (HBv) Donated blood is screened for HBV using assays for hepatitis B surface antigen (HbsAg). NAAT testing is not practical because of slow viral replication and lower levels of viremia. The risk of transfusion-associated HBV infection is several times greater than for HCV. Vaccination of individuals who require longterm transfusion therapy can prevent this complication.

1	otHer Hepatitis viruses Hepatitis A virus is rarely transmitted by transfusion; infection is typically asymptomatic and does not lead to chronic disease. Other transfusion-transmitted viruses—TT virus, SEN virus, and GB virus C—do not cause chronic hepatitis or other disease states. Routine testing does not appear to be warranted. West nile virus (Wnv) Transfusion-transmitted WNV infections were documented in 2002. This RNA virus can be detected using NAAT; routine screening began in 2003. WNV infections range in severity from asymptomatic to fatal, with the older population at greater risk.

1	Cytomegalovirus This ubiquitous virus infects ≥50% of the general population and is transmitted by the infected “passenger” WBCs found in transfused PRBCs or platelet components. Cellular components that are leukocyte-reduced have a decreased risk of transmitting CMV, regardless of the serologic status of the donor. Groups at risk for CMV infections include immunosuppressed patients, CMV-seronegative transplant recipients, and neonates; these patients should receive leukocyte-depleted components or CMV seronegative products. Human t lympHotropiC virus (Htlv) type 1 Assays to detect HTLV-1 and -2 are used to screen all donated blood. HTLV-1 is associated with adult T cell leukemia/lymphoma and tropical spastic paraparesis in a small percentage of infected persons (Chap. 225e). The risk of HTLV-1 infection via transfusion is 1 in 641,000 transfusion episodes. HTLV-2 is not clearly associated with any disease.

1	parvovirus B19 Blood components and pooled plasma products can transmit this virus, the etiologic agent of erythema infectiosum, or fifth disease, in children. Parvovirus B19 shows tropism for erythroid precursors and inhibits both erythrocyte production and maturation. Pure red cell aplasia, presenting either as acute aplastic crisis or chronic anemia with shortened RBC survival, may occur in individuals with an underlying hematologic disease, such as sickle cell disease or thalassemia (Chap. 130). The fetus of a seronegative woman is at risk for developing hydrops from this virus. Bacterial Contamination The relative risk of transfusion-transmitted bacterial infection has increased as the absolute risk of viral infections has dramatically decreased.

1	Most bacteria do not grow well at cold temperatures; thus, PRBCs and FFP are not common sources of bacterial contamination. However, some gram-negative bacteria can grow at 1–6°C. Yersinia, Pseudomonas, Serratia, Acinetobacter, and Escherichia species have all been implicated in infections related to PRBC transfusion. Platelet concentrates, which are stored at room temperature, are more likely to contain skin contaminants such as gram-positive organisms, including coagulase-negative staphylococci. It is estimated that 1 in 1000–2000 platelet components is contaminated with bacteria. The risk of death due to transfusion-associated sepsis has been calculated at 1 in 17,000 for single-unit platelets derived from whole blood donation and 1 in 61,000 for apheresis product. Since 2004, blood banks have instituted methods to detect contaminated platelet components.

1	Recipients of transfusion contaminated with bacteria may develop fever and chills, which can progress to septic shock and DIC. These reactions may occur abruptly, within minutes of initiating the transfusion, or after several hours. The onset of symptoms and signs is often sudden and fulminant, which distinguishes bacterial contamination from an FNHTR. The reactions, particularly those related to gram-negative contaminants, are the result of infused endotoxins formed within the contaminated stored component. When these reactions are suspected, the transfusion must be stopped immediately. Therapy is directed at reversing any signs of shock, and broad-spectrum antibiotics should be given. The blood bank should be notified to identify any clerical or serologic error. The blood component bag should be sent for culture and Gram stain.

1	Other Infectious Agents Various parasites, including those causing malaria, babesiosis, and Chagas’ disease, can be transmitted by blood transfusion. Geographic migration and travel of donors shift the incidence of these rare infections. Other agents implicated in transfusion transmission include dengue, chikungunya virus, variant Creutzfeldt-Jakob disease, A. phagocytophilum, and yellow fever vaccine virus, and the list will grow. Tests for some pathogens are available, such as T. cruzi, but not universally required, whereas others are being developed (B. microti). These infections should be considered in the transfused patient in the appropriate clinical setting.

1	Alternatives to allogeneic blood transfusions that avoid homologous donor exposures with attendant immunologic and infectious risks remain attractive. Autologous blood is the best option when transfusion is anticipated. However, the cost-benefit ratio of autologous transfusion remains high. No transfusion is a zero-risk event; clerical errors and bacterial contamination remain potential complications even with autologous transfusions. Additional methods of autologous transfusion in the surgical patient include preoperative hemodilution, recovery of shed blood from sterile surgical sites, and postoperative drainage collection. Directed or designated donation from friends and family of the potential recipient has not been safer than volunteer donor component transfusions. Such directed donations may in fact place the recipient at higher risk for complications such as GVHD and alloimmunization.

1	Granulocyte and granulocyte-macrophage colony-stimulating factors are clinically useful to hasten leukocyte recovery in patients with leukopenia related to high-dose chemotherapy. Erythropoietin stimulates erythrocyte production in patients with anemia of chronic renal failure and other conditions, thus avoiding or reducing the need for transfusion. This hormone can also stimulate erythropoiesis in the autologous donor to enable additional donation.

1	hematopoietic Cell transplantation Frederick R. Appelbaum Bone marrow transplantation was the original term used to describe the collection and transplantation of hematopoietic stem cells, but with 139e the demonstration that peripheral blood and umbilical cord blood are also useful sources of stem cells, hematopoietic cell transplantation has become the preferred generic term for this process. The procedure is usually carried out for one of two purposes: (1) to replace an abnormal but nonmalignant lymphohematopoietic system with one from a normal donor or (2) to treat malignancy by allowing the administration of higher doses of myelosuppressive therapy than would otherwise be possible. The use of hematopoietic cell transplantation has been increasing, both because of its efficacy in selected diseases and because of increasing availability of donors. The Center for International Blood and Marrow Transplant Research (http://www.cibmtr.org) estimates that about 65,000 transplants are

1	diseases and because of increasing availability of donors. The Center for International Blood and Marrow Transplant Research (http://www.cibmtr.org) estimates that about 65,000 transplants are performed each year.

1	Several features of the hematopoietic stem cell make transplantation clinically feasible, including its remarkable regenerative capacity, its ability to home to the marrow space following intravenous injection, and the ability of the stem cell to be cryopreserved (Chap. 89e). Transplantation of a single stem cell can replace the entire lymphohematopoietic system of an adult mouse. In humans, transplantation of a small percentage of a donor’s bone marrow volume regularly results in complete and sustained replacement of the recipient’s entire lymphohematopoietic system, including all red cells, granulocytes, B and T lymphocytes, and platelets, as well as cells comprising the fixed macrophage population, including Kupffer cells of the liver, pulmonary alveolar macrophages, osteoclasts, Langerhans cells of the skin, and brain microglial cells. The ability of the hematopoietic stem cell to home to the marrow following intravenous injection is mediated, in part, by an interaction between

1	cells of the skin, and brain microglial cells. The ability of the hematopoietic stem cell to home to the marrow following intravenous injection is mediated, in part, by an interaction between CXCL12, also known as stromal cell–derived factor 1, produced by marrow stromal cells and the alpha-chemokine receptor CXCR4 found on stem cells. Homing is also influenced by the interaction of cell-surface molecules, termed selectins, including Eand L-selectin, on bone marrow endothelial cells with ligands, termed integrins, such as VLA-4, on early hematopoietic cells. Human hematopoietic stem cells can survive freezing and thawing with little, if any, damage, making it possible to remove and store a portion of the patient’s own bone marrow for later reinfusion following treatment of the patient with high-dose myelotoxic therapy.

1	Hematopoietic cell transplantation can be described according to the relationship between the patient and the donor and by the anatomic source of stem cells. In ~1% of cases, patients have identical twins who can serve as donors. With the use of syngeneic donors, there is no risk of graft-versus-host disease (GVHD), which often complicates allogeneic transplantation, and unlike the use of autologous marrow, there is no risk that the stem cells are contaminated with tumor cells.

1	Allogeneic transplantation involves a donor and a recipient who are not genetically identical. Following allogeneic transplantation, immune cells transplanted with the stem cells or developing from them can react against the patient, causing GVHD. Alternatively, if the immunosuppressive preparative regimen used to treat the patient before transplant is inadequate, immunocompetent cells of the patient can cause graft rejection. The risks of these complications are greatly influenced by the degree of matching between donor and recipient for antigens encoded by genes of the major histocompatibility complex.

1	The human leukocyte antigen (HLA) molecules are responsible for binding antigenic proteins and presenting them to T cells. The antigens presented by HLA molecules may derive from exogenous sources (e.g., during active infections) or may be endogenous proteins. 139e-1 If individuals are not HLA-matched, T cells from one individual will react strongly to the mismatched HLA, or “major antigens,” of the second. Even if the individuals are HLA-matched, the T cells of the donor may react to differing endogenous or “minor antigens” presented by the HLA of the recipient. Reactions to minor antigens tend to be less vigorous. The genes of major relevance to transplantation include HLA-A, -B, -C, and -D; they are closely linked and therefore tend to be inherited as haplotypes, with only rare crossovers between them. Thus, the odds that any one full sibling will match a patient are one in four, and the probability that the patient has an HLA-identical sibling is 1 − (0.75)n, where n equals the

1	between them. Thus, the odds that any one full sibling will match a patient are one in four, and the probability that the patient has an HLA-identical sibling is 1 − (0.75)n, where n equals the number of siblings.

1	With current techniques, the risk of graft rejection is 1–3%, and the risk of severe, life-threatening acute GVHD is ~15% following transplantation between HLA-identical siblings. The incidence of graft rejection and GVHD increases progressively with the use of family member donors mismatched for one, two, or three antigens. Although survival following a one-antigen mismatched transplant is not markedly altered, survival following twoor three-antigen mismatched transplants is significantly reduced. Since the formation of the National Marrow Donor Program and other registries, it has become possible to identify HLA-matched unrelated donors for many patients. The genes encoding HLA antigens are highly polymorphic, and thus the odds of any two unrelated individuals being HLA identical are extremely low, somewhat less than 1 in 10,000. However, by identifying and typing >20 million volunteer donors, HLA-matched donors can now be found for ~60% of patients for whom a search is initiated,

1	low, somewhat less than 1 in 10,000. However, by identifying and typing >20 million volunteer donors, HLA-matched donors can now be found for ~60% of patients for whom a search is initiated, with higher rates among whites and lower rates among minorities and patients of mixed race. It takes, on average, 3–4 months to complete a search and schedule and initiate an unrelated donor transplant. With improvements in HLA typing and supportive care measures, survival following matched unrelated donor transplantation is essentially the same as that seen with HLA-matched siblings.

1	Autologous transplantation involves the removal and storage of the patient’s own stem cells with subsequent reinfusion after the patient receives high-dose myeloablative therapy. Unlike allogeneic transplantation, there is no risk of GVHD or graft rejection with autologous transplantation. On the other hand, autologous transplantation lacks a graft-versus-tumor (GVT) effect, and the autologous stem cell product can be contaminated with tumor cells, which could lead to relapse. A variety of techniques have been developed to “purge” autologous products of tumor cells. Some use antibodies directed at tumor-associated antigens plus complement, antibodies linked to toxins, or antibodies conjugated to immunomagnetic beads. Another technique is positive selection of stem cells using antibodies to CD34, with subsequent column adherence or flow techniques to select normal stem cells while leaving tumor cells behind. All of these approaches can reduce the number of tumor cells from 1000to

1	to CD34, with subsequent column adherence or flow techniques to select normal stem cells while leaving tumor cells behind. All of these approaches can reduce the number of tumor cells from 1000to 10,000-fold and are clinically feasible; however, no prospective randomized trials have yet shown that any of these approaches results in a decrease in relapse rates or improvements in disease-free or overall survival.

1	Bone marrow aspirated from the posterior and anterior iliac crests initially was the source of hematopoietic stem cells for transplantation. Typically, anywhere from 1.5 to 5 × 108 nucleated marrow cells per kilogram are collected for allogeneic transplantation. Several studies have found improved survival in the settings of both matched sibling and unrelated transplantation by transplanting higher numbers of bone marrow cells.

1	Hematopoietic stem cells circulate in the peripheral blood but in very low concentrations. Following the administration of certain hematopoietic growth factors, including granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), and during recovery from intensive chemotherapy, the concentration of hematopoietic progenitor cells in blood, as measured either by colony-forming units or expression of the CD34 antigen, increases markedly. This has made it possible to harvest adequate numbers of stem cells from the peripheral blood for transplantation. Donors are typically treated with 4 or 5 days of hematopoietic growth factor, following which stem cells are collected in one or two 4-h pheresis sessions. In the autologous setting, transplantation of >2.5 × 106 CD34 cells per kilogram, a number that can be collected in most circumstances, leads to rapid and sustained engraftment in virtually all cases. In the 10–20% of patients who fail to

1	of >2.5 × 106 CD34 cells per kilogram, a number that can be collected in most circumstances, leads to rapid and sustained engraftment in virtually all cases. In the 10–20% of patients who fail to mobilize sufficient CD34+ cells with growth factor alone, the addition of plerixafor, an antagonist of CXCR4, may be useful. Compared to the use of autologous marrow, use of peripheral blood stem cells results in more rapid hematopoietic recovery, with granulocytes recovering to 500/μL by day 12 and platelets recovering to 20,000/μL by day 14. Although this more rapid recovery diminishes the morbidity rate of transplantation, no studies show improved survival.

1	Hesitation in studying the use of peripheral blood stem cells for allogeneic transplantation occurred because peripheral blood stem cell products contain as much as 1 log more T cells than are contained in the typical marrow harvest; in animal models, the incidence of GVHD is related to the number of T cells transplanted. Nonetheless, clinical trials have shown that the use of growth factor–mobilized peripheral blood stem cells from HLA-matched family members leads to faster engraftment without an increase in acute GVHD. Chronic GVHD may be increased with peripheral blood stem cells, but in trials conducted so far, this has been more than balanced by reductions in relapse rates and nonrelapse mortality rates, with the use of peripheral blood stem cells resulting in improved overall survival. However, in the setting of matched unrelated donor transplantation, use of peripheral blood results in more chronic GVHD without a compensatory survival advantage, favoring the use of bone marrow

1	However, in the setting of matched unrelated donor transplantation, use of peripheral blood results in more chronic GVHD without a compensatory survival advantage, favoring the use of bone marrow in this setting.

1	Umbilical cord blood contains a high concentration of hematopoietic progenitor cells, allowing for its use as a source of stem cells for transplantation. Cord blood transplantation from family members has been explored in the setting where the immediate need for transplantation precludes waiting the 9 or so months generally required for the baby to mature to the point of donating marrow. Use of cord blood results in slower engraftment and peripheral count recovery than seen with marrow but a lower incidence of GVHD, perhaps reflecting the low number of T cells in cord blood. Multiple cord blood banks have been developed to harvest and store cord blood for possible transplantation to unrelated patients from material that would otherwise be discarded. Currently more than 500,000 units are cryopreserved and available for use. The advantages of unrelated cord blood are rapid availability and decreased immune reactivity allowing for the use of partially matched units, which is of

1	are cryopreserved and available for use. The advantages of unrelated cord blood are rapid availability and decreased immune reactivity allowing for the use of partially matched units, which is of particular importance for those without matched unrelated donors. The risks of graft failure and transplant-related mortality are related to the dose of cord blood cells per kilogram, which previously limited the application of single cord blood transplantation to pediatric and smaller adult patients. Subsequent trials have found that the use of double cord transplants diminishes the risk of graft failure and early mortality even though only one of the donors ultimately engrafts. Survival rates are now similar with unrelated donor and cord blood transplantation.

1	The treatment regimen administered to patients immediately preceding transplantation is designed to eradicate the patient’s underlying disease and, in the setting of allogeneic transplantation, immunosuppress the patient adequately to prevent rejection of the transplanted marrow. The appropriate regimen therefore depends on the disease setting and source of marrow. For example, when transplantation is performed to treat severe combined immunodeficiency and the donor is a histocompatible sibling, no treatment is needed because no host cells require eradication and the patient is already too immunoincompetent to reject the transplanted marrow. For aplastic anemia, there is no large population of cells to eradicate, and high-dose cyclophosphamide plus antithymocyte globulin are sufficient to immunosuppress the patient adequately to accept the marrow graft. In the setting of thalassemia and sickle cell anemia, high-dose busulfan is frequently added to cyclophosphamide in order to

1	to immunosuppress the patient adequately to accept the marrow graft. In the setting of thalassemia and sickle cell anemia, high-dose busulfan is frequently added to cyclophosphamide in order to eradicate hyperplastic host hematopoiesis. A variety of different regimens have been developed to treat malignant diseases. Most of these regimens include agents that have high activity against the tumor in question at conventional doses and have myelosuppression as their predominant dose-limiting toxicity. Therefore, these regimens commonly include busulfan, cyclophosphamide, melphalan, thiotepa, carmustine, etoposide, and total-body irradiation in various combinations.

1	Although high-dose treatment regimens have typically been used in transplantation, the understanding that much of the antitumor effect of transplantation derives from an immunologically mediated GVT response has led investigators to ask if reduced-intensity conditioning regimens might be effective and more tolerable. Evidence for a GVT effect comes from studies showing that posttransplant relapse rates are lowest in patients who develop acute and chronic GVHD, higher in those without GVHD, and higher still in recipients of T cell–depleted allogeneic or syngeneic marrow. The demonstration that complete remissions can be obtained in many patients who have relapsed after transplant by simply administering viable lymphocytes from the original donor further strengthens the argument for a potent GVT effect. Accordingly, a variety of reduced-intensity regimens have been studied, ranging from the very minimum required to achieve engraftment (e.g., fludarabine plus 200 cGy total-body

1	a potent GVT effect. Accordingly, a variety of reduced-intensity regimens have been studied, ranging from the very minimum required to achieve engraftment (e.g., fludarabine plus 200 cGy total-body irradiation) to regimens of more immediate intensity (e.g., fludarabine plus melphalan). Studies to date document that engraftment can be readily achieved with less toxicity than seen with conventional transplantation. Furthermore, the severity of acute GVHD appears to be somewhat decreased. Complete sustained responses have been documented in many patients, particularly those with more indolent hematologic malignancies. In general, relapse rates are higher following reduced-intensity conditioning, but transplant-related mortality is lower, favoring the use of reduced-intensity conditioning in older patients and those with significant comorbidities. High-dose regimens are favored in younger, fitter patients.

1	Marrow is usually collected from the donor’s posterior and sometimes anterior iliac crests, with the donor under general or spinal anesthesia. Typically, 10–15 mL/kg of marrow is aspirated, placed in heparinized media, and filtered through 0.3and 0.2-mm screens to remove fat and bony spicules. The collected marrow may undergo further processing depending on the clinical situation, such as the removal of red cells to prevent hemolysis in ABO-incompatible transplants, the removal of donor T cells to prevent GVHD, or attempts to remove possible contaminating tumor cells in autologous transplantation. Marrow donation is safe, with only very rare complications reported.

1	Peripheral blood stem cells are collected by leukapheresis after the donor has been treated with hematopoietic growth factors or, in the setting of autologous transplantation, sometimes after treatment with a combination of chemotherapy and growth factors. Stem cells for transplantation are infused through a large-bore central venous catheter. Such infusions are usually well tolerated, although occasionally patients develop fever, cough, or shortness of breath. These symptoms typically resolve with slowing of the infusion. When the stem cell product has been cryopreserved using dimethyl sulfoxide, patients more often experience short-lived nausea or vomiting due to the odor and taste of the cryoprotectant.

1	Peripheral blood counts usually reach their nadir several days to a week after transplant as a consequence of the preparative regimen; then cells produced by the transplanted stem cells begin to appear in the peripheral blood. The rate of recovery depends on the source of stem cells, the use of posttransplant growth factors, and the form of GVHD prophylaxis used. If marrow is the source of stem cells, recovery to 100 granulocytes/μL occurs on average by day 16 and to 500/μL by day 22. Use of G-CSF–mobilized peripheral blood stem cells speeds the rate of recovery by ~1 week when compared to marrow, whereas engraftment following cord blood transplantation is typically delayed by ~1 week compared to marrow. Use of a myeloid growth factor (G-CSF or GM-CSF) after transplant can accelerate recovery by 3–5 days, whereas use of methotrexate to prevent GVHD delays engraftment by a similar

1	FIGURE 139e-1 Major syndromes complicating marrow transplantation. CMV, cytomegalovirus; GVHD, graft-versus-host disease; HSV, herpes simplex virus; SOS, sinusoidal obstructive syndrome (formerly venoocclusive disease); VZV, varicella-zoster virus. The size of the shaded area roughly reflects the period of risk of the complication. period. Following allogeneic transplantation, engraftment can be documented using fluorescence in situ hybridization of sex chromosomes if donor and recipient are sex-mismatched or by analysis of a variable number of tandem repeats or short tandem repeat polymorphisms after DNA amplification.

1	COMPLICATIONS FOLLOWING HEMATOPOIETIC CELL TRANSPLANT Early Direct Chemoradiotoxicities The transplant preparative regimen may cause a spectrum of acute toxicities that vary according to intensity of the regimen and the specific agents used, but frequently results in nausea, vomiting, and mild skin erythema (Fig. 139e-1). Regimens that include high-dose cyclophosphamide can result in hemorrhagic cystitis, which can usually be prevented by bladder irrigation or with the sulfhydryl compound mercaptoethanesulfonate (MESNA); rarely, acute hemorrhagic carditis is seen. Most high-dose preparative regimens will result in oral mucositis, which typically develops 5–7 days after transplant and often requires narcotic analgesia. Use of a patient-controlled analgesic pump provides the greatest patient satisfaction and results in a lower cumulative dose of narcotic. Keratinocyte growth factor (palifermin) can shorten the duration of mucositis by several days following autologous transplantation.

1	satisfaction and results in a lower cumulative dose of narcotic. Keratinocyte growth factor (palifermin) can shorten the duration of mucositis by several days following autologous transplantation. Patients begin losing their hair 5–6 days after transplant and by 1 week are usually profoundly pancytopenic.

1	Depending on the intensity of the conditioning regimen, 3–10% of patients will develop sinusoidal obstruction syndrome (SOS) of the liver (formerly called venoocclusive disease), a syndrome that results from direct cytotoxic injury to hepatic-venular and sinusoidal endothelium, with subsequent deposition of fibrin and the development of a local hypercoagulable state. This chain of events leads to the clinical symptoms of tender hepatomegaly, ascites, jaundice, and fluid retention. These symptoms can develop any time during the first month after transplant, with the peak incidence at day 16. Predisposing factors include prior exposure to intensive chemotherapy, pretransplant hepatitis of any cause, and use of more intense conditioning regimens. The mortality rate of sinusoidal obstruction syndrome is ~30%, with progressive hepatic failure culminating in a terminal hepatorenal syndrome. Both thrombolytic and antithrombotic agents, such as tissue plasminogen activator, heparin, and

1	syndrome is ~30%, with progressive hepatic failure culminating in a terminal hepatorenal syndrome. Both thrombolytic and antithrombotic agents, such as tissue plasminogen activator, heparin, and prostaglandin E, have been studied as therapy, but none has proven of consistent major benefit in controlled trials, and all have significant toxicity. Studies with defibrotide, a polydeoxyribonucleotide, seem encouraging.

1	Although most pneumonias developing early after transplant are caused by infectious agents, in ~5% of patients a diffuse interstitial pneumonia will develop that is thought to be the result of direct toxicity of high-dose preparative regimens. Bronchoalveolar lavage 139e-3 usually shows alveolar hemorrhage, and biopsies are typically characterized by diffuse alveolar damage, although some cases may have a more clearly interstitial pattern. High-dose glucocorticoids or antitumor necrosis factor therapies are sometimes used as treatment, although randomized trials testing their utility have not been reported.

1	Late Direct Chemoradiotoxicities Late complications of the preparative regimen include decreased growth velocity in children and delayed development of secondary sex characteristics. These complications can be partly ameliorated with the use of appropriate growth and sex hormone replacement. Most men become azoospermic, and most postpubertal women will develop ovarian failure, which should be treated. However, pregnancy is possible after transplantation, and patients should be counseled accordingly. Thyroid dysfunction, usually well compensated, is sometimes seen. Cataracts develop in 10–20% of patients and are most common in patients treated with total-body irradiation and those who receive glucocorticoid therapy after transplant for treatment of GVHD. Aseptic necrosis of the femoral head is seen in 10% of patients and is particularly frequent in those receiving chronic glucocorticoid therapy. Both acute and late chemoradiotoxicities (except those due to glucocorticoids and other

1	head is seen in 10% of patients and is particularly frequent in those receiving chronic glucocorticoid therapy. Both acute and late chemoradiotoxicities (except those due to glucocorticoids and other agents used to treat GVHD) are considerably less frequent in recipients of reduced-intensity compared to high-dose preparative regimens.

1	Graft Failure Although complete and sustained engraftment is usually seen after transplant, occasionally marrow function either does not return or, after a brief period of engraftment, is lost. Graft failure after autologous transplantation can be the result of inadequate numbers of stem cells being transplanted, damage during ex vivo treatment or storage, or exposure of the patient to myelotoxic agents after transplant. Infections with cytomegalovirus (CMV) or human herpesvirus type 6 have also been associated with loss of marrow function. Graft failure after allogeneic transplantation can also be due to immunologic rejection of the graft by immunocompetent host cells. Immunologically based graft rejection is more common following use of less immunosuppressive preparative regimens, in recipients of T cell–depleted stem cell products, and in patients receiving grafts from HLA-mismatched donors or cord blood.

1	Treatment of graft failure usually involves removing all potentially myelotoxic agents from the patient’s regimen and attempting a short trial of a myeloid growth factor. Persistence of lymphocytes of host origin in allogeneic transplant recipients with graft failure indicates immunologic rejection. Reinfusion of donor stem cells in such patients is usually unsuccessful unless preceded by a second immunosuppressive preparative regimen. Standard high-dose preparative regimens are generally tolerated poorly if administered within 100 days of a first transplant because of cumulative toxicities. However, use of regimens combining, for example, fludarabine plus low-dose total-body irradiation, or cyclophosphamide plus antithymocyte globulin, has been effective in some cases.

1	Graft-Versus-Host Disease GVHD is the result of allogeneic T cells that are transferred with the donor’s stem cell inoculum reacting with antigenic targets on host cells. Acute GVHD usually occurs within the first 3 months after transplant with a peak onset around 4 weeks and is characterized by an erythematous maculopapular rash; by persistent anorexia or diarrhea, or both; and by liver disease with increased serum levels of bilirubin, alanine and aspartate aminotransferase, and alkaline phosphatase. Because many conditions can mimic acute GVHD, the diagnosis usually requires skin, liver, or endoscopic biopsy for confirmation. In all these organs, endothelial damage and lymphocytic infiltrates are seen. In skin, the epidermis and hair follicles are damaged; in liver, the small bile ducts show segmental disruption; and in intestines, destruction of the crypts and mucosal ulceration may be noted. A commonly used rating system for acute GVHD is shown in Table 139e-1. Grade I acute GVHD

1	show segmental disruption; and in intestines, destruction of the crypts and mucosal ulceration may be noted. A commonly used rating system for acute GVHD is shown in Table 139e-1. Grade I acute GVHD is of little clinical significance, does not affect the likelihood of survival, and does not require treatment. In contrast, grades II to IV GVHD are associated with significant symptoms and a poorer probability of survival, and they require aggressive therapy.

1	infection. Early after transplantation, patients are profoundly neutropenic, and because the risk of Liver—Bilirubin, bacterial infection is so great, most centers initiate falls to <500/μL. Fluconazole prophylaxis at a dose of 200–400 mg/d reduces the risk of can-Diarrhea >1500 mL/d didal infections. Patients seropositive for herpes Ileus simplex should receive acyclovir prophylaxis. One approach to infection prophylaxis is shown in Gut Stage Table 139e-2. Despite these prophylactic measures, The incidence of acute GVHD is higher in recipients of stem cells from mismatched or unrelated donors, in older patients, and in patients unable to receive full doses of drugs used to prevent the disease.

1	One general approach to the prevention of GVHD is the administration of immunosuppressive drugs early after transplant. Combinations of methotrexate and either cyclosporine or tacrolimus are among the most effective and widely used regimens. Prednisone, anti–T cell antibodies, mycophenolate mofetil, sirolimus, and other immunosuppressive agents have also been or are being studied in various combinations. A second general approach to GVHD prevention is removal of T cells from the stem cell inoculum. While effective in preventing GVHD, T cell depletion has been associated with an increased incidence of graft failure, infectious complications, and tumor recurrence after transplant; as yet, it is unsettled whether T cell depletion improves cure rates in any specific setting.

1	Despite prophylaxis, significant acute GVHD will develop in ~30% of recipients of stem cells from matched siblings and in as many as 60% of those receiving stem cells from unrelated donors. The disease is usually treated with glucocorticoids, additional immunosuppressants, or monoclonal antibodies targeted against T cells or T cell subsets.

1	Chronic GVHD occurs most commonly between 3 months and 2 years after allogeneic transplant, developing in 20–50% of recipients. The disease is more common in older patients, in recipients of mismatched or unrelated stem cells, and in those with a preceding episode of acute GVHD. The disease resembles an autoimmune disorder with malar rash, sicca syndrome, arthritis, obliterative bronchiolitis, and bile duct degeneration and cholestasis. Single-agent prednisone or cyclosporine is standard treatment at present, although trials of other agents are under way. Mortality rates from chronic GVHD average around 15%, but range from 5–50% depending on severity. In most patients, chronic GVHD resolves, but it may require 1–3 years of immunosuppressive treatment before these agents can be withdrawn without the disease recurring. Because patients with chronic GVHD are susceptible to significant infection, they should receive prophylactic trimethoprim-sulfamethoxazole, and all suspected infections

1	the disease recurring. Because patients with chronic GVHD are susceptible to significant infection, they should receive prophylactic trimethoprim-sulfamethoxazole, and all suspected infections should be investigated and treated aggressively.

1	Although onset before or after 3 months after transplant is often used to discriminate between acute and chronic GVHD, occasional patients will develop signs and symptoms of acute GVHD after 3 months (late-onset acute GVHD), whereas others will exhibit signs and symptoms of both acute and chronic GVHD (overlap syndrome). There are as yet no data to suggest that these patients should be treated differently than those with classic acute or chronic GVHD. From 3–5% of patients will develop an autoimmune disorder following allogeneic HCT, most commonly autoimmune hemolytic anemia or idiopathic thrombocytopenic purpura. Unrelated donor source and chronic GVHD are risk factors, but autoimmune disorders have been reported in patients with no obvious GVHD. Treatment is with prednisone, cyclosporine, or rituximab.

1	most patients will develop fever and signs of infection after transplant. The management of patients who become febrile despite bacterial and fungal prophylaxis is a difficult challenge and is guided by individual aspects of the patient and by the institution’s experience. The general problem of infection in the immunocompromised host is discussed in Chap. 169.

1	Once patients engraft, the incidence of bacterial infection diminishes; however, patients, particularly allogeneic transplant recipients, remain at significant risk of infection. During the period from engraftment until about 3 months after transplant, the most common causes of infection are gram-positive bacteria, fungi (particularly Aspergillus), and viruses including CMV. CMV infection, which in the past was frequently seen and often fatal, can be prevented in seronegative patients transplanted from seronegative donors by the use of either seronegative blood products or products from which the white blood cells have been removed. In seropositive patients or patients transplanted from seropositive donors, the use of ganciclovir, either as prophylaxis beginning at the time of engraftment or initiated when CMV first reactivates as evidenced by development of antigenemia or viremia, can significantly reduce the risk of CMV disease. Foscarnet is effective for some patients who develop

1	or initiated when CMV first reactivates as evidenced by development of antigenemia or viremia, can significantly reduce the risk of CMV disease. Foscarnet is effective for some patients who develop CMV antigenemia or infection despite the use of ganciclovir or who cannot tolerate the drug.

1	Pneumocystis jiroveci pneumonia, once seen in 5–10% of patients, can be prevented by treating patients with oral trimethoprimsulfamethoxazole for 1 week before transplant and resuming the treatment once patients have engrafted. The risk of infection diminishes considerably beyond 3 months after transplant unless chronic GVHD develops, requiring continuous immunosuppression. Most transplant centers recommend continuing trimethoprim-sulfamethoxazole prophylaxis while patients are receiving any immunosuppressive drugs and also recommend careful monitoring for late CMV reactivation. In addition, many centers recommend prophylaxis against varicella-zoster, using acyclovir for 1 year after transplant. Patients should be revaccinated against tetanus, diphtheria, Haemophilus influenzae, polio, and pneumococcal pneumonia starting at 12 months after transplant and against measles, mumps, and rubella (MMR), varicella-zoster virus, and possibly pertussis at 24 months.

1	400 mg PO qd to day 75 posttransplant 800 mg PO bid to day 30 800 mg PO bid to day 365 5 mg/kg IV bid for 7 days, then 5 (mg/kg)/d 5 days/week to day 100 Infection Posttransplant patients, particularly recipients of alloge neic transplantation, require unique approaches to the problem of TREATMENT OF SPECIFIC DISEASES USING HEMATOPOIETIC CELL be expected if patients are transplanted before they develop hepa-139e-5 TRANSPLANTATION tomegaly or portal fibrosis and if they have been given adequate iron chelation therapy. Among such patients, the probabilities of 5-year survival and disease-free survival are 95 and 90%, respectively. Although prolonged survival can be achieved with aggressive

1	By replacing abnormal stem cells with cells from a normal donor, hematopoietic cell transplantation can cure patients of a variety of immunodeficiency disorders including severe combined immunodeficiency, Wiskott-Aldrich syndrome, and Chédiak-Higashi syndrome. The widest experience has been with severe combined immunodeficiency disease, where cure rates of 90% can be expected with HLA-identical donors and success rates of 50–70% have been reported using haplotype-mismatched parents as donors (Table 139e-3).

1	Transplantation from matched siblings after a preparative regimen of high-dose cyclophosphamide and antithymocyte globulin can cure up to 90% of patients age <40 years with severe aplastic anemia. Results in older patients and in recipients of mismatched family member or unrelated marrow are less favorable; therefore, a trial of immunosuppressive therapy is generally recommended for such patients before considering transplantation. Transplantation is effective in all forms of aplastic anemia including, for example, the syndromes associated with paroxysmal nocturnal hemoglobinuria and Fanconi’s anemia. Patients with Fanconi’s anemia are abnormally sensitive to the toxic effects of alkylating agents, and so less intensive preparative regimens must be used in their treatment (Chap. 130). Marrow transplantation from an HLA-identical sibling following a preparative regimen of busulfan and cyclophosphamide can cure 80–90% of patients with thalassemia major. The best outcomes can

1	Marrow transplantation from an HLA-identical sibling following a preparative regimen of busulfan and cyclophosphamide can cure 80–90% of patients with thalassemia major. The best outcomes can Disease Allogeneic, % Autologous, % aThese estimates are generally based on data reported by the International Bone Marrow Transplant Registry. The analysis has not been reviewed by their Advisory Committee. Abbreviations: ID, insufficient data; N/A, not applicable.

1	Abbreviations: ID, insufficient data; N/A, not applicable. chelation therapy, transplantation is the only curative treatment for thalassemia. Transplantation is being studied as a curative approach to patients with sickle cell anemia. Two-year survival and disease-free survival rates of 90 and 80%, respectively, have been reported following matched sibling or cord blood transplantation. Decisions about patient selection and the timing of transplantation remain difficult, but transplantation represents a reasonable option for younger patients who suffer repeated crises or other significant complications and who have not responded to other interventions (Chap. 127).

1	Theoretically, hematopoietic cell transplantation should be able to cure any disease that results from an inborn error of the lymphohematopoietic system. Transplantation has been used successfully to treat congenital disorders of white blood cells such as Kostmann’s syndrome, chronic granulomatous disease, and leukocyte adhesion deficiency. Congenital anemias such as Blackfan-Diamond anemia can also be cured with transplantation. Infantile malignant osteopetrosis is due to an inability of the osteoclast to resorb bone, and because osteoclasts derive from the marrow, transplantation can cure this rare inherited disorder.

1	Hematopoietic cell transplantation has been used as treatment for a number of storage diseases caused by enzymatic deficiencies, such as Gaucher’s disease, Hurler’s syndrome, Hunter’s syndrome, and infantile metachromatic leukodystrophy. Transplantation for these diseases has not been uniformly successful, but treatment early in the course of these diseases, before irreversible damage to extramedullary organs has occurred, increases the chance for success. Transplantation is being explored as a treatment for severe acquired autoimmune disorders. These trials are based on studies demonstrating that transplantation can reverse autoimmune disorders in animal models and on the observation that occasional patients with coexisting autoimmune disorders and hematologic malignancies have been cured of both with transplantation.

1	Allogeneic hematopoietic cell transplantation cures 15–20% of patients who do not achieve complete response from induction chemotherapy for acute myeloid leukemia (AML) and is the only form of therapy that can cure such patients. Cure rates of 30–35% are seen when patients are transplanted in second remission or in first relapse. The best results with allogeneic transplantation are achieved when applied during first remission, with disease-free survival rates averaging 55–60%. Meta-analyses of studies comparing matched related donor transplantation to chemotherapy for adult AML patients age <60 years show a survival advantage with transplantation. This advantage is greatest for those with unfavorable-risk AML and is lost in those with favorable-risk disease. The role of autologous transplantation in the treatment of AML is less well defined. The rates of disease recurrence with autologous transplantation are higher than those seen after allogeneic transplantation, and cure rates are

1	in the treatment of AML is less well defined. The rates of disease recurrence with autologous transplantation are higher than those seen after allogeneic transplantation, and cure rates are somewhat less.

1	Similar to patients with AML, adults with acute lymphocytic leukemia who do not achieve a complete response to induction chemotherapy can be cured in 15–20% of cases with immediate transplantation. Cure rates improve to 30–50% in second remission, and therefore transplantation can be recommended for adults who have persistent disease after induction chemotherapy or who have subsequently relapsed. Transplantation in first remission results in cure rates about 55%. Transplantation appears to offer a clear advantage over chemotherapy for patients with high-risk disease, such as those with Philadelphia chromosome–positive disease. Debate continues about whether adults with standard-risk disease should be transplanted in first remission or whether transplantation should be reserved until relapse. Autologous transplantation is associated with a higher relapse rate but a somewhat lower risk of nonrelapse mortality when compared to allogeneic transplantation. There is no obvious role of

1	relapse. Autologous transplantation is associated with a higher relapse rate but a somewhat lower risk of nonrelapse mortality when compared to allogeneic transplantation. There is no obvious role of autologous transplantation for acute lymphocytic leukemia in first remission, and for second-remission patients, most experts recommend use of allogeneic stem cells if an appropriate donor is available.

1	Allogeneic hematopoietic cell transplantation is the only therapy shown to cure a substantial portion of patients with chronic myeloid leukemia (CML). Five-year disease-free survival rates are 15–20% for patients transplanted for blast crisis, 25–50% for accelerated-phase patients, and 60–70% for chronic-phase patients, with cure rates as high as 80% at selected centers. However, with the availability of imatinib mesylate and other highly active tyrosine kinase inhibitors (TKIs), transplantation is generally reserved for those who fail to achieve a complete cytogenetic response with a TKI, relapse after an initial response, or are intolerant of the drugs (Chap. 133).

1	Allogeneic transplantation using a high-dose preparative regimen has rarely been used for chronic lymphocytic leukemia (CLL), in large part because of the chronic nature of the disease and because of the age profile of patients. In those cases where it was studied, complete remissions were achieved in the majority of patients, with disease-free survival rates of ~50% at 3 years, despite the advanced stage of the disease at the time of transplant. The marked antitumor effects have resulted in the increased use and study of allogeneic transplantation using reduced-intensity conditioning for the treatment of CLL.

1	Between 20 and 65% of patients with myelodysplasia appear to be cured with allogeneic transplantation. Results are better among younger patients and those with less advanced disease. However, patients with early-stage myelodysplasia can live for extended periods without intervention, and so transplantation is generally reserved for patients with an International Prognostic Scoring System (IPSS) score of Int-2 or for selected patients with an IPSS score of Int-1 who have other poor prognostic features (Chap. 130).

1	Patients with disseminated intermediateor high-grade non-Hodgkin’s lymphoma who have not been cured by first-line chemotherapy and are transplanted in first relapse or second remission can still be cured in 40–50% of cases. This represents a clear advantage over results obtained with conventional-dose salvage chemotherapy. It is unsettled whether patients with high-risk disease benefit from transplantation in first remission. Most experts favor the use of autologous rather than allogeneic transplantation for patients with intermediateor high-grade non-Hodgkin’s lymphoma, because fewer complications occur with this approach and survival appears equivalent. For patients with recurrent disseminated indolent nonHodgkin’s lymphoma, autologous transplantation results in high response rates and improved progression-free survival compared to salvage chemotherapy. However, late relapses are seen after transplantation. The role of autologous transplantation in the initial treatment of patients

1	improved progression-free survival compared to salvage chemotherapy. However, late relapses are seen after transplantation. The role of autologous transplantation in the initial treatment of patients is debated. It may be indicated in the small subset of patients presenting with high-risk prognostic factors but is not clearly more effective in those in lower risk groups. Reduced-intensity conditioning regimens followed by allogeneic transplantation result in high response rates in patients with indolent lymphomas, but the exact role of this approach remains to be defined.

1	The role of transplantation in Hodgkin’s disease is similar to that in intermediateand high-grade non-Hodgkin’s lymphoma. With transplantation, 5-year disease-free survival is 20–30% in patients who never achieve a first remission with standard chemotherapy and up to 70% for those transplanted in second remission. Transplantation has no defined role in first remission in Hodgkin’s disease. Patients with myeloma who have progressed on first-line therapy can sometimes benefit from allogeneic or autologous transplantation. Prospective randomized studies demonstrate that the inclusion of autologous transplantation as part of the initial therapy of patients results in improved disease-free survival and overall survival. Further benefit is seen with the use of lenalidomide maintenance therapy following transplantation. The use of autologous transplantation followed by nonmyeloablative allogeneic transplantation has yielded mixed results.

1	Randomized trials evaluating autologous transplantation as treatment for primary or metastatic breast cancer have failed to show a consistent survival advantage with this approach, and therefore, there is no established role for transplantation in this disease. Patients with testicular cancer in whom first-line platinum-containing chemotherapy has failed can still be cured in ~50% of cases if treated with high-dose chemotherapy with autologous stem cell support, an outcome better than that seen with low-dose salvage chemotherapy. The use of high-dose chemotherapy with autologous stem cell support is being studied for several other solid tumors, including neuroblastoma and pediatric sarcomas. As in most other settings, the best results have been obtained in patients with limited amounts of disease and where the remaining tumor remains sensitive to conventional-dose chemotherapy. Few randomized trials of transplantation in these diseases have been completed.

1	Partial and complete responses have been reported following nonmyeloablative allogeneic transplantation for some solid tumors, most notably renal cell cancers. The GVT effect, well documented in the treatment of hematologic malignancies, may apply to selected solid tumors under certain circumstances.

1	Patients who relapse following autologous transplantation sometimes respond to further chemotherapy and may be candidates for possible allogeneic transplantation, particularly if the remission following the initial autologous transplant was long. Several options are available for patients who relapse following allogeneic transplantation. Of particular interest are the response rates seen with infusion of unirradiated donor lymphocytes. Complete responses in as many as 75% of patients with chronic myeloid leukemia, 40% in myelodysplasia, 25% in AML, and 15% in myeloma have been reported. Major complications of donor lymphocyte infusions include transient myelosuppression and the development of GVHD. These complications depend on the number of donor lymphocytes given and the schedule of infusions, with less GVHD seen with lower dose, fractionated schedules.

1	Disorders of Platelets and Vessel wall Barbara A. Konkle Hemostasis is a dynamic process in which the platelet and the blood vessel wall play key roles. Platelets become activated upon adhesion to 140 sEC TION 3 Disorders of Platelets and Vessel Wall von Willebrand factor (VWF) and collagen in the exposed subendothelium after injury. Platelet activation is also mediated through shear forces imposed by blood flow itself, particularly in areas where the vessel wall is diseased, and is also affected by the inflammatory state of the endothelium. The activated platelet surface provides the major physiologic site for coagulation factor activation, which results in further platelet activation and fibrin formation. Genetic and acquired influences on the platelet and vessel wall, as well as on the coagulation and fibrinolytic systems, determine whether normal hemostasis or bleeding or clotting symptoms will result.

1	Platelets are released from the megakaryocyte, likely under the influence of flow in the capillary sinuses. The normal blood platelet count is 150,000–450,000/μL. The major regulator of platelet production is the hormone thrombopoietin (TPO), which is synthesized in the liver. Synthesis is increased with inflammation and specifically by interleukin 6. TPO binds to its receptor on platelets and megakaryocytes, by which it is removed from the circulation. Thus a reduction in platelet and megakaryocyte mass increases the level of TPO, which then stimulates platelet production. Platelets circulate with an average life span of 7–10 days. Approximately one-third of the platelets reside in the spleen, and this number increases in proportion to splenic size, although the platelet count rarely decreases to <40,000/μL as the spleen enlarges. Platelets are physiologically very active, but are anucleate, and thus have limited capacity to synthesize new proteins.

1	Normal vascular endothelium contributes to preventing thrombosis by inhibiting platelet function (Chap. 78). When vascular endothelium is injured, these inhibitory effects are overcome, and platelets adhere to the exposed intimal surface primarily through VWF, a large multimeric protein present in both plasma and in the extracellular matrix of the subendothelial vessel wall. Platelet adhesion results in the generation of intracellular signals that lead to activation of the platelet glycoprotein (Gp) IIb/IIIa (αIIbβ3) receptor and resultant platelet aggregation.

1	Activated platelets undergo release of their granule contents, which include nucleotides, adhesive proteins, growth factors, and procoagulants that serve to promote platelet aggregation and blood clot formation and influence the environment of the forming clot. During platelet aggregation, additional platelets are recruited to the site of injury, leading to the formation of an occlusive platelet thrombus. The platelet plug is stabilized by the fibrin mesh that develops simultaneously as the product of the coagulation cascade. Endothelial cells line the surface of the entire circulatory tree, totaling 1–6 × 1013 cells, enough to cover a surface area equivalent to about six tennis courts. The endothelium is physiologically active, controlling vascular permeability, flow of biologically active molecules and nutrients, blood cell interactions with the vessel wall, the inflammatory response, and angiogenesis.

1	The endothelium normally presents an antithrombotic surface (Chap. 78) but rapidly becomes prothrombotic when stimulated, which promotes coagulation, inhibits fibrinolysis, and activates platelets. In many cases, endothelium-derived vasodilators are also platelet inhibitors (e.g., nitric oxide) and, conversely, endothelium-derived vasoconstrictors (e.g., endothelin) can also be platelet activators. The net effect of vasodilation and inhibition of platelet function is to promote blood fluidity, whereas the net effect of vasoconstriction and platelet activation is to promote thrombosis. Thus, blood fluidity and hemostasis are regulated by the balance of antithrombotic/prothrombotic and vasodilatory/vasoconstrictor properties of endothelial cells.

1	Thrombocytopenia results from one or more of three processes: (1) decreased bone marrow production; (2) sequestration, usually in an enlarged spleen; and/or (3) increased platelet destruction. Disorders of production may be either inherited or acquired. In evaluating a patient with thrombocytopenia, a key step is to review the peripheral blood smear and to first rule out “pseudothrombocytopenia,” particularly in a patient without an apparent cause for the thrombocytopenia. Pseudothrombocytopenia (Fig. 140-1B) is an in vitro artifact resulting from platelet agglutination via antibodies (usually IgG, but also IgM and IgA) when the calcium content is decreased by blood collection in ethylenediamine tetraacetic (EDTA) (the anticoagulant present in tubes [purple top] used to collect blood for complete blood counts [CBCs]). If a low platelet count is obtained in EDTA-anticoagulated blood, a blood smear should be evaluated and a platelet count determined in blood collected into sodium

1	for complete blood counts [CBCs]). If a low platelet count is obtained in EDTA-anticoagulated blood, a blood smear should be evaluated and a platelet count determined in blood collected into sodium citrate (blue top tube) or heparin (green top tube), or a smear of freshly obtained unanticoagulated blood, such as from a finger stick, can be examined.

1	FIGURE 140-1 Photomicrographs of peripheral blood smears. A. Normal peripheral blood. B. Platelet clumping in pseudothrombocytopenia. C. Abnormal large platelet in autosomal dominant macrothrombocytopenia. D. Schistocytes and decreased platelets in microangiopathic hemolytic anemia. APPROACH TO THE PATIENT:

1	The history and physical examination, results of the CBC, and review of the peripheral blood smear are all critical components in the initial evaluation of thrombocytopenic patients (Fig. 140-2). The overall health of the patient and whether he or she is receiving drug treatment will influence the differential diagnosis. A healthy young adult with thrombocytopenia will have a much more limited differential diagnosis than an ill hospitalized patient who is receiving multiple medications. Except in unusual inherited disorders, decreased platelet production usually results from bone marrow disorders that also affect red blood cell (RBC) and/or white blood cell (WBC) production. Because myelodysplasia can present with isolated thrombocytopenia, the bone marrow should be examined in patients presenting with isolated thrombocytopenia who are older than 60 years of age. While inherited thrombocytopenia is rare, any prior platelet counts should be retrieved and a family history regarding

1	presenting with isolated thrombocytopenia who are older than 60 years of age. While inherited thrombocytopenia is rare, any prior platelet counts should be retrieved and a family history regarding thrombocytopenia obtained. A careful history of drug ingestion should be obtained, including nonprescription and herbal remedies, because drugs are the most common cause of thrombocytopenia.

1	The physical examination can document an enlarged spleen, evidence of chronic liver disease, and other underlying disorders. Mild to moderate splenomegaly may be difficult to appreciate in many individuals due to body habitus and/or obesity but can be easily assessed by abdominal ultrasound. A platelet count of approxi- FIGURE 140-2 Algorithm for evaluating the thrombocytopenic mately 5000–10,000 is required to maintain vascular integrity in the patient. DIC, disseminated intravascular coagulation; RBC, red blood microcirculation. When the count is markedly decreased, petechiae cell; TTP, thrombotic thrombocytopenic purpura.

1	Platelet count < 150,000/˜L Hemoglobin and white blood count Normal Abnormal Bone marrow examination Peripheral blood smear Platelets clumped: Redraw in sodium citrate or heparin Fragmented red blood cells Normal RBC morphology; platelets normal or increased in size Microangiopathic hemolytic anemias (e.g., DIC, TTP) Consider: Drug-induced thrombocytopenia Infection-induced thrombocytopenia Idiopathic immune thrombocytopenia Congenital thrombocytopenia first appear in areas of increased venous pressure, the ankles and feet in an ambulatory patient. Petechiae are pinpoint, nonblanching hemorrhages and are usually a sign of a decreased platelet number and not platelet dysfunction. Wet purpura, blood blisters that form on the oral mucosa, are thought to denote an increased risk of life-threatening hemorrhage in the thrombocytopenic patient. Excessive bruising is seen in disorders of both platelet number and function.

1	Infection-Induced Thrombocytopenia Many viral and bacterial infections result in thrombocytopenia and are the most common noniatrogenic cause of thrombocytopenia. This may or may not be associated with laboratory evidence of disseminated intravascular coagulation (DIC), which is most commonly seen in patients with systemic infections with gram-negative bacteria. Infections can affect both platelet production and platelet survival. In addition, immune mechanisms can be at work, as in infectious mononucleosis and early HIV infection. Late in HIV infection, pancytopenia and decreased and dysplastic platelet production are more common. Immune-mediated thrombocytopenia in children usually follows a viral infection and almost always resolves spontaneously. This association of infection with immune thrombocytopenic purpura is less clear in adults.

1	Bone marrow examination is often requested for evaluation of occult infections. A study evaluating the role of bone marrow examination in fever of unknown origin in HIV-infected patients found that for 86% of patients, the same diagnosis was established by less invasive techniques, notably blood culture. In some instances, however, the diagnosis can be made earlier; thus, a bone marrow examination and culture are recommended when the diagnosis is needed urgently or when other, less invasive methods have been unsuccessful.

1	Drug-Induced Thrombocytopenia Many drugs have been associated with thrombocytopenia. A predictable decrease in platelet count occurs after treatment with many chemotherapeutic drugs due to bone marrow suppression (Chap. 103e). Drugs that cause isolated thrombocytopenia and have been confirmed with positive laboratory testing are listed in Table 140-1, but all drugs should be suspect in a patient with thrombocytopenia without an apparent cause and should be stopped, or substituted, if possible. A helpful website, Platelets on the Internet (http://www.ouhsc.edu/platelets/ditp.html), lists drugs and supplements reported to have caused thrombocytopenia and the level of evidence supporting the association. Although not as well studied, herbal and over-the-counter preparations may also result in thrombocytopenia and should be discontinued in patients who are thrombocytopenic.

1	aBased on scoring requiring a compatible clinical picture and positive laboratory testing. Source: Adapted from DM Arnold et al: J Thromb Hemost 11:169, 2013.

1	Classic drug-dependent antibodies are antibodies that react with 727 specific platelet surface antigens and result in thrombocytopenia only when the drug is present. Many drugs are capable of inducing these antibodies, but for some reason, they are more common with quinine and sulfonamides. Drug-dependent antibody binding can be demonstrated by laboratory assays, showing antibody binding in the presence of, but not without, the drug present in the assay. The thrombocytopenia typically occurs after a period of initial exposure (median length 21 days), or upon reexposure, and usually resolves in 7–10 days after drug withdrawal. The thrombocytopenia caused by the platelet Gp IIb/ IIIa inhibitory drugs, such as abciximab, differs in that it may occur within 24 h of initial exposure. This appears to be due to the presence of naturally occurring antibodies that cross-react with the drug bound to the platelet.

1	Heparin-Induced Thrombocytopenia Drug-induced thrombocytopenia due to heparin differs from that seen with other drugs in two major ways. (1) The thrombocytopenia is not usually severe, with nadir counts rarely <20,000/μL. (2) Heparin-induced thrombocytopenia (HIT) is not associated with bleeding and, in fact, markedly increases the risk of thrombosis. HIT results from antibody formation to a complex of the platelet-specific protein platelet factor 4 (PF4) and heparin. The anti-heparin/PF4 antibody can activate platelets through the FcγRIIa receptor and also activate monocytes and endothelial cells. Many patients exposed to heparin develop antibodies to heparin/PF4, but do not appear to have adverse consequences. A fraction of those who develop antibodies will develop HIT, and a portion of those (up to 50%) will develop thrombosis (HITT).

1	HIT can occur after exposure to low-molecular-weight heparin (LMWH) as well as unfractionated heparin (UFH), although it is more common with the latter. Most patients develop HIT after exposure to heparin for 5–14 days (Fig. 140-3). It occurs before 5 days in those who were exposed to heparin in the prior few weeks or months (<~100 days) and have circulating anti-heparin/PF4 antibodies. Rarely, thrombocytopenia and thrombosis begin several days after all heparin has been stopped (termed delayed-onset HIT). The “4T’s” have been recommended to be used in a diagnostic algorithm for HIT: thrombocytopenia, timing of platelet count drop, thrombosis and other sequelae such as localized skin reactions, and other causes of thrombocytopenia not evident. Application of the 4T scoring system is very useful in excluding a diagnosis of HIT but will result in overdiagnosis of HIT in situations where thrombocytopenia and thrombosis due to other etiologies are common, such as in the intensive care

1	useful in excluding a diagnosis of HIT but will result in overdiagnosis of HIT in situations where thrombocytopenia and thrombosis due to other etiologies are common, such as in the intensive care unit. A scoring model based on broad expert opinion (the HIT Expert Probability [HEP] Score) has improved operating characteristics and may provide better utility as a scoring system.

1	laboratory testing for Hit HIT (anti-heparin/PF4) antibodies can be detected using two types of assays. The most widely available is an enzyme-linked immunoassay (ELISA) with PF4/polyanion complex as the antigen. Because many patients develop antibodies but do not develop clinical HIT, the test has a low specificity for the diagnosis Days of heparin (UFH or LMWH) exposure

1	Days of heparin (UFH or LMWH) exposure FIGURE 140-3 Time course of heparin-induced thrombocytopenia (HIT) development after heparin exposure. The timing of development after heparin exposure is a critical factor in determining the likelihood of HIT in a patient. HIT occurs early after heparin exposure in the presence of preexisting heparin/platelet factor 4 (PF4) antibodies, which disappear from circulation by ~100 days following a prior exposure. Rarely, HIT may occur later after heparin exposure (termed delayed-onset HIT). In this setting, heparin/PF4 antibody testing is usually markedly positive. HIT can occur after exposure to either unfractionated (UFH) or low-molecular-weight heparin (LMWH).

1	Disorders of Platelets and Vessel Wall 728 of HIT. This is especially true in patients who have undergone cardiopulmonary bypass surgery, where approximately 50% of patients develop these antibodies postoperatively. IgG-specific ELISAs increase specificity but may decrease sensitivity. The other assay is a platelet activation assay, most commonly the serotonin release assay, which measures the ability of the patient’s serum to activate platelets in the presence of heparin in a concentration-dependent manner. This test has lower sensitivity but higher specificity than the ELISA. However, HIT remains a clinical diagnosis.

1	Early recognition is key in treatment of HIT, with prompt discontinuation of heparin and use of alternative anticoagulants if bleeding risk does not outweigh thrombotic risk. Thrombosis is a common complication of HIT, even after heparin discontinuation, and can occur in both the venous and arterial systems. Patients with higher anti-heparin/PF4 antibody titers may have a higher risk of thrombosis. In patients diagnosed with HIT, imaging studies to evaluate the patient for thrombosis (at least lower extremity duplex Doppler imaging) are recommended. Patients requiring anticoagulation should be switched from heparin to an alternative anticoagulant. The direct thrombin inhibitors (DTIs) argatroban and lepirudin are effective in HITT. The DTI bivalirudin and the antithrombinbinding pentasaccharide fondaparinux are also effective but not yet approved by the U.S. Food and Drug Administration (FDA) for this indication. Danaparoid, a mixture of glycosaminoglycans with anti-Xa activity, has

1	fondaparinux are also effective but not yet approved by the U.S. Food and Drug Administration (FDA) for this indication. Danaparoid, a mixture of glycosaminoglycans with anti-Xa activity, has been used extensively for the treatment of HITT; it is no longer available in the United States but is in other countries. HIT antibodies cross-react with LMWH, and these preparations should not be used in the treatment of HIT.

1	Because of the high rate of thrombosis in patients with HIT, anticoagulation should be considered, even in the absence of thrombosis. In patients with thrombosis, patients can be transitioned to warfarin, with treatment usually for 3–6 months. In patients without thrombosis, the duration of anticoagulation needed is undefined. An increased risk of thrombosis is present for at least 1 month after diagnosis; however, most thromboses occur early, and whether thrombosis occurs later if the patient is initially anticoagulated is unknown. Options include continuing anticoagulation until a few days after platelet recovery or for 1 month. Introduction of warfarin alone in the setting of HIT or HITT may precipitate thrombosis, particularly venous gangrene, presumably due to clotting activation and severely reduced levels of proteins C and S. Warfarin therapy, if started, should be overlapped with a DTI or fondaparinux and started after resolution of the thrombocytopenia and lessening of the

1	severely reduced levels of proteins C and S. Warfarin therapy, if started, should be overlapped with a DTI or fondaparinux and started after resolution of the thrombocytopenia and lessening of the prothrombotic state.

1	Immune Thrombocytopenic Purpura Immune thrombocytopenic purpura (ITP; also termed idiopathic thrombocytopenic purpura) is an acquired disorder in which there is immune-mediated destruction of platelets and possibly inhibition of platelet release from the megakaryocyte. In children, it is usually an acute disease, most commonly following an infection, and with a self-limited course. In adults, it is a more chronic disease, although in some adults, spontaneous remission occurs, usually within months of diagnosis. ITP is termed secondary if it is associated with an underlying disorder; autoimmune disorders, particularly systemic lupus erythematosus (SLE), and infections, such as HIV and hepatitis C, are common causes. The association of ITP with Helicobacter pylori infection is unclear.

1	ITP is characterized by mucocutaneous bleeding and a low, often very low, platelet count, with an otherwise normal peripheral blood cells and smear. Patients usually present either with ecchymoses and petechiae, or with thrombocytopenia incidentally found on a routine CBC. Mucocutaneous bleeding, such as oral mucosa, gastrointestinal, or heavy menstrual bleeding, may be present. Rarely, life-threatening, including central nervous system, bleeding can occur. Wet purpura (blood blisters in the mouth) and retinal hemorrhages may herald life-threatening bleeding.

1	laboratory testing in itp Laboratory testing for antibodies (serologic testing) is usually not helpful due to the low sensitivity and specificity of the current tests. Bone marrow examination can be reserved for those who have other signs or laboratory abnormalities not explained by ITP or in patients who do not respond to initial therapy. The peripheral blood smear may show large platelets, with otherwise normal morphology. Depending on the bleeding history, iron-deficiency anemia may be present.

1	Laboratory testing is performed to evaluate for secondary causes of ITP and should include testing for HIV infection and hepatitis C (and other infections if indicated). Serologic testing for SLE, serum protein electrophoresis, immunoglobulin levels to potentially detect hypogammaglobulinemia, selective testing for IgA deficiency or monoclonal gammopathies, and testing for H. pylori infection should be considered, depending on the clinical circumstance. If anemia is present, direct antiglobulin testing (Coombs’ test) should be performed to rule out combined autoimmune hemolytic anemia with ITP (Evans’ syndrome). The treatment of ITP uses drugs that decrease reticuloendothelial uptake of the antibody-bound platelet, decrease antibody production, and/or increase platelet production. The diagnosis of ITP does not necessarily mean that treatment must be instituted. Patients with platelet counts >30,000/μL appear not to have increased mortality related to the thrombocytopenia.

1	Initial treatment in patients without significant bleeding symptoms, severe thrombocytopenia (<5000/μL), or signs of impending bleeding (such as retinal hemorrhage or large oral mucosal hemorrhages) can be instituted as an outpatient using single agents. Traditionally, this has been prednisone at 1 mg/kg, although Rh0(D) immune globulin therapy (WinRho SDF), at 50–75 μg/kg, is also being used in this setting. Rh0(D) immune globulin must be used only in Rh-positive patients because the mechanism of action is production of limited hemolysis, with antibody-coated cells “saturating” the Fc receptors, inhibiting Fc receptor function. Monitoring patients for 8 h after infusion is now advised by the FDA because of the rare complication of severe intravascular hemolysis. Intravenous gamma globulin (IVIgG), which is pooled, primarily IgG antibodies, also blocks the Fc receptor system, but appears to work primarily through different mechanism(s). IVIgG has more efficacy than anti-Rh0(D) in

1	(IVIgG), which is pooled, primarily IgG antibodies, also blocks the Fc receptor system, but appears to work primarily through different mechanism(s). IVIgG has more efficacy than anti-Rh0(D) in postsplenectomized patients. IVIgG is dosed at 1–2 g/kg total, given over 1–5 days. Side effects are usually related to the volume of infusion and infrequently include aseptic meningitis and renal failure. All immunoglobulin preparations are derived from human plasma and undergo treatment for viral inactivation.

1	For patients with severe ITP and/or symptoms of bleeding, hospital admission and combined-modality therapy is given using high-dose glucocorticoids with IVIgG or anti-Rh0(D) therapy and, as needed, additional immunosuppressive agents. Rituximab, an anti-CD20 (B cell) antibody, has shown efficacy in the treatment of refractory ITP, although long-lasting remission only occurs in approximately 30% of patients.

1	Splenectomy has been used for treatment of patients who relapse after glucocorticoids are tapered. Splenectomy remains an important treatment option; however, more patients than previously thought will go into a remission over time. Observation, if the platelet count is high enough, or intermittent treatment with anti-Rh0(D) or IVIgG, or initiation of treatment with a TPO receptor agonist (see below) may be a reasonable approach to see if the ITP will resolve. Vaccination against encapsulated organisms (especially pneumococcus, but also meningococcus and Haemophilus influenzae, depending on patient age and potential exposure) is recommended before splenectomy. Accessory spleen(s) are a very rare cause of relapse.

1	TPO receptor agonists are now available for the treatment of ITP. This approach stems from the finding that many patients with ITP do not have increased TPO levels, as was previously hypothesized. TPO levels reflect megakaryocyte mass, which is usually normal in ITP. VWF and Platelet Adhesion TPO levels are not increased in the setting of platelet destruction. Two agents, one administered subcutaneously (romiplostim) and another orally (eltrombopag), are effective in raising platelet counts in patients with ITP and are recommended for adults at risk of bleeding who relapse after splenectomy or who have been unresponsive to at least one other therapy, particularly in those who have a contraindication to splenectomy. However, with the recognition that ITP will resolve spontaneously in some adult patients, short-term treatment with a TPO agonist can be considered before splenectomy in patients who need therapy.

1	Inherited Thrombocytopenia Thrombocytopenia is rarely inherited, either as an isolated finding or as part of a syndrome, and may be inherited in an autosomal dominant, autosomal recessive, or X-linked pattern. Many forms of autosomal dominant thrombocytopenia are now known to be associated with mutations in the nonmuscle myosin heavy chain MYH9 gene. Interestingly, these include the May-Hegglin anomaly, and Sebastian, Epstein’s, and Fechtner syndromes, all of which have distinct distinguishing features. A common feature of these disorders is large platelets (Fig. 140-1C). Autosomal recessive disorders include congenital amegakaryocytic thrombocytopenia, thrombocytopenia with absent radii, and Bernard-Soulier syndrome. The latter is primarily a functional platelet disorder due to absence of Gp Ib-IX-V, the VWF adhesion receptor. X-linked disorders include Wiskott-Aldrich syndrome and a dyshematopoietic syndrome resulting from a mutation in GATA-1, an important transcriptional regulator

1	Gp Ib-IX-V, the VWF adhesion receptor. X-linked disorders include Wiskott-Aldrich syndrome and a dyshematopoietic syndrome resulting from a mutation in GATA-1, an important transcriptional regulator of hematopoiesis.

1	Thrombotic thrombocytopenic microangiopathies are a group of disorders characterized by thrombocytopenia, a microangiopathic hemolytic anemia evident by fragmented RBCs (Fig. 140-1D) and laboratory evidence of hemolysis, and microvascular thrombosis. They include thrombotic thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome (HUS), as well as syndromes complicating bone marrow transplantation, certain medications and infections, pregnancy, and vasculitis. In DIC, although thrombocytopenia and microangiopathy are seen, a coagulopathy predominates, with consumption of clotting factors and fibrinogen resulting in an elevated prothrombin time (PT) and often activated partial thromboplastin time (aPTT). The PT and aPTT are characteristically normal in TTP or HUS.

1	Thrombotic Thrombocytopenic Purpura TTP and HUS were previously considered overlap syndromes. However, in the past few years, the pathophysiology of inherited and idiopathic TTP has become better understood and clearly differs from HUS. TTP was first described in 1924 by Eli Moschcowitz and characterized by a pentad of findings that include microangiopathic hemolytic anemia, thrombocytopenia, renal failure, neurologic findings, and fever. The full-blown syndrome is less commonly seen now, probably due to earlier diagnosis. The introduction of treatment with plasma exchange markedly improved the prognosis in patients, with a decrease in mortality from 85–100% to 10–30%.

1	The pathogenesis of inherited (Upshaw-Schulman syndrome) and idiopathic TTP is related to a deficiency of, or antibodies to, the metalloprotease ADAMTS13, which cleaves VWF. VWF is normally secreted as ultra-large multimers, which are then cleaved by ADAMTS13. The persistence of ultra-large VWF molecules is thought to contribute to pathogenic platelet adhesion and aggregation (Fig. 140-4). This defect alone, however, is not sufficient to result in TTP because individuals with a congenital absence of ADAMTS13 develop TTP only episodically. Additional provocative factors have not been defined. The level of ADAMTS13 activity, as well as antibodies, can now be detected by laboratory assays. Although assays with sufficient sensitivity and specificity to direct clinical management have yet to be clearly defined, ADAMTS13 activity levels of <10% are more clearly associated with idiopathic TTP. Normal “Ultralarge” TTP? multimers multimers

1	Normal “Ultralarge” TTP? multimers multimers FIGURE 140-4 Pathogenesis of thrombotic thrombocytopenic purpura (TTP). Normally the ultra-high-molecular-weight multimers of von Willebrand factor (VWF) produced by the endothelial cells are processed into smaller multimers by a plasma metalloproteinase called ADAMTS13. In TTP, the activity of the protease is inhibited, and the ultra-high-molecular-weight multimers of VWF initiate platelet aggregation and thrombosis.

1	Idiopathic TTP appears to be more common in women than in men. No geographic or racial distribution has been defined. TTP is more common in patients with HIV infection and in pregnant women. TTP in pregnancy is not clearly related to ADAMTS13. Medication-related microangiopathic hemolytic anemia may be secondary to antibody formation (ticlopidine and possibly clopidogrel) or direct endothelial toxicity (cyclosporine, mitomycin C, tacrolimus, quinine), although this is not always so clear, and fear of withholding treatment, as well as lack of other treatment alternatives, results in broad application of plasma exchange. However, withdrawal, or reduction in dose, of endothelial toxic agents usually decreases the microangiopathy.

1	TTP is a devastating disease if not diagnosed and treated promptly. In patients presenting with new thrombocytopenia, with or without evidence of renal insufficiency and other elements of classic TTP, laboratory data should be obtained to rule out DIC and to evaluate for evidence of microangiopathic hemolytic anemia. Findings to support the TTP diagnosis include an increased lactate dehydrogenase and indirect bilirubin, decreased haptoglobin, and increased reticulocyte count, with a negative direct antiglobulin test. The peripheral smear should be examined for evidence of schistocytes (Fig. 140-1D). Polychromasia is usually also present due to the increased number of young red blood cells, and nucleated RBCs are often present, which is thought to be due to infarction in the micro-circulatory system of the bone marrow.

1	Plasma exchange remains the mainstay of treatment of TTP. ADAMTS13 antibody-mediated TTP (idiopathic TTP) appears to respond best to plasma exchange. Plasma exchange is continued CHAPTEr140 Disorders of Platelets and Vessel Wall 730 until the platelet count is normal and signs of hemolysis are resolved for at least 2 days. Although never evaluated in clinical trials, the use of glucocorticoids seems a reasonable approach, but should only be used as an adjunct to plasma exchange. Additionally, other immunomodulatory therapies have been reported to be successful in refractory or relapsing TTP, including rituximab, vincristine, cyclophosphamide, and splenectomy. A significant relapse rate is noted; 25–45% of patients relapse within 30 days of initial “remission,” and 12–40% of patients have late relapses. Relapses are more frequent in patients with severe ADAMTS13 deficiency at presentation.

1	Hemolytic-Uremic Syndrome HUS is a syndrome characterized by acute renal failure, microangiopathic hemolytic anemia, and thrombocytopenia. It is seen predominantly in children and in most cases is preceded by an episode of diarrhea, often hemorrhagic in nature. Escherichia coli O157:H7 is the most frequent, although not only, etiologic serotype. HUS not associated with diarrhea is more heterogeneous in presentation and course. Atypical HUS (aHUS) due to genetic defects that result in chronic complement activation has been defined, and screening for mutations in complement regulatory genes is available.

1	Treatment of HUS is primarily supportive. In HUS associated with diarrhea, many (~40%) children require at least some period of support with dialysis; however, the overall mortality is <5%. In HUS not associated with diarrhea, the mortality is higher, approximately 26%. Plasma infusion or plasma exchange has not been shown to alter the overall course. ADAMTS13 levels are generally reported to be normal in HUS, although occasionally they have been reported to be decreased. In patients with atypical HUS, eculizumab therapy increases the platelet count and preserves renal function.

1	Thrombocytosis is almost always due to (1) iron deficiency; (2) inflammation, cancer, or infection (reactive thrombocytosis); or (3) an underlying myeloproliferative process (essential thrombocythemia or polycythemia vera) (Chap. 131) or, rarely, the 5q– myelodysplastic process (Chap. 130). Patients presenting with an elevated platelet count should be evaluated for underlying inflammation or malignancy, and iron deficiency should be ruled out. Thrombocytosis in response to acute or chronic inflammation has not been clearly associated with an increased thrombotic risk. In fact, patients with markedly elevated platelet counts (>1.5 million), usually seen in the setting of a myeloproliferative disorder, have an increased risk of bleeding. This appears to be due, at least in part, to acquired von Willebrand disease (VWD) due to platelet-VWF binding and removal from the circulation.

1	QUALITATIVE DISORDERS OF PLATELET FUNCTION Inherited Disorders of Platelet Function Inherited platelet function disorders are thought to be relatively rare, although the prevalence of mild disorders of platelet function is unclear, in part because our testing for such disorders is suboptimal. Rare qualitative disorders include the autosomal recessive disorders Glanzmann’s thrombasthenia (absence of the platelet Gp IIb/IIIa receptor) and Bernard-Soulier syndrome (absence of the platelet Gp Ib-IX-V receptor). Both are inherited in an autosomal recessive fashion and present with bleeding symptoms in childhood.

1	Platelet storage pool disorder (SPD) is the classic autosomal dominant qualitative platelet disorder. This results from abnormalities of platelet granule formation. It is also seen as a part of inherited disorders of granule formation, such as Hermansky-Pudlak syndrome. Bleeding symptoms in SPD are variable, but often are mild. The most common inherited disorders of platelet function prevent normal secretion of granule content and are termed secretion defects. Few of these abnormalities have been dissected at the molecular level but they likely result from various mutations..

1	Bleeding symptoms or prevention of bleeding in patients with severe platelet dysfunction frequently requires platelet transfusion. Care is taken to limit the risk of alloimmunization by limiting exposure, using HLA-matched leuko-depleted platelet concentrates for transfusion. Platelet disorders associated with milder bleeding symptoms frequently respond to desmopressin (1-deamino-8-Darginine vasopressin [DDAVP]). DDAVP increases plasma VWF and factor VIII levels; it may also have a direct effect on platelet function. Particularly for mucosal bleeding symptoms, antifibrinolytic therapy (ε-aminocaproic acid or tranexamic acid) is used alone or in conjunction with DDAVP or platelet therapy.

1	Acquired Disorders of Platelet Function Acquired platelet dysfunction is common, usually due to medications, either intentionally as with antiplatelet therapy or unintentionally as with high-dose penicillins. Acquired platelet dysfunction occurs in uremia. This is likely multifactorial, but the resultant effect is defective adhesion and activation. The platelet defect is improved most by dialysis but may also be improved by increasing the hematocrit to 27–32%, giving DDAVP (0.3 μg/kg), or use of conjugated estrogens. Platelet dysfunction also occurs with cardiopulmonary bypass due to the effect of the artificial circuit on platelets, and bleeding symptoms respond to platelet transfusion. Platelet dysfunction seen with underlying hematologic disorders can result from nonspecific interference by circulating paraproteins or intrinsic platelet defects in myeloproliferative and myelodysplastic syndromes.

1	VWD is the most common inherited bleeding disorder. Estimates from laboratory data suggest a prevalence of approximately 1%, but data based on symptomatic individuals suggest that it is closer to 0.1% of the population. VWF serves two roles: (1) as the major adhesion molecule that tethers the platelet to the exposed subendothelium; and (2) as the binding protein for factor VIII (FVIII), resulting in significant prolongation of the FVIII half-life in circulation. The platelet-adhesive function of VWF is critically dependent on the presence of large VWF multimers, whereas FVIII binding is not. Most of the symptoms of VWD are “platelet-like” except in more severe VWD when the FVIII is low enough to produce symptoms similar to those found in FVIII deficiency (hemophilia A).

1	VWD has been classified into three major types, with four subtypes of type 2 (Table 140-2; Fig. 140-5). By far the most common type of VWD is type 1 disease, with a parallel decrease in VWF protein, VWF function, and FVIII levels, accounting for at least 80% of cases. Patients have predominantly mucosal bleeding symptoms, although postoperative bleeding can also be seen. Bleeding symptoms are very uncommon in infancy and usually manifest later in childhood with excessive bruising and epistaxis. Because these symptoms occur commonly in childhood, the clinician should particularly note bruising at sites unlikely to be traumatized and/or prolonged epistaxis requiring medical attention. Menorrhagia is a common manifestation of VWD. Menstrual bleeding resulting in anemia should warrant an evaluation for VWD and, if negative, functional platelet disorders. Frequently, mild type 1 VWD first manifests with dental extractions, particularly wisdom tooth extraction, or tonsillectomy.

1	Not all patients with low VWF levels have bleeding symptoms. Whether patients bleed or not will depend on the overall hemostatic balance they have inherited, along with environmental influences and the type of hemostatic challenges they experience. Although the inheritance of VWD is autosomal, many factors modulate both VWF levels and bleeding symptoms. These have not all been defined, but include blood type, thyroid hormone status, race, stress, exercise, and hormonal (both endogenous and exogenous) influences. Patients with type O blood have VWF protein levels of approximately one-half that of patients with AB blood type; and, in fact, the normal range for 731resulting in loss of intermediateand high-molecular-weight multimers, or to TABLE 140-2 LABOrATOrY DIAgNOsIs Of VON wILLEBrAND DIsEAsE (VwD) decreased secretion of these multimers 2M Nl or ↑↓ ↓ ↓ ↓ 2N ↑↑ Nl or ↓b Nl or ↓b ↓↓ 3 ↑↑↓↓ ↓↓ ↓↓ Normal distribution, decreased in by the cell. Type 2B VWD results from

1	Loss of highand intermediate-MW increased spontaneous binding of VWF to platelets in circulation, with subse- Loss of high-MW multimers quent clearance of this complex by the Normal distribution, decreased in reticuloendothelial system. The resultingquantity VWF in the patients’ plasma lacks the Normal distribution highest molecular-weight multimers, and Absent the platelet count is usually modestly reduced. Type 2M occurs as a conse aUsually also decreased platelet count. bFor type 2N, in the homozygous state, FVIII is very low; in the heterozygous state, it is only seen in conjunction with type 1 VWD. quence of a group of mutations that cause dysfunction but do not affect multimer Abbreviations: aPTT, activated partial thromboplastin time; F, factor; MW, molecular weight; Nl, normal; VWF, von Willebrand factor.

1	Abbreviations: aPTT, activated partial thromboplastin time; F, factor; MW, molecular weight; Nl, normal; VWF, von Willebrand factor. patients with type O blood overlaps that which has been considered diagnostic for VWD. A mildly decreased VWF level should be viewed more as a risk factor for bleeding than as an actual disease. Patients with type 2 VWD have functional defects; thus, the VWF antigen measurement is significantly higher than the test of function. For types 2A, 2B, and 2M VWD, platelet-binding and/or collagen binding VWF activity is decreased. In type 2A VWD, the impaired function is due either to increased susceptibility to cleavage by ADAMTS13,

1	FIGURE 140-5 Pattern of inheritance and laboratory findings in von Willebrand disease (VWD). The assays of platelet function include a coagulation assay of factor VIII bound and carried by von Willebrand factor (VWF), abbreviated as VIII; immunoassay of total VWF protein (VWF:Ag); bioassay of the ability of patient plasma to support ristocetin-induced agglutination of normal platelets (VWF:RCoF); and ristocetin-induced aggregation of patient platelets, abbreviated RIPA. The multimer pattern illustrates the protein bands present when plasma is electrophoresed in a polyacrylamide gel. The II-1 and II-2 columns refer to the phenotypes of the second-generation offspring. structure.

1	structure. Type 2N VWD is due to mutations in VWF that affect binding of FVIII. As FVIII is stabilized by binding to VWF, the FVIII in patients with type 2N VWD has a very short half-life, and the FVIII level is markedly decreased. This is sometimes termed autosomal hemophilia. Type 3 VWD, or severe VWD, describes patients with virtually no VWF protein and FVIII levels <10%. Patients experience mucosal and joint bleeding, surgery-related bleeding, and other bleeding symptoms. Some patients with type 3 VWD, particularly those with large VWF gene deletions, are at risk of developing antibodies to infused VWF.

1	Acquired VWD is a rare disorder, most commonly seen in patients with underlying lymphoproliferative disorders, including monoclonal gammopathies of underdetermined significance (MGUS), multiple myeloma, and Waldenström’s macroglobulinemia. It is seen most commonly in the setting of MGUS and should be suspected in patients, particularly elderly patients, with a new onset of severe mucosal bleeding symptoms. Laboratory evidence of acquired VWD is found in some patients with aortic valvular disease. Heyde’s syndrome (aortic stenosis with gastrointestinal bleeding) is attributed to the presence of angiodysplasia of the gastrointestinal tract in patients with aortic steno-sis. The shear stress on blood passing through the stenotic aortic valve appears to produce a change in VWF, making it susceptible to serum proteases. Consequently, large multimer forms are lost, leading to an acquired type 2 VWD, but return when the stenotic valve is replaced.

1	The mainstay of treatment for type 1 VWD is DDAVP (desmopressin), which results in release of VWF and FVIII from endothelial stores. DDAVP can be given intravenously or by a high-concentration intranasal spray (1.5 mg/mL). The peak activity when given intravenously is approximately 30 min, whereas it is 2 h when given intranasally. The usual dose is 0.3 μg/kg intravenously or two squirts (one in each nostril) for patients >50 kg (one squirt for those <50 kg). It is recommended that patients with VWD be tested with DDAVP to assess their response before using it. In patients who respond well (increase in laboratory values of twoto fourfold), it can be used for procedures with minor to moderate risk of bleeding. Depending on the procedure, additional doses may be needed; it is usually given every 12–24 h. Less frequent dosing may result in less tachyphylaxis, which occurs when synthesis cannot compensate for the released stores. The major side effect of DDAVP is hyponatremia due to

1	every 12–24 h. Less frequent dosing may result in less tachyphylaxis, which occurs when synthesis cannot compensate for the released stores. The major side effect of DDAVP is hyponatremia due to decreased free water clearance. This occurs most commonly in the very young and the very old, but fluid restriction should be advised for all patients for the 24 h following each dose.

1	Some patients with types 2A and 2M VWD respond to DDAVP such that it can be used for minor procedures. For the other subtypes, for type 3 disease, and for major procedures requiring longer periods of normal hemostasis, VWF replacement can be given. Virally inactivated VWF-containing factor concentrates are safer than cryoprecipitate as the replacement product. Antifibrinolytic therapy using either ε-aminocaproic acid or tranexamic acid is an important therapy, either alone or in an Disorders of Platelets and Vessel Wall 732 adjunctive capacity, particularly for the prevention or treatment of mucosal bleeding. These agents are particularly useful in prophylaxis for dental procedures, with DDAVP for dental extractions and tonsillectomy, menorrhagia, and prostate procedures. It is contraindicated in the setting of upper urinary tract bleeding, due to the risk of ureteral obstruction.

1	The vessel wall is an integral part of hemostasis, and separation of a fluid phase is artificial, particularly in disorders such as TTP or HIT that clearly involve the endothelium as well. Inflammation localized to the vessel wall, such as vasculitis, and inherited connective tissue disorders are abnormalities inherent to the vessel wall.

1	METABOLIC AND INFLAMMATORY DISORDERS Acute febrile illnesses may result in vascular damage. This can result from immune complexes containing viral antigens or the viruses themselves. Certain pathogens, such as the rickettsiae causing Rocky Mountain spotted fever, replicate in endothelial cells and damage them. Vascular purpura may occur in patients with polyclonal gammopathies but more commonly in those with monoclonal gammopathies, including Waldenström’s macroglobulinemia, multiple myeloma, and cryoglobulinemia. Patients with mixed cryoglobulinemia develop a more extensive maculopapular rash due to immune complex–mediated damage to the vessel wall.

1	Patients with scurvy (vitamin C deficiency) develop painful episodes of perifollicular skin bleeding as well as more systemic bleeding symptoms. Vitamin C is needed to synthesize hydroxyproline, an essential constituent of collagen. Patients with Cushing’s syndrome or on chronic glucocorticoid therapy develop skin bleeding and easy bruising due to atrophy of supporting connective tissue. A similar phenomenon is seen with aging, where following minor trauma, blood spreads superficially under the epidermis. This has been termed senile purpura. It is most common on skin that has been previously damaged by sun exposure.

1	Henoch-Schönlein, or anaphylactoid, purpura is a distinct, self-limited type of vasculitis that occurs in children and young adults. Patients have an acute inflammatory reaction with IgA and complement components in capillaries, mesangial tissues, and small arterioles leading to increased vascular permeability and localized hemorrhage. The syndrome is often preceded by an upper respiratory infection, commonly with streptococcal pharyngitis, or is triggered by drug or food allergies. Patients develop a purpuric rash on the extensor surfaces of the arms and legs, usually accompanied by polyarthralgias or arthritis, abdominal pain, and hematuria from focal glomerulonephritis. All coagulation tests are normal, but renal impairment may occur. Glucocorticoids can provide symptomatic relief but do not alter the course of the illness.

1	INHERITED DISORDERS OF THE VESSEL WALL Patients with inherited disorders of the connective tissue matrix, such as Marfan’s syndrome, Ehlers-Danlos syndrome, and pseudoxanthoma elasticum, frequently report easy bruising. Inherited vascular abnormalities can result in increased bleeding. This is notably seen in hereditary hemorrhagic telangiectasia (HHT, or Osler-Weber-Rendu disease), a disorder where abnormal telangiectatic capillaries result in frequent bleeding episodes, primarily from the nose and gastrointestinal tract. Arteriovenous malformation (AVM) in the lung, brain, and liver may also occur in HHT. The telangiectasia can often be visualized on the oral and nasal mucosa. Signs and symptoms develop over time. Epistaxis begins, on average, at the age of 12 and occurs in >95% of affected individuals by middle age. Two genes involved in the pathogenesis are eng (endoglin) on chromosome 9q33-34 (so-called HHT type 1), associated with pulmonary AVM in 40% of cases, and alk1

1	of affected individuals by middle age. Two genes involved in the pathogenesis are eng (endoglin) on chromosome 9q33-34 (so-called HHT type 1), associated with pulmonary AVM in 40% of cases, and alk1 (activin-receptor-like kinase 1) on chromosome 12q13, associated with a much lower risk of pulmonary AVM.

1	Robert Handin, MD, contributed this chapter in the 16th edition and some materials from his chapter are included here. Valder R. Arruda, Katherine A. High

1	Deficiencies of coagulation factors have been recognized for centuries. Patients with genetic deficiencies of plasma coagulation factors exhibit life-long recurrent bleeding episodes into joints, muscles, and closed spaces, either spontaneously or following an injury. The most common inherited factor deficiencies are the hemophilias, X-linked diseases caused by deficiency of factor (F) VIII (hemophilia A) or FIX (hemophilia B). Rare congenital bleeding disorders due to deficiencies of other factors, including FII (prothrombin), FV, FVII, FX, FXI, and FXIII, and fibrinogen are commonly inherited in an autosomal recessive manner (Table 141-1). Advances in characterization of the molecular bases of clotting factor deficiencies have contributed to better understanding of the disease phenotypes and may eventually allow more targeted therapeutic approaches through the development of small molecules, recombinant proteins, or cell and gene-based therapies.

1	Commonly used tests of hemostasis provide the initial screening for clotting factor activity (Fig. 141-1), and disease phenotype often correlates with the level of clotting activity. An isolated abnormal prothrombin time (PT) suggests FVII deficiency, whereas a prolonged activated partial thromboplastin time (aPTT) indicates most commonly hemophilia or FXI deficiency (Fig. 141-1). The prolongation of both PT and aPTT suggests deficiency of FV, FX, FII, or fibrinogen abnormalities. The addition of the missing factor at a range of doses to the subject’s plasma will correct the abnormal clotting times; the result is expressed as a percentage of the activity observed in normal subjects.

1	Acquired deficiencies of plasma coagulation factors are more frequent than congenital disorders; the most common disorders include hemorrhagic diathesis of liver disease, disseminated intravascular coagulation (DIC), and vitamin K deficiency. In these disorders, blood coagulation is hampered by the deficiency of more than one clotting factor, and the bleeding episodes are the result of perturbation of both primary (coagulation) and secondary (e.g., platelet and vessel wall interactions) hemostasis.

1	The development of antibodies to coagulation plasma proteins, clinically termed inhibitors, is a relatively rare disease that often affects hemophilia A or B and FXI-deficient patients on repetitive exposure to the missing protein to control bleeding episodes. Inhibitors also occur among subjects without genetic deficiency of clotting factors (e.g., in the postpartum setting as a manifestation of underlying autoimmune or neoplastic disease or idiopathically). Rare cases of inhibitors to thrombin or FV have been reported in patients receiving topical bovine thrombin preparation as a local hemostatic agent in complex surgeries. The diagnosis of inhibitors is based on the same tests as those used to diagnose inherited plasma coagulation factor deficiencies. However, the addition of the missing protein to the plasma of a subject with an inhibitor does not correct the abnormal aPTT and/or PT tests (known as mixing tests). This is the major laboratory difference between deficiencies and

1	protein to the plasma of a subject with an inhibitor does not correct the abnormal aPTT and/or PT tests (known as mixing tests). This is the major laboratory difference between deficiencies and inhibitors. Additional tests are required to measure the specificity of the inhibitor and its titer.

1	The treatment of these bleeding disorders often requires replacement of the deficient protein using recombinant or purified plasma-derived products or fresh-frozen plasma (FFP). Therefore, it is imperative to arrive at a proper diagnosis to optimize patient care without unnecessary exposure to suboptimal treatment and the risks of bloodborne disease. Hemophilia is an X-linked recessive hemorrhagic disease due to mutations in the F8 gene (hemophilia A or classic hemophilia) or F9 gene (hemophilia B). The disease affects 1 in 10,000 males worldwide, in all ethnic groups; hemophilia A represents 80% of all cases. Male subjects

1	Prothrombin AR 1 in 2,000,000 + + − 20–30% Factor V AR 1 in 1,000,000 +/− +/− − 15–20% Factor VII AR 1 in 500,000 − + − 15–20% Factor VIII X-linked 1 in 5,000 + − − 30% Factor IX X-linked 1 in 30,000 + − − 30% Factor X AR 1 in 1,000,000 +/− +/− − 15–20% Factor XI AR 1 in 1,000,000 + − − 15–20% Factor XII AR ND + − − b HK ARND +−− b Prekallikrein AR ND + − − b Factor XIII AR 1 in 2,000,000 − − +/− 2–5% aValues within normal range (−) or prolonged (+). bNo risk for bleeding; treatment is not indicated. Abbreviations: aPTT, activated partial thromboplastin time; AR, autosomal recessive; FFP, fresh-frozen plasma; HK, high-molecular-weight kininogen; ND, not determined; PCC, prothrom bin complex concentrates; PT, prothrombin time; TT, thrombin time.

1	are clinically affected; women, who carry a single mutated gene, are generally asymptomatic. Family history of the disease is absent in ~30% of cases, and in these cases, 80% of the mothers are carriers of the de novo mutated allele. More than 500 different mutations have been identified in the F8 or F9 genes of patients with hemophilia A or B, respectively. One of the most common hemophilia A mutations results from an inversion of the intron 22 sequence, and it is present in 40% of cases of severe hemophilia A. Advances in molecular diagnosis now permit precise identification of mutations, allowing accurate diagnosis of women carriers of the hemophilia gene in affected families.

1	Clinically, hemophilia A and hemophilia B are indistinguishable. The disease phenotype correlates with the residual activity of FVIII or FIX and can be classified as severe (<1%), moderate (1–5%), or mild (6–30%). In the severe and moderate forms, the disease is characterized by bleeding into the joints (hemarthrosis), soft tissues, and Intrinsic Pathway Extrinsic Pathway aPTT PT aPTT/PT TT Ca2+ Contact phase FXIIa Fibrinogen Fibrin polymer Fibrin monomer XIIIa Thrombin V X Va XaX Ca2+ PL Ca2+ PL Common Pathway XIaXI PK HMWH VIII VIIIa IX IXa Ca2+ VIIa/tissue factor VII Prothrombin Cross-linked fibrin clot FIGURE 141-1 Coagulation cascade and laboratory assessment of clotting factor deficiency by activated partial prothrombin time (aPTT), prothrombin time (PT), thrombin time (TT), and phospholipid (PL).

1	muscles after minor trauma or even spontaneously. Patients with mild disease experience infrequent bleeding that is usually secondary to trauma. Among those with residual FVIII or FIX activity >25% of normal, the disease is discovered only by bleeding after major trauma or during routine presurgery laboratory tests. Typically, the global tests of coagulation show only an isolated prolongation of the aPTT assay. Patients with hemophilia have normal bleeding times and platelet counts. The diagnosis is made after specific determination of FVIII or FIX clotting activity.

1	Early in life, bleeding may present after circumcision or rarely as intracranial hemorrhages. The disease is more evident when children begin to walk or crawl. In the severe form, the most common bleeding manifestations are the recurrent hemarthroses, which can affect every joint but mainly affect knees, elbows, ankles, shoulders, and hips. Acute hemarthroses are painful, and clinical signs are local swelling and erythema. To avoid pain, the patient may adopt a fixed position, which leads eventually to muscle contractures. Very young children unable to communicate verbally show irritability and a lack of movement of the affected joint. Chronic hemarthroses are debilitating, with synovial thickening and synovitis in response to the intraarticular blood. After a joint has been damaged, recurrent bleeding episodes result in the clinically recognized “target joint,” which then establishes a vicious cycle of bleeding, resulting in progressive joint deformity that in critical cases requires

1	bleeding episodes result in the clinically recognized “target joint,” which then establishes a vicious cycle of bleeding, resulting in progressive joint deformity that in critical cases requires surgery as the only therapeutic option. Hematomas into the muscle of distal parts of the limbs may lead to external compression of arteries, veins, or nerves that can evolve to a compartment syndrome.

1	Bleeding into the oropharyngeal spaces, central nervous system (CNS), or retroperitoneum is life threatening and requires immediate therapy. Retroperitoneal hemorrhages can accumulate large quantities of blood with formation of masses with calcification and inflammatory tissue reaction (pseudotumor syndrome) and also result in damage to the femoral nerve. Pseudotumors can also form in bones, especially long bones of the lower limbs. Hematuria is frequent among hemophilia patients, even in the absence of genitourinary pathology. It is often self-limited and may not require specific therapy. The FVIII half-life of 8–12 h requires injections twice a day to main-

1	Without treatment, severe hemophilia has a limited life expectancy. Advances in the blood fractionation industry during World War II resulted in the realization that plasma could be used to treat hemophilia, but the volumes required to achieve even modest elevation of circulating factor levels limit the utility of plasma infusion as an approach to disease management. The discovery in the 1960s that the cryoprecipitate fraction of plasma was enriched for FVIII, and the eventual purification of FVIII and FIX from plasma, led to the introduction of home infusion therapy with factor concentrates in the 1970s. The availability of factor concentrates resulted in a dramatic improvement in life expectancy and in quality of life for people with severe hemophilia. However, the contamination of the blood supply with hepatitis viruses and, subsequently, HIV resulted in widespread transmission of these bloodborne infections within the hemophilia population; complications of HIV and of hepatitis C

1	blood supply with hepatitis viruses and, subsequently, HIV resulted in widespread transmission of these bloodborne infections within the hemophilia population; complications of HIV and of hepatitis C are now the leading causes of death among U.S. adults with severe hemophilia. The introduction of viral inactivation steps in the preparation of plasma-derived products in the mid-1980s greatly reduced the risk of HIV and hepatitis, and the risks were further reduced by the successful production of recombinant FVIII and FIX proteins, both licensed in the 1990s. It is uncommon for hemophilic patients born after 1985 to have contracted either hepatitis or HIV, and for these individuals, life expectancy is approximately 65 years. In fact, since 1998, no evidence of new infections with viral hepatitis or HIV has been reported in patients using blood products. Factor replacement therapy for hemophilia can be provided either in response to a bleeding episode or as a prophylactic treatment.

1	hepatitis or HIV has been reported in patients using blood products. Factor replacement therapy for hemophilia can be provided either in response to a bleeding episode or as a prophylactic treatment. Primary prophylaxis is defined as a strategy for maintaining the missing clotting factor at levels ~1% or higher on a regular basis in order to prevent bleeds, especially the onset of hemarthroses. Hemophilic boys receiving regular infusions of FVIII (3 days/week) or FIX (2 days/week) can reach puberty without detectable joint abnormalities. Prophylaxis has become gradually more common in young patients. The Centers for Disease Control and Prevention reported that 51% of children with severe hemophilia who are younger than age 6 years receive prophylaxis, increasing considerably from 33% in 1995. Although highly recommended, the high cost and difficulties in accessing peripheral veins in young patients and the potential infectious and thrombotic risks of long-term central vein catheters

1	1995. Although highly recommended, the high cost and difficulties in accessing peripheral veins in young patients and the potential infectious and thrombotic risks of long-term central vein catheters are important limiting factors for many young patients. Emerging data show that prophylaxis is also increasing among adults with severe hemophilia.

1	General considerations regarding the treatment of bleeds in hemophilia include the following: (1) Treatment should begin as soon as possible because symptoms often precede objective evidence of bleeding; because of the superior efficacy of early therapeutic intervention, classic symptoms of bleeding into the joint in a reliable patient, headaches, or automobile or other accidents require prompt replacement and further laboratory investigation.

1	(2) Drugs that hamper platelet function, such as aspirin or aspirin-containing drugs, should be avoided; to control pain, drugs such as ibuprofen or propoxyphene are preferred. FVIII and FIX are dosed in units. One unit is defined as amount of FVIII (100 ng/mL) or FIX (5 μg/mL) in 1 mL of normal plasma. One unit of FVIII per kilogram of body weight increases the plasma FVIII level by 2%. One can calculate the dose needed to increase FVIII levels to 100% in a 70-kg severe hemophilia patient (<1%) using the simple formula below. Thus, 3500 units of FVIII will raise the circulating level to 100%. FVIII dose (IU) = Target FVIII levels – FVIII baseline levels × body weight (kg) × 0.5 unit/kg The doses for FIX replacement are different from those for FVIII, because FIX recovery after infusion is usually only 50% of the predicted value. Therefore, the formula for FIX replacement is as follows:

1	FIX dose (IU) = Target FIX levels – FIX baseline levels × body weight (kg) × 1 unit/kg tain therapeutic levels, whereas the FIX half-life is longer, ~24 h, so that once-a-day injection is sufficient. In specific situations such as after surgery, continuous infusion of factor may be desirable because of its safety in achieving sustained factor levels at a lower total cost. Cryoprecipitate is enriched with FVIII protein (each bag contains ~80 IU of FVIII) and was commonly used for the treatment of hemophilia A decades ago; it is still in use in some developing countries, but because of the risk of bloodborne diseases, this product should be avoided in hemophilia patients when factor concentrates are available.

1	Mild bleeds such as uncomplicated hemarthroses or superficial hematomas require initial therapy with factor levels of 30–50%. Additional doses to maintain levels of 15–25% for 2 or 3 days are indicated for severe hemarthroses, especially when these episodes affect the “target joint.” Large hematomas, or bleeds into deep muscles, require factor levels of 50% or even higher if the clinical symptoms do not improve, and factor replacement may be required for a period of 1 week or longer. The control of serious bleeds including those that affect the oropharyngeal spaces, CNS, and the retroperitoneum require sustained protein levels of 50–100% for 7–10 days. Prophylactic replacement for surgery is aimed at achieving normal factor levels (100%) for a period of 7–10 days; replacement can then be tapered depending on the extent of the surgical wounds. Oral surgery is associated with extensive tissue damage that usually requires factor replacement for 1–3 days coupled with oral antifibrinolytic

1	depending on the extent of the surgical wounds. Oral surgery is associated with extensive tissue damage that usually requires factor replacement for 1–3 days coupled with oral antifibrinolytic drugs.

1	NONTRANSFUSION THERAPY IN HEMOPHILIA DDAVP (1-Amino-8-D-Arginine Vasopressin) DDAVP is a synthetic vasopressin analog that causes a transient rise in FVIII and von Willebrand factor (VWF), but not FIX, through a mechanism involving release from endothelial cells. Patients with moderate or mild hemophilia A should be tested to determine if they respond to DDAVP before a therapeutic application. DDAVP at doses of 0.3 μg/kg body weight, over a 20-min period, is expected to raise FVIII levels by twoto threefold over baseline, peaking between 30 and 60 min after infusion. DDAVP does not improve FVIII levels in severe hemophilia A patients, because there are no stores to release. Repeated dosing of DDAVP results in tachyphylaxis because the mechanism is an increase in release rather than de novo synthesis of FVIII and VWF. More than three consecutive doses become ineffective, and if further therapy is indicated, FVIII replacement is required to achieve hemostasis.

1	Antifibrinolytic Drugs Bleeding in the gums, gastrointestinal tract, and during oral surgery requires the use of oral antifibrinolytic drugs such as ε-amino caproic acid (EACA) or tranexamic acid to control local hemostasis. The duration of the treatment depending on the clinical indication is 1 week or longer. Tranexamic acid is given at doses of 25 mg/kg three to four times a day. EACA treatment requires a loading dose of 200 mg/kg (maximum of 10 g) followed by 100 mg/kg per dose (maximum 30 g/d) every 6 h. These drugs are not indicated to control hematuria because of the risk of formation of an occlusive clot in the lumen of genitourinary tract structures.

1	COMPLICATIONS Inhibitor Formation The formation of alloantibodies to FVIII or FIX is currently the major complication of hemophilia treatment. The prevalence of inhibitors to FVIII is estimated to be between 5 and 10% of all cases and ~20% of severe hemophilia A patients. Inhibitors to FIX are detected in only 3–5% of all hemophilia B patients. The high-risk group for inhibitor formation includes severe deficiency (>80% of all cases of inhibitors), familial history of inhibitor, African descent, mutations in the FVIII or FIX gene resulting in deletion of large coding regions, or gross gene rearrangements. Inhibitors usually appear early in life, at a median of 2 years of age, and after 10 cumulative days of exposure. However, intensive replacement therapy such as for major surgery, intracranial bleeding, or trauma increases the risk of inhibitor formation for patients of all ages and degree of clinical severity, which requires close laboratory monitoring in the following weeks.

1	The clinical diagnosis of an inhibitor is suspected when patients do not respond to factor replacement at therapeutic doses. Inhibitors increase both morbidity and mortality in hemophilia. Because early detection of an inhibitor is critical to a successful correction of the bleeding or to eradication of the antibody, most hemophilia centers perform annual screening for inhibitors. The laboratory test required to confirm the presence of an inhibitor is an aPTT with a mix (with normal plasma). In most hemophilia patients, a 1:1 mix with normal plasma completely corrects the aPTT. In inhibitor patients, the aPTT on a 1:1 mix is abnormally prolonged, because the inhibitor neutralizes the FVIII clotting activity of the normal plasma. The Bethesda assay uses a similar principle and defines the specificity of the inhibitor and its titer. The results are expressed in Bethesda units (BU), in which 1 BU is the amount of antibody that neutralizes 50% of the FVIII or FIX present in normal plasma

1	specificity of the inhibitor and its titer. The results are expressed in Bethesda units (BU), in which 1 BU is the amount of antibody that neutralizes 50% of the FVIII or FIX present in normal plasma after 2 h of incubation at 37°C. Clinically, inhibitor patients are classified as low responders or high responders, which provides guidelines for optimal therapy. Therapy for inhibitor patients has two goals: the control of acute bleeding episodes and the eradication of the inhibitor. For the control of bleeding episodes, low responders, those with titer <5 BU, respond well to high doses of human or porcine FVIII (50–100 U/kg), with minimal or no increase in the inhibitor titers. However, high-responder patients, those with initial inhibitor titer >10 BU or an anamnestic response in the antibody titer to >10 BU even if low titer initially, do not respond to FVIII or FIX concentrates. The control of bleeding episodes in high-responder patients can be achieved by using concentrates

1	antibody titer to >10 BU even if low titer initially, do not respond to FVIII or FIX concentrates. The control of bleeding episodes in high-responder patients can be achieved by using concentrates enriched for prothrombin, FVII, FIX, FX (prothrombin complex concentrates [PCCs] or activated PCCs [aPCCs]), and more recently recombinant activated factor VII (FVIIa) known as “bypass agents” (Fig. 141-1). The rates of therapeutic success have been higher for FVIIa than for PCC or aPCC. For eradication of the inhibitory antibody, immunosuppression alone is not effective. The most effective strategy is the immune tolerance induction (ITI) based on daily infusion of missing protein until the inhibitor disappears, typically requiring periods longer than 1 year, with success rates of approximately 60%. The management of patients with severe hemophilia and inhibitors resistant to ITI is challenging. The use of anti-CD20 monoclonal antibody (rituximab) combined with ITI was thought to be

1	60%. The management of patients with severe hemophilia and inhibitors resistant to ITI is challenging. The use of anti-CD20 monoclonal antibody (rituximab) combined with ITI was thought to be effective. Although this therapy may reduce the inhibitor titers in some cases, sustained eradication is uncommon and may require two to three infusions weekly of clotting factor concentrates.

1	Novel Therapeutic Approaches in Development for Hemophilia Clinical studies using long-acting clotting factors with prolonged half-lives are in the late phase of clinical testing, and these new-generation products (for FVIII and FIX) may facilitate prophylaxis by requiring fewer injections to maintain circulating levels above 1%. The use of recombinant interleukin 11 in patients with moderate or mild hemophilia A unresponsive to DDAVP has been tested in early-phase clinical trials and may be an alternate therapeutic strategy for clinical situations that require transient increases in FVIII levels. Gene therapy trials for hemophilia B using adeno-associated viral vectors are ongoing, and initial data are promising (Chap. 91e).

1	Hepatitis C virus (HCV) infection is the major cause of morbidity and the second leading cause of death in hemophilia patients exposed to older clotting factor concentrates. The vast majority of young patients treated with plasma-derived products from 1970 to 1985 became infected with HCV. It has been estimated that >80% of patients older than 20 years of age are HCV antibody positive as of 2006. The comorbidity of the underlying liver disease in hemophilia patients is clear when these individuals require invasive procedures; correction of both genetic and acquired (secondary to liver disease) deficiencies may be needed. Infection with HIV also swept the population of patients using plasma-derived concentrates two decades ago. Co-infection of HCV and HIV, present in almost 50% of hemo-735 philia patients, is an aggravating factor for the evolution of liver disease. The response to HCV antiviral therapy in hemophilia is restricted to <30% of patients and even poorer among those with

1	philia patients, is an aggravating factor for the evolution of liver disease. The response to HCV antiviral therapy in hemophilia is restricted to <30% of patients and even poorer among those with both HCV and HIV infection. End-stage liver disease requiring organ transplantation may be curative for both the liver disease and for hemophilia.

1	There has been continuous improvement of the management of hemophilia since the increase in the population of adults living beyond middle age in the developing world. The life expectancy of a patient with severe hemophilia is only ~10 years shorter than the general male population. In patients with mild or moderate hemophilia, life expectancy is approaching that of the male population without coagulopathy. Elderly hemophilia patients have different problems compared to the younger generation; they have more severe arthropathy and chronic pain, due to suboptimal treatment, and high rates of HCV and/or HIV infections.

1	Early data indicate that mortality from coronary artery disease is lower in hemophilia patients than the general male population. The underlying hypocoagulability probably provides a protective effect against thrombus formation, but it does not prevent atherogenesis. Similar to the general population, these patients are exposed to cardiovascular risk factors such as age, obesity, and smoking. Moreover, physical inactivity, hypertension, and chronic renal disease are commonly observed in hemophilia patients. In HIV patients on combined antiretroviral therapy, there may be a further increase in the risk of cardiovascular disease. Therefore, these patients should be carefully considered for preventive and therapeutic approaches to minimize the risk of cardiovascular disease.

1	Excessive replacement therapy should be avoided, and it is prudent to slowly infuse factor concentrates. Continuous infusion of clotting factor is preferable to bolus dosing in patients with cardiovascular risk factors undergoing invasive procedures. The management of an acute ischemic event and coronary revascularization should include the collaboration of hematologists and internists. The early assumption that hemophilia would protect against occlusive vascular disease may change in this aging population. Cancer is a common cause of mortality in aging hemophilia patients because they are at risk for HIVand HCV-related malignancies. Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer and a common cause of death in HIV-negative patients. The recommendations for cancer screening for the general population should be the same for age-matched hemophilia patients. Among those with high-risk HCV, a semiannual or annual ultrasound and α fetoprotein are recommended for

1	screening for the general population should be the same for age-matched hemophilia patients. Among those with high-risk HCV, a semiannual or annual ultrasound and α fetoprotein are recommended for HCC. Screening for urogenital neoplasm in the presence of hematuria or hematochezia may be delayed due to the underlying bleeding disease, thus preventing early intervention. Multidisciplinary interaction should facilitate the attempts to ensure optimal cancer prevention and treatment recommendations for those with hemophilia.

1	Usually hemophilia carriers, with factor levels of ~50% of normal, have not been considered to be at risk for bleeding. However, a wide range of values (22–116%) have been reported due to random inactivation of the X chromosomes (lyonization). Therefore, it is important to measure the factor level of carriers to recognize those at risk of bleeding and to optimize preoperative and postoperative management. During pregnancy, both FVIII and FIX levels increase gradually until delivery. FVIII levels increase approximately twoto threefold compared to nonpregnant women, whereas an FIX increase is less pronounced. After delivery, there is a rapid fall in the pregnancy-induced rise of maternal clotting factor levels. This represents an imminent risk of bleeding that can be prevented by infusion of factor concentrate to levels of 50–70% for 3 days in the setting of vaginal delivery and up to 5 days for cesarean section. In mild cases, the use of DDAVP and/or antifibrinolytic drugs is

1	of factor concentrate to levels of 50–70% for 3 days in the setting of vaginal delivery and up to 5 days for cesarean section. In mild cases, the use of DDAVP and/or antifibrinolytic drugs is recommended.

1	Factor XI is a zymogen of an active serine protease (FIXa) in the intrinsic pathway of blood coagulation that activates FIX (Fig. 141-1). There are two pathways for the formation of FXIa. In an aPTT-based assay, the protease is the result of activation by FXIIa in conjunction with high-molecular-weight kininogen and kallikrein. In vivo data suggest that thrombin is the physiologic activator of FXI. The generation of thrombin by the tissue factor/factor VIIa pathway activates FXI on the platelet surface that contributes to additional thrombin generation after the clot has formed and thus augments resistance to fibrinolysis through a thrombin-activated fibrinolytic inhibitor (TAFI).

1	Factor XI deficiency is a rare bleeding disorder that occurs in the general population at a frequency of one in a million. However, the disease is highly prevalent among Ashkenazi and Iraqi Jewish populations, reaching a frequency of 6% as heterozygotes and 0.1–0.3% as homozygotes. More than 65 mutations in the FXI gene have been reported, whereas fewer mutations (two to three) are found among affected Jewish populations.

1	Normal FXI clotting activity levels range from 70 to 150 U/dL. In heterozygous patients with moderate deficiency, FXI ranges from 20 to 70 U/dL, whereas in homozygous or double heterozygote patients, FXI levels are <1–20 U/dL. Patients with FXI levels <10% of normal have a high risk of bleeding, but the disease phenotype does not always correlate with residual FXI clotting activity. A family history is indicative of the risk of bleeding in the propositus. Clinically, the presence of mucocutaneous hemorrhages such as bruises, gum bleeding, epistaxis, hematuria, and menorrhagia are common, especially following trauma. This hemorrhagic phenotype suggests that tissues rich in fibrinolytic activity are more susceptible to FXI deficiency. Postoperative bleeding is common but not always present, even among patients with very low FXI levels.

1	FXI replacement is indicated in patients with severe disease required to undergo a surgical procedure. A negative history of bleeding complications following invasive procedures does not exclude the possibility of an increased risk for hemorrhage.

1	The treatment of FXI deficiency is based on the infusion of FFP at doses of 15–20 mL/kg to maintain trough levels ranging from 10 to 20%. Because FXI has a half-life of 40–70 h, the replacement therapy can be given on alternate days. The use of antifibrinolytic drugs is beneficial to control bleeds, with the exception of hematuria or bleeds in the bladder. The development of an FXI inhibitor was observed in 10% of severely FXI-deficient patients who received replacement therapy. Patients with severe FXI deficiency who develop inhibitors usually do not bleed spontaneously. However, bleeding following a surgical procedure or trauma can be severe. In these patients, FFP and FXI concentrates should be avoided. The use of PCC/aPCC or recombinant activated FVII has been effective.

1	Collectively, the inherited disorders resulting from deficiencies of clotting factors other than FVIII, FIX, and FXI (Table 141-1) represent a group of rare bleeding diseases. The bleeding symptoms in these patients vary from asymptomatic (dysfibrinogenemia or FVII deficiency) to life-threatening (FX or FXIII deficiency). There is no pathognomonic clinical manifestation that suggests one specific disease, but overall, in contrast to hemophilia, hemarthrosis is a rare event and bleeding in the mucosal tract or after umbilical cord clamping is common. Individuals heterozygous for plasma coagulation deficiencies are often asymptomatic. The laboratory assessment for the specific deficient factor following screening with general coagulation tests (Table 141-1) will define the diagnosis.

1	Replacement therapy using FFP or prothrombin complex concentrates (containing prothrombin, FVII, FIX, and FX) provides adequate hemostasis in response to bleeds or as prophylactic treatment. The use of PCC should be carefully monitored and avoided in patients with underlying liver disease, or those at high risk for thrombosis because of the risk of DIC. There are several bleeding disorders characterized by the inherited deficiency of more than one plasma coagulation factor. To date, the genetic defects in two of these diseases have been characterized, and they provide new insights into the regulation of hemostasis by gene-encoding proteins outside blood coagulation.

1	Combined Deficiency of FV and FVIII Patients with combined FV and FVIII deficiency exhibit ~5% of residual clotting activity of each factor. Interestingly, the disease phenotype is a mild bleeding tendency, often following trauma. An underlying mutation has been identified in the endoplasmic reticulum/Golgi intermediate compartment (ERGIC-53) gene, a mannose-binding protein localized in the Golgi apparatus that functions as a chaperone for both FV and FVIII. In other families, mutations in the multiple coagulation factor deficiency 2 (MCFD2) gene have been defined; this gene encodes a protein that forms a Ca2+ -dependent complex with ERGIC-53 and provides cofactor activity in the intracellular mobilization of both FV and FVIII.

1	Multiple Deficiencies of Vitamin K–Dependent Coagulation Factors Two enzymes involved in vitamin K metabolism have been associated with combined deficiency of all vitamin K–dependent proteins, including the procoagulant proteins prothrombin, VII, IX, and X and the anticoagulant proteins C and S. Vitamin K is a fat-soluble vitamin that is a cofactor for carboxylation of the gamma carbon of the glutamic acid residues in the vitamin K–dependent factors, a critical step for calcium and phospholipid binding of these proteins (Fig. 141-2). The enzymes γ-glutamylcarboxylase and epoxide reductase are critical for the metabolism and regeneration of vitamin K. Mutations in the genes encoding the γ-carboxylase (GGCX) or vitamin K epoxide reductase complex 1 (VKORC1) result in defective enzymes and thus in vitamin K–dependent factors with reduced activity, varying from 1 to 30% of normal. The disease phenotype is characterized by mild to severe bleeding episodes present from birth. Some patients

1	in vitamin K–dependent factors with reduced activity, varying from 1 to 30% of normal. The disease phenotype is characterized by mild to severe bleeding episodes present from birth. Some patients respond to high doses of vitamin K. For severe bleeding, replacement therapy with FFP or PCC may be necessary to achieve full hemostatic control.

1	DIC is a clinicopathologic syndrome characterized by widespread intravascular fibrin formation in response to excessive blood protease Warfarin Carboxylase Vitamin K reduced Glutamic acid Epoxide reductase Vitamin K epoxide ˜-Carboxyglutamic acid FIGURE 141-2 The vitamin K cycle. Vitamin K is a cofactor for the formation of γ-carboxyglutamic acid residues on coagulation proteins. Vitamin K–dependent γ-glutamylcarboxylase, the enzyme that cata-lyzes the vitamin K epoxide reductase, regenerates reduced vitamin K. Warfarin blocks the action of the reductase and competitively inhibits the effects of vitamin K.

1	• Large vessel aneurysms (e.g., aorta) liver of pregnancy systemic bleeding. This is further aggravated by secondary hyperfibri-737 nolysis. Studies in animals demonstrate that the fibrinolytic system is indeed suppressed at the time of maximal activation of coagulation. Interestingly, in patients with acute promyelocytic leukemia, a severe hyperfibrinolytic state often occurs in addition to the coagulation activation. The release of several proinflammatory cytokines such as interleukin 6 and tumor necrosis factor α plays a central role in mediating the coagulation defects in DIC and symptoms associated with systemic inflammatory response syndrome (SIRS).

1	Clinical manifestations of DIC are related to the magnitude of the imbalance of hemostasis, to the underlying disease, or to both. The most common findings are bleeding ranging from oozing from venipuncture sites, petechiae, and ecchymoses to severe hemorrhage from the gastrointestinal tract, lung, or into the CNS. In chronic DIC, the bleeding symptoms are discrete and restricted to skin or mucosal surfaces. The hypercoagulability of DIC manifests as the occlusion of vessels in the microcirculation and resulting organ failure. Thrombosis of large vessels and cerebral embolism can also occur. Hemodynamic complications and shock are common among patients with acute DIC. The mortality ranges from 30 to >80% depending on the underlying disease, the severity of the DIC, and the age of the patient.

1	The mortality ranges from 30 to >80% depending on the underlying disease, the severity of the DIC, and the age of the patient. The diagnosis of clinically significant DIC is based on the presence of clinical and/or laboratory abnormalities of coagulation or thrombocytopenia. The laboratory diagnosis of DIC should prompt a search for the underlying disease if it is not already apparent. There is no single test that establishes the diagnosis of DIC. The laboratory investigation should include coagulation tests (aPTT, PT, thrombin time [TT]) and markers of fibrin degradation products (FDPs), in addition to platelet and red cell count and analysis of the blood smear. These tests should be repeated over a period of 6–8 h because an initially mild abnormality can change dramatically in patients with severe DIC.

1	Common findings include the prolongation of PT and/or aPTT; platelet counts μ100,000/μL, or a rapid decline in platelet numbers; the presence of schistocytes (fragmented red cells) in the blood smear; and elevated levels of FDP. The most sensitive test for DIC is the FDP level. DIC is an unlikely diagnosis in the presence of normal levels of FDP. The D-dimer test is more specific for detection of fibrin—but not fibrinogen—degradation products and indicates that the cross-linked fibrin has been digested by plasmin. Because fibrinogen has a prolonged half-life, plasma levels diminish acutely only in severe cases of DIC. High-grade DIC is also associated with levels of antithrombin III or plasminogen activity <60% of normal. • Adenocarcinoma (prostate, pancreas, etc.) activity that overcomes the natural anticoagulant mechanisms. There are several underlying pathologies associated with DIC (Table 141-2).

1	• Adenocarcinoma (prostate, pancreas, etc.) activity that overcomes the natural anticoagulant mechanisms. There are several underlying pathologies associated with DIC (Table 141-2). The most common causes are bacterial sepsis, malignant disorders such as solid tumors or acute promyelocytic leukemia, and obstetric causes. DIC is diagnosed in almost one-half of pregnant women with abruptio placentae or with amniotic fluid embolism. Trauma, particularly to the brain, can also result in

1	DIC. The exposure of blood to phospholipids from damaged tissue, hemolysis, and endothelial damage are all contributing factors to the development of DIC in this setting. Purpura fulminans is a severe form of DIC resulting from thrombosis of extensive areas of the skin; it affects predominantly young children following viral or bacterial infection, particularly those with inherited or acquired hypercoagulability due to deficiencies of the components of the protein C pathway. Neonates homozygous for protein C deficiency also present high risk for purpura fulminans with or without thrombosis of large vessels.

1	The central mechanism of DIC is the uncontrolled generation of thrombin by exposure of the blood to pathologic levels of tissue factor (Fig. 141-3). Simultaneous suppression of physiologic anticoagulant mechanisms and abnormal fibrinolysis further accelerate the process. Together, these abnormalities contribute to systemic fibrin deposition in small and midsize vessels. The duration and intensity of the fibrin deposition can compromise the blood supply of many organs, especially the lung, kidney, liver, and brain, with consequent organ failure. The sustained activation of coagulation results in consumption of clotting factors and platelets, which in turn leads to

1	DIC Uncontrolled thrombin generation Fibrin deposits in the microcirculation Secondary fibrinolysis Diffuse bleeding Consumption of platelets and coagulation factors Red blood cell damage and hemolysis Ischemic tissue damage Failure of multiple organs Vessel patency FDP D-dimer FIGURE 141-3 The pathophysiology of disseminated intravascular coagulation (DIC). Interactions between coagulation and fibrinolytic pathways result in bleeding and thrombosis in the microcirculation in patients with DIC. FDP, fibrin degradation product. 738 Chronic DIC Low-grade, compensated DIC can occur in clinical situations including giant hemangioma, metastatic carcinoma, or the dead fetus syndrome. Plasma levels of FDP or D-dimers are elevated. aPTT, PT, and fibrinogen values are within the normal range or high. Mild thrombocytopenia or normal platelet counts are also common findings. Red cell fragmentation is often detected but at a lower degree than in acute DIC.

1	Differential Diagnosis The differential diagnosis between DIC and severe liver disease is challenging and requires serial measurements of the laboratory parameters of DIC. Patients with severe liver disease are at risk for bleeding and manifest laboratory features including thrombocytopenia (due to platelet sequestration, portal hypertension, or hypersplenism), decreased synthesis of coagulation factors and natural anticoagulants, and elevated levels of FDP due to reduced hepatic clearance. However, in contrast to DIC, these laboratory parameters in liver disease do not change rapidly. Other important differential findings include the presence of portal hypertension or other clinical or laboratory evidence of an underlying liver disease.

1	Microangiopathic disorders such as thrombotic thrombocytopenic purpura present an acute clinical onset of illness accompanied by thrombocytopenia, red cell fragmentation, and multiorgan failure. However, there is no consumption of clotting factors or hyperfibrinolysis. Over the last few years, several clinical trials on immune therapies for neoplasias using monoclonal antibodies or gene-modified T cells targeting tumor-specific antigens showed unwanted inflammatory responses with increased cytokine release. These complications are sometimes associated with increased D-dimers and decreased fibrinogen levels, cytopenias, and liver dysfunction; thus, careful screening tests for DIC are indicated.

1	The morbidity and mortality associated with DIC are primarily related to the underlying disease rather than the complications of the DIC. The control or elimination of the underlying cause should therefore be the primary concern. Patients with severe DIC require control of hemodynamic parameters, respiratory support, and sometimes invasive surgical procedures. Attempts to treat DIC without accompanying treatment of the causative disease are likely to fail.

1	Administration of FFP and/or platelet concentrates is indicated for patients with active bleeding or at high risk of bleeding, such as in preparation for invasive procedures or after chemotherapy. The control of bleeding in DIC patients with marked thrombocytopenia (platelet counts <10,000–20,000/μL) and low levels of coagulation factors will require replacement therapy. The PT (>1.5 times the normal) provides a good indicator of the severity of the clotting factor consumption. Replacement with FFP is indicated (1 unit of FFP increases most coagulation factors by 30% in an adult without DIC). Low levels of fibrinogen (<100 mg/dL) or brisk hyperfibrinolysis will require infusion of cryoprecipitate (plasma fraction enriched for fibrinogen, FVIII, and VWF). The replacement of 10 U of cryoprecipitate for every 2–3 U of FFP is sufficient to correct the hemostasis. The transfusion scheme must be adjusted according to the patient’s clinical and laboratory evolution. Platelet concentrates at

1	for every 2–3 U of FFP is sufficient to correct the hemostasis. The transfusion scheme must be adjusted according to the patient’s clinical and laboratory evolution. Platelet concentrates at a dose of 1–2 U/10 kg body weight are sufficient for most DIC patients with severe thrombocytopenia. Clotting factor concentrates are not recommended for control of bleeding in DIC because of the limited efficacy afforded by replacement of single factors (FVIII or FIX concentrates) and the high risk of products containing traces of aPCCs that further aggravate the disease.

1	Drugs to control coagulation such as heparin, antithrombin III (ATIII) concentrates, or antifibrinolytic drugs have all been tried in the treatment of DIC. Low doses of continuous-infusion heparin (5–10 U/kg per h) may be effective in patients with low-grade DIC associated with solid tumor, acute promyelocytic leukemia, or in a setting with recognized thrombosis. Heparin is also indicated for the treatment of purpura fulminans during the surgical resection of giant hemangiomas and during removal of a dead fetus. In acute DIC, the use of heparin is likely to aggravate bleeding. To date, the use of heparin in patients with severe DIC has no proven survival benefit. The use of antifibrinolytic drugs, EACA, or tranexamic acid to prevent fibrin degradation by plasmin may reduce bleeding episodes in patients with DIC and confirmed hyperfibrinolysis. However, these drugs can increase the risk of thrombosis, and concomitant use of heparin is indicated. Patients with acute promyelocytic

1	episodes in patients with DIC and confirmed hyperfibrinolysis. However, these drugs can increase the risk of thrombosis, and concomitant use of heparin is indicated. Patients with acute promyelocytic leukemia or those with chronic DIC associated with giant hemangiomas are among the few patients who may benefit from this therapy. The use of protein C concentrates to treat purpura fulminans associated with acquired protein C deficiency or meningococcemia has been proven efficacious. The results from the replacement of ATIII in early-phase studies are promising but require further study.

1	Guidance for diagnosis and treatment of DIC had been proposed by the International Society of Thrombosis and Haemostasis. This initiative will permit more detailed clinical data on diagnosis and treatment of DIC. The clinical utility of these scoring systems and therapeutic recommendations contained in these guidelines is not yet known.

1	VITAMIN K DEFICIENCY Vitamin K–dependent proteins are a heterogenous group, including clotting factor proteins and also proteins found in bone, lung, kidney, and placenta. Vitamin K mediates posttranslational modification of glutamate residues to γ-carboxylglutamate, a critical step for the activity of vitamin K–dependent proteins for calcium binding and proper assembly to phospholipid membranes (Fig. 141-2). Inherited deficiency of the functional activity of the enzymes involved in vitamin K metabolism, notably the GGCX or VKORC1 (see above), results in bleeding disorders. The amount of vitamin K in the diet is often limiting for the carboxylation reaction; thus recycling of the vitamin K is essential to maintain normal levels of vitamin K–dependent proteins. In adults, low dietary intake alone is seldom reason for severe vitamin K deficiency but may become common in association with the use of broad-spectrum antibiotics. Disease or surgical interventions that affect the ability of

1	alone is seldom reason for severe vitamin K deficiency but may become common in association with the use of broad-spectrum antibiotics. Disease or surgical interventions that affect the ability of the intestinal tract to absorb vitamin K, either through anatomic alterations or by changing the fat content of bile salts and pancreatic juices in the proximal small bowel, can result in significant reduction of vitamin K levels. Chronic liver diseases such as primary biliary cirrhosis also deplete vitamin K stores. Neonatal vitamin K deficiency and the resulting hemorrhagic disease of the newborn have been almost entirely eliminated by routine administration of vitamin K to all neonates. Prolongation of PT values is the most common and earliest finding in vitamin K–deficient patients due to reduction in prothrombin, FVII, FIX, and FX levels. FVII has the shortest half-life among these factors that can prolong the PT before changes in the aPTT. Parenteral administration of vitamin K at a

1	reduction in prothrombin, FVII, FIX, and FX levels. FVII has the shortest half-life among these factors that can prolong the PT before changes in the aPTT. Parenteral administration of vitamin K at a total dose of 10 mg is sufficient to restore normal levels of clotting factor within 8–10 h. In the presence of ongoing bleeding or a need for immediate correction before an invasive procedure, replacement with FFP or PCC is required. The latter should be avoided in patients with severe underlying liver disorders due to high risk of thrombosis. The reversal of excessive anticoagulant therapy with warfarin or warfarinlike drugs can be achieved by minimal doses of vitamin K (1 mg orally or by intravenous injection) for asymptomatic patients. This strategy can diminish the risk of bleeding while maintaining therapeutic anticoagulation for an underlying prothrombotic state.

1	In patients with life-threatening bleeds, the use of recombinant factor VIIa in nonhemophilia patients on anticoagulant therapy has been shown to be effective at restoring hemostasis rapidly, allowing emergency surgical intervention. However, patients with underlying vascular disease, vascular trauma and other comorbidities are at risk for thromboembolic complications that affect both arterial and venous Decreased synthesis of clotting factors Hepatocyte failure Vitamin K deficiency Decreased synthesis of coagulation inhibitors: protein C, protein S, antithrombin Hepatocyte failure Vitamin K deficiency (protein C, protein S) Failure to clear activated coagulation proteins (DIC) Dysfibrinogenemia Iatrogenic: Transfusion of prothrombin complex concentrates Antifibrinolytic agents: EACA, tranexamic acid Abbreviations: DIC, disseminated intravascular coagulation; EACA, ε-aminocaproic acid.

1	systems. Thus, the use of factor VIIa in this setting is limited to administration of low doses given for only a limited number of injections. Close monitoring for vascular complications is highly indicated.

1	COAGULATION DISORDERS ASSOCIATED WITH LIVER FAILURE The liver is central to hemostasis because it is the site of synthesis and clearance of most procoagulant and natural anticoagulant proteins and of essential components of the fibrinolytic system. Liver failure is associated with a high risk of bleeding due to deficient synthesis of procoagulant factors and enhanced fibrinolysis. Thrombocytopenia is common in patients is not a vitamin K–dependent protein, reduced levels of FV may be an 739 indicator of hepatocyte failure. Normal levels of FV and low levels of FVII suggest vitamin K deficiency. Vitamin K levels may be reduced in patients with liver failure due to compromised storage in hepatocellular disease, changes in bile acids, or cholestasis that can diminish the absorption of vitamin K. Replacement of vitamin K may be desirable (10 mg given by slow intravenous injection) to improve hemostasis.

1	Treatment with FFP is the most effective to correct hemostasis in patients with liver failure. Infusion of FFP (5–10 mL/kg; each bag contains ~200 mL) is sufficient to ensure 10–20% of normal levels of clotting factors but not correction of PT or aPTT. Even high doses of FFP (20 mL/kg) do not correct the clotting times in all patients. Monitoring for clinical symptoms and clotting times will determine if repeated doses are required 8–12 h after the first infusion. Platelet concentrates are indicated when platelet counts are <10,000–20,000/μL to control an ongoing bleed or immediately before an invasive procedure if counts are <50,000/μL. Cryoprecipitate is indicated only when fibrinogen levels are less than 100 mg/mL; dosing is six bags for a 70-kg patient daily. Prothrombin complex concentrate infusion in patients with liver failure should be avoided due to the high risk of thrombotic complications. The safety of the use of antifibrinolytic drugs to control bleeding in patients with

1	infusion in patients with liver failure should be avoided due to the high risk of thrombotic complications. The safety of the use of antifibrinolytic drugs to control bleeding in patients with liver failure is not yet well defined and should be avoided.

1	liver disease and tHromboembolism The clinical bleeding phenotype of hemostasis in patients with stable liver disease is often mild or even asymptomatic. However, as the disease progresses, the hemostatic bal ance is less stable and more easily disturbed than in healthy individuals. Furthermore, the hemostatic balance is compromised by comorbid complications such as infections and renal failure (Fig. 141-4). Based on the clinical bleeding complications in patients with cirrhosis and laboratory evidence of hypocoagulation such as a prolonged PT/aPTT, it has long been assumed that these patients are protected against thrombotic disease. Cumulative clinical experience, however, has demonstrated that these patients are at risk for thrombosis, especially those with advanced liver disease. Although hypercoagulability could explain the occurrence of venous thrombosis, according to Virchow’s with liver disease, and may be due to con-BLEEDING THROMBOSIS gestive splenomegaly (hypersplenism) or

1	hypercoagulability could explain the occurrence of venous thrombosis, according to Virchow’s with liver disease, and may be due to con-BLEEDING THROMBOSIS gestive splenomegaly (hypersplenism) or immune-mediated shortened platelet lifespan (primary biliary cirrhosis). In addition, several anatomic abnormalities secondary to underlying liver disease further promote the occurrence of hemorrhage (Table 141-3). Dysfibrinogenemia is a relatively common finding in patients with liver disease due to impaired fibrin polymerization. The development of DIC concomitant to chronic liver disease is not uncommon and may enhance the risk for bleeding. Laboratory evaluation is mandatory for an optimal therapeutic strategy, either to control ongoing bleeding or to prepare patients with liver disease for invasive procedures. Typically, these patients present with prolonged PT, aPTT, and TT depending on the degree of liver damage, thrombocytopenia, and normal or slight increase of FDP. Fibrinogen levels

1	procedures. Typically, these patients present with prolonged PT, aPTT, and TT depending on the degree of liver damage, thrombocytopenia, and normal or slight increase of FDP. Fibrinogen levels are diminished only in fulminant hepatitis, decompensated cirrhosis, or advanced liver disease, or in the presence of DIC. The presence of prolonged TT and normal fibrinogen and FDP levels suggest dysfibrinogenemia. FVIII levels are often normal or elevated in patients with liver failure, and decreased levels suggest superimposing DIC. Because FIGURE 141-4 Balance of hemostasis in liver disease. TAFI, thrombin-activated fibrinolytic FV is only synthesized in the hepatocyte and inhibitor; t-PA, tissue plasminogen activator; VWF, von Willebrand factor.

1	740 triad, hemodynamic changes and damaged vasculature may also be a contributing factor, and both processes may potentially also occur in patients with liver disease. Liver-related thrombosis, in particular, thrombosis of the portal and mesenteric veins, is common in patients with advanced cirrhosis. Hemodynamic changes, such as decreased portal flow, and evidence that inherited thrombophilia may enhance the risk for portal vein thrombosis in patients with cirrhosis suggest that hypercoagulability may play a role as well. Patients with liver disease develop deep vein thrombosis and pulmonary embolism at appreciable rates (ranging from 0.5 to 1.9%). The implication of these findings is relevant to the erroneous exclusion of thrombosis in patients with advanced liver disease, even in the presence of prolongation of routine clotting times, and caution should be advised on overcorrection of these laboratory abnormalities.

1	ACQUIRED INHIBITORS OF COAGULATION FACTORS An acquired inhibitor is an immune-mediated disease characterized by the presence of an auto-antibody against a specific clotting factor. FVIII is the most common target of antibody formation, and is sometimes referred to as acquired hemophilia A, but inhibitors to prothrombin, FV, FIX, FX, and FXI are also reported. Acquired inhibitor to FVIII occurs predominantly in older adults (median age of 60 years), but occasionally in pregnant or postpartum women with no previous history of bleeding. In 50% of patients with inhibitors, no underlying disease is identified at the time of diagnosis. In the remaining patients, the causes are autoimmune diseases, malignancies (lymphomas, prostate cancer), dermatologic diseases, and pregnancy. Bleeding episodes occur commonly in soft tissues, the gastrointestinal or urinary tracts, and skin. In contrast to hemophilia, hemarthrosis is rare in these patients. Retroperitoneal hemorrhages and other

1	episodes occur commonly in soft tissues, the gastrointestinal or urinary tracts, and skin. In contrast to hemophilia, hemarthrosis is rare in these patients. Retroperitoneal hemorrhages and other life-threatening bleeding may appear suddenly. The overall mortality in untreated patients ranges from 8 to 22%, and most deaths occur within the first few weeks after presentation. The diagnosis is based on the prolonged aPTT with normal PT and TT. The aPTT remains prolonged after mixture of the test plasma with equal amounts of pooled normal plasma for 2 h at 37°C. The Bethesda assay using FVIII-deficient plasma as performed for inhibitor detection in hemophilia will confirm the diagnosis. Major bleeding is treated with bypass products such as PCC/aPCC or recombinant FVIIa. In contrast to hemophilia, inhibitors in nonhemophilic patients are typically responsive to immune suppression, and therapy should be initiated early for most cases. The first choice includes steroid or a combination of

1	inhibitors in nonhemophilic patients are typically responsive to immune suppression, and therapy should be initiated early for most cases. The first choice includes steroid or a combination of steroid with cytotoxic therapy (e.g., cyclophosphamide), with complete eradication of the inhibitors in more than 70% of patients. High-dose intravenous γ-globulin and anti-CD20 monoclonal antibody have been reported to be effective in patients with autoantibodies to FVIII; however, there is no firm evidence that these alternatives are superior to the first line of immunosuppressive drugs. Notably, relapse of the inhibitor to FVIII is relatively common (up to 20%) within the first 6 months following withdrawal of immunosuppression. Thus, after eradication, patients should be followed up regularly for early therapeutic intervention when indicated or prior to invasive procedure.

1	Topical plasma-derived bovine and human thrombin are commonly used in the United States and worldwide. These effective hemostatic sealants are used during major surgery such as for cardiovascular, thoracic, neurologic, pelvic, and trauma indications, as well as in the setting of extensive burns. The development of antibody formation to the xenoantigen or its contaminant (bovine clotting protein) has the potential to show cross-reactivity with human clotting factors that may hamper their function and induce bleeding. Clinical features of these antibodies include bleeding from a primary hemostatic defect or coagulopathy that sometimes can be life threatening. The clinical diagnosis of these acquired coagulopathies is often complicated by the fact that the bleeding episodes may be detectable during or immediately following major surgery and could be assumed to be due to the procedure itself.

1	Notably, the risk of this complication is further increased by repeated exposure to topical thrombin preparations. Thus, a careful medical history of previous surgical interventions that may have occurred even decades earlier is critical to assessing risk. The laboratory abnormalities are reflected by combined prolongation of the aPTT and PT that often fails to improve by transfusion of FFP and vitamin K. The abnormal laboratory tests cannot be corrected by mixing a test with equal parts of normal plasma that denotes the presence of inhibitory antibodies. The diagnosis of a specific antibody is obtained by the determination of the residual activity of human FV or other suspected human clotting factor. There are no commercially available assays specific for bovine thrombin coagulopathy.

1	There are no established treatment guidelines. Platelet transfusions have been used as a source of FV replacement for patients with FV inhibitors. Frequent injections of FFP and vitamin K supplementation may function as co-adjuvant rather than an effective treatment of the coagulopathy itself. Experience with recombinant FVIIa as a bypass agent is limited, and outcomes have been generally poor. Specific treatments to eradicate the antibodies based on immunosuppression with steroids, intravenous immunoglobulin, or serial plasmapheresis have been sporadically reported. Patients should be advised to avoid any topical thrombin sealant in the future. Novel plasma-derived and recombinant human thrombin preparations for topical hemostasis have been approved by the U.S. Food and Drug Administration. These preparations have demonstrated hemostatic efficacy with reduced immunogenicity compared to the first generation of bovine thrombin products.

1	The presence of lupus anticoagulant can be associated with venous or arterial thrombotic disease. However, bleeding has also been reported in lupus anticoagulant; it is due to the presence of antibodies to prothrombin, which results in hypoprothrombinemia. Both disorders show a prolonged PTT that does not correct on mixing. To distinguish acquired inhibitors from lupus anticoagulant, note that the dilute Russell’s viper venom test and the hexagonal-phase phospholipids test will be negative in patients with an acquired inhibitor and positive in patients with lupus anticoagulants. Moreover, lupus anticoagulant interferes with the clotting activity of many factors (FVIII, FIX, FXII, FXI), whereas acquired inhibitors are specific to a single factor. Jane E. Freedman, Joseph Loscalzo

1	Thrombosis, the obstruction of blood flow due to the formation of clot, may result in tissue anoxia and damage, and it is a major cause of morbidity and mortality in a wide range of arterial and venous diseases and patient populations. In 2009 in the United States, an estimated 785,000 people had a new coronary thrombotic event, and about 470,000 had a recurrent ischemic episode. Each year, approximately 795,000 people have a new or recurrent stroke. It is estimated that 300,000–600,000 people each year have a pulmonary embolism or deep venous thrombotic event. In the nondiseased state, physiologic hemostasis reflects a delicate interplay between factors that promote and inhibit blood clotting, favoring the former. This response is crucial as it prevents uncontrolled hemorrhage and exsanguination following injury. In specific settings, the same processes that regulate normal hemostasis can cause pathologic thrombosis, leading to arterial or venous occlusion. Importantly, many commonly

1	following injury. In specific settings, the same processes that regulate normal hemostasis can cause pathologic thrombosis, leading to arterial or venous occlusion. Importantly, many commonly used therapeutic interventions may also alter the thrombotic–hemostatic balance adversely.

1	Hemostasis and thrombosis primarily involve the interplay among three factors: the vessel wall, coagulation proteins, and platelets. Many prevalent acute vascular diseases are due to thrombus formation within a vessel, including myocardial infarction, thrombotic cerebrovascular events, and venous thrombosis. Although the end result is vessel occlusion and tissue ischemia, the pathophysiologic processes governing these pathologies have similarities as well as distinct differences. While many of the pathways regulating thrombus formation are similar to those that regulate hemostasis, the processes triggering thrombosis and, often, perpetuating the thrombus may be distinct and can vary in different clinical and genetic settings. In venous thrombosis, primary hypercoagulable states reflecting defects in the proteins governing coagulation and/or fibrinolysis or secondary hypercoagulable states involving abnormalities of blood vessels and blood flow or stasis lead to thrombosis. By

1	defects in the proteins governing coagulation and/or fibrinolysis or secondary hypercoagulable states involving abnormalities of blood vessels and blood flow or stasis lead to thrombosis. By contrast, arterial thrombosis is highly dependent on the state of the vessel wall, the platelet, and factors related to blood flow.

1	In arterial thrombosis, the platelets and abnormalities of the vessel wall typically play a key role in vessel occlusion. Arterial thrombus forms via a series of sequential steps in which platelets adhere to the vessel wall, additional platelets are recruited, and thrombin is activated (Fig. 142-1). The regulation of platelet adhesion, activation, aggregation, and recruitment will be described in detail below. In addition, while the primary function of platelets is regulation of hemostasis, our understanding of their role in other processes, such as immunity, wound healing, and inflammation, continues to grow.

1	Arterial thrombosis is a major cause of morbidity and mortality both in the United States and, increasingly, worldwide. Although the rates have declined in the United States, the overall burden remains high and accounts for approximately 33% of deaths. Overall, coronary heart disease is estimated to cause about 1 of every 5 deaths in the United States. In addition to the 785,000 Americans who will have a new coronary event, an additional 195,000 silent first myocardial infarctions are projected to occur annually. Although the rate of strokes has fallen by 741 a third, each year, about 795,000 people experience a new or recurrent stroke, although not all are caused by thrombotic occlusion of the vessel. Approximately 610,000 strokes are first events and 185,000 are recurrent events; it is estimated that 1 of every 18 deaths in the United States is due to stroke.

1	Many processes in platelets have parallels with other cell types, such as the presence of specific receptors and signaling pathways; however, unlike most cells, platelets lack a nucleus and are unable to adapt to changing biologic settings by altered gene transcription. Platelets sustain limited protein synthetic capacity from megakaryocyte-derived and intracellularly transported microRNA (miRNA) and messenger RNA (mRNA). Most of the molecules needed to respond to various stimuli, however, are maintained in storage granules and membrane compartments.

1	Platelets are disc-shaped, very small, anucleate cells (1–5 μm in diameter) that circulate in the blood at concentrations of 200–400,000/ μL, with an average lifespan of 7–10 days. Platelets are derived from megakaryocytes, polyploidal hematopoietic cells found in the bone marrow. The primary regulator of platelet formation is thrombopoietin (TPO). The precise mechanism by which megakaryocytes produce and release fully formed platelets is unclear, but the process likely involves formation of proplatelets, pseudopod-like structures generated by the evagination of the cytoplasm from which platelets bud. Platelet granules are synthesized in megakaryocytes before thrombo poiesis and contain an array of prothrombotic, proinflammatory, and antimicrobial mediators. The two major types of platelet granules, alpha and dense, are distinguished by their size, abundance, and con tent. Alpha-granules contain soluble coagulation proteins, adhesion molecules, growth factors, integrins, cytokines,

1	granules, alpha and dense, are distinguished by their size, abundance, and con tent. Alpha-granules contain soluble coagulation proteins, adhesion molecules, growth factors, integrins, cytokines, and inflammatory modulators. Platelet dense-granules are smaller than alpha-granules and less abundant. Whereas alpha-granules contain proteins that may be more important in the inflammatory response, dense-granules contain high concentrations of small molecules, including adenosine diphosphate (ADP) and serotonin, that influence platelet aggregation.

1	Platelet Adhesion (See Fig. 142-1) The formation of a thrombus is initiated by the adherence of platelets to the damaged vessel wall. Damage exposes sub-endothelial components responsible for triggering platelet reactivity, including collagen, von Willebrand factor, fibronectin, and other adhesive proteins, such as vitronectin and thrombospondin. The hemostatic response may vary, depending on the extent of damage, the specific proteins exposed, and flow conditions. Certain proteins are expressed on the platelet surface that subsequently regulate collagen-induced platelet adhesion, particularly under flow conditions, and include glycoprotein (GP) IV, GPVI, and the integrin α2β1. The platelet GPIb-IX-V complex adhesive receptor is central both to platelet adhesion and to the initiation of platelet activation. Damage to the

1	FIGURE 142-1 Platelet activation and thrombosis. Platelets circulate in an inactive form in the blood vessel wall exposes subendothelial vasculature. Damage to the endothelium and/or external stimuli activates platelets that adhere von Willebrand factor and collagen to the to the exposed subendothelial von Willebrand factor and collagen. This adhesion leads to activa-circulating blood. The GPIb-IX-V comtion of the platelet, shape change, and the synthesis and release of thromboxane (TxA2), serotonin plex binds to the exposed von Willebrand (5-HT), and adenosine diphosphate (ADP). Platelet stimuli cause conformational change in the factor, causing platelets to adhere (Fig. platelet integrin glycoprotein (GP) IIb/IIIa receptor, leading to the high-affinity binding of fibrino-142-1). In addition, the engagement of the gen and the formation of a stable platelet thrombus. GPIb-IX-V complex with ligand induces 742 signaling pathways that lead to platelet activation. von Willebrand

1	In addition, the engagement of the gen and the formation of a stable platelet thrombus. GPIb-IX-V complex with ligand induces 742 signaling pathways that lead to platelet activation. von Willebrand factor–bound GPIb-IX-V promotes a calcium-dependent conformational change in the GPIIb/IIIa receptor, transforming it from an inactive low-affinity state to an active high-affinity receptor for fibrinogen.

1	Platelet Activation The activation of platelets is controlled by a variety of surface receptors that regulate various functions in the activation process. Platelet receptors control many distinct processes and are stimulated by a wide variety of agonists and adhesive proteins that result in variable degrees of activation. In general terms, the stimulation of platelet receptors triggers two specific processes: (1) activation of internal signaling pathways that lead to further platelet activation and granule release and (2) the capacity of the platelet to bind to other adhesive proteins/platelets. Both of these processes contribute to the formation of a thrombus. Stimulation of nonthrombotic receptors results in platelet adhesion or interaction with other vascular cells including endothelial cells, neutrophils, and mononuclear cells.

1	Many families and subfamilies of receptors are found on platelets that regulate a variety of platelet functions. These include the seven transmembrane receptor family, which is the main agoniststimulated receptor family. Several seven transmembrane receptors are found on platelets, including the ADP receptors, prostaglandin receptors, lipid receptors, and chemokine receptors. Receptors for thrombin comprise the major seven transmembrane receptors found on platelets. Among this last group, the first identified was the protease activation receptor 1 (PAR1). The PAR class of receptors has a distinct mechanism of activation that involves specific cleavage of the N-terminus of thrombin, which, in turn, acts as a ligand for the receptor. Other PAR receptors are present on platelets, including PAR2 (not activated by thrombin) and PAR4. Adenosine receptors are responsible for transduction of ADP-induced signaling events, which are initiated by the binding of ADP to purinergic receptors on the

1	(not activated by thrombin) and PAR4. Adenosine receptors are responsible for transduction of ADP-induced signaling events, which are initiated by the binding of ADP to purinergic receptors on the platelet surface. There are several distinct ADP receptors, classified as P2X1, P2Y1, and P2Y12. The activation of both the P2Y12 and P2Y1 receptors is essential for ADP-induced platelet aggregation. The thienopyridine derivatives, clopidogrel and prasugrel, are clinically used inhibitors of ADP-induced platelet aggregation.

1	Platelet Aggregation Activation of platelets results in a rapid series of signal transduction events, including tyrosine kinase, serine/threonine kinase, and lipid kinase activation. In unstimulated platelets, the major platelet integrin GPIIb/IIIa is maintained in an inactive conformation and functions as a low-affinity adhesion receptor for fibrinogen. This integrin is unique as it is only expressed on platelets. After stimulation, the interaction between fibrinogen and GPIIb/IIIa forms intercellular connections between platelets, leading to the formation of a platelet aggregate (Fig. 142-1). A calcium-sensitive conformational change in the extracellular domain of GPIIb/IIIa enables the high-affinity binding of soluble plasma fibrinogen as a result of a complex network of inside-out signaling events. The GPIIb/IIIa receptor serves as a bidirectional conduit with GPIIb/IIIa-mediated signaling (outside-in) occurring immediately after the binding of fibrinogen. This leads to additional

1	events. The GPIIb/IIIa receptor serves as a bidirectional conduit with GPIIb/IIIa-mediated signaling (outside-in) occurring immediately after the binding of fibrinogen. This leads to additional intracellular signaling that further stabilizes the platelet aggregate and transforms platelet aggregation from a reversible to an irreversible process (inside-out).

1	Inflammation plays an important role during the acute thrombotic phase of acute coronary syndromes. In the setting of acute upper respiratory infections, people are at higher risk of myocardial infarction and thrombotic stroke. Patients with acute coronary syndromes have not only increased interactions between platelets (homotypic aggregates), but also increased interactions between platelets and leukocytes (heterotypic aggregates) detectable in circulating blood. These latter aggregates form when platelets are activated and adhere to circulating leukocytes. Platelets bind via P-selectin (CD62P) expressed on the surface of activated platelets to the leukocyte receptor, P-selectin glycoprotein ligand 1 (PSGL-1). This association leads to increased expression of CD11b/CD18 (Mac-1) on leukocytes, which itself supports interactions with platelets partially via bivalent fibrinogen linking this integrin with its platelet surface counterpart, GPIIb/IIIa. Platelet surface P-selectin also

1	which itself supports interactions with platelets partially via bivalent fibrinogen linking this integrin with its platelet surface counterpart, GPIIb/IIIa. Platelet surface P-selectin also induces the expression of tissue factor on monocytes, which promotes fibrin formation.

1	In addition to platelet–monocyte aggregates, the immunomodulator, soluble CD40 ligand (CD40L or CD154), also reflects a link between thrombosis and inflammation. The CD40 ligand is a trimeric transmembrane protein of the tumor necrosis factor family and, with its receptor CD40, is an important contributor to the inflammatory process leading both to thrombosis and atherosclerosis. While many immunologic and vascular cells have been found to express CD40 and/or CD40 ligand, in platelets, CD40 ligand is rapidly translocated to the surface after stimulation and is upregulated in the newly formed thrombus. The surface-expressed CD40 ligand is cleaved from the platelet to generate a soluble fragment (soluble CD40 ligand).

1	Links have also been established among platelets, infection, immunity, and inflammation. Bacterial and viral infections are associated with a transient increase in the risk of acute thrombotic events, such as acute myocardial infarction and stroke. In addition, platelets contribute significantly to the pathophysiology and high mortality rates of sepsis. The expression, functionality, and signaling pathways of toll-like receptors (TLRs) have been established in platelets. Stimulation of platelet TLR2, TLR3, and TLR4 directly and indirectly activates the platelet’s thrombotic and inflammatory responses, and live bacteria induce a proinflammatory response in platelets in a TLR2-dependent manner, suggesting a mechanism by which specific bacteria and bacterial components can directly activate platelet-dependent thrombosis.

1	Some studies have associated arterial thrombosis with genetic variants (Table 142-1 A); however, the associations have been weak and not confirmed in larger series. Platelet count and mean platelet volume have been studied by genome-wide association studies (GWAS), and this approach identified signals located to noncoding regions. Of 15 quantitative trait loci associated with mean platelet volume and platelet count, one located at 12q24 is also a risk locus for coronary artery disease. In the area of genetic variability and platelet function, studies have primarily dealt with pharmacogenetics, the field of pharmacology dealing with the interindividual variability in drug response based on genetic determinants (Table 142-2). This focus has been driven by the wide variability among individuals in terms of response to antithrombotic drugs and the lack of a common explanation for this variance. The best described is the issue of “aspirin resistance,” although heterogeneity for other

1	in terms of response to antithrombotic drugs and the lack of a common explanation for this variance. The best described is the issue of “aspirin resistance,” although heterogeneity for other antithrombotics (e.g., clopidogrel) has also been extensively examined. Primarily, platelet-dependent genetic determinants have been defined at the level of (1) drug effect, (2) drug compliance, and (3) drug metabolism. Many candidate platelet genes have been studied for their interaction with antiplatelet and antithrombotic agents.

1	Many patients have an inadequate response to the inhibitory effects of aspirin. Heritable factors contribute to the variability; however, ex vivo tests of residual platelet responsiveness after aspirin administration have not provided firm evidence for a pharmacogenetic interaction between aspirin and COX1 or other relevant platelet receptors. As such, currently, there is no clinical indication for genotyping to optimize aspirin’s antiplatelet efficiency. For the platelet P2Y12 receptor inhibitor clopidogrel, additional data suggest that genetics may affect the drug’s responsiveness and utility. The responsible genetic variant appears not to be the expected P2Y12 receptor but an enzyme responsible for drug metabolism. Clopidogrel is a prodrug, and liver metabolism by specific cytochrome P450 enzymes is required for activation. The genes encoding the CYP-dependent oxidative steps are polymorphic, and carriers of specific alleles of the CYP2C19 and CYP3A4 loci have increased platelet

1	P450 enzymes is required for activation. The genes encoding the CYP-dependent oxidative steps are polymorphic, and carriers of specific alleles of the CYP2C19 and CYP3A4 loci have increased platelet aggregability. Increased platelet activity has also been specifically associated with the CYP2C19*2 allele, which causes loss of platelet function in select patients. Because these are common genetic variants, this observation has been shown to be clinically relevant in large studies. In summary, although the loss-of-function polymorphisms in CYP2C19 is the strongest individual variable affecting pharmacokinetics and antiplatelet response to clopidogrel, it only

1	A. Arterial Thrombosis Plasma glutathione peroxidase H2 promoter haplotype Endothelial nitric oxide synthase −786T/C, −922A/G, −1468T/A Paraoxonase −107T allele, 192R allele Cystathionine β-synthase 833T → C 5,10-Methylene tetrahydrofolate reductase (MTHFR) 677C → T B. Venous Thrombosis Fibrinogen −455G/A, –854G/A Prothrombin (20210G → A) Factor V Leiden: 1691G → A (Arg506Gln) Thrombomodulin 1481C → T (Ala455Val) Fibrinolytic Proteins with Known Polymorphisms Tissue plasminogen activator (tPA) 7351C/T, 20 099T/C in exon 6, 27 445T/A in intron 10 Plasminogen activator inhibitor (PAI-1) 4G/5G insertion/deletion polymorphism at position –675 Cystathionine β-synthase 833T → C 5,10-MTHFR 677C → T accounts for 5–12% of the variability in ADP-induced platelet aggregation on clopidogrel. In addition, genetic variables do not appear to significantly contribute to the clinical outcomes of patients treated with the P2Y12 receptor antagonists prasugrel or ticagrelor.

1	Coagulation is the process by which thrombin is activated and soluble plasma fibrinogen is converted into insoluble fibrin. These steps account for both normal hemostasis and the pathophysiologic processes influencing the development of venous thrombosis. The primary forms of venous thrombosis are deep vein thrombosis (DVT) in the extremities and the subsequent embolization to the lungs (pulmonary embolism), referred to together as venous thromboembolic disease. Venous thrombosis occurs due to heritable causes (Table 142-1 B) and acquired causes (Table 142-3).

1	More than 200,000 new cases of venous thromboembolism occur each year. Of these cases, up to 30% of patients die within 30 days and one-fifth suffer sudden death due to pulmonary embolism; 30% go on to develop recurrent venous thromboembolism within 10 years. Data from the Atherosclerosis Risk in Communities (ARIC) study reported a 9% 28-day fatality rate from DVT and a 15% fatality rate from pulmonary embolism. Pulmonary embolism in the setting of cancer has a 25% fatality rate. The mean incidence of first DVT in the general population is 5 per 10,000 person-years; the incidence is similar in males and females when adjusting for factors related to reproduction and birth control and increases dramatically with age from 2 to 3 per 10,000 person-years at 30–49 years of age to 20 at 70–79 years of age.

1	Coagulation is defined as the formation of fibrin by a series of linked enzymatic reactions in which each reaction product converts the subsequent inactive zymogen into an active serine protease (Fig. 142-2). This coordinated sequence is called the coagulation cascade and is a key mechanism for regulating hemostasis. Central to the function of the coagulation cascade is the principle of amplification: due to a series of linked enzymatic reactions, a small stimulus can lead to much greater quantities of fibrin, the end product that prevents hemorrhage at the site of vascular injury. In addition to the known risk factors relevant to hypercoagulopathy, stasis, and vascular dysfunction, newer areas of research have identified contributions from procoagulant microparticles, inflammatory cells, microvesicles, and fibrin structure.

1	The coagulation cascade is primarily initiated by vascular injury exposing tissue factor to blood components (Fig. 142-2). Tissue factor may also be found in bloodborne cell-derived microparticles and, under pathophysiologic conditions, in leukocytes or platelets. Plasma factor VII (FVII) is the ligand for and is activated (FVIIa) by binding to tissue factor exposed at the site of vessel damage. The binding of FVII/ VIIa to tissue factor activates the downstream conversion of factor X (FX) to active FX (FXa). In an alternative reaction, the FVII/FVIIa–tissue factor complex initially converts FIX to FIXa, which then activates FX in conjunction with its cofactor factor VIII (FVIIIa). Factor Xa with its cofactor FVa converts prothrombin to thrombin, which then converts soluble plasma fibrinogen to insoluble fibrin, leading to clot or thrombus formation. Thrombin also activates FXIII to FXIIIa, a transglutaminase that covalently cross-links and stabilizes the fibrin clot. Formation of

1	to insoluble fibrin, leading to clot or thrombus formation. Thrombin also activates FXIII to FXIIIa, a transglutaminase that covalently cross-links and stabilizes the fibrin clot. Formation of thrombi is affected by mechanisms governing fibrin structure and stability including specific fibrinogen variants and how they alter fibrin formation, strength and structure.

1	744 XII significantly increase the risk of venous thrombosis. While homozygous protein C or protein S deficiencies are rare and may lead to fatal purpura fulminans, heterozygous deficiencies are associated with a moderate risk of thrombosis. Activated protein C impairs coagulation by proteolytic degradation of FVa. Patients resistant to the activity of activated protein C may have a point mutation in the FV gene located on chromosome 1, a mutant denoted factor V Leiden. Mildly increased risk has been attributed to elevated levels of procoagulant factors, as well as low levels of tissue factor pathway inhibitor. Polymorphisms of methylene tetrahydrofolate reductase as well as hyperhomocysteinemia have been shown to be independent risk factors for venous thrombosis, as well as arterial vascular disease; however, many of the initial descriptions of genetic variants and their associations with thromboembolism are being questioned in larger, more current studies.

1	Specific abnormalities in the fibrinolytic system have been associated with enhanced thrombosis. Factors such as elevated levels of tissue plasminogen activator (tPA) and plasminogen activator FIGURE 142-2 Summary of the coagulation pathways. Specific coagulation factors (“a” inhibitor type 1 (PAI-1) have been associated with indicates activated form) are responsible for the conversion of soluble plasma fibrinogen decreased fibrinolytic activity and an increased into insoluble fibrin. This process occurs via a series of linked reactions in which the enzy-risk of arterial thrombotic disease. Specific genetic matically active product subsequently converts the downstream inactive protein into an variants have been associated with decreased fibriactive serine protease. In addition, the activation of thrombin leads to stimulation of plate-nolytic activity, including the 4G/5G insertion/ lets. HK, high-molecular-weight kininogen; PK, prekallikrein; TF, tissue factor. deletion polymorphism

1	of thrombin leads to stimulation of plate-nolytic activity, including the 4G/5G insertion/ lets. HK, high-molecular-weight kininogen; PK, prekallikrein; TF, tissue factor. deletion polymorphism in the (plasminogen activator type 1) PAI-1 gene. Additionally, the 311-bp Alu insertion/deletion in tPA’s intron 8 has been

1	Several antithrombotic factors also regulate coagulation; these associated with enhanced thrombosis; however, genetic abnormalities include antithrombin, tissue factor pathway inhibitor (TFPI), heparin have not been associated consistently with altered function or tPA levcofactor II, and protein C/protein S. Under normal conditions, these els, raising questions about the relevant pathophysiologic mechanism. factors limit the production of thrombin to prevent the perpetuation Thrombin-activatable fibrinolysis inhibitor (TAFI) is a carboxypeptiof coagulation and thrombus formation. Typically, after the clot has dase that regulates fibrinolysis; elevated plasma TAFI levels have been caused occlusion at the damaged site and begins to expand toward associated with an increased risk of both DVT and cardiovascular adjacent uninjured vessel segments, the anticoagulant reactions gov-disease. erned by the normal endothelium become pivotal in limiting the extent The metabolic syndrome also is

1	and cardiovascular adjacent uninjured vessel segments, the anticoagulant reactions gov-disease. erned by the normal endothelium become pivotal in limiting the extent The metabolic syndrome also is accompanied by altered fibrinoof this hemostatically protective clot. lytic activity. This syndrome, which comprises abdominal fat (cen tral obesity), altered glucose and insulin metabolism, dyslipidemia, and hypertension, has been associated with atherothrombosis. The mechanism for enhanced thrombosis appears to be due both to alteredThe risk factors for venous thrombosis are primarily related to hyper-platelet function and to a procoagulant and hypofibrinolytic state.

1	coagulability, which can be genetic (Table 142-1) or acquired, or due One of the most frequently documented prothrombotic abnormalitiesto immobilization and venous stasis. Independent predictors for recur-reported in this syndrome is an increase in plasma levels of PAI-1. rence include increasing age, obesity, malignant neoplasm, and acute In addition to contributing to platelet function, inflammation playsextremity paresis. It is estimated that 5–8% of the U.S. population has a role in both coagulation-dependent thrombus formation and throma genetic risk factor known to predispose to venous thrombosis. Often, bus resolution. Both polymorphonuclear neutrophils and monocytes/multiple risk factors are present in a single individual. Significant risk macrophages contribute to multiple overlapping thrombotic funcis incurred by major orthopedic, abdominal, or neurologic surgeries.

1	tions, including fibrinolysis, chemokine and cytokine production, and Moderate risk is promoted by prolonged bedrest; certain types of canphagocytosis. cer, pregnancy, hormone replacement therapy, or oral contraceptive use; and other sedentary conditions such as long-distance plane travel. It has been reported that the risk of developing a venous thromboem-THE DISTINCTION BETWEEN ARTERIAL AND VENOUS bolic event doubles after air travel lasting 4 h, although the absolute THROMBOSIS risk remains low (1 in 6000). The relative risk of venous thromboem- Although there is overlap, venous thrombosis and arterial thrombosis bolism among pregnant or postpartum women is 4.3, and the overall are initiated differently, and clot formation progresses by somewhatincidence (absolute risk) is 199.7 per 100,000 woman-years.

1	distinct pathways. In the setting of stasis or states of hypercoagulability, venous thrombosis is activated with the initiation of the coagulation GENETICS OF VENOUS THROMBOSIS cascade primarily due to exposure of tissue factor; this leads to the(See Table 142-2) Less common causes of venous thrombosis are formation of thrombin and the subsequent conversion of fibrinogen tothose due to genetic variants. These abnormalities include loss-of-fibrin. In the artery, thrombin formation also occurs, but thrombosisfunction mutations of endogenous anticoagulants as well as gain-is primarily promoted by the adhesion of platelets to an injured vesselof-function mutations of procoagulant proteins. Heterozygous anti-and stimulated by exposed extracellular matrix (Figs. 142-1 and 142-2).thrombin deficiency and homozygosity of the factor V Leiden mutation There is wide variation in individual responses to vascular injury, an important determinant of which is the predisposition an individual has to

1	and homozygosity of the factor V Leiden mutation There is wide variation in individual responses to vascular injury, an important determinant of which is the predisposition an individual has to arterial or venous thrombosis. This concept has been supported indirectly in prothrombotic animal models in which there is poor correlation between the propensity to develop venous versus arterial thrombosis.

1	Despite considerable progress in understanding the role of hyper-coagulable states in venous thromboembolic disease, the contribution of hypercoagulability to arterial vascular disease is much less well understood. Although specific thrombophilic conditions, such as factor V Leiden and the prothrombin G20210A mutation, are risk factors for DVT, pulmonary embolism, and other venous thromboembolic events, their contribution to arterial thrombosis is less well defined. In fact, to the contrary, many of these thrombophilic factors have not been found to be clinically important risk factors for arterial thrombotic events, such as acute coronary syndromes.

1	Clinically, although the pathophysiology is distinct, arterial and venous thrombosis do share common risk factors, including age, obesity, cigarette smoking, diabetes mellitus, arterial hypertension, hyperlipidemia, and metabolic syndrome. Select genetic variants, including those of the glutathione peroxidase gene, have also been associated with arterial and venous thrombo-occlusive disease. Importantly, arterial and venous thrombosis may both be triggered by pathophysiologic stimuli responsible for activating inflammatory and oxidative pathways. The diagnosis and treatment of ischemic heart disease are discussed in Chap. 293. Stroke diagnosis and management are discussed in Chap. 330. The diagnosis and management of DVT and pulmonary embolus are discussed in Chap. 300. Jeffrey I. Weitz 143 Antiplatelet, Anticoagulant, and fibrinolytic Drugs

1	Antiplatelet, Anticoagulant, and fibrinolytic Drugs Thromboembolic disorders are major causes of morbidity and mortal ity. Thrombosis can occur in arteries or veins. Arterial thrombosis is the most common cause of acute myocardial infarction (MI), ischemic stroke, and limb gangrene. Venous thromboembolism encompasses deep vein thrombosis (DVT), which can lead to postthrombotic syn drome, and pulmonary embolism (PE), which can be fatal or can result in chronic thromboembolic pulmonary hypertension.

1	Most arterial thrombi are superimposed on disrupted atherosclerotic plaque because plaque rupture exposes thrombogenic material in the plaque core to the blood. This material then triggers platelet aggregation and fibrin formation, which results in the generation of a platelet-rich thrombus that can temporarily or permanently occlude blood flow. In contrast, venous thrombi rarely form at sites of obvious vascular disruption. Although they can develop after surgical trauma to veins or secondary to indwelling venous catheters, venous thrombi usually originate in the valve cusps of the deep veins of the calf or in the muscular sinuses. Sluggish blood flow reduces the oxygen supply to the avascular valve cusps. Endothelial cells lining these valve cusps become activated and express adhesion molecules on their surface. Tissue factor–bearing leukocytes and microparticles adhere to these activated cells and induce coagulation. DNA extruded from neutrophils forms neutrophil extracelluar traps

1	on their surface. Tissue factor–bearing leukocytes and microparticles adhere to these activated cells and induce coagulation. DNA extruded from neutrophils forms neutrophil extracelluar traps (NETs) that provide a scaffold that traps red blood cells, promotes platelet adhesion and activation, and augments coagulation. Local thrombus formation is exacerbated by reduced clearance of activated clotting factors as a result of impaired blood flow. If the thrombi extend from the calf veins into the popliteal and more proximal veins of the leg, thrombus fragments can dislodge, travel to the lungs, and produce a PE.

1	Arterial and venous thrombi are composed of platelets, fibrin, and trapped red blood cells, but the proportions differ. Arterial thrombi FIGURE 143-1 Classification of antithrombotic drugs. are rich in platelets because of the high shear in the injured arteries. In contrast, venous thrombi, which form under low shear conditions, contain relatively few platelets and are predominantly composed of fibrin and trapped red cells. Because of the predominance of platelets, arterial thrombi appear white, whereas venous thrombi are red in color, reflecting the trapped red cells.

1	Antithrombotic drugs are used for prevention and treatment of thrombosis. Targeting the components of thrombi, these agents include (1) antiplatelet drugs, (2) anticoagulants, and (3) fibrinolytic agents (Fig. 143-1). With the predominance of platelets in arterial thrombi, strategies to attenuate arterial thrombosis focus mainly on antiplatelet agents, although, in the acute setting, often include anticoagulants and fibrinolytic agents. Anticoagulants are the mainstay of prevention and treatment of venous thromboembolism because fibrin is the predominant component of venous thrombi. Antiplatelet drugs are less effective than anticoagulants in this setting because of the limited platelet content of venous thrombi. Fibrinolytic therapy is used in selected patients with venous thromboembolism. For example, patients with massive or submassive PE can benefit from systemic or catheter-directed fibrinolytic therapy. Pharmaco-mechanical therapy also is used to restore blood flow in patients

1	For example, patients with massive or submassive PE can benefit from systemic or catheter-directed fibrinolytic therapy. Pharmaco-mechanical therapy also is used to restore blood flow in patients with extensive DVT involving the iliac and/or femoral veins.

1	In healthy vasculature, circulating platelets are maintained in an inactive state by nitric oxide (NO) and prostacyclin released by endothelial cells lining the blood vessels. In addition, endothelial cells also express CD39 on their surface, a membrane-associated ecto-adenosine diphosphatase (ADPase) that degrades ADP released from activated platelets. When the vessel wall is damaged, release of these substances is impaired and subendothelial matrix is exposed. Platelets adhere to exposed collagen via α2β1 and glycoprotein (Gp) V1 and to von Willebrand factor (VWF) via Gp Ibα and Gp IIb/IIIa (αIIbβ3)—receptors that are constitutively expressed on the platelet surface. Adherent platelets undergo a change in shape, secrete ADP from their dense granules, and synthesize and release thromboxane A2. Released ADP and thromboxane A2, which are platelet agonists, activate ambient platelets and recruit them to the site of vascular injury (Fig. 143-2).

1	Disruption of the vessel wall also exposes tissue factor–expressing cells to the blood. Tissue factor binds factor VIIa and initiates coagulation. Activated platelets potentiate coagulation by providing a surface that binds clotting factors and supports the assembly of activation complexes that enhance thrombin generation. In addition to converting fibrinogen to fibrin, thrombin serves as a potent platelet agonist and recruits more platelets to the site of vascular injury. Thrombin also amplifies its own generation by feedback activation of factors V, VIII, and XI and solidifies the fibrin network by activating factor XIII, which then cross-links the fibrin strands.

1	When platelets are activated, Gp IIb/IIIa, the most abundant receptor on the platelet surface, undergoes a conformational change that enables it to bind fibrinogen and, under high shear conditions, VWF. Divalent fibrinogen or multivalent VWF molecules bridge adjacent platelets together to form platelet aggregates. Fibrin strands, generated through the action of thrombin, then weave these aggregates together to form a platelet/fibrin mesh. Antiplatelet drugs target various steps in this process. The commonly used drugs include aspirin, ADP receptor inhibitors, which Antiplatelet, Anticoagulant, and Fibrinolytic Drugs

1	FIGURE 143-2 Coordinated role of platelets and the coagulation system in thrombogenesis. Vascular injury simultaneously triggers platelet activation and aggregation and activation of the coagulation system. Platelet activation is initiated by exposure of subendothelial collagen and von Willebrand factor (VWF), onto which platelets adhere. Adherent platelets become activated and release ADP and thromboxane A2, platelet agonists that activate ambient platelets and recruit them to the site of injury. When platelets are activated, glycoprotein IIb/IIIa on their surface undergoes a conformational change that enables it to ligate fibrinogen and/or VWF and mediate platelet aggregation. Coagulation is triggered by tissue factor exposed at the site of injury. Tissue factor triggers thrombin generation. As a potent platelet agonist, thrombin amplifies platelet recruitment to the site of injury. Thrombin also converts fibrinogen to fibrin, and the fibrin strands then weave the platelet

1	generation. As a potent platelet agonist, thrombin amplifies platelet recruitment to the site of injury. Thrombin also converts fibrinogen to fibrin, and the fibrin strands then weave the platelet aggregates together to form a platelet/fibrin thrombus.

1	include the thienopyridines (clopidogrel and prasugrel) and ticagrelor, dipyridamole, and Gp IIb/IIIa antagonists. The most widely used antiplatelet agent worldwide is aspirin. As a cheap and effective antiplatelet drug, aspirin serves as the foundation of most antiplatelet strategies. Mechanism of Action Aspirin produces its antithrombotic effect by irreversibly acetylating and inhibiting platelet cyclooxygenase (COX)-1 (Fig. 143-3), a critical enzyme in the biosynthesis of thromboxane A2. At high doses (~1 g/d), aspirin also inhibits COX-2, an inducible COX isoform found in endothelial cells and inflammatory cells. In endothelial cells, COX-2 initiates the synthesis of prostacyclin, a potent vasodilator and inhibitor of platelet aggregation.

1	Indications Aspirin is widely used for secondary prevention of cardiovascular events in patients with coronary artery, cerebrovascular, or peripheral vascular disease. Compared with placebo, aspirin produces a 25% reduction in the risk of cardiovascular death, MI, or stroke. Aspirin is also used for primary prevention in patients whose estimated annual risk of MI is >1%, a point where its benefits are likely to outweigh harms. This includes patients older than age 40 years with two or more major risk factors for cardiovascular disease or men older than age 45 years and women over the age of 55 years with one or more such risk factors. Aspirin is equally effective in men and women. In men, aspirin mainly reduces the risk of MI, whereas in women, aspirin lowers the risk of stroke.

1	Dosages Aspirin is usually administered at doses of 75–325 mg once daily. Higher doses of aspirin are not more effective than lower aspirin doses, and some analyses suggest reduced efficacy with higher doses. Because the side effects of aspirin are dose-related, daily aspirin doses of 75–100 mg are recommended for most indications. When rapid

1	FIGURE 143-3 Site of action of antiplatelet drugs. Aspirin inhibits thromboxane A2 (TXA2) synthesis by irreversibly acetylating cyclooxygenase-1 (COX-1). Reduced TXA2 release attenuates platelet activation and recruitment to the site of vascular injury. Clopidogrel and prasugrel irreversibly block P2Y12, a key ADP receptor on the platelet surface; cangrelor and ticagrelor are reversible inhibitors of P2Y12. Abciximab, eptifibatide, and tirofiban inhibit the final common pathway of platelet aggregation by blocking fibrinogen and von Willebrand factor binding to activated glycoprotein (Gp) IIb/IIIa. Vorapaxar inhibits thrombin-mediated platelet activation by targeting protease-activated receptor-1 (PAR-1), the major thrombin receptor on human platelets. platelet inhibition is required, an initial aspirin dose of at least 160 mg should be given.

1	platelet inhibition is required, an initial aspirin dose of at least 160 mg should be given. Side Effects The most common side effects are gastrointestinal and range from dyspepsia to erosive gastritis or peptic ulcers with bleeding and perforation. These side effects are dose-related. Use of entericcoated or buffered aspirin in place of plain aspirin does not eliminate gastrointestinal side effects. The overall risk of major bleeding with aspirin is 1–3% per year. The risk of bleeding is increased twoto threefold when aspirin is given in conjunction with other antiplatelet drugs, such as clopidogrel, or with anticoagulants, such as warfarin. When dual or triple therapy is prescribed, low-dose aspirin should be given (75–100 mg daily). Eradication of Helicobacter pylori infection and administration of proton pump inhibitors may reduce the risk of aspirin-induced upper gastrointestinal bleeding in patients with peptic ulcer disease.

1	Aspirin should not be administered to patients with a history of aspirin allergy characterized by bronchospasm. This problem occurs in ~0.3% of the general population but is more common in those with chronic urticaria or asthma, particularly in individuals with nasal polyps or chronic rhinitis. Hepatic and renal toxicity are observed with aspirin overdose. Aspirin Resistance Clinical aspirin resistance is defined as the failure of aspirin to protect patients from ischemic vascular events. This is not a helpful definition because it is made after the event occurs. Furthermore, it is not realistic to expect aspirin, which only blocks thromboxane A2–induced platelet activation, to prevent all vascular events.

1	Aspirin resistance has also been described biochemically as failure of the drug to produce its expected inhibitory effects on tests of platelet function, such as thromboxane A2 synthesis or arachidonic acid-induced platelet aggregation. Potential causes of aspirin resistance include poor compliance, reduced absorption, drug-drug interaction with ibuprofen, and overexpression of COX-2. Unfortunately, the tests for aspirin resistance have not been well standardized, and there is little evidence that they identify patients at increased risk of recurrent vascular events, or that resistance can be reversed by giving higher doses of aspirin or by adding other antiplatelet drugs. Until such information is available, testing for aspirin resistance remains a research tool. The ADP receptor antagonists include the thienopyridines (clopidogrel and prasugrel) and ticagrelor. All of these drugs target P2Y12, the key ADP receptor on platelets.

1	The ADP receptor antagonists include the thienopyridines (clopidogrel and prasugrel) and ticagrelor. All of these drugs target P2Y12, the key ADP receptor on platelets. Thienopyridines • mecHanism of action The thienopyridines are structurally related drugs that selectively inhibit ADP-induced platelet aggregation by irreversibly blocking P2Y12 (Fig. 143-3). Clopidogrel and prasugrel are prodrugs that require metabolic activation by the hepatic cytochrome P450 (CYP) enzyme system. Prasugrel is about 10-fold more potent than clopidogrel and has a more rapid onset of action because of better absorption and more streamlined metabolic activation.

1	indications When compared with aspirin in patients with recent ischemic stroke, recent MI, or a history of peripheral arterial disease, clopidogrel reduced the risk of cardiovascular death, MI, and stroke by 8.7%. Therefore, clopidogrel is more effective than aspirin but is also more expensive. In some patients, clopidogrel and aspirin are combined to capitalize on their capacity to block complementary pathways of platelet activation. For example, the combination of aspirin plus clopidogrel is recommended for at least 4 weeks after implantation of a bare metal stent in a coronary artery and for at least a year in those with a drug-eluting stent. Concerns about late in-stent thrombosis with drug-eluting stents have led some experts to recommend longterm use of clopidogrel plus aspirin for the latter indication. However, these recommendations are likely to change because the risk of late stent thrombosis is decreasing with the newer generation of drug-eluting coronary stents.

1	The combination of clopidogrel and aspirin is also effective in patients with unstable angina. Thus, in 12,562 such patients, the risk of cardiovascular death, MI, or stroke was 9.3% in those randomized to the combination of clopidogrel and aspirin and 11.4% in those given aspirin alone. This 20% relative risk reduction with combination therapy was highly statistically significant. However, combining clopidogrel with aspirin increases the risk of major bleeding to about 2% per year. This bleeding risk persists even if the daily dose of aspirin is ≤100 mg. Therefore, the combination of clopidogrel and aspirin should only be used when there is a clear benefit. For example, this combination has not proven to be superior to clopidogrel alone in patients with acute ischemic stroke or to aspirin alone for primary prevention in those at risk for cardiovascular events.

1	Prasugrel was compared with clopidogrel in 13,608 patients with acute coronary syndromes who were scheduled to undergo percutaneous coronary intervention. The incidence of the primary efficacy endpoint, a composite of cardiovascular death, MI, or stroke, was significantly lower with prasugrel than with clopidogrel (9.9% and 12.1%, respectively), mainly reflecting a reduction in the incidence of nonfatal MI. The incidence of stent thrombosis also was significantly lower with prasugrel (1.1% and 2.4%, respectively). However, these advantages were at the expense of significantly higher rates of fatal bleeding (0.4% and 0.1%, respectively) and life-threatening bleeding (1.4% and 0.9%, respectively) with prasugrel. Because patients older than age 75 years and those with a history of prior stroke or transient ischemic attack have a particularly high risk of bleeding, prasugrel should generally be avoided in older patients, and the drug is contraindicated in those with a history of

1	stroke or transient ischemic attack have a particularly high risk of bleeding, prasugrel should generally be avoided in older patients, and the drug is contraindicated in those with a history of cerebrovascular disease. Caution is required if prasugrel is used in patients weighing less than 60 kg or in those with renal impairment.

1	When prasugrel was compared with clopidogrel in 7243 patients with unstable angina or MI without ST-segment elevation, prasugrel failed to reduce the rate of the primary efficacy endpoint, which was a composite of cardiovascular death, MI, and stroke. Because of the negative results of this study, prasugrel is reserved for patients undergoing percutaneous coronary intervention. In this setting, prasugrel is usually given in conjunction with aspirin. To reduce the risk of bleeding, the daily aspirin dose should be ≤100 mg.

1	dosing Clopidogrel is given once daily at a dose of 75 mg. Loading 747 doses of clopidogrel are given when rapid ADP receptor blockade is desired. For example, patients undergoing coronary stenting are often given a loading dose of 300 mg, which produces inhibition of ADP-induced platelet aggregation in about 6 h; loading doses of 600 or 900 mg produce an even more rapid effect. After a loading dose of 60 mg, prasugrel is given once daily at a dose of 10 mg. Patients older than age 75 years or weighing less than 60 kg should receive a lower daily prasugrel dose of 5 mg. side effects The most common side effect of clopidogrel and prasugrel is bleeding. Because of its greater potency, bleeding is more common with prasugrel than clopidogrel. To reduce the risk of bleeding, clopidogrel and prasugrel should be stopped 5–7 days before major surgery. In patients taking clopidogrel or prasugrel who present with serious bleeding, platelet transfusion may be helpful.

1	Hematologic side effects, including neutropenia, thrombocytopenia, and thrombotic thrombocytopenic purpura, are rare.

1	tHienopyridine resistance The capacity of clopidogrel to inhibit ADP-induced platelet aggregation varies among subjects. This variability reflects, at least in part, genetic polymorphisms in the CYP isoenzymes involved in the metabolic activation of clopidogrel. Most important of these is CYP2C19. Clopidogrel-treated patients with the loss-offunction CYP2C19*2 allele exhibit reduced platelet inhibition compared with those with the wild-type CYP2C19*1 allele and experience a higher rate of cardiovascular events. This is important because estimates suggest that up to 25% of whites, 30% of African Americans, and 50% of Asians carry the loss-of-function allele, which would render them resistant to clopidogrel. Even patients with the reduced function CYP2C19*3, *4, or *5 alleles may derive less benefit from clopidogrel than those with the full-function CYP2C19*1 allele. Concomitant administration of clopidogrel with proton pump inhibitors, which are inhibitors of CYP2C19, produces a small

1	benefit from clopidogrel than those with the full-function CYP2C19*1 allele. Concomitant administration of clopidogrel with proton pump inhibitors, which are inhibitors of CYP2C19, produces a small reduction in the inhibitory effects of clopidogrel on ADP-induced platelet aggregation. The extent to which this interaction increases the risk of cardiovascular events remains controversial.

1	In contrast to their effect on the metabolic activation of clopidogrel, CYP2C19 polymorphisms appear to be less important determinants of the activation of prasugrel. Thus, no association was detected between the loss-of-function allele and decreased platelet inhibition or increased rate of cardiovascular events with prasugrel. The observation that genetic polymorphisms affecting clopidogrel absorption or metabolism influence clinical outcomes raises the possibilities that pharmacogenetic profiling may be useful to identify clopidogrelresistant patients and that point-of-care assessment of the extent of clopidogrel-induced platelet inhibition may help detect patients at higher risk for subsequent cardiovascular events. Clinical trials designed to evaluate these possibilities have thus far been negative. Although administration of higher doses of clopidogrel can overcome a reduced response to clopidogrel, the clinical benefit of this approach is uncertain. Instead, prasugrel or

1	far been negative. Although administration of higher doses of clopidogrel can overcome a reduced response to clopidogrel, the clinical benefit of this approach is uncertain. Instead, prasugrel or ticagrelor may be better choices for these patients.

1	Ticagrelor As an orally active inhibitor of P2Y12, ticagrelor differs from the thienopyridines in that ticagrelor does not require metabolic activation and it produces reversible inhibition of the ADP receptor. mecHanism of action Like the thienopyridines, ticagrelor inhibits P2Y12. Because it does not require metabolic activation, ticagrelor has a more rapid onset and offset of action than clopidogrel, and it produces greater and more predictable inhibition of ADP-induced platelet aggregation than clopidogrel. indications When compared with clopidogrel in patients with acute coronary syndromes, ticagrelor produced a greater reduction in the primary efficacy endpoint—a composite of cardiovascular death, MI, and stroke at 1 year—than clopidogrel (9.8% and 11.7%, respectively; p = .001). This difference reflected a significant reduction in both cardiovascular death (4.0% and 5.1%, respectively; p = .001) and MI (5.8% and 6.9%, respectively; p = .005) with ticagrelor compared with

1	Antiplatelet, Anticoagulant, and Fibrinolytic Drugs 748 clopidogrel. Rates of stroke were similar with ticagrelor and clopido-Indications Dipyridamole plus aspirin was compared with aspirin grel (1.5% and 1.3%, respectively), and no difference in rates of major or dipyridamole alone, or with placebo, in patients with an ischemic bleeding was noted. When minor bleeding was added to the major stroke or transient ischemic attack. The combination reduced the risk bleeding results, however, ticagrelor showed an increase relative to of stroke by 22.1% compared with aspirin and by 24.4% compared clopidogrel (16.1% and 14.6%, respectively; p = .008). Ticagrelor also with dipyridamole. A second trial compared dipyridamole plus aspirin was superior to clopidogrel in patients with acute coronary syndrome with aspirin alone for secondary prevention in patients with ischemic who underwent percutaneous coronary intervention or cardiac sur-stroke. Vascular death, stroke, or MI occurred in 13% of

1	syndrome with aspirin alone for secondary prevention in patients with ischemic who underwent percutaneous coronary intervention or cardiac sur-stroke. Vascular death, stroke, or MI occurred in 13% of patients gery. Based on these observations, some guidelines give ticagrelor given combination therapy and in 16% of those treated with aspirin preference over clopidogrel, particularly in higher risk patients. alone. Another trial randomized 20,332 patients with noncardioembolic ischemic stroke to either Aggrenox or clopidogrel. The primarydosing Ticagrelor is initiated with an oral loading dose of 180 mg efficacy endpoint of recurrent stroke occurred in 9.0% of those given followed by 90 mg twice daily. The dose does not require adjustment Aggrenox and in 8.8% of patients treated with clopidogrel. Althoughin patients with renal impairment, but the drug should be used with this difference was not statistically significant, the study failed to meet caution in patients with hepatic disease

1	Althoughin patients with renal impairment, but the drug should be used with this difference was not statistically significant, the study failed to meet caution in patients with hepatic disease and in those receiving potent the prespecified margin to claim noninferiority of Aggrenox relativeinhibitors or inducers of CYP3A4 because ticagrelor is metabolized in to clopidogrel. These results have dampened enthusiasm for the usethe liver via CYP3A4. Ticagrelor is usually administered in conjunc-of Aggrenox.tion with aspirin; the daily aspirin dose should not exceed 100 mg. Because of its vasodilatory effects and the paucity of data supporting the use of dipyridamole in patients with symptomatic coronary artery side effects In addition to bleeding, the most common side effects of disease, Aggrenox should not be used for stroke prevention in such ticagrelor are dyspnea, which can occur in up to 15% of patients, and patients. Clopidogrel is a better choice in this setting.

1	asymptomatic ventricular pauses. The dyspnea, which tends to occur soon after initiating ticagrelor, is usually self-limiting and mild in Dosing Aggrenox is given twice daily. Each capsule contains 200 mg intensity. The mechanism responsible for this side effect is unknown. of extended-release dipyridamole and 25 mg of aspirin. To reduce the risk of bleeding, ticagrelor should be stopped 5–7 Side Effects Because dipyridamole has vasodilatory effects, it must days prior to major surgery. Platelet transfusions are unlikely to be of be used with caution in patients with coronary artery disease. benefit in patients with ticagrelor-related bleeding because the drug Gastrointestinal complaints, headache, facial flushing, dizziness, and will bind to P2Y12 on the transfused platelets. hypotension can also occur. These symptoms often subside with continued use of the drug.

1	hypotension can also occur. These symptoms often subside with continued use of the drug. Dipyridamole is a relatively weak antiplatelet agent on its own, but an extended-release formulation of dipyridamole combined with low- As a class, parenteral Gp IIb/IIIa receptor antagonists have an estab dose aspirin, a preparation known as Aggrenox, is used for prevention lished niche in patients with acute coronary syndromes. The three of stroke in patients with transient ischemic attacks. agents in this class are abciximab, eptifibatide, and tirofiban.

1	Mechanism of Action By inhibiting phosphodiesterase, dipyridamole Mechanism of Action A member of the integrin family of adhesion blocks the breakdown of cyclic adenosine monophosphate (AMP). receptors, Gp IIb/IIIa is found on the surface of platelets and mega-Increased levels of cyclic AMP reduce intracellular calcium and inhibit karyocytes. With about 80,000 copies per platelet, Gp IIb/IIIa is the platelet activation. Dipyridamole also blocks the uptake of adenosine most abundant receptor. Consisting of a noncovalently linked hetby platelets and other cells. This produces a further increase in local erodimer, Gp IIb/IIIa is inactive on resting platelets. When platelets cyclic AMP levels because the platelet adenosine A2 receptor is coupled are activated, inside-outside signal transduction pathways trigger a to adenylate cyclase (Fig. 143-4). conformational activation of the receptor. Once activated, Gp IIb/ IIIa binds adhesive molecules, such as fibrinogen and, under high shear

1	pathways trigger a to adenylate cyclase (Fig. 143-4). conformational activation of the receptor. Once activated, Gp IIb/ IIIa binds adhesive molecules, such as fibrinogen and, under high shear conditions, VWF. Binding is mediated by the Arg-Gly-Asp (RGD) sequence found on the α chains of fibrinogen and on VWF, and by the Lys-Gly-Asp (KGD) sequence located within a unique dodecapeptide domain on the γ chains of fibrinogen. Once bound, fibrinogen and/or VWF bridge adjacent platelets together to induce platelet aggregation. Although abciximab, eptifibatide, and tirofiban all target the Gp IIb/ IIIa receptor, they are structurally and pharmacologically distinct (Table 143-1). Abciximab is a Fab fragment of a humanized murine monoclonal antibody directed against the activated form of Gp IIb/IIIa. Abciximab binds to the activated receptor with high affinity and blocks the binding of adhesive molecules. In contrast, eptifibatide and tirofiban are synthetic small molecules. Eptifibatide is a

1	Abciximab binds to the activated receptor with high affinity and blocks the binding of adhesive molecules. In contrast, eptifibatide and tirofiban are synthetic small molecules. Eptifibatide is a cyclic heptapeptide that

1	FIGURE 143-4 Mechanism of action of dipyridamole. Dipyridamole increases levels of cyclic AMP binds Gp IIb/IIIa because it incorpo(cAMP) in platelets by (1) blocking the reuptake of adenosine and (2) inhibiting phosphodiesterase-rates the KGD motif, whereas tirofiban mediated cyclic AMP degradation. By promoting calcium uptake, cyclic AMP reduces intracellular is a nonpeptidic tyrosine derivative that levels of calcium. This, in turn, inhibits platelet activation and aggregation. acts as an RGD mimetic. Abciximab has clopidogrel (3.8% and 4.7%, respec a long half-life and can be detected on the surface of platelets for up to 2 weeks; eptifibatide and tirofiban have short half-lives.

1	Whereas eptifibatide and tirofiban are specific for Gp IIb/IIIa, abciximab also inhibits the closely related αvβ3 receptor, which binds vitronectin, and α β , a leukocyte integrin. Inhibition of α β and α β may endow abciximab with anti-inflammatory and/or antiproliferative properties that extend beyond platelet inhibition. Indications Abciximab and eptifibatide are used in patients undergoing percutaneous coronary interventions, particularly those who have not been pretreated with an ADP receptor antagonist. Tirofiban is used in high-risk patients with unstable angina. Eptifibatide also can be used for this indication.

1	Dosing All of the Gp IIb/IIIa antagonists are given as an IV bolus followed by an infusion. The recommended dose of abciximab is a bolus of 0.25 mg/kg followed by an infusion of 0.125 μg/kg per minute to a maximum of 10 μg/kg for 12 h. Eptifibatide is given as two 180 μg/kg boluses given 10 min apart, followed by an infusion of 2.0 μg/kg per minute for 18–24 h. Tirofiban is started at a rate of 0.4 μg/kg per minute for 30 min; the drug is then continued at a rate of 0.1 μg/kg per minute for up to 18 h. Because these agents are cleared by the kidneys, the doses of eptifibatide and tirofiban must be reduced in patients with renal insufficiency. Thus, the eptifibatide infusion is reduced to 1 μg/kg per minute in patients with a creatinine clearance below 50 mL/min, whereas the dose of tirofiban is cut in half for patients with a creatinine clearance below 30 mL/min.

1	Side Effects In addition to bleeding, thrombocytopenia is the most serious complication. Thrombocytopenia is immune-mediated and is caused by antibodies directed against neoantigens on Gp IIb/IIIa that are exposed upon antagonist binding. With abciximab, thrombocytopenia occurs in up to 5% of patients. Thrombocytopenia is severe in ~1% of these individuals. Thrombocytopenia is less common with the other two agents, occurring in ~1% of patients. New agents in advanced stages of development include cangrelor, a parenteral, rapidly acting, reversible inhibitor of P2Y12, and vorapaxar, an orally active inhibitor of protease-activated receptor 1 (PAR-1), the major thrombin receptor on platelets (Fig. 143-3).

1	Cangrelor An adenosine analogue, cangrelor binds reversibly to P2Y12 and inhibits its activity. The drug has a half-life of 3–6 min and is given IV as a bolus followed by an infusion. When stopped, platelet function recovers within 60 min. A trial comparing cangrelor with placebo during percutaneous coronary interventions and a study comparing cangrelor with clopidogrel after such procedures revealed little or no advantage of cangrelor. A third trial compared cangrelor (given as an IV bolus of 30 μg/kg followed by an infusion of 4 μg/kg per minute for at least 2 h, or for the duration of the procedure, whichever was longer) with a loading dose of clopidogrel (300 or 600 mg) in 11,145 patients undergoing urgent or elective percutaneous coronary intervention. The rate of the primary efficacy endpoint, a composite of death, MI, ischemia-driven revascularization, and stent thrombosis, was 4.7% in the cangrelor group and 5.9% in the clopidogrel group (p = .005). The rates of severe

1	efficacy endpoint, a composite of death, MI, ischemia-driven revascularization, and stent thrombosis, was 4.7% in the cangrelor group and 5.9% in the clopidogrel group (p = .005). The rates of severe bleeding, the primary safety endpoint, were 0.16% and 0.11% in the cangrelor and clopidogrel groups, respectively. Using the same efficacy endpoint, a prespecified meta-analysis of the three trials thrombosis (0.5% and 0.8%, respec-

1	Yes tively) with no significant increase in Long (2.0 h) serious bleeding. Based on these data, Short (s) cangrelor is currently under regula- Yes tory review. antagonist, vorapaxar is slowly eliminated with a half-life of about 200 h. When compared with placebo in 12,944 patients with acute coronary syndrome without ST-segment elevation, vorapaxar failed to significantly reduce the primary efficacy endpoint, a composite of cardiovascular death, MI, stroke, recurrent ischemia requiring rehospitalization, and urgent coronary revascularization. Moreover, vorapaxar was associated with increased rates of bleeding, including intracranial bleeding.

1	In a second trial, vorapaxar was compared with placebo for secondary prevention in 26,449 patients with prior MI, ischemic stroke, or peripheral arterial disease. Overall, vorapaxar reduced the risk for cardiovascular death, MI, or stroke by 13%, but doubled the risk of intracranial bleeding. In the prespecified subgroup of 17,779 patients with prior MI, however, vorapaxar reduced the risk for cardiovascular death, MI, or stroke by 20% compared with placebo (from 9.7% to 8.1%, respectively). The rate of intracranial hemorrhage was higher with vorapaxar than with placebo (0.6% and 0.4%, respectively; p = .076) as was the rate of moderate or severe bleeding (3.4% and 2.1%, respectively; P <0.0001). Based on these data, the drug is under consideration for regulatory approval in MI patients under the age of 75 years who have no history of stroke or transient ischemic attack and have a weight over 60 kg.

1	There are both parenteral and oral anticoagulants. The parenteral anticoagulants include heparin, low-molecular-weight heparin (LMWH), fondaparinux (a synthetic pentasaccharide), lepirudin, desirudin, bivalirudin, and argatroban. Currently available oral anticoagulants include warfarin; dabigatran etexilate, an oral thrombin inhibitor; and rivaroxaban and apixaban, oral factor Xa inhibitors. Edoxaban, a third oral factor Xa inhibitor, is undergoing regulatory review. PARENTERAL ANTICOAGULANTS Heparin A sulfated polysaccharide, heparin is isolated from mammalian tissues rich in mast cells. Most commercial heparin is derived from porcine intestinal mucosa and is a polymer of alternating d-glucuronic acid and N-acetyl-d-glucosamine residues.

1	mecHanism of action Heparin acts as an anticoagulant by activating antithrombin (previously known as antithrombin III) and accelerating the rate at which antithrombin inhibits clotting enzymes, particularly thrombin and factor Xa. Antithrombin, the obligatory plasma cofactor for heparin, is a member of the serine protease inhibitor (serpin) superfamily. Synthesized in the liver and circulating in plasma at a concentration of 2.6 ± 0.4 μM, antithrombin acts as a suicide substrate for its target enzymes.

1	To activate antithrombin, heparin binds to the serpin via a unique pentasaccharide sequence that is found on one-third of the chains of commercial heparin (Fig. 143-5). Heparin chains without this pentasaccharide sequence have little or no anticoagulant activity. Once bound to antithrombin, heparin induces a conformational change in the reactive center loop of antithrombin that renders it more readily accessible to its target proteases. This conformational change enhances the rate at which antithrombin inhibits factor Xa by at least two orders of magnitude but has little effect on the rate of thrombin inhibition. To catalyze thrombin inhibition, heparin serves as a template that binds antithrombin and thrombin simultaneously. Formation of this ternary complex brings the enzyme in close apposition to the inhibitor, thereby

1	Antiplatelet, Anticoagulant, and Fibrinolytic Drugs promoting the formation of a stable covalent thrombin-antithrombin may render patients at risk for recurrent thrombosis, whereas excescomplex. sive anticoagulation increases the risk of bleeding.

1	Only pentasaccharide-containing heparin chains composed of at least 18 saccharide units (which correspond to a molecular weight monitoring tHe anticoagulant effect Heparin therapy can be moniof 5400) are of sufficient length to bridge thrombin and antithrom-tored using the activated partial thromboplastin time (aPTT) or bin together. With a mean molecular weight of 15,000, and a range anti-factor Xa level. Although the aPTT is the test most often used for of 5000–30,000, almost all of the chains of unfractionated heparin this purpose, there are problems with this assay. aPTT reagents vary are long enough to do so. Consequently, by definition, heparin has in their sensitivity to heparin, and the type of coagulometer used for equal capacity to promote the inhibition of thrombin and factor Xa testing can influence the results. Consequently, laboratories must by antithrombin and is assigned an anti-factor Xa to anti-factor IIa establish a therapeutic aPTT range with each

1	and factor Xa testing can influence the results. Consequently, laboratories must by antithrombin and is assigned an anti-factor Xa to anti-factor IIa establish a therapeutic aPTT range with each reagent-coagulometer (thrombin) ratio of 1:1. combination by measuring the aPTT and anti-factor Xa level in plasma

1	Heparin causes the release of tissue factor pathway inhibitor (TFPI) samples collected from heparin-treated patients. For most of the aPTT from the endothelium. A factor Xa–dependent inhibitor of tissue reagents and coagulometers in current use, therapeutic heparin levels factor–bound factor VIIa, TFPI may contribute to the antithrombotic are achieved with a twoto threefold prolongation of the aPTT. activity of heparin. Longer heparin chains induce the release of more Anti-factor Xa levels also can be used to monitor heparin therapy. TFPI than shorter ones. With this test, therapeutic heparin levels range from 0.3 to 0.7 units/ mL. Although this test is gaining in popularity, anti-factor Xa assays pHarmacology Heparin must be given parenterally. It is usually have yet to be standardized, and results can vary widely between administered SC or by continuous IV infusion. When used for thera-laboratories. peutic purposes, the IV route is most often employed. If heparin is Up to 25% of

1	and results can vary widely between administered SC or by continuous IV infusion. When used for thera-laboratories. peutic purposes, the IV route is most often employed. If heparin is Up to 25% of heparin-treated patients with venous thromboemgiven SC for treatment of thrombosis, the dose of heparin must be bolism require >35,000 units/d to achieve a therapeutic aPTT. These patients are considered heparin resistant. It is useful to measure anti-factor Xa levels in heparin-resistant patients because many will have a therapeutic anti-factor Xa level despite a subtherapeutic aPTT. This dissociation in test results occurs because elevated plasma levels of fibrinogen and factor VIII, both of which are acute-phase proteins, shorten the aPTT but have no effect on anti-factor Xa levels. Heparin therapy in patients who exhibit this phenomenon is best monitored using anti-factor Xa levels instead of the aPTT. Patients with congenital or acquired antithrombin deficiency and those with elevated

1	therapy in patients who exhibit this phenomenon is best monitored using anti-factor Xa levels instead of the aPTT. Patients with congenital or acquired antithrombin deficiency and those with elevated levels of heparin-binding proteins may also need high doses of heparin to achieve a therapeutic aPTT or anti-factor Xa level. If there is good correlation between the aPTT and the anti-factor Xa levels, either test can be used to monitor heparin therapy.

1	dosing For prophylaxis, heparin is usually given in fixed doses of 5000 units SC two or three times daily. With these low doses, coagulation monitoring is unnecessary. In contrast, monitoring is essential when the drug is given in therapeutic doses. Fixed-dose or weight-based heparin nomograms are used to standardize heparin dosing and to shorten the time required to achieve a therapeutic anticoagulant response. At least two heparin nomograms have been validated in patients with venous thromboembolism and reduce the time required to achieve a therapeutic aPTT. Weight-adjusted heparin nomograms have also been evaluated in patients with acute coronary syndromes. After an IV heparin bolus of 5000 units or 70 units/kg, a heparin infusion rate of 12–15 units/kg per hour is usually administered. In contrast, weight-adjusted heparin nomograms for patients with venous thromboembolism use an initial bolus of 5000 units or 80 units/kg, followed by an infusion of 18 units/kg per h. Thus,

1	In contrast, weight-adjusted heparin nomograms for patients with venous thromboembolism use an initial bolus of 5000 units or 80 units/kg, followed by an infusion of 18 units/kg per h. Thus, patients with venous thromboembolism appear to require higher doses of heparin to achieve a therapeutic aPTT than do patients with acute coronary syndromes. This may reflect differences in the thrombus burden. Heparin binds to fibrin, and the amount of fibrin in patients with extensive DVT is greater than that in those with coronary thrombosis.

1	Heparin manufacturers in North America have traditionally measured heparin potency in USP units, with a unit defined as the concentration of heparin that prevents 1 mL of citrated sheep plasma from clotting for 1 h after calcium addition. In contrast, manufacturers in Europe measure heparin potency with anti-Xa assays using an international heparin standard for comparison. Because of problems with heparin contamination with oversulfated chondroitin sulfate, which the USP assay system does not detect, North American heparin manufacturers now use the anti-Xa assay to assess heparin potency. The use of international units in place of USP units results in a 10% reduction in heparin doses, which is a difference unlikely to affect patient care because monitoring will help to ensure that a therapeutic anticoagulant response has been achieved.

1	limitations Heparin has pharmacokinetic and biophysical limitations (Table 143-2). The pharmacokinetic limitations reflect heparin’s propensity to bind in a pentasaccharide-independent fashion to cells and plasma proteins. Heparin binding to endothelial cells explains its dose-dependent clearance, whereas binding to plasma proteins results in a variable anticoagulant response and can lead to heparin resistance. Poor bioavailability at low doses Binds to endothelial cells and macrophages Dose-dependent clearance Binds to macrophages Variable anticoagulant response Binds to plasma proteins whose levels vary from patient to patient Reduced activity in the vicinity of Neutralized by platelet factor 4 platelet-rich thrombi released from activated platelets

1	Reduced activity in the vicinity of Neutralized by platelet factor 4 platelet-rich thrombi released from activated platelets Limited activity against factor Xa Reduced capacity of heparin-incorporated in the prothrombinase antithrombin complex to inhibit complex and thrombin bound to factor Xa bound to activated platelets fibrin and thrombin bound to fibrin

1	The biophysical limitations of heparin reflect the inability of the 751 heparin-antithrombin complex to inhibit factor Xa when it is incorporated into the prothrombinase complex, the complex that converts prothrombin to thrombin, and to inhibit thrombin bound to fibrin. Consequently, factor Xa bound to activated platelets within platelet-rich thrombi has the potential to generate thrombin, even in the face of heparin. Once this thrombin binds to fibrin, it too is protected from inhibition by the heparin-antithrombin complex. Clot-associated thrombin can then trigger thrombus growth by locally activating platelets and amplifying its own generation through feedback activation of factors V, VIII, and XI. Further compounding the problem is the potential for heparin neutralization by the high concentrations of PF4 released from activated platelets within the platelet-rich thrombus.

1	side effects The most common side effect of heparin is bleeding. Other complications include thrombocytopenia, osteoporosis, and elevated levels of transaminases. bleeding The risk of bleeding rises as the dose of heparin is increased. Concomitant administration of drugs that affect hemostasis, such as antiplatelet or fibrinolytic agents, increases the risk of bleeding, as does recent surgery or trauma. Heparin-treated patients with serious bleeding can be given protamine sulfate to neutralize the heparin. Protamine sulfate, a mixture of basic polypeptides isolated from salmon sperm, binds heparin with high affinity, and the resultant protamine-heparin complexes are then cleared. Typically, 1 mg of protamine sulfate neutralizes 100 units of heparin. Protamine sulfate is given IV. Anaphylactoid reactions to protamine sulfate can occur, and drug administration by slow IV infusion is recommended to reduce the risk.

1	tHrombocytopenia Heparin can cause thrombocytopenia. Heparin-induced thrombocytopenia (HIT) is an antibody-mediated process that is triggered by antibodies directed against neoantigens on PF4 that are exposed when heparin binds to this protein. These antibodies, which are usually of the IgG isotype, bind simultaneously to the heparin-PF4 complex and to platelet Fc receptors. Such binding activates the platelets and generates platelet microparticles. Circulating microparticles are prothrombotic because they express anionic phospholipids on their surface and can bind clotting factors and promote thrombin generation.

1	The clinical features of HIT are illustrated in Table 143-3. Typically, HIT occurs 5–14 days after initiation of heparin therapy, but it can manifest earlier if the patient has received heparin within the past 3 months. A platelet count below 100,000/μL or a 50% decrease in the platelet count from the pretreatment value should raise the suspicion of HIT in those receiving heparin. HIT is more common in surgical patients than in medical patients and, like many autoimmune disorders, occurs more frequently in females than in males. HIT can be associated with thrombosis, either arterial or venous. Venous thrombosis, which manifests as DVT and/or PE, is more common than arterial thrombosis. Arterial thrombosis can manifest as ischemic stroke or acute MI. Rarely, platelet-rich thrombi in the distal aorta or iliac arteries can cause critical limb ischemia. Thrombocytopenia Platelet count of ≤100,000/μL or a decrease in platelet count of ≥50%

1	Thrombocytopenia Platelet count of ≤100,000/μL or a decrease in platelet count of ≥50% Type of heparin More common with unfractionated heparin than low-molecular-weight heparin Type of patient More common in surgical patients and patients with cancer than general medical patients; more common in women than in men Antiplatelet, Anticoagulant, and Fibrinolytic Drugs Stop all heparin. Give an alternative anticoagulant, such as lepirudin, argatroban, bivalirudin, or fondaparinux. Do not give platelet transfusions. Do not give warfarin until the platelet count returns to its baseline level. If warfarin was administered, give vitamin K to restore the INR to normal. Evaluate for thrombosis, particularly deep vein thrombosis. Abbreviation: INR, international normalized ratio.

1	Evaluate for thrombosis, particularly deep vein thrombosis. Abbreviation: INR, international normalized ratio. The diagnosis of HIT is established using enzyme-linked assays to detect antibodies against heparin-PF4 complexes or with platelet activation assays. Enzyme-linked assays are sensitive but can be positive in the absence of any clinical evidence of HIT. The most specific diagnostic test is the serotonin release assay. This test is performed by quantifying serotonin release when washed platelets loaded with labeled serotonin are exposed to patient serum in the absence or presence of varying concentrations of heparin. If the patient serum contains the HIT antibody, heparin addition induces platelet activation and serotonin release.

1	Management of HIT is outlined in Table 143-4. Heparin should be stopped in patients with suspected or documented HIT, and an alternative anticoagulant should be administered to prevent or treat thrombosis. The agents most often used for this indication are parenteral direct thrombin inhibitors, such as lepirudin, argatroban, or bivalirudin, or factor Xa inhibitors, such as fondaparinux.

1	Patients with HIT, particularly those with associated thrombosis, often have evidence of increased thrombin generation that can lead to consumption of protein C. If these patients are given warfarin without a concomitant parenteral anticoagulant to inhibit thrombin or thrombin generation, the further decrease in protein C levels induced by the vitamin K antagonist can trigger skin necrosis. To avoid this problem, patients with HIT should be treated with a direct thrombin inhibitor or fondaparinux until the platelet count returns to normal levels. At this point, low-dose warfarin therapy can be introduced, and the thrombin inhibitor can be discontinued when the anticoagulant response to warfarin has been therapeutic for at least 2 days.

1	osteoporosis Treatment with therapeutic doses of heparin for >1 month can cause a reduction in bone density. This complication has been reported in up to 30% of patients given long-term heparin therapy, and symptomatic vertebral fractures occur in 2–3% of these individuals. Heparin causes bone loss both by decreasing bone formation and by enhancing bone resorption. Thus, heparin affects the activity of both osteoblasts and osteoclasts. elevated levels of transaminases Therapeutic doses of heparin are frequently associated with modest elevations in the serum levels of hepatic transaminases without a concomitant increase in the level of bilirubin. The levels of transaminases rapidly return to normal when the drug is stopped. The mechanism responsible for this phenomenon is unknown.

1	Low-Molecular-Weight Heparin Consisting of smaller fragments of heparin, LMWH is prepared from unfractionated heparin by controlled enzymatic or chemical depolymerization. The mean molecular weight of LMWH is about 5000, one-third the mean molecular weight of unfractionated heparin. LMWH has advantages over heparin (Table 143-5) and has replaced heparin for most indications. mecHanism of action Like heparin, LMWH exerts its anticoagulant activity by activating antithrombin. With a mean molecular weight of 5000, which corresponds to about 17 saccharide units, at least half of the pentasaccharide-containing chains of LMWH are too short to bridge thrombin to antithrombin (Fig. 143-5). However, these chains retain the capacity to accelerate factor Xa inhibition by antithrombin because this activity is largely the result of the conformational changes in antithrombin evoked by pentasaccharide binding. Consequently, LMWH catalyzes factor Xa inhibition by antithrombin more than

1	Predictable anticoagulant response Coagulation monitoring is unnecessary in most patients Lower risk of heparin-induced throm-Safer than heparin for shortor longbocytopenia term administration Lower risk of osteoporosis Safer than heparin for extended administration Abbreviation: LMWH, low-molecular-weight heparin. thrombin inhibition. Depending on their unique molecular weight distributions, LMWH preparations have anti-factor Xa to anti-factor IIa ratios ranging from 2:1 to 4:1.

1	thrombin inhibition. Depending on their unique molecular weight distributions, LMWH preparations have anti-factor Xa to anti-factor IIa ratios ranging from 2:1 to 4:1. pHarmacology Although usually given SC, LMWH also can be administered IV if a rapid anticoagulant response is needed. LMWH has pharmacokinetic advantages over heparin. These advantages reflect the fact that shorter heparin chains bind less avidly to endothelial cells, macrophages, and heparin-binding plasma proteins. Reduced binding to endothelial cells and macrophages eliminates the rapid, dose-dependent, and saturable mechanism of clearance that is a characteristic of unfractionated heparin. Instead, the clearance of LMWH is dose-independent and its plasma half-life is longer. Based on measurement of anti-factor Xa levels, LMWH has a plasma half-life of ~4 h. LMWH is cleared almost exclusively by the kidneys, and the drug can accumulate in patients with renal insufficiency.

1	LMWH exhibits about 90% bioavailability after SC injection. Because LMWH binds less avidly to heparin-binding proteins in plasma than heparin, LMWH produces a more predictable dose response, and resistance to LMWH is rare. With a longer half-life and more predictable anticoagulant response, LMWH can be given SC once or twice daily without coagulation monitoring, even when the drug is given in treatment doses. These properties render LMWH more convenient than unfractionated heparin. Capitalizing on this feature, studies in patients with venous thromboembolism have shown that home treatment with LMWH is as effective and safe as in-hospital treatment with continuous IV infusions of heparin. Outpatient treatment with LMWH streamlines care, reduces health care costs, and increases patient satisfaction.

1	monitoring In the majority of patients, LMWH does not require coagulation monitoring. If monitoring is necessary, anti-factor Xa levels must be measured because most LMWH preparations have little effect on the aPTT. Therapeutic anti-factor Xa levels with LMWH range from 0.5 to 1.2 units/mL when measured 3–4 h after drug administration. When LMWH is given in prophylactic doses, peak anti-factor Xa levels of 0.2–0.5 units/mL are desirable.

1	Indications for LMWH monitoring include renal insufficiency and obesity. LMWH monitoring in patients with a creatinine clearance of ≤50 mL/min is advisable to ensure that there is no drug accumulation. Although weight-adjusted LMWH dosing appears to produce therapeutic anti-factor Xa levels in patients who are overweight, this approach has not been extensively evaluated in those with morbid obesity. It may also be advisable to monitor the anticoagulant activity of LMWH during pregnancy because dose requirements can change, particularly in the third trimester. Monitoring should also be considered in high-risk settings, such as in patients with mechanical heart valves who are given LMWH for prevention of valve thrombosis, and when LMWH is used in treatment doses in infants or children.

1	dosing The doses of LMWH recommended for prophylaxis or treatment vary depending on the LMWH preparation. For prophylaxis, once-daily SC doses of 4000–5000 units are often used, whereas doses of 2500–3000 units are given when the drug is administered twice daily. For treatment of venous thromboembolism, a dose of 150–200 units/kg is given if the drug is administered once daily. If a twice-daily regimen is used, a dose of 100 units/kg is given. In patients with unstable angina, LMWH is given SC on a twice-daily basis at a dose of 100–120 units/kg. side effects The major complication of LMWH is bleeding. Meta-analyses suggest that the risk of major bleeding is lower with LMWH than with unfractionated heparin. HIT and osteoporosis are less common with LMWH than with unfractionated heparin.

1	bleeding Like the situation with heparin, bleeding with LMWH is more common in patients receiving concomitant therapy with anti-platelet or fibrinolytic drugs. Recent surgery, trauma, or underlying hemostatic defects also increase the risk of bleeding with LMWH. Although protamine sulfate can be used as an antidote for LMWH, protamine sulfate incompletely neutralizes the anticoagulant activity of LMWH because it only binds the longer chains of LMWH. Because longer chains are responsible for catalysis of thrombin inhibition by antithrombin, protamine sulfate completely reverses the anti-factor IIa activity of LMWH. In contrast, protamine sulfate only partially reverses the anti-factor Xa activity of LMWH because the shorter pentasaccharide-containing chains of LMWH do not bind to protamine sulfate. Consequently, patients at high risk for bleeding may be more safely treated with continuous IV unfractionated heparin than with SC LMWH.

1	thrombocytopenia The risk of HIT is about fivefold lower with LMWH than with heparin. LMWH binds less avidly to platelets and causes less PF4 release. Furthermore, with lower affinity for PF4 than heparin, LMWH is less likely to induce the conformational changes in PF4 that trigger the formation of HIT antibodies. LMWH should not be used to treat HIT patients because most HIT antibodies exhibit cross-reactivity with LMWH. This in vitro cross-reactivity is not simply a laboratory phenomenon because there are case reports of thrombosis when HIT patients were switched from heparin to LMWH. osteoporosis Because the risk of osteoporosis is lower with LMWH than with heparin, LMWH is the better choice for extended treatment.

1	osteoporosis Because the risk of osteoporosis is lower with LMWH than with heparin, LMWH is the better choice for extended treatment. Fondaparinux A synthetic analogue of the antithrombin-binding pentasaccharide sequence, fondaparinux differs from LMWH in several ways (Table 143-6). Fondaparinux is licensed for thromboprophylaxis in general medical or surgical patients and in high-risk orthopedic patients and as an alternative to heparin or LMWH for initial treatment of patients with established venous thromboembolism. Although widely used in Europe, as an alternative to heparin or LMWH in patients with acute coronary syndromes, fondaparinux is not licensed for this indication in the United States.

1	mecHanism of action As a synthetic analogue of the antithrombinbinding pentasaccharide sequence found in heparin and LMWH, fondaparinux has a molecular weight of 1728. Fondaparinux binds only to antithrombin (Fig. 143-5) and is too short to bridge thrombin to antithrombin. Consequently, fondaparinux catalyzes factor Xa inhibition by antithrombin and does not enhance the rate of thrombin inhibition. pHarmacology Fondaparinux exhibits complete bioavailability after 753 SC injection. With no binding to endothelial cells or plasma proteins, the clearance of fondaparinux is dose independent and its plasma half-life is 17 h. The drug is given SC once daily. Because fondaparinux is cleared unchanged via the kidneys, it is contraindicated in patients with a creatinine clearance <30 mL/min and should be used with caution in those with a creatinine clearance <50 mL/min.

1	Fondaparinux produces a predictable anticoagulant response after administration in fixed doses because it does not bind to plasma proteins. The drug is given at a dose of 2.5 mg once daily for prevention of venous thromboembolism. For initial treatment of established venous thromboembolism, fondaparinux is given at a dose of 7.5 mg once daily. The dose can be reduced to 5 mg once daily for those weighing <50 kg and increased to 10 mg for those >100 kg. When given in these doses, fondaparinux is as effective as heparin or LMWH for initial treatment of patients with DVT or PE and produces similar rates of bleeding.

1	Fondaparinux is used at a dose of 2.5 mg once daily in patients with acute coronary syndromes. When this prophylactic dose of fondaparinux was compared with treatment doses of enoxaparin in patients with non-ST-segment elevation acute coronary syndrome, there was no difference in the rate of cardiovascular death, MI, or stroke at 9 days. However, the rate of major bleeding was 50% lower with fondaparinux than with enoxaparin, a difference that likely reflects the fact that the dose of fondaparinux was lower than that of enoxaparin. In acute coronary syndrome patients who require percutaneous coronary intervention, there is a risk of catheter thrombosis with fondaparinux unless adjunctive heparin is given.

1	side effects Fondaparinux does not cause HIT because it does not bind to PF4. In contrast to LMWH, there is no cross-reactivity of fondaparinux with HIT antibodies. Consequently, fondaparinux appears to be effective for treatment of HIT patients, although large clinical trials supporting its use are lacking. The major side effect of fondaparinux is bleeding. There is no antidote for fondaparinux. Protamine sulfate has no effect on the anticoagulant activity of fondaparinux because it fails to bind to the drug. Recombinant activated factor VII reverses the anticoagulant effects of fondaparinux in volunteers, but it is unknown whether this agent controls fondaparinux-induced bleeding.

1	Parenteral Direct Thrombin Inhibitors Direct thrombin inhibitors bind directly to thrombin and block its interaction with its substrates. Approved parenteral direct thrombin inhibitors include recombinant hirudins (lepirudin and desirudin), argatroban, and bivalirudin (Table 143-7). Lepirudin and argatroban are licensed for treatment of patients with HIT, desirudin is licensed for thromboprophylaxis after elective hip arthroplasty, and bivalirudin is approved as an alternative to heparin in patients undergoing percutaneous coronary intervention, including those with HIT. lepirudin and desirudin Recombinant forms of hirudin, lepirudin, and desirudin are bivalent direct thrombin inhibitors that interact with the active site and exosite 1, the substrate-binding site on thrombin. For rapid anticoagulation, lepirudin is given by continuous IV infusion, but the drug can be given SC. Lepirudin has a plasma half-life of 60 min after IV infusion and is cleared by the kidneys. Consequently,

1	TABLE 143-7 COMPArIsON Of THE PrOPErTIEs Of LEPIruDIN, BIVALIruDIN, AND ArgATrOBAN

1	Antiplatelet, Anticoagulant, and Fibrinolytic Drugs 754 lepirudin accumulates in patients with renal insufficiency. For thromboprophylaxis, desirudin is given SC twice daily in fixed doses; the half-life of desirudin is 2–3 h after SC injection. A high proportion of lepirudin-treated patients develop antibodies against the drug; antibody formation is rare with SC desirudin. Although lepirudin-directed antibodies rarely cause problems, in a small subset of patients, they can delay lepirudin clearance and enhance its anticoagulant activity. Serious bleeding has been reported in some of these patients. Lepirudin is usually monitored using the aPTT, and the dose is adjusted to maintain an aPTT that is 1.5–2.5 times the control. The aPTT is not an ideal test for monitoring lepirudin therapy because the clotting time plateaus with higher drug concentrations. Although the clotting time with ecarin, a snake venom that converts prothrombin to meizothrombin, provides a better index of lepirudin

1	the clotting time plateaus with higher drug concentrations. Although the clotting time with ecarin, a snake venom that converts prothrombin to meizothrombin, provides a better index of lepirudin dose than the aPTT, the ecarin clotting time has yet to be standardized. When used for thromboprophylaxis, desirudin does not require monitoring.

1	argatroban A univalent inhibitor that targets the active site of thrombin, argatroban is metabolized in the liver. Consequently, this drug must be used with caution in patients with hepatic insufficiency. Argatroban is not cleared via the kidneys, so this drug is safer than lepirudin for HIT patients with renal insufficiency. Argatroban is administered by continuous IV infusion and has a plasma half-life of ~45 min. The aPTT is used to monitor its anticoagulant effect, and the dose is adjusted to achieve an aPTT 1.5–3 times the baseline value, but not to exceed 100 s. Argatroban also prolongs the international normalized ratio (INR), a feature that can complicate the transitioning of patients to warfarin. This problem can be circumvented by using the levels of factor X to monitor warfarin in place of the INR. Alternatively, argatroban can be stopped for 2–3 h before INR determination.

1	bivalirudin A synthetic 20-amino-acid analogue of hirudin, bivalirudin is a divalent thrombin inhibitor. Thus, the N-terminus of bivalirudin interacts with the active site of thrombin, whereas its C-terminus binds to exosite 1. Bivalirudin has a plasma half-life of 25 min, the shortest half-life of all the parenteral direct thrombin inhibitors. Bivalirudin is degraded by peptidases and is partially excreted via the kidneys. When given in high doses in the cardiac catheterization laboratory, the anticoagulant activity of bivalirudin is monitored using the activated clotting time. With lower doses, its activity can be assessed using the aPTT. Bivalirudin is licensed as an alternative to heparin in patients undergoing percutaneous coronary intervention. Bivalirudin also has been used successfully in HIT patients who require percutaneous coronary intervention or cardiac bypass surgery.

1	Current oral anticoagulant practice dates back almost 60 years to when the vitamin K antagonists were discovered as a result of investigations into the cause of hemorrhagic disease in cattle. Characterized by a decrease in prothrombin levels, this disorder is caused by ingestion of hay containing spoiled sweet clover. Hydroxycoumarin, which was isolated from bacterial contaminants in the hay, interferes with vitamin K metabolism, thereby causing a syndrome similar to vitamin K deficiency. Discovery of this compound provided the impetus for development of other vitamin K antagonists, including warfarin. For many years, the vitamin K antagonists were the only available oral anticoagulants. This situation changed with the introduction of new oral anticoagulants, including dabigatran, which targets thrombin, and rivaroxaban, apixaban, and edoxaban, which target factor Xa.

1	Warfarin A water-soluble vitamin K antagonist initially developed as a rodenticide, warfarin is the coumarin derivative most often prescribed in North America. Like other vitamin K antagonists, warfarin interferes with the synthesis of the vitamin K–dependent clotting proteins, which include prothrombin (factor II) and factors VII, IX, and X. The synthesis of the vitamin K–dependent anticoagulant proteins, proteins C and S, is also reduced by vitamin K antagonists.

1	mecHanism of action All of the vitamin K–dependent clotting factors possess glutamic acid residues at their N termini. A posttranslational modification adds a carboxyl group to the γ-carbon of these residues to generate γ-carboxyglutamic acid. This modification is essential for expression of the activity of these clotting factors because it permits their calcium-dependent binding to negatively charged phospholipid surfaces. The γ-carboxylation process is catalyzed by a vitamin K– dependent carboxylase. Thus, vitamin K from the diet is reduced to vitamin K hydroquinone by vitamin K reductase (Fig. 143-6). Vitamin K hydroquinone serves as a cofactor for the carboxylase enzyme, which in the presence of carbon dioxide replaces the hydrogen on the γ-carbon of glutamic acid residues with a carboxyl group. During this process, vitamin K hydroquinone is oxidized to vitamin K epoxide, which is then reduced to vitamin K by vitamin K epoxide reductase.

1	Warfarin inhibits vitamin K epoxide reductase (VKOR), thereby blocking the γ-carboxylation process. This results in the synthesis of vitamin K–dependent clotting proteins that are only partially γ-carboxylated. Warfarin acts as an anticoagulant because these partially γ-carboxylated proteins have reduced or absent biologic activity. The onset of action of warfarin is delayed until the newly synthesized clotting factors with reduced activity gradually replace their fully active counterparts. The antithrombotic effect of warfarin depends on a reduction in the functional levels of factor X and prothrombin, clotting factors that have half-lives of 24 and 72 h, respectively. Because the antithrombotic effect of warfarin is delayed, patients with established thrombosis or at high risk for thrombosis require concomitant treatment with a rapidly acting parenteral anticoagulant, such as heparin, LMWH, or fondaparinux, for at least 5 days.

1	pHarmacology Warfarin is a racemic mixture of R and S isomers. Warfarin is rapidly and almost completely absorbed from the FIGURE 143-6 Mechanism of action of warfarin. A racemic mixture of Sand R-enantiomers, S-warfarin is most active. By blocking vitamin K epoxide reductase, warfarin inhibits the conversion of oxidized vitamin K into its reduced form. This inhibits vitamin K–dependent γ-carboxylation of factors II, VII, IX, and X because reduced vitamin K serves as a cofactor for a γ-glutamyl carboxylase that catalyzes the γ-carboxylation process, thereby converting prozymogens to zymogens capable of binding calcium and interacting with anionic phospholipid surfaces. S-warfarin is metabolized by CYP2C9. Common genetic polymorphisms in this enzyme can influence warfarin metabolism. Polymorphisms in the C1 subunit of vitamin K reductase (VKORC1) also can affect the susceptibility of the enzyme to warfarininduced inhibition, thereby influencing warfarin dosage requirements.

1	tion. Thromboplastins vary in their sensitivity to TABLE 143-8 frEquENCIEs Of CYP2C9gENOTYPEs AND VKORC1 HAPLOTYPEs IN DIffErENT POPuLATIONs AND THEIr EffECT ON wArfArIN DOsE rEquIrEMENTs monitoring Warfarin therapy is most often moni-755 tored using the prothrombin time, a test that is sensitive to reductions in the levels of prothrombin, Frequency, % factor VII, and factor X. The test is performed by adding thromboplastin, a reagent that contains tis- Compared with sue factor, phospholipid, and calcium, to citrated plasma and determining the time to clot forma gastrointestinal tract. Levels of warfarin in the blood peak about 90 min after drug administration. Racemic warfarin has a plasma half-life of 36–42 h, and more than 97% of circulating warfarin is bound to albumin. Only the small fraction of unbound warfarin is biologically active.

1	Warfarin accumulates in the liver where the two isomers are metabolized via distinct pathways. CYP2C9 mediates oxidative metabolism of the more active S isomer (Fig. 143-6). Two relatively common variants, CYP2C9*2 and CYP2C9*3, encode an enzyme with reduced activity. Patients with these variants require lower maintenance doses of warfarin. Approximately 25% of Caucasians have at least one variant allele of CYP2C9*2 or CYP2C9*3, whereas those variant alleles are less common in African Americans and Asians (Table 143-8). Heterozygosity for CYP2C9*2 or CYP2C9*3 decreases the warfarin dose requirement by 20–30% relative to that required in subjects with the wild-type CYP2C9*1/*1 alleles, whereas homozygosity for the CYP2C9*2 or CYP2C9*3 alleles reduces the warfarin dose requirement by 50–70%.

1	Consistent with their decreased warfarin dose requirement, subjects with at least one CYP2C9 variant allele are at increased risk for bleeding. Compared with individuals with no variant alleles, the relative risks for warfarin-associated bleeding in CYP2C9*2 or CYP2C9*3 carriers are 1.9 and 1.8, respectively.

1	Polymorphisms in VKORC1 also can influence the anticoagulant response to warfarin. Several genetic variations of VKORC1 are in strong linkage disequilibrium and have been designated as non-A haplotypes. VKORC1 variants are more prevalent than variants of CYP2C9. Asians have the highest prevalence of VKORC1 variants, followed by Caucasians and African Americans (Table 143-8). Polymorphisms in VKORC1 likely explain 30% of the variability in warfarin dose requirements. Compared with VKORC1 non-A/non-A homozygotes, the warfarin dose requirement decreases by 25 and 50% in A haplotype heterozygotes and homozygotes, respectively. These findings prompted the Food and Drug Administration to amend the prescribing information for warfarin to indicate that lower initiation doses should be considered for patients with CYP2C9 and VKORC1 genetic variants. In addition to genotype data, other pertinent patient information has been incorporated into warfarin dosing algorithms. Although such algorithms

1	for patients with CYP2C9 and VKORC1 genetic variants. In addition to genotype data, other pertinent patient information has been incorporated into warfarin dosing algorithms. Although such algorithms help predict suitable warfarin doses, it remains unclear whether better dose identification improves patient outcome in terms of reducing hemorrhagic complications or recurrent thrombotic events.

1	In addition to genetic factors, the anticoagulant effect of warfarin is influenced by diet, drugs, and various disease states. Fluctuations in dietary vitamin K intake affect the activity of warfarin. A wide variety of drugs can alter absorption, clearance, or metabolism of warfarin. Because of the variability in the anticoagulant response to warfarin, coagulation monitoring is essential to ensure that a therapeutic response is obtained.

1	reductions in the levels of the vitamin K–dependent22 clotting factors. Thus, less sensitive thromboplastins 34 will trigger the administration of higher doses of 43 warfarin to achieve a target prothrombin time. This is problematic because higher doses of warfarin increase the risk of bleeding.76 The INR was developed to circumvent many of the problems associated with the prothrombin time. To calculate the INR, the patient’s prothrombin time is 26 divided by the mean normal prothrombin time, and 50 this ratio is then multiplied by the international sensitivity index (ISI), which is an index of the sensitivity of the thromboplastin used for prothrombin time determination to reductions in the levels of the vitamin K–dependent clotting factors. Highly sensitive thromboplastins have an ISI of 1.0. Most current thromboplastins have ISI values that range from 1.0 to 1.4. Although the INR has helped to standardize anticoagulant practice, problems persist. The precision of INR determination

1	1.0. Most current thromboplastins have ISI values that range from 1.0 to 1.4. Although the INR has helped to standardize anticoagulant practice, problems persist. The precision of INR determination varies depending on reagent-coagulometer combinations. This leads to variability in the INR results. Also complicating INR determination is unreliable reporting of the ISI by thromboplastin manufacturers. Furthermore, every laboratory must establish the mean normal prothrombin time with each new batch of thromboplastin reagent. To accomplish this, the prothrombin time must be measured in fresh plasma samples from at least 20 healthy volunteers using the same coagulometer that is used for patient samples. For most indications, warfarin is administered in doses that produce a target INR of 2.0–3.0. An exception is patients with mechanical heart valves, particularly those in the mitral position or older ball and cage valves in the aortic position, where a target INR of 2.5–3.5 is recommended.

1	An exception is patients with mechanical heart valves, particularly those in the mitral position or older ball and cage valves in the aortic position, where a target INR of 2.5–3.5 is recommended. Studies in atrial fibrillation demonstrate an increased risk of cardioembolic stroke when the INR falls to <1.7 and an increase in bleeding with INR values >4.5. These findings highlight the fact that vitamin K antagonists have a narrow therapeutic window. In support of this concept, a study in patients receiving long-term warfarin therapy for unprovoked venous thromboembolism demonstrated a higher rate of recurrent venous thromboembolism with a target INR of 1.5–1.9 compared with a target INR of 2.0–3.0.

1	dosing Warfarin is usually started at a dose of 5–10 mg. Lower doses are used for patients with CYP2C9 or VKORC1 polymorphisms, which affect the pharmacodynamics or pharmacokinetics of warfarin and render patients more sensitive to the drug. The dose is then titrated to achieve the desired target INR. Because of its delayed onset of action, patients with established thrombosis or those at high risk for thrombosis are given concomitant initial treatment with a rapidly acting parenteral anticoagulant, such as heparin, LMWH, or fondaparinux. Early prolongation of the INR reflects reduction in the functional levels of factor VII. Consequently, concomitant treatment with the parenteral anticoagulant should be continued until the INR has been therapeutic for at least 2 consecutive days. A minimum 5-day course of parenteral anticoagulation is recommended to ensure that the levels of factor Xa and prothrombin have been reduced into the therapeutic range with warfarin.

1	Because warfarin has a narrow therapeutic window, frequent coagulation monitoring is essential to ensure that a therapeutic anticoagulant response is maintained. Even patients with stable warfarin dose requirements should have their INR determined every 3–4 weeks. More frequent monitoring is necessary when new medications are introduced because so many drugs enhance or reduce the anticoagulant effects of warfarin.

1	Antiplatelet, Anticoagulant, and Fibrinolytic Drugs 756 side effects Like all anticoagulants, the major side effect of warfarin trimester of pregnancy. Central nervous system abnormalities can also is bleeding. A rare complication is skin necrosis. Warfarin crosses the occur with exposure to warfarin at any time during pregnancy. Finally, placenta and can cause fetal abnormalities. Consequently, warfarin maternal administration of warfarin produces an anticoagulant effect should not be used during pregnancy. in the fetus that can cause bleeding. This is of particular concern at delivery when trauma to the head during passage through the birth bleeding At least half of the bleeding complications with warfarin canal can lead to intracranial bleeding. Because of these potential prob-occur when the INR exceeds the therapeutic range. Bleeding compli-lems, warfarin is contraindicated in pregnancy, particularly in the firstcations may be mild, such as epistaxis or hematuria, or more severe,

1	the INR exceeds the therapeutic range. Bleeding compli-lems, warfarin is contraindicated in pregnancy, particularly in the firstcations may be mild, such as epistaxis or hematuria, or more severe, and third trimesters. Instead, heparin, LMWH, or fondaparinux can such as retroperitoneal or gastrointestinal bleeding. Life-threatening be given during pregnancy for prevention or treatment of thrombosis.intracranial bleeding can also occur. Warfarin does not pass into the breast milk. Consequently, warfarin

1	To minimize the risk of bleeding, the INR should be maintained can safely be given to nursing mothers.in the therapeutic range. In asymptomatic patients whose INR is special problems Patients with a lupus anticoagulant and those whobetween 3.5 and 10, warfarin should be withheld until the INR returns need urgent or elective surgery present special challenges. Although to the therapeutic range. If the INR is over 10, oral vitamin K should observational studies suggested that patients with thrombosis com-be administered, at a dose of 2.5–5 mg, although there is no evidence plicating the antiphospholipid antibody syndrome required higherthat doing so reduces the bleeding risk. Higher doses of oral vitamin intensity warfarin regimens to prevent recurrent thromboembolicK (5–10 mg) produce more rapid reversal of the INR but may render events, two randomized trials showed that targeting an INR of 2.0–3.0 patients temporarily resistant to warfarin when the drug is restarted.

1	is as effective as higher intensity treatment and produces less bleed-Patients with serious bleeding need more aggressive treatment. These ing. Monitoring warfarin therapy can be problematic in patients with patients should be given 5–10 mg of vitamin K by slow IV infusion. antiphospholipid antibody syndrome if the lupus anticoagulant pro-Additional vitamin K should be given until the INR is in the nor-longs the baseline INR; factor X levels can be used instead of the INRmal range. Treatment with vitamin K should be supplemented with in such patients.

1	fresh-frozen plasma as a source of the vitamin K–dependent clotting There is no need to stop warfarin before procedures associated withproteins. Four factor prothrombin complex concentrates, which con-a low risk of bleeding; these include dental cleaning, simple dentaltain all four vitamin K–dependent clotting proteins, are the treatment extraction, cataract surgery, or skin biopsy. For procedures associatedof choice for (1) life-threatening bleeds, (2) rapid restoration of the with a moderate or high risk of bleeding, warfarin should be stoppedINR into the normal range in patients requiring urgent surgery or 5 days before the procedure to allow the INR to return to normal levels. intervention, and (3) patients who cannot tolerate the volume load of Patients at high risk for thrombosis, such as those with mechanicalfresh-frozen plasma.

1	intervention, and (3) patients who cannot tolerate the volume load of Patients at high risk for thrombosis, such as those with mechanicalfresh-frozen plasma. heart valves, can be bridged with onceor twice-daily SC injections ofWarfarin-treated patients who experience bleeding when their INR LMWH when the INR falls to <2.0. The last dose of LMWH should beis in the therapeutic range require investigation into the cause of the given 12–24 h before the procedure, depending on whether LMWH isbleeding. Those with gastrointestinal or genitourinary bleeding often administered twice or once daily. After the procedure, treatment with have an underlying lesion. warfarin can be restarted. skin necrosis A rare complication of warfarin, skin necrosis usually is

1	warfarin can be restarted. skin necrosis A rare complication of warfarin, skin necrosis usually is New Oral Anticoagulants New oral anticoagulants are now availableseen 2–5 days after initiation of therapy. Well-demarcated erythemaas alternatives to warfarin. These include dabigatran, which targetstous lesions form on the thighs, buttocks, breasts, or toes. Typically, thrombin, and rivaroxaban, apixaban, and edoxaban, which targetthe center of the lesion becomes progressively necrotic. Examination factor Xa. All of these drugs have a rapid onset and offset of action of skin biopsies taken from the border of these lesions reveals thrombi and have half-lives that permit onceor twice-daily administration. in the microvasculature.

1	in the microvasculature. Designed to produce a predictable level of anticoagulation, the newWarfarin-induced skin necrosis is seen in patients with congenital oral agents are more convenient to administer than warfarin becauseor acquired deficiencies of protein C or protein S. Initiation of warfarin they are given in fixed doses without routine coagulation monitoring.

1	therapy in these patients produces a precipitous fall in plasma levels of proteins C or S, thereby eliminating this important anticoagulant mecHanism of action The new oral anticoagulants are small molecules pathway before warfarin exerts an antithrombotic effect through lower-that bind reversibly to the active site of their target enzyme. Table 143-9 ing of the functional levels of factor X and prothrombin. The resultant summarizes the distinct pharmacologic properties of these agents. procoagulant state triggers thrombosis. Why the thrombosis is localized to the microvasculature of fatty tissues is unclear. indications The new oral anticoagulants have been compared with

1	Treatment involves discontinuation of warfarin and reversal with warfarin for stroke prevention in patients with nonvalvular atrial vitamin K, if needed. An alternative anticoagulant, such as heparin or fibrillation in four randomized trials that enrolled 71,683 patients. A LMWH, should be given in patients with thrombosis. Protein C con-meta-analysis of these data demonstrates that compared with warfarin, centrate can be given to protein C–deficient patients to accelerate heal-the new agents significantly reduce stroke or systemic embolism by ing of the skin lesions; fresh-frozen plasma may be of value if protein 19% (p = .001), primarily driven by a 51% reduction in hemorrhagic C concentrate is unavailable and for those with protein S deficiency. stroke (p <.0001), and are associated with a 10% reduction in mortality Occasionally, skin grafting is necessary when there is extensive skin loss. (p <.0001). New oral anticoagulants reduce intracranial hemorrhage by

1	Because of the potential for skin necrosis, patients with known protein C or protein S deficiency require overlapping treatment with a parenteral anticoagulant when initiating war- farin therapy. Warfarin should be started in low doses in these patients, and the parenteral anticoagulant should be continued until the INR is therapeutic for at least 2–3 consecutive days. pregnancy Warfarin crosses the placenta and can cause fetal abnormalities or bleeding. The fetal abnormalities include a characteristic embryopathy, which consists of nasal hypopla-Monitoring No No No No sia and stippled epiphyses. The risk of embry-Interactions 3A4/P-gp 3A4/P-gp P-gp P-gp opathy is highest if warfarin is given in the first Abbreviations: bid, twice a day; P-gp, P-glycoprotein; qd., once a day.

1	52% compared with warfarin (p <.0001), but increase gastrointestinal bleeding by about 24% (p = .04). Overall, the new agents demonstrate a favorable benefit-to-risk profile compared with warfarin, and their relative efficacy and safety are maintained across a wide spectrum of atrial fibrillation patients, including those over the age of 75 years and those with a prior history of stroke. Based on these findings, dabigatran, rivaroxaban, and apixaban are licensed as alternatives to warfarin for stroke prevention in nonvalvular atrial fibrillation, and edoxaban is under regulatory consideration for this indication. Nonvalvular atrial fibrillation is defined as that occurring in patients without mechanical heart valves or severe rheumatic valvular disease, particularly mitral stenosis and/or regurgitation.

1	Dabigatran, rivaroxaban, and apixaban have been compared with enoxaparin for thromboprophylaxis after elective hip or knee arthroplasty. Currently, only rivaroxaban and apixaban are licensed for this indication in the United States. Rivaroxaban and dabigatran are also licensed for treatment of DVT or PE. Apixaban and edoxaban have also been investigated for treatment of patients with venous thromboembolism, but have not yet been approved for this indication. Rivaroxaban is licensed in Europe for prevention of recurrent ischemic events in patients who have been stabilized after an acute coronary syndrome. In this setting, rivaroxaban is usually administered in conjunction with dual antiplatelet therapy with aspirin and clopidogrel.

1	dosing For stroke prevention in patients with nonvalvular atrial fibrillation, rivaroxaban is given at a dose of 20 mg once daily with a dose reduction to 15 mg once daily in patients with a creatinine clearance of 15–49 mL/min; dabigatran is given at a dose of 150 mg twice daily with a dose reduction to 75 mg twice daily in those with a creatinine clearance of 15–30 mL/min; and apixaban is given at a dose of 5 mg twice daily with a dose reduction to 2.5 mg twice daily for patients with a creatinine >1.5 g/dL, for those 80 years of age or older, or for patients who weigh <60 kg.

1	For thromboprophylaxis after elective hip or knee replacement surgery, rivaroxaban is given at a dose of 10 mg once daily, whereas apixaban is given at a dose of 2.5 mg twice daily. For treatment of patients with DVT or PE, rivaroxaban is started at a dose of 15 mg twice daily for 3 weeks; the dose is then reduced to 20 mg once daily thereafter. After a minimum of a 5 day course of treatment with heparin or LMWH, dabigatran is given at a dose of 150 mg twice daily.

1	monitoring Although designed to be administered without routine monitoring, there are situations where determination of the anticoagulant activity of the new oral anticoagulants can be helpful. These include assessment of adherence, detection of accumulation or overdose, identification of bleeding mechanisms, and determination of activity prior to surgery or intervention. For qualitative assessment of anticoagulant activity, the prothrombin time can be used for factor Xa inhibitors and the aPTT for dabigatran. Rivaroxaban and edoxaban prolong the prothrombin time more than apixaban. In fact, because apixaban has such a limited effect on the prothrombin time, anti-factor Xa assays are needed to assess its activity. The effect of the drugs on tests of coagulation varies depending on the time that the blood is drawn relative to the timing of the last dose of the drug and the reagents used to perform the tests. Chromogenic anti-factor Xa assays and a dilute thrombin clotting time with

1	time that the blood is drawn relative to the timing of the last dose of the drug and the reagents used to perform the tests. Chromogenic anti-factor Xa assays and a dilute thrombin clotting time with appropriate calibrators provide quantitative assays to measure the plasma levels of the factor Xa inhibitors and dabigatran, respectively.

1	side effects Like all anticoagulants, bleeding is the most common side effect of the new oral anticoagulants. The new agents are associated with less intracranial bleeding than warfarin. The increased risk of intracranial bleeding with warfarin likely reflects the reduction in functional levels of factor VII, which precludes efficient thrombin generation at sites of microvascular bleeding in the brain. Because the new oral anticoagulants target downstream coagulation enzymes, they produce less impairment of hemostatic plug formation at sites of vascular injury. A downside of the new oral anticoagulants is the increased risk of gastrointestinal bleeding. This likely occurs because unabsorbed active drug in the gut exacerbates bleeding from lesions. Although dabigatran 757 etexilate is a prodrug, only 7% is absorbed. Although the remainder passes through the gut, at least two-thirds is metabolically activated to dabigatran by gut esterases.

1	Dyspepsia occurs in up to 10% of patients treated with dabigatran; this problem improves with time and can be minimized by administering the drug with food. Dyspepsia is rare with rivaroxaban, apixaban, and edoxaban. periprocedural management Like warfarin, the new oral anticoagulants must be stopped before procedures associated with a moderate or high risk of bleeding. The drugs should be held for 1–2 days, or longer if renal function is impaired. Assessment of residual anticoagulant activity before procedures associated with a high bleeding risk is prudent.

1	management of bleeding There are no specific antidotes for the new oral anticoagulants. With minor bleeding, holding one or two doses of drug is usually sufficient. The approach to serious bleeding is similar to that with warfarin except that vitamin K administration is of no benefit. Thus, the anticoagulant and antiplatelet drugs should be held, the patient should be resuscitated with fluids and blood products as necessary, and, if possible, the bleeding site should be identified and managed. Coagulation testing will determine the extent of anticoagulation. and renal function should be assessed so that the half-life of the drug can be calculated. Timing of the last dose of anticoagulant is important; administration of oral activated charcoal may help to prevent absorption of drug administered in the past 2–4 h. If bleeding continues or is life-threatening, procoagulants, such as prothrombin complex concentrate (either unactivated or activated) or factor VIIa, can be administered,

1	in the past 2–4 h. If bleeding continues or is life-threatening, procoagulants, such as prothrombin complex concentrate (either unactivated or activated) or factor VIIa, can be administered, although the evidence of their effectiveness is limited. Dialysis removes dabigatran from the circulation in patients with renal impairment; dialysis does not remove rivaroxaban, apixaban, or edoxaban because unlike dabigatran, these drugs are highly protein-bound.

1	pregnancy As small molecules, the new oral anticoagulants can all pass through the placenta. Consequently, these agents are contraindicated in pregnancy, and when used by women of childbearing potential, appropriate contraception is important. ongoing investigations Although the lack of antidotes has created concern about the risk of bleeding events in patients taking the new oral anticoagulants, emerging postmarketing data suggest that the rates of bleeding in the real-world setting are similar to those reported in the trials. Nonetheless, specific antidotes are under development. These include a humanized mouse monoclonal antibody fragment against dabigatran and a recombinant variant of factor Xa that serves as a decoy for the oral factor Xa inhibitors. Neither agent is currently available for clinical use.

1	Fibrinolytic drugs can be used to degrade thrombi and are administered systemically or can be delivered via catheters directly into the substance of the thrombus. Systemic delivery is used for treatment of acute MI, acute ischemic stroke, and most cases of massive PE. The goal of therapy is to produce rapid thrombus dissolution, thereby restoring antegrade blood flow. In the coronary circulation, restoration of blood flow reduces morbidity and mortality rates by limiting myocardial damage, whereas in the cerebral circulation, rapid thrombus dissolution decreases the neuronal death and brain infarction that produce irreversible brain injury. For patients with massive PE, the goal of thrombolytic therapy is to restore pulmonary artery perfusion.

1	Peripheral arterial thrombi and thrombi in the proximal deep veins of the leg are most often treated using catheter-directed thrombolytic therapy. Catheters with multiple side holes can be used to enhance drug delivery. In some cases, intravascular devices that fragment and extract the thrombus are used to hasten treatment. These devices can be used alone or in conjunction with fibrinolytic drugs. Antiplatelet, Anticoagulant, and Fibrinolytic Drugs FIGURE 143-7 The fibrinolytic system and its regulation. Plasminogen activators convert plasminogen to plasmin. Plasmin then degrades fibrin into soluble fibrin degradation products. The system is regulated at two levels. Type 1 plasminogen activator inhibitor (PAI-1) regulates the plasminogen activators, whereas α2-antiplasmin serves as the major inhibitor of plasmin.

1	Currently approved fibrinolytic agents include streptokinase; acylated plasminogen streptokinase activator complex (anistreplase); urokinase; recombinant tissue-type plasminogen activator (rtPA), which is also known as alteplase or activase; and two recombinant derivatives of rtPA, tenecteplase and reteplase. All of these agents act by converting plasminogen, the zymogen, to plasmin, the active enzyme (Fig. 143-7). Plasmin then degrades the fibrin matrix of thrombi and produces soluble fibrin degradation products.

1	Endogenous fibrinolysis is regulated at two levels. Plasminogen activator inhibitors, particularly the type 1 form (PAI-1), prevent excessive plasminogen activation by regulating the activity of tPA and urokinasetype plasminogen activator (uPA). Once plasmin is generated, it is regulated by plasmin inhibitors, the most important of which is α2antiplasmin. The plasma concentration of plasminogen is twofold higher than that of α2-antiplasmin. Consequently, with pharmacologic doses of plasminogen activators, the concentration of plasmin that is generated can exceed that of α2-antiplasmin. In addition to degrading fibrin, unregulated plasmin can also degrade fibrinogen and other clotting factors. This process, which is known as the systemic lytic state, reduces the hemostatic potential of the blood and increases the risk of bleeding.

1	The endogenous fibrinolytic system is geared to localize plasmin generation to the fibrin surface. Both plasminogen and tPA bind to fibrin to form a ternary complex that promotes efficient plasminogen activation. In contrast to free plasmin, plasmin generated on the fibrin surface is relatively protected from inactivation by α2-antiplasmin, a feature that promotes fibrin dissolution. Furthermore, C-terminus lysine residues, exposed as plasmin degrades fibrin, serve as binding sites for additional plasminogen and tPA molecules. This creates a positive feedback that enhances plasmin generation. When used pharmacologically, the various plasminogen activators capitalize on these mechanisms to a lesser or greater extent.

1	Plasminogen activators that preferentially activate fibrin-bound plasminogen are considered fibrin-specific. In contrast, nonspecific plasminogen activators do not discriminate between fibrin-bound and circulating plasminogen. Activation of circulating plasminogen results in the generation of unopposed plasmin that can trigger the systemic lytic state. Alteplase and its derivatives are fibrin-specific plasminogen activators, whereas streptokinase, anistreplase, and urokinase are nonspecific agents. Unlike other plasminogen activators, streptokinase is not an enzyme and does not directly convert plasminogen to plasmin. Instead, streptokinase forms a 1:1 stoichiometric complex with plasminogen. Formation of this complex induces a conformational change in plasminogen that exposes its active site (Fig. 143-8). The streptokinase-plasminogen complex then converts additional plasminogen to plasmin.

1	Streptokinase has no affinity for fibrin, and the streptokinaseplasminogen complex activates both free and fibrin-bound plasminogen. Activation of circulating plasminogen generates sufficient amounts of plasmin to overwhelm α2-antiplasmin. Unopposed plasmin not only degrades fibrin in the occlusive thrombus but also induces a systemic lytic state. StreptokinasePlasminogenSStreptokinasePlasminogenSFIGURE 143-8 Mechanism of action of streptokinase. Streptokinase binds to plasminogen and induces a conformational change in plas-minogen that exposes its active site. The streptokinase/plasmin(ogen) complex then serves as the activator of additional plasminogen.

1	When given systemically to patients with acute MI, streptokinase reduces mortality. For this indication, the drug is usually given as an IV infusion of 1.5 million units over 30–60 min. Patients who receive streptokinase can develop antibodies against the drug, as can patients with prior streptococcal infection. These antibodies can reduce the effectiveness of streptokinase. Allergic reactions occur in ~5% of patients treated with streptokinase. These may manifest as a rash, fever, chills, and rigors. Although anaphylactic reactions can occur, these are rare. Transient hypotension is common with streptokinase and has been attributed to plasminmediated release of bradykinin from kininogen. The hypotension usually responds to leg elevation and administration of IV fluids and low doses of vasopressors, such as dopamine or norepinephrine.

1	To generate this drug, streptokinase is combined with equimolar amounts of Lys-plasminogen, a plasmin-cleaved form of plasminogen with a Lys residue at its N terminus. The active site of Lys-plasminogen that is exposed upon combination with streptokinase is then masked with an anisoyl group. After IV infusion, the anisoyl group is slowly removed by deacylation, giving the complex a half-life of ~100 min. This allows drug administration via a single bolus infusion. Although it is more convenient to administer, anistreplase offers few mechanistic advantages over streptokinase. Like streptokinase, anistreplase does not distinguish between fibrin-bound and circulating plasminogen. Consequently, it too produces a systemic lytic state. Likewise, allergic reactions and hypotension are just as frequent with anistreplase as they are with streptokinase.

1	When anistreplase was compared with alteplase in patients with acute MI, reperfusion was obtained more rapidly with alteplase than with anistreplase. Improved reperfusion was associated with a trend toward better clinical outcomes and reduced mortality rate with alteplase. These results and the high cost of anistreplase have dampened the enthusiasm for its use. Urokinase is a two-chain serine protease derived from cultured fetal kidney cells with a molecular weight of 34,000. Urokinase converts plasminogen to plasmin directly by cleaving the Arg560-Val561 bond. Unlike streptokinase, urokinase is not immunogenic and allergic reactions are rare. Urokinase produces a systemic lytic state because it does not discriminate between fibrin-bound and circulating plasminogen.

1	Despite many years of use, urokinase has never been systemically evaluated for coronary thrombolysis. Instead, urokinase is often employed for catheter-directed lysis of thrombi in the deep veins or the peripheral arteries. Because of production problems, the availability of urokinase is limited. A recombinant form of single-chain tPA, alteplase has a molecular weight of 68,000. Alteplase is rapidly converted into its two-chain form by plasmin. Although singleand two-chain forms of tPA have equivalent activity in the presence of fibrin, in its absence, single-chain tPA has tenfold lower activity.

1	Alteplase consists of five discrete domains (Fig. 143-9); the N-terminus A chain of two-chain alteplase contains four of these domains. Residues 4 through 50 make up the finger domain, a region that resembles the finger domain of fibronectin; residues 50 through 87 are homologous with epidermal growth factor, whereas residues 92 through 173 and 180 through 261, which have homology to the kringle domains of plasminogen, are designated as the first and second kringle, respectively. The fifth alteplase domain is the protease domain; it is located on the C-terminus B chain of two-chain alteplase.

1	The interaction of alteplase with fibrin is mediated by the finger domain and, to a lesser extent, by the second kringle domain. The affinity of alteplase for fibrin is considerably higher than that for fibrinogen. Consequently, the catalytic efficiency of plasminogen activation by alteplase is two to three orders of magnitude higher in the presence of fibrin than in the presence of fibrinogen. This phenomenon helps to localize plasmin generation to the fibrin surface. Although alteplase preferentially activates plasminogen in the presence of fibrin, alteplase is not as fibrin-selective as was first predicted. Its fibrin specificity is limited because like fibrin, (DD)E, the major

1	FIGURE 143-9 Domain structures of alteplase (tPA), tenecteplase (TNK-tPA), desmoteplase (b-PA), and reteplase (r-PA). The finger (F), epidermal growth factor (EGF), first and second kringles (K1 and K2, respectively), and protease (P) domains are illustrated. The glycosylation site (Y) on K1 has been repositioned in tenecteplase to endow it with a longer half-life. In addition, a tetra-alanine substitution in the protease domain renders tenecteplase resistant to type 1 plasminogen activator inhibitor (PAI-1) inhibition. Desmoteplase differs from alteplase and tenecteplase in that it lacks a K2 domain. Reteplase is a truncated variant that lacks the F, EGF, and K1 domains.

1	soluble degradation product of cross-linked fibrin, binds alteplase and 759 plasminogen with high affinity. Consequently, (DD)E is as potent as fibrin as a stimulator of plasminogen activation by alteplase. Whereas plasmin generated on the fibrin surface results in thrombolysis, plasmin generated on the surface of circulating (DD)E degrades fibrinogen. Fibrinogen degradation results in the accumulation of fragment X, a high-molecular-weight clottable fibrinogen degradation product. Incorporation of fragment X into hemostatic plugs formed at sites of vascular injury renders them susceptible to lysis. This phenomenon may contribute to alteplase-induced bleeding. A trial comparing alteplase with streptokinase for treatment of patients with acute MI demonstrated significantly lower mortality with alteplase than with streptokinase, although the absolute difference was small. The greatest benefit was seen in patients age <75 years with anterior MI who presented <6 h after symptom onset.

1	For treatment of acute MI or acute ischemic stroke, alteplase is given as an IV infusion over 60–90 min. The total dose of alteplase usually ranges from 90 to 100 mg. Allergic reactions and hypotension are rare, and alteplase is not immunogenic. Tenecteplase is a genetically engineered variant of tPA and was designed to have a longer half-life than tPA and to be resistant to inactivation by PAI-1. To prolong its half-life, a new glycosylation site was added to the first kringle domain (Fig. 143-9). Because addition of this extra carbohydrate side chain reduced fibrin affinity, the existing glycosylation site on the first kringle domain was removed. To render the molecule resistant to inhibition by PAI-1, a tetra-alanine substitution was introduced at residues 296–299 in the protease domain, the region responsible for the interaction of tPA with PAI-1.

1	Tenecteplase is more fibrin-specific than tPA. Although both agents bind to fibrin with similar affinity, the affinity of tenecteplase for (DD) E is significantly lower than that of tPA. Consequently, (DD)E does not stimulate systemic plasminogen activation by tenecteplase to the same extent as tPA. As a result, tenecteplase produces less fibrinogen degradation than tPA. For coronary thrombolysis, tenecteplase is given as a single IV bolus. In a large phase III trial that enrolled >16,000 patients, the 30-day mortality rate with single-bolus tenecteplase was similar to that with accelerated-dose tPA. Although rates of intracranial hemorrhage were also similar with both treatments, patients given tenecteplase had fewer noncerebral bleeds and a reduced need for blood transfusions than those treated with tPA. The improved safety profile of tenecteplase likely reflects its enhanced fibrin specificity.

1	Reteplase is a is a single-chain, recombinant tPA derivative that lacks the finger, epidermal growth factor, and first kringle domains (Fig. 143-9). This truncated derivative has a molecular weight of 39,000. Reteplase binds fibrin more weakly than tPA because it lacks the finger domain. Because it is produced in Escherichia coli, reteplase is not glycosylated. This endows it with a plasma half-life longer than that of tPA. Consequently, reteplase is given as two IV boluses, which are separated by 30 min. Clinical trials have demonstrated that reteplase is at least as effective as streptokinase for treatment of acute MI, but the agent is not superior to tPA.

1	Two new drugs are under investigation. These include desmoteplase (Fig. 143-9), a recombinant form of the full-length plasminogen activator isolated from the saliva of the vampire bat, and alfimeprase, a truncated form of fibrolase, an enzyme isolated from the venom of the southern copperhead snake. Clinical studies with these agents have been disappointing. Desmoteplase, which is more fibrin-specific than tPA, was investigated for treatment of acute ischemic stroke. Patients presenting 3–9 h after symptom onset were randomized to one of two doses of desmoteplase or to placebo. Overall response rates were low and no different with desmoteplase than with placebo. The mortality rate was higher in the desmoteplase arms.

1	Antiplatelet, Anticoagulant, and Fibrinolytic Drugs 760 Alfimeprase is a metalloproteinase that degrades fibrin and fibrinogen in a plasmin-independent fashion. In the circulation, alfimeprase is rapidly inhibited by α2-macroglobulin. Consequently, the drug must be delivered via a catheter directly into the thrombus. Studies of alfimeprase for treatment of peripheral arterial occlusion or for restoration of flow in blocked central venous catheters were stopped due to lack of efficacy. The disappointing results with desmoteplase and alfimeprase highlight the challenges of introducing new fibrinolytic drugs.

1	Thrombosis involves a complex interplay among the vessel wall, platelets, the coagulation system, and the fibrinolytic pathways. Activation of coagulation also triggers inflammatory pathways that may exacerbate thrombosis. A better understanding of the biochemistry of blood coagulation and advances in structure-based drug design have identified new targets and resulted in the development of novel antithrombotic drugs. Well-designed clinical trials have provided detailed information on which drugs to use and when to use them. Despite these advances, however, thromboembolic disorders remain a major cause of morbidity and mortality. Therefore, the search for better targets and more potent antiplatelet, anticoagulant, and fibrinolytic drugs continues.

1	Approach to the Patient with an Infectious Disease Neeraj K. Surana, Dennis L. Kasper HISTORICAL PERSPECTIVE The origins of the field of infectious diseases are humble. The notion that communicable diseases were due to a miasma (“bad air”) can be traced back to at least the mid-sixteenth century. Not until the work of Louis Pasteur and Robert Koch in the late nineteenth century was there credible evidence supporting the germ theory of disease—i.e., that microorganisms are the direct cause of infections. In contrast to this relatively slow start, the twentieth century saw remarkable advances in the field of infectious diseases, and the etiologic agents of numerous infectious diseases were soon identified. Furthermore, the discovery of antibiotics and the advent of vaccines against some of the most deadly and debilitating infections greatly altered the landscape of human health. Indeed, the twentieth century saw the elimination of smallpox, one of the great scourges in the history of

1	the most deadly and debilitating infections greatly altered the landscape of human health. Indeed, the twentieth century saw the elimination of smallpox, one of the great scourges in the history of humanity. These remarkable successes prompted noted scholar Aidan Cockburn to write in a 1963 publication entitled The Evolution and Eradication of Infectious Diseases: “It seems reasonable to anticipate that within some measurable time . . . all the major infections will have disappeared.” Professor Cockburn was not alone in this view. Robert Petersdorf, a renowned infectious disease expert and former editor of this textbook, wrote in 1978 that “even with my great personal loyalties to infectious diseases, I cannot conceive a need for 309 more [graduating trainees in infectious diseases] unless they spend their time culturing each other.” Given the enormous growth of interest in the microbiome in the past 5 years, Dr. Petersdorf’s statement might have been ironically clairvoyant, although

1	they spend their time culturing each other.” Given the enormous growth of interest in the microbiome in the past 5 years, Dr. Petersdorf’s statement might have been ironically clairvoyant, although he could have had no idea what was in store for humanity, with an onslaught of new, emerging, and re-emerging infectious diseases. Clearly, even with all the advances of the twentieth century, infec-tious diseases continue to represent a formidable challenge for patients and physicians alike. Furthermore, during the latter half of the century, several chronic diseases were demonstrated to be directly or indirectly caused by infectious microbes; perhaps the most notable examples are the associations of Helicobacter pylori with peptic ulcer disease and gastric carcinoma, human papillomavirus with cervical cancer, and hepatitis B and C viruses with liver cancer. In fact, ~16% of all malignancies are now known to be associated with an infectious cause. In addition, numerous emerging and

1	with cervical cancer, and hepatitis B and C viruses with liver cancer. In fact, ~16% of all malignancies are now known to be associated with an infectious cause. In addition, numerous emerging and re-emerging infectious diseases continue to have a dire impact on global health: HIV/AIDS, pandemic influenza, and severe acute respiratory syndrome (SARS) are but a few examples. The fear of weaponizing pathogens for bioterrorism is ever present and poses a potentially enormous threat to public health. Moreover, escalating antimicrobial resistance in clinically relevant microbes (e.g., Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumoniae, Plasmodium species, and HIV) signifies that the administration of antimicrobial agents—once thought to be a panacea—requires appropriate stewardship. For all these reasons, infectious diseases continue to exert grim effects on individual patients as well as on international public health. Even with all the successes of the past

1	stewardship. For all these reasons, infectious diseases continue to exert grim effects on individual patients as well as on international public health. Even with all the successes of the past century, physicians must be as thoughtful about infectious diseases now as they were at the beginning of the twentieth century. 144 SEC TIon 1 BASIC ConSIDERATIonS In InfEC TIouS DISEASES PART 8: Infectious Diseases

1	Infectious diseases remain the second leading cause of death worldwide. Although the rate of infectious disease–related deaths has decreased dramatically over the past 20 years, the absolute numbers of such deaths have remained relatively constant, totaling just over 12 million in 2010 (Fig. 144-1A). As shown in Fig. 144-1B, these deaths disproportionately affect lowand middle-income countries (Chap. 13e); in 2010, 23% of all deaths worldwide were related to infectious diseases, with rates >60% in most sub-Saharan African countries.

1	Given that infectious diseases are still a major cause of global mortality, understanding the local epidemiology of disease is critically important in evaluating patients. Diseases such as HIV/AIDS have decimated sub-Saharan Africa, with HIV-infected adults representing 15–26% of the total population in countries like Zimbabwe, Botswana, and Swaziland. Moreover, drug-resistant tuberculosis is rampant throughout the former Soviet-bloc countries, India, China, and South Africa. The ready availability of this type of information allows physicians to develop appropriate differential diagnoses and treatment plans for individual patients. Programs such as the Global Burden of Disease seek to quantify human losses (e.g., deaths, disability-adjusted life years) due to diseases by age, sex, and country over time; these data not only help inform local, national, and international health policy but can also help guide local medical decision-making. Even though some diseases (e.g., pandemic

1	country over time; these data not only help inform local, national, and international health policy but can also help guide local medical decision-making. Even though some diseases (e.g., pandemic influenza, SARS) are seemingly geographically restricted, the increasing ease of rapid worldwide travel has raised concern about their swift spread around the globe. The world’s increasing interconnectedness has profound implications not only for the global economy but also for medicine and the spread of infectious diseases.

1	Normal, healthy humans are colonized with over 100 trillion bacteria as well as countless viruses, fungi, and archaea; taken together, these microorganisms outnumber human cells by 10–100 times (Chap. 86e). The major reservoir of these microbes is the gastrointestinal tract, but very substantial numbers of microbes live in the female genital tract, the oral cavity, and the nasopharynx. There is increasing interest in the skin and even the lungs as sites where microbial colonization might be highly relevant to the biology and disease susceptibility of the host. These commensal organisms provide the host with myriad benefits, from aiding in metabolism to shaping the immune system. With regard to infectious diseases, the vast majority of infections are caused by organisms that are part of the normal flora (e.g., S. aureus,

1	S. pneumoniae, Pseudomonas aeruginosa), with relatively few infections due to organisms that are strictly pathogens (e.g, Neisseria gonorrhoeae, rabies virus). Perhaps it is not surprising that a general understanding of the microbiota is essential in the evaluation of infectious diseases. Individuals’ microbiotas likely have a major impact on their susceptibility to infectious diseases and even their responses to vaccines. Site-specific knowledge of the indigenous flora may facilitate appropriate interpretation of culture results, aid in selection of empirical antimicrobial therapy based on the likely causative agents, and provide additional impetus for rational antibiotic use to minimize the untoward effects of these drugs on the “beneficial” microbes that inhabit the body. The title of this chapter may appear to presuppose that the physician knows when a patient has an infectious disease. In reality, this chapter can serve only as a guide to the evaluation of a patient in whom an

1	Approach to the Patient with an Infectious Disease Number of Deaths (in millions) 1990101519952000200520105Rate of Death (per 100,000) 200350150250300 FIGURE 144-1 Magnitude of infectious disease–related deaths globally. A. The absolute number (blue line; left axis) and rate (red line; right axis) of infectious disease–related deaths throughout the world since 1990. B. A map depicting country-specific data for the percentages of total deaths that were attributable to communicable, maternal, neonatal, and nutritional disorders in 2010. (Source: Global Burden of Disease Study,

1	Institute for Health Metrics and Evaluation.) infectious disease is a possibility. Once a specific diagnosis is made, the reader should consult the subsequent chapters that deal with specific microorganisms in detail. The challenge for the physician is to recognize which patients may have an infectious disease as opposed to some other underlying disorder. This task is greatly complicated by the fact that infections have an infinite range of presentations, from acute life-threatening conditions (e.g., meningococcemia) to chronic diseases of varying severity (e.g., H. pylori–associated peptic ulcer disease) to no symptoms at all (e.g., latent M. tuberculosis infection). While it is impossible to generalize about a presentation that encompasses all infections, common findings in the history, physical examination, and basic laboratory testing often suggest that the patient either has an infectious disease or should be more closely evaluated for one. This chapter focuses on these common

1	physical examination, and basic laboratory testing often suggest that the patient either has an infectious disease or should be more closely evaluated for one. This chapter focuses on these common findings and how they may direct the ongoing evaluation of the patient.

1	APPROACH TO THE PATIENT: See also Chap. 147. As in all of medicine, obtaining a complete and thorough history is paramount in the evaluation of a patient with a possible infectious disease. The history is critical for developing a focused differential diagnosis and for guiding the physical exam and initial diagnostic testing. Although detailing all the elements of a history is beyond the scope of this chapter, specific components relevant to infectious diseases require particular attention. In general, these aspects focus on two areas: (1) an exposure history that may identify microorganisms with which the patient may have come into contact and (2) host-specific factors that may predispose to the development of an infection.

1	Exposure History • History of infections or exposure to drug-resistant microbes Knowledge about a patient’s previous infections, with the associated microbial susceptibility profiles, is very helpful in determining possible etiologic agents. Specifically, knowing whether a patient has a history of infection with drug-resistant organisms (e.g., methicillin-resistant S. aureus, vancomycin-resistant Enterococcus species, enteric organisms that produce an extended-spectrum β-lactamase or carbapenemase) or may have been exposed to drug-resistant microbes (e.g., during a recent stay in a hospital, nursing home, or long-term acute-care facility) may alter the choice of empirical antibiotics. For example, a patient presenting with sepsis who is known to have a history of invasive infection with a multidrug-resistant isolate of P. aeruginosa should be treated empirically with an antimicrobial regimen that will cover this strain.

1	social History Although the social history taken by physicians is often limited to inquiries about a patient’s alcohol and tobacco use, a complete social history can offer a number of clues to the underlying diagnosis. Knowing whether the patient has any high-risk behaviors (e.g., unsafe sexual behaviors, IV drug use), potential hobby-associated exposures (e.g., avid gardening, with possible Sporothrix schenckii exposure), or occupational exposures (e.g., increased risk for M. tuberculosis exposure in funeral service workers) can facilitate diagnosis. The importance of the social history is exemplified by a case in 2009 in which a laboratory researcher died of a Yersinia pestis infection acquired during his work; although this patient had visited both an outpatient clinic and an emergency department, his records at both sites failed to include his occupation—information that potentially could have led quickly to appropriate treatment and infection control measures.

1	dietary Habits As certain pathogens are associated with specific dietary habits, inquiring about a patient’s diet can provide insight into possible exposures. For example, Shiga toxin–producing strains of Escherichia coli and Toxoplasma gondii are associated with the consumption of raw or undercooked meat; Salmonella typhimurium, Listeria monocytogenes, and Mycobacterium bovis with unpasteurized milk; Leptospira species, parasites, and enteric bacteria with unpurified water; and Vibrio species, norovirus, helminths, and protozoa with raw seafood.

1	animal exposures Because animals are often important vectors of infectious diseases, patients should be asked about exposures to any animals, including contact with their own pets, visits to petting zoos, or random encounters (e.g., home rodent infestation). For example, dogs can carry ticks that serve as agents for the transmission of several infectious diseases, including Lyme disease, Rocky Mountain spotted fever, and ehrlichiosis. Cats are associated with Bartonella henselae infection, reptiles with Salmonella infection, rodents with leptospirosis, and rabbits with tularemia (Chap. 167e).

1	travel History Attention should be paid to both international and domestic travel. Fever in a patient who has recently returned from abroad significantly broadens the differential diagnosis (Chap. 149); even a remote history of international travel may reflect patients’ exposure to infections with pathogens such as M. tuberculosis or Strongyloides stercoralis. Similarly, domestic travel may have exposed patients to pathogens that are not normally found in their local environment and therefore may not routinely be considered in the differential diagnosis. For example, a patient who has recently visited California or Martha’s Vineyard may have been exposed to Coccidioides immitis or Francisella tularensis, respectively. Beyond simply identifying locations that a patient may have visited, the physician needs to delve deeper to learn what kinds of activities and behaviors the patient engaged in during travel (e.g., the types of food and sources of water consumed, freshwater swimming,

1	the physician needs to delve deeper to learn what kinds of activities and behaviors the patient engaged in during travel (e.g., the types of food and sources of water consumed, freshwater swimming, animal exposures) and whether the patient had the necessary immunizations and/or took the necessary prophylactic medications prior to travel; these additional exposures, which the patient may not think to report without specific prompting, are as important as exposures during a patient’s routine daily living.

1	Host-Specific Factors Because many opportunistic infections (e.g., with Pneumocystis jirovecii, Aspergillus species, or JC virus) affect only immunocompromised patients, it is of vital importance to determine the immune status of the patient. Defects in the immune system may be due to an underlying disease (e.g., malignancy, HIV infection, malnutrition), a medication (e.g., chemotherapy, glucocorticoids, monoclonal antibodies to components of the immune system), a treatment modality (e.g., total body irradiation, splenectomy), or a primary immunodeficiency. The type of infection for which the patient is at increased risk varies with the specific type of immune defect (Chap. 375e). In concert with determining whether a patient is immunocompromised for any reason, the physician should review the immunization record to ensure that the patient is adequately protected against vaccine-preventable diseases (Chap. 148).

1	Similar to the history, a thorough physical examination is crucial in evaluating patients with an infectious disease. Some elements of the physical exam (e.g., skin, lymphatics) that are often performed in a cursory manner as a result of the ever-increasing pace of medical practice may help identify the underlying diagnosis. Moreover, serial exams are critical since new findings may appear as the illness progresses. A description of all the elements of a physical exam is beyond the scope of this chapter, but the following components have particular relevance to infectious diseases.

1	Vital Signs Given that elevations in temperature are often a hallmark of infection, paying close attention to the temperature may be of value in diagnosing an infectious disease. The idea that 37°C (98.6°F) is the normal human body temperature dates back to the nineteenth century and was initially based on axillary measurements. Rectal temperatures more accurately reflect the core body temperature and are 0.4°C (0.7°F) and 0.8°C (1.4°F) higher than oral and axillary temperatures, respectively. Although the definition of fever varies greatly throughout the medical literature, the most common definition, which is based on studies defining fever of unknown origin (Chap. 26), uses a temperature ≥38.3°C (101°F). Although fever is very commonly associated with infection, it is also documented in many other diseases (Chap. 23). For every 1°C (1.8°F) increase in core temperature, the heart rate typically rises by 15–20 beats/min. Table 144-1 lists infections that are associated with relative

1	many other diseases (Chap. 23). For every 1°C (1.8°F) increase in core temperature, the heart rate typically rises by 15–20 beats/min. Table 144-1 lists infections that are associated with relative bradycardia (Faget’s sign), where patients have a lower heart rate than might be expected for a given body temperature. Although this pulse-temperature dissociation is not highly sensitive or specific for establishing a diagnosis, it is potentially useful in low-resource settings given its ready availability and simplicity.

1	Lymphatics There are ~600 lymph nodes throughout the body, and infections are an important cause of lymphadenopathy. A physical examination should include evaluation of lymph nodes in multiple aPrimarily early in the course of infection with Marburg or Ebola virus.

1	Approach to the Patient with an Infectious Disease regions (e.g., popliteal, inguinal, epitrochlear, axillary, multiple cervical regions), with notation of the location, size (normal, <1 cm), presence or absence of tenderness, and consistency (soft, firm, or shotty) and of whether the nodes are matted (i.e., connected and moving together). Of note, palpable epitrochlear nodes are always pathologic. Of patients presenting with lymphadenopathy, 75% have localized findings, and the remaining 25% have generalized lymphadenopathy (i.e., that involving more than one anatomic region). Localized lymphadenopathy in the head and neck region is found in 55% of patients, inguinal lymphadenopathy in 14%, and axillary lymphadenopathy in 5%. Determining whether the patient has generalized versus localized lymphadenopathy can help narrow the differential diagnosis, as various infections present differently.

1	Skin The fact that many infections have cutaneous manifestations gives the skin examination particular importance in the evaluation of patients (Chaps. 24, 25e, 72, and 156). It is important to perform a complete skin exam, with attention to both front and back. Specific rashes are often extremely helpful in narrowing the differential diagnosis of an infection (Chaps. 24 and 25e). In numerous anecdotal instances, patients in the intensive care unit have had “fever of unknown origin” that was actually due to unrecognized pressure ulcers. Moreover, close examination of the distal extremities for splinter hemorrhages, Janeway lesions, or Osler’s nodes may yield evidence of endocarditis or other causes of septic emboli.

1	Foreign Bodies As previously mentioned, many infections are caused by members of the indigenous microbiota. These infections typically occur when these microbes escape their normal habitat and enter a new one. Thus, maintenance of epithelial barriers is one of the most important mechanisms in protection against infection. However, hospitalization of patients is often associated with breaches of these barriers—e.g., due to placement of IV lines, surgical drains, or tubes (such as endotracheal tubes and Foley catheters) that allow microorganisms to localize in sites to which they normally would not have access (Chap. 168). Accordingly, knowing what lines, tubes, and drains are in place is helpful in ascertaining what body sites might be infected.

1	Laboratory and radiologic testing has advanced greatly over the past few decades and has become an important component in the evaluation of patients. The dramatic increase in the number of serologic diagnostics, antigen tests, and molecular diagnostics available to the physician has, in fact, revolutionized medical care. However, all of these tests should be viewed as adjuncts to the history and physical examination—not a replacement for them. The selection of initial tests should be based directly on the patient’s history and physical exam findings. Moreover, diagnostic testing should generally be limited to those conditions that are reasonably likely and treatable, important in terms of public health considerations, and/ or capable of providing a definitive diagnosis that will consequently limit other testing.

1	White Blood Cell (WBC) Count Elevations in the WBC count are often associated with infection, though many viral infections are associated with leukopenia. It is important to assess the WBC differential, given that different classes of microbes are associated with various leukocyte types. For example, bacteria are associated with an increase in polymorphonuclear neutrophils, often with elevated levels of earlier developmental forms such as bands; viruses are associated with an increase in lymphocytes; and certain parasites are associated with an increase in eosinophils. Table 144-2 lists the major infectious causes of eosinophilia.

1	Inflammatory Markers The erythrocyte sedimentation rate (ESR) and the C-reactive protein (CRP) level are indirect and direct measures of the acute-phase response, respectively, that can be used to assess a patient’s general level of inflammation. Moreover, these markers can be followed serially over time to monitor disease progress/resolution. It is noteworthy that the ESR changes relatively slowly, and its measurement more often than weekly usually is not useful; in contrast, CRP concentrations change rapidly, and daily measurements can be useful in the appropriate context. Although these markers are sensitive indicators of inflammation, neither is very specific. An extremely elevated ESR (>100 mm/h) has a 90% predictive value for a serious underlying disease (Table 144-3). Work is ongoing to identify other potentially useful inflammatory markers (e.g., procalcitonin, serum amyloid A protein); however, their clinical utility requires further validation.

1	Analysis of Cerebrospinal Fluid (CSF) Assessment of CSF is critical for patients with suspected meningitis or encephalitis. An opening pressure should always be recorded, and fluid should routinely be sent for cell counts, Gram’s stain and culture, and determination of glucose and protein levels. A CSF Gram’s stain typically requires >105 bacteria/mL for reliable positivity; its specificity approaches 100%. Table 144-4 lists the typical CSF profiles for various infections. In general, CSF with a lymphocytic pleocytosis and a low glucose concentration suggests either infection (e.g., with Listeria,

1	M. tuberculosis, or a fungus) or a noninfectious disorder (e.g, neoplastic meningitis, sarcoidosis). Bacterial antigen testing of CSF (e.g., latex agglutination tests for Haemophilus influenzae type b, group B Streptococcus, S. pneumoniae, and Neisseria meningitidis) is not recommended as a screening assay, given that these tests are no more sensitive than Gram’s stain; however, these assays can be helpful in presumptively identifying organisms seen on Gram’s stain. In contrast, other antigen tests (e.g., for Cryptococcus) and some CSF serologic testing (e.g., for Treponema pallidum, Coccidioides) are highly sensitive and are useful for select patients. In addition, polymerase chain reaction (PCR) analysis of CSF is increasingly being used for the diagnosis of bacterial (e.g., N. meningitidis, S. pneumoniae, mycobacteria) and viral (e.g., herpes simplex virus, enterovirus) infections; while these molecular tests permit rapid diagnosis with a high degree of sensitivity and specificity,

1	S. pneumoniae, mycobacteria) and viral (e.g., herpes simplex virus, enterovirus) infections; while these molecular tests permit rapid diagnosis with a high degree of sensitivity and specificity, they often do not allow determination of antimicrobial resistance profiles.

1	Cultures The mainstays of infectious disease diagnosis include the culture of infected tissue (e.g., surgical specimens) or fluid (e.g., blood, urine, sputum, purulence from a wound). Samples can be sent for culture of bacteria (aerobic or anaerobic), fungi, or viruses. Ideally, specimens are collected before the administration of antimicrobial therapy; in instances where this order of events is not clinically feasible, microscopic examination of the specimen (e.g., Gram-stained or potassium hydroxide [KOH]–treated preparations) is particularly important. Culture of the organism(s) allows identification of the etiologic agent, determination of the antimicrobial susceptibility profile, and—when there is concern about an outbreak—isolate typing. While cultures are extremely useful in the evaluation of patients, determining whether culture results are clinically meaningful or represent contamination (e.g., a non-aureus, non-lugdunensis staphylococcal species growing in a blood culture)

1	of patients, determining whether culture results are clinically meaningful or represent contamination (e.g., a non-aureus, non-lugdunensis staphylococcal species growing in a blood culture) can sometimes be challenging and requires an understanding of the patient’s immune status, exposure history, and microbiota. In some cases, serial cultures to demonstrate clearance of the organism may be helpful.

1	Pathogen-Specific Testing Numerous pathogen-specific tests (e.g., serology, antigen testing, PCR testing) are commercially available, and many hospitals now offer some of these tests in-house to facilitate rapid turnaround that ultimately enhances patient care. The reader is directed to relevant chapters on the pathogens of interest for specific details. Some of these tests (e.g., universal PCRs) identify organisms that currently are not cultivable and have unclear relationships to disease, thereby complicating diagnosis. As these tests become more commonplace and the work of the Human Microbiome Project progresses, the relevance of some of these previously unrecognized bacteria to human health will likely become more apparent.

1	aThere are numerous noninfectious causes of eosinophilia, such as atopic disease, DRESS (drug reaction with eosinophilia and systemic symptoms) syndrome, and pernicious anemia, which can cause mild eosinophilia; drug hypersensitivity and serum sickness, which can cause mild to moderate eosinophilia; collagen vascular disease, which can cause moderate eosinophilia; and malignancy, Churg-Strauss syndrome, and hyper-IgE syndromes, which can cause moderate to extreme eosinophilia. bMild: 500–1500 cells/μL; moderate: 1500–5000 cells/μL; extreme: >5000 cells/μL. cCan also affect the liver and the eyes. dCan also affect the lungs. eCan also affect the eyes and the central nervous system. fLevels are typically higher with pulmonary infections. Approach to the Patient with an Infectious Disease

1	Approach to the Patient with an Infectious Disease Radiology Imaging provides an important adjunct to the physical examination, allowing evaluation for lymphadenopathy in regions that are not externally accessible (e.g., mediastinum, intraabdominal sites), assessment of internal organs for evidence of infection, and facilitation of image-guided percutaneous sampling of deep spaces. The choice of imaging modality (e.g., CT, MRI, ultrasound, nuclear medicine, use of contrast) is best made in consultation with a radiologist to ensure that the results will address the physician’s specific concerns.

1	Physicians often must balance the need for empirical antibiotic treatment with the patient’s clinical condition. When clinically feasible, it is best to obtain relevant samples (e.g., blood, CSF, tissue, purulent exudate) for culture prior to the administration of antibiotics, as antibiotic treatment often makes subsequent diagnosis more difficult. Although a general maxim for antibiotic treatment is to use a regimen with as narrow a spectrum as possible (Chap. 170), empirical regimens are necessarily somewhat broad, given that a specific diagnosis has not yet been made. Table 144-5 lists empirical antibiotic treatment regimens for commonly encountered infectious presentations. These regimens should be narrowed as appropriate once a specific diagnosis is made. In addition to antibiotics, there is sometimes a role for adjunctive therapies, such as intravenous immunoglobulin G (IVIG) pooled from healthy adults or hyperimmune globulin prepared from the blood of individuals with high

1	there is sometimes a role for adjunctive therapies, such as intravenous immunoglobulin G (IVIG) pooled from healthy adults or hyperimmune globulin prepared from the blood of individuals with high titers of specific antibodies to select pathogens (e.g., cytomegalovirus, hepatitis B virus, rabies virus, vaccinia virus,

1	Etiologic Category (% of Cases) Specific Causes Clostridium tetani, varicella-zoster virus, Clostridium botulinum toxin). Although the data suggesting efficacy are limited, IVIG is often used for patients with suspected staphylococcal or streptococcal toxic shock syndrome. When evaluating a patient with a suspected infectious disease, the physician must consider what infection control methods are necessary to prevent transmission of any possible infection to other people. In 2007, the U.S. Centers for Disease Control and Prevention published guidelines for isolation precautions that are available for download at www.cdc.gov/hicpac/2007IP/2007isolationPrecautions .html. Persons exposed to certain pathogens (e.g., N. meningitidis, HIV, Bacillus anthracis) should receive postexposure prophylaxis to prevent disease acquisition. (See relevant chapters for details on specific pathogens.)

1	At times, primary physicians need assistance with patient management, from a diagnostic and/or therapeutic perspective. Multiple studies have demonstrated that an infectious disease consult is associated with positive outcomes for patients with various diseases. For example, in a prospective cohort study of patients with S. aureus bacteremia, infectious disease consultation was independently associated with a 56% reduction in 28-day mortality. In addition, infectious disease specialists provide other services (e.g., infection control, antimicrobial stewardship, management of outpatient antibiotic therapy, occupational exposure programs) that have been shown to benefit patients. Whenever such assistance would be advantageous to a patient with a possible infection, the primary physician should opt for an infectious disease consult. Specific situations that might prompt a consult include (1) difficult-todiagnose patients with presumed infections, (2) patients who are not responding to

1	opt for an infectious disease consult. Specific situations that might prompt a consult include (1) difficult-todiagnose patients with presumed infections, (2) patients who are not responding to treatment as expected, (3) patients with a complicated medical history (e.g., organ transplant recipients, patients immunosuppressed due to autoimmune or inflammatory conditions), and (4) patients with “exotic” diseases (i.e., diseases that are not typically seen within the region).

1	The study of infectious diseases is really a study of host-bacterial interactions and represents evolution by both the host and the bacteria—an endless struggle in which microbes have generally been more creative and adaptive. Given that nearly one-quarter of deaths worldwide are still related to infectious diseases, it is clear that the war against infectious diseases has not been won. For example, a cure for HIV infection is still lacking, there have been only marginal improvements in the methods for detection and treatment of tuberculosis after more than a half century of research, new infectious diseases (e.g., pandemic influenza, viral hemorrhagic fevers) continue to emerge, and the threat of microbial bioterrorism remains high. The subsequent chapters in Part 8 detail— on both a syndrome and a microbe-by-microbe basis—the current state of medical knowledge about infectious diseases. At their core, all of these chapters carry a similar message: Despite numerous advances in the

1	and a microbe-by-microbe basis—the current state of medical knowledge about infectious diseases. At their core, all of these chapters carry a similar message: Despite numerous advances in the diagnosis, treatment, and prevention of infectious diseases, much work and research are required before anyone can confidently claim that “all the major infections have disappeared.” In reality, this goal will never be attained, given the rapid adaptability of microbes.

1	aNumbers indicate typical results, but actual results may vary. bCerebrospinal fluid characteristics depend greatly on the specific organism. cNeutrophils may predominate early in the disease course. dPatients typically have striking eosinophilia as well. eSensitivity can be increased by examination of a smear of protein coagulum (pellicle) and the use of acid-fast stains. Abbreviations: PMNs, polymorphonuclear neutrophils; WBC, white blood cell. Septic shock Staphylococcus aureus, Streptococcus pneumoniae, enteric gram-negative bacilli Meningitis S. pneumoniae, Neisseria meningitidis CNS abscess Streptococcus spp., Staphylococcus spp., anaerobes, gram-negative bacilli Endocarditis S. aureus, Streptococcus spp., coagulasenegative staphylococci Community-S. pneumoniae, acquired, Mycoplasma pneuoutpatient moniae, Haemophilus influenzae, Chlamydia pneumoniae Inpatient, non-ICU Above plus Legionella spp. Inpatient, ICU Above plus S. aureus

1	Community-S. pneumoniae, acquired, Mycoplasma pneuoutpatient moniae, Haemophilus influenzae, Chlamydia pneumoniae Inpatient, non-ICU Above plus Legionella spp. Inpatient, ICU Above plus S. aureus Hospital-acquired S. pneumoniae, pneumoniad H. influenzae, S. aureus, gram-negative bacilli (e.g., Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter spp.) Mild to moderate Anaerobes (Bacteroides severity spp., Clostridium spp.), gram-negative bacilli (Escherichia coli), Streptococcus spp. High-risk patient Same as above or high degree of severity Vancomycin, 15 mg/kg q12hb; A broad-spectrum antipseudomonal β-lactam (piperacillin-tazobactam, 4.5 g q6h; imipenem, 1 g q8h; meropenem, 1 g q8h; or cefepime, 1–2 g q8–12h) Vancomycin, 15 mg/kg q12hb; Ceftriaxone, 2 g q12h Vancomycin, 15 mg/kg q12hb; Ceftriaxone, 2 g q12h; Metronidazole, 500 mg q8h Vancomycin, 15 mg/kg q12hb; Ceftriaxone, 2 g q12h Azithromycin, 500 mg PO × 1, then 250 mg PO qd × 4 days

1	Vancomycin, 15 mg/kg q12hb; Ceftriaxone, 2 g q12h; Metronidazole, 500 mg q8h Vancomycin, 15 mg/kg q12hb; Ceftriaxone, 2 g q12h Azithromycin, 500 mg PO × 1, then 250 mg PO qd × 4 days A respiratory fluoroquinolone (moxifloxacin, 400 mg IV/PO qd; gemifloxacin, 320 mg PO qd; or levofloxacin, 750 mg IV/PO qd); A β-lactam (cefotaxime, ceftriaxone, or ampicillinsulbactam) plus azithromycin Azithromycin or a respiratory fluoroquinolone An antipseudomonal β-lactam (cefepime, 1–2 g q8–12 h; ceftazidime, 2 g q8h; imipenem, 1 g q8h; meropenem, 1 g q8h; or piperacillin-tazobactam, 4.5 g q6h); An antipseudomonal fluoroquinolone (levofloxacin or ciprofloxacin, 400 mg q8h) or an aminoglycoside (amikacin, 20 mg/kg q24hc; gentamicin, 7 mg/kg q24he; or tobramycin, 7 mg/kg q24he) Cefoxitin, 2 g q6h; A combination of metronidazole (500 mg q8–12h) plus cefazolin (1–2 g q8h) or cefuroxime (1.5 g q8h) or ceftriaxone (1–2 g q12–24h) or cefotaxime (1–2 g q6–8h)

1	Cefoxitin, 2 g q6h; A combination of metronidazole (500 mg q8–12h) plus cefazolin (1–2 g q8h) or cefuroxime (1.5 g q8h) or ceftriaxone (1–2 g q12–24h) or cefotaxime (1–2 g q6–8h) A carbapenem (imipenem, 1 g q8h; meropenem, 1 g q8h; doripenem, 500 mg q8h); Piperacillin-tazobactam, 3.375 g q6hf; A combination of metronidazole (500 mg q8–12h) plus an antipseudomonal cephalosporin (cefepime, 2 g q8–12h; ceftazidime, 2 g q8h) or an antipseudomonal fluoroquinolone (ciprofloxacin, 400 mg q12h; levofloxacin, 750 mg q24h) — Dexamethasone (0.15 mg/ kg IV q6h for 2–4 d) should be added for patients with suspected or proven pneumococcal meningitis, with the first dose administered 10–20 min before the first dose of antibiotics. If MRSA is a consideration, add vancomycin (15 mg/kg q12hb) or linezolid (600 mg q12h); daptomycin should not be used in patients with pneumonia. If MRSA is a consideration, add vancomycin (15 mg/kg q12hb). 159, 201, and pathogen-specific chapters

1	If MRSA is a consideration, add vancomycin (15 mg/kg q12hb). 159, 201, and pathogen-specific chapters Approach to the Patient with an Infectious Disease Skin and soft tissue S. aureus, Streptococcus Dicloxacillin, 250–500 mg PO qid; If MRSA is a consideration, 156 and infection pyogenes clindamycin, vancomycin pathogen-specific (15 mg/kg q12hb), linezolid chapters Cephalexin, 250–500 mg PO qid; (600 mg IV/PO q12h), or or TMP-SMX (1–2 double-Clindamycin, 300–450 mg PO tid; strength tablets PO bidg) can be used.

1	Nafcillin/oxacillin, 1–2 g q4h aThis table refers to immunocompetent adults with normal renal and hepatic function. All doses listed are for parenteral administration unless indicated otherwise. Local antimicrobial susceptibility profiles may influence the choice of antibiotic. Therapy should be tailored once a specific etiologic agent and its susceptibilities are identified. bTrough levels for vancomycin should be 15–20 ≥g/mL. cTrough levels for amikacin should be <4 ≥g/mL. dIn patients with late onset (i.e., after ≤5 days of hospitalization) or risk factors for multidrug-resistant organisms. eTrough levels for gentamicin and tobramycin should be <1 ≥g/mL. fIf P. aeruginosa is a concern, the dosage may be increased to 3.375 g IV q4h or 4.5 g IV q6h. gData on the efficacy of TMP-SMX in skin and soft tissue infections are limited. Abbreviations: CNS, central nervous system; ICU, intensive care unit; MRSA, methicillin-resistant S. aureus; TMP-SMX, trimethoprim-sulfamethoxazole.

1	Molecular Mechanisms of Microbial Pathogenesis Gerald B. Pier Over the past four decades, molecular studies of the pathogenesis of microorganisms have yielded an explosion of information about the 145e various microbial and host molecules that contribute to the processes of infection and disease. These processes can be classified into several stages: microbial encounter with and entry into the host; microbial growth after entry; avoidance of innate host defenses; tissue invasion and tropism; tissue damage; and transmission to new hosts. Virulence is the measure of an organism’s capacity to cause disease and is a function of the pathogenic factors elaborated by microbes. These factors promote colonization (the simple presence of potentially pathogenic microbes in or on a host), infection (attachment and growth of pathogens and avoidance of host defenses), and disease (often, but not always, the result of activities of secreted toxins or toxic metabolites). In addition, the host’s

1	(attachment and growth of pathogens and avoidance of host defenses), and disease (often, but not always, the result of activities of secreted toxins or toxic metabolites). In addition, the host’s inflammatory response to infection greatly contributes to disease and its attendant clinical signs and symptoms. The recent surge of interest in the role of the microbiota and its associated microbiome—the collection of microbial genomes residing in or on mammalian organisms—in the physiology of, susceptibility to, and response to infection and in immune system development has had an enormous impact on our understanding of host-pathogen interaction.

1	(See also Chap. 86e) We now understand that the indigenous microbial organisms living in close association with almost all animals are organized into complex communities that strongly modulate the ability of pathogenic microbes to become established in or on host surfaces. The sheer numbers of these microbes and their genomic variability vastly exceed the numbers of host cells and genes in a typical animal. Changes and differences in microbiomes within and between individuals, currently characterized by high-throughput DNA sequencing techniques and bioinformatic analysis, affect the development and control of the immune system as well as such diverse conditions as obesity, type 1 diabetes, cognition, neurologic states, autoimmune diseases, and infectious diseases of the skin, gastrointestinal tract, respiratory tract, and vagina. It has been more difficult to directly associate specific types of microbiomes with pathophysiologic states and to assess how conserved or variable microbial

1	tract, respiratory tract, and vagina. It has been more difficult to directly associate specific types of microbiomes with pathophysiologic states and to assess how conserved or variable microbial species within human and animal microbiomes are evolving. Defining clusters of organisms associated with diseases may become more feasible as more data are obtained. Complicating this task are the results from the Human Microbiome Project suggesting a high level of variability among individuals in the components of the microbiome, although many individuals appear to maintain a fairly conserved microbiome throughout their lives. In the context of infectious diseases, clear changes and disruptions of the indigenous microbiome have a strong and often fundamental impact on the progression of infection. Such alterations can be associated with the effects of antibiotic and immunosuppressive drug use on the normal flora, with environmental changes, and with the impact of microbial virulence factors

1	Such alterations can be associated with the effects of antibiotic and immunosuppressive drug use on the normal flora, with environmental changes, and with the impact of microbial virulence factors that displace the indigenous microbial flora to facilitate pathogen colonization. As the available technology for defining the microbiome expands, there is no doubt that the resulting data will markedly affect our concepts of and approaches to microbial pathogenesis and infectious disease treatment.

1	MICROBIAL ENTRY AND ADHERENCE Entry Sites A microbial pathogen can potentially enter any part of a host organism. In general, the type of disease produced by a particular microbe is often a direct consequence of its route of entry into the body. The most common sites of entry are mucosal surfaces (the respiratory, alimentary, and urogenital tracts) and the skin. Ingestion, inhalation, and sexual contact are typical routes of microbial entry. Other portals of entry include sites of skin injury (cuts, bites, burns, trauma) along with injection via natural (i.e., vector-borne) or artificial (i.e., needle-stick injury) routes. A few pathogens, such as Schistosoma 145e-1 species, can penetrate unbroken skin. The conjunctiva can serve as an entry point for pathogens of the eye, which occasionally spread systemically from that site.

1	Microbial entry usually relies on the presence of specific factors needed for persistence and growth in a tissue. Fecal-oral spread via the alimentary tract requires a biologic profile consistent with survival in the varied environments of the gastrointestinal tract (including the low pH of the stomach and the high bile content of the intestine) as well as in contaminated food or water outside the host. Organisms that gain entry via the respiratory tract survive well in small moist droplets produced during sneezing and coughing. Pathogens that enter by venereal routes often survive best in the warm moist environment of the urogenital mucosa and have restricted host ranges (e.g., Neisseria gonorrhoeae, Treponema pallidum, and HIV).

1	The biology of microbes entering through the skin is highly varied. Some of these organisms can survive in a broad range of environments, such as the salivary glands or alimentary tracts of arthropod vectors, the mouths of larger animals, soil, and water. A complex biology allows protozoan parasites such as Plasmodium, Leishmania, and Trypanosoma species to undergo morphogenic changes that permit transmission to mammalian hosts during insect feeding for blood meals. Plasmodia are injected as infective sporozoites from the salivary glands during mosquito feeding. Leishmania parasites are regurgitated as promastigotes from the alimentary tract of sandflies and injected by bite into a susceptible host. Trypanosomes are first ingested from infected hosts by reduviid bugs; the pathogens then multiply in the gastrointestinal tract of the insects and are released in feces onto the host’s skin during subsequent feedings. Most microbes that land directly on intact skin are destined to die, as

1	in the gastrointestinal tract of the insects and are released in feces onto the host’s skin during subsequent feedings. Most microbes that land directly on intact skin are destined to die, as survival on the skin or in hair follicles requires resistance to fatty acids, low pH, and other antimicrobial factors on the skin. Once it is damaged (and particularly if it becomes necrotic), the skin can be a major portal of entry and growth for pathogens and elaboration of their toxic products. Burn wound infections and tetanus are clear examples. After animal bites, pathogens resident in the animal’s saliva gain access to the victim’s tissues through the damaged skin. Rabies is the paradigm for this pathogenic process; rabies virus grows in striated muscle cells at the site of inoculation.

1	Microbial Adherence Once in or on a host, most microbes must anchor themselves to a tissue or tissue factor; the possible exceptions are organisms that directly enter the bloodstream and multiply there. Specific ligands or adhesins for host receptors constitute a major area of study in the field of microbial pathogenesis. Adhesins comprise a wide range of surface structures, not only anchoring the microbe to a tissue and promoting cellular entry where appropriate but also eliciting host responses critical to the pathogenic process (Table 145e-1). Most microbes produce multiple adhesins specific for multiple host receptors. These adhesins are often redundant, are serologically variable, and act additively or synergistically with other microbial factors to promote microbial sticking to host tissues. In addition, some microbes adsorb host proteins onto their surface and utilize the natural host protein receptor for microbial binding and entry into target cells.

1	VIRAL ADHESINS All viral pathogens must bind to host cells, enter them, and replicate within them. Viral coat proteins serve as the ligands for cellular entry, and more than one ligand-receptor interaction may be needed; for example, HIV utilizes its envelope glycoprotein (gp) 120 to enter host cells by binding both to CD4 and to one of two receptors for chemokines (designated CCR5 and CXCR4). Similarly, the measles virus H glycoprotein binds to both CD46 and the membrane-organizing protein moesin on host cells. The gB and gC proteins on herpes simplex virus bind to heparan sulfate, although this adherence is not essential for entry but rather serves to concentrate virions close to the cell surface; this step is followed by attachment to mammalian cells mediated by the viral gD protein, with subsequent formation of a homotrimer of viral gB protein or a heterodimer of viral gH and gL proteins that permits fusion of the viral envelope with the host cell membrane. Herpes simplex virus

1	with subsequent formation of a homotrimer of viral gB protein or a heterodimer of viral gH and gL proteins that permits fusion of the viral envelope with the host cell membrane. Herpes simplex virus can use a number of eukaryotic cell

1	CHAPTER 145e Molecular Mechanisms of Microbial Pathogenesis Type of Microbial Microorganism Ligand Host Receptor (SLAM) Human herpesvirus ? CD46 type 6 Neisseria spp. Pili Membrane cofactor protein (CD46) Yersinia spp. Invasin/accessory β1 Integrins invasin locus aA novel dendritic cell–specific C-type lectin. surface receptors for entry, including the herpesvirus entry mediator (related to the tumor necrosis factor receptor), members of the immunoglobulin superfamily, the proteins nectin-1 and nectin-2, and modified heparan sulfate.

1	BACTERIAL ADHESINS Among the microbial adhesins studied in greatest detail are bacterial pili and flagella (Fig. 145e-1). Pili or fimbriae are commonly used by gram-negative bacteria for attachment to host cells and tissues; studies have identified similar factors produced by gram-positive organisms such as group B streptococci. In electron micro-graphs, these hairlike projections (up to several hundred per cell) may be confined to one end of the organism (polar pili) or distributed more evenly over the surface. An individual cell may have pili with a variety of functions. Most pili are made up of a major pilin protein subunit

1	FIguRE 145e-1 Bacterial surface structures. A and B. Traditional electron micrographic images of fixed cells of Pseudomonas aeruginosa. Flagella (A) and pili (B) project out from the bacterial poles. C and D. Atomic force microscopic image of live P. aeruginosa freshly planted onto a smooth mica surface. This technology reveals the fine, three-dimensional detail of the bacterial surface structures. (Images courtesy of Drs. Martin Lee and Milan Bajmoczi, Harvard Medical School.) (molecular weight, 17,000–30,000) that polymerizes to form the pilus. Many strains of Escherichia coli isolated from urinary tract infections express mannose-binding type 1 pili, whose binding to integral membrane glycoproteins called uroplakins that coat the cells in the bladder epithelium is inhibited by d-mannose. Other strains produce the Pap (pyelonephritis-associated) or P pilus adhesin that mediates binding to digalactose (gal-gal) residues on globosides of the human P blood groups. Both of these types

1	Other strains produce the Pap (pyelonephritis-associated) or P pilus adhesin that mediates binding to digalactose (gal-gal) residues on globosides of the human P blood groups. Both of these types of pili have proteins located at the tips of the main pilus unit that are critical to the binding specificity of the whole pilus unit. Although immunization with the mannose-binding tip protein (FimH) of type 1 pili prevents experimental E. coli bladder infections in mice and monkeys, a human trial of this vaccine was not successful. E. coli cells causing diarrheal disease express pilus-like receptors for enterocytes on the small bowel, along with other receptors termed colonization factors.

1	The type IV pilus, a common type of pilus found in Neisseria species, Moraxella species, Vibrio cholerae, Legionella pneumophila, Salmonella enterica serovar Typhi, enteropathogenic E. coli, and Pseudomonas aeruginosa, often mediates adherence of organisms to target surfaces. Type IV pili tend to have a relatively conserved aminoterminal region and a more variable carboxyl-terminal region. For some species (e.g.,

1	N. gonorrhoeae, Neisseria meningitidis, and enteropathogenic E. coli), the pili are critical for attachment to mucosal epithelial cells. For others, such as P. aeruginosa, the pili only partially mediate the cells’ adherence to host tissues and may in some circumstances inhibit colonization. For example, a recent study of P. aeruginosa colonization of the gastrointestinal tract of mice evaluated a bank of mutants in which all nonessential genes were interrupted; those mutants that were unable to produce the type IVa pili were actually better able to colonize the gastrointestinal mucosa, although the basis for this observation was not identified. V. cholerae cells appear to use two different types of pili for intestinal colonization. Whereas interference with this stage of colonization would appear to be an effective antibacterial strategy, attempts to develop pilus-based vaccines for human diseases have not been highly successful to date.

1	Flagella are long appendages attached at either one or both ends of the bacterial cell (polar flagella) or distributed over the entire cell surface (peritrichous flagella). Flagella, like pili, are composed of a polymerized or aggregated basic protein. In flagella, the protein subunits form a tight helical structure and vary serologically with the species. Spirochetes such as T. pallidum and Borrelia burgdorferi have axial filaments similar to flagella running down the long axis of the center of the cell, and they “swim” by rotation around these filaments. Some bacteria can glide over a surface in the absence of obvious motility structures.

1	Other bacterial structures involved in adherence to host tissues include specific staphylococcal and streptococcal proteins that bind to human extracellular matrix proteins such as fibrin, fibronectin, fibrinogen, laminin, and collagen. Fibronectin appears to be a commonly used receptor for various pathogens; a particular amino acid sequence in fibronectin, Arg-Gly-Asp or RGD, is a critical target used by bacteria to bind to host tissues. Binding of a highly conserved Staphylococcus aureus surface protein, clumping factor A (ClfA), to fibrinogen has been implicated in many aspects of pathogenesis. Attempts to interrupt this interaction and prevent S. aureus sepsis in low-birth-weight infants by administering an intravenous IgG preparation derived from the plasma of individuals with high titers of antibody to ClfA failed to show efficacy in a clinical trial; however, this approach is being pursued in some vaccine formulations targeting this organism. The conserved outer-core portion of

1	of antibody to ClfA failed to show efficacy in a clinical trial; however, this approach is being pursued in some vaccine formulations targeting this organism. The conserved outer-core portion of the lipopolysaccharide (LPS) of P. aeruginosa mediates binding to the cystic fibrosis trans-membrane conductance regulator (CFTR) on airway epithelial cells— an event that appears to play a critical role in normal host resistance to infection by initiating recruitment of polymorphonuclear neutrophils (PMNs) to the lung mucosa to kill the cells via opsonophagocytosis. A large number of microbial pathogens encompassing major gram-positive bacteria (staphylococci and streptococci), gram-negative bacteria (major enteric species and coccobacilli), fungi (Candida, Fusobacterium, Aspergillus), and even eukaryotes (Trichomonas vaginalis and Plasmodium falciparum) express a surface polysaccharide composed of β-1-6-linked-poly-N-acetyl-d-glucosamine (PNAG). One of the functions of PNAG for some of these

1	(Trichomonas vaginalis and Plasmodium falciparum) express a surface polysaccharide composed of β-1-6-linked-poly-N-acetyl-d-glucosamine (PNAG). One of the functions of PNAG for some of these organisms is to promote binding to materials used in catheters and other types of implanted devices. This polysaccharide may be a critical factor in the establishment of device-related infections by pathogens such as staphylococci and E. coli. High-powered imaging techniques (e.g., atomic force microscopy) have revealed that bacterial cells have a nonhomogeneous surface that is probably attributable to different concentrations of cell surface molecules, including microbial adhesins, at specific places on the cell surface (Figs. 120-1C and 120-1D).

1	FUNGAL ADHESINS Several fungal adhesins have been described that mediate colonization of epithelial surfaces, particularly adherence to structures like fibronectin, laminin, and collagen. The product of the Candida albicans INT1 gene, Int1p, bears similarity to mammalian integrins that bind to extracellular matrix proteins. The agglutinin-like sequence (ALS) adhesins are large cell-surface glycoproteins mediating adherence of pathogenic Candida to host tissues. These adhesins possess a conserved three-domain structure composed of an N-terminal domain that mediates adherence to host tissue receptors, a central motif consisting of a number of repeats of a conserved sequence of 36 amino acids, and a C-terminal domain that varies in length and sequence and contains a glycosylphosphatidylinositol (GPI) anchor addition site that allows binding of the adhesin to the fungal cell wall. Variability in the number of central domains in different ALS proteins characterizes different adhesins with

1	(GPI) anchor addition site that allows binding of the adhesin to the fungal cell wall. Variability in the number of central domains in different ALS proteins characterizes different adhesins with specificity for different host receptors. The ALS adhesins are expressed under certain environmental conditions and are crucial for pathogenesis of fungal infections.

1	For several fungal pathogens that initiate infections after inhalation 145e-3 of infectious material, the inoculum is ingested by alveolar macrophages, in which the fungal cells transform to pathogenic phenotypes. Like C. albicans, Blastomyces dermatitidis binds to CD11b/CD18 integrins as well as to CD14 on macrophages. B. dermatitidis produces a 120-kDa surface protein, designated WI-1, that mediates this adherence. An unidentified factor on Histoplasma capsulatum also mediates binding of this fungal pathogen to the integrin surface proteins.

1	EUKARYOTIC PATHOGEN ADHESINS Eukaryotic parasites use complicated surface glycoproteins as adhesins, some of which are lectins (proteins that bind to specific carbohydrates on host cells). For example, Plasmodium vivax, one of six Plasmodium species causing malaria, binds (via Duffy-binding protein) to the Duffy blood group carbohydrate antigen Fy on erythrocytes. Entamoeba histolytica, the third leading cause of death from parasitic diseases, expresses two proteins that bind to the disaccharide galactose/N-acetyl galactosamine. Reports indicate that children with mucosal IgA antibody to one of these lectins are resistant to reinfection with virulent E. histolytica. A major surface glycoprotein (gp63) of Leishmania promastigotes is needed for these parasites to enter human macrophages—the principal target cell of infection. This glycoprotein promotes complement binding but inhibits complement lytic activity, allowing the parasite to use complement receptors for entry into macrophages;

1	principal target cell of infection. This glycoprotein promotes complement binding but inhibits complement lytic activity, allowing the parasite to use complement receptors for entry into macrophages; gp63 also binds to fibronectin receptors on macrophages. In addition, the pathogen can express a carbohydrate that mediates binding to host cells. Evidence suggests that, as part of hepatic granuloma formation, Schistosoma mansoni expresses a carbohydrate epitope related to the Lewis X blood group antigen that promotes adherence of helminthic eggs to vascular endothelial cells under inflammatory conditions.

1	Host Receptors Host receptors are found both on target cells (such as epithelial cells lining mucosal surfaces) and within the mucus layer covering these cells. Microbial pathogens bind to a wide range of host receptors to establish infection (Table 145e-1). Selective loss of host receptors for a pathogen may confer natural resistance to an otherwise susceptible population. For example, 70% of individuals in West Africa lack Fy antigens and are resistant to P. vivax infection. S. enterica serovar Typhi, the etiologic agent of typhoid fever, produces a pilus protein that binds to CFTR to enter the gastrointestinal submucosa after being ingested by enterocytes. As homozygous mutations in CFTR are the cause of the life-shortening disease cystic fibrosis, heterozygote carriers (e.g., 4–5% of individuals of European ancestry) may have had a selective advantage due to decreased susceptibility to typhoid fever.

1	Numerous virus–target cell interactions have been described, and it is now clear that different viruses can use similar host cell receptors for entry. The list of certain and likely host receptors for viral pathogens is long. Among the host membrane components that can serve as receptors for viruses are sialic acids, gangliosides, glycosaminoglycans, integrins and other members of the immunoglobulin superfamily, histocompatibility antigens, and regulators and receptors for complement components. A notable example of the effect of host receptors on the pathogenesis of infection has emerged from studies comparing the binding of avian influenza A subtype H5N1 with that of influenza A strains expressing the H1 subtype of hemagglutinin. The H1 subtypes tend to be highly pathogenic and transmissible from human to human, and they bind to a receptor composed of two sugar molecules: sialic acid linked α-2-6 to galactose. This receptor is expressed at high levels in the airway epithelium; when

1	from human to human, and they bind to a receptor composed of two sugar molecules: sialic acid linked α-2-6 to galactose. This receptor is expressed at high levels in the airway epithelium; when virus is shed from this surface, its transmission via coughing and aerosol droplets is facilitated. In contrast, the H5N1 avian influenza virus binds to sialic acid linked α-2-3 to galactose, and this receptor is expressed at high levels in pneumocytes in the alveoli. Infection in the alveoli is thought to underlie the high mortality rate associated with avian influenza but also the low interhuman transmissibility of this strain, which is not readily transported to the airways from which it can be expelled by coughing. Nonetheless, it was recently shown that H5 hemagglutinins can acquire mutations that vastly increase their transmissibility while not affecting their high level of lethality.

1	CHAPTER 145e Molecular Mechanisms of Microbial Pathogenesis

1	Once established on a mucosal or skin site, pathogenic microbes must replicate before causing full-blown infection and disease. Within cells, viral particles release their nucleic acids, which may be directly translated into viral proteins (positive-strand RNA viruses), transcribed from a negative strand of RNA into a complementary mRNA (negative-strand RNA viruses), or transcribed into a complementary strand of DNA (retroviruses); for DNA viruses, mRNA may be transcribed directly from viral DNA, either in the cell nucleus or in the cytoplasm. To grow, bacteria must acquire specific nutrients or synthesize them from precursors in host tissues. Many infectious processes are usually confined to specific epithelial surfaces—e.g., H1 subtype influenza to the respiratory mucosa, gonorrhea to the urogenital epithelium, shigellosis to the gastrointestinal epithelium. While there are multiple reasons for this specificity, one important consideration is the ability of these pathogens to obtain

1	urogenital epithelium, shigellosis to the gastrointestinal epithelium. While there are multiple reasons for this specificity, one important consideration is the ability of these pathogens to obtain from these specific environments the nutrients needed for growth and survival.

1	Temperature restrictions also play a role in limiting certain pathogens to specific tissues. Rhinoviruses, a cause of the common cold, grow best at 33°C and replicate in cooler nasal tissues but not in the lung. Leprosy lesions due to Mycobacterium leprae are found in and on relatively cool body sites. Fungal pathogens that infect the skin, hair follicles, and nails (dermatophyte infections) remain confined to the cooler, exterior, keratinous layer of the epithelium. A topic of major interest is the ability of many bacterial, fungal, and protozoal species to grow in multicellular masses referred to as biofilms. These masses are biochemically and morphologically quite distinct from the free-living individual cells referred to as planktonic cells. Growth in biofilms leads to altered microbial metabolism, production of extracellular virulence factors, and decreased susceptibility to biocides, antimicrobial agents, and host defense molecules and cells.

1	P. aeruginosa growing on the bronchial mucosa during chronic infection, staphylococci and other pathogens growing on implanted medical devices, and dental pathogens growing on tooth surfaces to form plaque are several examples of microbial biofilm growth associated with human disease. Many other pathogens can form biofilms during in vitro growth. It is increasingly accepted that this mode of growth contributes to microbial virulence and induction of disease and that biofilm formation can also be an important factor in microbial survival outside the host, promoting transmission to additional susceptible individuals.

1	As microbes have interacted with mucosal/epithelial surfaces since the emergence of multicellular organisms, it is not surprising that multicellular hosts have a variety of innate surface defense mechanisms that can sense when pathogens are present and contribute to their elimination. The skin is acidic and is bathed with fatty acids toxic to many microbes. Skin pathogens such as staphylococci must tolerate these adverse conditions. Mucosal surfaces are covered by a barrier composed of a thick mucus layer that entraps microbes and facilitates their transport out of the body by such processes as mucociliary clearance, coughing, and urination. Mucous secretions, saliva, and tears contain antibacterial factors such as lysozyme and antimicrobial peptides as well as antiviral factors such as interferons (IFNs). Gastric acidity and bile salts are inimical to the survival of many ingested pathogens, and most mucosal surfaces—particularly the nasopharynx, the vaginal tract, and the

1	such as interferons (IFNs). Gastric acidity and bile salts are inimical to the survival of many ingested pathogens, and most mucosal surfaces—particularly the nasopharynx, the vaginal tract, and the gastrointestinal tract—contain a resident flora of commensal microbes that interfere with the ability of pathogens to colonize and infect a host. Major advances in the use of nucleic acid sequencing now allow extensive identification and characterization of the vast array of commensal organisms that have come to be referred to as the microbiota. In addition to its role in providing competition for mucosal colonization, acquisition of a normal microbiota is critical for proper development of the immune system, influencing maturation and differentiation of components of both the innate and acquired arms.

1	Pathogens that survive local antimicrobial factors must still contend with host endocytic, phagocytic, and inflammatory responses as well as with host genetic factors that determine the degree to which a pathogen can survive and grow. The list of genes whose variants, usually by single-nucleotide polymorphisms, can affect host susceptibility and resistance to infection is rapidly expanding. A classic example is a 32-bp deletion in the gene for the HIV-1 co-receptor known as chemokine receptor 5 (CCR5), which, when present in the homozygous state, confers high-level resistance to HIV-1 infection. The growth of viral pathogens entering skin or mucosal epithelial cells can be limited by a variety of host genetic factors, including production of IFNs, modulation of receptors for viral entry, and ageand hormone-related susceptibility factors; by nutritional status; and even by personal habits such as smoking and exercise.

1	Encounters with Epithelial Cells Over the past two decades, many pathogens have been shown to enter epithelial cells (Fig. 145e-2); they often use specialized surface structures that bind to receptors, with consequent internalization. However, the exact role and the importance of this process in infection and disease are not well defined for most of these pathogens. Microbial entry into host epithelial cells is seen as a means for dissemination to adjacent or deeper tissues or as a route to sanctuary to avoid ingestion and killing by professional FIguRE 145e-2 Entry of bacteria into epithelial cells. A. Internalization of Pseudomonas aeruginosa by cultured airway epithelial cells expressing wild-type cystic fibrosis transmembrane conductance regulator, the cell receptor for bacterial ingestion. B. Entry of P. aeruginosa into murine tracheal epithelial cells after murine infection by the intranasal route.

1	B. Entry of P. aeruginosa into murine tracheal epithelial cells after murine infection by the intranasal route. phagocytes. Epithelial cell entry appears, for instance, to be a critical aspect of dysentery induction by Shigella.

1	Curiously, the less virulent strains of many bacterial pathogens are more adept at entering epithelial cells than are more virulent strains; examples include pathogens that lack the surface polysaccharide capsule needed to cause serious disease. Thus, for Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus agalactiae (group B Streptococcus), and Streptococcus pyogenes, isogenic mutants or variants lacking capsules enter epithelial cells better than the wild-type, encapsulated parental forms that cause disseminated disease. These observations have led to the proposal that epithelial cell entry may be primarily a manifestation of host defense, resulting in bacterial clearance by both shedding of epithelial cells containing internalized bacteria and initiation of a protective and nonpathogenic inflammatory response. However, a possible consequence of this process could be the opening of a hole in the epithelium, potentially allowing uningested organisms to enter the

1	and nonpathogenic inflammatory response. However, a possible consequence of this process could be the opening of a hole in the epithelium, potentially allowing uningested organisms to enter the submucosa. This scenario has been documented in murine S. enterica serovar Typhimurium infections and in experimental bladder infections with uropathogenic E. coli. In the latter system, bacterial pilus-mediated attachment to uroplakins induces exfoliation of the cells with attached bacteria. Subsequently, infection is produced by residual bacterial cells that invade the superficial bladder epithelium, where they can grow intracellularly into biofilm-like masses encased in an extracellular polysaccharide-rich matrix and surrounded by uroplakin. This mode of growth produces structures that have been referred to as bacterial pods. It is likely that at low bacterial inocula epithelial cell ingestion and subclinical inflammation are efficient means to eliminate pathogens, while at higher inocula a

1	referred to as bacterial pods. It is likely that at low bacterial inocula epithelial cell ingestion and subclinical inflammation are efficient means to eliminate pathogens, while at higher inocula a proportion of surviving bacterial cells enter the host tissue through the damaged mucosal surface and multiply, producing disease. Alternatively, failure of the appropriate epithelial cell response to a pathogen may allow the organism to survive on a mucosal surface where, if it avoids other host defenses, it can grow and cause a local infection. Along these lines, as noted above, P. aeruginosa is taken into epithelial cells by CFTR, a protein missing or nonfunctional in most severe cases of cystic fibrosis. The major clinical consequence of this disease is chronic airway-surface infection with P. aeruginosa in 80–90% of patients. The failure of airway epithelial cells to ingest and promote the removal of

1	P. aeruginosa via a properly regulated inflammatory response has been proposed as a key component of the hypersusceptibility of cystic fibrosis patients to chronic airway infection with this organism. Encounters with Phagocytes • PHAGOCYTOSIS AND INFLAMMATION

1	Phagocytosis of microbes is a major innate host defense that limits the growth and spread of pathogens. Phagocytes appear rapidly at sites of infection in conjunction with the initiation of inflammation. Ingestion of microbes by both tissue-fixed macrophages and migrating phagocytes probably accounts for the limited ability of most microbial agents to cause disease. A family of related molecules called collectins, soluble defense collagens, or pattern-recognition molecules are found in blood (mannose-binding lectins), in lung (surfactant proteins A and D), and most likely in other tissues as well and bind to carbohydrates on microbial surfaces to promote phagocyte clearance. Bacterial pathogens seem to be ingested principally by PMNs, while eosinophils are frequently found at sites of infection by protozoan or multicellular parasites. Successful pathogens, by definition, must avoid being cleared by professional phagocytes. One of several antiphagocytic strategies employed by bacteria

1	by protozoan or multicellular parasites. Successful pathogens, by definition, must avoid being cleared by professional phagocytes. One of several antiphagocytic strategies employed by bacteria and by the fungal pathogen Cryptococcus neoformans is to elaborate large-molecular-weight surface polysaccharide antigens, often in the form of a capsule that coats the cell surface. Most pathogenic bacteria produce such antiphagocytic capsules. On occasion, proteins or polypeptides form capsule-like coatings for organisms such as group A streptococci and Bacillus anthracis.

1	As activation of local phagocytes in tissues is a key step in initiating inflammation and migration of additional phagocytes into infected sites, much attention has been paid to microbial factors that initiate inflammation. These are usually conserved factors critical to the microbes’ survival and are referred to as pathogen-associated molecular patterns (PAMPs). Cellular responses to microbial encounters with phagocytes are governed largely by the structure of the microbial 145e-5 PAMPs that elicit inflammation, and detailed knowledge of these structures of bacterial pathogens has contributed greatly to our understanding of molecular mechanisms of microbial pathogenesis mediated by activation of host cell molecules such as TLRs (Fig. 145e-3). One of the best-studied systems involves the interaction of LPS from gram-negative bacteria and the GPI-anchored membrane protein CD14 found on the surface of professional phagocytes, including migrating and tissue-fixed macrophages and PMNs. A

1	interaction of LPS from gram-negative bacteria and the GPI-anchored membrane protein CD14 found on the surface of professional phagocytes, including migrating and tissue-fixed macrophages and PMNs. A soluble form of CD14 is also found in plasma and on mucosal surfaces. A plasma protein, LPS-binding protein, transfers LPS to membrane-bound CD14 on myeloid cells and promotes binding of LPS to soluble CD14. Soluble CD14/LPS/LPS-binding protein complexes bind to many cell types and may be internalized to initiate cellular responses to microbial pathogens. It has been shown that peptidoglycan and lipoteichoic acid from gram-positive bacteria as well as cell-surface products of mycobacteria and spirochetes can interact with CD14 (Fig. 145e-3). Additional molecules, such as MD-2, also participate in the recognition of bacterial activators of inflammation.

1	GPI-anchored receptors do not have intracellular signaling domains; therefore, it is the TLRs that transduce signals for cellular activation due to LPS binding. Binding of microbial factors to TLRs to activate signal transduction occurs in the phagosome—and not on the surface—of dendritic cells that have internalized the microbe. This binding is probably due to the release of the microbial surface factor from the cell in the environment of the phagosome, where the liberated factor can bind to its cognate TLRs. TLRs initiate cellular activation through a series of signal-transducing molecules (Fig. 145e-3) that lead to nuclear translocation of the transcription factor NF-κB (nuclear factor κB), a master-switch for production of important inflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin (IL) 1.

1	The initiation of inflammation can occur not only with LPS and peptidoglycan but also with viral particles and other microbial products such as polysaccharides, enzymes, and toxins. Bacterial flagella activate inflammation by binding of a conserved sequence to TLR5. Some pathogens (e.g., Campylobacter jejuni, Helicobacter pylori, and Bartonella bacilliformis) make flagella that lack this sequence and do not bind to TLR5; thus efficient host responses to infection are prevented. Bacteria also produce a high proportion of DNA molecules with unmethylated CpG residues that activate inflammation through TLR9. TLR3 recognizes double-stranded RNA, a pattern-recognition molecule produced by many viruses during their replicative cycle. TLR1 and TLR6 associate with TLR2 to promote recognition of acylated microbial proteins and peptides.

1	The myeloid differentiation factor 88 (MyD88) molecule and the Toll/IL-1R (TIR) domain-containing adapter protein (TIRAP) bind to the cytoplasmic domains of TLRs and also to receptors that are part of the IL-1 receptor families. Numerous studies have shown that MyD88/TIRAP-mediated transduction of signals from TLRs and other receptors is critical for innate resistance to infection, activating MAPkinases and NF-κB and thereby leading to production of cytokines/ chemokines. Mice lacking MyD88 are more susceptible than normal mice to infections with a broad range of pathogens. In one study, nine children homozygous for defective MyD88 genes had recurrent infections with S. pneumoniae, S. aureus, and P. aeruginosa—three bacterial species showing increased virulence in MyD88-deficient mice; however, unlike these mice, the MyD88-deficient children seemed to have no greater susceptibility to other bacteria, viruses, fungi, or parasites. Another component of the MyD88-dependent signaling

1	however, unlike these mice, the MyD88-deficient children seemed to have no greater susceptibility to other bacteria, viruses, fungi, or parasites. Another component of the MyD88-dependent signaling pathway is a molecule known as IL-1 receptor–associated kinase 4 (IRAK-4). Individuals with a homozygous deficiency in genes encoding this protein are at increased risk for S. pneumoniae and S. aureus infections and, to some degree, for P. aeruginosa infections as well.

1	In addition to their role in MyD88-mediated signaling, some TLRs (e.g., TLR3 and TLR4) can activate signal transduction via a MyD88-independent pathway involving TIR domain–containing, adapter-inducing IFN-β (TRIF) and the TRIF-related adapter molecule (TRAM). Signaling through TRIF and TRAM activates the production of both NF-κB-dependent cytokines/chemokines and type 1 IFNs. CHAPTER 145e Molecular Mechanisms of Microbial Pathogenesis Anti-viral compounds, Inflammation, immune regulation, survival, proliferation

1	FIguRE 145e-3 Cellular signaling pathways for production of inflammatory cytokines in response to microbial products. Microbial cell-surface constituents interact with Toll-like receptors (TLRs), in some cases requiring additional factors such as MD-2, which facilitates the response to lipopolysaccharide (LPS) via TLR4. Although these constituents are depicted as interacting with the TLRs on the cell surface, TLRs contain extracellular leucine-rich domains that become localized to the lumen of the phagosome upon uptake of bacterial cells. The internalized TLRs can bind to microbial products. The TLRs are oligomerized, usually forming homodimers, and then bind to the general adapter protein MyD88 via the C-terminal Toll/IL-1R (TIR) domains, which also bind to TIRAP (TIR domain-containing adapter protein), a molecule that participates in the transduction of signals from TLRs 1, 2, 4, and 6. The MyD88/TIRAP complex activates signal-transducing molecules such as IRAK-4 (IL-1Rc-associated

1	protein), a molecule that participates in the transduction of signals from TLRs 1, 2, 4, and 6. The MyD88/TIRAP complex activates signal-transducing molecules such as IRAK-4 (IL-1Rc-associated kinase 4), which in turn activates IRAK-1. This activation can be blocked by IRAK-M and Toll-interacting protein (TOLLIP). IRAK1 activates TRAF6 (tumor necrosis factor receptor–associated factor 6), TAK1 (transforming growth factor β–activating kinase 1), and TAB1/2

1	The type 1 IFNs bind to the IFN-α receptor composed of two protein chains, IFNAR1 and IFNAR2. Humans produce three type 1 IFNs: IFN-α, IFN-β, and IFN-γ. These molecules activate another class of proteins known as the signal transducer and activator of transcription (STAT) complexes. The STAT factors are important in regulating immune system genes and thus play a critical role in responding to microbial infections.

1	Another intracellular complex of proteins found to be a major factor in the host cell response to infection is the inflammasome (Fig. 145e-4), in which inflammatory cytokines IL-1 and IL-18 are changed from their precursor to their active forms prior to secretion by the cysteine protease caspase-1. Within the inflammasome are additional proteins that are members of the nucleotide binding and oligomerization domain (NOD)–like receptor (NLR) family. Like the TLRs, NOD proteins sense the presence of the conserved microbial factors released inside a cell. Recognition of these PAMPs by NLRs leads to caspase-1 activation and to secretion of active IL-1 and IL-18 by an unknown mechanism. Studies of mice indicate that as many as four inflammasomes with different components are formed: the IPAF inflammasome, the NALP1 inflammasome, the cryopyrin/NALP3 inflammasome, and an inflammasome triggered by Francisella tularensis infection (Fig. 145e-4). The components depend on the type of stimulus

1	inflammasome, the NALP1 inflammasome, the cryopyrin/NALP3 inflammasome, and an inflammasome triggered by Francisella tularensis infection (Fig. 145e-4). The components depend on the type of stimulus driving inflammasome formation and activation.

1	A recent addition to the identified intracellular components responding to microbial infection is autophagy, initially described as an intracellular process for degradation and recycling of cellular components for reuse. Now it is clear that autophagy constitutes an early defense mechanism in which, after ingestion, microbial pathogens either within vacuoles or in the cytoplasm are delivered to lysosomal compartments for degradation. Avoidance of this process is critical if pathogens are to cause disease and can be achieved by multiple mechanisms, such as inhibition of proteins within the autophagic vacuole by shigellae, recruitment of host proteins to mask Listeria monocytogenes, and inhibition of formation of the vacuole by L. pneumophila.

1	ADDITIONAL INTERACTIONS OF MICROBIAL PATHOGENS AND PHAGOCYTES Other ways that microbial pathogens avoid destruction by phagocytes include production of factors that are toxic to these cells or that interfere with their chemotactic and ingestion function. Hemolysins, leukocidins, and the like are microbial proteins that can kill phagocytes that are attempting to ingest organisms elaborating these substances. For example, S. aureus elaborates a family of bicomponent leukocidins that bind to host receptors such as the HIV co-receptor CCR5 (which is also used by the LukE/D toxin) or—in the case of the Panton-Valentine leukocidin—the receptor of the C5a component of activated complement (which is used by LukF/S). Streptolysin O made by S. pyogenes binds to cholesterol in phagocyte membranes and initiates a process of internal degranulation, with the release of normally granule-sequestered toxic components into the phagocyte’s cytoplasm.

1	E. histolytica, an intestinal protozoan that causes amebic dysentery, can disrupt phagocyte membranes after direct contact via the release of protozoal phospholipase A and pore-forming peptides.

1	MICROBIAL SURVIVAL INSIDE PHAGOCYTES Many important microbial 145e-7 pathogens use a variety of strategies to survive inside phagocytes (particularly macrophages) after ingestion. Inhibition of fusion of the phagocytic vacuole (the phagosome) containing the ingested microbe with the lysosomal granules containing antimicrobial substances (the lysosome) allows Mycobacterium tuberculosis, S. enterica serovar Typhi, and Toxoplasma gondii to survive inside macrophages. Some organisms, such as L. monocytogenes, escape into the phagocyte’s cytoplasm to grow and eventually spread to other cells. Resistance to killing within the macrophage and subsequent growth are critical to successful infection by herpes-type viruses, measles virus, poxviruses, Salmonella, Yersinia, Legionella, Mycobacterium, Trypanosoma, Nocardia, Histoplasma, Toxoplasma, and Rickettsia. Salmonella species use a master regulatory system—in which the PhoP/PhoQ genes control other genes—to enter and survive within cells,

1	Trypanosoma, Nocardia, Histoplasma, Toxoplasma, and Rickettsia. Salmonella species use a master regulatory system—in which the PhoP/PhoQ genes control other genes—to enter and survive within cells, with intracellular survival entailing structural changes in the cell envelope LPS.

1	TISSuE INVASION AND TISSuE TROPISM Tissue Invasion Most viral pathogens cause disease by growth at skin or mucosal entry sites, but some pathogens spread from the initial site to deeper tissues. Virus can spread via the nerves (rabies virus) or plasma (picornaviruses) or within migratory blood cells (poliovirus, Epstein-Barr virus, and many others). Specific viral genes determine where and how individual viral strains can spread.

1	Bacteria may invade deeper layers of mucosal tissue via intracellular uptake by epithelial cells, traversal of epithelial cell junctions, or penetration through denuded epithelial surfaces. Among virulent Shigella strains and invasive strains of E. coli, outer-membrane proteins are critical to epithelial cell invasion and bacterial multiplication. Neisseria and Haemophilus species penetrate mucosal cells by poorly understood mechanisms before dissemination into the bloodstream. Staphylococci and streptococci elaborate a variety of extracellular enzymes, such as hyaluronidase, lipases, nucleases, and hemolysins, that are probably important in breaking down cellular and matrix structures and allowing the bacteria access to deeper tissues and blood. For example, staphylococcal α-hemolysin binds to a receptor, A-disintegrin and metalloprotease 10 (ADAM-10), to cause endothelial cell damage and disruption of vascular barrier function— events that are likely critical for systemic spread of

1	to a receptor, A-disintegrin and metalloprotease 10 (ADAM-10), to cause endothelial cell damage and disruption of vascular barrier function— events that are likely critical for systemic spread of S. aureus from an initial infectious site. Organisms that colonize the gastrointestinal tract can often translocate through the mucosa into the blood and, under circumstances in which host defenses are inadequate, cause bacteremia. Yersinia enterocolitica can invade the mucosa through the activity of the invasin protein. The complex milieu of the basement membrane–containing structures, such as laminin and collagen, that anchor epithelial cells to mucosal surfaces must often be breached. Numerous organisms express factors known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). These MSCRAMMS promote bacterial attachment to factors in the host extracellular matrix, such as laminin, collagen, and fibronectin. Additional microbial proteases, along with the host’s

1	molecules). These MSCRAMMS promote bacterial attachment to factors in the host extracellular matrix, such as laminin, collagen, and fibronectin. Additional microbial proteases, along with the host’s own surface-bound

1	CHAPTER 145e Molecular Mechanisms of Microbial Pathogenesis (TAK1-binding protein 1/2). This signaling complex associates with the ubiquitin-conjugating enzyme Ubc13 and the Ubc-like protein UEV1A to catalyze the formation of a polyubiquitin chain on TRAF6. Polyubiquitination of TRAF6 activates TAK1, which, along with TAB1/2 (a protein that binds to lysine residue 63 in polyubiquitin chains via a conserved zinc-finger domain), phosphorylates the inducible kinase complex: IKKα, IKKβ, and IKKγ. IKKγ is also called NEMO (nuclear factor κB [NF-κB] essential modulator). This large complex phosphorylates the inhibitory component of NF-κB, IκBα, resulting in release of IκBα from NF-κB. Phosphorylated (PP) IκB is then ubiquitinated (ub) and degraded, and the two components of NF-κB, p50 or Rel and p65, translocate to the nucleus, where they bind to regulatory transcriptional sites on target genes, many of which encode inflammatory proteins. In addition to inducing NF-κB nuclear translocation,

1	p65, translocate to the nucleus, where they bind to regulatory transcriptional sites on target genes, many of which encode inflammatory proteins. In addition to inducing NF-κB nuclear translocation, the TAK1/TAB1/2 complex activates MAP kinase transducers such as MKK 4/7 and MKK 3/6, which can lead to nuclear translocation of transcription factors such as AP1. TLR4 can also activate NF-κB nuclear translocation via the MyD88-independent TRIF (TIR domain–containing adapter-inducing IFN-β) and TRAM (TRIF-related adapter molecule) cofactors. Intracellular TLRs 3, 7, 8, and 9 also use MyD88 and TRIF to activate IFN response factors 3 and 7 (IRF-3 and IRF-7), which also function as transcriptional factors in the nucleus. ATP, adenosine 5’-triphosphate; ECSIT, evolutionarily conserved signaling intermediate in Toll pathways; FADD, Fas-associated protein with death domain; JNK, c-Jun N-terminal kinase; MAVS, mitochondrial antiviral signaling protein; MEKK-1, MAP/ERK kinase kinase 1; p38 MAPK,

1	in Toll pathways; FADD, Fas-associated protein with death domain; JNK, c-Jun N-terminal kinase; MAVS, mitochondrial antiviral signaling protein; MEKK-1, MAP/ERK kinase kinase 1; p38 MAPK, p38 mitogen-activated protein kinase; RIG-1, retinoic acid–inducible gene 1; TBK1, TANK-binding kinase 1. (Pathway diagram reproduced courtesy of Cell Signaling Technology, Inc. [www.cellsignal.com].)

1	PAMPs PMA TLR p50 p65 p50 p65 NF-˜B Cathepsin °pro-IL-1°pro-IL-18 pro-IL-1°pro-Casp-5 CARD8 pro-Casp-1 NLRP1 ASC pro-Casp-1 Caspase-1 NLRP3 ASC NALPs pro-Casp-1 NLRC4 ASC AIM2 pro-Casp-1 ASC pro-IL-18 IL-1 IL-18 IL-1 IL-18 I˜B Lysosome Phagosome Pannexin K+ K+ PAMPs + ATP Nigericin MDP Anthrax toxin Flagellin dsDNA P2X7 A°, asbestos, alum, cholesterols, silica, uric acid Phagolysosome

1	FIguRE 145e-4 Inflammasomes. The nucleotide-binding oligomerization domain-like receptor (NLR) family of proteins is involved in the regulation of innate immune responses. These proteins sense pathogen-associated molecular patterns (PAMPs) in the cytosol as well as the host-derived signals known as damage-associated molecular patterns (DAMPs). Certain NLRs induce the assembly of large caspase-1-activating complexes called inflammasomes. Activation of caspase-1 through autoproteolytic maturation leads to the processing and secretion of the proinflammatory cytokines interleukin 1β (IL-1β) and IL-18. So far, four inflammasomes have been identified and defined by the NLR protein that they contain: the NLRP1/NALP1b inflammasome; the NLRC4/IPAF inflammasome; the NLRP3/NALP3 inflammasome; and the AIM2 (absent in melanoma 2)–containing inflammasome. Aβ, amyloid β; ASC, apoptosis-associated speck-like protein containing CARD; ATP, adenosine 5’-triphosphate; CARD8, caspase recruitment

1	and the AIM2 (absent in melanoma 2)–containing inflammasome. Aβ, amyloid β; ASC, apoptosis-associated speck-like protein containing CARD; ATP, adenosine 5’-triphosphate; CARD8, caspase recruitment domain–containing protein 8; IκB, inhibitor of κB; IPAF, interleukin-converting enzyme protease-activating factor; MDP, muramyl dipeptide; NF-κB, nuclear factor κB; P2X7, purinergic P2X7 (receptor); PMA, phorbol myristate acetate; TLR, Toll-like receptor. (Pathway diagram reproduced with permission from Invivogen [www.invivogen.com/review-inflammasome].) plasminogen and host matrix metalloproteases, then combine to degrade the extracellular matrix and promote microbial spread. Some bacteria (e.g., brucellae) can be carried from a mucosal site to a distant site by phagocytic cells that ingest but fail to kill the bacteria.

1	Fungal pathogens almost always take advantage of host immunocompromise to spread hematogenously to deeper tissues. The AIDS epidemic has resoundingly illustrated this principle: the immunodeficiency of many HIV-infected patients permits the development of life-threatening fungal infections of the lung, blood, and brain. Other than the capsule of C. neoformans, specific fungal antigens involved in tissue invasion are not well characterized. Both fungal pathogens and protozoal pathogens (e.g., Plasmodium species and E. histolytica) undergo morphologic changes to spread within a host. C. albicans undertakes a yeast-hyphal transformation wherein the hyphal forms are found where the fungus is infiltrating the mucosal barrier of tissues, while the yeast form grows on epithelial cell surfaces as well as on the tips of hyphae that have infiltrated tissues. Malarial parasites grow in liver cells as merozoites and are released into the blood to invade erythrocytes and become trophozoites. E.

1	as well as on the tips of hyphae that have infiltrated tissues. Malarial parasites grow in liver cells as merozoites and are released into the blood to invade erythrocytes and become trophozoites. E. histolytica is found as both a cyst and a trophozoite in the intestinal lumen, through which this pathogen enters the host, but only the trophozoite form can spread systemically to cause amebic liver abscesses. Other protozoal pathogens, such as T. gondii, Giardia lamblia, and Cryptosporidium, also undergo extensive morphologic changes after initial infection to spread to other tissues.

1	Tissue Tropism The propensity of certain microbes to cause disease by infecting specific tissues has been known since the early days of bacteriology, yet the molecular basis for this propensity is understood somewhat better for viral pathogens than for other agents of infectious disease. Specific receptor-ligand interactions clearly underlie the ability of certain viruses to enter cells within tissues and disrupt normal tissue function, but the mere presence of a receptor for a virus on a target tissue is not sufficient for tissue tropism. Factors in the cell, route of viral entry, viral capacity to penetrate into cells, viral genetic elements that regulate gene expression, and pathways of viral spread in a tissue all affect tissue tropism. Some viral genes are best transcribed in specific target cells, such as hepatitis B genes in liver cells and Epstein-Barr virus genes in B lymphocytes. The route of inoculation of poliovirus determines its neurotropism, although the molecular basis

1	target cells, such as hepatitis B genes in liver cells and Epstein-Barr virus genes in B lymphocytes. The route of inoculation of poliovirus determines its neurotropism, although the molecular basis for this circumstance is not understood.

1	Compared with viral tissue tropism, the tissue tropism of bacterial and parasitic infections has not been as clearly elucidated, but studies of Neisseria species have provided insights. Both N. gonorrhoeae, which colonizes and infects the human genital tract, and N. meningitidis, which principally colonizes the human oropharynx but can spread to the brain, produce type IV pili (Tfp) that mediate adherence to host tissues. In the case of N. gonorrhoeae, the Tfp bind to a glucosamine-galactose-containing adhesin on the surface of cervical and urethral cells; in the case of N. meningitidis, the Tfp bind to cells in the human meninges and thus cross the blood-brain barrier. N. meningitidis expresses a capsular polysaccharide, while N. gonorrhoeae does not; however, there is no indication that this property plays a role in the different tissue tropisms displayed by these two bacterial species.

1	N. gonorrhoeae can use cytidine monophosphate N-acetylneuraminic acid from host tissues to add N-acetylneuraminic acid (sialic acid) to its lipooligosaccharide O side chain, and this alteration appears to make the organism resistant to host defenses. Lactate, present at high levels on genital mucosal surfaces, stimulates sialylation of gonococcal lipooligosaccharide. Bacteria with sialic acid sugars in their capsules, such as N. meningitidis, E. coli K1, and group B streptococci, have a propensity to cause meningitis, but this generalization has many exceptions. For example, all recognized serotypes of group B streptococci contain sialic acid in their capsules, but only one serotype (III) is responsible for most cases of group B streptococcal meningitis. Moreover, both H. influenzae and S. pneumoniae can readily cause meningitis, but these organisms do not have sialic acid in their capsules.

1	Disease is a complex phenomenon resulting from tissue invasion and destruction, toxin elaboration, and host response. Viruses cause much of their damage by exerting a cytopathic effect on host cells and inhibiting host defenses. The growth of bacterial, fungal, and protozoal parasites in tissue, which may or may not be accompanied by toxin elaboration, can compromise tissue function and lead to disease. For some bacterial and possibly some fungal pathogens, toxin production is one of the best-characterized molecular mechanisms of pathogenesis, while host factors such as IL-1, TNF-α, kinins, inflammatory proteins, products of complement activation, and mediators derived from arachidonic acid metabolites (leukotrienes) and cellular degranulation (histamines) readily contribute to the severity of disease.

1	Viral Disease Viral pathogens are well known to inhibit host immune responses by a variety of mechanisms. Immune responses can be affected by decreasing production of most major histocompatibility complex molecules (adenovirus E3 protein), by diminishing cytotoxic T cell recognition of virus-infected cells (Epstein-Barr virus EBNA1 antigen and cytomegalovirus IE protein), by producing virus-encoded complement receptor proteins that protect infected cells from complement-mediated lysis (herpesvirus and vaccinia virus), by making proteins that interfere with the action of IFN (influenza virus and poxvirus), and by elaborating superantigen-like proteins (mouse mammary tumor virus and related retroviruses and the rabies nucleocapsid). Superantigens activate large populations of T cells that express particular subsets of the T cell receptor β protein, causing massive cytokine release and subsequent host reactions. Another molecular mechanism of viral virulence involves the production of

1	express particular subsets of the T cell receptor β protein, causing massive cytokine release and subsequent host reactions. Another molecular mechanism of viral virulence involves the production of peptide growth factors for host cells, which disrupt normal cellular growth, proliferation, and differentiation. In addition, viral factors can bind to and interfere with the function of host receptors for signaling molecules. Modulation of cytokine production during viral infection can stimulate viral growth inside cells with receptors for the cytokine, and virus-encoded cytokine homologues (e.g., the Epstein-Barr virus BCRF1 protein, which is highly homologous to the immunoinhibitory IL-10 molecule) can potentially prevent immune-mediated clearance of viral particles. Viruses can cause disease in neural cells by interfering with levels of neurotransmitters without necessarily destroying the cells, or they may induce either programmed cell death (apoptosis) to destroy tissues or

1	disease in neural cells by interfering with levels of neurotransmitters without necessarily destroying the cells, or they may induce either programmed cell death (apoptosis) to destroy tissues or inhibitors of apoptosis to allow prolonged viral infection of cells. For infection to spread, many viruses must be released from cells. In a newly identified function, viral protein U (Vpu) of HIV facilitates the release of virus, a process that is specific to certain cells. Mammalian cells produce a restriction factor involved in inhibiting the release of virus; for HIV, this factor is designated BST-2 (bone marrow stromal antigen 2)/HM1.24/CD317, or tetherin. Vpu of HIV interacts with tetherin, promoting release of infectious virus. Overall, disruption of normal cellular and tissue function due to viral infection, replication, and release promotes clinical disease.

1	Bacterial Toxins Among the first infectious diseases to be understood were those due to toxin-elaborating bacteria. Diphtheria, botulism, and tetanus toxins are responsible for the diseases associated with local infections due to Corynebacterium diphtheriae, Clostridium botulinum, and Clostridium tetani, respectively. Clostridium difficile is an anaerobic gram-positive organism that elaborates two toxins, A and B, responsible for disruption of the intestinal mucosa when organism numbers expand in the intestine, leading to antibiotic-associated diarrhea and potentially to pseudomembranous colitis. Enterotoxins produced by E. coli, Salmonella, Shigella, Staphylococcus, and V. cholerae contribute to diarrheal disease caused by these organisms. 145e-9 Staphylococci, streptococci, P. aeruginosa, and Bordetella elaborate various toxins that cause or contribute to disease, including toxic shock syndrome toxin 1; erythrogenic toxin; exotoxins A, S, T, and U; and pertussis toxin. A number of

1	and Bordetella elaborate various toxins that cause or contribute to disease, including toxic shock syndrome toxin 1; erythrogenic toxin; exotoxins A, S, T, and U; and pertussis toxin. A number of bacterial toxins (e.g., cholera toxin, diphtheria toxin, pertussis toxin, E. coli heat-labile toxin, and P. aeruginosa exotoxin) have adenosine diphosphate ribosyl transferase activity; i.e., the toxins enzymatically catalyze the transfer of the adenosine diphosphate ribosyl portion of nicotinamide adenine diphosphate to target proteins and inactivate them. The staphylococcal enterotoxins, toxic shock syndrome toxin 1, and the streptococcal pyogenic exotoxins behave as superantigens, stimulating certain T cells to proliferate without processing of the protein toxin by antigen-presenting cells. Part of this process involves stimulation of the antigen-presenting cells to produce IL-1 and TNF-α, which have been implicated in many clinical features of diseases like toxic shock syndrome and

1	Part of this process involves stimulation of the antigen-presenting cells to produce IL-1 and TNF-α, which have been implicated in many clinical features of diseases like toxic shock syndrome and scarlet fever. A number of gram-negative pathogens (Salmonella, Yersinia, and P. aeruginosa) can inject toxins directly into host target cells by means of a complex set of proteins referred to as the type III secretion system. Loss or inactivation of this virulence system usually greatly reduces the capacity of a bacterial pathogen to cause disease.

1	Endotoxin The lipid A portion of gram-negative LPS has potent biologic activities that cause many of the clinical manifestations of gram-negative bacterial sepsis, including fever, muscle proteolysis, uncontrolled intravascular coagulation, and shock. The effects of lipid A appear to be mediated by the production of potent cytokines due to LPS binding to CD14 and signal transduction via TLRs, particularly TLR4. Cytokines exhibit potent hypothermic activity through effects on the hypothalamus; they also increase vascular permeability, alter the activity of endothelial cells, and induce endothelial-cell procoagulant activity. Numerous therapeutic strategies aimed at neutralizing the effects of endotoxin are under investigation, but so far the results have been disappointing. It has been suggested that this lack of success may be due to substantial differences between mouse and human inflammatory responses to factors such as endotoxin; thus drugs developed in mouse models of infection

1	that this lack of success may be due to substantial differences between mouse and human inflammatory responses to factors such as endotoxin; thus drugs developed in mouse models of infection may not be applicable to the human response.

1	Invasion Many diseases are caused primarily by pathogens growing in tissue sites that are normally sterile. Pneumococcal pneumonia is mostly attributable to the growth of S. pneumoniae in the lung and the attendant host inflammatory response, although specific factors that enhance this process (e.g., pneumolysin) may be responsible for some of the pathogenic potential of the pneumococcus. Disease that follows bloodstream infection and invasion of the meninges by meningitis-producing bacteria such as N. meningitidis, H. influenzae, E. coli K1, and group B streptococci appears to be due solely to the ability of these organisms to gain access to these tissues, multiply in them, and provoke cytokine production leading to tissue-damaging host inflammation. Specific molecular mechanisms accounting for tissue invasion by fungal and protozoal pathogens are less well described. Except for studies pointing to factors like capsule and melanin production by

1	Specific molecular mechanisms accounting for tissue invasion by fungal and protozoal pathogens are less well described. Except for studies pointing to factors like capsule and melanin production by C. neoformans and possibly levels of cell wall glucans in some pathogenic fungi, the molecular basis for fungal invasiveness is not well defined. Melanism has been shown to protect the fungal cell against death caused by phagocyte factors such as nitric oxide, superoxide, and hypochlorite. Morphogenic variation and production of proteases (e.g., the Candida aspartyl proteinase) have been implicated in fungal invasion of host tissues.

1	If pathogens are to effectively invade host tissues (particularly the blood), they must avoid the major host defenses represented by complement and phagocytic cells. Bacteria most often elude these defenses through their surface polysaccharides—either capsular polysaccharides or long O-side-chain antigens characteristic of the smooth LPS of gram-negative bacteria. These molecules can prevent the activation and/or deposition of complement opsonins or can limit the access of phagocytic cells with receptors for complement opsonins to these molecules when they are deposited on the bacterial surface CHAPTER 145e Molecular Mechanisms of Microbial Pathogenesis below the capsular layer. Another potential mechanism of microbial virulence is the ability of some organisms to present the capsule as an apparent self antigen through molecular mimicry. For example, the polysialic acid capsule of group B N. meningitidis is chemically identical to an oligosaccharide found on human brain cells.

1	Immunochemical studies of capsular polysaccharides have led to an appreciation of the tremendous chemical diversity that can result from the linking of a few monosaccharides. For example, three hexoses can link up in more than 300 different, potentially serologically distinct ways, while three amino acids have only six possible peptide combinations. Capsular polysaccharides have been used as effective vaccines against meningococcal meningitis as well as against pneumococcal and H. influenzae infections and may prove to be of value as vaccines against any organisms that express a nontoxic, immunogenic capsular polysaccharide. In addition, most encapsulated pathogens become virtually avirulent when capsule production is interrupted by genetic manipulation; this observation emphasizes the importance of this structure in pathogenesis. It is noteworthy that the capsule-like surface polysaccharide PNAG has been found as a conserved structure shared by many microbes but generally is a poor

1	of this structure in pathogenesis. It is noteworthy that the capsule-like surface polysaccharide PNAG has been found as a conserved structure shared by many microbes but generally is a poor target for antibody-mediated immunity because of the propensity of most humans and animals—all colonized by PNAG-producing microbes—to produce a nonprotective type of antibody. Altering the structure of PNAG by removing the acetate substituents on the N-acetylglucosamine monomers yields an immunogenic form, deacetylated PNAG, that reportedly induces antibodies that protect animals against diverse microbial pathogens.

1	Host Response The inflammatory response of the host is critical for interruption and resolution of the infectious process but is often responsible for the signs and symptoms of disease. Infection promotes a complex series of host responses involving the complement, kinin, and coagulation pathways. The production of cytokines such as IL-1, IL-18, TNF-α, IFN-γ, and other factors regulated in part by the NF-κB transcription factor leads to fever, muscle proteolysis, and other effects. An inability to kill or contain the microbe usually results in further damage due to the progression of inflammation and infection. For example, in many chronic infections, degranulation of host inflammatory cells can lead to release of host proteases, elastases, histamines, and other toxic substances that can degrade host tissues. Chronic inflammation in any tissue can lead to the destruction of that tissue and to clinical disease associated with loss of organ function, such as sterility from pelvic

1	can degrade host tissues. Chronic inflammation in any tissue can lead to the destruction of that tissue and to clinical disease associated with loss of organ function, such as sterility from pelvic inflammatory disease caused by chronic infection with N. gonorrhoeae.

1	The nature of the host response elicited by the pathogen often determines the pathology of a particular infection. Local inflammation produces local tissue damage, while systemic inflammation, such as that seen during sepsis, can result in the signs and symptoms of septic shock. The severity of septic shock is associated with the degree of production of host effectors. Disease due to intracellular parasitism results from the formation of granulomas, wherein the host attempts to wall off the parasite inside a fibrotic lesion surrounded by fused epithelial cells that make up so-called multinucleated giant cells. A number of pathogens, particularly anaerobic bacteria, staphylococci, and streptococci, provoke the formation of an abscess, probably because of the presence of zwitterionic surface polysaccharides such as the capsular polysaccharide of Bacteroides fragilis. The outcome of an infection depends on the balance between an effective host response that eliminates a pathogen and an

1	polysaccharides such as the capsular polysaccharide of Bacteroides fragilis. The outcome of an infection depends on the balance between an effective host response that eliminates a pathogen and an excessive inflammatory response that is associated with an inability to eliminate a pathogen and with the resultant tissue damage that leads to disease.

1	As part of the pathogenic process, most microbes are shed from the host, often in a form infectious for susceptible individuals. However, the rate of transmissibility may not necessarily be high, even if the disease is severe in the infected individual, as these traits are not linked. Most pathogens exit via the same route by which they entered: respiratory pathogens by aerosols from sneezing or coughing or through salivary spread, gastrointestinal pathogens by fecal-oral spread, sexually transmitted diseases by venereal spread, and vector-borne organisms by either direct contact with the vector through a blood meal or indirect contact with organisms shed into environmental sources such as water. Microbial factors that specifically promote transmission are not well characterized. Respiratory shedding is facilitated by overproduction of mucous secretions, with consequently enhanced sneezing and coughing. Diarrheal toxins such as cholera toxin, E. coli heat-labile toxins, and Shigella

1	shedding is facilitated by overproduction of mucous secretions, with consequently enhanced sneezing and coughing. Diarrheal toxins such as cholera toxin, E. coli heat-labile toxins, and Shigella toxins probably facilitate fecal-oral spread of microbial cells in the high volumes of diarrheal fluid produced during infection. The ability to produce phenotypic variants that resist hostile environmental factors (e.g., the highly resistant cysts of E. histolytica shed in feces) represents another mechanism of pathogenesis relevant to transmission. Blood parasites such as Plasmodium species change phenotype after ingestion by a mosquito—a prerequisite for the continued transmission of this pathogen. Venereally transmitted pathogens may undergo phenotypic variation due to the production of specific factors to facilitate transmission, but shedding of these pathogens into the environment does not result in the formation of infectious foci.

1	In summary, the molecular mechanisms used by pathogens to colonize, invade, infect, and disrupt the host are numerous and diverse. Each phase of the infectious process involves a variety of microbial and host factors interacting in a manner that can result in disease. Recognition of the coordinated genetic regulation of virulence factor elaboration when organisms move from their natural environment into the mammalian host emphasizes the complex nature of the host-parasite interaction. Fortunately, the need for diverse factors in successful infection and disease implies that a variety of therapeutic strategies may be developed to interrupt this process and thereby to prevent and treat microbial infections.

1	microbes with unprecedented resolution, thereby illuminating their complex and dynamic interactions with one another, the environment, and human health. The field of infectious disease genomics encompasses a vast frontier of active research that has the potential to transform clinical practice in relation to infectious diseases. While genetics has long played a key role in elucidating the process of infection and managing clinical infectious diseases, the ability to extend our thinking and our approaches beyond the study of single genes to an examination of the sequence, structure, and function of entire genomes is identifying new possibilities for research and opportunities to change clinical practice. From the development of diagnostics with unprecedented sensitivity, specificity, and speed to the design of novel public health interventions, technical and statistical genomic innovations are reshaping our understanding of the influence of the microbial world on human health and

1	and speed to the design of novel public health interventions, technical and statistical genomic innovations are reshaping our understanding of the influence of the microbial world on human health and providing us with new tools to combat infection. This chapter explores the application of genomics methods to microbial pathogens and the infections they cause (Table 146-1). It discusses innovations that are driving the development of diagnostic approaches and the discovery of new pathogens; providing insight into novel therapeutic approaches and paradigms; and advancing methods in infectious disease epidemiology and the study of pathogen evolution that can inform infection control measures, public health responses to outbreaks, and vaccine development. We draw on examples in current practice and from the recent scientific literature as signposts that point toward the ways in which the insights from pathogen genomics may influence infectious diseases in the short and long terms. Table

1	and from the recent scientific literature as signposts that point toward the ways in which the insights from pathogen genomics may influence infectious diseases in the short and long terms. Table 146-2 provides definitions for a selection of important terms used in genomics.

1	genomics and Infectious Disease Roby P. Bhattacharyya, Yonatan H. Grad, Deborah T. Hung Just as microscopy opened up the worlds of microbiology by providing a tool with which to visualize microorganisms, technological advances in genomics are now providing microbiologists with powerful new 146 methods with which to characterize the genetic map underlying all

1	The basic goals of a clinical microbiology laboratory are to establish the presence of a pathogen in a clinical sample, to identify the pathogen, and, when possible, to provide other information that can help guide clinical management and even prognosis, such as antibiotic susceptibility profiles or the presence of virulence factors. To date, clinical microbiology laboratories have largely approached these goals phenotypically by growth-based assays and biochemical testing. Bacteria, for instance, are algorithmically grouped into species by their characteristic microscopic appearance, nutrient requirements for growth, and ability to catalyze certain reactions. Antibiotic susceptibility is determined in most cases by assessing growth in the presence of antibiotic.

1	With the sequencing revolution paving the way to easy access of complete pathogen genomes (Fig. 146-1), we are now able to more systematically clarify the genetic basis of these observable phenotypes. Compared with traditional growth-based methods for bacterial diagnostics that dominate the clinical microbiology laboratory, nucleic Abbreviations: CDC, Centers for Disease Control and Prevention; HBV, hepatitis B virus; HCV, hepatitis C virus; MDR, multidrug-resistant; MERS, Middle East respiratory syndrome; MRSA, methicillin-resistant Staphylococcus aureus; NAAT, nucleic acid amplification test; PCR, polymerase chain reaction; SARS, severe acute respiratory syndrome; TB, tuberculosis; VRE, vancomycin-resistant enterococci. Contig A DNA sequence representing a continuous fragment of a genome, assembled from overlapping sequences; relevant for de novo assembly of sequence data that do not align to previously sequenced genomes

1	Genome The entire set of heritable genetic material within an organism Horizontal gene The transfer of genes between organisms through mechanisms other than by clonal descent, such as through transformation, conjugatransfer tion, or transduction Metagenomics Analysis of genetic material from multiple species directly from primary samples without requiring prior culture steps Microarray A collection of DNA oligonucleotides (“oligos”) spatially arranged on a solid surface and used to detect or quantify sequences in a sample of interest that are complementary (and therefore bind) to one or more of the arrayed oligos Mobile genetic DNA elements that can move within a genome and can be transferred between genomes through horizontal gene transfer (e.g., plaselement mids, bacteriophages, and transposons) Multilocus sequence A methodology for typing organisms based on DNA sequence fragments from a prespecified set of genes typing

1	Multilocus sequence A methodology for typing organisms based on DNA sequence fragments from a prespecified set of genes typing Next-generation High-throughput sequencing using a parallelized sequencing process that produces millions of sequences concurrently, far beyond the sequencing capacity of prior dye-terminator methods Nucleic acid amplifi-Biochemical assay that evaluates for the presence of a particular string of nucleic acids through amplification by one of several methods, cation test (NAAT) including polymerase and ligase chain reactions Polymerase chain A subset of NAAT used to amplify a specific region of DNA with specific oligonucleotide primers and a DNA polymerase reaction (PCR) Transcriptome The catalog of the full set of messenger RNA (mRNA) transcripts from a cell or organism, which are typically measured by microarray or by next-generation sequencing of complementary DNA (cDNA) via a process called RNA-Seq

1	Whole-genome A process that determines the full DNA sequence of an organism’s genome; has been greatly facilitated by next-generation sequencing sequencing technology Completed bacterial genomes from 1995 to 2012 Number of genomes FIGURE 146-1 Completed bacterial genome sequences by year, through 2012. (Data compiled from www.genomesonline.org.) acid–based diagnostics promise improved speed, sensitivity, specificity, and breadth of information. Bridging clinical and research laboratories, adaptations of genomic technologies have begun to deliver on this promise.

1	The molecular diagnostics revolution in the clinical microbiology laboratory is well under way, borne of necessity in the effort to identify microbes that are refractory to traditional culture methods. Historically, diagnosis of many so-called unculturable pathogens has relied largely on serology and antigen detection. However, these methods provide only limited clinical information because of their suboptimal sensitivity and specificity as well as the long delays that diminish their utility for real-time patient management. Newer tests to detect pathogens based on nucleic acid content have already offered improvements in the select cases to which they have been applied thus far.

1	Unlike direct pathogen detection, serologic diagnosis—measurement of the host’s response to pathogen exposure—can typically be made only in retrospect, requiring both acuteand convalescent-phase sera. For chronic infections, distinguishing active from latent infection or identifying repeat exposure by serology alone can be difficult or impossible, depending on the syndrome. In addition, the sensitivity of serologic diagnosis varies with the organism and the patient’s immune status. For instance, tuberculosis is notoriously difficult to identify by serologic methods; tuberculin skin testing using purified protein derivative (PPD) is especially insensitive in active disease and may be cross-reactive with vaccines or other mycobacteria. Even the newer interferon γ release assays (IGRAs), which measure cytokine release from T lymphocytes in response to Mycobacterium tuberculosis– specific antigens in vitro, have limited sensitivity in immunodeficient hosts. Neither PPD testing nor IGRAs

1	measure cytokine release from T lymphocytes in response to Mycobacterium tuberculosis– specific antigens in vitro, have limited sensitivity in immunodeficient hosts. Neither PPD testing nor IGRAs can distinguish latent from active infection. Serologic Lyme disease diagnostics suffer similar limitations: in patients from endemic regions, the presence of IgG antibodies to Borrelia burgdorferi may reflect prior exposure rather than active disease, while IgM antibodies are imperfectly sensitive and specific (50% and 80%, respectively, in early disease). The complex nature of these tests, particularly in view of the nonspecific symptoms that may accompany Lyme disease, has had substantial implications on public perceptions of Lyme disease and antibiotic misuse in endemic areas. Similarly, syphilis, a chronic infection caused by Treponema pallidum, is notoriously difficult to stage by serology alone, requiring the use of multiple different nontreponemal (e.g., rapid protein reagin) and

1	syphilis, a chronic infection caused by Treponema pallidum, is notoriously difficult to stage by serology alone, requiring the use of multiple different nontreponemal (e.g., rapid protein reagin) and treponemal (e.g., fluorescent treponemal antibody) tests in conjunction with clinical suspicion. Complementing serology, antigen detection can improve sensitivity and specificity in select cases but has been validated only for a limited set of infections. Typically, structural elements of pathogens are detected, including components of viral envelopes (e.g., hepatitis B surface antigen, HIV p24 antigen), cell surface markers in certain bacteria (e.g., Streptococcus pneumoniae, Legionella pneumophila serotype 1) or fungi (e.g., Cryptococcus, Histoplasma), and less specific fungal cell-wall components such as galactomannan and β-glucan (e.g., Aspergillus and other dimorphic fungi).

1	Given the impracticality of culture and the lack of sensitivity or sufficient clinical information afforded by serologic and antigenic methods, the push toward nucleic acid–based diagnostics originated in pursuit of viruses and fastidious bacteria, becoming part of the standard of care for select organisms in U.S. hospitals. Such tests, including polymerase chain reaction (PCR) and other nucleic acid amplification tests (NAATs), are now widely used for many viral infections, both chronic (e.g., HIV infection) and acute (e.g., influenza). This technique provides essential information about both the initial diagnosis and the response to therapy and in some cases genotypically predicts drug resistance. Indeed, progression from antigen detection to PCR transformed our understanding of the natural course of HIV infection, with profound implications for treatment (Fig. 146-2). In the early years of the AIDS pandemic, p24 antigenemia was detected in acute HIV infection but then disappeared

1	course of HIV infection, with profound implications for treatment (Fig. 146-2). In the early years of the AIDS pandemic, p24 antigenemia was detected in acute HIV infection but then disappeared for years before emerging again with progression to AIDS (Fig. 146-2B). Without a marker demonstrating viremia, the role of treatment during HIV infection prior to the development of clinical AIDS was uncertain, and monitoring treatment efficacy was challenging. With the emergence of PCR as a progressively more sensitive test (now able to detect as few as 20 copies of virus per milliliter of blood), viremia was recognized as a near-universal feature of HIV infection. This recognition has been transformative in guiding the initiation of therapy as well as adjustments in therapy and, together with the development of less toxic therapies, has helped to shape guidelines that now favor earlier introduction of antiretroviral therapy for HIV infection.

1	As they are for viruses, nucleic acid–based tests have become the diagnostic tests of choice for fastidious bacteria, including the common sexually transmitted intracellular bacterial pathogens Neisseria gonorrhoeae and Chlamydia trachomatis as well as the tick-borne Ehrlichia chaffeensis and Anaplasma phagocytophilum. More recently, nucleic acid amplification–based detection has offered improved sensitivity for diagnosis of the important nosocomial pathogen Clostridium difficile; NAATs can provide clinically relevant information on the presence of cytotoxins A and B as well as molecular markers of hyper-virulence such as those characterizing the recently recognized North American pulsotype 1 (NAP1), which is found more frequently in

1	FIGURE 146-2 A. Timeline of select milestones in HIV management. Genomic advances are shown in bold type. The approvals and recommendations indicated apply to the United States. ARV, antiretroviral; AZT, zidovudine; NRTI, nucleoside reverse transcriptase (RT) inhibitor; NNRTI, non-nucleoside RT inhibitor; PI, protease inhibitor. B. Viral dynamics in the natural history of HIV infection. Three diagnostic markers are shown: HIV antibody (Ab), p24 antigen (p24), and viral load (VL). Dashed gray line represents limit of detection. (Adapted from data in HH Fiebig et al: Dynamics of HIV viremia and antibody seroconversion in plasma donors: Implications for diagnosis and staging of primary HIV infection. AIDS 17:1871, 2003.) cases of severe illness. The importance of genomics in selecting loci for diagnostic assays and in monitoring test sensitivity was recently highlighted by the emergence in Sweden of a new variant of C. trachomatis containing a deletion that includes the gene targeted by

1	diagnostic assays and in monitoring test sensitivity was recently highlighted by the emergence in Sweden of a new variant of C. trachomatis containing a deletion that includes the gene targeted by a set of commercial NAATs. By evading detection through this deletion (which would have prompted the initiation of treatment), this strain came to be highly prevalent in some areas of Sweden. While nucleic acid–based tests remain the diagnostic approach of choice for fastidious bacteria, this example serves as a reminder of the need for careful development and ongoing monitoring of molecular diagnostics.

1	In contrast, for typical bacterial pathogens for which culture methods are well established, growth-based assays followed by biochemical tests still dominate in the clinical laboratory. Informed by decades of clinical microbiology, these tests have served clinicians well, yet the limitations of growth-based tests—in particular, the delays associated with waiting for growth—have left open opportunities for improvements. Molecular diagnostics, greatly informed by the vast quantity of microbial genome sequences generated in recent years, offers a way forward. First, sequencing studies may identify key genes (or noncoding nucleic acids) that can be developed into targets for clinical assays using PCR or hybridization platforms. Second, sequencing itself may eventually become inexpensive and rapid enough to be performed routinely on clinical specimens, with consequent unbiased detection of pathogens.

1	In order to adapt nucleic acid detection to diagnostic tests and thus to identify pathogens on a wide scale, sequences must be identified that are conserved enough within a species to identify the diversity of strains that may be encountered in various clinical settings, yet divergent enough to distinguish one species from another. Until recently, this problem has been solved for bacteria by targeting the element of a bacterial genome that is most highly conserved within a species: the 16S ribosomal RNA (rRNA) subunit. At present, 16S PCR amplification from 772 tissue specimens can be performed by specialty laboratories, though its sensitivity and clinical utility to date have remained somewhat limited because, for instance, of inhibitory molecules often found in clinical tissue samples that prevent reliable, sensitive PCR amplification. As such barriers are reduced through technological advances and as the causes of culture-negative infection are clarified (perhaps in part through

1	that prevent reliable, sensitive PCR amplification. As such barriers are reduced through technological advances and as the causes of culture-negative infection are clarified (perhaps in part through sequencing efforts), these tests may become both more accessible and more helpful. With the wealth of sequencing data now available, other regions beyond 16S rRNA can be targeted for bacterial species identification. These other genomic loci can provide additional information about a clinical isolate that is relevant to patient management. For instance, detection of the presence—or potentially even the expression—of toxin genes such as those for C. difficile toxins A and B or Shiga toxin may provide clinicians with additional information that will help distinguish commensals or colonizing bacteria from pathogens and thus aid in prognostication as well as diagnosis. While amplification tests such as PCR exemplify one approach to nucleic acid detection, other approaches exist, including

1	bacteria from pathogens and thus aid in prognostication as well as diagnosis. While amplification tests such as PCR exemplify one approach to nucleic acid detection, other approaches exist, including detection by hybridization. Although not currently used in the clinical realm, techniques for detection and identification of pathogens by hybridization to microarrays are being developed for other purposes. Of note, these different detection techniques require different degrees of conservation. Highly sensitive amplification methods require a high degree of sequence identity between PCR primer pairs and their short, specific target sequences; even a single base-pair mismatch (particularly near the 3′ end of the primer) may interfere with detection. In contrast, hybridization-based tests are more tolerant of mismatch and thus can be used to detect important regions that may be less precisely conserved within a species, thus potentially allowing detection of clinical isolates from a given

1	more tolerant of mismatch and thus can be used to detect important regions that may be less precisely conserved within a species, thus potentially allowing detection of clinical isolates from a given species with greater diversity between isolates. Such assays take advantage of the predictable binding interactions of nucleic acids. The applicability of hybridization-based methods toward either DNA or RNA opens up the possibility of expression profiling, which can uncover phenotypic information from nucleic acid content. Both PCR and hybridization methods target specific, known organisms. At the other extreme, as sequencing costs and turnaround times decrease, direct metagenomic sequencing from patient samples is becoming increasingly feasible. This shotgun sequencing approach is unbiased—i.e., is able to detect any microbial sequence, however divergent or unexpected. This new approach brings its own set of challenges, however, including the need to recognize pathogenic sequences

1	is able to detect any microbial sequence, however divergent or unexpected. This new approach brings its own set of challenges, however, including the need to recognize pathogenic sequences against a background of expected host and commensal sequences and to distinguish true pathogens from either colonizers or laboratory contaminants. In a powerful example of this new frontier of sequencing-based clinical diagnosis, investigators diagnosed neuroleptospirosis in a child with an unexplained encephalitis syndrome by finding sequences corresponding to the Leptospira genus in cerebrospinal fluid from the patient. Rapid (<48-h) sequencing and analysis informed the patient’s care in real time, leading to life-saving targeted antibiotic therapy for an unexpected diagnosis that was impossible to make through standard laboratory testing. The diagnosis was retrospectively confirmed through both convalescent serologies and PCR using primers designed on the basis of sequencing data.

1	In addition to clinical diagnostic applications, novel genomic technologies, including whole-genome sequencing, are being applied to clinical research specimens with a goal of identifying new pathogens in a variety of circumstances. The tremendous sensitivity and unbiased nature of sequencing is also ideal in searching clinical samples for unknown or unsuspected pathogens.

1	Causal inference in infectious diseases has progressed since the time of Koch, whose historical postulates provided a rigorous framework for attributing a disease to a microorganism. According to an updated version of Koch’s postulates, an organism, whether it can be cultured or not, should induce disease upon introduction into a healthy host if it is to be implicated as a causative pathogen. Current sequencing technologies are ideal for advancing this modern version of Koch’s postulates because they can identify candidate causal pathogens with unprecedented sensitivity and in an unbiased way, unencumbered by limitations such as culturability. Yet, as direct sequencing on primary patient samples greatly expands our ability to recognize associations between microbes and disease states, critical thinking and experimentation will continue to be vital to establishing causality.

1	Virus discovery in particular has been greatly facilitated by new nucleic acid technology. These frontiers were first notably explored with high-density microarrays containing spatially arrayed sequences from a phylogenetically diverse collection of viruses. Although biased toward those with homology to known viruses, novel viruses in clinical samples were successfully identified on the basis of their ability to hybridize to these prespecified sequences. This methodology famously contributed to identification of the coronavirus causing severe acute respiratory syndrome (SARS). Once discovered, this SARS corona-virus was rapidly sequenced: the full genome was assembled in April 2003, less than 6 months after recognition of the first case. This accomplishment illustrated the advancing power and speed of new diagnostic technologies.

1	With the advent of next-generation sequencing, unbiased pathogen discovery is now possible through a process known as metagenomic assembly (Fig. 146-3). Sequences of random nucleotide fragments can be generated from clinical specimens with no a priori knowledge of pathogen identity through a process called shotgun sequencing. This collection of sequences can then be computationally aligned to host (i.e., human) sequences, with aligned sequences removed and remaining sequences compared with other known genomes to detect the presence of known microorganisms. Sequence fragments that remain unaligned suggest the presence of an additional organism that cannot be matched to a known, characterized genome; these reads can be assembled into contiguous nucleic acid stretches that can be compared to known sequences to construct the genome of a potentially novel organism. Assembled genomes (or parts of genomes) can then be compared to known genomes to infer the phylogeny of new organisms and

1	to known sequences to construct the genome of a potentially novel organism. Assembled genomes (or parts of genomes) can then be compared to known genomes to infer the phylogeny of new organisms and identify related classes or traits. Thus, not only can this process identify unanticipated pathogens; it can even identify undiscovered organisms. Some early applications of sequencing on clinical samples have centered around the discovery of novel viruses, including such emerging pathogens as West Nile virus, SARS coronavirus, and the Middle East respiratory syndrome coronavirus (MERS-CoV) that has caused severe respiratory illnesses in healthy adults, as well as viral causes of myriad other conditions, from tropical hemorrhagic fevers to diarrhea in newborns.

1	More recently, metagenomic assembly has been successfully extended to bacterial pathogen discovery. Investigators identified a new bacterial species associated with “cord colitis”—a rare antibiotic-responsive, culture-negative colitis in recipients of umbilical cord-blood stem cells—by sequencing colon biopsy samples from affected patients and matched controls. A single dominant species emerged from metagenomic assembly in samples from patients that was absent from control samples. The presence of this species was confirmed by PCR and fluorescence in situ hybridization on primary tissue samples. On the basis of its similarity to other known species, the organism was named Bradyrhizobium enterica, a novel species from a genus that has proved difficult to culture and thus would have been hard to identify by other means. Correlation versus causation remains an open question; therefore, further efforts will be required to make such links.

1	As metagenomic sequencing and assembly techniques become more robust, this technology holds great promise for identifying microorganisms that are associated with clinical conditions of unknown etiology. Conventional methods already have unexpectedly linked numerous conditions with specific agents of infection—e.g., cervical and oropharyngeal cancers with human papillomavirus, Kaposi’s sarcoma with human herpesvirus 8, and certain lymphomas with Epstein-Barr virus. Sequencing techniques offer unprecedented sensitivity and specificity for identifying foreign nucleic acid sequences that may suggest other conditions—from malignancies to inflammatory conditions to unexplained fevers or other clinical syndromes—associated with organisms ...CCTAAGGGCTCCAGA + CTCCAGAGTTCAGTC... taxonomic assignment of reads ...CCTAAGGGCTCCAGAGTTCAGTC... host known genomes comparison to

1	host known genomes comparison to FIGURE 146-3 Workflow of metagenomic assembly for pathogen discovery. DNA is isolated from a specimen of interest (e.g., tissue, body fluid) containing a mixture of host DNA and nucleic acids from coexisting microbes, either commensal or pathogenic. All DNA (and RNA if a reverse transcription step is added) is then sequenced, yielding a mixture of DNA sequence fragments (“reads”) from organisms present. These reads are then aligned to existing reference genomes for the host or any known microbes, leaving reads that do not align (“map”) to any known sequence. These unmapped reads are then computationally assembled de novo into the largest contiguous stretches of DNA possible (“contigs”), representing fragments of previously unsequenced genomes. These genome fragments (contigs) are then mapped onto a phylogenetic tree based on their sequence. Some may represent known but as-yet-unsequenced organisms, while others will represent novel species.

1	(Figure prepared with valuable input from Dr. Ami S. Bhatt, personal communication.) from viruses to bacteria to parasites. As sequencing-based discovery expands, microbes may be found to be associated with conditions not classically thought of as infectious. Studies of bowel flora in laboratory animals and even humans are already beginning to suggest correlations between microbe composition and various aspects of metabolic and cardiovascular health. Improved methods for pathogen detection will continue to uncover unexpected correlations between microbes and disease states, but the mere presence of a microbe does not establish causality. Fortunately, once the relatively laborious and computationally intensive metagenomic sequencing and assembly efforts have identified a pathogen, further detection can easily be undertaken with targeted methods such as PCR or hybridization, which are much more straightforward and scalable. This capacity should facilitate the additional careful

1	further detection can easily be undertaken with targeted methods such as PCR or hybridization, which are much more straightforward and scalable. This capacity should facilitate the additional careful investigation that will be required to progress beyond correlation and to draw causal inference.

1	At present, antibiotic resistance in bacteria and fungi is determined by isolating a single colony from a cultured clinical specimen and testing its growth in the presence of a drug. The requirement for multiple growth steps in these conventional assays has several consequences. First, only culturable pathogens can be readily processed. Second, this process requires considerable infrastructure to support the sterile environment required for culture-based testing of diverse organisms. Finally, and perhaps most significantly, even the fastest-growing organisms require 1–2 days of processing for identification and 2–3 days for determination of susceptibilities. Slower-growing organisms take even longer: for instance, weeks must pass before drug-resistant

1	M. tuberculosis can be identified by growth phenotype. Given the clinical imperative in serious illness to begin effective therapy early, this inherent delay in susceptibility determination has obvious implications for empirical antibiotic use: broad-spectrum antibiotics often must be chosen up front in situations where it is later shown that preferred narrower-spectrum drugs would have been effective or even that no antibiotics were appropriate (i.e., in viral infections). With this strategy, the empirical choice can be incorrect, often with devastating consequences. Real-time identification of the infecting organism and information on its susceptibility profile would guide initial therapy and support judicious antibiotic use, ideally improving patient outcomes while aiding in the ever-escalating struggle with antibiotic resistance by reserving the use of broad-spectrum agents for cases in which they are truly needed.

1	Molecular diagnostics and sequencing offer a way to accelerate detection of a pathogen’s antibiotic susceptibility profile. If a genotype that confers resistance can be identified, this genotype can be targeted for molecular detection. In infectious disease, this approach has most convincingly come to fruition for HIV (Fig. 146-2A). (In a conceptually parallel application of genomic analysis, molecular detection of certain resistance determinants in cancers is beginning to inform chemotherapeutic selection.) Extensive sequencing of HIV strains and correlations drawn between viral genotypes and phenotypic resistance have delineated the majority of mutations in key HIV genes, such as reverse transcriptase, protease, and integrase, that confer resistance to the antiretroviral agents that target these proteins. For instance, the singleamino-acid substitution K103N in the HIV reverse transcriptase gene predicts resistance to the first-line nonnucleoside reverse transcriptase inhibitor

1	target these proteins. For instance, the singleamino-acid substitution K103N in the HIV reverse transcriptase gene predicts resistance to the first-line nonnucleoside reverse transcriptase inhibitor efavirenz, and its detection thus informs a clinician to choose a different agent. The effects of these common mutations on HIV susceptibility to various drugs—as well as on viral fitness—are curated in publically available databases. Thus, genotypes are now routinely used to predict drug resistance in HIV, as phenotypic resistance assays are far more cumbersome than targeted sequencing. Indeed, current recommendations in the United States are to sequence virus from a patient’s blood before initiating antiretroviral therapy, which is then tailored to the predicted resistance phenotype. As new targeted therapies are introduced, this targeted sequencing–based approach to drug resistance will likely prove important in other viral infections (e.g., hepatitis C).

1	For several reasons, the challenge of predicting antibiotic susceptibility from genotype has not yet been met in bacteria to the same degree as in HIV. In general, bacteria have evolved diverse resistance mechanisms to most antibiotics; thus, the task cannot be reduced to probing for a single genetic lesion, target, or mechanism. For instance, at least five distinct modes of resistance to fluoroquinolones are known: reduced import, increased efflux, mutated target sites, drug modification, and shielding of the target sites by expression of another protein. Further, we lack a comprehensive compendium of genetic elements conferring resistance, and new mechanisms and genes emerge regularly in the face of antibiotic deployment. As bacteria have far more complex genomes than viruses, with thousands of genes on their chromosomes and the capacity for acquiring many more through 774 horizontal gene transfer of plasmids and mobile genetic elements within and even between species, the task of

1	of genes on their chromosomes and the capacity for acquiring many more through 774 horizontal gene transfer of plasmids and mobile genetic elements within and even between species, the task of not only defining all current but also predicting all future mechanisms at a genetic level is daunting or perhaps impossible. Despite these challenges, in a few select cases where the genotypic basis for resistance has been well defined, genotypic assays for antibiotic resistance are already being introduced into clinical practice. One important example is the detection of methicillin-resistant Staphylococcus aureus (MRSA). S. aureus is one of the most common and serious bacterial pathogens of humans, particularly in health care settings. Resistance to methicillin, the most effective antistaphylococcal antibiotic, has become very common even in community-acquired strains. The alternative to methicillin, vancomycin, is effective against MRSA but measurably inferior to methicillin against

1	antibiotic, has become very common even in community-acquired strains. The alternative to methicillin, vancomycin, is effective against MRSA but measurably inferior to methicillin against methicillinsusceptible S. aureus (MSSA). Analysis of clinical MRSA isolates has demonstrated that the molecular basis for resistance to methicillin in essentially all cases stems from the expression of an alternative penicillin-binding protein (PBP2A) encoded by the gene mecA, which is found within a transferable genetic element called mec. This mobile cassette has spread rapidly through the S. aureus population via horizontal gene transfer and selection from widespread antibiotic use. Because resistance is essentially always due to the presence of the mec cassette, MRSA is amenable to molecular detection. In recent years, a PCR test for the presence of the mec cassette, which saves hours to days compared with standard culture-based methods, has been approved by the U.S. Food and Drug Administration.

1	In recent years, a PCR test for the presence of the mec cassette, which saves hours to days compared with standard culture-based methods, has been approved by the U.S. Food and Drug Administration. Additional molecular diagnostics are being implemented in the evaluation of bacterial antibiotic resistance. Vancomycin-resistant enterococci (VRE) harbor one of a limited number of van genes responsible for resistance to this important antibiotic by altering the mechanism for cell wall cross-linking that vancomycin inhibits. Detection of one of these genes by PCR indicates resistance. Identification of two carbapenemase-encoding plasmids—NDM-1 and KPC—responsible for a significant fraction of carbapenem resistance (though not for all such resistance) has led to the development of a multiplexed PCR assay to detect these important resistance elements. Because carbapenems are broad-spectrum antibiotics frequently reserved for multidrugresistant bacteria (particularly enteric gram-negative

1	PCR assay to detect these important resistance elements. Because carbapenems are broad-spectrum antibiotics frequently reserved for multidrugresistant bacteria (particularly enteric gram-negative bacilli) and are often used as antibiotics of last resort, the initial appearance of resistance and the subsequent increase in its prevalence have caused considerable concern. Therefore, even though other mechanisms for carbapenem resistance exist, a rapid PCR test for the two plasmids encoding these two carbapenemases has been developed to aid in both diagnostic and infection control efforts. Efforts are under way to extend this multiplexed PCR assay to other plasmid-borne carbapenemases and thus to make it more comprehensive. The power and application of molecular genetic tests are not limited to high-income settings. With the increasing burden of drug-resistant tuberculosis in the developing world, a molecular diagnostic test has now been developed to detect rifampin-resistant

1	not limited to high-income settings. With the increasing burden of drug-resistant tuberculosis in the developing world, a molecular diagnostic test has now been developed to detect rifampin-resistant tuberculosis. The genetic basis for rifampin resistance has been well defined by targeted sequencing: characteristic mutations in the molecular target of rifampin, RNA polymerase, account for the vast majority of rifampinresistant strains of M. tuberculosis. A PCR assay that can detect both the M. tuberculosis organism and a rifampin-resistant allele of RNA polymerase from clinical samples has recently been approved. Since rifampin resistance frequently accompanies resistance to other antibiotics, this test can suggest the possible presence of multidrug-resistant

1	M. tuberculosis within hours instead of weeks. Despite differences in relative genome complexity, HIV genotypic screening for antiretroviral resistance offers one framework for broadening efforts at genotypic assays for nonviral antibiotic resistance. As whole-genome pathogen sequencing has become increasingly feasible and inexpensive (Fig. 146-1), significant efforts have been launched to sequence hundreds to thousands of antibiotic-sensitive and -resistant isolates of a given pathogen in order to more comprehensively define resistance-conferring genetic elements. In parallel with advancing sequencing technologies, progress in computational techniques, bioinformatics and statistics, and data storage as well as experimental confirmatory testing of hypotheses will be needed to move toward the ambitious goal of a comprehensive compendium of antibiotic resistance determinants. Open sharing and careful curation of new sequence information will be of paramount importance.

1	Yet no matter how thorough and carefully curated such a genotype-phenotype database is, history suggests that comprehensively cataloguing resistance in nonviral pathogens, with new mechanisms continuously emerging, will be challenging at best. Even identifying and itemizing current resistance mutations is a daunting prospect: nonviral genomes are much larger than viral ones, and their abundance and diversity are such that hundreds to thousands of genetic differences often exist between clinical isolates, of which perhaps only one may cause resistance. For example, increasing resistance to artemisinin, one of the most effective new agents for malaria, has prompted recent large-scale efforts to identify the basis for resistance. While such studies have identified promising leads, no clear mechanism has emerged; in fact, a single genetic lesion alone may not fully account for resistance. Especially with multiple possible resistance mechanisms for a given antibiotic as well as ongoing

1	mechanism has emerged; in fact, a single genetic lesion alone may not fully account for resistance. Especially with multiple possible resistance mechanisms for a given antibiotic as well as ongoing evolutionary pressure resulting in the development and acquisition of new modes of resistance, a genotypic approach to diagnosing antibiotic resistance is likely to be imperfect.

1	We have already observed the accumulation of new or unanticipated modes of resistance from ongoing evolutionary pressure caused by the widespread clinical use of antibiotics. Even with MRSA, perhaps the best-studied case of antibiotic resistance and a model of relative simplicity with a single known monogenic resistance determinant (mecA), a genotype-based approach to resistance detection proved flawed. One limitation was a recall of the initial commercial genotypic resistance assay that was deployed for the identification of MRSA. A clinical isolate of S. aureus emerged in Belgium that expressed a variant of the mec cassette not detected by the assay’s PCR primers. New primers were added to detect this new variant, and the assay was re-approved for use. More recently, an even more divergent but functionally analogous gene called mecC, which confers methicillin resistance but evades PCR detection by this assay, was found. This series of events exemplifies the need for ongoing

1	divergent but functionally analogous gene called mecC, which confers methicillin resistance but evades PCR detection by this assay, was found. This series of events exemplifies the need for ongoing monitoring of any genotypic resistance assay. A second limitation is that a contradiction can occur between genotypic and phenotypic evidence for resistance. Up to 5% of MSSA strains carry a copy of the mecA gene that is either nonfunctional or not expressed. Thus, the erroneous identification of these strains as MRSA by genotypic detection would lead to administration of the inferior antibiotic vancomycin rather than the preferred β-lactam therapy.

1	These examples illustrate one of the prime challenges of moving beyond growth-based assays: genotype is merely a proxy for the resistance phenotype that directly informs patient care. One alternative approach currently under development attempts to circumvent the limitations of genotypic resistance testing by returning to a phenotypic approach, albeit one informed by genomic methods: transcriptional profiles serve as a rapid phenotypic signature for antibiotic response. Conceptually, since dying cells are transcriptionally distinct from cells fated to survive, susceptible bacteria enact different transcriptional profiles after antibiotic exposure that are different from the profiles of resistant strains, independent of the mechanism of resistance. These differences can be measured and, since transcription is one of the most rapid responses to cell stress (minutes to hours), can be used to determine whether cells are resistant or susceptible much more rapidly than is possible if one

1	since transcription is one of the most rapid responses to cell stress (minutes to hours), can be used to determine whether cells are resistant or susceptible much more rapidly than is possible if one waits for growth in the presence of antibiotics (days). Like DNA, RNA can be readily detected through predictable rules governing base pairing via either amplification or hybridization-based methods. Changes in a carefully selected set of transcripts form an expression signature that can represent the total cellular response to antibiotic without requiring full characterization of the entire transcriptome. Preliminary proof-of-concept studies suggest that this approach may identify antibiotic susceptibility on the basis of transcriptional phenotype much more quickly than is possible with growth-based assays.

1	Because of its sensitivity in detecting even very rare nucleic acid fragments, sequencing is now permitting studies of unprecedented depth into complex populations of cells and tissues. The strength of this depth and sensitivity applies not only to the detection of rare, novel pathogens in a sea of host signal but also to the identification of heterogeneous pathogen subpopulations in a single host that may differ, for example, in drug resistance profiles or pathogenesis determinants. Future studies will be needed to elucidate the clinical significance of these variable subpopulations, even as deep sequencing is now providing unprecedented levels of detail about majority and minority members of this population.

1	While pathogen-based diagnostics continue to be the mainstay for diagnosing infection, serologic testing has long been the basis of a strategy to diagnose infection by measuring host responses. Here, too, the application of genomics is now being explored to improve upon this approach, given the previously described limitations of serologic testing. Rather than using antibody responses as a retrospective biomarker for infection, recent efforts have focused on transcriptomic analysis of the host response as a new direction with diagnostic implications for human disease. For instance, while pathogen-based diagnostic tests to distinguish active from latent tuberculosis infection have proved elusive, recent work shows that the transcriptional profile of circulating white blood cells exhibits a differential pattern of expression of nearly 400 transcripts that distinguish active from latent tuberculosis; this expression pattern is driven in part by changes in interferon-inducible genes in

1	a differential pattern of expression of nearly 400 transcripts that distinguish active from latent tuberculosis; this expression pattern is driven in part by changes in interferon-inducible genes in the myeloid lineage. In a validation cohort, this transcriptional signature was able to distinguish patients with active versus latent disease, to distinguish tuberculosis infection from other pulmonary inflammatory states or infections, and to track responses to treatment in as little as 2 weeks, with normalization of expression toward that of patients without active disease over 6 months of effective therapy. Such a test could play an important role not only in the management of patients but also as a marker of efficacy in clinical trials of new therapeutic agents. Similarly, other investigators have been trying to identify host transcriptional signatures in circulating blood cells that are distinct in influenza A infection from those in upper respiratory infections caused by certain

1	have been trying to identify host transcriptional signatures in circulating blood cells that are distinct in influenza A infection from those in upper respiratory infections caused by certain other viruses or bacteria. These signatures also varied with phase of infection and showed promise in distinguishing exposed subjects who will become symptomatic from those who will not. These results suggest that profiling of host transcriptional dynamics could augment the information obtained from studies of pathogens, both enhancing diagnosis and monitoring the progression of illness and the response to therapy.

1	In this era of genome-wide association studies and attempts to move toward personalized medicine, genomic approaches are also being applied to the identification of host genetic loci and factors that contribute to infection susceptibility. Such loci will have undergone strong selection among populations in which the disease is endemic. By identifying the beneficial genetic alleles among individuals who survive in such settings, markers for susceptibility or resistance are being discovered; these markers can be translated into diagnostic tests to identify susceptible individuals in order to implement preventive or prophylactic interventions. Further, such studies may offer mechanistic insight into the pathogenesis of infection and inform new methods of therapeutic intervention. Such beneficial genetic associations were recognized long before the advent of genomics, as in the protective effects of the negative Duffy blood group or heterozygous hemoglobin abnormalities against Plasmodium

1	genetic associations were recognized long before the advent of genomics, as in the protective effects of the negative Duffy blood group or heterozygous hemoglobin abnormalities against Plasmodium infection. Genomic methods enable more systematic and widespread investigations of the host to identify not only people with altered susceptibility to numerous diseases (e.g., HIV infection, tuberculosis, and cholera) but also host factors that contribute to and thus might predict the severity of disease.

1	Genomics has the potential to impact infectious disease therapeutics in two ways. By transforming the speed of diagnostic information acquisition or the type of diagnostic information that can be attained, it can influence therapeutic decision-making. Alternatively, by opening up new avenues to understanding pathogenesis, providing new ways to disrupt infection, and delineating new approaches to antibiotic 775 discovery, it can facilitate the development of new therapeutic agents.

1	Efforts at antibiotic discovery are declining, with few new agents in the pipeline and even fewer entering the market. This phenomenon is due in part to the lack of economic incentives for the private sector; however, it is also attributable in part to the enormous challenges involved in the discovery and development of antibiotics. For obvious market-related reasons, nearly all efforts have focused on broad-spectrum antibiotics; the development of a chemical entity that works across an extremely diverse set of organisms (i.e., more divergent from each other than a human is from an amoeba) is far more challenging than the development of an agent that is designed to target a single bacterial species. Nevertheless, the concept of narrow-spectrum antibiotics has heretofore been rejected because of the lack of early diagnostic information that would guide the selection of such agents. Thus, rapid diagnostics providing antibiotic susceptibility information that can guide antibiotic

1	because of the lack of early diagnostic information that would guide the selection of such agents. Thus, rapid diagnostics providing antibiotic susceptibility information that can guide antibiotic selection in real time have the potential to alter and simplify antibiotic strategies by allowing a paradigm shift away from broad-spectrum drugs and toward narrow-spectrum agents. Such a paradigm shift clearly would have additional implications for the current escalation of antibiotic resistance.

1	In yet another diagnostic paradigm with the potential to impact therapeutic interventions, genomics is opening new avenues to a better understanding not only of different host susceptibilities to infection but also of different host responses to therapy. In a sense, the promise of “personalized medicine” has been a tantalizing holy grail. Some signs now point to the realization of this goal. For example, the role of glucocorticoids in tuberculous meningitis has been long debated. Recently, polymorphisms in the human genetic locus LTA4H, which encodes a leukotriene-modifying enzyme, were found to modulate the inflammatory response to tuberculosis. Patients with tuberculous meningitis who were homozygous for the proinflammatory LTA4H allele were most helped by adjunctive glucocorticoid treatment, while those who were homozygous for the anti-inflammatory allele were negatively affected by steroid treatment. This anti-inflammatory adjunct has become the standard of care in tuberculous

1	while those who were homozygous for the anti-inflammatory allele were negatively affected by steroid treatment. This anti-inflammatory adjunct has become the standard of care in tuberculous meningitis, but this study suggests that perhaps only a subset of patients benefit and further suggests a genetic means of prospectively identifying this subset. Thus, genomic diagnostic tests may eventually inform diagnosis, prognosis, and treatment decisions by revealing the pathogenic potential of the microbe and detecting host responses to both infection and therapy.

1	Genomic technologies are already dramatically changing research on host–pathogen interactions, with a goal of increasingly influencing the process of therapeutic discovery and development. Sequencing offers several possible avenues into antimicrobial therapeutic discovery. First, genomic-scale molecular methods have paved the way for comprehensive identification of all essential genes encoded within a pathogen’s genome, with consequent systematic identification of all possible vulnerabilities within a pathogen that could be targeted therapeutically. Second, transcriptional profiling can offer insights into mechanisms of action of new candidate drugs that emerge from screens. For instance, the transcriptional signature of cell wall disruptors (e.g., β-lactams) is distinct from that of DNA-damaging agents (e.g., fluoroquinolones) or protein synthesis inhibitors (e.g., aminoglycosides). Thus, transcriptional analysis of a pathogen’s response to a new antibiotic can either suggest a

1	DNA-damaging agents (e.g., fluoroquinolones) or protein synthesis inhibitors (e.g., aminoglycosides). Thus, transcriptional analysis of a pathogen’s response to a new antibiotic can either suggest a mechanism of action or flag compounds for prioritization because of a potentially novel activity. In an alternative genomic strategy for determining mechanisms of action, an RNA interference approach followed by targeted sequencing has been used to identify genes required for antitrypanosomal drug efficacy. This approach provided new insights into the mechanism of action of drugs that have been in use for decades for human African trypanosomiasis. Third, sequencing can readily identify the most conserved regions of a pathogen’s genomes and corresponding gene products; this information is invaluable in narrowing antigen candidates for vaccine development. These surface 776 proteins can be expressed recombinantly and tested for the ability to elicit a serologic response and protective

1	invaluable in narrowing antigen candidates for vaccine development. These surface 776 proteins can be expressed recombinantly and tested for the ability to elicit a serologic response and protective immunity. This process, termed reverse vaccinology, has proved particularly useful for pathogens that are difficult to culture or poorly immunogenic, as was the case with the development of a vaccine for Neisseria meningitidis serogroup B. Large-scale gene content analysis from sequencing or expression profiling enables new research directions that provide novel insights into the interplay of pathogen and host during infection or colonization. One important goal of such research is to suggest new therapeutic approaches to disrupt this interaction in favor of the host. Indeed, one of the most immediate applications of next-generation sequencing technology has come from simply characterizing human pathogens and related commensal or environmental strains and then finding genomic correlates

1	immediate applications of next-generation sequencing technology has come from simply characterizing human pathogens and related commensal or environmental strains and then finding genomic correlates for pathogenicity. For instance, as Escherichia coli varies from a simple nonpathogenic, lab-adapted strain (K-12) to a Shiga toxin–producing enterohemorrhagic gastrointestinal pathogen (O157:H7), it displays up to a 25% difference in gene content, even though its phylogenetic classification stays the same. Although this is an extreme example, it is not an isolated case. Some isolates of Enterococcus—notorious for its increasing incidence of resistance to common antibiotics such as ampicillin, vancomycin, and aminoglycosides—also contain recently acquired genetic material comprising up to 25% of the genome on mobile genetic elements. This fact suggests that horizontal gene transfer may play an important role in the organism’s adaptation as a nosocomial pathogen. On closer study, this

1	25% of the genome on mobile genetic elements. This fact suggests that horizontal gene transfer may play an important role in the organism’s adaptation as a nosocomial pathogen. On closer study, this genome expansion has been demonstrated to be associated with loss of regulatory elements called CRISPRs (clustered, regularly interspaced short palindromic repeats). Loss of CRISPR elements, which protect the bacterial genome from invasion by certain foreign genetic materials, may thus facilitate the acquisition of antibiotic resistance–conferring genetic elements. While loss of this regulation appears to impose a competitive disadvantage in antibiotic-free environments, these drug-resistant strains thrive in the presence of even some of the most useful antienterococcal therapies. In addition to insights gained from genome sequencing, extension of unbiased whole-transcriptome sequencing (RNA-Seq) efforts to bacteria is beginning to identify unexpected regulatory, noncoding RNAs in many

1	to insights gained from genome sequencing, extension of unbiased whole-transcriptome sequencing (RNA-Seq) efforts to bacteria is beginning to identify unexpected regulatory, noncoding RNAs in many diverse species. While the functional implications of these new transcripts are as yet largely unknown, the presence of such features—conserved across many bacterial species— implies evolutionary importance and suggests areas for future study and possible new therapeutic avenues. Thus, genomic studies are already beginning to transform our understanding of infection, offering evidence of virulence factors or toxins and providing insight into ongoing evolution of pathogenicity and drug resistance. One goal of such studies is to identify therapeutic agents that can disrupt the pathogenic process; there is currently much interest in the theoretical concept of antivirulence drugs that inhibit virulence factors rather than killing the pathogen outright as a means to intervene in infection.

1	there is currently much interest in the theoretical concept of antivirulence drugs that inhibit virulence factors rather than killing the pathogen outright as a means to intervene in infection. Further, as sequencing becomes increasingly accessible and efficient, large-scale studies with unprecedented statistical power to associate clinical outcomes with pathogen and host genotypes and thus to further reveal vulnerabilities in the infection process that can be targeted for disruption are being initiated. Although this is just the beginning, such studies point to a tantalizing future in which the clinician is armed with genomic predictors of infection outcome and therapeutic response to guide clinical decision-making.

1	Epidemiologic studies of infectious diseases have several main goals: to identify and characterize outbreaks, to describe the pattern and dynamics of an infectious disease as it spreads through populations, and to identify interventions that can limit or reduce the burden of disease. One classic, paradigmatic example is John Snow’s elucidation of the origin of the 1854 London cholera outbreak. Snow used careful geographic mapping of cases to determine that the likely source of the outbreak was contaminated water from the Broad Street pump, and, by removing the pump handle, he aborted the outbreak. Whereas that intervention was undertaken without knowledge of the causative agent of cholera, advances in microbiology and genomics have expanded the purview of epidemiology, which now considers not just the disease but also the pathogen, its virulence factors, and the complex relationships between microbial and host populations.

1	Through the use of novel genomic tools such as high-throughput sequencing, the diversity of a microbial population can now be rapidly described with unprecedented resolution, with discrimination between isolates that have single-nucleotide differences across the entire genome and advancement beyond prior approaches that relied on phenotypes (such as antibiotic resistance testing) or genetic markers (such as multilocus sequence typing). The development of statistical methods grounded in molecular genetics and evolutionary theory has established analytical approaches that translate descriptions of microbial population diversity and structure into insights into the origin and history of pathogen spread. By linking phylogenetic reconstruction with epidemiologic and demographic data, genomic epidemiology provides the opportunity to track transmission from person to person, to infer transmission patterns of both pathogens and sequence elements that confer phenotypes of interest, and to

1	epidemiology provides the opportunity to track transmission from person to person, to infer transmission patterns of both pathogens and sequence elements that confer phenotypes of interest, and to estimate the transmission dynamics of outbreaks.

1	The use of comparisons of whole-genome sequencing to infer person-to-person transmission and identify point-source outbreaks of pathogens has proved useful in hospital infection control settings. As reported in a seminal paper in 2010, a study of MRSA in a Thai hospital demonstrated that whole-genome sequencing can be used to infer transmission of a pathogen from patient to patient within a hospital setting through integration of the analysis of accumulation of mutations over time with dates and hospital locations of the infections. Since that time, multiple instances of the use of whole-genome sequencing to define and motivate interventions aimed at interrupting transmission chains have been reported. In another MRSA outbreak in a special-care baby unit in Cambridge, United Kingdom, whole-genome sequencing extended the traditional infection control analysis, which relies on typing organisms by their antibiotic susceptibilities, to sequencing of isolates from clinical samples. This

1	sequencing extended the traditional infection control analysis, which relies on typing organisms by their antibiotic susceptibilities, to sequencing of isolates from clinical samples. This approach identified an otherwise unrecognized outbreak of a specific MRSA strain that was occurring against a background of the usual pattern of infections caused by a diverse circulating population of MRSA strains. The analysis showed evidence of transmission among mothers within the special-care baby unit and in the community and demonstrated the key role of MRSA carriage in a single health care provider in the persistence of the outbreak. MRSA decolonization of the health care provider terminated the outbreak. In yet another example, in response to the observation of 18 cases of infection by carbapenemase-producing Klebsiella pneumoniae over 6 months at the National Institutes of Health Clinical Research Center, genome sequencing of the isolates was used to discriminate between the possibilities

1	Klebsiella pneumoniae over 6 months at the National Institutes of Health Clinical Research Center, genome sequencing of the isolates was used to discriminate between the possibilities that these cases represented multiple, independent introductions into the health care system or a single introduction with subsequent transmission. On the basis of network and phylogenetic analysis of genomic and epidemiologic data, the authors reconstructed the likely relationships among the isolates from patient to patient, demonstrating that the spread of resistant Klebsiella infection was in fact due to nosocomial transmission of a single strain.

1	Uncovering of unexpected transmission events by genomic epidemiology studies is motivating renewed questioning of pathogen ecology and modes of transmission. For example, the rise in prevalence of infections with nontuberculous mycobacteria, including Mycobacterium abscessus, among patients with cystic fibrosis (CF) has led to speculation about the possible role of patient-to-patient transmission in the CF community; however, conventional typing approaches have lacked the resolution to define population structure accurately, a critical component of inferring transmission. Past infection control guidelines discounted the possibility of acquisition of nontuberculous mycobacteria in health care settings, as no strong evidence for such transmission had been described. In a whole-genome sequencing study of M. abscessus isolates from patients with CF, an analytical approach using genome sequencing, epidemiology, and Bayesian modeling examined the likelihood of transmission between patients

1	study of M. abscessus isolates from patients with CF, an analytical approach using genome sequencing, epidemiology, and Bayesian modeling examined the likelihood of transmission between patients within a CF center; the authors found nearly identical isolates in a number of patients and observed that these isolates were less diverse than isolates from a single individual. Because no clear epidemiologic link places the infected patients in the same place at the same time, this finding highlights a need to explore preexisting notions of circumstances required for transmission and a reconsideration of M. abscessus infection control guidelines. Similar studies of other pathogens—particularly those that share human, other animal host, and environmental reservoirs—will continue to advance our insight into the relative roles and prominence of sources of infection as well as the modes of spread through populations, thereby establishing evidence-based strategies for prevention and intervention.

1	As increasing numbers of studies aim to carefully define the origins and spread of infectious agents using the high-resolution lens of whole-genome sequencing, fundamental questions are arising with regard to our understanding of infection in a single individual and the process of a single transmission event. For example, a better understanding of a pathogen population’s diversity within a single infected individual is a critical component in interpreting the relationship among isolates from different patients. While we have traditionally thought of individuals as infected with a single bacterial strain, a recent sequencing study of multiple colonies of S. aureus from a single individual showed a “cloud” of diversity; this finding raises a number of questions that will be important to address as this field develops: What is the clinical significance of this diversity? What are the processes that generate and limit diversity? What amount of diversity is transmitted under different

1	address as this field develops: What is the clinical significance of this diversity? What are the processes that generate and limit diversity? What amount of diversity is transmitted under different conditions and routes of transmission? How do the answers to these questions vary by infectious organism, type of infection, and host and in response to treatment? More comprehensive descriptions of diversity, population dynamics, transmission bottlenecks, and the forces that shape and influence the growth and spread of microbial populations will be a critically important focus of future investigations.

1	In addition to reconstructing the transmission chains of local outbreaks, genomics-based epidemiologic methods are providing insight into broad-scale geographic and temporal spread of pathogens. A classic example has been the study of cholera, the dehydrating diarrheal illness caused by infection with Vibrio cholerae. Cholera first spread worldwide from the Indian subcontinent in the 1800s and has since caused seven pandemics; the seventh pandemic has been ongoing since the 1960s. An investigation into the geographic patterns of cholera spread in the seventh pandemic used genome sequences from 777 a global collection of 154 V. cholerae strains representing isolates from 1957–2010. This investigation revealed that the seventh pandemic has comprised at least three overlapping waves spreading out from the Indian subcontinent (Fig. 146-4). Further, analysis of the genome of an isolate of V. cholerae from the 2010 outbreak of cholera in Haiti showed it to be more closely related to

1	out from the Indian subcontinent (Fig. 146-4). Further, analysis of the genome of an isolate of V. cholerae from the 2010 outbreak of cholera in Haiti showed it to be more closely related to isolates from South Asia than to isolates from neighboring Latin America, a result supporting the hypothesis that the outbreak was derived from V. cholerae introduced into Haiti by human travel (likely from Nepal) rather than by environmental or more geographically proximal sources. A subsequent study that dated the time to the most recent common ancestor of a population of V. cholerae isolates from Haiti provided further support for a single point-source introduction from Nepal.

1	Increasing numbers of investigations into the spread of many pathogens—thus far including strains of S. aureus, S. pneumoniae, Chlamydia, Salmonella, Shigella, E. coli, C. difficile, West Nile virus, rabies virus, and dengue virus—are contributing to a growing atlas of maps describing routes, patterns, and tempos of microbial diversification and dissemination. Large-scale efforts like the 100K Foodborne Pathogen Genome Project, which aims to sequence the genomes of 100,000 strains of food-borne pathogens collected from sources including food, the environment, and farm animals, are possible because of advances in sequencing technologies. Such studies will yield a vast amount of data that can be used to investigate diversity and microbiologic links within distinct niches and the patterns of spread from one niche to another. The increasingly broad adoption of genome sequencing by health care and public health institutions will ensure that the available catalog of genome sequences and

1	of spread from one niche to another. The increasingly broad adoption of genome sequencing by health care and public health institutions will ensure that the available catalog of genome sequences and associated epidemiologic data will grow very rapidly. With higher-resolution description of microbial diversity and of the dynamics of that diversity over time and across epidemiologic and demographic boundaries and evolutionary niches, we will gain even greater insights into the relationships of transmission routes and patterns of historic spread.

1	Defining pathogen transmissibility is a critical step in the development of public health surveillance and intervention strategies as this information can help predict the epidemic potential of an outbreak. Transmissibility can be estimated by a variety of methods, including inference from the growth rate of an epidemic together with the generation time of an infection (the mean interval between infection of an index case and of the people infected by that index case). Genome

1	FIGURE 146-4 Transmission events inferred from phylogenetic reconstruction of 154 Vibrio cholerae isolates from the seventh cholera pandemic. Date ranges represent estimated time to the most recent common ancestor for strains transmitted from source to destination locations, based on a Bayesian model of the phylogeny. (Reprinted with permission from A Mutreja et al: Evidence for several waves of global transmission in the seventh cholera pandemic. Nature 477:462, 2011.) 778 sequencing and analysis of a well-sampled population provide another method by which to derive similar fundamental epidemiologic parameters. One key measure of transmissibility is the basic reproduction number (R0), defined as the number of secondary infections generated from a single primary infectious case. When the basic reproduction number is greater than 1 (R0 >1), an outbreak has epidemic potential; when it is less than 1 (R0 <1), the outbreak will become extinct. On the basis of sequences from influenza

1	the basic reproduction number is greater than 1 (R0 >1), an outbreak has epidemic potential; when it is less than 1 (R0 <1), the outbreak will become extinct. On the basis of sequences from influenza samples obtained from infected patients very early in the 2009 H1N1 influenza pandemic, the basic reproduction number was estimated through a population genomic analysis at 1.2; this compared to estimates of 1.4–1.6 based on several epidemiologic analyses. In addition, with the assumption of a molecular clock model, sequences of H1N1 samples together with information about when and where the samples were obtained have been used to estimate the date and location of the pandemic’s origin, providing insight into disease origins and dynamics. Because the magnitude and intensity of the public health response are guided by the predicted size of an outbreak, the ability of genomic methods to elucidate a pathogen’s origin and epidemic potential adds an important dimension to the contributions of

1	response are guided by the predicted size of an outbreak, the ability of genomic methods to elucidate a pathogen’s origin and epidemic potential adds an important dimension to the contributions of these methods to infectious disease epidemiology.

1	Beyond describing transmission and dynamics, pathogen genomics can provide insight into the evolution of pathogens and the interaction of selective pressures, the host, and pathogen populations, which can have implications for vaccine or therapeutic development. From a clinical perspective, this process is central to the acquisition of antibiotic resistance, the generation of increasing pathogenicity or new virulence traits, the evasion of host immunity and clearance (leading to chronic infection), and vaccine efficacy.

1	Microbial genomes evolve through a variety of mechanisms, including mutation, duplication, insertion, deletion, recombination, and horizontal gene transfer. Segmented viruses (e.g., influenza virus) can reassort gene segments within multiply infected cells. The pandemic 2009 H1N1 influenza A virus, for example, appears to have been generated through reassortment of several avian, swine, and human influenza strains. Such potential for the evolution of novel pandemic strains has precipitated concern about the possible evolution to transmissibility of virulent strains that have been associated with high mortality rates but have not yet exhibited efficient human infectivity. Controversial experiments with H5N1 avian influenza virus, for example, have defined five mutations that render the virus transmissible, at least in ferrets—the animal model system for human influenza.

1	The continual antigenic evolution of seasonal influenza offers an example of how studies of pathogen evolution can impact surveillance and vaccine development. Frequent updates to the annual influenza vaccine are needed to ensure protection against the dominant strains. These updates are based on an ability to anticipate which viral populations from a pool of substantial locally and globally diverse circulating viruses will predominate in the upcoming season. Toward that end, sequencing-based studies of influenza virus dynamics have shed light on the global spread of influenza, providing concrete data on patterns of spread and helping to elucidate the origins, emergence, and circulation of novel strains. Through analysis of more than 1000 influenza A H3N2 virus isolates over the 2002–2007 influenza seasons, Southeast Asia was identified as the usual site from which diversity originates and spreads worldwide. Further studies of global isolate collections have shed further light on the

1	influenza seasons, Southeast Asia was identified as the usual site from which diversity originates and spreads worldwide. Further studies of global isolate collections have shed further light on the diversity of circulating virus, showing that some strains persist and circulate outside of Asia for multiple seasons.

1	Not only do genomic epidemiology studies have the potential to help guide vaccine selection and development; they are also helping to track what happens to pathogens circulating in the population in response to vaccination. By describing pathogen evolution under the selective pressure of a vaccinated population, such studies can play a key role in surveillance and identification of virulence determinants and perhaps may even help to predict the future evolution of escape from vaccine protection. The 7-valent pneumococcal conjugate vaccine (PCV-7) targeted the seven serotypes of S. pneumoniae responsible for the majority of cases of invasive disease at the time of its introduction in 2000; since then, PCV-7 has dramatically reduced the incidence of pneumococcal disease and mortality. Population genomic analysis of the sequences of more than 600 Massachusetts pneumococcal isolates from 2001–2007 has shown that preexisting rare nonvaccine serotypes are replacing vaccine serotypes and

1	genomic analysis of the sequences of more than 600 Massachusetts pneumococcal isolates from 2001–2007 has shown that preexisting rare nonvaccine serotypes are replacing vaccine serotypes and that some strains have persisted despite vaccination by recombining the vaccine-targeted capsule locus with a cassette of capsule genes from non-vaccine-targeted serotypes.

1	While cutting-edge genomic technologies are largely imple mented in the developed world, their application to infectious diseases offers perhaps the biggest potential impact in less developed regions where the burden of these infections is greatest. This globalization of genomic technology and its extensions has already begun in each of the areas of focus highlighted in this chapter; it has occurred both through the application of advanced technologies to samples collected in the developing world and through the adaptation and importation of technologies directly to the developing world for on-site implementation as they become more globally accessible. Genomic characterization of the pathogens responsible for important global illnesses such as tuberculosis, malaria, trypanosomiasis, and cholera has led to insights in diagnosis, treatment, and infection control. For instance, the nucleic acid–based test developed for rapid diagnosis of M. tuberculosis infection and detection of

1	and cholera has led to insights in diagnosis, treatment, and infection control. For instance, the nucleic acid–based test developed for rapid diagnosis of M. tuberculosis infection and detection of rifampin resistance is being priced for implementation in field settings in Africa and Asia where tuberculosis is most prevalent. The potential to diagnose multidrug-resistant tuberculosis in hours instead of weeks or months may truly revolutionize treatment and control of this common and devastating illness. High-resolution genomic tracking of the spread of cholera has yielded insights into which public health measures may prove most effective in controlling local epidemics. Overall, sequencing efforts have become exponentially cheaper with each passing year. As these technologies synergize with efforts to globalize information-technology resources, global implementation of genomic methods promises to spread state-of-the-art methods for diagnosis, treatment, and epidemic tracking of

1	with efforts to globalize information-technology resources, global implementation of genomic methods promises to spread state-of-the-art methods for diagnosis, treatment, and epidemic tracking of infections to areas that need these capabilities the most.

1	By illuminating the genetic information that encodes the most fundamental processes of life, genomic technologies are transforming many aspects of medicine. In infectious diseases, methods such as next-generation sequencing and genome-scale expression analysis offer information of unprecedented depth about individual microbes as well as microbial communities. This information is expanding our understanding of the interactions of these microorganisms and communities with one another, with their human hosts, and with the environment. Despite significant progress and the abundant genomic data now available, technological and financial barriers continue to impede the widespread adoption of large-scale pathogen sequencing in clinical, public health, and research settings. As even vaster amounts of data are generated, innovations in storage, development of bioinformatics tools to manipulate the data, standardization of methods, and training of end-users in both the research and clinical

1	of data are generated, innovations in storage, development of bioinformatics tools to manipulate the data, standardization of methods, and training of end-users in both the research and clinical realms will be required. The cost-effectiveness and applicability of whole-genome sequencing, particularly in the clinic, remain to be studied, and studies of the impact of genome sequencing on patient outcomes will be needed to clarify the contexts in which these new methodologies can make the greatest contributions to patient well-being. The ongoing efforts to overcome limitations through collaboration, teaching, and reduction of financial obstacles should be applauded and expanded. With advances in genomic technologies and computational analysis, our ability to detect, characterize, treat, monitor, prevent, and control infections has progressed rapidly in recent years and will continue to do so, with the hope of heralding a new era where the clinician is better armed to combat infection and

1	prevent, and control infections has progressed rapidly in recent years and will continue to do so, with the hope of heralding a new era where the clinician is better armed to combat infection and promote human health.

1	Approach to the Acutely Ill Infected febrile Patient Tamar F. Barlam, Dennis L. Kasper The physician treating the acutely ill febrile patient must be able to recognize infections that require emergent attention. If such infections 147 Approach to the Acutely Ill Infected Febrile Patient are not adequately evaluated and treated at initial presentation, the opportunity to alter an adverse outcome may be lost. In this chapter, the clinical presentations of and approach to patients with relatively common infectious disease emergencies are discussed. These infectious processes and their treatments are discussed in detail in other chapters. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: Before the history is elicited and a physical examination is performed, an immediate assessment of the patient’s general appearance can yield valuable information. The perceptive physician’s subjective sense that a patient is septic or toxic often proves accurate. Visible agitation or anxiety in a febrile patient can be a harbinger of critical illness.

1	HISTORY Presenting symptoms are frequently nonspecific. Detailed questions should be asked about the onset and duration of symptoms and about changes in severity or rate of progression over time. Host factors and comorbid conditions may increase the risk of infection with certain organisms or of a more fulminant course than is usually seen. Lack of splenic function, alcoholism with significant liver disease, IV drug use, HIV infection, diabetes, malignancy, organ transplantation, and chemotherapy all predispose to specific infections and frequently to increased severity. The patient should be questioned about factors that might help identify a nidus for invasive infection, such as recent upper respiratory tract infections, influenza, or varicella; prior trauma; disruption of cutaneous barriers due to lacerations, burns, surgery, body piercing, or decubiti; and the presence of foreign bodies, such as nasal packing after rhinoplasty, tampons, or prosthetic joints. Travel, contact with

1	due to lacerations, burns, surgery, body piercing, or decubiti; and the presence of foreign bodies, such as nasal packing after rhinoplasty, tampons, or prosthetic joints. Travel, contact with pets or other animals, or activities that might result in tick or mosquito exposure can lead to diagnoses that would not otherwise be considered. Recent dietary intake, medication use, social or occupational contact with ill individuals, vaccination history, recent sexual contacts, and menstrual history may be relevant. A review of systems should focus on any neurologic signs or sensorium alterations, rashes or skin lesions, and focal pain or tenderness and should also include a general review of respiratory, gastrointestinal, or genitourinary symptoms.

1	PHYSICAL EXAMINATION A complete physical examination should be performed, with special attention to several areas that are sometimes given short shrift in routine examinations. Assessment of the patient’s general appearance and vital signs, skin and soft tissue examination, and the neurologic evaluation are of particular importance. The patient may appear either anxious and agitated or lethargic and apathetic. Fever is usually present, although elderly patients and compromised hosts (e.g., patients who are uremic or cirrhotic and those who are taking glucocorticoids or nonsteroidal anti-inflammatory drugs) may be afebrile despite serious underlying infection. Measurement of blood pressure, heart rate, and respiratory rate helps determine the degree of hemodynamic and metabolic compromise. The patient’s airway must be evaluated to rule out the risk of obstruction from an invasive oropharyngeal infection.

1	The etiologic diagnosis may become evident in the context of a thorough skin examination (Chap. 24). Petechial rashes are typically seen with meningococcemia or Rocky Mountain spotted fever (RMSF; see Fig. 25e-16); erythroderma is associated with toxic shock syndrome (TSS) and drug fever. The soft tissue and muscle examination is critical. Areas of erythema or duskiness, edema, and tenderness may indicate underlying necrotizing fasciitis, myositis, or myonecrosis. The neurologic examination must include a careful assessment of mental status for signs of early encephalopathy. Evidence of nuchal rigidity or focal neurologic findings should be sought.

1	DIAGNOSTIC WORKUP After a quick clinical assessment, diagnostic material should be obtained rapidly and antibiotic and supportive treatment begun. Blood (for cultures; baseline complete blood count with differential; measurement of serum electrolytes, blood urea nitrogen, serum creatinine, and serum glucose; and liver function tests) can be obtained at the time an IV line is placed and before antibiotics are administered. The blood lactate concentration also should be measured. Three sets of blood cultures should be performed for patients with possible acute endocarditis. Asplenic patients should have a buffy coat examined for bacteria; these patients can have >106 organisms per milliliter of blood (compared with 104/mL in patients with an intact spleen). Blood smears from patients at risk for severe parasitic disease, such as malaria or babesiosis (Chap. 250e), must be examined for the diagnosis and quantitation of parasitemia. Blood smears may also be diagnostic in ehrlichiosis and

1	for severe parasitic disease, such as malaria or babesiosis (Chap. 250e), must be examined for the diagnosis and quantitation of parasitemia. Blood smears may also be diagnostic in ehrlichiosis and anaplasmosis.

1	Patients with possible meningitis should have cerebrospinal fluid (CSF) drawn before the initiation of antibiotic therapy. Focal findings, depressed mental status, or papilledema should be evaluated by brain imaging prior to lumbar puncture, which, in this setting, could initiate herniation. Antibiotics should be administered before imaging but after blood for cultures has been drawn. If CSF cultures are negative, blood cultures will provide the diagnosis in 50–70% of cases. Molecular diagnostic techniques (e.g., broad-range 16S rRNA gene polymerase chain reaction testing for bacterial meningitis pathogens) are of increasing importance in the rapid diagnosis of life-threatening infections.

1	Focal abscesses necessitate immediate CT or MRI as part of an evaluation for surgical intervention. Other diagnostic procedures, such as wound cultures, should not delay the initiation of treatment for more than minutes. Once emergent evaluation, diagnostic procedures, and (if appropriate) surgical consultation (see below) have been completed, other laboratory tests can be conducted. Appropriate radiography, computed axial tomography, MRI, urinalysis, erythrocyte sedimentation rate and C-reactive protein determination, and transthoracic or transesophageal echocardiography all may prove important.

1	In the acutely ill patient, empirical antibiotic therapy is critical and should be administered without undue delay. Increased prevalence of antibiotic resistance in community-acquired bacteria must be considered when antibiotics are selected. Table 147-1 lists first-line empirical regimens for infections considered in this chapter. In addition to the rapid initiation of antibiotic therapy, several of these infections require urgent surgical attention. Neurosurgical evaluation for subdural empyema, otolaryngologic surgery for possible mucormycosis, and cardiothoracic surgery for critically ill patients with acute endocarditis are as important as antibiotic therapy. For infections such as necrotizing fasciitis and clostridial myonecrosis, rapid surgical intervention supersedes other diagnostic or therapeutic maneuvers.

1	Adjunctive treatments may reduce morbidity and mortality rates and include dexamethasone for bacterial meningitis or IV immunoglobulin for TSS and necrotizing fasciitis caused by group A Streptococcus. Adjunctive therapies should usually be initiated within the first hours of treatment; however, dexamethasone for Clinical Syndrome Possible Etiologies Treatment Comments See Chap(s). Meningococcemia N. meningitidis Penicillin (4 mU q4h) or ceftriaxone Consider protein C replacement, if available, in 180 (2 g q12h) fulminant meningococcemia. Drotrecogin alfa (activated) is no longer produced. Rocky Mountain Rickettsia rickettsii Doxycycline (100 mg bid) If both meningococcemia and RMSF are being 211 spotted fever (RMSF) considered, use ceftriaxone (2 g q12h) plus doxycycline (100 mg bid). If RMSF is diagnosed, doxycycline is the proven superior agent. Purpura fulminans S. pneumoniae, H. influenzae, Ceftriaxone (2 g q12h) plus If a β-lactam-sensitive strain is identified, 171, 180,

1	Purpura fulminans S. pneumoniae, H. influenzae, Ceftriaxone (2 g q12h) plus If a β-lactam-sensitive strain is identified, 171, 180, N. meningitidis vancomycin (15 mg/kg q12h)b vancomycin can be discontinued. 182, 325 Erythroderma: toxic Group A Streptococcus, Vancomycin (15 mg/kg q12h)b plus If a penicillinor oxacillin-sensitive strain is 172, 173 shock syndrome Staphylococcus aureus clindamycin (600 mg q8h isolated, these agents are superior to vancomycin (penicillin, 2 mU q4h; or oxacillin, 2 g IV q4h). The site of toxigenic bacteria should be debrided; IV immunoglobulin can be used in severe cases.d Sepsis with Soft Tissue Findings Necrotizing fasciitis Group A Streptococcus, mixed Vancomycin (15 mg/kg q12h)b plus Urgent surgical evaluation is critical. Adjust 156, 172, aerobic/anaerobic flora, clindamycin (600 mg q8h) plus treatment when culture data become available. 173 CA-MRSAe gentamicin (5 mg/kg q8h)

1	Clostridial myonecrosis Clostridium perfringens Penicillin (2 mU q4h) plus clindamy-Urgent surgical evaluation is critical. 179 cin (600 mg q8h) Bacterial meningitis S. pneumoniae, N. meningitidis Brain abscess, Streptococcus spp., suppurative intracranial Staphylococcus spp., infections anaerobes, gram-negative Spinal epidural abscess Staphylococcus spp., gram-negative bacilli Artesunate (2.4 mg/kg IV at 0, 12, and 24 h; then once daily)f or quinine (IV loading dose of 20 mg salt/kg; then 10 mg/kg q8h) Vancomycin (15 mg/kg q12h)b plus ceftriaxone (2 g q24h) If a β-lactam-sensitive strain is identified, vancomycin can be discontinued. If the patient is >50 years old or has comorbid disease, add ampicillin (2 g q4h) for Listeria coverage. Dexamethasone (10 mg q6h × 4 days) improves outcome in adults with meningitis (especially pneumococcal) and cloudy CSF, positive CSF Gram’s stain, or a CSF leukocyte count >1000/mL.

1	Urgent surgical evaluation is critical. If a penicillinor oxacillin-sensitive strain is isolated, these agents are superior to vancomycin (penicillin, 4 mU q4h; or oxacillin, 2 g q4h). Do not use glucocorticoids. Use IV quinidine if IV quinine is not available. During IV quinidine treatment, blood pressure and cardiac function should be monitored continuously and blood glucose periodically. Surgical evaluation is essential. If a penicillinor oxacillin-sensitive strain is isolated, these agents are superior to vancomycin (penicillin, 4 mU q4h; or oxacillin, 2 g q4h). 246e, 248 Acute bacterial S. aureus, β-hemolytic Ceftriaxone (2 g q12h) plus Adjust treatment when culture data become 155 endocarditis streptococci, HACEK group,g vancomycin (15 mg/kg q12h)b available. Surgical evaluation is essential.

1	Neisseria spp., S. pneumoniae aThese empirical regimens include coverage for gram-positive pathogens that are resistant to β-lactam antibiotics. Local resistance patterns should be considered and may alter the need for empirical vancomycin. bA vancomycin loading dose of 20–25 mg/kg can be considered in critically ill patients. cβ-Lactam antibiotics may exhibit unpredictable pharmacodynamics in sepsis. Prolonged or continuous infusions can be considered. dThe optimal dose of IV immunoglobulin has not been determined, but the median dose in observational studies is 2 g/kg (total dose administered for 1–5 days). eCommunity-acquired methicillin-resistant S. aureus. fIn the United States, artesunate must be obtained through the Centers for Disease Control and Prevention. For patients diagnosed with severe malaria, full doses of parenteral antimalarial treatment should be started with whichever recommended antimalarial agent is first available. gHaemophilus species, Aggregatibacter species,

1	with severe malaria, full doses of parenteral antimalarial treatment should be started with whichever recommended antimalarial agent is first available. gHaemophilus species, Aggregatibacter species, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae.

1	bacterial meningitis must be given before or at the time of the first dose of antibiotic. Glucocorticoids can also be harmful, sometimes resulting in worse outcomes—e.g., when given in the setting of cerebral malaria or viral hepatitis. The infections considered below according to common clinical presentation can have rapidly catastrophic outcomes, and their immediate recognition and treatment can be life-saving. Recommended empirical therapeutic regimens are presented in Table 147-1. Patients initially have a brief prodrome of nonspecific symptoms and signs that progresses quickly to hemodynamic instability with hypo-tension, tachycardia, tachypnea, respiratory distress, and altered mental status. Disseminated intravascular coagulation (DIC) with clinical evidence of a hemorrhagic diathesis is a poor prognostic sign.

1	Septic Shock (See also Chap. 325) Patients with bacteremia leading to septic shock may have a primary site of infection (e.g., pneumonia, pyelonephritis, or cholangitis) that is not evident initially. Elderly patients with comorbid conditions, hosts compromised by malignancy and neutropenia, and patients who have recently undergone a surgical procedure or hospitalization are at increased risk for an adverse outcome. Gram-negative bacteremia with organisms such as Pseudomonas aeruginosa or Escherichia coli and gram-positive infection with organisms such as Staphylococcus aureus (including methicillin-resistant S. aureus [MRSA]) or group A streptococci can present as intractable hypotension and multiorgan failure. Treatment can usually be initiated empirically on the basis of the presentation, host factors (Chap. 325), and local patterns of bacterial resistance. Outcomes are worse when antimicrobial treatment is delayed or when the responsible pathogen ultimately proves not to be

1	host factors (Chap. 325), and local patterns of bacterial resistance. Outcomes are worse when antimicrobial treatment is delayed or when the responsible pathogen ultimately proves not to be susceptible to the initial regimen. Broad-spectrum antimicrobial agents are therefore recommended and should be instituted rapidly, preferably within the first hour after presentation. Risk factors for fungal infection should be assessed, as the incidence of fungal septic shock is increasing. Biomarkers such as C-reactive protein and procalcitonin have not proved reliable diagnostically but, when measured over time, can facilitate appropriate de-escalation of therapy. Glucocorticoids should be considered only for patients with severe sepsis who do not respond to fluid resuscitation and vasopressor therapy.

1	Overwhelming Infection in Asplenic Patients (See also Chap. 325) Patients without splenic function are at risk for overwhelming bacterial sepsis. Asplenic adult patients succumb to sepsis at 58 times the rate of the general population. Most infections are thought to occur within the first 2 years after splenectomy, with a mortality rate of ~50%, but the increased risk persists throughout life. In asplenia, encapsulated bacteria cause the majority of infections. Adults, who are more likely to have antibody to these organisms, are at lower risk than children. Streptococcus pneumoniae is the most common isolate, causing 50–70% of cases, but the risk of infection with Haemophilus influenzae or Neisseria meningitidis is also high. Severe clinical manifestations of infections due to E. coli, S. aureus, group B streptococci, P. aeruginosa, Bordetella holmesii, and Capnocytophaga, Babesia, and Plasmodium species have been described.

1	Babesiosis (See also Chap. 249) A history of recent travel to endemic areas raises the possibility of infection with Babesia. Between 1 and 4 weeks after a tick bite, the patient experiences chills, fatigue, anorexia, myalgia, arthralgia, shortness of breath, nausea, and headache; ecchymosis and/or petechiae are occasionally seen. The tick that most commonly transmits Babesia, Ixodes scapularis, also transmits Borrelia burgdorferi (the agent of Lyme disease) and Anaplasma; co-infection can occur, resulting in more severe disease. Infection with the European species Babesia divergens is more frequently fulminant than that due to the U.S. species Babesia microti. B. divergens causes a febrile syndrome with hemolysis, jaundice, hemoglobinemia, and renal failure and is associated with a mortality rate of >40%. Severe babesiosis is especially common in asplenic hosts but does occur in hosts with 781 normal splenic function, particularly those >60 years of age and those with underlying

1	rate of >40%. Severe babesiosis is especially common in asplenic hosts but does occur in hosts with 781 normal splenic function, particularly those >60 years of age and those with underlying immunosuppressive conditions such as HIV infection or malignancy. Complications include renal failure, acute respiratory failure, and DIC.

1	Other Sepsis Syndromes Tularemia (Chap. 195) is seen throughout the United States but occurs primarily in Arkansas, Missouri, South Dakota, and Oklahoma. This disease is associated with wild rabbit, tick, and tabanid fly contact. It can be transmitted by arthropod bite, handling of infected animal carcasses, consumption of contaminated food and water, or inhalation. The typhoidal form can be associated with gram-negative septic shock and a mortality rate of >30%, especially in patients with underlying comorbid or immunosuppressive conditions. Plague occurs infrequently in the United States (Chap. 196), primarily after contact with ground squirrels, prairie dogs, or chipmunks, but is endemic in other parts of the world, with >90% of all cases occurring in Africa. The septic form is particularly rare and is associated with shock, multiorgan failure, and a 30% mortality rate. These infections should be considered in the appropriate epidemiologic setting. The Centers for Disease Control

1	rare and is associated with shock, multiorgan failure, and a 30% mortality rate. These infections should be considered in the appropriate epidemiologic setting. The Centers for Disease Control and Prevention lists Francisella tularensis and Yersinia pestis (the agents of tularemia and plague, respectively) along with Bacillus anthracis (the agent of anthrax) as important organisms that might be used for bioterrorism (Chap. 261e).

1	(See also Chap. 24) Maculopapular rashes may reflect early meningococcal or rickettsial disease but are usually associated with nonemergent infections. Exanthems are usually viral. Primary HIV infection commonly presents with a rash that is typically maculopapular and involves the upper part of the body but can spread to the palms and soles. The patient is usually febrile and can have lymphadenopathy, severe headache, dysphagia, diarrhea, myalgias, and arthralgias. Recognition of this syndrome provides an opportunity to prevent transmission and to institute treatment and monitoring early on. Petechial rashes caused by viruses are seldom associated with hypotension or a toxic appearance, although there can be exceptions (e.g., severe measles or arboviral infection). Petechial rashes limited to the distribution of the superior vena cava are rarely associated with severe disease. In other settings, petechial rashes require more urgent attention.

1	Meningococcemia (See also Chap. 180) Almost three-quarters of patients with N. meningitidis bacteremia have a rash.

1	Meningococcemia most often affects young children (i.e., those 6 months to 5 years old). In sub-Saharan Africa, the high prevalence of serogroup A meningococcal disease has been a threat to public health for more than a century. Thousands of deaths occur annually in this area, which is known as the “meningitis belt,” and large epidemic waves occur approximately every 8–12 years. Serogroups W135 and X are also important emerging pathogens in Africa. In the United States, sporadic cases and outbreaks occur in day-care centers, schools (grade school through college, particularly among college freshmen living in residential halls), and army barracks. Household contacts of index cases are at 400–800 times greater risk of disease than the general population. Patients may exhibit fever, headache, nausea, vomiting, myalgias, changes in mental status, and meningismus. However, the rapidly progressive form of disease is not usually associated with meningitis. The rash is initially pink,

1	nausea, vomiting, myalgias, changes in mental status, and meningismus. However, the rapidly progressive form of disease is not usually associated with meningitis. The rash is initially pink, blanching, and maculopapular, appearing on the trunk and extremities, but then becomes hemorrhagic, forming petechiae. Petechiae are first seen at the ankles, wrists, axillae, mucosal surfaces, and palpebral and bulbar conjunctiva, with subsequent spread on the lower extremities and to the trunk. A cluster of petechiae may be seen at pressure points—e.g., where a blood pressure cuff has been inflated. In rapidly progressive meningococcemia (10–20% of cases), the petechial rash quickly becomes purpuric (see Fig. 70-5), and patients develop DIC, multiorgan failure, and shock; 50–60% of these patients die, and survivors often require extensive debridement or amputation of gangrenous extremities.

1	Approach to the Acutely Ill Infected Febrile Patient 782 Hypotension with petechiae for <12 h is associated with significant mortality. Cyanosis, coma, oliguria, metabolic acidosis, and elevated partial thromboplastin time also are associated with a fatal outcome. Correction of protein C deficiency may improve outcome. Antibiotics given in the office by the primary care provider before hospital evaluation and admission may improve prognosis; this observation suggests that early initiation of treatment may be life-saving. Meningococcal conjugate vaccines are protective against serogroups A, C, Y and W135 and are recommended for children 11–18 years of age and for other high-risk patients.

1	Rocky Mountain Spotted Fever (See also Chap. 211) RMSF is a tick-borne disease caused by Rickettsia rickettsii that occurs throughout North and South America. Up to 40% of patients do not report a history of a tick bite, but a history of travel or outdoor activity (e.g., camping in tick-infested areas) can often be ascertained. For the first 3 days, headache, fever, malaise, myalgias, nausea, vomiting, and anorexia are documented. By day 3, half of patients have skin findings. Blanching macules develop initially on the wrists and ankles and then spread over the legs and trunk. The lesions become hemorrhagic and are frequently petechial. The rash spreads to palms and soles later in the course. The centripetal spread is a classic feature of RMSF but occurs in a minority of patients. Moreover, 10–15% of patients with RMSF never develop a rash. The patient can be hypotensive and develop noncardiogenic pulmonary edema, confusion, lethargy, and encephalitis progressing to coma. The CSF

1	10–15% of patients with RMSF never develop a rash. The patient can be hypotensive and develop noncardiogenic pulmonary edema, confusion, lethargy, and encephalitis progressing to coma. The CSF contains 10–100 cells/μL, usually with a predominance of mononuclear cells. The CSF glucose level is often normal; the protein concentration may be slightly elevated. Renal and hepatic injury as well as bleeding secondary to vascular damage are noted. For untreated infections, mortality rates are 20–30%. Delayed recognition and treatment are associated with a greater risk of death; Native Americans, children 5–9 years of age, adults >70 years old, and persons with underlying immunosuppression also are at increased risk of death.

1	Other rickettsial diseases cause significant morbidity and mortality worldwide. Mediterranean spotted fever caused by Rickettsia conorii is found in Africa, southwestern and south-central Asia, and southern Europe. Patients have fever, flu-like symptoms, and an inoculation eschar at the site of the tick bite. A maculopapular rash develops within 1–7 days, involving the palms and soles but sparing the face. Elderly patients or those with diabetes, alcoholism, uremia, or congestive heart failure are at risk for severe disease characterized by neurologic involvement, respiratory distress, and gangrene of the digits. Mortality rates associated with this severe form of disease approach 50%. Epidemic typhus, caused by Rickettsia prowazekii, is transmitted in louse-infested environments and emerges in conditions of extreme poverty, war, and natural disaster. Patients experience a sudden onset of high fevers, severe headache, cough, myalgias, and abdominal pain. A maculopapular rash develops

1	in conditions of extreme poverty, war, and natural disaster. Patients experience a sudden onset of high fevers, severe headache, cough, myalgias, and abdominal pain. A maculopapular rash develops (primarily on the trunk) in more than half of patients and can progress to petechiae and purpura. Serious signs include delirium, coma, seizures, noncardiogenic pulmonary edema, skin necrosis, and peripheral gangrene. Mortality rates approached 60% in the preantibiotic era and continue to exceed 10–15% in contemporary outbreaks. Scrub typhus, caused by Orientia tsutsugamushi (a separate genus in the family Rickettsiaceae), is transmitted by larval mites or chiggers and is one of the most common infections in southeastern Asia and the western Pacific. The organism is found in areas of heavy scrub vegetation (e.g., along riverbanks). Patients may have an inoculation eschar and may develop a maculopapular rash. Severe cases progress to pneumonia, meningoencephalitis, DIC, and renal failure.

1	vegetation (e.g., along riverbanks). Patients may have an inoculation eschar and may develop a maculopapular rash. Severe cases progress to pneumonia, meningoencephalitis, DIC, and renal failure. Mortality rates range from 1% to 35%.

1	If recognized in a timely fashion, rickettsial disease is very responsive to treatment. Doxycycline (100 mg twice daily for 3–14 days) is the treatment of choice for both adults and children. The newer macrolides and chloramphenicol may be suitable alternatives, but mortality rates are higher when a tetracycline-based treatment is not given. Purpura Fulminans (See also Chaps. 180 and 325) Purpura fulminans is the cutaneous manifestation of DIC and presents as large ecchymotic areas and hemorrhagic bullae. Progression of petechiae to purpura, ecchymoses, and gangrene is associated with congestive heart failure, septic shock, acute renal failure, acidosis, hypoxia, hypotension, and death. Purpura fulminans has been associated primarily with N. meningitidis but, in splenectomized patients, may be associated with S. pneumoniae, H. influenzae, and S. aureus.

1	N. meningitidis but, in splenectomized patients, may be associated with S. pneumoniae, H. influenzae, and S. aureus. Ecthyma Gangrenosum Septic shock caused by P. aeruginosa or Aeromonas hydrophila can be associated with ecthyma gangrenosum (see Figs. 189-1 and 25e-35): hemorrhagic vesicles surrounded by a rim of erythema with central necrosis and ulceration. These gram-negative bacteremias are most common among patients with neutropenia, extensive burns, and hypogammaglobulinemia.

1	Other Emergent Infections Associated with Rash Vibrio vulnificus and other noncholera Vibrio bacteremic infections (Chap. 193) can cause focal skin lesions and overwhelming sepsis in hosts with chronic liver disease, iron storage disorders, diabetes, renal insufficiency, or other immunocompromising conditions. After ingestion of contaminated raw shellfish, typically oysters from the Gulf Coast, there is a sudden onset of malaise, chills, fever, and hypotension. The patient develops bullous or hemorrhagic skin lesions, usually on the lower extremities, and 75% of patients have leg pain. The mortality rate can be as high as 50–60%, particularly when the patient presents with hypotension. Outcomes are improved when patients are treated with tetracycline-containing regimens. Other infections, caused by agents such as Aeromonas, Klebsiella, and E. coli, can cause hemorrhagic bullae and death due to overwhelming sepsis in cirrhotic patients. Capnocytophaga canimorsus can cause septic shock

1	caused by agents such as Aeromonas, Klebsiella, and E. coli, can cause hemorrhagic bullae and death due to overwhelming sepsis in cirrhotic patients. Capnocytophaga canimorsus can cause septic shock in asplenic patients. Infection typically follows a dog bite. Patients present with fever, chills, myalgia, vomiting, diarrhea, dyspnea, confusion, and headache. Findings can include an exanthem or erythema multiforme (see Figs. 70-9 and 25e-25), cyanotic mottling or peripheral cyanosis, petechiae, and ecchymosis. About 30% of patients with this fulminant form die of overwhelming sepsis and DIC, and survivors may require amputation because of gangrene.

1	Erythroderma TSS (Chaps. 172 and 173) is usually associated with erythroderma. The patient presents with fever, malaise, myalgias, nausea, vomiting, diarrhea, and confusion. There is a sunburn-type rash that may be subtle and patchy but is usually diffuse and is found on the face, trunk, and extremities. Erythroderma, which desquamates after 1–2 weeks, is more common in Staphylococcus-associated than in Streptococcus-associated TSS. Hypotension develops rapidly—often within hours—after the onset of symptoms. Multiorgan failure occurs. Early renal failure may precede hypotension and distinguishes this syndrome from other septic shock syndromes. There may be no indication of a primary focal infection, although possible cutaneous or mucosal portals of entry for the organism can be ascertained when a careful history is taken. Colonization rather than overt infection of the vagina or a postoperative wound, for example, is typical with staphylococcal TSS, and the mucosal areas appear

1	when a careful history is taken. Colonization rather than overt infection of the vagina or a postoperative wound, for example, is typical with staphylococcal TSS, and the mucosal areas appear hyperemic but not infected. Streptococcal TSS is more often associated with skin or soft tissue infection (including necrotizing fasciitis), and patients are more likely to be bacteremic. TSS caused by Clostridium sordellii is associated with childbirth or with skin injection of black-tar heroin. The diagnosis of TSS is defined by the clinical criteria of fever, rash, hypotension, and multiorgan involvement. The mortality rate is 5% for menstruation-associated TSS, 10–15% for nonmenstrual TSS, 30–70% for streptococcal TSS, and up to 90% for obstetric C. sordellii TSS.

1	Viral Hemorrhagic Fevers Viral hemorrhagic fevers (Chaps. 233 and 234) animal reservoirs or arthropod vectors. These diseases occur worldwide and are restricted to areas where the host species live. They are caused by four major groups of viruses: Arenaviridae (e.g., Lassa fever in Africa), Bunyaviridae (e.g., Rift Valley fever in Africa; hantavirus hemorrhagic fever with renal syndrome in Asia; or Crimean-Congo hemorrhagic fever, which has an extensive geographic distribution), Filoviridae (e.g., Ebola and Marburg virus infections in Africa), and Flaviviridae (e.g., yellow fever in Africa and South America and dengue in Asia, Africa, and the Americas). Lassa fever and Ebola and Marburg virus infections are also transmitted from person to person. The vectors for most viral fevers are found in rural areas; dengue and yellow fever are important exceptions. After a prodrome of fever, myalgias, and malaise, patients develop evidence of vascular damage, petechiae, and local hemorrhage.

1	in rural areas; dengue and yellow fever are important exceptions. After a prodrome of fever, myalgias, and malaise, patients develop evidence of vascular damage, petechiae, and local hemorrhage. Shock, multifocal hemorrhaging, and neurologic signs (e.g., seizures or coma) predict a poor prognosis. Dengue (Chap. 233) is the most common arboviral disease worldwide. More than half a million cases of dengue hemorrhagic fever occur each year, with at least 12,000 deaths. Patients have a triad of symptoms: hemorrhagic manifestations, evidence of plasma leakage, and platelet counts of <100,000/μL. Mortality rates are 10–20%. If dengue shock syndrome develops, mortality rates can reach 40%. Supportive care to maintain blood pressure and intravascular volume with careful volume-replacement therapy is key to survival. Ribavirin also may be useful against Arenaviridae and Bunyaviridae.

1	See also Chap. 156.

1	Necrotizing Fasciitis This infection is characterized by extensive necrosis of the subcutaneous tissue and fascia. It may arise at a site of minimal trauma or postoperative incision and may also be associated with recent varicella, childbirth, or muscle strain. The most common causes of necrotizing fasciitis are group A streptococci alone (Chap. 173), the incidence of which has been increasing for the past two decades, and a mixed facultative and anaerobic flora (Chap. 156). Diabetes mellitus, IV drug use, chronic liver or renal disease, and malignancy are associated risk factors. Physical findings are initially minimal compared with the severity of pain and the degree of fever. The examination is often unremarkable except for soft tissue edema and erythema. The infected area is red, hot, shiny, swollen, and exquisitely tender. In untreated infection, the overlying skin develops blue-gray patches after 36 h, and cutaneous bullae and necrosis develop after 3–5 days. Necrotizing

1	hot, shiny, swollen, and exquisitely tender. In untreated infection, the overlying skin develops blue-gray patches after 36 h, and cutaneous bullae and necrosis develop after 3–5 days. Necrotizing fasciitis due to a mixed flora, but not that due to group A streptococci, can be associated with gas production. Without treatment, pain decreases because of thrombosis of the small blood vessels and destruction of the peripheral nerves—an ominous sign. The mortality rate is 15–34% overall, >70% in association with TSS, and nearly 100% without surgical intervention. Necrotizing fasciitis may also be due to Clostridium perfringens (Chap. 179); in this condition, the patient is extremely toxic and the mortality rate is high. Within 48 h, rapid tissue invasion and systemic toxicity associated with hemolysis and death ensue. The distinction between this entity and clostridial myonecrosis is made by muscle biopsy. Necrotizing fasciitis caused by community-acquired MRSA also has been reported.

1	Clostridial Myonecrosis (See also Chap. 179) Myonecrosis is often associated with trauma or surgery but can develop spontaneously. The incubation period is usually 12–24 h long, and massive necrotizing gangrene develops within hours of onset. Systemic toxicity, shock, and death can occur within 12 h. The patient’s pain and toxic appearance are out of proportion to physical findings. On examination, the patient is febrile, apathetic, tachycardic, and tachypneic and may express a feeling of impending doom. Hypotension and renal failure develop later, and hyperalertness is evident preterminally. The skin over the affected area is bronze-brown, mottled, and edematous. Bullous lesions with serosanguineous drainage and a mousy or sweet odor can develop. Crepitus can occur secondary to gas production in muscle tissue. The mortality rate is >65% for spontaneous myonecrosis, which is often associated with Clostridium septicum or C. tertium and underlying malignancy. The mortality rates

1	production in muscle tissue. The mortality rate is >65% for spontaneous myonecrosis, which is often associated with Clostridium septicum or C. tertium and underlying malignancy. The mortality rates associated with trunk and limb infection are 63% and 12%, respectively, and any delay in surgical treatment increases the risk of death.

1	NEUROLOGIC INFECTIONS WITH OR WITHOUT SEPTIC SHOCK Bacterial Meningitis (See also Chap. 164) Bacterial meningitis is one of the most common infectious disease emergencies involving the central nervous system. Although hosts with cell-mediated immune deficiency (including transplant recipients, diabetic patients, elderly patients, and cancer patients receiving certain chemotherapeutic agents) are at particular risk for Listeria monocytogenes meningitis, 783 most cases in adults are due to S. pneumoniae (30–60%) and N. meningitidis (10–35%). The classic presentation of fever, meningismus, and altered mental status is seen in only one-half to two-thirds of patients. The elderly can present without fever or meningeal signs. Cerebral dysfunction is evidenced by confusion, delirium, and lethargy that can progress to coma. In some cases, the presentation is fulminant, with sepsis and brain edema; papilledema at presentation is unusual and suggests another diagnosis (e.g., an intracranial

1	that can progress to coma. In some cases, the presentation is fulminant, with sepsis and brain edema; papilledema at presentation is unusual and suggests another diagnosis (e.g., an intracranial lesion). Focal signs, including cranial nerve palsies (IV, VI, VII), can be seen in 10–20% of cases; 50–70% of patients have bacteremia. A poor outcome is associated with coma, hypotension, a pneumococcal etiology, respiratory distress, a CSF glucose level of <0.6 mmol/L (<<0 mg/dL), a CSF protein level of >2.5 g/L, a peripheral white blood cell count of <5000/μL, and a serum sodium level of <135 mmol/L. Rapid initiation of treatment is essential; the odds of an unfavorable outcome may increase by 30% for each hour that treatment is delayed. Mortality also increases linearly with age of the patient.

1	Suppurative Intracranial Infections (See also Chap. 164) In suppurative intracranial infections, rare intracranial lesions present along with sepsis and hemodynamic instability. Rapid recognition of the toxic patient with central neurologic signs is crucial to improvement of the dismal prognosis of these entities. Subdural empyema arises from the paranasal sinus in 60–70% of cases. Microaerophilic streptococci and staphylococci are the predominant etiologic organisms. The patient is toxic, with fever, headache, and nuchal rigidity. Of all patients, 75% have focal signs and 6–20% die. Despite improved survival rates, 15–44% of patients are left with permanent neurologic deficits. Septic cavernous sinus thrombosis follows a facial or sphenoid sinus infection; 70% of cases are due to staphylococci (including MRSA), and the remainder are due primarily to aerobic or anaerobic streptococci. A unilateral or retroorbital headache progresses to a toxic appearance and fever within days.

1	staphylococci (including MRSA), and the remainder are due primarily to aerobic or anaerobic streptococci. A unilateral or retroorbital headache progresses to a toxic appearance and fever within days. Three-quarters of patients have unilateral periorbital edema that becomes bilateral and then progresses to ptosis, proptosis, ophthalmoplegia, and papilledema. The mortality rate is as high as 30%. Septic thrombosis of the superior sagittal sinus spreads from the ethmoid or maxillary sinuses and is caused by S. pneumoniae, other streptococci, and staphylococci. The fulminant course is characterized by headache, nausea, vomiting, rapid progression to confusion and coma, nuchal rigidity, and brainstem signs. If the sinus is totally thrombosed, the mortality rate exceeds 80%.

1	Brain Abscess (See also Chap. 164) Brain abscess often occurs without systemic signs. Almost half of patients are afebrile, and presentations are more consistent with a space-occupying lesion in the brain; 70% of patients have headache and/or altered mental status, 50% have focal neurologic signs, and 25% have papilledema. Abscesses can present as single or multiple lesions resulting from contiguous foci or hematogenous infection, such as endocarditis. The infection progresses over several days from cerebritis to an abscess with a mature capsule. More than half of infections are polymicrobial, with an etiology consisting of aerobic bacteria (primarily streptococcal species) and anaerobes. Abscesses arising hematogenously are especially apt to rupture into the ventricular space, causing a sudden and severe deterioration in clinical status and a high mortality rate. Otherwise, mortality is low but morbidity is high (30–55%). Patients presenting with stroke and a parameningeal infectious

1	and severe deterioration in clinical status and a high mortality rate. Otherwise, mortality is low but morbidity is high (30–55%). Patients presenting with stroke and a parameningeal infectious focus, such as sinusitis or otitis, may have a brain abscess, and physicians must maintain a high level of suspicion. Prognosis worsens in patients with a fulminant course, delayed diagnosis, abscess rupture into the ventricles, multiple abscesses, or abnormal neurologic status at presentation.

1	Cerebral Malaria (See also Chap. 248) This entity should be urgently considered if patients who have recently traveled to areas endemic for malaria present with a febrile illness and lethargy or other neurologic signs. Fulminant malaria is caused by Plasmodium falciparum and is associated with temperatures of >40°C (>104°F), hypotension, jaundice, adult respiratory distress syndrome, and bleeding. By definition, any patient with a change in mental status or repeated

1	Approach to the Acutely Ill Infected Febrile Patient 784 seizure in the setting of fulminant malaria has cerebral malaria. In adults, this nonspecific febrile illness progresses to coma over several days; occasionally, coma occurs within hours and death within 24 h. Nuchal rigidity and photophobia are rare. On physical examination, symmetric encephalopathy is typical, and upper motor neuron dysfunction with decorticate and decerebrate posturing can be seen in advanced disease. Unrecognized infection results in a 20–30% mortality rate.

1	Intracranial and Spinal Epidural Abscesses (See also Chap. 456) Spinal and intracranial epidural abscesses (SEAs and ICEAs) can result in permanent neurologic deficits, sepsis, and death. At-risk patients include those with diabetes mellitus; IV drug use; chronic alcohol abuse; recent spinal trauma, surgery, or epidural anesthesia; and other comorbid conditions, such as HIV infection. Fungal epidural abscess and meningitis can follow epidural or paraspinal glucocorticoid infections. In the United States and Canada, where early treatment of otitis and sinusitis is typical, ICEA is rare but the number of cases of SEA is on the rise. In Africa and areas with limited access to health care, SEAs and ICEAs cause significant morbidity and mortality. ICEAs typically present as fever, mental status changes, and neck pain, while SEAs often present as fever, localized spinal tenderness, and back pain. ICEAs are typically polymicrobial, whereas SEAs are most often due to hematogenous seeding,

1	changes, and neck pain, while SEAs often present as fever, localized spinal tenderness, and back pain. ICEAs are typically polymicrobial, whereas SEAs are most often due to hematogenous seeding, with staphylococci the most common etiologic agent. Early diagnosis and treatment, which may include surgical drainage, minimize rates of mortality and permanent neurologic sequelae. Outcomes are worse for SEA due to MRSA, infection at a higher vertebral-body level, impaired neurologic status on presentation, and dorsal rather than ventral location of the abscess. Elderly patients and persons with renal failure, malignancy, and other comorbidities also have less favorable outcomes.

1	Other Focal Syndromes with a Fulminant Course Infection at virtually any primary focus (e.g., osteomyelitis, pneumonia, pyelonephritis, or cholangitis) can result in bacteremia and sepsis. Lemierre’s disease— jugular septic thrombophlebitis caused by Fusobacterium necrophorum—is associated with metastatic infectious emboli (primarily to the lung) and sepsis, with mortality rates of >15%. TSS has been associated with focal infections such as septic arthritis, peritonitis, sinusitis, and wound infection. Rapid clinical deterioration and death can be associated with destruction of the primary site of infection, as is seen in endocarditis and in infections of the oropharynx (e.g., Ludwig’s angina or epiglottitis, in which edema suddenly compromises the airway).

1	Rhinocerebral Mucormycosis (See also Chap. 242) Individuals with diabetes or immunocompromising conditions are at risk for invasive rhinocerebral mucormycosis. Patients present with low-grade fever, dull sinus pain, diplopia, decreased mental status, decreased ocular motion, chemosis, proptosis, dusky or necrotic nasal turbinates, and necrotic hard-palate lesions that respect the midline. Without rapid recognition and intervention, the process continues on an inexorable invasive course, with high mortality rates.

1	Acute Bacterial Endocarditis (See also Chap. 155) This entity presents with a much more aggressive course than subacute endocarditis. Bacteria such as S. aureus, S. pneumoniae, L. monocytogenes, Haemophilus species, and streptococci of groups A, B, and G attack native valves. Native-valve endocarditis caused by S. aureus (including MRSA strains) is increasing, particularly in health care settings. Mortality rates range from 10% to 40%. The host may have comorbid conditions such as underlying malignancy, diabetes mellitus, IV drug use, or alcoholism. The patient presents with fever, fatigue, and malaise <2 weeks after onset of infection. On physical examination, a changing murmur and congestive heart failure may be noted. Hemorrhagic macules on palms or soles (Janeway lesions) sometimes develop. Petechiae, Roth’s spots, splinter hemorrhages, and splenomegaly are unusual. Rapid valvular destruction, particularly of the aortic valve, results in pulmonary edema and hypotension. Myocardial

1	Petechiae, Roth’s spots, splinter hemorrhages, and splenomegaly are unusual. Rapid valvular destruction, particularly of the aortic valve, results in pulmonary edema and hypotension. Myocardial abscesses can form, eroding through the septum or into the conduction system and causing life-threatening arrhythmias or high-degree conduction block. Large friable vegetations can result in major arterial emboli, metastatic infection, or tissue infarction. Older patients with S. aureus endocarditis are especially likely to present with nonspecific symptoms—a circumstance that delays diagnosis and worsens prognosis. Rapid intervention is crucial for a successful outcome.

1	Inhalational Anthrax (See also Chap. 261e) Inhalational anthrax, the most severe form of disease caused by B. anthracis, had not been reported in the United States for more than 25 years until the use of this organism as an agent of bioterrorism in 2001. Patients presented with malaise, fever, cough, nausea, drenching sweats, shortness of breath, and headache. Rhinorrhea was unusual. All patients had abnormal chest roentgenograms at presentation. Pulmonary infiltrates, mediastinal widening, and pleural effusions were the most common findings. Hemorrhagic meningitis was seen in 38% of these patients. Survival was more likely when antibiotics were given during the prodromal period and when multidrug regimens were used. In the absence of urgent intervention with antimicrobial agents and supportive care, inhalational anthrax progresses rapidly to hypotension, cyanosis, and death.

1	Avian and Swine Influenza (See also Chap. 224) Human cases of avian influenza have occurred primarily in Southeast Asia, particularly Vietnam (H5N1) and China (H7N9). Avian influenza should be considered in patients with severe respiratory tract illness, particularly if they have been exposed to poultry. Patients present with high fever, an influenza-like illness, and lower respiratory tract symptoms; this illness can progress rapidly to bilateral pneumonia, acute respiratory distress syndrome, multiorgan failure, and death. Early antiviral treatment with neuraminidase inhibitors should be initiated along with aggressive supportive measures. Unlike avian influenza, for which human-to-human transmission has been rare so far and has not been sustained, a novel swine-associated influenza A/H1N1 virus has spread rapidly throughout the world. Patients most at risk of severe disease are children <5 years of age, elderly persons, patients with underlying chronic conditions, and pregnant

1	A/H1N1 virus has spread rapidly throughout the world. Patients most at risk of severe disease are children <5 years of age, elderly persons, patients with underlying chronic conditions, and pregnant women. Obesity also has been identified as a risk factor for severe illness.

1	Hantavirus Pulmonary Syndrome (See also Chap. 233) Hantavirus pulmonary syndrome has been documented in the United States (primarily the southwestern states), Canada, and South America. Most cases occur in rural areas and are associated with exposure to rodents. Patients present with a nonspecific viral prodrome of fever, malaise, myalgias, nausea, vomiting, and dizziness that may progress to pulmonary edema and respiratory failure. Hantavirus pulmonary syndrome causes myocardial depression and increased pulmonary vascular permeability; therefore, careful fluid resuscitation and use of pressor agents are crucial. Aggressive cardiopulmonary support during the first few hours of illness can be life-saving. The early onset of thrombocytopenia may help distinguish this syndrome from other febrile illnesses in an appropriate epidemiologic setting.

1	Acutely ill febrile patients with the syndromes discussed in this chapter require close observation, aggressive supportive measures, and—in most cases—admission to intensive care units. The most important task of the physician is to distinguish these patients from other infected febrile patients whose illness will not progress to fulminant disease. The alert physician must recognize the acute infectious disease emergency and then proceed with appropriate urgency.

1	Immunization Principles and vaccine use Anne Schuchat, Lisa A. Jackson Few medical interventions of the past century can rival the effect that immunization has had on longevity, economic savings, and quality 148 of life. Seventeen diseases are now preventable through vaccines routinely administered to children and adults in the United States (Table 148-1), and most vaccine-preventable diseases of childhood are at historically low levels (Table 148-2). Health care providers deliver the vast majority of vaccines in the United States in the course of providing routine health services and therefore play an integral role in the nation’s public health system.

1	VACCINE IMPACT Direct and Indirect Effects Immunizations against specific infectious diseases protect individuals against infection and thereby prevent symptomatic illnesses. Specific vaccines may blunt the severity of clinical illness (e.g., rotavirus vaccines and severe gastroenteritis) or reduce complications (e.g., zoster vaccines and postherpetic neuralgia). Some immunizations also reduce transmission of infectious disease agents from immunized people to others, thereby reducing the impact of infection spread. This indirect impact is known as herd immunity. The level of immunization in a population that is required to achieve indirect protection of unimmunized people varies substantially with the specific vaccine.

1	Since childhood vaccines have become widely available in the United States, major declines in rates of vaccine-preventable diseases among both children and adults have become evident (Table 148-2). For example, vaccination of children <5 years of age against seven types of Streptococcus pneumoniae led to a >90% overall reduction in invasive disease caused by those types. A series of childhood vaccines targeting 13 vaccine-preventable diseases in a single birth cohort leads to prevention of 42,000 premature deaths and 20 million illnesses and saves nearly $70 billion (U.S.). Control, Elimination, and Eradication of Vaccine-Preventable Diseases Immunization programs are associated with the goals of controlling,

1	Control, Elimination, and Eradication of Vaccine-Preventable Diseases Immunization programs are associated with the goals of controlling, Pertussis Children, adolescents, adults Diphtheria Children, adolescents, adults Tetanus Children, adolescents, adults Poliomyelitis Children Measles Children Mumps Children Rubella, congenital rubella syndrome Children Hepatitis B Children Haemophilus influenzae type b Children infection Hepatitis A Children Influenza Children, adolescents, adults Varicella Children Invasive pneumococcal disease Children, older adults Meningococcal disease Adolescents Rotavirus infection Infants Human papillomavirus infection, Adolescents and young adults cervical and anogenital cancers Zoster Older adults Smallpox 29,005 0 100 Diphtheria 21,053 1 ≥99 Measles 530,217 55 ≥99 Mumps 162,344 229 ≥99 Pertussis 200,752 48,277 76 Polio (paralytic) 16,316 0 100 Rubella 47,745 9 >99 Haemophilus influenzae 20,000 30b 99 type b infection

1	Measles 530,217 55 ≥99 Mumps 162,344 229 ≥99 Pertussis 200,752 48,277 76 Polio (paralytic) 16,316 0 100 Rubella 47,745 9 >99 Haemophilus influenzae 20,000 30b 99 type b infection Hepatitis A 117,333 2,890c 98 Hepatitis B (acute) 66,232 18,800c 72 Invasive pneumococcal 63,067 31,600d 50 infection: all ages Invasive pneumococ-16,069 1,800d 89 cal infection: <5 years of age Varicella 4,085,120 216,511 95 aExcept for cases of hepatitis A and hepatitis B, for which 2011 figures are shown. bAn additional 13 type b infections are estimated to have occurred among 210 reports of H. influenzae infection caused by unknown types among children <5 years of age. cData are from the CDC’s Viral Hepatitis Surveillance, 2011. dData are from the CDC’s Active Bacterial Core Surveillance 2012 Provisional Report. Source: Adapted from SW Roush et al: JAMA 298:2155, 2007; and MMWR 62(33); 669, 2013.

1	Source: Adapted from SW Roush et al: JAMA 298:2155, 2007; and MMWR 62(33); 669, 2013. eliminating, or eradicating a disease. Control of a vaccine-preventable disease reduces poor illness outcomes and often limits the disruptive impacts associated with outbreaks of disease in communities, schools, and institutions. Control programs can also reduce absences from work for ill persons and for parents caring for sick children, decrease absences from school, and limit health care utilization associated with treatment visits.

1	Elimination of a disease is a more demanding goal than control, usually requiring the reduction to zero of cases in a defined geographic area but sometimes defined as reduction in the indigenous sustained transmission of an infection in a geographic area. As of 2013, the United States had eliminated indigenous transmission of measles, rubella, poliomyelitis, and diphtheria. Importation of pathogens from other parts of the world continues to be important, and public health efforts are intended to react promptly to such cases and to limit forward spread of the infectious agent.

1	Eradication of a disease is achieved when its elimination can be sustained without ongoing interventions. The only vaccine- preventable disease of humans that has been globally eradicated thus far is smallpox. Although smallpox vaccine is no longer given routinely, the disease has not reemerged naturally because all chains of human transmission were interrupted through earlier vaccination efforts and humans were the only natural reservoir of the virus. Currently, a major health initiative is targeting the global eradication of polio. Sustained transmission of polio has been eliminated from most nations but has never been interrupted in three countries— Afghanistan, Nigeria, and Pakistan—while recent outbreaks in Syria and the Horn of Africa underscore that other countries remain at risk for importation until these reservoirs have been addressed. Detection of a case of disease that has been targeted for eradication or elimination is considered a sentinel event that could permit the

1	for importation until these reservoirs have been addressed. Detection of a case of disease that has been targeted for eradication or elimination is considered a sentinel event that could permit the infectious agent to become reestablished in the community or region. Therefore, such episodes must be promptly reported to public health authorities.

1	786 Outbreak Detection and Control Clusters of cases of a vaccine-preventable disease detected in an institution, a medical practice, or a community may signal important changes in the pathogen, vaccine, or environment. Several factors can give rise to increases in vaccine-preventable disease, including (1) low rates of immunization that result in an accumulation of susceptible people (e.g., measles resurgence among vaccination abstainers); (2) changes in the infectious agent that permit it to escape vaccine-induced protection (e.g., non-vaccine-type pneumococci); (3) waning of vaccine-induced immunity (e.g., pertussis among adolescents and adults vaccinated in early childhood); and (4) point-source introductions of large inocula (e.g., food-borne exposure to hepatitis A virus). Reporting episodes of outbreak-prone diseases to public health authorities can facilitate recognition of clusters that require further interventions.

1	public HealtH reporting Recognition of suspected cases of diseases targeted for elimination or eradication—along with other diseases that require urgent public health interventions, such as contact tracing, administration of chemoor immunoprophylaxis, or epidemiologic investigation for common-source exposure—is typically associated with special reporting requirements. Many diseases against which vaccines are routinely used, including measles, pertussis, Haemophilus influenzae type b invasive disease, and varicella, are nationally notifiable. Clinicians and laboratory staff have a responsibility to report some vaccine-preventable disease occurrences to local or state public health authorities according to specific case-definition criteria. All providers should be aware of state or city disease-reporting requirements and the best ways to contact public health authorities. A prompt response to vaccine-preventable disease outbreaks can greatly enhance the effectiveness of control

1	city disease-reporting requirements and the best ways to contact public health authorities. A prompt response to vaccine-preventable disease outbreaks can greatly enhance the effectiveness of control measures.

1	global considerations Several international health initiatives currently focus on reducing vaccine-preventable diseases in regions throughout the world. These efforts include improving access to new and underutilized vaccines, such as pneumococcal conjugate, rotavirus, human papillomavirus (HPV), and meningococcal A conjugate vaccines. The American Red Cross, the World Health Organization (WHO), the United Nations Foundation, the United Nations Children’s Fund (UNICEF), and the Centers for Disease Control and Prevention (CDC) are partners in the Measles & Rubella Initiative, which targets reduction of worldwide measles deaths by 95% from 2000 to 2015. During 2000–2011, global measles mortality rates declined by 71%—i.e., from an estimated 548,000 deaths in 2000 to 158,000 deaths in 2011. Rotary International, UNICEF, the CDC, and the WHO are leading partners in the global eradication of polio, an endeavor that reduced the annual number of paralytic polio cases from 350,000 in 1988 to

1	Rotary International, UNICEF, the CDC, and the WHO are leading partners in the global eradication of polio, an endeavor that reduced the annual number of paralytic polio cases from 350,000 in 1988 to <250 in 2012. The GAVI Alliance and the Bill and Melinda Gates Foundation have brought substantial momentum to global efforts to reduce vaccine-preventable diseases, expanding on earlier efforts by the WHO, UNICEF, and governments in developed and developing countries.

1	Enhancing Immunization in Adults Although immunization has become a centerpiece of routine pediatric medical visits, it has not been as well integrated into routine health care visits for adults. This chapter focuses on immunization principles and vaccine use in adults. Accumulating evidence suggests that immunization coverage can be increased through efforts directed at consumer-, provider-, institution-, and system-level factors. The literature suggests that the application of multiple strategies is more effective at raising coverage rates than is the use of any single strategy.

1	recommendations for adult immunizations The CDC’s Advisory Committee on Immunization Practices (ACIP) is the main source of recommendations for administration of vaccines approved by the U.S. Food and Drug Administration (FDA) for use in children and adults in the U.S. civilian population. The ACIP is a federal advisory committee that consists of 15 voting members (experts in fields associated with immunization) appointed by the Secretary of the U.S. Department of Health and Human Services; 8 ex officio members representing federal agencies; and 26 nonvoting representatives of various liaison organizations, including major medical societies and managed-care organizations. The ACIP recommendations are available at www.cdc .gov/vaccines/hcp/acip-recs/. These recommendations are harmonized to the greatest extent possible with vaccine recommendations made by other organizations, including the American College of Obstetricians and Gynecologists, the American Academy of Family Physicians,

1	to the greatest extent possible with vaccine recommendations made by other organizations, including the American College of Obstetricians and Gynecologists, the American Academy of Family Physicians, and the American College of Physicians.

1	adult immunization scHedules Immunization schedules for adults in the United States are updated annually and can be found online (www .cdc.gov/vaccines/schedules/hcp/adult.html). In January, the schedules are published in American Family Physician, Annals of Internal Medicine, and Morbidity and Mortality Weekly Report (www.cdc.gov/ mmwr). The adult immunization schedules for 2013 are summarized in Fig. 148-1. Additional information and specifications are contained in the footnotes to these schedules. In the time between annual publications, additions and changes to schedules are published as Notices to Readers in Morbidity and Mortality Weekly Report.

1	Administering immunizations to adults involves a number of processes, such as deciding whom to vaccinate, assessing vaccine contraindications and precautions, providing vaccine information statements (VISs), ensuring appropriate storage and handling of vaccines, administering vaccines, and maintaining vaccine records. In addition, provider reporting of adverse events that follow vaccination is an essential component of the vaccine safety monitoring system.

1	Deciding Whom to Vaccinate Every effort should be made to ensure that adults receive all indicated vaccines as expeditiously as possible. When adults present for care, their immunization history should be assessed and recorded, and this information should be used to identify needed vaccinations according to the most current version of the adult immunization schedule. Decision-support tools incorporated into electronic health records can provide prompts for needed vaccinations. Standing orders, which are often used for routinely indicated vaccines (e.g., influenza and pneumococcal vaccines), permit a nurse or another approved licensed practitioner to administer vaccines without a specific physician order, thus lowering barriers to adult immunization.

1	Assessing Contraindications and Precautions Before vaccination, all patients should be screened for contraindications and precautions. A contraindication is a condition that increases the risk of a serious adverse reaction to vaccination. A vaccine should not be administered when a contraindication is documented. For example, a history of an anaphylactic reaction to a dose of vaccine or to a vaccine component is a contraindication for further doses. A precaution is a condition that may increase the risk of an adverse event or that may compromise the ability of the vaccine to evoke immunity (e.g., administering measles vaccine to a person who has recently received a blood transfusion and may consequently have transient passive immunity to measles virus). Normally, a vaccine is not administered when a precaution is noted. However, situations may arise when the benefits of vaccination outweigh the estimated risk of an adverse event, and the provider may decide to vaccinate the patient

1	when a precaution is noted. However, situations may arise when the benefits of vaccination outweigh the estimated risk of an adverse event, and the provider may decide to vaccinate the patient despite the precaution.

1	In some cases, contraindications and precautions are temporary and may lead to mere deferral of vaccination until a later time. For example, moderate or severe acute illness with or without fever is generally considered a transient precaution to vaccination and results in postponement of vaccine administration until the acute phase has resolved; thus the superimposition of adverse effects of vaccination on the underlying illness and the mistaken attribution of a manifestation of the underlying illness to the vaccine are avoided. Contraindications and precautions to vaccines licensed in the United States for use in civilian adults are summarized in Table 148-3. It is important to recognize conditions that are not contraindications in order not to miss opportunities for vaccination. For example, in most cases, mild acute illness (with or without fever), a history of a mild to moderate local reaction to a previous dose of the vaccine, and breast-feeding are not contraindications to

1	example, in most cases, mild acute illness (with or without fever), a history of a mild to moderate local reaction to a previous dose of the vaccine, and breast-feeding are not contraindications to vaccination.

1	FOOTNOTES: (Influenza vaccine)1 There are several flu vaccines available—talk to your healthcare professional about which flu vaccine is right for you. (Tdap vaccine)2 Pregnant women are recommended to get Tdap vaccine with each pregnancy to increase protection for infants who are too young for vaccination but at highest risk for severe illness and death from pertussis (whooping cough). (HPV vaccine)3 There are two HPV vaccines but only one HPV vaccine (Gardasil®) should be given to men. Gay men or men who have sex with men who are 22 through 26 years old should get HPV vaccine if they haven’t already started or completed the series.

1	(MMR vaccine)4 If you were born in 1957 or after, and don’t have a record of being vaccinated or having had these infections, talk to your healthcare professional about how many doses you may need. (Pneumococcal vaccine)5 There are two different types of pneumococcal vaccine: PCV13 and PPSV23. Talk with your healthcare professional to find out if one or both pneumococcal vaccines are recommended for you. If you are traveling outside of the United States, you may need additional vaccines. Ask your healthcare professional which vaccines you may need. For more information, call toll free 1-800-CDC-INFO (1-800-232-4636) or visit http://www.cdc.gov/vaccines FIGURE 148-1 Recommended adult immunization schedules, United States, 2013. For complete statements by the Advisory Committee on Immunization Practices (ACIP), visit www.cdc.gov/ vaccines/hcp/acip-recs/.

1	FOOTNOTES: (Influenza vaccine)1 There are several flu vaccines available—talk to your healthcare professional about which flu vaccine is right for you. (HPV vaccine)2 There are two HPV vaccines but only one HPV vaccine (Gardasil®) should be given to men. Gay men or men who have sex with men who are 22 through 26 years old should get HPV vaccine if they haven’t already started or completed the series. (Zoster)3 You should get zoster vaccine even if you’ve had shingles before. (MMR vaccine)4 If you were born in 1957 or after, and don’t have a record of being vaccinated or having had these infections, talk to your healthcare professional about how many doses you may need. All vaccines Contraindication Severe allergic reaction (e.g., anaphylaxis) after a previous vaccine dose or to a vaccine component Moderate or severe acute illness with or without fever. Defer vaccination until illness resolves.

1	Moderate or severe acute illness with or without fever. Defer vaccination until illness resolves. Td Precautions GBS within 6 weeks after a previous dose of TT-containing vaccine History of arthus-type hypersensitivity reactions after a previous dose of TDor DT-containing vaccines (including MCV4). Defer vac cination until at least 10 years have elapsed since the last dose. Tdap Contraindication History of encephalopathy (e.g., coma or prolonged seizures) not attributable to another identifiable cause within 7 days of administration of a vaccine with pertussis components, such as DTaP or Tdap GBS within 6 weeks after a previous dose of TT-containing vaccine

1	GBS within 6 weeks after a previous dose of TT-containing vaccine Progressive or unstable neurologic disorder, uncontrolled seizures, or progressive encephalopathy. Defer vaccination until a treatment regimen has been established and the condition has stabilized. History of arthus-type hypersensitivity reactions after a previous dose of TTor DT-containing vaccines (including MCV4). Defer vaccina tion until at least 10 years have elapsed since the last dose. HPV Contraindication History of immediate hypersensitivity to yeast (for Gardasil) Pregnancy. If a woman is found to be pregnant after initiation of the vaccination series, the remainder of the 3-dose regimen should be delayed until after completion of the pregnancy. If a vaccine dose has been administered during pregnancy, no intervention is needed. Exposure to Gardasil during pregnancy should be reported to Merck (800-986-8999); exposure to Cervarix during pregnancy should be reported to GlaxoSmithKline (888-452-9622).

1	History of immediate hypersensitivity reaction to gelatina or neomycin Known severe immunodeficiency (e.g., hematologic and solid tumors; chemotherapy; congenital immunodeficiency; long-term immunosuppressive therapy; severe immunocompromise due to HIV infection) Recent receipt (within 11 months) of antibody-containing blood product History of thrombocytopenia or thrombocytopenic purpura Varicella Contraindications Pregnancy Known severe immunodeficiency History of immediate hypersensitivity reaction to gelatina or neomycin Recent receipt (within 11 months) of antibody-containing blood product Influenza, inactivated, Precautions injectable History of severe allergic reaction (e.g., anaphylaxis) to egg proteinb (note: not a precaution for Flublok recombinant influenza vaccine, which is approved for persons 18–49 years of age and is manufactured without the use of eggs) History of GBS within 6 weeks after a previous influenza vaccine dose

1	History of GBS within 6 weeks after a previous influenza vaccine dose Influenza, live Contraindications attenuated nasal spray History of severe allergic reaction (e.g., anaphylaxis) to egg proteinb Immunosuppression, including that caused by medications or by HIV infection; known severe immunodeficiency (e.g., hematologic and solid tumors; chemotherapy; congenital immunodeficiency; long-term immunosuppressive therapy; severe immunocompromise due to HIV infection) Certain chronic medical conditions, such as diabetes mellitus; chronic pulmonary disease (including asthma); chronic cardiovascular disease (except hypertension); renal, hepatic, neurologic/neuromuscular, hematologic, or metabolic disorders Close contact with severely immunosuppressed persons who require a protected environment, such as isolation in a bone marrow transplantation unit

1	Close contact with severely immunosuppressed persons who require a protected environment, such as isolation in a bone marrow transplantation unit Close contact with persons with lesser degrees of immunosuppression (e.g., persons receiving chemotherapy or radiation therapy who are not being cared for in a protective environment; persons with HIV infection) is not a contraindication or a precaution. Health care personnel in neonatal intensive care units or oncology clinics may receive live attenuated influenza vaccine. History of GBS within 6 weeks of a previous influenza vaccine dose Receipt of specific antiviral agents (i.e., amantadine, rimantadine, zanamivir, or oseltamivir) with 48 h before vaccination Pneumococcal None, other than those listed for all vaccines Pneumococcal None, other than those listed for all vaccines History of immediate hypersensitivity to yeast Meningococcal Contraindication conjugate

1	Pneumococcal None, other than those listed for all vaccines History of immediate hypersensitivity to yeast Meningococcal Contraindication conjugate History of severe allergic reaction to dry natural rubber (latex) (certain vaccine formulations; see text) History of severe allergic reaction to dry natural rubber (latex) History of immediate hypersensitivity reaction to gelatina or neomycin

1	History of severe allergic reaction to dry natural rubber (latex) History of immediate hypersensitivity reaction to gelatina or neomycin Receipt of specific antiviral agents (i.e., acyclovir, famciclovir, or valacyclovir) within 24 h before vaccination aExtreme caution must be exercised in administering MMR, varicella, or zoster vaccine to persons with a history of anaphylactic reaction to gelatin or gelatin-containing products. Before administration, skin testing for sensitivity to gelatin can be considered. However, no specific protocols for this purpose have been published. bRecommendations for safely administering influenza vaccine to persons with egg allergies are reported in the annual ACIP recommendations for influenza vaccination (www.cdc.gov/vaccines/hcp/acip-recs/vacc-specific/flu.html).

1	Abbreviations: DT, diphtheria toxoid; DTaP, diphtheria, tetanus, and pertussis; GBS, Guillain-Barré syndrome; HPV, human papillomavirus; MCV4, quadrivalent meningococcal conjugate vaccine; MMR, measles, mumps, and rubella; Td, tetanus and diphtheria toxoids; Tdap, tetanus and diphtheria toxoids and acellular pertussis; TT, tetanus toxoid.

1	History of immediate Hypersensitivity to a vaccine component A severe allergic reaction (e.g., anaphylaxis) to a previous dose of a vaccine or to one of its components is a contraindication to vaccination. While most vaccines have many components, substances to which individuals are most likely to have had a severe allergic reaction include egg protein, gelatin, and yeast. In addition, although natural rubber (latex) is not a vaccine component, some vaccines are supplied in vials or syringes that contain natural rubber latex. These vaccines can be identified by the product insert and should not be administered to persons who report a severe (anaphylactic) allergy to latex unless the benefit of vaccination clearly outweighs the risk for a potential allergic reaction. The much more common local or contact hypersensitivity to latex, such as to medical gloves (which contain synthetic latex that is not linked to allergic reactions), is not a contraindication to administration of a vaccine

1	or contact hypersensitivity to latex, such as to medical gloves (which contain synthetic latex that is not linked to allergic reactions), is not a contraindication to administration of a vaccine supplied in a vial or syringe that contains natural rubber latex. Vaccines routinely indicated for adults that, as of December 2012, were sometimes supplied in a vial or syringe containing natural rubber include Havrix hepatitis A vaccine (syringe); Vaqta hepatitis A vaccine (vial and syringe); Engerix-B hepatitis B vaccine (syringe); Recombivax HB hepatitis B vaccine (vial); Cervarix HPV vaccine (syringe); Fluarix, Fluvirin, Agriflu, and Flucelvax influenza vaccines (syringe); Adacel and Boostrix Tdap (tetanus and diphtheria toxoids and acellular pertussis) vaccines (syringe); Td (tetanus and diphtheria toxoids) vaccines (syringe); Twinrix hepatitis A and B vaccine (syringe); and Menomune meningococcal polysaccharide vaccine (vial).

1	pregnancy Live-virus vaccines are contraindicated during pregnancy because of the theoretical risk that vaccine virus replication will cause congenital infection or have other adverse effects on the fetus. Most live-virus vaccines, including varicella vaccine, are not secreted in breast milk; therefore, breast-feeding is not a contraindication for live-virus or other vaccines. Pregnancy is not a contraindication to administration of inactivated vaccines, but most are avoided during pregnancy because relevant safety data are limited. Two inactivated vaccines, Tdap vaccine and inactivated influenza vaccine, are routinely recommended for pregnant women in the United States. Tdap vaccine is recommended during each pregnancy, regardless of prior vaccination status, in order to prevent pertussis in neonates. Annual influenza vaccination is recommended for all persons 6 months of age and older, regardless of pregnancy status. Some other vaccines, such as meningococcal vaccines, may be given

1	in neonates. Annual influenza vaccination is recommended for all persons 6 months of age and older, regardless of pregnancy status. Some other vaccines, such as meningococcal vaccines, may be given to pregnant women in certain circumstances.

1	immunosuppression Live-virus vaccines elicit an immune response due to replication of the attenuated (weakened) vaccine virus that is contained by the recipient’s immune system. In persons with compromised immune function, enhanced replication of vaccine viruses is possible and could lead to disseminated infection with the vaccine virus. For this reason, live-virus vaccines are contraindicated for persons with severe immunosuppression, the definition of which may vary with the vaccine. Severe immunosuppression may be caused by many disease conditions, including HIV infection and hematologic or generalized malignancy. In some of these conditions, all affected persons are severely immunocompromised. In others (e.g., HIV infection), the degree to which the immune system is compromised depends on the severity of the condition, which in turn depends on the stage of disease or treatment. For example, measles-mumps-rubella (MMR) vaccine may be given to HIV-infected persons who are not

1	depends on the severity of the condition, which in turn depends on the stage of disease or treatment. For example, measles-mumps-rubella (MMR) vaccine may be given to HIV-infected persons who are not severely immunocompromised. Severe immunosuppression may also be due to therapy with immunosuppressive agents, including high-dose glucocorticoids. In this situation, the dose, duration, and route of administration may influence the degree of immunosuppression.

1	A VIS is a one-page (two-sided) information sheet produced by the CDC that informs vaccine recipients (or their parents or legal representatives) about the benefits and risks of a vaccine. VISs are mandated by the National Childhood Vaccine Injury Act (NCVIA) of 1986 and— whether the vaccine recipient is a child or an adult—must be provided for any vaccine covered by the Vaccine Injury Compensation Program. As of July 2011, vaccines that are covered by the NCVIA and that are licensed for use in adults include Td, Tdap, hepatitis A, hepatitis B, HPV, trivalent inactivated influenza, trivalent live intranasal influenza, MMR, 13-valent pneumococcal conjugate, meningococcal, polio, and varicella vaccines. When combination vaccines for which no separate VIS exists are given (e.g., hepatitis A and B combination vaccine), all relevant VISs should be provided. VISs also exist for some vaccines not covered by the NCVIA, such as pneumococcal polysaccharide, Japanese encephalitis, rabies,

1	A and B combination vaccine), all relevant VISs should be provided. VISs also exist for some vaccines not covered by the NCVIA, such as pneumococcal polysaccharide, Japanese encephalitis, rabies, zoster, typhoid, and yellow fever vaccines. The use of these VISs is encouraged but is not mandated.

1	All current VISs are available on the Internet at two websites: the CDC’s Vaccines & Immunizations site (www.cdc.gov/vaccines/hcp/vis/) and the Immunization Action Coalition’s site (www.immunize.org/vis/). (The latter site also includes translations of the VISs.) VISs from these sites can be downloaded and printed.

1	Injectable vaccines are packaged in multidose vials, single-dose vials, or manufacturer-filled single-dose syringes. The live attenuated nasal-spray influenza vaccine is packaged in single-dose sprayers. Oral typhoid vaccine is packaged in capsules. Some vaccines, such as MMR, varicella, zoster, and meningococcal polysaccharide vaccines, come as lyophilized (freeze-dried) powders that must be reconstituted (i.e., mixed with a liquid diluent) before use. The lyophilized powder and the diluent come in separate vials. Diluents are not interchangeable but rather are specifically formulated for each type of vaccine; only the specific diluent provided by the manufacturer for each type of vaccine should be used. Once lyophilized vaccines have been reconstituted, their shelf-life is limited and they must be stored under appropriate temperature and light conditions. For example, varicella and zoster vaccines must be protected from light and administered within 30 min of reconstitution; MMR

1	they must be stored under appropriate temperature and light conditions. For example, varicella and zoster vaccines must be protected from light and administered within 30 min of reconstitution; MMR vaccine likewise must be protected from light but can be used up to 8 h after reconstitution. Single-dose vials of meningococcal polysaccharide vaccine must be used within 30 min of reconstitution, while multidose vials must be used within 35 days.

1	Vaccines are stored either at refrigerator temperature (2–8°C) or at freezer temperature (–15°C or colder). In general, inactivated vaccines (e.g., inactivated influenza, pneumococcal polysaccharide, and meningococcal conjugate vaccines) are stored at refrigerator temperature, while vials of lyophilized-powder live-virus vaccines (e.g., varicella, zoster, and MMR vaccines) are stored at freezer temperature. Diluents for lyophilized vaccines may be stored at refrigerator or room temperature. Live attenuated influenza vaccine—a live-virus liquid formulation administered by nasal spray—is stored at refrigerator temperature.

1	Vaccine storage and handling errors can result in the loss of vaccines worth millions of dollars, and administration of improperly stored vaccines may elicit inadequate immune responses in patients. To improve the standard of vaccine storage and handling practices, the CDC has published detailed guidance (available at www.cdc.gov/vaccines/recs/ storage/toolkit/storage-handling-toolkit.pdf). For vaccine storage, the CDC recommends stand-alone units—i.e., self-contained units that either refrigerate or freeze but do not do both—as these units maintain the required temperatures better than combination refrigerator/freezer units. Dormitory-style combined refrigerator/freezer units should never be used for vaccine storage.

1	The temperature of refrigerators and freezers used for vaccine storage must be monitored and the temperature recorded at least twice each workday. Ideally, continuous thermometers that measure and record temperature all day and all night are used, and minimum and maximum temperatures are read and documented each workday. The CDC recommends the use of calibrated digital thermometers with a probe in a glycol-filled bottle; more detailed information on specifications of storage units and temperature-monitoring devices is provided at the link given above.

1	Most parenteral vaccines recommended for routine administration to adults in the United States are given by either the IM or the SC route; one influenza vaccine formulation approved for use in adults 18–64 years of age is given intradermally. Live-virus vaccines such as varicella, zoster, and MMR are given SC. Most inactivated vaccines are given IM, except for meningococcal polysaccharide vaccine, which is given SC. The 23-valent pneumococcal polysaccharide vaccine may be given either IM or SC, but IM administration is preferred because it is associated with a lower risk of injection-site reactions.

1	Vaccines given to adults by the SC route are administered with a 5/8-inch needle into the upper outer-triceps area. Vaccines administered to adults by the IM route are injected into the deltoid muscle (Fig. 148-2) with a needle whose length should be selected on the basis of the recipient’s sex and weight to ensure adequate penetration into the muscle. Current guidelines indicate that, for men and women weighing <152 lbs (<70 kg), a 1-inch needle is sufficient; for women weighing 152–200 lbs (70–90 kg) and men weighing 152–260 lbs (70–118 kg), a 1to 1.5-inch needle is needed; and for women weighing >200 lbs (>90 kg) and men weighing >260 lbs (>118 kg), a 1.5-inch needle is required. Additional illustrations of vaccine injection locations and techniques may be found at www.immunize.org/ catg.d/p2020a.pdf.

1	FIGURE 148-2 Technique for IM administration of vaccine. (Photo credit: James Gathany, Centers for Disease Control and Prevention; acces-sible at Public Health Image Library, www.cdc.gov. PHIL ID#9420.) Aspiration, the process of pulling back on the plunger of the syringe after skin penetration but prior to injection, is not necessary because no large blood vessels are present at the recommended vaccine injection sites.

1	Multiple vaccines can be administered at the same visit; indeed, administration of all needed vaccines at one visit is encouraged. Studies have shown that vaccines are as effective when administered simultaneously as they are individually, and simultaneous administration of multiple vaccines is not associated with an increased risk of adverse effects. If more than one vaccine must be administered in the same limb, the injection sites should be separated by 1–2 inches so that any local reactions can be differentiated. If a vaccine and an immune globulin preparation are administered simultaneously (e.g., Td vaccine and tetanus immune globulin), a separate anatomic site should be used for each injection.

1	For certain vaccines (e.g., HPV vaccine and hepatitis B vaccine), multiple doses are required for an adequate and persistent antibody response. The recommended vaccination schedule specifies the interval between doses. Many adults who receive the first dose in a multiple-dose vaccine series do not complete the series or do not receive subsequent doses within the recommended interval. For example, at least one-third of adults who receive the first dose of hepatitis B vaccine in the three-dose series do not complete the series. In these circumstances, vaccine efficacy and/or the duration of protection may be compromised. Providers should implement recall systems that will prompt patients to return for subsequent doses in a vaccination series at the appropriate intervals. With the exception of oral typhoid vaccination, an interruption in the schedule does not require restarting of the entire series or the addition of extra doses.

1	Syncope may follow vaccination, especially in adolescents and young adults. Serious injuries, including skull fracture and cerebral hemorrhage, have occurred. Adolescents and adults should be seated or lying down during vaccination. The majority of reported syncope episodes after vaccination occur within 15 min. The ACIP recommends that vaccine providers strongly consider observing patients, particularly adolescents, with patients seated or lying down for 15 min after vaccination. If syncope develops, patients should be observed until the symptoms resolve. 792 Anaphylaxis is a rare complication of vaccination. All facilities providing immunizations should have an emergency kit containing aqueous epinephrine for administration in the event of a systemic anaphylactic reaction.

1	All vaccines administered should be fully documented in the patient’s permanent medical record. Documentation should include the date of administration, the name or common abbreviation of the vaccine, the vaccine lot number and manufacturer, the administration site, the VIS edition, the date the VIS was provided, and the name, address, and title of the person who administered the vaccine. Increasing use of two-dimensional bar codes on vaccine vials and syringes that can be scanned for data entry into compatible electronic medical records and immunization information systems may facilitate more complete and accurate recording of required information.

1	VACCINE SAFETY MONITORING AND ADVERSE EVENT REPORTING Prelicensure Evaluations of Vaccine Safety Before vaccines are licensed by the FDA, they are evaluated in clinical trials with volunteers. These trials are conducted in three progressive phases. Phase 1 trials are small, usually involving fewer than 100 volunteers. Their purposes are to provide a basic evaluation of safety and to identify common adverse events. Phase 2 trials, which are larger and may involve several hundred participants, collect additional information on safety and are usually designed to evaluate immunogenicity as well. Data gained from phase 2 trials can be used to determine the composition of the vaccine, the number of doses required, and a profile of common adverse events. Vaccines that appear promising are evaluated in phase 3 trials, which typically involve several hundred to several thousand volunteers and are generally designed to demonstrate vaccine efficacy and provide additional information on vaccine

1	in phase 3 trials, which typically involve several hundred to several thousand volunteers and are generally designed to demonstrate vaccine efficacy and provide additional information on vaccine safety.

1	Postlicensure Monitoring of Vaccine Safety After licensure, a vaccine’s safety is assessed by several mechanisms. The NCVIA of 1986 requires health care providers to report certain adverse events that follow vaccination. As a mechanism for that reporting, the Vaccine Adverse Event Reporting System (VAERS) was established in 1990 and is jointly managed by the CDC and the FDA. This safety surveillance system collects reports of adverse events associated with vaccines currently licensed in the United States. Adverse events are defined as untoward events that occur after immunization and that might be caused by the vaccine product or vaccination process. While the VAERS was established in response to the NCVIA, any adverse event following vaccination— whether in a child or an adult, and whether or not it is believed to have actually been caused by vaccination—may be reported through the VAERS. The adverse events that health care providers are required to report are listed in the

1	and whether or not it is believed to have actually been caused by vaccination—may be reported through the VAERS. The adverse events that health care providers are required to report are listed in the reportable-events table on the VAERS website at vaers.hhs.gov/reportable.htm. Approximately 30,000 VAERS reports are filed annually, with ~13% reporting serious events resulting in hospitalization, life-threatening illness, disability, or death.

1	Anyone can file a VAERS report, including health care providers, manufacturers, and vaccine recipients or their parents or guardians. VAERS reports may be submitted online (vaers.hhs.gov/esub/step1) or by completing a paper form requested by email (info@vaers.org), phone (800-822-7967), or fax (877-721-0366). The VAERS form asks for the following information: the type of vaccine received; the timing of vaccination; the time of onset of the adverse event; and the recipient’s current illnesses or medications, history of adverse events following vaccination, and demographic characteristics (e.g., age and sex). This information is entered into a database. The individual who reported the adverse event then receives a confirmation letter by mail with a VAERS identification number that can be used if additional information is submitted later. In selected cases of serious adverse reaction, the patient’s recovery status may be followed up at 60 days and 1 year after vaccination. The FDA and

1	used if additional information is submitted later. In selected cases of serious adverse reaction, the patient’s recovery status may be followed up at 60 days and 1 year after vaccination. The FDA and the CDC have access to VAERS data and use this information to monitor vaccine safety and conduct research studies. VAERS data (minus personal information) are also available to the public.

1	While the VAERS provides useful information on vaccine safety, this passive reporting system has important limitations. One is that events following vaccination are merely reported; the system cannot assess whether a given type of event occurs more often than expected after vaccination. A second is that event reporting is incomplete and is biased toward events that are believed to be more likely to be due to vaccination and that occur relatively soon after vaccination. To obtain more systematic information on adverse events occurring in both vaccinated and unvaccinated persons, the Vaccine Safety Datalink project was initiated in 1991. Directed by the CDC, this project includes nine managed-care organizations in the United States; member databases include information on immunizations, medical conditions, demographics, laboratory results, and medication prescriptions. The Department of Defense oversees a similar system monitoring the safety of immunizations among active-duty military

1	conditions, demographics, laboratory results, and medication prescriptions. The Department of Defense oversees a similar system monitoring the safety of immunizations among active-duty military personnel. In addition, postlicensure evaluations of vaccine safety may be conducted by the vaccine manufacturer. In fact, such evaluations are often required by the FDA as a condition of vaccine licensure.

1	By removing barriers to the consumer or patient, providers and health care institutions can improve vaccine use. Financial barriers have traditionally been important constraints, particularly among uninsured adults. Even for insured adults, out-of-pocket costs associated with newer, more expensive adult vaccines (e.g., zoster vaccine) are an obstacle to be overcome. After influenza vaccine was included by Medicare for all beneficiaries in 1993, coverage among persons ≥65 years of age doubled (from ~30% in 1989 to >60% in 1997). Other strategies that enhance patients’ access to vaccination include extended office hours (e.g., evening and weekend hours) and scheduled vaccination-only clinics where waiting times are reduced. Provision of vaccines outside the “medical home” (e.g., through occupational clinics, universities, pharmacies, and retail settings) can expand access for adults who do not make medical visits frequently. Increasing proportions of adults are being vaccinated in these

1	clinics, universities, pharmacies, and retail settings) can expand access for adults who do not make medical visits frequently. Increasing proportions of adults are being vaccinated in these settings.

1	Health promotion efforts aimed at increasing the demand for immunization are common. Direct-to-consumer advertising by pharmaceutical companies has been used for some newer adolescent and adult vaccines. Efforts to raise consumer demand for vaccines have not increased immunization rates unless implemented in conjunction with other strategies that target strengthening of provider practices or reduction of consumer barriers. Attitudes and beliefs related to vaccination can be considerable impediments to consumer demand. Many adults view vaccines as important for children but are less familiar with vaccinations targeting disease prevention in adults. Several vaccines are recommended for adults with certain medical risk factors, but self-identification as a high-risk individual is relatively rare. Communication research suggests that many adults with chronic diseases may be more motivated to receive a vaccine by a desire to protect their family members rather than to reduce their own

1	rare. Communication research suggests that many adults with chronic diseases may be more motivated to receive a vaccine by a desire to protect their family members rather than to reduce their own risk. Some vaccines are explicitly recommended for persons at relatively low risk of serious complications, with the goal of reducing the risk of transmission to higher-risk contacts. For example, for protection of newborns, vaccinations against influenza and pertussis are recommended for pregnant women and for others who will be around the infant.

1	STRATEGIES FOR PROVIDERS AND HEALTH CARE FACILITIES Recommendation from the Provider Health care providers can have great influence on patients with regard to immunization. A recommendation from a doctor or nurse carries more weight than do recommendations from professional societies or endorsements by celebrities. Providers should be well informed about vaccine risks and benefits so that they can address patients’ common concerns. The CDC, the American College of Physicians, and the American Academy of Family Physicians review and update the schedule for adult immunization on an annual basis and also have developed educational materials to facilitate provider–patient discussions about vaccination (www.cdc .gov/vaccines/hcp.htm).

1	System Supports Medical offices can incorporate a variety of methods to ensure that providers consistently offer specific immunizations to patients with indications for specific vaccines. Decision-support tools have been incorporated into some electronic health records to alert the provider when specific vaccines are indicated. Manual or automated reminders and standing orders have been discussed (see “Deciding Whom to Vaccinate,” above) and have consistently improved vaccination coverage in both office and hospital settings. Most clinicians’ estimates of their own performance diverge from objective measurements of their patients’ immunization coverage; quantitative assessment and feedback have been shown in pediatric and adolescent practices to increase immunization performance significantly. Some health plans have instituted incentives for providers with high rates of immunization coverage. Specialty providers, including obstetrician–gynecologists, may be the only providers serving

1	Some health plans have instituted incentives for providers with high rates of immunization coverage. Specialty providers, including obstetrician–gynecologists, may be the only providers serving some high-risk patients with indications for selected vaccines (e.g., Tdap, influenza, or pneumococcal polysaccharide vaccine).

1	Immunization Requirements Vaccination against selected communicable diseases is required for attendance at many universities and colleges as well as for service in the U.S. military or in some occupational settings (e.g., child care, laboratory, veterinary, and health care). Immunizations are recommended and sometimes required for travel to certain countries (Chap. 149).

1	Vaccination of Health Care Staff A particular area of focus for medical settings is vaccination of health care workers, including those with and without direct patient-care responsibilities. The Joint Commission (which accredits health care organizations), the CDC’s Healthcare Infection Control Practices Advisory Committee, and the ACIP all recommend influenza vaccination of all health care personnel; recommendations also focus on requiring documentation of declination for providers who do not accept annual influenza vaccination. As part of their participation in the Centers for Medicare and Medicaid Services’ Hospital Inpatient Quality Reporting program, acute-care hospitals are required to report the proportion of their health care personnel who have received seasonal influenza vaccine. Some institutions and jurisdictions have added mandates on influenza vaccination of health care workers and have expanded on earlier requirements related to vaccination or proof of immunity for

1	Some institutions and jurisdictions have added mandates on influenza vaccination of health care workers and have expanded on earlier requirements related to vaccination or proof of immunity for hepatitis B, measles, mumps, rubella, and varicella.

1	Receipt of vaccination in medical offices is most frequent among young children and adults ≥65 years of age. People in these age groups make more office visits and are more likely to receive care in a consistent “medical home” than are older children, adolescents, and nonelderly adults. Vaccination outside the medical home can expand access to those whose health care visits are limited and reduce the burden on busy clinical practices. In some locations, financial constraints related to inventory and storage requirements have led providers to stock few or no vaccines. Outside private office and hospital settings, vaccination may also occur at health department venues, workplaces, retail sites (including pharmacies and supermarkets), and schools or colleges.

1	When vaccines are given in nonmedical settings, it remains important for standards of immunization practice to be followed. Consumers should be provided with information on how to report adverse events (e.g., via provision of a VIS), and procedures should ensure that documentation of vaccine administration is forwarded to the primary care provider and the state or city public health immunization registry. Detailed documentation may be required for employment, school attendance, and travel. Personalized health records can help consumers keep track of their immunizations, and some occupational health clinics have incorporated automated immunization reports that help employees stay up-to-date with recommended vaccinations. Some pharmacy chain establishments are using automated systems to report immunization information to the state or local immunization information system.

1	Tracking of immunization coverage at national, state, institution, and practice levels can yield feedback to practitioners and programs and facilitate quality improvement. Healthcare Effectiveness Data 793 and Information Set (HEDIS) measures related to adult immunization facilitate comparison of health plans. The CDC’s National Immunization Survey and National Health Interview Survey provide selected information on immunization coverage among adults and track progress toward achievement of Healthy People 2020 targets for immunization coverage. Influenza and pneumococcal vaccine coverage rates have been higher among persons ≥65 years of age (60–70%) than among high-risk 18to 64-year-olds. Figures on state-specific immunization coverage with pneumococcal polysaccharide and influenza vaccines (as measured through the CDC’s Behavioral Risk Factor Surveillance System) reveal substantial geographic variation in coverage. There are persistent disparities in adult immunization coverage rates

1	(as measured through the CDC’s Behavioral Risk Factor Surveillance System) reveal substantial geographic variation in coverage. There are persistent disparities in adult immunization coverage rates between whites and racial and ethnic minorities. In contrast, racial and economic disparities in immunization of young children have been dramatically reduced during the past 20 years. Much of this progress is attributed to the Vaccines for Children Program, which since 1994 has entitled uninsured children to receive free vaccines.

1	Although most vaccines developed in the twentieth century targeted common acute infectious diseases of childhood, more recently developed vaccines prevent chronic conditions prevalent among adults. Hepatitis B vaccine prevents hepatitis B–related cirrhosis and hepatocellular carcinoma, zoster vaccine prevents shingles and postherpetic neuralgia, and HPV vaccine prevents some types of cervical cancer, genital warts, and anogenital cancers and may also prevent some oropharyngeal cancers (although this outcome was not studied in prelicensure randomized controlled trials). New targets of vaccine development and research may further broaden the definition of vaccine-preventable disease. Research is ongoing on vaccines to prevent insulin-dependent diabetes mellitus, nicotine addiction, and Alzheimer’s disease. Expanding strategies for vaccine development are incorporating molecular approaches such as DNA, vector, and peptide vaccines. New technologies, such as the use of transdermal and

1	disease. Expanding strategies for vaccine development are incorporating molecular approaches such as DNA, vector, and peptide vaccines. New technologies, such as the use of transdermal and other needle-less routes of administration, are being applied to vaccine delivery.

1	Jay S. Keystone, Phyllis E. Kozarsky

1	According to the World Tourism Organization, international tourist arrivals grew dramatically from 25 million in 1950 to >1 billion in 2012. Not only are more people traveling; travelers are seeking more exotic and remote destinations. Travel from industrialized to developing regions has been increasing, with Asia and the Pacific, Africa, and the Middle East now emerging destinations. Figure 149-1 summarizes the monthly incidence of health problems during travel in developing countries. Studies continue to show that 50–75% of short-term travelers to the tropics or subtropics report some health impairment. Most of these health problems are minor: only 5% require medical attention, and <1% require hospitalization. Although infectious agents contribute substantially to morbidity among travelers, these pathogens account for only ~1% of deaths in this population. Cardiovascular disease and injuries are the most frequent causes of death among travelers from the United States, accounting for

1	these pathogens account for only ~1% of deaths in this population. Cardiovascular disease and injuries are the most frequent causes of death among travelers from the United States, accounting for 49% and 22% of deaths, respectively. Age-specific rates of death due to cardiovascular disease are similar among travelers and nontravelers. In contrast, rates of death due to injury (the majority from 794 10.001% 0.0001% 0.01% 0.1% Cholera Meningococcal disease Typhoid (other areas) Legionella infection Typhoid (India, northern/ northwestern Africa, Peru) HIV infection Hepatitis B (expatriates) Hepatitis A Gonorrhea Animal bites with rabies risk Dengue infection (Southeast Asia) Acute febrile respiratory tract infection Malaria (no chemoprophylaxis, West Africa) Travelers’ diarrhea ETEC diarrhea 10% 100% 30–80% 100,000 10,000 10 100 1% 1,000 recommended (immunizations that are desirable because of travel-related risks).

1	Any health problem: used medication or felt ill Felt subjectively ill commonly given to travelers are listed in Table 149-1. Consulted MD abroad or back home Routine Immunizations • dipHtHeria, teta- Stayed in bed nus, and polio Diphtheria (Chap. 175) continues to be a problem worldwide. Large outbreaks have occurred in countries that do not have rigorous vaccination programs or that have reduced their public vaccination programs. Serologic Incapacity to work after return surveys show that tetanus (Chap. 177) antibodies are lacking in many North Americans, especially in women over the age of 50. The risk of polio (Chap. 228) to the international traveler is extremely low, and wild-type poliovirus Died in high-altitude trekking has been eradicated from the Western Hemisphere and Europe. However, studies in the United States suggest that 12% of adult travelers are unprotected against at least one poliovirus serogroup. In addition, challenges continue to be faced

1	Died abroad (average) by polio eradication programs. Foreign travel offers an ideal opportunity to have these immunizations updated. With the recent increase in pertussis among adults, the diphtheria–tetanus–acellular pertussis (Tdap) combination is now recommended for adults as a once-only replacement for the 10-year tetanus–diphtheria FIGURE 149-1 Monthly incidence rates of health problems during stays in developing countries. (Td) booster.

1	FIGURE 149-1 Monthly incidence rates of health problems during stays in developing countries. (Td) booster. ETEC, enterotoxigenic Escherichia coli. (From R Steffen et al: Int J Antimicrob Agents 21:89, 2003.) measles Measles (rubeola) continues to be a major cause of morbidity and death motor vehicle, drowning, or aircraft accidents) are several times higher in the developing world (Chap. 229). Several outbreaks of measles among travelers. Motor vehicle accidents account for >40% of travelers’ in the United States and Canada have been linked to imported cases, deaths that are not due to cardiovascular disease or preexisting illness. especially from Europe, where large outbreaks have occurred recently. The group at highest risk consists of persons born after 1956 and vac-GENERAL ADVICE cinated before 1980, in many of whom primary vaccination failed. The measles–mumps–rubella (MMR) vaccine is typically used; its coverage

1	Health maintenance recommendations are based not only on the travof rubella also addresses a growing concern in some areas of Eastern eler’s destination but also on assessment of risk, which is determined Europe and Asia. by such variables as health status, specific itinerary, purpose of travel, season, and lifestyle during travel. Detailed information regarding influenza Influenza (Chap. 224)—possibly the most common vaccinecountry-specific risks and recommendations may be obtained from the preventable infection in travelers—occurs year-round in the tropicsCenters for Disease Control and Prevention (CDC) publication Health and during the summer months in the Southern Hemisphere (coin-Information for International Travel (available at www.cdc.gov/travel).

1	ciding with the winter months in the Northern Hemisphere). OneFitness for travel is an issue of growing concern in view of the prospective study showed that influenza developed in 1% of travelersincreased numbers of elderly and chronically ill individuals journeying to Southeast Asia per month of stay. Annual vaccination should beto exotic destinations (see “Travel and Special Hosts,” below). Since considered for all travelers who do not have a contraindication. Travel-most commercial aircraft are pressurized to 2500 m (8000 ft) above related influenza continues to occur during summer months in Alaska sea level (corresponding to a Pa02 of ~55 mmHg), individuals with seri-and the Northwest Territories of Canada among cruise-ship passenous cardiopulmonary problems or anemia should be evaluated before gers and staff. The speed of global spread of the pandemic H1N1 virustravel. In addition, those who have recently had surgery, a myocardial once again illustrates why influenza immunization

1	before gers and staff. The speed of global spread of the pandemic H1N1 virustravel. In addition, those who have recently had surgery, a myocardial once again illustrates why influenza immunization is so important forinfarction, a cerebrovascular accident, or a deep-vein thrombosis may travelers. be at high risk for adverse events during flight. A summary of current recommendations regarding fitness to fly has been published by the pneumococcal infection Regardless of travel, pneumococcal vaccine Aerospace Medical Association Air Transport Medicine Committee (Chap. 171) should be administered routinely to persons over the age (www.asma.org/publications/medical-publications-for-airline-travel). of 65 and to persons at high risk of serious infection, including those A pretravel health assessment may be advisable for individuals consid-with chronic heart, lung, or kidney disease; those who have been spleering particularly adventurous recreational activities, such as moun-nectomized; and

1	may be advisable for individuals consid-with chronic heart, lung, or kidney disease; those who have been spleering particularly adventurous recreational activities, such as moun-nectomized; and those who have sickle cell disease. tain climbing and scuba diving.

1	Required Immunizations • yellow fever Documentation of vaccina-IMMUNIZATIONS FOR TRAVEL tion against yellow fever (Chap. 233) may be required or recom-Immunizations for travel fall into three broad categories: mended as a condition for entry into or passage through countries of routine (childhood/adult boosters that are necessary regardless of sub-Saharan Africa and equatorial South America, where the disease travel), required (immunizations that are mandated by international is endemic or epidemic, or (by the International Health Regulations) regulations for entry into certain areas or for border crossings), and for entry into countries at risk of having the infection introduced. This aCross-protects against enterotoxigenic Escherichia coli and provides 30–50% protection against travelers’ diarrhea.

1	vaccine is given only by state-authorized yellow fever centers, and its administration must be documented on an official International Certificate of Vaccination. A registry of U.S. clinics that provide the vaccine is available from the CDC (www.cdc.gov/travel). Recent data suggest that fewer than 50% of travelers entering areas endemic for yellow fever are immunized; this lack of coverage is a serious problem, as 13 countries in Central and South America and 30 countries in Africa harbor the illness. Severe adverse events associated with this vaccine have recently increased in incidence. First-time vaccine recipients may present with a syndrome characterized as either neurotropic (1 case per 125,000 doses) or viscerotropic (overall, 1 case per 250,000 doses; among persons 60–69 years of age, 1 case per 100,000 doses; and among persons ≥ 70 years of age, 1 case per 40,000 doses). Immunosuppression and thymic disease increase the risk of these adverse events

1	persons 60–69 years of age, 1 case per 100,000 doses; and among persons ≥ 70 years of age, 1 case per 40,000 doses). Immunosuppression and thymic disease increase the risk of these adverse events (www.cdc.gov/vaccines/hcp/vis/vis-statements/yf.pdf).

1	meningococcal meningitis Protection against meningitis with one of the quadrivalent (preferably conjugate) vaccines is required for entry into Saudi Arabia during the Hajj (Chap. 180). influenza Both seasonal and pandemic H1N1 vaccines (the latter, where available) were required for entry into Saudi Arabia during the Hajj in 2013.

1	influenza Both seasonal and pandemic H1N1 vaccines (the latter, where available) were required for entry into Saudi Arabia during the Hajj in 2013. Recommended Immunizations • Hepatitis a and b Hepatitis A (Chap. 795 360) is one of the most common vaccine-preventable infections of travelers. The risk is six times greater for travelers who stray from the usual tourist routes. The mortality rate for hepatitis A increases with age, reaching almost 2% among individuals over age 50. Of the four hepatitis A vaccines currently available in North America (two in the United States), all are interchangeable and have an efficacy of >95%. Hepatitis A vaccine is currently given to all children in the United States. Since the most frequently identified risk factor for hepatitis A in the United States is international travel, and since morbidity and mortality increase with age, it seems appropriate that all adults be immune prior to travel.

1	Long-stay overseas workers appear to be at considerable risk for hepatitis B infection (Chap. 360). The recommendation that all travelers be immunized against hepatitis B before departure is supported by two studies showing that 17% of the assessed travelers who received health care abroad had some type of injection; according to the World Health Organization, nonsterile equipment is used for up to 75% of all injections given in parts of the developing world. A 3-week accelerated schedule of the combined hepatitis A and B vaccine has been approved in the United States. Although no data are available on the specific risk of infection with hepatitis B virus among U.S. travelers, ~240 million people in the world have chronic infection. All children and adolescents in the United States are immunized against this illness. Hepatitis B vaccination should be considered for all travelers.

1	typHoid fever Most cases of typhoid fever in North America are due to travel, with ~300 cases seen per year in the United States. The attack rate for typhoid fever (Chap. 190) is 1 case per 30,000 travelers per month of travel to the developing world. However, attack rates in India, Senegal, and North Africa are tenfold higher; rates are especially high among travelers to relatively remote destinations and among immigrants and their families who have returned to their homelands to visit friends or relatives (VFRs). Between 1999 and 2006 in the United States, 66% of imported cases involved the latter group. Unfortunately, data show that the causative organism has become increasingly resistant to fluoroquinolone antibiotics (especially in those cases acquired on the Indian subcontinent). Both of the available vaccines—one oral (live) and the other injectable (polysaccharide)— have efficacy rates of ~70%. In some countries, a combined hepatitis A/typhoid vaccine is available.

1	meningococcal meningitis Although the risk of meningococcal disease among travelers has not been quantified, it is likely to be higher among travelers who live with poor indigenous populations in overcrowded conditions (Chap. 180). Because of its enhanced ability to prevent nasal carriage (compared with the older polysaccharide vaccine), a quadrivalent conjugate vaccine is the product of choice (regardless of age) for immunization of persons traveling to sub-Saharan Africa during the dry season or to areas of the world where there are epidemics. The vaccine, which protects against serogroups A, C, Y, and W-135, has an efficacy rate of >90%.

1	Japanese encepHalitis The risk of Japanese encephalitis (Chap. 233), an infection transmitted by mosquitoes in rural Asia and Southeast Asia, can be as high as ~1 case per 5000 travelers per month of stay in an endemic area. Most infections are asymptomatic, with a very small proportion of infected persons becoming ill. However, among those who do become ill, severe neurologic sequelae are common. Most symptomatic infections among U.S. residents have involved military personnel or their families. The vaccine efficacy rate is >90%. The vaccine is recommended for persons staying >1 month in rural endemic areas or for shorter periods if their activities (e.g., camping, bicycling, hiking) in these areas will increase exposure risk.

1	cHolera The risk of cholera (Chap. 193) is extremely low, with ~1 case per 500,000 journeys to endemic areas. Cholera vaccine, not currently available in the United States, was rarely recommended but was considered for aid and health care workers in refugee camps or in disaster-stricken/war-torn areas. A more effective oral cholera vaccine is available in other countries.

1	796 rabies Domestic animals, primarily dogs, are the major transmitters of rabies in developing countries (Chap. 232). Several studies have shown that the risk of rabies posed by a dog bite in an endemic area translates into 1–3.6 cases per 1000 travelers per month of stay. Countries where canine rabies is highly endemic include Mexico, the Philippines, Sri Lanka, India, Thailand, China, and Vietnam. The two vaccines available in the United States provide >90% protection. Rabies vaccine is recommended for long-stay travelers, particularly children (who tend to play with animals and may not report bites), and for persons who may be occupationally exposed to rabies in endemic areas; however, in a large-scale study, almost 50% of potential exposures occurred within the first month of travel. Even after receipt of a preexposure rabies vaccine series, two postexposure doses are required. Travelers who have had the preexposure series do not require rabies immune globulin (which is often

1	Even after receipt of a preexposure rabies vaccine series, two postexposure doses are required. Travelers who have had the preexposure series do not require rabies immune globulin (which is often unavailable in developing countries) if they are exposed to the disease.

1	It is estimated that more than 30,000 American and European travelers develop malaria each year (Chap. 248). The risk to travelers is highest in Oceania and sub-Saharan Africa (estimated at 1:5 and 1:50 per month of stay, respectively, among persons not using chemoprophylaxis); intermediate in malarious areas on the Indian subcontinent and in Southeast Asia (1:250–1:1000 per month); and low in South and Central America (1:2500–1:10,000 per month). Of the 1925 cases of malaria reported in 2011 in the United States (the highest figure in 40 years), 90% of those due to Plasmodium falciparum occurred in travelers returning or emigrating from Africa and Oceania. VFRs are at the highest risk of acquiring malaria and may die of the disease if their immunity has waned after living outside an endemic area for a number of years. According to data from the CDC, VFRs accounted for 59% of severe malaria cases in the United States in 2011. With the worldwide increase in chloroquineand

1	an endemic area for a number of years. According to data from the CDC, VFRs accounted for 59% of severe malaria cases in the United States in 2011. With the worldwide increase in chloroquineand multidrug-resistant falciparum malaria, decisions about chemoprophylaxis have become more difficult. The case-fatality rate for falciparum malaria in the United States is 4%; however, in only one-third of patients who die is the diagnosis of malaria considered before death.

1	Several studies indicate that fewer than 50% of travelers adhere to basic recommendations for malaria prevention. Keys to the prevention of malaria include both personal protection measures against mosquito bites (especially between dusk and dawn) and malaria chemoprophylaxis. The former measures entail the use of DEET-containing insect repellents, permethrin-impregnated bed nets and clothing, screened sleeping accommodations, and protective clothing. Thus, in regions where infections such as malaria are transmitted, DEET products (25–50%) are recommended, even for children and infants at birth. Studies suggest that concentrations of DEET above ~50% do not offer a marked increase in protection time against mosquitoes. The CDC also recommends picaridin, oil of lemon eucalyptus (PMD, paramenthane-3,8-diol), and IR3535 (3-[N-butyl-N-acetyl]-aminopropionic acid, ethyl ester). In general, higher concentrations of any active ingredient provide a longer duration of protection. Personal

1	and IR3535 (3-[N-butyl-N-acetyl]-aminopropionic acid, ethyl ester). In general, higher concentrations of any active ingredient provide a longer duration of protection. Personal protection measures also help prevent other insect-transmitted illnesses, such as dengue fever (Chap. 233). Over the past decade, the incidence of dengue has markedly increased, particularly in the Caribbean region, Latin America, Southeast Asia, and (more recently) Africa. Chikungunya, another mosquito-borne infection that clinically resembles dengue fever but with arthralgia and arthritis instead of myalgia, has recently crossed to the Western Hemisphere; many thousands of cases are now occurring in the Caribbean. Both dengue and chikungunya viruses are transmitted by an urban-dwelling mosquito that bites primarily at dawn and dusk.

1	Table 149-2 lists the currently recommended drugs of choice for prophylaxis of malaria, by destination. Diarrhea, the leading cause of illness in travelers (Chap. 160), is usually a short-lived, self-limited condition. However, 40% of affected individuals need to alter their scheduled activities, and another 20% are confined to bed. The most important determinant of risk is the destination. Incidence rates per 2-week stay have been reported to be TABLE 149-2 MALARIA CHEMoSuPPRESSIvE REgIMEnS, ACCoRDIng To gEogRAPHIC AREAa Geographic Area Drug of Choiceb Alternatives aSee CDC’s Health Information for International Travel 2014 (www.cdc.gov/travel). bIn all areas where chloroquine can still be used, the other drugs listed may be used as alternatives. cMalarone. Note: See also Chap. 248.

1	Note: See also Chap. 248. as low as 8% in industrialized countries and as high as 55% in parts of Africa, Central and South America, and Southeast Asia. Infants and young adults are at particularly high risk for gastrointestinal illness and for complications such as dehydration. Recent reviews suggest that there is little correlation between dietary indiscretions and the occurrence of travelers’ diarrhea. Earlier studies of U.S. students in Mexico showed that eating meals in restaurants and cafeterias or consuming food from street vendors was associated with increased risk. For further discussion, see “Precautions,” below.

1	Etiology (See also Table 160-3) The most frequently identified pathogens causing travelers’ diarrhea are enterotoxigenic and enteroaggregative Escherichia coli (Chap. 186), although in some parts of the world (notably northern Africa and Southeast Asia) Campylobacter infections (Chap. 192) appear to predominate. Other common causative organisms include Salmonella (Chap. 190), Shigella (Chap. 191), rotavirus (Chap. 227), and norovirus (Chap. 227). The latter virus has caused numerous outbreaks on cruise ships. Except for giardiasis (Chap. 254), parasitic infections are uncommon causes of travelers’ diarrhea in short-term travelers. A growing problem for travelers is the development of antibiotic resistance among many bacterial pathogens. Examples include strains of Campylobacter resistant to quinolones and strains of E. coli, Shigella, and Salmonella resistant to trimethoprimsulfamethoxazole E. coli. O157 is very rarely a cause of travelers’ diarrhea.

1	Precautions Some experts think that it is not only what travelers eat but also where they eat that puts them at risk of illness. Food sold by street vendors can carry a high risk, and restaurant hygiene can be a major problem over which the traveler has no control. In addition to discretion in choosing the source of food and water, general precautions include eating foods piping hot; avoiding foods that are raw or poorly cooked; and drinking only boiled or commercially bottled beverages, particularly those that are carbonated. Heating kills diarrhea-causing organisms, whereas freezing does not; therefore, ice cubes made from unpurified water should be avoided. In spite of these recommendations, the literature has repeatedly documented dietary indiscretions by 98% of travelers within the first 72 h after arrival at their destination. The maxim “Boil it, cook it, peel it, or forget it!” is easy to remember but apparently difficult to follow.

1	Self-Treatment (See also Table 160-5) As travelers’ diarrhea often occurs despite rigorous food and water precautions, travelers should carry medications for self-treatment. An antibiotic is useful in reducing the frequency of bowel movements and the duration of illness in moderate to severe diarrhea. The standard regimen is a 3-day course of a quinolone taken twice daily (or, in the case of some newer formulations, once daily). However, studies have shown that one double dose of a quinolone may be equally effective. For diarrhea acquired in areas such as Thailand, where >90% of Campylobacter infections are quinolone resistant, azithromycin may be a better alternative. Rifaximin, a poorly absorbed rifampin derivative, is highly effective against noninvasive bacterial pathogens such as enterotoxigenic and enteroaggregative E. coli. The current approach to self-treatment of travelers’ diarrhea for the typical short-term traveler is to carry three once-daily doses of an antibiotic and to

1	and enteroaggregative E. coli. The current approach to self-treatment of travelers’ diarrhea for the typical short-term traveler is to carry three once-daily doses of an antibiotic and to use as many doses as necessary to resolve the illness. If neither high fever nor blood in the stool accompanies the diarrhea, loperamide should be taken in combination with the antibiotic; studies have shown that this combination is more effective than an antibiotic alone and does not prolong illness.

1	Prophylaxis Prophylaxis of travelers’ diarrhea with bismuth subsalicylate is widely used but only ~60% effective. For certain individuals (e.g., athletes, persons with a repeated history of travelers’ diarrhea, and persons with chronic diseases), a single daily dose of a quinolone, azithromycin, or rifaximin during travel of <1 month’s duration is 75–90% efficacious in preventing travelers’ diarrhea. Probiotics have been only ~20% effective as prophylaxis. In Europe and Canada, an oral subunit cholera vaccine that cross-protects against enterotoxigenic E. coli (Dukoral) has been shown to provide 30–50% protection against travelers’ diarrhea.

1	Illness After Return Although extremely common, acute travelers’ diarrhea is usually self-limited or amenable to antibiotic therapy. Persistent bowel problems after the traveler returns home have a less well-defined etiology and may require medical attention from a specialist. Infectious agents (e.g., Giardia lamblia, Cyclospora cayetanensis, Entamoeba histolytica) appear to be responsible for only a small proportion of cases with persistent bowel symptoms. By far the most common causes of persistent diarrhea after travel are postinfectious sequelae such as lactose intolerance and irritable bowel syndrome. A meta-analysis showed that postinfectious irritable bowel syndrome lasting months to years may occur in as many as 4–13% of cases. When no infectious etiology can be identified, a trial of metronidazole therapy for presumed giardiasis, a strict lactose-free diet for 1 week, or a several-week trial of high-dose hydrophilic mucilloid (plus an osmotic laxative such as lactulose or PEG

1	of metronidazole therapy for presumed giardiasis, a strict lactose-free diet for 1 week, or a several-week trial of high-dose hydrophilic mucilloid (plus an osmotic laxative such as lactulose or PEG 3350 for persons with alternating diarrhea and constipation) relieves the symptoms of many patients.

1	Travelers are at high risk for sexually transmitted diseases (Chap. 163). Surveys have shown that large numbers of travelers engage in casual sex, and there is a reluctance to use condoms consistently. An increasing number of travelers are being diagnosed with illnesses such as schistosomiasis (Chap. 259), dengue (Chap. 233), chikungunya (Chap. 233), and tick-borne rickettsial disease (Chap. 211). Travelers should be cautioned to avoid bathing, swimming, or wading in freshwater lakes, streams, or rivers in parts of northeastern South America, the Caribbean, Africa, and Southeast Asia. Insect repellents are important for prevention not only of malaria but also of other vector-borne diseases. Prevention of travel-associated injury depends mostly on common-sense precautions. Riding on motorcycles (especially without helmets) and in overcrowded public vehicles is not recommended; in developing countries, individuals should never travel by road in rural areas after dark. Of persons who die

1	(especially without helmets) and in overcrowded public vehicles is not recommended; in developing countries, individuals should never travel by road in rural areas after dark. Of persons who die during travel, fewer than 1% die of infection, whereas 40% die in motor vehicle accidents. Excessive alcohol use has been a significant factor in motor vehicle accidents, drownings, assaults, and injuries. Travelers are cautioned to avoid walking barefoot because of the risk of hookworm and Strongyloides infections (Chap. 257) and snakebites (Chap. 474).

1	A traveler’s medical kit is strongly advisable. The contents may vary widely, depending on the itinerary, duration of stay, style of travel, and local medical facilities. While many medications are available abroad (often over the counter), directions for their use may be nonexistent or in a foreign language, or a product may be outdated or counterfeit. For example, a multicountry study in Southeast Asia 797 showed that a mean of 53% (range, 21–92%) of antimalarial products were counterfeit or contained inadequate amounts of active drug. The sale and marketing of such medications constitute a growing industry. In the medical kit, the short-term traveler should consider carrying an analgesic; an antidiarrheal agent and an antibiotic for self-treatment of travelers’ diarrhea; antihistamines; a laxative; oral rehydration salts; a sunscreen with broad-spectrum protection (UVA and UVB, with the latter at a level of at least 30 SPF); a DEET-containing or equivalent insect repellent for the

1	a laxative; oral rehydration salts; a sunscreen with broad-spectrum protection (UVA and UVB, with the latter at a level of at least 30 SPF); a DEET-containing or equivalent insect repellent for the skin; an insecticide for clothing (permethrin); and, if necessary, an antimalarial drug. To these medications, the long-stay traveler might add a broad-spectrum general-purpose antibiotic (levofloxacin or azithromycin), an antibacterial eye and skin ointment, and a topical antifungal cream. Regardless of the duration of travel, a first-aid kit containing such items as scissors, tweezers, and bandages should be considered. A practical approach to self-treatment of infections in the long-stay traveler who carries a once-daily dose of antibiotics (e.g., levofloxacin) is to use 3 tablets “below the waist” (bowel and bladder infections) and 6 tablets “above the waist” (skin and respiratory infections).

1	(See also Chap. 8) A woman’s medical history and itinerary, the quality of medical care at her destinations, and her degree of flexibility determine whether travel is wise during pregnancy. According to the American College of Obstetrics and Gynecology, the safest part of pregnancy in which to travel is between 18 and 24 weeks, when there is the least danger of spontaneous abortion or premature labor. Some obstetricians prefer that women stay within a few hundred miles of home after the 28th week of pregnancy in case problems arise. In general, however, healthy women may be advised that it is acceptable to travel.

1	Relative contraindications to international travel during pregnancy include a history of miscarriage, premature labor, incompetent cervix, or toxemia. General medical problems such as diabetes, heart failure, severe anemia, or a history of thromboembolic disease also should prompt the pregnant woman to postpone her travels. Finally, regions in which the pregnant woman and her fetus may be at excessive risk (e.g., those at high altitudes, those where live-virus vaccines are required, and those where multidrug-resistant malaria is endemic) are not ideal destinations during any trimester.

1	Malaria Malaria during pregnancy carries a significant risk of morbidity and death. Levels of parasitemia are highest and failure to clear the parasites after treatment is most frequent among primigravidae. Severe disease, with complications such as cerebral malaria, massive hemolysis, and renal failure, is especially likely in pregnancy. Fetal sequelae include spontaneous abortion, stillbirth, preterm delivery, and congenital infection. Chloroquine and mefloquine are considered to be safe in all trimesters. Enteric Infections Pregnant travelers must be extremely cautious regarding their food and beverage intake. Dehydration due to travelers’ diarrhea can lead to inadequate placental blood flow. Infections such as toxoplasmosis, hepatitis E, and listeriosis also can cause serious sequelae in pregnancy.

1	The mainstay of therapy for travelers’ diarrhea is rehydration. Loperamide may be used if necessary. For self-treatment, azithromycin may be the best option. Although quinolones are increasingly being used safely during pregnancy and rifaximin is poorly absorbed from the gastrointestinal tract, these drugs are not approved for this indication. Because of the serious problems encountered when infants are given local foods and beverages, women are strongly encouraged to breast-feed when traveling with a neonate. A nursing mother with travelers’ diarrhea should not stop breast-feeding but should increase her fluid intake.

1	Air Travel and High-Altitude Destinations Commercial air travel is not a risk to the healthy pregnant woman or to the fetus. The higher radiation levels reported at altitudes of >10,500 m (>35,000 ft) should pose 798 no problem for the healthy pregnant traveler. Since each airline has a policy regarding pregnancy and flying, it is best to check with the specific carrier when booking reservations. Domestic air travel is usually permitted until the 36th week, whereas international air travel is generally curtailed after the 32nd week. There are no known risks for pregnant women who travel to high-altitude destinations and stay for short periods. However, there are likewise no data on the safety of pregnant women at altitudes of >4500 m (15,000 ft).

1	(See also Chap. 226) The HIV-infected traveler is at special risk of serious infections due to a number of pathogens that may be more prevalent at travel destinations than at home. However, the degree of risk depends primarily on the state of the immune system at the time of travel. For persons whose CD4+ T cell counts are normal or >500/ μL, data suggest no greater risk during travel than for persons without HIV infection. Individuals with AIDS (CD4+ T cell counts of <200/μL) and others who are symptomatic need special counseling and should visit a travel medicine practitioner before departure, especially when traveling to the developing world.

1	Several countries routinely deny entry to HIV-positive individuals for prolonged stay, even though these restrictions do not appear to decrease rates of transmission of the virus. In general, HIV testing is required for individuals who wish to stay abroad >3 months or who intend to work or study abroad. Some countries will accept an HIV serologic test done within 6 months of departure, whereas others will not accept a blood test done at any time in the traveler’s home country. Border officials often have the authority to make inquiries of individuals entering a country and to check the medications they are carrying. If antiretroviral drugs are identified, the person may be barred from entering the country. Information on testing requirements for specific countries is available from consular offices but is subject to frequent change.

1	Immunizations All of the HIV-infected traveler’s routine immunizations should be up to date (Chap. 148). The response to immunization may be impaired at CD4+ T cell counts of <200/μL and in some cases at even higher counts. Thus HIV-infected persons should be vaccinated as early as possible to ensure adequate immune responses. For patients receiving antiretroviral therapy, at least 3 months must elapse before regenerated CD4+ T cells can be considered fully functional; therefore, vaccination of these patients should be delayed. However, when the risk of illness is high or the sequelae of illness are serious, immunization is recommended. In certain circumstances, it may be prudent to check the adequacy of the serum antibody response before departure.

1	Because of the increased risk of infections due to Streptococcus pneumoniae and other bacterial pathogens that cause pneumonia after influenza, the conjugate pneumococcal vaccine (Prevnar 13) followed by the 23-valent polysaccharide vaccine (Pneumovax) as well as influenza vaccine should be administered. The estimated rates of response to influenza vaccine are >80% among persons with asymptomatic HIV infection and <50% among those with AIDS. In general, live attenuated vaccines are contraindicated for persons with immune dysfunction. Because measles (rubeola) can be a severe or lethal infection in HIV-positive patients, these patients should receive the measles vaccine (or the combination MMR vaccine) unless the CD4+ T cell count is <200/μL. Between 18% and 58% of symptomatic HIV-infected vaccinees develop adequate measles antibody titers, and 50–100% of asymptomatic HIV-infected persons seroconvert.

1	It is recommended that the live yellow fever vaccine not be given to HIV-infected travelers. Although the potential adverse effects of a live vaccine in an HIV-infected individual are always a consideration, there appear to have been no reported cases of illness in those who have inadvertently received this vaccine. Nonetheless, if the CD4+ T cell count is <200/μL, an alternative itinerary that poses no risk of exposure to yellow fever is recommended. If the traveler is passing through or traveling to an area where the vaccine is required but the disease risk is low, a physician’s waiver should be issued. A transient increase in HIV viremia (lasting days to weeks) has been demonstrated in HIV-infected individuals after immunization against influenza, pneumococcal infection, and tetanus (Chap. 226). At this point, however, no evidence indicates that this transient increase is detrimental.

1	Gastrointestinal Illness Decreased levels of gastric acid, abnormal gastrointestinal mucosal immunity, other complications of HIV infection, and medications taken by HIV-infected patients make travelers’ diarrhea especially problematic in these individuals. Travelers’ diarrhea is likely to occur more frequently, to be more severe, to be accompanied by bacteremia, and to be more difficult to treat. Cryptosporidium, Isospora belli, and Microsporidium infections, although uncommon, are associated with increased morbidity and mortality rates in AIDS patients. The HIV-infected traveler must be careful to consume only appropriately prepared foods and beverages and may benefit from antibiotic prophylaxis for travelers’ diarrhea. Sulfonamides (as used to prevent pneumocystosis) are ineffective because of widespread resistance.

1	Other Travel-Related Infections Data are lacking on the severity of many vector-borne diseases in HIV-infected individuals. Malaria is especially severe in asplenic persons and in those with AIDS. The HIV load doubles during malaria, with subsidence in ~8–9 weeks; the significance of this increase in viral load is unknown. Visceral leishmaniasis (Chap. 251) has been reported in numerous HIV-infected travelers. Diagnosis may be difficult, given that splenomegaly and hyperglobulinemia are often lacking and serologic results are frequently negative. Sandfly bites may be prevented by evening use of insect repellents.

1	Certain respiratory illnesses, such as histoplasmosis and coccidioidomycosis, cause greater morbidity and mortality among patients with AIDS. Although tuberculosis is common among HIV-infected persons (especially in developing countries), its acquisition by the short-term HIV-infected traveler has not been reported as a major problem. From a prospective study, it is estimated that for travelers not engaged in health care the risk of tuberculosis infection is ~3% per year of travel.

1	Medications Adverse events due to medications and drug interactions are common and raise complex issues for HIV-infected persons. Rates of cutaneous reaction (e.g., increased cutaneous sensitivity to sulfonamides) are unusually high among patients with AIDS. Since zidovudine is metabolized by hepatic glucuronidation, inhibitors of this process may elevate serum levels of the drug. Concomitant administration of the antimalarial drug mefloquine and the antiretroviral agent ritonavir may result in decreased plasma levels of ritonavir; mefloquine may also interact with many of the other protease inhibitors. In contrast, no significant influence of concomitant mefloquine administration on plasma levels of indinavir or nelfinavir was detected in two HIV-infected travelers. Serum levels of mefloquine may be lowered with the use of efavirenz or nevirapine. There are also potential interactions between atovaquone-proguanil (Malarone) and many of the protease inhibitors as well as between

1	mefloquine may be lowered with the use of efavirenz or nevirapine. There are also potential interactions between atovaquone-proguanil (Malarone) and many of the protease inhibitors as well as between Malarone and the nonnucleoside reverse transcriptase inhibitors (NNRTIs). Because of the increase in antiretroviral agents and the lack of accumulated data on their interactions with antimalarial agents, decisions about malaria chemoprophylaxis continue to be difficult; with a short duration of travel, an interaction may be inconsequential. However, doxycycline appears to have no clinically significant interactions with either the protease inhibitors or the NNRTIs. With regard to malaria treatment, a great hypothetical concern is that the antimalarial drugs lumefantrine (combined with artemisinin in Coartem) and halofantrine may interact with HIV protease inhibitors and NNRTIs since drugs in the latter two categories are known to be potent inhibitors of cytochrome P450. In keeping current

1	in Coartem) and halofantrine may interact with HIV protease inhibitors and NNRTIs since drugs in the latter two categories are known to be potent inhibitors of cytochrome P450. In keeping current with antiretroviral drug interactions, a website from the University of Liverpool (www.hiv-druginteractions.org) is helpful.

1	CHRONIC ILLNESS, DISABILITY, AND TRAVEL Chronic health problems need not prevent travel, but special measures can make the journey safer and more comfortable.

1	Heart Disease Cardiovascular events are the main cause of deaths among travelers and of in-flight emergencies on commercial aircraft. Extra supplies of all medications should be kept in carry-on luggage, along with a copy of a recent electrocardiogram and the name and telephone number of the traveler’s physician at home. Pacemakers are not affected by airport security devices, although electronic telephone checks of pacemaker function cannot be transmitted by international satellites. Travelers with electronic defibrillators should carry a note to that effect and ask for hand screening. A traveler may benefit from supplemental oxygen; since oxygen delivery systems are not standard, supplementary oxygen should be ordered by the traveler’s physician well before flight time. Travelers may benefit from aisle seating and should walk, perform stretching and flexing exercises, consider wearing support hose, and remain hydrated during the flight to prevent venous thrombosis and pulmonary

1	benefit from aisle seating and should walk, perform stretching and flexing exercises, consider wearing support hose, and remain hydrated during the flight to prevent venous thrombosis and pulmonary embolism.

1	Chronic Lung Disease Chronic obstructive pulmonary disease is one of the most common diagnoses in patients who require emergency-department evaluation for symptoms occurring during airline flights. The best predictor of the development of in-flight problems is the sea-level PaO2. A PaO2 of at least 72 mmHg corresponds to an in-flight arterial PaO2 of ~55 mmHg when the cabin is pressurized to 2500 m (8000 ft). If the traveler’s baseline PaO2 is <72 mmHg, the provision of supplemental oxygen should be considered. Contraindications to flight include active bronchospasm, lower respiratory infection, lower-limb deep-vein phlebitis, pulmonary hypertension, and recent thoracic surgery (within the preceding 3 weeks) or pneumothorax. Decreased outdoor activity at the destination should be considered if air pollution is excessive.

1	Diabetes Mellitus Alterations in glucose control and changes in insulin requirements are common problems among patients with diabetes who travel. Changes in time zone, in the amount and timing of food intake, and in physical activity demand vigilant assessment of metabolic control. Because of the risk of foot ulcers, travelers should wear closed footwear that has been proven to be comfortable. The traveler with diabetes should pack medication (including a bottle of regular insulin for emergencies), insulin syringes and needles, equipment and supplies for glucose monitoring, and snacks in carry-on luggage. Insulin is stable for ~3 months at room temperature but should be kept as cool as possible. The name and telephone number of the home physician and a card and bracelet listing the patient’s medical problems and the type and dose of insulin used should accompany the traveler. In order to facilitate international border crossings, travelers should carry a physician’s letter authorizing

1	medical problems and the type and dose of insulin used should accompany the traveler. In order to facilitate international border crossings, travelers should carry a physician’s letter authorizing the carriage of needles and syringes. In traveling eastward (e.g., from the United States to Europe), the morning insulin dose on arrival may need to be decreased. The blood glucose can then be checked during the day to determine whether additional insulin is required. For flights westward, with lengthening of the day, an additional dose of regular insulin may be required.

1	Other Special Groups Other groups for whom special travel measures are encouraged include patients undergoing dialysis, those with transplants, and those with other disabilities. Up to 13% of travelers have some disability, but few advocacy groups and tour companies dedicate themselves to this growing population. Medication interactions are a source of serious concern for these travelers, and appropriate medical information should be carried, along with the home physician’s name and telephone number. Some travelers taking glucocorticoids carry stress doses in case they become ill. Immunization of these immunocompromised travelers may result in less than adequate protection. Thus the traveler and the physician must carefully consider which destinations are appropriate.

1	Today, more elderly or chronically ill individuals travel and more of these individuals journey to remote locations and enjoy adventurous activities. Illness or injury abroad is not uncommon and is best considered before the journey. Persons who develop health problems abroad may incur enormous out-of-pocket expenses. Thus prospective travelers should consider purchasing additional travel health insurance and 799 should check with their health insurance company regarding whether they have coverage for illness or injury overseas. Unfortunately, many insurance companies will not cover pre-existing illness if it is the reason for trip cancellation or illness abroad. Most countries do not accept routine health insurance from other countries unless there is a special traveler supplement. In most circumstances, travelers are asked to pay in cash for services rendered on an emergency basis, whether in a physician’s office, in an emergency or urgent care center, or even in a hospital. There

1	circumstances, travelers are asked to pay in cash for services rendered on an emergency basis, whether in a physician’s office, in an emergency or urgent care center, or even in a hospital. There are several types of travel insurance. It is wise to purchase trip cancellation insurance, especially, for example, if the traveler has an underlying chronic illness and may need to cancel a trip due to an exacerbation of disease. Travel health insurance will cover expenses in the event that medical care abroad is needed. Evacuation insurance will cover medical evacuation, usually to a medical center in another location where it is deemed that the care is similar to that available in the traveler’s home country. The cost of medical evacuation can easily exceed $100,000 US. There are a number of travel insurance providers, and it is very important to read the fine print carefully and to determine exactly what each company provides, thereby ensuring an appropriate fit for the individual’s

1	of travel insurance providers, and it is very important to read the fine print carefully and to determine exactly what each company provides, thereby ensuring an appropriate fit for the individual’s particular circumstances. The U.S. Department of State website lists travel health insurance companies (http://travel.state.gov/travel/tips/ emergencies/emergencies_5981.html).

1	Travel for the purpose of obtaining health care abroad has received a great deal of attention in the medical literature and the media. According to the annual U.S. Department of Commerce In-Flight Survey, there were ~500,000 overseas trips during 2006 in which health treatment was at least one purpose of travel. Lower cost is usually cited as the motivation for this type of tourism, and an entire industry has flourished as a result of this phenomenon. However, the quality of facilities, assistance services, and care is neither uniform nor regulated; thus, in most instances, responsibility for assessing the suitability of an individual program or facility lies solely with the traveler. Persons considering this option must recognize that they are almost always at a disadvantage when being treated in a foreign country, particularly if there are complications. Concerns to be addressed include the quality of the health care facility and its staff; language and cultural differences that may

1	in a foreign country, particularly if there are complications. Concerns to be addressed include the quality of the health care facility and its staff; language and cultural differences that may impede accurate interpretation of both verbal and nonverbal communication; religious and ethical differences that may be encountered over issues such as efforts to preserve life and limb or the provision of care for the terminally ill; lack of familiarity with the local medical system; limited access of the care provider to the patient’s medical history; the use of unfamiliar drugs and medicines; the relative difficulty of arranging follow-up care back in the United States; and the possibility that such follow-up care may be fraught with problems should there be complications. If serious issues arise, legal recourse may be difficult or impossible. Patients planning to travel abroad to obtain health care, particularly when surgery is involved, should be immunized for hepatitis B and should

1	arise, legal recourse may be difficult or impossible. Patients planning to travel abroad to obtain health care, particularly when surgery is involved, should be immunized for hepatitis B and should consider having baseline hepatitis C and HIV tests preoperatively. Prevalence rates of hepatitis B and C and HIV infection vary considerably around the world and are generally higher in developing regions than in the United States and Western Europe. The latest information available on the safety of the blood supply outside the United States is the World Health Organization’s Global Database on Blood Safety based on data from 2011 (www.who.int/bloodsafety/global_database/en). Persons researching the accreditation status of overseas facilities should note that, although these facilities may be part of a chain, they are surveyed and accredited individually. Accreditation resources include (1) the Joint Commission International (www.jointcommissioninternational.org), (2) the Australian Council

1	of a chain, they are surveyed and accredited individually. Accreditation resources include (1) the Joint Commission International (www.jointcommissioninternational.org), (2) the Australian Council for Healthcare Standards International (www .achs.org.au/achs-international/), and (3) the Canadian Council on Health Services (www.cchsa.ca). The American Medical Association also offers guidelines for medical tourism (www.ama-assn.org/ama1/ pub/upload/mm/31/medicaltourism.pdf).

1	800 PROBLEMS AFTER RETURN particular the epidemiology and clinical presentation of infectious disorders. A geographic history should focus on the traveler’s exactThe most common medical problems encountered by travelers after itinerary, including dates of arrival and departure; exposure his-their return home are diarrhea, fever, respiratory illnesses, and skin tory (food indiscretions, drinking-water sources, freshwater contact,diseases (Fig. 149-2). Frequently ignored problems are fatigue and sexual activity, animal contact, insect bites); location and style of travel emotional stress, especially in long-stay travelers. The approach (urban vs. rural, first-class hotel accommodation vs. camping); immuto diagnosis requires some knowledge of geographic medicine, in nization history; and use of antimalarial chemosuppression. Recently,

1	FIGURE 149-2 Proportionate morbidity among ill travelers returning from the developing world, according to region of travel. The proportions (not incidence rates) are shown for each of the top 22 specific diagnoses among all ill returned travelers within each region. STDs, sexually transmitted diseases. Asterisks indicate syndromic diagnoses for which specific etiologies could not be assigned. (Reprinted from DO Freedman et al: N Engl J Med 354:119, 2006.) some travelers who have been hospitalized abroad have been shown on return to be colonized with multidrug-resistant bacteria such as Enterobacteriaceae producing extended-spectrum β-lactamases and bacteria producing NDM-1 (New Delhi metallo-β-lactamase 1). See “Prevention of Gastrointestinal Illness,” above.

1	See “Prevention of Gastrointestinal Illness,” above. Fever in a traveler who has returned from a malarious area should be considered a medical emergency because death from P. falciparum malaria can follow an illness of only several days’ duration. Although “fever from the tropics” does not always have a tropical cause, malaria should be the first diagnosis considered. The risk of P. falciparum malaria is highest among travelers returning from Africa or Oceania The growth of global travel and migration now Percentage of Casesa demand that the clinician become as familiar Central South Sub-Saharan South-Central Southeast as possible with travel medicine. Practitioners tiology Caribbean America America Africa Asia Asia may choose either to refer their patients to aBold type is for emphasis only. Source: Revised from Table 2 in DO Freedman et al: N Engl J Med 354:119, 2006.

1	and among those who become symptomatic within the first 2 months after return. Other important causes of fever after travel include viral hepatitis (A and E), typhoid and paratyphoid fever, bacterial enteritis, arboviral infections (e.g., dengue fever), rickettsial infections (including tick typhus, scrub typhus, and fever), and—in rare instances— leptospirosis, acute HIV infection, and amebic liver abscess. A cooperative study by GeoSentinel (an emerging infectious disease surveillance group established by the CDC and the International Society of Travel Medicine) showed that, among 3907 febrile returned travelers, malaria was acquired most often in Africa, dengue in Southeast Asia and the Caribbean, typhoid fever in southern Asia, and rickettsial infections (tick typhus) in South Africa (Table 149-3). Outbreaks of dengue, previously considered to be very rare in Africa, have been documented recently in Angola, Kenya, and Tanzania. However, in at least 25% of cases, no etiology of

1	(Table 149-3). Outbreaks of dengue, previously considered to be very rare in Africa, have been documented recently in Angola, Kenya, and Tanzania. However, in at least 25% of cases, no etiology of the fever can be found and it resolves spontaneously. Clinicians should keep in mind that no present-day antimalarial agent guarantees protection from malaria and that some immunizations (notably, that against typhoid fever) are only partially protective.

1	When no specific diagnosis is forthcoming, the following investigations, where applicable, are suggested: complete blood count, liver function tests, thick/thin blood films or rapid diagnostic testing for malaria (repeated several times if necessary), urinalysis, urine and blood cultures (repeated once), chest x-ray, and collection of an acute-phase serum sample to be held for subsequent examination along with a paired convalescent-phase serum sample. Pyodermas, sunburn, insect bites, skin ulcers, and cutaneous larva migrans are the most common skin conditions affecting travelers after their return home. In those with persistent skin ulcers, a diagnosis of cutaneous leishmaniasis, mycobacterial infection, or fungal infection should be considered. Careful, complete inspection of the skin is important in detecting the rickettsial eschar in a febrile patient or the central breathing hole in a “boil” due to myiasis.

1	In recent years, travel and commerce have fostered the worldwide spread of HIV infection, led to the reemergence of cholera as a global health threat, and created considerable fear about the possible spread of novel respiratory diseases, including those caused by influenza viruses (H5N1, H1N1, and H7N9). For travelers, there are more common, everyday concerns. One of the largest outbreaks of dengue fever ever documented is now raging in Latin America and Southeast Asia; chikungunya virus has spread rapidly from Africa to southern Asia, southern Europe, and, for the first time in the Western Hemisphere, the Caribbean; schistosomiasis is being described in previously unaffected lakes in Africa; and antibiotic-resistant strains of sexually transmitted and enteric pathogens are emerging at an alarming rate in the developing world. In addition, concerns have been raised about the potential for bioterrorism involving not only standard strains of unusual agents but mutant strains as well.

1	plex post-travel illnesses. The CDC publishes a biennial text, Health Information for International Travel (accessed through their website at www.cdc.gov/travel) that provides pretravel health recommendations. The International Society of Travel Medicine (www.istm .org) publishes a list of travel clinics, and the American Society of Tropical Medicine and Hygiene (www.astmh.org) publishes a list of clinical tropical medicine specialists. As Nobel Laureate Dr. Joshua Lederberg pointed out, “The microbe that felled one child in a distant continent yesterday can reach yours today and seed a global pandemic tomorrow.” The vigilant clinician understands that the importance of a thorough travel history cannot be overemphasized.

1	Laboratory Diagnosis of Infectious Diseases Alexander J. McAdam, Andrew B. Onderdonk The laboratory diagnosis of infection requires the demonstration— either direct or indirect—of viral, bacterial, fungal, or parasitic agents 150e in tissues, fluids, or excreta of the host. Clinical microbiology laboratories are responsible for processing these specimens and also for determining the antibiotic susceptibility of bacterial and fungal pathogens. Traditionally, detection of pathogenic agents has relied largely on either the microscopic visualization of pathogens in clinical material or the growth of microorganisms in the laboratory. Identification generally is based on phenotypic characteristics such as fermentation profiles for bacteria, cytopathic effects in tissue culture for viral agents, and microscopic morphology for fungi and parasites. These techniques are reliable but are often time-consuming. Increasingly, the use of nucleic acid probes is becoming a standard method for

1	agents, and microscopic morphology for fungi and parasites. These techniques are reliable but are often time-consuming. Increasingly, the use of nucleic acid probes is becoming a standard method for detection, quantitation, and/or identification in the clinical microbiology laboratory, gradually replacing phenotypic characterization and microscopic visualization methods. This chapter discusses general concepts of diagnostic testing, with an emphasis on detection of bacteria. Detection of viral, fungal, and parasitic pathogens is discussed in greater detail in separate chapters (see Chaps. 214e, 235, and 245e, respectively).

1	Reappraisal of the methods employed in the clinical microbiology laboratory has led to the development of strategies for detection of pathogenic agents through nonvisual biologic signal detection systems. A biologic signal is generated by detection of a material that can be reproducibly differentiated from other substances present in the sample. Key issues in the use of a biologic signal are distinguishing it from background noise and translating it into meaningful information. Examples of useful materials for detection of biologic signals applicable to clinical microbiology include structural components of bacteria, fungi, and viruses; specific antigens; metabolic end products; unique DNA or RNA base sequences; enzymes; toxins or other proteins; and surface polysaccharides.

1	A detector is used to sense a signal and discriminate between that signal and background noise. Detection systems range from the trained eyes of a technologist assessing morphologic variations to electronic instruments such as gas-liquid chromatographs or mass spectrometers. The sensitivity with which signals can be detected varies widely. It is essential to use a detection system that discerns small amounts of signal even when biologic background noise is present—i.e., that is both sensitive and specific. Common detection systems include immunofluorescence; chemiluminescence for DNA/ RNA probes; flame ionization detection of shortor long-chain fatty acids; and detection of substrate utilization or end-product formation as color changes, of enzyme activity as a change in light absorbance, of turbidity changes as a measure of growth, of cytopathic effects in cell lines, and of particle agglutination as a measure of antigen presence.

1	Amplification enhances the sensitivity with which weak signals can be detected. The most common microbiologic amplification technique is growth of a single bacterium into a discrete, visible colony on an agar plate or into a suspension containing many identical organisms. The advantage of growth as an amplification method is that it requires only an appropriate growth medium; the disadvantage is the amount of time required. More rapid amplification of biologic signals can be achieved with techniques such as polymerase chain reaction (PCR), ligase chain reaction (LCR), and transcription-mediated amplification (TMA), all of which target the pathogen’s DNA/RNA; enzyme immunoassays (EIAs) for antigens and antibodies; electronic amplification (for gas-liquid chromatography assays); antibody capture methods (for concentration and/or separation); and selective filtration or centrifugation.

1	Direct detection refers to detection of pathogens without the use of culture. Molecular methods of direct detection are discussed below. The field of microbiology was defined largely by the development and use of the microscope. The examination of specimens by microscopic methods rapidly provides useful diagnostic information. Staining techniques permit organisms to be seen more clearly.

1	The simplest method for microscopic evaluation is the wet mount, which is used, for example, to examine cerebrospinal fluid (CSF) for the presence of Cryptococcus neoformans, with India ink as a background against which to visualize large-capsuled yeast cells. Wet mounts with dark-field illumination also are used to detect spirochetes in genital lesions and Borrelia or Leptospira in blood. Skin scrapings and hair samples can be examined with the use of either 10% KOH wet-mount preparations or the calcofluor white method and ultraviolet illumination to detect fungal elements as fluorescing structures. Staining of wet mounts—e.g., with lactophenol cotton blue stain for fungal elements—often is used for morphologic identification. Bacteria are difficult to see by light microscopy unless they are stained. Although simple one-step stains can be used, differential stains are more common.

1	Gram’s Stain Gram’s stain differentiates between organisms with thick peptidoglycan cell walls (gram-positive) and those with thin peptidoglycan cell walls and outer membranes that can be dissolved with alcohol or acetone (gram-negative). Morphology and Gram’s stain characteristics often can be used to categorize stained organisms into groups such as streptococci, staphylococci, and clostridia (Table 150e-1). Gram’s stain is particularly useful for examining sputum for polymorphonuclear leukocytes (PMNs) and bacteria. Sputum specimens from immunocompetent patients with ≥25 PMNs and <10 epithelial cells per low-power field often provide clinically useful information. However, the presence in “sputum” samples of >10 epithelial cells per low-power field and of multiple bacterial types suggests contamination with oral microflora. Despite the difficulty of discriminating between normal microflora and pathogens, Gram’s stain may prove useful for specimens from areas with a large resident

1	contamination with oral microflora. Despite the difficulty of discriminating between normal microflora and pathogens, Gram’s stain may prove useful for specimens from areas with a large resident microflora if a useful biologic marker (signal) is available. Gram’s staining of vaginal swab specimens can be used to detect epithelial cells covered with gram-positive bacteria in the absence of lactobacilli and the presence of gram-negative rods—a scenario regarded as a sign of bacterial vaginosis. Similarly, examination of stained stool specimens for leukocytes is useful as a screening procedure before testing for Clostridium difficile toxin or other enteric pathogens.

1	The examination of samples from normally sterile body sites (e.g., CSF or joint, pleural, or peritoneal fluid) with Gram’s stain is useful for determining whether bacteria and/or PMNs are present. The sensitivity is such that >104 bacteria/mL should be detected. Centrifugation often is performed before staining to concentrate specimens thought to contain low numbers of organisms. This simple method is particularly useful for examination of CSF for bacteria and white blood cells or examination of sputum for mycobacteria.

1	Acid-Fast Stain The acid-fast stain identifies acid-fast bacteria (AFB; mycobacteria) by their retention of carbol fuchsin dye after acid/ organic solvent disruption. Modifications of this procedure allow the differentiation of Actinomyces from Nocardia or other weakly (or partially) acid-fast organisms. The acid-fast stain is applied to sputum, other fluids, and tissue samples when Mycobacterium species are suspected. Because few AFB may be present in an entire smear, even when the specimen has been concentrated by centrifugation, identification of the pink/red AFB against the blue background of the counterstain requires a trained eye. An alternative method for detection of AFB is the auramine-rhodamine fluorescent dye stain.

1	CHAPTER 150e Laboratory Diagnosis of Infectious Diseases aSome important bacteria cannot be seen with Gram’s stain because they are too small or too slender or do not retain the stain. These bacteria include Treponema pallidum, Borrelia burgdorferi, Chlamydia spp., Mycoplasma spp., and Ureaplasma spp. Fluorochrome Stains Fluorochrome stains such as acridine orange are used to identify white blood cells, yeasts, and bacteria in body fluids. Capsular, flagellar, and spore stains are used for identification or demonstration of characteristic structures.

1	Immunofluorescent Stains The direct immunofluorescent antibody technique uses antibody coupled to a fluorescent compound (e.g., fluorescein) and directed at a specific antigenic target to visualize organisms. When samples are examined under appropriate conditions, the fluorescing compound absorbs ultraviolet light and re-emits light at a higher wavelength that is visible to the human eye. In the indirect immunofluorescent antibody technique, an unlabeled (target) antibody binds a specific antigen. The specimen is then stained with a fluorochrome-labeled antibody directed at the target antibody. Because each unlabeled target antibody attached to the appropriate antigen has multiple sites for attachment of the second antibody, the visual signal is amplified. Immunofluorescence is used to detect viral antigens (e.g., cytomegalovirus, herpes simplex virus, and respiratory viruses) within cultured cells or clinical specimens as well as many difficult-to-grow bacterial agents (e.g.,

1	to detect viral antigens (e.g., cytomegalovirus, herpes simplex virus, and respiratory viruses) within cultured cells or clinical specimens as well as many difficult-to-grow bacterial agents (e.g., Legionella pneumophila) in clinical specimens.

1	Latex agglutination assays and EIAs are rapid and inexpensive methods for identifying organisms, extracellular toxins, and viral agents by means of protein and polysaccharide antigens. Such assays may be performed directly on clinical samples or after growth of organisms on agar plates or in viral cell cultures. Antibodies coupled to a reporter (such as latex particles or an enzyme) are used for detection of antibody–antigen binding reactions.

1	Direct agglutination of bacterial cells with specific antibody is simple but relatively insensitive; latex agglutination and EIAs are more sensitive. Some cell-associated antigens, such as capsular polysaccharides and lipopolysaccharides, can be detected by agglutination of a suspension of bacterial cells when antibody is added; this method is useful for typing of the somatic antigens of Shigella and Salmonella. EIAs employ antibodies coupled to an enzyme, and an antigen–antibody reaction results in the conversion of a colorless substrate to a colored product. Most of these assays provide information about whether antigen is present but do not quantify the antigen. EIAs are also useful for detecting bacterial toxins—e.g., toxins produced by Shiga toxin– producing Escherichia coli.

1	Rapid and simple immunoassays for antigens of group A Streptococcus, influenza virus, and respiratory syncytial virus can be used in the clinical setting without a specialized diagnostic laboratory. Such tests usually are reasonably specific but may have only modest sensitivity.

1	Measurement of serum antibody provides an indirect marker for past or current infection with a specific viral agent or other pathogens, including Brucella, Legionella, Rickettsia, and Helicobacter pylori. Serologic methods can be used to determine whether an individual has protective antibody levels or is infected by a specific pathogen. Determination of an antibody level as a measure of current immunity is important in the case of viral agents for which there are vaccines, such as rubella virus and varicella-zoster virus; assays for this purpose normally use one or two dilutions of serum for a qualitative determination of protective antibody levels. Quantitative serologic assays to detect increases in antibody titers most often employ paired serum samples obtained at the onset of illness and 10–14 days later (i.e., acuteand convalescent-phase samples). Since the incubation period before symptoms are noted may be long enough for an antibody response to occur, the demonstration of

1	and 10–14 days later (i.e., acuteand convalescent-phase samples). Since the incubation period before symptoms are noted may be long enough for an antibody response to occur, the demonstration of acute-phase antibody alone is often insufficient to establish the diagnosis of active infection as opposed to past exposure. A fourfold increase in total antibody titer between the acuteand convalescent-phase samples is regarded as evidence for active infection. In addition, IgM may be useful as a measure of an early, acute-phase antibody response. For certain viral agents, such as Epstein-Barr virus, the antibodies produced may be directed at different antigens during different phases of the infection. For this reason, most laboratories test for antibody directed at both viral capsid antigens and antigens associated with recently infected host cells to determine the stage of infection.

1	To culture bacterial, fungal, or viral pathogens, an appropriate sample must be placed into the proper medium for growth. The success of efforts to identify a specific pathogen often depends on the collection and transport process coupled to a laboratory-processing algorithm suitable for the specific sample/agent. In some instances, it is better for specimens to be plated at the time of collection rather than first being transported to the laboratory (e.g., urethral swabs being cultured for Neisseria gonorrhoeae). In general, the more rapidly a specimen is plated onto appropriate media, the better the chance is for isolating bacterial pathogens. Deep tissue or fluid (pus) samples are more likely to give useful culture results than are superficial swab specimens. Table 150e-2 lists procedures for collection and transport of common specimens. Because there are many pathogen-specific paradigms for these procedures, it is important to seek advice from the microbiology laboratory when in

1	for collection and transport of common specimens. Because there are many pathogen-specific paradigms for these procedures, it is important to seek advice from the microbiology laboratory when in doubt about a particular situation.

1	Isolation of pathogens from clinical material relies on the use of artificial media that support bacterial growth. Such media are composed of agar, nutrients, and sometimes substances that inhibit the growth of other bacteria. Broth is employed for growth of organisms from specimens with few bacteria, such as peritoneal dialysis fluid or CSF, or from samples in which anaerobes or other fastidious organisms may be present. Broths that allow the growth of small numbers of organisms may be subcultured onto solid medium once growth is detected. The use of liquid medium for all specimens is not worthwhile.

1	Two basic strategies are used to isolate pathogenic bacteria. The first is to employ enriched media that support the growth of any bacteria that may be present at a site that is normally sterile, such as blood or CSF. The second strategy is to use selective media to isolate specific bacterial species from samples that contain many bacteria under normal conditions (e.g., stool or genital tract secretions). Antimicrobial agents or other substances are incorporated into the agar medium to inhibit growth of all but the bacteria of interest. After incubation, organisms that grow on such media are characterized further to determine whether they are pathogens (Fig. 150e-1).

1	The detection of microbial pathogens in blood is difficult because the number of organisms present in the sample is often low and the organisms’ integrity and ability to replicate may be damaged by humoral defense mechanisms or antimicrobial agents. Automated blood culture systems detect production of gas (mainly CO2) by bacteria or yeasts growing in broth in a blood culture bottle. Because the bottles are monitored frequently, a positive culture often is detected more rapidly than by manual techniques, and important information, including the results of Gram’s stain and preliminary susceptibility assays, can be obtained sooner. Several factors affect the yield of blood culture from bacteremic patients. Increasing the volume of blood tested increases the chance of a positive culture. For example, a sample-size increase from 10 to 20 mL of blood increases the proportion of positive cultures by ~30%, although this effect is less pronounced in patients with bacterial endocarditis.

1	For example, a sample-size increase from 10 to 20 mL of blood increases the proportion of positive cultures by ~30%, although this effect is less pronounced in patients with bacterial endocarditis. Obtaining multiple samples for culture (up to three per 24-h period) also increases the chance of detecting a bacterial pathogen. Prolonged culture and blind subculture for detection of most fastidious bacteria (e.g., HACEK organisms) are not needed with automated blood culture systems.

1	Automated systems also have been applied to the detection of microbial growth from specimens other than blood, such as peritoneal and other normally sterile fluids. Mycobacterium species can be detected in certain automated systems if appropriate liquid media are used for culture. Although automated blood culture systems are more sensitive than lysis-centrifugation methods (e.g., Isolator) for yeasts and most bacteria, lysis-centrifugation culture is recommended for filamentous fungi, Histoplasma capsulatum, and some fastidious bacteria (Legionella and Bartonella).

1	Once bacteria are isolated, characteristics that are readily detectable after growth on agar media (colony size, color, hemolytic reactions, odor, microscopic appearance) may suggest a species, but defini-150e-3 tive identification requires additional tests. Identification methods include classic biochemical phenotyping, which is still the most common approach, as well as more sophisticated methods such as mass spectrometry, gas chromatography, and nucleic acid tests (see below).

1	Biochemical Phenotyping Classic biochemical identification of bacteria entails tests for protein or carbohydrate antigens, the production of specific enzymes, the ability to metabolize specific substrates and carbon sources (such as carbohydrates), or the production of certain metabolites. Rapid versions of some of these tests are available, and many common organisms can be identified on the first day of growth. Other organisms, particularly gram-negative bacteria, require more extensive testing, either manual or automated.

1	Automated systems allow rapid phenotypic identification of bacterial pathogens. Most of these systems are based on biotyping techniques in which isolates are grown on multiple substrates and the reaction pattern is compared with known patterns for various bacterial species. This procedure is relatively fast. Commercially available systems include miniaturized fermentation, coding to simplify recording of results, and probability calculations for the most likely pathogens. If the biotyping approach is automated and the reading process is coupled to computer-based data analysis, rapidly growing organisms (such as Enterobacteriaceae) can be identified within hours of detection on agar plates.

1	Several systems use substrates for preformed bacterial enzymes for identification within 2–3 h. These systems do not rely on bacterial growth per se to determine whether a substrate has been used. They employ a heavy inoculum in which specific bacterial enzymes are present in amounts sufficient to convert substrate to product rapidly. In addition, some systems use fluorogenic substrate/endproduct detection methods to increase sensitivity through signal amplification. Gas-Liquid Chromatography Gas-liquid chromatography often is used to detect metabolic end products of bacterial fermentation. One common application is identification of short-chain fatty acids produced by obligate anaerobes during glucose fermentation. Because the types and relative concentrations of volatile acids differ among the various genera and species that make up this group of organisms, such information serves as a metabolic fingerprint for a particular isolate.

1	Gas-liquid chromatography can be coupled to a sophisticated signal-analysis software system for identification and quantitation of long-chain fatty acids (LCFAs) in the outer membranes and cell walls of bacteria and fungi. For any particular species, the types and relative concentrations of LCFAs are distinctive enough to allow differentiation even from closely related species. An organism may be identified definitively within a few hours after detection of growth on appropriate media.

1	Matrix-Assisted Laser Desorption/Ionization/Time-of-Flight Mass Spectrometry (MALDI-TOF MS) MALDI-TOF MS is a rapid and accurate method for identifying microorganisms by protein analysis. The organism is mixed with a chemical matrix, dried on a target plate, and then pulsed with a laser. The laser ionizes and vaporizes the microbial proteins, which then travel through a charged vacuum chamber to a detector. The time of flight to the detector is measured for the individual proteins, and the resulting pattern (or fingerprint) is compared with a library of known patterns for various microorganisms to identify the test organism.

1	The primary clinical advantage of MALDI-TOF is that it takes only minutes to identify an organism, whereas several hours are needed for conventional phenotyping. MALDI-TOF is highly accurate for identification of most bacteria and yeasts grown on solid agar or in blood culture broth. Other potential uses of MALDI-TOF include identification of bacteria and yeasts directly from clinical specimens (e.g., urine), detection of β-lactamase activity, and strain typing of bacteria; however, these applications are still under development. CHAPTER 150e Laboratory Diagnosis of Infectious Diseases Note: It is absolutely essential that the microbiology laboratory be informed of the site of origin of the sample to be cultured and the infections that are suspected. This information determines the selection of culture media and the duration of culture. Type of Culture (Synonyms) Specimen Minimal Volume Container Other Considerations

1	Type of Culture (Synonyms) Specimen Minimal Volume Container Other Considerations Stool for routine culture; stool Fresh, randomly collected stool (pref-1 g of stool or 2 rectal swabs Plastic-coated cardboard cup or plastic cup for Salmonella, Shigella, and erably) or rectal swab with tight-fitting lid. Other leakproof contain- ers also are acceptable. Stool for Yersinia, Escherichia Fresh, randomly collected stool 1 g Plastic-coated cardboard cup or plastic cup coli O157 with tight-fitting lid Stool for Aeromonas and Fresh, randomly collected stool 1 g Plastic-coated cardboard cup or plastic cup Plesiomonas If Vibrio spp. are suspected, the laboratory must be notified, and appropriate collection/transport methods should be used. Limitations: Procedure requires enrichment techniques. Limitations: Stool should not be cultured for these organisms unless also cultured for other enteric pathogens.

1	Limitations: Procedure requires enrichment techniques. Limitations: Stool should not be cultured for these organisms unless also cultured for other enteric pathogens. Urine Clean-voided urine specimen or urine 0.5 mL Sterile, leakproof container with screw cap or See below.d collected by catheter special urine transfer tube Urogenital secretions Vaginal or urethral secretions, cervical 1 swab or 0.5 mL of fluid Vaginal and rectal swabs transported in Vaginal swab samples for “routine culture” should be discouraged swabs, uterine fluid, prostatic fluid, etc. Amies transport medium or similar holding whenever possible unless a particular pathogen is suspected. medium for group B Streptococcus; direct For detection of multiple organisms (e.g., group B Streptococcus, inoculation preferred for Neisseria gonor-Trichomonas, Chlamydia, or Candida spp.), 1 swab per test should be rhoeae obtained. Body Fluids, Aspirates, and Tissues

1	Body Fluids, Aspirates, and Tissues Cerebrospinal fluid (lumbar Spinal fluid 1 mL for routine cultures; ≥5 mL Sterile tube with tight-fitting cap Do not refrigerate; transfer to laboratory as soon as possible. puncture) Body fluids Aseptically aspirated body fluids 1 mL for routine cultures Sterile tube with tight-fitting cap. Specimen For some body fluids (e.g., peritoneal lavage samples), increased vol-may be left in syringe used for collection if umes are helpful for isolation of small numbers of bacteria. the syringe is capped before transport. Tissue removed at surgery, bone, anticoagulated bone marrow, biopsy samples, or other specimens from normally sterile areas Purulent material or abscess contents obtained from wound or abscess without contamination by normal microflora 1 mL of fluid or a 1-g piece of tissue 2 swabs or 0.5 mL of aspirated pus

1	Purulent material or abscess contents obtained from wound or abscess without contamination by normal microflora 1 mL of fluid or a 1-g piece of tissue 2 swabs or 0.5 mL of aspirated pus Sterile Culturette-type swab or similar transport system containing holding medium. Sterile bottle or jar should be used for tissue specimens. Culturette swab or similar transport system or sterile tube with tight-fitting screw cap. For simultaneous anaerobic cultures, send specimen in anaerobic transport device or closed syringe. Accurate identification of specimen and source is critical. Enough tissue should be collected for both microbiologic and histopathologic evaluations. Collection: When possible, abscess contents or other fluids should be collected in a syringe (rather than with a swab) to provide an adequate sample volume and an anaerobic environment. Specimen types listed above may be used. When urine or sputum is cultured for fungi, a first morning specimen usually is preferred.

1	Specimen types listed above may be used. When urine or sputum is cultured for fungi, a first morning specimen usually is preferred. Sputum, tissue, urine, body fluids Pleural fluid, lung biopsy, bronchoalveolar lavage fluid, bronchial/ transbronchial biopsy Respiratory secretions, wash aspirates from respiratory tract, nasal swabs, blood samples (including buffy coats), vaginal and rectal swabs, swab specimens from suspicious skin lesions, stool samples (in some cases) 1 mL or as specified above for individual listing of specimens. Large volumes may be useful for urinary fungi. 10 mL of fluid or small piece of tissue. Swabs should not be used. 1 mL of fluid; any size tissue sample, although a 0.5-g sample should be obtained when possible 1 mL of aspirated fluid, 1 g of tissue, or 2 swabs 1 mL of fluid, 1 swab, or 1 g of stool in each appropriate transport medium Sterile, leakproof container with tight-fitting cap Sterile container with tight-fitting cap

1	Sterile container with tight-fitting cap An appropriate anaerobic transport device is required.e Fluid or stool samples in sterile containers or swab samples in viral Culturette devices (kept on ice but not frozen) are generally suitable. Plasma samples and buffy coats in sterile collection tubes should be kept at 4–8°C. If specimens are to be shipped or kept for a long time, freezing at –80°C is usually adequate. Collection: Specimen should be transported to microbiology laboratory within 1 h of collection. Contamination with normal flora from skin, rectum, vaginal tract, or other body surfaces should be avoided. Detection of Mycobacterium spp. is improved by use of concentration techniques. Smears and cultures of pleural, peritoneal, and pericardial fluids often have low yields. Multiple cultures from the same patient are encouraged. Culturing in liquid media shortens time to detection. Rapid transport to laboratory is critical.

1	Rapid transport to laboratory is critical. Specimens cultured for obligate anaerobes should be cultured for facultative bacteria as well. Fluid or tissue is preferred to swabs. Most samples for culture are transported in holding medium containing antibiotics to prevent bacterial overgrowth and viral inactivation. Many specimens should be kept cool but not frozen, provided they are transported promptly to the laboratory. Procedures and transport media vary with the agent to be cultured and the duration of transport.

1	aFor samples from adults, two bottles (smaller for pediatric samples) should be used: one with dextrose phosphate, tryptic soy, or another appropriate broth and the other with thioglycollate or another broth containing reducing agents appropriate for isolation of obligate anaerobes. For children, from whom only limited volumes of blood can be obtained, only an aerobic culture should be done unless there is specific concern about anaerobic sepsis (e.g., with abdominal infections). For special situations (e.g., suspected fungal infection, culture-negative endocarditis, or mycobacteremia), different blood collection systems may be used (Isolator systems; see table).

1	bCollection: An appropriate disinfecting technique should be used on both the bottle septum and the patient. Do not allow air bubbles to get into anaerobic broth bottles. Special considerations: There is no more important clinical microbiology test than the detection of bloodborne pathogens. The rapid identification of bacterial and fungal agents is a major determinant of patients’ survival. Bacteria may be present in blood either continuously (as in endocarditis, overwhelming sepsis, and the early stages of salmonellosis and brucellosis) or intermittently (as in most other bacterial infections, in which bacteria are shed into the blood on a sporadic basis). Most blood culture systems employ two separate bottles containing broth medium: one that is vented in the laboratory for the growth of facultative and aerobic organisms and one that is maintained under anaerobic conditions. In cases of suspected continuous bacteremia/fungemia, two or three samples should be drawn before the start

1	of facultative and aerobic organisms and one that is maintained under anaerobic conditions. In cases of suspected continuous bacteremia/fungemia, two or three samples should be drawn before the start of therapy, with additional sets obtained if fastidious organisms are thought to be involved. For intermittent bacteremia, two or three samples should be obtained at least 1 h apart during the first 24 h.

1	cNormal microflora in the throat includes α-hemolytic streptococci, saprophytic Neisseria spp., diphtheroids, and Staphylococcus spp. Aerobic culture of the throat (“routine”) includes screening for and identification of β-hemolytic Streptococcus spp. and other potentially pathogenic organisms. Although considered components of the normal microflora, organisms such as Staphylococcus aureus, Haemophilus influenzae, and Streptococcus pneumoniae will be identified by most laboratories, if requested. When Neisseria gonorrhoeae or Corynebacterium diphtheriae is suspected, a special culture request is recommended.

1	d(1) When clean-voided specimens, midvoid specimens, and Foley or indwelling catheter specimens yield 50,000 organisms/mL and no more than three species are isolated, the organisms should be identified. Neither indwelling catheter tips nor urine from the bag of a catheterized patient should be cultured. (2) Straight-catheterized, bladder-tap, and similar urine specimens should undergo a complete workup (identification and susceptibility testing) for all potentially pathogenic organisms regardless of colony count. (3) Certain clinical problems (e.g., acute dysuria in women) may warrant identification and susceptibility testing of isolates present at concentrations of >50,000 organisms/mL.

1	eAspirated specimens in capped syringes or other transport devices designed to limit oxygen exposure are suitable for the cultivation of obligate anaerobes. A variety of commercially available transport devices may be used. Contamination of specimens with normal microflora from the skin, rectum, vaginal vault, or another body site should be avoided. Collection containers for aerobic culture (such as dry swabs) and inappropriate specimens (such as refrigerated samples; expectorated sputum; stool; gastric aspirates; and vaginal, throat, nose, and rectal swabs) should be rejected as unsuitable. f Laboratories generally use diverse methods to detect viral agents, and the specific requirements for each specimen should be checked before a sample is sent. CHAPTER 150e Laboratory Diagnosis of Infectious Diseases

1	Suspected Infectious Agent Obtain Appropriate Specimen Bacteriology specimen for rapid diagnosis or routine culture methods for common and fastidious pathogens Rapid diagnosis: Latex agglutination for Cryptococcus; direct DNA/RNA probes; Gram’s stain for sputum or vaginal swab DNA/RNA amplification for Chlamydia, GC, TB; direct stain for infectious agents such as Legionella, Pneumocystis Blood: Specify site and time of collection; use Isolator cultures for fungus, Mycobactrium Urine, wound, tissue, or sputum: Specify site and collection method; prepare sample for culture; use enrichment and selective agar Stool: Gram’s stain for fecal leukocytes; selective agar for common pathogens; specialized media for other pathogens Evaluate MacConkey’s, HE, BAP, Tergitol agars for pathogens; serogroup Salmonella, Shigella; examine specialized media for other pathogens Evaluate MacConkey’s, BAP, and chocolate agar for pathogens; use liquid medium for fastidious pathogens; use Gram’s stain or other

1	Salmonella, Shigella; examine specialized media for other pathogens Evaluate MacConkey’s, BAP, and chocolate agar for pathogens; use liquid medium for fastidious pathogens; use Gram’s stain or other rapid tests Examine both aerobic and anaerobic liquid medium; subculture to chocolate agar or 7H10 for TB; use other enrichment media for HACEK

1	Isolation from subculture; identification of pathogens; perform susceptibility tests; report MRSA, VREF, ESBL Anaerobes: Group with Gram’s stain, spore test, and GLC; use fermentation profile for identification Isolation; identification of pathogens; perform susceptibility tests; report MRSA, VREF, ESBL C. difficile: Cell culture assay or EIA for toxins A and B Mycology specimen: Use fluorochrome stain for fungal elements in tissue, hair, and skin scrapings; culture and identify yeast isolates from blood and CSF Sample preparation: Culture to Sabouraud’s and other media; stain with lactophenol cotton blue or other stain; examine wet mount Virology specimen: Use cell culture or serologic methods; samples include buffy coat, serum, blood, bronchial wash, stool, urine Examine cell cultures for CPE; use serology to detect antibody in acute and convalescent sera; use rapid DFA where possible

1	Examine cell cultures for CPE; use serology to detect antibody in acute and convalescent sera; use rapid DFA where possible Use immunofluorescence for viral agents in cultures; use other identification methods, such as direct DNA/RNA probes Viral load testing: Use for CMV, HIV, HepC; use DNA/RNA amplification for genotyping

1	FIGURE 150e-1 Common specimen-processing algorithms used in clinical microbiology laboratories. BAP, blood agar plate; CMV, cytomegalovirus; CPE, cytopathic effects; CSF, cerebrospinal fluid; DFA, direct fluorescent antibody; EIA, enzyme immunoassay; ESBL, extended-spectrum β-lactamase; GBS, group B Streptococcus; GC, Neisseria gonorrhoeae; GLC, gas-liquid chromatography; HACEK, Haemophilus aphrophilus/parainfluenzae/paraphrophilus, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae; HE, Hektoen enteric medium; HepC, hepatitis C virus; HIV, human immunodeficiency virus; MRSA, methicillin-resistant Staphylococcus aureus; TB, Mycobacterium tuberculosis; VREF, vancomycin-resistant Enterococcus faecium.

1	Techniques for the detection and quantitation of specific DNA and RNA base sequences in clinical specimens have become powerful tools for the diagnosis of bacterial, viral, parasitic, and fungal infections. Nucleic acid tests are used for four purposes. First, they are used to detect, and sometimes to quantify, specific pathogens in clinical specimens. Second, such tests are used for identification of organisms (usually bacteria) that are difficult to identify by conventional methods. Third, nucleic acid tests are used to determine whether two or more isolates of the same pathogen are closely related (i.e., whether they belong to the same “clone” or “strain”). Fourth, these tests are used to predict the sensitivity of organisms to chemotherapeutic agents. Current technology encompasses a wide array of methods for amplification and signal detection, some of which have been approved by the U.S. Food and Drug Administration (FDA) for clinical diagnosis.

1	Use of nucleic acid tests generally involves lysis of intact cells or viruses and denaturation of the DNA or RNA to render it single-stranded. Probe(s) or primer(s) complementary to the pathogen-specific target sequence are hybridized to the target sequence in a solution or on a solid support, depending on the system employed. In situ hybridization of a probe to a target also is possible and allows the use of probes with agents present in tissue specimens. Once the probe(s) or primer(s) have been hybridized to the target (biologic signal), a variety of strategies may be employed to detect, amplify, and/or quantify the target–probe complex (Fig. 150e-2).

1	Nucleic acid probes are used for direct detection of pathogens in clinical specimens without amplification of the target strand of DNA or RNA. Such tests detect a relatively short sequence of bases specific for a particular pathogen on single-stranded DNA or RNA by hybridization of a complementary sequence of bases (probe) coupled to a reporter system that serves as the signal for detection. Nucleic acid probes are available commercially for direct detection of various bacterial and parasitic pathogens, including Chlamydia trachomatis,

1	N. gonorrhoeae, and group A Streptococcus. A combined assay to detect and differentiate agents of vaginitis/vaginosis (Gardnerella vaginalis, Trichomonas vaginalis, and Candida species) also has been approved. An assortment of probes is available for confirming the identity of cultured pathogens, including some dimorphic molds, Mycobacterium species, and other bacteria (e.g., Campylobacter species, Streptococcus species, and Staphylococcus aureus). Probes for the direct detection of bacterial pathogens often are aimed at highly conserved 16S ribosomal RNA sequences, of which there are many more copies than there are of any single genomic DNA sequence in a bacterial cell. The sensitivity and specificity of probe assays for direct detection are comparable to those of more traditional assays, including EIA and culture.

1	In an alternative probe assay called hybrid capture, an RNA probe anneals to a DNA target, and the resulting DNA/RNA hybrid is captured on a solid support by antibody specific for DNA/RNA hybrids (concentration/amplification) and detected by chemiluminescent-labeled antibody specific for DNA/RNA hybrids. Hybrid capture

1	FIGURE 150e-2 Strategies for amplification and/or detection of a target–probe complex. DNA or RNA extracted from microorganisms is heated to create single-stranded (ss) DNA/RNA containing appropriate target sequences. These target sequences may be hybridized directly (direct detection) with probes attached to reporter molecules; they may be amplified by repetitive cycles of complementary strand extension (polymerase chain reaction) before attachment of a reporter probe; or the original target–probe signal may be amplified via hybridization with an additional probe containing multiple copies of a secondary reporter target sequence (branched-chain DNA, or bDNA). DNA/RNA hybrids also can be “captured” on a solid support (hybrid capture), with antibody to the DNA/RNA hybrids used to concentrate them and a second antibody CHAPTER 150e Laboratory Diagnosis of Infectious Diseases coupled to a reporter molecule attached to the captured hybrid.

1	CHAPTER 150e Laboratory Diagnosis of Infectious Diseases coupled to a reporter molecule attached to the captured hybrid. assays are available for C. trachomatis, N. gonorrhoeae, and human papillomavirus. Many laboratories have developed their own probes for pathogens; however, unless a method-validation protocol for diagnostic testing has been performed, federal law in the United States restricts the use of such probes to research.

1	There are several methods for amplification (copying) of small numbers of molecules of nucleic acid to readily detectable levels. These NAATs include PCR, LCR, strand displacement amplification, and self-sustaining sequence replication. In each case, exponential amplification of a pathogen-specific DNA or RNA sequence depends on primers that anneal to the target sequence. The amplified nucleic acid can be detected after the reaction is complete or (in real-time detection) as amplification proceeds. The sensitivity of NAATs is far greater than that of traditional assay methods such as culture. PCR, the first and still the most common NAAT, requires repeated heating of the DNA to separate the two complementary strands of the double helix, hybridization of a primer sequence to the appropriate target sequence, target amplification using PCR for complementary strand extension, and signal detection via a labeled probe. Methods for the monitoring of PCR after each amplification cycle—via

1	target sequence, target amplification using PCR for complementary strand extension, and signal detection via a labeled probe. Methods for the monitoring of PCR after each amplification cycle—via either incorporation of fluorescent dyes into the DNA during primer extension or use of fluorescent probes capable of fluorescence resonance energy transfer—have decreased the time required to detect a specific target. An alternative NAAT employs transcription-mediated amplification, in which an RNA target sequence is converted to DNA, which then is exponentially transcribed into an RNA target. The advantage of this method is that only a single heating/annealing step is required for amplification. Identification of otherwise difficult-to-identify bacteria involves an initial amplification of a highly conserved region of 16S rDNA by PCR. Automated sequencing of several hundred bases is then performed, and the sequence information is compared with large databases containing sequence information

1	conserved region of 16S rDNA by PCR. Automated sequencing of several hundred bases is then performed, and the sequence information is compared with large databases containing sequence information for thousands of different organisms. Although 16S sequencing is not as rapid as other methods and is still relatively expensive for routine use in a clinical microbiology laboratory, it is becoming the definitive method for identification of unusual or difficult-to-cultivate organisms.

1	With the advent of newer therapeutic regimens for HIV-associated disease, cytomegalovirus infection, and hepatitis B and C virus infections, the response to therapy has been monitored by determining both genotype and viral load at various times after treatment initiation. Quantitative NAATs are available for HIV (PCR), cytomegalovirus (PCR), hepatitis B virus (PCR), and hepatitis C virus (PCR and TMA). Many laboratories have validated and perform quantitative assays for these and other pathogens (e.g., Epstein-Barr virus), using analytespecific reagents for NAATs.

1	Branched-chain DNA (bDNA) testing is an alternative to NAATs for quantitative nucleic acid testing. In such testing, bDNA attaches to a site different from the target-binding sequence of the original probe. Chemiluminescence-labeled oligonucleotides can then bind to multiple repeating sequences on the bDNA. The amplified bDNA signal is detected by chemiluminescence. bDNA assays for viral load of HIV, hepatitis B virus, and hepatitis C virus have been approved by the FDA. The advantage of bDNA assays over PCR is that only a single heating/ annealing step is required to hybridize the target-binding probe to the target sequence for amplification. The number of FDA-approved NAATS has increased dramatically and includes tests for Mycobacterium tuberculosis, N. gonorrhoeae,

1	The number of FDA-approved NAATS has increased dramatically and includes tests for Mycobacterium tuberculosis, N. gonorrhoeae, C. trachomatis, group B Streptococcus, and methicillin-resistant S. aureus. FDA-approved multiplex NAAT panels for detection of several respiratory or gastrointestinal pathogens also are available. Again, many laboratories have used commercially available reagents and analytespecific reagents to create laboratory-developed tests for diagnostic use. Nucleic acid tests are useful to detect and identify difficult-to-grow or noncultivable bacterial pathogens such as Legionella, Ehrlichia, Rickettsia, Babesia, Borrelia, and Tropheryma whipplei. In addition, methods for rapid detection of agents of public health concern, such as Franciscella tularensis, Bacillus anthracis, smallpox virus, and Yersinia pestis, have been developed and are available in regional (state) laboratories and at the Centers for Disease Control and Prevention.

1	Nucleic acid tests are also used to determine how close the relationship is among different isolates of the same species of pathogen. The demonstration that bacteria of a single clone have infected multiple patients in the context of a possible source of transmission (e.g., a health care provider) offers confirmatory evidence for an outbreak. Pulsed-field gel electrophoresis remains the gold standard for bacterial strain analysis. This method involves the use of restriction enzymes that recognize rare sequences of nucleotides to digest bacterial DNA, resulting in large DNA fragments. These fragments are separated by gel electrophoresis with variable polarity of the electrophoretic current and then are visualized. Similar band patterns (i.e., differences in ≤3 bands) suggest that different bacterial isolates are closely related, or clonal. Simpler methods of strain typing include sequencing of single or multiple genes and PCR-based amplification of repetitive DNA sequences in the

1	bacterial isolates are closely related, or clonal. Simpler methods of strain typing include sequencing of single or multiple genes and PCR-based amplification of repetitive DNA sequences in the bacterial chromosome. Whole bacterial genome sequencing has been used in investigation of some outbreaks, but this method is still under development.

1	Future applications of nucleic acid testing probably will include the replacement of culture for identification of many pathogens with additional multiplex NAAT and solid-state DNA/RNA chip technology, in which thousands of unique nucleic acid sequences can be detected on a single silicon chip.

1	A principal responsibility of the clinical microbiology laboratory is to determine which antimicrobial agents inhibit a specific bacterial isolate. Such testing is used for patient care and for monitoring of infection control problems, such as methicillin-resistant S. aureus or vancomycin-resistant Enterococcus faecium. Two approaches are useful. The first is a qualitative assessment of susceptibility, with responses categorized as susceptible, resistant, or intermediate. This approach can involve either the placement of paper disks containing antibiotics on an agar surface inoculated with the bacterial strain to be tested (Kirby-Bauer or disk-diffusion method), with measurement of the zones of growth inhibition after incubation, or the use of broth cultures containing a set concentration of antibiotic (breakpoint method). These methods have been calibrated carefully against quantitative methods and clinical experience with each antibiotic, and zones of inhibition and breakpoints have

1	of antibiotic (breakpoint method). These methods have been calibrated carefully against quantitative methods and clinical experience with each antibiotic, and zones of inhibition and breakpoints have been determined on a species-byspecies basis.

1	The second approach is to inoculate the test strain of bacteria into a series of cultures with increasing concentrations of antibiotic. The lowest concentration of antibiotic that inhibits visible microbial growth of the bacteria is known as the minimal inhibitory concentration (MIC). If tubes in which no growth is seen are subcultured, the minimal concentration of antibiotic required to kill 99.9% of the starting inoculum also can be determined (minimal bactericidal concentration, or MBC). The MIC value can be given a categorical interpretation of susceptible, resistant, or intermediate and so is more widely used than the MBC. Quantitative susceptibility testing using microbroth dilution in microwell plates or other miniaturized testing platforms has been automated and is used commonly in clinical laboratories. The epsilometer test (E-test), a novel method for determination of the MIC, uses a plastic strip with a known gradient of antibiotic concentrations along its length. When the

1	clinical laboratories. The epsilometer test (E-test), a novel method for determination of the MIC, uses a plastic strip with a known gradient of antibiotic concentrations along its length. When the strip is placed on the surface of an agar plate seeded with the bacterial strain to be tested, antibiotic diffuses into the medium, and bacterial growth is inhibited. For some organisms, such as obligate anaerobes and some β-hemolytic streptococci, routine susceptibility testing generally is not performed because of the difficulty of growing the organisms or the predictable sensitivity of most isolates to specific antibiotics.

1	With the advent of many new agents for treating yeasts and systemic fungal agents, the need for testing of individual isolates for susceptibility to specific antifungal agents has increased. In the past, few laboratories participated in such testing because of a lack of standard methods like those available for testing bacterial agents. However, several systems have been approved for antifungal susceptibility testing. These methods, which determine the minimal fungicidal concentration (MFC), are similar to the broth microdilution methods used to determine the MIC for bacteria. The E-test method is approved for testing the susceptibility of yeasts to fluconazole, itraconazole, and flucytosine, and disk diffusion can be used to test the susceptibility of Candida species to fluconazole and voriconazole. Methods for determining the MFC of a drug against fungal agents such as Aspergillus species are technically difficult, and most clinical laboratories refer requests for such testing to

1	voriconazole. Methods for determining the MFC of a drug against fungal agents such as Aspergillus species are technically difficult, and most clinical laboratories refer requests for such testing to reference laboratories.

1	See Chaps. 214e and 215e. Climate Change and Infectious Disease Aaron S. Bernstein The release of greenhouse gases—principally carbon dioxide—into Earth’s atmosphere since the late nineteenth century has contributed 151e unprecedented in the last 50 million years. Climate science, although 151e-1 still a relatively new discipline, has provided an ever-clearer picture of how the changing chemistry of the atmosphere has influenced, and will continue to influence, the global climate.

1	Greenhouse gases (Table 151e-1 and Fig. 151e-2) are a group of gases in Earth’s atmosphere that absorb infrared radiation and thus retain heat inside the atmosphere. In the absence of these gases, the earth’s to a climate unfamiliar to our species, Homo sapiens. This new climate has already altered the epidemiology of some infectious diseases. average temperature would be about 33°C colder. Carbon dioxide, released into the atmosphere primarily from fossil fuel combustionContinued accumulation of greenhouse gases in the atmosphere will and deforestation, has had the greatest effect on climate since thefurther alter the planet’s climate. In some cases climate change may establish conditions favoring the emergence of infectious diseases, Industrial Revolution. Of note, the Swedish scientist Svante Arrhenius first suggested in the late nineteenth century that the addition ofwhile in others it may render areas that are presently suitable for carbon dioxide to the earth’s atmosphere would

1	Svante Arrhenius first suggested in the late nineteenth century that the addition ofwhile in others it may render areas that are presently suitable for carbon dioxide to the earth’s atmosphere would increase the planet’s certain diseases unsuitable. This chapter presents the current state of surface temperature. Water vapor is the most abundant and a highly knowledge regarding the known and prospective infectious-disease potent greenhouse gas but, given its short atmospheric life span and consequences of climate change.

1	The term climate change refers to long-term alterations in tempera- sensitivity to temperature, is not a major factor in recently observed climate change. The atmosphere, some of the aerosols suspended in it, and clouds reflect a portion of incoming solar radiation back toward space. The ture, precipitation, wind, humidity, and other components of weather. Over the past 2.5 million years, the earth has warmed and cooled, cycling between glacial and interglacial periods during which average global temperatures moved up and down by 4–7°C. During the last glacial period, which ended roughly 12,000 years ago, global temperatures were, on average, 5°C cooler than in the mid-twentieth century (Fig. 151e-1).

1	The present climate period, known as the Holocene, is remarkable for its stability: temperatures have largely remained within a range of 2–3°C. This stability has enabled the successful population and cultivation of much of the earth’s landmass by humanity. Current climate change differs from that in the past not only because its primary cause is human activities but also because its pace is faster. The 5°C of warming that occurred at the end of the last ice age took roughly 5000 years, whereas such a temperature increment may occur within the next 150 years unless the release of greenhouse gases is substantially reduced in coming decades. The current rate of warming on Earth is remainder reaches Earth’s surface, where it is absorbed and some is then emitted back at the atmosphere. The earth emits energy absorbed from the sun at longer wavelengths, primarily infrared, that greenhouse gases are able to absorb. The change in wavelength that occurs as solar radiation is absorbed and

1	earth emits energy absorbed from the sun at longer wavelengths, primarily infrared, that greenhouse gases are able to absorb. The change in wavelength that occurs as solar radiation is absorbed and re-emitted from the earth’s surface is fundamental to the greenhouse effect (Fig. 151e-3).

1	Climate change has become nearly synonymous with global warming, as a clear signal from rising greenhouse gas concentrations has been an increase in the mean global surface temperature of ~0.85°C since 1880. However, this mean warming belies warming that is occurring much faster in certain regions. The Arctic has warmed twice as fast overall, and winters are warming faster than summers. Nighttime minimum temperatures are also rising faster than daytime high temperatures. Each of these nuances bears upon the incidence of infectious diseases in general and vector-borne disease specifically. 600 500 400 300 200 Time (before 2005), in thousands of years 2.8 3.2 3.6 4.0 4.4 4.8 5.2

1	FIGURE 151e-1 Overview of the earth’s temperature and primary greenhouse gases over the last 600,000 years. Variations of deuterium (δD; black) serve as a proxy for temperature. Atmospheric concentrations of greenhouse gases—CO2 (red), CH4 (blue), and nitrous oxide (N2O; green)—were derived from air trapped within Antarctic ice cores and from recent atmospheric measurements. Shaded areas indicate interglacial periods. Benthic δ18O marine records (dark gray) are a proxy for global ice-volume fluctuations and can be compared to the ice core data. Downward trends in the benthic δ18O curve reflect increasing ice volumes on land. The stars and labels indicate atmospheric concentrations as of the year 2000. CO2 levels surpassed 400 ppm as of 2013 and are rising at a rate of 2–2.5 ppm per year. (From Intergovernmental Panel on Climate Change Fourth Assessment Report. Working Group I, Chapter 6, Figure 6.3. Cambridge University Press, 2007.)

1	TABLE 151e-1 GREEnHousE GAsEs: souRCEs, sInks, AnD FoRCInGs aIn this table, a sink refers to the place where greenhouse gases are naturally stored or the mechanism through which they are destroyed. bRadiative forcing, measured in watts per meter squared, refers to how much an entity can alter the balance of incoming and outgoing radiation to and from Earth’s atmosphere. It is measured relative to a preindustrial (i.e., 1750) baseline. Greenhouse gases have a positive “forcing”; that is, on balance, they increase the amount of radiation (and specifically infrared radiation) that is retained in Earth’s atmosphere. (Sources: Intergovernmental Panel on Climate Change Fifth Assessment Report, Working Group 1, Chapter 8; American Chemical Society “Greenhouse gas sources and sinks,” available at www.acs .org/content/acs/en/climatescience/greenhousegases/sourcesandsinks.html.) 0.04 0.03 0.02 0.01 0.00

1	A moderate projection based on the best available scientific evidence suggests that average global temperatures will warm an additional 1.4–3.1°C by 2100 as compared to the period 1986–2005. Because of climate change, extreme heat waves have already become more common and are expected to be even more frequent later in this century. Besides contributing directly to morbidity and mortality in human populations, heat waves wilt crops and are expected to contribute substantially to predicted agricultural losses. For example, the 2010 heat wave in Russia, which was unprecedented in its severity, contributed to hundreds of forest fires that generated enough air pollution to kill an estimated 56,000 people and that burned 300,000 acres of crops, including roughly 25% of the nation’s wheat fields. Nutritional deficiencies underlie a substantial portion of the global burden of

1	FIGURE 151e-2 Acceleration of radiative forcing (RF) from release many infectious diseases. of major greenhouse gases, 1850–2011. For definition of radiative forcing, see footnote b to Table 151e-1. (From Intergovernmental Panel PRECIPITATION on Climate Change Fifth Assessment Report, Working Group 1, Chapter 8, In addition to changing temperature, the emission of greenhouse gases Figure 8.6, p. 677.) and the consequent increase in energy in Earth’s atmosphere have influenced the planet’s water cycle. Since 1950, substantial increases in the heaviest precipitation events (i.e., those above the 95th percentile) have been observed in Europe and North America. While trends over the surface transpiration radiation the surface

1	FIGURE 151e-3 Earth’s energy balance. (Courtesy of NASA CERES project. Data from Trenberth KE et al: Earth’s global energy budget. Bull Am Meteor Soc 90:311, 2009.) that same interval are less clear in other regions because of limited data, regions of Southeast Asia and southern South America have likely experienced increases in heavy precipitation as well. Other areas have seen greater drought, notably southern Australia and the southwestern United States. A warmer atmosphere holds more water vapor; specifically, there is 6–7.5% more water vapor per degree (Celsius) of warming in the lower atmosphere. For areas that have traditionally had more precipitation on average, warming tends to promote heavier precipitation events. In contrast, in regions prone to drought, warming tends to result in greater periods between rainfalls and in the risk of drought.

1	The world’s oceans have absorbed 90% of the excess heat that greenhouse gases have kept in Earth’s atmosphere since the 1960s. Ocean heat provides energy for hurricanes, and warmer years tend to have greater hurricane activity. Atlantic hurricanes are the best studied and have the most data available. An analysis of satellite observations from 1983 to 2005 has shown a trend toward increasing severity—albeit decreasing frequency—of Atlantic hurricanes. Modeling of future tropical cyclones suggests that their intensity may increase 2–11% by 2100 and that the average storm will bring 20% more rainfall.

1	El Niño events drive alterations in weather worldwide (Fig. 151e-4) 151e-3 and are associated with extreme events and consequently higher rates of morbidity and mortality. Hurricane Mitch, one of the most powerful hurricanes ever observed, with winds reaching 290 km/h, dropped 1–1.8 m (3–6 feet) of rain over 72 h on parts of Honduras and Nicaragua. As a result of this storm, 11,000 people died and 2.7 million were displaced. Outbreaks of cholera, leptospirosis, and dengue occurred in the storm’s aftermath.

1	The final common outcome of all climate change effects is population migration. Sea level rise, extreme heat and precipitation, droughts, and salinization of water supplies all conspire to make regions (including some inhabited by humans for millennia) uninhabitable. Among climate change migrants in the near future may be the inhabitants of low-lying South Pacific islands that are vulnerable to sea level rise and residents of the Alaskan archipelago, where melting of permafrost has rendered traditional means of cold food storage difficult. Climate change may also be contributing to humanitarian crises and conflicts. A severe 2011 drought in East Africa may have incited the Somali famine that resulted in 1 million refugees; mortality rates reached 7.4/10,000 in some refugee camps. Crop losses associated with the 2010

1	Between 1901 and 2010, the global sea level rose ~200 mm, or ~1.7 mm per year on average. From 1993 to 2010, the rate of rise nearly doubled—i.e., to 3.2 mm annually. Most of this sea level rise has resulted from the thermal expansion of water. Glacial ice melt is the second greatest factor, and its contribution is accelerating. By 2100, global sea level may rise by ≥500 mm from 1986–2005 levels, with an annual rate of rise of 8–16 mm at the century’s end. A large section of the West Antarctic ice sheet has begun to fall apart, and its melting alone may cause sea level to rise by ≥3 m in coming centuries.

1	Sea level rise is not uniform. The rate of rise on the eastern seaboard of North America has been roughly double the global rate. Compounding sea level rise is the subsidence of coastal areas due to human settlement. In the absence of levee upgrades, an estimated 170 million people living near coasts worldwide will be at risk of flooding in 2100 because of the combined effects of subsidence, erosion, and sea level rise. Along with extreme storms and overuse of coastal aquifers, rising seas also contribute to salinization of coastal groundwater. About 1 billion people rely on coastal aquifers for potable water.

1	Along with extreme storms and overuse of coastal aquifers, rising seas also contribute to salinization of coastal groundwater. About 1 billion people rely on coastal aquifers for potable water. The El Niño Southern Oscillation (ENSO) refers to periodic changes in water temperature in the eastern Pacific Ocean that occur roughly every 5 years. ENSO cycles have dramatic effects on weather around the globe. Warmer-than-average water temperatures in the eastern Pacific define El Niño events (see below), whereas cooler-than-average water temperatures define La Niña periods. Evidence is accruing that climate change may be increasing the frequency and severity of FIGURE 151e-4 Characteristic weather anomalies, by season, during El Ni events. (Source: El Niño events. www.cpc.ncep.noaa.gov/products/precip/CWlink/ENSO/ENSO-Global-Impacts/High-Resolution/.) Russian heat wave led Russia to halt grain exports, causing higher grain prices on the world market and food riots in developing nations.

1	Russian heat wave led Russia to halt grain exports, causing higher grain prices on the world market and food riots in developing nations. The incidence of most, if not all, infectious diseases depends on climate. For any given infection, however, climate change is but one of many factors that determine disease epidemiology, and often it is not the most influential factor. Even in instances in which climate change creates conditions favorable to the spread of infections, diseases may be kept in check through interventions such as vector control or antibiotic treatment.

1	Detecting climate-change influence on an emerging human disease can be challenging. Research with animal pathogens, which in most instances is less well monitored and intervened upon than that with their human counterparts, has suggested how climate change may influence disease spread. For example, the life cycle of nematode parasites of caribou and musk oxen shortens as temperatures rise. As the Arctic has warmed, higher nematode burdens and consequently higher rates of morbidity and mortality have been observed. Other examples from animals, such as the spread of the protozoan parasite Perkinsus marinus in oysters, demonstrate how warming can enable range expansion of pathogens previously held in check by colder temperatures. As these and other examples from studies of animals make clear, the influence of climate change on infectious diseases can be pronounced. The following sections deal with the infectious diseases for which research has explored the influence of climate change.

1	Because insects are cold-blooded, ambient temperature dictates their geographic distribution. With increases in temperatures (in particular, nighttime minimum temperatures), insects are freed to move poleward and up mountainsides. At the same time, as new areas become climatically suitable, current mosquito habitats may become unsuitable as a result of heat extremes. In addition, insects tend to be sensitive to water availability. Mosquitoes that transmit malaria, dengue, and other infections may breed in pools of water created by heavy downpours. As has been observed in the Amazon, breeding pools can also appear during periods of drought when rivers recede and leave behind stagnant pools of water for Anopheles mosquitoes. These circumstances have raised interest in the potentially favorable impact of water-cycle intensification on the spread of mosquito-borne disease.

1	Malaria • TEMPERATURE Higher temperatures promote higher mosquito-biting rates, shorter parasite reproductive cycles, and the potential for the survival of mosquito vectors of Plasmodium infection in locations previously too cold to sustain them. Recent modeling experiments identify highland areas of East Africa and South America as perhaps most vulnerable to increased malarial incidence as a result of rising temperatures. In addition, a recent analysis of interannual malaria in Ecuador and Colombia has documented a greater incidence of malaria at higher altitudes in warmer years. Highland populations may be more vulnerable to malaria epidemics because they lack immunity.

1	Although rising temperature has the potential to expand the viable range of disease, malaria incidence is not associated with temperature in a strictly linear fashion. While mosquitoes and parasites may adapt to a warming climate, the present optimal temperature for malaria transmission is ~25°C, with a range of transmission temperatures between 16°C and 34°C. Rising temperatures also can have differential effects on parasite development during external incubation and on the mosquitoes’ gonotrophic cycle. Asynchrony between these two temperature-sensitive processes has been shown to decrease the vectorial capacity of mosquitoes.1 1rVc is the vectorial capacity relative to the vector-to-human population ratio and is defined by the equation rVc = a2bhbme–μmn/μm where a is the vector biting rate; bh is the probability of vector-to-human transmission per bite; bm is the probability of human-to-vector infection per bite; n is the duration of the extrinsic incubation period; and μm is the

1	rate; bh is the probability of vector-to-human transmission per bite; bm is the probability of human-to-vector infection per bite; n is the duration of the extrinsic incubation period; and μm is the vector mortality rate.

1	PRECIPITATION The abundance of Anopheles mosquitoes is strongly correlated with the availability of surface water pools for mosquito breeding, and biting rates have been linked to soil moisture (a surrogate for breeding pools). Research in the East African highlands has documented that increased variance in rainfall over time has strengthened the association between precipitation and disease incidence. These disease-promoting effects of precipitation may be countered by the potential for extreme rainfall to flush mosquito larvae from breeding sites.

1	PROJECTIONS Climate models have begun to deliver output on regional scales, permitting projections of climate-suitable regions to assist national and local health authorities. Climate models speak to the temperature and precipitation ranges necessary for malaria transmission but do not—and cannot—account for the capacity of malaria control programs to halt the spread of disease. The global reduction in malaria distribution over the past century makes it clear that, even with climate change, malaria occurs in far fewer places today because of public health interventions.

1	Despite intensive efforts, malaria remains the single greatest vector-borne disease cause of morbidity and death in the world. Particularly in regions that are most affected by malaria and where the public health infrastructure is inadequate to contain it, climate modeling may provide a useful tool in determining where the disease may spread. Recent modeling studies in sub-Saharan Africa have suggested that, although East African nations may encompass regions that will become more climatically suitable for malaria over this century, West African nations may not. By 2100, temperatures in West Africa may largely exceed those optimal for malaria transmission, and the climate may become drier; in contrast, higher temperatures and changes in precipitation may allow malaria to move up the mountainsides of East African countries. Climate change may create conditions favorable to malaria in subtropical and temperate regions of the Americas, Europe, and Asia as well.

1	Dengue Like malaria epidemics, dengue fever epidemics depend on temperature (Fig. 151e-5). Higher temperatures increase the rate of larval development and accelerate the emergence of adult Aedes mosquitoes. The daily temperature range may also influence dengue virus transmission, with a smaller range corresponding to a higher transmission potential. Temperatures <15°C or >36°C also substantially reduce mosquito feeding. In a Rhesus model of dengue, viral replication can occur in as little as 7 days with temperatures of >32–35°C; at 30°C, replication takes ≥12 days; and replication does not reliably occur at 26°C. Research on dengue in New Caledonia has shown peak transmission at ~32°C, reflecting combined effects of a shorter extrinsic incubation period, a higher feeding frequency, and more rapid development of mosquitoes. Along with temperature, peak relative humidity is a strong predictor of dengue outbreaks.

1	The association between dengue epidemics and precipitation is less consistent in the peer-reviewed literature, possibly because of the mosquito vector’s greater reliance on domestic breeding sites than on natural pools of water. For instance, in some studies, increased access to a piped water supply has been linked to dengue epidemics, presumably because of associated increased domestic water storage. Nonetheless, several studies have established rainfall as a predictor of the seasonal timing of dengue epidemics.

1	The current global distribution of dengue largely overlaps the geographic spread of Aedes mosquitoes (Fig. 151e-6). The presence of Aedes without dengue endemicity in large regions of North and South America and Africa illustrates the relevance of variables other than climate to disease incidence. Nevertheless, coupled climatic– epidemiologic modeling suggests dramatic shifts in the relative vectorial capacity for dengue by the end of this century should little or no mitigation of greenhouse gas emissions occur (Fig. 151e-7). Other Arbovirus Infections Climate change may favor increased geographic spread of other arboviral diseases, including Chikungunya virus disease, West Nile virus disease, and eastern equine encephalitis. Chikungunya virus disease emerged in Italy in 2007, having previously Days (EIP length and development to adult)

1	Days (EIP length and development to adult) FIGURE 151e-5 Effects of temperature on variables associated with dengue transmission. Shown are the number of days required for development of immature Aedes aegypti mosquitoes to adults, the length of the dengue virus type 2 extrinsic incubation period (EIP), the percentage of A. aegypti mosquitoes that complete a blood meal within 30 min after a blood source is made available, and the percentage of hatched A. aegypti larvae surviving to adulthood. (Reproduced from CW Morin et al: Climate and dengue transmission: evidence and implications.

1	Environ Health Perspect 121:1264, 2013.) been mostly a disease of African nations. Climate models predict that, should competent vectors be present, conditions will be suitable for Chikungunya virus to gain a foothold in Western Europe, especially France, in the first half of the twenty-first century. In North America, areas favorable to West Nile virus outbreaks are expected to shift northward in this century. Current hotspots in North America are the California Central Valley, southwestern Arizona, southern Texas, and Louisiana, which have both compatible climates and avian reservoirs for the disease. By mid-century, the upper Midwest and New England will be more climatically suited to West Nile virus; by the end of the century, the area of risk may shift even further north to southern Canada. Whether the disease will ultimately move northward will depend on reservoir availability and mosquito control programs, among other factors.

1	Whether the disease will ultimately move northward will depend on reservoir availability and mosquito control programs, among other factors. Lyme Disease In the past few decades, Ixodes scapularis, the primary tick vector for Lyme disease as well as for anaplasmosis and babesiosis in New England, has become established in Canada because of warming temperatures. With climate change, the range of the tick is expected to expand further (Fig. 151e-8). Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most commonly reported vector-borne disease in North America, with ~30,000 cases per year. The model used in Fig. 151e-8 showed 95% FIGURE 151e-6 Distribution of Aedes aegypti mosquitoes (turquoise) and dengue fever epidemics (red). (Map produced by the Agricultural Research Service of the U.S. Department of Agriculture.) 0.21 0.12 0.06 0.03 –0.03 –0.06 0.52 0.41 0.26 0.12 0.06 0.03 –0.03 –0.06 –0.12 –0.26 –0.41 –0.52

1	FIGURE 151e-7 Trend of annually averaged global dengue epidemic potential (rVc). Differences in rVc are based on 30-year averages of temperature and daily temperature range. A. Differences between 1980–2009 and 1901–1930. B. Differences between 2070–2099 and 1980–2009. The mean value of rVc was averaged from five global climate models under RCP8.5, a scenario of high greenhouse-gas emission. The color bar describes the values of the rVc. (From J Liu-Helmersson et al: Vectorial capacity of Aedes aegypti: effects of temperature and implications for global dengue epidemic potential. PLoS ONE 9:e89783, 2014 [doi:10.1371/journal.pone.0089783].) accuracy in predicting current I. scapularis distribution and suggests substantial expansion of tick habitat and consequently of populations at risk for the diseases this tick transmits, particularly in Quebec, Iowa, and Arkansas, by 2080. Of note, some areas on the Gulf Coast may become less suitable for ticks by the end of the century.

1	Outbreaks of waterborne disease are associated with heavy rainfall events. A review of 548 waterborne disease outbreaks in the United States found that 51% were preceded by precipitation levels above the 90th percentile. Since 1900, most regions of the United States except the Southwest and Hawaii have experienced an increase in heavy downpours (Fig. 151e-9), with the greatest intensification of the water cycle in New England and Alaska. Climate models suggest that by 2100 daily heavy-precipitation events, which are defined as a cumulative daily amount that now occurs once every 20 years, will increase nationwide (Fig. 151e-10). This scenario may be from two to as much as five times more likely, depending on the extent of greenhouse gas emission reductions achieved early in the twenty-first century.

1	FIGURE 151e-8 Present and projected probability of establishment of Ixodes scapularis. (From U.S. National Climate Assessment 2014, adapted from JS Brownstein et al: Effect of climate change on Lyme disease risk in North America. Ecohealth 2:38, 2005.) U.S. average 1900s20s 40s60s 80s00s Change (%) –50 0 50 Decade Northeast 1900s20s 40s60s 80s00s Change (%) –50 0 50 DecadeMidwest 1900s20s 40s60s 80s00s Change (%) –50 0 50 Decade Great Plains, north 1900s20s 40s60s 80s00s Change (%) –50 0 50 Decade Great Plains, south 1900s20s 40s60s 80s00s Change (%) –50 0 50 Decade Southeast 1900s20s 40s60s 80s00s Change (%) –50 0 50 Decade Southwest 1900s20s 40s60s 80s00s Change (%) –50 0 50 Decade Northwest 1900s20s 40s60s 80s00s Change (%)–50 0 50 Decade Hawaii 1900s20s 40s60s 80s00s Change (%) –50 0 50 Decade Alaska 1900s20s 40s60s 80s00s Change (%) –50 0 50 Decade Observed change in very heavy precipitation

1	FIGURE 151e-9 Percentage changes in the annual amount of precipitation falling in very heavy events, defined as the heaviest 1% of all daily events from 1901 to 2012 for each region. Changes are relative to a 1901–1960 average for all regions except values for Alaska and Hawaii, which are relative to the 1951–1980 average. (From U.S. National Climate Assessment 2014, NOAA National Climate Data Center/Cooperative Climate Change and Infectious Disease Institute for Climate and Satellites, North Carolina.) Projected change in heavy precipitation events Rapid emissions reductions (RCP 2.6) Continued emissions increases (RCP 8.5)

1	FIGURE 151e-10 Increased frequency of extreme daily precipitation events (defined as a daily amount that now occurs once in 20 years) by the latter part of the twenty-first century (2081–2100) compared to the frequency in the latter part of the twentieth century (1981–2000). A representative concentration pathway (RCP) describes a plausible climate future based on a net radiative forcing (e.g., 2.6 or 8.5) in 2100. (From U.S. National Climate Assessment 2014, NOAA National Climate Data Center/Cooperative Institute for Climate and Satellites, North Carolina.) 151e-8 Most disease outbreaks after heavy precipitation occur through contamination of drinking-water supplies. While outbreaks related to surface-water contamination generally occur within a month of the precipitation event, disease outbreaks from groundwater contamination tend to occur ≥2 months later. According to a review of published reports of waterborne disease outbreaks, Vibrio and Leptospira species are the pathogens most

1	from groundwater contamination tend to occur ≥2 months later. According to a review of published reports of waterborne disease outbreaks, Vibrio and Leptospira species are the pathogens most commonly involved in the wake of heavy precipitation.

1	Combined Sewer Systems Roughly 40 million people in the United States and millions more around the world rely on combined sewer systems in which storm water and sanitary wastewater are conveyed in the same pipe to treatment facilities. These systems were designed on the basis of the nineteenth-century climate, in which heavy downpours were less frequent than they are today. The frequency of combined sewer overflows resulting in untreated sewage discharge, usually into freshwater bodies, has been increasing in cities worldwide. Overflows are associated with discharges of heavy metals and other chemical pollutants as well as a variety of pathogens. Outbreaks of hepatitis A, Escherichia coli O157:H7 infection, and cryptosporidial disease have been associated with sewer overflows in the United States.

1	Rising Temperatures and Vibrio Species Warmer temperatures favor proliferation of Vibrio species and disease outbreaks, as has been demonstrated in countries surrounding the Baltic Sea, Chile, Israel, northwestern Spain, and the U.S. Pacific Northwest. Around the Baltic Sea, outbreaks of Vibrio infection may be particularly likely because of faster warming near the poles and the sea’s relatively low salt content. In 2004, a Vibrio parahaemolyticus outbreak occurred from consumption of Alaskan oysters. This pathogen was unknown in Alaskan oysters prior to this event and extended the known geographic range of the disease 1000 km northward.

1	In the past, El Niño events were used as a model to investigate the potential for extreme weather–related infectious disease epidemics occurring in association with climate change. Recent evidence has indicated that climate change itself may be strengthening El Niño events. These events tend to promote epidemic infections in certain regions (Fig. 151e-11). Associations of El Niño with outbreaks of Rift Valley fever in east ern and southern Africa have been known since the 1950s. El Niño favors wet conditions suitable for the insect vectors of the disease in these regions. Given the strong association between El Niño conditions and disease incidence, models have successfully predicted Rift Valley fever epidemics in humans and animals. In the 2006–2007 El Niño season, for example, outbreaks of Rift Valley fever were accurately predicted 2–6 weeks prior to epidemics in Somalia, Kenya, and Tanzania.

1	El Niño has had inconsistent associations with malaria incidence in African countries. Some of the strongest associations between El Niño and malaria have been identified in South Africa and Swaziland, where available data on incidence are relatively robust; however, even in these instances, the observed increased risk did not reach statistical significance. A stronger link to El Niño has been found in several studies done in South America. Research on malaria incidence in Colombia between 1960 and 2006 found that a 1°C temperature rise contributed to a 20% increase in incidence. El Niño years are often associated with an increased incidence of dengue. Research on dengue outbreaks in Thailand from 1996 to 2005 revealed that 15–22% of the variance in monthly dengue disease incidence was attributable to El Niño. In South America, data on dengue outbreaks between 1995 and 2010 showed an increased incidence during the El Niño events of 1997–1998 and 2006–2007.

1	CLIMATE CHANGE, POPULATION DISPLACEMENT, AND INFECTIOUS DISEASE EPIDEMICS For many reasons, including freshwater shortages, flooding, food shortages, and climate change–driven conflicts, climate change has and will continue to put pressure on human populations to move. Human migrations have long been associated with epidemic disease in the migrating populations themselves and in the communities in which they settle. The specific pathogens and patterns of disease that may appear after population migration relate to endemic diseases present in the migrant populations.

1	Large-scale migrations are common after extreme precipitation events. Hurricane Katrina, for instance, displaced about 1 million people from the U.S. Gulf Coast. Among Katrina refugees, outbreaks of respiratory, diarrheal, and skin diseases were most common. While attribution of a single weather event to increased greenhouse gas emissions is difficult, research can provide information on the likelihood of such events. It is expected, for example, that warming by 1°C increases the odds of a storm as strong as or stronger than Katrina by twoto sevenfold. In the developing world, infectious disease outbreaks associated with population displacement due to extreme weather events may be

1	In the developing world, infectious disease outbreaks associated with population displacement due to extreme weather events may be FIGURE 151e-11 Characteristic patterns of disease outbreaks associated with El Ni events, determined on the basis of 2006–2007 conditions. (From A Anyamba et al: Developing global climate anomalies suggest potential disease risks for 2006–2007. Int J Health Geogr 5:60, 2006.) especially hard to detect and respond to. Mitigation of disease risk requires overlaying of climate-related migration risk with foci of disease epidemics.

1	Climate change has far-reaching implications for the distribution and spread of infectious diseases worldwide. However, the greatest disease burdens related to climate change may not be due to infections. Because climate change disrupts the foundations of health, such as access to safe drinking water and food, it has the potential to undermine progress against major existing health problems such as malnutrition. In addition, resource scarcity and climate instability are increasingly associated with conflicts. Scholars have argued that events related to climate change were a factor in the revolutions of the Arab Spring and the Syrian civil war.

1	The public health response to climate change entails both mitigation 151e-9 and adaptation measures. Mitigation represents primary prevention and involves the reduction of greenhouse gas emissions into the atmosphere. Although no clear safety threshold of greenhouse gas emissions has been agreed upon, national governments from the major industrialized countries have agreed to set a warming target of <2°C above preindustrial levels by 2050; the attainment of this goal will require reducing greenhouse gas emissions by 40–70% below 2010 levels. To date, no international agreement exists to facilitate this reduction. Adaptation represents secondary prevention and is aimed at reducing the harms associated with sea level rise, heat waves, floods, droughts, wildfires, and other greenhouse gas–driven events. The efficacy of adaptation is constrained by the challenges inherent in predicting the precise location, duration, and severity of extreme weather events and flooding related to sea level

1	events. The efficacy of adaptation is constrained by the challenges inherent in predicting the precise location, duration, and severity of extreme weather events and flooding related to sea level rise, among other considerations.

1	Infections in Veterans Returning from Foreign Wars Andrew W. Artenstein Wars are an unfortunate but apparently inescapable consequence of human socialization. In the past quarter-century alone, there have 152e been dozens of major armed conflicts worldwide. Several of these wars have been multinational in scope and have involved the deployment of large numbers of ground forces from their home countries to distinct areas of conflict in developing portions of the world, such as southwest and central Asia (e.g., Iraq, Afghanistan) and Africa.

1	Troops who are deployed as combatants or in other military capacities on foreign soil are at risk of acquiring infectious diseases endemic to that region on the basis of intimate human and environmental exposures and their immunologic naiveté with regard to local endemic or enzootic pathogens. This risk is magnified by the crowded social conditions engendered by mass troop deployments, infrastructure destruction, and population displacement; it is further amplified by vulnerabilities in public health, such as the lapses in hygiene and sanitation that invariably accompany armed conflicts. The clinical spectrum of infectious illness acquired in this setting includes acute infections in the combat theater, acute infections with delayed symptoms, and chronic or relapsing infections. The latter two scenarios have the potential to cause illness in veterans returning from foreign wars.

1	The impact of acute infectious diseases of war, which were once a major cause of noncombat mortality, has significantly lessened in modern conflicts, largely because of the use of preventive vaccines and the early institution of antimicrobial therapy. Nonetheless, these acute diseases remain an important cause of morbidity in deployed military personnel (Table 152e-1). Many acute infectious diseases, such as influenza, meningococcal meningitis, hepatitis A, and adenoviral respiratory disease, can largely be prevented by routine vaccination of troops. Others, such as bacterial gastroenteritis and viral respiratory tract infections, continue to represent common causes of minor morbidity among deployed forces. The incidence of several other acute infections, such as malaria and dengue, can be favorably impacted— although not completely abrogated—by the use of chemoprophylaxis, personal protective measures, and vector control. Uncommonly, infections that have short incubation periods and

1	favorably impacted— although not completely abrogated—by the use of chemoprophylaxis, personal protective measures, and vector control. Uncommonly, infections that have short incubation periods and are acquired just days before leaving a combat theater may become clinically manifest only upon the return of troops to their countries of origin. Such was the case in a cluster of African tick typhus that occurred during short-term

1	U.S. troop deployments to Somalia and Botswana in the early 1990s. However, because most acute infections with brief clinical incubation periods are self-limited or responsive to treatment, they are not typically seen in returning war veterans and will not be addressed further in this chapter. A number of infectious diseases acquired in a theater of military operations may become apparent in veterans only after they have returned to their home countries. Although their incidences have not been precisely defined, these complications of military service—manifesting clinically with either acute or chronic signs and symptoms— may cause significant morbidity in affected veterans and in some settings may endanger public health through secondary transmission or contamination of the blood supply. Of a sample of nearly 53,000 U.S.

1	U.S. veterans of the Persian Gulf War (1990–1991), 7% were diagnosed with an infectious disease in the aftermath of the war; no excess risk of mortality was observed over a 6-year follow-up comparison with nondeployed veterans. This chapter focuses on infectious diseases that have occurred or have been a source of concern in returning veterans of foreign wars over the past quarter-century. During this period, several pathogens have been associated with disease in this population, as discussed below. Some pathogens have been associated with only rare case reports in war veterans, and some, given their epidemiology, may pose Gastroenteritis (enterotoxigenic or enteroinvasive Escherichia coli, Shigella, Salmonella, Campylobacter) Typhus (epidemic, murine, scrub) Sexually transmitted diseases (gonorrhea, chlamydial infection, genital her pes, genital warts, chancroid, syphilis) Combat wound infections Q fever

1	Typhus (epidemic, murine, scrub) Sexually transmitted diseases (gonorrhea, chlamydial infection, genital her pes, genital warts, chancroid, syphilis) Combat wound infections Q fever Norovirus gastroenteritis West Nile virus infection Crimean-Congo hemorrhagic fever Influenza Adenoviral respiratory disease Viral upper respiratory tract infections Dengue Alphavirus infections (e.g., Chikungunya, O'nyong-nyong, or Sindbis virus) Tickborne encephalitis Sandfly fever Hantavirus syndromes

1	Gastroenteritis (cryptosporidiosis, giardiasis, amebiasis) a risk in future conflicts. In general, it is practical to classify infections with delayed signs and symptoms related to prolonged incubation periods or significant clinical latency in terms of their potential to manifest clinically as acute illnesses or chronic/relapsing diseases. Table 152e-2 provides details regarding the epidemiology, clinical characteristics, diagnosis, therapy, and prevention of infectious diseases of concern in returning war veterans. Figure 152e-1 illustrates a differential diagnostic approach—based on prominent signs or symptoms—to suspected infections in this population.

1	ACUTE INFECTIOUS DISEASES WITH DELAYED CLINICAL PRESENTATIONS Malaria (See Chap. 248) Malaria, which is due to infection with Plasmodium species of protozoa, has historically caused significant battlefield morbidity and lost duty time among armed forces; these phenomena have been affirmed by recent U.S. military experiences in Africa and central Asia. Because of its worldwide prevalence and its pathophysiology, malaria remains an important cause of infection both during military operations and in returning war veterans.

1	The risk of malaria is exacerbated by several factors inherent to war: inadequate shelter promoting increased troop exposure to vectors; abeyance of governmental programs for vector control; and ecologic changes leading to an increased vector presence in the contested environment. Because of the complex life cycle of the parasite and the predilection of P. vivax and P. ovale to remain latent in their liver stages of development for prolonged periods, malaria in foreign-stationed troops may become clinically apparent only after their return home. In the aftermath of the Vietnam War, more than 13,000 cases of malaria—the vast majority due to P. vivax—were imported into the United States. Of the 7683 Soviet troops diagnosed with P. vivax InFECTIous DIsEAsEs AssoCIATED WITH DElAyED ClInICAl mAnIFEsTATIons THAT mAy PREsEnT WITH ACuTE, CHRonIC, oR RElAPsIng CouRsEs In VETERAns RETuRnIng FRom RECEnT FoREIgn WARs

1	InFECTIous DIsEAsEs AssoCIATED WITH DElAyED ClInICAl mAnIFEsTATIons THAT mAy PREsEnT WITH ACuTE, CHRonIC, oR RElAPsIng CouRsEs In VETERAns RETuRnIng FRom RECEnT FoREIgn WARs InFECTIous DIsEAsEs AssoCIATED WITH DElAyED ClInICAl mAnIFEsTATIons THAT mAy PREsEnT WITH ACuTE, CHRonIC, oR RElAPsIng CouRsEs In VETERAns RETuRnIng FRom RECEnT FoREIgn WARs (CONTINUED) Cryptosporidiosis Cryptosporidium Worldwide Fecal-oral 3–6 days Symptoms as above; Fecal microscopy or No specific anti-Food and water hygiene 254 spp. chronic watery diarrhea, intestinal biopsy; stool parasitic therapy for with or without fever, antigen assay postinfectious syn-abdominal pain, nausea

1	chronic watery diarrhea, intestinal biopsy; stool parasitic therapy for with or without fever, antigen assay postinfectious syn-abdominal pain, nausea Strongyloidiasis Strongyloides Tropical and subtropi-Fecal-oral as 11–28 days Abdominal pain, persis-Stool antigen detec-Ivermectin; thiaben-Personal protective 257 stercoralis cal climates initial route; per- tent diarrhea, urticaria; tion assay; serology dazole or albendazole measures, includsistent infection cause wasting, pulmonary symptoms, eosinophilia Sandfly fever Phleboviruses Africa, Asia, South and Vector Weeks to Depression, fatigue, gener-Serology No specific therapy Personal protective 233 (convalescence) Central America (Phlebotomus spp. months for alized weakness

1	Central America (Phlebotomus spp. months for alized weakness Relapsing fever Borrelia recurren-Worldwide Vector (body 4–18 days Recurrent episodes of fever, Spirochetes on stained Tetracycline or eryth-Personal protective 209 tis (louse-borne, louse; soft tick) initially, with rigors, diaphoresis, head-peripheral-blood romycin; antibiotic measures; vector epidemic), Borrelia relapses after ache, myalgias, arthralgias, smear during febrile treatment may lead control spp. (tick-borne, 7to 10-day asthenia lasting 3–6 days episodes to Jarisch-Herxheimer endemic) intervals and alternating with symp reaction with fever, tom-free periods rigor, hypotension within 2 h of initiation Brill-Zinsser Rickettsia prowazekii Worldwide Vector (body Recrudescent Mild febrile illness with Serology Doxycycline or Vector control; personal 211 disease appropriate treatment episode of epi

1	Brill-Zinsser Rickettsia prowazekii Worldwide Vector (body Recrudescent Mild febrile illness with Serology Doxycycline or Vector control; personal 211 disease appropriate treatment episode of epi Chronic wound Acinetobacter Worldwide Inoculation via Weeks to Chronic pain, swelling, ± Culture of tissue Guided by results of Adequate initial wound Miscellaneous infection spp., other gram- and penetrating gression of lent drainage of infected antibiotic susceptibil-treatment of acute soft- injury acute infection site with or without consti ity testing; carbape-tissue infection; removal aureus, including nem ± amikacin as of foreign bodies; strict MRSA; invasive empirical therapy for adherence to infection molds (Aspergillus, multidrug-resistant control precautions to Fusarium, Mucor, Acinetobacter; colistin prevent nosocomial Absidia spp.); atypi (M. chelonei, M. abscessus) associated with draining sinuses

1	Acinetobacter; colistin prevent nosocomial Absidia spp.); atypi (M. chelonei, M. abscessus) associated with draining sinuses InFECTIous DIsEAsEs AssoCIATED WITH DElAyED ClInICAl mAnIFEsTATIons THAT mAy PREsEnT WITH ACuTE, CHRonIC, oR RElAPsIng CouRsEs In VETERAns RETuRnIng FRom RECEnT FoREIgn WARs (CONTINUED) • Schistosomiasis, including Katayama fever • Strongyloidiasis • Visceral larva migrans • Filariasis • Echinococcal disease FIGURE 152e-1 Syndromic approach to the differential diagnosis of suspected infection in a veteran who has returned from a foreign war in southwest Asia, central Asia, or Africa at least 2 weeks prior to clinical presentation. HBV, hepatitis B virus. malaria acquired during the U.S.S.R.’s war in Afghanistan in the 1980s, 76% developed clinical manifestations >1 month after their return to the U.S.S.R., with some cases developing as long as 3 years later.

1	Imported malaria with prolonged clinical latency periods remains a problem among veterans returning from wars in endemic areas. In a cluster of 112 cases of imported disease in U.S. Marines returning to the United States from deployment to Somalia in 1993, falciparum malaria was diagnosed as late as 12 weeks after return; some cases due to P. vivax were diagnosed after an additional 2 months. In an outbreak of imported P. vivax malaria among U.S. Army Rangers deployed to Afghanistan in 2002, the median time to diagnosis was nearly 8 months after return.

1	Although malaria is largely preventable through the combined use of vector control, personal protective measures (e.g., bed nets, insect repellent, long sleeves, permethrin-treated clothing), and chemoprophylaxis, nonadherence to these measures and/or to chemotherapy (including terminal primaquine prophylaxis to eradicate the liver stage of P. vivax) appears to be responsible for the majority of cases in recent U.S. military experiences. However, there is also evidence to support chemoprophylaxis failures in a small subgroup of cases of P. falciparum and P. vivax malaria acquired during U.S. deployments to Somalia. Thus, imported malaria in returning war veterans remains possible despite appropriate adherence to chemoprophylaxis.

1	Viral Hepatitis (See Chap. 360) The incidence of viral hepatitis, once a major scourge of military campaigns and their aftermath, has declined considerably over the past half-century of military engagements. Although more than 115,000 cases of viral hepatitis, most due to hepatitis A virus, were reported among Soviet troops serving in Afghanistan in the 1980s, only rare reports of hepatitis A and B were noted during the massive, short-term deployment of U.S. troops to the Persian Gulf in the early 1990s. Hepatitis A and E, endemic in many parts of the developing world, present clinically as acute infections transmitted by the fecal-oral route and can be largely controlled with interventions practiced widely among military forces from industrialized countries: appropriate food and water hygiene and pre-deployment vaccination against hepatitis A. Hepatitis B contamination of serum-stabilized yellow fever vaccine caused a large outbreak of the disease among

1	U.S. forces during World War II; such events are currently unlikely, given the use of modern virus-inactivation techniques in vaccine manufacturing. Despite their potential—as a consequence of their long clinical incubation and latency periods—to cause disease in returning veterans, hepatitis B and hepatitis C have been acquired relatively rarely in theater because of risk factor mitigation: routine drug testing of modern armies and screening of the blood supply to exclude viral contamination.

1	Rabies (See Chap. 232) Deployed soldiers are often in close contact with feral dogs and other potentially rabid animals in both urban and remote combat environments. In the period 2001–2010, 643 animal bites were documented during medical encounters among U.S. forces in the combat theaters of southwest and central Asia; the majority of these potential exposures were dog bites. Of bitten personnel, 18% received rabies postexposure prophylaxis. Recently, a U.S. soldier developed rapidly progressive signs and symptoms of rabies 8 months after a dog bite in Afghanistan and died within 17 days of illness onset. This case, which represents the first rabies death in nearly 40 years in a member of the U.S. military who acquired the infection overseas, reinforces both the lethality of the disease and the need to practice vigilant exposure precautions among deployed military personnel and to administer postexposure prophylaxis to troops who are bitten by animals in high-risk areas.

1	CHRONIC OR RELAPSING INFECTIOUS DISEASES ACQUIRED IN THE THEATER OF WAR Leishmaniasis (See Chap. 251) Various forms of leishmaniasis may be acquired during military deployment and may present with myriad chronic clinical manifestations in war veterans. Because the protozoal pathogens (Leishmania species) are endemic throughout much of southwest and central Asia, their associated diseases have occurred in veterans returning from several recent conflicts there. The widespread distribution of various species of Leishmania elsewhere throughout the developing world suggests that leishmaniasis may complicate future conflicts as well.

1	Leishmaniasis may present clinically as cutaneous, mucocutaneous, or visceral disease; all forms are transmitted to humans by the bite of infected phlebotomine sandflies via zoonotic (small mammal) reservoirs. In rare circumstances, infection may be transmitted through blood transfusion. Transmission to humans is enhanced by factors that bring them into close proximity to animal reservoirs, such as life in dense, mobile populations; disruption of ecologic niches; and infrastructural breakdown. All these factors are common sequelae of war. At least 1300 cases of cutaneous leishmaniasis caused by L. major or

1	At least 1300 cases of cutaneous leishmaniasis caused by L. major or L. tropica have been diagnosed in American soldiers deployed to Iraq and Afghanistan over the past decade; however, the actual burden of infection may be higher due to underreporting, as lesions spontaneously resolve in many cases. The infection manifests clinically as one or more chronic, painless skin ulcers or nodules that may persist for 6–12 months. Rarely, lesions of cutaneous leishmaniasis may disseminate locally or diffusely.

1	Visceral disease (kala-azar) is typically caused by L. donovani and may be life-threatening. There have been at least five confirmed reports of U.S. veterans returning from recent deployments with classic visceral leishmaniasis associated with chronic fever, weight loss, pancytopenia, hypergammaglobulinemia, and organomegaly. As systemic leishmanial infection is known to manifest clinically years after exposure and may recrudesce if host immunity wanes due to unrelated causes, it remains possible that additional cases may yet surface.

1	Chronic Diarrhea Although acute bacterial gastroenteritis remains a major noncombat cause of morbidity and duty days lost during troop deployments, chronic illness is unusual. However, selected bacterial and parasitic enteric pathogens may cause chronic infections or illnesses in returning veterans. Although such infections have been uncommonly noted in recent deployments, they pose potential threats in future wars because of their worldwide prevalence.

1	Giardiasis (Chap. 254), amebiasis (Chap. 247), and cryptosporidiosis (Chap. 254), which usually cause self-limited protozoal gastroenteritis in immunocompetent hosts, may result in persistent symptoms in immunocompromised populations or when complicated by secondary illness. Giardia infection has been associated with chronic diarrhea due to postinfectious irritable bowel syndrome and with chronic signs and symptoms of systemic illness in association with postinfection fatigue or protein-losing enteropathy. Cryptosporidiosis also may cause chronic diarrhea or malabsorptive syndromes in immunocompromised individuals. Amebic infection of the colon may be associated with several serious complications, including perforation, fistulae, and obstruction; extraintestinal spread of amebiasis may result in hepatic invasion leading to abscess formation.

1	Systemic Illness Due to Enteric Pathogens Certain helminthic infections are endemic in many parts of the developing world and may pose continuing risks to infected military personnel after their return. Larvae of the intestinal nematode Strongyloides stercoralis (Chap. 257) either may be passed in the feces and develop into the infective stage in the external environment or may persist in the human small intestine and initiate new infective cycles in a process known as autoinfection. Autoinfection with Strongyloides may result in chronic clinical manifestations such as pruritus, rash, abdominal pain, weight loss, diarrhea, and eosinophilia. In immunocompromised hosts, chronic strongyloidiasis may cause a life-threatening hyperinfection syndrome, likely triggered by high parasite burdens and resulting in a multiorgan, systemic illness consistent with severe inflammatory response syndrome. In some cases, Strongyloides hyperinfection syndrome may be complicated by gram-negative sepsis or

1	resulting in a multiorgan, systemic illness consistent with severe inflammatory response syndrome. In some cases, Strongyloides hyperinfection syndrome may be complicated by gram-negative sepsis or meningitis related to bacterial seeding from parasitic involvement of the lungs or gastrointestinal tract. Although not described in association with recent wars, chronic strongyloidiasis was an uncommon phenomenon affecting a small number of World War II and Vietnam War veterans; one study estimated that there were up to 400 affected individuals still living in Great Britain. The pathogen may pose a risk to troops deployed in the future to tropical and subtropical regions of the world where the parasite is endemic.

1	Chronic schistosomiasis (Chap. 259) results from intravascular infection by trematode parasites whose larval forms penetrate the skin of humans through contact with freshwater inhabited by the snail intermediate host. The pathogens are widely distributed throughout large portions of the developing world. A chronic inflammatory response in the portal circulation to the eggs of S. mansoni and S. japonicum leads to fibrosing disease in the liver and eventually to cirrhosis. Similar pathophysiologic events occur in the vascular plexus of the human genitourinary tract in response to chronic S. haematobium infection, leading to fibrosing changes in the human bladder and ureters—a precursor to bladder cancer. Rarely, individuals with chronic schistosomiasis develop a syndrome of persistent or relapsing bacteremia with Salmonella typhi, which is the etiologic agent of typhoid fever and is not otherwise a typical infectious cause of chronic disease in veterans.

1	Other Chronic Infections/Syndromes Awareness of the potential threat of troop exposure to agents of biological warfare (Chap. 261e) has been heightened over the past two decades by revelations regarding Iraq’s state-sponsored chemical weapons program in the 1990s, the known broad availability of such technology, and escalations of global and geopolitical conflicts as well as of international acts of terrorism. Most of the high-risk pathogens posing a threat of bioterrorism cause acute clinical manifestations; however, selected agents, such as those causing Q fever and brucellosis, may result in chronic diseases, whether exposure is natural or intentional. Isolated cases of naturally acquired Q fever and brucellosis have been reported in recent U.S. veterans of the wars in Iraq and Afghanistan. To date, there has been no confirmed evidence of infections related to exposure to biological weapons in returning war veterans.

1	HIV-1 infection (Chap. 226), ubiquitous throughout the world, continues to pose a potential bloodborne and sexually transmitted risk to soldiers engaged in armed conflict in high-prevalence areas. Several reports describe war veterans returning to their countries of origin harboring HIV-1; in some of these cases, novel viral genotypes have been imported into the population. Tuberculosis (Chap. 202) also is endemic throughout much of the developing world and is prevalent in several areas of recent multinational conflicts. Although there is no evidence that active, chronic tuberculosis has affected veterans of recent wars, the rate of tuberculin skin test conversion, which indicates new infections, was noted to be 2.5% among U.S. military personnel deployed to southwest Asia in the early 2000s.

1	Several chronic infections that pose a risk to war veterans tend to recur or become clinically active in immunocompromised individuals and may be particularly aggressive in this population. Latent infections such as leishmaniasis, tuberculosis, histoplasmosis, brucellosis, Q fever, and strongyloidiasis in otherwise healthy veterans returning from war may become clinically expressed only much later in the setting of chronic glucocorticoid use, monoclonal antibody therapy, organ transplantation, cytotoxic chemotherapy, advanced HIV-1 disease, hematologic malignancy, or other immunosuppressive conditions. Thus, physicians should remain vigilant regarding the potential development of symptomatic disease due to such latent microbes in immunologically compromised veterans who may have initially acquired an infection years previously while serving in the military.

1	A number of syndromes of possible infectious etiology, some of which may present with chronic clinical manifestations, have been noted in veterans returning from recent wars. After the Gulf War in 1990–1991, numerous veterans from several countries experienced constellations of various common, nonspecific symptoms, including fatigue, musculoskeletal pain, sleep disturbances, and difficulty concentrating. Despite exhaustive investigations and several hypotheses regarding potential etiologies of this chronic multisystemic illness, including infectious agents, no unifying or single cause has been identified. In a randomized, placebo-controlled trial, a prolonged course of doxycycline failed to alleviate such symptoms at 1 year. In 2003, a small outbreak of acute idiopathic eosinophilic pneumonia was reported among U.S. troops serving in southwest Asia. Although a thorough investigation failed to elucidate an infectious etiology, it is noteworthy that symptoms developed up to 11 months

1	was reported among U.S. troops serving in southwest Asia. Although a thorough investigation failed to elucidate an infectious etiology, it is noteworthy that symptoms developed up to 11 months after arrival in the theater of combat; this time frame suggests that such cases may become clinically manifest after return to the home front.

1	Chronic Wound Infections and Osteomyelitis War wounds, an important cause of morbidity in all armed conflicts, are at high risk of infection due to contamination with environmental bacteria and the presence of retained foreign bodies. In recent conflicts, improved survival rates due to enhanced and expedited care of combat casualties have had the unintended consequence of increasing the potential for infectious complications, a situation exacerbated by repeated and at times prolonged exposure to health care environments and their associated pathogens. Many wounds sustained in recent wars have resulted from penetrating soft-tissue trauma and open fractures of the extremities— injuries attributable to improvised explosive devices used as antipersonnel weapons and to body armor that leaves the limbs unprotected. Cultures of samples taken at the time of injury at a combat support hospital in Iraq revealed that most contaminated wounds harbored gram-positive commensal skin bacteria; other

1	limbs unprotected. Cultures of samples taken at the time of injury at a combat support hospital in Iraq revealed that most contaminated wounds harbored gram-positive commensal skin bacteria; other investigators, however, have noted an early predominance of gram-negative bacteria, including multidrug-resistant (MDR) pathogens.

1	Approximately 3% of nearly 17,000 combat injuries sustained between 2003 and 2009 in U.S. military operations in Iraq and Afghanistan involved soft tissue infections. Although it is not clear how many of these infections became chronic or progressed to involve deeper tissue structures, a significant number were managed in tertiary care facilities, many on the home front. The bacteriology of infected combat wounds comprises predominantly gram-negative bacilli and MDR organisms. Broad-spectrum antimicrobial prophylaxis administered at the time of injury appears to be a risk factor for subsequent infection; nosocomial acquisition of health care–associated MDR pathogens likely contributes as well. Invasive fungal infections have recently emerged as a significant cause of morbidity and death in the context of combat wounds.

1	During the past decade of wars in Iraq and Afghanistan, MDR strains of Acinetobacter baumannii (Chap. 187) have emerged as important pathogens in both wound and bloodstream infections 152e-9 among returning veterans treated at U.S. health care facilities. The majority of isolates display in vitro susceptibility to amikacin and variable susceptibility to carbapenems but are largely resistant to other commonly used antimicrobial agents. Antimicrobial treatment should be guided by in vitro susceptibility data; patients who are critically ill, are immunocompromised, or have significant medical co-morbidities may benefit from combination therapy. Colistin (polymyxin E) has been shown to be clinically effective against Acinetobacter infections caused by isolates resistant to both aminoglycosides and carbapenems. Mortality rates have been low among immunocompetent hosts receiving appropriate antimicrobial treatment and undergoing debridement; however, Acinetobacter infections in

1	and carbapenems. Mortality rates have been low among immunocompetent hosts receiving appropriate antimicrobial treatment and undergoing debridement; however, Acinetobacter infections in immunocompromised individuals are associated with higher mortality risk. Strict adherence to hand-washing and other infection control procedures is important to limit the nosocomial spread of MDR organisms.

1	Chronic osteomyelitis related to either extension of a contiguous soft tissue infection or an infected prosthesis also represents a burgeoning problem for wounded veterans of recent wars. Limited micro-biologic data have shown a predominance of gram-negative etiologic agents—most often Acinetobacter and Pseudomonas aeruginosa—in the initial episodes of osteomyelitis but a shift to staphylococcal isolates in the majority of recurrent cases—a change that may perhaps be related to nosocomial acquisition. Relapses have been noted to occur 1 month to 1 year after treatment of the initial infection.

1	Veterans with traumatic brain injury, who have accounted for 22% of American casualties in recent wars in Iraq and Afghanistan, are at risk for infectious complications due to several factors: the presence of foreign bodies or prosthetic material related to their traumatic wounds; acquisition of a wide range of nosocomial infections during repeated interactions with the health care system; and injury-induced cognitive changes that may increase impulsivity and risk-taking behaviors. In line with the last-mentioned factor, this subgroup of veterans may be at heightened risk for substance abuse and other practices that expose them to various bloodborne and sexually transmitted infections. Moreover, they may be at risk for post-neurosurgical complications, such as pyogenic meningitis due to multidrug-resistant Acinetobacter species.

1	Pneumonia Lionel A. Mandell, Richard G. Wunderink DEFINITION Pneumonia is an infection of the pulmonary parenchyma. Despite being the cause of significant morbidity and mortality, pneumonia 153 SECTIon 2 Pseudomonas Acinetobacter MDR Condition MRSA aeruginosa spp. Enterobacteriaceae √ Chronic dialysis √ Home infusion therapy √ Home wound care √ Family member with MDR infection √ √ Abbreviations: MDR, multidrug-resistant; MRSA, methicillin-resistant Staphylococcus aureus.

1	is often misdiagnosed, mistreated, and underestimated. In the past, pneumonia was typically classified as community-acquired (CAP), hospital-acquired (HAP), or ventilator-associated (VAP). Over the past two decades, however, some persons presenting with onset of pneumonia as outpatients have been found to be infected with the multidrug-resistant (MDR) pathogens previously associated with HAP. Factors responsible for this phenomenon include the development and widespread use of potent oral antibiotics, earlier transfer of patients out of acute-care hospitals to their homes or various lower-acuity facilities, increased use of outpatient IV antibiotic therapy, general aging of the population, and more extensive immunomodulatory therapies. The potential involvement of these MDR pathogens has led to a designation for a new category of pneumonia—health care–associated pneumonia (HCAP)—that is distinct from CAP. Conditions associated with HCAP and the likely pathogens are listed in Table

1	led to a designation for a new category of pneumonia—health care–associated pneumonia (HCAP)—that is distinct from CAP. Conditions associated with HCAP and the likely pathogens are listed in Table 153-1.

1	Although the new classification system has been helpful in designing empirical antibiotic strategies, it is not without its disadvantages. Not all MDR pathogens are associated with all risk factors (Table 153-1). Moreover, HCAP is a distillation of multiple risk factors, and each patient must be considered individually. For example, the risk of infection with MDR pathogens for a nursing home resident who has dementia but can independently dress, ambulate, and eat is quite different from the risk for a patient who is in a chronic vegetative state with a tracheostomy and a percutaneous feeding tube in place. In addition, risk factors for MDR infection do not preclude the development of pneumonia caused by the usual CAP pathogens.

1	This chapter deals with pneumonia in patients who are not considered to be immunocompromised. Pneumonia in severely immunocompromised patients, some of whom overlap with the groups 804 of patients considered in this chapter, warrants separate discussion (see Chaps. 104, 169, and 226). Pneumonia results from the proliferation of microbial pathogens at the alveolar level and the host’s response to those pathogens. Microorganisms gain access to the lower respiratory tract in several ways. The most common is by aspiration from the oropharynx. Small-volume aspiration occurs frequently during sleep (especially in the elderly) and in patients with decreased levels of consciousness. Many pathogens are inhaled as contaminated droplets. Rarely, pneumonia occurs via hematogenous spread (e.g., from tricuspid endocarditis) or by contiguous extension from an infected pleural or mediastinal space.

1	Mechanical factors are critically important in host defense. The hairs and turbinates of the nares capture larger inhaled particles before they reach the lower respiratory tract. The branching architecture of the tracheobronchial tree traps microbes on the airway lining, where mucociliary clearance and local antibacterial factors either clear or kill the potential pathogen. The gag reflex and the cough mechanism offer critical protection from aspiration. In addition, the normal flora adhering to mucosal cells of the oropharynx, whose components are remarkably constant, prevents pathogenic bacteria from binding and thereby decreases the risk of pneumonia caused by these more virulent bacteria.

1	When these barriers are overcome or when microorganisms are small enough to be inhaled to the alveolar level, resident alveolar macrophages are extremely efficient at clearing and killing pathogens. Macrophages are assisted by proteins that are produced by the alveolar epithelial cells (e.g., surfactant proteins A and D) and that have intrinsic opsonizing properties or antibacterial or antiviral activity. Once engulfed by the macrophage, the pathogens—even if they are not killed—are eliminated via either the mucociliary elevator or the lymphatics and no longer represent an infectious challenge. Only when the capacity of the alveolar macrophages to ingest or kill the microorganisms is exceeded does clinical pneumonia become manifest. In that situation, the alveolar macrophages initiate the inflammatory response to bolster lower respiratory tract defenses. The host inflammatory response, rather than proliferation of microorganisms, triggers the clinical syndrome of pneumonia. The

1	the inflammatory response to bolster lower respiratory tract defenses. The host inflammatory response, rather than proliferation of microorganisms, triggers the clinical syndrome of pneumonia. The release of inflammatory mediators, such as interleukin 1 and tumor necrosis factor, results in fever. Chemokines, such as interleukin 8 and granulocyte colony-stimulating factor, stimulate the release of neutrophils and their attraction to the lung, producing both peripheral leukocytosis and increased purulent secretions. Inflammatory mediators released by macrophages and the newly recruited neutrophils create an alveolar capillary leak equivalent to that seen in the acute respiratory distress syndrome, although in pneumonia this leak is localized (at least initially). Even erythrocytes can cross the alveolar-capillary membrane, with consequent hemoptysis. The capillary leak results in a radiographic infiltrate and rales detectable on auscultation, and hypoxemia results from alveolar

1	cross the alveolar-capillary membrane, with consequent hemoptysis. The capillary leak results in a radiographic infiltrate and rales detectable on auscultation, and hypoxemia results from alveolar filling. Moreover, some bacterial pathogens appear to interfere with the hypoxemic vasoconstriction that would normally occur with fluid-filled alveoli, and this interference can result in severe hypoxemia. Increased respiratory drive in the systemic inflammatory response syndrome (Chap. 325) leads to respiratory alkalosis. Decreased compliance due to capillary leak, hypoxemia, increased respiratory drive, increased secretions, and occasionally infection-related bronchospasm all lead to dyspnea. If severe enough, the changes in lung mechanics secondary to reductions in lung volume and compliance and the intrapulmonary shunting of blood may cause respiratory failure and the patient’s death.

1	Classic pneumonia evolves through a series of pathologic changes. The initial phase is one of edema, with the presence of a proteinaceous exudate—and often of bacteria—in the alveoli. This phase is rarely evident in clinical or autopsy specimens because of the rapid transition to the red hepatization phase. The presence of erythrocytes in the cellular intraalveolar exudate gives this second stage its name, but neutrophil influx is more important with regard to host defense. Bacteria are occasionally seen in pathologic specimens collected during this phase. In the third phase, gray hepatization, no new erythrocytes are extravasating, and those already present have been lysed and degraded. The neutrophil is the predominant cell, fibrin deposition is abundant, and bacteria have disappeared. This phase corresponds with successful containment of the infection and improvement in gas exchange. In the final phase, resolution, the macrophage reappears as the dominant cell type in the alveolar

1	This phase corresponds with successful containment of the infection and improvement in gas exchange. In the final phase, resolution, the macrophage reappears as the dominant cell type in the alveolar space, and the debris of neutrophils, bacteria, and fibrin has been cleared, as has the inflammatory response.

1	This pattern has been described best for lobar pneumococcal pneumonia and may not apply to pneumonia of all etiologies, especially viral or Pneumocystis pneumonia. In VAP, respiratory bronchiolitis may precede the development of a radiologically apparent infiltrate. Because of the microaspiration mechanism, a bronchopneumonia pattern is most common in nosocomial pneumonias, whereas a lobar pattern is more common in bacterial CAP. Despite the radiographic appearance, viral and Pneumocystis pneumonias represent alveolar rather than interstitial processes.

1	The extensive list of potential etiologic agents in CAP includes bacteria, fungi, viruses, and protozoa. Newly identified pathogens include metapneumoviruses, the coronaviruses responsible for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome, and community-acquired strains of methicillin-resistant Staphylococcus aureus (MRSA). Most cases of CAP, however, are caused by relatively few pathogens (Table 153-2). Although Streptococcus pneumoniae is most common, other organisms also must be considered in light of the patient’s risk factors and severity of illness. Separation of potential agents into either “typical” bacterial pathogens or “atypical” organisms may be helpful. The former category includes S. pneumoniae, Haemophilus influenzae, and (in selected patients) S. aureus and gram-negative bacilli such as Klebsiella pneumoniae and Pseudomonas aeruginosa. The “atypical” organisms include Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella species

1	S. aureus and gram-negative bacilli such as Klebsiella pneumoniae and Pseudomonas aeruginosa. The “atypical” organisms include Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella species (in inpatients) as well as respiratory viruses such as influenza viruses, adenoviruses, human metapneumovirus, and respiratory syncytial viruses. Viruses may be responsible for a large proportion of CAP cases that require hospital admission, even in adults. Atypical organisms cannot be cultured on standard media or seen on Gram’s stain. The frequency and importance of atypical pathogens have significant implications for therapy. These organisms are intrinsically resistant to all β-lactam agents and must be treated with a macrolide, a fluoroquinolone, or a tetracycline. In the ∼10–15% of CAP cases that are polymicrobial, the etiology usually includes a combination of typical and atypical pathogens.

1	Anaerobes play a significant role only when an episode of aspiration has occurred days to weeks before presentation of pneumonia. The TABLE 153-2 MICRoBIAL CAuSES of CoMMunITy-ACquIRED PnEuMonIA, By SITE of CARE aInfluenza A and B viruses, human metapneumovirus, adenoviruses, respiratory syncytial viruses, parainfluenza viruses. Note: Pathogens are listed in descending order of frequency. ICU, intensive care unit. combination of an unprotected airway (e.g., in patients with alcohol or drug overdose or a seizure disorder) and significant gingivitis constitutes the major risk factor. Anaerobic pneumonias are often complicated by abscess formation and by significant empyemas or parapneumonic effusions.

1	S. aureus pneumonia is well known to complicate influenza infection. However, MRSA has been reported as the primary etiologic agent of CAP. While this entity is still relatively uncommon, clinicians must be aware of its potentially serious consequences, such as necrotizing pneumonia. Two important developments have led to this problem: the spread of MRSA from the hospital setting to the community and the emergence of genetically distinct strains of MRSA in the community. The former circumstance is more likely to result in HCAP, whereas the novel community-acquired MRSA (CA-MRSA) strains may infect healthy individuals with no association with health care.

1	Unfortunately, despite a careful history and physical examination as well as routine radiographic studies, the causative pathogen in a case of CAP is difficult to predict with any degree of certainty; in more than one-half of cases, a specific etiology is never determined. Nevertheless, epidemiologic and risk factors may suggest the involvement of certain pathogens (Table 153-3).

1	More than 5 million CAP cases occur annually in the United States; usually, 80% of the affected patients are treated as outpatients and 20% as inpatients. The mortality rate among outpatients is usually ≤1%, whereas among hospitalized patients the rate can range from ∼12% to 40%, depending on whether treatment is provided in or outside of the intensive care unit (ICU). CAP results in more than 1.2 million hospitalizations and more than 55,000 deaths annually. The overall yearly cost associated with CAP is estimated at $12 billion. The incidence rates are highest at the extremes of age. The overall annual rate in the United States is 12 cases/1000 persons, but the figure increases to 12–18/1000 among children <4 years of age and to 20/1000 among persons >60 years of age. Structural lung disease (e.g., bronchiectasis) Dementia, stroke, decreased level of consciousness Travel to Ohio or St. Lawrence river valleys Travel to southwestern United States Travel to Southeast Asia

1	Dementia, stroke, decreased level of consciousness Travel to Ohio or St. Lawrence river valleys Travel to southwestern United States Travel to Southeast Asia Stay in hotel or on cruise ship in previous 2 weeks Local influenza activity Exposure to bats or birds Exposure to birds Exposure to rabbits Exposure to sheep, goats, parturient cats Streptococcus pneumoniae, oral anaerobes, Klebsiella pneumoniae, Acinetobacter spp., Mycobacterium tuberculosis Haemophilus influenzae, Pseudomonas aeruginosa, Legionella spp., S. pneumoniae, Moraxella catarrhalis, Chlamydia pneumoniae P. aeruginosa, Burkholderia cepacia, Staphylococcus aureus Oral anaerobes, gram-negative enteric bacteria CA-MRSA, oral anaerobes, endemic fungi, M. tuberculosis, atypical mycobacteria Histoplasma capsulatum Hantavirus, Coccidioides spp. Burkholderia pseudomallei, avian influenza virus Legionella spp. Influenza virus, S. pneumoniae, S. aureus

1	Hantavirus, Coccidioides spp. Burkholderia pseudomallei, avian influenza virus Legionella spp. Influenza virus, S. pneumoniae, S. aureus H. capsulatum Chlamydia psittaci Francisella tularensis Coxiella burnetii Abbreviations: CA-MRSA, community-acquired methicillin-resistant Staphylococcus aureus; COPD, chronic obstructive pulmonary disease.

1	The risk factors for CAP in general and for pneumococcal pneu-805 monia in particular have implications for treatment regimens. Risk factors for CAP include alcoholism, asthma, immunosuppression, institutionalization, and an age of ≥70 years. In the elderly, factors such as decreased cough and gag reflexes as well as reduced antibody and Toll-like receptor responses increase the likelihood of pneumonia. Risk factors for pneumococcal pneumonia include dementia, seizure disorders, heart failure, cerebrovascular disease, alcoholism, tobacco smoking, chronic obstructive pulmonary disease, and HIV infection. CA-MRSA pneumonia is more likely in patients with skin colonization or infection with CA-MRSA. Enterobacteriaceae tend to infect patients who have recently been hospitalized and/or received antibiotic therapy or who have comorbidities such as alcoholism, heart failure, or renal failure. P. aeruginosa is a particular problem in patients with severe structural lung disease, such as

1	antibiotic therapy or who have comorbidities such as alcoholism, heart failure, or renal failure. P. aeruginosa is a particular problem in patients with severe structural lung disease, such as bronchiectasis, cystic fibrosis, or severe chronic obstructive pulmonary disease. Risk factors for Legionella infection include diabetes, hematologic malignancy, cancer, severe renal disease, HIV infection, smoking, male gender, and a recent hotel stay or ship cruise. (Many of these risk factors would now reclassify as HCAP some cases that were previously designated CAP.)

1	CAP can vary from indolent to fulminant in presentation and from mild to fatal in severity. Manifestations of progression and severity include both constitutional findings and those limited to the lung and associated structures. In light of the pathobiology of the disease, many of the findings are to be expected. The patient is frequently febrile with tachycardia or may have a history of chills and/or sweats. Cough may be either nonproductive or productive of mucoid, purulent, or blood-tinged sputum. Gross hemoptysis is suggestive of CA-MRSA pneumonia. Depending on severity, the patient may be able to speak in full sentences or may be very short of breath. If the pleura is involved, the patient may experience pleuritic chest pain. Up to 20% of patients may have gastrointestinal symptoms such as nausea, vomiting, and/or diarrhea. Other symptoms may include fatigue, headache, myalgias, and arthralgias.

1	Findings on physical examination vary with the degree of pulmonary consolidation and the presence or absence of a significant pleural effusion. An increased respiratory rate and use of accessory muscles of respiration are common. Palpation may reveal increased or decreased tactile fremitus, and the percussion note can vary from dull to flat, reflecting underlying consolidated lung and pleural fluid, respectively. Crackles, bronchial breath sounds, and possibly a pleural friction rub may be heard on auscultation. The clinical presentation may not be so obvious in the elderly, who may initially display new-onset or worsening confusion and few other manifestations. Severely ill patients may have septic shock and evidence of organ failure.

1	When confronted with possible CAP, the physician must ask two questions: Is this pneumonia, and, if so, what is the likely etiology? The former question is typically answered by clinical and radiographic methods, whereas the latter requires the aid of laboratory techniques. Clinical Diagnosis The differential diagnosis includes both infectious and noninfectious entities such as acute bronchitis, acute exacerbations of chronic bronchitis, heart failure, pulmonary embolism, hypersensitivity pneumonitis, and radiation pneumonitis. The importance of a careful history cannot be overemphasized. For example, known cardiac disease may suggest worsening pulmonary edema, while underlying carcinoma may suggest lung injury secondary to irradiation.

1	Unfortunately, the sensitivity and specificity of the findings on physical examination are less than ideal, averaging 58% and 67%, respectively. Therefore, chest radiography is often necessary to differentiate CAP from other conditions. Radiographic findings may include risk factors for increased severity (e.g., cavitation or multilobar involvement). Occasionally, radiographic results suggest an etiologic diagnosis. For example, pneumatoceles suggest infection with S. aureus, and an upper-lobe cavitating lesion suggests tuberculosis. CT may be of value in a patient with suspected postobstructive 806 pneumonia caused by a tumor or foreign body or suspected cavitary disease. For outpatients, the clinical and radiologic assessments are usually all that is done before treatment for CAP is started since most laboratory results are not available soon enough to influence initial management significantly. In certain cases, the availability of rapid point-of-care outpatient diagnostic tests

1	since most laboratory results are not available soon enough to influence initial management significantly. In certain cases, the availability of rapid point-of-care outpatient diagnostic tests can be very important; for example, rapid diagnosis of influenza virus infection can prompt specific anti-influenza drug treatment and secondary prevention.

1	Etiologic Diagnosis The etiology of pneumonia usually cannot be determined solely on the basis of clinical presentation. Except for CAP patients admitted to the ICU, no data exist to show that treatment directed at a specific pathogen is statistically superior to empirical therapy. The benefit of establishing a microbial etiology can therefore be questioned, particularly in light of the cost of diagnostic testing. However, a number of reasons can be advanced for attempting an etiologic diagnosis. Identification of an unexpected pathogen allows narrowing of the initial empirical regimen, thereby decreasing antibiotic selection pressure and lessening the risk of resistance. Pathogens with important public safety implications, such as Mycobacterium tuberculosis and influenza virus, may be found in some cases. Finally, without culture and susceptibility data, trends in resistance cannot be followed accurately, and appropriate empirical therapeutic regimens are harder to devise.

1	GRAM’S STAIN AND CULTURE OF SPUTUM The main purpose of the sputum Gram’s stain is to ensure that a sample is suitable for culture. However, Gram’s staining may also identify certain pathogens (e.g., S. pneumoniae, S. aureus, and gram-negative bacteria) by their characteristic appearance. To be adequate for culture, a sputum sample must have >25 neutrophils and <10 squamous epithelial cells per low-power field. The sensitivity and specificity of the sputum Gram’s stain and culture are highly variable. Even in cases of proven bacteremic pneumococcal pneumonia, the yield of positive cultures from sputum samples is ≤50%.

1	Some patients, particularly elderly individuals, may not be able to produce an appropriate expectorated sputum sample. Others may already have started a course of antibiotics that can interfere with culture results at the time a sample is obtained. Inability to produce sputum can be a consequence of dehydration, and the correction of this condition may result in increased sputum production and a more obvious infiltrate on chest radiography. For patients admitted to the ICU and intubated, a deep-suction aspirate or bronchoalveolar lavage sample (obtained either via bronchoscopy or non-bronchoscopically) has a high yield on culture when sent to the microbiology laboratory as soon as possible. Since the etiologies in severe CAP are somewhat different from those in milder disease (Table 153-2), the greatest benefit of staining and culturing respiratory secretions is to alert the physician of unsuspected and/or resistant pathogens and to permit appropriate modification of therapy. Other

1	the greatest benefit of staining and culturing respiratory secretions is to alert the physician of unsuspected and/or resistant pathogens and to permit appropriate modification of therapy. Other stains and cultures (e.g., specific stains for M. tuberculosis or fungi) may be useful as well.

1	BLOOD CULTURES The yield from blood cultures, even when samples are collected before antibiotic therapy, is disappointingly low. Only 5–14% of cultures of blood from patients hospitalized with CAP are positive, and the most frequently isolated pathogen is S. pneumoniae. Since recommended empirical regimens all provide pneumococcal coverage, a blood culture positive for this pathogen has little, if any, effect on clinical outcome. However, susceptibility data may allow narrowing of antibiotic therapy in appropriate cases. Because of the low yield and the lack of significant impact on outcome, blood cultures are no longer considered de rigueur for all hospitalized CAP patients. Certain high-risk patients—including those with neutropenia secondary to pneumonia, asplenia, complement deficiencies, chronic liver disease, or severe CAP—should have blood cultured.

1	URINARY ANTIGEN TESTS Two commercially available tests detect pneumococcal and Legionella antigen in urine. The test for Legionella pneumophila detects only serogroup 1, but this serogroup accounts for most community-acquired cases of Legionnaires’ disease in the United States. The sensitivity and specificity of the Legionella urine antigen test are as high as 90% and 99%, respectively. The pneumococcal urine antigen test also is quite sensitive and specific (80% and >90%, respectively). Although false-positive results can be obtained with samples from pneumococcus-colonized children, the test is generally reliable. Both tests can detect antigen even after the initiation of appropriate antibiotic therapy.

1	POLYMERASE CHAIN REACTION Polymerase chain reaction (PCR) tests, which amplify a microorganism’s DNA or RNA, are available for a number of pathogens. PCR of nasopharyngeal swabs has become the standard for diagnosis of respiratory viral infection. In addition, PCR can detect the nucleic acid of Legionella species, M. pneumoniae, C. pneumoniae, and mycobacteria. In patients with pneumococcal pneumonia, an increased bacterial load in whole blood documented by PCR is associated with an increased risk of septic shock, the need for mechanical ventilation, and death. Clinical availability of such a test could conceivably help identify patients suitable for ICU admission.

1	SEROLOGY A fourfold rise in specific IgM antibody titer between acuteand convalescent-phase serum samples is generally considered diagnostic of infection with the pathogen in question. In the past, serologic tests were used to help identify atypical pathogens as well as selected unusual organisms such as Coxiella burnetii. Recently, however, they have fallen out of favor because of the time required to obtain a final result for the convalescent-phase sample.

1	BIOMARKERS A number of substances can serve as markers of severe inflammation. The two currently in use are C-reactive protein (CRP) and procalcitonin (PCT). Levels of these acute-phase reactants increase in the presence of an inflammatory response, particularly to bacterial pathogens. CRP may be of use in the identification of worsening disease or treatment failure, and PCT may play a role in determining the need for antibacterial therapy. These tests should not be used on their own but, when interpreted in conjunction with other findings from the history, physical examination, radiology, and laboratory tests, may help with antibiotic stewardship and appropriate management of seriously ill patients with CAP.

1	The cost of inpatient management exceeds that of outpatient treatment by a factor of 20, and hospitalization accounts for most CAP-related expenditures. Thus the decision to admit a patient with CAP to the hospital has considerable implications. Certain patients clearly can be managed at home, and others clearly require treatment in the hospital, but the choice is sometimes difficult. Tools that objectively assess the risk of adverse outcomes, including severe illness and death, can minimize unnecessary hospital admissions. There are currently two sets of criteria: the Pneumonia Severity Index (PSI), a prognostic model used to identify patients at low risk of dying; and the CURB-65 criteria, a severity-of-illness score.

1	To determine the PSI, points are given for 20 variables, including age, coexisting illness, and abnormal physical and laboratory findings. On the basis of the resulting score, patients are assigned to one of five classes with the following mortality rates: class 1, 0.1%; class 2, 0.6%; class 3, 2.8%; class 4, 8.2%; and class 5, 29.2%. Determination of the PSI is often impractical in a busy emergency-department setting because of the number of variables that must be assessed. However, clinical trials demonstrate that routine use of the PSI results in lower admission rates for class 1 and class 2 patients. Patients in class 3 could ideally be admitted to an observation unit until a further decision can be made.

1	The CURB-65 criteria include five variables: confusion (C); urea >7 mmol/L (U); respiratory rate ≥30/min (R); blood pressure, systolic ≤90 mmHg or diastolic ≤60 mmHg (B); and age ≥65 years. Patients with a score of 0, among whom the 30-day mortality rate is 1.5%, can be treated outside the hospital. With a score of 2, the 30-day mortality rate is 9.2%, and patients should be admitted to the hospital. Thrombocytopenia Severe acidosis (pH <7.30) Among patients with scores of ≥3, mortality rates are 22% overall; these patients may require ICU admission. It is not clear which assessment tool is superior. Whichever system is used, these objective criteria must always be tempered by careful consideration of factors relevant to individual patients, including the ability to comply reliably with an oral antibiotic regimen and the resources available to the patient outside the hospital.

1	Neither PSI nor CURB-65 is accurate in determining the need for ICU admission. Septic shock or respiratory failure in the emergency department is an obvious indication for ICU care. However, mortality rates are higher among less ill patients who are admitted to the floor and then deteriorate than among equally ill patients monitored in the ICU. A variety of scores have been proposed to identify patients most likely to have early deterioration (Table 153-4). Most factors in these scores are similar to the minor severity criteria proposed by the Infectious Diseases Society of America (IDSA) and the American Thoracic Society (ATS) in their guidelines for the management of CAP.

1	Antimicrobial resistance is a significant problem that threatens to diminish our therapeutic armamentarium. Misuse of antibiotics results in increased antibiotic selection pressure that can affect resistance locally or even globally by clonal dissemination. For CAP, the main resistance issues currently involve S. pneumoniae and CA-MRSA. S. pneumoniae In general, pneumococcal resistance is acquired (1) by direct DNA incorporation and remodeling resulting from contact with closely related oral commensal bacteria, (2) by the process of natural transformation, or (3) by mutation of certain genes.

1	The minimal inhibitory concentration (MIC) cutoffs for penicillin in pneumonia are ≤2 μg/mL for susceptibility, >2–4 μg/mL for intermediate, and ≥8 μg/mL for resistant. A change in susceptibility thresholds resulted in a dramatic decrease in the proportion of pneumococcal isolates considered nonsusceptible. For meningitis, MIC thresholds remain at the former higher levels. Fortunately, resistance to penicillin appeared to plateau even before the change in MIC thresholds. Pneumococcal resistance to β-lactam drugs is due solely to low-affinity penicillin-binding proteins. Risk factors for penicillin-resistant pneumococcal infection include recent antimicrobial therapy, an age of <2 years or >65 years, attendance at day-care centers, recent hospitalization, and HIV infection.

1	In contrast to penicillin resistance, resistance to macrolides is increasing through several mechanisms. Target-site modifica tion caused by ribosomal methylation in 23S rRNA encoded by the ermB gene results in high-level resistance (MICs, ≥64 μg/mL) to macrolides, lincosamides, and streptogramin B–type antibiotics. The efflux mechanism encoded by the mef gene (M phenotype) is usually associated with low-level resistance (MICs, 1–32 μg/mL). These two mechanisms account for ∼45% and ∼65%, respectively, of resistant pneumococcal isolates in the United States. High-level resistance to macrolides is more common in Europe, whereas lower-level resistance predominates in North America.

1	Pneumococcal resistance to fluoroquinolones (e.g., ciprofloxacin and levofloxacin) has been reported. Changes can occur in one or both target sites (topoisomerases II and IV) from mutations in the gyrA and parC genes, respectively. In addition, an efflux pump may play a role in pneumococcal resistance to fluoroquinolones. Isolates resistant to drugs from three or more antimicrobial 807 classes with different mechanisms of action are considered MDR strains. The propensity for an association of pneumococcal resistance to penicillin with reduced susceptibility to other drugs, such as macrolides, tetracyclines, and trimethoprim-sulfamethoxazole, also is of concern. In the United States, 58.9% of penicillin-resistant pneumococcal isolates from blood are also resistant to macrolides.

1	The most important risk factor for antibiotic-resistant pneumococcal infection is use of a specific antibiotic within the previous 3 months. Therefore, a patient’s history of prior antibiotic treatment is a critical factor in avoiding the use of an inappropriate antibiotic. CA-MRSA CAP due to MRSA may be caused by the classic hospital-acquired strains or by the more recently identified, genotypically and phenotypically distinct community-acquired strains. Most infections with the former strains have been acquired either directly or indirectly by contact with the health care environment and would now be classified as HCAP. However, in some hospitals, the CA-MRSA strains are displacing the classic hospital-acquired strains—a trend suggesting that the newer strains may be more robust and blurring this distinction.

1	Methicillin resistance in S. aureus is determined by the mecA gene, which encodes for resistance to all β-lactam drugs. At least five staphylococcal chromosomal cassette mec (SCCmec) types have been described. The typical hospital-acquired strain usually has type II or III, whereas CA-MRSA has a type IV SCCmec element. CA-MRSA isolates tend to be less resistant than the older hospital-acquired strains and are often susceptible to trimethoprimsulfamethoxazole, clindamycin, and tetracycline in addition to vancomycin and linezolid. However, the most important distinction is that CA-MRSA strains also carry genes for superantigens, such as enterotoxins B and C and Panton-Valentine leukocidin, a membrane-tropic toxin that can create cytolytic pores in polymorphonuclear neutrophils, monocytes, and macrophages.

1	Gram-Negative Bacilli A detailed discussion of resistance among gram-negative bacilli is beyond the scope of this chapter (Chap. 186). Fluoroquinolone resistance among isolates of Escherichia coli from the community appears to be increasing. Enterobacter species are typically resistant to cephalosporins; the drugs of choice for use against these bacteria are usually fluoroquinolones or carbapenems. Similarly, when infections due to bacteria producing extended-spectrum β-lactamases are documented or suspected, a fluoroquinolone or a carbapenem should be used; these MDR strains are more likely to be involved in HCAP.

1	Since the etiology of CAP is rarely known at the outset of treatment, initial therapy is usually empirical, designed to cover the most likely pathogens (Table 153-5). In all cases, antibiotic treatment should be initiated as expeditiously as possible. The CAP treatment guidelines in the United States (summarized in Table 153-5) represent joint statements from the IDSA and the ATS; the Canadian guidelines come from the Canadian Infectious Disease Society and the Canadian Thoracic Society. In all these guidelines, coverage is always provided for the pneumococcus and the atypical pathogens. In contrast, guidelines from some European countries do not always include atypical coverage based on local epidemiologic data. The U.S./Canadian approach is supported by retrospective data derived from administrative databases including thousands of patients. Atypical pathogen coverage provided by the addition of a macrolide to a cephalosporin or by the use of a fluoroquinolone alone has been

1	from administrative databases including thousands of patients. Atypical pathogen coverage provided by the addition of a macrolide to a cephalosporin or by the use of a fluoroquinolone alone has been consistently associated with a significant reduction in mortality rates compared with those for β-lactam coverage alone.

1	Therapy with a macrolide or a fluoroquinolone within the previous 3 months is associated with an increased likelihood of infection with a resistant strain of S. pneumoniae. For this reason, a fluoroquinolone-based regimen should be used for patients recently given a macrolide, and vice versa (Table 153-5). 1. Previously healthy and no antibiotics in past 3 months PO once, then 250 mg qd]) or 2. Comorbidities or antibiotics in past 3 months: select an alternative from a different class • A respiratory fluoroquinolone (moxifloxacin [400 mg PO qd], gemifloxacin [320 mg PO qd], levofloxacin [750 mg PO qd]) or • A β-lactam (preferred: high-dose amoxicillin [1 g tid] or amoxicillin/ clavulanate [2 g bid]; alternatives: ceftriaxone [1–2 g IV qd], cefpodoxime [200 mg PO bid], cefuroxime [500 mg PO bid]) plus a macrolidea 3. In regions with a high rate of “high-level” pneumococcal macrolide resistance,b consider alternatives listed above for patients with comorbidities.

1	Inpatients, Non-ICU • A respiratory fluoroquinolone (e.g., moxifloxacin [400 mg PO or IV qd] or • A β-lactamc (e.g., ceftriaxone [1–2 g IV qd], ampicillin [1–2 g IV q4–6h], cefotaxime [1–2 g IV q8h], ertapenem [1 g IV qd]) plus a macrolided (e.g., oral clarithromycin or azithromycin [as listed above] or IV azithromycin [1 g once, then 500 mg qd]) Inpatients, ICU • A β-lactame (e.g., ceftriaxone [2 g IV qd], ampicillin-sulbactam [2 g IV q8h], or cefotaxime [1–2 g IV q8h]) plus either azithromycin or a fluoroquinolone (as listed above for inpatients, non-ICU) If Pseudomonas is a consideration: • An antipseudomonal β-lactam (e.g., piperacillin/tazobactam [4. 5 g IV q6h], cefepime [1–2 g IV q12h], imipenem [500 mg IV q6h], meropenem [1 g IV q8h]) plus either ciprofloxacin (400 mg IV q12h) or levofloxacin (750 mg IV qd) • The above β-lactams plus an aminoglycoside (amikacin [15 mg/kg qd] or tobramycin [1. 7 mg/kg qd]) plus azithromycin • The

1	If CA-MRSA is a consideration: • Add linezolid (600 mg IV q12h) or vancomycin (15 mg/kg q12h initially, aDoxycycline (100 mg PO bid) is an alternative to the macrolide. bMICs of >16 μg/mL in 25% of isolates. cA respiratory fluoroquinolone should be used for penicillin-allergic patients. dDoxycycline (100 mg IV q12h) is an alternative to the macrolide. eFor penicillin-allergic patients, use a respiratory fluoroquinolone and aztreonam (2 g IV q8h). fFor penicillin-allergic patients, substitute aztreonam. Abbreviations: CA-MRSA, community-acquired methicillin-resistant Staphylococcus aureus; ICU, intensive care unit. Once the etiologic agent(s) and susceptibilities are known, therapy may be altered to target the specific pathogen(s). However, this decision is not always straightforward. If blood cultures yield

1	Once the etiologic agent(s) and susceptibilities are known, therapy may be altered to target the specific pathogen(s). However, this decision is not always straightforward. If blood cultures yield S. pneumoniae sensitive to penicillin after 2 days of treatment with a macrolide plus a β-lactam or with a fluoroquinolone alone, should therapy be switched to penicillin alone? The concern here is that a β-lactam alone would not be effective in the potential 15% of cases with atypical co-infection. No standard approach exists. In all cases, the individual patient and the various risk factors must be considered.

1	Management of bacteremic pneumococcal pneumonia also is controversial. Data from nonrandomized studies suggest that combination therapy (especially macrolide/β-lactam) is associated with a lower mortality rate than monotherapy, particularly in severely ill patients. The exact reason is unknown, but possible explanations include an additive or synergistic antibacterial effect, antimicrobial tolerance, atypical co-infection, or the immunomodulatory effects of the macrolides. For CAP patients admitted to the ICU, the risk of infection with

1	For CAP patients admitted to the ICU, the risk of infection with P. aeruginosa or CA-MRSA is increased. Empirical coverage should be considered when a patient has risk factors or a Gram’s stain suggestive of these pathogens (Table 153–5). If CA-MRSA is suspected, either linezolid or vancomycin can be added to the initial empirical regimen; however, there is increasing concern about vancomycin’s loss of potency against MRSA, poor penetration into epithelial lining fluid, and lack of effect on toxin production relative to linezolid. Although hospitalized patients have traditionally received initial therapy by the IV route, some drugs—particularly the fluoroquinolones—are very well absorbed and can be given orally from the outset to select patients. For patients initially treated IV, a switch to oral treatment is appropriate as long as the patient can ingest and absorb the drugs, is hemodynamically stable, and is showing clinical improvement.

1	The duration of treatment for CAP has generated considerable interest. Patients were previously treated for 10–14 days, but studies with fluoroquinolones and telithromycin suggest that a 5-day course is sufficient for otherwise uncomplicated CAP. Even a single dose of ceftriaxone has been associated with a significant cure rate. A longer course may be required for patients with bacteremia, metastatic infection, or infection with a virulent pathogen such as P. aeruginosa or CA-MRSA.

1	In addition to appropriate antimicrobial therapy, certain general considerations apply in dealing with CAP, HCAP, or HAP/VAP. Adequate hydration, oxygen therapy for hypoxemia, and assisted ventilation when necessary are critical to successful treatment. Patients with severe CAP who remain hypotensive despite fluid resuscitation may have adrenal insufficiency and may respond to glucocorticoid treatment. The value of adjunctive therapy, such as glucocorticoids, statins, and angiotensin-converting enzyme inhibitors, remains unproven in the management of CAP.

1	Failure to Improve Patients slow to respond to therapy should be reevaluated at about day 3 (sooner if their condition is worsening rather than simply not improving), and several possible scenarios should be considered. A number of noninfectious conditions mimic pneumonia, including pulmonary edema, pulmonary embolism, lung carcinoma, radiation and hypersensitivity pneumonitis, and connective tissue disease involving the lungs. If the patient truly has CAP and empirical treatment is aimed at the correct pathogen, lack of response may be explained in a number of ways. The pathogen may be resistant to the drug selected, or a sequestered focus (e.g., lung abscess or empyema) may be blocking access of the antibiotic(s) to the pathogen. The patient may be getting either the wrong drug or the correct drug at the wrong dose or frequency of administration. Another possibility is that CAP is the correct diagnosis but an unsuspected pathogen (e.g., CA-MRSA, M. tuberculosis, or a fungus) is the

1	drug at the wrong dose or frequency of administration. Another possibility is that CAP is the correct diagnosis but an unsuspected pathogen (e.g., CA-MRSA, M. tuberculosis, or a fungus) is the cause. Nosocomial superinfections—both pulmonary and extrapulmonary—are other possible explanations for a patient’s failure to improve or deterioration. In all cases of delayed response or deteriorating condition, the patient must be carefully reassessed and appropriate studies initiated, possibly including such diverse procedures as CT or bronchoscopy.

1	Complications As in other severe infections, common complications of severe CAP include respiratory failure, shock and multiorgan failure, coagulopathy, and exacerbation of comorbid illnesses. Three particularly noteworthy conditions are metastatic infection, lung abscess, and complicated pleural effusion. Metastatic infection (e.g., brain abscess or endocarditis) is very unusual and will require a high degree of suspicion and a detailed workup for proper treatment. Lung abscess may occur in association with aspiration or with infection caused by a single CAP pathogen, such as CA-MRSA,

1	P. aeruginosa, or (rarely) S. pneumoniae. Aspiration pneumonia is typically a polymicrobial infection involving both aerobes and anaerobes. A significant pleural effusion should be tapped for both diagnostic and therapeutic purposes. If the fluid has a pH of <7, a glucose level of <2. 2 mmol/L, and a lactate dehydrogenase concentration of >1000 U/L or if bacteria are seen or cultured, then it should be completely drained; a chest tube is often required and video-assisted thoracoscopy may be needed for late treatment or difficult cases.

1	Follow-Up Fever and leukocytosis usually resolve within 2–4 days in otherwise healthy patients with CAP, but physical findings may persist longer. Chest radiographic abnormalities are slowest to resolve (4–12 weeks), with the speed of clearance depending on the patient’s age and underlying lung disease. Patients may be discharged from the hospital once their clinical conditions, including comorbidities, are stable. The site of residence after discharge (nursing home, home with family, home alone) is an important discharge timing consideration, particularly for elderly patients. For a hospitalized patient, a follow-up radiograph ∼4–6 weeks later is recommended. If relapse or recurrence is documented, particularly in the same lung segment, the possibility of an underlying neoplasm must be considered.

1	The prognosis of CAP depends on the patient’s age, comorbidities, and site of treatment (inpatient or outpatient). Young patients without comorbidity do well and usually recover fully after ~2 weeks. Older patients and those with comorbid conditions can take several weeks longer to recover fully. The overall mortality rate for the outpatient group is <1%. For patients requiring hospitalization, the overall mortality rate is estimated at 10%, with ~50% of deaths directly attributable to pneumonia. The main preventive measure is vaccination (Chap. 148). Recommendations of the Advisory Committee on Immunization Practices should be followed for influenza and pneumococcal vaccines.

1	A pneumococcal polysaccharide vaccine (PPV23) and a protein conjugate pneumococcal vaccine (PCV13) are available in the United States. The former product contains capsular material from 23 pneumococcal serotypes; in the latter, capsular polysaccharide from 13 of the most frequent pneumococcal pathogens affecting children is linked to an immunogenic protein. PCV13 produces T cell–dependent antigens that result in long-term immunologic memory. Administration of this vaccine to children has led to an overall decrease in the prevalence of antimicrobial-resistant pneumococci and in the incidence of invasive pneumococcal disease among both children and adults. However, vaccination can be followed by the replacement of vaccine serotypes with nonvaccine serotypes, as was seen with serotypes 19A and 35B after introduction of the original 7-valent conjugate vaccine. PCV13 now is also recommended for the elderly and for younger immunocompromised patients. Because of an increased risk of

1	19A and 35B after introduction of the original 7-valent conjugate vaccine. PCV13 now is also recommended for the elderly and for younger immunocompromised patients. Because of an increased risk of pneumococcal infection, even among patients without obstructive lung disease, smokers should be strongly encouraged to stop smoking.

1	Two forms of influenza vaccine are available: intramuscular inactivated vaccine and intranasal live-attenuated cold-adapted vaccine. The latter is contraindicated in immunocompromised patients. In the event of an influenza outbreak, unprotected patients at risk from complications should be vaccinated immediately and given chemoprophylaxis with either oseltamivir or zanamivir for 2 weeks—i.e., until vaccine-induced antibody levels are sufficiently high. HCAP represents a transition between classic CAP and typical HAP. The definition of HCAP is still in some flux because of a lack of consistent large-scale studies. Several early studies were limited to patients with culture-positive pneumonia. In these studies, the incidence of MDR pathogens in HCAP was as high as or higher than in HAP/VAP. MRSA in particular was more common in HCAP than in traditional 809 HAP/VAP. Conversely, prospective studies in nontertiary-care centers have found a low incidence of MDR pathogens in HCAP.

1	The patients at greatest risk for HCAP are not well defined. Patients from nursing homes are not always at elevated risk for infection with MDR pathogens. Careful evaluation of nursing home residents with pneumonia suggests that their risk of MDR infection is low if they have not recently received antibiotics and are independent in most activities of daily living. Recent hospitalization (i.e., in the preceding 90 days) is also a major risk factor for infection with MDR pathogens. Conversely, nursing home patients are at increased risk of infection with influenza virus and other atypical pneumonia pathogens. Undue concern about MDR pathogens occasionally results in failure to cover atypical pathogens when treating nursing home patients. In addition, patients receiving home infusion therapy or undergoing chronic dialysis are probably at particular risk for MRSA pneumonia but may not be at greater risk for infection with Pseudomonas or Acinetobacter than are other patients who develop

1	or undergoing chronic dialysis are probably at particular risk for MRSA pneumonia but may not be at greater risk for infection with Pseudomonas or Acinetobacter than are other patients who develop CAP.

1	In general, the management of HCAP due to MDR pathogens is similar to that of MDR HAP/VAP. This topic will therefore be covered in subsequent sections on HAP and VAP. The prognosis of HCAP is intermediate between that of CAP and VAP and is closer to that of HAP. Most hospital-acquired pneumonia research has focused on VAP. However, the information and principles based on this research can be applied to non-ICU HAP and HCAP as well. The greatest difference between VAP and HCAP/HAP studies is the dependence on expectorated sputum for a microbiologic diagnosis of VAP (as for that of CAP), which is further complicated by frequent colonization by pathogens in patients with HAP or HCAP. Therefore, most of the literature has focused on HCAP or HAP resulting in intubation, where, once again, access to the lower respiratory tract facilitates an etiologic diagnosis.

1	Etiology Potential etiologic agents of VAP include both MDR and non-MDR bacterial pathogens (Table 153-6). The non-MDR group is nearly identical to the pathogens found in severe CAP (Table 153-2); it is not surprising that such pathogens predominate if VAP develops in the first 5–7 days of the hospital stay. However, if patients have other risk factors for HCAP, MDR pathogens are a consideration, even early in the hospital course. The relative frequency of individual MDR pathogens can vary significantly from hospital to hospital and even between different critical care units within the same institution. Most hospitals have problems with P. aeruginosa and MRSA, but other MDR pathogens are often institution-specific. Less commonly, fungal and viral pathogens cause VAP, usually affecting severely immunocompromised patients. Rarely, community-associated viruses cause mini-epidemics, usually when introduced by ill health care workers.

1	Streptococcus pneumoniae Pseudomonas aeruginosa Other Streptococcus spp. MRSA Haemophilus influenzae Acinetobacter spp. MSSA Antibiotic-resistant Antibiotic-sensitive Enterobacteriaceae Enterobacteriaceae Enterobacter spp. Escherichia coli ESBL-positive strains Klebsiella pneumoniae Klebsiella spp. Proteus spp. Legionella pneumophila Enterobacter spp. Burkholderia cepacia Serratia marcescens Aspergillus spp. Abbreviations: ESBL, extended-spectrum β-lactamase; MDR, multidrug-resistant; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-sensitive S. aureus.

1	810 Epidemiology Pneumonia is a common complication among patients requiring mechanical ventilation. Prevalence estimates vary between 6 and 52 cases per 100 patients, depending on the population studied. On any given day in the ICU, an average of 10% of patients will have pneumonia—VAP in the overwhelming majority of cases. The frequency of diagnosis is not static but changes with the duration of mechanical ventilation, with the highest hazard ratio in the first 5 days and a plateau in additional cases (1% per day) after ~2 weeks. However, the cumulative rate among patients who remain ventilated for as long as 30 days is as high as 70%. These rates often do not reflect the recurrence of VAP in the same patient. Once a ventilated patient is transferred to a chronic-care facility or to home, the incidence of pneumonia drops significantly, especially in the absence of other risk factors for pneumonia. However, in chronic ventilator units, purulent tracheobronchitis becomes a significant

1	the incidence of pneumonia drops significantly, especially in the absence of other risk factors for pneumonia. However, in chronic ventilator units, purulent tracheobronchitis becomes a significant issue, often interfering with efforts to wean patients off mechanical ventilation (Chap. 323). Three factors are critical in the pathogenesis of VAP: colonization of the oropharynx with pathogenic microorganisms, aspiration of these organisms from the oropharynx into the lower respiratory tract, and compromise of the normal host defense mechanisms. Most risk factors and their corresponding prevention strategies pertain to one of these three factors (Table 153-7). The most obvious risk factor is the endotracheal tube, which bypasses the normal mechanical factors preventing aspiration. While the presence of an endotracheal tube may prevent large-volume aspiration, microaspiration is actually exacerbated by secretions pooling above the cuff. The endotracheal tube and the concomitant need

1	Oropharyngeal colonization with pathogenic bacteria Elimination of normal flora Avoidance of prolonged antibiotic courses Large-volume oropharyngeal Short course of prophylactic aspiration around time of antibiotics for comatose patientsa intubation Gastroesophageal reflux Postpyloric enteral feedingb; avoidance of high gastric residuals, prokinetic agents Bacterial overgrowth of stomach Avoidance of prophylactic agents that raise gastric pHb; selective decontamination of digestive tract with nonabsorbable antibioticsb Cross-infection from other colonized Hand washing, especially with patients alcohol-based hand rub; intensive infection control educationa; isolation; proper cleaning of reusable equipment Large-volume aspiration Endotracheal intubation; rapid-sequence intubation technique; avoidance of sedation; decompression of small-bowel obstruction Prolonged duration of ventilation Daily awakening from sedation,a weaning protocolsa

1	Prolonged duration of ventilation Daily awakening from sedation,a weaning protocolsa Secretions pooled above Head of bed elevateda; continuous endotracheal tube aspiration of subglottic secretions with specialized endotracheal tubea; avoidance of reintubation; minimization of sedation and patient transport Altered lower respiratory host Tight glycemic controlb; lowering of defenses hemoglobin transfusion threshold aStrategies demonstrated to be effective in at least one randomized controlled trial. bStrategies with negative randomized trials or conflicting results. for suctioning can damage the tracheal mucosa, thereby facilitating tracheal colonization. In addition, pathogenic bacteria can form a glycocalyx biofilm on the tube’s surface that protects them from both antibiotics and host defenses. The bacteria can also be dislodged during suctioning and can reinoculate the trachea, or tiny fragments of glycocalyx can embolize to distal airways, carrying bacteria with them.

1	In a high percentage of critically ill patients, the normal oropharyngeal flora is replaced by pathogenic microorganisms. The most important risk factors are antibiotic selection pressure, cross-infection from other infected/colonized patients or contaminated equipment, and malnutrition. Of these factors, antibiotic exposure poses the greatest risk by far. Pathogens such as P. aeruginosa almost never cause infection in patients without prior exposure to antibiotics. The recent emphasis on hand hygiene has lowered the cross-infection rate.

1	How the lower respiratory tract defenses become overwhelmed remains poorly understood. Almost all intubated patients experience microaspiration and are at least transiently colonized with pathogenic bacteria. However, only around one-third of colonized patients develop VAP. Colony counts increase to high levels, sometimes days before the development of clinical pneumonia; these increases suggest that the final step in VAP development, independent of aspiration and oropharyngeal colonization, is the overwhelming of host defenses. Severely ill patients with sepsis and trauma appear to enter a state of immunoparalysis several days after admission to the ICU—a time that corresponds to the greatest risk of developing VAP. The mechanism of this immunosuppression is not clear, although several factors have been suggested. Hyperglycemia affects neutrophil function, and trials suggest that keeping the blood sugar level close to normal with exogenous insulin may have beneficial effects,

1	factors have been suggested. Hyperglycemia affects neutrophil function, and trials suggest that keeping the blood sugar level close to normal with exogenous insulin may have beneficial effects, including a decreased risk of infection. More frequent transfusions also adversely affect the immune response.

1	Clinical Manifestations The clinical manifestations are generally the same in VAP as in all other forms of pneumonia: fever, leukocytosis, increase in respiratory secretions, and pulmonary consolidation on physical examination, along with a new or changing radiographic infiltrate. The frequency of abnormal chest radiographs before the onset of pneumonia in intubated patients and the limitations of portable radiographic technique make interpretation of radiographs more difficult than in patients who are not intubated. Other clinical features may include tachypnea, tachycardia, worsening oxygenation, and increased minute ventilation. Diagnosis No single set of criteria is reliably diagnostic of pneumonia in a ventilated patient. The inability to identify such patients compromises efforts to prevent and treat VAP and even calls into question estimates of the impact of VAP on mortality rates.

1	Application of clinical criteria consistently results in overdiagnosis of VAP, largely because of three common findings in at-risk patients: (1) tracheal colonization with pathogenic bacteria in patients with endotracheal tubes, (2) multiple alternative causes of radiographic infiltrates in mechanically ventilated patients, and (3) the high frequency of other sources of fever in critically ill patients. The differential diagnosis of VAP includes a number of entities such as atypical pulmonary edema, pulmonary contusion, alveolar hemorrhage, hypersensitivity pneumonitis, acute respiratory distress syndrome, and pulmonary embolism. Clinical findings in ventilated patients with fever and/or leukocytosis may have alternative causes, including antibiotic-associated diarrhea, sinusitis, urinary tract infection, pancreatitis, and drug fever. Conditions mimicking pneumonia are often documented in patients in whom VAP has been ruled out by accurate diagnostic techniques. Most of these

1	urinary tract infection, pancreatitis, and drug fever. Conditions mimicking pneumonia are often documented in patients in whom VAP has been ruled out by accurate diagnostic techniques. Most of these alternative diagnoses do not require antibiotic treatment; require antibiotics different from those used to treat VAP; or require some additional intervention, such as surgical drainage or catheter removal, for optimal management.

1	This diagnostic dilemma has led to debate and controversy. The major question is whether a quantitative-culture approach as a means of eliminating false-positive clinical diagnoses is superior to the clinical approach enhanced by principles learned from quantitative-culture studies. The most recent IDSA/ATS guidelines for HAP/VAP suggest that either approach is clinically valid.

1	Quantitative-culture approacH The essence of the quantitative-culture approach is to discriminate between colonization and true infection by determining the bacterial burden. The more distal in the respiratory tree the diagnostic sampling, the more specific the results and therefore the lower the threshold of growth necessary to diagnose pneumonia and exclude colonization. For example, a quantitative endotracheal aspirate yields proximate samples, and the diagnostic threshold is 106 cfu/mL. The protected specimen brush method, in contrast, obtains distal samples and has a threshold of 103 cfu/mL. Conversely, sensitivity declines as more distal secretions are obtained, especially when they are collected blindly (i.e., by a technique other than bronchoscopy). Additional tests that may increase the diagnostic yield include Gram’s staining, differential cell counts, staining for intracellular organisms, and detection of local protein levels elevated in response to infection.

1	The Achilles heel of the quantitative approach is the effect of antibiotic therapy. With sensitive microorganisms, a single antibiotic dose can reduce colony counts below the diagnostic threshold. Recent changes in antibiotic therapy are the most significant. After 3 days, the operating characteristics of the tests are almost the same as if no antibiotic therapy has been given. Conversely, colony counts above the diagnostic threshold during antibiotic therapy suggest that the current antibiotics are ineffective. Even the normal host response may be sufficient to reduce quantitative-culture counts below the diagnostic threshold if sampling is delayed. In short, expertise in quantitative-culture techniques is critical, with a specimen obtained as soon as pneumonia is suspected and before antibiotic therapy is initiated or changed.

1	In a study comparing the quantitative with the clinical approach, use of bronchoscopic quantitative cultures resulted in significantly less antibiotic use at 14 days after study entry and in lower rates of mortality and severity-adjusted mortality at 28 days. In addition, more alternative sites of infection were found in patients randomized to the quantitative-culture strategy. A critical aspect of this study was that antibiotic treatment was initiated only in patients whose gram-stained respiratory sample was positive or who displayed signs of hemodynamic instability. Fewer than one-half as many patients were treated for pneumonia in the bronchoscopy group, and only one-third as many microorganisms were cultured.

1	clinical approacH The lack of specificity of a clinical diagnosis of VAP has led to efforts to improve the diagnostic criteria. The Clinical Pulmonary Infection Score (CPIS) was developed by weighting of the various clinical criteria usually used for the diagnosis of VAP (Table 153-8). Use of the CPIS allows the selection of low-risk patients who may need only short-course antibiotic therapy or no treatment at all. Moreover, studies have demonstrated that the absence of bacteria in gram-stained endotracheal aspirates makes pneumonia an unlikely cause of fever or pulmonary infiltrates. These findings, coupled with a heightened awareness of the alternative diagnoses possible in patients with suspected VAP, can prevent inappropriate overtreatment for pneumonia. Furthermore, data show that the absence of an MDR pathogen in tracheal aspirate cultures eliminates the need for MDR coverage when empirical antibiotic therapy is narrowed. Since the most likely explanations for the mortality

1	the absence of an MDR pathogen in tracheal aspirate cultures eliminates the need for MDR coverage when empirical antibiotic therapy is narrowed. Since the most likely explanations for the mortality benefit of bronchoscopic quantitative cultures are decreased antibiotic selection pressure (which reduces the risk of subsequent infection with MDR pathogens) and identification of alternative sources of infection, a clinical diagnostic approach that incorporates such principles may result in similar outcomes.

1	Other large randomized studies that did not demonstrate a similar beneficial impact of quantitative culture on outcomes did not tightly link antibiotic treatment to the results of quantitative culture and other tests. Given the conflicting results only partially explained by methodologic issues, the IDSA/ATS guidelines therefore suggest that the choice depends on availability and local expertise. aAt the time of the original diagnosis, the progression of the infiltrate is not known and results of tracheal aspirate culture are often unavailable; thus, the maximal score is initially 8–10. Abbreviations: ARDS, acute respiratory distress syndrome; CHF, congestive heart failure. Many studies have demonstrated higher mortality rates with initially inappropriate empirical antibiotic therapy. The key to appropriate antibiotic management of VAP is an appreciation of the resistance patterns of the most likely pathogens in a given patient.

1	If not for the higher risk of infection with MDR pathogens (Table 153-1), VAP could be treated with the same antibiotics used for severe CAP. However, antibiotic selection pressure leads to the frequent involvement of MDR pathogens by selecting either for drug-resistant isolates of common pathogens (MRSA and Enterobacteriaceae producing extended-spectrum β-lactamases or carbapenemases) or for intrinsically resistant pathogens (P. aeruginosa and Acinetobacter species). Frequent use of β-lactam drugs, especially cephalosporins, appears to be the major risk factor for infection with MRSA and extended-spectrum β-lactamase–positive strains.

1	P. aeruginosa has demonstrated the ability to develop resistance to all routinely used antibiotics. Unfortunately, even if initially sensitive, P. aeruginosa isolates also have a propensity to develop resistance during treatment. Either de-repression of resistance genes or selection of resistant clones within the large bacterial inoculum associated with most pneumonias may be the cause. Acinetobacter species, Stenotrophomonas maltophilia, and Burkholderia cepacia are intrinsically resistant to many of the empirical antibiotic regimens employed (see later in this chapter). VAP caused by these pathogens emerges during treatment of other infections, and resistance is always evident at initial diagnosis.

1	Recommended options for empirical therapy are listed in Table 153-9. Treatment should be started once diagnostic specimens have been obtained. The major factor in the selection of agents is the presence of risk factors for MDR pathogens. Choices among the various options listed depend on local patterns of resistance and—a very important factor—the patient’s prior antibiotic exposure. The majority of patients without risk factors for MDR infection can be treated with a single agent. The major difference from CAP is the markedly lower incidence of atypical pathogens in VAP; the exception is Legionella, which can be a nosocomial pathogen, especially with breakdowns in the treatment of potable water in the hospital. Moxifloxacin (400 mg IV q24h), ciprofloxacin (400 mg IV q8h), or levofloxacin (750 mg IV q24h) or Ampicillin/sulbactam (3 g IV q6h) or Ertapenem (1 g IV q24h) Patients with Risk Factors for MDR Pathogens 1. A β-lactam: Piperacillin/tazobactam (4. 5 g IV q6h) or

1	Patients with Risk Factors for MDR Pathogens 1. A β-lactam: Piperacillin/tazobactam (4. 5 g IV q6h) or Imipenem (500 mg IV q6h or 1 g IV q8h), or meropenem (1 g IV q8h) 2. A second agent active against gram-negative bacterial pathogens: 3. An agent active against gram-positive bacterial pathogens: Vancomycin (15 mg/kg q12h initially with adjusted doses) Abbreviation: MDR, multidrug-resistant. The standard recommendation for patients with risk factors for MDR infection is for three antibiotics: two directed at P. aeruginosa and one at MRSA. The choice of a β-lactam agent provides the greatest variability in coverage, yet the use of the broadest-spectrum agent—a carbapenem, even in an antibiotic combination—still represents inappropriate initial therapy in 10–15% of cases.

1	Once an etiologic diagnosis is made, broad-spectrum empirical therapy can be modified to specifically address the known pathogen. For patients with MDR risk factors, antibiotic regimens can be reduced to a single agent in more than one-half of cases and to a two-drug combination in more than one-quarter of cases. Only a minority of cases require a complete course with three drugs. A negative tracheal-aspirate culture or growth below the threshold for quantitative cultures, especially if the sample was obtained before any antibiotic change, strongly suggests that antibiotics should be discontinued. Identification of other confirmed or suspected sites of infection may require ongoing antibiotic therapy, but the spectrum of pathogens (and the corresponding antibiotic choices) may be different from those for VAP. If the CPIS decreases over the first 3 days, antibiotics should be stopped after 8 days. An 8-day course of therapy is just as effective as a 2-week course and is associated with

1	from those for VAP. If the CPIS decreases over the first 3 days, antibiotics should be stopped after 8 days. An 8-day course of therapy is just as effective as a 2-week course and is associated with less frequent emergence of antibiotic-resistant strains.

1	The major controversy regarding specific therapy for VAP concerns the need for ongoing combination treatment of Pseudomonas infection. No randomized controlled trials have demonstrated a benefit of combination therapy with a β-lactam and an aminoglycoside, nor have subgroup analyses in other trials found a survival benefit with such a regimen. The unacceptably high rates of clinical failure and death for VAP caused by P. aeruginosa despite combination therapy (see “Failure to Improve,” later) indicate that better regimens are needed—including, perhaps, aerosolized antibiotics. VAP caused by MRSA is associated with a 40% clinical failure rate when treated with standard-dose vancomycin. One proposed solution is the use of high-dose individualized treatment, although the risk of renal toxicity increases with this strategy. In addition, the MIC of vancomycin has been increasing, and a high percentage of clinical failures occur when the MIC is in the upper range of sensitivity (i.e., 1.

1	increases with this strategy. In addition, the MIC of vancomycin has been increasing, and a high percentage of clinical failures occur when the MIC is in the upper range of sensitivity (i.e., 1. 5–2 μg/mL). Linezolid appears to be 15% more efficacious than even adjusted-dose vancomycin and is clearly preferred in patients with renal insufficiency and those infected with high-MIC isolates of MRSA.

1	Treatment failure is not uncommon in VAP, especially that caused by MDR pathogens. In addition to the 40% failure rate for MRSA infection treated with vancomycin, VAP due to Pseudomonas has a 50% failure rate, no matter what the regimen. Causes of clinical failure vary with the pathogen(s) and the antibiotic(s). Inappropriate therapy can usually be minimized by use of the recommended triple-drug regimen (Table 153-9). However, the emergence of β-lactam resistance during therapy is an important problem, especially in infection with Pseudomonas and Enterobacter species. Recurrent VAP caused by the same pathogen is possible because the biofilm on endotracheal tubes allows reintroduction of the microorganism. However, studies of VAP caused by Pseudomonas show that approximately half of recurrent cases are caused by a new strain. Inadequate local levels of vancomycin are the likely cause of treatment failure in VAP due to MRSA.

1	Treatment failure is very difficult to diagnose. Pneumonia due to a new superinfection, the presence of extrapulmonary infection, and drug toxicity must be considered in the differential diagnosis of treatment failure. Serial CPIS calculations appear to track the clinical response accurately, while repeat quantitative cultures may clarify the microbiologic response. A persistently elevated or rising CPIS by day 3 of therapy is likely to indicate treatment failure. The most sensitive component of the CPIS is improvement in oxygenation. Apart from death, the major complication of VAP is prolongation of mechanical ventilation, with corresponding increases in length of stay in the ICU and in the hospital. In most studies, an additional week of mechanical ventilation resulting from VAP is common. The additional expense of this complication often warrants costly and aggressive efforts at prevention.

1	In rare cases, some types of necrotizing pneumonia (e.g., that due to P. aeruginosa) result in significant pulmonary hemorrhage. More commonly, necrotizing infections result in the long-term complications of bronchiectasis and parenchymal scarring leading to recurrent pneumonias. The long-term complications of pneumonia are underappreciated. Pneumonia results in a catabolic state in a patient already nutritionally at risk. The muscle loss and general debilitation from an episode of VAP often require prolonged rehabilitation and, in the elderly, commonly result in an inability to return to independent function and the need for nursing home placement.

1	Clinical improvement, if it occurs, is usually evident within 48–72 h of the initiation of antimicrobial treatment. Because findings on chest radiography often worsen initially during treatment, they are less helpful than clinical criteria as an indicator of clinical response in severe pneumonia. Seriously ill patients with pneumonia often undergo follow-up chest radiography daily, at least until they are being weaned off mechanical ventilation.

1	Prognosis VAP is associated with significant mortality. Crude mortality rates of 50–70% have been reported, but the real issue is attributable mortality. Many patients with VAP have underlying diseases that would result in death even if VAP did not occur. Attributable mortality exceeded 25% in one matched-cohort study, while more recent studies have suggested much lower rates. Patients who develop VAP are at least twice as likely to die as those who do not. Some of the variability in VAP mortality rates is clearly related to the type of patient and ICU studied. VAP in trauma patients is not associated with attributable mortality, possibly because many of the patients were otherwise healthy before being injured. However, the causative pathogen also plays a major role. Generally, MDR pathogens are associated with significantly greater attributable mortality than non-MDR pathogens. Pneumonia caused by some pathogens (e.g., S. maltophilia) is simply a marker for a patient whose immune

1	are associated with significantly greater attributable mortality than non-MDR pathogens. Pneumonia caused by some pathogens (e.g., S. maltophilia) is simply a marker for a patient whose immune system is so compromised that death is almost inevitable.

1	Prevention (Table 153-7) Because of the significance of the endotracheal tube as a risk factor for VAP, the most important preventive intervention is to avoid endotracheal intubation or minimize its duration. Successful use of noninvasive ventilation via a nasal or full-face mask avoids many of the problems associated with endotracheal tubes. Strategies that minimize the duration of ventilation through daily holding of sedation and formal weaning protocols also have been highly effective in preventing VAP.

1	Unfortunately, a tradeoff in risks is sometimes required. Aggressive attempts to extubate early may result in reintubation(s) and increase aspiration, posing a risk of VAP. Heavy continuous sedation increases the risk, but self-extubation because of insufficient sedation also is a risk. The tradeoffs also apply to antibiotic therapy. Short-course antibiotic prophylaxis can decrease the risk of VAP in comatose patients requiring intubation, and data suggest that antibiotics decrease VAP rates in general. However, the major benefit appears to be a decrease in the incidence of early-onset VAP, which is usually caused by the less pathogenic non-MDR microorganisms. Conversely, prolonged courses of antibiotics consistently increase the risk of VAP caused by the more lethal MDR pathogens. Despite its virulence and associated mortality, VAP caused by Pseudomonas is rare among patients who have not recently received antibiotics.

1	Minimizing the amount of microaspiration around the endotracheal tube cuff also is a strategy for avoidance of VAP. Simply elevating the head of the bed (at least 30° above horizontal but preferably 45°) decreases VAP rates. Specially modified endotracheal tubes that allow removal of the secretions pooled above the cuff also may prevent VAP. The risk-to-benefit ratio of transporting the patient outside the ICU for diagnostic tests or procedures should be carefully considered, since VAP rates are increased among transported patients.

1	Emphasis on the avoidance of agents that raise gastric pH and on oropharyngeal decontamination has been diminished by the equivocal and conflicting results of recent clinical trials. The role in the pathogenesis of VAP that is played by the overgrowth of bacterial components of the bowel flora in the stomach also has been downplayed. MRSA and the nonfermenters P. aeruginosa and Acinetobacter species are not normally part of the bowel flora but reside primarily in the nose and on the skin, respectively. Therefore, emphasis on controlling overgrowth of the bowel flora may be relevant only in certain populations, such as liver transplant recipients and patients who have undergone other major intraabdominal procedures or who have bowel obstruction.

1	In outbreaks of VAP due to specific pathogens, the possibility of a breakdown in infection control measures (particularly contamination of reusable equipment) should be investigated. Even high rates of pathogens that are already common in a particular ICU may be a result of cross-infection. Education and reminders of the need for consistent hand washing and other infection-control practices can minimize this risk. While significantly less well studied than VAP, HAP in nonintubated patients—both inside and outside the ICU—is similar to VAP. The main differences are the higher frequency of non-MDR pathogens and the better underlying host immunity in nonintubated patients. The lower frequency of MDR pathogens allows monotherapy in a larger proportion of cases of HAP than of VAP.

1	The only pathogens that may be more common in the non-VAP population are anaerobes. The greater risk of macroaspiration by nonintubated patients and the lower oxygen tensions in the lower respiratory tract of these patients increase the likelihood of a role for anaerobes. While more common in patients with HAP, anaerobes are usually only contributors to polymicrobial pneumonias except in patients with large-volume aspiration or in the setting of bowel obstruction/ileus. As in the management of CAP, specific therapy targeting anaerobes probably is not indicated (unless gross aspiration 813 is a concern) since many of the recommended antibiotics are active against anaerobes.

1	Diagnosis is even more difficult for HAP in the nonintubated patient than for VAP. Lower respiratory tract samples appropriate for culture are considerably more difficult to obtain from nonintubated patients. Many of the underlying diseases that predispose a patient to HAP are also associated with an inability to cough adequately. Since blood cultures are infrequently positive (<15% of cases), the majority of patients with HAP do not have culture data on which antibiotic modifications can be based. Therefore, de-escalation of therapy is less likely in patients with risk factors for MDR pathogens. Despite these difficulties, the better host defenses in non-ICU patients result in lower mortality rates than are documented for VAP. In addition, the risk of antibiotic failure is lower in HAP. Rebecca M. Baron, Miriam Baron Barshak

1	Rebecca M. Baron, Miriam Baron Barshak Lung abscess represents necrosis and cavitation of the lung following microbial infection. Lung abscesses can be single or multiple but usually are marked by a single dominant cavity >2 cm in diameter. The low prevalence of lung abscesses makes them difficult to study in randomized controlled trials. Although the incidence of lung abscesses has decreased in the postantibiotic era, they are still a source of significant morbidity and mortality.

1	Lung abscesses are usually characterized as either primary (~80% of cases) or secondary. Primary lung abscesses usually arise from aspiration, are often caused principally by anaerobic bacteria, and occur in the absence of an underlying pulmonary or systemic condition. Secondary lung abscesses arise in the setting of an underlying condition, such as a postobstructive process (e.g., a bronchial foreign body or tumor) or a systemic process (e.g., HIV infection or another immunocompromising condition). Lung abscesses can also be characterized as acute (<4–6 weeks in duration) or chronic (~40% of cases).

1	The majority of the existing epidemiologic information involves primary lung abscesses. In general, middle-aged men are more commonly affected than middle-aged women. The major risk factor for primary lung abscesses is aspiration. Patients at particular risk for aspiration, such as those with altered mental status, alcoholism, drug overdose, seizures, bulbar dysfunction, prior cerebrovascular or cardiovascular events, or neuromuscular disease, are most commonly affected. In addition, patients with esophageal dysmotility or esophageal lesions (strictures or tumors) and those with gastric distention and/or gastroesophageal reflux, especially those who spend substantial time in the recumbent position, are at risk for aspiration.

1	It is widely thought that colonization of the gingival crevices by anaerobic bacteria or microaerophilic streptococci (especially in patients with gingivitis and periodontal disease), combined with a risk of aspiration, is important in the development of lung abscesses. In fact, many physicians consider it extremely rare for lung abscesses to develop in the absence of teeth as a nidus for bacterial colonization. The importance of these risk factors in the development of lung abscesses is highlighted by a significant reduction in abscess incidence in the late 1940s that coincided with a change in oral surgical technique: beginning at that time, these operations were no longer performed with the patient in the seated position without a cuffed endotracheal tube, and the frequency of perioperative aspiration events was thus decreased. In addition, the introduction of penicillin around the same time significantly reduced the incidence of and mortality rate from lung abscess.

1	PATHOGENESIS Primary Lung Abscesses The development of primary lung abscesses is thought to originate when chiefly anaerobic bacteria (as well as microaerophilic streptococci) in the gingival crevices are aspirated into the lung parenchyma in a susceptible host (Table 154-1). Thus, patients who develop primary lung abscesses usually carry an overwhelming burden of aspirated material or are unable to clear the bacterial load. Pneumonitis develops initially (exacerbated in part by tissue damage caused by gastric acid); then, over a period of 7–14 days, the anaerobic bacteria produce parenchymal necrosis and cavitation whose extent depends on the host–pathogen interaction (Fig. 154-1). Anaerobes are thought to produce more extensive tissue necrosis in polymicrobial infections in which virulence factors of the various bacteria can act synergistically to cause more significant tissue destruction.

1	Secondary Lung Abscesses The pathogenesis of secondary abscesses depends on the predisposing factor. For example, in cases of bronchial

1	FIGURE 154-1 Representative chest CT scans demonstrating development of lung abscesses. This patient was immunocompromised due to underlying lymphoma and developed severe Pseudomonas aeruginosa pneumonia, as represented by a left lung infiltrate with concern for central regions of necrosis ( panel A, black arrow). Two weeks later, areas of cavitation with air fluid levels were visible in this region and were consistent with the development of lung abscesses ( panel B, white arrow). (Images provided by Dr. Ritu Gill, Division of Chest Radiology, Brigham and Women’s Hospital, Boston.) obstruction from malignancy or a foreign body, the obstructing lesion prevents clearance of oropharyngeal secretions, leading to abscess development. With underlying systemic conditions (e.g., immunosuppression after bone marrow or solid organ transplantation), impaired host defense mechanisms lead to increased susceptibility to development of lung abscesses caused by a broad range of pathogens, including

1	after bone marrow or solid organ transplantation), impaired host defense mechanisms lead to increased susceptibility to development of lung abscesses caused by a broad range of pathogens, including opportunistic organisms (Table 154-1).

1	Lung abscesses also arise from septic emboli, either in tricuspid valve endocarditis (often involving Staphylococcus aureus) or in Lemierre’s syndrome, in which an infection begins in the pharynx (classically involving Fusobacterium necrophorum) and then spreads to the neck and the carotid sheath (which contains the jugular vein) to cause septic thrombophlebitis. PATHOLOGY AND MICROBIOLOGY Primary Lung Abscesses In primary lung abscesses, the dependent segments (posterior upper lobes and superior lower lobes) are the most common locations, given the predisposition of aspirated materials to be deposited in these areas. Generally, the right lung is affected more commonly than the left because the right mainstem bronchus is less angulated. In secondary abscesses, the location of the abscess may vary with the underlying cause.

1	The microbiology of primary lung abscesses is often polymicrobial, primarily including anaerobic organisms as well as microaerophilic streptococci (Table 154-1). The retrieval and culture of anaerobes can be complicated by the contamination of samples with microbes from the oral cavity, the need for expeditious transport of the cultures to the laboratory, the need for early plating with special culture techniques, the prolonged time required for culture growth, and the need for collection of specimens prior to administration of antibiotics. When attention is paid to these factors, rates of recovery of specific isolates have been reported to be as high as 78%.

1	Because it is not clear that knowing the identity of the causative anaerobic isolate alters the response to treatment of a primary lung abscess, practice has shifted away from the use of specialized techniques to obtain material for culture, such as transtracheal aspiration and bronchoalveolar lavage with protected brush specimens that allow recovery of culture material while avoiding contamination from the oral cavity. When no pathogen is isolated from a primary lung abscess (which is the case as often as 40% of the time), the abscess is termed a nonspecific lung abscess, and the presence of anaerobes is often presumed. A putrid lung abscess refers to foul-smelling breath, sputum, or empyema and is essentially diagnostic of an anaerobic lung abscess.

1	Secondary Lung Abscesses In contrast, the microbiology of secondary lung abscesses can encompass quite a broad bacterial spectrum, with infection by Pseudomonas aeruginosa and other gram-negative rods most common. In addition, a broad array of pathogens can be identified in patients from certain endemic areas and in specific clinical scenarios (e.g., a significant incidence of fungal infections among immunosuppressed patients following bone marrow or solid organ transplantation). Because immunocompromised hosts and patients without the classic presentation of a primary lung abscess can be infected with a wide array of unusual organisms (Table 154-1), it is of special importance to obtain culture material in order to target therapy.

1	Clinical manifestations may initially be similar to those of pneumonia, with fevers, cough, sputum production, and chest pain; a more chronic and indolent presentation that includes night sweats, fatigue, and anemia is often observed with anaerobic lung abscesses. A subset of patients with putrid lung abscesses may report discolored phlegm and foul-tasting or foul-smelling sputum. Patients with lung abscesses due to non-anaerobic organisms, such as S. aureus, may present with a more fulminant course characterized by high fevers and rapid progression. Findings on physical examination may include fevers, poor dentition, and/or gingival disease as well as amphoric and/or cavernous breath sounds on lung auscultation. Additional findings may include digital clubbing and the absence of a gag reflex.

1	The differential diagnosis of lung abscesses includes other noninfectious processes that result in cavitary lung lesions, including lung infarction, malignancy, sequestration, vasculitides (e.g., granulomatosis with polyangiitis), lung cysts or bullae containing fluid, and septic emboli (e.g., from tricuspid valve endocarditis). The presence of a lung abscess is determined by chest imaging. Although a chest radiograph usually detects a thick-walled cavity with an air-fluid level, computed tomography (CT) permits better definition and may provide earlier evidence of cavitation. CT may also yield additional information regarding a possible underlying cause of lung abscess, such as malignancy, and may help distinguish a peripheral lung abscess from a pleural infection. This distinction has important implications for treatment, because a pleural space infection, such as an empyema, may require urgent drainage.

1	As described earlier (see “Pathology and Microbiology,” above), more invasive diagnostics (such as transtracheal aspiration) were traditionally undertaken for primary lung abscesses, whereas empirical therapy that includes drugs targeting anaerobic organisms currently is used more often. While sputum can be collected noninvasively for Gram’s stain and culture, which may yield a pathogen, it is likely that the infection will be polymicrobial, and culture results may not reflect the presence of anaerobic organisms. Many physicians consider putrid-smelling sputum to be virtually diagnostic of an anaerobic infection.

1	When a secondary lung abscess is present or empirical therapy fails to elicit a response, sputum and blood cultures are advised in addition to serologic studies for opportunistic pathogens (e.g., viruses and fungi causing infections in immunocompromised hosts). Additional diagnostics, such as bronchoscopy with bronchoalveolar lavage or protected brush specimen collection and CT-guided percutaneous needle aspiration, can be undertaken. Risks posed by these more invasive diagnostics include spillage of abscess contents into the other lung (with bronchoscopy) and pneumothorax and bronchopleural fistula development (with CT-guided needle aspiration). However, early diagnostics in secondary abscesses, especially in immunocompromised hosts, are particularly important, because the patients involved may be especially fragile and at risk for infection with a broad array of pathogens and, therefore, less likely than other patients to respond to empirical therapy.

1	The availability of antibiotics in the 1940s and 1950s established therapy with this drug class as the primary approach to the treatment of lung abscess. Previously, surgery had been relied upon much more frequently. For many decades, penicillin was the antibiotic of choice for primary lung abscesses in light of its anaerobic coverage; however, because oral anaerobes can produce β-lactamases, clindamycin has proved superior to penicillin in clinical trials. For primary lung abscesses, the recommended regimens are (1) clindamycin (600 mg IV three times daily; then, with the disappearance of fever and clinical improvement, 300 mg PO four times daily) or (2) an IV-administered β-lactam/β-lactamase combination, followed—once the patient’s condition is stable—by orally administered amoxicillin-clavulanate. This therapy should be continued until imaging demonstrates that the lung abscess has cleared or regressed to a small scar. Treatment duration may range from 3–4 weeks to as long as 14

1	This therapy should be continued until imaging demonstrates that the lung abscess has cleared or regressed to a small scar. Treatment duration may range from 3–4 weeks to as long as 14 weeks. One small study suggested that moxifloxacin (400 mg/d PO) is as effective and well tolerated as ampicillin-sulbactam. Notably, metronidazole is not effective as a single agent: it covers anaerobic organisms but not the microaerophilic streptococci that are often components of the mixed flora of primary lung abscesses.

1	In secondary lung abscesses, antibiotic coverage should be 815 directed at the identified pathogen, and a prolonged course (until resolution of the abscess is documented) is often required. Treatment regimens and courses vary widely, depending on the immune state of the host and the identified pathogen. Other interventions may be necessary as well, such as relief of an obstructing lesion or treatment directed at the underlying condition predisposing the patient to lung abscess. Similarly, if the condition of patients with presumed primary lung abscess fails to improve, additional studies to rule out an underlying predisposing cause for a secondary lung abscess are indicated.

1	Although it can take as long as 7 days for patients receiving appropriate therapy to defervesce, as many as 10–20% of patients may not respond at all, with continued fevers and progression of the abscess cavity on imaging. An abscess >6–8 cm in diameter is less likely to respond to antibiotic therapy without additional interventions. Options for patients who do not respond to antibiotics and whose additional diagnostic studies fail to identify an additional pathogen that can be treated include surgical resection and percutaneous drainage of the abscess, especially in poor surgical candidates. Possible complications of percutaneous drainage include bacterial contamination of the pleural space as well as pneumothorax and hemothorax.

1	Larger cavity size on presentation may correlate with the development of persistent cystic changes (pneumatoceles) or bronchiectasis. Additional possible complications include recurrence of abscesses despite appropriate therapy, extension to the pleural space with development of empyema, life-threatening hemoptysis, and massive aspiration of lung abscess contents. Reported mortality rates for primary abscesses have been as low as 2%, while rates for secondary abscesses are generally higher—as high as 75% in some case series. Other poor prognostic factors include an age >60, the presence of aerobic bacteria, sepsis at presentation, symptom duration of >8 weeks, and abscess size >6 cm.

1	Mitigation of underlying risk factors may be the best approach to prevention of lung abscesses, with attention directed toward airway protection, oral hygiene, and minimized sedation with elevation of the head of the bed for patients at risk for aspiration. Prophylaxis against certain pathogens in at-risk patients (e.g., recipients of bone marrow or solid organ transplants or patients whose immune systems are significantly compromised by HIV infection) may be undertaken. APPROACH TO THE PATIENT:

1	For patients with a lung abscess and a low likelihood of malignancy (e.g., smokers <45 years old) and with risk factors for aspiration, it is reasonable to administer empirical treatment and then to pursue further evaluation if therapy does not elicit a response. However, some clinicians may opt for up-front cultures, even in primary lung abscesses. In patients with risk factors for malignancy or other underlying conditions (especially immunocompromised hosts) or with an atypical presentation, earlier diagnostics should be considered, such as bronchoscopy with biopsy or CT-guided needle aspiration. Bronchoscopy should be performed early in patients whose history, symptoms, or imaging findings are consistent with possible bronchial obstruction. In patients from areas endemic for tuberculosis or patients with other risk factors for tuberculosis (e.g., underlying HIV infection), induced sputum samples should be examined early in the workup to rule out this disease. Adolf W. Karchmer

1	Adolf W. Karchmer The prototypic lesion of infective endocarditis, the vegetation (Fig. 155-1), is a mass of platelets, fibrin, microcolonies of microorganisms, and scant inflammatory cells. Infection most commonly involves heart valves but may also occur on the low-pressure side of a ventricular septal defect, on mural endocardium damaged by aberrant jets of blood or foreign bodies, or on intracardiac devices themselves. The analogous process involving arteriovenous shunts, arterio-arterial shunts (patent ductus arteriosus), or a coarctation of the aorta is called infective endarteritis.

1	Endocarditis can be classified according to the temporal evolution of disease, the site of infection, the cause of infection, or the predisposing risk factor (e.g., injection drug use). While each classification criterion provides therapeutic and prognostic insight, none is sufficient alone. Acute endocarditis is a hectically febrile illness that rapidly damages cardiac structures, seeds extracardiac sites, and, if untreated, progresses to death within weeks. Subacute endocarditis follows an indolent course; causes structural cardiac damage only slowly, if at all; rarely metastasizes; and is gradually progressive unless complicated by a major embolic event or a ruptured mycotic aneurysm.

1	In developed countries, the incidence of endocarditis ranges from 4 to 7 cases per 100,000 population per year and has remained relatively stable during recent decades. While congenital heart diseases remain a constant predisposition, predisposing conditions in developed countries have shifted from chronic rheumatic heart disease (still a common predisposition in developing countries) to illicit IV drug use, degenerative valve disease, and intracardiac devices. The incidence of endocarditis is notably increased among the elderly. In developed countries, 25–35% of cases of native valve endocarditis (NVE) are associated with health care, and 16–30% of all cases of endocarditis involve prosthetic valves. The risk of prosthesis infection is greatest during the first 6–12 months after valve replacement; gradually declines to a low, stable rate thereafter; and is similar for mechanical and bioprosthetic devices. The incidence of endocarditis involving cardiovascular implantable electronic

1	gradually declines to a low, stable rate thereafter; and is similar for mechanical and bioprosthetic devices. The incidence of endocarditis involving cardiovascular implantable electronic devices (CIED), primarily permanent pacemakers and implantable cardioverter-defibrillators, ranges from 0.5 to 1.14 cases per 1000 device recipients and is higher among patients with an implantable cardioverter-defibrillator than among those with a permanent pacemaker.

1	Although many species of bacteria and fungi cause sporadic episodes of endocarditis, a few bacterial species cause the majority of cases (Table 155-1). The oral cavity, skin, and upper respiratory tract are the respective primary portals for viridans streptococci, staphylococci, and HACEK organisms (Haemophilus species, Aggregatibacter aphrophilus, A. actinomycetemcomitans, Cardiobacterium species, Eikenella species, and Kingella species). Streptococcus gallolyticus subspecies gallolyticus (formerly S. bovis biotype 1) originates from the gastrointestinal tract, where it is associated with polyps and colonic tumors, and enterococci enter the bloodstream from the genitourinary tract. Health care–associated NVE, most commonly caused by Staphylococcus aureus, coagulase-negative staphylococci (CoNS), and enterococci, may have either a nosocomial onset (55%) or a community onset (45%); community-onset cases develop in patients who have had extensive contact with the health care system over

1	(CoNS), and enterococci, may have either a nosocomial onset (55%) or a community onset (45%); community-onset cases develop in patients who have had extensive contact with the health care system over the preceding 90 days. Endocarditis complicates 6–25% of episodes of catheter-associated S. aureus bacteremia; the higher rates are detected in high-risk patients studied by transesophageal echocardiography (TEE) (see “Echocardiography,” later).

1	FIGURE 155-1 Vegetations (arrows) due to viridans streptococcal endocarditis involving the mitral valve. Prosthetic valve endocarditis (PVE) arising within 2 months of valve surgery is generally nosocomial, the result of intraoperative contamination of the prosthesis or a bacteremic postoperative complication. This nosocomial origin is reflected in the primary microbial causes: S. aureus, CoNS, facultative gram-negative bacilli, diphtheroids, and fungi. The portals of entry and organisms causing cases beginning >12 months after surgery are similar to those in community-acquired NVE. PVE due to CoNS that presents 2–12 months after surgery often represents delayed-onset nosocomial infection. Regardless of the time of onset after surgery, at least 68–85% of CoNS strains that cause PVE are resistant to methicillin.

1	Endocarditis related to a permanent pacemaker or an implantable cardioverter-defibrillator involves the device or the endothelium at points of device contact. Occasionally, there is concurrent aortic or mitral valve infection. One-third of cases of CIED endocarditis present within 3 months after device implantation or manipulation, one-third present at 4–12 months, and one-third present at >1 year. S. aureus and CoNS, both of which are commonly resistant to methicillin, cause the majority of cases.

1	Injection drug use–associated endocarditis, especially that involving the tricuspid valve, is commonly caused by S. aureus, which in many cases is resistant to methicillin. Left-sided valve infections in addicts have a more varied etiology. In addition to the usual causes of endocarditis, these cases can be due to Pseudomonas aeruginosa and Candida species, and sporadic cases can be caused by unusual organisms such as Bacillus, Lactobacillus, and Corynebacterium species. Polymicrobial endocarditis occurs among injection drug users. HIV infection in drug users does not significantly influence the causes of endocarditis.

1	From 5% to 15% of patients with endocarditis have negative blood cultures; in one-third to one-half of these cases, cultures are negative because of prior antibiotic exposure. The remainder of these patients are infected by fastidious organisms, such as nutritionally variant bacteria (now designated Granulicatella and Abiotrophia species), HACEK organisms, Coxiella burnetii, and Bartonella species. Some fastidious organisms occur in characteristic geographic settings (e.g., C. burnetii and Bartonella species in Europe, Brucella species in the Middle East). Tropheryma whipplei causes an indolent, culture-negative, afebrile form of endocarditis.

1	The undamaged endothelium is resistant to infection by most bacteria and to thrombus formation. Endothelial injury (e.g., at the site of impact of high-velocity blood jets or on the low-pressure side of a cardiac structural lesion) allows either direct infection by virulent organisms or the development of a platelet-fibrin thrombus—a condition called nonbacterial thrombotic endocarditis (NBTE). This thrombus serves as a site of bacterial attachment during transient bacteremia. The cardiac conditions most commonly resulting in NBTE are mitral regurgitation, aortic stenosis, aortic regurgitation, ventricular septal defects, and complex congenital heart disease. NBTE also arises as a result of a hypercoagulable state; this gives rise to marantic endocarditis (uninfected vegetations seen in patients with malignancy and chronic diseases) and to bland vegetations

1	Percentage of Cases aThe total number of cases is larger than the sum of rightand left-sided cases because the location of infection was not specified in some cases. bIncludes viridans streptococci; Streptococcus gallolyticus; other non–group A, groupable streptococci; and Abiotrophia and Granulicatella spp. (nutritionally variant, pyridoxal-requiring streptococci). cPrimarily E. faecalis or nonspeciated isolates; occasionally E. faecium or other, less likely species. dMethicillin resistance is common among these S. aureus strains. eIncludes Haemophilus spp., Aggregatibacter aphrophilus, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella spp., and Kingella spp. Note: Data are compiled from multiple studies. complicating systemic lupus erythematosus and the antiphospholipid antibody syndrome.

1	Organisms that cause endocarditis enter the bloodstream from mucosal surfaces, the skin, or sites of focal infection. Except for more virulent bacteria (e.g., S. aureus) that can adhere directly to intact endothelium or exposed subendothelial tissue, microorganisms in the blood adhere at sites of NBTE. The organisms that commonly cause endocarditis have surface adhesin molecules, collectively called microbial surface components recognizing adhesin matrix molecules (MSCRAMMs), that mediate adherence to NBTE sites or injured endothelium. Adherence is facilitated by fibronectin-binding proteins present on many gram-positive bacteria; by clumping factor (a fibrinogenand fibrin-binding surface protein) on S. aureus; by fibrinogen-binding surface proteins (Fss2), collagen-binding surface protein (Ace), and Ebp pili (the latter mediating platelet adherence) in Enterococcus faecalis; and by glucans or FimA (a member of the family of oral mucosal adhesins) on streptococci. Fibronectin-binding

1	(Ace), and Ebp pili (the latter mediating platelet adherence) in Enterococcus faecalis; and by glucans or FimA (a member of the family of oral mucosal adhesins) on streptococci. Fibronectin-binding proteins are required for S. aureus invasion of intact endothelium; thus these surface proteins may facilitate infection of previously normal valves. If resistant to the bactericidal activity of serum and the microbicidal peptides released locally by platelets, adherent organisms proliferate to form dense microcolonies. Microorganisms also induce platelet deposition and a localized procoagulant state by eliciting tissue factor from the endothelium or, in the case of S. aureus, from monocytes as well. Fibrin deposition combines with platelet aggregation and microorganism proliferation to generate an infected vegetation. Organisms deep in vegetations are metabolically inactive (nongrowing) and relatively resistant to killing by antimicrobial agents. Proliferating surface organisms are shed

1	an infected vegetation. Organisms deep in vegetations are metabolically inactive (nongrowing) and relatively resistant to killing by antimicrobial agents. Proliferating surface organisms are shed into the bloodstream continuously.

1	The clinical manifestations of endocarditis—other than constitutional symptoms, which probably result from cytokine production— arise from damage to intracardiac structures; embolization of vegetation fragments, leading to infection or infarction of remote tissues; hematogenous infection of sites during bacteremia; and tissue injury due to the deposition of circulating immune complexes or immune responses to deposited bacterial antigens.

1	The clinical endocarditis syndrome is highly variable and spans a continuum between acute and subacute presentations. NVE, PVE, and endocarditis due to injection drug use share clinical and laboratory manifestations (Table 155-2). The causative microorganism is primarily responsible for the temporal course of endocarditis. β-Hemolytic streptococci, S. aureus, and pneumococci typically result in an acute course, although S. aureus occasionally causes subacute disease. Endocarditis caused by Staphylococcus lugdunensis (a coagulasenegative species) or by enterococci may present acutely. Subacute endocarditis is typically caused by viridans streptococci, enterococci, CoNS, and the HACEK group. Endocarditis caused by Bartonella species, T. whipplei, or C. burnetii is exceptionally indolent. Feature Frequency, %

1	Feature Frequency, % Fever 80–90 Chills and sweats 40–75 Anorexia, weight loss, malaise 25–50 Myalgias, arthralgias 15–30 Back pain 7–15 Heart murmur 80–85 New/worsened regurgitant murmur 20–50 Arterial emboli 20–50 Splenomegaly 15–50 Clubbing 10–20 Neurologic manifestations 20–40 Peripheral manifestations (Osler’s nodes, subungual 2–15 hemorrhages, Janeway lesions, Roth’s spots) Petechiae 10–40 Laboratory manifestations 818 In patients with subacute presentations, fever is typically low-grade and rarely exceeds 39.4°C (103°F); in contrast, temperatures of 39.4°–40°C (103°–104°F) are often noted in acute endocarditis. Fever may be blunted in patients who are elderly, are severely debilitated, or have renal failure.

1	Cardiac Manifestations Although heart murmurs are usually indicative of the predisposing cardiac pathology rather than of endocarditis, valvular damage and ruptured chordae may result in new regurgitant murmurs. In acute endocarditis involving a normal valve, murmurs may be absent initially but ultimately are detected in 85% of cases. Congestive heart failure (CHF) develops in 30–40% of patients as a consequence of valvular dysfunction. Occasionally, CHF is due to endocarditis-associated myocarditis or an intracardiac fistula. Heart failure due to aortic valve dysfunction progresses more rapidly than does that due to mitral valve dysfunction. Extension of infection beyond valve leaflets into adjacent annular or myocardial tissue results in perivalvular abscesses, which in turn may cause intracardiac fistulae with new murmurs. Abscesses may burrow from the aortic valve annulus through the epicardium, causing pericarditis, or into the upper ventricular septum, where they may interrupt

1	intracardiac fistulae with new murmurs. Abscesses may burrow from the aortic valve annulus through the epicardium, causing pericarditis, or into the upper ventricular septum, where they may interrupt the conduction system, leading to varying degrees of heart block. Mitral perivalvular abscesses, which are usually more distant from the conduction system, only rarely cause conduction abnormalities; if such abnormalities occur in this setting, the conduction pathway is most likely disrupted near the atrioventricular node or in the proximal bundle of His. Emboli to a coronary artery occur in 2% of patients and may result in myocardial infarction.

1	Noncardiac Manifestations The classic nonsuppurative peripheral manifestations of subacute endocarditis (e.g., Janeway lesions; Fig. 155-2A) are related to prolonged infection; with early diagnosis and treatment, these have become infrequent. In contrast, septic embolization mimicking some of these lesions (subungual hemorrhage, Osler’s nodes) is common in patients with acute S. aureus endocarditis (Fig. 155-2B). Musculoskeletal pain usually remits promptly with treatment but must be distinguished from focal metastatic infections (e.g., spondylodiscitis), which may complicate 10–15% of cases. Hematogenously seeded focal infection occurs most often in the skin, spleen, kidneys, skeletal system, and meninges. Arterial emboli, one-half of which precede the diagnosis, are clinically apparent in up to 50% of patients. Endocarditis caused by S. aureus, vegetations >10 mm in diameter (as measured by echocardiography), and infection involving the mitral valve, especially the anterior leaflet,

1	up to 50% of patients. Endocarditis caused by S. aureus, vegetations >10 mm in diameter (as measured by echocardiography), and infection involving the mitral valve, especially the anterior leaflet, are independently associated with an increased risk of embolization. Symptoms, pain, or ischemia-induced dysfunction relate to the organ or area suffering embolic arterial occlusion (e.g., kidney, spleen, bowel, extremity). Cerebrovascular emboli presenting as strokes or occasionally as encephalopathy complicate 15–35% of cases of endocarditis. Again, one-half of these events precede the diagnosis of endocarditis. The frequency of stroke is 8 per 1000 patient-days during the week prior to diagnosis; the figure falls to 4.8 and 1.7 per 1000 patient-days during the first and second weeks of effective antimicrobial therapy, respectively. This decline exceeds that which can be attributed to change in vegetation size. Only 3% of strokes occur after 1 week of effective therapy. Emboli occurring

1	antimicrobial therapy, respectively. This decline exceeds that which can be attributed to change in vegetation size. Only 3% of strokes occur after 1 week of effective therapy. Emboli occurring late during or after effective therapy do not in themselves constitute evidence of failed antimicrobial treatment.

1	FIGURE 155-2 A. Janeway lesions on toe (left) and plantar surface (right) of the foot in subacute Neisseria mucosa endocarditis. (Image courtesy of Rachel Baden, MD.) B. Septic emboli with hemorrhage and infarction due to acute Staphylococcus aureus endocarditis. gitis, intracranial hemorrhage due to hemorrhagic infarcts or ruptured mycotic aneurysms, and seizures. (Mycotic aneurysms are focal dilations of arteries occurring at points in the artery wall that have been weakened by infection in the vasa vasorum or where septic emboli have lodged.) Microabscesses in brain and meninges occur commonly in S. aureus endocarditis; surgically drainable intracerebral abscesses are infrequent. Immune complex deposition on the glomerular basement membrane causes diffuse hypocomplementemic glomerulonephritis and renal dysfunction, which typically improve with effective antimicrobial therapy. Embolic renal infarcts cause flank pain and hematuria but rarely cause renal dysfunction.

1	Manifestations of Specific Predisposing Conditions Almost 50% of endocarditis associated with injection drug use is limited to the tricuspid valve and presents with fever but with faint or no murmur and no peripheral manifestations. Septic pulmonary emboli, which are common with tricuspid endocarditis, cause cough, pleuritic chest pain, nodular pulmonary infiltrates, or occasionally pyopneumothorax. Infection of the aortic or mitral valves presents with the typical clinical features of endocarditis, including peripheral manifestations.

1	If not associated with a retained intracardiac device or masked by the symptoms of concurrent comorbid illness, health care–associated endocarditis has typical manifestations. CIED endocarditis may be associated with obvious or cryptic generator pocket infection and results in fever, minimal murmur, and pulmonary symptoms due to septic emboli. Late-onset PVE presents with typical clinical features. In cases arising within 60 days of valve surgery (early onset), typical symptoms may be obscured by comorbidity associated with recent surgery. In both early-onset and more delayed presentations, paravalvular infection is common and often results in partial valve dehiscence, regurgitant murmurs, CHF, or disruption of the conduction system.

1	In order to avoid delayed or missed diagnosis, careful clinical, micro-biologic, and echocardiographic evaluation should be pursued when febrile patients have endocarditis predispositions, cardiac or noncardiac features of endocarditis, or microbiologic findings consistent with endocarditis (e.g., a stroke or splenic infarct, multiple positive blood cultures for an endocarditis-associated organism).

1	The Duke Criteria The diagnosis of infective endocarditis is established with certainty only when vegetations are examined histologically and microbiologically. Nevertheless, a highly sensitive and specific diagnostic schema—known as the modified Duke criteria—is based on clinical, laboratory, and echocardiographic findings commonly encountered in patients with endocarditis (Table 155-3). While developed as a research tool rather than for patient management, the criteria can be a helpful diagnostic tool. If the criteria are to be maximally helpful in evaluating patients, appropriate data must be collected. Furthermore, clinical judgment must be exercised in order to use the criteria effectively. Documentation of two major criteria, of one major criterion and three minor criteria, or of five minor criteria allows a clinical diagnosis of definite endocarditis. The diagnosis of endocarditis is rejected if an alternative diagnosis is established, if symptoms resolve and do not recur with

1	minor criteria allows a clinical diagnosis of definite endocarditis. The diagnosis of endocarditis is rejected if an alternative diagnosis is established, if symptoms resolve and do not recur with ≤4 days of antibiotic therapy, or if surgery or autopsy after ≤4 days of antimicrobial therapy yields no histologic evidence of endocarditis. Illnesses not classified as definite endocarditis or rejected as such are considered cases of possible infective endocarditis when either one major and one minor criterion or three minor criteria are fulfilled. Requiring some clinical features of endocarditis for classification as possible infective endocarditis increases the specificity of the schema without significantly reducing its sensitivity. Unless there are extenuating circumstances, patients with definite or possible endocarditis are treated as such.

1	The criteria emphasize bacteremia and echocardiographic findings typical of endocarditis. The requirement for multiple positive blood cultures over time is consistent with the continuous low-density 1. Positive blood culture Typical microorganism for infective endocarditis from two separate blood cultures Viridans streptococci, Streptococcus gallolyticus, HACEK group organisms, Staphylococcus aureus, or Community-acquired enterococci in the absence of a primary focus, Persistently positive blood culture, defined as recovery of a microorganism consistent with infective endocarditis from: All of 3 or a majority of ≥4 separate blood cultures, with first and last

1	Persistently positive blood culture, defined as recovery of a microorganism consistent with infective endocarditis from: All of 3 or a majority of ≥4 separate blood cultures, with first and last Single positive blood culture for Coxiella burnetii or phase I IgG antibody titer of >1:800 2. Evidence of endocardial involvement Positive echocardiogramb Oscillating intracardiac mass on valve or supporting structures or in the path of regurgitant jets or in implanted material, in the absence of an alternative anatomic explanation, or Abscess, or New partial dehiscence of prosthetic valve, 1. Predisposition: predisposing heart conditionsc or injection drug use 2. Fever ≥38.0°C (≥100.4°F) 3. Vascular phenomena: major arterial emboli, septic pulmonary infarcts, mycotic aneurysm, intracranial hemorrhage, conjunctival hemorrhages, Janeway lesions 4. Immunologic phenomena: glomerulonephritis, Osler’s nodes, Roth’s spots, rheumatoid factor 5.

1	Microbiologic evidence: positive blood culture but not meeting major criterion, as noted previously,d or serologic evidence of active infection with an organism consistent with infective endocarditis aDefinite endocarditis is defined by documentation of two major criteria, of one major criterion and three minor criteria, or of five minor criteria. See text for further details. bTransesophageal echocardiography is required for optimal assessment of possible prosthetic valve endocarditis or complicated endocarditis. cValvular disease with stenosis or regurgitation, presence of a prosthetic valve, congenital heart disease including corrected or partially corrected conditions (except isolated atrial septal defect, repaired ventricular septal defect, or closed patent ductus arteriosus), prior endocarditis, or hypertrophic cardiomyopathy. dExcluding single positive cultures for coagulase-negative staphylococci and diphtheroids, which are common culture contaminants, or for organisms that do

1	or hypertrophic cardiomyopathy. dExcluding single positive cultures for coagulase-negative staphylococci and diphtheroids, which are common culture contaminants, or for organisms that do not cause endocarditis frequently, such as gram-negative bacilli.

1	Source: Adapted from JS Li et al: Clin Infect Dis 30:633, 2000. With permission from Oxford University Press. bacteremia characteristic of endocarditis. Among patients with untreated endocarditis who ultimately have a positive blood culture, 95% of all blood cultures are positive. The diagnostic criteria attach significance to the species of organism isolated from blood cultures. To fulfill a major criterion, the isolation of an organism that causes both endocarditis and bacteremia in the absence of endocarditis (e.g., S. aureus, enterococci) must take place repeatedly (i.e., persistent bacteremia) and in the absence of a primary focus of infection. Organisms that rarely cause endocarditis but commonly contaminate blood cultures (e.g., diphtheroids, CoNS) must be isolated repeatedly if their isolation is to serve as a major criterion.

1	Blood Cultures Isolation of the causative microorganism from blood cultures is critical for diagnosis and for planning treatment. In patients with suspected NVE, PVE, or CIED endocarditis who have not received antibiotics during the prior 2 weeks, three 2-bottle blood 820 culture sets, separated from one another by at least 2 h, should be obtained from different venipuncture sites over 24 h. If the cultures remain negative after 48–72 h, two or three additional blood culture sets should be obtained, and the laboratory should be consulted for advice regarding optimal culture techniques. Pending culture results, empirical antimicrobial therapy should be withheld initially from hemodynamically stable patients with suspected subacute endocarditis, especially those who have received antibiotics within the preceding 2 weeks. Thus, if necessary, additional blood culture sets can be obtained without the confounding effect of empirical treatment. Patients with acute endocarditis or with

1	within the preceding 2 weeks. Thus, if necessary, additional blood culture sets can be obtained without the confounding effect of empirical treatment. Patients with acute endocarditis or with deteriorating hemodynamics who may require urgent surgery should receive empirical treatment immediately after three sets of blood cultures are obtained over several hours.

1	Non-Blood-Culture Tests Serologic tests can be used to implicate organisms that are difficult to recover by blood culture: Brucella, Bartonella, Legionella, Chlamydia psittaci, and C. burnetii. Pathogens can also be identified in vegetations by culture, microscopic examination with special stains (i.e., the periodic acid–Schiff stain for T. whipplei), or direct fluorescence antibody techniques and by the use of polymerase chain reaction to recover unique microbial DNA or DNA encoding the 16S or 28S ribosomal unit (16S rRNA or 28S rRNA); sequencing of these DNAs allows identification of bacteria and fungi, respectively.

1	Echocardiography Echocardiography anatomically confirms and measures vegetations, detects intracardiac complications, and assesses cardiac function (Fig. 155-3). Transthoracic echocardiography (TTE) is noninvasive and exceptionally specific; however, it cannot image vegetations <2 mm in diameter, and in 20% of patients it is technically inadequate because of emphysema or body habitus. TTE detects vegetations in 65–80% of patients with definite clinical endocarditis but is not optimal for evaluating prosthetic valves or detecting intracardiac complications. TEE is safe and detects vegetations in >90% of patients with definite endocarditis; nevertheless, initial studies may yield false-negative results in 6–18% of endocarditis patients. When endocarditis is likely, a negative TEE result does not exclude the diagnosis but rather warrants repetition of the study once or twice in 7–10 days. TEE is the optimal method for the diagnosis of PVE, the detection of myocardial abscess, valve

1	does not exclude the diagnosis but rather warrants repetition of the study once or twice in 7–10 days. TEE is the optimal method for the diagnosis of PVE, the detection of myocardial abscess, valve perforation, or intracardiac fistulae and for the detection of vegetations in patients with CIED. In patients with CIED and negative blood cultures, a mass adherent to the lead is likely to be a bland thrombosis rather than an infected vegetation.

1	Because S. aureus bacteremia is associated with a high prevalence of endocarditis, routine echocardiographic evaluation (TTE or preferably TEE) is recommended in these patients. Patients with nosocomial S. aureus bacteremia are at increased risk of endocarditis if one or more of the following are present: positive blood cultures for 2–4 days, hemodialysis dependency, a permanent intracardiac device, spine infection, nonvertebral osteomyelitis, or an endocarditis-predisposing valve abnormality. Ideally, these patients should be evaluated with TEE. In patients with none of these findings, the risk of endocarditis is low and evaluation with TTE may suffice.

1	Experts favor echocardiographic evaluation of all patients with a clinical diagnosis of endocarditis; however, the test should not be used to screen patients with a low probability of endocarditis (e.g., patients with unexplained fever). An American Heart Association approach to the use of echocardiography for evaluation of patients with suspected endocarditis is illustrated in (Fig. 155-4). Other Studies Many studies that are not diagnostic—i.e., complete blood count, creatinine determination, liver function tests, chest radiography, and electrocardiography—are important in the management of patients with endocarditis. The erythrocyte sedimentation rate, C-reactive protein level, and circulating immune complex titer are commonly increased in endocarditis (Table 155-2). Cardiac catheterization is useful primarily to assess coronary artery patency in older individuals who are to undergo surgery for endocarditis.

1	FIGURE 155-3 Imaging of a mitral valve infected with Staphylococcus aureus by low-esophageal, four-chamber-view, transesophageal echocardiography (TEE). A. Two-dimensional echocardiogram showing a large vegetation with an adjacent echo-lucent abscess cavity. B. Color-flow Doppler image showing severe mitral regurgitation through both the abscess-fistula and the central valve orifice. A, abscess; A-F, abscess-fistula; L, valve leaflets; LA, left atrium; LV, left ventricle; MR, mitral central valve regurgitation; RV, right ventricle; veg, vegetation. (With permission of Andrew Burger, MD.)

1	To cure endocarditis, all bacteria in the vegetation must be killed. However, it is difficult to eradicate these bacteria because local host defenses are deficient and because the bacteria are largely nongrowing and metabolically inactive and thus are less easily killed by antibiotics. Accordingly, therapy must be bactericidal and prolonged. Antibiotics are generally given parenterally to achieve serum concentrations that, through passive diffusion, result in effective concentrations in the depths of the vegetation. To select effective therapy requires knowledge of the susceptibility of the causative microorganisms. The decision to initiate treatment empirically must balance the need to establish a microbiologic diagnosis against the potential progression of disease or the need for urgent surgery (see “Blood Cultures,” earlier). Simultaneous infection at other sites (such as the meninges), allergies, end-organ dysfunction, interactions with concomitantly administered medications, and

1	surgery (see “Blood Cultures,” earlier). Simultaneous infection at other sites (such as the meninges), allergies, end-organ dysfunction, interactions with concomitantly administered medications, and risks of adverse events must be considered in the selection of therapy.

1	Although given for several weeks longer, the regimens recommended for the treatment of PVE (except that caused by staphylococci) are similar to those used to treat NVE (Table 155-4). Recommended doses and durations of therapy should be followed Initial TTE Initial TEE High suspicion persists Repeat TEE Alternative diagnosis established Follow-up TEE or TTE to reassess vegetations, complications, or Rx response as clinically indicated Low suspicion persists Increased suspicion during clinical course Rx Rx Rx Rx High-risk echo features* No high-risk echo features TEE for detection of complications No TEE unless clinical status deteriorates TEE Look for other source Look for other source + + + +– – – – –+ Look for other source of symptoms

1	FIGURE 155-4 The diagnostic use of transesophageal and transtracheal echocardiography (TEE and TTE, respectively). †High initial patient risk for infective endocarditis (IE), as listed in Table 155-8, or evidence of intracardiac complications (new regurgitant murmur, new electrocardiographic conduction changes, or congestive heart failure). *High-risk echocardiographic features include large vegetations, valve insufficiency, paravalvular infection, or ventricular dysfunction. Rx indicates initiation of antibiotic therapy. (Reproduced with permission from Diagnosis and Management of Infective Endocarditis and Its Complications. Circulation 98:2936, 1998. © 1998 American Heart Association.) unless alterations are required by end-organ dysfunction or adverse events.

1	Organism-Specific Therapies • streptococci Optimal therapy for streptococcal endocarditis is based on the minimal inhibitory concentration (MIC) of penicillin for the causative isolate (Table 155-4). The 2-week penicillin/gentamicin or ceftriaxone/gentamicin regimens should not be used to treat PVE or complicated NVE. Caution should be exercised in considering aminoglycoside-containing regimens for the treatment of patients at increased risk for aminoglycoside toxicity. The regimens recommended for relatively penicillin-resistant streptococci are advocated for treatment of group B, C, or G streptococcal endocarditis. Nutritionally variant organisms (Granulicatella or Abiotrophia species) and Gemella species are treated with the regimens for moderately penicillin-resistant streptococci, as is PVE caused by these organisms or by streptococci with a penicillin MIC of >0.1 μg/mL (Table 155-4).

1	enterococci Enterococci are resistant to oxacillin, nafcillin, and the cephalosporins and are only inhibited—not killed—by penicillin, ampicillin, teicoplanin (not available in the United States), and vancomycin. To kill enterococci requires the synergistic interaction of a cell wall–active antibiotic that is effective at achievable serum concentrations (penicillin, ampicillin, vancomycin, or teicoplanin) and an aminoglycoside (gentamicin or streptomycin) to which the isolate does not exhibit high-level resistance. An isolate’s resistance to cell wall–active agents or its ability to replicate in the presence of gentamicin at ≥500 μg/mL or streptomycin at 1000–2000 μg/mL—a phenomenon called high-level aminoglycoside resistance—indicates that the ineffective antimicrobial agent cannot participate in the interaction to produce killing. High-level resistance to gentamicin predicts that tobramycin, netilmicin, amikacin, and kanamycin also will be ineffective. In fact, even when enterococci

1	in the interaction to produce killing. High-level resistance to gentamicin predicts that tobramycin, netilmicin, amikacin, and kanamycin also will be ineffective. In fact, even when enterococci are not highly resistant to gentamicin, it is difficult to predict the ability of these other aminoglycosides to participate in synergistic killing; consequently, they should not, in general, be used to treat enterococcal endocarditis. High concentrations of ampicillin plus ceftriaxone or cefotaxime, by expanded binding of penicillin-binding proteins, also kill E. faecalis in vitro and in animal models of endocarditis.

1	Enterococci must be tested for high-level resistance to streptomycin and gentamicin, β-lactamase production, and susceptibility to penicillin and ampicillin (MIC, <8 μg/mL) and to vancomycin (MIC, ≤4 μg/mL) and teicoplanin (MIC ≤2 μg/ml). If the isolate produces β-lactamase, ampicillin/sulbactam or vancomycin can be used as the cell wall–active component; if the penicillin/ampicillin MIC is ≥8 μg/mL, vancomycin can be considered; and if the vancomycin MIC is ≥8 μg/mL, penicillin or ampicillin can be considered. In the absence of high-level resistance, gentamicin or streptomycin should be used as the aminoglycoside (Table 155-4). Although the dose of gentamicin used to achieve bactericidal synergy in treating enterococcal endocarditis is smaller than that used in standard therapy, nephrotoxicity (or vestibular toxicity with streptomycin) is not uncommon during treatment lasting 4–6 weeks. Regimens in which the aminoglycoside component is given for only 2–3 weeks have been curative and

1	(or vestibular toxicity with streptomycin) is not uncommon during treatment lasting 4–6 weeks. Regimens in which the aminoglycoside component is given for only 2–3 weeks have been curative and associated with less nephrotoxicity than those using longer courses of gentamicin. Thus regimens wherein gentamicin is administered for only 2–3 weeks are preferred by some.

1	If there is high-level resistance to both gentamicin and streptomycin, a synergistic bactericidal effect cannot be achieved by the addition of an aminoglycoside; thus no aminoglycoside should be given. Instead, an 8to 12-week course of a single cell wall–active agent can be considered; for E. faecalis endocarditis, high doses of ampicillin combined with ceftriaxone or cefotaxime are suggested (Table 155-4). Nonrandomized comparative studies suggest that ampicillin-ceftriaxone may be as effective as (and less nephrotoxic than) penicillin or ampicillin plus an aminoglycoside in the treatment of E. faecalis endocarditis. Given the reduced risk G (4–5 mU IV q4h) plus gentamicind (1 mg/kg IV q8h), both for 4–6 weeks (2 g IV q4h) plus gentamicind (1 mg/kg IV q8h), both for 4–6 weeks (15 mg/kg IV q12h) plus gentamicind (1 mg/kg IV q8h), both for 4–6 weeks (2 g IV q4h) plus ceftriaxone (2 g IV q12h), both for 6 weeks

1	Can use streptomycin (7.5 mg/kg q12h) in lieu of gentamicin if there is not high-level resistance to streptomycin. Use vancomycin plus gentamicin for penicillin-allergic patients (or desensitize to penicillin) and for isolates resistant to penicillin/ ampicillin. Use for E. faecalis isolates with high-level resistance to gentamicin and streptomycin or for patients at high risk for aminoglycoside nephrotoxicity (see text). • Nafcillin, oxacillin, or flucloxacillin (2 g IV q4h for 4–6 weeks) • Nafcillin, oxacillin, or flucloxacillin (2 g IV q4h for 6–8 weeks) Rifampini (300 mg PO q8h for 6–8 weeks) Can use penicillin (4 mU q4h) if isolate is penicillin-susceptible (does not produce β-lactamase). Can use cefazolin regimen for patients with nonimmediate penicillin allergy. Use vancomycin for patients with immediate (urticarial) or severe penicillin allergy; see text regarding addition of gentamicin, fusidic acid, or rifampin.

1	Use vancomycin for patients with immediate (urticarial) or severe penicillin allergy; see text regarding addition of gentamicin, fusidic acid, or rifampin. No role for routine use of rifampin (see text). Consider alternative treatment (see text) for MRSA with vancomycin MIC >1.0 or persistent bacteremia during vancomycin therapy. Use gentamicin during initial 2 weeks; determine susceptibility to gentamicin before initiating rifampin (see text); if patient is highly allergic to penicillin, use regimen for MRSA; if β-lactam allergy is of the minor nonimmediate type, cefazolin can be substituted for oxacillin/nafcillin. Use gentamicin during initial 2 weeks; determine gentamicin susceptibility before initiating rifampin (see text). (2 g/d IV as a single dose for 4 weeks) Can use another third-generation cephalosporin at comparable dosage.

1	• Ceftriaxone (2 g IV q24h) or ampicillin (2 g IV q4h) or If patient is highly allergic to β-lactams, use doxycycline. doxycycline (100 mg q12h PO) for 6 weeks aDoses are for adults with normal renal function. Doses of gentamicin, streptomycin, and vancomycin must be adjusted for reduced renal function. Ideal body weight is used to calculate doses of gentamicin and streptomycin per kilogram (men = 50 kg + 2.3 kg per inch over 5 feet; women = 45.5 kg + 2.3 kg per inch over 5 feet). bMIC, ≤0.1 μg/mL. cVancomycin dose is based on actual body weight. Adjust for trough level of 10–15 μg/mL for streptococcal and enterococcal infections and 15–20 μg/mL for staphylococcal infections. dAminoglycosides should not be administered as single daily doses for enterococcal endocarditis and should be introduced as part of the initial treatment. Target peak and trough serum concentrations of divided-dose gentamicin 1 h after a 20to 30-min infusion or IM injection are ~3.5 μg/mL and ≤1 μg/mL,

1	be introduced as part of the initial treatment. Target peak and trough serum concentrations of divided-dose gentamicin 1 h after a 20to 30-min infusion or IM injection are ~3.5 μg/mL and ≤1 μg/mL, respectively; target peak and trough serum concentrations of streptomycin (timing as with gentamicin) are 20–35 μg/mL and <10 μg/mL, respectively. eNetilmicin (4 mg/kg qd, as a single dose) can be used in lieu of gentamicin. fMIC, >0.1 μg/mL and <0.5 μg/mL. gMIC, ≥0.5 μg/mL and <8 μg/mL. hAntimicrobial susceptibility must be evaluated; see text. iRifampin increases warfarin and dicumarol requirements for anticoagulation.

1	Abbreviations: MIC, minimal inhibitory concentration; MRSA, methicillin-resistant S. aureus; MSSA, methicillin-sensitive S. aureus. of nephrotoxicity with ampicillin-ceftriaxone therapy, this regimen may also be preferred in patients who are at increased risk for aminoglycoside nephrotoxicity. If the enterococcal isolate is resistant to all of the commonly used agents, suppression of bacteremia followed by surgical treatment should be considered. The role of newer agents potentially active against multidrug-resistant enterococci (quinupristin/dalfopristin [E. faecium only], linezolid, and daptomycin) in the treatment of endocarditis has not been established.

1	stapHylococci The regimens used to treat staphylococcal endocarditis (Table 155-4) are based not on coagulase production but rather on the presence or absence of a prosthetic valve or foreign device, the native valve(s) involved, and the susceptibility of the isolate to penicillin, methicillin, and vancomycin. All staphylococci are considered penicillin-resistant until shown not to produce penicillinase. Similarly, methicillin resistance has become so prevalent among staphylococci that empirical therapy should be initiated with a regimen that covers methicillin-resistant organisms and should later be revised if the isolate proves to be susceptible to methicillin. The addition of 3–5 days of gentamicin to a β-lactam antibiotic or vancomycin to enhance therapy for native mitral or aortic valve endocarditis has not improved survival rates and may be associated with nephrotoxicity. Neither this addition nor the addition of fusidic acid or rifampin is recommended.

1	For treatment of endocarditis caused by methicillin-resistant S. aureus (MRSA), vancomycin, dosed to achieve trough concentrations of 15–20 μg/mL, is recommended, with the caveat that this regimen may be associated with nephrotoxicity. Although resistance to vancomycin among staphylococci is rare, reduced vancomycin susceptibility among MRSA strains is increasingly encountered. Isolates with a vancomycin MIC of 4–16 μg/mL have intermediate susceptibility and are referred to as vancomycin-intermediate S. aureus (VISA). Isolates with an MIC of 2 μg/mL may harbor subpopulations with higher MICs. These heteroresistant VISA (hVISA) isolates are not detectable by routine susceptibility testing. Because of the pharmacokinetics/pharmacodynamics of vancomycin, killing of MRSA with a vancomycin MIC of >1.0 μg/mL is unpredictable, even with aggressive vancomycin dosing. Although not approved by the

1	U.S. Food and Drug Administration for this indication, daptomycin (6 mg/kg [or, as some experts prefer, 8–10 mg/kg] IV once daily) has been recommended as an alternative to vancomycin, particularly for left-sided endocarditis caused by VISA, hVISA, or isolates with a vancomycin MIC of >1.0 μg/mL. These isolates should be tested to document daptomycin susceptibility. Daptomycin activity against MRSA—even against some isolates with reduced daptomycin susceptibility—is enhanced by the addition of nafcillin or ceftaroline. Case reports suggest that either the latter combinations or ceftaroline alone (600 mg IV q8h) may be effective in recalcitrant MRSA endocarditis. Nevertheless, a discussion of treatment of endocarditis in which MRSA bacteremia persists despite therapy is beyond the scope of this chapter and requires consultation with an infectious disease specialist. The efficacy of linezolid for left-sided MRSA endocarditis has not been established. Although not widely adopted by other

1	this chapter and requires consultation with an infectious disease specialist. The efficacy of linezolid for left-sided MRSA endocarditis has not been established. Although not widely adopted by other groups, the recommendation of the British Society for Antimicrobial Chemotherapy is that a second drug be added to vancomycin (rifampin) or to daptomycin (rifampin, gentamicin, or linezolid) for the treatment of NVE due to MRSA.

1	Methicillin-susceptible S. aureus endocarditis that is uncomplicated and limited to the tricuspid or pulmonic valve can often be treated with a 2-week course that combines oxacillin or nafcillin (but not vancomycin) with gentamicin. However, patients with prolonged fever (≥5 days) during therapy or multiple septic pulmonary emboli should receive standard-duration therapy. Vancomycin plus gentamicin for 2 weeks as treatment for right-sided endocarditis caused by MRSA yields suboptimal results; thus this entity is treated for 4 weeks with vancomycin or daptomycin (6 mg/kg as a single daily dose).

1	Staphylococcal PVE is treated for 6–8 weeks with a multidrug regimen. Rifampin is an essential component because it kills staphylococci that are adherent to foreign material in a biofilm. Two other agents (selected on the basis of susceptibility testing) are combined with rifampin to prevent in vivo emergence of resistance. Because many staphylococci (particularly MRSA and Staphylococcus epidermidis) are resistant to gentamicin, the isolate’s susceptibility to gentamicin or an alternative agent should be established before rifampin treatment is begun. If the isolate is resistant to gentamicin, then another aminoglycoside, a fluoroquinolone (chosen on the basis of susceptibility), or another active agent should be substituted for gentamicin.

1	otHer organisms In the absence of meningitis, endocarditis caused by Streptococcus pneumoniae isolates with a penicillin MIC of ≤1 μg/mL can be treated with IV penicillin (4 million units every 4 h), ceftriaxone (2 g/d as a single dose), or cefotaxime (at a comparable dosage). Infection caused by pneumococcal strains with a penicillin MIC of ≥2 μg/mL should be treated with vancomycin. If meningitis is suspected or present, treatment with vancomycin plus ceftriaxone—at the doses advised for meningitis—should be initiated until susceptibility results are known. Definitive therapy should then be selected on the basis of meningitis breakpoints (penicillin MIC, 0.06 μg/mL; or ceftriaxone MIC, 0.5 μg/mL). P. aeruginosa endocarditis is treated with an antipseudomonal penicillin (ticarcillin or piperacillin) and high doses of tobramycin (8 mg/kg per day in three divided doses). Endocarditis caused by Enterobacteriaceae is treated with a potent β-lactam antibiotic plus an aminoglycoside.

1	or piperacillin) and high doses of tobramycin (8 mg/kg per day in three divided doses). Endocarditis caused by Enterobacteriaceae is treated with a potent β-lactam antibiotic plus an aminoglycoside. Corynebacterial 824 endocarditis is treated with a penicillin plus an aminoglycoside (if the organism is susceptible to the aminoglycoside) or with vancomycin, which is highly bactericidal for most strains. Therapy for Candida endocarditis consists of amphotericin B plus flucytosine and early surgery; long-term (if not indefinite) suppression with an oral azole is advised. Echinocandin treatment of Candida endocarditis has been effective in sporadic cases; nevertheless, the role of echinocandins in this setting has not been established.

1	Empirical Therapy In designing therapy (largely with antimicrobials and doses from Table 155-4 to target putative microorganisms) to be administered before culture results are known or when cultures are negative, clinical clues (e.g., acute vs. subacute presentation, site of infection, patient’s predispositions) as well as epidemiologic clues to etiology must be considered. Thus empirical therapy for acute endocarditis in an injection drug user should cover MRSA and gram-negative bacilli. Treatment with vancomycin plus gentamicin, initiated immediately after blood samples are obtained for culture, covers these organisms as well as many other potential causes. Similarly, treatment of health care–associated endocarditis must cover MRSA. In the treatment of culture-negative episodes, marantic endocarditis must be excluded and fastidious organisms sought by serologic testing. In the absence of prior antibiotic therapy, it is unlikely that S. aureus, CoNS, or enterococcal infection will

1	endocarditis must be excluded and fastidious organisms sought by serologic testing. In the absence of prior antibiotic therapy, it is unlikely that S. aureus, CoNS, or enterococcal infection will present with negative blood cultures; thus, in this situation, recommended empirical therapy targets not these organisms but rather nutritionally variant organisms, the HACEK group, and Bartonella species. Pending the availability of diagnostic data, blood culture–negative subacute NVE is treated with gentamicin plus ampicillin-sulbactam (12 g every 24 h) or ceftriaxone; doxycycline (100 mg twice daily) is added for enhanced Bartonella coverage. For culture-negative PVE, vancomycin, gentamicin, cefepime, and rifampin should be used if the prosthetic valve has been in place for ≤1 year. Empirical therapy for infected prosthetic valves in place for >1 year is similar to that for culture-negative NVE. If cultures may be negative because of confounding by prior antibiotic administration, broader

1	therapy for infected prosthetic valves in place for >1 year is similar to that for culture-negative NVE. If cultures may be negative because of confounding by prior antibiotic administration, broader empirical therapy may be indicated, with particular attention to pathogens that are likely to be inhibited by the specific prior therapy.

1	CIED Endocarditis Antimicrobial therapy for CIED endocarditis is adjunctive to complete device removal. The antimicrobial selected is based on the causative organism and should be used as recommended for NVE (Table 155-4). Bacteremic CIED infection may be complicated by coincident NVE or remote-site infection (e.g., osteomyelitis). A 4to 6-week course of endocarditis-targeted therapy is recommended for patients with CIED endocarditis and for those with bacteremia that continues during ongoing antimicrobial therapy after device removal. Although S. aureus bacteremia (and persistent CoNS bacteremia) in patients who have a CIED in place is likely—in the absence of another source—to reflect endocarditis and should be managed accordingly, not all bloodstream infections in these patients indicate endocarditis. If evidence suggesting endocarditis is lacking, bloodstream infection due to gram-negative bacilli, streptococci, enterococci, and Candida species may not indicate device infection.

1	endocarditis. If evidence suggesting endocarditis is lacking, bloodstream infection due to gram-negative bacilli, streptococci, enterococci, and Candida species may not indicate device infection. However, in the absence of another source, relapse after antimicrobial therapy increases the likelihood of CIED endocarditis and warrants treatment as such.

1	Outpatient Antimicrobial Therapy Fully compliant, clinically stable patients who are no longer bacteremic, are not febrile, and have no clinical or echocardiographic findings that suggest an impending complication may complete therapy as outpatients. Careful follow-up and a stable home setting are necessary, as are predictable IV access and use of antimicrobial agents that are stable in solution. Recommended regimens should not be compromised to accommodate outpatient therapy. Monitoring Antimicrobial Therapy Measurement of the serum bactericidal titer—the highest dilution of the patient’s serum during therapy that kills 99.9% of the standard inoculum of the infecting organism—is not recommended for assessment of standard regimens but may be useful for assessment of the treatment of endocarditis caused by unusual organisms. Serum concentrations of aminoglycosides and vancomycin should be monitored and doses adjusted to avoid or address toxicity.

1	Antibiotic toxicities, including allergic reactions, occur in 25–40% of patients and commonly arise after several weeks of therapy. Blood tests to detect renal, hepatic, and hematologic toxicity should be performed periodically.

1	Blood cultures should be repeated daily until sterile in patients with endocarditis due to S. aureus or difficult-to-treat organisms, rechecked if there is recrudescent fever, and performed again 4–6 weeks after therapy to document cure. Blood cultures become sterile within 2 days after the start of appropriate therapy when infection is caused by viridans streptococci, enterococci, or HACEK organisms. In S. aureus endocarditis, β-lactam therapy results in sterile cultures in 3–5 days, whereas in MRSA endocarditis, positive cultures may persist for 7–9 days with vancomycin or daptomycin treatment. MRSA bacteremia persisting despite an adequate dosage of vancomycin may indicate infection due to a strain with reduced vancomycin susceptibility and therefore may point to a need for alternative therapy. When fever persists for 7 days despite appropriate antibiotic therapy, patients should be evaluated for paravalvular abscess, extracardiac abscesses (spleen, kidney), or complications

1	therapy. When fever persists for 7 days despite appropriate antibiotic therapy, patients should be evaluated for paravalvular abscess, extracardiac abscesses (spleen, kidney), or complications (embolic events). Recrudescent fever raises the possibility of these complications but also of drug reactions or complications of hospitalization. Vegetations become smaller with effective therapy; however, 3 months after cure, 50% are unchanged and 25% are slightly larger.

1	Intracardiac and central nervous system complications are important causes of morbidity and death due to infective endocarditis. In some cases, effective treatment for these complications requires surgery. The indications for cardiac surgical treatment of endocarditis (Table 155-5) have been derived from observational studies and expert opinion. The strength of individual indications varies; thus the risks and benefits as well as the timing of surgery must be individualized (Table 155-6). From 25% to 40% of patients with left-sided endocarditis undergo cardiac surgery during active infection, with slightly higher surgery rates for PVE than NVE. Intracardiac complications (which are most reliably detected by TEE) and CHF are the most commonly cited indications for surgery. The benefit of surgery has been assessed primarily in studies comparing populations of medically and surgically treated patients matched for the necessity of

1	Moderate to severe congestive heart failure due to valve dysfunction Partially dehisced unstable prosthetic valve Persistent bacteremia despite optimal antimicrobial therapy Lack of effective microbicidal therapy (e.g., fungal or Brucella endocarditis) S. aureus prosthetic valve endocarditis with an intracardiac complication Relapse of prosthetic valve endocarditis after optimal antimicrobial therapy Surgery to Be Strongly Considered for Improved Outcomea Perivalvular extension of infection Poorly responsive S. aureus endocarditis involving the aortic or mitral valve Large (>10 mm in diameter) hypermobile vegetations with increased risk of embolism, particularly with prior embolic event or with significant valve dysfunction Poorly responsive or relapsed endocarditis due to highly antibiotic-resistant enterococci or gram-negative bacilli aSurgery must be carefully considered; findings are often combined with other indications to prompt surgery.

1	aSupported by a single-institution randomized trial showing benefit from early surgery. Implementation requires clinical judgment. Source: Adapted from L Olaison, G Pettersson: Infect Dis Clin North Am 16:453, 2002. surgery (indications assessed in studies as propensity), with adjustments for predictors of death (comorbidities) and timing of the surgical intervention. Although study results vary, surgery for currently advised indications appears to convey a significant survival benefit (27–55%) that becomes apparent only with follow-up for ≥6 months. During the initial weeks after surgery, mortality risk may appear increased (disease + surgery–related mortality).

1	Indications • congestive Heart failure Moderate to severe refractory CHF caused by new or worsening valve dysfunction is the major indication for cardiac surgery. At 6 months of follow-up, patients with left-sided endocarditis and moderate to severe heart failure due to valve dysfunction who are treated medically have a 50% mortality rate, while among matched patients who undergo surgery the mortality rate is 15%. The survival benefit with surgery, which is most predictable among patients with the most weighty indications (propensity), is seen in both NVE and PVE. Surgery can relieve functional stenosis due to large vegetations or restore competence to damaged regurgitant valves by repair or replacement.

1	perivalvular infection This complication, which is most common with aortic valve infection, occurs in 10–15% of native valve and 45–60% of prosthetic valve infections. It is suggested by persistent unexplained fever during appropriate therapy, new electrocardiographic conduction disturbances, or pericarditis. TEE with color Doppler is the test of choice to detect perivalvular abscesses (sensitivity, ≥85%). For optimal outcome, surgery is required, especially when fever persists, fistulae develop, prostheses are dehisced and unstable, or infection relapses after appropriate treatment. Cardiac rhythm must be monitored since high-grade heart block may require insertion of a pacemaker.

1	uncontrolled infection Continued positive blood cultures or otherwise-unexplained persistent fevers (in patients with either blood culture–positive or –negative endocarditis) despite optimal antibiotic therapy may reflect uncontrolled infection and may warrant surgery. Surgical treatment is also advised for endocarditis caused by organisms against which effective antimicrobial therapy is lacking (e.g., yeasts, fungi, P. aeruginosa, other highly resistant gram-negative bacilli, Brucella species).

1	s. aureus endocarditis The mortality rate for S. aureus PVE exceeds 50% with medical treatment but is reduced to 25% with surgical treatment. In patients with intracardiac complications associated with S. aureus PVE, surgical treatment reduces the mortality rate twentyfold. Surgical treatment should be considered for patients with S. aureus native aortic or mitral valve infection who have TTE-demonstrable vegetations and remain septic during the initial week of therapy. Isolated tricuspid valve endocarditis, even with persistent fever, rarely requires surgery.

1	prevention of systemic emboli Death and persisting morbidity may result from cerebral or coronary artery emboli. Predicting a high risk of systemic embolization by echocardiographic determination of vegetation size and anatomy does not by itself identify those patients in whom surgery to prevent emboli will result in increased chances of survival. Net benefits from surgery to prevent emboli are most likely when other surgical benefits can be achieved simultaneously—e.g., repair of a moderately dysfunctional valve or debridement of a paravalvular abscess. Only 3.5% of patients undergo surgery solely to prevent systemic emboli. Valve repair, with the consequent avoidance of prosthesis insertion, improves the benefitto-risk ratio of surgery performed to address vegetations.

1	cied endocarditis Removal of all hardware is recommended for patients with established CIED infection (pocket or intracardiac lead) or erosion of the device through the skin. Percutaneous lead extraction is preferred. With lead vegetations of >3 cm and the resulting risk of a pulmonary embolus or with retained hardware after attempted percutaneous extraction, surgical removal should be considered. Removal of the infected CIED during the initial hospitalization is associated with increased 30-day and 1-year survival rates over those attained with antibiotic therapy and device retention. If necessary, the CIED can be reimplanted percutaneously or surgically (epicardial leads) at a new site after at least 10–14 days of effective antimicrobial therapy. CIEDs should be removed and replaced subsequently when patients undergo valve surgery for endocarditis.

1	Timing of Cardiac Surgery With the more life-threatening indications for surgery (valve dysfunction and severe CHF, paravalvular abscess, major prosthesis dehiscence), early surgery—i.e., during the initial week of therapy—is associated with a greater chance of survival than later surgery. With less compelling indications, surgery may reasonably be delayed to allow further treatment as well as improvement in overall health (Table 155-6). After 14 days of recommended antibiotic therapy, excised valves are culture-negative in 99% and 50% of patients with streptococcal and S. aureus endocarditis, 826 respectively. Recrudescent endocarditis on a new implanted prosthetic valve follows surgery for active NVE and PVE in 2% and 6–15% of patients, respectively. These frequencies do not justify the risk of an adverse outcome due to a delay in surgery, particularly in patients with severe heart failure, valve dysfunction, and uncontrolled staphylococcal infections. Delay is justified when

1	the risk of an adverse outcome due to a delay in surgery, particularly in patients with severe heart failure, valve dysfunction, and uncontrolled staphylococcal infections. Delay is justified when infection is controlled and CHF is resolved with medical therapy. Neurologic complications of endocarditis may be exacerbated as a consequence of cardiac surgery. The risk of neurologic deterioration is related to the type of neurologic complication and the interval between the complication and surgery. Whenever feasible, cardiac surgery should be delayed for 2–3 weeks after a nonhemorrhagic embolic infarction and for 4 weeks after a cerebral hemorrhage. A ruptured mycotic aneurysm should be treated before cardiac surgery.

1	Antibiotic Therapy after Cardiac Surgery Organisms have been detected on Gram’s stain—or their DNA has been detected by polymerase chain reaction—in excised valves from 45% of patients who have successfully completed the recommended therapy for endocarditis. In only 7% of these patients are the organisms, most of which are unusual and antibiotic resistant, cultured from the valve. Detection of organisms or their DNA does not necessarily indicate antibiotic failure; in fact, relapse of endocarditis after surgery is uncommon. Thus, when valve cultures are negative in uncomplicated NVE caused by susceptible organisms, the duration of preoperative plus postoperative treatment should equal the total duration of recommended therapy, with ~2 weeks of treatment administered after surgery. For endocarditis complicated by paravalvular abscess, partially treated PVE, or cases with culture-positive valves, a full course of therapy should be given postoperatively.

1	Extracardiac Complications Splenic abscess develops in 3–5% of patients with endocarditis. Effective therapy requires either image-guided percutaneous drainage or splenectomy. Mycotic aneurysms occur in 2–15% of endocarditis patients; one-half of these cases involve the cerebral arteries and present as headaches, focal neurologic symptoms, or hemorrhage. Cerebral aneurysms should be monitored by angiography. Some will resolve with effective antimicrobial therapy, but those that persist, enlarge, or leak should be treated surgically if possible. Extracerebral aneurysms present as local pain, a mass, local ischemia, or bleeding; these aneurysms are treated surgically.

1	Factors that can adversely affect outcome include older age, severe comorbid conditions and diabetes, delayed diagnosis, involvement of prosthetic valves or the aortic valve, an invasive (S. aureus) or antibiotic-resistant (P. aeruginosa, yeast) pathogen, intracardiac and major neurologic complications, and an association with health care. Death and poor outcome often are related not to failure of antibiotic therapy but rather to the interactions of comorbidities and endocarditis-related end-organ complications. In developed countries, overall survival rates are 80–85%; however, rates vary considerably among subpopulations of endocarditis patients. Survival rates for patients with NVE caused by viridans streptococci, HACEK organisms, or enterococci (susceptible to synergistic therapy) are 85–90%. For S. aureus NVE in patients who do not inject drugs, survival rates are 55–70%, whereas 85–90% of injection drug users survive this infection. PVE beginning within 2 months of valve

1	are 85–90%. For S. aureus NVE in patients who do not inject drugs, survival rates are 55–70%, whereas 85–90% of injection drug users survive this infection. PVE beginning within 2 months of valve replacement results in mortality rates of 40–50%, whereas rates are only 10–20% in later-onset cases.

1	To prevent endocarditis (long a goal in clinical practice), past expert committees have supported systemic antibiotic administration prior to many bacteremia-inducing procedures. A reappraisal of the evidence for antibiotic prophylaxis for endocarditis by the American Heart Association and the European Society of Cardiology culminated in guidelines advising its more restrictive use. At best, the AnTIBIoTIC REgIMEnS foR PRoPHyLAxIS of EnDoCARDITIS In ADuLTS wITH HIgH-RISk CARDIAC LESIonSa,b A. Standard oral regimen Amoxicillin: 2 g PO 1 h before procedure B. Inability to take oral medication Ampicillin: 2 g IV or IM within 1 h before procedure C. Penicillin allergy 1. Clarithromycin or azithromycin: 500 mg PO 1 h before procedure 2. Cephalexinc: 2 g PO 1 h before procedure 3. Clindamycin: 600 mg PO 1 h before procedure D. Penicillin allergy, inability to take oral medication 1. Cefazolinc or ceftriaxonec: 1 g IV or IM 30 min before procedure 2.

1	Clindamycin: 600 mg PO 1 h before procedure D. Penicillin allergy, inability to take oral medication 1. Cefazolinc or ceftriaxonec: 1 g IV or IM 30 min before procedure 2. Clindamycin: 600 mg IV or IM 1 h before procedure aDosing for children: for amoxicillin, ampicillin, cephalexin, or cefadroxil, use 50 mg/kg PO; cefazolin, 25 mg/kg IV; clindamycin, 20 mg/kg PO or 25 mg/kg IV; clarithromycin, 15 mg/ kg PO; and vancomycin, 20 mg/kg IV. bFor high-risk lesions, see Table 155-8. Prophylaxis is not advised for other lesions. cDo not use cephalosporins in patients with immediate hypersensitivity (urticaria, angioedema, anaphylaxis) to penicillin. Source: Table created using the guidelines published by the American Heart Association and the European Society of Cardiology (W Wilson et al: Circulation 116:1736, 2007; and G Habib et al: Eur Heart J 30:2369, 2009).

1	benefit of antibiotic prophylaxis is minimal. Most endocarditis cases do not follow a procedure. Although dental treatments have been widely considered to predispose to endocarditis, such infection occurs no more frequently in patients who are undergoing dental treatment than in matched controls who are not. Furthermore, the frequency and magnitude of bacteremia associated with dental procedures and a routine day’s activities (e.g., tooth brushing and flossing) are similar; because dental procedures are infrequent events, exposure of cardiac structures to bacteremic oral-cavity organisms is notably greater from routine daily activities than from dental care. The relation of gastrointestinal and genitourinary procedures to subsequent endocarditis is even more tenuous than that of dental procedures. In addition, cost-effectiveness and cost-benefit estimates suggest that antibiotic prophylaxis represents a poor use of resources.

1	Nevertheless, studies in animal models suggest that antibiotic prophylaxis may be effective. Thus it is possible that rare cases of endocarditis are prevented. Weighing the potential benefits, potential adverse events, and costs associated with antibiotic prophylaxis, the American Heart Association and the European Society of Cardiology now recommend prophylactic antibiotics (Table 155-7) only for those patients at highest risk for severe morbidity or death from endocarditis (Table 155-8). Maintaining good dental hygiene is essential. Prophylaxis is recommended only when there is manipulation of gingival tissue or the periapical region of the teeth or perforation of the oral mucosa (including surgery on the respiratory tract). Prophylaxis is not advised for patients undergoing gastrointestinal or genitourinary tract procedures. High-risk patients should be treated before or

1	Prosthetic heart valves Prior endocarditis Unrepaired cyanotic congenital heart disease, including palliative shunts or Completely repaired congenital heart defects during the 6 months after repair Incompletely repaired congenital heart disease with residual defects adjacent to prosthetic material Valvulopathy developing after cardiac transplantationa aNot a target population for prophylaxis according to recommendations of the European Society for Cardiology. Source: Table created using the guidelines published by the American Heart Association and the European Society of Cardiology (W Wilson et al: Circulation 116:1736, 2007; and G Habib et al: Eur Heart J 30:2369, 2009). when they undergo procedures on an infected genitourinary tract or on infected skin and soft tissue.

1	when they undergo procedures on an infected genitourinary tract or on infected skin and soft tissue. In patients with aortic or mitral valve regurgitation or a prosthetic valve, treatment of acute Q fever with doxycycline plus hydroxychloroquine (for doses, see Table 155-4) for 12 months is highly effective in preventing C. burnetii endocarditis. The National Institute for Health and Clinical Excellence in the United Kingdom has advised discontinuation of all antibiotic prophylaxis for endocarditis. Limited surveillance studies have not detected increased viridans streptococcal endocarditis subsequent to the promulgation of guidelines that are more restrictive or advise no prophylaxis. Infections of the Skin, Muscles, Dennis L. Stevens

1	Skin and soft tissue infections occur in all races, all ethnic groups, and all geographic locations, although some have unique geographic niches. In modern times, the frequency and severity of some skin and soft tissue infections have increased for several reasons. First, microbes are rapidly disseminated throughout the world via efficient air travel, acquiring genes for virulence factors and antibiotic resistance. Second, natural disasters, such as earthquakes, tsunamis, tornadoes, and hurricanes, appear to be increasing in frequency, and the injuries sustained during these events commonly cause major skin and soft-tissue damage that predisposes to infection. Third, trauma and casualties resulting from combat and terrorist activities can markedly damage or destroy tissues and provide both endogenous and exogenous pathogens with ready access to deeper structures. Unfortunately, because the marvels of modern medicine may not be available during human-instigated and natural disasters,

1	endogenous and exogenous pathogens with ready access to deeper structures. Unfortunately, because the marvels of modern medicine may not be available during human-instigated and natural disasters, primary treatment may be delayed and the likelihood of severe infection and death increased.

1	ANATOMIC RELATIONSHIPS: CLUES TO THE DIAGNOSIS OF SOFT TISSUE INFECTIONS Skin and soft tissue infections have been common human afflictions for centuries. However, between 2000 and 2004, hospital admissions for skin and soft tissue infections rose by 27%, a remarkable increase that was attributable largely to the emergence of the USA300 clone of methicillin-resistant Staphylococcus aureus (MRSA). This chapter provides an anatomic approach to understanding the types of soft tissue infections and the diverse microbes responsible.

1	Protection against infection of the epidermis depends on the mechanical barrier afforded by the stratum corneum, since the epidermis itself is devoid of blood vessels (Fig. 156-1). Disruption of this layer by burns or bites, abrasions, foreign bodies, primary dermatologic disorders (e.g., herpes simplex, varicella, ecthyma gangrenosum), surgery, or vascular or pressure ulcer allows penetration of bacteria to the deeper structures. Similarly, the hair follicle can serve as a portal either for components of the normal flora (e.g., Staphylococcus) or for extrinsic bacteria (e.g., Pseudomonas in hot-tub folliculitis). Intracellular infection of the squamous epithelium with vesicle formation may arise from cutaneous inoculation, as in infection with herpes simplex virus (HSV) type 1; from the dermal capillary plexus, as in varicella and infections due to other viruses associated with viremia; or from cutaneous nerve roots, as in herpes zoster. Bacteria infecting the epidermis, such as

1	the dermal capillary plexus, as in varicella and infections due to other viruses associated with viremia; or from cutaneous nerve roots, as in herpes zoster. Bacteria infecting the epidermis, such as Streptococcus pyogenes, may be translocated laterally to deeper structures via lymphatics, an event that results in the rapid superficial spread of erysipelas. Later, engorgement or obstruction of

1	FIGURE 156-1 Structural components of the skin and soft tissue, superficial infections, and infections of the deeper structures. The rich capillary network beneath the dermal papillae plays a key role in the localization of infection and in the development of the acute inflammatory reaction. lymphatics causes flaccid edema of the epidermis, another characteristic of erysipelas.

1	The rich plexus of capillaries beneath the dermal papillae provides nutrition to the stratum germinativum, and physiologic responses of this plexus produce important clinical signs and symptoms. For example, infective vasculitis of the plexus results in petechiae, Osler’s nodes, Janeway lesions, and palpable purpura, which, if present, are important clues to the existence of endocarditis (Chap. 155). In addition, metastatic infection within this plexus can result in cutaneous manifestations of disseminated fungal infection (Chap. 240), gonococcal infection (Chap. 181), Salmonella infection (Chap. 190), Pseudomonas infection (i.e., ecthyma gangrenosum; Chap. 189), meningococcemia (Chap. 180), and staphylococcal infection (Chap. 172). The plexus also provides bacteria with access to the circulation, thereby facilitating local spread or bacteremia. The postcapillary venules of this plexus are a prominent site of polymorphonuclear leukocyte sequestration, diapedesis, and chemotaxis to the

1	thereby facilitating local spread or bacteremia. The postcapillary venules of this plexus are a prominent site of polymorphonuclear leukocyte sequestration, diapedesis, and chemotaxis to the site of cutaneous infection.

1	Amplification of these physiologic mechanisms by excessive levels of cytokines or bacterial toxins causes leukostasis, venous occlusion, and pitting edema. Edema with purple bullae, ecchymosis, and cutaneous anesthesia suggests loss of vascular integrity and necessitates exploration of the deeper structures for evidence of necrotizing fasciitis or myonecrosis. An early diagnosis requires a high level of suspicion in instances of unexplained fever and of pain and tenderness in the soft tissue, even in the absence of acute cutaneous inflammation. Table 156-1 indicates the chapters in which the infections described below are discussed in greater detail. Many of these infections are illustrated in the chapters cited or in Chap. 25e (Atlas of Rashes Associated with Fever).

1	(Table 156-1) Vesicle formation due to infection is caused by viral proliferation within the epidermis. In varicella and variola, viremia precedes the onset of a diffuse centripetal rash that progresses from macules to vesicles, then to pustules, and finally to scabs over the course of 1–2 weeks. Vesicles of varicella have a “dewdrop” appearance and develop in crops randomly about the trunk, extremities, and face over 3–4 days. Herpes zoster occurs in a single dermatome; the appearance of vesicles is preceded by pain for several days. Zoster may occur in persons of any age but is most common among immunosuppressed individuals and elderly patients, whereas most cases of varicella occur in young children. Vesicles due to HSV are found on or around the

1	CHAPTER 156 Infections of the Skin, Muscles, and Soft Tissues lips (HSV-1) or genitals (HSV-2) but also may appear on the head and neck of young wrestlers (herpes gladiatorum) or on the digits of health care workers (herpetic whitlow). Recurrent herpes labialis (HSV-1) and herpes genitalis commonly follow primary infection. Coxsackievirus A16 characteristically causes vesicles on the hands, feet, and mouth of children. Orf is caused by a DNA virus related to smallpox virus and infects the fingers of individuals who work around goats and sheep. Molluscum contagiosum virus induces flaccid vesicles on the skin of healthy and immunocompromised individuals. Although variola (smallpox) in nature was eradicated as of 1977, post-millennial terrorist events have renewed interest in this devastating infection (Chap. 261e). Viremia beginning after an incubation period of 12 days is followed by a diffuse maculopapular rash, with rapid evolution to vesicles, pustules, and then scabs. Secondary

1	infection (Chap. 261e). Viremia beginning after an incubation period of 12 days is followed by a diffuse maculopapular rash, with rapid evolution to vesicles, pustules, and then scabs. Secondary cases can occur among close contacts.

1	Rickettsialpox begins after mite-bite inoculation of Rickettsia akari into the skin. A papule with a central vesicle evolves to form a 1to 2.5-cm painless crusted black eschar with an erythematous halo and proximal adenopathy. While more common in the northeastern United States and the Ukraine in 1940–1950, rickettsialpox has recently been described in Ohio, Arizona, and Utah. Blistering dactylitis is a painful, vesicular, localized S. aureus or group A streptococcal infection of the pulps of the distal digits of the hands.

1	(Table 156-1) Staphylococcal scalded-skin syndrome (SSSS) in neonates is caused by a toxin (exfoliatin) from phage group II S. aureus. SSSS must be distinguished from toxic epidermal necrolysis (TEN), which occurs primarily in adults, is drug-induced, and is associated with a higher mortality rate. Punch biopsy with frozen section is useful in making this distinction since the cleavage plane is the stratum corneum in SSSS and the stratum germinativum in TEN (Fig. 156-1). Intravenous γ-globulin is a promising treatment for TEN. Necrotizing fasciitis and gas gangrene also induce bulla formation (see “Necrotizing Fasciitis,” below). Halophilic vibrio infection can be as aggressive and fulminant as necrotizing fasciitis; a helpful clue in its diagnosis is a history of exposure to waters of the Gulf of Mexico or the Atlantic seaboard or (in a patient with cirrhosis) the ingestion of raw seafood. The etiologic organism (Vibrio vulnificus) is highly susceptible to tetracycline.

1	(Table 156-1) Impetigo contagiosa is caused by S. pyogenes, and bullous impetigo is due to S. aureus. Both skin lesions may have an early bullous stage but then appear as thick crusts with a golden-brown color. Epidemics of impetigo caused by MRSA have been reported. Streptococcal lesions are most common among children 2–5 years of age, and epidemics may occur in settings of poor hygiene, particularly among children in lower socioeconomic settings in tropical climates. It is important to recognize impetigo contagiosa because of its relationship to poststreptococcal glomerulonephritis. Rheumatic fever is not a complication of skin infection caused by S. pyogenes. Superficial dermatophyte infection (ringworm) can occur on any skin surface, and skin scrapings with KOH staining are diagnostic. Primary infections with dimorphic fungi such as Blastomyces dermatitidis and Sporothrix schenckii can initially present as crusted skin lesions resembling ringworm. Disseminated infection with

1	Primary infections with dimorphic fungi such as Blastomyces dermatitidis and Sporothrix schenckii can initially present as crusted skin lesions resembling ringworm. Disseminated infection with Coccidioides immitis can also involve the skin, and biopsy and culture should be performed on crusted lesions in patients from endemic areas. Crusted nodular lesions caused by Mycobacterium chelonei have been described in HIVseropositive patients. Treatment with clarithromycin looks promising.

1	(Table 156-1) Hair follicles serve as portals for a number of bacteria, although S. aureus is the most common cause of localized folliculitis. Sebaceous glands empty into hair follicles and ducts and, if these portals are blocked, form sebaceous cysts that may resemble staphylococcal abscesses or may become secondarily infected. Infection of sweat glands (hidradenitis suppurativa) also can mimic infection of hair 829 follicles, particularly in the axillae. Chronic folliculitis is uncommon except in acne vulgaris, where constituents of the normal flora (e.g., Propionibacterium acnes) may play a role.

1	Diffuse folliculitis occurs in two settings. Hot-tub folliculitis is caused by Pseudomonas aeruginosa in waters that are insufficiently chlorinated and maintained at temperatures of 37–40°C. Infection is usually self-limited, although bacteremia and shock have been reported. Swimmer’s itch occurs when a skin surface is exposed to water infested with freshwater avian schistosomes. Warm water temperatures and alkaline pH are suitable for mollusks that serve as intermediate hosts between birds and humans. Free-swimming schistosomal cercariae readily penetrate human hair follicles or pores but quickly die and elicit a brisk allergic reaction, causing intense itching and erythema.

1	(Table 156-1) Raised lesions of the skin occur in many different forms. Mycobacterium marinum infections of the skin may present as cellulitis or as raised erythematous nodules. Similar lesions caused by Mycobacterium abscessus and M. chelonei have been described among patients undergoing cosmetic laser surgery and tattooing, respectively. Erythematous papules are early manifestations of cat-scratch disease (with lesions developing at the primary site of inoculation of Bartonella henselae) and bacillary angiomatosis (also caused by B. henselae). Raised serpiginous or linear eruptions are characteristic of cutaneous larva migrans, which is caused by burrowing larvae of dog or cat hookworms (Ancylostoma braziliense) and which humans acquire through contact with soil that has been contaminated with dog or cat feces. Similar burrowing raised lesions are present in dracunculiasis caused by migration of the adult female nematode Dracunculus medinensis. Nodules caused by Onchocerca volvulus

1	with dog or cat feces. Similar burrowing raised lesions are present in dracunculiasis caused by migration of the adult female nematode Dracunculus medinensis. Nodules caused by Onchocerca volvulus measure 1–10 cm in diameter and occur mostly in persons bitten by Simulium flies in Africa. The nodules contain the adult worm encased in fibrous tissue. Migration of microfilariae into the eyes may result in blindness. Verruga peruana is caused by Bartonella bacilliformis, which is transmitted to humans by the sandfly Phlebotomus. This condition can take the form of single gigantic lesions (several centimeters in diameter) or multiple small lesions (several millimeters in diameter). Numerous subcutaneous nodules may also be present in cysticercosis caused by larvae of Taenia solium. Multiple erythematous papules develop in schistosomiasis; each represents a cercarial invasion site. Skin nodules as well as thickened subcutaneous tissue are prominent features of lepromatous leprosy. Large

1	erythematous papules develop in schistosomiasis; each represents a cercarial invasion site. Skin nodules as well as thickened subcutaneous tissue are prominent features of lepromatous leprosy. Large nodules or gummas are features of tertiary syphilis, whereas flat papulosquamous lesions are characteristic of secondary syphilis. Human papillomavirus may cause singular warts (verruca vulgaris) or multiple warts in the anogenital area (condylomata acuminata). The latter are major problems in HIV-infected individuals.

1	(Table 156-1) Cutaneous anthrax begins as a pruritic papule, which develops within days into an ulcer with surrounding vesicles and edema and then into an enlarging ulcer with a black eschar. Cutaneous anthrax may cause chronic nonhealing ulcers with an overlying dirty-gray membrane, although lesions may also mimic psoriasis, eczema, or impetigo. Ulceroglandular tularemia may have associated ulcerated skin lesions with painful regional adenopathy. Although buboes are the major cutaneous manifestation of plague, ulcers with eschars, papules, or pustules are also present in 25% of cases.

1	Mycobacterium ulcerans typically causes chronic skin ulcers on the extremities of individuals living in the tropics. Mycobacterium leprae may be associated with cutaneous ulcerations in patients with lepromatous leprosy related to Lucio’s phenomenon, in which immune-mediated destruction of tissue bearing high concentrations of M. leprae bacilli occurs, usually several months after initiation of effective therapy. Mycobacterium tuberculosis also may cause ulcerations, papules, or erythematous macular lesions of the skin in both immunocompetent and immunocompromised patients.

1	Infections of the Skin, Muscles, and Soft Tissues 830 Decubitus ulcers are due to tissue hypoxemia secondary to pressure-induced vascular insufficiency and may become secondarily infected with components of the skin and gastrointestinal flora, including anaerobes. Ulcerative lesions on the anterior shins may be due to pyoderma gangrenosum, which must be distinguished from similar lesions of infectious etiology by histologic evaluation of biopsy sites. Ulcerated lesions on the genitals may be either painful (chancroid) or painless (primary syphilis).

1	(Table 156-1) Erysipelas is due to S. pyogenes and is characterized by an abrupt onset of fiery-red swelling of the face or extremities. The distinctive features of erysipelas are well-defined indurated margins, particularly along the nasolabial fold; rapid progression; and intense pain. Flaccid bullae may develop during the second or third day of illness, but extension to deeper soft tissues is rare. Treatment with penicillin is effective; swelling may progress despite appropriate treatment, although fever, pain, and the intense red color diminish. Desquamation of the involved skin occurs 5–10 days into the illness. Infants and elderly adults are most commonly afflicted, and the severity of systemic toxicity varies.

1	(Table 156-1) Cellulitis is an acute inflammatory condition of the skin that is characterized by localized pain, erythema, swelling, and heat. It may be caused by indigenous flora colonizing the skin and appendages (e.g., S. aureus and S. pyogenes) or by a wide variety of exogenous bacteria. Because the exogenous bacteria involved in cellulitis occupy unique niches in nature, a thorough history (including epidemiologic data) provides important clues to etiology. When there is drainage, an open wound, or an obvious portal of entry, Gram’s stain and culture provide a definitive diagnosis. In the absence of these findings, the bacterial etiology of cellulitis is difficult to establish, and in some cases staphylococcal and streptococcal cellulitis may have similar features. Even with needle aspiration of the leading edge or a punch biopsy of the cellulitis tissue itself, cultures are positive in only 20% of cases. This observation suggests that relatively low numbers of bacteria may cause

1	of the leading edge or a punch biopsy of the cellulitis tissue itself, cultures are positive in only 20% of cases. This observation suggests that relatively low numbers of bacteria may cause cellulitis and that the expanding area of erythema within the skin may be a direct effect of extracellular toxins or of the soluble mediators of inflammation elicited by the host.

1	Bacteria may gain access to the epidermis through cracks in the skin, abrasions, cuts, burns, insect bites, surgical incisions, and IV catheters. Cellulitis caused by S. aureus spreads from a central localized infection, such as an abscess, folliculitis, or an infected foreign body (e.g., a splinter, a prosthetic device, or an IV catheter). MRSA is rapidly replacing methicillin-sensitive S. aureus (MSSA) as a cause of cellulitis in both inpatient and outpatient settings. Cellulitis caused by MSSA or MRSA is usually associated with a focal infection, such as a furuncle, a carbuncle, a surgical wound, or an abscess; the U.S. Food and Drug Administration preferentially refers to these types of infection as purulent cellulitis. In contrast, cellulitis due to S. pyogenes is a more rapidly spreading, diffuse process that is frequently associated with lymphangitis and fever and should be referred to as nonpurulent cellulitis. Recurrent streptococcal cellulitis of the lower extremities may be

1	diffuse process that is frequently associated with lymphangitis and fever and should be referred to as nonpurulent cellulitis. Recurrent streptococcal cellulitis of the lower extremities may be caused by organisms of group A, C, or G in association with chronic venous stasis or with saphenous venectomy for coronary artery bypass surgery. Streptococci also cause recurrent cellulitis among patients with chronic lymphedema resulting from elephantiasis, lymph node dissection, or Milroy’s disease. Recurrent staphylococcal cutaneous infections are more common among individuals who have eosinophilia and elevated serum levels of IgE (Job’s syndrome) and among nasal carriers of staphylococci. Cellulitis caused by Streptococcus agalactiae (group B Streptococcus) occurs primarily in elderly patients and those with diabetes mellitus or peripheral vascular disease. Haemophilus influenzae typically causes periorbital cellulitis in children in association with sinusitis, otitis media, or

1	patients and those with diabetes mellitus or peripheral vascular disease. Haemophilus influenzae typically causes periorbital cellulitis in children in association with sinusitis, otitis media, or epiglottitis. It is unclear whether this form of cellulitis will (like meningitis) become less common as a result of the impressive efficacy of the H. influenzae type b vaccine.

1	Many other bacteria also cause cellulitis. It is fortunate that these organisms occur in such characteristic settings that a good history provides useful clues to the diagnosis. Cellulitis associated with cat bites and, to a lesser degree, with dog bites is commonly caused by Pasteurella multocida, although in the latter case Staphylococcus intermedius and Capnocytophaga canimorsus also must be considered. Sites of cellulitis and abscesses associated with dog bites and human bites also contain a variety of anaerobic organisms, including Fusobacterium, Bacteroides, aerobic and anaerobic streptococci, and Eikenella corrodens. Pasteurella is notoriously resistant to dicloxacillin and nafcillin but is sensitive to all other β-lactam antimicrobial agents as well as to quinolones, tetracycline, and erythromycin. Ampicillin/clavulanate, ampicillin/sulbactam, and cefoxitin are good choices for the treatment of animal or human bite infections. Aeromonas hydrophila causes aggressive cellulitis

1	and erythromycin. Ampicillin/clavulanate, ampicillin/sulbactam, and cefoxitin are good choices for the treatment of animal or human bite infections. Aeromonas hydrophila causes aggressive cellulitis in tissues surrounding lacerations sustained in freshwater (lakes, rivers, and streams). This organism remains sensitive to aminoglycosides, fluoroquinolones, chloramphenicol, trimethoprim-sulfamethoxazole, and third-generation cephalosporins; it is resistant to ampicillin, however.

1	P. aeruginosa causes three types of soft tissue infection: ecthyma gangrenosum in neutropenic patients, hot-tub folliculitis, and cellulitis following penetrating injury. Most commonly, P. aeruginosa is introduced into the deep tissues when a person steps on a nail. Treatment includes surgical inspection and drainage, particularly if the injury also involves bone or joint capsule. Choices for empirical treatment while antimicrobial susceptibility data are awaited include an aminoglycoside, a third-generation cephalosporin (ceftazidime, cefoperazone, or cefotaxime), a semisynthetic penicillin (ticarcillin, mezlocillin, or piperacillin), or a fluoroquinolone (although drugs of the last class are not indicated for the treatment of children <13 years old).

1	Gram-negative bacillary cellulitis, including that due to P. aeruginosa, is most common among hospitalized, immunocompromised hosts. Cultures and sensitivity tests are critically important in this setting because of multidrug resistance (Chap. 189).

1	The gram-positive aerobic rod Erysipelothrix rhusiopathiae is most often associated with fish and domestic swine and causes cellulitis primarily in bone renderers and fishmongers. E. rhusiopathiae remains susceptible to most β-lactam antibiotics (including penicillin), erythromycin, clindamycin, tetracycline, and cephalosporins but is resistant to sulfonamides, chloramphenicol, and vancomycin. Its resistance to vancomycin, which is unusual among gram-positive bacteria, is of potential clinical significance since this agent is sometimes used in empirical therapy for skin infection. Fish food containing the water flea Daphnia is sometimes contaminated with M. marinum, which can cause cellulitis or granulomas on skin surfaces exposed to the water in aquariums or injured in swimming pools. Rifampin plus ethambutol has been an effective therapeutic combination in some cases, although no comprehensive studies have been undertaken. In addition, some strains of M. marinum are susceptible to

1	plus ethambutol has been an effective therapeutic combination in some cases, although no comprehensive studies have been undertaken. In addition, some strains of M. marinum are susceptible to tetracycline or to trimethoprim-sulfamethoxazole.

1	(Table 156-1) Necrotizing fasciitis, formerly called streptococcal gangrene, may be associated with group A Streptococcus or mixed aerobic– anaerobic bacteria or may occur as a component of gas gangrene caused by Clostridium perfringens. Strains of MRSA that produce the Panton-Valentine leukocidin (PVL) toxin have been reported to cause necrotizing fasciitis. Early diagnosis may be difficult when pain or unexplained fever is the only presenting manifestation. Swelling then develops and is followed by brawny edema and tenderness. With progression, dark-red induration of the epidermis appears, along with bullae filled with blue or purple fluid. Later the skin becomes friable and takes on a bluish, maroon, or black color. By this stage, thrombosis of blood vessels in the dermal papillae (Fig. 156-1) is extensive. Extension of infection to the level of the deep fascia causes this tissue to take on a brownish-gray appearance. Rapid spread occurs along fascial planes, through venous

1	(Fig. 156-1) is extensive. Extension of infection to the level of the deep fascia causes this tissue to take on a brownish-gray appearance. Rapid spread occurs along fascial planes, through venous channels and lymphatics. Patients in the later stages are toxic and frequently manifest shock and multiorgan failure.

1	Necrotizing fasciitis caused by mixed aerobic-anaerobic bacteria begins with a breach in the integrity of a mucous membrane barrier, such as the mucosa of the gastrointestinal or genitourinary tract. The portal can be a malignancy, a diverticulum, a hemorrhoid, an anal fissure, or a urethral tear. Other predisposing factors include peripheral vascular disease, diabetes mellitus, surgery, and penetrating injury to the abdomen. Leakage into the perineal area results in a syndrome called Fournier’s gangrene, characterized by massive swelling of the scrotum and penis with extension into the perineum or the abdominal wall and the legs.

1	Necrotizing fasciitis caused by S. pyogenes has increased in frequency and severity since 1985. There are two distinct clinical presentations: those with no portal of entry and those with a defined portal of entry. Infections in the first category often begin deep at the site of a non-penetrating minor trauma, such as a bruise or a muscle strain. Seeding of the site via transient bacteremia is likely, although most patients deny antecedent streptococcal infection. The affected patients present with only severe pain and fever. Late in the course, the classic signs of necrotizing fasciitis, such as purple (violaceous) bullae, skin sloughing, and progressive toxicity, develop. In infections of the second type, S. pyogenes may reach the deep fascia from a site of cutaneous infection or penetrating trauma. These patients have early signs of superficial skin infection with progression to necrotizing fasciitis. In either case, toxicity is severe, and renal impairment may precede the

1	penetrating trauma. These patients have early signs of superficial skin infection with progression to necrotizing fasciitis. In either case, toxicity is severe, and renal impairment may precede the development of shock. In 20–40% of cases, myositis occurs concomitantly, and, as in gas gangrene (see below), serum creatine phosphokinase levels may be markedly elevated. Necrotizing fasciitis due to mixed aerobic-anaerobic bacteria may be associated with gas in deep tissue, but gas usually is not present when the cause is S. pyogenes or MRSA. Prompt surgical exploration down to the deep fascia and muscle is essential. Necrotic tissue must be surgically removed, and Gram’s staining and culture of excised tissue are useful in establishing whether group A streptococci, mixed aerobic-anaerobic bacteria, MRSA, or Clostridium species are present (see “Treatment,” below).

1	(Table 156-1) Muscle involvement can occur with viral infection (e.g., influenza, dengue, or coxsackievirus B infection) or parasitic invasion (e.g., trichinellosis, cysticercosis, or toxoplasmosis). Although myalgia develops in most of these infections, severe muscle pain is the hallmark of pleurodynia (coxsackievirus B), trichinellosis, and bacterial infection. Acute rhabdomyolysis predictably occurs with clostridial and streptococcal myositis but may also be associated with influenza virus, echovirus, coxsackievirus, Epstein-Barr virus, and Legionella infections.

1	Pyomyositis is usually due to S. aureus, is common in tropical areas, and generally has no known portal of entry. Cases of pyomyositis caused by MRSA producing the PVL toxin have been described among children in the United States. Muscle infection begins at the exact site of blunt trauma or muscle strain. Infection remains localized, and shock does not develop unless organisms produce toxic shock syndrome toxin 1 or certain enterotoxins and the patient lacks antibodies to the toxin produced by the infecting organisms. In contrast, S. pyogenes may induce primary myositis (referred to as streptococcal necrotizing myositis) in association with severe systemic toxicity. Myonecrosis occurs concomitantly with necrotizing fasciitis in ~50% of cases. Both are part of the streptococcal toxic shock syndrome.

1	Gas gangrene usually follows severe penetrating injuries that result in interruption of the blood supply and introduction of soil into wounds. Such cases of traumatic gangrene are usually caused by the clostridial species C. perfringens, C. septicum, and C. histolyticum. Rarely, latent or recurrent gangrene can occur years after penetrating trauma; dormant spores that reside at the site of previous injury are most likely responsible. Spontaneous nontraumatic gangrene among patients with neutropenia, gastrointestinal malignancy, diverticulosis, or recent radiation therapy to the abdomen is caused by several clostridial species, of which C. septicum is the most commonly involved. The tolerance of this anaerobe to oxygen probably explains why it can initiate infection spontaneously in normal tissue anywhere in the body.

1	Gas gangrene of the uterus, especially that due to Clostridium sor-831 dellii, historically occurred as a consequence of illegal or self-induced abortion and nowadays also follows spontaneous abortion, vaginal delivery, and cesarean section. C. sordellii has also been implicated in medically induced abortion. Postpartum C. sordellii infections in young, previously healthy women present as a unique clinical picture: little or no fever, lack of a purulent discharge, refractory hypotension, extensive peripheral edema and effusions, hemoconcentration, and a markedly elevated white blood cell count. The infection is almost uniformly fatal, with death ensuing rapidly. C. sordellii and C. novyi have also been associated with cutaneous injection of black tar heroin; mortality rates are lower among the affected individuals, probably because their aggressive injection-site infections are readily apparent and diagnosis is therefore prompt.

1	Synergistic nonclostridial anaerobic myonecrosis, also known as necrotizing cutaneous myositis and synergistic necrotizing cellulitis, is a variant of necrotizing fasciitis caused by mixed aerobic and anaerobic bacteria with the exclusion of clostridial organisms (see “Necrotizing Fasciitis,” above).

1	This chapter emphasizes the physical appearance and location of lesions within the soft tissues as important diagnostic clues. Other crucial considerations in narrowing the differential diagnosis are the temporal progression of the lesions as well as the patient’s travel history, animal exposure or bite history, age, underlying disease status, and lifestyle. However, even the astute clinician may find it challenging to diagnose all infections of the soft tissues by history and inspection alone. Soft tissue radiography, CT (Fig. 156-2), and MRI may be useful in determining the depth of infection and should be performed when the patient has rapidly progressing lesions or evidence of a systemic inflammatory response syndrome. These tests are particularly valuable for defining a localized abscess or detecting gas in tissue. Unfortunately, they may reveal only soft tissue swelling and thus are not specific for fulminant infections such as necrotizing fasciitis or myonecrosis caused by

1	abscess or detecting gas in tissue. Unfortunately, they may reveal only soft tissue swelling and thus are not specific for fulminant infections such as necrotizing fasciitis or myonecrosis caused by group A Streptococcus (Fig. 156-2), where gas is not found in lesions.

1	Aspiration of the leading edge or punch biopsy with frozen section may be helpful if the results of imaging tests are positive, but false-negative results occur in ~80% of cases. There is some evidence that aspiration alone may be superior to injection and aspiration with normal saline. Frozen sections are especially useful in distinguishing SSSS from TEN and are quite valuable in cases of necrotizing fasciitis. Open surgical inspection, with debridement as indicated, is clearly the best way to determine the extent and severity of infection and to obtain material for Gram’s staining and culture. Such an aggressive approach is important and may be lifesaving if undertaken early in the course of fulminant infections where there is evidence of systemic toxicity. FIGURE 156-2 CT showing edema and inflammation of the left chest wall in a patient with necrotizing fasciitis and myonecrosis caused by group A Streptococcus. Infections of the Skin, Muscles, and Soft Tissues

1	Infections of the Skin, Muscles, and Soft Tissues TREATMEnT InfecTIons of The skIn, muscles, And sofT TIssues A full description of the treatment of all the clinical entities described herein is beyond the scope of this chapter. As a guide to the clinician in selecting appropriate treatment, the antimicrobial agents useful in the most common and the most fulminant cutaneous infections are listed in Table 156-2.

1	Furuncles, carbuncles, and abscesses caused by MRSA and MSSA are common, and their treatment depends upon the size of the lesion. Furuncles <2.5 cm in diameter are usually treated with moist heat. Those that are larger (4.5 cm of erythema and induration) require surgical drainage, and the occurrence of these larger lesions in association with fever, chills, or leukocytosis requires both drainage and antibiotic treatment. A study in children demonstrated that surgical drainage of abscesses (mean diameter, 3.8 cm) was as effective when used alone as when combined with trimethoprimsulfamethoxazole treatment. However, the rate of recurrence of new lesions was lower in the group undergoing both drainage and antibiotic treatment.

1	Early and aggressive surgical exploration is essential in cases of suspected necrotizing fasciitis, myositis, or gangrene in order to (1) visualize the deep structures, (2) remove necrotic tissue, (3) reduce compartment pressure, and (4) obtain suitable material for Gram’s staining and for aerobic and anaerobic cultures. Appropriate empirical antibiotic treatment for mixed aerobic–anaerobic infections could consist of ampicillin/sulbactam, cefoxitin, or the following combination: (1) clindamycin (600–900 mg IV every 8 h) or metronidazole (500 mg every 6 h) plus (2) ampicillin or ampicillin/sulbactam (1.5–3 g IV every 6 h) plus (3) gentamicin (1–1.5 mg/kg every 8 h). Group A streptococcal and clostridial infection of the fascia and/or muscle carries a mortality rate of 20–50% with penicillin treatment. In experimental models of streptococcal and clostridial necrotizing fasciitis/myositis, clindamycin has exhibited markedly superior efficacy, but no comparative clinical trials have been

1	treatment. In experimental models of streptococcal and clostridial necrotizing fasciitis/myositis, clindamycin has exhibited markedly superior efficacy, but no comparative clinical trials have been performed. A retrospective study of children with invasive group A streptococcal

1	See Also Diagnosis/Condition Primary Treatment Alternative Treatment Chap(s).

1	aPasteurella multocida, a species commonly associated with both dog and cat bites, is resistant to cephalexin, dicloxacillin, clindamycin, and erythromycin. Eikenella corrodens, a bacterium commonly associated with human bites, is resistant to clindamycin, penicillinase-resistant penicillins, and metronidazole but is sensitive to trimethoprim-sulfamethoxazole and fluoroquinolones. bThe frequency of erythromycin resistance in group A Streptococcus is currently ~5% in the United States but has reached 70–100% in some other countries. Most, but not all, erythromycin-resistant group A streptococci are susceptible to clindamycin. Approximately 90% of Staphylococcus aureus strains are sensitive to clindamycin, but resistance—both intrinsic and inducible—is increasing. cSevere hospital-acquired S. aureus infections or community-acquired S. aureus infections that are not responding to the β-lactam antibiotics recommended in this table may be caused by methicillin-resistant strains, requiring

1	S. aureus infections or community-acquired S. aureus infections that are not responding to the β-lactam antibiotics recommended in this table may be caused by methicillin-resistant strains, requiring a switch to vancomycin, daptomycin, or linezolid. dSome strains of methicillin-resistant S. aureus (MRSA) remain sensitive to tetracycline and trimethoprim-sulfamethoxazole. Daptomycin (4 mg/kg IV q24h) or tigecycline (100-mg loading dose followed by 50 mg IV q12h) is an alternative treatment for MRSA.

1	infection demonstrated higher survival rates with clindamycin treatment than with β-lactam antibiotic therapy. Hyperbaric oxygen treatment also may be useful in gas gangrene due to clostridial species. Antibiotic treatment should be continued until all signs of systemic toxicity have resolved, all devitalized tissue has been removed, and granulation tissue has developed (Chaps. 173, 179, and 201). In summary, infections of the skin and soft tissues are diverse in presentation and severity and offer a great challenge to the clinician. This chapter provides an approach to diagnosis and understanding of the pathophysiologic mechanisms involved in these infections. More in-depth information is found in chapters on Lawrence C. Madoff

1	Lawrence C. Madoff Although Staphylococcus aureus, Neisseria gonorrhoeae, and other bacteria are the most common causes of infectious arthritis, various mycobacteria, spirochetes, fungi, and viruses also infect joints (Table 157-1). Since acute bacterial infection can destroy articular cartilage rapidly, all inflamed joints must be evaluated without delay to exclude noninfectious processes and determine appropriate antimicrobial therapy and drainage procedures. For more detailed information on infectious arthritis caused by specific organisms, the reader is referred to the chapters on those organisms.

1	Acute bacterial infection typically involves a single joint or a few joints. Subacute or chronic monarthritis or oligoarthritis suggests mycobacterial or fungal infection; episodic inflammation is seen in syphilis, Lyme disease, and the reactive arthritis that follows enteric infections and chlamydial urethritis. Acute polyarticular inflammation occurs as an immunologic reaction during the course of endocarditis, rheumatic fever, disseminated neisserial infection, and acute hepatitis B. Bacteria and viruses occasionally infect multiple joints, the former most commonly in persons with rheumatoid arthritis. APPROACH TO THE PATIENT:

1	Aspiration of synovial fluid—an essential element in the evaluation of potentially infected joints—can be performed without difficulty in most cases by the insertion of a large-bore needle into the site of maximal fluctuance or tenderness or by the route of easiest access. Ultrasonography or fluoroscopy may be used to guide aspiration of difficult-to-localize effusions of the hip and, occasionally, the shoulder and other joints. Normal synovial fluid contains <180 cells (predominantly mononuclear cells) per microliter. Synovial cell counts averaging 100,000/μL (range, 25,000–250,000/μL), with >90% neutrophils, are characteristic of acute bacterial infections. Crystal-induced, rheumatoid, and other noninfectious inflammatory arthritides usually are associated with <30,000–50,000 cells/μL; cell counts of 10,000–30,000/μL, with 50–70% neutrophils and the remainder lymphocytes, are common in mycobacterial and fungal infections. Definitive diagnosis of an infectious process relies on

1	cell counts of 10,000–30,000/μL, with 50–70% neutrophils and the remainder lymphocytes, are common in mycobacterial and fungal infections. Definitive diagnosis of an infectious process relies on identification of the pathogen in stained smears of synovial fluid, isolation of the pathogen from cultures of synovial fluid and blood, or detection of microbial nucleic acids and proteins by nucleic acid amplification (NAA)–based assays and immunologic techniques.

1	Bacteria enter the joint from the bloodstream; from a contiguous site of infection in bone or soft tissue; or by direct inoculation during surgery, injection, animal or human bite, or trauma. In hematogenous infection, bacteria escape from synovial capillaries, which have no limiting basement membrane, and within hours provoke neutrophilic infiltration of the synovium. Neutrophils and bacteria enter the joint space; later, bacteria adhere to articular cartilage. Degradation of cartilage begins within 48 h as a result of increased intraarticular pressure, release of proteases and cytokines from chondrocytes and synovial macrophages, and invasion of the cartilage by bacteria and inflammatory cells. Histologic studies reveal bacteria lining the synovium and 834 cartilage as well as abscesses extending into the synovium, cartilage, and—in severe cases—subchondral bone. Synovial proliferation results in the formation of a pannus over the cartilage, and thrombosis of inflamed synovial

1	extending into the synovium, cartilage, and—in severe cases—subchondral bone. Synovial proliferation results in the formation of a pannus over the cartilage, and thrombosis of inflamed synovial vessels develops. Bacterial factors that appear important in the pathogenesis of infective arthritis include various surface-associated adhesins in S. aureus that permit adherence to cartilage and endotoxins that promote chondrocyte-mediated breakdown of cartilage.

1	The hematogenous route of infection is the most common route in all age groups, and nearly every bacterial pathogen is capable of causing septic arthritis. In infants, group B streptococci, gram-negative enteric bacilli, and S. aureus are the most common pathogens. Since the advent of the Haemophilus influenzae vaccine, the predominant causes among children <5 years of age have been S. aureus, Streptococcus pyogenes (group A Streptococcus), and (in some centers) Kingella kingae. Among young adults and adolescents, N. gonorrhoeae is the most commonly implicated organism. S. aureus accounts for most nongonococcal isolates in adults of all ages; gram-negative bacilli, pneumococci, and β-hemolytic streptococci—particularly groups A and B but also groups C, G, and F—are involved in up to one-third of cases in older adults, especially those with underlying comorbid illnesses.

1	Infections after surgical procedures or penetrating injuries are due most often to S. aureus and occasionally to other gram-positive bacteria or gram-negative bacilli. Infections with coagulase-negative staphylococci are unusual except after the implantation of prosthetic joints or arthroscopy. Anaerobic organisms, often in association with aerobic or facultative bacteria, are found after human bites and when decubitus ulcers or intraabdominal abscesses spread into adjacent joints. Polymicrobial infections complicate traumatic injuries with extensive contamination. Bites and scratches from cats and other animals may introduce Pasteurella multocida or Bartonella henselae into joints either directly or hematogenously, and bites from humans may introduce Eikenella corrodens or other components of the oral flora. Penetration of a sharp object through a shoe is associated with Pseudomonas aeruginosa arthritis in the foot.

1	NONGONOCOCCAL BACTERIAL ARTHRITIS Epidemiology Although hematogenous infections with virulent organisms such as S. aureus, H. influenzae, and pyogenic streptococci occur in healthy persons, there is an underlying host predisposition in many cases of septic arthritis. Patients with rheumatoid arthritis have the highest incidence of infective arthritis (most often secondary to S. aureus) because of chronically inflamed joints; glucocorticoid therapy; and frequent breakdown of rheumatoid nodules, vasculitic ulcers, and skin overlying deformed joints. Diabetes mellitus, glucocorticoid therapy, hemodialysis, and malignancy all carry an increased risk of infection with

1	S. aureus and gram-negative bacilli. Tumor necrosis factor inhibitors (e.g., etanercept, infliximab), which increasingly are used for the treatment of rheumatoid arthritis, predispose to mycobacterial infections and possibly to other pyogenic bacterial infections and could be associated with septic arthritis in this population. Pneumococcal infections complicate alcoholism, deficiencies of humoral immunity, and hemoglobinopathies. Pneumococci, Salmonella species, and H. influenzae cause septic arthritis in persons infected with HIV. Persons with primary immunoglobulin deficiency are at risk for mycoplasmal arthritis, which results in permanent joint damage if tetracycline and replacement therapy with IV immunoglobulin are not administered promptly. IV drug users acquire staphylococcal and streptococcal infections from their own flora and acquire pseudomonal and other gram-negative infections from drugs and injection paraphernalia.

1	Clinical Manifestations Some 90% of patients present with involvement of a single joint—most commonly the knee; less frequently the hip; and still less often the shoulder, wrist, or elbow. Small joints of the hands and feet are more likely to be affected after direct inoculation or a bite. Among IV drug users, infections of the spine, sacroiliac joints, and sternoclavicular joints (Fig. 157-1) are more common than infections of the appendicular skeleton. Polyarticular infection is most FIGURE 157-1 Acute septic arthritis of the sternoclavicular joint.

1	FIGURE 157-1 Acute septic arthritis of the sternoclavicular joint. A man in his forties with a history of cirrhosis presented with a new onset of fever and lower neck pain. He had no history of IV drug use or previous catheter placement. Jaundice and a painful swollen area over his left sternoclavicular joint were evident on physical examination. Cultures of blood drawn at admission grew group B Streptococcus. The patient recovered after treatment with IV penicillin. (Courtesy of Francisco M. Marty, MD, Brigham and Women’s Hospital, Boston; with permission.) common among patients with rheumatoid arthritis and may resemble a flare of the underlying disease.

1	The usual presentation consists of moderate to severe pain that is uniform around the joint, effusion, muscle spasm, and decreased range of motion. Fever in the range of 38.3–38.9°C (101–102°F) and sometimes higher is common but may not be present, especially in persons with rheumatoid arthritis, renal or hepatic insufficiency, or conditions requiring immunosuppressive therapy. The inflamed, swollen joint is usually evident on examination except in the case of a deeply situated joint such as the hip, shoulder, or sacroiliac joint. Cellulitis, bursitis, and acute osteomyelitis, which may produce a similar clinical picture, should be distinguished from septic arthritis by their greater range of motion and less-than-circumferential swelling. A focus of extraarticular infection, such as a boil or pneumonia, should be sought. Peripheral-blood leukocytosis with a left shift and elevation of the erythrocyte sedimentation rate or C-reactive protein level are common.

1	Plain radiographs show evidence of soft-tissue swelling, joint-space widening, and displacement of tissue planes by the distended capsule. Narrowing of the joint space and bony erosions indicate advanced infection and a poor prognosis. Ultrasound is useful for detecting effusions in the hip, and CT or MRI can demonstrate infections of the sacroiliac joint, the sternoclavicular joint, and the spine very well.

1	Laboratory Findings Specimens of peripheral blood and synovial fluid should be obtained before antibiotics are administered. Blood cultures are positive in up to 50–70% of S. aureus infections but are less frequently positive in infections due to other organisms. The synovial fluid is turbid, serosanguineous, or frankly purulent. Gram-stained smears confirm the presence of large numbers of neutrophils. Levels of total protein and lactate dehydrogenase in synovial fluid are elevated, and the glucose level is depressed; however, these findings are not specific for infection, and measurement of these levels is not necessary for diagnosis. The synovial fluid should be examined for crystals, because gout and pseudogout can resemble septic arthritis clinically, and infection and crystal-induced disease occasionally occur together. Organisms are seen on synovial fluid smears in nearly three-quarters of infections with S. aureus and streptococci and in 30–50% of infections due to

1	disease occasionally occur together. Organisms are seen on synovial fluid smears in nearly three-quarters of infections with S. aureus and streptococci and in 30–50% of infections due to gram-negative and other bacteria. Cultures of synovial fluid are positive in >90% of cases. Inoculation of synovial fluid into bottles containing liquid media for blood cultures increases the yield of a culture, especially if the pathogen is a fastidious organism or the patient is taking an antibiotic. NAA-based assays for bacterial DNA, when available, can be useful for the diagnosis of partially treated or culture-negative bacterial arthritis.

1	Prompt administration of systemic antibiotics and drainage of the involved joint can prevent destruction of cartilage, postinfectious degenerative arthritis, joint instability, or deformity. Once samples of blood and synovial fluid have been obtained for culture, empirical antibiotics should be given that are directed against the bacteria visualized on smears or the pathogens that are likely in light of the patient’s age and risk factors. Initial therapy should consist of IV administration of bactericidal agents; direct instillation of antibiotics into the joint is not necessary to achieve adequate levels in synovial fluid and tissue. An IV third-generation cephalosporin such as cefotaxime (1 g every 8 h) or ceftriaxone (1–2 g every 24 h) provides adequate empirical coverage for most community-acquired infections in adults when smears show no organisms. IV vancomycin (1 g every 12 h) is used if there are gram-positive cocci on the smear. If methicillin-resistant S. aureus is an

1	community-acquired infections in adults when smears show no organisms. IV vancomycin (1 g every 12 h) is used if there are gram-positive cocci on the smear. If methicillin-resistant S. aureus is an unlikely pathogen (e.g., when it is not widespread in the community), either oxacillin or nafcillin (2 g every 4 h) should be given. In addition, an aminoglycoside or third-generation cephalosporin should be given to IV drug users and to other patients in whom P. aeruginosa may be the responsible agent.

1	Definitive therapy is based on the identity and antibiotic susceptibility of the bacteria isolated in culture. Infections due to staphylococci are treated with oxacillin, nafcillin, or vancomycin for 4 weeks. Pneumococcal and streptococcal infections due to penicillin-susceptible organisms respond to 2 weeks of therapy with penicillin G (2 million units IV every 4 h); infections caused by

1	H. influenzae and by strains of Streptococcus pneumoniae that are resistant to penicillin are treated with cefotaxime or ceftriaxone for 2 weeks. Most enteric gram-negative infections can be cured in 3–4 weeks by a secondor third-generation cephalosporin given IV or by a fluoroquinolone such as levofloxacin (500 mg IV or PO every 24 h). P. aeruginosa infection should be treated for at least 2 weeks with a combination regimen composed of an aminoglycoside plus either an extended-spectrum penicillin such as mezlocillin (3 g IV every 4 h) or an antipseudomonal cephalosporin such as ceftazidime (1 g IV every 8 h). If tolerated, this regimen is continued for an additional 2 weeks; alternatively, a fluoroquinolone such as ciprofloxacin (750 mg PO twice daily) is given by itself or with the penicillin or cephalosporin in place of the aminoglycoside.

1	Timely drainage of pus and necrotic debris from the infected joint is required for a favorable outcome. Needle aspiration of readily accessible joints such as the knee may be adequate if loculations or particulate matter in the joint does not prevent its thorough decompression. Arthroscopic drainage and lavage may be employed initially or within several days if repeated needle aspiration fails to relieve symptoms, decrease the volume of the effusion and the synovial white cell count, and clear bacteria from smears and cultures. In some cases, arthrotomy is necessary to remove loculations and debride infected synovium, cartilage, or bone. Septic arthritis of the hip is best managed with arthrotomy, particularly in young children, in whom infection threatens the viability of the femoral head. Septic joints do not require immobilization except for pain control before symptoms are alleviated by treatment. Weight bearing should be avoided until signs of inflammation have subsided, but

1	head. Septic joints do not require immobilization except for pain control before symptoms are alleviated by treatment. Weight bearing should be avoided until signs of inflammation have subsided, but frequent passive motion of the joint is indicated to maintain full mobility. Although addition of glucocorticoids to antibiotic treatment improves the outcome of S. aureus arthritis in experimental animals, no clinical trials have evaluated this approach in humans.

1	GONOCOCCAL ARTHRITIS Epidemiology Although its incidence has declined in recent years, gonococcal arthritis (Chap. 181) has accounted for up to 70% of episodes of infectious arthritis in persons <40 years of age in the United States. Arthritis due to N. gonorrhoeae is a consequence of 835 bacteremia arising from gonococcal infection or, more frequently, from asymptomatic gonococcal mucosal colonization of the urethra, cervix, or pharynx. Women are at greatest risk during menses and during pregnancy and overall are two to three times more likely than men to develop disseminated gonococcal infection (DGI) and arthritis. Persons with complement deficiencies, especially of the terminal components, are prone to recurrent episodes of gonococcemia. Strains of gonococci that are most likely to cause DGI include those which produce transparent colonies in culture, have the type IA outer-membrane protein, or are of the AUH-auxotroph type.

1	Clinical Manifestations and Laboratory Findings The most common manifestation of DGI is a syndrome of fever, chills, rash, and articular symptoms. Small numbers of papules that progress to hemorrhagic pustules develop on the trunk and the extensor surfaces of the distal extremities. Migratory arthritis and tenosynovitis of the knees, hands, wrists, feet, and ankles are prominent. The cutaneous lesions and articular findings are believed to be the consequence of an immune reaction to circulating gonococci and immune-complex deposition in tissues. Thus, cultures of synovial fluid are consistently negative, and blood cultures are positive in fewer than 45% of patients. Synovial fluid may be difficult to obtain from inflamed joints and usually contains only 10,000–20,000 leukocytes/μL.

1	True gonococcal septic arthritis is less common than the DGI syndrome and always follows DGI, which is unrecognized in one-third of patients. A single joint such as the hip, knee, ankle, or wrist is usually involved. Synovial fluid, which contains >50,000 leukocytes/μL, can be obtained with ease; the gonococcus is evident only occasionally in Gram-stained smears, and cultures of synovial fluid are positive in fewer than 40% of cases. Blood cultures are almost always negative.

1	Because it is difficult to isolate gonococci from synovial fluid and blood, specimens for culture should be obtained from potentially infected mucosal sites. NAA-based urine tests also may be positive. Cultures and Gram-stained smears of skin lesions are occasionally positive. All specimens for culture should be plated onto Thayer-Martin agar directly or in special transport media at the bedside and transferred promptly to the microbiology laboratory in an atmosphere of 5% CO2, as generated in a candle jar. NAA-based assays are extremely sensitive in detecting gonococcal DNA in synovial fluid. A dramatic alleviation of symptoms within 12–24 h after the initiation of appropriate antibiotic therapy supports a clinical diagnosis of the DGI syndrome if cultures are negative.

1	Initial treatment consists of ceftriaxone (1 g IV or IM every 24 h) to cover possible penicillin-resistant organisms. Once local and systemic signs are clearly resolving, the 7-day course of therapy can be completed with an oral fluoroquinolone such as ciprofloxacin (500 mg twice daily) if the organism is known to be susceptible. If penicillin-susceptible organisms are isolated, amoxicillin (500 mg three times daily) may be used. Suppurative arthritis usually responds to needle aspiration of involved joints and 7–14 days of antibiotic treatment. Arthroscopic lavage or arthrotomy is rarely required. Patients with DGI should be treated for Chlamydia trachomatis infection unless this infection is ruled out by appropriate testing. It is noteworthy that arthritis symptoms similar to those seen in DGI occur in meningococcemia. A dermatitis-arthritis syndrome, purulent monarthritis, and reactive polyarthritis have been described. All respond to treatment with IV penicillin.

1	Lyme disease (Chap. 210) due to infection with the spirochete Borrelia burgdorferi causes arthritis in up to 60% of persons who are not treated. Intermittent arthralgias and myalgias—but not arthritis—occur within days or weeks of inoculation of the spirochete by the Ixodes tick. Later, 836 there are three patterns of joint disease: (1) Fifty percent of untreated persons experience intermittent episodes of monarthritis or oligoarthritis involving the knee and/or other large joints. The symptoms wax and wane without treatment over months, and each year 10–20% of patients report loss of joint symptoms. (2) Twenty percent of untreated persons develop a pattern of waxing and waning arthralgias. (3) Ten percent of untreated patients develop chronic inflammatory synovitis that results in erosive lesions and destruction of the joint. Serologic tests for IgG antibodies to B. burgdorferi are positive in more than 90% of persons with Lyme arthritis, and an NAA-based assay detects Borrelia DNA

1	lesions and destruction of the joint. Serologic tests for IgG antibodies to B. burgdorferi are positive in more than 90% of persons with Lyme arthritis, and an NAA-based assay detects Borrelia DNA in 85%.

1	Lyme arthritis generally responds well to therapy. A regimen of oral doxycycline (100 mg twice daily for 30 days), oral amoxicillin (500 mg four times daily for 30 days), or parenteral ceftriaxone (2 g/d for 2–4 weeks) is recommended. Patients who do not respond to a total of 2 months of oral therapy or 1 month of parenteral therapy are unlikely to benefit from additional antibiotic therapy and are treated with anti-inflammatory agents or synovectomy. Failure of therapy is associated with host features such as the human leukocyte antigen DR4 (HLA-DR4) genotype, persistent reactivity to OspA (outer-surface protein A), and the presence of hLFA-1 (human leukocyte function–associated antigen 1), which cross-reacts with OspA.

1	Articular manifestations occur in different stages of syphilis (Chap. 206). In early congenital syphilis, periarticular swelling and immobilization of the involved limbs (Parrot’s pseudoparalysis) complicate osteochondritis of long bones. Clutton’s joint, a late manifestation of congenital syphilis that typically develops between ages 8 and 15 years, is caused by chronic painless synovitis with effusions of large joints, particularly the knees and elbows. Secondary syphilis may be associated with arthralgias, with symmetric arthritis of the knees and ankles and occasionally of the shoulders and wrists, and with sacroiliitis. The arthritis follows a subacute to chronic course with a mixed mononuclear and neutrophilic synovial-fluid pleocytosis (typical cell counts, 5000–15,000/μL). Immunologic mechanisms may contribute to the arthritis, and symptoms usually improve rapidly with penicillin therapy. In tertiary syphilis, Charcot’s joint results from sensory loss due to tabes dorsalis.

1	mechanisms may contribute to the arthritis, and symptoms usually improve rapidly with penicillin therapy. In tertiary syphilis, Charcot’s joint results from sensory loss due to tabes dorsalis. Penicillin is not helpful in this setting.

1	Tuberculous arthritis (Chap. 202) accounts for ~1% of all cases of tuberculosis and 10% of extrapulmonary cases. The most common presentation is chronic granulomatous monarthritis. An unusual syndrome, Poncet’s disease, is a reactive symmetric form of polyarthritis that affects persons with visceral or disseminated tuberculosis. No mycobacteria are found in the joints, and symptoms resolve with antituberculous therapy.

1	Unlike tuberculous osteomyelitis (Chap. 158), which typically involves the thoracic and lumbar spine (50% of cases), tuberculous arthritis primarily involves the large weight-bearing joints, in particular the hips, knees, and ankles, and only occasionally involves smaller non-weight-bearing joints. Progressive monarticular swelling and pain develop over months or years, and systemic symptoms are seen in only half of all cases. Tuberculous arthritis occurs as part of a disseminated primary infection or through late reactivation, often in persons with HIV infection or other immunocompromised hosts. Coexistent active pulmonary tuberculosis is unusual.

1	Aspiration of the involved joint yields fluid with an average cell count of 20,000/μL, with ~50% neutrophils. Acid-fast staining of the fluid yields positive results in fewer than one-third of cases, and cultures are positive in 80%. Culture of synovial tissue taken at biopsy is positive in ~90% of cases and shows granulomatous inflammation in most. NAA methods can shorten the time to diagnosis to 1 or 2 days. Radiographs reveal peripheral erosions at the points of synovial attachment, periarticular osteopenia, and eventually joint-space narrowing. Therapy for tuberculous arthritis is the same as that for tuberculous pulmonary disease, requiring the administration of multiple agents for 6–9 months. Therapy is more prolonged in immunosuppressed individuals such as those infected with HIV.

1	Various atypical mycobacteria (Chap. 204) found in water and soil may cause chronic indolent arthritis. Such disease results from trauma and direct inoculation associated with farming, gardening, or aquatic activities. Smaller joints, such as the digits, wrists, and knees, are usually involved. Involvement of tendon sheaths and bursae is typical. The mycobacterial species involved include Mycobacterium marinum, M. avium-intracellulare, M. terrae, M. kansasii, M. fortuitum, and M. chelonae. In persons who have HIV infection or are receiving immunosuppressive therapy, hematogenous spread to the joints has been reported for M. kansasii, M. avium complex, and M. haemophilum. Diagnosis usually requires biopsy and culture, and therapy is based on antimicrobial susceptibility patterns.

1	Fungi are an unusual cause of chronic monarticular arthritis. Granulomatous articular infection with the endemic dimorphic fungi Coccidioides immitis, Blastomyces dermatitidis, and (less commonly) Histoplasma capsulatum (Fig. 157-2) results from hematogenous seeding or direct extension from bony lesions in persons with disseminated disease. Joint involvement is an unusual complication of sporotrichosis (infection with Sporothrix schenckii) among gardeners and other persons who work with soil or sphagnum moss. Articular sporotrichosis is six times more common among men than among women, and alcoholics and other debilitated hosts are at risk for polyarticular infection.

1	Candida infection involving a single joint—usually the knee, hip, or shoulder—results from surgical procedures, intraarticular injections, or (among critically ill patients with debilitating illnesses such as diabetes mellitus or hepatic or renal insufficiency and patients receiving immunosuppressive therapy) hematogenous spread. Candida infections in IV drug users typically involve the spine, sacroiliac joints, or other fibrocartilaginous joints. Unusual cases of arthritis due to Aspergillus species, Cryptococcus neoformans, Pseudallescheria boydii, and the dematiaceous fungi also have resulted from direct inoculation or disseminated hematogenous infection in immunocompromised persons. In the United States, a 2012 national outbreak of fungal arthritis (and meningitis) caused by Exserohilum rostratum was linked to intraspinal and intraarticular injection of a contaminated preparation of methylprednisolone acetate.

1	The synovial fluid in fungal arthritis usually contains 10,000–40,000 cells/μL, with ~70% neutrophils. Stained specimens and cultures of synovial tissue often confirm the diagnosis of fungal arthritis when studies of synovial fluid give negative results. Treatment consists of drainage and lavage of the joint and systemic administration of an antifungal agent directed at a specific pathogen. The doses and duration of therapy are the same as for disseminated disease (see Part 8, Section 16). Intraarticular instillation of amphotericin B has been used in addition to IV therapy.

1	Viruses produce arthritis by infecting synovial tissue during systemic infection or by provoking an immunologic reaction that involves joints. As many as 50% of women report persistent arthralgias, and 10% report frank arthritis within 3 days of the rash that follows natural infection with rubella virus and within 2–6 weeks after receipt of live-virus vaccine. Episodes of symmetric inflammation of fingers, wrists, and knees uncommonly recur for >1 year, but a syndrome of chronic fatigue, low-grade fever, headaches, and myalgias can persist for months or years. IV immunoglobulin has been helpful in selected cases. Self-limited monarticular or migratory polyarthritis may develop within 2 weeks of the parotitis of mumps; this sequela is more common among men than among women. Approximately

1	FIGURE 157-2 Chronic arthritis caused by Histoplasma capsulatum in the left knee. A. A man in his sixties from El Salvador presented with a history of progressive knee pain and difficulty walking for several years. He had undergone arthroscopy for a meniscal tear 7 years before presentation (without relief) and had received several intraarticular glucocorticoid injections. The patient developed significant deformity of the knee over time, including a large effusion in the lateral aspect. B. An x-ray of the knee showed multiple abnormalities, including severe medial femorotibial joint-space narrowing, several large subchondral cysts within the tibia and the patellofemoral compartment, a large suprapatellar joint effusion, and a large soft tissue mass projecting laterally over the knee. C. MRI further defined these abnormalities and demonstrated the cystic nature of the lateral knee abnormality. Synovial biopsies demonstrated chronic inflammation with giant cells, and cultures grew H.

1	MRI further defined these abnormalities and demonstrated the cystic nature of the lateral knee abnormality. Synovial biopsies demonstrated chronic inflammation with giant cells, and cultures grew H. capsulatum after 3 weeks of incubation. All clinical cystic lesions and the effusion resolved after 1 year of treatment with itraconazole. The patient underwent a left total knee replacement for definitive treatment. (Courtesy of Francisco M. Marty, MD, Brigham and Women’s Hospital, Boston; with permission.) 10% of children and 60% of women develop arthritis after infection with parvovirus B19. In adults, arthropathy sometimes occurs without fever or rash. Pain and stiffness, with less prominent swelling (primarily of the hands but also of the knees, wrists, and ankles), usually resolve within weeks, although a small proportion of patients develop chronic arthropathy.

1	About 2 weeks before the onset of jaundice, up to 10% of persons with acute hepatitis B develop an immune complex–mediated, serum sickness–like reaction with maculopapular rash, urticaria, fever, and arthralgias. Less common developments include symmetric arthritis involving the hands, wrists, elbows, or ankles and morning stiffness that resembles a flare of rheumatoid arthritis. Symptoms resolve at the time jaundice develops. Many persons with chronic hepatitis C infection report persistent arthralgia or arthritis, both in the presence and in the absence of cryoglobulinemia.

1	Painful arthritis involving larger joints often accompanies the fever and rash of several arthropod-borne viral infections, including those caused by chikungunya, O’nyong-nyong, Ross River, Mayaro, and Barmah Forest viruses (Chap. 233). Symmetric arthritis involving the hands and wrists may occur during the convalescent phase of infection with lymphocytic choriomeningitis virus. Patients infected with an enterovirus frequently report arthralgias, and echovirus has been isolated from patients with acute polyarthritis.

1	Several arthritis syndromes are associated with HIV infection. Reactive arthritis with painful lower-extremity oligoarthritis often follows an episode of urethritis in HIV-infected persons. HIV-associated reactive arthritis appears to be extremely common among persons with the HLA-B27 haplotype, but sacroiliac joint disease is unusual and is seen mostly in the absence of HLA-B27. Up to one-third of HIV-infected persons with psoriasis develop psoriatic arthritis. Painless monarthropathy and persistent symmetric polyarthropathy occasionally complicate HIV infection. Chronic persistent oligoarthritis of the shoulders, wrists, hands, and knees occurs in women infected with human T-lymphotropic virus type I. Synovial thickening, destruction of articular cartilage, and leukemic-appearing atypical lymphocytes in synovial fluid are characteristic, but progression to T cell leukemia is unusual.

1	Arthritis due to parasitic infection is rare. The guinea worm Dracunculus medinensis may cause destructive joint lesions in the lower extremities as migrating gravid female worms invade joints or cause ulcers in adjacent soft tissues that become secondarily infected. Hydatid cysts infect bones in 1–2% of cases of infection with Echinococcus granulosus. The expanding destructive cystic lesions may spread to and destroy adjacent joints, particularly the hip and pelvis. In rare cases, chronic synovitis has been associated with the presence of schistosomal eggs in synovial biopsies. Monarticular arthritis in children with lymphatic filariasis appears to respond to therapy with diethylcarbamazine even in the absence of microfilariae in synovial fluid. Reactive arthritis has been attributed to hookworm, Strongyloides, Cryptosporidium, and Giardia infection in case reports, but confirmation is required.

1	Reactive polyarthritis develops several weeks after ~1% of cases of nongonococcal urethritis and 2% of enteric infections, particularly those due to Yersinia enterocolitica, Shigella flexneri, Campylobacter jejuni, and Salmonella species. Only a minority of these patients have the other findings of classic reactive arthritis, including urethritis, conjunctivitis, uveitis, oral ulcers, and rash. Studies have identified microbial DNA or antigen in synovial fluid or blood, but the pathogenesis of this condition is poorly understood.

1	Reactive arthritis is most common among young men (except after Yersinia infection) and has been linked to the HLA-B27 locus as a potential genetic predisposing factor. Patients report painful, asymmetric oligoarthritis that affects mainly the knees, ankles, and feet. Low-back pain is common, and radiographic evidence of sacroiliitis is found in patients with long-standing disease. Most patients recover within 6 months, but prolonged recurrent disease is more common in cases that follow chlamydial urethritis. Anti-inflammatory agents help relieve symptoms, but the role of prolonged antibiotic therapy in eliminating microbial antigen from the synovium is controversial.

1	Migratory polyarthritis and fever constitute the usual presentation of acute rheumatic fever in adults (Chap. 381). This presentation is distinct from that of poststreptococcal reactive arthritis, which also follows infections with group A Streptococcus but is not migratory, lasts beyond the typical 3-week maximum of acute rheumatic fever, and responds poorly to aspirin.

1	Infection complicates 1–4% of total joint replacements. The majority of infections are acquired intraoperatively or immediately postoperatively as a result of wound breakdown or infection; less commonly, 838 these joint infections develop later after joint replacement and are the result of hematogenous spread or direct inoculation. The presentation may be acute, with fever, pain, and local signs of inflammation, especially in infections due to S. aureus, pyogenic streptococci, and enteric bacilli. Alternatively, infection may persist for months or years without causing constitutional symptoms when less virulent organisms, such as coagulase-negative staphylococci or diphtheroids, are involved. Such indolent infections usually are acquired during joint implantation and are discovered during evaluation of chronic unexplained pain or after a radiograph shows loosening of the prosthesis; the erythrocyte sedimentation rate and C-reactive protein level are usually elevated in such cases. The

1	evaluation of chronic unexplained pain or after a radiograph shows loosening of the prosthesis; the erythrocyte sedimentation rate and C-reactive protein level are usually elevated in such cases. The diagnosis is best made by needle aspiration of the joint; accidental introduction of organisms during aspiration must be avoided meticulously. Synovial fluid pleocytosis with a predominance of polymorphonuclear leukocytes is highly suggestive of infection, since other inflammatory processes uncommonly affect prosthetic joints. Culture and Gram’s stain usually yield the responsible pathogen. Sonication of explanted prosthetic material can improve the yield of culture, presumably by breaking up bacterial biofilms on the surfaces of prostheses. Use of special media for unusual pathogens such as fungi, atypical mycobacteria, and Mycoplasma may be necessary if routine and anaerobic cultures are negative.

1	Treatment includes surgery and high doses of parenteral antibiotics, which are given for 4–6 weeks because bone is usually involved. In most cases, the prosthesis must be replaced to cure the infection. Implantation of a new prosthesis is best delayed for several weeks or months because relapses of infection occur most commonly within this time frame. In some cases, reimplantation is not possible, and the patient must manage without a joint, with a fused joint, or even with amputation. Cure of infection without removal of the prosthesis is occasionally possible in cases that are due to streptococci or pneumococci and that lack radiologic evidence of loosening of the prosthesis. In these cases, antibiotic therapy must be initiated within several days of the onset of infection, and the joint should be drained vigorously by open arthrotomy or arthroscopically. In selected patients who prefer to avoid the high morbidity rate associated with joint removal and reimplantation, suppression of

1	should be drained vigorously by open arthrotomy or arthroscopically. In selected patients who prefer to avoid the high morbidity rate associated with joint removal and reimplantation, suppression of the infection with antibiotics may be a reasonable goal. A high cure rate with retention of the prosthesis has been reported when the combination of oral rifampin and ciprofloxacin is given for 3–6 months to persons with staphylococcal prosthetic joint infection of short duration. This approach, which is based on the ability of rifampin to kill organisms adherent to foreign material and in the stationary growth phase, requires confirmation in prospective trials.

1	To avoid the disastrous consequences of infection, candidates for joint replacement should be selected with care. Rates of infection are particularly high among patients with rheumatoid arthritis, persons who have undergone previous surgery on the joint, and persons with medical conditions requiring immunosuppressive therapy. Perioperative antibiotic prophylaxis, usually with cefazolin, and measures to decrease intraoperative contamination, such as laminar flow, have lowered the rates of perioperative infection to <1% in many centers. After implantation, measures should be taken to prevent or rapidly treat extra-articular infections that might give rise to hematogenous spread to the prosthesis. The effectiveness of prophylactic antibiotics for the prevention of hematogenous infection after dental procedures has not been demonstrated; in fact, viridans streptococci and other components of the oral flora are extremely unusual causes of prosthetic joint infection. Accordingly, the

1	after dental procedures has not been demonstrated; in fact, viridans streptococci and other components of the oral flora are extremely unusual causes of prosthetic joint infection. Accordingly, the American Dental Association and the American Academy of Orthopaedic Surgeons do not recommend antibiotic prophylaxis for most dental patients with total joint replacements and have stated that there is no convincing evidence to support its use. Similarly, guidelines issued by the American Urological Association and the American Academy of Orthopaedic Surgeons do not recommend the use of prophylactic antibiotics for most patients with prosthetic joints who are undergoing urologic procedures but state that prophylaxis should be considered in certain situations—e.g., for patients (especially immunocompromised patients) who are undergoing a procedure posing a relatively high risk of bacteremia (such as lithotripsy or surgery involving bowel segments).

1	The contributions of James H. Maguire and the late Scott J. Thaler to this chapter in earlier editions are gratefully acknowledged. Osteomyelitis, an infection of bone, can be caused by various microorganisms that arrive at bone through different routes. Spontaneous hematogenous osteomyelitis may occur in otherwise healthy individuals, whereas local microbial spread mainly affects either individuals who have underlying disease (e.g., vascular insufficiency) or patients who have compromised skin or other tissue barriers, with consequent exposure of bone. The latter situation typically follows surgery involving bone, such as sternotomy or orthopedic repair. The manifestations of osteomyelitis are different in children and adults. In children circulating microorganisms seed mainly long bones, whereas in adults the vertebral column is the most commonly affected site.

1	Management of osteomyelitis differs greatly depending on whether an implant is involved. The most important aim of the management of either type of osteomyelitis is to prevent progression to chronic osteomyelitis by rapid diagnosis and prompt treatment. Device-related bone and joint infection necessitates a multidisciplinary approach requiring antibiotic therapy and, in many cases, surgical removal of the device. The optimal duration of antibiotic treatment has not been established for any type of osteomyelitis in clinical trials. Therefore, the recommendations for therapy in this chapter reflect only expert opinions.

1	There is no generally accepted, comprehensive system for classification of osteomyelitis, primarily because of the multifaceted presentation of this infection. Different specialists are confronted with different facets of bone disease. Most often, however, general practitioners or internists are the first to encounter patients with the initial signs and symptoms of osteomyelitis. These primary care physicians should be able to recognize this disease in any of its forms. Osteomyelitis cases can be classified by various criteria, including pathogenesis, duration of infection, location of infection, and presence or absence of foreign material. The widely used Cierny-Mader staging system classifies osteomyelitis according to anatomic site, comorbidity, and radiographic findings, with stratification of long-bone osteomyelitis to optimize surgical management; this system encompasses both systemic and local factors affecting immune status, metabolism, and local vascularity.

1	Any of three mechanisms can underlie osteomyelitis: (1) hematogenous spread; (2) spread from a contiguous site following surgery; and (3) secondary infection in the setting of vascular insufficiency or concomitant neuropathy. Hematogenous osteomyelitis in adults typically involves the vertebral column. In only about half of patients can a primary focus be detected. The most common primary foci of infection are the urinary tract, skin/soft tissue, intravascular catheterization sites, and the endocardium. Spread from a contiguous source follows either bone trauma or surgical intervention. Wound infection leading to osteomyelitis typically occurs after cardiovascular intervention involving the sternum, orthopedic repair, or prosthetic joint insertion. Osteomyelitis secondary to vascular insufficiency or peripheral neuropathy most often follows chronic, progressively deep skin and soft tissue infection of the foot. The most common underlying condition is diabetes. In diabetes that is

1	insufficiency or peripheral neuropathy most often follows chronic, progressively deep skin and soft tissue infection of the foot. The most common underlying condition is diabetes. In diabetes that is poorly controlled, the diabetic foot syndrome is caused by skin, soft tissue, and bone ischemia combined with motor, sensory, and autonomic neuropathy.

1	Classification of osteomyelitis according to the duration of infection, although ill defined (because there is no clear time limit for the transition from acute to chronic osteomyelitis), is useful because the management of acute and chronic osteomyelitis differs. Whereas acute osteomyelitis can generally be treated with antibiotics alone, antibiotic treatment for chronic osteomyelitis should be combined with debridement surgery. Acute hematogenous or contiguous osteomyelitis evolves over a short period—i.e., a few days or weeks. In contrast, subacute or chronic osteomyelitis lasts for weeks or months before treatment is started. Typical examples of a subacute course are vertebral osteomyelitis due to tuberculosis or brucellosis and delayed implant-associated infections caused mainly by low-virulence microorganisms (coagulase-negative staphylococci, Propionibacterium acnes). Chronic osteomyelitis develops when insufficient therapy leads to persistence or recurrence, most often after

1	low-virulence microorganisms (coagulase-negative staphylococci, Propionibacterium acnes). Chronic osteomyelitis develops when insufficient therapy leads to persistence or recurrence, most often after sternal, mandibular, or foot infection.

1	Classification by location distinguishes among cases in the long bones, the vertebral column, and the periarticular bones. Long bones are generally involved after hematogenous seeding in children or contiguous spread following trauma or surgery. The risk of vertebral osteomyelitis in adults increases with age. Periarticular osteomyelitis, which complicates septic arthritis that has not been adequately treated, is especially common in periprosthetic joint infection. Osteomyelitis involving a foreign device requires surgical management for cure. Even acute implant-associated infection calls for prolonged antimicrobial therapy. Therefore, identification of this type of disease is of practical importance.

1	Vertebral osteomyelitis, also referred to as disk-space infection, septic diskitis, spondylodiskitis, or spinal osteomyelitis, is the most common manifestation of hematogenous bone infection in adults. This designation reflects a pathogenic process leading to involvement of the adjacent vertebrae and the corresponding intervertebral disk. In adults, the disk is avascular. Microorganisms invade via the segmental arterial circulation in adjacent endplates and then spread into the disk. Alternative routes of infection are retrograde seeding through the prevertebral venous plexus and direct inoculation during spinal surgery, epidural infiltration, or trauma. In the setting of implant surgery, microorganisms are inoculated either during the procedure or, if wound healing is impaired, in the early postoperative period.

1	Vertebral osteomyelitis occurs more often in male than in female patients (ratio, 1.5:1). The overall incidence is 2.4 cases/100,000 population. There is a clear age-dependent increase from 0.3 cases/100,000 at ages <20 years to 6.5 cases/100,000 at ages >70 years. The observed increase in reported cases during the past two decades may reflect improvements in diagnosis resulting from the broad availability of MRI technology. In addition, the fraction of cases of vertebral osteomyelitis acquired in association with health care is certainly increasing as a consequence of the rising number of spinal interventions and local infiltrations.

1	Vertebral osteomyelitis is typically classified as pyogenic or nonpyogenic. However, this distinction is arbitrary because, in “nonpyogenic” cases (tuberculous, brucellar), macroscopic pus formation (caseous necrosis, abscess) is quite common. A more accurate scheme is to classify cases as acute or subacute/chronic. Whereas the micro-839 biologic spectrum of acute cases is similar in different parts of the world, the spectrum of subacute/chronic cases varies according to the geographic region. The great majority of cases are monomicrobial in etiology. Of episodes of acute vertebral osteomyelitis, 40–50% are caused by Staphylococcus aureus, 12% by streptococci, and 20% by gram-negative bacilli—mainly Escherichia coli (9%) and Pseudomonas aeruginosa (6%). Subacute vertebral osteomyelitis is typically caused by Mycobacterium tuberculosis or Brucella species in regions where these microorganisms are endemic. Osteomyelitis due to viridans streptococci also has a subacute presentation;

1	is typically caused by Mycobacterium tuberculosis or Brucella species in regions where these microorganisms are endemic. Osteomyelitis due to viridans streptococci also has a subacute presentation; these infections most often occur as secondary foci in patients with endocarditis. In vertebral osteomyelitis due to Candida species, the diagnosis is often delayed by several weeks; this etiology should be suspected in IV drug users who do not use sterile paraphernalia. In implant-associated spinal osteomyelitis, coagulase-negative staphylococci and P. acnes—which, in the absence of an implant, are generally considered contaminants—typically cause low-grade (chronic) infections. As an exception, coagulasenegative staphylococci can cause native spinal osteomyelitis in cases of prolonged bacteremia (e.g., in patients with infected pacemaker electrodes or implanted vascular catheters that are not promptly removed).

1	The signs and symptoms of vertebral osteomyelitis are nonspecific. Only about half of patients develop fever >38°C (100.4°F), perhaps because analgesic drugs are frequently used by these patients. Back pain is the leading initial symptom (>85% of cases). The location of the pain corresponds to the site of infection: the cervical spine in ~10% of cases, the thoracic spine in 30%, and the lumbar spine in 60%. One exception is involvement at the thoracic level in two-thirds of cases of tuberculous osteomyelitis and at the lumbar level in only one-third. This difference is due to direct mycobacterial spread via pleural or mediastinal lymph nodes in pulmonary tuberculosis.

1	Neurologic deficits, such as radiculopathy, weakness, or sensory loss, are observed in about one-third of cases of vertebral osteomyelitis. In brucellar vertebral osteomyelitis, neurologic impairment is less frequent; in tuberculous osteomyelitis, it is about twice as frequent as in cases of other etiologies. Neurologic signs and symptoms are caused mostly by spinal epidural abscess. This complication starts with severe localized back pain and progresses to radicular pain, reflex changes, sensory abnormalities, motor weakness, bowel and bladder dysfunction, and paralysis. A primary focus should always be sought but is found in only half of cases. Overall, endocarditis is identified in ~10% of patients. In osteomyelitis caused by viridans streptococci, endocarditis is the source in about half of patients.

1	Implant-associated spinal osteomyelitis can present as either earlyor late-onset infection. Early-onset infection is diagnosed within 30 days after implant placement. S. aureus is the most common pathogen. Wound healing impairment and fever are the leading findings. Late-onset infection is diagnosed beyond 30 days after surgery, with low-virulence organisms such as coagulase-negative staphylococci or P. acnes as typical infecting agents. Fever is rare. One-quarter of patients have a sinus tract. Because of the delayed course and the lack of classic signs of infection, rapid diagnosis requires a high degree of suspicion.

1	Leukocytosis and neutrophilia have low levels of diagnostic sensitivity (only 65% and 40%, respectively). In contrast, an increased erythrocyte sedimentation rate or C-reactive protein (CRP) level has been reported in 98% and 100% of cases, respectively; thus, these tests are helpful in excluding vertebral osteomyelitis. The fraction of blood cultures that yield positive results depends heavily on whether the patient has been pretreated with antibiotics; across studies, the range is from 30% to 78%. In view of this low rate of positive blood culture after antibiotic treatment, such therapy should be withheld until microbial growth is proven unless the patient has sepsis syndrome. In patients with negative blood cultures, CT-guided or open biopsy is needed. Whether a CT-guided biopsy with a negative result is repeated or followed by 840 open biopsy depends on the experience of personnel at the specific center. Bone samples should be cultured for aerobic, anaerobic, and fungal agents,

1	a negative result is repeated or followed by 840 open biopsy depends on the experience of personnel at the specific center. Bone samples should be cultured for aerobic, anaerobic, and fungal agents, with a portion of the sample sent for histopathologic study. In cases with a subacute/chronic presentation, a suggestive history, or a granuloma detected during histopathologic analysis, mycobacteria and brucellae also should be sought. When blood and tissue cultures are negative despite suggestive histopathology, broad-range polymerase chain reaction analysis of biopsy specimens or aspirated pus should be considered. This technique allows detection of unusual pathogens such as Tropheryma whipplei. Given that signs and symptoms of osteomyelitis are nonspecific, the clinical differential diagnosis of febrile back pain is broad, including pyelonephritis, pancreatitis, and viral syndromes. In addition, multiple noninfectious pathologies of the vertebral column, such as osteoporotic fracture,

1	of febrile back pain is broad, including pyelonephritis, pancreatitis, and viral syndromes. In addition, multiple noninfectious pathologies of the vertebral column, such as osteoporotic fracture, seronegative spondylitis (ankylosing spondylitis, psoriasis, reactive arthritis, enteropathic arthritis), and spinal stenosis must be considered. Imaging procedures are the most important tools not only for the diagnosis of vertebral osteomyelitis but also for the detection of pyogenic complications and alternative conditions (e.g., bone metastases or osteoporotic fractures). Plain radiography is a reasonable first step in evaluating patients without neurologic symptoms and may reveal an alternative diagnosis. Because of its low sensitivity, plain radiography generally is not helpful in acute osteomyelitis, but it can be useful in subacute or chronic cases. The gold standard is MRI, which should be performed expeditiously in patients with neurologic impairment in order to rule out a herniated

1	but it can be useful in subacute or chronic cases. The gold standard is MRI, which should be performed expeditiously in patients with neurologic impairment in order to rule out a herniated disk or to detect pyogenic complications in a timely manner. Even if the pathologic findings on MRI suggest vertebral osteomyelitis, alternative diagnoses should be considered, especially when blood cultures are negative. The most common alternative diagnosis is erosive osteochondrosis. Septic bone necrosis, gouty spondylodiskitis, and erosive diskovertebral lesions (Andersson lesions) in ankylosing spondylitis may likewise mimic vertebral osteomyelitis. CT is less sensitive than MRI but may be helpful in guiding a percutaneous biopsy. In the future, positron-emission tomography (PET) with 18F-fluorodeoxyglucose, which has a high degree of diagnostic accuracy, may be an alternative imaging procedure when MRI is contraindicated. 18F-fluorodeoxyglucose PET should be considered for patients with

1	which has a high degree of diagnostic accuracy, may be an alternative imaging procedure when MRI is contraindicated. 18F-fluorodeoxyglucose PET should be considered for patients with implants and patients in whom several foci are suspected.

1	The aims of therapy for vertebral osteomyelitis are (1) elimination of the pathogen(s), (2) protection from further bone loss, (3) relief of back pain, (4) prevention of complications, and (5) stabilization, if needed.

1	Table 158-1 summarizes suggested antimicrobial regimens for infections attributable to the most common etiologic agents. For optimal antimicrobial therapy, identification of the infecting agent is required. Therefore, in patients without sepsis syndrome, antibiotics should not be administered until the pathogen is identified in a blood culture, a bone biopsy, or an aspirated pus collection. Traditionally, bone infections are at least initially treated by the IV route. Unfortunately, relevant controlled trials are lacking, and the preference for the IV route is not evidence based. There are no good arguments for the assumption that IV therapy is superior to oral administration if the following requirements are met: (1) optimal antibiotic spectrum, (2) excellent bioavailability of the oral drug, (3) clinical studies confirming efficacy of the oral drug, (4) normal intestinal function, and (5) no vomiting. However, a short initial course of parenteral therapy with a β-lactam antibiotic

1	drug, (3) clinical studies confirming efficacy of the oral drug, (4) normal intestinal function, and (5) no vomiting. However, a short initial course of parenteral therapy with a β-lactam antibiotic may lower the risk of emergence of fluoroquinolone resistance, especially if P. aeruginosa infection is treated with ciprofloxacin or staphylococcal infection with the combination of a fluoroquinolone plus rifampin. These suggestions are based on observational studies and expert opinion. There are no data from controlled trials on the optimal duration of therapy. Most experts suggest 6 weeks for patients

1	Microorganism Antimicrobial Agent (Dose,b Route) Staphylococcus spp. Streptococcus spp. Penicillin Gc (5 million units IV q6h) or ceftriaxone (2 g IV q24h)

1	Piperacillin-tazobactam (4.5 g IV q8h) plus an aminoglycosideg for 2–4 weeks aUnless otherwise indicated, the total duration of antimicrobial treatment is generally 6 weeks. bAll dosages are for adults with normal renal function. cWhen the patient has delayed-type penicillin hypersensitivity, cefuroxime (1.5 g IV q6–8h) can be administered. When the patient has immediate-type penicillin hypersensitivity, the penicillin should be replaced by vancomycin (1 g IV q12h). dTarget vancomycin trough level: 15–20 μg/ mL.e Trimethoprim-sulfamethoxazole. A double-strength tablet contains 160 mg of trimethoprim and 800 mg of sulfamethoxazole.f Including isolates producing extended-spectrum β-lactamase. gThe need for addition of an aminoglycoside has not yet been proven. However, this addition may decrease the risk of emergence of resistance to the β-lactam. hThe rationale for starting ciprofloxacin treatment only after pretreatment with a β-lactam is the increased risk of emergence of quinolone

1	the risk of emergence of resistance to the β-lactam. hThe rationale for starting ciprofloxacin treatment only after pretreatment with a β-lactam is the increased risk of emergence of quinolone resistance in the presence of a heavy bacterial load. iAlternatively, penicillin G (5 million units IV q6h) or ceftriaxone (2 g IV q24h) can be used against gram-positive anaerobes (e.g., Propionibacterium acnes), and metronidazole (500 mg IV/PO q8h) can be used against gram-negative anaerobes (e.g., Bacteroides spp.).

1	Source: From W Zimmerli: N Engl J Med 362:1022, 2010. © Massachusetts Medical Society. Reprinted with permission. who have acute osteomyelitis without an implant. According to an observational study, prolonging antibiotic therapy beyond 6 weeks does not improve the rate of recovery or lower the risk of recurrence. However, prolonged antibiotic therapy is recommended for patients with abscesses that have not been drained and patients with spinal implants. Treatment efficacy should be regularly monitored through inquiries about signs and symptoms (fever, pain) and assessment for signs of inflammation (elevated CRP concentrations). Follow-up MRI is appropriate only for patients with pyogenic complications, since the correlation between clinical healing and improvement on MRI is very poor.

1	Surgical treatment generally is not needed in acute hematogenous vertebral osteomyelitis. However, it is always necessary in implant-associated spinal infection. Early infections (those occurring up to 30 days after internal stabilization) can be cured with debridement, implant retention, and a 3-month course of antibiotics (Table 158-2). In contrast, in late infection with a duration of >30 days, implant removal and a 6-week-course of antibiotics (Table 158-1) Microorganism Antimicrobial Agenta (Dose, Route) Staphylococcus spp. Recommendation for initial treatment phase (2 weeks with implant) Methicillin-susceptible Rifampin (450 mg PO/IV q12hb) Vancomycin (15 mg/kg IV q12h) or daptomycin (6–8 mg/kg IV q24h) Staphylococcus spp. Recommendation after completion of initial treatment phase Streptococcus spp.e Penicillin Gc (18–24 million units/d IV in 6 divided doses) or ceftriaxone (2 g IV q24h) for 4 weeks Enterococcus spp.f

1	Streptococcus spp.e Penicillin Gc (18–24 million units/d IV in 6 divided doses) or ceftriaxone (2 g IV q24h) for 4 weeks Enterococcus spp.f Enterobacteriaceae A β-lactam selected in light of in vitro susceptibility profile for 2 weeksh Enterobacter spp.i and Cefepime or ceftazidime (2 g IV q8h) or meropenem (1 g IV q8hk) for 2–4 weeks nonfermenters j (e.g., aeruginosa) Propionibacterium spp. Penicillin Gc (18–24 million units/d IV in 6 divided doses) or clindamycin (600–900 mg IV q8h) for 2–4 weeks Gram-negative anaer-Metronidazole (500 mg IV/PO q8h) obes (e.g., Bacteroides spp.) Mixed bacteria (without Ampicillin-sulbactam (3 g IV q6h) or amoxicillin-clavulanatel (2.2 g IV q6h) or piperacillin-tazobactam (4.5 g IV q8h) or imipenem (500 methicillin-resistant mg IV q6h) or meropenem (1 g IV q8hk) for 2–4 weeks staphylococci)

1	Individualized oral regimens chosen in light of antimicrobial susceptibility aAntimicrobial agents should be chosen in light of the isolate’s in vitro susceptibility, the patient’s drug allergies and intolerances, potential drug interactions, and contraindications to specific drugs. All dosages recommended are for adults with normal renal and hepatic function. See text for total durations of antibiotic treatment. bOther dosages and intervals of administration with equivalent success rates have been reported. cWhen the patient has delayed-type penicillin hypersensitivity, cefazolin (2 g IV q8h) can be administered. When the patient has immediate-type penicillin hypersensitivity, the penicillin should be replaced by vancomycin (1 g IV q12h). dTrimethoprim-sulfamethoxazole. A double-strength tablet contains 160 mg of trimethoprim and 800 mg of sulfamethoxazole. eDetermination of the minimal inhibitory concentration (MIC) of penicillin is advisable. fCombination therapy with an

1	tablet contains 160 mg of trimethoprim and 800 mg of sulfamethoxazole. eDetermination of the minimal inhibitory concentration (MIC) of penicillin is advisable. fCombination therapy with an aminoglycoside is optional since its superiority to monotherapy for prosthetic joint infection is unproved. When using combination therapy, monitor signs of aminoglycoside ototoxicity and nephrotoxicity; the latter is potentiated by other nephrotoxic agents (e.g., vancomycin). gFor patients with hypersensitivity to penicillin, see treatment options for penicillin-resistant enterococci. hCiprofloxacin (PO or IV) can be administered to patients with hypersensitivity to β-lactams. iCeftriaxone and ceftazidime should not be administered for treatment targeting Enterobacter species, even strains that test susceptible in the laboratory, but can be used against nonfermenters. Strains producing extended-spectrum β-lactamases should not be treated with any cephalosporin, including cefepime. Enterobacter

1	susceptible in the laboratory, but can be used against nonfermenters. Strains producing extended-spectrum β-lactamases should not be treated with any cephalosporin, including cefepime. Enterobacter infections can also be treated with ertapenem (1 g IV q24h); however, ertapenem is not effective against Pseudomonas spp. and other nonfermenters. jAddition of an aminoglycoside is optional. Use of two active drugs can be considered in light of the patient’s clinical condition. kThe recommended dosage is in line with the guidelines of the Infectious Diseases Society of America. In Europe, 2 g IV q8h is suggested for P. aeruginosa infections. lNot available as an IV formulation in the United States.

1	Source: Modified from W Zimmerli et al: N Engl J Med 351:1645, 2004. © Massachusetts Medical Society. Reprinted with permission. are required for complete elimination of the infection. If implants (12%). Persistent pain despite normalization of CRP values indicates cannot be removed, oral suppressive long-term treatment should mechanical complications such as severe osteonecrosis or spinal instafollow the initial course of IV antibiotics. The optimal duration of bility. These patients require a consult with an experienced orthopedic suppressive therapy is unknown. However, if antibiotic therapy is surgeon. discontinued after, for example, 1 year, close clinical and laboratory (CRP) follow-up is needed.

1	Osteomyelitis in long bones is a consequence of hematogenous seed-COMPLICATIONS ing, exogenous contamination during trauma (open fracture), or peri-Complications include persistent pain, persistently increased CRP operative contamination during orthopedic repairs. Its presentation levels, and new-onset or persistent neurologic impairment. In cases is either acute (with a duration of days to a few weeks) or chronic. of persistent pain with or without signs of inflammation, paraver-Hematogenous infection in long bones typically occurs in children. tebral, epidural, or psoas abscesses (Fig. 158-1) must be sought. Ineffectively treated hematogenous osteomyelitis during childhood Epidural abscesses occur in 15–20% of cases. This complication is can progress to chronic disease. In adults, the leading pathogenic more common in the cervical column (30%) than in the lumbar spine source is exogenous infection, mainly associated with internal fixation devices. Chronic osteomyelitis can recur

1	leading pathogenic more common in the cervical column (30%) than in the lumbar spine source is exogenous infection, mainly associated with internal fixation devices. Chronic osteomyelitis can recur after a symptom-free interval of >50 years. Such recurrences are most common among elderly patients who developed osteomyelitis in the preantibiotic era.

1	FIGURE 158-1 CT scan of acute vertebral osteomyelitis (L1/L2) due to Staphylococcus aureus in a 64-year-old man. Low-grade fever persisted despite appropriate IV antibiotic therapy. The scan revealed a psoas abscess on the right side. In adults, most cases of long-bone osteomyelitis are posttraumatic or postsurgical; less frequently, late recurrence arises from hematogenous infections during childhood. The risk of infection depends on the type of fracture. After closed fracture, implant-associated infection occurs in fewer than 1% of patients. In contrast, after open fracture, the risk of osteomyelitis ranges from ~2% up to 16%, with the precise figure depending on the degree of tissue damage during trauma.

1	The spectrum of microorganisms causing hematogenous long-bone osteomyelitis does not differ from that in vertebral osteomyelitis. S. aureus is most commonly isolated from adult patients. In rare cases, mycobacteria or fungal agents such as Cryptococcus species, Sporothrix schenckii, Blastomyces dermatitidis, or Coccidioides species are found in patients who live or have traveled in endemic regions. Impaired cellular immunity (e.g., in HIV infection or after transplantation) predisposes to these etiologies. Coagulase-negative staphylococci are the second most common etiologic agents (after S. aureus) in implant-associated osteomyelitis. After open fracture, contiguous long-bone osteomyelitis is typically caused by gram-negative bacilli or a polymicrobial mixture of organisms.

1	The leading symptoms in adults with primary or recurrent hematogenous long-bone osteomyelitis are pain and low-grade fever. Infection occasionally manifests as clinical sepsis and local signs of inflammation (erythema and swelling). After internal fixation, osteomyelitis can be classified as acute (≤3 weeks) or chronic. Acute long-bone osteomyelitis manifests as signs of surgical site infection, such as erythema and impaired wound healing. Acute implant-associated infection may also follow hematogenous seeding at any time after implantation of a device. Typical symptoms are new-onset pain and signs of sepsis. Chronic infections are usually caused by low-virulence microorganisms or occur after ineffective treatment of early-onset infection.

1	FIGURE 158-2 A 42-year-old man who had had a malleolar frac-ture 6 weeks previously had persistent pain and slight inflam-mation after orthopedic repair. His infection was treated with oral antibiotics without debridement surgery. This insufficient manage-ment of an implant-associated Staphylococcus aureus infection was complicated by a sinus tract. Patients may present with persisting pain, subtle local signs of inflammation, intermittent discharge of pus, or fluctuating erythema over the scar (Fig. 158-2).

1	Patients may present with persisting pain, subtle local signs of inflammation, intermittent discharge of pus, or fluctuating erythema over the scar (Fig. 158-2). The diagnostic workup for acute hematogenous long-bone osteomyelitis is similar to that for vertebral osteomyelitis. Bone remodeling and thus marker uptake are increased for at least 1 year after surgery. Therefore, the three-phase bone scan is not useful during this interval. However, in late recurrences it allows rapid diagnosis at low cost. If the results are positive, CT is required in order to estimate the extent of inflamed tissue and to detect bone necrosis (sequesters). Implant-associated infection should be suspected if CRP values do not return to the normal range or rise after an initial decrease. Clinical and laboratory suspicion should prompt surgical exploration and sampling.

1	In chronic osteomyelitis of >1 year’s duration, single-photon emission CT plus conventional CT (SPECT/CT) is a good option, either with 99mTc methylene diphosphonate (99mTc-MDP)–labeled leukocytes or with labeled monoclonal antibodies to granulocytes. Surgical debridement is needed for diagnostic (biopsy culture, histology) and therapeutic reasons.

1	Treatment for acute hematogenous infection in long bones is identical to that for acute vertebral osteomyelitis (Table 158-1). The suggested duration of antibiotic therapy is 4–6 weeks. In contrast to chronic or implant-associated osteomyelitis, acute hematogenous infection does not require surgical intervention. Initial IV administration of antimicrobial agents is followed by long-term oral treatment. The duration of the initial IV phase of therapy has not been defined. The IV course can be as short as a couple of days if a drug with excellent bioavailability is available. In case of recurrence of chronic osteomyelitis as well as in each type of exogenous osteomyelitis (acute, chronic, with or without an implant), a combination of surgical debridement, obliteration of dead space, and long-term antibiotic therapy is needed.

1	The therapeutic aims in patients whose infections are associated with internal fixation devices are consolidation of the fracture and prevention of chronic osteomyelitis. Stable implants can be maintained except in patients with uncontrolled sepsis. Appropriate antimicrobial therapies are listed in Table 158-2. The cure rate for early staphylococcal implant-associated infections treated with a fluoroquinolone plus rifampin is >90%. Rifampin is efficacious against staphylococcal biofilms of ≤3 weeks’ duration. Similarly, fluoroquinolones are active against biofilms formed by gram-negative bacilli. In these cases, an initial 2-week course of IV therapy with a β-lactam is suggested in order to minimize the risk of emergence of resistance to the oral drugs. The total duration of treatment is 3 months, and the device can be retained even after antibiotics have been discontinued. In contrast, in cases caused by rifampin-resistant staphylococci or fluoroquinolone-resistant gram-negative

1	is 3 months, and the device can be retained even after antibiotics have been discontinued. In contrast, in cases caused by rifampin-resistant staphylococci or fluoroquinolone-resistant gram-negative bacilli, the hardware should be removed after consolidation of the fracture and before discontinuation of antibiotics. These patients are treated with an oral antibiotic (suppressive therapy) as long as retention of the hardware is necessary.

1	The main complication of long-bone osteomyelitis is the persistence of infection with progression to chronic osteomyelitis. This risk is especially high after internal fixation of an open fracture and among patients with implant-associated osteomyelitis that is treated without surgical debridement. In chronic osteomyelitis, recurrent sinus tracts result in severe damage to skin and soft tissue (Fig. 158-2). Patients who have chronic open wounds need a therapeutic approach combining orthopedic repair and plastic reconstructive surgery.

1	Implanted foreign material is highly susceptible to local infection due to local immunodeficiency around the device. Infection occurs by either the exogenous or the hematogenous route. More rarely, contiguous spread from adjacent sites of osteomyelitis or deep soft-tissue infection may cause periprosthetic joint infection (PJI). The fact that foreign devices are covered with host proteins such as fibronectin favors the adherence of staphylococci and the formation of a biofilm that resists phagocytosis. The risk of infection manifesting during the first 2 postoperative years varies according to the joint. It is lowest after hip and knee arthroplasty (0.3–1.5%) and highest after ankle and elbow replacement (4–10%). The risk of hematogenous PJI is highest in the early postoperative period. However, hematogenous seeding occurs throughout life, and most cases therefore develop >2 years after implantation.

1	About 70% of cases of PJI are caused by staphylococci (S. aureus and coagulase-negative staphylococci), 10% by streptococci, 10% by gram-negative bacilli, and the rest by various other microorganisms. All microorganisms can cause PJI, including fungi and mycobacteria. P. acnes causes up to one-third of episodes of periprosthetic shoulder infection. PJI is traditionally classified as early (<3 months after implantation), delayed (3–24 months after surgery), or late (>2 years after implantation). For therapeutic decision-making (see below), it is more useful to classify PJI as (1) acute hematogenous PJI with <3 weeks of symptoms, (2) early postinterventional PJI manifesting within 1 month after surgery, and (3) chronic PJI with symptom duration of >3 weeks.

1	Acute exogenous PJI typically presents with local signs of infection (Fig. 158-3). In contrast, acute hematogenous PJI, most often caused by S. aureus, is characterized by new-onset pain that initially is not accompanied by prominent local inflammatory signs. In most cases, an ongoing sepsis syndrome dominates the clinical picture. Key findings in chronic PJI are joint effusion, local pain, implant loosening, and occasionally a sinus tract. Chronic PJI is most commonly caused by low-virulence microorganisms such as coagulase-negative staphylococci or P. acnes. These infections are characterized by nonspecific symptoms, such as chronic pain caused by low-grade inflammation or early loosening. FIGURE 158-3 Early periprosthetic joint infection of the left hip caused by group B streptococci in a 68-year-old woman.

1	FIGURE 158-3 Early periprosthetic joint infection of the left hip caused by group B streptococci in a 68-year-old woman. Blood tests such as the measurement of CRP (elevated levels, ≥10 mg/L) and erythrocyte sedimentation rate (elevated rates, ≥30 mm/h) are sensitive (91–97%) but not specific (70–78%). Synovial fluid cell counts are ~90% sensitive and specific, with threshold values of 1700 leukocytes/μL in periprosthetic knee infection and 4200 leukocytes/μL in periprosthetic hip infection. During debridement surgery, at least three but optimally six tissue samples should be obtained for culture and histopathology. If implant material (modular parts, screws, or the prosthesis) is removed, sonication of this material followed by culture and/or use of molecular methods to examine the sonicate fluid allows the detection of microorganisms in biofilms.

1	The three-phase bone scan is very sensitive for detecting PJI but is not specific. As mentioned above, this test does not differentiate bone remodeling from infection and therefore is not useful during at least the first year after implantation. CT and MRI detect soft tissue infection, prosthetic loosening, and bone erosion, but imaging artifacts caused by metal implants limit their use. 18F-fluorodeoxyglucose PET is an alternative method with fair sensitivity and specificity for the detection of PJI. However, this technique is not yet an established procedure for this purpose.

1	Treatment of PJI requires a multidisciplinary approach involving an experienced orthopedic surgeon, an infectious disease specialist, a plastic reconstructive surgeon, and a microbiologist. Therefore, most patients are referred to a specialized center. In general, the goal of treatment is cure—i.e., a pain-free functional joint with complete eradication of the infecting pathogen(s). However, for patients with severe comorbidity, lifelong suppressive antimicrobial therapy may be preferred. As a rule, antimicrobial therapy without surgical intervention is not curative but merely suppressive. There are four curative surgical options: debridement and implant retention, one-stage implant exchange, two-stage implant exchange, and implant removal without replacement. Implant retention offers a good chance of infection-free survival (>80%) only if the following conditions are fulfilled: (1) acute infection, (2) stable implant, (3) pathogen susceptible to a biofilm-active antimicrobial agent

1	good chance of infection-free survival (>80%) only if the following conditions are fulfilled: (1) acute infection, (2) stable implant, (3) pathogen susceptible to a biofilm-active antimicrobial agent (see below), and (4) skin and soft tissue in good condition.

1	Table 158-2 summarizes pathogen-specific antimicrobial therapy for PJI. Initial IV therapy is followed by long-term oral antibiotics. Efficacious treatment is best defined in staphylococcal implant-associated infections. Rifampin exhibits excellent activity against 844 biofilms composed of susceptible staphylococci. Because of the risk of rapid emergence of resistance, rifampin must always be combined with another effective antibiotic. If gram-negative infections are treated with implant retention, fluoroquinolones should be used because of their activity against gram-negative biofilms.

1	As mentioned above, hematogenous seeding may occur throughout life. This risk is highest during S. aureus bacteremia from a distant focus. Therefore, documented bacterial infections should be promptly treated in patients with prosthetic joints. However, according to a large, prospective, case-control study, the risk of prosthetic hip or knee infection is not increased following dental procedures. Therefore, antibiotic prophylaxis is not needed during dental work.

1	Sternal osteomyelitis occurs primarily after sternal surgery (with the entry of exogenous organisms) and more rarely by hematogenous seeding or contiguous extension from adjacent sites of sternocostal arthritis. Exogenous sternal osteomyelitis after open sternal surgery is also called deep sternal wound infection. Exogenous infection may also follow minor sternal trauma, sternal fracture, and manubriosternal septic arthritis. Tuberculous sternal osteomyelitis typically manifests during hematogenous seeding in children or as reactivated infection in adults. Reactivation is sometimes preceded by blunt trauma. In rare cases, tuberculous sternal osteomyelitis is caused by continuous infection from an infected internal mammary lymph node.

1	The incidence of poststernotomy wound infection varies from 0.5% to 5%, but figures are even higher among patients with risk factors such as diabetes, obesity, chronic renal failure, emergency surgery, use of bilateral internal mammary arteries, and reexploration for bleeding. Rapid diagnosis and correct management of superficial sternal wound infection prevent its progression to sternal osteomyelitis. Primary (hematogenous) sternal osteomyelitis accounts for only 0.3% of all cases of osteomyelitis. Risk factors are IV drug use, HIV infection, radiotherapy, blunt trauma, cardiopulmonary resuscitation, alcohol abuse, liver cirrhosis, and hemoglobinopathy.

1	Poststernotomy osteomyelitis is generally caused by S. aureus (40–50% of cases), coagulase-negative staphylococci (15–30%), enterococci (5–12%), or gram-negative bacilli (15–25%). Fungal infections caused by Candida species also play a role. The fact that ~20% of cases are polymicrobial is indicative of exogenous superinfection during therapy. Hematogenous sternal osteomyelitis is caused most commonly by S. aureus. Other microorganisms play a role in special populations— e.g., P. aeruginosa in IV drug users, Salmonella species in individuals with sickle cell anemia, and M. tuberculosis in patients from endemic areas who have previously had tuberculosis.

1	Exogenous sternal osteomyelitis manifests as fever, increased local pain, erythema, wound discharge, and sternal instability (Fig. 158-4). Contiguous mediastinitis is a feared complication, occurring in ~10–30% of patients with sternal osteomyelitis. Hematogenous sternal osteomyelitis is characterized by sternal pain, swelling, and erythema. In addition, most patients have systemic signs and symptoms of sepsis. The differential diagnosis of hematogenous sternal osteomyelitis includes immunologic processes typically presenting as systemic or multifocal inflammation of the sternum or the sternoclavicular or sternocostal joints (e.g., SAPHO [synovitis, acne, pustulosis, hyperostosis, osteitis], vasculitis, and chronic multifocal relapsing osteomyelitis). FIGURE 158-4 Sternal osteomyelitis caused by Staphylococcus epi-dermidis 5 weeks after sternotomy for aortocoronary bypass in a 72-year-old man.

1	In primary sternal osteomyelitis, the diagnostic workup does not differ from that in other types of hematogenous osteomyelitis (see above). When a patient has grown up in regions where tuberculosis is endemic, a specific workup for mycobacterial infection should be performed, especially if osteomyelitis had its onset after a blunt sternal trauma. In secondary sternal osteomyelitis, leukocyte counts may be normal, but the CRP level is >100 mg/L in most cases. Tissue sampling for microbiologic studies is crucial. In osteomyelitis associated with sternal wires, low-virulence microorganisms, such as coagulasenegative staphylococci, play an important role. In order to differentiate between colonization and infection, samples from at least three deep biopsies should be subjected to microbiologic examination. Superficial swab cultures are not diagnostic and may be misleading. No studies have compared the value of the various imaging modalities in suspected primary sternal osteomyelitis.

1	examination. Superficial swab cultures are not diagnostic and may be misleading. No studies have compared the value of the various imaging modalities in suspected primary sternal osteomyelitis. However, MRI is the current gold standard for detection of each type of osteomyelitis.

1	In cases of deep sternal wound infection, antibiotic therapy should be started immediately after samples have been obtained for microbiologic analyses in order to control clinical sepsis. To protect a newly inserted heart valve, initial treatment should be directed against staphylococci, with consideration of the local susceptibility pattern. In centers with a high prevalence of methicillin-resistant

1	S. aureus, vancomycin or daptomycin should be added to a broad-spectrum β-lactam drug. As soon as cultures of blood and/or deep wound biopsies have confirmed the pathogen’s identity and susceptibility pattern, treatment should be optimized and narrowed accordingly. Tables 158-1 and 158-2 show appropriate therapeutic choices for the most frequently identified microorganisms causing sternal osteomyelitis in the absence and presence, respectively, of an implanted device. In a recent observational study of patients with staphylococcal deep sternal wound infection, the use of a rifampin-containing regimen was predictive of success. The optimal duration of antibiotic therapy has not been established. In acute sternal osteomyelitis without hardware, a 6-week course is the rule. In patients with remaining sternal wires, treatment duration is generally prolonged to 3 months (Table 158-2). Like other types of tuberculous bone infection, tuberculous sternal osteomyelitis is treated for 6–12

1	remaining sternal wires, treatment duration is generally prolonged to 3 months (Table 158-2). Like other types of tuberculous bone infection, tuberculous sternal osteomyelitis is treated for 6–12 months.

1	Primary sternal osteomyelitis can generally be treated without surgery. In contrast, in secondary sternal osteomyelitis, debridement is always required. This procedure should be performed by a team of experienced surgeons, since mediastinitis, bone infection, and skin and soft tissue damage may need to be treated during the same intervention. Primary sternal osteomyelitis poses a minimal mortality risk. In contrast, the in-hospital mortality rates from secondary sternal osteomyelitis are 15–30% after sternal surgery.

1	Primary sternal osteomyelitis poses a minimal mortality risk. In contrast, the in-hospital mortality rates from secondary sternal osteomyelitis are 15–30% after sternal surgery. Osteomyelitis of the foot usually occurs in patients with diabetes, peripheral arterial insufficiency, or peripheral neuropathy and after foot surgery. These entities are often linked to each other, especially in diabetic patients with late complications. However, foot osteomyelitis is also seen in patients with isolated peripheral neuropathy and can manifest as implant-associated osteomyelitis in patients without comorbidity due to a deep wound infection after foot surgery (hallux valgus surgery, arthrodesis, total ankle arthroplasty). Foot osteomyelitis is acquired almost exclusively by the exogenous route. It is a complication of deep pressure ulcers and of impaired wound healing after surgery.

1	The incidence of diabetic foot infection is 30–40 cases/1000 persons with diabetes per year. The condition starts with skin and soft tissue lesions and progresses to osteomyelitis, especially in patients with risk factors. About 60–80% of patients with diabetic foot infection have confirmed osteomyelitis. Diabetic foot osteomyelitis increases the risk of amputation. With adequate management of the early stage of diabetic foot infections, the rate of amputation can be lowered. Risk factors for diabetic foot infection are (1) peripheral motor, sensory, and autonomic neuropathy; (2) neuro-osteoarthropathic deformities (Charcot foot; Fig. 158-5); (3) arterial insufficiency; (4) uncontrolled hyperglycemia; (5) disabilities such as reduced vision; and (6) maladaptive behavior.

1	The correlation between cultures from bone biopsy and those from wound swabs or even deep soft tissue punctures is poor. Consistent results have been found in only 13–43% of cases in various studies. The 845 correlation is better when S. aureus is isolated (40–50%) than when anaerobes (20–35%), gram-negative bacilli (20–30%), or coagulasenegative staphylococci (0–20%) are identified. When only bone biopsy samples are considered, the leading pathogens are S. aureus (30–40%), anaerobes (10–20%), and various gram-negative bacilli (30–40%). The precise distribution depends on whether the patient already has been treated with antibiotics. Anaerobes are especially prevalent in chronic wounds. Pretreatment typically selects for P. aeruginosa or enterococci. FIGURE 158-5 Neuropathic joint disease (Charcot foot) compli-cated by chronic foot osteomyelitis in a 78-year old woman with diabetes mellitus complicated by severe neuropathy.

1	FIGURE 158-5 Neuropathic joint disease (Charcot foot) compli-cated by chronic foot osteomyelitis in a 78-year old woman with diabetes mellitus complicated by severe neuropathy. In many cases, foot osteomyelitis can be diagnosed clinically, without imaging procedures. Most clinicians rely on the “probe-to-bone” test, which has a positive predictive value of ~90% in populations with a high pretest probability. Thus, in a patient with diabetes who is hospitalized for a chronic deep foot ulcer, the diagnosis of foot osteomyelitis is highly probable if bone can be directly touched with a metal instrument. In a patient with a lower pretest probability, MRI should be performed because of its high degree of sensitivity (80–100%) and specificity (80–90%). Plain radiography has a sensitivity of only 30–90% and a specificity of only 50–90%; it may be considered for follow-up of patients with confirmed diabetic foot osteomyelitis.

1	As mentioned above, correlation between cultures of bone and those of wound swabs or wound punctures is poor. Antibiotic treatment should be based on bone culture. If no bone biopsy is performed, empirical therapy chosen in light of the most common infecting agents and the type of clinical syndrome should be given. Wound debridement combined with a 4to 6-week course of antibiotics has been shown to render amputation unnecessary in about two-thirds of patients. According to the 2012 Infectious Diseases Society of America Clinical Practice Guideline for the Diagnosis and Treatment of Diabetic Foot Infections, the following management strategies should be considered. If a foot ulcer is clinically infected, prompt empirical antimicrobial therapy may prevent progression to osteomyelitis. When the risk of methicillin-resistant S. aureus is considered high, an agent active against these strains (e.g., vancomycin) should be chosen. If the patient has not recently received antibiotics, the

1	the risk of methicillin-resistant S. aureus is considered high, an agent active against these strains (e.g., vancomycin) should be chosen. If the patient has not recently received antibiotics, the spectrum of the selected antibiotic must include gram-positive cocci (e.g., clindamycin, ampicillin-sulbactam). If the patient has received antibiotics within the past month, the spectrum of empirical antibiotics should include gram-negative bacilli (e.g., clindamycin plus a fluoroquinolone). If the patient has risk factors for Pseudomonas infection (previous colonization, residence in a warm climate, frequent exposure of the foot to water), an empirical antipseudomonal agent (e.g., piperacillin-tazobactam, cefepime) is indicated. If osteomyelitis is suspected either on clinical grounds (probe to bone) or on the basis of imaging procedures (MRI), bone biopsy should be performed. If not all infected bone is surgically removed, the patient should be treated for 4–6 weeks in line with the

1	to bone) or on the basis of imaging procedures (MRI), bone biopsy should be performed. If not all infected bone is surgically removed, the patient should be treated for 4–6 weeks in line with the identified pathogen(s) and their susceptibility. Treatment should initially be given by the IV route. Whether therapy can later be administered by the oral route depends on the bioavailability of oral drugs that cover the infecting agents. If dead bone cannot be removed, longterm therapy (at least 3 months) should be considered. In such cases, cure of osteomyelitis is usually the exception, and repetitive suppressive treatment may be needed.

1	846 Intraabdominal Infections and Abscesses Miriam Baron Barshak, Dennis L. Kasper Intraperitoneal infections generally arise because a normal anatomic barrier is disrupted. This disruption may occur when the appendix, a 159 diverticulum, or an ulcer ruptures; when the bowel wall is weakened by ischemia, tumor, or inflammation (e.g., in inflammatory bowel disease); or with adjacent inflammatory processes, such as pancreatitis or pelvic inflammatory disease, in which enzymes (in the former case) or organisms (in the latter) may leak into the peritoneal cavity. Whatever the inciting event, once inflammation develops and organisms usually contained within the bowel or another organ enter the normally sterile peritoneal space, a predictable series of events takes place. Intraabdominal infections occur in two stages: peritonitis and—if the patient survives this stage and goes untreated—abscess formation. The types of microorganisms predominating in each stage of infection are responsible

1	occur in two stages: peritonitis and—if the patient survives this stage and goes untreated—abscess formation. The types of microorganisms predominating in each stage of infection are responsible for the pathogenesis of disease.

1	Peritonitis is a life-threatening event that is often accompanied by bacteremia and sepsis syndrome (Chap. 325). The peritoneal cavity is large but is divided into compartments. The upper and lower peritoneal cavities are divided by the transverse mesocolon; the greater omentum extends from the transverse mesocolon and from the lower pole of the stomach to line the lower peritoneal cavity. The pancreas, duodenum, and ascending and descending colon are located in the anterior retroperitoneal space; the kidneys, ureters, and adrenals are found in the posterior retroperitoneal space. The other organs, including liver, stomach, gallbladder, spleen, jejunum, ileum, transverse and sigmoid colon, cecum, and appendix, are within the peritoneal cavity. The cavity is lined with a serous membrane that can serve as a conduit for fluids—a property exploited in peritoneal dialysis (Fig. 159-1).

1	FIGURE 159-1 Diagram of the intraperitoneal spaces, showing the circulation of fluid and potential areas for abscess formation. Some compartments collect fluid or pus more often than others. These compartments include the pelvis (the lowest portion), the subphrenic spaces on the right and left sides, and Morrison’s pouch, which is a posterosuperior extension of the subhepatic spaces and is the lowest part of the paravertebral groove when a patient is recumbent. The falciform ligament separating the right and left subphrenic spaces appears to act as a barrier to the spread of infection; consequently, it is unusual to find bilateral subphrenic collections. (Reprinted with permission from B Lorber [ed]: Atlas of Infectious Diseases, vol VII: Intra-abdominal Infections, Hepatitis, and Gastroenteritis. Philadelphia, Current Medicine, 1996, p 1.13.)

1	A small amount of serous fluid is normally present in the peritoneal space, with a protein content (consisting mainly of albumin) of <30 g/L and <300 white blood cells (WBCs, generally mononuclear cells) per microliter. In bacterial infections, leukocyte recruitment into the infected peritoneal cavity consists of an early influx of polymorphonuclear leukocytes (PMNs) and a prolonged subsequent phase of mononuclear cell migration. The phenotype of the infiltrating leukocytes during the course of inflammation is regulated primarily by resident-cell chemokine synthesis.

1	Peritonitis is either primary (without an apparent source of contamination) or secondary. The types of organisms found and the clinical presentations of these two processes are different. In adults, primary bacterial peritonitis (PBP) occurs most commonly in conjunction with cirrhosis of the liver (frequently the result of alcoholism). However, the disease has been reported in adults with metastatic malignant disease, postnecrotic cirrhosis, chronic active hepatitis, acute viral hepatitis, congestive heart failure, systemic lupus erythematosus, and lymph-edema as well as in patients with no underlying disease. Although PBP virtually always develops in patients with preexisting ascites, it is, in general, an uncommon event, occurring in ≤10% of cirrhotic patients. The cause of PBP has not been established definitively but is believed to involve hematogenous spread of organisms in a patient in whom a diseased liver and altered portal circulation result in a defect in the usual

1	has not been established definitively but is believed to involve hematogenous spread of organisms in a patient in whom a diseased liver and altered portal circulation result in a defect in the usual filtration function. Organisms multiply in ascites, a good medium for growth. The proteins of the complement cascade have been found in peritoneal fluid, with lower levels in cirrhotic patients than in patients with ascites of other etiologies. The opsonic and phagocytic properties of PMNs are diminished in patients with advanced liver disease. Cirrhosis is associated with alterations in the gut microbiota, including an increased prevalence of potentially pathogenic bacteria such as Enterobacteriaceae. Small-intestinal bacterial overgrowth is frequently present in advanced stages of liver cirrhosis and has been linked with pathologic bacterial translocation and PBP. Factors promoting these changes in cirrhosis may include deficiencies in Paneth cell defensins, reduced intestinal motility,

1	and has been linked with pathologic bacterial translocation and PBP. Factors promoting these changes in cirrhosis may include deficiencies in Paneth cell defensins, reduced intestinal motility, decreased pancreatobiliary secretions, and portal-hypertensive enteropathy.

1	The presentation of PBP differs from that of secondary peritonitis. The most common manifestation is fever, which is reported in up to 80% of patients. Ascites is found but virtually always predates infection. Abdominal pain, an acute onset of symptoms, and peritoneal irritation during physical examination can be helpful diagnostically, but the absence of any of these findings does not exclude this often-subtle diagnosis. Nonlocalizing symptoms (such as malaise, fatigue, or encephalopathy) without another clear etiology should also prompt consideration of PBP in a susceptible patient. It is vital to sample the peritoneal fluid of any cirrhotic patient with ascites and fever. The finding of >250 PMNs/μL is diagnostic for PBP, according to Conn (http://jac.oxfordjournals.org/cgi/content/full/47/3/369). This criterion does not apply to secondary peritonitis (see below). The microbiology of PBP is also distinctive. While enteric gram-negative bacilli such as Escherichia coli are most

1	This criterion does not apply to secondary peritonitis (see below). The microbiology of PBP is also distinctive. While enteric gram-negative bacilli such as Escherichia coli are most commonly encountered, gram-positive organisms such as streptococci, enterococci, or even pneumococci are sometimes found. In an important development, widespread use of quinolones to prevent PBP in high-risk subgroups of patients, frequent hospitalizations, and exposure to broad-spectrum antibiotics have led to a change in flora of infections in patients with cirrhosis, with more gram-positive bacteria and extended-spectrum β-lactamase–producing Enterobacteriaceae in recent years. Risk factors for multiresistant infections include nosocomial origin of infection, long-term norfloxacin prophylaxis, recent infection with multiresistant bacteria, and recent use of β-lactam antibiotics. In PBP, a single organism is typically isolated; anaerobes are found less frequently in PBP than in secondary peritonitis, in

1	with multiresistant bacteria, and recent use of β-lactam antibiotics. In PBP, a single organism is typically isolated; anaerobes are found less frequently in PBP than in secondary peritonitis, in which a mixed flora including anaerobes is the rule. In fact, if PBP is suspected and multiple organisms including anaerobes are recovered from the peritoneal fluid, the diagnosis must be reconsidered and a source of secondary peritonitis sought.

1	FIGURE 159-2 Pneumoperitoneum. Free air under the diaphragm on an upright chest film suggests the presence of a bowel perfora-tion and associated peritonitis. (Image courtesy of Dr. John Braver; with permission.)

1	The diagnosis of PBP is not easy. It depends on the exclusion of a primary intraabdominal source of infection. Contrast-enhanced CT is useful in identifying an intraabdominal source for infection. It may be difficult to recover organisms from cultures of peritoneal fluid, presumably because the burden of organisms is low. However, the yield can be improved if 10 mL of peritoneal fluid is placed directly into a blood culture bottle. Because bacteremia frequently accompanies PBP, blood should be cultured simultaneously. To maximize the yield, culture samples should be collected prior to administration of antibiotics. No specific radiographic studies are helpful in the diagnosis of PBP. A plain film of the abdomen would be expected to show ascites. Chest and abdominal radiography should be performed in patients with abdominal pain to exclude free air, which signals a perforation (Fig. 159-2).

1	Treatment for PBP is directed at the isolate from blood or peritoneal fluid. Gram’s staining of peritoneal fluid often gives negative results in PBP. Therefore, until culture results become available, therapy should cover gram-negative aerobic bacilli and gram-positive cocci. Third-generation cephalosporins such as cefotaxime (2 g q8h, administered IV) provide reasonable initial coverage in moderately ill patients. Broad-spectrum antibiotics, such as penicillin/ β-lactamase inhibitor combinations (e.g., piperacillin/tazobactam, 3.375 g q6h IV for adults with normal renal function) or ceftriaxone (2 g q24h IV), are also options. Broader empirical coverage aimed at resistant hospital-acquired gram-negative bacteria (e.g., treatment with carbapenem) may be appropriate for nosocomially acquired PBP until culture results become available. Empirical coverage for anaerobes is not necessary. A mortality benefit from albumin (1.5 g/ kg of body weight within 6 h of detection and 1.0 g/kg on day

1	PBP until culture results become available. Empirical coverage for anaerobes is not necessary. A mortality benefit from albumin (1.5 g/ kg of body weight within 6 h of detection and 1.0 g/kg on day 3) has been demonstrated for patients who present with serum creatinine levels ≥1 mg/dL, blood urea nitrogen levels ≥30 mg/dL, or total bilirubin levels ≥4 mg/dL but not for patients who do not meet these criteria. After the infecting organism is identified, therapy should be narrowed to target the specific pathogen. Patients with PBP usually respond within 72 h to appropriate antibiotic therapy. Antimicrobial treatment can be administered for as little as 5 days if rapid improve-847 ment occurs and blood cultures are negative, but a course of up to 2 weeks may be required for patients with bacteremia and for those whose improvement is slow. Persistence of WBCs in the ascitic fluid after therapy should prompt a search for additional diagnoses.

1	Prevention • primary prevention Several observational studies and a meta-analysis raise the concern that proton pump inhibitor therapy may increase the risk of PBP. No prospective studies have yet addressed whether avoidance of such therapy may prevent PBP. Nonselective beta blockers may prevent secondary bacterial peritonitis. A 2012 guideline from the American Association for the Study of Liver Diseases recommends chronic antibiotic prophylaxis with a regimen described in the next section for patients who are at highest risk for PBP—that is, those with an ascitic-fluid total protein level <1.5 g/ dL along with impaired renal function (creatinine, ≥1.2 mg/dL; blood urea nitrogen, ≥25 mg/dL; or serum sodium, ≤130 mg/dL) and/or liver failure (Child-Pugh score, ≥9; and bilirubin, ≥3 mg/dL). A 7-day course of antibiotic prophylaxis is recommended for patients with cirrhosis and gastrointestinal bleeding.

1	secondary prevention PBP has a high rate of recurrence. Up to 70% of patients experience a recurrence within 1 year. Antibiotic prophylaxis is recommended for patients with a history of PBP to reduce this rate to <20% and improve short-term survival rates. Prophylactic regimens for adults with normal renal function include fluoroquinolones (ciprofloxacin, 750 mg weekly; norfloxacin, 400 mg/d) or trimethoprimsulfamethoxazole (one double-strength tablet daily). However, longterm administration of broad-spectrum antibiotics in this setting has been shown to increase the risk of severe staphylococcal infections.

1	Secondary peritonitis develops when bacteria contaminate the peritoneum as a result of spillage from an intraabdominal viscus. The organisms found almost always constitute a mixed flora in which facultative gram-negative bacilli and anaerobes predominate, especially when the contaminating source is colonic. Early in the course of infection, when the host response is directed toward containment, exudate containing fibrin and PMNs is found. Early death in this setting is attributable to gram-negative bacillary sepsis and to potent endotoxins circulating in the bloodstream (Chap. 325). Gram-negative bacilli, particularly

1	E. coli, are common bloodstream isolates, but Bacteroides fragilis bacteremia also occurs. The severity of abdominal pain and the clinical course depend on the inciting process. The organisms isolated from the peritoneum also vary with the source of the initial process and the normal flora at that site. Secondary peritonitis can result primarily from chemical irritation and/or bacterial contamination. For example, as long as the patient is not achlorhydric, a ruptured gastric ulcer will release low-pH gastric contents that will serve as a chemical irritant. The normal flora of the stomach comprises the same organisms found in the oropharynx but in lower numbers. Thus, the bacterial burden in a ruptured ulcer is negligible compared with that in a ruptured appendix. The normal flora of the colon below the ligament of Treitz contains ~1011 anaerobic organisms/g of feces but only 108 aerobes/g; therefore, anaerobic species account for 99.9% of the bacteria. Leakage of colonic contents (pH

1	below the ligament of Treitz contains ~1011 anaerobic organisms/g of feces but only 108 aerobes/g; therefore, anaerobic species account for 99.9% of the bacteria. Leakage of colonic contents (pH 7–8) does not cause significant chemical peritonitis, but infection is intense because of the heavy bacterial load.

1	Depending on the inciting event, local symptoms may occur in secondary peritonitis—for example, epigastric pain from a ruptured gastric ulcer. In appendicitis (Chap. 356), the initial presenting symptoms are often vague, with periumbilical discomfort and nausea followed in a number of hours by pain more localized to the right lower quadrant. Unusual locations of the appendix (including a retrocecal position) can complicate this presentation further. Once infection has spread to the peritoneal cavity, pain increases, particularly with infection involving the parietal peritoneum, which is innervated extensively. Patients usually lie motionless, often with knees drawn up to avoid stretching the nerve fibers of the peritoneal cavity. Coughing and sneezing, which increase pressure within the peritoneal cavity, are associated with sharp 848 pain. There may or may not be pain localized to the infected or diseased organ from which secondary peritonitis has arisen. Patients with secondary

1	peritoneal cavity, are associated with sharp 848 pain. There may or may not be pain localized to the infected or diseased organ from which secondary peritonitis has arisen. Patients with secondary peritonitis generally have abnormal findings on abdominal examination, with marked voluntary and involuntary guarding of the anterior abdominal musculature. Later findings include tenderness, especially rebound tenderness. In addition, there may be localized findings in the area of the inciting event. In general, patients are febrile, with marked leukocytosis and a left shift of the WBCs to band forms. While recovery of organisms from peritoneal fluid is easier in secondary than in primary peritonitis, a tap of the abdomen is rarely the procedure of choice in secondary peritonitis. An exception is in cases involving trauma, where the possibility of a hemoperitoneum may need to be excluded early. Emergent studies (such as abdominal CT) to find the source of peritoneal contamination should be

1	is in cases involving trauma, where the possibility of a hemoperitoneum may need to be excluded early. Emergent studies (such as abdominal CT) to find the source of peritoneal contamination should be undertaken if the patient is hemodynamically stable; unstable patients may require surgical intervention without prior imaging.

1	Treatment for secondary peritonitis includes early administration of antibiotics aimed particularly at aerobic gram-negative bacilli and anaerobes (see below). Mild to moderate disease can be treated with many drugs covering these organisms, including broad-spectrum penicillin/β-lactamase inhibitor combinations (e.g., ticarcillin/clavulanate, 3.1 g q4–6h IV), cefoxitin (2 g q4–6h IV), or a combination of either a fluoroquinolone (e.g., levofloxacin, 750 mg q24h IV) or a third-generation cephalosporin (e.g., ceftriaxone, 2 g q24h IV) plus metronidazole (500 mg q8h IV). Patients in intensive care units should receive imipenem (500 mg q6h IV), meropenem (1 g q8h IV), or combinations of drugs, such as ampicillin plus metronidazole plus ciprofloxacin. The role of enterococci and Candida species in mixed infections is controversial. Secondary peritonitis usually requires both surgical intervention to address the inciting process and antibiotics to treat early bacteremia, to decrease the

1	in mixed infections is controversial. Secondary peritonitis usually requires both surgical intervention to address the inciting process and antibiotics to treat early bacteremia, to decrease the incidence of abscess formation and wound infection, and to prevent distant spread of infection. Although surgery is rarely indicated in PBP in adults, it may be life-saving in secondary peritonitis. Recombinant human activated protein C (APC) was considered at one time for treatment of severe sepsis from causes including secondary peritonitis but was withdrawn from the market in 2011 after it was determined that the drug was associated with an increased risk of bleeding and that evidence for its beneficial effects was inadequate. Thus APC should not be used for sepsis or septic shock outside randomized clinical trials.

1	Peritonitis may develop as a complication of abdominal surgeries. These infections may be accompanied by localizing pain and/ or nonlocalizing signs or symptoms such as fever, malaise, anorexia, and toxicity. As a nosocomial infection, postoperative peritonitis may be associated with organisms such as staphylococci, components of the gram-negative hospital microflora, and the microbes that cause PBP and secondary peritonitis, as described above.

1	A third type of peritonitis arises in patients who are undergoing continuous ambulatory peritoneal dialysis (CAPD). Unlike PBP and secondary peritonitis, which are caused by endogenous bacteria, CAPD-associated peritonitis usually involves skin organisms. The pathogenesis of infection is similar to that of intravascular device– related infection, in which skin organisms migrate along the catheter, which both serves as an entry point and exerts the effects of a foreign body. Exit-site or tunnel infection may or may not accompany CAPD-associated peritonitis. Like PBP, CAPD-associated peritonitis is usually caused by a single organism. Peritonitis is, in fact, the most common reason for discontinuation of CAPD. Improvements in equipment design, especially the Y-set connector, have resulted in a decrease from one case of peritonitis per 9 months of CAPD to one case per 24 months.

1	The clinical presentation of CAPD peritonitis resembles that of secondary peritonitis in that diffuse pain and peritoneal signs are common. The dialysate is usually cloudy and contains >100 WBCs/μL, >50% of which are neutrophils. However, the number of cells depends in part on dwell time. According to a guideline from the International Society for Peritoneal Dialysis (2010), for patients undergoing automated peritoneal dialysis who present during their nighttime treatment and whose dwell time is much shorter than with CAPD, the clinician should use the percentage of PMNs rather than the absolute number of WBCs to diagnose peritonitis. As the normal peritoneum has very few PMNs, a proportion above 50% is strong evidence of peritonitis even if the absolute WBC count does not reach 100/μL. Meanwhile, patients undergoing automated peritoneal dialysis without a daytime exchange who present with abdominal pain may have no fluid to withdraw, in which case 1 L of dialysate should be infused

1	Meanwhile, patients undergoing automated peritoneal dialysis without a daytime exchange who present with abdominal pain may have no fluid to withdraw, in which case 1 L of dialysate should be infused and permitted to dwell a minimum of 1–2 h, then drained, examined for turbidity, and sent for cell count with differential and culture. The differential (with a shortened dwell time) may be more useful than the absolute WBC count. In equivocal cases or in patients with systemic or abdominal symptoms in whom the effluent appears clear, a second exchange is performed, with a dwell time of at least 2 h. Clinical judgment should guide initiation of therapy.

1	The most common organisms are Staphylococcus species, which accounted for ~45% of cases in one series. Historically, coagulasenegative staphylococcal species were identified most commonly in these infections, but these isolates have more recently been decreasing in frequency. Staphylococcus aureus is more often involved among patients who are nasal carriers of the organism than among those who are not, and this organism is the most common pathogen in overt exit-site infections. Gram-negative bacilli and fungi such as Candida species are also found. Vancomycin-resistant enterococci and vancomycinintermediate S. aureus have been reported to produce peritonitis in CAPD patients. The finding of more than one organism in dialysate culture should prompt evaluation for secondary peritonitis. As with PBP, culture of dialysate fluid in blood culture bottles improves the yield. To facilitate diagnosis, several hundred milliliters of removed dialysis fluid should be concentrated by

1	As with PBP, culture of dialysate fluid in blood culture bottles improves the yield. To facilitate diagnosis, several hundred milliliters of removed dialysis fluid should be concentrated by centrifugation before culture.

1	S. aureus, coagulase-negative Staphylococcus, and gram-negative bacilli until the results of cultures become available. Guidelines suggest that agents should be chosen on the basis of local experience with resistant organisms. In some centers, a first-generation cephalosporin such as cefazolin (for gram-positive bacteria) and a fluoroquinolone or a third-generation cephalosporin such as ceftazidime (for gram-negative bacteria) may be reasonable; in areas with high rates of infection with methicillin-resistant S. aureus, vancomycin should be used instead of cefazolin, and gram-negative coverage may need to be broadened—e.g., with an aminoglycoside, ceftazidime, cefepime, or carbapenem. Broad coverage including vancomycin should be particularly considered for toxic patients and for those with exit-site infections. Vancomycin should also be included in the regimen if the patient has a history of colonization or infection with methicillin-resistant S. aureus or has a history of severe

1	with exit-site infections. Vancomycin should also be included in the regimen if the patient has a history of colonization or infection with methicillin-resistant S. aureus or has a history of severe allergy to penicillins and cephalosporins. Loading doses are administered intraperitoneally; doses depend on the dialysis method and the patient’s renal function. Antibiotics are given either continuously (i.e., with each exchange) or intermittently (i.e., once daily, with the dose allowed to remain in the peritoneal cavity for at least 6 h). If the patient is severely ill, IV antibiotics should be added at doses appropriate for the patient’s degree of renal failure. The clinical response to an empirical treatment regimen should be rapid; if the patient has not responded after 48–96 h of treatment, new samples should be collected for cell counts and cultures, and catheter removal should be considered. For patients who lack exit-site or tunnel infection, the typical duration of antibiotic

1	new samples should be collected for cell counts and cultures, and catheter removal should be considered. For patients who lack exit-site or tunnel infection, the typical duration of antibiotic treatment is 14 days. For patients with exit-site or tunnel infection, catheter removal should be considered, and a longer duration of antibiotic therapy (up to 21 days) may be appropriate. In fungal infections, the catheter should be removed immediately.

1	See Chap. 202. Abscess formation is common in untreated peritonitis if overt gram-negative sepsis either does not develop or develops but is not fatal. In experimental models of abscess formation, mixed aerobic and anaerobic organisms have been implanted intraperitoneally. Without therapy directed at anaerobes, animals develop intraabdominal abscesses. As in humans, these experimental abscesses may stud the peritoneal cavity, lie within the omentum or mesentery, or even develop on the surface of or within viscera such as the liver.

1	Pathogenesis and Immunity There is often disagreement about whether an abscess represents a disease state or a host response. In a sense, it represents both: while an abscess is an infection in which viable infecting organisms and PMNs are contained in a fibrous capsule, it is also a process by which the host confines microbes to a limited space, thereby preventing further spread of infection. In any event, abscesses do cause significant symptoms, and patients with abscesses can be quite ill. Experimental work has helped to define both the host cells and the bacterial virulence factors responsible—most notably in the case of B. fragilis. This organism, although accounting for only 0.5% of the normal colonic flora, is the anaerobe most frequently isolated from intraabdominal infections, is especially prominent in abscesses, and is the most common anaerobic bloodstream isolate. On clinical grounds, therefore, B. fragilis appears to be uniquely virulent. Moreover, B. fragilis acts alone

1	especially prominent in abscesses, and is the most common anaerobic bloodstream isolate. On clinical grounds, therefore, B. fragilis appears to be uniquely virulent. Moreover, B. fragilis acts alone to cause abscesses in animal models of intraabdominal infection, whereas most other Bacteroides species must act synergistically with a facultative organism to induce abscess formation.

1	Of the several virulence factors identified in B. fragilis, one is critical: the capsular polysaccharide complex found on the bacterial surface. This complex comprises at least eight distinct surface polysaccharides. Structural analysis of these polysaccharides has shown an unusual motif of oppositely charged sugars. Polysaccharides having these zwitterionic characteristics, such as polysaccharide A, evoke a host response in the peritoneal cavity that localizes bacteria into abscesses. B. fragilis and polysaccharide A have been found to adhere to primary mesothelial cells in vitro; this adherence, in turn, stimulates the production of tumor necrosis factor α and intercellular adhesion molecule 1 by peritoneal macrophages. Although abscesses characteristically contain PMNs, the process of abscess induction depends on the stimulation of T lymphocytes by these unique zwitterionic polysaccharides. The stimulated CD4+ T lymphocytes secrete leukoattractant cytokines and chemokines. The

1	of abscess induction depends on the stimulation of T lymphocytes by these unique zwitterionic polysaccharides. The stimulated CD4+ T lymphocytes secrete leukoattractant cytokines and chemokines. The alternative pathway of complement and fibrinogen also participate in abscess formation.

1	While antibodies to the capsular polysaccharide complex enhance bloodstream clearance of B. fragilis, CD4+ T cells are critical in immunity to abscesses. When administered subcutaneously, B. fragilis polysaccharide A has immunomodulatory characteristics and stimulates CD4+ T regulatory cells via an interleukin 2–dependent mechanism to produce interleukin 10. Interleukin 10 downregulates the inflammatory response, thereby preventing abscess formation.

1	Clinical Presentation Of all intraabdominal abscesses, 74% are intra-peritoneal or retroperitoneal and are not visceral. Most intraperitoneal abscesses result from fecal spillage from a colonic source, such as an inflamed appendix. Abscesses can also arise from other processes. They usually form within weeks of the development of peritonitis and may be found in a variety of locations from omentum to mesentery, pelvis to psoas muscles, and subphrenic space to a visceral organ such as the liver, where they may develop either on the surface of the organ or within it. Periappendiceal and diverticular abscesses occur commonly. Diverticular abscesses are least likely to rupture. Infections of the female genital tract and pancreatitis are also among the more 849 common causative events. When abscesses occur in the female genital tract—either as a primary infection (e.g., tuboovarian abscess) or as an infection extending into the pelvic cavity or peritoneum—B. fragilis figures prominently

1	abscesses occur in the female genital tract—either as a primary infection (e.g., tuboovarian abscess) or as an infection extending into the pelvic cavity or peritoneum—B. fragilis figures prominently among the organisms isolated. B. fragilis is not found in large numbers in the normal vaginal flora. For example, it is encountered less commonly in pelvic inflammatory disease and endometritis without an associated abscess. In pancreatitis with leakage of damaging pancreatic enzymes, inflammation is prominent. Therefore, clinical findings such as fever, leukocytosis, and even abdominal pain do not distinguish pancreatitis itself from complications such as pancreatic pseudocyst, pancreatic abscess (Chap. 371), or intraabdominal collections of pus. Especially in cases of necrotizing pancreatitis, in which the incidence of local pancreatic infection may be as high as 30%, needle aspiration under CT guidance is performed to sample fluid for culture. Many centers prescribe preemptive

1	in which the incidence of local pancreatic infection may be as high as 30%, needle aspiration under CT guidance is performed to sample fluid for culture. Many centers prescribe preemptive antibiotics for patients with necrotizing pancreatitis. Imipenem is frequently used for this purpose because it reaches high tissue levels in the pancreas (although it is not unique in this regard). Recent randomized controlled studies have not demonstrated a benefit from this practice, and some guidelines no longer recommend preemptive antibiotics for patients with acute pancreatitis. If needle aspiration yields infected fluid in the setting of acute necrotizing pancreatitis, antibiotic treatment is appropriate in conjunction with surgical and/or percutaneous drainage of infected material. Infected pseudocysts that occur remotely from acute pancreatitis are unlikely to be associated with significant amounts of necrotic tissue and may be treated with either surgical or percutaneous catheter drainage

1	that occur remotely from acute pancreatitis are unlikely to be associated with significant amounts of necrotic tissue and may be treated with either surgical or percutaneous catheter drainage in conjunction with appropriate antibiotic therapy. Diagnosis Scanning procedures have considerably facilitated the diagnosis of intraabdominal abscesses. Abdominal CT probably has the highest yield, although ultrasonography is particularly useful for the right upper quadrant, kidneys, and pelvis. Both indium-labeled WBCs and gallium tend to localize in abscesses and may be useful in finding a collection. Because gallium is taken up in the bowel, indium-labeled WBCs may have a slightly greater yield for abscesses near the bowel. Neither indium-labeled WBC nor gallium scans serve as a basis for a definitive diagnosis, however; both need to be followed by other, more specific studies, such as CT, if an area of possible abnormality is identified. Abscesses contiguous with or contained within

1	a definitive diagnosis, however; both need to be followed by other, more specific studies, such as CT, if an area of possible abnormality is identified. Abscesses contiguous with or contained within diverticula are particularly difficult to diagnose with scanning procedures. Although barium should not be injected if a perforation is suspected, a barium enema occasionally may detect a diverticular abscess not diagnosed by other procedures. If one study is negative, a second study sometimes reveals a collection. Although exploratory laparotomy has been less commonly used since the advent of CT, this procedure still must be undertaken on occasion if an abscess is strongly suspected on clinical grounds.

1	An algorithm for the management of patients with intraabdominal (including intraperitoneal) abscesses by percutaneous drainage is presented in Fig. 159-3. The treatment of intraabdominal infections involves the determination of the initial focus of infection, the administration of broad-spectrum antibiotics targeting the organisms involved, and the performance of a drainage procedure if one or more definitive abscesses have formed. Antimicrobial therapy, in general, is adjunctive to drainage and/or surgical correction of an underlying lesion or process in intraabdominal abscesses. Unlike the intraabdominal abscesses resulting from most causes, for which drainage of some kind is generally required, abscesses associated with diverticulitis usually wall off locally after rupture of a diverticulum, so that surgical intervention is not routinely required.

1	A number of agents exhibit excellent activity against aerobic gram-negative bacilli. Because death in intraabdominal sepsis is linked to gram-negative bacteremia, empirical therapy for intra-abdominal infection always needs to include adequate coverage of gram-negative aerobic, facultative, and anaerobic organisms. Even 850 Percutaneous drainage No improvement by 48 h Defervescence by 24–48 h Drain out when criteria for catheter removal satisfied Repeat CT scan with dilute Hypaque injection into cavity and attempt further drainage No drainage or no improvement Successful drainage and defervescence Surgery FIGURE 159-3 Algorithm for the management of patients with intraabdominal abscesses using percutaneous drainage. Antimicrobial therapy should be administered concomitantly.

1	FIGURE 159-3 Algorithm for the management of patients with intraabdominal abscesses using percutaneous drainage. Antimicrobial therapy should be administered concomitantly. (Reprinted with permission from B Lorber [ed]: Atlas of Infectious Diseases, vol VII: Intra-abdominal Infections, Hepatitis, and Gastroenteritis. Philadelphia, Current Medicine, 1996, p 1.30, as adapted from OD Rotstein, RL Simmons, in SL Gorbach et al [eds]: Infectious Diseases. Philadelphia, Saunders, 1992, p 668.) if anaerobes are not cultured from clinical specimens, they still must be covered by the therapeutic regimen. Empirical antibiotic therapy should be the same as that discussed above for secondary peritonitis.

1	VISCERAL ABSCESSES Liver Abscesses The liver is the organ most subject to the development of abscesses. In one study of 540 intraabdominal abscesses, 26% were visceral. Liver abscesses made up 13% of the total number, or 48% of all visceral abscesses. Liver abscesses may be solitary or multiple; they may arise from hematogenous spread of bacteria or from local spread from contiguous sites of infection within the peritoneal cavity. In the past, appendicitis with rupture and subsequent spread of infection was the most common source for a liver abscess. Currently, associated disease of the biliary tract is most common. Pylephlebitis (suppurative thrombosis of the portal vein), usually arising from infection in the pelvis but sometimes from infection elsewhere in the peritoneal cavity, is another common source for bacterial seeding of the liver.

1	Fever is the most common presenting sign of liver abscess. Some patients, particularly those with associated disease of the biliary tract, have symptoms and signs localized to the right upper quadrant, including pain, guarding, punch tenderness, and even rebound tenderness. Nonspecific symptoms, such as chills, anorexia, weight loss, nausea, and vomiting, may also develop. Only 50% of patients with liver abscesses, however, have hepatomegaly, right-upper-quadrant tenderness, or jaundice; thus, one-half of patients have no symptoms or signs to direct attention to the liver. Fever of unknown origin may be the only manifestation of liver abscess, especially in the elderly. Diagnostic studies of the abdomen, especially the right upper quadrant, should be a part of any workup for fever of unknown origin. The single most reliable laboratory finding is an elevated serum concentration of alkaline phosphatase, which is documented in 70% of patients with liver abscesses. Other tests of liver

1	origin. The single most reliable laboratory finding is an elevated serum concentration of alkaline phosphatase, which is documented in 70% of patients with liver abscesses. Other tests of liver function may yield normal results, but 50% of patients have elevated serum levels of bilirubin, and 48% have elevated concentrations of aspartate aminotransferase. Other laboratory findings include leukocytosis in 77% of patients, anemia (usually normochromic, normocytic) in 50%, and hypoalbuminemia in 33%. Concomitant bacteremia is found in one-third to one-half of patients. A liver abscess is sometimes suggested by chest radiography, especially if a new elevation of the right hemidiaphragm is seen; other suggestive findings include a right basilar infiltrate and a right pleural effusion.

1	Imaging studies are the most reliable methods for diagnosing liver abscesses. These studies include ultrasonography, CT (Fig. 159-4), indium-labeled WBC or gallium scan, and MRI. More than one such FIGURE 159-4 Multilocular liver abscess on CT scan. Multiple or multilocular abscesses are more common than solitary abscesses. (Reprinted with permission from B Lorber [ed]: Atlas of Infectious Diseases, vol VII: Intra-abdominal Infections, Hepatitis, and Gastroenteritis. Philadelphia, Current Medicine, 1996, Fig. 1.22.) study may be required.

1	Organisms recovered from liver abscesses vary with the source. In liver infection arising from the biliary tree, enteric gram-negative aerobic bacilli and enterococci are common isolates. Klebsiella pneumoniae liver abscess has been well described in Southeast Asia for more than 20 years and has become an emerging syndrome in North America and elsewhere. These community-acquired infections have been linked to a virulent hypermucoviscous K. pneumoniae phenotype and to a specific genotype. The typical syndrome includes liver abscess, bacteremia, and metastatic infection. Ampicillin/amoxicillin therapy started within the previous 30 days has been associated with increased risk for this syndrome, presumably because of selection for the causative strain. Unless previous surgery has been performed, anaerobes are not generally involved in liver abscesses arising from biliary infections. In contrast, in liver abscesses arising from pelvic and other intraperitoneal sources, a mixed flora

1	anaerobes are not generally involved in liver abscesses arising from biliary infections. In contrast, in liver abscesses arising from pelvic and other intraperitoneal sources, a mixed flora including both aerobic and anaerobic species is common; B. fragilis is the species most frequently isolated. With hematogenous spread of infection, usually only a single organism is encountered; this species may be S. aureus or a streptococcal species such as one in the Streptococcus milleri group. Results of cultures obtained from drain sites are not reliable for defining the etiology of infections. Liver abscesses may also be caused by Candida species; such abscesses usually follow fungemia in patients receiving chemotherapy for cancer and often present when PMNs return after a period of neutropenia. Amebic liver abscesses are not an uncommon problem (Chap. 247). Amebic serologic testing gives positive results in >95% of cases. In addition, polymerase chain reaction (PCR) testing has been used in

1	Amebic liver abscesses are not an uncommon problem (Chap. 247). Amebic serologic testing gives positive results in >95% of cases. In addition, polymerase chain reaction (PCR) testing has been used in recent years. Negative results from these studies help to exclude this diagnosis.

1	(Fig. 159-3) Drainage is the mainstay of therapy for intraabdominal abscesses, including liver abscesses; the approach can be either percutaneous (with a pigtail catheter kept in place or possibly with a device that can perform pulse lavage to fragment and evacuate the semisolid contents of a liver abscess) or surgical. However, there is growing interest in medical management alone for pyogenic liver abscesses. The drugs used for empirical therapy include the same ones used in intraabdominal sepsis and secondary bacterial peritonitis. Usually, blood cultures and a diagnostic aspirate of abscess contents should be obtained before the initiation of empirical therapy, with antibiotic choices adjusted when the results of Gram’s staining and culture become available. Cases treated without definitive drainage generally require longer courses of antibiotic therapy. When percutaneous drainage was compared with open surgical drainage, the average length of hospital stay for the former was

1	definitive drainage generally require longer courses of antibiotic therapy. When percutaneous drainage was compared with open surgical drainage, the average length of hospital stay for the former was almost twice that for the latter, although both the time required for fever to resolve and the mortality rate were the same for the two procedures. The mortality rate was appreciable despite treatment, averaging 15%. Several factors predict the failure of percutaneous drainage and therefore may favor primary surgical intervention. These factors include the presence of multiple, sizable abscesses; viscous abscess contents that tend to plug the catheter; associated disease (e.g., disease of the biliary tract) requiring surgery; the presence of yeast; communication with an untreated obstructed biliary tree; or the lack of a clinical response to percutaneous drainage in 4–7 days.

1	Treatment of candidal liver abscesses often entails initial administration of amphotericin B or liposomal amphotericin, with subsequent fluconazole therapy (Chap. 240). In some cases, therapy with fluconazole alone (6 mg/kg daily) may be used—e.g., in clinically stable patients whose infecting isolate is susceptible to this drug.

1	Splenic Abscesses Splenic abscesses are much less common than liver abscesses. The incidence of splenic abscesses has ranged from 0.14% to 0.7% in various autopsy series. The clinical setting and the organisms isolated usually differ from those for liver abscesses. The degree of clinical suspicion for splenic abscess needs to be high because this condition is frequently fatal if left untreated. Even in the most recently published series, diagnosis was made only at autopsy in 37% of cases. Although splenic abscesses may arise occasionally from contiguous spread of infection or from direct trauma to the spleen, hematogenous spread of infection is more common. Bacterial endocarditis is the most common associated infection (Chap. 155). Splenic abscesses can develop in patients who have received extensive immunosuppressive therapy (particularly those with malignancy involving the spleen) and in patients with hemoglobinopathies or other hematologic disorders (especially sickle cell anemia).

1	Although ~50% of patients with splenic abscesses have abdominal pain, the pain is localized to the left upper quadrant in only one-half of these cases. Splenomegaly is found in ~50% of cases. Fever and leukocytosis are generally present; the development of fever preceded diagnosis by an average of 20 days in one series. Left-sided chest findings may include abnormalities to auscultation, and chest radiographic findings may include an infiltrate or a left-sided pleural effusion. CT scan of the abdomen has been the most sensitive diagnostic tool. Ultrasonography can yield the diagnosis but is less sensitive. Liver-spleen scan or gallium scan may also be useful. Streptococcal species are the most common bacterial isolates from splenic abscesses, followed by S. aureus—presumably reflecting the associated endocarditis. An increase in the prevalence of gram-negative aerobic isolates from splenic abscesses has been reported; these organisms often derive from a urinary tract focus, with

1	the associated endocarditis. An increase in the prevalence of gram-negative aerobic isolates from splenic abscesses has been reported; these organisms often derive from a urinary tract focus, with associated bacteremia, or from another intraabdominal source. Salmonella species are seen fairly commonly, especially in patients with sickle cell hemoglobinopathy. Anaerobic species accounted for only 5% of isolates in the largest collected series, but the reporting of a number of “sterile abscesses” may indicate that optimal techniques for the isolation of anaerobes were not used.

1	Because of the high mortality figures reported for splenic abscesses, splenectomy with adjunctive antibiotics has traditionally been considered standard treatment and remains the best approach for complex, multilocular abscesses or multiple abscesses. However, percutaneous drainage has worked well for single, small (<3-cm) abscesses in some studies and may also be useful for patients with high surgical risk. Patients undergoing splenectomy should be vaccinated against encapsulated organisms (Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis). The most important factor in successful treatment of splenic abscesses is early diagnosis.

1	Perinephric and Renal Abscesses Perinephric and renal abscesses are 851 not common. The former accounted for only ~0.02% of hospital admissions and the latter for ~0.2% in Altemeier’s series of 540 intraabdominal abscesses. Before antibiotics became available, most renal and perinephric abscesses were hematogenous in origin, usually complicating prolonged bacteremia, with S. aureus most commonly recovered. Now, in contrast, >75% of perinephric and renal abscesses arise from a urinary tract infection. Infection ascends from the bladder to the kidney, with pyelonephritis preceding abscess development. Bacteria may directly invade the renal parenchyma from medulla to cortex. Local vascular channels within the kidney may also facilitate the transport of organisms. Areas of abscess developing within the parenchyma may rupture into the perinephric space. The kidneys and adrenal glands are surrounded by a layer of perirenal fat that, in turn, is surrounded by Gerota’s fascia, which extends

1	within the parenchyma may rupture into the perinephric space. The kidneys and adrenal glands are surrounded by a layer of perirenal fat that, in turn, is surrounded by Gerota’s fascia, which extends superiorly to the diaphragm and inferiorly to the pelvic fat. Abscesses extending into the perinephric space may track through Gerota’s fascia into the psoas or transversalis muscles, into the anterior peritoneal cavity, superiorly to the subdiaphragmatic space, or inferiorly to the pelvis. Of the risk factors that have been associated with the development of perinephric abscesses, the most important is concomitant nephrolithiasis obstructing urinary flow. Of patients with perinephric abscess, 20–60% have renal stones. Other structural abnormalities of the urinary tract, prior urologic surgery, trauma, and diabetes mellitus have also been identified as risk factors.

1	The organisms most frequently encountered in perinephric and renal abscesses are E. coli, Proteus species, and Klebsiella species. E. coli, the aerobic species most commonly found in the colonic flora, seems to have unique virulence properties in the urinary tract, including factors promoting adherence to uroepithelial cells. The urease of Proteus species splits urea, thereby creating a more alkaline and more hospitable environment for bacterial proliferation. Proteus species are frequently found in association with large struvite stones caused by the precipitation of magnesium ammonium sulfate in an alkaline environment. These stones serve as a nidus for recurrent urinary tract infection. Although a single bacterial species is usually recovered from a perinephric or renal abscess, multiple species may also be found. If a urine culture is not contaminated with periurethral flora and is found to contain more than one organism, a perinephric abscess or renal abscess should be considered

1	species may also be found. If a urine culture is not contaminated with periurethral flora and is found to contain more than one organism, a perinephric abscess or renal abscess should be considered in the differential diagnosis. Urine cultures may also be polymicrobial in cases of bladder diverticulum.

1	Candida species can cause renal abscesses. This fungus may spread to the kidney hematogenously or by ascension from the bladder. The hallmark of the latter route of infection is ureteral obstruction with large fungal balls.

1	The presentation of perinephric and renal abscesses is quite nonspecific. Flank pain and abdominal pain are common. At least 50% of patients are febrile. Pain may be referred to the groin or leg, particularly with extension of infection. The diagnosis of perinephric abscess, like that of splenic abscess, is frequently delayed, and the mortality rate in some series is appreciable, although lower than in the past. Perinephric or renal abscess should be most seriously considered when a patient presents with symptoms and signs of pyelonephritis and remains febrile after 4 or 5 days of treatment. Moreover, when a urine culture yields a polymicrobial flora, when a patient is known to have renal stones, or when fever and pyuria coexist with a sterile urine culture, these diagnoses should be entertained.

1	Renal ultrasonography and abdominal CT are the most useful diagnostic modalities. If a renal or perinephric abscess is diagnosed, nephrolithiasis should be excluded, especially when a high urinary pH suggests the presence of a urea-splitting organism. Treatment for perinephric and renal abscesses, like that for other intraabdominal abscesses, includes drainage of pus and antibiotic therapy directed at the organism(s) recovered. For perinephric abscesses, percutaneous drainage is usually successful.

1	852 Psoas Abscesses The psoas muscle is another location in which abscesses are encountered. Psoas abscesses may arise from a hematogenous source, by contiguous spread from an intraabdominal or pelvic process, or by contiguous spread from nearby bony structures (e.g., vertebral bodies). Associated osteomyelitis due to spread from bone to muscle or from muscle to bone is common in psoas abscesses. When Pott’s disease was common, Mycobacterium tuberculosis was a frequent cause of psoas abscess. Currently, either S. aureus or a mixture of enteric organisms including aerobic and anaerobic gram-negative bacilli is usually isolated from psoas abscesses in the United States.

1	S. aureus is most likely to be isolated when a psoas abscess arises from hematogenous spread or a contiguous focus of osteomyelitis; a mixed enteric flora is the most likely etiology when the abscess has an intra-abdominal or pelvic source. Patients with psoas abscesses frequently present with fever, lower abdominal or back pain, or pain referred to the hip or knee. CT is the most useful diagnostic technique. Treatment includes surgical drainage and the administration of an antibiotic regimen directed at the inciting organism(s). Pancreatic Abscesses See Chap. 371. The substantial contributions of Dori F. Zaleznik, MD, to this chapter in previous editions are gratefully acknowledged. PART 8 Acute Infectious Diarrheal Diseases and Bacterial food Poisoning Regina C. LaRocque, Edward T. Ryan, Stephen B. Calderwood 160

1	PART 8 Acute Infectious Diarrheal Diseases and Bacterial food Poisoning Regina C. LaRocque, Edward T. Ryan, Stephen B. Calderwood 160 Acute diarrheal disease is a leading cause of illness globally and is associated with an estimated 1.4 million deaths per year. Among children <5 years of age, diarrheal disease is second only to lower respiratory infection as the most common infectious cause of death. The incidence rate of diarrheal disease among children in lowand middle-income countries is estimated to be 2.9 episodes per child per year, for a total of 1.7 billion episodes annually. The morbidity from diarrhea is also significant. Recurrent intestinal infections are associated with physical and mental stunting, wasting, micronutrient deficiencies, and malnutrition. In short, diarrheal disease is a driving factor in global morbidity and mortality.

1	The wide range of clinical manifestations of acute gastrointestinal illnesses is matched by the wide variety of infectious agents involved, including viruses, bacteria, and parasites (Table 160-1). This chapter discusses factors that enable gastrointestinal pathogens to cause disease, reviews host defense mechanisms, and delineates an approach to the evaluation and treatment of patients presenting with acute diarrhea. Individual organisms causing acute gastrointestinal illnesses are discussed in detail in subsequent chapters. Enteric pathogens have developed a variety of tactics to overcome host defenses. Understanding the virulence factors employed by these organisms is important in the diagnosis and treatment of clinical disease.

1	The number of microorganisms that must be ingested to cause disease varies considerably from species to species. For Shigella, enterohemorrhagic Escherichia coli, Giardia lamblia, or Entamoeba, as few as 10–100 bacteria or cysts can produce infection, while 105−108 Vibrio cholerae organisms must be ingested to cause disease. The infective dose of Salmonella varies widely, depending on the species, host, and food vehicle. The ability of organisms to overcome host defenses has important implications for transmission; Shigella, enterohemorrhagic E. coli, Entamoeba, and Giardia can spread by person-to-person contact, whereas under some circumstances Salmonella may have to grow in food for several hours before reaching an effective infectious dose.

1	Many organisms must adhere to the gastrointestinal mucosa as an initial step in the pathogenic process; thus, organisms that can compete with the normal bowel flora and colonize the mucosa have an important advantage in causing disease. Specific cell-surface proteins involved in attachment of bacteria to intestinal cells are important virulence determinants. V. cholerae, for example, adheres to the brush border of small-intestinal enterocytes via specific surface adhesins, including the toxin-coregulated pilus and other accessory colonization factors. Enterotoxigenic E. coli, which causes watery diarrhea, produces an adherence protein called colonization factor antigen that is necessary for colonization of the upper small intestine by the organism prior to the production of enterotoxin. Enteropathogenic E. coli, an agent of diarrhea in young children, and enterohemorrhagic E. coli, which causes hemorrhagic colitis and the hemolytic-uremic syndrome, produce virulence determinants that

1	E. coli, an agent of diarrhea in young children, and enterohemorrhagic E. coli, which causes hemorrhagic colitis and the hemolytic-uremic syndrome, produce virulence determinants that allow these organisms to attach to and efface the brush border of the intestinal epithelium.

1	The production of one or more exotoxins is important in the pathogenesis of numerous enteric organisms. Such toxins include enterotoxins, which cause watery diarrhea by acting directly on secretory mechanisms Mechanism Location Illness Stool Findings Examples of Pathogens Involved Vibrio cholerae, enterotoxigenic Escherichia coli (LT and/or ST), enteroaggregative E. coli, Clostridium perfringens, Bacillus cereus, Staphylococcus aureus, Aeromonas hydrophila, Plesiomonas shigelloides, rotavirus, norovirus, enteric adenoviruses, Giardia lamblia, Cryptosporidium spp., Cyclospora spp., microsporidia Shigella spp., Salmonella spp., Campylobacter jejuni, enterohemorrhagic E. coli, enteroinvasive E. coli, Yersinia enterocolitica, Listeria monocytogenes, Vibrio parahaemolyticus, Clostridium difficile, A. hydrophila, P. shigelloides, Entamoeba histolytica, Klebsiella oxytoca Salmonella typhi, Y. enterocolitica Abbreviations: LT, heat-labile enterotoxin; ST, heat-stable enterotoxin.

1	Salmonella typhi, Y. enterocolitica Abbreviations: LT, heat-labile enterotoxin; ST, heat-stable enterotoxin. in the intestinal mucosa; cytotoxins, which cause destruction of mucosal cells and associated inflammatory diarrhea; and neurotoxins, which act directly on the central or peripheral nervous system.

1	in the intestinal mucosa; cytotoxins, which cause destruction of mucosal cells and associated inflammatory diarrhea; and neurotoxins, which act directly on the central or peripheral nervous system. The prototypical enterotoxin is cholera toxin, a heterodimeric protein composed of one A and five B subunits. The A subunit contains the enzymatic activity of the toxin, while the B subunit pentamer binds holotoxin to the enterocyte surface receptor, the ganglioside GM1. After the binding of holotoxin, a fragment of the A subunit is translocated across the eukaryotic cell membrane into the cytoplasm, where it catalyzes the adenosine diphosphate ribosylation of a guanosine triphosphate–binding protein and causes persistent activation of adenylate cyclase. The end result is an increase of cyclic adenosine monophosphate in the intestinal mucosa, which increases Cl– secretion and decreases Na+ absorption, leading to a loss of fluid and the production of diarrhea.

1	Enterotoxigenic strains of E. coli may produce a protein called heat-labile enterotoxin (LT) that is similar to cholera toxin and causes secretory diarrhea by the same mechanism. Alternatively, enterotoxigenic strains of E. coli may produce heat-stable enterotoxin (ST), one form of which causes diarrhea by activation of guanylate cyclase and elevation of intracellular cyclic guanosine monophosphate. Some enterotoxigenic strains of E. coli produce both LT and ST. Bacterial cytotoxins, in contrast, destroy intestinal mucosal cells and produce the syndrome of dysentery, with bloody stools containing inflammatory cells. Enteric pathogens that produce such cytotoxins include Shigella dysenteriae type 1, Vibrio parahaemolyticus, and Clostridium difficile. S. dysenteriae type 1 and Shiga toxin–producing strains of E. coli produce potent cytotoxins and have been associated with outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome.

1	Neurotoxins are usually produced by bacteria outside the host and therefore cause symptoms soon after ingestion. Included are the staphylococcal and Bacillus cereus toxins, which act on the central nervous system to produce vomiting.

1	Dysentery may result not only from the production of cytotoxins but also from bacterial invasion and destruction of intestinal mucosal cells. Infections due to Shigella and enteroinvasive E. coli are characterized by the organisms’ invasion of mucosal epithelial cells, intraepithelial multiplication, and subsequent spread to adjacent cells. Salmonella causes inflammatory diarrhea by invasion of the bowel mucosa but generally is not associated with the destruction of enterocytes or the full clinical syndrome of dysentery. Salmonella typhi and Yersinia enterocolitica can penetrate intact intestinal mucosa, multiply intracellularly in Peyer’s patches and intestinal lymph nodes, and then disseminate through the bloodstream to cause enteric fever, a syndrome characterized by fever, headache, relative bradycardia, abdominal pain, splenomegaly, and leukopenia.

1	Given the enormous number of microorganisms ingested with every meal, the normal host must combat a constant influx of potential enteric pathogens. Studies of infections in patients with alterations in defense mechanisms have led to a greater understanding of the variety of ways in which the normal host can protect itself against disease.

1	The large numbers of bacteria that normally inhabit the intestine (the intestinal microbiota) act as an important host defense mechanism, preventing colonization by potential enteric pathogens. Persons with fewer intestinal bacteria, such as infants who have not yet developed normal enteric colonization or patients receiving antibiotics, are at significantly greater risk of developing infections with enteric pathogens. The composition of the intestinal microbiota is as important as the number of organisms present. More than 99% of the normal colonic microbiota is made up of anaerobic bacteria, and the acidic pH and volatile fatty acids produced by these organisms appear to be critical elements in resistance to colonization.

1	The acidic pH of the stomach is an important barrier to enteric pathogens, and an increased frequency of infections due to Salmonella, G. lamblia, and a variety of helminths has been reported among patients who have undergone gastric surgery or are achlorhydric for some other reason. Neutralization of gastric acid with antacids, proton pump inhibitors, or H2 blockers—a common practice in the management of hospitalized patients—similarly increases the risk of enteric colonization. In addition, some microorganisms can survive the extreme acidity of the gastric environment; rotavirus, for example, is highly stable to acidity.

1	Normal peristalsis is the major mechanism for clearance of bacteria from the proximal small intestine. When intestinal motility is impaired (e.g., by treatment with opiates or other antimotility drugs, anatomic abnormalities, or hypomotility states), the frequency of bacterial overgrowth and infection of the small bowel with enteric pathogens is increased. Some patients whose treatment for Shigella infection consists of diphenoxylate hydrochloride with atropine (Lomotil) experience prolonged fever and shedding of organisms, while patients treated with opiates for mild Salmonella gastroenteritis have a higher frequency of bacteremia than those not treated with opiates.

1	Both cellular immune responses and antibody production play important roles in protection from enteric infections. Humoral immunity to enteric pathogens consists of systemic IgG and IgM as well as secretory IgA. The mucosal immune system may be the first line of defense against many gastrointestinal pathogens. The binding of bacterial antigens to the luminal surface of M cells in the distal small bowel and the subsequent presentation of antigens to subepithelial lymphoid tissue lead to the proliferation of sensitized lymphocytes. These lymphocytes circulate and populate all of the mucosal tissues of the body as IgAsecreting plasma cells.

1	Host genetic variation influences susceptibility to diarrheal diseases. People with blood group O show increased susceptibility to disease due to V. cholerae, Shigella, E. coli O157, and norovirus. Polymorphisms in genes encoding inflammatory mediators have been associated with the outcome of infection with enteroaggregative E. coli, enterotoxin-producing E. coli, Salmonella, C. difficile, and V. cholerae. APPROACH TO THE PATIENT: The approach to the patient with possible infectious diarrhea or bacterial food poisoning is shown in Fig. 160-1. The answers to questions with high discriminating value can quickly narrow the range of potential causes of diarrhea and help determine whether treatment is needed. Important elements of the narrative history are detailed in Fig. 160-1.

1	The examination of patients for signs of dehydration provides essential information about the severity of the diarrheal illness and the need for rapid therapy. Mild dehydration is indicated by thirst, dry mouth, decreased axillary sweat, decreased urine output, and slight weight loss. Signs of moderate dehydration include an orthostatic fall in blood pressure, skin tenting, and sunken eyes (or, in infants, a sunken fontanelle). Signs of severe dehydration include lethargy, obtundation, feeble pulse, hypotension, and frank shock. After the severity of illness is assessed, the clinician must distinguish between inflammatory and noninflammatory disease. Using the history and epidemiologic features of the case as guides, the clinician Resolution Continued illness Assess: Duration (>1 day) Severity (see text) Symptomatic therapy Oral rehydration therapy (see Table 160-5) Diarrhea, Nausea, or Vomiting No

1	Resolution Continued illness Assess: Duration (>1 day) Severity (see text) Symptomatic therapy Oral rehydration therapy (see Table 160-5) Diarrhea, Nausea, or Vomiting No Obtain stool to be examined for WBCs (and, if >10 days, for parasites) Continue symptomatic therapy (Table 160-5); further evaluation if no resolution Specific antiparasitic therapy Culture for: Shigella, Salmonella, C. jejuni Consider: C.difficile cytotoxin Noninflammatory (no WBCs; see Table 160-1) Inflammatory (WBCs; see Table 160-1) Examine stool for parasites Consider: Empirical antimicrobial therapy (Table 160-5) FIGURE 160-1 Clinical algorithm for the approach to patients with community-acquired infectious diarrhea or bacterial food poisoning.

1	Key to superscripts: 1. Diarrhea lasting >2 weeks is generally defined as chronic; in such cases, many of the causes of acute diarrhea are much less likely, and a new spectrum of causes needs to be considered. 2. Fever often implies invasive disease, although fever and diarrhea may also result from infection outside the gastrointestinal tract, as in malaria. 3. Stools that contain blood or mucus indicate ulceration of the large bowel. Bloody stools without fecal leukocytes should alert the laboratory to the possibility of infection with Shiga toxin–producing enterohemorrhagic Escherichia coli. Bulky white stools suggest a small-intestinal process that is causing malabsorption. Profuse “rice-water” stools suggest cholera or a similar toxigenic process. 4. Frequent stools over a given period can provide the first warning of impending dehydration. 5. Abdominal pain may be most severe in inflammatory processes like those due to Shigella, Campylobacter, and necrotizing toxins. Painful

1	can provide the first warning of impending dehydration. 5. Abdominal pain may be most severe in inflammatory processes like those due to Shigella, Campylobacter, and necrotizing toxins. Painful abdominal muscle cramps, caused by electrolyte loss, can develop in severe cases of cholera. Bloating is common in giardiasis. An appendicitis-like syndrome should prompt a culture for Yersinia enterocolitica with cold enrichment. 6. Tenesmus (painful rectal spasms with a strong urge to defecate but little passage of stool) may be a feature of cases with proctitis, as in shigellosis or amebiasis. 7. Vomiting implies an acute infection (e.g., a toxin-mediated illness or food poisoning) but can also be prominent in a variety of systemic illnesses (e.g., malaria) and in intestinal obstruction. 8. Asking patients whether anyone else they know is sick is a more efficient means of identifying a common source than is constructing a list of recently eaten foods. If a common source seems likely,

1	8. Asking patients whether anyone else they know is sick is a more efficient means of identifying a common source than is constructing a list of recently eaten foods. If a common source seems likely, specific foods can be investigated. See text for a discussion of bacterial food poisoning. 9. Current antibiotic therapy or a recent history of treatment suggests Clostridium difficile diarrhea (Chap. 161). Stop antibiotic treatment if possible and consider tests for C. difficile toxins. Antibiotic use may increase the risk of chronic intestinal carriage following salmonellosis. 10. See text (and Chap. 149) for a discussion of traveler’s diarrhea.

1	(After TS Steiner, RL Guerrant: Principles and syndromes of enteric infection, in Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases, 7th ed, GL Mandell et al [eds]. Philadelphia, Churchill Livingstone, 2010, pp 1335–1351; RL Guerrant, DA Bobak: N Engl J Med 325:327, 1991; with permission.) can then rapidly evaluate the need for further efforts to define a specific etiology and for therapeutic intervention. Examination of a stool sample may supplement the narrative history. Grossly bloody or mucoid stool suggests an inflammatory process. A test for fecal leukocytes (preparation of a thin smear of stool on a glass slide, addition of a drop of methylene blue, and examination of the wet mount) can suggest inflammatory disease in patients with diarrhea, although the predictive value of this test is still debated. A test for fecal lactoferrin, which is a marker of fecal leukocytes, is more sensitive and is available in latex agglutination and enzyme-linked

1	the predictive value of this test is still debated. A test for fecal lactoferrin, which is a marker of fecal leukocytes, is more sensitive and is available in latex agglutination and enzyme-linked immunosorbent assay formats. Causes of acute infectious diarrhea, categorized as inflammatory and noninflammatory, are listed in Table 160-1.

1	Chronic complications may follow the resolution of an acute diarrheal episode. The clinician should inquire about prior diarrheal illness if the conditions listed in Table 160-2 are observed. Chronic diarrhea Occurs in ~1% of travelers with acute diarrhea • Protozoa account for ~1/3 of cases Initial presentation or May be precipitated by traveler’s diarrhea exacerbation of inflammatory bowel disease Irritable bowel syndrome Occurs in ~10% of travelers with traveler’s diarrhea Reactive arthritis Particularly likely after infection with invasive organisms (Shigella, Salmonella, Campylobacter, Yersinia) Hemolytic-uremic syndrome Follows infection with Shiga toxin– (hemolytic anemia, producing bacteria (Shigella dysenteriae type thrombocytopenia, and renal 1 and enterohemorrhagic Escherichia coli) failure) Of the several million people who travel from temperate industrialized countries to tropical regions of Asia, Africa, and

1	Of the several million people who travel from temperate industrialized countries to tropical regions of Asia, Africa, and Central and South America each year, 20–50% experience a sudden onset of abdominal cramps, anorexia, and watery diarrhea; thus traveler’s diarrhea is the most common travel-related infectious illness (Chap. 149). The time of onset is usually 3 days to 2 weeks after the traveler’s arrival in a resource-poor area; most cases begin within the first 3–5 days. The illness is generally self-limited, lasting 1–5 days. The high rate of diarrhea among travelers to underdeveloped areas is related to the ingestion of contaminated food or water.

1	The organisms that cause traveler’s diarrhea vary considerably with location (Table 160-3), as does the pattern of antimicrobial resistance. In all areas, enterotoxigenic and enteroaggregative strains of E. coli are the most common isolates from persons with the classic secretory traveler’s diarrhea syndrome. Infection with Campylobacter jejuni is especially common in areas of Asia. Closed and semi-closed communities, including day-care centers, schools, residential facilities, and cruise ships, are important settings for outbreaks of enteric infections. Norovirus, which is highly contagious and robust in surviving on surfaces, is the most common etiologic agent associated with outbreaks of acute gastroenteritis. Other common organisms, often spread by fecal-oral contact in such communities, are Shigella,

1	C. jejuni, and Cryptosporidium. Rotavirus is rarely a cause of pediatric diarrheal outbreaks in the United States since rotavirus vaccination was broadly recommended in 2006. Similarly, hospitals are sites in which enteric infections are concentrated. Diarrhea is one of the most common manifestations of nosocomial infections. C. difficile is the predominant cause of nosocomial diarrhea among adults in the United States, and outbreaks of norovirus infection are common in health care settings. Klebsiella oxytoca has been identified as a cause of antibiotic-associated hemorrhagic colitis. Enteropathogenic E. coli has been associated with outbreaks of diarrhea in nurseries for newborns. One-third of elderly patients in chronic-care institutions develop a significant diarrheal illness each year; more than one-half of these cases are caused by cytotoxin-producing C. difficile. Antimicrobial therapy can predispose to pseudomembranous colitis by altering the normal colonic flora and allowing

1	more than one-half of these cases are caused by cytotoxin-producing C. difficile. Antimicrobial therapy can predispose to pseudomembranous colitis by altering the normal colonic flora and allowing the multiplication of C. difficile (Chap. 161).

1	aFor etiologic agents, see Table 160-4. Source: After DR Hill et al: The practice of travel medicine: Guidelines by the Infectious Diseases Society of America. Clin Infect Dis 43:1499, 2006. Globally, most morbidity and mortality from enteric pathogens involves children <5 years of age. Breast-fed infants are protected from contaminated food and water and derive some protection from maternal antibodies, but their risk of infection rises dramatically when they begin to eat solid foods. Exposure to rotavirus is universal, with most children experiencing their first infection in the first or second year of life if not vaccinated. Older children and adults are more commonly infected with norovirus. Other organisms with higher attack rates among children than among adults include enterotoxigenic, enteropathogenic, and enterohemorrhagic E. coli; Shigella; C. jejuni; and G. lamblia.

1	Immunocompromised hosts are at elevated risk of acute and chronic infectious diarrhea. Individuals with defects in cell-mediated immunity (including those with AIDS) are at particularly high risk of invasive enteropathies, including salmonellosis, listeriosis, and cryptosporidiosis. Individuals with hypogammaglobulinemia are at particular risk of C. difficile colitis and giardiasis. Patients with cancer are more likely to develop C. difficile infection as a result of chemotherapy and frequent hospitalizations. Infectious diarrhea can be life-threatening in immunocompromised hosts, with complications including bacteremia and metastatic seeding of infection. Furthermore, dehydration may compromise renal function and increase the toxicity of immunosuppressive drugs.

1	If the history and the stool examination indicate a noninflammatory etiology of diarrhea and there is evidence of a common-source outbreak, questions concerning the ingestion of specific foods and the time of onset of the diarrhea after a meal can provide clues to the bacterial cause of the illness. Potential causes of bacterial food poisoning are shown in Table 160-4.

1	Bacterial disease caused by an enterotoxin elaborated outside the host, such as that due to Staphylococcus aureus or B. cereus, has the shortest incubation period (1–6 h) and generally lasts <12 h. Most cases of staphylococcal food poisoning are caused by contamination from infected human carriers. Staphylococci can multiply at a wide range of temperatures; thus, if food is left to cool slowly and remains at room temperature after cooking, the organisms will have the opportunity to form enterotoxin. Outbreaks following picnics where potato salad, mayonnaise, and cream pastries have been served offer classic examples of staphylococcal food poisoning. Diarrhea, nausea, vomiting, and abdominal cramping are common, while fever is less so.

1	B. cereus can produce either a syndrome with a short incubation period—the emetic form, mediated by a staphylococcal type of enterotoxin—or one with a longer incubation period (8–16 h)—the diarrheal form, caused by an enterotoxin resembling E. coli LT, in which diarrhea and abdominal cramps are characteristic but vomiting is uncommon. The emetic form of B. cereus food poisoning is associated with contaminated fried rice; the organism is common in uncooked rice, and its heat-resistant spores survive boiling. If cooked rice is not refrigerated, the spores can germinate and produce toxin. Frying before serving may not destroy the preformed, heat-stable toxin.

1	Food poisoning due to Clostridium perfringens also has a slightly longer incubation period (8–14 h) and results from the survival of heat-resistant spores in inadequately cooked meat, poultry, or legumes. After ingestion, toxin is produced in the intestinal tract, causing moderately severe abdominal cramps and diarrhea; vomiting is rare, as is fever. The illness is self-limited, rarely lasting >24 h. Not all food poisoning has a bacterial cause. Nonbacterial agents of short-incubation food poisoning include capsaicin, which is found in hot peppers, and a variety of toxins found in fish and shellfish (Chap. 474).

1	Many cases of noninflammatory diarrhea are self-limited or can be treated empirically, and in these instances the clinician may not need to determine a specific etiology. Potentially pathogenic E. coli cannot be distinguished from normal fecal flora by routine culture, and tests to detect enterotoxins are not available in most clinical laboratories. In situations in which cholera is a concern, stool should be cultured on selective media such as thiosulfate–citrate–bile salts–sucrose (TCBS) or tellurite-taurocholate-gelatin (TTG) agar. A latex agglutination test has made the rapid detection of rotavirus in stool practical for many laboratories, while reverse-transcriptase polymerase chain reaction (PCR) and specific antigen enzyme immunoassays have been developed for the identification of norovirus. Stool specimens should be examined by immunofluorescence-based rapid assays or (less sensitive) standard microscopy for Giardia cysts or Cryptosporidium if the level of clinical suspicion

1	norovirus. Stool specimens should be examined by immunofluorescence-based rapid assays or (less sensitive) standard microscopy for Giardia cysts or Cryptosporidium if the level of clinical suspicion regarding the involvement of these organisms is high.

1	All patients with fever and evidence of inflammatory disease acquired outside the hospital should have stool cultured for Salmonella, Shigella, and Campylobacter. Salmonella and Shigella can be selected on MacConkey agar as non-lactose-fermenting (colorless) colonies or can be grown on Salmonella-Shigella agar or in selenite enrichment broth, both of which inhibit most organisms except these pathogens. Evaluation of nosocomial diarrhea should initially focus on C. difficile; stool culture for other pathogens in this setting has an extremely low yield and is not cost-effective. Toxins A and B produced by pathogenic strains of C. difficile can be detected by rapid enzyme immunoassays, latex agglutination tests, or PCR (Chap. 161). Isolation of C. jejuni requires inoculation of fresh stool onto selective growth medium and incubation at 42°C in a microaerophilic atmosphere. In many laboratories in the United States, E. coli O157:H7 is among the most common pathogens isolated from visibly

1	selective growth medium and incubation at 42°C in a microaerophilic atmosphere. In many laboratories in the United States, E. coli O157:H7 is among the most common pathogens isolated from visibly bloody stools. Strains of this enterohemorrhagic serotype can be identified in specialized laboratories by serotyping but also can be identified presumptively in hospital laboratories as lactose-fermenting, indole-positive colonies of sorbitol nonfermenters (white colonies) on sorbitol MacConkey plates. If the clinical presentation suggests the possibility of intestinal amebiasis, stool should be examined by a rapid antigen detection assay or by (less sensitive and less specific) microscopy.

1	In many cases, a specific diagnosis is not necessary or not available to guide treatment. The clinician can proceed with the information obtained from the history, stool examination, and evaluation of dehydration severity. Empirical regimens for the treatment of traveler’s diarrhea are listed in Table 160-5.

1	The mainstay of treatment is adequate rehydration. The treatment of cholera and other dehydrating diarrheal diseases was revolutionized by the promotion of oral rehydration solution (ORS), the efficacy of which depends on the fact that glucose-facilitated absorption of sodium and water in the small intestine remains intact in the presence of cholera toxin. The use of ORS has reduced mortality rates for cholera from >50% (in untreated cases) to <1%. A number of ORS formulas have been used. Initial preparations were based on the treatment of patients with cholera and included a solution containing 3.5 g of sodium chloride, 2.5 g of sodium bicarbonate (or 2.9 g of sodium citrate), 1.5 g of potassium chloride, and 20 g of glucose (or 40 g of sucrose) per liter of water. Such a preparation can still be used for the treatment of severe cholera. Many causes of secretory diarrhea, however, are associated with less electrolyte loss than occurs in cholera. Beginning in 2002, the World Health

1	still be used for the treatment of severe cholera. Many causes of secretory diarrhea, however, are associated with less electrolyte loss than occurs in cholera. Beginning in 2002, the World Health Organization recommended a “reduced-osmolarity/reduced-salt” ORS that is better tolerated and more effective than classic ORS. This preparation contains 2.6 g of sodium chloride, 2.9 g of trisodium citrate, 1.5 g of potassium chloride, and 13.5 g of glucose (or 27 g of sucrose) per liter of water. ORS formulations containing rice or cereal as the carbohydrate source may be even more effective than glucose-based solutions. Patients who are severely dehydrated or in whom vomiting precludes the use of oral therapy should receive IV solutions such as Ringer’s lactate.

1	Although most secretory forms of traveler’s diarrhea (usually due to enterotoxigenic or enteroaggregative E. coli or to Campylobacter) can be treated effectively with rehydration, bismuth subsalicylate, Watery diarrhea (no blood in stool, Oral fluids (oral rehydration solution, no fever), 1 or 2 unformed stools Pedialyte, Lytren, or flavored mineral per day without distressing enteric water) and saltine crackers symptoms Watery diarrhea (no blood in stool, Bismuth subsalicylate (for adults): no fever), 1 or 2 unformed stools per 30 mL or 2 tablets (262 mg/tablet) day with distressing enteric every 30 min for 8 doses; or symptoms loperamideb: 4 mg initially followed by 2 mg after passage of each unformed stool, not to exceed Watery diarrhea (no blood in stool, Antibacterial drugc plus (for adults) no distressing abdominal pain, no loperamideb (see dose above) fever), >2 unformed stools per day Dysentery (passage of bloody stools) Antibacterial drugc or fever (>37.8°C)

1	Dysentery (passage of bloody stools) Antibacterial drugc or fever (>37.8°C) Vomiting, minimal diarrhea Bismuth subsalicylate (for adults; see dose above) rehydration solution, Pedialyte, Lytren); continue feeding, especially attention for moderate dehydration, fever lasting >24 h, bloody stools, or aAll patients should take oral fluids (Pedialyte, Lytren, or flavored mineral water) plus saltine crackers. If diarrhea becomes moderate or severe, if fever persists, or if bloody stools or dehydration develops, the patient should seek medical attention. bLoperamide should not be used by patients with fever or dysentery; its use may prolong diarrhea in patients with infection due to Shigella or other invasive organisms. cThe recommended antibacterial drugs are as follows: If the level of suspicion is low for fluoroquinolone-resistant Campylobacter:

1	If the level of suspicion is low for fluoroquinolone-resistant Campylobacter: Adults: (1) A fluoroquinolone such as ciprofloxacin, 750 mg as a single dose or 500 mg bid for 3 days; levofloxacin, 500 mg as a single dose or 500 mg qd for 3 days; or norfloxacin, 800 mg as a single dose or 400 mg bid for 3 days. (2) Azithromycin, 1000 mg as a single dose or 500 mg qd for 3 days. (3) Rifaximin, 200 mg tid or 400 mg bid for 3 days (not recommended for use in dysentery). Children: Azithromycin, 10 mg/kg on day 1, 5 mg/kg on days 2 and 3 if diarrhea persists. If fluoroquinolone-resistant Campylobacter is suspected (for example, following travel to Southeast Asia): Adults: Azithromycin (at above dose for adults). Children: other areas (see above). Source: Diseases Society of America. Clin Infect Dis 43:1499, 2006. be treated empirically with an antimicrobial agent (e.g.,

1	Source: Diseases Society of America. Clin Infect Dis 43:1499, 2006. be treated empirically with an antimicrobial agent (e.g., Individuals Individuals with Campylobacter infection often benefit from antimicrobial treatment as well. Because of widespread resistance of Campylobacter to fluoroquinolones, especially in parts of Asia, a macrolide antibiotic such as erythromycin or azithromycin may be preferred for this infection.

1	Treatment of salmonellosis must be tailored to the individual patient. Since administration of antimicrobial agents often prolongs intestinal colonization with Salmonella, these drugs are usually reserved for individuals at high risk of complications from disseminated salmonellosis, such as young children, patients with prosthetic devices, elderly patients, and immunocompromised persons. Antimicrobial agents should not be administered to individuals (especially children) in whom enterohemorrhagic E. coli infection is 857 suspected. Laboratory studies of enterohemorrhagic E. coli strains have demonstrated that a number of antibiotics induce replication of Shiga toxin–producing lambdoid bacteriophages, thereby significantly increasing toxin production by these strains. Clinical studies have supported these laboratory results, and antibiotics may increase by twentyfold the risk of hemolytic-uremic syndrome and renal failure during enterohemorrhagic E. coli infection. A clinical clue in

1	supported these laboratory results, and antibiotics may increase by twentyfold the risk of hemolytic-uremic syndrome and renal failure during enterohemorrhagic E. coli infection. A clinical clue in the diagnosis of the latter infection is bloody diarrhea with low fever or none at all.

1	Improvements in hygiene to limit fecal-oral spread of enteric pathogens will be necessary if the prevalence of diarrheal diseases is to be significantly reduced in developing countries. Travelers can reduce their risk of diarrhea by eating only hot, freshly cooked food; by avoiding raw vegetables, salads, and unpeeled fruit; and by drinking only boiled or treated water and avoiding ice. Historically, few travelers to tourist destinations adhere to these dietary restrictions. Bismuth subsalicylate is an inexpensive agent for the prophylaxis of traveler’s diarrhea; it is taken at a dosage of 2 tablets (525 mg) four times a day. Treatment appears to be effective and safe for up to 3 weeks, but adverse events such as temporary darkening of the tongue and tinnitus can occur. A meta-analysis suggests that probiotics may lessen the likelihood of traveler’s diarrhea by ~15%. Prophylactic antimicrobial agents, although effective, are not generally recommended for the prevention of traveler’s

1	that probiotics may lessen the likelihood of traveler’s diarrhea by ~15%. Prophylactic antimicrobial agents, although effective, are not generally recommended for the prevention of traveler’s diarrhea except when travelers are immunosuppressed or have other underlying illnesses that place them at high risk for morbidity from gastrointestinal infection. The risk of side effects and the possibility of developing an infection with a drug-resistant organism or with more harmful, invasive bacteria make it more reasonable to institute an empirical short course of treatment if symptoms develop. If prophylaxis is indicated, the nonabsorbed antibiotic rifaximin can be considered.

1	The possibility of exerting a major impact on the worldwide morbidity and mortality associated with diarrheal diseases has led to intense efforts to develop effective vaccines against the common bacterial and viral enteric pathogens. An effective rotavirus vaccine is currently available. Vaccines against S. typhi and V. cholerae also are available, although the protection they offer is incomplete and/or short lived. At present, there is no effective commercially available vaccine against Shigella, enterotoxigenic E. coli, Campylobacter, nontyphoidal Clostridium difficile Infection, 161 Including Pseudomembranous Colitis Dale N. Gerding, Stuart Johnson

1	Clostridium difficile Infection, 161 Including Pseudomembranous Colitis Dale N. Gerding, Stuart Johnson Clostridium difficile infection (CDI) is a unique colonic disease that is acquired most often in association with antimicrobial use and the consequent disruption of the normal colonic microbiota. The most commonly diagnosed diarrheal illness acquired in the hospital, CDI results from the ingestion of spores of C. difficile that vegetate, multiply, and secrete toxins, causing diarrhea and pseudomembranous colitis (PMC) in the most severe cases. C. difficile is an obligately anaerobic, gram-positive, spore-forming bacillus whose spores are found widely in nature, particularly in the environment of hospitals and chronic-care facilities. CDI occurs

1	Clostridium difficile Infection, Including Pseudomembranous Colitis 858 frequently in hospitals and nursing homes (or shortly after discharge from these facilities) where the level of antimicrobial use is high and the environment is contaminated by C. difficile spores. Clindamycin, ampicillin, and cephalosporins were the first antibiotics associated with CDI. The secondand third-generation cephalosporins, particularly cefotaxime, ceftriaxone, cefuroxime, and ceftazidime, are frequently responsible for this condition, and the fluoroquinolones (ciprofloxacin, levofloxacin, and moxifloxacin) are the most recent drug class to be implicated in hospital outbreaks. Penicillin/ β-lactamase-inhibitor combinations such as ticarcillin/clavulanate and piperacillin/tazobactam pose significantly less risk. However, all antibiotics, including vancomycin and metronidazole (the agents most commonly used to treat CDI), carry a risk of subsequent CDI. Rare cases are reported in patients without prior

1	risk. However, all antibiotics, including vancomycin and metronidazole (the agents most commonly used to treat CDI), carry a risk of subsequent CDI. Rare cases are reported in patients without prior antibiotic exposure.

1	C. difficile is acquired exogenously—most frequently in the hospital or nursing home, but also possibly in the outpatient setting—and is carried in the stool of both symptomatic and asymptomatic patients. The rate of fecal colonization is often ≥20% among adult patients hospitalized for >1 week; in contrast, the rate is 1–3% among community residents. Community-onset CDI without recent hospitalization, nursing home residence, or outpatient health-care contact probably accounts for ≤10% of all cases. The risk of C. difficile acquisition increases in proportion to the length of hospital stay. Asymptomatic fecal carriage of C. difficile in healthy neonates is very common, with repeated colonization by multiple strains in infants (<1 year old), but associated disease in these infants is extremely rare if it occurs at all. Spores of C. difficile are found on environmental surfaces (where the organism can persist for months) and on the hands of hospital personnel who fail to practice good

1	rare if it occurs at all. Spores of C. difficile are found on environmental surfaces (where the organism can persist for months) and on the hands of hospital personnel who fail to practice good hand hygiene. Hospital epidemics of CDI have been attributed to a single C. difficile strain and to multiple strains present simultaneously. Other identified risk factors for CDI include older age, greater severity of underlying illness, gastrointestinal surgery, use of electronic rectal thermometers, enteral tube feeding, and antacid treatment. Use of proton pump inhibitors may be a risk factor, but this risk is probably modest, and no firm data have implicated these agents in patients who are not already receiving antibiotics.

1	Spores of toxigenic C. difficile are ingested, survive gastric acidity, germinate in the small bowel, and colonize the lower intestinal tract, where they elaborate two large toxins: toxin A (an enterotoxin) and toxin B (a cytotoxin). These toxins initiate processes resulting in the disruption of epithelial-cell barrier function, diarrhea, and pseudo-membrane formation. Toxin A is a potent neutrophil chemoattractant, and both toxins glucosylate the guanosine triphosphate (GTP)–binding proteins of the Rho subfamily that regulate the actin cell cytoskeleton. Data from studies using molecular disruption of toxin genes in isogenic mutants suggest that toxin B is the more important virulence factor. This possibility, if confirmed, might account for the occurrence of clinical disease caused by toxin A–negative strains. Disruption of the cytoskeleton results in loss of cell shape, adherence, and tight junctions, with consequent fluid leakage. A third toxin, binary toxin CDT, was previously

1	toxin A–negative strains. Disruption of the cytoskeleton results in loss of cell shape, adherence, and tight junctions, with consequent fluid leakage. A third toxin, binary toxin CDT, was previously found in only ~6% of strains but is present in all isolates of the widely recognized epidemic NAP1/BI/027 strain (see “Global Considerations,” below); this toxin is related to C. perfringens iota toxin. Its role in the pathogenesis of CDI has not yet been defined.

1	The pseudomembranes of PMC are confined to the colonic mucosa and initially appear as 1to 2-mm whitish-yellow plaques. The intervening mucosa appears unremarkable, but, as the disease progresses, the pseudomembranes coalesce to form larger plaques and become confluent over the entire colon wall (Fig. 161-1). The whole colon is usually involved, but 10% of patients have rectal sparing. Viewed microscopically, the pseudomembranes have a mucosal attachment point and contain necrotic leukocytes, fibrin, mucus, and cellular debris. The epithelium is eroded and necrotic in focal areas, with neutrophil infiltration of the mucosa.

1	Patients colonized with C. difficile were initially thought to be at high risk for CDI. However, four prospective studies have shown that colonized patients who have not previously had CDI actually have a decreased risk of CDI. At least three events are proposed as essential for the development of CDI (Fig. 161-2). Exposure to antimicrobial agents is the first event and establishes susceptibility to C. difficile infection, most likely through disruption of the normal gastrointestinal microbiota. The second event is exposure to toxigenic C. difficile. Given that the majority of patients do not develop CDI after the first two events, a third event is clearly essential for its occurrence. Candidate third events include exposure to a C. difficile strain of particular virulence, exposure to antimicrobial agents especially likely

1	FIGURE 161-1 Autopsy specimen showing confluent pseudo-membranes covering the cecum of a patient with pseudomembra-nous colitis. Note the sparing of the terminal ileum (arrow). Pathogenesis model for C. difficile enteric disease Antimicrobial(s) Hospitalization CDI Asymptomatic C. difficile colonization Acquisition of a toxigenic strain of C. difficile and failure to mount an anamnestic toxin A antibody response result in CDI. C. difficile acquisition C. difficile acquisition

1	FIGURE 161-2 Pathogenesis model for hospital-acquired Clostridium difficile infection (CDI). At least three events are integral to C. difficile pathogenesis: (1) Exposure to antibiotics establishes susceptibility to infection. (2) Once susceptible, the patient may acquire nontoxigenic (nonpathogenic) or toxigenic strains of C. difficile as a second event. (3) Acquisition of toxigenic C. difficile may be followed by asymptomatic colonization or CDI, depending on one or more additional events (e.g., an inadequate host anamnestic IgG response to C. difficile toxin A).

1	to cause CDI, and an inadequate host immune response. The host anamnestic serum IgG antibody response to toxin A of C. difficile is the most likely third event that determines which patients develop diarrhea and which patients remain asymptomatic. In all probability, the majority of people first develop antibody to C. difficile toxins when colonized asymptomatically during the first year of life or after CDI in childhood. Infants are thought not to develop symptomatic CDI because they lack suitable mucosal toxin receptors that develop later in life. In adulthood, serum levels of IgG antibody to toxin A increase more in response to infection in individuals who become asymptomatic carriers than in those who develop CDI. For persons who develop CDI, development of increasing levels of antitoxin A during treatment correlates with a lower risk of recurrence of CDI. A clinical trial using monoclonal antibodies to both toxin A and toxin B in addition to standard therapy showed rates of

1	A during treatment correlates with a lower risk of recurrence of CDI. A clinical trial using monoclonal antibodies to both toxin A and toxin B in addition to standard therapy showed rates of recurrence significantly lower than those obtained with placebo plus standard therapy.

1	Rates and severity of CDI in the United States, Canada, and Europe increased markedly after the year 2000. Rates in U.S.

1	hospitals tripled between 2000 and 2005. In 2005, hospitals in Montreal, Quebec, reported rates four times higher than the 1997 baseline, with directly attributable mortality of 6.9% (increased from 1.5%). An epidemic strain, variously known as toxinotype III, REA type BI, polymerase chain reaction (PCR) ribotype 027, and pulsed-field type NAP1 and thus collectively designated NAP1/BI/027, is thought to account for much of the increase in incidence and has been found in North America, Europe, and Asia. It is now recognized that two clones of NAP1/BI/027 originated in the United States and Canada and spread to the United Kingdom, Europe, and Asia. The epidemic organism is characterized by (1) an ability to produce 16–23 times as much toxin A and toxin B as control strains in vitro; (2) the presence of a third toxin (binary toxin CDT); and (3) high-level resistance to all fluoroquinolones. New strains have been and probably will continue to be implicated in outbreaks, including a strain

1	presence of a third toxin (binary toxin CDT); and (3) high-level resistance to all fluoroquinolones. New strains have been and probably will continue to be implicated in outbreaks, including a strain (toxinotype V, ribotype 078) commonly found in food animals that also carries binary toxin and has been associated with high mortality risk in human infections. In the past 5 years, rates of CDI in the United Kingdom have markedly decreased, and the frequency of the NAP1/BI/027 strain in the countries of the European Union has likewise decreased. However, there has been no evidence of decreased rates of CDI or a decreased incidence of NAP1/BI/027 in North America; the latter strain still causes 25–35% of all CDIs in most regions of the United States.

1	Diarrhea is the most common manifestation caused by C. difficile. Stools are almost never grossly bloody and range from soft and unformed to watery or mucoid in consistency, with a characteristic odor. Patients may have as many as 20 bowel movements per day. Clinical and laboratory findings include fever in 28% of cases, abdomi-859 nal pain in 22%, and leukocytosis in 50%. When adynamic ileus (which is seen on x-ray in ~20% of cases) results in cessation of stool passage, the diagnosis of CDI is frequently overlooked. A clue to the presence of unsuspected CDI in these patients is unexplained leukocytosis, with ≥15,000 white blood cells (WBCs)/μL. Such patients are at high risk for complications of C. difficile infection, particularly toxic megacolon and sepsis.

1	C. difficile diarrhea recurs after treatment in ~15–30% of cases, and this figure may be increasing. Recurrences may represent either relapses due to the same strain or reinfections with a new strain. Susceptibility to recurrence of clinical CDI is likely a result of continued fecal-microbiota disruption caused by the antibiotic used to treat CDI.

1	The diagnosis of CDI is based on a combination of clinical criteria: (1) diarrhea (≥3 unformed stools per 24 h for ≥2 days) with no other recognized cause plus (2) toxin A or B detected in the stool, toxin-producing C. difficile detected in the stool by PCR or culture, or pseudomembranes seen in the colon. PMC is a more advanced form of CDI and is visualized at endoscopy in only ~50% of patients with diarrhea who have a positive stool culture and toxin assay for C. difficile. Endoscopy is a rapid diagnostic tool in seriously ill patients with suspected PMC and an acute abdomen, but a negative result in this examination does not rule out CDI.

1	Despite the array of tests available for C. difficile and its toxins (Table 161-1), no single traditional test has high sensitivity, high specificity, and rapid turnaround. Most laboratory tests for toxins, including enzyme immunoassays, lack sensitivity. However, testing of multiple additional stool specimens is not recommended. Nucleic acid amplification tests, including PCR assays, have now been approved for diagnostic purposes and appear to be both rapid and sensitive while retaining high specificity. Testing of asymptomatic patients is not recommended except for epidemiologic study purposes. In particular, so-called tests of cure following treatment are not recommended because >50% of patients continue to harbor the organism and toxin after diarrhea has ceased and test results do not always predict recurrence of CDI. Thus these results should not be used to restrict placement of patients in long-term-care or nursing home facilities.

1	When possible, discontinuation of any ongoing antimicrobial administration is recommended as the first step in treatment of CDI. Earlier studies indicated that 15–23% of patients respond to this simple measure. However, with the advent of the current epidemic strain and the associated rapid clinical deterioration of some patients, Clostridium difficile Infection, Including Pseudomembranous Colitis Type of Test Relative Sensitivitya Relative Specificitya Comment Stool culture for C. difficile ++++ +++ Most sensitive test; specificity of ++++ if the C. difficile isolate tests positive for toxin; with clinical data, is diagnostic of CDI; turnaround time too slow for practical use Cell culture cytotoxin test on stool +++ ++++ With clinical data, is diagnostic of CDI; highly specific but not as sensitive as stool culture; slow turnaround time

1	Cell culture cytotoxin test on stool +++ ++++ With clinical data, is diagnostic of CDI; highly specific but not as sensitive as stool culture; slow turnaround time Enzyme immunoassay for toxin A or ++ to +++ +++ With clinical data, is diagnostic of CDI; rapid results, but not as sen-toxins A and B in stool sitive as stool culture or cell culture cytotoxin test Enzyme immunoassay for C. difficile +++ to ++++ +++ Detects glutamate dehydrogenase found in toxigenic and non-common antigen in stool toxigenic strains of C. difficile and other stool organisms; more sensitive and less specific than enzyme immunoassay for toxins; rapid results Nucleic acid amplification tests for ++++ ++++ Detect toxigenic C. difficile in stool; newly approved for clinical test- C. difficile toxin A or B gene in stool ing, but appears to be more sensitive than enzyme immunoassay toxin testing and at least as specific

1	C. difficile toxin A or B gene in stool ing, but appears to be more sensitive than enzyme immunoassay toxin testing and at least as specific Colonoscopy or sigmoidoscopy + ++++ Highly specific if pseudomembranes are seen; insensitive compared with other tests aAccording to both clinical and test-based criteria. ++++, >90%; +++, 71–90%; ++, 51–70%; +, ~50%.

1	860 prompt initiation of specific CDI treatment has become the standard. Empirical treatment is appropriate if CDI is strongly suspected on clinical grounds. General treatment guidelines include hydration and the avoidance of antiperistaltic agents and opiates, which may mask symptoms and possibly worsen disease. Nevertheless, antiperistaltic agents have been used safely with vancomycin or metronidazole for mild to moderate CDI. Oral administration of vancomycin, fidaxomicin, or metronidazole is recommended for CDI treatment. IV vancomycin is ineffective for CDI, and fidaxomicin is available only for oral administration; when IV metronidazole is administered, fecal bactericidal drug concentrations are achieved during acute diarrhea; however, in the presence of adynamic ileus, IV metronidazole treatment of CDI has failed. Two large clinical trials comparing vancomycin and fidaxomicin indicated comparable resolution of diarrhea (~90% of patients) as well as significantly reduced rates

1	treatment of CDI has failed. Two large clinical trials comparing vancomycin and fidaxomicin indicated comparable resolution of diarrhea (~90% of patients) as well as significantly reduced rates of recurrent CDI with fidaxomicin from rates with vancomycin. In previous randomized trials, diarrhea response rates to oral therapy with vancomycin or metronidazole were ≥94%, but four observational studies found that response rates for metronidazole had declined to 62–78%. Although the mean time to resolution of diarrhea is 2–4 days, the response to metronidazole may be much slower. Treatment should not be deemed a failure until a drug has been given for at least 6 days. On the basis of data for shorter courses of vancomycin and the results of two large-scale clinical trials, it is recommended that vancomycin, fidaxomicin, and metronidazole be given for at least 10 days. Metronidazole is not approved for CDI by the U.S. Food and Drug Administration (FDA), but most patients with mild to

1	that vancomycin, fidaxomicin, and metronidazole be given for at least 10 days. Metronidazole is not approved for CDI by the U.S. Food and Drug Administration (FDA), but most patients with mild to moderate illness respond to 500 mg given by mouth three times a day for 10 days; extension of the treatment period may be needed for slow responders. In addition to the reports of increases in metronidazole failures, a prospective, randomized, double-blind, placebo-controlled study has demonstrated the superiority of vancomycin over metronidazole for treatment of severe CDI. The severity assessment score in that study included age as well as laboratory parameters (elevated temperature, low albumin level, or elevated WBC count), documentation of PMC by endoscopy, and treatment of CDI in the intensive care unit. Although a validated severity score is not available, it is important to initiate treatment with oral vancomycin for patients who appear seriously ill, particularly if they have a high

1	care unit. Although a validated severity score is not available, it is important to initiate treatment with oral vancomycin for patients who appear seriously ill, particularly if they have a high WBC count (>15,000/μL) or a creatinine level that is ≥1.5 times higher than the premorbid value (Table 161-2). In addition, a randomized blinded trial compared a toxin-binding polymer, tolevamer, with two antibiotic regimens for treatment of CDI and showed that vancomycin was superior to metronidazole for all patients regardless of severity. Small randomized trials of nitazoxanide, bacitracin, rifaximin, and fusidic acid for treatment of CDI have been conducted. These drugs have not been extensively studied, shown to be superior, or approved by the FDA for CDI, but they provide potential alternatives to vancomycin, fidaxomicin, and metronidazole.

1	Overall, ~15–30% of successfully treated patients experience recurrences of CDI, either as relapses caused by the original organism or as reinfections following treatment. Rates of CDI recurrence are significantly lower among patients treated with fidaxomicin rather than vancomycin. Rates of recurrence are comparable with vancomycin and metronidazole. Recurrence rates are higher among patients ≥65 years old, those who continue to take antibiotics while being treated for CDI, and those who remain in the hospital after the initial episode of CDI. Patients who have a first recurrence of CDI have a high rate of second recurrence (33–65%). In the first recurrence, re-treatment with metronidazole is comparable to treatment with vancomycin (Table 161-2), and fidaxomicin is superior to vancomycin in reducing the risk of further recurrences in patients who have had one recurrence. Recurrent CDI, once thought to be relatively mild, has now been documented to pose a significant (11%) risk of

1	in reducing the risk of further recurrences in patients who have had one recurrence. Recurrent CDI, once thought to be relatively mild, has now been documented to pose a significant (11%) risk of serious complications (shock, megacolon, perforation, colectomy, or death within 30 days). There is no standard treatment for multiple recurrences, but long or repeated metronidazole courses should be avoided because of potential neurotoxicity. The use of vancomycin in tapering doses or with pulse dosing every other day for 2–8 weeks may be the most practical approach to treatment of patients with multiple recurrences. Other approaches include the administration of vancomycin followed by the yeast Saccharomyces boulardii; the administration of vancomycin followed by a fecal microbiota transplant given via nasoduodenal tube, colonoscope, or enema; and the intentional colonization of the patient with a nontoxigenic

1	Initial episode, mild to moderate Metronidazole (500 mg tid × 10–14 d) Vancomycin (125 mg qid × 10–14 d) may be more effective than metronidazole. Fidaxomicin (200 mg bid × 10 d) is another alternative. Initial episode, severe Vancomycin (125 mg qid × 10–14 d) Indicators of severe disease may include leukocytosis (≥15,000 white blood cells/μL) and a creatinine level ≥1.5 times the premorbid value. Fidaxomicin is an alternative. Initial episode, severe compli-Vancomycin (500 mg PO or via nasogastric tube) plus Severe complicated or fulminant CDI is defined as severe CDI cated or fulminant metronidazole (500 mg IV q8h) plus consider with the addition of hypotension, shock, ileus, or toxic megacolon. The duration of treatment may need to be >2 weeks and is Rectal instillation of vancomycin (500 mg in 100 mL of dictated by response. Consider using tigecycline (50 mg IV q12h normal saline as a retention enema q6–8h) after a 100-mg loading dose) in place of metronidazole.

1	First recurrence Same as for initial episode Adjust treatment if severity of CDI has changed with recurrence. Consider fidaxomicin, which significantly decreases the likelihood of additional recurrences. Second recurrence Vancomycin in taper/pulse regimen Typical taper/pulse regimen: 125 mg qid × 10–14 d, then bid × 1 week, then daily × 1 week, then q2–3d for 2–8 weeks. Multiple recurrences Consider the following options: The only controlled studies that included patients with one or more recurrent CDI episodes were with vancomycin and S. boulardii, which showed borderline significance compared with vancomycin plus placebo, and fecal microbiota transplantation, boulardii (500 mg bid × 28 d) which was highly significant compared with a high-dose course (125 mg qid × 10–14 d); then stop vanco-of vancomycin. (The vancomycin taper was not compared.) mycin and start rifaximin (400 mg bid × 2 weeks) aAll agents are given orally unless otherwise specified.

1	strain of C. difficile. None of these biotherapeutic approaches has been approved by the FDA for use in the United States. Other nonFDA-approved antibiotic strategies include (1) sequential treatment with vancomycin (125 mg four times daily for 10–14 days) followed by rifaximin (400 mg twice daily for 14 days) and (2) treatment with nitazoxanide (500 mg twice daily for 7 days). IV immunoglobulin, which has also been used with variable success, presumably provides antibodies to C. difficile toxins.

1	Fulminant (rapidly progressive and severe) CDI presents the most difficult treatment challenge. Patients with fulminant disease often do not have diarrhea, and their illness mimics an acute surgical abdomen. Sepsis (hypotension, fever, tachycardia, leukocytosis) may result from severe CDI. An acute abdomen (with or without toxic megacolon) may include signs of obstruction, ileus, colon-wall thickening and ascites on abdominal CT, and peripheral-blood leukocytosis (≥20,000 WBCs/μL). With or without diarrhea, the differential diagnosis of an acute abdomen, sepsis, or toxic megacolon should include CDI if the patient has received antibiotics in the past 2 months. Cautious sigmoidoscopy or colonoscopy to visualize PMC and abdominal CT are the best diagnostic tests in patients without diarrhea.

1	Medical management of fulminant CDI is suboptimal because of the difficulty of delivering oral fidaxomicin, metronidazole, or vancomycin to the colon in the presence of ileus (Table 161-2). The combination of vancomycin (given via nasogastric tube and by retention enema) plus IV metronidazole has been used with some success in uncontrolled studies, as has IV tigecycline in small-scale uncontrolled studies. Surgical colectomy may be life-saving if there is no response to medical management. If possible, colectomy should be performed before the serum lactate level reaches 5 mmol/L. The incidence of fulminant CDI requiring colectomy appears to be increasing in the evolving epidemic; however, morbidity and death associated with colectomy may be reduced by performing instead a laparoscopic ileostomy followed by colon lavage with polyethylene glycol and vancomycin infusion into the colon via the ileostomy.

1	The mortality rate attributed to CDI, previously found to be 0.6–3.5%, has reached 6.9% in recent outbreaks and rises progressively with increasing age. Most patients recover, but recurrences are common.

1	Strategies for the prevention of CDI are of two types: those aimed at preventing transmission of the organism to the patient and those aimed at reducing the risk of CDI if the organism is transmitted. Transmission of C. difficile in clinical practice has been prevented by gloving of personnel, elimination of the use of contaminated electronic thermometers, and use of hypochlorite (bleach) solution for environmental decontamination of patients’ rooms. Hand hygiene is critical; hand washing is recommended in CDI outbreaks because alcohol hand gels are not sporicidal. CDI outbreaks have been best controlled by restricting the use of specific antibiotics, such as clindamycin and secondand third-generation cephalosporins. Outbreaks of CDI due to clindamycin-resistant strains have resolved promptly when clindamycin use is restricted. Future preventive strategies are likely to include use of monoclonal antibodies, vaccines, and biotherapeutics containing live organisms that will restore

1	promptly when clindamycin use is restricted. Future preventive strategies are likely to include use of monoclonal antibodies, vaccines, and biotherapeutics containing live organisms that will restore colonization protection in the microbiota.

1	urinary Tract Infections, 162 Pyelonephritis, and Prostatitis Kalpana Gupta, Barbara W. Trautner Urinary tract infection (UTI) is a common and painful human illness that, fortunately, is rapidly responsive to modern antibiotic therapy. In the preantibiotic era, UTI caused significant morbidity. Hippocrates, writing about a disease that appears to have been acute cystitis, said that the illness could last for a year before either resolving or worsening to involve the kidneys. When chemotherapeutic agents used to treat UTI were introduced in the early twentieth century, they were relatively ineffective, and persistence of infection after 3 weeks of therapy was common. Nitrofurantoin, which became available in the 1950s, was the first tolerable and effective agent for the treatment of UTI.

1	Since the most common manifestation of UTI is acute cystitis and since acute cystitis is far more prevalent among women than among men, most clinical research on UTI has involved women. Many studies have enrolled women from college campuses or large health maintenance organizations in the United States. Therefore, when reviewing the literature and recommendations concerning UTI, clinicians must consider whether the findings are applicable to their patient populations.

1	UTI may be asymptomatic (subclinical infection) or symptomatic (disease). Thus, the term urinary tract infection encompasses a variety of clinical entities, including asymptomatic bacteriuria (ASB), cystitis, prostatitis, and pyelonephritis. The distinction between symptomatic UTI and ASB has major clinical implications. Both UTI and ASB connote the presence of bacteria in the urinary tract, usually accompanied by white blood cells and inflammatory cytokines in the urine. However, ASB occurs in the absence of symptoms attributable to the bacteria in the urinary tract and does not usually require treatment, while UTI has more typically been assumed to imply symptomatic disease that warrants antimicrobial therapy. Much of the literature concerning UTI, particularly catheter-associated infection, does not differentiate between UTI and ASB. In this chapter, the term UTI denotes symptomatic disease; cystitis, symptomatic infection of the bladder; and pyelonephritis, symptomatic infection

1	does not differentiate between UTI and ASB. In this chapter, the term UTI denotes symptomatic disease; cystitis, symptomatic infection of the bladder; and pyelonephritis, symptomatic infection of the kidneys. Uncomplicated UTI refers to acute cystitis or pyelonephritis in nonpregnant outpatient women without anatomic abnormalities or instrumentation of the urinary tract; the term complicated UTI encompasses all other types of UTI. Recurrent UTI is not necessarily complicated; individual episodes can be uncomplicated and treated as such. Catheter-associated bacteriuria can be either symptomatic (CAUTI) or asymptomatic.

1	Except among infants and the elderly, UTI occurs far more commonly in females than in males. During the neonatal period, the incidence of UTI is slightly higher among males than among females because male infants more commonly have congenital urinary tract anomalies. After 50 years of age, obstruction from prostatic hypertrophy becomes common in men, and the incidence of UTI is almost as high among men as among women. Between 1 year and ~50 years of age, UTI and recurrent UTI are predominantly diseases of females. The prevalence of ASB is ~5% among women between ages 20 and 40 and may be as high as 40–50% among elderly women and men.

1	As many as 50–80% of women in the general population acquire at least one UTI during their lifetime—uncomplicated cystitis in most cases. Recent use of a diaphragm with spermicide, frequent sexual intercourse, and a history of UTI are independent risk factors for acute cystitis. Cystitis is temporally related to recent sexual intercourse in a dose-response manner, with an increased relative risk ranging from 1.4 with one episode of intercourse to 4.8 with five episodes of intercourse in the preceding week. In healthy postmenopausal women, sexual activity, diabetes mellitus, and incontinence are risk factors for UTI.

1	Urinary Tract Infections, Pyelonephritis, and Prostatitis 862 Many factors predisposing women to cystitis also increase the risk of pyelonephritis. Factors independently associated with pyelonephritis in young healthy women include frequent sexual intercourse, a new sexual partner, a UTI in the previous 12 months, a maternal history of UTI, diabetes, and incontinence. The common risk factors for cystitis and pyelonephritis are not surprising given that pyelonephritis typically arises through the ascent of bacteria from the bladder to the upper urinary tract. However, pyelonephritis can occur without clear antecedent cystitis. About 20–30% of women who have had one episode of UTI will have recurrent episodes. Early recurrence (within 2 weeks) is usually regarded as relapse rather than reinfection and may indicate the need to evaluate the patient for a sequestered focus. Intracellular pods of infecting organisms within the bladder epithelium have been demonstrated in animal models of

1	and may indicate the need to evaluate the patient for a sequestered focus. Intracellular pods of infecting organisms within the bladder epithelium have been demonstrated in animal models of UTI, but the importance of this phenomenon in humans is not yet clear. The rate of recurrence ranges from 0.3 to 7.6 infections per patient per year, with an average of 2.6 infections per year. It is not uncommon for multiple recurrences to follow an initial infection, resulting in clustering of episodes. Clustering may be related temporally to the presence of a new risk factor or to the sloughing of the protective outer bladder epithelial layer in response to bacterial attachment during acute cystitis. The likelihood of a recurrence decreases with increasing time since the last infection. A case-control study of predominantly white premenopausal women with recurrent UTI identified frequent sexual intercourse, use of spermicide, a new sexual partner, a first UTI before 15 years of age, and a

1	study of predominantly white premenopausal women with recurrent UTI identified frequent sexual intercourse, use of spermicide, a new sexual partner, a first UTI before 15 years of age, and a maternal history of UTI as independent risk factors for recurrent UTI. The only consistently documented behavioral risk factors for recurrent UTI include frequent sexual intercourse and spermicide use. In postmenopausal women, major risk factors for recurrent UTI include a history of premenopausal UTI and anatomic factors affecting bladder emptying, such as cystoceles, urinary incontinence, and residual urine. In pregnant women, ASB has clinical consequences, and both screening for and treatment of this condition are indicated. Specifically, ASB during pregnancy is associated with preterm birth and perinatal death of the fetus and with pyelonephritis in the mother. A Cochrane meta-analysis found that treatment of ASB in pregnant women decreased the risk of pyelonephritis by 75%. The majority of

1	perinatal death of the fetus and with pyelonephritis in the mother. A Cochrane meta-analysis found that treatment of ASB in pregnant women decreased the risk of pyelonephritis by 75%. The majority of men with UTI have a functional or anatomic abnormality of the urinary tract, most commonly urinary obstruction secondary to prostatic hypertrophy. That said, not all men with UTI have detectable urinary abnormalities; this point is particularly relevant for men ≤45 years of age. Lack of circumcision is also associated with an increased risk of UTI because Escherichia coli is more likely to colonize the glans and prepuce and subsequently migrate into the urinary tract. Women with diabetes have been found to have a twoto threefold higher rate of ASB and UTI than women without diabetes; there is insufficient evidence to make a corresponding statement about men. Increased duration of diabetes and the use of insulin rather than oral medication are also associated with a higher risk of UTI

1	is insufficient evidence to make a corresponding statement about men. Increased duration of diabetes and the use of insulin rather than oral medication are also associated with a higher risk of UTI among women with diabetes. Poor bladder function, obstruction in urinary flow, and incomplete voiding are additional factors commonly found in patients with diabetes that increase the risk of UTI. Impaired cytokine secretion may contribute to ASB in diabetic women.

1	The uropathogens causing UTI vary by clinical syndrome but are usually enteric gram-negative rods that have migrated to the urinary tract. The susceptibility patterns of these organisms vary by clinical syndrome and by geography. In acute uncomplicated cystitis in the United States, the etiologic agents are highly predictable: E. coli accounts for 75–90% of isolates; Staphylococcus saprophyticus for 5–15% (with particularly frequent isolation from younger women); and Klebsiella, Proteus, Enterococcus, and Citrobacter species, along with other organisms, for 5–10%. Similar etiologic agents are found in Europe and Brazil. The spectrum of agents causing uncomplicated pyelonephritis is similar, with E. coli predominating. In complicated UTI (e.g., CAUTI), E. coli remains the predominant organism, but other aerobic gram-negative rods, such as Pseudomonas aeruginosa and Klebsiella, Proteus, Citrobacter, Acinetobacter, and Morganella species, also are frequently isolated. Gram-positive

1	organism, but other aerobic gram-negative rods, such as Pseudomonas aeruginosa and Klebsiella, Proteus, Citrobacter, Acinetobacter, and Morganella species, also are frequently isolated. Gram-positive bacteria (e.g., enterococci and Staphylococcus aureus) and yeasts are also important pathogens in complicated UTI. Data on etiology and resistance are generally obtained from laboratory surveys and should be understood in the context that organism identification is performed only in cases in which urine is sent for culture—i.e., typically, when complicated UTI or pyelonephritis is suspected. The available data demonstrate a worldwide increase in the resistance of E. coli to antibiotics commonly used to treat UTI. North American and European surveys from women with acute cystitis have documented resistance rates of >20% to trimethoprim-sulfamethoxazole (TMP-SMX) and to ciprofloxacin in some regions. In community-acquired infections, the increased prevalence of uropathogens producing

1	resistance rates of >20% to trimethoprim-sulfamethoxazole (TMP-SMX) and to ciprofloxacin in some regions. In community-acquired infections, the increased prevalence of uropathogens producing extended-spectrum β-lactamases has left few oral options for therapy. Since resistance rates vary by local geographic region, with individual patient characteristics, and over time, it is important to use current and local data when choosing a treatment regimen.

1	The urinary tract can be viewed as an anatomic unit united by a continuous column of urine extending from the urethra to the kidneys. In the majority of UTIs, bacteria establish infection by ascending from the urethra to the bladder. Continuing ascent up the ureter to the kidney is the pathway for most renal parenchymal infections. However, introduction of bacteria into the bladder does not inevitably lead to sustained and symptomatic infection. The interplay of host, pathogen, and environmental factors determines whether tissue invasion and symptomatic infection will ensue (Fig. 162-1). For example, bacteria often enter the bladder after sexual intercourse, but normal voiding and innate host defense mechanisms in the bladder eliminate these organisms. Any foreign body in the urinary tract, such as a urinary catheter or stone, provides an inert surface for bacterial colonization. Abnormal micturition and/or significant residual urine volume promotes true infection. In the simplest of

1	such as a urinary catheter or stone, provides an inert surface for bacterial colonization. Abnormal micturition and/or significant residual urine volume promotes true infection. In the simplest of terms, anything that increases the likelihood of bacteria entering the bladder and staying there increases the risk of UTI.

1	Bacteria can also gain access to the urinary tract through the bloodstream. However, hematogenous spread accounts for <2% of documented UTIs and usually results from bacteremia caused by relatively virulent organisms, such as Salmonella and S. aureus. Indeed, the isolation of either of these pathogens from a patient without a catheter or other instrumentation warrants a search for a bloodstream source. Hematogenous infections may produce focal abscesses or areas of pyelonephritis within a kidney and result in positive urine cultures. The pathogenesis of candiduria is distinct in that the hematogenous route is common. The presence of Candida in the urine of a noninstrumented Type of organism Genetic background Infection, colonization, or elimination HostOrganism Behavioral factors Underlying disease Tissue-specific receptors Presence of virulence factors Expression of virulence factors

1	Infection, colonization, or elimination HostOrganism Behavioral factors Underlying disease Tissue-specific receptors Presence of virulence factors Expression of virulence factors FIGURE 162-1 Pathogenesis of urinary tract infection. The relationship among specific host, pathogen, and environmental factors determines the clinical outcome. immunocompetent patient implies either genital contamination or potentially widespread visceral dissemination.

1	Environmental Factors • vaginal ecology In women, vaginal ecology is an important environmental factor affecting the risk of UTI. Colonization of the vaginal introitus and periurethral area with organisms from the intestinal flora (usually E. coli) is the critical initial step in the pathogenesis of UTI. Sexual intercourse is associated with an increased risk of vaginal colonization with E. coli and thereby increases the risk of UTI. Nonoxynol-9 in spermicide is toxic to the normal vaginal microflora and thus is likewise associated with an increased risk of E. coli vaginal colonization and bacteriuria. In postmenopausal women, the previously predominant vaginal lactobacilli are replaced with colonizing gram-negative bacteria. The use of topical estrogens to prevent UTI in postmenopausal women is controversial; given the side effects of systemic hormone replacement, oral estrogens should not be used to prevent UTI.

1	anatomic and functional abnormalities Any condition that permits urinary stasis or obstruction predisposes the individual to UTI. Foreign bodies such as stones or urinary catheters provide an inert surface for bacterial colonization and formation of a persistent biofilm. Thus, vesicoureteral reflux, ureteral obstruction secondary to prostatic hypertrophy, neurogenic bladder, and urinary diversion surgery create an environment favorable to UTI. In persons with such conditions, E. coli strains lacking typical urinary virulence factors are often the cause of infection. Inhibition of ureteral peristalsis and decreased ureteral tone leading to vesicoureteral reflux are important in the pathogenesis of pyelonephritis in pregnant women. Anatomic factors—specifically, the distance of the urethra from the anus—are considered to be the primary reason why UTI is predominantly an illness of young women rather than of young men.

1	Host Factors The genetic background of the host influences the individual’s susceptibility to recurrent UTI, at least among women. A familial disposition to UTI and to pyelonephritis is well documented. Women with recurrent UTI are more likely to have had their first UTI before the age of 15 years and to have a maternal history of UTI. A component of the underlying pathogenesis of this familial predisposition to recurrent UTI may be persistent vaginal colonization with E. coli, even during asymptomatic periods. Vaginal and periurethral mucosal cells from women with recurrent UTI bind threefold more uropathogenic bacteria than do mucosal cells from women without recurrent infection. Epithelial cells from women who are non-secretors of certain blood group antigens may possess specific types of receptors to which E. coli can bind, thereby facilitating colonization and invasion. Mutations in host response genes (e.g., those coding for Toll-like receptors and the interleukin 8 receptor)

1	of receptors to which E. coli can bind, thereby facilitating colonization and invasion. Mutations in host response genes (e.g., those coding for Toll-like receptors and the interleukin 8 receptor) also have been linked to recurrent UTI and pyelonephritis. Polymorphisms in the interleukin 8–specific receptor gene CXCR1 are associated with increased susceptibility to pyelonephritis. Lower-level expression of CXCR1 on the surface of neutrophils impairs neutrophil-dependent host defense against bacterial invasion of the renal parenchyma.

1	a stronger barrier to infection than a compromised urinary tract. Thus, strains of E. coli that cause invasive symptomatic infection of the urinary tract in otherwise normal hosts often possess and express genetic virulence factors, including surface adhesins that mediate binding to specific receptors on the surface of uroepithelial cells. The best-studied adhesins are the P fimbriae, hairlike protein structures that interact with a specific receptor on renal epithelial cells. (The letter P denotes the ability of these fimbriae to bind to blood group antigen P, which contains a D-galactose-D-galactose residue.) P fimbriae are important in the pathogenesis of pyelonephritis and subsequent bloodstream invasion from the kidney.

1	Another adhesin is the type 1 pilus (fimbria), which all E. coli strains possess but not all E. coli strains express. Type 1 pili are thought to play a key role in initiating E. coli bladder infection; they mediate binding to uroplakins on the luminal surface of bladder uroepithelial cells. The binding of type 1 fimbriae of E. coli to receptors on uroepithelial cells initiates a complex series of signaling events that leads to apoptosis and 863 exfoliation of uroepithelial cells, with the attached E. coli organisms carried away in the urine. APPROACH TO THE PATIENT: The most important issue to be addressed when a UTI is suspected is the characterization of the clinical syndrome as ASB, uncomplicated cystitis, pyelonephritis, prostatitis, or complicated UTI. This information will shape the diagnostic and therapeutic approach.

1	A diagnosis of ASB can be considered only when the patient does not have local or systemic symptoms referable to the urinary tract. The clinical presentation is usually that of a patient who undergoes a screening urine culture for a reason unrelated to the genitourinary tract and is incidentally found to have bacteriuria. The presence of systemic signs or symptoms such as fever, altered mental status, and leukocytosis in the setting of a positive urine culture does not merit a diagnosis of symptomatic UTI unless other potential etiologies have been considered. The typical symptoms of cystitis are dysuria, urinary frequency, and urgency. Nocturia, hesitancy, suprapubic discomfort, and gross hematuria are often noted as well. Unilateral back or flank pain is generally an indication that the upper urinary tract is involved. Fever also is an indication of invasive infection of either the kidney or the prostate.

1	Mild pyelonephritis can present as low-grade fever with or without lower-back or costovertebral-angle pain, whereas severe pyelonephritis can manifest as high fever, rigors, nausea, vomiting, and flank and/or loin pain. Symptoms are generally acute in onset, and symptoms of cystitis may not be present. Fever is the main feature distinguishing cystitis and pyelonephritis. The fever of pyelonephritis typically exhibits a high spiking “picket-fence” pattern and resolves over 72 h of therapy. Bacteremia develops in 20–30% of cases of pyelonephritis. Patients with diabetes may present with obstructive uropathy associated with acute papillary necrosis when the sloughed papillae obstruct the ureter. Papillary necrosis may also be evident in some cases of pyelonephritis complicated by obstruction, sickle cell disease, analgesic nephropathy, or combinations of these conditions. In the rare cases of bilateral papillary necrosis, a rapid rise in the serum creatinine level may be the first

1	sickle cell disease, analgesic nephropathy, or combinations of these conditions. In the rare cases of bilateral papillary necrosis, a rapid rise in the serum creatinine level may be the first indication of the condition. Emphysematous pyelonephritis is a particularly severe form of the disease that is associated with the production of gas in renal and perinephric tissues and occurs almost exclusively in diabetic patients (Fig. 162-2). Xanthogranulomatous pyelonephritis occurs when chronic urinary obstruction (often by stag-horn calculi), together with chronic infection, leads to suppurative destruction of renal tissue (Fig. 162-3). On pathologic examination, the residual renal tissue frequently has a yellow coloration, with infiltration by lipid-laden macrophages. Pyelonephritis can also be complicated by intraparenchymal abscess formation; this situation should be suspected when a patient has continued fever and/or bacteremia despite antibacterial therapy.

1	Prostatitis includes both infectious and noninfectious abnormalities of the prostate gland. Infections can be acute or chronic, are almost always bacterial in nature, and are far less common than the noninfectious entity chronic pelvic pain syndrome (formerly known as chronic prostatitis). Acute bacterial prostatitis presents as dysuria, frequency, and pain in the prostatic pelvic or perineal area. Fever and chills are usually present, and symptoms of bladder outlet obstruction are common. Chronic bacterial prostatitis presents Urinary Tract Infections, Pyelonephritis, and Prostatitis FIGURE 162-2 Emphysematous pyelonephritis. Infection of the right kidney of a diabetic man by Escherichia coli, a gas-forming, fac-ultative anaerobic uropathogen, has led to destruction of the renal parenchyma (arrow) and tracking of gas through the retroperitoneal space (arrowhead).

1	more insidiously as recurrent episodes of cystitis, sometimes with associated pelvic and perineal pain. Men who present with recurrent cystitis should be evaluated for a prostatic focus. Complicated UTI presents as a symptomatic episode of cystitis or pyelonephritis in a man or woman with an anatomic predisposition to infection, with a foreign body in the urinary tract, or with factors predisposing to a delayed response to therapy.

1	DIAGNOSTIC TOOLS History The diagnosis of any of the UTI syndromes or ASB begins with a detailed history (Fig. 162-4). The history given by the patient has a high predictive value in uncomplicated cystitis. A meta-analysis evaluating the probability of acute UTI on the basis of history and physical findings concluded that, in women presenting with at least one symptom of UTI (dysuria, frequency, hematuria, or back pain) and without complicating factors, the probability of acute cystitis or pyelonephritis is 50%. The even higher rates of accuracy of self-diagnosis among women with recurrent UTI probably account for the success of patient-initiated treatment of recurrent cystitis. If vaginal discharge and complicating factors are absent and risk factors for UTI are present, then the probability of UTI is close to 90%, and no laboratory evaluation is needed. Similarly, a combination of dysuria and urinary frequency in the absence of vaginal discharge increases the probability of UTI to

1	of UTI is close to 90%, and no laboratory evaluation is needed. Similarly, a combination of dysuria and urinary frequency in the absence of vaginal discharge increases the probability of UTI to 96%. Further laboratory evaluation with dipstick testing or urine culture is not necessary in such patients before the initiation of definitive therapy.

1	When the patient’s history is applied as a diagnostic tool, it is important to recall that the studies included in the meta-analysis cited above did not enroll children, adolescents, pregnant women, men, or patients with complicated UTI. One significant concern is that sexually transmitted disease—that caused by Chlamydia trachomatis in particular—may be inappropriately treated as UTI. This concern is particularly relevant for female patients under the age of 25. The differential diagnosis to be considered when women present with dysuria includes cervicitis (C. trachomatis, Neisseria gonorrhoeae), vaginitis (Candida albicans, Trichomonas vaginalis), herpetic urethritis, interstitial cystitis, and noninfectious vaginal or vulvar irritation. Women with more than one sexual partner and inconsistent use of condoms are at high risk for both UTI and sexually transmitted disease, and symptoms alone do not always distinguish between these conditions.

1	The Urine Dipstick Test, Urinalysis, and Urine Culture Useful diagnostic tools include the urine dipstick test and urinalysis, both of which provide point-of-care information, and the urine culture, which can retrospectively confirm a prior diagnosis. Understanding the parameters of the dipstick test is important in interpreting its results. Only members of the family Enterobacteriaceae convert nitrate to nitrite, and enough nitrite must accumulate in the urine to reach the threshold of detection. If a woman with acute cystitis is forcing fluids and voiding frequently, the dipstick test for nitrite is less likely to be positive, even when E. coli is present. The leukocyte esterase test detects this enzyme in the host’s polymorphonuclear leukocytes in the urine, whether the cells are intact or lysed. Many reviews have attempted to describe the diagnostic accuracy of dipstick testing. The bottom line for clinicians is that a urine dipstick test can confirm the diagnosis of uncomplicated

1	or lysed. Many reviews have attempted to describe the diagnostic accuracy of dipstick testing. The bottom line for clinicians is that a urine dipstick test can confirm the diagnosis of uncomplicated cystitis in a patient with a reasonably high pretest probability of this

1	FIGURE 162-3 Xanthogranulomatous pyelonephritis. A. This photograph shows extensive destruction of renal parenchyma due to longstanding suppurative inflammation. The precipitating factor was obstruction by a staghorn calculus, which has been removed, leaving a depression (arrow). The mass effect of xanthogranulomatous pyelonephritis can mimic renal malignancy. B. A large staghorn calculus (arrow) is seen obstructing the renal pelvis and calyceal system. The lower pole of the kidney shows areas of hemorrhage and necrosis with collapse of cortical areas. (Images courtesy of Dharam M. Ramnani, MD, Virginia Urology Pathology Laboratory, Richmond, VA.)

1	Acute onset of Back pain Nausea/vomiting Fever Cystitis symptoms Acute onset of urinary symptoms Dysuria Frequency Urgency Non-localizing systemic symptoms of infection Fever Altered mental status Leukocytosis Positive urine culture in the absence of Urinary symptoms Systemic symptoms related to the urinary tract Recurrent acute urinary symptoms Male with perineal, pelvic, or prostatic pain All other patients Woman with unclear history or risk factors for STD Otherwise healthy woman who is not pregnant, clear history Patient who is pregnant, is a renal transplant recipient, or will undergo an invasive urologic procedure Otherwise healthy woman who is not pregnant Patient with urinary catheter All other patients All other patients Otherwise healthy woman who is not pregnant Male No obvious non-urinary cause Consider acute prostatitis Urinalysis and culture Consider urology evaluation Consider uncomplicated cystitis or STD Dipstick, urinalysis, and culture STD evaluation, pelvic exam

1	non-urinary cause Consider acute prostatitis Urinalysis and culture Consider urology evaluation Consider uncomplicated cystitis or STD Dipstick, urinalysis, and culture STD evaluation, pelvic exam Consider uncomplicated cystitis No urine culture needed Consider telephone management Consider complicated UTI, CAUTI, or pyelonephritis Urine culture Blood cultures Exchange or remove catheter if present Consider complicated UTI Urinalysis and culture Address any modifiable anatomic or functional abnormalities Consider uncomplicated pyelonephritis Urine culture Consider outpatient management Consider ASB Screening and treatment warranted Consider pyelonephritis Urine culture Blood cultures Consider ASB No additional workup or treatment needed Consider CA-ASB No additional workup or treatment needed Remove unnecessary catheters Consider recurrent cystitis Urine culture to establish diagnosis Consider prophylaxis or patient-initiated management Consider chronic bacterial prostatitis

1	needed Remove unnecessary catheters Consider recurrent cystitis Urine culture to establish diagnosis Consider prophylaxis or patient-initiated management Consider chronic bacterial prostatitis Meares-Stamey 4-glass test Consider urology consult

1	Urinary Tract Infections, Pyelonephritis, and Prostatitis FIGURE 162-4 Diagnostic approach to urinary tract infection (UTI). STD, sexually transmitted disease; CAUTI, catheter-associated UTI; ASB, asymptomatic bacteriuria; CA-ASB, catheter-associated ASB.

1	disease. Either nitrite or leukocyte esterase positivity can be interpreted differ in men (highly specific) and in noncatheterized nursing home as a positive result. Blood in the urine also may suggest a diagnosis of residents (highly sensitive). UTI. A dipstick test negative for both nitrite and leukocyte esterase Urine microscopy reveals pyuria in nearly all cases of cystitis and in the same type of patient should prompt consideration of other hematuria in ~30% of cases. In current practice, most hospital laboratoexplanations for the patient’s symptoms and collection of urine for ries use an automated system rather than manual examination for urine culture. A negative dipstick test is not sufficiently sensitive to rule out microscopy. A machine aspirates a sample of the urine and then classifies bacteriuria in pregnant women, in whom it is important to detect all the particles in the urine by size, shape, contrast, light scatter, volume, and episodes of bacteriuria. Performance

1	classifies bacteriuria in pregnant women, in whom it is important to detect all the particles in the urine by size, shape, contrast, light scatter, volume, and episodes of bacteriuria. Performance characteristics of the dipstick test other properties. These automated systems can be overwhelmed by high 866 numbers of dysmorphic red blood cells, white blood cells, or crystals; in general, counts of bacteria are less accurate than are counts of red and white blood cells. The authors’ clinical recommendation is that the patient’s symptoms and presentation should outweigh an incongruent result on automated urinalysis. The detection of bacteria in a urine culture is the diagnostic “gold standard” for UTI; unfortunately, however, culture results do not become available until 24 h after the patient’s presentation. Identifying specific organism(s) can require an additional 24 h. Studies of women with symptoms of cystitis have found that a colony count threshold of >102 bacteria/mL is more

1	presentation. Identifying specific organism(s) can require an additional 24 h. Studies of women with symptoms of cystitis have found that a colony count threshold of >102 bacteria/mL is more sensitive (95%) and specific (85%) than a threshold of 105/mL for the diagnosis of acute cystitis in women. In men, the minimal level indicating infection appears to be 103/mL. Urine specimens frequently become contaminated with the normal microbial flora of the distal urethra, vagina, or skin. These contaminants can grow to high numbers if the collected urine is allowed to stand at room temperature. In most instances, a culture that yields mixed bacterial species is contaminated except in settings of long-term catheterization, chronic urinary retention, or the presence of a fistula between the urinary tract and the gastrointestinal or genital tract.

1	The approach to diagnosis is influenced by which of the clinical UTI syndromes is suspected (Fig. 162-4). Uncomplicated Cystitis in Women Uncomplicated cystitis in women can be treated on the basis of history alone. However, if the symptoms are not specific or if a reliable history cannot be obtained, then a urine dipstick test should be performed. A positive nitrite or leukocyte esterase result in a woman with one symptom of UTI increases the probability of UTI from 50% to ~80%, and empirical treatment can be considered without further testing. In this setting, a negative dipstick result does not rule out UTI, and a urine culture, close clinical follow-up, and possibly a pelvic examination are recommended. These recommendations are made with the caveat that no factors associated with complicated UTI, such as pregnancy, are known to be present.

1	Cystitis in Men The signs and symptoms of cystitis in men are similar to those in women, but this disease differs in several important ways in the male population. Collection of urine for culture is strongly recommended when a man has symptoms of UTI, as the documentation of bacteriuria can differentiate the less common syndromes of acute and chronic bacterial prostatitis from the very common entity of chronic pelvic pain syndrome, which is not associated with bacteriuria and thus is not usually responsive to antibacterial therapy. If the diagnosis is unclear, localization cultures using the twoor four-glass Meares-Stamey test (urine collection after prostate massage) should be undertaken to differentiate between bacterial and nonbacterial prostatic syndromes, and the patient should be referred to a urologist. Men with febrile UTI often have an elevated serum level of prostate-specific antigen as well as an enlarged prostate and enlarged seminal vesicles on ultrasound—findings

1	be referred to a urologist. Men with febrile UTI often have an elevated serum level of prostate-specific antigen as well as an enlarged prostate and enlarged seminal vesicles on ultrasound—findings indicative of prostate involvement. In 85 men with febrile UTI, symptoms of urinary retention, early recurrence of UTI, hematuria at follow-up, and voiding difficulties were predictive of surgically correctable disorders. Men with none of these symptoms had normal upper and lower urinary tracts on urologic workup.

1	Asymptomatic Bacteriuria The diagnosis of ASB involves both micro-biologic and clinical criteria. The microbiologic criterion is usually ≥105 bacterial CFU/mL except in catheter-associated disease, in which ≥102 CFU/mL is the cutoff. The clinical criterion is that the person has no signs or symptoms referable to UTI. Antimicrobial therapy is warranted for any symptomatic UTI. The choice of antimicrobial agent and the dose and duration of therapy depend on the site of infection and the presence or absence of complicating conditions. Each category of UTI warrants a different approach based on the particular clinical syndrome.

1	region to region and impacts the approach to empirical treatment of UTI. E. coli ST131 is the predominant multilocus sequence type found worldwide as the cause of multidrug-resistant UTI. Recommendations for treatment must be considered in the context of local resistance patterns and national differences in some agents’ availability. For example, fosfomycin and pivmecillinam are not available in all countries but are considered first-line options where they are available because they retain activity against a majority of uropathogens that produce extended-spectrum β-lactamases. Thus, therapeutic choices should depend on local resistance, drug availability, and individual patient factors such as recent travel and antimicrobial use.

1	Since the species and antimicrobial susceptibilities of the bacteria that cause acute uncomplicated cystitis are highly predictable, many episodes of uncomplicated cystitis can be managed over the telephone (Fig. 162-4). Most patients with other UTI syndromes require further diagnostic evaluation. Although the risk of serious complications with telephone management appears to be low, studies of telephone management algorithms generally have involved otherwise healthy white women who are at low risk for complications of UTI.

1	In 1999, TMP-SMX was recommended as the first-line agent for treatment of uncomplicated UTI in the published guidelines of the Infectious Diseases Society of America. Antibiotic resistance among uropathogens causing uncomplicated cystitis has since increased, appreciation of the importance of collateral damage (as defined below) has increased, and newer agents have been studied. Unfortunately, there is no longer a single best agent for acute uncomplicated cystitis.

1	Collateral damage refers to the adverse ecologic effects of antimicrobial therapy, including killing of the normal flora and selection of drug-resistant organisms. Outbreaks of Clostridium difficile infection offer an example of collateral damage in the hospital environment. The implication of collateral damage in this context is that a drug that is highly efficacious for the treatment of UTI is not necessarily the optimal first-line agent if it also has pronounced secondary effects on the normal flora or is likely to change resistance patterns. Drugs used for UTI that have a minimal effect on fecal flora include pivmecillinam, fosfomycin, and nitrofurantoin. In contrast, trimethoprim, TMP-SMX, quinolones, and ampicillin affect the fecal flora more significantly; these drugs are notably the agents for which rising resistance levels have been documented.

1	Several effective therapeutic regimens are available for acute uncomplicated cystitis in women (Table 162-1). Well- studied first-line agents include TMP-SMX and nitrofurantoin. Second-line agents include fluoroquinolone and β-lactam compounds. Single-dose fosfomycin treatment for acute cystitis is widely used in Europe but has produced mixed results in randomized trials. There is increasing experience with the use of fosfomycin against UTIs (including complicated infections) caused by multidrugresistant E. coli. Pivmecillinam is not currently available in the United States or Canada but is a popular agent in some European countries. The pros and cons of other therapies are discussed briefly below.

1	Traditionally, TMP-SMX has been recommended as first-line treatment for acute cystitis, and it remains appropriate to consider the use of this drug in regions with resistance rates not exceeding 20%. TMP-SMX resistance has clinical significance: in TMP-SMX-treated patients with resistant isolates, the time to symptom resolution is longer and rates of both clinical and microbiologic failure are higher. Individual host factors associated with an elevated risk of UTI caused by a strain of E. coli resistant to TMP-SMX include recent use of TMPSMX or another antimicrobial agent and recent travel to an area with high rates of TMP-SMX resistance. The optimal setting for empirical use of TMP-SMX is uncomplicated UTI in a female patient who has an established relationship with the practitioner and who can thus seek further care if her symptoms do not respond promptly. aMicrobial response as measured by reduction of bacterial counts in the urine.

1	aMicrobial response as measured by reduction of bacterial counts in the urine. Note: Efficacy rates are averages or ranges calculated from the data and studies included in the 2010 Infectious Diseases Society of America/European Society of Clinical Microbiology and Infectious Diseases Guideline for Treatment of Uncomplicated UTI. TMP-SMX, trimethoprim-sulfamethoxazole; DS, double-strength.

1	Resistance to nitrofurantoin remains low despite >60 years of use. Since this drug affects bacterial metabolism in multiple pathways, several mutational steps are required for the development of resistance. Nitrofurantoin remains highly active against E. coli and most non–E. coli isolates. Proteus, Pseudomonas, Serratia, Enterobacter, and yeasts are all intrinsically resistant to this drug. Although nitrofurantoin has traditionally been prescribed as a 7-day regimen, similar microbiologic and clinical efficacies are noted with a 5-day course of nitrofurantoin or a 3-day course of TMP-SMX for treatment of women with acute cystitis; 3-day courses of nitrofurantoin are not recommended for acute cystitis. Nitrofurantoin does not reach significant levels in tissue and cannot be used to treat pyelonephritis.

1	Most fluoroquinolones are highly effective as short-course therapy for cystitis; the exception is moxifloxacin, which may not reach adequate urinary levels. The fluoroquinolones commonly used for UTI include ofloxacin, ciprofloxacin, and levofloxacin. The main concern about fluoroquinolone use for acute cystitis is the propagation of fluoroquinolone resistance, not only among uropathogens but also among other organisms causing more serious and difficult-totreat infections at other sites. Fluoroquinolone use is also a factor driving the emergence of C. difficile outbreaks in hospital settings. Most experts now call for restricting fluoroquinolones to specific instances of uncomplicated cystitis in which other antimicrobial agents are not suitable. Quinolone use in certain populations, including adults >60 years of age, has been associated with an increased risk of Achilles tendon rupture.

1	Except for pivmecillinam, β-lactam agents generally have not performed as well as TMP-SMX or fluoroquinolones in acute cystitis. Rates of pathogen eradication are lower and relapse rates are higher with β-lactam drugs. The generally accepted explanation is that β-lactams fail to eradicate uropathogens from the vaginal reservoir. A proposed role for intracellular biofilm communities is intriguing. Many strains of E. coli that are resistant to TMP-SMX are also resistant to amoxicillin and cephalexin; thus, these drugs should be used only for patients infected with susceptible strains. Urinary analgesics are appropriate in certain situations to speed resolution of bladder discomfort. The urinary tract analgesic phenazopyridine is widely used but can cause significant nausea. Combination analgesics containing urinary antiseptics (methenamine, methylene blue), a urine-acidifying agent (sodium phosphate), and an antispasmodic agent (hyoscyamine) also are available.

1	Since patients with pyelonephritis have tissue-invasive disease, the treatment regimen chosen should have a very high likelihood of eradicating the causative organism and should reach therapeutic blood levels quickly. High rates of TMP-SMX-resistant E. coli in patients with pyelonephritis have made fluoroquinolones the first-line therapy for acute uncomplicated pyelonephritis. Whether the fluoroquinolones are given orally or parenterally depends on the patient’s tolerance for oral intake. A randomized clinical trial demonstrated that a 7-day course of therapy with oral ciprofloxacin (500 mg twice daily, with or without an initial IV 400-mg dose) was highly effective for the initial management of pyelonephritis in the outpatient setting. Oral TMP-SMX (one double-strength tablet twice daily for 14 days) also is effective for treatment of acute uncomplicated pyelonephritis if the uropathogen is known to be susceptible. If the pathogen’s susceptibility is not known and TMPSMX is used, an

1	for 14 days) also is effective for treatment of acute uncomplicated pyelonephritis if the uropathogen is known to be susceptible. If the pathogen’s susceptibility is not known and TMPSMX is used, an initial IV 1-g dose of ceftriaxone is recommended. Oral β-lactam agents are less effective than the fluoroquinolones and should be used with caution and close follow-up. Options for parenteral therapy for uncomplicated pyelonephritis include fluoroquinolones, an extended-spectrum cephalosporin with or without an aminoglycoside, or a carbapenem. Combinations of a β-lactam and a β-lactamase inhibitor (e.g., ampicillin-sulbactam, ticarcillinclavulanate, piperacillin-tazobactam) or imipenem-cilastatin can be used in patients with more complicated histories, previous episodes of pyelonephritis, or recent urinary tract manipulations; in general, the treatment of such patients should be guided by urine culture results. Once the patient has responded clinically, oral therapy should be substituted

1	recent urinary tract manipulations; in general, the treatment of such patients should be guided by urine culture results. Once the patient has responded clinically, oral therapy should be substituted for parenteral therapy.

1	Nitrofurantoin, ampicillin, and the cephalosporins are considered relatively safe in early pregnancy. One retrospective case-control study suggesting an association between nitrofurantoin and birth defects has not been confirmed. Sulfonamides should clearly be avoided both in the first trimester (because of possible teratogenic effects) and near term (because of a possible role in the development of kernicterus). Fluoroquinolones are avoided because of possible adverse effects on fetal cartilage development. Ampicillin and the cephalosporins have been used extensively in pregnancy and are the drugs of choice for the treatment of asymptomatic or symptomatic UTI in this group of patients. For pregnant women with overt pyelonephritis, parenteral β-lactam therapy with or without aminoglycosides is the standard of care.

1	Since the prostate is involved in the majority of cases of febrile UTI in men, the goal in these patients is to eradicate the prostatic infection as well as the bladder infection. A 7to 14-day course of a fluoroquinolone or TMP-SMX is recommended if the uropathogen is susceptible. If acute bacterial prostatitis is suspected, antimicrobial therapy should be initiated after urine and blood are obtained for cultures. Therapy can be tailored to urine culture results and should be continued for 2–4 weeks. For documented chronic bacterial prostatitis, a 4to 6-week course of antibiotics is often necessary. Recurrences, which are not uncommon in chronic prostatitis, often warrant a 12-week course of treatment.

1	Complicated UTI (other than that discussed above) occurs in a heterogeneous group of patients with a wide variety of structural and functional abnormalities of the urinary tract and kidneys. The range of species and their susceptibility to antimicrobial agents are likewise heterogeneous. As a consequence, therapy for complicated UTI must be individualized and guided by urine culture results. Frequently, a patient with complicated UTI will have prior urine culture data that can be used to guide empirical therapy while current culture results are awaited. Xanthogranulomatous pyelonephritis is treated with nephrectomy. Percutaneous drainage can be used Urinary Tract Infections, Pyelonephritis, and Prostatitis 868 as the initial therapy in emphysematous pyelonephritis and can be followed by elective nephrectomy as needed. Papillary necrosis with obstruction requires intervention to relieve the obstruction and to preserve renal function.

1	Treatment of ASB does not decrease the frequency of symptomatic infections or complications except in pregnant women, persons undergoing urologic surgery, and perhaps neutropenic patients and renal transplant recipients. Treatment of ASB in pregnant women and patients undergoing urologic procedures should be directed by urine culture results. In all other populations, screening for and treatment of ASB are discouraged. The majority of cases of catheter-associated bacteriuria are asymptomatic and do not warrant antimicrobial therapy. Multiple institutions have released guidelines for the treatment of CAUTI, which is defined by bacteriuria and symptoms in a catheterized patient. The signs and symptoms either are localized to the urinary tract or can include otherwise unexplained systemic manifestations, such as fever. The accepted threshold for bacteriuria to meet the definition of CAUTI is ≥103 CFU/mL, while the threshold for bacteriuria to meet the definition of ASB is ≥105 CFU/mL.

1	The formation of biofilm—a living layer of uropathogens—on the urinary catheter is central to the pathogenesis of CAUTI and affects both therapeutic and preventive strategies. Organisms in a biofilm are relatively resistant to killing by antibiotics, and eradication of a catheter-associated biofilm is difficult without removal of the device itself. Furthermore, because catheters provide a conduit for bacteria to enter the bladder, bacteriuria is inevitable with longterm catheter use. The typical signs and symptoms of UTI, including pain, urgency, dysuria, fever, peripheral leukocytosis, and pyuria, have less predictive value for the diagnosis of infection in catheterized patients. Furthermore, the presence of bacteria in the urine of a patient who is febrile and catheterized does not necessarily predict CAUTI, and other explanations for the fever should be considered.

1	The etiology of CAUTI is diverse, and urine culture results are essential to guide treatment. Fairly good evidence supports the practice of catheter change during treatment for CAUTI. The goal is to remove biofilm-associated organisms that could serve as a nidus for reinfection. Pathology studies reveal that many patients with long-term catheters have occult pyelonephritis. A randomized trial in persons with spinal cord injury who were undergoing intermittent catheterization found that relapse was more common after 3 days of therapy than after 14 days. In general, a 7to 14-day course of antibiotics is recommended, but further studies on the optimal duration of therapy are needed.

1	In the setting of long-term catheter use, systemic antibiotics, bladder-acidifying agents, antimicrobial bladder washes, topical disinfectants, and antimicrobial drainage-bag solutions have all been ineffective at preventing the onset of bacteriuria and have been associated with the emergence of resistant organisms. The best strategy for prevention of CAUTI is to avoid insertion of unnecessary catheters and to remove catheters once they are no longer necessary. Evidence is insufficient to recommend suprapubic catheters and condom catheters as alternatives to indwelling urinary catheters as a means to prevent CAUTI. However, intermittent catheterization may be preferable to long-term indwelling urethral catheterization in certain populations (e.g., spinal cord–injured persons) to prevent both infectious and anatomic complications. Antimicrobial catheters impregnated with silver or nitrofurazone have not been shown to provide significant clinical benefit in terms of reducing rates of

1	both infectious and anatomic complications. Antimicrobial catheters impregnated with silver or nitrofurazone have not been shown to provide significant clinical benefit in terms of reducing rates of symptomatic UTI.

1	The appearance of Candida in the urine is an increasingly common complication of indwelling catheterization, particularly for patients in the intensive care unit, those taking broad-spectrum antimicrobial drugs, and those with underlying diabetes mellitus. In many studies, >50% of urinary Candida isolates have been found to be non-albicans species. The clinical presentation varies from an asymptomatic laboratory finding to pyelonephritis and even sepsis. Removal of the urethral catheter results in resolution of candiduria in more than one-third of asymptomatic cases. Treatment of asymptomatic patients does not appear to decrease the frequency of recurrence of candiduria. Treatment is recommended for patients who have symptomatic cystitis or pyelonephritis and for those who are at high risk for disseminated disease. High-risk patients include those with neutropenia, those who are undergoing urologic manipulation, those who are clinically unstable, and low-birth-weight infants.

1	high risk for disseminated disease. High-risk patients include those with neutropenia, those who are undergoing urologic manipulation, those who are clinically unstable, and low-birth-weight infants. Fluconazole (200–400 mg/d for 14 days) reaches high levels in urine and is the first-line regimen for Candida infections of the urinary tract. Although instances of successful eradication of candiduria by some of the newer azoles and echinocandins have been reported, these agents are characterized by only low-level urinary excretion and thus are not recommended. For Candida isolates with high levels of resistance to fluconazole, oral flucytosine and/ or parenteral amphotericin B are options. Bladder irrigation with amphotericin B generally is not recommended.

1	Recurrence of uncomplicated cystitis in reproductive-age women is common, and a preventive strategy is indicated if recurrent UTIs are interfering with a patient’s lifestyle. The threshold of two or more symptomatic episodes per year is not absolute; decisions about interventions should take the patient’s preferences into account.

1	Three prophylactic strategies are available: continuous, postcoital, and patient-initiated therapy. Continuous prophylaxis and postcoital prophylaxis usually entail low doses of TMP-SMX, a fluoroquinolone, or nitrofurantoin. These regimens are all highly effective during the period of active antibiotic intake. Typically, a prophylactic regimen is prescribed for 6 months and then discontinued, at which point the rate of recurrent UTI often returns to baseline. If bothersome infections recur, the prophylactic program can be reinstituted for a longer period. Selection of resistant strains in the fecal flora has been documented in studies of women taking prophylactic antibiotics for 12 months.

1	Patient-initiated therapy involves supplying the patient with materials for urine culture and with a course of antibiotics for self-medication at the first symptoms of infection. The urine culture is refrigerated and delivered to the physician’s office for confirmation of the diagnosis. When an established and reliable patient-provider relationship exists, the urine culture can be omitted as long as the symptomatic episodes respond completely to short-course therapy and are not followed by relapse.

1	Cystitis is a risk factor for recurrent cystitis and pyelonephritis. ASB is common among elderly and catheterized patients but does not in itself increase the risk of death. The relationships among recurrent UTI, chronic pyelonephritis, and renal insufficiency have been widely studied. In the absence of anatomic abnormalities, recurrent infection in children and adults does not lead to chronic pyelonephritis or to renal failure. Moreover, infection does not play a primary role in chronic interstitial nephritis; the primary etiologic factors in this condition are analgesic abuse, obstruction, reflux, and toxin exposure. In the presence of underlying renal abnormalities (particularly obstructing stones), infection as a secondary factor can accelerate renal parenchymal damage. In spinal cord–injured patients, use of a longterm indwelling bladder catheter is a well-documented risk factor for bladder cancer. Chronic bacteriuria resulting in chronic inflammation is one possible explanation

1	patients, use of a longterm indwelling bladder catheter is a well-documented risk factor for bladder cancer. Chronic bacteriuria resulting in chronic inflammation is one possible explanation for this observation.

1	Sexually Transmitted Infections: overview and Clinical Approach Jeanne M. Marrazzo, King K. Holmes CLASSIFICATION AND EPIDEMIOLOGY Worldwide, most adults acquire at least one sexually transmitted infec-163 tion (STI), and many remain at risk for complications. Each year, for example, an estimated 14 million persons in the United States acquire a new genital human papillomavirus (HPV) infection, and many of these individuals are at risk for genital neoplasias. Certain STIs, such as syphilis, gonorrhea, HIV infection, hepatitis B, and chancroid, are most concentrated within “core populations” characterized by high rates of partner change, multiple concurrent partners, or “dense,” highly connected sexual networks—e.g., involving sex workers and their clients, some men who have sex with men (MSM), and persons involved in the use of illicit drugs, particularly crack cocaine and methamphetamine. Other STIs are distributed more evenly throughout societies. For example, chlamydial infections,

1	and persons involved in the use of illicit drugs, particularly crack cocaine and methamphetamine. Other STIs are distributed more evenly throughout societies. For example, chlamydial infections, genital infections with HPV, and genital herpes can spread widely, even in relatively low-risk populations.

1	In general, the product of three factors determines the initial rate of spread of any STI within a population: rate of sexual exposure of susceptible to infectious people, efficiency of transmission per exposure, and duration of infectivity of those infected. Accordingly, efforts to prevent and control STIs aim to decrease the rate of sexual exposure of susceptibles to infected persons (e.g., through individual counseling and efforts to change the norms of sexual behavior and through a variety of STI control efforts aimed at reducing the proportion of the population infected), to decrease the duration of infectivity (through early diagnosis and curative or suppressive treatment), and to decrease the efficiency of transmission (e.g., through promotion of condom use and safer sexual practices, through use of effective vaccines, and recently through male circumcision).

1	In all societies, STIs rank among the most common of all infectious diseases, with >30 infections now classified as pre dominantly sexually transmitted or as frequently sexually transmissible (Table 163-1). In developing countries, with three-quarters of the world’s population and 90% of the world’s STIs, factors such as population growth (especially in adolescent and young-adult age groups), rural-to-urban migration, wars, limited or no provision of reproductive health services for women, and poverty create exceptional vulnerability to disease resulting from unprotected sex. During the 1990s in China, Russia, the other states of the former Soviet Union, and South Africa, internal social structures changed rapidly as borders opened to the West, unleashing enormous new epidemics of HIV infection and other STIs. Despite advances in the provision of highly effective antiretroviral therapy worldwide, HIV remains the leading cause of death in some developing countries, and HPV and

1	HIV infection and other STIs. Despite advances in the provision of highly effective antiretroviral therapy worldwide, HIV remains the leading cause of death in some developing countries, and HPV and hepatitis B virus (HBV) remain important causes of cervical and hepatocellular carcinoma, respectively—two of the most common malignancies in the developing world. Sexually transmitted herpes simplex virus (HSV) infections now cause most genital ulcer disease throughout the world and an increasing proportion of cases of genital herpes in developing countries with generalized HIV epidemics, where the positive-feedback loop between HSV and HIV transmission is a growing, intractable problem. Despite this consistent link, randomized trials evaluating the efficacy of antiviral therapy in suppressing HSV in both HIV-uninfected and HIV-infected persons have not demonstrated a protective effect against acquisition or transmission of HIV. The World Health Organization estimated that 448 million new

1	HSV in both HIV-uninfected and HIV-infected persons have not demonstrated a protective effect against acquisition or transmission of HIV. The World Health Organization estimated that 448 million new cases of four curable STIs—gonorrhea, chlamydial infection, syphilis, and trichomoniasis—occurred in 2005. Up to 50% of women of reproductive age in developing countries have bacterial vaginosis (arguably acquired sexually). All of these curable infections have been associated with increased risk of HIV transmission or acquisition.

1	Sexual Transmission Repeatedly Described but Not Well Defined or Not the Predominant Mode Transmitted by Sexual Contact Involving Oral-Fecal Exposure; of Declining Importance in Men Who Have Sex with Men Shigella spp. Hepatitis A virus Giardia lamblia Campylobacter spp. aIncludes protozoa, ectoparasites, and fungi. bAmong U.S. patients for whom a risk factor can be ascertained, most hepatitis B virus infections are transmitted sexually.

1	In the United States, the prevalence of antibody to HSV-2 began to fall in the late 1990s, especially among adolescents and young adults; the decline is presumably due to delayed sexual debut, increased condom use, and lower rates of multiple (four or more) sex partners, as is well documented by the U.S. Youth Risk Behavior Surveillance System. The estimated annual incidence of HBV infection has also declined dramatically since the mid-1980s; this decrease is probably attributable more to adoption of safer sexual practices and reduced needle sharing among injection drug users than to use of hepatitis B vaccine, for which coverage among young adults (including those at high risk for this infection) initially was very limited. Genital HPV remains the most common sexually transmitted pathogen in this country, infecting 60% of a cohort of initially HPV-negative, sexually active Washington state college women within 5 years in a study conducted from 1990 to 2000. The scale-up of HPV

1	in this country, infecting 60% of a cohort of initially HPV-negative, sexually active Washington state college women within 5 years in a study conducted from 1990 to 2000. The scale-up of HPV vaccine coverage among young women has already shown promise in reducing the incidence of infection with the HPV types included in the vaccines and of conditions associated with these viruses.

1	In industrialized countries, fear of HIV infection since the mid1980s, coupled with widespread behavioral interventions and better-organized systems of care for the curable STIs, initially helped curb the transmission of the latter diseases. However, foci of hyperendemic transmission persist in the southeastern United States and in most large U.S. cities. Rates of gonorrhea and syphilis remain higher in the United States than in any other Western industrialized country.

1	In the United States, the Centers for Disease Control and Prevention (CDC) has compiled reported rates of STIs since 1941. The incidence of reported gonorrhea peaked at 468 cases per 100,000 population in the mid-1970s and fell to a low of 98 cases per 100,000 in 2012. With increased testing and more sensitive tests, the incidence of reported Chlamydia trachomatis infection has been increasing steadily since reporting began in 1984, reaching an all-time peak of 457.6 cases per 100,000 in 2011. The incidence of primary and secondary syphilis per 100,000 peaked at 71 cases in 1946, fell rapidly to 3.9 cases in 1956, ranged from ~10 to 15 cases through 1987 (with markedly increased

1	Sexually Transmitted Infections: Overview and Clinical Approach 870 rates among MSM and African Americans), and then fell to a nadir of 2.1 cases in 2000–2001 (with rates falling most rapidly among heterosexual African Americans). Unfortunately, since 1996, with the introduction of highly active antiretroviral therapy, the increased use of “serosorting” (i.e., the avoidance of unprotected sex with HIVserodiscordant partners but not with HIV-seroconcordant partners, a strategy that provides no protection against STIs other than HIV infection), and an ongoing epidemic of methamphetamine use, gonorrhea, syphilis, and chlamydial infection have had a remarkable resurgence among MSM in North America and Europe, where outbreaks of a rare type of chlamydial infection (lymphogranuloma venereum [LGV]) that had virtually disappeared during the AIDS era have occurred. In 2012, ~75% of primary and secondary syphilis cases reported to the CDC were in MSM. These developments have resulted in a high

1	that had virtually disappeared during the AIDS era have occurred. In 2012, ~75% of primary and secondary syphilis cases reported to the CDC were in MSM. These developments have resulted in a high degree of co-infection with HIV and other sexually transmitted pathogens (particularly syphilis and LGV), primarily among MSM.

1	Although other chapters discuss management of specific STIs, delineating treatment based on diagnosis of a specific infection, most patients are actually managed (at least initially) on the basis of presenting symptoms and signs and associated risk factors, even in industrialized countries. Table 163-2 lists some of the most common clinical STD syndromes and their microbial etiologies. Strategies for their management are outlined below. Chapters 225e and 226 address the management of infections with human retroviruses.

1	STD care and management begin with risk assessment and proceed to clinical assessment, diagnostic testing or screening, treatment, and prevention. Indeed, the routine care of any patient begins with risk assessment (e.g., for risk of heart disease, cancer). STD/HIV risk assessment is important in primary care, urgent care, and emergency care settings as well as in specialty clinics providing adolescent, HIV/ AIDS, prenatal, and family planning services. STD/HIV risk assessment guides detection and interpretation of symptoms that could reflect an STD; decisions on screening or prophylactic/preventive treatment; risk reduction counseling and intervention (e.g., hepatitis B vaccination); and treatment of partners of patients with known infections. Consideration of routine demographic data (e.g., gender, age, area of residence) is a simple first step in STD/HIV risk assessment. For example, national guidelines strongly recommend routine screening of sexually active females ≤25 years of

1	(e.g., gender, age, area of residence) is a simple first step in STD/HIV risk assessment. For example, national guidelines strongly recommend routine screening of sexually active females ≤25 years of age for C. trachomatis infection. Table 163-3 provides a set of 11 STD/HIV risk-assessment questions that clinicians can pose verbally or that health care systems can adapt (with yes/no responses) into a routine self-administered questionnaire for use in clinics. The initial framing statement gives permission to discuss topics that may be perceived as sensitive or socially unacceptable by providers and patients alike.

1	Risk assessment is followed by clinical assessment (elicitation of information on specific current symptoms and signs of STDs). Confirmatory diagnostic tests (for persons with symptoms or signs) or screening tests (for those without symptoms or signs) may involve microscopic examination, culture, antigen detection tests, nucleic acid amplification tests (NAATs), or serology. Initial syndrome-based treatment should cover the most likely causes. For certain syndromes, results of rapid tests can narrow the spectrum of this initial therapy (e.g., saline microscopy of vaginal fluid for women with vaginal discharge, Gram’s stain of urethral discharge for men with urethral discharge, rapid plasma reagin test for genital ulcer). After the institution of treatment, STD management proceeds to the “4 Cs” of prevention and control: contact tracing (see “Prevention and Control of STIs,” below), ensuring compliance with therapy, and counseling on risk reduction, including condom promotion and

1	the “4 Cs” of prevention and control: contact tracing (see “Prevention and Control of STIs,” below), ensuring compliance with therapy, and counseling on risk reduction, including condom promotion and provision.

1	Consistent with current guidelines, all adults should be screened for infection with HIV-1 at least once and more frequently if they are at elevated risk for acquisition of this infection. Urethritis in men produces urethral discharge, dysuria, or both, usually without frequency of urination. Causes include Neisseria gonorrhoeae, AIDS HIV types 1 and 2 Urethritis: males Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Ureaplasma urealyticum (subspecies urealyticum), Trichomonas vaginalis, HSV Epididymitis C. trachomatis, N. gonorrhoeae Lower genital tract infections: females Cystitis/urethritis C. trachomatis, N. gonorrhoeae, HSV Mucopurulent cervicitis C. trachomatis, N. gonorrhoeae, M. genitalium Vulvitis Candida albicans, HSV Vulvovaginitis C. albicans, T. vaginalis Acute pelvic inflammatory N. gonorrhoeae, C. trachomatis, disease BV-associated bacteria, M. genitalium, group B streptococci

1	Vulvovaginitis C. albicans, T. vaginalis Acute pelvic inflammatory N. gonorrhoeae, C. trachomatis, disease BV-associated bacteria, M. genitalium, group B streptococci Infertility N. gonorrhoeae, C. trachomatis, BV-associated bacteria Ulcerative lesions of the HSV-1, HSV-2, Treponema pallidum, genitalia Haemophilus ducreyi, C. trachomatis (LGV strains), Klebsiella (Calymmatobacterium) granulomatis Complications of pregnancy/ Several agents implicated puerperium Intestinal infections Proctitis C. trachomatis, N. gonorrhoeae, HSV, T. pallidum Proctocolitis or enterocolitis Campylobacter spp., Shigella spp., Entamoeba histolytica, Helicobacter spp., other enteric pathogens Enteritis Giardia lamblia Acute arthritis with urogenital N. gonorrhoeae (e.g., DGI), C. trachomatis infection or viremia (e.g., reactive arthritis), HBV Genital and anal warts HPV (30 genital types) Mononucleosis syndrome CMV, HIV, EBV Hepatitis Hepatitis viruses, T. pallidum, CMV, EBV Neoplasias

1	Squamous cell dysplasias and HPV (especially types 16, 18, 31, 45) cancers of the cervix, anus, vulva, vagina, or penis Kaposi’s sarcoma, body-cavity HHV-8 lymphomas T cell leukemia HTLV-1 Hepatocellular carcinoma HBV Tropical spastic paraparesis HTLV-1 Scabies Sarcoptes scabiei Pubic lice Pthirus pubis Abbreviations: BV, bacterial vaginosis; CMV, cytomegalovirus; DGI, disseminated gonococcal infection; EBV, Epstein-Barr virus; HBV, hepatitis B virus; HHV-8, human herpesvirus type 8; HPV, human papillomavirus; HSV, herpes simplex virus; HTLV, human T cell lymphotropic virus; LGV, lymphogranuloma venereum. C. trachomatis, Mycoplasma genitalium, Ureaplasma urealyticum, Trichomonas vaginalis, HSV, and adenovirus.

1	C. trachomatis, Mycoplasma genitalium, Ureaplasma urealyticum, Trichomonas vaginalis, HSV, and adenovirus. Until recently, C. trachomatis caused ~30–40% of cases of nongonococcal urethritis (NGU), particularly in heterosexual men; however, the proportion of cases due to this organism has probably declined in some populations served by effective chlamydial-control programs, and older men with urethritis appear less likely to have chlamydial infection. HSV and T. vaginalis each cause a small proportion of NGU cases in the United States. Recently, multiple studies have consistently implicated M. genitalium as a probable cause of many Framing Statement: In order to provide the best care for you today and to understand your risk for certain infections, it is necessary for us to talk about your sexual behavior. Screening Questions: Do you have any reason to think you might have a sexually transmitted infection? If so, what reason?

1	Screening Questions: Do you have any reason to think you might have a sexually transmitted infection? If so, what reason? For all adolescents <18 years old: Have you begun having any kind of sex yet? STD History: (3) Have you ever had any sexually transmitted infections or any genital infections? If so, which ones? Sexual Preference: (4) Have you had sex with men, women, or both? Injection Drug Use: Have you ever injected yourself (“shot up”) with drugs? (If yes, have you ever shared needles or injection equipment?) Have you ever had sex with a gay or bisexual man or with anyone who had ever injected drugs? Characteristics of Partner(s): Has your sex partner had any sexually transmitted infections? If so, which ones? Has your sex partner had other sex partners during the time you’ve been together? STD Symptoms Checklist: (9) Have you recently developed any of these symptoms?

1	Has your sex partner had other sex partners during the time you’ve been together? STD Symptoms Checklist: (9) Have you recently developed any of these symptoms? Sexual Practices, Past 2 Months (for patients answering yes to any of the above questions, to guide examination and testing): Now I’d like to ask what parts of your body may have been sexually exposed to an STD (e.g., your penis, mouth, vagina, anus). Query about Interest in STD Screening Tests (for patients answering no to all of the above questions): Would you like to be tested for HIV or any other STDs today? (If yes, clinician can explore which STD and why.) Source: Adapted from JR Curtis, KK Holmes, in KK Holmes et al (eds): Sexually Transmitted Diseases, 4th ed. New York, McGraw-Hill, 2008. Chlamydia-negative cases. Fewer studies than in the past have implicated Ureaplasma; the ureaplasmas have been differentiated into U. urealyticum and Ureaplasma parvum, and a few studies suggest that

1	U. urealyticum—but not U. parvum—is associated with NGU. Coliform bacteria can cause urethritis in men who practice insertive anal intercourse. The initial diagnosis of urethritis in men currently includes specific tests only for N. gonorrhoeae and C. trachomatis; it does not yet include testing for Mycoplasma or Ureaplasma species. The following summarizes the approach to the patient with suspected urethritis: 1. Establish the presence of urethritis. If proximal-to-distal “milking” of the urethra does not express a purulent or mucopurulent discharge, even after the patient has not voided for several hours (or preferably overnight), a Gram’s-stained smear of an anterior urethral specimen obtained by passage of a small urethrogenital swab 2–3 cm into the urethra usually reveals ≥5 neutrophils per 1000× field in areas containing cells; in gonococcal infection, such a smear usually reveals gram-negative intracellular diplococci as well. Alternatively, the centrifuged sediment of the

1	per 1000× field in areas containing cells; in gonococcal infection, such a smear usually reveals gram-negative intracellular diplococci as well. Alternatively, the centrifuged sediment of the first 20–30 mL of voided urine—ideally collected as the first morning specimen— can be examined for inflammatory cells, either by microscopy showing ≥10 leukocytes per high-power field or by the leukocyte esterase test. Patients with symptoms who lack objective evidence of urethritis may have functional rather than organic problems and generally do not benefit from repeated courses of antibiotics.

1	2. Evaluate for complications or alternative diagnoses. A brief history 871 and examination will exclude epididymitis and systemic complications, such as disseminated gonococcal infection (DGI) and reactive arthritis. Although digital examination of the prostate gland seldom contributes to the evaluation of sexually active young men with urethritis, men with dysuria who lack evidence of urethritis as well as sexually inactive men with urethritis should undergo prostate palpation, urinalysis, and urine culture to exclude bacterial prostatitis and cystitis. 3.

1	Evaluate for gonococcal and chlamydial infection. An absence of typical gram-negative diplococci on Gram’s-stained smear of urethral exudate containing inflammatory cells warrants a preliminary diagnosis of NGU, as this test is 98% sensitive for the diagnosis of gonococcal urethral infection. However, an increasing proportion of men with symptoms and/or signs of urethritis are simultaneously assessed for infection with N. gonorrhoeae and C. trachomatis by “multiplex” NAATs of first-voided urine. The urine specimen tested should consist of the first 10–15 mL of the stream, and, if possible, patients should not have voided for the prior 2 h. Culture or NAAT for N. gonorrhoeae may yield positive results when Gram’s staining is negative; certain strains of N. gonorrhoeae can result in negative urethral Gram’s stains in up to 30% of cases of urethral infection. Results of tests for gonococcal and chlamydial infection predict the patient’s prognosis (with greater risk for recurrent NGU if

1	urethral Gram’s stains in up to 30% of cases of urethral infection. Results of tests for gonococcal and chlamydial infection predict the patient’s prognosis (with greater risk for recurrent NGU if neither chlamydiae nor gonococci are found than if either is detected) and can guide both the counseling given to the patient and the management of the patient’s sexual partner(s).

1	4. Treat urethritis promptly while test results are pending. Table 163-4 summarizes the steps in management of sexually active men with urethral discharge and/or dysuria.

1	In practice, if Gram’s stain does not reveal gonococci, urethritis is treated with a regimen effective for NGU, such as azithromycin or doxycycline. Both are effective, although azithromycin may give better results in M. genitalium infection. If gonococci are demonstrated by Gram’s stain or if no diagnostic tests are performed to exclude gonorrhea definitively, treatment should include parenteral cephalosporin therapy for gonorrhea (Chap. 181) plus oral azithromycin, primarily for additive activity against N. gonorrhoeae given concerns about evolving antibiotic resistance. Azithromycin also treats C. trachomatis, which often causes urethral co-infection in men with gonococcal urethritis. Ideally, sexual partners should be tested for gonorrhea and chlamydial infection; regardless of whether they are tested for these infections, however, they should receive the same regimen given to the male index case. Patients with confirmed persistence or recurrence of urethritis after treatment

1	they are tested for these infections, however, they should receive the same regimen given to the male index case. Patients with confirmed persistence or recurrence of urethritis after treatment should be re-treated with the initial regimen if they did not comply with the original treatment or were reexposed to an untreated partner. Otherwise, an intraurethral swab specimen and a first-voided urine sample should be tested for T. vaginalis (currently done by culture, although NAATs are more sensitive and are approved for the diagnosis of trichomoniasis in women). If compliance with initial treatment is confirmed and reexposure to an untreated sex partner is deemed unlikely, the recommended treatment is with metronidazole or tinidazole (2 g by mouth in a single dose) plus azithromycin (1 g by mouth in a single dose); the azithromycin component is especially important if this drug has not been given during initial therapy.

1	Acute epididymitis, almost always unilateral, produces pain, swelling, and tenderness of the epididymis, with or without symptoms or signs of urethritis. This condition must be differentiated from testicular torsion, tumor, and trauma. Torsion, a surgical emergency, usually occurs in Sexually Transmitted Infections: Overview and Clinical Approach Chlamydia trachomatis Demonstration of urethral discharge or pyuria Exclusion of local or systemic Urethral Gram’s stain to confirm urethritis, detect gram-negative Herpes simplex virus diplococci Test for N. gonorrhoeae, C. trachomatis (unless excluded): Ceftriaxone, 250 mg IMa plus Azithromycin, 1 g PO Management of Recurrence Confirm objective evidence of urethritis. If patient was reexposed to untreated or new partner, repeat treatment of patient and partner.

1	Azithromycin, 1 g PO Management of Recurrence Confirm objective evidence of urethritis. If patient was reexposed to untreated or new partner, repeat treatment of patient and partner. If patient was not reexposed, consider infection with T. vaginalisb or doxycycline-resistant M. genitaliumc or Ureaplasma, and consider treatment with metronidazole, azithromycin, or both.

1	aNeither oral cephalosporins nor fluoroquinolones are recommended for treatment of gonorrhea in the United States because of the emergence of increasing fluoroquinolone resistance in N. gonorrhoeae, especially (but not only) among men who have sex with men, and decreasing susceptibility of a still-small proportion of gonococci to ceftriaxone (Fig. 163-1). Updates on the emergence of antimicrobial resistance in N. gonorrhoeae can be obtained from the Centers for Disease Control and Prevention at http://www.cdc.gov/std. bIn men, the diagnosis of T. vaginalis infection requires culture, DNA testing, or nucleic acid amplification testing (where available) of early-morning first-voided urine sediment or of a urethral swab specimen obtained before voiding. cM. genitalium is often resistant to doxycycline and azithromycin but is usually susceptible to the fluoroquinolone moxifloxacin. Until nucleic acid amplification testing for M. genitalium becomes commercially available, moxifloxacin can

1	and azithromycin but is usually susceptible to the fluoroquinolone moxifloxacin. Until nucleic acid amplification testing for M. genitalium becomes commercially available, moxifloxacin can be considered for treatment of refractory nongonococcal, nonchlamydial urethritis.

1	the second or third decade of life and produces a sudden onset of pain, elevation of the testicle within the scrotal sac, rotation of the epididymis from a posterior to an anterior position, and absence of blood flow on Doppler examination or 99mTc scan. Persistence of symptoms after a course of therapy for epididymitis suggests the possibility of testicular tumor or of a chronic granulomatous disease, such as tuberculosis. In sexually active men under age 35, acute epididymitis is caused most frequently by C. trachomatis and less commonly by N. gonorrhoeae and is usually associated with overt or subclinical urethritis. Acute epididymitis occurring in older men or following urinary tract instrumentation is usually caused by urinary pathogens. Similarly, epididymitis in men who have practiced insertive rectal intercourse is often caused by Enterobacteriaceae. These older men usually have no urethritis but do have bacteriuria.

1	Ceftriaxone (250 mg as a single dose IM) followed by doxycycline (100 mg by mouth twice daily for 10 days) constitutes effective treatment for epididymitis caused by N. gonorrhoeae or C. trachomatis. Neither oral cephalosporins nor fluoroquinolones are recommended for treatment of gonorrhea in the United States because of resistance in N. gonorrhoeae, especially (but not only) among MSM (Fig. 163-1). Oral levofloxacin (500 mg once daily for 10 days) is also effective for syndrome-based initial treatment of epididymitis when infection with Enterobacteriaceae is suspected; however, this regimen should be combined with effective therapy for possible gonococcal or chlamydial infection unless bacteriuria with Enterobacteriaceae is confirmed.

1	C. trachomatis, N. gonorrhoeae, and occasionally HSV cause symptomatic urethritis—known as the urethral syndrome in women—that is characterized by “internal” dysuria (usually without urinary urgency or frequency), pyuria, and an absence of Escherichia coli and other Percentage of isolates 1 0.8 0.6 0.4 0.2 FIGURE 163-1 Proportion of Neisseria gonorrhoeae isolates with elevated ceftriaxone minimum inhibitory concentrations (MICs), United States 2008–2012. Elevated resistance is defined by ceftriaxone MICs of ≥0.125 μg/mL. *Preliminary (January–June).

1	(From the Centers for Disease Control and Prevention: Gonococcal Isolate Surveillance Project [GISP], 2013.) uropathogens at counts of ≥102/mL in urine. In contrast, the dysuria associated with vulvar herpes or vulvovaginal candidiasis (and perhaps with trichomoniasis) is often described as “external,” being caused by painful contact of urine with the inflamed or ulcerated labia or introitus. Acute onset, association with urinary urgency or frequency, hematuria, or suprapubic bladder tenderness suggests bacterial cystitis. Among women with symptoms of acute bacterial cystitis, costovertebral pain and tenderness or fever suggests acute pyelonephritis. The management of bacterial urinary tract infection (UTI) is discussed in Chap. 162.

1	Signs of vulvovaginitis, coupled with symptoms of external dysuria, suggest vulvar infection (e.g., with HSV or Candida albicans). Among dysuric women without signs of vulvovaginitis, bacterial UTI must be differentiated from the urethral syndrome by assessment of risk, evaluation of the pattern of symptoms and signs, and specific microbiologic testing. An STI etiology of the urethral syndrome is suggested by young age, more than one current sexual partner, a new partner within the past month, a partner with urethritis, or coexisting mucopurulent cervicitis (see below). The finding of a single urinary pathogen, such as E. coli or Staphylococcus saprophyticus, at a concentration of ≥102/mL in a properly collected specimen of midstream urine from a dysuric woman with pyuria indicates probable bacterial UTI, whereas pyuria with <102 conventional uropathogens per milliliter of urine (“sterile” pyuria) suggests acute urethral syndrome due to C. trachomatis or

1	N. gonorrhoeae. Gonorrhea and chlamydial infection should be sought by specific tests (e.g., NAATs of vaginal secretions collected with a swab). Among dysuric women with sterile pyuria caused by infection with N. gonorrhoeae or C. trachomatis, appropriate treatment alleviates dysuria. The role of M. genitalium in the urethral syndrome in women remains undefined. VULVOVAGINAL INFECTIONS Abnormal Vaginal Discharge If directly questioned about vaginal discharge during routine health checkups, many women acknowledge having nonspecific symptoms of vaginal discharge that do not correlate with objective signs of inflammation or with actual infection. However, unsolicited reporting of abnormal vaginal discharge does suggest bacterial vaginosis or trichomoniasis. Specifically, an abnormally increased amount or an abnormal odor of the discharge is associated with one or both of these conditions. Cervical infection with N. gonorrhoeae or

1	C. trachomatis does not often cause an increased amount or abnormal odor of discharge; however, when these pathogens cause cervicitis, they—like T. vaginalis—often result in an increased number of neutrophils in vaginal fluid, which thus takes on a yellow color. Vulvar conditions such as genital herpes or vulvovaginal candidiasis can cause vulvar pruritus, burning, irritation, or lesions as well as external dysuria (as urine passes over the inflamed vulva or areas of epithelial disruption) or vulvar dyspareunia.

1	Certain vulvovaginal infections may have serious sequelae. Trichomoniasis, bacterial vaginosis, and vulvovaginal candidiasis have all been associated with increased risk of acquisition of HIV infection; bacterial vaginosis may promote HIV transmission from HIV-infected women to their male sex partners. Vaginal trichomoniasis and bacterial vaginosis early in pregnancy independently predict premature onset of labor. Bacterial vaginosis can also lead to anaerobic bacterial infection of the endometrium and salpinges. Vaginitis may be an early and prominent feature of toxic shock syndrome, and recurrent or chronic vulvovaginal candidiasis develops with increased frequency among women who have systemic illnesses, such as diabetes mellitus or HIV-related immunosuppression (although only a very small proportion of women with recurrent vulvovaginal candidiasis in industrialized countries actually have a serious predisposing illness).

1	Thus vulvovaginal symptoms or signs warrant careful evaluation, including speculum and pelvic examination, simple rapid diagnostic tests, and appropriate therapy specific for the anatomic site and type of infection. Unfortunately, a survey in the United States indicated that clinicians seldom perform the tests required to establish the cause of such symptoms. Further, comparison of telephone and office management of vulvovaginal symptoms has documented the inaccuracy of the former, and comparison of evaluations by nurse-midwives with those by physician-practitioners showed that the practitioners’ clinical evaluations correlated poorly both with the nurses’ evalua-873 tions and with diagnostic tests. The diagnosis and treatment of the three most common types of vaginal infection are summarized in Table 163-5.

1	Inspection of the vulva and perineum may reveal tender genital ulcerations or fissures (typically due to HSV infection or vulvovaginal candidiasis) or discharge visible at the introitus before insertion of a speculum (suggestive of bacterial vaginosis or trichomoniasis). Speculum examination permits the clinician to discern whether the discharge in fact looks abnormal and whether any abnormal discharge in the vagina emanates from the cervical os (mucoid and, if abnormal, yellow) or from the vagina (not mucoid, since the vaginal epithelium does not produce mucus). Symptoms or signs of abnormal vaginal discharge should prompt testing of vaginal fluid for pH, for a fishy odor when mixed with 10% KOH, and for certain microscopic features when mixed with saline (motile trichomonads and/or “clue cells”) and with 10% KOH (pseudohyphae or hyphae indicative of vulvovaginal candidiasis). Additional objective laboratory tests useful for establishing the cause of abnormal vaginal discharge

1	“clue cells”) and with 10% KOH (pseudohyphae or hyphae indicative of vulvovaginal candidiasis). Additional objective laboratory tests useful for establishing the cause of abnormal vaginal discharge include rapid point-ofcare tests for bacterial vaginosis and T. vaginalis, as described below; a DNA probe test (the Affirm test) to detect T. vaginalis and C. albicans as well as the increased concentrations of Gardnerella vaginalis associated with bacterial vaginosis; and a NAAT for T. vaginalis. Gram’s

1	Sexually Transmitted Infections: Overview and Clinical Approach aColor of discharge is best determined by examination against the white background of a swab. bA pH determination is not useful if blood is present or if the test is performed on endocervical secretions. cTo detect fungal elements, vaginal fluid is digested with 10% KOH prior to microscopic examination; to examine for other features, fluid is mixed (1:1) with physiologic saline. Gram’s stain is also excellent for detecting yeasts (less predictive of vulvovaginitis) and pseudomycelia or mycelia (strongly predictive of vulvovaginitis) and for distinguishing normal flora from the mixed flora seen in bacterial vaginosis, but it is less sensitive than the saline preparation for detection of T. vaginalis. dNAAT, nucleic acid amplification test (where available).

1	874 staining of vaginal fluid can be used to score alterations in the vaginal microbiota but is used primarily for research purposes and requires familiarity with the morphotypes and scale involved.

1	Patterns of treatment for vaginal discharge vary widely. In developing countries, where clinics or pharmacies often dispense treatment based on symptoms alone without examination or testing, oral treatment with metronidazole—particularly with a 7-day regimen— provides reasonable coverage against both trichomoniasis and bacterial vaginosis, the usual causes of symptoms of vaginal discharge. Metronidazole treatment of sex partners prevents reinfection of women with trichomoniasis, even though it does not help prevent the recurrence of bacterial vaginosis. Guidelines for syndromic management promulgated by the World Health Organization suggest consideration of treatment for cervical infection and for trichomoniasis, bacterial vaginosis, and vulvovaginal candidiasis in women with symptoms of abnormal vaginal discharge. However, it is important to note that the majority of chlamydial and gonococcal cervical infections produce no symptoms.

1	In industrialized countries, clinicians treating symptoms and signs of abnormal vaginal discharge should, at a minimum, differentiate between bacterial vaginosis and trichomoniasis, because optimal management of patients and partners differs for these two conditions (as discussed briefly below).

1	Vaginal Trichomoniasis (See also Chap. 254) Symptomatic trichomoniasis characteristically produces a profuse, yellow, purulent, homogeneous vaginal discharge and vulvar irritation, sometimes with visible inflammation of the vaginal and vulvar epithelium and petechial lesions on the cervix (the so-called strawberry cervix, usually evident only by colposcopy). The pH of vaginal fluid—normally <4.7—usually rises to ≥5. In women with typical symptoms and signs of trichomoniasis, microscopic examination of vaginal discharge mixed with saline reveals motile trichomonads in most culture-positive cases. However, saline microscopy probably detects only one-half of all cases, and, especially in the absence of symptoms or signs, culture or NAAT is usually required for detection of the organism. NAAT for T. vaginalis is as sensitive as or more sensitive than culture, and NAAT of urine has disclosed surprisingly high prevalences of this pathogen among men at several STD clinics in the United

1	NAAT for T. vaginalis is as sensitive as or more sensitive than culture, and NAAT of urine has disclosed surprisingly high prevalences of this pathogen among men at several STD clinics in the United States. Treatment of asymptomatic as well as symptomatic cases reduces rates of transmission and prevents later development of symptoms.

1	Only nitroimidazoles (e.g., metronidazole and tinidazole) consistently cure trichomoniasis. A single 2-g oral dose of metronidazole is effective and much less expensive than the alternatives. Tinidazole has a longer half-life than metronidazole, causes fewer gastrointestinal symptoms, and may be useful in treating trichomoniasis that fails to respond to metronidazole. Treatment of sexual partners— facilitated by dispensing metronidazole to the female patient to give to her partner(s), with a warning about avoiding the concurrent use of alcohol—significantly reduces both the risk of reinfection and the reservoir of infection; treating the partner is the standard of care. Intravaginal treatment with 0.75% metronidazole gel is not reliable for vaginal trichomoniasis. Systemic use of metronidazole is recommended throughout pregnancy. In a large randomized trial, metronidazole treatment of trichomoniasis during pregnancy did not reduce—and in fact actually increased—the frequency of

1	is recommended throughout pregnancy. In a large randomized trial, metronidazole treatment of trichomoniasis during pregnancy did not reduce—and in fact actually increased—the frequency of perinatal morbidity; thus routine screening of asymptomatic pregnant women for trichomoniasis is not recommended.

1	Bacterial Vaginosis Bacterial vaginosis (formerly termed nonspecific vaginitis, Haemophilus vaginitis, anaerobic vaginitis, or Gardnerella-associated vaginal discharge) is a syndrome of complex etiology that is characterized by symptoms of vaginal malodor and a slightly to moderately increased white discharge, which appears homogeneous, is low in viscosity, and evenly coats the vaginal mucosa. Bacterial vaginosis has been associated with increased risk of acquiring several other genital infections, including those caused by HIV, C. trachomatis, and N. gonorrhoeae. Other risk factors include recent unprotected vaginal intercourse, having a female sex partner, and vaginal douching. Although bacteria associated with bacterial vaginosis have been detected under the foreskin of uncircumcised men, metronidazole treatment of male partners has not reduced the rate of recurrence among affected women.

1	Among women with bacterial vaginosis, culture of vaginal fluid has shown markedly increased prevalences and concentrations of

1	G. vaginalis, Mycoplasma hominis, and several anaerobic bacteria (e.g., Mobiluncus, Prevotella [formerly Bacteroides], and some Peptostreptococcus species) as well as an absence of hydrogen peroxide– producing Lactobacillus species that constitute most of the normal vaginal microbiota and help protect against certain cervical and vaginal infections. Broad-range polymerase chain reaction (PCR) amplification of 16S rDNA in vaginal fluid, with subsequent identification of specific bacterial species by various methods, has documented an even greater and unexpected bacterial diversity, including several unique species not previously cultivated (e.g., three species in the order Clostridiales that appear to be specific for bacterial vaginosis and are associated with metronidazole treatment failure [Fig. 163-2]). Also detected are DNA sequences related to Atopobium vaginae, an organism that is strongly associated with bacterial vaginosis, is resistant to metronidazole, and is also associated

1	[Fig. 163-2]). Also detected are DNA sequences related to Atopobium vaginae, an organism that is strongly associated with bacterial vaginosis, is resistant to metronidazole, and is also associated with recurrent bacterial vaginosis after metronidazole treatment. Other genera newly implicated in bacterial vaginosis include Megasphaera, Leptotrichia, Eggerthella, and Dialister.

1	Bacterial vaginosis is conventionally diagnosed clinically with the Amsel criteria, which include any three of the following four clinical abnormalities: (1) objective signs of increased white homogeneous vaginal discharge; (2) a vaginal discharge pH of >4.5; (3) liberation of a distinct fishy odor (attributable to volatile amines such as trimethylamine) immediately after vaginal secretions are mixed with a 10% solution of KOH; and (4) microscopic demonstration of “clue cells” (vaginal epithelial cells coated with coccobacillary organisms, which have a granular appearance and indistinct borders; Fig. 163-3) on a wet mount prepared by mixing vaginal secretions with normal saline in a ratio of ~1:1.

1	FIGURE 163-2 Broad-range polymerase chain reaction amplification of 16S rDNA in vaginal fluid from a woman with bacterial vaginosis shows a field of bacteria hybridizing with probes for bacterial vaginosis– associated bacterium 1 (BVAB-1, visible as a thin, curved green rod) and for BVAB-2 (red). The inset shows that BVAB-1 has a morphology similar to that of Mobiluncus (curved rod). (Reprinted with permission from DN Fredricks et al: N Engl J Med 353:1899, 2005.) FIGURE 163-3 Wet mount of vaginal fluid showing typical clue cells from a woman with bacterial vaginosis. Note the obscured epithelial cell margins and the granular appearance attributable to many adherent bacteria (×400). (Photograph provided by Lorna K. Rabe, reprinted with permission from S Hillier et al, in KK Holmes et al [eds]: Sexually Transmitted Diseases, 4th ed. New York, McGraw-Hill, 2008.)

1	The standard dosage of oral metronidazole for the treatment of bacterial vaginosis is 500 mg twice daily for 7 days. The single 2-g oral dose of metronidazole recommended for trichomoniasis produces significantly lower short-term cure rates and should not be used. Intravaginal treatment with 2% clindamycin cream (one full applicator [5 g containing 100 mg of clindamycin phosphate] each night for 7 nights) or with 0.75% metronidazole gel (one full applicator [5 g containing 37.5 mg of metronidazole] twice daily for 5 days) is also approved for use in the United States and does not elicit systemic adverse reactions; the response to both of these treatments is similar to the response to oral metronidazole. Other alternatives include oral clindamycin (300 mg twice daily for 7 days), clindamycin ovules (100 g intravaginally once at bedtime for 3 days), and oral tinidazole (1 g daily for 5 days or 2 g daily for 3 days). Unfortunately, recurrence over the long term (i.e., several months

1	ovules (100 g intravaginally once at bedtime for 3 days), and oral tinidazole (1 g daily for 5 days or 2 g daily for 3 days). Unfortunately, recurrence over the long term (i.e., several months later) is distressingly common after either oral or intravaginal treatment. A randomized trial comparing intravaginal gel containing 37.5 mg of metronidazole with a suppository containing 500 mg of metronidazole plus nystatin (the latter not marketed in the United States) showed significantly higher rates of recurrence with the 37.5-mg regimen; this result suggests that higher metronidazole dosages may be important in topical intravaginal therapy. Recurrences can be significantly lessened with the twice-weekly use of suppressive intravaginal metronidazole gel. As stated above, treatment of male partners with metronidazole does not prevent recurrence of bacterial vaginosis.

1	Efforts to replenish numbers of vaginal lactobacilli that produce hydrogen peroxide and probably sustain vaginal health have been unsuccessful. While one randomized trial of orally ingested lactobacilli found reduced rates of recurrent bacterial vaginosis, this result has not yet been either confirmed or refuted, and a randomized multicenter trial in the United States found no benefit of repeated intravaginal inoculation of a vaginal peroxide-producing Lactobacillus species following treatment of bacterial vaginosis with metronidazole. A meta-analysis of 18 studies concluded that bacterial vaginosis during pregnancy substantially increased the risk of preterm delivery and of spontaneous abortion. However, most studies of topical intravaginal treatment of bacterial vaginosis with clindamycin during pregnancy have not reduced adverse pregnancy outcomes. Numerous trials of oral metronidazole treatment during pregnancy have given inconsistent results, and a 2013 Cochrane review concluded

1	during pregnancy have not reduced adverse pregnancy outcomes. Numerous trials of oral metronidazole treatment during pregnancy have given inconsistent results, and a 2013 Cochrane review concluded that antenatal treatment of women with bacterial vaginosis—even those with previous preterm deliv-875 ery—did not reduce the risk of preterm delivery. The U.S. Preventive Services Task Force thus recommends against routine screening of pregnant women for bacterial vaginosis.

1	Vulvovaginal Pruritus, Burning, or Irritation Vulvovaginal candidiasis produces vulvar pruritus, burning, or irritation, generally without symptoms of increased vaginal discharge or malodor. Genital herpes can produce similar symptoms, with lesions sometimes difficult to distinguish from the fissures and inflammation caused by candidiasis. Signs of vulvovaginal candidiasis include vulvar erythema, edema, fissures, and tenderness. With candidiasis, a white scanty vaginal discharge sometimes takes the form of white thrush-like plaques or cottage cheese–like curds adhering loosely to the vaginal epithelium. C. albicans accounts for nearly all cases of symptomatic vulvovaginal candidiasis, which probably arise from endogenous strains of C. albicans that have colonized the vagina or the intestinal tract. Complicated vulvovaginal candidiasis includes cases that recur four or more times per year; are unusually severe; are caused by non-albicans Candida species; or occur in women with

1	intestinal tract. Complicated vulvovaginal candidiasis includes cases that recur four or more times per year; are unusually severe; are caused by non-albicans Candida species; or occur in women with uncontrolled diabetes, debilitation, immunosuppression, or pregnancy.

1	In addition to compatible clinical symptoms, the diagnosis of vulvovaginal candidiasis usually involves the demonstration of pseudohyphae or hyphae by microscopic examination of vaginal fluid mixed with saline or 10% KOH or subjected to Gram’s staining. Microscopic examination is less sensitive than culture but correlates better with symptoms. Culture is typically reserved for cases that do not respond to standard first-line antimycotic agents and is undertaken to rule out imidazole or azole resistance (often associated with Candida glabrata) or before the initiation of suppressive antifungal therapy for recurrent disease. TREATMEnT VulVoVAgInAl PrurITus, burnIng, or IrrITATIon

1	Symptoms and signs of vulvovaginal candidiasis warrant treatment, usually intravaginal administration of any of several imidazole antibiotics (e.g., miconazole or clotrimazole) for 3–7 days or of a single dose of oral fluconazole (Table 163-5). Over-the-counter marketing of such preparations has reduced the cost of care and made treatment more convenient for many women with recurrent yeast vulvovaginitis. However, most women who purchase these preparations do not have vulvovaginal candidiasis, whereas many do have other vaginal infections that require different treatment. Therefore, only women with classic symptoms of vulvar pruritus and a history of previous episodes of yeast vulvovaginitis documented by an experienced clinician should self-treat. Short-course topical intravaginal azole drugs are effective for the treatment of uncomplicated vulvovaginal candidiasis (e.g., clotrimazole, two 100-mg vaginal tablets daily for 3 days; or miconazole, a 1200-mg vaginal suppository as a

1	drugs are effective for the treatment of uncomplicated vulvovaginal candidiasis (e.g., clotrimazole, two 100-mg vaginal tablets daily for 3 days; or miconazole, a 1200-mg vaginal suppository as a single dose). Single-dose oral treatment with fluconazole (150 mg) is also effective and is preferred by many patients. Management of complicated cases (see above) and those that do not respond to the usual intravaginal or single-dose oral therapy often involves prolonged or periodic oral therapy; this situation is discussed extensively in the 2010 CDC STD treatment guidelines (http://www.cdc.gov/std/treatment). Treatment of sexual partners is not routinely indicated.

1	Other Causes of Vaginal Discharge or Vaginitis In the ulcerative vaginitis associated with staphylococcal toxic shock syndrome, Staphylococcus aureus should be promptly identified in vaginal fluid by Gram’s stain and by culture. In desquamative inflammatory vaginitis, smears of vaginal fluid reveal neutrophils, massive vaginal epithelial-cell exfoliation with increased numbers of parabasal cells, and gram-positive cocci; this syndrome may respond to treatment with 2% clindamycin cream, often given in combination with topical steroid preparations for several weeks. Additional causes

1	Sexually Transmitted Infections: Overview and Clinical Approach 876 of vaginitis and vulvovaginal symptoms include retained foreign bodies (e.g., tampons), cervical caps, vaginal spermicides, vaginal antiseptic preparations or douches, vaginal epithelial atrophy (in postmenopausal women or during prolonged breast-feeding in the postpartum period), allergic reactions to latex condoms, vaginal aphthae associated with HIV infection or Behçet’s syndrome, and vestibulitis (a poorly understood syndrome).

1	Mucopurulent cervicitis (MPC) refers to inflammation of the columnar epithelium and subepithelium of the endocervix and of any contiguous columnar epithelium that lies exposed in an ectopic position on the ectocervix. MPC in women represents the “silent partner” of urethritis in men, being equally common and often caused by the same agents (N. gonorrhoeae, C. trachomatis, or—as shown by case–control studies—M. genitalium); however, MPC is more difficult than urethritis to recognize. As the most common manifestation of these serious bacterial infections in women, MPC can be a harbinger or sign of upper genital tract infection, also known as pelvic inflammatory disease (PID; see below). In pregnant women, MPC can lead to obstetric complications. In a prospective study in Seattle of 167 consecutive patients with MPC (defined on the basis of yellow endocervical mucopus or ≥30 polymorphonuclear leukocytes [PMNs]/1000× microscopic field) who were seen at STD clinics during the 1980s,

1	consecutive patients with MPC (defined on the basis of yellow endocervical mucopus or ≥30 polymorphonuclear leukocytes [PMNs]/1000× microscopic field) who were seen at STD clinics during the 1980s, slightly more than one-third of cervicovaginal specimens tested for C. trachomatis,

1	N. gonorrhoeae, M. genitalium, HSV, and T. vaginalis revealed no identifiable etiology (Fig. 163-4). More recently, a study in Baltimore using NAATs for these pathogens still failed to identify a microbiologic etiology in nearly one-half of the 133 women with MPC.

1	The diagnosis of MPC rests on the detection of cardinal signs at the cervix, including yellow mucopurulent discharge from the cervical os, endocervical bleeding upon gentle swabbing, and edematous cervical ectopy (see below); the latter two findings are somewhat more common with MPC due to chlamydial infection, but signs alone do not allow a distinction between the causative pathogens. Unlike the endocervicitis produced by gonococcal or chlamydial infection, cervicitis caused by HSV produces ulcerative lesions on the stratified squamous epithelium of the ectocervix as well as on the columnar epithelium. Yellow cervical mucus on a white swab removed from the endocervix indicates the presence of PMNs. Gram’s staining may confirm their

1	No organism 35% CT 17% GC 13% TV 10% MG 8% HSV 5% MG/CT 2% MG/GC/CT 1% GC/CT 7% FIGURE 163-4 Organisms detected among female sexually transmitted disease clinic patients with mucopurulent cervicitis (n = 167). CT, Chlamydia trachomatis; GC, gonococcus; MG, Mycoplasma genitalium; TV, Trichomonas vaginalis; HSV, herpes simplex virus. (Courtesy of Dr. Lisa Manhart; with permission.) presence, although it adds relatively little to the diagnostic value of assessment for cervical signs. The presence of ≥20 PMNs/1000× microscopic field within strands of cervical mucus not contaminated by vaginal squamous epithelial cells or vaginal bacteria indicates endocervicitis. Detection of intracellular gram-negative diplococci in carefully collected endocervical mucus is quite specific but ≤50% sensitive for gonorrhea. Therefore, specific and sensitive tests for N. gonorrhoeae as well as for C. trachomatis (e.g., NAATs) are always indicated in the evaluation of MPC.

1	Although the above criteria for MPC are neither highly specific nor highly predictive of gonococcal or chlamydial infection in some settings, the 2010 CDC STD guidelines call for consideration of empirical treatment for MPC, pending test results, in most patients. Presumptive treatment with antibiotics active against C. trachomatis should be provided for women at increased risk for this common STI (risk factors: age <25 years, new or multiple sex partners, and unprotected sex), especially if follow-up cannot be ensured. Concurrent therapy for gonorrhea is indicated if the prevalence of this infection is substantial in the relevant patient population (e.g., young adults, a clinic with documented high prevalence). In this situation, therapy should include a single-dose regimen effective for gonorrhea plus treatment for chlamydial infection, as outlined in Table 163-4 for the treatment of urethritis. In settings where gonorrhea is much less common than chlamydial infection, initial

1	for gonorrhea plus treatment for chlamydial infection, as outlined in Table 163-4 for the treatment of urethritis. In settings where gonorrhea is much less common than chlamydial infection, initial therapy for chlamydial infection alone suffices, pending test results for gonorrhea. The etiology and potential benefit of treatment for endocervicitis not associated with gonorrhea or chlamydial infection have not been established. Although the antimicrobial susceptibility of

1	M. genitalium is not yet well defined, the organism frequently persists after doxycycline therapy, and it currently seems reasonable to use azithromycin to treat possible M. genitalium infection in such cases. With resistance of M. genitalium to azithromycin now recognized, moxifloxacin may be a reasonable alternative. The sexual partner(s) of a woman with MPC should be examined and given a regimen similar to that chosen for the woman unless results of tests for gonorrhea or chlamydial infection in either partner warrant different therapy or no therapy.

1	Cervical ectopy, often mislabeled “cervical erosion,” is easily confused with infectious endocervicitis. Ectopy represents the presence of the one-cell-thick columnar epithelium extending from the endocervix out onto the visible ectocervix. In ectopy, the cervical os may contain clear or slightly cloudy mucus but usually not yellow mucopus. Colposcopy shows intact epithelium. Normally found during adolescence and early adulthood, ectopy gradually recedes through the second and third decades of life, as squamous metaplasia replaces the ectopic columnar epithelium. Oral contraceptive use favors the persistence or reappearance of ectopy, while smoking apparently accelerates squamous metaplasia. Cauterization of ectopy is not warranted. Ectopy may render the cervix more susceptible to infection with N. gonorrhoeae, C. trachomatis, or HIV.

1	The term pelvic inflammatory disease usually refers to infection that ascends from the cervix or vagina to involve the endometrium and/ or fallopian tubes. Infection can extend beyond the reproductive tract to cause pelvic peritonitis, generalized peritonitis, perihepatitis, perisplenitis, or pelvic abscess. Rarely, infection not related to specific sexually transmitted pathogens extends secondarily to the pelvic organs (1) from adjacent foci of inflammation (e.g., appendicitis, regional ileitis, or diverticulitis) or bacterial vaginosis, (2) as a result of hematogenous dissemination (e.g., of tuberculosis or staphylococcal bacteremia), or (3) as a complication of certain tropical diseases (e.g., schistosomiasis). Intrauterine infection can be primary (spontaneously occurring and usually sexually transmitted) or secondary to invasive intrauterine surgical procedures (e.g., dilation and curettage, termination of pregnancy, insertion of an intrauterine device [IUD], or

1	and usually sexually transmitted) or secondary to invasive intrauterine surgical procedures (e.g., dilation and curettage, termination of pregnancy, insertion of an intrauterine device [IUD], or hysterosalpingography) or to parturition.

1	Etiology The agents most often implicated in acute PID include the primary causes of endocervicitis (e.g., N. gonorrhoeae and C. trachomatis) and organisms that can be regarded as components of an altered vaginal microbiota. In general, PID is most often caused by N. gonorrhoeae where there is a high incidence of gonorrhea—e.g., in inner-city populations in the United States. In case–control studies, M. genitalium has also been significantly associated with histopathologic diagnoses of endometritis and with salpingitis.

1	Anaerobic and facultative organisms (especially Prevotella species, peptostreptococci, E. coli, Haemophilus influenzae, and group B streptococci) as well as genital mycoplasmas have been isolated from the peritoneal fluid or fallopian tubes in a varying proportion (typically one-fourth to one-third) of women with PID studied in the United States. The difficulty of determining the exact microbial etiology of an individual case of PID—short of using invasive procedures for specimen collection—has implications for the approach to empirical antimicrobial treatment of this infection.

1	Epidemiology In the United States, the estimated annual number of initial visits to physicians’ offices for PID by women 15–44 years of age fell from an average of 400,000 during the 1980s to 250,000 in 1999 and then to 90,000 in 2011. Hospitalizations for acute PID in the United States also declined steadily throughout the 1980s and early 1990s but have remained fairly constant at 70,000–100,000 per year since 1995. Important risk factors for acute PID include the presence of endocervical infection or bacterial vaginosis, a history of salpingitis or of recent vaginal douching, and recent insertion of an IUD. Certain other iatrogenic factors, such as dilation and curettage or cesarean section, can increase the risk of PID, especially among women with endocervical gonococcal or chlamydial infection or bacterial vaginosis. Symptoms of N. gonorrhoeae–associated and C. trachomatis–associated PID often begin during or soon after the menstrual period; this timing suggests that menstruation

1	infection or bacterial vaginosis. Symptoms of N. gonorrhoeae–associated and C. trachomatis–associated PID often begin during or soon after the menstrual period; this timing suggests that menstruation is a risk factor for ascending infection from the cervix and vagina. Experimental inoculation of the fallopian tubes of nonhuman primates has shown that repeated exposure to

1	C. trachomatis leads to the greatest degree of tissue inflammation and damage; thus, immunopathology probably contributes to the pathogenesis of chlamydial salpingitis. Women using oral contraceptives appear to be at decreased risk of symptomatic PID, and tubal sterilization reduces the risk of salpingitis by preventing intraluminal spread of infection into the tubes. Clinical Manifestations • endometritis: a clinical patHologic syndrome

1	A study of women with clinically suspected PID who were undergoing both endometrial biopsy and laparoscopy showed that those with endometritis alone differed from those who also had salpingitis in significantly less often having lower-quadrant, adnexal, or cervical motion or abdominal rebound tenderness; fever; or elevated C-reactive protein levels. In addition, women with endometritis alone differed from those with neither endometritis nor salpingitis in more often having gonorrhea, chlamydial infection, and risk factors such as douching or IUD use. Thus, women with endometritis alone were intermediate between those with neither endometritis nor salpingitis and those with salpingitis with respect to risk factors, clinical manifestations, cervical infection prevalence, and elevated C-reactive protein level. Women with endometritis alone are at lower risk of subsequent tubal occlusion and resulting infertility than are those with salpingitis.

1	salpingitis Symptoms of nontuberculous salpingitis classically evolve from a yellow or malodorous vaginal discharge caused by MPC and/ or bacterial vaginosis to midline abdominal pain and abnormal vaginal bleeding caused by endometritis and then to bilateral lower abdominal and pelvic pain caused by salpingitis, with nausea, vomiting, and increased abdominal tenderness if peritonitis develops. The abdominal pain in nontuberculous salpingitis is usually described as dull or aching. In some cases, pain is lacking or atypical, but active inflammatory changes are found in the course of an unrelated evaluation or procedure, such as a laparoscopic evaluation 877 for infertility. Abnormal uterine bleeding precedes or coincides with the onset of pain in ~40% of women with PID, symptoms of urethritis (dysuria) occur in 20%, and symptoms of proctitis (anorectal pain, tenesmus, and rectal discharge or bleeding) are occasionally seen in women with gonococcal or chlamydial infection.

1	Speculum examination shows evidence of MPC (yellow endocervical discharge, easily induced endocervical bleeding) in the majority of women with gonococcal or chlamydial PID. Cervical motion tenderness is produced by stretching of the adnexal attachments on the side toward which the cervix is pushed. Bimanual examination reveals uterine fundal tenderness due to endometritis and abnormal adnexal tenderness due to salpingitis that is usually, but not necessarily, bilateral. Adnexal swelling is palpable in about one-half of women with acute salpingitis, but evaluation of the adnexae in a patient with marked tenderness is not reliable. The initial temperature is >38°C in only about one-third of patients with acute salpingitis. Laboratory findings include elevation of the erythrocyte sedimentation rate (ESR) in 75% of patients with acute salpingitis and elevation of the peripheral white blood cell count in up to 60%.

1	Unlike nontuberculous salpingitis, genital tuberculosis often occurs in older women, many of whom are postmenopausal. Presenting symptoms include abnormal vaginal bleeding, pain (including dysmenorrhea), and infertility. About one-quarter of these women have had adnexal masses. Endometrial biopsy shows tuberculous granulomas and provides optimal specimens for culture.

1	periHepatitis and periappendicitis Pleuritic upper-abdominal pain and tenderness, usually localized to the right upper quadrant (RUQ), develop in 3–10% of women with acute PID. Symptoms of perihepatitis arise during or after the onset of symptoms of PID and may overshadow lower abdominal symptoms, thereby leading to a mistaken diagnosis of cholecystitis. In perhaps 5% of cases of acute salpingitis, early laparoscopy reveals perihepatic inflammation ranging from edema and erythema of the liver capsule to exudate with fibrinous adhesions between the visceral and parietal peritoneum. When treatment is delayed and laparoscopy is performed late, dense “violin-string” adhesions can be seen over the liver; chronic exertional or positional RUQ pain ensues when traction is placed on the adhesions. Although perihepatitis, also known as the Fitz-Hugh–Curtis syndrome, was for many years specifically attributed to gonococcal salpingitis, most cases are now attributed to chlamydial salpingitis. In

1	Although perihepatitis, also known as the Fitz-Hugh–Curtis syndrome, was for many years specifically attributed to gonococcal salpingitis, most cases are now attributed to chlamydial salpingitis. In patients with chlamydial salpingitis, serum titers of microimmunofluorescent antibody to C. trachomatis are typically much higher when perihepatitis is present than when it is absent.

1	Physical findings include RUQ tenderness and usually include adnexal tenderness and cervicitis, even in patients whose symptoms do not suggest salpingitis. Results of liver function tests and RUQ ultrasonography are nearly always normal. The presence of MPC and pelvic tenderness in a young woman with subacute pleuritic RUQ pain and normal ultrasonography of the gallbladder points to a diagnosis of perihepatitis. Periappendicitis (appendiceal serositis without involvement of the intestinal mucosa) has been found in ~5% of patients undergoing appendectomy for suspected appendicitis and can occur as a complication of gonococcal or chlamydial salpingitis.

1	Among women with salpingitis, HIV infection is associated with increased severity of salpingitis and with tuboovarian abscess requiring hospitalization and surgical drainage. Nonetheless, among women with HIV infection and salpingitis, the clinical response to conventional antimicrobial therapy (coupled with drainage of tuboovarian abscess, when found) has usually been satisfactory. Diagnosis Treatment appropriate for PID must not be withheld from patients who have an equivocal diagnosis; it is better to err on the side of overdiagnosis and overtreatment. On the other hand, it is essential to differentiate between salpingitis and other pelvic pathology, particularly surgical emergencies such as appendicitis and ectopic pregnancy. Nothing short of laparoscopy definitively identifies salpingitis, but routine laparoscopy to confirm suspected salpingitis is generally

1	Sexually Transmitted Infections: Overview and Clinical Approach 878 impractical. Most patients with acute PID have lower abdominal pain of <3 weeks’ duration, pelvic tenderness on bimanual pelvic examination, and evidence of lower genital tract infection (e.g., MPC). Approximately 60% of such patients have salpingitis at laparoscopy, and perhaps 10–20% have endometritis alone. Among the patients with these findings, a rectal temperature >38°C, a palpable adnexal mass, and elevation of the ESR to >15 mm/h also raise the probability of salpingitis, which has been found at laparoscopy in 68% of patients with one of these additional findings, 90% of patients with two, and 96% of patients with three. However, only 17% of all patients with laparoscopy-confirmed salpingitis have had all three additional findings. In a woman with pelvic pain and tenderness, increased numbers of PMNs (30 per 1000× microscopic field in strands of cervical mucus) or leukocytes outnumbering epithelial cells in

1	additional findings. In a woman with pelvic pain and tenderness, increased numbers of PMNs (30 per 1000× microscopic field in strands of cervical mucus) or leukocytes outnumbering epithelial cells in vaginal fluid (in the absence of trichomonal vaginitis, which also produces PMNs in vaginal discharge) increase the predictive value of a clinical diagnosis of acute PID, as do onset with menses, history of recent abnormal menstrual bleeding, presence of an IUD, history of salpingitis, and sexual exposure to a male with urethritis. Appendicitis or another disorder of the gut is favored by the early onset of anorexia, nausea, or vomiting; the onset of pain later than day 14 of the menstrual cycle; or unilateral pain limited to the right or left lower quadrant. Whenever the diagnosis of PID is being considered, serum assays for human β-chorionic gonadotropin should be performed; these tests are usually positive with ectopic pregnancy. Ultrasonography and magnetic resonance imaging (MRI) can

1	considered, serum assays for human β-chorionic gonadotropin should be performed; these tests are usually positive with ectopic pregnancy. Ultrasonography and magnetic resonance imaging (MRI) can be useful for the identification of tuboovarian or pelvic abscess. MRI of the tubes can also show increased tubal diameter, intratubal fluid, or tubal wall thickening in cases of salpingitis. The primary and uncontested value of laparoscopy in women with lower abdominal pain is for the exclusion of other surgical problems. Some of the most common or serious problems that may be confused with salpingitis (e.g., acute appendicitis, ectopic pregnancy, corpus luteum bleeding, ovarian tumor) are unilateral. Unilateral pain or pelvic mass, although not incompatible with PID, is a strong indication for laparoscopy unless the clinical picture warrants laparotomy instead. Atypical clinical findings such as the absence of lower genital tract infection, a missed menstrual period, a positive pregnancy

1	laparoscopy unless the clinical picture warrants laparotomy instead. Atypical clinical findings such as the absence of lower genital tract infection, a missed menstrual period, a positive pregnancy test, or failure to respond to appropriate therapy are other common indications for laparoscopy. Endometrial biopsy is relatively sensitive and specific for the diagnosis of endometritis, which correlates well with the presence of salpingitis. Vaginal or endocervical swab specimens should be examined by NAATs for N. gonorrhoeae and C. trachomatis. At a minimum, vaginal fluid should be evaluated for the presence of PMNs, and endocervical secretions ideally should be assessed by Gram’s staining for PMNs and gram-negative diplococci, which indicate gonococcal infection. The clinical diagnosis of PID made by expert gynecologists is confirmed by laparoscopy or endometrial biopsy in ~90% of women who also have cultures positive for N. gonorrhoeae or

1	C. trachomatis. Even among women with no symptoms suggestive of acute PID who were attending an STD clinic or a gynecology clinic in Pittsburgh, endometritis was significantly associated with endocervical gonorrhea or chlamydial infection or with bacterial vaginosis, being detected in 26%, 27%, and 15% of women with these conditions, respectively. Recommended combination regimens for ambulatory or parenteral management of PID are presented in Table 163-6. Women managed as outpatients should receive a combined regimen with broad activity, such as ceftriaxone (to cover possible gonococcal infection) followed by doxycycline (to cover possible chlamydial infection). Metronidazole can be added, if tolerated, to enhance activity against anaerobes; this addition should be strongly considered if bacterial vaginosis is documented. Although few methodologically sound clinical trials (especially with prolonged follow-up) have been

1	Ceftriaxone (250 mg IM once) Initiate parenteral therapy with either of the following regimens; continue to outpatient therapy, as described plusb in the text Gentamicin (loading dose of 2 mg/kg IV or IM, then maintenance dose of 1.5 mg/kg q8h) aSee text for discussion of options in the patient who is intolerant of cephalosporins. bThe addition of metronidazole is recommended by some experts, particularly if bacterial vaginosis is present. Source: Adapted from Centers for Disease Control and Prevention: MMWR Recomm Rep 59(RR-12):1, 2010. conducted, one meta-analysis suggested a benefit of providing good coverage against anaerobes.

1	The CDC STD treatment guidelines recommend initiation of empirical treatment for PID in sexually active young women and other women at risk for PID if they are experiencing pelvic or lower abdominal pain, if no other cause for the pain can be identified, and if pelvic examination reveals one or more of the following criteria for PID: cervical motion tenderness, uterine tenderness, or adnexal tenderness. Women with suspected PID can be treated as either outpatients or inpatients. In the multicenter Pelvic Inflammatory Disease Evaluation and Clinical Health (PEACH) trial, 831 women with mild to moderately severe symptoms and signs of PID were randomized to receive either inpatient treatment with IV cefoxitin and doxycycline or outpatient treatment with a single IM dose of cefoxitin plus oral doxycycline. Short-term clinical and microbiologic outcomes and long-term outcomes were equivalent in the two groups. Nonetheless, hospitalization should be considered when (1) the diagnosis is

1	oral doxycycline. Short-term clinical and microbiologic outcomes and long-term outcomes were equivalent in the two groups. Nonetheless, hospitalization should be considered when (1) the diagnosis is uncertain and surgical emergencies such as appendicitis and ectopic pregnancy cannot be excluded, (2) the patient is pregnant, (3) pelvic abscess is suspected, (4) severe illness or nausea and vomiting preclude outpatient management, (5) the patient has HIV infection, (6) the patient is assessed as unable to follow or tolerate an outpatient regimen, or (7) the patient has failed to respond to outpatient therapy. Some experts also prefer to hospitalize adolescents with PID for initial therapy, although younger women do as well as older women on outpatient therapy.

1	Currently, oral cephalosporins, doxycycline, and the fluoroquinolones do not provide reliable coverage for gonococcal infection. Thus, adequate oral treatment of women with serious intolerance to cephalosporins is a challenge. If penicillins are an option, amoxicillin/ clavulanic acid combined with doxycycline has elicited a short-term clinical response in one trial. If fluoroquinolones are the only option and if the community prevalence and individual risk of gonorrhea are known to be low, oral levofloxacin (500 mg once daily) or ofloxacin (400 mg twice daily) for 14 days, with or without metronidazole, may be considered; moreover, clinical trials performed outside the United States support the effectiveness of oral moxifloxacin. In this case, it is imperative to perform a sensitive diagnostic test for gonorrhea (ideally, culture to test for antimicrobial susceptibility) before initiation of therapy. For women whose PID involves quinolone-resistant

1	N. gonorrhoeae, treatment is uncertain but could include parenteral gentamicin or oral azithromycin, although the latter agent has not been studied for this purpose. For hospitalized patients, the following two parenteral regimens (Table 163-6) have given nearly identical results in a multicenter randomized trial: 1. Doxycycline plus either cefotetan or cefoxitin: Administration of these drugs should be continued by the IV route for at least 48 h after the patient’s condition improves and then followed with oral doxycycline (100 mg twice daily) to complete 14 days of therapy. 2.

1	2. Clindamycin plus gentamicin in patients with normal renal function: Once-daily administration of gentamicin (with combination of the total daily dose into a single daily dose) has not been evaluated in PID but has been efficacious in other serious infections and could be substituted. Treatment with these drugs should be continued for at least 48 h after the patient’s condition improves and then followed with oral doxycycline (100 mg twice daily) or clindamycin (450 mg four times daily) to complete 14 days of therapy. In cases with tuboovarian abscess, clindamycin rather than doxycycline for continued therapy provides better coverage for anaerobic infection.

1	Hospitalized patients should show substantial clinical improvement within 3–5 days. Women treated as outpatients should be clinically reevaluated within 72 h. A follow-up telephone survey of women seen in an emergency department and given a prescription for 10 days of oral doxycycline for PID found that 28% never filled the prescription and 41% stopped taking the medication early (after an average of 4.1 days), often because of persistent symptoms, lack of symptoms, or side effects. Women not responding favorably to ambulatory therapy should be hospitalized for parenteral therapy and further diagnostic evaluations, including a consideration of laparoscopy. Male sex partners should be evaluated and treated empirically for gonorrhea and chlamydial infection. After completion of treatment, tests for persistent or recurrent infection with

1	N. gonorrhoeae or C. trachomatis should be performed if symptoms persist or recur or if the patient has not complied with therapy or has been reexposed to an untreated sex partner. Surgery is necessary for the treatment of salpingitis only in the face of life-threatening infection (such as rupture or threatened rupture of a tuboovarian abscess) or for drainage of an abscess. Conservative surgical procedures are usually sufficient. Pelvic abscesses can often be drained by posterior colpotomy, and peritoneal lavage can be used for generalized peritonitis.

1	Prognosis Late sequelae include infertility due to bilateral tubal occlusion, ectopic pregnancy due to tubal scarring without occlusion, chronic pelvic pain, and recurrent salpingitis. The overall post-salpingitis risk of infertility due to tubal occlusion in a large study in Sweden was 11% after one episode of salpingitis, 23% after two episodes, and 54% after three or more episodes. A University of Washington study found a sevenfold increase in the risk of ectopic pregnancy and an eightfold increase in the rate of hysterectomy after PID.

1	Prevention A randomized controlled trial designed to determine whether selective screening for chlamydial infection reduces the risk of subsequent PID showed that women randomized to undergo screening had a 56% lower rate of PID over the following year than did women receiving the usual care without screening. This report helped prompt U.S. national guidelines for risk-based chlamydial screening of young women to reduce the incidence of PID and the prevalence of post-PID sequelae, while also reducing sexual transmission of C. trachomatis. The CDC and the U.S. Preventive Services Task Force recommend that sexually active women ≤25 years of age be screened for genital chlamydial infection annually. Despite this recommenda-879 tion, screening coverage in many primary care settings remains low.

1	Genital ulceration reflects a set of important STIs, most of which sharply increase the risk of sexual acquisition and shedding of HIV. In a 1996 study of genital ulcers in 10 of the U.S. cities with the highest rates of primary syphilis, PCR testing of ulcer specimens demonstrated HSV in 62% of patients, Treponema pallidum (the cause of syphilis) in 13%, and Haemophilus ducreyi (the cause of chancroid) in 12–20%. Today, genital herpes represents an even higher proportion of genital ulcers in the United States and other industrialized countries.

1	In Asia and Africa, chancroid (Fig. 163-5) was once considered the most common type of genital ulcer, followed in frequency by primary syphilis and then genital herpes (Fig. 163-6). With increased efforts to control chancroid and syphilis and widespread use of broad-spectrum antibiotics to treat STI-related syndromes, together with more frequent recurrences or persistence of genital herpes attributable to HIV infection, PCR testing of genital ulcers now clearly implicates genital herpes as by far the most common cause of genital ulceration in most developing countries. LGV due to C. trachomatis (Fig. 163-7) and donovanosis (granuloma inguinale, due to Klebsiella granulomatis; see Fig. 198e-1) continue to cause genital ulceration in some developing countries. LGV virtually disappeared in industrialized countries during the first 20 years of the HIV pandemic, but outbreaks are again occurring in Europe (including the United Kingdom), in North America, and in Australia. In these

1	in industrialized countries during the first 20 years of the HIV pandemic, but outbreaks are again occurring in Europe (including the United Kingdom), in North America, and in Australia. In these outbreaks, LGV typically presents as proctitis, with or without anal lesions, in men who report unprotected receptive anal intercourse, very often in association with HIV and/or hepatitis C virus infection; the latter may be an acute infection acquired through the same exposure. Other causes of genital ulcers include (1) candidiasis and traumatized genital warts—both readily recognized; (2) lesions due to genital involvement by more widespread dermatoses; (3) cutaneous manifestations of systemic diseases such as genital mucosal ulceration in Stevens-Johnson syndrome or Behçet’s disease; (4) superinfections of lesions that may originally have been sexually acquired (for example, methicillin-resistant S. aureus complicating a genital ulcer due to HSV-2); and (5) localized drug reactions, such

1	of lesions that may originally have been sexually acquired (for example, methicillin-resistant S. aureus complicating a genital ulcer due to HSV-2); and (5) localized drug reactions, such as the ulcers occasionally seen with topical paromomycin cream or boric acid preparations.

1	Diagnosis Although most genital ulcerations cannot be diagnosed confidently on clinical grounds alone, clinical findings (Table 163-7) FIGURE 163-5 Chancroid: multiple, painful, punched-out ulcers with undermined borders on the labia occurring after autoinoculation. Sexually Transmitted Infections: Overview and Clinical Approach FIGURE 163-6 Genital herpes. A relatively mild, superficial ulcer is typically seen in episodic outbreaks. (Courtesy of Michael Remington, University of Washington Virology Research Clinic.) FIGURE 163-7 Lymphogranuloma venereum (LGV): striking ten-der lymphadenopathy occurring at the femoral and inguinal lymph nodes, separated by a groove made by Poupart’s ligament. This “sign-of-the-groove” is not considered specific for LGV; for example, lym-phomas may present with this sign. InITIAL MAnAgEMEnT of gEnITAL oR PERIAnAL uLCER

1	InITIAL MAnAgEMEnT of gEnITAL oR PERIAnAL uLCER Dark-field exam (if available), direct FA, or PCR for T. pallidum RPR, VDRL, or EIA serologic test for syphilisa Culture, direct FA, ELISA, or PCR for HSV HSV-2-specific serology (consider) In chancroid-endemic area: PCR or culture for H. ducreyi Herpes confirmed or suspected (history or sign of vesicles): Treat for genital herpes with acyclovir, valacyclovir, or famciclovir. Syphilis confirmed (dark-field, FA, or PCR showing T. pallidum, or RPR reactive): Benzathine penicillin (2.4 million units IM once to patient, to recent [e.g., within 3 months] seronegative partner[s], and to all seropositive partners)b Chancroid confirmed or suspected (diagnostic test positive, or HSV and syphilis excluded, and persistent lesion): Ciprofloxacin (500 mg PO as single dose) or Ceftriaxone (250 mg IM as single dose) or

1	Ciprofloxacin (500 mg PO as single dose) or Ceftriaxone (250 mg IM as single dose) or Azithromycin (1 g PO as single dose) aIf results are negative but primary syphilis is suspected, treat presumptively when indicated by epidemiologic and sexual risk assessment; repeat in 1 week. bThe same treatment regimen is also effective in HIV-infected persons with early syphilis. Abbreviations: EIA, enzyme immunoassay; ELISA, enzyme-linked immunosorbent assay; FA, fluorescent antibody; HSV, herpes simplex virus; PCR, polymerase chain reaction; RPR, rapid plasma reagin; VDRL, Venereal Disease Research Laboratory.

1	and epidemiologic considerations can usually guide initial management (Table 163-8) pending results of specific tests. Clinicians should order a rapid serologic test for syphilis in all cases of genital ulcer. To evaluate lesions except those highly characteristic of infection with HSV (i.e., those with herpetic vesicles), dark-field microscopy, direct immunofluorescence, and PCR for T. pallidum can be useful but are rarely available today in most countries. It is important to note that 30% of syphilitic chancres—the primary ulcer of syphilis—are associated with an initially nonreactive syphilis serology. All patients presenting with genital ulceration should be counseled and tested for HIV infection. Typical vesicles or pustules or a cluster of painful ulcers preceded by vesiculopustular lesions suggests genital herpes. These typical clinical manifestations make detection of the virus optional; however, many patients want confirmation of the diagnosis, and differentiation of

1	Source: From RM Ballard, in KK Holmes et al (eds): Sexually Transmitted Diseases, 4th ed. New York, McGraw-Hill, 2008. HSV-1 from HSV-2 has prognostic implications, because the latter causes more frequent genital recurrences. Painless, nontender, indurated ulcers with firm, nontender inguinal adenopathy suggest primary syphilis. If results of dark-field examination and a rapid serologic test for syphilis are initially negative, presumptive therapy should be provided on the basis of the individual’s risk. For example, with increasing rates of syphilis among MSM in the United States, most experts would not withhold therapy for this infection pending watchful waiting and/or subsequent detection of seroconversion. Repeated serologic testing for syphilis 1 or 2 weeks after treatment of seronegative primary syphilis usually demonstrates seroconversion.

1	“Atypical” or clinically trivial ulcers may be more common manifestations of genital herpes than classic vesiculopustular lesions. Specific tests for HSV in such lesions are therefore indicated (Chap. 216). Commercially available type-specific serologic tests for serum antibody to HSV-2 may give negative results, especially when patients present early with the initial episode of genital herpes or when HSV-1 is the cause of genital herpes (as is often the case today). Furthermore, a positive test for antibody to HSV-2 does not prove that the current lesions are herpetic, because nearly one-fifth of the general population of the United States (and no doubt a higher proportion of those at risk for other STIs) becomes seropositive for HSV-2 during early adulthood. Although even “type-specific” tests for HSV-2 that are commercially available in the United States are not 100% specific, a positive HSV-2 serology does enable the clinician to tell the patient that he or she has probably had

1	tests for HSV-2 that are commercially available in the United States are not 100% specific, a positive HSV-2 serology does enable the clinician to tell the patient that he or she has probably had genital herpes, should learn to recognize symptoms, and should avoid sex during recurrences. In addition, because genital shedding and sexual transmission of HSV-2 often occur in the absence of symptoms and signs of recurrent herpetic lesions, persons who have a history of genital herpes or who are seropositive for HSV-2 should consider the use of condoms or suppressive antiviral therapy, both of which can reduce the risk of HSV-2 transmission to a sexual partner.

1	Demonstration of H. ducreyi by culture (or by PCR, where available) is most useful when ulcers are painful and purulent, especially if inguinal lymphadenopathy with fluctuance or overlying erythema is noted; if chancroid is prevalent in the community; or if the patient has recently had a sexual exposure elsewhere in a chancroid-endemic area (e.g., a developing country). Enlarged, fluctuant lymph nodes should be aspirated for culture or PCR to detect H. ducreyi as well as for Gram’s staining and culture to rule out the presence of other pyogenic bacteria.

1	When genital ulcers persist beyond the natural history of initial episodes of herpes (2–3 weeks) or of chancroid or syphilis (up to 6 weeks) and do not resolve with syndrome-based antimicrobial therapy, then— in addition to the usual tests for herpes, syphilis, and chancroid—biopsy is indicated to exclude donovanosis, carcinoma, and other nonvenereal dermatoses. If not performed previously, HIV serology should be standard because chronic, persistent genital herpes is common in AIDS.

1	Immediate syndrome-based treatment for acute genital ulcerations (after collection of all necessary diagnostic specimens at the first visit) is often appropriate before all test results become available because patients with typical initial or recurrent episodes of genital or anorectal herpes can benefit from prompt oral antiviral therapy (Chap. 216); because early treatment of sexually transmitted causes of genital ulcers decreases further transmission; and because many patients do not return for test results and treatment. A thorough assessment of the patient’s sexual-risk profile and medical history is critical in determining the course of initial management. The patient who has risk factors consistent with exposure to syphilis (e.g., a male patient who reports sex with other men or who has HIV infection) should generally receive initial treatment for syphilis. Empirical therapy for chancroid should be considered if there has been an exposure in an area of the world where chancroid

1	has HIV infection) should generally receive initial treatment for syphilis. Empirical therapy for chancroid should be considered if there has been an exposure in an area of the world where chancroid occurs or if regional lymph node suppuration is evident. In resource-poor settings lacking ready access to diagnostic tests, this approach to 881 syndromic treatment for syphilis and chancroid has helped bring these two diseases under control. Finally, empirical antimicrobial therapy may be indicated if ulcers persist and the diagnosis remains unclear after a week of observation despite attempts to diagnose herpes, syphilis, and chancroid.

1	PROCTITIS, PROCTOCOLITIS, ENTEROCOLITIS, AND ENTERITIS

1	Sexually acquired proctitis, with inflammation limited to the rectal mucosa (the distal 10–12 cm), results from direct rectal inoculation of typical STD pathogens. In contrast, inflammation extending from the rectum to the colon (proctocolitis), involving both the small and the large bowel (enterocolitis), or involving the small bowel alone (enteritis) can result from ingestion of typical intestinal pathogens through oral–anal exposure during sexual contact. Anorectal pain and mucopurulent, bloody rectal discharge suggest proctitis or protocolitis. Proctitis commonly produces tenesmus (causing frequent attempts to defecate, but not true diarrhea) and constipation, whereas proctocolitis and enterocolitis more often cause true diarrhea. In all three conditions, anoscopy usually shows mucosal exudate and easily induced mucosal bleeding (i.e., a positive “wipe test”), sometimes with petechiae or mucosal ulcers. Exudate should be sampled for Gram’s staining and other microbiologic studies.

1	exudate and easily induced mucosal bleeding (i.e., a positive “wipe test”), sometimes with petechiae or mucosal ulcers. Exudate should be sampled for Gram’s staining and other microbiologic studies. Sigmoidoscopy or colonoscopy shows inflammation limited to the rectum in proctitis or disease extending at least up into the sigmoid colon in proctocolitis.

1	The AIDS era brought an extraordinary shift in the clinical and etiologic spectrum of intestinal infections among MSM. The number of cases of the acute intestinal STIs described above fell as high-risk sexual behaviors became less common in this group. At the same time, the number of AIDS-related opportunistic intestinal infections increased rapidly, many associated with chronic or recurrent symptoms. The incidence of these opportunistic infections has since fallen with increasingly widespread coverage of HIV-infected persons with effective antiretroviral therapy. Two species initially isolated in association with intestinal symptoms in MSM—now known as Helicobacter cinaedi and H. fennelliae—have both been isolated from the blood of HIV-infected men and other immunosuppressed persons, often in association with a syndrome of multifocal dermatitis and arthritis.

1	H. fennelliae—have both been isolated from the blood of HIV-infected men and other immunosuppressed persons, often in association with a syndrome of multifocal dermatitis and arthritis. Acquisition of HSV, N. gonorrhoeae, or C. trachomatis (including LGV strains of C. trachomatis) during receptive anorectal intercourse causes most cases of infectious proctitis in women and MSM. Primary and secondary syphilis can also produce anal or anorectal lesions, with or without symptoms. Gonococcal or chlamydial proctitis typically involves the most distal rectal mucosa and the anal crypts and is clinically mild, without systemic manifestations. In contrast, primary proctitis due to HSV and proctocolitis due to the strains of

1	C. trachomatis that cause LGV usually produce severe anorectal pain and often cause fever. Perianal ulcers and inguinal lymphadenopathy, most commonly due to HSV, can also occur with LGV or syphilis. Sacral nerve root radiculopathies, usually presenting as urinary retention, laxity of the anal sphincter, or constipation, may complicate primary herpetic proctitis. In LGV, rectal biopsy typically shows crypt abscesses, granulomas, and giant cells—findings resembling those in Crohn’s disease; such findings should always prompt rectal culture and serology for LGV, which is a curable infection. Syphilis can also produce rectal granulomas, usually in association with infiltration by plasma cells or other mononuclear cells. Syphilis, LGV, and HSV infection involving the rectum can produce perirectal adenopathy that is sometimes mistaken for malignancy; syphilis, LGV, HSV infection, and chancroid involving the anus can produce inguinal adenopathy because anal lymphatics drain to inguinal

1	adenopathy that is sometimes mistaken for malignancy; syphilis, LGV, HSV infection, and chancroid involving the anus can produce inguinal adenopathy because anal lymphatics drain to inguinal lymph nodes.

1	Diarrhea and abdominal bloating or cramping pain without anorectal symptoms and with normal findings on anoscopy and sigmoidoscopy occur with inflammation of the small intestine (enteritis) or with proximal colitis. In MSM without HIV infection, enteritis is often attributable to Giardia lamblia. Sexually acquired proctocolitis is most often due to Campylobacter or Shigella species. Sexually Transmitted Infections: Overview and Clinical Approach TREATMEnT ProcTITIs, ProcTocolITIs, enTerocolITIs, And enTerITIs

1	Acute proctitis in persons who have practiced receptive anorectal intercourse is usually sexually acquired. Such patients should undergo anoscopy to detect rectal ulcers or vesicles and petechiae after swabbing of the rectal mucosa; to examine rectal exudates for PMNs and gram-negative diplococci; and to obtain rectal swab specimens for testing for rectal gonorrhea, chlamydial infection, herpes, and syphilis. Pending test results, patients with proctitis should receive empirical syndromic treatment—e.g., with ceftriaxone (a single IM dose of 250 mg for gonorrhea) plus doxycycline (100 mg by mouth twice daily for 7 days) for possible chlamydial infection plus treatment for herpes or syphilis if indicated. If LGV proctitis is proven or suspected, the recommended treatment is doxycycline (100 mg by mouth twice daily for 21 days); alternatively, 1 g of azithromycin once a week for 3 weeks is likely to be effective but is little studied.

1	Prevention and control of STIs require the following: 1. Reduction of the average rate of sexual exposure to STIs through alteration of sexual risk behaviors and behavioral norms among both susceptible and infected persons in all population groups. The necessary changes include reduction in the total number of sexual partners and the number of concurrent sexual partners. 2.

1	2. Reduction of the efficiency of transmission through the promotion of safer sexual practices, the use of condoms during casual or commercial sex, vaccination against HBV and HPV infection, male circumcision (which reduces risk of acquisition of HIV infection, chancroid, and perhaps other STIs), and a growing number of other approaches (e.g., early detection and treatment of other STIs to reduce the efficiency of sexual transmission of HIV). Longitudinal studies have shown that consistent condom use is associated with significant protection of both males and females against all STIs that have been examined, including HIV, HPV, and HSV infections as well as gonorrhea and chlamydial infection. The only exceptions are probably sexually transmitted Pthirus pubis and Sarcoptes scabiei infestations. 3. Shortening of the duration of infectivity of STIs through early detection and curative or suppressive treatment of patients and their sexual partners.

1	3. Shortening of the duration of infectivity of STIs through early detection and curative or suppressive treatment of patients and their sexual partners. Financial and time constraints imposed by many clinical practices, along with the reluctance of some clinicians to ask questions about stigmatized sexual behaviors, often curtail screening and prevention services. As outlined in Fig. 163-8, the success of clinicians’ efforts to detect and treat STIs depends in part on societal efforts to teach young people how to recognize symptoms of STIs; to motivate individuals with symptoms to seek care promptly; to educate persons who are at risk but have no symptoms about what tests they should undergo routinely; and to make high-quality, appropriate care accessible, affordable, and acceptable, especially to the young indigent patients most likely to acquire an STI.

1	Because many infected individuals develop no symptoms or fail to recognize and report symptoms, clinicians should routinely perform an STI risk assessment for teenagers and young adults as a guide to selective screening. As stated earlier, U.S. Preventive Services Task Force Guidelines recommend screening sexually active female patients ≤25 years of age for C. trachomatis whenever they present for health care (at least once a year); older women should be tested if they have more than one sexual partner, have begun a new sexual relationship since the previous test, or have another STI diagnosed. In women 25–29 years of age, chlamydial infection is uncommon but still may reach a prevalence of 3–5% in some settings; information provided by women in this age group on a sex partner’s concurrency (whether a male partner has had another sex partner during the time they have been together) is helpful in identifying women at increased risk. In some regions of the United States, widespread

1	(whether a male partner has had another sex partner during the time they have been together) is helpful in identifying women at increased risk. In some regions of the United States, widespread selective screening and treatment of young women for cervical C. trachomatis infection have been associated with a 50–60% drop in prevalence. Such screening and treatment also protect the individual woman from PID. Sensitive urine-based genetic amplification tests permit expansion of screening to men, teenage boys, and girls in settings where examination is not planned or is impractical (e.g., during preparticipation sports examinations or during initial medical evaluation of adolescent girls). Vaginal swabs—collected either by the health care provider at a pelvic examination or by the woman herself—are highly sensitive and specific for the diagnosis of chlamydial and gonococcal infection; they are now the preferred type of specimen for screening and diagnosis of these infections.

1	Number whose behaviors and ecologic settings result in exposure to STDs Number who acquire STDs Number who develop symptoms of STDs Number who perceive the symptoms of STDs Number who promptly seek medical care when symptomatic Number seeking care who have ready access to care Number perceived by clinicians as possibly having STDs Number perceived as possibly having STDs who can be tested for STDs Number with objective evidence of STDs who get proper treatment for STDs Number who comply with treatment Number whose partners are treated and who are not reinfected FIGURE 163-8 Critical control points for preventive and clini-cal interventions against sexually transmitted diseases (STDs). (Adapted from HT Waller and MA Piot: Bull World Health Organ 41:75, 1969 and 43:1, 1970; and from “Resource allocation model for public health planning—a case study of tuberculosis control,” Bull World Health Organ 48 [Suppl], 1973.)

1	Although gonorrhea is now substantially less common than chlamydial infection in industrialized countries, screening tests for N. gonorrhoeae are still appropriate for women and teenage girls attending STD clinics and for sexually active teens and young women from areas of high gonorrhea prevalence. Multiplex NAATs that combine screening for N. gonorrhoeae and C. trachomatis—and, more recently, for T. vaginalis—in a single low-cost assay now facilitate the prevention and control of these infections for populations at high risk.

1	All patients who have newly detected STIs or are at high risk for STIs according to routine risk assessment as well as all pregnant women should be encouraged to undergo serologic testing for syphilis and HIV infection, with appropriate HIV counseling before and after testing. Randomized trials have shown that risk-reduction counseling of patients with STIs significantly lowers subsequent risk of acquiring an STI; such counseling should now be considered a standard component of STI management. Preimmunization serologic testing for antibody to HBV is indicated for unvaccinated persons who are known to be at high risk, such as MSM and people who use injection drugs. In most young persons, however, it is more cost-effective to vaccinate against HBV without serologic screening. It is important to recognize that, while immunization against HBV has contributed to marked reductions in the incidence of infection with this virus, the majority of new cases that do occur are acquired through

1	to recognize that, while immunization against HBV has contributed to marked reductions in the incidence of infection with this virus, the majority of new cases that do occur are acquired through sex. In 2006, the Advisory Committee on Immunization Practices (ACIP) of the CDC recommended the following: (1) Universal hepatitis B vaccination should be implemented for all unvaccinated adults in settings in which a high proportion of adults have risk factors for HBV infection (e.g., STD clinics, HIV testing and treatment facilities, drug-abuse treatment and prevention settings, health care settings targeting services to injection drug users or MSM, and correctional facilities). (2) In other primary care and specialty medical settings in which adults at risk for HBV infection receive care, health care providers should inform all patients about the health benefits of vaccination, the risk factors for HBV infection, and the persons for whom vaccination is recommended and should vaccinate

1	health care providers should inform all patients about the health benefits of vaccination, the risk factors for HBV infection, and the persons for whom vaccination is recommended and should vaccinate adults who report risk factors for HBV infection as well as any adult who requests protection from HBV infection. To promote vaccination in all settings, health care providers should implement standing orders to identify adults recommended for hepatitis B vaccination, should administer hepatitis B vaccine as part of routine clinical services, should not require acknowledgment of an HBV infection risk factor for adult vaccination, and should use available reimbursement mechanisms to remove financial barriers to hepatitis B vaccination.

1	In 2007, the ACIP recommended routine immunization of 9to 26-year-old girls and women with the quadrivalent HPV vaccine (against HPV types 6, 11, 16, and 18) approved by the U.S. Food and Drug Administration; the optimal age for recommended vaccination is 11–12 years because of the very high risk of HPV infection after sexual debut. In 2009, the ACIP added bivalent HPV vaccine (against types 6 and 11) as an option and expanded the groups in which immunization (with either quadrivalent or bivalent vaccine) is safe and effective to include boys and men 9–26 years old. HPV vaccines offering broader protection against additional oncogenic HPV types are anticipated. Since 2011, the ACIP has recommended routine administration of quadrivalent HPV vaccine to boys at 11 or 12 years of age and to males 13–21 years of age who have not yet been vaccinated or who have not completed the three-dose vaccine series; men 22–26 years of age may also be vaccinated.

1	Partner notification is the process of identifying and informing partners of infected patients about possible exposure to an STI and of examining, testing, and treating partners as appropriate. In a series of 22 reports concerning partner notification during the 1990s, index patients with gonorrhea or chlamydial infection named a mean of 0.75–1.6 partners, of whom one-fourth to one-third were infected; those with syphilis named 1.8–6.3 partners, with one-third to one-half infected; and those with HIV infection named 0.76–5.31 partners, with up to one-fourth infected. Persons who transmit infection or who have recently been infected and are still in the incubation period usually have no symptoms or only mild symptoms and seek medical attention only when notified of their exposure. Therefore, the clinician must encourage patients to participate in partner notification, must ensure that exposed persons are notified and treated, and must guarantee confidentiality to all involved. In the

1	the clinician must encourage patients to participate in partner notification, must ensure that exposed persons are notified and treated, and must guarantee confidentiality to all involved. In the United States, local health departments often offer assistance in partner notification, treatment, and/or counseling. It seems both feasible and most useful to notify those partners exposed within the patient’s likely period of infectiousness, which is often considered the preceding 1 month for gonorrhea, 1–2 months for chlamydial infection, and up to 3 months for early syphilis.

1	Persons with a new-onset STI always have a source contact who gave them the infection; in addition, they may have a secondary (spread or exposed) contact with whom they had sex after becoming infected. The identification and treatment of these two types of contacts have different objectives. Treatment of the source contact (often a casual contact) benefits the community by preventing further transmission and benefits the source contact; treatment of the recently exposed secondary contact (typically a spouse or another steady sexual partner) prevents the development of serious complications (such as PID) in the partner, reinfection of the index patient, and further spread of infection. A survey of a random sample of U.S. physicians found that most instructed patients to abstain from sex during treatment, to use condoms, and to inform their sex partners after being diagnosed with gonorrhea, chlamydial infection, or syphilis; physicians sometimes gave the patients drugs for their

1	sex during treatment, to use condoms, and to inform their sex partners after being diagnosed with gonorrhea, chlamydial infection, or syphilis; physicians sometimes gave the patients drugs for their partners. However, follow-up of the partners by physicians was infrequent. A randomized trial compared patients’ delivery of therapy to partners exposed to gonorrhea or chlamydial infection with conventional notification and advice to partners to seek evaluation for 883 STD; patients’ delivery of partners’ therapy, also known as expedited partner therapy (EPT), significantly reduced combined rates of reinfection of the index patient with N. gonorrhoeae or C. trachomatis. State-by-state variations in regulations governing this approach have not been well defined, but the 2010 CDC STD treatment guidelines and the EPT final report of 2006 (http://www.cdc.gov/std/treatment/ EPTFinalReport2006.pdf) describe its potential use. Currently, EPT is commonly used by many practicing physicians. Its

1	guidelines and the EPT final report of 2006 (http://www.cdc.gov/std/treatment/ EPTFinalReport2006.pdf) describe its potential use. Currently, EPT is commonly used by many practicing physicians. Its legal status varies by state, but EPT is now permissible in 38 states and potentially allowable in another 9. (Updated information on the legal status of EPT is available at http://www.cdc.gov/std/ept.)

1	In summary, clinicians and public health agencies share responsibility for the prevention and control of STIs. In the current health care environment, the role of primary care clinicians has become increasingly important in STI prevention as well as in diagnosis and treatment, and the resurgence of bacterial STIs like syphilis and LGV among MSM—particularly those co-infected with HIV—emphasizes Meningitis, Encephalitis, Brain 164 Abscess, and Empyema Karen L. Roos, Kenneth L. Tyler

1	Meningitis, Encephalitis, Brain 164 Abscess, and Empyema Karen L. Roos, Kenneth L. Tyler Acute infections of the nervous system are among the most important problems in medicine because early recognition, efficient decision making, and rapid institution of therapy can be lifesaving. These distinct clinical syndromes include acute bacterial meningitis, viral meningitis, encephalitis, focal infections such as brain abscess and subdural empyema, and infectious thrombophlebitis. Each may present with a nonspecific prodrome of fever and headache, which in a previously healthy individual may initially be thought to be benign, until (with the exception of viral meningitis) altered consciousness, focal neurologic signs, or seizures appear. Key goals of early management are to emergently distinguish between these conditions, identify the responsible pathogen, and initiate appropriate antimicrobial therapy. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: Meningitis, Encephalitis, Brain Abscess, and Empyema (Figure 164-1) The first task is to identify whether an infection predominantly involves the subarachnoid space (meningitis) or whether there is evidence of either generalized or focal involvement of brain tissue in the cerebral hemispheres, cerebellum, or brainstem. When brain tissue is directly injured by a bacterial or viral infection, the disease is referred to as encephalitis, whereas focal infections involving brain tissue are classified as either cerebritis or abscess, depending on the presence or absence of a capsule.

1	Nuchal rigidity (“stiff neck”) is the pathognomonic sign of meningeal irritation and is present when the neck resists passive flexion. Kernig’s and Brudzinski’s signs are also classic signs of meningeal irritation. Kernig’s sign is elicited with the patient in the supine position. The thigh is flexed on the abdomen, with the knee flexed; attempts to passively extend the knee elicit pain when meningeal irritation is present. Brudzinski’s sign is elicited with the patient in the supine position and is positive when passive flexion of the neck results in spontaneous flexion of the hips and knees. Although commonly tested on physical examinations, the sensitivity and specificity of Kernig’s and Brudzinski’s signs are uncertain. Both may be absent or reduced in very young or elderly patients, immunocompromised individuals, or patients with a severely depressed

1	Meningitis, Encephalitis, Brain Abscess, and Empyema mental status. The high prevalence of cervical spine disease in older individuals may result in false-positive tests for nuchal rigidity.

1	Initial management can be guided by several considerations: (1) Empirical therapy should be initiated promptly whenever bacterial meningitis is a significant diagnostic consideration. (2) All patients who have had recent head trauma, are immunocompromised, have known malignant lesions or central nervous system (CNS) neoplasms, or have focal neurologic findings, papilledema, or a depressed level of consciousness should undergo computed tomography (CT) or magnetic resonance imaging (MRI) of the brain prior to lumbar puncture (LP). In these cases empirical antibiotic therapy should not be delayed pending test results but should be administered prior to neuroimaging and LP. (3) A significantly depressed level of consciousness (e.g., somnolence, coma), seizures, or focal neurologic deficits do not occur in viral meningitis; patients with these symptoms should be hospitalized for further evaluation and treated empirically for bacterial and viral meningoencephalitis. (4) Immunocompetent

1	do not occur in viral meningitis; patients with these symptoms should be hospitalized for further evaluation and treated empirically for bacterial and viral meningoencephalitis. (4) Immunocompetent patients with a normal level of consciousness, no prior antimicrobial treatment, and a cerebrospinal fluid (CSF) profile consistent with viral meningitis (lymphocytic pleocytosis and a normal glucose concentration) can often be treated as outpatients if appropriate contact and monitoring can be ensured. Failure of a patient with suspected viral meningitis to improve within 48 h should prompt a reevaluation including follow-up neurologic and general medical examination and repeat imaging and laboratory studies, often including a second LP.

1	ADEM Encephalitis Bacterial process Appropriate medical and/or surgical interventions Pleocytosis with PMNs Elevated protein Decreased glucose Gram’s stain positive Tier 1 Eval (no unusual historic points or exposures): Viral: CSF PCR for enterovirus, HSV, VZVPleocytosis with MNCs Normal or increased protein Normal or decreased glucose Gram’s stain negative Abscess or tumor White matter abnormalities Focal or generalized gray matter abnormalities or normal No mass lesion Yes Yes No No Headache, Fever, ±Nuchal Rigidity Altered mental status? Immediate blood culture and lumbar puncture Meningoencephalitis, ADEM, encephalopathy, or mass lesion Imaging: Head CT or MRI (preferred) Mass lesion Obtain blood culture and start empirical antimicrobial therapy Meningitis Papilledema and/or focal neurologic deficit? Immunocompromised? History of recent head trauma, known cancer, sinusitis?

1	FIGURE 164-1 The management of patients with suspected central nervous system (CNS) infection. ADEM, acute disseminated encephalomyelitis; AFB, acid-fast bacillus; Ag, antigen; CSF, cerebrospinal fluid; CT, computed tomography; CTFV, Colorado tick fever virus; CXR, chest x-ray; DFA, direct fluorescent antibody; EBV, Epstein-Barr virus; HHV, human herpesvirus; HSV, herpes simplex virus; LCMV, lymphocytic choriomeningitis virus; MNCs, mononuclear cells; MRI, magnetic resonance imaging; PCR, polymerase chain reaction; PMNs, polymorphonuclear leukocytes; PPD, purified protein derivative; TB, tuberculosis; VDRL, Venereal Disease Research Laboratory; VZV, varicella-zoster virus; WNV, West Nile virus. Viral culture: CSF, throat, stool If skin lesions DFA for HSV, VZV HIV serology Serology for enteroviruses and arthropod-borne viruses Fungal: CSF cryptococcal Ag, fungal cultures Bacterial: VDRL and bacterial culture, PCR Mycobacterial: CSF AFB stain and TB PCR, TB culture, CXR, PPD

1	Fungal: CSF cryptococcal Ag, fungal cultures Bacterial: VDRL and bacterial culture, PCR Mycobacterial: CSF AFB stain and TB PCR, TB culture, CXR, PPD Tier 2 Evaluation (if above negative): EBV: Serum serology, CSF PCR Mycoplasma: Serum serology, CSF PCR Influenza A, B: Serology, respiratory culture, CSF PCR Adenovirus: Serology, throat swab. CSF PCR Fungal: CSF & serum coccidioidal antibody, Histoplasma antigen & antibody Rabies Raccoon exposure or Hx of pica Baylisascaris procyonis Pet bird (Psittacine) exposure Chlamydia psittaci (Psittacosis) Exposure to cattle or unpasteurized dairy products Brucella spp. (Brucellosis) Coxiella burnetii (Q fever) Wild rodent or hamster exposure Bat exposure Animal bite Cat exposure LCMV Swimming in lakes or ponds or nonchlorinated water Acanthamoeba or Naegleria fowleri (amebic meningoencephalitis) Bartonella spp. (cat scratch fever) B

1	Bacterial meningitis is an acute purulent infection within the subarachnoid space. It is associated with a CNS inflammatory reaction that may result in decreased consciousness, seizures, raised intracranial pressure (ICP), and stroke. The meninges, subarachnoid space, and brain parenchyma are all frequently involved in the inflammatory reaction (meningoencephalitis). Bacterial meningitis is the most common form of suppurative CNS infection, with an annual incidence in the United States of >2.5 cases/100,000 population. The organisms most often responsible for community-acquired bacterial meningitis are Streptococcus pneumoniae (~50%), Neisseria meningitidis (~25%), group B streptococci (~15%), and Listeria monocytogenes (~10%). Haemophilus influenzae type b accounts for <10% of cases of bacterial meningitis in most series. N. meningitidis is the causative organism of recurring epidemics of meningitis every 8 to 12 years.

1	N. meningitidis is the causative organism of recurring epidemics of meningitis every 8 to 12 years. S. pneumoniae (Chap. 173) is the most common cause of meningitis in adults >20 years of age, accounting for nearly half the reported cases (1.1 per 100,000 persons per year). There are a number of predisposing conditions that increase the risk of pneumococcal meningitis, the most important of which is pneumococcal pneumonia. Additional risk factors include coexisting acute or chronic pneumococcal sinusitis or otitis media, alcoholism, diabetes, splenectomy, hypogammaglobulinemia, complement deficiency, and head trauma with basilar skull fracture and CSF rhinorrhea. The mortality rate remains ~20% despite antibiotic therapy.

1	The incidence of meningitis due to N. meningitidis (Chap. 180) has decreased with the routine immunization of 11to 18-year-olds with the quadrivalent (serogroups A, C, W-135, and Y) meningococcal glycoconjugate vaccine. The vaccine does not contain serogroup B, which is responsible for one-third of cases of meningococcal disease. The presence of petechial or purpuric skin lesions can provide an important clue to the diagnosis of meningococcal infection. In some patients the disease is fulminant, progressing to death within hours of symptom onset. Infection may be initiated by nasopharyngeal colonization, which can result in either an asymptomatic carrier state or invasive meningococcal disease. The risk of invasive disease following nasopharyngeal colonization depends on both bacterial virulence factors and host immune defense mechanisms, including the host’s capacity to produce antimeningococcal antibodies and to lyse meningococci by both classic and alternative complement pathways.

1	factors and host immune defense mechanisms, including the host’s capacity to produce antimeningococcal antibodies and to lyse meningococci by both classic and alternative complement pathways. Individuals with deficiencies of any of the complement components, including proper-din, are highly susceptible to meningococcal infections.

1	Gram-negative bacilli cause meningitis in individuals with chronic and debilitating diseases such as diabetes, cirrhosis, or alcoholism and in those with chronic urinary tract infections. Gram-negative meningitis can also complicate neurosurgical procedures, particularly craniotomy, and head trauma associated with CSF rhinorrhea or otorrhea. Otitis, mastoiditis, and sinusitis are predisposing and associated conditions for meningitis due to Streptococci sp., gram-negative anaerobes,

1	Meningitis, Encephalitis, Brain Abscess, and Empyema 886 Staphylococcus aureus, Haemophilus sp., and Enterobacteriaceae. and complications of bacterial meningitis result from the immune Meningitis complicating endocarditis may be due to viridans strepto-response to the invading pathogen rather than from direct bacteria-cocci, S. aureus, Streptococcus bovis, the HACEK group (Haemophilus induced tissue injury. As a result, neurologic injury can progress even sp., Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, after the CSF has been sterilized by antibiotic therapy. Eikenella corrodens, Kingella kingae), or enterococci. The lysis of bacteria with the subsequent release of cell-wall compo-Group B Streptococcus, or Streptococcus agalactiae, was previously nents into the subarachnoid space is the initial step in the induction of responsible for meningitis predominantly in neonates, but it has been the inflammatory response and the formation of a purulent exudate in reported

1	space is the initial step in the induction of responsible for meningitis predominantly in neonates, but it has been the inflammatory response and the formation of a purulent exudate in reported with increasing frequency in individuals >50 years of age, the subarachnoid space (Fig. 164-2). Bacterial cell-wall components, particularly those with underlying diseases. such as the lipopolysaccharide (LPS) molecules of gram-negative bac-

1	L. monocytogenes (Chap. 176) is an increasingly important cause teria and teichoic acid and peptidoglycans of S. pneumoniae, induce of meningitis in neonates (<1 month of age), pregnant women, indi-meningeal inflammation by stimulating the production of inflammaviduals >60 years, and immunocompromised individuals of all ages. tory cytokines and chemokines by microglia, astrocytes, monocytes, Infection is acquired by ingesting foods contaminated by Listeria. microvascular endothelial cells, and CSF leukocytes. In experimental Foodborne human listerial infection has been reported from contami-models of meningitis, cytokines including tumor necrosis factor nated coleslaw, milk, soft cheeses, and several types of “ready-to-eat” alpha (TNF-α) and interleukin 1β (IL-1β) are present in CSF within foods, including delicatessen meat and uncooked hotdogs. 1–2 h of intracisternal inoculation of LPS. This cytokine response is

1	The frequency of H. influenzae type b (Hib) meningitis in children quickly followed by an increase in CSF protein concentration and has declined dramatically since the introduction of the Hib conjugate leukocytosis. Chemokines (cytokines that induce chemotactic vaccine, although rare cases of Hib meningitis in vaccinated children migration in leukocytes) and a variety of other proinflammatory cytohave been reported. More frequently, H. influenzae causes meningitis kines are also produced and secreted by leukocytes and tissue cells that in unvaccinated children and older adults, and non-b H. influenzae is are stimulated by IL-1β and TNF-α. In addition, bacteremia and the an emerging pathogen. inflammatory cytokines induce the production of excitatory amino

1	S. aureus and coagulase-negative staphylococci (Chap. 172) are acids, reactive oxygen and nitrogen species (free oxygen radicals, nitric important causes of meningitis that occurs following invasive neu-oxide, and peroxynitrite), and other mediators that can induce death of rosurgical procedures, particularly shunting procedures for hydro-brain cells, especially in the dentate gyrus of the hippocampus. cephalus, or as a complication of the use of subcutaneous Ommaya Much of the pathophysiology of bacterial meningitis is a direct consereservoirs for administration of intrathecal chemotherapy. quence of elevated levels of CSF cytokines and chemokines. TNF-α and

1	The most common bacteria that cause meningitis, S. pneumoniae and N. meningitidis, initially colonize the nasopharynx by attaching to nasopharyngeal epithelial cells. Bacteria are transported across epithelial cells in membrane-bound vacuoles to the intravascular space or invade the intravascular space by creating separations in the apical tight junctions of columnar epithelial cells. Once in the bloodstream, bacteria are able to avoid phagocytosis by neutrophils and classic complement-mediated bactericidal activity because of the presence of a polysaccharide capsule. Bloodborne bacteria can reach the intraventricular choroid plexus, directly infect choroid plexus epithelial cells, and gain access to the CSF. Some bacteria, such as S. pneumoniae, can adhere to cerebral capillary endothelial cells and subsequently migrate through or between these cells to reach the CSF. Bacteria are able to multiply rapidly within CSF because of the absence of effective host immune defenses. Normal CSF

1	cells and subsequently migrate through or between these cells to reach the CSF. Bacteria are able to multiply rapidly within CSF because of the absence of effective host immune defenses. Normal CSF contains few white blood cells (WBCs) and relatively small amounts of complement proteins and immunoglobulins. The paucity of the latter two prevents effective opsonization of bacteria, an essential prerequisite for bacterial phagocytosis by neutrophils. Phagocytosis of bacteria is further impaired by the fluid nature of CSF, which is less conducive to phagocytosis than a solid tissue substrate.

1	A critical event in the pathogenesis of bacterial meningitis is the inflammatory reaction induced by the invading bacteria. FIGURE 164-2 The pathophysiology of the neurologic complications of bacterial meningitis. Many of the neurologic manifestations CSF, cerebrospinal fluid; SAS, subarachnoid space.

1	Invasion of SAS by meningeal pathogens Multiplication of organisms and lysis of organisms by bactericidal antibiotics Release of bacterial cell-wall components (endotoxin, teichoic acid) Production of inflammatory cytokines Altered blood-brain barrier permeability Adherence of leukocytes to cerebral capillary endothelial cells Permeability of blood vessels with leakage of plasma proteins into CSF Leukocytes migrate into CSF, degranulate, and release toxic metabolites Alterations in cerebral blood flow Production of excitatory amino acids and reactive oxygen and nitrogen species Cell injury and death blood flow Cerebral ischemia Vasogenic edema Exudate in SAS obstructs outflow and resorption of CSF and surrounds and infiltrates cerebral vasculature Obstructive and communicating hydrocephalus and interstitial edema Cytotoxic edema, stroke, seizures blood flow

1	IL-1β act synergistically to increase the permeability of the blood-brain barrier, resulting in induction of vasogenic edema and the leakage of serum proteins into the subarachnoid space (Fig. 164-2). The sub-arachnoid exudate of proteinaceous material and leukocytes obstructs the flow of CSF through the ventricular system and diminishes the resorptive capacity of the arachnoid granulations in the dural sinuses, leading to obstructive and communicating hydrocephalus and concomitant interstitial edema.

1	Inflammatory cytokines upregulate the expression of selectins on cerebral capillary endothelial cells and leukocytes, promoting leukocyte adherence to vascular endothelial cells and subsequent migration into the CSF. The adherence of leukocytes to capillary endothelial cells increases the permeability of blood vessels, allowing for the leakage of plasma proteins into the CSF, which adds to the inflammatory exudate. Neutrophil degranulation results in the release of toxic metabolites that contribute to cytotoxic edema, cell injury, and death. Contrary to previous beliefs, CSF leukocytes probably do little to contribute to the clearance of CSF bacterial infection.

1	During the very early stages of meningitis, there is an increase in cerebral blood flow, soon followed by a decrease in cerebral blood flow and a loss of cerebrovascular autoregulation (Chap. 330). Narrowing of the large arteries at the base of the brain due to encroachment by the purulent exudate in the subarachnoid space and infiltration of the arterial wall by inflammatory cells with intimal thickening (vasculitis) also occur and may result in ischemia and infarction, obstruction of branches of the middle cerebral artery by thrombosis, thrombosis of the major cerebral venous sinuses, and thrombophlebitis of the cerebral cortical veins. The combination of interstitial, vasogenic, and cytotoxic edema leads to raised ICP and coma. Cerebral herniation usually results from the effects of cerebral edema, either focal or generalized; hydrocephalus and dural sinus or cortical vein thrombosis may also play a role.

1	Meningitis can present as either an acute fulminant illness that progresses rapidly in a few hours or as a subacute infection that progressively worsens over several days. The classic clinical triad of meningitis is fever, headache, and nuchal rigidity, but the classic triad may not be present. A decreased level of consciousness occurs in >75% of patients and can vary from lethargy to coma. Fever and either headache, stiff neck, or an altered level of consciousness will be present in nearly every patient with bacterial meningitis. Nausea, vomiting, and photophobia are also common complaints.

1	Seizures occur as part of the initial presentation of bacterial meningitis or during the course of the illness in 20–40% of patients. Focal seizures are usually due to focal arterial ischemia or infarction, cortical venous thrombosis with hemorrhage, or focal edema. Generalized seizure activity and status epilepticus may be due to hyponatremia, cerebral anoxia, or, less commonly, the toxic effects of antimicrobial agents.

1	Raised ICP is an expected complication of bacterial meningitis and the major cause of obtundation and coma in this disease. More than 90% of patients will have a CSF opening pressure >180 mmH2O, and 20% have opening pressures >400 mmH2O. Signs of increased ICP include a deteriorating or reduced level of consciousness, papilledema, dilated poorly reactive pupils, sixth nerve palsies, decerebrate posturing, and the Cushing reflex (bradycardia, hypertension, and irregular respirations). The most disastrous complication of increased ICP is cerebral herniation. The incidence of herniation in patients with bacterial meningitis has been reported to occur in as few as 1% to as many as 8% of cases.

1	Specific clinical features may provide clues to the diagnosis of individual organisms and are discussed in more detail in specific chapters devoted to individual pathogens. The most important of these clues is the rash of meningococcemia, which begins as a diffuse erythematous maculopapular rash resembling a viral exanthem; however, the skin lesions of meningococcemia rapidly become petechial. Petechiae are found on the trunk and lower extremities, in the mucous membranes and conjunctiva, and occasionally on the palms and soles.

1	When bacterial meningitis is suspected, blood cultures should be immediately obtained and empirical antimicrobial and adjunctive dexamethasone therapy initiated without delay (Table 164-1). The diagnosis of bacterial meningitis is made by examination of the CSF (Table 164-2). The need to obtain neuroimaging studies (CT or MRI) prior to LP requires clinical judgment. In an immunocompetent patient with no known history of recent head trauma, a normal level of consciousness, and no evidence of papilledema or focal neurologic deficits, it is considered safe to perform LP without prior neuroimaging studies. If LP is delayed in order to obtain neuroimaging studies, empirical antibiotic therapy should be initiated after blood cultures are obtained. Antibiotic therapy initiated a few hours prior to LP will not significantly alter the CSF WBC count or glucose concentration, nor is it likely to prevent visualization of organisms by Gram’s stain or detection of bacterial nucleic acid by

1	prior to LP will not significantly alter the CSF WBC count or glucose concentration, nor is it likely to prevent visualization of organisms by Gram’s stain or detection of bacterial nucleic acid by polymerase chain reaction (PCR) assay.

1	The classic CSF abnormalities in bacterial meningitis (Table 164-2) are (1) polymorphonuclear (PMN) leukocytosis (>100 cells/μL in 90%), (2) decreased glucose concentration (<2.2 mmol/L [<40 mg/dL] and/ or CSF/serum glucose ratio of <0.4 in ~60%), (3) increased protein concentration (>0.45 g/L [>45 mg/dL] in 90%), and (4) increased opening pressure (>180 mmH2O in 90%). CSF bacterial cultures are positive in >80% of patients, and CSF Gram’s stain demonstrates organisms in >60%. CSF glucose concentrations <2.2 mmol/L (<40 mg/dL) are abnormal, and a CSF glucose concentration of zero can be seen in bacterial men ingitis. Use of the CSF/serum glucose ratio corrects for hyperglycemia Preterm infants to infants Ampicillin + cefotaxime <1 month Immunocompetent Cefotaxime, ceftriaxone, or cefepime + children >3 months and vancomycin adults <55 Adults >55 and adults of Ampicillin + cefotaxime, ceftriaxone or any age with alcoholism cefepime + vancomycin or other debilitating illnesses

1	Adults >55 and adults of Ampicillin + cefotaxime, ceftriaxone or any age with alcoholism cefepime + vancomycin or other debilitating illnesses Hospital-acquired Ampicillin + ceftazidime or meropenem + meningitis, posttraumatic vancomycin or postneurosurgery meningitis, neutropenic patients, or patients with impaired cell-mediated immunity

1	Ampicillin 300 (mg/kg)/d, q6h 12 g/d, q4h Cefepime 150 (mg/kg)/d, q8h 6 g/d, q8h Cefotaxime 225-300 (mg/kg)/d, q6h 12 g/d, q4h Ceftriaxone 100 (mg/kg)/d, q12h 4 g/d, q12h Ceftazidime 150 (mg/kg)/d, q8h 6 g/d, q8h Gentamicin 7.5 (mg/kg)/d, q8hb 7.5 (mg/kg)/d, q8h Meropenem 120 (mg/kg)/d, q8h 6 g/d, q8h Metronidazole 30 (mg/kg)/d, q6h 1500–2000 mg/d, q6h Nafcillin 100–200 (mg/kg)/d, q6h 9–12 g/d, q4h Penicillin G 400,000 (U/kg)/d, q4h 20–24 million U/d, q4h Vancomycin 45-60 (mg/kg)/d, q6h 45-60 (mg/kg)d, q6–12hb aAll antibiotics are administered intravenously; doses indicated assume normal renal and hepatic function. bDoses should be adjusted based on serum peak and trough levels: gentamicin therapeutic level: peak: 5–8 μg/mL; trough: <2 μg/mL; vancomycin therapeutic level: peak: 25–40 μg/mL; trough: 5–15 μg/mL. Meningitis, Encephalitis, Brain Abscess, and Empyema White blood cells 10/μL to 10,000/μL; neutrophils predominate Glucose <2.2 mmol/L (<40 mg/dL) CSF/serum glucose <0.4

1	Meningitis, Encephalitis, Brain Abscess, and Empyema White blood cells 10/μL to 10,000/μL; neutrophils predominate Glucose <2.2 mmol/L (<40 mg/dL) CSF/serum glucose <0.4 Protein >0.45 g/L (>45 mg/dL) Latex agglutination May be positive in patients with meningitis due to Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae type b, Escherichia coli, group B streptococci Limulus lysate Positive in cases of gram-negative meningitis

1	Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae type b, Escherichia coli, group B streptococci Limulus lysate Positive in cases of gram-negative meningitis Abbreviation: PCR, polymerase chain reaction that may mask a relative decrease in the CSF glucose concentration. The CSF glucose concentration is low when the CSF/serum glucose ratio is <0.6. A CSF/serum glucose ratio <0.4 is highly suggestive of bacterial meningitis but may also be seen in other conditions, including fungal, tuberculous, and carcinomatous meningitis. It takes from 30 min to several hours for the concentration of CSF glucose to reach equilibrium with blood glucose levels; therefore, administration of 50 mL of 50% glucose (D50) prior to LP, as commonly occurs in emergency room settings, is unlikely to alter CSF glucose concentration significantly unless more than a few hours have elapsed between glucose administration and LP.

1	A 16S rRNA conserved sequence broad-based bacterial PCR can detect small numbers of viable and nonviable organisms in CSF and is expected to be useful for making a diagnosis of bacterial meningitis in patients who have been pretreated with oral or parenteral antibiotics and in whom Gram’s stain and CSF culture are negative. When the broad-range PCR is positive, a PCR that uses specific bacterial primers to detect the nucleic acid of S. pneumoniae, N. meningitidis, Escherichia coli, L. monocytogenes, H. influenzae, and S. agalactiae can be obtained based on the clinical suspicion of the meningeal pathogen. The latex agglutination (LA) test for the detection of bacterial antigens of S. pneumoniae, N. meningitidis, H. influenzae type b, group B Streptococcus, and E. coli K1 strains in the CSF has been useful for making a diagnosis of bacterial meningitis but is being replaced by the CSF bacterial PCR assay. The CSF LA test has a specificity of 95–100% for S. pneumoniae and N.

1	in the CSF has been useful for making a diagnosis of bacterial meningitis but is being replaced by the CSF bacterial PCR assay. The CSF LA test has a specificity of 95–100% for S. pneumoniae and N. meningitidis, so a positive test is virtually diagnostic of bacterial meningitis caused by these organisms. However, the sensitivity of the CSF LA test is only 70–100% for detection of S. pneumoniae and 33–70% for detection of N. meningitidis antigens, so a negative test does not exclude infection by these organisms. The Limulus amebocyte lysate assay is a rapid diagnostic test for the detection of gram-negative endotoxin in CSF and thus for making a diagnosis of gram-negative bacterial meningitis. The test has a specificity of 85–100% and a sensitivity approaching 100%. Thus, a positive Limulus amebocyte lysate assay occurs in virtually all patients with gram-negative bacterial meningitis, but false positives may occur.

1	Almost all patients with bacterial meningitis will have neuroimaging studies performed during the course of their illness. MRI is preferred over CT because of its superiority in demonstrating areas of cerebral edema and ischemia. In patients with bacterial meningitis, diffuse meningeal enhancement is often seen after the administration of gadolinium. Meningeal enhancement is not diagnostic of meningitis but occurs in any CNS disease associated with increased blood-brain barrier permeability. Petechial skin lesions, if present, should be biopsied. The rash of meningococcemia results from the dermal seeding of organisms with vascular endothelial damage, and biopsy may reveal the organism on Gram’s stain.

1	Viral meningoencephalitis, and particularly herpes simplex virus (HSV) encephalitis, can mimic the clinical presentation of bacterial meningitis (see “Viral Encephalitis,” below). HSV encephalitis typically presents with headache, fever, altered consciousness, focal neurologic deficits (e.g., dysphasia, hemiparesis), and focal or generalized seizures. The findings on CSF studies, neuroimaging, and electroencephalogram (EEG) distinguish HSV encephalitis from bacterial meningitis. The typical CSF profile with viral CNS infections is a lymphocytic pleocytosis with a normal glucose concentration, in contrast to the PMN pleocytosis and hypoglycorrhachia characteristic of bacterial meningitis. MRI abnormalities (other than meningeal enhancement) are not seen in uncomplicated bacterial meningitis. By contrast, in HSV encephalitis, on T2-weighted, fluid-attenuated inversion recovery (FLAIR) and diffusion-weighted MRI images, high signal intensity lesions are seen in the orbitofrontal,

1	By contrast, in HSV encephalitis, on T2-weighted, fluid-attenuated inversion recovery (FLAIR) and diffusion-weighted MRI images, high signal intensity lesions are seen in the orbitofrontal, anterior, and medial temporal lobes in the majority of patients within 48 h of symptom onset. Some patients with HSV encephalitis have a distinctive periodic pattern on EEG (see below).

1	Rickettsial disease can resemble bacterial meningitis (Chap. 211). Rocky Mountain spotted fever (RMSF) is transmitted by a tick bite and caused by the bacteria Rickettsia rickettsii. The disease may present acutely with high fever, prostration, myalgia, headache, nausea, and vomiting. Most patients develop a characteristic rash within 96 h of the onset of symptoms. The rash is initially a diffuse erythematous maculopapular rash that may be difficult to distinguish from that of meningococcemia. It progresses to a petechial rash, then to a purpuric rash, and if untreated, to skin necrosis or gangrene. The color of the lesions changes from bright red to very dark red, then yellowish-green to black. The rash typically begins in the wrist and ankles and then spreads distally and proximally within a matter of a few hours, involving the palms and soles. Diagnosis is made by immunofluorescent staining of skin biopsy specimens. Ehrlichioses are also transmitted by a tick bite. These are small

1	a matter of a few hours, involving the palms and soles. Diagnosis is made by immunofluorescent staining of skin biopsy specimens. Ehrlichioses are also transmitted by a tick bite. These are small gram-negative coccobacilli of which two species cause human disease. Anaplasma phagocytophilum causes human granulocytic ehrlichiosis (anaplasmosis), and Ehrlichia chaffeensis causes human monocytic ehrlichiosis. The clinical and laboratory manifestations of the infections are similar. Patients present with fever, headache, confusion, nausea, and vomiting. Twenty percent of patients have a maculopapular or petechial rash. There is laboratory evidence of leukopenia, thrombocytopenia, and anemia, and mild to moderate elevations in alanine aminotransferases, alkaline phosphatase, and lactate dehydrogenase. Patients with RMSF and those with ehrlichial infections may have an altered level of consciousness ranging from mild lethargy to coma, confusion, focal neurologic signs, cranial nerve palsies,

1	Patients with RMSF and those with ehrlichial infections may have an altered level of consciousness ranging from mild lethargy to coma, confusion, focal neurologic signs, cranial nerve palsies, hyperreflexia, and seizures.

1	Focal suppurative CNS infections (see below), including subdural and epidural empyema and brain abscess, should also be considered, especially when focal neurologic findings are present. MRI should be performed promptly in all patients with suspected meningitis who have focal features, both to detect the intracranial infection and to search for associated areas of infection in the sinuses or mastoid bones.

1	A number of noninfectious CNS disorders can mimic bacterial meningitis. Subarachnoid hemorrhage (SAH; Chap. 330) is generally the major consideration. Other possibilities include chemical meningitis due to rupture of tumor contents into the CSF (e.g., from a cystic glioma or craniopharyngioma epidermoid or dermoid cyst); drug-induced hypersensitivity meningitis; carcinomatous or lymphomatous meningitis; meningitis associated with inflammatory disorders such as sarcoid, systemic lupus erythematosus (SLE), and Behçet’s syndrome; pituitary apoplexy; and uveomeningitic syndromes (Vogt-Koyanagi-Harada syndrome). On occasion, subacutely evolving meningitis (Chap. 165) may be considered in the differential diagnosis of acute meningitis. The principal causes include Mycobacterium tuberculosis (Chap. 202), Cryptococcus neoformans (Chap. 239), Histoplasma capsulatum (Chap. 236), Coccidioides immitis (Chap. 237), and Treponema pallidum (Chap. 206).

1	(Table 164-1) Bacterial meningitis is a medical emergency. The goal is to begin antibiotic therapy within 60 min of a patient’s arrival in the emergency room. Empirical antimicrobial therapy is initiated in patients with suspected bacterial meningitis before the results of CSF Gram’s stain and culture are known. S. pneumoniae (Chap. 171) and N. meningitidis (Chap. 180) are the most common etiologic organisms of community-acquired bacterial meningitis. Due to the emergence of penicillinand cephalosporin-resistant S. pneumoniae, empirical therapy of community-acquired suspected bacterial meningitis in children and adults should include a combination of dexamethasone, a thirdor fourth-generation cephalosporin (e.g., ceftriaxone, cefotaxime, or cefepime), and vancomycin, plus acyclovir, as HSV encephalitis is the leading disease in the differential diagnosis, and doxycycline during tick season to treat tick-borne bacterial infections. Ceftriaxone or cefotaxime provides good coverage for

1	encephalitis is the leading disease in the differential diagnosis, and doxycycline during tick season to treat tick-borne bacterial infections. Ceftriaxone or cefotaxime provides good coverage for susceptible S. pneumoniae, group B streptococci, and H. influenzae and adequate coverage for N. meningitidis. Cefepime is a broad-spectrum fourth-generation cephalosporin with in vitro activity similar to that of cefotaxime or ceftriaxone against S. pneumoniae and N. meningitidis and greater activity against Enterobacter species and Pseudomonas aeruginosa. In clinical trials, cefepime has been demonstrated to be equivalent to cefotaxime in the treatment of penicillin-sensitive pneumococcal and meningococcal meningitis, and it has been used successfully in some patients with meningitis due to Enterobacter species and P. aeruginosa. Ampicillin should be added to the empirical regimen for coverage of L. monocytogenes in individuals <3 months of age, those >55, or those with suspected impaired

1	species and P. aeruginosa. Ampicillin should be added to the empirical regimen for coverage of L. monocytogenes in individuals <3 months of age, those >55, or those with suspected impaired cell-mediated immunity because of chronic illness, organ transplantation, pregnancy, malignancy, or immunosuppressive therapy. Metronidazole is added to the empirical regimen to cover gram-negative anaerobes in patients with otitis, sinusitis, or mastoiditis. In hospital-acquired meningitis, and particularly meningitis following neurosurgical procedures, staphylococci and gram-negative organisms including P. aeruginosa are the most common etiologic organisms. In these patients, empirical therapy should include a combination of vancomycin and ceftazidime, cefepime, or meropenem. Ceftazidime, cefepime, or meropenem should be substituted for ceftriaxone or cefotaxime in neurosurgical patients and in neutropenic patients, because ceftriaxone and cefotaxime do not provide adequate activity against CNS

1	or meropenem should be substituted for ceftriaxone or cefotaxime in neurosurgical patients and in neutropenic patients, because ceftriaxone and cefotaxime do not provide adequate activity against CNS infection with P. aeruginosa. Meropenem is a carbapenem antibiotic that is highly active in vitro against L. monocytogenes, has been demonstrated to be effective in cases of meningitis caused by P. aeruginosa, and shows good activity against penicillin-resistant pneumococci. In experimental pneumococcal meningitis, meropenem was comparable to ceftriaxone and inferior to vancomycin in sterilizing CSF cultures. The number of patients with bacterial meningitis enrolled in clinical trials of meropenem has not been sufficient to definitively assess the efficacy of this antibiotic.

1	SPECIFIC ANTIMICROBIAL THERAPY Meningococcal Meningitis (Table 164-3) Although ceftriaxone and cefotaxime provide adequate empirical coverage for N. meningitidis, penicillin G remains the antibiotic of choice for meningococcal meningitis caused by susceptible strains. Isolates of N. meningitidis with moderate resistance to penicillin have been identified and are increasing in incidence worldwide. CSF isolates of N. meningitidis should be tested for penicillin and ampicillin susceptibility, and if resistance is found, cefotaxime or ceftriaxone should be substituted for penicillin. A 7-day course of intravenous antibiotic therapy is adequate for uncomplicated meningococcal meningitis. The index case and all close contacts should receive chemoprophylaxis with a 2-day regimen of rifampin (600 mg every 12 h for 2 days in adults and 10 mg/kg every 12 h for 2 days in children >1 year). Rifampin is not recommended in pregnant women. Alternatively, adults can be

1	Gram-negative bacilli (except Ceftriaxone or cefotaxime Pseudomonas spp.) Staphylococci spp. Fusobacterium spp. Metronidazole aDoses are as indicated in Table 164-1. treated with one dose of azithromycin (500 mg) or one intramuscular dose of ceftriaxone (250 mg). Close contacts are defined as those individuals who have had contact with oropharyngeal secretions, either through kissing or by sharing toys, beverages, or cigarettes.

1	Pneumococcal Meningitis Antimicrobial therapy of pneumococcal meningitis is initiated with a cephalosporin (ceftriaxone, cefotaxime, or cefepime) and vancomycin. All CSF isolates of S. pneumoniae should be tested for sensitivity to penicillin and the cephalosporins. Once the results of antimicrobial susceptibility tests are known, therapy can be modified accordingly (Table 164-3). For S. pneumoniae meningitis, an isolate of S. pneumoniae is considered to be susceptible to penicillin with a minimal inhibitory concentration (MIC) <0.06 μg/mL and to be resistant when the MIC is >0.12 μg/ mL. Isolates of S. pneumoniae that have cephalosporin MICs ≤0.5 μg/mL are considered sensitive to the cephalosporins (cefotaxime, ceftriaxone, cefepime). Those with MICs of 1 μg/mL are considered to have intermediate resistance, and those with MICs ≥2 μg/mL are considered resistant. For meningitis due to pneumococci, with cefotaxime or ceftriaxone MICs ≤0.5 μg/mL, treatment with cefotaxime or ceftriaxone

1	resistance, and those with MICs ≥2 μg/mL are considered resistant. For meningitis due to pneumococci, with cefotaxime or ceftriaxone MICs ≤0.5 μg/mL, treatment with cefotaxime or ceftriaxone is usually adequate. For MIC >1 μg/mL, vancomycin is the antibiotic of choice. Rifampin can be added to vancomycin for its synergistic effect but is inadequate as monotherapy because resistance develops rapidly when it is used alone.

1	A 2-week course of intravenous antimicrobial therapy is recommended for pneumococcal meningitis. Patients with S. pneumoniae meningitis should have a repeat LP performed 24–36 h after the initiation of antimicrobial therapy to document sterilization of the CSF. Failure to sterilize the CSF after 24–36 h of antibiotic therapy should be considered presumptive evidence of antibiotic resistance. Patients with penicillinand cephalosporin-resistant strains of S. pneumoniae who do not respond to intravenous vancomycin alone may benefit from the addition of intraventricular vancomycin. The intraventricular route of administration is preferred over the intrathecal route because adequate concentrations of vancomycin in the cerebral ventricles are not always achieved with intrathecal administration.

1	Listeria Meningitis Meningitis due to L. monocytogenes is treated with ampicillin for at least 3 weeks (Table 164-3). Gentamicin is added in critically ill patients (2 mg/kg loading dose, then 7.5 mg/kg per day Meningitis, Encephalitis, Brain Abscess, and Empyema 890 given every 8 h and adjusted for serum levels and renal function). The combination of trimethoprim (10–20 mg/kg per day) and sulfamethoxazole (50–100 mg/kg per day) given every 6 h may provide an alternative in penicillin-allergic patients.

1	Staphylococcal Meningitis Meningitis due to susceptible strains of S. aureus or coagulase-negative staphylococci is treated with nafcillin (Table 164-3). Vancomycin is the drug of choice for methicillinresistant staphylococci and for patients allergic to penicillin. In these patients, the CSF should be monitored during therapy. If the CSF is not sterilized after 48 h of intravenous vancomycin therapy, then either intraventricular or intrathecal vancomycin, 20 mg once daily, can be added. Gram-Negative Bacillary Meningitis The third-generation cephalosporins— cefotaxime, ceftriaxone, and ceftazidime—are equally efficacious for the treatment of gram-negative bacillary meningitis, with the exception of meningitis due to P. aeruginosa, which should be treated with ceftazidime, cefepime, or meropenem (Table 164-3). A 3-week course of intravenous antibiotic therapy is recommended for meningitis due to gram-negative bacilli.

1	The release of bacterial cell-wall components by bactericidal antibiotics leads to the production of the inflammatory cytokines IL-1β and TNF-α in the subarachnoid space. Dexamethasone exerts its beneficial effect by inhibiting the synthesis of IL-1β and TNF-α at the level of mRNA, decreasing CSF outflow resistance, and stabilizing the blood-brain barrier. The rationale for giving dexamethasone 20 min before antibiotic therapy is that dexamethasone inhibits the production of TNF-α by macrophages and microglia only if it is administered before these cells are activated by endotoxin. Dexamethasone does not alter TNF-α production once it has been induced. The results of clinical trials of dexamethasone therapy in meningitis due to H. influenzae, S. pneumoniae, and N. meningitidis have demonstrated its efficacy in decreasing meningeal inflammation and neurologic sequelae such as the incidence of sensorineural hearing loss.

1	A prospective European trial of adjunctive therapy for acute bacterial meningitis in 301 adults found that dexamethasone reduced the number of unfavorable outcomes (15 vs. 25%, p = .03) including death (7 vs. 15%, p = .04). The benefits were most striking in patients with pneumococcal meningitis. Dexamethasone (10 mg intravenously) was administered 15–20 min before the first dose of an antimicrobial agent, and the same dose was repeated every 6 h for 4 days. These results were confirmed in a second trial of dexamethasone in adults with pneumococcal meningitis. Therapy with dexamethasone should ideally be started 20 min before, or not later than concurrent with, the first dose of antibiotics. It is unlikely to be of significant benefit if started >6 h after antimicrobial therapy has been initiated. Dexamethasone may decrease the penetration of vancomycin into CSF, and it delays the sterilization of CSF in experimental models of S. pneumoniae meningitis. As a result, to assure reliable

1	Dexamethasone may decrease the penetration of vancomycin into CSF, and it delays the sterilization of CSF in experimental models of S. pneumoniae meningitis. As a result, to assure reliable penetration of vancomycin into the CSF, children and adults are treated with vancomycin in a dose of 45–60 mg/kg per day. Alternatively, vancomycin can be administered by the intra-ventricular route.

1	One of the concerns for using dexamethasone in adults with bacterial meningitis is that in experimental models of meningitis, dexamethasone therapy increased hippocampal cell injury and reduced learning capacity. This has not been the case in clinical series. The efficacy of dexamethasone therapy in preventing neurologic sequelae is different between highand low-income countries. Three large randomized trials in low-income countries (sub-Saharan Africa, Southeast Asia) failed to show benefit in subgroups of patients. The lack of efficacy of dexamethasone in these trials has been attributed to late presentation to the hospital with more advanced disease, antibiotic pretreatment, malnutrition, infection with HIV, and treatment of patients with probable, but not micro-biologically proven, bacterial meningitis. The results of these clinical trials suggest that patients in sub-Saharan Africa and those in low-income countries with negative CSF Gram’s stain and culture should not be treated

1	meningitis. The results of these clinical trials suggest that patients in sub-Saharan Africa and those in low-income countries with negative CSF Gram’s stain and culture should not be treated with dexamethasone.

1	Emergency treatment of increased ICP includes elevation of the patient’s head to 30–45°, intubation and hyperventilation (Paco2 25–30 mmHg), and mannitol. Patients with increased ICP should be managed in an intensive care unit; accurate ICP measurements are best obtained with an ICP monitoring device. Treatment of increased intracranial pressure is discussed in detail in Chap. 330.

1	Mortality rate is 3–7% for meningitis caused by H. influenzae, N. meningitidis, or group B streptococci; 15% for that due to L. monocytogenes; and 20% for S. pneumoniae. In general, the risk of death from bacterial meningitis increases with (1) decreased level of consciousness on admission, (2) onset of seizures within 24 h of admission, (3) signs of increased ICP, (4) young age (infancy) and age >50, (5) the presence of comorbid conditions including shock and/or the need for mechanical ventilation, and (6) delay in the initiation of treatment. Decreased CSF glucose concentration (<2.2 mmol/L [<40 mg/dL]) and markedly increased CSF protein concentration (>3 g/L [> 300 mg/dL]) have been predictive of increased mortality and poorer outcomes in some series. Moderate or severe sequelae occur in ~25% of survivors, although the exact incidence varies with the infecting organism. Common sequelae include decreased intellectual function, memory impairment, seizures, hearing loss and dizziness,

1	~25% of survivors, although the exact incidence varies with the infecting organism. Common sequelae include decreased intellectual function, memory impairment, seizures, hearing loss and dizziness, and gait disturbances.

1	Immunocompetent adult patients with viral meningitis usually present with headache, fever, and signs of meningeal irritation coupled with an inflammatory CSF profile (see below). Headache is almost invariably present and often characterized as frontal or retroorbital and frequently associated with photophobia and pain on moving the eyes. Nuchal rigidity is present in most cases but may be mild and present only near the limit of neck anteflexion. Constitutional signs can include malaise, myalgia, anorexia, nausea and vomiting, abdominal pain, and/or diarrhea. Patients often have mild lethargy or drowsiness; however, profound alterations in consciousness, such as stupor, coma, or marked confusion, do not occur in viral meningitis and suggest the presence of encephalitis or other alternative diagnoses. Similarly, seizures or focal neurologic signs or symptoms or neuroimaging abnormalities indicative of brain parenchymal involvement are not typical of viral meningitis and suggest the

1	diagnoses. Similarly, seizures or focal neurologic signs or symptoms or neuroimaging abnormalities indicative of brain parenchymal involvement are not typical of viral meningitis and suggest the presence of encephalitis or another CNS infectious or inflammatory process.

1	Using a variety of diagnostic techniques, including CSF PCR, culture, and serology, a specific viral cause can be found in 60–90% of cases of viral meningitis. The most important agents are enteroviruses (including echoviruses and coxsackieviruses in addition to numbered enteroviruses), varicella-zoster virus (VZV), HSV (HSV-2 > HSV-1), HIV, and arboviruses (Table 164-4). CSF cultures are positive in 30–70% of patients, with the frequency of isolation depending on the specific viral agent. Approximately two-thirds of culture-negative cases of “aseptic” meningitis have a specific viral etiology identified by CSF PCR testing (see below).

1	Viral meningitis is not a nationally reportable disease; however, it has been estimated that the incidence is ~60,000–75,000 cases per year. In temperate climates, there is a substantial increase in cases during the nonwinter months, reflecting the seasonal predominance of enterovirus and arthropod-borne virus (arbovirus) infections in the summer and fall, with a peak monthly incidence of about 1 reported case per 100,000 population. Enteroviruses (coxsackieviruses, echo-Herpes simplex virus 1 viruses, and human enteroviruses aImmunocompromised host. bThe most common cause of sporadic encephalitis. cThe most common cause of epidemic encephalitis.

1	LABORATORY DIAGNOSIS CSF Examination The most important laboratory test in the diagnosis of viral meningitis is examination of the CSF. The typical profile is a pleocytosis, a normal or slightly elevated protein concentration (0.2–0.8 g/L [20–80 mg/dL]), a normal glucose concentration, and a normal or mildly elevated opening pressure (100–350 mmH2O). Organisms are not seen on Gram’s stain of CSF. The total CSF cell count in viral meningitis is typically 25–500/μL, although cell counts of several thousand/μL are occasionally seen, especially with infections due to lymphocytic choriomeningitis virus (LCMV) and mumps virus. Lymphocytes are typically the predominant cell. Rarely, PMNs may predominate in the first 48 h of illness, especially with infections due to echovirus 9, West Nile virus, eastern equine encephalitis (EEE) virus, or mumps. A PMN pleocytosis occurs in 45% of patients with West Nile virus (WNV) meningitis and can persist for a week or longer before shifting to a

1	virus, eastern equine encephalitis (EEE) virus, or mumps. A PMN pleocytosis occurs in 45% of patients with West Nile virus (WNV) meningitis and can persist for a week or longer before shifting to a lymphocytic pleocytosis. PMN pleocytosis with low glucose may also be a feature of cytomegalovirus (CMV) infections in immunocompromised hosts. Despite these exceptions, the presence of a CSF PMN pleocytosis in a patient with suspected viral meningitis in whom a specific diagnosis has not been established should prompt consideration of alternative diagnoses, including bacterial meningitis or parameningeal infections. The CSF glucose concentration is typically normal in viral infections, although it may be decreased in 10–30% of cases due to mumps or LCMV. Rare instances of decreased CSF glucose concentration occur in cases of meningitis due to echo-viruses and other enteroviruses, HSV-2, and VZV. As a rule, a lymphocytic pleocytosis with a low glucose concentration should suggest fungal or

1	concentration occur in cases of meningitis due to echo-viruses and other enteroviruses, HSV-2, and VZV. As a rule, a lymphocytic pleocytosis with a low glucose concentration should suggest fungal or tuberculous meningitis, Listeria meningoencephalitis, or noninfectious disorders (e.g., sarcoid, neoplastic meningitis).

1	A number of tests measuring levels of various CSF proteins, enzymes, and mediators—including C-reactive protein, lactic acid, lactate dehydrogenase, neopterin, quinolinate, IL-1β, IL-6, soluble IL-2 receptor, β2-microglobulin, and TNF—have been proposed as potential discriminators between viral and bacterial meningitis or as markers of specific types of viral infection (e.g., infection with HIV), but they remain of uncertain sensitivity and specificity and are not 891 widely used for diagnostic purposes.

1	Polymerase Chain Reaction Amplification of Viral Nucleic Acid Amplification of viral-specific DNA or RNA from CSF using PCR amplification has become the single most important method for diagnosing CNS viral infections. In both enteroviral and HSV infections of the CNS, CSF PCR has become the diagnostic procedure of choice and is substantially more sensitive than viral cultures. HSV CSF PCR is also an important diagnostic test in patients with recurrent episodes of “aseptic” meningitis, many of whom have amplifiable HSV DNA in CSF despite negative viral cultures. CSF PCR is also used routinely to diagnose CNS viral infections caused by CMV, Epstein-Barr virus (EBV), VZV, and human herpesvirus 6 (HHV-6). CSF PCR tests are available for WNV but are not as sensitive as detection of WNV-specific CSF IgM. PCR is also useful in the diagnosis of CNS infection caused by Mycoplasma pneumoniae, which can mimic viral meningitis and encephalitis. PCR of throat washings may assist in diagnosis of

1	CSF IgM. PCR is also useful in the diagnosis of CNS infection caused by Mycoplasma pneumoniae, which can mimic viral meningitis and encephalitis. PCR of throat washings may assist in diagnosis of enteroviral and mycoplasmal CNS infections. PCR of stool specimens may also assist in diagnosis of enteroviral infections (see below).

1	Viral Culture The sensitivity of CSF cultures for the diagnosis of viral meningitis and encephalitis, in contrast to its utility in bacterial infections, is generally poor. In addition to CSF, specific viruses may also be isolated from throat swabs, stool, blood, and urine. Enteroviruses and adenoviruses may be found in feces; arboviruses, some enteroviruses, and LCMV in blood; mumps and CMV in urine; and enteroviruses, mumps, and adenoviruses in throat washings. During enteroviral infections, viral shedding in stool may persist for several weeks. The presence of enterovirus in stool is not diagnostic and may result from residual shedding from a previous enteroviral infection; it also occurs in some asymptomatic individuals during enteroviral epidemics.

1	Serologic Studies For many arboviruses including WNV, serologic studies remain important diagnostic tools. Serum antibody determination is less useful for viruses with high seroprevalence rates in the general population such as HSV, VZV, CMV, and EBV. For viruses with low seroprevalence rates, diagnosis of acute viral infection can be made by documenting seroconversion between acute-phase and convalescent sera (typically obtained after 2–4 weeks) or by demonstrating the presence of virus-specific IgM antibodies. For viruses with high seroprevalence such as VZV and HSV, demonstration of synthesis of virus-specific antibodies in CSF, as shown by an increased IgG index or the presence of CSF IgM antibodies, may be useful and can provide presumptive evidence of CNS infection. Although serum and CSF IgM antibodies generally persist for only a few months after acute infection, there are exceptions to this rule. For example, WNV serum IgM has been shown to persist in some patients for >1

1	and CSF IgM antibodies generally persist for only a few months after acute infection, there are exceptions to this rule. For example, WNV serum IgM has been shown to persist in some patients for >1 year following acute infection. Unfortunately, the delay between onset of infection and the host’s generation of a virus-specific antibody response often means that serologic data are useful mainly for the retrospective establishment of a specific diagnosis, rather than in aiding acute diagnosis or management. In the case of EBV, demonstration of antibody responses consistent with recent/acute infection (e.g., IgM viral capsid antibody, antibody against early antigen, absence of antibody against EBV-associated nuclear antigen) may assist in diagnosis.

1	CSF oligoclonal gamma globulin bands occur in association with a number of viral infections. The associated antibodies are often directed against viral proteins. Oligoclonal bands also occur commonly in certain noninfectious neurologic diseases (e.g., multiple sclerosis) and may be found in nonviral infections (e.g., neurosyphilis, Lyme neuroborreliosis). Other Laboratory Studies All patients with suspected viral meningitis should have a complete blood count and differential, liver and renal function tests, erythrocyte sedimentation rate (ESR), and C-reactive protein, electrolytes, glucose, creatine kinase, aldolase, amylase, and lipase. Neuroimaging studies (MRI preferable to CT) are not absolutely necessary in patients with uncomplicated viral meningitis but should be performed in patients with altered consciousness, seizures, focal neurologic signs or symptoms, atypical CSF profiles, or underlying immunocompromising treatments or conditions.

1	Meningitis, Encephalitis, Brain Abscess, and Empyema 892 DIFFERENTIAL DIAGNOSIS The most important issue in the differential diagnosis of viral meningitis is to consider diseases that can mimic viral meningitis, including (1) untreated or partially treated bacterial meningitis; (2) early stages of meningitis caused by fungi, mycobacteria, or Treponema pallidum (neurosyphilis), in which a lymphocytic pleocytosis is common, cultures may be slow growing or negative, and hypoglycorrhachia may not be present early; (3) meningitis caused by agents such as Mycoplasma, Listeria spp., Brucella spp., Coxiella spp., Leptospira spp., and Rickettsia spp.; (4) parameningeal infections; (5) neoplastic meningitis; and (6) meningitis secondary to noninfectious inflammatory diseases, including autoimmune and hypersensitivity meningitis, SLE and other rheumatologic diseases, sarcoidosis, Behçet’s syndrome, and the uveomeningitic syndromes. Studies in children >28 days of age suggest that the presence of

1	hypersensitivity meningitis, SLE and other rheumatologic diseases, sarcoidosis, Behçet’s syndrome, and the uveomeningitic syndromes. Studies in children >28 days of age suggest that the presence of CSF protein >0.5 g/L (sensitivity 89%, specificity 78%) and elevated serum procalcitonin levels >0.5 ng/mL (sensitivity 89%, specificity 89%) were clues to the presence of bacterial as opposed to “aseptic” meningitis. A variety of clinical algorithms for differentiating bacterial from aseptic meningitis have been developed. One such prospectively validated system, the bacterial meningitis score, suggests that the probability of bacterial meningitis is 0.3% or less (negative predictive value 99.7%, 95% confidence interval 99.6–100%) in children with CSF pleocytosis who have (1) a negative CSF Gram’s stain, (2) CSF neutrophil count <1000 cells/μL, (3) CSF protein <80 mg/dL, (4) peripheral absolute neutrophil count of <10,000 cells/μL, and (5) no prior history or current presence of seizures.

1	Enteroviruses (EV) (Chap. 228) are the most common cause of viral meningitis, accounting for >85% of cases in which a specific etiology can be identified. Cases may either be sporadic or occur in clusters. EV71 has produced large epidemics of neurologic disease outside the United States, especially in Southeast Asia, but most recently reported cases in the United States have been sporadic. Enteroviruses are the most likely cause of viral meningitis in the summer and fall months, especially in children (<15 years), although cases occur at reduced frequency year round. Although the incidence of enteroviral meningitis declines with increasing age, some outbreaks have preferentially affected older children and adults. Meningitis outside the neonatal period is usually benign. Patients present with sudden onset of fever; headache; nuchal rigidity; and often constitutional signs, including vomiting, anorexia, diarrhea, cough, pharyngitis, and myalgias. The physical examination should include

1	sudden onset of fever; headache; nuchal rigidity; and often constitutional signs, including vomiting, anorexia, diarrhea, cough, pharyngitis, and myalgias. The physical examination should include a careful search for stigmata of enterovirus infection, including exanthems, hand-foot-mouth disease, herpangina, pleurodynia, myopericarditis, and hemorrhagic conjunctivitis. The CSF profile is typically a lymphocytic pleocytosis (100–1000 cells/μL) with normal glucose and normal or mildly elevated protein concentration. However, up to 15% of patients, most commonly young infants rather than older children or adults, have a normal CSF leukocyte count. In rare cases, PMNs may predominate during the first 48 h of illness. CSF reverse transcriptase PCR (RT-PCR) is the diagnostic procedure of choice and is both sensitive (>95%) and specific (>100%). CSF PCR has the highest sensitivity if performed within 48 h of symptom onset, with sensitivity declining rapidly after day 5 of symptoms. PCR of

1	and is both sensitive (>95%) and specific (>100%). CSF PCR has the highest sensitivity if performed within 48 h of symptom onset, with sensitivity declining rapidly after day 5 of symptoms. PCR of throat washings or stool specimens may be positive for several weeks, and positive results can help support the diagnosis of an acute enteroviral infection. The sensitivity of routine enteroviral PCRs for detecting EV71 is low, and specific testing may be required. Treatment is supportive, and patients usually recover without sequelae. Chronic and severe infections can occur in neonates and in individuals with hypoor agammaglobulinemia.

1	Arbovirus infections (Chap. 233) occur predominantly in the summer and early fall. Arboviral meningitis should be considered when clusters of meningitis and encephalitis cases occur in a restricted geographic region during the summer or early fall. In the United States, the most important causes of arboviral meningitis and encephalitis are WNV, St. Louis encephalitis virus, and the California encephalitis group of viruses. In WNV epidemics, avian deaths may serve as sentinel infections for subsequent human disease. A history of tick exposure or travel or residence in the appropriate geographic area should suggest the possibility of Colorado tick fever virus or Powassan virus infection, although nonviral tick-borne diseases, including RMSF and Lyme neuroborreliosis, may present similarly. Arbovirus meningoencephalitis is typically associated with a CSF lymphocytic pleocytosis, normal glucose concentration, and normal or mildly elevated protein concentration. However, ~45% of patients

1	Arbovirus meningoencephalitis is typically associated with a CSF lymphocytic pleocytosis, normal glucose concentration, and normal or mildly elevated protein concentration. However, ~45% of patients with WNV meningoencephalitis have CSF neutrophilia, which can persist for a week or more. The rarity of hypoglycorrhachia in WNV infection, the absence of positive Gram’s stains, and the negative cultures help distinguish these patients from those with bacterial meningitis. Definitive diagnosis of arboviral meningoencephalitis is based on demonstration of viral-specific IgM in CSF or seroconversion. The prevalence of CSF IgM increases progressively during the first week after infection, peaking at >80% in patients with neuroinvasive disease; as a result, repeat studies may be needed when disease suspicion is high and an early study is negative. CSF PCR tests are available for some viruses in selected diagnostic laboratories and at the Centers for Disease Control and Prevention (CDC), but

1	suspicion is high and an early study is negative. CSF PCR tests are available for some viruses in selected diagnostic laboratories and at the Centers for Disease Control and Prevention (CDC), but in the case of WNV, sensitivity (~70%) of CSF PCR is less than that of CSF serology. WNV CSF PCR may be useful in immunocompromised patients who may have absent or reduced antibody responses.

1	HSV meningitis (Chap. 216) has been increasingly recognized as a major cause of viral meningitis in adults, and overall, it is probably second in importance to enteroviruses as a cause of viral meningitis, accounting for 5% of total cases overall and undoubtedly a higher frequency of those cases occurring in adults and/or outside of the summer-fall period when enterovirus infections are increasingly common. In adults, the majority of cases of uncomplicated meningitis are due to HSV-2, whereas HSV-1 is responsible for 90% of cases of HSV encephalitis. HSV meningitis occurs in ~25–35% of women and ~10–15% of men at the time of an initial (primary) episode of genital herpes. Of these patients, 20% go on to have recurrent attacks of meningitis. Diagnosis of HSV meningitis is usually by HSV CSF PCR because cultures may be negative, especially in patients with recurrent meningitis. Demonstration of intrathecal synthesis of HSV-specific antibody may also be useful in diagnosis, although

1	CSF PCR because cultures may be negative, especially in patients with recurrent meningitis. Demonstration of intrathecal synthesis of HSV-specific antibody may also be useful in diagnosis, although antibody tests are less sensitive and less specific than PCR and may not become positive until after the first week of infection. Although a history of or the presence of HSV genital lesions is an important diagnostic clue, many patients with HSV meningitis give no history and have no evidence of active genital herpes at the time of presentation. Most cases of recurrent viral or “aseptic” meningitis, including cases previously diagnosed as Mollaret’s meningitis, are due to HSV.

1	VZV meningitis should be suspected in the presence of concurrent chickenpox or shingles. However, it is important to recognize that VZV is being increasingly identified as an important cause of both meningitis and encephalitis in patients without rash. The frequency of VZV as a cause of meningitis is extremely variable, ranging from as low as 3% to as high as 20% in different series. Diagnosis is usually based on CSF PCR, although the sensitivity of this test may not be as high as for the other herpesviruses. VZV serologic studies complement PCR testing, and the diagnosis of VZV CNS infection can be made by the demonstration of VZV-specific intrathecal antibody synthesis and/or the presence of VZV CSF IgM antibodies, or by positive CSF cultures.

1	EBV infections may also produce aseptic meningitis, with or without associated infectious mononucleosis. The presence of atypical lymphocytes in the CSF or peripheral blood is suggestive of EBV infection but may occasionally be seen with other viral infections. EBV is almost never cultured from CSF. Serum and CSF serology can help establish the presence of acute infection, which is characterized by IgM viral capsid antibodies (VCAs), antibodies to early antigens (EAs), and the absence of antibodies to EBV-associated nuclear antigen (EBNA). CSF PCR is another important diagnostic test, although false-positive results may reflect viral reactivation associated with other infectious or inflammatory processes or the presence of latent viral DNA in lymphocytes recruited due to other inflammatory conditions. HIV meningitis should be suspected in any patient presenting with a viral meningitis with known or suspected risk factors for HIV infection.

1	HIV meningitis should be suspected in any patient presenting with a viral meningitis with known or suspected risk factors for HIV infection. Meningitis may occur following primary infection with HIV in 5–10% of cases and less commonly at later stages of illness. Cranial nerve palsies, most commonly involving cranial nerves V, VII, or VIII, are more common in HIV meningitis than in other viral infections. Diagnosis can be confirmed by detection of HIV genome in blood or CSF. Seroconversion may be delayed, and patients with negative HIV serologies who are suspected of having HIV meningitis should be monitored for delayed seroconversion. For further discussion of HIV infection, see Chap. 226.

1	Mumps (Chap. 231e) should be considered when meningitis occurs in the late winter or early spring, especially in males (male-to-female ratio 3:1). With the widespread use of the live attenuated mumps vaccine in the United States since 1967, the incidence of mumps meningitis has fallen by >95%; however, mumps remains a potential source of infection in nonimmunized individuals and populations. Rare cases (10–100:100,000 vaccinated individuals) of vaccine-associated mumps meningitis have been described, with onset typically 2–4 weeks after vaccination. The presence of parotitis, orchitis, oophoritis, pancreatitis, or elevations in serum lipase and amylase is suggestive of mumps meningitis; however, their absence does not exclude the diagnosis. Clinical meningitis was previously estimated to occur in 10–30% of patients with mumps parotitis; however, in a recent U.S. outbreak of nearly 2600 cases of mumps, only 11 cases of meningitis were identified, suggesting the incidence may be lower

1	occur in 10–30% of patients with mumps parotitis; however, in a recent U.S. outbreak of nearly 2600 cases of mumps, only 11 cases of meningitis were identified, suggesting the incidence may be lower than previously suspected. Mumps infection confers lifelong immunity, so a documented history of previous infection excludes this diagnosis. Patients with meningitis have a CSF pleocytosis that can exceed 1000 cells/μL in 25%. Lymphocytes predominate in 75%, although CSF neutrophilia occurs in 25%. Hypoglycorrhachia, occurs in 10–30% of patients and may be a clue to the diagnosis when present. Diagnosis is typically made by culture of virus from CSF or by detecting IgM antibodies or seroconversion. CSF PCR is available in some diagnostic and research laboratories.

1	LCMV infection (Chap. 233) should be considered when aseptic meningitis occurs in the late fall or winter and in individuals with a history of exposure to house mice (Mus musculus), pet or laboratory rodents (e.g., hamsters, rats, mice), or their excreta. Some patients have an associated rash, pulmonary infiltrates, alopecia, parotitis, orchitis, or myopericarditis. Laboratory clues to the diagnosis of LCMV, in addition to the clinical findings noted above, may include the presence of leukopenia, thrombocytopenia, or abnormal liver function tests. Some cases present with a marked CSF pleocytosis (>1000 cells/μL) and hypoglycorrhachia (<30%). Diagnosis is based on serology and/or culture of virus from CSF.

1	Treatment of almost all cases of viral meningitis is primarily symptomatic and includes use of analgesics, antipyretics, and antiemetics. Fluid and electrolyte status should be monitored. Patients with suspected bacterial meningitis should receive appropriate empirical therapy pending culture results (see above). Hospitalization may not be required in immunocompetent patients with presumed viral meningitis and no focal signs or symptoms, no significant alteration in consciousness, and a classic CSF profile (lymphocytic pleocytosis, normal glucose, negative Gram’s stain) if adequate provision for monitoring at home and medical follow-up can be ensured. Immunocompromised patients; patients with significant alteration in consciousness, seizures, or the presence of focal signs and symptoms suggesting the possibility of encephalitis or parenchymal brain involvement; and patients who have an atypical CSF profile should be hospitalized. Oral or intravenous acyclovir may be of benefit in

1	suggesting the possibility of encephalitis or parenchymal brain involvement; and patients who have an atypical CSF profile should be hospitalized. Oral or intravenous acyclovir may be of benefit in patients with meningitis caused by HSV-1 or -2 and in cases of severe EBV or VZV infection. Data concerning treatment of HSV, EBV, and VZV meningitis are extremely limited. Seriously ill patients should probably receive intravenous acyclovir (15–30 mg/kg per day in three divided doses), which can be followed by an oral drug such as acyclovir (800 mg five times daily), famciclovir (500 mg tid), or valacyclovir (1000 mg tid) for a total course of 7–14 days. Patients who are less ill can be treated with oral drugs alone. Patients with 893 HIV meningitis should receive highly active antiretroviral therapy (Chap. 226). There is no specific therapy of proven benefit for patients with arboviral encephalitis, including that caused by WNV.

1	Patients with viral meningitis who are known to have deficient humoral immunity (e.g., X-linked agammaglobulinemia) and who are not already receiving either intramuscular gamma globulin or intravenous immunoglobulin (IVIg) should be treated with these agents. Intraventricular administration of immunoglobulin through an Ommaya reservoir has been tried in some patients with chronic enteroviral meningitis who have not responded to intramuscular or intravenous immunoglobulin.

1	Vaccination is an effective method of preventing the development of meningitis and other neurologic complications associated with poliovirus, mumps, measles, rubella, and varicella infection. A live attenuated VZV vaccine (Varivax) is available in the United States. Clinical studies indicate an effectiveness rate of 70–90% for this vaccine, but a booster may be required after ~10 years to maintain immunity. A related vaccine (Zostavax) is recommended for prevention of herpes zoster (shingles) in adults over the age of 60. An inactivated varicella vaccine is available for transplant recipients and others for whom live viral vaccines are contraindicated

1	In adults, the prognosis for full recovery from viral meningitis is excellent. Rare patients complain of persisting headache, mild mental impairment, incoordination, or generalized asthenia for weeks to months. The outcome in infants and neonates (<1 year) is less certain; intellectual impairment, learning disabilities, hearing loss, and other lasting sequelae have been reported in some studies. In contrast to viral meningitis, where the infectious process and associated inflammatory response are limited largely to the meninges, in encephalitis the brain parenchyma is also involved. Many patients with encephalitis also have evidence of associated meningitis (meningoencephalitis) and, in some cases, involvement of the spinal cord or nerve roots (encephalomyelitis, encephalomyeloradiculitis).

1	In addition to the acute febrile illness with evidence of meningeal involvement characteristic of meningitis, the patient with encephalitis commonly has an altered level of consciousness (confusion, behavioral abnormalities), or a depressed level of consciousness ranging from mild lethargy to coma, and evidence of either focal or diffuse neurologic signs and symptoms. Patients with encephalitis may have hallucinations, agitation, personality change, behavioral disorders, and, at times, a frankly psychotic state. Focal or generalized seizures occur in many patients with encephalitis. Virtually every possible type of focal neurologic disturbance has been reported in viral encephalitis; the signs and symptoms reflect the sites of infection and inflammation. The most commonly encountered focal findings are aphasia, ataxia, upper or lower motor neuron patterns of weakness, involuntary movements (e.g., myoclonic jerks, tremor), and cranial nerve deficits (e.g., ocular palsies, facial

1	focal findings are aphasia, ataxia, upper or lower motor neuron patterns of weakness, involuntary movements (e.g., myoclonic jerks, tremor), and cranial nerve deficits (e.g., ocular palsies, facial weakness). Involvement of the hypothalamic-pituitary axis may result in temperature dysregulation, diabetes insipidus, or the development of the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Even though neurotropic viruses typically cause pathologic injury in distinct regions of the CNS, variations in clinical presentations make it impossible to reliably establish the etiology of a specific case of encephalitis on clinical grounds alone (see “Differential Diagnosis,” below).

1	In the United States, there are an estimated ~20,000 cases of encephalitis per year, although the actual number of cases is likely to be significantly larger. Despite comprehensive diagnostic efforts, the

1	Meningitis, Encephalitis, Brain Abscess, and Empyema 894 majority of cases of acute encephalitis of suspected viral etiology remain of unknown cause. Hundreds of viruses are capable of causing encephalitis, although only a limited subset is responsible for most cases in which a specific cause is identified (Table 164-4). The most commonly identified viruses causing sporadic cases of acute encephalitis in immunocompetent adults are herpesviruses (HSV, VZV, EBV). Epidemics of encephalitis are caused by arboviruses, which belong to several different viral taxonomic groups including Alphaviruses (e.g., EEE virus, western equine encephalitis virus), Flaviviruses (e.g., WNV, St. Louis encephalitis virus, Japanese encephalitis virus, Powassan virus), and Bunyaviruses (e.g., California encephalitis virus serogroup, La Crosse virus). Historically, the largest number of cases of arbovirus encephalitis in the United States has been due to St. Louis encephalitis virus and the California

1	virus serogroup, La Crosse virus). Historically, the largest number of cases of arbovirus encephalitis in the United States has been due to St. Louis encephalitis virus and the California encephalitis virus serogroup. However, since 2002, WNV has been responsible for the majority of arbovirus meningitis and encephalitis cases in the United States. WNV caused 2873 confirmed cases of neuroinvasive disease (encephalitis, meningitis, or myelitis) in 2012 with 286 deaths. States reporting >200 cases included Texas (1868 cases), California (479), Louisiana (335), Illinois (290), Mississippi (249), South Dakota (203), and Michigan (202). In 2013, there were 1140 neuroinvasive cases with 100 deaths. States reporting >100 cases included California (357 cases), Colorado (315), Nebraska (213), Texas (157), South Dakota (148), North Dakota (123), and Illinois (106). It is important to recognize that WNV epidemics are unpredictable and that cases have occurred in every state in the continental

1	(157), South Dakota (148), North Dakota (123), and Illinois (106). It is important to recognize that WNV epidemics are unpredictable and that cases have occurred in every state in the continental United States. New causes of viral CNS infections are constantly appearing, as evidenced by the outbreak of cases of encephalitis in Southeast Asia caused by Nipah virus, a newly identified member of the Paramyxoviridae family; of meningitis in Europe caused by Toscana virus, an arbovirus belonging to the Bunyavirus family; and of neurologic disorders associated with major epidemics of Chikungunya virus, a togavirus, in Africa, India, and Southeast Asia. Parechoviruses including human parechovirus 3 (HPeV3), members of the Picornavirus family, have recently been reported as causes of fever, sepsis, and meningitis in infants (age <3 months) in the United States and abroad.

1	LABORATORY DIAGNOSIS CSF Examination CSF examination should be performed in all patients with suspected viral encephalitis unless contraindicated by the presence of severely increased ICP. Ideally at least 20 mL should be collected with 5–10 mL stored frozen for later studies as needed. The characteristic CSF profile is indistinguishable from that of viral meningitis and typically consists of a lymphocytic pleocytosis, a mildly elevated protein concentration, and a normal glucose concentration. A CSF pleocytosis (>5 cells/μL) occurs in >95% of immunocompetent patients with documented viral encephalitis. In rare cases, a pleocytosis may be absent on the initial LP but present on subsequent LPs. Patients who are severely immunocompromised by HIV infection, glucocorticoid or other immunosuppressant drugs, chemotherapy, or lymphoreticular malignancies may fail to mount a CSF inflammatory response. CSF cell counts exceed 500/μL in only about 10% of patients with encephalitis. Infections

1	drugs, chemotherapy, or lymphoreticular malignancies may fail to mount a CSF inflammatory response. CSF cell counts exceed 500/μL in only about 10% of patients with encephalitis. Infections with certain arboviruses (e.g., EEE virus or California encephalitis virus), mumps, and LCMV may occasionally result in cell counts >1000/μL, but this degree of pleocytosis should suggest the possibility of nonviral infections or other inflammatory processes. Atypical lymphocytes in the CSF may be seen in EBV infection and less commonly with other viruses, including CMV, HSV, and enteroviruses. Increased numbers of plasmacytoid or Mollaret-like large mononuclear cells have been reported in WNV encephalitis. Polymorphonuclear pleocytosis occurs in ~45% of patients with WNV encephalitis and is also a common feature in CMV myeloradiculitis in immunocompromised patients. Large numbers of CSF PMNs may be present in patients with encephalitis due to EEE virus, echovirus 9, and, more rarely, other

1	a common feature in CMV myeloradiculitis in immunocompromised patients. Large numbers of CSF PMNs may be present in patients with encephalitis due to EEE virus, echovirus 9, and, more rarely, other enteroviruses. However, persisting CSF neutrophilia should prompt consideration of bacterial infection, leptospirosis, amebic infection, and noninfectious processes such as acute hemorrhagic leukoencephalitis. About 20% of patients with encephalitis will have a significant number of red blood cells (>500/μL) in the CSF in a nontraumatic tap. The pathologic correlate of this finding may be a hemorrhagic encephalitis of the type seen with HSV; however, CSF red blood cells occur with similar frequency and in similar numbers in patients with nonherpetic focal encephalitides. A decreased CSF glucose concentration is distinctly unusual in viral encephalitis and should suggest the possibility of bacterial, fungal, tuberculous, parasitic, leptospiral, syphilitic, sarcoid, or neoplastic meningitis.

1	concentration is distinctly unusual in viral encephalitis and should suggest the possibility of bacterial, fungal, tuberculous, parasitic, leptospiral, syphilitic, sarcoid, or neoplastic meningitis. Rare patients with mumps, LCMV, or advanced HSV encephalitis and many patients with CMV myeloradiculitis have low CSF glucose concentrations.

1	CSF PCR has become the primary diagnostic test for CNS infections caused by CMV, EBV, HHV-6, and enteroviruses (see “Viral Meningitis,” above). In the case of VZV CNS infection, CSF PCR and detection of virus-specific IgM or intrathecal antibody synthesis both provide important aids to diagnosis. The sensitivity and specificity of CSF PCRs vary with the virus being tested. The sensitivity (~96%) and specificity (~99%) of HSV CSF PCR are equivalent to or exceed those of brain biopsy. It is important to recognize that HSV CSF PCR results need to be interpreted after considering the likelihood of disease in the patient being tested, the timing of the test in relationship to onset of symptoms, and the prior use of antiviral therapy. A negative HSV CSF PCR test performed by a qualified laboratory at the appropriate time during illness in a patient with a high likelihood of HSV encephalitis based on clinical and laboratory abnormalities significantly reduces the likelihood of HSV

1	laboratory at the appropriate time during illness in a patient with a high likelihood of HSV encephalitis based on clinical and laboratory abnormalities significantly reduces the likelihood of HSV encephalitis but does not exclude it. For example, in a patient with a pretest probability of 35% of having HSV encephalitis, a negative HSV CSF PCR reduces the posttest probability to ~2%, and for a patient with a pretest probability of 60%, a negative test reduces the posttest probability to ~6%. In both situations, a positive test makes the diagnosis almost certain (98–99%). There have been several recent reports of initially negative HSV CSF PCR tests that were obtained early (≤72 h) following symptom onset and that became positive when repeated 1–3 days later. The frequency of positive HSV CSF PCRs in patients with herpes encephalitis also decreases as a function of the duration of illness, with only ~20% of cases remaining positive after ≥14 days. PCR results are generally not affected

1	PCRs in patients with herpes encephalitis also decreases as a function of the duration of illness, with only ~20% of cases remaining positive after ≥14 days. PCR results are generally not affected by ≤1 week of antiviral therapy. In one study, 98% of CSF specimens remained PCR-positive during the first week of initiation of antiviral therapy, but the numbers fell to ~50% by 8–14 days and to ~21% by >15 days after initiation of antiviral therapy.

1	The sensitivity and specificity of CSF PCR tests for viruses other than HSV have not been definitively characterized. Enteroviral (EV) CSF PCR appears to have a sensitivity and specificity of >95%. EV PCR sensitivity for EV71 may be considerably lower (~30% in some reports). Parechoviruses are also not detected by standard EV RT-PCRs. The specificity of EBV CSF PCR has not been established. Positive EBV CSF PCRs associated with positive tests for other pathogens have been reported and may reflect reactivation of EBV latent in lymphocytes that enter the CNS as a result of an unrelated infectious or inflammatory process. In patients with CNS infection due to VZV, CSF antibody and PCR studies should be considered complementary, because patients may have evidence of intrathecal synthesis of VZV-specific antibodies and negative CSF PCRs. In the case of WNV infection, CSF PCR appears to be less sensitive (~70% sensitivity) than detection of WNV-specific CSF IgM, although PCR testing remains

1	antibodies and negative CSF PCRs. In the case of WNV infection, CSF PCR appears to be less sensitive (~70% sensitivity) than detection of WNV-specific CSF IgM, although PCR testing remains useful in immunocompromised patients who may not mount an effective anti-WNV antibody response.

1	CSF Culture CSF culture is generally of limited utility in the diagnosis of acute viral encephalitis. Culture may be insensitive (e.g., >95% of patients with HSV encephalitis have negative CSF cultures as do virtually all patients with EBV-associated CNS disease) and often takes too long to significantly affect immediate therapy.

1	Serologic Studies and Antigen Detection The basic approach to the serodiagnosis of viral encephalitis is identical to that discussed earlier for viral meningitis. Demonstration of WNV IgM antibodies is diagnostic of WNV encephalitis because IgM antibodies do not cross the blood-brain barrier, and their presence in CSF is therefore indicative of intrathecal synthesis. Timing of antibody collection may be important because the rate of CSF WNV IgM seropositivity increases by ~10% per day during the first week after illness onset, reaching 80% or higher on day 7 after symptom onset. In patients with HSV encephalitis, both antibodies to HSV-1 glycoproteins and glycoprotein antigens have been detected in the CSF. Optimal detection of both HSV antibodies and antigen typically occurs after the first week of illness, limiting the utility of these tests in acute diagnosis. Nonetheless, HSV CSF antibody testing is of value in selected patients whose illness is >1 week in duration and who are CSF

1	week of illness, limiting the utility of these tests in acute diagnosis. Nonetheless, HSV CSF antibody testing is of value in selected patients whose illness is >1 week in duration and who are CSF PCR–negative for HSV. In the case of VZV infection, CSF antibody tests may be positive when PCR fails to detect viral DNA, and both tests should be considered complementary rather than mutually exclusive.

1	MRI, CT, and EEG Patients with suspected encephalitis almost invariably undergo neuroimaging studies and often EEG. These tests help identify or exclude alternative diagnoses and assist in the differentiation between a focal, as opposed to a diffuse, encephalitic process. Focal findings in a patient with encephalitis should always raise the possibility of HSV encephalitis. Examples of focal findings include: (1) areas of increased signal intensity in the frontotemporal, cingulate, or insular regions of the brain on T2-weighted, FLAIR, or diffusion-weighted MRI (Fig. 164-3); (2) focal areas of low absorption, mass effect, and contrast enhancement on CT; or (3) periodic focal temporal lobe spikes on a background of slow or low-amplitude (“flattened”) activity on EEG. Approximately 10% of patients with PCR-documented HSV encephalitis will have a normal MRI, although nearly 80% will have abnormalities in the temporal lobe, and an additional 10% in extratemporal regions. The lesions are

1	patients with PCR-documented HSV encephalitis will have a normal MRI, although nearly 80% will have abnormalities in the temporal lobe, and an additional 10% in extratemporal regions. The lesions are typically hyperintense on T2-weighted images. The addition of FLAIR and diffusion-weighted images to the standard MRI sequences enhances sensitivity. Children with HSV encephalitis may have atypical patterns of MRI lesions and often show involvement of brain regions outside the frontotemporal areas. CT is less sensitive than MRI and is normal in up to 20–35% of patients. EEG abnormalities occur in >75% of PCR-documented cases of HSV encephalitis; they typically involve the temporal lobes but are often nonspecific. Some

1	FIGURE 164-3 Coronal fluid-attenuated inversion recovery (FLAIR) magnetic resonance image from a patient with herpes simplex encephalitis. Note the area of increased signal in the right temporal lobe (left side of image) confined predominantly to the gray matter. This patient had predominantly unilateral disease; bilateral lesions are more common but may be quite asymmetric in their intensity. patients with HSV encephalitis have a distinctive EEG pattern consist-895 ing of periodic, stereotyped, sharp-and-slow complexes originating in one or both temporal lobes and repeating at regular intervals of 2–3 s. The periodic complexes are typically noted between days 2 and 15 of the illness and are present in two-thirds of pathologically proven cases of HSV encephalitis.

1	Significant MRI abnormalities are found in only approximately two-thirds of patients with WNV encephalitis, a frequency less than that with HSV encephalitis. When present, abnormalities often involve deep brain structures, including the thalamus, basal ganglia, and brainstem, rather than the cortex and may only be apparent on FLAIR images. EEGs in patients with WNV encephalitis typically show generalized slowing that may be more anteriorly prominent rather than the temporally predominant pattern of sharp or periodic discharges more characteristic of HSV encephalitis. Patients with VZV encephalitis may show multifocal areas of hemorrhagic and ischemic infarction, reflecting the tendency of this virus to produce a CNS vasculopathy rather than a true encephalitis. Immunocompromised adult patients with CMV often have enlarged ventricles with areas of increased T2 signal on MRI outlining the ventricles and subependymal enhancement on T1-weighted postcontrast images. Table 164-5 highlights

1	with CMV often have enlarged ventricles with areas of increased T2 signal on MRI outlining the ventricles and subependymal enhancement on T1-weighted postcontrast images. Table 164-5 highlights specific diagnostic test results in encephalitis that can be useful in clinical decision making.

1	Brain Biopsy Brain biopsy is now generally reserved for patients in whom CSF PCR studies fail to lead to a specific diagnosis, who have focal abnormalities on MRI, and who continue to show progressive clinical deterioration despite treatment with acyclovir and supportive therapy. Infection by a variety of other organisms can mimic viral encephalitis. In studies of biopsy-proven HSV encephalitis, common infectious uSE of DIAgnoSTIC TESTS In EnCEPHALITIS The best test for WNV encephalitis is the CSF IgM antibody test. The prevalence of positive CSF IgM tests increases by about 10% per day after illness onset and reaches 70–80% by the end of the first week. Serum WNV IgM can provide evidence for recent WNV infection, but in the absence of other findings does not establish the diagnosis of neuroinvasive disease (meningitis, encephalitis, acute flaccid paralysis).

1	Approximately 80% of patients with proven HSV encephalitis have MRI abnormalities involving the temporal lobes. This percentage likely increases to >90% when FLAIR and diffusion-weighted MRI sequences are also used. The absence of temporal lobe lesions on MRI reduces the likelihood of HSV encephalitis and should prompt consideration of other diagnostic possibilities. The CSF HSV PCR test may be negative in the first 72 h of symptoms of HSV encephalitis. A repeat study should be considered in patients with an initial early negative PCR in whom diagnostic suspicion of HSV encephalitis remains high and no alternative diagnosis has yet been established.

1	Detection of intrathecal synthesis (increased CSF/serum HSV antibody ratio corrected for breakdown of the blood-brain barrier) of HSV-specific antibody may be useful in diagnosis of HSV encephalitis in patients in whom only late (>1 week after onset) CSF specimens are available and PCR studies are negative. Serum serology alone is of no value in diagnosis of HSV encephalitis due to the high seroprevalence rate in the general population. Negative CSF viral cultures are of no value in excluding the diagnosis of HSV or EBV encephalitis. VZV CSF IgM antibodies may be present in patients with a negative VZV CSF PCR. Both tests should be performed in patients with suspected VZV CNS disease.

1	VZV CSF IgM antibodies may be present in patients with a negative VZV CSF PCR. Both tests should be performed in patients with suspected VZV CNS disease. The specificity of EBV CSF PCR for diagnosis of CNS infection is unknown. Positive tests may occur in patients with a CSF pleocytosis due to other causes. Detection of EBV CSF IgM or intrathecal synthesis of antibody to VCA supports the diagnosis of EBV encephalitis. Serologic studies consistent with acute EBV infection (e.g., IgM VCA, presence of antibodies against EA but not against EBNA) can help support the diagnosis.

1	Abbreviations: CNS, central nervous system; CSF, cerebrospinal fluid; DWI, diffusion-weighted imaging; EA, early antigen; EBNA, EBV-associated nuclear antigen; EBV, Epstein-Barr virus; FLAIR, fluid-attenuated inversion recovery; HSV, herpes simplex virus; IgM, immunoglobulin M; MRI, magnetic resonance imaging; PCR, polymerase chain reaction; VCA, viral capsid antibody; VZV, varicella-zoster virus; WNV, West Nile virus.

1	Meningitis, Encephalitis, Brain Abscess, and Empyema 896 mimics of focal viral encephalitis included mycobacteria, fungi, rickettsiae, Listeria, Mycoplasma, and other bacteria (including Bartonella sp.). Autoimmune causes of encephalitis, including those associated with antibodies against N-methyl-d-aspartate (NMDA) receptor, voltage-gated potassium channels (VGKC), α-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA), and γ-aminobutyric acid (GABA) receptors, and GAD-65, have been increasingly recognized as causes of encephalitis that can mimic that caused by viral infection. In most cases, diagnosis is made by detection of the specific autoantibodies in serum and/or CSF. NMDA receptor antibodies have recently been reported in some patients with HSE encephalitis, and their presence should not exclude appropriate testing and treatment for HSV encephalitis. Autoimmune encephalitis may also be associated with specific cancers (paraneoplastic) and onconeuronal antibodies (e.g.,

1	should not exclude appropriate testing and treatment for HSV encephalitis. Autoimmune encephalitis may also be associated with specific cancers (paraneoplastic) and onconeuronal antibodies (e.g., anti-Hu, Yo, Ma2, amphiphysin, CRMP5, CV2) (Chap. 122). Subacute or chronic forms of encephalitis may occur in association with auto-antibodies against thyroglobulin and thyroperoxidase (Hashimoto’s encephalopathy) and with prion diseases. Infection caused by the ameba Naegleria fowleri can also cause acute meningoencephalitis (primary amebic meningoencephalitis), whereas that caused by Acanthamoeba and Balamuthia more typically produces subacute or chronic granulomatous amebic meningoencephalitis. Naegleria thrive in warm, iron-rich pools of water, including those found in drains, canals, and both natural and human-made outdoor pools. Infection has typically occurred in immunocompetent children with a history of swimming in potentially infected water. The CSF, in contrast to the typical

1	both natural and human-made outdoor pools. Infection has typically occurred in immunocompetent children with a history of swimming in potentially infected water. The CSF, in contrast to the typical profile seen in viral encephalitis, often resembles that of bacterial meningitis with a neutrophilic pleocytosis and hypoglycorrhachia. Motile trophozoites can be seen in a wet mount of warm, fresh CSF. There have been an increasing number of cases of Balamuthia mandrillaris amebic encephalitis mimicking acute viral encephalitis in children and immunocompetent adults. This organism has also been associated with encephalitis in recipients of transplanted organs from a donor with unrecognized infection. No effective treatment has been identified, and mortality approaches 100%. Encephalitis can be caused by the raccoon pinworm Baylisascaris procyonis. Clues to the diagnosis include a history of raccoon exposure, especially of playing in or eating dirt potentially contaminated with raccoon

1	be caused by the raccoon pinworm Baylisascaris procyonis. Clues to the diagnosis include a history of raccoon exposure, especially of playing in or eating dirt potentially contaminated with raccoon feces. Most patients are children, and many have an associated eosinophilia. Once nonviral causes of encephalitis have been excluded, the major diagnostic challenge is to distinguish HSV from other viruses that cause encephalitis. This distinction is particularly important because in virtually every other instance the therapy is supportive, whereas specific and effective antiviral therapy is available for HSV, and its efficacy is enhanced when it is instituted early in the course of infection. HSV encephalitis should be considered when clinical features suggesting involvement of the inferomedial frontotemporal regions of the brain are present, including prominent olfactory or gustatory hallucinations, anosmia, unusual or bizarre behavior or personality alterations, or memory disturbance.

1	frontotemporal regions of the brain are present, including prominent olfactory or gustatory hallucinations, anosmia, unusual or bizarre behavior or personality alterations, or memory disturbance. HSV encephalitis should always be suspected in patients with signs and symptoms consistent with acute encephalitis with focal findings on clinical examination, neuroimaging studies, or EEG. The diagnostic procedure of choice in these patients is CSF PCR analysis for HSV. A positive CSF PCR establishes the diagnosis, and a negative test dramatically reduces the likelihood of HSV encephalitis (see above). The anatomic distribution of lesions may provide an additional clue to diagnosis. Patients with rapidly progressive encephalitis and prominent brainstem signs, symptoms, or neuroimaging abnormalities may be infected by flaviviruses (WNV, St. Louis encephalitis virus, Japanese encephalitis virus), HSV, rabies, or

1	L. monocytogenes. Significant involvement of deep gray matter structures, including the basal ganglia and thalamus, should also suggest possible flavivirus infection. These patients may present clinically with prominent movement disorders (tremor, myoclonus) or parkinsonian features. Patients with WNV infection can also present with a polio-myelitis-like acute flaccid paralysis, as can patients infected with EV71 and, less commonly, other enteroviruses. Acute flaccid paralysis is characterized by the acute onset of a lower motor neuron type of weakness with flaccid tone, reduced or absent reflexes, and relatively preserved sensation. The complete eradication of polio remains an ongoing challenge despite a continuing World Health Organization poliovirus elimination campaign. Three hundred forty-one cases of polio (almost all due to serotype 1) have been reported in 2013 from eight countries (Somalia 183 cases, Pakistan 63, Nigeria 51, Kenya 14, Syria 13, Afghanistan 9, Ethiopia 6, and

1	forty-one cases of polio (almost all due to serotype 1) have been reported in 2013 from eight countries (Somalia 183 cases, Pakistan 63, Nigeria 51, Kenya 14, Syria 13, Afghanistan 9, Ethiopia 6, and Cameroon 2). There have been small outbreaks of poliomyelitis associated with vaccine strains of virus that have reverted to virulence through mutation or recombination with circulating wild-type enteroviruses in Hispaniola, China, the Philippines, Indonesia, Nigeria, and Madagascar.

1	Epidemiologic factors may provide important clues to the diagnosis of viral meningitis or encephalitis. Particular attention should be paid to the season of the year; the geographic location and travel history; and possible exposure to animal bites or scratches, rodents, and ticks. Although transmission from the bite of an infected dog remains the most common cause of rabies worldwide, in the United States very few cases of dog rabies occur, and the most common risk factor is exposure to bats—although a clear history of a bite or scratch is often lacking. The classic clinical presentation of encephalitic (furious) rabies is fever, fluctuating consciousness, and autonomic hyperactivity. Phobic spasms of the larynx, pharynx, neck muscles, and diaphragm can be triggered by attempts to swallow water (hydrophobia) or by inspiration (aerophobia). Patients may also present with paralytic (dumb) rabies characterized by acute ascending paralysis. Rabies due to the bite of a bat has a different

1	water (hydrophobia) or by inspiration (aerophobia). Patients may also present with paralytic (dumb) rabies characterized by acute ascending paralysis. Rabies due to the bite of a bat has a different clinical presentation than classic rabies due to a dog or wolf bite. Patients present with focal neurologic deficits, myoclonus, seizures, and hallucinations; phobic spasms are not a typical feature. Patients with rabies have a CSF lymphocytic pleocytosis and may show areas of increased T2 signal abnormality in the brainstem, hippocampus, and hypothalamus. Diagnosis can be made by finding rabies virus antigen in brain tissue or in the neural innervation of hair follicles at the nape of the neck. PCR amplification of viral nucleic acid from CSF and saliva or tears may also enable diagnosis. Serology is frequently negative in both serum and CSF in the first week after onset of infection, which limits its acute diagnostic utility. No specific therapy is available, and cases are almost

1	Serology is frequently negative in both serum and CSF in the first week after onset of infection, which limits its acute diagnostic utility. No specific therapy is available, and cases are almost invariably fatal, with isolated survivors having devastating neurologic sequelae.

1	State public health authorities provide a valuable resource concerning isolation of particular agents in individual regions. Regular updates concerning the number, type, and distribution of cases of arboviral encephalitis can be found on the CDC and U.S. Geological Survey (USGS) websites (http://www.cdc.gov and http://diseasemaps.usgs.gov).

1	Specific antiviral therapy should be initiated when appropriate. Vital functions, including respiration and blood pressure, should be monitored continuously and supported as required. In the initial stages of encephalitis, many patients will require care in an intensive care unit. Basic management and supportive therapy should include careful monitoring of ICP, fluid restriction, avoidance of hypotonic intravenous solutions, and suppression of fever. Seizures should be treated with standard anticonvulsant regimens, and prophylactic therapy should be considered in view of the high frequency of seizures in severe cases of encephalitis. As with all seriously ill, immobilized patients with altered levels of consciousness, encephalitis patients are at risk for aspiration pneumonia, stasis ulcers and decubiti, contractures, deep venous thrombosis and its complications, and infections of indwelling lines and catheters.

1	Acyclovir is of benefit in the treatment of HSV and should be started empirically in patients with suspected viral encephalitis, especially if focal features are present, while awaiting viral diagnostic studies. Treatment should be discontinued in patients found not to have HSV encephalitis, with the possible exception of patients with severe encephalitis due to VZV or EBV. HSV, VZV, and EBV all encode an enzyme, deoxypyrimidine (thymidine) kinase, that phosphorylates acyclovir to produce acyclovir-5’-monophosphate.

1	Host cell enzymes then phosphorylate this compound to form a triphosphate derivative. It is the triphosphate that acts as an antiviral agent by inhibiting viral DNA polymerase and by causing premature termination of nascent viral DNA chains. The specificity of action depends on the fact that uninfected cells do not phosphorylate significant amounts of acyclovir to acyclovir-5’-monophosphate. A second level of specificity is provided by the fact that the acyclovir triphosphate is a more potent inhibitor of viral DNA polymerase than of the analogous host cell enzymes.

1	Adults should receive a dose of 10 mg/kg of acyclovir intravenously every 8 h (30 mg/kg per day total dose) for 14–21 days. CSF PCR can be repeated at the completion of this course, with PCR-positive patients receiving additional treatment, followed by a repeat CSF PCR test. Neonatal HSV CNS infection is less responsive to acyclovir therapy than HSV encephalitis in adults; it is recommended that neonates with HSV encephalitis receive 20 mg/kg of acyclovir every 8 h (60 mg/kg per day total dose) for a minimum of 21 days.

1	Prior to intravenous administration, acyclovir should be diluted to a concentration ≤7 mg/mL. (A 70-kg person would receive a dose of 700 mg, which would be diluted in a volume of 100 mL.) Each dose should be infused slowly over 1 h, rather than by rapid or bolus infusion, to minimize the risk of renal dysfunction. Care should be taken to avoid extravasation or intramuscular or subcutaneous administration. The alkaline pH of acyclovir can cause local inflammation and phlebitis (9%). Dose adjustment is required in patients with impaired renal glomerular filtration. Penetration into CSF is excellent, with average drug levels ~50% of serum levels. Complications of therapy include elevations in blood urea nitrogen and creatinine levels (5%), thrombocytopenia (6%), gastrointestinal toxicity (nausea, vomiting, diarrhea) (7%), and neurotoxicity (lethargy or obtundation, disorientation, confusion, agitation, hallucinations, tremors, seizures) (1%). Acyclovir resistance may be mediated by

1	(nausea, vomiting, diarrhea) (7%), and neurotoxicity (lethargy or obtundation, disorientation, confusion, agitation, hallucinations, tremors, seizures) (1%). Acyclovir resistance may be mediated by changes in either the viral deoxypyrimidine kinase or DNA polymerase. To date, acyclovir-resistant isolates have not been a significant clinical problem in immunocompetent individuals. However, there have been reports of clinically virulent acyclovir-resistant HSV isolates from sites outside the CNS in immunocompromised individuals, including those with AIDS.

1	Oral antiviral drugs with efficacy against HSV, VZV, and EBV, including acyclovir, famciclovir, and valacyclovir, have not been evaluated in the treatment of encephalitis either as primary therapy or as supplemental therapy following completion of a course of parenteral acyclovir. A recently completed National Institute of Allergy and Infectious Disease (NIAID)/National Institute of Neurological Disorders and Stroke–sponsored phase III trial of supplemental oral valacyclovir therapy (2 g tid for 3 months) following the initial 14to 21-day course of therapy with parenteral acyclovir (www.clinicaltrials.gov, identifier NCT00031486) was terminated early due to low enrollment. Although analysis was compromised due to low numbers, no differences were seen in the 12-month endpoints including dementia rating scale, mini-mental state exam, and Glasgow coma score in patients receiving valacyclovir versus placebo. The role of adjunctive intravenous glucocorticoids in treatment of HSV and VZV

1	dementia rating scale, mini-mental state exam, and Glasgow coma score in patients receiving valacyclovir versus placebo. The role of adjunctive intravenous glucocorticoids in treatment of HSV and VZV infection remains unclear, with most guidelines considering the existing supportive evidence weak and recommendation for possible use based on expert opinion only.

1	Ganciclovir and foscarnet, either alone or in combination, are often used in the treatment of CMV-related CNS infections, although their efficacy remains unproven. Cidofovir (see below) may provide an alternative in patients who fail to respond to ganciclovir and foscarnet, although data concerning its use in CMV CNS infections are extremely limited.

1	Ganciclovir is a synthetic nucleoside analogue of 2’-deoxyguanosine. The drug is preferentially phosphorylated by virus-induced cellular kinases. Ganciclovir triphosphate acts as a competitive inhibitor of the CMV DNA polymerase, and its incorporation into nascent viral DNA results in premature chain termination. Following intravenous administration, CSF concentrations of ganciclovir are 25–70% of coincident plasma levels. The usual dose for treatment of severe 897 neurologic illnesses is 5 mg/kg every 12 h given intravenously at a constant rate over 1 h. Induction therapy is followed by maintenance therapy of 5 mg/kg every day for an indefinite period. Induction therapy should be continued until patients show a decline in CSF pleocytosis and a reduction in CSF CMV DNA copy number on quantitative PCR testing (where available). Doses should be adjusted in patients with renal insufficiency. Treatment is often limited by the development of granulocytopenia and thrombocytopenia (20–25%),

1	PCR testing (where available). Doses should be adjusted in patients with renal insufficiency. Treatment is often limited by the development of granulocytopenia and thrombocytopenia (20–25%), which may require reduction in or discontinuation of therapy. Gastrointestinal side effects, including nausea, vomiting, diarrhea, and abdominal pain, occur in ~20% of patients. Some patients treated with ganciclovir for CMV retinitis have developed retinal detachment, but the causal relationship to ganciclovir treatment is unclear. Valganciclovir is an orally bioavailable prodrug that can generate high serum levels of ganciclovir, although studies of its efficacy in treating CMV CNS infections are limited.

1	Foscarnet is a pyrophosphate analogue that inhibits viral DNA polymerases by binding to the pyrophosphate-binding site. Following intravenous infusion, CSF concentrations range from 15 to 100% of coincident plasma levels. The usual dose for serious CMV-related neurologic illness is 60 mg/kg every 8 h administered by constant infusion over 1 h. Induction therapy for 14–21 days is followed by maintenance therapy (60–120 mg/kg per day). Induction therapy may need to be extended in patients who fail to show a decline in CSF pleocytosis and a reduction in CSF CMV DNA copy number on quantitative PCR tests (where available). Approximately one-third of patients develop renal impairment during treatment, which is reversible following discontinuation of therapy in most, but not all, cases. This is often associated with elevations in serum creatinine and proteinuria and is less frequent in patients who are adequately hydrated. Many patients experience fatigue and nausea. Reductions in serum

1	is often associated with elevations in serum creatinine and proteinuria and is less frequent in patients who are adequately hydrated. Many patients experience fatigue and nausea. Reductions in serum calcium, magnesium, and potassium occur in ~15% of patients and may be associated with tetany, cardiac rhythm disturbances, or seizures.

1	Cidofovir is a nucleotide analogue that is effective in treating CMV retinitis and equivalent to or better than ganciclovir in some experimental models of murine CMV encephalitis, although data concerning its efficacy in human CMV CNS disease are limited. The usual dose is 5 mg/kg intravenously once weekly for 2 weeks, then biweekly for two or more additional doses, depending on clinical response. Patients must be prehydrated with normal saline (e.g., 1 L over 1–2 h) prior to each dose and treated with probenecid (e.g., 1 g 3 h before cidofovir and 1 g 2 and 8 h after cidofovir). Nephrotoxicity is common; the dose should be reduced if renal function deteriorates.

1	Intravenous ribavirin (15–25 mg/kg per day in divided doses given every 8 h) has been reported to be of benefit in isolated cases of severe encephalitis due to California encephalitis (La Crosse) virus. Ribavirin might be of benefit for the rare patients, typically infants or young children, with severe adenovirus or rotavirus encephalitis and in patients with encephalitis due to LCMV or other arenaviruses. However, clinical trials are lacking. Hemolysis, with resulting anemia, has been the major side effect limiting therapy.

1	No specific antiviral therapy of proven efficacy is currently available for treatment of WNV encephalitis. Patients have been treated with a-interferon, ribavirin, an Israeli IVIg preparation that contains high-titer anti-WNV antibody (Omr-IgG-am) (www.clinicaltrials.gov, identifier NCT00069316 and 0068055), and humanized monoclonal antibodies directed against the viral envelope glycoprotein (www .clinicaltrials.gov, identifier NCT00927953 and 00515385). WNV chimeric vaccines, in which WNV envelope and premembrane proteins are inserted into the background of another flavivirus, are already undergoing human clinical testing and have been found to be both safe and immunogenic in healthy adults but have not yet been tested for disease prevention in humans (www.clinicaltrials .gov, identifier NCT00746798, 00442169, 00094718, and 00537147). Both chimeric and killed inactivated WNV vaccines have been found to be safe and effective in preventing equine WNV infection, and

1	Meningitis, Encephalitis, Brain Abscess, and Empyema 898 several effective flavivirus vaccines are already in human use, creating optimism that a safe and effective human WNV vaccine can also be developed.

1	There is considerable variation in the incidence and severity of sequelae in patients surviving viral encephalitis. In the case of EEE virus infection, nearly 80% of survivors have severe neurologic sequelae. At the other extreme are infections due to EBV, California encephalitis virus, and Venezuelan equine encephalitis virus, where severe sequelae are unusual. For example, approximately 5–15% of children infected with La Crosse virus have a residual seizure disorder, and 1% have persistent hemiparesis. Detailed information about sequelae in patients with HSV encephalitis treated with acyclovir is available from the NIAID-Collaborative Antiviral Study Group (CASG) trials. Of 32 acyclovir-treated patients, 26 survived (81%). Of the 26 survivors, 12 (46%) had no or only minor sequelae, 3 (12%) were moderately impaired (gainfully employed but not functioning at their previous level), and 11 (42%) were severely impaired (requiring continuous supportive care). The incidence and severity

1	(12%) were moderately impaired (gainfully employed but not functioning at their previous level), and 11 (42%) were severely impaired (requiring continuous supportive care). The incidence and severity of sequelae were directly related to the age of the patient and the level of consciousness at the time of initiation of therapy. Patients with severe neurologic impairment (Glasgow coma score 6) at initiation of therapy either died or survived with severe sequelae. Young patients (<30 years) with good neurologic function at initiation of therapy did substantially better (100% survival, 62% with no or mild sequelae) compared with their older counterparts (>30 years; 64% survival, 57% no or mild sequelae). Some recent studies using quantitative HSV CSF PCR tests indicate that clinical outcome following treatment also correlates with the amount of HSV DNA present in CSF at the time of presentation. Many patients with WNV infection have sequelae, including cognitive impairment; weakness; and

1	treatment also correlates with the amount of HSV DNA present in CSF at the time of presentation. Many patients with WNV infection have sequelae, including cognitive impairment; weakness; and hyperor hypokinetic movement disorders, including tremor, myoclonus, and parkinsonism. In a large longitudinal study of prognosis in 156 patients with WNV infection, the mean time to achieve recovery (defined as 95% of maximal predicted score on specific validated tests) was 112–148 days for fatigue, 121–175 days for physical function, 131–139 days for mood, and 302–455 days for mental function (the longer interval in each case representing patients with neuroinvasive disease).

1	Patients with subacute meningitis typically have an unrelenting headache, stiff neck, low-grade fever, and lethargy for days to several weeks before they present for evaluation. Cranial nerve abnormalities and night sweats may be present. This syndrome overlaps that of chronic meningitis, discussed in detail in Chap. 165. Common causative organisms include M. tuberculosis, C. neoformans,

1	H. capsulatum, C. immitis, and T. pallidum. Initial infection with M. tuberculosis is acquired by inhalation of aerosolized droplet nuclei. Tuberculous meningitis in adults does not develop acutely from hematogenous spread of tubercle bacilli to the meninges. Rather, millet seed–sized (miliary) tubercles form in the parenchyma of the brain during hematogenous dissemination of tubercle bacilli in the course of primary infection. These tubercles enlarge and are usually caseating. The propensity for a caseous lesion to produce meningitis is determined by its proximity to the subarachnoid space (SAS) and the rate at which fibrous encapsulation develops. Subependymal caseous foci cause meningitis via discharge of bacilli and tuberculous antigens into the SAS. Mycobacterial antigens produce an intense inflammatory reaction that leads to the production of a thick exudate that fills the basilar cisterns and surrounds the cranial nerves and major blood vessels at the base of the brain.

1	Fungal infections are typically acquired by the inhalation of airborne fungal spores. The initial pulmonary infection may be asymptomatic or present with fever, cough, sputum production, and chest pain. The pulmonary infection is often self-limited. A localized pulmonary fungal infection can then remain dormant in the lungs until there is an abnormality in cell-mediated immunity that allows the fungus to reactivate and disseminate to the CNS. The most common pathogen causing fungal meningitis is C. neoformans. This fungus is found worldwide in soil and bird excreta. H. capsulatum is endemic to the Ohio and Mississippi River valleys of the central United States and to parts of Central and South America. C. immitis is endemic to the desert areas of the southwest United States, northern Mexico, and Argentina.

1	Syphilis is a sexually transmitted disease that is manifest by the appearance of a painless chancre at the site of inoculation. T. pallidum invades the CNS early in the course of syphilis. Cranial nerves VII and VIII are most frequently involved.

1	The classic CSF abnormalities in tuberculous meningitis are as follows: (1) elevated opening pressure, (2) lymphocytic pleocytosis (10–500 cells/μL), (3) elevated protein concentration in the range of 1–5 g/L, and (4) decreased glucose concentration in the range of 1.1–2.2 mmol/L (20–40 mg/dL). The combination of unrelenting headache, stiff neck, fatigue, night sweats, and fever with a CSF lymphocytic pleocytosis and a mildly decreased glucose concentration is highly suspicious for tuberculous meningitis. The last tube of fluid collected at LP is the best tube to send for a smear for acid-fast bacilli (AFB). If there is a pellicle in the CSF or a cobweb-like clot on the surface of the fluid, AFB can best be demonstrated in a smear of the clot or pellicle. Positive smears are typically reported in only 10–40% of cases of tuberculous meningitis in adults. Cultures of CSF take 4–8 weeks to identify the organism and are positive in ~50% of adults. Culture remains the gold standard to make

1	in only 10–40% of cases of tuberculous meningitis in adults. Cultures of CSF take 4–8 weeks to identify the organism and are positive in ~50% of adults. Culture remains the gold standard to make the diagnosis of tuberculous meningitis. PCR for the detection of M. tuberculosis DNA should be sent on CSF if available, but the sensitivity and specificity on CSF have not been defined. The CDC recommends the use of nucleic acid amplification tests for the diagnosis of pulmonary tuberculosis.

1	The characteristic CSF abnormalities in fungal meningitis are a mononuclear or lymphocytic pleocytosis, an increased protein concentration, and a decreased glucose concentration. There may be eosinophils in the CSF in C. immitis meningitis. Large volumes of CSF are often required to demonstrate the organism on India ink smear or grow the organism in culture. If spinal fluid examined by LP on two separate occasions fails to yield an organism, CSF should be obtained by high-cervical or cisternal puncture.

1	The cryptococcal polysaccharide antigen test is a highly sensitive and specific test for cryptococcal meningitis. A reactive CSF cryptococcal antigen test establishes the diagnosis. The detection of the Histoplasma polysaccharide antigen in CSF establishes the diagnosis of a fungal meningitis but is not specific for meningitis due to H. capsulatum. It may be falsely positive in coccidioidal meningitis. The CSF complement fixation antibody test is reported to have a specificity of 100% and a sensitivity of 75% for coccidioidal meningitis.

1	The diagnosis of syphilitic meningitis is made when a reactive serum treponemal test (fluorescent treponemal antibody absorption test [FTA-ABS] or microhemagglutination assay–T. pallidum [MHA-TP]) is associated with a CSF lymphocytic or mononuclear pleocytosis and an elevated protein concentration, or when the CSF Venereal Disease Research Laboratory (VDRL) test is positive. A reactive CSF FTA-ABS is not definitive evidence of neurosyphilis. The CSF FTA-ABS can be falsely positive from blood contamination. A negative CSF VDRL does not rule out neurosyphilis. A negative CSF FTA-ABS or MHA-TP rules out neurosyphilis.

1	Empirical therapy of tuberculous meningitis is often initiated on the basis of a high index of suspicion without adequate laboratory support. Initial therapy is a combination of isoniazid (300 mg/d), rifampin (10 mg/kg per day), pyrazinamide (30 mg/kg per day in divided doses), ethambutol (15–25 mg/kg per day in divided doses), and pyridoxine (50 mg/d). When the antimicrobial sensitivity of the

1	M. tuberculosis isolate is known, ethambutol can be discontinued. If the clinical response is good, pyrazinamide can be discontinued after 8 weeks and isoniazid and rifampin continued alone for the next 6–12 months. A 6-month course of therapy is acceptable, but therapy should be prolonged for 9–12 months in patients who have an inadequate resolution of symptoms of meningitis or who have positive mycobacterial cultures of CSF during the course of therapy. Dexamethasone therapy is recommended for HIV-negative patients with tuberculous meningitis. The dose is 12–16 mg/d for 3 weeks, and then tapered over 3 weeks.

1	Meningitis due to C. neoformans in non-HIV, nontransplant patients is treated with induction therapy with amphotericin B (AmB) (0.7 mg/kg IV per day) plus flucytosine (100 mg/kg per day in four divided doses) for at least 4 weeks if CSF culture results are negative after 2 weeks of treatment. Therapy should be extended for a total of 6 weeks in the patient with neurologic complications. Induction therapy is followed by consolidation therapy with fluconazole 400 mg/d for 8 weeks. Organ transplant recipients are treated with liposomal AmB (3–4 mg/kg per day) or AmB lipid complex (ABLC) 5 mg/kg per day plus flucytosine (100 mg/kg per day in four divided doses) for at least 2 weeks or until CSF culture is sterile. Follow CSF yeast cultures for sterilization rather than the cryptococcal antigen titer. This treatment is followed by an 8to 10-week course of fluconazole (400–800 mg/d [6–12 mg/kg] PO). If the CSF culture is sterile after 10 weeks of acute therapy, the dose of fluconazole is

1	titer. This treatment is followed by an 8to 10-week course of fluconazole (400–800 mg/d [6–12 mg/kg] PO). If the CSF culture is sterile after 10 weeks of acute therapy, the dose of fluconazole is decreased to 200 mg/d for 6 months to a year. Patients with HIV infection are treated with AmB or a lipid formulation plus flucytosine for at least 2 weeks, followed by fluconazole for a minimum of 8 weeks. HIV-infected patients may require indefinite maintenance therapy with fluconazole 200 mg/d. Meningitis due to H. capsulatum is treated with AmB (0.7–1.0 mg/kg per day) for 4–12 weeks. A total dose of 30 mg/kg is recommended. Therapy with AmB is not discontinued until fungal cultures are sterile. After completing a course of AmB, maintenance therapy with itraconazole 200 mg two or three times daily is initiated and continued for at least 9 months to a year. C. immitis meningitis is treated with either high-dose fluconazole (1000 mg daily) as monotherapy or intravenous AmB (0.5–0.7 mg/kg per

1	is initiated and continued for at least 9 months to a year. C. immitis meningitis is treated with either high-dose fluconazole (1000 mg daily) as monotherapy or intravenous AmB (0.5–0.7 mg/kg per day) for >4 weeks. Intrathecal AmB (0.25–0.75 mg/d three times weekly) may be required to eradicate the infection. Lifelong therapy with fluconazole (200–400 mg daily) is recommended to prevent relapse. AmBisome (5 mg/kg per day) or AmB lipid complex (5 mg/kg per day) can be substituted for AmB in patients who have or who develop significant renal dysfunction. The most common complication of fungal meningitis is hydrocephalus. Patients who develop hydrocephalus should receive a CSF diversion device. A ventriculostomy can be used until CSF fungal cultures are sterile, at which time the ventriculostomy is replaced by a ventriculoperitoneal shunt.

1	Syphilitic meningitis is treated with aqueous penicillin G in a dose of 3–4 million units intravenously every 4 h for 10–14 days. An alternative regimen is 2.4 million units of procaine penicillin G intramuscularly daily with 500 mg of oral probenecid four times daily for 10–14 days. Either regimen is followed with 2.4 million units of benzathine penicillin G intramuscularly once a week for 3 weeks. The standard criterion for treatment success is reexamination of the CSF. The CSF should be reexamined at 6-month intervals for 2 years. The cell count is expected to normalize within 12 months, and the VDRL titer to decrease by two dilutions or revert to nonreactive within 2 years of completion of therapy. Failure of the CSF pleocytosis to resolve or an increase in the CSF VDRL titer by two or more dilutions requires retreatment.

1	PROGRESSIVE MULTIFOCAL LEUKOENCEPHALOPATHY Clinical Features and Pathology Progressive multifocal leukoencephalopathy (PML) is characterized pathologically by multifocal areas of demyelination of varying size distributed throughout the brain but sparing the spinal cord and optic nerves. In addition to demyelination, 899 there are characteristic cytologic alterations in both astrocytes and oligodendrocytes. Astrocytes are enlarged and contain hyperchromatic, deformed, and bizarre nuclei and frequent mitotic figures. Oligodendrocytes have enlarged, densely staining nuclei that contain viral inclusions formed by crystalline arrays of JC virus (JCV) particles. Patients often present with visual deficits (45%), typically a homonymous hemianopia; mental impairment (38%) (dementia, confusion, personality change); weakness, including hemior monoparesis; and ataxia. Seizures occur in ~20% of patients, predominantly in those with lesions abutting the cortex.

1	Almost all patients have an underlying immunosuppressive disorder or are receiving immunomodulatory therapy. In recent series, the most common associated conditions were AIDS (80%), hematologic malignancies (13%), transplant recipients (5%), and chronic inflammatory diseases (2%). It has been estimated that up to 5% of AIDS patients will develop PML. There have been over 400 reported cases of PML occurring in patients being treated for multiple sclerosis and inflammatory bowel disease with natalizumab, a humanized monoclonal antibody that inhibits lymphocyte trafficking into CNS and bowel mucosa by binding to α4 integrins. Overall risk in these patients has been estimated at ~3.4 PML cases per 1000 treated patients, but the risk depends on a variety of factors including anti-JCV antibody serostatus, prior immunosuppressive therapy use, and duration of natalizumab therapy. Patients who lack detectable JCV antibody have a risk of developing PML of <0.1 case/1000 patients, whereas those

1	serostatus, prior immunosuppressive therapy use, and duration of natalizumab therapy. Patients who lack detectable JCV antibody have a risk of developing PML of <0.1 case/1000 patients, whereas those who are JCV seropositive and have been exposed to prior immunosuppressive therapy and have received >24 months of natalizumab therapy have a risk of >1 case/100 treated patients. PML cases have also been reported in patients receiving other humanized monoclonal antibodies with immunomodulatory activity including efalizumab and rituximab, although the relative risks have not been clearly established. The basic clinical and diagnostic features appear to be similar in HIV-associated PML and PML associated with immunomodulatory drugs with the exception of an increased likelihood of peripheral enhancement in MRIs of PML lesions in immunomodulatory cases. In natalizumab-associated PML, patients will also almost invariably develop clinical and radiographic worsening of lesions with

1	enhancement in MRIs of PML lesions in immunomodulatory cases. In natalizumab-associated PML, patients will also almost invariably develop clinical and radiographic worsening of lesions with discontinuation of therapy, attributed to development of immune reconstitution inflammatory syndrome (IRIS).

1	Diagnostic Studies The diagnosis of PML is frequently suggested by MRI. MRI reveals multifocal asymmetric, coalescing white matter lesions located periventricularly, in the centrum semiovale, in the parietal-occipital region, and in the cerebellum. These lesions have increased signal on T2 and FLAIR images and decreased signal on T1-weighted images. HIV-PML lesions are classically nonenhancing (90%), but patients with immunomodulatory drug associated PML may have peripheral ring enhancement. PML lesions are not typically associated with edema or mass effect. CT scans, which are less sensitive than MRI for the diagnosis of PML, often show hypodense nonenhancing white matter lesions.

1	The CSF is typically normal, although mild elevation in protein and/ or IgG may be found. Pleocytosis occurs in <25% of cases, is predominantly mononuclear, and rarely exceeds 25 cells/μL. PCR amplification of JCV DNA from CSF has become an important diagnostic tool. The presence of a positive CSF PCR for JCV DNA in association with typical MRI lesions in the appropriate clinical setting is diagnostic of PML, reflecting the assay’s relatively high specificity (92–100%); however, sensitivity is variable, and a negative CSF PCR does not exclude the diagnosis. In HIV-negative patients and HIV-positive patients not receiving highly active antiviral therapy (HAART), sensitivity is likely 70–90%. In HAART-treated patients, sensitivity may be closer to 60%, reflecting the lower JCV CSF viral load in this relatively more immunocompetent group. Studies with quantitative JCV CSF PCR indicate that patients with low JCV loads (<100 copies/μL) have a generally better prognosis than those with

1	load in this relatively more immunocompetent group. Studies with quantitative JCV CSF PCR indicate that patients with low JCV loads (<100 copies/μL) have a generally better prognosis than those with higher viral loads. Patients with negative CSF PCR studies may require brain biopsy for definitive diagnosis. In biopsy or necropsy specimens of brain, JCV antigen and nucleic acid can be detected by immunocytochemistry, in situ hybridization, or PCR amplification.

1	Meningitis, Encephalitis, Brain Abscess, and Empyema 900 Serologic studies are of no utility in diagnosis due to high basal seroprevalence level, but may contribute to risk stratification in patients contemplating therapy with immunomodulatory drugs such as natalizumab.

1	No effective therapy for PML is available. There are case reports of potential beneficial effects of the 5-HT2a receptor antagonist mirtazapine, which may inhibit binding of JCV to its receptor on oligodendrocytes. Retrospective noncontrolled studies have also suggested a possible beneficial effect of treatment with interferon-α. Neither of these agents has been tested in randomized controlled clinical trials. A prospective multicenter clinical trial to evaluate the efficacy of the antimalarial drug mefloquine failed to show benefit. Intravenous and/or intrathecal cytarabine were not shown to be of benefit in a randomized controlled trial in HIV-associated PML, although some experts suggest that cytarabine may have therapeutic efficacy in situations where breakdown of the blood-brain barrier allows sufficient CSF penetration. A randomized controlled trial of cidofovir in HIV-associated PML also failed to show significant benefit. Because PML almost invariably occurs in

1	barrier allows sufficient CSF penetration. A randomized controlled trial of cidofovir in HIV-associated PML also failed to show significant benefit. Because PML almost invariably occurs in immunocompromised individuals, any therapeutic interventions designed to enhance or restore immunocompetence should be considered. Perhaps the most dramatic demonstration of this is disease stabilization and, in rare cases, improvement associated with the improvement in the immune status of HIV-positive patients with AIDS following institution of HAART. In HIV-positive PML patients treated with HAART, 1-year survival is ~50%, although up to 80% of survivors may have significant neurologic sequelae. HIV-positive PML patients with higher CD4 counts (>300/μL) and low or nondetectable HIV viral loads have a better prognosis than those with lower CD4 counts and higher viral loads. Although institution of HAART enhances survival in HIV-positive PML patients, the associated immune reconstitution in

1	have a better prognosis than those with lower CD4 counts and higher viral loads. Although institution of HAART enhances survival in HIV-positive PML patients, the associated immune reconstitution in patients with an underlying opportunistic infection such as PML may also result in a severe CNS inflammatory syndrome (IRIS) associated with clinical worsening, CSF pleocytosis, and the appearance of new enhancing MRI lesions. Patients receiving natalizumab or other immunomodulatory antibodies, who are suspected of having PML, should have therapy immediately halted and circulating antibodies removed by plasma exchange. Patients should be closely monitored for development of IRIS, which is generally treated with intravenous glucocorticoids, although controlled clinical trials of efficacy remain lacking.

1	SSPE is a rare chronic, progressive demyelinating disease of the CNS associated with a chronic nonpermissive infection of brain tissue with measles virus. The frequency has been estimated at 1 in 100,000– 500,000 measles cases. An average of five cases per year are reported in the United States. The incidence has declined dramatically since the introduction of a measles vaccine. Most patients give a history of primary measles infection at an early age (2 years), which is followed after a latent interval of 6–8 years by the development of a progressive neurologic disorder. Some 85% of patients are between 5 and 15 years old at diagnosis. Initial manifestations include poor school performance and mood and personality changes. Typical signs of a CNS viral infection, including fever and headache, do not occur. As the disease progresses, patients develop progressive intellectual deterioration, focal and/or generalized seizures, myoclonus, ataxia, and visual disturbances. In the late stage

1	do not occur. As the disease progresses, patients develop progressive intellectual deterioration, focal and/or generalized seizures, myoclonus, ataxia, and visual disturbances. In the late stage of the illness, patients are unresponsive, quadriparetic, and spastic, with hyperactive tendon reflexes and extensor plantar responses.

1	Diagnostic Studies MRI is often normal early, although areas of increased T2 signal develop in the white matter of the brain and brain-stem as disease progresses. The EEG may initially show only nonspecific slowing, but with disease progression, patients develop a characteristic periodic pattern with bursts of high-voltage, sharp, slow waves every 3–8 s, followed by periods of attenuated (“flat”) background. The CSF is acellular with a normal or mildly elevated protein concentration and a markedly elevated gamma globulin level (>20% of total CSF protein). CSF antimeasles antibody levels are invariably elevated, and oligoclonal antimeasles antibodies are often present. Measles virus can be cultured from brain tissue using special cocultivation techniques. Viral antigen can be identified immunocytochemically, and viral genome can be detected by in situ hybridization or PCR amplification.

1	No definitive therapy for SSPE is available. Treatment with isoprinosine (Inosiplex, 100 mg/kg per day), alone or in combination with intrathecal or intraventricular interferon-α, has been reported to prolong survival and produce clinical improvement in some patients but has never been subjected to a controlled clinical trial.

1	This is an extremely rare disorder that primarily affects males with congenital rubella syndrome, although isolated cases have been reported following childhood rubella. After a latent period of 8–19 years, patients develop progressive neurologic deterioration. The manifestations are similar to those seen in SSPE. CSF shows a mild lymphocytic pleocytosis, slightly elevated protein concentration, markedly increased gamma globulin, and rubella virus–specific oligoclonal bands. No therapy is available. Universal prevention of both congenital and childhood rubella through the use of the available live attenuated rubella vaccine would be expected to eliminate the disease. A brain abscess is a focal, suppurative infection within the brain parenchyma, typically surrounded by a vascularized capsule. The term cerebritis is often employed to describe a nonencapsulated brain abscess.

1	A bacterial brain abscess is a relatively uncommon intracranial infection, with an incidence of ~0.3–1.3:100,000 persons per year. Predisposing conditions include otitis media and mastoiditis, paranasal sinusitis, pyogenic infections in the chest or other body sites, penetrating head trauma or neurosurgical procedures, and dental infections. In immunocompetent individuals the most important pathogens are Streptococcus spp. (anaerobic, aerobic, and viridans [40%]), Enterobacteriaceae (Proteus spp., E. coli sp., Klebsiella spp. [25%]), anaerobes (e.g., Bacteroides spp., Fusobacterium spp. [30%]), and staphylococci (10%). In immunocompromised hosts with underlying HIV infection, organ transplantation, cancer, or immunosuppressive therapy, most brain abscesses are caused by Nocardia spp., Toxoplasma gondii, Aspergillus spp., Candida spp., and C. neoformans. In Latin America and in immigrants from Latin America, the most common cause of brain abscess is Taenia solium (neurocysticercosis).

1	gondii, Aspergillus spp., Candida spp., and C. neoformans. In Latin America and in immigrants from Latin America, the most common cause of brain abscess is Taenia solium (neurocysticercosis). In India and East Asia, mycobacterial infection (tuberculoma) remains a major cause of focal CNS mass lesions.

1	A brain abscess may develop (1) by direct spread from a contiguous cranial site of infection, such as paranasal sinusitis, otitis media, mastoiditis, or dental infection; (2) following head trauma or a neurosurgical procedure; or (3) as a result of hematogenous spread from a remote site of infection. In up to 25% of cases, no obvious primary source of infection is apparent (cryptogenic brain abscess). Approximately one-third of brain abscesses are associated with otitis media and mastoiditis, often with an associated cholesteatoma.

1	Otogenic abscesses occur predominantly in the temporal lobe (55– 75%) and cerebellum (20–30%). In some series, up to 90% of cerebellar abscesses are otogenic. Common organisms include streptococci, Bacteroides spp., Pseudomonas spp., Haemophilus spp., and Enterobacteriaceae. Abscesses that develop as a result of direct spread of infection from the frontal, ethmoidal, or sphenoidal sinuses and those that occur due to dental infections are usually located in the frontal lobes. Approximately 10% of brain abscesses are associated with paranasal sinusitis, and this association is particularly strong in young males in their second and third decades of life. The most common pathogens in brain abscesses associated with paranasal sinusitis are streptococci (especially Streptococcus milleri), Haemophilus spp., Bacteroides spp., Pseudomonas spp., and S. aureus. Dental infections are associated with ~2% of brain abscesses, although it is often suggested that many “cryptogenic” abscesses are in

1	spp., Bacteroides spp., Pseudomonas spp., and S. aureus. Dental infections are associated with ~2% of brain abscesses, although it is often suggested that many “cryptogenic” abscesses are in fact due to dental infections. The most common pathogens in this setting are streptococci, staphylococci, Bacteroides spp., and Fusobacterium spp.

1	Hematogenous abscesses account for ~25% of brain abscesses. Hematogenous abscesses are often multiple, and multiple abscesses often (50%) have a hematogenous origin. These abscesses show a predilection for the territory of the middle cerebral artery (i.e., posterior frontal or parietal lobes). Hematogenous abscesses are often located at the junction of the gray and white matter and are often poorly encapsulated. The microbiology of hematogenous abscesses is dependent on the primary source of infection. For example, brain abscesses that develop as a complication of infective endocarditis are often due to viridans streptococci or S. aureus. Abscesses associated with pyogenic lung infections such as lung abscess or bronchiectasis are often due to streptococci, staphylococci, Bacteroides spp., Fusobacterium spp., or Enterobacteriaceae. Abscesses that follow penetrating head trauma or neurosurgical procedures are frequently due to methicillin-resistant S. aureus (MRSA), S. epidermidis,

1	Fusobacterium spp., or Enterobacteriaceae. Abscesses that follow penetrating head trauma or neurosurgical procedures are frequently due to methicillin-resistant S. aureus (MRSA), S. epidermidis, Enterobacteriaceae, Pseudomonas spp., and Clostridium spp. Enterobacteriaceae and P. aeruginosa are important causes of abscesses associated with urinary sepsis. Congenital cardiac malformations that produce a right-to-left shunt, such as tetralogy of Fallot, patent ductus arteriosus, and atrial and ventricular septal defects, allow bloodborne bacteria to bypass the pulmonary capillary bed and reach the brain. Similar phenomena can occur with pulmonary arteriovenous malformations. The decreased arterial oxygenation and saturation from the right-to-left shunt and polycythemia may cause focal areas of cerebral ischemia, thus providing a nidus for microorganisms that bypassed the pulmonary circulation to multiply and form an abscess. Streptococci are the most common pathogens in this setting.

1	Results of experimental models of brain abscess formation suggest that for bacterial invasion of brain parenchyma to occur, there must be preexisting or concomitant areas of ischemia, necrosis, or hypoxemia in brain tissue. The intact brain parenchyma is relatively resistant to infection. Once bacteria have established infection, brain abscess frequently evolves through a series of stages, influenced by the nature of the infecting organism and by the immunocompetence of the host. The early cerebritis stage (days 1–3) is characterized by a perivascular infiltration of inflammatory cells, which surround a central core of coagulative necrosis. Marked edema surrounds the lesion at this stage. In the late cerebritis stage (days 4–9), pus formation leads to enlargement of the necrotic center, which is surrounded at its border by an inflammatory infiltrate of macrophages and fibroblasts. A thin capsule of fibroblasts and reticular fibers gradually develops, and the surrounding area of

1	which is surrounded at its border by an inflammatory infiltrate of macrophages and fibroblasts. A thin capsule of fibroblasts and reticular fibers gradually develops, and the surrounding area of cerebral edema becomes more distinct than in the previous stage. The third stage, early capsule formation (days 10–13), is characterized by the formation of a capsule that is better developed on the cortical than on the ventricular side of the lesion. This stage correlates with the appearance of a ring-enhancing capsule on neuroimaging studies. The final stage, late capsule formation (day 14 and beyond), is defined by a well-formed necrotic center surrounded by a dense collagenous capsule. The surrounding area of cerebral edema has regressed, but marked gliosis with large numbers of reactive astrocytes has developed outside the capsule. This gliotic process may 901 contribute to the development of seizures as a sequela of brain abscess.

1	A brain abscess typically presents as an expanding intracranial mass lesion rather than as an infectious process. Although the evolution of signs and symptoms is extremely variable, ranging from hours to weeks or even months, most patients present to the hospital 11–12 days following onset of symptoms. The classic clinical triad of headache, fever, and a focal neurologic deficit is present in <50% of cases. The most common symptom in patients with a brain abscess is headache, occurring in >75% of patients. The headache is often characterized as a constant, dull, aching sensation, either hemicranial or generalized, and it becomes progressively more severe and refractory to therapy. Fever is present in only 50% of patients at the time of diagnosis, and its absence should not exclude the diagnosis. The new onset of focal or generalized seizure activity is a presenting sign in 15–35% of patients. Focal neurologic deficits including hemiparesis, aphasia, or visual field defects are part of

1	The new onset of focal or generalized seizure activity is a presenting sign in 15–35% of patients. Focal neurologic deficits including hemiparesis, aphasia, or visual field defects are part of the initial presentation in >60% of patients.

1	The clinical presentation of a brain abscess depends on its location, the nature of the primary infection if present, and the level of the ICP. Hemiparesis is the most common localizing sign of a frontal lobe abscess. A temporal lobe abscess may present with a disturbance of language (dysphasia) or an upper homonymous quadrantanopia. Nystagmus and ataxia are signs of a cerebellar abscess. Signs of raised ICP—papilledema, nausea and vomiting, and drowsiness or confusion—can be the dominant presentation of some abscesses, par ticularly those in the cerebellum. Meningismus is not present unless the abscess has ruptured into the ventricle or the infection has spread to the subarachnoid space.

1	Diagnosis is made by neuroimaging studies. MRI (Fig. 164-4) is better than CT for demonstrating abscesses in the early (cerebritis) stages and is superior to CT for identifying abscesses in the posterior fossa. Cerebritis appears on MRI as an area of low-signal intensity on T1-weighted images with irregular postgadolinium enhancement and as an area of increased signal intensity on T2-weighted images. Cerebritis is often not visualized by CT scan, but when present, appears as an area of hypodensity. On a contrast-enhanced CT scan, a mature brain abscess appears as a focal area of hypodensity surrounded by ring enhancement with surrounding edema (hypodensity). On contrast-enhanced T1-weighted MRI, a mature brain abscess has a capsule that enhances surrounding a hypodense center and surrounded by a hypodense area of edema. On T2-weighted MRI, there is a hyperintense central area of pus surrounded by a well-defined hypointense capsule and a hyperintense surrounding area of edema. It is

1	by a hypodense area of edema. On T2-weighted MRI, there is a hyperintense central area of pus surrounded by a well-defined hypointense capsule and a hyperintense surrounding area of edema. It is important to recognize that the CT and MRI appearance, particularly of the capsule, may be altered by treatment with glucocorticoids. The distinction between a brain abscess and other focal CNS lesions such as primary or metastatic tumors may be facilitated by the use of diffusion-weighted imaging sequences on which a brain abscess typically shows increased signal due to restricted diffusion of the abscess cavity with corresponding low signal on apparent diffusion coefficient images.

1	Microbiologic diagnosis of the etiologic agent is most accurately determined by Gram’s stain and culture of abscess material obtained by CT-guided stereotactic needle aspiration. Aerobic and anaerobic bacterial cultures and mycobacterial and fungal cultures should be obtained. Up to 10% of patients will also have positive blood cultures. LP should not be performed in patients with known or suspected focal intracranial infections such as abscess or empyema; CSF analysis contributes nothing to diagnosis or therapy, and LP increases the risk of herniation. Additional laboratory studies may provide clues to the diagnosis of brain abscess in patients with a CNS mass lesion. About 50% of patients have a peripheral leukocytosis, 60% an elevated ESR, and 80% an elevated C-reactive protein. Blood cultures are positive in ~10% of cases overall but may be positive in >85% of patients with abscesses due to Listeria. Meningitis, Encephalitis, Brain Abscess, and Empyema

1	Meningitis, Encephalitis, Brain Abscess, and Empyema FIGURE 164-4 Pneumococcal brain abscess. Note that the abscess wall has hyperintense signal on the axial T1-weighted magnetic resonance imaging (MRI) (A, black arrow), hypointense signal on the axial proton density images (B, black arrow), and enhances prominently after gado-linium administration on the coronal T1-weighted image (C). The abscess is surrounded by a large amount of vasogenic edema and has a small “daughter” abscess (C, white arrow). (Courtesy of Joseph Lurito, MD; with permission.)

1	Conditions that can cause headache, fever, focal neurologic signs, and seizure activity include brain abscess, subdural empyema, bacterial meningitis, viral meningoencephalitis, superior sagittal sinus thrombosis, and acute disseminated encephalomyelitis. When fever is absent, primary and metastatic brain tumors become the major differential diagnosis. Less commonly, cerebral infarction or hematoma can have an MRI or CT appearance resembling brain abscess.

1	Optimal therapy of brain abscesses involves a combination of high-dose parenteral antibiotics and neurosurgical drainage. Empirical therapy of community-acquired brain abscess in an immunocompetent patient typically includes a thirdor fourth-generation cephalosporin (e.g., cefotaxime, ceftriaxone, or cefepime) and metronidazole (see Table 164-1 for antibiotic dosages). In patients with penetrating head trauma or recent neurosurgical procedures, treatment should include ceftazidime as the third-generation cephalosporin to enhance coverage of Pseudomonas spp. and vancomycin for coverage of staphylococci. Meropenem plus vancomycin also provides good coverage in this setting.

1	Aspiration and drainage of the abscess under stereotactic guidance are beneficial for both diagnosis and therapy. Empirical antibiotic coverage should be modified based on the results of Gram’s stain and culture of the abscess contents. Complete excision of a bacterial abscess via craniotomy or craniectomy is generally reserved for multiloculated abscesses or those in which stereotactic aspiration is unsuccessful. Medical therapy alone is not optimal for treatment of brain abscess and should be reserved for patients whose abscesses are neurosurgically inaccessible, for patients with small (<2–3 cm) or nonencapsulated abscesses (cerebritis), and for patients whose condition is too tenuous to allow performance of a neurosurgical procedure. All patients should receive a minimum of 6–8 weeks of parenteral antibiotic therapy. The role, if any, of supplemental oral antibiotic therapy following completion of a standard course of parenteral therapy has never been adequately studied.

1	In addition to surgical drainage and antibiotic therapy, patients should receive prophylactic anticonvulsant therapy because of the high risk (~35%) of focal or generalized seizures. Anticonvulsant therapy is continued for at least 3 months after resolution of the abscess, and decisions regarding withdrawal are then based on the EEG. If the EEG is abnormal, anticonvulsant therapy should be continued. If the EEG is normal, anticonvulsant therapy can be slowly withdrawn, with close follow-up and repeat EEG after the medication has been discontinued. Glucocorticoids should not be given routinely to patients with brain abscesses. Intravenous dexamethasone therapy (10 mg every 6 h) is usually reserved for patients with substantial periabscess edema and associated mass effect and increased ICP. Dexamethasone should be tapered as rapidly as possible to avoid delaying the natural process of encapsulation of the abscess.

1	Serial MRI or CT scans should be obtained on a monthly or twice-monthly basis to document resolution of the abscess. More frequent studies (e.g., weekly) are probably warranted in the subset of patients who are receiving antibiotic therapy alone. A small amount of enhancement may remain for months after the abscess has been successfully treated. The mortality rate of brain abscess has declined in parallel with the development of enhanced neuroimaging techniques, improved neurosurgical procedures for stereotactic aspiration, and improved antibiotics. In modern series, the mortality rate is typically <15%. Significant sequelae, including seizures, persisting weakness, aphasia, or mental impairment, occur in ≥20% of survivors.

1	Neurocysticercosis is the most common parasitic disease of the CNS worldwide. Humans acquire cysticercosis by the ingestion of food contaminated with the eggs of the parasite T. solium. Toxoplasmosis is a parasitic disease caused by T. gondii and acquired from the ingestion of undercooked meat and from handling cat feces.

1	The most common manifestation of neurocysticercosis is new-onset partial seizures with or without secondary generalization. Cysticerci may develop in the brain parenchyma and cause seizures or focal neurologic deficits. When present in the subarachnoid or ventricular spaces, cysticerci can produce increased ICP by interference with CSF flow. Spinal cysticerci can mimic the presentation of intraspinal tumors. When the cysticerci first lodge in the brain, they frequently cause little in the way of an inflammatory response. As the cysticercal cyst degenerates, it elicits an inflammatory response that may present clinically as a seizure. Eventually the cyst dies, a process that may take several years and is typically associated with resolution of the inflammatory response and, often, abatement of seizures. Primary Toxoplasma infection is often asymptomatic. However,

1	Primary Toxoplasma infection is often asymptomatic. However, Meningitis, Encephalitis, Brain Abscess, and Empyema during this phase parasites may spread to the CNS, where they become latent. Reactivation of CNS infection is almost exclusively associated with immunocompromised hosts, particularly those with HIV infection. During this phase patients present with headache, fever, seizures, and focal neurologic deficits.

1	The lesions of neurocysticercosis are readily visualized by MRI or CT scans. Lesions with viable parasites appear as cystic lesions. The scolex can often be visualized on MRI. Lesions may appear as contrast-enhancing lesions surrounded by edema. A very early sign of cyst death is hypointensity of the vesicular fluid on T2-weighted images when compared with CSF. Parenchymal brain calcifications are the most common finding and evidence that the parasite is no longer viable. MRI findings of toxoplasmosis consist of multiple lesions in the deep white matter, the thalamus, and basal ganglia and at the gray-white junction in the cerebral hemispheres. With contrast administration, the majority of the lesions enhance in a ringed, nodular, or homogeneous pattern and are surrounded by edema. In the presence of the characteristic neuroimaging abnormalities of T. gondii infection, serum IgG antibody to T. gondii should be obtained and, when positive, the patient should be treated.

1	Anticonvulsant therapy is initiated when the patient with neurocysticercosis presents with a seizure. There is controversy about whether or not anthelmintic therapy should be given to all patients, and recommendations are based on the stage of the lesion. Cysticerci appearing as cystic lesions in the brain parenchyma with or without pericystic edema or in the subarachnoid space at the convexity of the cerebral hemispheres should be treated with anticysticidal therapy. Cysticidal drugs accelerate the destruction of the parasites, resulting in a faster resolution of the infection. Albendazole and praziquantel are used in the treatment of neurocysticercosis. Approximately 85% of parenchymal cysts are destroyed by a single course of albendazole, and ~75% are destroyed by a single course of praziquantel. The dose of albendazole is 15 mg/kg per day in two doses for 8 days. The dose of praziquantel is 50 mg/kg per day for 15 days, although a number of other dosage regimens are also

1	of praziquantel. The dose of albendazole is 15 mg/kg per day in two doses for 8 days. The dose of praziquantel is 50 mg/kg per day for 15 days, although a number of other dosage regimens are also frequently cited. Prednisone or dexamethasone is given with anticysticidal therapy to reduce the host inflammatory response to degenerating parasites. Many, but not all, experts recommend anticysticidal therapy for lesions that are surrounded by a contrast-enhancing ring. There is universal agreement that calcified lesions do not need to be treated with anticysticidal therapy. Antiepileptic therapy can be stopped once the follow-up CT scan shows resolution of the lesion. Long-term antiepileptic therapy is recommended when seizures occur after resolution of edema and resorption or calcification of the degenerating cyst.

1	CNS toxoplasmosis is treated with a combination of sulfadiazine, 1.5–2.0 g orally qid, plus pyrimethamine, 100 mg orally to load, then 75–100 mg orally qd, plus folinic acid, 10–15 mg orally qd. Folinic acid is added to the regimen to prevent megaloblastic anemia. Therapy is continued until there is no evidence of active disease on neuroimaging studies, which typically takes at least 6 weeks, and then the dose of sulfadiazine is reduced to 2–4 g/d and pyrimethamine to 50 mg/d. Clindamycin plus pyrimethamine is an alternative therapy for patients who cannot tolerate sulfadiazine, but the combination of pyrimethamine and sulfadiazine is more effective. A subdural empyema (SDE) is a collection of pus between the dura and arachnoid membranes (Fig. 164-5). SDE is a rare disorder that accounts for 15–25% of focal suppurative CNS infections. Sinusitis is the most common predisposing condition FIGURE 164-5 Subdural empyema.

1	SDE is a rare disorder that accounts for 15–25% of focal suppurative CNS infections. Sinusitis is the most common predisposing condition FIGURE 164-5 Subdural empyema. and typically involves the frontal sinuses, either alone or in combination with the ethmoid and maxillary sinuses. Sinusitis-associated empyema has a striking predilection for young males, possibly reflecting sex-related differences in sinus anatomy and development. It has been suggested that SDE may complicate 1–2% of cases of frontal sinusitis severe enough to require hospitalization. As a consequence of this epidemiology, SDE shows an ~3:1 male/female predominance, with 70% of cases occurring in the second and third decades of life. SDE may also develop as a complication of head trauma or neurosurgery. Secondary infection of a subdural effusion may also result in empyema, although secondary infection of hematomas, in the absence of a prior neurosurgical procedure, is rare.

1	Aerobic and anaerobic streptococci, staphylococci, Enterobacteriaceae, and anaerobic bacteria are the most common causative organisms of sinusitis-associated SDE. Staphylococci and gram-negative bacilli are often the etiologic organisms when SDE follows neurosurgical procedures or head trauma. Up to one-third of cases are culture-negative, possibly reflecting difficulty in obtaining adequate anaerobic cultures.

1	Sinusitis-associated SDE develops as a result of either retrograde spread of infection from septic thrombophlebitis of the mucosal veins draining the sinuses or contiguous spread of infection to the brain from osteomyelitis in the posterior wall of the frontal or other sinuses. SDE may also develop from direct introduction of bacteria into the subdural space as a complication of a neurosurgical procedure. The evolution of SDE can be extremely rapid because the subdural space is a large compartment that offers few mechanical barriers to the spread of infection. In patients with sinusitis-associated SDE, suppuration typically begins in the upper and anterior portions of one cerebral hemisphere and then extends posteriorly. SDE is often associated with other intracranial infections, including epidural empyema (40%), cortical thrombophlebitis (35%), and intracranial abscess or cerebritis (>25%). Cortical venous infarction produces necrosis of underlying cerebral cortex and subcortical

1	epidural empyema (40%), cortical thrombophlebitis (35%), and intracranial abscess or cerebritis (>25%). Cortical venous infarction produces necrosis of underlying cerebral cortex and subcortical white matter, with focal neurologic deficits and seizures (see below).

1	A patient with SDE typically presents with fever and a progressively worsening headache. The diagnosis of SDE should always be suspected in a patient with known sinusitis who presents with new CNS signs or symptoms. Patients with underlying sinusitis frequently have symptoms related to this infection. As the infection progresses, focal neurologic deficits, seizures, nuchal rigidity, and signs of increased ICP commonly occur. Headache is the most common complaint at the time of presentation; initially it is localized to the side of the subdural infection, but then it becomes more severe and generalized. Contralateral hemiparesis or hemiplegia is the most common focal neurologic deficit and can occur from the direct effects of the SDE on the cortex or as a consequence of venous infarction. Seizures begin as partial motor seizures that then become secondarily generalized. Seizures may be due to the direct irritative effect of the SDE on the underlying cortex or result from cortical

1	Seizures begin as partial motor seizures that then become secondarily generalized. Seizures may be due to the direct irritative effect of the SDE on the underlying cortex or result from cortical venous infarction (see above). In untreated SDE, the increasing mass effect and increase in ICP cause progressive deterioration in consciousness, leading ultimately to coma.

1	FIGURE 164-6 Subdural empyema. There is marked enhancement of the dura and leptomeninges (A, B, straight arrows) along the left medial hemisphere. The pus is hypointense on T1-weighted images (A, B) but markedly hyperintense on the proton density–weighted (C, curved arrow) image. (Courtesy of Joseph Lurito, MD; with permission.)

1	MRI (Fig. 164-6) is superior to CT in identifying SDE and any associated intracranial infections. The administration of gadolinium greatly improves diagnosis by enhancing the rim of the empyema and allowing the empyema to be clearly delineated from the underlying brain parenchyma. Cranial MRI is also extremely valuable in identifying sinusitis, other focal CNS infections, cortical venous infarction, cerebral edema, and cerebritis. CT may show a crescent-shaped hypodense lesion over one or both hemispheres or in the interhemispheric fissure. Frequently the degree of mass effect, exemplified by midline shift, ventricular compression, and sulcal effacement, is far out of proportion to the mass of the SDE. CSF examination should be avoided in patients with known or suspected SDE because it adds no useful information and is associated with the risk of cerebral herniation.

1	CSF examination should be avoided in patients with known or suspected SDE because it adds no useful information and is associated with the risk of cerebral herniation. The differential diagnosis of the combination of headache, fever, focal neurologic signs, and seizure activity that progresses rapidly to an altered level of consciousness includes subdural hematoma, bacterial meningitis, viral encephalitis, brain abscess, superior sagittal sinus thrombosis, and acute disseminated encephalomyelitis. The presence of nuchal rigidity is unusual with brain abscess or epidural empyema and should suggest the possibility of SDE when associated with significant focal neurologic signs and fever. Patients with bacterial meningitis also have nuchal rigidity but do not typically have focal deficits of the severity seen with SDE.

1	SDE is a medical emergency. Emergent neurosurgical evacuation of the empyema, either through craniotomy, craniectomy, or burr-hole drainage, is the definitive step in the management of this infection. Empirical antimicrobial therapy for community-acquired SDE should include a combination of a third-generation cephalosporin (e.g., cefotaxime or ceftriaxone), vancomycin, and metronidazole (see Table 164-1 for dosages). Patients with hospital-acquired SDE may have infections due to Pseudomonas spp. or MRSA and should receive coverage with a carbapenem (e.g., meropenem) and vancomycin. Metronidazole is not necessary for antianaerobic therapy when meropenem is being used. Parenteral antibiotic therapy should be continued for a minimum of 3–4 weeks after SDE drainage. Patients with associated cranial osteomyelitis may require longer therapy. Specific diagnosis of the etiologic organisms is made based on Gram’s stain and culture of fluid obtained via either burr holes or craniotomy; the

1	cranial osteomyelitis may require longer therapy. Specific diagnosis of the etiologic organisms is made based on Gram’s stain and culture of fluid obtained via either burr holes or craniotomy; the initial empirical antibiotic coverage can be modified accordingly.

1	Prognosis is influenced by the level of consciousness of the patient at the time of hospital presentation, the size of the empyema, and the speed with which therapy is instituted. Long-term neurologic sequelae, which include seizures and hemiparesis, occur in up to 50% of cases. Cranial epidural abscess is a suppurative infection occurring in the potential space between the inner skull table and dura (Fig. 164-7).

1	Cranial epidural abscess is less common than either brain abscess or SDE and accounts for <2% of focal suppurative CNS infections. A cranial epidural abscess develops as a complication of a craniotomy or compound skull fracture or as a result of spread of infection from the frontal sinuses, middle ear, mastoid, or orbit. An epidural abscess may develop contiguous to an area of osteomyelitis, when craniotomy is complicated by infection of the wound or bone flap, or as a result of direct infection of the epidural space. Infection in the frontal sinus, middle ear, mastoid, or orbit can reach the epidural space through retrograde spread of infection from septic thrombophlebitis in the emissary veins that drain these areas or by way of direct spread of infection through areas of osteomyelitis. Unlike the subdural space, the epidural space is really a potential rather than an actual compartment. The dura is normally tightly adherent to the inner skull table, and infection must dissect the

1	Unlike the subdural space, the epidural space is really a potential rather than an actual compartment. The dura is normally tightly adherent to the inner skull table, and infection must dissect the dura away from the skull table as it spreads. As a result, epidural abscesses are often smaller than SDEs. Cranial epidural abscesses, unlike brain abscesses, only rarely result from hematogenous spread of infection from extracranial primary sites. The bacteriology of a cranial epidural abscess is similar to that of SDE (see above). The etiologic organisms of an epidural abscess that arises from frontal sinusitis, middle-ear infections, or mastoiditis are usually streptococci or anaerobic organisms. Staphylococci or gram-negative organisms are the usual cause of an epidural abscess that develops as a complication of craniotomy or compound skull fracture.

1	FIGURE 164-7 Cranial epidural abscess is a collection of pus between the dura and the inner table of the skull. Patients present with fever (60%), headache (40%), nuchal rigidity (35%), seizures (10%), and focal deficits (5%). Development of symptoms may be insidious, as the empyema usually enlarges slowly in the confined anatomic space between the dura and the inner table of the skull. Periorbital edema and Pott’s puffy tumor, reflecting underlying associated frontal bone osteomyelitis, are present in ~40%. In patients with a recent neurosurgical procedure, wound infection is invariably present, but other symptoms may be subtle and can include altered mental status (45%), fever (35%), and headache (20%). The diagnosis should be considered when fever and headache follow recent head trauma or occur in the setting of frontal sinusitis, mastoiditis, or otitis media.

1	Cranial MRI with gadolinium enhancement is the procedure of choice to demonstrate a cranial epidural abscess. The sensitivity of CT is limited by the presence of signal artifacts arising from the bone of the inner skull table. The CT appearance of an epidural empyema is that of a lens or crescent-shaped hypodense extraaxial lesion. On MRI, an epidural empyema appears as a lentiform or crescent-shaped fluid collection that is hyperintense compared to CSF on T2-weighted images. On T1-weighted images, the fluid collection may be either isointense or hypointense compared to brain. Following the administration of gadolinium, there is linear enhancement of the dura on T1-weighted images. In distinction to subdural empyema, signs of mass effect or other parenchymal abnormalities are uncommon.

1	Immediate neurosurgical drainage is indicated. Empirical antimicrobial therapy, pending the results of Gram’s stain and culture of the purulent material obtained at surgery, should include a combination of a third-generation cephalosporin, vancomycin, and metronidazole (Table 164-1). Ceftazidime or meropenem should be substituted for ceftriaxone or cefotaxime in neurosurgical patients. Metronidazole is not necessary for antianaerobic coverage in patients receiving meropenem. When the organism has been identified, antimicrobial therapy can be modified accordingly. Antibiotics should be continued for 3–6 weeks after surgical drainage. Patients with associated osteomyelitis may require additional therapy. The mortality rate is <5% in modern series, and full recovery is the rule in most survivors.

1	The mortality rate is <5% in modern series, and full recovery is the rule in most survivors. Suppurative intracranial thrombophlebitis is septic venous thrombosis of cortical veins and sinuses. This may occur as a complication of bacterial meningitis; SDE; epidural abscess; or infection in the skin of the face, paranasal sinuses, middle ear, or mastoid.

1	The cerebral veins and venous sinuses have no valves; therefore, blood within them can flow in either direction. The superior sagittal sinus is the largest of the venous sinuses (Fig. 164-8). It receives blood from the frontal, parietal, and occipital superior cerebral veins and the diploic veins, which communicate with the meningeal veins. Bacterial meningitis is a common predisposing condition for septic thrombosis of the superior sagittal sinus. The diploic veins, which drain into the superior sagittal sinus, provide a route for the spread of infection from the meninges, especially in cases where there is purulent exudate near areas of the superior sagittal sinus. Infection can also spread to the superior sagittal sinus from nearby SDE or epidural abscess. Dehydration from vomiting, hypercoagulable states, and immunologic abnormalities, including the presence of circulating antiphospholipid antibodies, also contribute to cerebral venous sinus thrombosis. Thrombosis may extend from

1	states, and immunologic abnormalities, including the presence of circulating antiphospholipid antibodies, also contribute to cerebral venous sinus thrombosis. Thrombosis may extend from one sinus to another, and at autopsy, thrombi of different histologic ages can often be detected in several sinuses. Thrombosis of the superior sagittal sinus is often associated with thrombosis of superior cortical veins and small parenchymal hemorrhages.

1	The superior sagittal sinus drains into the transverse sinuses (Fig. 164-8). The transverse sinuses also receive venous drainage from small veins from both the middle ear and mastoid cells. The transverse sinus becomes the sigmoid sinus before draining into the internal jugular vein. Septic transverse/sigmoid sinus thrombosis can be a complication of acute and chronic otitis media or mastoiditis. Infection spreads from the mastoid air cells to the transverse sinus via the emissary veins or by direct invasion. The cavernous sinuses are inferior to the superior sagittal sinus at the base of the skull. The cavernous sinuses receive blood from the facial veins via the superior and inferior ophthalmic veins. Bacteria in the facial veins enter the cavernous sinus via these veins. Bacteria in the sphenoid and ethmoid sinuses can spread to the cavernous sinuses via the small emissary veins. The sphenoid and ethmoid sinuses are the most common sites of primary infection resulting in septic

1	the sphenoid and ethmoid sinuses can spread to the cavernous sinuses via the small emissary veins. The sphenoid and ethmoid sinuses are the most common sites of primary infection resulting in septic cavernous sinus thrombosis.

1	Septic thrombosis of the superior sagittal sinus presents with headache, fever, nausea and vomiting, confusion, and focal or generalized seizures. There may be a rapid development of stupor and coma. FIGURE 164-8 Anatomy of the cerebral venous sinuses.

1	Meningitis, Encephalitis, Brain Abscess, and Empyema 906 Weakness of the lower extremities with bilateral Babinski’s signs or hemiparesis is often present. When superior sagittal sinus thrombosis occurs as a complication of bacterial meningitis, nuchal rigidity and Kernig’s and Brudzinski’s signs may be present. The oculomotor nerve, the trochlear nerve, the abducens nerve, the ophthalmic and maxillary branches of the trigeminal nerve, and the internal carotid artery all pass through the cavernous sinus (see Fig. 455-4). The symptoms of septic cavernous sinus thrombosis are fever, headache, frontal and retroorbital pain, and diplopia. The classic signs are ptosis, proptosis, chemosis, and extraocular dysmotility due to deficits of cranial nerves III, IV, and VI; hyperesthesia of the ophthalmic and maxillary divisions of the fifth cranial nerve and a decreased corneal reflex may be detected. There may be evidence of dilated, tortuous retinal veins and papilledema. Headache and earache

1	and maxillary divisions of the fifth cranial nerve and a decreased corneal reflex may be detected. There may be evidence of dilated, tortuous retinal veins and papilledema. Headache and earache are the most frequent symptoms of transverse sinus thrombosis. A transverse sinus thrombosis may also present with otitis media, sixth nerve palsy, and retroorbital or facial pain (Gradenigo’s syndrome). Sigmoid sinus and internal jugular vein thrombosis may present with neck pain.

1	The diagnosis of septic venous sinus thrombosis is suggested by an absent flow void within the affected venous sinus on MRI and confirmed by magnetic resonance venography, CT angiogram, or the venous phase of cerebral angiography. The diagnosis of thrombophlebitis of intracerebral and meningeal veins is suggested by the presence of intracerebral hemorrhage but requires cerebral angiography for definitive diagnosis.

1	Septic venous sinus thrombosis is treated with antibiotics, hydration, and removal of infected tissue and thrombus in septic lateral or cavernous sinus thrombosis. The choice of antimicrobial therapy is based on the bacteria responsible for the predisposing or associated condition. Optimal duration of therapy is unknown, but antibiotics are usually continued for 6 weeks or until there is radiographic evidence of resolution of thrombosis. Anticoagulation with dose-adjusted intravenous heparin is recommended for aseptic venous sinus thrombosis and in the treatment of septic venous sinus thrombosis complicating bacterial meningitis in patients who have progressive neurologic deterioration despite antimicrobial therapy and intravenous fluids. The presence of a small intracerebral hemorrhage from septic thrombophlebitis is not an absolute contraindication to heparin therapy. Successful management of aseptic venous sinus thrombosis has been reported with surgical thrombectomy,

1	hemorrhage from septic thrombophlebitis is not an absolute contraindication to heparin therapy. Successful management of aseptic venous sinus thrombosis has been reported with surgical thrombectomy, catheter-directed urokinase therapy, and a combination of intrathrombus recombinant tissue plasminogen activator (rtPA) and intravenous heparin, but there are not enough data to recommend these therapies in septic venous sinus thrombosis.

1	Walter J. Koroshetz, Avindra Nath Chronic inflammation of the meninges (pia, arachnoid, and dura) can produce profound neurologic disability and may be fatal if not successfully treated. Chronic meningitis is diagnosed when a characteristic neurologic syndrome exists for >4 weeks and is associated with a persistent inflammatory response in the cerebrospinal fluid (CSF) (white blood cell count >5/μL). The causes are varied, and appropriate treatment depends on identification of the etiology. Five categories of disease account for most cases of chronic meningitis: (1) meningeal infections, (2) malignancy, (3) autoimmune inflammatory disorders, (4) chemical meningitis, and (5) parameningeal infections. Neck or back pain/stiffness Brudzinski’s or Kernig’s sign of meningeal irritation Change in personality Altered mental status—drowsiness, inat tention, disorientation, memory loss, frontal release signs (grasp, suck, snout), perseveration Double vision Paresis of CNs III, IV, VI

1	Change in personality Altered mental status—drowsiness, inat tention, disorientation, memory loss, frontal release signs (grasp, suck, snout), perseveration Double vision Paresis of CNs III, IV, VI Diminished Vision Papilledema, optic atrophy Abbreviation: CN, cranial nerve.

1	Neurologic manifestations of chronic meningitis (Table 165-1) are determined by the anatomic location of the inflammation and its consequences. Persistent headache with or without a stiff neck, hydrocephalus, cranial neuropathies, radiculopathies, and cognitive or personality changes are the cardinal features. These can occur alone or in combination. When they appear in combination, widespread dissemination of the inflammatory process along CSF pathways has occurred. In some cases, the presence of an underlying systemic illness points to a specific agent or class of agents as the probable cause. The diagnosis of chronic meningitis is usually made when the clinical presentation prompts the physician to examine the CSF for signs of inflammation. CSF is produced by the choroid plexus of the cerebral ventricles, exits through narrow foramina into the subarachnoid space surrounding the brain and spinal cord, circulates around the base of the brain and over the cerebral hemispheres, and is

1	cerebral ventricles, exits through narrow foramina into the subarachnoid space surrounding the brain and spinal cord, circulates around the base of the brain and over the cerebral hemispheres, and is resorbed by arachnoid villi projecting into the superior sagittal sinus. CSF flow provides a pathway for rapid spread of infectious and other infiltrative processes over the brain, spinal cord, and cranial and spinal nerve roots. Spread from the subarachnoid space into brain parenchyma may occur via the arachnoid cuffs that surround blood vessels that penetrate brain tissue (Virchow-Robin spaces).

1	Intracranial Meningitis Nociceptive nerve fibers of the meninges are stimulated by the inflammatory process, resulting in headache, neck pain, or back pain. Obstruction of CSF pathways at the foramina or arachnoid villi may produce hydrocephalus and symptoms of raised intracranial pressure (ICP), including headache, vomiting, apathy or drowsiness, gait instability, papilledema, visual loss, impaired upgaze, or palsy of the sixth cranial nerve (CN) (Chap. 455). Cognitive and behavioral changes during the course of chronic meningitis may also result from vascular damage, which may similarly produce seizures, stroke, or myelopathy. Inflammatory deposits seeded via the CSF circulation are often prominent around the brainstem and cranial nerves and along the undersurface of the frontal and temporal lobes. Such cases, termed basal meningitis, often present as multiple cranial neuropathies, with decreased vision (CN II), facial weakness (CN VII), decreased hearing (CN VIII), diplopia (CNs

1	lobes. Such cases, termed basal meningitis, often present as multiple cranial neuropathies, with decreased vision (CN II), facial weakness (CN VII), decreased hearing (CN VIII), diplopia (CNs III, IV, and VI), sensory or motor abnormalities of the oropharynx (CNs IX, X, and XII), decreased olfaction (CN I), or decreased facial sensation and masseter weakness (CN V).

1	Spinal Meningitis Injury may occur to motor and sensory roots as they traverse the subarachnoid space and penetrate the meninges. These cases present as multiple radiculopathies with combinations of radicular pain, sensory loss, motor weakness, and urinary or fecal incontinence. Meningeal inflammation can encircle the cord, resulting in a myelopathy. Patients with slowly progressive involvement of multiple cranial nerves and/or spinal nerve roots are likely to have chronic meningitis. Electrophysiologic testing (electromyography, nerve conduction studies, and evoked response testing) may be helpful in determining whether there is involvement of cranial and spinal nerve roots.

1	Systemic Manifestations In some patients, evidence of systemic disease provides clues to the underlying cause of chronic meningitis. A complete history of travel, sexual practice, and exposure to infectious agents should be sought. Infectious causes are often associated with fever, malaise, anorexia, and signs of localized or disseminated infection outside the nervous system. Infectious causes are of major concern in the immunosuppressed patient, especially in patients with AIDS, in whom chronic meningitis may present without headache or fever. Noninfectious inflammatory disorders often produce systemic manifestations, but meningitis may be the initial manifestation. Carcinomatous meningitis may or may not be accompanied by clinical evidence of the primary neoplasm. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: The occurrence of chronic headache, hydrocephalus, cranial neuropathy, radiculopathy, and/or cognitive decline in a patient should prompt consideration of a lumbar puncture for evidence of meningeal inflammation. On occasion, the diagnosis is made when an imaging study (CT or MRI) shows contrast enhancement of the meninges, which is always concerning with the exception of dural enhancement after lumbar puncture, neurosurgical procedures, or spontaneous CSF leakage. Once chronic meningitis is confirmed by CSF examination, effort is focused on identifying the cause (Tables 165-2 and 165-3) by (1) further analysis of the CSF, (2) diagnosis of an underlying systemic infection or noninfectious inflammatory condition, or (3) pathologic examination of meningeal biopsy specimens.

1	Two clinical forms of chronic meningitis exist. In the first, the symptoms are chronic and persistent, whereas in the second there are recurrent, discrete episodes of illness. In the latter group, all symptoms, signs, and CSF parameters of meningeal inflammation resolve completely between episodes without specific therapy. In such patients, the likely etiologies include herpes simplex virus (HSV) type 2; chemical meningitis due to episodic leakage from an epidermoid tumor, craniopharyngioma, or cholesteatoma into CSF; primary autoimmune inflammatory conditions, including VogtKoyanagi-Harada syndrome, Behçet’s syndrome, systemic lupus erythematosus (SLE), and Mollaret’s meningitis; and drug hypersensitivity with repeated administration of the offending agent.

1	The epidemiologic history is of considerable importance and may provide direction for selection of laboratory studies. Pertinent features include a history of tuberculosis or exposure to a likely case; past travel to areas endemic for fungal infections (the San Joaquin Valley in California and southwestern states for coccidioidomycosis, midwestern states for histoplasmosis, southeastern states for blastomycosis); travel to the Mediterranean region or ingestion of imported unpasteurized dairy products (Brucella); time spent in wooded areas endemic for Lyme disease; exposure to sexually transmitted disease (syphilis); exposure of an immunocompromised host to pigeons and their droppings (Cryptococcus); gardening (Sporothrix schenckii); ingestion of poorly cooked meat or contact with a household cat (Toxoplasma gondii); residence in Thailand or Japan (Gnathostoma spinigerum), Latin America (Paracoccidioides brasiliensis), or the South Pacific (Angiostrongylus cantonensis); rural residence

1	cat (Toxoplasma gondii); residence in Thailand or Japan (Gnathostoma spinigerum), Latin America (Paracoccidioides brasiliensis), or the South Pacific (Angiostrongylus cantonensis); rural residence and raccoon exposure (Baylisascaris procyonis); and residence in Latin America, the Philippines, or Southeast Asia (Taenia solium/cysticercosis).

1	The presence of focal cerebral signs in a patient with chronic meningitis suggests the possibility of a brain abscess or other Syphilis (secondary, tertiary) Treponema pallidum Mononuclear or mixed mononuclear-polymorphonuclear cells Mononuclear cells except polymorphonuclear cells in early infection (commonly <500 WBC/μL); low CSF glucose, high protein Contrast-enhanced CT or MRI to detect parenchymal, subdural, epidural, or sinus infection Tuberculin skin test may be negative; AFB culture of CSF (sputum, urine, gastric contents if indicated); tuberculostearic acid detection in CSF; identify tubercle bacillus on acid-fast stain of CSF or protein pellicle; PCR Serum Lyme antibody titer; Western blot confirmation; (patients with syphilis may have false-positive Lyme titer)

1	Serum Lyme antibody titer; Western blot confirmation; (patients with syphilis may have false-positive Lyme titer) CSF VDRL; serum VDRL (or RPR); fluorescent treponemal antibody-absorbed (FTA) or MHA-TP; serum VDRL may be negative in tertiary syphilis History consistent with acute bacterial meningitis and incomplete treatment Otitis media, pleuropulmonary infection, right-to-left cardiopulmonary shunt for brain abscess; focal neurologic signs; neck, back, ear, or sinus tenderness Exposure history; previous tuberculous illness; immunosuppressed, anti-TNF therapy or AIDS; young children; fever, meningismus, night sweats, miliary TB on x-ray or liver biopsy; stroke due to arteritis History of tick bite or appropriate exposure history; erythema chronicum migrans skin rash; arthritis, radiculopathy, Bell’s palsy, meningoencephalitis–multiple sclerosis-like syndrome

1	History of tick bite or appropriate exposure history; erythema chronicum migrans skin rash; arthritis, radiculopathy, Bell’s palsy, meningoencephalitis–multiple sclerosis-like syndrome Appropriate exposure history; HIV-seropositive individuals at increased risk of aggressive infection; “dementia”; cerebral infarction due to endarteritis Xylohypha (formerly Cladosporium) trichoides and other dark-walled (dematiaceous) fungi such as Curvularia, Drechslera; Mucor, and, after water aspiration, Pseudallescheria boydii, iatrogenic Exserohilum rostratum infection following spinal blocks Acanthamoeba sp. causing granulomatous amebic encephalitis and meningoencephalitis in immunocompromised and debilitated individuals. Balamuthia mandrillaris causing chronic meningoencephalitis in immunocompetent hosts.

1	Trichinella spiralis (trichinosis); Fasciola hepatica (liver fluke), Echinococcus cysts; Schistosoma sp. The former may produce a lymphocytic pleocytosis whereas the latter two may produce an eosinophilic response in CSF associated with cerebral cysts (Echinococcus) or granulomatous lesions of brain or spinal cord Abbreviations: AFB, acid-fast bacillus; CMV, cytomegalovirus; CSF, cerebrospinal fluid; CT, computed tomography; EBV, Epstein-Barr virus; ELISA, enzyme-linked immunosorbent assay; EM, electron microscopy; FTA, fluorescent treponemal antibody absorption test; HSV, herpes simplex virus; MHA-TP, microhemagglutination assay–T. pallidum; MRI, magnetic resonance imaging; PAS, periodic acid–Schiff; PCR, polymerase chain reaction; RPR, rapid plasma reagin test; TB, tuberculosis; VDRL, Venereal Disease Research Laboratories test.

1	Malignancy Mononuclear cells, elevated Repeated cytologic examination of Metastatic cancer of breast, lung, stomach, or pancreas; mela protein, low glucose large volumes of CSF; CSF exam by noma, lymphoma, leukemia; meningeal gliomatosis; meninpolarizing microscopy; clonal lympho-geal sarcoma; cerebral dysgerminoma; meningeal melanoma cyte markers; deposits on nerve roots or B cell lymphoma or meninges seen on myelogram or contrast-enhanced MRI; meningeal biopsy

1	Chemical Mononuclear or PMNs, low Contrast-enhanced CT scan or MRI; History of recent injection into the subarachnoid space; his-compounds (may glucose, elevated protein; xan-cerebral angiogram to detect tory of sudden onset of headache; recent resection of acouscause recurrent thochromia from subarachnoid aneurysm tic neuroma or craniopharyngioma; epidermoid tumor of meningitis) hemorrhage in week prior to brain or spine, sometimes with dermoid sinus tract; pituitary presentation with “meningitis” apoplexy Primary inflammation CNS sarcoidosis Mononuclear cells; elevated Serum and CSF angiotensin-CN palsy, especially of CN VII; hypothalamic dysfunction, protein; often low glucose converting enzyme levels; biopsy of especially diabetes insipidus; abnormal chest radiograph; extraneural affected tissues or brain peripheral neuropathy or myopathy lesion/meningeal biopsy

1	Recurrent meningoencephalitis with uveitis, retinal detach-Harada syndrome ment, alopecia, lightening of eyebrows and lashes, dysacou(recurrent meningitis) sia, cataracts, glaucoma Isolated granuloma-Mononuclear cells, elevated Angiography or meningeal biopsy Subacute dementia; multiple cerebral infarctions; recent tous angiitis of the protein nervous system Systemic lupus Mononuclear or PMNs Anti-DNA antibody, antinuclear Encephalopathy; seizures; stroke; transverse myelopathy; rash; erythematosus antibodies arthritis Behçet’s syndrome Mononuclear or PMNs, elevated hemorrhages; pathergic lesions at site of skin puncture Chronic benign lym-Mononuclear cells

1	Recovery in 2–6 months, diagnosis by exclusion phocytic meningitis Mollaret’s meningitis Large endothelial cells and PCR for herpes; MRI/CT to rule out Recurrent meningitis; exclude HSV-2; rare cases due to HSV-1; (recurrent meningitis) PMNs in first hours, followed by epidermoid tumor or dural cyst occasional case associated with dural cyst mononuclear cells Drug hypersensitivity PMNs; occasionally mononu-Complete blood count (eosinophilia) Exposure to nonsteroidal anti-inflammatory agents, sulfonclear cells or eosinophils amides, isoniazid, tolmetin, ciprofloxacin, penicillin, carbamazepine, lamotrigine, IV immunoglobulin, OKT3 antibodies, phenazopyridine; improvement after discontinuation of drug; recurrence with repeat exposure

1	Granulomatosis Mononuclear cells Chest and sinus radiographs; urinaly-Associated sinus, pulmonary, or renal lesions; CN palsies; skin with polyangiitis sis; ANCA antibodies in serum lesions; peripheral neuropathy (Wegener’s) Other: multiple sclerosis, Sjögren’s syndrome, monogenic autoinflammatory disorders, and rarer forms of vasculitis (e.g., Cogan’s syndrome) Abbreviations: ANCA, antineutrophil cytoplasmic antibodies; CN, cranial nerve; CSF, cerebrospinal fluid; CT, computed tomography; HSV, herpes simplex virus; MRI, magnetic resonance imaging; PCR, polymerase chain reaction; PMNs, polymorphonuclear cells.

1	parameningeal infection; identification of a potential source of outflow (obstructive hydrocephalus), then lumbar puncture carries infection (chronic draining ear, sinusitis, right-to-left cardiac or the potential risk of brain herniation. Obstructive hydrocephalus pulmonary shunt, chronic pleuropulmonary infection) supports this usually requires direct ventricular drainage. In patients with open diagnosis. In some cases, diagnosis may be established by recogni-CSF flow pathways, elevated ICP can still occur due to impaired tion and biopsy of unusual skin lesions (Behçet’s syndrome, SLE, resorption of CSF by arachnoid villi. In such patients, lumbar punccryptococcosis, blastomycosis, Lyme disease, sporotrichosis, trypano-ture is usually safe, but repetitive or continuous lumbar drainage may somiasis, IV drug use) or enlarged lymph nodes (lymphoma, sarcoid, be necessary to prevent abrupt deterioration and death from raised tuberculosis, HIV, secondary syphilis, or Whipple’s disease). A

1	somiasis, IV drug use) or enlarged lymph nodes (lymphoma, sarcoid, be necessary to prevent abrupt deterioration and death from raised tuberculosis, HIV, secondary syphilis, or Whipple’s disease). A careful ICP. In some patients, especially those with cryptococcal meningiophthalmologic examination may reveal uveitis (Vogt-Koyanagi-tis, fatal levels of raised ICP can occur without enlarged ventricles. Harada syndrome, sarcoid, or central nervous system [CNS] lym-Contrast-enhanced MRI or CT studies of the brain and spinal phoma), keratoconjunctivitis sicca (Sjögren’s syndrome), or iridocy-cord can identify meningeal enhancement, parameningeal infecclitis (Behçet’s syndrome) and is essential to assess visual loss from tions (including brain abscess), encasement of the spinal cord papilledema. Aphthous oral lesions, genital ulcers, and hypopyon (malignancy, inflammation or infection), or nodular deposits on the suggest Behçet’s syndrome. Hepatosplenomegaly suggests lymphoma, meninges or

1	Aphthous oral lesions, genital ulcers, and hypopyon (malignancy, inflammation or infection), or nodular deposits on the suggest Behçet’s syndrome. Hepatosplenomegaly suggests lymphoma, meninges or nerve roots (malignancy or sarcoidosis) (Fig. 165-1). sarcoid, tuberculosis, or brucellosis. Herpetic lesions in the genital Imaging studies are also useful to localize areas of meningeal dis-area or on the thighs suggest HSV-2 infection. A breast nodule, a sus-ease prior to meningeal biopsy. picious pigmented skin lesion, focal bone pain, or an abdominal mass Angiographic studies can identify evidence of cerebral arteritis directs attention to possible carcinomatous meningitis. in patients with chronic meningitis and stroke.

1	Once the clinical syndrome is recognized as a potential manifesta-The CSF pressure should be measured and samples sent for bactetion of chronic meningitis, proper analysis of the CSF is essential. rial, fungal, and tuberculous culture; Venereal Disease Research However, if the possibility of raised ICP exists, a brain imaging study Laboratories (VDRL) test; cell count and differential; Gram’s should be performed before lumbar puncture. If ICP is elevated stain; and measurement of glucose and protein. Wet mount for because of a mass lesion, brain swelling, or a block in ventricular CSF fungus and parasites, india ink preparation and culture, culture

1	FIGURE 165-1 Primary central nervous system lymphoma. A 24-year-old man, immunosuppressed due to intestinal lymphangiectasia, developed multiple cranial neuropathies. CSF findings consisted of 100 lymphocytes/μL and a protein of 2.5 g/L (250 mg/dL); cytology and cultures were negative. Gadolinium-enhanced T1 MRI revealed diffuse, multifocal meningeal enhancement surrounding the brain-stem (A), spinal cord, and cauda equina (B).

1	for fastidious bacteria and fungi, assays for cryptococcal antigen and oligoclonal immunoglobulin bands, and cytology should be performed. Other specific CSF tests (Tables 165-2 and 165-3) or blood tests and cultures should be ordered as indicated on the basis of the history, physical examination, or preliminary CSF results (i.e., eosinophilic, mononuclear, or polymorphonuclear meningitis). Rapid diagnosis may be facilitated by serologic tests and polymerase chain reaction (PCR) testing to identify DNA sequences in the CSF that are specific for the suspected pathogen. In patients with suspected fungal infections, when other tests are negative, assays for beta-glucans may be a useful adjunct in establishing the diagnosis.

1	In most categories of chronic (not recurrent) meningitis, mononuclear cells predominate in the CSF. When neutrophils predominate after 3 weeks of illness, the principal etiologic considerations are Nocardia asteroides, Actinomyces israelii, Brucella, Mycobacterium tuberculosis (5–10% of early cases only), various fungi (Blastomyces dermatitidis, Candida albicans, Histoplasma capsulatum, Aspergillus spp., Pseudallescheria boydii, Cladophialophora bantiana), and noninfectious causes (SLE, exogenous chemical meningitis). When eosinophils predominate or are present in limited numbers in a primarily mononuclear cell response in the CSF, the differential diagnosis includes parasitic diseases (A. cantonensis, G. spinigerum, B. procyonis, or Toxocara canis infection, cysticercosis, schistosomiasis, echinococcal disease, T. gondii infection), fungal infections (6–20% eosinophils along with a predominantly lymphocyte pleocytosis, particularly with coccidioidal meningitis), neoplastic disease

1	echinococcal disease, T. gondii infection), fungal infections (6–20% eosinophils along with a predominantly lymphocyte pleocytosis, particularly with coccidioidal meningitis), neoplastic disease (lymphoma, leukemia, metastatic carcinoma), or other inflammatory processes (sarcoidosis, hypereosinophilic syndrome).

1	It is often necessary to broaden the number of diagnostic tests if the initial workup does not reveal the cause. In addition, repeated samples of large volumes of CSF may be required to diagnose certain infectious and malignant causes of chronic meningitis. Flow cytometry for malignant cells may be useful in patients with suspected carcinomatous meningitis. Lymphomatous or carcinomatous meningitis may be diagnosed by examination of sections cut from a cell block formed by spinning down the sediment from a large volume of CSF. The diagnosis of fungal meningitis may require large volumes of CSF for culture of sediment. If standard lumbar puncture is unrewarding, a cervical cisternal tap to sample CSF near to the basal meninges may be fruitful.

1	In addition to the CSF examination, an attempt should be made to uncover pertinent underlying illnesses. Tuberculin skin test, chest radiograph, urine analysis and culture, blood count and differential, renal and liver function tests, alkaline phosphatase, sedimentation rate, antinuclear antibody, anti-Ro antibody, anti-La antibody, and serum angiotensin-converting enzyme level are often indicated. In some cases, a thorough search for a systemic site of infection is indicated. Pulmonary foci of infection may be present, particularly with fungal or tuberculous disease. Hence a CT or MRI of the chest and a sputum examination may be helpful. Abnormalities can be pursued by bronchoscopy or transthoracic needle biopsy. A tuberculin skin test is often placed, although the test has limited specificity and sensitivity for diagnosis of active disease. Liver or bone marrow biopsy may be diagnostic in some cases of miliary tuberculosis, disseminated fungal infection, sarcoidosis, or metastatic

1	and sensitivity for diagnosis of active disease. Liver or bone marrow biopsy may be diagnostic in some cases of miliary tuberculosis, disseminated fungal infection, sarcoidosis, or metastatic malignancy. Positron emission tomography with fluorodeoxyglucose may be useful in identifying a systemic site for biopsy in patients with suspected carcinomatous meningitis or sarcoidosis when other tests are unrevealing. Genetic testing can identify mutations that cause rare monogenic autoinflammatory disorders.

1	If CSF is not diagnostic then a meningeal biopsy should be strongly considered in patients who are severely disabled, who need chronic ventricular decompression, or whose illness is progressing rapidly. The activities of the surgeon, pathologist, microbiologist, and cytologist should be coordinated so that a large enough sample is obtained and the appropriate cultures and histologic and molecular studies, including electron-microscopic and PCR studies, are performed. The diagnostic yield of meningeal biopsy can be increased by targeting regions that enhance with contrast on MRI or CT. With current microsurgical techniques, most areas of the basal meninges can be accessed for biopsy via a limited craniotomy. In a series from the Mayo Clinic reported by TM Cheng et al. (Neurosurgery 34:590, 1994), MRI demonstrated meningeal enhancement in 47% of patients undergoing meningeal biopsy. Biopsy of an enhancing region was diagnostic in 80% of cases; biopsy of nonenhancing regions was

1	34:590, 1994), MRI demonstrated meningeal enhancement in 47% of patients undergoing meningeal biopsy. Biopsy of an enhancing region was diagnostic in 80% of cases; biopsy of nonenhancing regions was diagnostic in only 9%; sarcoid (31%) and metastatic adenocarcinoma (25%) were the most common conditions identified. Tuberculosis is the most common condition identified in many reports from outside the United States.

1	In approximately one-third of cases, the diagnosis is not known despite careful evaluation of CSF and potential extraneural sites of disease. A number of the organisms that cause chronic meningitis may take weeks to be identified by cultures. In enigmatic cases, several options are available, determined by the extent of the clinical deficits and rate of progression. It is prudent to wait until cultures are finalized if the patient is asymptomatic or symptoms are mild and not progressive. Unfortunately, in many cases progressive neurologic deterioration occurs, and rapid treatment is required. Ventricular-peritoneal shunts may be placed to relieve hydrocephalus, but the risk of disseminating the undiagnosed inflammatory process into the abdomen must be considered.

1	Empirical Treatment Diagnosis of the causative agent is essential because effective therapies exist for many etiologies of chronic meningitis, but if the condition is left untreated, progressive damage to the CNS and cranial nerves and roots is likely to occur. Occasionally, empirical therapy must be initiated when all attempts at diagnosis fail. In general, empirical therapy in the United States consists of antimycobacterial agents, amphotericin for fungal infection, or glucocorticoids for noninfectious inflammatory causes. It is important to direct empirical therapy of lymphocytic meningitis at tuberculosis, particularly if the condition is associated with low CSF glucose and sixth and other CN palsies, since untreated disease can be devastating within weeks. Patients on prolonged anti–tumor necrosis factor therapy who develop chronic meningitis also should be treated empirically with antituberculous therapy if the etiology is uncertain. In the Mayo Clinic series, the most useful

1	necrosis factor therapy who develop chronic meningitis also should be treated empirically with antituberculous therapy if the etiology is uncertain. In the Mayo Clinic series, the most useful empirical therapy was administration of glucocorticoids rather than antituberculous therapy. Carcinomatous or lymphomatous meningitis may be difficult to diagnose initially, but the diagnosis becomes evident with time.

1	Chronic meningitis is not uncommon in the course of HIV infection. Pleocytosis and mild meningeal signs often occur at the onset of HIV infection, and occasionally low-grade meningitis persists. Toxoplasmosis commonly presents as intracranial abscesses and also may be associated with meningitis. Other important causes of chronic meningitis in AIDS include infection with Cryptococcus, Nocardia, Candida, or other fungi; syphilis; and lymphoma (Fig. 165-1). Toxoplasmosis, cryptococcosis, nocardiosis, and other fungal infections are important etiologic considerations in individuals with immunodeficiency states other than AIDS, including those due to immunosuppressive medications. Because of the increased risk of chronic meningitis and the attenuation of clinical signs of meningeal irritation in immunosuppressed individuals, CSF examination should be performed for any persistent headache or unexplained change in mental state.

1	Morton N. Swartz contributed to earlier editions of this chapter. Infectious Complications of Burns Lawrence C. Madoff, Florencia Pereyra The skin is an essential component of immunity, protecting the host from potential pathogens in the environment. Breaches in this 166e protective barrier thus represent a form of immunocompromise that predisposes the patient to infection. Thermal burns may cause massive destruction of the integument as well as derangements in humoral and cellular immunity, permitting the development of infection caused by environmental opportunists and components of the host’s skin flora.

1	Over the past decade, the estimated incidence of burn injuries in the United States has steadily declined; still, however, >1 million burn injuries are brought to medical attention each year. While many burn injuries are minor and require little or no intervention, 183,000 cases were reported between 2002 and 2011 to the National Burn Repository from specialized burn care facilities; of the 45,000 persons hospitalized for these injuries, 60% required intensive care and 20,000 had major burns involving at least 25% of the total body surface area. The majority of burn patients are men. Children under the age of 5 account for ~20% of all reported cases. Scalds, structural fires, and flammable liquids and gases are the major causes of burns, but electrical, chemical, and smoking-related sources also are important. Burns predispose to infection by damaging the protective barrier function of the skin, thus facilitating the entry of pathogenic microorganisms, and by inducing systemic

1	sources also are important. Burns predispose to infection by damaging the protective barrier function of the skin, thus facilitating the entry of pathogenic microorganisms, and by inducing systemic immunosuppression. It is therefore not surprising that multiorgan failure and infectious complications are the major causes of morbidity and death in serious burn injury. More than 3000 patients in the United States die of burn-related infections each year, and 6 of the top 10 complications recently identified by the American Burn Association’s 10-year review are infectious: pneumonia (4.6%), septicemia (2.7%), cellulitis/traumatic injury (2.6%), respiratory failure (2.5%), wound infection (2.2%), another infection (2.0%), renal failure (1.5%), line infection (1.4%), acute respiratory distress syndrome (1.2%), and arrhythmia (1.0%).

1	Loss of the cutaneous barrier facilitates entry of the patient’s own flora and of organisms from the hospital environment into the burn wound. Initially, the wound is colonized with gram-positive bacteria from the surrounding tissue, but the number of bacteria grows rapidly beneath the burn eschar, reaching ~8.4 × 103 cfu/g on day 4 after the burn. The avascularity of the eschar, along with the impairment of local immune responses, favors further bacterial colonization and proliferation. By day 7, the wound is colonized with other microbes, including gram-positive bacteria, gram-negative bacteria, and yeasts derived from the gastrointestinal and upper respiratory flora. Invasive infection—localized and/or systemic—occurs when these bacteria penetrate viable tissue. In addition, a role for biofilms has been recognized in experimental animal models of burn-wound infection. (Biofilms are surface-associated communities of bacteria, often embedded in a matrix, that allow the microbes to

1	biofilms has been recognized in experimental animal models of burn-wound infection. (Biofilms are surface-associated communities of bacteria, often embedded in a matrix, that allow the microbes to persist and to resist the effects of host immunity and antimicrobial agents.)

1	Streptococci and staphylococci were the predominant causes of burn-wound infection in the preantibiotic era and remain important pathogens at present. With the advent of antimicrobial agents, Pseudomonas aeruginosa became a major problem in burn-wound management. In animal models of cutaneous thermal injury and wound infection with Pseudomonas, there is an early, steady increase of neutrophils in the skin and bacterial dissemination to lungs and spleen within 72 h. Less common anaerobic bacteria are typically found in infections of electrical burns or when open wound dressings are used. The widespread use of topical and more effective antimicrobial agents has resulted in a decline in bacterial wound infections and the emergence of fungi (particularly Candida albicans, Aspergillus 166e-1 species, and the agents of mucormycosis) as increasingly important pathogens in burn-wound patients. Herpes simplex virus also has been found in burn wounds, especially those on the neck and face and

1	species, and the agents of mucormycosis) as increasingly important pathogens in burn-wound patients. Herpes simplex virus also has been found in burn wounds, especially those on the neck and face and those associated with inhalation injury. Cytomegalovirus viremia has been described in up to 71% of seropositive burn patients in prospective studies, and high levels (>1000 copies/mL) have been associated with increased duration of mechanical ventilation and longer stay in the intensive care unit (ICU).

1	Autopsy reports from patients with severe thermal burns over the last decade have identified P. aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus in association with mortality, independent of the percentage of total body surface area burned, the percentage of full-thickness burn, inhalation injury, and day of death after the burn. Indeed, burn trauma patients who acquire secondary P. aeruginosa infection have a fourfold greater mortality rate than those without P. aeruginosa. Historically, mortality rates among burn patients infected with P. aeruginosa have been as high as 77% over a 25-year period. In addition, Acinetobacter calcoaceticus-baumannii is among the top pathogens in some burn centers.

1	The cascade of events that follow a severe burn injury and that lead to multiorgan system failure and death is thought to represent a two-step process. The burn injury itself, with ensuing hypovolemia and tissue hypoxia, is followed by invasive infection arising from large amounts of devitalized tissue. The frequency of infection parallels the extent and severity of the burn injury. Severe burn injuries cause a state of immunosuppression that affects innate and adaptive immune responses. The substantial impact of immunocompromise on infection is due to effects on both the cellular and the humoral arms of the immune system. For example, decreases in the number and activity of circulating helper T cells, increases in suppressor T cells, decreases in production and release of monocytes and macrophages, and diminution in levels of immunoglobulin follow major burns. Neutrophil and complement functions also are impaired after burns. The increased levels of multiple cytokines detected in

1	macrophages, and diminution in levels of immunoglobulin follow major burns. Neutrophil and complement functions also are impaired after burns. The increased levels of multiple cytokines detected in burn patients are compatible with the widely held belief that the inflammatory response becomes dysregulated in these individuals; bacterial cell products play a potent role in inducing proinflammatory mediators that contribute to this uncontrolled systemic inflammatory response. Increased permeability of the gut wall to bacteria and their components (e.g., endotoxin) also contributes to immune dysregulation and sepsis. Thus, the burn patient is predisposed to infection at remote sites (see below) as well as at the sites of burn injury. Another contributor to secondary immunosuppression after burn injuries is the endocrine system; increasing levels of vasopressin, aldosterone, cortisol, glucagon, growth hormone, catecholamines, and other hormones that directly affect lymphocyte

1	after burn injuries is the endocrine system; increasing levels of vasopressin, aldosterone, cortisol, glucagon, growth hormone, catecholamines, and other hormones that directly affect lymphocyte proliferation, secretion of proinflammatory cytokines, natural killer cell activity, and suppressive T cells are seen.

1	Since clinical indications of wound infection are difficult to interpret, wounds must be monitored carefully for changes that may reflect infection. A margin of erythema frequently surrounds the sites of burns and by itself is not usually indicative of infection. Signs of infection include the conversion of a partial-thickness to a full-thickness burn, color changes (e.g., the appearance of a dark brown or black discoloration of the wound), the new appearance of erythema or violaceous edema in normal tissue at the wound margins, the sudden separation of the eschar from subcutaneous tissues, and the degeneration of the wound with the appearance of a new eschar.

1	Early surgical excision of devitalized tissue is now widely used, and burn-wound infections can be classified in relation to the excision site as (1) burn-wound impetigo (infection characterized by loss of epithelium from a previously re-epithelialized surface, as seen in a partial-thickness burn that is allowed to close by secondary intention, a grafted burn, or a healed skin donor site); (2) burn-related surgical-wound infection (purulent infection of excised burn and donor sites that have not yet epithelialized, accompanied by positive CHAPTER 166e Infectious Complications of Burns FIGurE 166e-1 Cellulitis complicating a burn wound of the arm, with extension of the infection to adjacent healthy tissue.

1	CHAPTER 166e Infectious Complications of Burns FIGurE 166e-1 Cellulitis complicating a burn wound of the arm, with extension of the infection to adjacent healthy tissue. (Courtesy of Dr. Robert L. Sheridan, Massachusetts General Hospital, Boston; with permission.) cultures); (3) burn-wound cellulitis (extension of infection to surrounding healthy tissue; Fig. 166e-1); and (4) invasive infection in unexcised burn wounds (infection that is secondary to a partialor full-thickness burn wound and is manifested by separation of the eschar or by violaceous, dark brown, or black discoloration of the eschar; Fig. 166e-2). The appearance of a green discoloration of the wound or subcutaneous fat (Fig. 166e-3) or the development of ecthyma gangrenosum (see Fig. 25e-35) at a remote site points to a diagnosis of invasive P. aeruginosa infection.

1	Changes in body temperature, hypotension, tachycardia, altered mentation, neutropenia or neutrophilia, thrombocytopenia, and renal failure may result from invasive burn wounds and sepsis. However, because profound alterations in homeostasis occur as a consequence of burns per se and because inflammation without infection is a normal component of these injuries, the assessment of these changes is complicated. Alterations in body temperature, for example, are attributable to thermoregulatory dysfunction; tachycardia and hyperventilation accompany the metabolic changes induced by extensive burn injury and are not necessarily indicative of bacterial sepsis. Given the difficulty of evaluating burn wounds solely on the basis of clinical observation and laboratory data, wound biopsies are necessary FIGurE 166e-2 A severe upper-extremity burn infected with Pseudomonas aeruginosa. The wound requires additional debride-ment. Note the dark brown to black discoloration of the eschar.

1	FIGurE 166e-2 A severe upper-extremity burn infected with Pseudomonas aeruginosa. The wound requires additional debride-ment. Note the dark brown to black discoloration of the eschar. (Courtesy of Dr. Robert L. Sheridan, Massachusetts General Hospital, Boston; with permission.)

1	FIGurE 166e-3 A burn wound infected with Pseudomonas aeruginosa, with liquefaction of tissue. Note the green discoloration at the wound margins, which is suggestive of Pseudomonas infection. (Courtesy of Dr. Robert L. Sheridan, Massachusetts General Hospital, Boston; with permission.) for definitive diagnosis of infection. The timing of these biopsies can be guided by clinical changes, but in some centers burn wounds are routinely biopsied at regular intervals. The biopsy specimen is examined for histologic evidence of bacterial invasion, and quantitative microbiologic cultures are performed. The presence of >105 viable bacteria per gram of tissue is highly suggestive of invasive infection and of a dramatically increased risk of sepsis. Histopathologic evidence of the invasion of viable tissue and the presence of microorganisms in unburned blood vessels and lymphatics is a more definitive indicator of infection. A blood culture positive for the same organism seen in large quantities

1	tissue and the presence of microorganisms in unburned blood vessels and lymphatics is a more definitive indicator of infection. A blood culture positive for the same organism seen in large quantities in biopsied tissue is a reliable indicator of burn sepsis. Surface cultures may provide some indication of the microorganisms present in the hospital environment but are not indicative of the etiology of infection. This noninvasive technique may be of use in determining the flora present in excised burn areas or in areas where the skin is too thin for biopsy (e.g., over the ears, eyes, or digits). Rapid identification of organisms and institution of appropriate therapy are critical to the survival of patients with severe burn injury; polymerase chain reaction (PCR) is now being used for rapid identification of specific pathogens, sometimes in <6 h, to allow earlier treatment interventions.

1	In addition to infection of the burn wound itself, a number of other infections due to the immunosuppression caused by extensive burns and the manipulations necessary for clinical care put burn patients at risk. Pneumonia, now the most common infectious complication among hospitalized burn patients, is most often acquired nosocomially via the respiratory route. The incidence of ventilator-associated pneumonia among burn patients is 22–30 cases per 1000 ventilator-days—more than double that among surgical or medical ICU cohorts; this infection usually results from colonization of the lower respiratory tract and parenchyma because of sustained microaspiration. Among the risk factors associated with secondary pneumonia are inhalation injury, intubation, full-thickness chest wall burns, cutaneous thermal injuries, immobility, blood transfusions, and uncontrolled wound sepsis with hematogenous spread. Septic pulmonary emboli also may occur. Suppurative thrombophlebitis may complicate the

1	thermal injuries, immobility, blood transfusions, and uncontrolled wound sepsis with hematogenous spread. Septic pulmonary emboli also may occur. Suppurative thrombophlebitis may complicate the vascular catheterization necessary for fluid and nutritional support in burns. Endocarditis, urinary tract infection, bacterial chondritis (particularly in patients with burned ears), and intraabdominal infection also complicate serious burn injury. Staphylococcal scalded skin syndrome due to burn-wound infection with S. aureus has been described as a rare complication. Finally, burn surgical-wound infections contribute to morbidity and have been found in up to 39% of patients; these infections often result in repeat skin grafting and prolonged hospitalization.

1	against gram-positive pathogens (e.g., oxacillin, 2 g IV every 4 h) and 166e-3 with a drug active against P. aeruginosa and other gram-negative

1	The ultimate goal of burn-wound management is closure and healing of the wound. Early surgical excision of burned tissue, with extensive debridement of necrotic tissue and grafting of skin or skin substitutes, greatly decreases mortality rates associated with severe burns. In addition, the four widely used topical antimicrobial agents—silver sulfadiazine cream, mafenide acetate cream, silver nitrate cream, and nanocrystalline silver dressings—dramatically decrease the bacterial burden of burn wounds and reduce the incidence of burn-wound infection; these agents are routinely applied to partial-and full-thickness burns. The bactericidal properties of silver are related to its effect on respiratory enzymes on bacterial cell walls; its interaction with structural proteins causes keratinocyte and fibroblast toxicity that can delay wound healing if silver-based compounds are used indiscriminately. All four agents are broadly active against many bacteria and some fungi and are useful before

1	and fibroblast toxicity that can delay wound healing if silver-based compounds are used indiscriminately. All four agents are broadly active against many bacteria and some fungi and are useful before bacterial colonization is established. Silver sulfadiazine is often used initially, but its value can be limited by bacterial resistance, poor wound penetration, or toxicity (leukopenia). Mafenide acetate has broader activity against gram-negative bacteria. The cream penetrates eschars and thus can prevent or treat infection beneath them; its use without dressings allows regular examination of the wound area. The foremost disadvantages of mafenide acetate are that it can inhibit carbonic anhydrase, resulting in metabolic acidosis, and that it elicits hypersensitivity reactions in up to 7% of patients. This agent is most often used when gram-negative bacteria invade the burn wound and when treatment with silver sulfadiazine fails. The activity of mafenide acetate against gram-positive

1	of patients. This agent is most often used when gram-negative bacteria invade the burn wound and when treatment with silver sulfadiazine fails. The activity of mafenide acetate against gram-positive bacteria is limited. Nanocrystalline silver dressings provide broader antimicrobial coverage than any other available topical preparation, exhibiting activity against methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci (VRE), moderate ability to penetrate eschars, and limited toxicity. In addition, this approach provides controlled and prolonged release of nanocrystalline silver into the wound, limiting the number of dressing changes and therefore reducing the risk of nosocomial infections as well as the cost of treatment. Mupirocin, a topical antimicrobial agent used to eradicate nasal colonization with MRSA, is increasingly being used in burn units where MRSA is prevalent. The efficacy of mupirocin in reducing burn-wound bacterial counts and preventing systemic

1	to eradicate nasal colonization with MRSA, is increasingly being used in burn units where MRSA is prevalent. The efficacy of mupirocin in reducing burn-wound bacterial counts and preventing systemic infections is comparable to that of silver sulfadiazine.

1	In recent years, rates of fungal infection have increased in burn patients. When superficial fungal infection occurs, nystatin may be mixed with silver sulfadiazine or mafenide acetate as topical therapy. A small study found that nystatin powder (6 million units/g) was effective for treatment of superficial and deep burn-wound infections caused by Aspergillus or Fusarium species. In addition to these products, moisture-retention ointments with antimicrobial properties can promote rapid autolysis, debridement, and moist healing of partial-thickness wounds.

1	When invasive wound infection is diagnosed, topical therapy should be changed to mafenide acetate. Subeschar clysis (the direct instillation of an antibiotic, often piperacillin, into wound tissues under the eschar) is a useful adjunct to surgical and systemic antimicrobial therapy. Systemic treatment with antibiotics active against the pathogens present in the wound should be instituted. In the absence of culture data, treatment should be broad in spectrum, covering organisms commonly encountered in that particular burn unit. Such coverage is usually achieved with an antibiotic active rods (e.g., mezlocillin, 3 g IV every 4 h; gentamicin, 5 mg/kg IV per day). In penicillin-allergic patients, vancomycin (1 g IV every 12 h) may be substituted for oxacillin (and is efficacious against MRSA), and ciprofloxacin (400 mg IV every 12 h) may be substituted for mezlocillin. Oxazolidinone antibiotics like linezolid have demonstrated efficacy in reducing bacterial growth and toxic shock syndrome

1	and ciprofloxacin (400 mg IV every 12 h) may be substituted for mezlocillin. Oxazolidinone antibiotics like linezolid have demonstrated efficacy in reducing bacterial growth and toxic shock syndrome toxin 1 levels in animal models of MRSA burn-wound infections.

1	Patients with burn wounds frequently have alterations in metabolism and renal clearance mechanisms that mandate the monitoring of serum antibiotic levels. The levels achieved with standard doses are often subtherapeutic. Treatment of infections caused by emerging resistant pathogens remains a challenge in the care of burn patients. MRSA, resistant enterococci, multidrug-resistant gram-negative rods, and Enterobacteriaceae producing extended-spectrum β-lactamases have been associated with burn-wound infections and identified in burn-unit outbreaks. Strict infection-control practices (including microbiologic surveillance in burn units) and appropriate antimicrobial therapy remain important measures in reducing rates of infection due to resistant organisms.

1	In general, prophylactic systemic antibiotics have no role in the management of burn wounds and can, in fact, lead to colonization with resistant microorganisms. In some studies, antibiotic prophylaxis has been associated with increases in secondary infections of the upper and lower respiratory tract and the urinary tract as well as with prolonged hospitalization. An exception involves cases requiring burn-wound manipulation. Since procedures such as debridement, excision, or grafting frequently result in bacteremia, prophylactic systemic antibiotics are administered at the time of wound manipulation; the specific agents used should be chosen on the basis of data obtained by wound culture or data on the hospital’s resident flora.

1	The use of oral antibiotics for selective digestive decontamination (SDD) to decrease bacterial colonization and the risk of burn-wound infection is controversial and has not been widely adopted. In a randomized, double-blind, placebo-controlled trial in patients with burns involving >20% of the total body surface area, SDD was associated with reduced mortality rates in the burn ICU and in the hospital and also with a reduced incidence of pneumonia. The effects of SDD on the normal anaerobic bowel flora must be taken into consideration before this approach is used.

1	Strategies to reduce or limit systemic spread of wound infections, particularly to the lung, may be useful adjuncts to therapy. Some of these strategies are aimed at reducing neutrophilic inflammation at the site of injury, which can accelerate biofilm formation, particularly by P. aeruginosa. For example, in animal models of cutaneous burns with P. aeruginosa wound inoculation, a single dose of azithromycin administered early reduces rates of Pseudomonas infection and systemic spread to lung and spleen and appears to have effects similar to those of classic anti-Pseudomonas agents, such as tobramycin. The extent to which azithromycin can be administered early in humans to prevent dissemination remains to be studied.

1	All burn-injury patients should undergo tetanus booster immunization if they have completed primary immunization but have not received a booster dose in the past 5 years. Patients without prior immunization should receive tetanus immune globulin and undergo primary immunization. CHAPTER 166e Infectious Complications of Burns Infectious Complications of Bites Lawrence C. Madoff, Florencia Pereyra The skin is an essential component of nonspecific immunity, protect-ing the host from potential pathogens in the environment. Breaches in 167e this protective barrier thus represent a form of immunocompromise that predisposes the patient to infection. Bites and scratches from animals and humans allow the inoculation of microorganisms past the skin’s protective barrier into deeper, susceptible host tissues.

1	Each year in the United States, millions of animal-bite wounds are sustained. The vast majority are inflicted by pet dogs and cats, which number >100 million; the annual incidence of dog and cat bites has been reported as 300 bites per 100,000 population. Other bite wounds are a consequence of encounters with animals in the wild or in occupational settings. While many of these wounds require minimal or no therapy, a significant number result in infection, which may be life-threatening. The microbiology of bite-wound infections in general reflects the oropharyngeal flora of the biting animal, although organisms from the soil, the skin of the animal and the victim, and the animal’s feces may also be involved.

1	In the United States, dogs bite >4.7 million people each year and are responsible for 80% of all animal-bite wounds, an estimated 15–20% of which become infected. Each year, 800,000 Americans seek medical attention for dog bites; of those injured, 386,000 require treatment in an emergency department, with >1000 emergency department visits each day and about a dozen deaths per year. Most dog bites are provoked and are inflicted by the victim’s pet or by a dog known to the victim. These bites are frequently sustained during efforts to break up a dogfight. Children are more likely than adults to sustain canine bites, with the highest incidence of 6 bites/1000 population among boys 5–9 years old. Victims are more often male than female, and bites most often involve an upper extremity. Among children <4 years old, two-thirds of all these injuries involve the head or neck. Infection typically manifests 8–24 h after the bite as pain at the site of injury with cellulitis accompanied by

1	children <4 years old, two-thirds of all these injuries involve the head or neck. Infection typically manifests 8–24 h after the bite as pain at the site of injury with cellulitis accompanied by purulent, sometimes foul-smelling discharge. Septic arthritis and osteomyelitis may develop if a canine tooth penetrates synovium or bone. Systemic manifestations (e.g., fever, lymphadenopathy, and lymphangitis) also may occur. The microbiology of dog-bite wound infections is usually mixed and includes β-hemolytic streptococci, Pasteurella species, Staphylococcus species (including methicillin-resistant Staphylococcus aureus [MRSA]), Eikenella corrodens, and Capnocytophaga canimorsus. Many wounds also include anaerobic bacteria such as Actinomyces, Fusobacterium, Prevotella, and Porphyromonas species.

1	While most infections resulting from dog-bite injuries are localized to the area of injury, many of the microorganisms involved are capable of causing systemic infection, including bacteremia, meningitis, brain abscess, endocarditis, and chorioamnionitis. These infections are particularly likely in hosts with edema or compromised lymphatic drainage in the involved extremity (e.g., after a bite on the arm in a woman who has undergone mastectomy) and in patients who are immunocompromised by medication or disease (e.g., glucocorticoid use, systemic lupus erythematosus, acute leukemia, or hepatic cirrhosis). In addition, dog bites and scratches may result in systemic illnesses such as rabies (Chap. 232) and tetanus (Chap. 177).

1	Infection with C. canimorsus following dog-bite wounds may result in fulminant sepsis, disseminated intravascular coagulation, and renal failure, particularly in hosts who have impaired hepatic function, who have undergone splenectomy, or who are immunosuppressed. This thin gram-negative rod is difficult to culture on most solid media but grows in a variety of liquid media. The bacteria are occasionally seen within polymorphonuclear leukocytes on Wright-stained smears of peripheral blood from septic patients. Tularemia (Chap. 195) also has 167e-1 been reported to follow dog bites.

1	Although less common than dog bites, cat bites and scratches result in infection in more than half of all cases. Because the cat’s narrow, sharp canine teeth penetrate deeply into tissue, cat bites are more likely than dog bites to cause septic arthritis and osteomyelitis; the development of these conditions is particularly likely when punctures are located over or near a joint, especially in the hand. Women sustain cat bites more frequently than do men. These bites most often involve the hands and arms. Both bites and scratches from cats are prone to infection from organisms in the cat’s oropharynx. Pasteurella multocida, a normal component of the feline oral flora, is a small gram-negative coccobacillus implicated in the majority of cat-bite wound infections. Like that of dog-bite wound infections, however, the microflora of cat-bite wound infections is usually mixed. Other microorganisms causing infection after cat bites are similar to those causing dog-bite wound infections.

1	The same risk factors for systemic infection following dog-bite wounds apply to cat-bite wounds. Pasteurella infections tend to advance rapidly, often within hours, causing severe inflammation accompanied by purulent drainage; Pasteurella may also be spread by respiratory droplets from animals, resulting in pneumonia or bacteremia. Like dog-bite wounds, cat-bite wounds may result in the transmission of rabies or in the development of tetanus. Infection with Bartonella henselae causes cat-scratch disease (Chap. 197) and is an important late consequence of cat bites and scratches. Tularemia (Chap. 195) also has been reported to follow cat bites.

1	Infections have been attributed to bites from many animal species. Often these bites are sustained as a consequence of occupational exposure (farmers, laboratory workers, veterinarians) or recreational exposure (hunters and trappers, wilderness campers, owners of exotic pets). Generally, the microflora of bite wounds reflects the oral flora of the biting animal. Most members of the cat family, including feral cats, harbor P. multocida. Bite wounds from aquatic animals such as alligators or piranhas may contain Aeromonas hydrophila. Venomous snakebites (Chap. 474) result in severe inflammatory responses and tissue necrosis—conditions that render these injuries prone to infection. The snake’s oral flora includes many species of aerobes and anaerobes, such as Pseudomonas aeruginosa, Serratia marcescens, Proteus species, Staphylococcus epidermidis, Bacteroides fragilis, and Clostridium species. Bites from nonhuman primates are highly susceptible to infection with pathogens similar to

1	marcescens, Proteus species, Staphylococcus epidermidis, Bacteroides fragilis, and Clostridium species. Bites from nonhuman primates are highly susceptible to infection with pathogens similar to those isolated from human bites (see below). Bites from Old World monkeys (Macaca) may also result in the transmission of B virus (Herpesvirus simiae, cercopithecine herpesvirus), a cause of serious infection of the human central nervous system. Bites of seals, walruses, and polar bears may cause a chronic suppurative infection known as seal finger, which is probably due to one or more species of Mycoplasma colonizing these animals.

1	Small rodents, including rats, mice, and gerbils, as well as ani iformis (a microaerophilic, pleomorphic gram-negative rod) or Spirillum minor (a spirochete); these organisms cause a clinical illness known as rat-bite fever. The vast majority of cases in the United States are streptobacillary, whereas Spirillum infection occurs mainly in Asia.

1	In the United States, the risk of rodent bites is usually greatest among laboratory workers or inhabitants of rodent-infested dwellings (particularly children). Rat-bite fever is distinguished from acute bite-wound infection by its typical manifestation after the initial wound has healed. Streptobacillary disease follows an incubation period of 3–10 days. Fever, chills, myalgias, headache, and severe migratory arthralgias are usually followed by a maculopapular rash, which characteristically involves the palms and soles and may become confluent or purpuric. Complications include endocarditis, myocarditis, meningitis, pneumonia, and abscesses in many organs. Haverhill fever is an

1	S. moniliformis infection acquired from contaminated milk or drinking water and has similar manifestations. Streptobacillary rat-bite fever was frequently fatal in the preantibiotic era. The differential diagnosis includes Rocky Mountain spotted fever, Lyme disease, leptospirosis, and secondary syphilis. The diagnosis is made by direct observation of the causative organisms in tissue or blood, by culture of the organisms on enriched media, or by serologic testing with specific agglutinins. Spirillum infection (referred to in Japan as sodoku) causes pain and purple swelling at the site of the initial bite, with associated lymphangitis and regional lymphadenopathy, after an incubation period of 1–4 weeks. The systemic illness includes fever, chills, and headache. The original lesion may eventually progress to an eschar. The infection is diagnosed by direct visualization of the spirochetes in blood or tissue or by animal inoculation.

1	Finally, NO-1 (CDC nonoxidizer group 1) is a bacterium associated with dogand cat-bite wounds. Infections in which NO-1 has been isolated have tended to manifest locally (i.e., as abscess and cellulitis). These infections have occurred in healthy persons with no underlying illness and in some instances have progressed from localized to systemic illnesses. The phenotypic characteristics of NO-1 are similar to those of asaccharolytic Acinetobacter species; i.e., NO-1 is oxidase-, indole-, and urease-negative. To date, all strains identified have been shown to be susceptible to aminoglycosides, β-lactam antibiotics, tetracyclines, quinolones, and sulfonamides.

1	Human bites may be self-inflicted; may be sustained by medical personnel caring for patients; or may take place during fights, domestic abuse, or sexual activity. Human-bite wounds become infected more frequently (~10–15% of the time) than do bites inflicted by other animals. These infections reflect the diverse oral microflora of humans, which includes multiple species of aerobic and anaerobic bacteria. Common aerobic isolates include viridans streptococci, S. aureus,

1	E. corrodens (which is particularly common in clenched-fist injury; see below), and Haemophilus influenzae. Anaerobic species, including Fusobacterium nucleatum and Prevotella, Porphyromonas, and Peptostreptococcus species, are isolated from 50% of wound infections due to human bites; many of these isolates produce β-lactamases. The oral flora of hospitalized and debilitated patients often includes Enterobacteriaceae in addition to the usual organisms. Hepatitis B, hepatitis C, herpes simplex virus infection, syphilis, tuberculosis, actinomycosis, and tetanus have been reported to be transmitted by human bites; it is biologically possible to transmit HIV through human bites, although this event is quite unlikely.

1	Human bites are categorized as either occlusional injuries, which are inflicted by actual biting, or clenched-fist injuries, which are sustained when the fist of one individual strikes the teeth of another, causing traumatic laceration of the hand. For several reasons, clenched-fist injuries, which are sometimes referred to as “fight bite” and which are more common than occlusional injuries, result in particularly serious infections. The deep spaces of the hand, including the bones, joints, and tendons, are frequently inoculated with organisms in the course of such injuries. The clenched position of the fist during injury, followed by extension of the hand, may further promote the introduction of bacteria as contaminated tendons retract beneath the skin’s surface. Moreover, medical attention is often sought only after frank infection develops. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: A careful history should be elicited, including the type of biting animal, the type of attack (provoked or unprovoked), and the amount of time elapsed since injury. Local and regional public-health authorities should be contacted to determine whether an individual species could be rabid and/or to locate and observe the biting animal when rabies prophylaxis may be indicated (Chap. 232).

1	Suspicious human-bite wounds should provoke careful questioning regarding domestic or child abuse. Details on antibiotic allergies, immunosuppression, splenectomy, liver disease, mastectomy, and immunization history should be obtained. The wound should be inspected carefully for evidence of infection, including redness, exudate, and foul odor. The type of wound (puncture, laceration, or scratch); the depth of penetration; and the possible involvement of joints, tendons, nerves, and bones should be assessed. It is often useful to include a diagram or photograph of the wound in the medical record. In addition, a general physical examination should be conducted and should include an assessment of vital signs as well as an evaluation for evidence of lymphangitis, lymphadenopathy, dermatologic lesions, and functional limitations. Injuries to the hand warrant consultation with a hand surgeon for the assessment of tendon, nerve, and muscular damage. Radiographs should be obtained when bone

1	lesions, and functional limitations. Injuries to the hand warrant consultation with a hand surgeon for the assessment of tendon, nerve, and muscular damage. Radiographs should be obtained when bone may have been penetrated or a tooth fragment may be present. Culture and Gram’s staining of all infected wounds are essential; anaerobic cultures should be undertaken if abscesses, devitalized tissue, or foul-smelling exudate is present. A small-tipped swab may be used to culture deep punctures or small lacerations. It is also reasonable to culture samples from apparently uninfected wounds due to bites inflicted by animals other than dogs and cats, since the microorganisms causing disease are less predictable in these cases. The white blood cell count should be determined and the blood cultured if systemic infection is suspected.

1	Wound closure is controversial in bite injuries. Many authorities prefer not to attempt primary closure of wounds that are or may become infected, choosing instead to irrigate these wounds copiously, debride devitalized tissue, remove foreign bodies, and approximate the wound edges. Delayed primary closure may be undertaken after the risk of infection is over. Small uninfected wounds may be allowed to close by secondary intention. Puncture wounds due to cat bites should be left unsutured because of the high rate at which they become infected. Facial wounds are usually sutured after thorough cleaning and irrigation because of the importance of a good cosmetic result in this area and because anatomic factors such as an excellent blood supply and the absence of dependent edema lessen the risk of infection.

1	ANTIBIOTIC THERAPY Established Infection Antibiotics should be administered for all established bite-wound infections and should be chosen in light of the most likely potential pathogens, as indicated by the biting species and by Gram’s stain and culture results (Table 167e-1). For dog and cat bites, antibiotics should be effective against S. aureus, Pasteurella species, C. canimorsus, streptococci, and oral anaerobes. For human bites, agents with activity against S. aureus,

1	H. influenzae, and β-lactamase-positive oral anaerobes should be used. The combination of an extended-spectrum penicillin with a β-lactamase inhibitor (amoxicillin/clavulanic acid, ticarcillin/ clavulanic acid, ampicillin/sulbactam) appears to offer the most reliable coverage for these pathogens. Second-generation cephalosporins (cefuroxime, cefoxitin) also offer substantial coverage. The choice of antibiotics for penicillin-allergic patients (particularly those in whom immediate-type hypersensitivity makes the use of cephalosporins hazardous) is more difficult and is based primarily on in vitro sensitivity since data on clinical efficacy are inadequate. The combination of an antibiotic active against gram-positive cocci and anaerobes (such as clindamycin) with trimethoprim-sulfamethoxazole or a fluoroquinolone, which is active against many of the other potential pathogens, would appear reasonable. In vitro data suggest that azithromycin alone provides coverage against most commonly

1	or a fluoroquinolone, which is active against many of the other potential pathogens, would appear reasonable. In vitro data suggest that azithromycin alone provides coverage against most commonly isolated bite-wound pathogens. As MRSA becomes

1	Rodent Streptobacillus moniliformis, Penicillin VK (500 mg PO Doxycycline (100 mg PO bid) Sometimes — Leptospira spp., P. multocida qid) aAntibiotic choices should be based on culture data when available. These suggestions for empirical therapy need to be tailored to individual circumstances and local conditions. IV regimens should be used for hospitalized patients. A single IV dose of antibiotics may be given to patients who will be discharged after initial management. bProphylactic antibiotics are suggested for severe or extensive wounds, facial wounds, and crush injuries; when bone or joint may be involved; and when comorbidity is present (see text). cMay be hazardous in patients with immediate-type hypersensitivity to penicillin. Abbreviations: DS, double-strength; TMP-SMX, trimethoprim-sulfamethoxazole.

1	Abbreviations: DS, double-strength; TMP-SMX, trimethoprim-sulfamethoxazole. more common in the community and evidence of its transmission between humans and their animal contacts increases, empirical use of agents active against MRSA should be considered in high-risk situations while culture results are awaited. Antibiotics are generally given for 10–14 days, but the response to therapy must be carefully monitored. Failure to respond should prompt a consideration of diagnostic alternatives and surgical evaluation for possible drainage or debridement. Complications such as osteomyelitis or septic arthritis mandate a longer duration of therapy. Management of C. canimorsus sepsis requires a 2-week course of IV penicillin G (2 million units IV every 4 h) and supportive measures. Alternative agents for the treatment of C. canimorsus infection include cephalosporins and fluoroquinolones. Serious infection with

1	P. multocida (e.g., pneumonia, sepsis, or meningitis) also should be treated with IV penicillin G. Alternative agents include secondor third-generation cephalosporins or ciprofloxacin. Bites by venomous snakes (Chap. 474) may not require antibiotic treatment. Because it is often difficult to distinguish signs of infection from tissue damage caused by the envenomation, many authorities continue to recommend treatment directed against the snake’s oral flora—i.e., the administration of broadly active agents such as ceftriaxone (1–2 g IV every 12–24 h) or ampicillin/sulbactam (1.5–3.0 g IV every 6 h). Seal finger appears to respond to doxycycline (100 mg twice daily for a duration guided by the response to therapy).

1	Presumptive or Prophylactic Therapy The use of antibiotics for patients presenting early (within 8 h) after bite injury is controversial. Although symptomatic infection frequently will not yet have manifested at this point, many early wounds will harbor pathogens, and many will become infected. Studies of antibiotic prophylaxis for wound infections are limited and have often included only small numbers of cases in which various types of wounds have been managed according to various protocols. A meta-analysis of eight randomized trials of prophylactic antibiotics in patients with dog-bite wounds demonstrated a reduction in the rate of infection by 50% with prophylaxis. However, in the absence of sound clinical trials, many clinicians base the decision to treat bite wounds with empirical antibiotics on the species of the biting animal; the location, severity, and extent of the bite wound; and the existence of comorbid conditions in the host. All humanand monkey-bite wounds should be

1	antibiotics on the species of the biting animal; the location, severity, and extent of the bite wound; and the existence of comorbid conditions in the host. All humanand monkey-bite wounds should be treated presumptively because of the high rate of infection. Most cat-bite wounds, particularly those involving the hand, should be treated. Other factors favoring treatment for bite wounds include severe injury, as in crush wounds; potential bone or joint involvement; involvement of the hands or genital region; host immunocompromise, including that due to liver disease or splenectomy; and prior mastectomy on the side of an involved upper extremity. When prophylactic antibiotics are administered, they are usually given for 3–5 days.

1	Rabies and Tetanus Prophylaxis Rabies prophylaxis, consisting of both passive administration of rabies immune globulin (with as much of the dose as possible infiltrated into and around the wound) and active immunization with rabies vaccine, should be given in consultation with local and regional public health authorities for some animal bites and scratches as well as for certain nonbite exposures (Chap. 232). Rabies is endemic in a variety of animals, including dogs and cats in many areas of the world. Many local health authorities require the reporting of all animal bites. A tetanus booster immunization should be given if the patient has undergone primary immunization but has not received a booster dose in the past 5 years. Patients who have not previously completed primary immunization should be immunized and should also receive tetanus immune globulin. Elevation of the site of injury is an important adjunct to antimicrobial therapy. Immobilization of the infected area, especially

1	should be immunized and should also receive tetanus immune globulin. Elevation of the site of injury is an important adjunct to antimicrobial therapy. Immobilization of the infected area, especially the hand, also is beneficial.

1	Infections Acquired in Health Care facilities Robert A. Weinstein The costs of hospital-acquired (nosocomial) and other health care– associated infections are great. These infections have affected as many 168 SEC TIon 3 as 1.7 million patients at a cost of ~$28–33 billion and 99,000 lives in

1	U.S. hospitals annually. Although efforts to lower infection risks have been challenged by the numbers of immunocompromised patients, antibiotic-resistant bacteria, fungal and viral superinfections, and invasive devices and procedures, a prevailing viewpoint—often termed “zero tolerance”—is that almost all health care–associated infections should be avoidable with strict application of evidence-based prevention guidelines (Table 168-1). In fact, rates of device-related infections—historically, the largest drivers of risk—have fallen steadily over the past few years. Unfortunately, at the same time, antimicrobial-resistant pathogens have risen in number and are estimated to contribute to ~23,000 deaths in and outside of hospitals annually. This chapter reviews health care–associated and device-related infections as well as basic surveillance, prevention, control, and treatment activities.

1	ORGANIZATION, RESPONSIBILITIES, AND INCREASING SCRUTINY OF HEALTH CARE–ASSOCIATED INFECTION PROGRAMS The standards of the Joint Commission require all accredited hospitals to have active programs for surveillance, prevention, and control of nosocomial infections. Education of physicians in infection control and health care epidemiology is required in infectious disease fellowship programs and is available in online courses. Concerns over “patient safety” have led to federal legislation that prevents U.S. hospitals from upgrading Medicare charges to pay for hospital costs resulting from at least 14 specific nosocomial events (Table 168-2) and have prompted national efforts to publicly

1	Abbreviations: AHRQ, Agency for Healthcare Research and Quality; APIC, Association for Professionals in Infection Control and Epidemiology; CAP, College of American Pathologists; CDC, Centers for Disease Control and Prevention; CMS, Centers for Medicare & Medicaid Services; CSTE, Council of State and Territorial Epidemiologists; DHQP, Division of Healthcare Quality Promotion; HHS, Health and Human Services; HICPAC, Healthcare Infection Control Practices Advisory Committee; HIS, Hospital Infection Society; IDSA, Infectious Diseases Society of America; IHI, Institute for Healthcare Improvement; IOM, Institute of Medicine; MedQIC, Medicare Quality Improvement Community; NIOSH, National Institute for Occupational Safety and Health; NQF, National Quality Forum; NSQIP, National Surgical Quality Improvement Program; OSHA, Occupational Safety & Health Administration; PQRI, Physician Quality Reporting Initiative; SHEA, Society for Healthcare Epidemiology of America.

1	report on processes of patient care (e.g., timely administration and of Health and Human Services released a major interagency Action appropriateness of perioperative antibiotic prophylaxis) and patient Plan to Prevent Healthcare-Associated Infections, including a outcomes (e.g., surgical wound infection rates). Neither the carrot list of 5-year national prevention targets that are mostly on track (pay-for-performance) nor the stick (nonpayment for preventable (Table 168-3). infections) appears to have impacted infection rates. The effect of public attention may be more positive; in 2009, the U.S. Department

1	Traditionally, infection preventionists have surveyed inpatients for infections acquired in hospitals (defined as those neither present nor incubating at the time of admission). Surveillance most often requires review of microbiology laboratory results, “shoe-leather” epidemiol- ogy on nursing wards, and application of standardized definitions of infection. Progressively more infection-control programs use comput-Blood incompatibilities erized hospital databases for algorithm-driven electronic surveillance Decubitus ulcers (stages III and IV) (e.g., of vascular catheter and surgical wound infections) that removes Fractures/other injuries from falls or trauma observer bias and, by so doing, provides data that are more reliable for Catheter-associated urinary tract infections interfacility comparisons. Although infection surveillance in nursing homes and some long-term acute-care hospitals (LTACHs) is still in its formative stage, the role of these facilities in the transmission of

1	Manifestations of poor glycemic control to infection surveillance and control. Surgical-site infection following certain orthopedic procedures Most hospitals aim surveillance at infections associated with high-Surgical-site infection following bariatric surgery for obesity level morbidity or expense. Quality-improvement activities in infecSurgical-site infection following cardiac electronic device implantation tion control have led to increased surveillance of personnel compliance Venous thromboembolism (after hip or knee replacement) with infection control policies (e.g., adherence to influenza vaccination recommendations). In the spirit of “what is measured improves,” the Iatrogenic pneumothorax with venous catheterization majority of states now require public reporting of processes for pre aBased on the U.S. Federal Deficit Reduction Act of 2005. As of October 2012, Medicare vention of health care–associated infection and/or patient outcomes.

1	stopped paying additional money to hospitals for these 14 health care–acquired conditions. See www.cms.gov/HospitalAcqCond/ (last accessed November 13, 2014). As a result, in some locales, the surveillance pendulum is swinging SuMMARy of PRogRESS TowARD THE nInE nATIonAL TARgETS foR ELIMInATIon of HEALTH CARE–ASSoCIATED InfECTIonS, u.S. DEPARTMEnT of HEALTH AnD HuMAn SERvICES: MIDPoInT EvALuATIon aExamples of changes: Catheter-related bloodstream infections decreased to ~1.7/1000 catheter-days; C. difficile infections increased to ~11.2 cases/10,000 discharges; catheter-related urinary tract infections decreased to ~3.1/1000 catheter-days; and hospital-onset MRSA invasive infections decreased to ~4.5/100,000 persons.

1	Abbreviations: EIP, CDC’s Emerging Infections Program; HCUP, Agency for Healthcare Research and Quality’s Healthcare Cost and Utilization Project; MRSA, methicillin-resistant Staphylococcus aureus; NHSN, CDC’s National Healthcare Safety Network; SCIP, Surgical Care Improvement Project. Source: Adapted from www.hhs.gov/ash/initiatives/hai/nationaltargets/ (last accessed November 13, 2014).

1	Source: Adapted from www.hhs.gov/ash/initiatives/hai/nationaltargets/ (last accessed November 13, 2014). back to use of “house-wide” surveillance, and many states now require that hospitals use the Centers for Disease Control and Prevention’s (CDC’s) National Healthcare Safety Network (NHSN) reporting system to provide uniform definitions and to facilitate transmission of data. Increasing reliance on the NHSN by states to facilitate public reporting has led to participation by more than 12,000 facilities (~4700 of the ~5700 acute-care hospitals in the United States, ~540 LTACHs, ~270 inpatient rehabilitation facilities, ~6000 outpatient dialysis facilities, ~300 ambulatory surgery centers, ~150 long-term-care facilities). This level of participation provides a nationwide view of health care– associated infections and represents a watershed in potential access to national rates of antimicrobial use and resistance.

1	Results of surveillance are expressed as rates. In general, for example, 5–10% of patients develop nosocomial infections. However, such broad statistics have little value unless qualified by duration of risk, by site of infection, by patient population, and by exposure to risk factors. To account for some of these variables, the CDC now uses a Standardized Infection Ratio (SIR; www.cdc.gov/hai/national-annual-sir/) as part of NHSN rate reporting. Meaningful denominators for infection rates include the number of patients exposed to a specific risk (e.g., patients using mechanical ventilators) or the number of intervention days (e.g., 1000 patient-days on a ventilator). As use of invasive devices such as indwelling bladder catheters has purposely been decreased, the denominators have become smaller, but the fact that patients who still require such devices may be those at intrinsically higher risk (potential numerators) may paradoxically increase rates when device-days account for the

1	smaller, but the fact that patients who still require such devices may be those at intrinsically higher risk (potential numerators) may paradoxically increase rates when device-days account for the denominator. Temporal trends in rates should be reviewed, and rates should be compared with regional and national benchmarks that incorporate the SIR. Interhospital comparisons still may be misleading because of the wide range in risk factors and severity of underlying illnesses. Process measures (e.g., adherence to hand hygiene) do not usually require risk adjustment, and outcome measures (e.g., cardiac surgery wound-infection rates) can identify hospitals with outlier infection rates (e.g., in the top deciles) for further evaluation. Moreover, temporal analysis of a hospital’s infection rates can help to determine whether control measures are succeeding and where increased efforts should be focused.

1	Nosocomial infections follow basic epidemiologic patterns that can help to direct prevention and control measures. Nosocomial pathogens have reservoirs, are transmitted by largely predictable routes, and require susceptible hosts. Reservoirs and sources exist in the inanimate environment (e.g., residual Clostridium difficile spores on frequently touched surfaces in patients’ rooms) and in the animate environment (e.g., infected or colonized health care workers, patients, and hospital visitors). The mode of transmission usually is either cross-infection (e.g., indirect spread of pathogens from one patient to another on the inadequately cleaned hands of hospital personnel) or autoinoculation (e.g., aspiration of oropharyngeal flora into the lungs along an endotracheal tube). Occasionally, pathogens (e.g., group A streptococci and many respiratory viruses) are spread from person to person via large infectious droplets released by coughing or sneezing. Much less common—but often

1	pathogens (e.g., group A streptococci and many respiratory viruses) are spread from person to person via large infectious droplets released by coughing or sneezing. Much less common—but often devastating in terms of epidemic risk—is true airborne spread of small or droplet nuclei (as in nosocomial chickenpox) or common-source spread (e.g., by contaminated IV fluids). Factors that increase host susceptibility include underlying conditions, abnormalities of innate defense (e.g., due to genetic polymorphisms; see Chap. 82), and medical-surgical interventions and procedures that compromise host defenses.

1	Hospitals’ infection-control programs must determine general and specific control measures. Given the prominence of cross-infection, hand hygiene is cited traditionally as the most important preventive measure. Health care workers’ rates of adherence to hand-hygiene recommendations are abysmally low (often <50%). Reasons cited include inconvenience, time pressures, and skin damage from frequent washing. Sinkless alcohol rubs are quick and highly effective and actually improve hand condition since they contain emollients and allow the retention of natural protective oils that would be removed with repeated rinsing. Use of alcohol hand rubs between patient contacts is recommended for all health care workers except when hands are visibly soiled or after care of a patient who is part of a health care facility outbreak of infection with C. difficile, whose spores resist killing by alcohol and require mechanical removal. In these cases, washing with soap and running water is recommended. A

1	care facility outbreak of infection with C. difficile, whose spores resist killing by alcohol and require mechanical removal. In these cases, washing with soap and running water is recommended. A number of innovative systems have been developed to track hand-hygiene adherence in real time and to provide feedback; although this approach is exciting, sustained improvements in rates remain to be seen.

1	The fact that >25–50% of nosocomial infections are due to the combined effect of the patient’s own flora and invasive devices highlights the importance of improvements in the use and design of such devices. Intensive education, “bundling” of evidence-based interventions (Table 168-4), and use of checklists to facilitate adherence have reduced infection rates (Table 168-3) through improved asepsis in handling and earlier removal of invasive devices. It is especially noteworthy that turnover or shortages of trained personnel jeopardize safe and effective patient care and have been associated with increased infection rates.

1	Urinary tract infections (UTIs) account for ~30–40% of nosocomial infections; up to 3% of bacteriuric patients develop bacteremia. Although UTIs contribute at most 15% to prolongation of hospital stay and may have an attributable cost in the range of only $1300, these infections are reservoirs and sources for spread of antibiotic-resistant bacteria. Most nosocomial UTIs are associated with preceding instrumentation or indwelling bladder catheters, which create a 3–7% risk of infection each day. UTIs generally are caused by pathogens that spread up the periurethral space from the patient’s perineum or gastrointestinal tract—the most common pathogenesis in women—or via intraluminal contamination of urinary catheters, usually due to cross-infection by caregivers who are irrigating catheters or emptying drainage bags. Pathogens come occasionally from Prevention of Central Venous Catheter Infections Catheter insertion bundle:

1	Prevention of Central Venous Catheter Infections Catheter insertion bundle: Educate personnel about catheter insertion and care. Use chlorhexidine to prepare the insertion site. Use maximal barrier precautions and asepsis during catheter insertion. Consolidate insertion supplies (e.g., in an insertion kit or cart). Use a checklist to enhance adherence to the “insertion bundle.” Empower nurses to halt insertion if asepsis is breached. Catheter maintenance bundle: Cleanse patients daily with chlorhexidine. Maintain clean, dry dressings. Enforce hand hygiene among health care workers. Ask daily: Is the catheter needed? Remove catheter if not needed or used. Prevention of Ventilator-Associated Events Elevate head of bed to 30–45 degrees. Decontaminate oropharynx regularly with chlorhexidine (controversial). Give “sedation vacation” and assess readiness to extubate daily. Use peptic ulcer disease prophylaxis. Use deep-vein thrombosis prophylaxis (unless contraindicated).

1	Prevention of Surgical-Site Infections Choose a surgeon wisely. Administer prophylactic antibiotics within 1 h before surgery; discontinue within 24 h. Limit any hair removal to the time of surgery; use clippers or do not remove hair at all. Prepare surgical site with chlorhexidine-alcohol. Maintain normal perioperative glucose levels (cardiac surgery patients).a Maintain perioperative normothermia (colorectal surgery patients).a Prevention of Urinary Tract Infections Place bladder catheters only when absolutely needed (e.g., to relieve obstruction), not solely for the provider’s convenience. Use aseptic technique for catheter insertion and urinary tract instrumentation. Minimize manipulation or opening of drainage systems. Ask daily: Is the bladder catheter needed? Remove catheter if not needed. Prevention of Pathogen Cross-Transmission Cleanse hands with alcohol hand rub before and after all contacts with patients or their environments.

1	Prevention of Pathogen Cross-Transmission Cleanse hands with alcohol hand rub before and after all contacts with patients or their environments. aThese components of care are supported by clinical trials and experimental evidence in the specified populations; they may prove valuable for other surgical patients as well. Source: Adapted from information presented at the following websites: www.cdc.gov/ hicpac/pubs.html; www.cdc.gov/HAI/prevent/prevention.html; www.ihi.org. inadequately disinfected urologic equipment and rarely from contaminated supplies.

1	inadequately disinfected urologic equipment and rarely from contaminated supplies. Hospitals should monitor essential performance measures for preventing nosocomial UTIs (Table 168-4). Prompts to clinicians to assess a patient’s need for continued use of an indwelling bladder catheter can improve removal rates and lessen the risk of UTI. Guidelines for managing postoperative urinary retention (e.g., with bladder scanners) also may limit the use or duration of catheterization. Other approaches to the prevention of UTIs have included the use of topical meatal antimicrobial agents, drainage bag disinfectants, and anti-infective catheters. None of the latter three measures is considered routine.

1	Administration of systemic antimicrobial agents for other purposes decreases the risk of UTI during the first 4 days of catheterization, after which resistant bacteria or yeasts emerge as pathogens. Prophylactic antibiotic administration at the time of catheter removal has been reported to decrease the risk of UTI. Selective decontamination of the gut also is associated with a reduced risk. Again, however, none of these approaches is routine. Irrigation of catheters, with or without antimicrobial agents, may actually increase the risk of infection. A condom catheter for men without bladder obstruction may be more acceptable than an indwelling catheter and may lessen the risk of UTI if maintained carefully. The role of suprapubic catheters in preventing infection is not well defined.

1	Treatment of UTIs is based on the results of quantitative urine cultures (Chap. 162). The most common pathogens are Escherichia coli, nosocomial gram-negative bacilli, enterococci, and Candida. Several caveats apply in the treatment of institutionally acquired infection. First, in patients with chronic indwelling bladder catheters, especially those in long-term-care facilities, “catheter flora”—microorganisms living on encrustations within the catheter lumen—may differ from actual urinary tract pathogens. Therefore, for suspected UTI in the setting of chronic catheterization (especially in women), it is useful to replace the bladder catheter and to obtain a freshly voided urine specimen. Second, as in all nosocomial infections, at the time treatment is initiated on the basis of a positive culture, it is useful to repeat the culture to verify the persistence of infection. Third, the frequency with which UTIs occur may lead to the erroneous assumption that the urinary tract alone is the

1	culture, it is useful to repeat the culture to verify the persistence of infection. Third, the frequency with which UTIs occur may lead to the erroneous assumption that the urinary tract alone is the source of infection in a febrile hospitalized patient. Fourth, recovery of Staphylococcus aureus from urine cultures may result from hematogenous seeding and may indicate an occult systemic infection. Finally, although Candida is now the most common pathogen in nosocomial UTIs among patients on intensive care units (ICUs), treatment of candiduria is often unsuccessful and is recommended only when there is upper-pole or bladder-wall invasion, obstruction, neutropenia, or immunosuppression.

1	Historically, pneumonia has accounted for ~10–15% of nosocomial infections; ventilator-associated pneumonia (VAP) occurred in 1 to >4 patients per 1000 ventilator-days, and these infections were reported as responsible for a mean of 10 extra hospital days and $23,000 in extra costs per episode. Most cases of bacterial nosocomial pneumonia are caused by aspiration of endogenous or hospital-acquired oropharyngeal (and occasionally gastric) flora. Nosocomial pneumonias are associated with more deaths than are infections at any other body site. However, attributable mortality rates suggest that the risk of dying from nosocomial pneumonia is affected greatly by other factors, including comorbidities, inadequate antibiotic treatment, and the involvement of specific pathogens (particularly Pseudomonas aeruginosa or Acinetobacter). Surveillance and accurate diagnosis of pneumonia have been problematic in hospitals because many patients, especially those in the ICU, have abnormal chest

1	Pseudomonas aeruginosa or Acinetobacter). Surveillance and accurate diagnosis of pneumonia have been problematic in hospitals because many patients, especially those in the ICU, have abnormal chest roentgenographs, fever, and leukocytosis potentially attributable to multiple causes. This diagnostic uncertainty has led to a refocus from VAP to “ventilatorassociated events” (VAEs), conditions, and complications, for which worsening physiologic parameters, such as oxygenation, are key metrics. Early data suggest that ~5–10% of patients using mechanical ventilators develop VAEs. Viral pneumonias, which are particularly important in pediatric and immunocompromised patients, are discussed in the virology section and in Chap. 153.

1	Risk factors for nosocomial pneumonia include those events that increase colonization by potential pathogens (e.g., prior antimicrobial therapy, contaminated ventilator circuits or equipment, or decreased gastric acidity); those that facilitate aspiration of oropharyngeal contents into the lower respiratory tract (e.g., intubation, decreased levels of consciousness, or presence of a nasogastric tube); and those that reduce host defense mechanisms in the lung and permit overgrowth of aspirated pathogens (e.g., chronic obstructive pulmonary disease, extremes of age, or upper abdominal surgery).

1	Control measures for pneumonia (Table 168-4) are aimed at frequent testing of readiness for extubation, remediation of risk factors in patient care (e.g., minimizing aspiration-prone supine positioning), and aseptic care of respirator equipment (e.g., disinfecting or sterilizing all inline reusable components such as nebulizers, replacing tubing/breathing circuits only if required because of malfunction or visible soiling—rather than on the basis of duration of use—to lessen the number of breaks in the system, and teaching aseptic technique for suctioning). Although the benefits of selective decontamination of the oropharynx and gut with nonabsorbable antimicrobial agents and/ or use of short-course postintubation systemic antibiotics have been controversial, a randomized multicenter trial demonstrated lowered ICU mortality rates among patients on mechanical ventilation who underwent oropharyngeal decontamination.

1	Among the logical preventive measures that require further investigation are placement of endotracheal tubes that provide channels for subglottic drainage of secretions, which has been associated with reduced infection risks during short-term postoperative use, and noninvasive mechanical ventilation whenever feasible. Use of silver-coated endotracheal tubes may lessen risk of VAP but is not considered routine. It is noteworthy that reducing the rate of VAP often has not reduced overall ICU mortality; this fact suggests that this infection is a marker for patients with an otherwise-heightened risk of death.

1	The most likely pathogens for nosocomial pneumonia and treatment options are discussed in Chap. 153. Several considerations regarding diagnosis and treatment are worth emphasizing. First, clinical criteria for diagnosis (e.g., fever, leukocytosis, development of purulent secretions, new or changing radiographic infiltrates, changes in oxygen requirement or ventilator settings) have high sensitivity but relatively low specificity. These criteria are most useful for selecting patients for bronchoscopic or nonbronchoscopic procedures that yield lower respiratory tract samples protected from upper-tract contamination; quantitative cultures of such specimens have diagnostic sensitivities in the range of 80%. Second, early-onset nosocomial pneumonia, which manifests within the first 4 days of hospitalization, is most often caused by community-acquired pathogens such as Streptococcus pneumoniae and Haemophilus species, although some studies have challenged this view. Late-onset pneumonias

1	hospitalization, is most often caused by community-acquired pathogens such as Streptococcus pneumoniae and Haemophilus species, although some studies have challenged this view. Late-onset pneumonias most commonly are due to

1	S. aureus, P. aeruginosa, Enterobacter species, Klebsiella pneumoniae, or Acinetobacter. When invasive techniques are used to diagnose VAP, the proportion of isolates accounted for by gram-negative bacilli decreases from 50–70% to 35–45%. Infection is polymicrobial in as many as 20–40% of cases. The role of anaerobic bacteria in VAP is not well defined. Third, one multicenter study suggested that 8 days is an appropriate duration of therapy for nosocomial pneumonia, with a longer duration (15 days in that study) when the pathogen is Acinetobacter or P. aeruginosa. Finally, in febrile patients (particularly those who have endotracheal or gastric tubes inserted through the nares), occult respiratory tract infections, especially bacterial sinusitis and otitis media, should be considered.

1	Wound infections occur in ~500,000 patients each year, account for ~15–20% of nosocomial infections, contribute up to 7–10 extra postoperative hospital days, and result in $3000 to $29,000 in extra costs, depending on the operative procedure and pathogen(s). The average wound infection has an incubation period of 5–7 days—longer than many postoperative stays. For this reason and because many procedures are now performed on an outpatient basis, the incidence of wound infections has become more difficult to assess. These infections usually are caused by the patient’s endogenous or hospital-acquired skin and mucosal flora and occasionally are due to airborne spread of skin squames that may be shed into the wound from members of the operating-room team. True airborne spread of infection through droplet nuclei is rare in operating rooms unless there is a “disseminator” (e.g., of group A streptococci or staphylococci) among the staff. In general, the common risks for postoperative wound

1	droplet nuclei is rare in operating rooms unless there is a “disseminator” (e.g., of group A streptococci or staphylococci) among the staff. In general, the common risks for postoperative wound infection are related to the surgeon’s technical skill, the patient’s underlying conditions (e.g., diabetes mellitus, obesity) or advanced age, and inappropriate timing of antibiotic prophylaxis. Additional risks include the presence of drains, prolonged preoperative hospital stays, shaving of operative sites by razor the day before surgery, long duration of surgery, and infection at remote sites (e.g., untreated UTI).

1	The substantial literature related to risk factors for surgical-site infections and the recognized morbidity and cost of these infections have led to national prevention efforts and to recommendations for “bundling” preventive measures (Table 168-4). Additional measures 915 include attention to technical surgical issues (e.g., avoiding open or prophylactic drains), operating-room asepsis, and preoperative therapy for active infection. Reporting surveillance results to surgeons has been associated with reductions in infection rates. Preoperative administration of intranasal mupirocin to patients colonized with S. aureus, preoperative antiseptic bathing, and intraand postoperative oxygen supplementation have been controversial because of conflicting study results, but evidence seems mostly to favor these interventions.

1	The process of diagnosing and treating wound infections begins with a careful assessment of the surgical site in the febrile postoperative patient. Diagnosis of deeper organ-space infections or subphrenic abscesses requires a high index of suspicion and the use of CT or MRI. Diagnosis of infections of prosthetic devices, such as orthopedic implants, may be particularly difficult and often requires the use of interventional radiographic techniques to obtain periprosthetic specimens for culture. Cultures of periprosthetic joint tissue obtained at surgery may miss pathogens that are cloistered in prosthesis-adherent biofilms; cultures of sonicates from explanted prosthetic joints have been more sensitive, particularly for patients who have received antimicrobial agents within 2 weeks of surgery.

1	The most common pathogens in postoperative wound infections are S. aureus, coagulase-negative staphylococci, and enteric and anaerobic bacteria. In rapidly progressing postoperative infections manifesting within 24–48 h of a surgical procedure, the level of suspicion regarding group A streptococcal or clostridial infection (Chaps. 173 and 179) should be high. Treatment of postoperative wound infections requires drainage or surgical excision of infected or necrotic material and antibiotic therapy aimed at the most likely or laboratory-confirmed pathogens.

1	Intravascular device–related bacteremias cause ~10–15% of nosocomial infections; central vascular catheters (CVCs) account for most of these bloodstream infections. Past national estimates indicated that as many as 200,000 bloodstream infections associated with CVCs occurred each year in the United States, with attributable mortality rates of 12–25%, an excess mean length of hospital stay of 12 days, and an estimated cost of $3700 to $29,000 per episode; one-third to one-half of these episodes occurred in ICUs. However, infection rates have dropped steadily (Table 168-3) since the publication of guidelines by the Healthcare Infection Control Practices Advisory Committee (HICPAC) in 2002. With increasing care of seriously ill patients in the community, vascular catheter–associated bloodstream infections acquired in outpatient settings are becoming more frequent. Broader surveillance for infections—outside ICUs and even outside hospitals—will be needed.

1	Catheter-related bloodstream infections derive largely from the cutaneous microflora of the insertion site, with pathogens migrating extraluminally to the catheter tip, usually during the first week after insertion. In addition, contamination of the hubs of CVCs or of the ports of “needle-less” systems may lead to intraluminal infection over longer periods, particularly with surgically implanted or cuffed catheters. Intrinsic (during the manufacturing process) or extrinsic (on-site in a health care facility) contamination of infusate, although rare, is the most common cause of epidemic device-related bloodstream infection; extrinsic contamination may cause up to half of endemic bacteremias related to arterial infusions used for hemodynamic monitoring. The most common pathogens isolated from vascular device–associated bacteremias include coagulase-negative staphylococci, S. aureus (with ≥50% of isolates in the United States resistant to methicillin), enterococci, nosocomial

1	isolated from vascular device–associated bacteremias include coagulase-negative staphylococci, S. aureus (with ≥50% of isolates in the United States resistant to methicillin), enterococci, nosocomial gram-negative bacilli, and Candida. Many pathogens, especially staphylococci, produce extracellular polysaccharide biofilms that facilitate attachment to catheters and provide sanctuary from antimicrobial agents. “Quorum-sensing” proteins, a target for future interventions, help bacterial cells communicate during biofilm development.

1	Evidence-based bundles of control measures (Table 168-4) have been strikingly effective, eliminating almost all CVC-associated infections in one ICU study. Additional control measures for infections associated with vascular access include use of a chlorhexidine-impregnated 916 patch at the skin-catheter junction; daily bathing of ICU patients with chlorhexidine; application of semitransparent access-site dressings (for ease of bathing and site inspection and protection of the site from secretions); avoidance of the femoral site for catheterization because of a higher risk of infection (most likely related to the density of the skin flora); rotation of peripheral catheters to a new site at specified intervals (e.g., every 72–96 h), which may be facilitated by use of an IV therapy team; and application of aseptic technique when accessing pressure transducers or other vascular ports. Unresolved issues include the role of gut translocation rather than vascular access sites as a cause of

1	application of aseptic technique when accessing pressure transducers or other vascular ports. Unresolved issues include the role of gut translocation rather than vascular access sites as a cause of primary bacteremia in immunocompromised patients and the implications for surveillance definitions; the best frequency for rotation of CVC sites (given that guidewire-assisted catheter changes at the same site do not lessen and can even increase infection risk); the appropriate role of mupirocin ointment, a topical antibiotic with excellent antistaphylococcal activity, in site care; the relative degrees of risk posed by peripherally inserted central catheters (PICC lines); and the risk-benefit of prophylactic use of heparin (to avoid catheter thrombi, which may be associated with increased risk of infection) or of vancomycin or alcohol (as catheter flushes or “locks”— i.e., concentrated anti-infective solutions instilled into the catheter lumen) for high-risk patients. Vascular

1	risk of infection) or of vancomycin or alcohol (as catheter flushes or “locks”— i.e., concentrated anti-infective solutions instilled into the catheter lumen) for high-risk patients. Vascular device–related infection is suspected on the basis of the appearance of the catheter site or the presence of fever or bacteremia without another source in patients with vascular catheters. The diagnosis is confirmed by the recovery of the same species of microorganism from peripheral-blood cultures (preferably two samples drawn from peripheral veins by separate venipunctures) and from semiquantitative or quantitative cultures of the vascular catheter tip. Less commonly used diagnostic measures include (1) differential (faster) time to positivity (>2 h) for blood drawn through the vascular access device than for a sample from a peripheral vein and (2) differences in quantitative cultures (a threefold or greater “step-up”) for blood samples drawn simultaneously from a peripheral vein and from a

1	than for a sample from a peripheral vein and (2) differences in quantitative cultures (a threefold or greater “step-up”) for blood samples drawn simultaneously from a peripheral vein and from a CVC, which should show the step-up if infected. When infusion-related sepsis is considered (e.g., because of the abrupt onset of fever or shock temporally related to infusion therapy), a sample of the infusate or blood product should be retained for culture. Therapy for vascular access–related infection is directed at the pathogen recovered from the blood and/or infected site. Important considerations in treatment are the need for an echocardiogram (to evaluate the patient for endocarditis), the duration of therapy, and the need to remove potentially infected catheters. In one report, approximately one-fourth of patients with intravascular catheter– associated S. aureus bacteremia who were studied by transesophageal echocardiography had evidence of endocarditis; this test may be useful in

1	one-fourth of patients with intravascular catheter– associated S. aureus bacteremia who were studied by transesophageal echocardiography had evidence of endocarditis; this test may be useful in determining the appropriate duration of treatment. Detailed consensus guidelines for the management of intravascular catheter–related infections have been published and recommend catheter removal in most cases of bacteremia or fungemia due to nontunneled CVCs. When attempting to salvage a potentially infected catheter, some clinicians use the “antibiotic lock” technique, which may facilitate penetration of infected biofilms, in addition to systemic antimicrobial therapy (see www.idsociety.org/Other_Guidelines/). The authors of the consensus treatment guidelines advise that the decision to remove a tunneled catheter or implanted device suspected of being the source of bacteremia or fungemia should be based on the severity of the patient’s illness, the strength of evidence that the device is

1	a tunneled catheter or implanted device suspected of being the source of bacteremia or fungemia should be based on the severity of the patient’s illness, the strength of evidence that the device is infected, the presence of local or systemic complications, an assessment of the specific pathogens, and the patient’s response to antimicrobial therapy if the catheter or device is initially retained. For patients with track-site infection, successful therapy without catheter removal is unusual. For patients with suppurative venous thrombophlebitis, excision of affected veins is usually required.

1	Written policies for the isolation of infectious patients are a standard component of infection control programs. To replace its prior pathogen-specific guidelines, the CDC published recommendations in 2006 for the control of multidrug-resistant organisms in health care settings; in 2007, the CDC published a revised edition of its basic isolation guidelines to provide updated recommendations for all components of health care, including acute-care hospitals and long-term, ambulatory, and home-care settings (see www.cdc.gov/hicpac/pdf/isolation/ Isolation2007.pdf).

1	Standard precautions are designed for the care of all patients in hospitals and aim to reduce the risk of transmission of microorganisms from both recognized and unrecognized sources. These precautions include gloving as well as hand cleansing for potential contact with (1) blood; (2) all other body fluids, secretions, and excretions, whether or not they contain visible blood; (3) nonintact skin; and (4) mucous membranes. Depending on exposure risks, standard precautions also include use of masks, eye protection, and gowns.

1	Precautions for the care of patients with potentially contagious clinical syndromes (e.g., acute diarrhea) or with suspected or diagnosed colonization or infection by transmissible pathogens are based on probable routes of transmission: airborne, droplet, or contact, for which personnel don, at a minimum, N95 respirators, surgical face masks, or glove and gown, respectively. Sets of precautions may be combined for diseases that have more than one route of transmission (e.g., contact and airborne isolation for varicella).

1	Some prevalent antibiotic-resistant pathogens, particularly those that colonize the gastrointestinal tract (e.g., vancomycin-resistant enterococci [VRE] and even multidrug-resistant gram-negative bacilli such as carbapenemase-producing strains of K. pneumoniae [KPCs]), may be present on intact skin of patients in hospitals (the “fecal patina”). This issue has led some experts to recommend gloving for all contact with patients who are acutely ill and/or in high-risk units, such as ICUs or LTACHs. Wearing gloves does not replace the need for hand hygiene because hands sometimes (in up to 20% of interactions) become contaminated during wearing or removal of gloves.

1	Outbreaks are always big news but probably account for <5% of nosocomial infections. The investigation and control of nosocomial epidemics require that infection control personnel (1) develop a case definition, (2) confirm that an outbreak really exists (since apparent epidemics may actually be pseudo-outbreaks due to surveillance or laboratory artifacts), (3) review aseptic practices and disinfectant use, (4) determine the extent of the outbreak, (5) perform an epidemiologic investigation to determine modes of transmission, (6) work closely with microbiology personnel to culture for common sources or personnel carriers as appropriate and to type epidemiologically important isolates, and (7) heighten surveillance to judge the effect of control measures. Control measures generally include reinforcing routine aseptic practices and hand hygiene, ensuring appropriate isolation of cases (and instituting cohort isolation and nursing if needed), and implementing further controls on the basis

1	routine aseptic practices and hand hygiene, ensuring appropriate isolation of cases (and instituting cohort isolation and nursing if needed), and implementing further controls on the basis of the investigation’s findings. Examples of some emerging and potential epidemic problems follow.

1	VIRAL RESPIRATORY INFECTIONS: PANDEMIC INFLUENZA Infections caused by the severe acute respiratory syndrome globally in 2003 (Chap. 223), and in 2012 Middle East respiratory syndrome coronavirus (MERS-CoV) emerged as a more geographically localized problem (Chap. 223). For SARS, basic infection-control measures helped to keep the worldwide case and death counts at ~8000 and ~800, respectively, although the virus was unforgiving of lapses in protocol adherence or laboratory biosafety. The epidemiology of SARS—spread largely in households once patients were ill or in hospitals—contrasts markedly with that of influenza (Chap. 224), which is often contagious a day before symptom onset; can spread rapidly in the community among nonimmune persons; and, even in its seasonal variety, kills as many as 35,000 persons each year in the United States.

1	Control of seasonal influenza has depended on (1) the use of effective vaccines, with increasingly broad evidence-based recommendations for vaccination of children, the general public, and health care workers; (2) the use of antiviral medications for early treatment and for prophylaxis as part of outbreak control, especially for high-risk patients and in high-risk settings like nursing homes or hospitals; and (3) infection control (surveillance and droplet precautions) for symptomatic patients. Controversial infection-control issues have been the questionable role of airborne spread of influenza and the need to mandate influenza vaccination of health care workers because of the embarrassingly low rates of vaccination in this high-risk group.

1	With the occurrence of localized outbreaks of avian (H5N1) influenza in Asia over the past few years, concerns about potential pandemic influenza led to (1) recommendations for universal “respiratory hygiene and cough etiquette” (basically, “cover your cough”), as described and promoted in the CDC’s 2007 Guideline for Isolation Precautions, and for “source containment” (e.g., use of face masks and spatial separation) for outpatients with potentially infectious respiratory illnesses; (2) re-examinations of the value in the 1918–1919 influenza pandemic of nonpharmacologic interventions, such as “social distancing” (e.g., closing of schools and community venues); and (3) debate about the level of respiratory protection required for health care workers (i.e., whether to use the higher-efficiency N95 respirators recommended for airborne isolation rather than the surgical masks used for droplet precautions).

1	In the spring of 2009, a novel strain of influenza virus—H1N1 or “swine flu” virus—caused the first influenza pandemic in four decades. Recombinant events that create new strains (e.g., H7N9) continue to challenge global efforts at infection control and vaccine development (Chap. 224). A new, more virulent strain of C. difficile—NAP1/BI/027—emerged in North America, and overall rates of C. difficile–associated diarrhea (Chap. 161) have increased, especially among older patients, in

1	U.S. hospitals during the past few years. C. difficile control measures include judicious use of all antibiotics, especially fluoroquinolone antibiotics that have been implicated in driving these changes; heightened suspicion for “atypical” presentations (e.g., toxic megacolon or leukemoid reaction without diarrhea); and early diagnosis, treatment, and contact precautions. To improve diagnosis, use of more sensitive polymerase chain reaction–based rather than enzyme immunoassay– based testing of diarrheal stool is now recommended, with resultant artificial doubling of infection rates in some hospitals. Preliminary data suggest a role for probiotics in the prevention of C. difficile– associated diarrhea in patients in whom systemic antibiotic therapy is being initiated. Fecal transplantation has had dramatic results in the treatment of relapsing cases of C. difficile–associated diarrhea (Chap. 161). Successes with fecal transplants and probiotics have called attention to the potential

1	has had dramatic results in the treatment of relapsing cases of C. difficile–associated diarrhea (Chap. 161). Successes with fecal transplants and probiotics have called attention to the potential role of manipulation of the intestinal microbiome as a broader infection-control strategy.

1	Outbreaks of norovirus infection (Chap. 227) in U.S. and European health care facilities appear to continue to increase in frequency or at least in reporting, with the virus often introduced by ill visitors or staff. This pathogen should be suspected when nausea and vomiting are prominent aspects of bacterial culture–negative diarrheal syndromes. Contact precautions may need to be augmented by aggressive environmental cleaning (given the persistence of norovirus on inanimate objects), prevention of secondary cases in cleaning staff through an emphasis on the use of personal protective equipment and hand hygiene, and active exclusion of ill staff and visitors.

1	Infection control practitioners institute a varicella exposure investigation and control plan whenever health care workers have been exposed to chickenpox (Chap. 217) or have worked while having or during the 24 h before developing chickenpox. The names of exposed workers and patients are obtained; medical histories are reviewed, and (if necessary) serologic tests for immunity are conducted; physicians are notified of susceptible exposed patients; postexposure prophylaxis with a preparation of varicella-zoster immune globulin (VZIG) is considered for immunocompromised or pregnant contacts, with administration as soon as possible (but as long as 10 days after expo-917 sure) (Table 217-1); varicella vaccine is recommended or preemptive use of acyclovir is considered as an alternative strategy in other susceptible persons; and susceptible exposed employees are furloughed during the at-risk period for disease (8–21 days or, if VZIG has been administered, 28 days). Routine varicella

1	strategy in other susceptible persons; and susceptible exposed employees are furloughed during the at-risk period for disease (8–21 days or, if VZIG has been administered, 28 days). Routine varicella vaccination of children and susceptible employees has made nosocomial spread less common and less problematic.

1	Important measures for the control of tuberculosis (Chap. 202) include prompt recognition, isolation, and treatment of cases; recognition of atypical presentations (e.g., lower-lobe infiltrates without cavitation); use of negative-pressure, 100% exhaust, private isolation rooms with closed doors and at least 6–12 air changes per hour; use of N95 respirators by caregivers entering isolation rooms; possible use of high-efficiency particulate air filter units and/or ultraviolet lights for disinfecting air when other engineering controls are not feasible or reliable; and follow-up testing of susceptible personnel who have been exposed to infectious patients before isolation. The use of serologic tests, rather than skin tests, in the diagnosis of latent tuberculosis for infection control purposes has become common, mostly for logistic reasons. As tuberculosis once again is on the decline in the United States, we need to remember that the price of freedom—in this instance, from a

1	purposes has become common, mostly for logistic reasons. As tuberculosis once again is on the decline in the United States, we need to remember that the price of freedom—in this instance, from a communicable disease—is eternal vigilance.

1	The potential for an outbreak of group A streptococcal infection (Chap. 173) should be considered when even one or two nosocomial cases occur. Most outbreaks involve surgical wounds and are due to the presence of an asymptomatic carrier in the operating room. Investigation can be confounded by carriage at extrapharyngeal sites such as the rectum and vagina. Health care workers in whom carriage has been linked to nosocomial transmission of group A streptococci are removed from the patient-care setting and are not permitted to return until carriage has been eliminated by antimicrobial therapy.

1	Fungal spores are common in the environment, particularly on dusty surfaces. When dusty areas are disturbed during hospital repairs or renovation, the spores become airborne. Inhalation of spores by immunosuppressed (especially neutropenic) patients creates a risk of pulmonary and/or paranasal sinus infection and disseminated aspergillosis (Chap. 241). Routine surveillance among neutropenic patients for infections with filamentous fungi, such as Aspergillus and Fusarium, helps hospitals to determine whether they are facing environmental risks. As a matter of routine, hospitals should inspect and clean air-handling equipment, review all planned renovations with infection control personnel and subsequently construct appropriate barriers, remove immunosuppressed patients from renovation sites, and consider the use of high-efficiency particulate air intake filters for rooms housing immunosuppressed patients.

1	A major multistate iatrogenic outbreak of meningitis, localized spinal or paraspinal infection, and arthritis due to Exserohilum rostratum was recognized in 2012 and traced to contamination of an injectable preservative-free steroid product produced by a single compounding pharmacy (Chap. 241).

1	Nosocomial Legionella pneumonia (Chap. 184) is most often due to contamination of potable water and predominantly affects immunosuppressed patients, particularly those receiving glucocorticoid medications. The risk varies greatly within and among geographic regions, depending on the extent of hospital water contamination and on specific hospital practices (e.g., inappropriate use of nonsterile water in respiratory therapy equipment). Laboratory-based surveillance for nosocomial Legionella should be performed, and a diagnosis of legionellosis should probably be considered more often than it is. If nosocomial cases are detected, environmental samples (e.g., tap water) 918 should be cultured. If cultures yield Legionella and if typing of clinical and environmental isolates reveals a correlation, eradication measures should be pursued. An alternative approach is to periodically culture tap water in wards housing high-risk patients. If Legionella is found, a concerted effort should be made

1	eradication measures should be pursued. An alternative approach is to periodically culture tap water in wards housing high-risk patients. If Legionella is found, a concerted effort should be made to culture samples from all patients with nosocomial pneumonia for Legionella.

1	Emerging multidrug-resistant bacteria like KPCs are harbingers of a potential “postantibiotic” era. Control of antibiotic resistance depends on close laboratory surveillance, with early detection of problems; on aggressive reinforcement of routine asepsis; on implementation of barrier precautions for all colonized and/or infected patients; on use of patient-surveillance cultures to more fully ascertain the extent of patient colonization; on antimicrobial stewardship to lessen ecologic pressures; and on timely initiation of an epidemiologic investigation when rates increase. Molecular typing (e.g., pulsed-field gel electrophoresis and, most recently, whole-genome sequencing) can help differentiate an outbreak due to a single strain (which necessitates an emphasis on hand hygiene and an evaluation of potential common-source exposures) from a polyclonal outbreak (which requires an emphasis on antibiotic prudence and device bundles; Table 168-4). Continuing emergence of

1	hygiene and an evaluation of potential common-source exposures) from a polyclonal outbreak (which requires an emphasis on antibiotic prudence and device bundles; Table 168-4). Continuing emergence of multidrug-resistant organisms suggests that control efforts have been insufficient and that regional or broader (national and global) strategies and interventions are urgently needed (see www.cdc.gov/drugresistance/threat-report-2013/ and www.gov.uk/ government/publications/uk-5-year-antimicrobial-resistancestrategy-2013-to-2018/).

1	Currently, several antibiotic resistance problems are of particular concern. First, over the past decade or so, the emergence of community-associated methicillin-resistant S. aureus (CA-MRSA) has been dramatic in many countries, with as many as 50% of community-acquired “staph infections” in some U.S. cities now caused by strains resistant to β-lactam antibiotics (Chap. 172). The incursion of CA-MRSA into hospitals is well documented and has impacted surveillance and control of nosocomial MRSA infections.

1	Second, in the ongoing global reemergence of nosocomial multidrug-resistant gram-negative bacilli, new problems include plasmid-mediated resistance to fluoroquinolones, metallo-β-lactamasemediated resistance to carbapenems, KPCs, and panresistant strains of Acinetobacter. The problematic New Delhi metallo-β-lactamase (NDM) is plasmid-mediated, has been highly successful in inter-genus transmission, and has quickly become a global threat (see wwwnc.cdc .gov/eid/article/17/10/11-0655_article.htm). For several years, KPCs were a very focal problem in the United States (predominantly in Brooklyn, NY), but more recently these strains have become a national threat. Many multidrug-resistant gram-negative bacilli are susceptible only to colistin, a drug that is consequently being “rediscovered,” or to no available agents.

1	Third, there has been renewed recognition of the role of nursing homes, and now LTACHs, in the spread of resistant gram-negative bacilli such as KPCs. In some LTACHs, as many as 30–50% of patients may be colonized with KPCs. Fourth, there has been increasing community-based spread of E. coli strains harboring an enzyme, CTX-M, that renders them broadly resistant to β-lactam antibiotics. Given the community focus of spread, these strains may be seen as a gram-negative version of CA-MRSA. Finally, clinical infections with MRSA strains exhibiting high-level vancomycin resistance due to VRE-derived plasmids have been reported in a few patients—almost all in the United States and most in Michigan—in the setting of prolonged or repeated treatment with vancomycin and/or VRE colonization. Much more common is vancomycin “MIC creep”: an increasing prevalence of MRSA strains that exhibit upper-limit susceptibility to vancomycin.

1	Colonized personnel who are implicated in nosocomial transmission of multidrug-resistant pathogens and patients who pose a threat can be decontaminated, depending on the pathogen. In a few ICUs, nonabsorbed antimicrobial agents for gastrointestinal decontamination of patients have been used successfully as a temporary emergency control measure for outbreaks of infection due to gram-negative bacilli. Potentially, manipulation of patients’ intestinal microbiome could be a more durable strategy to control outbreaks of multidrugresistant pathogens that have a gastrointestinal reservoir.

1	In several trials over the past 10 years, source control—i.e., removal of patients’ fecal patina—by daily bathing with chlorhexidine has reduced the risk of bacteremia in ICU patients. “Search-and-destroy” methods—i.e., active surveillance cultures to detect and isolate the “resistance iceberg” of patients colonized with MRSA—in nonoutbreak settings are credited with elimination of nosocomial MRSA in the Netherlands and Denmark. In a recent multicenter trial in the United States, universal source control with chlorhexidine and nasal mupirocin was significantly more effective for controlling MRSA than was a search-and-destroy approach and led to control of other pathogens as well, providing a broad (“horizontal”) rather than a narrower (“vertical”) intervention (see www.ahrq.gov/ professionals/systems/hospital/universal_icu_decolonization/). For some pathogens, such as VRE, enforcement of environmental cleaning also reduces cross-transmission risk.

1	Because the excessive use of broad-spectrum antibiotics underlies many resistance problems, “antibiotic stewardship” has been promulgated actively. The main tenets are to restrict the use of particular agents to narrowly defined indications in order to limit selective pressure on the nosocomial flora and, when broad-spectrum therapy is begun empirically in critically ill patients, to “de-escalate” treatment as soon as possible on the basis of the results of culture and susceptibility tests. The horrific attack on the World Trade Center in New York

1	The horrific attack on the World Trade Center in New York City on September 11, 2001; the subsequent mailings of anthrax spores in the United States; the Boston Marathon bombing in 2013; and ongoing revelations of terrorist plans and activities in many other countries as well as the United States have made bioterrorism a prominent source of concern to hospital infection-control programs. The essentials for hospital preparedness entail education, internal and external communication, and risk assessment. Up-to-date information is available from the CDC (see www.bt.cdc.gov).

1	An institution’s employee health service is a critical component of its infection control efforts. New employees should be processed through the service, where a contagious-disease history can be taken; evidence of immunity to a variety of diseases, such as hepatitis B, chickenpox, measles, mumps, and rubella, can be sought; immunizations for hepatitis B, measles, mumps, rubella, varicella, and pertussis (the only vaccine-preventable childhood disease that is on the rise again in the United States) can be given as needed; baseline tuberculosis testing can be performed; and education about personal responsibility for infection control can be initiated. Evaluations of employees should be codified to meet the requirements of accrediting and regulatory agencies.

1	The employee health service must have protocols for dealing with workers exposed to contagious diseases (e.g., influenza) and those percutaneously or mucosally exposed to the blood of patients infected with HIV or hepatitis B or C virus. For example, postexposure HIV prophylaxis (PEP) with combination antiretroviral agents is recommended, as indicated; free consultation is available from the CDC-funded PEPLine (888-HIV-4911). Protocols are also needed for dealing with caregivers who have common contagious diseases (such as chickenpox, group A streptococcal infection, influenza or another respiratory infection, or infectious diarrhea) and for those who have less common but high-visibility public health problems (such as chronic hepatitis B or C or HIV infection) for which exposure-control guidelines have been published by the CDC and by the Society for Healthcare Epidemiology of America.

1	Infections in Transplant Recipients Robert W. Finberg, Joyce Fingeroth This chapter considers aspects of infection unique to patients receiv-ing transplanted tissue. The evaluation of infections in transplant 169 recipients involves consideration of both the donor and the recipient of the transplanted cells or organ. Two central issues are of paramount importance: (1) infectious agents (particularly viruses, but also bacteria, fungi, and parasites) can be introduced into the recipient by the donor; and (2) treatment of the recipient with medicine to prevent rejection can suppress normal immune responses, greatly increasing susceptibility to infection. Thus, what might have been a latent or asymptomatic infection in an immunocompetent donor or in the recipient prior to therapy can become a life-threatening problem when the recipient becomes immunosuppressed. The pretransplantation evaluation of each patient should be guided by an analysis of both (1) what infections the recipient is

1	a life-threatening problem when the recipient becomes immunosuppressed. The pretransplantation evaluation of each patient should be guided by an analysis of both (1) what infections the recipient is currently harboring, since organisms that exist in a state of latency or dormancy before the procedure may cause fatal disease when the patient receives immunosuppressive treatment; and (2) what organisms are likely to be transmitted by the donor, particularly those to which the recipient may be naïve.

1	PRETRANSPLANTATION EVALUATION The Donor A variety of organisms have been transmitted by organ transplantation. Transmission of infections that may have been latent or not clinically apparent in the donor has resulted in the development of specific donor-screening protocols. Results from routine blood bank studies, including those for antibodies to Treponema pallidum (syphilis), Trypanosoma cruzi, hepatitis B and C viruses, HIV-1 and -2, human T-lymphotropic virus types 1 and 2 (HTLV-1 and -2), and West Nile virus (WNV), should be documented. Serologic studies should be ordered to identify latent infection with viruses such as herpes simplex virus types 1 and 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), Kaposi’s sarcoma–associated herpes-virus (KSHV); acute infection with hepatitis A virus; and infection with the common parasite Toxoplasma gondii. Donors should be screened, when relevant, for viruses such as rabies virus and

1	herpes-virus (KSHV); acute infection with hepatitis A virus; and infection with the common parasite Toxoplasma gondii. Donors should be screened, when relevant, for viruses such as rabies virus and lymphocytic choriomeningitis virus as well as for parasites such as Strongyloides stercoralis and Schistosoma species. Clinicians caring for prospective organ donors should examine chest radiographs for evidence of granulomatous disease (e.g., caused by mycobacteria or fungi) and should perform skin testing or obtain blood for immune cell–based assays that detect active or latent Mycobacterium tuberculosis infection. An investigation of the donor’s dietary habits (e.g., consumption of raw meat or fish or of unpasteurized dairy products), occupations or avocations (e.g., gardening or spelunking), and travel history (e.g., travel to areas with endemic fungi) also is indicated and may mandate additional testing. Creutzfeldt-Jakob disease has been transmitted through corneal transplants.

1	and travel history (e.g., travel to areas with endemic fungi) also is indicated and may mandate additional testing. Creutzfeldt-Jakob disease has been transmitted through corneal transplants. Whether it can be transmitted by transfused blood is not known. Variant Creutzfeldt-Jakob disease can be transmitted with transfused non-leukodepleted blood, posing a theoretical risk to transplant recipients.

1	The Recipient It is expected that the recipient will have been even more comprehensively assessed than the donor. Additional studies recommended for the recipient include evaluation for acute respiratory viruses and gastrointestinal pathogens in the immediate pretransplantation period. An important caveat is that, because of immune dysfunction resulting from chemotherapy or underlying chronic disease, serologic testing of the recipient may prove less reliable than usual.

1	The Donor Cells/Organ Careful attention to the sterility of the medium used to process the donor organ, combined with meticulous microbiologic evaluation, reduces rates of transmission of bacteria (or, rarely, yeasts) that may be present or grow in the organ culture medium. From 2% to >20% of donor kidneys are estimated to be contaminated with bacteria—in most cases, with the organisms that colonize the skin or grow in the tissue culture medium used to bathe the donor organ 919 while it awaits implantation. The reported rate of bacterial contamination of transplanted stem cells (bone marrow, peripheral blood, cord blood) is as high as 17% but most commonly is ∼1%. The use of enrichment columns and monoclonal antibody depletion procedures results in a higher incidence of contamination. In one series of patients receiving contaminated stem cells, 14% had fever or bacteremia, but none died. Results of cultures performed at the time of cryopreservation and at the time of thawing were

1	In one series of patients receiving contaminated stem cells, 14% had fever or bacteremia, but none died. Results of cultures performed at the time of cryopreservation and at the time of thawing were helpful in guiding therapy for the recipient.

1	Transplantation of hematopoietic stem cells (HSCs) from bone marrow or from peripheral or cord blood for cancer, immunodeficiency, or autoimmune disease most often results in a transient state of complete immunologic incompetence. Immediately after myeloablative chemotherapy and transplantation, both innate immune cells (phagocytes, dendritic cells, natural killer cells) and adaptive immune cells (T and B cells) are absent, and the host is extremely susceptible to infection. The reconstitution that follows transplantation has been likened to maturation of the immune system in neonates. The analogy does not entirely predict infections seen in HSC transplant recipients, however, because the stem cells mature in an old host who has several latent infections already. The choice among the current variety of methods for obtaining stem cells is determined by availability and by the need to optimize the chances of a cure for an individual recipient. One strategy is autologous HSC

1	the current variety of methods for obtaining stem cells is determined by availability and by the need to optimize the chances of a cure for an individual recipient. One strategy is autologous HSC transplantation, in which the donor and the recipient are the same. After chemotherapy, stem cells are collected and are purged (ex vivo) of residual neoplastic populations. Allogeneic HSC transplantation has the advantage of providing a graft-versus-tumor effect. In this case, the recipient is matched to varying degrees for human leukocyte antigens (HLAs) with a donor who may be related or unrelated. In some individuals, nonmyeloablative therapy (mini-allo transplantation) is used and permits recipient cells to persist for some time after transplantation while preserving the graft-versus-tumor effect and sparing the recipient myeloablative therapy. Cord-blood transplantation is increasingly utilized in adults; two independent cord-blood units are typically required for suitable neutrophil

1	effect and sparing the recipient myeloablative therapy. Cord-blood transplantation is increasingly utilized in adults; two independent cord-blood units are typically required for suitable neutrophil engraftment early after transplantation, even though only one of the units is likely to provide long-term engraftment. In each circumstance, a different balance is struck among the toxicity of conditioning therapy, the need for a maximal graft-versus-target effect, short-term and long-term infectious complications, and the risk of graft-versus-host disease (GVHD; acute versus chronic). The various approaches differ in terms of reconstitution speed, cell lineages introduced, and likelihood of GVHD—all factors that can produce distinct effects on the risk of infection after transplantation (Table 169-1). Despite these caveats, most infections occur in a predictable time frame after transplantation (Table 169-2).

1	In the first month after HSC transplantation, infectious complications are similar to those in granulocytopenic patients receiving chemotherapy for acute leukemia (Chap. 104). Because of the anticipated 1to 4-week duration of neutropenia and the high rate of bacterial infection in this population, many centers give prophylactic antibiotics to patients upon initiation of myeloablative therapy. Quinolones decrease the incidence of gram-negative bacteremia among these patients. Bacterial infections are common in the first few days after HSC transplantation. The organisms involved are predominantly those found on skin, mucosa, or IV catheters (Staphylococcus aureus, coagulase-negative staphylococci, streptococci) or aerobic bacteria that colonize the bowel (Escherichia coli, Klebsiella, Pseudomonas). Bacillus cereus, although rare, has emerged as a pathogen early after transplantation and can cause meningitis, which is unusual in these patients. Chemotherapy, use of broad-spectrum

1	Pseudomonas). Bacillus cereus, although rare, has emerged as a pathogen early after transplantation and can cause meningitis, which is unusual in these patients. Chemotherapy, use of broad-spectrum antibiotics, and delayed reconstitution of humoral immunity place HSC transplant patients at risk for diarrhea and colitis caused by Clostridium difficile overgrowth and toxin production.

1	920 TABLE 169-1 RISk of InfECTIon, By TyPE of HEMAToPoIETIC STEM CELL TRAnSPLAnT aDepending on the disparity of the match (major and minor histocompatibility antigens), GVHD may be severe or mild, the requirement for immunosuppression intense or minimal, and the risk of severe late infections coordinate with the degree of immunosuppression. Abbreviation: GVHD, graft-versus-host disease.

1	Beyond the first few days of neutropenia, infections with nosocomial pathogens (e.g., vancomycin-resistant enterococci, Stenotrophomonas maltophilia, Acinetobacter species, and extended-spectrum β-lactamase–producing gram-negative bacteria) as well as with filamentous bacteria (e.g., Nocardia species) become more common. Vigilance is indicated, particularly for patients with a history of active or known latent tuberculosis, even when they have been appropriately pretreated. A form of bacterial colitis among cord-blood recipients has occurred 90–300 days after transplantation, responds to antimicrobial agents such as metronidazole, and—as determined by polymerase chain reaction (PCR) of biopsy specimens—may be attributed to the bacterium Bradyrhizobium enterica (related to B. japonicum). Episodes of bacteremia due to encapsulated organisms mark the late posttransplantation period (>6 months after HSC reconstitution); patients who have undergone splenectomy and those with persistent

1	Episodes of bacteremia due to encapsulated organisms mark the late posttransplantation period (>6 months after HSC reconstitution); patients who have undergone splenectomy and those with persistent hypogammaglobulinemia are at particular risk.

1	Beyond the first week after transplantation, fungal infections become increasingly common, particularly among patients who have received broad-spectrum antibiotics. As in most granulocytopenic patients, Candida infections are most commonly seen in this setting. However, with increased use of prophylactic fluconazole, infections with resistant fungi—in particular, Aspergillus and other non-Aspergillus molds (Rhizopus, Fusarium, Scedosporium, Penicillium)—have become more common, prompting some centers to replace fluconazole with agents such as micafungin, voriconazole, or posaconazole. The role of anti-fungal prophylaxis with these different agents, in contrast to empirical treatment for suspected infection that is based on a positive β-dglucan assay or galactomannan antigen test, remains controversial (Chap. 104). Documented infection should be aggressively treated, ideally with agents of proven activity. In patients with GVHD who require prolonged or indefinite courses of

1	remains controversial (Chap. 104). Documented infection should be aggressively treated, ideally with agents of proven activity. In patients with GVHD who require prolonged or indefinite courses of glucocorticoids and other immunosuppressive agents (e.g., cyclosporine, tacrolimus [FK 506, Prograf], mycophenolate mofetil [Cellcept], rapamycin [sirolimus, Rapamune], antithymocyte globulin, or anti-CD52 antibody [alemtuzumab, Campath—an antilymphocyte and antimonocyte monoclonal antibody]), there is a high risk of fungal infection (usually with Candida or Aspergillus) even after engraftment and resolution of neutropenia. These patients are also at high risk for reactivation of latent fungal infection (histoplasmosis, coccidioidomycosis, or blastomycosis) in areas where endemic fungi reside and after involvement

1	Abbreviations: CMV, cytomegalovirus; EBV, Epstein-Barr virus; HHV-6, human herpesvirus type 6; HPV, human papillomavirus; HSV, herpes simplex virus; VZV, varicella-zoster virus. in activities such as gardening or caving. Prolonged use of central venous catheters for parenteral nutrition (lipids) increases the risk of fungemia with Malassezia. Some centers administer prophylactic anti-fungal agents to these patients. Because of the high and prolonged risk of Pneumocystis jirovecii pneumonia (especially among patients being treated for hematologic malignancies), most patients receive maintenance prophylaxis with trimethoprim-sulfamethoxazole (TMP-SMX) starting 1 month after engraftment and continuing for at least 1 year.

1	The regimen just described for the fungal pathogen Pneumocystis may also protect patients seropositive for the parasite T. gondii, which can cause pneumonia, visceral disease (occasionally), and central nervous system (CNS) lesions (more commonly). The advantages of maintaining HSC transplant recipients on daily TMP-SMX for 1 year after transplantation include some protection against Listeria monocytogenes and nocardial disease as well as late infections with Streptococcus pneumoniae and Haemophilus influenzae, which stem from the inability of the immature immune system to respond to polysaccharide antigens.

1	With increasing international travel, parasitic diseases typically restricted to particular environmental niches may pose a risk of reactivation in certain patients after HSC transplantation. Thus, in recipients with an appropriate history who were not screened and/ or treated before transplantation or in patients with recent exposures, evaluation for infection with Strongyloides, Leishmania, schistosomes, trypanosomes, or various parasitic causes of diarrheal illness (Giardia, Entamoeba, Cryptosporidium, microsporidia) may be warranted. HSC transplant recipients are susceptible to infection with a variety of viruses, including primary and reactivation syndromes caused by most human herpesviruses (Table 169-3) and acute infections caused by viruses that circulate in the community. Hepatitis (rare) Varicella-zoster virus Zoster (can disseminate) Cytomegalovirus Associated with graft rejection Abbreviation: HSC, hematopoietic stem cell.

1	Hepatitis (rare) Varicella-zoster virus Zoster (can disseminate) Cytomegalovirus Associated with graft rejection Abbreviation: HSC, hematopoietic stem cell. Herpes Simplex Virus Within the first 2 weeks after transplanta-921 tion, most patients who are seropositive for HSV-1 excrete the virus from the oropharynx. The ability to isolate HSV declines with time. Administration of prophylactic acyclovir (or valacyclovir) to seropositive HSC transplant recipients has been shown to reduce mucositis and prevent HSV pneumonia (a rare condition reported almost exclusively in allogeneic HSC transplant recipients). Both esophagitis (usually due to HSV-1) and anogenital disease (commonly caused by HSV-2) may be prevented with acyclovir prophylaxis. For further discussion, see Chap. 216.

1	Varicella-Zoster Virus Reactivation of VZV manifests as herpes zoster and may occur within the first month but more commonly occurs several months after transplantation. Reactivation rates are ∼40% for allogeneic HSC transplant recipients and 25% for autologous recipients. Localized zoster can spread rapidly in an immunosuppressed patient. Fortunately, disseminated disease can usually be controlled with high doses of acyclovir. Because of frequent dissemination among patients with skin lesions, acyclovir is given prophylactically in some centers to prevent severe disease. Low doses of acyclovir appear to be effective in preventing reactivation of VZV. However, acyclovir can also suppress the development of VZV-specific immunity. Thus, its administration for only 6 months after transplantation does not prevent zoster from occurring when treatment is stopped. Administration of low doses of acyclovir for an entire year after transplantation is effective and may eliminate most cases of

1	does not prevent zoster from occurring when treatment is stopped. Administration of low doses of acyclovir for an entire year after transplantation is effective and may eliminate most cases of posttransplantation zoster, even among cord-blood recipients. For further discussion, see Chap. 217.

1	Cytomegalovirus The onset of CMV disease (interstitial pneumonia, bone marrow suppression, graft failure, hepatitis/colitis) usually begins 30–90 days after HSC transplantation, when the granulocyte count is adequate but immunologic reconstitution has not occurred. CMV disease rarely develops earlier than 14 days after transplantation and may become evident as late as 4 months after the procedure. It is of greatest concern in the second month after transplantation, particularly in allogeneic HSC transplant recipients. In cases in which the donor marrow is depleted of T cells (to prevent GVHD or eliminate a T cell tumor) and in cord-blood recipients, the disease may manifest earlier. The use of alemtuzumab to prevent GVHD in nonmyeloablative transplantation has been associated with an increase in CMV disease. Patients who receive ganciclovir for prophylaxis, preemptive treatment, or treatment (see below) may develop recurrent CMV infection even later than 4 months after

1	with an increase in CMV disease. Patients who receive ganciclovir for prophylaxis, preemptive treatment, or treatment (see below) may develop recurrent CMV infection even later than 4 months after transplantation, as treatment appears to delay the development of the normal immune response to CMV infection. Although CMV disease may present as isolated fever, granulocytopenia, thrombocytopenia, or gastrointestinal disease, the foremost cause of death from CMV infection in the setting of HSC transplantation is pneumonia.

1	With the standard use of CMV-negative or filtered blood products, CMV infection should be a major risk in allogeneic transplantation only when the recipient is CMV-seropositive and the donor is CMV-seronegative. This situation is the reverse of that in solid organ transplant recipients. CMV reactivates from latent reservoirs present in the recipient at a time when donor T cells (especially cord-blood T cells) are too immature to control CMV replication. If the T cells from the donor have never encountered CMV and the recipient carries the virus, the patient is at maximal risk of severe disease. Reactivation disease or superinfection with another strain from the donor also can occur in CMV-positive recipients, but clinical manifestations are typically less severe, presumably because of CMV-specific memory in transplanted donor T cells. Most patients infected with CMV who undergo HSC transplantation excrete virus, with or without clinical findings. Serious CMV disease is much more

1	CMV-specific memory in transplanted donor T cells. Most patients infected with CMV who undergo HSC transplantation excrete virus, with or without clinical findings. Serious CMV disease is much more common among allogeneic than autologous recipients and is often associated with GVHD. In addition to pneumonia and marrow suppression (and, less often, graft failure), manifestations of CMV disease in HSC transplant recipients include fever with or without arthralgias, myalgias, hepatitis, and esophagitis. CMV ulcerations occur in both the lower and the upper gastrointestinal tract, and it may be difficult to distinguish diarrhea due to GVHD from that due to CMV infection. The finding of CMV in 922 the liver of a patient with GVHD does not necessarily mean that CMV is responsible for hepatic enzyme abnormalities. It is interesting that the ocular and neurologic manifestations of CMV infections, which are common in patients with AIDS, are uncommon in patients who develop disease after

1	enzyme abnormalities. It is interesting that the ocular and neurologic manifestations of CMV infections, which are common in patients with AIDS, are uncommon in patients who develop disease after transplantation. Management of CMV disease in HSC transplant recipients includes strategies directed at prophylaxis, preemptive therapy (suppression of silent replication), and treatment of disease. Prophylaxis results in a lower incidence of disease at the cost of treating many patients who otherwise would not require therapy. Because of the high fatality rate associated with CMV pneumonia in these patients and the difficulty of early diagnosis of CMV infection, prophylactic IV ganciclovir (or oral valganciclovir) has been used in some centers and has been shown to prevent CMV disease during the period of maximal vulnerability (from engraftment to day 120 after transplantation). Ganciclovir also prevents HSV reactivation and reduces the risk of VZV reactivation; thus acyclovir prophylaxis

1	period of maximal vulnerability (from engraftment to day 120 after transplantation). Ganciclovir also prevents HSV reactivation and reduces the risk of VZV reactivation; thus acyclovir prophylaxis should be discontinued when ganciclovir is administered. The foremost problem with the administration of ganciclovir relates to adverse effects, which include dose-related bone marrow suppression (thrombocytopenia, leukopenia, anemia, and pancytopenia). Because the frequency of CMV pneumonia is lower among autologous HSC transplant recipients (2–7%) than among allogeneic HSC transplant recipients (10–40%), prophylaxis in the former group will not become the rule until a less toxic oral antiviral agent becomes available. Several are under study. Preemptive treatment of CMV—that is, initiation of therapy with drugs only after CMV is detected in blood by a nucleic acid amplification test (NAAT)—is used at most centers. To limit variability between tests, the World Health Organization (WHO) has

1	therapy with drugs only after CMV is detected in blood by a nucleic acid amplification test (NAAT)—is used at most centers. To limit variability between tests, the World Health Organization (WHO) has developed an international reference standard for measurement of CMV load by NAAT-based assays. Because of toxic drug side effects (e.g., neutropenia and bone marrow suppression), the preemptive approach has supplanted prophylactic therapy; it has also replaced treatment of all seropositive (recipient and/or donor) HSC transplants with an antiviral agent (typically ganciclovir). A positive test (or increasing viral load) prompts the initiation of preemptive therapy with ganciclovir. Preemptive approaches that target patients who have quantitative NAAT evidence of CMV infection can still lead to unnecessary treatment of many individuals with drugs that have adverse effects on the basis of a laboratory test that is not highly predictive of disease; however, invasive disease, particularly in

1	unnecessary treatment of many individuals with drugs that have adverse effects on the basis of a laboratory test that is not highly predictive of disease; however, invasive disease, particularly in the form of pulmonary infection, is difficult to treat and is associated with high mortality rates. When prophylaxis or preemptive therapy is stopped, late manifestations of CMV replication may occur, although by then the HSC transplant patient is often equipped with improved graft function and is better able to combat disease. Cord-blood transplant recipients are especially vulnerable to disease caused by members of the human herpesvirus family, including CMV. Implementation of the WHO standard for CMV load measurement will facilitate large-scale comparative studies and thus the establishment of optimal guidelines for distinct patient subsets. CMV pneumonia in HSC transplant recipients (unlike that in other clinical settings) is often treated with both IV immunoglobulin (IVIg) and

1	of optimal guidelines for distinct patient subsets. CMV pneumonia in HSC transplant recipients (unlike that in other clinical settings) is often treated with both IV immunoglobulin (IVIg) and ganciclovir. In patients who cannot tolerate ganciclovir, foscarnet is a useful alternative, although it may produce nephrotoxicity and electrolyte imbalance. When neither ganciclovir nor foscarnet is clinically tolerated, cidofovir can be used; however, its efficacy is less well established, and its side effects include nephrotoxicity. A lipid-conjugate form of cidofovir and an oral antiviral agent, maribavir, are in clinical trials. Case reports have suggested that the immunosuppressive agent leflunomide may be active in this setting, but controlled studies are lacking. Transfusion of CMV-specific T cells from the donor has decreased viral load in a small series of patients; this result suggests that immunotherapy (e.g., banked T cells) may play a role in the management of this disease in the

1	T cells from the donor has decreased viral load in a small series of patients; this result suggests that immunotherapy (e.g., banked T cells) may play a role in the management of this disease in the future. For further discussion, see Chap. 219.

1	Human Herpesviruses 6 and 7 Human herpesvirus type 6 (HHV-6), the cause of roseola in children, is a ubiquitous herpesvirus that is reactivated (as determined by quantitative plasma PCR) in ∼50% of HSC transplant recipients 2–4 weeks after transplantation. Reactivation is more common among patients requiring glucocorticoids for GVHD and among those receiving second transplants. Reactivation of HHV6, primarily type B, may be associated with delayed monocyte and platelet engraftment. Limbic encephalitis developing after transplantation has been associated with HHV-6 in cerebrospinal fluid (CSF). The causality of the association is not well defined; in several cases, plasma viremia was detected long before the onset of encephalitis. Nevertheless, most patients with encephalitis had very high viral loads in plasma at the time of CNS illness, and viral antigen has been detected in hippocampal astrocytes. HHV-6 DNA is sometimes found in lung samples after transplantation. However, its role

1	viral loads in plasma at the time of CNS illness, and viral antigen has been detected in hippocampal astrocytes. HHV-6 DNA is sometimes found in lung samples after transplantation. However, its role in pneumonitis is unclear, as co-pathogens are frequently present. While HHV-6 is susceptible to foscarnet or cidofovir (and possibly to ganciclovir) in vitro, the efficacy of antiviral treatment has not been well studied. Little is known about the related herpesvirus HHV-7 or its role in posttransplantation infection. For further discussion, see Chap. 219.

1	Epstein-Barr Virus Primary EBV infection can be fatal to HSC transplant recipients; EBV reactivation can cause EBV–B cell lymphoproliferative disease (EBV-LPD), which may also be fatal to patients taking immunosuppressive drugs. Latent EBV infection of B cells leads to several interesting phenomena in HSC transplant recipients. The marrow ablation that occurs as part of the HSC transplantation procedure may sometimes eliminate latent EBV from the host. Infection can then be reacquired immediately after transplantation by transfer of infected donor B cells. Rarely, transplantation from a seronegative donor may result in a cure. The recipient is then at risk for a second primary infection.

1	EBV-LPD can develop in the recipient’s B cells (if any survive marrow ablation) but is more likely to be a consequence of outgrowth of infected donor cells. Both lytic replication and latent replication of EBV are more likely during immunosuppression (e.g., they are associated with GVHD and the use of antibodies to T cells). Although less likely in autologous transplantation, reactivation can occur in T cell–depleted autologous recipients (e.g., patients being given antibodies to T cells for the treatment of a T cell lymphoma with marrow depletion). EBV-LPD, which can become apparent as early as 1–3 months after engraftment, can cause high fevers and cervical adenopathy resembling the symptoms of infectious mononucleosis but more commonly presents as an extranodal mass. The incidence of EBVLPD among allogeneic HSC transplant recipients is 0.6–1%, which contrasts with figures of ∼5% for renal transplant recipients and up to 20% for cardiac transplant patients. In all cases, EBV-LPD is

1	among allogeneic HSC transplant recipients is 0.6–1%, which contrasts with figures of ∼5% for renal transplant recipients and up to 20% for cardiac transplant patients. In all cases, EBV-LPD is more likely to occur with high-dose, prolonged immunosuppression, especially that caused by the use of antibodies to T cells, glucocorticoids, and calcineurin inhibitors (e.g., cyclosporine, tacrolimus). Cord-blood recipients constitute another high-risk group because of delayed T cell function. Ganciclovir, administered to preempt CMV disease, may reduce EBV lytic replication and thereby diminish the pool of B cells that can become newly infected and give rise to LPD. Increasing evidence indicates that replacement of calcineurin inhibitors with mTor inhibitors (e.g., rapamycin) exerts an antiproliferative effect on EBV-infected B cells that decreases the likelihood of development of LPD or unrelated proliferative disorders associated with transplant-related immunosuppression.

1	PCR can be used to monitor EBV production after HSC transplantation. High or increasing viral loads predict an enhanced likelihood of EBV-LPD development and should prompt rapid reduction of immunosuppression and a search for nodal or extranodal disease. If reduction of immunosuppression does not have the desired effect, administration of a monoclonal antibody to CD20 (e.g., rituximab) for the treatment of B cell lymphomas that express this surface protein has elicited dramatic responses and currently constitutes first-line therapy for CD20-positive EBV-LPD. However, long-term suppression of new antibody responses accompanies therapy, and recurrences are not infrequent. Additional B cell–directed antibodies, including anti-CD22, are under study. The role of antiviral drugs is uncertain because no available agents have been documented to have activity against the different forms of latent EBV infection. Diminishing lytic replication and virion production in these patients would

1	because no available agents have been documented to have activity against the different forms of latent EBV infection. Diminishing lytic replication and virion production in these patients would theoretically produce a statistical decrease in the frequency of latent disease by decreasing the number of virions available to cause additional infection. In case reports and animal studies, ganciclovir and/or high-dose zidovudine, together with other agents, has been used to eradicate EBV-LPD and CNS lymphomas, another EBV-associated complication of transplantation. Both interferon and retinoic acid have been employed in the treatment of EBV-LPD, as has IVIg, but no large-scale prospective studies have assessed the efficacy of any of these agents. Several additional drugs are undergoing preclinical evaluation. Standard chemotherapeutic regimens are used if disease persists after reduction of immunosuppressive agents and administration of antibodies. EBV-specific T cells generated from the

1	evaluation. Standard chemotherapeutic regimens are used if disease persists after reduction of immunosuppressive agents and administration of antibodies. EBV-specific T cells generated from the donor have been used experimentally to prevent and treat EBV-LPD in allogeneic recipients, and efforts are under way to increase the activity and specificity of ex vivo–generated T cells. For further discussion, see Chap. 218.

1	Human Herpesvirus 8 (KSHV) The EBV-related gammaherpesvirus KSHV, which is causally associated with Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease, has rarely resulted in disease in HSC transplant recipients, although some cases of virus-associated marrow aplasia have been reported in the peritransplantation period. The relatively low seroprevalence of KSHV in the population and the limited duration of profound T cell suppression after HSC transplantation provide a plausible explanation for the currently low incidence of KSHV disease compared with that in recipients of solid organ transplants and patients with HIV infection. For further discussion, see Chap. 219.

1	Other (Non-Herpes) Viruses The diagnosis of pneumonia in HSC transplant recipients poses special problems. Because patients have undergone treatment with multiple chemotherapeutic agents and sometimes irradiation, their differential diagnosis should include—in addition to bacterial and fungal pneumonia—CMV pneumonitis, pneumonia of other viral etiologies, parasitic pneumonia, diffuse alveolar hemorrhage, and chemicalor radiation-associated pneumonitis. Since fungi and viruses (e.g., influenza A and B viruses, respiratory syncytial virus [RSV], parainfluenza virus [types 1–4], adenovirus, enterovirus, bocavirus, human metapneumovirus, coronavirus, and rhinovirus [increasingly detected by multiplex PCR]) also can cause pneumonia in this setting, it is important to obtain a specific diagnosis. Diagnostic modalities include Gram’s stain, microbiologic culture, antigen testing, and—increasingly—multipathogen PCR and mass spectrometry assays. M. tuberculosis has been an uncommon cause of

1	Diagnostic modalities include Gram’s stain, microbiologic culture, antigen testing, and—increasingly—multipathogen PCR and mass spectrometry assays. M. tuberculosis has been an uncommon cause of pneumonia among HSC transplant recipients in Western countries (accounting for <0.1–0.2% of cases) but is common in Hong Kong (5.5%) and in countries where the prevalence of tuberculosis is high. The recipient’s exposure history is clearly critical in an assessment of posttransplantation infections.

1	Both RSV and parainfluenza viruses, particularly type 3, can cause severe or even fatal pneumonia in HSC transplant recipients. Infections with both of these agents sometimes occur as disastrous nosocomial epidemics. Therapy with palivizumab or ribavirin for RSV infection remains controversial. New agents, some host-directed, are under study. Influenza also occurs in HSC transplant recipients and generally mirrors the presence of infection in the community. Progression to pneumonia is more common when infection occurs early after transplantation and when the recipient is lymphopenic. The neuraminidase inhibitors oseltamivir (oral) and zanamivir (aerosolized) are active against both influenza A virus and influenza B virus and are a reasonable treatment option. Parenteral forms of neuraminidase inhibitors such as peramivir (intravenous) and several new oral agents remain in trial status. An important preventive measure is immunization of household members, hospital staff members, and

1	inhibitors such as peramivir (intravenous) and several new oral agents remain in trial status. An important preventive measure is immunization of household members, hospital staff members, and other frequent contacts. Adenoviruses can be isolated from HSC transplant recipients at rates varying from 5% to ≥18%. Like CMV infection, adenovirus infection usually occurs in the first to third month after transplantation and is often asymptomatic, although pneumonia, hemorrhagic cystitis/nephritis, severe gastroenteritis with hemorrhage, and fatal disseminated infection have been reported and may be strain-specific. 923 A role for cidofovir therapy has been suggested, but the efficacy of this agent in adenovirus infection remains to be determined. Banked virus-specific T cell therapy is under study for adenovirus infection (as well as for CMV and EBV infections).

1	Although diverse respiratory viruses can sometimes cause severe pneumonia and respiratory failure in HSC transplant recipients, mild or even asymptomatic infection may be more common. For example, rhinoviruses and coronaviruses are frequent co-pathogens in HSC transplant recipients; however, whether they independently contribute to significant pulmonary infection is not known. At present, the overall contribution of these viral respiratory pathogens to the burden of lower respiratory tract disease in HSC transplant recipients requires further study. Infections with parvovirus B19 (presenting as anemia or occasionally as pancytopenia) and disseminated enteroviruses (sometimes fatal) can occur. Parvovirus B19 infection can be treated with IVIg (Chap. 221).

1	Rotaviruses are a cause of gastroenteritis in HSC transplant recipients, more frequently in children. Norovirus is a common cause of vomiting and diarrhea, and symptoms can be prolonged in HSC recipients. The polyomavirus BK virus is found at high titers in the urine of patients who are profoundly immunosuppressed. BK viruria may be associated with hemorrhagic cystitis in these patients. In contrast to its incidence among patients with impaired T cell function due to AIDS (4–5%), progressive multifocal leukoencephalopathy caused by the related JC virus is relatively rare among HSC transplant recipients (Chap. 164). When transmitted by mosquitoes or by blood transfusion, WNV can cause encephalitis and death after HSC transplantation.

1	Rates of morbidity and mortality among recipients of solid organ transplants (SOTs) are reduced by the use of effective antibiotics. The organisms that cause acute infections in recipients of SOTs are different from those that infect HSC transplant recipients because SOT recipients do not go through a period of neutropenia. As the transplantation procedure involves major surgery, however, SOT recipients are subject to infections at anastomotic sites and to wound infections. Compared with HSC transplant recipients, SOT patients are immunosuppressed for longer periods (often permanently). Thus they are susceptible to many of the same organisms as patients with chronically impaired T cell immunity (Chap. 104, especially Table 104-1). Moreover, the persistent HLA mismatch between recipient immune cells (e.g., effector T cells) and the donor organ (allograft) places the organ at permanently increased risk of infection.

1	During the early period (<1 month after transplantation; Table 169-4), infections are most commonly caused by extracellular bacteria (staphylococci, streptococci, enterococci, and E. coli and other gram-negative organisms, including nosocomial organisms with broad antibiotic resistance), which often originate in surgical wound or anastomotic sites. The type of transplant largely determines the spectrum of infection. In subsequent weeks, the consequences of the administration of agents that suppress cell-mediated immunity become apparent, and acquisition—or, more commonly, reactivation—of viruses, mycobacteria, endemic fungi, and parasites (from the recipient or from the transplanted organ) can occur. CMV infection is often a problem, particularly in the first 6 months after transplantation, and may present as severe systemic disease or as infection of the transplanted organ. HHV-6 reactivation (assessed by plasma PCR) occurs within the first 2–4 weeks after transplantation and may be

1	and may present as severe systemic disease or as infection of the transplanted organ. HHV-6 reactivation (assessed by plasma PCR) occurs within the first 2–4 weeks after transplantation and may be associated with fever, leukopenia, and very rare cases of encephalitis. Data suggest that replication of HHV-6 and HHV-7 may exacerbate CMV-induced disease. CMV is associated not only with generalized immunosuppression but also with organ-specific, rejection-related syndromes: glomerulopathy in kidney transplant recipients, bronchiolitis obliterans in lung transplant recipients, vasculopathy in heart transplant recipients, and the vanishing bile duct syndrome in liver transplant recipients. A complex interplay between increased CMV 924 TABLE 169-4 CoMMon InfECTIonS AfTER SoLID oRgAn TRAnSPLAnTATIon, By SITE of InfECTIon

1	Central nervous system Listeria infection (meningitis); T. gondii Listerial meningitis; cryptococcal meningitis; infection; CMV infection nocardial abscess; JC virus–associated PML Abbreviations: CMV, cytomegalovirus; EBV, Epstein-Barr virus; PML, progressive multifocal leukoencephalopathy.

1	replication and enhanced graft rejection is well established: elevated organ, have been noted. High organ-specific content of B lymphoid immunosuppression leads to increased CMV replication, which is tissues (e.g., bronchus-associated lymphoid tissue in the lung), anaassociated with graft rejection. For this reason, considerable atten-tomic factors (e.g., lack of access of host T cells to the transplanted tion has been focused on the diagnosis, prophylaxis, and treatment organ because of disturbed lymphatics), and differences in major of CMV infection in SOT recipients. Early transmission of WNV to histocompatibility loci between the host T cells and the organ (e.g., transplant recipients from a donated organ or transfused blood has lack of cell migration or lack of effective T cell/macrophage/dendritic been reported; however, the risk of WNV acquisition has been reduced cell cooperation) may result in defective elimination of EBV-infected by implementation of screening procedures. In

1	been reported; however, the risk of WNV acquisition has been reduced cell cooperation) may result in defective elimination of EBV-infected by implementation of screening procedures. In rare instances, rabies B cells. SOT recipients are also highly susceptible to the development virus and lymphocytic choriomeningitis virus also have been acutely of Kaposi’s sarcoma and, less frequently, to the B cell–proliferative transmitted in this setting; although accompanied by distinct clinical disorders associated with KSHV, such as primary effusion lymphoma syndromes, both viral infections have resulted in fatal encephalitis. As and multicentric Castleman’s disease. Kaposi’s sarcoma is 550–1000 screening for unusual viruses is not routine, only vigilant assessment of times more common among SOT recipients than in the general poputhe prospective donor is likely to prevent the use of an infected organ. lation, can develop very rapidly after transplantation, and can also

1	Beyond 6 months after transplantation, infections characteristic of occur in the allograft. However, because the seroprevalence of KSHV patients with defects in cell-mediated immunity—e.g., infections with is very low in Western countries, Kaposi’s sarcoma is not common. Listeria, Nocardia, Rhodococcus, mycobacteria, various fungi, and other Recipients (or donors) from Iceland, the Middle East, Mediterranean intracellular pathogens—may be a problem. International patients and countries, and Africa are at highest risk of disease. Data suggest that global travelers may experience reactivation of dormant infections a switch of immunosuppressive agents—from calcineurin inhibitors with trypanosomes, Leishmania, Plasmodium, Strongyloides, and other (cyclosporine, tacrolimus) to mTor pathway–active agents (sirolimus, parasites. Reactivation of latent M. tuberculosis infection, while rare in everolimus)—after adequate wound healing may significantly reduce Western nations, is far more common

1	agents (sirolimus, parasites. Reactivation of latent M. tuberculosis infection, while rare in everolimus)—after adequate wound healing may significantly reduce Western nations, is far more common among persons from developing the likelihood of development of Kaposi’s sarcoma and perhaps of countries. The recipient is typically the source, although reactivation EBV-LPD and certain other posttransplantation malignancies. and spread from the donor organ can occur. While pulmonary disease remains most common, atypical sites can be involved and mortality KIDNEY TRANSPLANTATION rates can be high (up to 30%). Vigilance, prophylaxis/preemptive ther-See Table 169-4. apy (when indicated), and rapid diagnosis and treatment of infections can be lifesaving in SOT recipients, who, unlike most HSC transplant Early Infections Bacteria often cause infections that develop in the recipients, continue to be immunosuppressed. period immediately after kidney transplantation. There is a role for

1	SOT recipients are susceptible to EBV-LPD from as early as 2 perioperative antibiotic prophylaxis, and many centers give cephalospomonths to many years after transplantation. The prevalence of this rins to decrease the risk of postoperative complications. Urinary tract complication is increased by potent and prolonged use of T cell–sup-infections developing soon after transplantation are usually related to pressive drugs. Decreasing the degree of immunosuppression may in anatomic alterations resulting from surgery. Such early infections may some cases reverse the condition. Among SOT patients, those with require prolonged treatment (e.g., 6 weeks of antibiotic administration heart and lung transplants—who receive the most intensive immuno-for pyelonephritis). Urinary tract infections that occur >6 months after suppressive regimens—are most likely to develop EBV-LPD, particu-transplantation may be treated for shorter periods because they do not larly in the lungs. Although the disease

1	occur >6 months after suppressive regimens—are most likely to develop EBV-LPD, particu-transplantation may be treated for shorter periods because they do not larly in the lungs. Although the disease usually originates in recipient seem to be associated with the high rate of pyelonephritis or relapse B cells, several cases of donor origin, particularly in the transplanted seen with infections that occur during the first 3 months.

1	History of exposure to active or latent Mycobacterium tuberculosis Isoniazid in patients with recent sero-Chest imaging; TST and/or cell-based tuberculosis aFor information on latent infection with hepatitis B or C virus, see Chap. 362. bSerologic examination, tuberculin skin test, and interferon assays may be less reliable after transplantation. Abbreviations: CMV, cytomegalovirus; EBV, Epstein-Barr virus; HHV-6, human herpesvirus type 6; HSC, hematopoietic stem cell; HSV, herpes simplex virus; KSHV, Kaposi’s sarcoma– associated herpesvirus; PCR, polymerase chain reaction; TST, tuberculin skin test; VZV, varicella-zoster virus. Prophylaxis with TMP-SMX for the first 4–6 months after transplantation decreases the incidence of early and middle-period infections (see below, Table 169-4, and Table 169-5).

1	Prophylaxis with TMP-SMX for the first 4–6 months after transplantation decreases the incidence of early and middle-period infections (see below, Table 169-4, and Table 169-5). Middle-Period Infections Because of continuing immunosuppression, kidney transplant recipients are predisposed to lung infections characteristic of those in patients with T cell deficiency (i.e., infections with intracellular bacteria, mycobacteria, nocardiae, fungi, viruses, and parasites). A high mortality rate associated with Legionella pneumophila infection (Chap. 184) led to the closing of renal transplant units in hospitals with endemic legionellosis. About 50% of all renal transplant recipients presenting with fever 1–4 months after transplantation have evidence of CMV disease; CMV itself accounts for the fever in more than two-thirds of cases and thus is the predominant pathogen during this period. CMV infection (Chap.

1	219) may also present as arthralgias, myalgias, or organ-specific symptoms. During this period, this infection may represent primary disease (in the case of a seronegative recipient of a kidney from a seropositive donor) or may represent reactivation disease or superinfection. Patients may have atypical lymphocytosis. Unlike immunocompetent patients, however, they rarely have lymphadenopathy or splenomegaly. Therefore, clinical suspicion and laboratory confirmation are necessary for diagnosis. The clinical syndrome may be accompanied by bone marrow suppression (particularly leukopenia). CMV also causes glomerulopathy and is associated with an increased incidence of other opportunistic infections. Because of the frequency and severity of disease, a considerable effort has been made to prevent and treat CMV infection in renal transplant recipients. An immune globulin preparation enriched with antibodies to CMV was used by many centers in the past in an effort to protect the group at

1	and treat CMV infection in renal transplant recipients. An immune globulin preparation enriched with antibodies to CMV was used by many centers in the past in an effort to protect the group at highest risk for severe infection (seronegative recipients of seropositive kidneys). However, with the development of effective oral antiviral agents, CMV immune globulin is no longer used. Ganciclovir (or valganciclovir) is beneficial for prophylaxis (when indicated) and for the treatment of serious CMV disease. The availability of valganciclovir has allowed most centers to move to oral prophylaxis for transplant recipients. Infection with the other herpesviruses may become evident within 6 months after transplantation or later. Early after transplantation, HSV may cause either oral or anogenital lesions that are usually responsive to acyclovir. Large ulcerating lesions in the anogenital area may lead to bladder and rectal dysfunction and may predispose the patient to bacterial infection. VZV

1	lesions that are usually responsive to acyclovir. Large ulcerating lesions in the anogenital area may lead to bladder and rectal dysfunction and may predispose the patient to bacterial infection. VZV may cause fatal disseminated infection in nonimmune kidney transplant recipients, but in immune patients reactivation zoster usually does not disseminate outside the dermatome; thus disseminated VZV infection is a less fearsome complication in kidney transplantation than in HSC transplantation. HHV-6 reactivation may take place and (although usually asymptomatic) may be associated with fever, rash, marrow suppression, or rare instances of renal impairment, hepatitis, colitis, or encephalitis.

1	EBV disease is more serious; it may present as an extranodal proliferation of B cells that invade the CNS, nasopharynx, liver, small bowel, heart, and other organs, including the transplanted kidney. The disease is diagnosed by the finding of a mass of proliferating EBV-positive B cells. The incidence of EBV-LPD is elevated among patients who acquire EBV infection from the donor and among patients given high doses of cyclosporine, tacrolimus, glucocorticoids, and anti–T cell antibodies. Disease may regress once immunocompetence is restored. KSHV infection can be transmitted with the donor kidney and result in development of Kaposi’s sarcoma, although it more often represents reactivation of latent infection of the recipient. Kaposi’s sarcoma often appears within 1 year after transplantation, although the time of onset ranges widely (1 month to ∼20 years). Avoidance of immunosuppressive agents that inhibit calcineurin has been associated with less Kaposi’s sarcoma, less EBV disease,

1	although the time of onset ranges widely (1 month to ∼20 years). Avoidance of immunosuppressive agents that inhibit calcineurin has been associated with less Kaposi’s sarcoma, less EBV disease, and even less CMV replication. The use of rapamycin (sirolimus) has independently led to regression of Kaposi’s sarcoma.

1	The papovaviruses BK virus and JC virus (polyomavirus hominis types 1 and 2) have been cultured from the urine of kidney transplant recipients (as they have from that of HSC transplant recipients) in the setting of profound immunosuppression. High levels of BK virus replication detected by PCR in urine and blood are predictive of pathology, especially in the setting of renal transplantation. JC virus may rarely cause similar disease in kidney transplantation. Urinary excretion of BK virus and BK viremia are associated with the development of ureteral strictures, polyomavirus-associated nephropathy (1–10% of renal transplant recipients), and (less commonly) generalized vasculopathy. Timely detection and early reduction of immunosuppression are critical and can reduce rates of graft loss related to polyomavirusassociated nephropathy from 90% to 10–30%. Therapeutic responses to IVIg, quinolones, leflunomide, and cidofovir have been reported, but the efficacy of these agents has not been

1	to polyomavirusassociated nephropathy from 90% to 10–30%. Therapeutic responses to IVIg, quinolones, leflunomide, and cidofovir have been reported, but the efficacy of these agents has not been substantiated through adequate clinical study. Most centers approach the problem by reducing immunosuppression in an effort to enhance host immunity and decrease viral titers. JC virus is associated with rare cases of progressive multifocal leukoencephalopathy. Adenoviruses may persist and cause hemorrhagic nephritis/cystitis with continued immunosuppression in these patients, but disseminated disease like that seen in HSC transplant recipients is much less common.

1	Kidney transplant recipients are also subject to infections with other intracellular organisms. These patients may develop pulmonary infections with Mycobacterium, Aspergillus, and Mucor species as well as infections with other pathogens in which the T cell/macrophage axis plays an important role. L. monocytogenes is a common cause of bacteremia ≥1 month after renal transplantation and should be seriously considered in renal transplant recipients presenting with fever and headache. Kidney transplant recipients may develop Salmonella bacteremia, which can lead to endovascular infections and require prolonged therapy. Pulmonary infections with Pneumocystis are common unless the patient is maintained on TMP-SMX prophylaxis.

1	926 Acute interstitial nephritis caused by TMP-SMX is rare. However, because transient increases in creatinine (artifactual) and hyperkalemia (manageable) can occur, early discontinuation of prophylaxis, especially after kidney transplantation, is recommended by some groups. Although additional monitoring is indicated, the benefits of TMP-SMX in kidney transplant recipients may outweigh the risks; otherwise, second-line prophylactic agents should be used. Nocardia infection (Chap. 199) may present in the skin, bones, and lungs or in the CNS, where it usually takes the form of single or multiple brain abscesses. Nocardiosis generally occurs ≥1 month after transplantation and may follow immunosuppressive treatment for an episode of rejection. Pulmonary manifestations most commonly consist of localized disease with or without cavities, but the disease may be disseminated. The diagnosis is made by culture of the organism from sputum or from the involved nodule. As it is for P. jirovecii

1	of localized disease with or without cavities, but the disease may be disseminated. The diagnosis is made by culture of the organism from sputum or from the involved nodule. As it is for P. jirovecii infection, prophylaxis with TMP-SMX is often efficacious in the prevention of nocardiosis. Toxoplasmosis can occur in seropositive patients but is less common than in other transplantation settings, usually developing in the first few months after kidney transplantation. Again, TMP-SMX is helpful in prevention. In endemic areas, histoplasmosis, coccidioidomycosis, and blastomycosis may cause pulmonary infiltrates or disseminated disease.

1	Late Infections Late infections (>6 months after kidney transplantation) may involve the CNS and include CMV retinitis as well as other CNS manifestations of CMV disease. Patients (particularly those whose immunosuppression has been increased) are at risk for subacute meningitis due to Cryptococcus neoformans. Cryptococcal disease may present in an insidious manner (sometimes as a skin infection before the development of clear CNS findings). Listeria meningitis may have an acute presentation and requires prompt therapy to avoid a fatal outcome. TMP-SMX prophylaxis may reduce the frequency of Listeria infections.

1	Patients who continue to take glucocorticoids are predisposed to ongoing infection. “Transplant elbow,” a recurrent bacterial infection in and around the elbow that is thought to result from a combination of poor tensile strength of the skin of steroid-treated patients and steroid-induced proximal myopathy, requires patients to push themselves up with their elbows to get out of chairs. Bouts of cellulitis (usually caused by S. aureus) recur until patients are provided with elbow protection.

1	Kidney transplant recipients are susceptible to invasive fungal infections, including those due to Aspergillus and Rhizopus, which may present as superficial lesions before dissemination. Mycobacterial infection (particularly that with Mycobacterium marinum) can be diagnosed by skin examination. Infection with Prototheca wickerhamii (an achlorophyllic alga) has been diagnosed by skin biopsy. Warts caused by human papillomaviruses (HPVs) are a late consequence of persistent immunosuppression; imiquimod or other forms of local therapy are usually satisfactory. Merkel cell carcinoma, a rare and aggressive neuroendocrine skin tumor whose frequency is increased fivefold in elderly SOT (especially kidney) recipients, is causally linked to a novel polyomavirus, Merkel cell polyomavirus.

1	Notably, although BK virus replication and virus-associated disease can be detected far earlier, polyomavirus-associated nephropathy is clinically diagnosed in a median of ∼300 days and thus qualifies as a late-onset disease. With the establishment of better screening procedures (e.g., urine cytology, urine nucleic acid load, plasma PCR), disease onset is being detected earlier (see “Middle-Period Infections,” above) and preemptive strategies (decrease or modification of immunosuppression) are being instituted more promptly, as the efficacy of antiviral therapy is not well established.

1	HEART TRANSPLANTATION Early Infections Sternal wound infection and mediastinitis are early complications of heart transplantation. An indolent course is common, with fever or a mildly elevated white blood cell count preceding the development of site tenderness or drainage. Clinical suspicion based on evidence of sternal instability and failure to heal may lead to the diagnosis. Common microbial residents of the skin (e.g., S. aureus, including methicillin-resistant strains, and Staphylococcus epidermidis) as well as gram-negative organisms (e.g., Pseudomonas aeruginosa) and fungi (e.g., Candida) are often involved. In rare cases, mediastinitis in heart transplant recipients can also be due to Mycoplasma hominis (Chap. 212); since this organism requires an anaerobic environment for growth and may be difficult to see on conventional medium, the laboratory should be alerted that its involvement is suspected. M. hominis mediastinitis has been cured with a combination of surgical

1	growth and may be difficult to see on conventional medium, the laboratory should be alerted that its involvement is suspected. M. hominis mediastinitis has been cured with a combination of surgical debridement (sometimes requiring muscle-flap placement) and the administration of clindamycin and tetracycline. Organisms associated with mediastinitis may sometimes be cultured from pericardial fluid.

1	Middle-Period Infections T. gondii (Chap. 253) residing in the heart of a seropositive donor may be transmitted to a seronegative recipient. Thus serologic screening for T. gondii infection is important before and in the months after cardiac transplantation. Rarely, active disease can be introduced at the time of transplantation. The overall incidence of toxoplasmosis is so high in the setting of heart transplantation that some prophylaxis is always warranted. Although alternatives are available, the most frequently used agent is TMP-SMX, which prevents infection with Pneumocystis as well as with Nocardia and several other bacterial pathogens. CMV also has been transmitted by heart transplantation. Toxoplasma, Nocardia, and Aspergillus can cause CNS infections. L. monocytogenes meningitis should be considered in heart transplant recipients with fever and headache.

1	CMV infection is associated with poor outcomes after heart transplantation. The virus is usually detected 1–2 months after transplantation, causes early signs and laboratory abnormalities (usually fever and atypical lymphocytosis or leukopenia and thrombocytopenia) at 2–3 months, and can produce severe disease (e.g., pneumonia) at 3–4 months. An interesting observation is that seropositive recipients usually develop viremia faster than patients whose primary CMV infection is a consequence of transplantation. Between 40% and 70% of patients develop symptomatic CMV disease in the form of (1) CMV pneumonia, the form most likely to be fatal; (2) CMV esophagitis and gastritis, sometimes accompanied by abdominal pain with or without ulcerations and bleeding; and (3) the CMV syndrome, consisting of CMV in the bloodstream along with fever, leukopenia, thrombocytopenia, and hepatic enzyme abnormalities. Ganciclovir is efficacious in the treatment of CMV infection; prophylaxis with ganciclovir

1	of CMV in the bloodstream along with fever, leukopenia, thrombocytopenia, and hepatic enzyme abnormalities. Ganciclovir is efficacious in the treatment of CMV infection; prophylaxis with ganciclovir or possibly with other antiviral agents, as described for renal transplantation, may reduce the overall incidence of CMV-related disease.

1	Late Infections EBV infection usually presents as a lymphoma-like proliferation of B cells late after heart transplantation, particularly in patients maintained on intense immunosuppressive therapy. A subset of heart and heart-lung transplant recipients may develop early fulminant EBV-LPD (within 2 months). Treatment includes the reduction of immunosuppression (if possible), the use of glucocorticoid and calcineurin inhibitor–sparing regimens, and the consideration of therapy with anti–B cell antibodies (rituximab and possibly others). Immunomodulatory and antiviral agents continue to be studied. Ganciclovir prophylaxis for CMV disease may indirectly reduce the risk of EBV-LPD through reduced spread of replicating EBV to naïve B cells. Aggressive chemotherapy is a last resort, as discussed earlier for HSC transplant recipients. KSHV-associated disease, including Kaposi’s sarcoma and primary effusion lymphoma, has been reported in heart transplant recipients. GVHD prophylaxis with

1	earlier for HSC transplant recipients. KSHV-associated disease, including Kaposi’s sarcoma and primary effusion lymphoma, has been reported in heart transplant recipients. GVHD prophylaxis with sirolimus may decrease the risk of both rejection and outgrowth of KSHV-infected cells. Antitumor therapy is discussed in Chap. 103e. Prophylaxis for Pneumocystis infection is required for these patients (see “Lung Transplantation, Late Infections,” below).

1	LUNG TRANSPLANTATION Early Infections It is not surprising that lung transplant recipients are predisposed to the development of pneumonia. The combination of ischemia and the resulting mucosal damage, together with accompanying denervation and lack of lymphatic drainage, probably contributes to the high rate of pneumonia (66% in one series). The prophylactic use of high doses of broad-spectrum antibiotics for the first 3–4 days after surgery may decrease the incidence of pneumonia. Gram-negative pathogens (Enterobacteriaceae and Pseudomonas species) are troublesome in the first 2 weeks after surgery (the period of maximal vulnerability). Pneumonia can also be caused by Candida (possibly as a result of colonization of the donor lung), Aspergillus, and Cryptococcus. Many centers use antifungal prophylaxis (typically fluconazole or liposomal amphotericin B) for the first 1–2 weeks.

1	Mediastinitis may occur at an even higher rate among lung transplant recipients than among heart transplant recipients and most commonly develops within 2 weeks of surgery. In the absence of prophylaxis, pneumonitis due to CMV (which may be transmitted as a consequence of transplantation) usually presents between 2 weeks and 3 months after surgery, with primary disease occurring later than reactivation disease.

1	Middle-Period Infections The incidence of CMV infection, either reactivated or primary, is 75–100% if either the donor or the recipient is seropositive for CMV. CMV-induced disease after solid organ transplantation appears to be most severe in recipients of lung and heart-lung transplants. Whether this severity relates to the mismatch in lung antigen presentation and host immune cells or is attributable to nonimmunologic factors is not known. More than half of lung transplant recipients with symptomatic CMV disease have pneumonia. Difficulty in distinguishing the radiographic picture of CMV infection from that of other infections or from organ rejection further complicates therapy. CMV can also cause bronchiolitis obliterans in lung transplants. The development of pneumonitis related to HSV has led to the prophylactic use of acyclovir. Such prophylaxis may also decrease rates of CMV disease, but ganciclovir is more active against CMV and is also active against HSV. The prophylaxis of

1	has led to the prophylactic use of acyclovir. Such prophylaxis may also decrease rates of CMV disease, but ganciclovir is more active against CMV and is also active against HSV. The prophylaxis of CMV infection with IV ganciclovir—or increasingly with valganciclovir, the oral alternative—is recommended for lung transplant recipients. Antiviral alternatives are discussed in the earlier section on HSC transplantation. Although the overall incidence of serious disease is decreased during prophylaxis, late disease may occur when prophylaxis is stopped—a pattern observed increasingly in recent years. With recovery from peritransplantation complications and, in many cases, a decrease in immunosuppression, the recipient is often better equipped to combat late infection.

1	Late Infections The incidence of Pneumocystis infection (which may present with a paucity of findings) is high among lung and heart-lung transplant recipients. Some form of prophylaxis for Pneumocystis pneumonia is indicated in all organ transplant situations (Table 169-5). Prophylaxis with TMP-SMX for 12 months after transplantation may be sufficient to prevent Pneumocystis disease in patients whose immunosuppression is not increased.

1	As in other transplant recipients, EBV infection in lung and heart-lung recipients may cause either a mononucleosis-like syndrome or EBV-LPD. The tendency of the B cell blasts to present in the lung appears to be greater after lung transplantation than after the transplantation of other organs, possibly because of a rich source of B cells in bronchus-associated lymphoid tissue. Reduction of immunosuppression and switching of regimens, as discussed in earlier sections, cause remission in some cases, but mTor inhibitors such as rapamycin may contribute to lung toxicity. Airway compression can be fatal, and rapid intervention may therefore become necessary. The approach to EBV-LPD is similar to that described in other sections.

1	LIVER TRANSPLANTATION Early Infections As in other transplantation settings, early bacterial infections are a major problem after liver transplantation. Many centers administer systemic broad-spectrum antibiotics for the first 24 h or sometimes longer after surgery, even in the absence of documented infection. However, despite prophylaxis, infectious complications are common and correlate with the duration of the surgical procedure and the type of biliary drainage. An operation lasting >12 h is associated with an increased likelihood of infection. Patients who have a choledochojejunostomy with drainage of the biliary duct to a Roux-en-Y jejunal bowel loop have more fungal infections than those whose bile is drained 927 via anastomosis of the donor common bile duct to the recipient common bile duct. Overall, liver transplant patients have a high incidence of fungal infections, and the occurrence of fungal (often candidal) infection in the setting of choledochojejunostomy correlates

1	bile duct. Overall, liver transplant patients have a high incidence of fungal infections, and the occurrence of fungal (often candidal) infection in the setting of choledochojejunostomy correlates with retransplantation, elevated creatinine levels, long procedures, transfusion of >40 units of blood, reoperation, preoperative use of glucocorticoids, prolonged treatment with antibacterial agents, and fungal colonization 2 days before and 3 days after surgery. Many centers give antifungal agents prophylactically in this setting.

1	Peritonitis and intraabdominal abscesses are common complications of liver transplantation. Bacterial peritonitis or localized abscesses may result from biliary leaks. Early leaks are especially common with live-donor liver transplants. Peritonitis in liver transplant recipients is often polymicrobial, frequently involving enterococci, aerobic gram-negative bacteria, staphylococci, anaerobes, or Candida and sometimes involving other invasive fungi. Only one-third of patients with intraabdominal abscesses have bacteremia. Abscesses within the first month after surgery may occur not only in and around the liver but also in the spleen, pericolic area, and pelvis. Treatment includes antibiotic administration and drainage as necessary.

1	Middle-Period Infections The development of postsurgical biliary stricture predisposes patients to cholangitis. The incidence of strictures is increased in live-donor liver transplantation. Transplant recipients who develop cholangitis may have high spiking fevers and rigors but often lack the characteristic signs and symptoms of classic cholangitis, including abdominal pain and jaundice. Although these findings may suggest graft rejection, rejection is typically accompanied by marked elevation of liver function enzymes. In contrast, in cholangitis in transplant recipients, results of liver function tests (with the possible exception of alkaline phosphatase levels) are often within the normal range. Definitive diagnosis of cholangitis in liver transplant recipients requires demonstration of aggregated neutrophils in bile duct biopsy specimens. Unfortunately, invasive studies of the biliary tract (either T-tube cholangiography or endoscopic retrograde cholangiopancreatography) may

1	of aggregated neutrophils in bile duct biopsy specimens. Unfortunately, invasive studies of the biliary tract (either T-tube cholangiography or endoscopic retrograde cholangiopancreatography) may themselves lead to cholangitis. For this reason, many clinicians recommend an empirical trial of therapy with antibiotics covering gram-negative organisms and anaerobes before these procedures are undertaken as well as antibiotic coverage if procedures are eventually performed.

1	Reactivation of viral hepatitis is a common complication of liver transplantation (Chap. 360). Recurrent hepatitis B and C infections, for which transplantation may be performed, are problematic. To prevent hepatitis B virus reinfection, prophylaxis with an optimal antiviral agent or combination of agents (lamivudine, adefovir, entecavir) and hepatitis B immune globulin is currently recommended, although the optimal dose, route, and duration of therapy remain controversial. Success in preventing reinfection with hepatitis B virus has increased in recent years. Complications related to hepatitis C infection are the most common reason for liver transplantation in the United States. Reinfection of the graft with hepatitis C virus occurs in all patients, with a variable time frame. Studies of aggressive pretransplantation treatment of selected recipients with antiviral agents and prophylactic/ preemptive regimens are ongoing. However, early posttransplantation initiation of treatment for

1	aggressive pretransplantation treatment of selected recipients with antiviral agents and prophylactic/ preemptive regimens are ongoing. However, early posttransplantation initiation of treatment for histologically documented disease with the classic combination of ribavirin and pegylated interferon has produced sustained responses at rates in the range of 25–40%. Several protease and polymerase inhibitors that block production of hepatitis C virus as well as regimens that spare interferon and a monoclonal antibody to the virus are undergoing preclinical and clinical trials for prevention and or control of infection after transplantation (Chap. 360).

1	As in other transplantation settings, reactivation disease with herpesviruses is common (Table 169-3). Herpesviruses can be transmitted in donor organs. Although CMV hepatitis occurs in ∼4% of liver transplant recipients, it is usually not so severe as to require retransplantation. Without prophylaxis, CMV disease develops in the majority of seronegative recipients of organs from CMV-positive donors, but fatality rates are lower among liver transplant recipients than among lung or heart-lung transplant recipients. Disease due to CMV can also 928 be associated with the vanishing bile duct syndrome after liver transplantation. Patients respond to treatment with ganciclovir; prophylaxis with oral forms of ganciclovir or high-dose acyclovir may decrease the frequency of disease. A role for HHV-6 reactivation in early posttransplantation fever and leukopenia has been proposed, although the more severe sequelae described in HSC transplantation are unusual. HHV-6 and HHV-7 appear to

1	for HHV-6 reactivation in early posttransplantation fever and leukopenia has been proposed, although the more severe sequelae described in HSC transplantation are unusual. HHV-6 and HHV-7 appear to exacerbate CMV disease in this setting. EBVLPD after liver transplantation shows a propensity for involvement of the liver, and such disease may be of donor origin. See previous sections for discussion of EBV infections in solid organ transplantation.

1	Pancreas transplantation is most frequently performed together with or after kidney transplantation, although it may be performed alone. Transplantation of the pancreas can be complicated by early bacterial and yeast infections. Most pancreatic transplants are drained into the bowel, and the rest are drained into the bladder. A cuff of duodenum is used in the anastomosis between the pancreatic graft and either the gut or the bladder. Bowel drainage poses a risk of early intraabdominal and allograft infections with enteric bacteria and yeasts. These infections can result in loss of the graft. Bladder drainage causes a high rate of urinary tract infection and sterile cystitis; however, such infection can usually be cured with appropriate antimicrobial agents. In both procedures, prophylactic antimicrobial agents are commonly used at the time of surgery. Aggressive immunosuppression, especially when the patient receives a kidney and a pancreas from different donors, is associated with

1	antimicrobial agents are commonly used at the time of surgery. Aggressive immunosuppression, especially when the patient receives a kidney and a pancreas from different donors, is associated with late-onset systemic fungal and viral infections; thus many centers administer an antifungal drug and an antiviral agent (ganciclovir or a congener) for extended prophylaxis.

1	Issues related to the development of CMV infection, EBV-LPD, and infections with opportunistic pathogens in patients receiving a pancreatic transplant are similar to those in other SOT recipients.

1	Composite tissue allotransplantation (CTA) is a new field in which, rather than a single organ, multiple tissue types composing a major body part are transplanted. The sites involved have included an upper extremity, the face, the trachea, the knee, and the abdominal wall. The numbers of recipients are limited. The different procedures and the associated infectious complications vary. Nevertheless, some early trends related to infectious complications have become apparent, as very intense and prolonged immunosuppression is typically required to prevent rejection. For example, in the early postoperative period, bacterial infections are especially frequent in facial transplant recipients. Perioperative prophylaxis is tailored to the organisms likely to complicate the different procedures. As in SOT recipients, complicated CMV infections have been observed in several CTA settings, particularly when the recipient is seronegative and the donor is seropositive. In some patients, anti-CMV

1	As in SOT recipients, complicated CMV infections have been observed in several CTA settings, particularly when the recipient is seronegative and the donor is seropositive. In some patients, anti-CMV immune globulin in addition to ganciclovir (as used in HSC transplant recipients with CMV pneumonia) was needed to control disease, and ganciclovir resistance requiring alternative therapies developed in several patients. Infectious complications from reactivation of other members of the human herpesvirus family and other latent viruses also caused significant morbidity, as discussed for SOT recipients. Prophylaxis for CMV infection, P. jirovecii infection, toxoplasmosis, and fungal infection is administered for several months on the basis of the limited studies available.

1	MISCELLANEOUS INFECTIONS IN SOLID ORGAN TRANSPLANTATION Indwelling IV Catheter Infections The prolonged use of indwelling IV catheters for administration of medications, blood products, and nutrition is common in diverse transplantation settings and poses a risk of local and bloodstream infections. Exit-site infection is most commonly caused by staphylococcal species. Bloodstream infection most frequently develops within 1 week of catheter placement or in patients who become neutropenic. Coagulase-negative staphylococci are the most common isolates from blood. Although infective endocarditis in HSC transplant recipients is uncommon, the incidence of endocarditis in SOT recipients has been estimated to be as high as 1%, and this infection is associated with excessive high mortality in this population. Although staphylococci predominate, the involvement of fungal and gram-negative organisms may be more common than in the general population.

1	For further discussion of differential diagnosis and therapeutic options, see Chap. 104.

1	Tuberculosis The incidence of tuberculosis within the first 12 months after solid organ transplantation is greater than that observed after HSC transplantation (0.23–0.79%) and ranges broadly worldwide (1.2– 15%), reflecting the prevalence of tuberculosis in local populations. Lesions suggesting prior tuberculosis on chest radiography, older age, diabetes, chronic liver disease, GVHD, and intense immunosuppression are predictive of tuberculosis reactivation and development of disseminated disease in a host with latent disease. Tuberculosis has rarely been transmitted from the donor organ. In contrast to the low mortality rate among HSC transplant recipients, mortality rates among SOT recipients are reported to be as high as 30%. Vigilance is indicated, as the presentation of disease is often extrapulmonary (gastrointestinal, genitourinary, central nervous, endocrine, musculoskeletal, laryngeal) and atypical; tuberculosis in this setting sometimes manifests as fever of unknown origin.

1	extrapulmonary (gastrointestinal, genitourinary, central nervous, endocrine, musculoskeletal, laryngeal) and atypical; tuberculosis in this setting sometimes manifests as fever of unknown origin. Careful elicitation of a history and direct evaluation of both the recipient and the donor prior to transplantation are optimal. Skin testing of the recipient with purified protein derivative may be unreliable because of chronic disease and/or immunosuppression. Cell-based assays that measure interferon γ and/or cytokine production may prove more sensitive in the future. Isoniazid toxicity has not been a significant problem except in the setting of liver transplantation. Therefore, appropriate prophylaxis should be used (see recommendations from the Centers for Disease Control and Prevention [CDC] at www.cdc.gov/tb/topic/treatment/ltbi.htm). An assessment of the need to treat latent disease should include careful consideration of the possibility of a false-negative test result. Pending final

1	at www.cdc.gov/tb/topic/treatment/ltbi.htm). An assessment of the need to treat latent disease should include careful consideration of the possibility of a false-negative test result. Pending final confirmation of suspected tuberculosis, aggressive multidrug treatment in accordance with the guidelines of the CDC, the Infectious Diseases Society of America, and the American Thoracic Society is indicated because of the high mortality rates among these patients. Altered drug metabolism (e.g., upon coadministration of antituberculous medications and certain immunosuppressive agents) can be managed with careful monitoring of drug levels and appropriate dose adjustment. Close follow-up of hepatic enzymes is warranted. Drug-resistant tuberculosis is especially problematic in these individuals (Chap. 202).

1	Virus-Associated Malignancies In addition to malignancy associated with gammaherpesvirus infection (EBV, KSHV) and simple warts (HPV), other tumors that are virus-associated or suspected of being virus-associated are more likely to develop in transplant recipients, particularly those who require long-term immunosuppression, than in the general population. The interval to tumor development is usually >1 year. Transplant recipients develop nonmelanoma skin or lip cancers that, in contrast to de novo skin cancers, have a high ratio of squamous cells to basal cells. HPV may play a major role in these lesions. Cervical and vulvar carcinomas, which are quite clearly associated with HPV, develop with increased frequency in female transplant recipients. The frequency of Merkel cell carcinoma associated with Merkel cell polyomavirus is also increased in transplant recipients; however, it is unclear whether recipients infected with HTLV-1 are at increased risk of leukemia. Among renal

1	associated with Merkel cell polyomavirus is also increased in transplant recipients; however, it is unclear whether recipients infected with HTLV-1 are at increased risk of leukemia. Among renal transplant recipients, rates of melanoma are modestly increased and rates of cancers of the kidney and bladder are increased.

1	(See also Chap. 148) In addition to receiving antibiotic prophylaxis, transplant recipients should be vaccinated against likely pathogens (Table 169-6). In the case of HSC transplant recipients, optimal responses cannot be achieved until after immune reconstitution, despite previous immunization of both donor and recipient. Recipients of an allogeneic HSC transplant must be reimmunized if they are to be protected against pathogens. The situation is less clear-cut in the case of autologous transplantation. T and B cells in the peripheral blood may reconstitute the immune response if they are transferred Type of Transplantation aImmunizations should be given before solid organ transplantation whenever possible.

1	Type of Transplantation aImmunizations should be given before solid organ transplantation whenever possible. Abbreviations: CDC, Centers for Disease Control and Prevention; ACIP, Advisory Committee on Immunization Practices; DTaP, full-level diphtheria and tetanus toxoids and acellular pertussis, adsorbed; GVHD, graft-versus-host disease; Tdap, tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis; IDSA, Infectious Diseases Society of America. Note: Recommendations from the CDC should be checked regularly as they frequently change upon receipt of new clinical information and new formulations of specific vaccines.

1	in adequate numbers. However, cancer patients (particularly those with Hodgkin’s disease, in whom vaccination has been extensively studied) who are undergoing chemotherapy do not respond normally to immunization, and titers of antibodies to infectious agents fall more rapidly than in healthy individuals. Therefore, even immunosuppressed patients who have not undergone HSC transplantation may need booster vaccine injections. If memory cells are specifically eliminated as part of a stem cell “cleanup” procedure, it will be necessary to reimmunize the recipient with a new primary series. Optimal times for immunizations of different transplant populations are being evaluated. Yearly immunization of household and other contacts (including health care personnel) against influenza benefits the patient by preventing local spread.

1	In the absence of compelling data as to optimal timing, it is reasonable to administer the pneumococcal and H. influenzae type b conjugate vaccines to both autologous and allogeneic HSC transplant recipients beginning 12 months after transplantation. A series that includes both the 13-valent pneumococcal conjugate vaccine (Prevnar) and the 23-valent pneumococcal polysaccharide vaccine (Pneumovax) is now recommended (according to CDC guidelines). The pneumococcal and

1	H. influenzae type b vaccines are particularly important for patients who have undergone splenectomy. The Neisseria meningitidis polysaccharide conjugate vaccine (Menactra or Menveo) also is recommended. In addition, diphtheria, tetanus, acellular pertussis, and inactivated polio vaccines can all be given at these same intervals (12 months and, as required, 24 months after transplantation). Some authorities recommend a new primary series for tetanus/diphtheria/pertussis and inactivated poliovirus vaccines beginning 12 months after transplantation. Vaccination to prevent hepatitis B and hepatitis A (both killed vaccines) also seems advisable. A formal recommendation regarding 929 immunization with the tetravalent HPV virus-like particle vaccine (Gardasil) after HSC transplantation has not been issued. However, HPV vaccination, which can prevent genital warts as well as specific cancers, is recommended through age 26 for healthy young adults who previously have not been vaccinated or

1	been issued. However, HPV vaccination, which can prevent genital warts as well as specific cancers, is recommended through age 26 for healthy young adults who previously have not been vaccinated or have not received the full series. Live-virus measles/mumps/rubella (MMR) vaccine can be given to autologous HSC transplant recipients 24 months after transplantation and to most allogeneic HSC transplant recipients at the same point if they are not receiving maintenance therapy with immunosuppressive drugs and do not have ongoing GVHD. The risk of spread from a household contact is low for MMR vaccine. In parts of the world where live poliovirus vaccine is used, patients as well as contacts should be advised to receive only the killed vaccine. In the rare setting where both donor and recipient are VZV naïve and the recipient is no longer receiving acyclovir or ganciclovir prophylaxis, the patient should be counseled to receive varicella-zoster immune globulin (VariZIG) up to 10 days after

1	are VZV naïve and the recipient is no longer receiving acyclovir or ganciclovir prophylaxis, the patient should be counseled to receive varicella-zoster immune globulin (VariZIG) up to 10 days after an exposure to a person with chickenpox or uncovered zoster; such patients should avoid close contact with persons recently vaccinated with Varivax. A formal recommendation regarding Varivax immunization of such patients is not currently available. Neither patients nor their household contacts should receive vaccinia vaccine unless they have been exposed to smallpox virus. Among patients who have active GVHD and/or are taking high maintenance doses of glucocorticoids, it may be prudent to avoid all live-virus vaccines.

1	In the case of SOT recipients, administration of all the usual vaccines and of the indicated booster doses should be completed before immunosuppression, if possible, to maximize responses. For patients taking immunosuppressive agents, the administration of pneumococcal vaccine should be repeated every 5 years. No data are available for the meningococcal vaccine, but it is probably reasonable to administer it along with the pneumococcal vaccine. H. influenzae conjugate vaccine is safe and should be efficacious in this population; therefore, its administration before transplantation is recommended. Booster doses of this vaccine are not recommended for adults. SOT recipients who continue to receive immunosuppressive drugs should not receive live-virus vaccines. A person in this group who is exposed to measles should be given measles immune globulin. Similarly, an immunocompromised patient who is seronegative for varicella and who comes into contact with a person who has chickenpox should

1	to measles should be given measles immune globulin. Similarly, an immunocompromised patient who is seronegative for varicella and who comes into contact with a person who has chickenpox should be given varicellazoster immune globulin as soon as possible (optimally within 96 h; up to 10 days after contact); if this is not possible, a 10to 14-day course of acyclovir therapy should be started immediately. Upon the discontinuation of treatment, clinical disease may still occur in a small number of patients; thus vigilance is indicated. Rapid re-treatment with acyclovir should limit the symptoms of disease. Household contacts of transplant recipients can receive live attenuated VZV vaccine, but vaccinees should avoid direct contact with the patient if a rash develops. Virus-like particle vaccines have been licensed for the prevention of infection with several HPV serotypes most commonly implicated in cervical and anal carcinomas and in anogenital and laryngeal warts. These vaccines are not

1	have been licensed for the prevention of infection with several HPV serotypes most commonly implicated in cervical and anal carcinomas and in anogenital and laryngeal warts. These vaccines are not live; however, no information is yet available about their immunogenicity or efficacy in transplant recipients.

1	Immunocompromised patients who travel may benefit from some but not all vaccines (Chaps. 148 and 149). In general, these patients should receive any killed or inactivated vaccine preparation appropriate to the area they are visiting; this recommendation includes the vaccines for Japanese encephalitis, hepatitis A and B, poliomyelitis, meningococcal infection, and typhoid. The live typhoid vaccines are not recommended for use in most immunocompromised patients, but an inactivated or purified polysaccharide typhoid vaccine can be used. Live yellow fever vaccine should not be administered. On the other hand, primary immunization or boosting with the purified-protein hepatitis B vaccine is indicated. Inactivated hepatitis A vaccine should also be used in the appropriate setting (Chap. 148). A vaccine is now available that provides dual protection against hepatitis A and hepatitis

1	B. If hepatitis A vaccine is not administered, travelers should consider receiving passive protection with immune globulin (the dose depending on the duration of travel in the high-risk area).

1	930 Treatment and Prophylaxis of Bacterial Infections David C. Hooper, Erica S. Shenoy, Christy A. Varughese Antimicrobial agents have had a major impact on human health. Together with vaccines, they have contributed to reduced mortality, extended lifespan, and enhanced quality of life. Among drugs used in human medicine, however, they are distinctive in that their use promotes the occurrence of drug resistance in the pathogens they are designed to treat as well as in other “bystander” organisms. Indeed, the history of antimicrobial development has been driven in large part by the medical need engendered by the emergence of resistance to each generation of agents. Thus, the careful and appropriate use of antimi-crobial drugs is particularly important not only for optimizing efficacy and minimizing adverse effects but also for minimizing the risk of resis-tance and preserving the value of existing agents. Although this chapter focuses on antibacterial agents, the optimal use of all

1	minimizing adverse effects but also for minimizing the risk of resis-tance and preserving the value of existing agents. Although this chapter focuses on antibacterial agents, the optimal use of all antimicrobials depends on an understanding of each drug’s mechanism of action, spectrum of activity, mechanisms of resistance, pharmacology, and adverse effect profile. This information is then applied in the context of the patient’s clinical presentation, underlying conditions, and epi-demiology to define the site and likely nature of the infection or other condition and thus to choose the best therapy. Gathering of microbio-logic information is important for refining therapeutic choices on the basis of documented pathogen and susceptibility data whenever pos-sible; this information also makes it possible to choose more targeted therapy, thereby reducing the risk of selection of resistant bacteria. Durations of therapy are chosen according to the nature of the infec-tion and the patient’s

1	it possible to choose more targeted therapy, thereby reducing the risk of selection of resistant bacteria. Durations of therapy are chosen according to the nature of the infec-tion and the patient’s response to treatment and are informed by clini-cal studies when they are available, with the understanding that shorter courses are less likely than longer ones to promote the emergence of resistance. This chapter provides specific information that is necessary for making informed choices among antibacterial agents. MECHANISMS OF ACTION AND RESISTANCE 170 SEC TIon 4 APPRoACH To THERAPy foR BACTERIAL DISEASES

1	The mechanisms of action of and resistance to antibacterial agents are discussed in detail in the text and are summarized for the most commonly used groups of agents in Table 170-1. A schematic of antibacterial targets is provided in Fig. 170-1.

1	Multiple essential components of bacterial cell structures and metabolism have been the targets of antibacterial agents used in clinical medicine, and the interaction of an agent with its target results in either inhibition of bacterial growth and replication (bacteriostatic effect) or bacterial killing (bactericidal effect). In general, targets have been chosen because they either do not exist in mammalian cells and physiology or are sufficiently different from their bacterial counterparts to allow selective bacterial targeting. Treatment with bacteriostatic agents is effective when the patient’s host defenses are sufficient to contribute to eradication of the infecting pathogen. In patients with impaired host defenses (e.g., neutropenia) or infections at body sites with impaired or limited host defenses (e.g., meningitis and endocarditis), bactericidal agents are generally preferred.

1	Inhibition of Cell Wall Synthesis The bacterial cell wall, which is external to the cytoplasmic membrane and has no counterpart in mammalian cells, protects bacterial cells from lysis under low osmotic conditions. The cell wall is a cross-linked peptidoglycan composed of a polymer of alternating units of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), four-amino-acid stem peptides linked to each NAM, and a peptide cross-bridge that links adjacent stem peptides to form a net-like structure. Several steps in peptidoglycan synthesis are targets of antibacterial agents. Inhibition of cell wall synthesis generally results in a bactericidal effect that is linked to cell lysis. This effect results not only from the blocking of new cell-wall formation but from the uninhibited action of cell wall–remodeling enzymes called autolysins, which cleave peptidoglycan as part of normal cell-wall growth.

1	In gram-positive bacteria the peptidoglycan is the most external cell structure, but in gram-negative bacteria an asymmetric lipid outer membrane is external to the peptidoglycan and contains diffusion channels called porins. The space between the cytoplasmic membrane peptidoglycan and the outer membrane is referred to as the periplasmic space. Most antibacterial drugs enter the gram-negative bacterial cell through a porin channel, since the outer membrane is a major diffusion barrier. Although the peptidoglycan layer is thicker in gram-positive (20–80 nm) than in gram-negative (1 nm) bacteria, peptidoglycan itself constitutes only a limited diffusion barrier for antibacterial agents. β-lactams The β-lactam drugs, including penicillins, cephalosporins, monobactams, and carbapenems, target transpeptidase enzymes (also called penicillin-binding proteins, or PBPs) involved in the stem-peptide cross-linking step.

1	glycopeptides The glycopeptides, including vancomycin, teicoplanin, telavancin, dalbavancin, and oritavancin, bind the two terminal d-alanine residues of the stem peptide, hindering the glycosyltransferase involved in polymerizing NAG–NAM units. Telavancin also binds to the lipid II intermediate that delivers cell-wall precursor subunits. Likewise, dalbavancin and oritavancin interact with the cell membrane, and oritavancin may also inhibit transpeptidases. Both β-lactams and glycopeptides interact with their targets external to the cytoplasmic membrane. bacitracin (topical) and fosfomycin These agents interrupt enzymatic steps in the production of peptidoglycan precursors in the cytoplasm.

1	bacitracin (topical) and fosfomycin These agents interrupt enzymatic steps in the production of peptidoglycan precursors in the cytoplasm. Inhibition of Protein Synthesis Most inhibitors of bacterial protein synthesis target bacterial ribosomes, whose difference from eukaryotic ribosomes allows selective antibacterial action. Some inhibitors bind to the 30S ribosomal subunit and others to the 50S subunit. Most protein synthesis–inhibiting agents are bacteriostatic; aminoglycosides are an exception and are bactericidal.

1	aminoglycosides Aminoglycosides (amikacin, gentamicin, kanamycin, netilmicin, streptomycin, tobramycin) bind irreversibly to 16S ribosomal RNA (rRNA) of the 30S ribosomal subunit, blocking the trans-location of peptidyl transfer RNA (tRNA) from the A (aminoacyl) to the P (peptidyl) site and, at low concentrations, causing misreading of messenger RNA (mRNA) codons and thus causing the introduction of incorrect amino acids into the peptide chain; at higher concentrations, translocation of the peptide chain is blocked. Cellular uptake of aminoglycosides is dependent on the electrochemical gradient across the bacterial membrane. Under anaerobic conditions, this gradient is reduced, with a consequent reduction in the uptake and activity of the aminoglycosides. Spectinomycin is a related aminocyclitol antibiotic that also binds to 16S rRNA of the 30S ribosomal subunit but at a different site. This drug inhibits translocation of the growing peptide chain but does not trigger codon misreading

1	antibiotic that also binds to 16S rRNA of the 30S ribosomal subunit but at a different site. This drug inhibits translocation of the growing peptide chain but does not trigger codon misreading and produces only a bacteriostatic effect.

1	tetracyclines and glycylcyclines Tetracyclines (doxycycline, minocycline, tetracycline) bind reversibly to the 16S rRNA of the 30S ribosomal subunit and block the binding of aminoacyl tRNA to the ribosomal A site, thereby inhibiting peptide elongation. Active transport of tetracyclines into bacterial but not mammalian cells contributes to the selectivity of these agents. Tigecycline, a derivative of minocycline and Antibacterial Agent(s) Major Target Mechanism(s) of Action Mechanism(s) of Resistance cytoplasmic membranes Daptomycin Cell membrane Produces membrane channel and membrane leakage Abbreviations: LPS, lipopolysaccharide; NAG, N-acetylglucosamine; PBP, penicillin-binding protein. 1. 2. 3. 1. 2. 1. 2. 1. 2. 3. Decreased permeation to target due to active efflux 1. 2. 1. 2. 1. Same as macrolides 2. 1. 2. Methylation of ribosome binding site 1. 2. 1. 2. 3. Protection of target from drug 4. 1. 2. 3.

1	2. 1. 2. 1. Same as macrolides 2. 1. 2. Methylation of ribosome binding site 1. 2. 1. 2. 3. Protection of target from drug 4. 1. 2. 3. Altered cell membrane charge with reduced drug binding Altered cell membrane with reduced drug binding Treatment and Prophylaxis of Bacterial Infections the only available glycylcycline, acts similarly to the tetracyclines but is distinctive for its ability to circumvent the most common mechanisms of resistance to the tetracyclines. macrolides and Ketolides In contrast to the aminoglycosides and tetracyclines, the macrolides (azithromycin, clarithromycin, erythromycin) and ketolides (telithromycin) bind to the 23S rRNA of the 50S ribosomal subunit. These agents block translocation of the growing peptide chain by binding to the tunnel from which the chain exits the ribosome.

1	lincosamides Clindamycin is the only lincosamide in clinical use. It binds to the 23S rRNA of the 50S ribosomal subunit, interacting with both the ribosomal A and P sites and blocking peptide bond formation. streptogramins The only streptogramin in clinical use is a combination of quinupristin, a group B streptogramin, and dalfopristin, a group A streptogramin. Both components bind to 23S rRNA of the 50S ribosome: dalfopristin binds to both the A and P sites of the peptidyl transferase center, and quinupristin binds to a site that overlaps the macrolide-binding site, blocking the emergence of nascent peptide FIGURE 170-1 Antibacterial targets. A, aminoacyl site; DHFR, dihydrofolate reductase; DHPS, dihydropteroate synthetase; P, peptidyl site; PBP, penicillin-binding protein; tRNA-aa, aminoacyl tRNA.

1	FIGURE 170-1 Antibacterial targets. A, aminoacyl site; DHFR, dihydrofolate reductase; DHPS, dihydropteroate synthetase; P, peptidyl site; PBP, penicillin-binding protein; tRNA-aa, aminoacyl tRNA. from the ribosome. The combination is bactericidal, but macrolideresistant bacteria exhibit cross-resistance to quinupristin, and the remaining activity of dalfopristin alone is bacteriostatic. cHlorampHenicol Chloramphenicol binds reversibly to the 23S rRNA of the 50S subunit in a manner that interferes with the proper positioning of the aminoacyl component of tRNA in the A site. This site of binding is near those of the macrolides and lincosamides. oxazolidinones Linezolid and tedizolid are the only oxazolidinones in clinical use. They bind directly to the A site in the 23S rRNA of the 50S ribosomal subunit and block binding of aminoacyl tRNA, inhibiting the initiation of protein synthesis.

1	mupirocin Mupirocin (pseudomonic acid) is used topically. It competes with isoleucine for binding to isoleucyl tRNA synthetase, depleting stores of isoleucyl tRNA and thereby inhibiting protein synthesis.

1	Inhibition of Bacterial Metabolism Available inhibitors (antimetabolites) target the pathway for synthesis of folate, which is a cofactor in a number of one-carbon transfer reactions involved in the synthesis of some nucleic acids, including pyrimidine, thymidine, and all purines (adenine and guanine), as well as some amino acids (methionine and serine) and acetyl coenzyme A (CoA). Two sequential steps in folate synthesis are targeted. The selective antibacterial effect stems from the inability of mammalian cells to synthesize folate; they depend instead on exogenous sources. Antibacterial activity, however, may be reduced in the presence of high exogenous concentrations of the end products of the folate pathway (e.g., thymidine and purines) that may occur in some infections, resulting from local breakdown of leukocytes and host tissues.

1	sulfonamides Sulfonamides, including sulfadiazine, sulfisoxazole, and sulfamethoxazole, inhibit dihydropteroate synthetase, which adds p-aminobenzoic acid (PABA) to pteridine, producing dihydropteroate. Sulfonamides are structural analogues of PABA and act as competing enzyme substrates. trimetHoprim Subsequent steps in folate synthesis are catalyzed by dihydrofolate synthase, which adds glutamate to dihydropteroate, and dihydrofolate reductase, which then generates the final product, tetrahydrofolate. Trimethoprim is a structural analogue of pteridine and inhibits dihydrofolate reductase. Trimethoprim is available alone but is most often used in combination products that also contain sulfamethoxazole and thus block two sequential steps in folate synthesis. Inhibition of DNA and RNA Synthesis or Activity A variety of antibacterial agents act on these processes.

1	Quinolones The quinolones include nalidixic acid, the first agent in the class, and newer, more widely used fluorinated derivatives (fluoroquinolones), including norfloxacin, ciprofloxacin, levofloxacin, moxifloxacin, and gemifloxacin. The quinolones are synthetic compounds that inhibit bacterial DNA synthesis by interacting with the DNA complexes of two essential enzymes, DNA gyrase and DNA topoisomerase IV, which alter DNA topology. Quinolones trap enzyme–DNA complexes in such a way that they block movement of the DNA replication apparatus and can generate lethal double-strand breaks in DNA, resulting in bactericidal activity. Although mammalian cells also have type II DNA topoisomerases like gyrase and topoisomerase IV, the structures of the mammalian enzymes are sufficiently different from those of the bacterial enzymes that quinolones have substantially selective antibacterial activity.

1	rifamycins Rifampin, rifabutin, and rifapentine are semisynthetic derivatives of rifamycin B and bind the β subunit of bacterial RNA polymerase, thereby blocking elongation of mRNA. Their action is highly selective for the bacterial enzyme over mammalian RNA polymerases. nitrofurantoin The reduction of nitrofurantoin, a nitrofuran compound, by bacterial enzymes produces highly reactive derivatives that are thought to cause DNA strand breakage. Nitrofurantoin is used only for the treatment of lower urinary tract infections.

1	metronidazole Metronidazole is a synthetic nitroimidazole with activity limited to anaerobic bacteria and certain anaerobic protozoa. Reduction of its nitro group by the electron-transport system in anaerobic bacteria produces reactive intermediates that damage DNA and result in bactericidal activity. Both nitrofurantoin and metronidazole have selective antibacterial activity because the reducing activity needed to generate active derivatives is generated only by bacterial and not mammalian enzymes. Disruption of Membrane Integrity The integrity of the bacterial cytoplasmic membrane—and, in gram-negative bacteria, the outer membrane—is important for bacterial viability. Two bactericidal drugs have membrane targets. polymyxins The polymyxins, including polymyxin B and polymyxin E (colistin), are cationic cyclic polypeptides that disrupt the cytoplasmic membrane and the outer membrane (the latter by binding lipopolysaccharide).

1	daptomycin Daptomycin is a lipopeptide that binds the cytoplasmic membrane of gram-positive bacteria in the presence of calcium, generating a channel that leads to leakage of cytoplasmic potassium ions and membrane depolarization.

1	Bacteria use a wide variety of mechanisms to block or circumvent the activity of antibacterial agents. Although myriad, these mechanisms can generally be grouped into three categories: (1) altered or bypass targets that exhibit reduced binding of the drug, (2) altered access of the drug to its target by reductions in uptake or increases in active efflux, and (3) a modification of the drug that reduces its activity. These mechanisms result from either mutations in bacterial chromosomal genes occurring spontaneously during bacterial DNA replication or the acquisition of new genes by DNA transfer from other bacteria or uptake of exogenous DNA. New genes are most often acquired on self-replicating plasmids or other DNA elements transferred from other bacteria. However, some bacteria, such as Streptococcus pneumoniae and Neisseria gonorrhoeae, can also take up fragments of environmental DNA from related species and recombine that DNA directly into their own chromosomes, a process called

1	Streptococcus pneumoniae and Neisseria gonorrhoeae, can also take up fragments of environmental DNA from related species and recombine that DNA directly into their own chromosomes, a process called transformation. Not uncommonly, resistant bacteria have combinations of resistance mechanisms either within one category or among categories, and many plasmids contain more than one resistance gene. Thus, plasmid acquisition itself can in many cases confer resistance to multiple antibacterial agents.

1	Many antibacterial drugs are derived from natural products of microbial species. Some genes encoding resistance to these drugs may have evolved and been mobilized onto plasmids from a protection mechanism in the producer organism or in other surviving bacteria in the exposed environment. Exposure to antibacterial agents either in nature or from human or animal use results in the selection of resistant strains within an otherwise susceptible bacterial population. Because the patterns and extent of resistance may differ among settings, initial choices of antibacterial drugs should be based, whenever possible, on local susceptibility data and should be modified as needed as soon as specific microbiology susceptibility data become available.

1	β-Lactams The most common mechanism of resistance to β-lactams 933 is their degradation by β-lactamases, enzymes that break down the core β-lactam ring and destroy drug activity. Different β-lactamases degrade different β-lactams. Some β-lactamases are encoded on the bacterial chromosome, and their activity contributes to the susceptibility profile of a particular species. Because other β-lactamases are encoded by acquired plasmids, their resistance profiles may be present in some strains of a species but not others. In gram-positive bacteria β-lactamases are secreted into the extracellular environment, whereas in gram-negative bacteria these enzymes are secreted into the periplasmic space between the cytoplasmic and outer membranes. Thus, in gram-negative bacteria, access of β-lactams both to their target PBPs and to β-lactamases requires diffusion across the outer membrane, generally through the porin channels.

1	Most strains of Staphylococcus aureus produce a plasmid-encoded β-lactamase that degrades penicillin but not semisynthetic penicillins, such as oxacillin and nafcillin. The most common plasmid-encoded β-lactamases of gram-negative bacteria are able to inactivate all penicillins and most earlier-generation cephalosporins. Extended-spectrum β-lactamase (ESBL) variants of these early enzymes that can degrade later-generation cephalosporins (ceftriaxone, cefotaxime, ceftazidime) as well as the monobactam aztreonam have now emerged and are widely disseminated. Some ESBLs also degrade the fourth-generation cephalosporin cefepime. Carbapenems (imipenem, meropenem, ertapenem, doripenem) are not degraded by ESBLs, but additional β-lactamases, called carbapenemases, that degrade carbapenems and most if not all other β-lactams have begun to emerge.

1	The chromosomal β-lactamase of Klebsiella pneumoniae preferentially degrades penicillins but not cephalosporins. In contrast, the chromosomal β-lactamase of Enterobacter and related genera, AmpC, can degrade almost all cephalosporins but is normally expressed in small amounts. Mutations that cause increased amounts of AmpC to be produced confer full resistance to penicillins and cephalosporins; the exceptions are cefoxitin and cefepime, which are relatively stable to AmpC. Resistance to cefepime can develop, however, through the combined effects of increased AmpC production and decreased porin diffusion channels. Genes encoding AmpC have also been found on plasmids but are less common than plasmid-encoded ESBLs.

1	Inhibitors of β-lactamases such as clavulanate, sulbactam, and tazobactam have been developed and paired with amoxicillin and ticarcillin, ampicillin, and piperacillin, respectively. These inhibitors have little or no antibacterial activity of their own but inhibit plasmid-mediated β-lactamases, including ESBLs but not AmpC enzymes. Resistance to β-lactams also occurs through alterations in their target PBPs. In S. pneumoniae, N. gonorrhoeae, and Neisseria meningitidis, resistance to penicillin occurs by recombination of transformed DNA from related species. In staphylococci, resistance to methicillin and other β-lactams occurs by the acquisition of the mec gene, which encodes PBP2a with reduced drug affinity. Ceftaroline is the only β-lactam that has affinity for PBP2a and is thus active against methicillin-resistant staphylococcal strains.

1	Glycopeptides Resistance to vancomycin in enterococci is due to the acquisition of a set of van genes that result in (1) the production of d-alanine-d-lactate—instead of the normal d-alanine-d-alanine—at the end of the peptidoglycan stem peptide and (2) the reduction of existing d-alanine-d-alanine terminated peptides. Vancomycin binds d-alanine-d-lactate with a thousandfold lower affinity than d-alanined-alanine. In a small number of cases, the van gene cassettes have been transferred from enterococci to S. aureus, with the consequent generation of full vancomycin resistance. Particularly in patients receiving prolonged courses of vancomycin, intermediate resistance to this drug has developed in S. aureus by a different mechanism: multiple chromosomal mutations that result in a thickened and poorly cross-linked cell wall in which multiple distant d-alanine-d-alanine stem peptide termini exist and bind vancomycin, impeding its access to the binding sites proximal to the cell membrane

1	and poorly cross-linked cell wall in which multiple distant d-alanine-d-alanine stem peptide termini exist and bind vancomycin, impeding its access to the binding sites proximal to the cell membrane where new cell-wall synthesis occurs and where binding would block transpeptidase and transglycosylase enzymes. Susceptibility to telavancin, dalbavancin, and oritavancin is

1	Treatment and Prophylaxis of Bacterial Infections 934 also reduced in strains that exhibit resistance or intermediate susceptibility to vancomycin, although in some cases strains may still be classified as susceptible on the basis of clinical interpretive criteria.

1	Aminoglycosides The most common mechanism of resistance is due to acquisition of plasmid genes encoding transferase enzymes that modify aminoglycosides by the addition of acetyl, adenyl, or phosphate groups; these added groups decrease the drugs’ binding affinity to their ribosomal target site. Transferases differ in which aminoglycosides they modify, and amikacin resistance occurs less often than resistance to gentamicin or tobramycin. More recently, plasmids encoding methyltransferases that modify the ribosomal site of aminoglycoside binding and confer resistance to all aminoglycosides have been found in enteric gram-negative bacteria. For streptomycin, a ribosomal protein mutation may cause resistance. In Pseudomonas aeruginosa, resistance may also occur through mutations causing increased expression of a chromosomally encoded efflux pump, MexXY.

1	Tetracyclines and Glycylcyclines For tetracyclines, resistance is most often plasmid mediated and attributable either to active efflux pumps, which are generally specific for tetracyclines, or to proteins that protect the ribosome from tetracycline action. Resistance to the glycylcycline tigecycline, which is not affected by the usual tetracycline resistance mechanisms, can occur through mutations that cause overexpression of certain broad-spectrum efflux pumps in Proteus species.

1	Macrolides, Ketolides, Lincosamides, and Streptogramins Resistance to macrolides, clindamycin, and quinupristin is most often due to plasmid-acquired methylases that modify the drug binding site on the ribosome. Resistance to quinupristin by this mechanism renders the quinupristin-dalfopristin combination bacteriostatic rather than bactericidal. Telithromycin, a ketolide, has an additional binding site on the ribosome and remains active in the presence of these methylases. Acquired genes encoding active efflux pumps can also contribute to resistance to macrolides in streptococci and resistance to macrolides, clindamycin, and dalfopristin in staphylococci. Plasmid-acquired drug-modifying enzymes in staphylococci can also cause resistance to quinupristin and dalfopristin. Macrolide resistance due to 23S rRNA mutations is uncommon in staphylococci and streptococci because of the multiple copies of the rRNA genes on the chromosomes of these species; such resistance may occur more

1	resistance due to 23S rRNA mutations is uncommon in staphylococci and streptococci because of the multiple copies of the rRNA genes on the chromosomes of these species; such resistance may occur more frequently, however, in mycobacteria and Helicobacter pylori, which have only single chromosomal copies of these rRNA genes.

1	Chloramphenicol Resistance to chloramphenicol is most often due to a plasmid-encoded drug-modifying acetyltransferase. Oxazolidinones Linezolid resistance has been seen in enterococci more often than in staphylococci and, in both organisms, is due to mutations in multiple copies of the 23S rRNA genes that reduce drug binding to the ribosome. A plasmid-acquired ribosomal methylase gene that confers resistance to both chloramphenicol and linezolid has also been found in some strains of staphylococci but is not yet widespread. Tedizolid may still be active against some but not all linezolidresistant strains. Mupirocin Resistance to mupirocin occurs by either mutation in the target leucyl-tRNA synthetase (low-level resistance) or the acquisition of a plasmid-encoded resistant tRNA synthetase (high-level resistance).

1	Sulfonamides and Trimethoprim Resistance to both of these antimetabolites is due to plasmid-acquired genes encoding resistant enzymes that bypass the inhibition of the native sensitive enzymes—a resistant dihydropteroate synthetase in the case of sulfonamides and a resistant dihydrofolate reductase in the case of trimethoprim.

1	Quinolones Resistance to quinolones is most often due either to chromosomal mutations altering the target enzymes DNA gyrase and DNA topoisomerase IV, with consequent reduction in drug binding, or to mutations that increase the expression of native broad-spectrum efflux pumps for which quinolones (among other compounds) are substrates. In addition, three types of genes can confer reduced susceptibility or low-level resistance by protecting target enzymes, modifying some quinolones, or pumping quinolones out of the cell (efflux). These genes are located on multidrug resistance plasmids that have spread worldwide. Their presence can promote the selection of higher levels of quinolone resistance linked to resistance to other antibacterial drugs that is encoded on the same plasmid.

1	Rifampin and Rifabutin Single mutations in the β subunit of RNA polymerase can cause high-level resistance to rifampin. Thus rifampin and other rifamycins are used for treatment of infections only in combination with other antibacterial drugs in order to prevent resistance. Metronidazole Acquired resistance to metronidazole in Bacteroides species is rare. Such resistance has been reported in strains that lack endogenous nitroreductase activity or that have acquired nim genes responsible for further reduction of DNA-damaging nitroso intermediates to an inactive derivative. Active efflux and enhanced DNA repair mechanisms also have been associated with resistance. Nitrofurantoin Resistance to nitrofurantoin in Escherichia coli can emerge through a series of mutations that progressively decrease the nitroreductase activity necessary for generating active nitrofuran metabolites.

1	Polymyxins Because of emerging multidrug resistance in gram-negative bacteria, colistin and polymyxin B are being used increasingly for infections due to resistant Enterobacteriaceae, P. aeruginosa, and Acinetobacter species. Rates of resistance vary. Resistance can emerge during therapy through mutations that cause reductions in the negative charge of the gram-negative bacterial cell surface, thereby reducing binding of the positively charged colistin. Daptomycin The mechanisms of resistance to daptomycin are complex and involve mutations in several genes that can alter cell membrane charge and reduce daptomycin binding. Resistance to daptomycin is relatively infrequent but has emerged in some S. aureus strains with intermediate vancomycin susceptibility from patients treated with vancomycin but not with daptomycin. In some methicillin-resistant

1	S. aureus (MRSA) strains, daptomycin resistance has been linked to acquired susceptibility to β-lactams; combinations of daptomycin and nafcillin have been successful for treatment of patients infected with resistant strains when daptomycin alone or in combination with other agents has failed. The mechanism of this effect is not yet clear. The term pharmacokinetics describes the disposition of a drug in the body, whereas pharmacodynamics describes the determinants of drug action on the pathogen in relation to pharmacokinetic factors. An understanding of the principles governing these two areas is required for effective drug selection, dosing, and prevention of toxicities. The process of drug disposition has four principal phases: absorption, distribution, metabolism, and excretion. These components determine the time course of drug concentrations in serum and subsequently the concentrations in other tissues and body fluids.

1	Absorption When a drug is given by a particular route, absorption is defined as the percentage of the dose that reaches the systemic circulation. For example, since IV administration provides direct access to the systemic circulation, 100% of a drug dose given IV is usually absorbed. The level of absorption becomes more relevant when non-IV routes are used—e.g., the oral, IM, SC, and topical routes. The percentage of a drug that is absorbed is termed its bioavailability. Examples of antibacterial agents with a high oral bioavailability include metronidazole, levofloxacin, and linezolid. IV administration and oral dosing for highly bioavailable agents usually give equivalent results. Many factors can affect a drug’s oral bioavailability, including the timing of food consumption relative to drug administration, drug-metabolizing enzymes, efflux transporters, concentration-dependent solubility, and acid degradation. Underlying conditions such as diarrhea or ileus can also affect the site

1	drug administration, drug-metabolizing enzymes, efflux transporters, concentration-dependent solubility, and acid degradation. Underlying conditions such as diarrhea or ileus can also affect the site of drug absorption and thereby alter bioavailability. Certain orally administered drugs have lower bioavailability because of the first-pass effect—the process by which drugs are absorbed in the small intestine through the portal circulation and then directly transported to the liver for metabolism.

1	Distribution Distribution describes the process by which a drug transfers reversibly between the general circulation and the tissues. After absorption into the general circulation and the central compartment (the extensively perfused organs), the drug will also distribute into the peripheral compartment (less well-perfused tissues). The volume of distribution (Vd) is a pharmacokinetic parameter that describes the amount of drug in the body at a given time relative to the measured serum concentration. Properties such as the drug’s lipophilicity, partition coefficient within different body tissues, and protein binding; blood flow; and pH can affect the volume of distribution. Drugs with a small volume of distribution are limited to certain areas within the body (typically extracellular fluid), whereas those with a higher volume of distribution penetrate extensively into tissues throughout the body. Antibacterial drugs can bind to serum proteins, and a given drug is usually described as

1	whereas those with a higher volume of distribution penetrate extensively into tissues throughout the body. Antibacterial drugs can bind to serum proteins, and a given drug is usually described as either poorly or highly protein bound. Only the unbound (free) drug is active and available to exert antibacterial effects. For example, because tigecycline is highly protein bound and also has a large volume of distribution, concentrations of free drug in the serum are low.

1	Metabolism Metabolism is the chemical transformation of a drug by the body. This modification can occur within several areas; the liver is the organ most commonly involved. Drugs are metabolized by enzymes, but enzyme systems have a finite capacity to metabolize a substrate drug. If a drug is given in a dose at which the concentration does not exceed the rate of metabolism, then the metabolic process is generally linear. If the dose exceeds the amount that can be metabolized, drug accumulation and potential toxicity may occur. Drugs are metabolized through phase I or phase II reactions. In phase I reactions, the drug is made more polar through dealkylation, hydroxylation, oxidation, and deamination. Polarity facilitates drug removal from the body. Phase II reactions, which include glucuronidation, sulfation, and acetylation, result in compounds larger and more polar than the parent drug. Both phases usually inactivate the parent drug, but some drugs are rendered more active. The

1	sulfation, and acetylation, result in compounds larger and more polar than the parent drug. Both phases usually inactivate the parent drug, but some drugs are rendered more active. The cytochrome P450 (CYP) enzyme system is responsible for phase I reactions and is generally found in the liver. CYP3A4 is a common subfamily within this system that is responsible for the majority of drug metabolism. Antibacterial drugs can be substrates, inhibitors, or inducers of a particular CYP enzyme. Inducers, such as rifampin, can increase the production of CYP enzymes and consequently increase the metabolism of other drugs. Inhibitors, such as quinupristin-dalfopristin, cause a decrease in enzyme activity (or competition for CYP substrate) and therefore an increase in the concentration of the interacting drug.

1	Excretion Excretion describes the body’s mechanisms of drug elimination. Drugs can be eliminated through more than one mechanism. Renal clearance is the most common route and includes elimination through glomerular filtration, tubular secretion, and/or passive diffusion. Some agents have nonrenal clearance and rely on the biliary tree or the intestine for excretion. Excretion affects the half-life of a drug—i.e., the time it takes for the blood concentration of a drug to decrease by one-half. This value can range from minutes to days. Approximately five to seven half-lives are required for a drug to reach steady state when multiple doses are given in a time frame shorter than the half-life itself. Drug half-life and overall clearance can be extended if the organ responsible for clearance is impaired. Patients with renal or hepatic impairment may require dose adjustments that take delayed clearance into account and prevent toxicities from drug accumulation. For example, imipenem is

1	is impaired. Patients with renal or hepatic impairment may require dose adjustments that take delayed clearance into account and prevent toxicities from drug accumulation. For example, imipenem is cleared predominantly through glomerular filtration, and in the presence of renal impairment the dosing interval is typically increased to account for the increased half-life.

1	The term pharmacodynamics describes the relationship between the serum concentrations that determine the efficacy of the drug and the serum concentrations that produce the toxic effects of the drug. For an antibacterial agent, the pharmacodynamic focus is the type of drug FIGURE 170-2 Pharmacokinetic and pharmacodynamic model predicting efficacy of antibacterial drugs. AUC, area under the time–concentration curve; Cmax, peak serum concentration of drug; MIC, minimal inhibitory concentration; T>MIC, duration of drug concentrations above the MIC.

1	exposure needed for optimal antibacterial effect in relation to the minimal inhibitory concentration (MIC)—the lowest drug concentration that inhibits the visible growth of a microorganism under standardized laboratory conditions. Antibacterial effect usually correlates with one of the following parameters: (1) ratio of peak serum concentration to the MIC (Cmax/MIC), (2) ratio of the area under the concentration– time curve to the MIC (AUC/MIC), or (3) duration of concentrations above the MIC (T>MIC) (Fig. 170-2).

1	For concentration-dependent killing agents, as the designation implies, the higher the drug concentration, the higher the rate and extent of bacterial killing. Aminoglycosides fit into the Cmax/MIC model of pharmacodynamics activity, and a particular peak serum concentration is often targeted to achieve optimal killing. Fluoroquinolones exemplify antibacterial agents for which the AUC/MIC is a predictor of efficacy. For example, studies have found that an AUC/MIC ratio of >30 will maximize killing of S. pneumoniae by fluoroquinolones. In contrast, time-dependent killing agents reach a ceiling at which higher concentrations do not result in increased effect. Instead, these agents are active against bacteria only when the drug concentration is above the MIC. The T>MIC predicts clinical efficacy for all β-lactams. The longer the concentration of the β-lactam remains above the MIC for an infecting pathogen during the dosing interval, the greater the killing effect. For some drug classes,

1	for all β-lactams. The longer the concentration of the β-lactam remains above the MIC for an infecting pathogen during the dosing interval, the greater the killing effect. For some drug classes, such as aminoglycosides, a postantibiotic effect—the delayed regrowth of surviving bacteria after exposure to an antibiotic—supports less frequent dosing.

1	The approach to antibiotic therapy is driven by host factors, site of infection, and local resistance profiles of suspected or known pathogens. Further, national and local drug shortages and formulary restrictions can affect available therapies. Regular monitoring of the patient and collection of laboratory data should be undertaken to streamline antibacterial therapy as appropriate and to investigate the possibility of treatment failure if the patient fails to respond appropriately.

1	Therapy is considered empirical when the causative agent has yet to be determined and therapeutic decisions are based on the severity of illness and the clinician’s assessment of likely pathogens in light of the clinical syndrome, the patient’s medical conditions and prior therapy, and relevant epidemiologic factors. For patients with severe illness, empirical therapy often takes the form of an antibacterial combination that provides broad coverage of diverse agents and thus ensures adequate treatment of possible pathogens while additional data are being collected. Directed therapy is predicated on identification of the

1	Treatment and Prophylaxis of Bacterial Infections 936 pathogen, determination of its susceptibility profile, and establishment of the extent of the infection. Directed therapy generally allows the use of more targeted and narrower-spectrum antibacterial agents than does empirical therapy. Information on epidemiology, exposures, and local antibacterial susceptibility patterns can help guide empirical therapy. When empirical treatment is clinically appropriate, care should be taken to obtain clinical specimens for microbiologic analysis before the initiation of therapy and to de-escalate therapy as new information is obtained about the patient’s clinical condition and the causal pathogens. De-escalation to the point of directed therapy can limit unnecessary risks to the patient as well as the risk of emergence of antibacterial resistance.

1	The site of infection is a consideration in antibacterial therapy, largely because of the differing abilities of drugs to penetrate and achieve adequate concentrations at particular body sites. For example, to be effective in the treatment of meningitis, an agent must (1) be able to cross the blood–brain barrier and reach adequate concentrations in the cerebrospinal fluid (CSF) and (2) be active against the relevant pathogen(s). Dexamethasone, administered with or 15–20 min before the first dose of an antibacterial drug, has been shown to improve outcomes in patients with acute bacterial meningitis, but its use may reduce penetration of some antibacterial agents, such as vancomycin, into the CSF. In this case, rifampin is added because its penetration is not reduced by dexamethasone. Infections at other sites where either pathogens are protected from normal host defenses or penetration of an antibacterial drug is suboptimal include osteomyelitis, prostatitis, intraocular infections,

1	at other sites where either pathogens are protected from normal host defenses or penetration of an antibacterial drug is suboptimal include osteomyelitis, prostatitis, intraocular infections, and abscesses. In such cases, consideration must be given to the mechanism of drug delivery (e.g., intravitreal injections) as well as to the role of interventions to drain, debride, or otherwise reduce the barriers to effective antibacterial therapy.

1	Host factors, including immune function, pregnancy, allergies, age, renal and hepatic function, drug–drug interactions, comorbid conditions, and occupational or social exposures, should be considered. Immune Dysfunction Patients with deficits in immune function that blunt the response to bacterial infection, including neutropenia, deficient humoral immunity, and asplenia (either surgical or functional), are all at increased risk of severe bacterial infection. Such patients should be treated aggressively and often broadly in the early stages of suspected infection pending results of microbiologic tests. For asplenic patients, treatment should include coverage of encapsulated organisms, particularly S. pneumoniae, that may cause rapidly life-threatening infection.

1	Pregnancy Pregnancy affects decisions regarding antibacterial therapy in two respects. First, pregnancy is associated with an increased risk of particular infections (e.g., those caused by Listeria). Second, the potential risks to the fetus that are posed by specific drugs must be considered. As for other drugs, the safety of the vast majority of antibacterial agents in pregnancy has not been established, and such agents are grouped in categories B and C by the U.S. Food and Drug Administration. Drugs in categories D and X are contraindicated in pregnancy or lactation due to established risks. The risks associated with antibacterial use in pregnancy and during lactation are summarized in Table 170-2.

1	Allergies Allergies to antibiotics are among the most common allergies reported, and an allergy history should be obtained whenever possible before therapy is chosen. A detailed allergy history can shed light on the type of reaction experienced previously and on whether rechallenge with the same or a related medication is advisable (and, if so, under what circumstances). Allergies to the penicillins are most common. Although as many as 10% of patients may report an allergy to penicillin, studies suggest that up to 90% of these patients could tolerate a penicillin or cephalosporin. Adverse effects (Table 170-3) should be distinguished from true allergies to ensure appropriate selection of antibacterial therapy. Drug–Drug Interactions Patients commonly receive other drugs that may interact with antibacterial agents. A summary of the most common drug–drug interactions, by antibacterial class, is provided in Table 170-4.

1	Exposures Exposures, both occupational and social, may provide clues to likely pathogens. When relevant, inquiries about exposure to ill contacts, animals, insects, and water should be included in the history, along with sites of residence and travel. Other Host Factors Age, renal and hepatic function, and comorbid conditions are all considerations in the choice of and schedule for therapy. Dose adjustments should be made accordingly. In patients with decreased or unreliable oral absorption, IV therapy may be preferred to ensure adequate blood levels of drug and delivery of the antibacterial agent to the site of infection.

1	Whether empirical or directed, the duration of therapy should be planned in most clinical situations. Guidelines that synthesize available literature and expert opinion provide recommendations on therapy duration that are based on infecting organism, organ system, and patient factors. For example, the American Heart Association has published guidelines endorsed by the Infectious Diseases Society of America (IDSA) on diagnosis, antibacterial therapy, and management of complications of infective endocarditis. Similar guidelines from the IDSA exist for bacterial meningitis, catheter-associated urinary tract infections, intraabdominal infections, communityand hospital-acquired pneumonia, and other infections.

1	If a patient does not respond to therapy, investigations often should include the collection of additional specimens for microbiologic testing and imaging as indicated. Failure to respond can be the result of an antibacterial regimen that does not address the underlying causative organism, the development of resistance during therapy, or the existence of a focus of infection at a site poorly penetrated by systemic therapy. Some infections may also require surgical interventions for cure (e.g., large abscesses, myonecrosis). Fever due to allergic drug reactions can sometimes complicate assessment of the patient’s response to antibacterial treatment. Selected websites with the most up-to-date information and guidance for the clinician include the following: Johns Hopkins ABX Guide (www.hopkins-abxguide.org) IDSA Practice Guidelines (www.idsociety.org/IDSA_Practice_Gui delines/) Center for Disease Dynamics, Economics and Policy Resistance Map (www.cddep.org/map)

1	IDSA Practice Guidelines (www.idsociety.org/IDSA_Practice_Gui delines/) Center for Disease Dynamics, Economics and Policy Resistance Map (www.cddep.org/map) CDC Antibiotic/Antimicrobial Resistance (www.cdc.gov/drug resistance/) The clinical application of antibacterial therapy is guided by the spectrum of the agent and the suspected or known target pathogen. Infections for which specific antibacterial agents are among the drugs of choice are listed, along with associated pathogens and susceptibility data, in Table 170-5. Resistance rates of specific organisms are dynamic and should be taken into account in the approach to antibacterial therapy. While national resistance rates can serve as a reference, the most useful reference for the clinician is the most recent local laboratory antibiogram, which provides details on local resistance patterns, often on an annual or semiannual basis.

1	The β-lactam class of antibiotics consists of penicillins, cephalosporins, carbapenems, and monobactams. The term β-lactam reflects the drugs’ four-membered lactam ring, which is their core structure. The differing aCategory B: Either animal reproduction studies have failed to demonstrate a risk to the fetus, and there are no adequate and well-controlled studies in pregnant women; or animal studies have shown an adverse effect, but adequate and well-controlled studies in pregnant women have failed to demonstrate a risk to the fetus in any trimester. Category C: Animal reproduction studies have shown an adverse effect on the fetus, and there are no adequate and well-controlled studies in humans, but potential benefits may warrant use of the drug in pregnant women despite potential risks. Category D: There is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans, but potential benefits may warrant use of

1	Category D: There is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans, but potential benefits may warrant use of the drug in pregnant women despite potential risks. bFetal risk recommendation and breast-feeding risk recommendation adapted from GG Briggs et al, eds: Drugs in Pregnancy and Lactation, 9th ed. Philadelphia, Lippincott Williams and Wilkins, 2011; and the U.S. Food and Drug Administration (Drugs@FDA). cA registry has been established to monitor pregnancy outcomes of pregnant women exposed to telavancin. Physicians are encouraged to register pregnant patients, or pregnant women may enroll themselves by calling 1-855-633-8479.

1	Abbreviation: G6PD, glucose-6-phosphate dehydrogenase.

1	side chains among the agents of this family determine the spectrum of as penicillin G, are active against non-β-lactamase-producing gram-activity. All β-lactams exert a bactericidal effect by inhibiting bacterial positive and gram-negative bacteria, anaerobes, and some gram-negative cell-wall synthesis. The β-lactams are classified as time-dependent kill-cocci. Penicillin G is used for penicillin-susceptible streptococcal infecing agents; therefore, their clinical efficacy is best correlated with the tions, pneumococcal and meningococcal meningitis, enterococcal endoproportion of the dosing interval during which the drug levels remain carditis, and syphilis. The antistaphyloccocal penicillins, which have above the MIC for the pathogenic organism. potent activity against methicillin-susceptible S. aureus (MSSA), include nafcillin, oxacillin, dicloxacillin, and flucloxacillin. Aminopenicillins, Penicillins and β-Lactamase Inhibitors Penicillin, the first β-lactam, was such as ampicillin

1	S. aureus (MSSA), include nafcillin, oxacillin, dicloxacillin, and flucloxacillin. Aminopenicillins, Penicillins and β-Lactamase Inhibitors Penicillin, the first β-lactam, was such as ampicillin and amoxicillin, provide added coverage beyond discovered in 1928 by Alexander Fleming. Natural penicillins, such penicillin against gram-negative cocci, such as Haemophilus influenzae,

1	Treatment and Prophylaxis of Bacterial Infections Note: All systemic antibiotics have the potential to alter abdominal flora and induce Clostridium difficile infection. Abbreviations: aPTT, activated partial thromboplastin time; CPK, creatine phosphokinase; INR, international normalized ratio; LFT, liver function test; PT, prothrombin time; TMP-SMX, trimethoprim-sulfamethoxazole.

1	and some Enterobacteriaceae, including E. coli, Proteus mirabilis, species (such as Bacteroides fragilis), which produce β-lactamases and Salmonella, and Shigella. The aminopenicillins are hydrolyzed by many are generally resistant. The rising prevalence of β-lactamase-producing common β-lactamases. These drugs are commonly used for otitis media, bacteria has led to the increased use of β-lactam/β-lactamase inhibitor respiratory tract infections, intraabdominal infections, endocarditis, combinations, such as ampicillin-sulbactam, amoxicillin-clavulanate, meningitis, and urinary tract infections. The antipseudomonal penicil-ticarcillin-clavulanate, and piperacillin-tazobactam. The β-lactamase lins include ticarcillin and piperacillin. These penicillin groups gener-inhibitors themselves do not have antibacterial activity (with the excepally offer adequate anaerobic coverage; the exceptions are Bacteroides tion of sulbactam, which has activity against Acinetobacter baumannii)

1	Nafcillin Warfarin, cyclosporine, tacrolimus Linezolid, tedizolid Serotonergic and adrenergic agents (e.g., SSRIs, vasopressors) Quinupristin dalfopristin Substrates of cytochrome CYP3A4 (e.g., warfarin, ritonavir, cyclosporine, diazepam, verapamil) Sucralfate; antacids containing aluminum, calcium, or magnesium; ferrous sulfate and zinc-containing multivitamins Rifampin Substrates of cytochrome CYP3A4 (e.g., warfarin, ritonavir, cyclosporine, diazepam, verapamil, protease inhibitors, voriconazole) Substrates of cytochrome CYP2C19 (e.g., omeprazole, lansoprazole) Substrates of cytochrome CYP2C9 (e.g., warfarin, tolbutamide) Substrates of cytochrome CYP2C8 (e.g., repaglinide, rosiglitazone) Substrates of cytochrome CYP2B6 (e.g., efavirenz) Hormone therapy (e.g., norethindrone) Tetracyclines Antacids or drugs containing calcium, magnesium, iron, or aluminum Macrolidesb Substrates of cytochrome CYP3A4 (e.g., warfarin, ritonavir, cyclosporine, diazepam, verapamil)

1	Tetracyclines Antacids or drugs containing calcium, magnesium, iron, or aluminum Macrolidesb Substrates of cytochrome CYP3A4 (e.g., warfarin, ritonavir, cyclosporine, diazepam, verapamil) QTc-prolonging agents (e.g., fluoroquinolones, sotalol) Protease inhibitors (e.g., ritonavir) Oritavancin Substrates of cytochrome CYP3A4 (e.g., cyclosporine, warfarin) and CYP2D6 (e.g., aripiprazole) Substrates of cytochrome CYP2C19 (e.g., omeprazole) and CYP2C9 (e.g., warfarin) Decreased levels of warfarin, cyclosporine via CYP3A4 induction. Monitor levels of affected drug closely if drugs are given concomitantly. Concomitant use is contraindicated in neonates (<28 days); the combination can lead to precipitation of ceftriaxone-calcium particulate. Ceftriaxone and calcium-containing solutions can be given to infants >28 days of age provided they are given sequentially and the lines are thoroughly flushed between infusions.

1	Ceftriaxone and calcium-containing solutions can be given to infants >28 days of age provided they are given sequentially and the lines are thoroughly flushed between infusions. Decreased levels of valproic acid. Monitor valproic acid levels closely if drugs are given concomitantly. Increased levels of serotonergic and adrenergic agents. Monitor for serotonin syndrome. Tedizolid may have less potential than linezolid to cause this drug interaction. Can result in increased levels of interacting drug. Erythromycin and clarithromycin are more potent CYP3A4 inhibitors than azithromycin. Avoid concomitant administration if possible. Can result in subtherapeutic fluoroquinolone levels. Administer fluoroquinolone 2 h before or 6 h after interacting drug. Can result in increased levels of tizanidine and hypotensive, sedative effects. Monitor for side effects if drugs are given concomitantly.

1	Can result in increased levels of tizanidine and hypotensive, sedative effects. Monitor for side effects if drugs are given concomitantly. Can result in decreased levels of interacting drug. Avoid concomitant use if possible. If giving drugs concomitantly, monitor drug levels if possible. Can result in decreased levels of hormone. If oral contraceptive and rifampin are given concomitantly, use alternative form of birth control. Can result in decreased absorption of tetracyclines. Administer tetracycline 2 h before or 6 h after interacting drug. Avoid concomitant administration if possible. Increased risk of cardiotoxicity and arrhythmias. Monitor QTc. Can result in increased levels of both macrolides and protease inhibitors. Avoid concomitant use if possible. Cimetidine can increase levels of macrolides. Can result in disulfiram-like reaction. Ethanol may be present in some for mulations of oral drug suspensions (e.g., ritonavir).

1	Can increase warfarin levels. Monitor INR closely if drugs are given concomitantly. Increased effect of warfarin. Monitor levels closely if drugs are given concomitantly. Increased levels of phenytoin. Monitor levels closely if drugs are given concomitantly. Increased levels of methotrexate. Monitor levels closely if drugs are given concomitantly. Can result in decreased levels of interacting drug. Avoid concomitant use if possible. If giving drugs concomitantly, monitor drug levels if possible. Can result in increased levels of interacting drug. Avoid concomitant use if possible. If giving drugs concomitantly, monitor drug levels if possible.

1	Treatment and Prophylaxis of Bacterial Infections aDrug reaction described with ciprofloxacin only. bClarithromycin and erythromycin are potent CYP3A4 inhibitors; the probability of a drug interaction with azithromycin is lower. Abbreviations: INR, international normalized ratio; SSRI, selective serotonin-reuptake inhibitor; TMP-SMX, trimethoprim-sulfamethoxazole. but typically inhibit the S. aureus class A β-lactamase, β-lactamases of H. influenzae and Bacteroides species, and a number of plasmid-encoded β-lactamases. These combination agents are typically used when broader-spectrum coverage is needed—e.g., in pneumonia and intraabdominal infections. Piperacillin-tazobactam is a useful agent for broad coverage in febrile neutropenic patients. The combination agents, however, are not effective against organisms that produce AmpC β-lactamases or carbapenemases.

1	Cephalosporins The cephalosporin drug class encompasses five generations determined by spectrum of antibacterial activity. The first generation (cefazolin, cefadroxil, cephalexin) largely has activity 940 TABLE 170-5 DRug InDICATIonS foR SPECIfIC InfECTIonS, ASSoCIATED PATHogEnS, AnD SAMPLE SuSCEPTIBILITy RATES Antimicrobial(s) Infections Common Pathogens (% Susceptible); Resistance as Noteda Ampicillin, amoxicillin Nafcillin, oxacillin Piperacillin-tazobactam Cefazolin Cefoxitin, cefotetan Ceftriaxone Ceftazidime, cefepime Ceftaroline Imipenem, meropenem Ertapenem Aztreonam Vancomycin Telavancin Dalbavancin, oritavancin Daptomycin Gentamicin, tobramycin, amikacin Azithromycin, clarithromycin, erythromycin Clindamycin Doxycycline, minocycline Intraabdominal infections (facultative enteric gram-negative bacilli and obligate anaerobes); infections caused by mixed flora (aspiration pneumonia, diabetic foot ulcers); infections caused by

1	Intraabdominal infections (facultative enteric gram-negative bacilli and obligate anaerobes); infections caused by mixed flora (aspiration pneumonia, diabetic foot ulcers); infections caused by E. coli UTI; surgical prophylaxis; MSSA bacteremia and endocarditis Hospital-acquired infections caused by facultative gram-negative bacilli and Pseudomonas spp. CAP caused by S. pneumoniae, MSSA, H. influenzae, K. pneumoniae, Klebsiella oxytoca, and E. coli; acute bacterial skin and skin-structure infections caused by MSSA, MRSA, Streptococcus pyogenes, Streptococcus agalactiae, E. coli, K. pneumoniae, and K. oxytoca Intraabdominal infections, infections caused by Enterobacter spp. and ESBL-producing gram-negative bacilli CAP; complicated UTIs, including pyelonephritis; acute pelvic infections; complicated intraabdominal infections; complicated skin and skin-structure infections, excluding diabetic foot infections accompanied by osteomyelitis or caused by P. aeruginosa

1	Bacteremia, endocarditis, and other invasive disease caused by MRSA; pneumococcal meningitis; oral formulation for CDAD Combined with penicillin for staphylococcal, enterococcal, or streptococcal endocarditis; combined with β-lactam for gram-negative bacteremia; pyelonephritis Legionella, Campylobacter, and Mycoplasma infections; CAP; GAS pharyngitis in penicillin-allergic patients; bacillary angiomatosis; gastric infections due to Helicobacter pylori; MAI infections Severe, invasive GAS infections (with β-lactam); infections caused by obligate anaerobes; infections caused by susceptible staphylococci

1	Severe, invasive GAS infections (with β-lactam); infections caused by obligate anaerobes; infections caused by susceptible staphylococci Acute bacterial exacerbations of chronic bronchitis; granuloma inguinale; brucellosis (with streptomycin); tularemia; glanders; melioidosis; spirochetal infections caused by Borrelia (Lyme disease and relapsing fever; doxycycline); infections caused by Vibrio vulnificus; some Aeromonas infections; infections due to Stenotrophomonas (minocycline); plague; ehrlichiosis; chlamydial infections (doxycycline); granulomatous infections due to Mycobacterium marinum (minocycline); rickettsial infections; mild CAP; skin and soft tissue infections caused by gram-positive cocci (e.g., CA-MRSA infections); leptospirosis; syphilis; and actinomycosis in the penicillin-allergic patient CAP caused by S. pneumoniae, H. influenzae, or Legionella pneumophila; complicated skin infections caused by E. coli, MRSA, MSSA,

1	CAP caused by S. pneumoniae, H. influenzae, or Legionella pneumophila; complicated skin infections caused by E. coli, MRSA, MSSA, S. pyogenes, Streptococcus anginosus, S. agalactiae, B. fragilis; complicated intraabdominal infections caused by E. coli, vancomycinsusceptible E. faecalis, Citrobacter freundii, Enterobacter cloacae, K. pneumoniae, K. oxytoca, Bacteroides spp., Clostridium perfringens, and Peptostreptococcus spp. Escherichia coli (52%); H. influenzae (70%); Salmonella spp. (91%) P. aeruginosa (88%)b E. coli (85%) Bacteroides fragilis (60%)c S. pneumoniae (93%);d E. coli (93%); Klebsiella pneumoniae (89%) P. aeruginosa (89%) Mostly susceptible; four strains of MRSA with ceftaroline MICs >4 μg/mL reported in isolates from a single Greek hospitale P. aeruginosa (76% and 83%); Acinetobacter calcoaceticusbaumannii complex (81% and 82%) Enterobacter cloacae (87%); K. pneumoniae (97%) P. aeruginosa (76%) S. aureus (100%); E. faecalis (89%); E. faecium (24%)

1	Enterobacter cloacae (87%); K. pneumoniae (97%) P. aeruginosa (76%) S. aureus (100%); E. faecalis (89%); E. faecium (24%) S. aureus (100%) S. aureus (100%) E. faecalis (99.9%);f E. faecium (99.7%);f S. aureus (99.9%)f E. coli (gentamicin, 91%); P. aeruginosa (amikacin, 87%; gentamicin, 81%); A. calcoaceticus-baumannii complex (amikacin, 68%; gentamicin, 83%) S. pneumoniae (59%); group A streptococci (78%); H. pylori (75%)g S. aureus (67%) S. pneumoniae (75%); S. aureus (94%) Mostly susceptible, though case reports of resistance in A. baumannii and K. pneumoniae 941TABLE 170-5 DRug InDICATIonS foR SPECIfIC InfECTIonS, ASSoCIATED PATHogEnS, AnD SAMPLE SuSCEPTIBILITy RATES (CONTINUED)

1	Antimicrobial(s) Infections Common Pathogens (% Susceptible); Resistance as Noteda aUnless otherwise noted, susceptibility rates are based on isolates from the Massachusetts General Hospital Clinical Microbiology Laboratory collected between January and December 2012. Local rates will vary. bThe Center for Disease Dynamics, Economics and Policy Resistance Map, Washington, DC. cS Sepehri et al: Prevalence of antimicrobial resistance among clinical isolates of Bacteroides fragilis group in Canada in 2010–2011: CANWARD Surveillance Study. Abstract C2-1814, presented at the 51st Interscience Conference on Antimicrobial Agents and Chemotherapy, 2011. Available at www.can-r.com/posters/ICAAC2011/Sepehri%20Prevalence%20Bfragilis%20ICAAC2011.pdf. dGV Doern et al: Clin Infect Dis 41:139, 2005. eRE Mendes et al: J Antimicrob Chemother 67:1321, 2012. fHS Sader et al: J Chemother 23:200, 2011. gJ Torres et al: J Clin Microbiol 39:2677, 2001. hWS Oh et al: Antimicrob Agents Chemother 49:5176,

1	eRE Mendes et al: J Antimicrob Chemother 67:1321, 2012. fHS Sader et al: J Chemother 23:200, 2011. gJ Torres et al: J Clin Microbiol 39:2677, 2001. hWS Oh et al: Antimicrob Agents Chemother 49:5176, 2005. iAE Simor et al: Antimicrob Agents Chemother 51:3880, 2007.

1	Abbreviations: CA-MRSA, community-acquired MRSA; CAP, community-acquired pneumonia; CA-UTI, community-acquired UTI; CDAD, Clostridium difficile–associated diarrhea; ESBL, extended-spectrum β-lactamase; GAS, group A streptococcal; HAI, hospital-acquired infection; MAI, Mycobacterium avium-intracellulare; MIC, minimal inhibitory concentration; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible S. aureus; TMP-SMX, trimethoprim-sulfamethoxazole; UTI, urinary tract infection; VRE, vancomycin-resistant Enterococcus. against gram-positive bacteria, with some additional activity against

1	E. coli, P. mirabilis, and K. pneumoniae. First-generation cephalosporins are commonly used for infections caused by MSSA and streptococci (e.g., skin and soft tissue infections). Cefazolin is a popular choice for surgical prophylaxis against skin organisms. The second generation (cefamandole, cefuroxime, cefaclor, cefprozil, cefuroxime axetil, cefoxitin, cefotetan) has additional activity against H. influenzae and Moraxella catarrhalis. Cefoxitin and cefotetan have potent activity against anaerobes as well. Second-generation cephalosporins are used to treat community-acquired pneumonia because of their activity against S. pneumoniae, H. influenzae, and M. catarrhalis. They are also used for other mild or moderate infections, such as acute otitis media and sinusitis. The third-generation cephalosporins are characterized by greater potency against gram-negative bacilli and reduced potency against gram-positive cocci. These cephalosporins, which include cefoperazone, cefotaxime,

1	cephalosporins are characterized by greater potency against gram-negative bacilli and reduced potency against gram-positive cocci. These cephalosporins, which include cefoperazone, cefotaxime, ceftazidime, ceftriaxone, cefdinir, cefixime, and cefpodoxime, are used for infections caused by Enterobacteriaceae, although resistance is an increasing concern. It is noteworthy that ceftazidime is the only third-generation cephalosporin with activity against P. aeruginosa but lacks activity against gram-positive bacteria. This drug is frequently used for pulmonary infections in cystic fibrosis and febrile neutropenia. Ceftriaxone penetrates the CSF and can be used to treat meningitis caused by H. influenzae, N. meningitidis, and susceptible strains of S. pneumoniae. It is also used for the treatment of later-stage Lyme disease. The fourth generation includes cefepime and cefpirome, broad-coverage agents that provide potent activity against both gram-negative bacilli, including P. aeruginosa,

1	of later-stage Lyme disease. The fourth generation includes cefepime and cefpirome, broad-coverage agents that provide potent activity against both gram-negative bacilli, including P. aeruginosa, and gram-positive cocci. The fourth generation has clinical applications similar to those of the third generation and can be used in bacteremia, pneumonia, skin and soft tissue infections, and urinary tract infections caused by susceptible bacteria. Cefepime is also commonly used in febrile neutropenia. Ceftaroline, a fifth-generation cephalosporin, differs from the other cephalosporins in its added activity against MRSA, which is resistant to all other β-lactams. Ceftaroline’s gram-negative activity is similar to that of the third-generation cephalosporins but does not include P. aeruginosa. Ceftaroline is efficacious in community-acquired pneumonia and skin infections, but few data are available on its use for more serious infections, such as bacteremia.

1	Carbapenems With a few exceptions for cefepime, all penicillins and cephalosporins are ineffective in the presence of ESBLs. Carbapenems, including doripenem, imipenem, meropenem, and ertapenem, offer the most reliable coverage for strains containing ESBLs. All carbapenems have broad activity against gram-positive cocci, gram-negative bacilli, and anaerobes. None is active against MRSA, but all are active against MSSA, Streptococcus species, and Enterobacteriaceae. Ertapenem is the only carbapenem that has poor activity against P. aeruginosa and Acinetobacter. Imipenem is active against penicillin-susceptible Enterococcus faecalis but not Enterococcus faecium. Carbapenems are not active against Enterobacteriaceae containing carbapenemases. Stenotrophomonas maltophilia and some Bacillus species are intrinsically resistant to carbapenems because of a zinc-dependent carbapenemase.

1	Monobactams Aztreonam is the sole monobactam. Its activity is limited to gram-negative bacteria and includes P. aeruginosa and most other Enterobacteriaceae. This drug is inactivated by ESBLs and carbapenemases. The principal use for aztreonam is as an alternative to penicillins, cephalosporins, or carbapenems in patients with serious β-lactam allergy. Aztreonam is structurally related to ceftazidime and should be used cautiously in individuals with a serious ceftazidime allergy. It is commonly used in febrile neutropenia and intraabdominal Treatment and Prophylaxis of Bacterial Infections 942 infections. Aztreonam does not penetrate the CSF and should not be used for treatment of meningitis.

1	Adverse Reactions to β-Lactam Drugs Agents within the β-lactam class are known for several adverse effects. Gastrointestinal side effects, mainly diarrhea, are common, but hypersensitivity reactions constitute the most common adverse effect of β-lactams. The reactions’ severity can range from rash to anaphylaxis, but the rate of true anaphylactic reactions is only 0.05%. An individual with an accelerated IgEmediated reaction to one β-lactam agent may still receive another agent within the class, but caution should be taken to choose a β-lactam that has a dissimilar side chain and a low level of cross-reactivity. For example, the second-, third-, and fourth-generation cephalosporins and the carbapenems display very low cross-reactivity in patients with penicillin allergy. Aztreonam is the only β-lactam that has no cross-reactivity with the penicillin group. In cases of severe allergy, desensitization (a graded challenge) to the indicated β-lactam, with close monitoring, may be

1	is the only β-lactam that has no cross-reactivity with the penicillin group. In cases of severe allergy, desensitization (a graded challenge) to the indicated β-lactam, with close monitoring, may be warranted if other antibacterial options are not suitable.

1	β-Lactams can rarely cause serum sickness, Stevens-Johnson syndrome, nephropathy, hematologic reactions, and neurotoxicity. Neutropenia appears to be related to high doses or prolonged use. Neutropenia and interstitial nephritis caused by β-lactams generally resolve upon discontinuation of the agent. Imipenem and cefepime are associated with an increased risk of seizure, but this risk is likely a class effect and related to high doses or doses that are not adjusted in renal impairment. The glycopeptide antibiotics include vancomycin and telavancin. Vancomycin has activity against staphylococci (including MRSA and coagulase-negative staphylococci), streptococci (including

1	S. pneumoniae), and enterococci. It is not active against gram-negative organisms. Vancomycin also displays activity against Bacillus species, Corynebacterium jeikeium, Listeria monocytogenes, and gram-positive anaerobes such as Peptostreptococcus, Actinomyces, Clostridium, and Propionibacterium species. Vancomycin has several important clinical uses. It is used for serious infections caused by MRSA, including health care–associated pneumonia, bacteremia, osteomyelitis, and endocarditis. It is also commonly used for skin and soft tissue infections. Oral vancomycin is not absorbed systemically and is reserved for the treatment of Clostridium difficile infection. Vancomycin is also an alternative for the treatment of infections caused by MSSA in patients who cannot tolerate β-lactams. Resistance to vancomycin is a rising concern. Strains of vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant enterococci (VRE) are not uncommon. Vancomycin appears to be a

1	β-lactams. Resistance to vancomycin is a rising concern. Strains of vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant enterococci (VRE) are not uncommon. Vancomycin appears to be a concentration-dependent killer, with AUC/MIC ratio being the best predictor of efficacy (Fig. 170-2). Guidelines recommend targeting a vancomycin trough level of 15–20 μg/mL in MRSA infections in order to maintain an AUC/MIC ratio >400. When using vancomycin, clinicians should monitor for nephrotoxicity. The risk increases when trough levels are >20 μg/ mL. Concomitant therapy with other nephrotoxic agents, such as aminoglycosides, also increases the risk of nephrotoxicity. Ototoxicity was reported with early formulations of vancomycin but is currently uncommon because purer formulations are available. Both of these adverse effects are reversible upon discontinuation of vancomycin. Clinicians should be aware of the “red man syndrome,” a common reaction that presents as a rapid onset of

1	available. Both of these adverse effects are reversible upon discontinuation of vancomycin. Clinicians should be aware of the “red man syndrome,” a common reaction that presents as a rapid onset of erythematous rash or pruritus on the head, face, neck, and upper trunk. This reaction is caused by histamine release from basophils and mast cells and can be treated with diphenhydramine and slowing of the vancomycin infusion.

1	Telavancin, dalbavancin, and oritavancin are structurally similar to vancomycin and are referred to as lipoglycopeptides. They have antibacterial activity against S. aureus (including MRSA and some strains of VISA and vancomycin-resistant S. aureus [VRSA]), streptococci, and enterococci. They also have good activity against anaerobic gram-positive organisms except for Lactobacillus and some Clostridium species. The clinical efficacy of telavancin has been demonstrated in both skin and soft tissue infections and nosocomial pneumonia, and the efficacy of dalbavancin and oritivancin has been shown in skin and soft tissue infections. The vancomycin resistance phenotype may reduce the potency of all three lipoglycopeptides, but the rate of resistance to these drugs among S. aureus and enterococci has been low. Adverse effects of telavancin include insomnia, a metallic taste, nephrotoxicity, and gastrointestinal side effects. Clinicians should be aware of the potential for

1	and enterococci has been low. Adverse effects of telavancin include insomnia, a metallic taste, nephrotoxicity, and gastrointestinal side effects. Clinicians should be aware of the potential for electrocardiographic QTc prolongation that can increase the risk of cardiac arrhythmias when telavancin is used concomitantly with other QTc-prolonging agents. Telavancin may interfere with certain coagulation tests (e.g., causing false elevations in prothrombin time). Dalbavancin and oritavancin have safety profiles similar to that of vancomycin.

1	Daptomycin is a lipopeptide antibiotic with activity against a broad range of gram-positive organisms. This drug is active against staphylococci (including MRSA and coagulase-negative staphylococci), streptococci, and enterococci. Daptomycin remains active against enterococci that are resistant to vancomycin. In addition, it exhibits activity against Bacillus, Corynebacterium, Peptostreptococcus, and Clostridium species. Daptomycin’s pharmacodynamic parameter for efficacy is concentration-dependent killing. Resistance to daptomycin is rare, but MICs may be higher for VISA strains. Daptomycin is efficacious in skin and soft tissue infections, bacteremia, endocarditis, and osteomyelitis. It is an important alternative for MRSA and other gram-positive infections when bactericidal therapy is needed and vancomycin cannot be used. Daptomycin is generally well tolerated, and its main toxicity consists of elevation of creatinine phosphokinase (CPK) levels and myopathy. CPK should be monitored

1	and vancomycin cannot be used. Daptomycin is generally well tolerated, and its main toxicity consists of elevation of creatinine phosphokinase (CPK) levels and myopathy. CPK should be monitored during daptomycin treatment, and the drug should be discontinued if muscular toxicities occur. There have also been case reports of reversible eosinophilic pneumonia associated with daptomycin use.

1	The aminoglycosides are a class of antibacterial agents with concentration-dependent activity against most gram-negative organisms. The most commonly used aminoglycosides are gentamicin, tobramycin, and amikacin, although others, such as streptomycin, kanamycin, neomycin, and paromomycin, may be used in special circumstances. Aminoglycosides have a significant dose-dependent post-antibiotic effect, meaning that they have an antibacterial effect even after serum drug levels are undetectable. The postantibiotic effect and concentration-dependent killing form the rationale behind extended-interval aminoglycoside dosing, in which a larger dose is given once daily rather than smaller doses multiple times daily. Aminoglycosides are active against gram-negative bacilli, such as Enterobacteriaceae,

1	P. aeruginosa, and Acinetobacter. They also enhance the activity of cell wall–active agents such as β-lactams or vancomycin in some gram-positive bacteria, including staphylococci and enterococci. This combination therapy is termed synergistic because the effect of both agents provides a killing effect greater than would be predicted from the effects of either agent alone. Amikacin and streptomycin have activity against Mycobacterium tuberculosis, and amikacin has activity against Mycobacterium avium-intracellulare. The aminoglycosides do not have activity against anaerobes, S. maltophilia, or Burkholderia cepacia. Aminoglycosides are used in clinical practice in a variety of infections caused by gram-negative organisms, including bacteremia and urinary tract infections. They are frequently used alone or in combination for the treatment of P. aeruginosa infection. When used in combination with a cell wall–active agent, gentamicin and streptomycin are also important for the treatment

1	used alone or in combination for the treatment of P. aeruginosa infection. When used in combination with a cell wall–active agent, gentamicin and streptomycin are also important for the treatment of gram-positive bacterial endocarditis. All aminoglycosides can cause nephrotoxicity and ototoxicity. The risk of nephrotoxicity is related to the dose and duration of therapy as well as the concomitant use of other nephrotoxic agents. Nephrotoxicity is usually reversible, but ototoxicity can be irreversible.

1	The macrolides (azithromycin, clarithromycin, erythromycin) and ketolides (telithromycin) are classes of antibiotics that inhibit protein synthesis. Compared with erythromycin (the older antibiotic), azithromycin and clarithromycin have better oral absorption and tolerability. Azithromycin, clarithromycin, and telithromycin all have broader spectra of activity than erythromycin, which is less frequently used. These agents are commonly used in the treatment of upper and lower respiratory tract infections caused by S. pneumoniae, H. influenzae, M. catarrhalis, and atypical organisms (e.g., Chlamydia pneumoniae, Legionella pneumophila, and Mycoplasma pneumoniae); group A streptococcal pharyngitis in penicillin-allergic patients; and nontuberculous mycobacterial infections (e.g., caused by M. marinum and

1	M. chelonae) as well as in the prophylaxis and treatment of M. avium-intracellulare infection in patients with HIV/AIDS and in combination therapy for H. pylori infection and bartonellosis. Enterobacteriaceae, Pseudomonas species, and Acinetobacter species are intrinsically resistant to macrolides as a result of decreased membrane permeability, although azithromycin is active against gram-negative diarrheal pathogens. The major adverse effects of this drug class include nausea, vomiting, diarrhea and abdominal pain, prolongation of QTc interval, exacerbation of myasthenia gravis, and tinnitus. Azithromycin specifically has been associated with an increased risk of death, especially among patients with underlying heart disease, because of the risk of QTc interval prolongation and torsades de pointes. Erythromycin, clarithromycin, and telithromycin inhibit the CYP3A4 hepatic drug-metabolizing enzyme and can result in increased levels of coadministered drugs, including benzodiazepines,

1	de pointes. Erythromycin, clarithromycin, and telithromycin inhibit the CYP3A4 hepatic drug-metabolizing enzyme and can result in increased levels of coadministered drugs, including benzodiazepines, statins, warfarin, cyclosporine, and tacrolimus. Azithromycin does not inhibit CYP3A4 and lacks these drug–drug interactions.

1	Clindamycin is a lincosamide antibiotic and is bacteriostatic against some organisms and bactericidal against others. It is used most often to treat bacterial infections caused by anaerobes (e.g., B. fragilis, Clostridium perfringens, Fusobacterium species, Prevotella melaninogenicus, and Peptostreptococcus species) and susceptible staphylococci and streptococci. Clindamycin is used for treatment of dental infections, anaerobic lung abscess, and skin and soft tissue infections. It is used together with bactericidal agents (penicillins or vancomycin) to inhibit new toxin synthesis in the treatment of streptococcal or staphylococcal toxic shock syndrome. Other uses include treatment of infections caused by Capnocytophaga canimorsus, a component of combination therapy for malaria and babesiosis, and therapy for toxoplasmosis. Clindamycin has excellent oral bioavailability. Adverse effects include nausea, vomiting, diarrhea, C. difficile–associated diarrhea and pseudomembranous colitis,

1	and therapy for toxoplasmosis. Clindamycin has excellent oral bioavailability. Adverse effects include nausea, vomiting, diarrhea, C. difficile–associated diarrhea and pseudomembranous colitis, maculopapular rash, and (rarely) Stevens-Johnson syndrome.

1	The tetracyclines (doxycycline, minocycline, and tetracycline) and the glycylcyclines (tigecycline) inhibit protein synthesis and are bacteriostatic. These drugs have wide clinical uses. They are used in the treatment of skin and soft tissue infections caused by gram-positive cocci (including MRSA), spirochetal infections (e.g., Lyme disease, syphilis, leptospirosis, and relapsing fever), rickettsial infections (e.g., Rocky Mountain spotted fever), atypical pneumonia, sexually transmitted infections (e.g., Chlamydia trachomatis infection, lymphogranuloma venereum, and granuloma inguinale), infections with Nocardia and Actinomyces, brucellosis, tularemia, Whipple’s disease, and malaria. Tigecycline, the only approved agent in the glycylcycline class, is a derivative of minocycline and is indicated in the treatment of infections due to MRSA, vancomycin-sensitive enterococci, many Enterobacteriaceae, and Bacteroides species. Tigecycline has no activity against P. aeruginosa. It has been

1	in the treatment of infections due to MRSA, vancomycin-sensitive enterococci, many Enterobacteriaceae, and Bacteroides species. Tigecycline has no activity against P. aeruginosa. It has been used in combination with colistin for the treatment of serious infections with multidrug-resistant gram-negative organisms. A pooled analysis of 13 clinical trials found an increased risk of death and treatment failure among patients treated with tigecycline alone. Tetracyclines have reduced absorption when coadministered with calciumand iron-containing compounds, including milk, and doses should be spaced at least 2 h apart. The major adverse reactions to both of these classes are nausea, vomiting, diarrhea, and photosensitivity. Tetracyclines have been associated with fetal bone-growth abnormalities and should be avoided during pregnancy and in the treatment of children <8 years old.

1	Trimethoprim-sulfamethoxazole (TMP-SMX) is an antibiotic whose two components both inhibit folate synthesis and produce antibacterial activity. TMP-SMX is active against gram-positive bacteria such as staphylococci and streptococci; however, its use against MRSA is usually limited to community-acquired infections, and its activity against Streptococcus pyogenes may not be reliable. TMP-SMX is also active against many gram-negative bacteria, including H. influenzae,

1	E. coli, P. mirabilis, N. gonorrhoeae, and S. maltophilia. TMP-SMX does not have activity against anaerobes or P. aeruginosa. It has many uses because of its wide spectrum of activity and high oral bioavailability. Urinary tract infections, skin and soft tissue infections, and respiratory tract infections are among the common uses. Another important indication is for both prophylaxis and treatment of Pneumocysitis jirovecii infections in immunocompromised patients. Resistance to TMP-SMX has limited its use against many Enterobacteriaceae. Resistance rates among urinary isolates of E. coli are almost 25% in the United States. The most common adverse reactions associated with TMP-SMX are gastrointestinal effects such as nausea, vomiting, and diarrhea. In addition, rash is a common allergic reaction and may preclude the subsequent use of other sulfonamides. With prolonged use, leukopenia, thrombocytopenia, and granulocytopenia can develop. TMP-SMX can also cause nephrotoxicity,

1	allergic reaction and may preclude the subsequent use of other sulfonamides. With prolonged use, leukopenia, thrombocytopenia, and granulocytopenia can develop. TMP-SMX can also cause nephrotoxicity, hyperkalemia, and hyponatremia, which are more common at high doses. TMP-SMX has several important interactions with other drugs (Table 170-4), including warfarin, phenytoin, and methotrexate.

1	The fluoroquinolones include norfloxacin, ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, and gemifloxacin. Ciprofloxacin and levofloxacin have the broadest spectrum of activity against gram-negative bacteria, including P. aeruginosa (similar to that of third-generation cephalosporins). Because of the risk of selection of resistance during fluoroquinolone treatment of serious pseudomonal infections, these agents are usually used in combination with an antipseudomonal β-lactam. Levofloxacin, moxifloxacin, and gemifloxacin have addi tional gram-positive activity, including that against S. pneumoniae and some strains of MSSA, and are used for treatment of community-acquired pneumonia. Strains of MRSA are commonly resistant to all fluoroquinolones. Moxifloxacin is used as one component of second-line regimens for multidrug-resistant tuberculosis. Fluoroquinolones exhibit concentration-dependent killing, are well absorbed orally, and have elimination half-lives that usually support

1	of second-line regimens for multidrug-resistant tuberculosis. Fluoroquinolones exhibit concentration-dependent killing, are well absorbed orally, and have elimination half-lives that usually support onceor twice-daily dosing. Oral coadministration with compounds containing high concentrations of aluminum, magnesium, or calcium can reduce fluoroquinolone absorption. Their penetration into prostate tissue supports their use for bacterial prostatitis. Fluoroquinolones are generally well tolerated but can cause CNS stimulatory effects, including seizures; glucose dysregulation; and tendinopathy associated with Achilles tendon rupture, particularly in older patients, organ transplant recipients, and patients taking glucocorticoids. Worsening of myasthenia gravis also has been associated with quinolone use. Moxifloxacin causes modest prolongation of the QTc interval and should be used with caution in patients receiving other QTc-prolonging drugs.

1	The rifamycins include rifampin, rifabutin, and rifapentine. Rifampin is the most commonly used rifamycin. For almost all therapeutic indications, it is used in combination with other agents to reduce the likelihood of selection of high-level rifampin resistance. Rifampin is used foremost in the treatment of mycobacterial infections—specifically, as a mainstay of combination therapy for M. tuberculosis infection or as a single agent in the treatment of latent M. tuberculosis infection. In addition, it is often used in the treatment of nontuberculous mycobacterial infection. Rifampin is used in combination regimens for the treatment of staphylococcal infections, particularly prosthetic valve endocarditis and bone infections with retained hardware. It is a component of combination therapy for brucellosis (with doxycycline) and leprosy (with dapsone for tuberculoid leprosy and with dapsone

1	Treatment and Prophylaxis of Bacterial Infections 944 and clofazimine for lepromatous disease). Rifampin can be used alone for prophylaxis in close contacts of patients with H. influenzae or N. meningitidis meningitis. The drug has high oral bioavailability, which is further enhanced when it is taken on an empty stomach. Rifampin has several adverse effects, including elevated aminotransferase levels (14%), rash (1–5%), and gastrointestinal events such as nausea, vomiting, and diarrhea (1–2%). Its many clinically relevant interactions with other drugs mandate the clinician’s careful review of the patient’s medications before rifampin initiation to assess safety and the need for additional monitoring.

1	Metronidazole is used in the treatment of anaerobic bacterial infections as well as infections caused by protozoa (e.g., amebiasis, giardiasis, trichomoniasis). It is the agent of choice as a component of combination therapy for polymicrobial abscesses in the lung, brain, or abdomen, the etiology of which often includes anaerobic bacteria, and for bacterial vaginosis, pelvic inflammatory disease, mild to moderate C. difficile–associated diarrhea, and anaerobic infections, such as those due to Bacteroides, Fusobacterium, and Prevotella species. Metronidazole is bactericidal against anaerobic bacteria and exhibits concentration-dependent killing. It has high oral bioavailability and tissue penetration, including penetration of the blood–brain barrier. The majority of Actinomyces, Propionibacterium, and Lactobacillus species are intrinsically resistant to metronidazole. The major adverse effects include nausea, diarrhea, and a metallic taste. Concomitant ingestion of alcohol may result

1	and Lactobacillus species are intrinsically resistant to metronidazole. The major adverse effects include nausea, diarrhea, and a metallic taste. Concomitant ingestion of alcohol may result in a disulfiram-like reaction, and patients are usually instructed to avoid alcohol during treatment. Long-term treatment carries the risk of leukopenia, neutropenia, peripheral neuropathy, and central nervous system toxicity manifesting as confusion, dysarthria, ataxia, nystagmus, and ophthalmoparesis. Through metronidazole’s effect on the CYP2C9 drug-metabolizing enzyme, its coadministration with warfarin can result in decreased metabolism and enhanced anticoagulant effects that require close monitoring. Concomitant administration of metronidazole with lithium can result in increased serum levels of lithium and associated toxicity; coadministration with phenytoin can result in phenytoin toxicity and possibly decreased levels of metronidazole.

1	Linezolid is a bacteriostatic agent and is indicated for serious infections due to resistant gram-positive bacteria, such as MRSA and VRE. The intrinsic resistance of gram-negative bacteria is mediated primarily by endogenous efflux pumps. Linezolid has excellent oral bioavailability. Adverse effects include myelosuppression and ocular and peripheral neuropathy with prolonged therapy. Peripheral neuropathy may be irreversible. Linezolid is a weak, reversible monoamine oxidase inhibitor, and coadministration with sympathomimetics and foods rich in tyramine should be avoided. Linezolid has been associated with serotonin syndrome when coadministered with selective serotonin-reuptake inhibitors. Tedizolid has properties similar to those of linezolid, but with lower dosing it may be less likely to cause adverse hematologic and neuropathic effects.

1	Nitrofurantoin’s antibacterial activity results from the drug’s conversion to highly reactive intermediates that can damage DNA and other macromolecules. Nitrofurantoin is bactericidal, and its action is concentration dependent. It displays activity against a range of gram-positive bacteria, including S. aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, E. faecalis, Streptococcus agalactiae, group D streptococci, viridans streptococci, and corynebacteria, as well as gram-negative organisms, including E. coli and Enterobacter, Neisseria, Salmonella, and Shigella species. Nitrofurantoin is used primarily in the treatment of urinary tract infections and is preferred in the treatment of such infections in pregnancy. It may be used for the prevention of recurrent cystitis. Recently, there has been interest in the use of nitrofurantoin for treatment of urinary tract infections caused by ESBL-producing Enterobacteriaceae such as E. coli, although resistance has been growing

1	there has been interest in the use of nitrofurantoin for treatment of urinary tract infections caused by ESBL-producing Enterobacteriaceae such as E. coli, although resistance has been growing in Latin America and parts of Europe. Coadministration with magnesium should be avoided because of decreased absorption, and patients should be encouraged to take the drug with food to increase its bioavailability and decrease the risk of adverse effects, which include nausea, vomiting, and diarrhea. Nitrofurantoin may also cause pulmonary fibrosis and drug-induced hepatitis. Because the risk of adverse reactions increases with age, the use of nitrofurantoin in elderly patients is not recommended. Patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency are at elevated risk for nitrofurantoin-associated hemolytic anemia.

1	Colistin and polymyxin B act by disrupting cell membrane integrity and are active against the nonenteric pathogens P. aeruginosa and A. baumannii but not against Burkholderia. These drugs also exhibit activity against many Enterobacteriaceae,with the exceptions of Proteus, Providencia, and Serratia species. They lack activity against gram-positive bacteria. Polymyxins are bactericidal and are available in IV formulations. Colistimethate is converted to the active form (colistin) in plasma. Polymyxins are most often used for infections due to pathogens resistant to multiple other antibacterial agents, including urinary tract infections, hospital-acquired pneumonia, and bloodstream infections. Nebulized formulations have been used for adjunctive treatment of refractory ventilator-associated pneumonia. The most important adverse effect is dose-dependent reversible nephrotoxicity. Neurotoxicity, including paresthesias, muscle weakness, and confusion, is reversible and less common than

1	pneumonia. The most important adverse effect is dose-dependent reversible nephrotoxicity. Neurotoxicity, including paresthesias, muscle weakness, and confusion, is reversible and less common than nephrotoxicity.

1	Quinupristin-dalfopristin is a member of the streptogramin class of antibiotics and kills bacteria by inhibiting protein synthesis. The antibacterial spectrum of quinupristin-dalfopristin includes staphylococci (including MRSA), streptococci, and E. faecium (but not

1	E. faecalis). This drug is also active against Corynebacterium species and L. monocytogenes. Quinupristin-dalfopristin is not reliably active against gram-negative organisms. It exhibits concentration-dependent killing, with an AUC/MIC ratio predicting efficacy. The clinical use of quinupristin-dalfopristin is largely for infections due to vancomycinresistant E. faecium and other gram-positive bacterial infections. The drug has demonstrated efficacy in a variety of infections, including urinary tract infections, bone and joint infections, and bacteremia. Adverse effects associated with quinupristin-dalfopristin include infusion-related reactions, arthralgias, and myalgias. The arthralgias and myalgias may be severe enough to warrant drug discontinuation. Quinupristin-dalfopristin inhibits the CYP3A4 drug-metabolizing enzyme, with consequent drug interactions (Table 170-4).

1	Fosfomycin is a phosphonic acid antibiotic that has greater activity in acidic environments and is excreted in its active form in the urine. Thus, its use is primarily for prophylaxis and treatment of uncomplicated cystitis. The drug is administered as a single 3-g dose that results in high urine concentrations for up to 48 h. Fosfomycin is active against S. aureus, vancomycin-susceptible and vancomycinresistant enterococci, and a wide range of gram-negative organisms, including E. coli, Enterobacter species, S. marcescens, P. aeruginosa, and K. pneumoniae. Notably, the vast majority of ESBL-producing Enterobacteriaceae are susceptible to fosfomycin. A. baumannii and Burkholderia species are resistant. The emergence of resistance to fosfomycin has not been observed during treatment of cystitis but has been documented during treatment of respiratory tract infections and osteomyelitis. The few adverse effects that have been reported include nausea and diarrhea.

1	The use of chloramphenicol is limited by its potentially serious toxicities. When other agents are contraindicated or ineffective, chloramphenicol represents an alternative treatment for infections, including meningitis caused by susceptible bacteria such as N. meningitidis, H. influenzae, and S. pneumoniae. It has also been used for the treatment of anthrax, brucellosis, Burkholderia infections, chlamydial infections, clostridial infections, erlichiosis, rickettsial infections, and typhoid fever. Adverse reactions include aplastic anemia, myelosuppression, and gray baby syndrome. Chloramphenicol inhibits the CYP2C19 and CYP3A4 drug-metabolizing enzymes and consequently increases levels of many classes of drugs.

1	Antibacterial prophylaxis is indicated only in selected circumstances (Table 170-6) and should be supported by well-designed studies or expert panel recommendations. In all cases the risk or severity of the infection to be prevented should be greater than the adverse consequences of antibacterial therapy, including the potential for selection of resistance. In addition, the timing and duration of antibacterial treatment should be targeted for maximal effect and minimal required exposure. Prophylaxis of surgical site infections targets bacteria that may contaminate the wound during the surgical procedure, including the skin flora of the patient or operating team and the air in the operating room. Delivery of the antibacterial drug within 1 h before the surgical incision is most effective. For prolonged procedures, redosing may be necessary to maintain effective blood and tissue levels until the wound is closed. In patients with nasal carriage of S. aureus, preopera-945 tive

1	effective. For prolonged procedures, redosing may be necessary to maintain effective blood and tissue levels until the wound is closed. In patients with nasal carriage of S. aureus, preopera-945 tive decolonization with nasal mupirocin reduces the rate of S. aureus surgical site infections and is generally recommended for high-risk procedures such as cardiac surgery and orthopedic implantation of prosthetic devices. For dental procedures, preprocedure antibacterial drugs are given to prevent transient bacteremia and the seeding of certain high-risk cardiac lesions. Prophylaxis is also used in nonprocedural settings in certain patients who have recurrent infections or who are at risk of serious infection from a specific exposure (e.g., close contact with a patient with meningococcal meningitis). Extension of prophylaxis beyond the period of infection risk (24 h in the case of surgical procedures) does not add further benefit and may increase the risk of resistance selection or C.

1	Extension of prophylaxis beyond the period of infection risk (24 h in the case of surgical procedures) does not add further benefit and may increase the risk of resistance selection or C. difficile disease.

1	In an era of increasing prevalence of multidrug-resistant bacteria and with a substantial amount of inappropriate antimicrobial use, the need for rational antimicrobial prescribing has never been greater. Antimicrobial stewardship describes the practice of promoting the selection of the appropriate drug, dosage, route, and duration of antimicrobial therapy. Antimicrobial stewardship programs implement a variety of strategies to (1) improve patient care through appropriate Condition Antibacterial Agentsa Timing or Duration of Prophylaxis Clean (cardiac, thoracic, neurologic, orthopedic, vascular, plastic) Clean (ophthalmic) Clean-contaminated (hysterectomy, gastroduodenal, biliary, unobstructed small intestine, urologic) Clean-contaminated (colorectal, appendectomy) Cefazolin (vancomycin,b clindamycin) Topical neomycin–polymyxin B–gramicidin, topical moxifloxacin Cefazolin + metronidazole, ampicillin-sulbactamc (clindamycin)

1	Cefazolin (vancomycin,b clindamycin) Topical neomycin–polymyxin B–gramicidin, topical moxifloxacin Cefazolin + metronidazole, ampicillin-sulbactamc (clindamycin) Cefazolin, ampicillin-sulbactamc (clindamycin + aminoglycoside, aztreonam, or fluoroquinolone) Cefazolin + metronidazole, ampicillin-sulbactam,c ertapenem (clindamycin + aminoglycoside, aztreonam, or fluoroquinolone) Therapeutic regimen directed at anaerobes and gram-negative bacteria (e.g., ceftriaxone + metronidazole) Therapeutic regimen: cefazolin (clindamycin ± aminoglycoside, aztreonam, or fluoroquinolone) 1 h before incision; redose with long procedures

1	Therapeutic regimen: cefazolin (clindamycin ± aminoglycoside, aztreonam, or fluoroquinolone) 1 h before incision; redose with long procedures Every 5–15 min for 5 doses immediately prior to procedure 1 h before incision; redose with long procedures 1 h before incision; redose with long procedures 1 h before incision; redose with long procedures 1 h before incision; redose with long procedures; continue for 3–5 days after procedure 1 h before incision; redose with long procedures; continue for 3–5 days after procedure Treatment and Prophylaxis of Bacterial Infections Dental, oral, or upper respiratory procedures in patients with high-risk cardiac lesions (prosthetic valves, congenital heart defects, prior endocarditis) Recurrent S. aureus skin infectionsd Recurrent cellulitis associated with lymphatic disruptiond Recurrent pneumococcal meningitis in patient with CSF leak or humoral immune defectd Exposure to patient with meningococcal meningitis

1	Recurrent cellulitis associated with lymphatic disruptiond Recurrent pneumococcal meningitis in patient with CSF leak or humoral immune defectd Exposure to patient with meningococcal meningitis High-risk neutropenia (ANC, ≤100/μL for >7 days)d Amoxicillin PO, ampicillin IM (clindamycin PO, IV) Benzathine penicillin IM monthly, oral penicillin or erythromycin twice daily Nitrofurantoin, TMP-SMX, fluoroquinolone Amoxicillin-clavulanate (doxycycline, moxifloxacin) Fluoroquinolonef Rifampin, ciprofloxacin Levofloxacin or ciprofloxacinf After sexual intercourse or 3 times weekly for up to 1 year 3–5 days Undefined 2 days (rifampin), single dose (ciprofloxacin)

1	Rifampin, ciprofloxacin Levofloxacin or ciprofloxacinf After sexual intercourse or 3 times weekly for up to 1 year 3–5 days Undefined 2 days (rifampin), single dose (ciprofloxacin) Until neutropenia resolves or fever dictates use of other antibacterials aRegimens in parentheses are alternatives for patients allergic to β-lactams. bVancomycin may be given together with cefazolin to patients known to be colonized with methicillinresistant Staphylococcus aureus. cCefoxitin or cefotetan may also be considered. dNot considered routine for all patients, but an acceptable consideration among alternative approaches. eUsually coupled with bathing with chlorhexidine-containing skin antiseptic. fChoice of fluoroquinolone prophylaxis must be balanced against the risk of selection of resistance. Abbreviations: ANC, absolute neutrophil count; CSF, cerebrospinal fluid; TMP-SMX, trimethoprim-sulfamethoxazole.

1	946 antimicrobial use; (2) decrease the development of resistance within patients and populations; (3) reduce the incidence of adverse effects; and (4) control costs. Infections caused by resistant pathogens result in significant morbidity and mortality as well as increased health care costs. Antimicrobial stewardship programs are typically multidisciplinary and often include infectious disease physicians, clinical pharmacists (usually with special training in infectious disease), clinical microbiologists, information systems specialists, infection prevention and control practitioners, and epidemiologists. These teams employ a variety of approaches to achieving the program’s goals. Established strategies of antimicrobial stewardship programs include (1) prospective audit of antimicrobial use, with intervention and feedback; (2) formulary restriction; and (3) preauthorization. Prospective audit and feedback are usually undertaken by an infectious disease physician or a pharmacist. In

1	use, with intervention and feedback; (2) formulary restriction; and (3) preauthorization. Prospective audit and feedback are usually undertaken by an infectious disease physician or a pharmacist. In this process, orders for broad-spectrum antimicrobials (e.g., carbapenems) or high-impact agents (e.g., linezolid, daptomycin) are reviewed on a regular basis for appropriateness. In circumstances in which an antimicrobial is used in the absence of an appropriate indication, the stewardship program team intervenes and recommends an alternative to the primary team caring for the patient. This process has been successful in several quasi-experimental studies, resulting in declines in use of broad-spectrum drugs and decreases in adverse events, such as C. difficile infection. Formulary restriction is the inclusion of a limited set of antimicrobial agents in a hospital formulary for the purpose of limiting indiscriminant use of antimicrobials in the absence of demonstrated benefit. Such

1	is the inclusion of a limited set of antimicrobial agents in a hospital formulary for the purpose of limiting indiscriminant use of antimicrobials in the absence of demonstrated benefit. Such restriction coincidentally serves to reduce costs. Preauthorization is the practice of requiring clinicians to obtain approval before using selected antimicrobials. Approval may be provided electronically with sophisticated Computerized Provider Order Entry (CPOE) software, after specific criteria for use are met, or after communication with an infectious disease specialist as designated by the stewardship program. These strategies have led to a decrease in C. difficile infections and to improvements in drug susceptibility patterns.

1	Additional strategies used in specific health-care settings are guidelines and pathways, dose optimization, parenteral-to-oral conversion, and de-escalation of therapy. Antimicrobial stewardship is an evolving area and an increasingly active area of research aimed at identifying the best practices. The IDSA, in collaboration with several other professional organizations, has published guidelines for developing institutional antimicrobial stewardship programs (www.idsociety.org/ Antimicrobial_Agents/). David Goldblatt, Katherine L. O’Brien

1	In the late nineteenth century, pairs of micrococci were first recognized in the blood of rabbits injected with human saliva by both Louis Pasteur, working in France, and George Sternberg, an American army physician. The important role of these micrococci in human disease was not appreciated at that time. By 1886, when the organism was designated “pneumokokkus” and Diplococcus pneumoniae, the pneumococcus had been isolated by many independent investigators, and its role in the etiology of pneumonia was well known. In the 1930s, pneumonia was the third leading cause of death in the United States (after heart disease and cancer) and was responsible for ~7% of all deaths both in the United States and in Europe. While pneumonia was caused by a host of pathogens, lobar pneumonia—a pattern more likely to be caused by the pneumococcus—accounted for approximately one-half of all pneumonia deaths in the United States in 1929. In 1974, the organism was reclassified as Streptococcus pneumoniae.

1	MICROBIOLOGY Etiologic Agent Pneumococci are spherical gram-positive bacteria of the genus Streptococcus. Within this genus, cell division occurs along a single axis, and bacteria grow in chains or pairs—hence the name Streptococcus, from the Greek streptos, meaning “twisted,” and kokkos, meaning “berry.” At least 22 streptococcal species are recognized and are divided further into groups based on their hemolytic properties. S. pneumoniae belongs to the α-hemolytic group that characteristically produces a greenish color on blood agar because of the reduction of iron in hemoglobin (Fig. 171-1). The bacteria are fastidious and grow best in 5% CO2 but require a source of catalase (e.g., blood) for growth on agar plates, where they develop mucoid (smooth/shiny) colonies. Pneumococci without a capsule produce colonies with a rough surface. Unlike that of other α-hemolytic streptococci, their growth is inhibited in the presence of optochin (ethylhydrocupreine hydrochloride), and they are

1	a capsule produce colonies with a rough surface. Unlike that of other α-hemolytic streptococci, their growth is inhibited in the presence of optochin (ethylhydrocupreine hydrochloride), and they are bile soluble.

1	In common with other gram-positive bacteria, pneumococci have a cell membrane beneath a cell wall, which in turn is covered by a polysaccharide capsule. Pneumococci are divided into serogroups or serotypes based on capsular polysaccharide structure, as distinguished with rabbit polyclonal antisera; capsules swell in the presence of specific antiserum (the Quellung reaction). The most recently discovered serotypes, 6C, 6D, and 11E, have been identified with monoclonal antibodies and by serologic, genetic, and biochemical means, respectively. The currently recognized 93 serotypes fall into 21 serogroups, and each serogroup contains two to five serotypes with closely related capsules. The capsule protects the bacteria from phagocytosis by host cells in the absence of type-specific antibody and is arguably the most FIGURE 171-1 Pneumococci growing on blood agar, illustrating α hemolysis and optochin sensitivity (zone around optochin disk).

1	FIGURE 171-1 Pneumococci growing on blood agar, illustrating α hemolysis and optochin sensitivity (zone around optochin disk). Inset: Gram’s stain, illustrating gram-positive diplococci. (Photographs courtesy of Paul Turner, Shoklo Malaria Research Unit, Thailand.) Pneumolysin: secreted cytolytic/cytotoxic protein; activates complement and stimulates proinflammatory cytokines 947 Polysaccharide capsule: prevents complement binding; therefore antiphagocytic, target for protective antibody Pneumococcal surface protein A: interferes with complement deposition by blocking alternative complement pathway activation Pneumococcal iron acquisition A and iron uptake A: lipoprotein components of iron ABC transporters, essential for iron uptake Pneumococcal surface protein C (choline-binding protein A): principal pneumococcal adhesion molecule PspA PspC/ CbpA PiaA and PiuA CbpG Pneumolysin Autolysin PsaA Neuraminidase (NanA, NanB) Hyal Enolase Phosphorylcholine Pili Histidine triad PBP

1	PspA PspC/ CbpA PiaA and PiuA CbpG Pneumolysin Autolysin PsaA Neuraminidase (NanA, NanB) Hyal Enolase Phosphorylcholine Pili Histidine triad PBP Choline-binding protein G: cleaves host extracellular matrix, aiding adhesion Pneumococcal surface antigen A: metal-binding lipoprotein (Zn and Mn); may have a role in adhesion IgA1 protease: degrades human IgA1 Hyaluronate lyase: degrades hyaluronan and chondroitin sulfate in extracellular matrix Binds to platelet-activating factor receptor on human epithelial cells Releases peptidoglycan, teichoic acid, pneumolysin, and other intracellular contents on autolysis Penicillin-binding proteins: catalyze polymerization of glycan chains and transpeptidation of pentapeptidic moieties within structure of peptidoglycan Neuraminidase: contributes to adherence; removes sialic acids on host glycopeptides and mucin to expose binding sites

1	Neuraminidase: contributes to adherence; removes sialic acids on host glycopeptides and mucin to expose binding sites Binds to fibronectin in host tissues PhtA, B, D, E: cell-surface exposed proteins, unknown function Pili: on cell surface; inhibit phagocytosis, promote invasion FIGURE 171-2 Schematic diagram of the pneumococcal cell surface, with key antigens and their roles highlighted. important determinant of pneumococcal virulence. Unencapsulated variants tend not to cause invasive disease.

1	Virulence Factors Within the cytoplasm, cell membrane, and cell wall, many molecules that may play a role in pneumococcal pathogenesis and virulence have been identified (Fig. 171-2). These proteins are often involved in direct interactions with host tissues or in concealment of the bacterial surface from host defense mechanisms. Pneumolysin is a secreted cytotoxin thought to result in cytolysis of cells and tissues, and LytA enhances pathogenesis. A number of cell wall proteins interfere with the complement pathway, thus inhibiting complement deposition and preventing lysis and/or opsonophagocytosis. The pneumococcal H inhibitor (Hic) impedes the formation of C3 convertase, while pneumococcal surface protein C (PspC), also known as choline-binding protein A (CbpA), binds factor H and is thought to accelerate the breakdown of C3. PspA and CbpA inhibit the deposition of or degrade C3b. The numerous pneumococcal proteins thought to be involved in adhesion include the ubiquitous

1	and is thought to accelerate the breakdown of C3. PspA and CbpA inhibit the deposition of or degrade C3b. The numerous pneumococcal proteins thought to be involved in adhesion include the ubiquitous surface-anchored sialidase (neuraminidase) NanA, which cleaves sialic acid on host cells and proteins, and pneumococcal surface adhesin A (PsaA). Pili recently recognized by electron microscopy also may play an important role in binding to cells. Some of the antigens mentioned above are potential vaccine candidates (see “Prevention,” below).

1	Although the capsule surrounding the cell wall of S. pneumoniae is the basis for categorization by serotype, the behavior and pathogenic potential of a serotype may also be related to the genetic origin of the strain. Molecular typing is therefore of considerable interest. Initially, techniques such as pulsed-field gel electrophoresis were used to determine genetic relatedness; such techniques have been superseded by sequencing of housekeeping genes to define a clone (multilocus sequence typing, MLST). For S. pneumoniae, alleles at each of the loci aroE, gdh, gki, recP, spi, xpt, and ddl are sequenced and compared with all of the known alleles at that locus. Sequences identical to a known allele are assigned the same allele number, whereas those differing from any known allele—even at a single nucleotide site—are assigned new numbers. Software for assignment of alleles at each locus is available on the pneumococcal MLST website (spneumoniae.mlst.net), and the allelic profile of each

1	single nucleotide site—are assigned new numbers. Software for assignment of alleles at each locus is available on the pneumococcal MLST website (spneumoniae.mlst.net), and the allelic profile of each isolate and its consequent sequence type are generated. With the advent of high-throughput and relatively inexpensive sequencing techniques, whole-genome sequencing will soon supersede MLST.

1	Pneumococcal infections remain a significant global cause of morbidity and death, particularly among children and the elderly. Rapid and dramatic changes in the epidemiology of this disease during the past decade in several developed countries followed the licensure and routine childhood administration of pneumococcal polysaccharide–protein conjugate vaccine (PCV). With PCV introduction in developing and middle-income countries, additional profound changes in pneumococcal ecology and disease epidemiology are likely. The disease burden and serotype distribution in the PCV era may be different than expected because of concomitant secular trends in pneumococcal disease, the impact of antibiotic use on pneumococcal strain ecology, and surveillance system attributes that can themselves affect analysis of epidemiologic features.

1	Serotype Distribution Not all pneumococcal serotypes are equally likely to cause disease; serotype distribution varies by age, disease syndrome, and geography. Geographic differences may be driven by variation in the burden of disease rather than by true serotype distribution differences. Most data on serotype distribution are related to pediatric invasive pneumococcal disease (IPD, defined as infection of a normally sterile site); much less information on global distribution is available for disease in adults. Among children <5 years of age, five to seven serotypes cause >60% of IPD cases in most parts of the world, seven serotypes (1, 5, 6A, 6B, 14, 19F, and 23F) account for ~60% of cases in all areas of the world, but in any given region these 100% <2 years of age and among adults ≥65 years of age (188 and 60 cases/100,000, respectively; Fig. 171-5). Since the introduction of PCV, 80% IPD rates among infants and children in the

1	United States have fallen by >75%, a decrease driven by the near elimination of vaccine serotype IPD. A similar impact of PCV on 948 18% 16% 14% 12% 10% 8% 6% 4% 2% 0% % of serotyped isolates 14 16B5 23F 19F6A19A 9V 18C Serotypes 2 4 37F12F 812A 15A9N29 FIGURE 171-3 Meta-analysis of available global pneumococcal serotype data, adjusted for regional disease incidence. The red line shows cumulative incidence, as indicated on the been introduced into the routine pediatric vaccination schedule. However, changes in the tries have been heterogeneous; the interpreta tion of this heterogeneity is a complex issue. In the United States, Canada, and Australia, rates of non-vaccine-serotype IPD have increased but the magnitude of the increase is generally small relative to the substantial reductions in vaccine-serotype IPD. In contrast, in other settings (e.g., Alaska Native communiright-hand Y axis. (Source: Global Serotype Project Report for the Pneumococcal Advance Market ties and the United

1	in vaccine-serotype IPD. In contrast, in other settings (e.g., Alaska Native communiright-hand Y axis. (Source: Global Serotype Project Report for the Pneumococcal Advance Market ties and the United Kingdom), the reduction Commitment Target Product Profile; available at http://www.gavi.org/library/gavi-documents/amc/ in vaccine-serotype IPD has been offset by tpp-codebook/.) seven serotypes may not all rank as the most common disease strains (Fig. 171-3). Some serotypes (e.g., types 1 and 5) not only tend to cause disease in areas with a high disease burden but also cause waves of disease in lower-burden areas (e.g., Europe) or outbreaks (e.g., in military barracks; meningitis in sub-Saharan Africa). The broader range of serotypes causing disease among adults than among children is apparent from a comparison of the coverage of existing multiserotype vaccines in different age groups. For example, data from the United States for 2006–2007 on the serotypes causing IPD indicated that a

1	from a comparison of the coverage of existing multiserotype vaccines in different age groups. For example, data from the United States for 2006–2007 on the serotypes causing IPD indicated that a polysaccharide vaccine containing 23 serotypes (PPSV23) would cover 84% of cases among children <5 years of age and 76% of those among persons 18–64 years of age but only 65% of those among persons ≥65 years of age.

1	Nasopharyngeal Carriage Pneumococci are intermittent inhabitants of the healthy human nasopharynx and are transmitted by respiratory droplets. In children, pneumococcal nasopharyngeal ecology varies by geographic region, socioeconomic status, climate, degree of crowding, and particularly intensity of exposure to other children, with children in day-care settings having higher rates of colonization. In developed-world settings, children serve as the major vectors of pneumococcal transmission. By 1 year of age, ~50% of children have had at least one episode of pneumococcal colonization. Cross-sectional prevalence data show rates of pneumococcal carriage ranging from 20% to 50% among children <5 years of age and from 5% to 15% among young notable increases in rates of disease caused by non-vaccine serotypes. Explanations for the heterogeneity of findings include replacement disease resulting from vaccine pressure, changes in clinical case investigation, secular trends unrelated to PCV

1	serotypes. Explanations for the heterogeneity of findings include replacement disease resulting from vaccine pressure, changes in clinical case investigation, secular trends unrelated to PCV use, antibiotic pressure selecting for resistant organisms, changes in surveillance or reporting systems, rapidity of introduction, and inclusion of a catch-up campaign. A recent systematic review concludes that serotype replacement in IPD follows the use of PCV7 but that the magnitude of this phenomenon is small relative to the reduction in disease from vaccine serotypes. The net effect of PCV is to reduce the rate of pneumococcal disease both in the age group targeted for vaccination and in unvaccinated age groups.

1	Pneumonia is the most common of the serious pneumococcal disease syndromes and poses special challenges from a clinical and public health perspective. Most cases of pneumococcal pneumonia are not associated with bacteremia, and in these cases a definitive etiologic diagnosis is difficult. As a result, estimates of disease burden focus primarily on IPD rates and fail to include the major portion of the burden of serious pneumococcal disease. Among children, PCV trials designed to collect efficacy data on syndrome-based outcomes (e.g., radiographically confirmed pneumonia, clinically diagnosed pneumonia) have revealed the burden of culture-negative pneumococcal pneumonia. The case-fatality ratios (CFRs) for pneumococcal pneumonia and IPD vary by age, underlying medical condition, and access to care. In addition, the CFR for pneumococcal pneumonia varies with the and middle-aged adults; Fig. 171-4 shows relevant data from the

1	United Kingdom. Data on colonization rates among healthy elderly individuals are limited. In developing-world settings, pneumococ cal acquisition occurs much earlier, sometimes within the first few days after birth, and nearly all infants have had at least one episode of colonization by 2 months of age. Cross-sectional studies show that up to the age of 5 years, 70–90% of children carry S. pneumoniae in the nasopharynx, and a significant proportion of adults (sometimes >40%) also are colonized. Their high rates of colonization make adults an important source of transmission and may affect community trans-10% mission dynamics. 0%

1	Invasive Disease and Pneumonia IPD develops when S. pneumoniae invades the bloodstream and seeds other organs or directly reaches the cerebrospinal fluid (CSF) by local extension. Pneumonia may fol low aspiration of pneumococci, although only 10–30% of such cases are associated with a positive blood culture (and thus contribute to the FIGURE 171-4 Prevalence of pneumococcal carriage in adults and measured burden of IPD). The dramatic variation of IPD rates with age children resident in the United Kingdom who had nasopharyngeal is illustrated by data from the United States for 1998–1999, a period swabs collected monthly for 10 months (no seasonal trend; t test trend, prior to PCV introduction. Rates of IPD were highest among children >.05). (Data adapted from D Goldblatt et al: J Infect Dis 192:387, 2005.)

1	FIGURE 171-5 Rates of invasive pneumococcal disease before the introduction of pneumococcal conjugate vaccine, by age group: United States, 1998. (Source: CDC, Active Bacterial Core Surveillance/ Emerging Infectious Program Network, 2000. Data adapted from MMWR 49[RR-9], 2000.) severity of disease at presentation (rather than according to whether the pneumonia episode is associated with bacteremia) and with the patient’s age (from <5% among hospitalized patients 18–44 years old to >12% among those >65 years old, even when appropriate and timely management is available). Notably, the likelihood of death in the first 24 h of hospitalization did not change substantially with the introduction of antibiotics; this surprising observation highlights the fact that the pathophysiology of severe pneumococcal pneumonia among adults reflects a rapidly progressive cascade of events that often unfolds irrespective of antibiotic administration. Management in an intensive care unit can provide

1	pneumococcal pneumonia among adults reflects a rapidly progressive cascade of events that often unfolds irrespective of antibiotic administration. Management in an intensive care unit can provide critical support for the patient through the acute period, with lower CFRs.

1	Rates of pneumococcal disease vary by season, with higher rates in colder than in warmer months in temperate climates; by sex, with males more often affected than females; and by risk group, with risk factors including underlying medical conditions, behavioral issues, and ethnic group. In the United States, some Native American populations (including Alaska natives) and African Americans have higher rates of disease than the general population; the increased risk is probably attributable to socioeconomic conditions and the prevalence of underlying risk factors for pneumococcal disease. Medical conditions that increase the risk of pneumococcal infection are listed in Table 171-1. Outbreaks of disease are well recognized in crowded settings with susceptible individuals, such as infant day-care facilities, military barracks, and nursing homes. Furthermore, there is a clear association between preceding viral respiratory disease (especially but not exclusively influenza) and risk of

1	day-care facilities, military barracks, and nursing homes. Furthermore, there is a clear association between preceding viral respiratory disease (especially but not exclusively influenza) and risk of secondary pneumococcal infections. The significant role of pneumococcal pneumonia in the morbidity and mortality associated with seasonal and pandemic influenza is increasingly recognized.

1	to penicillin was first noted in 1967, but not until the 1990s did reduced antibiotic susceptibility emerge as a significant clinical and public health issue, with an increasing prevalence of pneumococcal isolates resistant to single or multiple classes of antibiotics and a rising absolute magnitude of minimal inhibitory concentrations (MICs). Strains with reduced susceptibility to penicillin G, cefotaxime, ceftriaxone, macrolides, and other antibiotics are now found worldwide and account for a significant proportion of disease-causing strains in many locations, especially among children. Vancomycin resistance has not yet been observed in clinical pneumococcal strains. Lack of antimicrobial susceptibility is clearly related to a subset of serotypes, many of which disproportionately cause disease among children. The vicious cycle of antibiotic exposure, selection of resistant organisms in the nasopharynx, and transmission of these organisms within the community, leading to

1	cause disease among children. The vicious cycle of antibiotic exposure, selection of resistant organisms in the nasopharynx, and transmission of these organisms within the community, leading to difficult-to-treat infections and increased antibiotic exposure, has been interrupted to some extent by the introduction and routine use of PCV. The clinical implications of pneumococcal antimicrobial nonsusceptibility are addressed below in the section on treatment.

1	Cases/100,000 population Asplenia or splenic Sickle cell disease, celiac disease dysfunction Chronic respiratory disease Chronic obstructive pulmonary disease, bronchiectasis, cystic fibrosis, interstitial lung fibrosis, pneumoconiosis, bronchopulmonary dysplasia, aspiration risk, neuromuscular dis ease (e.g., cerebral palsy), severe asthma Chronic heart disease Ischemic heart disease, congenital heart dis ease, hypertension with cardiac complications, Chronic kidney disease Nephrotic syndrome, chronic renal failure, renal transplantation Chronic liver disease Cirrhosis, biliary atresia, chronic hepatitis Immunocompromise/ HIV infection, common variable immuno immunosuppression deficiency, leukemia, lymphoma, Hodgkin’s disease, multiple myeloma, generalized malig nancy, chemotherapy, organ or bone marrow transplantation, systemic glucocorticoid treat ment for >1 month at a dose equivalent to ≥20 mg/d (children, ≥1 mg/kg per day) training camps, prisons, homeless shelters

1	Note: Groups for whom pneumococcal vaccines are recommended by the Advisory Committee on Immunization Practices can be found at www.cdc.gov/vaccines/schedules/.

1	Pneumococci colonize the human nasopharynx from an early age; colonization acquisition events are generally described as asymptomatic, but evidence exists to associate acquisition with mild respiratory symptoms, especially in the very young. From the nasopharynx, the bacteria spread either via the bloodstream to distant sites (e.g., brain, joint, bones, peritoneal cavity) or locally to mucosal surfaces where they can cause otitis media or pneumonia. Direct spread from the nasopharynx to the central nervous system (CNS) can occur in rare cases of skull base fracture, although most cases of pneumococcal meningitis are secondary to hematogenous spread. Pneumococci can cause disease in almost any organ or part of the body; however, otitis media, pneumonia, bacteremia, and meningitis are most common. Colonization is a relatively frequent event, yet disease is rare. In the nasopharynx, pneumococci survive in mucus secreted by epithelial cells, where they can avoid local immune factors such

1	common. Colonization is a relatively frequent event, yet disease is rare. In the nasopharynx, pneumococci survive in mucus secreted by epithelial cells, where they can avoid local immune factors such as leukocytes and complement. The mucus itself is a component of local defense mechanisms, and the flow of mucus (driven in part by cilia in what is known as the mucociliary escalator) effects mechanical clearance of pneumococci. While many colonization episodes are of short duration, longitudinal studies in adults and children have revealed persistent colonization with a specific serotype over many months. Colonization eventually results in the development of capsule-specific serum IgG, which is thought to play a role in mediating clearance of bacteria from the nasopharynx. IgG antibodies to surface-exposed cell wall or secreted proteins also appear in the circulation in an age-dependent fashion or after colonization; the biologic role of these antibodies is less clear. Recent

1	to surface-exposed cell wall or secreted proteins also appear in the circulation in an age-dependent fashion or after colonization; the biologic role of these antibodies is less clear. Recent acquisition of a new colonizing serotype is more likely to be associated with subsequent invasion, presumably as a result of the absence of type-specific immunity. Intercurrent viral infections make the host more susceptible to pneumococcal colonization, and pneumococcal disease in a colonized individual often follows perturbation of the nasopharyngeal mucosa by such infections. Local cytokine production after a viral infection is thought to upregulate adhesion factors in the respiratory epithelium, allowing pneumococci to adhere via a variety of surface adhesin molecules, including PsaA, PspA, 950 CbpA, PspC, Hyl, pneumolysin, and the neuraminidases (Fig. 171-2). Adhesion coupled with inflammation induced by pneumococcal factors such as peptidoglycans and teichoic acids results in invasion. It

1	CbpA, PspC, Hyl, pneumolysin, and the neuraminidases (Fig. 171-2). Adhesion coupled with inflammation induced by pneumococcal factors such as peptidoglycans and teichoic acids results in invasion. It is the inflammation induced by various bacterium-derived factors that is responsible for the pathology associated with pneumococcal infection. Cell wall–derived teichoic acids and peptidoglycans induce a variety of cytokines, including the proinflammatory cytokines interleukin (IL) 1, IL-6, and tumor necrosis factor, and activate complement via the alternative pathway. Polymorphonuclear leukocytes are thus attracted, and an intense inflammatory response is initiated. Pneumolysin also is important in local pathology, inducing proinflammatory cytokine production by local monocytes. The pneumococcal capsule, consisting of polysaccharides with antiphagocytic properties due to resistance to the deposition of complement, plays an important role in pathogenesis. While most capsular types can

1	capsule, consisting of polysaccharides with antiphagocytic properties due to resistance to the deposition of complement, plays an important role in pathogenesis. While most capsular types can cause human disease, certain capsular types are more commonly isolated from sites of infection. The reason for the dominance of some serotypes over others in IPD, as depicted in Fig. 171-3, is unclear.

1	HOST DEFENSE MECHANISMS Innate Immunity As described above, intact respiratory epithelium and a host of nonspecific or innate immune factors (e.g., mucus, splenic function, complement, neutrophils, and macrophages) constitute the first line of defense against pneumococci. Physical factors such as the cough reflex and the mucociliary escalator are important in clearing bacteria from the lungs. Immunologic factors are critical as well: C-reactive protein (CRP) binds phosphorylcholine in the pneumococcal cell wall, inducing complement activation and leading to bacterial clearance; Toll-like receptor 2 (TLR2) recognizes both pneumococcal lipoteichoic acid and cell wall peptidoglycan; and in animal models, the absence of host TLR2 leads to more severe infection and impaired clearance of nasopharyngeal colonization. TLR4 appears to be necessary for the proinflammatory effect of pneumolysin on macrophages. The importance of TLR recognition is underlined by descriptions of an inherited

1	nasopharyngeal colonization. TLR4 appears to be necessary for the proinflammatory effect of pneumolysin on macrophages. The importance of TLR recognition is underlined by descriptions of an inherited deficiency of human IL-1 receptor–associated kinase 4 (IRAK-4) that manifests as an unusual susceptibility to infection with bacteria, including S. pneumoniae. IRAK-4 is essential for the normal functioning of several TLRs. Other factors that interfere with these nonspecific mechanisms (e.g., viral infections, cystic fibrosis, bronchiectasis, complement deficiency, and chronic obstructive pulmonary disease) all predispose to the development of pneumococcal pneumonia. Patients who lack a spleen or have abnormal splenic function (e.g., persons with sickle cell disease) are at high risk of developing overwhelming pneumococcal disease.

1	Acquired Immunity Acquired immunity induced via contact following colonization or through cross-reactive antigens rests largely on the development of serum IgG antibody specific for the pneumococcal capsular polysaccharide. Nearly all polysaccharides are T cell– independent antigens; B cells can make antibodies to such antigens without T cell help. However, in children <1–2 years old, such B cell responses are poorly developed. This delayed ontogeny of capsule-specific IgG in young children is associated with susceptibility to pneumococcal infection (Fig. 171-5). The extremely high risk of pneumococcal infection in the absence of serum immunoglobulin (i.e., in conditions such as agammaglobulinemia) highlights the important role of capsular antibody in protection against disease. Each serotype’s capsule is chemically distinct; thus immunity tends to be serotype specific, although some cross-immunity exists. For example, conjugate vaccine–induced antibodies to serotype 6B prevent

1	serotype’s capsule is chemically distinct; thus immunity tends to be serotype specific, although some cross-immunity exists. For example, conjugate vaccine–induced antibodies to serotype 6B prevent infection due to serotype 6A. However, cross-protection against serotypes within serogroups is not universal; for instance, antibodies to serotype 19F do not appear to confer protection against disease caused by serotype 19A. Antibodies to surface-exposed or secreted pneumococcal proteins (such as pneumolysin, PsaA, and PspA) also appear in the circulation with increasing age of the host, but their functional significance remains unclear. Data from murine models suggest that CD4+ T cells may play a role in preventing pneumococcal colonization and disease, and recent experimental data derived from humans suggest that IL-17-secreting CD4+ T cells may be relevant.

1	APPROACH TO THE PATIENT: There is no pathognomonic presentation of pneumococcal disease; patients may present with a range of syndromes and with more than one clinical syndrome (e.g., pneumonia and meningitis). S. pneumoniae can infect nearly any body tissue, manifesting as disease ranging in severity from mild and self-limited to life-threatening. The differential diagnosis of common clinical syndromes such as pneumonia, otitis media, fever of unknown origin, and meningitis should always include pneumococcal infection. A microbiologically confirmed diagnosis is made in only a minority of pneumococcal cases since, in most circumstances (and especially in pneumonia and otitis media), fluid from the site of infection is not available for etiologic determination. Empirical therapy that includes appropriate treatment for S. pneumoniae is often indicated.

1	Algorithms for assessment and management of ill children have been developed for use in the developing world or in other settings where evaluation by a trained physician may not be feasible. Children who present with ominous signs such as an inability to drink, convulsions, lethargy, and severe malnutrition are categorized as having very severe disease without further evaluation by the community health care worker, are given antibiotics, and are immediately referred to a hospital for diagnosis and management. Children who present with cough and tachypnea (the latter defined according to specific age strata) are further stratified into severity categories based on the presence or absence of lower chest wall indrawing and are managed accordingly with either antibiotics alone or antibiotics and referral to a hospital facility. Children with cough but no tachypnea are categorized as having a nonpneumonia respiratory illness.

1	The clinical manifestations of pneumococcal disease depend on the site of infection and the duration of illness. Clinical syndromes are classified as noninvasive (e.g., otitis media and nonbacteremic pneumonia) or invasive (e.g., bacteremic pneumonia). The pathogenesis of noninvasive illness involves contiguous spread from the nasopharynx or skin; invasive disease involves infection of a normally sterile body fluid or follows bacteremia. Pneumonia Pneumonia is the most common serious pneumococcal syndrome and is considered invasive when associated with a positive blood culture. Pneumococcal pneumonia can present as a mild community-acquired infection at one extreme and as a life-threatening disease requiring intubation and intensive support at the other.

1	presenting manifestations The presentation of pneumococcal pneumonia does not reliably distinguish it from pneumonia of other etiologies. In a subset of cases, pneumococcal pneumonia is recognized at the outset as associated with a viral upper respiratory infection and is characterized by the abrupt onset of cough and dyspnea accompanied by fever, shaking chills, and myalgias. The cough evolves from nonpurulent to productive of sputum that is purulent and sometimes tinged with blood. Patients may describe stabbing pleuritic chest pain and significant dyspnea indicating involvement of the parietal pleura. Among the elderly, the presenting clinical symptoms may be less specific, with confusion or malaise but without fever or cough. In such cases, a high index of suspicion is required because failure to treat pneumococcal pneumonia promptly in an elderly patient is likely to result in rapid evolution of the infection, with increased severity, morbidity, and risk of death.

1	findings on pHysical examination The clinical signs associated with pneumococcal pneumonia among adults include tachypnea (>30 breaths/min) and tachycardia, hypotension in severe cases, and fever in most cases (although not in all elderly patients). Respiratory signs are varied, including dullness to percussion in areas of the chest with significant consolidation, crackles on auscultation, reduced expansion of the chest in some cases as a result of splinting to reduce pain, bronchial breathing in a minority of cases, pleural rub in occasional cases, and cyanosis in cases with significant hypoxemia. Among infants with severe pneumonia, chest wall indrawing and nasal flaring are common. Nonrespiratory findings can include upper abdominal pain if the diaphragmatic pleura is involved as well as mental status changes, particularly confusion in elderly patients.

1	differential diagnosis The differential diagnosis of pneumococcal pneumonia includes cardiac conditions such as myocardial infarction and heart failure with atypical pulmonary edema; pulmonary conditions such as atelectasis; and pneumonia caused by viral pathogens, mycoplasmas, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Legionella, or (in HIV-infected and otherwise immunocompromised hosts) Pneumocystis. In cases with abdominal symptoms, the differential diagnosis includes cholecystitis, appendicitis, perforated peptic ulcer disease, and subphrenic abscesses. The challenge in cases with abdominal symptoms is to remember to include pneumococcal pneumonia—a nonabdominal process—in the differential diagnosis.

1	diagnosis Some authorities advocate treating uncomplicated, non-severe, community-acquired pneumonia without determining the microbiologic etiology, given that this information is unlikely to alter clinical management. However, efforts to identify the cause of pneumonia are important when the disease is more severe and when the diagnosis of pneumonia is not clearly established. The gold standard for etiologic diagnosis of pneumococcal pneumonia is pathologic examination of lung tissue. In lieu of that procedure, evidence of an infiltrate on chest radiography warrants a diagnosis of pneumonia. However, cases of pneumonia without radiographic evidence do occur. An infiltrate can be absent either early in the course of the illness or with dehydration; upon rehydration, an infiltrate usually appears. The radiographic appearance of pneumococcal pneumonia is varied; it classically consists of lobar or segmental consolidation (Fig. 171-6) but in some cases is patchy. More than one lobe is

1	appears. The radiographic appearance of pneumococcal pneumonia is varied; it classically consists of lobar or segmental consolidation (Fig. 171-6) but in some cases is patchy. More than one lobe is involved in ~30% of cases. Consolidation may be associated with a small pleural effusion

1	FIGURE 171-6 Chest radiograph depicting classic lobar pneumococcal pneumonia in the right lower lobe of an elderly patient’s lung. or empyema in complicated cases. In children, “round pneumonia,” 951 a distinctly spherical consolidation on chest radiography, is associated with a pneumococcal etiology. Round pneumonia is uncommon in adults. S. pneumoniae is not the only cause of such lesions; other causes, especially cancer, should be considered.

1	Blood drawn from patients with suspected pneumococcal pneumonia can be used for supportive or definitive diagnostic tests. Blood cultures are positive for pneumococci in a minority (<30%) of cases of pneumococcal pneumonia. Nonspecific findings include an elevated polymorphonuclear leukocyte count (>15,000/μL in most cases and upward of 40,000/μL in some), leukopenia in <10% of cases (a poor prognostic sign associated with a fatal outcome), and elevated values in liver function tests (e.g., both conjugated and unconjugated hyperbilirubinemia). Anemia, low serum albumin levels, hyponatremia, and elevated serum creatinine levels are all found in ~20–30% of patients.

1	Urinary pneumococcal antigen assays have facilitated etiologic diagnosis. In adults, among whom the prevalence of pneumococcal nasopharyngeal colonization is relatively low, a positive pneumococcal urinary antigen test has a high predictive value. The same is not true for children, in whom a positive urinary antigen test can reflect the mere presence of S. pneumoniae in the nasopharynx. Most cases of pneumococcal pneumonia are diagnosed by Gram’s staining and culture of sputum. The utility of a sputum specimen is directly related to its quality and the patient’s antibiotic treatment status.

1	complications Empyema is the most common focal complication of pneumococcal pneumonia, occurring in <5% of cases. When fluid in the pleural space is accompanied by fever and leukocytosis (even low-grade) after 4–5 days of appropriate antibiotic treatment for pneumococcal pneumonia, empyema should be considered. Parapneumonic effusions are more common than empyema, representing a self-limited inflammatory response to pneumonia. Pleural fluid with frank pus, bacteria (detected by microscopic examination), or a pH of ≤7.1 indicates empyema and demands aggressive and complete drainage, usually through chest tube insertion. Meningitis Pneumococcal meningitis typically presents as a pyogenic condition that is clinically indistinguishable from meningitis of other bacterial etiologies. Meningitis can be the primary presenting pneumococcal syndrome or a complication of other conditions such as skull fracture, otitis media, bacteremia, or mastoiditis. Now that

1	H. influenzae type b vaccine is routinely used, S. pneumoniae and Neisseria meningitidis are the most common bacterial causes of meningitis in both adults and children. Pyogenic meningitis, including that due to S. pneumoniae, is associated clinically with findings that include severe, generalized, gradual-onset headache, fever, and nausea as well as specific CNS manifestations such as stiff neck, photophobia, seizures, and confusion. Clinical signs include a toxic appearance, altered consciousness, bradycardia, and hypertension indicative of increased intracranial pressure. A small proportion of adult patients have Kernig’s or Brudzinski’s sign or cranial nerve palsies (particularly of the third and sixth cranial nerves).

1	A definitive diagnosis of pneumococcal meningitis rests on the examination of CSF for (1) evidence of turbidity (visual inspection); (2) elevated protein level, elevated white blood cell count, and reduced glucose concentration (quantitative measurement); and (3) specific identification of the etiologic agent (culture, Gram’s staining, antigen testing, or polymerase chain reaction [PCR]). A blood culture positive for S. pneumoniae in conjunction with clinical manifestations of meningitis also is considered confirmatory. Among adults, detection of pneumococcal antigen in urine is considered highly specific because of the low prevalence of nasopharyngeal colonization in this age group.

1	The mortality rate for pneumococcal meningitis is ~20%. In addition, up to 50% of survivors experience acute or chronic complications, including deafness, hydrocephalus, and mental retardation in children and diffuse brain swelling, subarachnoid bleeding, hydrocephalus, cerebrovascular complications, and hearing loss in adults. Other Invasive Syndromes S. pneumoniae can cause other invasive syndromes involving virtually any body site. These syndromes include 952 primary bacteremia without other sites of infection (bacteremia without a source; occult bacteremia), osteomyelitis, septic arthritis, endocarditis, pericarditis, and peritonitis. The essential diagnostic approach is collection of fluid from the site of infection by sterile technique and examination by Gram’s staining, culture, and—when relevant—capsular antigen assay or PCR. Hemolytic-uremic syndrome can complicate invasive pneumococcal disease.

1	Noninvasive Syndromes The two major noninvasive syndromes caused by S. pneumoniae are sinusitis and otitis media; the latter is the most common pneumococcal syndrome and most often affects young children. The manifestations of otitis media include the acute onset of severe pain, fever, deafness, and tinnitus, most frequently in the setting of a recent upper respiratory tract infection. Clinical signs include a red, swollen, often bulging tympanic membrane with reduced movement on insufflation or tympanography. Redness of the tympanic membrane is not sufficient for the diagnosis of otitis media. Pneumococcal sinusitis is also a complication of upper respiratory tract infections and presents with facial pain, congestion, fever, and— in many cases—persistent nighttime cough. A definitive diagnosis is made by aspiration and culture of sinus material; however, presumptive treatment is most commonly initiated after application of a strict set of clinical diagnostic criteria.

1	Historically, the activity of penicillin against pneumococci made parenteral penicillin G the drug of choice for disease caused by susceptible organisms, including community-acquired pneumonia. For susceptible strains, penicillin G remains the most commonly used agent, with daily doses ranging from 50,000 U/kg for minor infections to 300,000 U/kg for meningitis. Other parenteral β-lactam drugs, such as ampicillin, cefotaxime, ceftriaxone, and cefuroxime, can be used against penicillin-susceptible strains but offer little advantage over penicillin. Macrolides and cephalosporins are alternatives for penicillin-allergic patients. While agents such as clindamycin, tetracycline, and trimethoprim-sulfamethoxazole exhibit some activity against pneumococci, resistance to these agents is frequently encountered in different parts of the world.

1	Penicillin-resistant pneumococci were first described in the mid1960s, at which point tetracyclineand macrolide-resistant strains had already been reported. Multidrug-resistant strains were first described in the 1970s, but it was during the 1990s that pneumococcal drug resistance reached pandemic proportions. The use of antibiotics selects for resistant pneumococci, and strains resistant to β-lactam agents and to multiple drugs are now found all over the world. The emergence of high rates of macrolide and fluoroquinolone resistance also has been described.

1	The molecular basis of penicillin resistance in S. pneumoniae is the alteration of penicillin-binding protein (PBP) genes by transformation and horizontal transfer of DNA from related streptococcal species. Such alteration of PBPs results in lower affinity for penicillins. Depending on the specific PBP(s) and the number of PBPs altered, the level of resistance ranges from intermediate to high. For many years, penicillin susceptibility breakpoints have been defined by MICs as follows: susceptible, ≤0.06 μg/mL; intermediate, 0.12–1.0 μg/mL; and resistant, ≥2.0 μg/mL. However, in vitro results often were not predictive of the response of a patient to treatment for pneumococcal diseases other than meningitis. New recommendations have been based on the revised penicillin G breakpoints established in 2008 by the Clinical and Laboratory Standards Institute. For IV treatment of meningitis with at least 24 million units per day in 8 divided doses, the susceptibility breakpoint remains ≤0.06

1	in 2008 by the Clinical and Laboratory Standards Institute. For IV treatment of meningitis with at least 24 million units per day in 8 divided doses, the susceptibility breakpoint remains ≤0.06 μg/mL, and MICs of ≥0.12 μg/mL indicate resistance. For IV treatment of nonmeningeal infections with 12 million units per day in 6 divided doses, the breakpoints are ≤2 μg/mL for susceptible organisms, 4 μg/mL for intermediate organisms, and ≥8 μg/mL for resistant organisms; a dosage of 18–24 million units per day is recommended for strains with MICs in the intermediate category. The original breakpoints remain the same for oral treatment of nonmeningeal infections with penicillin V.

1	Although guidelines for antibiotic therapy should be driven in part by local patterns of resistance, guidelines from national organizations in many countries (e.g., the Infectious Diseases Society of America/American Thoracic Society, the British Thoracic Society, and the European Respiratory Society) lay out evidence-based approaches. The following guidelines for the treatment of individual sepsis syndromes are based on those advocated by the American Academy of Pediatrics and published in the 2012 Red Book. MENINGITIS LIKELY OR PROVEN TO BE DUE TO S. PNEUMONIAE

1	As a result of the increased prevalence of resistant pneumococci, first-line therapy for persons ≥1 month of age is a combination of vancomycin (adults, 30–60 mg/kg per day; infants and children, 60 mg/kg per day) and cefotaxime (adults, 8–12 g/d in 4–6 divided doses; children, 225–300 mg/kg per day in 1 dose or 2 divided doses) or ceftriaxone (adults, 4 g/d in 1 dose or 2 divided doses; children, 100 mg/kg per day in 1 dose or 2 divided doses). If children are hypersensitive to β-lactam agents (penicillins and cephalosporins), rifampin (adults, 600 mg/d; children, 20 mg/d in 1 dose or 2 divided doses) can be substituted for cefotaxime or ceftriaxone. A repeat lumbar puncture should be considered after 48 h if the organism is not susceptible to penicillin and information on cephalosporin sensitivity is not yet available, if the patient’s clinical condition does not improve or deteriorates, or if dexamethasone has been administered and may be compromising clinical evaluation. When

1	sensitivity is not yet available, if the patient’s clinical condition does not improve or deteriorates, or if dexamethasone has been administered and may be compromising clinical evaluation. When antibiotic sensitivity data become available, treatment should be modified accordingly. If the isolate is sensitive to penicillin, vancomycin can be discontinued and penicillin can replace the cephalosporin, or cefotaxime or ceftriaxone can be continued alone. If the isolate displays any resistance to penicillin but is susceptible to the cephalosporins, vancomycin can be discontinued and cefotaxime or ceftriaxone continued. If the isolate exhibits any resistance to penicillin and is not susceptible to cefotaxime and ceftriaxone, vancomycin and high-dose cefotaxime or ceftriaxone can be continued; rifampin may be added as well if the isolate is susceptible and the patient’s clinical condition is worsening, if the CSF remains positive for bacteria, or if the MIC of the cephalosporin in question

1	rifampin may be added as well if the isolate is susceptible and the patient’s clinical condition is worsening, if the CSF remains positive for bacteria, or if the MIC of the cephalosporin in question against the infecting strain is high. Some physicians advocate the use of glucocorticoids in children >6 months old, but this recommendation remains controversial and is not universally considered the standard of care. Glucocorticoids significantly reduce rates of mortality, severe hearing loss, and neurologic sequelae in adults and should be administered to those with community-acquired bacterial meningitis. If dexamethasone is given to either adults or children, it should be administered before or in conjunction with the first antibiotic dose.

1	In previously well children with noncritical illness, therapy with a recommended antibiotic should be instigated at the following dosages: penicillin G, 250,000–400,000 units/kg per day (in divided doses 4–6 h apart); cefotaxime, 75–100 mg/d (doses 8 h apart); or ceftriaxone, 50–75 mg/d (doses 12–24 h apart). For critically ill children, including those who have myocarditis or multilobular pneumonia with hypoxia or hypotension, vancomycin may be added if the isolate may possibly be resistant to β-lactam drugs, with its use reviewed once susceptibility data become available. If the organism is resistant to β-lactam agents, therapy should be modified on the basis of clinical response and susceptibility to other antibiotics. Clindamycin or vancomycin can be used as a first-line agent for children with severe β-lactam hypersensitivity, but vancomycin should not be continued if the organism is shown to be sensitive to other non-β-lactam antibiotics.

1	For outpatient management, amoxicillin (1 g every 8 h) provides effective treatment for virtually all cases of pneumococcal pneumonia. Neither cephalosporins nor quinolones, which are far more expensive, offer any advantage over amoxicillin. Levofloxacin (500–750 mg/d as a single dose) and moxifloxacin (400 mg/d as a single dose) also are highly likely to be effective in the United States except in patients who come from closed populations where these drugs are used widely or who have themselves been treated recently with a quinolone. Clindamycin (600–1200 mg/d every 6 h) is effective in 90% of cases and azithromycin (500 mg on day 1 followed by 250–500 mg/d) or clarithromycin (500–750 mg/d as a single dose) in 80% of cases. Treatment failure resulting in bacteremic disease due to macrolide-resistant isolates has been amply documented in patients given azithromycin empirically. As noted above, rates of resistance to all these antibiotics are relatively low in some countries and much

1	isolates has been amply documented in patients given azithromycin empirically. As noted above, rates of resistance to all these antibiotics are relatively low in some countries and much higher in others; high-dose amoxicillin remains the best option worldwide.

1	The optimal duration of treatment for pneumococcal pneumonia is uncertain, but its continuation for at least 5 days once the patient becomes afebrile appears to be a prudent approach. Cases with a second focus of infection (e.g., empyema or septic arthritis) require longer therapy.

1	Amoxicillin (80–90 mg/kg per day) is recommended for children with acute otitis media except in situations where observation and symptom-based treatment without antibiotics are advocated. These situations include nonsevere illness and an uncertain diagnosis in children 6 months to 2 years of age and nonsevere illness (even if the diagnosis seems certain) in children >2 years of age. Although the optimal duration of therapy has not been conclusively established, a 10-day course is recommended for younger children and for children with severe disease at any age. For children >6 years old who have mild or moderate disease, a course of 5–7 days is considered adequate. Patients whose illness fails to respond should be reassessed at 48–72 h. If acute otitis media is confirmed and antibiotic treatment has not been started, administration of amoxicillin should be commenced. If antibiotic therapy fails, a change is indicated. Failure to respond to second-line antibiotics as well indicates that

1	has not been started, administration of amoxicillin should be commenced. If antibiotic therapy fails, a change is indicated. Failure to respond to second-line antibiotics as well indicates that myringotomy or tympanocentesis may need to be undertaken in order to obtain samples for culture.

1	The above recommendations can also be followed for the treatment of sinusitis. Detailed information on the further management of these conditions in children has been published by the American Academy of Pediatrics and the American Academy of Family Physicians. Measures to prevent pneumococcal disease include vaccination against S. pneumoniae and influenza viruses, reduction of comorbidities that increase the risk of pneumococcal disease, and prevention of antibiotic overuse, which fuels pneumococcal resistance.

1	Capsular Polysaccharide Vaccines The 23-valent pneumococcal polysaccharide vaccine (PPSV23), containing 25 μg of each capsular polysaccharide, has been licensed for use since 1983. Recommendations for its use vary by country. The U.S. Advisory Committee on Immunization Practices recommends PPSV23 for all persons ≥65 years of age and for those 2–64 years of age who have underlying medical conditions that put them at increased risk for pneumococcal disease or, if infected, disease of increased severity (Table 171-1; see also www.cdc .gov/vaccines/schedules/). The committee recently updated their recommendations to include the combined use of PPSV23 and a conjugate vaccine in at-risk individuals (see “Polysaccharide–Protein Conjugate Vaccines,” below). Revaccination 5 years after the first dose is recommended for persons >2 years of age who have underlying medical conditions but not routinely for those whose only indication is an age of ≥65 years. PPSV23 does not induce an anamnestic

1	dose is recommended for persons >2 years of age who have underlying medical conditions but not routinely for those whose only indication is an age of ≥65 years. PPSV23 does not induce an anamnestic response, and antibody concentrations wane over time; thus revaccination is particularly important for individuals with conditions resulting in loss of antibody. Concerns about repeated revaccination have focused on safety (i.e., local reactions) and the induction of immune hyporesponsiveness.

1	Neither the clinical relevance nor the biologic basis of hyporesponsive-953 ness is clear, but, given the possibility of its occurrence, more than one revaccination has not been recommended.

1	The effectiveness of PPSV23 against IPD, pneumococcal pneumonia, all-cause pneumonia, and death is controversial, with wide variation in observations. The many published meta-analyses of PPSV efficacy have often reached opposing conclusions with regard to a given clinical entity. Generally, observational studies cite greater effectiveness than do controlled clinical trials. The consensus is that PPSV is effective against IPD but is less effective or ineffective against nonbacteremic pneumococcal pneumonia. However, published trials, observational studies, and meta-analyses contradict this view. Efficacy is often lower in the elderly and in immunodeficient patients whose condition is associated with reduced antibody responses to vaccines than in younger, healthier populations. When PPSV is effective, the duration of protection following a single dose of vaccine is estimated to be ~5 years.

1	What is not disputed is that improved pneumococcal vaccines are needed for adults. Even in the setting of routine pneumococcal conjugate vaccination of infants (which indirectly protects adults from vaccine-serotype strains), disease caused by serotypes not represented in the conjugate vaccine continues to be a significant burden among adults.

1	Polysaccharide–Protein Conjugate Vaccines Infants and young children respond poorly to PPSV, which contains T cell–independent antigens. Consequently, another class of pneumococcal vaccines, the PCVs, were developed specifically for infants and young children. The first product, a 7-valent PCV, was licensed in 2000 in the United States. Three PCV products—containing 7, 10, and 13 serotypes, respectively—are currently (2014) commercially available. The serotypes included in these PCV formulations are important causes of IPD and antibiotic resistance among young children. Randomized controlled trials have demonstrated a high degree of efficacy of PCVs against vaccine-serotype IPD as well as efficacy against pneumonia, otitis media, nasopharyngeal colonization, and all-cause mortality. PCVs are recommended by the World Health Organization for inclusion in routine childhood immu nization schedules worldwide, especially in countries with high infant mortality rates.

1	The United States was the first country to introduce PCV and therefore has the longest experience with its community-wide effects. The introduction of PCV in the United States has resulted in a >90% reduction in vaccine-serotype IPD among the whole population (Fig. 171-7). This decline has been noted not only in those age groups immunized but also in adults and is attributable to the near elimination of vaccineserotype nasopharyngeal colonization in immunized infants, which reduces spread to adults. This protection of unimmunized community members through vaccination of a subset of the community is termed the indirect effect. Increases in colonization with—and concomitantly in disease due to—non-vaccine-serotype strains (i.e., replacement colonization and disease) have been seen; however, the absolute rate increases in IPD caused by non-vaccine serotypes are generally small, especially relative to decreases in vaccine-serotype IPD (see “Epidemiology,” above). Since vaccine-serotype

1	the absolute rate increases in IPD caused by non-vaccine serotypes are generally small, especially relative to decreases in vaccine-serotype IPD (see “Epidemiology,” above). Since vaccine-serotype strains are more commonly resistant to antibiotics than are non-vaccine serotypes, use of PCV has also resulted in dramatic declines in the proportion and absolute rates of drug-resistant pneumococcal disease. The recommendations of the Advisory Committee on Immunization Practices for the use of conjugate vaccines can be found at www.cdc.gov/MMWR/pdf/ wk/mm5909.pdf. Recently, PCV has been shown to prevent pneumococcal infection in HIV-infected adults. In the United States, PCV13 followed by a dose of PPSV23 is now recommended for all immunocompromised children and adults.

1	Other Prevention Strategies Pneumococcal disease can also be averted through the prevention of illnesses that predispose individuals to pneumococcal infections. Relevant measures include influenza vaccination and improved management and control of diabetes, HIV infection, heart disease, and lung disease. Finally, the reduction of antibiotic misuse is a strategy for the prevention of pneumococcal disease in that

1	FIGURE 171-7 Changes in invasive pneumococcal disease (IPD) incidence, by serotype group, among children <5 years old (top) and adults >65 years old (bottom), 1998–2009. 7-Valent pneumococcal conjugate vaccine (PCV7) was introduced in the United States for routine administration to infants and young children during the second half of 2000, while PCV13 was introduced in 2010, the year following this surveillance period. PCV7 serotypes include serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F as well as cross-reactive serotype 6A. PCV13 serotypes include the PCV7 serotypes as well as serotypes 1, 3, 5, 6A, 7F, and 19A. (Reprinted with permission from Dr. M. Moore, Centers for Disease Control and Prevention.) antimicrobial resistance directly and indirectly perpetuates organism transmission and disease in the community.

1	American Academy of Pediatrics RED BOOK. The report of the Committee on Infectious Diseases: aapredbook.aappublications.org; Pneumococcal Regional Serotype Distribution for Pneumococcal AMC TPP: www.gavialliance.org/library/documents/amc/tpp-codebook/ Cases per 100,000 population Cases per 100,000 population Staphylococcal Infections Franklin D. Lowy Staphylococcus aureus, the most virulent of the many staphylococcal species, has demonstrated its versatility by remaining a major cause of morbidity and mortality worldwide despite the availability of numer-ous effective antistaphylococcal antibiotics. S. aureus is a pluripotent 172 pathogen, causing disease through both toxinand non-toxin-mediated mechanisms. This organism is responsible for numerous nosocomial and community-based infections that range from relatively minor skin and soft tissue infections to life-threatening systemic infections.

1	The “other” staphylococci, collectively designated coagulase-negative staphylococci (CoNS), are considerably less virulent than S. aureus but remain important pathogens in infections that are primarily associated with prosthetic devices. Staphylococci, gram-positive cocci in the family Micrococcaceae, form grapelike clusters on Gram’s stain (Fig. 172-1). These organisms (~1 μm in diameter) are catalase-positive (unlike streptococcal species), non-motile, aerobic, and facultatively anaerobic. They are capable of prolonged survival on environmental surfaces under varying conditions. Some species have a relatively broad host range, including mammals and birds, whereas for others the host range is quite narrow—i.e., limited to one or two closely related animals.

1	More than 30 staphylococcal species are pathogenic. Identification of the more clinically important species has generally relied on a series of biochemical tests. Automated diagnostic systems, kits for biochemical characterization, and DNA-based assays are available for species identification. With few exceptions, S. aureus is distinguished from other staphylococcal species by its production of coagulase, a surface enzyme that converts fibrinogen to fibrin. Latex kits that detect both protein A and clumping factor also distinguish S. aureus from most other staphylococcal species. S. aureus ferments mannitol, is positive for protein A, and produces DNAse. On blood agar plates, S. aureus tends to form golden β-hemolytic colonies; in contrast, CoNS produce small white nonhemolytic colonies. Increasingly, sequence-based methods (e.g., 16S rRNA) are being used to identify different staphylococcal species.

1	Determining whether multiple staphylococcal isolates from different patients are the same or different is often relevant when there is concern that a nosocomial outbreak is due to a common point source (e.g., a contaminated medical instrument). Molecular typing methods, such as pulsed-field gel electrophoresis and sequence-based techniques (e.g., staphylococcal protein A [SpA] typing), have increasingly been used for this purpose. More recently, whole-genome sequencing has enhanced the ability to discriminate among clinical isolates. S.AUREUS INFECTIONS

1	S.AUREUS INFECTIONS S. aureus is both a commensal and an opportunistic pathogen. Approximately 30% of healthy persons are colonized with S. aureus, with a smaller percentage (~10%) persistently colonized. The rate of colonization is elevated among insulin-dependent diabetics, HIV-infected patients, patients undergoing hemodialysis, injection drug users, and individuals with skin damage. The anterior nares and oropharynx are frequent sites of human colonization, although the skin (especially when damaged), vagina, axilla, and perineum may also be colonized. These colonization sites serve as a reservoir for future infections.

1	Transmission of S. aureus most frequently results from direct personal contact. Colonization of different body sites allows transfer from one person to another during contact. Spread of staphylococci in aerosols of respiratory or nasal secretions from heavily colonized individuals has also been reported. Most individuals who develop S. aureus infections become infected with a strain that is already a part of their own commensal flora. Breaches of the skin or mucosal membrane allow S. aureus to initiate infection. FIGURE 172-1 Gram’s stain of S. aureus in a sputum sample. (From ASM MicrobeLibrary.org.© Pfizer, Inc.)

1	FIGURE 172-1 Gram’s stain of S. aureus in a sputum sample. (From ASM MicrobeLibrary.org.© Pfizer, Inc.) Some diseases increase the risk of S. aureus infection; diabetes, for example, combines an increased rate of S. aureus colonization and the use of injectable insulin with the possibility of impaired leukocyte function. Individuals with congenital or acquired qualitative or quantitative defects of polymorphonuclear leukocytes (PMNs) are at increased risk of S. aureus infections; this group includes neutropenic patients (e.g., those receiving chemotherapeutic agents), those with chronic granulomatous disease, and those with Job’s or Chédiak-Higashi syndrome. Other groups at risk include individuals with end-stage renal disease, HIV infection, skin abnormalities, or prosthetic devices.

1	S. aureus is a leading cause of health care–associated infections (Chap. 168). It is the most common cause of surgical wound infections and is second only to CoNS as a cause of primary bacteremia. These isolates are generally resistant to multiple antibiotics; thus available therapeutic options are limited. In the community, S. aureus remains an important cause of skin and soft tissue infections, respiratory infections, and (among injection drug users) infective endocarditis. The increasing use of home infusion therapy is another cause of community-acquired staphylococcal infections. In the past two decades, there has been a dramatic change in the epidemiology of infections due to methicillin-resistant

1	S. aureus (MRSA). In addition to its major role as a nosocomial pathogen, MRSA has become an established community-based pathogen. Numerous outbreaks of community-associated MRSA (CA-MRSA) infections have been reported in both rural and urban settings in widely separated regions throughout the world. The outbreaks have occurred among such diverse groups as children, prisoners, athletes, Native Americans, and drug users. Risk factors common to these outbreaks include poor hygienic conditions, close contact, contaminated material, and damaged skin. These infections have been caused by a limited number of MRSA strains. In the United States, strain USA300 (defined by pulsed-field gel electrophoresis) has been the predominant clone. In other geographic regions of the world, different strains of CA-MRSA have been responsible for these community-based outbreaks. Although the majority of infections caused by these strains have involved the skin and soft tissue, 5–10% have been invasive and

1	of CA-MRSA have been responsible for these community-based outbreaks. Although the majority of infections caused by these strains have involved the skin and soft tissue, 5–10% have been invasive and potentially life-threatening. CA-MRSA strains have also been responsible for an increasing number of nosocomial infections. Of concern has been the apparent capacity of CA-MRSA to cause disease in immunocompetent individuals.

1	PATHOGENESIS General Concepts S. aureus is a pyogenic pathogen known for its capacity to induce abscess formation at sites of both local and metastatic infections. This classic pathologic response to S. aureus defines the framework within which the infection will progress. The bacteria elicit an inflammatory response characterized by an initial intense infiltration of PMNs and a subsequent infiltration of macrophages and fibroblasts. Either the host cellular response (including the deposition of fibrin and collagen) contains the infection, or infection spreads to the adjoining tissue or the bloodstream.

1	In toxin-mediated staphylococcal disease, infection is not invariably present. For example, once toxin has been elaborated into food, staphylococcal food poisoning can develop in the absence of viable bacteria. In staphylococcal toxic shock syndrome (TSS), conditions allowing toxin elaboration at colonization sites (e.g., the presence of a superabsorbent tampon) suffice for initiation of clinical illness.

1	The S. aureus Genome The complete genomes of numerous strains of S. aureus have now been fully sequenced. Among the interesting revelations are (1) the high degree of nucleotide sequence similarity of the core genomes of different strains; (2) acquisition of a relatively large amount of genetic information by horizontal transfer from other bacterial species; and (3) the presence of unique “pathogenicity” or “genomic” islands—mobile genetic elements that contain clusters of enterotoxin and exotoxin genes and/or antimicrobial 955 resistance determinants. Among the genes in these islands are those carrying mecA, the gene responsible for methicillin resistance. Methicillin resistance–containing islands have been designated staphylococcal cassette chromosome mec (SCCmec) types and range in size from ~20 to 60 kb. To date, 11 SCCmec types have been identified. Among the more common types, types 1–3 are traditionally associated with nosocomial MRSA isolates, whereas types 4–6 have been

1	size from ~20 to 60 kb. To date, 11 SCCmec types have been identified. Among the more common types, types 1–3 are traditionally associated with nosocomial MRSA isolates, whereas types 4–6 have been associated with the epidemic CA-MRSA strains.

1	A limited number of MRSA clones have been responsible for most communityand hospital-associated infections worldwide. A comparison of these strains with those from earlier outbreaks (e.g., the phage 80/81 strains from the 1950s) has revealed preservation of the nucleotide sequence over time. This observation suggests that these strains possess determinants that facilitate survival and spread.

1	Regulation of Virulence Gene Expression In both toxin-mediated and non-toxin-mediated diseases due to S. aureus, the expression of virulence determinants associated with infection depends on a series of regulatory genes (e.g., accessory gene regulator [agr] and staphylococcal accessory regulator [sar]) that coordinately control the expression of many virulence genes. The regulatory gene agr is part of a quorum-sensing signal transduction pathway that senses and responds to bacterial density. Staphylococcal surface proteins are synthesized during the bacterial exponential growth phase in vitro. In contrast, many secreted proteins, such as α toxin, the enterotoxins, and assorted enzymes, are released during the postexponential growth phase in response to transcription of the effector molecule of agr, RNAIII.

1	It has been hypothesized that these regulatory genes serve a similar function in vivo. Successful invasion requires the sequential expression of these different bacterial elements. Bacterial adhesins are needed to initiate colonization of host tissue surfaces. The subsequent release of various enzymes enables the colony to obtain nutritional support and permits bacteria to spread to adjacent tissues. Studies with strains in which these regulatory genes are inactivated show reduced virulence in several animal models of S. aureus infection.

1	Pathogenesis of Invasive S. aureus Infection Staphylococci are opportunists. For these organisms to invade the host and cause infection, some or all of the following steps are necessary: contamination and colonization of host tissue surfaces, breach of cutaneous or mucosal barriers, establishment of a localized infection, invasion, evasion of the host response, and metastatic spread. Colonizing strains or strains transferred from other individuals are introduced into damaged skin, a wound, or the bloodstream. Recurrences of S. aureus infections are common, apparently because of the capacity of these pathogens to survive, to persist in a quiescent state in various tissues, and then to cause recrudescent infections when suitable conditions arise.

1	s.aureus colonization of body surfaces The anterior nares is one of the primary sites of staphylococcal colonization in humans. Colonization appears to involve the attachment of S. aureus to keratinized epithelial cells of the anterior nares. Other factors that may contribute to colonization include the influence of other resident nasal flora and their bacterial density, host factors, and nasal mucosal damage (e.g., that resulting from inhalational drug use). Other colonized body sites, such as damaged skin, the groin, and the oropharynx, may be particularly important reservoirs for CA-MRSA strains. inoculation and colonization of tissue surfaces Staphylococci may be introduced into tissue as a result of minor abrasions, administration of medications such as insulin, or establishment of IV access with catheters. After their introduction into a tissue site, bacteria replicate and colonize the host tissue surface. A family of structurally related

1	S. aureus surface proteins referred to as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) plays an important role in mediating adherence to these sites. By adhering to exposed matrix molecules (e.g., fibrinogen, fibronectin), MSCRAMMs such as clumping factor and collagen-binding protein enable the bacteria to colonize different tissue surfaces; these proteins contribute to 956 the pathogenesis of invasive infections such as endocarditis and septic arthritis by facilitating the adherence of S. aureus to surfaces with exposed fibrinogen or collagen. Although CoNS are classically known for their ability to elaborate biofilms and to colonize prosthetic devices, S. aureus also possesses the genes responsible for biofilm formation, such as the intercellular adhesion (ica) locus. Binding to these devices occurs in a stepwise fashion, involving staphylococcal adherence to serum constituents that have coated the device surface and subsequent biofilm elaboration.

1	(ica) locus. Binding to these devices occurs in a stepwise fashion, involving staphylococcal adherence to serum constituents that have coated the device surface and subsequent biofilm elaboration. S. aureus is thus a frequent cause of biomedical-device infections.

1	invasion After colonization, staphylococci replicate at the initial site of infection, elaborating enzymes that include serine proteases, hyaluronidases, thermonucleases, and lipases. These enzymes facilitate bacterial survival and local spread across tissue surfaces, although their precise role in infections is not well defined. The lipases may facilitate survival in lipid-rich areas such as the hair follicles, where S. aureus infections are often initiated. The S. aureus toxin Panton-Valentine leukocidin is cytolytic to PMNs, macrophages, and monocytes. Strains elaborating this toxin have been epidemiologically linked with cutaneous and more serious infections caused by strains of CA-MRSA. MSCRAMMs also appear to play an important role in the ability of S. aureus to spread and cause disease at other tissue sites.

1	S. aureus to spread and cause disease at other tissue sites. Constitutional findings may result from either localized or systemic infections. The staphylococcal cell wall—consisting of alternating N-acetyl muramic acid and N-acetyl glucosamine units in combination with an additional cell wall component, lipoteichoic acid—can initiate an inflammatory response that includes the sepsis syndrome. Staphylococcal α toxin, which causes pore formation in various eukaryotic cells, can also initiate an inflammatory response with findings suggestive of sepsis.

1	evasion of Host defense mecHanisms Staphylococci have a multitude of immune evasion strategies that are critical to their success as invasive pathogens. They possess an antiphagocytic polysaccharide microcapsule. Most human S. aureus infections are due to capsular types 5 and 8. The zwitterionic (both negatively and positively charged) S. aureus capsule plays a critical role in the induction of abscess formation. Protein A, an MSCRAMM unique to S. aureus, acts as an Fc receptor, binding the Fc portion of IgG subclasses 1, 2, and 4 and preventing opsonophagocytosis by PMNs. Both chemotaxis inhibitory protein of staphylococci (CHIPS, a secreted protein) and extracellular adherence protein (EAP, a surface protein) interfere with PMN migration to sites of infection.

1	An additional potential mechanism of S. aureus evasion is its capacity for intracellular survival. Both professional and nonprofessional phagocytes internalize staphylococci. Internalization by these cells may provide a sanctuary that protects bacteria against the host’s defenses. The intracellular environment favors the phenotypic expression of S. aureus small-colony variants. Small-colony variants are found in patients receiving antimicrobial therapy (e.g., with aminoglycosides) and in those with cystic fibrosis or osteomyelitis. These variants, whether intraor extracellular, may facilitate prolonged staphylococcal survival in different tissue sites and enhance the likelihood of recurrences. Finally, S. aureus can survive within PMNs and may use these cells to spread and to seed other tissue sites.

1	patHogenesis of community-acQuired mrsa infections A number of virulence determinants have been identified as contributing to the pathogenesis of CA-MRSA infections. There is a strong epidemiologic association linking the presence of the gene for the Panton-Valentine leukocidin with skin and soft tissue infections as well as with necrotizing postinfluenza pneumonia. Other determinants that play a role in the pathogenesis of these infections include the arginine catabolic mobile element (ACME), a cluster of unique genes that may facilitate evasion of host defense mechanisms; phenol-soluble modulins, a family of cytolytic peptides; and α toxin.

1	Host Response to S. aureus Infection The primary host response to S. aureus infection is the recruitment of PMNs. These cells are attracted to infection sites by bacterial components such as formylated peptides or peptidoglycan as well as by the cytokines tumor necrosis factor (TNF) and interleukins (ILs) 1 and 6, which are released by activated macrophages and endothelial cells. Although most individuals have antibodies to staphylococci, it is not clear that antibody levels are qualitatively or quantitatively sufficient to protect against infection. Although anticapsular and anti-MSCRAMM antibodies facilitate opsonization in vitro and have been protective against infection in several animal models, they have not yet successfully prevented staphylococcal infections in clinical trials.

1	Pathogenesis of Toxin-Mediated Disease S. aureus produces three types of toxin: cytotoxins, pyrogenic toxin superantigens, and exfoliative toxins. Both epidemiologic data and studies in animals suggest that antitoxin antibodies are protective against illness in TSS, staphylococcal food poisoning, and staphylococcal scalded-skin syndrome (SSSS). Illness develops after toxin synthesis and absorption and the subsequent toxin-initiated host response.

1	enterotoxin and toxic sHocK syndrome toxin 1 (tsst-1) The pyrogenic toxin superantigens are a family of small-molecular-size, structurally similar proteins that are responsible for two diseases: TSS and food poisoning. TSS results from the ability of enterotoxins and TSST-1 to function as T cell mitogens. In the normal process of antigen presentation, the antigen is first processed within the cell, and peptides are then presented in the major histocompatibility complex (MHC) class II groove, initiating a measured T cell response. In contrast, enterotoxins bind directly to the invariant region of MHC—outside the MHC class II groove. The enterotoxins can then bind T cell receptors via the vβ chain; this binding results in a dramatic overexpansion of T cell clones (up to 20% of the total T cell population). The consequence of this T cell expansion is a “cytokine storm,” with the release of inflammatory mediators that include interferon γ, IL-1, IL-6, TNF-α, and TNF-β. The resulting

1	T cell population). The consequence of this T cell expansion is a “cytokine storm,” with the release of inflammatory mediators that include interferon γ, IL-1, IL-6, TNF-α, and TNF-β. The resulting multisystem disease produces a constellation of findings that mimic those in endotoxin shock; however, the pathogenic mechanisms differ. The release of endotoxin from the gastrointestinal tract may synergistically enhance the toxin’s effects.

1	A different region of the enterotoxin molecule is responsible for the symptoms of food poisoning. The enterotoxins are heat stable and can survive conditions that kill the bacteria. Illness results from the ingestion of preformed toxin. As a result, the incubation period is short (1–6 h). The toxin stimulates the vagus nerve and the vomiting center of the brain. It also appears to stimulate intestinal peristaltic activity. exfoliative toxins and ssss The exfoliative toxins are responsible for SSSS. The toxins that produce disease in humans are of two serotypes: ETA and ETB. These toxins are serine proteases, which cleave desmosomal cadherins in the superficial layer of the skin, triggering exfoliation. The result is a split in the epidermis at the granular level, which is responsible for the superficial desquamation of the skin that typifies this illness.

1	Staphylococcal infections are readily diagnosed by Gram’s stain (Fig. 172-1) and microscopic examination of abscess contents or of infected tissue. Routine culture of infected material usually yields positive results, and blood cultures are sometimes positive even when infections are localized to extravascular sites. S. aureus is rarely a blood culture contaminant. Polymerase chain reaction (PCR)–based assays have been applied to the rapid diagnosis of S. aureus infection and are increasingly used in clinical microbiology laboratories. A number of point-of-care tests are now available to screen patients for colonization with MRSA. Determining whether patients with documented S. aureus bacteremia also have infective endocarditis or a metastatic focus of infection remains a diagnostic challenge. Uniformly positive blood cultures suggest an endovascular infection such as endocarditis (see “Bacteremia, Sepsis, and Infective Endocarditis,” below).

1	Skin and Soft Tissue Infections S. aureus causes a variety of cutaneous infections, many of which can also be caused by group A streptococci Folliculitis Abscess, furuncle, carbuncle Cellulitis Impetigo Mastitis Surgical wound infections Ventilator-associated or nosocomial pneumonia Septic pulmonary emboli Postviral pneumonia (e.g., influenza) Empyema Sepsis, septic shock Metastatic foci of infection (kidney, joints, bone, lung) Infective endocarditis Device-Related Infections (e.g., intravascular catheters, prosthetic joints) Toxin-Mediated Illnesses

1	Staphylococcal scalded-skin syndrome Invasive Infections Associated with Community-Acquired Methicillin-Resistant S. aureus or (less commonly) other streptococcal species. Common factors predisposing to S. aureus cutaneous infection include chronic skin conditions (e.g., eczema), skin damage (e.g., insect bites, minor trauma), injections (e.g., in diabetes, injection drug use), and poor personal hygiene. These infections are characterized by the formation of pus-containing blisters, which often begin in hair follicles and spread to adjoining tissues. Folliculitis is a superficial infection that involves the hair follicle, with a central area of purulence (pus) surrounded by induration and erythema. Furuncles (boils) are more extensive, painful lesions that tend to occur in hairy, moist regions of the body and extend from the hair follicle to become a true abscess with an area of central purulence. Carbuncles are most often located in the lower neck and are even more severe and

1	regions of the body and extend from the hair follicle to become a true abscess with an area of central purulence. Carbuncles are most often located in the lower neck and are even more severe and painful, resulting from the coalescence of other lesions that extend to a deeper layer of the subcutaneous tissue. In general, furuncles and carbuncles are readily apparent, with pus often expressible or discharging from the abscess. Other cutaneous

1	S. aureus infections include impetigo and cellulitis. S. aureus is one of the most common causes of surgical wound infection. Mastitis develops in 1–3% of nursing mothers. This infection of the breast, which generally presents within 2–3 weeks after delivery, is characterized by findings that range from cellulitis to abscess formation. Systemic signs, such as fever and chills, are often present in more severe cases.

1	Musculoskeletal Infections S. aureus is among the most common 957 causes of bone infections—both those resulting from hematogenous dissemination and those arising from contiguous spread from a soft tissue site. Hematogenous osteomyelitis in children most often involves the long bones. Infections present as fever and bone pain or with a child’s reluctance to bear weight. The white blood cell count and erythrocyte sedimentation rate are often elevated. Blood cultures are positive in ~50% of cases. When necessary, bone biopsies for culture and histopathologic examination are usually diagnostic. Routine x-rays may be normal for up to 14 days after the onset of symptoms. 99mTc-phosphonate scanning often detects early evidence of infection. MRI is more sensitive than other techniques in establishing a radiologic diagnosis.

1	In adults, hematogenous osteomyelitis involving the long bones is less common. However, vertebral osteomyelitis is among the more common clinical presentations. Vertebral bone infections are most often seen in patients with endocarditis, those undergoing hemodialysis, diabetics, and injection drug users. These infections may present as intense back pain and fever but may also be clinically occult, presenting as chronic back pain and low-grade fever. S. aureus is the most common cause of epidural abscess, a complication that can result in neurologic compromise. Patients complain of difficulty voiding or walking and of radicular pain in addition to the symptoms associated with their osteomyelitis. Surgical intervention in this setting often constitutes a medical emergency. MRI most reliably establishes the diagnosis (Fig. 172-2).

1	Bone infections that result from contiguous spread tend to develop from soft tissue infections, such as those associated with diabetic or vascular ulcers, surgery, or trauma. Exposure of bone, a draining fistulous tract, failure to heal, or continued drainage suggests involvement of underlying bone. Bone involvement is established by bone culture and histopathologic examination (revealing evidence of PMN infiltration). Contamination of culture material from adjacent tissue can make the diagnosis of osteomyelitis difficult in the absence of pathologic confirmation. In addition, it is sometimes hard to distinguish radiologically between osteomyelitis and overlying soft tissue infection with underlying osteitis.

1	In both children and adults, S. aureus is the most common cause of septic arthritis in native joints. This infection is rapidly progressive and may be associated with extensive joint destruction if left untreated. It presents as intense pain on motion of the affected joint, swelling, and fever. Aspiration of the joint reveals turbid fluid, with >50,000 PMNs/μL and gram-positive cocci in clusters on Gram’s stain (Fig. 172-1). In FIGURE 172-2 S. aureus vertebral osteomyelitis and epidural abscess involving the thoracic disk between T9 and T10. Sagittal postcontrast MRI of the spine illustrates destruction of the T9–T10 intervertebral space with enhancement (arrow). There is impingement on the thoracic cord and an epidural collection extending from T9 through T11 (short arrows).

1	958 adults, septic arthritis may result from trauma, surgery, or hematogenous dissemination. The most commonly involved joints include the knees, shoulders, hips, and phalanges. Infection frequently develops in joints previously damaged by osteoarthritis or rheumatoid arthritis. Iatrogenic infections resulting from aspiration or injection of agents into the joint also occur. In these settings, the patient experiences increased pain and swelling in the involved joint in association with fever. Pyomyositis is an unusual infection of skeletal muscles that is seen primarily in tropical climates but also occurs in immunocompromised and HIV-infected patients. It is believed to arise from occult bacteremia. Pyomyositis presents as fever, swelling, and pain overlying the involved muscle. Aspiration of fluid from the involved tissue yields pus. Although a history of trauma may be associated with the infection, its pathogenesis is poorly understood.

1	S. aureus occur in selected clinical settings. S. aureus is a cause of serious respiratory tract infections in newborns and infants; these infections present with shortness of breath, fever, and respiratory failure. Chest x-ray may reveal pneumatoceles (shaggy, thin-walled cavities). Pneumothorax and empyema are recognized complications.

1	In adults, nosocomial S. aureus pulmonary infections are common among intubated patients in intensive care units. Nasally colonized patients are at increased risk of these infections. The clinical presentation is no different from that encountered in pulmonary infections of other bacterial etiologies. Patients produce increased volumes of purulent sputum and develop respiratory distress, fever, and new pulmonary infiltrates. Distinguishing bacterial pneumonia from respiratory failure or other causes of new pulmonary infiltrates in critically ill patients is often difficult and relies on a constellation of clinical, radiologic, and laboratory findings.

1	Community-acquired respiratory tract infections due to S. aureus usually follow viral infections—most commonly influenza. Patients may present with fever, bloody sputum production, and midlung-field pneumatoceles or multiple, patchy pulmonary infiltrates. Diagnosis is made by sputum Gram’s stain and culture. Blood cultures, although useful, are usually negative. Bacteremia, Sepsis, and Infective Endocarditis S. aureus bacteremia may be complicated by sepsis, endocarditis, vasculitis, or metastatic seeding (establishment of suppurative collections at other tissue sites). The frequency of metastatic seeding during bacteremia has been estimated to be as high as 31%. Among the more commonly seeded tissue sites are bones, joints, kidneys, and lungs.

1	Recognition of these complications by clinical and laboratory diagnostic methods alone is often difficult. Comorbid conditions that are frequently seen in association with S. aureus bacteremia and that increase the risk of complications include diabetes, HIV infection, and renal insufficiency. Other host factors associated with an increased risk of complications include presentation with community-acquired S. aureus bacteremia (except in injection drug users), lack of an identifiable primary focus of infection, and the presence of prosthetic devices or material. Clinically, S. aureus sepsis presents in a manner similar to that documented for sepsis due to other bacteria. The well-described progression of hemodynamic changes—beginning with respiratory alkalosis and clinical findings of hypotension and fever—is commonly seen. The microbiologic diagnosis is established by positive blood cultures.

1	The overall incidence of S. aureus endocarditis has increased over the past 20 years. S. aureus is now the leading cause of endocarditis worldwide, accounting for 25–35% of cases. This increase is due, at least in part, to the increased use of intravascular devices. Studies of patients with S. aureus bacteremia and intravascular catheters that used transesophageal echocardiography found an infective endocarditis incidence of ~25%. Other factors associated with an increased risk of endocarditis are injection drug use, hemodialysis, the presence of intravascular prosthetic devices at the time of bacteremia, and immunosuppression. Patients with implantable cardiac devices (e.g., permanent pacemakers) are at increased risk of endocarditis or device-related infections. Despite the availability of effective antibiotics, mortality rates from these infections continue to range from 20% to 40%, depending on both the host and the nature of the infection. Complications of

1	S. aureus endocarditis include cardiac valvular insufficiency, peripheral emboli, metastatic seeding, and central nervous system (CNS) involvement (e.g., mycotic aneurysms, embolic strokes).

1	S. aureus endocarditis is encountered in four clinical settings: (1) right-sided endocarditis in association with injection drug use, (2) left-sided native-valve endocarditis, (3) prosthetic-valve endocarditis, and (4) nosocomial endocarditis. In each of these settings, the diagnosis is suspected by recognition of clinical stigmata suggestive of endocarditis. These findings include cardiac manifestations, such as new or changing cardiac valvular murmurs; cutaneous evidence, such as vasculitic lesions, Osler’s nodes, or Janeway lesions; evidence of rightor left-sided embolic disease; and a history suggesting a risk for S. aureus bacteremia. In the absence of antecedent antibiotic therapy, blood cultures are almost uniformly positive. Transthoracic echocardiography, while less sensitive than transesophageal echocardiography, is less invasive and may establish the presence of valvular vegetations. The Duke criteria (see Table 155-3) are now commonly used to help establish the likelihood

1	transesophageal echocardiography, is less invasive and may establish the presence of valvular vegetations. The Duke criteria (see Table 155-3) are now commonly used to help establish the likelihood of this diagnosis.

1	Acute right-sided tricuspid valvular S. aureus endocarditis is most often seen in injection drug users. The classic presentation includes a high fever, a toxic clinical appearance, pleuritic chest pain, and the production of purulent (sometimes bloody) sputum. Chest x-rays or CT scans reveal evidence of septic pulmonary emboli (small, peripheral, circular lesions that may cavitate with time) (Fig. 172-3). A high percentage of affected patients have no history of antecedent valvular damage. At the outset of their illness, patients may present with fever alone, without cardiac or other localizing findings. As a result, a high index of clinical suspicion is essential for diagnosis.

1	Individuals with antecedent cardiac valvular damage more commonly present with left-sided native-valve endocarditis involving the damaged valve. These patients tend to be older than those with right-sided endocarditis, their prognosis is worse, and their incidence of complications (including peripheral emboli, cardiac decompensation, and metastatic seeding) is higher. S. aureus is one of the more common causes of prosthetic-valve endocarditis. This infection is especially fulminant in the early postoperative period and is associated with a high mortality rate. In most instances, medical therapy alone is not sufficient and urgent valve replacement is necessary. Patients are prone to develop valvular insufficiency or myocardial abscesses originating from the region of valve implantation. FIGURE 172-3 CT scan illustrating septic pulmonary emboli in a patient with methicillin-resistant Staphylococcus aureus bacteremia.

1	FIGURE 172-3 CT scan illustrating septic pulmonary emboli in a patient with methicillin-resistant Staphylococcus aureus bacteremia. The increased frequency of nosocomial endocarditis (15–30% of cases, depending on the series) reflects in part the increased use of intravascular devices. This form of endocarditis is most commonly caused by S. aureus. Because patients often are critically ill, are receiving antibiotics for various other indications, and have comorbid conditions, the diagnosis is often missed. Urinary Tract Infections Urinary tract infections (UTIs) are infrequently caused by S. aureus. The presence of S. aureus in the urine generally suggests hematogenous dissemination. Ascending S. aureus infections occasionally result from instrumentation of the genitourinary tract.

1	Prosthetic Device–Related Infections S. aureus accounts for a large proportion of prosthetic device–related infections. These infections often involve intravascular catheters, prosthetic valves, orthopedic devices, peritoneal catheters, pacemakers, left-ventricular-assist devices, and vascular grafts. In contrast with the more indolent presentation of CoNS infections, S. aureus device-related infections are often more acute, with both localized and systemic manifestations. The latter infections also tend to progress more rapidly. It is relatively common for a pyogenic collection to be present at the device site. Aspiration of these collections and performance of blood cultures are important components in establishing a diagnosis. S. aureus infections tend to occur more commonly soon after implantation unless the device is used for access (e.g., intravascular or hemodialysis catheters). In the latter instance, infections can occur at any time. As in most prosthetic-device infections,

1	implantation unless the device is used for access (e.g., intravascular or hemodialysis catheters). In the latter instance, infections can occur at any time. As in most prosthetic-device infections, successful therapy usually involves removal of the device. Left in place, the device is a potential nidus for either persistent or recurrent infections.

1	Infections Associated with Community-Acquired MRSA Although the skin and soft tissues are the most common sites of infection associated with CA-MRSA, 5–10% of these infections are invasive and can even be life-threatening. The latter unique infections, including necrotizing fasciitis, necrotizing pneumonia, and sepsis with Waterhouse-Friderichsen syndrome or purpura fulminans, were rarely associated with S. aureus prior to the emergence of CA-MRSA. These life-threatening infections reflect the increased virulence of CA-MRSA strains.

1	Toxin-Mediated Diseases • food poisoning S. aureus is among the most common causes of foodborne outbreaks of infection in the United States. Staphylococcal food poisoning results from the inoculation of toxin-producing S. aureus into food by colonized food handlers. Toxin is then elaborated in such growth-promoting food as custards, potato salad, or processed meats. Even if the bacteria are killed by warming, the heat-stable toxin is not destroyed. The onset of illness is rapid, occurring within 1–6 h of ingestion. The illness is characterized by nausea and vomiting, although diarrhea, hypotension, and dehydration may also occur. The differential diagnosis includes diarrhea of other etiologies, especially that caused by similar toxins (e.g., the toxins elaborated by Bacillus cereus). The rapidity of onset, the absence of fever, and the epidemic nature of the presentation (without second-degree spread) arouse suspicion of staphylococcal food poisoning. Symptoms generally resolve within

1	of onset, the absence of fever, and the epidemic nature of the presentation (without second-degree spread) arouse suspicion of staphylococcal food poisoning. Symptoms generally resolve within 8–10 h. The diagnosis can be established by the demonstration of bacteria or the documentation of enterotoxin in the implicated food. Treatment is entirely supportive.

1	toxic sHocK syndrome TSS gained attention in the early 1980s, when a nationwide outbreak occurred in the United States among young, otherwise healthy, menstruating women. Epidemiologic investigation demonstrated that these cases were associated with the use of a highly absorbent tampon that had recently been introduced to the market. Subsequent studies established the role of TSST-1 in these illnesses. Withdrawal of the tampon from the market resulted in a rapid decline in the incidence of this disease. However, menstrual and nonmenstrual cases continue to be reported. Nonmenstrual cases are frequently seen in patients with surgical or postpartum wound infections. The clinical presentation is similar in menstrual and nonmenstrual TSS. Evidence of clinical S. aureus infection is not a prerequisite. CASE DEfInITIon of S. AUREUS ToxIC SHoCk SynDRoME 1. Fever: temperature of ≥38.9°C ( ≥102°F) 2.

1	CASE DEfInITIon of S. AUREUS ToxIC SHoCk SynDRoME 1. Fever: temperature of ≥38.9°C ( ≥102°F) 2. Hypotension: systolic blood pressure of ≤90 mmHg or orthostatic hypo-tension (orthostatic drop in diastolic blood pressure by ≥15 mmHg, orthostatic syncope, or orthostatic dizziness) 3. Diffuse macular rash, with desquamation 1–2 weeks after onset (including the palms and soles) 4. a. Hepatic: bilirubin or aminotransferase levels ≥2 times normal b. Hematologic: platelet count ≤100,000/μL c. Renal: blood urea nitrogen or serum creatinine level ≥2 times the normal upper limit d. Mucous membranes: vaginal, oropharyngeal, or conjunctival hyperemia e. Gastrointestinal: vomiting or diarrhea at onset of illness f. Muscular: severe myalgias or serum creatine phosphokinase level ≥2 times the normal upper limit g.

1	Gastrointestinal: vomiting or diarrhea at onset of illness f. Muscular: severe myalgias or serum creatine phosphokinase level ≥2 times the normal upper limit g. Central nervous system: disorientation or alteration in consciousness without focal neurologic signs and in the absence of fever and hypo-tension 5. Negative serologic or other tests for measles, leptospirosis, and Rocky Mountain spotted fever as well as negative blood or cerebrospinal fluid cultures for organisms other than S. aureus Source: M Wharton et al: Case definitions for public health surveillance. MMWR 39:1, 1990; with permission.

1	TSS results from the elaboration of an enterotoxin or the structurally related enterotoxin-like TSST-1. More than 90% of menstrual cases are caused by TSST-1, whereas a high percentage of nonmenstrual cases are caused by enterotoxins. TSS begins with relatively nonspecific flu-like symptoms. In menstrual cases, the onset usually comes 2 or 3 days after the start of menstruation. Patients present with fever, hypotension, and erythroderma of variable intensity. Mucosal involvement is common (e.g., conjunctival hyperemia). The illness can rapidly progress to symptoms that include vomiting, diarrhea, confusion, myalgias, and abdominal pain. These symptoms reflect the multisystemic nature of the disease, with involvement of the liver, kidneys, gastrointestinal tract, and/or CNS. Desquamation of the skin occurs during convalescence, usually 1–2 weeks after the onset of illness. Laboratory findings may include azotemia, leukocytosis, hypoalbuminemia, thrombocytopenia, and liver function

1	of the skin occurs during convalescence, usually 1–2 weeks after the onset of illness. Laboratory findings may include azotemia, leukocytosis, hypoalbuminemia, thrombocytopenia, and liver function abnormalities.

1	Diagnosis of TSS still depends on a constellation of findings rather than one specific finding and on a lack of evidence of other possible infections (Table 172-2). Other diagnoses to be considered are drug toxicities, viral exanthems, Rocky Mountain spotted fever, sepsis, and Kawasaki disease. Illness occurs only in persons who lack antibody to TSST-1. Recurrences are possible if antibody fails to develop after the illness.

1	stapHylococcal scalded-sKin syndrome SSSS primarily affects newborns and children. The illness may vary from a localized blister to exfoliation of much of the skin surface. The skin is usually fragile and often tender, with thin-walled, fluid-filled bullae. Gentle pressure results in rupture of the lesions, leaving denuded underlying skin (Nikolsky’s sign; Fig. 172-4). The mucous membranes are usually spared. In more generalized infection, there are often constitutional symptoms, including fever, lethargy, and irritability with poor feeding. Significant amounts of fluid can be lost in more extensive cases. Illness usually follows localized infection at one of a number of possible sites. SSSS is much less common among adults but can follow infections caused by exfoliative toxin–producing strains.

1	Primary prevention of S. aureus infections in the hospital setting involves hand washing and careful attention to appropriate isolation procedures. Through careful screening for MRSA carriage and strict isolation practices, several Scandinavian countries have been remarkably FIGURE 172-4 Evidence of staphylococcal scalded-skin syn-drome in a 6-year-old boy. Nikolsky’s sign, with separation of the superficial layer of the outer epidermal layer, is visible. (Reprinted with permission from LA Schenfeld et al: N Engl J Med 342:1178, 2000. © 2000 Massachusetts Medical Society. All rights reserved.) successful at preventing the introduction and dissemination of MRSA in hospitals.

1	Decolonization strategies, using both universal and targeted approaches with topical agents (e.g., mupirocin) to eliminate nasal colonization and/or chlorhexidine to eliminate cutaneous colonization with S. aureus, have been successful in some clinical settings (e.g., intensive care units) where the risk of infection is high. An analysis of clinical trials suggests that there may also be a reduction in the incidence of postsurgical infections among persons who are nasally colonized with S. aureus.

1	“Bundling” (the application of selected medical interventions in a sequence of prescribed steps) has reduced rates of nosocomial infections related to such procedures as the insertion of intravenous catheters, in which staphylococci are among the most common pathogens (see Table 168-4). A number of immunization strategies to prevent S. aureus infections—both active (e.g., capsular polysaccharide–protein conjugate vaccine) and passive (e.g., clumping factor antibody)—have been investigated. However, none has been successful for either prophylaxis or therapy in clinical trials.

1	Although considerably less virulent than S. aureus, CoNS are among the most common causes of prosthetic-device infections. Approximately half of the identified CoNS species have been associated with human infections. Of these species, Staphylococcus epidermidis is the most common human pathogen. This component of the normal human flora is found on the skin (where it is the most abundant bacterial species) as well as in the oropharynx and vagina. Staphylococcus saprophyticus, a novobiocin-resistant species, is a common pathogen in UTIs. S. epidermidis is the CoNS species most often associated with pros-thetic-device infections. Infection is a two-step process, with initial adhesion to the device followed by colonization. S. epidermidis is uniquely adapted to colonize these devices by its capacity to elaborate the extracellular polysaccharide (glycocalyx or slime) that facilitates formation of a protective biofilm on the device surface.

1	Implanted prosthetic material is rapidly coated with host serum or tissue constituents such as fibrinogen or fibronectin. These molecules serve as potential bridging ligands, facilitating initial bacterial attachment to the device surface. A number of staphylococcal surface-associated proteins, such as autolysin (AtlE), fibrinogen-binding protein, and accumulation-associated protein (AAP), may play a role in attachment to either modified or unmodified prosthetic surfaces. The polysaccharide intercellular adhesin facilitates subsequent staphylococcal colonization and accumulation on the device surface. In S. epidermidis, intercellular adhesin (ica) genes are more commonly found in strains associated with device infections than in strains associated with colonization of mucosal surfaces. Biofilm appears to act as a barrier protecting bacteria from host defense mechanisms as well as from antibiotics, while providing a suitable environment for bacterial survival. Poly-γ-DL-glutamic acid

1	appears to act as a barrier protecting bacteria from host defense mechanisms as well as from antibiotics, while providing a suitable environment for bacterial survival. Poly-γ-DL-glutamic acid is secreted by S. epidermidis and provides protection against neutrophil phagocytosis.

1	Two additional staphylococcal species, Staphylococcus lugdunensis and Staphylococcus schleiferi, produce more serious infections (nativevalve endocarditis and osteomyelitis) than do other CoNS. The basis for this enhanced virulence is not known, although both species appear to share more virulence determinants with S. aureus (e.g., clumping factor and lipase) than do other CoNS. The capacity of S. saprophyticus to cause UTIs in young women appears to be related to its enhanced capacity to adhere to uroepithelial cells. A 160-kDa hemagglutinin/adhesin may contribute to this affinity.

1	Although the detection of CoNS at sites of infection or in the bloodstream is not difficult by standard microbiologic culture methods, interpretation of these results is frequently problematic. Because these organisms are present in large numbers on the skin, they often contaminate cultures. It has been estimated that only 10–25% of blood cultures positive for CoNS reflect true bacteremia. Similar problems arise with cultures obtained from other sites. Among the clinical findings suggestive of true bacteremia are fever, evidence of local infection (e.g., erythema or purulent drainage at the IV catheter site), leukocytosis, and systemic signs of sepsis. Laboratory findings suggestive of true bacteremia include multiple isolations of the same strain (i.e., the same species with the same antibiogram or a closely related DNA fingerprint) from separate cultures, growth of the strain within 48 h, and bacterial growth in both aerobic and anaerobic bottles.

1	CoNS cause a diverse array of prosthetic device–related infections, including those that involve prosthetic cardiac valves and joints, vascular grafts, intravascular devices, and CNS shunts. In all of these settings, the clinical presentation is similar. The signs of localized infection are often subtle, the rate of disease progression is slow, and the systemic findings are often limited. Signs of infection, such as purulent drainage, pain at the site, or loosening of prosthetic implants, are sometimes evident. Fever is frequently but not always present, and there may be mild leukocytosis. Acute-phase reactant levels, the erythrocyte sedimentation rate, and the C-reactive protein concentration may be elevated.

1	Infections that are not associated with prosthetic devices are infrequent, although native-valve endocarditis due to CoNS has accounted for ~5% of cases in some reviews. S. lugdunensis appears to be a more aggressive pathogen in this setting, causing greater mortality and rapid valvular destruction with abscess formation.

1	Surgical incision and drainage of all suppurative collections constitute the most important therapeutic intervention for staphylococcal infections. The emergence of MRSA in the community has increased the importance of culturing all collections in order to identify pathogens and to determine antimicrobial susceptibility. Successful therapy for prosthetic-device infections generally requires device removal. In situations in which removal is not possible or the infection is due to CoNS, an initial attempt at medical therapy without device removal may be warranted. Because of the well-recognized risk of complications associated with S. aureus bacteremia (e.g., endocarditis, metastatic foci of infection), therapy is generally prolonged (4–6 weeks) unless the patient is identified as being among those individuals who are at low risk for complications. Debate continues regarding the duration of therapy for bacteremic

1	Debate continues regarding the duration of therapy for bacteremic S. aureus infections. Patients with “complicated” bacteremia are at increased risk of endocarditis and metastatic infections. Among the findings associated with an increased risk of complicated bacteremia are persistently positive blood cultures 96 h after institution of therapy, acquisition of the infection in the community, failure to remove a removable focus of infection (i.e., an intravascular catheter), and infection with cutaneous or embolic manifestations. For immunocompetent patients in whom short-course therapy is planned, transesophageal echocardiography to rule out endocarditis is warranted because neither clinical nor laboratory findings can reliably detect cardiac involvement. In addition, an aggressive radiologic investigation to identify potential metastatic collections is indicated. All symptomatic body sites must be carefully evaluated.

1	The choice of antimicrobial agents to treat both coagulasepositive and coagulase-negative staphylococcal infections has become increasingly problematic because of the prevalence of multidrug-resistant strains. Staphylococcal resistance to most antibiotic families, including β-lactams, aminoglycosides, fluoroquinolones, and (to a lesser extent) glycopeptides, has increased. This trend is more apparent with CoNS: >80% of nosocomial isolates are resistant to methicillin, and these methicillin-resistant strains are usually resistant to most other antibiotics as well. Because the selection of antimicrobial agents for S. aureus infections is similar to that for CoNS infections, treatment options for these pathogens are discussed together and are summarized in Table 172-3.

1	As a result of the widespread dissemination of plasmids containing the enzyme penicillinase, few strains of staphylococci (≤5%) remain susceptible to penicillin. However, penicillin remains the drug of choice against susceptible strains if the laboratory can reliably test for penicillin susceptibility. Penicillin-resistant isolates are treated with semisynthetic penicillinase-resistant penicillins (SPRPs), such as oxacillin or nafcillin. Methicillin, the first of the SPRPs, is now used infrequently. Cephalosporins are alternative therapeutic agents for these infections. Secondand third-generation cephalosporins do not offer a therapeutic advantage over first-generation cephalosporins for the treatment of staphylococcal infections. The carbapenems have excellent activity against methicillin-sensitive S. aureus but not against MRSA.

1	The isolation of MRSA was reported within 1 year of the introduction of methicillin. Since then, the prevalence of MRSA has steadily increased. In many hospitals, 40–50% of S. aureus isolates are now resistant to methicillin. Resistance to methicillin indicates resistance to all SPRPs as well as to all cephalosporins (except ceftaroline). Production of a novel penicillin-binding protein (PBP2a) is responsible for methicillin resistance. This protein is synthesized by the mecA gene, which (as stated above) is part of a large mobile genetic element—a pathogenicity or genomic island—called SCCmec. It is hypothesized that this genetic material was acquired via horizontal transfer from a related staphylococcal species, such as Staphylococcus sciuri. Phenotypic expression of methicillin resistance may be constitutive (i.e., expressed in all organisms in a population) or heterogeneous (i.e., displayed by only a proportion of the total organism population). Detection of methicillin resistance

1	may be constitutive (i.e., expressed in all organisms in a population) or heterogeneous (i.e., displayed by only a proportion of the total organism population). Detection of methicillin resistance in the clinical microbiology laboratory can be difficult if the strain expresses heterogeneous resistance. Therefore, susceptibility studies are routinely performed at reduced temperatures (≤35°C for 24 h), with increased concentrations of salt in the medium to enhance the expression of resistance. In addition to PCR-based techniques, a number of rapid methods for the detection of methicillin resistance have been developed.

1	In light of decreasing susceptibility of MRSA isolates to vancomycin, both vancomycin and daptomycin are now recommended as the drugs of choice for the treatment of MRSA infections. Vancomycin is less effective than SPRPs for the treatment of infections due to methicillin-susceptible strains. Alternatives to SPRPs should be used only after careful consideration in patients with a history of serious β-lactam allergies.

1	Three types of staphylococcal resistance to vancomycin have 961 emerged. (1) Minimal inhibitory concentration (MIC) “creep” refers to the incremental increase in vancomycin MICs that has been detected in various geographic areas. Studies suggest that infections due to S. aureus strains with vancomycin MICs of >1 μg/mL may not respond as well to vancomycin therapy as those due to strains with MICs of <1 μg/mL. Some authorities (e.g., The Medical Letter) have recommended choosing an alternative agent in this setting. (2) In 1997, an S. aureus strain with reduced susceptibility to vancomycin (vancomycin-intermediate S. aureus [VISA]) was reported from Japan. Subsequently, additional VISA clinical isolates were reported. These strains were all resistant to methicillin and many other antimicrobial agents. The VISA strains appear to evolve (under vancomycin selective pressure) from strains that are susceptible to vancomycin but are heterogeneous, with a small proportion of the bacterial

1	agents. The VISA strains appear to evolve (under vancomycin selective pressure) from strains that are susceptible to vancomycin but are heterogeneous, with a small proportion of the bacterial population expressing the resistance phenotype. The mechanism of VISA resistance is in part due to an abnormally thick cell wall. Vancomycin is trapped by the abnormal peptidoglycan cross-linking and is unable to gain access to its target site.

1	(3) In 2002, the first clinical isolate of fully vancomycin-resistant S. aureus was reported. Resistance in this and several additional clinical isolates was due to the presence of vanA, the gene responsible for expression of vancomycin resistance in enterococci. This observation suggested that resistance was acquired as a result of horizontal conjugal transfer from a vancomycin-resistant strain of Enterococcus faecalis. Several patients had both MRSA and vancomycin-resistant enterococci cultured from infection sites. The vanA gene is respon sible for the synthesis of the dipeptide D-Ala-D-Lac in place of D-AlaD-Ala. Vancomycin cannot bind to the altered peptide.

1	Daptomycin, a parenteral bactericidal agent with antistaphylococcal activity, is approved for the treatment of bacteremia (including right-sided endocarditis) and complicated skin infections. It is not effective in respiratory infections. This drug has a novel mechanism of action: it disrupts the cytoplasmic membrane. Staphylococcal resistance to daptomycin, sometimes developing during therapy, has been reported. Linezolid—the first oxazolidinone—is bacteriostatic against staphylococci and offers the advantage of comparable bioavailability after oral or parenteral administration. Cross-resistance with other inhibitors of protein synthesis has not been detected. However, resistance to linezolid has been reported. Serious adverse reactions to linezolid include thrombocytopenia, occasional cases of neutropenia, and rare instances of peripheral and optic neuropathy.

1	Tedizolid, a second oxazolidinone released in 2014, is available as both oral and parenteral preparations. It has enhanced in vitro activity against antibiotic-resistant gram-positive bacteria, including staphylococci. Tedizolid is administered once a day. Ceftaroline is a fifth-generation cephalosporin with bactericidal activity against MRSA (including strains with reduced susceptibility to vancomycin and daptomycin). It is approved for use in nosocomial pneumonias and for skin and soft tissue infections. The parenteral streptogramin antibiotic quinupristin/dalfopristin displays bactericidal activity against all staphylococci, including VISA strains. This drug has been used successfully to treat serious MRSA infections. In cases of resistance to erythromycin or clindamycin, quinupristin/dalfopristin is bacteriostatic against staphylococci. There are limited data on the efficacy of either quinupristin/dalfopristin or linezolid for the treatment of infective endocarditis.

1	Telavancin is a parenteral lipoglycopeptide derivative of vancomycin that is approved for the treatment of complicated skin and soft tissue infections and for nosocomial pneumonia. The drug has two targets: the cell wall and the cell membrane. It remains active against VISA strains. Because of its nephrotoxicity, it should be avoided in patients with renal disease. Dalbavancin is a long-acting, parenterally administered lipoglycopeptide that has been used to treat skin and soft tissue infections. Because of its long half-life, it can be administered on a weekly basis. There are limited data on its use in the treatment of invasive staphylococcal infections. Sensitivity/Resistance of Isolate Drug of Choice Alternative(s) Comments

1	Sensitivity/Resistance of Isolate Drug of Choice Alternative(s) Comments Sensitive to methicillin Dicloxacillin (500 mg qid), cephalexin Minocycline or doxycycline (100 mg It is important to know the antibiotic susceptibility of (500 mg qid) q12hb), TMP-SMX (1 or 2 ds tablets isolates in the specific geographic region. All drain-bid), clindamycin (300–450 mg/kg tid), age should be cultured. linezolid (600 mg PO q12h), tedizolid (200 mg PO q24h)

1	Resistant to methicillin Clindamycin (300–450 mg/kg tid), Same options as under “Drug of It is important to know the antibiotic susceptibility of TMP-SMX (1 or 2 ds tablets bid), mino-Choice” isolates in the specific geographic region. All draincycline or doxycycline (100 mg q12hb), age should be cultured. linezolid (600 mg bid) or tedizolid (200 mg once daily) aRecommended dosages are for adults with normal renal and hepatic function. bThe dosage must be adjusted for patients with reduced creatinine clearance. cFor the treatment of prosthetic-valve endocarditis, the addition of gentamicin (1 mg/kg q8h) and rifampin (300 mg PO q8h) is recommended, with adjustment of the gentamicin dosage if the creatinine clearance rate is reduced. dDaptomycin cannot be used for the treatment of pneumonia. eVancomycin-resistant S. aureus isolates from clinical infections have been reported.

1	Abbreviations: ds, double-strength; TMP-SMX, trimethoprim-sulfamethoxazole; VISA, vancomycin-intermediate S. aureus; VRSA, vancomycin-resistant S. aureus. Source: Modified with permission from FD Lowy: N Engl J Med 339:520, 1998 (© 1998 Massachusetts Medical Society. All rights reserved.); C Liu et al: Clin Infect Dis 52:285, 2011; DL Stevens et al: Clin Infect Dis 59:148, 2014; and Med Lett Drugs Ther 56:39, 2014.

1	Although the quinolones are active against staphylococci in vitro, Tigecycline, a broad-spectrum minocycline analogue, has bactethe frequency of staphylococcal resistance to these agents has riostatic activity against MRSA and is approved for use in skin and increased progressively, especially among methicillin-resistant isolates. soft tissue infections as well as intraabdominal infections caused by Of particular concern in MRSA is the possible emergence of quinolone S. aureus. Other antibiotics, such as minocycline and trimethoprimresistance during therapy. Therefore, quinolones are not recom-sulfamethoxazole, have been used successfully to treat MRSA infecmended for the treatment of MRSA infections. Resistance to the quino-tions in cases of vancomycin toxicity or intolerance. lones is most commonly chromosomal and results from mutations of Combinations of antistaphylococcal agents have been used to the topoisomerase IV or DNA gyrase genes, although multidrug efflux enhance

1	is most commonly chromosomal and results from mutations of Combinations of antistaphylococcal agents have been used to the topoisomerase IV or DNA gyrase genes, although multidrug efflux enhance bactericidal activity in the treatment of serious infecpumps may also contribute. Although the newer quinolones exhibit tions such as endocarditis or osteomyelitis. In selected instances increased in vitro activity against staphylococci, it is uncertain whether (e.g., right-sided endocarditis), drug combinations are also used to this increase translates into enhanced in vivo activity. shorten the duration of therapy. Among the antimicrobial agents used in combinations are rifampin, aminoglycosides (e.g., gentamicin), and fusidic acid (not readily available in the United States). To date, clinical studies have not documented a therapeutic benefit; recent reports have raised concern about the potential nephrotoxicity of gentamicin and about adverse drug reactions from the addition of rifampin.

1	studies have not documented a therapeutic benefit; recent reports have raised concern about the potential nephrotoxicity of gentamicin and about adverse drug reactions from the addition of rifampin. As a result, the use of gentamicin in combination with β-lactams or other antimicrobial agents is no longer routinely recommended for the treatment of endocarditis. Rifampin continues to be used for the treatment of prosthetic device–related infections and for osteomyelitis.

1	The combination of daptomycin with a β-lactam antibiotic has been successfully used to treat patients with persistent MRSA bacteremia, even those infected with isolates that exhibit reduced susceptibility to daptomycin. The combination appears to enhance the bactericidal activity of daptomycin, perhaps by reducing the bacterial cell surface charge and thus allowing more daptomycin binding. When necessary, the use of oral antistaphylococcal agents for uncomplicated skin and soft tissue infections is usually successful. For other infections, parenteral therapy is indicated. S. aureus endocarditis is usually an acute, life-threatening infection. Thus, prompt collection of blood for cultures must be followed immediately by empirical antimicrobial therapy. For life-threatening

1	S. aureus native-valve endocarditis, therapy with a β-lactam is recommended. If a MRSA strain is isolated, vancomycin (15–20 mg/kg every 8–12 h, given in equal doses up to a total of 2 g) or daptomycin (6 mg/kg every 24 h) is recommended. The vancomycin dose should be adjusted on the basis of trough vancomycin levels. Patients are generally treated for 4–6 weeks, with duration depending on whether there are complications. For prosthetic-valve endocarditis, surgery in addition to antibiotic therapy is often necessary. The combination of a β-lactam agent—or, if the isolate is β-lactam-resistant, vancomycin (30 mg/kg every 24 h, given in doses up to a total of 2 g) or daptomycin (6 mg/kg every 24 h)—with an aminoglycoside (gentamicin, 1 mg/kg IV every 8 h) and rifampin (300 mg orally or IV every 8 h) for ≥6 weeks is recommended.

1	For hematogenous osteomyelitis or septic arthritis in children, a 4-week course of therapy is usually adequate. In adults, treatment is often more prolonged. For chronic forms of osteomyelitis, surgical debridement is necessary in combination with antimicrobial therapy. For joint infections, a critical component of therapy is the repeated aspiration or arthroscopy of the affected joint to prevent damage from leukocytes. The combination of rifampin with ciprofloxacin has been used successfully to treat prosthetic joint infections, especially when the device cannot be removed. The efficacy of this combination may reflect enhanced activity against staphylococci in biofilms as well as the attainment of effective intracellular concentrations.

1	The choice of empirical therapy for staphylococcal infections depends in part on susceptibility data for the local geographic area. Increasingly, vancomycin and daptomycin are the drugs of choice for both communityand hospital-acquired infections. The increase in CA-MRSA skin and soft tissue infections has drawn attention to the need for initiation of appropriate empirical therapy. Oral agents that have been effective against these isolates include clindamycin, trimethoprim-sulfamethoxazole, doxycycline, linezolid, and tedizolid.

1	Supportive therapy with reversal of hypotension is the mainstay of therapy for TSS. Both fluids and pressors may be necessary. Tampons or other packing material should be promptly removed. The role of antibiotics is less clear. Some investigators recommend a combination of clindamycin and a semisynthetic penicillin or vancomycin (if the isolate is resistant to methicillin). Clindamycin is advocated because, as a protein synthesis inhibitor, it reduces toxin synthesis in vitro. Linezolid also appears to be effective. A semisynthetic penicillin or glycopeptide is suggested to eliminate any potential focus of infection as well as to eradicate persistent carriage that might increase the likelihood of recurrent illness. Anecdotal reports document the successful use of IV immuno-963 globulin to treat TSS. The role of glucocorticoids in the treatment of this disease is uncertain.

1	Anecdotal reports document the successful use of IV immuno-963 globulin to treat TSS. The role of glucocorticoids in the treatment of this disease is uncertain. Therapy for staphylococcal food poisoning is entirely supportive. For SSSS, antistaphylococcal therapy targets the primary site of infection. Michael R. Wessels

1	Many varieties of streptococci are found as part of the normal flora colonizing the human respiratory, gastrointestinal, and genitourinary tracts. Several species are important causes of human disease. Group A Streptococcus (GAS, Streptococcus pyogenes) is responsible for streptococcal pharyngitis, one of the most common bacterial infections of school-age children, and for the postinfectious syndromes of acute rheumatic fever (ARF) and poststreptococcal glomerulonephritis (PSGN). Group B Streptococcus (GBS, Streptococcus agalactiae) is the leading cause of bacterial sepsis and meningitis in newborns and a major cause of endometritis and fever in parturient women. Viridans streptococci are the most common cause of bacterial endocarditis. Enterococci, which are morphologically similar to streptococci, are now considered a separate genus on the basis of DNA homology studies. Thus, the species previously designated as Streptococcus faecalis and Streptococcus faecium have been renamed

1	streptococci, are now considered a separate genus on the basis of DNA homology studies. Thus, the species previously designated as Streptococcus faecalis and Streptococcus faecium have been renamed Enterococcus faecalis and Enterococcus faecium, respectively. The enterococci are discussed in Chap. 174.

1	Streptococci are gram-positive, spherical to ovoid bacteria that characteristically form chains when grown in liquid media. Most streptococci that cause human infections are facultative anaerobes, although some are strict anaerobes. Streptococci are relatively fastidious organisms, requiring enriched media for growth in the laboratory. Clinicians and clinical microbiologists identify streptococci by several classification systems, including hemolytic pattern, Lancefield group, species name, and common or trivial name. Many streptococci associated with human infection produce a zone of complete (β) hemolysis around the bacterial colony when cultured on blood agar. The β-hemolytic streptococci can be classified by the Lancefield system, a serologic grouping based on the reaction of specific antisera with bacterial cell-wall carbohydrate antigens. With rare exceptions, organisms belonging to Lancefield groups A, B, C, and G are all β-hemolytic, and each is associated with characteristic

1	antisera with bacterial cell-wall carbohydrate antigens. With rare exceptions, organisms belonging to Lancefield groups A, B, C, and G are all β-hemolytic, and each is associated with characteristic patterns of human infection. Other streptococci produce a zone of partial (α) hemolysis, often imparting a greenish appearance to the agar. These α-hemolytic streptococci are further identified by biochemical testing and include Streptococcus pneumoniae (Chap. 171), an important cause of pneumonia, meningitis, and other infections, and the several species referred to collectively as the viridans streptococci, which are part of the normal oral flora and are important agents of subacute bacterial endocarditis. Finally, some streptococci are nonhemolytic, a pattern sometimes called γ hemolysis. Among the organisms classified serologically as group D streptococci, the enterococci are classified as a distinct genus (Chap. 174). The classification of the major streptococcal groups causing human

1	the organisms classified serologically as group D streptococci, the enterococci are classified as a distinct genus (Chap. 174). The classification of the major streptococcal groups causing human infections is outlined in Table 173-1.

1	Lancefield’s group A consists of a single species, S. pyogenes. As its species name implies, this organism is associated with a variety of suppurative infections. In addition, GAS can trigger the postinfectious syndromes of ARF (which is uniquely associated with S. pyogenes infection; Chap. 381) and PSGN (Chap. 338). TABLE 173-1 CLASSIfICATIon of STREPToCoCCI A S. pyogenes β Pharyngitis, impetigo, cellulitis, scarlet fever B S. agalactiae β Neonatal sepsis and meningitis, puerperal infection, urinary tract infection, diabetic ulcer infection, endocarditis C, G S. dysgalactiae subsp. equisimilis β Cellulitis, bacteremia, endocarditis D Enterococcia: E. faecalis, E. faecium Usually nonhemolytic Urinary tract infection, nosocomial bacteremia, endocarditis Nonenterococci: S. gallolyticus Usually nonhemolytic Bacteremia, endocarditis (formerly S. bovis) Variable or nongroupable Viridans streptococci: S. sanguis, α Endocarditis, dental abscess, brain abscess S. mitis

1	Variable or nongroupable Viridans streptococci: S. sanguis, α Endocarditis, dental abscess, brain abscess S. mitis Intermedius or milleri group: S. interme-Variable Brain abscess, visceral abscess dius, S. anginosus, S. constellatus Anaerobic streptococcib: Usually nonhemolytic Sinusitis, pneumonia, empyema, brain abscess, liver abscess aSee Chap. 174. bSee Chap. 201.

1	Worldwide, GAS infections and their postinfectious sequelae the emm gene, which encodes M protein. DNA sequence analysis of (primarily ARF and rheumatic heart disease) account for an the amplified gene segment can be compared with an extensive data-estimated 500,000 deaths per year. Although data are incom-base (developed at the Centers for Disease Control and Prevention plete, the incidence of all forms of GAS infection and that of rheumatic [CDC]) for assignment of emm type. This method eliminates the need heart disease are thought to be tenfold higher in resource-limited for typing sera, which are available in only a few reference laboratories. countries than in developed countries (Fig. 173-1). The presence of M protein on a GAS isolate correlates with its capacity to resist phagocytic killing in fresh human blood. This phenomenon PATHOGENESIS appears to be due, at least in part, to the binding of plasma fibrinogen GAS elaborates a number of cell-surface components and

1	phagocytic killing in fresh human blood. This phenomenon PATHOGENESIS appears to be due, at least in part, to the binding of plasma fibrinogen GAS elaborates a number of cell-surface components and extracel-to M protein molecules on the streptococcal surface, which interferes lular products important in both the pathogenesis of infection and with complement activation and deposition of opsonic complement the human immune response. The cell wall contains a carbohydrate fragments on the bacterial cell. This resistance to phagocytosis may antigen that may be released by acid treatment. The reaction of such be overcome by M protein–specific antibodies; thus individuals with acid extracts with group A–specific antiserum is the basis for defini-antibodies to a given M type acquired as a result of prior infection are tive identification of a streptococcal strain as S. pyogenes. The major protected against subsequent infection with organisms of the same M surface protein of GAS is M protein,

1	of prior infection are tive identification of a streptococcal strain as S. pyogenes. The major protected against subsequent infection with organisms of the same M surface protein of GAS is M protein, which occurs in more than 100 type but not against that with different M types.

1	antigenically distinct types and is the basis for the serotyping of strains GAS also elaborates, to varying degrees, a polysaccharide capsule with specific antisera. The M protein molecules are fibrillar structures composed of hyaluronic acid. The production of large amounts of anchored in the cell wall of the organism that extend as hairlike pro-capsule by certain strains imparts a characteristic mucoid appearance jections away from the cell surface. The amino acid sequence of the to the colonies. The capsular polysaccharide plays an important role in distal or amino-terminal portion of the M protein molecule is quite protecting GAS from ingestion and killing by phagocytes. In contrast variable, accounting for the antigenic variation of the different M to M protein, the hyaluronic acid capsule is a weak immunogen, and types, while more proximal regions of the protein are relatively con-antibodies to hyaluronate have not been shown to be important in pro- served. A newer technique for

1	is a weak immunogen, and types, while more proximal regions of the protein are relatively con-antibodies to hyaluronate have not been shown to be important in pro- served. A newer technique for assignment of M type to GAS isolates tective immunity. The presumed explanation is the apparent structural uses the polymerase chain reaction to amplify the variable region of identity between streptococcal hyaluronic acid and the hyaluronic

1	Presence of rheumatic heart disease (cases per 1000) 0.3 1.01.3 3.5 0.8 1.8 2.2 5.7 FIGURE 173-1 Prevalence of rheumatic heart disease in children 5–14 years old. The circles within Australia and New Zealand represent indigenous populations (and also Pacific Islanders in New Zealand). (From JR Carapetis et al: Lancet Infect Dis 5:685, 2005, with permission.) acid of mammalian connective tissues. The capsular polysaccharide may also play a role in GAS colonization of the pharynx by binding to CD44, a hyaluronic acid–binding protein expressed on human pharyngeal epithelial cells.

1	GAS produces a large number of extracellular products that may be important in local and systemic toxicity and in the spread of infection through tissues. These products include streptolysins S and O, toxins that damage cell membranes and account for the hemolysis produced by the organisms; streptokinase; DNAses; SpyCEP, a serine protease that cleaves and inactivates the chemoattractant cytokine interleukin 8, thereby inhibiting neutrophil recruitment to the site of infection; and several pyrogenic exotoxins. Previously known as erythrogenic toxins, the pyrogenic exotoxins cause the rash of scarlet fever. Since the mid1980s, pyrogenic exotoxin–producing strains of GAS have been linked to unusually severe invasive infections, including necrotizing fasciitis and the streptococcal toxic shock syndrome (TSS). Several extracellular products stimulate specific antibody responses useful for serodiagnosis of recent streptococcal infection. Tests for these antibodies are used primarily for

1	syndrome (TSS). Several extracellular products stimulate specific antibody responses useful for serodiagnosis of recent streptococcal infection. Tests for these antibodies are used primarily for detection of preceding streptococcal infection in cases of suspected ARF or PSGN.

1	CLINICAL MANIFESTATIONS Pharyngitis Although seen in patients of all ages, GAS pharyngitis is one of the most common bacterial infections of childhood, accounting for 20–40% of all cases of exudative pharyngitis in children; it is rare among those under the age of 3. Younger children may manifest streptococcal infection with a syndrome of fever, malaise, and lymphadenopathy without exudative pharyngitis. Infection is acquired through contact with another individual carrying the organism. Respiratory droplets are the usual mechanism of spread, although other routes, including food-borne outbreaks, have been well described. The incubation period is 1–4 days. Symptoms include sore throat, fever and chills, malaise, and sometimes abdominal complaints and vomiting, particularly in children. Both symptoms and signs are quite variable, ranging from mild throat discomfort with minimal physical findings to high fever and severe sore throat associated with intense erythema and swelling of the

1	symptoms and signs are quite variable, ranging from mild throat discomfort with minimal physical findings to high fever and severe sore throat associated with intense erythema and swelling of the pharyngeal mucosa and the presence of purulent exudate over the posterior pharyngeal wall and tonsillar pillars. Enlarged, tender anterior cervical lymph nodes commonly accompany exudative pharyngitis.

1	The differential diagnosis of streptococcal pharyngitis includes the many other bacterial and viral etiologies (Table 173-2). Streptococcal infection is an unlikely cause when symptoms and signs suggestive of viral infection are prominent (conjunctivitis, coryza, cough, hoarseness, or discrete ulcerative lesions of the buccal or pharyngeal mucosa). Because of the range of clinical presentations of streptococcal pharyngitis and the large number of other agents that can produce the same clinical picture, diagnosis of streptococcal pharyngitis on clinical grounds alone is not reliable. The throat culture remains the diagnostic gold standard. Culture of a throat specimen that is properly collected (i.e., by vigorous rubbing of a sterile swab over both tonsillar pillars) and properly processed is the most sensitive and specific means of definitive diagnosis. A rapid diagnostic kit for latex agglutination or enzyme immunoassay of swab specimens is a useful adjunct to throat culture. While

1	is the most sensitive and specific means of definitive diagnosis. A rapid diagnostic kit for latex agglutination or enzyme immunoassay of swab specimens is a useful adjunct to throat culture. While precise figures on sensitivity and specificity vary, rapid diagnostic kits generally are >95% specific. Thus a positive result can be relied upon for definitive diagnosis and eliminates the need for throat culture. However, because rapid diagnostic tests are less sensitive than throat culture (relative sensitivity in comparative studies, 55–90%), a negative result should be confirmed by throat culture.

1	In the usual course of uncomplicated streptococcal pharyngitis, symptoms resolve after 3–5 days. The course is shortened little by treatment, which is given primarily to prevent suppurative complications and ARF. Prevention of ARF depends on eradication of the organism from the pharynx, not simply on resolution of symptoms, Common cold Common cold Pharyngoconjunctival fever Influenza Cold, croup Herpangina, hand-foot-and-mouth Group A streptococci Pharyngitis, scarlet fever Group C or G streptococci Pharyngitis Mixed anaerobes Vincent’s angina Arcanobacterium haemolyticum Pharyngitis, scarlatiniform rash Neisseria gonorrhoeae Pharyngitis Treponema pallidum Secondary syphilis Francisella tularensis Pharyngeal tularemia Corynebacterium diphtheriae Diphtheria Yersinia enterocolitica Pharyngitis, enterocolitis Yersinia pestis Plague Chlamydiae

1	Chlamydia pneumoniae Bronchitis, pneumonia Chlamydia psittaci Psittacosis Mycoplasmas Mycoplasma pneumoniae Bronchitis, pneumonia and requires 10 days of penicillin treatment (Table 173-3). A first-generation cephalosporin, such as cephalexin or cefadroxil, may be substituted for penicillin in cases of penicillin allergy if the nature of the allergy is not an immediate hypersensitivity reaction (anaphylaxis or urticaria) or another potentially life-threatening manifestation (e.g., severe rash and fever).

1	aPenicillin allergy: A first-generation cephalosporin, such as cephalexin or cefadroxil, may be substituted for penicillin in cases of penicillin allergy if the nature of the allergy is not an immediate hypersensitivity reaction (anaphylaxis or urticaria) or another potentially life-threatening manifestation (e.g., severe rash and fever). Alternative agents for oral therapy are erythromycin (10 mg/kg PO qid, up to a maximum of 250 mg per dose) and azithromycin (a 5-day course at a dose of 12 mg/kg once daily, up to a maximum of 500 mg/d). Vancomycin is an alternative for parenteral therapy. bEfficacy unproven, but recommended by several experts. See text for discussion.

1	Alternative agents are erythromycin and azithromycin. Azithromycin is more expensive but offers the advantages of better gastrointestinal tolerability, once-daily dosing, and a 5-day treatment course. Resistance to erythromycin and other macrolides is common among isolates from several countries, including Spain, Italy, Finland, Japan, and Korea. Macrolide resistance may be becoming more prevalent elsewhere with the increasing use of this class of antibiotics. In areas with resistance rates exceeding 5–10%, macrolides should be avoided unless results of susceptibility testing are known. Follow-up culture after treatment is no longer routinely recommended but may be warranted in selected cases, such as those involving patients or families with frequent streptococcal infections or those occurring in situations in which the risk of ARF is thought to be high (e.g., when cases of ARF have recently been reported in the community).

1	complications Suppurative complications of streptococcal pharyngitis have become uncommon with the widespread use of antibiotics for most symptomatic cases. These complications result from the spread of infection from the pharyngeal mucosa to deeper tissues by direct extension or by the hematogenous or lymphatic route and may include cervical lymphadenitis, peritonsillar or retropharyngeal abscess, sinusitis, otitis media, meningitis, bacteremia, endocarditis, and pneumonia. Local complications, such as peritonsillar or parapharyngeal abscess formation, should be considered in a patient with unusually severe or prolonged symptoms or localized pain associated with high fever and a toxic appearance. Nonsuppurative complications include ARF (Chap. 381) and PSGN (Chap. 338), both of which are thought to result from immune responses to streptococcal infection. Penicillin treatment of streptococcal pharyngitis has been shown to reduce the likelihood of ARF but not that of PSGN.

1	bacteriologic treatment failure and tHe asymptomatic carrier state Surveillance cultures have shown that up to 20% of individuals in certain populations may have asymptomatic pharyngeal colonization with GAS. There are no definitive guidelines for management of these asymptomatic carriers or of asymptomatic patients who still have a positive throat culture after a full course of treatment for symptomatic pharyngitis. A reasonable course of action is to give a single 10-day course of penicillin for symptomatic pharyngitis and, if positive cultures persist, not to re-treat unless symptoms recur. Studies of the natural history of streptococcal carriage and infection have shown that the risk both of developing ARF and of transmitting infection to others is substantially lower among asymptomatic carriers than among individuals with symptomatic pharyngitis. Therefore, overly aggressive attempts to eradicate carriage probably are not justified under most circumstances. An exception is the

1	carriers than among individuals with symptomatic pharyngitis. Therefore, overly aggressive attempts to eradicate carriage probably are not justified under most circumstances. An exception is the situation in which an asymptomatic carrier is a potential source of infection to others. Outbreaks of food-borne infection and nosocomial puerperal infection have been traced to asymptomatic carriers who may harbor the organisms in the throat, vagina, or anus or on the skin.

1	When a carrier is transmitting infection to others, attempts to eradicate carriage are warranted. Data are limited on the best regimen to clear GAS after penicillin alone has failed. Regimens reported to have efficacy superior to that of penicillin alone for eradication of carriage include (1) oral clindamycin (7 mg/kg; 300 mg maximum) three times daily for 10 days or (2) penicillin (as recommended for treatment of pharyngitis in Table 173-3) plus oral rifampin (10 mg/kg; 300 mg maximum) twice daily for the first 4 days of treatment. A 10-day course of oral vancomycin (250 mg four times daily) and rifampin (600 mg twice daily) has eradicated rectal colonization. Scarlet Fever Scarlet fever consists of streptococcal infection, usually pharyngitis, accompanied by a characteristic rash (Fig. 173-2).

1	Scarlet Fever Scarlet fever consists of streptococcal infection, usually pharyngitis, accompanied by a characteristic rash (Fig. 173-2). FIGURE 173-2 Scarlet fever exanthem. Finely punctate erythema has become confluent (scarlatiniform); petechiae can occur and have a linear configuration within the exanthem in body folds (Pastia’s lines). (From Fitzpatrick, Johnson, Wolff: Color Atlas and Synopsis of Clinical Dermatology, 4th ed, New York, McGraw-Hill, 2001, with permission.)

1	The rash arises from the effects of one of several toxins, currently designated streptococcal pyrogenic exotoxins and previously known as erythrogenic or scarlet fever toxins. In the past, scarlet fever was thought to reflect infection of an individual lacking toxin-specific immunity with a toxin-producing strain of GAS. Susceptibility to scarlet fever was correlated with results of the Dick test, in which a small amount of erythrogenic toxin injected intradermally produced local erythema in susceptible individuals but elicited no reaction in those with specific immunity. Subsequent studies have suggested that development of the scarlet fever rash may reflect a hypersensitivity reaction requiring prior exposure to the toxin. For reasons that are not clear, scarlet fever has become less common in recent years, although strains of GAS that produce pyrogenic exotoxins continue to be prevalent in the population. The symptoms of scarlet fever are the same as those of pharyngitis alone. The

1	in recent years, although strains of GAS that produce pyrogenic exotoxins continue to be prevalent in the population. The symptoms of scarlet fever are the same as those of pharyngitis alone. The rash typically begins on the first or second day of illness over the upper trunk, spreading to involve the extremities but sparing the palms and soles. The rash is made up of minute papules, giving a characteristic “sandpaper” feel to the skin. Associated findings include circumoral pallor, “strawberry tongue” (enlarged papillae on a coated tongue, which later may become denuded), and accentuation of the rash in skinfolds (Pastia’s lines). Subsidence of the rash in 6–9 days is followed after several days by desquamation of the palms and soles. The differential diagnosis of scarlet fever includes other causes of fever and generalized rash, such as measles and other viral exanthems, Kawasaki disease, TSS, and systemic allergic reactions (e.g., drug eruptions).

1	Skin and Soft Tissue Infections GAS—and occasionally other streptococcal species—can cause a variety of infections involving the skin, subcutaneous tissues, muscles, and fascia. While several clinical syndromes offer a useful means for classification of these infections, not all cases fit exactly into one category. The classic syndromes are general guides to predicting the level of tissue involvement in a particular patient, the probable clinical course, and the likelihood that surgical intervention or aggressive life support will be required.

1	impetigo (pyoderma) Impetigo, a superficial infection of the skin, is caused primarily by GAS and occasionally by other streptococci or Staphylococcus aureus. Impetigo is seen most often in young children, tends to occur during warmer months, and is more common in semitropical or tropical climates than in cooler regions. Infection is more common among children living under conditions of poor hygiene. Prospective studies have shown that colonization of unbroken skin with GAS precedes clinical infection. Minor trauma, such as a scratch or an insect bite, may then serve to inoculate organisms into the skin. Impetigo is best prevented, therefore, by attention to adequate hygiene. The usual sites of involvement are the face (particularly around the nose and mouth) and the legs, although lesions may occur at other locations. Individual lesions begin as red papules, which evolve quickly into vesicular and then pustular lesions that break down and coalesce to form characteristic

1	lesions may occur at other locations. Individual lesions begin as red papules, which evolve quickly into vesicular and then pustular lesions that break down and coalesce to form characteristic honeycomb-like crusts (Fig. 173-3). Lesions generally are not painful, and patients do not appear ill. Fever is not a feature of impetigo and, if present, suggests either infection extending to deeper tissues or another diagnosis. The classic presentation of impetigo usually poses little diagnostic difficulty. Cultures of impetiginous lesions often yield S. aureus as well as GAS. In almost all cases, streptococci are isolated initially and staphylococci appear later, presumably as secondary colonizing flora. In the past, penicillin was nearly always effective against these infections. However, an increasing frequency of penicillin treatment failure suggests that S. aureus may have become more prominent as a cause of impetigo. Bullous impetigo due to S. aureus is distinguished from typical

1	an increasing frequency of penicillin treatment failure suggests that S. aureus may have become more prominent as a cause of impetigo. Bullous impetigo due to S. aureus is distinguished from typical streptococcal infection by more extensive, bullous lesions that break down and leave thin paper-like crusts instead of the thick amber crusts of streptococcal impetigo. Other skin lesions that may be confused with impetigo include herpetic lesions—either those of orolabial herpes simplex or those of chickenpox or zoster. Herpetic lesions can generally be distinguished by their appearance as more discrete, grouped vesicles and by a positive Tzanck test. In difficult cases, cultures of vesicular fluid should yield GAS in impetigo and the responsible virus in herpesvirus infections.

1	FIGURE 173-3 Impetigo contagiosa is a superficial streptococcal or Staphylococcus aureus infection consisting of honey-colored crusts and erythematous weeping erosions. Occasionally, bullous lesions may be seen. (Courtesy of Mary Spraker, MD; with permission.)

1	Treatment of streptococcal impetigo is the same as that for streptococcal pharyngitis. In view of evidence that S. aureus has become a relatively frequent cause of impetigo, empirical regimens should cover both streptococci and S. aureus. For example, either dicloxacillin or cephalexin can be given at a dose of 250 mg four times daily for 10 days. Topical mupirocin ointment is also effective. Culture may be indicated to rule out methicillin-resistant S. aureus, especially if the response to empirical treatment is unsatisfactory. ARF is not a sequela to streptococcal skin infections, although PSGN may follow either skin or throat infection. The reason for this difference is not known. One hypothesis is that the immune response necessary 967 for development of ARF occurs only after infection of the pharyngeal mucosa. In addition, the strains of GAS that cause pharyngitis are generally of different M protein types than those associated with skin infections; thus the strains that cause

1	of the pharyngeal mucosa. In addition, the strains of GAS that cause pharyngitis are generally of different M protein types than those associated with skin infections; thus the strains that cause pharyngitis may have rheumatogenic potential, while the skin-infecting strains may not.

1	cellulitis Inoculation of organisms into the skin may lead to cellulitis: infection involving the skin and subcutaneous tissues. The portal of entry may be a traumatic or surgical wound, an insect bite, or any other break in skin integrity. Often, no entry site is apparent. One form of streptococcal cellulitis, erysipelas, is characterized by a bright red appearance of the involved skin, which forms a plateau sharply demarcated from surrounding normal skin (Fig. 173-4). The lesion is warm to the touch, may be tender, and appears shiny and swollen. The skin often has a peau d’orange texture, which is thought to reflect involvement of superficial lymphatics; superficial blebs or bullae may form, usually 2–3 days after onset. The lesion typically develops over a few hours and is associated with fever and chills. Erysipelas tends to occur on the malar area of the face (often with extension over the bridge of the nose to the contralateral malar region) and the lower extremities. After one

1	fever and chills. Erysipelas tends to occur on the malar area of the face (often with extension over the bridge of the nose to the contralateral malar region) and the lower extremities. After one episode, recurrence at the same site—sometimes years later— is not uncommon. Classic cases of erysipelas, with typical features, are almost always due to β-hemolytic streptococci, usually GAS and occasionally group C or G. Often, however, the appearance of streptococcal cellulitis is not sufficiently distinctive to permit a specific diagnosis on clinical grounds. The area involved may not be typical for erysipelas, the lesion may be less intensely red than usual and may fade into surrounding skin, and/or the patient may appear only mildly ill. In such cases, it is prudent to broaden the spectrum of empirical antimicrobial therapy to include other pathogens, particularly S. aureus, that can produce cellulitis with the same appearance. Staphylococcal infection should be suspected if cellulitis

1	of empirical antimicrobial therapy to include other pathogens, particularly S. aureus, that can produce cellulitis with the same appearance. Staphylococcal infection should be suspected if cellulitis develops around a wound or an ulcer.

1	Streptococcal cellulitis tends to develop at anatomic sites in which normal lymphatic drainage has been disrupted, such as sites of prior cellulitis, the arm ipsilateral to a mastectomy and axillary lymph node dissection, a lower extremity previously involved in deep venous thrombosis or chronic lymphedema, or the leg from which a saphenous vein has been harvested for coronary artery bypass grafting. The organism may enter via a dermal breach some distance from the eventual site of clinical cellulitis. For example, some patients with recurrent leg cellulitis following saphenous vein removal stop having recurrent episodes only after treatment of tinea pedis on the affected FIGURE 173-4 Erysipelas is a streptococcal infection of the superficial dermis and consists of well-demarcated, erythematous, edematous, warm plaques.

1	FIGURE 173-4 Erysipelas is a streptococcal infection of the superficial dermis and consists of well-demarcated, erythematous, edematous, warm plaques. 968 extremity. Fissures in the skin presumably serve as a portal of entry for streptococci, which then produce infection more proximally in the leg at the site of previous injury. Streptococcal cellulitis may also involve recent surgical wounds. GAS is among the few bacterial pathogens that typically produce signs of wound infection and surrounding cellulitis within the first 24 h after surgery. These wound infections are usually associated with a thin exudate and may spread rapidly, either as cellulitis in the skin and subcutaneous tissue or as a deeper tissue infection (see below). Streptococcal wound infection or localized cellulitis may also be associated with lymphangitis, manifested by red streaks extending proximally along superficial lymphatics from the infection site. See Table 173-3 and Chap. 156.

1	deep soft-tissue infections Necrotizing fasciitis (hemolytic streptococcal gangrene) involves the superficial and/or deep fascia investing the muscles of an extremity or the trunk. The source of the infection is either the skin, with organisms introduced into tissue through trauma (sometimes trivial), or the bowel flora, with organisms released during abdominal surgery or from an occult enteric source, such as a diverticular or appendiceal abscess. The inoculation site may be inapparent and is often some distance from the site of clinical involvement; e.g., the introduction of organisms via minor trauma to the hand may be associated with clinical infection of the tissues overlying the shoulder or chest. Cases associated with the bowel flora are usually polymicrobial, involving a mixture of anaerobic bacteria (such as Bacteroides fragilis or anaerobic streptococci) and facultative organisms (usually gram-negative bacilli). Cases unrelated to contamination from bowel organisms are most

1	anaerobic bacteria (such as Bacteroides fragilis or anaerobic streptococci) and facultative organisms (usually gram-negative bacilli). Cases unrelated to contamination from bowel organisms are most commonly caused by GAS alone or in combination with other organisms (most often S. aureus). Overall, GAS is implicated in ~60% of cases of necrotizing fasciitis. The onset of symptoms is usually quite acute and is marked by severe pain at the site of involvement, malaise, fever, chills, and a toxic appearance. The physical findings, particularly early on, may not be striking, with only minimal erythema of the overlying skin. Pain and tenderness are usually severe. In contrast, in more superficial cellulitis, the skin appearance is more abnormal, but pain and tenderness are only mild or moderate. As the infection progresses (often over several hours), the severity and extent of symptoms worsen, and skin changes become more evident, with the appearance of dusky or mottled erythema and edema.

1	As the infection progresses (often over several hours), the severity and extent of symptoms worsen, and skin changes become more evident, with the appearance of dusky or mottled erythema and edema. The marked tenderness of the involved area may evolve into anesthesia as the spreading inflammatory process produces infarction of cutaneous nerves.

1	Although myositis is more commonly due to S. aureus infection, GAS occasionally produces abscesses in skeletal muscles (streptococcal myositis), with little or no involvement of the surrounding fascia or overlying skin. The presentation is usually subacute, but a fulminant form has been described in association with severe systemic toxicity, bacteremia, and a high mortality rate. The fulminant form may reflect the same basic disease process seen in necrotizing fasciitis, but with the necrotizing inflammatory process extending into the muscles themselves rather than remaining limited to the fascial layers.

1	Once necrotizing fasciitis is suspected, early surgical exploration is both diagnostically and therapeutically indicated. Surgery reveals necrosis and inflammatory fluid tracking along the fascial planes above and between muscle groups, without involvement of the muscles themselves. The process usually extends beyond the area of clinical involvement, and extensive debridement is required. Drainage and debridement are central to the management of necrotizing fasciitis; antibiotic treatment is a useful adjunct (Table 173-3), but surgery is life-saving. Treatment for streptococcal myositis consists of surgical drainage—usually by an open procedure that permits evaluation of the extent of infection and ensures adequate debridement of involved tissues—and high-dose penicillin (Table 173-3).

1	Pneumonia and Empyema GAS is an occasional cause of pneumonia, generally in previously healthy individuals. The onset of symptoms may be abrupt or gradual. Pleuritic chest pain, fever, chills, and dyspnea are the characteristic manifestations. Cough is usually present but may not be prominent. Approximately one-half of patients with GAS pneumonia have an accompanying pleural effusion. In contrast to the sterile parapneumonic effusions typical of pneumococcal pneumonia, those complicating streptococcal pneumonia are almost always infected. The empyema fluid is usually visible by chest radiography on initial presentation, and its volume may increase rapidly. These pleural collections should be drained early, as they tend to become loculated rapidly, resulting in a chronic fibrotic reaction that may require thoracotomy for removal.

1	Bacteremia, Puerperal Sepsis, and Streptococcal Toxic Shock Syndrome GAS bacteremia is usually associated with an identifiable local infection. Bacteremia occurs rarely with otherwise uncomplicated pharyngitis, occasionally with cellulitis or pneumonia, and relatively frequently with necrotizing fasciitis. Bacteremia without an identified source raises the possibility of endocarditis, an occult abscess, or osteomyelitis. A variety of focal infections may arise secondarily from streptococcal bacteremia, including endocarditis, meningitis, septic arthritis, osteomyelitis, peritonitis, and visceral abscesses. GAS is occasionally implicated in infectious complications of childbirth, usually endometritis and associated bacteremia. In the preantibiotic era, puerperal sepsis was commonly caused by GAS; currently, it is more often caused by GBS. Several nosocomial outbreaks of puerperal GAS infection have been traced to an asymptomatic carrier, usually someone present at delivery. The site of

1	GAS; currently, it is more often caused by GBS. Several nosocomial outbreaks of puerperal GAS infection have been traced to an asymptomatic carrier, usually someone present at delivery. The site of carriage may be the skin, throat, anus, or vagina.

1	Beginning in the late 1980s, several reports described patients with GAS infections associated with shock and multisystem organ failure. This syndrome was called streptococcal TSS because it shares certain features with staphylococcal TSS. In 1993, a case definition for streptococcal TSS was formulated (Table 173-4). The general features of the illness include fever, hypotension, renal impairment, and respiratory distress syndrome. Various types of rash have been described, but rash usually does not develop. Laboratory abnormalities include a marked shift to the left in the white blood cell differential, with many immature granulocytes; hypocalcemia; hypoalbuminemia; and thrombocytopenia, which usually becomes more pronounced on the second or third day of illness. In contrast to patients with staphylococcal TSS, the majority with streptococcal TSS are bacteremic. The most common associated infection is a soft tissue infection—necrotizing

1	I. Isolation of group A streptococci (Streptococcus pyogenes) A. From a normally sterile site B. From a nonsterile site II. Clinical signs of severity A. B. ≥2 of the following signs 1. 2. 3. 4. 5. 6. Soft tissue necrosis, including necrotizing fasciitis or myositis; or gangrene aAn illness fulfilling criteria IA, IIA, and IIB is defined as a definite case. An illness fulfilling criteria IB, IIA, and IIB is defined as a probable case if no other etiology for the illness is identified. Source: Modified from Working Group on Severe Streptococcal Infections: JAMA 269:390, 1993.

1	fasciitis, myositis, or cellulitis—although a variety of other associated local infections have been described, including pneumonia, peritonitis, osteomyelitis, and myometritis. Streptococcal TSS is associated with a mortality rate of ≥30%, with most deaths secondary to shock and respiratory failure. Because of its rapidly progressive and lethal course, early recognition of the syndrome is essential. Patients should receive aggressive supportive care (fluid resuscitation, pressors, and mechanical ventilation) in addition to antimicrobial therapy and, in cases associated with necrotizing fasciitis, surgical debridement. Exactly why certain patients develop this fulminant syndrome is not known. Early studies of the streptococcal strains isolated from these patients demonstrated a strong association with the production of pyrogenic exotoxin A. This association has been inconsistent in subsequent case series. Pyrogenic exotoxin A and several other streptococcal exotoxins act as

1	strong association with the production of pyrogenic exotoxin A. This association has been inconsistent in subsequent case series. Pyrogenic exotoxin A and several other streptococcal exotoxins act as superantigens to trigger release of inflammatory cytokines from T lymphocytes. Fever, shock, and organ dysfunction in streptococcal TSS may reflect, in part, the systemic effects of superantigen-mediated cytokine release.

1	In light of the possible role of pyrogenic exotoxins or other streptococcal toxins in streptococcal TSS, treatment with clindamycin has been advocated by some authorities (Table 173-3), who argue that, through its direct action on protein synthesis, clindamycin is more effective in rapidly terminating toxin production than is penicillin—a cell-wall agent. Support for this view comes from studies of an experimental model of streptococcal myositis, in which mice given clindamycin had a higher rate of survival than those given penicillin. Comparable data on the treatment of human infections are not available, although retrospective analysis has suggested a better outcome when patients with invasive soft-tissue infection are treated with clindamycin rather than with cell wall-active antibiotics. Although clindamycin resistance in GAS is uncommon (<2% among U.S. isolates), it has been documented. Thus, if clindamycin is used for initial treatment of a critically ill patient, penicillin

1	Although clindamycin resistance in GAS is uncommon (<2% among U.S. isolates), it has been documented. Thus, if clindamycin is used for initial treatment of a critically ill patient, penicillin should be given as well until the antibiotic susceptibility of the streptococcal isolate is known. IV immunoglobulin has been used as adjunctive therapy for streptococcal TSS (Table 173-3). Pooled immunoglobulin preparations contain antibodies capable of neutralizing the effects of streptococcal toxins. Anecdotal reports and case series have suggested favorable clinical responses to IV immunoglobulin, but no adequately powered, prospective, controlled trials have been reported.

1	No vaccine against GAS is commercially available. A formulation that consists of recombinant peptides containing epitopes of 26 M-protein types has undergone phase 1 and 2 testing in volunteers. Early results indicate that the vaccine is well tolerated and elicits type-specific antibody responses. Vaccines based on a conserved region of M protein or on a mixture of other conserved GAS protein antigens are in earlier stages of development.

1	Household contacts of individuals with invasive GAS infection (e.g., bacteremia, necrotizing fasciitis, or streptococcal TSS) are at greater risk of invasive infection than the general population. Asymptomatic pharyngeal colonization with GAS has been detected in up to 25% of persons with >4 h/d of same-room exposure to an index case. However, antibiotic prophylaxis is not routinely recommended for contacts of patients with invasive disease because such an approach (if effective) would require treatment of hundreds of contacts to prevent a single case.

1	Group C and group G streptococci are β-hemolytic bacteria that occasionally cause human infections similar to those caused by GAS. Strains that form small colonies on blood agar (<0.5 mm) are generally members of the Streptococcus milleri (Streptococcus intermedius, Streptococcus anginosus) group (see “Viridans Streptococci,” below). 969 Large-colony group C and G streptococci of human origin are now considered a single species, Streptococcus dysgalactiae subspecies equisimilis. These organisms have been associated with pharyngitis, cellulitis and soft tissue infections, pneumonia, bacteremia, endocarditis, and septic arthritis. Puerperal sepsis, meningitis, epidural abscess, intraabdominal abscess, urinary tract infection, and neonatal sepsis have also been reported. Group C or G streptococcal bacteremia most often affects elderly or chronically ill patients and, in the absence of obvious local infection, is likely to reflect endocarditis. Septic arthritis, sometimes involving

1	bacteremia most often affects elderly or chronically ill patients and, in the absence of obvious local infection, is likely to reflect endocarditis. Septic arthritis, sometimes involving multiple joints, may complicate endocarditis or develop in its absence. Distinct streptococcal species of Lancefield group C cause infections in domesticated animals, especially horses and cattle; some human infections are acquired through contact with animals or consumption of unpasteurized milk. These zoonotic organisms include Streptococcus equi subspecies zooepidemicus and S. equi subspecies equi.

1	Penicillin is the drug of choice for treatment of group C or G streptococcal infections. Antibiotic treatment is the same as for similar syndromes due to GAS (Table 173-3). Patients with bacteremia or septic arthritis should receive IV penicillin (2–4 mU every 4 h). All group C and G streptococci are sensitive to penicillin; nearly all are inhibited in vitro by concentrations of ≤0.03 μg/mL. Occasional isolates exhibit tolerance: although inhibited by low concentrations of penicillin, they are killed only by significantly higher concentrations. The clinical significance of tolerance is unknown. Because of the poor clinical response of some patients to penicillin alone, the addition of gentamicin (1 mg/kg every 8 h for patients with normal renal function) is recommended by some authorities for treatment of endocarditis or septic arthritis due to group C or G streptococci; however, combination therapy has not been shown to be superior to penicillin treatment alone. Patients with joint

1	for treatment of endocarditis or septic arthritis due to group C or G streptococci; however, combination therapy has not been shown to be superior to penicillin treatment alone. Patients with joint infections often require repeated aspiration or open drainage and debridement for cure; the response to treatment may be slow, particularly in debilitated patients and those with involvement of multiple joints. Infection of prosthetic joints almost always requires prosthesis removal in addition to antibiotic therapy.

1	Identified first as a cause of mastitis in cows, streptococci belonging to Lancefield’s group B have since been recognized as a major cause of sepsis and meningitis in human neonates. GBS is also a frequent cause of peripartum fever in women and an occasional cause of serious infection in nonpregnant adults. Since the widespread institution of prenatal screening for GBS in the 1990s, the incidence of neonatal infection per 1000 live births has fallen from ~2–3 cases to ~0.6 case. During the same period, GBS infection in adults with underlying chronic illnesses has become more common; adults now account for a larger proportion of invasive GBS infections than do newborns. Lancefield group B consists of a single species, S. agalactiae, which is definitively identified with specific antiserum to the group B cell wall–associated carbohydrate antigen. A streptococcal isolate can be classified presumptively as GBS on the basis of biochemical tests, including hydrolysis of sodium hippurate

1	to the group B cell wall–associated carbohydrate antigen. A streptococcal isolate can be classified presumptively as GBS on the basis of biochemical tests, including hydrolysis of sodium hippurate (in which 99% of isolates are positive), hydrolysis of bile esculin (in which 99–100% are negative), bacitracin susceptibility (in which 92% are resistant), and production of CAMP factor (in which 98–100% are positive). CAMP factor is a phospholipase produced by GBS that causes synergistic hemolysis with β lysin produced by certain strains of S. aureus. Its presence can be demonstrated by cross-streaking of the test isolate and an appropriate staphylococcal strain on a blood agar plate. GBS organisms causing human infections are encapsulated by one of ten antigenically distinct polysaccharides. The capsular polysaccharide is an important virulence factor. Antibodies to the capsular polysaccharide afford protection against GBS of the same (but not of a different) capsular type.

1	Two general types of GBS infection in infants are defined by the age of the patient at presentation. Early-onset infections occur within the first week of life, with a median age of 20 h at onset. Approximately half of these infants have signs of GBS disease at birth. The infection is acquired during or shortly before birth from the colonized maternal genital tract. Surveillance studies have shown that 5–40% of women are vaginal or rectal carriers of GBS. Approximately 50% of infants delivered vaginally by carrier mothers become colonized, although only 1–2% develop clinically evident infection. Prematurity, prolonged labor, obstetric complications, and maternal fever are risk factors for early-onset infection. The presentation of early-onset infection is the same as that of other forms of neonatal sepsis. Typical findings include respiratory distress, lethargy, and hypotension. Essentially all infants with early-onset disease are bacteremic, one-third to one-half have pneumonia

1	of neonatal sepsis. Typical findings include respiratory distress, lethargy, and hypotension. Essentially all infants with early-onset disease are bacteremic, one-third to one-half have pneumonia and/or respiratory distress syndrome, and one-third have meningitis.

1	Late-onset infections occur in infants 1 week to 3 months old and, in rare instances, in older infants (mean age at onset, 3–4 weeks). The infecting organism may be acquired during delivery (as in early-onset cases) or during later contact with a colonized mother, nursery personnel, or another source. Meningitis is the most common manifestation of late-onset infection and in most cases is associated with a strain of capsular type III. Infants present with fever, lethargy or irritability, poor feeding, and seizures. The various other types of late-onset infection include bacteremia without an identified source, osteomyelitis, septic arthritis, and facial cellulitis associated with submandibular or preauricular adenitis.

1	Penicillin is the agent of choice for all GBS infections. Empirical broad-spectrum therapy for suspected bacterial sepsis, consisting of ampicillin and gentamicin, is generally administered until culture results become available. If cultures yield GBS, many pediatricians continue to administer gentamicin, along with ampicillin or penicillin, for a few days until clinical improvement becomes evident. Infants with bacteremia or soft tissue infection should receive penicillin at a dosage of 200,000 units/kg per day in divided doses. For meningitis, infants ≤7 days of age should receive 250,000–450,000 units/kg per day in three divided doses; infants >7 days of age should receive 450,000–500,000 units/kg per day in four divided doses. Meningitis should be treated for at least 14 days because of the risk of relapse with shorter courses.

1	The incidence of GBS infection is unusually high among infants of women with risk factors: preterm delivery, early rupture of membranes (>24 h before delivery), prolonged labor, fever, or chorioamnionitis. Because the usual source of the organisms infecting a neonate is the mother’s birth canal, efforts have been made to prevent GBS infections by the identification of high-risk carrier mothers and their treatment with various forms of antibiotic prophylaxis or immunoprophylaxis. Prophylactic administration of ampicillin or penicillin to such patients during delivery reduces the risk of infection in the newborn. This approach has been hampered by logistical difficulties in identifying colonized women before delivery; the results of vaginal cultures early in pregnancy are poor predictors of carrier status at delivery. The CDC recommends screening for anogenital colonization at 35–37 weeks of pregnancy by a swab culture of the lower vagina and anorectum; intrapartum chemoprophylaxis is

1	carrier status at delivery. The CDC recommends screening for anogenital colonization at 35–37 weeks of pregnancy by a swab culture of the lower vagina and anorectum; intrapartum chemoprophylaxis is recommended for culture-positive women and for women who, regardless of culture status, have previously given birth to an infant with GBS infection or have a history of GBS bacteriuria during pregnancy. Women whose culture status is unknown and who develop premature labor (<37 weeks), prolonged rupture of membranes (>18 h), or intrapartum fever or who have a positive intrapartum nucleic acid amplification test for GBS should also receive intrapartum chemoprophylaxis. The recommended regimen for chemoprophylaxis is a loading dose of 5 million units of penicillin G followed by 2.5 million units every 4 h until delivery. Cefazolin is an alternative for women with a history of penicillin allergy who are thought not to be at high risk for anaphylaxis. For women with a history of immediate

1	every 4 h until delivery. Cefazolin is an alternative for women with a history of penicillin allergy who are thought not to be at high risk for anaphylaxis. For women with a history of immediate hypersensitivity, clindamycin may be substituted, but only if the colonizing isolate has been demonstrated to be susceptible. If susceptibility testing results are not available or indicate resistance, vancomycin should be used in this situation.

1	Treatment of all pregnant women who are colonized or have risk factors for neonatal infection will result in exposure of up to one-third of pregnant women and newborns to antibiotics, with the attendant risks of allergic reactions and selection for resistant organisms. Although still in the developmental stages, a GBS vaccine may ultimately offer a better solution to prevention. Because transplacental passage of maternal antibodies produces protective antibody levels in newborns, efforts are under way to develop a vaccine against GBS that can be given to childbearing-age women before or during pregnancy. Results of phase 1 clinical trials of GBS capsular polysaccharide–protein conjugate vaccines suggest that a multivalent conjugate vaccine would be safe and highly immunogenic.

1	The majority of GBS infections in otherwise healthy adults are related to pregnancy and parturition. Peripartum fever, the most common manifestation, is sometimes accompanied by symptoms and signs of endometritis or chorioamnionitis (abdominal distention and uterine or adnexal tenderness). Blood and vaginal swab cultures are often positive. Bacteremia is usually transitory but occasionally results in meningitis or endocarditis. Infections in adults that are not associated with the peripartum period generally involve individuals who are elderly or have an underlying chronic illness, such as diabetes mellitus or a malignancy. Among the infections that develop with some frequency in adults are cellulitis and soft tissue infection (including infected diabetic skin ulcers), urinary tract infection, pneumonia, endocarditis, and septic arthritis. Other reported infections include meningitis, osteomyelitis, and intraabdominal or pelvic abscesses. Relapse or recurrence of invasive infection

1	pneumonia, endocarditis, and septic arthritis. Other reported infections include meningitis, osteomyelitis, and intraabdominal or pelvic abscesses. Relapse or recurrence of invasive infection weeks to months after a first episode is documented in ~4% of cases.

1	GBS is less sensitive to penicillin than GAS, requiring somewhat higher doses. Adults with serious localized infections (pneumonia, pyelonephritis, abscess) should receive doses of ~12 million units of penicillin G daily; patients with endocarditis or meningitis should receive 18–24 million units per day in divided doses. Vancomycin is an acceptable alternative for penicillin-allergic patients. The main nonenterococcal group D streptococci that cause human infections were previously considered a single species, Streptococcus bovis. The organisms encompassed by S. bovis have been reclassified into two species, each of which has two subspecies: Streptococcus gallolyticus subspecies gallolyticus, S. gallolyticus subspecies pasteurianus, Streptococcus infantarius subspecies infantarius, and

1	S. infantarius subspecies coli. Endocarditis caused by these organisms is often associated with neoplasms of the gastrointestinal tract—most frequently, a colon carcinoma or polyp—but is also reported in association with other bowel lesions. When occult gastrointestinal lesions are carefully sought, abnormalities are found in >60% of patients with endocarditis due to S. gallolyticus or S. infantarius. In contrast to the enterococci, nonenterococcal group D streptococci like these organisms are reliably killed by penicillin as a single agent, and penicillin is the agent of choice for the infections they cause.

1	Consisting of multiple species of α-hemolytic streptococci, the viridans streptococci are a heterogeneous group of organisms that are important agents of bacterial endocarditis (Chap. 155). Several species of viridans streptococci, including Streptococcus salivarius, Streptococcus mitis, Streptococcus sanguis, and Streptococcus mutans, are part of the normal flora of the mouth, where they live in close association with the teeth and gingiva. Some species contribute to the development of dental caries. Previously known as Streptococcus morbillorum, Gemella morbillorum has been placed in a separate genus, along with Gemella haemolysans, on the basis of genetic-relatedness studies. These species resemble viridans streptococci with respect to habitat in the human host and associated infections.

1	The transient viridans streptococcal bacteremia induced by eating, toothbrushing, flossing, and other sources of minor trauma, together with adherence to biologic surfaces, is thought to account for the predilection of these organisms to cause endocarditis (see Fig. 155-1). Viridans streptococci are also isolated, often as part of a mixed flora, from sites of sinusitis, brain abscess, and liver abscess. Viridans streptococcal bacteremia occurs relatively frequently in neutropenic patients, particularly after bone marrow transplantation or high-dose chemotherapy for cancer. Some of these patients develop a sepsis syndrome with high fever and shock. Risk factors for viridans streptococcal bacteremia include chemotherapy with high-dose cytosine arabinoside, prior treatment with trimethoprimsulfamethoxazole or a fluoroquinolone, treatment with antacids or histamine antagonists, mucositis, and profound neutropenia.

1	The S. milleri group (also referred to as the S. intermedius or S. anginosus group) includes three species that cause human disease: S. intermedius, S. anginosus, and Streptococcus constellatus. These organisms are often considered viridans streptococci, although they differ somewhat from other viridans streptococci in both their hemolytic pattern (they may be α-, β-, or nonhemolytic) and the disease syndromes they cause. This group commonly produces suppurative infections, particularly abscesses of brain and abdominal viscera, and infections related to the oral cavity or respiratory tract, such as peritonsillar abscess, lung abscess, and empyema. Isolates from neutropenic patients with bacteremia are often resistant to penicillin; thus these patients should be treated presumptively with vancomycin until the results of susceptibility testing become available. Viridans streptococci isolated in other clinical settings usually are sensitive to penicillin.

1	Occasional isolates cultured from the blood of patients with endocarditis fail to grow when subcultured on solid media. These nutritionally variant streptococci require supplemental thiol compounds or active forms of vitamin B6 (pyridoxal or pyridoxamine) for growth in the laboratory. The nutritionally variant streptococci are generally grouped with the viridans streptococci because they cause similar types of infections. However, they have been reclassified on the basis of 16S ribosomal RNA sequence comparisons into two separate genera: Abiotrophia, with a single species (Abiotrophia defectiva), and Granulicatella, with three species associated with human infection (Granulicatella adiacens, Granulicatella para-adiacens, and Granulicatella elegans).

1	Treatment failure and relapse appear to be more common in cases of endocarditis due to nutritionally variant streptococci than in those due to the usual viridans streptococci. Thus the addition of gentamicin (1 mg/kg every 8 h for patients with normal renal func-971 tion) to the penicillin regimen is recommended for endocarditis due to the nutritionally variant organisms.

1	Streptococcus suis is an important pathogen in swine and has been reported to cause meningitis in humans, usually in individuals with occupational exposure to pigs. Strains of S. suis associated with human infections have generally reacted with Lancefield group R typing serum and sometimes with group D typing serum as well. Isolates may be α-or β-hemolytic and are sensitive to penicillin. Streptococcus iniae, a pathogen of fish, has been associated with infections in humans who have handled live or freshly killed fish. Cellulitis of the hand is the most common form of human infection, although bacteremia and endocarditis have been reported. Anaerobic streptococci, or peptostreptococci, are part of the normal flora of the oral cavity, bowel, and vagina. Infections caused by the anaerobic streptococci are discussed in Chap. 201. Cesar A. Arias, Barbara E. Murray

1	Enterococci have been recognized as potential human pathogens for more than a century, but only in recent years have these organisms acquired prominence as important causes of nosocomial infections. The ability of enterococci to survive and/or disseminate in the hospital environment and to acquire antibiotic resistance determinants makes the treatment of some enterococcal infections in critically ill patients a difficult challenge. Enterococci were first mentioned in the French literature in 1899; the “entérocoque” was found in the human gastrointestinal tract and was noted to have the potential to produce significant disease. Indeed, the first pathologic description of an enterococcal infection dates to the same year. A clinical isolate from a patient who died as a consequence of endocarditis was initially designated Micrococcus zymogenes, was later named Streptococcus faecalis subspecies zymogenes, and would now be classified as Enterococcus faecalis. The ability of this isolate to

1	was initially designated Micrococcus zymogenes, was later named Streptococcus faecalis subspecies zymogenes, and would now be classified as Enterococcus faecalis. The ability of this isolate to cause severe disease in both rabbits and mice illustrated its potential lethality in the appropriate settings.

1	Enterococci are gram-positive organisms. In clinical specimens, they are usually observed as single cells, diplococci, or short chains (Fig. 174-1), although long chains are noted with some strains. Enterococci were originally classified as streptococci because organisms of the two genera share many morphologic and phenotypic characteristics, including a generally negative catalase reaction. Only DNA hybridization studies and later 16S rRNA sequencing clearly demonstrated that enterococci should be grouped as a genus distinct from the streptococci. Nonetheless, unlike the majority of streptococci, enterococci hydrolyze esculin in the presence of 40% bile salts and grow at high salt concentrations (e.g., 6.5%) and at high temperatures (46°C). Enterococci are usually reported by the clinical laboratory to be nonhemolytic on the basis of their inability to lyse the ovine or bovine red blood cells (RBCs) commonly used in agar plates; however, some strains of E. faecalis do lyse RBCs from

1	laboratory to be nonhemolytic on the basis of their inability to lyse the ovine or bovine red blood cells (RBCs) commonly used in agar plates; however, some strains of E. faecalis do lyse RBCs from humans, horses, and rabbits. The majority of clinically relevant enterococcal species hydrolyze pyrrolidonyl-β-naphthylamide (PYR); this characteristic is helpful in differentiating enterococci from organisms of the Streptococcus gallolyticus group (formerly known as S. bovis), which includes

1	S. gallolyticus, Streptococcus pasteurianus, and Streptococcus infantarius, and from Leuconostoc species. Although at least 18 species of enterococci have been isolated from human infections, the overwhelming FIGURE 174-1 Gram’s stain of cultured blood from a patient with enterococcal bacteremia. Oval gram-positive bacterial cells are arranged as diplococci and short chains. (Courtesy of Audrey Wanger, PhD.) majority of cases are caused by two species, E. faecalis and Enterococcus faecium. Less frequently isolated species include Enterococcus gallinarum, Enterococcus durans, Enterococcus hirae, and Enterococcus avium.

1	Enterococci are normal inhabitants of the large bowel of human adults, although they usually make up <1% of the culturable intestinal microflora. In the healthy human gastrointestinal tract, enterococci are typical symbionts that coexist with other gastrointestinal bacteria; in fact, the utility of certain enterococcal strains as probiotics in the treatment of diarrhea suggests their possible role in maintaining the homeostatic equilibrium of the bowel. Enterococci are intrinsically resistant to a variety of commonly used antibacterial drugs. One of the most important factors that disrupts this equilibrium and promotes increased gastrointestinal colonization by enterococci is the administration of antimicrobial agents. In particular, antibiotics that are excreted in the bile and have broad-spectrum activity (e.g., certain cephalosporins that target anaerobes and gram-negative bacteria) are usually associated with the recovery of higher numbers of enterococci from feces. This increased

1	activity (e.g., certain cephalosporins that target anaerobes and gram-negative bacteria) are usually associated with the recovery of higher numbers of enterococci from feces. This increased colonization appears to be due not only to the simple enterococcal replacement in a “biologic niche” after the eradication of competing components of the flora, but also (at least in mice) to the suppression—upon reduction of the gram-negative microflora by antibiotics—of important immunologic signals (e.g., by the lectin RegIIIγ) that help keep enterococcal counts low in the normal human bowel. Several studies have shown that higher levels of gastrointestinal colonization are a critical factor in the pathogenesis of enterococcal infections. However, the mechanisms by which enterococci successfully colonize the bowel and gain access to the lymphatics and/or bloodstream remain incompletely understood.

1	Several vertebrate, worm, and insect models have been developed to study the role of possible pathogenic determinants in both E. faecalis and E. faecium. Three main groups of virulence factors may increase the ability of enterococci to colonize the gastrointestinal tract and/or cause disease. The first group, enterococcal secreted factors, are molecules released outside the bacterial cell that contribute to the process of infection. The best-studied of these molecules include enterococcal hemolysin/cytolysin and two enterococcal proteases (gelatinase and serine protease). Enterococcal cytolysin is a heterodimeric toxin produced by some strains of E. faecalis that is capable of lysing human RBCs as well as polymorphonuclear leukocytes and macrophages.

1	E. faecalis gelatinase and serine protease are thought to mediate virulence by several mechanisms, including the degradation of host tissues and the modification of critical components of the immune system. Mutants lacking the genes corresponding to these proteins are highly attenuated in experimental peritonitis, endocarditis, and endophthalmitis.

1	A second group of virulence factors, enterococcal surface components, are thought to contribute to bacterial attachment to extracellular matrix molecules in the human host. Several molecules on the surface of enterococci have been characterized and shown to play a role in the pathogenesis of enterococcal infections. Among the characterized adhesins is aggregation substance of E. faecalis, which mediates the attachment of bacterial cells to each other, thereby facilitating conjugative plasmid exchange. Several lines of evidence indicate that aggregation substance and enterococcal cytolysin act synergistically to increase the virulence potential of E. faecalis strains in experimental endocarditis. The surface protein adhesin of collagen of E. faecalis (Ace) and its E. faecium homologue (Acm) recognize adhesive matrix molecules (MSCRAMMs) involved in bacterial attachment to host proteins such as collagen, fibronectin, and fibrinogen; both Ace and Acm are important in the pathogenesis of

1	recognize adhesive matrix molecules (MSCRAMMs) involved in bacterial attachment to host proteins such as collagen, fibronectin, and fibrinogen; both Ace and Acm are important in the pathogenesis of experimental endocarditis. Pili of gram-positive bacteria have been shown to be important mediators of attachment to and invasion of host tissues and are considered potential targets for immunotherapy. Both E. faecalis and E. faecium have surface pili. Mutants of E. faecalis lacking pili are attenuated in biofilm production, experimental endocarditis, and urinary tract infections (UTIs). Other surface proteins that share structural homology with MSCRAMMs and appear to play a role in enterococcal attachment to the host and in virulence include the E. faecalis surface protein Esp and its E. faecium homologue Espfm, the second collagen adhesin of E. faecium (Scm), the surface proteins of E. faecium (Fms), SgrA (which binds to components of the basal lamina), and EcbA (which binds to collagen

1	homologue Espfm, the second collagen adhesin of E. faecium (Scm), the surface proteins of E. faecium (Fms), SgrA (which binds to components of the basal lamina), and EcbA (which binds to collagen type V). Additional surface components apparently associated with pathogenicity include the Elr protein (a protein from the WxL family) and polysaccharides, which are thought to interfere with phagocytosis of the organism by host immune cells. Some E. faecalis strains appear to harbor at least three distinct classes of capsular polysaccharide; some of these polysaccharides play a role in virulence and are potential targets for immunotherapy.

1	The third group of virulence factors has not been well characterized but consists of the E. faecalis stress protein Gls24, which has been associated with enterococcal resistance to bile salts and appears to be important in the pathogenesis of endocarditis, and the hylEfm-containing plasmids of E. faecium, which are transferable between strains and increase gastrointestinal colonization by E. faecium. In mouse peritonitis, acquisition of these plasmids increased the lethality of a commensal strain of E. faecium. Recently, a gene encoding a regulator of oxidative stress (AsrR) has been identified as an important virulence factor of E. faecium.

1	The ability to sequence bacterial genomes has increased our understanding of bacterial diversity, evolution, pathogenesis, and mechanisms of antibiotic resistance. The genome sequences of more than 560 enterococcal strains are currently available, and some have been entirely closed and annotated. Sequence analysis has shown that the genetic diversity of enterococci is related in large part to the acquisition of exogenous DNA and the mobilization of large chromosomal regions, resulting in recombination of the “core” genomes. In addition, analyses indicate that E. faecium harbors a malleable accessory genome incorporating a substantial content of exogenous elements, including DNA from phages. Indeed, a hospital-associated E. faecium clade that contains most clinical and outbreak-associated strains is the predominant genetic lineage circulating in hospitals around the world. This clade appears to be evolving rapidly, and genomic comparisons suggest that this lineage emerged 75 years

1	strains is the predominant genetic lineage circulating in hospitals around the world. This clade appears to be evolving rapidly, and genomic comparisons suggest that this lineage emerged 75 years ago—a time point that coincides with the introduction of antimicrobial drugs—and evolved from animal strains, not from human commensal isolates. An initial genomic separation within E. faecium appears to have occurred ~3000 years ago, simultaneous with urbanization and domestication of animals. This genomic information provides new clues with regard to the evolution of enterococci from commensal organisms to important nosocomial pathogens.

1	According to the National Healthcare Safety Network of the Centers for Disease Control and Prevention, enterococci are the second most common organisms (after staphylococci) isolated from hospital-associated infections in the United States. Although E. faecalis remains the predominant species recovered from nosocomial infections, the isolation of E. faecium has increased substantially in the past 20 years. In fact, E. faecium is now almost as common as E. faecalis as an etiologic agent of hospital-associated infections. This point is important, because E. faecium is by far the most resistant and challenging enterococcal species to treat; indeed, more than 80% of E. faecium isolates recovered in U.S. hospitals are resistant to vancomycin, and more than 90% are resistant to ampicillin (historically the most effective β-lactam agent against enterococci). Resistance to vancomycin and ampicillin in E. faecalis isolates is much less common (~7% and ~4%, respectively).

1	The dynamics of enterococcal transmission and dissemination in the hospital environment have been extensively studied, with a focus on vancomycin-resistant enterococci (VRE). These studies have revealed that VRE colonization of the gastrointestinal tract is a critical step in the natural history of enterococcal disease and that a substantial proportion of patients colonized with VRE remain colonized for prolonged periods (sometimes >1 year) and are more likely to develop an Enterococcus-related illness (e.g., bacteremia). The most important factors associated with VRE colonization and persistence in the gut include prolonged hospitalization; long courses of antibiotic therapy; hospitalization in long-term-care facilities, surgical units, and/or intensive care units; organ transplantation; renal failure (particularly in patients undergoing hemodialysis) and/or diabetes; high Acute Physiology and Chronic Health Evaluation (APACHE) scores; and physical proximity to patients infected or

1	renal failure (particularly in patients undergoing hemodialysis) and/or diabetes; high Acute Physiology and Chronic Health Evaluation (APACHE) scores; and physical proximity to patients infected or colonized with VRE or these patients’ rooms. Once a patient becomes colonized with VRE, several key factors are involved in the organisms’ dissemination in the hospital environment. VRE can survive exposure to heat and certain disinfectants and have been found on numerous inanimate objects in the hospital, including bed rails, medical equipment, doorknobs, gloves, telephones, and computer keyboards. Thus health care workers and the environment play pivotal roles in enterococcal transmission from patient to patient, and infection control measures are crucial in breaking the chain of transmission. Moreover, two meta-analyses have found that, independent of the patient’s clinical status, VRE infection increases the risk of death over that among individuals infected with a

1	chain of transmission. Moreover, two meta-analyses have found that, independent of the patient’s clinical status, VRE infection increases the risk of death over that among individuals infected with a glycopeptide-susceptible enterococcal strain.

1	The epidemiology of enterococcal disease and the emergence of VRE have followed slightly different trends in other parts of the world than in the United States. In Europe, the emergence of VRE in the mid-1980s was seen primarily in isolates recovered from animals and healthy humans rather than from hospitalized patients. The presence of VRE was associated with the use of the glycopeptide avoparcin as a growth promoter in animal feeds; this association prompted the European Union to ban the use of this compound in animal husbandry in 1996. However, after an initial decrease in the isolation of VRE from animals and humans, the prevalence of hospital-associated VRE infections has slowly increased in certain European countries, with important regional differences. For example, rates of vancomycin resistance among E. faecium clinical isolates in Europe are highest in Greece, the United Kingdom, and Portugal (10–30%), whereas rates in the Scandinavian countries and the Netherlands are <1%.

1	resistance among E. faecium clinical isolates in Europe are highest in Greece, the United Kingdom, and Portugal (10–30%), whereas rates in the Scandinavian countries and the Netherlands are <1%. These regional differences have been attributed in part to the implementation of aggressive “search-and-destroy” policies of infection control in countries such as the Netherlands; these policies have kept the frequency of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) and VRE very low. Despite regional differences, rates 973 of VRE continue to be much lower in Europe than in the United States. The reasons are not totally understood, although it has been postulated that this difference is related to the higher levels of human antibiotic use in the United States. Rates of enterococcal resistance to vancomycin in some Latin American countries are also lower (~4%) than those in the United States. Conversely, in Asia, rates of vancomycin resistance among enterococci appear to be

1	resistance to vancomycin in some Latin American countries are also lower (~4%) than those in the United States. Conversely, in Asia, rates of vancomycin resistance among enterococci appear to be similar to those in U.S. hospitals.

1	As mentioned above, genomic analyses of vancomycin-resistant E. faecium in different parts of the world suggest that the emergence and dissemination of these organisms in the hospital environment worldwide are due to the success of a unique hospital-associated genetic clade that acquired the genes responsible for vancomycin resistance as well as other antibiotic resistance determinants.

1	Enterococci are well-known causes of nosocomial UTI—the most common infection caused by these organisms (Chap. 162). Enterococcal UTIs are usually associated with indwelling catheters, instrumentation, or anatomic abnormalities of the genitourinary tract, and it is often challenging to differentiate between true infection and colonization (particularly in patients with chronic indwelling catheters). The presence of leukocytes in the urine in conjunction with systemic manifestations (e.g., fever) or local signs and symptoms of infection with no other explanation and a positive urine culture (≥105 colony-forming units [CFU]/mL) suggests the diagnosis. Moreover, enterococcal UTIs often occur in critically ill patients whose comorbidities may obscure the diagnosis. In many cases, removal of the indwelling catheter may suffice to eradicate the organism without specific antimicrobial therapy. In rare circumstances, UTIs caused by enterococci may run a complicated course, with the

1	of the indwelling catheter may suffice to eradicate the organism without specific antimicrobial therapy. In rare circumstances, UTIs caused by enterococci may run a complicated course, with the development of pyelonephritis and perinephric abscesses that may be a portal of entry for bloodstream infections (see below). Enterococci are also known causes of chronic prostatitis, particularly in patients whose urinary tract has been manipulated surgically or endoscopically. These infections can be difficult to treat because the agents most potent against enterococci (i.e., aminopenicillins and glycopeptides) penetrate prostatic tissue poorly. Chronic prostatic infection can be a source of recurrent enterococcal bacteremia.

1	Bacteremia without endocarditis is one of the most common presentations of enterococcal disease. Intravascular catheters and other devices are commonly associated with these bacteremic episodes (Chap. 168). Other well-known sources of enterococcal bacteremia include the gastrointestinal and hepatobiliary tracts; pelvic and intraabdominal foci; and, less frequently, wound infections, UTIs, and bone infections. In the United States, enterococci are ranked second (after coagulasenegative staphylococci) as etiologic agents of central line–associated bacteremia. Patients with enterococcal bacteremia usually have comorbidities and have been in the hospital for prolonged periods; they commonly have received several courses of antibiotics. Several studies indicate that the isolation of E. faecium from the blood may lead to worse outcomes and higher mortality rates than when other enterococcal species are isolated; this finding may be related to the higher prevalence of vancomycin and

1	faecium from the blood may lead to worse outcomes and higher mortality rates than when other enterococcal species are isolated; this finding may be related to the higher prevalence of vancomycin and ampicillin resistance in E. faecium than in other enterococcal species, with the consequent reduction of therapeutic options. In many cases (usually when the gastrointestinal tract is the source), enterococcal bacteremia may be polymicrobial, with gram-negative organisms isolated at the same time. In addition, several cases have now been documented in which enterococcal bacteremia was associated with Strongyloides stercoralis hyperinfection syndrome in immunocompromised patients.

1	Enterococci are important causes of communityand health care– associated endocarditis, ranking second after staphylococci in the 974 latter infections. The presumed initial source of bacteremia leading to endocarditis is the gastrointestinal or genitourinary tract—e.g., in patients who have malignant and inflammatory conditions of the gut or have undergone procedures in which these tracts are manipulated. The affected patients tend to be male and elderly and to have other debilitating diseases and heart conditions. Both prosthetic and native valves can be involved; mitral and aortic valves are affected most often. Community-associated endocarditis (usually caused by E. faecalis) also occurs in patients with no apparent risk factors or cardiac abnormalities. Endocarditis in women of childbearing age has been well described. The typical presentation of enterococcal endocarditis is a subacute course of fever, weight loss, malaise, and cardiac murmur; typical stigmata of endocarditis

1	age has been well described. The typical presentation of enterococcal endocarditis is a subacute course of fever, weight loss, malaise, and cardiac murmur; typical stigmata of endocarditis (e.g., petechiae, Osler’s nodes, Roth’s spots) are found in only a minority of patients. Atypical manifestations include arthralgias and manifestations of metastatic disease (splenic abscesses, hiccups, pain in the left flank, pleural effusion, and spondylodiscitis). Embolic complications are variable and can affect the brain. Heart failure is a common complication of enterococcal endocarditis, and valve replacement may be critical in curing this infection, particularly when multidrug-resistant organisms or major complications are involved. The duration of therapy is usually 4–6 weeks, with more prolonged courses suggested for multidrug-resistant isolates in the absence of valvular replacement.

1	Enterococcal meningitis is an uncommon disease (accounting for only ~4% of meningitis cases) that is usually associated with neurosurgical interventions and conditions such as shunts, central nervous system (CNS) trauma, and cerebrospinal fluid (CSF) leakage. In some instances—usually in patients with a debilitating condition, such as cardiovascular or congenital heart disease, chronic renal failure, malignancy, receipt of immunosuppressive therapy, or HIV/AIDS— presumed hematogenous seeding of the meninges is seen in infections such as endocarditis or bacteremia. Fever and changes in mental status are common, whereas overt meningeal signs are less so. CSF findings are consistent with bacterial infection—i.e., pleocytosis with a predominance of polymorphonuclear leukocytes (average, ~500/μL), an elevated serum protein level (usually >100 mg/dL), and a decreased glucose concentration (average, 28 mg/dL). Gram’s staining yields a positive result in about half of cases, with a high rate

1	an elevated serum protein level (usually >100 mg/dL), and a decreased glucose concentration (average, 28 mg/dL). Gram’s staining yields a positive result in about half of cases, with a high rate of organism recovery from CSF cultures; the most common species isolated are

1	E. faecalis and E. faecium. Complications include hydrocephalus, brain abscesses, and stroke. As mentioned before for bacteremia, an association with Strongyloides hyperinfection has also been documented. INTRAABDOMINAL, PELVIC, AND SOFT TISSUE INFECTIONS

1	As mentioned earlier, enterococci are part of the commensal flora of the gastrointestinal tract and can produce spontaneous peritonitis in cirrhotic individuals and in patients undergoing chronic ambulatory peritoneal dialysis (Chap. 159). These organisms are commonly found (usually along with other bacteria, including enteric gram-negative species and anaerobes) in clinical samples from intraabdominal and pelvic collections. The presence of enterococci in intraabdominal infections is sometimes considered to be of little clinical relevance. Several studies have shown that the role of enterococci in intraabdominal infections originating in the community and involving previously healthy patients is minor, because surgery and broad-spectrum antimicrobial drugs that do not target enterococci are often sufficient to treat these infections successfully. In the last few decades, however, these organisms have become prominent as a cause of intraabdominal infections in hospitalized patients

1	are often sufficient to treat these infections successfully. In the last few decades, however, these organisms have become prominent as a cause of intraabdominal infections in hospitalized patients because of the emergence and spread of vancomycin resistance among enterococci and the increase in rates of nosocomial infections due to multidrug-resistant E. faecium isolates. In fact, several studies have now documented treatment failures due to enterococci, with consequently increased rates of postoperative complications and death among patients with intraabdominal infections. Thus, anti-enterococcal therapy is recommended for nosocomial peritonitis in immunocompromised and severely ill patients who have had a prolonged hospital stay, have undergone multiple procedures, have persistent abdominal sepsis and collections, or have risk factors for the development of endocarditis (e.g., prosthetic or damaged heart valves). Conversely, specific treatment for enterococci in the first episode

1	sepsis and collections, or have risk factors for the development of endocarditis (e.g., prosthetic or damaged heart valves). Conversely, specific treatment for enterococci in the first episode of intraabdominal infections originating in the community and affecting previously healthy patients with no important cardiac risk factors for endocarditis does not appear to be beneficial.

1	Enterococci are commonly isolated from soft tissue infections (Chap. 156), particularly those involving surgical wounds (Chap. 168). In fact, these organisms rank third as agents of nosocomial surgical-site infections, with E. faecalis the most frequently isolated species. The clinical relevance of enterococci in some of these infections—as in intraabdominal infections—is a matter of debate; differentiating between colonization and true infection is sometimes challenging, although in some cases enterococci have been recovered from lung, liver, and skin abscesses. Diabetic foot and decubitus ulcers are often colonized with enterococci and may be the portal of entry for bone infections.

1	Enterococci are well-known causes of neonatal infections, including sepsis (mostly late-onset), bacteremia, meningitis, pneumonia, and UTI. Outbreaks of enterococcal sepsis in neonatal units have been well documented. Risk factors for enterococcal disease in newborns include prematurity, low birth weight, indwelling devices, and abdominal surgery. Enterococci have also been described as etiologic agents of bone and joint infections, including vertebral osteomyelitis, usually in patients with underlying conditions such as diabetes or endocarditis. Similarly, enterococci have been isolated from bone infections in patients who have undergone arthroplasty or reconstruction of fractures with the placement of hardware. Because enterococci can produce a biofilm that is likely to alter the efficacy of otherwise active anti-enterococcal agents, treatment of infections that involve foreign material is challenging, and removal of the hardware may be necessary to eradicate the infection. Rare

1	of otherwise active anti-enterococcal agents, treatment of infections that involve foreign material is challenging, and removal of the hardware may be necessary to eradicate the infection. Rare cases of enterococcal pneumonia, lung abscess, and spontaneous empyema have been described.

1	Enterococci are intrinsically resistant and/or tolerant to several antimicrobial agents (with tolerance defined as lack of killing by drug concentrations 32 times higher than the minimal inhibitory concentration [MIC]). Monotherapy for endocarditis with a β-lactam antibiotic (to which many enterococci are tolerant) has produced disappointing results, with low cure rates at the end of therapy. However, the addition of an aminoglycoside to a cell wall–active agent (a β-lactam or a glycopeptide) increases cure rates and eradicates the organisms; moreover, this combination is synergistic and bactericidal in vitro. Therefore, for many decades, combination therapy with a cell wall–active agent and an aminoglycoside has been the standard of care for endovascular infections caused by enterococci. This synergistic effect can be explained, at least in part, by the increased penetration of the aminoglycoside into the bacterial cell, presumably as a result of cell wall alterations produced by the

1	This synergistic effect can be explained, at least in part, by the increased penetration of the aminoglycoside into the bacterial cell, presumably as a result of cell wall alterations produced by the β-lactam (or glycopeptide). Nonetheless, attaining synergistic bactericidal activity in the treatment of severe enterococcal infections has become increasingly difficult because of the development of resistance to virtually all antibiotics available for this purpose.

1	The treatment of E. faecalis differs substantially from that of E. faecium (Tables 174-1 and 174-2), mainly because of differences in resistance profiles (see below). For example, resistance to ampicillin and vancomycin is rare in E. faecalis, whereas these antibiotics are only infrequently useful against current isolates of E. faecium. Moreover, as a consequence of the challenges and therapeutic limitations posed by the emergence of drug resistance in enterococci, Meningitis • Ampicillin (20–24 g/d IV in divided doses q4h) or penicillin (24 million units/d IV in divided doses q4h) plus an aminoglycosidec,h or ceftriaxone

1	Urinary tract infections • Fosfomycin (3 g PO, one dose)i (uncomplicated) aAuthors’ preferences are underlined for each category; many of these regimens are off-label. bIn rare cases, β-lactamase-producing isolates may be found. Because these isolates are not detected by conventional minimal inhibitory concentration determination, additional tests (e.g., the nitrocefin disk) are recommended for isolates from endocarditis. The use of ampicillin/sulbactam (12–24 g/d) is suggested in these cases. cOnly if the organism does not exhibit high-level resistance (HLR) to aminoglycosides. HLR is assessed by the clinical microbiology laboratory only for gentamicin or streptomycin, because gentamicin (1–1.5 mg/kg IV q8h) and streptomycin (15 mg/kg per day IV/IM, in two divided doses) are the only two recommended aminoglycosides. The test used to detect HLR is the growth of enterococci on agar containing gentamicin (500 μg/mL) or streptomycin (2000 μg/mL). If HLR is documented, the aminoglycoside

1	recommended aminoglycosides. The test used to detect HLR is the growth of enterococci on agar containing gentamicin (500 μg/mL) or streptomycin (2000 μg/mL). If HLR is documented, the aminoglycoside will not act synergistically with the other agent in the combination. HLR to gentamicin implies lack of synergism with tobramycin and with amikacin. d Vancomycin is recommended only as an alternative to β-lactam agents in cases of allergy, toxicity, and inability to desensitize. Cerebrospinal fluid (CSF) concentrations should be determined in meningitis. Vancomycin-resistant strains of E. faecalis have been reported. eConsider doses of 8–10 mg/kg per day if used in combination and 10–12 mg/kg per day if used alone. Close monitoring of creatine phosphokinase levels is recommended throughout therapy because of possible rhabdomyolysis. fPotentially active agents may include an aminoglycoside (if HLR is not detected), ampicillin, ceftaroline, tigecycline, or a fluoroquinolone (which, if the

1	because of possible rhabdomyolysis. fPotentially active agents may include an aminoglycoside (if HLR is not detected), ampicillin, ceftaroline, tigecycline, or a fluoroquinolone (which, if the isolate is susceptible, may be favored in meningitis). gIn selected cases of catheter-associated bacteremia, removal of the catheter and a short course of therapy (~5–7 days) may be sufficient. A single positive blood culture that is likely to be associated with a catheter in a patient who is otherwise doing well may not require therapy after removal of the catheter. Patients at high risk for endovascular infections or with severe disease may benefit from synergistic combination therapy. hThe addition of intrathecal or intraventricular therapy with gentamicin (2–10 mg/d if the organism does not exhibit HLR) or vancomycin (10–20 mg/d when the isolate is susceptible) has been suggested by some authorities. The addition of systemic rifampin (a good CSF-penetrating agent) may be considered. The

1	exhibit HLR) or vancomycin (10–20 mg/d when the isolate is susceptible) has been suggested by some authorities. The addition of systemic rifampin (a good CSF-penetrating agent) may be considered. The combination of ampicillin and ceftriaxone may have clinical benefit (by analogy with endocarditis), but no cases treated with this combination have been reported. iApproved by the Food and Drug Administration only for uncomplicated urinary tract infections caused by vancomycin-susceptible E. faecalis.

1	(22.5 mg/kg per day in divided doses q8h) ± another active agentf ampicillin (if MIC is ≤64 μg/mL) ± an aminoglycosided (22.5 mg/kg per day in divided doses q8h) ± another active agentf (22.5 mg/kg per day in divided doses q8h plus intraventricular Q/D)i ± another active agenth daptomycinb (plus intraventricular daptomycin) ± another CSF-penetrating active agenth,j

1	Urinary tract infections • Fosfomycin (3 g PO, one dose)k aAuthors’ preferences are underlined for each category; many of these regimens are off-label. bConsider doses of 8–10 mg/kg per day if used in combination and 10–12 mg/kg per day if used alone (off-label). Close monitoring of creatine phosphokinase levels is recommended throughout therapy because of possible rhabdomyolysis. cPotentially active agents may include ampicillin or ceftaroline (even if the infecting strain is resistant in vitro) or tigecycline. In vitro synergism of daptomycin with some β-lactam agents is observed against some isolates that subsequently become nonsusceptible to daptomycin during therapy. Consider combination therapy if the daptomycin minimal inhibitory concentration (MIC) is ≥3 μg/ mL. dOnly if the organism does not exhibit high-level resistance to aminoglycosides (see Table 174–1, footnote c). eQuinupristin-dalfopristin (Q/D) and linezolid are listed in the American Heart Association’s

1	if the organism does not exhibit high-level resistance to aminoglycosides (see Table 174–1, footnote c). eQuinupristin-dalfopristin (Q/D) and linezolid are listed in the American Heart Association’s recommendations for the treatment of endocarditis caused by vancomycinand ampicillin-resistant E. faecium. fAgents that may be useful in combination with Q/D (if the isolate is susceptible to each agent) include doxycycline with rifampin (one reported case) and fluoroquinolones (one reported case). gIn selected cases of catheter-associated bacteremia, removal of the catheter and a short course of therapy (~5–7 days) may be sufficient. A single positive blood culture that is likely to be associated with a catheter in a patient who is otherwise doing well may not require therapy after removal of the catheter. hFluoroquinolone antibiotics (e.g., moxifloxacin) and rifampin (if the isolate is susceptible to each agent) reach therapeutic levels in the cerebrospinal fluid (CSF). iIntrathecal Q/D

1	the catheter. hFluoroquinolone antibiotics (e.g., moxifloxacin) and rifampin (if the isolate is susceptible to each agent) reach therapeutic levels in the cerebrospinal fluid (CSF). iIntrathecal Q/D (1–5 mg/d) has been used in combination with Q/D systemic therapy in meningitis. If Q/D is chosen, simultaneous use of both systemic and intrathecal therapy is suggested. jIntrathecal gentamicin (2–10 mg/d) if high-level resistance is not detected. Intraventricular daptomycin has been used in two cases of meningitis. kApproved by the Food and Drug Administration only for uncomplicated urinary tract infections caused by vancomycin-susceptible E. faecalis. lConcentrations of amoxicillin and ampicillin in urine far exceed those in serum and may be potentially effective even against isolates with high MICs. Doses up to 12 g/d are suggested for isolates with MICs of ≥64 μg/mL.

1	976 valve replacement may need to be considered in the treatment of endocarditis caused by multidrug-resistant enterococci. Less severe infections are often related to indwelling intravascular catheters; removal of the catheter increases the likelihood of enterococcal eradication by a subsequent short course of appropriate antimicrobial therapy.

1	Among the β-lactams, the most active are the aminopenicillins (ampicillin, amoxicillin) and ureidopenicillins (i.e., piperacillin); next most active are penicillin G and imipenem. For E. faecium, a combination of high-dose ampicillin (up to 30 g/d) plus an aminoglycoside has been suggested—even for ampicillin-resistant strains if the MIC is ≤64 μg/mL—because a plasma ampicillin concentration of >100 μg/mL can be achieved at high doses. The only two aminoglycosides recommended for synergistic therapy in severe enterococcal infections are gentamicin and streptomycin. The use of amikacin is discouraged, tobramycin should never be used against E. faecium, and aminoglycoside monotherapy is not effective. Vancomycin is an alternative to β-lactam drugs for the treatment of E. faecalis infections but is less useful against E. faecium because resistance is common.

1	As mentioned above, use of the aminoglycoside–ampicillin combination for E. faecalis infections has become increasingly problematic because of toxicity in critically ill patients and increased rates of high-level resistance to aminoglycosides. A recent observational, nonrandomized, comparative study encompassing a multicenter cohort was conducted in 17 Spanish hospitals and 1 Italian hospital; this study found that the combination of ampicillin and ceftriaxone is as effective as ampicillin plus gentamicin in the treatment of E. faecalis endocarditis, with less risk of toxicity. Therefore, this regimen should be considered in patients at risk for aminoglycoside toxicity and could be considered for all patients.

1	Linezolid and quinupristin/dalfopristin (Q/D) are two agents approved by the U.S. Food and Drug Administration (FDA) for the treatment of some VRE infections (Table 174–2). Linezolid is not bactericidal, and its use in severe endovascular infections has produced mixed results; therefore, it is recommended only as an alternative to other agents. In addition, linezolid may cause significant toxicities (thrombocytopenia, peripheral neuropathy, and optic neuritis) when used in regimens given for >2 weeks. Nonetheless, linezolid may play a role in the treatment of enterococcal meningitis and other CNS infections, although clinical data are limited. Q/D is not active against most E. faecalis isolates, and its in vivo efficacy against

1	E. faecium may often be compromised by resistance (see below). Adverse reactions to Q/D are common, including pain and inflammation at the infusion site and severe arthralgias and myalgias leading to discontinuation of treatment. Thus, Q/D should be used with caution and probably combined with other agents (Table 174–2).

1	The lipopeptide daptomycin is a bactericidal antibiotic with potent in vitro activity against all enterococci. Although daptomycin is not approved by the FDA for the treatment of VRE or E. faecium infections, it has been used alone (at high dosage) or in combination with other agents (ampicillin, ceftaroline, and tigecycline) with apparent success against multidrug-resistant enterococcal infections (Tables 174–1 and 174–2). The main adverse reactions to daptomycin are elevated creatine phosphokinase levels and eosinophilic pneumonitis (rare). Daptomycin is not useful against pulmonary infections because the pulmonary surfactant inhibits its antibacterial activity. Although the glycylcycline drug tigecycline is active in vitro against all enterococci (regardless of the isolates’ vancomycin susceptibility), its use as monotherapy for endovascular or severe enterococcal infections is not recommended because of low attainable blood levels. Telavancin, a lipoglycopeptide approved by the

1	susceptibility), its use as monotherapy for endovascular or severe enterococcal infections is not recommended because of low attainable blood levels. Telavancin, a lipoglycopeptide approved by the FDA for the treatment of skin and soft tissue infections as well as hospital-associated pneumonia, is active against vancomycinsusceptible enterococci but not VRE. Oritavancin, a compound of the same class that is active against VRE, has recently been approved by the FDA for the treatment of bacterial skin and soft tissue infections and may offer promise for the treatment of VRE in the future.

1	As mentioned above, resistance to β-lactam agents continues to be observed only infrequently in E. faecalis, although rare outbreaks caused by β-lactamase-producing isolates have occurred in the United States and Argentina. However, ampicillin resistance is common in E. faecium. The mechanism of this resistance is related to a penicillin-binding protein (PBP) designated PBP5, which is the target of β-lactam antibiotics. PBP5 exhibits lower affinity for ampicillin and can synthesize cell wall in the presence of this antibiotic, even when other PBPs are inhibited. Two common mechanisms of high-level ampicillin resistance (MIC, >64 μg/mL) in clinical strains are (1) mutations in the PBP5-encoding gene that further decrease the protein’s affinity for ampicillin and (2) hyperproduction of PBP5. These factors preclude the use of all β-lactam agents in the treatment of E. faecium infections.

1	Vancomycin is a glycopeptide antibiotic that inhibits cell wall peptidoglycan synthesis in susceptible enterococci and has been widely used against enterococcal infections in clinical practice when the utility of β-lactams is limited by resistance, allergy, or adverse reactions. This effect is mediated by binding of the antibiotic to peptidoglycan precursors (UDP-MurNAc-pentapeptides) upon their exit from the bacterial cell cytoplasm. The interaction of vancomycin with the peptidoglycan is specific and involves the last two D-alanine residues of the precursor. The first isolates of VRE were documented in 1986, and vancomycin resistance (particularly in E. faecium) has since increased considerably around the world. The mechanism involves the replacement of the last D-alanine residue of peptidoglycan precursors with D-lactate or D-serine, with consequent highand low-level resistance, respectively. There is significant heterogeneity among isolates, but either substitution substantially

1	precursors with D-lactate or D-serine, with consequent highand low-level resistance, respectively. There is significant heterogeneity among isolates, but either substitution substantially decreases the affinity of vancomycin for the peptidoglycan; with the D-lactate substitution, the MIC is increased by up to 1000-fold. Vancomycin-resistant organisms also produce enzymes that destroy the D-alanine-D-alanine ending precursors, ensuring that additional binding sites for vancomycin are not available.

1	High-level resistance to aminoglycosides (of which gentamicin and streptomycin are the only two tested by clinical laboratories) abolishes the synergism observed between cell wall–active agents and the aminoglycoside. This important phenotype is routinely sought in isolates from serious infections (Tables 174–1 and 174–2). The laboratory reports high-level resistance as gentamicin and streptomycin MICs of >500 μg/mL and >2000 μg/mL, respectively (agar dilution method) or as “SYN-R” (resistance to synergism). Genes encoding aminoglycoside-modifying enzymes are usually the cause of high-level resistance to these compounds and are widely disseminated among enterococci, decreasing the options for the treatment of severe enterococcal infections. The aforementioned enterococcal resistance to newer antibiotics such as linezolid (usually due to mutations in the 23S rRNA genes and the presence of an rRNA methylase), Q/D, daptomycin (involving major changes in cell membrane homeostasis), and

1	newer antibiotics such as linezolid (usually due to mutations in the 23S rRNA genes and the presence of an rRNA methylase), Q/D, daptomycin (involving major changes in cell membrane homeostasis), and tigecycline further reduces therapeutic alternatives.

1	Diphtheria and other Corynebacterial Infections William R. Bishai, John R. Murphy DIPHTHERIA Diphtheria is a nasopharyngeal and skin infection caused by 175 Corynebacterium diphtheriae. Toxigenic strains of C. diphtheriae produce a protein toxin that causes systemic toxicity, myocarditis, and polyneuropathy. The toxin is associated with the formation of pseudomembranes in the pharynx during respiratory diphtheria. While toxigenic strains most frequently cause pharyngeal diphtheria, nontoxigenic strains commonly cause cutaneous disease.

1	C. diphtheriae is a gram-positive bacillus that is unencapsulated, nonmotile, and nonsporulating. The organism was first identified microscopically in 1883 by Klebs and a year later was isolated in pure culture by Löffler in Robert Koch’s laboratory. The bacteria have a characteristic club-shaped bacillary appearance and typically form clusters of parallel rays, or palisades, that are referred to as “Chinese characters.” The specific laboratory media recommended for the cultivation of C. diphtheriae rely upon tellurite, colistin, or nalidixic acid for the organism’s selective isolation from other autochthonous pharyngeal microbes. C. diphtheriae may be isolated from individuals with both nontoxigenic (tox–) and toxigenic (tox+) phenotypes. Uchida and Pappenheimer demonstrated that corynebacteriophage beta carries the structural gene tox, which encodes diphtheria toxin, and that a family of closely related corynebacteriophages are responsible for toxigenic conversion of tox– C.

1	corynebacteriophage beta carries the structural gene tox, which encodes diphtheria toxin, and that a family of closely related corynebacteriophages are responsible for toxigenic conversion of tox– C. diphtheriae to the tox+ phenotype. Moreover, lysogenic conversion from a nontoxigenic to a toxigenic phenotype has been shown to occur in situ. Growth of toxigenic strains of C. diphtheriae under iron-limiting conditions leads to the optimal expression of diphtheria toxin and is believed to be a pathogenic mechanism during human infection.

1	recent years with effective vaccination, there have been spo radic outbreaks in the United States and Europe. Diphtheria is still common in the Caribbean, Latin America, and the Indian subcontinent, where mass immunization programs are not enforced. Large-scale epidemics of diphtheria have occurred in the post-Soviet independent states. Additional outbreaks have been reported in Algeria, China, and Ecuador.

1	C. diphtheriae is transmitted via the aerosol route, usually during close contact with an infected person. There are no significant reservoirs other than humans. The incubation period for respiratory diphtheria is 2–5 days, but disease onset has occurred as late as 10 days after exposure. Prior to the vaccination era, most individuals over the age of 10 were immune to C. diphtheriae; infants were protected by maternal IgG antibodies but became susceptible after ~6 months of age. Thus, the disease primarily affected children and nonimmune young adults. In temperate regions, respiratory diphtheria occurs year-round but is most common during winter months.

1	The development of diphtheria antitoxin in 1898 by von Behring and of the diphtheria toxoid vaccine in 1924 by Ramon led to the near-elimination of diphtheria in Western countries. The annual incidence rate in the United States peaked in 1921 at 191 cases per 100,000 population. In contrast, since 1980, the annual figure in the United States has been <5 cases per 100,000. Nevertheless, pockets of colonization persist in North America, particularly in South Dakota, Ontario, and recently the state of Washington. Immunity to diphtheria induced by childhood vaccination gradually decreases in adulthood. An estimated 30% of men 60–69 years old have antitoxin titers below the protective level. In addition to older age and lack of vaccination, risk factors for 977 diphtheria outbreaks include alcoholism, low socioeconomic status, crowded living conditions, and Native American ethnic background. An outbreak of diphtheria in Seattle, Washington, between 1972 and 1982 comprised 1100 cases, most

1	low socioeconomic status, crowded living conditions, and Native American ethnic background. An outbreak of diphtheria in Seattle, Washington, between 1972 and 1982 comprised 1100 cases, most of which were cutaneous. During the 1990s in the states of the former Soviet Union, a much larger diphtheria epidemic included more than 150,000 cases and more than 5000 deaths. Clonally related toxigenic C. diphtheriae strains of the ET8 complex were associated with this outbreak. Given that the ET8 complex expressed a toxin against which the prevalent diphtheria toxoid vaccine was effective, the epidemic was attributed to failure of the public health infrastructure to effectively vaccinate the population. Beginning in 1998, this epidemic was controlled by mass vaccination programs. During the epidemic, the incidence rate was high among individuals between 16 and 50 years of age. Socioeconomic instability, migration, deteriorating public health programs, frequent vaccine shortages, delayed

1	epidemic, the incidence rate was high among individuals between 16 and 50 years of age. Socioeconomic instability, migration, deteriorating public health programs, frequent vaccine shortages, delayed implementation of vaccination and treatment in response to cases, and lack of public education and awareness were contributing factors.

1	Significant outbreaks of diphtheria and diphtheria-related mortality continue to be reported from many developing countries, particularly in Africa and Asia. Statistics collected by the World Health Organization indicated the occurrence of ~7000 reported diphtheria cases in 2008 and ~5000 diphtheria deaths in 2004. Although ~82% of the global population has been adequately vaccinated, only 26% of countries have successfully vaccinated >80% of individuals in all districts.

1	Cutaneous diphtheria is usually a secondary infection that follows a primary skin lesion due to trauma, allergy, or autoimmunity. Most often, these isolates lack the tox gene and thus do not express diphtheria toxin. In tropical latitudes, cutaneous diphtheria is more common than respiratory diphtheria. In contrast to respiratory disease, cutaneous diphtheria is not reportable in the United States. Nontoxigenic strains of C. diphtheriae have also been associated with pharyngitis in Europe, causing outbreaks among men who have sex with men and persons who use illicit IV drugs.

1	Diphtheria toxin produced by tox+ strains of C. diphtheriae is the primary virulence factor in clinical disease. The toxin is synthesized in precursor form; is released as a 535-amino-acid, single-chain protein; and, in sensitive species (e.g., guinea pigs and humans, but not mice or rats), has a 50% lethal dose of ~100 ng/kg of body weight. The toxin is produced in the pseudomembranous lesion and is taken up in the bloodstream, from which it is distributed to all organ systems in the body. Once bound to its cell surface receptor (a heparin-binding epidermal growth factor–like precursor), the toxin is internalized by receptor-mediated endocytosis and enters the cytosol from an acidified early endosomal compartment. In vitro, the toxin may be separated into two chains by digestion with serine proteases: the N-terminal A fragment and the C-terminal B fragment. Delivery of the A fragment into the eukaryotic cell cytosol results in irreversible inhibition of protein synthesis by

1	with serine proteases: the N-terminal A fragment and the C-terminal B fragment. Delivery of the A fragment into the eukaryotic cell cytosol results in irreversible inhibition of protein synthesis by NAD+-dependent ADP-ribosylation of elongation factor 2. The eventual result is the death of the cell.

1	In 1926, Ramon at the Institut Pasteur found that formalinization of diphtheria toxin resulted in the production of a nontoxic but highly immunogenic diphtheria toxoid. Subsequent studies showed that immunization with diphtheria toxoid elicited antibodies that neutralized the toxin and prevented most disease manifestations. In the 1930s, mass immunization of children and susceptible adults with diphtheria toxoid commenced in the United States and Europe. Individuals with a diphtheria antitoxin titer of >0.01 U/mL are at low risk of disease. In populations where a majority of individuals have protective antitoxin titers, the carrier rate for toxigenic strains of C. diphtheriae decreases and the overall risk of diphtheria among susceptible individuals is reduced. Nevertheless, individuals with nonprotective titers may contract diphtheria through either travel or exposure to individuals who have recently returned from regions where the disease is endemic.

1	978 Characteristic pathologic findings of diphtheria include mucosal ulcers with a pseudomembranous coating composed of an inner band of fibrin and a luminal band of neutrophils. Initially white and firmly adherent, in advanced diphtheria the pseudomembranes turn gray or even green or black as necrosis progresses. Mucosal ulcers result from toxin-induced necrosis of the epithelium accompanied by edema, hyperemia, and vascular congestion of the submucosal base. A significant fibrinosuppurative exudate from the ulcer develops into the pseudomembrane. Ulcers and pseudomembranes in severe respiratory diphtheria may extend from the pharynx into medium-sized bronchial airways. Expanding and sloughing membranes may result in fatal airway obstruction. APPROACH TO THE PATIENT:

1	Diphtheria, though rare in the United States and other developed countries, should be considered when a patient has severe pharyngitis, particularly when there is difficulty swallowing, respiratory compromise, or signs of systemic disease (e.g., myocarditis or generalized weakness). The leading causes of pharyngitis are respiratory viruses (rhinoviruses, influenza viruses, parainfluenza viruses, coronaviruses, adenoviruses; ~25% of cases), group A streptococci (15–30%), group C streptococci (~5%), atypical bacteria such as Mycoplasma pneumoniae and Chlamydia pneumoniae (15–20% in some series), and other viruses such as herpes simplex virus (~4%) and Epstein-Barr virus (<1% in infectious mononucleosis). Less common causes are acute HIV infection, gonorrhea, fusobacterial infection (e.g., Lemierre’s syndrome), thrush due to Candida albicans or other Candida species, and diphtheria. The presence of a pharyngeal pseudomembrane or an extensive exudate should prompt consideration of

1	Lemierre’s syndrome), thrush due to Candida albicans or other Candida species, and diphtheria. The presence of a pharyngeal pseudomembrane or an extensive exudate should prompt consideration of diphtheria (Figure 175-1).

1	FIGURE 175-1 Respiratory diphtheria due to toxigenic C. diphtheriae producing exudative pharyngitis in a 47-year-old female patient displaying neck edema and a pseudomembrane extending from the uvula to the pharyngeal wall. The characteristic white pseudomembrane is caused by diphtheria toxin–mediated necrosis of the respiratory epithelial layer, producing a fibrinous coagulative exudate. Submucosal edema adds to airway narrowing. The pharyngitis is acute in onset, and respiratory obstruction from the pseudomembrane may occur in severe cases. Inoculation of pseudomembrane fragments or submembranous swabs onto Löffler’s or tellurite selective medium reveals C. diphtheriae. (Photograph by P. Strebel, MD, used by permission. From R. Kadirova et al: J Infect Dis 181:S110, 2000. With permission of Oxford University Press.)

1	CLINICAL MANIFESTATIONS Respiratory Diphtheria The clinical diagnosis of diphtheria is based on the constellation of sore throat; adherent tonsillar, pharyngeal, or nasal pseudomembranous lesions; and low-grade fever. In addition, diagnosis requires the isolation of C. diphtheriae or histopathologic isolation of compatible gram-positive organisms. The Centers for Disease Control and Prevention (CDC) recognizes confirmed respiratory diphtheria (laboratory proven or epidemiologically linked to a culture-confirmed case) and probable respiratory diphtheria (clinically compatible but not laboratory proven or epidemiologically linked). Carriers are defined as individuals who have positive cultures for C. diphtheriae and who either are asymptomatic or have symptoms but lack pseudomembranes. Most patients seek medical care for sore throat and fever several days into the illness. Occasionally, weakness, dysphagia, headache, and voice change are the initial manifestations. Neck edema and

1	Most patients seek medical care for sore throat and fever several days into the illness. Occasionally, weakness, dysphagia, headache, and voice change are the initial manifestations. Neck edema and difficulty breathing are evident in more advanced cases and carry a poor prognosis.

1	The systemic manifestations of diphtheria stem from the effects of diphtheria toxin and include weakness as a result of neurotoxicity and cardiac arrhythmias or congestive heart failure due to myocarditis. Most commonly, the pseudomembranous lesion is located in the tonsillopharyngeal region. Less commonly, the lesions are located in the larynx, nares, and trachea or bronchial passages. Large pseudo-membranes are associated with severe disease and a poor prognosis. A few patients develop massive swelling of the tonsils and present with “bull-neck” diphtheria, which results from massive edema of the submandibular and paratracheal region and is further characterized by foul breath, thick speech, and stridorous breathing. The diphtheritic pseudomembrane is gray or whitish and sharply demarcated. Unlike the exudative lesion associated with streptococcal pharyngitis, the pseudomembrane in diphtheria is tightly adherent to the underlying tissues. Attempts to dislodge the membrane may cause

1	Unlike the exudative lesion associated with streptococcal pharyngitis, the pseudomembrane in diphtheria is tightly adherent to the underlying tissues. Attempts to dislodge the membrane may cause bleeding. Hoarseness suggests laryngeal diphtheria, in which laryngoscopy may be diagnostically helpful.

1	Cutaneous Diphtheria This dermatosis is characterized by punched-out ulcerative lesions with necrotic sloughing or pseudomembrane formation (Figure 175-2). The diagnosis requires cultivation of C. diphtheriae from lesions, which most commonly occur on the lower and upper extremities, head, and trunk. Infections Due to Non-diphtheriae Corynebacterium Species and Nontoxigenic C. diphtheriae Non-diphtheriae species of Corynebacterium and related genera (discussed below) as well as nontoxigenic strains of C. diphtheriae itself have been found in bloodstream and respiratory infections, often in individuals with immunosuppression or chronic respiratory disease. These organisms can cause disease manifestations and should not necessarily be dismissed as colonizers. FIGURE 175-2 Cutaneous diphtheria due to nontoxigenic C. diphtheriae on the lower extremity. (From the Centers for Disease Control and Prevention.)

1	FIGURE 175-2 Cutaneous diphtheria due to nontoxigenic C. diphtheriae on the lower extremity. (From the Centers for Disease Control and Prevention.) Other Clinical Manifestations C. diphtheriae causes rare cases of endo carditis and septic arthritis, most often in patients with preexisting risk factors, such as abnormal cardiac valves, injection drug use, or cirrhosis. Airway obstruction poses a significant early risk in patients presenting with advanced diphtheria. Pseudomembranes may slough and obstruct the airway or may advance to the larynx or into the tracheobronchial tree. Children are particularly prone to obstruction because of their small airways. Polyneuropathy and myocarditis are late toxic manifesta tions of diphtheria. During a diphtheria outbreak in the

1	Polyneuropathy and myocarditis are late toxic manifesta tions of diphtheria. During a diphtheria outbreak in the Kyrgyz Republic in 1999, myocarditis was found in 22% and neuropathy in 5% of 656 hospitalized patients. The mortality rate was 7% among patients with myocarditis as opposed to 2% among those without myocardial manifestations. The median time to death in hospitalized patients was 4.5 days. Myocarditis is typically associated with dysrhythmia of the conduction tract and dilated cardiomyopathy.

1	Polyneuropathy is seen 3–5 weeks after the onset of diphtheria and has a slow indolent course. However, patients may develop severe and prolonged neurologic abnormalities. The disorders typically occur in the mouth and neck, with lingual or facial numbness as well as dysphonia, dysphagia, and cranial nerve paresthesias. More ominous signs include weakness of respiratory and abdominal muscles and paresis of the extremities. Sensory manifestations and sensory ataxia also are observed. Cranial nerve dysfunction typically precedes disturbances of the trunk and extremities because of proximity to the site of infection. Autonomic dysfunction also is associated with polyneuropathy and can lead to hypotension. Polyneuropathy is typically reversible in patients who survive the acute phase. Other complications of diphtheria include pneumonia, renal failure, encephalitis, cerebral infarction, pulmonary embolism, and serum sickness from antitoxin therapy.

1	Other complications of diphtheria include pneumonia, renal failure, encephalitis, cerebral infarction, pulmonary embolism, and serum sickness from antitoxin therapy. The diagnosis of diphtheria is based on clinical signs and symptoms plus laboratory confirmation. Respiratory diphtheria should be considered in patients with sore throat, pharyngeal exudates, and fever. Other symptoms may include hoarseness, stridor, or palatal paralysis. The presence of a pseudomembrane should prompt strong consideration of diphtheria. Once a clinical diagnosis of diphtheria is made, diphtheria antitoxin should be obtained and administered as rapidly as possible. Laboratory diagnosis of diphtheria is based either on cultivation of

1	Laboratory diagnosis of diphtheria is based either on cultivation of C. diphtheriae or toxigenic Corynebacterium ulcerans from the site of infection or on the demonstration of local lesions with characteristic histopathology. Corynebacterium pseudodiphtheriticum, a nontoxigenic organism, is a common component of the normal throat flora and does not pose a significant risk. Throat samples should be submitted to the laboratory for culture with the notation that diphtheria is being considered. This information should prompt cultivation on special selective medium and subsequent biochemical testing to differentiate C. diphtheriae from other nasopharyngeal commensal corynebacteria. All laboratory isolates of C. diphtheriae, including nontoxigenic strains, should be submitted to the CDC.

1	A diagnosis of cutaneous diphtheria requires laboratory confirmation since the lesions are not characteristic and are indistinguishable from other dermatoses. Diphtheritic ulcers occasionally—but not consistently—have a punched-out appearance (Fig. 175-2). Patients in whom cutaneous diphtheria is identified should have the nasopharynx cultured for C. diphtheriae. The laboratory medium for cutaneous diphtheria specimens is the same as that used for respiratory diphtheria: Löffler’s or Tinsdale’s selective medium in addition to nonselective medium such as blood agar. As has been mentioned, respiratory diphtheria remains a notifiable disease in the United States, whereas cutaneous diphtheria is not.

1	Prompt administration of diphtheria antitoxin is critical in the management of respiratory diphtheria. Diphtheria antitoxin, a horse antiserum, is effective in reducing the extent of local disease as well as the risk of complications of myocarditis and neuropathy. Rapid institution of antitoxin therapy is also associated with a significant reduction in mortality risk. Because diphtheria antitoxin cannot neutralize cell-bound toxin, prompt initiation is important. This product, which is no longer commercially available in the United States, can be obtained from the CDC by calling the Bacterial Vaccine Preventable Disease Branch of the National Immunization Program at 404-639-8257 (8:00 A.M. to 4:30 P.M., U.S. Eastern time) or, at other hours, the Emergency Operations Center at 770-488-7100; the relevant website is www.cdc.gov/diphtheria/dat.html. The current protocol for the use of diphtheria antitoxin involves a test dose to rule out immediate hypersensitivity. Patients who

1	the relevant website is www.cdc.gov/diphtheria/dat.html. The current protocol for the use of diphtheria antitoxin involves a test dose to rule out immediate hypersensitivity. Patients who demonstrate hypersensitivity require desensitization before a full therapeutic dose of antitoxin is administered.

1	Antibiotics are used in the management of diphtheria primarily to prevent transmission to susceptible contacts. Antibiotics also prevent further toxin production and reduce the severity of local infection. Recommended treatment options for patients with respiratory diphtheria are as follows: • Procaine penicillin G, 600,000 U IM q12h (for children: 12,500–25,000 U/kg IM q12h) until the patient can swallow comfortably; then oral penicillin V, 125–250 mg qid to complete a 14-day course • Erythromycin, 500 mg IV q6h (for children: 40–50 mg/kg per day

1	IV in two or four divided doses) until the patient can swallow com fortably; then 500 mg PO qid to complete a 14-day course ciated with a more rapid resolution of fever and a lower rate of bacterial resistance than erythromycin; however, relapses were more common in the penicillin group. Erythromycin therapy targets protein synthesis and thus offers the presumed benefit of stopping toxin synthesis more quickly than a cell wall– active β-lactam agent. Alternative therapeutic agents for patients who are allergic to penicillin or cannot take erythromycin include rifampin and clindamycin. Eradication of C. diphtheriae should be documented after antimicrobial therapy is complete. A repeat throat culture 2 weeks later is recommended. For patients in whom the organism is not eradicated after a 14-day course of erythromycin or penicillin, an additional 10-day course followed by repeat culture is recommended. Drug-resistant strains of C. diphtheriae exist, and several reports have described

1	14-day course of erythromycin or penicillin, an additional 10-day course followed by repeat culture is recommended. Drug-resistant strains of C. diphtheriae exist, and several reports have described multidrugresistant strains, predominantly in Southeast Asia. Drug resistance should be considered when efforts at pathogen eradication fail.

1	Cutaneous diphtheria should be treated as described above for respiratory disease. Individuals infected with toxigenic strains should receive antitoxin. It is important to treat the underlying cause of the dermatoses in addition to the superinfection with C. diphtheriae. Patients who recover from respiratory or cutaneous diphtheria should have antitoxin levels measured. If diphtheria antitoxin has been administered, this test should be performed 6 months later. Patients who recover from respiratory or cutaneous diphtheria should receive the appropriate vaccine to ensure the development of protective antibody titers.

1	Patients in whom diphtheria is suspected should be hospitalized in respiratory isolation rooms, with close monitoring of cardiac and respiratory function. A cardiac workup is recommended to assess the possibility of myocarditis. In patients with extensive pseudomembranes, an anesthesiology or an ear, nose, and throat consultation is recommended because of the possible need for 980 tracheostomy or intubation. In some settings, pseudomembranes can be removed surgically. Treatment with glucocorticoids has not been shown to reduce the risk of myocarditis or polyneuropathy.

1	Fatal pseudomembranous diphtheria typically occurs in patients with nonprotective antibody titers and in unimmunized patients. The pseudomembrane may actually increase in size from the time it is first noted. Risk factors for death include bullneck diphtheria; myocarditis with ventricular tachycardia; atrial fibrillation; complete heart block; an age of >60 years or <6 months; alcoholism; extensive pseudo-membrane elongation; and laryngeal, tracheal, or bronchial involvement. Another important predictor of fatal outcome is the interval between the onset of local disease and the administration of antitoxin. Cutaneous diphtheria has a low mortality rate and is rarely associated with myocarditis or peripheral neuropathy.

1	PREVENTION Vaccination Sustained campaigns for vaccination of children and adequate boosting vaccination of adults are responsible for the exceedingly low incidence of diphtheria in most developed nations. Currently, diphtheria toxoid vaccine is coadministered with tetanus vaccine (with or without acellular pertussis). DTaP (a full-level diphtheria and tetanus toxoids and acellular pertussis vaccine) is currently recommended for children up to the age of 7; DTaP replaced the earlier whole-cell pertussis vaccine DTP in 1997. Tdap is a tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine formulated for adolescents and adults. Tdap was licensed for use in the United States in 2005 and is the recommended booster vaccine for children 11–12 years old and the recommended catch-up vaccine for children 7–10 and 13–18 years of age. It is recommended that all adults (i.e., persons >19 years old) receive a single dose of Tdap if they have not received it previously,

1	catch-up vaccine for children 7–10 and 13–18 years of age. It is recommended that all adults (i.e., persons >19 years old) receive a single dose of Tdap if they have not received it previously, regardless of the interval since the last dose of Td (tetanus and reduced-dose diphtheria toxoids, adsorbed). Tdap vaccination is a priority for health care workers, pregnant women, adults anticipating contact with infants, and adults not previously vaccinated for pertussis. Adults who have received acellular pertussis vaccine should continue to receive decennial Td booster vaccinations.

1	The vaccine schedule is detailed in Chap. 148. Prophylaxis Administration to Contacts Close contacts of diphtheria patients should undergo throat culture to determine whether they are carriers. After samples for throat culture are obtained, antimicrobial prophylaxis should be considered for all contacts, even those whose cultures are negative. The options are 7–10 days of oral erythromycin or one dose of IM benzathine penicillin G (1.2 million units for persons ≥6 years of age or 600,000 units for children <6 years of age). Contacts of diphtheria patients whose immunization status is uncertain should receive the appropriate diphtheria toxoid–containing vaccine. The Tdap vaccine (rather than Td) is now the booster vaccine of choice for adults who have not recently received an acellular pertussis– containing vaccine. Carriers of C. diphtheriae in the community should be treated and vaccinated when identified.

1	Nondiphtherial corynebacteria, referred to as diphtheroids or coryneforms, are frequently considered colonizers or contaminants; however, they have been associated with invasive disease, particularly in immunocompromised patients. These organisms have been isolated from the bloodstream, particularly in association with catheter infection, endocarditis, prosthetic valve infection, meningitis, neurosurgical shunt infection, brain abscess, and peritonitis and often in the setting of chronic ambulatory peritoneal dialysis, osteomyelitis, septic arthritis, urinary tract infection, empyema, and pneumonia, among other infections. Patients infected with these organisms usually have significant medical comorbidity or are immunosuppressed. The nondiphtherial coryneforms are a diverse collection of bacteria that are taxonomically grouped together in the genus Corynebacterium on the basis of their 16S rDNA signature nucleotides. Despite the shared rDNA signatures, these isolates are quite

1	of bacteria that are taxonomically grouped together in the genus Corynebacterium on the basis of their 16S rDNA signature nucleotides. Despite the shared rDNA signatures, these isolates are quite diverse. For example, their guanine-cytosine content ranges from 45% to 70%. Several nondiphtheroid corynebacteria, including Corynebacterium jeikeium and Corynebacterium urealyticum, are associated with resistance to multiple antibiotics. Rhodococcus equi is associated with necrotizing pneumonia and granulomatous infection, particularly in immunocompromised individuals.

1	These organisms are non-acid-fast, catalase-positive, aerobic or facultatively anaerobic rods. Their colonial morphologies vary widely; some species are small and α-hemolytic (similar to lactobacilli), whereas others form large white colonies (similar to yeasts). Many nondiphtherial coryneforms require special media, such as Löffler’s, Tinsdale’s, or telluride medium. These cultivation idiosyncrasies have led to a complex taxonomic categorization of the organisms. Humans are the natural reservoirs for several nondiphtherial coryneforms, including C. xerosis, C. pseudodiphtheriticum, C. striatum, C. minutissimum, C. jeikeium, C. urealyticum, and Arcanobacterium haemolyticum. Animal reservoirs are responsible for carriage of Arcanobacterium pyogenes, C. ulcerans, and C. pseudotuberculosis. Soil is the natural reservoir for R. equi.

1	C. pseudodiphtheriticum is a component of the normal flora of the human pharynx and skin. C. xerosis is found on the skin, nasopharynx, and conjunctiva; C. auris in the external auditory canal; and C. striatum in the anterior nares and on the skin. C. jeikeium and C. urealyticum are found in the axilla, groin, and perineum, particularly in hospitalized patients. Infections with C. ulcerans and C. pseudotuberculosis have been associated with the consumption of raw milk from infected cattle.

1	C. ulcerans This organism causes a diphtheria-like illness and produces both diphtheria toxin and a dermonecrotic toxin. The organism is a commensal in horses and cattle and has been isolated from cow’s milk. C. ulcerans causes exudative pharyngitis, primarily during summer months, in rural areas, and among individuals exposed to cattle. In contrast to diphtheria, this infection is considered a zoonosis whose person-to-person transmission has not been documented. Nevertheless, treatment with antitoxin and antibiotics should be initiated when respiratory C. ulcerans is identified, and a contact investigation (including throat cultures to determine the need for antimicrobial prophylaxis and, in unimmunized contacts, administration of the appropriate diphtheria toxoid–containing vaccine) should be conducted. The organism grows on Löffler’s, Tinsdale’s, and telluride agars as well as blood agar. In addition to exudative pharyngitis, cutaneous disease due to C. ulcerans has been reported.

1	be conducted. The organism grows on Löffler’s, Tinsdale’s, and telluride agars as well as blood agar. In addition to exudative pharyngitis, cutaneous disease due to C. ulcerans has been reported. C. ulcerans is susceptible to a wide panel of antibiotics. Erythromycin and macrolides appear to be the first-line agents.

1	C. pseudotuberculosis (ovis) Infection caused by C. pseudotuberculosis is rare and is reported almost exclusively from Australia. C. pseudo-tuberculosis causes suppurative granulomatous lymphadenitis and an eosinophilic pneumonia syndrome among individuals who handle horses, cattle, goats, and deer or who drink raw milk. The organism is an important veterinary pathogen, causing suppurative lymphadenitis, abscesses, and pneumonia, but is rarely a human pathogen. Successful treatment with erythromycin or tetracycline has been reported, with surgery also performed when indicated. C. jeikeium (Group JK) Originally described in American hospitals,

1	C. jeikeium (Group JK) Originally described in American hospitals, C. jeikeium infection was subsequently reported in Europe. After a 1976 survey of diseases caused by nondiphtherial corynebacteria, CDC group JK emerged as an important opportunistic pathogen among neutropenic and HIV-infected patients. The organism has now been designated a separate species. C. jeikeium forms small, gray to white, glistening, nonhemolytic colonies on blood agar. It lacks urease and nitrate reductase and does not ferment most carbohydrates. The predominant syndrome associated with C. jeikeium is sepsis with pneumonia, endocarditis, meningitis, osteomyelitis, and epidural abscess. Risk factors for C. jeikeium infection include hematologic malignancy, neutropenia from comorbid conditions, prolonged hospitalization, exposure to multiple antibiotics, and skin disruption. There is evidence that C. jeikeium is part of the inguinal, axillary, genital, and perirectal flora of hospitalized patients.

1	Broad-spectrum antimicrobial therapy appears to select for colonization. Gram’s staining shows gram-positive coccobacillary forms slightly resembling streptococci. Moreover, C. jeikeium is resistant to all antibiotics tested except vancomycin. Effective therapy involves removal of the infectious source, whether a catheter, prosthetic joint, or prosthetic valve. Efforts have been made to prevent C. jeikeium infection by improving hygienic conditions for high-risk patients in intensive care settings with antibacterial soap.

1	C. urealyticum (Group D2) Identified as a urease-positive nondiphtherial Corynebacterium in 1972, C. urealyticum is an opportunistic pathogen causing sepsis and urinary tract infection. C. urealyticum appears to be the etiologic agent of a severe urinary tract syndrome known as alkaline-encrusted cystitis, a chronic inflammatory bladder infection associated with deposition of ammonium magnesium phosphate on the surface and walls of ulcerating lesions in the bladder. In addition, C. urealyticum has been associated with pneumonia, peritonitis, endocarditis, osteomyelitis, and wound infection. It is similar to C. jeikeium in its resistance to most antibiotics except vancomycin. Vancomycin therapy has been used successfully in severe infections.

1	C. minutissimum (Erythrasma) Erythrasma is a cutaneous infection producing reddish-brown, macular, scaly, pruritic intertriginous patches. The dermatologic presentation under the Wood’s lamp is of coral red fluorescence. C. minutissimum appears to be a common cause of erythrasma, although there is evidence for a polymicrobial etiology in certain settings. This microbe has also been associated with bacteremia in patients with hematologic malignancy. Erythrasma responds to topical erythromycin, clarithromycin, clindamycin, or fusidic acid, although more severe infections may require oral macrolide therapy.

1	Other Nondiphtherial Corynebacterial Infections C. xerosis is a human commensal found in the conjunctiva, nasopharynx, and skin. This nontoxigenic organism is occasionally identified as a source of invasive infection in immunocompromised or postoperative patients and prosthetic joint recipients. C. striatum is found in the anterior nares, skin, face, and upper torso of healthy individuals. Also nontoxigenic, this organism has been associated with invasive opportunistic infections in severely ill or immunocompromised patients. C. amycolatum is isolated from human skin and is identified on the basis of a unique 16S ribosomal RNA sequence associated with opportunistic infection. C. glucuronolyticum is a nonlipophilic species that causes male genitourinary tract infections such as prostatitis and urethritis. These infections may be successfully treated with a wide variety of antibacterial agents, including β-lactams, rifampin, aminoglycosides, or vancomycin; however, the organism appears

1	and urethritis. These infections may be successfully treated with a wide variety of antibacterial agents, including β-lactams, rifampin, aminoglycosides, or vancomycin; however, the organism appears to be resistant to fluoroquinolones, macrolides, and tetracyclines. C. imitans has been identified in eastern Europe as a nontoxigenic cause of pharyngitis. C. auris has been identified in children with otitis media; it is susceptible to fluoroquinolones, rifampin, tetracycline, and vancomycin but resistant to penicillin G and variably susceptible to macrolides. C. pseudodiphtheriticum (C. hoffmanii) is a nontoxigenic species that is part of the normal human flora. Human infections—particularly endocarditis of either prosthetic or natural valves and invasive pneumonia—have been reported only rarely. Although C. pseudodiphtheriticum may be isolated from the nasopharynx of patients with suspected diphtheria, it is part of the normal flora and does not produce diphtheria toxin. 981

1	C. propinquum, a close relative of C. pseudodiphtheriticum, is part of CDC group ANF-3 and is isolated from the human respiratory tract and blood. C. afermentans subspecies lipophilum belongs to CDC group ANF-1 and has been isolated from human blood and abscesses. C. accolens has been isolated from wound drainage, throat swabs, and sputum and is typically identified as a satellite of staphylococcal organisms; this species has been associated with endocarditis. C. bovis is a veterinary commensal that has not been clearly associated with human disease. C. aquaticum is a water-dwelling organism that is occasionally isolated from patients using medical devices (e.g., for chronic ambulatory peritoneal dialysis or venous access).

1	Rhodococcus Rhodococcus species are phylogenetically related to the corynebacteria. These gram-positive coccobacilli have been associated with tuberculosis-like infections in humans with granulomatous pathology. While R. equi is best known, other species have been identified, including R. (Gordonia) bronchialis, R. (Tsukamurella) aurantiacus, R. luteus, R. erythropolis, R. rhodochrous, and R. rubropertinctus.

1	R. equi has been recognized as a cause of pneumonia in horses since the 1920s and as a cause of related infections in cattle, sheep, and swine. It is found in soil as an environmental microbe. The organisms vary in length; appear as spherical to long, curved, clubbed rods; and produce large irregular mucoid colonies. R. equi cannot ferment carbohydrates or liquefy gelatin and is often acid fast. An intracellular pathogen of macrophages, R. equi can cause granulomatous necrosis and caseation. This organism has most commonly been identified in pulmonary infection, but infections of brain, bone, and skin also have been reported. Most commonly, R. equi disease manifests as nodular cavitary pneumonia of the upper lobe—a picture similar to that seen in tuberculosis or nocardiosis. Most patients are immunocompromised, often by HIV infection. Subcutaneous nodular lesions have also been identified. The involvement of R. equi should be considered when any patient presents with a

1	Most patients are immunocompromised, often by HIV infection. Subcutaneous nodular lesions have also been identified. The involvement of R. equi should be considered when any patient presents with a tuberculosis-like syndrome.

1	Infection due to R. equi has been treated successfully with antibiotics that penetrate intracellularly, including macrolides, clindamycin, rifampin, and trimethoprim-sulfamethoxazole. β-Lactam antibiotics have not been useful. The organism is routinely susceptible to vancomycin, which is considered the drug of choice. Other Related Species • actinomyces pyogenes This organism, a well-known pathogen of cattle, sheep, goats, and pigs, causes seasonal leg ulcers in rural Thailand. A few human cases of sepsis, endocarditis, septic arthritis, pneumonia, meningitis, and empyema have been reported. This species is susceptible to β-lactams, tetracyclines, aminoglycosides, and fluoroquinolones.

1	arcanobacterium Haemolyticum A. haemolyticum was identified as an agent of wound infections in U.S. soldiers in the South Pacific during World War II. It appears to be a human commensal of the nasopharynx and skin, but has also been implicated in pharyngitis and chronic skin ulcers. In contrast to the much more common pharyngitis caused by Streptococcus pyogenes, A. haemolyticum pharyngitis is associated with a scarlatiniform rash on the trunk and proximal extremities in about half of cases; this illness is occasionally confused with toxic shock syndrome. Because A. haemolyticum pharyngitis primarily affects teenagers, it has been postulated that the rash-pharyngitis syndrome may represent copathogenicity, synergy, or opportunistic secondary infection with Epstein-Barr virus. A. haemolyticum has also been reported as a cause of bacteremia, soft tissue infections, osteomyelitis, and cavitary pneumonia, predominantly in the setting of underlying diabetes mellitus. The organism is

1	has also been reported as a cause of bacteremia, soft tissue infections, osteomyelitis, and cavitary pneumonia, predominantly in the setting of underlying diabetes mellitus. The organism is susceptible to β-lactams, macrolides, fluoroquinolones, clindamycin, vancomycin, and doxycycline. Penicillin resistance has been reported.

1	982 Listeria monocytogenes Infections Elizabeth L. Hohmann, Daniel A. Portnoy Listeria monocytogenes is a food-borne pathogen that can cause serious infections, particularly in pregnant women and immunocompromised 176 individuals. A ubiquitous saprophytic environmental bacterium, L. monocytogenes is also a facultative intracellular pathogen with a broad host range. Humans are probably accidental hosts for this microorganism. L. monocytogenes is of interest not only to clinicians but also to basic scientists as a model intracellular pathogen that is used to study basic mechanisms of microbial pathogenesis and host immunity. L. monocytogenes is a facultatively anaerobic, nonsporulating, gram-positive rod that grows over a broad temperature range, including refrigeration temperatures. This organism is motile during growth at low temperatures but much less so at 37°C. The vast majority of cases of human listerial disease can be traced to serotypes 1/2a, 1/2b, and 4.

1	L. monocytogenes is weakly β-hemolytic on blood agar, and (as detailed below) its β-hemolysin is an essential determinant of its pathogenicity.

1	Infections with L. monocytogenes follow ingestion of contaminated food that contains the bacteria at high concentrations. The conversion from environmental saprophyte to pathogen involves the coordinate regulation of bacterial determinants of pathogenesis that mediate entry into cells, intracellular growth, and cell-to-cell spread. Many of the organism’s pathogenic strategies can be examined experimentally in tissue culture models of infection (Fig. 176-1). Like other enteric pathogens, L. monocytogenes induces its own internalization by cells that are not normally phagocytic. Its entry into cells is mediated by host surface proteins classified as internalins. Internalin-mediated entry is important in the crossing of intestinal, blood-brain, and fetoplacental barriers, although how L. monocytogenes traffics from the intestine to the brain or fetus is only beginning to be investigated. In a pregnant guinea pig model of infection, L. monocytogenes was shown

1	FIGURE 176-1 Stages in the intracellular life cycle of Listeria monocytogenes. The central diagram depicts cell entry, escape from a vacuole, actin nucleation, actin-based motility, and cell-to-cell spread. Surrounding the diagram are representative electron micro-graphs from which it was derived. ActA, surface protein mediating nucleation of host actin filaments to propel bacteria intraand inter-cellularly; LLO, listeriolysin O; PLCs, phospholipases C; Inl, internalin. See text for further details. (Adapted with permission from LG Tilney, DA Portnoy: J Cell Biol 109:1597, 1989. © Rockefeller University Press.) to traffic from maternal organs to the placenta; surprisingly, however, it also trafficked from the placenta back to maternal organs. These data are consistent with a model in which miscarriage can be viewed as a host defense strategy to eliminate a nidus of infection.

1	An essential determinant of the pathogenesis of L. monocytogenes is its β-hemolysin, listeriolysin O (LLO). LLO is a pore-forming, cholesterol-dependent cytolysin. (Related cytolysins include streptolysin O, pneumolysin, and perfringolysin O, all of which are produced by extracellular pathogens.) LLO is largely responsible for mediating the rupture of the phagosomal membrane that forms after phagocytosis of

1	L. monocytogenes. LLO probably acts by insertion into an acidifying phagosome, which prevents the vesicle’s maturation. In addition, LLO acts as a translocation pore for one or both of the L. monocytogenes phospholipases that also contribute to vacuolar lysis. LLO synthesis and activity are controlled at multiple levels to ensure that its lytic activity is limited to acidic vacuoles and does not affect the cytosol. Mutations in LLO that influence its synthesis, cytosolic half-life, or pH optimum cause premature toxicity to infected cells. There is an inverse relationship between toxicity and virulence—i.e., the more cytotoxic the strain, the less virulent it is in animals. This relationship may seem paradoxical, but, as an intracellular pathogen, L. monocytogenes benefits from leaving its host cell unharmed.

1	Shortly after exposure to the mammalian-cell cytosol, L. monocytogenes expresses a surface protein, ActA, that mediates the nucleation of host actin filaments to propel the bacteria intraand intercellularly. ActA mimics host proteins of the Wiskott-Aldrich syndrome protein (WASP) family by promoting the actin nucleation properties of the Arp2/3 complex. Thus, L. monocytogenes can enter the cytosol of almost any eukaryotic cell or cell extract and can exploit a conserved and essential actin-based motility system. Other pathogens as diverse as certain Shigella, Mycobacterium, Rickettsia, and Burkholderia species use a related pathogenic strategy that allows cell-to-cell spread without exposure to the extracellular milieu.

1	The innate and acquired immune responses to L. monocytogenes have been studied extensively in mice. Shortly after IV injection, most bacteria are found in Kupffer cells in the liver, with some organisms in splenic dendritic cells and macrophages. Listeriae that survive the bactericidal activity of initially infected macrophages grow in the cytosol and spread from cell to cell. L. monocytogenes triggers three innate immune pathways: a MyD88-dependent pathway leading to inflammatory cytokines, a STING/IRF3 pathway leading to a type I interferon response; and low-level inflammasome activation. Neutrophils are crucial to host defense during the first 24 h of infection, whereas an influx of activated macrophages from the bone marrow is critical subsequently. Mice that survive sublethal infection clear the organisms within a week, with consequent sterile immunity. Studies with knockout mice have been instrumental in dissecting the roles played by chemokines and cytokines during infection.

1	clear the organisms within a week, with consequent sterile immunity. Studies with knockout mice have been instrumental in dissecting the roles played by chemokines and cytokines during infection. For example, interferon γ, tumor necrosis factor, and CCR2 are essential in controlling infection. While innate immunity is sufficient to control infection, the acquired immune response is required for sterile immunity. Immunity is cell mediated; antibody plays no measurable role. The critical effector cells are cytotoxic (CD8+) T cells that recognize and lyse infected cells; the resulting extracellular bacteria are killed by circulating activated phagocytes.

1	A hallmark of the L. monocytogenes model is that killed vaccines do not provide protective immunity. The explanation for this fundamental observation is multifactorial, involving the generation of appropriate cytokines and the compartmentalization of bacterial proteins for antigen processing and presentation. Because the organism has the capacity to induce a robust cell-mediated immune response, attenuated strains have been engineered to express foreign antigens and are undergoing clinical studies as therapeutic vaccines for cancer.

1	L. monocytogenes usually enters the body via the gastrointestinal tract in foods. Listeriosis is most often sporadic, although outbreaks do occur. No epidemiologic or clinical evidence supports person-toperson transmission (other than vertical transmission from mother to fetus) or waterborne infection. In line with its survival and multiplication at refrigeration temperatures, L. monocytogenes is commonly found in processed and unprocessed foods of animal and plant origin, especially soft cheeses, delicatessen meats, hot dogs, milk, and cold salads; fresh fruits and vegetables can also transmit the organism. Because food supplies are increasingly centralized and normal hosts tolerate the organism well, outbreaks may not be immediately apparent. The U.S. Food and Drug Administration has a zero-tolerance policy for L. monocytogenes in ready-to-eat foods.

1	Symptoms of listerial infection overlap greatly with those of other infectious diseases. Timely diagnosis requires that the illness be considered in groups at risk: pregnant women; elderly persons; neonates; individuals immunocompromised by organ transplantation, cancer, or treatment with tumor necrosis factor antagonists or glucocorticoids; and patients with a variety of chronic medical conditions, including alcoholism, diabetes, renal disease, and rheumatologic and hepatic illnesses. Meningitis in older adults (especially with parenchymal brain involvement or subcortical brain abscess) should trigger consideration of L. monocytogenes infection and treatment. Listeriosis occasionally affects healthy, young, nonpregnant individuals. HIV-infected patients are at risk; however, listeriosis seems to be prevented by trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis targeting other AIDS-related infections. The diagnosis is typically made by culture of blood, cerebrospinal fluid (CSF), or

1	to be prevented by trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis targeting other AIDS-related infections. The diagnosis is typically made by culture of blood, cerebrospinal fluid (CSF), or amniotic fluid.

1	L. monocytogenes may be confused with “diphtheroids” or pneumococci in Gram-stained CSF or may be gram-variable and confused with Haemophilus species. Polymerase chain reaction diagnostics have been described but are not widely available, and serology is not clinically useful. Listerial infections present as several clinical syndromes, of which meningitis and septicemia are most common. Monocytosis is seen in infected rabbits but is not a hallmark of human infection.

1	Gastroenteritis Appreciated only since the outbreaks of the late 1980s, listerial gastroenteritis typically develops within 48 h of ingestion of a large inoculum of bacteria in contaminated foods. Attack rates are high (50–100%). L. monocytogenes is neither sought nor found in routine fecal cultures, but its involvement should be considered in outbreaks when cultures for other likely pathogens are negative. Sporadic intestinal illness appears to be uncommon. Manifestations include fever, diarrhea, headache, and constitutional symptoms. The largest reported outbreak occurred in an Italian school system and included 1566 individuals; ~20% of patients were hospitalized, but only one person had a positive blood culture. Isolated gastrointestinal illness does not require antibiotic treatment. Surveillance studies show that 0.1–5% of healthy asymptomatic adults may have stool cultures positive for the organism.

1	Bacteremia L. monocytogenes septicemia presents with fever, chills, and myalgias/arthralgias and cannot be differentiated from septicemia involving other organisms. Meningeal symptoms, focal neurologic findings, or mental status changes may suggest the diagnosis. Bacteremia is documented in 70–90% of cancer patients with listeriosis. A nonspecific flulike illness with fever is a common presentation in pregnant women. Endocarditis of prosthetic and native valves is an uncommon complication, with reported fatality rates of 35–50% in case series. A lumbar puncture is often prudent, although not necessary, in pregnant women without central nervous system (CNS) symptoms.

1	Meningitis L. monocytogenes causes ~5–10% of all cases of community-acquired bacterial meningitis in adults in the United States. Case-fatality rates are reported to be 15–26% and do not appear to have changed over time. This diagnosis should be considered in all older or chronically ill adults with “aseptic” meningitis. The presentation is 983 more frequently subacute (with illness developing over several days) than in meningitis of other bacterial etiologies, and nuchal rigidity and meningeal signs are less common. Photophobia is infrequent. Focal findings and seizures are common in some but not all series. The CSF profile in listerial meningitis most often shows white blood cell counts in the range of 100–5000/μL (rarely higher); 75% of patients have counts below 1000/μL, usually with a neutrophil predominance more modest than that in other bacterial meningitides. Low glucose levels and positive results on Gram’s staining are found ~30–40% of the time. Hydrocephalus can occur.

1	Meningoencephalitis and Focal CNS Infection L. monocytogenes can directly invade the brain parenchyma, producing either cerebritis or focal abscess. Approximately 10% of cases of CNS infection are macroscopic abscesses resulting from bacteremic seeding; the affected patients often have positive blood cultures. Concurrent meningitis can exist, but the CSF may appear normal. Abscesses can be misdiagnosed as metastatic or primary tumors and, in rare instances, occur in the cerebellum and the spinal cord. Invasion of the brainstem results in a characteristic severe rhombencephalitis, usually in otherwise healthy older adults (although there are numerous other infectious and noninfectious causes of this syndrome). The presentation may be biphasic, with a prodrome of fever and headache followed by asymmetric cranial nerve deficits, cerebellar signs, and hemiparetic and hemisensory deficits. Respiratory failure can occur. The subacute course and the often minimally abnormal CSF findings may

1	by asymmetric cranial nerve deficits, cerebellar signs, and hemiparetic and hemisensory deficits. Respiratory failure can occur. The subacute course and the often minimally abnormal CSF findings may delay the diagnosis, which may be suggested by MRI showing ring-enhancing lesions after gadolinium contrast and hyperintense lesions on diffusion-weighted imaging. MRI is superior to CT for the diagnosis of these infections.

1	Infection in Pregnant Women and Neonates Listeriosis in pregnancy is a severe and important infection. The usual presentation is a nonspecific acute or subacute febrile illness with myalgias, arthralgias, backache, and headache. Pregnant women with listeriosis are usually bacteremic. This syndrome should prompt blood cultures, especially if there is no other reasonable explanation. Involvement of the CNS is rare in the absence of other risk factors. Preterm delivery is a common complication, and the diagnosis may be made only post-partum. As many as 70–90% of fetuses from infected women can become infected. Prepartum treatment of bacteremic women enhances the chances of delivery of a healthy infant. Women usually do well after delivery: maternal deaths are very rare, even when the diagnosis is made late in pregnancy or post-partum. Overall mortality rates for fetuses infected in utero approach 50% in some series; among live-born neonates treated with antibiotics, mortality rates are

1	is made late in pregnancy or post-partum. Overall mortality rates for fetuses infected in utero approach 50% in some series; among live-born neonates treated with antibiotics, mortality rates are much lower (~20%). Granulomatosis infantiseptica is an overwhelming listerial fetal infection with miliary microabscesses and granulomas, most often in the skin, liver, and spleen. Less severe neonatal infection acquired in utero presents at birth. “Late-onset” neonatal illness typically develops ~10–30 days post-partum. Mothers of infants with late-onset disease are not ill.

1	No clinical trials have compared antimicrobial agents for the treatment of L. monocytogenes infections. Data from studies conducted in vitro and in animals as well as observational clinical data indicate that ampicillin is the drug of choice, although penicillin also is highly active. Adults should receive IV ampicillin at high doses (2 g every 6 h). Many experts recommend the addition of gentamicin for synergy (1.0–1.7 mg/kg every 8 h); retrospective uncontrolled trials are not conclusive, but one study suggests that gentamicin may not help. TMP-SMX, given IV, is the best alternative for the penicillin-allergic patient (15–20 mg of TMP/kg per day in divided doses every 6–8 h). The dosages recommended cover CNS infection and 984 bacteremia (see below for duration); dosages must be reduced for patients with renal insufficiency. One small nonrandomized study supports a combination of ampicillin and TMP-SMX. Case reports document success with vancomycin, imipenem, meropenem, linezolid,

1	for patients with renal insufficiency. One small nonrandomized study supports a combination of ampicillin and TMP-SMX. Case reports document success with vancomycin, imipenem, meropenem, linezolid, tetracycline, and macrolides, although there are also reports of clinical failure or disease development with some of these agents. Acquired resistance to antimicrobial agents has been sought but not found in large strain collections. Cephalosporins are not effective and should not be used. Neonates should receive ampicillin and gentamicin at doses based on weight.

1	The duration of therapy depends on the syndrome: 2 weeks for bacteremia, 3 weeks for meningitis, 6–8 weeks for brain abscess/ encephalitis, and 4–6 weeks for endocarditis in both neonates and adults. Early-onset neonatal disease may be more severe and should be treated for >2 weeks. Many individuals who are promptly diagnosed and treated recover fully, but permanent neurologic sequelae are common in patients with brain abscess or rhombencephalitis. Focal infections of visceral organs; the eye; the pleural, peritoneal, and pericardial spaces; the bones; and both native and prosthetic joints have all been reported. Of 100 live-born, treated neonates in one series, 60% recovered fully, 24% died, and 13% had long-term neurologic or other complications.

1	Healthy persons should take standard precautions to prevent food-borne illness: fully cooking meats, washing fresh vegetables, carefully cleaning utensils, and avoiding unpasteurized dairy products. In addition, persons at risk for listeriosis, including pregnant women, should avoid soft cheeses (hard cheeses and yogurt are not problematic) and should avoid or thoroughly reheat ready-to-eat and delicatessen foods. Tetanus C. Louise Thwaites, Lam Minh Yen Tetanus is an acute disease manifested by skeletal muscle spasm and autonomic nervous system disturbance. It is caused by a powerful neurotoxin produced by the bacterium Clostridium tetani and is completely preventable by vaccination. C. 177 Infectious Diseases tetani is found throughout the world, and tetanus commonly occurs where the vaccination coverage rate is low. In developed countries, the disease is seen occasionally in individuals who are incompletely vaccinated. In any setting, established tetanus is a severe disease with a

1	coverage rate is low. In developed countries, the disease is seen occasionally in individuals who are incompletely vaccinated. In any setting, established tetanus is a severe disease with a high mortality rate.

1	Tetanus is diagnosed on clinical grounds (sometimes with supportive laboratory confirmation of the presence of C. tetani; see “Diagnosis,” below), and case definitions are often used to facilitate clinical and epidemiologic assessments. The Centers for Disease Control and Prevention (CDC) defines tetanus as “the acute onset of hypertonia or … painful muscular contractions (usually of the muscles of the jaw and neck) and generalized muscle spasms without other apparent medical cause.” Neonatal tetanus is defined by the World Health Organization (WHO) as “an illness occurring in a child who has the normal ability to suck and cry in the first 2 days of life but who loses this ability between days 3 and 28 of life and becomes rigid and has spasms.” Given the unique presentation of neonatal tetanus, the history generally permits accurate classification of the illness with a high degree of probability.

1	Maternal tetanus is defined by the WHO as tetanus occurring during pregnancy or within 6 weeks after the conclusion of pregnancy (whether with birth, miscarriage, or abortion). C. tetani is an anaerobic, gram-positive, spore-forming rod whose spores are highly resilient and can survive readily in the environment throughout the world. Spores resist boiling and many disinfectants. In addition, C. tetani spores and bacilli survive in the intestinal systems of many animals, and fecal carriage is common. The spores or bacteria enter the body through abrasions, wounds, or (in the case of neonates) the umbilical stump. Once in a suitable anaerobic environment, the organisms grow, multiply, and release tetanus toxin, an exotoxin that enters the nervous system and causes disease. Very low concentrations of this highly potent toxin can result in tetanus (minimum lethal human dose, 2.5 ng/kg).

1	In 20–30% of cases of tetanus, no puncture entry wound is found. Superficial abrasions to the limbs are the commonest infection sites in adults. Deeper infections (e.g., attributable to open fracture, abortion, or drug injection) are associated with more severe disease and worse outcomes. In neonates, infection of the umbilical stump can result from inadequate umbilical cord care; in some cultures, for example, the cord is cut with grass or animal dung is applied to the stump. Circumcision or ear-piercing also can result in neonatal tetanus.

1	Tetanus is a rare disease in the developed world. Two cases of neonatal tetanus have occurred in the United States since 1989. Between 2001 and 2008, a total of 231 cases of tetanus were reported to the U.S. national surveillance system. Most cases occur in incompletely vaccinated or unvaccinated individuals. Vaccination status is known in 50% of cases reported in the United States between 1972 and 2009; among these cases, only 16% of patients had had three or more doses of tetanus toxoid.

1	Persons >60 years of age are at greater risk of tetanus because antibody levels decrease over time. One-third of recent cases in the United States were in persons >65 years old. Injection drug users—particularly those injecting heroin subcutaneously (“skin-popping”)— are increasingly recognized as a high-risk group (15% of all cases in 2001–2008). In 2004, an outbreak of tetanus occurred in the United Kingdom, which had previously reported low rates among drug users. The reasons for this outbreak remain unclear but are thought to involve a combination of heroin contamination, skin-popping, and incomplete vaccination. Since then, only seven sporadic cases have been reported in the United Kingdom.

1	Genome sequencing of C. tetani has allowed identification of several exotoxins and virulence factors. Only those bacteria producing tetanus toxin (tetanospasmin) can cause tetanus. Although closely related to the botulinum toxins in structure and mode of action, tetanus toxin undergoes retrograde transport into the central nervous system and thus produces clinical effects different from those caused by the botulinum toxins, which remain at the neuromuscular junction.

1	Toxin is transported by intra-axonal transport to motor nuclei of the cranial nerves or ventral horns of the spinal cord. Tetanus toxin is produced as a single 150-kDa protein that is cleaved to produce heavy (100-kDa) and light (50-kDa) chains linked by a disulfide bond and noncovalent forces. The carboxy terminal of the heavy chain binds to specific membrane components in presynaptic α-motor nerve terminals; evidence suggests binding to both polysialogangliosides and membrane proteins. This binding results in toxin internalization and uptake into the nerves. Once inside the neuron, the toxin enters a retrograde transport pathway, whereby it is transported proximally to the motor neuron body in what appears to be a highly specific process. Unlike other components of the endosomal contents, which undergo acidification following internalization, tetanus toxin is transported in a carefully regulated pH-neutral environment that prevents an acid-induced conformational change that would

1	which undergo acidification following internalization, tetanus toxin is transported in a carefully regulated pH-neutral environment that prevents an acid-induced conformational change that would result in light-chain expulsion into the surrounding cytosol.

1	The next stage in toxin trafficking is less clearly understood but involves tetanus toxin’s escaping normal lysosomal degradation processes and undergoing translocation across the synapse to the GABA-ergic presynaptic inhibitory interneuron terminals. Here the light chain, which is a zinc-dependent endopeptidase, cleaves vesicle-associated membrane protein 2 (VAMP2, also known as synaptobrevin). This molecule is necessary for presynaptic binding and release of neurotransmitter; thus tetanus toxin prevents transmitter release and effectively blocks inhibitory interneuron discharge. The result is unregulated activity in the motor nervous system. Similar activity in the autonomic system accounts for the characteristic features of skeletal muscle spasm and autonomic system disturbance. The increased circulating catecholamine levels in severe tetanus are associated with cardiovascular complications.

1	Relatively little is known about the processes of recovery from tetanus. Recovery can take several weeks. Peripheral nerve sprouting is involved in recovery from botulism, and similar central nervous system sprouting may occur in tetanus. Other evidence suggests toxin degradation as a mechanism of recovery. APPROACH TO THE PATIENT: The clinical manifestations of tetanus occur only after tetanus toxin has reached presynaptic inhibitory nerves. Once these effects become apparent, there may be little that can be done to affect disease progression. Treatment should not be delayed while the results of laboratory tests are awaited. Management strategies aim to neutralize remaining unbound toxin and support vital functions until the effects of the toxin have worn off. Recent interest has focused on intrathecal methods of antitoxin administration to neutralize toxin within the central nervous system and limit disease progression (see “Treatment,” below).

1	Tetanus produces a wide spectrum of clinical features that are broadly divided into generalized (including neonatal) and local. In the usually mild form of local tetanus, only isolated areas of the body are affected and only small areas of local muscle spasm may be apparent. If the cra-985 nial nerves are involved in localized cephalic tetanus, the pharyngeal or laryngeal muscles may spasm, with consequent aspiration or airway obstruction, and the prognosis may be poor. In the typical progression of generalized tetanus (Fig. 177-1), muscles of the face and jaw often are affected first, presumably because of the shorter distances toxin must travel up motor nerves to reach presynaptic terminals. Neonates typically present with inability to suck.

1	In assessing prognosis, the speed at which tetanus develops is important. The incubation period (time from wound to first symptom) and the period of onset (time from first symptom to first generalized spasm) are of particular significance; shorter times are associated with worse outcome. In neonatal tetanus, the younger the infant is when symptoms occur, the worse the prognosis.

1	The commonest initial symptoms are trismus (lockjaw), muscle pain and stiffness, back pain, and difficulty swallowing. In neonates, difficulty in feeding is the usual presentation. As the disease progresses, muscle spasm develops. Generalized muscle spasm can be very painful. Commonly, the laryngeal muscles are involved early or even in isolation. This is a life-threatening event as complete airway obstruction may ensue. Spasm of the respiratory muscles results in respiratory failure. Without ventilatory support, respiratory failure is the commonest cause of death in tetanus. Spasms strong enough to produce tendon avulsions and crush fractures have been reported, but this outcome is rare.

1	Autonomic disturbance is maximal during the second week of severe tetanus, and death due to cardiovascular events becomes the major risk. Blood pressure is usually labile, with rapid fluctuations from high to low accompanied by tachycardia. Episodes of bradycardia and heart block can also occur. Autonomic involvement is evidenced by gastrointestinal stasis, sweating, increased tracheal secretions, and acute (often high-output) renal failure. The diagnosis of tetanus is based on clinical findings. As stated above, treatment should not be delayed while laboratory tests are conducted. Culture of C. tetani from a wound provides supportive evidence. Serum anti-tetanus immunoglobulin G may also be measured in a sample taken before the administration of antitoxin or immunoglobulin. Serum levels >0.1 IU/mL are deemed protective and do not support the diagnosis of tetanus. If levels are below this threshold, a Wound infection, multiplication of Ctetani

1	Wound infection, multiplication of Ctetani Initial symptoms: muscle aches, trismus, myalgia Tetanus toxin uptake into nervous system and VAMP cleavage in GABA inhibitory neurons Cardiovascular instability: labile BP, tachyor bradycardia Pyrexia, increased respiratory and GI secretions Muscle spasm: local and generalized Cardiovascular and autonomic control Cessation of spasms, restoration of normal muscle tone Toxin degradation Further toxin effects causing widespread disinhibition of motor and autonomic nervous system 4–6 weeks FIGURE 177-1 Clinical and pathologic progression of tetanus. BP, blood pressure; GABA, γ-aminobutyric acid; GI, gastrointestinal; VAMP, vesicle-associated membrane protein (synaptobrevin).

1	FIGURE 177-1 Clinical and pathologic progression of tetanus. BP, blood pressure; GABA, γ-aminobutyric acid; GI, gastrointestinal; VAMP, vesicle-associated membrane protein (synaptobrevin). 986 bioassay for serum tetanus toxin may be helpful, but a negative result does not exclude the diagnosis. Polymerase chain reaction also has been used for detection of tetanus toxin, but its sensitivity is unknown. The few conditions that mimic generalized tetanus include strychnine poisoning and dystonic reactions to antidopaminergic drugs. Abdominal muscle rigidity is characteristically continuous in tetanus but is episodic in the latter two conditions. Cephalic tetanus can be confused with other causes of trismus, such as oropharyngeal infection. Hypocalcemia and meningoencephalitis are included in the differential diagnosis of neonatal tetanus.

1	If possible, the entry wound should be identified, cleaned, and debrided of necrotic material in order to remove anaerobic foci of infection and prevent further toxin production. Metronidazole (400 mg rectally or 500 mg IV every 6 h for 7 days) is the preferred antibiotic. An alternative is penicillin (100,000–200,000 IU/kg per day), although this drug theoretically may exacerbate spasms. Failure to remove pockets of ongoing infection may result in recurrent or prolonged tetanus.

1	Antitoxin should be given early in an attempt to deactivate any circulating tetanus toxin and prevent its uptake into the nervous system. Two preparations are available: human tetanus immune globulin (TIG) and equine antitoxin. TIG is the preparation of choice, as it is less likely to be associated with anaphylactoid reactions. Recommended therapy is 3000–5000 IU of TIG as a single IM dose, a portion of which should be injected around the wound. Equine-derived antitoxin is available widely and is used in low-income countries at a dosage of 10,000–20,000 U administered IM as a single dose or as divided doses after testing for hypersensitivity. Some evidence indicates that intrathecal administration of TIG inhibits disease progression and leads to a better outcome. The results of relevant studies have been supported by a meta-analysis of trials involving both adults and neonates, with TIG doses of 50–1500 IU administered intrathecally.

1	Spasms are controlled by heavy sedation with benzodiazepines. Chlorpromazine and phenobarbital are commonly used worldwide, and IV magnesium sulfate has been used as a muscle relaxant. A significant problem with all these treatments is that the doses necessary to control spasms also cause respiratory depression; thus, in resource-limited settings without mechanical ventilators, controlling spasms while maintaining adequate ventilation is problematic, and respiratory failure is a common cause of death. In locations with ventilation equipment, severe spasms are best controlled with a combination of sedatives or magnesium and relatively short-acting, cardiovascularly inert, nondepolarizing neuromuscular blocking agents that allow titration against spasm intensity. Infusions of propofol have also been used successfully to control spasms and provide sedation.

1	It is important to establish a secure airway early in severe tetanus. Ideally, patients should be nursed in calm, quiet environments because light and noise can trigger spasms. Tracheal secretions are increased in tetanus, and dysphagia due to pharyngeal involvement combined with hyperactivity of laryngeal muscles makes endotracheal intubation difficult. Patients may need ventilator support for several weeks. Thus tracheostomy is the usual method of securing the airway in severe tetanus.

1	Cardiovascular instability in severe tetanus is notoriously difficult to treat. Rapid fluctuations in blood pressure and heart rate can occur. Cardiovascular stability is improved by increasing sedation with IV magnesium sulfate (plasma concentration, 2–4 mmol/L or titrated against disappearance of the patella reflex), morphine, or other sedatives. In addition, drugs acting specifically on the cardiovascular system (e.g., esmolol, calcium antagonists, and inotropes) may be required. Short-acting drugs that allow rapid titration are preferred; particular care should be taken when longer-acting β antagonists are administered, as their use has been associated with hypotensive cardiac arrest. Complications arising from treatment are common and include thrombophlebitis associated with diazepam injection, ventilator-associated pneumonia, central-line infections, and septicemia. In some centers, prophylaxis against deep-vein thrombosis and thromboembolism is routine.

1	Recovery from tetanus may take 4–6 weeks. Patients must be given a full primary course of immunization, as tetanus toxin is poorly immunogenic and the immune response following natural infection is inadequate. Rapid development of tetanus is associated with more severe disease and poorer outcome; it is important to note time of onset and length of incubation period. More sophisticated modeling has revealed other important predictors of prognosis (Table 177-1). Few studies have formally addressed long-term outcomes of tetanus. However, it is generally accepted that recovery is typically complete unless periods of hypoventilation have been prolonged or other complications have ensued. Studies of children and neonates have suggested a higher incidence of neurologic sequelae. Neonates may be at increased risk of learning disabilities, behavioral problems, cerebral palsy, and deafness.

1	Tetanus is prevented by good wound care and immunization (Chap. 148). In neonates, use of safe, clean delivery and cord-care practices as well as maternal vaccination are essential. The WHO guidelines for tetanus vaccination consist of a primary course of three doses in infancy, boosters at 4–7 and 12–15 years of age, and one booster in adulthood. In the United States, the CDC suggests an additional dose at 14–16 months and boosters every 10 years. “Catch-up” schedules recommend a three-dose primary course for unimmunized adolescents followed by two further doses. For persons who have received a complete primary course in childhood but no further boosters, two doses at least 4 weeks apart are recommended.

1	Standard WHO recommendations for prevention of maternal and neonatal tetanus call for administration of two doses of tetanus toxoid at least 4 weeks apart to previously unimmunized pregnant women. However, in high-risk areas, a more intensive approach has been successful, with all women of childbearing age receiving a primary course along with education on safe delivery and postnatal practices. Individuals sustaining tetanus-prone wounds should be immunized if their vaccination status is incomplete or unknown or if their last booster was given >10 years earlier. Patients sustaining wounds not classified as clean or minor should also undergo passive immunization with TIG. It is recommended that tetanus toxoid be given in conjunction with diphtheria toxoid in a preparation with or without acellular pertussis: DTaP for children <7 years old, Td for 7to 9-year olds, and Tdap for children >9 years old and adults.

1	In the early 1980s, tetanus caused more than 1 million deaths a year, accounting for an estimated 5% of maternal deaths and 14% of all neonatal deaths. In 1989, the World Health Age >70 years Younger age, premature birth Incubation period <7 days Incubation period <6 days Short time from first symptom to Delay in hospital admission Grass used to cut cord Puerperal, IV, postsurgery, burn entry Period of onseta <48 h Heart rate >140 beats/minb Systolic blood pressure >140 mmHgb Severe disease or spasmsb Temperature >38.5°Cb aTime from first symptom to first generalized spasm. bAt hospital admission.

1	Assembly adopted a resolution to eliminate neonatal tetanus by the year 2000; elimination was defined as <1 case/1000 live births in every district in every country. By 1999, elimination was still to be achieved in 57 countries and the deadline was extended until 2005, with the additional target of eliminating maternal tetanus (tetanus occurring during pregnancy or within 6 weeks of its end). Ratification of the Millennium Development Goals, in particular goal 4 (achieving a two-thirds reduction in the mortality rate among children under 5 by 2015), has further focused attention on reducing deaths from vaccine-preventable disease, particularly in the first 4 weeks of life.

1	Because vaccination reduces the incidence of neonatal tetanus by an estimated 94%, immunization of pregnant women with two doses of tetanus toxoid at least 4 weeks apart has been the primary method of maternal and neonatal tetanus elimination. In some areas, all women of childbearing age have been targeted as a means of increasing vaccination coverage. In addition, educational programs have focused on improving hygiene during the birth process, an intervention that in itself is estimated to reduce neonatal tetanus deaths by up to 40%.

1	The latest available data show that 34 countries have yet to eliminate maternal and neonatal tetanus, although incidence has declined significantly. Worldwide, deaths from neonatal tetanus fell by 92% between 1990 and 2008; in the latter year, with 84% of newborns protected from the disease by maternal vaccination, there were an estimated 59,000 neonatal tetanus deaths. Despite this relative success, immunization programs need to be ongoing as there is no tetanus herd immunity effect and C. tetani contamination of soil and feces is widespread.

1	Botulism Susan Maslanka, Agam K. Rao Botulism, recognized at least since the eighteenth century, is a neu-roparalytic disease caused by botulinum toxin, one of the most toxic substances known. While initially thought to be caused only by the ingestion of botulinum toxin in contaminated food (food-borne 178 botulism), three additional forms caused by in situ toxin production after germination of spores in either a wound or the intestine are now recognized worldwide: wound botulism, infant botulism, and adult intestinal colonization botulism. In addition to occurring in these recognized natural forms of the disease, botulism symptoms have been reported in patients receiving injections of botulinum toxin for cosmetic or therapeutic purposes (iatrogenic botulism). Moreover, botulism was reported after inhalation of botulinum toxin in a laboratory setting. All forms of botulism manifest as a relatively distinct clinical syndrome of symmetric cranial-nerve palsies followed by descending

1	after inhalation of botulinum toxin in a laboratory setting. All forms of botulism manifest as a relatively distinct clinical syndrome of symmetric cranial-nerve palsies followed by descending bilateral flaccid paralysis of voluntary muscles, which may progress to respiratory compromise and death. The mainstays of therapy are meticulous intensive care and treatment with antitoxin as soon as botulism is suspected and before other illnesses have been ruled out.

1	Seven serologically distinct confirmed serotypes of botulinum toxin (A through G) have been confirmed. Botulinum toxin is produced by four recognized species of clostridia: Clostridium botulinum, Clostridium argentinense, Clostridium baratii, and Clostridium butyricum. Certain strains may produce more than one serotype. All are anaerobic gram-positive organisms that form subterminal spores; C. botulinum and C. argentinense spores have been recovered from the environment. The spores survive environmental conditions and ordinary cooking procedures. Toxin production, however, requires a rare confluence of product storage conditions: an anaerobic environment, a pH of >4.6, low salt and sugar concentrations, and temperatures of >4°C. Although commonly ingested, spores do not normally germinate and produce toxin in the adult human intestine.

1	Food-borne botulism is caused by consumption of foods contami-987 nated with botulinum toxin; no confirmed host-specific factors are involved in the disease. Wound botulism is caused by contamination of wounds with C. botulinum spores, subsequent spore germination, and toxin production in the anaerobic milieu of an abscess or a wound. Infant botulism results from absorption of toxin produced in situ by toxigenic clostridia colonizing the intestine of children ≤1 year of age. Colonization is thought to occur because the normal bowel microbiota is not yet fully established; this theory is supported by studies in animals. Adult intestinal colonization botulism, a very rare form that is poorly understood, has a pathology similar to that of infant botulism but occurs in adults; typically, patients have some anatomic or functional bowel abnormality or have recently used antibiotics that may help toxigenic clostridia compete more successfully against the normal bowel microbiota. Despite

1	patients have some anatomic or functional bowel abnormality or have recently used antibiotics that may help toxigenic clostridia compete more successfully against the normal bowel microbiota. Despite antitoxin treatment, relapse due to intermittent intraluminal production of toxin may be observed in patients with adult intestinal colonization botulism.

1	Regardless of how exposure occurs, botulinum neurotoxin enters the vascular system and is transported to peripheral cholinergic nerve terminals, including neuromuscular junctions, postganglionic parasympathetic nerve endings, and peripheral ganglia. Botulinum toxin is a zinc-endopeptidase protein of ~150 kDa, consisting of a 100-kDa heavy chain and a 50-kDa light chain. Steps in neurotoxin activity include (1) heavy-chain binding to nerve terminals, (2) internalization in endocytic vesicles, (3) translocation of the light chain to cytosol, and (4) light-chain serotype-specific cleavage of one of several proteins involved in the release of the neurotransmitter acetylcholine. Inhibition of acetylcholine release by any of the seven toxin serotypes results in characteristic flaccid paralysis. Recovery follows sprouting of new nerve terminals.

1	All botulinum toxin serotypes have been demonstrated to cause botulism in nonhuman primates. Human cases associated with serotypes A, B, E, and F are reported each year. Serotype A produces the most severe syndrome, with the greatest proportion of patients requiring mechanical ventilation. Serotype B appears to cause milder disease than type A in both food-borne and infant botulism. Serotype E, most often associated with foods of aquatic origin, produces a syndrome of variable severity. The rare cases of illness caused by toxin serotype F, whether in infants or adults, are characterized by rapid progression to quadriplegia and respiratory failure but also by relatively rapid recovery. Recent studies have shown that at least some serotypes can be differentiated into subtypes through neurotoxin gene sequencing; however, the impact of these subtype differences on clinical illness is not yet known.

1	Botulism occurs worldwide, but the number of cases reported varies among countries and regions. The variation may be due not only to actual differences in incidence but also to (1) availability of resources to identify botulism, a rare disease; (2) differences in reporting requirements; and (3) limited external access to data collections. There is no universal surveillance system to capture worldwide botulism incidence. However, 30 countries currently participate in voluntary reporting of botulism cases to the European Union through an established surveillance system that includes standardized case definitions similar to those used in the United States and Canada. Other countries (e.g., Argentina, China, Thailand, Japan) maintain independent botulism surveillance.

1	Food-Borne Botulism From 1899 to 2011, 1225 food-borne botulism events (single cases or outbreaks) were reported in the United States; from 1990 to 2000, a median of 23 cases were reported annually. Most such events (~80%) involve vegetables or fish/ aquatic animals, usually home-preserved (canned, jarred). Native communities in both the United States (Alaska) and Canada have a high incidence of food-borne botulism due to traditional food-preparation practices; 85% of all cases in Canada occur in Native communities. Outbreaks typically involve two or three cases; however, one restaurant-associated outbreak in 1977 affected 59 persons. Worldwide, the highest incidence rate is reported from Georgia and Armenia in the southern Caucasus region, where illness is also associated with home-canning practices. Outbreaks in Asia are 988 attributable to consumption of home-preserved fish or vegetable products such as bean curd and bamboo shoots. In parts of Europe, including Poland, France, and

1	practices. Outbreaks in Asia are 988 attributable to consumption of home-preserved fish or vegetable products such as bean curd and bamboo shoots. In parts of Europe, including Poland, France, and Germany, illness is often associated with home-preserved meat such as ham or sausage. Since 1950, commercial products have rarely been implicated in botulism in the United States, and botulism from commercial products is most often attributed to consumer error in storage or cooking. However, manufacturer deficiencies do occur. In 2007, botulism developed in eight persons in the United States who consumed a commercially canned hot-dog chili sauce. Significant deficiencies discovered by regulatory authorities involved 91 different products and resulted in the recall of 111 million cans of food.

1	Wound Botulism This form of disease was first recognized in 1951 as a result of a review of the clinical records on an accidental injury in 1943. Between 1943 and 2011, 491 cases of wound botulism were reported in the United States; 97% of cases reported after 1990 were associated with injection drug use. The typical patient was a 30to 50-year-old resident of the western United States with a long history of black-tar heroin injection. In the early 2000s, wound botulism associated with drug use emerged in Europe, and at least two case clusters have been reported. Infant Botulism More than 3900 infant botulism cases have been reported worldwide (84% in the United States alone) since this form of the disease was first recognized in 1976; ~80–100 cases (commonly caused by serotypes A and B) are reported annually in the United States.

1	Adult Intestinal Colonization Botulism This form of botulism is difficult to confirm because it is poorly understood and because no clear guidelines are available to help differentiate it from other adult botulism cases. Often these cases are caused by C. baratii type F, but the involvement of both C. botulinum type A and C. butyricum type E has been reported.

1	Iatrogenic Botulism Paralysis of variable severity has followed injection of licensed botulinum toxin products for treatment of conditions involving hypertonicity of large muscle groups. The U.S. Food and Drug Administration received 658 reports of adverse events related to botulinum toxin use—some very serious—between 1997 and 2006. Although some patients had symptoms consistent with botulism, no cases were laboratory confirmed. Injection of approved doses of licensed products for cosmetic purposes has not been associated with botulism. However, four cases of laboratory-confirmed botulism resulted from illegal injection of research-grade toxin for cosmetic purposes in a U.S. medical facility in 2004. Inhalational Botulism Inhalational botulism does not occur naturally. One report from Germany has described botulism resulting from possible inhalational exposure to botulinum toxin in a laboratory incident.

1	Intentional Botulism Botulinum toxin has been “weaponized” by governments and terrorist organizations. An attack might use aerosolization of toxin or contamination of foods or beverages ranging in scope from small-scale tampering to contamination of a widely distributed food item. An unnatural event may be suggested by unusual relationships between patients (e.g., a visit to the same building), atypical exposure vehicles, or atypical toxin serotypes.

1	The distinctive clinical syndrome of botulism consists of symmetric cranial-nerve palsies followed by bilateral descending flaccid paralysis that may progress to respiratory failure and death. The incubation period from ingestion of contaminated food to onset of symptoms in food-borne botulism is usually 8–36 h but can be as long as 10 days and is dose dependent. Incubation periods of 4–17 days have been documented in wound botulism associated with accidental injury. However, estimation is difficult in cases involving injection drug users because most of these patients inject drugs several times daily. Similarly, the incubation period for infant botulism has not been established, but the fact that the illness has affected infants <3 days old suggests that this interval may be very short.

1	Cranial nerve deficits may include some of the following: diplopia, dysarthria, dysphonia, ptosis, ophthalmoplegia, facial paralysis, and impaired gag reflex. Pupillary reflexes may be depressed, and fixed or dilated pupils are sometimes noted. Autonomic symptoms such as dizziness, dry mouth, and very dry, occasionally sore throat are common. Constipation due to paralytic ileus is nearly universal, and urinary retention is also common. Patients are afebrile and remain alert and oriented. Respiratory failure may occur due to either paralysis of the diaphragm and accessory breathing muscles or pharyngeal collapse secondary to cranial nerve paralysis. Weakness descends, often rapidly, from the head to involve the neck, arms, thorax, and legs; occasionally, weakness is asymmetric. Deep tendon reflexes may be normal or may progressively disappear. Paresthesias, while rare, have been reported and may represent secondary nerve compression from immobility due to paralysis. Absence of cranial

1	reflexes may be normal or may progressively disappear. Paresthesias, while rare, have been reported and may represent secondary nerve compression from immobility due to paralysis. Absence of cranial nerve palsies or their onset after the appearance of other true neurologic symptoms makes botulism highly unlikely. Nausea, vomiting, and abdominal pain may precede or follow the onset of paralysis in food-borne botulism. Infants with botulism typically present with reduced ability to suck and swallow, constipation, weakened voice, ptosis, sluggish pupils, hypotonia, and floppy neck; as in adults, illness can progress to generalized flaccidity and respiratory compromise.

1	Even when intubated, patients can respond to questions by moving their fingers or toes unless paralysis has affected the digits. In some instances, unfortunately, the severe ptosis, expressionless face, and weak phonation of patients with botulism have been interpreted as signs of mental status changes due to alcohol intoxication, drug overdose, encephalitis, or meningitis—a conclusion that delays an accurate diagnosis. Because of skeletal muscle paralysis, patients experiencing respiratory distress may appear placid and detached even as they near respiratory arrest. Death in untreated botulism is usually due to airway obstruction from pharyngeal muscle paralysis and inadequate tidal volume resulting from paralysis of diaphragmatic and accessory respiratory muscles. Death can also result from hospital-associated infections and other sequelae of long-term paralysis, hospitalization, and mechanical ventilation.

1	A history of preparing home-canned foods may assist with the diagnosis. Patients with wound botulism may or may not have a discernible wound or abscess. A history of injection drug use or the presence of track marks may raise suspicion of the diagnosis. Clinical improvement follows sprouting of new nerve terminals and may take weeks to months. Patients often require outpatient rehabilitation therapy and may experience residual deficits.

1	Botulism is diagnosed primarily on clinical grounds, with laboratory confirmation by specific tests that identify botulinum toxin in clinical and food samples. In the setting of an outbreak with multiple patients presenting to the same treatment facility, the diagnosis is apparent as long as physicians recognize that cases within a cluster may have varied signs and symptoms. The temporal occurrence of two or more cases with symptoms compatible with botulism is essentially pathognomonic because other illnesses that resemble botulism do not usually occur in clusters. In lone (sporadic) cases, the diagnosis is often missed. The rarity of this disease prevents many physicians from gaining experience with its clinical diagnosis, and some patients present with signs and symptoms that do not fit the classic pattern. Assessing clinical characteristics of other paralytic illnesses in single cases is sometimes critical to rule in or rule out the diagnosis of botulism.

1	In adults, a food history and the names of contacts who may have shared foods should be obtained before illness progresses to respiratory failure; specific questions should include information about the consumption of home-preserved and/or exotic foods and of products requiring refrigeration that have been left at room temperature in sealed plastic containers or bags. A history of recent consumption of home-canned food substantially enhances the probability of food-borne botulism. Ascertainment of the patient’s behavioral history related to injection drug use is critical to the diagnosis of wound botulism unless an accidental wound is evident. Caretakers’ observations up to and including the onset of symptoms are vital to the diagnosis of infant botulism. A history of recent abdominal surgery or antibiotic use may be important in the diagnosis of adult intestinal colonization botulism.

1	Differential Diagnosis The illnesses most commonly considered in the differential diagnosis of adult botulism cases include Guillain-Barré syndrome (GBS), myasthenia gravis, stroke syndromes, Eaton-Lambert syndrome, and tick paralysis. Less likely possibilities are poisoning by tetrodotoxin, shellfish, or a host of rarer agents and antimicrobial drug–associated paralysis. A thorough history and a meticulous physical examination can effectively eliminate most alternative diagnoses, but a workup for other diagnoses should not delay treatment with botulinum antitoxin.

1	GBS, a rare autoimmune demyelinating polyneuropathy that often follows an acute infection, presents most often as an ascending paralysis. Case clusters are rare. Occasional GBS cases present as the Miller Fisher variant, whose characteristic triad of ophthalmoplegia, ataxia, and areflexia is easily mistaken for the early descending paralysis of botulism. Protein levels in cerebrospinal fluid (CSF) are elevated in GBS; because this increase may be delayed until several days after symptom onset, an early lumbar puncture with a negative result may need to be repeated. In contrast, CSF findings are generally normal in botulism, although marginally elevated CSF protein concentrations have been reported. In experienced hands, electromyography may differentiate GBS from botulism. The edrophonium (Tensilon) test is sometimes of value in distinguishing botulism (usually a negative result) from myasthenia gravis (usually a positive result).

1	The edrophonium (Tensilon) test is sometimes of value in distinguishing botulism (usually a negative result) from myasthenia gravis (usually a positive result). In most cerebrovascular accidents, physical examination reveals asymmetry of paralysis and upper motor neuron signs. Brain imaging can reveal the rare basilar stroke that produces symmetric bulbar palsies. Eaton-Lambert syndrome usually manifests as proximal limb weakness in a patient already debilitated by cancer. Tick paralysis is a rare flaccid condition closely resembling botulism and caused by neurotoxins of certain ticks.

1	Botulism-Specific Laboratory Tests Botulism is confirmed in the laboratory by demonstration of toxin in clinical specimens (e.g., serum, stool, gastric aspirate, and sterile-water enema samples) or in samples of ingested foods. Isolation of toxigenic clostridia from stool also provides evidence of botulism. Wound cultures yielding the organism are highly suggestive in symptomatic cases. The universally accepted method for confirmation of botulism is the mouse bioassay, which is available only in specialized laboratories. Specific guidance about what specimens to collect should be obtained from the testing laboratory because the requirements vary with the form of botulism suspected. Clinical specimens collected early in the hospital admission process should be submitted for testing; toxin results may be negative if specimens are collected >7 days after symptom onset. Because of the extreme potency of botulinum toxin, a test may yield a negative result even when a patient has botulism;

1	results may be negative if specimens are collected >7 days after symptom onset. Because of the extreme potency of botulinum toxin, a test may yield a negative result even when a patient has botulism; thus, a negative result does not exclude this diagnosis. In suspected wound botulism, material from abscesses should be collected in anaerobic culture tubes. New laboratory tests for botulism are being developed but remain experimental. Nerve conduction studies showing reduced amplitude of motor potentials—with or without potentiation by rapid repetitive stimulation in weak muscles—and needle electromyography showing small-motor-unit action potentials are consistent with botulism; these results and those that make alternative diagnoses more likely may be useful. Standard blood work and radiologic studies are not useful in diagnosing botulism.

1	The cornerstones of treatment for botulism are meticulous intensive care and immediate administration of botulinum antitoxin. Because antitoxin is most beneficial early in the course of clinical illness, it should be administered as soon as botulism is suspected and before the time-consuming workup for other illnesses is complete. Persons of all ages (including infants) in whom botulism is suspected should be hospitalized immediately so that respiratory failure—the usual cause of death—can be detected and man-989 aged promptly. Vital capacity should be monitored frequently and mechanical ventilation provided as needed. Botulinum antitoxin can limit the progression of illness because it neutralizes toxin molecules in the circulation that have not yet bound to nerve endings. However, antitoxin does not reverse existing paralysis, which may take weeks to improve. In the United States, there are two licensed antitoxin products: Botulism Antitoxin Heptavalent® (BAT; Emergent Biosolutions,

1	does not reverse existing paralysis, which may take weeks to improve. In the United States, there are two licensed antitoxin products: Botulism Antitoxin Heptavalent® (BAT; Emergent Biosolutions, Rockville, MD), an equine-derived heptavalent (A through G) product enzymatically de-speciated for treatment of all forms of adult botulism and infant cases not involving serotypes A and B; and Botulism Immune Globulin Intravenous (BabyBIG®; California Department of Public Health, Sacramento, CA), a human-derived product for treating infant botulism caused by serotype A and/or B only. Antitoxin is also available in some other countries. Aminoglycosides and other medications that block the neuromuscular junction may potentiate botulism and should be avoided.

1	In wound botulism, suspect wounds and abscesses should be cleaned, debrided, and drained promptly. The role of penicillin and metronidazole in treatment and decolonization is unclear. It has been hypothesized that antimicrobial agents may increase circulating botulinum toxin from lysis of bacterial cells. Person-to-person transmission of botulism does not occur. Universal precautions are the only infection-control measures required during inpatient care. NOTIFICATION, EXPERT CONSULTATION, AND ANTITOXIN PROVISION Every botulism case is a public health emergency. Antitoxin is not universally available. Some countries maintain stockpiles of antitoxin for immediate response, whereas others must access supplies from other nations when an outbreak occurs.

1	In the United States, clinicians must report every suspected case, regardless of form, on an emergency basis to their state health department. The state health department may put the physician in contact with the 24/7 botulism consultation service at the Centers for Disease Control and Prevention (CDC) through the CDC Emergency Operations Center (770-488-7100) or a locally available service. The botulism consultant will review the case and determine whether botulism is likely. If indicated, the consultant will coordinate laboratory confirmation at appropriate testing facilities and facilitate emergency shipment of antitoxin for all adult cases and for infant cases not involving serotypes A and B. In this country, botulinum antitoxin for noninfant cases is available exclusively from the CDC. Physicians who see suspected infant botulism cases should contact the California Department of Public Health Infant Botulism Treatment and Prevention Program (510-231-7600), which provides 24-h

1	CDC. Physicians who see suspected infant botulism cases should contact the California Department of Public Health Infant Botulism Treatment and Prevention Program (510-231-7600), which provides 24-h consultation and distributes antitoxin (BabyBIG) for the treatment of infant botulism nationwide. Except in cases involving infants who reside in California, laboratory testing requests must still be authorized by the state health department where the infant is located or by the CDC.

1	No prophylaxis or licensed vaccine is available against botulism. Home-canning instructions and equipment have changed over the years. Up-to-date canning instructions from reliable sources (e.g., the U.S. Department of Agriculture or the U.S. Food and Drug Administration) should be followed to ensure food safety. Processed food should be stored properly and heated thoroughly prior to consumption. Because of the possible presence of spores, honey should not be given to infants (≤12 months of age). Injection of illicit drugs should be avoided. All wounds should be meticulously cleaned to eliminate possible contamination with bacterial spores. Clinicians should educate individuals or family members of at-risk individuals, including infants, illegal drug users, and preparers of home-preserved foods. The authors thank Dr. Jeremy Sobel for his valued contributions to the previous version of this chapter.

1	The authors thank Dr. Jeremy Sobel for his valued contributions to the previous version of this chapter. 990 gas gangrene and other Clostridial Infections Amy E. Bryant, Dennis L. Stevens The genus Clostridium encompasses more than 60 species that may be commensals of the gut microflora or may cause a variety of infections in humans and animals through the production of a plethora of pro-179 teinaceous exotoxins. C. tetani and C. botulinum, for example, cause specific clinical disease by elaborating single but highly potent toxins. In contrast, C. perfringens and C. septicum cause aggressive necrotizing infections that are attributable to multiple toxins, including bacterial proteases, phospholipases, and cytotoxins.

1	Vegetative cells of Clostridium species are pleomorphic, rod-shaped, and arranged singly or in short chains (Fig. 179-1); the cells have rounded or sometimes pointed ends. Although clostridia stain gram-positive in the early stages of growth, they may appear to be gram-negative or gram-variable later in the growth cycle or in infected tissue specimens. Most strains are motile by means of peritrichous flagella; C. septicum swarms on solid media. Nonmotile species include C. perfringens, C. ramosum, and C. innocuum. Most species are obligately anaerobic, although clostridial tolerance to oxygen varies widely; some species (e.g., C. septicum, C. tertium) will grow but will not sporulate in air.

1	Clostridia produce more protein toxins than any other bacterial genus, and more than 25 clostridial toxins lethal to mice have been identified. These proteins include neurotoxins, enterotoxins, cytotoxins, collagenases, permeases, necrotizing toxins, lipases, lecithinases, hemolysins, proteinases, hyaluronidases, DNases, ADPribosyltransferases, and neuraminidases. Botulinum and tetanus neurotoxins are the most potent toxins known, with lethal doses of 0.2–10 ng/kg for humans. Epsilon toxin, a 33-kDa protein produced by C. perfringens types B and D, causes edema and hemorrhage in the brain, heart, spinal cord, and kidneys of animals. It is among the most lethal of the clostridial toxins and is considered a potential agent of bioterrorism (Chap. 261e). The genomic sequences of some pathogenic clostridia are now available and are likely to facilitate a comprehensive approach to understanding the virulence factors involved in clostridial pathogenesis.

1	Clostridium species are widespread in nature, forming endospores that are commonly found in soil, feces, sewage, and marine sediments. The ecology of C. perfringens in soil is greatly influenced by the degree and duration of animal husbandry in a given location and is relevant to the incidence of gas gangrene caused by contamination of war wounds with soil. For example, the incidence of clostridial gas gangrene is higher in agricultural regions of Europe than in the Sahara Desert of Africa. Similarly, the incidences of tetanus and food-borne botulism are clearly related to the presence of clostridial spores in soil, water, and many foods. Clostridia are present in large numbers in the indigenous microbiota of the intestinal tract of humans and animals, in the female genital tract, and on the oral mucosa. It should be noted that not all commensal clostridia are toxigenic. FIGURE 179-1 Scanning electron micrograph of C. perfringens.

1	worldwide. In developing nations, food poisoning, necrotizing enterocolitis, and gas gangrene are common because large portions of the population are poor and have little or no immediate access to health care. These infections remain prevalent in developed countries as well. Gas gangrene commonly follows knife or gunshot wounds or vehicular accidents or develops as a complication of surgery or gastrointestinal carcinoma. Severe clostridial infections have emerged as a health threat to injection drug users and to women undergoing childbirth or abortion. Historically, clostridial gas gangrene has been the scourge of the battlefield. The global political situation portends another possible scenario involving mass casualties of war or terrorism, with extensive injuries conducive to gas gangrene. Thus there is an ongoing need to develop novel strategies to prevent or attenuate the course of clostridial infections in both civilians and military personnel. Vaccination against exotoxins

1	Thus there is an ongoing need to develop novel strategies to prevent or attenuate the course of clostridial infections in both civilians and military personnel. Vaccination against exotoxins important in pathogenesis would be of great benefit in developing nations and could also be used safely in at-risk populations such as the elderly, patients with diabetes who may require lower-limb surgery due to trauma or poor circulation, and those undergoing intestinal surgery. Moreover, a hyperimmune globulin would be a valuable tool for prophylaxis in victims of acute traumatic injury or for attenuation of the spread of infection in patients with established gas gangrene.

1	Life-threatening clostridial infections range from intoxications (e.g., food poisoning, tetanus) to necrotizing enteritis/colitis, bacteremia, myonecrosis, and toxic shock syndrome (TSS). Tetanus and botulism are discussed in Chaps. 177 and 178, respectively. Colitis due to C. difficile is discussed in Chap. 161. Of open traumatic wounds, 30–80% reportedly are contaminated with clostridial species. In the absence of devitalized tissue, the presence of clostridia does not necessarily lead to infection. In traumatic injuries, clostridia are isolated with equal frequency from both suppurative and well-healing wounds. Thus, diagnosis and treatment of clostridial infection should be based on clinical signs and symptoms and not solely on bacteriologic findings.

1	Clostridial species may be found in polymicrobial infections also involving microbial components of the indigenous flora. In these infections, clostridia often appear in association with non-spore-forming anaerobes and facultative or aerobic organisms. Head and neck infections, conjunctivitis, brain abscess, sinusitis, otitis, aspiration pneumonia, lung abscess, pleural empyema, cholecystitis, septic arthritis, and bone infections all may involve clostridia. These conditions are often associated with severe local inflammation but may lack the characteristic systemic signs of toxicity and rapid progression seen in other clostridial infections. In addition, clostridia are isolated from ~66% of intraabdominal infections in which the mucosal integrity of the bowel or respiratory system has been compromised. In this setting, C. ramosum, C. perfringens, and C. bifermentans are the most commonly isolated species. Their presence does not invariably lead to a poor outcome. Clostridia have been

1	compromised. In this setting, C. ramosum, C. perfringens, and C. bifermentans are the most commonly isolated species. Their presence does not invariably lead to a poor outcome. Clostridia have been isolated from suppurative infections of the female genital tract (e.g., ovarian or pelvic abscess) and from diseased gallbladders. Although the most frequently isolated species is C. perfringens, gangrene is not typically observed; however, gas formation in the biliary system can lead to emphysematous cholecystitis, especially in diabetic patients. C. perfringens in association with mixed aerobic and anaerobic microbes can cause aggressive life-threatening type I necrotizing fasciitis or Fournier’s gangrene.

1	The treatment of mixed aerobic/anaerobic infection of the abdomen, perineum, or gynecologic organs should be based on Gram’s staining, culture, and antibiotic sensitivity information. Reasonable empirical treatment consists of ampicillin or ampicillin/sulbactam combined with either clindamycin or metronidazole (Table 179-1). Broader gram-negative coverage may be necessary if the patient has recently been hospitalized or treated with antibiotics. Such coverage can be obtained by substituting ticarcillin/clavulanic acid, piperacillin/sulbactam, or a penem antibiotic for ampicillin or by adding a fluoroquinolone or an aminoglycoside to the regimen. Empirical treatment should be given for 10–14 days or until the patient’s clinical condition improves.

1	C. perfringens type A is one of the most common bacterial causes of food-borne illness in the United States and Canada. The foods typically implicated include improperly cooked meat and meat products (e.g., gravy) in which residual spores germinate and proliferate during slow cooling or insufficient reheating. Illness results from the ingestion of food containing at least ~108 viable vegetative cells, which sporulate in the alkaline environment of the small intestine, producing

1	C. perfringens enterotoxin in the process. The diarrhea that develops within 7–30 h of ingestion of contaminated food is generally mild and self-limiting; however, in the very young, the elderly, and the immunocompromised, symptoms are more severe and occasionally fatal. Enterotoxin-producing C. perfringens has been implicated as an etiologic agent of persistent diarrhea in elderly patients in nursing homes and tertiary-care institutions and has been considered to play a role in antibiotic-associated diarrhea without pseudomembranous colitis.

1	C. perfringens strains associated with food poisoning possess the gene (cpe) coding for enterotoxin, which acts by forming pores in host cell membranes. C. perfringens strains isolated from non food-borne diseases, such as antibiotic-associated and sporadic diarrhea, carry cpe on a plasmid that may be transmitted to other strains. Several methods have been described for the detection of C. perfringens enterotoxin in feces, including cell culture assay (Vero cells), enzyme-linked immunosorbent assay, reversed-phase latex agglutination, and polymerase chain reaction (PCR) amplification of cpe. Each method has its advantages and limitations.

1	Enteritis necroticans (gas gangrene of the bowel) is a fulminating clinical illness characterized by extensive necrosis of the intestinal mucosa and wall. Cases can occur sporadically in adults or as epidemics in people of all ages. Enteritis necroticans is 991 caused by α toxin– and β toxin–producing strains of C. perfringens type C; β toxin is located on a plasmid and is mainly responsible for pathogenesis. This life-threatening infection causes ischemic necrosis of the jejunum. In Papua New Guinea during the 1960s, enteritis necroticans (known in that locale as pigbel) was found to be the most common cause of death in childhood; it was associated with pig feasts and occurred both sporadically and in outbreaks. Intramuscular immunization against the β toxin resulted in a decreased incidence of the disease in Papua New Guinea, although the condition remains common. Enteritis necroticans has also been recognized in the United States, the United Kingdom, Germany (where it is known as

1	of the disease in Papua New Guinea, although the condition remains common. Enteritis necroticans has also been recognized in the United States, the United Kingdom, Germany (where it is known as darmbrand), and other developed nations; especially affected are adults who are malnourished or who have diabetes, alcoholic liver disease, or neutropenia.

1	Necrotizing enterocolitis, a disease resembling enteritis necroticans but associated with C. perfringens type A, has been found in North America in previously healthy adults. It is also a serious gastrointestinal disease of low-birth-weight (premature) infants hospitalized in neonatal intensive care units. The etiology and pathogenesis of this disease have remained enigmatic for more than four decades. Pathologic similarities between necrotizing enterocolitis and enteritis necroticans include the pattern of small-bowel necrosis involving the submucosa, mucosa, and muscularis; the presence of gas dissecting the tissue planes; and the degree of inflammation. In contrast to enteritis necroticans, which most commonly involves the jejunum, necrotizing enterocolitis affects the ileum and frequently the ileocecal valve. Both diseases may manifest as intestinal gas cysts, although this feature is more common in necrotizing enterocolitis. The sources of the gas, which contains hydrogen,

1	frequently the ileocecal valve. Both diseases may manifest as intestinal gas cysts, although this feature is more common in necrotizing enterocolitis. The sources of the gas, which contains hydrogen, methane, and carbon dioxide, are probably the fermentative activities of intestinal bacteria, including clostridia. Epidemiologic data support an important role for C. perfringens or other gas-producing microorganisms (e.g., C. neonatale, certain other clostridia, or Klebsiella species) in the pathogenesis of necrotizing enterocolitis.

1	Patients with suspected clostridial enteric infection should undergo nasogastric suction and receive IV fluids. Pyrantel is given by mouth, and the bowel is rested by fasting. Benzylpenicillin (1 mU) is given IV every 4 h, and the patient is observed for complications requiring surgery. Patients with mild cases recover without surgical intervention. If surgical indications are present (gas in the peritoneal cavity, absent bowel sounds, rebound tenderness, abdominal rigidity), however, the mortality rate ranges from 35% to 100%; a fatal outcome is due in part to perforation of the intestine. As pigbel continues to be a common disease in Papua New Guinea, consideration should be given to the use of a C. perfringens Polymicrobial anaerobic infections involving clostridia (e.g., abdominal wall, gynecologic) Ciprofloxacin (400 mg IV q6–8 h) Penicillin, 3–4 mU IV q4–6h

1	Polymicrobial anaerobic infections involving clostridia (e.g., abdominal wall, gynecologic) Ciprofloxacin (400 mg IV q6–8 h) Penicillin, 3–4 mU IV q4–6h Clindamycin (600–900 mg IV q6–8h) Treatment should be based on clinical signs and symptoms as listed below and not solely on bacteriologic findings. Empirical therapy should be initiated. Therapy should be based on Gram’s stain and culture results and on sensitivity data when available. Add gram-negative coverage if indicated (see text). Transient bacteremia without signs of systemic toxicity may be clinically insignificant. Emergent surgical exploration and thorough debridement are extremely important. Hyperbaric oxygen therapy may be considered after surgery and antibiotic initiation. 992 type C β toxoid vaccine in local areas. Two doses given 3–4 months apart are preventive.

1	992 type C β toxoid vaccine in local areas. Two doses given 3–4 months apart are preventive. Clostridium species are important causes of bloodstream infections. Molecular epidemiologic studies of anaerobic bacteremia have identified C. perfringens and C. tertium as the two most frequently isolated species; these organisms cause up to 79% and 5%, respectively, of clostridial bacteremias. Occasionally, C. perfringens bacteremia occurs in the absence of an identifiable infection at another site. When associated with myonecrosis, bacteremia has a grave prognosis.

1	C. septicum is also commonly associated with bacteremia. This species is isolated only rarely from the feces of healthy individuals but may be found in the normal appendix. More than 50% of patients whose blood cultures are positive for this organism have some gastrointestinal anomaly (e.g., diverticular disease) or underlying malignancy (e.g., carcinoma of the colon). In addition, a clinically important association of C. septicum bacteremia with neutropenia of any origin—and, more specifically, with neutropenic enterocolitis involving the terminal ileum or cecum—has been observed. Patients with diabetes mellitus, severe atherosclerotic cardiovascular disease, or anaerobic myonecrosis (gas gangrene) also may develop C. septicum bacteremia. C. septicum has been recovered from the bloodstream of cirrhotic patients, as have C. perfringens, C. bifermentans, and other clostridia. Infections of the bloodstream by C. sordellii and C. perfringens have been associated with TSS.

1	Bloodstream infection by C. tertium, either alone or in combination with C. septicum or C. perfringens, can be found in patients with serious underlying disease such as malignancy or acute pancreatitis, with or without neutropenic enterocolitis; the frequency has not been systematically studied. C. tertium may present special problems in terms of both identification and treatment. This organism may stain gram-negative; is aerotolerant; and is resistant to metronidazole, clindamycin, and cephalosporins. Other clostridia from the C. clostridioforme group (including C. clostridioforme, C. hathewayi, and C. bolteae) can cause bacteremia.

1	Other clostridia from the C. clostridioforme group (including C. clostridioforme, C. hathewayi, and C. bolteae) can cause bacteremia. The clinical importance of recognizing clostridial bacteremia— especially that due to C. septicum—and starting appropriate treatment immediately (Table 179-1) cannot be overemphasized. Patients with this condition usually are gravely ill, and infection may metastasize to distant anatomic sites, resulting in spontaneous myonecrosis (see next section). Alternative methods to identify bacteremia-causing clostridial species, such as PCR or other rapid diagnostic tests, are not currently available. Anaerobic blood cultures and Gram’s stain interpretation remain the best diagnostic tests at this point. Histotoxic clostridial species such as C. perfringens, C. histolyticum,

1	Histotoxic clostridial species such as C. perfringens, C. histolyticum, C. septicum, C. novyi, and C. sordellii cause aggressive necrotizing infections of the skin and soft tissues. These infections are attributable in part to the elaboration of bacterial proteases, phospholipases, and cytotoxins. Necrotizing clostridial soft-tissue infections are rapidly progressive and are characterized by marked tissue destruction, gas in the tissues, and shock; they frequently end in death. Severe pain, crepitus, brawny induration with rapid progression to skin sloughing, violaceous bullae, and marked tachycardia are characteristics found in the majority of patients.

1	Clostridial Myonecrosis (Gas Gangrene) • traumatic gas gangrene C. perfringens myonecrosis (gas gangrene) is one of the most fulminant gram-positive bacterial infections of humans. Even with appropriate antibiotic therapy and management in an intensive care unit, tissue destruction can progress rapidly. Gas gangrene is accompanied by bacteremia, hypotension, and multiorgan failure and is invariably fatal if untreated. Gas gangrene is a true emergency and requires immediate surgical debridement.

1	The development of gas gangrene requires an anaerobic environment and contamination of a wound with spores or vegetative organisms. Devitalized tissue, foreign bodies, and ischemia reduce locally available oxygen levels and favor outgrowth of vegetative cells and spores. Thus conditions predisposing to traumatic gas gangrene include crush-type injury, laceration of large or medium-sized arteries, and open fractures of long bones that are contaminated with soil or bits of clothing containing the bacterial spores. Gas gangrene of the abdominal wall and flanks follows penetrating injuries such as knife or gunshot wounds that are sufficient to compromise intestinal integrity, with resultant leakage of the bowel contents into the soft tissues. Proximity to fecal sources of bacteria is a risk factor for cases following hip surgery, adrenaline injections into the buttocks, or amputation of the leg for ischemic vascular disease. In the last decade, cutaneous gas gangrene caused by C.

1	a risk factor for cases following hip surgery, adrenaline injections into the buttocks, or amputation of the leg for ischemic vascular disease. In the last decade, cutaneous gas gangrene caused by C. perfringens, C. novyi, and C. sordellii has been described in the United States and northern Europe among persons injecting black-tar heroin subcutaneously.

1	The incubation period for traumatic gas gangrene can be as short as 6 h and is usually <4 days. The infection is characterized by the sudden onset of excruciating pain at the affected site and the rapid development of a foul-smelling wound containing a thin serosanguineous discharge and gas bubbles. Brawny edema and induration develop and give way to cutaneous blisters containing bluish to maroon-colored fluid. Such tissue later may become liquefied and slough. The margin between healthy and necrotic tissue often advances several inches per hour despite appropriate antibiotic therapy, and radical amputation remains the single best life-saving intervention. Shock and organ failure frequently accompany gas gangrene; when patients become bacteremic, the mortality rate exceeds 50%.

1	Diagnosis of traumatic gas gangrene is not difficult because the infection always begins at the site of significant trauma, is associated with gas in the tissue, and is rapidly progressive. Gram’s staining of drainage or tissue biopsy is usually definitive, demonstrating large gram-positive (or gram-variable) rods, an absence of inflammatory cells, and widespread soft-tissue necrosis.

1	spontaneous (nontraumatic) gas gangrene Spontaneous gas gangrene generally occurs via hematogenous seeding of normal muscle with histotoxic clostridia—principally C. perfringens, C. septicum, and C. novyi and occasionally C. tertium—from a gastrointestinal tract portal of entry (as in colonic malignancy, inflammatory bowel disease, diverticulitis, necrotizing enterocolitis, cecitis, or distal ileitis or after gastrointestinal surgery). These gastrointestinal pathologies permit bacterial access to the bloodstream; consequently, aerotolerant C. septicum can proliferate in normal tissues. Patients surviving bacteremia or spontaneous gangrene due to C. septicum should undergo aggressive diagnostic studies to rule out gastrointestinal pathology.

1	Additional predisposing host factors include leukemia, lymphoproliferative disorders, cancer chemotherapy, radiation therapy, and AIDS. Cyclic, congenital, or acquired neutropenia also is strongly associated with an increased incidence of spontaneous gas gangrene due to C. septicum; in such cases, necrotizing enterocolitis, cecitis, or distal ileitis is common, particularly among children.

1	The first symptom of spontaneous gas gangrene may be confusion followed by the abrupt onset of excruciating pain in the absence of trauma. These findings, along with fever, should heighten suspicion of spontaneous gas gangrene. However, because of the lack of an obvious portal of entry, the correct diagnosis is frequently delayed or missed. The infection is characterized by rapid progression of tissue destruction with demonstrable gas in the tissue (Fig. 179-2). Swelling increases, and bullae filled with clear, cloudy, hemorrhagic, or purplish fluid appear. The surrounding skin has a purple hue, which may reflect vascular compromise resulting from the diffusion of bacterial toxins into surrounding tissues. Invasion of healthy tissue rapidly ensues, with quick progression to shock and multiple-organ failure. Mortality rates in this setting range from 67 to 100% among adults; among children, the mortality rate is 59%, with the majority of deaths occurring within 24 h of onset.

1	patHogenesis of gas gangrene In traumatic gas gangrene, organisms are introduced into devitalized tissue. It is important to recognize that for C. perfringens and C. novyi, trauma must be sufficient to interrupt the blood supply and thereby to establish an optimal anaerobic FIGURE 179-2 Radiograph of patient with spontaneous gas gan-grene due to C. septicum, demonstrating gas in the affected arm and shoulder. environment for growth of these species. These conditions are not strictly required for the more aerotolerant species such as C. septicum and C. tertium, which can seed normal tissues from gastrointestinal lesions. Once introduced into an appropriate niche, the organisms proliferate locally and elaborate exotoxins.

1	The major C. perfringens extracellular toxins implicated in gas gangrene are α toxin and θ toxin. A lethal hemolysin that has both phospholipase C and sphingomyelinase activities, α toxin has been implicated as the major virulence factor of C. perfringens: immunization of mice with the C-terminal domain of α toxin provides protection against lethal challenge with C. perfringens, and isogenic α toxin–deficient mutant strains of C. perfringens are not lethal in a murine model of gas gangrene. It has been shown in experimental models that the severe pain, rapid progression, marked tissue destruction, and absence of neutrophils in C. perfringens gas gangrene are attributable in large part to α toxin–induced occlusion of blood vessels by heterotypic aggregates of platelets and neutrophils. The formation of these aggregates, which occurs within minutes, is largely mediated by α toxin’s ability to activate the platelet adhesion molecule gpIIb/ IIIa (Fig. 179-3); the implication is that

1	The formation of these aggregates, which occurs within minutes, is largely mediated by α toxin’s ability to activate the platelet adhesion molecule gpIIb/ IIIa (Fig. 179-3); the implication is that platelet glycoprotein inhibitors (e.g., eptifibatide, abciximab) may be therapeutic for maintaining tissue blood flow.

1	C. perfringens θ toxin (perfringolysin) is a member of the thiolactivated cytolysin family known as cholesterol-dependent cytolysins, which includes streptolysin O from group A Streptococcus, pneumolysin from Streptococcus pneumoniae, and several other toxins. Cholesterol-dependent cytolysins bind as oligomers to cholesterol in host cell membranes. At high concentrations, these toxins form ringlike pores resulting in cell lysis. At sublytic concentrations, θ toxin hyperactivates phagocytes and vascular endothelial cells. Cardiovascular collapse and end-organ failure occur late in the course of C. perfringens gas gangrene and are largely attributable to both direct and indirect effects of α and θ toxins. In experimental models, θ toxin causes markedly reduced systemic vascular resistance but increased cardiac output (i.e., “warm shock”), probably via induction of endogenous mediators (e.g., prostacyclin, platelet-activating factor)

1	FIGURE 179-3 Schematic illustration of the molecular mechanisms of C. perfringens α toxin–induced platelet/neutrophil aggregates. Homotypic aggregates of platelets (not shown) and heterotypic aggregates of platelets and leukocytes are due to α toxin–induced activation of the platelet fibrinogen receptor gpIIb/ IIIa and upregulation of leukocyte CD11b/CD18. Binding of fibrinogen (red) bridges the connection between these adhesion molecules on adjacent cells. An auxiliary role for α toxin–induced upregulation of platelet P-selectin and its binding to leukocyte P-selectin glycoprotein ligand 1 (PSGL-1) or other leukocyte surface carbohydrates also has been demonstrated.

1	that cause vasodilation. This effect is similar to that observed in gram-negative sepsis. In sharp contrast, α toxin directly suppresses myocardial contractility; the consequence is profound hypotension due to a sudden reduction in cardiac output. The roles of other endogenous mediators, such as cytokines (e.g., tumor necrosis factor, interleukin 1, interleukin 6) and vasodilators (e.g., bradykinin) have not been fully elucidated. C. septicum produces four main toxins—α toxin (lethal, hemolytic, necrotizing activity), β toxin (DNase), γ toxin (hyaluronidase), and Δ toxin (septicolysin, an oxygen-labile hemolysin)—as well as a protease and a neuraminidase. Unlike the α toxin of C. perfringens, that of C. septicum does not possess phospholipase activity. The mechanisms remain to be fully elucidated, but it is likely that each of these toxins contributes uniquely to C. septicum gas gangrene.

1	Patients with suspected gas gangrene (either traumatic or spontaneous) should undergo prompt surgical inspection of the infected site. Direct examination of a Gram-stained smear of the involved tissues is of major importance. Characteristic histologic findings in clostridial gas gangrene include widespread tissue destruction, a paucity of leukocytes in infected tissues in conjunction with an accumulation of leukocytes in adjacent vessels (Fig. 179-4), and the presence of gram-positive rods (with or without spores). CT and MRI are invaluable for determining whether the infection is localized or is spreading along fascial planes, and needle aspiration or punch biopsy may provide an etiologic diagnosis in at least 20% of cases. However, these techniques should not replace surgical exploration, Gram’s staining, and histopathologic examination. When spontaneous gas gangrene is suspected, blood should be cultured since bacteremia usually precedes cutaneous manifestations by several hours.

1	For patients with evidence of clostridial gas gangrene, thorough emergent surgical debridement is of extreme importance. All devitalized tissue should be widely resected back to healthy viable muscle and skin so as to remove conditions that allow anaerobic organisms to continue proliferating. Closure of traumatic wounds or compound fractures should be delayed for 5–6 days until it is certain that these sites are free of infection. FIGURE 179-4 Histopathology of experimental gas gangrene due to C. perfringens, demonstrating widespread muscle necrosis, a pau-city of leukocytes in infected tissues, and accumulation of leukocytes in adjacent vessels (arrows). These features are due to the effects of α and θ toxins on muscle cells, platelets, leukocytes, and endothelial cells.

1	Antibiotic treatment of traumatic or spontaneous gas gangrene (Table 179-1) consists of the administration of penicillin and clindamycin for 10–14 days. Penicillin is recommended on the basis of in vitro sensitivity data; clindamycin is recommended because of its superior efficacy over penicillin in animal models of C. perfringens gas gangrene and in some clinical reports. Controlled clinical trials comparing the efficacy of these agents in humans have not been performed. In the penicillin-allergic patient, clindamycin may be used alone. The superior efficacy of clindamycin is probably due to its ability to inhibit bacterial protein toxin production, its insensitivity to the size of the bacterial load or the stage of bacterial growth, and its ability to modulate the host’s immune response.

1	C. tertium is resistant to penicillin, cephalosporins, and clindamycin. Appropriate antibiotic therapy for C. tertium infection is vancomycin (1 g every 12 h IV) or metronidazole (500 mg every 8 h IV). The value of adjunctive treatment with hyperbaric oxygen (HBO) for gas gangrene remains controversial. Basic science studies suggest that HBO can inhibit the growth of C. perfringens but not that of the more aerotolerant C. septicum. In vitro, blood and macerated muscle inhibit the bactericidal potential of HBO. Numerous studies in animals demonstrate little efficacy of HBO alone, whereas antibiotics alone—especially those that inhibit bacterial protein synthesis—confer marked benefits. Addition of HBO to the therapeutic regimen provides some additional benefit, but only if surgery and antibiotic administration precede HBO treatment.

1	In conclusion, gas gangrene is a rapidly progressive infection whose outcome depends on prompt recognition, emergent surgery, and timely administration of antibiotics that inhibit toxin production. Gas gangrene associated with bacteremia probably represents a later stage of illness and is associated with the worst outcomes. Emergent surgical debridement is crucial to ensure survival, and ancillary procedures (e.g., CT or MRI) or transport to HBO units should not delay this intervention. Some trauma centers associated with HBO units may have special expertise in managing these aggressive infections, but proximity and speed of transfer must be carefully weighed against the need for haste.

1	prognosis of gas gangrene The prognosis for patients with gas gangrene is more favorable when the infection involves an extremity rather than the trunk or visceral organs, since debridement of the latter sites is more difficult. Gas gangrene is most likely to progress to shock and death in patients with associated bacteremia and intravascular hemolysis. Mortality rates are highest for patients in shock at the time of diagnosis. Mortality rates are relatively high among patients with spontaneous gas gangrene, especially that due to C. septicum. Survivors of gas gangrene may undergo multiple debridements and face long periods of hospitalization and rehabilitation.

1	prevention of gas gangrene Initial aggressive debridement of devitalized tissue can reduce the risk of gas gangrene in contaminated deep wounds. Interventions to be avoided include prolonged application of tourniquets and surgical closure of traumatic wounds; patients with compound fractures are at significant risk for gas gangrene if the wound is closed surgically. Vaccination against α toxin is protective in experimental animal models of C. perfringens gas gangrene but has not been investigated in humans. In addition, as mentioned above, a hyperimmune globulin would represent a significant advance for prophylaxis in victims of acute traumatic injury or for attenuation of the spread of infection in patients with established gas gangrene.

1	Toxic Shock Syndrome Clostridial infection of the endometrium, particularly that due to C. sordellii, can develop after gynecologic procedures, childbirth, or abortion (spontaneous or elective, surgical or medical) and, once established, proceeds rapidly to TSS and death. Systemic manifestations, including edema, effusions, profound leukocytosis, and hemoconcentration, are followed by the rapid onset of hypotension and multiple-organ failure. Elevation of the hematocrit to 75–80% and leukocytosis of 50,000–200,000 cells/ μL, with a left shift, are characteristic of C. sordellii infection. Pain may not be a prominent feature, and fever is typically absent. In one series, 18% of 45 cases of C. sordellii infection were associated with normal childbirth, 11% with medically induced abortion, and 0.4% with spontaneous abortion; the case-fatality rate was 100% in these groups. Of the infections in this series that were not related to gynecologic procedures or childbirth, 22% occurred in

1	and 0.4% with spontaneous abortion; the case-fatality rate was 100% in these groups. Of the infections in this series that were not related to gynecologic procedures or childbirth, 22% occurred in injection drug users, and 50% of these patients died. Other infections followed trauma or surgery (42%), mostly in healthy persons, and 53% of these patients died. Overall, the mortality rate was 69% (31 of 45 cases). Of patients who succumbed, 85% died within 2–6 days after infection onset or following procedures.

1	Early diagnosis of C. sordellii infections often proves difficult for several reasons. First, the prevalence of these infections is low. Second, the initial symptoms are nonspecific and frankly misleading. Early in the course, the illness resembles any number of infectious diseases, including viral syndromes. Given these vague symptoms and an absence of fever, physicians usually do not aggressively pursue additional diagnostic tests. The absence of local evidence of infection and the lack of fever make early diagnosis of C. sordellii infection particularly problematic in patients who develop deep-seated infection following childbirth, therapeutic abortion, gastrointestinal surgery, or trauma. Such patients are frequently evaluated for pulmonary embolization, gastrointestinal bleeding, pyelonephritis, or cholecystitis. Unfortunately, such delays in diagnosis increase the risk of death, and, as in most necrotizing soft-tissue infections, patients are hypotensive with evidence of organ

1	or cholecystitis. Unfortunately, such delays in diagnosis increase the risk of death, and, as in most necrotizing soft-tissue infections, patients are hypotensive with evidence of organ dysfunction by the time local signs and symptoms become apparent. In contrast, infection is more readily suspected in injection drug users presenting with local swelling, pain, and redness at injection sites; early recognition probably contributes to the lower mortality rates in this group.

1	Physicians should suspect C. sordellii infection in patients who present within 2–7 days after injury, surgery, drug injection, childbirth, or abortion and who report pain, nausea, vomiting, and diarrhea but are afebrile. There is little information regarding appropriate treatment for C. sordellii infections. In fact, the interval between onset of symptoms and death is often so short that there is little time to initiate empirical antimicrobial therapy. Indeed, anaerobic cultures of blood and wound aspirates are time-consuming, and many hospital laboratories do not routinely perform antimicrobial sensitivity testing on anaerobes. Antibiotic susceptibility data from older studies suggest that C. sordellii, like most clostridia, is susceptible to β-lactam antibiotics, clindamycin, tetracycline, and chloramphenicol but is resistant to aminoglycosides and sulfonamides. Antibiotics that suppress toxin synthesis (e.g., clindamycin) may possibly prove useful as therapeutic adjuncts since

1	and chloramphenicol but is resistant to aminoglycosides and sulfonamides. Antibiotics that suppress toxin synthesis (e.g., clindamycin) may possibly prove useful as therapeutic adjuncts since they are effective in necrotizing infections due to other toxin-producing gram-positive organisms.

1	Other Clostridial Skin and Soft-Tissue Infections Crepitant cellulitis (also called anaerobic cellulitis) occurs principally in diabetic patients and characteristically involves subcutaneous tissues or retroperitoneal tissues, whereas the muscle and fascia are not involved. This infection can progress to fulminant systemic disease. Cases of C. histolyticum infection with cellulitis, abscess forma-995 tion, or endocarditis have also been documented in injection drug users. Endophthalmitis due to C. sordellii or C. perfringens has been described. C. ramosum is also isolated frequently from clinical specimens, including blood and both intraabdominal and soft tissues. This species may be resistant to clindamycin and multiple cephalosporins.

1	Meningococcal Infections Andrew J. Pollard DEFINITION Infection with Neisseria meningitidis most commonly manifests as asymptomatic colonization in the nasopharynx of healthy adolescents and adults. Invasive disease occurs rarely, usually presenting as either 180 SECTIon 6 bacterial meningitis or meningococcal septicemia. Patients may also present with occult bacteremia, pneumonia, septic arthritis, conjunctivitis, and chronic meningococcemia. N. meningitidis is a gram-negative aerobic diplococcus that colonizes humans only and that causes disease after transmission to a susceptible individual. Several related organisms have been recognized, including the pathogen N. gonorrhoeae and the commensals N. lactamica, N. flavescens, N. mucosa, N. sicca, and N. subflava. N. meningitidis is a catalaseand oxidase-positive organism that utilizes glucose and maltose to produce acid.

1	Meningococci associated with invasive disease are usually encapsulated with polysaccharide, and the antigenic nature of the capsule determines an organism’s serogroup (Table 180-1). In total, 13 serogroups have been identified (A–D, X–Z, 29E, W, H–J, and L), but just 6 serogroups—A, B, C, X, Y, and W (formerly W135)—account for the majority of cases of invasive disease. Acapsular meningococci are commonly isolated from the nasopharynx in studies of carriage; the lack of capsule often is a result of phase variation of capsule expression, but as many as 16% of isolates lack the genes for capsule synthesis and assembly. These “capsule-null” meningococci and those that express capsules other than A, B, C, X, Y, and W are only rarely associated with invasive disease and are most commonly identified in the nasopharynx of asymptomatic carriers.

1	Beneath the capsule, meningococci are surrounded by an outer phospholipid membrane containing lipopolysaccharide (LPS, endotoxin) and multiple outer-membrane proteins (Figs. 180-1 and 180-2). Antigenic variability in porins expressed in the outer membrane defines the serotype (PorB) and serosubtype (PorA) of the organism, and structural differences in LPS determine the immunotype. Serologic methods for typing of meningococci are restricted by the limited availability of serologic reagents that can distinguish among the organisms’ highly variable surface proteins. Where available, high-throughput antigen gene sequencing has superseded serology for meningococcal typing. A large database of antigen gene sequences for the outer-membrane proteins PorA, PorB, FetA, Opa, and factor H–binding protein is available online (www.neisseria.org). The number of specialized iron-regulated proteins found in the meningococcal outer membrane (e.g., FetA and transferrin-binding proteins) highlights the

1	is available online (www.neisseria.org). The number of specialized iron-regulated proteins found in the meningococcal outer membrane (e.g., FetA and transferrin-binding proteins) highlights the organisms’ dependence on iron from human sources. A thin peptidoglycan cell wall separates the outer membrane from the cytoplasmic membrane.

1	The structure of meningococcal populations involved in local and global spread has been studied with multilocus enzyme electrophoresis (MLEE), which characterizes isolates according to differences in the electrophoretic mobility of cytoplasmic enzymes. However, this technique has mostly been replaced by multi-locus sequence typing (MLST), in which meningococci are characterized by sequence types assigned on the basis of sequences of internal fragments of seven housekeeping genes. The online MLST database currently includes more than 27,000 meningococcal isolates and 10,500 unique sequence types (pubmlst.org/neisseria/). A limited number of hyperinvasive lineages of N. meningitidis have been recognized

1	FIGURE 180-1 Electron micrograph of Neisseria meningitidis. Black dots are gold-labeled polyclonal antibodies binding surface opacity proteins. Blebs of outer membrane can be seen being released from the bacterial surface (arrow). (Photo courtesy of D. Ferguson, Oxford University.) e.g.FbpA, SodC Periplasmic space e.g.FbpB, FbpC

1	FIGURE 180-2 Cross-section through surface structures of Neisseria meningitidis. LPS, lipopolysaccharide. (Reprinted with permission from M Sadarangani, AJ Pollard: Lancet Infect Dis 10:112, 2010.) and are responsible for the majority of cases of invasive meningococcal disease worldwide. The apparent genetic stability of these meningococcal clones over decades and during wide geographic spread indicates that they are well adapted to the nasopharyngeal environment of the host and to efficient transmission. While MLST has become established as the main method of genotyping meningococci in many reference laboratories over the past decade, whole-genome sequencing is set to replace this approach in the decade ahead, with almost 1000 genomes already available in the United Kingdom’s national library (www.meningitis.org/genome-library).

1	The group B meningococcal genome is >2 megabases in length and contains 2158 coding regions. Many genes undergo phase variation that makes it possible to control their expression; this capacity is likely to be important in meningococcal adaptation to the host environment and evasion of the immune response. Meningococci can obtain DNA from their environment and can acquire new genes—including the capsular operon—such that capsule switching from one serogroup to another can occur. Patterns of Disease Up to 500,000 cases of meningococcal disease are thought to occur worldwide each year, and ~10% of the individuals affected die. There are several patterns of disease: epidemic, outbreak (small clusters of cases), hyperendemic, and sporadic or endemic. islands), and the province of Normandy in France.

1	islands), and the province of Normandy in France. Most countries now experience predominantly sporadic cases (0.3–5 cases per 100,000 population), with many different disease-causing clones involved and usually no clear epidemiologic link between one case and another. The disease rate and the distribution of meningococcal strains vary in different regions of the world and also in any one location over time. For example, in the United States, the rate of meningococcal disease fell from 1.2 cases per 100,000 population in 1997 to <0.15 case per 100,000 in 2012 (Fig. 180-3). Meningococcal disease in the United States was previously dominated by serogroups B and C; however, serogroup Y emerged during the 1990s and became more common than serogroup C in 2007. In contrast, rates of disease in England and Wales rose to >5 cases per 100,000 during the 1990s because of an increase in cases caused by the

1	ST11 serogroup C clone. As a result of a mass immunization program against serogroup C in 1999, almost all cases in the United Kingdom are now attributed to serogroup B (Fig. 180-4). Over the last decade, most industrialized nations have seen a general decrease in meningococcal disease; this decrease is linked to immunization against serogroup C meningococci in Europe, Canada, and Australia and to adolescent immunization programs for A, C, Y and W in the United States. However, other factors, including changes in population immunity (probably the explanation for the cyclic nature of meningococcal disease rates) as well as a reduction in smoking and passive exposure to tobacco smoke (driven by bans on smoking in buildings and public spaces) across wealthy countries are likely to have contributed to the fall in cases.

1	Factors Associated with Disease Risk and Susceptibility The principal determinant of disease susceptibility is age, with the peak incidence in the first year of life (Fig. 180-5). The susceptibility of the very young presumably results from an absence of specific adaptive immunity in combination with very close contact with colonized individuals, including parents. Compared with other age groups, infants appear to be particularly susceptible to serogroup B disease: >30% of serogroup B cases in the United States occur during the first year of life. In the early 1.4

1	Epidemics have continued since the original descriptions of menin gococcal disease, especially affecting the sub-Saharan meningitis belt of Africa, where tens to hundreds of thousands of cases (caused mainly by serogroup A but also by serogroups W and X) may be reported over a season and rates may be as high as 1000 cases per 100,000 popula tion. Serogroup A epidemics took place in Europe and North America after the First and Second World Wars, and serogroup A outbreaks have been documented over the past 30 years in New Zealand, China, Nepal, Mongolia, India, Pakistan, Poland, and Russia. Clusters of cases occur where there is an opportunity for increased transmission—i.e., in (semi-)closed communities such as schools, Cases per 100,000 population 1.2 0.8 0.6 0.4 0.2 colleges, universities, military training centers, and refugee camps.

1	Cases per 100,000 population 1.2 0.8 0.6 0.4 0.2 colleges, universities, military training centers, and refugee camps. Recently, such clusters have been especially strongly linked with a particular clone (sequence type 11) that is mainly associated with the serogroup C or W capsule but was first described in association Year with serogroup B. Wider and more prolonged community outbreaks FIGURE 180-3 Meningococcal disease in the United States over (hyperendemic disease) due to single clones of serogroup B menin time. ABCs, active bacterial cores. (Adapted from ABC Surveillance data, gococci account for ≥10 cases per 100,000. Regions affected in the Centers for Disease Control and Prevention; www.cdc.gov.) past decade include the U.S. Pacific Northwest, New Zealand (both B,C,YB,CB,C,YBBB,CB,CB,CB,CA,B,CA,B,CB,WB,WAW,AB,CA,W(X)A,W(X)FIGURE 180-4 Global distribution of meningococcal serogroups, 1999–2009.

1	B,C,YB,CB,C,YBBB,CB,CB,CB,CA,B,CA,B,CB,WB,WAW,AB,CA,W(X)A,W(X)FIGURE 180-4 Global distribution of meningococcal serogroups, 1999–2009. 1990s in North America, the median ages for patients with disease due to serogroups B, C, Y, and W were 6, 17, 24, and 33 years, respectively. After early childhood, a second peak of disease occurs among adolescents and young adults (15–25 years of age) in Europe and North America. It is thought that this peak relates to social behaviors and environmental exposures in this age group, as discussed below. Most cases of infection with N. meningitidis in developed countries today are sporadic, and the rarity of the disease suggests that individual susceptibility may be important. A number of factors probably contribute to individual susceptibility, including the host’s genetic constitution, environment, and contact with a carrier or a case.

1	The best-documented genetic association with meningococcal disease is complement deficiency, chiefly of the terminal complement components (C5–9), properdin, or factor D; such a deficiency increases the risk of disease by up to 600-fold and may result in recurrent attacks. Complement components are believed to be important for the bactericidal activity of serum, which is considered the principal mechanism of immunity against invasive meningococcal disease. However, when investigated, complement deficiency is found in only a very small proportion of individuals with meningococcal disease (0.3%). Conversely, 7–20% of persons whose disease is caused by the less common serogroups (W, X, Y, Z, 29E) have a complement deficiency. Complement deficiency appears to be associated with serogroup B disease only rarely. Individuals with recurrences of meningococcal disease, particularly those caused by non-B serogroups, should be assessed for complement deficiency by measurement of total hemolytic

1	only rarely. Individuals with recurrences of meningococcal disease, particularly those caused by non-B serogroups, should be assessed for complement deficiency by measurement of total hemolytic complement activity. There is also limited evidence that hyposplenism (through reduction in phagocytic capacity) and hypogammaglobulinemia (through absence of specific antibody) increase the risk of meningococcal disease. Genetic studies have revealed various associations with disease susceptibility, including complement and mannose-binding lectin deficiency, single-nucleotide polymorphisms in Toll-like receptor (TLR) 4 and complement factor H, and variants of Fc gamma receptors.

1	Factors that increase the chance of a susceptible individual’s acquiring N. meningitidis via the respiratory route also increase the risk of meningococcal disease. Acquisition occurs through close contact with carriers as a result of overcrowding (e.g., in poor socioeconomic settings, in refugee camps, during the Hajj pilgrimage to Mecca, and during freshman-year residence in college dormitories) and certain social behaviors (e.g., attendance at bars and nightclubs, kissing). Secondary cases may occur in close contacts of an index case (e.g., household members and persons kissing the infected individual); the risk to these contacts may be as high as 1000 times the background rate in the population. Factors that damage the nasopharyngeal epithelium also increase the risk of both colonization with N. meningitidis and invasive disease. The most important of these factors are cigarette smoking (odds ratio, 4.1) and passive exposure to cigarette smoke. In addition, recent viral respiratory

1	with N. meningitidis and invasive disease. The most important of these factors are cigarette smoking (odds ratio, 4.1) and passive exposure to cigarette smoke. In addition, recent viral respiratory tract infection, infection with Mycoplasma species, and winter or the dry season have been associated with meningococcal disease; all of these factors presumably either increase the expression of adhesion molecules in the nasopharynx, thus

1	Number of cases FIGURE 180-5 Age distribution of serogroups B and C meningococcal disease in enhancing meningococcal adhesion, or facilitate England and Wales, 1998–1999. (Health Protection Agency, UK; www.hpa.org.uk.) meningococcal invasion of the bloodstream.

1	N. meningitidis has evolved as an effective colonizer of the human nasopharynx, with asymptomatic infection rates of >25% described in some series of adolescents and young adults and among residents of crowded communities. Point-prevalence studies reveal widely divergent rates of carriage for different types of meningococci. This variation suggests that some types may be adapted to a short duration of carriage with frequent transmission to maintain the population, while others may be less efficiently transmitted but may overcome this disadvantage by colonizing for a long period. Despite the high rates of carriage among adolescents and young adults, only ~10% of adults carry meningococci, and colonization is very rare in early childhood. Many of the same factors that increase the risk of meningococcal disease also increase the risk of carriage, including smoking, crowding, and respiratory viral infection. Colonization of the nasopharynx involves a series of interactions of

1	risk of meningococcal disease also increase the risk of carriage, including smoking, crowding, and respiratory viral infection. Colonization of the nasopharynx involves a series of interactions of meningococcal adhesins (e.g., Opa proteins and pili) with their ligands on the epithelial mucosa. N. meningitidis produces an IgA1 protease that is likely to reduce interruption of colonization by mucosal IgA.

1	Colonization should be considered the normal state of meningococcal infection, with an increased risk of invasion being the unfortunate consequence (for both host and organism) of adaptations of hyperinvasive meningococcal lineages. The meningococcal capsule is an important virulence factor: acapsular strains only very rarely cause invasive disease. The capsule provides resistance to phagocytosis and may be important in preventing desiccation during transmission between hosts. Antigenic diversity in surface structures and an ability to vary levels of their expression have probably evolved as important factors in maintaining meningococcal populations within and between individual hosts.

1	Invasion through the mucosa into the blood occurs rarely, usually within a few days of acquisition of an invasive strain by a susceptible individual. Only occasional cases of prolonged colonization prior to invasion have been documented. Once the organism is in the bloodstream, its growth may be limited if the individual is partially immune, although bacteremia may allow seeding of another site, such as the meninges or the joints. Alternatively, unchecked proliferation may continue, resulting in high bacterial counts in the circulation. During growth, meningococci release blebs of outer membrane (Fig. 180-1) containing outer-membrane proteins and LPS. Endotoxin binds cell-bound CD14 in association with TLR4 to initiate an inflammatory cascade with the release of high levels of various mediators, including tumor necrosis factor (TNF) α, soluble TNF receptor, interleukin (IL) 1, IL-1 receptor antagonist, IL-1β, IL-6, IL-8, IL-10, plasminogenactivator inhibitor 1 (PAI-1), and leukemia

1	including tumor necrosis factor (TNF) α, soluble TNF receptor, interleukin (IL) 1, IL-1 receptor antagonist, IL-1β, IL-6, IL-8, IL-10, plasminogenactivator inhibitor 1 (PAI-1), and leukemia inhibitory factor. Soluble CD14-bound endotoxin acts as a mediator of endothelial activation. The severity of meningococcal disease is related both to the levels of endotoxin in the blood and to the magnitude of the inflammatory response. The latter is determined to some extent by polymorphisms in the inflammatory response genes (and their inhibitors), and the release of the inflammatory cascade heralds the development of meningococcal septicemia (meningococcemia). Endothelial injury is central to many clinical features of meningococcemia, including increased vascular permeability, pathologic changes in vascular tone, loss of thromboresistance, intravascular coagulation, and myocardial dysfunction. Endothelial injury leads to increased vascular permeability (attributed to loss of glycosaminoglycans

1	vascular tone, loss of thromboresistance, intravascular coagulation, and myocardial dysfunction. Endothelial injury leads to increased vascular permeability (attributed to loss of glycosaminoglycans and endothelial proteins), with subsequent gross proteinuria. Leakage of fluid and electrolytes into the tissues from capillaries (“capillary leak syndrome”) leads to hypovolemia, tissue edema, and pulmonary edema. Initial compensation results in vasoconstriction and tachycardia, although cardiac output eventually falls. While resuscitation fluids may restore circulating volume, tissue edema will continue to increase, and, in the lung, the consequence may be respiratory failure.

1	Intravascular thrombosis (caused by activation of procoagulant pathways in association with upregulation of tissue factor on the endothelium) occurs in some patients with meningococcal disease and results in purpura fulminans and infarction of areas of skin or even of whole limbs. At the same time, multiple anticoagulant pathways are downregulated through loss of endothelial thrombomodulin and protein C receptors and decreases in levels of antithrombin III, protein C, protein S, and tissue factor pathway inhibitor. Thrombolysis is also profoundly impaired in meningococcal sepsis through the release of high levels of PAI-1.

1	Shock in meningococcal septicemia appears to be attributable to a combination of factors, including hypovolemia, which results from the capillary leak syndrome secondary to endothelial injury, and myocardial depression, which is driven by hypovolemia, hypoxia, metabolic derangements (e.g., hypocalcemia), and cytokines (e.g., IL-6). Decreased perfusion of tissues as a result of intravascular thrombosis, vasoconstriction, tissue edema, and reduced cardiac output in meningococcal septicemia can cause widespread organ dysfunction, including renal impairment and—later in the disease—a decreased level of consciousness due to central nervous system involvement.

1	Bacteria that reach the meninges cause a local inflammatory response—with release of a spectrum of cytokines similar to that seen in septicemia—that presents clinically as meningitis and is thought to determine the severity of neuronal injury. Local endothelial injury may result in cerebral edema and rapid onset of raised intracranial pressure in some cases.

1	As discussed above, the most common form of infection with N. meningitidis is asymptomatic carriage of the organism in the nasopharynx. Despite the location of infection in the upper airway, meningococcal pharyngitis is rarely reported; however, upper respiratory tract symptoms are common prior to presentation with invasive disease. It is not clear whether these symptoms relate to preceding viral infection (which may promote meningococcal acquisition) or to meningococcal acquisition itself. After acquiring the organism, susceptible individuals develop disease manifestations in 1–10 days (usually <4 days, although colonization for 11 weeks has been documented).

1	Along the spectrum of presentations of meningococcal disease, the most common clinical syndromes are meningitis and meningococcal septicemia. In fulminant cases, death may occur within hours of the first symptoms. Occult bacteremia is also recognized and, if untreated, progresses in two-thirds of cases to focal infection, including meningitis or septicemia. Meningococcal disease may also present as pneumonia, pyogenic arthritis or osteomyelitis, purulent pericarditis, endophthalmitis, conjunctivitis, primary peritonitis, or (rarely) urethritis. Perhaps because it is difficult to diagnose, pneumococcal pneumonia is not commonly reported but is associated with serogroups Y, W, and Z and appears most often to affect individuals >10 years of age.

1	Rash A nonblanching rash (petechial or purpuric) develops in >80% of cases of meningococcal disease; however, the rash is often absent early in the illness. Usually initially blanching in nature (macules, maculopapules, or urticaria) and indistinguishable from more common viral rashes, the rash of meningococcal infection becomes petechial or frankly purpuric over the hours after onset. In the most severe cases, large purpuric lesions develop (purpura fulminans). Some patients (including those with overwhelming sepsis) may have no rash. While petechial rash and fever are important signs of meningococcal disease, fewer than 10% of children (and, in some clinical settings, fewer than 1% of patients) with this presentation are found to have meningococcal disease. Most patients presenting with a petechial or purpuric rash have a viral infection (Table 180-2). The skin lesions exhibit widespread endothelial necrosis and occlusion of small vessels in the dermis and subcutaneous tissues, with

1	a petechial or purpuric rash have a viral infection (Table 180-2). The skin lesions exhibit widespread endothelial necrosis and occlusion of small vessels in the dermis and subcutaneous tissues, with a neutrophilic infiltrate.

1	Meningitis Meningococcal meningitis commonly presents as nonspecific manifestations, including fever, vomiting, and (especially in infants and young children) irritability, and is indistinguishable from other forms of bacterial meningitis unless there is an associated petechial or purpuric rash, which occurs in two-thirds of cases. Headache is rarely reported in early childhood but is more common in later childhood and adulthood. When headache is present, the following features, in association with fever or a history of fever, are suggestive of bacterial meningitis: neck stiffness, photophobia, decreased CoMMon CAuSES of PETECHIAL oR PuRPuRIC RASHES Deficiency of protein C or S (including postvaricella protein S deficiency) Platelet disorders (e.g., idiopathic thrombocytopenic purpura, drug effects, bone marrow infiltration) Henoch-Schönlein purpura, connective tissue disorders, trauma (including nonaccidental injuries in children)

1	Henoch-Schönlein purpura, connective tissue disorders, trauma (including nonaccidental injuries in children) Pneumococcal, streptococcal, staphylococcal, or gram-negative bacterial sepsis level of consciousness, seizures or status epilepticus, and focal neurologic signs. Classic signs of meningitis, such as neck stiffness and photophobia, are often absent in infants and young children with bacterial meningitis, who more usually present with fever and irritability and may have a bulging fontanelle.

1	While 30–50% of patients present with a meningitis syndrome alone, up to 40% of meningitis patients also present with some features of septicemia. Most deaths from meningococcal meningitis alone (i.e., without septicemia) are associated with raised intracranial pressure presenting as a reduced level of consciousness, relative bradycardia and hypertension, focal neurologic signs, abnormal posturing, and signs of brainstem involvement—e.g., unequal, dilated, or poorly reactive pupils; abnormal eye movement; and impaired corneal responses (Chap. 328).

1	Septicemia Meningococcal septicemia alone accounts for up to 20% of cases of meningococcal disease. The condition may progress from early nonspecific symptoms to death within hours. Mortality rates among children with this syndrome have been high (25–40%), but early aggressive management (as discussed below) may reduce the figure to <10%. Early symptoms are nonspecific and suggest an influenza-like illness with fever, headache, and myalgia accompanied by vomiting and abdominal pain. As discussed above, the rash, if present, may appear to be viral early in the course until petechiae or purpuric lesions develop. Purpura fulminans occurs in severe cases, with multiple large purpuric lesions and signs of peripheral ischemia. Surveys of patients have indicated that limb pain, pallor (including a mottled appearance and cyanosis), and cold hands and feet may be prominent. Shock is manifested by tachycardia, poor peripheral perfusion, tachypnea, and oliguria. Decreased cerebral perfusion

1	a mottled appearance and cyanosis), and cold hands and feet may be prominent. Shock is manifested by tachycardia, poor peripheral perfusion, tachypnea, and oliguria. Decreased cerebral perfusion leads to confusion, agitation, or decreased level of consciousness. With progressive shock, multiorgan failure ensues; hypotension is a late sign in children, who more commonly present with compensated shock (tachycardia, poor peripheral perfusion, and normal blood pressure). Poor outcome is associated with an absence of meningism, hypotension, young age, coma, relatively low temperature (<38°C), leukopenia, and thrombocytopenia. Spontaneous hemorrhage (pulmonary, gastric, or cerebral) may result from consumption of coagulation factors and thrombocytopenia.

1	Chronic Meningococcemia Chronic meningococcemia, which is rarely recognized, presents as repeated episodes of petechial rash associated with fever, joint pain, features of arthritis, and splenomegaly that may progress to acute meningococcal septicemia if untreated. During the relapsing course, bacteremia characteristically clears without treatment and then recurs. The differential diagnosis includes bacterial endocarditis, acute rheumatic fever, Henoch-Schönlein purpura, infectious mononucleosis, disseminated gonococcal infection, and immune-mediated vasculitis. This condition has been associated with complement deficiencies in some cases and with inadequate sulfonamide therapy in others. A study from the Netherlands found that half of isolates from patients with chronic meningococcemia had an underacylated lipid A (part of the surface LPS molecule) due to an lpxL1 gene mutation, which markedly reduces the inflammatory response to 999 endotoxin.

1	Postmeningococcal Reactive Disease In a small proportion of patients, an immune complex disease develops ~4–10 days after the onset of meningococcal disease, with manifestations that include a maculopapular or vasculitic rash (2% of cases), arthritis (up to 8% of cases), iritis (1%), pericarditis, and/or polyserositis associated with fever. The immune complexes involve meningococcal polysaccharide antigen and result in immunoglobulin and complement deposition with an inflammatory infiltrate. These features resolve spontaneously without sequelae. It is important to recognize this condition since a new onset of fever and rash can lead to concerns about relapse of meningococcal disease and unnecessarily prolonged antibiotic treatment.

1	Like other invasive bacterial infections, meningococcal disease may produce elevations of the white blood cell (WBC) count and of values for inflammatory markers (e.g., C-reactive protein and procalcitonin levels or the erythrocyte sedimentation rate). Values may be normal or low in rapidly progressive disease, and a lack of rise in these laboratory test values does not exclude the diagnosis. However, in the presence of fever and a petechial rash, these elevations are suggestive of meningococcal disease. In patients with severe meningococcal septicemia, common laboratory findings include hypoglycemia, acidosis, hypokalemia, hypocalcemia, hypomagnesemia, hypophosphatemia, anemia, and coagulopathy.

1	Although meningococcal disease is often diagnosed on clinical grounds, in suspected meningococcal meningitis or meningococcemia, blood should routinely be sent for culture to confirm the diagnosis and to facilitate public health investigations; blood cultures are positive in up to 75% of cases. Culture media containing sodium polyanethol sulfonate, which may inhibit meningococcal growth, should be avoided. Meningococcal viability is reduced if there is a delay in transport of the specimen to the microbiology laboratory for culture or in plating of cerebrospinal fluid (CSF) samples. In countries where treatment with antibiotics before hospitalization is recommended for meningococcal disease, the majority of clinically suspected cases are culture negative. Real-time polymerase chain reaction (PCR) analysis of whole-blood samples increases the diagnostic yield by >40%, and results obtained with this method may remain positive for several days after administration of antibiotics. Indeed,

1	(PCR) analysis of whole-blood samples increases the diagnostic yield by >40%, and results obtained with this method may remain positive for several days after administration of antibiotics. Indeed, in the United Kingdom, more than half of clinically suspected cases are currently identified by PCR.

1	Unless contraindications exist (raised intracranial pressure, uncorrected shock, disordered coagulation, thrombocytopenia, respiratory insufficiency, local infection, ongoing convulsions), lumbar puncture should be undertaken to identify and confirm the etiology of suspected meningococcal meningitis, whose presentation cannot be distinguished from that of meningitis of other bacterial causes. Some authorities have recommended a CT brain scan prior to lumbar puncture because of the risk of cerebral herniation in patients with raised intracranial pressure. However, a normal CT scan is not uncommon in the presence of raised intracranial pressure in meningococcal meningitis, and the decision to perform a lumbar puncture should be made on clinical grounds. CSF features of meningococcal meningitis (elevated protein level and WBC count, decreased glucose level) are indistinguishable from those of other types of bacterial meningitis unless a gram-negative diplococcus is identified. (Gram’s

1	(elevated protein level and WBC count, decreased glucose level) are indistinguishable from those of other types of bacterial meningitis unless a gram-negative diplococcus is identified. (Gram’s staining is up to 80% sensitive for meningococcal meningitis.) CSF should be submitted for culture (sensitivity, 90%) and (where available) PCR analysis. CSF antigen testing with latex agglutination is insensitive and should be replaced by molecular diagnosis when possible.

1	Lumbar puncture should generally be avoided in meningococcal septicemia, as positioning for the procedure may critically compromise the patient’s circulation in the context of hypovolemic shock. Delayed lumbar puncture may still be useful when the diagnosis is uncertain, particularly if molecular diagnostic technology is available. In other types of focal infection, culture and PCR analysis of normally sterile body fluids (e.g., synovial fluid) may aid in the diagnosis. Although some authorities have recommended cultures of scrapings 1000 or aspirates from skin lesions, this procedure adds little to the diagnostic yield when compared with a combination of blood culture and PCR analysis. Urinary antigen testing also is insensitive, and serologic testing for meningococcal infection has not been adequately studied. Because N. meningitidis is a component of the normal human nasopharyngeal flora, identification of the organism on throat swabs has no diagnostic value.

1	Death from meningococcal disease is associated most commonly with hypovolemic shock (meningococcemia) and occasionally with raised intracranial pressure (meningococcal meningitis). Therefore, management should focus on the treatment of these urgent clinical issues in addition to the administration of specific antibiotic therapy. Delayed recognition of meningococcal disease or its associated physiologic derangements, together with inadequate emergency management, is associated with poor outcome. Since the disease is rare, protocols for emergency management have been developed (see www.meningitis.org).

1	Airway patency may be compromised if the level of consciousness is depressed as a result of shock (impaired cerebral perfusion) or raised intracranial pressure; this situation may require intervention. In meningococcemia, pulmonary edema and pulmonary oligemia (presenting as hypoxia) require oxygen therapy or elective endotracheal intubation. In cases with shock, aggressive fluid resuscitation (with replacement of the circulating volume several times in severe cases) and inotropic support may be necessary to maintain cardiac output. If shock persists after volume resuscitation at 40 mL/kg, the risk of pulmonary edema is high, and elective intubation is recommended to improve oxygenation and decrease the work of breathing. Metabolic derangements, including hypoglycemia, acidosis, hypokalemia, hypocalcemia, hypomagnesemia, hypophosphatemia, anemia, and coagulopathy, should be anticipated and corrected. In the presence of raised intracranial pressure, management includes correction of

1	hypocalcemia, hypomagnesemia, hypophosphatemia, anemia, and coagulopathy, should be anticipated and corrected. In the presence of raised intracranial pressure, management includes correction of coexistent shock and neurointensive care to maintain cerebral perfusion.

1	Empirical antibiotic therapy for suspected meningococcal disease consists of a third-generation cephalosporin such as ceftriaxone (75–100 mg/kg per day [maximum, 4 g/d] in one or two divided IV doses) or cefotaxime (200 mg/kg per day [maximum, 8 g/d] in four divided IV doses) to cover the various other (potentially penicillin-resistant) bacteria that may produce an indistinguishable clinical syndrome. Although unusual in most isolates, reduced meningococcal sensitivity to penicillin (a minimal inhibitory concentration of 0.12–1.0 μg/mL) has been reported widely.

1	Both meningococcal meningitis and meningococcal septicemia are conventionally treated for 7 days, although courses of 3–5 days may be equally effective. Furthermore, a single dose of ceftriaxone or an oily suspension of chloramphenicol has been used successfully in resource-poor settings. No data are available to guide the duration of treatment for meningococcal infection at other foci (e.g., pneumonia, arthritis); antimicrobial therapy is usually continued until clinical and laboratory evidence of infection has resolved. Cultures usually become sterile within 24 h of initiation of appropriate antibiotic chemotherapy.

1	The use of glucocorticoids for adjunctive treatment of meningococcal meningitis remains controversial since no relevant studies have had sufficient power to determine true efficacy. One large study in adults did indicate a trend toward benefit, and in clinical practice a decision to use glucocorticoids usually must precede a definite microbiologic diagnosis. Therapeutic doses of glucocorticoids are not recommended in meningococcal septicemia, but many intensivists recommend replacement glucocorticoid doses for patients who have refractory shock in association with impaired adrenal gland responsiveness.

1	Various other adjunctive therapies for meningococcal disease have been considered, but few have been subjected to clinical trials and none can currently be recommended. An antibody to LPS (HA1A) failed to confer a demonstrable benefit. Recombinant bactericidal/permeability-increasing protein (which is not currently available) was tested in a study that had inadequate power to show an effect on mortality rates; however, there were trends toward lower mortality rates among patients who received a complete infusion, and this group also had fewer amputations, fewer blood-product transfusions, and a significantly improved functional outcome. Given that protein C concentrations are reduced in meningococcal disease, the use of activated protein C has been considered since a survival benefit was demonstrated in adult sepsis trials; however, trials in pediatric sepsis (of particular relevance for meningococcal disease) found no benefit and indicated a potential risk of bleeding complications

1	demonstrated in adult sepsis trials; however, trials in pediatric sepsis (of particular relevance for meningococcal disease) found no benefit and indicated a potential risk of bleeding complications with use of activated protein C.

1	The postmeningococcal immune-complex inflammatory syndrome has been treated with nonsteroidal anti-inflammatory agents until spontaneous resolution occurs.

1	About 10% of patients with meningococcal disease die despite the availability of antimicrobial therapy and other intensive medical interventions. The most common complication of meningococcal disease (10% of cases) is scarring after necrosis of purpuric skin lesions, for which skin grafting may be necessary. The lower limbs are most often affected; next in frequency are the upper limbs, the trunk, and the face. On average, 13% of the skin surface area is involved. Amputations are necessary in 1–2% of survivors of meningococcal disease because of a loss of tissue viability after peripheral ischemia or compartment syndromes. Unless there is local infection, amputation should usually be delayed to allow the demarcation between viable and nonviable tissue to become apparent. Approximately 5% of patients with meningococcal disease suffer hearing loss, and 7% have neurologic complications. In one study pain was reported by 21% of survivors, and in a recent analysis of serogroup B

1	5% of patients with meningococcal disease suffer hearing loss, and 7% have neurologic complications. In one study pain was reported by 21% of survivors, and in a recent analysis of serogroup B meningococcal disease (the MOSAIC study) as many as one-quarter of survivors had psychological disorders. In some investigations, the rate of complications is higher for serogroup C disease (mostly associated with the ST11 clone) than for serogroup B disease. In patients with severe hypovolemic shock, renal perfusion may be impaired and prerenal failure is common, but permanent renal replacement therapy is rarely needed.

1	Several studies suggest adverse psychosocial outcomes after meningococcal disease, with reduced quality of life, lowered self-esteem, and poorer neurologic development, including increased rates of attention deficit/hyperactivity disorder and special educational needs. Other studies have not found evidence of such outcomes.

1	Several prognostic scoring systems have been developed to identify patients with meningococcal disease who are least likely to survive. Factors associated with a poorer prognosis are shock; young age (infancy), old age, and adolescence; coma; purpura fulminans; disseminated intravascular coagulation; thrombocytopenia; leukopenia; absence of meningitis; metabolic acidosis; low plasma concentrations of antithrombin and proteins S and C; high blood levels of PAI-1; and a low erythrocyte sedimentation rate or C-reactive protein level. The Glasgow Meningococcal Septicaemia Prognostic Score (GMSPS) is probably the best-performing scoring system studied so far and may be clinically useful for severity assessment in meningococcal disease. However, scoring systems do not direct the clinician to specific interventions, and the priority in management should be recognition of compromised airways, breathing, or circulation and direct, urgent intervention. Most patients improve rapidly with

1	to specific interventions, and the priority in management should be recognition of compromised airways, breathing, or circulation and direct, urgent intervention. Most patients improve rapidly with appropriate antibiotics and supportive therapy. Fulminant meningococcemia is more likely to result in death or ischemic skin loss than is meningitis; optimal emergency management may reduce mortality rates among the most severely affected patients.

1	Since mortality rates in meningococcal disease remain high despite improvements in intensive care management, immunization is the only rational approach to prevention at a population level. Secondary cases are common among household and “kissing” contacts of cases, and secondary prophylaxis with antibiotic therapy is widely recommended for these contacts (see below).

1	Polysaccharide Vaccines Purified meningococcal capsular polysaccharide has been used for immunization since the 1960s. Meningococcal polysaccharide vaccines are currently formulated as either bivalent (serogroups A and C) or quadrivalent (serogroups A, C, Y, and W), with 50 μg of each polysaccharide per dose. Local reactions (erythema, induration, and tenderness) may occur in up to 40% of vaccinees, but serious adverse events (including febrile convulsions in young children) are very rarely reported. In adults, the vaccines are immunogenic, but immunity appears to be relatively short-lived (with antibody levels above baseline for only 2–10 years), and booster doses do not induce a further rise in antibody concentration. Indeed, a state of immunologic hyporesponsiveness has been widely reported to follow booster doses of plain polysaccharide vaccines. The repeating units of these vaccines cross-link B cell receptors to drive specific memory B cells to become plasma cells and produce

1	to follow booster doses of plain polysaccharide vaccines. The repeating units of these vaccines cross-link B cell receptors to drive specific memory B cells to become plasma cells and produce antibody. Because meningococcal polysaccharides are T cell–independent antigens, no memory B cells are produced after immunization, and the memory B-cell pool is depleted such that fewer polysaccharide-specific cells are available to respond to a subsequent dose of vaccine (Fig. 180-6). The clinical relevance of hyporesponsiveness is unknown. Plain polysaccharide vaccines generally are not immunogenic in early childhood, pos-1001 sibly because marginal-zone B cells are involved in polysaccharide responses and maturation of the splenic marginal zone is not complete until 18 months to 2 years of age. The efficacy of the meningococcal serogroup C component is >90% in young adults; no efficacy data are available for the serogroup Y and W polysaccharides in this age group.

1	Group A meningococcal polysaccharides are exceptional in that they have been found to be effective in preventing disease at all ages. Two doses administered 2–3 months apart to children 3–18 months of age or a single dose administered to older children or adults has a protective efficacy rate of >95%. The vaccine has been widely used in the control of meningococcal disease in the African meningitis belt. The duration of protection appears to be only 3–5 years. There is no meningococcal serogroup B plain polysaccharide vaccine because α-2,8-N-acetylneuraminic acid is expressed on the surface of neural cells in the fetus such that the B polysaccharide is perceived as “self” and therefore is not immunogenic in humans.

1	Conjugate Vaccines The poor immunogenicity of plain polysaccharide vaccines in infancy has been overcome by chemical conjugation of the polysaccharides to a carrier protein (CRM197, tetanus toxoid, or diphtheria toxoid). Conjugates that contain monovalent serogroup C polysaccharide and quadrivalent vaccines with A, C, Y, and W polysaccharides have been developed, as have vaccines including various other antigen combinations (e.g., tetanus conjugates with serogroup C

1	Polysaccharide-specific B cell Polysaccharide-specific memory B cell CD40 CD80 or CD86 CD28CD40L TCR Carrier peptide– specific T cell Polysaccharide-specific plasma cell IgG1 and IgG3 BCR Polysaccharide Carrier protein Antibody production Memory response Internalization and processing of carrier protein T-cell help MHC Class II FIGURE 180-6 A. Polysaccharides from the encapsulated bacteria that cause disease in early childhood stimulate B cells by cross-linking the BCR and driving the production of immunoglobulins. There is no production of memory B cells, and the B-cell pool may be depleted by this process such that subsequent immune responses are decreased. B. The carrier protein from protein-polysaccharide conjugate vaccines is processed by the polysaccharide-specific B cell, and peptides are presented to carrier peptide–specific T cells, with the consequent production of both plasma cells and memory B cells. BCR, B-cell receptor; MHC, major histocompatibility complex; TCR, T-cell

1	are presented to carrier peptide–specific T cells, with the consequent production of both plasma cells and memory B cells. BCR, B-cell receptor; MHC, major histocompatibility complex; TCR, T-cell receptor. (Reprinted from AJ Pollard et al: Nat Rev Immunol 9:213, 2009.) 1002 and/or Y polysaccharide with Haemophilus influenzae type b polysaccharide). After immunization, peptides from the carrier protein are conventionally thought to be presented to peptide-specific T cells in association with major histocompatibility complex (MHC) class II molecules (some recent data suggesting that carrier protein peptide may actually be presented in association with an oligosaccharide and MHCII) by polysaccharide-specific B cells; the result is a T cell– dependent immune response that allows production of antibody and generation of an expanded B-cell memory pool. Unlike responses to booster doses of plain polysaccharides, responses to booster doses of conjugate vaccines have the characteristics of

1	of antibody and generation of an expanded B-cell memory pool. Unlike responses to booster doses of plain polysaccharides, responses to booster doses of conjugate vaccines have the characteristics of memory responses. Indeed, conjugate vaccines overcome the hyporesponsiveness induced by plain polysaccharides by replenishing the memory pool. The reactogenicity of conjugate vaccines is similar to that of plain polysaccharide vaccines. The first widespread use of serogroup C meningococcal conjugate vaccine (MenC) came in 1999 in the United Kingdom after a rise in serogroup C disease. A mass vaccination campaign involving all individuals <19 years of age was undertaken, and the number of laboratory-confirmed serogroup C cases fell from 955 in 1998–1999 to just 29 in 2011–2012. The effectiveness of the immunization program was attributed both to direct protection of immunized persons and to reduced transmission of the organism in the population as a result of decreased rates of colonization

1	of the immunization program was attributed both to direct protection of immunized persons and to reduced transmission of the organism in the population as a result of decreased rates of colonization among the immunized (herd immunity). Data on immunogenicity and effectiveness have shown that the duration of protection is short when the vaccine is administered in early childhood; thus booster doses are needed to maintain population immunity. In contrast, immunity after a dose of vaccine given in adolescence appears to be prolonged. The first quadrivalent conjugate meningococcal vaccine containing A, C, Y, and W polysaccharides conjugated to diphtheria toxoid was initially recommended for all children >11 years of age in the United States in 2005. In 2007 the license was extended to high-risk children 2–10 years of age. In the same year, the vaccine was licensed in Canada for persons 2–55 years of age. Uptake was slow, but current U.S. data suggest an efficacy rate of 82% in the first

1	children 2–10 years of age. In the same year, the vaccine was licensed in Canada for persons 2–55 years of age. Uptake was slow, but current U.S. data suggest an efficacy rate of 82% in the first year after vaccination, with waning to 59% at 3–6 years after vaccination. Limited data from the

1	U.S. Vaccine Adverse Events Reporting System indicated that there might be a short-term increase in the risk of Guillain-Barré syndrome after immunization with the diphtheria conjugate vaccine; however, further investigation has not confirmed this finding. Quadrivalent conjugate vaccines with tetanus or CRM197 as carrier protein are now available in many countries. A monovalent serogroup A vaccine, manufactured in India, was licensed in 2010 and rolled out to countries in the sub-Saharan African meningitis belt. There is strong evidence that this vaccine has been highly effective in controlling epidemic meningococcal disease in the region, with some evidence of a >90% reduction in disease in vaccinated populations. However, disease caused by serogroup X and W persists.

1	Vaccines Based on Subcapsular Antigens The lack of immunogenicity of the serogroup B capsule has led to the development of vaccines based on subcapsular antigens. Various surface components have been studied in early-phase clinical trials. Outer-membrane vesicles (OMVs) containing outer-membrane proteins, phospholipid, and LPS can be extracted from cultures of N. meningitidis by detergent treatment (Fig. 180-7). OMVs prepared in this way were used in efficacy trials with a Norwegian outbreak strain and reduced the incidence of group B disease among 14to 16-year-old schoolchildren by 53%. Similarly, OMV vaccines constructed from local outbreak strains in Cuba and New Zealand have had reported efficacy rates of >70%. These OMV vaccines appear to produce strain-specific immune responses, with only limited cross-protection, and are therefore best suited to clonal outbreaks (e.g., those in Cuba and New Zealand as well as others in Norway and the province of Normandy in France).

1	Several purified surface proteins have been evaluated in phase 1 clinical trials but have not yet been developed further because of antigenic variability or poor immunogenicity (e.g., transferrin-binding FIGURE 180-7 Illustration of meningococcal outer-membrane vesicle containing outer-membrane structures. proteins, neisserial surface protein A). Other vaccine candidates have been identified since sequencing of the meningococcal genome. A combination vaccine that includes the New Zealand OMV vaccine and three recombinant proteins (neisserial adhesin A, factor H–binding protein, and neisserial heparin-binding antigen) is immunogenic in infancy and has been licensed for use in Europe and Australia. Recommendations for its use are pending. Finally, a highly immunogenic vaccine based on two variants of the lipoprotein factor H–binding protein is undergoing clinical evaluation

1	Close (household and kissing) contacts of individuals with meningococcal disease are at increased risk (up to 1000 times the rate for the general population) of developing secondary disease; a secondary case follows as many as 3% of sporadic cases. About one-fifth of secondary cases are actually co-primary cases—i.e., cases that occur soon after the primary case and in which transmission is presumed to have originated from the same third party. The rate of secondary cases is highest during the week after presentation of the index case. The risk falls rapidly but remains above baseline for up to 1 year after the index case; 30% of secondary cases occur in the first week, 20% in the second week, and most of the remainder over the next 6 weeks. In outbreaks of meningococcal disease, mass prophylaxis has been used; however, limited data support population intervention, and significant concerns have arisen about adverse events and the development of resistance. For these reasons,

1	mass prophylaxis has been used; however, limited data support population intervention, and significant concerns have arisen about adverse events and the development of resistance. For these reasons, prophylaxis is usually restricted to (1) persons at greatest risk who are intimate and/or household contacts of the index case and (2) health care workers who have been directly exposed to respiratory secretions. In most cases, members of wider communities (e.g., at schools or colleges) are not offered prophylaxis.

1	The aim of prophylaxis is to eradicate colonization of close contacts with the strain that has caused invasive disease in the index case. Prophylaxis should be given to all contacts at the same time to avoid recolonization by meningococci transmitted from untreated contacts and should also be used as soon as possible to treat early disease in secondary cases. If the index patient is treated with an antibiotic that does not reliably clear colonization (e.g., penicillin), he or she should be given a prophylactic agent at the end of treatment to prevent relapse or onward transmission. Although rifampin has been gonococcal Infections Sanjay Ram, Peter A. Rice DEFINITION Gonorrhea is a sexually transmitted infection (STI) of epithelium and commonly manifests as cervicitis, urethritis, proctitis, and conjunctivi-tis. If untreated, infections at these sites can lead to local complications 181 most widely used and studied, it is not the optimal agent because it fails to eradicate carriage in

1	and conjunctivi-tis. If untreated, infections at these sites can lead to local complications 181 most widely used and studied, it is not the optimal agent because it fails to eradicate carriage in 15–20% of cases, rates of adverse events have been high, compliance is affected by the need for four doses, and emerging resistance has been reported. Ceftriaxone as a single IM or IV injection is highly (97%) effective in carriage eradication and can be used at all ages and in pregnancy. Reduced susceptibility of isolates to ceftriaxone has occasionally been reported. Ciprofloxacin or ofloxacin is preferred in some countries; these agents are highly effective and can be administered by mouth but are not recommended in pregnancy. Resistance to fluoroquinolones has been reported in some meningococci in North America, Europe, and Asia.

1	In documented serogroup A, C, Y, or W disease, contacts may be offered immunization (preferably with a conjugate vaccine) in addition to chemoprophylaxis to provide protection beyond the duration of antibiotic therapy. Mass vaccination has been used successfully to control disease during outbreaks in closed communities (educational and military establishments) as well as during epidemics in open communities. such as endometritis, salpingitis, tuboovarian abscess, bartholinitis, peritonitis, and perihepatitis in female patients; periurethritis and epididymitis in male patients; and ophthalmia neonatorum in newborns. Disseminated gonococcemia is an uncommon event whose manifestations include skin lesions, tenosynovitis, arthritis, and (in rare cases) endocarditis or meningitis.

1	Neisseria gonorrhoeae is a gram-negative, nonmotile, non-sporeforming organism that grows singly and in pairs (i.e., as monococci and diplococci, respectively). Exclusively a human pathogen, the gonococcus contains, on average, three genome copies per coccal unit; this polyploidy permits a high level of antigenic variation and the survival of the organism in its host. Gonococci, like all other Neisseria species, are oxidase positive. They are distinguished from other neisseriae by their ability to grow on selective media and to use glucose but not maltose, sucrose, or lactose. The incidence of gonorrhea has declined significantly in the United States, but there were still ~311,000 newly reported cases in 2012. Gonorrhea remains a major public health problem worldwide, is a significant cause of morbidity in developing countries, and may play a role in enhancing transmission of HIV.

1	Gonorrhea predominantly affects young, nonwhite, unmarried, less educated members of urban populations. The number of reported cases probably represents half of the true number of cases—a discrepancy resulting from underreporting, self-treatment, and nonspecific treatment without a laboratory-proven diagnosis. The number of reported new cases of gonorrhea in the United States rose from ~250,000 in the early 1960s to a high of 1.01 million in 1978. The recorded incidence of gonorrhea in modern times peaked in 1975, with 468 reported new cases per 100,000 population in the United States. This peak was attributable to the interaction of several variables, including improved accuracy of diagnosis, changes in patterns of contraceptive use, and changes in sexual behavior. The incidence of the disease has since declined gradually and is currently estimated at 120 cases per 100,000, a figure that is still the highest among industrialized countries. A fur-1003 ther decline in the overall

1	the disease has since declined gradually and is currently estimated at 120 cases per 100,000, a figure that is still the highest among industrialized countries. A fur-1003 ther decline in the overall incidence of gonorrhea in the United States over the past quarter-century may reflect increased condom use resulting from public health efforts to curtail HIV transmission. At present, the attack rate in the United States is highest among 15to 19-year-old women and 20to 24-year-old men; 60% of all reported cases occur in the preceding two groups together. From the standpoint of ethnicity, rates are highest among African Americans and lowest among persons of Asian or Pacific Island descent.

1	The incidence of gonorrhea is higher in developing countries than in industrialized nations. The exact incidence of any STI is difficult to ascertain in developing countries because of limited surveillance and variable diagnostic criteria. Studies in Africa have clearly demonstrated that nonulcerative STIs such as gonorrhea (in addition to ulcerative STIs) are an independent risk factor for the transmission of HIV (Chap. 226). Gonorrhea is transmitted from males to females more efficiently than in the opposite direction. The rate of transmission to a woman during a single unprotected sexual encounter with an infected man is ~50–70%. Oropharyngeal gonorrhea occurs in ~20% of women who practice fellatio with infected partners. Transmission in either direction by cunnilingus is rare.

1	In any population, there exists a small minority of individuals who have high rates of new-partner acquisition. These “core-group members” or “high-frequency transmitters” are vital in sustaining STI transmission at the population level. Another instrumental factor in sustaining gonorrhea in the population is the large number of infected individuals who are asymptomatic or have minor symptoms that are ignored. These persons, unlike symptomatic individuals, may not cease sexual activity and therefore continue to transmit the infection. This situation underscores the importance of contact tracing and empirical treatment of the sex partners of index cases.

1	PATHOGENESIS, IMMUNOLOGY, AND ANTIMICROBIAL RESISTANCE Outer-Membrane Proteins • pili Fresh clinical isolates of N. gonor rhoeae initially form piliated (fimbriated) colonies distinguishable on translucent agar. Pilus expression is rapidly switched off with unselected subculture because of rearrangements in pilus genes. This change is a basis for antigenic variation of gonococci. Piliated strains adhere better to cells derived from human mucosal surfaces and are more virulent in organ culture models and human inoculation experiments than nonpiliated variants. In a fallopian tube explant model, pili mediate gonococcal attachment to nonciliated columnar epithelial cells. This event initiates gonococcal phagocytosis and transport through these cells to intercellular spaces near the basement membrane or directly into the subepithelial tissue. Pili are also essential for genetic competence and transformation of N. gonorrhoeae, which permit horizontal transfer of genetic material between

1	membrane or directly into the subepithelial tissue. Pili are also essential for genetic competence and transformation of N. gonorrhoeae, which permit horizontal transfer of genetic material between different gonococcal lineages in vivo.

1	opacity-associated protein Another gonococcal surface protein that is important in adherence to epithelial cells is opacity-associated protein (Opa, formerly called protein II). Opa contributes to intergonococcal adhesion, which is responsible for the opaque nature of gonococcal colonies on translucent agar and the organism’s adherence to a variety of eukaryotic cells, including polymorphonuclear leukocytes (PMNs). Certain Opa variants promote invasion of epithelial cells, and this effect has been linked with the ability of Opa to bind vitronectin, glycosaminoglycans, and several members of the carcinoembryonic antigen–related cell adhesion molecule (CEACAM) receptor family.

1	N. gonorrhoeae Opa proteins that bind CEACAM1, which is expressed by primary CD4+ T lymphocytes, suppress the activation and proliferation of these lymphocytes. This phenomenon may serve to explain the transient decrease in CD4+ T lymphocyte counts associated with gonococcal infection. Select Opa proteins can engage CEACAM3, which is expressed on neutrophils, with consequent nonopsonic phagocytosis (i.e., phagocytosis independent of antibody and complement) and killing of bacteria.

1	1004 porin Porin (previously designated protein I) is the most abundant gonococcal surface protein, accounting for >50% of the organism’s total outer-membrane protein. Porin molecules exist as trimers that provide anion-transporting aqueous channels through the otherwise hydrophobic outer membrane. Porin exhibits stable interstrain antigenic variation and forms the basis for gonococcal serotyping. Two main serotypes have been identified: PorB.1A strains are often associated with disseminated gonococcal infection (DGI), whereas PorB.1B strains usually cause local genital infections only. DGI strains are generally resistant to the killing action of normal human serum and do not incite a significant local inflammatory response; therefore, they may not cause symptoms at genital sites. These characteristics may be related to the ability of PorB.1A strains to bind to complement-inhibitory molecules, resulting in a diminished inflammatory response. Porin can translocate to the cytoplasmic

1	characteristics may be related to the ability of PorB.1A strains to bind to complement-inhibitory molecules, resulting in a diminished inflammatory response. Porin can translocate to the cytoplasmic membrane of host cells—a process that could initiate gonococcal endocytosis and invasion.

1	otHer outer-membrane proteins Other notable outer-membrane proteins include H.8, a lipoprotein that is present in high concentration on the surface of all gonococcal strains and is an excellent target for antibody-based diagnostic testing. Transferrin-binding proteins (Tbp1 and Tbp2) and lactoferrin-binding protein are required for scavenging iron from transferrin and lactoferrin in vivo. Transferrin and iron have been shown to enhance the attachment of iron-deprived N. gonorrhoeae to human endometrial cells. IgA1 protease is produced by N. gonorrhoeae and may protect the organism from the action of mucosal IgA.

1	N. gonorrhoeae to human endometrial cells. IgA1 protease is produced by N. gonorrhoeae and may protect the organism from the action of mucosal IgA. Lipooligosaccharide Gonococcal lipooligosaccharide (LOS) consists of a lipid A and a core oligosaccharide that lacks the repeating O-carbohydrate antigenic side chain seen in other gram-negative bacteria (Chap. 145e). Gonococcal LOS possesses marked endotoxic activity and contributes to the local cytotoxic effect in a fallopian tube model. LOS core sugars undergo a high degree of phase variation under different conditions of growth; this variation reflects genetic regulation and expression of glycotransferase genes that dictate the carbohydrate structure of LOS. These phenotypic changes may affect interactions of

1	N. gonorrhoeae with elements of the humoral immune system (antibodies and complement) and may also influence direct binding of organisms to both professional phagocytes and nonprofessional phagocytes (epithelial cells). For example, gonococci that are sialylated at their LOS sites bind complement factor H and inhibit the alternative pathway of complement. LOS sialylation may also decrease nonopsonic Opamediated association with neutrophils and inhibit the oxidative burst in PMNs. The binding of the unsialylated terminal lactosamine residue of LOS to an asialoglycoprotein receptor on male epithelial cells facilitates adherence and subsequent gonococcal invasion of these cells. Moreover, oligosaccharide structures in LOS can modulate host immune responses. For example, the terminal monosaccharide expressed by LOS determines the C-type lectin receptor on dendritic cells that is targeted by the bacteria. In turn, the specific C-type lectin receptor engaged influences whether a TH1or

1	expressed by LOS determines the C-type lectin receptor on dendritic cells that is targeted by the bacteria. In turn, the specific C-type lectin receptor engaged influences whether a TH1or TH2-type response is elicited; the latter response may be less favorable for clearance of gonococcal infection.

1	Host Factors In addition to gonococcal structures that interact with epithelial cells, host factors seem to be important in mediating entry of gonococci into nonphagocytic cells. Activation of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase by N. gonorrhoeae, which results in the release of diacylglycerol and ceramide, is a requirement for the entry of N. gonorrhoeae into epithelial cells. Ceramide accumulation within cells leads to apoptosis, which may disrupt epithelial integrity and facilitate entry of gonococci into subepithelial tissue. Release of chemotactic factors as a result of complement activation contributes to inflammation, as does the toxic effect of LOS in provoking the release of inflammatory cytokines.

1	The importance of humoral immunity in host defenses against neisserial infections is best illustrated by the predisposition of persons deficient in terminal complement components (C5 through C9) to recurrent bacteremic gonococcal infections and to recurrent meningococcal meningitis or meningococcemia. Gonococcal porin induces T cell–proliferative responses in persons with urogenital gonococcal disease. A significant increase in porin-specific interleukin (IL) 4– producing CD4+ as well as CD8+ T lymphocytes is seen in individuals with mucosal gonococcal disease. A portion of these lymphocytes that show a porin-specific TH2-type response could traffic to mucosal surfaces and play a role in immune protection against the disease. Few data clearly indicate that protective immunity is acquired from a previous gonococcal infection, although bactericidal and opsonophagocytic antibodies to porin and LOS may offer partial protection. On the other hand, women who are infected and acquire high

1	from a previous gonococcal infection, although bactericidal and opsonophagocytic antibodies to porin and LOS may offer partial protection. On the other hand, women who are infected and acquire high levels of antibody to another outer-membrane protein, Rmp (reduction modifiable protein, formerly called protein III), may be especially likely to become reinfected with N. gonorrhoeae because Rmp antibodies block the effect of bactericidal antibodies to porin and LOS. Rmp shows little, if any, interstrain antigenic variation; therefore, Rmp antibodies potentially may block antibody-mediated killing of all gonococci. The mechanism of blocking has not been fully characterized, but Rmp antibodies may noncompetitively inhibit binding of porin and LOS antibodies because of the proximity of these structures in the gonococcal outer membrane. In male volunteers who have no history of gonorrhea, the net effect of these events may influence the outcome of experimental challenge with N. gonorrhoeae.

1	in the gonococcal outer membrane. In male volunteers who have no history of gonorrhea, the net effect of these events may influence the outcome of experimental challenge with N. gonorrhoeae. Because Rmp bears extensive homology to enterobacterial OmpA and meningococcal class 4 proteins, it is possible that these blocking antibodies result from prior exposure to cross-reacting proteins from these species and also play a role in first-time infection with N. gonorrhoeae.

1	Gonococcal Resistance to Antimicrobial Agents It is no surprise that N. gonorrhoeae, with its remarkable capacity to alter its antigenic structure and adapt to changes in the microenvironment, has become resistant to numerous antibiotics. The first effective agents against gonorrhea were the sulfonamides, which were introduced in the 1930s and became ineffective within a decade. Penicillin was then used as the drug of choice for the treatment of gonorrhea. By 1965, 42% of gonococcal isolates had developed low-level resistance to penicillin G. Resistance due to the production of penicillinase arose later. Gonococci become fully resistant to antibiotics either by chro mosomal mutations or by acquisition of R factors (plasmids).

1	Gonococci become fully resistant to antibiotics either by chro mosomal mutations or by acquisition of R factors (plasmids). Two types of chromosomal mutations have been described. The first type, which is drug specific, is a single-step mutation leading to high-level resistance. The second type involves mutations at several chromosomal loci that combine to determine the level as well as the pattern of resistance. Strains with mutations in chromosomal genes were first observed in the late 1950s. As recently as 2007, chromosomal mutations accounted for resistance to penicillin, tetracycline, or both in ~16% of strains surveyed in the United States.

1	β-Lactamase (penicillinase)–producing strains of N. gonorrhoeae (PPNG) carrying plasmids with the Pcr determinant had rapidly spread worldwide by the early 1980s. N. gonorrhoeae strains with plasmid-borne tetracycline resistance (TRNG) can mobilize some β-lactamase plasmids, and PPNG and TRNG occur together, sometimes along with strains exhibiting chromosomally mediated resistance (CMRNG). Penicillin, ampicillin, and tetracycline are no longer reliable for the treatment of gonorrhea and should not be used.

1	treatment of gonococcal infections; the fluoroquinolones offered the advantage of antichlamydial activity when administered for 7 days. However, quinolone-resistant N. gonorrhoeae (QRNG) appeared soon after these agents were first used to treat gonorrhea. QRNG is particularly common in the Pacific Islands (including Hawaii) and Asia, where, in certain areas, all gonococcal strains are now resistant to quinolones. At present, QRNG is also common in parts of Europe and the Middle East. In the United States, QRNG has been identified in midwestern and eastern areas as well as in states on the Pacific coast, where resistant strains were first seen. Alterations in DNA gyrase and topoisomerase IV have been implicated as mechanisms of fluoroquinolone resistance.

1	Resistance to spectinomycin, which has been used in the past as an alternative agent, has been reported. Because this agent usually is not associated with resistance to other antibiotics, spectinomycin can be reserved for use against multidrug-resistant strains of N. gonorrhoeae. Nevertheless, outbreaks caused by strains resistant to spectinomycin have been documented in Korea and England when the drug has been used for primary treatment of gonorrhea.

1	Third-generation cephalosporins have remained highly effective as single-dose therapy for gonorrhea, but the recent isolation of strains highly resistant to ceftriaxone (minimal inhibitory concentrations [MICs], 2 μg/mL) in Japan and some European countries is cause for concern. Even though the MICs of ceftriaxone against certain strains may reach 0.015–0.125 μg/mL (higher than the MICs of 0.0001–0.008 μg/mL for fully susceptible strains), these levels are greatly exceeded in the blood, the urethra, and the cervix when the routinely recommended parenteral dose of ceftriaxone is administered. The rising MICs of oral cefixime (the previously recommended alternative oral third-generation cephalosporin) against N. gonorrhoeae, combined with this drug’s limited capacity to reach levels sufficiently higher than MICs in the blood, the urethra, the cervix, and especially the pharynx, have resulted in the removal of cefixime from the list of first-line agents for treatment of uncomplicated

1	higher than MICs in the blood, the urethra, the cervix, and especially the pharynx, have resulted in the removal of cefixime from the list of first-line agents for treatment of uncomplicated gonorrhea. All

1	N. gonorrhoeae strains with reduced susceptibility to ceftriaxone and cefixime (i.e., cephalosporin-intermediate/resistant strains) contain (1) a mosaic penA allele, which is the principal resistance determinant and encodes a penicillin-binding protein (PBP2) whose sequence differs in 60 amino acids from that of wild-type PBP2, and (2) additional genetic resistance determinants that are also required for high-level penicillin resistance.

1	CLINICAL MANIFESTATIONS Gonococcal Infections in Men Acute urethritis is the most common clinical manifestation of gonorrhea in male patients. The usual incubation period after exposure is 2–7 days, although the interval can be longer and some men remain asymptomatic. Strains of the PorB.1A serotype tend to cause a greater proportion of cases of mild and asymptomatic urethritis than do PorB.1B strains. Urethral discharge and dysuria, usually without urinary frequency or urgency, are the major symptoms. The discharge initially is scant and mucoid but becomes profuse and purulent within a day or two. Gram’s staining of the urethral discharge may reveal PMNs and gram-negative intracellular monococci and diplococci (Fig. 181-1). The clinical manifestations of gonococcal urethritis are usually more severe and overt than those of nongonococcal urethritis, including urethritis caused by Chlamydia trachomatis (Chap. 213); however, exceptions are common, and it is often impossible to

1	usually more severe and overt than those of nongonococcal urethritis, including urethritis caused by Chlamydia trachomatis (Chap. 213); however, exceptions are common, and it is often impossible to differentiate the causes of urethritis on clinical grounds alone. The majority of cases of urethritis seen in the United States today are not caused by N. gonorrhoeae and/or C. trachomatis. Although a number of other organisms may be responsible, many cases do not have a specific etiologic agent identified.

1	Most symptomatic men with gonorrhea seek treatment and cease to be infectious. The remaining men, who are largely asymptomatic, accumulate in number over time and constitute about two-thirds of all infected men at any point in time; together with men incubating

1	FIGURE 181-1 Gram’s stain of urethral discharge from a male patient with gonorrhea shows gram-negative intracellular mono-cocci and diplococci. (From the Public Health Agency of Canada.) the organism (who shed the organism but are asymptomatic), they 1005 serve as the source of spread of infection. Before the antibiotic era, symptoms of urethritis persisted for ~8 weeks. Epididymitis is now an uncommon complication, and gonococcal prostatitis occurs rarely, if at all. Other unusual local complications of gonococcal urethritis include edema of the penis due to dorsal lymphangitis or thrombophlebitis, submucous inflammatory “soft” infiltration of the urethral wall, periurethral abscess or fistula, inflammation or abscess of Cowper’s gland, and seminal vesiculitis. Balanitis may develop in uncircumcised men.

1	Gonococcal Infections in Women • gonococcal cervicitis Mucopurulent cervicitis is a common STI diagnosis in American women and may be caused by N. gonorrhoeae, C. trachomatis, and other organisms, including Mycoplasma genitalium (Chap. 212). Cervicitis may coexist with candidal or trichomonal vaginitis. N. gonorrhoeae primarily infects the columnar epithelium of the cervical os. Bartholin’s glands occasionally become infected.

1	Women infected with N. gonorrhoeae usually develop symptoms. However, the women who either remain asymptomatic or have only minor symptoms may delay in seeking medical attention. These minor symptoms may include scant vaginal discharge issuing from the inflamed cervix (without vaginitis or vaginosis per se) and dysuria (often without urgency or frequency) that may be associated with gonococcal urethritis. Although the incubation period of gonorrhea is less well defined in women than in men, symptoms usually develop within 10 days of infection and are more acute and intense than those of chlamydial cervicitis.

1	The physical examination may reveal a mucopurulent discharge (mucopus) issuing from the cervical os. Because Gram’s stain is not sensitive for the diagnosis of gonorrhea in women, specimens should be submitted for culture or a nonculture assay (see “Laboratory Diagnosis,” below). Edematous and friable cervical ectopy and endocervical bleeding induced by gentle swabbing are more often seen in chlamydial infection. Gonococcal infection may extend deep enough to produce dyspareunia and lower abdominal or back pain. In such cases, it is imperative to consider a diagnosis of pelvic inflammatory disease (PID) and to administer treatment for that disease (Chaps. 163 and 213).

1	N. gonorrhoeae may also be recovered from the urethra and rectum of women with cervicitis, but these are rarely the only infected sites. Urethritis in women may produce symptoms of internal dysuria, which is often attributed to “cystitis.” Pyuria in the absence of bacteriuria seen on Gram’s stain of unspun urine, accompanied by urine cultures that fail to yield >102 colonies of bacteria usually associated with urinary tract infection, signifies the possibility of urethritis due to C. trachomatis. Urethral infection with N. gonorrhoeae may also occur in this context, but in this instance urethral cultures are usually positive.

1	gonococcal vaginitis The vaginal mucosa of healthy women is lined by stratified squamous epithelium and is rarely infected by N. gonorrhoeae. However, gonococcal vaginitis can occur in anestrogenic women (e.g., prepubertal girls and postmenopausal women), in whom the vaginal stratified squamous epithelium is often thinned down to the basilar layer, which can be infected by N. gonorrhoeae. The intense inflammation of the vagina makes the physical (speculum and bimanual) examination extremely painful. The vaginal mucosa is red and edematous, and an abundant purulent discharge is often present. Infection in the urethra and in Skene’s and Bartholin’s glands often accompanies gonococcal vaginitis. Inflamed cervical erosion or abscesses in nabothian cysts may also occur. Coexisting cervicitis may result in pus in the cervical os.

1	Anorectal Gonorrhea Because the female anatomy permits the spread of cervical exudate to the rectum, N. gonorrhoeae is sometimes recovered from the rectum of women with uncomplicated gonococcal cervicitis. The rectum is the sole site of infection in only 5% of women with gonorrhea. Such women are usually asymptomatic but occasionally have acute proctitis manifested by anorectal pain or pruritus, tenesmus, purulent rectal discharge, and rectal bleeding. Among men 1006 who have sex with men (MSM), the frequency of gonococcal infection, including rectal infection, fell by ≥90% throughout the United States in the early 1980s, but a resurgence of gonorrhea among MSM has been documented in several cities since the 1990s. Gonococcal isolates from the rectum of MSM tend to be more resistant to antimicrobial agents than are gonococcal isolates from other sites. Gonococcal isolates with a mutation in mtrR (multiple transferable resistance repressor) or in the promoter region of the gene that

1	antimicrobial agents than are gonococcal isolates from other sites. Gonococcal isolates with a mutation in mtrR (multiple transferable resistance repressor) or in the promoter region of the gene that encodes for this transcriptional repressor develop increased resistance to antimicrobial hydrophobic agents such as bile acids and fatty acids in feces and thus are found with increased frequency in MSM. This situation may have been responsible for higher rates of failure of treatment for rectal gonorrhea with older regimens consisting of penicillin or tetracyclines.

1	Pharyngeal Gonorrhea Pharyngeal gonorrhea is usually mild or asymptomatic, although symptomatic pharyngitis does occasionally occur with cervical lymphadenitis. The mode of acquisition is oral-genital sexual exposure, with fellatio being a more efficient means of transmission than cunnilingus. In certain female adolescent populations in the United States, pharyngeal gonorrhea has become as common as genital gonorrhea. Most cases resolve spontaneously, and transmission from the pharynx to sexual contacts is rare. Pharyngeal infection almost always coexists with genital infection. Swabs from the pharynx should be plated directly onto gonococcal selective media. Pharyngeal colonization with Neisseria meningitidis needs to be differentiated from that with other Neisseria species.

1	Ocular Gonorrhea in Adults Ocular gonorrhea in an adult usually results from autoinoculation of N. gonorrhoeae from an infected genital site. As in genital infection, the manifestations range from severe to occasionally mild or asymptomatic disease. The variability in clinical manifestations may be attributable to differences in the ability of the infecting strain to elicit an inflammatory response. Infection may result in a markedly swollen eyelid, severe hyperemia and chemosis, and a profuse purulent discharge. The massively inflamed conjunctiva may be draped over the cornea and limbus. Lytic enzymes from the infiltrating PMNs occasionally cause corneal ulceration and rarely cause perforation. Prompt recognition and treatment of this condition are of paramount importance. Gram’s stain and culture of the purulent discharge establish the diagnosis. Genital cultures should also be performed.

1	Gonorrhea in Pregnant Women, Neonates, and Children Gonorrhea in pregnancy can have serious consequences for both the mother and the infant. Recognition of gonorrhea early in pregnancy also identifies a population at risk for other STIs, particularly chlamydial infection, syphilis, and trichomoniasis. The risks of salpingitis and PID—conditions associated with a high rate of fetal loss—are highest during the first trimester. Pharyngeal infection, most often asymptomatic, may be more common during pregnancy because of altered sexual practices. Prolonged rupture of the membranes, premature delivery, chorioamnionitis, funisitis (infection of the umbilical cord stump), and sepsis in the infant (with N. gonorrhoeae detected in the newborn’s gastric aspirate during delivery) are common complications of maternal gonococcal infection at term. Other conditions and microorganisms, including Mycoplasma hominis, Ureaplasma urealyticum, C. trachomatis, and bacterial vaginosis (often accompanied by

1	of maternal gonococcal infection at term. Other conditions and microorganisms, including Mycoplasma hominis, Ureaplasma urealyticum, C. trachomatis, and bacterial vaginosis (often accompanied by infection with Trichomonas vaginalis), have been associated with similar complications.

1	The most common form of gonorrhea in neonates is ophthalmia neonatorum, which results from exposure to infected cervical secretions during parturition. Ocular neonatal instillation of a prophylactic agent (e.g., 1% silver nitrate eye drops or ophthalmic preparations containing erythromycin or tetracycline) prevents ophthalmia neonatorum but is not effective for its treatment, which requires systemic antibiotics. The clinical manifestations are acute and usually begin 2–5 days after birth. An initial nonspecific conjunctivitis with a serosanguineous discharge is followed by tense edema of the eyelids, chemosis, and a profuse, thick, purulent discharge. Corneal ulcerations that result in nebulae or perforation may lead to anterior synechiae, anterior staphyloma, panophthalmitis, and blindness. Infections described at other mucosal sites in infants, including vaginitis, rhinitis, and anorectal infection, are likely to be asymptomatic. Pharyngeal colonization has been demonstrated in 35%

1	Infections described at other mucosal sites in infants, including vaginitis, rhinitis, and anorectal infection, are likely to be asymptomatic. Pharyngeal colonization has been demonstrated in 35% of infants with gonococcal ophthalmia, and coughing is the most prominent symptom in these cases. Septic arthritis (see below) is the most common manifestation of systemic infection or DGI in the newborn. The onset usually comes at 3–21 days of age, and polyarticular involvement is common. Sepsis, meningitis, and pneumonia are seen in rare instances.

1	Any STI in children beyond the neonatal period raises the possibility of sexual abuse. Gonococcal vulvovaginitis is the most common manifestation of gonococcal infection in children beyond infancy. Anorectal and pharyngeal infections are common in these children and are frequently asymptomatic. The urethra, Bartholin’s and Skene’s glands, and the upper genital tract are rarely involved. All children with gonococcal infection should also be evaluated for chlamydial infection, syphilis, and possibly HIV infection.

1	Gonococcal Arthritis (DGI) DGI (gonococcal arthritis) results from gonococcal bacteremia. In the 1970s, DGI occurred in ~0.5–3% of persons with untreated gonococcal mucosal infection. The lower incidence of DGI at present is probably attributable to a decline in the prevalence of particular strains that are likely to disseminate. DGI strains resist the bactericidal action of human serum and generally do not incite inflammation at genital sites, probably because of limited generation of chemotactic factors. Strains recovered from DGI cases in the 1970s were often of the PorB.1A serotype, were highly susceptible to penicillin, and had special growth requirements—including arginine, hypoxanthine, and uracil—that made the organism more fastidious and more difficult to isolate.

1	Menstruation is a risk factor for dissemination, and approximately two-thirds of cases of DGI are in women. In about half of affected women, symptoms of DGI begin within 7 days of onset of menses. Complement deficiencies, especially of the components involved in the assembly of the membrane attack complex (C5 through C9), predispose to neisserial bacteremia, and persons with more than one episode of DGI should be screened with an assay for total hemolytic complement activity.

1	The clinical manifestations of DGI have sometimes been classified into two stages: a bacteremic stage, which is less common today, and a joint-localized stage with suppurative arthritis. A clear-cut progression usually is not evident. Patients in the bacteremic stage have higher temperatures, and chills more frequently accompany their fever. Painful joints are common and often occur together with tenosynovitis and skin lesions. Polyarthralgias usually include the knees, elbows, and more distal joints; the axial skeleton is generally spared. Skin lesions are seen in ~75% of patients and include papules and pustules, often with a hemorrhagic component (Fig. 181-2; see also Fig. 25e-44). Other manifestations of noninfectious dermatitis, such as nodular lesions, urticaria, and erythema multiforme, have been described. These lesions are usually on the extremities and number between 5 and 40. The differential diagnosis of the bacteremic stage of DGI includes reactive arthritis, acute

1	multiforme, have been described. These lesions are usually on the extremities and number between 5 and 40. The differential diagnosis of the bacteremic stage of DGI includes reactive arthritis, acute rheumatoid arthritis, sarcoidosis, erythema nodosum, drug-induced arthritis, and viral infections (e.g., hepatitis B and acute HIV infection). The distribution of joint symptoms in reactive arthritis differs from that in DGI (Fig. 181-3), as do the skin and genital manifestations (Chap. 384).

1	Suppurative arthritis involves one or two joints, most often the knees, wrists, ankles, and elbows (in decreasing order of frequency); other joints occasionally are involved. Most patients who develop gonococcal septic arthritis do so without prior polyarthralgias or skin lesions; in the absence of symptomatic genital infection, this disease cannot be distinguished from septic arthritis caused by other pathogens. The differential diagnosis of acute arthritis in young adults is discussed in Chap. 157. Rarely, osteomyelitis complicates septic arthritis involving small joints of the hand. Gonococcal endocarditis, although rare today, was a relatively common complication of DGI in the preantibiotic era, accounting for about one-quarter of reported cases of endocarditis. Another unusual complication of DGI is meningitis. Gonococcal Infections in HIV-Infected Persons The association between gonorrhea and the acquisition of HIV has been demonstrated in

1	Gonococcal Infections in HIV-Infected Persons The association between gonorrhea and the acquisition of HIV has been demonstrated in FIGURE 181-2 Characteristic skin lesions in patients with proven gonococcal bacteremia. The lesions are in various stages of evolution. A. Very early petechia on finger. B. Early papular lesion, 7 mm in diameter, on lower leg. C. Pustule with central eschar resulting from early petechial lesion. D. Pustular lesion on finger. E. Mature lesion with central necrosis (black) on hemorrhagic base. F. Bullae on anterior tibial surface.

1	(Reprinted with permission from KK Holmes et al: Disseminated gonococcal infection. Ann Intern Med 74:979, 1971.) several well-controlled studies, mainly in Kenya and Zaire. The non-ulcerative STIs enhance the transmission of HIV by threeto fivefold; transmission of HIV-infected immune cells and increased viral shedding by persons with urethritis or cervicitis may contribute (Chap. 226). HIV has been detected by polymerase chain reaction (PCR) more commonly in ejaculates from HIV-positive men with gonococcal urethritis than in those from HIV-positive men with nongonococcal urethritis. PCR positivity diminishes by twofold after appropriate therapy for urethritis. Not only does gonorrhea enhance the transmission of HIV, but it may also increase the individual’s risk for acquisition of HIV. A proposed mechanism is the significantly greater number of CD4+ T lymphocytes and dendritic cells that can be infected by HIV in endocervical secretions Percent of patients

1	Percent of patients FIGURE 181-3 Distribution of joints with arthritis in 102 patients with disseminated gonococcal infection and 173 patients with reactive arthritis. *Includes the sternoclavicular joints. †SI, sacroiliac joint. from women with nonulcerative STIs than in those from women with ulcerative STIs.

1	A rapid diagnosis of gonococcal infection in men may be obtained by Gram’s staining of urethral exudates (Fig. 181-1). The detection of gram-negative intracellular monococci and diplococci is usually highly specific and sensitive in diagnosing gonococcal urethritis in symptomatic males but is only ~50% sensitive in diagnosing gonococcal cervicitis. Samples should be collected with Dacron or rayon swabs. Part of the sample should be inoculated onto a plate of modified Thayer-Martin or other gonococcal selective medium for culture. It is important to process all samples immediately because gonococci do not tolerate drying. If plates cannot be incubated immediately, they can be held safely for several hours at room temperature in candle extinction jars prior to incubation. If processing is to occur within 6 h, transport of specimens may be facilitated by the use of nonnutritive swab transport systems such as Stuart or Amies medium. For longer holding periods (e.g., when specimens for

1	to occur within 6 h, transport of specimens may be facilitated by the use of nonnutritive swab transport systems such as Stuart or Amies medium. For longer holding periods (e.g., when specimens for culture are to be mailed), culture media with self-contained CO2-generating systems (such as the JEMBEC or Gono-Pak systems) may be used. Specimens should also be obtained for the diagnosis of chlamydial infection (Chap. 213).

1	PMNs are often seen in the endocervix on a Gram’s stain, and an abnormally increased number (≥30 PMNs per field in five 1000× oil-immersion microscopic fields) establishes the presence of an inflammatory discharge. Unfortunately, the presence or absence of gram-negative intracellular monococci or diplococci in cervical smears does not accurately predict which patients have gonorrhea, and the diagnosis in this setting should be made by culture or another suitable nonculture diagnostic method. The sensitivity of a single endocervical culture is ~80–90%. If a history of rectal sex is elicited, a rectal wall swab (uncontaminated with feces) should be cultured. A presumptive diagnosis of gonorrhea cannot be made on the basis of gram-negative diplococci in smears from the pharynx, where other Neisseria species are components of the normal flora.

1	Increasingly, nucleic acid probe tests are being substituted for culture for the direct detection of N. gonorrhoeae in urogenital 1008 specimens. A common assay uses a nonisotopic chemiluminescent DNA probe that hybridizes specifically with gonococcal 16S ribosomal RNA; this assay is as sensitive as conventional culture techniques. A disadvantage of non-culture-based assays is that N. gonorrhoeae cannot be grown from the transport systems. Thus a culture-confirmatory test and formal antimicrobial susceptibility testing, if needed, cannot be performed. Nucleic acid amplification tests (NAATs), including the Roche Cobas® Amplicor, Gen-Probe APTIMA COMBO 2®, and BD ProbeTec™ ET, also detect C. trachomatis and are more sensitive than culture identification of either

1	N. gonorrhoeae or C. trachomatis. The Gen-Probe and BD tests offer the advantage that urine samples can be tested with a sensitivity similar to or greater than that obtained when urethral or cervical swab samples are assessed by other non-NAATs or culture, respectively. Several amplification tests are now available on semiautomated or fully automated platforms.

1	Because of the legal implications, the preferred method for the diagnosis of gonococcal infection in children is a standardized culture. Two positive NAATs, each targeting a different nucleic acid sequence, may be substituted for culture of the cervix or the urethra as legal evidence of infection in children. Although nonculture tests for gonococcal infection have not been approved by the U.S. Food and Drug Administration for use with specimens obtained from the pharynx and rectum of infected children, NAATs from these sites are preferred for diagnostic evaluation in adult victims of suspected sexual abuse, especially if the NAATs have been evaluated by the local laboratory and found to be superior. Cultures should be obtained from the pharynx and anus of both girls and boys, the urethra of boys, and the vagina of girls; cervical specimens are not recommended for prepubertal girls. For boys with a urethral discharge, a meatal specimen of the discharge is adequate for culture.

1	urethra of boys, and the vagina of girls; cervical specimens are not recommended for prepubertal girls. For boys with a urethral discharge, a meatal specimen of the discharge is adequate for culture. Presumptive colonies of N. gonorrhoeae should be identified definitively by at least two independent methods.

1	Blood should be cultured in suspected cases of DGI. The use of Isolator blood culture tubes may enhance the yield. The probability of positive blood cultures decreases after 48 h of illness. Synovial fluid should be inoculated into blood culture broth medium and plated onto chocolate agar rather than selective medium because this fluid is not likely to be contaminated with commensal bacteria. Gonococci are infrequently recovered from early joint effusions containing <20,000 leukocytes/μL but may be recovered from effusions containing >80,000 leukocytes/μL. The organisms are seldom recovered from blood and synovial fluid of the same patient.

1	Treatment failure can lead to continued transmission and the emergence of antibiotic resistance. The importance of adequate treatment with a regimen that the patient will adhere to cannot be overemphasized. Thus highly effective single-dose regimens have been developed for uncomplicated gonococcal infections. The modified 2010 treatment guidelines for gonococcal infections from the Centers for Disease Control and Prevention (CDC) are summarized in Table 181-1. Rising MICs of cefixime worldwide have led the CDC to discontinue its recommendation of this agent as first-line treatment for uncomplicated gonorrhea. The recommendations for uncomplicated gonorrhea apply to HIV-infected as well as HIV-uninfected patients.

1	Currently, a single IM dose of the third-generation cephalosporin ceftriaxone is the mainstay of therapy for uncomplicated gonococcal infection of the urethra, cervix, rectum, or pharynx and almost always results in an effective cure. Quinolone-containing regimens are no longer recommended in the United States as first-line treatment because of widespread resistance. A recent multicenter trial of treatment for uncomplicated gonorrhea in the United States showed ≥99.5% efficacy of two combination regimens: (1) gemifloxacin (320 mg, single oral dose) plus azithromycin (2 g, single oral dose) or (2) azithromycin (2 g, single oral dose) plus gentamicin (a single IM dose of 240 mg or, in individuals who weigh ≤45 kg, 5 mg/kg).

1	Because co-infection with C. trachomatis occurs frequently, initial treatment regimens must also incorporate an agent (e.g., azithromycin or doxycycline) that is effective against chlamydial infection. Pregnant women with gonorrhea, who should not take doxycycline, should receive concurrent treatment with a macrolide antibiotic for possible chlamydial infection. A single 1-g dose of azithromycin, which is effective therapy for uncomplicated chlamydial infections, results in an unacceptably low cure rate (93%) for gonococcal infections and should not be used alone. A single 2-g dose of azithromycin, particularly in the extended-release microsphere formulation, delivers azithromycin to the lower gastrointestinal tract, thereby improving tolerability. Azithromycin is effective against sensitive strains, but this drug is expensive, causes gastrointestinal distress, and is not recommended for routine or first-line treatment of gonorrhea. Spectinomycin has been used as an alternative agent

1	strains, but this drug is expensive, causes gastrointestinal distress, and is not recommended for routine or first-line treatment of gonorrhea. Spectinomycin has been used as an alternative agent for the treatment of uncomplicated gonococcal infections in penicillin-allergic persons outside the United States but is not currently available in this country. Of note, the limited effectiveness of spectinomycin for the treatment of pharyngeal infection reduces its utility in populations among whom such infection is common, such as MSM.

1	Persons with uncomplicated infections who receive ceftriaxone do not need a test of cure; however, cultures for N. gonorrhoeae should be performed if symptoms persist after therapy with an established regimen, and any gonococci isolated should be tested for antimicrobial susceptibility. Persons given an alternative regimen should return for a test of cure targeting the infected anatomic site. This test ideally should be a culture. If culture is not readily available and a NAAT is positive, every effort should be made to perform a confirmatory culture. All positive cultures for test of cure should undergo antimicrobial susceptibility testing. Because of high rates of reinfection with N. gonorrhoeae and C. trachomatis within 6 months, repeat testing is recommended 3 months after treatment.

1	Symptomatic gonococcal pharyngitis is more difficult to eradicate than genital infection. Persons who cannot tolerate ceftriaxone and those in whom quinolones are contraindicated may be treated with spectinomycin if it is available, but this agent results in a cure rate of ≤52%. Persons given spectinomycin should have a pharyngeal sample cultured 3–5 days after treatment as a test of cure. A single 2-g dose of azithromycin may be used in areas where rates of resistance to azithromycin are low. Treatments for gonococcal epididymitis and PID are discussed in Chap. 163. Ocular gonococcal infections in older children and adults should be managed with a single dose of ceftriaxone combined with saline irrigation of the conjunctivae (both undertaken expeditiously), and patients should undergo a careful ophthalmologic evaluation that includes a slit-lamp examination.

1	DGI may require higher dosages and longer durations of therapy (Table 181-1). Hospitalization is indicated if the diagnosis is uncertain, if the patient has localized joint disease that requires aspiration, or if the patient cannot be relied on to comply with treatment. Open drainage is necessary only occasionally—e.g., for management of hip infections that may be difficult to drain percutaneously. Nonsteroidal anti-inflammatory agents may be indicated to alleviate pain and hasten clinical improvement of affected joints. Gonococcal meningitis and endocarditis should be treated in the hospital with high-dose IV ceftriaxone (1–2 g every 12 h); therapy should continue for 10–14 days for meningitis and for at least 4 weeks for endocarditis. All persons who experience more than one episode of DGI should be evaluated for complement deficiency. RECoMMEnDED TREATMEnT foR gonoCoCCAL InfECTIonS: ADAPTED fRoM THE 2010 guIDELInES of THE CEnTERS foR DISEASE ConTRoL AnD PREvEnTIon

1	Diagnosis Treatment of Choicea aTrue failure of treatment with a recommended regimen is rare and should prompt an evaluation for reinfection, infection with a drug-resistant strain, or an alternative diagnosis. bCeftriaxone is the only agent recommended for treatment of pharyngeal infection. cSee text for follow-up of persons with infection who are treated with alternative regimens. dSpectinomycin, cefotetan, and cefoxitin, which are alternative agents, currently are unavailable or in short supply in the United States. eSpectinomycin may be ineffective for the treatment of pharyngeal gonorrhea. fPlus lavage of the infected eye with saline solution (once). gProphylactic regimens are discussed in the text. hHospitalization is indicated if the diagnosis is uncertain, if the patient has frank arthritis with an effusion, or if the patient cannot be relied on to adhere to treatment. iAll initial regimens should be continued for 24–48 h after clinical improvement begins, at which time the

1	arthritis with an effusion, or if the patient cannot be relied on to adhere to treatment. iAll initial regimens should be continued for 24–48 h after clinical improvement begins, at which time the switch may be made to an oral agent (e.g., cefixime or a quinolone) if antimicrobial susceptibility can be documented by culture of the causative organism. If no organism is isolated and the diagnosis is secure, then treatment with ceftriaxone should be continued for at least 1 week. Treatment for chlamydial infection (as above) should be given if this infection has not been ruled out. jHospitalization is indicated to exclude suspected meningitis or endocarditis.

1	PREVENTION AND CONTROL medications to treat gonorrhea and chlamydial infection diminish the Condoms, if properly used, provide effective protection against the likelihood of reinfection (or relapse) in the infected patient. In states transmission and acquisition of gonorrhea as well as other infec-where it is legal, this approach is an option for partner management. tions that are transmitted to and from genital mucosal surfaces. Patients should be instructed to abstain from sexual intercourse until Spermicidal preparations used with a diaphragm or cervical sponges therapy is completed and until they and their sex partners no longer impregnated with nonoxynol 9 offer some protection against gonor-have symptoms. Greater emphasis must be placed on prevention by rhea and chlamydial infection. However, the frequent use of prepara-public health education, individual patient counseling, and behavior tions that contain nonoxynol 9 is associated with mucosal disruption modification. Sexually

1	However, the frequent use of prepara-public health education, individual patient counseling, and behavior tions that contain nonoxynol 9 is associated with mucosal disruption modification. Sexually active persons, especially adolescents, should that paradoxically may enhance the risk of HIV infection in the event be offered screening for STIs. For male patients, a NAAT on urine of exposure. All patients should be instructed to refer sex partners for or a urethral swab may be used for screening. Preventing the spread evaluation and treatment. All sex partners of persons with gonorrhea of gonorrhea may help reduce the transmission of HIV. No effective should be evaluated and treated for N. gonorrhoeae and C. trachoma-vaccine for gonorrhea is yet available, but efforts to test several can-tis infections if their last contact with the patient took place within didates are under way. 60 days before the onset of symptoms or the diagnosis of infection in the patient. If the patient’s last

1	infections if their last contact with the patient took place within didates are under way. 60 days before the onset of symptoms or the diagnosis of infection in the patient. If the patient’s last sexual encounter was >60 days before Acknowledgmentonset of symptoms or diagnosis, the patient’s most recent sex partner The authors acknowledge the contributions of Dr. King K. Holmes and should be treated. Partner-delivered medications or prescriptions for Dr. Stephen A. Morse to the chapter on this subject in earlier editions.

1	1010 Haemophilus and Moraxella Infections Timothy F. Murphy HAEMOPHILUS INFLUENZAE MICROBIOLOGY 182 Haemophilus influenzae was first recognized in 1892 by Pfeiffer, who erroneously concluded that the bacterium was the cause of influenza. H. influenzae is a small (1× 0.3-μm) gram-negative organism of variable shape; thus, it is often described as a pleomorphic coccobacillus. In clinical specimens such as cerebrospinal fluid (CSF) and sputum, H. influenzae frequently stains only faintly with safranin and therefore can easily be overlooked.

1	H. influenzae frequently stains only faintly with safranin and therefore can easily be overlooked. H. influenzae grows both aerobically and anaerobically. Its aerobic growth requires two factors: hemin (X factor) and nicotinamide adenine dinucleotide (V factor). These requirements are used in the clinical laboratory to identify the bacterium. Caution must be used to distinguish H. influenzae from H. haemolyticus, a respiratory tract commensal that has identical growth requirements. H. haemolyticus has classically been distinguished from H. influenzae by the hemolysis of the former species on horse blood agar. However, a significant proportion of isolates of H. haemolyticus have now been recognized as nonhemolytic. Analysis of various genotypic and phenotypic markers, including16S ribosomal sequences, superoxide dismutase, outer-membrane protein P6, protein D, and fuculose kinase, can be used to distinguish these two species.

1	Six major serotypes of H. influenzae have been identified; designated a through f, they are based on antigenically distinct polysaccharide capsules. In addition, some strains lack a polysaccharide capsule and are referred to as nontypable strains. Type b and nontypable strains are the most relevant strains clinically (Table 182-1), although encapsulated strains other than type b can cause disease. H. influenzae was the first free-living organism to have its entire genome sequenced. The antigenically distinct type b capsule is a linear polymer composed of ribosyl-ribitol phosphate. Strains of H. influenzae type b (Hib) cause disease primarily in infants and children <6 years of age. Nontypable strains are primarily mucosal pathogens but occasionally cause invasive disease.

1	H. influenzae, an exclusively human pathogen, is spread by airborne droplets or by direct contact with secretions or fomites. Colonization with nontypable H. influenzae is a dynamic process; new strains are acquired and other strains are replaced periodically.

1	The widespread use of Hib conjugate vaccines in many industrialized countries has resulted in striking decreases in the rate of nasopharyngeal colonization by Hib and in the incidence of Hib infection (Fig. 182-1). However, the majority of the world’s children remain unimmunized. Worldwide, invasive Hib disease occurs predominantly in unimmunized children and in those who have not completed the primary immunization series. Certain groups have a higher incidence of invasive Hib disease than the general population, including African-American children and Native American groups. Although this increased incidence has not yet been accounted for, several factors may be relevant, including age at exposure to the bacterium, socioeconomic conditions, and genetic differences.

1	FIGURE 182-1 Estimated incidence (rate per 100,000) of invasive disease due to Haemophilus influenzae type b among children <5 years of age: 1987–2000. Fewer than 40 cases per year have been reported since 2000. (Data from the Centers for Disease Control and Prevention.) Hib strains cause systemic disease by invasion and hematogenous spread from the respiratory tract to distant sites such as the meninges, bones, and joints. The type b polysaccharide capsule is an important virulence factor affecting the bacterium’s ability to avoid opsonization and cause systemic disease.

1	Nontypable strains cause disease by local invasion of mucosal surfaces. Otitis media results when bacteria reach the middle ear by way of the eustachian tube. Adults with chronic bronchitis experience recurrent lower respiratory tract infection due to nontypable strains. In addition, persistent nontypable H. influenzae colonization of the lower airways of adults with chronic obstructive pulmonary disease (COPD) contributes to the airway inflammation that is a hallmark of the disease. Nontypable strains that cause infection in adults with COPD differ in pathogenic potential and genome content from strains that cause otitis media. In the middle ear, nontypable strains form biofilms. More resistant to host clearance mechanisms and to antibiotics than are planktonic bacteria, biofilms are associated with chronic and recurrent otitis media. The incidence of invasive disease caused by nontypable strains is low. Strains that cause invasive disease are genetically and phenotypically diverse.

1	Antibody to the capsule is important in protection from infection by Hib strains. The level of (maternally acquired) serum antibody to the capsular polysaccharide, which is a polymer of polyribitol ribose phosphate (PRP), declines from birth to 6 months of age and, in the absence of vaccination, remains low until ~2 or 3 years of age. The age at the antibody nadir correlates with that of the peak incidence of type b disease. Antibody to PRP then appears partly as a result of exposure to Hib or cross-reacting antigens. Systemic Hib disease is unusual after the age of 6 years because of the presence of protective antibody. Vaccines in which PRP is conjugated to protein carrier molecules have been developed and are now used widely. These vaccines generate an antibody response to PRP in infants and effectively prevent invasive infections in infants and children.

1	Since nontypable strains lack a capsule, the immune response to infection is directed at noncapsular antigens. These antigens have generated considerable interest as immune targets and potential vaccine components. The human immune response to nontypable strains appears to be strain-specific, a characteristic that accounts in part for the propensity of these strains to cause recurrent otitis media and recurrent exacerbations of chronic bronchitis in immunocompetent hosts.

1	CLINICAL MANIFESTATIONS Hib The most serious manifestation of infection with Hib is meningitis (Chap. 164), which primarily affects children <2 years of age. The clinical manifestations of Hib meningitis are similar to those of meningitis caused by other bacterial pathogens. Fever and altered central nervous system function are the most common features at presentation. Nuchal rigidity may or may not be evident. Subdural effusion, the most common complication, is suspected when, despite 2 or 3 days of appropriate antibiotic therapy, the infant has seizures, hemiparesis, or continued obtundation. The overall mortality rate from Hib meningitis is ~5%, and the morbidity rate is high. Of survivors, 6% have permanent sensorineural hearing loss, and about one-fourth have a significant handicap of some type. If more subtle handicaps are sought, up to half of survivors are found to have some neurologic sequelae, such as partial hearing loss and delayed language development.

1	Epiglottitis (Chap. 44) is a life-threatening Hib infection involving cellulitis of the epiglottis and supraglottic tissues. It can lead to acute upper airway obstruction. Its unique epidemiologic features are its occurrence in an older age group (2–7 years old) than other Hib infections and its absence among Navajo Indians and Alaskan Eskimos. Sore throat and fever rapidly progress to dysphagia, drooling, and airway obstruction. Epiglottitis also occurs in adults. Cellulitis (Chap. 156) due to Hib occurs in young children. The most common location is on the head or neck, and the involved area sometimes takes on a characteristic bluish-red color. Most patients have bacteremia, and 10% have an additional focus of infection.

1	Hib causes pneumonia in infants. The infection is clinically indistinguishable from other types of bacterial pneumonia (e.g., pneumococcal pneumonia) except that Hib is more likely to involve the pleura. Several less common invasive conditions can be important clinical manifestations of Hib infection in children. These include osteomyelitis, septic arthritis, pericarditis, orbital cellulitis, endophthalmitis, urinary tract infection, abscesses, and bacteremia without an identifiable focus. Non–type b encapsulated strains of H. influenzae (types a, c, d, e, and f) are unusual causes of invasive infection manifested predominantly by bacteremia and pneumonia. Most such infections occur in the setting of underlying conditions.

1	Nontypable H. influenzae Nontypable H. influenzae is the most common bacterial cause of exacerbations of COPD; these exacerbations are characterized by increased cough, sputum production, and shortness of breath. Fever is low-grade, and no infiltrates are evident on chest x-ray. Nontypable strains also cause community-acquired bacterial pneumonia in adults, especially among patients with COPD or AIDS. The clinical features of H. influenzae pneumonia are similar to those of other types of bacterial pneumonia, including pneumococcal pneumonia.

1	Nontypable H. influenzae is one of the three most common causes of childhood otitis media (the other two being Streptococcus pneumoniae and Moraxella catarrhalis) (Chap. 44). Infants are febrile and irritable, while older children report ear pain. Symptoms of viral upper respiratory infection often precede otitis media. The diagnosis is made by pneumatic otoscopy. An etiologic diagnosis, although not routinely sought, can be established by tympanocentesis and culture of middle-ear fluid. Clinical features associated with H. influenzae otitis media include a history of recurrent episodes, treatment failure, concomitant conjunctivitis, bilateral otitis media, and recent antimicrobial therapy. The increasing use of pneumococcal polysaccharide conjugate vaccines in infants is resulting in a relative increase in the proportion of otitis media cases that are caused by H. influenzae.

1	Nontypable H. influenzae also causes puerperal sepsis and is an important cause of neonatal bacteremia. These nontypable strains, which are closely related to H. haemolyticus, tend to be of biotype IV and cause invasive disease after colonizing the female genital tract.

1	Nontypable H. influenzae causes sinusitis (Chap. 44) in adults and children. In addition, the bacterium is a less common cause of various invasive infections. These infections include empyema, adult epiglottitis, pericarditis, cellulitis, septic arthritis, osteomyelitis, endocarditis, cholecystitis, intraabdominal infections, urinary tract infections, mastoiditis, aortic graft infection, and bacteremia without a detectable 1011 focus. While most H. influenzae invasive infections in countries where Hib vaccines are used widely are caused by nontypable strains, there is no convincing evidence of an increased incidence of infection by nontypable H. influenzae as a result of use of Hib vaccines. Continued monitoring will be important. Many patients with H. influenzae bacteremia have an underlying condition, such as HIV infection, cardiopulmonary disease, alcoholism, or cancer.

1	The most reliable method for establishing a diagnosis of Hib infection is recovery of the organism in culture. The presence of gram-negative coccobacilli in Gram-stained CSF is strong evidence for Hib meningitis. Recovery of the organism from CSF confirms the diagnosis. Cultures of other normally sterile body fluids, such as blood, joint fluid, pleural fluid, pericardial fluid, and subdural effusion, are confirmatory in other infections. Detection of PRP is an important adjunct to culture in rapid diagnosis of Hib meningitis. Immunoelectrophoresis, latex agglutination, coagglutination, and enzyme-linked immunosorbent assay are effective in detecting PRP. These assays are particularly helpful when patients have received prior antimicrobial therapy and thus are especially likely to have negative cultures.

1	Because nontypable H. influenzae is primarily a mucosal pathogen, it is a component of a mixed flora; thus etiologic diagnosis is challenging. Nontypable H. influenzae infection is strongly suggested by the predominance of gram-negative coccobacilli among abundant polymorphonuclear leukocytes in a Gram-stained sputum specimen from a patient in whom pneumonia is suspected. Although bacteremia is detectable in a small proportion of patients with pneumonia due to nontypable H. influenzae, most such patients have negative blood cultures. A diagnosis of otitis media is based on the detection by pneumatic otoscopy of fluid in the middle ear. An etiologic diagnosis requires tympanocentesis but is not routinely sought. An invasive procedure is also required to determine the etiology of sinusitis; thus, treatment is often empirical once the diagnosis is suspected in light of clinical symptoms and sinus radiographs.

1	Initial therapy for meningitis due to Hib should consist of a cephalosporin such as ceftriaxone or cefotaxime. For children, the dosage of ceftriaxone is 75–100 mg/kg daily given in two doses 12 h apart. The pediatric dosage of cefotaxime is 200 mg/kg daily given in four doses 6 h apart. Adult dosages are 2 g every 12 h for ceftriaxone and 2 g every 4–6 h for cefotaxime. An alternative regimen for initial therapy is ampicillin (200–300 mg/kg daily in four divided doses) plus chloramphenicol (75–100 mg/kg daily in four divided doses). Therapy should continue for a total of 1–2 weeks.

1	Administration of glucocorticoids to patients with Hib meningitis reduces the incidence of neurologic sequelae. The presumed mechanism is reduction of the inflammation induced by bacterial cell-wall mediators of inflammation when cells are killed by antimicrobial agents. Dexamethasone (0.6 mg/kg per day intravenously in four divided doses for 2 days) is recommended for the treatment of Hib meningitis in children >2 months of age. Invasive infections other than meningitis are treated with the same antimicrobial agents. For epiglottitis, the dosage of ceftriaxone is 50 mg/kg daily, and the dosage of cefotaxime is 150 mg/kg daily, given in three divided doses 8 h apart. Epiglottitis constitutes a medical emergency, and maintenance of an airway is critical. The duration of therapy is determined by the clinical response. A course of 1–2 weeks is usually appropriate.

1	Many infections caused by nontypable strains of H. influenzae, such as otitis media, sinusitis, and exacerbations of COPD, can be treated with oral antimicrobial agents. Approximately 20–35% of nontypable strains produce β-lactamase (with the exact proportion 1012 depending on geographic location), and these strains are resistant to ampicillin. Several agents have excellent activity against nontypable H. influenzae, including amoxicillin/clavulanic acid, various extended-spectrum cephalosporins, and the macrolides azithromycin and clarithromycin. Fluoroquinolones are highly active against H. influenzae and are useful in adults with exacerbations of COPD. However, fluoroquinolones are not currently recommended for the treatment of children or pregnant women because of possible effects on articular cartilage.

1	In addition to β-lactamase production, alteration of penicillin-binding proteins—a second mechanism of ampicillin resistance—has been detected in isolates of H. influenzae. Although rare in the United States, these β-lactamase-negative ampicillin-resistant strains are common in Japan and are increasing in prevalence in Europe. Continued monitoring of the evolving antimicrobial susceptibility patterns of H. influenzae will be important.

1	Vaccination (See also Chap. 148) Two conjugate vaccines that prevent invasive infections with Hib in infants and children are licensed in the United States. In addition to eliciting protective antibody, these vaccines prevent disease by reducing rates of pharyngeal colonization with Hib. The widespread use of conjugate vaccines has dramatically reduced the incidence of Hib disease in developed countries. Even though the manufacture of Hib vaccines is costly, vaccination is cost-effective. The Global Alliance for Vaccines and Immunizations has recognized the underuse of Hib conjugate vaccines. The disease burden has been reduced in developing countries that have implemented routine vaccination (e.g., The Gambia, Chile). An important obstacle to more widespread vaccination is the lack of data on the epidemiology and burden of Hib disease in many developing countries.

1	All children should be immunized with an Hib conjugate vaccine, receiving the first dose at ~2 months of age, the rest of the primary series at 2–6 months of age, and a booster dose at 12–15 months of age. Specific recommendations vary for the different conjugate vaccines. The reader is referred to the recommendations of the American Academy of Pediatrics (Chap. 148 and www.cispimmunize.org). Currently, no vaccines are available specifically for the prevention of disease caused by nontypable H. influenzae. However, a vaccine that contains protein D—a surface protein of H. influenzae—conjugated to pneumococcal polysaccharides is licensed in other countries and is used widely in Europe. The vaccine has shown partial efficacy in preventing H. influenzae otitis media in clinical trials. Additional progress in the development of vaccines against nontypable H. influenzae is anticipated.

1	Chemoprophylaxis The risk of secondary disease is greater than normal among household contacts of patients with Hib disease. Therefore, all children and adults (except pregnant women) in households with an index case and at least one incompletely immunized contact <4 years of age should receive prophylaxis with oral rifampin. When two or more cases of invasive Hib disease have occurred within 60 days at a child-care facility attended by incompletely vaccinated children, administration of rifampin to all attendees and personnel is indicated, as is recommended for household contacts. Chemoprophylaxis is not indicated in nursery and child-care contacts of a single index case. The reader is referred to the recommendations of the American Academy of Pediatrics.

1	Haemophilus ducreyi is the etiologic agent of chancroid (Chap. 163), a sexually transmitted disease characterized by genital ulceration and inguinal adenitis. In addition to being a cause of morbidity in itself, chancroid is associated with HIV infection because of the role played by genital ulceration in HIV transmission. Chancroid increases the efficiency of transmission of and the degree of susceptibility to HIV infection.

1	H. ducreyi is a highly fastidious coccobacillary gram-negative bacterium whose growth requires X factor (hemin). Although, in light of this requirement, the bacterium has been classified in the genus Haemophilus, DNA homology and chemotaxonomic studies have established substantial differences between H. ducreyi and other Haemophilus species. Taxonomic reclassification of the organism is likely in the future but awaits further study. Ulcers contain predominantly T cells. The fact that patients who have had chancroid may have repeated infections indicates that infection does not confer protection.

1	The prevalence of chancroid has declined in the United States and worldwide. However, prevalence data must be interpreted with caution because of the difficulty of establishing a diagnosis. The infection appears to be more common in developing countries. Transmission is predominantly heterosexual, and cases in males have outnumbered those in females by ratios of 3:1 to 25:1 during outbreaks. Contact with commercial sex workers and illicit drug use are strongly associated with chancroid.

1	Infection is acquired as the result of a break in the epithelium during sexual contact with an infected individual. After an incubation period of 4–7 days, the initial lesion—a papule with surrounding erythema— appears. In 2 or 3 days, the papule evolves into a pustule, which spontaneously ruptures and forms a sharply circumscribed ulcer that generally is not indurated (Fig. 182-2). The ulcers are painful and bleed easily; little or no inflammation of the surrounding skin is evident. Approximately half of patients develop enlarged, tender inguinal lymph nodes, which frequently become fluctuant and spontaneously rupture. Patients usually seek medical care after 1–3 weeks of painful symptoms.

1	The presentation of chancroid does not usually include all of the typical clinical features and is sometimes atypical. Multiple ulcers can coalesce to form giant ulcers. Ulcers can appear and then resolve, with inguinal adenitis (Fig. 182-2) and suppuration following 1–3 weeks later; this clinical picture can be confused with that of lymphogranuloma venereum (Chap. 213). Multiple small ulcers can resemble folliculitis. Other differential diagnostic considerations include the various infections causing genital ulceration, such as primary syphilis, secondary syphilis (condyloma latum), genital herpes, and donovanosis. In rare cases, chancroid lesions become secondarily infected with bacteria; the result is extensive inflammation. FIGURE 182-2 Chancroid with characteristic penile ulcers and associated left inguinal adenitis (bubo).

1	FIGURE 182-2 Chancroid with characteristic penile ulcers and associated left inguinal adenitis (bubo). Clinical diagnosis of chancroid is often inaccurate, and laboratory confirmation should be attempted in suspected cases. An accurate diagnosis of chancroid relies on culture of H. ducreyi from the lesion or from an aspirate of suppurative lymph nodes. Since the organism can be difficult to grow, the use of selective and supplemented media is necessary. No polymerase chain reaction assay for H. ducreyi is commercially available; such tests can be performed by Clinical Laboratory Improvement Amendment (CLIA)–certified clinical laboratories that have developed their own assays.

1	A probable diagnosis of chancroid can be made when the following criteria are met: (1) one or more painful genital ulcers; (2) no evidence of Treponema pallidum infection by dark-field examination of ulcer exudate or by a negative serologic test for syphilis performed at least 7 days after ulcer onset; (3) a typical clinical presentation for chancroid; and (4) a negative test for herpes simplex virus in the ulcer exudate.

1	Treatment regimens recommended by the Centers for Disease Control and Prevention include (1) a single 1-g oral dose of azithromycin; (2) ceftriaxone (250 mg intramuscularly in a single dose); (3) ciprofloxacin (500 mg by mouth twice a day for 3 days); and (4) erythromycin base (500 mg by mouth three times a day for 7 days). Isolates from patients who do not respond promptly to treatment should be tested for antimicrobial resistance. In patients with HIV infection, healing may be slow and longer courses of treatment may be necessary. Clinical treatment failure in HIV-seropositive patients may reflect co-infection, especially with herpes simplex virus. Contacts of patients with chancroid should be identified and treated, whether or not symptoms are present, if they have had sexual contact with the patient during the 10 days preceding the patient’s onset of symptoms.

1	M. catarrhalis is an unencapsulated gram-negative diplococcus whose ecologic niche is the human respiratory tract. The organism was initially designated Micrococcus catarrhalis. Its name was changed to Neisseria catarrhalis in 1970 because of phenotypic similarities to commensal Neisseria species. On the basis of more rigorous analysis of genetic relatedness, Moraxella catarrhalis is now the widely accepted name for this species.

1	Nasopharyngeal colonization by M. catarrhalis is common in infancy, with colonization rates ranging between 33% and 100% and depending on geographic location. Several factors probably account for this geographic variation, including living conditions, day-care attendance, hygiene, household smoking, and population genetics. The prevalence of colonization decreases steadily with age. The widespread use of pneumococcal conjugate vaccines in some countries has resulted in alterations in patterns of nasopharyngeal colonization in resident populations. A relative increase in colonization by nonvaccine pneumococcal serotypes, nontypable H. influenzae, and M. catarrhalis has occurred. These changes in colonization patterns may be altering the distribution of pathogens of both otitis media and sinusitis in children.

1	M. catarrhalis has occurred. These changes in colonization patterns may be altering the distribution of pathogens of both otitis media and sinusitis in children. M. catarrhalis causes mucosal infections of the respiratory tract by contiguous spread from its colonizing site in the upper airway. A preceding viral upper respiratory tract infection is a common incit-1013 ing event for otitis media. In exacerbations of COPD, the acquisition of new strains is critical for pathogenesis. Strains exhibit substantial genetic diversity and differences in virulence properties. The expression of several adhesin molecules with differing specificities for various host cell receptors reflects the importance of adherence to the respiratory epithelial surface in the pathogenesis of infection. M. catarrhalis invades multiple cell types. Its intracellular residence in lymphoid tissue provides a potential reservoir for persistence in the human respiratory tract. Like many gram-negative bacteria,

1	M. catarrhalis sheds vesicles into the surrounding environment. The vesicles are internalized by host cells and mediate several virulence mechanisms, including induction of inflammation and delivery of β-lactamase, that can promote the survival of co-pathogens. In children, M. catarrhalis causes predominantly mucosal infections when the bacterium migrates from the nasopharynx to the middle ear or the sinuses (Chap. 44). The inciting event for both otitis media and sinusitis is often a preceding viral infection. Overall, cultures of middle-ear fluid obtained by tympanocentesis indicate that M. catarrhalis causes 15–20% of cases of acute otitis media. Acute otitis media caused by M. catarrhalis or nontypable H. influenzae is clinically milder than otitis media caused by S. pneumoniae, with less fever and a lower prevalence of a red bulging tympanic membrane. However, substantial overlap makes it impossible to predict etiology in an individual child on the basis of clinical features.

1	A small proportion of viral upper respiratory tract infections are complicated by bacterial sinusitis. Cultures of sinus puncture aspirates show that M. catarrhalis accounts for ~20% of cases of acute bacterial sinusitis in children and for a smaller proportion in adults.

1	M. catarrhalis is a common cause of exacerbations in adults with COPD. The bacterium has been overlooked in this clinical setting because it has long been considered to be a commensal and because it is easily mistaken for commensal Neisseria species in cultures of respiratory secretions (see “Diagnosis,” below). Several independent lines of evidence have established M. catarrhalis as a pathogen in COPD. These include (1) the demonstration of M. catarrhalis in the lower airways during exacerbations, (2) the association of exacerbation with acquisition of new strains, (3) elevations of inflammatory markers in association with M. catarrhalis, and (4) the development of specific immune responses following infection. M. catarrhalis is the second most common bacterial cause of COPD exacerbations (after

1	H. influenzae), as shown in a 10-year prospective study; the distribution of exacerbations associated with new-strain acquisitions is shown in Fig. 182-3. Not included are culture-negative cases or cases from which a pathogen had been previously isolated. With the application of rigorous clinical criteria for defining the etiology of exacerbations (both culture-positive and culture-negative), ~10% of all exacerbations in the same study were caused by M. catarrhalis. The clinical features of an exacerbation due to M. catarrhalis are similar to those of exacerbations due to other bacterial pathogens, including H. influenzae and S. pneumoniae. The cardinal symptoms are cough with increased sputum production, sputum purulence, and dyspnea in comparison with baseline symptoms.

1	Pneumonia due to M. catarrhalis occurs in the elderly, particularly in the setting of underlying cardiopulmonary disease, but is infrequent. Invasive infections, such as bacteremia, endocarditis, neonatal meningitis, and septic arthritis, are rare. Tympanocentesis is required for etiologic diagnosis of otitis media, but this procedure is not performed routinely. Therefore, treatment of otitis media is generally empirical. Similarly, an etiologic diagnosis of sinusitis requires an invasive procedure and thus is usually not available to the clinician. Isolation of M. catarrhalis from an expectorated sputum sample from an adult experiencing clinical symptoms of an exacerbation is suggestive, but not diagnostic, of M. catarrhalis as the cause. 1014 Exacerbations associated with new isolates

1	1014 Exacerbations associated with new isolates FIgURE 182-3 Cumulatie results of a prospectie study (14004) of bacterial infection in chronic obstructive pulmonary disease (COPD) showing etiology of exacerbations. The numbers of exacerbations shown indicate the acquisition of a new strain simultaneous with clinical symptoms of an exacerbation. NTHI, nontypable H. influenzae; M.cat, M. catarrhalis; S.pn, Streptococcus pneumoniae; PA, Pseudomonas aeruginosa. (Adapted from TF Murphy, GI Parameswaran: Clin Infect Dis 49:124, 2009, with permission. 2009 Infectious Diseases Society of America.) No. of COPD exacerbations NTHI M.cat S.pn PA

1	No. of COPD exacerbations NTHI M.cat S.pn PA Upon culture, colonies of M. catarrhalis resemble commensal neisseriae that are part of the normal upper airway flora. As mentioned above, the difficulty in distinguishing colonies of M. catarrhalis from neisserial colonies in cultures of respiratory secretions explains in part why M. catarrhalis has been overlooked as a pathogen. In contrast to these Neisseria species, M. catarrhalis colonies can be slid across the agar surface without disruption (the “hockey puck sign”). In addition, after 48 h of growth, M. catarrhalis colonies take on a pink color and tend to be larger than neisserial colonies. A variety of biochemical tests can distinguish M. catarrhalis from neisseriae. Kits that rely on these biochemical reactions are commercially available.

1	M. catarrhalis rapidly acquired Δ-lactamases during the 1970s and 1980s; antimicrobial susceptibility patterns have remained relatively stable since that time, with >90% of strains now producing Δ-lactamase and thus resistant to amoxicillin. Otitis media in children and exacerbations of COPD in adults are generally managed empirically with antimicrobial agents that are active against S. pneumoniae, H. influenzae, and M. catarrhalis. Most strains of M. catarrhalis are susceptible to amoxicillin/clavulanic acid, extended-spectrum cephalosporins, newer macrolides (azithromycin, clarithromycin), trimethoprimsulfamethoxazole, and fluoroquinolones. Infections Due to the HACEK Group and Miscellaneous Gram-Negative Bacteria Tamar F. Barlam, Dennis L. Kasper THE HACEK GROUP 183e

1	Infections Due to the HACEK Group and Miscellaneous Gram-Negative Bacteria Tamar F. Barlam, Dennis L. Kasper THE HACEK GROUP 183e HACEK organisms are a group of fastidious, slow-growing, gram-negative bacteria whose growth requires an atmosphere of carbon dioxide. Species belonging to this group include several Haemophilus species, Aggregatibacter (formerly Actinobacillus) species, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae. HACEK bacteria normally reside in the oral cavity and have been associated with local infections in the mouth. They are also known to cause severe systemic infections—most often bacterial endocarditis, which can develop on either native or prosthetic valves (Chap. 155).

1	In large series, 0.8–6% of cases of infective endocarditis are attributable to HACEK organisms, most often Aggregatibacter species, Haemophilus species, and C. hominis. Invasive infection typically occurs in patients with a history of cardiac valvular disease, often in the setting of a recent dental procedure or nasopharyngeal infection. The aortic and mitral valves are most commonly affected. Compared with non-HACEK endocarditis, HACEK endocarditis occurs in younger patients and is more frequently associated with embolic, vascular, and immunologic manifestations but less commonly associated with congestive heart failure. The clinical course of HACEK endocarditis tends to be subacute, particularly with Aggregatibacter or Cardiobacterium. However, K. kingae endocarditis may have a more aggressive presentation. Systemic embolization is common. The overall prevalence of major emboli associated with HACEK endocarditis ranges from 28% to 71% in different series. On echocardiography,

1	aggressive presentation. Systemic embolization is common. The overall prevalence of major emboli associated with HACEK endocarditis ranges from 28% to 71% in different series. On echocardiography, valvular vegetations are seen in up to 85% of patients. Aggregatibacter and Haemophilus species cause mitral valve vegetations most often; Cardiobacterium is associated with aortic valve vegetations. The microbiology laboratory should be alerted when a HACEK organism is being considered; however, most cultures that ultimately yield a HACEK organism become positive within the first week, especially with improved culture systems such as BACTEC. In addition, polymerase chain reaction (PCR) techniques (e.g., of cardiac valves) and other tools, such as matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry performed directly on agar colonies, are facilitating the diagnosis of HACEK infections. Because of the organisms’ slow growth, antimicrobial testing may be

1	of flight (MALDI-TOF) mass spectrometry performed directly on agar colonies, are facilitating the diagnosis of HACEK infections. Because of the organisms’ slow growth, antimicrobial testing may be difficult, and β-lactamase production may not be detected. Resistance is most commonly noted in Haemophilus and Aggregatibacter species. E-test methodology may increase the accuracy of susceptibility testing. The overall prognosis in HACEK endocarditis is excellent and significantly better than that in endocarditis caused by non-HACEK pathogens.

1	Haemophilus Species Haemophilus parainfluenzae is the most common species isolated from cases of HACEK endocarditis. Of patients with HACEK endocarditis due to Haemophilus species, 60% have been ill for <2 months before presentation, and 19–50% develop congestive heart failure. Mortality rates as high as 30–50% were reported in older series; however, more recent studies have documented mortality rates of <5%. H. parainfluenzae has been isolated from other infections, such as meningitis; brain, dental, pelvic, and liver abscess; pneumonia; urinary tract infection; and septicemia.

1	Aggregatibacter Species The species of Aggregatibacter that most frequently cause infective endocarditis are A. actinomycetemcomitans, A. (formerly Haemophilus) aphrophilus, and A. paraphrophilus. Aggregatibacter is associated with prosthetic valve endocarditis more often than are Haemophilus species. A. actinomycetemcomitans can be isolated from soft tissue infections and abscesses in association with 183e-1 Actinomyces israelii. Typically, patients who develop endocarditis with Aggregatibacter have periodontal disease or have recently undergone dental procedures in the setting of underlying cardiac valvular damage. The disease is insidious; patients may be sick for several months before diagnosis. Frequent complications include embolic phenomena, congestive heart failure, and renal failure. A. actinomycetemcomitans has been isolated from patients with brain abscess, meningitis, endophthalmitis, parotitis, osteomyelitis, urinary tract infection, pneumonia, and empyema, among other

1	A. actinomycetemcomitans has been isolated from patients with brain abscess, meningitis, endophthalmitis, parotitis, osteomyelitis, urinary tract infection, pneumonia, and empyema, among other infections, while A. aphrophilus is often associated with bone and joint infection.

1	Cardiobacterium hominis C. hominis primarily causes endocarditis in patients with underlying valvular heart disease or with prosthetic valves. This organism most frequently affects the aortic valve. Many patients have signs and symptoms of long-standing infection before diagnosis, with evidence of arterial embolization, vasculitis, cerebrovascular accidents, immune complex glomerulonephritis, or arthritis at presentation. Embolization, mycotic aneurysms, and congestive heart failure are common complications. A second species, C. valvarum, has now been described in association with endocarditis.

1	Eikenella corrodens E. corrodens is most frequently recovered from sites of infection in conjunction with other bacterial species. Clinical sources of E. corrodens include sites of human bite wounds (clenchedfist injuries), endocarditis, soft tissue infections, osteomyelitis, head and neck infections, respiratory infections, chorioamnionitis, gynecologic infections associated with intrauterine devices, meningitis, brain abscesses, and visceral abscesses. This organism is the least common cause of HACEK endocarditis.

1	Kingella kingae Because of improved microbiologic methodology and molecular methods such as real-time PCR, the isolation of K. kingae is increasingly common. Inoculation of clinical specimens (e.g., synovial fluid) into aerobic blood culture bottles enhances recovery of this organism. More than half of cases of K. kingae infection are bone and joint infections; the majority of the remaining infections are infective endocarditis and bacteremia. PCR studies of joint fluid can identify K. kingae in culture-negative cases. Some studies now demonstrate that K. kingae has surpassed Staphylococcus aureus as the leading cause of septic arthritis in children. Invasive K. kingae infections with bacteremia are associated with upper respiratory tract infections and stomatitis in 80% of cases. Rates of oropharyngeal colonization with

1	K. kingae are highest in the first 3 years of life (detected in ~10% of children), coinciding with an increased incidence of skeletal infections due to this organism. K. kingae bacteremia can present with a petechial rash similar to that seen in Neisseria meningitidis sepsis. Infective endocarditis, unlike other infections with K. kingae, occurs in older children and adults. The majority of patients have preexisting valvular disease. There is a high incidence of complications, including arterial emboli, cerebrovascular accidents, tricuspid insufficiency, and congestive heart failure with cardiovascular collapse. (Table 183e-1) Ceftriaxone (2 g/d) is first-line therapy for HACEK endocarditis. Data on the use of levofloxacin (500–750 mg/d) for HACEK endocarditis remain limited, but this drug can be considered an alternative for treatment of patients intolerant of β-lactam therapy. Of note, Eikenella is resistant to clindamycin, metronidazole, and aminoglycosides.

1	Native-valve endocarditis should be treated for 4 weeks with antibiotics, whereas prosthetic-valve endocarditis requires 6 weeks of therapy. The cure rates for HACEK prosthetic-valve endocarditis appear to be high. Unlike prosthetic-valve endocarditis caused by other gram-negative organisms, HACEK endocarditis is often cured with antibiotic treatment alone—i.e., without surgical intervention. CHAPTER 183e Infections Due to the HACEK Group and Miscellaneous Gram-Negative Bacteria Haemophilus spp. Ceftriaxone (2 g/d) Ampicillin/sulbactam (3 g of ampicillin q6h) Aggregatibacter actinomycetemcomitans, A. aphrophilus, A. paraphrophilus, other species units q4h) or ampicillin (2 g q4h) Ampicillin/sulbactam resistance has been described in Haemophilus and Aggregatibacter spp. Data on use of levofloxacin for endocarditis therapy are limited. Fluoroquinolones are not recommended for treatment of patients <18 years of age.

1	Data on use of levofloxacin for endocarditis therapy are limited. Fluoroquinolones are not recommended for treatment of patients <18 years of age. Penicillin or ampicillin can be used if the organism is susceptible. However, because of the slow growth of HACEK bacteria, antimicrobial testing may be difficult, and β-lactamase production may not be detected.

1	Achromobacter xylosoxidans Achromobacter (previously Alcaligenes) xylosoxidans is probably part of the endogenous intestinal flora and has been isolated from a variety of water sources, including well water, IV fluids, and humidifiers. Immunocompromised hosts, including patients with cancer and postchemotherapy neutropenia, cirrhosis, chronic renal failure, and cystic fibrosis, are at increased risk. Nosocomial outbreaks and pseudo-outbreaks of A. xylosoxidans infection have been attributed to contaminated fluids, and clinical illness has been associated with isolates from many sites, including blood (often in the setting of intravascular devices). Community-acquired A. xylosoxidans bacteremia usually occurs in the setting of pneumonia. Metastatic skin lesions are present in one-fifth of cases. The reported mortality rate is as high as 67%—a figure similar to rates for other bacteremic gram-negative pneumonias.

1	carbapenems, and aminoglycosides, but resistance has been described to all those agents. Because Aeromonas can produce various β-lactamases, including carbapenemases, susceptibility testing must be used to guide therapy. Antibiotic prophylaxis (e.g., with ciprofloxacin) is indicated when medicinal leeches are used. Capnocytophaga Species This genus of fastidious, fusiform, gram-negative coccobacilli is facultatively anaerobic and requires an atmosphere enriched in carbon dioxide for optimal growth. C. ochracea, C. gingivalis, C. haemolytica, and C. sputigena have been associated with sepsis in immunocompromised hosts, particularly neutropenic patients with oral ulcerations. These species have been isolated from many other sites as well, usually as part of a polymicrobial infection. Most Capnocytophaga infections are contiguous with the oropharynx (e.g., periodontal disease, respiratory tract infections, cervical abscesses, and endophthalmitis).

1	(Table 183e-2) Treatment is based on in vitro susceptibility testing of all clinically relevant isolates; multidrug resistance is common. Meropenem, tigecycline, and colistin are typically the most active agents. Aeromonas Species More than 85% of Aeromonas infections are caused by A. hydrophila, A. caviae, and A. veronii biovar sobria. Aeromonas proliferates in potable water, freshwater, and soil. It remains controversial whether Aeromonas is a cause of bacterial gastroenteritis; asymptomatic colonization of the intestinal tract with Aeromonas occurs frequently. However, rare cases of hemolytic-uremic syndrome following bloody diarrhea have been shown to be secondary to the presence of Aeromonas.

1	Aeromonas causes sepsis and bacteremia in infants with multiple medical problems and in immunocompromised hosts, particularly those with cancer or hepatobiliary disease. A. caviae is associated with health care–related bacteremia. Aeromonas infection and sepsis can occur in patients with trauma (including severe trauma with myonecrosis) and in burn patients exposed to Aeromonas by environmental (freshwater or soil) contamination of their wounds. Reported mortality rates range from 25% among immunocompromised adults with sepsis to >90% among patients with myonecrosis. Aeromonas can produce ecthyma gangrenosum (hemorrhagic vesicles surrounded by a rim of erythema with central necrosis and ulceration; see Fig. 25e-35) resembling the lesions seen in Pseudomonas aeruginosa infection. This organism causes nosocomial infections related to catheters, surgical incisions, or use of leeches. Other manifestations include necrotizing fasciitis, meningitis, peritonitis, pneumonia, and ocular

1	organism causes nosocomial infections related to catheters, surgical incisions, or use of leeches. Other manifestations include necrotizing fasciitis, meningitis, peritonitis, pneumonia, and ocular infections.

1	(Table 183e-2) Aeromonas species are generally susceptible to fluoroquinolones (e.g., ciprofloxacin at a dosage of 500 mg every 12 h PO or 400 mg every 12 h IV), thirdand fourth-generation cephalosporins, Aeromonas spp. Elizabethkingia/ Chryseobacterium spp. Shewanella spp. Capnocytophaga spp. Meropenem, Treatment should tigecycline, colistin be based on in vitro susceptibility testing; multidrug resistance is common among these organisms. Fluoroquinolones (e.g., ciprofloxacin), thirdand fourth-generation cephalosporins, carbapenems, aminoglycosides Fluoroquinolones, trimethoprimsulfamethoxazole, piperacillin/tazobactam Fluoroquinolones, thirdand fourth-generation cephalosporins, carbapenems Fluoroquinolones, thirdand fourth-generation cephalosporins, β-lactam/βlactamase inhibitors, carbapenems, aminoglycosides Ampicillin/sulbactam Penicillin should be used if the isolate is known to be susceptible.

1	Ampicillin/sulbactam Penicillin should be used if the isolate is known to be susceptible. ceftriaxone used if the isolate is known to be susceptible. P. multocida is also susceptible to tetracyclines and fluoroquinolones. C. canimorsus and C. cynodegmi are endogenous to the canine mouth (Chap. 167e). Patients infected with these species frequently have a history of dog bites or of canine exposure without scratches or bites. Asplenia, glucocorticoid therapy, and alcohol abuse are predisposing conditions that can be associated with severe sepsis with shock and disseminated intravascular coagulation. Patients typically have a petechial rash that can progress from purpuric lesions to gangrene. Meningitis, endocarditis, cellulitis, osteomyelitis, and septic arthritis also have been associated with these organisms.

1	(Table 183e-2) Because of increasing β-lactamase production, a penicillin derivative plus a β-lactamase inhibitor—such as ampicillin/ sulbactam (1.5–3.0 g of ampicillin every 6 h)—is currently recommended for empirical treatment of infections caused by Capnocytophaga species. If the isolate is known to be susceptible, infections with C. canimorsus should be treated with penicillin (12– 18 million units every 4 h). Capnocytophaga is also susceptible to clindamycin (600–900 mg every 6–8 h). This regimen or ampicillin/ sulbactam should be given prophylactically to asplenic patients who have sustained dog-bite injuries.

1	Elizabethkingia/Chryseobacterium Species Elizabethkingia meningoseptica (formerly Chryseobacterium meningosepticum) is an important cause of nosocomial infections, including outbreaks due to contaminated fluids (e.g., disinfectants and aerosolized antibiotics) and sporadic infections due to indwelling devices, feeding tubes, and other fluid-associated apparatuses. Nosocomial E. meningoseptica infection usually involves neonates or patients with underlying immunosuppression (e.g., related to malignancy or diabetes). E. meningoseptica has been reported to cause meningitis (primarily in neonates), pneumonia, sepsis, endocarditis, bacteremia, and soft tissue infections. Most published reports have originated from Taiwan. Chryseobacterium indologenes has caused bacteremia, sepsis, and pneumonia, typically in immunocompromised patients with indwelling devices.

1	(Table 183e-2) These organisms are often susceptible to fluoroquinolones and trimethoprim-sulfamethoxazole. They may be susceptible to β-lactam/β-lactamase inhibitor combinations such as piperacillin/ tazobactam but can possess extended-spectrum β-lactamases and metallo-β-lactamases. Susceptibility testing should be performed.

1	Pasteurella multocida P. multocida is a bipolar-staining, gram-negative coccobacillus that colonizes the respiratory and gastrointestinal tracts of domestic animals; oropharyngeal colonization rates are 70–90% in cats and 50–65% in dogs. P. multocida can be transmitted to humans 183e-3 through bites or scratches, via the respiratory tract from contact with contaminated dust or infectious droplets, or via deposition of the organism on injured skin or mucosal surfaces during licking. Most human infections affect skin and soft tissue; almost two-thirds of these infections are caused by cats. Patients at the extremes of age or with serious underlying disorders (e.g., cirrhosis, diabetes) are at increased risk for systemic manifestations, including meningitis, peritonitis, osteomyelitis and septic arthritis, endocarditis, and septic shock, but cases have also occurred in healthy individuals of all ages. If inhaled,

1	P. multocida can cause acute respiratory tract infection, particularly in patients with underlying sinus and pulmonary disease. P. multocida is susceptible to penicillin, ampicillin, ampicillin/sulbactam, secondand third-generation cephalosporins, tetracyclines, and fluoroquinolones. β-lactamase-producing strains have been reported. Rhizobium (formerly Agrobacterium) radiobacter has usually been associated with infection in the presence of medical devices, including intravascular catheter–related infections, prosthetic-joint and prosthetic-valve infections, and peritonitis caused by dialysis catheters. Cases of endophthalmitis after cataract surgery also have been described. Most R. radiobacter infections occur in immunocompromised hosts, especially individuals with malignancy or HIV infection. Strains are usually susceptible to fluoroquinolones, thirdand fourth-generation cephalosporins, and carbapenems (Table 183e-2).

1	Shewanella putrefaciens and S. algae are ubiquitous organisms found primarily in seawater. Devitalized tissues can become colonized with Shewanella and serve as a nidus for systemic infection. Shewanella species cause skin and soft tissue infections, chronic ulcers of the lower extremities, ear infections, biliary tract infections, pneumonia, necrotizing fasciitis, bacteremia, and sepsis. A fulminant course is associated with cirrhosis, diabetes mellitus, malignancy, or other severe underlying conditions. Organisms are often susceptible to fluoroquinolones, thirdand fourth-generation cephalosporins, β-lactam/β-lactamase inhibitors, carbapenems, and aminoglycosides (Table 183e-2).

1	Chromobacterium violaceum has been responsible for life-threatening infections with severe sepsis and metastatic abscesses, particularly in children with defective neutrophil function (e.g., those with chronic granulomatous disease). Ochrobactrum anthropi causes infections related to central venous catheters in compromised hosts; other invasive infections have been described. Other organisms implicated in human infections include Weeksella species; various CDC groups, such as Ve-1 and Ve-2; Flavimonas species; Sphingobacterium species; and Oligella urethralis. The reader is advised to consult subspecialty texts and references for further guidance on these organisms. CHAPTER 183e Infections Due to the HACEK Group and Miscellaneous Gram-Negative Bacteria Victor L. Yu, M. Luisa Pedro-Botet, Yusen E. Lin

1	CHAPTER 183e Infections Due to the HACEK Group and Miscellaneous Gram-Negative Bacteria Victor L. Yu, M. Luisa Pedro-Botet, Yusen E. Lin Legionellosis refers to the two clinical syndromes caused by bacteria of the genus Legionella. Pontiac fever is an acute, febrile, self-limited illness that has been serologically linked to Legionella species, whereas Legionnaires’ disease is the designation for pneumonia caused by these species. Legionnaires’ disease was first recognized in 1976, when an outbreak of pneumonia took place at a Philadelphia hotel during an American Legion convention. The family Legionellaceae comprises more than 50 species with more than 70 serogroups. The species L. pneumophila causes 80–90% of human infections and includes at least 16 serogroups; serogroups 1, 4, and 6 are most commonly implicated in human infections. To date, 18 species other than L. pneumophila have been associated with human infections, among which L. micdadei (Pittsburgh pneumonia agent),

1	L. bozemanii, L. dumoffii, and L. longbeachae are the most common. Members of the Legionellaceae are aerobic gram-negative bacilli that do not grow on routine microbiologic media. Buffered charcoal yeast extract (BCYE) agar is the medium used to grow Legionella.

1	The natural habitats for L. pneumophila are aquatic bodies, including lakes and streams. L. longbeachae has been isolated from natural soil. Commercial potting soil has been suggested as the reservoir for L. longbeachae infections in Australia and New Zealand. Legionella can survive under a wide range of environmental conditions; for example, the organisms can live for years in refrigerated water samples. Natural bodies of water contain only small numbers of legionellae. However, once the organisms enter human-constructed aquatic reservoirs (such as drinking-water systems), they can grow and proliferate. Factors known to enhance colonization by and amplification of legionellae include warm temperatures (25°–42°C) and the presence of scale and sediment. L. pneumophila can form micro-colonies within biofilms; its eradication from drinking-water systems requires disinfectants that can penetrate the biofilm. The presence of symbiotic microorganisms, including algae, amebas, ciliated

1	within biofilms; its eradication from drinking-water systems requires disinfectants that can penetrate the biofilm. The presence of symbiotic microorganisms, including algae, amebas, ciliated protozoa, and other water-dwelling bacteria, promotes the growth of Legionella. The organisms can invade and multiply within free-living protozoa.

1	Heavy rainfall and flooding can result in the entry of high numbers of legionellae into water-distribution systems, leading to an upsurge of cases. Large buildings over three stories high are commonly colonized with Legionella. Sporadic community-acquired Legionnaires’ disease has been linked to colonization of hotels, office buildings, factories, and even private homes. Drinking-water systems in hospitals and extended-care facilities have been the source for health care–associated Legionnaires’ disease.

1	In contrast, cooling towers and evaporative condensers have been overestimated as sources of Legionella causing human illness. Early investigations that implicated cooling towers antedated the discovery that the organism could also exist in drinking water. In many outbreaks attributed to cooling towers, cases of Legionnaires’ disease continued to occur despite disinfection of the towers; drinking water was found to be the actual source. Koch’s postulates have never been fulfilled for Legionella links to cooling tower–associated outbreaks as they have been for hospital-acquired Legionnaires’ disease. Nevertheless, cooling towers have, in rare instances, been implicated in community-acquired outbreaks, including an outbreak in Murcia, Spain. As mentioned above, L. longbeachae infections have been linked to potting soil, but the mode of transmission remains to be clarified.

1	Multiple modes of transmission of Legionella to humans exist, including aerosolization, aspiration, and direct instillation into the lungs during respiratory tract manipulations. Aspiration is now known to be the predominant mode of transmission, but it is unclear whether Legionella enters the lungs via oropharyngeal colonization or directly via the drinking of contaminated water. Oropharyngeal colonization with Legionella has been demonstrated in patients undergoing transplantation. Nasogastric tubes have been linked to hospital-acquired Legionnaires’ disease; microaspiration of contaminated water was the hypothesized mode of transmission. Surgery with general anesthesia is a known risk factor that is consistent with aspiration. Especially compelling is the reported 30% incidence of postoperative Legionnaires’ disease among patients undergoing head and neck surgery at a hospital with a contaminated water supply; aspiration is a recognized postoperative complication in such cases. One

1	Legionnaires’ disease among patients undergoing head and neck surgery at a hospital with a contaminated water supply; aspiration is a recognized postoperative complication in such cases. One observational study showed that patients with hospital-acquired Legionnaires’ disease underwent endotracheal intubation significantly more often and for a significantly longer duration than patients with hospital-acquired pneumonias of other etiologies.

1	Aerosolization of Legionella by devices filled with tap water, including whirlpools, nebulizers, and humidifiers, has been linked to cases in patients. An ultrasonic mist machine in the produce section of a grocery store has been the source in community outbreaks. Pontiac fever has been linked to Legionella-containing aerosols from water-using machinery, a cooling tower, air conditioners, and whirlpools.

1	Community-Acquired Pneumonia The incidence of Legionnaires’ disease depends on the degree of contamination of the aquatic reservoir, the immune status of the persons exposed to water from that reservoir, the intensity of exposure, and the availability of specialized laboratory tests on which the correct diagnosis can be based. Numerous prospective studies have ranked Legionella among the top four microbial causes of community-acquired pneumonia, finding that it accounts for 2–13% of cases. (Streptococcus pneumoniae, Haemophilus influenzae, and Chlamydia pneumoniae are usually ranked first, second, and third, respectively.) On the basis of a multihospital study of community-acquired pneumonia in Ohio, the Centers for Disease Control and Prevention (CDC) estimated that only 3% of community-acquired cases of Legionnaires’ disease are diagnosed as such. Observational studies of community-acquired pneumonia showed that Legionnaires’ disease was largely unrecognized unless Legionella

1	cases of Legionnaires’ disease are diagnosed as such. Observational studies of community-acquired pneumonia showed that Legionnaires’ disease was largely unrecognized unless Legionella diagnostic testing was routinely applied to all patients with pneumonia; such studies in Spain and Germany resulted 1015 in detection of increased numbers of cases throughout Europe. It is likely that observational studies in Taiwan and Australia will have a similar result, with more cases identified throughout Asia as the index of suspicion rises.

1	Hospital-Acquired Pneumonia Legionella is responsible for 10–50% of cases of nosocomial pneumonia when a hospital’s water system is colonized with the organism. The incidence of hospital-acquired Legionnaires’ disease depends on the degree of contamination of drinking water, as defined by the rate of positivity of distal water sites; in contrast, the use of quantitative criteria of the number of colony-forming units per milliliter has proven useless.

1	Proactive culture of the hospital water supply has increased the detection of hospital-acquired Legionnaires’ disease and simul taneously allowed expeditious diagnosis resulting in early administration of antibiotic therapy. In the early years after its recognition, Legionnaires’ disease was documented primarily in the United States. As diagnostic modalities (especially the urinary antigen test) became more widely used, cases were documented in European hospitals. Likewise, following the enactment of public health guidelines in Taiwan, cases attributable to hospital tap water were found in Taiwanese hospitals. Risk factors for Legionnaires’ disease include cigarette smoking, chronic lung disease, advanced age, prior hospitalization with discharge within 10 days before onset of pneumonia symptoms, and immunosuppression. Immunosuppressive conditions that predispose to Legionnaires’ disease include transplantation, HIV infection, and treatment with glucocorticoids or tumor necrosis

1	symptoms, and immunosuppression. Immunosuppressive conditions that predispose to Legionnaires’ disease include transplantation, HIV infection, and treatment with glucocorticoids or tumor necrosis factor α antagonists. However, in a large prospective study of community-acquired pneumonia, 28% of patients with Legionnaires’ disease did not have these classic risk factors. Hospital-acquired cases are now being recognized among neonates and immunosuppressed children.

1	Pneumonia in Transplant Recipients Transplant recipients appear to be at unusually high risk of Legionella pneumonia. This elevated risk may be due to diagnostic bias, given the extensive workup for opportunistic pathogens with pneumonic symptoms as well as the long-standing immunosuppression in this population of patients. Legionnaires’ disease usually occurs in the 3 months after transplantation. Cavitation is seen on chest radiograph more frequently in transplant recipients, and mortality rates are higher. Pontiac Fever Pontiac fever occurs in epidemics. The high attack rate (>90%) reflects airborne transmission.

1	Pontiac Fever Pontiac fever occurs in epidemics. The high attack rate (>90%) reflects airborne transmission. Legionella enters the lungs through aspiration or direct inhalation. Attachment to host cells is mediated by bacterial type IV pili, heat-shock proteins, a major outer-membrane protein, and complement. Because the organism possesses pili that mediate adherence to respiratory tract epithelial cells, conditions that impair mucociliary clearance, including cigarette smoking, lung disease, or alcoholism, predispose to Legionnaires’ disease. Both innate and adaptive immune responses play a role in host defense. Toll-like receptors mediate recognition of L. pneumophila in alveolar macrophages and enhance early neutrophil recruitment to the site of infection. Alveolar macrophages phagocytose legionellae by a conventional or a coiling mechanism. After phagocytosis,

1	L. pneumophila evades intracellular killing by inhibiting phagosome– lysosome fusion. Although many legionellae are killed, some proliferate intracellularly until the cells rupture; the bacteria are then phagocytosed again by newly recruited phagocytes, and the cycle begins anew. The role of neutrophils in immunity appears to be minimal: neutropenic patients are not predisposed to Legionnaires’ disease. Although L. pneumophila is susceptible to oxygen-dependent microbiologic systems in vitro, it resists killing by neutrophils. The humoral immune system is active against Legionella. Type-specific IgM and IgG antibodies are measurable within weeks of infection. In vitro, antibodies promote killing of Legionella by phagocytes (neutrophils, monocytes, and alveolar macrophages). Immunized animals 1016 develop a specific antibody response, with subsequent resistance to Legionella challenge. However, antibodies neither enhance lysis by complement nor inhibit intracellular multiplication

1	animals 1016 develop a specific antibody response, with subsequent resistance to Legionella challenge. However, antibodies neither enhance lysis by complement nor inhibit intracellular multiplication within phagocytes. The genome of L. pneumophila has been sequenced. A broad range of membrane transporters within the genome are thought to optimize the use of nutrients in water and soil. Some L. pneumophila strains are clearly more virulent than others, although the precise factors mediating virulence remain uncertain. For example, although multiple strains may colonize water-distribution systems, only a few cause disease in patients exposed to water from these systems. At least one surface epitope of L. pneumophila serogroup 1 is associated with virulence. Monoclonal antibody subtype mAb2 has been linked to virulence. L. pneumophila serogroup 6 is more commonly involved in hospital-acquired Legionnaires’ disease and is especially likely to be associated with a poor outcome.

1	CLINICAL AND LABORATORY FEATURES Pontiac Fever Pontiac fever is an acute, self-limiting, flu-like illness with an incubation period of 24–48 h. Pneumonia does not develop. Malaise, fatigue, and myalgias are the most common symptoms, occurring in 97% of cases. Fever (usually with chills) develops in 80–90% of cases and headache in 80%. Other symptoms (seen in fewer than 50% of cases) include arthralgias, nausea, cough, abdominal pain, and diarrhea. Modest leukocytosis with a neutrophilic predominance is sometimes detected. Complete recovery occurs within a few days; antibiotic therapy is unnecessary. A few patients may experience lassitude for some weeks after recovery. The diagnosis is established by antibody seroconversion. Pontiac fever due to L. longbeachae has been reported in individuals exposed to potting soil.

1	Legionnaires’ Disease (Pneumonia) Legionnaires’ disease is often included in the differential diagnosis of “atypical pneumonia,” along with pneumonia due to C. pneumoniae, Chlamydia psittaci, Mycoplasma pneumoniae, Coxiella burnetii, and some viruses. The clinical similarities among “atypical” pneumonias include a nonproductive cough with a low frequency of grossly purulent sputum. The clinical manifestations of Legionnaires’ disease are usually more severe than those of most “atypical” pneumonias. The course and prognosis of Legionella pneumonia more closely resemble those of bacteremic pneumococcal pneumonia than those of pneumonia due to other “atypical” pathogens. Patients with community-acquired Legionnaires’ disease are significantly more likely than patients with pneumonia of other etiologies to be admitted to an intensive care unit (ICU) on presentation.

1	The incubation period for Legionnaires’ disease is usually 2–10 days, although slightly longer incubation periods have been documented. Fever is almost universal. In one observational study, 20% of patients had temperatures in excess of 40°C (104°F). The symptoms and signs may range from a mild cough and a slight fever to stupor with widespread pulmonary infiltrates and multisystem failure. The mild cough of Legionnaires’ disease is only slightly productive. Sometimes the sputum is streaked with blood. Chest pain—either pleuritic or nonpleuritic—can be a prominent feature and, when coupled with hemoptysis, can lead to an incorrect diagnosis of pulmonary embolism. Shortness of breath is reported by one-third to one-half of patients. Gastrointestinal difficulties are often pronounced; abdominal pain, nausea, and vomiting affect 10–20% of patients. Diarrhea (watery rather than bloody) is reported in 25–50% of cases. The most common neurologic abnormalities are confusion or changes in

1	pain, nausea, and vomiting affect 10–20% of patients. Diarrhea (watery rather than bloody) is reported in 25–50% of cases. The most common neurologic abnormalities are confusion or changes in mental status; however, the multitudinous neurologic symptoms reported range from headache and lethargy to encephalopathy. Nonspecific symptoms—malaise, fatigue, anorexia, and headache—are reported early in the illness. Myalgias and arthralgias are uncommon but are prominent in a few patients. Upper respiratory symptoms, including coryza, are rare.

1	Relative bradycardia has been overemphasized as a useful diagnostic finding; it occurs primarily in older patients with severe pneumonia. Rales are detected by chest examination early in the course, and evidence of consolidation is found as the disease progresses. Abdominal examination may reveal generalized or local tenderness. CLInICAL CLuES SuggESTIvE of LEgIonnAIRES’ DISEASE Numerous neutrophils but no organisms revealed by Gram’s staining of respiratory secretions Failure to respond to β-lactam drugs (penicillins or cephalosporins) and aminoglycoside antibiotics Occurrence of illness in an environment in which the potable water supply is known to be contaminated with Legionella Onset of symptoms within 10 days after discharge from the hospital (hospital-acquired legionellosis manifesting after discharge or transfer)

1	Onset of symptoms within 10 days after discharge from the hospital (hospital-acquired legionellosis manifesting after discharge or transfer) Although the clinical manifestations often considered classic for Legionnaires’ disease may suggest the diagnosis (Table 184-1), prospective comparative studies have shown that clinical manifestations are generally nonspecific and that Legionnaires’ disease is not readily distinguishable from pneumonia of other etiologies. In a review of 13 studies of community-acquired pneumonia, clinical manifestations that occurred significantly more often in Legionnaires’ disease included diarrhea, neurologic findings (including confusion), and a temperature of >39°C. Hyponatremia, elevated values in liver function tests, and hematuria also occurred more frequently in Legionnaires’ disease. Other laboratory abnormalities include creatine phosphokinase elevation, hypophosphatemia, serum creatinine elevation, and proteinuria.

1	Sporadic cases of Legionnaires’ disease appear to be more severe than outbreak-associated and hospital-acquired cases, presumably because their diagnosis is delayed. Results of the German CAPNETZ Study showed that, among cases of community-acquired Legionella pneumonia, ambulatory patients were as common as hospitalized patients. Extrapulmonary Legionellosis Because the portal of entry for Legionella is the lung in virtually all cases, extrapulmonary manifestations usually result from bloodborne dissemination from the lung. Legionella has been identified in lymph nodes, spleen, liver, or kidneys in autopsied cases. Sinusitis, peritonitis, pyelonephritis, skin and soft tissue infection, septic arthritis, and pancreatitis have developed predominantly in immunosuppressed patients. The most severe sequela, neurologic dysfunction, is rare but can be debilitating. The most common neurologic deficits in the long term—ataxia and speech difficulties—result from cerebellar dysfunction.

1	We speculate that cardiac abnormalities in patients without pneumonia are caused by Legionella-contaminated water entering through an intravenous site, chest tube, or surgical wound, with subsequent seeding of a prosthetic valve, the myocardium, or the pericardium. This scenario is supported by cases occurring at Stanford University Hospital in which sternal wound infections and prosthetic valve endocarditis due to L. pneumophila were observed. The source was a sink in the postoperative surgical recovery ward.

1	Chest Radiography Virtually all patients with Legionnaires’ disease have abnormal chest radiographs showing pulmonary infiltrates at the time of clinical presentation. In a few cases of hospital-acquired disease, fever and respiratory tract symptoms have preceded the radiographic appearance of the infiltrate. Radiologic findings are nonspecific. Pleural effusion is evident in 28–63% of patients on hospital admission. In immunosuppressed patients, especially those receiving glucocorticoids, distinctive rounded nodular opacities may be seen; these lesions may expand and cavitate (Fig. 184-1). Likewise, abscesses can occur in immunosuppressed hosts. The progression of infiltrates and pleural effusion on chest radiography despite appropriate antibiotic therapy within the first week is common, and radiographic improvement lags behind clinical improvement by several days. Complete clearing of infiltrates requires 1–4 months. Computed tomography (CT) is more sensitive than chest radiography,

1	and radiographic improvement lags behind clinical improvement by several days. Complete clearing of infiltrates requires 1–4 months. Computed tomography (CT) is more sensitive than chest radiography, may show more extensive disease, and should be performed if fever persists during treatment with presumably effective antibiotics (Fig. 184-2).

1	FIGURE 184-1 Chest radiographic findings in a 52-year-old man who presented with pneumonia subsequently diagnosed as Legionnaires’ disease. The patient was a cigarette smoker with chronic obstructive pulmonary disease and alcoholic cardiomyopathy; he had received glucocor-ticoids. Legionella pneumophila was identified by direct fluorescent antibody staining and culture of sputum. Left: Baseline chest radiograph show-ing long-standing cardiomegaly. Center: Admission chest radiograph showing new rounded opacities. Right: Chest radiograph taken 3 days after admission, during treatment with erythromycin.

1	Given the nonspecific clinical manifestations of Legionnaires’ disease and the high mortality rates for untreated Legionnaires’ disease, Legionella testing—especially the Legionella urinary antigen test—is recommended for all patients with pneumonia, including patients with ambulatory pneumonia and hospitalized children. Legionella cultures should be made more widely available because the urinary antigen test can diagnose only L. pneumophila serogroup 1. Hospitals in which the drinking water is known to be colonized with Legionella species should have Legionella cultures routinely available.

1	The diagnosis of Legionnaires’ disease requires special microbiologic tests (Table 184-2). The sensitivity of bronchoscopy specimens is similar to that of sputum samples for culture on selective media; if sputum is not available, bronchoscopy specimens may yield the organism. Bronchoalveolar lavage fluid gives higher yields than bronchial wash specimens. Thoracentesis should be performed if pleural effusion is found, and the fluid should be evaluated by direct fluorescent antibody (DFA) staining, culture, and the antigen assay designed for use with urine. Stains Gram’s staining of material from normally sterile sites, such as pleural fluid or lung tissue, occasionally suggests the diagnosis; efforts to detect Legionella in sputum by Gram’s staining typically reveal numerous leukocytes but no organisms. When they are visualized, the organisms appear as small, pleomorphic, faint, gram-negative bacilli.

1	L. micdadei organisms can be detected as weakly or partially acid-fast bacilli in clinical specimens. The DFA stain is rapid and highly specific but is less sensitive than culture because large numbers of organisms are required for microscopic visualization. This test is more likely to be positive in advanced than in early disease.

1	Culture The definitive method for diagnosis of Legionella infection is isolation of the organism from respiratory secretions, although culture for 3–5 days is required. Antibiotics added to the medium suppress the growth of competing flora from nonsterile sites, and dyes color the colonies and assist in identification. The use of multiple selective BCYE media is necessary for maximal sensitivity. When culture plates are overgrown with other microflora, pretreatment of the specimen with acid or heat can markedly improve the yield. L. pneumophila is often isolated from sputum that is not grossly or microscopically purulent; sputum containing more than 25 epithelial cells per high-power field (a finding that classically suggests contamination) may still yield L. pneumophila.

1	L. pneumophila. Antibody Detection Antibody testing of both acuteand convalescent-phase sera is necessary. A fourfold rise in titer is diagnostic; 12 weeks are often required for the detection of an antibody response. A single titer of 1:128 in a patient with pneumonia constitutes circumstantial evidence for Legionnaires’ disease. The CDC uses a titer of 1:256 as presumptive evidence for Legionnaires’ disease. Serology is of use primarily in epidemiologic studies. The specificity of serology for Legionella species other than L. pneumophila is uncertain; there is cross-reactivity within Legionella species and with some gram-negative bacilli. Serology is used as the criterion for the diagnosis of Pontiac fever.

1	Urinary Antigen The assay for Legionella soluble antigen in urine is second only to culture in terms of sensitivity and is highly specific. A rapid immunochromatographic assay is commercially available (BinaxNOW; Alere, Waltham, MA). This assay is relatively inexpensive and easy to perform. Its drawback is that the urinary antigen test is reliable only for L. pneumophila serogroup 1, which causes ~80% of Legionella infections. Cross-reactivity with other L. pneumophila serogroups and other Legionella species has been detected in up to 22% of urine samples from patients with culture-proven cases. Antigen in urine is detectable 3 days after the onset of clinical disease and disappears over 2 months; positivity can be prolonged when patients receive glucocorticoids. The test is not affected by antibiotic administration.

1	Molecular Methods DFA stains can identify a number of Legionella species. Both polyclonal and monoclonal antibody stains are commercially available. Polymerase chain reaction (PCR) with DNA probes is being applied in-house in selected hospitals but is not yet commercially available. PCR has proven somewhat useful in the identification of Legionella from environmental water specimens. Epidemiologic links cannot easily be made with PCR because the infecting pathogen is not available for molecular subtyping. Procalcitonin can be used as an indicator of severity of illness in patients in ICUs. Clinical response to antibiotics can be monitored by procalcitonin levels. Because Legionella is an intracellular pathogen, antibiotics that can attain high intracellular concentrations are most likely to be effective. The dosages for various drugs used in the treatment of Legionella infection are listed in Table 184-3.

1	The macrolides (especially azithromycin) and the respiratory quinolones are now the antibiotics of choice and are effective as mono-therapy. Compared with erythromycin, the newer macrolides have superior in vitro activity, display greater intracellular activity, reach higher concentrations in respiratory secretions and lung tissue, and have fewer adverse effects. The pharmacokinetics of the newer macrolides and quinolones also allow onceor twice-daily dosing. Quinolones are the preferred antibiotics for transplant recipients because both macrolides and rifampin interact pharmacologically with cyclosporine and tacrolimus. Retrospective uncontrolled studies have shown that complications of pneumonia are fewer and clinical response is more rapid in patients receiving quinolones than in those receiving macrolides. Initial therapy should be given by the

1	FIGURE 184-2 Computed tomography (CT) scans of a 49-year-old woman with no underlying conditions who presented with community-acquired pneumonia. CT revealed multilobar infiltrates, some of which were not as prominent on chest x-ray. Cultures of both the patient’s sputum and her home water supply yielded Legionella pneumophila serogroup 1. (Images courtesy of Dr. Wen-Chien Ko, National Cheng Kung University Hospital, Tainan, Taiwan.) Test Sensitivity, % Specificity, % aUse of multiple selective media with dyes. bReliable only for L. pneumophila serogroup 1. cIgG and IgM testing of both acuteand convalescent-phase sera. A single titer of ≥1:256 is considered presumptive, whereas a fourfold rise in titer between the acute and convalescent phases is considered definitive. Titers peak at 3 months. IV route. A clinical response usually occurs within 3–5 days, after which oral therapy can be substituted. The total duration of therapy in the immunocompetent host is 10–14 days.

1	IV route. A clinical response usually occurs within 3–5 days, after which oral therapy can be substituted. The total duration of therapy in the immunocompetent host is 10–14 days. Alternative agents include tetracycline and its analogues doxycycline and minocycline. Tigecycline is active in vitro, but clinical experience with this drug is minimal. Anecdotal reports have described both successes and failures with trimethoprim-sulfamethoxazole, imipenem, and clindamycin.

1	For critically ill patients, the authors use combination regimens of azithromycin, a quinolone, and/or rifampin. This practice is empirical and is not supported by comparative studies. Rifampin is highly active in vitro and in cell models. Its interaction with other medications and its side effect of reversible hyperbilirubinemia can be minimized by limiting the duration of therapy to 3–5 days. A longer course of therapy (3 weeks) may be appropriate for immunosuppressed patients and those with advanced disease. For azithromycin, with its long half-life, a 5to 10-day course is sufficient. Pontiac fever requires only symptom-based treatment, not antimicrobial therapy. Mortality rates for Legionnaires’ disease vary with the patient’s underlying disease, the patient’s immune status, the severity of pneumonia, and the timing of administration of appropriate antimicrobial therapy.

1	Trimethoprim-sulfamethoxazole 160/800 mg IV q8h 160/800 mg PO q12h Rifampind 300–600 mg PO or IV q12h aDosages are derived from clinical experience. bThe authors recommend doubling the first dose. cThe IV formulation is not available in some countries. dRifampin should be used only in combination with a macrolide or a quinolone. Mortality rates are highest (80%) among immunosuppressed patients who do not receive appropriate antimicrobial therapy early in the course of illness. With timely antibiotic treatment, mortality rates from community-acquired Legionnaires’ disease among immunocompetent patients range from 0 to 11%; without treatment, the figure may be as high as 31%. In a study of survivors of an outbreak of community-acquired Legionnaires’ disease, sequelae of fatigue, neurologic symptoms, and weakness were found in 63–75% of patients 17 months after receipt of antibiotics.

1	Routine environmental culture of hospital water supplies for Legionella is recommended as an approach to the prevention of hospital-acquired Legionnaires’ disease. Guidelines mandating this proactive approach have been adopted throughout Europe and in several U.S. states. The presence of Legionella in the water supply mandates the use of specialized laboratory tests (especially culture on selective media and the urinary antigen test) for patients with hospital-acquired pneumonia. A 30% cutoff for the presence of Legionella in water from multiple hospital sites prompts an increased index of suspicion. When the 30% cutoff point is exceeded, diagnostic tests for Legionella need to be applied in all cases of hospital-acquired pneumonia, and measures directed at eliminating the organism from the water supply should be considered. Quantitative criteria at a given water site (colony-forming units [CFU]/mL) have proven unreliable and inconsistent in the prediction of disease.

1	Studies have shown that neither a high degree of outward cleanliness of the water system nor routine application of maintenance measures decreases the frequency or intensity of Legionella contamination. Thus, engineering guidelines and building codes, although routinely advocated as preventive measures, have little impact on the presence of Legionella. Environmental cultures for Legionella from cold-water taps, hot-water taps, the hot-water recirculating line, and water-storage tanks will reveal the source of hospital-acquired infections. Disinfection of the hospital drinking-water system is an effective preventive measure for hospital-acquired cases of Legionnaires’ diseases because this system is the reservoir for Legionella. In geographic areas where the climate is semitropical, cold-water lines may be colonized by Legionella.

1	Copper-silver ionization is a reliable method for eradication of Legionella. Unlike the efficacy of chlorine dioxide decontamination and chlorination, that of ionization is not affected by high water temperature. Ionization systems are easy to install, and the ions are odorless, with minimal adverse effects. The efficacy of copper-silver ionization has been documented in hospitals worldwide. A comprehensive review of 10 published studies concluded that copper-silver ionization is effective for Legionella control as long as ion levels are monitored. If cold-water colonization by Legionella is the source of an outbreak, chlorine dioxide and monochloramine offer advantages. Chlorine dioxide, often the least expensive option, penetrates biofilms better and is less corrosive than chlorine. The major disadvantage of chlorine dioxide is the need to maintain an effective residual throughout the drinking-water system, especially in the hot-water system. Eradication of Legionella by chlorine

1	major disadvantage of chlorine dioxide is the need to maintain an effective residual throughout the drinking-water system, especially in the hot-water system. Eradication of Legionella by chlorine dioxide may require several months—a drawback in outbreak situations. Monochloramine is a promising approach in disinfection. Hyperchlorination is no longer recommended because of its expense, carcinogenicity, corrosive effects on piping, and unreliable efficacy.

1	Point-of-use disposable water filters (0.2 μm) may be an economical and effective option in high-risk areas (e.g., ICUs and transplantation units). These filters can be used in an outbreak situation for a limited period. Ineffective yet expensive methods that are often promulgated include removal of stagnation (“dead legs”) in the water-distribution system and replacement or disinfection/cleaning of distal outlets. Infection control personnel should oversee the selection of disinfection technology and should apply evidence-based criteria when making their choice. Managers of health care facilities should not be given the primary responsibility for selection and subsequent monitoring of measures to eliminate and control Legionella.

1	Pertussis and Other Bordetella Infections Karina A. Top, Scott A. Halperin Pertussis is an acute infection of the respiratory tract caused by Bordetella pertussis. The name pertussis means “violent cough,” which aptly describes the most consistent and prominent feature of the ill-185 ness. The inspiratory sound made at the end of an episode of paroxys mal coughing gives rise to the common name for the illness, “whoop ing cough.” However, this feature is variable: it is uncommon among infants ≤6 months of age and is frequently absent in older children and adults. The Chinese name for pertussis is “the 100-day cough,” which accurately describes the clinical course of the illness. The identification of B. pertussis was first reported by Bordet and Gengou in 1906, and vaccines were produced over the following two decades. Of the 10 identified species in the genus Bordetella, only four are of major medical significance. B. pertussis infects only humans and is the

1	B. pertussis produces a wide array of toxins and biologically active 1021 products that are important in its pathogenesis and in immunity. Most of these virulence factors are under the control of a single genetic locus that regulates their production, resulting in antigenic modulation and phase variation. Although these processes occur both in vitro and in vivo, their importance in the pathobiology of the organism is unknown; they may play a role in intracellular persistence and person-to-person spread. The organism’s most important virulence factor is pertussis toxin, which is composed of a B oligomer–binding subunit and an enzymatically active A protomer that ADP-ribosylates get cells, producing a variety of biologic effects. Pertussis toxin has important mitogenic activity, affects the circulation of lymphocytes, and serves as an adhesin for bacterial binding to respiratory ciliated cells. Other important virulence factors and adhesins are filamentous hemagglutinin, a component of

1	circulation of lymphocytes, and serves as an adhesin for bacterial binding to respiratory ciliated cells. Other important virulence factors and adhesins are filamentous hemagglutinin, a component of the cell wall, and pertactin, an outer- membrane protein. Fimbriae, bacterial appendages that play a role in bacterial attachment, are the major antigens against which agglu tinating antibodies are directed. These agglutinating antibodies have historically been the primary means of serotyping B. pertussis strains.

1	Other virulence factors include tracheal cytotoxin, which causes respi ratory epithelial damage; adenylate cyclase toxin, which impairs host most important Bordetella species causing human disease. B. paraper tussis causes an illness in humans that is similar to pertussis but is typiimmune-cell function; dermonecrotic toxin, which may contribute to respiratory mucosal damage; and lipooligosaccharide, which has propcally milder; co-infections with B. parapertussis and B. pertussis have erties similar to those of other gram-negative bacterial endotoxins.

1	been documented. With improved polymerase chain reaction (PCR) diagnostic methodology, up to 20% of patients with a pertussis-like syndrome have been found to be infected with B. holmesii, formerly thought to be an unusual cause of bacteremia. B. bronchiseptica is an important pathogen of domestic animals that causes kennel cough in dogs, atrophic rhinitis and pneumonia in pigs, and pneumonia in cats. Both respiratory infection and opportunistic infection due to B. bronchiseptica are occasionally reported in humans. B. petrii, B. hinzii, and B. ansorpii have been isolated from patients who are immunocompromised. Bordetella species are gram-negative pleomorphic aerobic bacilli that share common genotypic characteristics. B. pertussis and B.

1	Bordetella species are gram-negative pleomorphic aerobic bacilli that share common genotypic characteristics. B. pertussis and B. parapertussis are the most similar of the species, but B. parapertussis does not express the gene coding for pertussis toxin. B. pertussis is a slow-growing fastidious organism that requires selective medium and forms small, glistening, bifurcated colonies. Suspicious colonies are presumptively identified as B. pertussis by direct fluorescent antibody testing or by agglutination with species-specific antiserum. B. pertussis is further differentiated from other Bordetella species by biochemical and motility characteristics.

1	Pertussis is a highly communicable disease, with attack rates of within households in well-immunized populations. The infection has a worldwide distribution, with cyclical outbreaks every 3–5 years (a pattern that has persisted despite widespread immunization). Pertussis occurs in all months; however, in North America, its activity peaks in summer and autumn. In developing countries, pertussis remains an important cause of infant morbidity and death. The reported incidence of pertussis worldwide has decreased as a result of improved vaccine coverage (Fig. 185-1). However, coverage rates are still <50% in many developing nations; the World Health Organization (WHO) estimates that 90% of the burden of pertussis is in developing regions. In addition, overreporting of immunization coverage and underreporting of disease result in substantial underestimation of the global burden of pertussis. The WHO estimates that there were 195,000 deaths from pertussis among children in 2008.

1	Pertussis and Other Bordetella Infections 0 500,000 1,000,000 1,500,000 2,000,000 2,500,000 Number of cases 0 10 20 30 40 50 60 70 80 90 100 Immunization coverage (%)Number of cases Official coverage WHO/UNICEF estimates Pertussis global annual reported cases and DTP3 coverage, 1980–2012 FIGuRE 185-1 Global annual reported cases of pertussis and rate of coverage with DTP3 (diphtheria toxoid, tetanus toxoid, and pertussis vaccine; 3 doses), 1980–2012. (© World Health Organization, 2013. All rights reserved. From www.who.int/immunization/monitoring_ surveillance/burden/vpd/surveillance_type/passive/Pertussis_coverage.JPG. Source: WHO/IVB database, 2013.) Number of reported cases FIGuRE 185-2 Reported cases of pertussis by year—United States, 1976–2012. (From the Centers for Disease Control and Prevention, www.cdc.gov/pertussis/surv-reporting/cases-by-year.html. Accessed December 17, 2013.)

1	Before the institution of widespread immunization programs in the developed world, pertussis was one of the most common infectious causes of morbidity and death. In the United States before the 1940s, between 115,000 and 270,000 cases of pertussis were reported annually, with an average yearly rate of 150 cases per 100,000 population. With universal childhood immunization, the number of reported cases fell by >95%, and mortality rates decreased even more dramatically. Only 1010 cases of pertussis were reported in 1976 (Fig. 185-2). After that historic low, rates of pertussis slowly increased. In recent years, pertussis epidemics have been reported with increasing frequency worldwide. The United States experienced widespread outbreaks of pertussis in 2005, 2010, and 2012 at levels not seen in 40–50 years (>40,000 reported cases in 2012).

1	Although thought of as a disease of childhood, pertussis can affect people of all ages and is increasingly being identified as a cause of prolonged coughing illness in adolescents and adults. In unimmunized populations, pertussis incidence peaks during the preschool years, and well over half of children have the disease before reaching adulthood. In highly immunized populations such as those in North America, the peak incidence is among infants <1 year of age who have not completed the three-dose primary immunization series. An increase in pertussis incidence among adolescents and adults began in the late 1990s and led to the introduction of an adolescent booster across North America by 2006. While the disease burden among adolescents has started to decrease, children 7–10 years of age have recently emerged as a high-risk group. In major outbreaks in 2010 and 2012, the incidence of pertussis among children 10 years of age, most of whom were fully immunized, was as high as that among

1	recently emerged as a high-risk group. In major outbreaks in 2010 and 2012, the incidence of pertussis among children 10 years of age, most of whom were fully immunized, was as high as that among infants <6 months of age. Although adults contribute a smaller proportion of reported cases of pertussis than do children and adolescents, this difference may be related to a greater degree of underrecognition and underreporting. A number of studies of prolonged coughing illness suggest that B. pertussis may be the etiologic agent in 12–30% of adults with cough that does not improve within 2 weeks. In one study of the efficacy of an acellular pertussis vaccine in adolescents and adults, the incidence of pertussis in the placebo group was 3.7–4.5 cases per 1000 person-years. Although this prospective cohort study yielded a lower estimate than the studies of cough illness, its results still translate to 600,000–800,000 cases of pertussis annually among adults in the United States.

1	Severe morbidity and high mortality rates, however, are restricted almost entirely to infants. In Canada, there were 16 deaths from pertussis between 1991 and 2001; all those who died were infants ≤6 months of age. Similarly, in the United States between 1993 and 2004, all pertussis deaths and 86% of hospitalizations for pertussis involved infants ≤3 months of age. Although school-age children are the source of infection for most households, adults are the likely source for cases in high-risk infants and may serve as the reservoir of infection between epidemic years.

1	Infection with B. pertussis is initiated by attachment of the organism to the ciliated epithelial cells of the nasopharynx. Attachment is mediated by surface adhesins (e.g., pertactin and filamentous hemagglutinin), which bind to the integrin family of cell-surface proteins, probably in conjunction with pertussis toxin. The role of fimbriae in adhesion and in maintenance of infection has not been fully delineated. At the site of attachment, the organism multiplies, producing a variety of other toxins that cause local mucosal damage (tracheal cytotoxin, dermonecrotic toxin). Impairment of host defense by B. pertussis is mediated by pertussis toxin and adenylate cyclase toxin. There is local cellular invasion, with intracellular bacterial persistence; however, systemic dissemination does not occur. Systemic manifestations (lymphocytosis) result from the effects of the toxins.

1	The pathogenesis of the clinical manifestations of pertussis is poorly understood. It is not known what causes the hallmark paroxysmal cough. A pivotal role for pertussis toxin has been proposed. Proponents of this position point to the efficacy of preventing clinical symptoms with a vaccine containing only pertussis toxoid. Detractors counter that pertussis toxin is not the critical factor because paroxysmal cough also occurs in patients infected with B. parapertussis, which does not produce pertussis toxin. It is thought that neurologic events in pertussis, such as seizures and encephalopathy, are due to hypoxia from coughing paroxysms or apnea rather than to the effects of specific bacterial products. B. pertussis pneumonia, which occurs in up to 10% of infants with pertussis, is usually a diffuse bilateral primary infection. In older children and adults with pertussis, pneumonia is often due to secondary bacterial infection with streptococci or staphylococci. Deaths from pertussis

1	a diffuse bilateral primary infection. In older children and adults with pertussis, pneumonia is often due to secondary bacterial infection with streptococci or staphylococci. Deaths from pertussis among young infants are frequently associated with very high levels of leukocytosis and pulmonary hypertension.

1	Both humoral and cell-mediated immunity are thought to be important in pertussis. Antibodies to pertussis toxin, filamentous hemagglutinin, pertactin, and fimbriae are all protective in animal models. Pertussis agglutinins were correlated with protection in early studies of whole-cell pertussis vaccines. Serologic correlates of protection conferred by acellular pertussis vaccines have not been established, although antibody to pertactin, fimbriae, and (to a lesser degree) pertussis toxin correlated best with protection in two efficacy trials. The duration of immunity after whole-cell pertussis vaccination is short-lived, with little protection remaining after 10–12 years. Recent studies have demonstrated early waning of immunity—i.e., within 2–4 years after the fifth dose of acellular pertussis vaccine in children who received acellular pertussis vaccine for their primary series in infancy. These data suggest that boosters may be needed more frequently than every 10 years, as

1	pertussis vaccine in children who received acellular pertussis vaccine for their primary series in infancy. These data suggest that boosters may be needed more frequently than every 10 years, as previously thought. Although immunity after natural infection was thought to be lifelong, seroepidemiologic evidence demonstrates that it clearly is not and that subsequent episodes of clinical pertussis are prevented by intermittent subclinical infection.

1	Pertussis is a prolonged coughing illness with clinical manifestations that vary by age (Table 185-1). Although not uncommon among adolescents and adults, classic pertussis is most often seen in preschool and school-age children. After an incubation period averaging 7–10 days, an illness develops that is indistinguishable from the common cold and is characterized by coryza, lacrimation, mild cough, low-grade fever, and malaise. After 1–2 weeks, this catarrhal phase evolves into the paroxysmal phase: the cough becomes more frequent and spasmodic with repetitive bursts of 5–10 coughs, often within a single expiration. Posttussive vomiting is frequent, with a mucous plug occasionally expelled at the end of an episode. The episode may be terminated by an audible whoop, which occurs upon rapid inspiration against a closed glottis at the end of a paroxysm. During a spasm, there may be impressive neck-vein distension, bulging eyes, tongue

1	TABLE 185-1 CLInICAL FEATuREs OF PERTussIs, By AgE gROuP And dIAgnOsTIC sTATus Percentage of Patients protrusion, and cyanosis. Paroxysms may be precipitated by noise, eating, or physical contact. Between attacks, the patient’s appearance is normal but increasing fatigue is evident. The frequency of paroxysmal episodes varies widely, from several per hour to 5–10 per day. Episodes are often worse at night and interfere with sleep. Weight loss is not uncommon as a result of the illness’s interference with eating. Most complications occur during the paroxysmal stage. Fever is uncommon and suggests bacterial superinfection. After 2–4 weeks, the coughing episodes become less frequent and less severe—changes heralding the onset of the convalescent phase. This phase can last 1–3 months and is characterized by gradual resolution of coughing episodes. For 6–12 months, intercurrent viral infections may be associated with a recrudescence of paroxysmal cough.

1	Not all individuals who develop pertussis have classic disease. The clinical manifestations in adolescents and adults are more often atypical. In a German study of pertussis in adults, more than two-thirds had paroxysmal cough and more than one-third had whoop. Adult illness in North America differs from this experience: the cough may be severe and prolonged but is less frequently paroxysmal, and a whoop is uncommon. Vomiting with cough is the best predictor of pertussis as the cause of prolonged cough in adults. Other predictive features are a cough at night, sweating episodes between paroxysms of coughing, and exposure to other individuals with a prolonged coughing illness.

1	Complications are frequently associated with pertussis and are more common among infants than among older children or adults. Subconjunctival hemorrhages, abdominal and inguinal hernias, pneumothoraces, and facial and truncal petechiae can result from increased intrathoracic pressure generated by severe fits of coughing. Weight loss can follow decreased caloric intake. In a series of more than 1100 children <2 years of age who were hospitalized with pertussis, 27.1% had apnea, 9.4% had pneumonia, 2.6% had seizures, and 0.4% had encephalopathy; 10 children (0.9%) died. Pneumonia is reported in <5% of adolescents and adults and increases in frequency after 50 years of age. In contrast to the primary B. pertussis pneumonia that develops in infants, pneumonia in adolescents and adults with pertussis is usually caused by a secondary infection with encapsulated organisms such as Streptococcus pneumoniae or Haemophilus influenzae. Pneumothorax, severe weight loss, inguinal hernia, rib

1	pertussis is usually caused by a secondary infection with encapsulated organisms such as Streptococcus pneumoniae or Haemophilus influenzae. Pneumothorax, severe weight loss, inguinal hernia, rib fracture, carotid artery aneurysm, and cough syncope have all been reported in adolescents and adults with pertussis.

1	If the classic symptoms of pertussis are present, clinical diagnosis is not difficult. However, particularly in older children and adults, it is difficult to differentiate infections caused by B. pertussis and B. parapertussis from other respiratory tract infections on clinical grounds. Therefore, laboratory confirmation should be attempted in all cases. Lymphocytosis (an absolute lymphocyte count of >108–109/L) is common among young children, in whom it is unusual with other infections, but not among adolescents and adults. Culture of nasopharyngeal secretions remains the gold standard of diagnosis, although DNA detection by PCR has replaced culture in many laboratories because 1023 of increased sensitivity and quicker results. Appropriate PCR methodology must include primers to differentiate among B. pertussis, B. parapertussis, and B. holmesii. The best specimen is collected by nasopharyngeal aspiration, in which a fine flexible plastic catheter attached to a 10-mL syringe is

1	among B. pertussis, B. parapertussis, and B. holmesii. The best specimen is collected by nasopharyngeal aspiration, in which a fine flexible plastic catheter attached to a 10-mL syringe is passed into the nasopharynx and withdrawn while gentle suction is applied. Since B. pertussis is highly sensitive to drying, secretions for culture should be inoculated without delay onto appropriate medium (Bordet-Gengou or Regan-Lowe), or the catheter should be flushed with a phosphate-buffered saline solution for culture and/or PCR. An alternative to the aspirate is a Dacron or rayon nasopharyngeal swab; again, inoculation of culture plates should be immediate or an appropriate transport medium (e.g., Regan-Lowe charcoal medium) should be used. Results of PCR can be available within hours; cultures become positive by day 5 of incubation. B. pertussis and B. parapertussis can be differentiated by agglutination with specific antisera or by direct immunofluorescence.

1	Nasopharyngeal cultures in untreated pertussis remain positive for a mean of 3 weeks after the onset of illness; these cultures become negative within 5 days of the institution of appropriate antimicrobial therapy. The duration of a positive PCR in untreated pertussis or after therapy is not known but exceeds that of positive cultures. Since much of the period during which the organism can be recovered from the nasopharynx falls into the catarrhal phase, when the etiology of the infection is not suspected, there is only a small window of opportunity for culture-proven diagnosis. Cultures from infants and young children are more frequently positive than those from older children and adults; this difference may reflect earlier presentation of the former age group for medical care. Direct fluorescent antibody tests of nasopharyngeal secretions for direct diagnosis may still be available in some laboratories but should not be used because of poor sensitivity and specificity.

1	Direct fluorescent antibody tests of nasopharyngeal secretions for direct diagnosis may still be available in some laboratories but should not be used because of poor sensitivity and specificity. Pseudo-outbreaks of pertussis have been reported as a result of false-positive PCR results. Greater standardization of PCR methodology can alleviate this problem.

1	As a result of the difficulties with laboratory diagnosis of pertussis in adolescents, adults, and patients who have been symptomatic for >4 weeks, increasing attention is being given to serologic diagnosis. Enzyme immunoassays detecting IgA and IgG antibodies to pertussis toxin, filamentous hemagglutinin, pertactin, and fimbriae have been developed and assessed for reproducibility. Twoor fourfold increases in antibody titer are suggestive of pertussis, although cross-reactivity of some antigens (such as filamentous hemagglutinin and pertactin) among Bordetella species makes it difficult to depend diagnostically on seroconversion involving a single type of antibody. Late presentation for medical care and prior immunization also complicate serologic diagnosis because the first sample obtained may in fact be a convalescent-phase specimen. Criteria for serologic diagnosis based on comparison of results for a single serum specimen with established population values are gaining acceptance,

1	may in fact be a convalescent-phase specimen. Criteria for serologic diagnosis based on comparison of results for a single serum specimen with established population values are gaining acceptance, and serologic measurement of antibody to pertussis toxin is becoming more widely standardized and available for diagnostic purposes, particularly in outbreak settings and for surveillance.

1	A child presenting with paroxysmal cough, posttussive vomiting, and whoop is likely to have an infection caused by B. pertussis or B. parapertussis; lymphocytosis increases the likelihood of a B. pertussis etiology. Viruses such as respiratory syncytial virus and adenovirus have been isolated from patients with clinical pertussis but probably represent co-infection. In adolescents and adults, who often do not have paroxysmal cough or whoop, the differential diagnosis of a prolonged coughing illness is more extensive. Pertussis should be suspected when any patient has a cough that does not improve within 14 days, a paroxysmal cough of any duration, a cough followed by vomiting (adolescents and adults), or any respiratory symptoms after contact with a laboratory-confirmed case of pertussis. Other etiologies to consider include infections caused by Mycoplasma pneumoniae, Chlamydia pneumoniae, adenovirus, influenza virus, and other respiratory viruses. Use of angiotensin-converting

1	Drug Adult Daily Dose Frequency Duration, Days Comments Erythromycin estolate 1–2 g 3 divided doses 7–14 Frequent gastrointestinal side effects Clarithromycin 500 mg 2 divided doses 7 — Azithromycin 500 mg on day 1, 250 mg 1 daily dose 5 — subsequently Trimethoprim-160 mg of trimethoprim, 800 mg of 2 divided doses 14 For patients allergic to macrolides; data on enzyme (ACE) inhibitors, reactive airway disease, and gastroesophageal reflux disease are well-described noninfectious causes of prolonged cough in adults. The purpose of antibiotic therapy for pertussis is to eradicate the infecting bacteria from the nasopharynx; therapy does not substantially alter the clinical course unless given early in the catarrhal phase. Macrolide antibiotics are the drugs of choice for treatment of pertussis (Table 185-2); macrolide-resistant B. pertussis strains have been reported but are rare. Trimethoprim-sulfamethoxazole is recommended as an alternative for individuals allergic to macrolides.

1	Young infants have the highest rates of complication and death from pertussis; therefore, most infants (and older children with severe disease) should be hospitalized. A quiet environment may decrease the stimulation that can trigger paroxysmal episodes. Use of β-adrenergic agonists and/or glucocorticoids has been advocated by some authorities but has not been proven to be effective. Cough suppressants are not effective and play no role in the management of pertussis. Hospitalized patients with pertussis should be placed in respiratory isolation, with the use of precautions appropriate for pathogens spread by large respiratory droplets. Isolation should continue for 5 days after initiation of macrolide therapy or, in untreated patients, for 3 weeks (i.e., until nasopharyngeal cultures are consistently negative).

1	PREVENTION Chemoprophylaxis Because the risk of transmission of B. pertussis within households is high, chemoprophylaxis is widely recommended for household contacts of pertussis cases. The effectiveness of chemoprophylaxis, although unproven, is supported by several epidemiologic studies of institutional and community outbreaks. In the only randomized placebo-controlled study, erythromycin estolate (50 mg/kg per day in three divided doses; maximum dose, 1 g/d) was effective in reducing the incidence of bacteriologically confirmed pertussis by 67%; however, there was no decrease in the incidence of clinical disease. Despite these disappointing results, many authorities continue to recommend chemoprophylaxis, particularly in households with members at high risk of severe disease (children <1 year of age, pregnant women). Data are not available on use of the newer macrolides for chemoprophylaxis, but these drugs are commonly used because of their increased tolerability and their

1	<1 year of age, pregnant women). Data are not available on use of the newer macrolides for chemoprophylaxis, but these drugs are commonly used because of their increased tolerability and their effectiveness.

1	Immunization (See also Chap. 148) The mainstay of pertussis prevention is active immunization. Pertussis vaccine became widely used in North America after 1940; the reported number of pertussis cases subsequently fell by >90%. Whole-cell pertussis vaccines are prepared through the heating, chemical inactivation, and purification of whole

1	B. pertussis organisms. Despite their efficacy (average estimate, 85%; range for different products, 30–100%), whole-cell pertussis vaccines are associated with adverse events—both common (fever; injection-site pain, erythema, and swelling; irritability) and uncommon (febrile seizures, hypotonic hyporesponsive episodes). Alleged associations of whole-cell pertussis vaccine with encephalopathy, sudden infant death syndrome, and autism, although not substantiated, have spawned an active anti-immunization lobby. The development of acellular pertussis vaccines, which are effective and less reactogenic, has greatly alleviated concerns about the inclusion of pertussis vaccine in the combined infant immunization series.

1	Although whole-cell vaccines are still used extensively in developing regions of the world, acellular pertussis vaccines are used exclusively for childhood immunization in much of the developed world. In North America, acellular pertussis vaccines for children are given as a three-dose primary series at 2, 4, and 6 months of age, with a reinforcing dose at 15–18 months of age and a booster dose at 4–6 years of age.

1	Although a wide variety of acellular pertussis vaccines were developed, only a few are still widely marketed; all contain pertussis toxoid and filamentous hemagglutinin. One acellular pertussis vaccine also contains pertactin, and another contains pertactin and two types of fimbriae. In light of phase 3 efficacy studies, most experts have concluded that two-component acellular pertussis vaccines are more effective than monocomponent vaccines and that the addition of pertactin increases efficacy still more. The further addition of fimbriae appears to enhance protective efficacy against milder disease. In two studies, protection conferred by pertussis vaccines correlated best with the production of antibody to pertactin, fimbriae, and pertussis toxin.

1	Adult formulations of acellular pertussis vaccines have been shown to be safe, immunogenic, and efficacious in clinical trials in adolescents and adults and are now recommended for routine immunization of these groups in several countries, including the United States. In this country, adolescents should receive a dose of the adult-formulation diphtheria–tetanus–acellular pertussis vaccine at the preadolescence physician’s visit, and all unvaccinated adults should receive a single dose of this combined vaccine. In addition, in the United States, pertussis immunization is specifically recommended for health care workers and for women during each pregnancy to increase passive transfer of maternal antibodies to the fetus. Pertussis vaccine coverage among

1	U.S. adolescents was 78.2% in 2011, but coverage among adults is low (2.1% as of 2007). Further improvements in adult vaccine coverage may permit better control of pertussis across the age spectrum, with collateral protection of infants too young to be immunized. However, more effective vaccines with longer-lasting protection will ultimately be needed to control this disease. diseases Caused by gram-negative Enteric Bacilli Thomas A. Russo, James R. Johnson GENERAL FEATuRES AND PRINCIPLES Escherichia coli, Klebsiella, Proteus, Enterobacter, Serratia, Citrobacter, 186

1	diseases Caused by gram-negative Enteric Bacilli Thomas A. Russo, James R. Johnson GENERAL FEATuRES AND PRINCIPLES Escherichia coli, Klebsiella, Proteus, Enterobacter, Serratia, Citrobacter, 186 Morganella, Providencia, Cronobacter, and Edwardsiella are gram-negative enteric bacilli that are members of the family Enterobacteriaceae. Salmonella, Shigella, and Yersinia, also in the family Enterobacteriaceae, are discussed in Chaps. 190, 191, and 196, respectively. These pathogens cause a wide variety of infections involving diverse anatomic sites in both healthy and compromised hosts. Increasing antimicrobial resistance in this group has put them at the forefront of an evolving public health crisis. In addition, new infectious syndromes have emerged. Therefore, a thorough knowledge of clinical presentations and appropriate therapeutic choices is necessary for optimal outcomes.

1	E. coli, Klebsiella, Proteus, Enterobacter, Serratia, Citrobacter, Morganella, Providencia, Cronobacter, and Edwardsiella are components of the normal animal and human colonic micro biota and/or the microbiota of a variety of environmental habitats, including long-term-care facilities (LTCFs) and hospitals. As a result, except for certain pathotypes of intestinal pathogenic E. coli, these genera are global pathogens. The incidence of infection due to these agents is increasing because of the combination of an aging population and increasing antimicrobial resistance. In healthy humans, E. coli is the predominant species of gram-negative bacilli (GNB) in the colonic flora; Klebsiella and Proteus are less prevalent. GNB (primarily E. coli, Klebsiella, and Proteus) only transiently colonize the oropharynx and skin of healthy individuals. In contrast, in LTCFs and hospital settings, a variety of GNB emerge as the dominant microbiota of both mucosal and skin surfaces, particularly in

1	the oropharynx and skin of healthy individuals. In contrast, in LTCFs and hospital settings, a variety of GNB emerge as the dominant microbiota of both mucosal and skin surfaces, particularly in association with antimicrobial use, severe illness, and extended length of stay. LTCFs are emerging as an important reservoir for resistant GNB. This colonization may lead to subsequent infection; for example, oropharyngeal colonization may lead to pneumonia. Interestingly, the use of ampicillin or amoxicillin was associated with an increased risk of subsequent infection due to the hypervirulent variant of Klebsiella pneumoniae in Taiwan; this association suggests that changes in the quantity or prevalence of colonizing bacteria may be important. Serratia and Enterobacter infection may be acquired through a variety of infusates (e.g., medications, blood products). Edwardsiella infections are acquired through freshwater and marine environment exposures and are most common in Southeast Asia.

1	Enteric GNB possess an extracytoplasmic outer membrane, which consists of a lipid bilayer with associated proteins, lipoproteins, and polysaccharides (capsule, lipopolysaccharide). The outer membrane 1025 interfaces with the external environment, including the human host. A variety of components of the outer membrane are critical determinants in pathogenesis (e.g., capsule) and antimicrobial resistance (e.g., permeability barrier, efflux pumps). Multiple bacterial virulence factors are required for the pathogenesis of infections caused by GNB. Possession of specialized virulence genes defines pathogens and enables them to infect the host efficiently. Hosts and their cognate pathogens have been co-adapting throughout evolutionary history. During the host-pathogen “chess match” over time, various and redundant strategies have emerged in both the pathogens and their hosts (Table 186-1).

1	Intestinal pathogenic mechanisms are discussed below. The members of the Enterobacteriaceae family that cause extraintestinal infections are primarily extracellular pathogens and therefore share certain pathogenic features. Innate immunity (including the activities of complement, antimicrobial peptides, and professional phagocytes) and humoral immunity are the principal host defense components. Both susceptibility to and severity of infection are increased with dysfunction or deficiencies of these components. By contrast, the virulence traits of intestinal pathogenic E. coli—i.e., the distinctive strains that can cause diarrheal disease—are for the most part different from those of extraintestinal pathogenic E. coli (ExPEC) and other GNB that cause extraintestinal infections. This distinction reflects site-specific differences in host environments and defense mechanisms.

1	A given strain usually possesses multiple adhesins for binding to a variety of host cells (e.g., in E. coli: type 1, S, and F1C fimbriae; P pili). Nutrient acquisition (e.g., of iron via siderophores) requires many genes that are necessary but not sufficient for pathogenesis. The ability to resist the bactericidal activity of complement and phagocytes in the absence of antibody (e.g., as conferred by capsule or O antigen of lipopolysaccharide) is one of the defining traits of an extracellular pathogen. Tissue damage (e.g., as mediated by hemolysin in the case of E. coli) may facilitate spread within the host. Without doubt, many important virulence genes await identification (Chap. 145e).

1	The ability to induce septic shock is another defining feature of these genera. GNB are the most common causes of this potentially lethal syndrome. Pathogen-associated molecular pattern molecules (PAMPs; e.g., the lipid A moiety of lipopolysaccharide) stimulate a proinflammatory host response via pattern recognition receptors (e.g., Toll-like or C-type lectin receptors) that activate host defense signaling pathways; if overly exuberant, this response results in shock (Chap. 325). Direct bacterial damage of host tissue (e.g., by toxins) or collateral damage from the host response can result in the release of damage-associated molecular pattern molecules (DAMPs; e.g., HMGB1) that can propagate a detrimental proinflammatory host response.

1	Many antigenic variants (serotypes) exist in most genera of GNB. For example, E. coli has more than 150 O-specific antigens and more than 80 capsular antigens. This antigenic variability, which permits immune evasion and allows recurrent infection by different strains of the same species, has impeded vaccine development (Chap. 148). InTERACTIOns OF ExTRAInTEsTInAL PATHOgEnIC ESCHERICHIA COLI wITH THE HuMAn HOsT: A PARAdIgM FOR ExTRACELLuLAR, ExTRAInTEsTInAL gRAM-nEgATIvE BACTERIAL PATHOgEns

1	E. coli can cause either intestinal or extraintestinal infection, depending on the particular pathotype, and Edwardsiella tarda can cause both intestinal and extraintestinal infection. Klebsiella primarily causes extraintestinal infection, but hemorrhagic colitis has been associated with a toxin-producing variant of Klebsiella oxytoca. Depending on both the host and the pathogen, nearly every organ or body cavity can be infected with GNB. E. coli and—to a lesser degree—Klebsiella account for most extraintestinal infections due to GNB and are the most virulent pathogens within this group; this virulence is demonstrated by the ability of E. coli and Klebsiella pneumoniae (primarily the hypervirulent variant) to cause severe infections in healthy, ambulatory hosts from the community. However, the other genera are also important, especially among LTCF residents and hospitalized patients, in large part because of the intrinsic or acquired antimicrobial resistance of these organisms and the

1	the other genera are also important, especially among LTCF residents and hospitalized patients, in large part because of the intrinsic or acquired antimicrobial resistance of these organisms and the increasing number of individuals with compromised host defenses. The mortality rate is substantial in many GNB infections and correlates with the severity of illness. Especially problematic are pneumonia and bacteremia (arising from any source), particularly when complicated by organ failure (severe sepsis) and/or shock, for which the associated mortality rates are 20–50%.

1	Isolation of GNB from sterile sites almost always implies infection, whereas their isolation from nonsterile sites, particularly from open soft-tissue wounds and the respiratory tract, requires clinical correlation to differentiate colonization from infection. Tentative laboratory identification based on lactose fermentation and indole production (described for each genus below), which usually is possible before final identification of the organism and determination of its antimicrobial susceptibilities, may help to guide empirical antimicrobial therapy.

1	(See also Chap. 170) Evidence indicates that initiation of appropriate empirical antimicrobial therapy early in the course of GNB infections (particularly serious infections) leads to improved outcomes. The ever-increasing prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) GNB; the lag between published (historical) and current resistance rates; and variations by species, geographic location, regional antimicrobial use, and hospital site (e.g., intensive care units [ICUs] versus wards) necessitate familiarity with evolving patterns of antimicrobial resistance for the selection of appropriate empirical therapy. Factors predictive of isolate resistance include recent antimicrobial use, a health care association (e.g., recent or ongoing hospitalization, dialysis, residence in an LTCF), or international travel (e.g., to Asia, Latin America, Africa, southern Europe). For appropriately selected patients, it may be prudent initially, while susceptibility results are

1	in an LTCF), or international travel (e.g., to Asia, Latin America, Africa, southern Europe). For appropriately selected patients, it may be prudent initially, while susceptibility results are awaited, to use two potentially active agents with the rationale that at least one agent will be active. If broad-spectrum treatment has been initiated, it is critical to switch to the most appropriate narrower-spectrum agent when information on antimicrobial susceptibility becomes available. Such responsible antimicrobial stewardship will slow down the ever-escalating cycle of selection for increasingly resistant bacteria, decrease the likelihood of Clostridium difficile infection, decrease costs, and maximize the useful longevity of available antimicrobial agents. Likewise, it is important to avoid treatment of patients who are colonized but not infected (e.g., who have a positive sputum culture without evidence of pneumonia). At present, the most reliably active agents against enteric GNB are

1	treatment of patients who are colonized but not infected (e.g., who have a positive sputum culture without evidence of pneumonia). At present, the most reliably active agents against enteric GNB are the carbapenems (e.g., imipenem), the aminoglycoside amikacin, the fourth-generation cephalosporin cefepime, the β-lactam/β-lactamase inhibitor combination piperacillin-tazobactam, and the polymyxins (e.g., colistin or polymyxin B). The number of antimicrobials effective against certain Enterobacteriaceae is shrinking. Truly pan-resistant GNB exist, and it is unlikely that new agents will come to market in the short term. Accordingly, the presently available antimicrobials must be used judiciously.

1	β-Lactamases, which inactivate β-lactam agents, are the most important mediators of resistance to these drugs in GNB. Decreased permeability and/or active efflux of β-lactam agents, although less common, may occur alone or in combination with β-lactamasemediated resistance. Broad-spectrum β-lactamases (e.g., TEM, SHV), which mediate resistance to many penicillins and first-generation cephalosporins, are frequently expressed in enteric GNB. These enzymes are inhibited by β-lactamase inhibitors (e.g., clavulanate, sulbactam, tazobactam). They usually do not hydrolyze thirdand fourth-generation cephalosporins or cephamycins (e.g., cefoxitin).

1	Extended-spectrum β-lactamases (ESBLs; e.g., CTX-M, SHV, TEM) are modified broad-spectrum enzymes that confer resistance to the same drugs as well as to third-generation cephalosporins, aztreonam, and (in some instances) fourth-generation cephalosporins. GNB that express ESBLs may also possess porin mutations that result in decreased uptake of cephalosporins and β-lactam/ β-lactamase inhibitor combinations. The prevalence of acquired ESBL production, particularly of CTX-M-type enzymes, is increasing in GNB worldwide, in large part due to the presence of the responsible genes on large transferable plasmids with linked or associated resistance to fluoroquinolones, trimethoprim-sulfamethoxazole (TMP-SMX), aminoglycosides, and tetracyclines. To date, ESBLs are most prevalent in

1	K. pneumoniae, K. oxytoca, and E. coli but also occur (and are probably underrecognized) in Enterobacter, Citrobacter, Proteus, Serratia, and other enteric GNB. At present, the rough regional prevalence of ESBL-producing GNB is India > China > rest of Asia, Latin America, Africa, southern Europe > northern Europe > United States, Canada, and Australia. International travel to high-prevalence regions increases the likelihood of colonization with these strains. ESBL-producing GNB were initially described in hospitals (ICUs > wards) and LTCFs, where outbreaks occurred in association with extensive use of third-generation cephalosporins. However, over the last decade, the incidence of uncomplicated cystitis due to CTX-M ESBL-containing E. coli has increased worldwide (including in the United States) among healthy ambulatory women without health care or antimicrobial exposure. Antimicrobial use in food animals has also been implicated in the rise of ESBLs.

1	The carbapenems are the most reliably active β-lactam agents against ESBL-expressing strains. Clinical experience with alternatives is more limited, but, for organisms susceptible to piperacillintazobactam (minimal inhibitory concentration [MIC], ≤4 μg/mL), this agent—at a dosage of 4.5 g q6h—may offer a carbapenemsparing alternative, at least for E. coli. The role of tigecycline is unclear despite its excellent in vitro activity; Proteus, Morganella, and Providencia are inherently resistant, and attainable serum and urine levels are low. Therefore, caution appears to be prudent, especially with serious infections, until more clinical data become available. Oral options for the treatment of strains expressing CTX-M ESBLs are limited, with fosfomycin being the most reliably active agent (see section below on the treatment of extraintestinal E. coli infections).

1	AmpC β-lactamases, when induced or stably derepressed to high levels of expression, confer resistance to the same substrates as ESBLs plus the cephamycins (e.g., cefoxitin and cefotetan). The genes encoding these enzymes are primarily chromosomally located and therefore may not exhibit the linked or associated resistance to fluoroquinolones, TMP-SMX, aminoglycosides, and tetracyclines that is common with ESBLs. These enzymes are problematic for the clinician: resistance may develop during therapy with third-generation cephalosporins, resulting in clinical failure, particularly in the setting of bacteremia. Although chromosomal AmpC β-lactamases are present in nearly all members of the Enterobacteriaceae family, the risk of clinically significant induction of high expression levels or selection of stably derepressed mutants with cephalosporin treatment is greatest with Enterobacter cloacae and Enterobacter aerogenes, lower with Serratia marcescens and Citrobacter freundii, and lowest

1	of stably derepressed mutants with cephalosporin treatment is greatest with Enterobacter cloacae and Enterobacter aerogenes, lower with Serratia marcescens and Citrobacter freundii, and lowest with Providencia and Morganella morganii. In addition, rare strains of E. coli, K. pneumoniae, and other Enterobacteriaceae have acquired plasmids containing inducible AmpC β-lactamase genes. Carbapenems are a viable treatment option. The fourth-generation cephalosporin cefepime may be an appropriate option if the concomitant presence of an ESBL can be excluded and source control is achieved. Other carbapenem-sparing alternatives to consider if isolates are susceptible in vitro are fluoroquinolones, piperacillintazobactam, TMP-SMX, tigecycline, and aminoglycosides, although clinical data are limited.

1	Carbapenemases (e.g., KPC [class A]; NDM-1, VIM, and IMP [class B]; and OXA-48 [class D]) confer resistance to the same drugs as ESBLs and also to cephamycins and carbapenems. Similar to ESBLs, carbapenemases are usually encoded on large transferable plasmids, which often encode linked resistance to fluoroquinolones, TMP-SMX, tetracyclines, and aminoglycosides. Unfortunately, carbapenemase-producing Enterobacteriaceae are becoming increasingly common, particularly in Asia, and infection with these strains is associated with elevated mortality rates. This reality has prompted the Centers for Disease Control and Prevention (CDC) to categorize carbapenem-resistant Enterobacteriaceae as an “urgent threat” to health care. Carbapenemase production by Enterobacteriaceae is most prevalent in K. pneumoniae and E. coli but has been described in nearly all members of the family. Automated susceptibility systems may be unreliable for detection of carbapenemases. An elevated MIC or a diminished

1	pneumoniae and E. coli but has been described in nearly all members of the family. Automated susceptibility systems may be unreliable for detection of carbapenemases. An elevated MIC or a diminished zone diameter for meropenem or imipenem should prompt genotypic confirmation, if available. Alternatively, the phenotype can be confirmed with a modified Hodge test (which detects classes A, B, and D, although results can be false positive) and/or inhibition tests with boronic acid (class A), EDTA (class B), or dipicolinic acid (class B). Carbapenem resistance may also occur in the absence of carbapenemase production and can be mediated by AmpC β-lactamase and ESBL production coupled with modifications in permeability/efflux.

1	For treatment of carbapenem-resistant Enterobacteriaceae, tigecycline and colistin are the parenteral agents with the most reliable in vitro activity. However, because tigecycline reaches only low serum and urine concentrations, caution is warranted in using it to treat bacteremia and perhaps urinary tract infection (UTI), although a few case reports describe some success with tigecycline therapy for UTI. Colistin has nephrotoxic and neurotoxic potential. Furthermore, increasing resistance has been described to both of these agents. Thus the clinician is left with few or no therapeutic options. Aminoglycosides may have some utility if active. Fosfomycin is often active in vitro, but clinical data are limited, concerns exist about the development of resistance with monotherapy, and no parenteral formulation is available in the United States. Although control data are lacking, combination therapy is being used in this setting with the goals of increasing efficacy and decreasing the

1	parenteral formulation is available in the United States. Although control data are lacking, combination therapy is being used in this setting with the goals of increasing efficacy and decreasing the emergence of resistance.

1	Resistance to fluoroquinolones usually is due to alterations of the target site (DNA gyrase and/or topoisomerase IV), with or without decreased permeability, active efflux, or protection of the target site. Resistance to this drug class is increasingly prevalent among GNB and is associated with resistance to other antimicrobial classes; for example, 20–80% of ESBL-producing enteric GNB are also resistant to fluoroquinolones. At present, quinolones should be considered unreliable as empirical therapy for infections due to GNB in critically ill patients.

1	In this era of increasing antimicrobial resistance, it is critical to culture the local site of infection before the initiation of antimicrobial therapy and, for systemically ill patients, to obtain blood samples for culture. Antimicrobial resistance may not always be identified by in vitro testing; therefore, it is important to assess the clinical response to treatment. Moreover, as discussed above, resistance may emerge during therapy through the induction or stable derepression of AmpC β-lactamases. In addition, drainage of abscesses, resection of necrotic tissue, and removal of infected foreign bodies are often required for cure. GNB are commonly involved in polymicrobial infections, in which the role of each individual pathogen is uncertain (Chap. 201). Although some GNB are more pathogenic than others, 1027 it is usually prudent, if possible, to design an antimicrobial regimen active against all of the GNB identified, because each is capable of pathogenicity in its own right.

1	than others, 1027 it is usually prudent, if possible, to design an antimicrobial regimen active against all of the GNB identified, because each is capable of pathogenicity in its own right. Lastly, for patients treated initially with a broad-spectrum empirical regimen, the regimen should be de-escalated as expeditiously as possible once susceptibility results are known and the patient has responded to therapy.

1	(See also Chap. 168) Avoidance of inappropriate antimicrobial use is a key measure in preventing infections due to antimicrobial-resistant strains and the further development of antimicrobial resistance. Antimicrobial stewardship programs should be adopted to facilitate achievement of this goal. Diligent adherence to hand-hygiene protocols by health care personnel, cleaning/disinfection of objects that come into contact with patients (e.g., stethoscopes and blood pressure cuffs), and contact precautions should be implemented for patients colonized or infected with carbapenem-resistant (and perhaps other XDR) GNB. Avoidance of the use of indwelling devices (e.g., urinary and intravascular catheters, endotracheal tubes) and, when such devices are necessary, placement according to an appropriate protocol decrease infection risk. Likewise, protocols for daily use evaluation and removal as soon as possible should be implemented. Patient positioning (e.g., head of bed at ≥30°) and good oral

1	protocol decrease infection risk. Likewise, protocols for daily use evaluation and removal as soon as possible should be implemented. Patient positioning (e.g., head of bed at ≥30°) and good oral hygiene decrease the incidence of pneumonia among ventilated patients. Increasing data support the implementation of universal decolonization to prevent infection in ICU patients.

1	Strains of E. coli are united by a core genome of ~2000 genes. A strain’s ability to cause infections and the nature of such infections are defined by ancillary genes that encode various virulence factors. This experiment of nature is fluid and ongoing, as demonstrated by the recent evolution of Shiga toxin–producing enteroaggregative E. coli.

1	For the most part, commensal E. coli variants, which constitute the bulk of the normal facultative intestinal flora in most humans, confer benefits to the host (e.g., resistance to colonization with pathogenic organisms). These strains generally lack the specialized virulence traits that enable extraintestinal and intestinal pathogenic E. coli strains to cause disease outside and within the gastrointestinal tract, respectively. However, even commensal E. coli strains can be involved in extraintestinal infections in the presence of an aggravating factor, such as a foreign body (e.g., a urinary catheter), host compromise (e.g., local anatomic or functional abnormalities, such as urinary or biliary tract obstruction or systemic immunocompromise), or an inoculum that is large or contains a mixture of bacterial species (e.g., fecal contamination of the peritoneal cavity).

1	ExPEC strains are the most common enteric GNB to cause infections. The emerging propensity of these strains to acquire new antimicrobial resistance mechanisms (e.g., ESBL and carbapenemase production) has posed challenges in managing ExPEC infection. One clonal group—ST131, the members of which are usually resistant to fluoroquinolones and increasingly express an ESBL (CTX-M)—has undergone global dissemination. Like commensal E. coli (but in contrast to intestinal pathogenic

1	E. coli), ExPEC strains are often found in the intestinal flora of healthy individuals and do not cause gastroenteritis in humans. Entry from their site of colonization (e.g., the colon, vagina, or oropharynx) into a normally sterile extraintestinal site (e.g., the urinary tract, peritoneal cavity, or lungs) is the rate-limiting step for infection. ExPEC strains have acquired genes encoding diverse extraintestinal virulence factors that enable the bacteria to cause infections outside the gastrointestinal 1028 tract in both normal and compromised hosts (Table 186-1). These virulence genes define ExPEC and, for the most part, are distinct from those that enable intestinal pathogenic strains to cause diarrheal disease (Table 186-2). All age groups, all types of hosts, and nearly all organs and anatomic sites are susceptible to infection by ExPEC. Even previously healthy hosts can become severely ill or die when infected with ExPEC; however, adverse outcomes are more common among hosts

1	and anatomic sites are susceptible to infection by ExPEC. Even previously healthy hosts can become severely ill or die when infected with ExPEC; however, adverse outcomes are more common among hosts with comorbid illnesses and host defense abnormalities. The diversity and the medical and economic impact of ExPEC infections are evident from consideration of the following specific syndromes.

1	Extraintestinal Infectious Syndromes • Urinary tract infection The urinary tract is the site most frequently infected by ExPEC. An exceedingly common infection among ambulatory patients, UTI accounts for 1% of ambulatory care visits in the United States and is second only to lower respiratory tract infection among infections responsible for hospitalization. UTIs are best considered by clinical syndrome (e.g., uncomplicated cystitis, pyelonephritis, and catheter-associated UTIs) and within the context of specific hosts (e.g., premenopausal women, compromised hosts; Chap. 162). E. coli is the single most common pathogen for all UTI syndrome/host group combinations. Each year in the United States, E. coli causes 80–90% of an estimated 6–8 million episodes of uncomplicated cystitis in premenopausal women. Furthermore, 20% of women with an initial cystitis episode develop frequent recurrences.

1	Uncomplicated cystitis, the most common acute UTI syndrome, is characterized by dysuria, urinary frequency, and suprapubic pain. Fever and/or back pain suggests progression to pyelonephritis. Even with appropriate treatment of pyelonephritis, fever may take 5–7 days to resolve completely. Persistently elevated or increasing fever and neutrophil counts should prompt evaluation for intrarenal or perinephric abscess and/or obstruction. Renal parenchymal damage and loss of renal function during pyelonephritis occur primarily with urinary obstruction, which can be preexisting or, rarely, occurs de novo in diabetic patients who develop renal papillary necrosis as a result of kidney infection. Pregnant women are at unusually high risk for developing pyelonephritis, which can adversely affect the outcome of pregnancy. As a result, prenatal screening for and treatment of asymptomatic bacteriuria are standard. Prostatic infection is a potential complication of UTI in men. The diagnosis and

1	the outcome of pregnancy. As a result, prenatal screening for and treatment of asymptomatic bacteriuria are standard. Prostatic infection is a potential complication of UTI in men. The diagnosis and treatment of UTI, as detailed in Chap. 162, should be tailored to the individual host, the nature and site of infection, and local patterns of antimicrobial susceptibility.

1	abdominal and pelvic infection The abdomen/pelvis is the second most common site of extraintestinal infection due to E. coli. A wide variety of clinical syndromes occur in this location, including acute peritonitis secondary to fecal contamination, spontaneous bacterial peritonitis, dialysis-associated peritonitis, diverticulitis, appendicitis, intraperitoneal or visceral abscesses (hepatic, pancreatic, splenic), infected pancreatic pseudocysts, and septic cholangitis and/or cholecystitis. In intraabdominal infections, E. coli can be isolated either alone or (as often occurs) in combination with other facultative and/ or anaerobic members of the intestinal flora (Chap. 159).

1	pneUmonia E. coli is not usually considered a cause of pneumonia (Chap. 153). Indeed, enteric GNB account for only 1–3% of cases of community-acquired pneumonia, in part because these organisms only transiently colonize the oropharynx in a minority of healthy individuals. However, rates of oral colonization with E. coli and other GNB increase with severity of illness and antibiotic use. Consequently, GNB are a more common cause of pneumonia among residents of LTCFs and are the most common cause (60–70% of cases) of hospital-acquired pneumonia (Chap. 168), particularly among postoperative and ICU patients (e.g., ventilator-associated pneumonia). Pulmonary infection is usually acquired by small-volume aspiration but occasionally occurs via hematogenous spread, in which case multifocal nodular infiltrates can be seen. Tissue necrosis, probably due to bacterial cytotoxins, is common. Despite significant institutional variation, E. coli is generally the third or fourth most commonly

1	nodular infiltrates can be seen. Tissue necrosis, probably due to bacterial cytotoxins, is common. Despite significant institutional variation, E. coli is generally the third or fourth most commonly isolated GNB in hospital-acquired pneumonia, accounting for 5–8% of episodes in both U.S.-based and Europe-based studies. Regardless of the host, pneumonia due to ExPEC is a serious disease, with high crude and attributable mortality rates (20–60% and 10–20%, respectively).

1	meningitis (See also Chap. 164) E. coli is one of the two leading causes of neonatal meningitis, the other being group B Streptococcus. Most E. coli strains that cause neonatal meningitis possess the K1 capsular antigen and derive from a limited number of meningitis-associated clonal groups. Ventriculomegaly commonly occurs. After the first month of life, E. coli meningitis is uncommon, occurring predominantly in the setting of surgical or traumatic disruption of the meninges or in the presence of cirrhosis. In patients with cirrhosis who develop meningitis, the meninges are presumably seeded as a result of poor hepatic clearance of portal vein bacteremia.

1	cellUlitis/mUscUloskeletal infection E. coli contributes frequently to infections of decubitus ulcers and occasionally to infections of ulcers and wounds of the lower extremity in diabetic patients and other hosts with neurovascular compromise. Osteomyelitis secondary to contiguous spread can occur in these settings. E. coli also causes cellulitis or infections of burn sites and surgical wounds (accounting for ~10% of surgical site infections), particularly when the infection originates TABLE 186-2 InTEsTInAL PATHOgEnIC E. COLI STEC/EHEC/STEAEC Food, water, person-toperson; all ages, industrialized countries ETEC Food, water; young children in and travelers to developing countries EIEC Food, water; children in and travelers to developing countries EAEC ?Food, water; children in and travelers to developing countries; all ages, industrialized countries Hemorrhagic colitis, hemolytic-uremic syndrome Watery diarrhea, persistent diarrhea

1	EAEC ?Food, water; children in and travelers to developing countries; all ages, industrialized countries Hemorrhagic colitis, hemolytic-uremic syndrome Watery diarrhea, persistent diarrhea Traveler’s diarrhea, acute diarrhea, persistent diarrhea Shiga toxin Heat-stable and labile enterotoxins, colonization factors Localized adherence, attaching and effacing lesion on intestinal epithelium Invasion of colonic epithelial cells, intracellular multiplication, cell-to-cell spread Aggregative/diffuse adherence, virulence factors regulated by AggR Lambda-like Stx1or Stx2-encoding bacteriophage

1	Aggregative/diffuse adherence, virulence factors regulated by AggR Lambda-like Stx1or Stx2-encoding bacteriophage Abbreviations: EAEC, enteroaggregative E. coli; EHEC, enterohemorrhagic E. coli; EIEC, enteroinvasive E. coli; EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic E. coli; STEAEC, Shiga toxin–producing enteroaggregative E. coli; STEC, Shiga toxin–producing E. coli. a Classic syndromes; see text for details on disease spectrum. b Pathogenesis involves multiple genes, including genes in addition to those listed.

1	close to the perineum. Hematogenously acquired osteomyelitis, especially of vertebral bodies, is more commonly caused by E. coli than is generally appreciated; this organism accounts for up to 10% of cases in some series (Chap. 158). E. coli occasionally causes orthopedic device– associated infection or septic arthritis and rarely causes hematogenous myositis. Upper-leg myositis or fasciitis due to E. coli should prompt an evaluation for an abdominal source with contiguous spread. endovascUlar infection Despite being one of the most common causes of bacteremia, E. coli rarely seeds native heart valves. When the organism does seed native valves, it usually does so in the setting of prior valvular disease. E. coli infections of aneurysms, the portal vein (pylephlebitis), and vascular grafts are quite uncommon.

1	miscellaneoUs infections E. coli can cause infection in nearly every organ and anatomic site. It occasionally causes postoperative mediastinitis or complicated sinusitis and uncommonly causes endophthalmitis, ecthyma gangrenosum, or brain abscess.

1	bacteremia E. coli bacteremia can arise from primary infection at any extraintestinal site. In addition, primary E. coli bacteremia can arise from percutaneous intravascular devices or transrectal prostate biopsy or from the increased intestinal mucosal permeability seen in neonates and in the settings of neutropenia and chemotherapy-induced mucositis, trauma, and burns. Roughly equal proportions of E. coli bacteremia cases originate in the community and in health care settings. In most studies, E. coli and Staphylococcus aureus are the two most common blood isolates of clinical significance. Isolation of E. coli from the blood is almost always clinically significant and is typically accompanied by the sepsis syndrome, severe sepsis (sepsis-induced dysfunction of at least one organ or system), or septic shock (Chap. 325).

1	The urinary tract is the most common source of E. coli bacteremia, accounting for one-half to two-thirds of episodes. Bacteremia from a urinary tract source is particularly common among patients with pyelonephritis, urinary tract obstruction, or urinary instrumentation in the presence of infected urine. The abdomen is the second most common source, accounting for 25% of episodes. Although biliary obstruction (stones, tumor) and overt bowel disruption, which typically are readily apparent, are responsible for many of these cases, some abdominal sources (e.g., abscesses) are remarkably silent clinically and require identification via imaging studies (e.g., CT). Therefore, the physician should be cautious in designating the urinary tract as the source of E. coli bacteremia in the absence of characteristic signs and symptoms of UTI. Soft tissue, bone, pulmonary, and intravascular catheter infections are other sources of E. coli bacteremia.

1	Diagnosis Strains of E. coli that cause extraintestinal infections usually grow both aerobically and anaerobically within 24 h on standard diagnostic media and are easily identified by the clinical microbiology laboratory according to routine biochemical criteria. More than 90% of ExPEC strains are rapid lactose fermenters and are indole positive. TREATMEnT extraintestinal e. coli infections

1	In the past, most E. coli isolates were highly susceptible to a broad range of antimicrobial agents. Unfortunately, this situation has changed. In general, the high prevalence of resistance precludes empirical use of ampicillin and amoxicillin-clavulanate, even for community-acquired infections. The prevalence of resistance to first-generation cephalosporins and TMP-SMX is increasing among community-acquired strains in the United States (with current rates of 10–40%) and is even higher outside North America. Until recently, TMP-SMX was the drug of choice for the treatment of uncomplicated cystitis in many locales. Although continued empirical use of TMP-SMX will predictably result in ever-diminishing cure rates, a wholesale switch to alternative agents (e.g., fluoroquinolones) will just as predictably accelerate the widespread emergence of resistance to these antimicrobial classes, as has already occurred in some areas. More than 90% of isolates that cause uncomplicated cystitis

1	as predictably accelerate the widespread emergence of resistance to these antimicrobial classes, as has already occurred in some areas. More than 90% of isolates that cause uncomplicated cystitis remain susceptible to nitrofurantoin and fosfomycin.

1	The prevalence of resistance to fluoroquinolones among E. coli 1029 isolates from U.S. outpatients has increased steadily over the last decade (i.e., from 3% in 2000 to 17.1% in 2010, according to one survey). Resistance rates are generally higher in the ambulatory setting outside the United States and are even higher in populations for which fluoroquinolone prophylaxis is used extensively (e.g., patients with leukemia, transplant recipients, and patients with cirrhosis) and among isolates from LTCFs and hospitals. For example, the National Healthcare Safety Network (NHSN) reported fluoroquinolone resistance in 41.8% of central line–associated bloodstream infection (CLABSI) E. coli isolates in 2009–2010, and the International Nosocomial Infection Control Consortium (INICC) reported that 53.4% of ICU E. coli isolates were resistant to quinolones in 2004–2009. Furthermore, the NHSN reported 19% resistance to thirdand fourth-generation cephalosporins in CLABSI E. coli isolates, and the

1	53.4% of ICU E. coli isolates were resistant to quinolones in 2004–2009. Furthermore, the NHSN reported 19% resistance to thirdand fourth-generation cephalosporins in CLABSI E. coli isolates, and the INICC found that 66.6% of ICU E. coli isolates were resistant to third-generation cephalosporins.

1	ESBL-producing strains are increasingly prevalent among both health care–associated (5–10%) and ambulatory isolates (regiondependent figures). An increasing number of reports describe com-munity-acquired UTIs caused by E. coli strains that produce CTX-M ESBLs. Data suggest that acquisition of CTX-M-producing, fluoroquinolone-resistant strains may result from consumption of meat products from food animals treated with thirdand fourth-generation cephalosporins and fluoroquinolones. Oral treatment options for such strains are limited; however, in vitro and limited clinical data indicate that, for cystitis, fosfomycin and nitrofurantoin appear to be useful options. Carbapenems and amikacin are the most predictably active agents overall, but carbapenemase-producing strains are on the rise (1–5% among health care–associated isolates in the United States and higher rates in many other countries). Tigecycline and the polymyxins, with or without a second agent, have been used most frequently

1	among health care–associated isolates in the United States and higher rates in many other countries). Tigecycline and the polymyxins, with or without a second agent, have been used most frequently against these extremely resistant isolates.

1	This evolving antimicrobial resistance—a source of serious concern—necessitates not only the increasing use of broad-spectrum agents but also the use of the most appropriate narrower-spectrum agent whenever possible and the avoidance of treatment of colonized but uninfected patients.

1	INTESTINAL PATHOGENIC STRAINS Pathotypes Certain strains of E. coli are capable of causing diarrheal disease. Other important intestinal pathogens are discussed in Chaps. 160, 161, and 190–193. At least in the industrialized world, intestinal pathogenic strains of E. coli are rarely encountered in the fecal flora of healthy persons and instead appear to be essentially obligate pathogens. These strains have evolved a special ability to cause enteritis, enterocolitis, and colitis when ingested in sufficient quantities by a naive host. At least five distinct pathotypes of intestinal pathogenic E. coli exist: (1) Shiga toxin–producing E. coli (STEC), which includes the subsets of enterohemorrhagic E. coli (EHEC) and the recently evolved Shiga toxin–producing enteroaggregative E. coli (STEAEC); (2) enterotoxigenic E. coli (ETEC); (3) enteropathogenic

1	E. coli (EPEC); (4) enteroinvasive E. coli (EIEC); and (5) enteroaggregative E. coli (EAEC). Diffusely adherent E. coli (DAEC) and cytodetaching E. coli are additional putative pathotypes. Lastly, a variant termed adherent invasive E. coli (AIEC) has been associated with Crohn’s disease (although a causal role remains unproven) but does not cause acute diarrheal disease. Transmission occurs predominantly via contaminated food and water for ETEC, STEC/EHEC/STEAEC, EIEC, and EAEC and by person-to-person spread for EPEC (and occasionally STEC/EHEC/STEAEC). Gastric acidity confers some protection against infection; therefore, persons with decreased stomach acid levels are especially susceptible. Humans are the major reservoir (except for STEC/EHEC, with regard to which bovines are the main concern); host range appears to be dictated by species-specific attachment factors. Although there is some overlap, each pathotype possesses a largely unique combination of virulence traits that results

1	host range appears to be dictated by species-specific attachment factors. Although there is some overlap, each pathotype possesses a largely unique combination of virulence traits that results in a distinctive intestinal pathogenic mechanism (Table 186-2). These strains are largely incapable of causing disease outside the 1030 intestinal tract. Except in the cases of STEC/EHEC/STEAEC and EAEC, disease due to this group of pathogens occurs primarily in developing countries.

1	enteroHemorrHagic e. coli/sHiga toXin–prodUcing enteroaggregative e. coli STEC/EHEC/STEAEC strains constitute an emerging group of pathogens that can cause hemorrhagic colitis and the hemolytic-uremic syndrome (HUS). Several large outbreaks resulting from the consumption of fresh produce (e.g., lettuce, spinach, sprouts) and of undercooked ground beef have received significant attention in the media. An outbreak in central Europe in 2011 due to STEAEC (O104:H4) that was probably transmitted by sprouts, with some subsequent human-to-human transmission, resulted in more than 800 cases of HUS and 54 deaths. Within this group of organisms, O157:H7 is the most prominent serotype, but many other serotypes have also been associated with these syndromes, including O6, O26, O45, O55, O91, O103, O111, O113, O121, O145, and OX3.

1	The ability of STEC/EHEC/STEAEC to produce Shiga toxin (Stx2 and/or Stx1) or related toxins is a critical factor in the occurrence of clinical disease. Shigella dysenteriae strains that produce the closely related Shiga toxin Stx can cause the same syndrome. Stx2 and its Stx2C variant (which may be variably present in combination with Stx2 and/or Stx1) appear to be more important than Stx1 in the development of HUS. All Shiga toxins studied to date are multimers comprising one enzymatically active A subunit and five identical B subunits that mediate binding to globosyl ceramides, which are membrane-associated glycolipids expressed on certain host cells. As in ricin, the A subunit cleaves an adenine from the host cell’s 28S rRNA, thereby irreversibly inhibiting ribosomal function and potentially leading to apoptosis. Stx2-mediated activation of complement may also play a role in the development of HUS.

1	Additional properties, such as acid tolerance and epithelial cell adherence, are necessary for full pathogenicity among STEC strains. Most disease-causing isolates possess the chromosomal locus for enterocyte effacement (LEE). This pathogenicity island was first described in EPEC strains and contains genes that mediate adherence to intestinal epithelial cells and a system that subverts host cells by the translocation of bacterial proteins (type III secretion system). EHEC strains make up the subgroup of STEC strains that possess stx1 and/ or stx2 as well as LEE. STEAEC (LEE-negative) evolved from EAEC via the acquisition of a number of genes, including those that encode Stx2, the Iha adhesin, tellurite resistance, a type VI secretion system, and the CTX-M-15 ESBL.

1	Domesticated ruminant animals, particularly cattle and young calves, serve as the major reservoir for STEC/EHEC. Ground beef—the most common food source of STEC/EHEC strains—is often contaminated during processing. Furthermore, manure from cattle or other animals (including that in the form of fertilizer) can contaminate produce (potatoes, lettuce, spinach, sprouts, fallen fruits, nuts, strawberries), and fecal runoff from this source can contaminate water systems. Dairy products and petting zoos are additional sources of infection. By contrast, humans appear to be the reservoir for STEAEC. It is estimated that <102 colony-forming units (CFU) of STEC/EHEC/STEAEC can cause disease. Therefore, not only can low levels of food or environmental contamination (e.g., in water swallowed while swimming) result in disease, but person-to-person transmission (e.g., at day-care centers and in institutions) is an important route for secondary spread. Laboratory-associated infections also occur.

1	swimming) result in disease, but person-to-person transmission (e.g., at day-care centers and in institutions) is an important route for secondary spread. Laboratory-associated infections also occur. Illness due to this group of pathogens occurs both as outbreaks and as sporadic cases, with a peak incidence in the summer months.

1	In contrast to other intestinal pathotypes, STEC/EHEC/ STEAEC causes infections more frequently in industrialized countries than in developing regions. O157:H7 strains are the fourth most commonly reported cause of bacterial diarrhea in the United States (after Campylobacter, Salmonella, and Shigella). Colonization of the colon and perhaps the ileum results in symptoms after an incubation period of 3 or 4 days. Colonic edema and an initial nonbloody secretory diarrhea may develop into the STEC/EHEC/ STEAEC hallmark syndrome of grossly bloody diarrhea (identified by history or examination) in >90% of cases. Significant abdominal pain and fecal leukocytes are common (70% of cases), whereas fever is not; absence of fever can incorrectly lead to consideration of noninfectious conditions (e.g., intussusception and inflammatory or ischemic bowel disease). Occasionally, infections caused by C. difficile, K. oxytoca (see “Klebsiella Infections,” below), Campylobacter, and Salmonella present

1	intussusception and inflammatory or ischemic bowel disease). Occasionally, infections caused by C. difficile, K. oxytoca (see “Klebsiella Infections,” below), Campylobacter, and Salmonella present in a similar fashion. STEC/EHEC disease is usually self-limited, lasting 5–10 days. An uncommon but feared complication of this infection is HUS, which occurs 2–14 days after diarrhea in 2–8% of cases, most often affecting very young or elderly patients. Distinctive features of STEAEC infection, as compared with classical STEC/EHEC disease, include a higher incidence among adults, especially young women, and a higher rate of HUS (~20%). It is estimated that >50% of all cases of HUS in the United States and 90% of HUS cases in children are caused by STEC/EHEC. This complication is mediated by the systemic translocation of Shiga toxins. Erythrocytes may serve as carriers of Stx to endothelial cells located in the small vessels of the kidney and brain. The subsequent development of thrombotic

1	systemic translocation of Shiga toxins. Erythrocytes may serve as carriers of Stx to endothelial cells located in the small vessels of the kidney and brain. The subsequent development of thrombotic microangiopathy (perhaps with direct toxin-mediated effects on various nonendothelial cells) commonly produces some combination of fever, thrombocytopenia, renal failure, and encephalopathy. Although the mortality rate with dialysis support is <10%, residual renal and neurologic dysfunction may persist.

1	enterotoXigenic e. coli In tropical or developing countries,

1	ETEC is a major cause of endemic diarrhea. After weaning, children in these locales commonly experience several episodes of ETEC infection during the first 3 years of life. The incidence of disease diminishes with age, a pattern that correlates with the development of mucosal immunity to colonization factors (i.e., adhesins). In industrialized countries, infection usually follows travel to endemic areas, although occasional food-borne outbreaks occur. ETEC is the most common agent of traveler’s diarrhea, causing 25–75% of cases. The incidence of infection may be decreased by prudent avoidance of potentially contaminated fluids and foods, particularly items that are poorly cooked, unpeeled, or unrefrigerated (Chap. 149). ETEC infection is uncommon in the United States, but outbreaks secondary to consumption of food products imported from endemic areas have occurred. A large inoculum (106–1010 CFU) is needed to produce disease, which usually develops after an incubation period of 12–72

1	to consumption of food products imported from endemic areas have occurred. A large inoculum (106–1010 CFU) is needed to produce disease, which usually develops after an incubation period of 12–72 h.

1	After adherence of ETEC via colonization factors (e.g., CFA/I, CS1-6), disease is mediated primarily by a heat-labile toxin (LT-1) and/ or a heat-stable toxin (STa) that causes net fluid secretion via activation of adenylate cyclase (LT-1) and/or guanylate cyclase (STa) in the jejunum and ileum. The result is watery diarrhea accompanied by cramps. LT-1 consists of an A and a B subunit and is structurally and functionally similar to cholera toxin. Strong binding of the B subunit to the GM1 ganglioside on intestinal epithelial cells leads to the intracellular translocation of the A subunit, which functions as an ADPribosyltransferase. Mature STa is an 18or 19-amino-acid secreted peptide whose biologic activity is mediated by binding to the guanylate cyclase C found in the brush-border membrane of enterocytes and results in increased intracellular concentrations of cyclic GMP. Characteristically absent in ETEC-mediated disease are histopathologic changes within the small bowel; mucus,

1	of enterocytes and results in increased intracellular concentrations of cyclic GMP. Characteristically absent in ETEC-mediated disease are histopathologic changes within the small bowel; mucus, blood, and inflammatory cells in stool; and fever. The disease spectrum ranges from a mild illness to a life-threatening cholera-like syndrome. Although symptoms are usually self-limited (typically lasting for 3 days), infection may result in significant morbidity and mortality (mostly from profound volume depletion) when access to health care or suitable rehydration fluids is limited and when small and/or undernourished children are affected.

1	enteropatHogenic e. coli EPEC causes disease primarily in young children, including neonates. The first E. coli pathotype recognized as an agent of diarrheal disease, EPEC was responsible for outbreaks of infantile diarrhea (including some outbreaks in hospital nurseries) in industrialized countries in the 1940s and 1950s. At present, EPEC infection is an uncommon cause of diarrhea in developed countries but is an important cause of diarrhea (both sporadic and epidemic) among infants in developing countries. Breast-feeding diminishes the incidence of EPEC infection. Rapid person-to-person spread may occur. Upon colonization of the small bowel, symptoms develop after a brief incubation period (1 or 2 days). Initial localized adherence via bundle-forming pili leads to a characteristic effacement of microvilli, with the formation of cuplike, actin-rich pedestals mediated by factors in the LEE. Diarrhea production is a complex and regulated process in which host cell modulation by a type

1	of microvilli, with the formation of cuplike, actin-rich pedestals mediated by factors in the LEE. Diarrhea production is a complex and regulated process in which host cell modulation by a type III secretion system plays an important role. Strains lacking bundle-forming pili have been categorized as atypical EPEC (aEPEC); increasing data support a role for these strains as intestinal pathogens. Diarrheal stool often contains mucus but not blood. Although EPEC diarrhea is usually self-limited (lasting 5–15 days), it may persist for weeks.

1	enteroinvasive e. coli EIEC, a relatively uncommon cause of diarrhea, is rarely identified in the United States, although a few food-related outbreaks have been described. In developing countries, sporadic disease is infrequently recognized in children and travelers. EIEC shares many genetic and clinical features with Shigella, both of which evolved from a common ancestor. However, unlike Shigella, EIEC produces disease only with a large inoculum (108–1010 CFU), with onset generally following an incubation period of 1–3 days. Initially, enterotoxins are believed to induce secretory small-bowel diarrhea. Subsequently, colonization and invasion of the colonic mucosa, followed by replication therein and cell-to-cell spread, result in the development of inflammatory colitis characterized by fever, abdominal pain, tenesmus, and scant stool containing mucus, blood, and inflammatory cells. Symptoms are usually self-limited (7–10 days).

1	enteroaggregative and diffUsely adHerent e. coli EAEC has young children. However, recent studies indicate that it may be a relatively common cause of diarrhea in all age groups in industrialized countries. EAEC has also been recognized increasingly as an important cause of traveler’s diarrhea. It is highly adapted to humans, the probable reservoir. A large inoculum is required for infection, which usually manifests as watery and sometimes persistent diarrhea in healthy, malnourished, and HIV-infected hosts. In vitro, the organisms exhibit a diffuse or “stacked-brick” pattern of adherence to small-intestine epithelial cells. Virulence factors that probably are necessary for disease are regulated in part by the transcriptional activator AggR and include the aggregative adherence fimbriae (AAF/I-III); the Hda adhesin; the mucinase Pic; the enterotoxins Pet, EAST-1, ShET1, and HlyE; and dispersin, an antiaggregation protein that promotes mucosal spread. Some strains of DAEC are capable

1	(AAF/I-III); the Hda adhesin; the mucinase Pic; the enterotoxins Pet, EAST-1, ShET1, and HlyE; and dispersin, an antiaggregation protein that promotes mucosal spread. Some strains of DAEC are capable of causing diarrheal disease, primarily in children 2–6 years of age in some developing countries, and may perhaps cause traveler’s diarrhea. The Afa/Dr adhesins may contribute to the pathogenesis of such infections.

1	Diagnosis A practical approach to the evaluation of diarrhea is to distinguish noninflammatory from inflammatory cases; the latter is suggested by grossly bloody or mucoid stool or a positive test for fecal leukocytes (Chap. 160). ETEC, EPEC, and DAEC cause noninflammatory diarrhea and are uncommon in the United States; in this country, the incidence of EAEC infection, which also causes noninflammatory diarrhea, may be underrecognized. The diagnosis of these infections requires specialized assays (e.g., polymerase chain reaction–based tests for pathotype-specific genes) that are not routinely available and are rarely needed because the diseases are self-limited. ETEC causes the majority and EAEC a minority of cases of noninflammatory traveler’s diarrhea. Definitive diagnosis generally is not necessary. Empirical antimicrobial (or symptom-based) treatment, along with rehydration therapy, is a reasonable approach. If diarrhea persists for >10 days despite treatment, Giardia or

1	is not necessary. Empirical antimicrobial (or symptom-based) treatment, along with rehydration therapy, is a reasonable approach. If diarrhea persists for >10 days despite treatment, Giardia or Cryptosporidium (or, in immunocompromised hosts, certain other microbial agents) should be sought. The diagnosis of infection with EIEC, a rare cause of inflammatory diarrhea in the United States, also requires specialized assays. The CDC now recommends that all patients with community-acquired diarrhea, whether inflammatory or not, be evaluated for STEC/EHEC/STEAEC infection by simultaneous culture (which is important for outbreak detection and control) and assay for the detection of Shiga toxin or its associated genes. The reasons for this recommendation are that bloody stool is not always present and detection of fecal white blood cells is 1031 not optimally sensitive for the diagnosis of STEC/EHEC/STEAEC infection. The use of both tests increases the rate of identification of infection over

1	and detection of fecal white blood cells is 1031 not optimally sensitive for the diagnosis of STEC/EHEC/STEAEC infection. The use of both tests increases the rate of identification of infection over rates obtained with either test alone. O157 STEC/EHEC may be identified via culture by screening for E. coli strains that do not ferment sorbitol, with subsequent serotyping and testing for Shiga toxin. Selective or screening media are not available for the culture of non-O157 strains. Detection of Shiga toxins or toxin genes via DNA-based, enzyme-linked immunosorbent, and cytotoxicity assays offers the advantages of rapidity plus detection of non-O157 STEC/EHEC/ STEAEC strains. Specimens positive for toxin but culture-negative for O157 should be forwarded to the local or state public health laboratory.

1	TREATMEnT intestinal e. coli infections (See also Chap. 128) The mainstay of treatment for all diarrheal syndromes is replacement of water and electrolytes. This measure is especially important for STEC/EHEC/STEAEC infection because appropriate volume expansion may decrease renal damage and improve outcome. The use of prophylactic antibiotics to prevent traveler’s diarrhea generally should be discouraged, especially in light of high rates of antimicrobial resistance. However, in selected patients (e.g., those who cannot afford a brief illness or are predisposed to infection), the use of rifaximin, which is nonabsorbable and is well tolerated, is reasonable. When stools are free of mucus and blood, early patient-initiated treatment of traveler’s diarrhea with a fluoroquinolone or azithromycin decreases the duration of illness, and the use of loperamide may halt symptoms within a few hours. Although dysentery caused by EIEC is self-limited, treatment hastens the resolution of symptoms,

1	decreases the duration of illness, and the use of loperamide may halt symptoms within a few hours. Although dysentery caused by EIEC is self-limited, treatment hastens the resolution of symptoms, particularly in severe cases. In contrast, antimicrobial therapy for STEC/ EHEC/STEAEC infection (the presence of which is suggested by grossly bloody diarrhea without fever) should be avoided because antibiotics may increase the incidence of HUS (possibly via increased production/ release of Stx). The role of plasmapheresis and inhibition of C5 (eculizumab) in the treatment of HUS is unresolved.

1	K. pneumoniae is the most important Klebsiella species from a medical standpoint, causing community-acquired, LTCF-acquired, and nosocomial infections. K. oxytoca is primarily a pathogen in LTCF and hospital settings. Klebsiella species are broadly prevalent in the environment and colonize mucosal surfaces of mammals. In healthy humans, the prevalence of K. pneumoniae colonization is 5–35% in the colon and 1–5% in the oropharynx; the skin is usually colonized only transiently. Person-to-person spread is the predominant mode of acquisition. Most Klebsiella infections in Western countries are caused by “classic” K. pneumoniae (cKP) and occur in hospitals and LTCFs. The most common clinical syndromes due to cKP are pneumonia, UTI, abdominal infection, intravascular device infection, surgical site infection, soft tissue infection, and subsequent bacteremia. cKP strains have gained notoriety because their propensity for acquiring antimicrobial resistance determinants makes treatment

1	site infection, soft tissue infection, and subsequent bacteremia. cKP strains have gained notoriety because their propensity for acquiring antimicrobial resistance determinants makes treatment challenging. Clonal group ST258, many members of which produce the KPC carbapenemase, is undergoing international dissemination. The spread of NDM-1 carbapenemase-producing strains from India in association with medical tourism has captured the attention of physicians and the lay press.

1	cKP strains appear to be phenotypically and clinically distinct from hypervirulent K. pneumoniae (hvKP), an emerging variant that was first recognized in Taiwan in 1986. Although hvKP infections have occurred globally in all ethnic groups, the majority have been reported in the Asian Pacific Rim. This concentration of cases raises the question of whether a geo-specific distribution of the organism or increased susceptibility of Asian hosts is responsible. In contrast to the usual health care–associated venue for cKP infections in the West, hvKP causes serious lifeand organ-threatening infections in younger, healthy individuals from the community and can spread metastatically 1032 from primary sites of infection. hvKP infection initially was characterized and distinguished from traditional infections due to cKP by presentation as community-acquired pyogenic liver abscess (Fig. 186-1, top), (2) occurrence in patients lacking a history of hepatobiliary disease, and (3) a propensity for

1	infections due to cKP by presentation as community-acquired pyogenic liver abscess (Fig. 186-1, top), (2) occurrence in patients lacking a history of hepatobiliary disease, and (3) a propensity for metastatic spread to distant sites (e.g., eyes, central nervous system, lungs), which occurred in 11–80% of cases. More recently, this variant has been recognized as the cause of a variety of serious community-acquired extrahepatic abscesses/ infections in the absence of liver involvement, including pneumonia, meningitis, endophthalmitis (Fig. 186-1, middle), splenic abscess, and necrotizing fasciitis. The affected individuals often have diabetes mellitus and are of Asian ethnicity; however, nondiabetics and all ethnic groups can be affected. Survivors often suffer catastrophic morbidity, such as loss of vision and neurologic sequelae.

1	K. pneumoniae subspecies rhinoscleromatis is the causative agent of rhinoscleroma, a granulomatous mucosal upper respiratory infection that progresses slowly (over months or years) and causes necrosis and occasionally obstruction of the nasal passages. K. pneumoniae subspecies ozaenae has been implicated as a cause of chronic atrophic rhinitis and rarely of invasive disease in compromised hosts. These two K. pneumoniae subspecies are usually isolated from patients in tropical climates and are genomically distinct from both cKP and hvKP.

1	INFECTIOuS SYNDROMES Pneumonia Although cKP accounts for only a small proportion of cases of community-acquired pneumonia in Western countries (Chap. 153), cKP and K. oxytoca are common causes of pneumonia among LTCF residents and hospitalized patients because of increased rates of oropharyngeal colonization. Mechanical ventilation is an important risk factor. In Asia and South Africa, community-acquired pneumonia due to hvKP is becoming increasingly common and often occurs in younger patients with no underlying disease. Klebsiella is also a common cause of pneumonia in severely malnourished children in developing countries.

1	As in all pneumonias due to enteric GNB, production of purulent sputum and evidence of airspace disease are typical. Presentation with earlier, less extensive infection is now more common than that with the classically described lobar infiltrate and bulging fissure. Pulmonary infection due to hvKP that has spread metastatically (e.g., from a hepatic abscess) usually includes nodular bilateral densities, more commonly in the lower lobes. Pulmonary necrosis, pleural effusion, and empyema can occur with disease progression. uTI cKP accounts for only 1–2% of UTI episodes among otherwise healthy adults but for 5–17% of episodes of complicated UTI, including infections associated with indwelling urinary catheters. UTI due to hvKP presents more commonly as renal or prostatic abscess due to bacteremic spread than as ascending infection.

1	Abdominal Infection cKP causes a spectrum of abdominal infections similar to that caused by E. coli but is less frequently isolated from these infections. hvKP is a common cause of monomicrobial community-acquired pyogenic liver abscess and in the Asian Pacific Rim has been recovered with steadily increasing frequency over the past two decades, replacing E. coli as the most common pathogen causing this syndrome. hvKP is increasingly described as a cause of spontaneous bacterial peritonitis and splenic abscess.

1	Other Infections cKPand K. oxytoca–mediated cellulitis or soft tissue infection most frequently affects devitalized tissue (e.g., decubitus and diabetic ulcers, burn sites) and immunocompromised hosts. cKP and K. oxytoca cause some cases of surgical site infection and nosocomial sinusitis in addition to occasional cases of osteomyelitis contiguous to soft tissue infection, nontropical myositis, and meningitis (both during the neonatal period and after neurosurgery). By contrast, hvKP has become an important cause of community-acquired

1	FIGuRE 186-1 New hypervirulent variant of K. pneumoniae (hvKP). Top: Abdominal CT scan of a previously healthy 24-year-old Vietnamese man shows a primary liver abscess (red arrow) with metastatic spread to the spleen (black arrow). (Courtesy of Drs. Chiu-Bin Hsaio and Diana Pomakova.) Middle: A previously healthy 33-year-old Chinese man presented with endophthalmitis. (From Virulence 4:2, 1-12 Feb. 15, 2013.) Bottom: A hypermucoviscous phenotype (which does not necessarily equate with a mucoid phenotype) has been associated with hvKP strains. This phenotype has been semiquantitatively defined by a positive “string test” (formation of a viscous string >5 mm long when bacterial colonies on an agar plate are stretched by an inoculation loop). (Courtesy of Dr. Russo.) monomicrobial necrotizing fasciitis; meningitis; brain, subdural, and epidural abscess; and endophthalmitis (Fig. 186-1, middle), particularly in the Asian Pacific Rim but also globally. Cytotoxin-producing strains of K.

1	fasciitis; meningitis; brain, subdural, and epidural abscess; and endophthalmitis (Fig. 186-1, middle), particularly in the Asian Pacific Rim but also globally. Cytotoxin-producing strains of K. oxytoca have been implicated as a cause of hemorrhagic antibiotic-associated non–C. difficile colitis.

1	Bacteremia Klebsiella infection at any site can produce bacteremia. Infections of the urinary tract, respiratory tract, and abdomen (especially hepatic abscess) each account for 15–30% of episodes of Klebsiella bacteremia. Intravascular device–related infections account for another 5–15% of episodes, and surgical site and miscellaneous infections account for the rest. Klebsiella is a cause of sepsis in neonates and of bacteremia in neutropenic patients. Like enteric GNB in general, Klebsiella rarely causes endocarditis or endovascular infection. Klebsiellae are readily isolated and identified in the laboratory. These organisms usually ferment lactose, although the subspecies rhinoscleromatis and ozaenae are nonfermenters and are indole negative. hvKP usually possesses a hypermucoviscous phenotype (Fig. 186-1, bottom), although the sensitivity and specificity of this test are undefined and probably less than optimal. A better diagnostic test for hvKP is desirable.

1	cKP and K. oxytoca have similar antibiotic resistance profiles. These species are intrinsically resistant to ampicillin and ticarcillin, and nitrofurantoin is inconsistently active against them. NHSN data for 2009–2010 documented resistance to thirdand fourth-generation cephalosporins in 28.9% of CLABSI isolates of cKP and K. oxytoca, and INICC data for 2004–2009 identified resistance to third-generation cephalosporins in 76.3% of ICU isolates of K. pneumoniae. This increasing resistance is mediated primarily by plasmid-encoded ESBLs. In addition, such plasmids usually encode resistance to aminoglycosides, tetracyclines, and TMP-SMX. Furthermore, isolates of cKP that produce CTX-M ESBLs have been obtained from ambulatory patients with no recent health care contact (see the section on the treatment of extraintestinal E. coli infections for treatment considerations). Resistance to β-lactam/β-lactamase inhibitor combinations and cephamycins independent of ESBL-encoding plasmids has also

1	treatment of extraintestinal E. coli infections for treatment considerations). Resistance to β-lactam/β-lactamase inhibitor combinations and cephamycins independent of ESBL-encoding plasmids has also been described with increasing frequency, particularly in Latin America. The prevalence of fluoroquinolone resistance is 15–20% overall and is 50% among ESBL-containing strains. Given both the undesirability of treating the latter strains with penicillins or cephalosporins and the fluoroquinolone resistance often associated with ESBLs, empirical treatment of serious or health care–associated cKP and K. oxytoca infections with amikacin or carbapenems is prudent, as dictated by local susceptibilities. Predictably, however, the ESBL-driven use of carbapenems has selected for strains of cKP and

1	K. oxytoca that express carbapenemases. NHSN data for 2009–2010 documented resistance to carbapenems in 12.8% of CLABSI isolates of cKP and K. oxytoca. Treatment of infections due to strains that produce carbapenemases is highly challenging; increasingly, these strains are nearly pan-resistant. The optimal choice for therapy is unclear. Tigecycline and the polymyxins (e.g., colistin) are the most active agents in vitro and are used most frequently. However, resistance to these agents is already emerging, and strains of cKP resistant to all known antimicrobial agents have been described in the United States and globally. Combination therapy is often used in this setting.

1	Proteus mirabilis causes 90% of Proteus infections, which occur in the community, LTCFs, and hospitals. Proteus vulgaris and Proteus penneri are associated primarily with infections acquired in LTCFs or hospitals. Proteus species are part of the colonic flora of a wide variety of mammals, birds, fish, and reptiles. The ability of these GNB to generate histamine from contaminated fish has implicated them in the pathogenesis of 1033 scombroid (fish) poisoning (Chap. 474). P. mirabilis colonizes healthy humans (prevalence, 50%), whereas P. vulgaris and P. penneri are isolated primarily from individuals with underlying disease. The urinary tract is by far the most common site of Proteus infection, with adhesins, flagella, IgA-IgG protease, iron acquisition systems, and urease representing the principal known urovirulence factors. Proteus less commonly causes infection at a variety of other extraintestinal sites.

1	INFECTIOuS SYNDROMES uTI Most Proteus infections arise from the urinary tract. P. mirabilis causes only 1–2% of UTIs in healthy women, and Proteus species collectively cause only 5% of hospital-acquired UTIs. However, Proteus is responsible for 10–15% of cases of complicated UTI, primarily those associated with catheterization; indeed, among UTI isolates from chronically catheterized patients, the prevalence of Proteus is 20–45%. This high prevalence is due in part to bacterial production of urease, which hydrolyzes urea to ammonia and results in alkalization of the urine. Alkalization of urine, in turn, leads to precipitation of organic and inorganic compounds, which contributes to formation of struvite and carbonate-apatite crystals, formation of biofilms on catheters, and/or development of frank calculi. Proteus becomes associated with the stones and biofilms; thereafter, it usually can be eradicated only by removal of the stones or the catheter. Over time, staghorn calculi may

1	of frank calculi. Proteus becomes associated with the stones and biofilms; thereafter, it usually can be eradicated only by removal of the stones or the catheter. Over time, staghorn calculi may form within the renal pelvis and lead to obstruction and renal failure. Thus, urine samples with unexplained alkalinity should be cultured for Proteus, and identification of a Proteus species in urine should prompt consideration of an evaluation for urolithiasis.

1	Other Infections Proteus occasionally causes pneumonia (primarily in LTCF residents or hospitalized patients), nosocomial sinusitis, intraabdominal abscesses, biliary tract infection, surgical site infection, soft tissue infection (especially decubitus and diabetic ulcers), and osteomyelitis (primarily contiguous); in rare cases, it causes nontropical myositis. In addition, Proteus uncommonly causes neonatal meningitis, with the umbilicus frequently implicated as the source; this disease is often complicated by development of a cerebral abscess. Otogenic brain abscess also occurs. Bacteremia The majority of Proteus bacteremia episodes originate from the urinary tract; however, any of the less common sites of infection as well as intravascular devices are also potential sources. Endovascular infection is rare. Proteus species are occasional agents of sepsis in neonates and of bacteremia in neutropenic patients.

1	Proteus is readily isolated and identified in the laboratory. Most strains are lactose negative, produce H2S, and demonstrate characteristic swarming motility on agar plates. P. mirabilis and P. penneri are indole negative, whereas P. vulgaris is indole positive. The inability to produce ornithine decarboxylase differentiates P. penneri from P. mirabilis.

1	P. mirabilis is usually susceptible to most antimicrobial agents except tetracycline, nitrofurantoin, the polymyxins, and tigecycline. Resistance to ampicillin and first-generation cephalosporins has been acquired by 10–50% of strains. Overall, 10–15% of P. mirabilis isolates are resistant to fluoroquinolones; 5% of isolates in the United States now produce ESBLs. Furthermore, isolates of P. mirabilis that produce CTX-M ESBLs have been recovered from ambulatory patients with no recent health care contact (see the section on the treatment of extraintestinal E. coli infections for treatment considerations). P. vulgaris and P. penneri exhibit more extensive drug resistance than does P. mirabilis. Resistance to ampicillin and first-generation cephalosporins is the rule, and 30–40% of isolates are resistant to fluoroquinolones. Induction or selection of variants with stable derepression of chromosomal AmpC β-lactamase may 1034 occur with P. vulgaris isolates. Carbapenems, fourth-generation

1	are resistant to fluoroquinolones. Induction or selection of variants with stable derepression of chromosomal AmpC β-lactamase may 1034 occur with P. vulgaris isolates. Carbapenems, fourth-generation cephalosporins (e.g., cefepime), amikacin, TMP-SMX, and fosfomycin display excellent activity against Proteus species (90–100% of isolates susceptible).

1	E. cloacae and E. aerogenes are responsible for most Enterobacter infections (65–75% and 15–25%, respectively); Cronobacter sakazakii (formerly Enterobacter sakazakii) and Enterobacter gergoviae are less commonly isolated (1% for each). Enterobacter species cause primarily health care–related infections. The organisms are widely prevalent in foods, environmental sources (including equipment at health care facilities), and a variety of animals. Few healthy humans are colonized, but the percentage increases significantly with LTCF residence or hospitalization. Although colonization is an important prelude to infection, direct introduction via IV lines (e.g., contaminated IV fluids or pressure monitors) also occurs. Extensive antibiotic resistance has developed in Enterobacter species and probably has contributed to the emergence of the organisms as prominent nosocomial pathogens. Individuals who have previously received antibiotic treatment, have comorbid disease, and are ICU residents

1	has contributed to the emergence of the organisms as prominent nosocomial pathogens. Individuals who have previously received antibiotic treatment, have comorbid disease, and are ICU residents are at greatest risk for infection. Enterobacter causes a spectrum of extraintestinal infections similar to that described for other GNB.

1	Pneumonia, UTI (particularly catheter-related), intravascular device– related infection, surgical site infection, and abdominal infection (primarily postoperative or related to devices such as biliary stents) are the most common syndromes encountered. Nosocomial sinusitis, meningitis related to neurosurgical procedures (including use of intra-cranial pressure monitors), osteomyelitis, and endophthalmitis after eye surgery are less frequent. C. sakazakii is associated with neonatal bacteremia, necrotizing enterocolitis, and meningitis (which is often complicated by brain abscess or ventriculitis); contaminated formula has been implicated as a source for such infections. Enterobacter bacteremia can result from infection at any anatomic site. In bacteremia of unclear origin, the contamination of IV fluids or medications, blood components or plasma derivatives, catheter-flushing fluids, pressure monitors, and dialysis equipment should be considered, particularly in an outbreak setting.

1	of IV fluids or medications, blood components or plasma derivatives, catheter-flushing fluids, pressure monitors, and dialysis equipment should be considered, particularly in an outbreak setting. Enterobacter can also cause bacteremia in neutropenic patients. Enterobacter endocarditis is rare, occurring primarily in association with illicit IV drug use or prosthetic valves.

1	Enterobacter is readily isolated and identified in the laboratory. Most strains are lactose positive and indole negative.

1	Enterobacter strains. Ampicillin and firstand second-gener ation cephalosporins have little or no activity. Extensive use of third-generation cephalosporins can induce or select for variants with stable derepression of AmpC β-lactamase, which confers resistance to these agents as well as monobactams (e.g., aztreonam) and—in many cases—β-lactam/β-lactamase inhibitor combinations. Resistance may emerge during therapy; in one study, this phenomenon was documented in 20% of clinical isolates. De novo resistance should be considered when clinical deterioration follows initial improvement, and third-generation cephalosporins should be avoided in the treatment of serious Enterobacter infections. Cefepime is stable in the presence of AmpC β-lactamases; thus, it is a suitable option for treatment of Enterobacter infections so long as no coexistent ESBL is present. Detection of ESBLs in Enterobacter is difficult because of the presence of AmpC β-lactamase; nonetheless, their prevalence

1	of Enterobacter infections so long as no coexistent ESBL is present. Detection of ESBLs in Enterobacter is difficult because of the presence of AmpC β-lactamase; nonetheless, their prevalence (particularly in E. cloacae) is known to be variable worldwide but is generally increasing and is now 5–50% overall. This increase is evidenced by NHSN data, which documented resistance to thirdand fourth-generation cephalosporins in 37.4% of CLABSI Enterobacter isolates in the United States; fortunately, carbapenems, amikacin, and tigecycline have generally retained excellent activity (90–99% susceptibility) and fluoroquinolones have good activity (85–95% susceptibility). Once susceptibility data become available, it is critical to de-escalate the antimicrobial regimen whenever possible.

1	S. marcescens causes the majority (>90%) of Serratia infections; Serratia liquefaciens, Serratia rubidaea, Serratia fonticola, Serratia grimesii, Serratia plymuthica, and Serratia odorifera are isolated occasionally. Serratiae are found primarily in the environment (including in health care institutions), particularly in moist settings. Serratiae have been isolated from a variety of animals, insects, and plants, but healthy humans are rarely colonized. In LTCFs or hospitals, reservoirs for the organisms include the hands and fingernails of health care personnel, food, milk (on neonatal units), sinks, respiratory and other medical equipment or devices, pressure monitors, IV solutions or parenteral medications (particularly those generated by compounding pharmacies), prefilled syringes and multiple-access medication vials (e.g., heparin, saline), blood products (e.g., platelets), hand soaps and lotions, irrigation solutions, and even disinfectants. Infection results from either direct

1	multiple-access medication vials (e.g., heparin, saline), blood products (e.g., platelets), hand soaps and lotions, irrigation solutions, and even disinfectants. Infection results from either direct inoculation (e.g., via IV fluid) or colonization (primarily of the respiratory tract). Sporadic infection is most common, but epidemics (often involving MDR strains in adult and neonatal ICUs) and common-source outbreaks also occur. The spectrum of extraintestinal infections caused by Serratia is similar to that for other GNB. Serratia species are usually considered causative agents of health care– associated infection and account for 1–3% of hospital-acquired infections. However, population-based laboratory surveillance studies in Canada and Australia demonstrated that community-acquired infections occur more commonly than was previously appreciated.

1	The respiratory tract, the genitourinary tract, intravascular devices, the eye (contact lens–associated keratitis and other ocular infections), surgical wounds, and the bloodstream (from contaminated infusions) are the most common sites of Serratia infection; the former five sites are the most common sources of Serratia bacteremia. Soft tissue infections (including myositis, fasciitis, mastitis), osteomyelitis, abdominal and biliary tract infection (postprocedural), and septic arthritis (primarily from intraarticular injections) occur less commonly. Serratiae are uncommon causes of neonatal or postsurgical meningitis and of bacteremia in neutropenic patients. Endocarditis is rare. Serratiae are readily cultured and identified by the laboratory and are usually lactose and indole negative. Some S. marcescens strains and S. rubidaea are red pigmented.

1	Serratiae are readily cultured and identified by the laboratory and are usually lactose and indole negative. Some S. marcescens strains and S. rubidaea are red pigmented. Most Serratia strains (>80%) are resistant to ampicillin, amoxi cillin-clavulanate, ampicillin-sulbactam, first-generation cepha losporins, cephamycins, nitrofurantoin, and colistin. In general, >90% of Serratia isolates are susceptible to other antibiotics appropriate for use against GNB. Induction or selection of variants with stable derepression of chromosomal AmpC β-lactamases may develop during therapy. Both in the United States and globally, the prevalence of ESBL-producing isolates is generally low (<5%), but rates of 20–30% have been reported in Asia and Latin America. Acquisition of carbapenemase-encoding genes is uncommon but increasing.

1	C. freundii and Citrobacter koseri cause most human Citrobacter infections, which are epidemiologically and clinically similar to Enterobacter infections. Citrobacter species are commonly present in water, food, soil, and certain animals. Citrobacter is part of the normal fecal flora in a minority of healthy humans, but colonization rates are higher in LTCFs and hospitals—the settings in which nearly all Citrobacter infections occur. Citrobacter species account for 1–2% of nosocomial infections. The affected hosts are usually immunocompromised or have comorbid disease. Citrobacter causes extraintestinal infections similar to those described for other GNB.

1	The urinary tract accounts for 40–50% of Citrobacter infections. Less commonly involved sites include the biliary tree (particularly with stones or obstruction), the respiratory tract, surgical sites, soft tissue (e.g., decubitus ulcers), the peritoneum, and intravascular devices. Osteomyelitis (usually from a contiguous focus), adult central nervous system infection (from neurosurgical or other types of meningeal disruption), and myositis occur rarely. Citrobacter (primarily C. koseri) also causes 1–2% of neonatal meningitis cases, of which 50–80% are complicated by brain abscess. Further, case reports in adults suggest that C. koseri infection has a predilection for abscess formation. Bacteremia is most often due to UTI, biliary/abdominal infection, or intravascular device infection. Citrobacter occasionally causes bacteremia in neutropenic patients. Endocarditis and endovascular infections are rare.

1	Citrobacter species are readily isolated and identified; 35–50% of isolates are lactose positive, and 100% are oxidase negative. C. freundii is indole negative, whereas C. koseri is indole positive.

1	C. freundii is more extensively resistant to antibiotics than is C. koseri. More than 90% of isolates are resistant to ampicillin and firstand sec-ond-generation cephalosporins. Citrobacter species (except C. koseri) possess AmpC β-lactamases; induction or selection of variants with stable derepression may develop during therapy. Resistance to antipseudomonal penicillins, aztreonam, fluoroquinolones, gentamicin, and third-generation cephalosporins is variable but increasing. The prevalence of ESBL-producing isolates is <5%. Carbapenems, amikacin, cefepime, tigecycline (with which clinical experience is limited), fosfomycin (which is available in the United States only as an oral formulation), and colistin (which is an agent of last resort because of potential toxicities) are most active, with >90% of strains susceptible.

1	M. morganii, Providencia stuartii, and (less frequently) Providencia rettgeri are the members of their respective genera that cause human infections. The epidemiologic associations, pathogenic properties, and clinical manifestations of these organisms resemble those of Proteus species. However, Morganella and Providencia occur more commonly among LTCF residents; to a lesser degree, they affect hospitalized patients. In settings with extensive use of polymyxins and tigecycline, these organisms may become increasingly common because of their intrinsic resistance to these agents.

1	These species are primarily urinary tract pathogens, causing UTIs that are most often associated with long-term (>30-day) catheterization. Such infections commonly lead to biofilm formation and catheter encrustation (sometimes causing catheter obstruction) or to the development of struvite bladder or renal stones (sometimes causing renal obstruction and serving as foci for relapse). Morganella is also commonly isolated from snakebite infection. Other, less common infectious syndromes include surgical site infection, soft tissue infection (primarily involving decubitus and diabetic ulcers), burn site infection, pneumonia (particularly ventilator-associated), intravascular device infection, and intraabdominal infection. Rarely, the other extraintestinal infections described for GNB also occur. Bacteremia is uncommon; any infected site can serve as the source, but the urinary tract accounts for most cases, with the next most common sources 1035 being surgical site, soft tissue, and

1	occur. Bacteremia is uncommon; any infected site can serve as the source, but the urinary tract accounts for most cases, with the next most common sources 1035 being surgical site, soft tissue, and hepatobiliary infections.

1	M. morganii and Providencia are readily isolated and identified. Nearly all isolates are lactose negative and indole positive.

1	Morganella and Providencia may be extensively resistant to antibiotics. Most isolates are resistant to ampicillin, first- generation cephalosporins, nitrofurantoin, fosfomycin, tigecycline, and the polymyxins; 40% are resistant to fluoroquinolones. Morganella and Providencia possess inducible AmpC β-lactamases; clinically significant induction or selection of stably derepressed mutants may develop during therapy. Resistance to antipseudomonal penicillins, aztreonam, gentamicin, TMP-SMX, and secondand third-generation cephalosporins is emerging but is still variably prevalent. The β-lactamase inhibitor tazobactam increases susceptibility to β-lactam agents, but sulbactam and clavulanic acid do not. Carbapenems, amikacin, and cefepime are the most active agents (>90% of isolates susceptible); however, resistance to the carbapenems, when present, is a concern because of the inherent resistance of Morganella and Providencia to the polymyxins and tigecycline. Removal of a colonized

1	however, resistance to the carbapenems, when present, is a concern because of the inherent resistance of Morganella and Providencia to the polymyxins and tigecycline. Removal of a colonized catheter or stone is critical for eradication of UTI.

1	E. tarda is the only member of the genus Edwardsiella that is associated with human disease. This organism is found predominantly in freshwater and marine environments and in the associated aquatic animal species. Human acquisition occurs primarily during interaction with these reservoirs and ingestion of inadequately cooked aquatic animals. E. tarda infection is rare in the United States; recently reported cases are mostly from Southeast Asia. This pathogen shares clinical features with Salmonella species (as an intestinal pathogen; Chap. 190), Vibrio vulnificus (as an extraintestinal pathogen; Chap. 193) and Aeromonas hydrophila (as both an intestinal and extraintestinal pathogen; Chap. 183e).

1	Gastroenteritis is the predominant infectious syndrome (50–80% of infections). Self-limiting watery diarrhea is most common, but severe colitis also occurs. The most common extraintestinal infection is wound infection due to direct inoculation, which is often associated with freshwater, marine, or snake-related injuries. Other infectious syndromes result from invasion of the gastrointestinal tract and subsequent bacteremia. Most afflicted hosts have comorbidities (e.g., hepatobiliary disease, iron overload, cancer, or diabetes mellitus). A primary bacteremic syndrome, sometimes complicated by meningitis, has a 40% case-fatality rate. Visceral (primarily hepatic) and intra-peritoneal abscesses also occur. Endocarditis and empyema have been described. Although E. tarda can readily be isolated and identified, most laboratories do not routinely seek to identify it in stool samples. Production of hydrogen sulfide is a characteristic biochemical property.

1	E. tarda is susceptible to most antimicrobial agents appropriate for use against GNB. Gastroenteritis is generally self-limiting, but treatment with a fluoroquinolone may hasten resolution. In the setting 1036 of severe sepsis, fluoroquinolones, thirdand fourth-generation cephalosporins, carbapenems, and amikacin—either alone or in combination—are the safest choices pending susceptibility data. Species of Hafnia, Kluyvera, Cedecea, Pantoea, Ewingella, Leclercia, and Photorhabdus are occasionally isolated from diverse clinical specimens, including blood, sputum, urine, cerebrospinal fluid, joint fluid, bile, and wounds. These organisms are rare and usually cause infection in a compromised host or in the setting of an invasive procedure or a foreign body. Cephalosporinases from Kluyvera have been implicated as the progenitors of CTX-M ESBLs.

1	Acinetobacter Infections David L. Paterson, Anton Y. Peleg Infections with bacteria of the genus Acinetobacter are established as a significant problem worldwide. Acinetobacter baumannii is par-ticularly formidable because of its propensity to acquire antibiotic resistance determinants. Endemic infections caused by strains of 187 A. baumannii resistant to multiple antibiotic classes, including carbapenems, are a serious concern in many specialized hospital units, especially intensive care units (ICUs). The foremost implication of infection with carbapenem-resistant A. baumannii is the need to use “last-line” antibiotics such as colistin, polymyxin B, or tigecycline; these options have the potential to render these bacteria resistant to all available antibiotics.

1	Acinetobacter species are oxidase-negative, nonfermenting, short, gram-negative bacilli. They were traditionally thought of as nonmotile—a characteristic from which the genus name was derived (from the Greek akineto, meaning “nonmotile”). However, recent work has shown that Acinetobacter organisms demonstrate motility under certain growth conditions. The bacteria grow well at 37°C in aerobic conditions on a range of laboratory media (e.g., blood agar). Some species may not grow on MacConkey agar. Differentiation of Acinetobacter species is difficult with the means typically available to most clinical microbiology laboratories, including commercial semiautomated identification systems. The commonly used matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) systems are undergoing evaluation for species-level identification of Acinetobacter. DNA–DNA hybridization is a method used for speciation in reference laboratories. Naturally occurring

1	MS) systems are undergoing evaluation for species-level identification of Acinetobacter. DNA–DNA hybridization is a method used for speciation in reference laboratories. Naturally occurring oxacillinase genes (blaOXA) have been identified in several Acinetobacter species, and their detection by polymerase chain reaction can aid in species identification.

1	Widely distributed in nature, Acinetobacter species can be found in water, in soil, and on vegetables. Acinetobacter is a component of the human skin flora and is sometimes identified as a contaminant in blood samples collected for culture. Fecal carriage can be detected in both healthy and hospitalized individuals. However, despite the ubiquity of some Acinetobacter species, the natural habitat of the most clinically important species, A. baumannii, remains to be fully defined. A. baumannii infections have been diagnosed in patients on all inhabited continents. The vast majority of infections occur in hospitalized patients and other patients with significant health-care contact. Outbreaks of carbapenem-resistant A. baumannii are particularly problematic. A significant issue is the introduction of carbapenem-resistant A. baumannii into hospitals as a result of medical transfers, especially from hospitals where the organism is highly endemic.

1	The Americas In 1991 and 1992, outbreaks of carbapenem-resistant A. baumannii infection occurred in a hospital in New York City. Subsequently, numerous other hospitals in the United States and South America have had outbreaks of carbapenem-resistant A. baumannii. Infections with A. baumannii among military personnel from the United States and Canada injured in Iraq or Afghanistan were widely observed beginning in 2002. Acinetobacter was one of the most common causes of bloodstream infections and bone and soft tissue infections after war-related injury. An epidemiologic investigation revealed that A. baumannii could be grown from environmental sites in field hospitals and that the environmental strains were closely related genotypically to clinical isolates. Europe A. baumannii infections have posed a substantial clinical challenge in many parts of Europe since the early 1980s. Three clones (European clones I, II, and III) have been the predominant causes of

1	A. baumannii infection in hospitals in Europe. Carbapenem resistance in A. baumannii is a significant issue in many European countries, most notably the United Kingdom, Greece, Italy, Spain, and Turkey. Asia, Australia, the Middle East, and Africa Although surveillance data are sparse from many countries in these regions, problems with carbapenem-resistant A. baumannii abound. Community-acquired infections are well described in northern Australia and some parts of Asia. These infections may be more likely in men >45 years of age who have histories of cigarette smoking, alcoholism, diabetes mellitus, or chronic obstructive airway disease. Community-acquired strains are more susceptible to antimicrobial agents than are hospital-acquired strains, but the clinical presentation of community-acquired disease is quite distinct and is characterized by overwhelming infection with severe pneumonia, septic shock, and multiorgan failure.

1	A. baumannii colonizes patients exposed to heavily contaminated hospital environments or to the hands of health care workers in these locations. Emerging data suggest that the organism can be found in the air in rooms of patients infected with Acinetobacter. Colonization of the upper airways in mechanically ventilated patients may lead to nosocomial pneumonia. Colonization of the skin may lead to central line–associated bloodstream infection, catheter-associated urinary tract infection (UTI), wound infection, or postneurosurgical meningitis. Throat carriage and microaspiration may be involved in the pathogenesis of community-acquired pneumonia due to A. baumannii.

1	Much less is known about the virulence mechanisms of and host responses to A. baumannii than about these aspects of other pathogenic gram-negative bacteria. Because of the emergence of multidrugresistant strains, including those resistant to all available antibiotics, the impetus to study A. baumannii pathogenesis has grown. Novel targets for antibacterial drug development are desperately required, and drugs that have antivirulence mechanisms may provide new therapeutic options. Specific virulence mechanisms in A. baumannii include iron acquisition and transport systems; outer-membrane protein A (OmpA), which mediates mammalian cell adhesion, invasion, and cytotoxicity through mitochondrial damage and initiation of caspase-dependent apoptosis; lipopolysaccharide (LPS); and proteins important in the formation of biofilm on abiotic and biotic surfaces. Biofilm formation on abiotic surfaces is dependent on a pilus assembly system, which in turn is controlled by a traditional

1	important in the formation of biofilm on abiotic and biotic surfaces. Biofilm formation on abiotic surfaces is dependent on a pilus assembly system, which in turn is controlled by a traditional two-component regulatory system mediated by bfmR. Also important in biofilm formation are a gene that encodes a biofilm-associated protein (Bap); OmpA; the quorum-sensing gene abaI, which controls the secretion of 3-hydroxy-C12-homoserine lactone; and the pga locus, which is essential for the production of the polysaccharide poly-β-1,6-Nacetylglucosamine. Most recently, a global virulence regulator known as GacSA was described as important in regulating A. baumannii de-stain. Given their small size, they may be misidentified as either 1037 biofilms, motility, growth in human serum, and virulence in a mam-gram-negative or gram-positive cocci. malian infection model. New model systems for the study of A. baumannii infection, including both nonmammalian (invertebrate) and mammalian models, have

1	in a mam-gram-negative or gram-positive cocci. malian infection model. New model systems for the study of A. baumannii infection, including both nonmammalian (invertebrate) and mammalian models, have been described. Furthermore, the use of A. baumannii transposongenerated mutant libraries to screen for mutants with attenuated growth in human biologic fluids (serum and ascites fluid) has allowed the identification of new virulence mechanisms. These include phospholipase D; capsule production mediated by ptk and epsA; penicillin-binding protein 7/8 encoded by the pbpG gene; and a glycosyltransferase important for LPS biosynthesis encoded by the lpsB gene.

1	The LPS of A. baumannii appears to play a significant role in eliciting host responses. In studies with knockout mice, Toll-like receptor 4 and CD14 were shown to be important in host recognition, signaling, and cytokine production in response to A. baumannii. Humoral responses targeting iron-regulated outer-membrane proteins and the O-polysaccharide component of LPS also have been described. APPROACH TO THE PATIENT: Acinetobacter must be considered in the differential diagnosis of hospital-acquired pneumonia, central line–associated bloodstream infection, posttraumatic wound infection in military personnel, and postneurosurgical meningitis.

1	CLINICAL MANIFESTATIONS Pneumonia It may be difficult to distinguish between upper-airway colonization with A. baumannii and hospital-acquired pneumonia. An estimated 5–10% of cases of ventilator-associated pneumonia are due to A. baumannii, although much regional variation exists. Typically, patients with A. baumannii ventilator-associated pneumonia have had a prolonged stay in an ICU; in outbreak situations, however, patients may acquire the infection within days of arrival in an ICU. Community-acquired pneumonia due to A. baumannii has been described in tropical regions of Australia and Asia. The disease typically occurs during the “wet” season among people with a history of alcohol abuse. Infection may result in fulminant pneumonia requiring admission to an ICU, with a mortality rate of ~50%.

1	Bloodstream Infection Although A. baumannii accounts for only ~1–2% of nosocomial bloodstream infections, crude mortality rates from these infections may be as high as 40%. Sources of bloodstream infection are typically a central line or underlying pneumonia, UTI, or wound infection. Traumatic Battlefield and Other Wounds A. baumannii is a well-known pathogen in burn units. This organism is commonly isolated from wounds of combat casualties; it was the most commonly isolated organism in one assessment of combat victims with open tibial fractures but did not appear to contribute directly to persistent nonunion or the need for amputation. Meningitis A. baumannii may cause meningitis following neurosurgical procedures. Patients typically have an external ventricular drain in situ. urinary Tract Infection A. baumannii is an occasional cause of catheter-associated UTI. It is highly unusual for this organism to cause uncomplicated UTI in healthy women.

1	urinary Tract Infection A. baumannii is an occasional cause of catheter-associated UTI. It is highly unusual for this organism to cause uncomplicated UTI in healthy women. Other Clinical Manifestations A small number of case reports describe Acinetobacter prosthetic-valve endocarditis and endophthalmitis/ keratitis. The latter is sometimes related to contact lens use or eye surgery. Acinetobacter infection should be suspected when plump coccobacilli are seen in Gram’s-stained respiratory tract secretions, blood cultures, or cerebrospinal fluid. Sometimes the organisms are difficult to

1	Treatment is hampered by the remarkable ability of A. baumannii to upregulate or acquire antibiotic resistance determinants. The most prominent example is that of β-lactamases, including those capable of inactivating carbapenems, cephalosporins, and penicillins. These enzymes, which include the OXA-type β-lactamases (e.g., OXA-23), the metallo-β-lactamases (e.g., NDM), and rarely KPC-type carbapenemases, are typically resistant to currently available β-lactamase inhibitors such as clavulanate or tazobactam. Plasmids that harbor genes encoding these β-lactamases may also harbor genes encoding resistance to aminoglycosides and sulfur antibiotics. The end result is that carbapenem-resistant A. baumannii may become truly multidrug resistant.

1	Selection of empirical antibiotic therapy when A. baumannii is suspected is challenging and must rely on a knowledge of local epidemiology. Receipt of prompt, effective antibiotic therapy is the goal. Given the diversity of resistance mechanisms in A. baumannii, definitive therapy should be based on the results of antimicrobial susceptibility testing. Carbapenems (imipenem, meropenem, and doripenem but not ertapenem) have long been thought of as the agents of choice for serious A. baumannii infections. However, the clinical utility of carbapenems is now widely jeopardized by the production of carbapenemases, as described above. Sulbactam may be an alternative to carbapenems. Unlike other β-lactamase inhibitors (e.g., clavulanic acid and tazobactam), sulbactam has intrinsic activity against Acinetobacter; this activity is mediated by the drug’s binding to penicillin-binding protein 2 rather than by its ability to inhibit β-lactamases. Sulbactam is commercially available in a combined

1	Acinetobacter; this activity is mediated by the drug’s binding to penicillin-binding protein 2 rather than by its ability to inhibit β-lactamases. Sulbactam is commercially available in a combined formulation with either ampicillin or cefoperazone and may also be available as a single agent in some countries. Despite the absence of randomized clinical trials, sulbactam seems to be equivalent to carbapenems in clinical effectiveness against susceptible strains.

1	Therapy for carbapenem-resistant A. baumannii is particularly problematic. The only currently available choices are polymyxins (colistin and polymyxin B) and tigecycline. Neither option is perfect. Polymyxins may be nephrotoxic and neurotoxic. Definition of the optimal dose and schedule for administration of polymyxins to patients in vulnerable groups (e.g., those requiring renal replacement therapy) remains challenging, and emergence of resistance in association with monotherapy is a concern. Conventional doses of tigecycline may not result in serum concentrations adequate to treat bloodstream infections. Resistance of A. baumannii to tigecycline may develop during treatment with this drug.

1	As a consequence of these issues with the polymyxins and tigecycline, combination therapy is now favored for carbapenem-resistant Acinetobacter. However, in a randomized controlled trial, 30-day mortality was not reduced by the addition of rifampin to colistin. Nevertheless, a significant increase in microbiologic eradication was observed in the colistin plus rifampin arm over that attained with Sulbactam Intrinsic activity against Acinetobacter, not linked to β-lactamase inhibition Tigecycline May be an option for carbapenem-resistant strains but inappropriate for urinary tract infection, bloodstream infection, or meningitis

1	Tigecycline May be an option for carbapenem-resistant strains but inappropriate for urinary tract infection, bloodstream infection, or meningitis Colistin or poly-May be an option for carbapenem-resistant strains, but myxin B pharmacokinetics not yet well understood 1038 colistin alone. Combinations of polymyxins with a carbapenem look more promising and are being evaluated in prospective clinical trials. Fosfomycin has poor activity against Acinetobacter and should not be relied upon for treatment. Clearly, new treatment options are needed for serious Acinetobacter infections.

1	Given the propensity of A. baumannii to cause infections in seriously ill patients in ICUs, it is not surprising that A. baumannii infections are associated with high mortality rates. Thus a pertinent question is whether A. baumannii infections are associated with high attributable mortality rates after the severity of illness is controlled for. A number of studies have addressed this issue but have had disparate results. Whether the discrepant results can be explained purely by methodologic differences is unknown at present.

1	Multidrug-resistant A. baumannii clearly causes outbreaks of infection and then establishes endemicity. In endemic situations, a small number of strain types predominate. In the 1991–1992 outbreaks in New York City, for example, two strain types accounted for more than 80% of carbapenem-resistant isolates. This “oligoclonality” plainly demonstrates the potential importance of infection control interventions in response to outbreaks of multidrug-resistant A. baumannii infection.

1	The hospital environment is an important reservoir of organisms capable of colonizing patients and causing infection. Environmental sources of A. baumannii include computer keyboards, glucometers, multidose medication vials, IV nutrition, inadequately sterilized reusable arterial pressure transducers, ventilator tubing, suction catheters, humidifiers, containers of distilled water, urine collection jugs, and moist bedding articles. Pulsatile-lavage wound treatment—a high-pressure irrigation system used to debride wounds—has been associated with an outbreak of A. baumannii infection.

1	Contaminated inanimate objects should be removed from the patient-care environment or subjected to enhanced environmental cleaning. Although contact-isolation procedures (use of gloves and gowns when dealing with colonized patients or their environment), accommodation of patients in single rooms, and improved hand hygiene are critical, attention to the patient-care environment may be the only measure that leads to control of outbreaks of A. baumannii infection. One study found that Acinetobacter can be cultured from the air in rooms of patients with A. baumannii infection; the infection-control implications are not yet clear. Helicobacter pylori Infections John C. Atherton, Martin J. Blaser DEFINITION Helicobacter pylori colonizes the stomach in ~50% of the world’s human population, essentially for life unless eradicated by antibiotic treatment. Colonization with this organism is the 188 main risk factor for peptic ulceration (Chap. 348) as well as for gastric adenocarcinoma and

1	essentially for life unless eradicated by antibiotic treatment. Colonization with this organism is the 188 main risk factor for peptic ulceration (Chap. 348) as well as for gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma (Chap. 109). Treatment for H. pylori has revolutionized the management of peptic ulcer disease, providing a permanent cure in most cases. Such treatment also represents first-line therapy for patients with low-grade gastric MALT lymphoma. Treatment of

1	H. pylori is of no benefit in the treatment of gastric adenocarcinoma, but prevention of H. pylori colonization could potentially prevent gastric malignancy and peptic ulceration. In contrast, increasing evidence indicates that lifelong H. pylori colonization may offer some protection against complications of gastroesophageal reflux disease (GERD), including esophageal adenocarcinoma. Recent research has focused on whether H. pylori colonization is also a risk factor for some extragastric diseases and whether it is protective against some recently emergent medical problems, such as childhood-onset asthma and obesity.

1	Helicobacter pylori H. pylori is a gram-negative bacillus that has naturally colonized humans for at least 100,000 years, and probably throughout human evolution. It lives in gastric mucus, with a small proportion of the bacteria adherent to the mucosa and possibly a very small number of the organisms entering cells or penetrating the mucosa; the organism’s distribution is never systemic. Its spiral shape and flagella render H. pylori motile in the mucus environment. The organism has several acid-resistance mechanisms, most notably a highly expressed urease that catalyzes urea hydrolysis to produce buffering ammonia. H. pylori is microaerophilic (i.e., requires low levels of oxygen), is slow-growing, and requires complex growth media in vitro.

1	Other Helicobacter Species A very small proportion of gastric Helicobacter infections are due to species other than H. pylori, possibly acquired as zoonoses. These non-pylori gastric helicobacters are associated with low-level inflammation and occasionally with disease. In immunocompromised hosts, several nongastric (intestinal) Helicobacter species can cause disease with clinical features resembling those of Campylobacter infections; these species are covered in Chap. 192.

1	Prevalence and Risk Factors The prevalence of H. pylori among adults is <30% in most parts of the United States and in other developed countries as opposed to >80% in most developing countries. In the United States, prevalence varies with age: up to 50% of 60-year-old persons, ~20% of 30-year-old persons, and fewer than 10% of children are colonized. H. pylori is usually acquired in childhood. The age association is due mostly to a birth-cohort effect whereby current 60-year-olds were more commonly colonized as children than are current children. Spontaneous acquisition or loss of H. pylori in adulthood is uncommon. Childhood acquisition explains why the main risk factors for infection are markers of crowding and social deprivation in childhood. Transmission Humans are the only important reservoir of

1	Transmission Humans are the only important reservoir of H. pylori. Children may acquire the organism from their par ents (most often the primary caregiver) or from other children. The former is more common in developed countries and the latter in less developed countries. Whether transmission takes place more often by the fecal-oral or the oral-oral route is unknown, but H. pylori is easily cultured from vomitus and gastroesophageal refluxate and is less easily cultured from stool.

1	H. pylori colonization induces chronic superficial gastritis, a tissue response in the stomach that includes infiltration of the mucosa by both mononuclear and polymorphonuclear cells. (The term gastritis should be used specifically to describe histologic features; it has also been used to describe endoscopic appearances and even symptoms, but these features do not correlate with microscopic findings or even with the presence of H. pylori.) Although H. pylori is capable of numerous adaptations that prevent excessive stimulation of the immune system, colonization is accompanied by a considerable persistent local and systemic immune response, including the production of antibodies and cell-mediated responses. However, these responses are ineffective in clearing the bacterium. This inefficient clearing appears to be due in part to H. pylori’s downregulation of the immune system, which fosters its own persistence.

1	Most H. pylori–colonized persons do not develop clinical sequelae. That some persons develop overt disease whereas others do not is related to a combination of factors: bacterial strain differences, host susceptibility to disease, and environmental factors.

1	Bacterial Virulence Factors Several H. pylori virulence factors are more common among strains that are associated with disease than among those that are not. The cag island is a group of genes that encodes a bacterial type IV secretion system. Through this system, an effector protein, CagA, is translocated into epithelial cells, where it may be transformed by phosphorylation and induces host cell signal transduction; proliferative, cytoskeletal, and inflammatory changes in the cell result. The protein at the tip of the secretory apparatus, CagL, binds to integrins on the cell surface, transducing further signaling. Finally, soluble components of the peptidoglycan cell wall enter the cell, mediated by the same secretory system. These components are recognized by the emergency intracellular bacterial receptor Nod1, which stimulates a proinflammatory cytokine response resulting in enhanced gastric inflammation. Carriage of cag-positive strains increases the risk of peptic ulcer or

1	bacterial receptor Nod1, which stimulates a proinflammatory cytokine response resulting in enhanced gastric inflammation. Carriage of cag-positive strains increases the risk of peptic ulcer or gastric adenocarcinoma. A second major virulence factor is the vacuolating cytotoxin VacA, which forms pores in cell membranes. VacA is polymorphic, and carriage of more active forms also increases the risk of disease. Other bacterial factors that are associated with increased disease risk include adhesins, such as BabA (which binds to blood group antigens on epithelial cells), and incompletely characterized factors, such as another recently described bacterial type 4 secretion system.

1	Host Genetic and Environmental Factors The best-characterized host determinants of disease are genetic polymorphisms leading to enhanced activation of the innate immune response, including polymorphisms in cytokine genes or in genes encoding bacterial recognition proteins such as Toll-like receptors. For example, colonized people with polymorphisms in the interleukin 1 gene that increase the production of this cytokine in response to H. pylori infection are at increased risk of gastric adenocarcinoma. In addition, environmental cofactors are important in pathogenesis. Smoking increases the risks of duodenal ulcers and gastric cancer in H. pylori–positive individuals. Diets high in salt and preserved foods increase cancer risk, whereas diets high in antioxidants and vitamin C are modestly protective.

1	Distribution of Gastritis and Differential Disease Risk The pattern of gastric inflammation is associated with disease risk: antral-predominant gastritis is most closely linked with duodenal ulceration, whereas pan-gastritis is linked with gastric ulceration and adenocarcinoma. This difference probably explains why patients with duodenal ulceration are not at high risk of developing gastric adenocarcinoma later in life, despite being colonized by H. pylori.

1	patHogenesis of dUodenal Ulceration How gastric colonization causes duodenal ulceration is now becoming more clear. H. pylori–induced inflammation of the gastric antrum diminishes the number of somatostatin-producing D cells. Because somatostatin inhibits gastrin release, gastrin levels are higher than in H. pylori–negative persons, and these higher levels lead to increased meal-stimulated acid secretion from the relatively spared gastric corpus. How this situation increases duodenal ulcer risk remains controversial, but the increased acid secretion may contribute to the formation of the potentially protective gastric metaplasia at the junction of antral and corpus-type mucosa, an area that is often 1039 particularly inflamed. Gastric cancer probably stems from progressive DNA damage and the survival of abnormal epithelial cell clones. The DNA damage is thought to be due principally to reactive oxygen and nitrogen species arising from inflammatory cells, perhaps in relation to other

1	the survival of abnormal epithelial cell clones. The DNA damage is thought to be due principally to reactive oxygen and nitrogen species arising from inflammatory cells, perhaps in relation to other bacteria that survive in a hypochlorhydric stomach. Longitudinal analyses of gastric biopsy specimens taken years apart from the same patient show that the common intestinal type of gastric adenocarcinoma follows stepwise changes from simple gastritis to gastric atrophy, intestinal metaplasia, and dysplasia. A second, diffuse type of gastric adenocarcinoma found more commonly in younger adults may arise directly from chronic gastritis without atrophic changes.

1	Primary phenomenon: Secondary phenomenon: Clinical outcome: Association with H. pylori (OR): Essentially all H. pylori–colonized persons have histologic gastritis, but only ~10–15% develop associated illnesses such as peptic ulceration, gastric adenocarcinoma, or gastric lymphoma (Fig. 188-1). Rates among women are less than half of those among men for both diseases. Peptic ulcer Disease Worldwide, >80% of duodenal ulcers and >60% of gastric ulcers are related to H. pylori colonization (Chap. 348). However, in particular, the proportion of gastric ulcers caused by aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) is increasing, and in many developed countries these drugs have overtaken H. pylori as a cause of gastric ulceration. The main lines of evidence supporting an ulcer-promoting role for H. pylori are that the presence of the organism is a risk factor for the development of ulcers, (2) non-NSAID-induced ulcers rarely develop in the absence of H.

1	H. pylori, (3) eradication of H. pylori virtually abolishes long-term ulcer relapse, and (4) experimental H. pylori infection of gerbils can cause gastric ulceration. Gastric Adenocarcinoma and Lymphoma Prospective nested case-control studies have shown that H. pylori colonization is a risk factor for adenocarcinomas of the distal (noncardia) stomach (Chap. 109). Long-term experimental infection of gerbils also may result in gastric adenocarcinoma. Moreover, H. pylori may induce primary gastric Hyperacidity Atrophic gastritis Antigenic stimulation ? Duodenal ulceration 3–83–6 6–50 0.2–0.6 B-cell lymphoma Noncardia gastric adenocarcinoma Reflux esophagitis and sequelae Tissue response (inflammation)

1	FIGuRE 188-1 Schematic of the relationships between colonization with Helicobacter found in the duodenum of duodenal ulcer pylori and diseases of the upper gastrointestinal tract. Essentially all persons colonized patients. Gastric metaplasia in the duodenum with H. pylori develop a host response, which is generally termed chronic gastritis. The nature may become colonized by H. pylori and subse of the host’s interaction with the particular bacterial population determines the clinical out quently inflamed and ulcerated.

1	come. H. pylori colonization increases the lifetime risk of peptic ulcer disease, noncardia gastric patHogenesis of gastric Ulceration and gastric cancer, and B-cell non-Hodgkin’s gastric lymphoma (odds ratios [ORs] for all, >3). In contrast, adenocarcinoma The pathogenesis of these a growing body of evidence indicates that H. pylori colonization (especially with cagA+ strains) conditions is less well understood, although protects against adenocarcinoma of the esophagus (and the sometimes related gastric cardia) both arise in association with panor corpus-and premalignant lesions such as Barrett’s esophagus (OR, <1). Although the incidences of predominant gastritis. The hormonal changes peptic ulcer disease (cases not due to nonsteroidal anti-inflammatory drugs) and noncardia described above still occur, but the inflam-gastric cancer are declining in developed countries, the incidence of adenocarcinoma of the mation in the gastric corpus means that it esophagus is increasing. (Adapted

1	still occur, but the inflam-gastric cancer are declining in developed countries, the incidence of adenocarcinoma of the mation in the gastric corpus means that it esophagus is increasing. (Adapted from MJ Blaser: Hypothesis: The changing relationships of produces less acid (hypochlorhydria) despite Helicobacter pylori and humans: Implications for health and disease. J Infect Dis 179:1523, 1999, hypergastrinemia. Gastric ulcers usually occur with permission.) 1040 lymphoma, although this condition is much less common. Many low-grade gastric B-cell lymphomas are dependent on H. pylori for continuing growth and proliferation, and these tumors may regress either fully or partially after H. pylori eradication. However, they require careful shortand long-term monitoring, and some necessitate additional treatment with chemotherapeutic agents.

1	Functional Dyspepsia Many patients have upper gastrointestinal symptoms but have normal results on upper gastrointestinal endoscopy (so-called functional or nonulcer dyspepsia; Chap. 348). Because H. pylori is common, some of these patients will be colonized with the organism. H. pylori eradication leads to symptom resolution a little more commonly (from 0 to 7% in different studies) than does placebo treatment. Whether such patients have peptic ulcers in remission at the time of endoscopy or whether a small subgroup of patients with “true” functional dyspepsia respond to H. pylori treatment is unclear.

1	Protection Against Peptic Esophageal Disease, Including Esophageal Adenocarcinoma Much interest has focused on a protective role for H. pylori against GERD (Chap. 347), Barrett’s esophagus (Chap. 347), and adenocarcinoma of the esophagus and gastric cardia (Chap. 109). The main lines of evidence for this role are (1) that there is a temporal relationship between a falling prevalence of gastric H. pylori colonization and a rising incidence of these conditions; (2) that, in most studies, the prevalence of H. pylori colonization (especially with proinflammatory cagA+ strains) is significantly lower among patients with these esophageal diseases than among control participants; and that, in prospective nested studies (see above), the presence of H.

1	H. pylori is inversely related to these cancers. The mechanism underlying this protective effect is likely H. pylori–induced hypochlorhydria. Because, at the individual level, GERD symptoms may decrease, worsen, or remain unchanged after H. pylori treatment, concerns about GERD should not affect decisions about whether to treat H. pylori when an indication exists.

1	Other Pathologies H. pylori has an increasingly recognized role in other gastric pathologies. It may be one initial precipitant of autoimmune gastritis and pernicious anemia and also may predispose some patients to iron deficiency through occult blood loss and/or hypochlorhydria and reduced iron absorption. In addition, several extragastrointestinal pathologies have been linked with H. pylori colonization, although evidence of causality is less strong. Studies of H. pylori treatment in idiopathic thrombocytopenic purpura have consistently described improvement in or even normalization of platelet counts. Potentially important but even more controversial associations are with ischemic heart disease and cerebrovascular disease. However, the strength of the latter associations is reduced if confounding factors are taken into account, and most authorities consider the associations to be noncausal. Several studies have shown an inverse association of cagA+

1	H. pylori with childhood-onset asthma, hay fever, and atopic disorders. These associations have been shown to be causal in animal models, but causality in humans and the size of any effect have not been established. Tests for H. pylori fall into two groups: tests that require upper gastrointestinal endoscopy and simpler tests that can be performed in the clinic (Table 188-1).

1	Endoscopy-Based Tests Endoscopy is usually unnecessary in the initial management of young patients with simple dyspepsia but is commonly used to exclude malignancy and make a positive diagnosis in older patients or those with “alarm” symptoms. If endoscopy is performed, the most convenient biopsy-based test is the biopsy urease test, in which one large or two small gastric biopsy specimens are placed into a gel containing urea and an indicator. The presence of H. pylori urease leads to a pH alteration and therefore to a color change, which often occurs within minutes but can require up to 24 h. Histologic examination of biopsy specimens for H. pylori also is accurate, provided that a special stain (e.g., a modified Giemsa or silver stain) permitting optimal visualization of the organism is used. If biopsy specimens are obtained from both antrum and corpus, histologic study yields additional information, including the degree and pattern of inflammation and the presence of any atrophy,

1	used. If biopsy specimens are obtained from both antrum and corpus, histologic study yields additional information, including the degree and pattern of inflammation and the presence of any atrophy, metaplasia, or dysplasia. Microbiologic culture is most specific but may be insensitive because of difficulty with H. pylori isolation. Once the organism is cultured, its identity as H. pylori can be confirmed by its typical appearance on Gram’s stain and its positive reactions in oxidase, catalase, and urease tests. Moreover, the organism’s susceptibility to antibiotics can be determined, and this information can be clinically useful in difficult cases. The occasional biopsy specimens containing the less common non-pylori gastric helicobacters give only weakly positive results in the biopsy urease test. Positive identification of these bacteria requires visualization of the characteristic long, tight spirals in histologic sections; they cannot easily be cultured.

1	Noninvasive Tests Noninvasive H. pylori testing is the norm if gastric cancer does not need to be excluded by endoscopy. The best-established test (and a very accurate one) is the urea breath test. In this simple test, the patient drinks a solution of urea labeled with the nonradioactive isotope 13C and then blows into a tube. If H. pylori urease is present, the urea is hydrolyzed, and labeled carbon dioxide is detected in breath samples. The stool antigen test, a simple and accurate test using monoclonal antibodies specific for H. pylori antigens, is more convenient and potentially less expensive than the urea breath test, but some patients dislike sampling stool. The simplest tests for ascertaining H. pylori status are serologic assays measuring specific IgG levels in serum by enzyme-linked immunosorbent assay or immunoblot. The best of these tests are as accurate as other diagnostic methods, but many commercial tests—especially rapid office tests—do not perform well.

1	use of Tests to Assess Treatment Success The urea breath test, the stool antigen test, and biopsy-based tests can all be used to assess the success of treatment (Fig. 188-2). However, because these tests are dependent on H. pylori load, their use <4 weeks after treatment may yield false-negative results. Furthermore, these tests are unreliable if performed within 4 weeks of intercurrent treatment with antibiotics or bismuth compounds or within 2 weeks of the discontinuation of proton pump inhibitor (PPI) treatment. In the assessment of treatment success, noninvasive tests are normally preferred; however, after gastric ulceration, endoscopy should be repeated to ensure healing and to exclude gastric carcinoma by further histologic sampling. Serologic tests are not used to monitor treatment success, as the gradual drop in titer of H. pylori– specific antibodies is too slow to be of practical use. Indication for H. pylori treatment (e.g., peptic ulcer disease or new-onset dyspepsia)

1	Indication for H. pylori treatment (e.g., peptic ulcer disease or new-onset dyspepsia) Test for H. pylori First-line treatment (Table 188-2) Positive H. pylori not the cause Wait at least 1 month after treatment finishes (no antibiotics, bismuth compounds, or proton pump inhibitors in the meantime) Third-line treatment; endoscopy with H. pylori culture and sensitivity testing; treat according to known antibiotic sensitivities† Refer to specialist Consider whether treatment is still indicated Any remaining symptoms are not due to H. pylori

1	Any remaining symptoms are not due to H. pylori FIGuRE 188-2 Algorithm for the management of Helicobacter pylori infection. *Note that either the urea breath test or the stool antigen test can be used in this algorithm. Occasionally, endoscopy and a biopsy-based test are used instead of either of these tests in follow-up after treatment. The main indication for these invasive tests is gastric ulceration; in this condition, as opposed to duodenal ulceration, it is important to check healing and to exclude underlying gastric adenocarcinoma. However, even in this situation, patients undergoing endoscopy may still be receiving proton pump inhibitor therapy, which precludes H. pylori testing. Thus a urea breath test or a stool antigen test is still required at a suitable interval after the end of therapy to determine whether treatment has been successful (see text). †Some authorities use empirical third-line regimens, of which several have been described.

1	The most clear-cut indications for treatment are H. pylori–related duodenal or gastric ulceration or low-grade gastric B-cell lymphoma. Whether or not the ulcers are currently active, H. pylori should be eradicated in patients with documented ulcer disease to prevent relapse (Fig. 188-2). Testing for H. pylori and treatment if the results are positive also have been advocated in uninvestigated simple dyspepsia, but only when the prevalence of H. pylori in the community is >20% are these measures more cost-effective than simply treating the dyspepsia with PPIs. Guidelines have recommended

1	H. pylori treatment in functional dyspepsia in case the patient is one of the perhaps 0–7% who will benefit from such treatment (beyond placebo effects). Some guidelines also recommend treatment of conditions not definitively known to respond to H. pylori eradication, including idiopathic thrombocytopenic purpura, vitamin B12 deficiency, and iron-deficiency anemia (in the last instance, only when other causes have been carefully excluded). Test-and-treat has emerged as a common clinical practice in recent years despite the lack of direct evidence that it is advantageous; whether this practice will survive the scrutiny of time and further study remains to be determined. For individuals with a strong family history of gastric cancer, treatment to eradicate H. pylori in the hope of reducing their cancer risk is reasonable but of unproven value. Currently, widespread community screening for and treatment of H. pylori as primary prophylaxis for gastric cancer and peptic ulcers are not

1	their cancer risk is reasonable but of unproven value. Currently, widespread community screening for and treatment of H. pylori as primary prophylaxis for gastric cancer and peptic ulcers are not recommended in most countries, mainly because the extent of the consequent reduction in cancer risk is not known. Several studies have found a modestly reduced cancer risk after treatment, but the period of follow-up is still fairly short and the size of the effect in different populations remains unclear. Other reasons not to treat H. pylori in asymptomatic populations at present include (1) the adverse side effects (which are common and can be severe in rare cases) of the multiple-antibiotic regimens used; (2) antibiotic resistance, which may emerge in H. pylori or other incidentally carried bacteria; (3) the anxiety that may arise in otherwise healthy people, especially if treatment is unsuccessful; and (4) the existence of a subset of people who will develop GERD symptoms after treatment,

1	(3) the anxiety that may arise in otherwise healthy people, especially if treatment is unsuccessful; and (4) the existence of a subset of people who will develop GERD symptoms after treatment, although, on average, H. pylori treatment does not affect GERD symptoms or severity. Despite the absence of screening strategies, many doctors treat H. pylori if it is known to be present (particularly in children and younger adults), even when the patient is asymptomatic. The rationale is that it reduces patient concern and may reduce future gastric cancer risk and that any reduction in risk is likely to be greater in younger patients. However, such practices do not factor in any potential benefits of H. pylori colonization. Overall, despite widespread clinical activity in this area, most treatment of asymptomatic H. pylori carriage is given without a firm evidence base.

1	Although H. pylori is susceptible to a wide range of antibiotics in vitro, monotherapy is not usually successful, probably because of inadequate antibiotic delivery to the colonization niche. Failure of monotherapy prompted the development of multidrug regimens, the most successful of which are triple and quadruple combinations. Current regimens consist of a PPI and two or three antimicrobial agents given for 7–14 days (Table 188-2). Research on optimizing drug combinations to increase efficacy continues, and guidelines are likely to change as the field develops and as countries increasingly tailor treatment to suit local antibiotic resistance patterns and economic needs.

1	aThe recommended first-line regimens for most of the world are shown in bold type. bThese regimens should be used only for populations in which the prevalence of clarithromycinresistant strains is known to be <20%. In practice, this restriction limits the regimens’ appropriate range mainly to northern Europe. Meta-analyses show that a 14-day course of therapy is slightly superior to a 7-day course. cMany authorities and some guidelines recommend doubling this dose of omeprazole, as trials show resultant increased efficacy with some antibiotic combinations. Omeprazole may be replaced with any proton pump inhibitor at an equivalent dosage. dData supporting this regimen come mainly from Europe and are based on the use of bismuth subcitrate (1 tablet qid) and metronidazole (400 mg tid). This is a recommended first-line regimen in most countries and is the recommended second-line regimen in northern Europe. eData supporting this regimen come mainly from Europe. This regimen may be used as

1	is a recommended first-line regimen in most countries and is the recommended second-line regimen in northern Europe. eData supporting this regimen come mainly from Europe. This regimen may be used as an alternative to regimen 3. f This regimen may be used as an alternative to regimen 3 or 4. gMetronidazole (500 mg bid) may be used as an alternative. hData supporting this regimen come mainly from Europe. It is used as second-line treatment in many countries (particularly where quadruple therapy is used as the first-line regimen) and as third-line treatment in others. This regimen may be less effective where rates of quinolone use are high.

1	The two most important factors in successful H. pylori treat ment are the patient’s close compliance with the regimen and the use of drugs to which the patient’s strain of H. pylori has not acquired resistance. Treatment failure following minor lapses in compliance is common and often leads to acquired resistance to metronidazole or clarithromycin. To stress the importance of compliance, written instructions should be given to the patient, and minor side effects of the regimen should be explained. Increasing levels of H. pylori resistance to clarithromycin, quinolones, and—to a lesser extent—metronidazole are of growing concern and are thought to be responsible for the reduced efficacy of previously popular clarithromycin-based triple-therapy regimens worldwide. Treatment with these regimens is now virtually confined to certain northern European countries where the use of clarithromycin (or azithromycin) for respiratory infections has not been widespread and resistance rates in H.

1	is now virtually confined to certain northern European countries where the use of clarithromycin (or azithromycin) for respiratory infections has not been widespread and resistance rates in H. pylori are still low. Strains of H. pylori with some degree of in vitro resistance to metronidazole are common but still may be eradicated with metronidazole-containing regimens, which have only slightly reduced efficacy in vivo. Assessment of antibiotic susceptibilities before treatment would be optimal but is not usually undertaken because endoscopy and mucosal biopsy are necessary to obtain H. pylori for culture and because most microbiology laboratories are inexperienced in H. pylori culture. In the absence of susceptibility information, the patient’s history of (even distant) antibiotic use for other conditions should be ascertained; use of the previously administered agent(s) should then be avoided if possible, particularly in the case of clarithromycin (e.g., previous use for upper

1	for other conditions should be ascertained; use of the previously administered agent(s) should then be avoided if possible, particularly in the case of clarithromycin (e.g., previous use for upper respiratory infection) and quinolones. If initial H. pylori treatment fails, the usual approach is empirical re-treatment with another drug regimen (Table 188-2). The third-line approach should ideally be endoscopy, biopsy, and culture plus treatment based on documented antibiotic sensitivities. However, empirical third-line therapies are often used.

1	Non-pylori gastric helicobacters are treated in the same way as H. pylori. However, in the absence of trials, it is unclear whether a positive outcome always represents successful treatment or whether it is sometimes due to natural clearance of the bacteria.

1	Carriage of H. pylori has considerable public health significance in developed countries, where it is associated with peptic ulcer disease and gastric adenocarcinoma, and in developing countries, where gastric adenocarcinoma may be an even more common cause of cancer death late in life. If mass prevention were contemplated, vaccination would be the most obvious method, and experimental immunization of animals has given promising results. However, given that H. pylori has co-evolved with its human host over millennia, preventing or eliminating colonization on a population basis may have biological and clinical costs. For example, lifelong absence of H. pylori is a risk factor for GERD complications, including esophageal adenocarcinoma. We have speculated that the disappearance of H. pylori may also be associated with an increased risk of other emergent diseases reflecting aspects of the current Western lifestyle, such as childhood-onset asthma and allergy.

1	Infections due to Pseudomonas 189 species and Related Organisms The pseudomonads are a heterogeneous group of gram-negative bacteria that have in common an inability to ferment lactose. Formerly classified in the genus Pseudomonas, the members of this group have been assigned to three medically important genera—Pseudomonas, Burkholderia, and Stenotrophomonas—whose biologic behaviors encompass both similarities and marked differences and whose genetic repertoires differ in many respects. The pathogenicity of most pseudomonads is based on opportunism; the exceptions are the organisms that cause melioidosis (Burkholderia pseudomallei) and glanders (Burkholderia mallei), which can be considered as primary pathogens.

1	Pseudomonas aeruginosa, the major pathogen of the group, is a significant cause of infections in hospitalized patients and in patients with cystic fibrosis (CF; Chap. 313). Cytotoxic chemotherapy, mechanical ventilation, and broad-spectrum antibiotic therapy probably paved the way for colonization and infection of increasing numbers of hospitalized patients by this organism. Thus most conditions predisposing to P. aeruginosa infections have involved host compromise and/ or broad-spectrum antibiotic use. The other members of the genus Pseudomonas—Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas stutzeri—infect humans infrequently.

1	The genus Burkholderia comprises more than 40 species, of which Burkholderia cepacia is most frequently encountered in Western countries. Like P. aeruginosa, B. cepacia is both a nosocomial pathogen and a cause of infection in CF. The other medically important members of this genus are B. pseudomallei and B. mallei, the etiologic agents of melioidosis and glanders, respectively. The genus Stenotrophomonas contains one species of medical significance, Stenotrophomonas maltophilia (previously classified in the genera Pseudomonas and Xanthomonas). This organism is strictly an opportunist that “overgrows” in the setting of potent broad-spectrum antibiotic use.

1	P. aeruginosa is found in most moist environments. Soil, plants, vegetables, tap water, and countertops are all potential reservoirs for this microbe, as it has simple nutritional needs. Given the ubiquity of P. aeruginosa, simple contact with the organism is not sufficient for colonization or infection. Clinical and experimental observations suggest that P. aeruginosa infection often occurs concomitantly with host defense compromise, mucosal trauma, physiologic derangement, and antibiotic-mediated suppression of normal flora. Thus, it comes as no surprise that the majority of P. aeruginosa infections occur in intensive care units (ICUs), where these factors frequently converge. The organism is initially acquired from environmental sources, but patient-to-patient spread also occurs in clinics and families. In the past, burned patients appeared to be unusually susceptible to

1	In the past, burned patients appeared to be unusually susceptible to P. aeruginosa. For example, in 1959–1963, Pseudomonas burn-wound sepsis was the principal cause of death in 60% of burned patients dying at the U.S. Army Institute of Surgical Research. For reasons that are unclear, P. aeruginosa infection in burns is no longer the major problem that it was during the 1950s and 1960s. Similarly, in the 1960s, P. aeruginosa appeared as a common pathogen in patients receiving cytotoxic chemotherapy at many institutions in the United States, but it subsequently diminished in importance. Despite this subsidence, P. aeruginosa remains one of the most feared pathogens in this popula tion because of its high attributable mortality rate. In some parts of Asia and Latin America, P. aeruginosa con tinues to be the most common cause of gram-negative bacte remia in neutropenic patients.

1	In some parts of Asia and Latin America, P. aeruginosa con tinues to be the most common cause of gram-negative bacte remia in neutropenic patients. In contrast to the trends for burned patients and neutropenic patients in the United States, the incidence of P. aeruginosa infections among patients with CF has not changed. P. aeruginosa remains the most common contributing factor to respiratory failure in CF and is responsible for the majority of deaths among CF patients.

1	P. aeruginosa is a nonfastidious, motile, gram-negative rod that grows on most common laboratory media, including blood and MacConkey agars. It is easily identified in the laboratory on primary-isolation agar plates by pigment production that confers a yellow to dark green or even bluish appearance. Colonies have a shiny “gun-metal” appearance and a characteristic fruity odor. Two of the identifying biochemical characteristics of P. aeruginosa are an inability to ferment lactose on MacConkey agar and a positive reaction in the oxidase test. Most strains are identified on the basis of these readily detectable laboratory features even before extensive biochemical testing is done. Some isolates from CF patients are easily identified by their mucoid appearance, which is due to the production of large amounts of the mucoid exopolysaccharide or alginate.

1	Unraveling the mechanisms that underlie disease caused by P. aeruginosa has proved challenging. Of the common gram-negative bacteria, no other species produces such a large number of putative virulence factors (Table 189-1). Yet P. aeruginosa rarely initiates an infectious process in the absence of host injury or compromise, and few of its putative virulence factors have been shown definitively to be involved in disease in humans. Despite its metabolic versatility and possession of multiple colonizing factors, P. aeruginosa exhibits no competitive advantage over enteric bacteria in the human gut; neither is it a normal inhabitant of the human gastrointestinal tract, despite the host’s continuous environmental exposure to the organism.

1	Virulence Attributes Involved in Acute P. aeruginosa Infections • motility and colonization A general tenet of bacterial pathogenesis is that most bacteria must adhere to surfaces or colonize a host niche in order to initiate disease. Most pathogens examined thus far possess adherence factors called adhesins. P. aeruginosa is no exception. Among its many adhesins are its pili, which demonstrate adhesive properties for a variety of cells and adhere best to injured cell surfaces. In the organism’s flagellum, the flagellin molecule binds to cells, and the flagellar cap attaches to mucins through the recognition of glycan chains. Other P. aeruginosa adhesins include the outer core of the lipopolysaccharide (LPS) molecule, which binds to the cystic fibrosis transmembrane conductance regulator (CFTR) and aids in internalization of the organism, and the alginate coat of mucoid strains, which enhances adhesion to cells and mucins. In addition, membrane proteins and lectins have been proposed

1	(CFTR) and aids in internalization of the organism, and the alginate coat of mucoid strains, which enhances adhesion to cells and mucins. In addition, membrane proteins and lectins have been proposed as colonization factors. The deletion of any given adhesin is not sufficient to abrogate the ability of P. aeruginosa to colonize surfaces. Motility is important in host invasion via mucosal surfaces in some animal models; however, nonmotile strains are not uniformly avirulent.

1	evasion of Host defenses The transition from bacterial colonization to disease requires the evasion of host defenses followed by invasion of the microorganism. P. aeruginosa appears to be well equipped for evasion. Attached bacteria inject four known toxins (ExoS or ExoU, ExoT, and ExoY) via a type III secretion system that allows the bacteria to evade phagocytic cells either by direct cytotoxicity or by inhibition of phagocytosis. Mutants with defects in this system fail to disseminate in some animal models of infection. The type II secretion system as a whole secretes toxins that can kill animals, and some of its secreted toxins, such as exotoxin A, have the potential to kill phagocytic cells. Multiple proteases secreted by this system may degrade host effector molecules, such as cytokines and chemokines, that are released in response to infection. Thus this system may also contribute to host evasion.

1	tissUe injUry Among gram-negative bacteria, P. aeruginosa probably produces the largest number of substances that are toxic to cells and thus may injure tissues. The toxins secreted by its type III secretion system are capable of tissue injury. However, their delivery requires the adherence of the organism to cells. Thus, the effects of these toxins are likely to be local or to depend on the presence of vast numbers of bacteria. On the other hand, diffusible toxins, secreted by the organism’s type II secretion system, can act freely wherever they come into contact with cells. Possible effectors include exotoxin A, four different proteases, and at least two phospholipases; in addition to these secreted toxins, rhamnolipids, pyocyanin, and hydrocyanic acid are produced by P. aeruginosa and are all capable of inducing host injury.

1	inflammatory components The inflammatory components of P. aeruginosa—e.g., the inflammatory responses to the lipid A component of LPSs and to flagellin, mediated through the Toll-like receptor (TLR) system (principally TLR4 and TLR5)—have been thought to represent important factors in disease causation. Although these inflammatory responses are required for successful defense against P. aeruginosa (i.e., in their absence, animals are defenseless against P. aeruginosa infection), florid responses are likely to result in disease. When the sepsis syndrome and septic shock develop in P. aeruginosa infection, they are probably the result of the host response to one or both of these substances, but injury to the lung by Pseudomonas toxins may also result in sepsis syndromes, possibly by causing cell death and the release of cellular components (e.g., heat-shock proteins) that may activate the TLR or another proinflammatory system.

1	Infections Due to Pseudomonas Species and Related Organisms 1044 Chronic P. aeruginosa Infections Chronic infection due to P. aeruginosa occurs mainly in the lungs in the setting of structural pulmonary diseases. The classic example is CF; others include bronchiectasis and chronic relapsing panbronchiolitis, a disease seen in Japan and some Pacific Islands. Hallmarks of these illnesses are altered mucociliary clearance leading to mucus stasis and mucus accumulation in the lungs. There is probably a common factor that selects for P. aeruginosa colonization in these lung diseases—perhaps the adhesiveness of P. aeruginosa for mucus, a phenomenon that is not noted for most other common gram-negative bacteria, and/or the ability of P. aeruginosa to evade host defenses in mucus. Furthermore, P. aeruginosa seems to evolve in ways that allow its prolonged survival in the lung without an early fatal outcome for the host. The strains found in CF patients exhibit minimal production of virulence

1	aeruginosa seems to evolve in ways that allow its prolonged survival in the lung without an early fatal outcome for the host. The strains found in CF patients exhibit minimal production of virulence factors. Some strains even lose the ability to produce pili and flagella, and most become complement-sensitive because of the loss of the O side chain of their LPS molecules. An example of the impact of these changes is found in the organism’s discontinuation of the production of flagellin (probably its most strongly proinflammatory molecule) when it encounters purulent mucus. This response probably dampens the host’s response, allowing the organism to survive in mucus. P. aeruginosa is also believed to lose the ability to secrete many of its injectable toxins during growth in mucus. Although the alginate coat is thought to play a role in the organism’s survival, alginate is not essential, as nonmucoid strains may also predominate for long periods. In short, virulence in chronic infections

1	alginate coat is thought to play a role in the organism’s survival, alginate is not essential, as nonmucoid strains may also predominate for long periods. In short, virulence in chronic infections may be mediated by the chronic but attenuated host inflammatory response, which injures the lungs over decades.

1	P. aeruginosa causes infections at almost all sites in the body but shows a rather strong predilection for the lungs. The infections encountered most commonly in hospitalized patients are described below. Bacteremia Crude mortality rates exceeding 50% have been reported among patients with P. aeruginosa bacteremia. Consequently, this clinical entity has been much feared, and its management has been attempted with the use of multiple antibiotics. Recent publications report attributable mortality rates of 28–44%, with the precise figure depending on the adequacy of treatment and the seriousness of the underlying disease. In the past, the patient with P. aeruginosa bacteremia classically was neutropenic or had a burn injury. Today, however, a minority of such patients have bacteremic P. aeruginosa infections. Rather, P. aeruginosa bacteremia is seen most often in patients in ICUs.

1	The clinical presentation of P. aeruginosa bacteremia rarely differs from that of sepsis in general (Chap. 324). Patients are usually febrile, but those who are most severely ill may be in shock or even hypothermic. The only point differentiating this entity from gram-negative sepsis of other causes may be the distinctive skin lesions (ecthyma gangrenosum) of Pseudomonas infection, which occur almost exclusively in markedly neutropenic patients and patients with AIDS. These small or large, painful, reddish, maculopapular lesions have a geographic margin; they are initially pink, then darken to purple, and finally become black and necrotic (Fig. 189-1). Histopathologic studies indicate that the lesions are due to vascular invasion and are teeming with bacteria. Although similar lesions may occur in aspergillosis and FIGuRE 189-1 Ecthyma gangrenosum in a neutropenic patient 3 days after onset.

1	FIGuRE 189-1 Ecthyma gangrenosum in a neutropenic patient 3 days after onset. mucormycosis, their presence suggests P. aeruginosa bacteremia as the most likely diagnosis. TREATMEnT p. aerUginosa bacteremia (Table 189-2) Antimicrobial treatment of P. aeruginosa bacteremia has been controversial. Before 1971, the outcome of Pseudomonas bacteremia in febrile neutropenic patients treated with the available agents—gentamicin and the polymyxins—was dismal. However, treatment with carbenicillin, with or without an aminoglycoside, significantly improved outcomes. Concurrently, several retrospective analyses suggested that the use of two agents that were synergistic against gram-negative pathogens in vitro resulted in better outcomes in neutropenic patients. Thus, combination therapy became the standard of care—first for P. aeruginosa bacteremia in febrile neutropenic patients and then for all P. aeruginosa infections in neutropenic or nonneutropenic patients.

1	With the introduction of newer antipseudomonal drugs, a number of studies have revisited the choice between combination treatment and monotherapy for Pseudomonas bacteremia. Although the majority of experts still favor combination therapy, most of these observational studies indicate that a single modern antipseudomonal β-lactam agent to which the isolate is sensitive is as efficacious as a combination. Even in patients at greatest risk of early death from P. aeruginosa bacteremia (i.e., those with fever and neutropenia), empirical antipseudomonal monotherapy is deemed to be as efficacious as empirical combination therapy by the practice guidelines of the Infectious Diseases Society of America. One firm conclusion is that monotherapy with an aminoglycoside is not optimal. There are, of course, institutions and countries where rates of susceptibility of P. aeruginosa to first-line antibiotics are <80%. Thus, when a septic patient with a high probability of

1	There are, of course, institutions and countries where rates of susceptibility of P. aeruginosa to first-line antibiotics are <80%. Thus, when a septic patient with a high probability of P. aeruginosa infection is encountered in such settings, empirical combination therapy should be administered until the pathogen is identified and susceptibility data become available. Thereafter, whether one or two agents should be continued remains a matter of individual preference. Recent studies suggest that extended infusions of β-lactams such as cefepime or piperacillin-tazobactam may result in better outcomes of Pseudomonas bacteremia and possibly Pseudomonas pneumonia.

1	Acute Pneumonia Respiratory infections are the most common of all infections caused by P. aeruginosa. This organism appears first or second among the causes of ventilator-associated pneumonia (VAP). However, much debate centers on the actual role of P. aeruginosa in VAP. Many of the relevant data are based on cultures of sputum or endotracheal tube aspirates and may represent nonpathogenic colonization of the tracheobronchial tree, biofilms on the endotracheal tube, or simple tracheobronchitis.

1	Older reports of P. aeruginosa pneumonia described patients with an acute clinical syndrome of fever, chills, cough, and necrotizing pneumonia indistinguishable from other gram-negative bacterial pneumonias. The traditional accounts described a fulminant infection. Chest radiographs demonstrated bilateral pneumonia, often with nodular densities with or without cavities. This picture is now remarkably rare. Today, the typical patient is on a ventilator, has a slowly progressive infiltrate, and has been colonized with P. aeruginosa for days. While some cases may progress rapidly over 48–72 h, they are the exceptions. Nodular densities are not commonly seen. However, infiltrates may go on to necrosis. Necrotizing pneumonia has also been seen in the community (e.g., after inhalation of hot-tub water contaminated with P. aeruginosa). The typical patient has fever, leukocytosis, and purulent sputum, and the chest radiograph shows a new infiltrate or the expansion of a preexisting

1	of hot-tub water contaminated with P. aeruginosa). The typical patient has fever, leukocytosis, and purulent sputum, and the chest radiograph shows a new infiltrate or the expansion of a preexisting infiltrate. Chest examination generally detects rales or dullness. Of course, such findings are quite common among ventilated patients in the ICU. A sputum Gram’s stain showing mainly polymorphonuclear leukocytes (PMNs) in conjunction with a culture positive for P. aeruginosa in this setting suggests a diagnosis of acute P. aeruginosa pneumonia. There is no consensus about whether

1	Abbreviations: IDSA, Infectious Diseases Society of America; TMP-SMX, trimethoprim-sulfamethoxazole. Add an aminoglycoside for patients in shock and in regions or hospitals where rates of resistance to the primary β-lactam agents are high. Tobramycin may be used instead of amikacin (susceptibility permitting). The duration of therapy is 7 days for nonneutropenic patients. Neutropenic patients should be treated until no longer neutropenic. Resistance during therapy is common. Surgery is required for relapse. IDSA guidelines recommend the addition of an aminoglycoside or ciprofloxacin. The duration of therapy is 10–14 days. Duration of therapy varies with the drug used (e.g., 6 weeks for a β-lactam agent; at least 3 months for oral therapy except in puncture-wound osteomyelitis, for which the duration should be 2–4 weeks). Abscesses or other closed-space infections may require drainage. The duration of therapy is ≥2 weeks.

1	Abscesses or other closed-space infections may require drainage. The duration of therapy is ≥2 weeks. Use maximal strengths available or compounded by pharmacy. Therapy should be administered for 2 weeks or until the resolution of eye lesions, whichever is shorter. Relapse may occur if an obstruction or a foreign body is present. The duration of therapy for complicated UTI is 7–10 days (up to 2 weeks for pyelonephritis). Doses used have varied. Dosage adjustment is required in renal failure. Inhaled colistin may be added for pneumonia (100 mg q12h). Resistance to all agents is increasing. Levofloxacin or tigecycline may be alternatives, but there is little published clinical experience with these agents. Resistance to both agents is increasing. Do not use them in combination because of possible antagonism.

1	Resistance to both agents is increasing. Do not use them in combination because of possible antagonism. Infections Due to Pseudomonas Species and Related Organisms an invasive procedure (e.g., bronchoalveolar lavage or protected-brush sampling of the distal airways) is superior to tracheal aspiration to obtain samples for lung cultures in order to substantiate the occurrence of P. aeruginosa pneumonia and prevent antibiotic overuse.

1	(Table 189-2) Therapy for P. aeruginosa pneumonia has been unsatisfactory. Reports suggest mortality rates of 40–80%, but how many of these deaths are attributable to underlying disease remains unknown. The drugs of choice for P. aeruginosa pneumonia are similar to those given for bacteremia. A potent antipseudomonal β-lactam drug is the mainstay of therapy. Failure rates were high when aminoglycosides were used as single agents, possibly because of their poor penetration into the airways and their binding to airway secretions. Thus a strong case cannot be made for the inclusion of the aminoglycoside component in regimens used against fully susceptible organisms, especially given the evidence that aminoglycosides are not optimally active in the lungs at concentrations normally reached after IV administration. Nonetheless, aminoglycosides are commonly used in clinical practice. Some experts suggest the combination of a β-lactam agent and an antipseudomonal fluoroquinolone instead when

1	IV administration. Nonetheless, aminoglycosides are commonly used in clinical practice. Some experts suggest the combination of a β-lactam agent and an antipseudomonal fluoroquinolone instead when combination therapy is desired.

1	Chronic Respiratory Tract Infections P. aeruginosa is responsi ble for chronic infections of the airways associated with a number of underlying or predisposing conditions—most commonly CF (Chap. 313). A state of chronic colonization beginning early in childhood is seen in some Asian populations with chronic or diffuse panbronchiolitis, a disease of unknown etiology. P. aeruginosa is one of the organisms that colonizes damaged bronchi in bronchiectasis, a disease secondary to multiple causes in which profound structural abnormalities of the airways result in mucus stasis.

1	Optimal management of chronic P. aeruginosa lung infection has not been determined. Patients respond clinically to antipseudomonal therapy, but the organism is rarely eradicated. Because 1046 eradication is unlikely, the aim of treatment for chronic infection is to quell exacerbations of inflammation. The regimens used are similar to those used for pneumonia, but an aminoglycoside is almost always added because resistance is common in chronic disease. However, it may be appropriate to use an inhaled aminoglycoside preparation in order to maximize airway drug levels.

1	Endovascular Infections Infective endocarditis due to P. aeruginosa is a disease of IV drug users whose native valves are involved. This organism has also been reported to cause prosthetic valve endocarditis. Sites of prior native-valve injury due to the injection of foreign material such as talc or fibers probably serve as niduses for bacterial attachment to the heart valve. The manifestations of P. aeruginosa endocarditis resemble those of other forms of endocarditis in IV drug users except that the disease is more indolent than Staphylococcus aureus endocarditis. While most disease involves the right side of the heart, left-sided involvement is not rare and multivalvular disease is common. Fever is a common manifestation, as is pulmonary involvement (due to septic emboli to the lungs). Hence, patients may also experience chest pain and hemoptysis. Involvement of the left side of the heart may lead to signs of cardiac failure, systemic emboli, and local cardiac involvement with

1	Hence, patients may also experience chest pain and hemoptysis. Involvement of the left side of the heart may lead to signs of cardiac failure, systemic emboli, and local cardiac involvement with sinus of Valsalva abscesses and conduction defects. Skin manifestations are rare in this disease, and ecthyma gangrenosum is not seen. The diagnosis is based on positive blood cultures along with clinical signs of endocarditis.

1	(Table 189-2) It has been customary to use synergistic antibiotic combinations in treating P. aeruginosa endocarditis because of the development of resistance during therapy with a single antipseudomonal β-lactam agent. Which combination therapy is preferable is unclear, as all combinations have failed. Cases of P. aeruginosa endocarditis that relapse during or fail to respond to therapy are often caused by resistant organisms and may require surgical therapy. Other considerations for valve replacement are similar to those in other forms of endocarditis (Chap. 155).

1	Bone and Joint Infections P. aeruginosa is an infrequent cause of bone and joint infections. However, Pseudomonas bacteremia or infective endocarditis caused by the injection of contaminated illicit drugs has been documented to result in vertebral osteomyelitis and sternoclavicular joint arthritis. The clinical presentation of vertebral P. aeruginosa osteomyelitis is more indolent than that of staphylococcal osteomyelitis. The duration of symptoms in IV drug users with vertebral osteomyelitis due to P. aeruginosa varies from weeks to months. Fever is not uniformly present; when present, it tends to be low grade. There may be mild tenderness at the site of involvement. Blood cultures are usually negative unless there is concomitant endocarditis. The erythrocyte sedimentation rate (ESR) is generally elevated. Vertebral osteomyelitis due to P. aeruginosa has also been reported in the elderly, in whom it originates from urinary tract infections (UTIs). The infection generally involves the

1	elevated. Vertebral osteomyelitis due to P. aeruginosa has also been reported in the elderly, in whom it originates from urinary tract infections (UTIs). The infection generally involves the lumbosacral area because of a shared venous drainage (Batson’s plexus) between the lumbosacral spine and the pelvis. Sternoclavicular septic arthritis due to P. aeruginosa is seen almost exclusively in IV drug users. This disease may occur with or without endocarditis, and a primary site of infection often is not found. Plain radiographs show joint or bone involvement. Treatment of these forms of disease is generally successful.

1	Pseudomonas osteomyelitis of the foot most often follows puncture wounds through sneakers and mostly affects children. The main manifestation is pain in the foot, sometimes with superficial cellulitis around the puncture wound and tenderness on deep palpation of the wound. Multiple joints or bones of the foot may be involved. Systemic symptoms are generally absent, and blood cultures are usually negative. Radiographs may or may not be abnormal, but the bone scan is usually positive, as are magnetic resonance imaging (MRI) studies. Needle aspiration usually yields a diagnosis. Prompt surgery, with exploration of the nail puncture tract and debridement of the involved bones and cartilage, is generally recommended in addition to antibiotic therapy.

1	Central Nervous System (CNS) Infections CNS infections due to P. aeruginosa are relatively rare. Involvement of the CNS is almost always secondary to a surgical procedure or head trauma. The entity seen most often is postoperative or posttraumatic meningitis. Subdural or epidural infection occasionally results from contamination of these areas. Embolic disease arising from endocarditis in IV drug users and leading to brain abscesses has also been described. The cerebrospinal fluid (CSF) profile of P. aeruginosa meningitis is no different from that of pyogenic meningitis of any other etiology.

1	(Table 189-2) Treatment of Pseudomonas meningitis is difficult; little information has been published, and no controlled trials in humans have been undertaken. However, the general principles involved in the treatment of meningitis apply, including the need for high doses of bactericidal antibiotics to attain high drug levels in the CSF. The agent with which there is the most published experience in P. aeruginosa meningitis is ceftazidime, but other antipseudomonal β-lactam drugs that reach high CSF concentrations, such as cefepime and meropenem, have also been used successfully. Other forms of P. aeruginosa CNS infection, such as brain abscesses and epidural and subdural empyema, generally require surgical drainage in addition to antibiotic therapy.

1	Eye Infections Eye infections due to P. aeruginosa occur mainly as a result of direct inoculation into the tissue during trauma or surface injury by contact lenses. Keratitis and corneal ulcers are the most common types of eye disease and are often associated with contact lenses (especially the extended-wear variety). Keratitis can be slowly or rapidly progressive, but the classic description is disease progressing over 48 h to involve the entire cornea, with opacification and sometimes perforation. P. aeruginosa keratitis should be considered a medical emergency because of the rapidity with which it can progress to loss of sight. P. aeruginosa endophthalmitis secondary to bacteremia is the most devastating of P. aeruginosa eye infections. The disease is fulminant, with severe pain, chemosis, decreased visual acuity, anterior uveitis, vitreous involvement, and panophthalmitis.

1	(Table 189-2) The usual therapy for keratitis is the administration of topical antibiotics. Therapy for endophthalmitis includes the use of high-dose local and systemic antibiotics (to achieve higher drug concentrations in the eye) and vitrectomy.

1	Ear Infections P. aeruginosa infections of the ears vary from mild swimmer’s ear to serious life-threatening infections with neurologic sequelae. Swimmer’s ear is common among children and results from infection of moist macerated skin of the external ear canal. Most cases resolve with treatment, but some patients develop chronic drainage. Swimmer’s ear is managed with topical antibiotic agents (otic solutions). The most serious form of Pseudomonas infection involving the ear has been given various names: two of these designations, malignant otitis externa and necrotizing otitis externa, are now used for the same entity. This disease was originally described in elderly diabetic patients, in whom the majority of cases still occur. However, it has also been described in patients with AIDS and in elderly patients without underlying diabetes or immunocompromise. The usual presenting symptoms are decreased hearing and ear pain, which may be severe and lancinating. The pinna is usually

1	and in elderly patients without underlying diabetes or immunocompromise. The usual presenting symptoms are decreased hearing and ear pain, which may be severe and lancinating. The pinna is usually painful, and the external canal may be tender. The ear canal almost always shows signs of inflammation, with granulation tissue and exudate. Tenderness anterior to the tragus may extend as far as the temporomandibular joint and mastoid process. A small minority of patients have systemic symptoms. Patients in whom the diagnosis is made late may present with cranial nerve palsies or even with cavernous venous sinus thrombosis. The ESR is invariably elevated (≥100 mm/h). The diagnosis is made on clinical grounds in severe cases; however, the “gold standard” is a positive technetium-99 bone scan in a patient with otitis externa due to P. aeruginosa. In diabetic patients, a positive bone scan constitutes presumptive evidence for this diagnosis and should prompt biopsy or empirical therapy.

1	(Table 189-2) Given the infection of the ear cartilage, sometimes with mastoid or petrous ridge involvement, patients with malignant (necrotizing) otitis externa are treated as for osteomyelitis. urinary Tract Infections UTIs due to P. aeruginosa generally occur as a complication of a foreign body in the urinary tract, an obstruction in the genitourinary system, or urinary tract instrumentation or surgery. However, UTIs caused by P. aeruginosa have been described in pediatric outpatients without stones or evident obstruction.

1	(Table 189-2) Most P. aeruginosa UTIs are considered complicated infections that must be treated longer than uncomplicated cystitis. In general, a 7to 10-day course of treatment suffices, with up to 2 weeks of therapy in cases of pyelonephritis. Urinary catheters, stents, or stones should be removed to prevent relapse, which is common and may be due not to resistance but rather to factors such as a foreign body that has been left in place or an ongoing obstruction.

1	Skin and Soft Tissue Infections Besides pyoderma gangrenosum in neutropenic patients, folliculitis and other papular or vesicular lesions due to P. aeruginosa have been extensively described and are collectively referred to as dermatitis. Multiple outbreaks have been linked to whirlpools, spas, and swimming pools. To prevent such outbreaks, the growth of P. aeruginosa in the home and in recreational environments must be controlled by proper chlorination of water. Most cases of hot-tub folliculitis are self-limited, requiring only the avoidance of exposure to the contaminated source of water.

1	Toe-web infections occur especially often in the tropics, and the “green nail syndrome” is caused by P. aeruginosa paro nychia, which results from frequent submersion of the hands in water. In the latter entity, the green discoloration results from diffusion of pyocyanin into the nail bed. P. aeruginosa remains a prominent cause of burn wound infections in some parts of the world. The management of these infections is best left to specialists in burn wound care.

1	nia, P. aeruginosa has historically been the organism against which empirical coverage is always essential. Although in Western countries these infections are now less common, their importance has not diminished because of persistently high mortality rates. In other parts of the world as well, P. aeruginosa continues to be a significant problem in febrile neutropenia, causing a larger proportion of infections in febrile neutropenic patients than any other single organism. For example, P. aeruginosa was responsible for 28% of documented infections in 499 febrile neutropenic patients in one study from the Indian subcontinent and for 31% of such infections in another. In a large study of infections in leukemia patients from Japan,

1	P. aeruginosa was the most frequently documented cause of bacterial infection. In studies performed in North America, northern Europe, and Australia, the incidence of P. aeruginosa bacteremia in febrile neutropenia was quite variable. In a review of 97 reports published in 1987–1994, the incidence was reported to be 1–2.5% among febrile neutropenic patients given empirical therapy and 5–12% among microbiologically documented infections. The most common clinical 1047 syndromes encountered were bacteremia, pneumonia, and soft tissue infections manifesting mainly as ecthyma gangrenosum.

1	(Table 189-2) Compared with rates three decades ago, improved rates of response to antibiotic therapy have been reported in many studies. A study of 127 patients demonstrated a reduction in the mortality rate from 71% to 25% with the introduction of ceftazidime and imipenem. Because neutrophils—the normal host defenses against this organism—are absent in febrile neutropenic patients, maximal doses of antipseudomonal β-lactam antibiotics should be used for the management of P. aeruginosa bacteremia in this setting.

1	Infections in Patients with AIDS Both communityand hospital-acquired P. aeruginosa infections were documented in patients with AIDS before the advent of antiretroviral therapy. Since the introduction of protease inhibitors, P. aeruginosa infections in AIDS patients have been seen less frequently but still occur, particularly in the form of sinusitis. The clinical presentation of Pseudomonas infection (especially pneumonia and bacteremia) in AIDS patients is remarkable in that, although the illness may appear not to be severe, the infection may nonetheless be fatal. Patients with bacteremia may have only a low-grade fever and may present with ecthyma gangrenosum. Pneumonia, with or without bacteremia, is perhaps the most common type of P. aeruginosa infection in AIDS patients. Patients with AIDS and P. aeruginosa pneumonia exhibit the classic clinical signs and symptoms of pneumonia, such as fever, productive cough, and chest pain. The infection may be lobar or multilobar and shows no

1	AIDS and P. aeruginosa pneumonia exhibit the classic clinical signs and symptoms of pneumonia, such as fever, productive cough, and chest pain. The infection may be lobar or multilobar and shows no predisposition for any particular location. The most striking feature is the high frequency of cavitary disease.

1	Therapy for any of these conditions in AIDS patients is no different from that in other patients. However, relapse is the rule unless the patient’s CD4+ T cell count rises to >50/μL or suppressive antibiotic therapy is given. In attempts to achieve cures and prevent relapses, therapy tends to be more prolonged than in the case of an immunocompetent patient.

1	Multidrug-Resistant Infections (Table 189-2) P. aeruginosa has a notorious propensity to develop antibiotic resistance. During three decades, the impact of resistance was minimized by the rapid development of potent antipseudomonal agents. However, the situation has recently changed, with the worldwide selection of strains carrying determinants that mediate resistance to β-lactams, fluoroquinolones, and aminoglycosides. This situation has been compounded by the lack of development of new classes of antipseudomonal drugs for nearly two decades. Physicians now resort to drugs such as colistin and polymyxin, which were discarded decades ago. These alternative approaches to the management of multiresistant P. aeruginosa infections were first used some time ago in CF patients, who receive colistin (polymyxin E) IV and by aerosol despite its renal toxicity. Colistin is rapidly becoming the last-resort agent of choice, even in non-CF patients infected with multiresistant P. aeruginosa.

1	The clinical outcome of multidrug-resistant P. aeruginosa infections treated with colistin is difficult to judge from case reports, especially given the many drugs used in the complicated management of these patients. Although earlier reports described marginal efficacy and serious nephrotoxicity and neurotoxicity, recent reports have been more encouraging. Because colistin shows synergy with other antimicrobial agents in vitro, it may be possible to reduce the dosage—and thus the toxicity—of this drug when it is combined with drugs such as rifampin Infections Due to Pseudomonas Species and Related Organisms 1048 and β-lactams; however, no studies in humans or animals support this lungs in CF, B. cepacia appears as an airway colonizer during broad-approach at this time. spectrum antibiotic therapy and is a cause of VAP, catheter-associated infections, and wound infections. TREATMEnT b. cepacia infections

1	TREATMEnT b. cepacia infections S. maltophilia is the only potential human pathogen among a genus of ubiquitous organisms found in the rhizosphere (i.e., the soil that surrounds the roots of plants). The organism is an opportunist that is acquired from the environment but is even more limited than

1	P. aeruginosa in its ability to colonize patients or cause infections. Immunocompromise is not sufficient to permit these events; rather, major perturbations of the human flora are usually necessary for the establishment of S. maltophilia. Accordingly, most cases of human infection occur in the setting of very broad-spectrum antibiotic therapy with agents such as advanced cephalosporins and carbapenems, which eradicate the normal flora and other pathogens. The remarkable ability of S. maltophilia to resist virtually all classes of antibiotics is attributable to the possession of antibiotic efflux pumps and of two β-lactamases (L1 and L2) that mediate β-lactam resistance, including that to carbapenems. It is fortunate that the virulence of S. maltophilia appears to be limited. Although a serine protease is present in some strains, virulence is probably a result of the host’s inflammatory response to components of the organism such as LPS and flagellin.

1	S. maltophilia is most commonly found in the respiratory tract of ventilated patients, where the distinction between its roles as a colonizer and as a pathogen is often difficult to make. However, S. maltophilia does cause pneumonia and bacteremia in such patients, and these infections have led to septic shock. Also common is central venous line–associated infection (with or without bacteremia), which has been reported most often in patients with cancer. S. maltophilia is a rare cause of ecthyma gangrenosum in neutropenic patients. It has been isolated from ~5% of CF patients but is not believed to be a significant pathogen in this setting. TREATMEnT s. maltopHilia infections

1	TREATMEnT s. maltopHilia infections The intrinsic resistance of S. maltophilia to most antibiotics renders infection difficult to treat. The antibiotics to which it is most often (although not uniformly) susceptible are trimethoprim-sulfamethoxazole (TMP-SMX), ticarcillin/clavulanate, levofloxacin, and tigecycline (Table 189-2). Consequently, a combination of TMPSMX and ticarcillin/clavulanate is recommended for initial therapy. Catheters must be removed in the treatment of bacteremia to hasten cure and prevent relapses. The treatment of VAP due to S. maltophilia is much more difficult than that of bacteremia, with the frequent development of resistance during therapy.

1	S. maltophilia is much more difficult than that of bacteremia, with the frequent development of resistance during therapy. B. cepacia gained notoriety as the cause of a rapidly fatal syndrome of respiratory distress and septicemia (the “cepacia syndrome”) in CF patients. Previously, it had been recognized as an antibiotic-resistant nosocomial pathogen (then designated P. cepacia) in ICU patients. Patients with chronic granulomatous disease are also predisposed to

1	B. cepacia lung disease. The organism has been reclassified into nine subgroups, only some of which are common in CF. B. cepacia is an environmental organism that inhabits moist environments and is found in the rhizosphere. This organism possesses multiple virulence factors that may play roles in disease as well as colonizing factors that are capable of binding to lung mucus—an ability that may explain the predilection of B. cepacia for the lungs in CF. B. cepacia secretes elastase and possesses components of an injectable toxin-secretion system like that of P. aeruginosa; its LPS is among the most potent of all LPSs in stimulating an inflammatory response in the lungs. Inflammation may be the major cause of the lung disease seen in the cepacia syndrome. The organism can penetrate epithelial surfaces by virtue of motility and inhibition of host innate immune defenses. Besides infecting the

1	B. cepacia is intrinsically resistant to many antibiotics. Therefore, treatment must be tailored according to sensitivities. TMP-SMX, meropenem, and doxycycline are the most effective agents in vitro and may be started as first-line agents (Table 189-2). Some strains are susceptible to third-generation cephalosporins and fluoroquinolones, and these agents may be used against isolates known to be susceptible. Combination therapy for serious pulmonary infection (e.g., in CF) is suggested when multidrug-resistant strains are implicated; the combination of meropenem and TMP-SMX may be antagonistic, however. Resistance to all agents used has been reported during therapy.

1	B. pseudomallei is the causative agent of melioidosis, a disease of humans and animals that is geographically restricted to Southeast Asia and northern Australia, with occasional cases in countries such as India and China. This organism may be isolated from individuals returning directly from these endemic regions and from military personnel who have served in endemic regions and then returned home after stops in Europe. Symptoms of this illness may develop only at a later date because of the organism’s ability to cause latent infections. B. pseudomallei is found in soil and water. Humans and animals are infected by inoculation, inhalation, or ingestion; only rarely is the organism transmitted from person to person. Humans are not colonized without being infected. Among the pseudomonads,

1	B. pseudomallei is perhaps the most virulent. Host compromise is not an essential prerequisite for disease, although many patients have common underlying medical diseases (e.g., diabetes or renal failure). B. pseudomallei is a facultative intracellular organism whose replication in PMNs and macrophages may be aided by the possession of a polysaccharide capsule. The organism also possesses elements of a type III secretion system that plays a role in its intracellular survival. During infection, there is a florid inflammatory response whose role in disease is unclear.

1	B. pseudomallei causes a wide spectrum of disease, ranging from asymptomatic infection to abscesses, pneumonia, and disseminated disease. It is a significant cause of fatal community-acquired pneumonia and septicemia in endemic areas, with mortality rates as high as 44% reported in Thailand. Acute pulmonary infection is the most commonly diagnosed form of melioidosis. Pneumonia may be asymptomatic (with routine chest radiographs showing mainly upper-lobe infiltrates) or may present as severe necrotizing disease. B. pseudomallei also causes chronic pulmonary infections with systemic manifestations that mimic those of tuberculosis, including chronic cough, fever, hemoptysis, night sweats, and cavitary lung disease. Besides pneumonia, the other principal form of B. pseudomallei disease is skin ulceration with associated lymphangitis and regional lymphadenopathy. Spread from the lungs or skin, which is most often documented in debilitated individuals, gives rise to septicemic forms of

1	skin ulceration with associated lymphangitis and regional lymphadenopathy. Spread from the lungs or skin, which is most often documented in debilitated individuals, gives rise to septicemic forms of melioidosis that carry a high mortality rate.

1	TREATMEnT b. pseUdomallei infections B. pseudomallei is susceptible to advanced penicillins and cephalosporins and to carbapenems (Table 189-2). Treatment is divided into two stages: an intensive 2-week phase of therapy with ceftazidime or a carbapenem followed by at least 12 weeks of oral TMP-SMX to eradicate the organism and prevent relapse. The recognition of this bacterium as a potential agent of biologic warfare has stimulated interest in the development of a vaccine. B. mallei causes the equine disease glanders in Africa, Asia, and South America. The organism was eradicated from Europe and North America decades ago. The last case seen in the United States occurred in 2001 in a laboratory worker; before that,

1	B. mallei had last been seen in this country in 1949. In contrast to the other organisms discussed in this chapter, B. mallei is not an environmental organism and does not persist outside its equine hosts. Consequently, B. mallei infection is an occupational risk for handlers of horses, equine butchers, and veterinarians in areas of the world where it still exists. The polysaccharide capsule is a critical virulence determinant; diabetics are thought to be especially susceptible to infection by this organism. The organism is transmitted from animals to humans by inoculation into the skin, where it causes local infection with nodules and lymphadenitis. Regional lymphadenopathy is common. Respiratory secretions from infected horses are extremely infectious. Inhalation results in clinical signs of typical pneumonia but may also cause an acute febrile illness with ulceration of the trachea. The organism may disseminate from the skin or lungs to cause septicemia with signs of sepsis. The

1	of typical pneumonia but may also cause an acute febrile illness with ulceration of the trachea. The organism may disseminate from the skin or lungs to cause septicemia with signs of sepsis. The septicemic form is frequently associated with shock and a high mortality rate. The infection may also enter a chronic phase and present as disseminated abscesses. B. mallei infection may present as early as 1–2 days after inhalation or (in cutaneous disease) may not become evident for months.

1	TREATMEnT b. mallei infections The antibiotic susceptibility pattern of B. mallei is similar to that of B. pseudomallei; in addition, the organism is susceptible to the newer macrolides azithromycin and clarithromycin. B. mallei infection should be treated with the same drugs and for the same duration as melioidosis. David A. Pegues, Samuel I. Miller Bacteria of the genus Salmonella are highly adapted for growth in both humans and animals and cause a wide spectrum of disease. The growth of serotypes Salmonella typhi and Salmonella paratyphi is restricted to human hosts, in whom these organisms cause enteric (typhoid) fever. The remaining serotypes (nontyphoidal Salmonella, or NTS) can colonize the gastrointestinal tracts of a broad range of animals, including mammals, reptiles, birds, and insects. More than 200 serotypes of Salmonella are pathogenic to humans, in whom they often cause gastroenteritis and can be associated with localized infections and/or bacteremia.

1	This large genus of gram-negative bacilli within the family Enterobacteriaceae consists of two species: Salmonella enterica, which contains six subspecies, and Salmonella bongori. S. enterica subspecies I includes almost all the serotypes pathogenic for humans. Members of the seven Salmonella subspecies are classified into >2500 serotypes (serovars); for simplicity, Salmonella serotypes (most of which are named for the city where they were identified) are often used as the species designation. For example, the full taxonomic designation S. enterica subspecies enterica serotype Typhimurium can be shortened to Salmonella serotype Typhimurium or simply S. typhimurium. Serotyping is based on the somatic O antigen (lipopolysaccharide cell-wall components), the surface Vi antigen (restricted to S. typhi and S. paratyphi C), and the flagellar H antigen.

1	S. paratyphi C), and the flagellar H antigen. Salmonellae are gram-negative, non-spore-forming, facultatively anaerobic bacilli that measure 2–3 μm by 0.4–0.6 μm. The initial identification of salmonellae in the clinical microbiology labora-1049 tory is based on growth characteristics. Salmonellae, like other Enterobacteriaceae, produce acid on glucose fermentation, reduce nitrates, and do not produce cytochrome oxidase. In addition, all salmonellae except Salmonella gallinarum-pullorum are motile by means of peritrichous flagella, and all but S. typhi produce gas (H2S) on sugar fermentation. Notably, only 1% of clinical isolates ferment lactose; a high level of suspicion must be maintained to detect these rare clinical lactose-fermenting isolates.

1	Although serotyping of all surface antigens can be used for formal identification, most laboratories perform a few simple agglutination reactions that define specific O-antigen serogroups, designated A, B, C1, C2, D, and E. Strains in these six serogroups cause ~99% of Salmonella infections in humans and other warm-blooded animals. Molecular typing methods, including pulsed-field gel electrophoresis, polymerase chain reaction fingerprinting, and genomic DNA microarray analysis, are used in epidemiologic investigations to differentiate Salmonella strains of a common serotype.

1	All Salmonella infections begin with ingestion of organisms, most commonly in contaminated food or water. The infectious dose ranges from 200 colony-forming units (CFU) to 106 CFU, and the ingested dose is an important determinant of incubation period and disease severity. Conditions that decrease either stomach acidity (an age of <1 year, antacid ingestion, or achlorhydric disease) or intestinal integ rity (inflammatory bowel disease, prior gastrointestinal surgery, or alteration of the intestinal flora by antibiotic administration) increase susceptibility to Salmonella infection.

1	Once S. typhi and S. paratyphi reach the small intestine, they penetrate the mucus layer of the gut and traverse the intestinal layer through phagocytic microfold (M) cells that reside within Peyer’s patches. Salmonellae can trigger the formation of membrane ruffles in normally nonphagocytic epithelial cells. These ruffles reach out and enclose adherent bacteria within large vesicles by bacterial- mediated endocytosis. This process is dependent on the direct delivery of Salmonella proteins into the cytoplasm of epithelial cells by the specialized bacterial type III secretion system. These bacterial proteins mediate alterations in the actin cytoskeleton that are required for Salmonella uptake. After crossing the epithelial layer of the small intestine, S. typhi and

1	After crossing the epithelial layer of the small intestine, S. typhi and S. paratyphi, which cause enteric (typhoid) fever, are phagocytosed by macrophages. These salmonellae survive the antimicrobial environment of the macrophage by sensing environmental signals that trigger alterations in regulatory systems of the phagocytosed bacteria. For example, PhoP/PhoQ (the best-characterized regulatory system) triggers the expression of outer-membrane proteins and mediates modifications in lipopolysaccharide so that the altered bacterial surface can resist microbicidal activities and potentially alter host cell signaling. In addition, salmonellae encode a second type III secretion system that directly delivers bacterial proteins across the phagosome membrane into the macrophage cytoplasm. This secretion system functions to remodel the Salmonella-containing vacuole, promoting bacterial survival and replication.

1	Once phagocytosed, typhoidal salmonellae disseminate throughout the body in macrophages via the lymphatics and colonize reticuloendothelial tissues (liver, spleen, lymph nodes, and bone marrow). Patients have relatively few or no signs and symptoms during this initial incubation stage. Signs and symptoms, including fever and abdominal pain, probably result from secretion of cytokines by macrophages and epithelial cells in response to bacterial products that are recognized by innate immune receptors when a critical number of organisms have replicated. Over time, the development of hepatosplenomegaly is likely to be related to the recruitment of mononuclear cells and the development of a specific acquired cell-mediated immune response to S. typhi colonization. The recruitment of additional mononuclear cells and lymphocytes to Peyer’s patches during the several weeks after initial colonization/infection can result in marked enlargement and necrosis 1050 of the Peyer’s patches, which may

1	mononuclear cells and lymphocytes to Peyer’s patches during the several weeks after initial colonization/infection can result in marked enlargement and necrosis 1050 of the Peyer’s patches, which may be mediated by bacterial products that promote cell death as well as the inflammatory response. In contrast to enteric fever, which is characterized by an infiltration of mononuclear cells into the small-bowel mucosa, NTS gastroenteritis is characterized by massive polymorphonuclear leukocyte infiltration into both the largeand small-bowel mucosa. This response appears to depend on the induction of interleukin 8, a strong neutrophil chemotactic factor, which is secreted by intestinal cells as a result of Salmonella colonization and translocation of bacterial proteins into host cell cytoplasm. The degranulation and release of toxic substances by neutrophils may result in damage to the intestinal mucosa, causing the inflammatory diarrhea observed with nontyphoidal gastroenteritis. An

1	The degranulation and release of toxic substances by neutrophils may result in damage to the intestinal mucosa, causing the inflammatory diarrhea observed with nontyphoidal gastroenteritis. An additional important factor in the persistence of nontyphoidal salmonellae in the intestinal tract and the organisms’ capacity to compete with endogenous flora is the ability to utilize the sulfur-containing compound tetrathionate for metabolism in a microaerophilic environment. In the presence of intestinal inflammation, tetrathionate is generated from thiosulfate produced by epithelial cells through inflammatory cell production of reactive oxygen species.

1	Enteric (typhoid) fever is a systemic disease characterized by fever and abdominal pain and caused by dissemination of S. typhi or S. paratyphi. The disease was initially called typhoid fever because of its clinical similarity to typhus. In the early 1800s, typhoid fever was clearly defined pathologically as a unique illness on the basis of its association with enlarged Peyer’s patches and mesenteric lymph nodes. In 1869, given the anatomic site of infection, the term enteric fever was proposed as an alternative designation to distinguish typhoid fever from typhus. However, to this day, the two designations are used interchangeably.

1	In contrast to other Salmonella serotypes, the etiologic agents of enteric fever—S. typhi and S. paratyphi serotypes A, B, and C—have no known hosts other than humans. Most commonly, food-borne or waterborne transmission results from fecal contamination by ill or asymptomatic chronic carriers. Sexual transmission between male partners has been described. Health care workers occasionally acquire enteric fever after exposure to infected patients or during processing of clinical specimens and cultures.

1	With improvements in food handling and water/sewage treatment, enteric fever has become rare in developed nations. Worldwide, however, there are an estimated 27 million cases of enteric fever, with 200,000–600,000 deaths annually. The annual incidence is highest (>100 cases/100,000 population) in south-central and Southeast Asia; medium (10–100 cases/100,000) in the rest of Asia, Africa, Latin America, and Oceania (excluding Australia and New Zealand); and low in other parts of the world (Fig. 190-1). A high incidence of enteric fever correlates with poor sanitation and lack of access to clean drinking water. In endemic regions, enteric fever is more common in urban than rural areas and among young children and adolescents than among other age groups. Risk factors include contaminated water or ice, flooding, food and drinks purchased from street vendors, raw fruits and vegetables grown in fields fertilized with sewage, ill household contacts, lack of hand washing and toilet access,

1	or ice, flooding, food and drinks purchased from street vendors, raw fruits and vegetables grown in fields fertilized with sewage, ill household contacts, lack of hand washing and toilet access, and evidence of prior Helicobacter pylori infection (an association probably related to chronically reduced gastric acidity). It is estimated that there is one case of paratyphoid fever for every four cases of typhoid fever, but the incidence of infection associated with S. paratyphi A appears to be increasing, especially in India; this increase may be a result of vaccination for S. typhi.

1	Multidrug-resistant (MDR) strains of S. typhi emerged in the 1980s in China and Southeast Asia and have since disseminated widely. These strains contain plasmids encoding resistance to chloramphenicol, ampicillin, and trimethoprim—antibiotics long used to treat enteric fever. With the increased use of fluoroquinolones to treat MDR enteric fever in the 1990s, strains of S. typhi and S. paratyphi with decreased ciprofloxacin susceptibility (DCS; minimal inhibitory concentration [MIC], 0.125–0.5 μg/mL) or ciprofloxacin resistance (MIC, ≥1 μg/mL) have emerged on the Indian subcontinent, in southern Asia, and (most recently) in sub-Saharan Africa and have been associated with clinical treatment failure. Testing of isolates for resistance to the first-generation quinolone nalidixic acid detects many but not all strains with reduced susceptibility to ciprofloxacin and is no longer recommended. Strains of S. typhi and S. paratyphi producing extended-spectrum β-lactamases have emerged

1	detects many but not all strains with reduced susceptibility to ciprofloxacin and is no longer recommended. Strains of S. typhi and S. paratyphi producing extended-spectrum β-lactamases have emerged recently, primarily in India and Nepal.

1	Approximately 300 cases of typhoid and 150 cases of paratyphoid fever are reported annually in the United States. Of 1902 cases of S. typhi–associated enteric fever reported to the Centers for Disease Control and Prevention in 1999–2006, 79% were associated with recent international travel, most commonly to India (47%), Pakistan (10%), Bangladesh (10%), Mexico (7%), and the Philippines (4%). Only 5% of travelers diagnosed with enteric fever had received S. typhi vaccine. Overall, 13% of S. typhi isolates in the United States were resistant to ampicillin, chloramphenicol, and trimethoprimsulfamethoxazole (TMP-SMX), and the proportion of DCS isolates increased from 19% in 1999 to 58% in 2006. Infection with DCS

1	S. typhi was associated with travel to the Indian subcontinent. Of the 25–30% of reported cases of enteric fever in the United States that are domestically acquired, the majority are sporadic, but outbreaks linked to contaminated food products and previously unrecognized chronic carriers continue to occur. Enteric fever is a misnomer, in that the hallmark features of this disease—fever and abdominal pain—are variable. While fever is documented at presentation in >75% of cases, abdominal pain is reported in only 30–40%. Thus, a high index of suspicion for this potentially fatal systemic illness is necessary when a person presents with fever and a history of recent travel to a developing country.

1	The incubation period for S. typhi averages 10–14 days but ranges from 5 to 21 days, depending on the inoculum size and the host’s health and immune status. The most prominent symptom is prolonged fever (38.8°–40.5°C; 101.8°–104.9°F), which can continue for up to 4 weeks if untreated. S. paratyphi A is thought to cause milder disease than S. typhi, with predominantly gastrointestinal symptoms. However, a prospective study of 669 consecutive cases of enteric fever in Kathmandu, Nepal, found that the infections caused by these organisms were clinically indistinguishable. In this series, symptoms reported on initial medical evaluation included headache (80%), chills (35–45%), cough (30%), sweating (20–25%), myalgias (20%), malaise (10%), and arthralgia (2–4%). Gastrointestinal manifestations included anorexia (55%), abdominal pain (30–40%), nausea (18–24%), vomiting (18%), and diarrhea (22–28%) more commonly than constipation (13–16%). Physical findings included coated tongue (51–56%),

1	included anorexia (55%), abdominal pain (30–40%), nausea (18–24%), vomiting (18%), and diarrhea (22–28%) more commonly than constipation (13–16%). Physical findings included coated tongue (51–56%), splenomegaly (5–6%), and abdominal tenderness (4–5%).

1	High (>100/100,000/year) Medium (10–100/100,000/year) Low (<10/100,000/year) FIGuRE 190-1 Annual incidence of typhoid fever per 100,000 population. (Adapted from JA Crump et al: The global burden of typhoid fever. Bull World Health Organ 82:346, 2004.) FIGuRE 190-2 “Rose spots,” the rash of enteric fever due to Salmonella typhi or Salmonella paratyphi. Early physical findings of enteric fever include rash (“rose spots”; 30%), hepatosplenomegaly (3–6%), epistaxis, and relative bradycardia at the peak of high fever (<50%). Rose spots (Fig. 190-2; see also Fig. 25e-9) make up a faint, salmon-colored, blanching, maculopapular rash located primarily on the trunk and chest. The rash is evident in ~30% of patients at the end of the first week and resolves without a trace after 2–5 days. Patients can have two or three crops of lesions, and Salmonella can be cultured from punch biopsies of these lesions. The faintness of the rash makes it difficult to detect in highly pigmented patients.

1	The development of severe disease (which occurs in ~10–15% of patients) depends on host factors (immunosuppression, antacid therapy, previous exposure, and vaccination), strain virulence and inoculum, and choice of antibiotic therapy. Gastrointestinal bleeding (10–20%) and intestinal perforation (1–3%) most commonly occur in the third and fourth weeks of illness and result from hyperplasia, ulceration, and necrosis of the ileocecal Peyer’s patches at the initial site of Salmonella infiltration (Fig. 190-3). Both complications are life-threatening and require immediate fluid resuscitation and surgical intervention, with broadened antibiotic coverage for polymicrobial peritonitis (Chap.

1	159) and treatment of gastrointestinal hemorrhages, including bowel resection. Neurologic manifestations occur in 2–40% of patients and include meningitis, Guillain-Barré syndrome, neuritis, and neuropsychiatric symptoms (described as “muttering delirium” or “coma vigil”), with picking at bedclothes or imaginary objects. Rare complications whose incidences are reduced by prompt antibiotic treatment include disseminated intravascular coagulation, hematophagocytic syndrome, pancreatitis, hepatic and splenic abscesses and granulomas, endocarditis, pericarditis, myocarditis, orchitis, hepatitis, glomerulonephritis, pyelonephritis and hemolytic-uremic syndrome, severe pneumonia, arthritis, osteomyelitis, endophthalmitis, and parotitis. Up to 10% of patients develop mild relapse, usually within 2–3 weeks of fever resolution and in association with the same strain type and susceptibility profile. Up to 10% of untreated patients with typhoid fever excrete

1	Up to 10% of untreated patients with typhoid fever excrete S. typhi in the feces for up to 3 months, and 1–4% develop chronic FIGuRE 190-3 Typical ileal perforation associated with Salmonella typhi infection. (From JM Saxe, R Cropsey: Is operative management effective in treatment of perforated typhoid? Am J Surg 189:342, 2005.) asymptomatic carriage, shedding S. typhi in either urine or stool for >1 year. Chronic carriage is more common among women, infants, and persons who have biliary abnormalities or concurrent bladder infection with Schistosoma haematobium. The anatomic abnormalities associated with the latter conditions presumably allow prolonged colonization.

1	Because the clinical presentation of enteric fever is relatively nonspecific, the diagnosis needs to be considered in any febrile traveler returning from a developing region, especially the Indian subcontinent, the Philippines, or Latin America. Other diagnoses that should be considered in these travelers include malaria, hepatitis, bacterial enteritis, dengue fever, rickettsial infections, leptospirosis, amebic liver abscesses, and acute HIV infection (Chap. 149). Other than a positive culture, no specific laboratory test is diagnostic for enteric fever. In 15–25% of cases, leukopenia and neutropenia are detectable. Leukocytosis is more common among children, during the first 10 days of illness, and in cases complicated by intestinal perforation or secondary infection. Other nonspecific laboratory findings include moderately elevated values in liver function tests and muscle enzyme levels. The definitive diagnosis of enteric fever requires the isolation of

1	The definitive diagnosis of enteric fever requires the isolation of S. typhi or S. paratyphi from blood, bone marrow, other sterile sites, rose spots, stool, or intestinal secretions. The sensitivity of blood culture is only 40–80%, probably because of high rates of antibiotic use in endemic areas and the small number of S. typhi organisms (i.e., <15/mL) typically present in the blood. Because almost all S. typhi organisms in blood are associated with the mononuclear cell/platelet fraction, centrifugation of blood and culture of the buffy coat can substantially reduce the time to isolation of the organism but do not increase sensitivity.

1	Bone marrow culture is 55–90% sensitive, and, unlike that of blood culture, its yield is not reduced by up to 5 days of prior antibiotic therapy. Culture of intestinal secretions (best obtained by a noninvasive duodenal string test) can be positive despite a negative bone marrow culture. If blood, bone marrow, and intestinal secretions are all cultured, the yield is >90%. Stool cultures, although negative in 60–70% of cases during the first week, can become positive during the third week of infection in untreated patients.

1	Serologic tests, including the classic Widal test for “febrile agglutinins,” and rapid tests to detect antibodies to outer- membrane proteins or O:9 antigen are available for detection of S. typhi in developing countries but have lower positive predictive values than blood culture. More sensitive antigen and nucleic acid amplification tests have been developed to detect S. typhi and 1052 S. paratyphi in blood but are not yet commercially available and remain impractical in many areas where enteric fever is endemic.

1	Prompt administration of appropriate antibiotic therapy prevents severe complications of enteric fever and results in a case-fatality rate of <1%. The initial choice of antibiotics depends on the susceptibility of the S. typhi and S. paratyphi strains in the area of residence or travel (Table 190-1). For treatment of drug-susceptible typhoid fever, fluoroquinolones are the most effective class of agents, with cure rates of ~98% and relapse and fecal carriage rates of <2%. Experience is most extensive with ciprofloxacin. Short-course ofloxacin therapy is similarly successful against infection caused by quinolone-susceptible strains. However, the increased incidence of DCS S. typhi in Asia, which is probably related to the widespread availability of fluoroquinolones over the counter, is now limiting the use of this drug class for empirical therapy. Patients infected with DCS S. typhi strains should be treated with ceftriaxone, azithromycin, or high-dose ciprofloxacin. A 7-day course of

1	limiting the use of this drug class for empirical therapy. Patients infected with DCS S. typhi strains should be treated with ceftriaxone, azithromycin, or high-dose ciprofloxacin. A 7-day course of high-dose fluoroquinolone therapy for DCS enteric fever has been associated with delayed resolution of fever and high rates of fecal carriage during convalescence. Thus, for DCS strains, a 10to 14-day course of high-dose ciprofloxacin is preferred.

1	Ceftriaxone, cefotaxime, and (oral) cefixime are effective for treatment of MDR enteric fever, including that caused by DCS and fluoroquinolone-resistant strains. These agents clear fever in ~1 week, with failure rates of ~5–10%, fecal carriage rates of <3%, and relapse rates of 3–6%. Oral azithromycin results in defervescence in 4–6 days, with rates of relapse and convalescent stool carriage of <3%. Against DCS strains, azithromycin is associated with lower rates of treatment failure and shorter durations of hospitalization than are fluoroquinolones. Despite efficient in vitro killing of Salmonella, first-and second-generation cephalosporins as well as aminoglycosides are ineffective in the treatment of clinical infections. aOr another third-generation cephalosporin (e.g., cefotaxime, 2 g q8h IV; or cefixime, 400 mg bid PO). bOr 1 g on day 1 followed by 500 mg/d PO for 6 days. cOr ofloxacin, 400 mg bid PO for 2–5 days.

1	aOr another third-generation cephalosporin (e.g., cefotaxime, 2 g q8h IV; or cefixime, 400 mg bid PO). bOr 1 g on day 1 followed by 500 mg/d PO for 6 days. cOr ofloxacin, 400 mg bid PO for 2–5 days. Most patients with uncomplicated enteric fever can be managed at home with oral antibiotics and antipyretics. Patients with persistent vomiting, diarrhea, and/or abdominal distension should be hospitalized and given supportive therapy as well as a parenteral third-generation cephalosporin or fluoroquinolone, depending on the susceptibility profile. Therapy should be administered for at least 10 days or for 5 days after fever resolution.

1	In a randomized, prospective, double-blind study of criti cally ill patients with enteric fever (i.e., those with shock and obtundation) in Indonesia in the early 1980s, the administration of dexamethasone (an initial dose of 3 mg/kg followed by eight doses of 1 mg/kg every 6 h) with chloramphenicol was associated with a substantially lower mortality rate than was treatment with chloramphenicol alone (10% vs 55%). Although this study has not been repeated in the “post-chloramphenicol era,” severe enteric fever remains one of the few indications for glucocorticoid treatment of an acute bacterial infection.

1	The 1–5% of patients who develop chronic carriage of Salmonella can be treated for 4–6 weeks with an appropriate oral antibiotic. Treatment with oral amoxicillin, TMP-SMX, ciprofloxacin, or norfloxacin is ~80% effective in eradicating chronic carriage of susceptible organisms. However, in cases of anatomic abnormality (e.g., biliary or kidney stones), eradication often requires both antibiotic therapy and surgical correction. Theoretically, it is possible to eliminate the salmonellae that cause enteric fever because they survive only in human hosts and are spread by contaminated food and water. However, given the high prevalence of the disease in developing countries that lack adequate sewage disposal and water treatment, this goal is currently unrealistic. Thus, travelers to developing countries should be advised to monitor their food and water intake carefully and to strongly consider immunization against S. typhi.

1	Two typhoid vaccines are commercially available: (1) Ty21a, an oral live attenuated S. typhi vaccine (given on days 1, 3, 5, and 7, with a booster every 5 years); and (2) Vi CPS, a parenteral vaccine consisting of purified Vi polysaccharide from the bacterial capsule (given in a single dose, with a booster every 2 years). The old parenteral whole-cell typhoid/paratyphoid A and B vaccine is no longer licensed, largely because of significant side effects, especially fever. An acetone-killed whole-cell vaccine is available only for use by the U.S. military. The minimal age for vaccination is 6 years for Ty21a and 2 years for Vi CPS. In a recent meta-analysis of vaccines for preventing typhoid fever in populations in endemic areas, the cumulative efficacy was 48% for Ty21a at 2.5–3.5 years and 55% for Vi CPS at 3 years. Although data on typhoid vaccines in travelers are limited, some evidence suggests that efficacy rates may be substantially lower than those for local populations in

1	and 55% for Vi CPS at 3 years. Although data on typhoid vaccines in travelers are limited, some evidence suggests that efficacy rates may be substantially lower than those for local populations in endemic areas. Currently, there is no licensed vaccine for paratyphoid fever.

1	Vi CPS typhoid vaccine is poorly immunogenic in children <5 years of age because of T cell–independent properties. In the more recently developed Vi-rEPA vaccine, Vi is bound to a nontoxic recombinant protein that is identical to Pseudomonas aeruginosa exotoxin A. In 2to 4-year-olds, two injections of Vi-rEPA induced higher T cell responses and higher levels of serum IgG antibody to Vi than did Vi CPS in 5to 14-year-olds. In a two-dose trial in 2to 5-year-old children in Vietnam, Vi-rEPA provided 91% efficacy at 27 months and 89% efficacy at 46 months and was very well tolerated. This vaccine is not yet commercially available in the United States. Efforts to improve the immunogenicity and reduce the number of doses of live attenuated oral vaccines are ongoing.

1	Typhoid vaccine is not required for international travel, but it is recommended for travelers to areas where there is a moderate to high risk of exposure to S. typhi, especially those who are traveling to southern Asia and other developing regions of Asia, Africa, the Caribbean, and Central and South America and who will be exposed to potentially contaminated food and drink. Typhoid vaccine should be considered even for persons planning <2 weeks of travel to high-risk areas. In addition, laboratory workers who deal with S. typhi and household contacts of known S. typhi carriers should be vaccinated. Because the protective efficacy of vaccine can be overcome by the high inocula that are commonly encountered in food-borne exposures, immunization is an adjunct and not a substitute for the avoidance of high-risk foods and beverages. Immunization is not recommended for adults residing in typhoid-endemic areas or for the management of persons who may have been exposed in a common-source

1	avoidance of high-risk foods and beverages. Immunization is not recommended for adults residing in typhoid-endemic areas or for the management of persons who may have been exposed in a common-source outbreak.

1	Enteric fever is a notifiable disease in the United States. Individual health departments have their own guidelines for allowing ill or colonized food handlers or health care workers to return to their jobs. The reporting system enables public health departments to identify potential source patients and to treat chronic carriers in order to prevent further outbreaks. In addition, because 1–4% of patients with S. typhi infection become chronic carriers, it is important to monitor patients (especially child-care providers and food handlers) for chronic carriage and to treat this condition if indicated.

1	In the United States, NTS causes ~12 million illnesses annually, and the incidence has remained relatively unchanged during the past two decades. In 2011, the incidence of NTS infection in this country was 16.5/100,000 persons—the highest rate among the 10 food-borne enteric pathogens under active surveillance. Five serotypes accounted for more than half of U.S. infections during the period 1996–2006: typhimurium (23%), enteritidis (16%), newport (10%), heidelberg (6%), and javiana (5%).

1	The incidence of nontyphoidal salmonellosis is highest during the rainy season in tropical climates and during the warmer months in temperate climates—a pattern coinciding with the peak in food-borne outbreaks. Rates of morbidity and mortality associated with NTS are highest among the elderly, infants, and immunocompromised individuals, including those with hemoglobinopathies, HIV infection, or infections that cause blockade of the reticuloendothelial system (e.g., bartonellosis, malaria, schistosomiasis, histoplasmosis). Unlike S. typhi and S. paratyphi, whose only reservoir is humans, NTS can be acquired from multiple animal reservoirs. Transmission is most commonly associated with food products of animal origin (especially eggs, poultry, undercooked ground meat, and dairy products), fresh produce contaminated with animal waste, and contact with animals or their environments.

1	S. enteritidis infection associated with chicken eggs emerged as a major cause of food-borne disease during the 1980s and 1990s. S. enteritidis infection of the ovaries and upper oviduct tissue of hens results in contamination of egg contents before shell deposition. Infection is spread to egg-laying hens from breeding flocks and through contact with rodents and manure. The percentage of Salmonella outbreaks attributed to eggs has declined significantly in the United States, from 33% during 1998–1999 to 15% during 2006–2008. This decrease probably reflects the impact of the coordinated public health response to

1	S. enteritidis infection attributed to eggs, including improved on-farm control measures, refrigeration, and education of consumers and food-service workers. Transmission via contaminated eggs can be prevented by cooking eggs until the yolk is solidified and pasteurizing egg products. Despite these control efforts, outbreaks of S. enteritidis infection associated with shell eggs continue to occur. In 2010, a national outbreak of S. enteritidis infection resulted in more than 1900 reported illnesses and the recall of 500 million eggs.

1	Centralization of food processing and widespread food distribution have contributed to the increased incidence of NTS in developed countries. Manufactured foods to which recent Salmonella outbreaks have been traced include peanut butter; milk products, including infant formula; and various processed foods, including packaged breakfast cereal, salsa, frozen prepared meals, and snack foods. Large outbreaks have also been linked to fresh produce, including alfalfa sprouts, cantaloupe, mangoes, papayas, and tomatoes; these items become contaminated by manure or water at a single site and then are widely 1053 distributed.

1	An estimated 6% of sporadic Salmonella infections in the United States are attributed to contact with reptiles or amphibians, especially iguanas, snakes, turtles, and lizards. Reptile-associated Salmonella infection more commonly leads to hospitalization and more frequently involves children, including infants, than do other Salmonella infections. Other pets, including African hedgehogs, birds, rodents, baby chicks, ducklings, dogs, and cats, are also potential sources of NTS.

1	Increasing antibiotic resistance in NTS species is a global prob lem and has been linked to the widespread use of antimicrobial agents in food animals and especially in animal feed. In the early 1990s, S. typhimurium definitive phage type 104 (DT104), characterized by resistance to at least five antibiotics (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracyclines; R-type ACSSuT), emerged worldwide. In 2010, resistance to at least ACSSuT was reported in 4.3% of NTS isolates, including 18.6% of S. typhimurium isolates. Acquisition is associated with exposure to ill farm animals and to various meat products, including uncooked or undercooked ground beef. Although probably no more virulent than susceptible S. typhimurium strains, DT104 strains are associated with an increased risk of bloodstream infection and hospitalization. DCS and trimethoprim-resistant DT104 strains are emerging, especially in the United Kingdom.

1	Because of increased resistance to conventional antibiotics such as ampicillin and TMP-SMX, extended-spectrum cephalosporins and fluoroquinolones have emerged as the agents of choice for the treatment of MDR NTS infections. In 2010, 2.8% of all NTS strains were resistant to ceftriaxone. Most ceftriaxone-resistant isolates were from children <18 years of age, in whom ceftriaxone is the antibiotic of choice for treatment of invasive NTS infection. These strains contained plasmid-encoded AmpC β-lactamases that were probably acquired by horizontal genetic transfer from Escherichia coli strains in food-producing animals—an event linked to the widespread use of the veterinary cephalosporin ceftiofur.

1	Over the last decade, strains of DCS NTS (MIC, 0.125–1 μg/mL) have emerged and have been associated with delayed response and treatment failure. In 2009, 2.4% of NTS isolates in the United States were DCS or resistant to ciprofloxacin. These strains have diverse resistance mechanisms, including single and multiple mutations in the DNA gyrase genes gyrA and gyrB and plasmid-encoded quinolone resistance determinants that may not be reliably detected by nalidixic acid susceptibility testing. In 2012, the U.S. Clinical Laboratory Standards Institute proposed a lower ciprofloxacin susceptibility breakpoint (≥0.06 μg/mL) for all Salmonella species to address this issue. Currently, because commercial test systems do not contain ciprofloxacin concentrations low enough to allow use of these breakpoints, laboratories need to determine the ciprofloxacin MIC by Etest or another alternative method.

1	CLINICAL MANIFESTATIONS Gastroenteritis Infection with NTS most often results in gastroenteritis indistinguishable from that caused by other enteric pathogens. Nausea, vomiting, and diarrhea occur 6–48 h after the ingestion of contaminated food or water. Patients often experience abdominal cramping and fever (38–39°C; 100.5–102.2°F). Diarrheal stools are usually loose, nonbloody, and of moderate volume. However, large-volume watery stools, bloody stools, or symptoms of dysentery may occur. Rarely, NTS causes pseudoappendicitis or an illness that mimics inflammatory bowel disease.

1	Gastroenteritis caused by NTS is usually self-limited. Diarrhea resolves within 3–7 days and fever within 72 h. Stool cultures remain positive for 4–5 weeks after infection and—in rare cases of chronic carriage (<1%)—for >1 year. Antibiotic treatment usually is not recommended and may prolong fecal carriage. Neonates, the elderly, and immunosuppressed patients (e.g., transplant recipients, HIV-infected persons) with NTS gastroenteritis are especially susceptible to dehydration and dissemination and may require hospitalization and antibiotic therapy. Acute NTS gastroenteritis was associated with a threefold increased risk of dyspepsia and irritable bowel syndrome at 1 year in a study from Spain.

1	1054 Bacteremia and Endovascular Infections Up to 8% of patients with NTS gastroenteritis develop bacteremia; of these, 5–10% develop localized infections. Bacteremia and metastatic infection are most common with Salmonella choleraesuis and Salmonella dublin and among infants, the elderly, and immunocompromised patients, especially those with HIV infection. NTS endovascular infection should be suspected in high-grade or persistent bacteremia, especially with preexisting valvular heart disease, atherosclerotic vascular disease, prosthetic vascular graft, or aortic aneurysm. Arteritis should be suspected in elderly patients with prolonged fever and back, chest, or abdominal pain developing after an episode of gastroenteritis. Endocarditis and arteritis are rare (<1% of cases) but are associated with potentially fatal complications, including valve perforation, endomyocardial abscess, infected mural thrombus, pericarditis, mycotic aneurysms, aneurysm rupture, aortoenteric fistula, and

1	with potentially fatal complications, including valve perforation, endomyocardial abscess, infected mural thrombus, pericarditis, mycotic aneurysms, aneurysm rupture, aortoenteric fistula, and vertebral osteomyelitis. In some areas of sub-Saharan Africa, NTS may be among the most common causes—or even the most common cause—of bacteremia in children. NTS bacteremia among these children is not associated with diarrhea and has been associated with nutritional status and HIV infection.

1	Localized Infections • intraabdominal infections Intraabdominal infections due to NTS are rare and usually manifest as hepatic or splenic abscesses or as cholecystitis. Risk factors include hepatobiliary anatomic abnormalities (e.g., gallstones), abdominal malignancy, and sickle cell disease (especially with splenic abscesses). Eradication of the infection often requires surgical correction of abnormalities and percutaneous drainage of abscesses. central nervoUs system infections NTS meningitis most commonly develops in infants 1–4 months of age. It often results in severe sequelae (including seizures, hydrocephalus, brain infarction, and mental retardation), with death in up to 60% of cases. Other rare central nervous system infections include ventriculitis, subdural empyema, and brain abscesses.

1	pUlmonary infections NTS pulmonary infections usually present as lobar pneumonia, and complications include lung abscess, empyema, and bronchopleural fistula formation. The majority of cases occur in patients with lung cancer, structural lung disease, sickle cell disease, or glucocorticoid use. Urinary and genital tract infections Urinary tract infections caused by NTS present as either cystitis or pyelonephritis. Risk factors include malignancy, urolithiasis, structural abnormalities, HIV infection, and renal transplantation. NTS genital infections are rare and include ovarian and testicular abscesses, prostatitis, and epididymitis. Like other focal infections, both genital and urinary tract infections can be complicated by abscess formation.

1	bone, joint, and soft tissUe infections Salmonella osteomyelitis most commonly affects the femur, tibia, humerus, or lumbar vertebrae and is most often seen in association with sickle cell disease, hemoglobinopathies, or preexisting bone disease (e.g., fractures). Prolonged antibiotic treatment is recommended to decrease the risk of relapse and chronic osteomyelitis. Septic arthritis occurs in the same patient population as osteomyelitis and usually involves the knee, hip, or shoulder joints. Reactive arthritis can follow NTS gastroenteritis and is seen most frequently in persons with the HLA-B27 histocompatibility antigen. NTS rarely can cause soft tissue infections, usually at sites of local trauma in immunosuppressed patients.

1	The diagnosis of NTS infection is based on isolation of the organism from freshly passed stool or from blood or another ordinarily sterile body fluid. All salmonellae isolated in clinical laboratories should be sent to local public health departments for serotyping. Blood cultures should be done whenever a patient has prolonged or recurrent fever. Endovascular infection should be suspected if there is high-grade bacteremia (>50% of three or more positive blood cultures). Echocardiography, computed tomography (CT), and indium-labeled white cell scanning are used to identify localized infection. When another localized infection is suspected, joint fluid, abscess drainage, or cerebrospinal fluid should be cultured, as clinically indicated.

1	Antibiotics should not be used routinely to treat uncomplicated NTS gastroenteritis. The symptoms are usually self-limited, and the duration of fever and diarrhea is not significantly decreased by antibiotic therapy. In addition, antibiotic treatment has been associated with increased rates of relapse, prolonged gastrointestinal carriage, and adverse drug reactions. Dehydration secondary to diarrhea should be treated with fluid and electrolyte replacement.

1	Preemptive antibiotic treatment (Table 190-2) should be considered for patients at increased risk for invasive NTS infection, including neonates (probably up to 3 months of age); persons >50 years of age with suspected atherosclerosis; and patients with immunosuppression, cardiac valvular or endovascular abnormalities, or significant joint disease. Treatment should consist of an oral or IV antibiotic administered for 48–72 h or until the patient becomes afebrile. Immunocompromised persons may require up to 7–14 days of therapy. The <1% of persons who develop chronic carriage of NTS should receive a prolonged antibiotic course, as described above for chronic carriage of S. typhi.

1	Because of the increasing prevalence of antibiotic resistance, empirical therapy for life-threatening NTS bacteremia or focal NTS infection should include a third-generation cephalosporin or a fluoroquinolone (Table 190-2). If the bacteremia is low-grade (<50% of positive blood cultures), the patient should be treated for 7–14 days. Patients with HIV/AIDS and NTS bacteremia should receive 1–2 weeks of IV antibiotic therapy followed by 4 weeks of oral therapy with a aConsider for neonates; persons >50 years of age with possible atherosclerotic vascular disease; and patients with immunosuppression, endovascular graft, or joint prosthesis. bOr ofloxacin, 400 mg bid (PO). cConsider on an individualized basis for patients with severe diarrhea and high fever who require hospitalization. dOr cefotaxime, 2 g q8h (IV).

1	fluoroquinolone. Patients whose infections relapse after this regimen should receive long-term suppressive therapy with a fluoroquinolone or TMP-SMX, as indicated by bacterial sensitivities.

1	If the patient has endocarditis or arteritis, treatment for 6 weeks with an IV β-lactam antibiotic (such as ceftriaxone or ampicillin) is indicated. IV ciprofloxacin followed by prolonged oral therapy is an option, but published experience is limited. Early surgical resection of infected aneurysms or other infected endovascular sites is recommended. Patients with infected prosthetic vascular grafts that cannot be resected have been maintained successfully on chronic suppressive oral therapy. For extraintestinal nonvascular infections, a 2to 4-week course of antibiotic therapy (depending on the infection site) is usually recommended. In chronic osteomyelitis, abscess, or urinary or hepatobiliary infection associated with anatomic abnormalities, surgical resection or drainage may be required in addition to prolonged antibiotic therapy for eradication of infection.

1	Despite widespread efforts to prevent or reduce bacterial contamination of animal-derived food products and to improve food-safety education and training, recent declines in the incidence of NTS in the United States have been modest compared with those of other food-borne pathogens. This observation probably reflects the complex epidemiology of NTS. Identifying effective risk-reduction strategies requires monitoring of every step of food production, from handling of raw animal or plant products to preparation of finished foods. Contaminated food can be made safe for consumption by pasteurization, irradiation, or proper cooking. All cases of NTS infection should be reported to local public health departments because tracking and monitoring of these cases can identify the source(s) of infection and help authorities anticipate large outbreaks. Lastly, the prudent use of antimicrobial agents in both humans and animals is needed to limit the emergence of MDR Salmonella.

1	shigellosis Philippe J. Sansonetti, Jean Bergounioux The discovery of Shigella as the etiologic agent of dysentery—a clinical syndrome of fever, intestinal cramps, and frequent passage of small, bloody, mucopurulent stools—is attributed to the Japanese microbi-ologist Kiyoshi Shiga, who isolated the Shiga bacillus (now known as 191 Shigella dysenteriae type 1) from patients’ stools in 1897 during a large and devastating dysentery epidemic. Shigella cannot be distinguished from Escherichia coli by DNA hybridization and remains a separate species only on historical and clinical grounds. Shigella is a non-spore-forming, gram-negative bacterium that, unlike

1	E. coli, is nonmotile and does not produce gas from sugars, decarboxylate lysine, or hydrolyze arginine. Some serovars produce indole, and occasional strains utilize sodium acetate. Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei (serogroups A, B, C, and D, respectively) can be differentiated on the basis of biochemical and serologic characteristics. Genome sequencing of E. coli K12, S. flexneri 2a, S. sonnei, S. dysenteriae type 1, and S. boydii has revealed that these species have ~93% of genes in common. The three major genomic “signatures” of Shigella are (1) a 215-kb virulence plasmid that carries most of the genes required for pathogenicity (particularly invasive capacity); (2) the lack or alteration of genetic sequences encoding products (e.g., lysine decarboxylase) that, if expressed, would attenuate pathogenicity; and (3) in S. dysenteriae type 1, the presence of genes 1055 encoding Shiga toxin, a potent cytotoxin.

1	The human intestinal tract represents the major reservoir of Shigella, which is also found (albeit rarely) in the higher pri mates. Because excretion of shigellae is greatest in the acute phase of disease, the bacteria are transmitted most efficiently by the fecal-oral route via hand carriage; however, some outbreaks reflect foodborne or waterborne transmission. In impoverished areas, Shigella can be transmitted by flies. The high-level infectivity of Shigella is reflected by the very small inoculum required for experimental infection of volunteers (100 colony-forming units [CFU]), by the very high attack rates during outbreaks in day-care centers (33–73%), and by the high rates of secondary cases among family members of sick children (26–33%). Shigellosis can also be transmitted sexually.

1	Throughout history, Shigella epidemics have often occurred in settings of human crowding under conditions of poor hygiene—e.g., among soldiers in campaigning armies, inhabitants of besieged cities, groups on pilgrimages, and refugees in camps. Epidemics follow a cyclical pattern in areas such as the Indian subcontinent and sub-Saharan Africa. These devastating epidemics, which are most often caused by S. dysenteriae type 1, are characterized by high attack and mortality rates. In Bangladesh, for instance, an epidemic caused by S. dysenteriae type 1 was associated with a 42% increase in mortality rate among children 1–4 years of age. Apart from these epidemics, shigellosis is mostly an endemic disease, with 99% of cases occurring in the developing world and the highest prevalences in the most impoverished areas, where personal and general hygiene is below standard. S. flexneri isolates predominate in the least developed areas, whereas

1	S. flexneri isolates predominate in the least developed areas, whereas S. sonnei is more prevalent in economically emerging countries and in the industrialized world.

1	Prevalence in the Developing World In a review published under the auspices of the World Health Organization (WHO), the total annual number of cases in 1966–1997 was estimated at 165 million, and 69% of these cases occurred in children <5 years of age. In this review, the annual number of deaths was calculated to range between 500,000 and 1.1 million. More recent data (2000–2004) from six Asian countries indicate that, even though the incidence of shigellosis remains stable, mortality rates associated with this disease may have decreased significantly, possibly as a result of improved nutritional status. However, extensive and essentially uncontrolled use of antibiotics, which may also account for declining mortality rates, has increased the rate of emergence of multidrug-resistant Shigella strains. A 2013 prospective matched case-control study of children <5 years of age emphasizes the importance of Shigella in the burden and etiology of diarrheal diseases in developing countries.

1	strains. A 2013 prospective matched case-control study of children <5 years of age emphasizes the importance of Shigella in the burden and etiology of diarrheal diseases in developing countries. Shigella is one of the top four pathogens associated with moderate to severe diarrhea and is now ranked first among children 12–59 months of age. These moderate to severe cases account for an 8.5-fold increase in mortality incidence over the average diarrheal disease–related mortality. The study’s authors conclude that Shigella remains a major pathogen to be targeted by health care programs.

1	An often-overlooked complication of shigellosis is the shortand long-term impairment of the nutritional status of infected children in endemic areas. Combined with anorexia, the exudative enteropathy resulting from mucosal abrasions contributes to rapid deterioration of the patient’s nutritional status. Shigellosis is thus a major contributor to stunted growth among children in developing countries. Peaking in incidence in the pediatric population, endemic shigellosis is rare among young and middle-aged adults, probably because of naturally acquired immunity. Incidence then increases again in the elderly population.

1	Prevalence in the Industrialized World In pediatric populations, local outbreaks occur when proper and adapted hygiene policies are not implemented in group facilities like day-care centers and institutions for the mentally retarded. In adults, as in children, sporadic cases occur among travelers returning from endemic areas, and rare outbreaks of varying size can follow waterborne or food-borne infections.

1	1056 PATHOGENESIS AND PATHOLOGY 28S ribosomal RNA. This process leads to inhibition of binding of the Shigella infection occurs essentially through oral contamination via amino-acyl-tRNA to the 60S ribosomal subunit and thus to a general direct fecal-oral transmission, the organism being poorly adapted to shutoff of cell protein biosynthesis. Shiga toxins are translocated from survive in the environment. Resistance to low-pH conditions allows the bowel into the circulation. After binding of the toxins to target cells shigellae to survive passage through the gastric barrier, an ability that in the kidney, pathophysiologic alterations may result in hemolytic-may explain in part why a small inoculum (as few as 100 CFU) is suf-uremic syndrome (HUS; see below). ficient to cause infection. The watery diarrhea that usually precedes the dysenteric syndrome CLINICAL MANIFESTATIONS is attributable to active secretion and abnormal water reabsorption—a The presentation and severity of shigellosis

1	diarrhea that usually precedes the dysenteric syndrome CLINICAL MANIFESTATIONS is attributable to active secretion and abnormal water reabsorption—a The presentation and severity of shigellosis depend to some extent secretory effect at the jejunal level described in experimentally infected on the infecting serotype but even more on the age and the immuno rhesus monkeys. This initial purge is probably due to the combined logic and nutritional status of the host. Poverty and poor standards of action of an enterotoxin (ShET-1) and mucosal inflammation. The hygiene are strongly related to the number and severity of diarrheal dysenteric syndrome, manifested by bloody and mucopurulent stools, episodes, especially in children <5 years old who have been weaned. reflects invasion of the mucosa. Shigellosis typically evolves through four phases: incubation, watery

1	The pathogenesis of Shigella is essentially determined by a large diarrhea, dysentery, and the postinfectious phase. The incubation virulence plasmid of 214 kb comprising ~100 genes, of which 25 period usually lasts 1–4 days but may be as long as 8 days. Typical iniencode a type III secretion system that inserts into the mem-tial manifestations are transient fever, limited watery diarrhea, malaise, brane of the host cell to allow effectors to transit from the bacterial and anorexia. Signs and symptoms may range from mild abdominal cytoplasm to the host cell cytoplasm (Fig. 191-1). Bacteria are thereby discomfort to severe cramps, diarrhea, fever, vomiting, and tenesmus. able to invade intestinal epithelial cells by inducing their own uptake The manifestations are usually exacerbated in children, with tempera-after the initial crossing of the epithelial barrier through M cells (the tures up to 40°–41°C (104.0°–105.8°F) and more severe anorexia and specialized translocating epithelial

1	with tempera-after the initial crossing of the epithelial barrier through M cells (the tures up to 40°–41°C (104.0°–105.8°F) and more severe anorexia and specialized translocating epithelial cells in the follicle-associated epi-watery diarrhea. This initial phase may represent the only clinical manithelium that covers mucosal lymphoid nodules). The organisms festation of shigellosis, especially in developed countries. Otherwise, induce apoptosis of subepithelial resident macrophages. Once inside dysentery follows within hours or days and is characterized by uninthe cytoplasm of intestinal epithelial cells, Shigella effectors trigger the terrupted excretion of small volumes of bloody mucopurulent stools cytoskeletal rearrangements necessary to direct uptake of the organism with increased tenesmus and abdominal cramps. At this stage, Shigella into the epithelial cell. The Shigella-containing vacuole is then quickly produces acute colitis involving mainly the distal colon and the rectum.

1	and abdominal cramps. At this stage, Shigella into the epithelial cell. The Shigella-containing vacuole is then quickly produces acute colitis involving mainly the distal colon and the rectum. lysed, releasing bacteria into the cytosol. Unlike most diarrheal syndromes, dysenteric syndromes rarely present Intracellular shigellae next use cytoskeletal components to propel with dehydration as a major feature. Endoscopy shows an edematous themselves inside the infected cell; when the moving organism and the and hemorrhagic mucosa, with ulcerations and possibly overlying host cell membrane come into contact, cellular protrusions form and exudates resembling pseudomembranes. The extent of the lesions corare engulfed by neighboring cells. This series of events permits bacte-relates with the number and frequency of stools and with the degree of rial cell-to-cell spread. protein loss by exudative mechanisms. Most episodes are self-limited Cytokines released by a growing number of infected

1	number and frequency of stools and with the degree of rial cell-to-cell spread. protein loss by exudative mechanisms. Most episodes are self-limited Cytokines released by a growing number of infected intestinal epi-and resolve without treatment in 1 week. With appropriate treatment, thelial cells attract increased numbers of immune cells (particularly recovery takes place within a few days to a week, with no sequelae. polymorphonuclear leukocytes [PMNs]) to the infected site, thus Acute life-threatening complications are seen most often in chilfurther destabilizing the epithelial barrier, exacerbating inflammation, dren <5 years of age (particularly those who are malnourished) and leading to the acute colitis that characterizes shigellosis. Evidence and in elderly patients. Risk factors for death in a clinically severe indicates that some type III secretion system–injected effectors can case include nonbloody diarrhea, moderate to severe dehydration, control the extent of

1	factors for death in a clinically severe indicates that some type III secretion system–injected effectors can case include nonbloody diarrhea, moderate to severe dehydration, control the extent of inflammation, thus facilitating bacterial survival. bacteremia, absence of fever, abdominal tenderness, and rectal pro-Shiga toxin produced by S. dysenteriae type 1 increases disease lapse. Major complications are predominantly intestinal (e.g., toxic severity. This toxin belongs to a group of A1-B5 protein toxins whose megacolon, intestinal perforations, rectal prolapse) or metabolic B subunit binds to the receptor globotriaosylceramide on the target (e.g., hypoglycemia, hyponatremia, dehydration). Bacteremia is rare cell surface and whose catalytic A subunit is internalized by receptor-and is reported most frequently in severely malnourished and HIV-mediated endocytosis and interacts with the subcellular machinery to infected patients. Alterations of consciousness, including seizures,

1	is reported most frequently in severely malnourished and HIV-mediated endocytosis and interacts with the subcellular machinery to infected patients. Alterations of consciousness, including seizures, inhibit protein synthesis by expressing RNA N-glycosidase activity on delirium, and coma, may occur, especially in children <5 years old, and are associated with a poor prognosis; fever and severe metabolic alterations are more often the major causes of altered consciousness than is meningitis or the Ekiri syndrome (toxic encepha- lopathy associated with bizarre posturing, cerebral edema, and fatty degeneration of viscera), which Activation of has been reported mostly in Japanese children.NF-°B caused by IcsA Pneumonia, vaginitis, and keratoconjunctivitis due to Shigella are rarely reported. In the absence of serious malnutrition, severe and very unusual clinical manifestations, such as meningitis, may be IpaB type III linked to genetic defects in innate immune func secretion tions (i.e.,

1	the absence of serious malnutrition, severe and very unusual clinical manifestations, such as meningitis, may be IpaB type III linked to genetic defects in innate immune func secretion tions (i.e., deficiency in interleukin 1 receptor–IL-1˜ IpaA associated kinase 4 [IRAK-4]) and may require

1	Disruption of epithelial genetic investigation. permeability barrier by PMNs Macrophage apoptosis Two complications of particular importance are toxic megacolon and HUS. Toxic megacolon Massive invasion of Bacterial survival is a consequence of severe inflammation extending epithelium

1	Initiation of inflammation to the colonic smooth-muscle layer and causing paralysis and dilation. The patient presents with abdominal distention and tenderness, with or FIGuRE 191-1 Invasive strategy of Shigella flexneri. IL, interleukin; NF-κB, nuclear factor without signs of localized or generalized peritoniκB; NLR, NOD-like receptor; PMN, polymorphonuclear leukocyte. tis. The abdominal x-ray characteristically shows marked dilation of the transverse colon (with the greatest distention in the ascending and descending segments); thumbprinting caused by mucosal inflammatory edema; and loss of the normal haustral pattern associated with pseudopolyps, often extending into the lumen. Pneumatosis coli is an occasional finding. If perforation occurs, radiographic signs of pneumoperitoneum may be apparent. Predisposing factors (e.g., hypokalemia and use of opioids, anticholinergics, loper-amide, psyllium seeds, and antidepressants) should be investigated.

1	Shiga toxin produced by S. dysenteriae type 1 has been linked countries, where enterohemorrhagic E. coli (EHEC) predominates as the etiologic agent of this syndrome. HUS is an early complication that most often develops after several days of diarrhea. Clinical examination shows pallor, asthenia, and irritability and, in some cases, bleeding of the nose and gums, oliguria, and increasing edema. HUS is a nonimmune (Coombs test–negative) hemolytic anemia defined by a diagnostic triad: microangiopathic hemolytic anemia (hemoglobin level typically <80 g/L [<8 g/dL]), thrombocytopenia (mild to moderate in severity; typically <60,000 platelets/μL), and acute renal failure due to thrombosis of the glomerular capillaries (with markedly elevated creatinine levels). Anemia is severe, with fragmented red blood cells (schizocytes) in the peripheral smear, high serum concentrations of lactate dehydrogenase and free circulating hemoglobin, and elevated reticulocyte counts. Acute renal failure occurs

1	red blood cells (schizocytes) in the peripheral smear, high serum concentrations of lactate dehydrogenase and free circulating hemoglobin, and elevated reticulocyte counts. Acute renal failure occurs in 55–70% of cases; however, renal function recovers in most of these cases (up to 70% in various series). Leukemoid reactions, with leukocyte counts of 50,000/ μL, are sometimes noted in association with HUS.

1	The postinfectious immunologic complication known as reactive arthritis can develop weeks or months after shigellosis, especially in patients expressing the histocompatibility antigen HLA-B27. About 3% of patients infected with S. flexneri later develop this syndrome, with arthritis, ocular inflammation, and urethritis—a condition that can last for months or years and can progress to difficult-to-treat chronic arthritis. Postinfectious arthropathy occurs only after infection with S. flexneri and not after infection with the other Shigella serotypes.

1	The differential diagnosis in patients with a dysenteric syn drome depends on the clinical and environmental context. In developing areas, infectious diarrhea caused by other invasive pathogenic bacteria (Salmonella, Campylobacter jejuni, Clostridium difficile, Yersinia enterocolitica) or parasites (Entamoeba histolytica) should be considered. Only bacteriologic and parasitologic examinations of stool can truly differentiate among these pathogens. A first flare of inflammatory bowel disease, such as Crohn’s disease or ulcerative colitis (Chap. 351), should be considered in patients in industrialized countries. Despite the similarity in symptoms, anamnesis discriminates between shigellosis, which usually follows recent travel in an endemic zone, and these other conditions.

1	Microscopic examination of stool smears shows erythrophagocytic trophozoites with very few PMNs in E. histolytica infection, whereas bacterial enteroinvasive infections (particularly shigellosis) are characterized by high PMN counts in each microscopic field. However, because shigellosis often manifests only as watery diarrhea, systematic attempts to isolate Shigella are necessary.

1	The “gold standard” for the diagnosis of Shigella infection remains the isolation and identification of the pathogen from fecal material. One major difficulty, particularly in endemic areas where laboratory facilities are not immediately available, is the fragility of Shigella and its common disappearance during transport, especially with rapid changes in temperature and pH. In the absence of a reliable enrichment medium, buffered glycerol saline or Cary-Blair medium can be used as a holding medium, but prompt inoculation onto isolation medium is essential. The probability of isolation is higher if the portion of stools that contains bloody and/or mucopurulent material is directly sampled. Rectal swabs can be used, as they offer the highest rate of successful isolation during the acute phase of disease. Blood cultures are positive in fewer than 5% of cases but should be done when a patient presents with a clinical picture of severe sepsis.

1	In addition to quick processing, the use of several media increases 1057 the likelihood of successful isolation: a nonselective medium such as bromocresol-purple agar lactose; a low-selectivity medium such as MacConkey or eosin-methylene blue; and a high-selectivity medium such as Hektoen, Salmonella-Shigella, or xylose-lysine-deoxycholate agar. After incubation on these media for 12–18 h at 37°C (98.6°F), shigellae appear as non-lactose-fermenting colonies that measure 0.5–1 mm in diameter and have a convex, translucent, smooth surface. Suspected colonies on nonselective or low-selectivity medium can be subcultured on a high-selectivity medium before being specifically identified or can be identified directly by standard commercial systems on the basis of four major characteristics: glucose positivity (usually without production of gas), lactose negativity, H2S negativity, and lack of motility. The four Shigella serogroups (A–D) can then be differentiated by additional

1	glucose positivity (usually without production of gas), lactose negativity, H2S negativity, and lack of motility. The four Shigella serogroups (A–D) can then be differentiated by additional characteristics. This approach adds time and difficulty to the identification process; however, after presumptive diagnosis, the use of serologic methods (e.g., slide agglutination, with group-and then type-specific antisera) should be considered. Group-specific antisera are widely available; in contrast, because of the large number of serotypes and subserotypes, type-specific antisera are rare and more expensive and thus are often restricted to reference laboratories.

1	As an enteroinvasive disease, shigellosis requires antibiotic treatment. Since the mid-1960s, however, increasing resis tance to multiple drugs has been a dominant factor in treatment decisions. Resistance rates are highly dependent on the geographic area. Clonal spread of particular strains and horizontal transfer of resistance determinants, particularly via plasmids and transposons, contribute to multidrug resistance. The current global status—i.e., high rates of resistance to classic first-line antibiotics such as amoxicillin—has led to a rapid switch to quinolones such as nalidixic acid. However, resistance to such early-generation quinolones has also emerged and spread quickly as a result of chromosomal mutations affecting DNA gyrase and topoisomerase IV; this resistance has necessitated the use of later-generation quinolones as first-line antibiotics in many areas. For instance, a review of the antibiotic resistance history of Shigella in India found that, after their

1	necessitated the use of later-generation quinolones as first-line antibiotics in many areas. For instance, a review of the antibiotic resistance history of Shigella in India found that, after their introduction in the late 1980s, the second-generation quinolones norfloxacin, ciprofloxacin, and ofloxacin were highly effective in the treatment of shigellosis, including cases caused by multidrug-resistant strains of S. dysenteriae type 1. However, investigations of subsequent outbreaks in India and Bangladesh detected resistance to norfloxacin, ciprofloxacin, and ofloxacin in 5% of isolates. The incidence of multidrug resistance parallels the widespread, uncontrolled use of antibiotics and calls for the rational use of effective drugs.

1	Because of the ready transmissibility of Shigella, current public health recommendations in the United States are that every case be treated with antibiotics. Ciprofloxacin is recommended as first-line treatment. A number of other drugs have been tested and shown to be effective, including ceftriaxone, azithromycin, pivmecillinam, and some fifth-generation quinolones. Whereas infections caused by non-dysenteriae Shigella in immunocompetent individuals are routinely treated with a 3-day course of antibiotics, it is recommended that S. dysenteriae type 1 infections be treated for 5 days and that Shigella infections in immunocompromised patients be treated for 7–10 days. Treatment for shigellosis must be adapted to the clinical context, with the recognition that the most fragile patients are children <5 years old, who represent two-thirds of all cases worldwide. There are few data on the use of quinolones in children, but Shigella-induced dysentery is a well-recognized indication

1	Azithromycin 6–20 mg/kg 1–1.5 g Cost Rapid emergence of resistance and spread to other bacteria Source: WHO Library Cataloguing-in-Publication Data: Guidelines for the control of shigellosis, including epidemics due to Shigella dysenteriae type 1 (www.who.int/cholera/ publications/shigellosis/en/). for their use. The half-life of ciprofloxacin is longer in infants than in older individuals. The ciprofloxacin dose generally recommended for children is 30 mg/kg per day in two divided doses. Adults living in areas with high standards of hygiene are likely to develop milder, shorter-duration disease, whereas infants in endemic areas can develop severe, sometimes fatal, dysentery. In the former setting, treatment will remain minimal and bacteriologic proof of infection will often come after symptoms have resolved; in the latter setting, antibiotic treatment and more aggressive measures, possibly including resuscitation, are often required.

1	Shigella infection rarely causes significant dehydration. industrialized countries) are uncommon. In developing countries, malnutrition remains the primary indicator for diarrhea-related death, highlighting the importance of nutrition in early management. Rehydration should be oral unless the patient is comatose or presents in shock. Because of the improved effectiveness of reduced-osmolarity oral rehydration solution (especially for children with acute noncholera diarrhea), the WHO and UNICEF now recommend a standard solution of 245 mOsm/L (sodium, 75 mmol/L; chloride, 65 mmol/L; glucose [anhydrous], 75 mmol/L; potassium, 20 mmol/L; citrate, 10 mmol/L). In shigellosis, the coupled transport of sodium to glucose may be variably affected, but oral rehydration therapy remains the easiest and most efficient form of rehydration, especially in severe cases.

1	Nutrition should be started as soon as possible after completion of initial rehydration. Early refeeding is safe, well tolerated, and clinically beneficial. Because breast-feeding reduces diarrheal losses and the need for oral rehydration in infants, it should be maintained in the absence of contraindications (e.g., maternal HIV infection). NONSPECIFIC, SYMPTOM-BASED THERAPY Antimotility agents have been implicated in prolonged fever in volunteers with shigellosis. These agents are suspected of increasing the risk of toxic megacolon and are thought to have been responsible for HUS in children infected by EHEC strains. For safety reasons, it is better to avoid antimotility agents in bloody diarrhea.

1	There is no consensus regarding the best treatment for toxic mega-colon. The patient should be assessed frequently by both medical and surgical teams. Anemia, dehydration, and electrolyte deficits (particularly hypokalemia) may aggravate colonic atony and should be actively treated. Nasogastric aspiration helps to deflate the colon. Parenteral nutrition has not been proven to be beneficial. Fever persisting beyond 48–72 h raises the possibility of local perforation or abscess. Most studies recommend colectomy if, after 48–72 h, colonic distention persists. However, some physicians recommend continuation of medical therapy for up to 7 days if the patient seems to be improving clinically despite persistent megacolon without free perforation. Intestinal perforation, either isolated or complicating toxic megacolon, requires surgical treatment and intensive medical support.

1	Rectal prolapse must be treated as soon as possible. With the health care provider using surgical gloves or a soft warm wet cloth and the patient in the knee-chest position, the prolapsed rectum is gently pushed back into place. If edema of the rectal mucosa is evident (rendering reintegration difficult), it can be osmotically reduced by the application of gauze impregnated with a warm solution of saturated magnesium sulfate. Rectal prolapse often relapses but usually resolves along with the resolution of dysentery. HUS must be treated by water restriction, including discontinuation of oral rehydration solution and potassium-rich alimentation. Hemofiltration is usually required.

1	HUS must be treated by water restriction, including discontinuation of oral rehydration solution and potassium-rich alimentation. Hemofiltration is usually required. Hand washing after defecation or handling of children’s feces and before handling of food is recommended. Stool decontamination (e.g., with sodium hypochlorite), together with a cleaning protocol for medical staff as well as for patients, has proven useful in limiting the spread of infection during Shigella outbreaks. Ideally, patients should have a negative stool culture before their infection is considered cured. Recurrences are rare if therapeutic and preventive measures are correctly implemented. Although several live attenuated oral and subunit parenteral vaccine candidates have been produced and are undergoing clinical trials, no vaccine against shigellosis is currently available. Especially given the rapid progression of antibiotic resistance in Shigella, a vaccine is urgently needed.

1	Infections due to Campylobacter 192 and Related Organisms Martin J. Blaser Bacteria of the genus Campylobacter and of the related genera Arcobacter and Helicobacter (Chap. 188) cause a variety of inflammatory conditions. Although acute diarrheal illnesses are most common, these organisms may cause infections in virtually all parts of the body, especially in compromised hosts, and these infections may have late nonsuppurative sequelae. The designation Campylobacter comes from the Greek for “curved rod” and refers to the organism’s vibrio-like morphology.

1	Campylobacters are motile, non-spore-forming, curved, gram-negative rods. Originally known as Vibrio fetus, these bacilli were reclassified as a new genus in 1973 after their dissimilarity to other vibrios was recognized. More than 15 species have since been identified. These species are currently divided into three genera: Campylobacter, Arcobacter, and Helicobacter. Not all of the species are pathogens of humans. The human pathogens fall into two major groups: those that primarily cause diarrheal disease and those that cause extraintestinal infection. The principal diarrheal pathogen is Campylobacter jejuni, which accounts for 80–90% of all cases of recognized illness due to campylobacters and related genera. Other organisms that cause diarrheal disease include Campylobacter coli, Campylobacter upsaliensis, Campylobacter lari, Campylobacter hyointestinalis, Campylobacter fetus, Arcobacter butzleri, Arcobacter cryaerophilus, Helicobacter cinaedi, and Helicobacter fennelliae. The two

1	upsaliensis, Campylobacter lari, Campylobacter hyointestinalis, Campylobacter fetus, Arcobacter butzleri, Arcobacter cryaerophilus, Helicobacter cinaedi, and Helicobacter fennelliae. The two Helicobacter species causing diarrheal disease, H. cinaedi and H. fennelliae, are intestinal rather than gastric organisms; in terms of the clinical features of the illnesses they cause, these species most closely resemble Campylobacter rather than Helicobacter pylori (Chap. 188) and thus are considered in this chapter. The pathogenic roles of Campylobacter concisus, Campylobacter ureolyticus, Campylobacter troglodytis, and Campylobacter pyloridis are uncertain. A new subspecies—C. fetus subspecies testudinum—has been described, chiefly in Asian patients; its close resemblance to strains isolated from reptiles suggests a food source.

1	The major species causing extraintestinal illnesses is C. fetus. However, any of the diarrheal agents listed above may cause systemic or localized infection as well, especially in compromised hosts. Neither aerobes nor strict anaerobes, these microaerophilic organisms are adapted for survival in the gastrointestinal mucous layer. This chapter focuses on C. jejuni and C. fetus as the major pathogens in and prototypes for their groups. The key features of infection are listed by species (excluding C. jejuni, described in detail in the text below) in Table 192-1.

1	Campylobacters are found in the gastrointestinal tract of many animals used for food (including poultry, cattle, sheep, and swine) and many household pets (including birds, dogs, and cats). These microorganisms usually do not cause illness in their animal hosts. In most cases, campylobacters are transmitted to humans in raw or undercooked food products or through direct contact with infected animals. In the United States and other developed countries, ingestion of contaminated poultry that has not been suffi-1059 ciently cooked is the most common mode of acquisition (30–70% of cases). Other modes include ingestion of raw (unpasteurized) milk or untreated water, contact with infected household pets, travel to developing countries (campylobacters being among the leading causes of traveler’s diarrhea; Chaps. 149 and 160), oral-anal sexual contact, and (occasionally) contact with an index case who is incontinent of stool (e.g., a baby).

1	Campylobacter infections are common. Several studies indicate that, in the United States, diarrheal disease due to campylobacters is more common than that due to Salmonella and Shigella combined. Infections occur throughout the year, but their incidence peaks during summer and early autumn. Persons of all ages are affected; however, attack rates for C. jejuni are highest among young children and young adults, whereas those for C. fetus are highest at the extremes of age. Systemic infections due to C. fetus (and to other Campylobacter and related species) are most common among compromised hosts. Persons at increased risk include those with AIDS, hypogammaglobulinemia, neoplasia, liver disease, diabetes mellitus, and generalized atherosclerosis as well as neonates and pregnant women. However, apparently healthy nonpregnant persons occasionally develop transient Campylobacter bacteremia as part of a gastrointestinal illness.

1	In contrast, in many developing countries, C. jejuni infections are hyperendemic, with the highest rates among children <2 years old. Infection rates fall with age, as does the illness-to-infection ratio. These observations suggest that frequent exposure to C. jejuni leads to the acquisition of immunity. C. jejuni infections may be subclinical, especially in hosts in developing countries who have had multiple prior infections and thus are partially immune. Symptomatic infections mostly occur within 2–4 days (range, 1–7 days) of exposure to the organism in food or water. The sites of tissue injury include the jejunum, ileum, and colon. Biopsies show an acute nonspecific inflammatory reaction, with neutrophils, monocytes, and eosinophils in the lamina propria, as well as damage to the epithelium, including loss of mucus, glandular degeneration, and crypt abscesses. Biopsy findings may be consistent with Crohn’s disease or ulcerative colitis, but these “idiopathic”

1	Infections Due to Campylobacter and Related Organisms axillary abscesses; diarrhea aIn immunocompromised hosts, especially HIV-infected persons. bIn children. Source: Adapted from BM Allos, MJ Blaser: Clin Infect Dis 20:1092, 1995.

1	1060 chronic inflammatory diseases should not be diagnosed unless infectious colitis, specifically including that due to infection with Campylobacter species and related organisms, has been ruled out. The high frequency of C. jejuni infections and their severity and recurrence among hypogammaglobulinemic patients suggest that antibodies are important in protective immunity. The pathogenesis of infection is uncertain. Both the motility of the strain and its capacity to adhere to host tissues appear to favor disease, but classic enterotoxins and cytotoxins (although they have been described and include cytolethal distending toxin, or CDT) appear not to play substantial roles in tissue injury or disease production. The organisms have been visualized within the epithelium, albeit in low numbers. The documentation of a significant tissue response and occasionally of C. jejuni bacteremia further suggests that tissue invasion is clinically significant, and in vitro studies are consistent

1	The documentation of a significant tissue response and occasionally of C. jejuni bacteremia further suggests that tissue invasion is clinically significant, and in vitro studies are consistent with this pathogenetic feature. The pathogenesis of C. fetus infections is better defined. Virtually all clinical isolates of C. fetus possess a proteinaceous capsule-like structure (an S-layer) that renders the organisms resistant to complement-mediated killing and opsonization. As a result, C. fetus can cause bacteremia and can seed sites beyond the intestinal tract. The ability of the organism to switch the S-layer proteins expressed—a phenomenon that results in antigenic variability—may contribute to the chronicity and high rate of recurrence of C. fetus infections in compromised hosts.

1	The clinical features of infections due to Campylobacter and the related Arcobacter and intestinal Helicobacter species causing enteric disease appear to be highly similar. C. jejuni can be considered the prototype, in part because it is by far the most common enteric pathogen in the group. A prodrome of fever, headache, myalgia, and/or malaise often occurs 12–48 h before the onset of diarrheal symptoms. The most common signs and symptoms of the intestinal phase are diarrhea, abdominal pain, and fever. The degree of diarrhea varies from several loose stools to grossly bloody stools; most patients presenting for medical attention have ≥10 bowel movements on the worst day of illness. Abdominal pain usually consists of cramping and may be the most prominent symptom. Pain is usually generalized but may become localized; C. jejuni infection may cause pseudoappendicitis. Fever may be the only initial manifestation of C. jejuni infection, a situation mimicking the early stages of typhoid

1	but may become localized; C. jejuni infection may cause pseudoappendicitis. Fever may be the only initial manifestation of C. jejuni infection, a situation mimicking the early stages of typhoid fever. Febrile young children may develop convulsions. Campylobacter enteritis is generally self-limited; however, symptoms persist for >1 week in 10–20% of patients seeking medical attention, and clinical relapses occur in 5–10% of such untreated patients. Studies of common-source epidemics indicate that milder illnesses or asymptomatic infections may commonly occur.

1	C. fetus may cause a diarrheal illness similar to that due to C. jejuni, especially in normal hosts. This organism also may cause either intermittent diarrhea or nonspecific abdominal pain without localizing signs. Sequelae are uncommon, and the outcome is benign. C. fetus may also cause a prolonged relapsing systemic illness (with fever, chills, and myalgias) that has no obvious primary source; this manifestation is especially common among compromised hosts. Secondary seeding of an organ (e.g., meninges, brain, bone, urinary tract, or soft tissue) complicates the course, which may be fulminant. C. fetus infections have a tropism for vascular sites: endocarditis, mycotic aneurysm, and septic thrombophlebitis may all occur. Infection during pregnancy often leads to fetal death. A variety of Campylobacter species and H. cinaedi can cause recurrent cellulitis with fever and bacteremia in immunocompromised hosts.

1	H. cinaedi can cause recurrent cellulitis with fever and bacteremia in immunocompromised hosts. Except in infection with C. fetus, bacteremia is uncommon, developing most often in immunocompromised hosts and at the extremes of age. Three patterns of extraintestinal infection have been noted: (1) transient bacteremia in a normal host with enteritis (benign course, no specific treatment needed); (2) sustained bacteremia or focal infection in a normal host (bacteremia originating from enteritis, with patients responding well to antimicrobial therapy); and (3) sustained bacteremia or focal infection in a compromised host. Enteritis may not be clinically apparent. Antimicrobial therapy, possibly prolonged, is necessary for suppression or cure of the infection.

1	Campylobacter, Arcobacter, and intestinal Helicobacter infections in patients with AIDS or hypogammaglobulinemia may be severe, persistent, and extraintestinal; relapse after cessation of therapy is common. Hypogammaglobulinemic patients also may develop osteomyelitis and an erysipelas-like rash or cellulitis.

1	Local suppurative complications of infection include cholecystitis, pancreatitis, and cystitis; distant complications include meningitis, endocarditis, arthritis, peritonitis, cellulitis, and septic abortion. All these complications are rare, except in immunocompromised hosts. Hepatitis, interstitial nephritis, and the hemolytic-uremic syndrome occasionally complicate acute infection. Reactive arthritis and other rheumatologic complaints may develop several weeks after infection, especially in persons with the HLA-B27 phenotype. Guillain-Barré syndrome or its Miller Fisher (cranial polyneuropathy) variant follows Campylobacter infections uncommonly—i.e., in 1 of every 1000–2000 cases or, for certain C. jejuni serotypes (such as O19), in 1 of every 100–200 cases. Despite the low frequency of this complication, it is now estimated that Campylobacter infections, because of their high incidence, may trigger 20–40% of all cases of Guillain-Barré syndrome. The presence of sialylated

1	of this complication, it is now estimated that Campylobacter infections, because of their high incidence, may trigger 20–40% of all cases of Guillain-Barré syndrome. The presence of sialylated lipopolysaccharides on C. jejuni strains is a form of molecular mimicry that promotes autoimmune recognition of sialylated cell surface molecules on axons. Asymptomatic Campylobacter infection also may trigger Guillain-Barré syndrome. Immunoproliferative small-intestinal disease (alpha chain disease), a form of lymphoma that originates in small-intestinal mucosa-associated lymphoid tissue, has been associated with C. jejuni; antimicrobial therapy has led to marked clinical improvement.

1	In patients with Campylobacter enteritis, peripheral leukocyte counts reflect the severity of the inflammatory process. However, stools from nearly all Campylobacter-infected patients presenting for medical attention in the United States contain leukocytes or erythrocytes. Gramor Wright-stained fecal smears should be examined in all suspected cases. When the diagnosis of Campylobacter enteritis is suspected on the basis of findings indicating inflammatory diarrhea (fever, fecal leukocytes), clinicians can ask the microbiology laboratory to attempt the visualization of organisms with characteristic vibrioid morphology by direct microscopic examination of stools with Gram’s staining or to use phase-contrast or dark-field microscopy to identify the organisms’ characteristic “darting” motility. Confirmation of the diagnosis of Campylobacter infection is based on identification of an isolate from cultures of stool, blood, or another site. Campylobacterspecific media should be used to

1	Confirmation of the diagnosis of Campylobacter infection is based on identification of an isolate from cultures of stool, blood, or another site. Campylobacterspecific media should be used to culture stools from all patients with inflammatory or bloody diarrhea. Because all Campylobacter species are fastidious, they will not be isolated unless selective media or other selective techniques are used. Not all media are equally useful for isolation of the broad array of campylobacters; therefore, failure to isolate campylobacters from stool does not entirely rule out their presence. Species-specific polymerase chain reaction techniques have been developed to facilitate exact diagnoses. The detection of the organisms in stool almost always implies infection; there is a brief period of post-convalescent fecal carriage and no obvious commensalism in humans. In contrast, Campylobacter sputorum and related organisms found in the oral cavity are commensals that only rarely have pathogenic

1	fecal carriage and no obvious commensalism in humans. In contrast, Campylobacter sputorum and related organisms found in the oral cavity are commensals that only rarely have pathogenic significance. Because of the low levels of metabolic activity of Campylobacter species in standard blood culture media, Campylobacter bacteremia may be difficult to detect unless laboratorians check for low-positive results in quantitative assays.

1	The symptoms of Campylobacter enteritis are not sufficiently unusual to distinguish this illness from that due to Salmonella, Shigella, Yersinia, and other pathogens. The combination of fever and fecal leukocytes or erythrocytes is indicative of inflammatory diarrhea, and definitive diagnosis is based on culture or demonstration of the characteristic organisms on stained fecal smears. Similarly, extraintestinal Campylobacter illness is diagnosed by culture. Infection due to Campylobacter should be suspected in the setting of septic abortion, and that due to C. fetus should be suspected specifically in the setting of septic thrombophlebitis. It is important to reiterate that (1) the presentation of Campylobacter enteritis may mimic that of ulcerative colitis or Crohn’s disease, (2) Campylobacter enteritis is much more common than either of the latter (especially among young adults), and (3) biopsy may not distinguish among these entities. Thus a diagnosis of inflammatory bowel disease

1	enteritis is much more common than either of the latter (especially among young adults), and (3) biopsy may not distinguish among these entities. Thus a diagnosis of inflammatory bowel disease should not be made until Campylobacter infection has been ruled out, especially in persons with a history of foreign travel, significant animal contact, immunodeficiency, or exposure incurring a high risk of transmission.

1	Fluid and electrolyte replacement is central to the treatment of diarrheal illnesses (Chap. 160). Even among patients presenting for medical attention with Campylobacter enteritis, not all clearly benefit from specific antimicrobial therapy. Indications for therapy include high fever, bloody diarrhea, severe diarrhea, persistence for >1 week, and worsening of symptoms. A 5to 7-day course of erythromycin (250 mg orally four times daily or—for children—30–50 mg/kg per day, in divided doses) is the regimen of choice. Both clinical trials and in vitro susceptibility testing indicate that other macrolides, including azithromycin (a 1or 3-day regimen), also are useful therapeutic agents. An alternative regimen for adults is ciprofloxacin (500 mg orally twice daily) or another fluoroquinolone for 5–7 days, but resistance to this class of agents as well as to tetracyclines is substantial; ~22% of U.S. isolates in 2010 were resistant to ciprofloxacin. Because macrolide resistance usually is

1	for 5–7 days, but resistance to this class of agents as well as to tetracyclines is substantial; ~22% of U.S. isolates in 2010 were resistant to ciprofloxacin. Because macrolide resistance usually is much less common (<10%), these drugs are the empirical agents of choice. Patients infected with antibiotic-resistant strains are at increased risk of adverse outcomes. Use of antimotility agents, which may prolong the duration of symptoms and have been associated with toxic megacolon and with death, is not recommended.

1	For systemic infections, treatment with gentamicin (1.7 mg/kg IV every 8 h after a loading dose of 2 mg/kg), imipenem (500 mg IV every 6 h), or chloramphenicol (50 mg/kg IV each day in three or four divided doses) should be started empirically, but susceptibility testing should then be performed. Ciprofloxacin and amoxicillin-clavulanate are alternative agents for susceptible strains. In the absence of immunocompromise or endovascular infections, therapy should be administered for 14 days. For immunocompromised patients with systemic infections due to C. fetus and for patients with endovascular infections, prolonged therapy (for up to 4 weeks) is usually necessary. For recurrent infections in immunocompromised hosts, lifelong therapy/prophylaxis is sometimes necessary.

1	Nearly all patients recover fully from Campylobacter enteritis, either spontaneously or after antimicrobial therapy. Volume depletion probably contributes to the few deaths that are reported. As stated above, occasional patients develop reactive arthritis or Guillain-Barré syndrome or its variants. Systemic infection with C. fetus is much more often fatal than that due to related species; this higher mortality rate reflects in part the population affected. Prognosis depends on the rapidity with which appropriate therapy is begun. Otherwise healthy hosts usually survive C. fetus infections without sequelae. Compromised hosts often have recurrent and/or life-threatening infections due to a variety of Campylobacter species. Matthew K. Waldor, Edward T. Ryan

1	Matthew K. Waldor, Edward T. Ryan Members of the genus Vibrio cause a number of important infectious syndromes. Classic among them is cholera, a devastating diarrheal disease caused by Vibrio cholerae that has been responsible for seven global pandemics and much suffering over the past two centuries. Epidemic cholera remains a significant public health concern in the developing world today. Other vibrioses caused by other Vibrio species include syndromes of diarrhea, soft tissue infection, or primary sepsis. All Vibrio species are highly motile, facultatively anaerobic, curved gram-negative rods with one or more flagella. In nature, vibrios most commonly reside in tidal rivers and bays under conditions of moderate salinity. They proliferate in the summer months when water temperatures exceed 20°C. As might be expected, the illnesses they cause also increase in frequency during the warm months.

1	Cholera is an acute diarrheal disease that can, in a matter of hours, result in profound, rapidly progressive dehydration and death. Accordingly, cholera gravis (the severe form) is a much-feared disease, particularly in its epidemic presentation. Fortunately, prompt aggressive fluid repletion and supportive care can obviate the high mortality that is historically associated with cholera. Although the term cholera has occasionally been applied to any severely dehydrating secretory diarrheal illness, whether infectious in etiology or not, it now refers to disease caused by V. cholerae serogroup O1 or O139—i.e., the serogroups with epidemic potential.

1	The species V. cholerae is classified into more than 200 serogroups based on the carbohydrate determinants of their lipopolysaccharide (LPS) O antigens. Although some non-O1 V. cholerae serogroups (strains that do not agglutinate in antisera to the O1 group antigen) have occasionally caused sporadic outbreaks of diarrhea, serogroup O1 was, until the emergence of serogroup O139 in 1992, the exclusive cause of epidemic cholera. Two biotypes of V. cholerae O1, classical and El Tor, are distinguished. Each biotype is further subdivided into two serotypes, termed Inaba and Ogawa. The natural habitat of V. cholerae is coastal salt water and brackish estuaries, where the organism lives in close relation to plankton. V. cholerae can also exist in freshwater in the presence of adequate nutrients and warmth. Humans become infected incidentally but, once infected, can act as vehicles for spread. Ingestion of water contaminated by human feces is the most common means of acquisition of

1	V. cholerae. Consumption of contaminated food also can contribute to spread. There is no known animal reservoir. Although the infectious dose is relatively high, it is markedly reduced in hypochlorhydric persons, in those using antacids, and when gastric acidity is buffered by a meal. Cholera is predominantly a pediatric disease in endemic areas, but it affects adults and children equally when newly introduced into a population. In endemic areas, the burden of disease is often greatest during “cholera seasons” associated with high temperatures, heavy rainfall, and flooding, but cholera can occur year-round. For unexplained reasons, susceptibility to cholera is significantly influenced by ABO blood group status; persons with type O blood are at greatest risk of severe disease if infected, whereas those with type AB are at least risk. Cholera is native to the Ganges delta in the Indian subcontinent. Since 1817, seven global pandemics have occurred. The current (seventh) pandemic—the

1	whereas those with type AB are at least risk. Cholera is native to the Ganges delta in the Indian subcontinent. Since 1817, seven global pandemics have occurred. The current (seventh) pandemic—the first due to the El Tor bio type—began in Indonesia in 1961 and spread in serial waves throughout Asia as V. cholerae El Tor displaced the endemic classical biotype, which is thought to have caused the previous six pandemics. In the early 1970s, El Tor cholera erupted in Africa, causing major epidemics

1	Confirmed epidemics, but not reported to WHO Yearly incidence rate [2010 2012], per 100,000 inhabitants >1000 [100–1000] [10–100] [1–10] [0.1–1] [0.01–0.1] <0.01 0 : No reported case FIGuRE 193-1 World distribution of cholera in 2010–2012. WHO, World Health Organization. (Courtesy of Drs. M. and R. Piarroux, Université de la Méditerranée, France; with permission.) before becoming a persistent endemic problem. Currently, >90% of cholera cases reported annually to the World Health Organization (WHO) are from Africa (Fig. 193-1), but the true burden in Africa as well as in Asia is unknown because diagnosis is often syndromic and because many countries with endemic cholera do not report cholera to the WHO. It is possible that >3 million cases of cholera occur yearly (of which only ~200,000 are reported to the WHO), resulting in >100,000 deaths annually (of which <5000 are reported to the WHO).

1	After a century without cholera in Latin America, the current cholera pandemic reached Central and South America in 1991. Following an initial explosive spread that affected millions, the burden of disease has markedly decreased in Latin America. In 2010, a severe cholera outbreak began in Haiti, a country with no recorded history of this disease. Several lines of evidence indicate that cholera was likely introduced into Haiti by United Nations security forces from Asia, raising the possibility that asymptomatic carriers of V. cholerae play an important role in transmitting cholera over long distances. To date, the outbreak has involved more than 700,000 individuals, resulting in thousands of deaths. The recent history of cholera has been punctuated by such severe outbreaks, especially among impoverished or displaced persons. These outbreaks are often precipitated by war or other circumstances that lead to the breakdown of public health measures. Such was the case in the camps for

1	among impoverished or displaced persons. These outbreaks are often precipitated by war or other circumstances that lead to the breakdown of public health measures. Such was the case in the camps for Rwandan refugees set up in 1994 around Goma, Zaire, and in 2008–2009 in Zimbabwe.

1	Sporadic endemic infections due to V. cholerae O1 strains related to the seventh-pandemic strain have been recognized along the U.S. Gulf Coast of Louisiana and Texas. These infections are typically associated with the consumption of contaminated, locally harvested shellfish. Occasionally, cases in U.S. locations remote from the Gulf Coast have been linked to shipped-in Gulf Coast seafood.

1	In October 1992, a large-scale outbreak of clinical cholera caused by a new serogroup, O139, occurred in southeastern India. The organism appears to be a derivative of El Tor O1 but has a distinct LPS and an immunologically related O-antigen polysaccharide capsule. (O1 organisms are not encapsulated.) After an initial spread across 11 Asian countries, V. cholerae O139 has once again been almost entirely replaced by O1 strains. The clinical manifestations of disease caused by V. cholerae O139 are indistinguishable from those of O1 cholera. Immunity to one, however, is not protective against the other.

1	In the final analysis, cholera is a toxin-mediated disease. The watery diarrhea characteristic of cholera is due to the action of cholera toxin, a potent protein enterotoxin elaborated by the organism in the small intestine. The toxin-coregulated pilus (TCP), so named because its synthesis is regulated in parallel with that of cholera toxin, is essential for V. cholerae to survive and multiply in (colonize) the small intestine. Cholera toxin, TCP, and several other virulence factors are coordinately regulated by ToxR. This protein modulates the expression of genes coding for virulence factors in response to environmental signals via a cascade of regulatory proteins. Additional regulatory processes, including bacterial responses to the density of the bacterial population (in a phenomenon known as quorum sensing), modulate the virulence of V. cholerae.

1	Once established in the human small bowel, the organism produces cholera toxin, which consists of a monomeric enzymatic moiety (the A subunit) and a pentameric binding moiety (the B subunit). The B pen-tamer binds to GM1 ganglioside, a glycolipid on the surface of epithelial cells that serves as the toxin receptor and makes possible the delivery of the A subunit to its cytosolic target. The activated A subunit (A1) irreversibly transfers ADP-ribose from nicotinamide adenine dinucleotide to its specific target protein, the GTP-binding regulatory component of adenylate cyclase. The ADP-ribosylated G protein upregulates the activity of adenylate cyclase; the result is the intracellular accumulation of high levels of cyclic AMP. In intestinal epithelial cells, cyclic AMP inhibits the absorptive sodium transport system in villus cells and activates the secretory chloride transport system in crypt cells, and these events lead to the accumulation of sodium chloride in the intestinal lumen.

1	sodium transport system in villus cells and activates the secretory chloride transport system in crypt cells, and these events lead to the accumulation of sodium chloride in the intestinal lumen. Because water moves passively to maintain osmolality, isotonic fluid accumulates in the lumen. When the volume of that fluid exceeds the capacity of the rest of the gut to resorb it, watery diarrhea results. Unless the wasted fluid and electrolytes are adequately replaced, shock (due to profound dehydration) and acidosis (due to loss of bicarbonate) follow. Although perturbation of the adenylate cyclase pathway is the primary mechanism by which cholera toxin causes excess fluid secretion, cholera toxin also enhances intestinal secretion via prostaglandins and/or neural histamine receptors.

1	The V. cholerae genome comprises two circular chromosomes. Lateral gene transfer has played a key role in the evolution of epidemic V. cholerae. The genes encoding cholera toxin (ctxAB) are part of the genome of a bacteriophage, CTXФ. The receptor for this phage on the V. cholerae surface is the intestinal colonization factor TCP. Because ctxAB is part of a mobile genetic element (CTXФ), horizontal transfer of this bacteriophage may account for the emergence of new toxigenic V. cholerae serogroups. Many of the other genes important for V. cholerae pathogenicity, including the genes encoding the biosynthesis of TCP, those encoding accessory colonization factors, and those regulating virulence gene expression, are clustered together in the V. cholerae pathogenicity island. Similar clustering of virulence genes is found in other bacterial pathogens. It is believed that pathogenicity islands are acquired by horizontal gene transfer.

1	V. cholerae O139 is probably derived from an El Tor O1 strain that acquired the genes for O139 O-antigen synthesis by horizontal gene transfer.

1	Individuals infected with V. cholerae O1 or O139 exhibit a range of clinical manifestations. Some individuals are asymptomatic or have only mild diarrhea; others present with the sudden onset of explosive and life-threatening diarrhea (cholera gravis). The reasons for the range in signs and symptoms of disease are incompletely understood but include the level of preexisting immunity, blood type, and nutritional status. In a nonimmune individual, after a 24to 48-h incubation period, cholera characteristically begins with the sudden onset of painless watery diarrhea that may quickly become voluminous. Patients often vomit. In severe cases, volume loss can exceed 250 mL/ kg in the first 24 h. If fluids and electrolytes are not replaced, hypovolemic shock and death may ensue. Fever is usually absent. Muscle cramps due to electrolyte disturbances are common. The stool has a characteristic appearance: a nonbilious, gray, slightly cloudy fluid with flecks of mucus, no blood, and a somewhat

1	absent. Muscle cramps due to electrolyte disturbances are common. The stool has a characteristic appearance: a nonbilious, gray, slightly cloudy fluid with flecks of mucus, no blood, and a somewhat fishy, inoffensive odor. It has been called “rice-water” stool because of its resemblance to the water in which rice has been washed (Fig. 193-2). Clinical symptoms

1	FIGuRE 193-2 Rice water cholera stool. Note floating mucus and gray watery appearance. (Courtesy of Dr. A. S. G. Faruque, International Centre for Diarrhoeal Disease Research, Dhaka; with permission.) parallel volume contraction: at losses of <5% of normal body weight, 1063 thirst develops; at 5–10%, postural hypotension, weakness, tachycardia, and decreased skin turgor are documented; and at >10%, oliguria, weak or absent pulses, sunken eyes (and, in infants, sunken fontanelles), wrinkled (“washerwoman”) skin, somnolence, and coma are characteristic. Complications derive exclusively from the effects of volume and electrolyte depletion and include renal failure due to acute tubular necrosis. Thus, if the patient is adequately treated with fluid and electrolytes, complications are averted and the process is self-limited, resolving in a few days.

1	Laboratory data usually reveal an elevated hematocrit (due to hemoconcentration) in nonanemic patients; mild neutrophilic leukocytosis; elevated levels of blood urea nitrogen and creatinine consistent with prerenal azotemia; normal sodium, potassium, and chloride levels; a markedly reduced bicarbonate level (<15 mmol/L); and an elevated anion gap (due to increases in serum lactate, protein, and phosphate). Arterial pH is usually low (~7.2).

1	Cholera should be suspected when a patient ≥2 years of age develops acute watery diarrhea in an area known to have cholera or when a patient ≥5 years of age develops severe dehydration or dies from acute watery diarrhea, even in an area where cholera is not known to be present. The clinical suspicion of cholera can be confirmed by the identification of V. cholerae in stool; however, the organism must be specifically sought. With experience, it can be detected directly by dark-field microscopy on a wet mount of fresh stool, and its serotype can be discerned by immobilization with specific antiserum. Laboratory isolation of the organism requires the use of a selective medium such as taurocholate-tellurite-gelatin (TTG) agar or thiosulfate–citrate–bile salts–sucrose (TCBS) agar. If a delay in sample processing is expected, Carey-Blair transport medium and/or alkaline-peptone water-enrichment medium may be used as well. In endemic areas, there is little need for biochemical confirmation

1	in sample processing is expected, Carey-Blair transport medium and/or alkaline-peptone water-enrichment medium may be used as well. In endemic areas, there is little need for biochemical confirmation and characterization, although these tasks may be worthwhile in places where V. cholerae is an uncommon isolate. Standard microbiologic biochemical testing for Enterobacteriaceae will suffice for identification of V. cholerae. All vibrios are oxidase-positive. A point-of-care antigen-detection cholera dipstick assay is now commercially available for use in the field or where laboratory facilities are lacking.

1	Death from cholera is due to hypovolemic shock; thus treatment of individuals with cholera first and foremost requires fluid resuscitation and management. In light of the level of dehydration (Table 193-1) and the patient’s age and weight, euvolemia should first be rapidly restored, and adequate hydration should then be maintained to replace ongoing fluid losses (Table 193-2). Administration of oral rehydration solution (ORS) takes advantage of the hexose-Na+ co-transport mechanism to move Na+ across the gut mucosa together with an actively transported molecule such as glucose (or galactose). Cl– and water follow. This transport mechanism remains intact even when cholera toxin is active. ORS may be made by adding safe water to prepackaged sachets containing salts and sugar or by adding 0.5 teaspoon of table salt and 6 teaspoons of table sugar to 1 L of safe water. Potassium intake in bananas or green coconut water should be encouraged. A number of ORS formulations are available, and

1	teaspoon of table salt and 6 teaspoons of table sugar to 1 L of safe water. Potassium intake in bananas or green coconut water should be encouraged. A number of ORS formulations are available, and the WHO now recommends “low-osmolarity” ORS for treatment of individuals with dehydrating diarrhea of any cause (Table 193-3). If available, rice-based ORS is considered superior to standard ORS in the treatment of cholera. ORS can be administered via a nasogastric tube to individuals who cannot ingest fluid; however, optimal management of individuals with severe dehydration includes the administration of IV fluid and electrolytes. Because profound acidosis (pH <7.2) is common in this group, Ringer’s lactate is the best choice among commercial products (Table 193-4); it must be used with additional potassium supplements, preferably given by mouth.

1	Degree of Dehydration Clinical Findings None or mild, but diarrhea Thirst in some cases; <5% loss of total body weight Moderate Thirst, postural hypotension, weakness, tachycardia, decreased skin turgor, dry mouth/ tongue, no tears; 5–10% loss of total body weight Severe Unconsciousness, lethargy, or “floppiness”; weak or absent pulse; inability to drink; sunken eyes (and, in infants, sunken fontanelles); >10% loss of total body weight TABLE 193-2 TREATMEnT OF CHOLERA, BAsEd On dEgREE OF dEHydRATIOna Degree of Dehydration, Patient’s Age (Weight) Treatmentb None or Mild, But Diarrheac <2 years 1/4–1/2 cup (50–100 mL) of ORS, to a maximum of 0.5 L/d 2–9 years 1/2–1 cup (100–200 mL) of ORS, to a maximum of 1 L/d ≥10 years As much ORS as desired, to a maximum of 2 L/d

1	Moderatec,d <4 months (<5 kg) 200–400 mL of ORS 4–11 months (5–<8 kg) 400–600 mL of ORS 12–23 months (8–<11 kg) 600–800 mL of ORS 2–4 years (11–<16 kg) 800–1200 mL of ORS 5–14 years (16–<30 kg) 1200–2200 mL of ORS ≥15 years (≥30 kg) 2200–4000 mL of ORS All ages and weights Undertake IV fluid replacement with Ringer’s lactate (or, if not available, normal saline). Give 100 mL/kg in the first 3-h period (or the first 6-h period for children <12 months old); start rapidly, then slow down. Give a total of 200 mL/ kg in the first 24 h. Continue until the patient is awake, can ingest ORS, and no longer has a weak pulse. aAdapted from World Health Organization: First steps for managing an outbreak of acute diarrhoea. Global Task Force on Cholera Control, 2009 (www.who.int/topics/cholera). bContinue normal feeding during treatment. cReassess regularly; monitor stool and vomit output. dVolumes of ORS listed should be given within the first 4 h. Abbreviation: ORS, oral rehydration solution.

1	Abbreviation: ORS, oral rehydration solution. TABLE 193-3 COMPOsITIOn OF wORLd HEALTH ORgAnIzATIOn REduCEd-OsMOLARITy ORAL REHydRATIOn sOLuTIOn (ORs)a,b Constituent Concentration, mmol/L aContains (per package, to be added to 1 L of drinking water): NaCl, 2.6 g; Na C H O ·2H O, 2.9 g; KCl, 1.5 g; and glucose (anhydrous), 13.5 g. bIf prepackaged ORS is unavailable, a simple homemade alternative can be prepared by combining 3.5 g (~1/2 teaspoon) of NaCl with either 50 g of precooked rice cereal or 6 teaspoons of table sugar (sucrose) in 1 L of drinking water. In that case, potassium must be supplied separately (e.g., in orange juice or coconut water). c10 mmol of citrate per liter, which supplies 30 mmol HCO3/L. Concentration, mmol/L aPotassium supplements, preferably administered by mouth, are required to replace the usual potassium losses from stool.

1	Concentration, mmol/L aPotassium supplements, preferably administered by mouth, are required to replace the usual potassium losses from stool. The total fluid deficit in severely dehydrated patients (>10% of body weight) can be replaced safely within the first 3–4 h of therapy, half within the first hour. Transient muscle cramps and tetany are common. Thereafter, oral therapy can usually be initiated, with the goal of maintaining fluid intake equal to fluid output. However, patients with continued large-volume diarrhea may require prolonged IV treatment to match gastrointestinal fluid losses. Severe hypokalemia can develop but will respond to potassium given either IV or orally. In the absence of adequate staff to monitor the patient’s progress, the oral route of rehydration and potassium replacement is safer than the IV route.

1	Although not necessary for cure, the use of an antibiotic to which the organism is susceptible diminishes the duration and volume of fluid loss and hastens clearance of the organism from the stool. Adjunctive antibiotics should therefore be administered to patients with moderate or severe dehydration due to cholera. In many areas, macrolides such as erythromycin (adults, 250 mg orally four times a day for 3 days; children, 12.5 mg/kg per dose four times a day for 3 days) or azithromycin (adults, a single 1-g dose; children, a single 20-mg/kg dose) are the agents of choice. Increasing resistance to tetracyclines is widespread; however, in areas with confirmed susceptibility, tetracycline (nonpregnant adults, 500 mg orally four times a day for 3 days; children >8 years old, 12.5 mg/kg per dose four times a day for 3 days) or doxycycline (nonpregnant adults, a 300-mg single dose; children >8 years old, a single dose of 4–6 mg/ kg) may be used. Similarly, increasing resistance to

1	per dose four times a day for 3 days) or doxycycline (nonpregnant adults, a 300-mg single dose; children >8 years old, a single dose of 4–6 mg/ kg) may be used. Similarly, increasing resistance to fluoroquinolones is being reported, but in areas with confirmed susceptibility, a fluoroquinolone such as ciprofloxacin may be used (adults, 500 mg twice a day for 3 days; children, 15 mg/kg twice a day for 3 days).

1	Provision of safe water and of facilities for sanitary disposal of feces, improved nutrition, and attention to food preparation and storage in the household can significantly reduce the incidence of cholera. In addition, precautions should be taken to prevent the spread of cholera via infected and potentially asymptomatic persons from endemic to nonendemic regions of the world (as was probably the case in the ongoing outbreak in Haiti; see “Microbiology and Epidemiology,” above).

1	Much effort has been devoted to the development of an effective cholera vaccine over the past few decades, with a particular focus on oral vaccine strains. In an attempt to maximize mucosal responses, two types of oral cholera vaccine have been developed: oral killed vaccines and live attenuated vaccines. Currently, two oral killed cholera vaccines have been prequalified by the WHO and are available internationally. WC-rBS (Dukoral®; Crucell, Stockholm, Sweden) contains several biotypes and serotypes of V. cholerae O1 supplemented with 1 mg of recombinant cholera toxin B subunit per dose. BivWC (Shanchol™; Shantha Biotechnics–Sanofi Pasteur, Mumbai, India) contains several biotypes and serotypes of V. cholerae O1 and

1	V. cholerae O139 without supplemental cholera toxin B subunit. The vaccines are administered as a twoor three-dose regimen, with doses usually separated by 14 days. They provide ~60–85% protection for the first few months. Booster immunizations of WC-rBS are recommended after 2 years for individuals ≥6 years of age and after 6 months for children 2–5 years of age. For BivWC, which was developed more recently, no formal recommendation regarding booster immunizations exists. However, BivWC was associated with ~60% protection over 5 years among recipients of all ages in a study in Kolkata, India; the rate of protection among children ≤5 years of age approximated 40%. Models predict significant herd immunity when vaccination coverage rates exceed 50%. The killed vaccines have been safely administered among populations with high rates of HIV.

1	Oral live attenuated vaccines for V. cholerae are also in development. These strains have in common the fact that they lack the genes encoding cholera toxin. One such vaccine, CVD 103-HgR, was safe and immunogenic in phase 1 and 2 studies but afforded minimal protection in a large field trial in Indonesia. Other live attenuated vaccine candidate strains have been prepared from El Tor and O139

1	V. cholerae and have been tested in studies of volunteers. A possible advantage of live attenuated cholera vaccines is that they may induce protection after a single oral dose. Conjugate and subunit cholera vaccines are also being developed. Recognizing that it may be decades before safe water and adequate sanitation become a reality for those most at risk of cholera, the WHO has now recommended incorporation of cholera vaccination into comprehensive control strategies and has established an international stockpile of oral killed cholera vaccine to assist in outbreak responses. No cholera vaccine is commercially available in the United States.

1	The genus Vibrio includes several human pathogens that do not cause cholera. Abundant in coastal waters throughout the world, noncholera vibrios can reach high concentrations in the tissues of filter-feeding mollusks. As a result, human infection commonly follows the ingestion of seawater or of raw or undercooked shellfish (Table 193-5). Most noncholera vibrios can be cultured on blood or MacConkey agar, which contains enough salt to support the growth of these halophilic species. In the microbiology laboratory, the species of noncholera vibrios are distinguished by standard biochemical tests. The most important of these organisms are Vibrio parahaemolyticus and Vibrio vulnificus.

1	The two major types of syndromes for which these species are responsible are gastrointestinal illness (due to V. parahaemolyticus, non-O1/O139 V. cholerae, Vibrio mimicus, Vibrio fluvialis, Vibrio hollisae, and Vibrio furnissii) and soft tissue infections (due to V. vulnificus, Vibrio alginolyticus, and Vibrio damselae). V. vulnificus is also a cause of primary sepsis in some compromised individuals.

1	V. parahaemolyticus Widespread in marine environments, the halophilic V. parahaemolyticus causes food-borne enteritis worldwide. This species was originally implicated in enteritis in Japan in 1953, accounting for 24% of reported cases in one study—a 1065 rate that presumably was due to the common practice of eating raw seafood in that country. In the United States, common-source outbreaks of diarrhea caused by this organism have been linked to the consumption of undercooked or improperly handled seafood or of other foods contaminated by seawater. Since the mid-1990s, the incidence of V. parahaemolyticus infections has increased in several countries, including the United States. Serotypes O3:K6, O4:K68, and O1:Kuntypable, which are genetically related to one another, account in part for this increase. Serotypes O4:K12 and O4:KUT were initially unique to the Pacific Northwest but caused recent outbreaks in the eastern United States and Spain. The enteropathogenicity of V.

1	in part for this increase. Serotypes O4:K12 and O4:KUT were initially unique to the Pacific Northwest but caused recent outbreaks in the eastern United States and Spain. The enteropathogenicity of V. parahaemolyticus is linked to its ability to cause hemolysis on Wagatsuma agar (i.e., the Kanagawa phenomenon). Although the mechanisms by which the organism causes diarrhea are not fully defined, the genome sequence of

1	V. parahaemolyticus contains two type III secretion systems, which directly inject toxic bacterial proteins into host cells. The activity of one of these secretion systems is required for intestinal colonization and virulence in animal models. V. parahaemolyticus should be considered a possible etiologic agent in all cases of diarrhea that can be linked epidemiologically to seafood consumption or to the sea itself.

1	Infections with V. parahaemolyticus can result in two distinct gastrointestinal presentations. The more common of the two presentations (including nearly all cases in North America) is characterized by watery diarrhea, usually occurring in conjunction with abdominal cramps, nausea, and vomiting and accompanied in ~25% of cases by fever and chills. After an incubation period of 4 h to 4 days, symptoms develop and persist for a median of 3 days. Dysentery, the less common presentation, is characterized by severe abdominal cramps, nausea, vomiting, and bloody or mucoid stools. V. parahaemolyticus also causes rare cases of wound infection and otitis and very rare cases of sepsis.

1	Most cases of V. parahaemolyticus–associated gastrointestinal illness, regardless of the presentation, are self-limited. Fluid replacement should be stressed. The role of antimicrobials is uncertain, but they may be of benefit in moderate or severe disease. Doxycycline, fluoroquinolones, or macrolides are usually used. Deaths are extremely rare among immunocompetent individuals. Severe infections are associated with underlying diseases, including diabetes, preexisting liver disease, iron-overload states, or immunosuppression. Non-O1/O139 (Noncholera) V. cholerae The heterogeneous non-O1/O139

1	Non-O1/O139 (Noncholera) V. cholerae The heterogeneous non-O1/O139 V. cholerae organisms cannot be distinguished from V. cholerae O1 or O139 by routine biochemical tests but do not agglutinate in O1 or O139 antiserum. Non-O1/O139 strains have caused several well-studied food-borne outbreaks of gastroenteritis and have also been responsible for sporadic cases of otitis media, wound infection, and bacteremia; although gastroenteritis outbreaks can occur, non-O1/O139 V. cholerae strains do not cause epidemics of cholera. Like other vibrios, non-O1/ O139 V. cholerae organisms are widely distributed in marine environments. In most instances, recognized cases in the United States have been associated with the consumption of raw oysters or with recent travel. The broad clinical spectrum of diarrheal illness caused by these aEspecially with liver disease or hemochromatosis. Source: Table 161-3 in Harrisons Principles of Internal Medicine, 14th edition.

1	1066 organisms is probably due to the group’s heterogeneous virulence attributes. In the United States, about half of all non-O1/O139 V. cholerae isolates are from stool samples. The typical incubation period for gastroenteritis due to these organisms is <2 days, and the illness lasts for ~2–7 days. Patients’ stools may be copious and watery or may be partly formed, less voluminous, and bloody or mucoid. Diarrhea can result in severe dehydration. Many cases include abdominal cramps, nausea, vomiting, and fever. Like those with cholera, patients who are seriously dehydrated should receive oral or IV fluids; the value of antibiotics is not clear. Extraintestinal infections due to non-O1/O139 V. cholerae commonly follow occupational or recreational exposure to seawater. Around 10% of non-O1/O139 V. cholerae isolates come from cases of wound infection, 10% from cases of otitis media, and 20% from cases of bacteremia (which is particularly likely to develop in patients with liver disease).

1	V. cholerae isolates come from cases of wound infection, 10% from cases of otitis media, and 20% from cases of bacteremia (which is particularly likely to develop in patients with liver disease). Extraintestinal infections should be treated with antibiotics. Information to guide antibiotic selection and dosing is limited, but most strains are sensitive in vitro to tetracycline, ciprofloxacin, and third-generation cephalosporins.

1	(See also Chap. 156)

1	V. vulnificus Infection with V. vulnificus is rare, but this organism is the most common cause of severe Vibrio infections in the United States. Like most vibrios, V. vulnificus proliferates in the warm summer months and requires a saline environment for growth. In the United States, infections in humans typically occur in coastal states between May and October and most commonly affect men >40 years of age. V. vulnificus has been linked to two distinct syndromes: primary sepsis, which usually occurs in patients with underlying liver disease, and primary wound infection, which generally affects people without underlying disease. (Vulnificus is Latin for “wound maker.”) Some authors have suggested that V. vulnificus also causes gastroenteritis independent of other clinical manifestations. V. vulnificus is endowed with a number of virulence attributes, including a capsule that confers resistance to phagocytosis and to the bactericidal activity of human serum as well as a cytolysin.

1	V. vulnificus is endowed with a number of virulence attributes, including a capsule that confers resistance to phagocytosis and to the bactericidal activity of human serum as well as a cytolysin. Measured as the 50% lethal dose in mice, the organism’s virulence is considerably increased under conditions of iron overload; this observation is consistent with the propensity of

1	V. vulnificus to infect patients who have hemochromatosis.

1	Primary sepsis most often develops in patients who have cirrhosis or hemochromatosis. However, V. vulnificus bacteremia can also affect individuals who have hematopoietic disorders or chronic renal insufficiency, those who are using immunosuppressive medications or alcohol, or (in rare instances) those who have no known underlying disease. After a median incubation period of 16 h, the patient develops malaise, chills, fever, and prostration. One-third of patients develop hypotension, which is often apparent at admission. Cutaneous manifestations develop in most cases (usually within 36 h of onset) and characteristically involve the extremities (the lower more often than the upper). In a common sequence, erythematous patches are followed by ecchymoses, vesicles, and bullae. In fact, sepsis and hemorrhagic bullous skin lesions suggest the diagnosis in appropriate settings. Necrosis and sloughing may also be evident. Laboratory studies reveal leukopenia more often than leukocytosis,

1	and hemorrhagic bullous skin lesions suggest the diagnosis in appropriate settings. Necrosis and sloughing may also be evident. Laboratory studies reveal leukopenia more often than leukocytosis, thrombocytopenia, or elevated levels of fibrin split products. V. vulnificus can be cultured from blood or cutaneous lesions. The mortality rate approaches 50%, with most deaths due to uncontrolled sepsis (Chap. 325). Accordingly, prompt treatment is critical and should include empirical antibiotic administration, aggressive debridement, and general supportive care. V. vulnificus is sensitive in vitro to a number of antibiotics, including tetracycline, fluoroquinolones, and third-generation cephalosporins. Data from animal models suggest that either a fluoroquinolone or the combination of a tetracycline and a third-generation cephalosporin should be used in the treatment of

1	V. vulnificus septicemia. V. vulnificus–associated soft tissue infection can complicate either a fresh or an old wound that comes into contact with seawater; the patient may or may not have underlying disease. After a short incubation period (4 h to 4 days; mean, 12 h), the disease begins with swelling, erythema, and (in many cases) intense pain around the wound. These signs and symptoms are followed by cellulitis, which spreads rapidly and is sometimes accompanied by vesicular, bullous, or necrotic lesions. Metastatic events are uncommon. Most patients have fever and leukocytosis. V. vulnificus can be cultured from skin lesions and occasionally from the blood. Prompt antibiotic therapy and debridement are usually curative. V. alginolyticus First identified as a pathogen of humans in 1973,

1	V. alginolyticus First identified as a pathogen of humans in 1973, V. alginolyticus occasionally causes eye, ear, and wound infections. This species is the most salt-tolerant of the vibrios and can grow in salt concentrations of >10%. Most clinical isolates come from superinfected wounds that presumably become contaminated at the beach. Although its severity varies, V. alginolyticus infection tends not to be serious and generally responds well to antibiotic therapy and drainage. A few cases of otitis externa, otitis media, and conjunctivitis due to this pathogen have been described. Tetracycline treatment usually results in cure. V. alginolyticus is a rare cause of bacteremia in immunocompromised hosts. The authors gratefully acknowledge the valuable contributions of Drs.≥Robert Deresiewicz and Gerald T. Keusch, coauthors of this chapter for previous editions.

1	The authors gratefully acknowledge the valuable contributions of Drs.≥Robert Deresiewicz and Gerald T. Keusch, coauthors of this chapter for previous editions. Brucellosis Nicholas J. Beeching, Michael J. Corbel DEFINITION Brucellosis is a bacterial zoonosis transmitted directly or indirectly to humans from infected animals, predominantly domesticated rumi-nants and swine. The disease is known colloquially as undulant fever 194e because of its remittent character. Although brucellosis commonly presents as an acute febrile illness, its clinical manifestations vary widely, and definitive signs indicative of the diagnosis may be lacking. Thus the clinical diagnosis usually must be supported by the results of bacteriologic and/or serologic tests.

1	Human brucellosis is caused by strains of Brucella, a bacterial genus that was previously suggested, on genetic grounds, to comprise a single species, B. melitensis, with a number of biologic variants exhibiting particular host preferences. This view was challenged on the basis of detailed differences in chromosomal structure and host preference. The traditional classification into nomen species is now favored both because of these differences and because this classification scheme closely reflects the epidemiologic patterns of the infection. The nomen system recognizes B. melitensis, which is the most common cause of symptomatic disease in humans and for which the main sources are sheep, goats, and camels; B. abortus, which is usually acquired from cattle or buffalo; B. suis, which generally is acquired from swine but has one variant enzootic in reindeer and caribou and another in rodents; and B. canis, which is acquired most often from dogs. B. ovis, which causes reproductive

1	generally is acquired from swine but has one variant enzootic in reindeer and caribou and another in rodents; and B. canis, which is acquired most often from dogs. B. ovis, which causes reproductive disease in sheep, and B. neotomae, which is specific for desert rodents, have not been clearly implicated in human disease. Two new species, B. ceti and B. pinnipedialis, have recently been identified in marine mammals, including seals and dolphins. At least one case of laboratory-acquired human disease due to one of these species has been described, and apparent cases of natural human infection have been reported. As infections in marine mammals appear to be widespread, more cases of zoonotic infection in humans may be identified. Other newly reported species include B. microti (isolated from field voles) and B. inopinata (isolated from a patient with a breast implant). Additional novel strains have been described from diverse species, including baboons, foxes, frogs, and various rodents,

1	field voles) and B. inopinata (isolated from a patient with a breast implant). Additional novel strains have been described from diverse species, including baboons, foxes, frogs, and various rodents, and the genus likely will expand further in forthcoming years. Moreover, it has become apparent that Brucella is closely related to the genus Ochrobactrum, which includes environmental bacteria sometimes associated with opportunistic infections. Genomics-based studies are beginning to elucidate the pathway of evolution from free-living soil bacteria to highly successful intracellular pathogens.

1	All brucellae are small, gram-negative, unencapsulated, nonsporulating, nonmotile rods or coccobacilli. They grow aerobically on peptonebased medium incubated at 37°C; the growth of some types is improved by supplementary CO2. In vivo, brucellae behave as facultative intracellular parasites. The organisms are sensitive to sunlight, ionizing radiation, and moderate heat; they are killed by boiling and pasteurization but are resistant to freezing and drying. Their resistance to drying renders brucellae stable in aerosol form, facilitating airborne transmission. The organisms can survive for up to 2 months in soft cheeses made from goat’s or sheep’s milk; for at least 6 weeks in dry soil contaminated with infected urine, vaginal discharge, or placental or fetal tissues; and for at least 6 months in damp soil or liquid manure kept under cool dark conditions. Brucellae are easily killed by a wide range of common disinfectants used under optimal conditions but are likely to be much more

1	months in damp soil or liquid manure kept under cool dark conditions. Brucellae are easily killed by a wide range of common disinfectants used under optimal conditions but are likely to be much more resistant at low temperatures or in the presence of heavy organic contamination.

1	Brucellosis is a zoonosis whose occurrence and control are closely related to its prevalence in domesticated animals. Its distribution is worldwide apart from the few countries where it has been eradicated from the animal reservoir. The true global 194e-1 prevalence of human brucellosis is unknown because of the imprecision of diagnosis and the inadequacy of reporting and surveillance systems in many countries. Recently, there has been increased recognition of the high incidence of brucellosis in India and parts of China and of importations to countries in Oceania, such as Fiji. In Europe, the incidence of brucellosis in a country is inversely related to gross domestic product, and, in both developed and less well-resourced settings, human brucellosis is related to rural poverty and inadequate access to medical care. Failure of veterinary control programs due to conflict or for economic reasons contributes further to the emergence and re-emergence of disease, as seen currently in some

1	access to medical care. Failure of veterinary control programs due to conflict or for economic reasons contributes further to the emergence and re-emergence of disease, as seen currently in some eastern Mediterranean countries.

1	Even in well-resourced settings, the true incidence of brucellosis in domesticated animals may be 10–20 times higher than the reported figures. Bovine brucellosis has been the target of control programs in many parts of the world and has been eradicated from the cattle populations of Australia, New Zealand, Bulgaria, Canada, Cyprus, Great Britain (including the Channel Islands), Japan, Luxembourg, Romania, the Scandinavian countries, Switzerland, and the Czech and Slovak Republics, among other nations. Its incidence has been reduced to a low level in the United States and most Western European countries, with a varied picture in other parts of the world. Efforts to eradicate B. melitensis infection from sheep and goat populations have been much less successful. These efforts have relied heavily on vaccination programs, which have tended to fluctuate with changing economic and political conditions. In some countries (e.g., Israel), B. melitensis has caused serious outbreaks in cattle.

1	on vaccination programs, which have tended to fluctuate with changing economic and political conditions. In some countries (e.g., Israel), B. melitensis has caused serious outbreaks in cattle. Infections with B. melitensis still pose a major public health problem in Mediterranean countries; in western, central, and southern Asia; and in parts of Africa and South and Central America.

1	Human brucellosis is usually associated with occupational or domestic exposure to infected animals or their products. Farmers, shepherds, goatherds, veterinarians, and employees in slaughterhouses and meat-processing plants in endemic areas are occupationally exposed to infection. Family members of individuals involved in animal husbandry may be at risk, although it is often difficult to differentiate food-borne infection from environmental contamination under these circumstances. Laboratory workers who handle cultures or infected samples also are at risk. Travelers and urban residents usually acquire the infection through consumption of contaminated foods. In countries that have eradicated the disease, new cases are most commonly acquired abroad. Dairy products, especially soft cheeses, unpasteurized milk, and ice cream, are the most frequently implicated sources of infection; raw meat and bone marrow may be sources under exceptional circumstances. Infections acquired through

1	unpasteurized milk, and ice cream, are the most frequently implicated sources of infection; raw meat and bone marrow may be sources under exceptional circumstances. Infections acquired through cosmetic treatments using materials of fetal origin have been reported. Person-to-person transmission is extremely rare, as is transfer of infection by blood or tissue donation. Although brucellosis is a chronic intracellular infection, there is no evidence for increased prevalence or severity among individuals with HIV infection or with immunodeficiency or immunosuppression of other etiologies.

1	Brucellosis may be acquired by ingestion, inhalation, or mucosal or percutaneous exposure. Accidental injection of the live vaccine strains of B. abortus (S19 and RB51) and B. melitensis (Rev 1) can cause disease. B. melitensis and B. suis have historically been developed as biological weapons by several countries and could be exploited for bioterrorism (Chap. 261e). This possibility should be borne in mind in the event of sudden unexplained outbreaks. Exposure to brucellosis elicits both humoral and cell-mediated immune responses. The mechanisms of protective immunity against human brucellosis are presumed to be similar to those documented in laboratory animals, but such generalizations must be interpreted with caution. The response to infection and its outcome are influenced by the virulence, phase, and species of the infecting strain. Differences have been reported between B. abortus and B. suis in modes of cellular entry and subsequent compartmentalization and processing.

1	Antibodies promote clearance of extracellular brucellae by bactericidal action and by facilitation of phagocytosis by polymorphonuclear and mononuclear phagocytes; however, antibodies alone cannot eradicate infection. Organisms taken up by macrophages and other cells can establish persistent intracellular infections. The key target cell is the macrophage, and bacterial mechanisms for suppressing intracellular killing and apoptosis result in very large intracellular populations. Opsonized bacteria are actively phagocytosed by neutrophilic granulocytes and by monocytes. In these and other cells, initial attachment takes place via specific receptors, including Fc, C3, fibronectin, and mannose-binding proteins. Opsonized—but not unopsonized—bacteria trigger an oxidative burst inside phagocytes. Unopsonized bacteria are internalized via similar receptors but at much lower efficiency. Smooth strains enter host cells via lipid rafts. Smooth lipopolysaccharide (LPS), β-cyclic glucan, and

1	Unopsonized bacteria are internalized via similar receptors but at much lower efficiency. Smooth strains enter host cells via lipid rafts. Smooth lipopolysaccharide (LPS), β-cyclic glucan, and possibly an invasion-attachment protein (IalB) are involved in this process. Tumor necrosis factor α (TNF-α) produced early in the course of infection stimulates cytotoxic lymphocytes and activates macrophages, which can kill intracellular brucellae (probably mainly through production of reactive oxygen and nitrogen intermediates) and may clear infection. However, virulent Brucella cells can suppress the TNF-α response, and control of infection in this situation depends on macrophage activation and interferon γ (IFN-γ) responses. Cytokines such as interleukin (IL) 12 promote production of IFN-γ, which drives TH1-type responses and stimulates macrophage activation. Inflammatory cytokines, including IL-4, IL-6, and IL-10, downregulate the protective response. As in other types of intracellular

1	which drives TH1-type responses and stimulates macrophage activation. Inflammatory cytokines, including IL-4, IL-6, and IL-10, downregulate the protective response. As in other types of intracellular infection, it is assumed that initial replication of brucellae takes place within cells of the lymph nodes draining the point of entry. Subsequent hematogenous spread may result in chronic localizing infection at almost any site, although the reticuloendothelial system, musculoskeletal tissues, and genitourinary system are most frequently targeted. Both acute and chronic inflammatory responses develop in brucellosis, and the local tissue response may include granuloma formation with or without necrosis and caseation. Abscesses may also develop, especially in chronic localized infection.

1	The determinants of pathogenicity of Brucella have not been fully characterized, and the mechanisms underlying the manifestations of brucellosis are incompletely understood. The organism is a “stealth” pathogen whose survival strategy is centered on several processes that avoid triggering innate immune responses and that permit survival within monocytic cells. The smooth Brucella LPS, which has an unusual O-chain and core-lipid composition, has relatively low endotoxin activity and plays a key role in pyrogenicity and in resistance to phagocytosis and serum killing in the nonimmune host. In addition, LPS is believed to play a role in suppressing phagosome–lysosome fusion and diverting the internalized bacteria into vacuoles located in endoplasmic reticulum, where intracellular replication takes place. Specific exotoxins have not been isolated, but a type IV secretion system (VirB) that regulates intracellular survival and trafficking has been identified. In B. abortus this system can

1	takes place. Specific exotoxins have not been isolated, but a type IV secretion system (VirB) that regulates intracellular survival and trafficking has been identified. In B. abortus this system can be activated extracellularly, but in B. suis it is activated (by low pH) only during intracellular growth. Brucellae then produce acid-stable proteins that facilitate the organisms’ survival in phagosomes and may enhance their resistance to reactive oxygen intermediates. A type III secretion system based on modified flagellar structures also has been inferred, although not yet confirmed. Virulent brucellae are resistant to defensins and produce a Cu-Zn superoxide dismutase that increases their resistance to reactive oxygen intermediates. A hemolysin-like protein may trigger the release of brucellae from infected cells.

1	Brucellosis almost invariably causes fever, which may be associated with profuse sweats, especially at night. In endemic areas, brucellosis may be difficult to distinguish from the many other causes of fever. However, two features recognized in the nineteenth century distinguish brucellosis from other tropical fevers, such as typhoid and malaria: (1) Left untreated, the fever of brucellosis shows an undulating pattern that persists for weeks before the commencement of an afebrile period that may be followed by relapse. (2) The fever of brucellosis is associated with musculoskeletal symptoms and signs in about one-half of all patients. The clinical syndromes caused by the different nomen species are similar, although B. melitensis tends to be associated with a more acute and aggressive presentation and B. suis with focal abscess induction.

1	B. abortus infections may be more insidious in onset and more likely to become chronic. B. canis infections are reported to present frequently with acute gastrointestinal symptoms. The incubation period varies from 1 week to several months, and the onset of fever and other symptoms may be abrupt or insidious. In addition to experiencing fever and sweats, patients become increasingly apathetic and fatigued; lose appetite and weight; and have nonspecific myalgia, headache, and chills. Overall, the presentation of brucellosis often fits one of three patterns: febrile illness that resembles typhoid but is less severe; fever and acute monoarthritis, typically of the hip or knee, in a young child; and long-lasting fever, misery, and low-back or hip pain in an older man. In an endemic area (e.g., much of the Middle East), a patient with fever and difficulty walking into the clinic would be regarded as having brucellosis until it was proven otherwise.

1	Diagnostic clues in the patient’s history include travel to an endemic area, employment in a diagnostic microbiology laboratory, consumption of unpasteurized milk products (including soft cheeses), contact with animals, accidental inoculation with veterinary Brucella vaccines, and—in an endemic setting—a history of similar illness in the family (documented in almost 50% of cases). Focal features are present in the majority of patients. The most common are musculoskeletal pain and physical findings in the peripheral and axial skeleton (~40% of cases). Osteomyelitis more commonly involves the lumbar and low thoracic vertebrae than the cervical and high thoracic spine. Individual joints that are most commonly affected by septic arthritis are the knee, hip, sacroiliac, shoulder, and sternoclavicular joints; the pattern may be one of monoarthritis or polyarthritis. Osteomyelitis may also accompany septic arthritis.

1	In addition to the usual causes of vertebral osteomyelitis or septic arthritis, the most important disease in the differential diagnosis is tuberculosis. This point influences the therapeutic approach as well as the prognosis, given that several antimicrobial agents used to treat brucellosis are also used to treat tuberculosis. Septic arthritis in brucellosis progresses slowly, starting with small pericapsular erosions. In the vertebrae, anterior erosions of the superior end plate are typically the first features to become evident, with eventual involvement and sclerosis of the whole vertebra. Anterior osteophytes eventually develop, but vertebral destruction or impingement on the spinal cord is rare and usually suggests tuberculosis (Table 194e-1).

1	Other systems may be involved in a manner that resembles typhoid. About one-quarter of patients have a dry cough, usually with few changes visible on the chest x-ray, although pneumonia, empyema, intrathoracic adenopathy, or lung abscess can occur. Sputum or pleural effusion cultures are rarely positive in such cases, which respond well to standard brucellosis treatment. One-quarter of patients have hepatosplenomegaly, and 10–20% have significant lymphadenopathy; the differential diagnosis includes glandular fever–like illness such as that caused by Epstein-Barr virus, Toxoplasma, cytomegalovirus, HIV, or Mycobacterium tuberculosis. Up to 10% of men have acute epididymoorchitis, which must be distinguished from mumps and from surgical problems such as torsion. Prostatitis, inflammation of the seminal vesicles, salpingitis, and pyelonephritis all occur. There is an increased incidence of fetal loss among infected pregnant women, although teratogenicity has not been described and the

1	of the seminal vesicles, salpingitis, and pyelonephritis all occur. There is an increased incidence of fetal loss among infected pregnant women, although teratogenicity has not been described and the tendency toward abortions is much less pronounced in humans than in farm animals.

1	Neurologic involvement is common, with depression and lethargy whose severity may not be fully appreciated by either the patient or the physician until after treatment. A small proportion of patients develop lymphocytic meningoencephalitis that mimics neurotuberculosis, atypical leptospirosis, or noninfectious conditions and that may be complicated by intracerebral abscess, a variety of cranial nerve deficits, or ruptured mycotic aneurysms. Endocarditis occurs in ~1% of cases, most often affecting the aortic valve (natural or prosthetic). Any site in the body may be involved in metastatic abscess formation or inflammation; the female breast and the thyroid gland are affected particularly often. Nonspecific maculopapular rashes and other skin manifestations are uncommon and are rarely noticed by the patient even if they develop.

1	Because the clinical picture of brucellosis is not distinctive, the diagnosis must be based on a history of potential exposure, a presentation consistent with the disease, and supporting laboratory findings. Results of routine biochemical assays are usually within normal limits, although serum levels of hepatic enzymes and bilirubin may be elevated. Peripheral leukocyte counts are usually normal or low, with relative lymphocytosis. Mild anemia may be documented. Thrombocytopenia and disseminated intravascular coagulation with raised levels of fibrinogen degradation products can develop. The erythrocyte sedimentation rate and C-reactive protein levels are often normal but may be raised.

1	In body fluids such as cerebrospinal fluid (CSF) or joint fluid, lymphocytosis and low glucose levels are the norm. Elevated CSF levels of adenosine deaminase cannot be used to distinguish tubercular meningitis, as they may also be found in brucellosis. Biopsied samples of tissues such as lymph node or liver may show noncaseating granulomas (Fig. 194e-1) without acid/alcohol-fast bacilli. The radiologic features of bony disease develop late and are much more subtle than those of FIGURE 194e-1 Liver biopsy specimen from a patient with brucellosis shows a noncaseating granuloma. (From Mandell’s Atlas of Infectious Diseases, Vol II, in DL Stevens [ed]: Skin, Soft Tissue, Bone and Joint Infections, Fig. 5-9; with permission.) tuberculosis or septic arthritis of other etiologies, with less bone and 194e-3 joint destruction. Isotope scanning is more sensitive than plain x-ray and continues to give positive results long after successful treatment.

1	Isolation of brucellae from blood, CSF, bone marrow, or joint fluid or from a tissue aspirate or biopsy sample is definitive, and attempts at isolation are usually successful in 50–70% of cases. Duplicate cultures should be incubated for up to 6 weeks (in air and 10% CO2, respectively). Concentration and lysis of buffy coat cells before culture may increase the isolation rate. Cultures in modern nonradiometric or similar signaling systems (e.g., Bactec) usually become positive within 7–10 days but should be maintained for at least 3 weeks before the results are declared negative. All cultures should be handled under containment conditions appropriate for dangerous pathogens. Brucella species may be misidentified as Agrobacterium, Ochrobactrum, or Psychrobacter (Moraxella) phenylpyruvicus by the gallery identification strips commonly used in the diagnostic laboratory. In recent years, matrix-assisted laser desorption ionization time-of-flight spectrometry (MALDI-TOF MS) has emerged as

1	by the gallery identification strips commonly used in the diagnostic laboratory. In recent years, matrix-assisted laser desorption ionization time-of-flight spectrometry (MALDI-TOF MS) has emerged as a powerful tool in bacterial identification. The relative homogeneity of classical Brucella species makes identification beyond the genus level by routine approaches challenging, although further improvements may facilitate discrimination at the species level. The place of this technique in routine diagnostic practice will depend on such refinements. Meanwhile, the authors are aware of cases in which blood culture isolates have been identified incorrectly using MALDI-TOF MS.

1	The peripheral blood–based polymerase chain reaction has enormous potential to detect bacteremia, to predict relapse, and to exclude “chronic brucellosis.” This method is probably more sensitive and is certainly quicker than blood culture, and it does not carry the attendant biohazard risk posed by culture. Nucleic acid amplification techniques are now quite widely used, although no single standardized procedure has been adopted. Primers for the spacer region between the genes encoding the 16S and 23S ribosomal RNAs (rrs-rrl), various outer-membrane protein–encoding genes, the insertion sequence IS711, and the protein BCSP31 are sensitive and specific. Blood and other tissues are the most suitable samples for analysis.

1	Serologic examination often provides the only positive laboratory findings in brucellosis. In acute infection, IgM antibodies appear early and are followed by IgG and IgA. All these antibodies are active in agglutination tests, whether performed by tube, plate, or microagglutination methods. The majority of patients have detectable agglutinins at this stage. As the disease progresses, IgM levels decline, and the avidity and subclass distribution of IgG and IgA change. The result is reduced or undetectable agglutinin titers. However, the antibodies are detectable by alternative tests, including the complement fixation test, Coomb’s antiglobulin test, and enzyme-linked immunosorbent assay. There is no clear cutoff value for a diagnostic titer. Rather, serology results must be interpreted in the context of exposure history and clinical presentation. In endemic areas or in settings of potential occupational exposure, agglutinin titers of 1:320–1:640 or higher are considered diagnostic; in

1	the context of exposure history and clinical presentation. In endemic areas or in settings of potential occupational exposure, agglutinin titers of 1:320–1:640 or higher are considered diagnostic; in nonendemic areas, a titer of ≥1:160 is considered significant. Repetition of tests after 2–4 weeks may demonstrate a rising titer.

1	In most centers, the standard agglutination test is still the mainstay of serologic diagnosis, although some investigators rely on the rose bengal test, which has not been fully validated for human diagnostic use. Dipstick assays for anti-Brucella IgM are useful for the diagnosis of acute infection but are less sensitive for infection with symptoms of several months’ duration. In an endemic setting, more than 90% of patients with acute bacteremia have standard agglutination titers of at least 1:320. Other screening tests are used in some centers.

1	Antibody to the Brucella LPS O chain—the dominant antigen—is detected by all the conventional tests that employ smooth B. abortus cells as antigen. Since B. abortus cross-reacts with B. melitensis and B. suis, there is no advantage in replicating the tests with these antigens. Cross-reactions also occur with the O chains of some other gram-negative bacteria, including Yersinia enterocolitica O:9, Escherichia coli O157, Francisella tularensis, Salmonella enterica group N, Stenotrophomonas maltophilia, and Vibrio cholerae. Cross-reactions do not occur with the cell-surface antigens of rough Brucella strains such as B. canis or B. ovis; serologic tests for these nomen species must employ an antigen prepared from either one. The live B. abortus vaccine strain RB51 does not elicit antibody responses in serologic tests that use smooth antigens, and this fact must be taken into account if serologic tests are employed in attempts to identify or follow the course of infections in persons

1	responses in serologic tests that use smooth antigens, and this fact must be taken into account if serologic tests are employed in attempts to identify or follow the course of infections in persons accidentally exposed to the vaccine.

1	The broad aims of antimicrobial therapy are to treat and relieve the symptoms of current infection and to prevent relapse. Focal disease presentations may require specific intervention in addition to more prolonged and tailored antibiotic therapy. In addition, tuberculosis must always be excluded, or—to prevent the emergence of resistance—therapy must be tailored to specifically exclude drugs active against tuberculosis (e.g., rifampin used alone) or to include a full antituberculous regimen.

1	Early experience with streptomycin monotherapy showed that relapse was common; thus dual therapy with tetracyclines became the norm. This is still the most effective combination, but alternatives may be used, with the options depending on local or national policy about the use of rifampin for the treatment of nonmycobacterial infection. For the several antimicrobial agents that are active in vivo, efficacy can usually be predicted by in vitro testing. However, numerous Brucella strains show in vitro sensitivity to a whole range of antimicrobials that are therapeutically ineffective, including assorted β-lactams. Moreover, the use of fluoroquinolones remains controversial despite the good in vitro activity and white-cell penetration of most agents of this class. Low intravacuolar pH is probably a factor in the poor performance of these drugs.

1	For adults with acute nonfocal brucellosis (duration, <1 month), a 6-week course of therapy incorporating at least two antimicrobial agents is required. Complex or focal disease necessitates ≥3 months of therapy. Adherence to the therapeutic regimen is very important, and poor adherence underlies almost all cases of apparent treatment failure; such failure is rarely due to the emergence of drug resistance, although increasing resistance to trimethoprimsulfamethoxazole (TMP-SMX) has been reported at one center. There is good retrospective evidence that a 3-week course of two agents is as effective as a 6-week course for treatment and prevention of relapse in children, but this point has not yet been proven in prospective studies.

1	The gold standard for the treatment of brucellosis in adults is IM streptomycin (0.75–1 g daily for 14–21 days) together with doxycycline (100 mg twice daily for 6 weeks). In both clinical trials and observational studies, relapse follows such treatment in 5–10% of cases. The usual alternative regimen (and the current World Health Organization recommendation) is rifampin (600–900 mg/d) plus doxycycline (100 mg twice daily) for 6 weeks. The relapse/failure rate is ~10% in trial conditions but rises to >20% in many non-trial situations, possibly because doxycycline levels are reduced and clearance rates increased by concomitant rifampin administration. Patients who cannot tolerate or receive tetracyclines (children, pregnant women) can be given high-dose TMP-SMX instead (two or three standard-strength tablets twice daily for adults, depending on weight).

1	Increasing evidence supports the use of an aminoglycoside such as gentamicin (5–6 mg/kg per day for at least 2 weeks) instead of streptomycin, although this regimen is not approved by the

1	U.S. Food and Drug Administration. Shorter courses have been associated with high failure rates in adults. A 5to 7-day course of therapy with gentamicin and a 3-week course of TMP-SMX may be adequate for children with uncomplicated disease, but prospective trials are still needed to support this recommendation. Early experience with fluoroquinolone monotherapy was disappointing, although it was suggested that ofloxacin or ciprofloxacin, given together with rifampin for 6 weeks, might be an acceptable alternative to the other 6-week regimens for adults. A substantial meta-analysis did not support the use of fluoroquinolones in first-line treatment regimens, and these drugs are not recommended by an expert consensus group (Ioannina) except in the context of well-designed clinical trials. However, a more recent meta-analysis is more supportive of the efficacy of these drugs, and an adequately powered prospective study will be needed to resolve their role in standard combination therapy.

1	a more recent meta-analysis is more supportive of the efficacy of these drugs, and an adequately powered prospective study will be needed to resolve their role in standard combination therapy. A triple-drug regimen—doxycycline and rifampin combined with an initial course of an aminoglycoside—was superior to double-drug regimens in a meta-analysis. The triple-drug regimen should be considered for all patients with complicated disease and for those for whom treatment adherence is likely to be a problem.

1	Significant neurologic disease due to Brucella species requires prolonged treatment (i.e., for 3–6 months), usually with ceftriaxone supplementation of a standard regimen. Brucella endocarditis is treated with at least three drugs (an aminoglycoside, a tetracycline, and rifampin), and many experts add ceftriaxone and/or a fluoroquinolone to reduce the need for valve replacement. Treatment is usually given for at least 6 months, and clinical endpoints for its discontinuation are often difficult to define. Surgery is still required for the majority of cases of infection of prosthetic heart valves and prosthetic joints.

1	There is no evidence base to guide prophylaxis after exposure to Brucella organisms (e.g., in the laboratory), inadvertent immunization with live vaccine intended for use in animals, or exposure to deliberately released brucellae. Most authorities have recommended the administration of rifampin plus doxycycline for 3 weeks after a low-risk exposure (e.g., an unspecified laboratory accident) and for 6 weeks after a major exposure to aerosol or injected material. However, such regimens are poorly tolerated, and doxycycline monotherapy of the same duration may be substituted. (Monotherapy is now the standard recommendation in the United Kingdom but not in the United States.) Rifampin should be omitted after exposure to vaccine strain RB51, which is resistant to rifampin but sensitive to doxycycline. After significant brucellosis exposure, expert consultation is advised for women who are (or may be) pregnant.

1	Relapse occurs in up to 30% of poorly compliant patients. Thus patients should ideally be followed clinically for up to 2 years to detect relapse, which responds to a prolonged course of the same therapy used originally. The general well-being and the body weight of the patient are more useful guides than serology to lack of relapse. IgG antibody levels detected by the standard agglutination test and its variants can remain in the diagnostic range for >2 years after successful treatment. Complement fixation titers usually fall to normal within 1 year of cure. Immunity is not solid; patients can be reinfected after repeated exposures. Fewer than 1% of patients die of brucellosis. When the outcome is fatal, death is usually a consequence of cardiac involvement; more rarely, it results from severe neurologic disease. Despite the low mortality rate, recovery from brucellosis is slow, and the illness can cause prolonged inactivity, with domestic and economic consequences.

1	The existence of a prolonged chronic brucellosis state after successful treatment remains controversial. Evaluation of patients in whom this state is considered (often those with work-related exposure to brucellae) includes careful exclusion of malingering, nonspecific chronic fatigue syndromes, and other causes of excessive sweating, such as alcohol abuse and obesity. In the future, the availability of more sensitive assays to detect Brucella antigen or DNA may help to identify patients with ongoing infection. Vaccines based on live attenuated Brucella strains, such as B. abortus strain 19BA or 104M, have been used in some countries to protect high-risk populations but have displayed only short-term efficacy and

1	Tularemia Richard F. Jacobs, Gordon E. Schutze Tularemia is a zoonosis caused by Francisella tularensis. Humans of any age, sex, or race are universally susceptible to this systemic infec-tion. Tularemia is primarily a disease of wild animals and persists in contaminated environments, ectoparasites, and animal carriers. 195 Human infection is incidental and usually results from interaction with biting or blood-sucking insects, contact with wild or domestic animals, ingestion of contaminated water or food, or inhalation of infective aerosols. The illness is characterized by various clinical syndromes, the most common of which consists of an ulcerative lesion at the site of inoculation, with regional lymphadenopathy and lymphadenitis. Systemic manifestations, including pneumonia, typhoidal tularemia, meningitis, and fever without localizing findings, pose a greater diagnostic challenge.

1	F. tularensis is a class A bioterrorism agent (Chap. 261e). With rare exceptions, tularemia is the only disease produced by F. tularensis—a small (0.2 μm by 0.2–0.7 μm), gram-negative, pleomorphic, nonmotile, non-spore-forming bacillus. Bipolar staining results in a coccoid appearance. The organism is a thinly encapsulated, nonpiliated strict aerobe that invades host cells. In nature, F. tularensis is a hardy organism that persists for weeks or months in mud, water, and decaying animal carcasses. Dozens of biting and blood-sucking insects, especially ticks and tabanid flies, serve as vectors. Ticks and wild rabbits are the source for most human cases in endemic areas of the southeastern United States. In Utah, Nevada, and California, tabanid flies are the most common vectors. Animal reservoirs include wild rabbits, squirrels, birds, sheep, beavers, muskrats, and domestic dogs and cats. Person-to-person transmission is rare or nonexistent.

1	The four subspecies of F. tularensis are tularensis, holarctica, novicida, and mediasiatica. The first three of these subspecies are found in North America; in fact, subspecies tularensis has been isolated only in North America, where it accounts for >70% of cases of tularemia and produces more serious human disease than other subspecies (although, with treatment, the associated fatality rate is <2%). The progression of illness depends on the infecting strain’s virulence, the inoculum size, the portal of entry, and the host’s immune status.

1	Ticks pass F. tularensis to their offspring transovarially. The organism is found in tick feces but not in large quantities in tick salivary glands. In the United States, the disease is carried by Dermacentor andersoni (Rocky Mountain wood tick), Dermacentor variabilis (American dog tick), Dermacentor occidentalis (Pacific Coast dog tick), and Amblyomma americanum (Lone Star tick). F. tularensis is transmitted frequently during blood meals taken by embedded ticks after hours of attachment. It is the taking of a blood meal through a fecally contaminated field that transmits the organism. Transmission by ticks and tabanid flies takes place mainly in the spring and summer. However, continued transmission in the winter by trapped or hunted animals has been documented. Tularemia is most common in the southeastern United States;

1	Arkansas, Missouri, and Oklahoma account for more than half of all reported cases in this country. Small outbreaks in higher-risk populations (e.g., professional landscapers cutting up brush, mowing, and using a leaf blower) have been reported. Although the irregular distribution of cases of tularemia makes worldwide estimates difficult, increasing numbers of cases have been reported between latitudes 30° and 71°N (the Holarctic region) in the Northern Hemisphere. Cases of tularemia have been reported from Europe, Turkey, Canada, Mexico, and Asia. If the disease is caused by subspecies tularensis, the clinical manifestations are similar to those in the United States. However, in areas where tularemia is due largely to subspecies holarctica, oropharyngeal disease is common. Disease acquisition results from the consumption of water contaminated by live organisms shed by animals (e.g., muskrats, beavers). Subspecies holarctica is known to cause milder disease than other subspecies and

1	results from the consumption of water contaminated by live organisms shed by animals (e.g., muskrats, beavers). Subspecies holarctica is known to cause milder disease than other subspecies and responds well to fluoroquinolones, especially ciprofloxacin.

1	The most common portal of entry for human infection is through skin or mucous membranes, either directly—through the bite of ticks, other arthropods, or other animals—or via inapparent abrasions. Inhalation or ingestion of F. tularensis also can result in infection. F. tularensis is extremely infectious: Although >108 organisms are usually required to produce infection via the oral route (oropharyngeal or gastrointestinal tularemia), as few as 10 organisms can result in infection when injected into the skin (ulceroglandular/glandular tularemia) or inhaled (pulmonary tularemia). After inoculation into the skin, the organism multiplies locally; within 2–5 days (range, 1–10 days), it produces an 1067 erythematous, tender, or pruritic papule. The papule rapidly enlarges and forms an ulcer with a black base (chancriform lesion). The bacteria spread to regional lymph nodes, producing lymphadenopathy (buboes). All forms can lead to bacteremia with spread to distant organs, including the

1	with a black base (chancriform lesion). The bacteria spread to regional lymph nodes, producing lymphadenopathy (buboes). All forms can lead to bacteremia with spread to distant organs, including the central nervous system.

1	Tularemia is characterized by mononuclear cell infiltration with pyogranulomatous pathology. The histopathologic findings can be quite similar to those in tuberculosis, although tularemia develops more rapidly. As a facultatively intracellular bacterium, F. tularensis can parasitize both phagocytic and nonphagocytic host cells and can survive intracellularly for prolonged periods. In the acute phase of infection, the primary organs affected (skin, lymph nodes, liver, and spleen) include areas of focal necrosis, which are initially surrounded by polymorphonuclear leukocytes (PMNs). Subsequently, granulomas form, with epithelioid cells, lymphocytes, and multinucleated giant cells surrounded by areas of necrosis. These areas may resemble caseation necrosis but later coalesce to form abscesses.

1	Conjunctival inoculation can result in infection of the eye, with regional lymph node enlargement (preauricular lymphadenopathy, Parinaud’s complex). Aerosolization and inhalation or hematogenous spread of organisms can result in pneumonia. In the lung, an inflammatory reaction develops, including foci of alveolar necrosis and cell infiltration (initially polymorphonuclear and later mononuclear) with granulomas. Chest roentgenograms usually reveal bilateral patchy infiltrates rather than large areas of consolidation. Pleural effusions are common and may contain blood. Lymphadenopathy occurs in regions draining infected organs. Therefore, in pulmonary infection, mediastinal adenopathy may be evident, whereas patients with oropharyngeal tularemia develop cervical lymphadenopathy. In gastrointestinal or typhoidal tularemia, mesenteric lymphadenopathy may follow the ingestion of large numbers of organisms. (The term typhoidal tularemia may be used to describe severe bacteremic disease,

1	or typhoidal tularemia, mesenteric lymphadenopathy may follow the ingestion of large numbers of organisms. (The term typhoidal tularemia may be used to describe severe bacteremic disease, irrespective of the mode of transmission or portal of entry.) Meningitis has been reported as a primary or secondary manifestation of bacteremia. Patients may also present with fever and no localizing signs.

1	Although a complete and widely accepted understanding of the protective immune response to F. tularensis is lacking, significant advances in the study of natural and protective immunity have been made in recent years and may ultimately result in a vaccine candidate. Complete genomic sequencing and the availability of attenuated F. tularensis strains developed through genetic manipulation are facilitating research that will expand our knowledge in this area. A number of investigators have studied various models and proposed various hypotheses regarding the induction of protective immunity to

1	A number of investigators have studied various models and proposed various hypotheses regarding the induction of protective immunity to F. tularensis. Although further research is needed, synergy between humoral and cell-mediated immune (CMI) responses appears to be critical in inducing effective immune protection. Elucidation of the molecular mechanisms for the organism’s evasion of the host response, pathogen-associated molecular patterns, and effective host immune protection has led to novel vaccination strategies tested in animal models. Antibodies to Fc receptors on antigen-presenting cells have been shown to be protective in animal models of pulmonary tularemia, resulting in both mucosal and CMI responses. This enhanced understanding of mucosal and serum antibodies in combination with a targeted CMI response holds great promise for future vaccine development.

1	Tularemia often starts with a sudden onset of fever, chills, headache, and generalized myalgias and arthralgias (Table 195-1). This onset takes place when the organism penetrates the skin, is ingested, or is inhaled. An incubation period of 2–10 days is followed by the formation of an ulcer at the site of penetration, with local inflammation. The ulcer may persist for several months as organisms are transported via the lymphatics to the regional lymph nodes. These nodes enlarge and may become necrotic and suppurative. If the organism enters the bloodstream, widespread dissemination can result. Rate of Occurrence, % Source: Adapted from RF Jacobs, JP Narain: Pediatrics 76:818, 1985; with permission.

1	Rate of Occurrence, % Source: Adapted from RF Jacobs, JP Narain: Pediatrics 76:818, 1985; with permission. In the United States, most patients with tularemia (75–85%) acquire the infection by inoculation of the skin. In adults, the most common localized form is inguinal/femoral lymphadenopathy; in children, it is cervical lymphadenopathy. About 20% of patients develop a generalized maculopapular rash, which occasionally becomes pustular. Erythema nodosum occurs infrequently. The clinical manifestations of tularemia have been divided into various syndromes, which are listed in Table 195-2.

1	ulceroglandular/Glandular Tularemia These two forms of tularemia account for ~75–85% of cases. The predominant form in children involves cervical or posterior auricular lymphadenopathy and is usually related to tick bites on the head and neck. In adults, the most common form is inguinal/femoral lymphadenopathy resulting from insect and tick exposures on the lower limbs. In cases related to wild game, the usual portal of entry for F. tularensis is either an injury sustained while skinning or cleaning an animal carcass or a bite (usually on the hand). Epitrochlear lymphadenopathy/lymphadenitis is common in patients with bite-related injuries.

1	In ulceroglandular tularemia, the ulcer is erythematous, indurated, and nonhealing, with a punched-out appearance that lasts 1–3 weeks. The papule may begin as an erythematous lesion that is tender or pruritic; it evolves over several days into an ulcer with sharply demarcated edges and a yellow exudate. The ulcer gradually develops a black base, and simultaneously the regional lymph nodes become tender and severely enlarged (Fig. 195-1). The affected lymph nodes may become fluctuant and drain spontaneously, but the condition usually resolves with effective treatment. Late suppuration of lymph nodes has been described in up to 25% of patients with ulceroglandular/glandular tularemia. Examination of material taken from these late fluctuant nodes after successful antimicrobial treatment reveals sterile necrotic tissue. In 5–10% of patients, the skin lesion may be inapparent, with lymphadenopathy plus systemic signs and symptoms the only physical findings (glandular tularemia).

1	reveals sterile necrotic tissue. In 5–10% of patients, the skin lesion may be inapparent, with lymphadenopathy plus systemic signs and symptoms the only physical findings (glandular tularemia). Conversely, a tick or deerfly bite on the trunk may result in an ulcer without evident lymphadenopathy.

1	Oculoglandular Tularemia In ~1% of patients, the portal of entry for F. tularensis is the conjunctiva, which the organism usually reaches through contact with contaminated fingers. The inflamed conjunctiva is painful, with numerous yellowish nodules and pinpoint ulcers. Rate of Occurrence, % Source: Adapted from RF Jacobs, JP Narain: Pediatrics 76:818, 1985; with permission. FIGuRE 195-1 An 8-year-old boy with inguinal lymphadenitis and associated tick-bite site characteristic of ulceroglandular tularemia. Purulent conjunctivitis with regional lymphadenopathy (preauricular, submandibular, or cervical) is evident. Because of debilitating pain, the patient may seek medical attention before regional lymphadenopathy develops. Painful preauricular lymphadenopathy is unique to tularemia and distinguishes it from tuberculosis, sporotrichosis, and syphilis. Corneal perforation may occur.

1	Oropharyngeal and Gastrointestinal Tularemia Rarely, tularemia follows ingestion of contaminated undercooked meat, oral inoculation of F. tularensis from the hands in association with the skinning and cleaning of animal carcasses, or consumption of contaminated food or water. Oral inoculation may result in acute, exudative, or membranous pharyngitis associated with cervical lymphadenopathy or in ulcerative intestinal lesions associated with mesenteric lymphadenopathy, diarrhea, abdominal pain, nausea, vomiting, and gastrointestinal bleeding. Infected tonsils become enlarged and develop a yellowish-white pseudomembrane, which can be confused with that of diphtheria. The clinical severity of gastrointestinal tularemia varies from mild, unexplained, persistent diarrhea with no other symptoms to a fulminant, fatal disease. In fatal cases, the extensive intestinal ulceration found at autopsy suggests an enormous inoculum.

1	Pulmonary Tularemia Pneumonia due to F. tularensis presents as variable parenchymal infiltrates that are unresponsive to treatment with β-lactam antibiotics. Tularemia must be considered in the differential diagnosis of atypical pneumonia in a patient with a history of travel to an endemic area. The disease can result from inhalation of an infectious aerosol or can spread to the lungs and pleura via bacteremia. Inhalation-related pneumonia has been described in laboratory workers after exposure to contaminated materials and, if untreated, can be associated with a relatively high mortality rate. Exposure to F. tularensis in aerosols from live domestic animals or dead wildlife (including birds) has been reported to cause pneumonia. Hematogenous dissemination to the lungs occurs in 10–15% of cases of ulceroglandular tularemia and in about half of cases of typhoidal tularemia. Previously, tularemia pneumonia was thought to be a disease of older patients, but as many as 10–15% of children

1	cases of ulceroglandular tularemia and in about half of cases of typhoidal tularemia. Previously, tularemia pneumonia was thought to be a disease of older patients, but as many as 10–15% of children with clinical manifestations of tularemia have parenchymal infiltrates detected by chest roentgenography. Patients with pneumonia usually have a nonproductive cough and may have dyspnea or pleuritic chest pain. Roentgenograms of the chest usually reveal bilateral patchy infiltrates (described as ovoid or lobar densities), lobar parenchymal infiltrates, and cavitary lesions. Pleural effusions may have a predominance of mononuclear leukocytes or PMNs and sometimes red blood cells. Empyema may develop. Blood cultures may be positive for F. tularensis.

1	Typhoidal Tularemia The typhoidal presentation is now considered rare in the United States. The source of infection in typhoidal tularemia is usually associated with pharyngeal and/or gastrointestinal inoculation or bacteremic disease. Fever usually develops without apparent skin lesions or lymphadenopathy. Some patients have cervical and mesenteric lymphadenopathy. In the absence of a history of possible contact with a vector, diagnosis can be extremely difficult. Blood cultures may be positive and patients may present with classic sepsis or septic shock in this acute systemic form of the infection. Typhoidal tularemia is usually associated with a huge inoculum or with a preexisting compromising condition. High continuous fevers, signs of sepsis, and severe headache are common. The patient may be delirious and may develop prostration and shock. If presumptive antibiotic therapy in culture-negative cases does not include an aminoglycoside, the estimated mortality rate is relatively

1	may be delirious and may develop prostration and shock. If presumptive antibiotic therapy in culture-negative cases does not include an aminoglycoside, the estimated mortality rate is relatively high.

1	Other Manifestations F. tularensis infection has been associated with meningitis, pericarditis, hepatitis, peritonitis, endocarditis, osteomyelitis, and sepsis and septic shock with rhabdomyolysis and acute renal failure. In cases of tularemia meningitis, a mean white blood cell count of 1788/ μL, a predominantly mononuclear cell response (70–100%), a depressed glucose level, an elevated protein concentration, and a negative Gram’s stain are typically found on examination of cerebrospinal fluid.

1	When patients in endemic areas present with fever, chronic ulcerative skin lesions, and large tender lymph nodes (Fig. 195-1), a diagnosis of tularemia should be made presumptively, and confirmatory diagnostic testing and appropriate therapy should be undertaken. When the possibility of tularemia is considered in a nonendemic area, an attempt should be made to identify contact with a potential animal vector. The level of suspicion should be especially high in hunters, trappers, game wardens, professional landscapers, veterinarians, laboratory workers, and individuals exposed to an insect or another animal vector. However, up to 40% of patients with tularemia have no known history of epidemiologic contact with an animal vector.

1	The characteristic presentation of ulceroglandular tularemia does not pose a diagnostic problem, but a less classic progression of regional lymphadenopathy or glandular tularemia must be differentiated from other diseases (Table 195-3). The skin lesion of tularemia may resemble those seen in various other diseases but is generally accompanied by more impressive regional lymphadenopathy. In children, the differentiation of tularemia from cat-scratch disease is made more difficult by the chronic papulovesicular lesion associated with Bartonella henselae infection (Chap. 197). Oropharyngeal tularemia can resemble and must be differentiated from pharyngitis due to other bacteria or viruses. Pulmonary tularemia may resemble any atypical pneumonia. Typhoidal tularemia and tularemia meningitis may resemble a variety of other infections.

1	The diagnosis of tularemia is most frequently confirmed by agglutination testing. Microagglutination and tube agglutination are the techniques most commonly used to detect antibody to F. tularensis. In the standard tube agglutination test, a single titer of ≥1:160 is interpreted as a presumptive positive result. A fourfold increase in titer between paired serum samples collected 2–3 weeks apart is considered diagnostic. False-negative serologic responses are obtained early in infection; up to 30% of patients infected for 3 weeks have sera that test negative. Late in infection, titers into the thousands are common, and titers of 1:20–1:80 may persist for years. Enzyme-linked immunosorbent assays have proved useful for the detection of both antibodies and antigens.

1	Culture and isolation of F. tularensis are difficult. In one study, the organism was isolated in only 10% of more than 1000 human cases, 84% of which were confirmed by serology. The medium of choice is cysteine-glucose-blood agar. F. tularensis can be isolated directly from infected ulcer scrapings, lymph node biopsy specimens, gastric washings, sputum, and blood cultures. Colonies are blue-gray, round, smooth, and slightly mucoid. On media containing blood, a small zone of α hemolysis usually surrounds the colony. Slide agglutination tests or direct fluorescent antibody tests with commercially available antisera can be applied directly to culture suspensions for identification. Most clinical laboratories will not attempt to culture F. tularensis because of the infectivity of the organism from the culture media and the consequent risk of a laboratory-acquired infection. Although tularemia is not spread from person to person, the organism can be inhaled from culture plates and infect

1	from the culture media and the consequent risk of a laboratory-acquired infection. Although tularemia is not spread from person to person, the organism can be inhaled from culture plates and infect unsuspecting laboratory workers. In most clinical laboratories, biosafety level 2 practices are recommended to handle clinical specimens thought to contain F. tularensis; however, biosafety level 3 conditions are required for procedures that produce aerosols or droplets during manipulation of cultures containing or possibly containing this organism.

1	A variety of polymerase chain reaction (PCR) methods have been used to detect F. tularensis DNA in many clinical specimens but mostly in ulceroglandular disease. The majority of these methods target the genes encoding outer-membrane proteins (e.g., fopA or tul4). A 16S rDNA sequence identification PCR may be helpful when the patient’s clinical information does not lead the clinician to suspect a diagnosis of tularemia. Only aminoglycosides, tetracyclines, chloramphenicol, and rifampin are currently approved by the U.S. Food and Drug Administration for the treatment of tularemia. Gentamicin is considered the drug of choice for both adults and children. The dosage for adults and children is 5 mg/kg daily in two divided doses. Gentamicin therapy is typically continued for 7–10 days; however, in mild to moderate

1	TABLE 195-3 TuLAREMIA: dIFFEREnTIAL dIAgnOsIs, By CLInICAL dIsEAsE CATEgORy aStaphylococcus aureus, Streptococcus pyogenes. bAdenovirus, enteroviruses, parainfluenza virus, influenza viruses A and B, respiratory syncytial virus. cHematologic and reticuloendothelial malignancies. dInfluenza viruses A and B, parainfluenza virus, respiratory syncytial virus, adenovirus, enteroviruses, hantavirus.

1	1070 cases of tularemia in which the patient becomes afebrile within the first 48–72 h of gentamicin treatment, a 5to 7-day course has been successful. If available, streptomycin given intramuscularly is also effective. The dosage for adults is 2 g/d in two divided doses. For children, the dosage is 30 mg/kg daily in two divided doses (maximal daily dose, 2 g). After a clinical response is demonstrated at 3–5 days, the dosage for children can be reduced to 10–15 mg/kg daily in two divided doses. The total duration of streptomycin therapy in both adults and children is usually 10 days. Unlike streptomycin and gentamicin, tobramycin is ineffective in the treatment of tularemia and should not be used. Because doxycycline is bacteriostatic against F. tularensis, there is a risk of relapse if the patient is not treated for a long enough period. Therefore, if doxycycline is used, it should be given for at least 14 days. The lack of availability of chloramphenicol limits the utility of this

1	the patient is not treated for a long enough period. Therefore, if doxycycline is used, it should be given for at least 14 days. The lack of availability of chloramphenicol limits the utility of this agent as a viable treatment option. Fluoroquinolones— specifically, ciprofloxacin and levofloxacin—have been used with good outcomes to treat infections caused by subspecies holarctica, which is most often found in Europe. The lack of data on the efficacy of these agents against subspecies tularensis limits their use in North America at this time.

1	F. tularensis cannot be subjected to standardized antimicrobial susceptibility testing because the organism will not grow on the media used. A wide variety of antibiotics, including all β-lactam antibiotics and the cephalosporins, are ineffective for the treatment of tularemia. Several studies indicated that third-generation cephalosporins were active against F. tularensis in vitro, but clinical case reports suggested nearly universal failure of ceftriaxone in pediatric patients with tularemia. Although in vitro data indicate that imipenem may be active, therapy with imipenem, sulfanilamides, and macrolides is not presently recommended because of the lack of relevant clinical data.

1	Virtually all strains of F. tularensis are susceptible to streptomycin and gentamicin. Hearing screening should be considered before initiation of streptomycin or gentamicin therapy. In successfully treated patients, defervescence usually occurs within 2 days, but skin lesions and lymph nodes may take 1–2 weeks to heal. When therapy is not initiated within the first several days of illness, defervescence may be delayed. Relapses are uncommon with streptomycin or gentamicin therapy. Late lymph-node suppuration, however, occurs in ~40% of children, regardless of the treatment received. These nodes have typically been found to contain sterile necrotic tissue without evidence of active infection. Patients with fluctuant nodes should receive several days of antibiotic therapy before drainage to minimize the risk to hospital personnel.

1	If tularemia goes untreated, symptoms usually last 1–4 weeks but may continue for months. The mortality rate from severe untreated infection (including all cases of untreated pulmonary and typhoidal tularemia) can be as high as 30%. However, the overall mortality rate for untreated tularemia is <8%. With appropriate treatment, the mortality rate is <1%. Poor outcomes are often associated with long delays in diagnosis and treatment. Lifelong immunity usually follows tularemia.

1	The prevention of tularemia is based on avoidance of exposure to biting and blood-sucking insects, especially ticks and deerflies. A wide range of approaches to vaccine development are being evaluated, but no vaccine against tularemia is yet licensed. Prophylaxis of tularemia has not proved effective in patients with embedded ticks or insect bites. However, in patients who are known to have been exposed to large quantities of organisms (e.g., in the laboratory) and who have incubating infection with F. tularensis, early treatment can prevent the development of significant clinical disease. Michael B. Prentice

1	Plague is a systemic zoonosis caused by Yersinia pestis. It predominantly affects small rodents in rural areas of Africa, Asia, and the Americas and is usually transmitted to humans by an arthropod vector (the flea). Less often, infection follows contact with animal tissues or respiratory droplets. Plague is an acute febrile illness that is treatable with antimicrobial agents, but mortality rates among untreated patients are high. Ancient DNA studies have confirmed that the fourteenth-century “Black Death” in Europe was Y. pestis infection. Patients can present with the bubonic, septicemic, or pneumonic form of the disease. Although there is concern among the general public about epidemic spread of plague by the respiratory route, this is not the usual route of plague transmission, and established infection-control measures for respiratory plague exist. However, the fatalities associated with plague and the capacity for infection via the respiratory tract mean that Y. pestis fits the

1	infection-control measures for respiratory plague exist. However, the fatalities associated with plague and the capacity for infection via the respiratory tract mean that Y. pestis fits the profile of a potential agent of bioterrorism. Consequently, measures have been taken to restrict access to the organism, including legislation affecting diagnostic and research procedures in some countries (e.g., the United States).

1	The genus Yersinia comprises gram-negative bacteria of the family Enterobacteriaceae (gamma proteobacteria). Overwhelming taxonomic evidence showing Y. pestis strains as a clonal group within Yersinia pseudotuberculosis suggests recent evolution from the latter organism—an enteric pathogen of mammals that is spread by the fecal-oral route and thus has a phenotype distinctly different from that of Y. pestis. When grown in vivo or at 37°C, Y. pestis forms an amorphous capsule made from a plasmid-specified fimbrial protein, Caf or fraction 1 (F1) antigen, which is an immunodiagnostic marker of infection.

1	Human plague generally follows an outbreak in a host rodent population (epizootic). Mass deaths among the rodent primary hosts lead to a search by fleas for new hosts, with consequent incidental infection of other mammals. The precipitating cause for an epizootic may ultimately be related to climate or other environmental factors. The reservoir for Y. pestis causing enzootic plague in natural endemic foci between epizootics (i.e., when the organism may be difficult to detect in rodents or fleas) is a topic of ongoing research and may not be the same in all regions. The enzootic/epizootic pattern may be the result of complex dynamic interactions of host rodents that have different plague susceptibilities and different flea vectors; alternatively, an environmental reservoir may be important. In general, the enzootic areas for plague are lightly populated regions of Africa, Asia, and the Americas (Fig. 196-1).

1	Between 2004 and 2009, 12,503 cases of plague, with a global case-fatality rate of 6.7%, were recorded by the World Health Organization (WHO); these figures were obtained by combining cases notified under the International Health Regulations with data from national surveillance programs and publications. More than 97% of these cases were in Africa; the majority of cases were reported from the Democratic Republic of the Congo and the island of Madagascar. The period covered spans a change in the International Health Regulations from a requirement for nations to notify the WHO of all cases of plague to a requirement to report pneumonic plague or any suspected case of plague in an area not known to be endemic for plague. In the past decade, outbreaks of pneumonic plague have been recorded in the Democratic Republic of the Congo, Uganda, Algeria, Madagascar, China, and Peru. Countries reporting human plague cases, 1970−2005 Probable sylvatic foci

1	Countries reporting human plague cases, 1970−2005 Probable sylvatic foci FIGuRE 196-1 Approximate global distribution of Yersinia pestis.(Compiled from WHO, CDC, and country sources. Reprinted with permission from DT Dennis, GL Campbell: Plague and other Yersinia infections, in Harrison’s Principles of Internal Medicine, 17th ed, AS Fauci et al [eds]. New York, McGraw-Hill, Chap. 152, 2008.)

1	Plague was introduced into North America via the port of San Francisco in 1900 as part of the Third Pandemic, which spread around the world from Hong Kong. The disease is presently enzootic on the western side of the continent from southwestern Canada to Mexico. Most human cases in the United States occur in two regions: “Four Corners” (the junction point of New Mexico, Arizona, Colorado, and Utah), especially northern New Mexico, northern Arizona, and southern Colorado; and further west in California, southern Oregon, and western Nevada (http://www.cdc.gov/plague/maps/index.html). From 1990 to 2011, 151 cases of plague were reported in the United States, a mean of seven cases per year. Most cases occurred from May to October—the time of year when people are outdoors and rodents and their fleas are most plentiful. The infection is most often acquired by fleabite in peridomestic environments; it can also be acquired through the handling of living or dead small mammals (e.g., rabbits,

1	fleas are most plentiful. The infection is most often acquired by fleabite in peridomestic environments; it can also be acquired through the handling of living or dead small mammals (e.g., rabbits, hares, and prairie dogs) or wild carnivores (e.g., wildcats, coyotes, or mountain lions). Dogs and cats may bring plague-infected fleas into the home, and infected cats may transmit plague directly to humans by the respiratory route. The last recorded case of person-to-person transmission in the United States occurred in 1925.

1	Plague most often develops in areas with poor sanitary conditions and infestations of rats—in particular, the widely distributed roof rat Rattus rattus and the brown rat Rattus norvegicus (which serves as a laboratory model of plague). Rat control in warehouses and shipping facilities has been recognized as important in preventing the spread of plague since the early twentieth century and features in the current WHO International Health Regulations. Urban rodents acquire infection from wild rodents, and the proximity of the former to humans increases the risk of transmission. The oriental rat flea Xenopsylla cheopis is the most efficient vector for transmission of plague among rats and onward to humans in Asia, Africa, and South America.

1	Worldwide, bubonic plague is the predominant form reported (80–95% of suspected cases), with mortality rates of 10–20%. The mortality rate is higher (22%) in the small proportion of patients (10–20%) with primary septicemic plague (i.e., systemic Y. pestis sepsis with no bubo; see “Clinical Manifestations,” below) and is highest with primary pulmonary plague; in this, the least common of the main plague presentations, the mortality rate approaches 100% without antimicrobial treatment and is >50% even with such treatment. Rare outbreaks of pharyngeal plague following consumption of raw or undercooked camel or goat meat have been reported. A total of 81 (76%) of the 107 plague cases reported in the United States from 1990 to 2005 were primary bubonic disease, 19 (18%) were primary septicemic disease, and 5 (5%) were primary pneumonic disease; 2 cases (2%) were not classified. Eleven cases (10%) were fatal.

1	As mentioned earlier, genetic evidence suggests that Y. pestis is a clone derived from the enteric pathogen Y. pseudotuberculosis in the recent evolutionary past (9000–40,000 years ago). The change from infection by the fecal-oral route to a two-stage life cycle, with alternate parasitization of arthropod and mammalian hosts, followed the acquisition of two plasmids (pFra and pPst) and the inactivation of remarkably few Y. pseudotuberculosis genes in conjunction with preexisting properties of the Y. pseudotuberculosis ancestor (e.g., the presence of a third plasmid, pYV, and the capacity to cause septicemia). In the arthropod-parasitizing portion of its life cycle, Y. pestis multiplies and forms biofilm-embedded aggregates in the flea midgut after ingestion of a blood meal containing bacteria. In some fleas, biofilm-embedded bacteria eventually fill the proventriculus (a valve connecting the esophagus to the midgut) and block normal blood feeding. Both “blocked” fleas and those

1	bacteria. In some fleas, biofilm-embedded bacteria eventually fill the proventriculus (a valve connecting the esophagus to the midgut) and block normal blood feeding. Both “blocked” fleas and those containing masses of biofilm-embedded Y. pestis without complete blockage inoculate Y. pestis into each bite site. The ability of Y. pestis to colonize and multiply in the flea requires phospholipase D encoded by the ymt gene on the pFra plasmid, and biofilm synthesis requires the chromosomal hms locus shared with Y. pseudotuberculosis. However, three Y. pseudotuberculosis genes inhibiting biofilm formation or promoting its degradation are inactivated in Y. pestis, together with urease, which causes acute flea gastrointestinal toxicity. Blockage takes days or weeks to come about after initial infection of the flea and is followed by the flea’s death. In addition, many flea vectors (including X. cheopis) are able to transmit plague in an early-phase unblocked state for up to 1 week after

1	infection of the flea and is followed by the flea’s death. In addition, many flea vectors (including X. cheopis) are able to transmit plague in an early-phase unblocked state for up to 1 week after feeding, but 10 fleas in this state are required to infect a mammalian host (mass transmission).

1	Y. pestis disseminates from the site of inoculation in the mammalian host in a process initially dependent on plasminogen activator Pla, which is encoded by the small pPst plasmid. This surface protease activates mammalian plasminogen, degrades complement, and adheres to the extracellular matrix component laminin. Pla is essential for the high-level virulence of Y. pestis in mice by subcutaneous or intradermal injection (laboratory proxies for fleabites) and for the development of primary pneumonic plague. When actual fleabite inoculation is used in mouse models, the fimbrial capsule-forming protein (Ca1 or fraction 1; F1 antigen) encoded on pFra increases the efficiency of transmission, and plasminogen activator is required for the formation 1072 of buboes. Because the antiphagocytic systems in Y. pestis are not fully operational at the time of inoculation into the mammalian host, the organism is taken up by macrophages from the inoculation site and transported to regional lymph

1	systems in Y. pestis are not fully operational at the time of inoculation into the mammalian host, the organism is taken up by macrophages from the inoculation site and transported to regional lymph nodes. After intracellular replication,

1	Y. pestis switches to extracellular replication with full expression of its antiphagocytic systems: the type III secretion machines and their effectors encoded by pYV as well as the F1 capsule. Overproduction of the type III secretion substrate and translocation protein LcrV exerts an anti-inflammatory effect, reducing host immune responses. Likewise, Y. pestis lipopolysaccharide is modified to minimize stimulation of host Toll-like receptor 4, thereby reducing protective host inflammatory responses during peripheral infection and prolonging host survival with high-grade bacteremia—an effect that probably enhances the pathogen’s subsequent transmission by fleabite.

1	Replication of Y. pestis in a regional lymph node results in the local swelling of the lymph node and periglandular region known as a bubo. On histology, the node is found to be hemorrhagic or necrotic, with thrombosed blood vessels, and the lymphoid cells and normal architecture are replaced by large numbers of bacteria and fibrin. Periglandular tissues are inflamed and also contain large numbers of bacteria in a serosanguineous, gelatinous exudate.

1	Continued spread through the lymphatic vessels to contiguous lymph nodes produces second-order primary buboes. Infection is initially contained in the infected regional lymph nodes, although transient bacteremia can be detected. As the infection progresses, spread via efferent lymphatics to the thoracic duct produces high-grade bacteremia. Hematogenous spread to the spleen, liver, and secondary buboes follows, with subsequent uncontrolled septicemia, endotoxic shock, and disseminated intravascular coagulation leading to death. In some patients, this septicemic phase occurs without obvious prior bubo development or lung disease (septicemic plague). Hematogenous spread to the lungs results in secondary plague pneumonia, with bacteria initially more prominent in the interstitium than in the air spaces (the reverse being the case in primary plague pneumonia). Hematogenous spread to other organs, including the meninges, can occur.

1	CLINICAL MANIFESTATIONS Bubonic Plague After an incubation period of 2–6 days, the onset of bubonic plague is sudden and is characterized by fever (>38°C), malaise, myalgia, dizziness, and increasing pain due to progressive lymphadenitis in the regional lymph nodes near the fleabite or other inoculation site. Lymphadenitis manifests as a tense, tender swelling (bubo) that, when palpated, has a boggy consistency with an underlying hard core. Generally, there is one painful and erythematous bubo with surrounding periganglionic edema. The bubo is most commonly inguinal but can also be crural, axillary (Fig. 196-2), cervical, or submaxillary, depending on the site of the bite. Abdominal pain from intraabdominal node involvement can occur without other visible signs. Children are most likely to present with cervical or axillary buboes.

1	The differential diagnosis includes acute focal lymphadenopathy of other etiologies, such as streptococcal or staphylococcal infection, tularemia, cat-scratch disease, tick typhus, infectious mononucleosis, or lymphatic filariasis. These infections do not progress as rapidly, are not as painful, and are associated with visible cellulitis or ascending lymphangitis—both of which are absent in plague. Without treatment, Y. pestis dissemination occurs and causes serious illness, including pneumonia (secondary pneumonic plague) and meningitis. Secondary pneumonic plague can be the source of person-to-person transmission of respiratory infection by productive cough (droplet infection), with the consequent development of primary plague pneumonia. Appropriate treatment of bubonic plague results in fever resolution within 2–5 days, but buboes may remain enlarged for >1 week after initial treatment and can become fluctuant. Primary Septicemic Plague A minority (10–25%) of infections with

1	Primary Septicemic Plague A minority (10–25%) of infections with Y. pestis present as gram-negative septicemia (hypotension, shock) without preceding lymphadenopathy. Septicemic plague occurs in all age groups, but persons older than age 40 years are at elevated risk. Some chronic conditions may predispose to septicemic plague: in 2009 in the United States, a fatal laboratory-acquired infection with an attenuated Y. pestis strain manifested as septicemic plague in a 60-yearold researcher with diabetes mellitus and undiagnosed hemochromatosis. These conditions also carry an increased risk of septicemia with other pathogenic Yersinia species. The term septicemic plague can be confusing since most patients with buboes have detectable bacteremia at some stage, with or without systemic signs of sepsis. In laboratory experiments, however, septicemic disease without histologic changes in lymph nodes is seen in a minority of mice infected via fleabites.

1	FIGuRE 196-2 Plague patient in the southwestern United States with a left axillary bubo and an unusual plague ulcer and eschar at the site of the infective flea bite. (Reprinted with permission from DT Dennis, GL Campbell: Plague and other Yersinia infections, in Harrison’s Principles of Internal Medicine, 17th ed, AS Fauci et al [eds]. New York, McGraw-Hill, Chap. 152, 2008.)

1	Pneumonic Plague Primary pneumonic plague results from inhalation of infectious bacteria in droplets expelled from another person or an animal with primary or secondary plague pneumonia. This syndrome has a short incubation period, averaging from a few hours to 2–3 days (range, 1–7 days), and is characterized by a sudden onset of fever, headache, myalgia, weakness, nausea, vomiting, and dizziness. Respiratory signs—cough, dyspnea, chest pain, and sputum production with hemoptysis—typically arise after 24 h. Progression of initial segmental pneumonitis to lobar pneumonia and then to bilateral lung involvement may occur (Fig. 196-3). The possible release of aerosolized Y. pestis bacteria in a bioterrorist attack, manifesting as an outbreak of primary pneumonic plague in nonendemic regions or in an urban setting where plague is rarely seen, has been a source of public health concern. Secondary pneumonic plague is a consequence of bacteremia occurring in ~10–15% of patients with bubonic

1	or in an urban setting where plague is rarely seen, has been a source of public health concern. Secondary pneumonic plague is a consequence of bacteremia occurring in ~10–15% of patients with bubonic plague. Bilateral alveolar infiltrates are seen on chest x-ray, and diffuse interstitial pneumonitis with scanty sputum production is typical.

1	Meningitis Meningeal plague is uncommon, occurring in ≤6% of plague cases reported in the United States. Presentation with headache and fever typically occurs >1 week after the onset of bubonic or septicemic plague and may be associated with suboptimal antimicrobial therapy (delayed therapy, penicillin administration, or low-dose tetracycline treatment) and cervical or axillary buboes. Pharyngitis Symptomatic plague pharyngitis can follow the consumption of contaminated meat from an animal dying of plague or contact with persons or animals with pneumonic plague. This condition can resemble tonsillitis, with peritonsillar abscess and cervical

1	FIGuRE 196-3 Sequential chest radiographs of a patient with fatal primary plague pneumonia. Left: Upright posteroanterior film taken at admission to the hospital emergency department on the third day of illness, showing segmental consolidation of the right upper lobe. Center: Portable anteroposterior film taken 8 h after admission, showing extension of pneumonia to the right middle and right lower lobes. Right: Portable anteroposterior film taken 13 h after admission (when the patient had clinical adult respiratory distress syndrome), showing diffuse infiltration throughout the right lung and patchy infiltration of the left lower lung. A cavity later developed at the site of the initial rightupper-lobe consolidation. (Reprinted with permission from DT Dennis, GL Campbell: Plague and other Yersinia infections, in Harrison’s Principles of Internal Medicine, 17th ed., AS Fauci et al [eds]. New York, McGraw-Hill, Chap. 152, 2008.) lymphadenopathy. Asymptomatic pharyngeal carriage of Y.

1	Yersinia infections, in Harrison’s Principles of Internal Medicine, 17th ed., AS Fauci et al [eds]. New York, McGraw-Hill, Chap. 152, 2008.) lymphadenopathy. Asymptomatic pharyngeal carriage of Y. pestis can also occur in close contacts of patients with pneumonic plague.

1	Because of the scarcity of laboratory facilities in regions where human Y. pestis infection is most common, and because of the potential significance of Y. pestis isolation in a nonendemic area or an area from which human plague has been absent for many years, the WHO recommends an initial presumptive diagnosis followed by reference laboratory confirmation (Table 196-1). In the United States, comprehensive national diagnostic facilities for plague have been in place since a federal Laboratory Response Network (LRN; www.bt.cdc.gov/lrn/) was set up in 1999 to detect possible use of biological terrorism agents, including Y. pestis. Routine diagnostic clinical microbiology laboratories that are included in this network as sentinel-level laboratories use joint protocols from the Centers for Disease Control and Prevention (CDC) and the American Society for Microbiology to identify suspected Y. pestis isolates and to refer these specimens to LRN reference laboratories for confirmatory tests

1	Control and Prevention (CDC) and the American Society for Microbiology to identify suspected Y. pestis isolates and to refer these specimens to LRN reference laboratories for confirmatory tests (http:// www.asm.org/index.php/issues/sentinel-laboratory-guidelines). Y. pestis is designated a “Tier 1 select agent” under the Public Health Security and Bioterrorism Preparedness and Response Act of 2002 and subsequent executive orders; the provisions of this act, the Patriot Act of 2001, and related executive orders apply to all U.S. laboratories and individuals working with Y. pestis. Details of the applicable regulations are available from the CDC.

1	Yersinia species are gram-negative coccobacilli (short rods with rounded ends) 1–3 μm in length and 0.5–0.8 μm in diameter. Y. pestis in particular appears bipolar (with a “closed safety pin” appearance) and pleomorphic when stained with a polychromatic stain (Wayson or Wright-Giemsa; Fig. 196–4). Its lack of motility distinguishes Y. pestis from other Yersinia species, which are motile at 25°C and nonmotile at 37°C. Transport medium (e.g., Cary-Blair medium) preserves the viability of Y. pestis if transport is delayed. Consistent epidemiologic features, such as exposure to infected animals or humans and/or evidence of fleabites and/or residence in or travel to a known endemic focus within the previous 10 days Presumptive case Meeting the definition of a suspected case

1	Presumptive case Meeting the definition of a suspected case Putative new or reemerging focus: ≥2 of the following tests positive • Microscopy: gram-negative coccobacilli in material from bubo, blood, or sputum; bipolar appearance on Wayson or Wright-Giemsa antigen detected in bubo aspirate, blood, or sputum single anti-F1 serology without evidence of previous Y. pestis infection or immunization chain reaction (PCR) detection of Y. pestis in bubo aspirate, blood, or sputum Known endemic focus: ≥1 of the following tests positive evidence of gram-negative or bipolar (Wayson, Wright-Giemsa) coccobacilli from bubo, blood, or sputum sample single anti-F1 serology without evidence of previous plague infection or immunization antigen detected in bubo aspirate, blood, or sputum

1	Confirmed case Meeting the definition of a suspected case • Identification of an isolate from a clinical sample as Y. pestis (colonial morphology and 2 of the 4 following tests positive: phage lysis of cultures at 20–25°C and 37°C; F1 antigen detection; PCR; Y. pestis biochemical profile) • In endemic areas when no other confirmatory test can be performed, a positive rapid diagnostic test with immunochromatography Source: Interregional Meeting on Prevention and Control of Plague, Antananarivo, Madagascar, 7–11 April 2006 (www.who.int/entity/csr/resources/publications/WHO_HSE_EPR_2008_3w .pdf ).

1	Source: Interregional Meeting on Prevention and Control of Plague, Antananarivo, Madagascar, 7–11 April 2006 (www.who.int/entity/csr/resources/publications/WHO_HSE_EPR_2008_3w .pdf ). FIGuRE 196-4 Peripheral blood smear from a patient with fatal plague septicemia and shock, showing characteristic bipolar-stain-ing Yersinia pestis bacilli (Wright’s stain, oil immersion). (Reprinted with permission from DT Dennis, GL Campbell: Plague and other Yersinia infec-tions, in Harrison’s Principles of Internal Medicine, 17th ed, AS Fauci et al [eds]. New York, McGraw-Hill, Chap. 152, 2008.)

1	The appropriate specimens for diagnosis of bubonic, pneumonic, and septicemic plague are bubo aspirate, bronchoalveolar lavage fluid or sputum, and blood, respectively. Culture of postmortem organ biopsy samples can also be diagnostic. A bubo aspirate is obtained by injection of 1 mL of sterile normal saline into a bubo under local anesthetic and aspiration of a small amount of (usually blood-stained) fluid. Gram’s staining of these specimens may reveal gram-negative rods, which are shown by Wayson or Wright-Giemsa staining to be bipolar. These bacteria may even be visible in direct blood smears in septicemic plague (Fig. 196-4); this finding indicates very high numbers of circulating bacteria and a poor prognosis.

1	Y. pestis grows on nutrient agar and other standard laboratory media but forms smaller colonies than do other Enterobacteriaceae. Specimens should be inoculated onto nutrient-rich media such as sheep blood agar (SBA), into nutrient-rich broth such as brain-heart infusion broth, and onto selective agar such as MacConkey or eosin methylene blue (EMB) agar. Yersinia-specific CIN (cefsulodin, triclosan [Irgasan], novobiocin) agar can be useful for culture of contaminated specimens, such as sputum. Blood should be cultured in a standard blood culture system. The optimal growth temperature is <37°C (25–29°C), with pinpoint colonies only on SBA at 24 h. Slower growth occurs at 37°C. Y. pestis is oxidase-negative, catalase-positive, urea-negative, indole-negative, and lactose-negative. Automated biochemical identification systems can misidentify Y. pestis as Y. pseudo-tuberculosis or other bacterial species.

1	Reference laboratory tests for definitive identification of isolates include direct immunofluorescence for F1 antigen; specific polymerase chain reaction (PCR) for targets such as F1 antigen, the pesticin gene, and the plasminogen activator gene; and specific bacteriophage lysis. PCR can also be applied to diagnostic specimens, as can direct immunofluorescence for F1 antigen (produced in large amounts by Y. pestis) by slide microscopy. An immunochromatographic test strip for F1 antigen detection by monoclonal antibodies in clinical specimens has been devised in Madagascar. This method is effective for both laboratory and near-patient use and is now widely used in endemic countries. A similar test strip for Pla antigen has recently been developed and could be used to detect wild-type or engineered F1-negative virulent strains. Many other rapid diagnostic kits for possible bioterrorism pathogens, including Y. pestis, have been described in recent years, but none is widely used for

1	or engineered F1-negative virulent strains. Many other rapid diagnostic kits for possible bioterrorism pathogens, including Y. pestis, have been described in recent years, but none is widely used for primary or reference laboratory identification, and only one (a field real-time PCR for a range of potential bioterrorism agents) is approved by the U.S. Food and Drug Administration (FDA). Detailed phylogeographic DNA sequence data based on culture collections have been accumulated to trace plague evolution, and this system could be adapted in the future to determine real-time clinical plague epidemiology.

1	In the absence of other positive laboratory diagnostic tests, a retrospective serologic diagnosis may be made on the basis of rising titers of hemagglutinating antibody to F1 antigen. Enzyme-linked immunosorbent assays (ELISAs) for IgG and IgM antibodies to F1 antigen are also available. The white blood cell (WBC) count is generally raised (to 10,000– 20,000/μL) in plague, with neutrophilic leukocytosis and a left shift (numerous immature neutrophils); in some cases, however, the WBC count is normal or leukopenia develops. WBC counts are occasionally very high, especially in children (>100,000/μL). Levels of fibrinogen degradation products are elevated in a majority of patients, but platelet counts are usually normal or low-normal. However, disseminated intravascular coagulation, with low platelet counts, prolonged prothrombin times, reduced fibrinogen, and elevated fibrinogen degradation product levels, occurs in a significant minority of patients.

1	Guidelines for the treatment of plague are given in Table 196-2. A 10-day course of antimicrobial therapy is recommended. Streptomycin has historically been the parenteral treatment of choice for plague and is approved for this indication by the FDA. Although not yet approved by the FDA for plague, gentamicin has proven safe and effective in clinical trials in Tanzania and Madagascar Drug Daily Dose Interval, h Route aAminoglycoside dose should be adjusted in light of renal function. There are no published trial data for once-daily gentamicin as plague therapy in adults or children, but this regimen is efficacious in gram-negative sepsis of other causes and was successful in a recent outbreak of pneumonic plague in the Democratic Republic of the Congo. Neonates up to 1 week of age and premature infants should receive gentamicin (2.5 mg/kg IV bid).

1	Source: Dennis DT, Campbell GL: Plague and other Yersinia infections, in AS Fauci et al (eds): Harrison’s Principles of Internal Medicine, 17th ed. 2008, p. 980; Inglesby TV et al: Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. JAMA 283:2281, 2000; and FDA Product Label Reference ID 3123374 (www.accessdata.fda.gov/drugsatfda_docs/label/2012/020634s061,020635s067,021721s028l bl.pdf ).

1	and in retrospectively reviewed cases in the United States. In view of streptomycin’s adverse-reaction profile and limited availability, some experts now recommend gentamicin over streptomycin. In 2012, the FDA approved levofloxacin for prophylaxis and treatment of plague (including septicemic and pneumonic disease), making it the first antibiotic approved for a new indication under a regulatory approach based on animal studies alone, known as the Animal Rule. An FDA decision on ciprofloxacin is pending. Levofloxacin is more efficacious than ciprofloxacin for postexposure prophylaxis of inhalational anthrax in animal models and also received FDA approval for this indication (Chap. 261e); thus it is approved for multiagent prophylaxis in possible bioterrorism exposures.

1	While systemic chloramphenicol therapy is available in the resource-poor countries primarily affected by plague, it is less likely to be available or used in high-income countries because of its adverse effect profile. Tetracyclines are also effective and can be given by mouth but are not recommended for children under the age of 7 years because of tooth discoloration. Doxycycline is the tetracycline of choice; at an oral dosage of 100 mg twice daily, this drug was as effective as IM gentamicin (2.5 mg/kg twice daily) in a trial in Tanzania.

1	Although Y. pestis is sensitive to β-lactam drugs in vitro and these drugs have been efficacious against plague in some animal models, the response to penicillins has been poor in some clinical cases; thus β-lactams and macrolides are not generally recommended as first-line therapy. Chloramphenicol, alone or in combination, is recommended for some focal complications of plague (e.g., meningitis, endophthalmitis, myocarditis) because of its tissue penetration properties. Fluoroquinolones, effective in vitro and in animal models, are recommended in guidelines for possible bioterrorism-associated pneumonic plague and are increasingly used in therapy, although the only human efficacy data available so far are from a case report. Animal and in vitro studies suggest that fluoroquinolones other than levofloxacin, at doses used in systemic gram-negative sepsis, should be effective as therapy for plague: e.g., ciprofloxacin (400 mg twice daily IV, 500 mg twice daily by mouth), ofloxacin (400

1	than levofloxacin, at doses used in systemic gram-negative sepsis, should be effective as therapy for plague: e.g., ciprofloxacin (400 mg twice daily IV, 500 mg twice daily by mouth), ofloxacin (400 mg twice daily IV or by mouth), or moxifloxacin (400 mg/d IV or by mouth).

1	In endemic areas, the control of plague in humans is based on reduction of the likelihood of being bitten by infected fleas or exposed to infected droplets from either humans or animals with plague pneumonia. In the United States, residence and outdoor activity in rural areas of western states where epizootics occur are the main risk factors for infection. To assess potential risks to humans in specific areas, surveillance for Y. pestis infection among animal plague hosts and vectors is carried out regularly as well as in response to observed animal die-offs. Personal protective measures include avoidance of areas where a plague epizootic has been identified and publicized (e.g., by warning signs or closure of campsites). Sick or dead animals should not be handled by the general public. Hunters and zoologists should wear gloves when handling wild-animal carcasses in endemic areas. General measures to avoid rodent fleabite during outdoor activity are appropriate and include the use of

1	and zoologists should wear gloves when handling wild-animal carcasses in endemic areas. General measures to avoid rodent fleabite during outdoor activity are appropriate and include the use of insect repellant, insecticide, and protective clothing. General measures to reduce peridomestic and occupational human contact with rodents are advised and include rodent-proofing of buildings and food-waste stores and removal of potential rodent habitats (e.g., woodpiles and junk heaps). Flea control by insecticide treatment of wild rodents is an effective means of minimizing human contact with plague if an epizootic is identified in an area close to human habitation. Any attempt to reduce rodent numbers must be preceded by flea suppression to reduce the migration of infected fleas to human hosts. An oral F1-V subunit vaccine using raccoon poxvirus (RCN) as a vector protects prairie dogs against Y. pestis injections and is being investigated for efficacy in preventing disease in wild animals,

1	An oral F1-V subunit vaccine using raccoon poxvirus (RCN) as a vector protects prairie dogs against Y. pestis injections and is being investigated for efficacy in preventing disease in wild animals, thus potentially reducing human exposure.

1	Patients in whom pneumonic plague is suspected should be man-1075 aged in isolation, with droplet precautions observed until pneumonia is excluded or effective antimicrobial therapy has been given for 48 h. Review of the literature published before the advent of antimicrobial agents suggests that the main infective risk is posed by patients in the final stages of disease who are coughing up sputum with plentiful visible blood and/or pus. Cotton and gauze masks were protective in these circumstances. Current surgical masks capable of barrier protection against droplets, including large respiratory particles, are considered protective; a particulate respirator (e.g., N95 or greater) is not required.

1	Antimicrobial Prophylaxis Postexposure antimicrobial prophylaxis lasting 7 days is recommended following household, hospital, or other close contact with persons with untreated pneumonic plague. (Close contact is defined as contact with a patient at <2 m.) In animal aerosol-infection studies, levofloxacin and ciprofloxacin are associated with higher survival rates than doxycycline (Table 196-3).

1	Immunization Studies with candidate plague vaccines in animal models show that neutralizing antibody provides protection against exposure but that cell-mediated immunity is critical for protection and clearance of Y. pestis from the host. A killed whole-cell vaccine used in humans required multiple doses, caused significant local and systemic reactions, and was not protective against pneumonic plague; this vaccine is not currently available in the United States. A live attenuated vaccine based on strain EV76 is still used in countries of the former Soviet Union but has significant side effects. The vaccines closest to licensure are subunit vaccines comprising recombinant F1 (rF1) and various recombinant V (rV) proteins produced in Escherichia coli, which are combined either as a fusion protein or as a mixture, purified, and adsorbed to aluminum hydroxide for injection. This combination protects mice and various nonhuman primates in laboratory models of bubonic and pneumonic plague and

1	or as a mixture, purified, and adsorbed to aluminum hydroxide for injection. This combination protects mice and various nonhuman primates in laboratory models of bubonic and pneumonic plague and has been evaluated in phase 2 clinical trials. Special ethical considerations with controlled clinical studies involving plague in humans make prelicensure field efficacy studies unlikely. In the United States, the FDA is therefore prepared to assess plague vaccines for human use under the Animal Rule, using efficacy data and other results from animal studies as well as antibodies and other correlates of immunity from human vaccine recipients (www.fda.gov/BiologicsBloodVaccines/ScienceRe search/BiologicsResearchAreas/ucm127288.htm). Live attenuated

1	Drug Daily Dose Interval, h Route Source: Dennis DT, Campbell GL: Plague and other Yersinia infections, in AS Fauci et al (eds): Harrison’s Principles of Internal Medicine. 2008, p. 980; Inglesby TV et al: Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. JAMA 283:2281, 2000; and FDA Drug Product Label Reference ID 3123374 (www .accessdata.fda.gov/drugsatfda_docs/label/2012/020634s061,020635s067,021721s028lbl.pdf ).

1	1076 Y. pseudotuberculosis and Salmonella strains expressing Y. pestis–specific antigens have been shown to be protective in laboratory animal models of bubonic and pneumonic plague and could be delivered by the oral route. A wide variety of other delivery mechanisms for Y. pestis antigens are being explored. Antigens other than F1 and V that could be added to subunit vaccines are being investigated. Advances providing impetus for exploration of these antigens are (1) the recovery of F1-negative Y. pestis strains from natural sources and (2) the observation that F1 antigen is not required for virulence in primate models of pneumonic plague.

1	Yersiniosis is a zoonotic infection with an enteropathogenic Yersinia species, usually Yersinia enterocolitica or Y. pseudotuberculosis. The usual hosts for these organisms are pigs and other wild and domestic animals; humans are usually infected by the oral route, and outbreaks from contaminated food occur. Yersiniosis is most common in childhood and in colder climates. Patients present with abdominal pain and sometimes with diarrhea (which may not occur in up to 50% of cases). Y. enterocolitica is more closely associated with terminal ileitis and Y. pseudotuberculosis with mesenteric adenitis, but both organisms may cause mesenteric adenitis and symptoms of abdominal pain and tenderness that result in pseudoappendicitis, with the surgical removal of a normal appendix. Diagnosis is based on culture of the organism or convalescent serology. Y. pseudotuberculosis and some rarer strains of

1	Y. enterocolitica are especially likely to cause systemic infection, which is also particularly common among patients with diabetes or iron overload. Systemic sepsis is treatable with antimicrobial agents, but postinfective arthropathy responds poorly to such therapy. Fourteen other Yersinia species are now recognized, but all lack the virulence plasmid pYV common to Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica and are generally considered to be, at most, opportunistic pathogens of humans (Y. aldovae, Y. aleksiciae, Y. bercovieri, Y. entomophaga, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. massiliensis, Y. mollaretii, Y. nurmii, Y. pekkanenii, Y. rohdei, Y. similis, and Y. ruckeri). Molecular phylogeny shows that Y. enterocolitica is more distantly related to Y. pseudotuberculosis than these other Yersinia species, and the similar virulence plasmid they share has probably been acquired independently by at least one of the two since the species diverged.

1	Y. enterocolitica Y. enterocolitica is found worldwide and has been isolated from a wide variety of wild and domestic animals and environmental samples, including samples of food and water. In vitro, Y. enterocolitica is resistant to predation by the protozoon Acanthamoeba castellanii and can survive inside it, suggesting a possible mode of environmental persistence. Strains are differentiated by combined biochemical reactions (biovar) and serogroup. Most clinical infections are associated with serogroups O:3, O:9, and O:5,27, with a declining number of O:8 infections in North America. Some O:8 infections, previously confined to North America, have been reported from Europe and Japan in recent years, and serogroup O:8 now causes a high percentage of yersiniosis cases in Poland. Yersiniosis, mostly due to Y. enterocolitica, is the third commonest zoonosis reported in Europe; most reports come from northern Europe, especially Germany and Scandinavia. The incidence is highest among

1	mostly due to Y. enterocolitica, is the third commonest zoonosis reported in Europe; most reports come from northern Europe, especially Germany and Scandinavia. The incidence is highest among children; children under the age of 4 years are more likely to present with diarrhea than are older children. Abdominal pain with mesenteric adenitis and terminal ileitis is more prominent among older children and adults. Septicemia is more likely in patients with preexisting conditions such as diabetes mellitus, liver disease, any condition involving iron overload (including thalassemia and hemochromatosis), advanced age, malignancy, or HIV/AIDS. As in enteritis of other bacterial etiologies, postinfective complications such as reactive arthritis occur mainly in individuals who are HLA-B27 positive. Erythema nodosum (see Fig. 25e-40) following Yersinia infection is not associated with HLAB27 and is more common among women than among men.

1	Consumption or preparation of raw pork products (such as chitterlings) and some processed pork products is strongly linked with infection because a high percentage of pigs carry pathogenic Y. enterocolitica strains. Outbreaks of Y. enterocolitica infection have been associated with consumption of milk (pasteurized, unpasteurized, and chocolate-flavored) and various foods contaminated with springwater. Person-to-person transmission is suspected in a few cases (e.g., in nosocomial and familial outbreaks) but is much less likely with Y. enterocolitica than with other causes of gastrointestinal infection, such as Salmonella. A multivariate analysis indicates that contact with companion animals is a risk factor for Y. enterocolitica infection among children in Sweden, and low-level colonization of dogs and cats with Y. enterocolitica has been reported. Transfusion-associated septicemia due to Y. enterocolitica, while recognized as a very rare but frequently fatal event for over 30 years,

1	of dogs and cats with Y. enterocolitica has been reported. Transfusion-associated septicemia due to Y. enterocolitica, while recognized as a very rare but frequently fatal event for over 30 years, has been difficult to eradicate.

1	Y. pseudotuberculosis Y. pseudotuberculosis is less frequently reported as a cause of human disease than Y. enterocolitica, and infection with Y. pseudotuberculosis is more likely to pres ent as fever and abdominal pain due to mesenteric lymphadenitis. This organism is associated with wild mammals (rodents, rabbits, and deer), birds, and domestic pigs. Strains are differentiated by combined biochemical reactions (biovar) and serogroup. Although outbreaks are generally rare, several have recently occurred in Finland in association with consumption of lettuce and raw carrots.

1	The usual route of infection is oral. Studies with both Y. enterocolitica and Y. pseudotuberculosis in animal models suggest that initial replication in the small intestine is followed by invasion of Peyer’s patches of the distal ileum via M cells, with onward spread to mesenteric lymph nodes. The liver and spleen can also be involved after oral infection. The characteristic histologic appearance of enteropathogenic yersiniae after invasion of host tissues is as extracellular microabscesses surrounded by an epithelioid granulomatous lesion.

1	Experiments involving oral infection of mice with tagged Y. enterocolitica show that only a very small proportion of bacteria in the gut invade tissues. Individual bacterial clones from an orally inoculated pool give rise to each microabscess in a Peyer’s patch, and the host restricts the invasion of previously infected Peyer’s patches. A prior model positing progressive bacterial spread from Peyer’s patches and mesenteric lymph nodes to the liver and spleen appears to be inaccurate: spread of individually tagged clones of Y. pseudotuberculosis to the liver and spleen of mice occurs independently of regional lymph node colonization and in mice lacking Peyer’s patches.

1	Invasion requires the expression of several nonfimbrial adhesins, such as invasin (Inv) and—in Y. pseudotuberculosis—Yersinia adhesin A (YadA). Inv interacts directly with β1 integrins, which are expressed on the apical surfaces of M cells but not enterocytes. YadA of Y. pseudotuberculosis interacts with extracellular matrix proteins such as collagen and fibronectin to facilitate host cell integrin association and invasion. YadA of Y. enterocolitica lacks a crucial N-terminal region and binds collagen and laminin but not fibronectin and does not cause invasion. Inv is chromosomally encoded, whereas YadA is encoded on the virulence plasmid pYV. YadA helps to confer serum resistance by binding host complement regulators such as factor H and C4-binding protein. Another chromosomal gene, ail (attachment and invasion locus), encodes the extracellular protein Ail, which also confers serum resistance by binding these complement regulators. By binding to host cell surfaces, YadA allows

1	ail (attachment and invasion locus), encodes the extracellular protein Ail, which also confers serum resistance by binding these complement regulators. By binding to host cell surfaces, YadA allows targeting of immune effector cells by the pYV plasmid–encoded type III secretion system (injectisome). As a consequence, the host’s innate immune response is altered; toxins (Yersinia outer proteins, or Yops) are injected into host macrophages, neutrophils, and dendritic cells, affecting signal transduction pathways, resulting in reduced phagocytosis and inhibited production of reactive oxygen species by neutrophils, and triggering apoptosis of macrophages. Other factors functional in invasive disease include yersiniabactin (Ybt), a siderophore produced by some strains of Y. pseudotuberculosis and Y. enterocolitica as well as other Enterobacteriaceae. Yersiniabactin allows bacteria to access iron from saturated lactoferrin during infection and reduces the production of reactive oxygen

1	and Y. enterocolitica as well as other Enterobacteriaceae. Yersiniabactin allows bacteria to access iron from saturated lactoferrin during infection and reduces the production of reactive oxygen species by innate immune effector cells, thereby decreasing bacterial killing. Y. pseudotuberculosis and Y. pestis make other siderophores in addition to yersiniabactin.

1	Self-limiting diarrhea is the most common reported presentation in infection with pathogenic Y. enterocolitica, especially in children under the age of 4, who form the single largest group in most case series. Blood may be detected in diarrheal stool. Older children and adults are more likely than younger children to present with abdominal pain, which can be localized to the right iliac fossa—a situation that often leads to laparotomy for presumed appendicitis (pseudoappendicitis). Appendectomy is not indicated for Yersinia infection causing pseudoappendicitis. Thickening of the terminal ileum and cecum is seen on endoscopy and ultrasound, with elevated round or oval lesions that may overlie Peyer’s patches. Mesenteric lymph nodes are enlarged. Ulcerations of the mucosa are noted on endoscopy. Gastrointestinal complications include granulomatous appendicitis, a chronic inflammatory condition affecting the appendix that is responsible for ≤2% of cases of appendicitis; Yersinia is

1	endoscopy. Gastrointestinal complications include granulomatous appendicitis, a chronic inflammatory condition affecting the appendix that is responsible for ≤2% of cases of appendicitis; Yersinia is involved in a minority of cases.

1	Y. enterocolitica infection can present as acute pharyngitis with or without other gastrointestinal symptoms. Fatal Y. enterocolitica pharyngitis has been recorded. Mycotic aneurysm can follow Y. enterocolitica bacteremia, as can focal infection (abscess) in many other sites and body compartments (liver, spleen, kidney, bone, meninges, endocardium).

1	In all age groups, Y. pseudotuberculosis infection is more likely to present as abdominal pain and fever than as diarrhea. A superantigenic toxin—Y. pseudotuberculosis mitogen (YPM)—is produced by strains seen in eastern Russia in association with Far Eastern scarlet-like fever, a childhood illness with desquamating rash, arthralgia, and toxic shock. A similar illness is recognized in Japan (Izumi fever) and Korea. Similarities have been noted with Kawasaki disease, the idiopathic acute systematic vasculitis of childhood. There is an epidemiologic link between exposure of populations to superantigen-positive Y. pseudotuberculosis and an elevated incidence of Kawasaki disease.

1	Y. enterocolitica or Y. pseudotuberculosis septicemia presents as a severe illness with fever and leukocytosis, often without localizing features, and is significantly associated with predisposing conditions such as diabetes mellitus, liver disease, and iron overload. Hemochromatosis combines several of these risk factors. Administration of iron chelators like desferrioxamine, which provide iron accessible to Yersinia (and have an inhibitory effect on neutrophil function), may result in Yersinia septicemia in patients with iron overload who presumably have an otherwise mild gastrointestinal infection. HIV/AIDS has been associated with Y. pseudotuberculosis septicemia. The unusual phenomenon of transfusion-associated septicemia is linked to the ability of Y. enterocolitica to multiply at refrigerator temperature (psychrotrophy). Typically, the transfused unit has been stored for >20 days, and it is believed that small numbers of yersiniae from an apparently healthy donor with

1	at refrigerator temperature (psychrotrophy). Typically, the transfused unit has been stored for >20 days, and it is believed that small numbers of yersiniae from an apparently healthy donor with subclinical bacteremia are amplified to very high numbers by growth inside the bag at ≤4°C, with consequent septic shock after transfusion. A method for preventing this very rare event (i.e., a range of 1 case in 500,000 to 1 case in several million transfused units in countries such as the United States and France) without unacceptable restriction in the blood supply has not yet been devised.

1	Like other invasive infections of intestinal origin (salmonello sis, shigellosis), reactive arthritis (articular arthritis of multiple joints developing within 2–4 weeks of a preceding infection) results from autoimmune activity initiated by the deposition of bacterial components (not viable bacteria) in joints in combination with the immune response to invading bacteria. The majority of individuals affected by reactive arthritis due to Yersinia are HLA-B27 positive. Myocarditis with electrocardiographic ST-segment abnormalities may occur with Yersinia-associated reactive arthritis. Most Yersiniaassociated cases follow Y. enterocolitica infection (presumably because it is more common than infection with other species), but Y. pseudotuberculosis–associated reactive arthritis is also well documented in Finland, where sporadic and outbreak infections with 1077

1	Y. pseudotuberculosis are more common than in other countries. Of infected individuals identified in a recent Y. pseudotuberculosis serotype O:3 outbreak in Finland, 12% developed reactive arthritis affecting the small joints of the hands and feet, knees, ankles, and shoulders and lasting >6 months in most cases. Erythema nodosum (see Fig. 25e-40) occurs after Yersinia infection (more commonly in women) with no evidence of HLA-B27 linkage.

1	There is a long-standing association between antithyroid and anti-Yersinia antibodies. Antibody evidence of prior Y. enterocolitica infection in Graves’ disease and increased levels of antithyroid antibody in patients with Y. enterocolitica antibodies were first noted in the 1970s. Y. enterocolitica contains a thyroid-stimulating hormone (TSH)–binding site that is recognized by anti-TSH antibodies from Graves’ disease patients. Raised titers of antibodies to Y. enterocolitica whole cells and Yops have been found in some series of Graves’ disease patients but not in others. One Danish study of twins found no evidence of an association between asymptomatic Yersinia infection (as evidenced by anti-Yop antibody titers) and antithyroid antibodies in euthyroid individuals, while another Danish study of twins with and without Graves’ disease found that increased anti-Yop antibody titers were associated with Graves’ disease. It remains unclear whether this cross-reactivity is significant in

1	study of twins with and without Graves’ disease found that increased anti-Yop antibody titers were associated with Graves’ disease. It remains unclear whether this cross-reactivity is significant in the etiology of Graves’ disease.

1	Standard laboratory culture methods can be used to isolate enteropathogenic Yersinia species from sterile samples, including blood and cerebrospinal fluid. Culture on specific selective media (CIN agar), with or without pre-enrichment in broth or phosphate-buffered saline at either 4°C or 16°C, is the basis of most schema for isolation of yersiniae from stool or other nonsterile samples. Outside known high-incidence areas, specific culture may be carried out by laboratories only upon request. Virulence plasmid–negative strains of Y. enterocolitica can be isolated from cultures of stool from asymptomatic individuals, especially after cold enrichment. These strains usually differ in biotype (typically biovar 1a) from virulence plasmid–possessing strains; although some display apparent pathogenicity in a mouse model, virulence plasmid–negative strains are not commonly accepted as human pathogens. Because of the frequency with which the virulence plasmid is lost on laboratory subculture,

1	in a mouse model, virulence plasmid–negative strains are not commonly accepted as human pathogens. Because of the frequency with which the virulence plasmid is lost on laboratory subculture, combined biochemical identification (with biotyping according to a standard schema) and serologic identification are usually required to interpret the significance of an isolate of Y. enterocolitica from a nonsterile site. Most pathogenic

1	Y. enterocolitica strains currently isolated from humans are of serogroup O:3/biovar 4 or serogroup O:9/biovar 2; this pattern holds even in the United States, where serogroup O:8/biovar 1B strains were previously predominant. Many self-validated multiplex PCR screens for detection of Y. enterocolitica in clinical samples—and rather more for its detection in food—have been described, but none of these assays is widely used outside its originating laboratory. Some CE-marked real-time PCR kits are now available in Europe for the diagnosis of yersiniosis in animals; as molecular diagnosis of enteric infection becomes more routine in human disease, it is likely that Y. enterocolitica will be included in diagnostic multiplex PCR screens of feces. Because of the presence of Ail in biovar 1a strains, this antigen cannot be used alone in diagnostic assays. A standard for PCR detection in food samples is being prepared by the International Organization for Standardization.

1	Agglutinating or ELISA antibody titers to specific O-antigen types are used in the retrospective diagnosis of both Y. enterocolitica and Y. pseudotuberculosis infections. IgA and IgG antibodies persist in patients with reactive arthritis. Serologic cross-reactions between Y. enterocolitica serogroup O:9 and Brucella are due to the similarity of their lipopolysaccharide structures. Multiple assays are required to cover even the predominant serogroups (Y. enterocolitica O:3, O:5,27, and O:9; Y. pseudotuberculosis O:1a, O:1b, and O:3), and these assays are generally available only in reference laboratories. ELISA and western blot tests for antibodies to Yops, which are expressed by all pathogenic strains of Y. enterocolitica and Y. pseudotuberculosis, are 1078 also available; most of the positivity in these assays probably relates to previous infection with Y. enterocolitica.

1	Most cases of diarrhea caused by enteropathogenic Yersinia are self-limiting. Data from clinical trials do not support antimicrobial treatment for adults or children with Y. enterocolitica diarrhea. Systemic infections with bacteremia or focal infections outside the gastrointestinal tract generally require antimicrobial therapy. Infants <3 months of age with documented Y. enterocolitica infection may require antimicrobial treatment because of the increased likelihood of bacteremia in this age group. Y. enterocolitica strains nearly always express β-lactamases. Because of the relative rarity of systemic Y. enterocolitica infection, there are no clinical trial data to guide antimicrobial choice or to suggest the optimal dose and duration of therapy. On the basis of retrospective case series and in vitro sensitivity data, fluoroquinolone therapy is effective for bacteremia in adults; for example, ciprofloxacin is given at a typical dose of 500 mg twice daily by mouth or 400 mg twice

1	and in vitro sensitivity data, fluoroquinolone therapy is effective for bacteremia in adults; for example, ciprofloxacin is given at a typical dose of 500 mg twice daily by mouth or 400 mg twice daily IV for at least 2 weeks (longer if positive blood cultures persist). A third-generation cephalosporin is an alternative—e.g., cefotaxime (typical dose, 6–8 g/d in three or four divided doses). In children, third-generation cephalosporins are effective; for example, cefotaxime is given to children ≥1 month of age at a typical dose of 75–100 mg/kg per day in three or four divided doses, with an increase to 150–200 mg/ kg per day in severe cases (maximal daily dose, 8–10 g). Amoxicillin and amoxicillin/clavulanate have shown poor efficacy in case series. Trimethoprim-sulfamethoxazole, gentamicin, and imipenem are all active in vitro. Y. pseudotuberculosis strains do not express β-lactamase but are intrinsically resistant to polymyxin. Because human infection with Y. pseudotuberculosis is

1	and imipenem are all active in vitro. Y. pseudotuberculosis strains do not express β-lactamase but are intrinsically resistant to polymyxin. Because human infection with Y. pseudotuberculosis is less common than that with Y. enterocolitica, less case information is available; however, studies in mice suggest that ampicillin is ineffective. Drugs similar to those used against Y. enterocolitica should be used. The best results have been obtained with a quinolone.

1	Some trials of treatment for reactive arthritis (with a large proportion of cases due to Yersinia) found that 3 months of oral ciprofloxacin therapy did not affect outcome. One trial in which the same therapy was given specifically for Y. enterocolitica–reactive arthritis found that, while outcome indeed was not affected, there was a trend toward faster remission of symptoms in the treated group. Follow-up 4–7 years after initial antibiotic treatment of reactive arthritis (predominantly following Salmonella and Yersinia infections) demonstrated apparent efficacy in the prevention of chronic arthritis in HLA-B27-positive individuals. A trial showing that azithromycin therapy did not affect outcome in reactive arthritis included cases believed to follow yersiniosis, although no breakdown of cases was provided. A Cochrane review evaluating the use of antibiotics for reactive arthritis is in progress.

1	Current control measures are similar to those used against other enteric pathogens like Salmonella and Campylobacter, which colonize the intestine of food animals. The focus is on safe handling and processing of food. No vaccine is effective in preventing intestinal colonization of food animals by enteropathogenic Yersinia. Consumption of food made from raw pork (which is popular in Germany and Belgium) should be discouraged at present because it is not possible to eliminate contamination with the enteropathogenic Yersinia strains found worldwide in pigs. Exposure of infants to raw pig intestine during domestic preparation of chitterlings is inadvisable. Modification of abattoir technique in Scandinavian countries from the 1990s onward included the removal of pig intestines in a closed plastic bag; levels of carcass contamination with Y. enterocolitica were reduced, but such contamination was not eliminated. Experimental pig herds free of pathogenic Y. enterocolitica O:3 (and also of

1	bag; levels of carcass contamination with Y. enterocolitica were reduced, but such contamination was not eliminated. Experimental pig herds free of pathogenic Y. enterocolitica O:3 (and also of Salmonella, Toxoplasma, and Trichinella) have been established in Norway and may be commercialized in the future because of their enhanced safety. In the food industry, vigilance is required because of the potential for large outbreaks if small numbers of enteropathogenic yersiniae contaminate any ready-to-eat food whose safe preservation is based on refrigeration before consumption.

1	The rare phenomenon of contamination of blood for transfusion has proved impossible to eradicate. However, leukodepletion is now practiced in most blood transfusion centers, primarily to prevent non-hemolytic febrile transfusion reactions and alloimmunization against HLA antigens. This measure reduces but does not eliminate the risk of Yersinia blood contamination. Notification of yersiniosis is now obligatory in some countries. Bartonella Infections, Including Michael Giladi, Moshe Ephros

1	Bartonella species are fastidious, facultative intracellular, slow-growing, gram-negative bacteria that cause a broad spectrum of diseases in humans. This genus includes more than 30 distinct species or subspecies, of which at least 16 have been recognized as confirmed or potential human pathogens; Bartonella bacilliformis, Bartonella quintana, and Bartonella henselae are most commonly identified (Table 197-1). Most Bartonella species have successfully adapted to survival in specific domestic or wild mammals. Prolonged intraerythrocytic infection in these animals creates a niche where the bacteria are protected from both innate and adaptive immunity and which serves as a reservoir for human infections. Bartonella characteristically evades the host immune system by modification of its virulence factors (e.g., lipopolysaccharides or flagella) and by attenuation of the immune response. B. bacilliformis and B. quintana, which are not zoonotic, are exceptions. Arthropod vectors are often

1	factors (e.g., lipopolysaccharides or flagella) and by attenuation of the immune response. B. bacilliformis and B. quintana, which are not zoonotic, are exceptions. Arthropod vectors are often involved. Isolation and characterization of Bartonella species are difficult and require special techniques. Clinical presentation generally depends on both the infecting Bartonella species and the immune status of the infected individual. Bartonella species are susceptible to many antibiotics in vitro; however, clinical responses to therapy and studies in animal models suggest that the minimal inhibitory concentrations of many antimicrobial agents correlate poorly with the drugs’ in vivo efficacies in patients with Bartonella infections.

1	Usually a self-limited illness, cat-scratch disease (CSD) has two general clinical presentations. Typical CSD, the more common, is characterized by subacute regional lymphadenopathy; atypical CSD is the collective designation for numerous extranodal manifestations involving various organs. B. henselae is the principal etiologic agent of CSD. Rare cases have been associated with Afipia felis and other Bartonella species.

1	CSD occurs worldwide, favoring warm and humid climates. In temperate climates, incidence peaks during fall and winter; in the tropics, disease occurs year-round. Adults are affected nearly as frequently as children. Intrafamilial clustering is rare, and person-to-person transmission does not occur. Apparently healthy cats constitute the major reservoir of B. henselae, and cat fleas (Ctenocephalides felis) may be responsible for cat-to-cat transmission. CSD usually follows contact with cats (especially kittens), but other animals (e.g., dogs) have been implicated as possible reservoirs in rare instances. In the United States, the estimated disease incidence is ~10 cases per 100,000 population. About 10% of patients are hospitalized.

1	aMany other Bartonella species exist but are not recognized as human pathogens. bB. henselae, B. vinsonii subsp. berkhoffii, B. koehlerae, Candidatus B. melophagi, or more than one Bartonella spp. (co-infection) were detected in blood samples from patients with extensive arthropod and other animal exposure who presented with various chronic neurologic or neurocognitive syndromes. The role of these pathogens in these patients needs further study. cAnimals are implicated when existing evidence supports their infection with Bartonella species. Data supporting animal-to-human transmission may be lacking. dRetinitis may also be associated with B. grahamii. eCandidatus is a taxonomic status for bacteria that cannot be described in sufficient detail to warrant establishment of a novel taxon or cannot be cultured or propagated in culture media. The phylogenetic relatedness of these bacteria has been determined by gene amplification and sequence analysis.

1	Inoculation of B. henselae, possibly via contaminated flea feces, usually results from a cat scratch or bite. Infection of mucous membranes or conjunctivae via droplets or licking may occur as well. With lymphatic drainage to one or more regional lymph nodes in immunocompetent hosts, a TH1 response can result in necrotizing granulomatous lymphadenitis. Dendritic cells, along with their associated chemokines, play a role in the host inflammatory response and granuloma formation.

1	Of patients with CSD, 85–90% have typical disease. The primary lesion, a small (0.3to 1-cm) painless erythematous papule or pustule, develops at the inoculation site (usually the site of a scratch or a bite) within days to 2 weeks in about one-third to two-thirds of patients (Fig. 197–1A, B). Lymphadenopathy develops 1–3 weeks or longer after cat contact. The affected lymph node(s) are enlarged and usually painful, sometimes have overlying erythema, and suppurate in 10–15% of cases (Fig. 197–1C, D, and E). Axillary/epitrochlear nodes are most commonly involved; next in frequency are head/neck nodes and then inguinal/femoral nodes. Approximately 50% of patients have fever, malaise, and anorexia. A smaller proportion experience weight loss and night sweats mimicking the presentation of lymphoma. Fever is usually low-grade but infrequently rises to ≥39°C. Resolution is slow, requiring weeks (for fever, pain, and accompanying signs and symptoms) to months (for node shrinkage).

1	Atypical CSD occurs in 10–15% of patients as extranodal or complicated disease in the absence or presence of lymphadenopathy. Atypical disease includes Parinaud’s oculoglandular syndrome (granulomatous conjunctivitis with ipsilateral preauricular lymphadenitis; Fig. 197–1E), granulomatous hepatitis/splenitis, neuroretinitis (often presenting as unilateral deterioration of vision; Fig. 197–1F), and other ophthalmologic manifestations. In addition, neurologic involvement (encephalopathy, seizures, myelitis, radiculitis, cerebellitis, facial and other cranial or peripheral palsies), fever of unknown origin, debilitating myalgia, arthritis or arthralgia (affecting mostly women >20 years old), osteomyelitis (including multifocal disease), tendinitis, neuralgia, and dermatologic manifestations (including erythema nodosum [see Fig. 25e-40], sometimes accompanying arthropathy) occur. Other manifestations and syndromes (pneumonitis, pleural effusion, idiopathic thrombocytopenic purpura,

1	(including erythema nodosum [see Fig. 25e-40], sometimes accompanying arthropathy) occur. Other manifestations and syndromes (pneumonitis, pleural effusion, idiopathic thrombocytopenic purpura, Henoch-Schönlein purpura, erythema multiforme [see Fig. 25e-25], hypercalcemia, glomerulonephritis, myocarditis) have also been associated with CSD. In elderly patients (>60 years old), lymphadenopathy is more often absent but encephalitis and fever of unknown origin are more common than in younger patients. In immunocompetent individuals, CSD—whether typical or atypical—usually resolves without treatment and without sequelae. Lifelong immunity is the rule.

1	Routine laboratory tests usually yield normal or nonspecific results. Histopathology initially shows lymphoid hyperplasia and later demonstrates stellate granulomata with necrosis, coalescing microabscesses, and occasional multinucleated giant cells—findings that, although nonspecific, may narrow the differential diagnosis. Serologic testing (immunofluorescence or enzyme immunoassay) is the most commonly used laboratory diagnostic approach, with variable sensitivity and specificity. Seroconversion may take a few weeks. Other tests are of low sensitivity (culture, Warthin-Starry silver staining), of low specificity (cytology, histopathology), or of limited availability in routine diagnostic laboratories (polymerase chain reaction [PCR], immunohistochemistry). PCR of lymph node tissue, pus, or the primary inoculation lesion is highly sensitive and specific and is particularly useful for definitive and rapid diagnosis in seronegative patients. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: A history of cat contact, a primary inoculation lesion, and regional lymphadenopathy are highly suggestive of CSD. A characteristic clinical course and corroborative laboratory tests make the diagnosis very likely. Conversely, when acuteand convalescent-phase sera are negative (as is the case in 10–20% of CSD patients), when spontaneous regression of lymph node size does not occur, and particularly when constitutional symptoms persist, malignancy must be ruled out. Pyogenic lymphadenitis, mycobacterial infection, brucellosis, syphilis, tularemia, plague, toxoplasmosis, sporotrichosis, and histoplasmosis should also be considered. In clinically suspected CSD in a seronegative individual, fine-needle aspiration may be adequate and PCR can confirm the diagnosis. When data are

1	Bartonella Infections, Including Cat-Scratch Disease less supportive of CSD, lymph node biopsy rather than fine-needle aspiration is preferred. In seronegative CSD patients with lymphadenopathy and severe complications (e.g., encephalitis or neuroretinitis), early biopsy is important to establish a specific diagnosis. (Table 197-2) Treatment regimens are based on only minimal data. Suppurative nodes should be drained by large-bore needle aspiration and not by incision and drainage in order to avoid chronic draining tracts. Immunocompromised patients must always be treated with systemic antimicrobials. Avoiding cats (especially kittens) and instituting flea control are options for immunocompromised patients and for patients with valvular heart disease.

1	Avoiding cats (especially kittens) and instituting flea control are options for immunocompromised patients and for patients with valvular heart disease. Trench fever, also known as 5-day fever or quintan fever, is a febrile illness caused by B. quintana. It was first described as an epidemic in the trenches of World War I and recently reemerged as chronic bacteremia seen most often in homeless people (also referred to in the latter setting as urban or contemporary trench fever).

1	FIGuRE 197-1 Manifestations of cat-scratch disease. A. Primary inoculation lesion. Axillary and epitrochlear lymphadenitis appeared 2 weeks later. B. Primary inoculation lesion. Submental lymphadenitis appeared 10 days later. C. Axillary lymphadenopathy of 2 weeks’ duration. The overlying skin appears normal. D. Cervical lymphadenopathy of 6 weeks’ duration. The overlying skin is red. Thick, odorless pus (12 mL) was aspirated. E. Preauricular lymphadenopathy. F. Left-eye neuroretinitis. Note papilledema and stellate macular exudates (“macular star”).

1	In addition to epidemics during World Wars I and II, sporadic outbreaks of trench fever have been reported in many regions of the world. The human body louse has been identified as the vector and humans as the only known reservoir. After a hiatus of several decades during which trench fever was almost forgotten, small clusters of cases of B. quintana chronic bacteremia were reported sporadically, primarily from the United States and France, in HIV-uninfected homeless people. Alcoholism and louse infestation were identified as risk factors.

1	The typical incubation period is 15–25 days (range, 3–38 days). “Classical” trench fever, as described in 1919, ranges from a mild febrile illness to a recurrent or protracted and debilitating disease. Onset may be abrupt or preceded by a prodrome of several days. Fever is often periodic, lasting 4–5 days with 5-day (range, 3to 8-day) intervals between episodes. Other symptoms and signs include headache, back and limb pain, profuse sweating, shivering, myalgia, arthralgia, splenomegaly, a maculopapular rash in occasional cases, and nuchal rigidity in some cases. Untreated, the disease usually lasts Typical cat-scratch disease Not routinely indicated; for patients with extensive lymphadenopathy, consider azithromycin (500 mg PO on day 1, then 250 mg PO qd for 4 days)

1	Typical cat-scratch disease Not routinely indicated; for patients with extensive lymphadenopathy, consider azithromycin (500 mg PO on day 1, then 250 mg PO qd for 4 days) Trench fever or chronic bacteremia Gentamicin (3 mg/kg IV qd for 14 days) plus doxycycline (200 mg PO qd or 100 mg PO bid for 6 weeks) with B. quintana Suspected Bartonella endocarditis Gentamicinb (1 mg/kg IV q8h for ≥14 days) plus doxycycline (100 mg PO/IV bid for 6 weeksc) plus ceftriaxone (2 g IV qd for 6 weeks) Confirmed Bartonella endocarditis As for suspected Bartonella endocarditis minus ceftriaxone Bacillary angiomatosis Erythromycind (500 mg PO qid for 3 months)

1	Ciprofloxacin (500 mg PO bid for 10 days) Verruga peruana Rifampin (10 mg/kg PO qd, to a maximum of 600 mg, for 14 days) aData on treatment efficacy for encephalitis and hepatosplenic CSD are lacking. Therapy similar to that given for retinitis is reasonable. bSome experts recommend gentamicin at 3 mg/ kg IV qd. If gentamicin is contraindicated, rifampin (300 mg PO bid) can be added to doxycycline for documented Bartonella endocarditis. cSome experts recommend extending oral doxycycline therapy for 3–6 months. dOther macrolides are probably effective and may be substituted for erythromycin or doxycycline. Source: Recommendations are modified from JM Rolain et al: Antimicrob Agents Chemother 48:1921, 2004.

1	Source: Recommendations are modified from JM Rolain et al: Antimicrob Agents Chemother 48:1921, 2004. Bartonella Infections, Including Cat-Scratch Disease 1082 4–6 weeks. Death is rare. The clinical spectrum of B. quintana bacteremia in homeless people ranges from asymptomatic infection to a febrile illness with headache, severe leg pain, and thrombocytopenia. Endocarditis sometimes develops. Definitive diagnosis requires isolation of B. quintana by blood culture. Some patients have positive blood cultures for several weeks. Patients with acute trench fever typically develop significant titers of antibody to Bartonella, whereas those with chronic B. quintana bacteremia may be seronegative. Patients with high titers of IgG antibodies should be evaluated for endocarditis. In epidemics, trench fever should be differentiated from epidemic louse-borne typhus and relapsing fever, which occur under similar conditions and share many features.

1	TREATMEnT b. QUintana bacteremia (Table 197–2) In a small, randomized, placebo-controlled trial involving homeless people with B. quintana bacteremia, therapy with gentamicin and doxycycline was superior to administration of placebo in eradicating bacteremia. Treatment of bacteremia is important even in clinically mild cases to prevent endocarditis. Optimal therapy for trench fever without documented bacteremia is uncertain. Coxiella burnetii (Chap. 211) and Bartonella species are the (Chap. 155). In France, for example, Bartonella species were identified as the etiologic agents in 28% of 348 cases of culture-negative endocarditis. Prevalence, however, varies by geographic location and epidemiologic setting. In addition to B. quintana and B. henselae (the most common Bartonella species implicated in endocarditis, the former more commonly than the latter), other Bartonella species have reportedly caused rare cases (Table 197-1).

1	B. henselae (the most common Bartonella species implicated in endocarditis, the former more commonly than the latter), other Bartonella species have reportedly caused rare cases (Table 197-1). Bartonella endocarditis has been reported worldwide. Most patients are adults; more are male than female. Risk factors associated with B. quintana endocarditis include homelessness, alcoholism, and body louse infestation; however, individuals with no risk factors have had Bartonella endocarditis diagnosed as well. B. henselae endocarditis is associated with exposure to cats. Most cases involve native rather than prosthetic valves; the aortic valve accounts for ~60% of cases. Patients with B. henselae endocarditis usually have preexisting valvulopathy, whereas B. quintana often infects normal valves.

1	Clinical manifestations are usually characteristic of subacute endocarditis of any etiology. However, a substantial number of patients have a prolonged, minimally febrile or even afebrile indolent illness, with mild nonspecific symptoms lasting weeks or months before the diagnosis is made. Initial echocardiography may not show vegetations. Acute, aggressive disease is rare. Blood cultures, even with use of special techniques (lysis centrifugation or EDTA-containing tubes), are positive in only ~25% of cases— mostly those caused by B. quintana and only rarely those caused by

1	B. henselae. Prolonged incubation of cultures (up to 6 weeks) is required. Serologic tests—either immunofluorescence or enzyme immunoassay—usually demonstrate high-titer IgG antibodies to Bartonella. Because of cross-antigenicity, serology does not distinguish between B. quintana and B. henselae and may also be low-titer cross-reactive with other pathogens, such as C. burnetii and Chlamydia species. Identification of Bartonella to the species level is usually accomplished by application of PCR-based methods to valve tissue.

1	(Table 197-2) For patients with culture-negative endocarditis suspected to be due to Bartonella species, empirical treatment consists of gentamicin, doxycycline, and ceftriaxone; the major role of ceftriaxone in this regimen is to adequately treat other potential causes of culture-negative endocarditis, including members of the HACEK group (Chap. 183e). Once a diagnosis of Bartonella endocarditis has been established, ceftriaxone is discontinued. Aminoglycosides, the only antibiotics known to be bactericidal against Bartonella, should be included in the regimen for ≥2 weeks. Indications for valvular surgery are the same as in subacute endocarditis due to other pathogens; however, the proportion of patients who undergo surgery (~60%) is high, probably as a consequence of delayed diagnosis.

1	Bacillary angiomatosis (sometimes called bacillary epithelioid angiomatosis or epithelioid angiomatosis) is a disease of severely immunocompromised patients, is caused by B. henselae or B. quintana, and is characterized by neovascular proliferative lesions involving the skin and other organs. Both species cause cutaneous lesions; hepatosplenic lesions are caused only by B. henselae, whereas subcutaneous and lytic bone lesions are more frequently associated with B. quintana. Bacillary peliosis is a closely related angioproliferative disorder caused by B. henselae and involving primarily the liver (peliosis hepatis) but also the spleen and lymph nodes. Bacillary peliosis is characterized by blood-filled cystic structures whose size ranges from microscopic to several millimeters.

1	Bacillary angiomatosis and bacillary peliosis occur primarily in HIV-infected persons (Chap. 226) with CD4+ T cell counts <100/μL but also affect other immunosuppressed patients and, in rare instances, immunocompetent patients. The previously reported incidence of ~1 case per 1000 HIV-infected persons is now lower; the decrease is most likely attributable to effective antiretroviral therapy and the routine use of rifabutin and macrolides to prevent Mycobacterium avium complex infection in AIDS patients. Contact with cats or cat fleas increases the risk of B. henselae infection. Risk factors for B. quintana infection are low income, homelessness, and body louse infestation.

1	Bacillary angiomatosis presents most commonly as one or more cutaneous lesions that are not painful and that may be tan, red, or purple in color. Subcutaneous masses or nodules, superficial ulcerated plaques (Fig. 197-2), and verrucous growths are also seen. Nodular forms resemble those seen in fungal or mycobacterial infections. Subcutaneous nodules are often tender. Painful osseous lesions, most often involving long bones, may underlie cutaneous lesions and occasionally develop in their absence. In rare cases, other organs are involved in bacillary angiomatosis. Patients usually have constitutional symptoms, including fever, chills, malaise, headache, anorexia, weight loss, and night sweats. In osseous disease, lytic lesions are generally seen on radiography, and technetium scan shows focal uptake. The differential diagnosis of cutaneous bacillary angiomatosis includes Kaposi’s sarcoma, pyogenic granuloma, subcutaneous tumors, and verruga peruana. In bacillary peliosis, hypodense

1	focal uptake. The differential diagnosis of cutaneous bacillary angiomatosis includes Kaposi’s sarcoma, pyogenic granuloma, subcutaneous tumors, and verruga peruana. In bacillary peliosis, hypodense hepatic areas are usually evident on imaging. In patients with advanced immunodeficiency, B. henselae and B. quintana are important causes of fever of unknown origin. Intermittent bacteremia with positive blood cultures can occur with or without endocarditis.

1	Bacillary angiomatosis consists of lobular proliferations of small blood vessels lined by enlarged endothelial cells interspersed with mixed infiltrates of neutrophils and lymphocytes, with predominance of the former. Histologic examination of organs with bacillary peliosis reveals small blood-filled cystic lesions partially lined by endothelial Donovanosis Nigel O’Farrell Donovanosis is a chronic, progressive bacterial infection that usually involves the genital region. The condition is generally regarded as a sexually transmitted infection of low infectivity. This infection has been known by many other names, the most common being granu-198e loma inguinale.

1	The causative organism has been reclassified as Klebsiella granulomatis comb nov on the basis of phylogenetic analysis, although there is ongoing debate about this decision. Some authorities consider the original nomenclature (Calymmatobacterium granulomatis) to be more appropriate in light of analysis of 16S rRNA gene sequences. Donovanosis was first described in Calcutta in 1882, and the causative organism was recognized by Charles Donovan in Madras in 1905. He identified the characteristic Donovan bodies, measuring 1.5 × 0.7 μm, in macrophages and the stratum malpighii. The organism was not reproducibly cultured until the mid-1990s, when its isolation in peripheral-blood monocytes and human epithelial cell lines was reported.

1	includes Papua New Guinea, parts of southern Africa, India, the Caribbean, French Guyana, Brazil, and aboriginal communities in Australia. In Australia, donovanosis has been almost entirely eliminated through a sustained program backed by strong political commitment and resources at the primary health care level. Although few cases are now reported in the United States, donovanosis was once prevalent in this country, with 5000–10,000 cases recorded in 1947. The largest epidemic recorded was in Dutch South Guinea, where 10,000 cases were identified in a population of 15,000 (the Marind-anim people) between 1922 and 1952. Donovanosis is associated with poor hygiene and is more common in lower socioeconomic groups than in those who are better off and in men than in women. Infection in sexual partners of index cases occurs to a limited extent. Donovanosis is a risk factor for HIV infection (Chap. 226).

1	Globally, the incidence of donovanosis has decreased significantly in recent times. This decline probably reflects a greater focus on effective management of genital ulcers because of their role in facilitating HIV transmission. A lesion starts as a papule or subcutaneous nodule that later ulcerates after trauma. The incubation period is uncertain, but experimental infections in humans indicate a duration of ~50 days. Four types of lesions have been described: (1) the classic ulcerogranulomatous lesion (Fig. 198e-1), a beefy red ulcer that bleeds readily when touched; (2) a hypertrophic or verrucous ulcer with a raised irregular edge; (3) a necrotic, offensive-smelling ulcer causing tissue destruction; and (4) a sclerotic or cicatricial lesion with fibrous and scar tissue.

1	The genitals are affected in 90% of patients and the inguinal region in 10%. The most common sites of infection are the prepuce, coronal sulcus, frenum, and glans in men and the labia minora and fourchette in women. Cervical lesions may mimic cervical carcinoma. In men, lesions are associated with lack of circumcision. Lymphadenitis is uncommon. Extragenital lesions occur in 6% of cases and may involve the lip, gums, cheek, palate, pharynx, larynx, and chest. Hematogenous spread with involvement of liver and bone has been reported. During pregnancy, lesions tend to develop more quickly and respond more slowly to treatment. Polyarthritis and osteomyelitis are rare complications. In newborn infants, donovanosis may present with ear infection. Cases in children have been attributed to sitting on the laps of infected adults. FIGURE 198e-1 Ulcerogranulomatous penile lesion of donovanosis, with some hypertrophic features.

1	FIGURE 198e-1 Ulcerogranulomatous penile lesion of donovanosis, with some hypertrophic features. As the incidence of donovanosis has decreased, the number of unusual case reports has appeared to be increasing. Complications include neoplastic changes, pseudo-elephantiasis, and stenosis of the urethra, vagina, or anus.

1	A clinical diagnosis of donovanosis made by an experienced practitioner on the basis of the lesion’s appearance usually has a high positive predictive value. The diagnosis is confirmed by microscopic identification of Donovan bodies (Fig. 198e-2) in tissue smears. Preparation of a good-quality smear is important. If donovanosis is suspected on clinical grounds, the smear for Donovan bodies should be taken before swab samples are collected to be tested for other causes of genital ulceration so that enough material can be collected from the ulcer. A swab should be rolled firmly over an ulcer previously cleaned with a dry swab to remove debris. Smears can be examined in a clinical setting by direct microscopy with a rapid Giemsa or Wright’s stain. Alternatively, a piece of granulation tissue crushed and spread between two slides can be used. Donovan bodies can be seen in large, mononuclear (Pund) cells as gram-negative intracytoplasmic cysts filled with deeply staining bodies that may

1	crushed and spread between two slides can be used. Donovan bodies can be seen in large, mononuclear (Pund) cells as gram-negative intracytoplasmic cysts filled with deeply staining bodies that may have a safety-pin appearance. These cysts eventually rupture and release the infective organisms. Histologic changes

1	FIGURE 198e-2 Pund cell stained by rapid Giemsa (RapiDiff) technique. Numerous Donovan bodies are visible. include chronic inflammation with infiltration of plasma cells and neutrophils. Epithelial changes include ulceration, microabscesses, and elongation of rete ridges. A diagnostic polymerase chain reaction (PCR) test was based on the observation that two unique base changes in the phoE gene eliminate Hae111 restriction sites, enabling differentiation of K. granulomatis comb nov from related Klebsiella species. PCR analysis with a colorimetric detection system can now be used in routine diagnostic laboratories. A genital ulcer multiplex PCR that includes K. granulomatis has been developed. Serologic tests are only poorly specific and are not currently used.

1	The differential diagnosis of donovanosis includes primary syphilitic chancres, secondary syphilis (condylomata lata), chancroid, lymphogranuloma venereum, genital herpes, neoplasm, and amebiasis. Mixed infections are common. Histologic appearances should be distinguished from those of rhinoscleroma, leishmaniasis, and histoplasmosis. Many patients with donovanosis present quite late with extensive ulceration. They may be embarrassed and have low self-esteem related to their disease. Reassurance that they have a treatable condition is important, as is the need to administer antibiotics and monitor patients for an adequate interval (see below). Epidemiologic treatment of sexual partners and advice about how to improve genital hygiene are recommended. Azithromycin 1 g on day 1, then 500 mg daily for 7 days or 1 g weekly for 4 weeks

1	Azithromycin 1 g on day 1, then 500 mg daily for 7 days or 1 g weekly for 4 weeks The recommended drug regimens for donovanosis are shown in Table 198e-1. Gentamicin can be added if the response is slow. Ceftriaxone, chloramphenicol, and norfloxacin are also effective. Patients treated for 14 days should be monitored until lesions have healed completely. Those treated with azithromycin probably do not need such rigorous follow-up. Surgery may be indicated for very advanced lesions. Donovanosis is probably the cause of genital ulceration that is most readily recognizable clinically. Donovanosis is now limited to a few specific locations, and its global eradication is a distinct possibility.

1	FIGuRE 197-2 Nodular lesion of bacillary angiomatosis with superficial ulceration in an AIDS patient with advanced immuno-deficiency. (Reprinted with permission from DH Spach and E Darby: Bartonella Infections, Including Cat-Scratch Disease, in Harrison’s Principles of Internal Medicine, 17th ed, AF Fauci et al [eds]. New York, McGraw-Hill, 2008, p 989.) cells that can be several millimeters in size. Peliotic lesions are surrounded by fibromyxoid stroma containing inflammatory cells, dilated capillaries, and clumps of granular material. Warthin-Starry silver staining of bacillary angiomatosis and peliosis lesions reveals clusters of bacilli. Cultures are usually negative. Bacillary angiomatosis and bacillary peliosis are diagnosed on histologic grounds. Blood cultures may be positive. (Table 197-2) Prolonged therapy with a macrolide or doxycycline is recommended for both bacillary angiomatosis and bacillary peliosis.

1	(Table 197-2) Prolonged therapy with a macrolide or doxycycline is recommended for both bacillary angiomatosis and bacillary peliosis. Control of cat-flea infestation and avoidance of cat scratches (for prevention of B. henselae) and avoidance and treatment of body louse infestation (for prevention of B. quintana) are reasonable strategies for HIV-infected persons. Primary prophylaxis is not recommended, but suppressive therapy with a macrolide or doxycycline is indicated in HIV-infected patients with bacillary angiomatosis or bacillary peliosis until CD4+ T cell counts are >200/μL. Relapse may necessitate lifelong suppressive therapy in individual cases. Carrión’s disease is a biphasic disease caused by B. bacilliformis. Oroya fever is the initial, bacteremic, systemic form, and verruga peruana is its late-onset, eruptive manifestation.

1	Carrión’s disease is a biphasic disease caused by B. bacilliformis. Oroya fever is the initial, bacteremic, systemic form, and verruga peruana is its late-onset, eruptive manifestation. Infection is endemic to the geographically restricted Andes valleys of Peru, Ecuador, and Colombia (~500–3200 m above sea level). Sporadic epidemics occur. The disease is transmitted by the phlebotomine sandfly Lutzomyia verrucarum. Humans are the only known reservoir of B. bacilliformis. Sandfly control measures (e.g., insecticides) and personal protection measures (e.g., repellents, screening, bed nets) may decrease the risk of infection.

1	After inoculation by the sandfly, bacteria invade the blood vessel endothelium and proliferate; the reticuloendothelial system and various organs may also be involved. Upon reentry into blood vessels, B. bacilliformis 1083 invades, replicates, and ultimately destroys erythrocytes, with consequent massive hemolysis and sudden, severe anemia. Microvascular thrombosis results in end-organ ischemia. Survivors sometimes develop cutaneous hemangiomatous lesions characterized by various inflammatory cells, endothelial proliferation, and the presence of B. bacilliformis.

1	The incubation period is 3 weeks (range, 2–14 weeks). Oroya fever may present as a nonspecific bacteremic febrile illness without anemia or as an acute, severe hemolytic anemia with hepatomegaly and jaundice of rapid onset leading to vascular collapse and clouded sensorium. Myalgia, arthralgia, lymphadenopathy, and abdominal pain may develop. Temperature is elevated but not extremely so; high fever may suggest intercurrent infection. Subclinical asymptomatic infection also occurs. In verruga peruana, red, hemangioma-like, cutaneous vascular lesions of various sizes appear either weeks to months after systemic illness or with no previous suggestive history. These lesions persist for months up to 1 year. Mucosal and internal lesions may also develop.

1	Systemic illness (with or without anemia) or the development of cutaneous lesions in a person who has been to an endemic area raises the possibility of B. bacilliformis infection. Severe anemia with exuberant reticulocytosis—and sometimes thrombocytopenia—can occur. In systemic illness, Giemsa-stained blood films show typical intraerythrocytic bacilli, and blood and bone marrow cultures are positive. Serologic assays may be helpful. Biopsy may be required to confirm the diagnosis of verruga peruana. Differential diagnosis includes the spectrum of coendemic systemic febrile illnesses (e.g., typhoid fever, malaria, brucellosis) as well as diseases producing cutaneous vascular lesions (e.g., hemangiomata, bacillary angiomatosis, Kaposi’s sarcoma).

1	(Table 197-2) Antibiotic therapy for systemic B. bacilliformis infection usually results in rapid defervescence. Additional antibiotic treatment of intercurrent infection (particularly salmonellosis) is often required. Blood transfusion may be necessary. Treatment of verruga peruana usually is not required, although large lesions or those interfering with function may require excision. Patients with numerous lesions, especially lesions that have been present for only a short period, may respond well to antibiotic therapy. Mortality rates associated with Oroya fever have been reported to be as high as 40% without treatment but are considerably lower (~10%) with treatment. Complications such as bacterial superinfection and neurologic and cardiac manifestations occur frequently. Generalized massive edema (anasarca) and petechiae are associated with poor outcome. Permanent immunity usually develops.

1	This is a digital-only chapter. It is available on the dvd that accompanies this book, as well as on Access Medicine/Harrison’s Online, and the eBook and “app” editions of HPIM 19e. Donovanosis is a chronic, progressive bacterial infection that usually involves the genital region. The condition is generally regarded as a sexually transmitted infection of low infectivity. This infection has been known by many other names, the most common being granuloma inguinale. Nocardia brasiliensis is usually associated with disease limited to the skin. Actinomycetoma—an indolent, slowly progressive disease of skin and underlying tissues with nodular swellings and draining sinuses— 199 Gregory A. Filice is often associated with N. brasiliensis, Nocardia otitidiscaviarum, N. transvalensis complex strains, or other actinomycetes.

1	N. transvalensis complex strains, or other actinomycetes. Nocardia, a genus of saprophytic aerobic actinomycetes that are common worldwide, resides in soil, contributing to the decay of organic matter. Nearly 100 species have been identified, mostly on the basis of 16S rRNA gene sequences. Nocardiae are relatively inactive in standard biochemical tests, and speciation is difficult without molecular phylogenetic techniques. Historically, the majority of isolates associated with pneumonia and systemic disease were identified as Nocardia asteroides, but the lineage of the type strain was muddled, and it is now clear that human disease is associated with several species. Most clinical laboratories cannot speciate isolates accurately and may identify them simply as N. asteroides or Nocardia species.

1	Nine species or species complexes are commonly associated with human disease (Table 199-1). Most systemic disease involves Nocardia cyriacigeorgica, Nocardia farcinica, Nocardia pseudobrasiliensis, and species in the Nocardia transvalensis and Nocardia nova complexes. Species Susceptible to Resistant to N. abscessus Amikacin, amoxicillin/ Ciprofloxacin, clarithroclavulanic acid, ampicil-mycin, erythromycin, lin, ceftriaxone, genta-imipenem (v)a micin, linezolid, minocycline, TMP-SMX N. brevicatena/pauci-Amikacin, amoxicillin/ Ciprofloxacin, clarithrovorans complex (N. brevi-clavulanic acid, ampicil-mycin, erythromycin, catena, N. paucivorans, lin, ceftriaxone, cipro-gentamicin, imipenem N. carnea, others) floxacin, linezolid, mino-(v) cycline (v), moxifloxacin, tobramycin, TMP-SMX N. nova complex Amikacin, ampicillin, Amoxicillin/clavulanic (N. nova, N. veterana, ceftriaxone, clarithro-acid, ciprofloxacin, gen- N.

1	N. nova complex Amikacin, ampicillin, Amoxicillin/clavulanic (N. nova, N. veterana, ceftriaxone, clarithro-acid, ciprofloxacin, gen- N. africana, N. kruczakiae, mycin, erythromycin, tamicin, tobramycin N. elegans, others) imipenem, linezolid, minocycline, TMP-SMX N. transvalensis complex Amoxicillin/clavulanic Amikacin, ampicillin, (N. blacklockiae, acid (v), ceftriaxone (v), clarithromycin, eryth- N. wallacei, others) ciprofloxacin, linezolid, romycin, gentamicin, minocycline (v), TMP-imipenem (v) SMX N. farcinica Amikacin, amoxicillin/ Ampicillin, ceftriaxone, clavulanic acid, imipenem ciprofloxacin, clarithro(v), linezolid, minocy-mycin, erythromycin, cline (v), TMP-SMX gentamicin, tobramycin N. cyriacigeorgica Amikacin, ceftriaxone Amoxicillin/clavulanic (v), imipenem, linezolid, acid, ampicillin (v), cipminocycline (v), rofloxacin, erythromycin, TMP-SMX gentamicin N.

1	N. cyriacigeorgica Amikacin, ceftriaxone Amoxicillin/clavulanic (v), imipenem, linezolid, acid, ampicillin (v), cipminocycline (v), rofloxacin, erythromycin, TMP-SMX gentamicin N. brasiliensis Amikacin, amoxicillin/ Ampicillin, ceftriaxone, clavulanic acid, mino-ciprofloxacin, clarithrocycline, moxifloxacin, mycin, imipenem TMP-SMX N. pseudobrasiliensis Amikacin, ceftriaxone Amoxicillin/clavulanic (v), ciprofloxacin, clar-acid, ampicillin, ithromycin, TMP-SMX imipenem, minocycline Nocardiosis occurs worldwide. The annual incidence, esti mated on three continents (North America, Europe, and

1	Nocardiosis occurs worldwide. The annual incidence, esti mated on three continents (North America, Europe, and Australia), is ~0.375 cases per 100,000 persons and may be increasing. The disease is more common among adults than among children and more common among males than among females. Nearly all cases are sporadic, but outbreaks have been associated with contamination of the hospital environment, cosmetic procedures, and parenteral illicit drug use. Person-to-person spread is not well documented. There is no known seasonality.

1	The majority of cases of pulmonary or disseminated disease occur in people with a host defense defect. Most have deficient cell-mediated immunity, especially that associated with lymphoma, transplantation, glucocorticoid therapy, or AIDS. The incidence is ~140-fold greater among patients with AIDS and ~340-fold greater among bone marrow transplant recipients than in general populations. In AIDS, nocardiosis usually affects persons with <250 CD4+ T lymphocytes/ μL. Nocardiosis has also been associated with pulmonary alveolar proteinosis, tuberculosis and other mycobacterial diseases, chronic granulomatous disease, interleukin 12 deficiency, and treatment with monoclonal antibodies that interfere with tumor necrosis factor. Any child with nocardiosis and no known cause of immunosuppression should undergo tests to determine the adequacy of the phagocytic respiratory burst.

1	Cases of actinomycetoma occur mainly in tropical and subtropical regions, especially those of Mexico, Central and South America, Africa, and India. The most important risk factor is frequent contact with soil or vegetable matter, especially in laborers. Pneumonia and disseminated disease are both thought to follow inhalation of fragmented bacterial mycelia. The characteristic histologic feature of nocardiosis is an abscess with extensive neutrophil infiltration and prominent necrosis. Granulation tissue usually surrounds the lesions, but extensive fibrosis or encapsulation is uncommon.

1	Actinomycetoma is characterized by suppurative inflammation with sinus tract formation. Granules—microcolonies composed of dense masses of bacterial filaments extending radially from a central core—are occasionally observed in histologic preparations. The granules are frequently found in discharges from lesions of actinomycetoma but almost never in discharges from lesions in other forms of nocardiosis. Nocardiae have evolved a number of properties that enable them to survive within phagocytes, including neutralization of oxidants, prevention of phagosome-lysosome fusion, and prevention of phagosome acidification. Neutrophils phagocytose the organisms and limit their growth but do not kill them efficiently. Cell-mediated immunity is important for definitive control and elimination of nocardiae.

1	CLINICAL MANIFESTATIONS Respiratory Tract Disease Pneumonia, the most common form of nocardial disease in the respiratory tract, is typically subacute; symptoms have usually been present for days or weeks at presentation. The onset is occasionally more acute in immunosuppressed patients. Cough is prominent and produces small amounts of thick, purulent sputum that is not malodorous. Fever, anorexia, weight loss, and malaise are common; dyspnea, pleuritic pain, and hemoptysis are less common. Remissions and exacerbations over several weeks are frequent. Roentgenographic patterns vary, but some are highly suggestive of nocardial pneumonia. Infiltrates vary in size and are typically dense. Single or multiple nodules are common (Figs. 199-1 and 199-2), sometimes suggesting tumors or metastases. Infiltrates and nodules tend to cavitate (Fig. 199-2). Empyema is present in one-quarter of cases. Co-infection with Nocardia and Mycobacterium tuberculosis has been reported from regions where

1	Infiltrates and nodules tend to cavitate (Fig. 199-2). Empyema is present in one-quarter of cases. Co-infection with Nocardia and Mycobacterium tuberculosis has been reported from regions where tuberculosis is common.

1	FIGuRE 199-1 Nocardial pneumonia. A dense infiltrate with a pos-sible cavity and several nodules are apparent in the right lung. Nocardiosis may spread directly from the lungs to adjacent tissues. Pericarditis, mediastinitis, and the superior vena cava syndrome have all been reported. Nocardial laryngitis, tracheitis, bronchitis, and sinusitis are much less common than pneumonia. In the major airways, disease often presents as a nodular or granulomatous mass. Nocardiae are sometimes isolated from respiratory secretions of persons without apparent nocardial disease, usually individuals who have underlying lung or airway abnormalities. FIGuRE 199-2 Nocardial pneumonia. A computed tomography scan shows bilateral nodules, with cavitation in the nodule in the left lung. FIGuRE 199-3 Nocardial abscesses in the right occipital lobe.

1	FIGuRE 199-2 Nocardial pneumonia. A computed tomography scan shows bilateral nodules, with cavitation in the nodule in the left lung. FIGuRE 199-3 Nocardial abscesses in the right occipital lobe. Extrapulmonary Disease In half of all cases of pulmonary nocardiosis, disease appears outside the lungs. In one-fifth of cases of disseminated disease, lung disease is not apparent. The most common site of dissemination is the brain. Other common sites include the skin and supporting structures, kidneys, bone, muscle, and eye, but almost any organ can be involved. Peritonitis has been reported in patients undergoing peritoneal dialysis. Nocardiae have been recovered from blood in a few cases of pneumonia, disseminated disease, or central venous catheter infection. Nocardial endocarditis occurs rarely and can affect either native or prosthetic valves.

1	The typical manifestation of extrapulmonary dissemination is a subacute abscess. A minority of abscesses outside the lungs or central nervous system (CNS) form fistulas and discharge small amounts of pus. In CNS infections, brain abscesses are usually supratentorial, are often multiloculated, and may be single or multiple (Fig. 199-3). Brain abscesses tend to burrow into the ventricles or extend out into the subarachnoid space. The symptoms and signs are somewhat more indolent than those of other types of bacterial brain abscess. Meningitis is uncommon and is usually due to spread from a nearby brain abscess. Nocardiae are not easily recovered from cerebrospinal fluid (CSF). Disease Following Transcutaneous Inoculation Disease that follows transcutaneous nocardial inoculation usually takes one of three forms: cellulitis, lymphocutaneous syndrome, or actinomycetoma.

1	Disease Following Transcutaneous Inoculation Disease that follows transcutaneous nocardial inoculation usually takes one of three forms: cellulitis, lymphocutaneous syndrome, or actinomycetoma. Cellulitis generally begins 1–3 weeks after a recognized breach of the skin, often with soil contamination. Subacute cellulitis, with pain, swelling, erythema, and warmth, develops over days to weeks. The lesions are usually firm and not fluctuant. Disease may progress to involve underlying muscles, tendons, bones, or joints. Dissemination is rare. N. brasiliensis and species in the N. otitidiscaviarum complex are most common in cellulitis cases.

1	Lymphocutaneous disease usually begins as a pyodermatous nodule at the site of inoculation, with central ulceration and purulent or honey-colored drainage. Subcutaneous nodules often appear along lymphatics that drain the primary lesion. Most cases of nocardial lymphocutaneous syndrome are associated with N. brasiliensis. Similar disease occurs with other pathogens, most notably Sporothrix schenckii (Chap. 243) and Mycobacterium marinum (Chap. 204). Actinomycetoma usually begins with a nodular swelling, sometimes at a site of local trauma. Lesions (Fig. 199-4A) typically develop on the feet or hands but may involve the posterior part of the neck, the upper back, the head, and other sites. The nodule eventually breaks down,

1	FIGuRE 199-4 Nocardia brasiliensis mycetoma. A. Draining sinuses and giant white grains with a seropurulent discharge. B. Radiography of the foot showing marked soft tissue enlargement and bony lytic lesions. C. Direct microscopy of grains stained with Lugol's iodine (×40).

1	D. Periodic acid–Schiff stain of skin biopsy (×40). (Image provided by Roberto Arenas and Mahreen Ameen, St. John's Institute of Dermatology, Guy's & St Thomas' NHS Trust, London, UK. Reprinted with permission from R Arenas, M Ameen: Lancet Infect Dis 10:66, 2010.) and a fistula appears, typically followed by others. The fistulas tend to come and go, with new ones forming as old ones disappear. The discharge is serous or purulent, may be bloody, and often contains 0.1to 2-mm white granules consisting of masses of mycelia (Figs. 199-4C and 199-4D). The lesions spread slowly along fascial planes to involve adjacent areas of skin, subcutaneous tissue, and bone. Over months or years, there may be extensive deformation of the affected part. Lesions involving soft tissues are only mildly painful; those affecting bones or joints are more so (Fig. 199-4B). Systemic symptoms are absent or minimal. Infection rarely disseminates from actinomycetoma, and lesions on the hands and feet usually

1	those affecting bones or joints are more so (Fig. 199-4B). Systemic symptoms are absent or minimal. Infection rarely disseminates from actinomycetoma, and lesions on the hands and feet usually cause only local disability. Lesions on the head, neck, and trunk can invade locally to involve deep organs, with consequent severe disability or death.

1	Eye Infections Nocardia species are uncommon causes of subacute keratitis, usually following eye trauma. Nocardial endophthalmitis can develop after eye surgery. In one series, nocardiae accounted for more than half of culture-proved cases of endophthalmitis after cataract surgery. Endophthalmitis can also occur during disseminated disease. Nocardial infection of lachrymal glands has been reported.

1	The first step in diagnosis is examination of sputum or pus for crooked, branching, beaded, gram-positive filaments 1 μm wide and up to 50 μm long (Fig. 199-5). Most nocardiae are acid-fast in direct smears if a weak acid is used for decolorization (e.g., in the modified Kinyoun, Ziehl-Neelsen, and Fite-Faraco methods). The organisms often take up silver stains. Recovery from specimens containing a mixed flora can be improved with selective media (colistin–nalidixic acid agar, modified Thayer-Martin agar, or buffered charcoal–yeast extract agar). Nocardiae grow well on most fungal and mycobacterial media, but procedures used for decontamination of specimens for mycobacterial culture can kill nocardiae and thus should not be used when nocardiae are suspected. Nocardiae grow relatively slowly; colonies may take up to 2 weeks to appear and may not develop their characteristic appearance—white, yellow, or orange, with aerial mycelia and delicate, dichotomously branched substrate

1	slowly; colonies may take up to 2 weeks to appear and may not develop their characteristic appearance—white, yellow, or orange, with aerial mycelia and delicate, dichotomously branched substrate mycelia—for up to 4 weeks. Several blood culture systems support nocardial growth, although nocardiae may not be detected for up to 2 weeks. The growth of nocardiae is so different from that of more common pathogens that the laboratory should be alerted when nocardiosis is suspected in order to maximize the likelihood of isolation.

1	In nocardial pneumonia, sputum smears are often negative. Unless the diagnosis can be made in smear-negative cases by sampling lesions in more accessible sites, bronchoscopy or lung aspiration is usually necessary. To evaluate the possibility of dissemination in patients with nocardial pneumonia, a careful history should be obtained and a thorough physical examination performed. Suggestive symptoms or signs should be pursued with further diagnostic tests. Computed tomography (CT) or magnetic resonance imaging (MRI) of the head, with and without contrast material, should be undertaken if signs or symptoms suggest brain involvement. Some authorities recommend brain imaging in all cases of pulmonary or disseminated disease. When clinically indicated, CSF or urine should be concentrated and then cultured. Actinomycetoma, eumycetoma (cases involving fungi; Chap. 243), and botryomycosis (cases involving cocci or bacilli, often Staphylococcus aureus) are difficult to distinguish clinically

1	then cultured. Actinomycetoma, eumycetoma (cases involving fungi; Chap. 243), and botryomycosis (cases involving cocci or bacilli, often Staphylococcus aureus) are difficult to distinguish clinically but are readily distinguished with microbiologic testing or biopsy. Granules should be sought in any discharge. Suspect particles should be washed in saline, examined microscopically, and cultured. Granules in actinomycetoma cases are usually white, pale yellow, pink, or red. Viewed microscopically, they consist of tight masses of fine filaments (0.5–1 μm wide) radiating outward from a central core (Fig. 199-5). Granules from eumycetoma cases are white, yellow, brown, black, or green. Under the microscope, they appear as masses of broader filaments (2–5 μm wide) encased in a matrix. Granules of botryomycosis consist of loose masses of cocci or bacilli. Organisms can also be seen in wound discharge or histologic specimens. The most reliable way to differentiate among the various organisms

1	of botryomycosis consist of loose masses of cocci or bacilli. Organisms can also be seen in wound discharge or histologic specimens. The most reliable way to differentiate among the various organisms associated with mycetoma is by culture.

1	FIGuRE 199-5 Gram-stained sputum from a patient with nocardial pneumonia. (Image provided by Charles Cartwright and Susan Nelson, Hennepin County Medical Center, Minneapolis, MN.) Isolation of nocardiae from sputum or blood occasionally represents colonization, transient infection, or contamination. In typical cases of respiratory tract colonization, Gram-stained specimens are negative and cultures are only intermittently positive. A positive sputum culture in an immunosuppressed patient usually reflects disease. When nocardiae are isolated from sputum of an immunocompetent patient without apparent nocardial disease, the patient should be observed carefully without treatment. A patient with a host-defense defect that increases the risk of nocardiosis should usually receive antimicrobial treatment.

1	For mild or moderate cases, therapy with drugs known to be effective against most isolates is usually adequate. For severe cases or cases that do not respond promptly to antimicrobial therapy, isolates should be sent to a laboratory experienced with Nocardia for identification and susceptibility testing. Identification of an isolate to the species level is accomplished with molecular testing, and susceptibility is assessed with a Clinical Laboratory Standards Institute (CLSI)–approved broth dilution test. Nocardial growth is slower than the growth of most clinically important bacteria, and nocardiae tend to clump in suspension so that susceptibility test endpoints are unusual; thus experience is necessary for reliable results. Because nocardiosis is uncommon, data on the relation between susceptibility test results for specific drugs and clinical outcomes in patients treated with these drugs are meager. Careful clinical monitoring is essential, and consultation with clinicians who

1	susceptibility test results for specific drugs and clinical outcomes in patients treated with these drugs are meager. Careful clinical monitoring is essential, and consultation with clinicians who have experience with nocardiosis is often needed.

1	Sulfonamides are the drugs of choice (Tables 199-1 and 199-2). The combination of sulfamethoxazole (SMX) and trimethoprim (TMP) is at least equivalent to a sulfonamide alone and may be slightly more effective, but the combination also poses a modestly greater risk of hematologic toxicity. At the outset, 10–20 mg/kg of TMP and 50–100 mg/kg of SMX are given each day in two divided doses. Later, daily doses can be decreased to as little as 5 mg/kg and 25 mg/kg, respectively. In persons with sulfonamide allergies, desensitization usually allows continuation of therapy with these effective and inexpensive drugs. Sulfonamide susceptibility testing is difficult. The CLSI standard methodology includes a technique for TMP-SMX but not for a sulfonamide alone. Reported rates of sulfonamide susceptibility have varied widely, and controversy has ensued about the reliability of sulfonamides for therapy. However, clinical responses to Cellulitis, lymphocutaneous 2 months syndrome

1	Cellulitis, lymphocutaneous 2 months syndrome Osteomyelitis, arthritis, laryngitis, 4 months sinusitis Keratitis Topical: until apparent cure Systemic: until 2–4 months after apparent cure aIn some patients with AIDS and CD4+ T lymphocyte counts of <200/μL or with chronic granulomatous disease, therapy for pulmonary or systemic disease must be continued indefinitely. bIf all apparent CNS disease has been excised, the duration of therapy may be reduced to 6 months. appropriate sulfonamide treatment are nearly always satisfactory. Sulfonamides remain the drugs of choice in nearly all cases.

1	appropriate sulfonamide treatment are nearly always satisfactory. Sulfonamides remain the drugs of choice in nearly all cases. Clinical experience with other oral drugs is limited. Minocycline (100–200 mg twice a day) is often effective; other tetracyclines are usually less effective. Linezolid is active against all species in vitro and in vivo, but adverse effects are common with long-term use. Tigecycline appears to be active in vitro against some species, but little clinical experience has been reported. Amoxicillin (875 mg) combined with clavulanic acid (125 mg), given twice a day, has been effective but should be avoided in cases involving strains of the N. nova complex, in which clavulanate induces β-lactamase production. Among the quinolones, moxifloxacin and gemifloxacin appear to be most active.

1	N. nova complex, in which clavulanate induces β-lactamase production. Among the quinolones, moxifloxacin and gemifloxacin appear to be most active. Amikacin, the best-established parenteral drug except in cases involving the N. transvalensis complex, is given in doses of 5–7.5 mg/ kg every 12 h or 15 mg/kg every 24 h. Serum drug levels should be monitored during prolonged therapy in patients with diminished renal function and in the elderly. Ceftriaxone and imipenem are usually effective except as indicated in Table 199-1.

1	Patients with severe disease are initially treated with a combination including TMP-SMX, amikacin, and ceftriaxone or imipenem. Clinical improvement is usually noticeable after 1–2 weeks of therapy but may take longer, especially with CNS disease. After definite clinical improvement, therapy can be continued with a single oral drug, usually TMP-SMX. Some experts use two or more drugs for the entire course of therapy, but whether multiple drugs are better than a single agent is not known, and additional drugs increase the risk of toxicity. In patients with nocardiosis who need immunosuppressive therapy for an underlying disease or prevention of transplant rejection, immunosuppressive therapy should be continued. Use of SMX and TMP in high-risk populations to prevent Pneumocystis disease or urinary tract infections appears to reduce but not eliminate the risk of nocardiosis. The incidence of nocardiosis is low enough that prophylaxis solely to prevent this disease is not recommended.

1	Surgical management of nocardial disease is similar to that of other bacterial diseases. Brain abscesses should be aspirated, drained, or excised if the diagnosis is unclear, if an abscess is large and accessible, or if an abscess fails to respond to chemotherapy. Small or inaccessible brain abscesses should be treated medically; clinical improvement should be noticeable within 1–2 weeks. Brain imaging should be repeated to document the resolution of lesions, although abatement on images often lags behind clinical improvement. Antimicrobial therapy usually suffices for nocardial actinomycetoma. In deep or extensive cases, drainage or excision of heavily involved tissue may facilitate healing, but structure and function should be preserved whenever possible. Keratitis is treated with topical sulfonamide or amikacin drops plus a sulfonamide or an alternative drug given by mouth.

1	Actinomycosis and whipple’s disease Thomas A. Russo Actinomycosis and Whipple’s disease share characteristics that confound even the skilled diagnostician. Because both diseases are 200 1088 Nocardial infections tend to relapse (particularly in patients with chronic granulomatous disease), and long courses of antimicrobial therapy are necessary (Table 199-2). If disease is unusually extensive or if the response to therapy is slow, the recommendations in Table 199-2 should be exceeded. With appropriate treatment, the mortality rate for pulmonary or disseminated nocardiosis outside the CNS should be <5%. CNS disease carries a higher mortality rate. Patients should be followed carefully for at least 6 months after therapy has ended.

1	uncommon, the physician’s personal experience with their clinical presentations is limited. The laboratory identification of the etiologic agents from the order Actinomycetales is not routine. Thus they remain a diagnostic challenge. However, both of these chronic infections are curable, usually with medical therapy alone. Therefore, an awareness of the full spectrum of these diseases, prompting clinical suspicion, can expedite their diagnosis and treatment and minimize unnecessary surgical interventions (especially with actinomycosis), morbidity, and mortality risk.

1	Actinomycosis is an indolent, slowly progressive infection caused by anaerobic or microaerophilic bacteria, primarily of the genus Actinomyces, that colonize the mouth, colon, and vagina. Mucosal disruption may lead to infection at virtually any site in the body. In vivo growth of actinomycetes usually results in the formation of characteristic clumps called grains or sulfur granules. The clinical presentations of actinomycosis are myriad. Common in the preantibiotic era, actinomycosis has diminished in incidence, as has its timely recognition. Actinomycosis has been called the most misdiagnosed disease, and it has been said that no disease is so often missed by experienced clinicians.

1	Three “classic” clinical presentations that should prompt consideration of this unique infection are (1) the combination of chronicity, progression across tissue boundaries, and mass-like features (mimicking malignancy, with which it is often confused); (2) the development of a sinus tract, which may spontaneously resolve and recur; and (3) a refractory or relapsing infection after a short course of therapy, since cure of established actinomycosis requires prolonged treatment. Actinomycosis is most commonly caused by A. israelii, A. naeslundii,

1	Actinomycosis is most commonly caused by A. israelii, A. naeslundii, A. odontolyticus, A. viscosus, A. meyeri, and A. gerencseriae. Most if not all actinomycotic infections are polymicrobial. Aggregatibacter (Actinobacillus) actinomycetemcomitans, Eikenella corrodens, Enterobacteriaceae, and species of Fusobacterium, Bacteroides, Capnocytophaga, Staphylococcus, and Streptococcus are commonly isolated with actinomycetes in various combinations, depending on the site of infection. Their contribution to the pathogenesis of actinomycosis is uncertain. Comparative 16S rRNA gene sequencing has led to the identification of an ever-expanding list of Actinomyces species and a reclassification of some species to other genera. At present, 46 species and 2 subspecies have been recognized (www.bacterio.cict.fr/a/actinomyces .html). A. europaeus, A. neuii, A. radingae, A. graevenitzii, A. turicensis,

1	A. cardiffensis, A. houstonensis, A. hongkongensis, A. lingnae, A. massiliensis, A. timonensis, and A. funkei as well as two former Actinomyces species—Arcanobacterium pyogenes and Arcanobacterium bernardiae— are additional causes of human actinomycosis, albeit not always with a “classic” presentation.

1	Actinomycosis has no geographic boundaries and occurs throughout life, with a peak incidence in the middle decades. Males have a threefold higher incidence than females, possibly because of poorer dental hygiene and/or more frequent trauma. Improved dental hygiene and the initiation of antimicrobial treatment before actinomycosis fully develops have probably contributed to a decrease in incidence since the advent of antibiotics. Individuals who do not seek or have access to health care, those who have an intrauterine contraceptive device (IUCD) in place for a prolonged period (see “Pelvic Disease,” below), and those who receive bisphosphonate treatment (see “Oral-Cervicofacial Disease,” below) are probably at higher risk.

1	The etiologic agents of actinomycosis are members of the normal oral flora and are often cultured from the bronchi, the gastrointestinal tract, and the female genital tract. The critical step in the development of actinomycosis is disruption of the mucosal barrier. Local infection may ensue. Once established, actinomycosis spreads contiguously in a slow, progressive manner, ignoring tissue planes. Although acute inflammation may initially develop at the infection site, the hallmark of actinomycosis is the characteristic chronic, indolent phase manifested by lesions that usually appear as single or multiple indurations. Central necrosis consisting of neutrophils and sulfur granules develops and is virtually diagnostic. The fibrotic walls of the mass are typically described as “wooden.” The responsible bacterial and/or host factors have not been identified. Over time, sinus tracts to the skin, adjacent organs, or bone may develop. In rare instances, distant hematogenous seeding may

1	The responsible bacterial and/or host factors have not been identified. Over time, sinus tracts to the skin, adjacent organs, or bone may develop. In rare instances, distant hematogenous seeding may occur. As mentioned above, these unique features of actinomycosis mimic malignancy, with which it is often confused.

1	Foreign bodies appear to facilitate infection. This association most frequently involves IUCDs. Reports have described an association of actinomycosis with HIV infection; transplantation; common variable immunodeficiency; chronic granulomatous disease; treatment with infliximab, glucocorticoids, or bisphosphonates; and radioor chemotherapy. Ulcerative mucosal infections (e.g., by herpes simplex virus or cytomegalovirus) may facilitate disease development.

1	CLINICAL MANIFESTATIONS Oral-Cervicofacial Disease Actinomycosis occurs most frequently at an oral, cervical, or facial site, usually as a soft tissue swelling, abscess, or mass lesion that is often mistaken for a neoplasm. The angle of the jaw is generally involved, but a diagnosis of actinomycosis should be considered with any mass lesion or relapsing infection in the head and neck (Chap. 44). Radiation therapy and especially bisphosphonate treatment have been recognized as contributing to an increasing incidence of actinomycotic infection of the mandible and maxilla (Fig. 200-1). Canaliculitis (also commonly due to Propionibacterium propionicum), otitis, and sinusitis also can develop. Pain, fever, and leukocytosis are variably reported. Contiguous extension to the cranium, cervical spine, or thorax is a potential sequela.

1	Thoracic Disease Thoracic actinomycosis, which may be facilitated by foreign material, usually follows an indolent progressive course, with involvement of the pulmonary parenchyma and/or the pleural space. Chest pain, fever, and weight loss are common. A cough, when present, is variably productive. The usual radiographic finding is either a mass lesion or pneumonia. On CT, central areas of low attenuation and ringlike rim enhancement may be seen. Cavitary disease or mediastinal or hilar adenopathy may develop. More than 50% of cases include pleural thickening, effusion, or empyema (Fig. 200-2). Rarely, pulmonary nodules or endobronchial lesions occur. Lesions suggestive of actinomycosis include those that cross fissures or pleura; extend into the mediastinum, contiguous bone, or chest wall; or are associated with a sinus tract. In the absence of these findings, thoracic actinomycosis is usually mistaken for a neoplasm or pneumonia due to more usual causes.

1	FIGuRE 200-1 Bisphosphonate-associated maxillary osteomy-elitis due to A. viscosus. A sulfur granule is seen within the bone. (Reprinted with permission from NH Naik, TA Russo: Bisphosphonate related osteonecrosis of the jaw: The role of Actinomyces. Clin Infect Dis 49:1729, 2009. © 2009 University of Chicago Press.) Mediastinal infection is uncommon, usually arising from thoracic extension but rarely from perforation of the esophagus, trauma, or extension of head and neck or abdominal disease. The structures within the mediastinum and the heart can be involved in various combinations; consequently, the possible presentations are diverse. Primary endocarditis (in which A. neuii has been increasingly described) and isolated disease of the breast occur.

1	Abdominal Disease Abdominal actinomycosis poses a great diagnostic challenge. Months or years usually pass from the inciting event (e.g., appendicitis, diverticulitis, peptic ulcer disease, spillage of gall stones or bile during laparoscopic cholecystectomy, foreign-body perforation, bowel surgery, or ascension from IUCD-associated pelvic disease) to clinical recognition. Because of the flow of peritoneal fluid and/ or the direct extension of primary disease, virtually any abdominal organ, region, or space can be involved. The disease usually presents as an abscess, a mass, or a mixed lesion that is often fixed to underlying tissue and mistaken for a tumor. On CT, enhancement is most often heterogeneous and adjacent bowel is thickened. Sinus tracts to the abdominal wall, to the perianal region, or between the bowel and other organs may develop and mimic inflammatory bowel disease (Chap. 351). Recurrent disease or a wound or fistula that fails to heal suggests actinomycosis.

1	Hepatic infection usually presents as one or more abscesses or 1089 masses (Fig. 200-3). Isolated disease presumably develops via hematogenous seeding from cryptic foci. Imaging and percutaneous techniques have resulted in improved diagnosis and treatment. All levels of the urogenital tract can be infected. Renal disease usually presents as pyelonephritis and/or renal and perinephric abscess. Bladder involvement, usually due to extension of pelvic disease, may result in ureteral obstruction or fistulas to bowel, skin, or uterus. Actinomyces can be detected in urine with appropriate stains and cultures.

1	Pelvic Disease Actinomycotic involvement of the pelvis occurs most commonly in association with an IUCD. When an IUCD is in place or has recently been removed, pelvic symptoms should prompt consideration of actinomycosis. The risk, although not quantified, appears small. The disease rarely develops when the IUCD has been in place for <1 year, but the risk increases with time. Actinomycosis can also present months after IUCD removal. Symptoms are typically indolent; fever, weight loss, abdominal pain, and abnormal vaginal bleeding or discharge are the most common. The earliest stage of disease—often endometritis—commonly progresses to pelvic masses or a tuboovarian abscess (Fig. 200-4). Unfortunately, because the diagnosis is often delayed, a “frozen pelvis” mimicking malignancy or endometriosis can develop by the time of recognition. Ca125 levels may be elevated, further contributing to misdiagnosis.

1	Actinomyces-like organisms (ALOs), which are identified in Papanicolaou-stained specimens in (on average) 7% of women using an IUCD, have a low positive predictive value for diagnosis. Nonetheless, although the risk appears small, the consequences of infection are significant. Therefore, until more quantitative data become available, it seems prudent to remove the IUCD in the presence of symptoms that cannot be accounted for, regardless of whether ALOs are detected, and—if advanced disease is excluded—to initiate a 14-day course of empirical treatment for possible early endometritis. The detection of ALOs in the asymptomatic patient warrants education and close follow-up but not removal of the IUCD unless a suitable contraceptive alternative is agreed on.

1	Central Nervous System Disease Actinomycosis of the central nervous system (CNS) is rare. Single or multiple brain abscesses are most common. An abscess usually appears on CT as a ring-enhancing lesion with a thick wall that may be irregular or nodular. Magnetic resonance perfusion and spectroscopy findings have also been described, as have primary meningitis, epidural or subdural space infection, and cavernous sinus syndrome. Musculoskeletal and Soft Tissue Infection Actinomycotic infection of bone and joints is usually due to adjacent soft-tissue infection but may be associated with trauma (e.g., fracture of the mandible), injections, FIGuRE 200-2 Thoracic actinomycosis. A. A chest wall mass from extension of pulmonary infection. B. Pulmonary infection is complicated by empyema (open arrow) and extension to the chest wall (closed arrow). (Courtesy of Dr. C. B. Hsiao, Division of Infectious Diseases, Department of Medicine, State University of New York at Buffalo.)

1	FIGuRE 200-3 Hepatic-splenic actinomycosis. A. Computed tomogram showing multiple hepatic abscesses and a small splenic lesion due to A. israelii. Arrow indicates extension outside the liver. Inset: Gram’s stain of abscess fluid demonstrating beaded filamentous gram-positive rods. B. Subsequent formation of a sinus tract. (Reprinted with permission from Saad M: Actinomyces hepatic abscess with cutaneous fistula. N Engl J Med 353:e16, 2005. © 2005 Massachusetts Medical Society. All rights reserved.) surgery, osteoradionecrosis and bisphosphonate osteonecrosis (limited to mandibular and maxillary bones), or hematogenous spread. Because of slow disease progression, new bone formation and bone destruction are seen concomitantly. Infection of an extremity is uncommon and is usually a result of trauma. Skin, subcutaneous tissue, muscle, and bone (with periostitis or acute or chronic osteomyelitis) are involved alone or in various combinations. Cutaneous sinus tracts frequently develop.

1	Disseminated Disease Hematogenous dissemination of disease from any location rarely results in multiple-organ involvement. A. meyeri is most commonly involved. The lungs and liver are most commonly affected, with the presentation of multiple nodules mimicking disseminated malignancy. The clinical presentation may be surprisingly indolent given the extent of disease.

1	The diagnosis of actinomycosis is rarely considered. All too often, actinomycosis is first mentioned by the pathologist after extensive surgery. Since medical therapy alone is frequently sufficient for cure, the challenge for the clinician is to consider the possibility of actinomycosis, to diagnose it in the least invasive fashion, and to avoid unnecessary surgery. The clinical and radiographic presentations that suggest actinomycosis are discussed above. Of note, hypermetabolism has been demonstrated by 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) in actinomycotic disease. Aspirations and biopsies (with or without CT or ultrasound guidance) are being used successfully to obtain clinical material for diagnosis, although surgery may be required. The diagnosis is most commonly made by microscopic identification of sulfur granules (an in vivo matrix of bacteria, calcium phosphate, and host material) in pus or tissues. Occasionally, these granules are identified grossly

1	made by microscopic identification of sulfur granules (an in vivo matrix of bacteria, calcium phosphate, and host material) in pus or tissues. Occasionally, these granules are identified grossly from draining sinus tracts or pus. Although sulfur granules are a defining characteristic of actinomycosis, granules also are found in mycetoma (Chaps. 199 and 243) and botryomycosis (a chronic suppurative bacterial infection of soft tissue or, in rare cases, visceral tissue that produces clumps of bacteria resembling granules). These entities can easily be differentiated from actinomycosis with appropriate histopathologic and microbiologic studies. Microbiologic identification of actinomycetes is often precluded by prior antimicrobial therapy or failure to perform appropriate micro-biologic cultures. For optimal yield, the avoidance of even a single dose of antibiotics is mandatory. Primary isolation usually requires 5–7 days under anaerobic conditions but may take as long as 2–4 weeks.

1	cultures. For optimal yield, the avoidance of even a single dose of antibiotics is mandatory. Primary isolation usually requires 5–7 days under anaerobic conditions but may take as long as 2–4 weeks. Although not routinely used, 16S rRNA gene amplification and sequencing have been successfully applied to increase diagnostic sensitivity and specificity. Because actinomycetes are components of the normal oral and genital-tract flora, their identification in the absence of sulfur granules in sputum, bronchial washings, and cervicovaginal secretions is of little significance.

1	FIGuRE 200-4 Computed tomogram showing pelvic actinomy-cosis associated with an intrauterine contraceptive device. The device is encased by endometrial fibrosis (solid arrow); also visible are paraendometrial fibrosis (open triangular arrowhead) and an area of suppuration (open arrow).

1	Decisions about treatment are based on the collective clinical experience of the past 65 years. Actinomycosis requires prolonged treatment with high doses of antimicrobial agents; suitable antimicrobial agents and those deemed unreliable are listed in Table 200-1. The need for intensive treatment is presumably due to the drugs’ poor penetration of the thick-walled masses common in this infection and/or the sulfur granules themselves, which may represent a biofilm. Although therapy must be individualized, the IV administration of 18–24 million units of penicillin daily for 2–6 weeks, followed by oral therapy with penicillin or amoxicillin (total duration, 6–12 months), is a reasonable guideline for serious infections and bulky disease. Less extensive disease, particularly that involving the oralcervicofacial region, may be cured with a shorter course. If therapy is extended beyond the resolution of measurable disease, the risk of relapse—a clinical hallmark of this infection—will be

1	the oralcervicofacial region, may be cured with a shorter course. If therapy is extended beyond the resolution of measurable disease, the risk of relapse—a clinical hallmark of this infection—will be minimized; CT and MRI are generally the most sensitive and objective techniques

1	Extensive successful clinical Penicillin: 3–4 million units IV q4hc experienceb Amoxicillin: 500 mg PO q6h Erythromycin: 500–1000 mg IV q6h or 500 mg PO q6h Tetracycline: 500 mg PO q6h Doxycycline: 100 mg IV or PO q12h Minocycline: 100 mg IV or PO q12h Clindamycin: 900 mg IV q8h or 300–450 mg PO q6h

1	Erythromycin: 500–1000 mg IV q6h or 500 mg PO q6h Tetracycline: 500 mg PO q6h Doxycycline: 100 mg IV or PO q12h Minocycline: 100 mg IV or PO q12h Clindamycin: 900 mg IV q8h or 300–450 mg PO q6h Agents predicted to be efficacious Moxifloxacin on the basis of in vitro activity aAdditional coverage for concomitant “companion” bacteria may be required. bControlled evaluations have not been performed. Dose and duration require individualization depending on the host, site, and extent of infection. As a general rule, a maximal parenteral antimicrobial dose for 2–6 weeks followed by oral therapy, for a total duration of 6–12 months, is required for serious infections and bulky disease, whereas a shorter course may suffice for less extensive disease, particularly in the oral-cervicofacial region. Monitoring the impact of therapy with CT or MRI is advisable when appropriate. cThese agents can be considered for at-home parenteral therapy; penicillin requires a continuous infusion pump.

1	by which to accomplish this goal. A similar approach is reasonable for immunocompromised patients, although refractory disease has been described in HIV-infected individuals. Although the role played by “companion” microbes in actinomycosis is unclear, many isolates are pathogens in their own right, and a regimen covering these organisms during the initial treatment course is reasonable.

1	Combined medical-surgical therapy is still advocated in some reports. However, an increasing body of literature now supports an initial attempt at cure with medical therapy alone, even in extensive disease. CT and MRI should be used to monitor the response to therapy. In most cases, either surgery can be avoided or a less extensive procedure can be used. This approach is particularly valuable in sparing critical organs, such as the bladder or the reproductive organs in women of childbearing age. For a well-defined abscess, percutaneous drainage in combination with medical therapy is a reasonable approach. When a critical location is involved (e.g., the epidural space, the CNS), when there is significant hemoptysis, or when suitable medical therapy fails, surgical intervention may be appropriate. In the absence of optimal data, the combination of a prolonged course of antimicrobial therapy and resection—at least of necrotic bone for bisphosphonate-related osteonecrosis of the jaw

1	appropriate. In the absence of optimal data, the combination of a prolonged course of antimicrobial therapy and resection—at least of necrotic bone for bisphosphonate-related osteonecrosis of the jaw (BRONJ)—is a reasonable approach.

1	Whipple’s disease, a chronic multiorgan infection caused by Tropheryma whipplei, was first described in 1907. The long-held belief that Whipple’s disease is an infection was supported by observations on its responsiveness to antimicrobial therapy in the 1950s and the identification of bacilli via electron microscopy in small-bowel biopsy 1091 specimens in the 1960s. This hypothesis was finally confirmed by amplification and sequencing of a partial 16S rRNA polymerase chain reaction (PCR)–generated amplicon from duodenal tissue in 1991. The subsequent successful cultivation of T. whipplei enabled whole-genome sequencing and the development of additional diagnostic tests. The development of PCR-based diagnostics has broadened our understanding of both the epidemiology and the clinical syndromes attributable to T. whipplei. Exposure to T. whipplei, which appears to be much more common than has been appreciated, can be followed by asymptomatic carriage, acute disease, or chronic

1	syndromes attributable to T. whipplei. Exposure to T. whipplei, which appears to be much more common than has been appreciated, can be followed by asymptomatic carriage, acute disease, or chronic infection. Chronic infection (Whipple’s disease) is a rare development after exposure. “Classic” Whipple’s disease is manifested variably by a combination of arthralgias/arthritis, weight loss, chronic diarrhea, abdominal pain, and fever; less commonly, involvement at sites other than the gastrointestinal tract is documented. Acute infection and chronic organ disease in the absence of intestinal involvement (see “Isolated Infection,” below) are described with increasing frequency. Since untreated Whipple’s disease is often fatal and delayed diagnosis may lead to irreparable organ damage (e.g., in the CNS), knowledge of the clinical scenarios in which Whipple’s should be considered and of an appropriate diagnostic strategy is mandatory.

1	T. whipplei is a weakly staining gram-positive bacillus. Genomic sequence data have revealed that the organism has a small (<1-megabase) chromosome, with many biosynthetic pathways absent or incomplete. This finding is consistent with a host-dependent intracellular pathogen or a pathogen that requires a nutritionally rich extracellular environment. A genotyping scheme based on a variable region has disclosed more than 70 genotypes (GTs) to date. GTs 1 and 3 are most commonly reported, but all GTs appear to be capable of causing similar clinical syndromes.

1	Whipple’s disease is rare but has been increasingly recognized since the advent of PCR-based diagnostic tools. It occurs in all parts of the globe, with an incidence presently estimated at 1 case per 1 million patient-years. Seroprevalence studies indicate that ~50% of Western Europeans and ~75% of Africans from rural Senegal have been exposed to T. whipplei. A predilection for chronic disease has been observed in middle-aged Caucasian men. Males are infected five to eight times more frequently than females. To date, no clear animal or environmental reservoir has been demonstrated. However, the organism has been identified by PCR in sewage water and human feces. Workers with direct exposure to sewage are more likely to be asymptomatically colonized than controls, a pattern suggesting fecal-oral spread. Recent data support oral-oral or fecal-oral spread among family members. Further, the development of acute T. whipplei pneumonia in children raises the possibility of droplet or

1	fecal-oral spread. Recent data support oral-oral or fecal-oral spread among family members. Further, the development of acute T. whipplei pneumonia in children raises the possibility of droplet or airborne transmission.

1	Since rates of exposure to T. whipplei appear to be much higher (e.g., ~50% in Western Europe, as stated above) than rates of chronic disease development (0.00001%), it has been hypothesized that chronically infected individuals possess a subtle host-defense abnormality that does not place them at risk for non–T. whipplei infection. The HLA alleles DRB1*13 and DQB1*06 may be associated with an increased risk of infection. Chronic infection results in a general state of immunosuppression characterized by low CD4+ T cell counts, high levels of interleukin 10 production, increased activity of regulatory T cells, alternative activation of macrophages with diminished antimicrobial activity (M2 polarization) and ensuing apoptosis, and blunted development of T. whipplei–specific T cells. Immunosuppressive glucocorticoid treatment or anti–tumor necrosis factor α therapy appears to accelerate progression of disease. Recently, asymptomatic HIV-infected individuals were found to have

1	Immunosuppressive glucocorticoid treatment or anti–tumor necrosis factor α therapy appears to accelerate progression of disease. Recently, asymptomatic HIV-infected individuals were found to have significantly higher levels of T. whipplei sequence in bronchoalveolar lavage fluid (BALF) than did non-HIV-infected individuals, and these levels decreased 1092 with antiretroviral therapy. A weak humoral response, perhaps due to bacterial glycosylation in patients with chronic disease, appears to differentiate persons who clear the bacillus from asymptomatic carriers. In the initiation of chronic infection, the relative importance of the host’s genetic background versus the modulation of the host response by T. whipplei is unknown.

1	T. whipplei has a tropism for myeloid cells, which it invades and in which it can avoid being killed. Infiltration of infected tissue by large numbers of foamy macrophages is a characteristic finding. In the intestine, villi are flat and wide with dilated lacteals. Involvement of lymphatic or hepatic tissue may manifest as noncaseating granulomas that can mimic sarcoid. CLINICAL MANIFESTATIONS Asymptomatic Colonization/Carriage Studies using primarily PCR have detected T. whipplei sequence in stool, saliva, duodenal tissue, and (rarely) blood in the absence of symptoms. Although prevalence rates are still being defined, in Western European countries, detection in saliva (0.2%) is less common than that in stool (1–11%) and appears to occur only with concomitant fecal carriage. The prevalence of fecal carriage is elevated in individuals with exposure to waste water or sewage (12–26%). However, in rural Senegal, 44% of children age 2–10 had

1	T. whipplei detected in fecal samples. The duration of carriage at these sites is still being examined but can be at least 1 year. It is not known how often the carrier state is associated with acute infection, but evolution into chronic disease is uncommon. Bacterial loads are lighter in asymptomatic carriage than in active disease. Acute Infection T. whipplei has been implicated as a cause of acute gastroenteritis in children. It was also detected via PCR in the blood of 6.4% of febrile patients (primarily children) from two villages in Senegal, often with concomitant cough and rhinorrhea. Further, T. whipplei has been implicated as a cause of acute pneumonia in the United States and France. These data suggest that primary acquisition can result in symptomatic pulmonary or intestinal infection, which may be more common than has been thought, and only rarely results in chronic disease.

1	Chronic Infection • “classic” wHipple’s disease So-called classic Whipple’s disease was the initial clinical syndrome recognized, with consequent identification of T. whipplei. This chronic infection is defined by involvement of the duodenum and/or jejunum that develops over years. In most individuals, the initial phase of disease manifests primarily as intermittent, occasionally chronic and destructive migratory oligoor polyarthralgias/seronegative arthritis. Spondylitis, sacroiliitis, and prosthetic hip infection also have been described. This initial stage is often confused with a variety of rheumatologic disorders and, on average, lasts 6–8 years before gastrointestinal symptoms commence. Treatment of presumed inflammatory arthritis with immunosuppressive agents (e.g., glucocorticoids, tumor necrosis factor α antagonists) can accelerate progression of the disease process. Alternatively, antimicrobial therapy used for another indication may reduce symptoms. In fact, the modulation

1	tumor necrosis factor α antagonists) can accelerate progression of the disease process. Alternatively, antimicrobial therapy used for another indication may reduce symptoms. In fact, the modulation of symptoms in these settings should prompt consideration of Whipple’s disease. The intestinal symptoms that develop in the majority of cases are characterized by diarrhea with accompanying weight loss and may be associated with fever and abdominal pain. Diagnostic misdirection can be caused by co-infection with Giardia lamblia, which is occasionally identified. Occult gastrointestinal blood loss, hepatosplenomegaly, and ascites are less common. Anemia and hypereosinophilia may be detected. Rheumatoid factor and antinuclear antibody tests are usually negative. The most common finding on abdominal CT is mesenteric and/ or retroperitoneal lymphadenopathy. The endoscopic or video capsule observation of pale, yellow, or shaggy mucosa with erythema or ulceration past the first portion of the

1	CT is mesenteric and/ or retroperitoneal lymphadenopathy. The endoscopic or video capsule observation of pale, yellow, or shaggy mucosa with erythema or ulceration past the first portion of the duodenum suggests Whipple’s disease (Fig. 200-5). In addition to rheumatologic and proximal intestinal disease, neurologic (6–63%), cardiac (17–55%), pulmonary (10–40%), lymphatic (10%), ocular (5–10%), dermal (1–5%), and (in rare instances) other sites are variably involved in classic Whipple’s disease.

1	FIGuRE 200-5 Endoscopic view of the jejunal mucosa demonstrating a thickened, granular mucosa and “white spots” due to dilated lacte-als. (Reprinted with permission from J Bureš et al: Whipple’s disease: Our own experience and review of the literature. Gastroenterol Res Pract , 2013. http://dx.doi.org/10.1155/2013/478349.) neurologic disease Asymptomatic neurologic involvement in Whipple’s disease has been documented by PCR-based detection in cerebrospinal fluid (CSF). A variety of neurologic manifestations have been reported, the most common of which are cognitive changes progressing to dementia; personality, mood, and sleep-cycle disorders; hypothalamic involvement; and supranuclear ophthalmoplegia. In addition to the latter, neuro-ophthalmologic manifestations of Whipple’s disease include supranuclear gaze palsy, oculomasticatory and oculofacial myorhythmia (highly suggestive of Whipple’s), nystagmus, and retrobulbar neuritis. Focal neurologic presentations (dependent on lesion

1	supranuclear gaze palsy, oculomasticatory and oculofacial myorhythmia (highly suggestive of Whipple’s), nystagmus, and retrobulbar neuritis. Focal neurologic presentations (dependent on lesion location), seizures, ataxia, meningitis, encephalitis, hydrocephalus, myelopathy, and distal polyneuropathy also have been described. Neurologic sequelae occur with CNS disease, and the mortality risk is significant.

1	MRI results may be normal. Identified lesions (solitary or multifocal) are usually T2 and fluid-attenuated inversion recovery (FLAIR) hyperintense and may enhance with gadolinium. Findings are myriad and not diagnostic, but the limbic system is commonly involved. FDG-PET may reveal increased uptake. CSF analysis may be abnormal; leukocytosis (generally lymphocyte-predominant) and an elevated protein concentration are common. A low CSF glucose level has been reported.

1	cardiac disease Endocarditis, which is increasingly recognized in Whipple’s disease, presents as culture-negative infection and/or congestive heart failure; hypotension occurs rarely. Embolic events or various arrhythmias may also be noted. Fever is often absent, and Duke clinical criteria are rarely met. Vegetations are identified by echocardiography in 50–75% of cases. All valves, alone or in combination, can be affected; most commonly involved are the aortic and mitral valves. Preexisting valvular disease is found in only a minority of cases, although infection of bioprosthetic valves has been described. Mural, myocardial, or pericardial disease also occurs alone or in combination with valvular involvement. Constrictive pericarditis develops infrequently.

1	pulmonary disease Some combination of interstitial disease, nodules, parenchymal infiltrate, and pleural effusion is observed. The clinical significance of T. whipplei dominating sequence reads in BALF from HIV-infected individuals is unresolved. lymphatic disease Mesenteric and retroperitoneal lymphadenopathy are common with intestinal disease, and mediastinal adenopathy may be associated with pulmonary infection. Peripheral adenopathy is less common.

1	ocular disease (non-neuro-ophthalmologic) Uveitis is the most common form of ocular disease, usually presenting as a change in vision or “floaters.” Anterior (anterior chamber), intermediate (vitreous), and posterior (retina/choroid) uveitis can occur alone or in combination. Postoperative acute or chronic ocular Whipple’s disease has been described in association with local or systemic glucocorticoid use; its detection in this setting raises the possibility that asymptomatic or subclinical disease has been unmasked. Keratitis and crystalline keratopathy also have been reported. Patients may be misdiagnosed with sarcoid or Behçet’s disease prior to the recognition of Whipple’s.

1	dermatologic disease Skin hyperpigmentation, particularly in light-exposed areas in the absence of adrenal dysfunction, should be suggestive of Whipple’s disease. A variety of other cutaneous manifestations have been described, including erythematous macular lesions, nonthrombocytopenic purpura, subcutaneous nodules, and hyperkeratosis. miscellaneous sites Thyroid, renal, testicular, epididymal, gallbladder, skeletal muscle, and bone marrow involvement have all been described. In fact, almost any organ can be involved in classic Whipple’s disease, with varying frequency, variable combinations, and myriad signs and symptoms. As a result, Whipple’s disease should be considered in the setting of a chronic multisystemic process. Despite its rarity, the combination of rheumatologic and intestinal disease with weight loss, with or without neurologic and cardiac involvement, warrants heightened suspicion.

1	isolated infection This entity has been defined as infection in the absence of intestinal symptoms, although an occasional small-bowel biopsy may be PCR-positive in this setting. “Isolated infection” is something of a misnomer since multiple nonintestinal sites of T. whipplei infection are not uncommon. Infection at the same nonintestinal sites (single or multiple) that are variably involved in classic Whipple’s disease may also present as “isolated infection.” Endocarditis, neurologic disease, uveitis, rheumatologic manifestations, and pulmonary involvement are most commonly described. Signs and symptoms are similar to those described for T. whipplei infection of these sites in classic Whipple’s disease. With enhanced PCR-based diagnostic capabilities, T. whipplei infection without concomitant intestinal involvement (of which endocarditis is the best example) will probably be diagnosed increasingly often.

1	reinfection/relapsing disease/immUne reconstitUtion inflammatory syndrome (iris) It has been suggested that, if an underlying host immune defect places an individual at risk for chronic infection, then that person may be at risk for reinfection due to occupational exposure or contact with family members who are asymptomatically colonized. One case of apparent relapse that was due to a different genotype supports this contention. Optimal treatment regimens and durations are still being defined. However, it is clear, especially in the setting of occult or overt CNS disease, that treatment with oral tetracycline or trimethoprimsulfamethoxazole (TMP-SMX) alone may result in disease relapse.

1	As in patients treated for HIV or mycobacterial disease, IRIS has been described in patients treated for T. whipplei infection. Prior immunosuppressive therapy increases the likelihood of IRIS, in which inflammation recurs after an initial clinical response to treatment and loss of PCR detection of T. whipplei. Manifestations include fever, arthritis, skin lesions, pleuritis, uveitis, and orbital and periorbital inflammation.

1	Considering T. whipplei infection and ensuring that the appropriate tests are performed are the critical steps in making the diagnosis, which otherwise will likely be missed. The clinical presentation will in part dictate which clinical specimens are most likely to enable the diagnosis. In the presence (and perhaps the absence) of gastrointestinal symptoms, postbulbar duodenal biopsies should be performed. As a 1093 general rule, diagnostic yield is greater for tissue specimens than for body fluids. Biopsy of normal-appearing skin may detect T. whipplei in the setting of classic Whipple’s disease and serve as a minimally invasive means to establish the diagnosis. It is unclear whether CSF should be obtained in the absence of CNS symptoms, but its collection should be considered: the CNS is the most common site for relapse, and thus the information gained by CSF examination could influence the design of the treatment regimen.

1	The development and implementation of PCR-based diagnostics have significantly increased the sensitivity and specificity of T. whipplei identification. PCR can be applied to affected tissues (fixed and nonfixed) and various body fluids (e.g., CSF; aqueous or vitreous humor; joint, pericardial, or pleural fluid; BALF; blood; feces). In some clinical scenarios, a generic 16S rRNA bacterial assay combined with amplicon sequencing can be used to detect and identify T. whipplei sequence. Delineation of the T. whipplei genomic sequence has enabled the development and broad availability of more sensitive and specific PCR-based assays. The interpretation of a PCR-based diagnostic approach must take into account limitations such as false-positive results due to sample contamination and false-negative results due to organism load, sample quality, and inadequate DNA extraction.

1	The diagnosis of classic Whipple’s disease was originally based on histologic findings in intestinal biopsy specimens, and this diagnostic procedure remains important. Infiltration of the lamina propria with macrophages containing inclusions (representing ingested bacteria) that are positive on periodic acid–Schiff (PAS) staining and resistant to diastase is observed. However, PAS is nonspecific, also yielding positive results with mycobacteria (which can be differentiated with Ziehl-Neelsen stain), Rhodococcus equi, Bacillus cereus, Corynebacterium species, and Histoplasma species. T. whipplei can also be detected by silver stain, Brown-Brenn (weakly positive), or acridine orange and is not stained by calcofluor. Staining of other tissues or fluids (e.g., ocular aspirations) for PAS-positive inclusions in macrophages can be performed to support the diagnosis. Electron microscopy can be used to identify the trilaminar cell wall of T. whipplei.

1	When available, immunohistochemistry has greater specificity and sensitivity than PAS staining and can be performed on archived fixed tissue. T. whipplei has been successfully cultured from blood, CSF, synovial fluid, BALF, valve tissue, duodenal tissue, skeletal muscle, and lymph nodes, but culture is not practical since it takes months to obtain a positive result. Likewise, serology is of limited value for the diagnosis of Whipple’s disease because the prevalence of exposure is much higher than that of chronic disease development and the antibody response to T. whipplei appears to be blunted in the disease state. Although histologic or cytologic detection of T. whipplei is less specific and sensitive than PCR, a positive result is strongly supportive within the appropriate clinical context and is definitive when combined with a more specific test (e.g., PCR, immunohistochemistry).

1	Data on treatment are emerging, but questions persist regarding the optimal regimen and duration, which may depend on the site of infection (e.g., CNS and heart valve). Appropriate treatment usually results in a rapid and at times remarkable clinical response (e.g., in CNS disease). Maintenance of a durable response has been more challenging. Rates of relapse, particularly of CNS disease, were unacceptable with oral tetracycline or TMP-SMX monotherapy. Sequence data now indicate that TMP is not active against T. whipplei due to the absence of dihydrofolate reductase, but this drug was used extensively before this fact was known. This information prompted a randomized controlled trial in 40 patients, who received either ceftriaxone (2 g IV q24h) or meropenem (1 g IV q8h) for 2 weeks followed by oral TMP-SMX (160/800 mg) twice a day for 1 year. The efficacy of these regimens was outstanding. The only instance of therapy failure—in a case of asymptomatic CNS infection that was

1	Infections due to Mixed Anaerobic Organisms Ronit Cohen-Poradosu, Dennis L. Kasper Anaerobes comprise the predominant class of bacteria of the normal human microbiota (formerly termed “the normal human flora”) that 201 1094 not eradicated by either regimen—was subsequently cured with oral minocycline and chloroquine (250 mg/d after a loading dose). A follow-up trial reported similar efficacy with a regimen of ceftriaxone (2 g IV q24h) for 2 weeks followed by oral TMP-SMX for 3 months. One issue in these trials was that the CNS doses—and perhaps the duration of ceftriaxone and meropenem treatment as well—were not optimal. Further, investigators have speculated that oral regimens with greater CNS penetrance, such as sulfadiazine (2–4 g/d in 3 or 4 divided doses) and/or doxycycline or minocycline (200 mg/d in 2 divided doses) plus hydroxychloroquine (200 mg three times a day, to raise phagosome pH and increase drug activity in vitro), might render the parenteral phase of treatment

1	minocycline (200 mg/d in 2 divided doses) plus hydroxychloroquine (200 mg three times a day, to raise phagosome pH and increase drug activity in vitro), might render the parenteral phase of treatment unnecessary, given that the one failure of therapy for CNS disease was cured with a similar regimen. Another issue is concern about the potential development of resistance to sulfa drugs. Lastly, it is unclear whether oral sulfaor tetracycline-based regimens will suffice in endocarditis. Until more data become available, it seems prudent—at least in asymptomatic/symptomatic CNS disease or cardiac infection—to administer CNS-optimized doses of IV ceftriaxone (2 g q12h) or meropenem (2 g q8h) for at least 2 weeks followed by oral doxycycline or minocycline plus hydroxychloroquine or chloroquine for at least 1 year, if tolerated. Although data on the use of PCR to guide therapy do not exist, it seems reasonable that continued T. whipplei detection by PCR, especially in the CSF, should

1	for at least 1 year, if tolerated. Although data on the use of PCR to guide therapy do not exist, it seems reasonable that continued T. whipplei detection by PCR, especially in the CSF, should dictate at least continuation of therapy and perhaps consideration of an alternative regimen. The occurrence of a Jarisch-Herxheimer reaction within 24 h of treatment initiation has been described, with rapid resolution. The addition of glucocorticoids may be beneficial in the management of clearly documented IRIS. Data on certain site-specific treatment issues are even more limited. Anecdotal reports describe successful treatment of uveitis with oral TMP-SMX with or without rifampin, whereas treatment with tetracycline alone has resulted in relapse. Although a role for adjunctive intraocular therapy has been reported, the data are unclear on this point. Surgery may be needed in the setting of endocarditis with significant valve dysfunction; however, timely recognition can result in cure with

1	has been reported, the data are unclear on this point. Surgery may be needed in the setting of endocarditis with significant valve dysfunction; however, timely recognition can result in cure with medical management alone. Although data on the treatment of foreign body–associated infection are virtually nonexistent, medical treatment for a prosthetic hip infection was apparently successful; however, follow-up was limited. Regardless of the therapeutic regimen chosen, an effort to ensure compliance and close follow-up for potential relapse (or perhaps reinfection), which can occur many years after an apparent cure, will maximize the chances for a good outcome.

1	reside on mucous membranes and predominate in many infectious processes, particularly those arising from mucosal surfaces. These organisms generally cause disease subsequent to the breakdown of mucosal barriers and the leakage of the microbiota into normally sterile sites. Infections resulting from contamination by the microbiota are usually polymicrobial and involve both aerobic and anaerobic bacteria. However, the difficulties encountered in handling specimens in which anaerobes may be important and the technical challenges entailed in cultivating and identifying these organisms in clinical microbiology laboratories continue to leave the anaerobic etiology of an infectious process unproven in many cases. Therefore, an understanding of the types of infections in which anaerobes can play a role is crucial in selecting appropriate microbiologic tools to identify the organisms in clinical specimens and in choosing the most appropriate treatment, including antibiotics and surgical

1	play a role is crucial in selecting appropriate microbiologic tools to identify the organisms in clinical specimens and in choosing the most appropriate treatment, including antibiotics and surgical drainage or debridement of the infected site.

1	This chapter focuses on infections caused by nonsporulating anaerobic bacteria. It does not address clostridial infections and syndromes, which are covered elsewhere (Chaps. 161 and 179).

1	Anaerobic bacteria are organisms that require reduced oxygen tension for growth, failing to grow on the surface of solid media in 10% CO2 in air. (In contrast, microaerophilic bacteria can grow in an atmosphere of 10% CO2 in air or under anaerobic or aerobic conditions, although they grow best in the presence of only a small amount of atmospheric oxygen, and facultative bacteria can grow in the presence or absence of air). Most clinically relevant anaerobes, such as Bacteroides fragilis, Prevotella melaninogenica, and Fusobacterium nucleatum, are relatively aerotolerant. Although they can survive for sustained periods in the presence of up to 2–8% oxygen, they generally do not multiply in this environment. A smaller number of pathogenic anaerobic bacteria (which are also part of the microbiota) die after brief contact with oxygen, even in low concentrations.

1	Most human mucocutaneous surfaces harbor a rich indigenous normal microbiota composed of aerobic and anaerobic bacteria. These surfaces are dominated by anaerobic bacteria, which often account for 99.0–99.9% of the culturable microbiota and range in concentration from 109/mL in saliva to 1012/mL in gingival scrapings and the colon. It is interesting that anaerobes inhabit many areas of the body that are exposed to air: skin, nose, mouth, and throat. Anaerobes are thought to reside in the portions of these sites that are relatively well protected from oxygen, such as gingival crevices. New technologies based on analyses of microbial DNA have expanded our knowledge of these bacterial populations. For example, in an analysis of 13,555 prokaryotic ribosomal RNA gene sequences from the colon, most bacteria identified were considered uncultivated and novel microorganisms. Two immense projects based on these new technologies, the Human Microbiome Project funded by the U.S. National

1	colon, most bacteria identified were considered uncultivated and novel microorganisms. Two immense projects based on these new technologies, the Human Microbiome Project funded by the U.S. National Institutes of Health and MetaHIT financed by the European Commission, aim to characterize the normal microbiota of healthy individuals.

1	The major reservoirs of anaerobic bacteria are the mouth, lower gastrointestinal tract, skin, and female genital tract (Table 201-1). In the oral cavity, the ratio of anaerobic to aerobic bacteria ranges from 1:1 on the surface of a tooth to 1000:1 in the gingival crevices. Prevotella and Porphyromonas species comprise much of the indigenous oral anaerobic microbiota. Fusobacterium and Bacteroides (non–B. fragilis group) are present in lower numbers. Anaerobic bacteria are not found in appreciable numbers in the normal stomach and upper small intestine. In the distal ileum, the microbiota begins to resemble that of the colon. In the colon, the ratio of anaerobes to facultative species is high; for example, there are 1011–1012 organisms/g of stool, and >99% of these organisms are anaerobic, with an anaerobe-to-aerobe ratio of ~1000:1. The predominant anaerobes in the human intestine belong to the phyla Bacteroidetes and Firmicutes and include a number of Bacteroides species (e.g.,

1	with an anaerobe-to-aerobe ratio of ~1000:1. The predominant anaerobes in the human intestine belong to the phyla Bacteroidetes and Firmicutes and include a number of Bacteroides species (e.g., members of the

1	B. fragilis group, such as B. fragilis, B. thetaiotaomicron, B. ovatus, B. vulgatus, B. uniformis, and Parabacteroides distasonis) as well as various clostridial, peptostreptococcal, and fusobacterial species. In the female genital tract, there are ~109 organisms/mL of secretions, with an anaerobe-to-aerobe ratio of 1:1 to 10:1. The predominant anaerobes in the female genital tract are Prevotella, Bacteroides, Fusobacterium, Clostridium, and the anaerobic Lactobacillus species. The skin microbiota contains anaerobes as well, the predominant species being Propionibacterium acnes and, in lower numbers, other species of propionibacteria and peptostreptococci.

1	Commensal bacteria in general and commensal anaerobes in particular have been implicated as crucial mediators of physiologic, metabolic, and immunologic functions in the mammalian host. One of the most important roles that anaerobes serve as components of the normal colonic microbiota is the promotion of resistance to colonization; the presence of anaerobic bacteria effectively interferes with colonization by potentially pathogenic bacterial species through the depletion of oxygen and nutrients, the production of enzymes and toxic end products, and the modulation of the host’s intestinal innate immune response. For example, B. thetaiotaomicron stimulates Paneth cells to produce RegIIIγ, a bactericidal lectin that can result in killing of gram-positive bacteria. The normal colonic microbiota plays an important role in protection against Clostridium difficile–associated diarrhea or colitis—a toxin-mediated, potentially life-threatening disease that results when C. difficile spores in

1	plays an important role in protection against Clostridium difficile–associated diarrhea or colitis—a toxin-mediated, potentially life-threatening disease that results when C. difficile spores in the colon transform into toxin-producing vegetative forms after antibiotic elimination of critical components of the competing colonic microbiota.

1	Bacteroides and other intestinal bacteria ferment carbohydrates and produce volatile fatty acids that are reabsorbed and used by the host as an energy source. The anaerobic intestinal microbiota is also responsible for the production of secreted products that promote human health (e.g., vitamin K and bile acids useful in fat absorption and cholesterol regulation). Moreover, the anaerobic intestinal microbiota influences the development of an intact mucosa and of mucosa-associated lymphoid tissue. Colonization of germ-free mice with a single species,

1	B. thetaiotaomicron, affects the expression of various host genes and corrects deficiencies of nutrient uptake, metabolism, angiogenesis, mucosal barrier function, and enteric nervous system development. The symbiosis factor polysaccharide A (PSA) of B. fragilis influences the normal development and function of the mammalian immune system and protects mice against colitis in a model of inflammatory bowel disease. It has also been shown that PSA can confer protection both prophylactically and therapeutically, restraining inflammatory processes at an extraintestinal site (the central nervous system [CNS]) and ameliorating disease in a mouse model of multiple sclerosis. Anaerobes can stimulate specific lymphocyte populations of the small and large intestine and can influence immunologic balance (including TH1/TH2 balance) as well as the number of TH17 and regulatory T cells in gut tissues.

1	Clearly, the gut microbiota confers many benefits, and its dysregulation may play a role in the pathogenesis of diseases characterized by inflammation and aberrant immune responses, such as inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, asthma, and type 1 diabetes. Furthermore, the gut microbiota has been associated with obesity and metabolic syndrome. An interesting association between certain microbes found in the microbiota and testosterone production has been suggested as well.

1	Thousands of species of anaerobic bacteria have been identified as components of the complete human microbiota, with each individual colonized by hundreds of these species. Despite the complex array of bacteria in the normal microbiota, relatively few species are isolated commonly from human infections. Anaerobic infections occur when the harmonious relationship between the host and the host’s microbiota is disrupted. Any site in the body is susceptible to infection with these indigenous organisms when a mucosal barrier or the skin is compromised by surgery, trauma, tumor, ischemia, or necrosis, all of which can reduce local tissue redox potentials. Because the sites that are colonized by anaerobes contain many species of bacteria, disruption of anatomic barriers allows contamination of normally sterile sites by many organisms, resulting in mixed infections involving multiple species of anaerobes in combination with synergistically acting facultative or microaerophilic organisms.

1	Severe mixed infections of the head and neck may arise from an abscessed tooth infected with commensal microbiota of the mouth. Examples of infections arising from an oral source are chronic sinusitis, chronic otitis media, Ludwig’s angina, and periodontal abscesses. Brain abscesses and subdural empyema are also commonly associated with the oral microbiota. Oral anaerobes are usually responsible for pleuropulmonary diseases such as aspiration pneumonia, necrotizing pneumonia, lung abscess, and empyema.

1	Anaerobes from the intestine play an important role in various intraabdominal infections, such as peritonitis and intraabdominal abscesses (Chap. 159). Colonic contents are the source of microorganisms in the case of these infections, which usually follow disruption of intestinal continuity and contamination of the peritoneal cavity. Anaerobic bacteria are isolated frequently in female genital tract infections, such as salpingitis, pelvic peritonitis, tuboovarian abscess, vulvovaginal abscess, septic abortion, and endometritis (Chap. 163). In addition, these bacteria are often found in bacteremia and in infections of the skin, soft tissues, and bones. Predominant among the anaerobic gram-positive cocci that produce disease are the peptostreptococci; the species of this genus that are most commonly involved in infections are P. micros,

1	Predominant among the anaerobic gram-positive cocci that produce disease are the peptostreptococci; the species of this genus that are most commonly involved in infections are P. micros, P. magnus, P. asaccharolyticus, P. anaerobius, and P. prevotii. Clostridia (Chap. 179) are anaerobic spore-forming gram-positive rods that are isolated from wounds, abscesses, sites of abdominal infection, and blood. Gram-positive anaerobic non-spore-forming bacilli are uncommon as etiologic agents of human infection. P. acnes, a component of the skin microbiota and a rare cause of foreign-body infections, is one of the few nonclostridial gram-positive rods associated with infections. The principal anaerobic gram-negative bacilli found in human infections belong to the B. fragilis group and to Fusobacterium, Prevotella, and Porphyromonas species. The most important potential anaerobic pathogens found in the upper airways and isolated from clinical specimens of oral and

1	Infections Due to Mixed Anaerobic Organisms 1096 pleuropulmonary infections are the Fusobacterium species F. necrophorum, F. nucleatum, and F. varium; P. melaninogenica; the Prevotella oralis group; Porphyromonas gingivalis; Porphyromonas asaccharolytica; Peptostreptococcus species; and the Bacteroides ureolyticus group. The B. fragilis group contains the anaerobic pathogens most frequently isolated from clinical infections. Members of this group are part of the normal bowel microbiota; they include several distinct species, such as B. fragilis, B. thetaiotaomicron, B. vulgatus, B. uniformis, B. ovatus, and P. distasonis. B. fragilis is the most important clinical isolate, although it is isolated in lower numbers than some other Bacteroides species from cultures of the commensal fecal microbiota. In female genital tract infections, organisms normally colonizing the vagina (e.g., Prevotella bivia and Prevotella disiens) are the most common isolates. However, B. fragilis is not

1	microbiota. In female genital tract infections, organisms normally colonizing the vagina (e.g., Prevotella bivia and Prevotella disiens) are the most common isolates. However, B. fragilis is not uncommon.

1	Anaerobic bacterial infections usually occur when an anatomic barrier is disrupted and constituents of the local microbiota enter a site that was previously sterile. Because of the specific growth requirements of anaerobic organisms and their presence as commensals on mucosal surfaces, conditions must arise that allow these organisms to penetrate mucosal barriers and enter tissue with a lowered oxidation-reduction potential. Therefore, tissue ischemia, trauma, surgery, perforated viscus, shock, and aspiration provide environments conducive to the proliferation of anaerobes. The introduction of many bacterial species into otherwise sterile sites leads to a polymicrobial infection in which certain organisms predominate. Three major factors are involved in the pathogenesis of anaerobic infections: bacterial synergy, bacterial virulence factors, and mechanisms of abscess formation. The ability of different anaerobic bacteria to act synergistically during polymicrobial infection

1	infections: bacterial synergy, bacterial virulence factors, and mechanisms of abscess formation. The ability of different anaerobic bacteria to act synergistically during polymicrobial infection contributes to the pathogenesis of anaerobic infections. It has been postulated that facultative organisms function in part to lower the oxidation-reduction potential in the microenvironment, allowing the propagation of obligate anaerobes. Anaerobes can produce compounds such as succinic acid and short-chain fatty acids that inhibit the ability of phagocytes to clear facultative organisms. In experimental models, facultative and obligate anaerobes synergistically potentiate abscess formation.

1	Virulence factors associated with anaerobes typically confer the ability to evade host defenses, adhere to cell surfaces, produce toxins and/or enzymes, or display surface structures such as capsular polysaccharides and lipopolysaccharide (LPS) that contribute to pathogenic potential. The ability of an organism to adhere to host tissues is important to the establishment of infection. Some oral species adhere to the epithelium in the oral cavity. P. melaninogenica actually attaches to other microorganisms. P. gingivalis, a common isolate in periodontal disease, has fimbriae that facilitate attachment. Some Bacteroides strains appear to be piliated, a characteristic that may account for their ability to adhere.

1	The most extensively studied virulence factor of the nonsporulating anaerobes is the capsular polysaccharide complex of B. fragilis. This organism is unique among anaerobes in its potential for virulence during growth at normally sterile sites. Although it constitutes only 0.5–1% of the normal colonic microbiota, B. fragilis is the anaerobe most commonly isolated from intraabdominal infections and bacteremia. In an animal model of intraabdominal sepsis, the capsular polysaccharide was identified as the major virulence factor of B. fragilis; this polymer plays a specific, central role in the induction of abscesses. A series of detailed biologic and molecular studies of this virulence factor showed that B. fragilis produces at least eight distinct capsular polysaccharides, far more than the number reported for any other encapsulated bacterium. B. fragilis can exhibit distinct surface polysaccharides either alone or in combination by regulating the expression of these different capsules

1	number reported for any other encapsulated bacterium. B. fragilis can exhibit distinct surface polysaccharides either alone or in combination by regulating the expression of these different capsules in an on–off manner through a reversible inversion of DNA segments within the promoters for operons containing the genes required for polysaccharide synthesis. Structural analysis of two of these polysaccharides, PSA and PSB, revealed that each polymer consists of repeating units with positively charged free amino groups and negatively charged groups. This structural feature is rare among bacterial polysaccharides, and the ability of PSA—and, to a lesser extent, PSB—to induce abscesses in animals depends on this zwitterionic charge motif. Intraabdominal abscess induction is related to the capacity of this polysaccharide to stimulate macrophages to release cytokines and chemokines—in particular, interleukin (IL) 8, IL-17, and tumor necrosis factor α (TNF-α)—from resident peritoneal cells

1	of this polysaccharide to stimulate macrophages to release cytokines and chemokines—in particular, interleukin (IL) 8, IL-17, and tumor necrosis factor α (TNF-α)—from resident peritoneal cells through a Toll-like receptor 2–dependent mechanism. The release of cytokines and chemokines results in the chemotaxis of polymorphonuclear neutrophils (PMNs) into the peritoneum, where they adhere to mesothelial cells induced by TNF-α to upregulate their expression of intercellular adhesion molecule 1 (ICAM-1). PMNs adherent to ICAM-1-expressing cells probably represent the nidus for an abscess. PSA also activates T cells to produce certain cytokines, including IL-17 and interferon γ, that are necessary for abscess formation.

1	B. fragilis produces other virulence factors that allow it to predominate in disease. This organism synthesizes pili, fimbriae, and hemagglutinins that aid in attachment to host cell surfaces. In addition, Bacteroides species produce many enzymes and toxins that contribute to pathogenicity. Enzymes such as neuraminidase, protease, glycoside hydrolases, and superoxide dismutases are all produced by B. fragilis. Anaerobic bacteria produce a number of exoproteins that can enhance the organisms’ virulence. The collagenase produced by

1	B. fragilis. Anaerobic bacteria produce a number of exoproteins that can enhance the organisms’ virulence. The collagenase produced by P. gingivalis may enhance tissue destruction. An association of B. fragilis strains positive for the enterotoxin BFT with clinical episodes of diarrhea in children and adults has been suggested. BFT is a metalloprotease that is cytopathic for intestinal epithelial cells and induces fluid secretion and tissue damage in ligated intestinal loops of experimental animals. Recent evidence from mouse models indicates that enterotoxin-producing strains of B. fragilis may play a role in colon carcinoma.

1	Exotoxins produced by clostridial species, including botulinum toxins, tetanus toxin, C. difficile toxins A and B, and five toxins produced by Clostridium perfringens, are among the most virulent bacterial toxins in mouse lethality assays. Anaerobic gram-negative bacteria such as B. fragilis possess LPSs (endotoxins) that are 100–1000 times less biologically potent than endotoxins associated with aerobic gram-negative bacteria. This relative biologic inactivity may account for the lower frequency of disseminated intravascular coagulation and purpura in Bacteroides bacteremia than in facultative and aerobic gram-negative bacillary bacteremia. An exception is the LPS from Fusobacterium, which may account for the severity of Lemierre’s syndrome (see “Complications of Anaerobic Head and Neck Infections,” below). APPROACH TO THE PATIENT: Infection due to Anaerobic Bacteria

1	APPROACH TO THE PATIENT: Infection due to Anaerobic Bacteria The physician must consider several points when approaching the patient with possible infection due to anaerobic bacteria. 1. Most of the organisms colonizing mucosal sites are commensals; very few cause disease. When these organisms do cause disease, it often occurs in proximity to the mucosal site they colonize. 2. For anaerobes to cause tissue infection, they must spread beyond the normal mucosal barriers. 3. Conditions favoring the propagation of anaerobic bacteria, particularly a lowered oxidation-reduction potential, are necessary. These conditions exist at sites of trauma, tissue destruction, compromised vascular supply, and complications of preexisting infection, which produce necrosis. 4. Frequently, a complex array of infecting microbes can be found. For example, as many as 12 species of organisms can be isolated from a suppurative site. 5.

1	4. Frequently, a complex array of infecting microbes can be found. For example, as many as 12 species of organisms can be isolated from a suppurative site. 5. Anaerobic organisms tend to be found in abscess cavities or in necrotic tissue. The failure of an abscess to yield organisms on routine culture is a clue that the abscess is likely to contain anaerobic bacteria. Often smears of this “sterile pus” are found to be teeming with bacteria when Gram’s stain is applied. Although some facultative organisms (e.g., Staphylococcus aureus) are also capable of causing abscesses, abscesses in organs or deeper body tissues should call anaerobic infection to mind. 6. Gas is found in many anaerobic infections of deep tissues but is not diagnostic because it can be produced by aerobic bacteria as well. 7.

1	6. Gas is found in many anaerobic infections of deep tissues but is not diagnostic because it can be produced by aerobic bacteria as well. 7. Although a putrid-smelling infection site or discharge is considered diagnostic for anaerobic infection, this manifestation usually develops late in the course and is present in only 30–50% of cases. 8. Some species (the best example being the B. fragilis group) require specific therapy. However, many synergistic infections can be cured with antibiotics directed at some but not all of the organisms involved. Antibiotic therapy, combined with debridement and drainage, disrupts the interdependent relationship among the bacteria, and some species that are resistant to the antibiotic do not survive without the co-infecting organisms. 9. Manifestations of severe sepsis and disseminated intravascular coagulation are unusual in patients with purely anaerobic infection.

1	9. Manifestations of severe sepsis and disseminated intravascular coagulation are unusual in patients with purely anaerobic infection. Difficulties in the performance of appropriate cultures, contamination of cultures by components of the normal microbiota, and the lack of readily available, reliable culture techniques have made it impossible to obtain accurate data on incidence or prevalence. However, anaerobic infections are encountered frequently in hospitals with active surgical, trauma, and obstetric and gynecologic services. Depending on the institution, anaerobic bacteria account for 0.5–12% of all cases of bacteremia.

1	CLINICAL MANIFESTATIONS Anaerobic Infections of the Mouth, Head, and Neck Anaerobic bacteria are commonly involved in infections of the mouth, head, and neck (Chap. 44). The predominant isolates are components of the normal microbiota of the upper airways—mainly the Bacteroides oralis group, pigmented Prevotella species, P. asaccharolytica, Fusobacterium species, peptostreptococci, and microaerophilic streptococci.

1	Soft tissue infections of the oral-facial area may or may not be odontogenic. Odontogenic infections—primarily dental caries and periodontal disease (gingivitis and periodontitis)—are common and have both local consequences (especially tooth loss) and the potential for life-threatening spread to the deep fascial spaces of the head and neck. Infections of the mouth can arise from either supragingival or subgingival dental plaque composed of bacteria colonizing the tooth surface. Supragingival plaque formation begins with the adherence of gram-positive bacteria to the tooth surface. This form of plaque is influenced by salivary and dietary components, oral hygiene, and local host factors. Supragingival plaque can lead to dental caries and, with further invasion, to pulpitis (endodontic infection) that can further perforate the alveolar bone, causing periapical abscess. Subgingival plaque is associated with periodontal infections (e.g., gingivitis, periodontitis, and periodontal abscess)

1	that can further perforate the alveolar bone, causing periapical abscess. Subgingival plaque is associated with periodontal infections (e.g., gingivitis, periodontitis, and periodontal abscess) that can further disseminate to adjacent structures such as the mandible, causing osteomyelitis of the maxillary sinuses. Periodontitis may also result in spreading infection that can involve adjacent bone or soft tissues. In the healthy periodontium, the sparse microbiota consists mainly of gram-positive organisms such as Streptococcus sanguinis and Actinomyces species. In the presence of gingivitis, there is a shift to a greater proportion of anaerobic gram-negative bacilli in the subgingival microbiota, with predominance of Prevotella intermedia. In well-established periodontitis, the complexity of the microbiota increases further. The predominant isolates are

1	P. gingivalis, P. intermedia, Aggregatibacter actinomycetemcomitans, Treponema denticola, and Tannerella forsythensis. Necrotizing ulcerative Gingivitis Gingivitis may become a necrotizing infection (trench mouth, Vincent’s stomatitis) (Chap. 44). The onset of disease is usually sudden and is associated with tender bleeding gums, foul breath, and a bad taste. The gingival mucosa, especially the 1097 papillae between the teeth, becomes ulcerated and may be covered by a gray exudate, which is removable with gentle pressure. Patients may become systemically ill, developing fever, cervical lymphadenopathy, and leukocytosis.

1	Noma (cancrum oris) is a necrotizing infection of the oral mucous membranes. It is characterized by destruction of soft tissue and bone and evolves rapidly from gingival inflammation to orofacial gangrene. Noma occurs most frequently in young children (1–4 years of age) with malnutrition or systemic disease. This infection occurs worldwide but is most common in sub-Saharan Africa.

1	Acute Necrotizing Infections of the Pharynx These infections usually occur in association with ulcerative gingivitis. Symptoms include an extremely sore throat, foul breath, and a bad taste accompanied by fever and a sensation of choking. Examination of the pharynx demonstrates that the tonsillar pillars are swollen, red, ulcerated, and covered with a grayish membrane that peels easily. Lymphadenopathy and leukocytosis are common. The disease may last for only a few days or, if not treated, may persist for weeks. Lesions begin unilaterally but may spread to the other side of the pharynx or the larynx. Aspiration of the infected material by the patient can result in lung abscesses. Peripharyngeal Space Infections These infections arise from the spread of organisms from the upper airways to potential spaces formed by the fascial planes of the head and neck. The etiology is typically polymicrobial and represents the normal microbiota of the mucosa of the originating site.

1	Peritonsillar abscess (quinsy) is a complication of acute tonsillitis caused mainly by a mixed flora containing anaerobes (e.g., F. necrophorum and Peptostreptococcus species) and the facultative anaerobe group A Streptococcus (Chap. 44). Of cases of submandibular space infection (Ludwig’s angina), 80% are caused by infection of the tissues surrounding the second and third molar teeth. This infection results in marked local swelling of tissues, with pain, trismus, and superior and posterior displacement of the tongue. Submandibular swelling of the neck can impair swallowing and cause respiratory obstruction. In some cases, tracheotomy is life-saving. Cervicofacial actinomycosis (Chap. 200) is caused by a branching, gram-positive, non-sporeforming, strict/facultative anaerobe that is a part of the normal oral microbiota. This chronic disease is characterized by abscesses, draining sinus tracts, fistula, bone destruction, and fibrosis. It can easily be mistaken for malignancy or

1	a part of the normal oral microbiota. This chronic disease is characterized by abscesses, draining sinus tracts, fistula, bone destruction, and fibrosis. It can easily be mistaken for malignancy or granulomatous disease. Actinomycosis less frequently involves the thorax, abdomen, pelvis, and CNS.

1	Sinusitis and Otitis Anaerobic bacteria have been implicated in chronic sinusitis but play little role in acute sinusitis. In several studies on chronic sinusitis, anaerobic bacteria were found in 0–52% of cases, depending on the method used to collect specimens. Polymicrobial infection is common, and the predominant anaerobic isolates are pigmented Prevotella, Fusobacterium, Peptostreptococcus, and P. acnes. Aerobic gram-negative bacilli and S. aureus have also been implicated in chronic sinusitis. Anaerobic bacteria have been isolated in a large percentage of cases of chronic suppurative otitis media in children. The role of anaerobes in acute otitis media is less clear.

1	Complications of Anaerobic Head and Neck Infections Contiguous cranial spread of these infections can result in osteomyelitis of the skull or mandible or in intracranial infections such as brain abscess and subdural empyema. Caudal spread can produce mediastinitis or pleuropulmonary infection. Hematogenous complications can also result from anaerobic infections of the head and neck. Bacteremia, which occasionally is polymicrobial, can lead to endocarditis or other distant infections. Lemierre’s syndrome (Chap. 44), which has been uncommon in the antimicrobial era, is an acute oropharyngeal infection with secondary septic thrombophlebitis of the internal jugular vein and frequent metastasis, most commonly to the lung. F. necrophorum is the usual cause. This infection typically begins with pharyngitis, which is followed by local invasion in the lateral pharyngeal space, with resultant internal jugular vein thrombophlebitis. A typical clinical

1	Infections Due to Mixed Anaerobic Organisms 1098 triad includes pharyngitis, a tender/swollen neck, and noncavitating pulmonary infiltrates. CNS Infections CNS infections associated with anaerobic bacteria are brain abscess, epidural abscess, and subdural empyema. Anaerobic meningitis is rare and is usually related to parameningeal collection or shunt infection. If optimal bacteriologic techniques are used, as many as 85% of brain abscesses yield anaerobic bacteria. Most anaerobic brain abscesses arise by direct extension from a site of otorhinolaryngeal infection such as otitis, sinusitis, or tooth infection. Hematogenous dissemination from a distant infected site, usually intraabdominal or pelvic, can occur. Common isolates are Peptostreptococcus, Fusobacterium, Bacteroides, Prevotella, Propionibacterium, Eubacterium, Veillonella, and Actinomyces species. Facultative or microaerophilic streptococci and coliforms are often part of a mixed infecting flora in brain abscesses.

1	Pleuropulmonary Infections Anaerobic pleuropulmonary infections result from the aspiration of oropharyngeal contents by patients with predisposing conditions such as dysphagia due to neurologic or esophageal disorders or transiently impaired consciousness due to conditions such as alcohol or drug abuse, seizures, head trauma, and cerebrovascular accidents. Clinical syndromes that are associated with anaerobic pleuropulmonary infection produced by aspiration include aspiration pneumonitis, which can be complicated by necrotizing pneumonia, lung abscess, and empyema. Many of these infections have an indolent course that may serve as a clinical clue differentiating them, for example, from pneumococcal pneumonia, which often presents with abrupt onset, shaking chills, and rapid progression.

1	The anaerobes most common in pleuropulmonary infections are indigenous to the oral cavity, especially the gingival crevice, and include pigmented and nonpigmented Prevotella, Peptostreptococcus, and Bacteroides species and F. nucleatum. Many of these infections are of mixed aerobic-anaerobic etiology, and the predominant aerobes isolated from community-acquired aspiration pneumonias are microaerophilic streptococci such as Streptococcus milleri. Studies using in-depth culture techniques in patients with community-acquired lung abscess showed aerobic and microaerophilic streptococci to be the most common pathogens (60% of patients) and anaerobes to be the second most common (26%). In a study on aspiration pneumonia from a long-term care facility, the most common isolates were gram-negative bacilli (49%), anaerobes (16%), and S. aureus (12%). Nosocomial aspiration pneumonia commonly involves a mixture of anaerobes and gram-negative bacilli or S. aureus.

1	aspiration pneUmonitis Bacterial aspiration pneumonitis must be distinguished from two other clinical syndromes associated with aspiration that are not of bacterial etiology. One syndrome results from aspiration of solids, usually food. Obstruction of major airways typically results in atelectasis and moderate nonspecific inflammation. Therapy consists of removal of the foreign body. The second aspiration syndrome is more easily confused with bacterial aspiration. Mendelson’s syndrome, a chemical pneumonitis, results from regurgitation of stomach contents and aspiration of chemical material, usually acidic gastric juices. Pulmonary inflammation—including the destruction of the alveolar lining, with transudation of fluid into the alveolar space—occurs with remarkable rapidity. Typically this syndrome develops within hours, often following anesthesia when the gag reflex is depressed. The patient becomes tachypneic, hypoxic, and febrile. The leukocyte count may rise, and the chest x-ray

1	syndrome develops within hours, often following anesthesia when the gag reflex is depressed. The patient becomes tachypneic, hypoxic, and febrile. The leukocyte count may rise, and the chest x-ray may evolve from normal to a complete bilateral “whiteout” within 8–24 h. Sputum production is minimal. The pulmonary signs and symptoms can resolve quickly with symptom-based therapy or can culminate in respiratory failure, with the subsequent development of bacterial superinfection over a period of days. Antibiotic therapy is not indicated unless bacterial infection supervenes.

1	In contrast to these syndromes, bacterial aspiration pneumonitis develops over a period of several days or weeks rather than hours. Patients who enter the hospital with this syndrome typically have been ill for several days and generally report low-grade fever, malaise, and sputum production. In some patients, weight loss and anemia reflect a more chronic process. Usually the history reveals factors predisposing to aspiration, such as alcohol overdose or residence in a nursing home. Examination sometimes yields evidence of periodontal disease. Sputum characteristically is not malodorous unless the process has been under way for at least a week. A mixed bacterial flora with many PMNs is evident on Gram’s staining of sputum. Expectorated sputum is unreliable for anaerobic cultures because of inevitable contamination by the normal oral microbiota. Reliable specimens for culture can be obtained by transtracheal or transthoracic aspiration—techniques that are rarely used at present.

1	of inevitable contamination by the normal oral microbiota. Reliable specimens for culture can be obtained by transtracheal or transthoracic aspiration—techniques that are rarely used at present. Culture of protected-brush specimens or bronchoalveolar lavage fluid obtained by bronchoscopy is controversial.

1	Chest x-rays show consolidation in dependent pulmonary segments: in the basilar segments of the lower lobes if the patient has aspirated while upright and in either the posterior segment of the upper lobe (usually on the right side) or the superior segment of the lower lobe if the patient has aspirated while supine. necrotizing pneUmonitis This form of anaerobic pneumonitis is characterized by numerous small abscesses that spread to involve several pulmonary segments. The process can be indolent or fulminating. This syndrome is less common than either aspiration pneumonitis or lung abscess and includes features of both types of infection.

1	anaerobic lUng abscesses (See also Chap. 154) These abscesses result from subacute anaerobic pulmonary infection. The clinical syndrome typically involves a history of constitutional signs and symptoms (including malaise, weight loss, fever, night sweats, and foul-smelling sputum), perhaps over a period of weeks (Chap. 153). Patients who develop lung abscesses characteristically have dental infection and periodontitis, but lung abscesses in edentulous patients have been reported. Abscess cavities may be single or multiple and generally occur in dependent pulmonary segments (Fig. 201-1). Anaerobic abscesses must be distinguished from lesions associated with tuberculosis, neoplasia, and other conditions. Septic pulmonary emboli may originate from intraabdominal or female genital tract infections and can produce anaerobic pneumonia and abscess.

1	Septic pulmonary emboli may originate from intraabdominal or female genital tract infections and can produce anaerobic pneumonia and abscess. empyema Empyema is a manifestation of long-standing anaerobic pulmonary infection complicated by bronchopleural fistula. The clinical presentation, which includes foul-smelling sputum, resembles that FIGuRE 201-1 Chest radiograph of right-lower-lobe lung abscess in a 60-year-old alcoholic patient. (From GL Mandell [ed]: Atlas of Infectious Diseases, Vol VI. Philadelphia, Current Medicine Inc, Churchill Livingstone, 1996; with permission.) of other anaerobic pulmonary infections. Patients may report pleuritic chest pain and marked chest-wall tenderness.

1	Empyema may be masked by overlying pneumonitis and should be considered especially in cases of persistent fever despite antibiotic therapy. Diligent physical examination and the use of ultrasound to localize a loculated empyema are important diagnostic tools. The collection of a foul-smelling exudate by thoracentesis is typical. Cultures of infected pleural fluid yield an average of 3.5 anaerobic and 0.6 facultative or aerobic bacterial species. Drainage is required. Defervescence, a return to a feeling of well-being, and resolution of the process may require several months. Extension from a subdiaphragmatic infection may also result in anaerobic empyema.

1	Intraabdominal Infections Intraabdominal infections—mainly peritonitis and abscesses—are usually polymicrobial and represent the normal intestinal (especially colonic) microbiota. These infections most often follow a breach in the mucosal barrier resulting from appendicitis, diverticulitis, neoplasm, inflammatory bowel disease, surgery, or trauma. On average, four to six bacterial species are isolated per specimen submitted to the microbiology laboratory, with a predominance of enteric aerobic/facultative gram-negative bacilli, anaerobes, and streptococci/enterococci. The most common isolates are Escherichia coli (found in ≥50% of patients) and B. fragilis (30– 50%). Other anaerobes commonly isolated from this type of infection include Peptostreptococcus, Prevotella, and Fusobacterium species. The involvement of clostridia can lead to severe infections. The dominance of four to six bacterial species out of the more than 500 colonic mucosal species is related both to the virulence

1	species. The involvement of clostridia can lead to severe infections. The dominance of four to six bacterial species out of the more than 500 colonic mucosal species is related both to the virulence factors of these species and to the inability of clinical laboratories to culture many other species residing in the colonic mucosa.

1	Disease originating from proximal-bowel perforation reflects the microbiota of this site, with a predominance of aerobic and anaerobic gram-positive bacteria and Candida. Neutropenic enterocolitis (typhlitis) has been associated with anaerobic infection of the cecum but—in the setting of neutropenia (Chap. 104)—may involve the entire bowel. Patients usually present with fever; abdominal pain, tenderness, and distention; and watery diarrhea. The bowel wall is edematous with hemorrhage and necrosis. The primary pathogen is thought by some authorities to be Clostridium septicum, but other clostridia and mixed anaerobes have also been implicated. More than 50% of patients developing early clinical signs can benefit from antibiotic therapy and bowel rest. Surgery is sometimes required to remove gangrenous bowel. See Chap. 159 for a complete discussion of intraabdominal infections.

1	Enterotoxigenic B. fragilis has been associated with watery diarrhea in a few young children and adults. In case–control studies of children with undiagnosed diarrheal disease, enterotoxigenic B. fragilis was isolated from significantly more children with diarrhea than children in the control group.

1	Pelvic Infections The vagina of a healthy woman is a major reservoir of anaerobic and aerobic bacteria. In the normal microbiota of the female genital tract, anaerobes outnumber aerobes by a ratio of ~10:1 and include anaerobic gram-positive cocci and Bacteroides species (Table 201-1). Anaerobes are isolated from most women with genital tract infections that are not caused by a sexually transmitted pathogen. The major anaerobic pathogens are B. fragilis, P. bivia, P. disiens, P. melaninogenica, anaerobic cocci, and Clostridium species. Anaerobes are frequently encountered in pelvic inflammatory disease, pelvic abscess, endometritis, tubo-ovarian abscess, septic abortion, and postoperative or postpartum infections. These infections are often of mixed etiology, involving both anaerobes and coliforms; pure anaerobic infections without coliform or other facultative bacterial species occur more often in pelvic than in intraabdominal sites. Septic pelvic thrombophlebitis may complicate the

1	coliforms; pure anaerobic infections without coliform or other facultative bacterial species occur more often in pelvic than in intraabdominal sites. Septic pelvic thrombophlebitis may complicate the infections and lead to repeated episodes of septic pulmonary emboli. See Chap. 163 for a complete discussion of pelvic inflammatory disease.

1	Anaerobic bacteria have been thought to be contributing factors in the etiology of bacterial vaginosis. This syndrome of unknown etiology is characterized by a profuse malodorous discharge and a change in the bacterial ecology that results in replacement of the Lactobacillus-1099 dominated normal microbiota with an overgrowth of bacterial species including Gardnerella vaginalis, Prevotella species, Mobiluncus species, peptostreptococci, and genital mycoplasmas. A study based on 16S rRNA identification found other anaerobes that were predominant in cases but not in controls: Atopobium, Leptotrichia, Megasphaera, and Eggerthella. Pelvic infections due to Actinomyces species have been associated with the use of intrauterine devices (Chap. 200).

1	Skin and Soft Tissue Infections Injury to skin, bone, or soft tissue by trauma, ischemia, or surgery creates a suitable environment for anaerobic infections. These infections are most frequently found in sites prone to contamination with feces or with upper airway secretions— e.g., wounds associated with intestinal surgery, decubitus ulcers, or human bites. Moreover, anaerobes have been isolated from cutaneous abscesses, rectal abscesses, and axillary sweat gland infections (hidradenitis suppurativa). Anaerobes also are often cultured from foot ulcers of diabetic patients. The deep soft-tissue infections associated with anaerobic bacteria are crepitant cellulitis, synergistic cellulitis, gangrene, and necrotizing fasciitis (Chaps. 156 and 179).

1	These soft tissue or skin infections are usually polymicrobial. A mean of 4.8 bacterial species are isolated, with an anaerobe-toaerobe ratio of ~3:2. The most frequently isolated organisms include Bacteroides, Peptostreptococcus, Clostridium, Enterococcus, and Proteus species. The involvement of anaerobes in these types of infections is associated with a higher frequency of fever, foul-smelling lesions, gas in the tissues, and visible foot ulcer.

1	Anaerobic bacterial synergistic gangrene (Meleney’s gangrene), a rare infection of the superficial fascia, is characterized by exquisite pain, redness, and swelling followed by induration. Erythema surrounds a central zone of necrosis. A granulating ulcer forms at the original center as necrosis and erythema extend outward. Symptoms are limited to pain; fever is not typical. These infections usually involve a combination of Peptostreptococcus species and S. aureus; the usual site of infection is an abdominal surgical wound or the area surrounding an ulcer on an extremity. Treatment includes surgical removal of necrotic tissue and antimicrobial administration.

1	Necrotizing fasciitis, a rapidly spreading destructive disease of the fascia, is usually attributed to group A streptococci (Chap. 173) but can also be a mixed infection involving anaerobes and aerobes, usually occurring after surgeries and in patients with diabetes or peripheral vascular disease. The most frequently isolated anaerobes in these infections are Peptostreptococcus and Bacteroides species. Gas may be found in the tissues. Similarly, myonecrosis can be associated with mixed anaerobic infection. Fournier’s gangrene consists of cellulitis involving the scrotum, perineum, and anterior abdominal wall, with mixed anaerobic organisms spreading along deep external fascial planes and causing extensive loss of skin.

1	Bone and Joint Infections Although actinomycosis (Chap. 200) accounts on a worldwide basis for most anaerobic infections in bone, organisms including peptostreptococci or microaerophilic cocci, Bacteroides species, Fusobacterium species, and Clostridium species can also be involved in osteomyelitis (Chap. 158). These infections frequently arise adjacent to soft tissue infections. Many patients with osteomyelitis due to anaerobic bacteria have evidence of an anaerobic infection elsewhere in the body; most commonly, infected adjacent soft-tissue sites are the source of the organisms involved. Examples are diabetic foot ulcers and decubitus ulcers that may be complicated by mixed aerobic-anaerobic osteomyelitis. Hematogenous seeding of bone is uncommon. Prevotella and Porphyromonas species are detected in infections involving the maxilla and mandible, whereas Clostridium species have been reported as anaerobic pathogens in cases of osteomyelitis of the long bones following fracture or

1	are detected in infections involving the maxilla and mandible, whereas Clostridium species have been reported as anaerobic pathogens in cases of osteomyelitis of the long bones following fracture or trauma. Fusobacteria have been isolated in pure culture from sites of osteomyelitis adjacent to the perinasal sinuses. Peptostreptococci and microaerophilic cocci have been reported as significant pathogens in infections involving the skull, mastoid, and prosthetic implants placed in bone. In patients with osteomyelitis, the most reliable culture specimen is a bone biopsy sample free of normal uninfected skin and subcutaneous tissue.

1	Infections Due to Mixed Anaerobic Organisms 1100 In contrast to anaerobic osteomyelitis, most cases of anaerobic arthritis (Chap. 157) involve a single isolate, and most cases are secondary to hematogenous spread. The most common isolates are Fusobacterium species. Most of the patients involved have uncontrolled peritonsillar infections progressing to septic cervical venous thrombophlebitis (Lemierre’s syndrome) and resulting in hematogenous dissemination with a predilection for the joints. Unlike anaerobic osteomyelitis, anaerobic pyoarthritis in most cases is not polymicrobial and may be acquired hematogenously. Anaerobes are important pathogens in infections involving prosthetic joints; in these infections, the causative organisms (such as Peptostreptococcus species and P. acnes) are part of the normal skin microbiota.

1	Bacteremia Transient bacteremia is a well-known event in healthy individuals whose anatomic mucosal barriers have been injured (e.g., during dental extractions or dental scaling). These bacteremic episodes, which are often due to anaerobes, have no pathologic consequences. However, anaerobic bacteria are found in cultures of blood from clinically ill patients when proper culture techniques are used. Anaerobes have accounted for 5% (range at various institutions, 0.5–12%) of cases of clinically significant bacteremia. The incidence of anaerobic bacteremia decreased from the 1970s through the early 1990s. This change may have been related to the administration of antibiotic prophylaxis before intestinal surgery, the earlier recognition of localized infections, and the empirical use of broad-spectrum antibiotics for presumed infection. Recent reports present conflicting data regarding rates of anaerobic bacteremia. A study from the Mayo Clinic compared three periods (1993–1996,

1	of broad-spectrum antibiotics for presumed infection. Recent reports present conflicting data regarding rates of anaerobic bacteremia. A study from the Mayo Clinic compared three periods (1993–1996, 1997–2000, and 2001–2004) and found a 74% increase in the mean incidence of anaerobic bacteremia; this finding contrasts with a 45% decrease in incidence from 1977 to 1988 at the same institution. In contrast, a report from Switzerland compared two periods (1997–2001 and 2002–2006) and found decreases in both the number of anaerobe-positive blood cultures and the proportion of all blood culture isolates that were anaerobes.

1	The majority of anaerobic bacteremias are due to gram-negative bacilli—mainly the B. fragilis group, with B. fragilis most commonly isolated (60–80% of cases). Other organisms causing bacteremia include Clostridium species (10%), Peptostreptococcus species (10%), and Fusobacterium species (5%).

1	Once the organism in the blood has been identified, both the portal of bloodstream entry and the underlying problem that probably led to seeding of the bloodstream can often be deduced from an understanding of the organism’s normal site of residence. For example, mixed anaerobic bacteremia including B. fragilis usually implies a colonic pathology with mucosal disruption from neoplasia, diverticulitis, or some other inflammatory lesion. Debilitating diseases such as malignancies, diabetes, organ transplantation, and abdominal and pelvic surgeries are among the predisposing factors for anaerobic bacteremia. In a retrospective nested case–control study, diabetes was identified as a risk factor for anaerobic bacteremia when the source of bacteremia was unknown. The initial manifestations are determined by the portal of entry and reflect the localized condition. When bloodstream invasion occurs, patients can become extremely ill, with rigors and hectic fevers. The clinical picture may be

1	determined by the portal of entry and reflect the localized condition. When bloodstream invasion occurs, patients can become extremely ill, with rigors and hectic fevers. The clinical picture may be quite similar to that seen in sepsis involving aerobic gram-negative bacilli. Although complications of anaerobic bacteremia (e.g., septic thrombophlebitis and septic shock) have been reported, their incidence in association with anaerobic bacteremia is low. Anaerobic bacteremia is potentially fatal and requires rapid diagnosis and appropriate therapy. Reported case–fatality rates are high, ranging from 25% to 44%, and appear to increase with the age of the patient (with reported rates of >66% among patients >60 years old), with the isolation of multiple species from the bloodstream, and with the failure to surgically remove a focus of infection. The attributable mortality rate for bacteremia associated with the B. fragilis group was examined in a matched case–control study. Patients with

1	the failure to surgically remove a focus of infection. The attributable mortality rate for bacteremia associated with the B. fragilis group was examined in a matched case–control study. Patients with B. fragilis–group bacteremia had a significantly higher mortality rate (28% vs 8%), with an attributable mortality rate of 19.3% and a mortality risk ratio of 3.2.

1	Endocarditis and Pericarditis (See also Chap. 155) Endocarditis due to anaerobes is uncommon. However, anaerobic streptococci, which are often classified incorrectly, are responsible for this disease more frequently than is generally appreciated. Gram-negative anaerobes are unusual causes of endocarditis. Signs and symptoms of anaerobic endocarditis are similar to those of endocarditis due to facultative organisms. Mortality rates of 21–43% have been reported for anaerobic endocarditis. Anaerobes, particularly B. fragilis and Peptostreptococcus species, are uncommonly found in infected pericardial fluids. Anaerobic pericarditis is associated with a mortality rate of >50%. Anaerobes can reach the pericardial space by hematogenous spread, by spread from a contiguous site of infection (e.g., heart or esophagus), or by direct inoculation arising from trauma or surgery.

1	There are three critical steps in the diagnosis of anaerobic infection: (1) proper collection of specimens; (2) rapid transport of the specimens to the microbiology laboratory, preferably in anaerobic transport media; and (3) proper handling of the specimens by the laboratory. Specimens must be collected by meticulous sampling of infected sites, with avoidance of contamination by the normal microbiota. When such contamination is likely, the specimen is unacceptable. Examples of specimens unacceptable for anaerobic culture include sputum collected by expectoration or nasal tracheal suction, bronchoscopy specimens, samples collected directly through the vaginal vault, urine collected by voiding, and feces. Specimens appropriate for anaerobic culture include sterile body fluids such as blood, pleural fluid, peritoneal fluid, cerebrospinal fluid, and aspirates or biopsy samples from normally sterile sites. As a general rule, liquid or tissue specimens are preferred; swab specimens should

1	pleural fluid, peritoneal fluid, cerebrospinal fluid, and aspirates or biopsy samples from normally sterile sites. As a general rule, liquid or tissue specimens are preferred; swab specimens should be avoided.

1	Because even brief exposure to oxygen may kill some anaerobic organisms and result in failure to isolate them in the laboratory, air must be expelled from the syringe used to aspirate the abscess cavity, and the needle must be capped with a sterile rubber stopper. It is also important to remember that prior antibiotic therapy reduces the cultivability of these bacteria. Specimens can be injected into transport bottles containing a reduced medium or taken immediately in syringes to the laboratory for direct culture on anaerobic media. Delays in transport may lead to a failure to isolate anaerobes due to exposure to oxygen or overgrowth of facultative organisms, which may eliminate or obscure any anaerobes that are present. All clinical specimens from suspected anaerobic infections should be subjected to Gram’s staining and examined for organisms with characteristic morphology. It is not unusual for organisms to be observed on Gram’s staining but not isolated in culture.

1	Because of the time and difficulty involved in the isolation of anaerobic bacteria, diagnosis of anaerobic infections must frequently be based on presumptive evidence. There are few clinical clues to the probable presence of anaerobic bacteria at infected sites. The involvement of certain sites with lowered oxidation-reduction potential (e.g., avascular necrotic tissues) and the presence of an abscess favor the diagnosis of an anaerobic infection. When infections occur in proximity to mucosal surfaces normally harboring an anaerobic microbiota, such as the gastrointestinal tract, female genital tract, or oropharynx, anaerobes should be considered as potential etiologic agents. A foul odor is often indicative of anaerobes, which produce certain organic acids as they proliferate in necrotic tissue. Although these odors are nearly pathognomonic for anaerobic infection, the absence of odor does not exclude an anaerobic etiology. The presence of gas in tissues is highly suggestive, but not

1	tissue. Although these odors are nearly pathognomonic for anaerobic infection, the absence of odor does not exclude an anaerobic etiology. The presence of gas in tissues is highly suggestive, but not diagnostic, of anaerobic infection. Because anaerobes often coexist with other bacteria and cause mixed or synergistic infection, Gram’s staining of exudate frequently reveals multiple morphotypes suggestive of anaerobes. Sometimes these organisms have morphologic characteristics associated with specific species.

1	When cultures of obviously infected sites or purulent material yield no growth, streptococci only, or a single aerobic species (such as E. coli) and Gram’s staining reveals a mixed flora, the involvement of anaerobes should be suspected; the implication is that the anaerobic microorganisms have failed to grow because of inadequate transport and/ or culture techniques. Failure of an infection to respond to antibiotics that are not active against anaerobes (e.g., aminoglycosides and—in some circumstances—penicillin, cephalosporins, or tetracyclines) suggests an anaerobic etiology.

1	Successful therapy for anaerobic infections requires the administration of a combination of appropriate antibiotics, surgical resection, debridement of devitalized tissues, and drainage either surgically or percutaneously (guided by an imaging technique such as CT , MRI, or ultrasound). Any anatomic breach must be closed promptly, closed spaces drained, tissue compartments decompressed, and an adequate blood supply established. Abscess cavities should be drained as soon as fluctuation or localization occurs.

1	The antibiotics used to treat anaerobic infections should be active against both aerobic and anaerobic organisms because many of these infections are of mixed etiology. Antibiotic regimens can usually be selected empirically on the basis of the type of infection, the species of the organisms usually present in such cases, the results of Gram’s staining, and a knowledge of antimicrobial resistance patterns (Chap. 170 and Table 201-2). Other factors influencing the selection of antibiotics include need for bactericidal activity and for penetration into certain organs (such as the brain), toxicity, and impact on the normal microbiota. Antibiotics active against clinically relevant anaerobes can be grouped into four categories based on their predicted activity (Table 201-2). Nearly all the drugs listed have toxic side effects, which are described in detail in Chap. 170.

1	Antibiotic susceptibility testing of anaerobic bacteria has been difficult and controversial. Because of the slow growth rate of many anaerobes, the lack of standardized testing methods and of clinically relevant standards for resistance, and the generally good results obtained with empirical therapy, there has been limited interest in testing these organisms for antibiotic susceptibility. However, one study of antibiotic-treated patients with Bacteroides isolates from blood found mortality rates of 45% among those whose isolates were deemed resistant to the agent used and 16% among those whose isolates were deemed sensitive. It is accepted that testing is important for patients with serious or prolonged infections or in cases in which antibiotics have not had an impact. Testing is also helpful in monitoring the activity of new drugs and recording current resistance patterns among anaerobic pathogens. The antibiotics with the greatest activity against nearly all anaerobic bacteria

1	helpful in monitoring the activity of new drugs and recording current resistance patterns among anaerobic pathogens. The antibiotics with the greatest activity against nearly all anaerobic bacteria include carbapenems, β-lactam/β-lactamase inhibitor combinations, metronidazole, and chloramphenicol.

1	Antibiotic resistance in anaerobic bacteria is an increasing problem. Resistance rates vary with the institution and the geographic region. In recent years, the activity of clindamycin, cefoxitin, cefotetan, and moxifloxacin has decreased against B. fragilis and related strains (B. distasonis, B. ovatus, B. thetaiotaomicron, B. uniformis, B. vulgatus). Multidrug-resistant B. fragilis has recently been reported. Nearly all organisms in the B. fragilis group (>97%) are 1101 resistant to penicillin G. The cephamycins cefoxitin and cefotetan display greater activity against this group, but rates of resistance have increased, with current figures at ~10% in the United States and higher in Argentina (28%) and Europe (17%). Rates of resistance to β-lactam agents among anaerobes other than Bacteroides are lower but are highly variable. β-Lactam/β-lactamase inhibitor combinations such as ampicillin/sulbactam, ticarcillin/clavulanic acid, and piperacillin/tazobactam are usually good therapeutic

1	are lower but are highly variable. β-Lactam/β-lactamase inhibitor combinations such as ampicillin/sulbactam, ticarcillin/clavulanic acid, and piperacillin/tazobactam are usually good therapeutic options against β-lactamase-producing anaerobes, including the B. fragilis group. Although resistance rates reported from most countries are still low, several studies have documented nonsusceptibility to ampicillin/sulbactam in 0.5–3% of isolates in the United States, 3–10% in Europe, and 1–8% in Argentina. Recently, up to 48% of B. fragilis isolates in Taiwan were found to be nonsusceptible to ampicillin/sulbactam, and a significant increase in resistance to this combination was also identified among other Bacteroides, Prevotella, and Fusobacterium species.

1	Carbapenems (ertapenem, doripenem, meropenem, and imipenem) are equally active against anaerobes, with <1% of B. fragilis strains showing resistance in the United States and Europe. Higher rates of carbapenem nonsusceptibility are being reported from some countries (5% in Germany, 8% [to doripenem] in Canada, and 7–12% in Taiwan).

1	Metronidazole is active against gram-negative anaerobes, including the B. fragilis group; resistance, although rare (<1%), has been reported in both Europe and the United States. Resistance to metronidazole is more common among gram-positive anaerobes, including P. acnes, Actinomyces species, lactobacilli, and anaerobic streptococci. Clindamycin is active against many anaerobes. However, rates of resistance to clindamycin among the B. fragilis group have increased in the United States from 3% in 1982 to 16% in 1996 and 26% in 2000, with rates as high as 40–50% in some series. Resistance to clindamycin among non-Bacteroides anaerobes is much less common (<10%). Tigecycline is active against some anaerobic bacteria, including

1	Tigecycline is active against some anaerobic bacteria, including Peptostreptococcus, Propionibacterium, Prevotella, Fusobacterium, and most Bacteroides species. Its efficacy for treatment of intraabdominal infections was comparable to that of imipenem in two phase 3 double-blind clinical trials. This drug is therefore recommended as single-agent treatment for complicated intraabdominal infections, but resistance (~6%) among Bacteroides and non-Bacteroides species has been reported.

1	Fluoroquinolones such as moxifloxacin have shown potential in the treatment of mixed aerobic-anaerobic infections. A survey in the United States found a 38% rate of resistance to moxifloxacin among the B. fragilis group; in Europe 14–30% of isolates were nonsusceptible to this drug, as were 7–25% of anaerobes isolated from blood cultures in Taiwan. Despite excellent in vitro activity against all clinically important anaerobes, chloramphenicol is less desirable than other active drugs for the treatment of anaerobic infection because of documented clinical failures.

1	Infections Due to Mixed Anaerobic Organisms aUsually needs to be given in combination with aerobic bacterial coverage. For infections originating below the diaphragm, aerobic gram-negative coverage is essential. For infections from an oral source, aerobic gram-positive coverage is added. Metronidazole also is not active against Actinomyces, Propionibacterium, or other gram-positive non-spore-forming bacilli (e.g., Eubacterium, Bifidobacterium) and is unreliable against peptostreptococci. bDespite excellent in vitro activity against all clinically important anaerobes, this drug is less desirable than other active drugs because of documented clinical failures. affected prehominids. This disease most often affects the lungs, although other organs are involved in up to one-third of cases. If properly treated, TB caused by drug-susceptible strains is curable in the vast majority of

1	TB caused by drug-susceptible strains is curable in the vast majority of Mario C. Raviglione cases. If untreated, the disease may be fatal within 5 years in 50–65% of cases. Transmission usually takes place through the airborne spread of droplet nuclei produced by patients with infectious pulmonary TB.

1	Tuberculosis (TB), which is caused by bacteria of the Mycobacterium tuberculosis complex, is one of the oldest diseases known to affect humans and a major cause of death worldwide. Recent population genomic studies suggest that M. tuberculosis may have emerged ~70,000 years ago in Mycobacteria belong to the family Mycobacteriaceae and the orderAfrica and subsequently disseminated along with anatomically modern Actinomycetales. Of the pathogenic species belonging to the M. tuber-humans, expanding globally during the Neolithic Age as human den-culosis complex, which comprises eight distinct subgroups, the mostsity started to increase. Progenitors of M. tuberculosis are likely to have common and important agent of human disease is M. tuberculosis. The complex includes M. bovis (the bovine tubercle bacillus—characteristically resistant to pyrazinamide, once an important cause of TB transmitted by unpasteurized milk, and currently the cause of a small percentage of human cases worldwide),

1	bacillus—characteristically resistant to pyrazinamide, once an important cause of TB transmitted by unpasteurized milk, and currently the cause of a small percentage of human cases worldwide), M. caprae (related to M. bovis), M. africanum (isolated from cases in West, Central, and East Africa), M. microti (the “vole” bacillus, a less virulent and rarely encountered organism), M. pinnipedii (a bacillus infecting seals and sea lions in the Southern Hemisphere and recently isolated from humans), M. mungi (isolated from banded mongooses in southern Africa), M. orygis (described recently in oryxes and other Bovidae in Africa and Asia and a potential cause of infection in humans), and M. canetti (a rare isolate from East African cases that produces unusual smooth colonies on solid media and is considered closely related to a supposed progenitor type).

1	FIGuRE 202-1 Acid-fast bacillus smear showing M. tuberculosis bacilli. (Courtesy of the Centers for Disease Control and Prevention, Atlanta.) M. tuberculosis is a rod-shaped, non-spore-forming, thin aerobic bacterium measuring 0.5 μm by 3 μm. Mycobacteria, including

1	M. tuberculosis, are often neutral on Gram’s staining. However, once stained, the bacilli cannot be decolorized by acid alcohol; this characteristic justifies their classification as acid-fast bacilli (AFB; Fig. 202-1). Acid fastness is due mainly to the organisms’ high content of mycolic acids, long-chain cross-linked fatty acids, and other cell-wall lipids. Microorganisms other than mycobacteria that display some acid fastness include species of Nocardia and Rhodococcus, Legionella micdadei, and the protozoa Isospora and Cryptosporidium. In the mycobacterial cell wall, lipids (e.g., mycolic acids) are linked to underlying arabinogalactan and peptidoglycan. This structure results in very low permeability of the cell wall, thus reducing the effectiveness of most antibiotics. Another molecule in the mycobacterial cell wall, lipoarabinomannan, is involved in the pathogen–host interaction and facilitates the survival of M. tuberculosis within macrophages. The complete genome sequence of

1	in the mycobacterial cell wall, lipoarabinomannan, is involved in the pathogen–host interaction and facilitates the survival of M. tuberculosis within macrophages. The complete genome sequence of M. tuberculosis comprises 4043 genes encoding 3993 proteins and 50 genes encoding RNAs; its high guanine-plus-cytosine content (65.6%) is indicative of an aerobic “lifestyle.” A large proportion of genes are devoted to the production of enzymes involved in cell wall metabolism.

1	More than 5.7 million new cases of TB (all forms, both pulmonary and extrapulmonary) were reported to the World Health Organization (WHO) in 2013; 95% of cases were reported from developing countries. However, because of insufficient case detection and incomplete notification, reported cases may represent only about two-thirds of the total estimated cases. The WHO estimated that 9 million (range, 8.6–9.4 million) new cases of TB occurred worldwide 1103 in 2013, 95% of them in developing countries of Asia (5 million), Africa (2.6 million), the Middle East (0.7 million), and Latin America (0.3 million). It is further estimated that 1.49 million (range, 1.32–1.67 million) deaths from TB, including 0.36 million among people living with HIV infection, occurred in 2013, 96% of them in developing countries. Estimates of TB incidence rates (per 100,000 population) and numbers of TB-related deaths in 2013 are depicted in Figs. 202-2 and 202-3, respectively. During the late 1980s and early

1	countries. Estimates of TB incidence rates (per 100,000 population) and numbers of TB-related deaths in 2013 are depicted in Figs. 202-2 and 202-3, respectively. During the late 1980s and early 1990s, numbers of reported cases of TB increased in industrialized countries. These increases were related largely to immigration from countries with a high incidence of TB; the spread of the HIV epidemic; social problems, such as increased urban poverty, homelessness, and drug abuse; and dismantling of TB services. During the past few years, numbers of reported cases have begun to decline again or have stabilized in most industrialized nations. In the United States, with the re-establishment of stronger control programs, the decline resumed in 1993 and has since been maintained. In 2013, 9582 cases of TB (3.0 cases/100,000 population) were reported to the Centers for Disease Control and Prevention (CDC).

1	In the United States, TB is uncommon among young adults of European descent, who have only rarely been exposed to M. tuberculosis infection during recent decades. In contrast, because of a high risk of transmission in the past, the prevalence of latent

1	M. tuberculosis infection (LTBI) is relatively high among elderly whites. In general, adults ≥65 years of age have the highest incidence rate per capita (4.9 cases/100,000 population in 2013) and children <14 years of age the lowest (0.8 case/100,000 population). Blacks account for the highest proportion of cases (37%; 1257 cases in 2013) among U.S.-born persons. TB in the United States is also a disease of adult members of the HIV-infected population, the foreign-born population (64.6% of all cases in 2013), and disadvantaged/marginalized populations. Of the 6193 cases reported among foreign-born persons in 2013, 37% occurred in persons from the Americas and 32% occurred in persons born in the Western Pacific region. Overall, the highest rates per capita were among Asian Americans (18.7 cases/100,000 population). A total of 536 deaths were caused by TB in the United States in 2011. In Canada in 2013, 1638 TB cases were reported (4.7 cases/100,000 population); 70% (1145) of these

1	cases/100,000 population). A total of 536 deaths were caused by TB in the United States in 2011. In Canada in 2013, 1638 TB cases were reported (4.7 cases/100,000 population); 70% (1145) of these cases occurred in foreign-born persons and 19% (309 cases) occurred in members of the Canadian aboriginal peoples, whose per capita rate is disproportionately high (23.4 cases/100,000 population) with a peak in the Nunavut territory of 143 cases/100,000 population—a rate similar to that in many highly endemic countries. Similarly, in Europe, TB has reemerged as an important public health problem, mainly as a result of cases among immigrants from high-incidence countries and among marginalized populations, often in large urban settings like London; in 2013, 41% of all cases reported from the United Kingdom occurred in London, and the rate per capita (36 cases/100,000 population) was similar to that in some middle-income countries. In most Western European countries, there are more cases

1	United Kingdom occurred in London, and the rate per capita (36 cases/100,000 population) was similar to that in some middle-income countries. In most Western European countries, there are more cases annually among foreign-born than native populations.

1	Recent data on global trends indicate that in 2013 the TB incidence was stable or falling in most regions; this trend began in the early 2000s and appears to have continued, with an average annual decline of 2% globally. This global decrease is explained largely by the simultaneous reduction in TB incidence in sub-Saharan Africa, where rates had risen steeply since the 1980s as a result of the HIV epidemic and the lack of capacity of health systems and services to deal with the problem effectively, and in Eastern Europe, where incidence increased rapidly during the 1990s because of a deterioration in socioeconomic conditions and the health care infrastructure (although, after peaking in 2001, incidence in Eastern Europe has since declined slowly).

1	Of the estimated 9 million new cases of TB in 2013, 13% (1.1 million) were associated with HIV infection, and 78% of these HIV-associated cases occurred in Africa. An estimated 0.36 million persons with HIV-associated TB died in 2013. Furthermore, an estimated 480,000 cases (range, 350,000–610,000) of multidrug-resistant TB (MDR-TB)—a form of the disease caused by bacilli resistant at least to isoniazid and rifampin—occurred in 2013. Only 28% of these FIGuRE 202-3 Estimated numbers of tuberculosis-related deaths in 2013. (See disclaimer in Fig. 202-2. Courtesy of the Global TB Programme, WHO; with permission.) per year 0–9.9 10–19 20–49 50–124 125–299 300–499 ˜500 No data Not applicable

1	FIGuRE 202-2 Estimated tuberculosis (TB) incidence rates (per 100,000 population) in 2013. The designations used and the presentation of material on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization (WHO) concerning the legal status of any country, territory, city, or area or of its authorities or concerning the delimitation of its frontiers or boundaries. Dotted, dashed, and white lines represent approximate border lines for which there may not yet be full agreement. (Courtesy of the Global TB Programme, WHO; with permission.) cases were diagnosed because of a lack of culture and drug-suscep-China, India, the Russian Federation, Pakistan, and Ukraine. Since tibility testing capacity in most settings worldwide. The countries of 2006, 100 countries, including the United States, have reported cases the former Soviet Union have reported the highest proportions of of extensively drug-resistant TB (XDR-TB), in which MDR-TB is MDR

1	100 countries, including the United States, have reported cases the former Soviet Union have reported the highest proportions of of extensively drug-resistant TB (XDR-TB), in which MDR-TB is MDR disease among new TB cases (up to 35–40% in some regions compounded by additional resistance to the most powerful second-of Russia and Belarus). Overall, 60% of all MDR-TB cases occur in line anti-TB drugs (fluoroquinolones and at least one of the injectable

1	Number of deaths drugs amikacin, kanamycin, and capreomycin). Up to 10% of the MDR-TB cases worldwide may actually be XDR-TB, but the vast majority of XDR-TB cases remain undiagnosed because reliable methods for drug susceptibility testing are lacking and laboratory capacity is limited. Lately, cases deemed resistant to all anti-TB drugs have been reported from countries such as India, Italy, and Iran; however, this information must be interpreted with caution because drug susceptibility testing for several second-line drugs is neither accurate nor reproducible.

1	M. tuberculosis is most commonly transmitted from a person with infectious pulmonary TB by droplet nuclei, which are aerosolized by coughing, sneezing, or speaking. The tiny droplets dry rapidly; the smallest (<5–10 μm in diameter) may remain suspended in the air for several hours and may reach the terminal air passages when inhaled. There may be as many as 3000 infectious nuclei per cough. Other routes of transmission of tubercle bacilli (e.g., through the skin or the placenta) are uncommon and of no epidemiologic significance. The probability of contact with a person who has an infectious form of TB, the intimacy and duration of that contact, the degree of infectiousness of the case, and the shared environment in which the contact takes place are all important determinants of the likelihood of transmission. Several studies of close-contact situations have clearly demonstrated that TB patients whose sputum contains AFB visible by microscopy (sputum smear–positive cases) are the most

1	of transmission. Several studies of close-contact situations have clearly demonstrated that TB patients whose sputum contains AFB visible by microscopy (sputum smear–positive cases) are the most likely to transmit the infection. The most infectious patients have cavitary pulmonary disease or, much less commonly, laryngeal TB and produce sputum containing as many as 105–107 AFB/mL. Patients with sputum smear–negative/culturepositive TB are less infectious, although they have been responsible for up to 20% of transmission in some studies in the United States. Those with culture-negative pulmonary TB and extrapulmonary TB are essentially noninfectious. Because persons with both HIV infection and TB are less likely to have cavitations, they may be less infectious than persons without HIV co-infection. Crowding in poorly ventilated rooms is one of the most important factors in the transmission of tubercle bacilli because it increases the intensity of contact with a case.

1	The risk of acquiring M. tuberculosis infection is determined mainly by exogenous factors. Because of delays in seeking care and in making a diagnosis, it is generally estimated that, in high-prevalence settings, up to 20 contacts may be infected by each AFB-positive case before the index case is diagnosed.

1	Unlike the risk of acquiring infection with M. tuberculosis, the risk of developing disease after being infected depends largely on endogenous factors, such as the individual’s innate immunologic and nonimmunologic defenses and the level at which the individual’s cell-mediated immunity (CMI) is functioning. Clinical illness directly following infection is classified as primary TB and is common among children in the first few years of life and among immunocompromised persons. Although primary TB may be severe and disseminated, it generally is not associated with high-level transmissibility. When infection is acquired later in life, the chance is greater that the mature immune system will contain it at least temporarily. Bacilli, however, may persist for years before reactivating to produce secondary (or postprimary) TB, which, because of frequent cavitation, is more often infectious than is primary disease. Overall, it is estimated that up to 10% of infected persons will eventually

1	secondary (or postprimary) TB, which, because of frequent cavitation, is more often infectious than is primary disease. Overall, it is estimated that up to 10% of infected persons will eventually develop active TB in their lifetime—half of them during the first 18 months after infection. The risk is much higher among HIV-infected persons. Reinfection of a previously infected individual, which is common in areas with high rates of TB transmission, may also favor the development of disease. At the height of the TB resurgence in the United States in the early 1990s, molecular typing and comparison of strains of M. tuberculosis suggested that up to one-third of cases of active TB in some inner-city communities were due to recent transmission rather than to reactivation of old latent infection. Age is an important determinant of the risk of disease after infection. Among infected persons, the incidence of TB is highest during late adolescence and early adulthood; the reasons are unclear.

1	Age is an important determinant of the risk of disease after infection. Among infected persons, the incidence of TB is highest during late adolescence and early adulthood; the reasons are unclear. The incidence among women peaks at 25–34 years of age. In this age group, rates among women may be higher than those among men, whereas at older ages the opposite is true. The risk increases in the elderly, possibly because of waning immunity and comorbidity.

1	A variety of diseases and conditions favor the development of active TB (Table 202-1). In absolute terms, the most potent risk factor for TB among infected individuals is clearly HIV co-infection, which suppresses cellular immunity. The risk that LTBI will proceed to active disease is directly related to the patient’s degree of immunosuppression. In a study of HIV-infected, tuberculin skin test (TST)–positive persons, this risk varied from 2.6 to 13.3 cases/100 person-years and increased as the CD4+ T cell count decreased.

1	Studies conducted in various countries before the advent of chemotherapy showed that untreated TB is often fatal. About one-third of patients died within 1 year after diagnosis, and more than 50% died within 5 years. The 5-year mortality rate among sputum smear–positive cases was 65%. Of the survivors at 5 years, ~60% had undergone spontaneous remission, while the remainder were still excreting tubercle bacilli. With effective, timely, and proper chemotherapy, patients have a very high chance of being cured. However, improper use of anti-TB drugs, while reducing mortality rates, may also result in large numbers of chronic infectious cases, often with drug-resistant bacilli.

1	The interaction of M. tuberculosis with the human host begins when droplet nuclei containing viable microorganisms propelled into the air by infectious patients are inhaled by a close bystander. Although the majority of inhaled bacilli are trapped in the upper airways and expelled by ciliated mucosal cells, a fraction (usually <10%) reach the alveoli, a unique immunoregulatory environment. There, alveolar macrophages that have not yet been activated (prototypic alternatively activated macrophages) phagocytose the bacilli. Adhesion of mycobacteria to macrophages results largely from binding of the bacterial cell wall to a variety of macrophage cell-surface molecules, including complement receptors, the mannose receptor, the immunoglobulin GFcγ receptor, and type A scavenger receptors. Phagocytosis is enhanced by complement activation leading to opsonization of bacilli with C3 activation products such as C3b and C3bi. (Bacilli are resistant to complement-mediated lysis.) Binding of

1	Phagocytosis is enhanced by complement activation leading to opsonization of bacilli with C3 activation products such as C3b and C3bi. (Bacilli are resistant to complement-mediated lysis.) Binding of certain receptors, such as the mannose receptor, regulates 1106 postphagocytic events such as phagosome–lysosome fusion and inflammatory cytokine production. After a phagosome forms, the survival of

1	M. tuberculosis within it seems to depend in part on reduced acidification due to lack of assembly of a complete vesicular proton-adenosine triphosphatase. A complex series of events is generated by the bacterial cell-wall lipoglycan lipoarabinomannan (ManLAM). ManLAM inhibits the intracellular increase of Ca2+. Thus, the Ca2+/calmodulin pathway (leading to phagosome–lysosome fusion) is impaired, and the bacilli survive within the phagosomes. The M. tuberculosis phagosome has been found to inhibit the production of phosphatidylinositol 3phosphate (PI3P). Normally, PI3P earmarks phagosomes for membrane sorting and maturation, including phagolysosome formation, which would destroy the bacteria. Bacterial factors have also been found to block the host defense of autophagy, in which the cell sequesters the phagosome in a double-membrane vesicle (autophagosome) that is destined to fuse with lysosomes. If the bacilli are successful in arresting phagosome maturation, then replication begins

1	sequesters the phagosome in a double-membrane vesicle (autophagosome) that is destined to fuse with lysosomes. If the bacilli are successful in arresting phagosome maturation, then replication begins and the macrophage eventually ruptures and releases its bacillary contents. Other uninfected phagocytic cells are then recruited to continue the infection cycle by ingesting dying macrophages and their bacillary content, thus in turn becoming infected themselves and expanding the infection.

1	M. tuberculosis must be viewed as a complex formed by a multitude of strains that differ in virulence and are capable of producing a variety of manifestations of disease. Since the elucidation of the

1	M. tuberculosis genome in 1998, large mutant collections have been generated, and many bacterial genes that contribute to M. tuberculosis virulence have been found. Different patterns of virulence defects have been defined in various animal models—predominantly mice but also guinea pigs, rabbits, and nonhuman primates. The katG gene encodes for a catalase/peroxidase enzyme that protects against oxidative stress and is required for isoniazid activation and subsequent bactericidal activity. Region of difference 1 (RD1) is a 9.5-kb locus that encodes two key small protein antigens—early secretory antigen-6 (ESAT-6) and culture filtrate protein-10 (CFP-10)—as well as a putative secretion apparatus that may facilitate their egress; the absence of this locus in the vaccine strain M. bovis bacille Calmette-Guérin (BCG) has been shown to be a key attenuating mutation. The validity of a recent observation in M. marinum needs to be confirmed in M. tuberculosis; in M. marinum, a mutation in the

1	Calmette-Guérin (BCG) has been shown to be a key attenuating mutation. The validity of a recent observation in M. marinum needs to be confirmed in M. tuberculosis; in M. marinum, a mutation in the RD1 virulence locus encoding the ESX1 secretion system impairs the capacity of apoptotic macrophages to recruit uninfected cells for further rounds of infection. The results are less replication and fewer new granulomas. Mutants lacking key enzymes of bacterial biosynthesis become auxotrophic for the missing substrate and often are totally unable to proliferate in animals; these include the leuCD and panCD mutants, which require leucine and pantothenic acid, respectively. The isocitrate lyase gene icl1 encodes a key step in the glyoxylate shunt that facilitates bacterial growth on fatty acid substrates; this gene is required for longterm persistence of M. tuberculosis infection in mice with chronic TB. M. tuberculosis mutants in regulatory genes such as sigma factor C and sigma factor H

1	substrates; this gene is required for longterm persistence of M. tuberculosis infection in mice with chronic TB. M. tuberculosis mutants in regulatory genes such as sigma factor C and sigma factor H (sigC and sigH) are associated with normal bacterial growth in mice, but they fail to elicit full tissue pathology. Finally, the mycobacterial protein CarD (expressed by the carD gene) seems essential for the control of rRNA transcription that is required for replication and persistence in the host cell. Its loss exposes mycobacteria to oxidative stress, starvation, DNA damage, and ultimately sensitivity to killing by a variety of host mutagens and defensive mechanisms.

1	Several observations suggest that genetic factors play a key role in innate nonimmune resistance to infection with M. tuberculosis and the development of disease. The existence of this resistance, which is polygenic in nature, is suggested by the differing degrees of susceptibility to TB in different populations. In mice, a gene called Nramp1 (natural resistance–associated macrophage protein 1) plays a regulatory role in resistance/susceptibility to mycobacteria. The human homologue NRAMP1, which maps to chromosome 2q, may play a role in determining susceptibility to TB, as is suggested by a study among West Africans. Studies of mouse genetics identified a novel host resistance gene, ipr1, which is encoded within the sst1 locus; ipr1 encodes an interferon (IFN)–inducible nuclear protein that interacts with other nuclear proteins in macrophages primed with IFNs or infected by M. tuberculosis. In addition, polymorphisms in multiple genes, such as those encoding for various major

1	that interacts with other nuclear proteins in macrophages primed with IFNs or infected by M. tuberculosis. In addition, polymorphisms in multiple genes, such as those encoding for various major histocompatibility complex (MHC) alleles, IFN-γ, T cell growth factor β, interleukin (IL) 10, mannose-binding protein, IFN-γ receptor, Toll-like receptor 2, vitamin D receptor, and IL-1, have been associated with susceptibility to TB.

1	THE HOST RESPONSE, GRANuLOMA FORMATION, AND “LATENCY”

1	In the initial stage of host–bacterium interaction, prior to the onset of an acquired CMI response, M. tuberculosis disseminates widely through the lymph vessels, spreading to other sites in the lungs and other organs, and undergoes a period of extensive growth within naïve unactivated macrophages; additional naïve macrophages are recruited to the early granuloma. Studies suggest that M. tuberculosis uses specific virulence mechanisms to subvert host cellular signaling and to elicit an early regulated proinflammatory response that promotes granuloma expansion and bacterial growth during this key early phase. A study of M. marinum infection in zebrafish has delineated one molecular mechanism by which mycobacteria induce granuloma formation. The mycobacterial protein ESAT-6 induces secretion of matrix metalloproteinase 9 (MMP9) by nearby epithelial cells that are in contact with infected macrophages. MMP9 in turn stimulates recruitment of naïve macrophages, thus inducing granuloma

1	of matrix metalloproteinase 9 (MMP9) by nearby epithelial cells that are in contact with infected macrophages. MMP9 in turn stimulates recruitment of naïve macrophages, thus inducing granuloma maturation and bacterial growth. Disruption of MMP9 function results in reduced bacterial growth. Another study has shown that M. tuberculosis–derived cyclic AMP is secreted from the phagosome into host macrophages, subverting the cell’s signal transduction pathways and stimulating an elevation in the secretion of tumor necrosis factor α (TNF-α) as well as further proinflammatory cell recruitment. Ultimately, the chemoattractants and bacterial products released during the repeated rounds of cell lysis and infection of newly arriving macrophages enable dendritic cells to access bacilli; these cells migrate to the draining lymph nodes and present mycobacterial antigens to T lymphocytes. At this point, the development of CMI and humoral immunity begins. These initial stages of infection are usually

1	to the draining lymph nodes and present mycobacterial antigens to T lymphocytes. At this point, the development of CMI and humoral immunity begins. These initial stages of infection are usually asymptomatic.

1	About 2–4 weeks after infection, two host responses to M. tuberculosis develop: a macrophage-activating CMI response and a tissue-damaging response. The macrophage-activating response is a T cell–mediated phenomenon resulting in the activation of macrophages that are capable of killing and digesting tubercle bacilli. The tissue-damaging response is the result of a delayed-type hypersensitivity (DTH) reaction to various bacillary antigens; it destroys unactivated macrophages that contain multiplying bacilli but also causes caseous necrosis of the involved tissues (see below). Although both of these responses can inhibit mycobacterial growth, it is the balance between the two that determines the forms of TB that will develop subsequently. With the development of specific immunity and the accumulation of large numbers of activated macrophages at the site of the primary lesion, granulomatous lesions (tubercles) are formed. These lesions consist of accumulations of lymphocytes and

1	the accumulation of large numbers of activated macrophages at the site of the primary lesion, granulomatous lesions (tubercles) are formed. These lesions consist of accumulations of lymphocytes and activated macrophages that evolve toward epithelioid and giant cell morphologies. Initially, the tissue-damaging response can limit mycobacterial growth within macrophages. As stated above, this response, mediated by various bacterial products, not only destroys macrophages but also produces early solid necrosis in the center of the tubercle. Although M. tuberculosis can survive, its growth is inhibited within this necrotic environment by low oxygen tension and low pH. At this point, some lesions may heal by fibrosis, with subsequent calcification, whereas inflammation and necrosis occur in other lesions. Some observations have challenged the traditional view that any encounter between mycobacteria and macrophages results in chronic infection. It is possible that an immune response capable

1	lesions. Some observations have challenged the traditional view that any encounter between mycobacteria and macrophages results in chronic infection. It is possible that an immune response capable of eradicating early infection may sometimes develop as a consequence, for instance, of disabling mutations in mycobacterial genomes rendering their replication ineffective. Individual granulomas that are formed during this phase of infection can vary in size and cell composition; some can contain the spread of mycobacteria, while others cannot. LTBI ensues as a result of this dynamic balance between the microorganism and the host. According to recent developments, latency may not be an accurate term because bacilli may remain active during this “latent” stage, forming biofilms in necrotic areas within which they temporarily hide. Thus, the term persister is probably more accurate to indicate the behavior of the bacilli in this phase. It is important to recognize that latent infection and

1	within which they temporarily hide. Thus, the term persister is probably more accurate to indicate the behavior of the bacilli in this phase. It is important to recognize that latent infection and disease represent not a binary state but rather a continuum along which infection will eventually move in the direction of full containment or disease. The ability to predict, through systemic biomarkers, which affected individuals will progress toward disease would be of immense value in devising prophylactic interventions.

1	CMI is critical at this early stage. In the majority of infected individuals, local macrophages are activated when bacillary antigens processed by macrophages stimulate T lymphocytes to release a variety of lymphokines. These activated macrophages aggregate around the lesion’s center and effectively neutralize tubercle bacilli without causing further tissue destruction. In the central part of the lesion, the necrotic material resembles soft cheese (caseous necrosis)—a phenomenon that may also be observed in other conditions, such as neoplasms. Even when healing takes place, viable bacilli may remain dormant within macrophages or in the necrotic material for many years. These “healed” lesions in the lung parenchyma and hilar lymph nodes may later undergo calcification.

1	In a minority of cases, the macrophage-activating response is weak, and mycobacterial growth can be inhibited only by intensified DTH reactions, which lead to lung tissue destruction. The lesion tends to enlarge further, and the surrounding tissue is progressively damaged. At the center of the lesion, the caseous material liquefies. Bronchial walls and blood vessels are invaded and destroyed, and cavities are formed. The liquefied caseous material, containing large numbers of bacilli, is drained through bronchi. Within the cavity, tubercle bacilli multiply, spill into the airways, and are discharged into the environment through expiratory maneuvers such as coughing and talking. In the early stages of infection, bacilli are usually transported by macrophages to regional lymph nodes, from which they gain access to the central venous return; from there they reseed the lungs and may also disseminate beyond the pulmonary vasculature throughout the body via the systemic circulation. The

1	which they gain access to the central venous return; from there they reseed the lungs and may also disseminate beyond the pulmonary vasculature throughout the body via the systemic circulation. The resulting extrapulmonary lesions may undergo the same evolution as those in the lungs, although most tend to heal. In young children with poor natural immunity, hematogenous dissemination may result in highly fatal miliary TB or tuberculous meningitis.

1	While CMI confers partial protection against M. tuberculosis, humoral immunity plays a less well-defined role in protection (although evidence is accumulating on the existence of antibodies to lipoarabinomannan, which may prevent dissemination of infection in children). In the case of CMI, two types of cells are essential: macrophages, which directly phagocytose tubercle bacilli, and T cells (mainly CD4+ T lymphocytes), which induce protection through the production of cytokines, especially IFN-γ. After infection with M. tuberculosis, alveolar macrophages secrete various cytokines responsible for a number of events (e.g., the formation of granulomas) as well as systemic effects (e.g., fever and weight loss). However, alternatively activated alveolar macrophages may be particularly susceptible to M. tuberculosis growth early on, given their more limited proinflammatory and bactericidal activity, which is related in part to being bathed in surfactant. New monocytes and macrophages

1	to M. tuberculosis growth early on, given their more limited proinflammatory and bactericidal activity, which is related in part to being bathed in surfactant. New monocytes and macrophages attracted to the site are key components of the immune response. Their primary mechanism is probably related to production of oxidants (such as reactive oxygen intermediates or nitric oxide) that have antimycobacterial activity and increase the synthesis of cytokines such as TNF-α and IL-1, which in turn regulate the release of reactive oxygen intermediates and reactive nitrogen intermediates. In addition, macrophages can undergo apoptosis—a defensive mechanism to prevent release of cytokines and bacilli via their sequestration in the apoptotic cell. Recent work also describes the involvement of neutrophils in the host response, although the timing 1107 of their appearance and their effectiveness remain uncertain.

1	Alveolar macrophages, monocytes, and dendritic cells are also critical in processing and presenting antigens to T lymphocytes, primarily CD4+ and CD8+ T cells; the result is the activation and proliferation of CD4+ T lymphocytes, which are crucial to the host’s defense against

1	M. tuberculosis. Qualitative and quantitative defects of CD4+ T cells explain the inability of HIV-infected individuals to contain mycobacterial proliferation. Activated CD4+ T lymphocytes can differentiate into cytokine-producing TH1 or TH2 cells. TH1 cells produce IFN-γ—an activator of macrophages and monocytes—and IL-2. TH2 cells produce IL-4, IL-5, IL-10, and IL-13 and may also promote humoral immunity. The interplay of these various cytokines and their cross-regulation determine the host’s response. The role of cytokines in promoting intracellular killing of mycobacteria, however, has not been entirely elucidated. IFN-γ may induce the generation of reactive nitrogen intermediates and regulate genes involved in bactericidal effects. TNF-α also seems to be important. Observations made originally in transgenic knockout mice and more recently in humans suggest that other T cell subsets, especially CD8+ T cells, may play an important role. CD8+ T cells have been associated with

1	made originally in transgenic knockout mice and more recently in humans suggest that other T cell subsets, especially CD8+ T cells, may play an important role. CD8+ T cells have been associated with protective activities via cytotoxic responses and lysis of infected cells as well as with production of IFN-γ and TNF-α. Finally, natural killer cells act as co-regulators of CD8+ T cell lytic activities, and γδ T cells are increasingly thought to be involved in protective responses in humans.

1	Lipids have been involved in mycobacterial recognition by the innate immune system, and lipoproteins (such as 19-kDa lipoprotein) have been proven to trigger potent signals through Toll-like receptors present in blood dendritic cells. M. tuberculosis possesses various protein antigens. Some are present in the cytoplasm and cell wall; others are secreted. That the latter are more important in eliciting a T lymphocyte response is suggested by experiments documenting the appearance of protective immunity in animals after immunization with live, protein-secreting mycobacteria. Among the antigens that may play a protective role are the 30-kDa (or 85B) and ESAT-6 antigens. Protective immunity is probably the result of reactivity to many different mycobacterial antigens. These antigens are being incorporated into newly designed vaccines on various platforms.

1	Coincident with the appearance of immunity, DTH to M. tuberculosis develops. This reactivity is the basis of the TST, which is used primarily for the detection of M. tuberculosis infection in persons without symptoms. The cellular mechanisms responsible for TST reactivity are related mainly to previously sensitized CD4+ T lymphocytes, which are attracted to the skin-test site. There, they proliferate and produce cytokines. Although DTH is associated with protective immunity (TST-positive persons are less susceptible to a new M. tuberculosis infection than TST-negative persons), it by no means guarantees protection against reactivation. In fact, cases of active TB are often accompanied by strongly positive skin-test reactions. There is also evidence of reinfection with a new strain of M. tuberculosis in patients previously treated for active disease. This evidence underscores the fact that previous latent or active TB may not confer fully protective immunity.

1	TB is classified as pulmonary, extrapulmonary, or both. Depending on several factors linked to different populations and bacterial strains, extrapulmonary TB may occur in 10–40% of patients. Furthermore, up to two-thirds of HIV-infected patients with TB may have both pulmonary and extrapulmonary TB or extrapulmonary TB alone. Pulmonary TB is conventionally categorized as primary or postprimary (adult-type, secondary). This distinction has been challenged 1108 by molecular evidence from TB-endemic areas indicating that a large percentage of cases of adult pulmonary TB result from recent infection (either primary infection or reinfection) and not from reactivation.

1	Primary Disease Primary pulmonary TB occurs soon after the initial infection with tubercle bacilli. It may be asymptomatic or may present with fever and occasionally pleuritic chest pain. In areas of high TB transmission, this form of disease is often seen in children. Because most inspired air is distributed to the middle and lower lung zones, these areas are most commonly involved in primary TB. The lesion forming after initial infection (Ghon focus) is usually peripheral and accompanied by transient hilar or paratracheal lymphadenopathy, which may or may not be visible on standard chest radiography (Fig. 202-4). Some patients develop erythema nodosum on the legs (see Fig. 25e-40) or phlyctenular conjunctivitis. In the majority of cases, the lesion heals spontaneously and becomes evident only as a small calcified nodule. Pleural reaction overlying a subpleural focus is also common. The Ghon focus, with or without overlying pleural reaction, thickening, and regional lymphadenopathy,

1	only as a small calcified nodule. Pleural reaction overlying a subpleural focus is also common. The Ghon focus, with or without overlying pleural reaction, thickening, and regional lymphadenopathy, is referred to as the Ghon complex.

1	In young children with immature CMI and in persons with impaired immunity (e.g., those with malnutrition or HIV infection), primary pulmonary TB may progress rapidly to clinical illness. The initial lesion increases in size and can evolve in different ways. Pleural effusion, which is found in up to two-thirds of cases, results from the penetration of bacilli into the pleural space from an adjacent subpleural focus. In severe cases, the primary site rapidly enlarges, its central portion undergoes necrosis, and cavitation develops (progressive primary TB). TB in young children is almost invariably accompanied by hilar or paratracheal lymphadenopathy due to the spread of bacilli from the lung parenchyma through lymphatic vessels. Enlarged lymph nodes may compress bronchi, causing total obstruction with distal collapse, partial obstruction with large-airway wheezing, or a ball-valve effect with segmental/lobar hyperinflation. Lymph nodes may also rupture into the airway with development

1	with distal collapse, partial obstruction with large-airway wheezing, or a ball-valve effect with segmental/lobar hyperinflation. Lymph nodes may also rupture into the airway with development of pneumonia, often including areas of necrosis and cavitation, distal to the obstruction. Bronchiectasis (Chap. 312) may develop in any segment/lobe damaged by progressive caseating pneumonia. Occult hematogenous dissemination commonly follows primary infection. However, in the absence of a sufficient acquired immune response, which usually contains the infection, disseminated or miliary disease may result (Fig. 202-5). Small granulomatous lesions develop in multiple organs and may cause locally progressive disease or result in tuberculous meningitis; this is a particular concern in very young children and immunocompromised persons (e.g., patients with HIV infection).

1	FIGuRE 202-4 Chest radiograph showing right hilar lymph node enlargement with infiltration into the surrounding lung tissue in a child with primary tuberculosis. (Courtesy of Prof. Robert Gie, Department of Paediatrics and Child Health, Stellenbosch University, South Africa; with permission.) FIGuRE 202-5 Chest radiograph showing bilateral miliary (millet-sized) infiltrates in a child. (Courtesy of Prof. Robert Gie, Department of Paediatrics and Child Health, Stellenbosch University, South Africa; with permission.)

1	Postprimary (Adult-Type) Disease Also referred to as reactivation or secondary TB, postprimary TB is probably most accurately termed adult-type TB because it may result from endogenous reactivation of distant LTBI or recent infection (primary infection or reinfection). It is usually localized to the apical and posterior segments of the upper lobes, where the substantially higher mean oxygen tension (compared with that in the lower zones) favors mycobacterial growth. The superior segments of the lower lobes are also more frequently involved. The extent of lung parenchymal involvement varies greatly, from small infiltrates to extensive cavitary disease. With cavity formation, liquefied necrotic contents are ultimately discharged into the airways and may undergo bronchogenic spread, resulting in satellite lesions within the lungs that may in turn undergo cavitation (Figs. 202-6 and 202-7). Massive involvement of pulmonary segments or lobes, with coalescence of lesions, produces caseating

1	in satellite lesions within the lungs that may in turn undergo cavitation (Figs. 202-6 and 202-7). Massive involvement of pulmonary segments or lobes, with coalescence of lesions, produces caseating pneumonia. While up to one-third of untreated patients reportedly succumb to severe pulmonary TB within a few months after onset (the classic “galloping consumption” of the past), others may undergo a process of spontaneous remission or proceed along a chronic, progressively debilitating course (“consumption” or phthisis). Under these circumstances, some pulmonary lesions become fibrotic and may later calcify, but cavities persist in other parts of the lungs. Individuals with such chronic disease continue to discharge tubercle bacilli into the environment. Most patients respond to treatment, with defervescence, decreasing cough, weight gain, and a general improvement in well-being within several weeks.

1	Early in the course of disease, symptoms and signs are often nonspecific and insidious, consisting mainly of diurnal fever and night sweats due to defervescence, weight loss, anorexia, general malaise, and weakness. However, in up to 90% of cases, cough eventually develops—often initially nonproductive and limited to the morning and subsequently accompanied by the production of purulent sputum, sometimes with blood streaking. Hemoptysis develops in 20–30% of cases, and massive hemoptysis may ensue as a consequence of the erosion of a blood vessel in the wall of a cavity. Hemoptysis, however, may also result from rupture of a dilated vessel in a cavity (Rasmussen’s aneurysm) or from aspergilloma formation in an old cavity. Pleuritic chest pain sometimes develops in patients with subpleural parenchymal lesions or pleural disease. Extensive disease may produce dyspnea and, in rare instances, adult respiratory distress syndrome. Physical findings are of limited use in pulmonary TB. Many

1	parenchymal lesions or pleural disease. Extensive disease may produce dyspnea and, in rare instances, adult respiratory distress syndrome. Physical findings are of limited use in pulmonary TB. Many patients have no abnormalities detectable by chest examination, whereas others have detectable rales in the involved areas during inspiration, especially after coughing. Occasionally, rhonchi due to partial bronchial obstruction and classic amphoric breath sounds in areas with large cavities may be heard. Systemic features include fever (often low-grade and intermittent) in up to 80% of cases and wasting. Absence of fever, however, does not exclude TB. In some cases, pallor and finger clubbing develop. The most common hematologic findings are mild anemia, leukocytosis, and thrombocytosis with a slightly elevated erythrocyte sedimentation rate and/or C-reactive protein level. None of these findings is consistent or sufficiently accurate for diagnostic purposes.

1	FIGuRE 202-6 Chest radiograph showing a right-upper-lobe infiltrate and a cavity with an air-fluid level in a patient with active tuberculosis. (Courtesy of Dr. Andrea Gori, Department of Infectious Diseases, S. Paolo University Hospital, Milan, Italy; with permission.) Hyponatremia due to the syndrome of inappropriate secretion of 1109 antidiuretic hormone has also been reported. In order of frequency, the extrapulmonary sites most commonly involved in TB are the lymph nodes, pleura, genitourinary tract, bones and joints, meninges, peritoneum, and pericardium. However, virtually all organ systems may be affected. As a result of hematogenous dissemination in HIV-infected individuals, extrapulmonary TB is seen more commonly today than in the past in settings of high HIV prevalence.

1	Lymph Node TB (Tuberculous Lymphadenitis) The most common presentation of extrapulmonary TB in both HIV-seronegative and HIV-infected patients (35% of cases worldwide and more than 40% of cases in the United States in recent series), lymph node disease is particularly frequent among HIV-infected patients and among children (Fig. 202-8). In the United States, besides children, women (particularly non-Caucasians) seem to be especially susceptible. Once caused mainly by M. bovis, tuberculous lymphadenitis today is due largely to M. tuberculosis. Lymph node TB presents as painless swelling of the lymph nodes, most commonly at posterior cervical and supraclavicular sites (a condition historically referred to as scrofula). Lymph nodes are usually discrete in early disease but develop into a matted nontender mass over time and may result in a fistulous tract draining caseous material. Associated pulmonary disease is present in fewer than 50% of cases, and systemic symptoms are uncommon

1	a matted nontender mass over time and may result in a fistulous tract draining caseous material. Associated pulmonary disease is present in fewer than 50% of cases, and systemic symptoms are uncommon except in HIV-infected patients. The diagnosis is established by fine-needle aspiration biopsy (with a yield of up to 80%) or surgical excision biopsy. Bacteriologic confirmation is achieved in the vast majority of cases, granulomatous lesions with or without visible AFBs are typically seen, and cultures are positive in 70–80% of cases. Among HIV-infected patients, granulomas are less well organized and are frequently absent entirely, but bacterial loads are heavier than in HIVseronegative patients, with higher yields from microscopy and culture. Differential diagnosis includes a variety of infectious conditions, neoplastic diseases such as lymphomas or metastatic carcinomas, and rare disorders like Kikuchi’s disease (necrotizing histiocytic lymphadenitis), Kimura’s disease, and

1	of infectious conditions, neoplastic diseases such as lymphomas or metastatic carcinomas, and rare disorders like Kikuchi’s disease (necrotizing histiocytic lymphadenitis), Kimura’s disease, and Castleman’s disease.

1	Pleural TB Involvement of the pleura accounts for ~20% of extra-pulmonary cases in the United States and elsewhere. Isolated pleural effusion usually reflects recent primary infection, and the collection of fluid in the pleural space represents a hypersensitivity response to mycobacterial antigens. Pleural disease may also result from contiguous parenchymal spread, as in many cases of pleurisy accompanying FIGuRE 202-7 CT scan showing a large cavity in the right lung of a patient with active tuberculosis. (Courtesy of Dr. Elisa Busi Rizzi, National Institute for Infectious Diseases, Spallanzani Hospital, Rome, Italy; with permission.)

1	FIGuRE 202-8 Tuberculous lymphadenitis affecting the cervical lymph nodes in a 2-year-old child from Malawi. (Courtesy of Prof. S. Graham, Centre for International Child Health, University of Melbourne, Australia; with permission.) 1110 postprimary disease. Depending on the extent of reactivity, the effusion may be small, remain unnoticed, and resolve spontaneously or may be sufficiently large to cause symptoms such as fever, pleuritic chest pain, and dyspnea. Physical findings are those of pleural effusion: dullness to percussion and absence of breath sounds. A chest radiograph reveals the effusion and, in up to one-third of cases, also shows a parenchymal lesion. Thoracentesis is required to ascertain the nature of the effusion and to differentiate it from manifestations of other etiologies. The fluid is straw colored and at times hemorrhagic; it is an exudate with a protein concentration >50% of that in serum (usually ~4–6 g/dL), a normal to low glucose concentration, a pH of ~7.3

1	The fluid is straw colored and at times hemorrhagic; it is an exudate with a protein concentration >50% of that in serum (usually ~4–6 g/dL), a normal to low glucose concentration, a pH of ~7.3 (occasionally <7.2), and detectable white blood cells (usually 500–6000/μL). Neutrophils may predominate in the early stage, but lymphocyte predominance is the typical finding later. Mesothelial cells are generally rare or absent. AFB are rarely seen on direct smear, and cultures often may be falsely negative for M. tuberculosis; positive cultures are more common among postprimary cases. Determination of the pleural concentration of adenosine deaminase (ADA) may be a useful screening test, and TB may be excluded if the value is very low. Lysozyme is also present in the pleural effusion. Measurement of IFN-γ, either directly or through stimulation of sensitized T cells with mycobacterial antigens, can be helpful. Needle biopsy of the pleura is often required for diagnosis and is recommended over

1	of IFN-γ, either directly or through stimulation of sensitized T cells with mycobacterial antigens, can be helpful. Needle biopsy of the pleura is often required for diagnosis and is recommended over pleural fluid; it reveals granulomas and/or yields a positive culture in up to 80% of cases. Pleural biopsy can yield a positive result in ~75% of cases when real-time automated nucleic acid amplification is used (the Xpert® MTB/RIF assay [Cepheid, Sunnyvale, CA]; see “Nucleic Acid Amplification Technology,” below), although pleural fluid testing with this assay is not recommended because of low sensitivity. This form of pleural TB responds rapidly to chemotherapy and may resolve spontaneously. Concurrent glucocorticoid administration may reduce the duration of fever and/or chest pain but is not of proven benefit. Tuberculous empyema is a less common complication of pulmonary TB. It is usually the result of the rupture of a cavity, with spillage of a large number of organisms into the

1	is not of proven benefit. Tuberculous empyema is a less common complication of pulmonary TB. It is usually the result of the rupture of a cavity, with spillage of a large number of organisms into the pleural space. This process may create a bronchopleural fistula with evident air in the pleural space. A chest radiograph shows hydropneumothorax with an air-fluid level. The pleural fluid is purulent and thick and contains large numbers of lymphocytes. Acid-fast smears and mycobacterial cultures are often positive. Surgical drainage is usually required as an adjunct to chemotherapy. Tuberculous empyema may result in severe pleural fibrosis and restrictive lung disease. Removal of the thickened visceral pleura (decortication) is occasionally necessary to improve lung function.

1	TB of the upper Airways Nearly always a complication of advanced cavitary pulmonary TB, TB of the upper airways may involve the larynx, pharynx, and epiglottis. Symptoms include hoarseness, dysphonia, and dysphagia in addition to chronic productive cough. Findings depend on the site of involvement, and ulcerations may be seen on laryngoscopy. Acid-fast smear of the sputum is often positive, but biopsy may be necessary in some cases to establish the diagnosis. Carcinoma of the larynx may have similar features but is usually painless.

1	Genitourinary TB Genitourinary TB, which accounts for ~10–15% of all extrapulmonary cases in the United States and elsewhere, may involve any portion of the genitourinary tract. Local symptoms predominate, and up to 75% of patients have chest radiographic abnormalities suggesting previous or concomitant pulmonary disease. Urinary frequency, dysuria, nocturia, hematuria, and flank or abdominal pain are common presentations. However, patients may be asymptomatic and their disease discovered only after severe destructive lesions of the kidneys have developed. Urinalysis gives abnormal results in 90% of cases, revealing pyuria and hematuria. The documentation of culture-negative pyuria in acidic urine should raise the suspicion of TB. IV pyelography, abdominal computed tomography (CT), or magnetic resonance imaging (MRI) (Fig. 202-9) may show deformities and obstructions; calcifications and ureteral strictures are suggestive findings. Culture of three morning urine specimens yields a

1	magnetic resonance imaging (MRI) (Fig. 202-9) may show deformities and obstructions; calcifications and ureteral strictures are suggestive findings. Culture of three morning urine specimens yields a definitive diagnosis in nearly 90% of cases. Severe ureteral strictures may lead to hydronephrosis and renal damage. Genital TB is diagnosed more commonly in female than in male patients. In female patients, it affects the fallopian tubes and the endometrium and may cause infertility, pelvic pain, and menstrual abnormalities. Diagnosis requires biopsy or culture of specimens obtained by dilation and curettage. In male patients, genital TB preferentially affects the epididymis, producing a slightly tender mass that may drain externally through a fistulous tract; orchitis and prostatitis may also develop. In almost half of cases of genitourinary TB, urinary tract disease is also present. Genitourinary TB responds well to chemotherapy.

1	FIGuRE 202-9 MRI of culture-confirmed renal tuberculosis. T2-weighted coronary plane: coronal sections showing several renal lesions in both the cortical and the medullary tissues of the right kidney. (Courtesy of Dr. Alberto Matteelli, Department of Infectious Diseases, University of Brescia, Italy; with permission.)

1	Skeletal TB In the United States, TB of the bones and joints is responsible for ~10% of extrapulmonary cases. In bone and joint disease, pathogenesis is related to reactivation of hematogenous foci or to spread from adjacent paravertebral lymph nodes. Weight-bearing joints (the spine in 40% of cases, the hips in 13%, and the knees in 10%) are most commonly affected. Spinal TB (Pott’s disease or tuberculous spondylitis; Fig. 202-10) often involves two or more adjacent vertebral bodies. Whereas the upper thoracic spine is the most common site of spinal TB in children, the lower thoracic and upper lumbar vertebrae are usually affected in adults. From the anterior superior or inferior angle of the vertebral body, the lesion slowly reaches the adjacent body, later affecting the intervertebral disk. With advanced disease, collapse of vertebral bodies results in kyphosis (gibbus). A paravertebral “cold” abscess may also form. In the upper spine, this abscess may track to and penetrate the

1	disk. With advanced disease, collapse of vertebral bodies results in kyphosis (gibbus). A paravertebral “cold” abscess may also form. In the upper spine, this abscess may track to and penetrate the chest wall, presenting as a soft tissue mass; in the lower spine, it may reach the inguinal ligaments or present as a psoas abscess. CT or MRI reveals the characteristic lesion and suggests its etiology. The differential diagnosis includes tumors and other infections. Pyogenic bacterial osteomyelitis, in particular, involves the disk very early and produces rapid sclerosis. Aspiration of the abscess or bone biopsy confirms the tuberculous etiology, as cultures are usually positive and histologic findings highly typical. A catastrophic complication of Pott’s disease is paraplegia, which is usually due to an abscess or a lesion compressing the spinal cord. Paraparesis due to a large abscess is a medical emergency and requires rapid drainage. TB of the hip joints, usually involving the head of

1	due to an abscess or a lesion compressing the spinal cord. Paraparesis due to a large abscess is a medical emergency and requires rapid drainage. TB of the hip joints, usually involving the head of the femur, causes pain; TB of the knee produces pain and swelling. If the disease goes unrecognized, the joints may be destroyed. Diagnosis requires examination of the synovial fluid, which is thick in appearance, with a high protein concentration and a variable cell count.

1	FIGuRE 202-10 CT scan demonstrating destruction of the right pedicle of T10 due to Pott’s disease. The patient, a 70-year-old Asian woman, presented with back pain and weight loss and had biopsy-proven tuberculosis. (Courtesy of Charles L. Daley, MD, University of California, San Francisco; with permission.) Although synovial fluid culture is positive in a high percentage of cases, synovial biopsy and tissue culture may be necessary to establish the diagnosis. Skeletal TB responds to chemotherapy, but severe cases may require surgery.

1	Tuberculous Meningitis and Tuberculoma TB of the central nervous system accounts for ~5% of extrapulmonary cases in the United States. It is seen most often in young children but also develops in adults, especially those infected with HIV. Tuberculous meningitis results from the hematogenous spread of primary or postprimary pulmonary TB or from the rupture of a subependymal tubercle into the subarachnoid space. In more than half of cases, evidence of old pulmonary lesions or a miliary pattern is found on chest radiography. The disease often presents subtly as headache and slight mental changes after a prodrome of weeks of low-grade fever, malaise, anorexia, and irritability. If not recognized, tuberculous meningitis may evolve acutely with severe headache, confusion, lethargy, altered sensorium, and neck rigidity. Typically, the disease evolves over 1–2 weeks, a course longer than that of bacterial meningitis. Because meningeal involvement is pronounced at the base of the brain,

1	sensorium, and neck rigidity. Typically, the disease evolves over 1–2 weeks, a course longer than that of bacterial meningitis. Because meningeal involvement is pronounced at the base of the brain, paresis of cranial nerves (ocular nerves in particular) is a frequent finding, and the involvement of cerebral arteries may produce focal ischemia. The ultimate evolution is toward coma, with hydrocephalus and intracranial hypertension.

1	Lumbar puncture is the cornerstone of diagnosis. In general, examination of cerebrospinal fluid (CSF) reveals a high leukocyte count (up to 1000/μL), usually with a predominance of lymphocytes but sometimes with a predominance of neutrophils in the early stage; a protein content of 1–8 g/L (100–800 mg/dL); and a low glucose concentration. However, any of these three parameters can be within the normal range. AFBs are infrequently seen on direct smear of CSF sediment, and repeated lumbar punctures increase the yield. Culture of CSF is diagnostic in up to 80% of cases and remains the gold standard. Real-time automated nucleic acid amplification (the Xpert MTB/RIF assay; see “Nucleic Acid Amplification Technology,” below) has a sensitivity of up to 80% and is the preferred initial diagnostic option. Treatment should be initiated immediately upon a positive Xpert MTB/RIF result. A negative result does not exclude a diagnosis of TB and requires further diagnostic workup. Imaging studies

1	option. Treatment should be initiated immediately upon a positive Xpert MTB/RIF result. A negative result does not exclude a diagnosis of TB and requires further diagnostic workup. Imaging studies (CT and MRI) may show hydrocephalus and abnormal enhancement of basal cisterns or ependyma. If unrecognized, tuberculous meningitis is uniformly fatal. This disease responds to chemotherapy; however, neurologic sequelae are documented in 25% of treated cases, in most of which the diagnosis has been delayed. Clinical trials have demonstrated that patients given adjunctive glucocorticoids may experience faster resolution of CSF abnormalities and elevated CSF pressure. In one study, adjunctive 1111 dexamethasone significantly enhanced the chances of survival among persons >14 years of age but did not reduce the frequency of neurologic sequelae. The dexamethasone schedule was (1) 0.4 mg/kg per day given IV with tapering by 0.1 mg/kg per week until the fourth week, when 0.1 mg/kg per day was

1	not reduce the frequency of neurologic sequelae. The dexamethasone schedule was (1) 0.4 mg/kg per day given IV with tapering by 0.1 mg/kg per week until the fourth week, when 0.1 mg/kg per day was administered; followed by (2) 4 mg/d given by mouth with tapering by 1 mg per week until the fourth week, when 1 mg/d was administered.

1	Tuberculoma, an uncommon manifestation of central nervous system TB, presents as one or more space-occupying lesions and usually causes seizures and focal signs. CT or MRI reveals contrast-enhanced ring lesions, but biopsy is necessary to establish the diagnosis.

1	Gastrointestinal TB Gastrointestinal TB is uncommon, making up 3.5% of extrapulmonary cases in the United States. Various pathogenetic mechanisms are involved: swallowing of sputum with direct seeding, hematogenous spread, or (largely in developing areas) ingestion of milk from cows affected by bovine TB. Although any portion of the gastrointestinal tract may be affected, the terminal ileum and the cecum are the sites most commonly involved. Abdominal pain (at times similar to that associated with appendicitis) and swelling, obstruction, hematochezia, and a palpable mass in the abdomen are common findings at presentation. Fever, weight loss, anorexia, and night sweats are also common. With intestinal-wall involvement, ulcerations and fistulae may simulate Crohn’s disease; the differential diagnosis of this entity is always difficult. Anal fistulae should prompt an evaluation for rectal TB. Because surgery is required in most cases, the diagnosis can be established by histologic

1	diagnosis of this entity is always difficult. Anal fistulae should prompt an evaluation for rectal TB. Because surgery is required in most cases, the diagnosis can be established by histologic examination and culture of specimens obtained intraoperatively.

1	Tuberculous peritonitis follows either the direct spread of tubercle bacilli from ruptured lymph nodes and intraabdominal organs (e.g., genital TB in women) or hematogenous seeding. Nonspecific abdominal pain, fever, and ascites should raise the suspicion of tuberculous peritonitis. The coexistence of cirrhosis (Chap. 363) in patients with tuberculous peritonitis complicates the diagnosis. In tuberculous peritonitis, paracentesis reveals an exudative fluid with a high protein content and leukocytosis that is usually lymphocytic (although neutrophils occasionally predominate). The yield of direct smear and culture is relatively low; culture of a large volume of ascitic fluid can increase the yield, but peritoneal biopsy (with a specimen best obtained by laparoscopy) is often needed to establish the diagnosis.

1	Pericardial TB (Tuberculous Pericarditis) Due either to direct extension from adjacent mediastinal or hilar lymph nodes or to hematogenous spread, pericardial TB has often been a disease of the elderly in countries with low TB prevalence. However, it also develops frequently in HIV-infected patients. Case–fatality rates are as high as 40% in some series. The onset may be subacute, although an acute presentation, with dyspnea, fever, dull retrosternal pain, and a pericardial friction rub, is possible. An effusion eventually develops in many cases; cardiovascular symptoms and signs of cardiac tamponade may ultimately appear (Chap. 288). In the presence of effusion, TB must be suspected if the patient belongs to a high-risk population (HIV-infected, originating in a high-prevalence country); if there is evidence of previous TB in other organs; or if echocardiography, CT, or MRI shows effusion and thickness across the pericardial space. A definitive diagnosis can be obtained by

1	country); if there is evidence of previous TB in other organs; or if echocardiography, CT, or MRI shows effusion and thickness across the pericardial space. A definitive diagnosis can be obtained by pericardiocentesis under echocardiographic guidance. The pericardial fluid must be submitted for biochemical, cytologic, and microbiologic evaluation. The effusion is exudative in nature, with a high count of lymphocytes and monocytes. Hemorrhagic effusion is common. Direct smear examination is very rarely positive. Culture of pericardial fluid reveals M. tuberculosis in up to two-thirds of cases, whereas pericardial biopsy has a higher yield. High levels of ADA, lysozyme, and IFN-γ may suggest a tuberculous etiology.

1	Without treatment, pericardial TB is usually fatal. Even with treatment, complications may develop, including chronic constrictive pericarditis with thickening of the pericardium, fibrosis, and sometimes calcification, which may be visible on a chest radiograph. Systematic reviews and meta-analyses show that adjunctive glucocorticoid treatment remains controversial, with no conclusive evidence of benefits 1112 for all principal outcomes of pericarditis—i.e., no significant impact on resolution of effusion, no significant difference in functional status after treatment, and no significant reduction in the frequency of development of constriction or death. However, in HIV-infected patients, glucocorticoids do improve functional status after treatment. Caused by direct extension from the pericardium or by retrograde lymphatic extension from affected mediastinal lymph nodes, tuberculous myocarditis is an extremely rare disease. Usually it is fatal and is diagnosed postmortem.

1	Miliary or Disseminated TB Miliary TB is due to hematogenous spread of tubercle bacilli. Although in children it is often the consequence of primary infection, in adults it may be due to either recent infection or reactivation of old disseminated foci. The lesions are usually yellowish granulomas 1–2 mm in diameter that resemble millet seeds (thus the term miliary, coined by nineteenth-century pathologists). Clinical manifestations are nonspecific and protean, depending on the predominant site of involvement. Fever, night sweats, anorexia, weakness, and weight loss are presenting symptoms in the majority of cases. At times, patients have a cough and other respiratory symptoms due to pulmonary involvement as well as abdominal symptoms. Physical findings include hepatomegaly, splenomegaly, and lymphadenopathy. Eye examination may reveal choroidal tubercles, which are pathognomonic of miliary TB, in up to 30% of cases. Meningismus occurs in fewer than 10% of cases.

1	A high index of suspicion is required for the diagnosis of miliary TB. Frequently, chest radiography (Fig. 202-5) reveals a miliary reticulonodular pattern (more easily seen on underpenetrated film), although no radiographic abnormality may be evident early in the course and among HIV-infected patients. Other radiologic findings include large infiltrates, interstitial infiltrates (especially in HIV-infected patients), and pleural effusion. Sputum-smear microscopy is negative in most cases. Various hematologic abnormalities may be seen, including anemia with leukopenia, lymphopenia, neutrophilic leukocytosis and leukemoid reactions, and polycythemia. Disseminated intravascular coagulation has been reported. Elevation of alkaline phosphatase levels and other abnormal values in liver function tests are detected in patients with severe hepatic involvement. The TST may be negative in up to half of cases, but reactivity may be restored during chemotherapy. Bronchoalveolar lavage and

1	function tests are detected in patients with severe hepatic involvement. The TST may be negative in up to half of cases, but reactivity may be restored during chemotherapy. Bronchoalveolar lavage and transbronchial biopsy are more likely to provide bacteriologic confirmation, and granulomas are evident in liver or bone-marrow biopsy specimens from many patients. If it goes unrecognized, miliary TB is lethal; with proper early treatment, however, it is amenable to cure. Glucocorticoid therapy has not proved beneficial.

1	A rare presentation seen in the elderly, cryptic miliary TB has a chronic course characterized by mild intermittent fever, anemia, and— ultimately—meningeal involvement preceding death. An acute septicemic form, nonreactive miliary TB, occurs very rarely and is due to massive hematogenous dissemination of tubercle bacilli. Pancytopenia is common in this form of disease, which is rapidly fatal. At postmortem examination, multiple necrotic but nongranulomatous (“nonreactive”) lesions are detected.

1	Less Common Extrapulmonary Forms TB may cause chorioretinitis, uveitis, panophthalmitis, and painful hypersensitivity-related phlyctenular conjunctivitis. Tuberculous otitis is rare and presents as hearing loss, otorrhea, and tympanic membrane perforation. In the nasopharynx, TB may simulate granulomatosis with polyangiitis. Cutaneous manifestations of TB include primary infection due to direct inoculation, abscesses and chronic ulcers, scrofuloderma, lupus vulgaris (a smoldering disease with nodules, plaques, and fissures), miliary lesions, and erythema nodosum. Tuberculous mastitis results from retrograde lymphatic spread, often from the axillary lymph nodes. Adrenal TB is a manifestation of disseminated disease presenting rarely as adrenal insufficiency. Finally, congenital TB results from transplacental spread of tubercle bacilli to the fetus or from ingestion of contaminated amniotic fluid. This rare disease affects the liver, spleen, lymph nodes, and various other organs.

1	Post-TB Complications TB may cause persisting pulmonary damage in patients whose infection has been considered cured on clinical grounds. Chronic impairment of lung functions, bronchiectasis, aspergillomas, and chronic pulmonary aspergillosis (CPA) have been associated with TB. CPA may manifest as simple aspergilloma (fungal ball) or chronic cavitary aspergillosis. Early studies revealed that, especially in the presence of large residual cavities, Aspergillus fumigatus may colonize the lesion and produce symptoms such as respiratory impairment, hemoptysis, persistent fatigue, and weight loss, often resulting in the erroneous diagnosis of TB recurrence. The detection of Aspergillus precipitins (IgG) in the blood suggests CPA, as do radiographic abnormalities such as thickening of the cavitary walls or the presence of a fungal ball inside the cavity. Treatment is difficult. Recent preliminary studies on the use of itraconazole for 6 months suggest that treatment with this agent may be

1	walls or the presence of a fungal ball inside the cavity. Treatment is difficult. Recent preliminary studies on the use of itraconazole for 6 months suggest that treatment with this agent may be superior to conservative treatment in improving radiologic and clinical manifestations of CPA. Surgical removal of lesions is risky.

1	HIV-Associated TB (See also Chap. 226) TB is one of the most common diseases among HIV-infected persons worldwide and a major cause of death in this population; more specifically, it is responsible for an estimated 24% of all HIV-related mortality. In certain urban settings in some African countries, the rate of HIV infection among TB patients reaches 70–80%. A person with a positive TST who acquires HIV infection has a 3–13% annual risk of developing active TB. A new TB infection acquired by an HIV-infected individual may evolve to active disease in a matter of weeks rather than months or years. TB can appear at any stage of HIV infection, and its presentation varies with the stage. When CMI is only partially compromised, pulmonary TB presents in a typical manner (Figs. 202-6 and 202-7), with upper-lobe infiltrates and cavitation and without significant lymphadenopathy or pleural effusion. In late stages of HIV infection, when the CD4+ T cell count is <200/μL, a primary TB–like

1	with upper-lobe infiltrates and cavitation and without significant lymphadenopathy or pleural effusion. In late stages of HIV infection, when the CD4+ T cell count is <200/μL, a primary TB–like pattern, with diffuse interstitial and subtle infiltrates, little or no cavitation, pleural effusion, and intrathoracic lymphadenopathy, is more common. However, these forms are becoming less common because of the expanded use of antiretroviral treatment (ART). Overall, sputum smears are less frequently positive among TB patients with HIV infection than among those without; thus, the diagnosis of TB may be difficult, especially in view of the variety of HIV-related pulmonary conditions mimicking TB. Extrapulmonary TB is common among HIV-infected patients. In various series, extrapulmonary TB—alone or in association with pulmonary disease—has been documented in 40–60% of all cases in HIV-co-infected individuals. The most common forms are lymphatic, disseminated, pleural, and pericardial.

1	or in association with pulmonary disease—has been documented in 40–60% of all cases in HIV-co-infected individuals. The most common forms are lymphatic, disseminated, pleural, and pericardial. Mycobacteremia and meningitis are also common, particularly in advanced HIV disease. The diagnosis of TB in HIV-infected patients may be complicated not only by the increased frequency of sputum-smear negativity (up to 40% in culture-proven pulmonary cases) but also by atypical radiographic findings, a lack of classic granuloma formation in the late stages, and a negative TST. The Xpert MTB/RIF assay (see “Nucleic Acid Amplification Technology,” below) is the preferred initial diagnostic option, and therapy should be started on the basis of a positive result because treatment delays may be fatal. A negative Xpert MTB/RIF result does not exclude a diagnosis of TB, and culture remains the gold standard.

1	Exacerbations in systemic (lymphadenopathy) or respiratory symptoms, signs, and laboratory or radiographic manifestations of TB—termed the immune reconstitution inflammatory syndrome (IRIS) or TB immune reconstitution disease (TB-IRD)—have been associated with the administration of ART and occur in ~10% of HIV-infected TB patients. Usually developing 1–3 months after initiation of ART, IRIS is more common among patients with advanced immunosuppression and extrapulmonary TB. “Unmasking IRIS” may also develop after the initiation of ART in patients with undiagnosed subclinical TB. The earlier ART is started and the lower the baseline CD4+ T cell count, the greater the risk of IRIS. Death due to IRIS is relatively infrequent and occurs mainly among patients who have a high preexisting mortality risk. The presumed pathogenesis of IRIS consists of an immune response that is elicited by antigens released as bacilli are killed during effective chemotherapy and that is temporally associated

1	risk. The presumed pathogenesis of IRIS consists of an immune response that is elicited by antigens released as bacilli are killed during effective chemotherapy and that is temporally associated with improving immune function. There is no diagnostic test for IRIS, and its confirmation relies heavily upon case definitions incorporating clinical and laboratory data; a variety of case definitions have been suggested. The first priority in the management of a possible case of IRIS is to ensure that the clinical syndrome does not represent a failure of TB treatment or the development of another infection. Mild paradoxical reactions can be managed with symptom-based treatment. Glucocorticoids have been used for more severe reactions, and prednisolone given for 4 weeks at a low dosage (1.5 mg/kg per day for 2 weeks and half that dose for the remaining 2 weeks) has reduced the need for hospitalization and therapeutic procedures and hastened alleviation of symptoms, as reflected by Karnofsky

1	per day for 2 weeks and half that dose for the remaining 2 weeks) has reduced the need for hospitalization and therapeutic procedures and hastened alleviation of symptoms, as reflected by Karnofsky performance scores, quality-of-life assessments, radiographic response, and C-reactive protein levels. The effectiveness of glucocorticoids in alleviating the symptoms of IRIS is probably linked to suppression of proinflammatory cytokine concentrations, as these medications reduce serum concentrations of IL-6, IL-10, IL-12p40, TNF-α, IFN-γ, and IFN-γ-inducible protein 10 (IP-10). Recommendations for the prevention and treatment of TB in HIV-infected individuals are provided below.

1	The key to the diagnosis of TB remains a high index of suspicion. Diagnosis is not difficult in persons belonging to high-risk populations who present with typical symptoms and a classic chest radio-graph showing upper-lobe infiltrates with cavities (Fig. 202-6). On the other hand, the diagnosis can easily be missed in an elderly nursing-home resident or a teenager with a focal infiltrate. Often, the diagnosis is first entertained when the chest radiograph of a patient being evaluated for respiratory symptoms is abnormal. If the patient has no complicating medical conditions that cause immunosuppression, the chest radiograph may show typical upper-lobe infiltrates with cavitation (Fig. 202-6). The longer the delay between the onset of symptoms and the diagnosis, the more likely is the finding of cavitary disease. In contrast, immunosuppressed patients, including those with HIV infection, may have “atypical” findings on chest radiography—e.g., lower-zone infiltrates without cavity

1	finding of cavitary disease. In contrast, immunosuppressed patients, including those with HIV infection, may have “atypical” findings on chest radiography—e.g., lower-zone infiltrates without cavity formation.

1	The several approaches to the diagnosis of TB require, above all, a well-organized laboratory network with an appropriate distribution of tasks at different levels of the health care system. At the peripheral and community levels, screening and referral are the principal tasks—besides clinical assessment and radiography—that can be accomplished through AFB microscopy and/or real-time automated nucleic acid amplification technology (the Xpert MTB/RIF assay; see below). At a secondary level (e.g., a traditional district hospital in a high-incidence setting), additional technology can be adopted, including rapid culture and drug susceptibility testing.

1	A presumptive diagnosis is commonly based on the finding of AFB on microscopic examination of a diagnostic specimen, such as a smear of expectorated sputum or of tissue (e.g., a lymph node biopsy). Although inexpensive, AFB microscopy has relatively low sensitivity (40–60%) in culture-confirmed cases of pulmonary TB. The traditional method— light microscopy of specimens stained with Ziehl-Neelsen basic fuchsin dyes—is nevertheless satisfactory, although time-consuming. Most modern laboratories processing large numbers of diagnostic specimens use auramine–rhodamine staining and fluorescence microscopy; this approach is more sensitive than the Ziehl-Neelsen method. However, it is expensive because it requires high-cost mercury vapor light sources and a dark room. Less expensive light-emitting diode (LED) fluorescence microscopes are now available. They are as sensitive as—or more sensitive than—traditional fluorescence microscopes. As a result, conventional light and fluorescence

1	diode (LED) fluorescence microscopes are now available. They are as sensitive as—or more sensitive than—traditional fluorescence microscopes. As a result, conventional light and fluorescence microscopes are being replaced with this more recent technology, especially in developing countries. For patients with suspected pulmonary TB, it has been recommended that two or three sputum specimens, preferably collected early in the morning, should be submitted to the laboratory for AFB smear and mycobacterial culture. Two specimens collected on the same visit may be as effective as three. If tissue is obtained, it is criti-1113 cal that the portion of the specimen intended for culture not be put in formaldehyde. The use of AFB microscopy in examining urine or gastric lavage fluid is limited by the presence of commensal mycobacteria that can cause false-positive results.

1	Several test systems based on amplification of mycobacterial nucleic acid have become available in the past few years. These tests are most useful for the rapid confirmation of TB in persons with AFB-positive specimens, but some also have utility for the diagnosis of AFB-negative pulmonary and extrapulmonary TB. One system that permits rapid diagnosis of TB with high specificity and sensitivity (approaching that of culture) is the fully automated, real-time nucleic acid amplification technology known as the Xpert MTB/RIF assay. Xpert MTB/RIF can simultaneously detect TB and rifampin resistance in <2 h and has minimal biosafety and training requirements. Therefore, it can be housed in nonconventional laboratory settings. The WHO recommends its use worldwide as the initial diagnostic test in adults and children presumed to have MDR-TB or HIV-associated TB. Taking into account the availability of resources, the test may also be used in any adult or child presumed to have TB or as a

1	in adults and children presumed to have MDR-TB or HIV-associated TB. Taking into account the availability of resources, the test may also be used in any adult or child presumed to have TB or as a follow-up test after microscopy in adults presumed to have TB but not at risk of MDR-TB or HIV-associated TB. Xpert MTB/RIF should be the initial test applied to CSF from patients in whom TB meningitis is suspected as well as a replacement test (over conventional microscopy, culture, and histopathology) for selected nonrespiratory specimens—obtained by gastric lavage, fine-needle aspiration, or pleural or other biopsies—from patients in whom extrapulmonary TB is suspected. This test has a sensitivity of 98% among AFB-positive cases and ~70% among AFB-negative specimens. Other tests, such as those based on manual amplification platforms, have not yet been deemed satisfactory for introduction into clinical practice as replacements for existing tests.

1	Definitive diagnosis depends on the isolation and identification of M. tuberculosis from a clinical specimen or the identification of specific DNA sequences in a nucleic acid amplification test. Specimens may be inoculated onto eggor agar-based medium (e.g., Löwenstein-Jensen or Middlebrook 7H10) and incubated at 37°C (under 5% CO2 for Middlebrook medium). Because most species of mycobacteria, including

1	M. tuberculosis, grow slowly, 4–8 weeks may be required before growth is detected. Although M. tuberculosis may be identified presumptively on the basis of growth time and colony pigmentation and morphology, a variety of biochemical tests have traditionally been used to speciate mycobacterial isolates. In modern, well-equipped laboratories, liquid culture for isolation and species identification by molecular methods or high-pressure liquid chromatography of mycolic acids has replaced isolation on solid media and identification by biochemical tests. A widely used technology is the mycobacterial growth indicator tube (BBL™ MGIT™; BD, Franklin Lakes, NJ), which uses a fluorescent compound sensitive to the presence of oxygen dissolved in the liquid medium. The appearance of fluorescence detected by fluorometric technology indicates active growth of mycobacteria. A low-cost, rapid immunochromatographic lateral-flow assay based on detection of MTP64 antigen may also be used for species

1	by fluorometric technology indicates active growth of mycobacteria. A low-cost, rapid immunochromatographic lateral-flow assay based on detection of MTP64 antigen may also be used for species identification of the M. tuberculosis complex in culture isolates. These new methods, which are also being introduced in low-income countries, have decreased the time required for bacteriologic confirmation of TB to 2–3 weeks.

1	Any initial isolate of M. tuberculosis should be tested for susceptibility to isoniazid and rifampin in order to detect drug resistance and/or MDR-TB, particularly if one or more risk factors for drug resistance are identified or if the patient either fails to respond to initial therapy or has a relapse after the completion of treatment (see “Treatment Failure and Relapse,” below). In addition, expanded susceptibility testing for second-line anti-TB drugs (especially the fluoroquinolones 1114 and the injectable drugs) is mandatory when MDR-TB is found. Susceptibility testing may be conducted directly (with the clinical specimen) or indirectly (with mycobacterial cultures) on solid or liquid medium. Results are obtained rapidly by direct susceptibility testing on liquid medium, with an average reporting time of 3 weeks. With indirect testing on solid medium, results may be unavailable for ≥8 weeks. Highly reliable genotypic methods for the rapid identification of genetic mutations in

1	reporting time of 3 weeks. With indirect testing on solid medium, results may be unavailable for ≥8 weeks. Highly reliable genotypic methods for the rapid identification of genetic mutations in gene regions known to be associated with resistance to rifampin (such as those in rpoB) and isoniazid (such as those in katG and inhA) have been developed and are being widely implemented for screening of patients at increased risk of drug-resistant TB. Apart from the Xpert MTB/RIF assay, which, as mentioned above, detects rifampin resistance, the most widely used are molecular line probe assays. After extraction of DNA from M. tuberculosis isolates or from clinical specimens, the resistance gene regions are amplified by polymerase chain reaction (PCR), and labeled and probe-hybridized PCR products are detected by colorimetric development. This assay reveals the presence of M. tuberculosis as well as mutations in target resistance gene regions. A similar approach has been developed for

1	products are detected by colorimetric development. This assay reveals the presence of M. tuberculosis as well as mutations in target resistance gene regions. A similar approach has been developed for second-line anti-TB drugs such as the fluoroquinolones, the aminoglycosides kanamycin and amikacin, and capreomycin, but the diagnostic accuracy of the current technology is not yet sufficient to recommend its use in clinical practice. Finally, a few noncommercial, inexpensive culture and drug-susceptibility testing methods (e.g., microscopically observed drug susceptibility, or MODS; nitrate reductase assays; and colorimetric redox indicator assays) may be useful in resource-limited settings. Their use is restricted to national reference laboratories with proven proficiency and adequate external quality control as an interim solution while genotypic or automated liquid culture technology is introduced.

1	As noted above, the initial suspicion of pulmonary TB is often based on abnormal chest radiographic findings in a patient with respiratory symptoms. Although the “classic” picture is that of upper-lobe disease with infiltrates and cavities (Fig. 202-6), virtually any radiographic pattern—from a normal film or a solitary pulmonary nodule to diffuse alveolar infiltrates in a patient with adult respiratory distress syndrome—may be seen. In the era of AIDS, no radiographic pattern can be considered pathognomonic. CT (Fig. 202-7) may be useful in interpreting questionable findings on plain chest radiography and may be helpful in diagnosing some forms of extrapulmonary TB (e.g., Pott’s disease; Fig. 202-10). MRI is useful in the diagnosis of intracranial TB.

1	Other diagnostic tests may be used when pulmonary TB is suspected. Sputum induction by ultrasonic nebulization of hypertonic saline may be useful for patients who cannot produce a sputum specimen spontaneously. Frequently, patients with radiographic abnormalities that are consistent with other diagnoses (e.g., bronchogenic carcinoma) undergo fiberoptic bronchoscopy with bronchial brushings and endobronchial or transbronchial biopsy of the lesion. Bronchoalveolar lavage of a lung segment containing an abnormality may also be performed. In all cases, it is essential that specimens be submitted for AFB smear, mycobacterial culture, and molecular testing with the Xpert MTB/RIF assay. For the diagnosis of primary pulmonary TB in children, who often do not expectorate sputum, induced sputum specimens and specimens from early-morning gastric lavage may yield positive cultures and/or Xpert MTB/RIF assay results.

1	Invasive diagnostic procedures are indicated for patients with suspected extrapulmonary TB. In addition to testing of specimens from involved sites (e.g., CSF for tuberculous meningitis, pleural fluid and biopsy samples for pleural disease), biopsy and culture of bone marrow and liver tissue have a good diagnostic yield in disseminated (miliary) TB, particularly in HIV-infected patients, who also have a high frequency of positive blood cultures. In some cases, culture or Xpert MTB/RIF assay results are negative but a clinical diagnosis of TB is supported by consistent epidemiologic evidence (e.g., a history of close contact with an infectious patient) and a compatible clinical and radiographic response to treatment. In the United States and other industrialized countries with low rates of TB, some patients with limited abnormalities on chest radiographs and sputum positive for AFB are infected with nontuberculous mycobacteria, most commonly organisms of the M. avium complex or M.

1	of TB, some patients with limited abnormalities on chest radiographs and sputum positive for AFB are infected with nontuberculous mycobacteria, most commonly organisms of the M. avium complex or M. kansasii (Chap. 204). Factors favoring the diagnosis of nontuberculous mycobacterial disease over TB include an absence of risk factors for TB and the presence of underlying chronic pulmonary disease.

1	Patients with HIV-associated TB pose several diagnostic problems (see “HIV-Associated TB,” above). Moreover, HIV-infected patients with sputum culture–positive, AFB-positive TB may present with a normal chest radiograph. The Xpert MTB/RIF assay is the preferred rapid diagnostic test in this population of patients because of simplicity of use and a sensitivity of ~60% among AFB-negative culture-positive cases and of 97% among AFB-positive cases. With the advent of ART, the occurrence of disseminated M. avium complex disease that can be confused with TB has become much less common.

1	A number of serologic tests based on detection of antibodies to a variety of mycobacterial antigens are marketed in developing countries but not in the United States. Careful independent assessments of these tests suggest that they are not useful as diagnostic aids—especially in persons with a low probability of TB—because of their low sensitivity and specificity and their poor reproducibility. After a rigorous evaluation of the tests, the WHO issued a “negative” recommendation in 2011 in order to prevent their abuse in the private sector of many resource-limited countries. Various methods aimed at detection of mycobacterial antigens in diagnostic specimens are being investigated but are limited at present by low sensitivity. Determinations of ADA and IFN-γ levels in pleural fluid may be useful adjunctive tests in the diagnosis of pleural TB; their utility in the diagnosis of other forms of extrapulmonary TB (e.g., pericardial, peritoneal, and meningeal) is less clear.

1	DIAGNOSIS OF LATENT M. TUBERCULOSIS INFECTION Tuberculin Skin Testing In 1891, Robert Koch discovered that components of M. tuberculosis in a concentrated liquid culture medium, subsequently named “old tuberculin,” were capable of eliciting a skin reaction when injected subcutaneously into patients with TB. In 1932, Seibert and Munday purified this product by ammonium sulfate precipitation to produce an active protein fraction known as tuberculin purified protein derivative (PPD). In 1941, PPD-S, developed by Seibert and Glenn, was chosen as the international standard. Later, the WHO and UNICEF sponsored large-scale production of a master batch of PPD (RT23) and made it available for general use. The greatest limitation of PPD is its lack of mycobacterial species specificity, a property due to the large number of proteins in this product that are highly conserved in the various species. In addition, subjectivity of the skin-reaction interpretation, deterioration of the product, and

1	due to the large number of proteins in this product that are highly conserved in the various species. In addition, subjectivity of the skin-reaction interpretation, deterioration of the product, and batch-to-batch variations limit the usefulness of PPD.

1	The skin test with tuberculin PPD (TST) is most widely used in screening for LTBI. It probably measures the response to antigenic stimulation by T cells that reside in the skin rather than the response of recirculating memory T cells. The test is of limited value in the diagnosis of active TB because of its relatively low sensitivity and specificity and its inability to discriminate between LTBI and active disease. False-negative reactions are common in immunosuppressed patients and in those with overwhelming TB. False-positive reactions may be caused by infections with nontuberculous mycobacteria (Chap. 204) and by BCG vaccination. Repeated TST can produce larger reaction sizes due to either boosting or true conversion. The “boosting phenomenon” is a spurious TST conversion resulting from boosting of reactivity on subsequent TST 1–5 weeks after the initial test. Distinguishing boosting from true conversion is difficult yet important and can be based on clinical and epidemiologic

1	from boosting of reactivity on subsequent TST 1–5 weeks after the initial test. Distinguishing boosting from true conversion is difficult yet important and can be based on clinical and epidemiologic considerations. For instance, true conversions are likely after BCG vaccination in a previously TST-negative person or in a close contact of an infectious patient.

1	IFN-γ Release Assays Two in vitro assays that measure T cell release of IFN-γ in response to stimulation with the highly TB-specific antigens ESAT-6 and CFP-10 are available. The T-SPOT®.TB test (Oxford Immunotec, Oxford, United Kingdom) is an enzyme-linked immunospot (ELISpot) assay, and the QuantiFERON®-TB Gold test (Qiagen GmbH, Hilden, Germany) is a whole-blood enzyme-linked immunosorbent assay (ELISA) for measurement of IFN-γ. The QuantiFERON®-TB Gold In-Tube assay, which facilitates blood collection and initial incubation, also contains another specific antigen, TB7.7. These tests likely measure the response of recirculating memory T cells—normally part of a reservoir in the spleen, bone marrow, and lymph nodes—to persisting bacilli producing antigenic signals.

1	In settings or population groups with low TB and HIV burdens, IFN-γ release assays (IGRAs) have previously been reported to be more specific than the TST as a result of less cross-reactivity due to BCG vaccination and sensitization by nontuberculous mycobacteria. Recent studies, however, suggest that IGRAs may not perform well in serial testing (e.g., among health care workers) and that interpretation of test results is dependent on cutoff values used to define positivity. Potential advantages of IGRAs include logistical convenience, the need for fewer patient visits to complete testing, and the avoidance of somewhat subjective measurements such as skin induration. However, IGRAs require that blood be drawn from the individual and then delivered to the laboratory in a timely fashion. IGRAs also require that testing be performed in a laboratory setting. These requirements pose challenges similar to those faced with the TST, including cold-chain requirements and batch-to-batch

1	IGRAs also require that testing be performed in a laboratory setting. These requirements pose challenges similar to those faced with the TST, including cold-chain requirements and batch-to-batch variations. Because of higher specificity and other potential advantages, IGRAs have usually replaced the TST for LTBI diagnosis in low-incidence, high-income settings. However, in high-incidence TB and HIV settings and population groups, there is limited and inconclusive evidence about the performance and usefulness of IGRAs. In view of higher costs and increased technical requirements, the WHO does not recommend the replacement of the TST by IGRAs in lowand middle-income countries.

1	A number of national guidelines on the use of IGRAs for LTBI testing have been issued. In the United States, an IGRA is preferred to the TST for most persons over the age of 5 years who are being screened for LTBI. However, for those at high risk of progression to active TB (e.g., HIV-infected persons), either test—or, to optimize sensitivity, both tests—may be used. Because of the paucity of data on the use of IGRAs in children, the TST is preferred for LTBI testing of children under age 5. In Canada and some European countries, a two-step approach for those with positive TSTs—i.e., initial TST followed by an IGRA—is recommended. However, a TST may boost an IGRA response if the interval between the two tests exceeds 3 days. Similar to the TST, current IGRAs have only modest predictive value for incident active TB, are not useful in identifying patients with the highest risk of progression toward disease, and cannot be used for diagnosis of active TB.

1	The two aims of TB treatment are (1) to prevent morbidity and death by curing TB while preventing the emergence of drug resistance and (2) to interrupt transmission by rendering patients noninfectious. Chemotherapy for TB became possible with the discovery of streptomycin in 1943. Randomized clinical trials clearly indicated that the administration of streptomycin to patients with chronic TB reduced mortality rates and led to cure in the majority of cases. However, monotherapy with streptomycin eventually was associated with the development of resistance to this drug and the resulting failure of treatment. With the introduction into clinical practice of paraaminosalicylic acid (PAS) and isoniazid, it became axiomatic in the early 1950s that cure of TB required the concomitant administration of at least two agents to which the organism was susceptible. Furthermore, early clinical trials demonstrated that a long period of treatment—i.e., 12–24 months—was required to prevent recurrence.

1	of at least two agents to which the organism was susceptible. Furthermore, early clinical trials demonstrated that a long period of treatment—i.e., 12–24 months—was required to prevent recurrence. The introduction of rifampin (rifampicin) in the early 1970s heralded the era of effective short-course chemotherapy, with a 1115 treatment duration of <12 months. The discovery that pyrazinamide, which was first used in the 1950s, augmented the potency of isoniazid/rifampin regimens led to the use of a 6-month course of this triple-drug regimen as standard therapy.

1	Four major drugs are considered first-line agents for the treatment of TB: isoniazid, rifampin, pyrazinamide, and ethambutol (Table 202-2). These drugs are well absorbed after oral administration, with peak serum levels at 2–4 h and nearly complete elimination within 24 h. These agents are recommended on the basis of their bactericidal activity (i.e., their ability to rapidly reduce the number of viable organisms and render patients noninfectious), their sterilizing activity (i.e., their ability to kill all bacilli and thus sterilize the affected tissues, measured in terms of the ability to prevent relapses), and their low rate of induction of drug resistance by selection of mutant bacilli. Two additional rifamycins, rifapentine and rifabutin, are also available in the United States; however, the level of cross-resistance with rifampin is high. For a detailed discussion of the drugs used for the treatment of TB, see Chap. 205e.

1	Because of a lower degree of efficacy and a higher degree of intolerability and toxicity, six classes of second-line drugs are generally used only for the treatment of patients with TB resistant to first-line drugs: (1) the fluoroquinolone antibiotics; (2) the injectable aminoglycosides kanamycin, amikacin, and streptomycin; (3) the injectable polypeptide capreomycin; and the oral agents (4) ethionamide and prothionamide, (5) cycloserine and terizidone (therizidone), and (6) PAS. Streptomycin, formerly a first-line agent, is now rarely used for drug-resistant TB because resistance levels worldwide are high and it is more toxic than the other drugs in the same class; however, the level of cross-resistance with the other injectables is low. Of the quinolones, later-generation agents such as levofloxacin and moxifloxacin are preferred. Gatifloxacin (no longer marketed in several countries, including the United States, because of previously observed dysglycemia) has recently been tested

1	levofloxacin and moxifloxacin are preferred. Gatifloxacin (no longer marketed in several countries, including the United States, because of previously observed dysglycemia) has recently been tested in a 4-month regi men that produced no detectable major side effects; thus, this drug could be reconsidered as a good alternative. Other drugs (referred to by the WHO as “group 5”) whose efficacy is not clearly defined are used in the treatment of patients with TB resistant to most of the firstand second-line agents; these drugs include clofazimine, linezolid, amoxicillin/clavulanic acid, clarithromycin, and carbapenems such as imipenem/cilastatin and meropenem. Today amithiozone (thiacetazone) is used very rarely because it has been associated with severe and at times fatal skin reactions among HIV-infected patients. Two novel drugs belonging to two new antibiotic classes—the diarylquinoline bedaquiline and the nitroimidazole delamanid—have recently been approved for use in severe cases of

1	HIV-infected patients. Two novel drugs belonging to two new antibiotic classes—the diarylquinoline bedaquiline and the nitroimidazole delamanid—have recently been approved for use in severe cases of MDR-TB by stringent regulatory authorities (the U.S. Food and Drug Administration

1	Isoniazid 5 mg/kg, max 300 mg 10 mg/kg, max 900 mg Rifampin 10 mg/kg, max 600 mg 10 mg/kg, max 600 mg Pyrazinamide 25 mg/kg, max 2 g 35 mg/kg, max 3 g aThe duration of treatment with individual drugs varies by regimen, as detailed in Table 202-3. bThe World Health Organization recommends the following dosages for children: isoniazid,10–15 mg/kg daily, max 300 mg/d; rifampin, 15 (range, 10–20) mg/kg daily, max 600 mg/d; pyrazinamide, 35 (range, 30–40) mg/kg daily; ethambutol, 20 (range, 15–25) mg/kg daily. cIn certain settings, streptomycin (15 mg/kg daily, with a maximum dose of 1 g; or 25–30 mg/kg thrice weekly, with a maximum dose of 1.5 g) can replace ethambutol in the initial phase of treatment. However, streptomycin generally is no longer considered a first-line drug. Source: Based on recommendations of the American Thoracic Society/Infectious Diseases Society of America/Centers for Disease Control and Prevention and the World Health Organization.

1	Source: Based on recommendations of the American Thoracic Society/Infectious Diseases Society of America/Centers for Disease Control and Prevention and the World Health Organization. 1116 [FDA] and the European Medicine Agency [EMA] in the case of bedaquiline; the EMA and the Pharmaceuticals and Medical Devices Agency of Japan in the case of delamanid).

1	Standard short-course regimens are divided into an initial, or bactericidal, phase and a continuation, or sterilizing, phase. During the initial phase, the majority of the tubercle bacilli are killed, symptoms resolve, and usually the patient becomes noninfectious. The continuation phase is required to eliminate persisting mycobacteria and prevent relapse. The treatment regimen of choice for virtually all forms of drug-susceptible TB in adults consists of a 2-month initial (or intensive) phase of isoniazid, rifampin, pyrazinamide, and ethambutol followed by a 4-month continuation phase of isoniazid and rifampin (Table 202-3). This regimen can cure TB in more than 90% of patients. In children, most forms of TB in the absence of HIV infection or suspected isoniazid resistance can be safely treated without ethambutol in the intensive phase. Treatment should be given daily throughout the course. However, daily treatment during the intensive phase and intermittently (three times weekly)

1	treated without ethambutol in the intensive phase. Treatment should be given daily throughout the course. However, daily treatment during the intensive phase and intermittently (three times weekly) during the continuation phase is an alternative for patients who can be directly supervised and properly supported. A fully supervised, three-timesweekly regimen throughout the course also can be offered in the absence of HIV infection, although the risk of acquired drug resistance is higher than that among patients treated daily for the full course. In addition, if the infecting strain is resistant to isoniazid, the risks of both acquired resistance and treatment failure are higher with three-times-weekly intensive therapy than with daily treatment in the intensive phase. HIV-infected patients should always receive their initial-phase regimen daily (see below). A continuation phase of once-weekly rifapentine and isoniazid has been shown to be equally effective for HIV-seronegative patients

1	should always receive their initial-phase regimen daily (see below). A continuation phase of once-weekly rifapentine and isoniazid has been shown to be equally effective for HIV-seronegative patients with noncavitary pulmonary TB who have negative sputum cultures at 2 months. Patients with cavitary pulmonary TB and delayed sputum-culture conversion (i.e., those who remain culture-positive at 2 months) should be tested immediately for drug-resistant TB, and a change of regimen should be considered. To prevent isoniazid-related neuropathy, pyridoxine (10–25 mg/d) should be added to the regimen given to persons at high risk of vitamin B6 deficiency (e.g., alcoholics; malnourished persons; pregnant and lactating women; and patients with conditions such as chronic renal failure, diabetes, and HIV infection, which are also associated with neuropathy). A full course of therapy (completion of treatment) is defined more accurately by the total number of doses taken than by the duration of

1	and HIV infection, which are also associated with neuropathy). A full course of therapy (completion of treatment) is defined more accurately by the total number of doses taken than by the duration of treatment, although the course should not include interruptions of longer than 4 weeks. Specific recommendations on the required number of doses for each of the various treatment regimens have been published jointly by the American Thoracic Society, the Infectious Diseases Society of America, and the CDC. In some developing countries where the ability to ensure adherence to treatment is limited, a continuation-phase regimen of daily isoniazid and ethambutol for 6 months was used in the past. However, this regimen is associated with a higher rate of relapse, failure, and death, especially among HIV-infected patients, and is no longer recommended by the WHO.

1	Lack of adherence to treatment is recognized worldwide as the most important impediment to cure. Moreover, the tubercle bacilli infecting patients who do not fully adhere to the prescribed regimen are likely to become drug resistant. Both patientand provider-related factors may affect adherence. Patient-related factors include a lack of belief that the illness is significant and/or that treatment will have a beneficial effect; the existence of concomitant medical conditions (notably alcohol or substance abuse); lack of social support; fear of stigma and discrimination associated with TB; and poverty, with attendant joblessness and homelessness. Provider-related factors that may promote adherence include the support, education, and encouragement of patients and the offering of convenient clinic hours. In addition to specific measures promoting adherence, two other strategic approaches are used: direct supervision of treatment with support to the patient, consisting of incentives and

1	clinic hours. In addition to specific measures promoting adherence, two other strategic approaches are used: direct supervision of treatment with support to the patient, consisting of incentives and enablers such as meals, travel vouchers, cash transfers, and grants to replace income loss; and provision of fixed-drug-combination products that reduce the number of tablets the patient needs to swallow. Because it is difficult to predict which patients will adhere to the recommended treatment for a disease that has important public as well as individual health implications, all patients should have their therapy directly supervised, especially during the initial phase, with

1	Resistance (or intolerance) to H Throughout (6–9) RZEh MDR-TB (resistance to at least H + R) Throughout (20 months in most cases) Q, Injj, Eto/Pto, Z, Cs/PAS

1	Intolerance to Z 2 HRE 7 HR aAll drugs can be given daily or intermittently (three times weekly throughout). A twice-weekly regimen after 2–8 weeks of daily therapy during the initial phase is sometimes used, although it is not recommended by the WHO. bStreptomycin can be used in place of ethambutol but is no longer considered a first-line drug. cSome experts suggest extending the continuation phase to 7 months for patients with cavitary pulmonary tuberculosis who remain sputum culture–positive after the initial phase of treatment. However, treatment in such patients must be guided by drug susceptibility testing to rule out drug-resistant TB. dA clinical trial showed that HIV-negative patients with noncavitary pulmonary tuberculosis who have negative sputum AFB smears after the initial phase of treatment can be given once-weekly rifapentine/isoniazid in the continuation phase. eThe 6-month regimen with pyrazinamide can probably be used safely during pregnancy and is recommended by the

1	of treatment can be given once-weekly rifapentine/isoniazid in the continuation phase. eThe 6-month regimen with pyrazinamide can probably be used safely during pregnancy and is recommended by the WHO and the International Union Against Tuberculosis and Lung Disease. If pyrazinamide is not included in the initial treatment regimen, the minimal duration of therapy is 9 months. fStreptomycin should be discontinued after 2 months. Drug susceptibility results will determine the best regimen option. gThe availability of rapid molecular methods to identify drug resistance allows initiation of a proper regimen at the start of treatment. hAlthough normally not recommended, a fluoroquinolone may strengthen the regimen for patients with extensive disease. A later-generation agent (such as levofloxacin, moxifloxacin, or possibly gatifloxacin; see text) is preferred. iIsoniazid is added if susceptibility to this agent is confirmed or presumed. jAmikacin and kanamycin (aminoglycosides) or

1	moxifloxacin, or possibly gatifloxacin; see text) is preferred. iIsoniazid is added if susceptibility to this agent is confirmed or presumed. jAmikacin and kanamycin (aminoglycosides) or capreomycin (polypeptide). Any of these injectable agents is recommended for the first 8 months in most patients, but the duration may be modified according to the clinical response to therapy. Continuation of treatment with the injectable drug for at least 4 months after culture conversion is advised.

1	Abbreviations: Cs/PAS, cycloserine or para-aminosalicylic acid; E, ethambutol; Eto/Pto, ethionamide or prothionamide; H, isoniazid; Inj, an injectable agent (the aminoglycosides amikacin and kanamycin or the polypeptide capreomycin); MDR-TB, multidrug-resistant tuberculosis; Q, a quinolone antibiotic; R, rifampin; S, streptomycin; WHO, World Health Organization; XDR-TB, extensively drug-resistant tuberculosis; Z, pyrazinamide.

1	proper social support including education, psychosocial counseling, and material sustainment. In an increasing number of countries, personnel to supervise therapy are usually available through TB control programs of local public health departments and from members of the community who are accepted by the patient to undertake that role and who have been properly educated by health workers. Direct supervision with patient support usually increases the proportion of patients completing treatment in all settings and greatly lessens the chances of failure, relapse, and acquired drug resistance. Fixed-drug-combination products (e.g., isoniazid/rifampin, isoniazid/rifampin/pyrazinamide, and isoniazid/rifampin/pyrazinamide/ ethambutol) are available and are strongly recommended as a means of minimizing the likelihood of prescription error and of the development of drug resistance as the result of monotherapy. In some formulations of these combination products, the bioavailability of rifampin

1	the likelihood of prescription error and of the development of drug resistance as the result of monotherapy. In some formulations of these combination products, the bioavailability of rifampin has been found to be substandard. Stringent regulatory authorities ensure that combination products are of good quality; however, this type of quality assurance is not always operative in low-income countries. Alternative regimens for patients who exhibit drug intolerance or adverse reactions are listed in Table 202-3. However, severe side effects prompting discontinuation of any of the first-line drugs and use of these alternative regimens are uncommon. The fluoroquinolones moxifloxacin and gatifloxacin have been tested as 4-month treatment-shortening regimens for drug-susceptible TB. Recently published results from these clinical trials failed to show that a 4-month regimen substituting gatifloxacin for ethambutol or moxifloxacin for either ethambutol or isoniazid is noninferior to the

1	published results from these clinical trials failed to show that a 4-month regimen substituting gatifloxacin for ethambutol or moxifloxacin for either ethambutol or isoniazid is noninferior to the standard 6-month regimen. Thus, currently there is no 4-month regimen available for TB treatment.

1	Bacteriologic evaluation through culture and/or smear microscopy is essential in monitoring the response to treatment for TB. In addition, the patient’s weight should be monitored regularly and the drug dosage adjusted with any significant weight change. Patients with pulmonary disease should have their sputum examined monthly until cultures become negative to allow early detection of treatment failure. With the recommended regimen, more than 80% of patients will have negative sputum cultures at the end of the second month of treatment. By the end of the third month, the sputum of virtually all patients should be culture negative. In some patients, especially those with extensive cavitary disease and large numbers of organisms, AFB smear conversion may lag behind culture conversion. This phenomenon is presumably due to the expectoration and microscopic visualization of dead bacilli. As noted above, patients with cavitary disease in whom sputum culture conversion does not occur by 2

1	phenomenon is presumably due to the expectoration and microscopic visualization of dead bacilli. As noted above, patients with cavitary disease in whom sputum culture conversion does not occur by 2 months require immediate testing for drug resistance. When a patient’s sputum cultures remain positive at ≥3 months, treatment failure and drug resistance or poor adherence to the regimen are likely, and testing of drug resistance should guide the choice of the best treatment option (see below). A sputum specimen should be collected by the end of treatment to document cure. If mycobacterial cultures are not practical, then monitoring by AFB smear examination should be undertaken at 2, 5, and 6 months. Smears that are positive after 3 months of treatment when the patient is known to be adherent are indicative of treatment failure and possible drug resistance. Therefore, if not done at the start of treatment, drug susceptibility testing is mandatory at this stage. Bacteriologic monitoring of

1	are indicative of treatment failure and possible drug resistance. Therefore, if not done at the start of treatment, drug susceptibility testing is mandatory at this stage. Bacteriologic monitoring of patients with extrapulmonary TB is more difficult and often is not feasible. In these cases, the response to treatment must be assessed clinically and radiographically.

1	Monitoring of the response during chemotherapy by nucleic acid amplification technology has not been shown to be suitable. Thus Xpert MTB/RIF should not be used to monitor treatment. Likewise, serial chest radiographs are not recommended because radiographic changes may lag behind bacteriologic response and are not highly sensitive. After the completion of treatment, neither sputum examination nor chest radiography is recommended for routine follow-up purposes. However, a chest radiograph obtained at the end of treatment may be useful for comparative purposes 1117 should the patient develop symptoms of recurrent TB months or years later. Patients should be instructed to report promptly for medical assessment if they develop any such symptoms. In addition, an end-of-treatment chest radiograph may reveal earlier the post-TB complications described above.

1	During treatment, patients should be monitored for drug toxicity. The most common adverse reaction of significance is hepatitis. Patients should be carefully educated about the signs and symptoms of drug-induced hepatitis (e.g., dark urine, loss of appetite) and should be instructed to discontinue treatment promptly and see their health care provider should these symptoms occur. Although biochemical monitoring is not routinely recommended, all adult patients should undergo baseline assessment of liver function (e.g., measurement of serum levels of hepatic aminotransferases and bilirubin). Older patients, those with concomitant diseases, those with a history of hepatic disease (especially hepatitis C), and those using alcohol daily should be monitored especially closely (i.e., monthly), with repeated measurements of aminotransferases, during the initial phase of treatment. Up to 20% of patients have small increases in aspartate aminotransferase (up to three times the upper limit of

1	with repeated measurements of aminotransferases, during the initial phase of treatment. Up to 20% of patients have small increases in aspartate aminotransferase (up to three times the upper limit of normal) that are not accompanied by symptoms and are of no consequence. For patients with symptomatic hepatitis and those with marked (fiveto sixfold) elevations in serum levels of aspartate aminotransferase, treatment should be stopped and drugs reintroduced one at a time after liver function has returned to normal. Hypersensitivity reactions usually require the discontinuation of all drugs and rechallenge to determine which agent is the culprit. Because of the variety of regimens available, it usually is not necessary—although it is possible—to desensitize patients. Hyperuricemia and arthralgia caused by pyrazinamide can usually be managed by the administration of acetylsalicylic acid; however, pyrazinamide treatment should be stopped if the patient develops gouty arthritis. Individuals

1	caused by pyrazinamide can usually be managed by the administration of acetylsalicylic acid; however, pyrazinamide treatment should be stopped if the patient develops gouty arthritis. Individuals who develop autoimmune thrombocytopenia secondary to rifampin therapy should not receive the drug thereafter. Similarly, the occurrence of optic neuritis with ethambutol is an indication for permanent discontinuation of this drug. Other common manifestations of drug intolerance, such as pruritus and gastrointestinal upset, can generally be managed without the interruption of therapy.

1	As stated above, treatment failure should be suspected when a patient’s sputum smears and/or cultures remain positive after 3 months of treatment. In the management of such patients, it is imperative that the current isolate be urgently tested for susceptibility to firstand second-line agents. Initial molecular testing for rifampin resistance should be done if the technology is available. When the results of susceptibility testing are based on molecular methods and are expected to become available within a few days, changes in the regimen can be postponed until that time. However, if the patient’s clinical condition is deteriorating, an earlier change in regimen may be indicated. A cardinal rule in the latter situation is always to add more than one drug at a time to a failing regimen: at least two and preferably three drugs that have never been used and to which the bacilli are likely to be susceptible should be added. The patient may continue to take isoniazid and rifampin along

1	at least two and preferably three drugs that have never been used and to which the bacilli are likely to be susceptible should be added. The patient may continue to take isoniazid and rifampin along with these new agents pending the results of susceptibility tests.

1	Patients who experience a recurrence after apparently successful treatment (relapse) are less likely to harbor drug-resistant strains (see below) than are patients in whom treatment has failed. Acquired resistance is uncommon among strains from patients in whom relapse follows the completion of a standard short-course regimen. However, pending the results of susceptibility testing, it is prudent to begin the treatment of all patients whose infections have relapsed with a standard regimen containing all four first-line drugs plus streptomycin. In less affluent countries and other settings where facilities for culture and drug susceptibility testing are not yet routinely available and where the prevalence of MDR-TB is low, the WHO recommends that a standard regimen with all four 1118 first-line drugs plus streptomycin be used in all instances of relapse and treatment default. Patients with treatment failure and those relapsing or defaulting with a high likelihood of MDR-TB should

1	first-line drugs plus streptomycin be used in all instances of relapse and treatment default. Patients with treatment failure and those relapsing or defaulting with a high likelihood of MDR-TB should receive a regimen that includes second-line agents and is based on their history of anti-TB treatment and the drug resistance patterns in the population (Table 202-3). Once drug susceptibility test results are available, the regimen can be adjusted accordingly.

1	Strains of M. tuberculosis resistant to individual drugs arise by spontaneous point mutations in the mycobacterial genome that occur at low but predictable rates (10–7–10–10 for the key drugs). Resistance to rifampin is associated with mutations in the rpoB gene in 95% of cases; that to isoniazid with mutations mainly in the katG (50–95% of cases) and inhA (up to 45%) genes; that to pyrazinamide in the pncA gene (up to 98%); that to ethambutol in the embB gene (50–65%); that to the fluoroquinolones in the gyrA–gyrB genes (75–95%); and that to the aminoglycosides mainly in the rrs gene (up to 80%). Because there is no cross-resistance among the commonly used drugs, the probability that a strain will be resistant to two drugs is the product of the probabilities of resistance to each drug and thus is low. The development of drug-resistant TB is almost invariably the result of monotherapy—i.e., the failure of the health care provider to prescribe at least two drugs to which tubercle

1	and thus is low. The development of drug-resistant TB is almost invariably the result of monotherapy—i.e., the failure of the health care provider to prescribe at least two drugs to which tubercle bacilli are susceptible or of the patient to take properly prescribed therapy. In addition, the use of drugs of substandard quality may cause the emergence of drug resistance. Drug-resistant TB may be either primary or acquired. Primary drug resistance is that which develops in a patient infected from the start by a drug-resistant strain. Acquired resistance is that which develops during treatment with an inappropriate regimen. In North America, Western Europe, most of Latin America, and the Persian Gulf States, rates of primary resistance are generally low and isoniazid resistance is most common. In the United States, although rates of primary isoniazid resistance have been stable at ~7–8%, the rate of primary MDR-TB has declined from 2.5% in 1993 to 1% since 2000. As described above,

1	common. In the United States, although rates of primary isoniazid resistance have been stable at ~7–8%, the rate of primary MDR-TB has declined from 2.5% in 1993 to 1% since 2000. As described above, MDR-TB is an increasingly serious problem in some regions, especially in the states of the former Soviet Union and some countries of Asia (Fig. 202-11). Even more serious is the recently described occurrence of XDR-TB due to MDR strains that are also resistant to any fluoroquinolones and to any of three second-line injectable agents (amikacin, kanamycin, and capreomycin). Creation of drug-resistant TB can be prevented by adherence to the principles of sound treatment: inclusion of at least two quality-assured, bactericidal drugs to which the organism is susceptible; use of fixed-drug-combination products; supervision of treatment with patient support; and verification that patients complete the prescribed course. Transmission of drug-resistant strains can be prevented by implementation of

1	products; supervision of treatment with patient support; and verification that patients complete the prescribed course. Transmission of drug-resistant strains can be prevented by implementation of respiratory infection-control measures (see below).

1	Although the 6-month regimen described in Table 202-3 is generally effective for patients with initial isoniazid-resistant disease, it is prudent to include at least ethambutol and possibly pyrazinamide for the full 6 months and to consider extending the treatment course to 9 months. In such cases, isoniazid probably does not contribute to a successful outcome and could be omitted. In case of documented resistance to both isoniazid and ethambutol, a 9to 12-month regimen of rifampin, pyrazinamide, and a fluoroquinolone can be used. Any patients whose isolates exhibit resistance to rifampin should be managed as if they had MDR-TB (see below), with the addition of isoniazid if susceptibility to this agent is confirmed via rapid testing or is presumed. MDR-TB, in which bacilli are resistant to (at least) isoniazid and rifampin, is more difficult to manage than is disease caused by drug-susceptible organisms because these two bactericidal drugs are the most potent agents available and

1	to (at least) isoniazid and rifampin, is more difficult to manage than is disease caused by drug-susceptible organisms because these two bactericidal drugs are the most potent agents available and because associated resistance to other first-line drugs as well (e.g., ethambutol) is not uncommon. For treatment of MDR-TB, the WHO recommends that in most patients five drugs be used in the initial phase of at least 8 months: a later-generation fluoroquinolone, an injectable agent (the aminoglycosides amikacin or kanamycin or the polypeptide capreomycin), ethionamide (or prothionamide), either cycloserine or PAS, and pyrazinamide. Ethambutol can be added (Table 202-3). Although the optimal duration of treatment is not known, a course of at least 20 months is recommended for previously untreated patients, including the initial phase with an injectable agent, which is usually discontinued at 4 months after culture conversion.

1	In late 2012, the FDA granted accelerated approval of bedaquiline, a diarylquinoline antibiotic. This new drug, when given for the first 24 weeks (400 mg daily for 2 weeks followed by 200 mg thrice weekly for 22 weeks), has been shown to increase the efficacy of the WHO standard regimen for MDR-TB with faster sputum conversion. Percentage of cases 0–2.9 3–5.9 6–11.9 12–17.9 FIGuRE 202-11 Percentage of new tuberculosis cases with multidrug resistance in all countries surveyed by the World Health Organization (WHO) Global Drug Resistance Surveillance Project during 1994–2013. (See disclaimer in Fig. 202-2. Courtesy of the Global TB Programme, WHO; with permission.)

1	Bedaquiline should be used with caution in people >65 years of age and in HIV-infected patients; its use is not advised in children and pregnant women. In early 2014, the European Medical Agency granted accelerated approval of another new agent, the nitroimidazole compound delamanid. Data from a phase 2B clinical trial in which delamanid was added to the WHO-recommended standard MDR-TB regimen have shown increased culture conversion at 2 months. Pending phase 3 trial results and in view of potential side effects of both new drugs (including QT interval prolongation in both cases and hepatotoxicity in the case of bedaquiline), the WHO recommends limiting the use of bedaquiline and delamanid to cases of MDR-TB when an effective WHO-recommended standard MDR-TB regimen cannot be designed because of known resistance, intolerance, or nonavailability of any second-line drugs in the regimen. Patients treated with bedaquiline or delamanid should be counseled, should give informed consent, and

1	of known resistance, intolerance, or nonavailability of any second-line drugs in the regimen. Patients treated with bedaquiline or delamanid should be counseled, should give informed consent, and should be closely monitored during treatment. In particular, patients with cardiac anomalies such as prolonged QT interval or a history of ventricular arrhythmias should not be given these drugs. Currently, there is no information about simultaneous use of these two agents; therefore, combining them is not recommended.

1	Finally, a shorter (9-month) regimen consisting of gatifloxacin or moxifloxacin, clofazimine, ethambutol, and pyrazinamide given throughout the treatment period and supplemented by prothionamide, kanamycin, and high-dose isoniazid during an intensive phase of at least 4 months is reportedly effective for MDR-TB in certain settings. Further investigations are necessary to elucidate the role of this shorter regimen in MDR-TB treatment.

1	Patients with XDR-TB have fewer treatment options and a much poorer prognosis. However, observational studies have shown that aggressive management of cases comprising early drug-susceptibility testing, rational combination of at least five drugs, readjustment of the regimen, strict directly observed therapy, monthly bacteriologic monitoring, and intensive patient support may result in cure and avert death. Table 202-4 summarizes the management of patients with XDR-TB. Some recently published studies regarding the use of linezolid in patients with XDR-TB suggest that, although it carries a high level of toxicity, this drug increases culture conversion.

1	For patients with localized disease and sufficient pulmonary reserve, lobectomy or pneumonectomy may be considered. Because the management of patients with MDRand XDR-TB is complicated by both social and medical factors, care of these patients is ideally provided in specialized centers or, in their absence, in the context of programs with adequate resources and capacity, including community support.

1	Several observational studies and randomized controlled trials have shown that treatment of HIV-associated TB with anti-TB drugs and simultaneous use of ART are associated with significant reductions in mortality risk and AIDS-related events. Evidence from randomized controlled trials shows that early initiation of ART during anti-TB treatment is associated with a 34–68% reduction in mortality rates, with especially good results in patients with CD4+ T cell counts of <50/μL. Therefore, the main aim in the management of HIV-associated TB is to initiate anti-TB treatment and to immediately consider initiating or continuing ART. All HIV-infected TB patients, regardless of CD4+ T cell count, are candidates for ART, which optimally is initiated as soon as possible after the diagnosis of TB and within the first 8 weeks of anti-TB therapy. However, ART should be started within the first 2 weeks of TB treatment for patients with CD4+ T cell counts of <50/μL. In general, the standard 6-month

1	within the first 8 weeks of anti-TB therapy. However, ART should be started within the first 2 weeks of TB treatment for patients with CD4+ T cell counts of <50/μL. In general, the standard 6-month daily regimen is equally efficacious in HIV-negative and HIV-positive patients for treatment of drug-susceptible TB. As for any other adult living with HIV (Chap. 226), first-line ART for TB patients should consist of two nucleoside reverse transcriptase inhibitors (NRTIs) plus a nonnucleoside reverse transcriptase inhibitor (NNRTI). Although TB treatment modalities are similar to those in HIV-negative patients, adverse drug effects may be more pronounced in HIV-infected patients. In this 1.

1	Use pyrazinamide and any first-line oral agents that may be effective. 2. Use an injectable agent to which the strain is susceptible, and consider an extended duration of use (12 months or possibly the whole treatment period). If the strain is resistant to all injectable agents, use of one that the patient has not previously received is recommended.a 3. Use a later-generation fluoroquinolone, such as moxifloxacin, high-dose levofloxacin, or possibly gatifloxacin.b 4. Use all second-line oral bacteriostatic agents (para-aminosalicylic acid, cycloserine, and ethionamide or prothionamide) that have not been used extensively in a previous regimen or any such agents that are likely to be effective. 5. Add bedaquiline or delamanid and one or more of the following drugsc: clofazimine, linezolid, amoxicillin/clavulanic acid, clarithromycin, and carbapenems such as imipenem/cilastatin and meropenem. 6.

1	6. The simultaneous use of bedaquiline and delamanid is not recommended at the moment in view of the current lack of information on the potential of adverse reactions when these drugs are administered together. 7. Consider treatment with high-dose isoniazid if low-level resistance to this drug is documented. 8. Consider adjuvant surgery if there is localized disease. 9. Enforce strong infection-control measures. 10. Implement strict directly observed therapy and full adherence support as well as comprehensive bacteriologic and clinical monitoring.

1	aThis recommendation is made because, although the reproducibility and reliability of susceptibility testing with injectable agents are good, few data are available on the correlation of clinical efficacy with test results. Options with XDR-TB are very limited, and some strains may be affected in vivo by an injectable agent even though they test resistant in vitro. bGatifloxacin (no longer marketed in several countries, including the United States, because of previously observed dysglycemia) has recently been tested in a 4-month regimen that produced no detectable major side effects; thus, this drug could be reconsidered as a good alternative. cThe number of drugs added is based on how many oral bacteriostatic drugs (see point 4 above) are believed to be effective: the advice is to add one drug if there is confidence in all three bacteriostatic drugs; two if there is confidence in only two bacteriostatic drugs; and three or more if there is confidence in only one bacteriostatic drug

1	drug if there is confidence in all three bacteriostatic drugs; two if there is confidence in only two bacteriostatic drugs; and three or more if there is confidence in only one bacteriostatic drug or none.

1	regard, three important considerations are relevant: an increased frequency of paradoxical reactions, interactions between ART components and rifamycins, and development of rifampin monoresistance with intermittent treatment. IRIS—i.e., the exacerbation of symptoms and signs of TB—has been described above. Rifampin, a potent inducer of enzymes of the cytochrome P450 system, lowers serum levels of many HIV protease inhibitors and some NNRTIs— essential drugs used in ART. In such cases, rifabutin, which has much less enzyme-inducing activity, has been used in place of rifampin. However, dosage adjustments for rifabutin and protease inhibitors are still being assessed. Several clinical trials have found that patients with HIV-associated TB whose degree of immunosuppression is advanced (e.g., CD4+ T cell counts of <100/μL) are prone to treatment failure and relapse with rifampin-resistant organisms when treated with “highly intermittent” (i.e., onceor twice-weekly) rifamycincontaining

1	CD4+ T cell counts of <100/μL) are prone to treatment failure and relapse with rifampin-resistant organisms when treated with “highly intermittent” (i.e., onceor twice-weekly) rifamycincontaining regimens. Consequently, it is recommended that all TB patients who are infected with HIV receive a rifampin-containing regimen on a daily basis. Because recommendations are frequently updated, consultation of the following websites is advised: www.who .int/hiv, www.who.int/tb, www.cdc.gov/hiv, and www.cdc.gov/tb.

1	Although comparative clinical trials of treatment for extrapulmonary TB are limited, the available evidence indicates that most forms of disease can be treated with the 6-month regimen recommended for patients with pulmonary disease. The WHO and the American Academy of Pediatrics recommend that children with bone and joint TB, tuberculous meningitis, or miliary TB receive up to 12 months of treatment. Treatment for TB may be complicated by underlying medical problems that require special consideration. As a rule, patients with chronic renal failure should not receive aminoglycosides and should receive ethambutol only if serum drug levels 1120 can be monitored. Isoniazid, rifampin, and pyrazinamide may be given in the usual doses in cases of mild to moderate renal failure, but the dosages of isoniazid and pyrazinamide should be reduced for all patients with severe renal failure except those undergoing hemodialysis. Patients with hepatic disease pose a special problem because of the

1	of isoniazid and pyrazinamide should be reduced for all patients with severe renal failure except those undergoing hemodialysis. Patients with hepatic disease pose a special problem because of the hepatotoxicity of isoniazid, rifampin, and pyrazinamide. Patients with severe hepatic disease may be treated with ethambutol, streptomycin, and possibly another drug (e.g., a fluoroquinolone); if required, isoniazid and rifampin may be administered under close supervision. The use of pyrazinamide by patients with liver failure should be avoided. Silicotuberculosis necessitates the extension of therapy by at least 2 months. The regimen of choice for pregnant women (Table 202-3) is 9 months of treatment with isoniazid and rifampin supplemented by ethambutol for the first 2 months. Although the WHO has recommended routine use of pyrazinamide for pregnant women, this drug has not been recommended in the United States because of insufficient data documenting its safety in pregnancy. Streptomycin

1	has recommended routine use of pyrazinamide for pregnant women, this drug has not been recommended in the United States because of insufficient data documenting its safety in pregnancy. Streptomycin is contraindicated because it is known to cause eighth-cranial-nerve damage in the fetus. Treatment for TB is not a contraindication to breast-feeding; most of the drugs administered will be present in small quantities in breast milk, albeit at concentrations far too low to provide any therapeutic or prophylactic benefit to the child. Medical consultation on difficult-to-manage cases is provided by the U.S. CDC Regional Training and Medical Consultation Centers (www.cdc.gov/tb/education/rtmc/).

1	The best way to prevent TB is to diagnose and isolate infectious cases rapidly and to administer appropriate treatment until patients are rendered noninfectious (usually 2–4 weeks after the start of proper treatment) and the disease is cured. Additional strategies include BCG vaccination and treatment of persons with LTBI who are at high risk of developing active disease.

1	BCG was derived from an attenuated strain of M. bovis and was first administered to humans in 1921. Many BCG vaccines are available worldwide; all are derived from the original strain, but the vaccines vary in efficacy, ranging from 80% to nil in randomized, placebo-controlled trials. A similar range of efficacy was found in recent observational studies (case–control, historic cohort, and cross-sectional) in areas where infants are vaccinated at birth. These studies and a meta-analysis also found higher rates of efficacy in the protection of infants and young children from serious disseminated forms of childhood TB, such as tuberculous meningitis and miliary TB. BCG vaccine is safe and rarely causes serious complications. The local tissue response begins 2–3 weeks after vaccination, with scar formation and healing within 3 months. Side effects—most commonly, ulceration at the vaccination site and regional lymphadenitis—occur in 1–10% of vaccinated persons. Some vaccine strains have

1	scar formation and healing within 3 months. Side effects—most commonly, ulceration at the vaccination site and regional lymphadenitis—occur in 1–10% of vaccinated persons. Some vaccine strains have caused osteomyelitis in ~1 case per million doses administered. Disseminated BCG infection (“BCGitis”) and death have occurred in 1–10 cases per 10 million doses administered, although this problem is restricted almost exclusively to persons with impaired immunity, such as children with severe combined immunodeficiency syndrome or adults with HIV infection. BCG vaccination induces TST reactivity, which tends to wane with time. The presence or size of TST reactions after vaccination does not predict the degree of protection afforded.

1	BCG vaccine is recommended for routine use at birth in countries with high TB prevalence. However, because of the low risk of transmission of TB in the United States and other high-income countries, the unreliable protection afforded by BCG, and its impact on the TST, the vaccine is not recommended for general use. HIV-infected adults and children should not receive BCG vaccine. Moreover, infants whose HIV status is unknown but who have signs and symptoms consistent with HIV infection or who are born to HIV-infected mothers should not receive BCG. Over the past decade, renewed research and development efforts have been made toward a new TB vaccine. In mid-2014, 16 candidates were in clinical trials and 12 were being field tested. The first new vaccine, for which results of a clinical trial became available in early 2013, is MVA85A/AERAS-485; unfortunately, this viral-vectored vaccine did not show clinical benefit as a booster to BCG.

1	It is estimated that about 2 billion people, or nearly one-third of the human population, have been infected with M. tuberculosis. Although only a small fraction of these infections will progress toward active disease, new active cases will continue to emerge from this pool of “latently” infected individuals. Unfortunately, there is no diagnostic test at present that can predict which individuals with LTBI will develop active TB. Treatment of selected persons with LTBI aims at preventing active disease. This intervention (also called preventive chemotherapy or chemoprophylaxis) is based on the results of a large number of randomized, placebo-controlled clinical trials demonstrating that a 6to 9-month course of isoniazid reduces the risk of active TB in infected people by up to 90%. Analysis of available data indicates that the optimal duration of treatment is ~9 months. In the absence of reinfection, the protective effect is believed to be lifelong. Clinical trials have shown that

1	of available data indicates that the optimal duration of treatment is ~9 months. In the absence of reinfection, the protective effect is believed to be lifelong. Clinical trials have shown that isoniazid reduces rates of TB among TST-positive persons with HIV infection. Studies in HIV-infected patients have also demonstrated the effectiveness of shorter courses of rifampinbased treatment.

1	Candidates for treatment of LTBI are listed in Table 202-5. They can be identified by TST or IGRA of persons in defined high-risk groups. For skin testing, 5 tuberculin units of polysorbate-stabilized PPD should be injected intradermally into the volar surface of the forearm (i.e., the Mantoux method). Multipuncture tests are not recommended. Reactions are read at 48–72 h as the transverse

1	Tuberculin Risk Group Reaction Size, mm aTuberculin-negative contacts, especially children, should receive prophylaxis for 2–3 months after contact ends and should then undergo repeat TST. Those whose results remain negative should discontinue prophylaxis. HIV-infected contacts should receive a full course of treatment regardless of TST results. bThese conditions include silicosis and end-stage renal disease managed by hemodialysis cThese settings include correctional facilities, nursing homes, homeless shelters, and hospitals and other health care facilities. dExcept for employment purposes where longitudinal TST screening is anticipated, TST is not indicated for these low-risk persons. A decision to treat should be based on individual risk/benefit considerations.

1	Source: Adapted from Centers for Disease Control and Prevention: TB elimination—treatment options for latent tuberculosis infection (2011). Available at http://www.cdc.gov/tb/ publications/factsheets/testing/skintestresults.pdf.

1	diameter (in millimeters) of induration; the diameter of erythema is not considered. In some persons, TST reactivity wanes with time but can be recalled by a second skin test administered ≥1 week after the first (i.e., two-step testing). For persons periodically undergoing the TST, such as health care workers and individuals admitted to long-term-care institutions, initial two-step testing may preclude subsequent misclassification of those who have boosted reactions as TST converters. The cutoff for a positive TST (and thus for treatment) is related both to the probability that the reaction represents true infection and to the likelihood that the individual, if truly infected, will develop TB. Table 202-5 suggests possible cutoff by risk group. Thus, positive reactions for persons with HIV infection, recent close contacts of infectious cases, organ transplant recipients, previously untreated persons whose chest radiograph shows fibrotic lesions consistent with old TB, and persons

1	HIV infection, recent close contacts of infectious cases, organ transplant recipients, previously untreated persons whose chest radiograph shows fibrotic lesions consistent with old TB, and persons receiving drugs that suppress the immune system are defined as an area of induration ≥5 mm in diameter. A 10-mm cutoff is used to define positive reactions in most other at-risk persons. For persons with a very low risk of developing TB if infected, a cutoff of 15 mm is used. (Except for employment purposes where longitudinal screening is anticipated, the TST is not indicated for these low-risk persons.) A positive IGRA is based on the manufacturers’ recommendations; however, good clinical practice requires that epidemiologic and clinical factors also guide the decision to implement treatment for LTBI and that active TB be definitively excluded before the initiation of chemoprophylaxis.

1	Some TSTand IGRA-negative individuals are also candidates for treatment. Once an appropriate clinical evaluation has excluded active TB, infants and children who have come into contact with infectious cases should be treated for presumed LTBI. HIV-infected persons who have been exposed to an infectious TB patient should receive treatment regardless of the TST result. Any HIV-infected candidate for LTBI treatment must be screened carefully to exclude active TB, which would necessitate full treatment. The use of a clinical algorithm based on four symptoms (current cough, fever, weight loss, and night sweats) helps to define which HIV-infected person is a candidate for LTBI treatment. The absence of all four symptoms tends to exclude active TB. The presence of one of the four symptoms, on the other hand, warrants further investigation for active TB before treatment of LTBI is started. Although administering a TST is prudent, this test is not an absolute requirement—given the logistical

1	other hand, warrants further investigation for active TB before treatment of LTBI is started. Although administering a TST is prudent, this test is not an absolute requirement—given the logistical challenges—among people living with HIV in high-TB-incidence and low-resource settings.

1	Before treatment of LTBI begins, it is mandatory to carefully exclude active TB. Several regimens can be used to treat LTBI. The most widely used is that based on isoniazid alone at a daily dose of 5 mg/kg (up to 300 mg/d) for 9 months. On the basis of cost-benefit analyses and concerns about feasibility, a 6-month period of treatment is currently recommended by the WHO, especially in highly TB-endemic countries. Isoniazid can be administered intermittently (twice weekly) at a dose of 15 mg/kg (up to 900 mg) but only as directly observed therapy. An alternative regimen for adults is 4 months of daily rifampin. A 3-month regimen of daily isoniazid and rifampin is used in some countries (e.g., the United Kingdom) for both adults and children who are known not to have HIV infection. A previously recommended regimen of 2 months of rifampin and pyrazinamide has been associated with serious or even fatal hepatotoxicity and now is generally not recommended. The rifampin-containing regimens

1	recommended regimen of 2 months of rifampin and pyrazinamide has been associated with serious or even fatal hepatotoxicity and now is generally not recommended. The rifampin-containing regimens should be considered for persons who are likely to have been infected with an isoniazid-resistant strain. A recent clinical trial showed that a regimen of isoniazid (900 mg) and rifapentine (900 mg) given once weekly for 12 weeks is as effective as the standard 9-month isoniazid regimen. This regimen was associated with higher treatment completion (82% vs 69%) and less hepatotoxicity (0.4% vs 2.7%) than isoniazid alone, although the rate of permanent discontinuation due to an adverse event was higher (4.9% vs 3.7%).

1	Currently, the isoniazid–rifapentine regimen is not recommended for children <2 years of age, people living with HIV infection who are receiving ART, or pregnant women. Rifampin and rifapentine are contraindicated in HIV-infected individuals receiv-1121 ing protease inhibitors and most NNRTIs. (Efavirenz is the safest agent in this class of antiretrovirals for simultaneous administration with a rifamycin.) Clinical trials to assess the efficacy of long-term isoniazid administration (i.e., for at least 3 years) among people living with HIV in high-TB-transmission settings have shown that this regimen can be more effective than 9 months of isoniazid and is therefore recommended under those circumstances. Isoniazid should not be given to persons with active liver disease. All persons at increased risk of hepatotoxicity (e.g., those abusing alcohol daily and those with a history of liver disease) should undergo baseline and then monthly assessment of liver function. All patients should be

1	risk of hepatotoxicity (e.g., those abusing alcohol daily and those with a history of liver disease) should undergo baseline and then monthly assessment of liver function. All patients should be carefully educated about hepatitis and instructed to discontinue use of the drug immediately should any symptoms develop. Moreover, patients should be seen and questioned monthly during therapy about adverse reactions and should be given no more than a 1-month supply of drug at each visit. Treatment of LTBI among persons likely to have been infected by a multidrug-resistant strain is a challenge because no regimens have yet been tested in clinical trials. Close observation for early signs of disease is one option; consultation with a TB expert is advised.

1	It may be more difficult to ensure compliance when treating persons with LTBI than when treating those with active TB. If family members of active cases are being treated, compliance and monitoring may be easier. When feasible, supervised therapy may increase the likelihood of completion. As in active cases, the provision of incentives may also be helpful.

1	The highest priority in any TB control program is the prompt detection of cases and the provision of short-course chemotherapy to all TB patients under proper case-management conditions, including directly observed therapy. In addition, screening of high-risk groups, including immigrants from high-prevalence countries, migratory workers, prisoners, homeless individuals, substance abusers, and HIVseropositive persons, is recommended. TST-positive high-risk persons should be treated for LTBI as described above. Contact investigation is an important component of efficient TB control. In the United States and other countries worldwide, a great deal of attention has been given to the transmission of TB (particularly in association with HIV infection) in institutional settings such as hospitals, homeless shelters, and prisons. Measures to limit such transmission include respiratory isolation of persons with suspected TB until they are proven to be noninfectious (at least by sputum AFB smear

1	homeless shelters, and prisons. Measures to limit such transmission include respiratory isolation of persons with suspected TB until they are proven to be noninfectious (at least by sputum AFB smear negativity), proper ventilation in rooms of patients with infectious TB, use of ultraviolet irradiation in areas of increased risk of TB transmission, and periodic screening of personnel who may come into contact with known or unsuspected cases of TB. In the past, radiographic surveys, especially those conducted with portable equipment and miniature films, were advocated for case finding. Today, however, the prevalence of TB in industrialized countries is sufficiently low that “mass miniature radiography” is not cost-effective.

1	In high-prevalence countries, most TB control programs have made remarkable progress in reducing morbidity and mortality since the mid-1990s by adopting and implementing the strategy promoted by the WHO. Between 2000 and 2013, 37 million lives were saved, and since 1995, 61 million TB cases have been successfully treated. The essential elements of good TB care and control (the DOTS strategy) are political commitment with increased and sustained financing; case detection through quality-assured bacteriology (starting with examination of sputum from patients with cough of >2–3 weeks’ duration); administration of standardized short-course chemotherapy, with direct supervision and patient support; (4) an effective drug supply and management system; and (5) a monitoring and evaluation system, with impact measurement (including assessment of treatment outcomes—e.g., cure, completion of treatment without bacteriologic proof of cure, death, treatment failure, and default—in all cases 1122

1	with impact measurement (including assessment of treatment outcomes—e.g., cure, completion of treatment without bacteriologic proof of cure, death, treatment failure, and default—in all cases 1122 registered and notified). In 2006, the WHO indicated that, although these essential elements remain the fundamental components of any Leprosy control strategy, additional steps must be undertaken to reach the

1	Robert H. Gelber 2015 international TB control targets set within the United Nations Millennium Development Goals. Thus, a new “Stop TB Strategy” with six components has been promoted since 2006: (1) Pursue high-quality DOTS expansion and enhancement. (2) Address HIV-associated TB, MDR-TB, and the needs of poor and vulnerable populations. (3) Contribute to health system strengthening. (4) Engage all care providers. (5) Empower people with TB and their communities. (6) Enable and promote research. As part of the fourth component, evidence-based International Standards for Tuberculosis Care— focused on diagnosis, treatment, and public health responsibilities— have been introduced for wide adoption by medical and professional societies, academic institutions, and all practitioners worldwide (http://www.who.int/tb/publications/ISTC_3rdEd.pdf?ua=1).

1	Care and control of HIV-associated TB are particularly challenging in developing countries because existing interventions require collaboration between HIV/AIDS and TB programs as well as standard services. While TB programs must test every patient for HIV in order to provide access to trimethoprim-sulfamethoxazole prophylaxis against common infections and ART, HIV/AIDS programs must regularly screen persons living with HIV/AIDS for active TB, provide treatment for LTBI, and ensure infection control in settings where people living with HIV congregate.

1	Early and active case detection is considered an important intervention not only among persons living with HIV/AIDS but also among other vulnerable populations, as it reduces transmission in a community and provides early effective care. For TB control efforts to succeed and for elimination to become a realistic target, programs must optimize their performance and include additional interventions as described. Moreover, the approach to TB control and care needs to become holistic and engage beyond dedicated programs. Therefore, the WHO’s “End TB” strategy has been designed and builds on three pillars for the post-2015 era of increased efforts by governments and national programs worldwide: (1) integrated, patient-centered care and prevention; (2) bold policies and supportive systems; and (3) intensified research and innovation. The first pillar incorporates all technological innovations, such as early diagnostic approaches (including universal drug-susceptibility testing and

1	and (3) intensified research and innovation. The first pillar incorporates all technological innovations, such as early diagnostic approaches (including universal drug-susceptibility testing and systematic screening of identified, setting-specific, high-risk groups); well-designed treatment regimens for all forms of TB; proper management of HIV-associated TB and other comorbidities; and preventive treatment of persons at high risk. The second pillar is fundamental and is normally beyond the control of dedicated programs, relying on policies forged by the highest-level health and governmental authorities: availability of adequate and well-identified human and financial resources; engagement of civil society organizations and all relevant public and private providers to facilitate care and prevention of all patients; a policy of universal health coverage (which implies avoidance of catastrophic expenditures caused by TB among the poorest); regulatory frameworks for case notifications,

1	prevention of all patients; a policy of universal health coverage (which implies avoidance of catastrophic expenditures caused by TB among the poorest); regulatory frameworks for case notifications, vital registration, quality and rational use of medicines, and infection control; social protection mechanisms; poverty alleviation strategies; and interventions on the broader determinants of TB. Finally, the third pillar of the new strategy emphasizes intensification of engagement in research and development of new tools and interventions as well as optimization of implementation and rapid adoption of new tools in endemic countries. In the end, besides specific clinical care and control interventions as described in this chapter, elimination of TB ultimately will require control and attenuation of the multitude of risk factors (e.g., HIV, smoking, and diabetes) and socioeconomic determinants (e.g., extreme poverty, inadequate living conditions and bad housing, alcoholism, malnutrition,

1	of the multitude of risk factors (e.g., HIV, smoking, and diabetes) and socioeconomic determinants (e.g., extreme poverty, inadequate living conditions and bad housing, alcoholism, malnutrition, and indoor air pollution) with clearly implemented policies within the health sector and other sectors linked to human development and welfare.

1	The contributions of Richard J. O’Brien to this chapter in previous editions are gratefully acknowledged.

1	Leprosy, first described in ancient Indian texts from the sixth century b.c., is a nonfatal, chronic infectious disease caused by Mycobacterium leprae, the clinical manifestations of which are largely confined to the skin, peripheral nervous system, upper respiratory tract, eyes, and testes. The unique tropism of M. leprae for peripheral nerves (from large nerve trunks to microscopic dermal nerves) and certain immunologically mediated reactional states are the major causes of morbidity in leprosy. The propensity of the disease, when untreated, to result in characteristic deformities and the recognition in most cultures that the disease is communicable from person to person have resulted historically in a profound social stigma. Today, with early diagnosis and the institution of appropriate and effective antimicrobial therapy, patients can lead productive lives in the community, and deformities and other visible manifestations can largely be prevented.

1	M. leprae is an obligate intracellular bacillus (0.3–1 μm wide and 1–8 μm long) that is confined to humans, armadillos in certain locales, and sphagnum moss. The organism is acid-fast, indistinguishable microscopically from other mycobacteria, and ideally detected in tissue sections by a modified Fite stain. Strain variability has been documented in this organism. M. leprae produces no known toxins and is well adapted to penetrate and reside within macrophages, yet it may survive outside the body for months. In untreated patients, only ~1% of M. leprae organisms are viable. The morphologic index (MI), a measure of the number of acid-fast bacilli (AFB) in skin scrapings that stain uniformly bright, correlates with viability. The bacteriologic index (BI), a logarithmic-scaled measure of the density of M. leprae in the dermis, may be as high as 4–6+ in untreated patients and falls by 1 unit per year during effective antimicrobial therapy; the rate of decrease is independent of the

1	of the density of M. leprae in the dermis, may be as high as 4–6+ in untreated patients and falls by 1 unit per year during effective antimicrobial therapy; the rate of decrease is independent of the relative potency of therapy. A rising MI or BI suggests relapse and perhaps—if the patient is being treated— drug resistance. Drug resistance can be confirmed or excluded in the mouse model of leprosy, and resistance to dapsone and rifampin can be documented by the recognition of mutant genes. However, the availability of these technologies is extremely limited.

1	As a result of reductive evolution, almost half of the M. leprae for proteins, and 1439 genes are shared with Mycobacterium tuberculosis. In contrast, M. tuberculosis uses 91% of its genome to encode for 4000 proteins. Among the lost genes in M. leprae are those for catabolic and respiratory pathways; transport systems; purine, methionine, and glutamine synthesis; and nitrogen regulation. The genome of M. leprae provides a metabolic rationale for its obligate intracellular existence and reliance on host biochemical support, a template for targets of drug development, and ultimately a pathway to cultivation. The finding of strain variability among M. leprae isolates has provided a powerful tool with which to address anew the organism’s epidemiology and pathobiology and to determine whether relapse represents reactivation or reinfection. The bacterium’s complex cell wall contains large amounts of an M. leprae–specific phenolic glycolipid (PGL-1), which is detected in serologic tests.

1	relapse represents reactivation or reinfection. The bacterium’s complex cell wall contains large amounts of an M. leprae–specific phenolic glycolipid (PGL-1), which is detected in serologic tests. The unique trisaccharide of M. leprae binds to the basal lamina of Schwann cells; this interaction is probably relevant to the fact that M. leprae is the only bacterium to invade peripheral nerves.

1	Although it was the first bacterium to be etiologically associated with human disease, M. leprae remains one of the few bacterial species that still has not been cultivated on artificial medium or tissue culture. The multiplication of M. leprae in mouse footpads (albeit limited, with a doubling time of ~2 weeks) has provided a means to evaluate antimicrobial agents, monitor clinical trials, and screen vaccines. M. leprae grows best in cooler tissues (the skin, peripheral nerves, anterior chamber of the eye, upper respiratory tract, and testes), sparing warmer areas of the skin (the axilla, groin, scalp, and midline of the back). Demographics Leprosy is almost exclusively a disease of the developing world, affecting areas of Asia, Africa, Latin

1	Demographics Leprosy is almost exclusively a disease of the developing world, affecting areas of Asia, Africa, Latin America, and the Pacific. While Africa has the highest disease prevalence, Asia has the most cases. More than 80% of the world’s cases occur in a few countries: India, China, Myanmar, Indonesia, Brazil, Nigeria, Madagascar, and Nepal. Within endemic locales, the distribution of leprosy is quite uneven, with areas of high prevalence bordering on areas with little or no disease. In Brazil the majority of cases occur in the Amazon basin and two western states, while in Mexico leprosy is mostly confined to the Pacific coast. Except as imported cases, leprosy is largely absent from the United States, Canada, and northwestern Europe. In the United States, ~4000 persons have leprosy and 100–200 new cases are reported annually, most of them in California, Texas, New York, and Hawaii among immigrants from Mexico, Southeast Asia, the Philippines, and the Caribbean.

1	The comparative genomics of single-nucleotide polymorphisms support the likelihood that four distinct strains exist, having originated in East Africa or Central Asia. A mutation spread to Europe and subsequently underwent two separate mutations that were then followed by spread to West Africa and the Americas.

1	The global prevalence of leprosy is difficult to assess, given that many of the locales with high prevalence lack a significant medical or public health infrastructure. Estimates range from 0.6 to 8 million affected individuals. The lower estimate includes only persons who have not completed chemotherapy, excluding those who may be physically or psychologically damaged from leprosy and who may yet relapse or develop immune-mediated reactions. The higher figure includes patients whose infections probably are already cured and many who have no leprosy-related deformity or disability. Although the figures on the worldwide prevalence of leprosy are debatable, incidence is not falling; there are still an estimated 500,000 new cases annually.

1	Leprosy is associated with poverty and rural residence. It appears not to be associated with AIDS, perhaps because of leprosy’s long incubation period. Most individuals appear to be naturally immune to leprosy and do not develop disease manifestations after exposure. The time of peak onset is in the second and third decades of life.

1	The most severe lepromatous form of leprosy is twice as com mon among men as among women and is rarely encountered in children. The frequency of the polar forms of leprosy in different countries varies widely and may in part be genetically determined; certain human leukocyte antigen (HLA) associations are known for both polar forms of leprosy (see below). Furthermore, variations in 1123 immunoregulatory genes are associated with an increased susceptibility to leprosy, particularly the multibacillary form. In India and Africa, 90% of cases are tuberculoid; in Southeast Asia, 50% are tuberculoid and 50% lepromatous; and in Mexico, 90% are lepromatous. (For definitions of disease types, see Table 203-1 and “Clinical, Histologic, and Immunologic Spectrum,” below.)

1	Transmission The route of transmission of leprosy remains uncertain, and transmission routes may in fact be multiple. Nasal droplet infection, contact with infected soil, and even insect vectors have been considered the prime candidates. Aerosolized M. leprae can cause infection in immunosuppressed mice, and a sneeze from an untreated lepromatous patient may contain >1010 AFB. Furthermore, both IgA antibody to M. leprae and genes of M. leprae—demonstrable by polymerase chain reaction (PCR)—have been found in the nose of individuals from endemic areas who have no signs of leprosy and in 19% of occupational contacts of lepromatous patients. Several lines of evidence implicate soil transmission. (1) In endemic countries such as India, leprosy is primarily a rural and not an urban disease. (2) M. leprae products reside in soil in endemic locales. (3) Direct dermal inoculation (e.g., during tattooing) may transmit M. leprae, and common sites of leprosy in children are the buttocks and

1	(2) M. leprae products reside in soil in endemic locales. (3) Direct dermal inoculation (e.g., during tattooing) may transmit M. leprae, and common sites of leprosy in children are the buttocks and thighs, suggesting that microinoculation of infected soil may transmit the disease. Evidence for insect vectors of leprosy includes the demonstration that bedbugs and mosquitoes in the vicinity of leprosaria regularly harbor

1	M. leprae and that experimentally infected mosquitoes can transmit the infection to mice. Skin-to-skin contact generally is not considered an important route of transmission. In endemic countries, ~50% of leprosy patients have a history of intimate contact with an infected person (often a household member), while, for unknown reasons, leprosy patients in nonendemic locales can identify such contact only 10% of the time. Moreover, household contact with an infected lepromatous case carries an eventual risk of disease acquisition of ~10% in endemic areas as opposed to only 1% in nonendemic locales. Contact with a tuberculoid case carries a very low risk. Physicians and nurses caring for leprosy patients and the coworkers of these patients are not at risk for leprosy. able variability among isolates, highly similar and even identical VNTR results have been obtained with isolates from a limited number

1	able variability among isolates, highly similar and even identical VNTR results have been obtained with isolates from a limited number TABLE 203-1 CLInICAL, BACTERIOLOgIC, PATHOLOgIC, And IMMunOLOgIC sPECTRuM OF LEPROsy Feature Tuberculoid (TT, BT) Leprosy Borderline (BB, BL) Leprosy Lepromatous (LL) Leprosy Skin lesions One or a few sharply defined annular Intermediate between BTand LL-type Symmetric, poorly marginated, asymmetric macules or plaques with a lesions; ill-defined plaques with an multiple infiltrated nodules and tendency toward central clearing, elevated occasional sharp margin; few or many plaques or diffuse infiltration; borders in number xanthoma-like or dermatofibroma Nerve lesions Skin lesions anesthetic early; nerve Hypesthetic or anesthetic skin Hypesthesia a late sign; nerve pal-near lesions sometimes enlarged; nerve lesions; nerve trunk palsies, at times sies variable; acral, distal, symmetric abscesses most common in BT symmetric anesthesia common

1	Acid-fast bacilli (BIa) 0–1+ 3–5+ 4–6+ Lymphocytes 2+ 1+ 0–1+ Macrophage differentiation Epithelioid Epithelioid in BB; usually undifferenti-Foamy change the rule; may be ated but may have foamy changes undifferentiated in early lesions in BL Langerhans giant cells 1–3+ — — Lepromin skin test +++ — — Lymphocyte transformation test Generally positive 1–10% 1–2% CD4+/CD8+ T cell ratio in lesions 1.2 BB: NT; BL: 0.48 0.50 M. leprae PGL-1 antibodies 60% 85% 95% aSee text. Abbreviations: BB, mid-borderline; BL, borderline lepromatous; BT, borderline tuberculoid; TT, polar tuberculoid; LL, polar lepromatous; BI, bacteriologic index; NT, not tested; PGL-1, phenolic glycolipid 1. 1124 of families with multiple cases. Moreover, VNTR results have been similar for isolates within certain geographic locales and divergent for isolates within others. These findings suggest that genomic analyses may prove useful in the future for defining M. leprae transmission patterns.

1	M. leprae causes disease primarily in humans. However, in Texas and Louisiana, 15% of nine-banded armadillos are infected, and armadillo contact occasionally results in human disease. Armadillos develop disseminated infection after IV inoculation of live M. leprae. CLINICAL, HISTOLOGIC, AND IMMuNOLOGIC SPECTRuM

1	The incubation period prior to manifestation of clinical disease can vary between 2 and 40 years, although it is generally 5–7 years in duration. This long incubation period is probably, at least in part, a consequence of the extremely long doubling time for M. leprae (14 days in mice versus in vitro doubling times of 1 day and 20 min for M. tuberculosis and Escherichia coli, respectively). Leprosy presents as a spectrum of clinical manifestations that have bacteriologic, pathologic, and immunologic counterparts. The spectrum from polar tuberculoid (TT) to borderline tuberculoid (BT) to mid-borderline (BB, which is rarely encountered) to borderline lepromatous (BL) to polar lepromatous (LL) disease is associated with an evolution from asymmetric localized macules and plaques to nodular and indurated symmetric generalized skin manifestations, an increasing bacterial load, and loss of M. leprae–specific cellular immunity (Table 203-1). Distinguishing dermatopathologic characteristics

1	and indurated symmetric generalized skin manifestations, an increasing bacterial load, and loss of M. leprae–specific cellular immunity (Table 203-1). Distinguishing dermatopathologic characteristics include the number of lymphocytes, giant cells, and AFB as well as the nature of epithelioid cell differentiation. Where a patient presents on the clinical spectrum largely determines prognosis, complications, reactional states, and the intensity of antimicrobial therapy required.

1	Tuberculoid Leprosy At the less severe end of the spectrum is tuberculoid leprosy, which encompasses TT and BT disease. In general, these forms of leprosy result in symptoms confined to the skin and peripheral nerves. TT leprosy is the most common form of the disease encountered in India and Africa but is virtually absent in Southeast Asia, where BT leprosy is frequent.

1	The skin lesions of tuberculoid leprosy consist of one or a few hypopigmented macules or plaques (Fig. 203-1) that are sharply demarcated and hypesthetic, often have erythematous or raised borders, and are devoid of the normal skin organs (sweat glands and hair follicles) and thus are dry, scaly, and anhidrotic. AFB are generally absent or few in number. Tuberculoid leprosy patients may have asymmetric enlargement of one or a few peripheral nerves. Indeed, leprosy and certain rare hereditary neuropathies are the only human diseases associated with peripheral-nerve enlargement. Although any peripheral nerve may be enlarged (including small digital and supraclavicular nerves), those most commonly affected are the ulnar, posterior auricular, peroneal, and posterior tibial nerves, with associated hypesthesia and myopathy. FIGuRE 203-1 Tuberculoid (TT) leprosy: a well-defined, hypopigmented, anesthetic macule with anhidrosis and a raised granular margin (arrowhead).

1	FIGuRE 203-1 Tuberculoid (TT) leprosy: a well-defined, hypopigmented, anesthetic macule with anhidrosis and a raised granular margin (arrowhead). In tuberculoid leprosy, T cells breach the perineurium, and destruction of Schwann cells and axons may be evident, resulting in fibrosis of the epineurium, replacement of the endoneurium with epithelial granulomas, and occasionally caseous necrosis. Such invasion and destruction of nerves in the dermis by T cells are pathognomonic for leprosy.

1	Circulating lymphocytes from patients with tuberculoid leprosy readily recognize M. leprae and its constituent proteins, patients have positive lepromin skin tests (see “Diagnosis,” below), and—owing to a type 1 cytokine pattern in tuberculoid tissues—strong T cell and macrophage activation results in a localized infection. In tuberculoid leprosy tissue, there is a 2:1 predominance of helper CD4+ over CD8+ T lymphocytes. Tuberculoid tissues are rich in the mRNAs of the pro-inflammatory TH1 family of cytokines: interleukin (IL) 2, interferon γ (IFN-γ), and IL-12; in contrast, IL-4, IL-5, and IL-10 mRNAs are scarce.

1	Lepromatous Leprosy Lepromatous leprosy patients present with symmetrically distributed skin nodules (Fig. 203-2), raised plaques, or diffuse dermal infiltration, which, when on the face, results in leonine facies. Late manifestations include loss of eyebrows (initially the lateral margins only) and eyelashes, pendulous earlobes, and dry scaling skin, particularly on the feet. In LL leprosy, bacilli are numerous in the skin (as many as 109/g), where they are often found in large clumps (globi), and in peripheral nerves, where they initially invade Schwann cells, resulting in foamy degenerative myelination and axonal degeneration and later in Wallerian degeneration. In addition, bacilli are plentiful in circulating blood and in all organ systems except the lungs and the central nervous system. Nevertheless, patients are afebrile, and there is no evidence of major organ system dysfunction.

1	Found almost exclusively in western Mexico and the Caribbean is a form of lepromatous leprosy without visible skin lesions but with diffuse dermal infiltration and a demonstrably thickened dermis, termed diffuse lepromatosis. In lepromatous leprosy, nerve enlargement and damage tend to be symmetric, result from actual bacillary invasion, and are more insidious but ultimately more extensive than in tuberculoid leprosy. Patients with LL leprosy have acral, distal, symmetric peripheral neuropathy and a tendency toward symmetric nerve-trunk enlargement. They may also have signs and symptoms related to involvement of the upper respiratory tract, the anterior chamber of the eye, and the testes.

1	In untreated LL patients, lymphocytes regularly fail to recognize either M. leprae or its protein constituents, and lepromin skin tests are negative (see “Diagnosis,” below). This loss of protective cellular immunity appears to be antigen-specific, as patients are not unusually susceptible to opportunistic infections, cancer, or AIDS and maintain delayed-type hypersensitivity to Candida, Trichophyton, mumps virus, tetanus toxoid, and even purified protein derivative of tuberculin. At times, M. leprae–specific anergy is reversible with effective chemotherapy. In LL tissues, there is a 2:1 ratio of CD8+ to CD4+ T lymphocytes. LL patients have a predominant TH2 response and hyperglobulinemia, and LL tissues demonstrate a TH2 cytokine profile, being rich in mRNAs for IL-4, IL-5, and IL-10 and poor in those for IL-2, IFN-γ, and IL-12. It appears that cytokines mediate a protective tissue response in leprosy, as injection of IFN-γ or IL-2 into lepromatous lesions causes a loss of AFB and

1	poor in those for IL-2, IFN-γ, and IL-12. It appears that cytokines mediate a protective tissue response in leprosy, as injection of IFN-γ or IL-2 into lepromatous lesions causes a loss of AFB and histopathologic conversion toward a tuberculoid pattern. Macrophages of lepromatous leprosy patients appear to be functionally intact; circulating monocytes exhibit normal microbicidal function and responsiveness to IFN-γ.

1	FIGuRE 203-2 Lepromatous (LL) leprosy: advanced nodular lesions. Reactional States Lepra reactions comprise several common immunologically mediated inflammatory states that cause considerable morbidity. Some of these reactions precede diagnosis and the institution of effective antimicrobial therapy; indeed, these reactions may precipitate presentation for medical attention and diagnosis. Other reactions follow the initiation of appropriate chemotherapy; these reactions may cause patients to perceive that their leprosy is worsening and to lose confidence in conventional therapy. Only by warning patients of the potential for these reactions and describing their manifestations can physicians treating leprosy patients ensure continued credibility.

1	type 1 lepra reactions (downgrading and reversal reactions) Type 1 lepra reactions occur in almost half of patients with borderline forms of leprosy but not in patients with pure lepromatous disease. Manifestations include classic signs of inflammation within previously involved macules, papules, and plaques and, on occasion, the appearance of new skin lesions, neuritis, and (less commonly) fever—generally low-grade. The nerve trunk most frequently involved in this process is the ulnar nerve at the elbow, which may be painful and exquisitely tender. If patients with affected nerves are not treated promptly with glucocorticoids (see below), irreversible nerve damage may result in as little as 24 h. The most dramatic manifestation is foot-drop, which occurs when the peroneal nerve is involved.

1	When type 1 lepra reactions precede the initiation of appropriate antimicrobial therapy, they are termed downgrading reactions, and the case becomes histologically more lepromatous; when they occur after the initiation of therapy, they are termed reversal reactions, and the case becomes more tuberculoid. Reversal reactions often occur in the first months or years after the initiation of therapy but may also develop several years thereafter. Edema is the most characteristic microscopic feature of type 1 lepra lesions, whose diagnosis is primarily clinical. Reversal reactions are typified by a TH1 cytokine profile, with an influx of CD4+ T helper cells and increased levels of IFN-γ and IL-2. In addition, type 1 reactions are associated with large numbers of T cells bearing γ/δ receptors—a unique feature of leprosy.

1	type 2 lepra reactions: erytHema nodosUm leprosUm Erythema nodosum leprosum (ENL) (Fig. 203-3) occurs exclusively in patients near the lepromatous end of the leprosy spectrum (BL/LL), affecting nearly 50% of this group. Although ENL may precede leprosy diagnosis and the initiation of therapy (sometimes, in fact, prompting the diagnosis), in 90% of cases it follows the institution of chemotherapy, generally within 2 years. The most common features of ENL are crops of painful erythematous papules that resolve spontaneously in a few days to a week but may recur; malaise; and fever that can be profound. However, patients may also experience symptoms of neuritis, lymphadenitis, uveitis, orchitis, and glomerulonephritis and may develop anemia, leukocytosis, and abnormal liver function tests (particularly increased aminotransferase levels). Individual patients may have either a single bout of ENL or chronic recurrent manifestations. Bouts may be either mild or severe and generalized; in rare

1	increased aminotransferase levels). Individual patients may have either a single bout of ENL or chronic recurrent manifestations. Bouts may be either mild or severe and generalized; in rare instances, ENL results in death. Skin biopsy of ENL papules reveals vasculitis or panniculitis, sometimes with many lymphocytes but characteristically with polymorphonuclear leukocytes as well.

1	FIGuRE 203-3 Moderately severe skin lesions of erythema nodosum leprosum, some with pustulation and ulceration. Elevated levels of circulating tumor necrosis factor (TNF) have been demonstrated in ENL; thus, TNF may play a central role in the pathobiology of this syndrome. ENL is thought to be a consequence of immune complex deposition, given its TH2 cytokine profile and its high levels of IL-6 and IL-8. However, in ENL tissue, the presence of HLA-DR framework antigen of epidermal cells—considered a marker for a delayed-type hypersensitivity response—and evidence of higher levels of IL-2 and IFN-γ than are usually seen in polar lepromatous disease suggest an alternative mechanism.

1	lUcio’s pHenomenon Lucio’s phenomenon is an unusual reaction seen exclusively in patients from the Caribbean and Mexico who have the diffuse lepromatosis form of lepromatous leprosy, most often those who are untreated. Patients with this reaction develop recurrent crops of large, sharply marginated, ulcerative lesions—particularly on the lower extremities—that may be generalized and, when so, are frequently fatal as a result of secondary infection and consequent septic bacteremia. Histologically, the lesions are characterized by ischemic necrosis of the epidermis and superficial dermis, heavy parasitism of endothelial cells with AFB, and endothelial proliferation and thrombus formation in the larger vessels of the deeper dermis. Like ENL, Lucio’s phenomenon is probably mediated by immune complexes.

1	Complications • tHe eXtremities Complications of the extrem ities in leprosy patients are primarily a consequence of neu ropathy leading to insensitivity and myopathy. Insensitivity affects fine touch, pain, and heat receptors but generally spares position and vibration appreciation. The most commonly affected nerve trunk is the ulnar nerve at the elbow, whose involvement results in clawing of the fourth and fifth fingers, loss of dorsal interosseous musculature in the affected hand, and loss of sensation in these distributions. Median nerve involvement in leprosy impairs thumb opposition and grasp; radial nerve dysfunction, although rare in leprosy, leads to wristdrop. Tendon transfers can restore hand function but should not be performed until 6 months after the initiation of antimicrobial therapy and the conclusion of episodes of acute neuritis.

1	Plantar ulceration, particularly at the metatarsal heads, is probably the most common complication of leprous neuropathy. Therapy requires careful debridement; administration of appropriate antibiotics; avoidance of weight-bearing until ulcerations are healed, with slowly progressive ambulation thereafter; and wearing of special shoes to prevent recurrence. Footdrop as a result of peroneal nerve palsy should be treated with a simple nonmetallic brace in the shoe or with surgical correction attained by tendon transfers. Although uncommon, Charcot’s joints, particularly of the foot and ankle, may result from leprosy.

1	The loss of distal digits in leprosy is a consequence of insensitivity, trauma, secondary infection, and—in lepromatous disease—a poorly understood and sometimes profound osteolytic process. Conscientious 1126 protection of the extremities during cooking and work and the early institution of therapy have substantially reduced the frequency and severity of distal digit loss in recent times. tHe nose In lepromatous leprosy, bacillary invasion of the nasal mucosa can result in chronic nasal congestion and epistaxis. Saline nose drops may relieve these symptoms. Long-untreated LL leprosy may further result in destruction of the nasal cartilage, with consequent saddle-nose deformity or anosmia (more common in the preantibiotic era than at present). Nasal reconstructive procedures can ameliorate significant cosmetic defects.

1	tHe eye Owing to cranial nerve palsies, lagophthalmos and corneal insensitivity may complicate leprosy, resulting in trauma, secondary infection, and (without treatment) corneal ulcerations and opacities. For patients with these conditions, eyedrops during the day and ointments at night provide some protection from such consequences. Furthermore, in LL leprosy, the anterior chamber of the eye is invaded by bacilli, and ENL may result in uveitis, with consequent cataracts and glaucoma. Thus leprosy is a major cause of blindness in the developing world. Slit-lamp evaluation of LL patients often reveals “corneal beading,” representing globi of M. leprae.

1	tHe testes M. leprae invades the testes, while ENL may cause orchitis. Thus males with lepromatous leprosy often manifest mild to severe testicular dysfunction, with an elevation of luteinizing and follicle-stimulating hormones, decreased testosterone, and aspermia or hypospermia in 85% of LL patients but in only 25% of BL patients. LL patients may become impotent and infertile. Impotence is sometimes responsive to testosterone replacement. amyloidosis Secondary amyloidosis is a complication of LL leprosy and ENL that is encountered infrequently in the antibiotic era. This complication may result in abnormalities of hepatic and particularly renal function.

1	nerve abscesses Patients with various forms of leprosy, but particularly those with the BT form, may develop abscesses of nerves (most commonly the ulnar), with a cellulitic appearance of adjacent skin. In such conditions, the affected nerve is swollen and exquisitely tender. Although glucocorticoids may reduce signs of inflammation, rapid surgical decompression is necessary to prevent irreversible sequelae.

1	Leprosy most commonly presents with both characteristic skin lesions and skin histopathology. Thus the disease should be suspected when a patient from an endemic area has suggestive skin lesions or peripheral neuropathy. The diagnosis should be confirmed by histopathology. In tuberculoid leprosy, lesional areas—preferably the advancing edge— must be biopsied because normal-appearing skin does not have pathologic features. In lepromatous leprosy, nodules, plaques, and indurated areas are optimal biopsy sites, but biopsies of normal-appearing skin also are generally diagnostic. Lepromatous leprosy is associated with diffuse hyperglobulinemia, which may result in false-positive serologic tests (e.g., Venereal Disease Research Laboratory, rheumatoid arthritis, and antinuclear antibody tests) and therefore may cause diagnostic confusion. On occasion, tuberculoid lesions may not (1) appear typical, (2) be hypesthetic, and (3) contain granulomas (instead containing only nonspecific

1	tests) and therefore may cause diagnostic confusion. On occasion, tuberculoid lesions may not (1) appear typical, (2) be hypesthetic, and (3) contain granulomas (instead containing only nonspecific lymphocytic infiltrates). In such instances, two of these three characteristics are considered sufficient for a diagnosis. It is preferable to overdiagnose leprosy rather than to allow a patient to remain untreated.

1	IgM antibodies to PGL-1 are found in 95% of patients with untreated lepromatous leprosy; the titer decreases with effective therapy. However, in tuberculoid leprosy—the form of disease most often associated with diagnostic uncertainty owing to the absence or paucity of AFB—patients have significant antibodies to PGL-1 only 60% of the time; moreover, in endemic locales, exposed individuals without clinical leprosy may harbor antibodies to PGL-1. Thus PGL-1 serology is of little diagnostic utility in tuberculoid leprosy. Heat-killed M. leprae (lepromin) has been used as a skin test reagent. It generally elicits a reaction in tuberculoid leprosy patients, may do so in individuals without leprosy, and gives negative results in lepromatous leprosy patients; consequently, it is likewise of little diagnostic value. Unfortunately, PCR of the skin for

1	M. leprae, although positive in LL and BL disease, yields negative results in 50% of tuberculoid cases, again offering little diagnostic assistance. Included in the differential diagnosis of lesions that resemble leprosy are sarcoidosis, leishmaniasis, lupus vulgaris, dermatofibroma, histiocytoma, lymphoma, syphilis, yaws, granuloma annulare, and various other disorders causing hypopigmentation (notably pityriasis alba, tinea, and vitiligo). Sarcoidosis may result in perineural inflammation, but actual granuloma formation within dermal nerves is pathognomonic for leprosy. In lepromatous leprosy, sputum specimens may be loaded with AFB—a finding that can be incorrectly interpreted as representing pulmonary tuberculosis.

1	ANTIMICROBIAL THERAPY Active Agents Established agents used to treat leprosy include dapsone (50–100 mg/d), clofazimine (50–100 mg/d, 100 mg three times weekly, or 300 mg monthly), and rifampin (600 mg daily or monthly; see “Choice of Regimens,” below). Of these drugs, only rifampin is bactericidal. The sulfones (folate antagonists), the foremost of which is dapsone, were the first antimicrobial agents found to be effective for the treatment of leprosy and are still the mainstays of therapy. With sulfone treatment, skin lesions resolve and numbers of viable bacilli in the skin are reduced. Although primarily bacteriostatic, dapsone monotherapy results in a resistance-related relapse rate of only 2.5%; after ≥18 years of therapy and subsequent discontinuation, only another 10% of patients relapse, developing new, usually asymptomatic, shiny, “histoid” nodules. Dapsone is generally safe and inexpensive. Individuals with glucose-6-phosphate dehydrogenase deficiency who are treated with

1	relapse, developing new, usually asymptomatic, shiny, “histoid” nodules. Dapsone is generally safe and inexpensive. Individuals with glucose-6-phosphate dehydrogenase deficiency who are treated with dapsone may develop severe hemolysis; those without this deficiency also have reduced red cell survival and a hemoglobin decrease averaging 1 g/dL. Dapsone’s usefulness is limited occasionally by allergic dermatitis and rarely by the sulfone syndrome (including high fever, anemia, exfoliative dermatitis, and a mononucleosis-type blood picture). It must be remembered that rifampin induces microsomal enzymes, necessitating increased doses of medications such as glucocorticoids and oral birth control regimens. Clofazimine is often cosmetically unacceptable to light-skinned leprosy patients because it causes a red-black skin discoloration that accumulates, particularly in lesional areas, and makes the patient’s diagnosis obvious to members of the community.

1	Other antimicrobial agents active against M. leprae in animal models and at the usual daily doses used in clinical trials include ethionamide/prothionamide; the aminoglycosides streptomycin, kanamycin, and amikacin (but not gentamicin or tobramycin); minocycline; clarithromycin; and several fluoroquinolones, particularly ofloxacin. Next to rifampin, minocycline, clarithromycin, and ofloxacin appear to be most bactericidal for M. leprae, but these drugs have not been used extensively in leprosy control programs. Most recently, rifapentine and moxifloxacin have been found to be especially potent against M. leprae in mice. In a clinical trial in lepromatous leprosy, moxifloxacin was profoundly bactericidal, matched in potency only by rifampin.

1	Choice of Regimens Antimicrobial therapy for leprosy must be individualized, depending on the clinical/pathologic form of the disease encountered. Tuberculoid leprosy, which is associated with a low bacterial burden and a protective cellular immune response, is the easiest form to treat and can be cured reliably with a finite course of chemotherapy. In contrast, lepromatous leprosy may have a higher bacillary load than any other human bacterial disease, and the absence of a salutary T cell repertoire requires prolonged or even lifelong chemotherapy. Hence, careful classification of disease prior to therapy is important.

1	In developed countries, clinical experience with leprosy classification is limited; fortunately, however, the resources needed for skin biopsy are highly accessible, and those for pathologic interpretation are readily available. In developing countries, clinical expertise is greater but is now waning substantially as the care of leprosy patients is integrated into general health services. In addition, access to dermatopathology services is often limited. In such instances, skin smears may prove useful, but in many locales access to the resources needed for their preparation and interpretation also may be unavailable. Use of skin smears is no longer encouraged by the World Health Organization (WHO) and is often replaced by mere counting of lesions, which, together with a lack of capacity for histopathologic assessment, may negatively affect decisions about chemotherapy, increase the potential for reactions, and worsen the ultimate prognosis. A reasoned approach to the treatment of

1	for histopathologic assessment, may negatively affect decisions about chemotherapy, increase the potential for reactions, and worsen the ultimate prognosis. A reasoned approach to the treatment of leprosy is confounded by these and several other issues: 1.

1	Even without therapy, TT leprosy may heal spontaneously, and prolonged dapsone monotherapy (even for LL leprosy) is generally curative in 80% of cases. 2. In tuberculoid disease, it is common for no bacilli to be found in the skin prior to therapy, and thus there is no objective measure of therapeutic success. Furthermore, despite adequate treatment, TT and particularly BT lesions often resolve minimally or incompletely, while relapse and late type 1 lepra reactions can be difficult to distinguish. 3. LL leprosy patients commonly harbor viable persistent M. leprae organisms after prolonged intensive therapy; the propensity of these organisms to initiate clinical relapse is unclear. Because relapse in LL patients after discontinuation of rifampin-containing regimens usually begins only after 7–10 years, follow-up over the very long term is necessary to assess ultimate clinical outcomes. 4.

1	4. Even though primary dapsone resistance is exceedingly rare and multidrug therapy is generally recommended (at least for lepromatous leprosy), there is a paucity of information from experimental animals and clinical trials on the optimal combination of antimicrobial agents, dosing schedule, and duration of therapy.

1	In 1982, the WHO made recommendations for leprosy chemotherapy administered in control programs. These recommendations came on the heels of the demonstration of the relative success of long-term dapsone monotherapy and in the context of concerns about dapsone resistance. Other complicating considerations included the limited resources available for leprosy care in the very areas where it is most prevalent and the frustration and discouragement of patients and program managers with the previous requirement for lifelong therapy for many leprosy patients. Thus, for the first time, the WHO advocated a finite duration of therapy for all forms of leprosy and—given the prohibitive cost of daily rifampin treatment in developing countries—encouraged the monthly administration of this agent as part of a multidrug regimen. Over the ensuing years, the WHO recommendations have been broadly implemented, and the duration of therapy required, particularly for lepromatous leprosy, has been

1	as part of a multidrug regimen. Over the ensuing years, the WHO recommendations have been broadly implemented, and the duration of therapy required, particularly for lepromatous leprosy, has been progressively shortened. For treatment purposes, the WHO classifies patients as paucibacillary or multibacillary. Previously, patients without demonstrable AFB in the dermis were classified as paucibacillary and those with AFB as multibacillary. Currently, in light of the perceived unreliability of skin smears in the field, patients are classified as multibacillary if they have six or more skin lesions and as paucibacillary if they have fewer. (Unfortunately, this classification method has been found wanting, as some patients near the lepromatous pole have only one or a few skin lesions.) The WHO recommends that paucibacillary adults be treated with 100 mg of dapsone daily and 600 mg of rifampin monthly (supervised) for 6 months (Table 203-2). For patients with single-lesion paucibacillary

1	recommends that paucibacillary adults be treated with 100 mg of dapsone daily and 600 mg of rifampin monthly (supervised) for 6 months (Table 203-2). For patients with single-lesion paucibacillary leprosy, the WHO recommends as an alternative a single dose of rifampin (600 mg), ofloxacin (400 mg), and minocycline (100 mg). Multibacillary adults should be treated with 100 mg of dapsone plus 50 mg of clofazimine daily (unsupervised)

1	Form of More Intensive WHO-Recommended Regimen Leprosy Regimen (1982) Tuberculoid Dapsone (100 mg/d) for Dapsone (100 mg/d, unsuper month, supervised) for 6 months (multibacillary) for 3 years plus dapsone mine (50 mg/d), unsupervised; Note: See text for discussion and comparison of the WHO recommendations with the more intensive approach as well as the alternative WHO regimen for single-lesion paucibacillary leprosy. and with 600 mg of rifampin plus 300 mg of clofazimine monthly (supervised). Originally, the WHO recommended that lepromatous patients be treated for 2 years or until smears became negative (generally in ~5 years); subsequently, the acceptable course was reduced to 1 year—a change that remains especially controversial in the absence of supporting clinical trials.

1	Several factors have caused many authorities to question the WHO recommendations and to favor a more intensive approach. Among these factors are—for multibacillary patients—a high (double-digit) relapse rate in several locales (reaching 20–40% in one locale, with the rate directly related to the initial bacterial burden) and—for paucibacillary patients—demonstrable lesional activity for years in fully half of patients after the completion of therapy. The more intensive approach (Table 203-2) calls for tuberculoid leprosy to be treated with dapsone (100 mg/d) for 5 years and for lepromatous leprosy to be treated with rifampin (600 mg/d) for 3 years and with dapsone (100 mg/d) throughout life.

1	With effective antimicrobial therapy, new skin lesions and signs and symptoms of peripheral neuropathy cease appearing. Nodules and plaques of lepromatous leprosy noticeably flatten in 1–2 months and resolve in 1 year or a few years, while tuberculoid skin lesions may disappear, ameliorate, or remain relatively unchanged. Although the peripheral neuropathy of leprosy may improve somewhat in the first few months of therapy, rarely is it significantly alleviated by treatment.

1	Despite the drawbacks of the WHO’s recommendations for multidrug therapy, these regimens have been used almost exclusively worldwide. Although two of the three recommended drugs (dapsone and clofazimine) are only bacteriostatic against M. leprae and bactericidal agents have been identified since the WHO formulated its recommendations, significant studies employing the available alternatives in newly designed regimens have not been initiated. Given the recent findings that moxifloxacin, like rifampin, is profoundly bactericidal in leprosy patients and that short-course chemotherapy for tuberculosis is possible only when two or more bactericidal agents are used, a moxifloxacin/rifamycin-based regimen including either minocycline or clarithromycin appears promising; such a regimen may prove to be more reliably curative than WHO-recommended multidrug therapy for lepromatous leprosy and may allow a considerably shorter course of treatment.

1	THERAPY FOR REACTIONS Type 1 Type 1 lepra reactions are best treated with glucocorticoids (e.g., prednisone, initially at doses of 40–60 mg/d). As inflammation subsides, the glucocorticoid dose can be tapered, but steroid therapy must be continued for at least 3–6 months lest recurrence supervene. Because of the myriad toxicities of prolonged glucocorticoid therapy, the indications for its initiation are strictly limited to lesions whose intense inflammation poses a threat of ulceration; lesions at cosmetically important sites, such as the face; and cases in which neuritis is present. Mild to moderate lepra reactions that do not meet these criteria should be tolerated and glucocorticoid treatment withheld. Thalidomide is ineffective against type 1 lepra 1128 reactions. Clofazimine (200–300 mg/d) is of questionable benefit but in any event is far less efficacious than glucocorticoids.

1	Type 2 Treatment of ENL must be individualized. If ENL is mild (i.e., if it occurs without fever or other organ involvement and with occasional crops of only a few skin papules), it may be treated with antipyretics alone. However, in cases with many skin lesions, fever, malaise, and other tissue involvement, brief courses (1–2 weeks) of glucocorticoid treatment (initially 40–60 mg/d) are often effective. With or without therapy, individual inflamed papules last for <1 week. Successful therapy is defined by the cessation of skin lesion development and the disappearance of other systemic signs and symptoms. If, despite two courses of glucocorticoid therapy, ENL appears to be recurring and persisting, treatment with thalidomide (100–300 mg nightly) should be initiated, with the dose depending on the initial severity of the reaction. Because even a single dose of thalidomide administered early in pregnancy may result in severe birth defects, including phocomelia, the use of this drug in

1	on the initial severity of the reaction. Because even a single dose of thalidomide administered early in pregnancy may result in severe birth defects, including phocomelia, the use of this drug in the United States for the treatment of fertile female patients is tightly regulated and requires informed consent, prior pregnancy testing, and maintenance of birth control measures. Although the mechanism of thalidomide’s dramatic action against ENL is not entirely clear, the drug’s efficacy is probably attributable to its reduction of TNF levels and IgM synthesis and its slowing of polymorphonuclear leukocyte migration. After the reaction is controlled, lower doses of thalidomide (50–200 mg nightly) are effective in preventing relapses of ENL. Clofazimine in high doses (300 mg nightly) has some efficacy against ENL, but its use permits only a modest reduction of the glucocorticoid dose necessary for ENL control.

1	Lucio’s Phenomenon Neither glucocorticoids nor thalidomide is effective against this syndrome. Optimal wound care and therapy for bacteremia are indicated. Ulcers tend to be chronic and heal poorly. In severe cases, exchange transfusion may prove useful. Vaccination at birth with bacille Calmette-Guérin (BCG) has proved variably effective in preventing leprosy: the results have ranged from total inefficacy to 80% efficacy. The addition of heat-killed M. leprae to BCG does not increase the effectiveness of the vaccine. Because whole mycobacteria contain large amounts of lipids and carbohydrates that have proved in vitro to be immunosuppressive for lymphocytes and macrophages, M. leprae proteins may prove to be superior vaccines. Data from a mouse model support this possibility.

1	Chemoprophylaxis with dapsone may reduce the number of tuberculoid leprosy cases but not the number of lepromatous cases and hence is not recommended, even for household contacts. In addition, single-dose rifampin prophylaxis is of doubtful efficacy. Because leprosy transmission appears to require close prolonged household contact, hospitalized patients need not be isolated.

1	In 1992, the WHO—on the basis of that organization’s treatment recommendations—launched a landmark campaign to eliminate leprosy as a public health problem by the year 2000 (goal, <1 case per 10,000 population). The campaign mobilized and energized nongovernmental organizations and national health services to treat leprosy with multiple drugs and to clean up outdated registries. In these respects, the effort has proved hugely successful, with >6 million patients completing therapy. However, the target of leprosy elimination has not yet been reached. In fact, the success of the WHO campaign in reducing the number of cases worldwide has been largely attributable to the redefinition of what constitutes a case of leprosy. Formerly calculated by disease prevalence, the count is now limited to cases not yet treated with multiple drugs. Worldwide, the annual incidence of leprosy has not fallen. Furthermore, after the completion of therapy, when a patient is no longer considered to represent

1	cases not yet treated with multiple drugs. Worldwide, the annual incidence of leprosy has not fallen. Furthermore, after the completion of therapy, when a patient is no longer considered to represent a “case,” half of all patients continue to manifest disease activity for years; relapse rates (at least for multibacillary patients) are unacceptably high; disabilities and deformities go unchecked; and the social stigma of the disease persists.

1	During most of the twentieth century, nongovernmental organizations, particularly Christian missionaries, provided a medical infrastructure devoted to the care and treatment of leprosy patients— the envy of those with other medical priorities in the developing world. With the public perception that leprosy is near eradication, resources for patient care are rapidly being diverted, and the burden of patient care is being transferred to nonexistent or overloaded national health services and to health workers who lack the tools and skills needed for the disease’s diagnosis and classification and for the selection of nuanced therapy (particularly in cases of reactional neuritis). Thus the prerequisites for a salutary outcome increasingly go unmet. Steven M. Holland

1	Steven M. Holland Several terms—nontuberculous mycobacteria (NTM), atypical mycobacteria, mycobacteria other than tuberculosis, and environmental mycobacteria—all refer to mycobacteria other than Mycobacterium tuberculosis, its close relatives (M. bovis, M. caprae, M. africanum, M. pinnipedii, M. canetti), and M. leprae. The number of identified species of NTM is growing and will continue to do so because of the use of DNA sequence typing for speciation. The number of known species currently exceeds 150. NTM are highly adaptable and can inhabit hostile environments, including industrial solvents.

1	NTM are ubiquitous in soil and water. Specific organisms have recurring niches, such as M. simiae in certain aquifers, M. fortuitum in pedicure baths, and M. immunogenum in metalworking fluids. Most NTM cause disease in humans only rarely unless some aspect of host defense is impaired, as in bronchiectasis, or breached, as by inoculation (e.g., liposuction, trauma). There are few instances of human-tohuman transmission of NTM, which occurs almost exclusively in cystic fibrosis. Because infections due to NTM are rarely reported to health agencies and because their identification is sometimes problematic, reliable data on incidence and prevalence are lacking. Disseminated disease denotes significant immune dysfunction (e.g., advanced HIV infection), whereas pulmonary disease, which is much more common, is highly associated with pulmonary epithelial defects but not with systemic immunodeficiency.

1	In the United States, the incidence and prevalence of pulmonary infection with NTM, mostly in association with bronchiectasis (Chap. 312), have for many years been several-fold higher than the corresponding figures for tuberculosis, and rates of the former are increasing among the elderly. Among patients with cystic fibrosis, who often have bronchiectasis, rates of clinical infection with NTM range from 3% to 15%, with even higher rates among older patients. Although NTM may be recovered from the sputa of many individuals, it is critical to differentiate active disease from commensal harboring of the organisms. A scheme to help with the proper diagnosis of pulmonary infection caused by NTM has been developed by the American Thoracic Society and is widely used. The bulk of nontuberculous mycobacterial disease in North America is due to M. kansasii, organisms of the M. avium complex (MAC), and M. abscessus.

1	In Europe, Asia, and Australia, the distribution of NTM in clinical specimens is roughly similar to that in North America, with MAC species and rapidly growing organisms such as M. abscessus encountered frequently. M. xenopi and M. malmoense are especially prominent in northern Europe. M. ulcerans causes the distinct clinical entity Buruli ulcer, which occurs throughout tropical zones, especially in western Africa. M. marinum is a common cause of cutaneous and tendon infections in coastal regions and among individuals exposed to fish tanks or swimming pools. The true international epidemiology of infections due to NTM is hard to determine because the isolation of these organisms often is not reported and speciation often is not performed for M. tuberculosis and NTM. The increasing ease of identification and speciation of these organisms is likely to have a major impact on the description of their international epidemiology in the next few years.

1	Because exposure to NTM is essentially universal and disease is rare, it can be assumed that normal host defenses against these organisms must be strong and that otherwise healthy individuals in whom significant disease develops are highly likely to have specific susceptibility factors that permit NTM to become established, multiply, and cause disease. At the advent of HIV infection, CD4+ T lymphocytes were recognized as key effector cells against NTM; the development of disseminated MAC disease was highly correlated with a decline in CD4+ T lymphocyte numbers. Such a decrease has also been implicated in disseminated MAC infection in patients with idiopathic CD4+ T lymphocytopenia. Potent inhibitors of tumor necrosis factor α (TNF-α), such as infliximab, adalimumab, certolizumab, golimumab, and etanercept, can neutralize this critical cytokine. The occasional result is severe mycobacterial or fungal infection; these associations indicate that TNF-α is a crucial element in

1	golimumab, and etanercept, can neutralize this critical cytokine. The occasional result is severe mycobacterial or fungal infection; these associations indicate that TNF-α is a crucial element in mycobacterial control. However, in cases without the above risk factors, much of the genetic basis of susceptibility to disseminated infection with NTM is accounted for by specific mutations in the interferon γ (IFN-γ)/interleukin 12 (IL-12) synthesis and response pathways.

1	Mycobacteria are typically phagocytosed by macrophages, which respond with the production of IL-12, a heterodimer composed of IL-12p35 and IL-12p40 moieties that together make up IL-12p70. IL-12 activates T lymphocytes and natural killer cells through binding to its receptor (composed of IL-12Rβ1 and IL-12Rβ2/IL-23R), with consequent phosphorylation of STAT4. IL-12 stimulation of STAT4 leads to secretion of IFN-γ, which activates neutrophils and macrophages to produce reactive oxidants, to increase expression of the major histocompatibility complex and Fc receptors, and to concentrate certain antibiotics intracellularly. Signaling by IFN-γ through its receptor (composed of IFN-γR1 and IFN-γR2) leads to phosphorylation of STAT1, which in turn regulates IFN-γ-responsive genes, such as those coding for IL-12 and TNF-α. TNF-α signals through its own receptor via a downstream complex containing the nuclear factor κB (NF-κB) essential modulator (NEMO). Therefore, the positive feedback loop

1	coding for IL-12 and TNF-α. TNF-α signals through its own receptor via a downstream complex containing the nuclear factor κB (NF-κB) essential modulator (NEMO). Therefore, the positive feedback loop between IFN-γ and IL-12/IL-23 drives the immune response to mycobacteria and other intracellular infections. These genes are known to be the critical ones in the pathway of mycobacterial control: specific Mendelian mutations have been identified in IFN-γR1, IFN-γR2, STAT1, GATA2, ISG15, IRF8, IL-12A, IL-12Rβ1, IL-12Rβ2, CYBB, and NEMO (Fig. 204-1). Despite the identification of genes associated with disseminated disease, only ~70% of cases of disseminated nontuberculous mycobacterial infections that are not associated with HIV infection have a genetic diagnosis; the implication is that more mycobacterial susceptibility genes and pathways remain to be identified.

1	In contrast to the recognized genes and mechanisms associated with disseminated nontuberculous mycobacterial infection, the best-recognized underlying condition for pulmonary infection with NTM is bronchiectasis (Chap. 312). Most of the well-characterized forms of bronchiectasis, including cystic fibrosis, primary ciliary dyskinesia, STAT3-deficient hyper-IgE syndrome, and idiopathic bronchiectasis, have high rates of association with nontuberculous mycobacterial infection. The precise mechanism by which bronchiectasis predisposes to locally destructive but not systemic involvement is unknown. Unlike disseminated or pulmonary infection, “hot-tub lung” represents pulmonary hypersensitivity to NTM—most commonly MAC organisms—growing in underchlorinated, often indoor hot tubs.

1	Unlike disseminated or pulmonary infection, “hot-tub lung” represents pulmonary hypersensitivity to NTM—most commonly MAC organisms—growing in underchlorinated, often indoor hot tubs. CLINICAL MANIFESTATIONS Disseminated Disease Disseminated MAC or M. kansasii infections in patients with advanced HIV infection are now uncommon in North America because of effective antimycobacterial prophylaxis T/NK12NEMONRAMP1ISG15STAT1GATA2IL-15IL-2IL-2RAFBIFN˛Salm.M˜TNF°RTLRIRF8CD14IFN˛RIL-12IL-18?˝1˝2

1	FIGuRE 204-1 Cytokine interactions of infected macrophages (Mф) with T and natural killer (NK) lymphocytes. Infection of macrophages by mycobacteria (AFB) leads to the release of heterodimeric interleukin 12 (IL-12). IL-12 acts on its receptor complex (IL-12R), with consequent STAT4 activation and production of homodimeric interferon γ (IFNγ). Through its receptor (IFNγR), IFNγ activates STAT1, stimulating the production of tumor necrosis factor α (TNFα) and leading to the killing of intracellular organisms such as mycobacteria, salmonellae (Salm), and some fungi. Homotrimeric TNFα acts through its receptor (TNFαR) and requires nuclear factor κB essential modulator (NEMO) to activate nuclear factor κB, which also contributes to the killing of intracellular bacteria. Both IFNγ and TNFα lead to upregulation of IL-12. TNFα-blocking antibodies work either by blocking the ligand (infliximab, adalimumab, certolizumab, golimumab) or by providing soluble receptor (etanercept). Mutations in

1	to upregulation of IL-12. TNFα-blocking antibodies work either by blocking the ligand (infliximab, adalimumab, certolizumab, golimumab) or by providing soluble receptor (etanercept). Mutations in IFNγR1, IFNγR2, IL-12p40, IL-12Rβ1, IL-12Rβ2, STAT1, GATA2, ISG15, IRF8, CYBB, and NEMO have been associated with a predisposition to mycobacterial infections. Other cytokines, such as IL-15 and IL-18, also contribute to IFNγ production. Signaling through the Toll-like receptor (TLR) complex and CD14 also upregulates TNFα production. LPS, lipopolysaccharide; NRAMP1, natural resistance-associated macrophage protein 1.

1	and improved treatment of HIV infection. When such mycobacterial disease was common, the portal of entry was the bowel, with spread to bone marrow and the bloodstream. Surprisingly, disseminated infections with rapidly growing NTM (e.g., M. abscessus, M. fortuitum) are very rare in HIV-infected patients, even those with very advanced HIV infection. Because these organisms are of low intrinsic virulence and disseminate only in conjunction with impaired immunity, disseminated disease can be indolent and progressive over weeks to months. Typical manifestations of malaise, fever, and weight loss are often accompanied by organomegaly, lymphadenopathy, and anemia. Because special cultures or stains are required to identify the organisms, the most critical step in diagnosis is to suspect infection with NTM. Blood cultures may be negative, but involved organs typically have significant organism burdens, sometimes with a grossly impaired granulomatous response. In a child, disseminated

1	infection with NTM. Blood cultures may be negative, but involved organs typically have significant organism burdens, sometimes with a grossly impaired granulomatous response. In a child, disseminated involvement (i.e., involvement of two or more organs) without an underlying iatrogenic cause should prompt an investigation of the IFN-γ/IL-12 pathway. Recessive mutations in IFN-γR1 and IFN-γR2 typically lead to severe infection with NTM. In contrast, dominant negative mutations in IFN-γR1, which lead to overaccumulation of a defective interfering mutant receptor on the cell surface, inhibit normal IFN-γ signaling and thus lead to nontuberculous mycobacterial osteomyelitis. Dominant negative mutations in STAT1 and recessive mutations in IL-12Rβ1 can produce variable phenotypes consistent with their residual capacities for IFN-γ synthesis and response. Male patients who have disseminated nontuberculous mycobacterial infections along with conical, peg, or missing teeth and an abnormal hair

1	their residual capacities for IFN-γ synthesis and response. Male patients who have disseminated nontuberculous mycobacterial infections along with conical, peg, or missing teeth and an abnormal hair pattern should be evaluated for defects in the pathway 1130 that activates NF-κB through NEMO. These patients may have associated immune globulin defects as well. Patients with myelodysplasia and mycobacterial disease should be investigated for GATA2 deficiency. A recently recognized group of patients that often develops disseminated infections with rapidly growing NTM (predominantly M. abscessus) as well as other opportunistic infections has high-titer neutralizing auto-antibodies to IFN-γ. Thus far, this syndrome has been reported most frequently in East Asian female patients. IV catheters can become infected with NTM, usually as a consequence of contaminated water. M. abscessus and M. fortuitum sometimes infect deep indwelling lines as well as fluids used in eye surgery, subcutaneous

1	can become infected with NTM, usually as a consequence of contaminated water. M. abscessus and M. fortuitum sometimes infect deep indwelling lines as well as fluids used in eye surgery, subcutaneous injections, and local anesthetics. Infected catheters should be removed.

1	Pulmonary Disease Lung disease is by far the most common form of nontuberculous mycobacterial infection in North America and the rest of the industrialized world. The clinical presentation typically consists of months or years of throat clearing, nagging cough, and slowly progressive fatigue. Patients will often have seen physicians multiple times and received symptom-based or transient therapy before the diagnosis is entertained and samples are sent for mycobacterial stains and cultures. Because not all patients can produce sputum, bronchoscopy may be required for diagnosis. The typical lag between onset of symptoms and diagnosis is ~5 years in older women. Predisposing factors include underlying lung diseases such as bronchiectasis (Chap. 312), pneumoconiosis (Chap. 311), chronic obstructive pulmonary disease (Chap. 314), primary ciliary dyskinesia (Chap. 312), α1 antitrypsin deficiency (Chap. 367e), and cystic fibrosis (Chap. 313). Bronchiectasis and nontuberculous mycobacterial

1	pulmonary disease (Chap. 314), primary ciliary dyskinesia (Chap. 312), α1 antitrypsin deficiency (Chap. 367e), and cystic fibrosis (Chap. 313). Bronchiectasis and nontuberculous mycobacterial infection often coexist and progress in tandem. This situation makes causality difficult to determine in a given index case, but bronchiectasis is certainly among the most critical predisposing factors that are exacerbated by infection.

1	MAC organisms are the most common causes of pulmonary non-tuberculous mycobacterial infection in North America, but rates vary somewhat by region. MAC infection most commonly develops during the sixth or seventh decade of life in women who have had months or years of nagging intermittent cough and fatigue, with or without sputum production or chest pain. The constellation of pulmonary disease due to NTM in a tall and thin woman who may have chest wall abnormalities is often referred to as Lady Windermere syndrome, after an Oscar Wilde character of the same name. In fact, pulmonary MAC infection does afflict older nonsmoking white women more than men, with onset at ~60 years. Patients tend to be taller and thinner than the general population, with high rates of scoliosis, mitral valve prolapse, and pectus anomalies. Whereas male smokers with upper-lobe cavitary disease tend to carry the same single strain of MAC indefinitely, nonsmoking females with nodular bronchiectasis tend to carry

1	and pectus anomalies. Whereas male smokers with upper-lobe cavitary disease tend to carry the same single strain of MAC indefinitely, nonsmoking females with nodular bronchiectasis tend to carry several strains of MAC simultaneously, with changes over the course of their disease.

1	M. kansasii can cause a clinical syndrome that strongly resembles tuberculosis, consisting of hemoptysis, chest pain, and cavitary lung disease. The rapidly growing NTM, such as M. abscessus, have been associated with esophageal motility disorders such as achalasia. Patients with pulmonary alveolar proteinosis are prone to pulmonary nontuberculous mycobacterial and Nocardia infections; the underlying mechanism may be inhibition of alveolar macrophage function due to the autoantibodies to granulocyte-macrophage colony-stimulating factor found in these patients.

1	Cervical Lymph Nodes The most common form of nontuberculous mycobacterial infection among young children in North America is isolated cervical lymphadenopathy, caused most frequently by MAC organisms but also by other NTM. The cervical swelling is typically firm and relatively painless, with a paucity of systemic signs. Because the differential diagnosis of painless adenopathy includes malignancy, many children have infection with NTM diagnosed inadvertently at biopsy; cultures and special stains may not have been requested because mycobacterial disease was not ranked high in the differential. Local fistulae usually resolve completely with resection and/or antibiotic therapy. Likewise, the entity of isolated pediatric intrathoracic nontuberculous mycobacterial infection, which is probably related to cervical lymph node infection, is usually mistaken for cancer. In neither isolated cervical nor isolated intrathoracic infections with NTM have children with underlying immune defects been

1	related to cervical lymph node infection, is usually mistaken for cancer. In neither isolated cervical nor isolated intrathoracic infections with NTM have children with underlying immune defects been identified, nor do the affected children go on to develop other opportunistic infections.

1	Skin and Soft Tissue Disease Cutaneous involvement with NTM usually requires a break in the skin for introduction of the bacteria. Pedicure bath–associated infection with M. fortuitum is more likely if skin abrasion (e.g., during leg shaving) has occurred just before the pedicure. Outbreaks of skin infection are often caused by rapidly growing NTM (especially M. abscessus, M. fortuitum, and M. chelonae) acquired via skin contamination from surgical instruments (especially in cosmetic surgery), injections, and other procedures. These infections are typically accompanied by painful, erythematous, draining subcutaneous nodules, usually without associated fever or systemic symptoms.

1	M. marinum lives in many water sources and can be acquired from fish tanks, swimming pools, barnacles, and fish scales. This organism typically causes papules or ulcers (“fish-tank granuloma”), but the infection can progress to tendinitis with significant impairment of manual dexterity. Lesions appear days to weeks after inoculation of organisms by a typically minor trauma (e.g., incurred during the cleaning of boats or the handling of fish). Tender nodules due to M. marinum can advance up the arm in a pattern also seen with Sporothrix schenckii (sporotricoid spread). The typical carpal tendon involvement may be the first presenting manifestation and may lead to surgical exploration or steroid injection. The index of suspicion for M. marinum infections must be high to ensure that proper specimens obtained during procedures are sent for culture.

1	M. marinum infections must be high to ensure that proper specimens obtained during procedures are sent for culture. M. ulcerans, another waterborne skin pathogen, is found mainly in the tropics, especially in tropical areas of Africa. Infection follows skin trauma or insect bites that allow admission to contaminated water. The skin lesions are typically painless, clean ulcers that slough and can cause osteomyelitis. The toxin mycolactone accounts for the modest host inflammatory response and the painless ulcerations.

1	NTM can be detected on acid-fast or fluorochrome smears of sputum or other body fluids. When the organism burden is high, the organisms may appear as gram-positive beaded rods, but this finding is unreliable. (In contrast, nocardiae may appear as gram-positive and beaded but filamentous bacteria.) Again, the requisite and most sensitive step in the diagnosis of any mycobacterial disease is to think of including it in the differential. In almost all laboratories, mycobacterial sample processing, staining, and culture are conducted separately from routine bacteriologic tests; thus many infections go undiagnosed because of the physician’s failure to request the appropriate test. In addition, mycobacteria usually require separate blood culture media. NTM are broadly differentiated into rapidly growing (<7 days) and slowly growing (≥7 days) forms. Because M. tuberculosis typically takes ≥2 weeks to grow, many laboratories refuse to consider culture results final until 6 weeks have elapsed.

1	growing (<7 days) and slowly growing (≥7 days) forms. Because M. tuberculosis typically takes ≥2 weeks to grow, many laboratories refuse to consider culture results final until 6 weeks have elapsed. Newer techniques using liquid culture media permit more rapid isolation of mycobacteria from specimens than is possible with traditional media. Species more readily detected with incubation at 30°C include M. marinum, M. haemophilum, and M. ulcerans. M. haemophilum prefers iron supplementation or blood, whereas M. genavense requires supplemented medium with the additive mycobactin J. Bacterial formation of pigment in light conditions (photochromogenicity) or dark conditions (scotochromogenicity) or a lack of bacterial pigment formation (nonchromogenicity) has been used to help categorize NTM. In contrast to NTM colonies, M. tuberculosis colonies are beige, rough, dry, and flat. Current identification schemes can reliably use biochemical, nucleic acid, or cell wall composition, as assessed

1	In contrast to NTM colonies, M. tuberculosis colonies are beige, rough, dry, and flat. Current identification schemes can reliably use biochemical, nucleic acid, or cell wall composition, as assessed by high-performance liquid chromatography or mass spectrometry, for speciation. With the remarkable decline in U.S. cases of tuberculosis over recent decades, NTM have become the mycobacteria most commonly isolated from humans in North America. However, not all isolations of NTM, especially from the lung, reflect pathology and require treatment. Whereas identification of an organism in a blood or organ biopsy specimen in a compatible clinical setting is diagnostic, the American Thoracic Society recommends that pulmonary infection due to NTM be diagnosed only when disease is clearly demonstrable—i.e., in an appropriate clinical and radiographic setting (nodules, bronchiectasis, cavities) and with repeated isolation of NTM from expectorated sputum or recovery of NTM from bronchoscopy or

1	in an appropriate clinical and radiographic setting (nodules, bronchiectasis, cavities) and with repeated isolation of NTM from expectorated sputum or recovery of NTM from bronchoscopy or biopsy specimens. Given the large number of species of NTM and the importance of accurate diagnosis for the implementation of proper therapy, identification of these organisms is ideally taken to the species level.

1	The purified protein derivative (PPD) of tuberculin is delivered intradermally to evoke a memory T cell response to mycobacterial antigens. This test is variously referred to as the PPD test, the tuberculin skin test, and the Mantoux test, among other designations. Unfortunately, the cutaneous immune response to these tuberculosis-derived filtrate proteins does not differentiate well between infection with NTM and that with M. tuberculosis. Because intermediate reactions (~10 mm) to PPD in latent tuberculosis and nontuberculous mycobacterial infections can overlap significantly, the progressive decline in active tuberculosis in the United States means that NTM probably account for increasing proportions of PPD reactivity. In addition, bacille Calmette-Guérin (BCG) can cause some degree of cross-reactivity, posing problems of interpretation for patients who have received BCG vaccine. Assays to measure the elaboration of IFN-γ in response to the relatively tuberculosis-specific proteins

1	cross-reactivity, posing problems of interpretation for patients who have received BCG vaccine. Assays to measure the elaboration of IFN-γ in response to the relatively tuberculosis-specific proteins ESAT6 and CFP10 form the basis for IFN-γ-release assays (IGRAs). These assays can be performed with whole blood or on membranes. It is important to note that M. marinum, M. kansasii, and M. szulgai also have ESAT6 and CFP10 and may cause false-positive reactions in IGRAs. Despite cross-reactivity with NTM, large PPD reactions (>15 mm) most commonly signify tuberculosis.

1	Isolation of NTM from blood specimens is clear evidence of disease. Whereas rapidly growing mycobacteria may proliferate in routine blood culture media, slow-growing NTM typically do not; thus it is imperative to suspect the diagnosis and to use the correct bottles for cultures. Isolation of NTM from a biopsy specimen constitutes strong evidence for infection, but cases of laboratory contamination do occur. Identification of organisms on stained sections of biopsy material confirms the authenticity of the culture. Certain NTM require lower incubation temperatures (M. genavense) or special additives (M. haemophilum) for growth. Some NTM (e.g., M. tilburgii) remain noncultivable but can be identified molecularly in clinical samples.

1	The radiographic appearance of nontuberculous mycobacterial disease in the lung depends on the underlying disease, the severity of the infection, and the imaging modality used. The advent and increase in the use of computed tomography (CT) has allowed the identification of characteristic changes that are highly consistent with nontuberculous mycobacterial infection, such as the “tree-in-bud” pattern of bronchiolar inflammation (Fig. 204-2). Involvement of the lingual and right-middle lobes is commonly seen on chest CT but is difficult to appreciate on plain film. Severe bronchiectasis and cavity formation are common in more advanced disease. Isolation of NTM from respiratory samples can be confusing. M. gordonae is often recovered from respiratory samples but is not usually seen on smear and is almost never a pathogen. Patients with bronchiectasis occasionally have NTM recovered from sputum culture with a negative smear. The American Thoracic Society has developed guidelines for the

1	and is almost never a pathogen. Patients with bronchiectasis occasionally have NTM recovered from sputum culture with a negative smear. The American Thoracic Society has developed guidelines for the diagnosis of infection with MAC, M. abscessus, and M. kansasii. A positive diagnosis requires the growth of NTM from two of three sputum samples, regardless of smear findings; a positive bronchoscopic alveolar sample, regardless of smear findings; or a pulmonary parenchyma biopsy sample with granulomatous inflammation or mycobacteria found on section and NTM found on culture. These guidelines probably apply to other NTM as well.

1	Although many laboratories use DNA probes to identify M. tuberculosis, MAC, M. gordonae, and M. kansasii, speciation of NTM helps determine the antimycobacterial therapy to be used. Only testing of MAC organisms for susceptibility to clarithromycin and of M. kansasii for susceptibility to rifampin is indicated; few data support other in vitro susceptibility tests, attractive though they appear. MAC isolates that have not been exposed to macrolides are almost always susceptible. NTM that have persisted beyond a course of antimicrobial therapy are often tested for antibiotic susceptibility, but the value and meaning of these tests are undetermined. FIGuRE 204-2 Chest computed tomography of a patient with pul-monary Mycobacterium avium complex infection. Arrows indicate the “tree-in-bud” pattern of bronchiolar inflammation (peripheral right lung) and bronchiectasis (central right and left lungs).

1	Prophylaxis of MAC disease in patients infected with HIV is started when the CD4+ T lymphocyte count falls to <50/μL. Azithromycin (1200 mg weekly), clarithromycin (1000 mg daily), or rifabutin (300 mg daily) is effective. Macrolide prophylaxis in immunodeficient patients who are susceptible to NTM (e.g., those with defects in the IFN-γ/IL-12 axis) has not been prospectively validated but seems prudent. NTM cause chronic infections that evolve relatively slowly over a period of weeks to years. Therefore, it is rarely necessary to initiate treatment on an emergent basis before the diagnosis is clear and the infecting species is known. Treatment of NTM is complex, often poorly tolerated, and potentially toxic. Just as in tuberculosis, inadequate single-drug therapy is almost always associated with the emergence of antimicrobial resistance and relapse.

1	MAC infection often requires multidrug therapy, the foundation of which is a macrolide (clarithromycin or azithromycin), ethambutol, and a rifamycin (rifampin or rifabutin). For disseminated nontuberculous mycobacterial disease in HIV-infected patients, the use of rifamycins poses special problems—i.e., rifamycin interactions with protease inhibitors. For pulmonary MAC disease, thrice-weekly administration of a macrolide, a rifamycin, and ethambutol has been successful. Therapy is prolonged, generally continuing for 12 months after culture conversion; typically, a course lasts for at least 18 months. Other drugs with activity against MAC organisms include IV and aerosolized aminoglycosides, fluoroquinolones, and clofazimine. In elderly patients, rifabutin can exert significant toxicity. However, with only modest efforts, most antimycobacterial regimens are well tolerated by most patients. Resection of cavitary lesions or severely bronchiectatic segments has been advocated for some

1	However, with only modest efforts, most antimycobacterial regimens are well tolerated by most patients. Resection of cavitary lesions or severely bronchiectatic segments has been advocated for some patients, especially those with macrolide-resistant infections. The success of therapy for pulmonary MAC infections depends on whether disease is nodular or cavitary and on whether it is early or advanced, ranging from 20% to 80%.

1	M. kansasii lung disease is similar to tuberculosis in many ways and is also effectively treated with isoniazid (300 mg/d), rifampin (600 mg/d), and ethambutol (15 mg/kg per day). Other drugs with very high-level activity against M. kansasii include clarithromycin, fluoroquinolones, and aminoglycosides. Treatment should continue until 1132 cultures have been negative for at least 1 year. In most instances, M. kansasii infection is easily cured.

1	apidly growing mycobacteria pose special therapeutic problems. Extrapulmonary disease in an immunocompetent host is usually due to inoculation (e.g., via surgery, injections, or trauma) or to line infection and is often treated successfully with a macrolide and another drug (with the choice based on in vitro susceptibility), along with removal of the offending focus. In contrast, pulmonary disease, especially that caused by M. abscessus, is extremely difficult to cure. epeated courses of treatment are usually effective in reducing the infectious burden and symptoms. Therapy generally includes a macrolide along with an Iadministered agent such as amikacin, a carbapenem, cefoxitin, or tigecycline. ther oral agents (used according to in vitro susceptibility testing and tolerance) include fluorouinolones, doxycycline, and linezolid. ecause nontuberculous mycobacterial infections are chronic, care must be taken in the long-term use of drugs with neurotoxicities, such as linezolid and

1	fluorouinolones, doxycycline, and linezolid. ecause nontuberculous mycobacterial infections are chronic, care must be taken in the long-term use of drugs with neurotoxicities, such as linezolid and ethambutol. Prophylactic pyridoxine has been suggested in these cases. Durations of therapy for M. abscessus lung disease are difficult to predict because so many cases are chronic and reuire intermittent therapy. Expert consultation and management are strongly recommended.

1	nce recognized, M. marinum infection is highly responsive to antimicrobial therapy and is cured relatively easily with any combination of a macrolide, ethambutol, and a rifamycin. Therapy should be continued for 1–2 months after clinical resolution of isolated soft tissue diseasetendon and bone involvement may reuire longer courses in light of clinical evolution. ther drugs with activity against M. marinum include sulfonamides, trimethoprim-sulfamethoxazole, doxycycline, and minocycline. Treatment of the other TM is less well defined, but macrolides and aminoglycosides are usually effective, with other agents added as indicated. Expert consultation is strongly encouraged for difficult or unusual infections due to TM.

1	The outcomes of nontuberculous mycobacterial infections are closely tied to the underlying condition (e.g., IFN-a/IL-12 pathway defect, cystic fibrosis) and can range from recovery to death. With no or inadequate treatment, symptoms and signs can be debilitating, including persistent cough, fever, anorexia, and severe lung destruction. With treatment, patients typically regain strength and energy. The optimal duration of therapy when NTM persist in sputum is unknown, but treatment in this situation can be prolonged.

1	In many countries, pulmonary tuberculosis is diagnosed by smear alone, which is also the method used for monitoring of response and relapse. However, examination of mycobacteria from the affected patients shows that a significant proportion of isolates are actually NTM. Overall, as rates of tuberculosis decline, the proportion of positive smears caused by NTM will increase. Advances in speciation will distinguish tuberculosis from nontuberculous mycobacterial infections and thereby affect rates of assumed relapse and resistance, leading to more targeted and appropriate therapy. Antimycobacterial Agents Max R. O'Donnell, Divya Reddy, Jussi J. Saukkonen Agents used for the treatment of mycobacterial infections, including tuberculosis (TB), leprosy, and infections due to nontuberculous mycobacteria (NTM), are administered in multiple-drug regimens for prolonged courses. Currently, more than 205e

1	Isoniazid 300 mg/d (5 mg/kg) 9 months Supplement with pyridoxine (25–50 mg daily). (6 months acceptable) Alternative: 900 mg twice weekly Twice-weekly regimens require directly observed therapy. (15 mg/kg) Rifampin 600 mg/d (10 mg/kg) 4 months Broader efficacy studies are needed. Isoniazid plus rifapentine 900 mg (15 mg/kg) weekly + 3 months Directly observed therapy is recommended for once-weekly 900 mg weekly treatment. This regimen may be supplemented with pyridoxine (25–50 mg/d). Sources: D Menzies et al: Ann Intern Med 149:689, 2008; American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America: Am J Respir Crit Care Med 167:603, 2003; T Sterling et al: N Engl J Med 365:2155, 2011.

1	160 species of mycobacteria have been identified, the majority of which do not cause disease in humans. While the incidence of disease caused by Mycobacterium tuberculosis has been declining in the United States, TB remains a leading cause of morbidity and mortality in developing countries—particularly in sub-Saharan Africa, where the HIV epidemic rages. Effective drug regimens are not all that is needed; without a well-organized infrastructure for diagnosis and treatment of TB, therapeutic and control efforts are severely hampered (Chaps. 2 and 13e). Infections with NTM have gained in clinical prominence in the United States and other developed countries. These largely environmental organisms often establish infection in immunocompromised patients or in persons with structural lung disease.

1	The earliest recorded human case of TB dates back 9000 years. Early treatment modalities, such as bloodletting, were replaced by sanatorium regimens in the late nineteenth century. The discovery of streptomycin in 1943 launched the era of antibiotic treatment for TB. Over subsequent decades, the discovery of additional agents and the use of multiple-drug regimens allowed progressive shortening of the treatment course from years to as little as 6 months with the regimen for drug-susceptible TB. Latent TB infection (LTBI) and active TB disease are diagnosed by history, physical examination, radiographic imaging, tuberculin skin test, interferon γ release assays, acid-fast staining, mycobacterial cultures, and/or new molecular diagnostics. LTBI is treated with isoniazid (optimally given daily or twice weekly for 9 months), rifampin (daily for 4 months), or isoniazid plus rifapentine (weekly for 3 months) (Table 205e-1).

1	For active or suspected TB disease, clinical factors, including HIV co-infection, symptom duration, radiographic appearance, and public health concerns about TB transmission, drive diagnostic testing and treatment initiation. Multiple-drug regimens are used for the treatment of TB disease (Table 205e-2). Initially, an intensive phase consisting of four drugs—isoniazid, rifampin, pyrazinamide, and ethambutol—given for 2 months is followed by a continuation phase of isoniazid and rifampin for 4 months, for a total treatment duration of 6 months. The continuation phase is extended to 7 months (for a total treatment duration of 9 months) if the 2-month course of pyrazinamide is not completed or, for patients with cavitary pulmonary TB, if sputum cultures remain positive beyond 2 months of treatment (delayed culture conversion).

1	Treatment of TB in individuals co-infected with HIV poses significant challenges, but some progress is being made. Recent data show improved survival when antiretroviral therapy (ART) is initiated early during TB treatment. Interactions of rifampin with protease inhibitors or non-nucleoside reverse transcriptase inhibitors are significant and require close monitoring and dose adjustments. The TB immune reconstitution inflammatory syndrome (IRIS) may appear as early as 1 week after initiation of ART and manifests as paradoxical worsening or unmasking of existing TB infection. Conservative management consists of continued administration of ART and TB medications. However, severe or debilitating IRIS has been anecdotally treated with varying doses of glucocorticoids. Intermittent therapy in patients co-infected with HIV and M. tuberculosis has been associated with low plasma levels of several key TB drugs and with higher rates of treatment failure or relapse; therefore, intermittent

1	in patients co-infected with HIV and M. tuberculosis has been associated with low plasma levels of several key TB drugs and with higher rates of treatment failure or relapse; therefore, intermittent twice-weekly therapy for TB in HIV-co-infected individuals is not recommended.

1	Adherence to medications is critical in achieving a cure with antimycobacterial therapy. Consequently, directly observed therapy (DOT) by trained staff, either in the clinic or at home, is recommended to ensure adherence. In addition, monthly dispensing of TB medicines is recommended because monthly clinical monitoring for hepatotoxicity due to these medications is essential for all patients. Discontinuation of suspected offending agents at the onset of hepatitis symptoms reduces the risk of progression to fatal hepatitis. Clinical monitoring includes at least monthly assessment for symptoms (nausea, vomiting, abdominal discomfort, and unexplained fatigue) and signs (jaundice, dark urine, light stools, diffuse pruritus) of hepatotoxicity, although the latter represent comparatively late manifestations (Table 205e-3). The presence of such symptoms and signs mandates provisional discontinuation of potentially hepatotoxic agents. Biochemical testing of at least serum alanine

1	late manifestations (Table 205e-3). The presence of such symptoms and signs mandates provisional discontinuation of potentially hepatotoxic agents. Biochemical testing of at least serum alanine aminotransferase and total bilirubin levels and exclusion of other causes of these abnormalities are also indicated during treatment for those at risk for hepatotoxicity (Table 205e-3). For patients with active TB, monthly mycobacterial cultures of sputum are recommended until it is certain that the organisms have been cleared and the patient has responded to therapy or until no sputum is available for culture.

1	If significant clinical improvement does not occur or the patient’s condition deteriorates over the course of therapy, possibilities include treatment failure due to nonadherence, poor medication absorption, or the development of resistance. For patients co-infected with HIV and M. tuberculosis, IRIS, which is a diagnosis of exclusion, should also be a consideration. Drug susceptibility testing should be repeated at this point. If resistance is documented or strongly suspected, at least two efficacious drugs to which the isolate is susceptible or which the patient has not already taken should be added to the therapeutic regimen.

1	Multidrug-resistant TB (MDR-TB) is defined as disease caused by a strain of M. tuberculosis that is resistant to both isoniazid and rifampin—the most efficacious of the first-line TB drugs. The risk of MDR-TB is elevated in patients presenting from geographic areas in which ≥5% of incident cases are MDR-TB and in patients previously treated for TB. Treatment regimens for MDR-TB generally include a late-generation fluoroquinolone and an injectable second-line agent (such as capreomycin, amikacin, or kanamycin). Regimens of at least five drugs are recommended for the treatment of MDR-TB. Both standardized and optimized/customized regimens are in use around the world. Extensively drug-resistant TB (XDR-TB) is defined as MDR-TB with additional resistance to any fluoroquinolone and at least one of the second-line injectable agents. Treatment of XDR-TB is individualized Culture Results Intensive Phase Continuation Phase Extension of Total Treatment

1	Culture Results Intensive Phase Continuation Phase Extension of Total Treatment Culture positive HRZE for 2 months, daily or intermit-HR for 4 months, daily or 5 d/wk To 9 months, if 2 months of Z is not completed or culture tent (with dose adjustment) or conversion is prolonged and cavitation is evident on plain radiographa HR for 4 months, intermittent (with dose adjustment) Culture negative HRZE for 2 months 2 months To 6 months, if patient is infected with HIV Extrapulmonary HRZE for 2 months HR for 4–7 months, daily or 5 d/wkb To 9–12 months in TB meningitis. Some recommend 9 months for bone/joint TB. Resistant to H QRZEc or, less often, RZES for 6 months … Prolonged culture conversion, cavitation Resistant to R HZEQc (IAd) for 2 months HEQ(S) for 10–16 months Prolonged culture conversion, delayed response

1	Resistant to HRe ZEQc (IAd) ± alternative agentsf for … Prolonged culture conversion 18–24 months a Culture conversion is prolonged if it occurs beyond 2 months. Some providers extend the continuation phase to 7 months if there is either prolonged culture conversion or cavitation. bMany experts recommend a continuation phase of 7 months for all extrapulmonary TB, including miliary disease. For TB pericarditis and meningitis, the addition of glucocorticoids is recommended. cLevofloxacin and moxifloxacin are the preferred fluoroquinolones. Gatifloxacin is associated with dysglycemia but may be an acceptable alternative; in a recent trial of TB treatment, this drug did not cause dysglycemia in patients who received it thrice weekly for 4 months. Ofloxacin and ciprofloxacin should generally be avoided because of resistance. dInjectable agents: streptomycin, amikacin, kanamycin, and capreomycin. eMultidrug-resistant TB should be managed by or in close consultation with an expert TB

1	be avoided because of resistance. dInjectable agents: streptomycin, amikacin, kanamycin, and capreomycin. eMultidrug-resistant TB should be managed by or in close consultation with an expert TB clinician. Surgical management should be considered. fAlternative agents: cycloserine, ethionamide, para-aminosalicylic acid, clarithromycin, linezolid, and amoxicillin-clavulanate.

1	Abbreviations: E, ethambutol; H, isoniazid; IA, injectable agent; Q, fluoroquinolone; R, rifampin; S, streptomycin; Z, pyrazinamide. Sources: American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America: Am J Respir Crit Care Med 167:603, 2003; C Mitnick et al: N Engl J Med 359:563, 2008; World Health Organization 2011 update: Guidelines for the programmatic management of drug-resistant tuberculosis (www.who.int/tb/challenges/mdr/programmatic _guidelines_for_mdrtb/en/index.html). on the basis of complete phenotypic and, if possible, genotypic antimi-actively investigated during the current remarkable period of drug crobial susceptibility testing. Therapeutic regimens for either MDR-TB development for TB treatment. or XDR-TB should be constructed with input from experienced clini- Isoniazid Isoniazid is a critical drug for treatment of both TB discians who should continue the management of the disease.

1	Isoniazid Isoniazid is a critical drug for treatment of both TB discians who should continue the management of the disease. ease and LTBI. Isoniazid has excellent bactericidal activity against both intracellular M. tuberculosis and extracellular, actively dividing FIRST-LINE ANTITUBERCULOSIS DRUGS organisms. This drug is bacteriostatic against slowly dividing organ-The following discussion of individual anti-TB agents focuses on treat-isms. In treatment of LTBI, isoniazid is considered the first-line agent ment of TB in adults, unless otherwise noted. Several agents are being because it is generally well tolerated, has well-established efficacy, and With hepatic risk factorsb, check ALT and bilirubin at baseline. If ALT is ≥3 × ULN or total bilirubin is >2 × ULN, defer treatment and reevaluate. Check ALT, bilirubin, platelets, creatinine, and hepatitis panel for all patients at baseline. If hepatic risk factors are present, check ALT and bilirubin monthly.

1	Check ALT, bilirubin, platelets, creatinine, and hepatitis panel for all patients at baseline. If hepatic risk factors are present, check ALT and bilirubin monthly. aAll regimens require monthly clinical monitoring. bHepatic risk factors: chronic alcohol use, viral hepatitis, preexisting liver disease, pregnancy or ≤3 months postpartum, hepatotoxic medications. cRelevant manifestations include nausea, vomiting, abdominal pain, jaundice, or unexplained fatigue. Abbreviations: ALT, alanine aminotransferase; BUN, blood urea nitrogen; ECG, electrocardiogram; ENT, ear, nose, and throat; LTBI, latent tuberculosis infection; MDR-TB, multidrug-resistant tuberculosis; ULN, upper limit of normal. Sources: JJ Saukkonen et al: Am J Respir Crit Care Med 174:935, 2006; American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America: Am J Respir Crit Care Med 167:603, 2003.

1	is inexpensive. In this setting, the drug is taken daily or intermittently (i.e., twice weekly) as DOT for 9 months. The 9-month course is more efficacious than the 6-month course (75–90% vs ≤65%), but extension of treatment to 12 months is not likely to provide further protection. A 6-month course of daily or intermittent isoniazid is considered second-line, but acceptable, therapy. A recent large open-label, multicenter, randomized, controlled trial showed that weekly DOT with isoniazid and rifapentine, administered over 3 months, was not inferior to daily isoniazid given for 9 months and had a higher treatment completion rate than the single-drug regimen.

1	For treatment of TB disease, isoniazid is used in combination with other agents to ensure killing of both actively dividing M. tuberculosis and slowly growing "persister" organisms. Unless the organism is resistant, the standard regimen includes isoniazid, rifampin, ethambutol, and pyrazinamide (Table 205e-2). Isoniazid is often given together with 25–50 mg of pyridoxine daily to prevent drug-related peripheral neuropathy.

1	MechanisM of action Isoniazid is a prodrug activated by the mycobacterial KatG catalase-peroxidase; isoniazid is coupled with reduced nicotinamide adenine dinucleotide (NADH). The resulting isonicotinic acyl–NADH complex blocks the mycobacterial ketoenoylreductase known as InhA, binding to its substrate and inhibiting fatty acid synthase and ultimately mycolic acid synthesis. Mycolic acids are essential components of the mycobacterial cell wall. KatG activation of isoniazid also results in the release of free radicals that have antimycobacterial activity, including nitric oxide. The minimal inhibitory concentrations (MICs) of isoniazid for wild-type (untreated) susceptible strains are <0.1 μg/mL for M. tuberculosis and 0.5–2 μg/mL for Mycobacterium kansasii.

1	The minimal inhibitory concentrations (MICs) of isoniazid for wild-type (untreated) susceptible strains are <0.1 μg/mL for M. tuberculosis and 0.5–2 μg/mL for Mycobacterium kansasii. PharMacology Isoniazid is the hydrazide of isonicotinic acid, a small, water-soluble molecule. The usual adult oral daily dose of 300 mg results in peak serum levels of 3–5 μg/mL within 30 min to 2 h after ingestion—well in excess of the MICs for most susceptible strains of M. tuberculosis. Both oral and IM preparations of isoniazid reach effective levels in the body, although antacids and high-carbohydrate meals may interfere with oral absorption. Isoniazid diffuses well throughout the body, reaching therapeutic concentrations in body cavities and fluids, with concentrations in cerebrospinal fluid (CSF) comparable to those in serum.

1	Isoniazid is metabolized in the liver via acetylation by N-acetyltransferase 2 (NAT2) and hydrolysis. Both fastand slowacetylation phenotypes occur; patients who are “fast acetylators” may have lower serum levels of isoniazid, whereas “slow acetylators” may have higher levels and experience more toxicity. Satisfactory isoniazid levels are attained in the majority of homozygous fast NAT2 acetylators given a dose of 6 mg/kg and in the majority of homozygous slow acetylators given only 3 mg/kg. Genotyping is increasingly being used to characterize isoniazid-related pharmacogenomic responses. Isoniazid’s interactions with other drugs are due primarily to its inhibition of the cytochrome P450 system. Among the drugs with significant isoniazid interactions are warfarin, carbamazepine, benzodiazepines, acetaminophen, clopidogrel, maraviroc, dronedarone, salmeterol, tamoxifen, eplerenone, and phenytoin.

1	Dosing The recommended daily dose for the treatment of TB in the United States is 5 mg/kg for adults and 10–20 mg/kg for children, with a maximal daily dose of 300 mg for both. For intermittent therapy in adults (usually twice per week), the dose is 15 mg/kg, with a maximal daily dose of 900 mg. Isoniazid does not require dosage adjustment in patients with renal disease. When the 12-dose, 3-month weekly LTBI regimen is used, the dose of isoniazid is 15 mg/kg, with a maximal dose of 900 mg, and the drug is coadministered with rifapentine.

1	resistance Although isoniazid, along with rifampin, is the mainstay of TB treatment regimens, ~7% of clinical M. tuberculosis isolates in the United States are resistant. Rates of primary isoniazid resistance among untreated patients are significantly higher in many populations born outside the United States. Five separate pathways for isoniazid resistance have been elucidated. Most strains have amino acid changes in either the catalase-peroxidase gene (katG) or the mycobacterial ketoenoylreductase gene (inhA). Less frequently, alterations in kasA, the gene for an enzyme involved in mycolic acid elongation, and loss of NADH dehydrogenase 2 activity confer isoniazid resistance. In 20–30% of isoniazid-resistant M. tuberculosis isolates, increased expression of efflux pump genes, such as efpA, mmpL7, mmr, p55, and the Tap-like gene Rv1258c, has been implicated as the underlying mechanism of resistance.

1	aDverse effects Although isoniazid is generally well tolerated, drug-induced liver injury and peripheral neuropathy are significant adverse effects associated with this agent. Isoniazid may cause asymptomatic transient elevation of aminotransferase levels (often termed hepatic adaptation) in up to 20% of recipients. Other adverse reactions include rash (2%), fever (1.2%), anemia, acne, arthritic symptoms, a systemic lupus erythematosus–like syndrome, optic atrophy, seizures, and psychiatric symptoms. Symptomatic hepatitis occurs in fewer than 0.1% of persons treated with isoniazid alone for LTBI, and fulminant hepatitis with hepatic failure occurs in fewer than 0.01%. Isoniazid-associated hepatitis is idiosyncratic, but its incidence increases with age, with daily alcohol consumption, and in women who are within 3 months postpartum.

1	In patients who have liver disorders or HIV infection, who are pregnant or in the 3-month postpartum period, who have a history of liver disease (e.g., hepatitis B or C, alcoholic hepatitis, or cirrhosis), who use alcohol regularly, who have multiple medical problems, or who have other risk factors for chronic liver disease, the risks and benefits of treatment for LTBI should be weighed. If treatment is undertaken, these patients should have serum concentrations of alanine aminotransferase (ALT) determined at baseline. Routine baseline hepatic ALT testing based solely on an age of >35 years is optional and depends on individual concerns. Monthly biochemical monitoring during isoniazid treatment is indicated for patients whose baseline liver function tests yield abnormal results and for persons at risk for hepatic disease, including the groups just mentioned. Guidelines recommend that isoniazid be discontinued in the presence of hepatitis symptoms or jaundice and an ALT level three

1	persons at risk for hepatic disease, including the groups just mentioned. Guidelines recommend that isoniazid be discontinued in the presence of hepatitis symptoms or jaundice and an ALT level three times the upper limit of normal or in the absence of symptoms with an ALT level five times the upper limit of normal (Table 205e-3).

1	Peripheral neuropathy associated with isoniazid occurs in up to 2% of patients given 5 mg/kg. Isoniazid appears to interfere with pyridoxine (vitamin B6) metabolism. The risk of isoniazid-related neurotoxicity is greatest for patients with preexisting disorders that also pose a risk of neuropathy, such as HIV infection; for those with diabetes mellitus, alcohol abuse, or malnutrition; and for those simultaneously receiving other potentially neuropathic medications, such as stavudine. These patients should be given prophylactic pyridoxine (25–50 mg/d).

1	Rifampin Rifampin is a semisynthetic derivative of Amycolatopsis rifamycinica (formerly known as Streptomyces mediterranei). The most active antimycobacterial agent available, rifampin is the keystone of first-line treatment for TB. Introduced in 1968, this drug eventually permitted dramatic shortening of the TB treatment course. Rifampin has both sterilizing and bactericidal activity against dividing and nondividing M. tuberculosis. The drug is also active against an array of other organisms, including some gram-positive and gram-negative bacteria, Legionella, M. kansasii, and Mycobacterium marinum.

1	Rifampin, administered for 4 months, is also an alternative agent to isoniazid for the treatment of LTBI, although efficacy data are scant at this time. A 3-month course of rifampin alone has been found to be similar in efficacy to a 6-month course of isoniazid. Although the efficacy of the 4-month regimen of rifampin is under study, comparison of this regimen with 9 months of isoniazid in randomized safety and tolerability studies suggests fewer adverse events, including hepatotoxicity; less treatment interruption; a higher completion rate; and greater cost-effectiveness. MechanisM of action Rifampin exerts both intracellular and extracellular bactericidal activity. Like other rifamycins, rifampin specifically binds to and inhibits mycobacterial DNA-dependent RNA polymerase, blocking RNA synthesis. Susceptible strains of M. tuberculosis as well as M. kansasii and M. marinum are inhibited by rifampin concentrations of 1 μg/mL.

1	PharMacology Rifampin is a fat-soluble, complex macrocyclic molecule readily absorbed after oral administration. Serum levels of 10–20 μg/mL are achieved 2.5 h after the usual adult oral dose of 10 mg/kg (given without food). Rifampin has a half-life of 1.5–5 h. The drug distributes well throughout most body tissues, including CSF. Rifampin turns body fluids such as urine, saliva, sputum, and tears a reddish-orange color—an effect that offers a simple means of assessing patients’ adherence to this medication. Rifampin is excreted primarily through the bile and enters the enterohepatic circulation; <30% of a dose is renally excreted.

1	As a potent inducer of the hepatic cytochrome P450 system, rifampin can decrease the half-life of some drugs, such as digoxin, warfarin, phenytoin, prednisone, cyclosporine, methadone, oral contraceptives, clarithromycin, azole antifungal agents, quinidine, antiretroviral protease inhibitors, and non-nucleoside reverse transcriptase inhibitors. The Centers for Disease Control and Prevention has issued guidelines for the management of drug interactions during treatment of HIV and M. tuberculosis co-infection (www.cdc.gov/tb/publications/ guidelines/TB_HIV_Drugs/default.htm). Dosing The daily dosage of rifampin is 10 mg/kg for adults and 10–20 mg/kg for children, with a maximum of 600 mg/d for both. The drug is given once daily, twice weekly, or three times weekly. No adjustments of dose or frequency are necessary in patients with renal insufficiency.

1	resistance Resistance to rifampin in M. tuberculosis, Mycobacterium leprae, and other organisms is the consequence of spontaneous, mostly missense point mutations in a core region of the bacterial gene coding for the β subunit of RNA polymerase (rpoB). RNA polymerase altered in this manner is no longer subject to inhibition by rifampin. Most rapidly and slowly growing NTM harbor intrinsic resistance to rifampin, for which the mechanism has yet to be determined.

1	aDverse effects Adverse events associated with rifampin are infrequent and generally mild. Hepatotoxicity due to rifampin alone is uncommon in the absence of preexisting liver disease and often consists of isolated hyperbilirubinemia rather than aminotransferase elevation. Other adverse reactions include rash, pruritus, gastrointestinal symptoms, and pancytopenia. Rarely, a hypersensitivity reaction may occur with intermittent therapy, manifesting as fever, chills, malaise, rash, and—in some instances—renal and hepatic failure.

1	Ethambutol Ethambutol is a bacteriostatic antimycobacterial agent first synthesized in 1961. A component of the standard first-line regimen, ethambutol provides synergy with the other drugs in the regimen and is generally well tolerated. Susceptible species include M. tuberculosis, M. marinum, M. kansasii, and organisms of the Mycobacterium avium complex (MAC); however, among first-line drugs, ethambutol is the least potent against M. tuberculosis. This agent is also used in combination with other agents in the continuation phase of treatment when patients cannot tolerate isoniazid or rifampin or are infected with organisms resistant to either of the latter drugs.

1	MechanisM of action Ethambutol is bacteriostatic against M. tuberculosis. Its primary mechanism of action is the inhibition of the arabinosyltransferases involved in cell wall synthesis, which probably inhibits the formation of arabinogalactan and lipoarabinomannan. The MIC of ethambutol for susceptible strains of M. tuberculosis is 0.5–2 μg/mL. PharMacology anD Dosing From a single dose of ethambutol, 75–80% is absorbed within 2–4 h of administration. Serum levels peak at 2–4 μg/mL after the standard adult daily dose of 15 mg/kg. Ethambutol is well distributed throughout the body except in the CSF; a dosage of 25 mg/kg is necessary for attainment of a CSF level half of that in serum. For intermittent therapy, the dosage is 50 mg/kg twice weekly. To prevent toxicity, the dosage must be lowered and the frequency of administration reduced for patients with renal insufficiency.

1	aDverse effects Ethambutol is usually well tolerated and has no significant interactions with other drugs. Optic neuritis, the most serious adverse effect reported, typically presents as reduced visual acuity, central scotoma, and loss of the ability to see green (or, less commonly, red). The cause of this neuritis is unknown, but it may be due to an effect of ethambutol on the amacrine and bipolar cells of the retina. Symptoms typically develop several months after initiation of therapy, but ocular toxicity soon after initiation of ethambutol has been described. The risk of ocular toxicity is dose dependent, occurring in 1–5% of patients, and can be increased by renal insufficiency. The routine use of ethambutol in younger children is not recommended because monitoring for visual complications can be difficult. If drug-resistant TB is suspected, ethambutol can be used for treatment of children.

1	All patients starting therapy with ethambutol should have a baseline test for visual acuity, visual fields, and color vision and should undergo an examination of the optic fundus. Visual acuity and color vision should be monitored monthly or less often as needed. Cessation of ethambutol in response to early symptoms of ocular toxicity usually results in reversal of the deficit within several months. Recovery of all visual function may take up to 1 year. In the elderly and in patients whose symptoms are not recognized early, deficits may be permanent. Some experts think that supplementation with hydroxocobalamin (vitamin B12) is beneficial for patients with ethambutol-related ocular toxicity. Other adverse effects of ethambutol are rare. Peripheral sensory neuropathy occurs in rare instances.

1	resistance Ethambutol resistance in M. tuberculosis and NTM is associated primarily with missense mutations in the embB gene that encodes for arabinosyltransferase. Mutations have been found in resistant strains at codon 306 in 50–70% of cases. Mutations at embB306 can cause significantly increased MICs of ethambutol, resulting in clinical resistance. Pyrazinamide A nicotinamide analog, pyrazinamide is an important bactericidal drug used in the initial phase of TB treatment. Its administration for the first 2 months of therapy with rifampin and isoniazid allows treatment duration to be shortened from 9 months to 6 months and decreases rates of relapse.

1	MechanisM of action Pyrazinamide’s antimycobacterial activity is essentially limited to M. tuberculosis. The drug is more active against slowly replicating organisms than against actively replicating organisms. Pyrazinamide is a prodrug that is converted by the mycobacterial pyrimidase to the active form, pyrazinoic acid (POA). This agent is active only in acidic environments (pH <6.0), as are found within phagocytes or granulomas. The exact mechanism of action of POA is unclear, but fatty acid synthetase I may be the primary target in M. tuberculosis. Susceptible strains of M. tuberculosis are inhibited by pyrazinamide concentrations of 16–50 μg/mL at pH 5.5.

1	M. tuberculosis. Susceptible strains of M. tuberculosis are inhibited by pyrazinamide concentrations of 16–50 μg/mL at pH 5.5. PharMacology anD Dosing Pyrazinamide is well absorbed after oral administration, with peak serum concentrations of 20–60 μg/mL at 1–2 h after ingestion of the recommended adult daily dose of 15–30 mg/kg (maximum, 2 g/d). It distributes well to various body compartments, including CSF, and is an important component of treatment for tuberculous meningitis. The serum half-life of the drug is 9–11 h with normal renal and hepatic function. Pyrazinamide is metabolized in the liver to POA, 5-hydroxypyrazinamide, and 5-hydroxy-POA. A high proportion of pyrazinamide and its metabolites (~70%) is excreted in the urine. The dosage must be adjusted according to the level of renal function in patients with reduced creatinine clearance.

1	aDverse effects At the higher dosages used previously, hepatotoxicity was seen in as many as 15% of patients treated with pyrazinamide. However, at the currently recommended dosages, hepatotoxicity now occurs less commonly when this drug is administered with isoniazid and rifampin during the treatment of TB. Older age, active liver disease, HIV infection, and low albumin levels may increase the risk of hepatotoxicity. The use of pyrazinamide with rifampin for the treatment of LTBI is no longer recommended because of unacceptable rates of hepatotoxicity and death in this setting. Hyperuricemia is a common adverse effect of pyrazinamide therapy that usually can be managed conservatively. Clinical gout is rare. Although pyrazinamide is recommended by international TB organizations for routine use in pregnancy, it is not recom mended in the United States because of inadequate teratogenicity data.

1	Although pyrazinamide is recommended by international TB organizations for routine use in pregnancy, it is not recom mended in the United States because of inadequate teratogenicity data. resistance The basis of pyrazinamide resistance in M. tuberculosis is a mutation in the pncA gene coding for pyrazinamidase, the enzyme that converts the prodrug to active POA. Resistance to pyrazinamide is associated with loss of pyrazinamidase activity, which prevents conversion of pyrazinamide to POA. Of pyrazinamide-resistant M. tuberculosis isolates, 72–98% have mutations in pncA. Conventional methods of testing for susceptibility to pyrazinamide may produce both false-negative and false-positive results because the high-acidity environment required for the drug's activation also inhibits the growth of M. tuberculosis. There is some controversy as to the clinical significance of in vitro pyrazinamide resistance.

1	OTHER FIRST-LINE DRUGS Rifabutin Rifabutin, a semisynthetic derivative of rifamycin S, inhibits mycobacterial DNA-dependent RNA polymerase. Rifabutin is recommended in place of rifampin for the treatment of HIV-co-infected individuals who are taking protease inhibitors or non-nucleoside reverse transcriptase inhibitors, particularly nevirapine. Rifabutin’s effect on hepatic enzyme induction is less pronounced than that of rifampin. Protease inhibitors may cause significant increases in rifabutin levels through inhibition of hepatic metabolism. Rifabutin is more active in vitro than rifampin against MAC organisms and other NTM, but its clinical superiority has not been established.

1	PharMacology Like rifampin, rifabutin is lipophilic and is absorbed rapidly after oral administration, reaching peak serum levels 2–4 h after ingestion. Rifabutin distributes best to tissues, reaching levels 5–10 times higher than those in plasma. Unlike rifampin, rifabutin and its metabolites are partially cleared by the hepatic microsomal system. Rifabutin’s slow clearance results in a mean serum half-life of 45 h— much longer than the 3to 5-h half-life of rifampin. Clarithromycin (but not azithromycin) and fluconazole appear to increase rifabutin levels by inhibiting hepatic metabolism.

1	aDverse effects Rifabutin is generally well tolerated, with adverse effects occurring at higher doses. The most common adverse events are gastrointestinal; other reactions include rash, headache, asthenia, chest pain, myalgia, and insomnia. Less common adverse reactions include fever, chills, a flulike syndrome, anterior uveitis, hepatitis, Clostridium difficile– associated diarrhea, a diffuse polymyalgia syndrome, and yellow skin discoloration (“pseudo-jaundice”). Laboratory abnormalities include neutropenia, leukopenia, thrombocytopenia, and increased levels of liver enzymes. Approximately 80% of patients who develop rifampin-related adverse events are able to complete TB treatment with rifabutin. resistance Similar to rifampin resistance, resistance to rifabutin is mediated by some mutations in rpoB.

1	Rifapentine Rifapentine is a semisynthetic cyclopentyl rifamycin, sharing a mechanism of action with rifampin. Rifapentine is lipophilic and has a prolonged half-life that permits weekly or twice-weekly dosing. Therefore, this drug is the subject of intensive clinical investigation aimed at determining optimal dosing and frequency of administration. Currently, rifapentine is an alternative to rifampin in the continuation phase of treatment for noncavitary drug-susceptible pulmonary TB in HIV-seronegative patients who have negative sputum smears at completion of the initial phase of treatment. When administered in these specific circumstances, rifapentine (10 mg/kg, up to 600 mg) is given once weekly with isoniazid. Because of higher rates of relapse, this regimen is not recommended for patients with TB disease and HIV co-infection. A large randomized controlled trial recently demonstrated that, for latent TB, a 12-dose (3-month) regimen of weekly DOT with a weight-based dose of

1	for patients with TB disease and HIV co-infection. A large randomized controlled trial recently demonstrated that, for latent TB, a 12-dose (3-month) regimen of weekly DOT with a weight-based dose of isoniazid and rifapentine was noninferior to daily isoniazid for 9 months. Although the rate of permanent drug discontinuation due to adverse events was higher with rifapentine/ isoniazid, this regimen had a higher treatment completion rate than daily isoniazid in this study. The efficacy of this combination regimen in HIV-infected individuals not receiving ART and in children <12 years of age is under study. The regimen is not recommended for pregnant women, for persons with hypersensitivity reactions to isoniazid or rifampin, or for HIV-infected individuals taking ART.

1	PharMacology Rifapentine’s absorption is improved when the drug is taken with food. After oral administration, rifapentine reaches peak serum concentrations in 5–6 h and achieves a steady state in 10 days. The half-life of rifapentine and its active metabolite, 25-desacetyl rifapentine, is ~13 h. The administered dose is excreted via the liver (70%). aDverse effects The adverse-effects profile of rifapentine is similar to that of other rifamycins. Rifapentine is teratogenic in animal models and is relatively contraindicated in pregnancy. resistance Rifapentine resistance is mediated by mutations in rpoB. Mutations that cause resistance to rifampin also cause resistance to rifapentine.

1	resistance Rifapentine resistance is mediated by mutations in rpoB. Mutations that cause resistance to rifampin also cause resistance to rifapentine. Streptomycin Streptomycin was the first antimycobacterial agent used for the treatment of TB. Derived from Streptomyces griseus, streptomycin is bactericidal against dividing M. tuberculosis organisms but has only low-level early bactericidal activity. This drug is administered only by the IM and IV routes. In developed nations, streptomycin is used infrequently because of its toxicity, the inconvenience of injections, and drug resistance. In developing countries, however, streptomycin is used because of its low cost. MechanisM of action Streptomycin inhibits protein synthesis by binding at a site on the 30S mycobacterial ribosome.

1	MechanisM of action Streptomycin inhibits protein synthesis by binding at a site on the 30S mycobacterial ribosome. PharMacology anD Dosing Serum levels of streptomycin peak at 25–45 μg/mL after a 1-g dose. This agent penetrates poorly into the CSF, reaching levels that are only 20% of serum levels. The usual daily dose of streptomycin (given IM either daily or 5 days per week) is 15 mg/kg for adults and 20–40 mg/kg for children, with a maximum of 1 g/d for both. For patients ≥60 years of age, 10 mg/kg is the recommended daily dose, with a maximum of 750 mg/d. Because streptomycin is eliminated almost exclusively by the kidneys, its use in patients with renal impairment should be avoided or implemented with caution, with lower doses and less frequent administration.

1	aDverse effects Adverse reactions occur frequently with streptomycin (10–20% of patients). Ototoxicity (primarily vestibulotoxicity), neuropathy, and renal toxicity are the most common and the most serious. Renal toxicity, usually manifested as nonoliguric renal failure, is less common with streptomycin than with other frequently used aminoglycosides, such as gentamicin. Manifestations of vestibular toxicity include loss of balance, vertigo, and tinnitus. Patients receiving streptomycin must be monitored carefully for these adverse effects, undergoing audiometry at baseline and monthly thereafter.

1	resistance Spontaneous mutations conferring resistance to streptomycin are relatively common, occurring in 1 in 106 organisms. In the two-thirds of streptomycin-resistant M. tuberculosis strains exhibiting high-level resistance, mutations have been identified in one of two genes: a 16S rRNA gene (rrs) or the gene encoding ribosomal protein S12 (rpsL). Both targets are believed to be involved in streptomycin ribosomal binding. However, low-level resistance, which is seen in about one-third of resistant isolates, has no associated resistance mutation. A gene (gidB) that confers low-level resistance to streptomycin has recently been identified. Strains of M. tuberculosis resistant to streptomycin generally are not cross-resistant to capreomycin or amikacin. Streptomycin is not used for the treatment of MDR-TB or XDR-TB because of (1) the high prevalence of streptomycin resistance among strains resistant to isoniazid and (2) the unreliability of drug susceptibility testing.

1	Second-line antituberculosis agents are indicated for treatment of drug-resistant TB, for patients who are intolerant or allergic to first-line agents, and when first-line supplemental agents are unavailable.

1	Fluoroquinolones Fluoroquinolones inhibit mycobacterial DNA gyrase and topoisomerase IV, preventing cell replication and protein synthesis, and are bactericidal. The later-generation fluoroquinolones levofloxacin and moxifloxacin are the most active against M. tuberculosis and are recommended for the treatment of MDR-TB. They are also being investigated for their potential to shorten the course of treatment for TB. In a recent trial, gatifloxacin, which had been withdrawn from the market because of significant dysglycemia, was assessed for treatment shortening; although its inclusion in the TB treatment regimen did not shorten the duration of therapy from 6 to 4 months, the drug did not cause dysglycemia in TB patients who took it thrice weekly for 4 months. Ciprofloxacin and ofloxacin are no longer recommended for the treatment of TB because of poor efficacy. Despite the documented resistance of the infecting strains to these and other early-generation fluoroquinolones, use of a

1	are no longer recommended for the treatment of TB because of poor efficacy. Despite the documented resistance of the infecting strains to these and other early-generation fluoroquinolones, use of a later-generation fluoroquinolone in patients with XDR-TB has been associated with favorable outcomes. Fluoroquinolones are also considered safe alternatives for patients who develop treatment-limiting adverse effects due to first-line agents. Levofloxacin and moxifloxacin have both been used effectively in the treatment of MDR-TB. The optimal dose of levofloxacin for this indication is being actively studied, but doses of at least 750 mg are commonly used.

1	The fluoroquinolones are well absorbed orally, reach high serum levels, and distribute well into body tissues and fluids. Their absorption is decreased by co-ingestion with products containing multivalent cations, such as antacids. Adverse effects are relatively infrequent (0.5–10% of patients) and include gastrointestinal intolerance, rashes, dizziness, and headache. Most studies of fluoroquinolone side effects have been based on relatively short-term administration for bacterial infections, but trials have now shown the relative safety and tolerability of fluoroquinolones administered for months during TB treatment in adults. Although the potential to prolong the QTc interval, leading to cardiac arrhythmias, has been a source of concern with fluoroquinolones, cessation of treatment due to this adverse effect is rare. There is increasing interest in the use of fluoroquinolones in children, which has traditionally been avoided because of the risks of tendon rupture and cartilage

1	due to this adverse effect is rare. There is increasing interest in the use of fluoroquinolones in children, which has traditionally been avoided because of the risks of tendon rupture and cartilage damage, because the benefits in treatment of drug-resistant TB may outweigh the risks.

1	Mycobacterial resistance can develop rapidly when a fluoroquinolone is inadvertently administered alone. Empirical fluoroquinolone therapy for presumed community-acquired pneumonia is associated with increased fluoroquinolone resistance in M. tuberculosis. Mutations in the genes encoding for DNA gyrase (gyrA and gyrB) are implicated in the majority of cases—but not all cases—of clinical resistance to fluoroquinolones.

1	Injectable Agents • caPreoMycin Capreomycin, a cyclic peptide antibiotic derived from Streptomyces capreolus, is an important first-choice second-line agent used for treatment of MDR-TB, particularly when additional resistance to aminoglycosides is documented. Capreomycin is administered by the IM route; an inhaled preparation is under study. A dose of 15 mg/kg per day is given five to seven times per week (maximal daily dose, 1 g) and results in peak blood levels of 20–40 μg/mL. The dosage may be reduced to 1 g two or three times per week 2–4 months after mycobacterial cultures become negative. For individuals ≥60 years of age, the dose should be reduced to 10 mg/kg per day (maximal daily dose, 750 mg). For patients with renal insufficiency, the drug should be given intermittently and at lower dosage (12–15 mg/kg two or three times per week). A minimal duration of 3 months is recommended for MDR-TB treatment. Penetration of capreomycin into the CSF is believed to be poor.

1	The mechanism of capreomycin’s action is not well understood but involves interference with the mycobacterial ribosome and inhibition of protein synthesis. Resistance to capreomycin is associated with mutations that inactivate a ribosomal methylase (tlyA) or that encode genes for the 16S ribosomal subunit (rrs). Cross-resistance to kanamycin and amikacin is common with rrs but not always with tylA mutations. However, some strains that are resistant to streptomycin, kanamycin, and amikacin generally remain susceptible to capreomycin. Adverse effects of capreomycin are relatively common. Significant hypokalemia and hypomagnesemia as well as otoand renal toxicity have been reported.

1	aMikacin anD kanaMycin Amikacin and kanamycin are aminoglycosides that exert mycobactericidal activity by binding to the 16S ribosomal subunit. The spectrum of antibiotic activity for amikacin and kanamycin includes M. tuberculosis, several NTM species, and aerobic gram-negative and gram-positive bacteria. Although amikacin is highly active against M. tuberculosis, it is used only infrequently because of its significant side effects. The usual daily adult dosage of both amikacin and kanamycin is 15–30 mg/kg given IM or IV (maximal daily dose, 1 g), with a reduction to 10 mg/kg for patients ≥60 years old. For patients with renal insufficiency, the dose and frequency should be reduced (12–15 mg/kg two or three times per week). Mycobacterial resistance is due to mutations in the genes encoding the 16S ribosomal RNA gene. Cross-resistance among kanamycin, amikacin, and capreomycin is common. Isolates resistant to streptomycin are frequently susceptible to amikacin or kanamycin. Adverse

1	the 16S ribosomal RNA gene. Cross-resistance among kanamycin, amikacin, and capreomycin is common. Isolates resistant to streptomycin are frequently susceptible to amikacin or kanamycin. Adverse effects of amikacin include ototoxicity (in up to 10% of recipients, with auditory dysfunction occurring more commonly than vestibulotoxicity), nephrotoxicity, and neurotoxicity. Kanamycin has a similar side-effects profile, but adverse reactions are thought to be less frequent and less severe.

1	Other Second-Line Agents • ethionaMiDe Ethionamide is a derivative of isonicotinic acid. Its mechanism of action is through inhibition of the inhA gene product enoyl–acyl carrier protein (acp) reductase, which is involved in mycolic acid synthesis. Ethionamide is bacteriostatic against metabolically active M. tuberculosis and some NTM. It is used in the treatment of drug-resistant TB, but its use is limited by severe gastrointestinal reactions (including abdominal pain, nausea, and vomiting) as well as significant central and peripheral neurologic side effects, reversible hepatitis (in ~5% of recipients), hypersensitivity reactions, and hypothyroidism. Ethionamide should be taken with food to reduce gastrointestinal effects and with pyridoxine (50–100 mg/d) to limit neuropathic side effects.

1	cycloserine Cycloserine is an analog of the amino acid d-alanine and prevents cell wall synthesis. It inhibits the action of enzymes, including alanine racemase, that are involved in the production of peptidoglycans. Cycloserine is active against a range of bacteria, including

1	M. tuberculosis. Mechanisms of mycobacterial resistance are not well understood, but overexpression of alanine racemase can confer resistance in Mycobacterium smegmatis. Cycloserine is well absorbed after oral administration and is widely distributed throughout body fluids, including CSF. The usual adult dosage is 250 mg two or three times per day. Serious potential side effects include seizures and psychosis (with suicide in some cases), peripheral neuropathy, headache, somnolence, and allergic reactions. Drug levels are monitored to achieve optimal dosing and to reduce the risk of adverse effects, especially in patients with renal failure. Cycloserine should be administered as DOT only with caution and with support from experienced TB physicians to patients with epilepsy, active alcohol abuse, severe renal insufficiency, or a history of depression or psychosis.

1	Para-aMinosalicylic aciD Para-aminosalicylic acid (PAS, 4-aminosalicylic acid) is an oral agent used in the treatment of MDRand XDR-TB. Its bacteriostatic activity is due to inhibition of folate synthesis and of iron uptake. PAS has relatively little activity as an anti-TB agent. Adverse effects may include high-level nausea, vomiting, and diarrhea. PAS may cause hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency. The drug should be taken with acidic foods to improve absorption. Enteric-coated PAS granules (4 g orally every 8 h) appear to be better tolerated than other formulations and produce higher therapeutic blood levels. PAS has a short half-life (1 h), and 80% of the dose is excreted in the urine.

1	clofaziMine Clofazimine is a fat-soluble riminophenazine dye used primarily in the treatment of leprosy worldwide. It is currently gaining popularity in the management of MDRand XDR-TB because of its low cost and intracellular and extracellular activity. By increasing reactive oxygen species and causing membrane destabilization, clofazimine may promote killing of antibiotic-tolerant M. tuberculosis persister organisms. In addition to antimicrobial activity, the drug has other pharmacologic properties—e.g., anti-inflammatory, pro-oxidative, and immunopharmacologic. Clofazimine has a half-life of ~70 days in humans, and average steady-state concentrations are achieved at ~1 month. Ingestion with fatty meals can improve its low and variable rates of absorption (45–62%). Common side effects include gastrointestinal intolerance and reversible orange-to-brownish discoloration of the skin, bodily fluids, and secretions. Dose adjustment may be necessary in patients with severe hepatic

1	include gastrointestinal intolerance and reversible orange-to-brownish discoloration of the skin, bodily fluids, and secretions. Dose adjustment may be necessary in patients with severe hepatic impairment. Clofazimine is being studied as part of a regimen developed in Bangladesh for potential shortening of the MDR-TB treatment course. A recent meta-analysis suggested that inclusion of clofazimine in a multidrug regimen for treatment of MDR-TB was associated with a favorable outcome. Newer analogues with improved pharmacokinetics and alternative formulations of clofazimine (liposomal, nanosuspension, inhalational) are being studied.

1	NEWER ANTITUBERCULOSIS DRUGS Oxazolidinones Linezolid is an oxazolidinone used primarily for the treatment of drug-resistant gram-positive bacterial infections. However, this drug is active in vitro against M. tuberculosis and NTM. Several case series have suggested that linezolid may help clear mycobacteria relatively rapidly when included in a regimen for the treatment of complex cases of MDRand XDR-TB. Linezolid’s mechanism of action is disruption of protein synthesis by binding to the 50S bacterial ribosome. Linezolid has nearly 100% oral bioavailability, with good penetration into tissues and fluids, including CSF. Clinical resistance to linezolid has been reported, but the mechanism is unclear. Adverse effects may include optic and peripheral neuropathy, pancytopenia, and lactic acidosis. Linezolid is a weak monoamine oxidase inhibitor and can be associated with the serotonin syndrome when given concomitantly with serotonergic drugs (primarily antidepressants such as selective

1	Linezolid is a weak monoamine oxidase inhibitor and can be associated with the serotonin syndrome when given concomitantly with serotonergic drugs (primarily antidepressants such as selective serotonin-reuptake inhibitors). A recent meta-analysis showed that ~80% of patients with MDRor XDR-TB can be successfully treated with linezolid-containing anti-TB regimens; however, significant adverse events attributed to linezolid were reported. For MDR-TB treatment, linezolid is usually administered at a dose of 600 mg (or less in some cases) once daily, which appears to be effective. The single daily dose is associated with fewer adverse events than twice-a-day dosing.

1	PNU 100480 and AZD 5847, modified versions of oxazolidinones and protein synthesis inhibitors, are undergoing phase 1 trials and appear to have greater efficacy than linezolid against M. tuberculosis. However, the adverse effect profiles of these compounds compared with that of linezolid need further investigation.

1	Amoxicillin-Clavulanate and Carbapenems β-Lactam agents are largely ineffective for the treatment of M. tuberculosis because of resistance conferred by a hydrolyzing class A β-lactamase. Because clavulanate may theoretically inhibit the β-lactamase, amoxicillin-clavulanate has been used in the treatment of MDR-TB; however, it is a comparatively weak agent. Carbapenems are poor substrates for class A β-lactamases found in M. tuberculosis. Accordingly, meropenem and imipenem have in vitro activity against M. tuberculosis, and their use to treat MDRand XDR-TB has been reported anecdotally. Nevertheless, the need to administer carbapenems by the IV route and lack of information on the drugs' long-term side effects have restricted their use to certain severe cases only.

1	Diarylquinolines Bedaquiline (TMC207 or R207910) is a new diarylquinoline with a novel mechanism of action: inhibition of the mycobacterial ATP synthetase proton pump. TMC207 is bactericidal for drug-susceptible and MDR strains of M. tuberculosis. Resistance has been reported and is due to point mutations in the atpE gene encoding for subunit c of ATP synthetase. A phase 2 randomized controlled clinical trial in MDR-TB patients demonstrated substantial improvement in 2-month culture-conversion rates as well as a reduction in acquired resistance to companion drugs. This drug is metabolized by the hepatic cytochrome CYP3A4. Rifampin lowers TMC207 levels by 50%, and protease inhibitors also interact significantly with this drug. The oral bioavailability of TMC207 appears to be excellent. The dosage is 400 mg/d for the first 2 weeks and then 200 mg thrice weekly. The elimination half-life is long (>14 days). A single dose of this drug can inhibit the growth of M. tuberculosis for up to 1

1	dosage is 400 mg/d for the first 2 weeks and then 200 mg thrice weekly. The elimination half-life is long (>14 days). A single dose of this drug can inhibit the growth of M. tuberculosis for up to 1 week through a combination of long plasma half-life, high-level tissue penetration, and long tissue half-life. Bedaquiline added to a background regimen improved the 2-month sputum culture conversion rate in multicenter, randomized placebo-controlled trials, and these results led to approval by the U.S. Food and Drug Administration (FDA). However, a higher death rate in one trial was observed in the bedaquiline arm than in the control arm (11.4% vs 2.5%); the result was a “black box” warning from the FDA, which also included QT prolongation. The Centers for Disease Control and Prevention has made a provisional recommendation for the use of bedaquiline for 24 weeks in adults with laboratory-confirmed pulmonary MDR-TB when no other effective treatment regimen can be provided.

1	Nitroimidazoles The prodrugs delamanid (OPC-67683) and PA 824 are novel nitro-dihydro-imidazooxazole derivatives that are activated by M. tuberculosis–specific flavin-dependent nitroreductases whose antimycobacterial activity is attributable to inhibition of mycolic acid biosynthesis. These drugs are currently in phase 2 clinical trials and show potential in shortening treatment duration through their activity against nonreplicating drug-susceptible and drug-resistant mycobacteria. Delamanid was shown in a randomized, placebo-controlled, multinational clinical trial to significantly improve the culture conversion rate at 2 months. QT prolongation occurred significantly more often in delamanid-treated patients, but no clinically relevant events were reported.

1	Diamines SQ109, an ethambutol analogue with a 1,2-diamine pharmacophore, is the most promising of the diamines for TB treatment. It is activated by mycobacterial cytochrome enzymes and inhibits mycobacterial cell-wall synthesis by an unknown mechanism. It has a high tissue protein-binding capacity with a very long half-life (~61 h) in humans. In vitro studies have demonstrated that SQ109 has low MICs against both susceptible and resistant M. tuberculosis strains as well as a synergistic effect when administered with isoniazid and rifampin. The drug is under study in clinical trials for TB treatment. Pyrroles LL3858, a pyrrole derivative, has entered clinical trials examining its utility in the treatment of drug-susceptible and drug-resistant TB. The drug’s mechanism of action is unknown. However, because it is active against M. tuberculosis strains that are resistant to available anti-TB drugs, its target is thought to differ from those of currently used agents.

1	More than 150 species of NTM have been identified. Only a minority of these environmental organisms, which are found in soil and water, are important human pathogens. NTM cause extensive disease, primarily in persons with preexisting pulmonary disease or immunocompromise, but also can cause nodular/bronchiectatic disease in otherwise seemingly healthy hosts. NTM are also important causes of infections in surgical settings. The two major classes of NTM are the slow-growing and rapidly growing species; subcultures of the latter grow within 1 week. The growth characteristics of NTM have diagnostic, therapeutic, and prognostic implications. The rate of growth can provide useful preliminary information within a specific clinical context, in that growth within 2–3 weeks is much more likely to indicate an NTM than M. tuberculosis. When NTM do grow from cultures, colonization should be distinguished from active disease in order to optimize the risk and benefit of prolonged treatment with

1	to indicate an NTM than M. tuberculosis. When NTM do grow from cultures, colonization should be distinguished from active disease in order to optimize the risk and benefit of prolonged treatment with multiple medications. According to the recommendations of the American Thoracic Society and the Infectious Diseases Society of America, significant clinical manifestations and/or sputum radiographic evidence of progressive disease consistent with NTM infection as well as either reproducible sputum culture results or a single positive culture are required for the diagnosis of NTM pulmonary disease. Isolation of NTM from blood or from an infected-appearing extrapulmonary site, such as soft tissue or bone, is usually indicative of disseminated or local NTM infection (Chap. 204). Treatment of NTM disease is prolonged and requires multiple medications. Side effects of the regimens employed are common, and intermittent therapy is often used to mitigate these adverse events. Treatment regimens

1	disease is prolonged and requires multiple medications. Side effects of the regimens employed are common, and intermittent therapy is often used to mitigate these adverse events. Treatment regimens depend on the NTM species, the extent or type of disease, and—to some degree—drug susceptibility test results. The nodular bronchiectatic form of MAC infection is generally treated three times per week, whereas fibrocavitary or disseminated MAC infection is treated daily.

1	M. avium Complex Among the NTM, MAC organisms most commonly cause human disease. In immunocompetent hosts, MAC species are most often found in conjunction with underlying significant lung disease, such as chronic obstructive pulmonary disease or bronchiectasis. For patients with nodular or bronchiectatic MAC lung disease, an initial regimen consisting of clarithromycin or azithromycin, rifampin or rifabutin, and ethambutol is given three times per week. Routine initial testing for macrolide resistance is recommended, as is testing at 6 months with a failing regimen (i.e., with cultures persistently positive for NTM).

1	In immunocompromised individuals, disseminated MAC infection is generally treated with clarithromycin, ethambutol, and rifabutin. Azithromycin may be substituted in patients unable to tolerate clarithromycin. Amikacin and fluoroquinolones are often used in salvage regimens. Treatment for disseminated MAC infection in AIDS patients may be lifelong in the absence of immune reconstitution. At least 12 months of MAC therapy and 6 months of effective immune reconstitution may be adequate. M. kansasii M. kansasii is the second most common NTM causing human disease. It is also the second most common cause of NTM pulmonary disease in the United States, where it is most often reported in the southeastern region. M. kansasii infection can be treated with isoniazid, rifampin, and ethambutol; therapy continues for 12 months after culture conversion. Rifampin-resistant M. kansasii has been treated with clarithromycin, trimethoprim-sulfamethoxazole, and streptomycin.

1	Rapidly Growing Mycobacteria Rapidly growing mycobacteria causing human disease include Mycobacterium abscessus, Mycobacterium fortuitum, and Mycobacterium chelonae. Treatment of these mycobacteria is complex and should be undertaken with input from experienced clinicians. Testing for macrolide resistance is recommended. However, in rapidly growing mycobacteria, an inducible erm gene may confer in vivo macrolide resistance to isolates that are susceptible in vitro. M. marinum M. marinum is an NTM found in salt water and freshwater, including swimming pools and fish tanks. It is a cause of localized soft-tissue infections, which may require surgical management. Combination regimens include clarithromycin and either ethambutol or rifampin. Other agents with activity against M. marinum include doxycycline, minocycline, and trimethoprim-sulfamethoxazole.

1	Clarithromycin is a macrolide antibiotic with broad activity against many gram-positive and gram-negative bacteria as well as NTM. This drug is active against MAC organisms and many other NTM species, inhibiting protein synthesis by binding to the 50S mycobacterial ribosomal subunit. NTM resistance to macrolides is probably caused by overexpression of the gene ermB, with consequent methylation of the binding site. Clarithromycin is well absorbed orally and distributes well to tissues. It is cleared both hepatically and renally; the dosage should be reduced in renal insufficiency. Clarithromycin is a substrate for and inhibits cytochrome 3A4 and should not be administered with cisapride, pimozide, or terfenadine because cardiac arrhythmias may occur. Numerous drugs interact with clarithromycin through the CYP3A4 metabolic pathway. Rifampin lowers clarithromycin levels; conversely, rifampin levels are increased by clarithromycin. However, the clinical relevance of this interaction does

1	through the CYP3A4 metabolic pathway. Rifampin lowers clarithromycin levels; conversely, rifampin levels are increased by clarithromycin. However, the clinical relevance of this interaction does not appear to be great.

1	For patients with nodular/bronchiectatic MAC infection, the dosage of clarithromycin is 500 mg, given morning and evening three times a week. For the treatment of fibrocavitary or severe nodular/ bronchiectatic MAC infection, a dose of 500–1000 mg is given daily. Disseminated MAC infection is treated with 1000 mg daily. Clarithromycin is used in combination regimens that typically include ethambutol and a rifamycin in order to avoid the development of macrolide resistance. Adverse effects include frequent gastrointestinal intolerance, hepatotoxicity, headache, rash, and rare instances of hypoglycemia. Clarithromycin is contraindicated during pregnancy because of its teratogenicity in animal models.

1	Azithromycin Azithromycin is a derivative of erythromycin. Although technically an azalide and not a macrolide, it works similarly to macrolides, inhibiting protein synthesis through binding to the 50S ribosomal subunit. Resistance to azithromycin is almost always associated with complete cross-resistance to clarithromycin. Azithromycin is well absorbed orally, with good tissue penetration and a prolonged half-life (~48 h). The usual dosage for treatment of MAC infection is 250 mg/d or 500 mg three times per week. Azithromycin is used in combination with other agents to avoid the development of resistance. For prophylaxis against disseminated MAC infection in immunocompromised individuals, a dose of 1200 mg once per week is given. Because azithromycin is not metabolized by cytochrome P450, it interacts with few drugs. Adjustment of the dosage on the basis of renal function is not necessary.

1	Cefoxitin Cefoxitin is a second-generation parenteral cephalosporin with activity against rapidly growing NTM, particularly M. abscessus, M. marinum, and M. chelonae. Its mechanism of action against NTM is unknown but may involve inactivation of cell wall synthesis enzymes. High doses are used for treatment of NTM: 200 mg/kg IV three or four times per day, with a maximal daily dose of 12 g. The half-life of cefoxitin is ~1 h, with primarily renal clearance that requires adjustment in renal insufficiency. Adverse effects are uncommon but include gastrointestinal manifestations, rash, eosinophilia, fever, and neutropenia.

1	Treatment of mycobacterial infections requires multiple-drug regimens that often exert significant side effects with the potential to limit tolerability. The prolonged duration of treatment has vastly improved results over those obtained in past decades, but drugs and regimens that will shorten treatment duration and limit adverse drug effects and interactions are needed.

1	syphilis Sheila A. Lukehart DEFINITION Syphilis, a chronic systemic infection caused by Treponema pallidum subspecies pallidum, is usually sexually transmitted and is characterized 206 sECTIOn 9 sPIROCHETAL dIsEAsEs by episodes of active disease interrupted by periods of latency. After an incubation period averaging 2–6 weeks, a primary lesion appears—often associated with regional lymphadenopathy—that resolves without treatment. The secondary stage, associated with generalized mucocutaneous lesions and generalized lymphadenopathy, is followed by a latent period of subclinical infection lasting years or decades. Central nervous system (CNS) involvement may occur early in infection and may be symptomatic or asymptomatic. In the preantibiotic era, about one-third of patients with untreated cases developed the tertiary stage, characterized by progressive destructive mucocutaneous, musculoskeletal, or parenchymal lesions; aortitis; or late CNS manifestations.

1	The Spirochaetales include four genera that are pathogenic for humans and for a variety of other animals: Leptospira species, which cause leptospirosis (Chap. 208); Borrelia species, which cause relapsing fever and Lyme disease (Chaps. 209 and 210); Brachyspira species, which cause intestinal infections; and Treponema species, which cause the diseases known collectively as treponematoses (see also Chap. 207e). The Treponema species include T. pallidum subspecies pallidum, which causes venereal syphilis; T. pallidum subspecies pertenue, which causes yaws; T. pallidum subspecies endemicum, which causes endemic syphilis or bejel; and T. carateum, which causes pinta. Until recently, the subspecies were distinguished primarily by the clinical syndromes they produce. Researchers have now identified molecular signatures sex with men (MSM), 20–70% of whom are co-infected with HIV 1133 (depending on geographic location). The number of primary and secondary cases among women in the United

1	molecular signatures sex with men (MSM), 20–70% of whom are co-infected with HIV 1133 (depending on geographic location). The number of primary and secondary cases among women in the United States increased from 2004 to 2008 but has since been declining in conjunction with a decline in congenital syphilis. Surveillance of the number of new cases of primary and secondary syphilis has revealed multiple 7to 10-year cycles, which may be attributed to herd immunity in at-risk populations, changing sexual behaviors, and changes in control efforts.

1	The populations at highest risk for acquiring syphilis have changed over time, with outbreaks among MSM in the pre-HIV era of the late 1970s and early 1980s as well as at present. It is speculated that recent increases in syphilis and other sexually transmitted infections in MSM may be due to unprotected sex between persons who are HIV concordant and to disinhibition caused by highly effective antiretroviral therapies. The syphilis epidemic that peaked in 1990 predominantly affected African-American heterosexual men and women and occurred largely in urban areas, where infectious syphilis was correlated with the exchange of sex for crack cocaine. The rate of primary and secondary syphilis among African Americans nearly doubled between 2003 and 2009, remains higher than rates for other racial/ethnic groups, but has since declined somewhat (Fig. 206-1).

1	The incidence of congenital syphilis roughly parallels that of infectious syphilis in women. In 2011, 360 cases in infants <1 year of age were reported, for a decline of 20% since 2008. The case definition for congenital syphilis was broadened in 1989 and now includes all live or stillborn infants delivered to women with untreated or inadequately treated syphilis. One-third to one-half of individuals named as sexual contacts of that can differentiate the three subspecies of T. pallidum by culture-independent methods based on polymerase chain reaction (PCR), but other sequence signatures cross subspecies boundaries in certain strains. Other Treponema species found in the human mouth, genital mucosa, and gastrointestinal tract have been associated with disease (e.g., periodontitis), but their role as primary etiologic agents is unclear.

1	T. pallidum subspecies pallidum (referred to hereafter as T. pallidum), a thin spiral organism, has a cell body surrounded by a trilaminar cytoplasmic membrane, a delicate peptidoglycan layer providing some structural rigidity, and a lipid-rich outer membrane containing relatively few integral membrane proteins. Endoflagella wind around the cell body in the periplasmic space and are responsible for motility.

1	T. pallidum cannot be cultured in vitro, and little was known about its metabolism until the genome was sequenced in 1998. This spirochete possesses severely limited metabolic capabilities, lacking the genes required for de novo synthesis of most amino acids, nucleotides, and lipids. In addition, T. pallidum lacks genes encoding the enzymes of the Krebs cycle and oxidative phosphorylation. The organism contains numerous compensatory genes predicted to encode transporters of amino acids, carbohydrates, and lipids. In addition, genome analyses and other studies have revealed the existence of a 12-member gene family (tpr) that bears similarities to variable outer-membrane antigens of other spirochetes. One member, TprK, has discrete variable (V) regions that undergo antigenic variation during infection, providing a mechanism for immune evasion.

1	The only known natural host for T. pallidum is the human. T. pallidum can infect many mammals, but only humans, higher apes, and a few laboratory animals regularly develop syphilitic lesions. Rabbits are used to propagate virulent strains of T. pallidum and serve as the animal model that best reflects human disease and immunopathology. Nearly all cases of syphilis are acquired by sexual contact with infectious lesions (i.e., the chancre, mucous patch, skin rash, or condylomata lata; see Fig. 25e-20). Less common modes of transmission include nonsexual personal contact, infection in utero, blood transfusion, and organ transplantation.

1	With the advent of penicillin therapy, the total number of cases of syphilis reported annually in the United States declined significantly to a low of 31,575 cases in 2000—a 95% decrease from 1943—with <6000 reported cases of infectious primary and secondary syphilis (the latter being a better indicator of disease activity than total syphilis cases). Since 2000, the number of cases of primary and secondary syphilis has more than doubled, with more than 14,000 cases reported in 2012 (Fig. 206-1). Approximately 70% of these cases were in men who have persons with infectious syphilis become infected. Many have already developed manifestations of syphilis when they are first seen, and ∼30% of asymptomatic contacts examined within 30 days of exposure actually have incubating infection and will later develop infectious syphilis if not treated. Thus, identification and treatment of all recently exposed sexual contacts continue to be important aspects of syphilis control.

1	Syphilis remains a significant health problem globally; the number of new infections is estimated at 11 million per year. The regions that are most affected include sub-Saharan Africa, South America, China, and Southeast Asia. During the past decade, the incidence rate in China has increased by approximately eightfold, and higher rates of infectious syphilis have been reported among MSM in many European countries. Worldwide, there are estimated to be 1.4 million cases of syphilis among pregnant women, with 500,000 adverse pregnancy outcomes annually (e.g., stillbirth, neonatal and early fetal death, prematurity/low birth weight, and infection in newborns). Congenital syphilis rates in China are ∼150 cases per 100,000 live births. FIGuRE 206-1 Primary and secondary syphilis in the United States, 1990–2012, by sex (A) and by race or ethnicity (B). (Data from the Centers for Disease Control and Prevention.) Number of cases 30,000 40,000

1	Number of cases 30,000 40,000 Number of cases 30,000 20,000 20,000 10,000 10,000 T. pallidum rapidly penetrates intact mucous membranes or microscopic abrasions in skin and, within a few hours, enters the lymphatics and blood to produce systemic infection and metastatic foci long before the appearance of a primary lesion. Blood from a patient with incubating or early syphilis is infectious. The generation time of T. pallidum during early active disease in vivo is estimated to be ∼30 h, and the incubation period of syphilis is inversely proportional to the number of organisms inoculated. The 50% infectious dose for intradermal inoculation in humans has been calculated to be 57 organisms, and the treponeme concentration generally reaches 107/g of tissue before a clinical lesion appears. The median incubation period in humans (∼21 days) suggests an average inoculum of 500–1000 infectious organisms for naturally acquired disease; the incubation period rarely exceeds 6 weeks.

1	The primary lesion appears at the site of inoculation, usually persists for 4–6 weeks, and then heals spontaneously. Histopathologic examination shows perivascular infiltration, chiefly by CD4+ and CD8+ T lymphocytes, plasma cells, and macrophages, with capillary endothelial proliferation and subsequent obliteration of small blood vessels. The cellular infiltration displays a TH1-type cytokine profile consistent with the activation of macrophages. Phagocytosis of opsonized organisms by activated macrophages ultimately causes their destruction, resulting in spontaneous resolution of the chancre.

1	The generalized parenchymal, constitutional, and mucocutaneous manifestations of secondary syphilis usually appear ∼6–8 weeks after the chancre heals, although primary and secondary manifestations may overlap. In contrast, some patients may enter the latent stage without ever recognizing secondary lesions. The histopathologic features of secondary maculopapular skin lesions include hyperkeratosis of the epidermis, capillary proliferation with endothelial swelling in the superficial dermis, dermal papillae with transmigration of polymorphonuclear leukocytes, and—in the deeper dermis—perivascular infiltration by CD8+ T lymphocytes, CD4+ T lymphocytes, macrophages, and plasma cells. Treponemes are found in many tissues, including the aqueous humor of the eye and the cerebrospinal fluid (CSF). T. pallidum invades the CNS during the first weeks or months of infection, and CSF abnormalities are detected in as many as 40% of patients during the secondary stage. Clinical hepatitis and immune

1	T. pallidum invades the CNS during the first weeks or months of infection, and CSF abnormalities are detected in as many as 40% of patients during the secondary stage. Clinical hepatitis and immune complex–induced glomerulonephritis are relatively rare but recognized manifestations of secondary syphilis; liver function tests may yield abnormal results in up to one-quarter of patients with early syphilis. Generalized non-tender lymphadenopathy is noted in 85% of patients with secondary syphilis. The paradoxical appearance of secondary manifestations despite high titers of antibody (including immobilizing antibody) to

1	T. pallidum may result from immune evasion due to antigenic variation or changes in expression of surface antigens. Secondary lesions generally subside within 2–6 weeks, and the infection enters the latent stage, which is detectable only by serologic testing. In the preantibiotic era, up to 25% of untreated patients experienced at least one generalized or localized mucocutaneous relapse, usually during the first year. Therefore, identification and examination of sexual contacts are most important for patients with syphilis of <1 year’s duration.

1	As stated earlier, about one-third of patients with untreated latent syphilis developed clinically apparent tertiary disease in the preantibiotic era, when the most common types of tertiary disease were the gumma (a usually benign granulomatous lesion); cardiovascular syphilis (usually involving the vasa vasorum of the ascending aorta and resulting in aneurysm); and late symptomatic neurosyphilis (tabes dorsalis and paresis). In Western countries today, specific treatment for early and latent syphilis and coincidental therapy (i.e., therapy with antibiotics that are given for other conditions but are active against treponemes) have nearly eliminated tertiary syphilis. Asymptomatic CNS involvement, however, is still demonstrable in up to 40% of persons with early syphilis and 25% of patients with late latent syphilis, and cases of general paresis and tabes dorsalis are being reported from China. The factors that contribute to the development and progression of tertiary disease are

1	with late latent syphilis, and cases of general paresis and tabes dorsalis are being reported from China. The factors that contribute to the development and progression of tertiary disease are unknown.

1	The course of untreated syphilis was studied retrospectively in a group of nearly 2000 patients with primary or secondary disease diagnosed clinically (the Oslo Study, 1891–1951) and was assessed prospectively in 431 African-American men with seropositive latent syphilis of ≥3 years’ duration (the notorious Tuskegee Study, 1932–1972). In the Oslo Study, 24% of patients developed relapsing secondary lesions within 4 years, and 28% eventually developed one or more manifestations of tertiary syphilis. Cardiovascular syphilis, including aortitis, was detected in 10% of patients; 7% of patients developed symptomatic neurosyphilis, and 16% developed benign tertiary gummatous syphilis. Syphilis was the primary cause of death in 15% of men and 8% of women. Cardiovascular syphilis was documented in 35% of men and 22% of women who eventually came to autopsy. In general, serious late complications were nearly twice as common among men as among women.

1	The Tuskegee Study showed that the death rate among untreated African-American men with syphilis (25–50 years old) was 17% higher than the rate among uninfected subjects and that 30% of all deaths were attributable to cardiovascular or, to a lesser extent, CNS syphilis. Anatomic evidence of aortitis was found in 40–60% of autopsied subjects with syphilis (vs 15% of control subjects), whereas CNS syphilis was found in only 4%. Rates of hypertension were also higher among the infected subjects. The ethical issues eventually raised by this study, begun in the preantibiotic era but continuing into the early 1970s, had a major influence on the development of current guidelines for human medical experimentation, and the history of the study may still contribute to a reluctance of some African Americans to participate as subjects in clinical research.

1	Primary Syphilis The typical primary chancre usually begins as a single painless papule that rapidly becomes eroded and usually becomes indurated, with a characteristic cartilaginous consistency on palpation of the edge and base of the ulcer. Multiple primary lesions are seen in a minority of patients. In heterosexual men the chancre is usually located on the penis (Fig. 206-2; see also Fig. 25e-17), whereas in MSM it may be found in the anal canal or rectum, in the mouth, or on the external genitalia. Oral sex has been identified as the source of infection in some MSM. In women, common primary sites are the cervix and labia. Consequently, primary syphilis goes unrecognized in women and homosexual men more often than in heterosexual men. FIGuRE 206-2 Primary syphilis with a firm, nontender chancre.

1	Atypical primary lesions are common. The clinical appearance depends on the number of treponemes inoculated and on the immunologic status of the patient. A large inoculum produces a dark-fieldpositive ulcerative lesion in nonimmune volunteers but may produce a small dark-field-negative papule, an asymptomatic but seropositive latent infection, or no response at all in some individuals with a history of syphilis. A small inoculum may produce only a papular lesion, even in nonimmune individuals. Therefore, syphilis should be considered even in the evaluation of trivial or atypical dark-field-negative genital lesions. The genital lesions that most commonly must be differentiated from those of primary syphilis include those caused by herpes simplex virus infection (Chap. 216), chancroid (Chap. 182), traumatic injury, and donovanosis (Chap. 198e). Regional (usually inguinal) lymphadenopathy accompanies the primary syphilitic lesion, appearing within 1 week of lesion onset. The nodes are

1	182), traumatic injury, and donovanosis (Chap. 198e). Regional (usually inguinal) lymphadenopathy accompanies the primary syphilitic lesion, appearing within 1 week of lesion onset. The nodes are firm, nonsuppurative, and painless. Inguinal lymphadenopathy is bilateral and may occur with anal as well as with external genital chancres. The chancre generally heals within 4–6 weeks (range, 2–12 weeks), but lymphadenopathy may persist for months.

1	Secondary Syphilis The protean manifestations of the secondary stage usually include mucocutaneous lesions and generalized nontender lymphadenopathy. The healing primary chancre may still be present in ∼15% of cases, and the stages may overlap more frequently in persons with concurrent HIV infection. The skin rash consists of macular, papular, papulosquamous, and occasionally pustular syphilides; often more than one form is present simultaneously. The eruption may be very subtle, and 25% of patients with a discernible rash may be unaware that they have dermatologic manifestations. Initial lesions are pale red or pink, nonpruritic, discrete macules distributed on the trunk and proximal extremities; these macules progress to papular lesions that are distributed widely and that frequently involve the palms and soles (Fig. 206-3; see also Figs. 25e-18 and 25e-19). Rarely, severe necrotic lesions (lues maligna) may appear; they are more commonly reported in HIV-infected individuals.

1	involve the palms and soles (Fig. 206-3; see also Figs. 25e-18 and 25e-19). Rarely, severe necrotic lesions (lues maligna) may appear; they are more commonly reported in HIV-infected individuals. Involvement of the hair follicles may result in patchy alopecia of the scalp hair, eyebrows, or beard in up to 5% of cases.

1	In warm, moist, intertriginous areas (commonly the perianal region, vulva, and scrotum), papules can enlarge to produce broad, moist, pink or gray-white, highly infectious lesions (condylomata lata; see Fig. 25e-20) in 10% of patients with secondary syphilis. Superficial mucosal erosions (mucous patches) occur in 10–15% of patients and commonly involve the oral or genital mucosa (see Fig. 25e-21). The typical mucous patch is a painless silver-gray erosion surrounded by a red periphery.

1	Constitutional signs and symptoms that may accompany or precede secondary syphilis include sore throat (15–30%), fever (5–8%), weight loss (2–20%), malaise (25%), anorexia (2–10%), headache (10%), and meningismus (5%). Acute meningitis occurs in only 1–2% of cases, but CSF cell and protein concentrations are increased in up to 40% of cases, and viable T. pallidum organisms have been recovered from CSF during primary and secondary syphilis in 30% of cases; the latter finding is 1135 often but not always associated with other CSF abnormalities.

1	Less common complications of secondary syphilis include hepatitis, nephropathy, gastrointestinal involvement (hypertrophic gastritis, patchy proctitis, or a rectosigmoid mass), arthritis, and periostitis. Ocular findings associated with secondary syphilis include pupillary abnormalities and optic neuritis as well as the classic iritis or uveitis. The diagnosis of ocular syphilis is often considered in affected patients only after they fail to respond to steroid therapy. Anterior uveitis has been reported in 5–10% of patients with secondary syphilis, and T. pallidum has been demonstrated in aqueous humor from such patients. Hepatic involvement is common in syphilis; although it is usually asymptomatic, up to 25% of patients may have abnormal liver function tests. Frank syphilitic hepatitis may be seen. Renal involvement usually results from immune complex deposition and produces proteinuria associated with an acute nephrotic syndrome. Like those of primary syphilis, the manifestations

1	may be seen. Renal involvement usually results from immune complex deposition and produces proteinuria associated with an acute nephrotic syndrome. Like those of primary syphilis, the manifestations of the secondary stage resolve spontaneously, usually within 1–6 months.

1	Latent Syphilis Positive serologic tests for syphilis, together with a normal CSF examination and the absence of clinical manifestations of syphilis, indicate a diagnosis of latent syphilis in an untreated person. The diagnosis is often suspected on the basis of a history of primary or secondary lesions, a history of exposure to syphilis, or the delivery of an infant with congenital syphilis. A previous negative serologic test or a history of lesions or exposure may help establish the duration of latent infection, which is an important factor in the selection of appropriate therapy. Early latent syphilis is limited to the first year after infection, whereas late latent syphilis is defined as that of ≥1 year’s duration (or of unknown duration). T. pallidum may still seed the bloodstream intermittently during the latent stage, and pregnant women with latent syphilis may infect the fetus in utero. Moreover, syphilis has been transmitted through blood transfusion or organ donation from

1	intermittently during the latent stage, and pregnant women with latent syphilis may infect the fetus in utero. Moreover, syphilis has been transmitted through blood transfusion or organ donation from patients with latent syphilis. It was previously thought that untreated late latent syphilis had three possible outcomes: (1) persistent lifelong infection; (2) development of late syphilis; or (3) spontaneous cure, with reversion of serologic tests to negative. It is now apparent, however, that the more sensitive treponemal antibody tests rarely, if ever, become nonreactive without treatment. Although progression to clinically evident late syphilis is very rare today, the occurrence of spontaneous cure is in doubt.

1	Involvement of the CNS Traditionally, neurosyphilis has been considered a late manifestation of syphilis, but this view is inaccurate. CNS syphilis represents a continuum encompassing early invasion (usually within the first weeks or months of infection), months to years of asymptomatic involvement, and, in some cases, development of early or late neurologic manifestations.

1	asymptomatic neUrosypHilis The diagnosis of asymptomatic neurosyphilis is made in patients who lack neurologic symptoms and signs but who have CSF abnormalities including mononuclear pleocytosis, increased protein concentrations, or CSF reactivity in the Venereal Disease Research Laboratory (VDRL) test. CSF abnormalities are demon- strated in up to 40% of cases of primary or secondary syphilis and in 25% of cases of latent syphilis. T. pallidum has been recovered by inoculation into rab-bits of CSF from up to 30% of patients with primary or secondary syphilis but less frequently by inoculation of CSF from patients with latent syphilis. The presence of T. pallidum in CSF is often associated with other CSF abnormali-ties, but organisms can be recovered from patients with otherwise normal CSF. Although the prognostic implica-tions of these findings in early syphilis

1	FIGuRE 206-3 Secondary syphilis. Left: Maculopapular truncal eruption. Middle: Papules on the are uncertain, it may be appropriate palms. Right: Papules on the soles. (Courtesy of Jill McKenzie and Christina Marra.) to conclude that even patients with 1136 early syphilis who have such findings do indeed have asymptomatic neurosyphilis and should be treated for neurosyphilis; such treatment is particularly important in patients with concurrent HIV infection. Before the advent of penicillin, the risk of development of clinical neurosyphilis in untreated asymptomatic persons was roughly proportional to the intensity of CSF changes, with the overall cumulative probability of progression to clinical neurosyphilis ∼20% in the first 10 years but increasing with time. Most experts agree that neurosyphilis is more common in HIV-infected persons, while immunocompetent patients with untreated latent syphilis and normal CSF probably run a very low risk of subsequent neurosyphilis. In several

1	neurosyphilis is more common in HIV-infected persons, while immunocompetent patients with untreated latent syphilis and normal CSF probably run a very low risk of subsequent neurosyphilis. In several recent studies, neurosyphilis was associated with a rapid plasma reagin (RPR) titer of ≥1:32, regardless of clinical stage or HIV infection status.

1	symptomatic neUrosypHilis The major clinical categories of symptomatic neurosyphilis include meningeal, meningovascular, and parenchymatous syphilis. The last category includes general paresis and tabes dorsalis. The onset of symptoms usually occurs <1 year after infection for meningeal syphilis, up to 10 years after infection for meningovascular syphilis, at ∼20 years for general paresis, and at 25–30 years for tabes dorsalis. Neurosyphilis is more frequently symptomatic in patients who are co-infected with HIV, particularly in the setting of a low CD4+ T lymphocyte count. In addition, recent evidence suggests that syphilis infection worsens the cognitive impairment seen in HIV-infected persons and that this effect persists even after treatment for syphilis.

1	Meningeal syphilis may present as headache, nausea, vomiting, neck stiffness, cranial nerve involvement, seizures, and changes in mental status. This condition may be concurrent with or may follow the secondary stage. Patients presenting with uveitis, iritis, or hearing loss often have meningeal syphilis, but these clinical findings can also be seen in patients with normal CSF. Meningovascular syphilis reflects meningitis together with inflammatory vasculitis of small, medium, or large vessels. The most common presentation is a stroke syndrome involving the middle cerebral artery of a relatively young adult. However, unlike the usual thrombotic or embolic stroke syndrome of sudden onset, meningovascular syphilis often becomes manifest after a subacute encephalitic prodrome (with headaches, vertigo, insomnia, and psychological abnormalities), which is followed by a gradually progressive vascular syndrome.

1	The manifestations of general paresis reflect widespread late parenchymal damage and include abnormalities corresponding to the mnemonic paresis: personality, affect, reflexes (hyperactive), eye (e.g., Argyll Robertson pupils), sensorium (illusions, delusions, hallucinations), intellect (a decrease in recent memory and in the capacity for orientation, calculations, judgment, and insight), and speech. Tabes dorsalis is a late manifestation of syphilis that presents as symptoms and signs of demyelination of the posterior columns, dorsal roots, and dorsal root ganglia. Symptoms include ataxic wide-based gait and foot drop; paresthesia; bladder disturbances; impotence; areflexia; and loss of positional, deep-pain, and temperature sensations. Trophic joint degeneration (Charcot’s joints) and perforating ulceration of the feet can result from loss of pain sensation. The small, irregular Argyll Robertson pupil, a feature of both tabes dorsalis and paresis, reacts to accommodation but not to

1	ulceration of the feet can result from loss of pain sensation. The small, irregular Argyll Robertson pupil, a feature of both tabes dorsalis and paresis, reacts to accommodation but not to light. Optic atrophy also occurs frequently in association with tabes.

1	Other Manifestations of Late Syphilis The slowly progressive inflammatory process leading to tertiary disease begins early during infection, although these manifestations may not become clinically apparent for years or decades. Early syphilitic aortitis becomes evident soon after secondary lesions subside, and treponemes that trigger the development of gummas may have seeded the tissue years earlier.

1	cardiovascUlar sypHilis Cardiovascular manifestations, usually appearing 10–40 years after infection, are attributable to endarteritis obliterans of the vasa vasorum, which provide the blood supply to large vessels; T. pallidum DNA has been detected by PCR in aortic tissue. Cardiovascular involvement results in uncomplicated aortitis, aortic regurgitation, saccular aneurysm (usually of the ascending aorta), or coronary ostial stenosis. In the preantibiotic era, symptomatic cardiovascular complications developed in ∼10% of persons with late untreated syphilis. Today, this form of late syphilis is rarely seen in the developed world. Linear calcification of the ascending aorta on chest x-ray films suggests asymptomatic syphilitic aortitis, as arteriosclerosis seldom produces this sign. Only 1 in 10 aortic aneurysms of syphilitic origin involves the abdominal aorta.

1	late benign sypHilis (gUmma) Gummas are usually solitary lesions ranging from microscopic to several centimeters in diameter. Histologic examination shows a granulomatous inflammation, with a central area of necrosis due to endarteritis obliterans. Although rarely demonstrated microscopically, T. pallidum has been detected by PCR or recovered from these lesions, and penicillin treatment results in rapid resolution, confirming the treponemal stimulus for the inflammation. Common sites include the skin and skeletal system; however, any organ (including the brain) may be involved. Gummas of the skin produce indolent, painless, indurated nodular or ulcerative lesions that may resemble other chronic granulomatous conditions, including tuberculosis, sarcoidosis, leprosy, and deep fungal infections. Skeletal gummas most frequently involve the long bones, although any bone may be affected. Upper respiratory gummas can lead to perforation of the nasal septum or palate.

1	Congenital Syphilis Transmission of T. pallidum across the placenta from a syphilitic woman to her fetus may occur at any stage of pregnancy, but fetal damage generally does not occur until after the fourth month of gestation, when fetal immunologic competence begins to develop. This timing suggests that the pathogenesis of congenital syphilis, like that of adult syphilis, depends on the host immune response rather than on a direct toxic effect of T. pallidum. The risk of fetal infection during untreated early maternal syphilis is ∼75–95%, decreasing to ∼35% for maternal syphilis of >2 years’ duration. Adequate treatment of the woman before the 16th week of pregnancy should prevent fetal damage, and treatment before the third trimester should adequately treat the infected fetus. Untreated maternal infection may result in a rate of fetal loss of up to 40% (with stillbirth more common than abortion because of the late onset of fetal pathology), prematurity, neonatal death, or nonfatal

1	maternal infection may result in a rate of fetal loss of up to 40% (with stillbirth more common than abortion because of the late onset of fetal pathology), prematurity, neonatal death, or nonfatal congenital syphilis. Among infants born alive, only fulminant congenital syphilis is clinically apparent at birth, and these babies have a very poor prognosis. The most common clinical problem is the healthy-appearing baby born to a mother with a positive serologic test

1	Routine serologic testing in early pregnancy is considered cost-effective in virtually all populations, even in areas with a low prenatal prevalence of syphilis. Low-tech point-of-care tests have been developed and are being widely implemented to facilitate antenatal testing in resource-poor settings. A recent study demonstrated the high cost-effectiveness of using these tests for screening (with subsequent treatment) in sub-Saharan Africa. Adverse outcomes were reduced, with 64,000 fewer stillbirths, 25,000 fewer neonatal deaths, and up to 25,000 fewer live births of infants with syphilis. The intervention would remain cost-effective even if the current syphilis seroprevalence among pregnant women declined from its present 3.1% to 0.4%. Where the prevalence of syphilis is high or when the patient is at high risk of reinfection, serologic testing should be repeated in the third trimester and at delivery. Neonatal congenital syphilis must be differentiated from other generalized

1	the patient is at high risk of reinfection, serologic testing should be repeated in the third trimester and at delivery. Neonatal congenital syphilis must be differentiated from other generalized congenital infections, including rubella, cytomegalovirus or herpes simplex virus infection, and toxoplasmosis, as well as from erythroblastosis fetalis.

1	The manifestations of congenital syphilis include (1) early manifestations, which appear within the first 2 years of life (often at 2–10 weeks of age), are infectious, and resemble the manifestations of secondary syphilis in the adult; (2) late manifestations, which appear after 2 years and are noninfectious; and (3) residual stigmata. The earliest manifestations of congenital syphilis include rhinitis, or “snuffles” (23%); mucocutaneous lesions (35–41%); bone changes (61%), including osteochondritis, osteitis, and periostitis detectable by x-ray examination of long bones; hepatosplenomegaly (50%); lymphadenopathy (32%); anemia (34%); jaundice (30%); thrombocytopenia; and leukocytosis. CNS invasion by T. pallidum is detectable in 22% of infected neonates. Neonatal death is usually due to pulmonary hemorrhage, secondary bacterial infection, or severe hepatitis.

1	Late congenital syphilis (untreated after 2 years of age) is subclinical in 60% of cases; the clinical spectrum in the remainder of cases may include interstitial keratitis (which occurs at 5–25 years of age), eighth-nerve deafness, and recurrent arthropathy. Bilateral knee effusions are known as Clutton’s joints. Neurosyphilis was present in about one-quarter of untreated patients with late congenital syphilis in the preantibiotic era. Gummatous periostitis occurs at 5–20 years of age and, as in nonvenereal endemic syphilis, tends to cause destructive lesions of the palate and nasal septum. Classic stigmata include Hutchinson’s teeth (centrally notched, widely spaced, peg-shaped upper central incisors), “mulberry” molars (sixth-year molars with multiple, poorly developed cusps), saddle nose, and saber shins.

1	Demonstration of the Organism T. pallidum cannot be detected by culture. Historically, dark-field microscopy and immunofluorescence antibody staining have been used to identify this spirochete in samples from moist lesions such as chancres or condylomata lata, but these tests are rarely available outside of research laboratories. Sensitive and specific PCR tests have been developed but are not commercially available, although a number of laboratories perform in-house validated PCR testing. T. pallidum can be found in tissue with appropriate silver stains, but these results should be interpreted with caution because artifacts resembling T. pallidum are often seen. Tissue treponemes can be demonstrated more reliably in research laboratories by PCR or by immunofluorescence or immunohistochemical methods using specific monoclonal or polyclonal antibodies to T. pallidum.

1	Serologic Tests for Syphilis There are two types of serologic test for syphilis: nontreponemal and treponemal. Both are reactive in persons with any treponemal infection, including yaws, pinta, and endemic syphilis.

1	The most widely used nontreponemal antibody tests for syphilis are the RPR and VDRL tests, which measure IgG and IgM directed against a cardiolipin-lecithin-cholesterol antigen complex. The RPR test is easier to perform and uses unheated serum or plasma; it is the test of choice for rapid serologic diagnosis in a clinical setting. The VDRL test remains the standard for examining CSF and is superior to the RPR for this purpose. The RPR and VDRL tests are recommended for screening or for quantitation of serum antibody. The titer reflects disease activity, rising during the evolution of early syphilis, often exceeding 1:32 in secondary syphilis, and declining thereafter without therapy. After treatment for early syphilis, a persistent fall by fourfold or more (e.g., a decline from 1:32 to 1:8) is considered an adequate response. VDRL titers do not correspond directly to RPR titers, and sequential quantitative testing (as for response to therapy) must employ a single test. As will be

1	1:8) is considered an adequate response. VDRL titers do not correspond directly to RPR titers, and sequential quantitative testing (as for response to therapy) must employ a single test. As will be discussed (see “Evaluation for Neurosyphilis,” below), the RPR titer may be useful in determining which patients will benefit from CSF examination.

1	Treponemal tests measure antibodies to native or recombinant T. pallidum antigens and include the fluorescent treponemal antibody– absorbed (FTA-ABS) test and the T. pallidum particle agglutination (TPPA) test, both of which are more sensitive for primary syphilis than the previously used hemagglutination tests. The T. pallidum hemagglutination (TPHA) test is widely used in Europe but is not available in the United States. When used to confirm positive nontreponemal test results, treponemal tests have a very high positive predictive value for diagnosis of syphilis. Treponemal enzyme or chemiluminescence immunoassays (EIAs/CIAs), based largely on reactivity to recombinant antigens, have also been developed and are now widely used as screening tests by large laboratories. In a screening setting, however, treponemal tests give false-positive results at rates as high as 1–2%, and the rate is higher with the EIA/CIA tests. Treponemal tests are likely to remain reactive even after adequate

1	however, treponemal tests give false-positive results at rates as high as 1–2%, and the rate is higher with the EIA/CIA tests. Treponemal tests are likely to remain reactive even after adequate treatment and cannot differentiate

1	EIA+ RPR+ Consistent with past or current syphilis EIA+ RPR– TPPA– Unconfirmed EIA. Unlikely to be syphilis. If pt is at risk for syphilis, retest in 1 month. EIA+ RPR– TPPA+ Possible syphilis infection. Requires historical and clinical evaluation. EIA+ RPR– TPPA or FTA-ABS (Different platform and target antigens) EIA– Negative for syphilis antibodies Syphilis EIA EIA+ Quantitative RPR or VDRL FIGuRE 206-4 Algorithm for interpretation of results from syphilis enzyme immunoassays (EIAs) used for screening. FTA-ABS, fluores-cent treponemal antibody–absorbed; RPR, rapid plasma reagin; TPPA, Treponema pallidum particle agglutination; VDRL, Venereal Disease Research Laboratory. (Based on the 2010 Sexually Transmitted Diseases Treatment Guidelines from the Centers for Disease Control and Prevention.) past from current T. pallidum infection. Figure 206-4 provides a suggested algorithm for management of such cases.

1	Both nontreponemal and treponemal tests may be nonreactive in early primary syphilis, although treponemal tests are slightly more sensitive (85–90%) during this stage than nontreponemal tests (∼80%). All tests are reactive during secondary syphilis. (Fewer than 1% of patients with high titers have a nontreponemal test that is nonreactive or weakly reactive with undiluted serum but is reactive with diluted serum—the prozone phenomenon.) VDRL and RPR sensitivity and titers may decline in untreated persons with late latent syphilis, but treponemal tests remain sensitive in these stages. After treatment for early syphilis, nontreponemal test titers will generally decline or the tests will become nonreactive, whereas treponemal tests often remain reactive after therapy and are not helpful in determining the infection status of persons with past syphilis.

1	For practical purposes, most clinicians need to be familiar with three uses of serologic tests for syphilis recommended by the Centers for Disease Control and Prevention (CDC): (1) screening or diagnosis (RPR or VDRL), (2) quantitative measurement of antibody to assess clinical syphilis activity or to monitor response to therapy (RPR or VDRL), and (3) confirmation of a syphilis diagnosis in a patient with a reactive RPR or VDRL test (FTA-ABS, TPPA, EIA/CIA). Studies have not demonstrated the utility of IgM testing for adult syphilis. Whereas IgM titers appear to decline after therapy, the presence or absence of specific IgM does not strictly correlate with T. pallidum infection. Moreover, no commercially available IgM test is recommended, even for evaluation of infants with suspected congenital syphilis.

1	False-Positive Serologic Tests for Syphilis The lipid antigens of nontreponemal tests are similar to those found in human tissues, and the tests may be reactive (usually with titers ≤1:8) in persons without treponemal infection. Among patients being screened for syphilis because of risk factors, clinical suspicion, or history of exposure, ∼1% of reactive tests are falsely positive. Modern VDRL and RPR tests are highly specific, and false-positive reactions are largely limited to persons with 1138 autoimmune conditions or injection drug use. The prevalence of false-positive results increases with advancing age, approaching 10% among persons >70 years old. In a patient with a false-positive nontreponemal test, syphilis is excluded by a nonreactive treponemal test. False-positive reactions may also occur with the treponemal tests, particularly the new, very sensitive EIA/CIA tests. When a low-prevalence population for syphilis is screened, the number of false-positive reactions may

1	may also occur with the treponemal tests, particularly the new, very sensitive EIA/CIA tests. When a low-prevalence population for syphilis is screened, the number of false-positive reactions may outnumber true positives, leading to unnecessary treatment. Although the precise reason is not known, it has been shown that sera from patients with periodontal disease react with antigens used in the EIA/CIA tests, presumably as a result of cross-reactive epitopes in the many treponemes that infect the gingival crevices during periodontal disease.

1	Evaluation for Neurosyphilis Involvement of the CNS is detected by examination of CSF for pleocytosis (>5 white blood cells/μL), increased protein concentration (>45 mg/dL), or VDRL reactivity. Elevated CSF cell counts and protein concentrations are not specific for neurosyphilis and may be confounded by HIV co-infection. Because CSF pleocytosis may also be due to HIV, some studies have suggested using a CSF white-cell cutoff of 20 cells/μL as diagnostic of neurosyphilis in HIV-infected patients with syphilis. The CSF VDRL test is highly specific and, when reactive, is considered diagnostic of neurosyphilis; however, this test is insensitive and may be nonreactive even in cases of symptomatic neurosyphilis. The FTA-ABS test on CSF is reactive far more often than the VDRL test on CSF in all stages of syphilis, but reactivity may reflect passive transfer of serum antibody into the CSF. A nonreactive FTA-ABS test on CSF, however, may be used to rule out asymptomatic neurosyphilis. The

1	all stages of syphilis, but reactivity may reflect passive transfer of serum antibody into the CSF. A nonreactive FTA-ABS test on CSF, however, may be used to rule out asymptomatic neurosyphilis. The utility of measuring CXCL13 in CSF to distinguish between neurosyphilis and HIV-related CSF abnormalities has been demonstrated.

1	Clearly, all T. pallidum–infected patients who have signs or symptoms consistent with neurologic disease (e.g., meningitis, hearing loss) or ophthalmic disease (e.g., uveitis, iritis) should have a CSF examination, regardless of disease stage. The appropriate management of asymptomatic persons is less clear. Lumbar puncture on all asymptomatic patients with untreated syphilis is impractical and unnecessary. Because standard therapy with penicillin G benzathine fails to result in treponemicidal drug levels in CSF, however, it is important to identify those persons at higher risk for having or developing neurosyphilis so that appropriate therapy may be given. Viable T. pallidum has been isolated from the CSF of several patients (with and without concurrent HIV infection) after penicillin G benzathine therapy for early syphilis. Large-scale prospective studies have now provided evidence-based guidelines for determining which syphilis patients may benefit most from CSF examination for

1	G benzathine therapy for early syphilis. Large-scale prospective studies have now provided evidence-based guidelines for determining which syphilis patients may benefit most from CSF examination for evidence of neurosyphilis. Specifically, patients with RPR titers of ≥1:32 are at higher risk of having neurosyphilis (11-fold and 6-fold higher in HIV-infected and HIV-uninfected persons, respectively), as are HIV-infected patients with CD4+ T cell counts of ≤350/μL. Guidelines for CSF examination are shown in Table 206-1.

1	Evaluation of HIV-Infected Patients for Syphilis Because persons at highest risk for syphilis are also at increased risk for HIV infection, these Signs or symptoms of nervous system involvement (e.g., meningitis, hearing loss, cranial nerve dysfunction, altered mental status, ophthalmic disease [e.g., uveitis, iritis, pupillary abnormalities], ataxia, loss of vibration sense), or RPR or VDRL titer ≥1:32, or Active tertiary syphilis, or Suspected treatment failure CD4+ T cell count ≤350/μL, or All HIV-infected persons (recommended by some experts) Source: Adapted from the 2010 Sexually Transmitted Diseases Treatment Guidelines from the Centers for Disease Control and Prevention.

1	All HIV-infected persons (recommended by some experts) Source: Adapted from the 2010 Sexually Transmitted Diseases Treatment Guidelines from the Centers for Disease Control and Prevention. two infections frequently coexist. There is evidence that syphilis and other genital ulcer diseases are important risk factors for acquisition and transmission of HIV infection. Some manifestations of syphilis may be altered in patients with concurrent HIV infection, and multiple cases of neurologic relapse after standard therapy have been reported in these patients.

1	Persons with newly diagnosed HIV infection should be tested for syphilis; conversely, all patients with newly diagnosed syphilis should be tested for HIV infection. Some authorities, persuaded by reports of persistent T. pallidum in CSF of HIV-infected persons after standard therapy for early syphilis, recommend CSF examination for evidence of neurosyphilis for all co-infected patients, regardless of the stage of syphilis, with treatment for neurosyphilis if CSF abnormalities are found. Others, on the basis of their own clinical experience, believe that standard therapy—without CSF examination—is sufficient for all cases of early syphilis in HIV-infected patients without neurologic signs or symptoms. As described above, RPR titer and CD4+ T cell count can be used to identify patients at higher risk of neurosyphilis for lumbar puncture, although some cases of neurosyphilis will be missed, even when these criteria are used. Table 206-1 summarizes guidelines suggested by published

1	at higher risk of neurosyphilis for lumbar puncture, although some cases of neurosyphilis will be missed, even when these criteria are used. Table 206-1 summarizes guidelines suggested by published studies. Serologic testing after treatment is important for all patients with syphilis, particularly for those also infected with HIV.

1	The CDC’s 2010 guidelines for the treatment of syphilis are summarized in Table 206-2 and are discussed below. Penicillin G is the drug of choice for all stages of syphilis. T. pallidum is killed by very low concentrations of penicillin G, although a long period of exposure to penicillin is required because of the unusually slow rate of multiplication of the organism. The efficacy of penicillin against syphilis remains undiminished after 60 years of use, and there is no evidence of penicillin resistance in T. pallidum. Other antibiotics effective in syphilis include the tetracyclines and the cephalosporins. Aminoglycosides and spectinomycin inhibit T. pallidum only in very large doses, and the sulfonamides and the quinolones are inactive. Azithromycin has shown significant promise as an effective oral agent against

1	T. pallidum; however, strains harboring 23S rRNA mutations that confer macrolide resistance are widespread; such strains represent >80% of recent isolates from Seattle and San Francisco and have now been identified in multiple North American and European sites. Macrolide resistance mutations have been identified in nearly all samples reported from some regions of China. In contrast, a study based in Madagascar documented the equivalence of benzathine penicillin and azithromycin for treatment of early syphilis, although a sample from one azithromycin clinical failure in that study showed the presence of a 23S rRNA resistance mutation. A more recent survey from South Africa showed a very low (1%) frequency of known 23s rRNA resistance mutations. In short, the prevalence of resistant strains varies widely by geographic location, and routine treatment of syphilis with azithromycin is not recommended. In all cases, careful follow-up of any patient treated for syphilis with azithromycin

1	varies widely by geographic location, and routine treatment of syphilis with azithromycin is not recommended. In all cases, careful follow-up of any patient treated for syphilis with azithromycin must be ensured.

1	Early Syphilis Patients and Their Contacts Penicillin G benzathine is the most widely used agent for the treatment of early syphilis; a single dose of 2.4 million units is recommended. Preventive treatment is also recommended for individuals who have been exposed to infectious syphilis within the previous 3 months. The regimens recommended for prevention are the same as those recommended for early syphilis. Penicillin G benzathine cures >95% of cases of early syphilis, although clinical relapse can follow treatment, particularly in patients with concurrent HIV infection. Because the risk of neurologic relapse may be higher in HIV-infected patients, CSF examination is recommended in HIV-seropositive individuals with syphilis of any stage, particularly those with a serum RPR titer of ≥1:32 or a Stage of Syphilis Patients without Penicillin Allergy Patients with Confirmed Penicillin Allergyb Primary, secondary, or early latent

1	Stage of Syphilis Patients without Penicillin Allergy Patients with Confirmed Penicillin Allergyb Primary, secondary, or early latent Late latent (or latent of uncertain duration), cardiovascular, or benign tertiary Syphilis in pregnancy CSF normal or not examined: Penicillin G benzathine (single dose of 2.4 mU IM) CSF abnormal: Treat as neurosyphilis CSF normal or not examined: Penicillin G benzathine (2.4 mU IM weekly for 3 weeks) CSF abnormal: Treat as neurosyphilis Aqueous crystalline penicillin G (18–24 mU/d IV, given as 3–4 mU q4h or continuous infusion) for 10–14 days Aqueous procaine penicillin G (2.4 mU/d IM) plus oral probenecid (500 mg qid), both for 10–14 days According to stage CSF normal or not examined: Tetracycline HCl (500 mg PO qid) or doxycycline (100 mg PO bid) for 2 weeks CSF abnormal: Treat as neurosyphilis CSF normal and patient not infected with HIV: Tetracycline HCl (500 mg PO qid) or doxycycline (100 mg PO bid) for 4 weeks

1	CSF abnormal: Treat as neurosyphilis CSF normal and patient not infected with HIV: Tetracycline HCl (500 mg PO qid) or doxycycline (100 mg PO bid) for 4 weeks CSF normal and patient infected with HIV: Desensitization and treatment with penicillin if compliance cannot be ensured CSF abnormal: Treat as neurosyphilis Desensitization and treatment with penicillinc Desensitization and treatment with penicillin aSee Table 206-1 and text for indications for CSF examination. bBecause of the documented presence of macrolide resistance in many T. pallidum strains in North America, Europe, and China, azithromycin or other macrolides should be used with caution only when treatment with penicillin or doxycycline is not feasible. Azithromycin should not be used for men who have sex with men or for pregnant women. cLimited data suggest that ceftriaxone (2 g/d either IM or IV for 10–14 days) can be used; however, cross-reactivity between penicillin and ceftriaxone is possible.

1	Abbreviations: CSF, cerebrospinal fluid; mU, million units. Source: Adapted from the 2010 Sexually Transmitted Diseases Treatment Guidelines from the Centers for Disease Control and Prevention. CD4+ T cell count of ≤350/μL. Therapy appropriate for neurosyphilis should be given if there is any evidence of CNS infection. Late Latent Syphilis or Syphilis of unknown Duration If the CSF is normal or is not examined, the recommended treatment is penicillin G benzathine (7.2 million units total; Table 206-2). If CSF abnormalities are found, the patient should be treated for neurosyphilis.

1	Tertiary Syphilis CSF examination should be performed. If the CSF is normal, the recommended treatment is penicillin G benzathine (7.2 million units total; Table 206-2). If CSF abnormalities are found, the patient should be treated for neurosyphilis. The clinical response to treatment for benign tertiary syphilis is usually impressive. However, responses to therapy for cardiovascular syphilis are not dramatic because aortic aneurysm and aortic regurgitation cannot be reversed by antibiotics.

1	Syphilis in Penicillin-Allergic Patients For penicillin-allergic patients with syphilis, a 2-week (early syphilis) or 4-week (late or late latent syphilis) course of therapy with doxycycline or tetracycline is recommended (Table 206-2). These regimens appear to be effective in early syphilis but have not been tested for late or late latent syphilis, and compliance may be problematic. Limited studies suggest that ceftriaxone (1 g/d, given IM or IV for 8–10 days) is effective for early syphilis. These nonpenicillin regimens have not been carefully evaluated in HIV-infected individuals and should be used with caution. If compliance and follow-up cannot be ensured, penicillin-allergic HIV-infected persons with late latent or late syphilis should be desensitized and treated with penicillin.

1	Neurosyphilis Penicillin G benzathine, given in total doses of up to 7.2 million units, does not produce detectable concentrations of penicillin G in CSF and should not be used for treatment of neurosyphilis. Asymptomatic neurosyphilis may relapse as symptomatic disease after treatment with benzathine penicillin, and the risk of relapse may be higher in HIV-infected patients. Both symptomatic and asymptomatic neurosyphilis should be treated with aqueous penicillin (Table 206-2). Administration either of IV aqueous crystalline penicillin G or of IM aqueous procaine penicillin G plus oral probenecid in recommended doses is thought to ensure treponemicidal concentrations of penicillin G in CSF. The clinical response to penicillin therapy for meningeal syphilis is dramatic, but treatment of neurosyphilis with existing parenchymal damage may only arrest disease progression. No data suggest that additional therapy (e.g., penicillin G benzathine for 3 weeks) is beneficial after treatment for

1	with existing parenchymal damage may only arrest disease progression. No data suggest that additional therapy (e.g., penicillin G benzathine for 3 weeks) is beneficial after treatment for neurosyphilis.

1	The use of antibiotics other than penicillin G for the treatment of neurosyphilis has not been studied, although very limited data suggest that ceftriaxone may be used. In patients with penicillin allergy demonstrated by skin testing, desensitization and treatment with penicillin are recommended. Management of Syphilis in Pregnancy Every pregnant woman should undergo a nontreponemal test at her first prenatal visit and, if at high risk of exposure, again in the third trimester and at delivery. In the untreated pregnant patient with presumed syphilis, expeditious treatment appropriate to the stage of the disease is essential. Patients should be warned of the risk of a Jarisch-Herxheimer reaction, which may be associated with mild premature contractions but rarely results in premature delivery.

1	Penicillin is the only recommended agent for the treatment of syphilis in pregnancy. If the patient has a documented penicillin allergy, desensitization and penicillin therapy should be undertaken according to the CDC’s 2010 guidelines. After treatment, a quantitative nontreponemal test should be repeated monthly throughout pregnancy to assess therapeutic efficacy. Treated women whose antibody titers rise by fourfold or whose titers do not decrease by fourfold over a 3-month period should be re-treated.

1	Whether or not they are infected, newborn infants of mothers with reactive serologic tests may themselves have reactive tests because of transplacental transfer of maternal IgG antibody. For asymptomatic infants born to women treated adequately with penicillin during the first or second trimester of pregnancy, monthly quantitative nontreponemal tests may be performed to monitor for appropriate reduction in antibody titers. Rising or persistent titers indicate infection, and the infant should be treated. Detection of neonatal IgM antibody may be useful, but no commercially available test is currently recommended.

1	An infant should be treated at birth if the treatment status of the seropositive mother is unknown; if the mother has received inadequate or nonpenicillin therapy; if the mother received penicillin therapy in the third trimester; or if the infant may be difficult to follow. The CSF should be examined to obtain baseline values before treatment. Penicillin is the only recommended drug for the treatment of syphilis in infants. Specific recommendations for the treatment of infants and older children are included in the CDC’s 2010 treatment guidelines.

1	1140 ARISCH-HERXHEIMER REACTIoN A dramatic although usually mild reaction consisting of fever, chills, myalgias, headache, tachycardia, increased respiratory rate, increased circulating neutrophil count, and vasodilation with mild hypotension may follow the initiation of treatment for syphilis. This reaction is thought to be a response to lipoproteins released by dying T. pallidum organisms. The arisch-Herxheimer reaction occurs in 50of patients with primary syphilis, of those with secondary syphilis, and a lower proportion of persons with later-stage disease. Defervescence takes place within 12–2 h. In patients with secondary syphilis, erythema and edema of the mucocutaneous lesions may increase. Patients should be warned to expect such symptoms, which can be managed with symptom-based treatment. Steroid or other anti-inflammatory therapy is not reuired for this mild transient reaction.

1	Efficacy of treatment should be assessed by clinical evaluation and monitoring of the uantitative L or P titer for a fourfold decline (e.g., from 12 to 1). Patients with primary or secondary syphilis should be examined and 12 months after treatment and persons with latent or late syphilis at , 12, and 2 months. More freuent clinical and serologic examination (, , 12, and 2 months) is recommended for patients concurrently infected with HI regardless of the stage of syphilis.

1	After successful treatment of seropositive first-episode primary or secondary syphilis, the L or P titer progressively declines, becoming negative by 12 months in 0–75of seropositive primary cases and in 20–0of secondary cases. Patients with HIinfection or a history of prior syphilis are less likely to become nonreactive in the L or P test. ates of decline of serologic titers appear to be slower, and serologically defined treatment failures more common, among HIinfected patients than among those without HI co-infectionhowever, effective antiretroviral therapy may reduce these differences. e-treatment should be considered if serologic responses are not adeuate or if clinical signs persist or recur. ecause it is difficult to differentiate treatment failure from reinfection, the CSF should be examined, with treatment for neurosyphilis if CSF is abnormal and treatment for late latent syphilis if CSF is normal. A minority of patients treated for early syphilis may experience a one-dilution

1	examined, with treatment for neurosyphilis if CSF is abnormal and treatment for late latent syphilis if CSF is normal. A minority of patients treated for early syphilis may experience a one-dilution titer increase within 1 days after treatmenthowever, this early elevation does not significantly affect the serologic outcome at months after treatment. Patients treated for late latent syphilis freuently have low initial L or P titers and may not have a fourfold decline after therapy with penicillin. In such patients, re-treatment is not warranted unless the titer rises or signs and symptoms of syphilis appear. ecause treponemal tests may remain reactive despite treatment for seropositive syphilis, these tests are not useful in following the response to therapy.

1	The activity of neurosyphilis (symptomatic or asymptomatic) correlates best with CSF pleocytosis, and this measure provides the most sensitive index of response to treatment. epeat CSF examinations should be performed every months until the cell count is normal. An elevated CSF cell count falls to normal in –12 months in adeuately treated HIuninfected patients. The persistence of mild pleocytosis in HIinfected patients may be due to the presence of HIin CSFthis scenario may be difficult to distinguish from treatment failure. Elevated levels of CSF protein fall more slowly, and the CSF L titer declines gradually over several years. In patients treated for neurosyphilis, a fourfold reduction in serum P titer has been positively correlated with normalization of CSF abnormalitiesthis correlation is stronger in HIuninfected patients and in HIinfected patients receiving effective antiretroviral therapy.

1	The rate of development of acquired resistance to T. pallidum after natural or experimental infection is related to the size of the antigenic stimulus, which depends on both the size of the infecting inoculum and the duration of infection before treatment. Both humoral and cellular responses are considered to be of major importance in immunity and in the healing of early lesions. Cellular infiltration, predominantly by T lymphocytes and macrophages, produces a TH1 cytokine milieu consistent with the clearance of organisms by activated macrophages. Specific antibody enhances phagocytosis and is required for macrophage-mediated killing of T. pallidum. Recent studies demonstrate antigenic variation of the TprK protein, which may lead to persistence of infection and determine susceptibility to reinfection with another strain. Comparative genomic studies have revealed some sequence variations among T. pallidum strains, which can be differentiated by molecular typing methods. Possible

1	to reinfection with another strain. Comparative genomic studies have revealed some sequence variations among T. pallidum strains, which can be differentiated by molecular typing methods. Possible correlations between molecular type and clinical manifestations are being examined.

1	are thought to persist in Haiti and other Caribbean islands, Peru, 207e-1 Endemic Treponematoses Colombia, Ecuador, Brazil, Guyana, and Surinam, although recent data are lacking. Pinta is limited to Central America and northern South Sheila A. Lukehart America, where it is found rarely and only in very remote villages.

1	The endemic treponematoses are chronic diseases that are transmitted by direct contact, usually during childhood, and, like syphilis, can cause severe late manifestations years after initial infection. These diseases are caused by very close relatives of Treponema pallidum subspecies pallidum, the etiologic agent of venereal syphilis (Chap. 206). Yaws, pinta, and endemic syphilis are traditionally distinguished from venereal syphilis by mode of transmission, age of acquisition, geographic distribution, and clinical features; however, there is some overlap for each of these factors. Generally, yaws flourishes in moist tropical areas of several regions, endemic syphilis is found primarily in arid climates, and pinta is found in temperate foci in the Americas (Fig. 207e-1). These infections are usually limited to rural areas of developing nations and are seen in developed countries only among recent immigrants from endemic regions. Our “knowledge” about the endemic treponematoses is

1	are usually limited to rural areas of developing nations and are seen in developed countries only among recent immigrants from endemic regions. Our “knowledge” about the endemic treponematoses is based on observations by health care workers who have visited endemic areas; virtually no well-designed studies of the natural history, diagnosis, or treatment of these infections have been conducted. The treponemal infections are compared and contrasted in Table 207e-1.

1	eradication campaign from 1952 to 1969, more than 160 mil lion people in Africa, Asia, and South America were examined for treponemal infections, and more than 50 million cases, contacts, and persons with latent infections were treated. This campaign reduced the prevalence of active yaws from >20% to <1% in many areas. In recent decades, lack of focused surveillance and diversion of resources have resulted in documented resurgence of these infections in some regions. The most recent WHO global estimate (1995) suggested that there are 460,000 new cases per year (mostly yaws) and a prevalence of 2.5 million infected persons; during subsequent years, an increased incidence was documented in some countries. Recent areas of resurgent yaws morbidity include West Africa (Ivory Coast, Ghana, Togo, Benin), the Central African Republic, Nigeria, and rural Democratic Republic of the Congo. The prevalence of endemic syphilis is estimated to be >10% in some regions of northern Ghana, Mali, Niger,

1	Benin), the Central African Republic, Nigeria, and rural Democratic Republic of the Congo. The prevalence of endemic syphilis is estimated to be >10% in some regions of northern Ghana, Mali, Niger, Burkina Faso, and Senegal. In Asia and the Pacific Islands, reports suggest active outbreaks of yaws in Indonesia, Papua New Guinea, the Solomon Islands, East Timor, Vanuatu, Laos, and Kampuchea. India actively renewed its focus on yaws control in 1996, achieved zero-case status in 2003, and declared elimination in 2006. In the Americas, foci of yaws

1	Evidence of yaws-like and venereal diseases, with treponemal seroreactivity, in wild gorillas and baboons in Africa has led to speculation that there may be an animal reservoir for yaws. The etiologic agents of the endemic treponematoses are listed in Table 207e-1. These little-studied organisms are morphologically identical to T. pallidum subspecies pallidum (the agent of venereal syphilis), and no definitive antigenic differences among them have been identified to date. A controversy has existed about whether the pathogenic treponemes are truly separate organisms, as genome sequencing indicates that yaws and syphilis treponemes are 99.8% identical. Three of the four organisms are classified as subspecies of

1	T. pallidum; the fourth (T. carateum) remains a separate species simply because no organisms have been available for genetic studies. Based on analysis of the small number of strains currently available, molecular signatures—assessed by polymerase chain reaction (PCR) amplification of tpr genes and restriction digestion—have been identified that can differentiate the T. pallidum subspecies. Whether these genetic differences are related to distinct clinical characteristics of these diseases has not been determined. Full genome sequencing of an unclassified strain (Fribourg-Blanc) isolated from a baboon in 1966 shows a very high degree of homology with available strains of T. pallidum subspecies pertenue. This observation is consistent with an earlier report that the Fribourg-Blanc strain can cause experimental infection of humans. Molecular analyses of additional samples from affected baboons suggests that the nonhuman primate samples diverge from the evolutionary tree prior to the

1	can cause experimental infection of humans. Molecular analyses of additional samples from affected baboons suggests that the nonhuman primate samples diverge from the evolutionary tree prior to the clade that contains the human isolates, but uncertainty remains about the importance of the nonhuman primate reservoir for human infection.

1	All of the treponemal infections, including syphilis, are chronic and are characterized by defined disease stages, with a localized primary lesion, disseminated secondary lesions, periods of latency, and possible late lesions. Primary and secondary stages are more frequently overlapping in yaws and endemic syphilis than in venereal syphilis, and the late manifestations of pinta are very mild relative to the destructive lesions of the other treponematoses. The current preference is to divide the clinical course of the endemic treponematoses into “early” and “late” stages.

1	The major clinical distinctions made between venereal syphilis and the nonvenereal infections are the apparent lack of congenital transmission and of central nervous system (CNS) involvement in the nonvenereal infections. It is not known whether these distinctions are entirely accurate. Because of the high degree of genetic relatedness among the organisms, there is little biological reason to think that T. pallidum subspecies endemicum and T. pallidum subspecies pertenue would be unable to cross the blood-brain barrier or to invade the placenta. These organisms are like T. pallidum subspecies pallidum in that they obviously disseminate from the site of initial infection and can persist for decades. The lack of recognized congenital infection may be due to the fact that childhood infections often reach the latent stage (low bacterial load) before girls reach sexual maturity. Neurologic involvement may go unrecognized because of the lack of trained

1	FIGURE 207e-1 Geographic distribution of endemic treponematoses.(Courtesy of the World medical personnel in endemic regions, Health Organization; updated from www.who.int/yaws/epidemiology/Map_yaws_90s.jpg.) the delay of many years between infection Organism T. pallidum subsp. pallidum Common modes of Sexual, transplacental transmission Usual age of acquisition Sexual maturity or in utero Primary lesion Cutaneous ulcer (chancre) Common location Genital, oral, anal Late complications Gummas, cardiovascular and central nervous system involvementa T. pallidum subsp. pertenue Skin-to-skin Early childhood Papilloma, often ulcerative Cutaneous papulosquamous lesions; condylomata lata, osteoperiostitis Destructive gummas of skin, bone, cartilage T. pallidum subsp. endemicum T. carateum

1	Cutaneous papulosquamous lesions; condylomata lata, osteoperiostitis Destructive gummas of skin, bone, cartilage T. pallidum subsp. endemicum T. carateum Mouth-to-mouth or via shared Skin-to-skin drinking/eating utensils Early childhood Late childhood Mucosal papule, rarely seen Nonulcerating papule with satellites, pruritic Oral Extremities, face Mucocutaneous lesions Pintides, pigmented, pruritic (mucous patch, split papule, condylomata lata); osteoperiostitis Unknown Unknown Destructive gummas of skin, Nondestructive, dyschromic, bone, cartilage achromic macules aCentral nervous system involvement and congenital infection in the endemic treponematoses have been postulated by some investigators (see text).

1	and possible CNS manifestations, or a low rate of symptomatic CNS disease. Some published evidence supports congenital transmission as well as cardiovascular, ophthalmologic, and CNS involvement in yaws and endemic syphilis. Although the reported studies have been small, have failed to control for other causes of CNS abnormalities, and in some instances have not included serologic confirmation, it may be erroneous to accept unquestioningly the frequently repeated belief that these organisms fail to cause such manifestations. Yaws Also known as pian, framboesia, or bouba, yaws is characterized by the development of one or several primary lesions (“mother yaw”) followed by multiple disseminated skin lesions. All early skin lesions are infectious and may persist for many months; cutaneous relapses are common during the first 5 years. Late manifestations, affecting ~10% of untreated persons, are destructive and can involve skin, bone, and joints.

1	The infection is transmitted by direct contact with infectious lesions, often during play or group sleeping, and may be enhanced by disruption of the skin by insect bites or abrasions. After an average of 3–4 weeks, the first lesion begins as a papule—usually on an extremity— and then enlarges (particularly during moist warm weather) to become papillomatous or “raspberry-like” (thus the name “framboesia”) (Fig. 207e-2A). Regional lymphadenopathy develops, and the lesion usually heals within 6 months; dissemination is thought to occur during the early weeks of infection. A generalized secondary eruption (Fig. 207e-2B), accompanied by generalized lymphadenopathy, appears either concurrent with or after the primary lesion; may take several forms (macular, papular, or papillomatous); and may become secondarily infected with other bacteria. Painful papillomatous lesions on the soles of the feet result in a crablike gait (“crab yaws”), and periostitis may result in nocturnal bone pain and

1	become secondarily infected with other bacteria. Painful papillomatous lesions on the soles of the feet result in a crablike gait (“crab yaws”), and periostitis may result in nocturnal bone pain and polydactylitis. Late yaws is manifested by gummas of the skin and long bones, hyperkeratoses of the palms and soles, osteitis and periostitis, and hydrarthrosis. The late gummatous lesions are characteristically extensive. Destruction of the nose, maxilla, palate, and pharynx is termed gangosa and is similar to the destructive lesions seen in leprosy and leishmaniasis.

1	Endemic Syphilis The early lesions of endemic syphilis (bejel, siti, dichuchwa, njovera, skerljevo) are localized primarily to mucocutaneous and mucosal surfaces. The infection is reportedly transmitted by direct contact, by kissing, by premastication of food, or by sharing of drinking and eating utensils. A role for insects in transmission has been suggested but is unproven. The initial lesion, usually an intraoral papule, often goes unrecognized and is followed by mucous patches (Fig. 207e-2C) on the oral mucosa and mucocutaneous lesions resembling the condylomata lata of secondary syphilis. This eruption may last for months or even years, and treponemes can readily be demonstrated in early lesions. Periostitis and regional lymphadenopathy are common. After a variable period of latency, late manifestations may appear, including osseous and cutaneous gummas. Destructive gummas, osteitis, and gangosa are more common in endemic syphilis than in yaws.

1	Pinta Pinta (mal del pinto, carate, azul, purupuru) is the most benign of the treponemal infections. This disease has three stages that are characterized by marked changes in skin color (Fig. 207e-2D), but pinta does not appear to cause destructive lesions or to involve

1	FIGURE 207e-2 Clinical manifestations of endemic treponematoses. A. Papillomatous initial lesion of early yaws. B. Disseminated lesions of early yaws. C. Mucous patches of endemic syphilis. D. Pigmented macules of pinta. (Photos published with permission from Dr. David Fegan, Brisbane, Australia [A and B]; and from PL Perine et al: Handbook of Endemic Treponematoses, Geneva, World Health Organization, 1984 [C and D].) tissues other than the skin. The initial papule is most often located on the extremities or face and is pruritic. After one to many months of infection, numerous disseminated secondary lesions (pintides) appear. These lesions are initially red but become deeply pigmented, ultimately turning a dark slate blue. The secondary lesions are infectious and highly pruritic and may persist for years. Late pigmented lesions are called dyschromic macules and contain treponemes. Over time, most pigmented lesions show varying degrees of depigmentation, becoming brown and eventually

1	persist for years. Late pigmented lesions are called dyschromic macules and contain treponemes. Over time, most pigmented lesions show varying degrees of depigmentation, becoming brown and eventually white and giving the skin a mottled appearance. White achromic lesions are characteristic of the late stage.

1	Diagnosis of the endemic treponematoses is based on clinical manifestations and, when available, dark-field microscopy and serologic testing. The same serologic tests that are used for venereal syphilis (Chap. 206) become reactive during all treponemal infections. Although several targets have been evaluated for specific serodiagnosis, to date there is no antibody test that can discriminate among the different infections. The nonvenereal treponemal infections should be considered in the evaluation of a reactive syphilis serology in any person who has emigrated from an endemic area. Sensitive PCR assays can be used to confirm treponemal infection and to identify the etiologic agent in research laboratories.

1	The WHO-recommended therapy for patients and their contacts is benzathine penicillin G (1.2 million units IM for adults; 600,000 units for children <10 years old). This dose is half of that recommended for early venereal syphilis, and no controlled efficacy studies have been conducted. Definitive evidence of resistance to penicillin is lacking, although relapsing lesions have been reported after penicillin treatment in Papua New Guinea. A recent study in that nation demonstrated equivalence between IM benzathine penicillin G and a single oral dose of azithromycin (30 mg/kg, up to a maximum of 2 g). This 207e-3 finding provided the WHO’s revitalized yaws eradication program with a much easier regimen for use in mass treatment. Although macrolide resistance mutations are common in circulating strains of

1	T. pallidum subspecies pallidum in many parts of the world, analysis of a limited number of yaws samples from Papua New Guinea has yielded no evidence of resistance mutations to date. Limited data suggest the efficacy of tetracycline for treatment of yaws, but no data exist for other endemic treponematoses. Solely on the basis of experience with venereal syphilis, it is thought that doxycycline or tetracycline (at doses appropriate for syphilis; Chap. 206) are alternatives for patients allergic to penicillin. A Jarisch-Herxheimer reaction (Chap. 206) may follow treatment of endemic treponematoses. Nontreponemal serologic titers (in the Venereal Disease Research Laboratory [VDRL] slide test or the rapid plasma reagin [RPR] test) usually decline after effective therapy, but patients may not become seronegative.

1	Buoyed by the successful elimination of yaws in India in 2006 and the availability of an inexpensive, single-dose oral drug for treatment, in 2012 the WHO renewed its efforts to eradicate yaws globally by 2020. Enthusiasm is high; several planning meetings have been held to develop country-specific plans of action; and resources are being sought. Some caution is warranted: The possible animal reservoir will need to be evaluated. There may be only a window of time during which countries can successfully use azithromycin for yaws eradication before resistance begins to appear in yaws organisms. Given the ongoing lower-dose azithromycin mass treatment campaigns against trachoma, often in populations also at high risk for yaws, development of macrolide resistance is likely at some point. Complete drug coverage and continued careful surveillance by health centers (the weak link in prior control efforts) will be essential for success.

1	Leptospirosis Rudy A. Hartskeerl, Jiři F. P. Wagenaar Leptospirosis is a globally important zoonotic disease whose apparent reemergence is illustrated by recent outbreaks on virtually all continents. The disease is caused by pathogenic Leptospira species and is charac-terized by a broad spectrum of clinical manifestations, varying from 208 asymptomatic infection to fulminant, fatal disease. In its mild form, leptospirosis may present as nonspecific symptoms such as fever, headache, and myalgia. Severe leptospirosis, characterized by jaundice, renal dysfunction, and hemorrhagic diathesis, is often referred to as Weil’s syndrome. With or without jaundice, severe pulmonary hemorrhage is increasingly recognized as an important presentation of severe disease. Leptospira species are spirochetes belonging to the order Spirochaetales and the family Leptospiraceae. Traditionally, the genus Leptospira comprised two species: the pathogenic L. interrogans and the free-living

1	L. biflexa, now designated L. interrogans sensu lato and L. biflexa sensu lato, respectively. Twenty-two Leptospira species with pathogenic (10 species), intermediate (5 species), and nonpathogenic (7 species) status have now been described on the basis of phylogenetic and virulence analyses (Fig. 208-1). Genome sequences of five Leptospira species 0.005 FIGuRE 208-1 Differentiation of pathogenic, intermediate, and nonpathogenic (saprophytic) Leptospira species by molecular phyloge-netic analysis using the rrs gene and including the potentially new pathogenic species Leptospira borgpetersenii group B and the saprophytic species Leptospira idonii. Scale bar indicates the rate of nucleotide substitutions per base pair. (Figure prepared and provided by Dr. A. Ahmed, KIT Biomedical Research, Amsterdam, The Netherlands.) (L. biflexa, L. interrogans, L. santarosai, L. borgpetersenii, and

1	L. licerasiae) have been published, and the availability of genome sequences of a wide variety of Leptospira strains will undoubtedly lead to a better understanding of the pathogenesis of leptospirosis. However, classification based on serologic differences better serves clinical, diagnostic, and epidemiologic purposes. Pathogenic Leptospira species are divided into serovars according to their antigenic composition. More than 250 serovars make up the 26 serogroups. Leptospires are coiled, thin, highly motile organisms that have hooked ends and two periplasmic flagella, with polar extrusions from the cytoplasmic membrane that are responsible for motility (Fig. 208-2). These organisms are 6–20 μm long and ~0.1 μm wide; they stain poorly but can be seen microscopically by dark-field examination and after silver impregnation staining of tissues. Leptospires require special media and conditions for growth; it may take weeks to months for cultures to become positive.

1	commonly in the tropics and subtropics because the climate and occasionally poor hygienic conditions favor the pathogen’s survival and distribution. In most countries, leptospirosis is an underappreciated problem. Most cases occur in men, with a peak incidence during the summer and fall in both the Northern and Southern Hemispheres and during the rainy season in the tropics. FIGuRE 208-2 Transmission electron microscopic image of Leptospira interrogans invading equine conjunctival tissue. (Image kindly provided by Dr. JE Nally, National Animal Disease Center, U.S. Department of Agriculture, Ames, IA. This image appears on the homepage of the European Leptospirosis Society website [http://eurolepto.ucd.ie/].) Reliable data on morbidity and mortality from leptospirosis have gradually started to appear. Current information on global human leptospirosis varies but indicates that approximately 1 million severe cases occur per year, with a mean case–fatality rate of nearly 10%.

1	As a zoonosis, leptospirosis affects almost all mammalian species and represents a significant veterinary burden. Rodents, especially rats, are the most important reservoir, although other wild mammals as well as domestic and farm animals may also harbor these microorganisms. Leptospires establish a symbiotic relationship with their host and can persist in the urogenital tract for years. Some serovars are generally associated with particular animals—e.g., Icterohaemorrhagiae and Copenhageni with rats, Grippotyphosa with voles, Hardjo with cattle, Canicola with dogs, and Pomona with pigs—but may occur in other animals as well.

1	Leptospirosis presents as both an endemic and an epidemic disease. Transmission of leptospires may follow direct contact with urine, blood, or tissue from an infected animal or, more commonly, exposure to environmental contamination. The dogma that human-to-human transmission is very rare is challenged by recent findings on household clustering, asymptomatic renal colonization, and prolonged excretion of leptospires. (Both of the latter features imply human infection sources that are not recognized.) Because leptospires can survive in a humid environment for many months, water is an important vehicle in their transmission. Epidemics of leptospirosis are not well understood. Outbreaks may result from exposure to flood waters contaminated by urine from infected animals, as has been reported from several countries. However, it is also true that outbreaks may occur without floods, and floods often occur without outbreaks.

1	The vast majority of infections with Leptospira cause no or only mild disease in humans. A small percentage of infections (~1%) lead to severe, potentially fatal complications. The proportion of leptospirosis cases that are mild is unknown because patients either do not seek or do not have access to medical care or because the nonspecific symptoms are interpreted as an influenza-like illness. Reported cases surely represent a significant underestimation of the total number. Certain occupational groups are at especially high risk, including veterinarians, agricultural workers, sewage workers, slaughterhouse employees, and workers in the fishing industry. Risk factors include direct or indirect contact with animals, including exposure to water and soil contaminated with animal urine. Leptospirosis has also been recognized in deteriorating inner cities and suburban areas where rat populations are expanding.

1	Recreational exposure and domestic-animal contact are prominent sources of leptospirosis. Recreational freshwater activities, such as canoeing, windsurfing, swimming, and waterskiing, place persons at risk for infection. Several outbreaks have followed sporting events. For example, an outbreak took place in 1998 among athletes after a triathlon in Springfield, Illinois. Ingestion of one or more swallows of lake water during the swimming leg of the triathlon was a prominent risk factor for illness. Heavy rains that preceded the triathlon, with consequent agricultural runoff, are likely to have increased the level of leptospiral contamination in the lake water. In another outbreak, 42% of participants contracted leptospirosis during the 2000 Eco-Challenge-Sabah multisport endurance race in Malaysian Borneo. Swimming in the Segama River was shown to be an independent risk factor.

1	In addition, leptospirosis is a traveler’s disease. Large proportions of patients acquire the infection while traveling in tropical countries, usually during adventurous activities such as whitewater rafting, jungle trekking, and caving. Transmission via laboratory accidents has been reported but is rare. New data indicate that leptospirosis may develop after unanticipated immersion in contaminated water (e.g., in an automobile accident) more frequently than has generally been thought and can also result from an animal bite.

1	Transmission occurs through cuts, abraded skin, or mucous membranes, especially the conjunctival and oral mucosa. After entry, the organisms proliferate, cross tissue barriers, and disseminate hematogenously to all organs (leptospiremic phase). During this initial incubation period, leptospires can be isolated from the bloodstream (Fig. 208-3). The organisms are able to survive in the nonimmune host: they evade complement-mediated killing by binding factor H, a strong inhibitor of the complement system, on their surface. Moreover, pathogenic leptospires resist ingestion and killing by neutrophils, monocytes, and macrophages. During the immune phase, the appearance of antibodies coincides with the disappearance of leptospires from the blood. However, the bacteria persist in various organs, including liver, lung, kidney, heart, and brain. Autopsy findings illustrate the involvement of multiple organ systems in severe disease. Renal pathology shows both acute tubular damage and

1	organs, including liver, lung, kidney, heart, and brain. Autopsy findings illustrate the involvement of multiple organ systems in severe disease. Renal pathology shows both acute tubular damage and interstitial nephritis. Acute tubular lesions progress in time to interstitial edema and acute tubular necrosis. Severe nephritis is observed in patients who survive long enough to develop it and seems to be a secondary response to acute epithelial damage. The reported deregulation of the expression of several transporters along the nephron, including the proximal sodium-hydrogen exchanger 3 (NHE3), aquaporins 1 and 2 (AQP1 and AQP2), Na+-K+ ATPase, and the Na-K-2Cl cotransporter NKCC2, contributes to tubular potassium wasting, hypokalemia, and polyuria. Histopathology of the liver shows focal necrosis, foci of inflammation, and plugging of bile canaliculi. Widespread hepatocellular necrosis is not found. Petechiae and hemorrhages are observed in the heart, lungs (Fig. 208-4), kidneys (and

1	necrosis, foci of inflammation, and plugging of bile canaliculi. Widespread hepatocellular necrosis is not found. Petechiae and hemorrhages are observed in the heart, lungs (Fig. 208-4), kidneys (and adrenals), pancreas, liver, gastrointestinal tract (including retro-peritoneal fat, mesentery, and omentum), muscles, prostate, testis, and brain (subarachnoid bleeding). Several studies show an association between hemorrhage and thrombocytopenia. Although the underlying mechanisms of thrombocytopenia have not been elucidated, it seems likely that platelet consumption plays an important role. A consumptive coagulopathy may occur, with elevated markers of coagulation activation (thrombin–antithrombin complexes, prothrombin fragments 1 and 2, d-dimer), diminished anticoagulant markers (antithrombin, protein C), and deregulated fibrinolytic activity. Overt disseminated intravascular coagulation (DIC) has been documented in patients from Thailand and Indonesia. Elevated plasma levels of

1	protein C), and deregulated fibrinolytic activity. Overt disseminated intravascular coagulation (DIC) has been documented in patients from Thailand and Indonesia. Elevated plasma levels of soluble E-selectin and von Willebrand factor in patients with leptospirosis reflect endothelial cell activation. Experimental models show that pathogenic leptospires or leptospiral proteins are able to activate endothelial cells in vitro and to disrupt endothelial-cell barrier function, promoting dissemination. Platelets have been shown to aggregate on activated endothelium in the human lung, whereas histology reveals swelling of activated endothelial cells but no evident vasculitis or necrosis. Immunoglobulin and complement deposition have been demonstrated in lung tissue involved in pulmonary hemorrhage.

1	Approximate time scale Week 1 Acute stage Convalescent stage Convalescent shedder Normal response fever Titers decline at varying rates Early treatment Leptospiremia Leptospiruria and immunity Anamnestic Delayed Reservoir host Uveitis ? Interstitial nephritis 2 3 4 months-years years Incubation period Inoculation Leptospires present in Blood CSF CSF 1 2 3 4 5 Urine Blood Urine Antibody titers High Low “Negative” Laboratory investigations Culture Serology Phases 2–20 days FIGuRE 208-3 Biphasic nature of leptospirosis and relevant investigations at different stages of disease. Note that an incubation period of up to 1 month has now been documented. Specimens 1 and 2 for serology are acute-phase serum samples; specimen 3 is a convalescent-phase serum sample that may facilitate detection of a delayed immune response; and specimens 4 and 5 are follow-up serum samples that can pro-vide epidemiologic information, such as the presumptive infecting serogroup. CSF, cerebrospinal fluid.

1	of a delayed immune response; and specimens 4 and 5 are follow-up serum samples that can pro-vide epidemiologic information, such as the presumptive infecting serogroup. CSF, cerebrospinal fluid. (Reprinted as adapted by PN Levett: Clin Microbiol Rev 14:296, 2001 [from LH Turner: Leptospirosis. BMJ 1:231, 1969] with permission from the American Society for Microbiology and the BMJ Publishing Group.)

1	Leptospira species have a typical double-membrane cell wall structure harboring a variety of membrane-associated proteins, including an unusually high number of lipoproteins. The peptidoglycan layer is located close to the cytoplasmic membrane. The lipopolysaccharide (LPS) in the outer membrane has an unusual structure with a relatively low endotoxic potency. Pathogenic leptospires contain a variety of genes coding for proteins involved in motility and in cell and tissue adhesion and invasion that represent potential virulence factors. Many of these are surface-exposed outer-membrane proteins (OMPs). To date, the only leptospiral virulence factor shown to satisfy Koch’s molecular postulates is loa22 encoding a surface-exposed protein with an unknown function. However, the gene is not confined to pathogenic Leptospira species.

1	Immunity to Leptospira depends on the production of circulating antibodies to serovar-specific LPS. It is unclear whether other antigens play a significant role in protective humoral immunity. Moreover, immunity may not be confined to antibody responses; involvement of the innate-immune Toll-like receptor 2 (TLR2) and TLR4 activation pathways in controlling infection has been demonstrated, whereas in vaccinated cattle a cell-mediated immune response is correlated with protection. It is likely that several surface-exposed proteins mediate leptospire–host cell interactions, and these proteins may represent candidate vaccine components. Although animal-model studies have shown various degrees of vaccine efficacy for various putative virulence-associated OMPs, it is not yet clear whether such proteins elicit acceptable levels of sterilizing immunity.

1	FIGuRE 208-4 Severe pulmonary hemorrhage in leptospirosis. Left panel: Chest x-ray. Right panel: Gross appearance of right lower lobes of lung at autopsy. This patient, a 15-year-old from the Peruvian Amazonian city of Iqitos, died several days after presentation with acute illness, jaundice, and hemoptysis. Blood culture yielded Leptospira interrogans serovar Copenhageni/Icterohaemorrhagiae. (Adapted with permission from E Segura et al: Clin Infect Dis 40:343, 2005. © 2005 by the Infectious Diseases Society of America.) 1144 CLINICAL MANIFESTATIONS Although leptospirosis is a potentially fatal disease with bleeding and multiorgan failure as its clinical hallmarks, the majority of cases are thought to be relatively mild, presenting as the sudden onset of a febrile illness. The incubation period is usually 1–2 weeks but ranges from 1 to 30 days. (Figure 208-3 indicates a slightly different range, but an incubation period of up to 1 month has now been documented.) Leptospirosis is

1	period is usually 1–2 weeks but ranges from 1 to 30 days. (Figure 208-3 indicates a slightly different range, but an incubation period of up to 1 month has now been documented.) Leptospirosis is classically described as biphasic. The acute leptospiremic phase is characterized by fever of 3–10 days’ duration, during which time the organism can be cultured from blood. During the immune phase, resolution of symptoms may coincide with the appearance of antibodies, and leptospires can be cultured from the urine. The distinction between the first and second phases is not always clear: milder cases do not always include the second phase, and severe disease may be monophasic and fulminant. The idea that distinct clinical syndromes are associated with specific serogroups has been refuted, although some serovars tend to cause more severe disease than others.

1	Mild Leptospirosis Most patients are asymptomatic or only mildly ill and do not seek medical attention. Serologic evidence of past inapparent infection is frequently found in persons who have been exposed but have not become ill. Mild symptomatic leptospirosis usually presents as a flu-like illness of sudden onset, with fever, chills, headache, nausea, vomiting, abdominal pain, conjunctival suffusion (redness without exudate), and myalgia. Muscle pain is intense and especially affects the calves, back, and abdomen. The headache is intense, localized to the frontal or retroorbital region (resembling that occurring in dengue), and sometimes accompanied by photophobia. Aseptic meningitis may be present and is more common among children than among adults. Although Leptospira can be cultured from the cerebrospinal fluid (CSF) in the early phase, the majority of cases follow a benign course with regard to the central nervous system; symptoms disappear within a few days but may persist for

1	from the cerebrospinal fluid (CSF) in the early phase, the majority of cases follow a benign course with regard to the central nervous system; symptoms disappear within a few days but may persist for weeks.

1	Physical examination may include any of the following findings, none of which is pathognomonic for leptospirosis: fever, conjunctival suffusion, pharyngeal injection, muscle tenderness, lymphadenopathy, rash, meningismus, hepatomegaly, and splenomegaly. If present, the rash is often transient; may be macular, maculopapular, erythematous, or hemorrhagic (petechial or ecchymotic); and may be misdiagnosed as due to scrub typhus or viral infection. Lung auscultation may reveal crackles, and mild jaundice may be present. The natural course of mild leptospirosis usually involves spontaneous resolution within 7–10 days, but persistent symptoms have been documented. In the absence of a clinical diagnosis and antimicrobial therapy, the mortality rate in mild leptospirosis is low.

1	Severe Leptospirosis Although the onset of severe leptospirosis may be no different from that of mild leptospirosis, severe disease is often rapidly progressive and is associated with a case–fatality rate ranging from 1 to 50%. Higher mortality rates are associated with an age >40, altered mental status, acute renal failure, respiratory insufficiency, hypotension, and arrhythmias. The classic presentation, often referred to as Weil's syndrome, encompasses the triad of hemorrhage, jaundice, and acute kidney injury.

1	Patients die of septic shock with multiorgan failure and/or severe bleeding complications that most commonly involve the lungs (pulmonary hemorrhage), gastrointestinal tract (melena, hemoptysis), urogenital tract (hematuria), and skin (petechiae, ecchymosis, and bleeding from venipuncture sites). Pulmonary hemorrhage (with or without jaundice) is now recognized as a widespread public health problem, presenting with cough, chest pain, respiratory distress, and hemoptysis that may not be apparent until patients are intubated. Jaundice occurs in 5–10% of all patients with leptospirosis; it can be profound and give an orange cast to the skin but usually is not associated with fulminant hepatic necrosis. Physical examination may reveal an enlarged and tender liver.

1	Acute kidney injury is common in severe disease, presenting after several days of illness, and can be either nonoliguric or oliguric. Typical electrolyte abnormalities include hypokalemia and hyponatremia. Loss of magnesium in the urine is uniquely associated with leptospiral nephropathy. Hypotension is associated with acute tubular necrosis, oliguria, or anuria, requiring fluid resuscitation and sometimes vasopressor therapy. Hemodialysis can be life-saving, with renal function typically returning to normal in survivors.

1	Other syndromes include (necrotizing) pancreatitis, cholecystitis, skeletal muscle involvement, rhabdomyolysis (with moderately elevated serum creatine kinase levels), and neurologic manifestations including aseptic meningitis. Cardiac involvement is commonly reflected on the electrocardiogram as nonspecific STand T-wave changes. Repolarization abnormalities and arrhythmias are considered poor prognostic factors. Myocarditis has been described. Rare hematologic complications include hemolysis, thrombotic thrombocytopenic purpura, and hemolytic-uremic syndrome. Long-term symptoms following severe leptospirosis include fatigue, myalgia, malaise, and headache and may persist for years. Autoimmune-associated uveitis, a potentially chronic condition, is a recognized sequela of leptospirosis.

1	The clinical diagnosis of leptospirosis should be based on an appropriate exposure history combined with any of the protean manifestations of the disease. Returning travelers from endemic areas usually have a history of recreational freshwater activities or other mucosal or percutaneous contact with contaminated surface waters or soil. For nontravelers, recreational water contact and occupational hazards that involve direct or indirect animal contact should be explored (see “Epidemiology,” above).

1	Although biochemical, hematologic, and urinalysis findings in acute leptospirosis are nonspecific, certain patterns may suggest the diagnosis. Laboratory results usually show signs of a bacterial infection, including leukocytosis with a left shift and elevated markers of inflammation (C-reactive protein level and erythrocyte sedimentation rate). Thrombocytopenia (platelet count ≤100 × 109/L) is common and is associated with bleeding and renal failure. In severe disease, signs of coagulation activation may be present, varying from borderline abnormalities to a serious derangement compatible with DIC as defined by international criteria. The kidneys are invariably involved in leptospirosis. Related findings range from urinary sediment changes (leukocytes, erythrocytes, and hyaline or granular casts) and mild proteinuria in mild disease to renal failure and azotemia in severe leptospirosis. Nonoliguric hypokalemic renal insufficiency (see “Clinical Manifestations,” above) is

1	or granular casts) and mild proteinuria in mild disease to renal failure and azotemia in severe leptospirosis. Nonoliguric hypokalemic renal insufficiency (see “Clinical Manifestations,” above) is characteristic of early leptospirosis. Serum bilirubin levels may be high, whereas rises in aminotransferase and alkaline phosphatase levels are usually moderate. Although clinical symptoms of pancreatitis are not a common finding, amylase levels are often elevated. When symptoms of aseptic meningitis develop, examination of the CSF shows pleocytosis that can range from a few cells to >1000 cells/μL, with a polymorphonuclear cell predominance. The protein concentration in the CSF may be elevated; CSF glucose levels are normal.

1	In severe leptospirosis, pulmonary radiographic abnormalities are more common than would be expected on the basis of physical examination (Fig. 208-4). The most common radiographic finding is a patchy bilateral alveolar pattern that corresponds to scattered alveolar hemorrhage. These abnormalities predominantly affect the lower lobes. Other findings include pleura-based densities (representing areas of hemorrhage) and diffuse ground-glass attenuation typical of acute respiratory distress syndrome (ARDS).

1	A definitive diagnosis of leptospirosis is based on isolation of the organism from the patient, on a positive result in the polymerase chain reaction (PCR), or on seroconversion or a rise in antibody titer. In cases with strong clinical evidence of infection, a single antibody titer of 1:200–1:800 (depending on whether the case occurs in a low-or high-endemic area) in the microscopic agglutination test (MAT) is required. Preferably, a fourfold or greater rise in titer is detected between acuteand convalescent-phase serum specimens. Antibodies generally do not reach detectable levels until the second week of illness. The antibody response can be affected by early treatment with antibiotics.

1	The MAT, which uses a battery of live leptospiral strains, and the enzyme-linked immunosorbent assay (ELISA), which uses a broadly reacting antigen, are the standard serologic procedures. The MAT usually is available only in specialized laboratories and is used for determination of the antibody titer and for tentative identification of the involved leptospiral serogroup—and, when epidemiologic background information is available, the putative serovar. This point underscores the importance of testing antigens representative of the serovars prevalent in the particular geographic area. However, cross-reactions occur frequently, and thus definitive identification of the infecting serovar or serogroup is not possible without isolation of the causative organism. Because serologic testing lacks sensitivity in the early acute phase of the disease (up to day 5), it cannot be used as the basis for a timely decision about whether to start treatment.

1	In addition to the MAT and the ELISA, various rapid tests with diagnostic value have been developed, and some of these are commercially available. These rapid tests mainly apply lateral flow, (latex) agglutination, or ELISA methodology and are reasonably sensitive and specific, although results reported in the literature vary, probably as a consequence of differences in test interpretation, (re)exposure risks, serovar distribution, and the use of biased serum panels. These methods do not require culture or MAT facilities and are useful in settings that lack a strong medical infrastructure. PCR methodologies, notably real-time PCR, have become increasingly widely implemented. Compared with serology, PCR offers a great advantage: the capacity to confirm the diagnosis of leptospirosis with a high degree of accuracy during the first 5 days of illness.

1	The differential diagnosis of leptospirosis is broad, reflecting the diverse clinical presentations of the disease. Although leptospirosis transmission is more common in tropical and subtropical regions, the absence of a travel history does not exclude the diagnosis. When fever, headache, and myalgia predominate, influenza and other common and less common (e.g., dengue and chikungunya) viral infections should be considered. Malaria, typhoid fever, ehrlichiosis, viral hepatitis, and acute HIV infection may mimic the early stages of leptospirosis and are important to recognize. Rickettsial diseases, hantavirus infections (hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome), and dengue share epidemiologic and clinical features with leptospirosis. Dual infections have been reported. In this light, it is advisable to conduct serologic testing for hantavirus, rickettsiae, and dengue virus when leptospirosis is suspected. When bleeding is detected, dengue

1	have been reported. In this light, it is advisable to conduct serologic testing for hantavirus, rickettsiae, and dengue virus when leptospirosis is suspected. When bleeding is detected, dengue hemorrhagic fever and other viral hemorrhagic fevers, including hantavirus infection, yellow fever, Rift Valley fever, filovirus infections, and Lassa fever, should be considered.

1	Severe leptospirosis should be treated with IV penicillin (Table 208-1) as soon as the diagnosis is considered. Leptospires are highly susceptible to a broad range of antibiotics, and early intervention may prevent the development of major organ system failure or lessen its severity. Although studies supporting antibiotic therapy have produced conflicting results, clinical trials are difficult to perform in settings where patients frequently present for medical care with late stages of disease. Antibiotics are less likely to benefit patients in whom organ damage has already occurred. Two open-label randomized studies comparing penicillin with parenteral cefotaxime, parenteral ceftriaxone, and doxycycline showed no significant differences among the antibiotics with regard to complications and mortality risk. Thus ceftriaxone, cefotaxime, or doxycycline is a satisfactory alternative to penicillin for the treatment of severe leptospirosis.

1	In mild cases, oral treatment with doxycycline, azithromycin, ampicillin, or amoxicillin is recommended. In regions where rickettsial diseases are coendemic, doxycycline or azithromycin is the drug of choice. In rare instances, a Jarisch-Herxheimer reaction develops within hours after the initiation of antimicrobial therapy. Moderate/severe leptospirosis Penicillin (1.5 million units IV or IM q6h) or Ceftriaxone (2 g/d IV) or Cefotaxime (1 g IV q6h) or Doxycycline (loading dose of 200 mg IV, aAll regimens are given for 7 days. bDoxycycline should not be given to pregnant women or children. c The efficacy of doxycycline prophylaxis in endemic or epidemic settings remains unclear. Experiments in animal models and a cost-effectiveness model indicate that azithromycin has a number of characteristics that may make it efficacious in treatment and prophylaxis.

1	Aggressive supportive care for leptospirosis is essential and can be life-saving. Patients with nonoliguric renal dysfunction require aggressive fluid and electrolyte resuscitation to prevent dehydration and precipitation of oliguric renal failure. Peritoneal dialysis or hemodialysis should be provided to patients with oliguric renal failure. Rapid initiation of hemodialysis has been shown to reduce mortality risk and typically is necessary only for short periods. Patients with pulmonary hemorrhage may have reduced pulmonary compliance (as seen in ARDS) and may benefit from mechanical ventilation with low tidal volumes to avoid high ventilation pressures. Evidence is contradictory for the use of glucocorticoids and desmopressin as adjunct therapy for pulmonary involvement associated with severe leptospirosis.

1	Most patients with leptospirosis recover. However, post-leptospirosis symptoms, mainly of a depression-like nature, may occur and persist for years after the acute disease. Mortality rates are highest among patients who are elderly and those who have severe disease (pulmonary hemorrhage, Weil’s syndrome). Leptospirosis during pregnancy is associated with high fetal mortality rates. Long-term follow-up of patients with renal failure and hepatic dysfunction has documented good recovery of renal and hepatic function. Individuals who may be exposed to Leptospira through their occupations or their involvement in recreational freshwater activities should be informed about the risks. Measures for controlling leptospirosis include avoidance of exposure to urine and tissues from infected animals through proper eyewear, footwear, and other protective equipment. Targeted rodent control strategies could be considered.

1	Vaccines for agricultural and companion animals are gener ally available, and their use should be encouraged. The veteri nary vaccine used in a given area should contain the serovars known to be present in that area. Unfortunately, some vaccinated animals still excrete leptospires in their urine. Vaccination of humans against a specific serovar prevalent in an area has been undertaken in some European and Asian countries and has proved effective. Although a large-scale trial of vaccine in humans has been reported from Cuba, no conclusions can be drawn about efficacy and adverse reactions because of insufficient details on study design. The efficacy of chemoprophylaxis with doxycycline (200 mg once a week) or azithromycin (in pregnant women and children) is being disputed, but focused pre-and postexposure administration is indicated in instances of well-defined short-term exposure (Table 208-1).

1	1146 Relapsing Fever Alan G. Barbour Relapsing fever is caused by infection with any of several species of Borrelia spirochetes. Physicians in ancient Greece distinguished relapsing fever from other febrile disorders by its characteristic clinical presentation: two or more fever episodes separated by varying peri-209 ods of well-being. In the nineteenth century, relapsing fever was one of the first diseases to be associated with a specific microbe by virtue of its characteristic laboratory finding: the presence of large numbers of spirochetes of the genus Borrelia in the blood.

1	The host responds with systemic inflammation that results in an illness ranging from a flulike syndrome to sepsis. Other manifestations are the consequences of central nervous system (CNS) involvement and coagulopathy. Antigenic variation of the spirochetes’ surface proteins accounts for the infection’s relapsing course. Acquired immunity follows the serial development of antibodies to each of the several variants appearing during an infection. Treatment with antibiotics results in rapid cure but at the risk of a moderate to severe Jarisch-Herxheimer reaction. the twentieth century and currently occurs in northeastern

1	the twentieth century and currently occurs in northeastern Africa. At present, however, most cases of relapsing fever are tick-borne in origin. Sporadic cases and small outbreaks are focally distributed on most continents, with Africa most affected. In North America, the majority of reports of relapsing fever have been from the western United States and Canada. Nevertheless, the recent discovery that another species in the relapsing fever group causes human disease in the same geographic distribution as Lyme disease (Chap. 210) confounds epidemiologic distinctions between the two major types of Borrelia infection.

1	Coiled, thin microscopic filaments that swim in one direction and then coil up before heading in another were first observed in the blood of patients with relapsing fever in the 1880s (www.youtube .com/watch?v=VxDPV2lBd9U). These microbes were categorized as spirochetes and grouped as several species in the genus Borrelia. It was not until the 1960s that the organisms were isolated in pure culture. The breakthrough cultivation medium and its derivatives are rich in their ingredients, ranging from simple (e.g., amino acids and N-acetylglucosamine) to more complex (e.g., serum and protein hydrolysates). The limited biosynthetic capacity of Borrelia cells is accounted for by a genome content one-quarter that of Escherichia coli.

1	Like other spirochetes, the helix-shaped Borrelia cells have two membranes, the outer of which is more loosely secured than in other double-membrane bacteria, such as E. coli. As a consequence, fixed organisms with damaged membranes can assume a variety of morphologies in smears and histologic preparations. The flagella of spirochetes run between the two membranes and are not on the cell surface. Although technically gram-negative in their staining properties, the 10to 20-μm-long Borrelia cells, with a diameter of 0.1–0.2 μm, are too narrow to be seen by bright-field microscopy of Gram-stained specimens.

1	The several species of Borrelia that cause relapsing fever have restricted geographic distributions (Table 209-1). The excep tion is Borrelia recurrentis, which is also the only species transmitted by the louse. Louse-borne relapsing fever (LBRF) is usually acquired from a body louse (Pediculus humanus corporis), with humans serving as the reservoir. Acquisition occurs not from the bite itself but from either rubbing the insect’s feces into the bite site with the fingers in response to irritation or inoculation of feces into the conjunctivae or an open wound. Although LBRF transmission is currently limited to Ethiopia and adjacent countries, the disease has had a global distribution in the past, and that potential remains. Epidemics with thousands of cases of LBRF can occur under circumstances of famine, natural disaster, refugee migration, and war.

1	All other known species of relapsing fever agents are tick-borne—in most cases, by soft ticks of the genus Ornithodoros (Fig. 209-1). Tick-borne relapsing fever (TBRF) is found on most continents but is absent or rare in tropical, low-desert, arctic, or alpine environments. For most species, the reservoirs of infection are small to medium-sized mammals, usually rodents but sometimes pigs and other domestic animals living in or around human habitats. However, one species, Borrelia duttonii in sub-Saharan Africa, is largely maintained by tick transmission between human hosts. In North America, TBRF occurs as single cases or small case clusters through transient exposure of persons to infested buildings or caves in less populated areas where the rodent reservoirs have nests. The two main Borrelia species involved in North America are Borrelia hermsii (in the mountainous west) and Borrelia turicatae (in the southwestern and south-central regions). The soft tick vectors typically feed for

1	species involved in North America are Borrelia hermsii (in the mountainous west) and Borrelia turicatae (in the southwestern and south-central regions). The soft tick vectors typically feed for no more than 30 min, usually without being noticed, while the victim is sleeping. Transovarial transmission from one generation of ticks to the next means that infection risk may persist in an area long after incriminated mammalian reservoirs have been eradicated.

1	A newly recognized pathogen, Borrelia miyamotoi, belongs to the clade of relapsing fever species but is transmitted to humans from other mammals by hard ticks (e.g., Ixodes scapularis in the eastern United States) that also transmit Lyme disease, babesiosis, anaplasmosis, ehrlichiosis, and arboviral encephalitis. B. miyamotoi is acquired through outdoor activities and through contact with ticks in forested and shrubby areas during recreation, work, or activities around the home. In residents of areas where B. miyamotoi and Borrelia burgdorferi coexist, the prevalence of antibodies to the former is about one-third of that to the latter. Unlike LBRF spirochetes, TBRF spirochetes enter the body in the tick’s saliva with the onset of feeding. From an inoculum of a few cells, the spirochetes proliferate in the blood, doubling every 6 h to numbers of 106–107/mL or more. Borrelia species are extracellular pathogens; their

1	TABLE 209-1 RELAPsIng FEvER BORRELIA sPECIEs, By gEOgRAPHIC REgIOn, vECTOR, And PRIMARy REsERvOIR aAlthough transmission is currently limited to Ethiopia and adjacent countries, B. recurrentis infection has had a global distribution in the past, and that potential remains. FIGuRE 209-1 Ornithodoros turicata soft ticks of different ages.

1	FIGuRE 209-1 Ornithodoros turicata soft ticks of different ages. presence inside cells likely represents a dead end for the bacteria after phagocytosis. Binding of the spirochetes to erythrocytes leads to aggregation of red blood cells, their sequestration in the spleen and liver, and hepatosplenomegaly and anemia. A bleeding disorder is probably the consequence of thrombocytopenia, impaired hepatic production of clotting factors, and/or blockage of small vessels by aggregates of spirochetes, erythrocytes, and platelets. Some species are neurotropic and frequently enter the brain, where they are comparatively sheltered from host immunity. Relapsing fever spirochetes can cross the maternal-fetal barrier and cause placental damage and inflammation, leading to intrauterine growth retardation and congenital infection.

1	Although Borrelia species do not have potent exotoxins or a lipopolysaccharide endotoxin, they have abundant membrane-associated lipoproteins whose recognition and binding by Toll-like receptors on host cells can lead to a proinflammatory process similar to that in endotoxemia, with elevations of tumor necrosis factor α, interleukin 6, and interleukin 8 concentrations.

1	IgM antibodies specific for the serotype-defining surface lipoprotein appear after a few days of infection and soon reach a concentration that causes lysis of bacteria in the blood through either direct bactericidal action or opsonization. The release of large amounts of lipoproteins and other bacterial products from dying bacteria provokes a “crisis,” during which there can be an increase in temperature, hypotension, and other signs of shock. A similar phenomenon occurring in some patients soon after the initiation of antibiotic treatment is characterized by the abrupt worsening of the condition, which is called a Jarisch-Herxheimer reaction (JHR).

1	Relapsing fever presents with the sudden onset of fever. Febrile periods are punctuated by intervening afebrile periods of a few days; this pattern occurs at least twice. The patient’s temperature is ≥39°C and may be as high as 43°C. The first fever episode often ends in a crisis lasting ~15–30 min and consisting of rigors, a further elevation in temperature, and increases in pulse and blood pressure. The crisis phase is followed by profuse diaphoresis, falling temperature, and hypotension, which usually persist for several hours. In LBRF, the first episode of fever is unremitting for 3–6 days; it is usually followed by a single milder episode. In TBRF, multiple febrile periods last 1–3 days each. In both forms, the interval between fevers ranges from 4 to 14 days, sometimes with symptoms of malaise and fatigue.

1	The symptoms that accompany the fevers are usually nonspecific. Headache, neck stiffness, arthralgia, myalgia, and nausea may accompany the first and subsequent febrile episodes. An enlarging spleen and liver cause abdominal pain. A nonproductive cough is common during LBRF and—in combination with fever and myalgias—may suggest influenza. Acute respiratory distress syndrome may occur during TBRF. On physical examination, the patient may be delirious or apathetic. There may be body lice in the patient’s clothes or signs of insect bites. In regions with B. miyamotoi infection, a hard tick may be embedded in the skin. Epistaxis, petechiae, and ecchymoses are common during LBRF but not in TBRF. Splenomegaly or spleen tenderness is common in both forms of relapsing fever. The majority of patients with LBRF and ~10% of patients with TBRF have discernible hepatomegaly.

1	Localizing neurologic findings are more common in TBRF than in LBRF. In North America, B. turicatae infection has neurologic manifestations more often than B. hermsii infection. Meningoencephalitis can result in residual hemiplegia or aphasia. Myelitis and radiculopathy may develop. Unilateral or bilateral Bell’s palsy and deafness from sev-1147 enth or eighth cranial nerve involvement are the most common forms of cranial neuritis and typically present in the second or third febrile episode, not the first. Visual impairment from unilateral or bilateral iridocyclitis or panophthalmitis may be permanent. In LBRF, neurologic manifestations such as altered mental state or stiff neck are thought to be secondary to spirochetemia and systemic inflammation rather than to direct invasion of the nervous system.

1	Myocarditis appears to be common in both forms of relapsing fever and accounts for some deaths. Most commonly, myocarditis is evidenced by gallops on cardiac auscultation, a prolonged QTc interval, and cardiomegaly and pulmonary edema on chest radiography. General laboratory studies are not specific. Mild to moderate normocytic anemia is common, but frank hemolysis and hemoglobinuria do not develop. Leukocyte counts are usually in the normal range or only slightly elevated, and leukopenia can occur during the crisis. Platelet counts can fall below 50,000/µL. Laboratory evidence of hepatitis can be found, with elevated serum concentrations of unconjugated bilirubin and aminotransferases; the prothrombin and partial thromboplastin times may be moderately prolonged.

1	Analysis of the cerebrospinal fluid (CSF) is indicated in cases of suspected relapsing fever with signs of meningitis or meningoencephalitis. The presence of mononuclear pleocytosis and mildly to moderately elevated protein levels justifies intravenous antibiotic therapy in TBRF. The manifestations and course of B. miyamotoi infection remain incompletely characterized, but reports to date indicate that the sign most often reported by patients at presentation is fever without respi ratory symptoms starting 1–2 weeks after a tick bite and recurring once or twice in some cases. Meningoencephalitis with spirochetes in the CSF was documented in one immunodeficient adult. Relapsing fever should be considered in a patient with the characteristic fever pattern and a history of recent exposure—i.e., within 1–2 weeks before illness onset—to body lice or soft-bodied ticks in geographic areas with documented current or past transmission.

1	Because of the longevity of the ticks and the transovarial transmission of the pathogen in the ticks, a case of relapsing fever may be diagnosed many years after the last case reported in that locale.

1	The bedrock for laboratory diagnosis remains the same as it has been for a century: direct detection of the spirochetes by microscopy of the blood. Manual differential counts of white blood cells by Wright or Giemsa stain usually reveal spirochetes in thin blood smears if their concentration is ≥105/mL and several oil-immersion fields are examined (Fig. 209-2). The preferred time to obtain a blood specimen is between the fever’s onset and its peak. Lower concentrations of spirochetes may be revealed by a thick blood smear that is either directly stained with acridine orange and then examined by fluorescence microscopy or treated with 0.5% acetic acid before Giemsa or Wright staining. An alternative to a fixed blood smear is a wet mount of anti-coagulated blood mixed with saline and examined by phase-contrast or dark-field microscopy for motile spirochetes.

1	Polymerase chain reaction (PCR) and similar DNA amplification procedures are increasingly used for examination of an extract of blood. PCR may reveal spirochetes between febrile episodes, since there are already escape variants in the population when the first wave of bacteria is neutralized. Culture of blood or CSF in Barbour-Stoenner-Kelly broth medium is an option for isolation of Borrelia species except for B. miyamotoi, which is noncultivable or poorly cultivable. However, few laboratories offer this service. An alternative for tick-borne Borrelia species, including B. miyamotoi, is inoculation of blood or CSF into immunodeficient mice and examination of the animal’s blood after a few days.

1	Options for serologic confirmation of infection are limited. Most assays that are available commercially or in reference laboratories are based on whole cells of a single Borrelia species. These assays may not detect the major variant antigens to which the patient is mainly responding or may yield false-positive results due to antibodies to cross-reactive antigens of related bacteria, including B. burgdorferi. The most FIGuRE 209-2 Photomicrograph of tick-borne relapsing fever spirochete (Borrelia turicatae) in a Wright-Giemsa-stained thin blood smear. Included in the figure are a polymorphonuclear leukocyte and two platelets. promising new assays under development are based on recombinant antigens such as GlpQ, a protein antigen of all relapsing fever Borrelia species (including B. miyamotoi) but not of any Lyme disease species.

1	Depending on the patient’s history of residential, occupational, travel, and recreational exposures, the differential diagnosis of relapsing fever includes one or more of the following infections that feature either periodicity in the fever pattern or an extended single febrile period with nonspecific constitutional symptoms: Colorado tick fever (which, along with dengue, can have a “saddleback” fever course), Rocky Mountain spotted fever and other rickettsioses, ehrlichiosis, anaplasmosis, tick-borne arbovirus infection, and babesiosis in North America, Europe, Russia, and northeastern Asia. Elsewhere in the Americas and Asia and in most of Africa, malaria, typhoid fever, typhus and other rickettsioses, dengue, brucellosis, and leptospirosis may also be considered. Depending on the geographic area and types of exposure, malaria, louse-borne typhus, typhoid fever, or Lyme disease may complicate relapsing fever.

1	Penicillins and tetracyclines have been the antibiotics of choice for relapsing fever for several decades. Erythromycin has been a longstanding second choice. There is no evidence of acquired resistance to these antibiotics. Borrelia species are also susceptible to most cephalosporins and chloramphenicol, but there is less clinical experience with these drugs. Borreliae are relatively resistant to rifampin, sulfonamides, fluoroquinolones, and aminoglycosides. Spirochetes are no longer detectable in the blood within a few hours after the first dose of an effective antibiotic.

1	A single dose of antibiotic is usually sufficient for the treatment of LBRF (Fig. 209-3). The recurrence rate after antibiotic treatment is ≤5%. For adults, a single dose of oral tetracycline (500 mg), oral doxycycline (200 mg), or intramuscular penicillin G procaine (400,000– 800,000 units) is effective. The corresponding doses for children are oral tetracycline at 12.5 mg/kg, oral doxycycline at 5 mg/kg, and intramuscular penicillin G procaine at 200,000–400,000 units. Oral therapy Single dose therapy Intravenous ceftriaxone 2 g qd or Na penicillin G, 5 million U q6h for 14 days First choiceFirst choiceMeningitis/encephalitis Tick-borne relapsing fever Louse-borne relapsing fever

1	Age š9 years, not pregnant: Age š9 years, not pregnant: doxcycline, 100 mg bid doxycycline 200 mg oral or Age <9 years: erythromycin, tetracycline 500 mg oral or 12.5 mg/kg per day 250–500 mg IV Second choice Age <9 years: erythromycin Age š9 years, not pregnant: 12.5 mg/kg oral tetracycline 500 mg qid Second choice Third choice Penicillin G procaine IM Age š9 years: erythromycin, 800,000 U adults 500 mg qid 200,000–400,000 U children Duration: 10 days Third choice Age š9 years: erythromycin 500 mg oral FIGuRE 209-3 Algorithm for treatment of relapsing fever. If it is not known whether the patient has tick-borne or louse-borne relapsing fever, the patient should be treated for the tick-borne form. The dashed line indicates that central nervous system invasion in louse-borne relapsing fever is uncommon.

1	When an adult patient is stuporous or nauseated, the intravenous dose is 250–500 mg. Tetracycline is contraindicated in pregnant and nursing women and in children <9 years old; for individuals in these groups who are allergic to penicillin, oral erythromycin (500 mg for adults and 12.5 mg/kg for children) is an alternative. Tetracycline is marginally superior to penicillin G in terms of time to fever clearance and relapse rate.

1	The accumulated anecdotal reports on TBRF therapy indicate a recurrence rate of ≥20% after single-dose treatment. This high rate of recurrence plausibly is due to the greater propensity of tick-borne species than of B. recurrentis to invade the CNS, from which they can reinvade the bloodstream after antibiotic levels decline. Accordingly, multiple antibiotic doses are recommended. The preferred treatment for adults is a 10-day course of tetracycline (500 mg or 12.5 mg/kg orally every 6 h) or doxycycline (100 mg twice daily). When tetracyclines are contraindicated, the alternative is erythromycin (500 mg or 12.5 mg/kg orally every 6 h) for 10 days. If a β-lactam antibiotic is given, it should be administered intravenously rather than orally, especially if CNS involvement is confirmed or suspected. For adults, the regimen is penicillin G (5 million units IV every 6 h) or ceftriaxone (2 g IV daily) for 10–14 days.

1	Experience with the treatment of B. miyamotoi infection is limited, but this organism likely has the same antibiotic susceptibilities as other Borrelia species. Until more is known about treatment efficacy, therapy for B. miyamotoi infection can follow the guidelines for Lyme disease—including parenteral therapy for CNS involvement— because it may be difficult to rule out co-infection. The JHR during treatment of relapsing fever can be severe and can even end in death if precautions are not in place for close monitoring and provision of cardiovascular and volume support as needed. Rigors, fever, and hypotension occur within 2–3 h of initiation of antibiotic treatment. The incidence of the JHR is ~80% in LBRF and ~50% in TBRF. Both penicillin and tetracycline can elicit the JHR. The mortality rates for untreated LBRF and TBRF are in the ranges of 10–70% and 4–10%, respectively, and are largely determined by coexisting conditions, such as malnutrition and dehydration.

1	Death from untreated relapsing fever is most common during the first fever episode. With prompt antibiotic treatment, the mortality rate is 2–5% for LBRF and <2% for TBRF. Features associated with a poor prognosis include concurrence with malaria, typhus, or typhoid; pregnancy; stupor or coma on admission; diffuse bleeding; poor liver function; myocarditis; and bronchopneumonia. The mortality rate from the JHR in LBRF, in the absence of adequate monitoring and resuscitation measures, is ~5%. Some patients have survived the crisis or the JHR only to die suddenly either later that day or on the next day. Relapsing fever during pregnancy frequently leads to abortion or stillbirth, but congenital malformations have not been reported. Although it is possible that spirochetes may persist in the CNS or other sequestered sites after bacteremia has resolved, chronic disease or disability from a persistent infection has not been attributed to relapsing fever. Partial immunity against

1	in the CNS or other sequestered sites after bacteremia has resolved, chronic disease or disability from a persistent infection has not been attributed to relapsing fever. Partial immunity against reinfection seems to develop in residents of endemic areas.

1	There is no vaccine for either LBRF or TBRF. Reduction of exposure to lice and ticks is the key strategy for prevention. LBRF can be prevented through improved personal hygiene, reduction of crowding, better access to washing facilities, and selected use of pesticides. Infested clothing is an important factor in maintaining body lice. The risk of TBRF can be reduced by construction of houses with concrete or sealed plank floors and without thatched roofs or mud walls. Log cabins pose a particular risk in North America when rodents nest in the roof or beneath the house or porch. Interiors of buildings infested with Ornithodoros ticks can be treated with pesticides. If residing in a high-risk environment, individuals should not sleep on the floor, and beds should be moved away from the wall. With an exposure to TBRF, postexposure treatment with doxycycline (200 mg on day 1 followed by 100 mg/d for 4 days) was efficacious in preventing infection in a placebo-controlled trial.

1	Lyme Borreliosis Allen C. Steere DEFINITION Lyme borreliosis is caused by a spirochete, Borrelia burgdorferi sensu lato, that is transmitted by ticks of the Ixodes ricinus complex. The infec-tion usually begins with a characteristic expanding skin lesion, erythema 210 migrans (EM; stage 1, localized infection). After several days or weeks, the spirochete may spread to many different sites (stage 2, disseminated infection). Possible manifestations of disseminated infection include secondary annular skin lesions, meningitis, cranial neuritis, radiculoneuritis, peripheral neuritis, carditis, atrioventricular nodal block, or migratory musculoskeletal pain. Months or years later (usually after periods of latent infection), intermittent or persistent arthritis, chronic encephalopathy or polyneuropathy, or acrodermatitis may develop (stage 3, persistent infection). Most patients experience early symptoms of the illness during the summer, but the infection may not become symptomatic until it

1	or acrodermatitis may develop (stage 3, persistent infection). Most patients experience early symptoms of the illness during the summer, but the infection may not become symptomatic until it progresses to stage 2 or 3.

1	Lyme disease was recognized as a separate entity in 1976 because of a geographic cluster of children in Lyme, Connecticut, who were thought to have juvenile rheumatoid arthritis. It became apparent that Lyme disease was a multisystemic illness that affected primarily the skin, nervous system, heart, and joints. Epidemiologic studies of patients with EM implicated certain Ixodes ticks as vectors of the disease. Early in the twentieth century, EM had been described in Europe and attributed to I. ricinus tick bites. In 1982, a previously unrecognized spirochete, now called Borrelia burgdorferi, was recovered from Ixodes scapularis ticks and then from patients with Lyme disease. The entity is now called Lyme disease or Lyme borreliosis.

1	B. burgdorferi, the causative agent of Lyme disease, is a fastidious microaerophilic bacterium. The spirochete’s genome is quite small (~1.5 Mb) and consists of a highly unusual linear chromo some of 950 kb as well as 17–21 linear and circular plasmids. The most remarkable aspect of the B. burgdorferi genome is that there are sequences for more than 100 known or predicted lipoproteins—a larger number than in any other organism. The spirochete has few proteins with biosynthetic activity and depends on its host for most of its nutritional requirements. It has no sequences for recognizable toxins.

1	Currently, 13 closely related borrelial species are collectively referred to as Borrelia burgdorferi sensu lato (i.e., “B. burgdor feri in the general sense”). The human infection Lyme borreliosis is caused primarily by three pathogenic genospecies: B. burgdorferi sensu stricto (“B. burgdorferi in the strict sense,” hereafter referred to simply as B. burgdorferi), Borrelia garinii, and Borrelia afzelii. B. burgdorferi is the sole cause of the infection in the United States; all three genospecies are found in Europe, and the latter two species occur in Asia.

1	Strains of B. burgdorferi have been subdivided according to several typing schemes: one based on sequence variation of outer-surface protein C (OspC), a second based on differences in the 16S–23S rRNA intergenic spacer region (RST or IGS), and a third called multilocus sequence typing. From these typing systems, it is apparent that strains of B. burgdorferi differ in pathogenicity. OspC type A (RST1) strains seem to be particularly virulent and may have played a role in the emergence of Lyme disease in epidemic form in the northeastern United States in the late twentieth century. The 13 known genospecies of B. burgdorferi sensu lato live in nature in enzootic cycles involving 14 species of ticks that are part of the I. ricinus complex. I. scapularis (Fig. 475-1) is the principal vector in the eastern United States from Maine to Georgia and in the midwestern states of Wisconsin, Minnesota, and Michigan.

1	I. pacificus is the vector in the western states of California and Oregon. The disease is acquired throughout Europe (from Great Britain to Scandinavia to European Russia), where I. ricinus is the vector, and in Asian Russia, China, and Japan, where I. persulcatus is the vector. These ticks may transmit other agents as well. In the United States, I. scapularis also transmits Babesia microti; Anaplasma phagocytophilum; Ehrlichia species Wisconsin; Borrelia miyamotoi; and, in rare instances, Powassan encephalitis virus (the deer tick virus) (see “Differential Diagnosis,” below). In Europe and Asia, I. ricinus and I. persulcatus also transmit tick-borne encephalitis virus.

1	Ticks of the I. ricinus complex have larval, nymphal, and adult stages. They require a blood meal at each stage. The risk of infection in a given area depends largely on the density of these ticks as well as their feeding habits and animal hosts, which have evolved differently in different locations. For I. scapularis in the northeastern United States, the white-footed mouse and certain other rodents are the preferred hosts of the immature larvae and nymphs. It is critical that both of the tick’s immature stages feed on the same host because the life cycle of the spirochete depends on horizontal transmission: in early summer from infected nymphs to mice and in late summer from infected mice to larvae, which then molt to become the infected nymphs that will begin the cycle again the following year. It is the tiny nymphal tick that is primarily responsible for transmission of the disease to humans during the early summer months. White-tailed deer, which are not involved in the life

1	year. It is the tiny nymphal tick that is primarily responsible for transmission of the disease to humans during the early summer months. White-tailed deer, which are not involved in the life cycle of the spirochete, are the preferred host for the adult stage of I. scapularis and seem to be critical to the tick’s survival.

1	Lyme disease is now the most common vector-borne infection in the United States and Europe. Since surveillance was begun by the Centers for Disease Control and Prevention (CDC) in 1982, the number of cases in the United States has increased dramatically. More than 30,000 new cases are now reported each summer, but the actual number of new cases is probably closer to 300,000 annually. In Europe, reported frequencies of the disease are highest in the middle of the continent and in Scandinavia.

1	1150 PATHOGENESIS AND IMMuNITY To maintain its complex enzootic cycle, B. burgdorferi must adapt to two markedly different environments: the tick and the mammalian host. The spirochete expresses outer-surface protein A (OspA) in the midgut of the tick, whereas OspC is upregulated as the organism travels to the tick’s salivary gland. There, OspC binds a tick salivary-gland protein (Salp15), which is required for infection of the mammalian host. The tick usually must be attached for at least 24 h for transmission of B. burgdorferi. After injection into the human skin, the spirochete downregulates OspC and upregulates the VlsE lipoprotein. This protein undergoes extensive antigenic variation, which is necessary for spirochetal survival. After several days to weeks, B. burgdorferi may migrate outward in the skin, producing EM, and may spread hematogenously or in the lymph to other organs. The only known virulence factors of B. burgdorferi are surface proteins that allow the spirochete to

1	outward in the skin, producing EM, and may spread hematogenously or in the lymph to other organs. The only known virulence factors of B. burgdorferi are surface proteins that allow the spirochete to attach to mammalian proteins, integrins, glycosaminoglycans, or glycoproteins. For example, spread through the skin and other tissue matrices may be facilitated by the binding of human plasminogen and its activators to the surface of the spirochete. Some Borrelia strains bind complement regulator–acquiring surface proteins (FHL-1/reconectin, or factor H), which help to protect spirochetes from complement-mediated lysis. Dissemination of the organism in the blood is facilitated by binding to the fibrinogen receptor on activated platelets (αIIbβ3) and the vitronectin receptor (αvβ3) on endothelial cells. As the name indicates, spirochetal decorin-binding proteins A and B bind decorin, a glycosaminoglycan on collagen fibrils; this binding may explain why the organism is commonly aligned with

1	cells. As the name indicates, spirochetal decorin-binding proteins A and B bind decorin, a glycosaminoglycan on collagen fibrils; this binding may explain why the organism is commonly aligned with collagen fibrils in the extracellular matrix in the heart, nervous system, or joints. To control and eradicate B. burgdorferi, the host mounts both innate and adaptive immune responses, resulting in macrophageand antibody-mediated killing of the spirochete. As part of the innate immune response, complement may lyse the spirochete in the skin. Cells at affected sites release potent proinflammatory cytokines, including interleukin 6, tumor necrosis factor α, interleukin 1β, and interferon γ. Patients who are homozygous for a Toll-like receptor 1 polymorphism (1805GG), particularly when infected with highly inflammatory B. burgdorferi RST1 strains, have exceptionally high levels of proinflammatory cytokines. The purpose of the adaptive immune response appears to be the production of specific

1	with highly inflammatory B. burgdorferi RST1 strains, have exceptionally high levels of proinflammatory cytokines. The purpose of the adaptive immune response appears to be the production of specific antibodies, which opsonize the organism—a step necessary for optimal spirochetal killing. Studies with protein arrays expressing ~1200 B. burgdorferi proteins detected antibody responses to a total of 120 spirochetal proteins (particularly outer-surface lipoproteins) in a population of patients with Lyme arthritis. Histologic examination of all affected tissues reveals an infiltration of lymphocytes, macrophages, and plasma cells with some degree of vascular damage, including mild vasculitis or hypervascular occlusion. These findings suggest that the spirochete may have been present in or around blood vessels. In enzootic infection, B. burgdorferi spirochetes must survive this immune assault only during the summer months before returning to larval ticks to begin the cycle again the

1	or around blood vessels. In enzootic infection, B. burgdorferi spirochetes must survive this immune assault only during the summer months before returning to larval ticks to begin the cycle again the following year. In contrast, infection of humans is a dead-end event for the spirochete. Within several weeks or months, innate and adaptive immune mechanisms— even without antibiotic treatment—control widely disseminated infection, and generalized systemic symptoms wane. However, without antibiotic therapy, spirochetes may survive in localized niches for several more years. For example, B. burgdorferi infection in the United States may cause persistent arthritis or, in rare cases, subtle encephalopathy or polyneuropathy. Thus, immune mechanisms seem to succeed eventually in the near or total eradication of B. burgdorferi from selected niches, including the joints or nervous system, and symptoms resolve in most patients.

1	CLINICAL MANIFESTATIONS Early Infection: Stage 1 (Localized Infection) Because of the small size of nymphal ixodid ticks, most patients do not remember the preceding tick bite. After an incubation period of 3–32 days, EM usually begins as a red macule or papule at the site of the tick bite that expands slowly to form a large annular lesion (Fig. 210-1). As the lesion increases in size, it often develops a bright red outer border and partial central clearing. The center of the lesion sometimes becomes intensely erythematous and indurated, vesicular, or necrotic. In other instances, the expanding lesion remains an even, intense red; several red rings are found within an outside ring; or the central area turns blue before the lesion clears. Although EM can be located anywhere, the thigh, groin, and axilla are particularly common sites. The lesion is warm but not often painful. Approximately 20% of patients do not exhibit this characteristic skin manifestation.

1	FIGuRE 210-1 A classic erythema migrans lesion (9 cm in diameter) is shown near the right axilla. The lesion has partial central clearing, a bright red outer border, and a target center. (Courtesy of Vijay K. Sikand, MD; with permission.)

1	Early Infection: Stage 2 (Disseminated Infection) In cases in the United States, B. burgdorferi often spreads hematogenously to many sites within days or weeks after the onset of EM. In these cases, patients may develop secondary annular skin lesions similar in appearance to the initial lesion. Skin involvement is commonly accompanied by severe headache, mild stiffness of the neck, fever, chills, migratory musculoskeletal pain, arthralgias, and profound malaise and fatigue. Less common manifestations include generalized lymphadenopathy or splenomegaly, hepatitis, sore throat, nonproductive cough, conjunctivitis, iritis, or testicular swelling. Except for fatigue and lethargy, which are often constant, the early signs and symptoms of Lyme disease are typically intermittent and changing. Even in untreated patients, the early symptoms usually become less severe or disappear within several weeks. In ~15% of patients, the infection presents with these nonspecific systemic symptoms.

1	Symptoms suggestive of meningeal irritation may develop early in Lyme disease when EM is present but usually are not associated with cerebrospinal fluid (CSF) pleocytosis or an objective neurologic deficit. After several weeks or months, ~15% of untreated patients develop frank neurologic abnormalities, including meningitis, subtle encephalitic signs, cranial neuritis (including bilateral facial palsy), motor or sensory radiculoneuropathy, peripheral neuropathy, mononeuritis multiplex, cerebellar ataxia, or myelitis—alone or in various combinations. In children, the optic nerve may be affected because of inflammation or increased intracranial pressure, and these effects may lead to blindness. In the United States, the usual pattern consists of fluctuating symptoms of meningitis accompanied by facial palsy and peripheral radiculoneuropathy. Lymphocytic pleocytosis (~100 cells/μL) is found in CSF, often along with elevated protein levels and normal or slightly low glucose

1	accompanied by facial palsy and peripheral radiculoneuropathy. Lymphocytic pleocytosis (~100 cells/μL) is found in CSF, often along with elevated protein levels and normal or slightly low glucose concentrations. In Europe and Asia, the first neurologic sign is characteristically radicular pain, which is followed by the development of CSF pleocytosis (meningopolyneuritis or Bannwarth’s syndrome); meningeal or encephalitic signs are frequently absent. These early neurologic abnormalities usually resolve completely within months, but in rare cases chronic neurologic disease may occur later.

1	Within several weeks after the onset of illness, ~8% of patients develop cardiac involvement. The most common abnormality is a fluctuating degree of atrioventricular block (first-degree, Wenckebach, or complete heart block). Some patients have more diffuse cardiac involvement, including electrocardiographic changes indicative of acute myopericarditis, left ventricular dysfunction evident on radionuclide scans, or (in rare cases) cardiomegaly or fatal pancarditis. Cardiac involvement lasts for only a few weeks in most patients but may recur in untreated patients. Chronic cardiomyopathy caused by B. burgdorferi has been reported in Europe. During this stage, musculoskeletal pain is common. The typical pattern consists of migratory pain in joints, tendons, bursae, muscles, or bones (usually without joint swelling) lasting for hours or days and affecting one or two locations at a time.

1	Late Infection: Stage 3 (Persistent Infection) Months after the onset of infection, ~60% of patients in the United States who have received no antibiotic treatment develop frank arthritis. The typical pattern comprises intermittent attacks of oligoarticular arthritis in large joints (especially the knees), lasting for weeks or months in a given joint. A few small joints or periarticular sites also may be affected, primarily during early attacks. The number of patients who continue to have recurrent attacks decreases each year. However, in a small percentage of cases, involvement of large joints—usually one or both knees—is persistent and may lead to erosion of cartilage and bone.

1	White cell counts in joint fluid range from 500 to 110,000/μL (average, 25,000/μL); most of these cells are polymorphonuclear leukocytes. Tests for rheumatoid factor or antinuclear antibodies usually give negative results. Examination of synovial biopsy samples reveals fibrin deposits, villous hypertrophy, vascular proliferation, microangiopathic lesions, and a heavy infiltration of lymphocytes and plasma cells.

1	Although most patients with Lyme arthritis respond well to antibiotic therapy, a small percentage in the northeastern United States have persistent (antibiotic-refractory) arthritis for months or even for several years after receiving oral and IV antibiotic therapy for 2 or 3 months. Although more often these patients are initially infected with RST1 strains of B. burgdorferi, this complication is not thought to result from persistent infection. Results of culture and polymerase chain reaction (PCR) for B. burgdorferi in synovial tissue obtained in the postantibiotic period have been uniformly negative. Rather, infection-induced autoimmunity, retained spirochetal antigens, or both may play a role in this outcome. Antibiotic-refractory arthritis is associated with a higher frequency of certain class II major histocompatibility complex molecules (particularly HLA-DRBI*0401 or -*0101 molecules); the Toll-like receptor 1 polymorphism 1805GG, which leads to exceptionally high levels of

1	certain class II major histocompatibility complex molecules (particularly HLA-DRBI*0401 or -*0101 molecules); the Toll-like receptor 1 polymorphism 1805GG, which leads to exceptionally high levels of cytokines and chemokines in affected joints; and low frequencies of FoxP3+ T regulatory cells in synovial fluid, which correlate with longer posttreatment durations of arthritis. The recent identification of a novel human autoantigen, endothelial cell growth factor, as a target of T and B cell responses in patients with Lyme disease provided the first direct evidence of autoimmune T and B cell responses in this illness. However, multiple spirochetal or additional yet-to-be identified auto-antigens may have a role in antibiotic-refractory arthritis.

1	Although rare, chronic neurologic involvement also may become apparent from months to several years after the onset of infection, sometimes after long periods of latent infection. The most common form of chronic central nervous system involvement is subtle encephalopathy affecting memory, mood, or sleep, and the most common form of peripheral neuropathy is an axonal polyneuropathy manifested as either distal paresthesia or spinal radicular pain. Patients with encephalopathy frequently have evidence of memory impairment in neuropsychological tests and abnormal results in CSF analyses. In cases of polyneuropathy, electromyography generally shows extensive abnormalities of proximal and distal nerve segments. Encephalomyelitis or leukoencephalitis, a rare manifestation of Lyme borreliosis associated primarily with B. garinii infection in Europe, is a severe neurologic disorder that may include spastic paraparesis, upper motor-neuron bladder dysfunction, and, rarely, lesions in the

1	associated primarily with B. garinii infection in Europe, is a severe neurologic disorder that may include spastic paraparesis, upper motor-neuron bladder dysfunction, and, rarely, lesions in the periventricular white matter.

1	Acrodermatitis chronica atrophicans, the late skin manifesta tion of Lyme borreliosis, has been associated primarily with B. afzelii infection in Europe and Asia. It has been observed especially often in elderly women. The skin lesions, which are usually 1151 found on the acral surface of an arm or leg, begin insidiously with reddish-violaceous discoloration; they become sclerotic or atrophic over a period of years.

1	The basic patterns of Lyme borreliosis are similar worldwide, but there are regional variations, primarily between the illness found in North America, which is caused exclusively by B. burgdorferi, and that found in Europe, which is caused primarily by B. afzelii and B. garinii. With each of the Borrelia species, the infection usually begins with EM. However, B. burgdorferi strains in the eastern United States often disseminate widely; they are particularly arthritogenic, and they may cause antibiotic-refractory arthritis. B. garinii typically disseminates less widely, but it is especially neurotropic and may cause borrelial encephalomyelitis. B. afzelii often infects only the skin but may persist in that site, where it may cause several different dermatoborrelioses, including acrodermatitis chronica atrophicans.

1	Post–Lyme Syndrome (Chronic Lyme Disease) Despite resolution of the objective manifestations of the infection with antibiotic therapy, ~10% of patients (although the reported percentages vary widely) continue to have subjective pain, neurocognitive manifestations, or fatigue symptoms. These symptoms usually improve and resolve within months but may last for years. At the far end of the spectrum, the symptoms may be similar to or indistinguishable from chronic fatigue syndrome (Chap. 464e) and fibromyalgia (Chap. 396). Compared with symptoms of active Lyme disease, post-Lyme symptoms tend to be more generalized or disabling. They include marked fatigue, severe headache, diffuse musculoskeletal pain, multiple symmetric tender points in characteristic locations, pain and stiffness in many joints, diffuse paresthesias, difficulty with concentration, and sleep disturbances. Patients with this condition lack evidence of joint inflammation, have normal neurologic test results, and may

1	joints, diffuse paresthesias, difficulty with concentration, and sleep disturbances. Patients with this condition lack evidence of joint inflammation, have normal neurologic test results, and may exhibit anxiety and depression. In contrast, late manifestations of Lyme disease, including arthritis, encephalopathy, and neuropathy, are usually associated with minimal systemic symptoms. Currently, no evidence indicates that persistent subjective symptoms after recommended courses of antibi otic therapy are caused by active infection.

1	The culture of B. burgdorferi in Barbour-Stoenner-Kelly (BSK) medium permits definitive diagnosis, but this method has been used primarily in research studies. Moreover, with a few exceptions, positive cultures have been obtained only early in the illness—particularly from biopsy samples of EM skin lesions, less often from plasma samples, and occasionally from CSF samples. Later in the infection, PCR is greatly superior to culture for the detection of B. burgdorferi DNA in joint fluid; this is the major use for PCR testing in Lyme disease. However, because B. burgdorferi DNA may persist for at least weeks after spirochetal killing with antibiotics, detection of spirochetal DNA in joint fluid is not an accurate test of active joint infection in Lyme disease and cannot be used reliably to determine the adequacy of antibiotic therapy. The sensitivity of PCR determinations in CSF from patients with neuroborreliosis has been much lower than that in joint fluid. There seems to be little if

1	the adequacy of antibiotic therapy. The sensitivity of PCR determinations in CSF from patients with neuroborreliosis has been much lower than that in joint fluid. There seems to be little if any role for PCR in the detection of B. burgdorferi DNA in blood or urine samples. Moreover, this procedure must be carefully controlled to prevent contamination.

1	Because of the problems associated with direct detection of B. burgdorferi, Lyme disease is usually diagnosed by the recognition of a characteristic clinical picture accompanied by serologic confirmation. Although serologic testing may yield negative results during the first several weeks of infection, almost all patients have a positive antibody response to B. burgdorferi after that time. The limitation of serologic tests is that they do not clearly distinguish between active and inactive infection. Patients with previous Lyme disease—particularly in cases progressing to late stages—often remain seropositive for years, even after adequate antibiotic treatment. In addition, ~10% of patients are seropositive because of asymptomatic infection. If these individuals subsequently develop another illness, the positive serologic test for Source: Adapted from the recommendations of the American College of Physicians (G Nichol et al: Ann Intern Med 128:37, 1998, with permission).

1	Source: Adapted from the recommendations of the American College of Physicians (G Nichol et al: Ann Intern Med 128:37, 1998, with permission). Lyme disease may cause diagnostic confusion. According to an algorithm published by the American College of Physicians (Table 210-1), serologic testing for Lyme disease is recommended only for patients with at least an intermediate pretest probability of Lyme disease, such as those with oligoarticular arthritis. It should not be used as a screening procedure in patients with pain or fatigue syndromes. In such patients, the probability of a false-positive serologic result is higher than that of a true-positive result.

1	For serologic analysis of Lyme disease in the United States, the CDC recommends a two-step approach in which samples are first tested by enzyme-linked immunosorbent assay (ELISA) and equivocal or positive results are then tested by western blotting. During the first weeks of infection, both IgM and IgG responses to the spirochete should be determined, preferably in both acuteand convalescent-phase serum samples. Approximately 20–30% of patients have a positive response detectable in acute-phase samples, whereas ~70–80% have a positive response during convalescence (2–4 weeks later). After 4–8 weeks of infection (by which time most patients with active Lyme disease have disseminated infection), the sensitivity and specificity of the IgG response to the spirochete are both very high—in the range of 99%—as determined by the two-test approach of ELISA and western blot. At this point and thereafter, a single test (that for IgG) is usually sufficient. In persons with illness of >2 months’

1	range of 99%—as determined by the two-test approach of ELISA and western blot. At this point and thereafter, a single test (that for IgG) is usually sufficient. In persons with illness of >2 months’ duration, a positive IgM test result alone is likely to be false-positive and therefore should not be used to support the diagnosis.

1	According to current criteria adopted by the CDC, an IgM western blot is considered positive if two of the following three bands are present: 23, 39, and 41 kDa. However, the combination of two such bands may still represent a false-positive result. Misuse or misinterpretation of IgM blots has been a factor in the incorrect diagnosis of Lyme disease in patients with other illnesses. An IgG blot is considered positive if 5 of the following 10 bands are present: 18, 23, 28, 30, 39, 41, 45, 58, 66, and 93 kDa. In European cases, no single set of criteria for the interpretation of immunoblots results in high levels of sensitivity and specificity in all countries.

1	The most promising second-generation serologic test is the VlsE C6 peptide IgG ELISA, which employs a 26-mer of the sixth invariant region of the VlsE lipoprotein of B. burgdorferi. The results achieved with this test are similar to those obtained with the standard two-test approach (sonicate IgM and IgG ELISA and western blot). The principal advantage of the C6 peptide ELISA is the early detection of an IgG response, which renders an IgM test unnecessary. However, not all patients with late Lyme disease have a response to the C6 peptide, and this test is not quite as specific as sonicate western blot. Thus, at present, a two-test approach that includes western blot is still recommended. Blotting can also be helpful in assessing the duration of current or past disease.

1	After successful antibiotic treatment, antibody titers decline slowly but responses (including that to the VlsE C6 peptide) may persist for years. Moreover, not only the IgG but also the IgM response may persist for years after therapy. Therefore, even a positive IgM response cannot be interpreted as confirmation of recent infection or reinfection unless the clinical picture is appropriate.

1	Classic EM is a slowly expanding erythema, often with partial central clearing. If the lesion expands little, it may represent the red papule of an uninfected tick bite. If the lesion expands rapidly, it may represent cellulitis (e.g., streptococcal cellulitis) or an allergic reaction, perhaps to tick saliva. Patients with secondary annular lesions may be thought to have erythema multiforme, but neither the development of blistering mucosal lesions nor the involvement of the palms or soles is a feature of B. burgdorferi infection. In the eastern United States, an EM-like skin lesion, sometimes with mild systemic symptoms, may be associated with Amblyomma americanum tick bites. However, the cause of this Southern tick-associated rash illness (STARI) has not yet been identified. This tick may also transmit Ehrlichia chaffeensis, a rickettsial agent (Chap. 211).

1	As stated above, I. scapularis ticks in the United States may transmit not only B. burgdorferi but also B. microti, a red blood cell parasite (Chap. 249); A. phagocytophilum, the agent of human granulocytotropic anaplasmosis (Chap. 211); Ehrlichia species Wisconsin; B. miyamotoi, a relapsing fever spirochete (Chap. 209); or (rarely) Powassan encephalitis virus (the deer tick virus, which is closely related to European tick-borne encephalitis virus) (Chap. 233). Although babesiosis and anaplasmosis are most often asymptomatic, infection with any of these agents may cause nonspecific systemic symptoms, particularly in the young or elderly, and co-infected patients may have more severe or persistent symptoms than patients infected with a single agent. Standard blood counts may yield clues regarding the presence of co-infection with Anaplasma or Babesia. Anaplasmosis may cause leukopenia or thrombocytopenia, and babesiosis may cause thrombocytopenia or (in severe cases) hemolytic anemia.

1	the presence of co-infection with Anaplasma or Babesia. Anaplasmosis may cause leukopenia or thrombocytopenia, and babesiosis may cause thrombocytopenia or (in severe cases) hemolytic anemia. IgM serologic responses may confuse the diagnosis. For example, A. phagocytophilum may elicit a positive IgM response to B. burgdorferi. The frequency of co-infection in different studies has been variable. In one prospective study, 4% of patients with EM had evidence of co-infection.

1	Facial palsy caused by B. burgdorferi, which occurs in the early disseminated phase of the infection (often in July, August, or September), is usually recognized by its association with EM. However, in rare cases, facial palsy without EM may be the presenting manifestation of Lyme disease. In such cases, both the IgM and the IgG responses to the spirochete are usually positive. The most common infectious agents that cause facial palsy are herpes simplex virus type 1 (Bell’s palsy; Chap. 216) and varicella-zoster virus (Ramsay Hunt syndrome; Chap. 217). Later in the infection, oligoarticular Lyme arthritis most resembles reactive arthritis in an adult or the pauciarticular form of juvenile idiopathic arthritis in a child. Patients with Lyme arthritis usually have the strongest IgG antibody responses seen in Lyme borreliosis, with reactivity to many spirochetal proteins.

1	The most common problem in diagnosis is to mistake Lyme disease for chronic fatigue syndrome (Chap. 464e) or fibromyalgia (Chap. 396). This difficulty is compounded by the fact that a small percentage of patients do in fact develop these chronic pain or fatigue syndromes in association with or soon after Lyme disease. Moreover, a counterculture has emerged that ascribes pain and fatigue syndromes to chronic Lyme disease when there is little or no evidence of B. burgdorferi infection. In such cases, the term chronic Lyme disease, which is equated with chronic B. burgdorferi infection, is a misnomer, and the use of prolonged, dangerous, and expensive antibiotic treatment is not warranted.

1	As outlined in the algorithm in Fig. 210-2, the various manifestations of Lyme disease can usually be treated successfully with orally administered antibiotics; the exceptions are objective neurologic abnormalities and third-degree atrioventricular heart block, which are generally treated with IV antibiotics, and arthritis that does not Joint Arthritis* Heart AV block Nervous system Facial palsy alone Meningitis Radiculoneuritis Encephalopathy Polyneuropathy Intravenous therapy First choice: ceftriaxone, 2 g qd Second choice: cefotaxime, 2 g q8h Third choice: Na penicillin G, 5 million U q6h 1°, 2° 3° First choice Age ˜ 9 years, not pregnant: doxycycline, 100 mg bid Age < 9 years: amoxicillin, 50 mg/kg per day Second choice for adults: amoxicillin, 500 mg tid Third choice for all ages: cefuroxime axetil, 500 mg bid Fourth choice for all ages: erythromycin, 250 mg qid Neurologic involvement: 14–28 days

1	Second choice for adults: amoxicillin, 500 mg tid Third choice for all ages: cefuroxime axetil, 500 mg bid Fourth choice for all ages: erythromycin, 250 mg qid Neurologic involvement: 14–28 days Cardiac involvement: 28 days complete course with oral therapy when patient is no longer in high-degree AV block FIGuRE 210-2 Algorithm for the treatment of the various early or late manifestations of Lyme borreliosis. AV, atrioventricular. *For arthritis, oral therapy should be tried first; if arthritis is unresponsive, IV therapy should be administered. **For Lyme arthritis, IV ceftriaxone (2 g given once a day for 14–28 days) also is effective and is necessary for a small percentage of patients; however, compared with oral treatment, this regimen is less convenient to administer, has more side effects, and is more expensive.

1	respond to therapy. For early Lyme disease, doxycycline is effective and can be administered to men and nonpregnant women. An advantage of this regimen is that it is also effective against A. phagocytophilum, which is transmitted by the same tick that transmits the Lyme disease agent. Amoxicillin, cefuroxime axetil, and erythromycin or its congeners are second-, third-, and fourth-choice alternatives, respectively. In children, amoxicillin is effective (not more than 2 g/d); in cases of penicillin allergy, cefuroxime axetil or erythromycin may be used. In contrast to secondor third-generation cephalosporin antibiotics, first-generation cephalosporins, such as cephalexin, are not effective. For patients with infection localized to the skin, a 14-day course of therapy is generally sufficient; in contrast, for patients with disseminated infection, a 21-day course is recommended. Approximately 15% of patients experience a Jarisch-Herxheimer-like reaction during the first 24 h of therapy.

1	in contrast, for patients with disseminated infection, a 21-day course is recommended. Approximately 15% of patients experience a Jarisch-Herxheimer-like reaction during the first 24 h of therapy. In multicenter studies, more than 90% of patients whose early Lyme disease was treated with these regimens had satisfactory outcomes. Although some patients reported symptoms after treatment, objective evidence of persistent infection or relapse was rare, and re-treatment was usually unnecessary.

1	Oral administration of doxycycline or amoxicillin for 30 days is recommended for the initial treatment of Lyme arthritis in patients who do not have concomitant neurologic involvement. Among patients with arthritis who do not respond to oral antibiotics, re-treatment with IV ceftriaxone for 28 days is appropriate. In patients with arthritis in whom joint inflammation persists for months or even several years after both oral and IV antibiotics, treatment with nonsteroidal anti-inflammatory agents, therapy with disease-1153 modifying antirheumatic drugs, or synovectomy may be successful.

1	In the United States, parenteral antibiotic therapy is usually used for objective neurologic abnormalities (with the exception of facial palsy alone). Patients with neurologic involvement are most commonly treated with IV ceftriaxone for 14–28 days, but IV cefotaxime or IV penicillin G for the same duration also may be effective. In Europe, similar results have been obtained with oral doxycycline and IV antibiotics in the treatment of acute neuroborreliosis. In patients with high-degree atrioventricular block or a PR interval of >0.3 s, IV therapy for at least part of the course and cardiac monitoring are recommended, but the insertion of a permanent pacemaker is not necessary.

1	It is unclear how and whether asymptomatic infection should be treated, but patients with such infection are often given a course of oral antibiotics. Because maternal-fetal transmission of B. burgdorferi seems to occur rarely (if at all), standard therapy for the manifestations of the illness is recommended for pregnant women. Long-term persistence of B. burgdorferi has not been documented in any large series of patients after treatment with currently recommended regimens. Although an occasional patient requires a second course of antibiotics, there is no indication for multiple, repeated antibiotic courses in the treatment of Lyme disease. After appropriately treated Lyme disease, a small percentage of patients continue to have subjective symptoms, primarily musculoskeletal pain, neurocognitive difficulties, or fatigue. This chronic

1	After appropriately treated Lyme disease, a small percentage of patients continue to have subjective symptoms, primarily musculoskeletal pain, neurocognitive difficulties, or fatigue. This chronic Lyme disease or post–Lyme syndrome is sometimes a disabling condition that is similar to chronic fatigue syndrome or fibromyalgia. In a large study, one group of patients with post–Lyme syndrome received IV ceftriaxone for 30 days followed by oral doxycycline for 60 days, while another group received IV and oral placebo preparations for the same durations. No significant differences were found between groups in the numbers of patients reporting that their symptoms had improved, become worse, or stayed the same. Such patients are best treated for the relief of symptoms rather than with prolonged courses of antibiotics.

1	The risk of infection with B. burgdorferi after a recognized tick bite is so low that antibiotic prophylaxis is not routinely indicated. However, if an attached, engorged I. scapularis nymph is found or if follow-up is anticipated to be difficult, a single 200-mg dose of doxycycline, which usually prevents Lyme disease when given within 72 h after the tick bite, may be administered. The response to treatment is best early in the disease. Later treatment of Lyme borreliosis is still effective, but the period of convalescence may be longer. Eventually, most patients recover with minimal or no residual deficits.

1	Reinfection may occur after EM when patients are treated with antimicrobial agents. In such cases, the immune response is not adequate to provide protection from subsequent infection. However, patients who develop an expanded immune response to the spirochete over a period of months (e.g., those with Lyme arthritis) have protective immunity for a period of years and rarely, if ever, acquire the infection again. Protective measures for the prevention of Lyme disease may include the avoidance of tick-infested areas, the use of repellents and acaricides, tick checks, and modification of landscapes in or near residential areas. Although a vaccine for Lyme disease used to be available, the manufacturer has discontinued its production. Therefore, no vaccine is now commercially available for the prevention of this infection.

1	1154 Rickettsial diseases David H. Walker, J. Stephen Dumler, Thomas Marrie The rickettsiae are a heterogeneous group of small, obligately intra-cellular, gram-negative coccobacilli and short bacilli, most of which are transmitted by a tick, mite, flea, or louse vector. Except in the case of louse-borne typhus, humans are incidental hosts. Among 211 Epidemiologic clues to the transmission of a particular pathogen include (1) environmental exposure to ticks, fleas, or mites during the season of activity of the vector species for the disease in the appropriate geographic region (spotted fever and typhus rickettsioses, scrub typhus, ehrlichioses, anaplasmosis); (2) travel to or residence in an endemic geographic region during the incubation period (Table 211-1); (3) expo-sure to parturient ruminants, cats, and dogs (Q fever); (4) exposure to flying squirrels (R. prowazekii infection); and (5) history of previous louse-borne typhus (recrudescent typhus). Clinical laboratory findings, such

1	cats, and dogs (Q fever); (4) exposure to flying squirrels (R. prowazekii infection); and (5) history of previous louse-borne typhus (recrudescent typhus). Clinical laboratory findings, such as thrombocytopenia (particularly sECTIOn 10 dIsEAsEs CAusEd By RICKETTsIAE, MyCOPLAsMAs, And CHLAMydIAE rickettsiae, Coxiella burnetii, Rickettsia prowazekii, and R. typhi have the well-documented ability to survive for an extended period outside the reservoir or vector and to be extremely infectious: inhalation of a single Coxiella microorganism can cause pneumonia. High-level infectivity and severe illness after inhalation make R. prowazekii, R. rickettsii, R. typhi, R. conorii, and C. burnetii bioterrorism threats.

1	Clinical infections with rickettsiae can be classified according to (1) the taxonomy and diverse microbial characteristics of the agents, which belong to seven genera (Rickettsia, Orientia, Ehrlichia, Anaplasma, Neorickettsia, Candidatus Neoehrlichia, and Coxiella); (2) epidemiology; or (3) clinical manifestations. The clinical manifestations of all the acute presentations are similar during the first 5 days: fever, headache, and myalgias with or without nausea, vomiting, and cough. As the course progresses, clinical manifestations—including occurrence of a macular, maculopapular, or vesicular rash; eschar; pneumonitis; and meningoencephalitis—vary from one disease to another. Given the 15 etiologic agents with varied mechanisms of transmission, geographic distributions, and associated disease manifestations, the consideration of rickettsial diseases as a single entity poses complex challenges (Table 211-1).

1	Establishing the etiologic diagnosis of rickettsioses is very difficult during the acute stage of illness, and definitive diagnosis usually requires the examination of paired serum samples after convalescence. Heightened clinical suspicion is based on epidemiologic data, history of exposure to vectors or reservoir animals, travel to endemic locations, clinical manifestations (sometimes including rash or eschar), and characteristic laboratory findings (including thrombocytopenia, normal or low white blood cell [WBC] counts, elevated hepatic enzyme levels, and hyponatremia). Such suspicion should prompt empirical treatment. Doxycycline is the drug of choice for most of these infections. Only one agent, C. burnetii, has been documented to cause chronic illness. One other species, R. prowazekii, causes recrudescent illness (Brill-Zinsser disease) when latent infection is reactivated years after resolution of the acute illness.

1	Rickettsial infections dominated by fever may resolve without further clinical evolution. However, after nonspecific early manifestations, the illnesses can also evolve along one or more of several principal clinical lines: (1) development of a macular or maculopapular rash; (2) development of an eschar at the site of tick or mite feeding; (3) development of a vesicular rash (often in rickettsialpox and African tick-bite fever); (4) development of pneumonitis with chest radiographic opacities and/or rales (Q fever and severe cases of Rocky Mountain spotted fever [RMSF], Mediterranean spotted fever [MSF], louse-borne typhus, human monocytotropic ehrlichiosis [HME], human granulocytotropic anaplasmosis [HGA], scrub typhus, and murine typhus); (5) development of meningoencephalitis (louse-borne typhus and severe cases of RMSF, scrub typhus, HME, murine typhus, MSF, and [rarely] Q fever); and (6) progressive hypotension and multiorgan failure as seen with sepsis or toxic shock syndromes

1	typhus and severe cases of RMSF, scrub typhus, HME, murine typhus, MSF, and [rarely] Q fever); and (6) progressive hypotension and multiorgan failure as seen with sepsis or toxic shock syndromes (RMSF, MSF, louse-borne typhus, murine typhus, scrub typhus, HME, HGA, and neoehrlichiosis).

1	in spotted fever and typhus rickettsioses, ehrlichioses, anaplasmosis, and scrub typhus), normal or low WBC counts, mild to moderate serum elevations of hepatic aminotransferases, and hyponatremia, suggest some common pathophysiologic mechanisms. Application of these clinical, epidemiologic, and laboratory principles requires consideration of a rickettsial diagnosis and knowledge of the individual diseases. TICK-, MITE-, LOuSE-, AND FLEA-BORNE RICKETTSIOSES

1	TICK-, MITE-, LOuSE-, AND FLEA-BORNE RICKETTSIOSES These diseases, caused by organisms of the genera Rickettsia and Orientia in the family Rickettsiaceae, result from endothelial cell infection and increased vascular permeability. Pathogenic rickettsial species are very closely related, have small genomes (as a result of reductive evolution, which eliminated many genes for biosynthesis of intracellularly available molecules), and are traditionally separated into typhus and spotted fever groups on the basis of lipopolysaccharide antigens. Some diseases and their agents (e.g., R. africae, R. parkeri, and R. sibirica) are too similar to require separate descriptions. Indeed, the similarities among MSF (R. conorii [all strains] and

1	R. parkeri, and R. sibirica) are too similar to require separate descriptions. Indeed, the similarities among MSF (R. conorii [all strains] and R. massiliae), North Asian tick typhus (R. sibirica), Japanese spotted fever (R. japonica), and Flinders Island spotted fever (R. honei) far outweigh the minor variations. The Rickettsiaceae that cause life-threatening infections are, in order of decreasing case-fatality rate, R. rickettsii (RMSF); R. prowazekii (louse-borne typhus); Orientia tsutsugamushi (scrub typhus); R. conorii (MSF); R. typhi (murine typhus); and, in rare cases, other spotted fever–group organisms. Some agents (e.g., R. parkeri, R. africae, R. akari, R. slovaca, R. honei, R. felis, R. massiliae, R. helvetica, R. heilongjiangensis, R. aeschlimannii, and R. monacensis) have never been documented to cause a fatal illness.

1	R. massiliae, R. helvetica, R. heilongjiangensis, R. aeschlimannii, and R. monacensis) have never been documented to cause a fatal illness. Epidemiology RMSF occurs in 47 states (with the highest prevalence in the south-central and southeastern states) as well as in Canada, Mexico, and Central and South America. The infection is transmitted by Dermacentor variabilis, the American dog tick, in the eastern two-thirds of the United States and California; by D. andersoni, the Rocky Mountain wood tick, in the western United States; by Rhipicephalus sanguineus in Mexico, Arizona, and probably Brazil; and by Amblyomma cajennense and A. aureolatum in Central and/or South America. Maintained principally by transovarian transmission from one generation of ticks to the next, R. rickettsii can be acquired by uninfected ticks through the ingestion of a blood meal from rickettsemic small mammals.

1	Humans become infected during tick season (in the Northern Hemisphere, from May to September), although some cases occur in winter. The mortality rate was 20–25% in the preantibiotic era and remains at ~3–5%, principally because of delayed diagnosis and treatment. The case-fatality ratio increases with each decade of life above age 20. Pathogenesis R. rickettsii organisms are inoculated into the dermis along with secretions of the tick’s salivary glands after ≥6 h of feeding.

1	The rickettsiae spread lymphohematogenously throughout the body course and treated appropriately as outpatients. In the tertiary-care and infect numerous foci of contiguous endothelial cells. The dose-setting, RMSF is all too often recognized only when late severe dependent incubation period is ~1 week (range, 2–14 days). Occlusive manifestations, developing at the end of the first week or during the thrombosis and ischemic necrosis are not the fundamental pathologic second week of illness in patients without appropriate treatment, bases for tissue and organ injury. Instead, increased vascular perme-prompt return to a physician or hospital and admission to an intenability, with resulting edema, hypovolemia, and ischemia, is responsi-sive care unit. ble. Consumption of platelets results in thrombocytopenia in 32–52% The progressive nature of the infection is clearly manifested in the of patients, but disseminated intravascular coagulation with hypofi-skin. Rash is evident in only 14%

1	thrombocytopenia in 32–52% The progressive nature of the infection is clearly manifested in the of patients, but disseminated intravascular coagulation with hypofi-skin. Rash is evident in only 14% of patients on the first day of illness brinogenemia is rare. Activation of platelets, generation of thrombin, and in only 49% during the first 3 days. Macules (1–5 mm) appear first and activation of the fibrinolytic system all appear to be homeostatic on the wrists and ankles and then on the remainder of the extremities physiologic responses to endothelial injury. and the trunk. Later, more severe vascular damage results in frank hemorrhage at the center of the maculopapule, producing a petechia Clinical Manifestations Early in the illness, when medical attention that does not disappear upon compression (Fig. 211-1). This sequence usually is first sought, RMSF is difficult to distinguish from many self-of events is sometimes delayed or aborted by effective treatment. limiting viral

1	upon compression (Fig. 211-1). This sequence usually is first sought, RMSF is difficult to distinguish from many self-of events is sometimes delayed or aborted by effective treatment. limiting viral illnesses. Fever, headache, malaise, myalgia, nausea, vom-However, the rash is a variable manifestation, appearing on day 6 iting, and anorexia are the most common symptoms during the first or later in 20% of cases and not appearing at all in 9–16% of cases. 3 days. The patient becomes progressively more ill as vascular Petechiae occur in 41–59% of cases, appearing on or after day 6 in 74% infection and injury advance. In one large series, only one-third of of cases that manifest a rash. Involvement of the palms and soles, often patients were diagnosed with presumptive RMSF early in the clinical considered diagnostically important, usually develops relatively late in

1	FIGuRE 211-1 Top: Petechial lesions of Rocky Mountain spotted fever on the lower legs and soles of a young, previously healthy patient. Bottom: Close-up of lesions from the same patient. (Photos courtesy of Dr. Lindsey Baden; with permission.) the course (after day 5 in 43% of cases) and does not develop at all in 18–64% of cases. Hypovolemia leads to prerenal azotemia and (in 17% of cases) hypotension. Infection of the pulmonary microcirculation leads to noncardiogenic pulmonary edema; 12% of patients have severe respiratory disease, and 8% require mechanical ventilation. Cardiac involvement manifests as dysrhythmia in 7–16% of cases.

1	Besides respiratory failure, central nervous system (CNS) involvement is the other important determinant of the outcome of RMSF. Encephalitis, presenting as confusion or lethargy, is apparent in 26–28% of cases. Progressively severe encephalitis manifests as stupor or delirium in 21–26% of cases, ataxia in 18%, coma in 10%, and seizures in 8%. Numerous focal neurologic deficits have been reported. Meningoencephalitis results in cerebrospinal fluid (CSF) pleocytosis in 34–38% of cases; usually there are 10–100 cells/μL and a mononuclear predominance, but occasionally there are >100 cells/μL and a polymorphonuclear predominance. The CSF protein concentration is increased in 30–35% of cases, but the CSF glucose concentration is usually normal.

1	Renal failure, often reversible with rehydration, is caused by acute tubular necrosis in severe cases with shock. Hepatic injury with increased serum aminotransferase concentrations (38% of cases) is due to focal death of individual hepatocytes without hepatic failure. Jaundice is recognized in 9% of cases and an elevated serum bilirubin concentration in 18–30%. Life-threatening bleeding is rare. Anemia develops in 30% of cases and is severe enough to require transfusions in 11%. Blood is detected in the stool or vomitus of 10% of patients, and death has followed massive upper-gastrointestinal hemorrhage.

1	Other characteristic clinical laboratory findings include increased plasma levels of proteins of the acute-phase response (C-reactive protein, fibrinogen, ferritin, and others), hypoalbuminemia, and hyponatremia (in 56% of cases) due to the appropriate secretion of antidiuretic hormone in response to the hypovolemic state. Myositis occurs occasionally, with marked elevations in serum creatine kinase levels and multifocal rhabdomyonecrosis. Ocular involvement includes conjunctivitis in 30% of cases and retinal vein engorgement, flame hemorrhages, arterial occlusion, and papilledema with normal CSF pressure in some instances.

1	In untreated cases, the patient usually dies 8–15 days after onset. A rare presentation, fulminant RMSF, is fatal within 5 days after onset. This fulminant presentation is seen most often in male black patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency and may be related to an undefined effect of hemolysis on the rickettsial infection. Although survivors of RMSF usually return to their previous state of health, permanent sequelae, including neurologic deficits and gangrene necessitating amputation of extremities, may follow severe illness. Diagnosis The diagnosis of RMSF during the acute stage is more difficult than is generally appreciated. The most important epidemiologic factor is a history of exposure to a potentially tick-infested environment within the 14 days preceding disease onset during a season of possible tick activity. However, only 60% of patients actually recall being bitten by a tick during the incubation period.

1	The differential diagnosis for early clinical manifestations of RMSF (fever, headache, and myalgia without a rash) includes influenza, enteroviral infection, infectious mononucleosis, viral hepatitis, leptospirosis, typhoid fever, gram-negative or gram-positive bacterial sepsis, HME, HGA, murine typhus, sylvatic flying-squirrel typhus, and rickettsialpox. Enterocolitis may be suggested by nausea, vomiting, and abdominal pain; prominence of abdominal tenderness has resulted in exploratory laparotomy. CNS involvement can masquerade as bacterial or viral meningoencephalitis. Cough, pulmonary signs, and chest radiographic opacities can lead to a diagnostic consideration of bronchitis or pneumonia.

1	At presentation during the first 3 days of illness, only 3% of patients exhibit the classic triad of fever, rash, and history of tick exposure. When a rash appears, a diagnosis of RMSF should be considered. However, many illnesses considered in the differential diagnosis also can be associated with a rash, including rubeola, rubella, meningococcemia, disseminated gonococcal infection, secondary syphilis, toxic shock syndrome, drug hypersensitivity, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, Kawasaki syndrome, and immune complex vasculitis. Conversely, any person in an endemic area with a provisional diagnosis of one of the above illnesses could have RMSF. Thus, if a viral infection is suspected during RMSF season in an endemic area, it should always be kept in mind that RMSF can mimic viral infection early in the course; if the illness worsens over the next couple of days after initial presentation, the patient should return for reevaluation.

1	The most common serologic test for confirmation of the diagnosis is the indirect immunofluorescence assay. Not until 7–10 days after onset is a diagnostic titer of ≥64 usually detectable. The sensitivity and specificity of the indirect immunofluorescence IgG assay are 89–100% and 99–100%, respectively. It is important to understand that serologic tests for RMSF are usually negative at the time of presentation for medical care and that treatment should not be delayed while a positive serologic result is awaited.

1	The only diagnostic test that has proven useful during the acute illness is immunohistologic examination of a cutaneous biopsy sample from a rash lesion for R. rickettsii. Examination of a 3-mm punch biopsy from such a lesion is 70% sensitive and 100% specific. The sensitivity of polymerase chain reaction (PCR) amplification and detection of R. rickettsii DNA in peripheral blood is improving. However, although rickettsiae are present in large quantities in heavily infected foci of endothelial cells, there are relatively low quantities in the circulation. Cultivation of rickettsiae in cell culture is feasible but is seldom undertaken because of biohazard concerns. The recent dramatic increase in the reported incidence of RMSF correlates with the use of single-titer spotted fever–group cross-reactive enzyme immunoassay serology. Few cases are specifically determined to be caused by R. rickettsii. Currently, many febrile persons who do not have RMSF present with cross-reactive

1	cross-reactive enzyme immunoassay serology. Few cases are specifically determined to be caused by R. rickettsii. Currently, many febrile persons who do not have RMSF present with cross-reactive antibodies, possibly because of previous exposure to the highly prevalent spotted fever–group rickettsia R. amblyommii.

1	The drug of choice for the treatment of both children and adults with RMSF is doxycycline, except when the patient is pregnant or allergic to this drug (see below). Because of the severity of RMSF, immediate empirical administration of doxycycline should be strongly considered for any patient with a consistent clinical presentation in the appropriate epidemiologic setting. Doxycycline is administered orally (or, in the presence of coma or vomiting, intravenously) at 200 mg/d in two divided doses. For children with suspected RMSF, up to five courses of doxycycline may be administered with minimal risk of dental staining. Other regimens include oral tetracycline (25–50 mg/kg per day) in four divided doses. Treatment with chloramphenicol, a less effective drug, is advised only for patients who are pregnant or allergic to doxycycline. The antirickettsial drug should be administered until the patient has been afebrile and improving clinically for 2–3 days. β-Lactam antibiotics,

1	patients who are pregnant or allergic to doxycycline. The antirickettsial drug should be administered until the patient has been afebrile and improving clinically for 2–3 days. β-Lactam antibiotics, erythromycin, and aminoglycosides have no role in the treatment of RMSF, and sulfa-containing drugs are associated with more adverse outcomes than no treatment at all. There is little clinical experience with fluoroquinolones, clarithromycin, and azithromycin, which are not recommended. The most seriously ill patients are managed in intensive care units, with careful administration of fluids to achieve optimal tissue perfusion without precipitating noncardiogenic pulmonary edema. In some severely ill patients, hypoxemia requires intubation and mechanical ventilation; oliguric or anuric acute renal failure requires hemodialysis; seizures necessitate the use of antiseizure medication; anemia or severe hemorrhage necessitates transfusions of packed red blood cells; or bleeding with severe

1	renal failure requires hemodialysis; seizures necessitate the use of antiseizure medication; anemia or severe hemorrhage necessitates transfusions of packed red blood cells; or bleeding with severe thrombocytopenia requires platelet transfusions. Heparin is not a useful component of treatment, and there is no evidence that glucocorticoids affect outcome.

1	Prevention Avoidance of tick bites is the only available preventive approach. Use of protective clothing and tick repellents, inspection of the body once or twice a day, and removal of ticks before they inoculate rickettsiae reduce the risk of infection. Prophylactic doxycycline treatment of tick bites has no proven role in preventing RMSF. MEDITERRANEAN SPOTTED FEVER (BOuTONNEuSE FEVER), AFRICAN TICKBITE FEVER, AND OTHER TICK-BORNE SPOTTED FEVERS Epidemiology R. conorii is prevalent in southern Europe,

1	MEDITERRANEAN SPOTTED FEVER (BOuTONNEuSE FEVER), AFRICAN TICKBITE FEVER, AND OTHER TICK-BORNE SPOTTED FEVERS Epidemiology R. conorii is prevalent in southern Europe, Africa, and southwestern and south-central Asia. Regional names for the disease caused by this organism include Mediterranean spotted fever, Kenya tick typhus, Indian tick typhus, Israeli spotted fever, and Astrakhan spotted fever. The disease is characterized by high fever, rash, and—in most geographic locales—an inoculation eschar (tâche noire) at the site of the tick bite. A severe form of the disease (mortality rate, 50%) occurs in patients with diabetes, alcoholism, or heart failure.

1	African tick-bite fever, caused by R. africae, occurs in rural areas of sub-Saharan Africa and in the Caribbean islands and is transmitted by Amblyomma hebraeum and A. variegatum ticks. The average incubation period is 4–10 days. The mild illness consists of headache, fever, eschar, and regional lymphadenopathy. Amblyomma ticks often feed in groups, with the consequent development of multiple eschars. Rash may be vesicular, sparse, or absent altogether. Because of tourism in sub-Saharan Africa, African tick-bite fever is the rickettsiosis most frequently imported into Europe and North America. A similar disease caused by the closely related species R. parkeri is transmitted by 1157 A. maculatum in the United States and by A. triste in South America. R. japonica causes Japanese spotted fever, which also occurs in Korea. Similar diseases in northern Asia are caused by R. sibirica and

1	R. japonica causes Japanese spotted fever, which also occurs in Korea. Similar diseases in northern Asia are caused by R. sibirica and R. heilongjiangensis. Queensland tick typhus due to R. australis is transmitted by Ixodes holocyclus ticks. Flinders Island spotted fever, found on the island for which it is named as well as in Tasmania, mainland Australia, and Asia, is caused by R. honei. In Europe, patients infected with R. slovaca after a wintertime Dermacentor tick bite manifest an afebrile illness with an eschar (usually on the scalp) and painful regional lymphadenopathy.

1	Diagnosis Diagnosis of these tick-borne spotted fevers is based on clinical and epidemiologic findings and is confirmed by serology, immunohistochemical demonstration of rickettsiae in skin biopsy specimens, cell-culture isolation of rickettsiae, or PCR of skin biopsy, eschar, or blood samples. Serologic diagnosis detects antibodies to antigens shared among spotted fever–group rickettsiae, hindering identification of the etiologic species. In an endemic area, a possible diagnosis of rickettsial spotted fevers should be considered when patients present with fever, rash, and/or a skin lesion consisting of a black necrotic area or a crust surrounded by erythema.

1	Successful therapeutic agents include doxycycline (100 mg bid orally for 1–5 days) and chloramphenicol (500 mg qid orally for 7–10 days). Pregnant patients may be treated with josamycin (3 g/d orally for 5 days). Data on the efficacy of treatment of mildly ill children with clarithromycin or azithromycin should not be extrapolated to adults or to patients with moderate or severe illness. R. akari infects mice and their mites (Liponyssoides sanguineus), which maintain the organisms by transovarial transmission. Epidemiology Rickettsialpox is recognized principally in New York City, but cases have also been reported in other urban and rural locations in the United States and in Ukraine, Croatia, Mexico, and Turkey. Investigation of eschars suspected of representing bioterrorism-associated cutaneous anthrax revealed that rickettsialpox occurs more frequently than previously realized.

1	Clinical Manifestations A papule forms at the site of the mite’s feeding, develops a central vesicle, and becomes a 1to 2.5-cm painless black crusted eschar surrounded by an erythematous halo (Fig. 211-2). Enlargement of the regional lymph nodes draining the eschar suggests initial lymphogenous spread. After an incubation period of FIGuRE 211-2 Eschar at the site of the mite bite in a patient with rickettsialpox. (Reprinted from A Krusell et al: Emerg Infect Dis 8:727, 2002. Photo obtained by Dr. Kenneth Kaye.)

1	FIGuRE 211-2 Eschar at the site of the mite bite in a patient with rickettsialpox. (Reprinted from A Krusell et al: Emerg Infect Dis 8:727, 2002. Photo obtained by Dr. Kenneth Kaye.) FIGuRE 211-3 Top: Papulovesicular lesions on the trunk of the patient with rickettsialpox shown in Fig. 211-2. Bottom: Close-up of lesions from the same patient. (Reprinted from A Krusell et al: Emerg Infect Dis 8:727, 2002. Photos obtained by Dr. Kenneth Kaye.) 10–17 days, during which the eschar and regional lymphadenopathy frequently go unnoticed, disease onset is marked by malaise, chills, fever, headache, and myalgia. A macular rash appears 2–6 days after onset and usually evolves sequentially into papules, vesicles, and crusts that heal without scarring (Fig. 211-3); in some cases, the rash remains macular or maculopapular. Some patients develop nausea, vomiting, abdominal pain, cough, conjunctivitis, or photophobia. Without treatment, fever lasts 6–10 days.

1	Diagnosis and Treatment Clinical, epidemiologic, and convalescent serologic data establish the diagnosis of a spotted fever–group rickettsiosis that is seldom pursued further. Doxycycline is the drug of choice for treatment. An emerging rickettsiosis caused by R. felis occurs worldwide. Maintained transovarially in the geographically widespread cat flea Ctenocephalides felis, the infection has been described as moderately severe, with fever, rash, and headache as well as CNS, gastrointestinal, and pulmonary symptoms.

1	EPIDEMIC (LOuSE-BORNE) TYPHuS Epidemiology The human body louse (Pediculus humanus corporis) lives in clothing under poor hygienic conditions and usually in impoverished cold areas. Lice acquire R. prowazekii when they ingest blood from a rickettsemic patient. The rickettsiae multiply in the louse’s mid-gut epithelial cells and are shed in its feces. The infected louse leaves a febrile person and deposits infected feces on its subsequent host during its blood meal; the patient autoinoculates the organisms by scratching. The louse is killed by the rickettsiae and does not pass R. prowazekii to its offspring. ters. An outbreak involved 100,000 people in refugee camps in Burundi in 1997. A small focus was documented in Russia in 1998; sporadic cases were reported from Algeria, and frequent outbreaks occurred in Peru. Eastern flying squirrels (Glaucomys volans) and their lice and fleas maintain R. prowazekii in a zoonotic cycle.

1	Brill-Zinsser disease is a recrudescent illness occurring years after acute epidemic typhus, probably as a result of waning immunity. R. prowazekii remains latent for years; its reactivation results in sporadic cases of disease in louse-free populations or in epidemics in louse-infested populations. Rickettsiae are potential agents of bioterrorism (Chap. 261e). Infections with R. prowazekii and R. rickettsii have high case–fatality ratios. These organisms cause difficult-to-diagnose diseases and are highly infectious when inhaled as aerosols. Organisms resistant to tetracycline or chloramphenicol have been developed in the laboratory.

1	Clinical Manifestations After an incubation period of ~1–2 weeks, the onset of illness is abrupt, with prostration, severe headache, and fever rising rapidly to 38.8°–40.0°C (102°–104°F). Cough is prominent, developing in 70% of patients. Myalgias are usually severe. A rash begins on the upper trunk, usually on the fifth day, and then becomes generalized, involving the entire body except the face, palms, and soles. Initially, this rash is macular; without treatment, it becomes maculopapular, petechial, and confluent. The rash often goes undetected in black skin; 60% of African patients have spotless epidemic typhus. Photophobia, with considerable conjunctival injection and eye pain, is common. The tongue may be dry, brown, and furred. Confusion and coma are common. Skin necrosis and gangrene of the digits as well as interstitial pneumonia may occur in severe cases. Untreated disease is fatal in 7–40% of cases, with outcome depending primarily on the condition of the host. Patients

1	of the digits as well as interstitial pneumonia may occur in severe cases. Untreated disease is fatal in 7–40% of cases, with outcome depending primarily on the condition of the host. Patients with untreated infections develop renal insufficiency and multiorgan involvement in which neurologic manifestations are frequently prominent. Overall, 12% of patients with epidemic typhus have neurologic involvement. Infection associated with North American flying squirrels is a milder illness; whether this milder disease is due to host factors (e.g., better health status) or attenuated virulence is unknown.

1	Diagnosis and Treatment Epidemic typhus is sometimes misdiagnosed as typhoid fever in tropical countries (Chap. 190). The means even for serologic studies are often unavailable in settings of louse-borne typhus. Epidemics can be recognized by the serologic or immunohistochemical diagnosis of a single case or by detection of R. prowazekii in a louse found on a patient. Doxycycline (200 mg/d, given in two divided doses) is administered orally or—if the patient is comatose or vomiting—intravenously. Although under epidemic conditions a single 200-mg oral dose is effective, treatment is generally continued until 2–3 days after defervescence. Pregnant patients should be evaluated individually and treated with chloramphenicol early in pregnancy or, if necessary, with doxycycline late in pregnancy. Prevention Prevention of epidemic typhus involves control of body lice. Clothes should be changed regularly, and insecticides should be used every 6 weeks to control the louse population.

1	Prevention Prevention of epidemic typhus involves control of body lice. Clothes should be changed regularly, and insecticides should be used every 6 weeks to control the louse population. ENDEMIC MuRINE TYPHuS Epidemiology R. typhi is maintained in mammalian host/flea cycles, with rats (Rattus rattus and R. norvegicus) and the Oriental rat flea (Xenopsylla cheopis) as the classic zoonotic niche. Fleas acquire R. typhi from rickettsemic rats and carry the organism throughout their life span. Nonimmune rats and humans are infected when rickettsia-laden flea feces contaminate pruritic bite lesions; less frequently, the flea bite transmits the organisms. Transmission can also occur via inhalation of aerosolized rickettsiae from flea feces. Infected rats appear healthy, although they are rickettsemic for ~2 weeks.

1	Murine typhus occurs mainly in Texas and southern California, where the classic rat/flea cycle is absent and an opossum/cat flea (C. felis) cycle is prominent. Globally, endemic typhus occurs mainly in warm (often coastal) areas throughout the tropics and subtropics, where it is highly prevalent though often unrecognized. The incidence peaks from April through June in southern Texas and during the warm months of summer and early fall in other geographic locations. Patients seldom recall exposure to fleas, although exposure to animals such as cats, opossums, and rats is reported in nearly 40% of cases. Clinical Manifestations The incubation period of experimental murine typhus averages 11 days (range, 8–16 days). Headache, myalgia, arthralgia, nausea, and malaise develop 1–3 days before onset of chills and fever. Nearly all patients experience nausea and vomiting early in the illness.

1	The duration of untreated illness averages 12 days (range, 9–18 days). Rash is present in only 13% of patients at presentation for medical care (usually ~4 days after onset of fever), appearing an average of 2 days later in half of the remaining patients and never appearing in the others. The initial macular rash is often detected by careful inspection of the axilla or the inner surface of the arm. Subsequently, the rash becomes maculopapular, involving the trunk more often than the extremities; it is seldom petechial and rarely involves the face, palms, or soles. A rash is detected in only 20% of patients with darkly pigmented skin.

1	Pulmonary involvement is frequently prominent; 35% of patients have a hacking, nonproductive cough, and 23% of patients who undergo chest radiography have pulmonary densities due to interstitial pneumonia, pulmonary edema, and pleural effusions. Bibasilar rales are the most common pulmonary sign. Less common clinical manifestations include abdominal pain, confusion, stupor, seizures, ataxia, coma, and jaundice. Clinical laboratory studies frequently reveal anemia and leukopenia early in the course, leukocytosis late in the course, thrombocytopenia, hyponatremia, hypoalbuminemia, mildly increased serum hepatic aminotransferases, and prerenal azotemia. Complications can include respiratory failure, hematemesis, cerebral hemorrhage, and hemolysis. Severe illness necessitates the admission of 10% of hospitalized patients to an intensive care unit. Greater severity is generally associated with old age, underlying disease, and treatment with a sulfonamide; the case-fatality rate is 1%. In a

1	10% of hospitalized patients to an intensive care unit. Greater severity is generally associated with old age, underlying disease, and treatment with a sulfonamide; the case-fatality rate is 1%. In a study of children with murine typhus, 50% suffered only nocturnal fevers, feeling well enough for active daytime play.

1	Diagnosis and Treatment Serologic studies of acuteand convalescent-phase sera can provide a diagnosis, and an immunohistochemical method for identification of typhus group-specific antigens in biopsy samples has been developed. Cultivation and PCR are used only infrequently and are not widely available. Nevertheless, most patients are treated empirically with doxycycline (100 mg bid orally for 7–15 days) on the basis of clinical suspicion. Ciprofloxacin provides an alternative if doxycycline is contraindicated.

1	SCRuB TYPHuS Epidemiology O. tsutsugamushi differs substantially from Rickettsia species both genetically and in cell wall composition (i.e., it lacks lipopolysaccharide). O. tsutsugamushi is maintained by transovarial transmission in trombiculid mites. After hatching, infected larval mites (chiggers, the only stage that feeds on a host) inoculate organisms into the skin. Infected chiggers are particularly likely to be found in areas of heavy scrub vegetation during the wet season, when mites lay eggs. Scrub typhus is endemic and reemerging in eastern and south ern Asia, northern Australia, and islands of the western Pacific and Indian Oceans. Infections are prevalent in these regions; in some areas, >3% of the population is infected or reinfected each month. Immunity wanes over 1–3 years, and the organism exhibits remarkable antigenic diversity.

1	Clinical Manifestations Illness varies from mild and self-limiting to fatal. After an incubation period of 6–21 days, onset is characterized by fever, headache, myalgia, cough, and gastrointestinal symptoms. Some patients recover spontaneously after a few days. The classic case description includes an eschar where the chigger has fed, regional lymphadenopathy, and a maculopapular rash—signs that are seldom seen in indigenous patients. Fewer than 50% of Westerners develop an eschar, and fewer than 40% develop a rash (on day 4–6 of illness). Severe 1159 cases typically manifest with encephalitis and interstitial pneumonia due to vascular injury. The case-fatality rate for untreated classic cases is 7% but would probably be lower if all mild cases were diagnosed.

1	Diagnosis and Treatment Serologic assays (indirect fluorescent antibody, indirect immunoperoxidase, and enzyme immunoassays) are the mainstays of laboratory diagnosis. PCR amplification of Orientia genes from eschars and blood also is effective. Patients are treated with doxycycline (100 mg bid orally for 7–15 days), azithromycin (500 mg orally for 3 days), or chloramphenicol (500 mg qid orally for 7–15 days). Some cases of scrub typhus in Thailand are caused by strains that have high doxycycline or chloramphenicol minimal inhibitory concentrations (MICs) but that are susceptible to azithromycin and rifampin.

1	Ehrlichioses are acute febrile infections caused by members of the family Anaplasmataceae, which is made up of obligately intracellular organisms of five genera: Ehrlichia, Anaplasma, Wolbachia, Candidatus Neoehrlichia, and Neorickettsia. The bacteria reside in vertebrate reservoirs and target vacuoles of hematopoietic cells (Fig. 211-4). Three Ehrlichia species and one Anaplasma species are transmitted by ticks to humans and cause infection that can be severe and prevalent. E. chaffeensis, the agent of HME, and an E. muris–like agent (EMLA) infect predominantly mononuclear phagocytes; E. ewingii and A. phagocytophilum infect neutrophils. Infection with Candidatus Neoehrlichia mikurensis is less well characterized, but the agent has been identified in human blood neutrophils.

1	Ehrlichia, Candidatus Neoehrlichia, and Anaplasma are maintained by horizontal tick-mammal-tick transmission, and humans are only inadvertently infected. Wolbachiae are associated with human filariasis, since they are important for filarial viability and pathogenicity; antibiotic treatment targeting wolbachiae is a strategy for filariasis control. Neorickettsiae parasitize flukes (trematodes) that in turn parasitize aquatic snails, fish, and insects. Only a single human neorickettsiosis has been described: sennetsu fever, an infectious mononucleosis–like illness that was first identified in 1953 and is associated with the ingestion of raw fish containing N. sennetsu–infected flukes. HuMAN MONOCYTOTROPIC EHRLICHIOSIS Epidemiology More than 8404 cases of E. chaffeensis infection had been reported to the Centers for Disease Control and Prevention (CDC) as of April 2013. However, active prospective surveillance has documented an incidence as high as 414 cases per 100,000 population

1	FIGuRE 211-4 Peripheral-blood smear from a patient with human granulocytotropic anaplasmosis. A neutrophil contains two morulae (vacuoles filled with A. phagocytophilum). (Photo courtesy of Dr. J. Stephen Dumler.) 1160 in some U.S. regions. Most E. chaffeensis infections are identified in the south-central, southeastern, and mid-Atlantic states, but cases have also been recognized in California and New York. All stages of the Lone Star tick (A. americanum) feed on white-tailed deer—a major reservoir. Dogs and coyotes also serve as reservoirs and often lack clinical signs. Tick bites and exposures are frequently reported by patients in rural areas, especially in May through July. The median age of HME patients is 52 years; however, severe and fatal infections in children also are well recognized. Of patients with HME, 60% are male. E. chaffeensis has been detected in South America, Africa, and Asia.

1	Clinical Manifestations E. chaffeensis disseminates hematogenously from the dermal blood pool created by the feeding tick. After a median incubation period of 8 days, illness develops. Clinical manifestations are undifferentiated and include fever (96% of cases), headache (72%), myalgia (68%), and malaise (77%). Less frequently observed are nausea, vomiting, and diarrhea (25–57%); cough (28%); rash (26% overall, 6% at presentation); and confusion (20%). HME can be severe: 49% of patients with documented cases are hospitalized, and ~2% die. Severe manifestations include a toxic shock–like or septic shock–like syndrome, adult respiratory distress syndrome, cardiac failure, hepatitis, meningoencephalitis, hemorrhage, and—in immunocompromised patients—overwhelming ehrlichial infection. Laboratory findings are valuable in the differential diagnosis of HME; 61% of patients have leukopenia (initially lymphopenia, later neutropenia), 73% have thrombocytopenia, and 84% have elevated serum

1	findings are valuable in the differential diagnosis of HME; 61% of patients have leukopenia (initially lymphopenia, later neutropenia), 73% have thrombocytopenia, and 84% have elevated serum levels of hepatic aminotransferases. Despite low blood cell counts, the bone marrow is hypercellular, and noncaseating granulomas can be present. Vasculitis is not a component of HME.

1	Diagnosis HME can be fatal. Early empirical antibiotic therapy based on clinical diagnosis diminishes adverse outcomes. This diagnosis is suggested by fever with a known tick exposure during the preceding 3 weeks, thrombocytopenia and/or leukopenia, and increased serum aminotransferase levels. Morulae are demonstrated in <10% of peripheral-blood smears. HME can be confirmed during active infection by PCR amplification of E. chaffeensis nucleic acids in blood obtained before the start of doxycycline therapy. Retrospective serodiagnosis requires a consistent clinical picture and a fourfold increase in E. chaffeensis antibody titer to ≥64 in paired sera obtained ~3 weeks apart. Separate specific diagnostic tests are necessary for HME and HGA.

1	Ehrlichia ewingii, originally a neutrophil pathogen causing fever and lameness in dogs, resembles E. chaffeensis in its tick vector (A. americanum) and vertebrate reservoirs (white-tailed deer and dogs). An E. muris–like agent (EMLA) has been discovered and identified as the cause of human infections in Wisconsin and Minnesota. E. ewingii and EMLA illnesses are similar to but less severe than HME. Many cases occur in immunocompromised patients. No specific serologic diagnostic tests for ewingii or EMLA ehrlichiosis are readily available.

1	Candidatus Neoehrlichia mikurensis, a bacterium in a phy logenetic clade between Ehrlichia and Anaplasma, was origi nally identified in Ixodes ricinus ticks from the Netherlands and in mice and Ixodes ovatus ticks from Japan. By means of broad-range 16S rRNA gene amplification and sequence analysis, this organism was identified as the cause of severe and sometimes prolonged febrile illnesses in European immunocompromised patients with tick bites or exposures and in Chinese patients with a mild febrile illness after being bitten by Ixodes persulcatus and Haemaphysalis concinna ticks. The clinical presentation is similar to those of HME and HGA. Specific diagnostic methods have been developed but are not widely available.

1	Doxycycline is effective for HME as well as for ewingii and EMLA ehrlichioses; the use of this drug in Candidatus N. mikurensis infection is associated with disease resolution. Therapy with doxycycline (100 mg given PO or IV twice daily) or tetracycline (250–500 mg given PO every 6 h) lowers hospitalization rates and shortens fever duration. E. chaffeensis is not susceptible to chloramphenicol in vitro, and the use of this drug is controversial. While a few reports document E. chaffeensis persistence in humans, this finding is rare; most infections are cured by short courses of doxycycline (continuing for 3–5 days after defervescence). Although poorly studied, rifampin may be suitable when doxycycline is contraindicated.

1	HME, ewingii ehrlichiosis, EMLA infection, and Candidatus N. mikurensis infection can be prevented by the avoidance of ticks in endemic areas. The use of protective clothing and tick repellents, careful post-exposure tick searches, and prompt removal of attached ticks probably diminish infection risk. Epidemiology As of April 2013, 10,181 cases of HGA had been reported to the CDC, most in the upper midwestern and northeastern United States; the geographic distribution is similar to that for Lyme disease because of the shared I. scapularis tick vector. White-footed mice, squirrels, and white-tailed deer in the United States and red deer in Europe are natural reservoirs for A. phagocytophilum. HGA incidence peaks in May through July, but the disease can occur throughout the year with exposure to Ixodes ticks. HGA often affects males (59%) and older persons (median age, 51 years).

1	Clinical Manifestations Seroprevalence rates are high in endemic regions; thus it seems likely that most individuals develop subclinical infections. The incubation period for HGA is 4–8 days, after which the disease manifests as fever (75–100% of cases), myalgia (77%), headache (82%), and malaise (97%). A minority of patients develop nausea, vomiting, or diarrhea (22–39%); cough (27%); or confusion (17%). Rash (6%) is almost invariably concurrent erythema migrans attributable to Lyme disease. Most patients develop thrombocytopenia (75%) and/or leukopenia (55%) with increased serum hepatic aminotransferase levels (83%).

1	Severe complications occur most often in the elderly and include adult respiratory distress syndrome, a toxic shock–like syndrome, and life-threatening opportunistic infections. Meningoencephalitis is rarely documented with HGA, but brachial plexopathy, cranial nerve involvement, and demyelinating polyneuropathy are reported. For HGA, 7% of patients require intensive care, and the case-fatality rate is 0.6%. Neither vasculitis nor granulomas are components of HGA. While co-infections with Borrelia burgdorferi and Babesia microti (transmitted by the same tick vector[s]) occur, there is little evidence of comorbidity or persistence. HGA is rarely acquired via transfusion.

1	Diagnosis HGA should be included in the differential diagnosis of influenza-like illnesses during seasons with Ixodes tick activity (May through December), especially with known tick bite or exposure. Concurrent thrombocytopenia, leukopenia, or elevated serum levels of alanine or aspartate aminotransferase further increase the likelihood of HGA. Many HGA patients develop Lyme disease antibodies in the absence of clinical findings consistent with that diagnosis. Thus, HGA should be considered in the differential diagnosis of atypical severe Lyme disease presentations. Peripheral-blood film examination for neutrophil morulae can yield a diagnosis in 20–75% of infections. PCR testing of blood from patients with active disease before doxycycline therapy is sensitive and specific. Serodiagnosis is retrospective, requiring a fourfold increase in A. phagocytophilum antibody titer (to ≥160) in paired serum samples obtained 1 month apart. Since seroprevalence is high in some regions, a single

1	is retrospective, requiring a fourfold increase in A. phagocytophilum antibody titer (to ≥160) in paired serum samples obtained 1 month apart. Since seroprevalence is high in some regions, a single acute-phase titer should not be used for diagnosis.

1	No prospective studies of therapy for HGA have been conducted. However, doxycycline (100 mg PO twice daily) is effective. Rifampin therapy is associated with improvement of HGA in pregnant women and children. Most treated patients defervesce within 24–48 h. Prevention HGA prevention requires tick avoidance. Transmission can be documented as few as 4 h after a tick bite.

1	Prevention HGA prevention requires tick avoidance. Transmission can be documented as few as 4 h after a tick bite. The agent of Q fever is Coxiella burnetii, a small intracellular prokaryote that only recently was grown in cell-free medium. C. burnetii, a pleomorphic coccobacillus with a gram-negative cell wall, survives in harsh environments; it escapes intracellular killing in macrophages by inhibiting the final step in phagosome maturation (cathepsin fusion) and has adapted to the acidic phagolysosome by producing superoxide dismutase. Infection with C. burnetii induces a range of immunomodulatory responses, from immunosuppression in chronic Q fever to the production of autoantibodies, particularly those to smooth muscle and cardiac muscle.

1	Q fever encompasses two broad clinical syndromes: acute and chronic infection. The host’s immune response (rather than the particular strain) most likely determines whether chronic Q fever develops. C. burnetii survives in monocytes from patients with chronic Q fever but not in monocytes from patients with acute Q fever or from uninfected subjects. Impairment of the bactericidal activity of the C. burnetii–infected monocyte is associated with overproduction of interleukin 10. The CD4+/CD8+ ratio is decreased in Q fever endocarditis. Very few organisms and a strong cellular response are observed in patients with acute Q fever, while many organisms and a moderate cellular response occur in chronic Q fever. Immune control of C. burnetii is T cell–dependent, but 80–90% of bone marrow aspirates obtained years after recovery from Q fever contain C. burnetii DNA. C. burnetii’s ready multiplication within trophoblasts accounts for the high concentrations it can reach in the placenta.

1	Epidemiology Q fever is a zoonosis. The primary sources of human infection are infected cattle, sheep, and goats. However, cats, rabbits, pigeons, and dogs also serve as sources for transmission of C. burnetii to humans. The wildlife reservoir is extensive and includes ticks, coyotes, gray foxes, skunks, raccoons, rabbits, deer, mice, bears, birds, and opossums. In female animals C. burnetii localizes to the uterus and mammary glands. Infection is reactivated during pregnancy and after radiotherapy in mouse models. High concentrations of C. burnetii are found in the placenta. At the time of parturition, the bacteria are released into the air, and infection follows inhalation of aerosolized organisms by a susceptible host. Windstorms can generate C. burnetii aerosols months after soil contamination during parturition. Individuals up to 18 km from the source have been infected. Because it is easily dispersed as an aerosol, C. burnetii is a potential agent of bioterrorism (Chap. 261e),

1	during parturition. Individuals up to 18 km from the source have been infected. Because it is easily dispersed as an aerosol, C. burnetii is a potential agent of bioterrorism (Chap. 261e), with a high infectivity rate and pneumonia as the major manifestation.

1	Determining the source of an outbreak of Q fever can be challenging. An outbreak of Q fever at a horse-boarding ranch in Colorado in 2005 was due to spread of infection from two herds of goats that had been acquired by the owners. PCR testing confirmed the presence of C. burnetii in the soil and among the goats. Of 138 persons who lived within 1 mile of the ranch and who were also tested, 11 (8%) had evidence of C. burnetii infection, and 8 of these 11 individuals had no direct contact with the ranch.

1	Persons at risk for Q fever include abattoir workers, veterinarians, farmers, and other individuals who have contact with infected animals (particularly newborn animals) or products of conception. The organism is shed in milk for weeks to months after parturition. The ingestion of contaminated milk in some geographic areas probably represents a major route of transmission to humans. A recent outbreak of Q fever associated with ingestion of raw milk confirms the oral route of transmission. In rare instances, person-to-person transmission follows labor and childbirth in an infected woman, autopsy of an infected individual, or blood transfusion. Some evidence suggests that C. bur-1161 netii can be sexually transmitted among humans. Crushing an infected tick between the fingers has resulted in Q fever; the implication is that percutaneous transmission can occur.

1	Infections due to C. burnetii occur in most geographic locations except New Zealand and Antarctica. Thus Q fever can be associ ated with travel. The number of reported cases of Q fever in the United States ranges from 28 to 54 per year. More than 70% of these cases occur in males, and April, May, and June are the most common months for acquisition. Q fever continues to be common in Australia, with 30 cases per 1 million population per year. Cases among abattoir workers in Australia declined dramatically as a result of a vaccination program. An outbreak of Q fever began in the Netherlands in 2007, and by 2010 more than 4000 cases had been reported. Pneumonia was a common manifestation in this outbreak. The outbreak was due to a combination of high-density goat farming in areas abutting large urban populations and environmental factors. Farms where spread did not occur had high vegetation densities and lower groundwater concentrations.

1	The primary manifestations of acute Q fever differ geographically (e.g., pneumonia in Nova Scotia and granulomatous hepatitis in Marseille). These differences could reflect the route of infection (i.e., ingestion of contaminated milk for hepatitis and inhalation of contaminated aerosols for pneumonia) or strain differences. In the Netherlands outbreak, sequelae of infection in pregnant women were rare; this was not the case among pregnant women elsewhere. Young age seems to be protective against disease caused by C. burnetii. In a large outbreak in Switzerland, symptomatic infection occurred five times more often among persons >15 years of age than among younger individuals. In many outbreaks, men are affected more commonly than women; the proposed explanation is that female hormones are partially protective.

1	Clinical Manifestations • acUte Q fever The symptoms of acute Q fever are nonspecific; common among them are fever, extreme fatigue, photophobia, and severe headache that is frequently retro-orbital. Other symptoms include chills, sweats, nausea, vomiting, and diarrhea, each occurring in 5–20% of cases. Cough develops in about half of patients with Q fever pneumonia. Neurologic manifestations of acute Q fever are uncommon; however, in one outbreak in the United Kingdom, 23% of 102 patients had neurologic signs and symptoms as the major manifestation. A nonspecific rash may be evident in 4–18% of patients. The WBC count is usually normal. Thrombocytopenia occurs in ~25% of patients, and reactive thrombocytosis (with platelet counts exceeding 106/μL) frequently develops during recovery. Chest radiography can show opacities similar to those seen in pneumonia caused by other pathogens, but multiple rounded opacities in patients in endemic areas suggest a diagnosis of Q fever pneumonia.

1	Acute Q fever occasionally complicates pregnancy. In one series, it resulted in premature birth in 35% of cases and in abortion or neonatal death in 43%. Neonatal death (previous or current) and lower infant birth weight are three times more likely among women seropositive for C. burnetii. After the usual incubation period of 3–30 days, 1070 patients with acute Q fever in southern France presented with hepatitis (40%), both pneumonia and hepatitis (20%), pneumonia (17%), isolated fever (14%), CNS involvement (2%), and pericarditis or myocarditis (1%). Acalculous cholecystitis, pancreatitis, lymphadenopathy, spontaneous rupture of the spleen, transient hypoplastic anemia, bone marrow necrosis, hemolytic anemia, histiocytic hemophagocytosis, optic neuritis, and erythema nodosum were less common manifestations.

1	post–Q fever fatigUe syndrome Prolonged fatigue can follow Q fever and can be accompanied by a constellation of symptoms including headaches, sweats, arthralgia, myalgias, blurred vision, muscle fasciculations, and enlarged and painful lymph nodes. Long-term persistence of a noninfective, nonbiodegraded complex of Coxiella cell components, with its antigens and specific lipopolysaccharide, has been detected in the affected persons. Patients who develop this syndrome have a higher frequency of carriage of HLA-DRB1*11 and of the 2/2 genotype of the interferon γ intron 1 microsatellite.

1	1162 cHronic Q fever Chronic Q fever almost always implies endocarditis and usually occurs in patients with previous valvular heart disease, immunosuppression, or chronic renal insufficiency. Fever is usually absent or low grade. Valvular vegetations are detected in only 12% of patients by transthoracic echocardiography, but the rate of detection is higher (21–50%) with transesophageal echocardiography. The vegetations in chronic Q fever endocarditis differ from those in bacterial endocarditis, manifesting as endothelium-covered nodules on the valves. A high index of suspicion is necessary for timely diagnosis. Patients with chronic Q fever are often ill for >1 year before the diagnosis is made. The disease should be suspected in all patients with culture-negative endocarditis. In addition, all patients with valvular heart disease and an unexplained purpuric eruption, renal insufficiency, stroke, and/ or progressive heart failure should be tested for C. burnetii infection. Patients

1	all patients with valvular heart disease and an unexplained purpuric eruption, renal insufficiency, stroke, and/ or progressive heart failure should be tested for C. burnetii infection. Patients with chronic Q fever have hepatomegaly and/or splenomegaly, which—in combination with rheumatoid factor, elevated erythrocyte sedimentation rate, high C-reactive protein level, and/or increased γ-globulin concentrations (up to 60–70 g/L)—suggests this diagnosis. Other manifestations of chronic Q fever include infection of vascular prostheses, aneurysms, and bone as well as chronic sternal wound infection. Unusual manifestations include chronic thrombocytopenia, mixed cryoglobulinemia, and livedo reticularis.

1	Diagnosis Isolation of C. burnetii from buffy-coat blood samples or tissue specimens by a shell-vial technique is easy but requires a biosafety level 3 laboratory. PCR detects C. burnetii DNA in tissue specimens, including paraffin-embedded samples. Serology is the most commonly used diagnostic tool. Indirect immunofluorescence is sensitive and specific and is the method of choice. Rheumatoid factor should be adsorbed from the specimen before testing. With chronic infection, the titer to phase I antigen is usually much higher than that to phase II antigen (i.e., C. burnetii that has truncated lipopolysaccharide associated with gene deletions during laboratory passages), and the diagnosis should not be based on serology alone. Rather, the entire clinical setting must be taken into consideration. An anti–phase I IgG titer of ≥6400 would be considered a major criterion for the diagnosis of chronic Q fever, while a titer of ≥800 but ≤6400 would be a minor criterion. In acute Q fever, a

1	An anti–phase I IgG titer of ≥6400 would be considered a major criterion for the diagnosis of chronic Q fever, while a titer of ≥800 but ≤6400 would be a minor criterion. In acute Q fever, a fourfold rise in titer can be demonstrated between acuteand convalescent-phase serum samples.

1	Fluorodeoxyglucose positron emission tomography combined with CT (FDG-PET/CT) can be useful because it can detect not only valvular infection but also intravascular infection elsewhere as well as osteomyelitis. Treatment of acute Q fever with doxycycline (100 mg twice daily for 14 days) is usually successful. Quinolones also are effective. When Q fever is diagnosed during pregnancy, treatment with trimethoprimsulfamethoxazole (TMP-SMX) is recommended for the duration of the pregnancy. One study showed no intrauterine fetal deaths and substantial reduction of obstetric complications in a group of Q fever patients treated with TMP-SMX.

1	The treatment of chronic Q fever is difficult and requires careful follow-up. Addition of hydroxychloroquine (to alkalinize the phagolysosome) renders doxycycline bactericidal against C. burnetii, and this combination is currently the favored regimen. Treatment with doxycycline (100 mg bid) and hydroxychloroquine (200 mg tid; plasma concentration maintained at 0.8–1.2 μg/mL) for 18 months is superior to a regimen of doxycycline and ofloxacin. Among 21 patients who received doxycycline and hydroxychloroquine, 1 died of a surgical complication, 2 were still being treated at the end of the study, 1 was still being evaluated, and 17 were cured. The mean duration of treatment was 31 months. In the ofloxacin and doxycycline group of 14 patients, 1 had died, 1 was still being treated, 7 had relapsed, and 5 had been cured by the end of the study. Optimal management of Q fever endocarditis entails determining the MIC of doxycycline for the patient’s isolate and measuring serum doxycycline

1	relapsed, and 5 had been cured by the end of the study. Optimal management of Q fever endocarditis entails determining the MIC of doxycycline for the patient’s isolate and measuring serum doxycycline levels. A serum level–to–doxycycline MIC ratio of ≥1 is associated with a rapid decline in phase I antibodies with the doxycycline-hydroxychloroquine regimen. Patients treated with this regimen must be advised about photosensitivity and retinal toxicity risks. The doxycycline-hydroxychloroquine regimen was successful in one patient with HIV infection and Q fever endocarditis. The Jarisch-Herxheimer reaction occasionally complicates the treatment of chronic Q fever. Treatment of C. burnetii–infected aortic aneurysms is the same as that for Q fever endocarditis. Surgical intervention is often required.

1	If doxycycline-hydroxychloroquine cannot be used, the regimen chosen should include at least two antibiotics active against C. burnetii. Rifampin (300 mg once daily) combined with doxycycline (100 mg twice daily) or ciprofloxacin (750 mg twice daily) has been used successfully. The management of patients with Q fever endocarditis is complex and should preferably be undertaken by individuals with experience in managing this illness. Monitoring of antibody titers on a quarterly basis is an essential part of the management of these patients. Thus the laboratory should be contacted and asked to save all serum samples from such patients so that the current sample can be run with the previous one. There is incomplete agreement on the antibody titer at which therapy can be stopped. However, it is reasonable to discontinue treatment if IgG antibody levels have decreased by fourfold at 1 year, if IgM antibody to phase II has disappeared, and if the patient is clinically stable.

1	Patients with acute Q fever and lesions of native heart valves (e.g., bicuspid aortic valve), prosthetic valves, or prosthetic intravascular material should undergo serologic monitoring every 4 months for 2 years. If the phase I IgG titer is >800, further investigation is warranted. Some authorities recommend that patients with valvulopathy and acute Q fever receive doxycycline and hydroxychloroquine to prevent chronic Q fever. For women who exhibit a serologic profile of chronic Q fever after childbirth, hydroxychloroquine and doxycycline should be given for 1 year. Interferon γ was successful in the treatment of a 3-year-old boy with prolonged fever, abdominal pain, and thrombocytopenia due to

1	Interferon γ was successful in the treatment of a 3-year-old boy with prolonged fever, abdominal pain, and thrombocytopenia due to C. burnetii that had not been eradicated with conventional antibiotic therapy. Many patients with granulomatous hepatitis due to Q fever have a prolonged febrile illness that is unresponsive to antibiotics. For these individuals, treatment with prednisone (0.5 mg/kg) has resulted in defervescence within 2–15 days. After defervescence, the glucocorticoid dose is tapered over the next month. Prevention A whole-cell vaccine (Q-Vax) licensed in Australia effectively prevents Q fever in abattoir workers. Before administration of the vaccine, skin testing with intradermal diluted C. burnetii vaccine is performed, serologic testing is undertaken, and a history of possible Q fever is sought. Vaccine is given only to patients with no history of Q fever and negative results in serologic and skin testing.

1	Good animal-husbandry practices are important in preventing widespread contamination of the environment by C. burnetii. These practices include isolating aborting animals for up to 14 days, raising feed bunks to prevent contamination of feed by excreta, destroying aborted materials (by burning and burying fetal membranes and stillborn animals), and wearing masks and gloves when handling aborted materials. Vaccination of sheep and goats and a culling program were effective in the Netherlands outbreak. Only seronegative pregnant animals should be used in research settings, and only seronegative animals should be permitted in petting zoos. During an outbreak of Q fever and for 4 weeks after it ceases, blood donations should not be accepted from individuals who live in the affected area. The contributions of Didier Raoult, MD, to this chapter in previous editions are gratefully acknowledged. Genuine second attacks of M. pneumoniae pneumonia have been 1163

1	The contributions of Didier Raoult, MD, to this chapter in previous editions are gratefully acknowledged. Genuine second attacks of M. pneumoniae pneumonia have been 1163 Infections due to Mycoplasmas reported infrequently. R. Doug Hardy EPIDEMIOLOGY Mycoplasmas are prokaryotes of the class Mollicutes. Their size (150–350 nm) is closer to that of viruses than to that of bacteria. Unlike viruses, however, mycoplasmas grow in cell-free culture media; in fact, they are the smallest organisms capable of independent replication.

1	The entire genomes of many Mycoplasma species have been sequenced and have been found to be among the smallest of all prokaryotic genomes. Sequencing information for these genomes has helped define the minimal set of genes necessary for cellular life. The absence of genes related to the synthesis of amino acids, fatty acid metabolism, and cholesterol dictates the mycoplasmas’ parasitic or saprophytic dependence on a host for exogenous nutrients and necessitates the use of complex fastidious media to culture these organisms. Mycoplasmas lack a cell wall and are bound only by a cell membrane. The absence of a cell wall explains the inactivity of β-lactam antibiotics (penicillins and cephalosporins) against infections caused by these organisms.

1	At least 13 Mycoplasma species, two Acholeplasma species, and two Ureaplasma species have been isolated from humans. Most of these species are thought to be normal inhabitants of oral and urogenital mucous membranes. Only four species—M. pneumoniae, M. hominis, U. urealyticum, and U. parvum—have been shown conclusively to be pathogenic in immunocompetent humans. M. pneumoniae primarily infects the respiratory tract, while M. hominis, U. urealyticum, and U. parvum are associated with a variety of genitourinary tract disorders and neonatal infections. Some data indicate that M. genitalium may be a cause of disease in humans. Other mycoplasmas may cause disease in immunocompromised persons.

1	M. pneumoniae is generally thought to act as an extracellular pathogen. Although the organism has been shown to exist and replicate within human cells, it is not known whether these intracellular events contribute to the pathogenesis of disease. M. pneumoniae attaches to ciliated respiratory epithelial cells by means of a complex terminal organelle at the tip of one end of the organism. Cytoadherence is mediated by interactive adhesins and accessory proteins clustered on this organelle. After extracellular attachment, M. pneumoniae causes injury to host respiratory tissue. The mechanism of injury is thought to be mediated by the production of hydrogen peroxide and of a recently identified ADP-ribosylating and vacuolating cytotoxin of M. pneumoniae that has many similarities to pertussis toxin. Because mycoplasmas lack a cell wall, they also lack cell wall–derived stimulators of the innate immune system, such as lipopolysaccharide, lipoteichoic acid, and murein (peptidoglycan)

1	toxin. Because mycoplasmas lack a cell wall, they also lack cell wall–derived stimulators of the innate immune system, such as lipopolysaccharide, lipoteichoic acid, and murein (peptidoglycan) fragments. However, lipoproteins from the mycoplasmal cell membrane appear to have inflammatory properties, probably acting through Toll-like receptors (primarily TLR2) on macrophages and other cells. Lung biopsy specimens from patients with

1	M. pneumoniae respiratory tract infection reveal an inflammatory process involving the trachea, bronchioles, and peribronchial tissue, with a monocytic infiltrate coinciding with a luminal exudate of polymorphonuclear leukocytes. Experimental evidence indicates that innate immunity provides most of the host’s defense against mycoplasmal infection in the lungs, whereas cellular immunity may actually play an immunopathogenic role, exacerbating mycoplasmal lung disease. Humoral immunity appears to provide protection against dissemination of M. pneumoniae infection; patients with humoral immunodeficiencies do not have more severe lung disease than do immunocompetent patients in the early stages of infection but more often develop disseminated infection resulting in syndromes such as arthritis, meningitis, and osteomyelitis. The immunity that follows severe M. pneumoniae infections is more protective and longer-lasting than that following mild infections.

1	M. pneumoniae infection occurs worldwide. It is likely that the incidence of upper respiratory illness due to M. pneumoniae is up to 20 times that of pneumonia caused by this organism. Infection is spread from one person to another by respiratory droplets expectorated during coughing and results in clinically apparent disease in an estimated 80% of cases. The incubation period for M. pneumoniae is 2–4 weeks; therefore, the time-course of infection in a specific population may be several weeks long. Intrafamilial attack rates are as high as 84% among children and 41% among adults. Outbreaks of M. pneumoniae illness often occur in institutional settings such as military bases, boarding schools, and summer camps. Infections tend to be endemic, with sporadic epidemics every 4–7 years. There is no seasonal pattern.

1	Most significantly, M. pneumoniae is a major cause of community-acquired respiratory illness in both children and adults and is often grouped with Chlamydia pneumoniae and Legionella species as being among the most important bacterial causes of “atypical” community-acquired pneumonia. For community-acquired pneumonia in adults, M. pneumoniae is the most frequently detected “atypical” organism. Analysis of 13 studies of community-acquired pneumonia published since 1995 (which included 6207 ambulatory and hospitalized adults) showed that the overall prevalence of M. pneumoniae was 22.7%; by comparison, the prevalence of C. pneumoniae was 11.7%, and that of Legionella species was 4.6%. M. pneumoniae pneumonia is also referred to as Eaton agent pneumonia (the organism having first been isolated in the early 1940s by Monroe Eaton), primary atypical pneumonia, and “walking” pneumonia.

1	CLINICAL MANIFESTATIONS upper Respiratory Tract Infections and Pneumonia Acute M. pneumoniae infections generally manifest as pharyngitis, tracheobronchitis, reactive airway disease/wheezing, or a nonspecific upper respiratory syndrome. Little evidence supports the commonly held belief that this organism is an important cause of otitis media, with or without bullous myringitis. Pneumonia develops in 3–13% of infected individuals; its onset is usually gradual, occurring over several days, but may be more abrupt. Although Mycoplasma pneumonia may begin with a sore throat, the most common presenting symptom is cough. The cough is typically nonproductive, but some patients produce sputum. Headache, malaise, chills, and fever are noted in the majority of patients.

1	On physical examination, wheezes or rales are detected in ∼80% of patients with M. pneumoniae pneumonia. In many patients, however, pneumonia can be diagnosed only by chest radiography. The most common radiographic pattern is that of peribronchial pneumonia with thickened bronchial markings, streaks of interstitial infiltration, and areas of subsegmental atelectasis. Segmental or lobar consolidation is not uncommon. While clinically evident pleural effusions are infrequent, lateral decubitus views reveal that up to 20% of patients have pleural effusions.

1	Overall, the clinical presentation of pneumonia in an individual patient is not useful for differentiating M. pneumoniae pneumonia from other types of community-acquired pneumonia. The possibility of M. pneumoniae infection deserves particular consideration when community-acquired pneumonia fails to respond to treatment with a penicillin or a cephalosporin—antibiotics that are ineffective against mycoplasmas. Symptoms usually resolve within 2–3 weeks after the onset of illness. Although M. pneumoniae pneumonia is generally self-limited, appropriate antimicrobial therapy significantly shortens the duration of clinical illness. Infection uncommonly results in critical illness and only rarely in death. In some patients, long-term recurrent wheezing or reactive airway disease may follow the resolution of acute pneumonia. The significance of chronic infection, especially as it relates to asthma, is an area of active investigation.

1	Extrapulmonary Manifestations An array of extrapulmonary manifestations may develop during M. pneumoniae infection. The most significant are neurologic, dermatologic, cardiac, rheumatologic, and

1	Infections Due to Mycoplasmas 1164 hematologic in nature. Extrapulmonary manifestations can be a result of disseminated infection, especially in patients with humoral immunodeficiencies (e.g., septic arthritis); postinfectious autoimmune phenomena (e.g., Guillain-Barré syndrome); or possibly ADPribosylating toxin. Overall, these manifestations are uncommon, given the frequency of M. pneumoniae infection. Notably, many patients with extrapulmonary M. pneumoniae disease do not have respiratory disease. Skin eruptions described with M. pneumoniae infection include erythematous (macular or maculopapular), vesicular, bullous, petechial, and urticarial rashes. In some reports, 17% of patients with M. pneumoniae pneumonia have had an exanthem. Erythema multiforme major (Stevens-Johnson syndrome) is the most clinically significant skin eruption associated with M. pneumoniae infection; it appears to occur more commonly with M. pneumoniae than with other infectious agents. A wide spectrum of

1	is the most clinically significant skin eruption associated with M. pneumoniae infection; it appears to occur more commonly with M. pneumoniae than with other infectious agents. A wide spectrum of neurologic manifestations has been reported with M. pneumoniae infection. The most common are meningoencephalitis, encephalitis, Guillain-Barré syndrome, and aseptic meningitis. M. pneumoniae has been implicated as a likely etiologic agent in 5–7% of cases of encephalitis. Other neurologic manifestations may include cranial neuropathy, acute psychosis, cerebellar ataxia, acute demyelinating encephalomyelitis, cerebrovascular thromboembolic events, and transverse myelitis. Hematologic manifestations of M. pneumoniae infection include hemolytic anemia, aplastic anemia, cold agglutinins, disseminated intravascular coagulation, and hypercoagulopathy. When anemia does occur, it generally develops in the second or third week of illness. In addition, hepatitis, glomerulonephritis, pancreatitis,

1	intravascular coagulation, and hypercoagulopathy. When anemia does occur, it generally develops in the second or third week of illness. In addition, hepatitis, glomerulonephritis, pancreatitis, myocarditis, pericarditis, rhabdomyolysis, and arthritis (septic and reactive) have been convincingly ascribed to M. pneumoniae infection. Septic arthritis has been described most commonly in hypogammaglobulinemic patients.

1	Clinical findings, nonmicrobiologic laboratory tests, and chest radiography are not useful for differentiating M. pneumoniae pneumonia from other types of community-acquired pneumonia. In addition, since M. pneumoniae lacks a cell wall, it is not visible on Gram’s stain. Although of historical interest, the measurement of cold agglutinin titers is no longer recommended for the diagnosis of M. pneumoniae infection because the findings are nonspecific and assays specific for M. pneumoniae are now available.

1	M. pneumoniae are now available. Acute M. pneumoniae infection can be diagnosed by polymerase chain reaction (PCR) detection of the organism in respiratory tract secretions or by isolation of the organism in culture (Table 212-1). Oropharyngeal, nasopharyngeal, and pulmonary specimens are all acceptable for diagnosing M. pneumoniae pneumonia. Other bodily fluids, such cerebrospinal fluid, are acceptable for extrapulmonary infection. M. pneumoniae culture (which requires special media) is not recommended for routine diagnosis because the organism may take weeks to grow and is often difficult to isolate from clinical specimens. In contrast, PCR allows rapid, specific diagnosis earlier in the course of clinical illness. The diagnosis can also be established by serologic tests for IgM and IgG antibodies to M. pneumoniae in paired (acuteand convalescent-phase) serum samples; enzyme-linked immunoassay is

1	The diagnosis can also be established by serologic tests for IgM and IgG antibodies to M. pneumoniae in paired (acuteand convalescent-phase) serum samples; enzyme-linked immunoassay is Test Sensitivity, % Specificity, % aA combination of PCR and serology is suggested for routine diagnosis. If macrolide resistance is suspected, M. pneumoniae culture may prove useful, providing an isolate for susceptibility testing. bAcuteand convalescent-phase serum samples are recommended. Abbreviation: PCR, polymerase chain reaction. the recommended serologic method. An acute-phase sample alone is not adequate for diagnosis, as antibodies to M. pneumoniae may not develop until 2 weeks into the illness; therefore, it is important to test paired samples. In addition, IgM antibody to M. pneumoniae can persist for up to 1 year after acute infection. Thus its presence may indicate recent rather than acute infection.

1	The combination of PCR of respiratory tract secretions and serologic testing constitutes the most sensitive and rapid approach to the diagnosis of M. pneumoniae infection.

1	Although in the majority of untreated cases symptoms resolve within 2–3 weeks without significant associated morbidity, M. pneumoniae pneumonia can be a serious illness that responds to appropriate antimicrobial therapy (Table 212-2). Randomized, double-blind, placebo-controlled trials in adults have demonstrated that antimicrobial treatment significantly decreases the duration of fever, cough, malaise, hospitalization, and radiologic abnormalities in M. pneumoniae pneumonia. Treatment options for acute M. pneumoniae infection include macrolides (e.g., oral azithromycin, 500 mg on day 1, then 250 mg/d on days 2–5), tetracyclines (e.g., oral doxycycline, 100 mg twice daily for 10–14 days), and respiratory fluoroquinolones. However, ciprofloxacin and ofloxacin are not recommended because of their high minimal inhibitory concentrations against M. pneumoniae isolates and their poor performance in experimental studies. A 10to 14-day course of quinolone therapy appears adequate.

1	In Japan and China, very high levels (up to ≥90%) of M. pneumoniae resistance to macrolides have been reported. In Europe and to a lesser degree in the United States, macrolide-resistant M. pneumoniae is emerging. In investigated outbreaks of respiratory illness due to M. pneumoniae in the United States, macrolide resistance has been reported in 8–27% of isolates. Clinical studies have demonstrated that, when treated with macrolides, patients with community-acquired pneumonia due to macrolideresistant M. pneumoniae experience a significantly longer duration of symptoms than do patients infected with macrolide-sensitive organisms; thus macrolide resistance in M. pneumoniae does appear to have clinical significance. If macrolide resistance is prominent in a particular geographic locale or is suspected, then a nonmacrolide antibiotic should be considered for treatment; in addition, culture of

1	M. pneumoniae may prove useful in these instances, providing an isolate for susceptibility testing. Clinical observations and experimental data suggest that the addition of glucocorticoids to an antibiotic regimen may be of value for the treatment of severe or refractory M. pneumoniae pneumonia. However, relevant clinical experience is limited. Even though appropriate antibiotic therapy significantly reduces the duration of respiratory illness, it does not appear to shorten the duration of detection of M. pneumoniae by culture or PCR; therefore, a test of cure or eradication is not suggested. The roles of antimicrobial drugs, glucocorticoids, and IV immunoglobulin in the treatment of neurologic disease due to M. pneumoniae remain unknown. aAntimicrobial resistance has been reported in mycoplasmas, as described in the text. uROGENITAL MYCOPLASMAS (SEE ALSO CHAP. 163)

1	aAntimicrobial resistance has been reported in mycoplasmas, as described in the text. uROGENITAL MYCOPLASMAS (SEE ALSO CHAP. 163) M. hominis, M. genitalium, U. urealyticum, and U. parvum can cause urogenital tract disease. The significance of isolation of these organisms in a variety of other syndromes is unknown and in some cases is being investigated. M. fermentans has not been shown convincingly to cause human disease.

1	While urogenital mycoplasmas may be transmitted to a fetus during passage through a colonized birth canal, sexual contact is the major mode of transmission, and the risk of colonization increases dramatically with increasing numbers of sexual partners. In asymptomatic women, these mycoplasmas may be found throughout the lower urogenital tract. The vagina yields the largest number of organisms; next most densely colonized are the periurethral area and the cervix. Ureaplasmas are isolated less often from urine than from the cervix, but M. hominis is found with approximately the same frequency at these two sites. Ureaplasmas are isolated from the vagina of 40–80% of sexually active, asymptomatic women and M. hominis from 21–70%. The two microorganisms are found concurrently in 31–60% of women. In men, colonization with each organism is less prevalent. Mycoplasmas have been isolated from urine, semen, and the distal urethra of asymptomatic men.

1	CLINICAL MANIFESTATIONS urethritis, Pyelonephritis, and urinary Calculi In many episodes of Chlamydia-negative nongonococcal urethritis, ureaplasmas may be the causative agent. These organisms may also cause chronic voiding symptoms in women. The common presence of ureaplasmas in the urethra of asymptomatic men suggests either that only certain serovars are pathogenic or that predisposing factors, such as lack of immunity, must exist in persons who develop symptomatic infection. Alternatively, disease may develop only upon initial exposure to urea-plasmas. Ureaplasmas have been implicated in epididymitis. M. genitalium also appears to cause urethritis. M. genitalium and ureaplasmas do not have a known role in prostatitis. M. hominis does not appear to play a primary etiologic role in urethritis, epididymitis, or prostatitis. Evidence suggests that M. hominis causes up to 5% of cases of acute pyelonephritis. Ureaplasmas have not been associated with this disease.

1	Evidence suggests that M. hominis causes up to 5% of cases of acute pyelonephritis. Ureaplasmas have not been associated with this disease. Ureaplasmas play a limited role in the production of urinary calculi. The frequency with which ureaplasmas reach the kidney, the predisposing factors that allow them to do so, and the relative frequency of urinary tract calculi induced by this organism (compared with other organisms) are not known. Pelvic Inflammatory Disease M. hominis can cause pelvic inflammatory disease. In most episodes, M. hominis occurs as part of a polymicrobial infection, but the organism may play an independent role in a limited number of cases. Some data also support an association of M. genitalium with pelvic inflammatory disease. Ureaplasmas are not thought to cause pelvic inflammatory disease.

1	Postpartum and Postabortal Infection Studies implicate M. hominis as the primary pathogen in ∼5–10% of women who have postpartum or postabortal fever; ureaplasmas have been implicated to a lesser degree. These infections are generally self-limited; however, if symptoms persist, specific antimicrobial therapy should be given. Ureaplasmas also appear to play a role in occasional postcesarean wound infections.

1	Non-urogenital Infection In rare instances, M. hominis causes non-urogenital infections, such as brain abscess, wound infection, poststernotomy mediastinitis, endocarditis, and neonatal meningitis. These infections are most common among immunocompromised and hypogammaglobulinemic patients. Ureaplasmas and M. hominis can cause septic arthritis in immunodeficient patients. Ureaplasmas probably cause neonatal pneumonitis; their significant role in the development of bronchopulmonary dysplasia—the chronic lung disease of premature infants—has been documented in a number of studies. It is unclear whether ureaplasmas and M. hominis cause infertility, spontaneous abortion, premature labor, low birth weight, 1165 or chorioamnionitis.

1	Culture and PCR are both appropriate methods for the isolation of urogenital mycoplasmas. Culture of these organisms, however, requires special techniques and media that generally are available only at larger medical centers and reference laboratories. Serologic testing is not recommended for the clinical diagnosis of urogenital Mycoplasma infections.

1	Because colonization with urogenital mycoplasmas is common, it appears at present that their isolation from the urogenital tract in the absence of disease generally does not warrant treatment. Macrolides and doxycycline are considered the antimicrobial agents of choice for Ureaplasma infections (Table 212-2). Ureaplasma resistance to macrolides, doxycycline, quinolones, and chloramphenicol has been reported. M. hominis is resistant to macrolides. Doxycycline is generally the drug of choice for M. hominis infections, although resistance has been reported. Clindamycin is generally active against M. hominis. Quinolones are active in vitro against M. hominis. For M. genitalium, the agent of choice appears to be azithromycin; treatment failures have been reported with other macrolides as well as with quinolones. Charlotte A. Gaydos, Thomas C. Quinn Chlamydiae are obligate intracellular bacteria that cause a wide variety of diseases in humans and animals.

1	Charlotte A. Gaydos, Thomas C. Quinn Chlamydiae are obligate intracellular bacteria that cause a wide variety of diseases in humans and animals. The chlamydiae were originally classified as four species in the genus Chlamydia: C. trachomatis, C. pneumoniae, C. psittaci, and C. pecorum (the last species being found in ruminants). The C. psittaci group has been separated into three species: C. psittaci, C. felis, and C. abortus. The mouse pneumonitis strain (MoPn) is now classified as C. muridarum, and the guinea pig inclusion conjunctivitis strain (GPIC) is now designated C. caviae.

1	C. trachomatis is divided into two biovars: trachoma and LGV (lymphogranuloma venereum). The trachoma biovar causes two major types of disease in humans: ocular trachoma, the leading infectious cause of preventable blindness in the developing world; and urogenital infections, which are sexually or neonatally transmitted. The 18 serovars of C. trachomatis fall into three groups: the trachoma serovars A, B, Ba, and C; the oculogenital serovars D–K; and the LGV serovars L1–L3. Serovars can be distinguished by serologic typing with monoclonal antibodies or by molecular gene typing. However, serovar identification usually is not important clinically since the antibiotic susceptibility pattern is the same for all three groups. The one exception applies when LGV is suspected on clinical grounds; in this situation, serovar determination is important because a longer treatment duration is required for LGV strains. BIOLOGY, GROWTH CYCLE, AND PATHOGENESIS

1	BIOLOGY, GROWTH CYCLE, AND PATHOGENESIS During their intracellular growth, chlamydiae produce characteristic intracytoplasmic inclusions that can be visualized by direct fluorescent antibody (DFA) or Giemsa staining of infected clinical material, such as conjunctival scrapings or cervical or urethral epithelial 1166 cells. Chlamydiae are nonmotile, gram-negative, obligate intracellular bacteria that replicate within the cytoplasm of host cells, forming the characteristic membrane-bound inclusions that are the basis for some diagnostic tests. Originally considered to be large viruses, chlamydiae differ from viruses in possessing RNA and DNA as well as a cell wall that is quite similar in structure to the cell wall of typical gram-negative bacteria. However, chlamydiae lack peptidoglycan; their structural integrity depends on disulfide binding of outer-membrane proteins.

1	Among the defining characteristics of chlamydiae is a unique growth cycle that involves alternation between two highly specialized morphologic forms (Figs. 213-1 and 213-2): the elementary body (EB), which is the infectious form and is specifically adapted for extracellular survival, and the metabolically active and replicating reticulate body (RB), which is not infectious, is adapted for an intracellular environment, and does not survive well outside the host cell. The biphasic growth cycle begins with attachment of the EB (diameter, 0.25–0.35 μm) at specific sites on the surface of the host cell. The EB enters the cell through a process similar to receptor-mediated endocytosis and resides in an inclusion, where the entire growth cycle is completed. The chlamydiae prevent phagosome-lysosome fusion. The inclusion membrane is modified by insertion of chlamydial antigens. Once the EB has entered the cell, it reorganizes into an RB, which is larger (0.5–1 μm) and contains more RNA. After

1	fusion. The inclusion membrane is modified by insertion of chlamydial antigens. Once the EB has entered the cell, it reorganizes into an RB, which is larger (0.5–1 μm) and contains more RNA. After ~8 h, the RB starts to divide by binary fission. The intracytoplasmic, membrane-bound inclusion body containing the RBs increases in size as the RBs multiply. Approximately 18–24 h after infection of the cell, these RBs begin to become EBs by a reorganization or condensation process that is poorly understood. After rupture of the inclusion body, the EBs are released to initiate another cycle of infection.

1	Chlamydiae are susceptible to many broad-spectrum antibiotics and possess a number of enzymes, but they have a very restricted FIGuRE 213-1 Chlamydial intracellular inclusions filled with smaller dense elementary bodies and larger reticulate bodies. (Reprinted with permission from WE Stamm: Chlamydial infections, in Harrison’s Principles of Internal Medicine, 17th ed, AS Fauci et al [eds]. New York, McGraw-Hill, 2008, p 1070.) 2.Initial inclusions 1.Uptake of 3. Fusion of inclusions; chlamydial EBs appearance of RBs 4.Multiplication of RBs; enlargement of inclusion 8. Return to normal removal 7. Persistence associated with IFN-˜ exposure; large aberrant RBs

1	FIGuRE 213-2 Chlamydial life cycle. EBs, elementary bodies; RBs, reticulate bodies; IFN-γ, interferon γ. (Reprinted with permission from WE Stamm: Chlamydial infections, in Harrison’s Principles of Internal Medicine, 17th ed, AS Fauci et al [eds]. New York, McGraw-Hill, 2008, p 1071.) 5. Conversion of RBs to EBs 6. Release of EBs cycle with IFN-˜ metabolic capacity. None of these metabolic reactions results in the production of energy. Chlamydiae have thus been considered to be energy parasites that use the ATP produced by the host cell for their own metabolic functions. Many aspects of chlamydial molecular biology are not well understood, but the sequencing of several chlamydial genomes and new proteomics research have provided researchers with many relevant tools for elucidating the biology of the life cycle.

1	Genital infections are mostly caused by C. trachomatis serovars D–K, with serovars D, E, and F involved most often. Molecular typing of the major outer-membrane protein gene (omp1) from which serovar differences arise has been used to demonstrate that polymorphisms can occur in isolates from patients who are exposed frequently to multiple infections, while less variation is observed in isolates from less sexually active populations. Polymorphisms in the major outer-membrane protein may provide antigenic variation, and the different forms allow persistence in the community because immunity to one is not protective against the others.

1	The trachoma biovar is essentially a parasite of squamocolumnar epithelial cells; the LGV biovar is more invasive and involves lymphoid cells. As is typical of chlamydiae, C. trachomatis strains are capable of causing chronic, clinically inapparent, asymptomatic infections. Because the duration of the chlamydial growth cycle is ~48–72 h, the incubation period of sexually transmitted chlamydial infections is relatively long—generally 1–3 weeks. C. trachomatis causes cell death as a result of its replicative cycle and can induce cell damage whenever it persists. However, few toxic effects are demonstrated, and cell death because of chlamydial replication is not sufficient to account for disease manifestations, the majority of which are due to immunopathologic mechanisms or nonspecific host responses to the organism or its byproducts.

1	In recent years, the entire genomes of various chlamydial species have been sequenced, the field of proteomics has become established, host innate immunity has been more precisely delineated, and innovative host cell–chlamydial interaction studies have been conducted. As a result, many insights have been gained into how chlamydiae adapt and replicate in their intracellular environment and produce disease. These insights into pathogenesis include information on the regulation of gene expression, protein localization, the type III secretion system, the roles of CD4+ and CD8+ T lymphocytes in the host response, and T lymphocyte trafficking.

1	The chlamydial heat-shock protein, which shares antigenic epitopes with similar proteins of other bacteria and with human heat-shock protein, may sensitize the host, and repeated infections may cause host cell damage. Persistent or recurrent chlamydial infections are associated with fibrosis, scarring, and complications following simple epithelial infections. A common endpoint of these late consequences is scarring of mucous membranes. Genital complications can lead to pelvic inflammatory disease (PID) and its late consequences of infertility, ectopic pregnancy, and chronic pelvic pain, while ocular infections may lead to blinding trachoma. High levels of antibody to human heat-shock protein have been associated with tubal factor infertility and ectopic pregnancy. Without adequate therapy, chlamydial infections may persist for several years, although symptoms—if present—usually abate.

1	The pathogenic mechanisms of C. pneumoniae have yet to be completely elucidated. The same is true for C. psittaci, except that this agent infects cells very efficiently and causes disease that may reflect direct cytopathic effects. GENITAL INFECTIONS Spectrum Although chlamydiae cause a number of human diseases, localized lower genital tract infections caused by C. trachomatis and the sequelae of such infections are the most important in terms of medical and economic impact. Oculogenital infections due to

1	C. trachomatis serovars D–K are transmitted during sexual contact or from mother to baby during childbirth and are associated with many syndromes, including cervicitis, salpingitis, acute urethral syndrome, endometritis, ectopic pregnancy, infertility, and PID in female patients; urethritis, proctitis, and epididymitis in male patients; and conjunctivitis and pneumonia in infants. Women bear the greatest burden of morbidity because of the serious sequelae of these infections. Untreated infections lead to PID, and multiple episodes of PID can lead to tubal factor infertility and chronic pelvic pain. Studies estimate that up to 80–90% of women and >50% of men with C. trachomatis genital infections lack symptoms; other patients have very mild symptoms. Thus a large reservoir of infected persons continues to transmit infection to sexual partners.

1	As their designations reflect, the LGV serovars (L1, L2, and L3) cause LGV, an invasive sexually transmitted disease (STD) characterized by acute lymphadenitis with bubo formation and/or acute hemorrhagic proctitis (see “Lymphogranuloma Venereum,” below). Epidemiology C. trachomatis genital infections are global in distribution. The World Health Organization (WHO) esti mated in 2008 that >106.4 million cases occur annually worldwide. This figure makes chlamydial infection the most prevalent sexually transmitted bacterial infection in the world. The associated morbidity is substantial, and economic costs are high.

1	In the United States, chlamydial infections are the most commonly reported of all infectious diseases. In 2012, 1.3 million cases were reported to the U.S. Centers for Disease Control and Prevention (CDC); however, the CDC estimates that 2–3 million new cases occur per year, with substantial underreporting due to lack of screening in some populations. Rates of infection have increased every year; higher rates among women than among men reflect the focus on expansion of screening programs for women during the past 20 years, the use of increasingly sensitive diagnostic tests, an increased emphasis on case reporting, and improvements in the information systems used for reporting. The CDC and other professional organizations recommend annual screening of all sexually active women ≤25 years of age as well as rescreening of previously infected individuals at 3 months. Young women have the highest infection rates; in 2012, the figures were 3416.5 and 3722.5 cases per 100,000 population at

1	of age as well as rescreening of previously infected individuals at 3 months. Young women have the highest infection rates; in 2012, the figures were 3416.5 and 3722.5 cases per 100,000 population at 15–19 and 20–24 years of age, respectively. Age-specific rates among men, while much lower than those among women, were highest in the 20-to 24-year-old age group, at 1343.3 cases per 100,000. In 2012, rates 1167 increased for all racial and ethnic groups, with the highest rates among African Americans. For example, the rate of chlamydial infection among African-American girls 15–19 years of age was 7507.1 cases per 100,000—almost six times the rate among Caucasian girls in the same age group (1301.5/100,000). The rate among African-American women 20–24 years old was 4.8 times the rate among Caucasian women in the same age group. Similar racial disparities in reported rates of chlamydial infection exist among men. For boys 15–19 years of age, the rate among African Americans was 11.1

1	Caucasian women in the same age group. Similar racial disparities in reported rates of chlamydial infection exist among men. For boys 15–19 years of age, the rate among African Americans was 11.1 times the rate among Caucasians. The rate among Native Americans/Alaska Natives was more than four times the rate among Caucasians (648.3), and the rate among Latinos (383.6) was two times higher than that among Caucasians. These disparities are important reflections of health inequities in the United States.

1	The above statistics are based on case reporting. Studies based on screening surveys estimate that the U.S. prevalence of C. trachomatis cervical infection is 5% among asymptomatic female college students and prenatal patients, >10% among women seen in family planning clinics, and >20% among women seen in STD clinics. The prevalence of genital C. trachomatis infections varies substantially by geographic locale, with the highest rates in the southeastern United States. However, asymptomatic infections have been detected in >8–10% of young female military recruits from all parts of the country. The prevalence of C. trachomatis in the cervix of pregnant women is 5–10 times higher than that of Neisseria gonorrhoeae. The prevalence of genital infection with either agent is highest among women who are between the ages of 18 and 24, single, and non-Caucasian (e.g., African-American, Latina, Asian, Pacific Islander). Infections recur frequently in these same risk groups and are often acquired

1	are between the ages of 18 and 24, single, and non-Caucasian (e.g., African-American, Latina, Asian, Pacific Islander). Infections recur frequently in these same risk groups and are often acquired from untreated sexual partners. The use of oral contraception and the presence of cervical ectopy also confer an increased risk. The proportion of infections that are asymptomatic appears to be higher for C. trachomatis than for

1	N. gonorrhoeae, and symptomatic C. trachomatis infections are clinically less severe. Mild or asymptomatic C. trachomatis infections of the fallopian tubes nonetheless cause ongoing tubal damage and infertility. The costs of C. trachomatis infections and their complications to the U.S. health care system have recently been estimated to exceed $516.7 million annually. C. trachomatis is the most common cause of nongonococcal urethritis (NGU) and postgonococcal urethritis (PGU). The designation PGU refers to NGU developing in men 2–3 weeks after treatment of gonococcal urethritis with single doses of agents such as penicillin or cephalosporins, which lack antimicrobial activity against chlamydiae. Since current treatment regimens for gonorrhea have evolved and now include combination therapy with tetracycline, doxycycline, or azithromycin—all of which are effective against concomitant chlamydial infection—both the incidence of PGU and the causative role of

1	C. trachomatis in this syndrome have declined.

1	In the United States, most of the estimated 2 million cases of acute urethritis are NGU, and C. trachomatis is implicated in 30–50% of these cases. The cause of most of the remaining cases of NGU is uncertain, but recent evidence suggests that Ureaplasma urealyticum, Mycoplasma genitalium, Trichomonas vaginalis, and herpes simplex virus (HSV) cause some cases. The rate of involvement of C. trachomatis in urethral infection ranges from 3–7% among asymptomatic men to 15–20% among symptomatic men attending STD clinics. A multisite study of men in Baltimore, Seattle, Denver, and San Francisco reported an overall chlamydial prevalence of 7% in urine samples assessed by nucleic acid amplification tests (NAATs). As in women, infection in men is age related, with young age as the greatest risk factor for chlamydial urethritis. The prevalence among men is highest at 20–24 years of age. In STD clinics, urethritis is usually less prevalent among men who have sex with men (MSM) than among

1	risk factor for chlamydial urethritis. The prevalence among men is highest at 20–24 years of age. In STD clinics, urethritis is usually less prevalent among men who have sex with men (MSM) than among heterosexual men and is almost always much more common among African-American men than among Caucasian men. One study reported prevalences of 19% and 9% among nonwhite and white heterosexual men, respectively.

1	1168 NGU is diagnosed by documentation of a leukocytic urethral exudate and by exclusion of gonorrhea by Gram’s staining or culture. C. trachomatis urethritis is generally less severe than gonococcal urethritis, although in any individual patient these two forms of urethritis cannot reliably be differentiated solely on clinical grounds. Symptoms include urethral discharge (often whitish and mucoid rather than frankly purulent), dysuria, and urethral itching. Physical examination may reveal meatal erythema and tenderness as well as a urethral exudate that is often demonstrable only by stripping of the urethra.

1	At least one-third of male patients with C. trachomatis urethral infection have no evident signs or symptoms of urethritis. The availability of NAATs for first-void urine specimens has facilitated broader-based testing for asymptomatic infection in male patients. As a result, asymptomatic chlamydial urethritis has been demonstrated in 5–10% of sexually active male adolescents screened at school-based clinics or community centers. Such patients generally have pyuria (≥15 leukocytes per 400× microscopic field in the sediment of first-void urine), a positive leukocyte esterase test, or an increased number of leukocytes on a Gram-stained smear prepared from a urogenital swab inserted 1–2 cm into the anterior urethra. To differentiate between true urethritis and functional symptoms in symptomatic patients or to make a presumptive diagnosis of C. trachomatis infection in high-risk but asymptomatic men (e.g., male patients in STD clinics, sex partners of women with nongonococcal salpingitis

1	patients or to make a presumptive diagnosis of C. trachomatis infection in high-risk but asymptomatic men (e.g., male patients in STD clinics, sex partners of women with nongonococcal salpingitis or mucopurulent cervicitis, fathers of children with inclusion conjunctivitis), the examination of an endourethral specimen for increased leukocytes is useful if specific diagnostic tests for chlamydiae are not available. Alternatively, urethritis can be assayed noninvasively by examination of a first-void urine sample for pyuria, either by microscopy or by the leukocyte esterase test. Urine (or a urethral swab) can also be tested directly for chlamydiae by DNA amplification methods, as described below (see “Detection Methods”).

1	epididymitis Chlamydial urethritis may be followed by acute epididymitis, but this condition is rare, generally occurring in sexually active patients <35 years of age; in older men, epididymitis is usually associated with gram-negative bacterial infection and/or instrumentation procedures. It is estimated that 50–70% of cases of acute epididymitis are caused by C. trachomatis. The condition usually presents as unilateral scrotal pain with tenderness, swelling, and fever in a young man, often occurring in association with chlamydial urethritis. The illness may be mild enough to treat with oral antibiotics on an outpatient basis or severe enough to require hospitalization and parenteral therapy. Testicular torsion should be excluded promptly by radionuclide scan, Doppler flow study, or surgical exploration in a teenager or young adult who presents with acute unilateral testicular pain without urethritis. The possibility of testicular tumor or chronic infection (e.g., tuberculosis)

1	surgical exploration in a teenager or young adult who presents with acute unilateral testicular pain without urethritis. The possibility of testicular tumor or chronic infection (e.g., tuberculosis) should be excluded when a patient with unilateral intrascrotal pain and swelling does not respond to appropriate antimicrobial therapy.

1	reactive artHritis Reactive arthritis consists of conjunctivitis, urethritis (or, in female patients, cervicitis), arthritis, and characteristic mucocutaneous lesions. It may develop in 1–2% of cases of NGU and is thought to be the most common type of peripheral inflammatory arthritis in young men. C. trachomatis has been recovered from the urethra of 16–44% of patients with reactive arthritis and from 69% of men who have signs of urogenital inflammation at the time of examination. Antibodies to C. trachomatis have also been detected in 46–67% of patients with reactive arthritis, and Chlamydia-specific cell-mediated immunity has been documented in 72%. In addition,

1	C. trachomatis has been isolated from synovial biopsy samples from 15 of 29 patients in a number of small series and from a smaller proportion of synovial fluid specimens. Chlamydial nucleic acids have been identified in synovial membranes and chlamydial EBs in joint fluid. The pathogenesis of reactive arthritis is unclear, but this condition probably represents an abnormal host response to a number of infectious agents, including those associated with bacterial gastroenteritis (e.g., Salmonella, Shigella, Yersinia, or Campylobacter), or to infection with

1	C. trachomatis or N. gonorrhoeae. Since >80% of affected patients have the HLA-B27 phenotype and since other mucosal infections produce an identical syndrome, chlamydial infection is thought to initiate an aberrant hyperactive immune response that produces inflammation of the involved target organs in these genetically predisposed individuals. Evidence of exaggerated cell-mediated and humoral immune responses to chlamydial antigens in reactive arthritis supports this hypothesis. The finding of chlamydial EBs and DNA in joint fluid and synovial tissue from patients with reactive arthritis suggests that chlamydiae may actually spread from genital to joint tissues in these patients—perhaps in macrophages.

1	NGU is the initial manifestation of reactive arthritis in 80% of patients, typically occurring within 14 days after sexual exposure. The urethritis may be mild and may even go unnoticed by the patient. Similarly, gonococcal urethritis may precede reactive arthritis, but co-infection with an agent of NGU is difficult to rule out. The urethral discharge may be purulent or mucopurulent, and patients may or may not report dysuria. Accompanying prostatitis, usually asymptomatic, has been described. Arthritis usually begins ~4 weeks after the onset of urethritis but may develop sooner or, in a small percentage of cases, may actually precede urethritis. The knees are most frequently involved; next most commonly affected are the ankles and small joints of the feet. Sacroiliitis, either symmetrical or asymmetrical, is documented in two-thirds of patients. Mild bilateral conjunctivitis, iritis, keratitis, or uveitis is sometimes present but lasts for only a few days. Finally, dermatologic

1	or asymmetrical, is documented in two-thirds of patients. Mild bilateral conjunctivitis, iritis, keratitis, or uveitis is sometimes present but lasts for only a few days. Finally, dermatologic manifestations occur in up to 50% of patients. The initial lesions— usually papules with a central yellow spot—most often involve the soles and palms and, in ~25% of patients, eventually epithelialize and thicken to produce keratoderma blenorrhagicum. Circinate balanitis is usually painless and occurs in fewer than half of patients. The initial episode of reactive arthritis usually lasts 2–6 months.

1	proctitis Primary anal or rectal infections with C. trachomatis have been described in women and MSM who practice anal intercourse. In these infections, rectal involvement is initially characterized by severe anorectal pain, a bloody mucopurulent discharge, and tenesmus. Oculogenital serovars D–K and LGV serovars L1, L2, and L3 have been found to cause proctitis. The LGV serovars are far more invasive and cause much more severely symptomatic disease, including severe ulcerative proctocolitis that can be clinically confused with HSV proctitis. Histologically, LGV proctitis may resemble Crohn’s disease in that giant cell formation and granulomas are detected. In the United States and Europe, cases of LGV proctitis occur almost exclusively in MSM, many of whom are positive for HIV infection.

1	The less invasive non-LGV serovars of C. trachomatis cause mild proctitis. Many infected individuals are asymptomatic, and in these cases infection is diagnosed only by routine culture or NAAT of rectal swabs. The number of fecal leukocytes is usually abnormal in both asymptomatic and symptomatic cases. Sigmoidoscopy may yield normal findings or may reveal mild inflammatory changes or small erosions or follicles in the lower 10 cm of the rectum. Histologic examination of rectal biopsies generally shows anal crypts and prominent follicles as well as neutrophilic infiltration of the lamina propria. Chlamydial proctitis is best diagnosed by isolation of C. trachomatis from the rectum and documentation of a response to appropriate therapy. NAATs are reportedly more sensitive than culture for diagnosis and are also specific.

1	mUcopUrUlent cervicitis Although many women with chlamydial infections of the cervix have no symptoms, almost half generally have local signs of infection on examination. Cervicitis is usually characterized by the presence of a mucopurulent discharge, with >20 neutrophils per microscopic field visible in strands of cervical mucus in a thinly smeared, gram-stained preparation of endocervical exudate. Hypertrophic ectopy of the cervix may also be evident as an edematous area near the cervical os that is congested and bleeds easily on minor trauma (e.g., when a specimen is collected with a swab). A Papanicolaou smear shows increased numbers of neutrophils as well as a characteristic pattern of mononuclear inflammatory cells including plasma cells, transformed lymphocytes, and histiocytes. Cervical biopsy shows a predominantly mononuclear cell infiltrate of the subepithelial stroma. Clinical experience and collaborative studies indicate that a cutoff of >30 polymorphonuclear neutrophils

1	biopsy shows a predominantly mononuclear cell infiltrate of the subepithelial stroma. Clinical experience and collaborative studies indicate that a cutoff of >30 polymorphonuclear neutrophils (PMNs) per 1000× field in a gram-stained smear of cervical mucus correlates best with chlamydial or gonococcal cervicitis.

1	Clinical recognition of chlamydial cervicitis depends on a high index of suspicion and careful cervical examination. No genital symptoms are specifically correlated with chlamydial cervical infection. The differential diagnosis of a mucopurulent discharge from the endocervical canal in a young, sexually active woman includes gonococcal endocervicitis, salpingitis, endometritis, and intrauterine contraceptive device–induced inflammation. Diagnosis of cervicitis is based on the presence of PMNs on a cervical swab as noted above; the presence of chlamydiae is confirmed by either culture or NAAT.

1	pelvic inflammatory disease Inflammation of sections of the fallopian tube is often referred to as salpingitis or PID. The proportion of acute salpingitis cases caused by C. trachomatis varies geographically and with the population studied. It has been estimated that C. trachomatis causes up to 50% of PID cases in the United States. PID occurs via ascending intraluminal spread of C. trachomatis or N. gonorrhoeae from the lower genital tract. Mucopurulent cervicitis is often followed by endometritis, endosalpingitis, and finally pelvic peritonitis. Evidence of mucopurulent cervicitis is often found in women with laparoscopically verified salpingitis. Similarly, endometritis, demonstrated by an endometrial biopsy showing plasma cell infiltration of the endometrial epithelium, is documented in most women with lap-aroscopy-verified chlamydial (or gonococcal) salpingitis. Chlamydial endometritis can also occur in the absence of clinical evidence of salpingitis. Histologic evidence of

1	in most women with lap-aroscopy-verified chlamydial (or gonococcal) salpingitis. Chlamydial endometritis can also occur in the absence of clinical evidence of salpingitis. Histologic evidence of endometritis has been correlated with a syndrome consisting of vaginal bleeding, lower abdominal pain, and uterine tenderness in the absence of adnexal tenderness. Chlamydial salpingitis produces milder symptoms than gonococcal salpingitis and may be associated with less marked adnexal tenderness. Thus, mild adnexal or uterine tenderness in a sexually active woman with cervicitis suggests chlamydial PID.

1	Chronic untreated endometrial and tubal inflammation can result in tubal scarring, impaired tubal function, tubal occlusion, and infertility, even among women who report no prior treatment for PID. C. trachomatis has been implicated particularly often in “subclinical” PID on the basis of (1) a lack of history of PID among Chlamydiaseropositive women with tubal damage or (2) detection of chlamydial DNA or antigen among asymptomatic women with tubal infertility. These data suggest that the best method to prevent PID and its sequelae is surveillance and control of lower genital tract infections along with diagnosis and treatment of sex partners and prevention of reinfections. Promotion of early symptom recognition and health care presentation may reduce the frequency and severity of sequelae of PID.

1	periHepatitis The Fitz-Hugh–Curtis syndrome was originally described as a complication of gonococcal PID. However, studies over the past several decades have suggested that chlamydial infection is more commonly associated with perihepatitis than is N. gonorrhoeae. Perihepatitis should be suspected in young, sexually active women who develop right-upper-quadrant pain, fever, or nausea. Evidence of salpingitis may or may not be found on examination. Frequently, perihepatitis is strongly associated with extensive tubal scarring, adhesions, and inflammation observed at laparoscopy, and high titers of antibody to the 57-kDa chlamydial heat-shock protein have been documented. Culture and/or serologic evidence of C. trachomatis is found in three-fourths of women with this syndrome. UretHral syndrome in women In the absence of infection with uropathogens such as coliforms or Staphylococcus saprophyticus,

1	C. trachomatis is the pathogen most commonly isolated from college women with dysuria, frequency, and pyuria. Screening studies can recover C. trachomatis from both the cervix and the urethra; in up to 25% of infected women, the organism is isolated only from the urethra. The urethral syndrome in women consists of dysuria and frequency in conjunction with chlamydial urethritis, pyuria, and no bacteriuria or urinary pathogens. Although symptoms of the urethral syndrome may develop in some women with chlamydial infection, the major-1169 ity of women attending STD clinics for urethral chlamydial infection do not have dysuria or frequency. Even in women with chlamydial urethritis causing the acute urethral syndrome, signs of urethritis such as urethral discharge, meatal redness, and swelling are uncommon. However, mucopurulent cervicitis in a woman presenting with dysuria and frequency strongly suggests C. trachomatis urethritis. Other correlates of chlamydial urethral syndrome include a

1	are uncommon. However, mucopurulent cervicitis in a woman presenting with dysuria and frequency strongly suggests C. trachomatis urethritis. Other correlates of chlamydial urethral syndrome include a duration of dysuria of >7–10 days, lack of hematuria, and lack of suprapubic tenderness. Abnormal urethral Gram’s stains showing >10 PMNs per 1000× field in women with dysuria but without coliform bacteriuria support the diagnosis of chlamydial urethritis. Other possible diagnoses include gonococcal or trichomonal infection of the urethra.

1	infection in pregnancy and tHe neonatal period Infections during pregnancy can be transmitted to infants during delivery. Approximately 20–30% of infants exposed to C. trachomatis in the birth canal develop conjunctivitis, and 10–15% subsequently develop pneumonia. Consequently, all newborn infants receive ocular prophylaxis at birth to prevent ophthalmia neonatorum. Without treatment, conjunctivitis usually develops at 5–19 days of life and often results in a profuse mucopurulent discharge. Roughly half of infected infants develop clinical evidence of inclusion conjunctivitis. However, it is impossible to differentiate chlamydial conjunctivitis from other forms of neonatal conjunctivitis (e.g., that due to N. gonorrhoeae, Haemophilus influenzae, Streptococcus pneumoniae, or HSV) on clinical grounds; thus laboratory diagnosis is required. Inclusions within epithelial cells are often detected in Giemsa-stained conjunctival smears, but these smears are considerably less sensitive than

1	grounds; thus laboratory diagnosis is required. Inclusions within epithelial cells are often detected in Giemsa-stained conjunctival smears, but these smears are considerably less sensitive than cultures or NAATs for chlamydiae. Gram-stained smears may show gonococci or occasional small gram-negative coccobacilli in Haemophilus conjunctivitis, but smears should be accompanied by cultures or NAATs for these agents.

1	C. trachomatis has also been isolated frequently and persistently from the nasopharynx, rectum, and vagina of infected infants—occasionally for >1 year in the absence of treatment. In some cases, otitis media results from perinatally acquired chlamydial infection. Pneumonia may develop in infants from 2 weeks to 4 months of age. C. trachomatis is estimated to cause 20–30% of pneumonia cases in infants <6 months of age. Epidemiologic studies have linked chlamydial pulmonary infection in infants with increased occurrence of subacute lung disease (bronchitis, asthma, wheezing) in later childhood.

1	lympHogranUloma venereUm C. trachomatis serovars L1, L2, and L3 cause LGV, an invasive systemic STD. The peak inci dence of LGV corresponds with the age of greatest sexual activity: the second and third decades of life. The worldwide incidence of LGV is falling, but the disease is still endemic and a major cause of morbidity in parts of Asia, Africa, South America, and the Caribbean. LGV is rare in industrialized countries; for more than a decade, the reported incidence in the United States has been only 0.1 case per 100,000 population. In the Bahamas, an apparent outbreak of LGV was described in association with a concurrent increase in heterosexual infection with HIV. Reports of outbreaks with the newly identified variant L2b in Europe, Australia, and the United States indicate that LGV is becoming more prevalent among MSM. These cases have usually presented as hemorrhagic proctocolitis in HIV-positive men. More widespread use of NAATs for identification of rectal infections may

1	LGV is becoming more prevalent among MSM. These cases have usually presented as hemorrhagic proctocolitis in HIV-positive men. More widespread use of NAATs for identification of rectal infections may have enhanced case recognition.

1	LGV begins as a small painless papule that tends to ulcerate at the site of inoculation, often escaping attention. This primary lesion heals in a few days without scarring and is usually recognized as LGV only in retrospect. LGV strains of C. trachomatis have occasionally been recovered from genital ulcers and from the urethra of men and the endocervix of women who present with inguinal adenopathy; these areas may be the primary sites of infection in some cases. Proctitis is more common among people who practice receptive anal intercourse, and an elevated white blood cell count in anorectal smears may predict LGV in these patients. Ulcer formation may facilitate transmission of HIV infection and other sexually transmitted and blood-borne diseases.

1	1170 As NAATs for C. trachomatis are being used more often, increasing numbers of cases of LGV proctitis are being recognized in MSM. Such patients present with anorectal pain and mucopurulent, bloody rectal discharge. Sigmoidoscopy reveals ulcerative proctitis or proctocolitis, with purulent exudate and mucosal bleeding. Histopathologic findings in the rectal mucosa include granulomas with giant cells, crypt abscesses, and extensive inflammation. These clinical, sigmoidoscopic, and histopathologic findings may closely resemble those of Crohn’s disease of the rectum. The most common presenting picture in heterosexual men and women is the inguinal syndrome, which is characterized by painful inguinal lymphadenopathy beginning 2–6 weeks after presumed exposure; in rare instances, the onset comes after a few months. The inguinal adenopathy is unilateral in two-thirds of cases, and palpable enlargement of the iliac and femoral nodes is often evident on the same side as the enlarged

1	onset comes after a few months. The inguinal adenopathy is unilateral in two-thirds of cases, and palpable enlargement of the iliac and femoral nodes is often evident on the same side as the enlarged inguinal nodes. The nodes are initially discrete, but progressive periadenitis results in a matted mass of nodes that becomes fluctuant and suppurative. The overlying skin becomes fixed, inflamed, and thin, and multiple draining fistulas finally develop. Extensive enlargement of chains of inguinal nodes above and below the inguinal ligament (“the sign of the groove”) is not specific and, although not uncommon, is documented in only a minority of cases. Spontaneous healing usually takes place after several months; inguinal scars or granulomatous masses of various sizes persist for life. Massive pelvic lymphadenopathy may lead to exploratory laparotomy.

1	Constitutional symptoms are common during the stage of regional lymphadenopathy and, in cases of proctitis, may include fever, chills, headache, meningismus, anorexia, myalgias, and arthralgias. Other systemic complications are infrequent but include arthritis with sterile effusion, aseptic meningitis, meningoencephalitis, conjunctivitis, hepatitis, and erythema nodosum (Fig. 25e-40). Complications of untreated anorectal infection include perirectal abscess; anal fistulas; and rectovaginal, rectovesical, and ischiorectal fistulas. Secondary bacterial infection probably contributes to these complications. Rectal stricture is a late complication of anorectal infection and usually develops 2–6 cm from the anal orifice—i.e., at a site within reach on digital rectal examination. A small percentage of cases of LGV in men present as chronic progressive infiltrative, ulcerative, or fistular lesions of the penis, urethra, or scrotum. Associated lymphatic obstruction may produce elephantiasis.

1	of cases of LGV in men present as chronic progressive infiltrative, ulcerative, or fistular lesions of the penis, urethra, or scrotum. Associated lymphatic obstruction may produce elephantiasis. When urethral stricture occurs, it usually involves the posterior urethra and causes incontinence or difficulty with urination.

1	Diagnosis • detection metHods Historically, chlamydiae were cultivated in the yolk sac of embryonated eggs. The organisms can be grown more easily in tissue culture, but cell culture—once considered the diagnostic gold standard—has been replaced by nonculture assays (Table 213-1). In general, culture for chlamydiae in clinical specimens is now performed only in specialized laboratories. The first nonculture assays, such as DFA staining of clinical material and enzyme immunoassay (EIA), have been replaced by NAATS, which are molecular Infection Suggestive Signs/Symptoms Presumptive Diagnosisa Confirmatory Test of Choice aA presumptive diagnosis of chlamydial infection is often made in the syndromes listed when gonococci are not found. A positive test for Neisseria gonorrhoeae does not exclude the involvement of C. trachomatis, which often is present in patients with gonorrhea.

1	Abbreviations: CF, complement-fixing; FA, fluorescent antibody; LGV, lymphogranuloma venereum; micro-IF, microimmunofluorescence; MPC, mucopurulent cervicitis; NAAT, nucleic acid amplification test; NGU, nongonococcal urethritis; PGU, postgonococcal urethritis. Source: Reprinted with permission from WE Stamm: Chlamydial infections, in Harrison’s Principles of Internal Medicine, 17th ed, AS Fauci et al (eds). New York, McGraw-Hill, 2008, p 1075. tests that amplify the nucleic acids in clinical specimens. NAATS are currently recommended by the CDC as the diagnostic assays of choice; four or five NAAT assays approved by the U.S. Food and Drug Administration (FDA) are commercially available, some as high-throughput robotic platforms. Point-of-care diagnostic assays (including NAATs), by which patients can be treated before leaving the clinic, are of increasing interest and are becoming available.

1	cHoice of specimen Cervical and urethral swabs have traditionally been used for the diagnosis of STDs in female and male patients, respectively. However, given the greatly increased sensitivity and specificity of NAATs, less invasive samples (e.g., urine for both sexes and vaginal swabs for women) can be used. For screening of asymptomatic women, the CDC now recommends that self-collected or clinician-collected vaginal swabs, which are slightly more sensitive than urine, be used. Urine screening tests are often used in outreach screening programs, however. For symptomatic women undergoing a pelvic examination, cervical swab samples are desirable because they have slightly higher chlamydial counts. For male patients, a urine specimen is the sample of choice, but self-collected penile-meatal swabs have been explored.

1	alternative specimen types Ocular samples from babies and adults can be assessed by NAATs. However, since commercial NAATs for this purpose have not yet been approved by the FDA, laboratories must perform their own verification studies. Samples from rectal and pharyngeal sites have been used successfully to detect chlamydiae, but laboratories must verify test performance.

1	otHer diagnostic issUes Because NAATs detect nucleic acids instead of live organisms, they should be used with caution as test-of-cure assays. Residual nucleic acid from cells rendered noninfective by antibiotics may continue to yield a positive result in NAATs until as long as 3 weeks after therapy, when viable organisms have actually been eradicated. Therefore, clinicians should not use NAATs for test of cure until after 3 weeks. The CDC currently does not recommend a test of cure after treatment for infection with C. trachomatis. However, because incidence studies have demonstrated that previous chlamydial infection increases the probability of becoming reinfected, the CDC does recommend that previously infected individuals be rescreened 3 months after treatment.

1	serology Serologic testing may be helpful in the diagnosis of LGV and neonatal pneumonia caused by C. trachomatis. The serologic test of choice is the microimmunofluorescence (MIF) test, in which high-titer purified EBs mixed with embryonated chicken yolk-sac material are affixed to a glass microscope slide to which dilutions of serum are applied. After incubation and washing, fluorescein-conjugated IgG or IgM antibody is applied. The test is read with an epifluorescence microscope, with the highest dilution of serum producing visible fluorescence designated as the titer. The MIF test is not widely available and is highly labor intensive. Although the complement fixation (CF) test also can be used, it employs only lipopolysaccharide (LPS) as the antigen and therefore identifies the pathogen only to the genus level. Single-point titers of >1:64 support a diagnosis of LGV, in which it is difficult to demonstrate rising antibody titers; i.e., paired serum samples are difficult to obtain

1	only to the genus level. Single-point titers of >1:64 support a diagnosis of LGV, in which it is difficult to demonstrate rising antibody titers; i.e., paired serum samples are difficult to obtain since, by its very nature, the disease results in the patient’s being seen by the physician after the acute stage. Any antibody titer of >1:16 is considered significant evidence of exposure to chlamydiae. However, serologic testing is never recommended for diagnosis of uncomplicated genital infections of the cervix, urethra, and lower genital tract or for C. trachomatis screening of asymptomatic individuals.

1	TREATMEnT c. tracHomatis Genital infections A 7-day course of tetracycline (500 mg four times daily), doxycycline (100 mg twice daily), erythromycin (500 mg four times daily), or a fluoroquinolone (ofloxacin, 300 mg twice daily; or levofloxacin, 500 mg/d) can be used for treatment of uncomplicated chlamydial infections. A single 1-g oral dose of azithromycin is as effective as a 7-day course of doxycycline for the treatment of uncomplicated genital 1171

1	C. trachomatis infections in adults. Azithromycin causes fewer adverse gastrointestinal reactions than do older macrolides such as erythromycin. The single-dose regimen of azithromycin has great appeal for the treatment of patients with uncomplicated chlamydial infection (especially those without symptoms and those with a likelihood of poor compliance) and of the sexual partners of infected patients. These advantages must be weighed against the considerably greater cost of azithromycin. Whenever possible, the single 1-g dose should be given as directly observed therapy. Although not approved by the FDA for use in pregnancy, this regimen appears to be safe and effective for this purpose. However, amoxicillin (500 mg three times daily for 7 days) also can be given to pregnant women. The fluoroquinolones are contraindicated in pregnancy. A 2-week course of treatment is recommended for complicated chlamydial infections (e.g., PID, epididymitis) and at least a 3-week course of doxycycline

1	are contraindicated in pregnancy. A 2-week course of treatment is recommended for complicated chlamydial infections (e.g., PID, epididymitis) and at least a 3-week course of doxycycline (100 mg orally twice daily) or erythromycin base (500 mg orally four times daily) for LGV. Failure of treatment with a tetracycline in genital infections usually indicates poor compliance or reinfection rather than involvement of a drug-resistant strain. To date, clinically significant drug resistance has not been observed in C. trachomatis.

1	Treatment or testing for chlamydiae should be considered among N. gonorrhoeae–infected patients because of the frequency of co-infection. Systemic treatment with erythromycin has been recommended for ophthalmia neonatorum and for C. trachomatis pneumonia in infants. For the treatment of adult inclusion conjunc tivitis, a single 1-g dose of azithromycin is as effective as standard 10-day treatment with doxycycline. Recommended treatment regimens for both bubonic and anogenital LGV include tetracycline, doxycycline, or erythromycin for 21 days.

1	The continued high prevalence of chlamydial infections in most parts of the United States is due primarily to the failure to diagnose—and therefore treat—patients with symptomatic or asymptomatic infection and their sex partners. Urethral or cervical infection with C. trachomatis has been well documented in a high proportion of the sex partners of patients with NGU, epididymitis, reactive arthritis, salpingitis, and endocervicitis. If possible, confirmatory laboratory tests for chlamydiae should be undertaken in these individuals, but even those without positive tests or evidence of clinical disease who have recently been exposed to proven or possible chlamydial infection (e.g., NGU) should be offered therapy. A novel approach is partner-delivered therapy, in which infected patients receive treatment and are also provided with single-dose azithromycin to give to their sex partner(s).

1	In neonates with conjunctivitis or infants with pneumonia, erythromycin ethylsuccinate or estolate can be given orally at a dosage of 50 mg/kg per day, preferably in four divided doses, for 2 weeks. Careful attention must be given to compliance with therapy—a frequent problem. Relapses of eye infection are common after topical treatment with erythromycin or tetracycline ophthalmic ointment and may also follow oral erythromycin therapy. Thus follow-up cultures should be performed after treatment. Both parents should be examined for C. trachomatis infection and, if diagnostic testing is not readily available, should be treated with doxycycline or azithromycin.

1	Prevention Since many chlamydial infections are asymptomatic, effective control and prevention must involve periodic screening of individuals at risk. Selective cost-effective screening criteria have been developed. Among women, young age (generally <25 years) is a critical risk factor for chlamydial infections in nearly all studies. Other risk factors include mucopurulent cervicitis; multiple, new, or symptomatic male sex partners; and lack of barrier contraceptive use. In some settings, screening based on young age may be as sensitive 1172 as criteria that incorporate behavioral and clinical measures. Another strategy is universal testing of all patients in high-prevalence clinic populations (e.g., STD clinics, juvenile detention facilities, and family planning clinics). The effectiveness of selective screening in reducing the prevalence of chlamydial infection among women has been demonstrated in several studies. In the Pacific Northwest, where extensive screening began in family

1	of selective screening in reducing the prevalence of chlamydial infection among women has been demonstrated in several studies. In the Pacific Northwest, where extensive screening began in family planning clinics in 1998 and in STD clinics in 1993, the prevalence declined from 10% in the 1980s to <5% in 2000. Similar trends have occurred in association with screening programs elsewhere. In addition, screening can effect a reduction in upper genital tract disease. In Seattle, women at a large health maintenance organization who were screened for chlamydial infection on a routine basis had a lower incidence of symptomatic PID than did women who received standard care and underwent more selective screening. In settings with low to moderate prevalence, the prevalence at which selective screening becomes more cost-effective than universal screening must be defined. Most studies have concluded that universal screening is preferable in settings with a chlamydial prevalence of >3–7%.

1	screening becomes more cost-effective than universal screening must be defined. Most studies have concluded that universal screening is preferable in settings with a chlamydial prevalence of >3–7%. Depending on the criteria used, selective screening is likely to be more cost-effective when prevalence falls below 3%. Nearly all regions of the United States have now initiated screening programs, particularly in family planning and STD clinics. Along with single-dose therapy, the availability of highly sensitive and specific diagnostic NAATs using urine specimens and self-obtained vaginal swabs makes it feasible to mount an effective nationwide Chlamydia control program, with screening of high-risk individuals in traditional health-care settings and in novel outreach and community-based settings. The

1	U.S. Preventive Services Task Force has given C. trachomatis screening a Grade A recommendation, which means that private insurance and Medicare will cover its cost under the Affordable Care Act. TRACHOMA Epidemiology Trachoma—a sequela of ocular disease in developing countries—continues to be a leading cause of preventable infectious blindness worldwide. The WHO estimates that ~6 million people have been blinded by trachoma and that ~1.3 million people in developing countries still suffer from preventable blindness due to trachoma; certainly hundreds of millions live in trachoma-endemic areas. Foci of trachoma persist in Australia, the South Pacific, and Latin America. Serovars A, B, Ba, and C are isolated from patients with clinical trachoma in areas of endemicity in developing countries in Africa, the Middle East, Asia, and South America.

1	The trachoma-hyperendemic areas of the world are in northern and sub-Saharan Africa, the Middle East, drier regions of the Indian subcontinent, and Southeast Asia. In hyperendemic areas, the prevalence of trachoma is essentially 100% by the second or third year of life. Active disease is most common among young children, who are the reservoir for trachoma. By adulthood, active infection is infrequent but sequelae result in blindness. In such areas, trachoma constitutes the major cause of blindness. Trachoma is transmitted through contact with discharges from the eyes of infected patients. Transmission is most common under poor hygienic conditions and most often takes place between family members or between families with shared facilities. Flies can also transfer the mucopurulent ocular discharges, carrying the organisms on their legs from one person to another. The International Trachoma Initiative founded by the WHO in 1998 aims to eliminate blinding trachoma globally by 2020.

1	Clinical Manifestations Both endemic trachoma and adult inclusion conjunctivitis present initially as conjunctivitis characterized by small lymphoid follicles in the conjunctiva. In regions with hyperendemic classic blinding trachoma, the disease usually starts insidiously before the age of 2 years. Reinfection is common and probably contributes to the pathogenesis of trachoma. Studies using polymerase chain reaction (PCR) or other NAATs indicate that chlamydial DNA is often present in the ocular secretions of patients with trachoma, even in the absence of positive cultures. Thus persistent infection may be more common than was previously thought.

1	The cornea becomes involved, with inflammatory leukocytic infiltrations and superficial vascularization (pannus formation). As the inflammation continues, conjunctival scarring eventually distorts the eyelids, causing them to turn inward so that the lashes constantly abrade the eyeball (trichiasis and entropion); eventually the corneal epithelium is abraded and may ulcerate, with subsequent corneal scarring and blindness. Destruction of the conjunctival goblet cells, lacrimal ducts, and lacrimal gland may produce a “dry-eye” syndrome, with resultant corneal opacity due to drying (xerosis) or secondary bacterial corneal ulcers.

1	Communities with blinding trachoma often experience seasonal epidemics of conjunctivitis due to H. influenzae that contribute to the intensity of the inflammatory process. In such areas, the active infectious process usually resolves spontaneously in affected persons at 10–15 years of age, but conjunctival scars continue to shrink, producing trichiasis and entropion with subsequent corneal scarring in adults. In areas with milder and less prevalent disease, the process may be much slower, with active disease continuing into adulthood; blindness is rare in these cases.

1	Eye infection with oculogenital C. trachomatis strains in sexually active young adults presents as an acute onset of unilateral follicular conjunctivitis and preauricular lymphadenopathy similar to that seen in acute conjunctivitis caused by adenovirus or HSV. If untreated, the disease may persist for 6 weeks to 2 years. It is frequently associated with corneal inflammation in the form of discrete opacities (“infiltrates”), punctate epithelial erosions, and minor degrees of superficial corneal vascularization. Very rarely, conjunctival scarring and eyelid distortion occur, particularly in patients treated for many months with topical glucocorticoids. Recurrent eye infections develop most often in patients whose sexual partners are not treated with antimicrobial agents.

1	The clinical diagnosis of classic trachoma can be made if two of the following signs are present: (1) lymphoid follicles on the upper tarsal conjunctiva; (2) typical conjunctival scarring; (3) vascular pannus; or (4) limbal follicles or their sequelae, Herbert’s pits. The clinical diagnosis of endemic trachoma should be confirmed by laboratory tests in children with relatively marked degrees of inflammation. Intracytoplasmic chlamydial inclusions are found in 10–60% of Giemsa-stained conjunctival smears in such populations, but chlamydial NAATs are more sensitive and are often positive when smears or cultures are negative. Follicular conjunctivitis in European or American adults living in trachomatous regions is rarely due to trachoma.

1	Adult inclusion conjunctivitis responds well to treatment with the same regimens used in uncomplicated genital infections—namely, azithromycin (a 1-g single oral dose) or doxycycline (100 mg twice daily for 7 days). Simultaneous treatment of all sexual partners is necessary to prevent ocular reinfection and chlamydial genital disease. Topical antibiotic treatment is not required for patients who receive systemic antibiotics.

1	Psittacine birds and many other avian species act as natural reservoirs for C. psittaci–type organisms, which are common pathogens in domestic mammals and birds. The species C. psittaci, which now includes only avian strains, affects humans only as a zoonosis. (The other strains previously included in this species have been placed into different species that generally reflect the animals they infect: C. abortus, C. muridarum, C. suis, C. felis, and C. caviae.) Although all birds are susceptible, pet birds (parrots, parakeets, macaws, and cockatiels) and poultry (turkeys and ducks) are most frequently involved in transmission of C. psittaci to humans. Exposure is greatest among poultry-processing workers and owners of pet birds. Infectious forms of the organisms are shed from both symptomatic and apparently healthy birds and may remain viable for several months. C. psittaci can be transmitted to humans by direct contact with infected birds or by inhalation of aerosols from avian nasal

1	and apparently healthy birds and may remain viable for several months. C. psittaci can be transmitted to humans by direct contact with infected birds or by inhalation of aerosols from avian nasal discharges and from infectious avian fecal or feather dust. Transmission from person to person has never been demonstrated.

1	The diagnosis is usually established serologically. Psittacosis in humans may present as acute primary atypical pneumonia (which can be fatal in up to 10% of untreated cases); as severe chronic pneumonia; or as a mild illness or asymptomatic infection in persons exposed to infected birds. Fewer than 50 confirmed cases of psittacosis are reported in the United States each year, although many more cases probably occur than are reported. Control of psittacosis depends on control of avian sources of infection. A pandemic of psittacosis was once stopped by banning shipment or importation of psittacine birds. Birds can receive prophylaxis in the form of a tetracycline-containing feed. Imported birds are currently quarantined for 30 days of treatment.

1	Typical symptoms include fever, chills, muscular aches and pains, severe headache, hepatoand/or splenomegaly, and gastrointestinal symptoms. Cardiac complications may involve endocarditis and myocarditis. Fatal cases were common in the preantibiotic era. As a result of quarantine of imported birds and improved veterinary-hygienic measures, outbreaks and sporadic cases of psittacosis are now rare. Severe pneumonia requiring management in an intensive care unit may develop. Endocarditis, hepatitis, and neurologic complications may occur, and fatal cases have been reported. The incubation period is usually 5–19 days but can last as long as 28 days.

1	Previously, the most widely used serologic test for diagnosing chlamydial infections was the genus-specific CF test, in which assay of paired serum specimens often shows fourfold or greater increases in antibody titer. The CF test remains useful, but the gold standard of serologic tests is now the MIF test, which is not widely available (see section on diagnosis of C. trachomatis genital infection, above). Any antibody titer above 1:16 is considered significant evidence of exposure to chlamydiae, and a fourfold titer rise in paired sera in combination with a clinically compatible syndrome can be used to diagnose psittacosis. Some commercially available serologic tests based on measurement of antibodies to LPS can be useful when the clinical diagnosis is consistent with bird exposure; however, since these tests are reactive for all chlamydiae (i.e., all chlamydiae contain LPS), caution must be used in their interpretation.

1	The antibiotic of choice is tetracycline; the dosage for adults is 250 mg four times a day, continued for at least 3 weeks to avoid relapse. Severely ill patients may need cardiovascular and respiratory support. Erythromycin (500 mg four times a day by mouth) is an alternative therapy. C. pneumoniae is a common cause of human respiratory diseases, such as pneumonia and bronchitis. This organism has been reported to account for as many as 10% of cases of community-acquired pneumonia, most of which are diagnosed by serology. Serologic studies have linked C. pneumoniae to atherosclerosis; isolation and PCR 1173 detection in cardiovascular tissues have also been reported. These findings suggest an expanded range of diseases and syndromes for

1	C. pneumoniae. The role of C. pneumoniae in the etiology of atherosclerosis has been discussed since 1988, when Finnish researchers presented serologic evidence of an association of this organism with coronary heart disease and acute myocardial infarction. Subsequently, the organism was identified in atherosclerotic lesions by culture, PCR, immunohistochemistry, and transmission electron microscopy; however, discrepant study results (including those of animal studies) and failure of large-scale treatment studies have raised doubts as to the etiologic role of C. pneumoniae in atherosclerosis. Large-scale case-cohort studies have demonstrated some association of C. pneumoniae with lung cancer, as evaluated by serology.

1	and reinfection in adults. Seroprevalence rates of 40–70% show that C. pneumoniae is widespread in both industrialized and developing countries. Seropositivity usually is first detected at school age, and rates generally increase by ~10% per decade. About 50% of individuals have detectable antibody at 30 years of age, and most have detectable antibody by the eighth decade of life. Although serologic evidence suggests that C. pneumoniae may be associated with up to 10% of cases of community-acquired pneumonia, most of this evidence is based not on paired serum samples but rather on a single high IgG titer. Some doubt exists about the true prevalence and etiologic role of C. pneumoniae in atypical pneumonia, especially since reports of cross-reactivity have raised questions about the specificity of serology when only a single serum sample is used for diagnosis.

1	Little is known about the pathogenesis of C. pneumoniae infection. It begins in the upper respiratory tract and, in many persons, persists as a prolonged asymptomatic condition of the upper respiratory mucosal surfaces. However, evidence of replication within vascular endothelium and synovial membranes of joints shows that, in at least some individuals, the organism is transported to distant sites, perhaps within macrophages. A C. pneumoniae outer-membrane protein may induce host immune responses whose cross-reactivity with human proteins results in an autoimmune reaction. As mentioned above, epidemiologic studies have demonstrated an association between serologic evidence of C. pneumoniae infection and atherosclerotic disease of the coronary and other arteries. In addition,

1	C. pneumoniae has been identified in atherosclerotic plaques by electron microscopy, DNA hybridization, and immunocytochemistry. The organism has been recovered in culture from atheromatous plaque—a result indicating the presence of viable replicating bacteria in vessels. Evidence from animal models supports the hypothesis that C. pneumoniae infection of the upper respiratory tract is followed by recovery of the organism from atheromatous lesions in the aorta and that the infection accelerates the process of atherosclerosis, especially in hypercholesterolemic animals. Antimicrobial treatment of the infected animals reverses the increased risk of atherosclerosis. In humans, two small trials in patients with unstable angina or recent myocardial infarction suggested that antibiotics reduce the likelihood of subsequent untoward cardiac events. However, larger-scale trials have not documented an effect of various antichlamydial regimens on the risk of these events.

1	C. pneumoniae was first reported as the etiologic agent of mild atypical pneumonia in military recruits and college students. The clinical spectrum of C. pneumoniae infection includes acute pharyngitis, sinusitis, bronchitis, and pneumonitis, primarily in young adults. The clinical manifestations of primary infection appear to be more severe 11 and prolonged than those of reinfection. The pneumonitis of C. pneumoniae pneumonia resembles that of Mycoplasma pneumonia in that leukocytosis is frequently lacking and patients often have prominent antecedent upper respiratory tract symptoms, fever, nonproductive cough, mild to moderate illness, minimal findings on chest auscultation, and small segmental infiltrates on chest x-ray. In elderly patients, pneumonia due to C. pneumoniae can be especially severe and may necessitate hospitalization and respiratory support. Chronic infection with C. pneumoniae has been reported among patients with chronic obstructive pulmonary disease and may also

1	severe and may necessitate hospitalization and respiratory support. Chronic infection with C. pneumoniae has been reported among patients with chronic obstructive pulmonary disease and may also play a role in the natural history of asthma, including exacerbations. The clinical symptoms of respiratory infections caused by C. pneumoniae are nonspecific and do not differ from those caused by other agents of atypical pneumonia, such as Mycoplasma pneumoniae.

1	Serology, PCR amplification, and culture can be used to diagnose

1	C. pneumoniae infection. Serology has been the traditional method of diagnosing infection by C. pneumoniae. The gold standard serologic test is the MIF test (see section on diagnosis of C. trachomatis genital infection, above). Any antibody titer above 1:16 is considered significant evidence of exposure to chlamydiae. According to a CDC-sponsored expert working group, the diagnosis of acute C. pneumoniae infection requires demonstration of a fourfold rise in titer in paired serum samples. There are no official recommendations for diagnosis of chronic infections, although many research studies have used high titers of IgA as an indicator. The older CF tests and EIAs for LPS are not recommended, as they are not specific for C. pneumoniae but identify the chlamydiae only to the genus level. The organism is very difficult to grow in tissue culture but has been cultivated in HeLa cells, HEp-2 cells, and HL cells. Although NAATs are commercially available for

1	C. trachomatis, only research-based PCR assays are available for C. pneumoniae. Although few controlled trials of treatment have been reported, C. pneumoniae is inhibited in vitro by erythromycin, tetracycline, azithromycin, clarithromycin, gatifloxacin, and gemifloxacin. ecommended therapy consists of 2 gd of either tetracycline or erythromycin for 10–1 days. ther macrolides (e.g., azithromycin) and some fluorouinolones (e.g., levofloxacin and gatifloxacin) also appear to be effective. The authors wish to acknowledge the late Walter E. Stamm, MD, for his significant contributions to the field of Chlamydia research. Dr. Stamm wrote the chapters on chlamydiae for previous editions of Harrison’s Principles of Internal Medicine, and we thank the editors for permission to reproduce Figs. 213-1 and 213-2 as well as Table 213-1 from his chapter in the 17th edition. Dr. Stamm died on December 14, 2009, and this chapter is dedicated to him.

1	Medical Virology Fred Wang, Elliott Kieff DEFINING A VIRUS Viruses are obligate intracellular parasites. They consist of a DNA or RNA genome surrounded by protein. They may also have an outer-214e Section 11 Viral DiSeaSeS: General conSiDerationS membrane lipoprotein envelope. Viruses can replicate only within cells because their nucleic acid does not encode many enzymes necessary for the metabolism of proteins, carbohydrates, or lipids or for the generation of high-energy phosphates. Typically, viral nucleic acids encode messenger RNA (mRNA) and proteins necessary for replicating, packaging, and releasing progeny virus from infected cells.

1	Viruses differ from virusoids, viroids, and prions. Virusoids are nucleic acids that depend on cells and helper viruses for packaging their nucleic acids into virus-like particles. Viroids are naked, cyclical, mostly double-strand small RNAs that appear to be restricted to plants, spread from cell to cell, and are replicated by cellular RNA polymerase II. Prions (Chap. 453e) are abnormal proteins that propagate and cause disease by altering the structure of a normal cell protein. Prions cause neurodegenerative diseases such as Creutzfeldt-Jakob disease, Gerstmann-Straüssler disease, kuru, and human or bovine spongiform encephalopathy (“mad cow disease”).

1	Viral genomes may consist of singleor double-strand DNA, singleor double-strand RNA, single-strand or segmented antisense RNA, or double-strand segmented RNA. Viral nucleic acids may encode only a few genes or more than 100. Sense-strand viral RNA genomes can be translated directly into protein, whereas antisense RNAs must be copied into translatable RNA. Sense and antisense genomes are also referred to as positive-strand and negative-strand genomes, respectively. Viral nucleic acid is usually associated with virus-encoded nucleoprotein(s) in the virus core. Viral nucleic acids and nucleoproteins are almost always enclosed in a protein capsid. Because of the limited genetic complexity of viruses, their capsids are usually composed of multimers of identical capsomeres made up of one or a few proteins. Capsids have icosahedral or helical symmetry. Icosahedral capsid structures approximate spheres and have two-, three-, or fivefold axes of symmetry, whereas helical capsid structures have

1	proteins. Capsids have icosahedral or helical symmetry. Icosahedral capsid structures approximate spheres and have two-, three-, or fivefold axes of symmetry, whereas helical capsid structures have only a twofold axis of symmetry. The nucleic acid, nucleoprotein(s), and protein capsid together are called a nucleocapsid.

1	Many viruses are composed of a nucleic acid core and a capsid. For these viruses, the outer capsid surface mediates contact with uninfected cells’ plasma membranes. Other viruses are more complex and have an outer phospholipid, cholesterol, glycoprotein, and glycolipid envelope that is derived from virus-modified infected cell membranes. Cell nuclear, endoplasmic reticulum, Golgi, or plasma membranes that become parts of the viral envelope have usually been modified during infection by the insertion of virus-encoded glycoproteins, which mediate contact of enveloped virus with uninfected cell surfaces. Matrix or tegument proteins may fill the space between the nucleocapsid and the outer envelope of the virus.

1	Enveloped viruses are usually sensitive to lipid solvents or detergents that can dissolve the envelope, whereas viruses with protein nucleocapsid exteriors may be somewhat detergent resistant. A schematic diagram of large and complex herpesviruses is shown in Fig. 214e-1. Structures of prototypical pathogenic human viruses are described in Table 214e-1. The relative sizes and structures of typical pathogenic human viruses are shown in Fig. 214e-2. FIGURE 214e-1 Schematic diagram of an enveloped herpesvirus with an icosahedral nucleocapsid. The approximate respective dimensions of the nucleocapsid and the enveloped particles are 110 and 180 nm. The capsid is composed of 162 capsomeres: 150 with sixfold and 12 with fivefold axes of symmetry.

1	As is apparent from Table 214e-1 and Fig. 214e-2, the classification of viruses into orders and families is based on nucleic acid composition, nucleocapsid size and symmetry, and presence or absence of an envelope. Viruses of a single family have similar structures and may be morphologically indistinguishable in electron micrographs. Subclassification into genera depends on similarity in epidemiology, biologic effects, and nucleic acid sequence. Most viruses that infect humans have a common name related to their pathologic effects or the circumstances of their discovery. In addition, formal species names—consisting of the name of the host followed by the family or genus of the virus and a number—have been assigned by the International Committee on Taxonomy of Viruses. This dual terminology can cause confusion when viruses are referred to by either name—e.g., varicella-zoster virus (VZV) or human herpesvirus 3 (HHV-3).

1	STAGES OF VIRAL INFECTION OF CELLS IN CULTURE Viral Interactions with Cell Surfaces and Cell Entry To deliver its nucleic acid payload to the cell cytoplasm or nucleoplasm, a virus must overcome barriers posed by the cell’s plasma and cytoplasmic membranes. Infection is frequently initiated by weak electrostatic or hydrophobic interactions with the cell surface. Subsequent stronger, more specific attachment to a cell plasma membrane protein, carbohydrate, glycolipid, heparan sulfate proteoglycan, or sialic acid enables stable binding to a specific cell surface “receptor” that mediates fusion with the cell plasma membrane (see Table 145e-1). Receptor binding is often augmented by a viral surface protein interaction with more than one cell surface protein or co-receptor. Receptors and co-receptors are important determinants of the species and cell type that a virus can infect. For example, the HIV envelope glycoprotein binds to the T cell surface protein CD4 and then engages a chemokine

1	are important determinants of the species and cell type that a virus can infect. For example, the HIV envelope glycoprotein binds to the T cell surface protein CD4 and then engages a chemokine receptor that is the definitive co-receptor for the virus and mediates entry into the cell cytoplasm. The Epstein-Barr virus (EBV) glycoprotein gp350 binds to the B lymphocyte complement receptor CD21 and then uses a major histocompatibility complex (MHC) class II molecule as a co-receptor and an integrin for definitive entry.

1	Family Representative Viruses Type of RNA/DNA Lipid Envelope Hepadnaviridae Hepatitis B virus ds DNA with ss portions Yes Parvoviridae Parvovirus B19 ss DNA No Papillomaviridae Human papillomaviruses ds DNA No aIncluding the coronaviruses causing severe acute respiratory syndrome (SARS) and Middle Eastern respiratory syndrome (MERS). bAlso called human herpesvirus 1 (HHV-1) and HHV-2, respectively. cAlso called HHV-3. dAlso called HHV-4. eAlso called HHV-5. fAlso called HHV-8. Abbreviations: ds, double-strand; ss, single-strand. Picornaviridae Genome size (kb) 7.2–8.4 Envelope No Capsid symmetry Icosahedral FIGURE 214e-2 Schematic diagrams of the major virus families including species that infect humans. The viruses are grouped by genome type and are drawn approximately to scale. Prototype viruses of each family that cause human disease are listed in Table 214e-1.

1	Viruses have evolved a wide range of strategies to enter cells. Influenza virus has an outer-membrane hemagglutinin glycoprotein that binds to sialic acid on respiratory tract cell plasma membranes. The hemagglutinin mediates adsorption to cell membranes, receptor aggregation, and endocytosis. As the endosome pH decreases in the cell cytoplasm, the influenza hemagglutinin conformation changes, enabling hydrophobic helices, which are initially at the base of the hemagglutinin, to extend, interacting and fusing with the endosome membrane and thereby releasing the viral genome into the cell cytoplasm. The influenza virus M2 membrane channel protein has a key role in lowering endosome pH and permitting virus and cell membrane fusion.

1	Nonenveloped viruses (e.g., human papillomaviruses [HPVs]) and some enveloped viruses have evolved to partially fuse with cell plasma membrane receptors and be internalized into endosomes. The low pH in an endosome can then trigger virus membrane or capsid fusion with the endocytic membrane, releasing viral DNA into the cytoplasm to initiate infection.

1	Hydrophobic interactions required for fusion can be susceptible to chemical inhibition or blockade. The HIV envelope glycoprotein gp120 is associated with gp41 on the viral surface. HIV gp120 binding to CD4 and then to specific chemokine receptors results in conformational changes that allow gp41 to initiate cell membrane fusion. The anti-HIV drug enfuvirtide is a small peptide derived from the gp41 structure. Enfuvirtide binds to gp41 and prevents conformational changes required for fusion. In contrast, maraviroc prevents virus entry by binding to the CCR5 receptor, thereby blocking gp120 binding to CCR5 and preventing gp120 fusion with CCR5.

1	Viral Gene Expression and Replication After uncoating and release of viral nucleoprotein into the cytoplasm, the viral genome is transported to sites of expression and replication. To produce infectious progeny, viruses must produce proteins necessary for replicating their nucleic acids as well as structural proteins necessary for coating their nucleic acids and for assembling nucleic acids and proteins into progeny virus. Different viruses use different strategies and gene repertoires to accomplish these goals. Most DNA viruses, except for poxviruses, replicate their nucleic acid and assemble into nucleocapsids in the cell nucleus. RNA viruses, except for influenza viruses, transcribe and replicate their RNA and assemble in the cytoplasm before envelopment at the cell plasma membrane. The replication strategies of DNA and RNA viruses and of positiveand negative-strand RNA viruses are presented and discussed separately below. Medically important viruses of each group are used for

1	The replication strategies of DNA and RNA viruses and of positiveand negative-strand RNA viruses are presented and discussed separately below. Medically important viruses of each group are used for illustrative purposes.

1	Positive-strand rna viruses RNA viruses of medical importance include positive-strand picornaviruses, flaviviruses, togaviruses, caliciviruses, and coronaviruses. Genome RNA from positive-strand RNA viruses is released into the cytoplasm without associated enzymes. Cell ribosomes recognize and associate with the viral genome’s internal ribosome entry sequence and translate a virus-encoded polyprotein. Proteases within the polyprotein cleave out the viral RNA polymerase and other viral proteins necessary for replication. Antigenomic RNA is next transcribed from the genome RNA template. Positive-strand genomes and mRNAs are then transcribed from the antigenome RNA by the viral RNA polymerase and are translated into capsid proteins. Genomic RNA is encapsidated in the cytoplasm and released as the infected cell undergoes lysis.

1	negative-strand rna viruses Medically important negative-strand RNA viruses include rhabdoviruses, filoviruses, paramyxoviruses, orthomyxoviruses, and bunyaviruses. The genomes of negative-strand viruses are frequently segmented. Negative-strand RNA viral genomes are released into the cytoplasm with an associated RNA polymerase and one or more polymerase accessory proteins. The viral RNA polymerase transcribes mRNAs as well as full-length antigenome RNA, which is the template for genome RNA replication. Viral mRNAs encode the viral RNA polymerase and accessory factors as well as viral structural proteins. Except for influenza virus, which transcribes its mRNAs and antigenome RNAs in the cell nucleus, negative-strand RNA viruses replicate entirely in the cytoplasm. All negative-strand RNA viruses, including influenza viruses, assemble in the cytoplasm.

1	double-strand segmented rna viruses Double-strand RNA viruses are taxonomically grouped in the family Reoviridae. The medically important viruses in this group are rotaviruses and Colorado tick fever virus. Reovirus genomes have 10–12 RNA segments. Reovirus particles contain an RNA polymerase complex. These viruses replicate and assemble in the cell cytoplasm.

1	dna viruses Medically important DNA viruses include parvoviruses, which have small single-strand DNA genomes and cause transient arthritis, and polyomaviruses, including the smaller polyomaviruses such as JC virus, which causes progressive multifocal leukoencephalopathy in immunocompromised patients; BK virus; and Merkel cell polyomavirus. The larger HPVs cause warts as well as cervical, penile, and oral carcinomas. The next larger DNA viruses are adenoviruses, which mostly cause transient respiratory tract and ocular inflammatory disease. The herpesviruses include eight viruses that cause a wide range of inflammatory and malignant diseases in humans. EBV is an important cause of lymphomas and Hodgkin’s disease in both immunocompromised and immunocompetent people and of nasopharyngeal carcinoma in southern Chinese and northern African populations. Cytomegalovirus (CMV) is an important cause of transplacental infections and neonatal neurologic impairment. Poxviruses, the largest DNA

1	in southern Chinese and northern African populations. Cytomegalovirus (CMV) is an important cause of transplacental infections and neonatal neurologic impairment. Poxviruses, the largest DNA viruses and the largest viruses that infect humans (barely visible by light microscopy), cause smallpox, monkeypox, and molluscum contagiosum. Aside from those of poxviruses, other DNA virus genomes enter the cell nucleus and are transcribed by cellular RNA polymerase II.

1	After receptor binding and fusion with plasma membranes or endocytic vesicle membranes, herpesvirus nucleocapsids are released into the cytoplasm with tegument proteins and are transported along microtubules to a nuclear pore. Capsids then release DNA into the nucleus.

1	DNA virus transcription and mRNA processing depend on both viral and cellular proteins. For herpes simplex virus (HSV), a viral tegument protein enters the nucleus and activates immediate-early genes, the first genes expressed after infection. Transcription of immediate-early genes requires the viral tegument protein and cell transcription factors. HSV becomes nonreplicating, or latent, in neurons because essential cell transcription factors for expression of viral immediate-early genes are docked in the cytoplasm in neurons. Heat shock or other cell stresses can cause these cell factors to enter the nucleus, activate viral gene expression, and initiate replication. This information explains HSV-1 latency in neurons and activation of replicative infection.

1	For adenoviruses and herpesviruses, transcription of immediate-early genes results in expression of early proteins necessary for viral DNA replication. Viral DNA synthesis is required to turn on late-gene expression and production of viral structural components. The HPVs, polyomaviruses, and parvoviruses are not dependent on transactivators encoded from the viral genome for early-gene transcription. Instead, their early genes have upstream enhancing elements that bind cell transcription factors. The early genes encode proteins that are necessary for viral DNA synthesis and late-gene transcription. DNA virus late genes encode structural proteins necessary for viral assembly and for viral egress from the infected cell. Late-gene transcription is continuously dependent on DNA replication. Therefore, inhibitors of DNA replication also stop late-gene transcription.

1	Each DNA virus family uses unique mechanisms for replicating its DNA. Adenovirus and herpesvirus DNAs are linear in the virion. Adenovirus DNA remains linear in infected cells and replicates as a linear genome, using an initiator protein–DNA complex. In contrast, herpesvirus DNA circularizes in the infected cell, and genomes replicate into linear concatemers through a “rolling-circle” mechanism. Full-length DNA genomes are cleaved and packaged into virus. Herpesviruses encode a DNA polymerase and at least six other viral proteins necessary for viral DNA replication. Acyclovir and ganciclovir prevent viral DNA synthesis when they are phosphorylated and incorporated into DNA by the viral polymerase. Herpesviruses also encode enzymes that increase the deoxynucleotide triphosphate pools. HPV and polyomavirus DNAs are circular both within the virus and in infected cells. These genomes are reproduced by cellular DNA replication enzymes and remain circular through replication and packaging.

1	and polyomavirus DNAs are circular both within the virus and in infected cells. These genomes are reproduced by cellular DNA replication enzymes and remain circular through replication and packaging. HPV and polyomavirus early proteins are necessary for DNA replication in both latent and viral replicative phases. Early viral proteins stimulate cells to remain in cycle, facilitating viral DNA replication.

1	Parvoviruses have negative single-strand DNA genomes and are the smallest DNA viruses. Their genomes are half the size of HPV genomes and include only two genes. The replication of autonomous parvoviruses, such as B19, depends on cellular DNA replication and requires the virus-encoded Rep protein. Other parvoviruses, such as adeno-associated virus (AAV), are not autonomous and require helper viruses of the adenovirus or herpesvirus family for their replication. AAV is being used as a potentially safe human gene therapy vector because its replication protein causes integration at a single chromosome site. The small genome size limits the range of proteins that can be expressed from AAV vectors.

1	As stated above, poxviruses are the largest DNA viruses. They are unique among DNA viruses in replicating and assembling in the cytoplasm. To accomplish cytoplasmic replication, poxviruses encode transcription factors, an RNA polymerase II orthologue, enzymes for RNA capping, enzymes for RNA polyadenylation, and enzymes for viral DNA synthesis. Poxvirus DNA also has a unique structure. The double-strand linear DNA is covalently linked at the ends, making a covalently closed double-strand circular genome. Replication of the circular genomes is initiated by nicking in inverted repeats at the ends of the linear DNA. During DNA replication, the genome is cleaved within the terminal inverted repeats, and the inverted repeats self-prime complementary-strand synthesis by the virus-encoded DNA polymerase. Like herpesviruses, poxviruses encode several enzymes that increase deoxynucleotide triphosphate precursor levels and thus facilitate viral DNA synthesis.

1	viruses that use both rna and dna genomes in their life cycle Retroviruses, including HIV, are RNA viruses that use a DNA intermediate to replicate their genomes. In contrast, hepatitis B virus (HBV) is a DNA virus that uses an RNA intermediate to replicate its genome. Thus these viruses are not purely RNA or DNA viruses. Retroviruses are RNA viruses with two identical sense-strand genomes and associated reverse transcriptase and integrase enzymes. Retroviruses differ from all other viruses in that they reverse-transcribe themselves into partially duplicated double-strand DNA copies and then routinely integrate into the host genome as part of their persistence and replication strategies. Inhibitors of reverse transcriptase (e.g., zidovudine) or integrase (e.g., raltegravir) are now commonly used as antiviral treatments for HIV infection. Integration of remnants and even complete copies of simple retrovirus DNAs into the human genome raises the possibility of replication-competent

1	used as antiviral treatments for HIV infection. Integration of remnants and even complete copies of simple retrovirus DNAs into the human genome raises the possibility of replication-competent simple human retroviruses. However, endogenous human retrovirus replication has not been documented or associated with any disease. Integrated, replication-competent retroviral DNAs are also present in many animal species, such as pigs. These porcine retroviruses are a potential cause for concern in xenotransplantation because retrovirus replication could cause disease in humans.

1	Cellular RNA polymerase II and transcription factors regulate transcription from the integrated provirus DNA genome. Some retroviruses also encode regulators of transcription and RNA processing, such as Tax and Rex in human T lymphotropic virus (HTLV) types 1 and 2. HIV-1 and HIV-2 have orthologous Tat and Rev genes as well as the additional accessory proteins Vpr, Vpu, and Vif, which are important for efficient infection and immune escape. Full-length pro-viral transcripts are made from a promoter in the viral terminal repeat and serve as both genome RNAs that are packaged in the nucleocapsids and differentially spliced mRNAs that encode for the virus Gag protein, polymerase/integrase protein, and envelope glycoprotein. The Gag protein includes a protease that cleaves it into several components, including a viral matrix protein that coats the viral RNA. Viral RNA polymerase/integrase, matrix protein, and cellular tRNAs are key components in the viral nucleocapsid. Protease inhibitors

1	including a viral matrix protein that coats the viral RNA. Viral RNA polymerase/integrase, matrix protein, and cellular tRNAs are key components in the viral nucleocapsid. Protease inhibitors have been developed as effective agents against infections caused by HIV (e.g., saquinavir) or hepatitis C virus (HCV) (e.g., telaprevir).

1	HBV replication is unique in several respects. The HBV genome is a partially double-strand DNA genome that is repaired in infected cells to a fully double-strand circular DNA by the virion polymerase. Viral mRNAs are transcribed from the closed circular viral episome by the cellular RNA polymerase II and are translated to yield HBV proteins, including core protein, surface antigen, and polymerase. In addition, a full-genome-length mRNA is packaged into viral core particles in the cytoplasm of infected cells as an intermediate for viral DNA replication. This RNA associates with the viral polymerase, which also has reverse transcriptase activity and converts the full-length encapsidated RNA genome into partially double-strand DNA. Thus, nucleos(t)ide analogs that inhibit reverse transcription (e.g., tenofovir) are commonly used to treat HBV infection. HBV is believed to mature by budding through the cell’s plasma membrane, which has been modified by the insertion of viral surface

1	(e.g., tenofovir) are commonly used to treat HBV infection. HBV is believed to mature by budding through the cell’s plasma membrane, which has been modified by the insertion of viral surface antigen protein.

1	Viral Assembly and Egress For most viruses, nucleic acid and structural protein synthesis is accompanied by the assembly of protein and nucleic acid complexes. The assembly and egress of mature infectious virus mark the end of the eclipse phase of infection, during which infectious virus cannot be recovered from the infected cell. Nucleic acids from RNA viruses and poxviruses assemble into nucleocapsids in the cytoplasm. For all DNA viruses except poxviruses, viral DNA assembles into nucleocapsids in the nucleus. In general, the capsid proteins of viruses with icosahedral nucleocapsids can self-assemble into densely packed and highly ordered capsid structures. Herpesviruses require an assemblin protein as a scaffold for capsid assembly. Viral nucleic acid then spools into the assembled capsid. For herpesviruses, a full unit of the viral DNA genome is packaged into the capsid, and a capsid-associated nuclease cleaves the viral DNA at both ends. In the case of viruses with helical

1	capsid. For herpesviruses, a full unit of the viral DNA genome is packaged into the capsid, and a capsid-associated nuclease cleaves the viral DNA at both ends. In the case of viruses with helical nucleocapsids, the protein component appears to assemble around the nucleic acid, which contributes to capsid organization.

1	Viruses must egress from the infected cell and not bind back to their receptor(s) on the outer surface of the plasma membrane. Viruses can acquire envelopes from cytoplasmic membranes or by budding through the cell’s plasma membrane. Excess viral membrane glycoproteins are synthesized to saturate cell receptors and facilitate separation of the virus from the infected cell. Some viruses encode membrane proteins with enzymatic activity for receptor destruction. Influenza virus, for example, encodes a glycoprotein with neuraminidase activity. Neuraminidase destroys sialic acid on the infected cell’s plasma membrane so that newly released virus does not get stuck to the dying cell. Oseltamivir and zanamivir are neuraminidase inhibitors that are used to treat or provide prophylaxis for influenza virus infection. Herpesvirus nucleocapsids acquire an initial envelope by assembling in the nucleus and then budding through the nuclear membrane into the endoplasmic reticular space. The initially

1	virus infection. Herpesvirus nucleocapsids acquire an initial envelope by assembling in the nucleus and then budding through the nuclear membrane into the endoplasmic reticular space. The initially enveloped herpesvirus is then de-enveloped and released from the cell either by exocytosis or by re-envelopment at the plasma membrane. Nonenveloped viruses depend on the death and dissolution of the infected cell for their release.

1	Hundreds or thousands of progeny may be produced from a single virus-infected cell. Many particles partially assemble and never mature into virions. Many mature-appearing virions are imperfect and have only incomplete or nonfunctional genomes. Despite the inefficiency of assembly, a typical virus-infected cell releases 10–1000 infectious progeny. Some of these progeny may contain genomes that differ from those of the virus that infected the cell. Smaller, “defective” viral genomes have been noted with the replication of many RNA and DNA viruses. Virions with defective genomes can be produced in large numbers through packaging of incompletely synthesized nucleic acid. Adenovirus packaging is notoriously inefficient, and a high ratio of particle to infectious virus may limit the amount of recombinant adenovirus that can be administered for gene therapy since the immunogenicity of defective particles may contribute to adverse effects.

1	Changes in viral genomes can lead to mutant viruses of medical significance. In general, viral nucleic acid replication is more error-prone than cellular nucleic acid replication. RNA polymerases and reverse transcriptases are significantly more error-prone than DNA polymerases. Mutations can also be introduced into the HIV genome by APOBEC3G, a cellular protein that is packaged in the virion. APOBEC3G deaminates cytidine in the virion RNA to uridine. When reverse transcriptase subsequently uses the altered virion RNA as a template in the infected cell, a guanosine-to-adenosine mutation is introduced into the proviral DNA. Mutations resulting in less efficient viral growth, or fitness, may be detrimental to the virus. HIV-encoded Vif blocks APOBEC3G activity in the virion, inhibiting the debilitating effects of hypermutation on genetic integrity. Nevertheless, mutations resulting in evasion of the host immune response or resistance to antiviral drugs are preferentially selected in

1	debilitating effects of hypermutation on genetic integrity. Nevertheless, mutations resulting in evasion of the host immune response or resistance to antiviral drugs are preferentially selected in patients, with the consequent perpetuation of infection. Viral genomes can also be altered by recombination or reassortment between two related viruses in a single infected cell. Although this occurrence is unusual under most circumstances of natural infection, the genome changes can be substantial and can significantly alter virulence or epidemiology. Reassortment of the avian or mammalian influenza A hemagglutinin gene into a human influenza background can result in the emergence of new epidemic or pandemic influenza A strains.

1	Viruses frequently have genes encoding proteins that are not directly involved in replication or packaging of the viral nucleic acid, in virion assembly, or in regulation of the transcription of viral genes involved in those processes. Most of these proteins fall into five classes: can be efficiently transcribed or translated; (3) proteins that promote cell survival or inhibit apoptosis so that progeny virus can mature and escape from the infected cell; (4) proteins that inhibit the host interferon response; and (5) proteins that downregulate host inflammatory or immune responses so that viral infection can proceed in an infected person to the extent consistent with the survival of the virus and its efficient transmission to a new host. More complex viruses of the poxvirus or herpesvirus family encode many proteins that serve these functions. Some of these viral proteins have motifs similar to those of cellular proteins, while others are quite novel. Virology has increasingly focused

1	family encode many proteins that serve these functions. Some of these viral proteins have motifs similar to those of cellular proteins, while others are quite novel. Virology has increasingly focused on these more sophisticated strategies evolved by viruses to permit the establishment of long-term infection in humans and other animals. These strategies often provide unique insights into the control of cell growth, cell survival, macromolecular synthesis, proteolytic processing, immune or inflammatory suppression, immune resistance, cytokine mimicry, or cytokine blockade.

1	MicroRNAs (miRNAs) are small noncoding RNAs that can regulate gene expression at the posttranscriptional level by targeting— and usually silencing—mRNAs. miRNAs were initially discovered in plants and plant viruses, where they alter expression of cell defensins. Herpesviruses are especially rich in miRNAs; for example, at least 23 miRNAs have been identified in EBV and 11 in CMV. Adenovirus and polyomavirus miRNAs have also been described. Increasing data indicate that animal viruses encode miRNAs to alter the growth and survival of host cells and the innate and acquired immune responses.

1	The concept of host range was originally based on the cell types in which a virus replicates in tissue culture. For the most part, the host range is limited by specific cell-surface proteins required for viral adsorption or penetration—i.e., to the cell types that express receptors or co-receptors for a specific virus. Another common basis for host-range limitation is the degree of transcriptional activity from viral promoters in different cell types. Most DNA viruses depend not only on cellular RNA polymerase II and the basal components of the cellular transcription complex but also on activated components and transcriptional accessory factors, both of which differ among differentiated tissues, among cells at various phases of the cell cycle, and between resting and cycling cells.

1	The importance of host range factors is illustrated by the effects of specific host determinants that limit the replication of influenza virus with avian or porcine hemagglutinins in humans. These viral proteins have adapted to bind avian or porcine sialic acids, and spread of avian or porcine influenza viruses in human populations is limited by their ability to infect human cells.

1	The replication of almost all viruses has adverse effects on the infected cell, inhibiting cellular synthesis of DNA, RNA, or proteins through efficient competition for key substrates and enzymatic processes. These general inhibitory effects enable viruses to nonspecifically limit components of innate host resistance, such as interferon (IFN) production. Viruses can specifically inhibit host protein synthesis by attacking a component of the translational initiation complex— frequently, a component that is not required for efficient translation of viral RNAs. Poliovirus protease 2A, for example, cleaves a cellular component of the complex that ordinarily facilitates translation of cellular mRNAs by interacting with their cap structure. Poliovirus RNA is efficiently translated without a cap because it has an internal ribosome entry sequence. Influenza virus inhibits the processing of mRNA by snatching cap structures from nascent cellular RNAs and using them as primers in the synthesis

1	because it has an internal ribosome entry sequence. Influenza virus inhibits the processing of mRNA by snatching cap structures from nascent cellular RNAs and using them as primers in the synthesis of viral mRNA. HSV has a virion tegument protein that inhibits cellular mRNA translation.

1	Apoptosis is the expected consequence of virus-induced inhibition of cellular macromolecular synthesis and viral nucleic acid replication. Although the induction of apoptosis may be important for the release of some viruses (particularly nonenveloped viruses), many viruses have acquired genes or parts of genes that enable them to forestall infected-cell death. This delay increases the yield from viral replication. Adenoviruses and herpesviruses encode analogues of the cellular Bc12 protein, which block mitochondrial enhancement of proapoptotic stimuli. Poxviruses and some herpesviruses also encode caspase inhibitors. Many viruses, including HPVs and adenoviruses, encode proteins that inhibit p53 or its downstream proapoptotic effects.

1	The capsid and envelope of a virus protect the genome and enable efficient transmission of the virus from cell to cell and to new prospective hosts. Most common viral infections are spread by direct contact, by ingestion of contaminated water or food, or by inhalation of aerosolized particles. In all these situations, infection begins on an epithelial or mucosal surface and spreads along the mucosa and into deeper tissues. Infection may spread to cells that can enter blood vessels, lymphatics, or neural circuits. HBV, HCV, HTLV, and HIV are dependent on transmission by parenteral inoculation. Some viruses are transmitted only between humans. The dependence of smallpox virus and poliovirus infections on interhuman transmission makes it feasible to eliminate these viruses from human circulation by mass vaccination. Herpesviruses also survive by interhuman transmission but may be more difficult to eliminate because they establish persistent latent infection in humans and continuously

1	by mass vaccination. Herpesviruses also survive by interhuman transmission but may be more difficult to eliminate because they establish persistent latent infection in humans and continuously reactivate to infect new and naïve generations.

1	Animals are also important reservoirs and vectors for transmission of viruses causing human disease. Insect vectors can mediate parenteral transfer of viruses that reach high titers in animal or human hosts. Arboviruses are parenterally transmitted from mammalian species to humans by mosquito vectors. Herpes B, monkeypox, rabies, and viral hemorrhagic fevers are other examples of zoonotic infections caused by direct contact with animals, animal tissues, or arthropod vectors.

1	Initial viral infections usually last for several days or weeks. During this period, the concentration of virus at sites of infection rises and then falls, usually to unmeasurable levels. The rise and fall of viral replication at a given site depend on local innate immune responses and the access of systemic antibody and cell immune effectors to the virus. Typically, primary infections with enteroviruses, mumps virus, measles virus, rubella virus, rotavirus, influenza virus, AAV, adenovirus, HSV, and VZV are cleared from almost all sites within 3–4 weeks. Some viruses are especially proficient in altering or evading innate and acquired immune responses. Primary infection with AAV, EBV, or CMV can last for several months. Characteristically, primary infections due to HBV, HCV, hepatitis D virus (HDV), HIV, HPV, and molluscum contagiosum virus (MCV) extend beyond several weeks. For some of these viruses (e.g., HPV, HBV, HCV, HDV, and MCV), the manifestations of primary infection are

1	D virus (HDV), HIV, HPV, and molluscum contagiosum virus (MCV) extend beyond several weeks. For some of these viruses (e.g., HPV, HBV, HCV, HDV, and MCV), the manifestations of primary infection are almost indistinguishable from the persistent phase.

1	Disease manifestations usually arise as a consequence of viral replication, infected-cell injury or death, and local inflammatory and innate immune responses. Disease severity may not necessarily correlate with the level of viral replication alone. For example, the clinical manifestations of intense primary infection with poliovirus, enterovirus, rabies virus, measles virus, mumps virus, or HSV at mucosal surfaces may be inapparent or relatively mild, whereas limited replication in neural cells can have dramatic consequences. Similarly, rubella virus or CMV infections in utero or neonatal HSV infections may have much more devastating effects than infections in adults.

1	Primary infections are cleared by nonspecific innate and specific adaptive immune responses. Thereafter, an immunocompetent host is usually immune to the disease manifestations of reinfection by the same virus. Immunity frequently does not prevent transient surface colonization on reexposure, persistent colonization, or even limited deeper infection.

1	Relatively few viruses cause persistent or latent infections. HBV, HCV, rabies virus, measles virus, HIV, HTLV, HPV, HHVs, and MCV are notable exceptions. The mechanisms for persistent infection vary. HCV RNA polymerase and HIV reverse transcriptase are error-prone and generate variant genomes. Genome variation can be sufficient to permit evasion of host immune responses, thereby allowing persistent infection. HIV is also directly immunosuppressive, depleting CD4+ T lymphocytes and compromising CD8+ cytotoxic T cell immune responsiveness. Moreover, HIV encodes the Nef protein, which downmodulates MHC class I expression, rendering HIV-infected cells partially resistant to immune CD8+ T cell lysis.

1	DNA viruses have low mutation rates. Their persistence in human populations usually depends on their ability to establish latent infection in some cells, to reactivate from latency, and then to replicate at epithelial surfaces. Latency is defined as a state of infection in which virus is not replicating, viral genes associated with lytic infection are not expressed, and infectious virus is not made. The complete viral genome is present and may be replicated by cellular DNA polymerase in conjunction with replication of the cell’s genome. HPVs establish latent infection in basal epithelial cells. The latently infected basal cell replicates, along with the HPV episome, by using cellular DNA polymerase. Some of the progeny cells provide new latently infected basal cells, whereas others go on to squamous differentiation. Infected cells that differentiate to squamous cells become permissive for lytic viral infection. Herpesviruses establish latent infection in nonreplicating neural cells

1	to squamous differentiation. Infected cells that differentiate to squamous cells become permissive for lytic viral infection. Herpesviruses establish latent infection in nonreplicating neural cells (HSV and VZV) or in replicating cells of hematopoietic lineages (EBV, CMV, HHV-6, HHV-7, and Kaposi’s sarcoma–associated herpesvirus [KSHV, also known as HHV-8]). In their latent stage, HPV and herpesvirus genomes are largely hidden from the normal immune response. Reactivated HPV and herpesvirus infections escape immediate and effective immune responses in highly immune hosts by inhibiting host innate immune and inflammatory responses. In addition, HPV, HSV, and VZV are somewhat protected because they replicate in the middle and upper layers of the squamous epithelium—sites not routinely visited by cells that mediate or amplify immune and inflammatory responses. HSV and CMV are also known to encode proteins that downregulate MHC class I expression and antigenic peptide presentation,

1	visited by cells that mediate or amplify immune and inflammatory responses. HSV and CMV are also known to encode proteins that downregulate MHC class I expression and antigenic peptide presentation, enabling infected cells to escape recognition by and cytotoxic effects of CD8+ T lymphocytes.

1	Like other poxviruses, MCV cannot establish latent infection. This virus causes persistent infection in hypertrophic skin lesions that last for months or years. MCV encodes a chemokine homologue that probably blocks inflammatory responses, an MHC class I analogue that blocks cytotoxic T lymphocyte attack, and inhibitors of cell death that prolong infected-cell viability. Persistent viral infection is estimated to be the root cause of as many as 20% of human malignancies. Cancer is an accidental and highly unusual or long-term effect of oncogenic human viral infection. With most “oncogenic viruses,” infection is a critical and ultimately determinative early step in carcinogenesis. Latent HPV infection can block cell death and cause cervical cells to proliferate. A virus-infected cell with an integrated HPV genome overexpressing E6 and E7 undergoes subsequent cellular genetic changes that enhance autonomous malignant cell growth.

1	Most hepatocellular carcinoma is believed to be caused by chronic inflammatory, immune, and regenerative responses to HBV or HCV infection. Epidemiologic data firmly link HBV and HCV infections to hepatocellular carcinoma. These infections elicit repetitive cycles of virus-induced liver injury followed by tissue repair and regeneration. Over decades, chronic viral infection, repetitive tissue regeneration, and acquired chromosomal changes can result in proliferative nodules. Further chromosomal mutations can lead to the degeneration of cells in a proliferating nodule into hepatocellular carcinoma. In rare instances, HBV DNA integrates into cellular DNA, promoting overexpression of a cell gene that can also contribute to oncogenesis.

1	Most cervical carcinoma is caused by persistent infection with “highrisk” HPV type 16 or 18. In contrast to HBV and HCV infections, which stimulate cell growth as a consequence of virus-induced cell death, HPV type 16 or 18 proteins E6 and E7 destroy p53 and pRB, respectively. Elimination of these key tumor-suppressive cell proteins increases cell growth, cell survival, and cell genome instability. However, like HBV and HCV infections, HPV infection alone is not sufficient for carcinogenesis. Cervical carcinoma is inevitably associated with persistent HPV infection and integration of the HPV genome into chromosomal DNA. Integrations that result in overexpression of E6 and E7 from HPV type 16 or 18 cause more profound changes in cell growth and survival and permit subsequent chromosomal changes that result in cervical carcinoma.

1	EBV is the most unusual oncogenic virus in that normal B cell infection results in latency with expression of viral proteins that can cause endless B lymphocyte growth. In almost all humans, strong CD4+ and CD8+ T cell immune responses to the antigenic EBV latent-infection nuclear proteins prevent uncontrolled B cell lymphoproliferation. However, when humans are severely immunosuppressed by transplantation-associated medication, HIV infection, or genetic immune deficiencies, EBV-induced B cell malignancies can emerge.

1	EBV infection also has a role in the long-term development of B lymphocyte and epithelial cell malignancies. Persistent EBV infection with expression of an EBV latency-associated integral membrane protein (LMP1) in latently infected epithelial cells appears to be a critical early step in the evolution of anaplastic nasopharyngeal carcinoma, a common malignancy in populations in southern China and northern Africa. Genomic instability and chromosomal abnormalities also contribute to the development of EBV-associated nasopharyngeal carcinoma. EBV is an important cause of Hodgkin’s lymphoma. High-level expression of LMP1 or LMP2 in Reed-Sternberg cells is a hallmark in up to 50% of Hodgkin’s lymphoma cases. LMP1-induced nuclear factor-κB (NF-κB) activity may prolong the survival of defective B cells that are normally eliminated by apoptosis, thereby allowing other genetic changes leading to the development of malignant Reed-Sternberg cells.

1	The HTLV-1 Tax and Rex proteins are critical to the initiation of cutaneous adult T cell lymphoma/leukemias that occur long after primary HTLV-1 infection. Tax-induced NF-κB activation may contribute to cytokine production, infected-cell survival, and eventual outgrowth of malignant cells. Molecular data confirm the presence of KSHV DNA in all Kaposi’s tumors, including those associated with HIV infection, transplantation, and familial transmission. KSHV infection is also etiologically implicated in pleural-effusion lymphomas and multicentric Castleman’s disease, which are more common among HIV-infected than among HIV-uninfected people. KSHV also has a virus-encoded cyclin, an IFN regulatory factor, and a latency-associated nuclear antigen that are implicated in increased-cell proliferation and survival.

1	Evidence supporting a causal role for viral infection in all of these malignancies includes (1) epidemiologic data, (2) the presence of viral DNA in all tumor cells, (3) the ability of the viruses to transform human cells in culture, (4) the results of in vitro cell culture–based assays that reveal transforming effects of specific viral genes on cell growth or survival, (5) pathologic data indicating the expression of transforming viral genes in premalignant or malignant cells in vivo, (6) the demonstration in animal models that these viral genes can cause malignant cell growth, and (7) the ability of virus-specific vaccines to reduce the incidence of virus-associated malignancy.

1	Virus-related malignancies provide an opportunity to expand our understanding of the biologic mechanisms important in the development of cancer. They also offer unique opportunities to develop diagnostics, vaccines, or therapeutics that could prevent or specifically treat cancers associated with viral infection. Widespread immunization against hepatitis B has resulted in a decreased prevalence of HBV-associated hepatitis and will probably prevent most HBV-related liver cancers. Current HPV vaccines can reduce rates of colonization with high-risk HPV strains and thereby decrease the risk of cervical cancer. The successful use of in vitro–expanded EBV-specific T cell populations to treat or prevent EBV-associated posttransplantation lymphoproliferative disease demonstrates the potential of immunoprevention or immunotherapy against virus-associated cancers.

1	Resistance to viral infections is initially provided by factors that are not virus-specific. Physical protection is afforded by the cornified layers of the skin and by mucous secretions that continuously sweep over mucosal surfaces. Once the first cell is infected, IFNs are induced and confer resistance to RNA virus replication. Viral infection may also trigger the release of other cytokines from infected cells. These cytokines may be chemotactic to inflammatory and immune cells. Viral protein epitopes expressed on the cell surface in the context of MHC class I and II proteins can stimulate the expansion of T cell populations with receptors that can recognize virus-encoded peptides presented on the cell surface by MHC class I proteins. Cytokines and antigens released by virus-induced cell death further attract inflammatory cells, dendritic cells, granulocytes, natural killer (NK) cells, and B lymphocytes to sites of infection and to draining lymph nodes. IFNs and NK cells are

1	cell death further attract inflammatory cells, dendritic cells, granulocytes, natural killer (NK) cells, and B lymphocytes to sites of infection and to draining lymph nodes. IFNs and NK cells are particularly important in containing viral infection for the first several days. Granulocytes and macrophages are also important in the phagocytosis and degradation of viruses, especially after an initial antibody response.

1	By 7–10 days after infection, virus-specific antibody responses, virus-specific human leukocyte antigen (HLA) class II–restricted CD4+ helper T lymphocyte responses, and virus-specific HLA class I–restricted CD8+ cytotoxic T lymphocyte responses develop. These responses, whose magnitude typically increases over the second and third weeks of infection, are important for rapid recovery. Also between the second and third weeks, the antibody type usually changes from IgM to IgG; IgG or IgA antibody can then be detected at infected mucosal surfaces. Antibody may directly neutralize virus by binding to its surface and preventing cell attachment or penetration. Complement can significantly enhance antibody-mediated virus neutralization. Antibody and complement can also lyse virus-infected cells that express viral membrane proteins on the cell surface. Cells infected with a replicating enveloped virus usually express the virus-envelope glycoproteins on the cell plasma membrane. Specific

1	that express viral membrane proteins on the cell surface. Cells infected with a replicating enveloped virus usually express the virus-envelope glycoproteins on the cell plasma membrane. Specific antibodies can bind to the glycoproteins, fix complement, and lyse the infected cell.

1	Antibody and CD4+/CD8+ T lymphocyte responses to viral infection can remain at high levels for several months after primary infection but usually wane over time. Low-level persistence of antibody-producing B lymphocytes and CD4+ or CD8+ T lymphocyte responses as memory cells can provide a rapid response to a second infection or an early barrier to reinfection with the same virus. Redevelopment of T cell immunity may take longer than secondary antibody responses, particularly when many years have elapsed between primary infection and reexposure. However, persistent infections or frequent reactivations from latency can result in sustained high-level T cell responses. EBV and CMV typically induce high-level CD4+ and CD8+ T cell responses that are maintained for decades after primary infection.

1	Some viruses have genes that alter innate and acquired host defenses. Adenoviruses encode small RNAs that inhibit IFN-induced, protein kinase R (PKR)–mediated shutoff of infected-cell protein synthesis. Adenovirus E1A can also directly inhibit IFN-mediated changes in cell gene transcription. Moreover, adenovirus E3 proteins prevent tumor necrosis factor (TNF)–induced cytolysis and block HLA class I synthesis by the infected cell. HSV ICP47 and CMV US11 also block class I antigen presentation. EBV encodes an interleukin (IL) 10 homologue that inhibits NK and T cell responses. Vaccinia virus encodes a soluble receptor for IFN-α and binding proteins for IFN-γ, IL-1, IL-18, and TNF, which inhibit host innate and adaptive immune responses. Vaccinia virus also encodes a caspase inhibitor that inhibits the ability of CD8+ cytotoxic T cells to kill virus-infected cells. Some poxviruses and herpes-viruses encode chemokine-binding proteins that inhibit cell inflammatory responses. The adoption

1	the ability of CD8+ cytotoxic T cells to kill virus-infected cells. Some poxviruses and herpes-viruses encode chemokine-binding proteins that inhibit cell inflammatory responses. The adoption of these strategies by viruses highlights the importance of the corresponding host resistance factors in containing viral infection and the importance of redundancy in host resistance.

1	The host inflammatory and immune responses to viral infection do not come without a price. These responses contribute to the symptoms, signs, and other pathophysiologic manifestations of viral infection. Inflammation at sites of viral infection can subvert an effective immune response and induce tissue death and dysfunction. Moreover, immune responses to viral infection could, in principle, result in immune attack upon cross-reactive epitopes on normal cells, with consequent autoimmunity.

1	All human cells can synthesize IFN-α or IFN-β in response to viral infection. These IFN responses are usually induced by the presence of double-strand viral RNA, which can be made by both RNA and DNA viruses and sensed by double-strand RNA binding proteins (e.g., PKR and RIG-I) in the cell cytoplasm. IFN-γ is not closely related to IFN-α or IFN-β and is produced mainly by NK cells and by immune T lymphocytes responding to IL-12. IFN-α and -β bind to the IFN-α receptor, whereas IFN-γ binds to a different but related receptor. Both receptors signal through receptor-associated JAK kinases and other cytoplasmic proteins, including “STAT” proteins, which are tyrosinephosphorylated by JAK kinases, translocate to the nucleus, and activate promoters for specific cell genes. Three types of antiviral effects are induced by IFN at the transcriptional level. The first effect is attributable to the induction of 2′-5′ oligo(A) synthetases, which require double-strand RNA for their activation.

1	effects are induced by IFN at the transcriptional level. The first effect is attributable to the induction of 2′-5′ oligo(A) synthetases, which require double-strand RNA for their activation. Activated synthetase polymerizes oligo(A) and thereby activates RNAse L, which in turn degrades single-strand RNA. A second effect results from the induction of PKR, a serine and threonine kinase that is also activated by double-strand RNA. PKR phosphorylates and negatively regulates the translational initiation factor eIF2α, shutting down protein synthesis in the infected cell. A third effect is initiated through the induction of Mx proteins, a family of GTPases that is particularly important in inhibiting the replication of influenza virus and vesicular stomatitis virus. These IFN effects are mostly directed against the infected cell, causing virus and cell dysfunction and thereby limiting viral replication.

1	A wide variety of methods are used to diagnose viral infection. Serology and virus isolation in tissue culture remain important standards. Acuteand convalescent-phase sera with rising titers of antibody to virus-specific antigens and a shift from IgM to IgG antibodies are generally accepted as diagnostic of acute viral infection. Serologic diagnosis is based on a more than fourfold rise in IgG antibody concentration when acuteand convalescent-phase serum samples are analyzed at the same time.

1	Immunofluorescence, hemadsorption, and hemagglutination assays for antiviral antibodies are labor-intensive and have been replaced by enzyme-linked immunosorbent assays (ELISAs), which generally use the specific viral proteins most frequently targeted by the antibody response. The proteins are purified from virus-infected cells or produced by recombinant DNA technology and are attached to a solid phase, where they can be incubated with serum, washed to eliminate nonspecific antibodies, and allowed to react with an enzyme-linked reagent to detect human IgG or IgM antibody specifically adhering to the viral antigen. The amount of antibody can then be quantitated by the intensity of a color reaction mediated by the linked enzyme. ELISAs can be sensitive and automated. Western blots can simultaneously confirm the presence of antibody to multiple specific viral proteins. The proteins are separated by size and transferred to an inert membrane, where they are incubated with serum antibodies.

1	confirm the presence of antibody to multiple specific viral proteins. The proteins are separated by size and transferred to an inert membrane, where they are incubated with serum antibodies. Western blots have an internal specificity control because the level of reactivity for viral proteins can be compared with that for cellular proteins in the same sample. Western blots require individual evaluation and are inherently difficult to quantitate or automate.

1	Isolation of virus in tissue culture depends on infection and replication in susceptible cells. Growth of virus in cell cultures can frequently be identified by effects on cell morphology under light microscopy. For example, HSV produces a typical cytopathic effect in rabbit kidney cells within 3 days. Other viral cytopathic effects may not be as diagnostically distinctive. Identification usually requires confirmation by staining with virus-specific monoclonal antibodies. The efficiency and speed of virus identification can be enhanced by combining short-term culture with immune detection. In assays with “shell vials” of tissue culture cells growing on a coverslip, viral infection can be detected by staining with a monoclonal antibody to a specific viral protein expressed early in viral replication. Thus, virus-infected cells can be detected within hours or days of inoculation, whereas several rounds of infection would be required to produce visible cytopathic effects.

1	Isolation of virus in tissue culture also depends on the collection of specimens from appropriate sites and the rapid transport of these specimens in appropriate medium to the virology laboratory (Chap. 150e). Rapid transport maintains viral viability and limits bacterial and fungal overgrowth. Enveloped viruses are generally more sensitive to freezing and thawing than nonenveloped viruses. The most appropriate site for culture depends on the pathogenesis of the virus in question. Nasopharyngeal, tracheal, or endobronchial aspirates are most appropriate for the identification of respiratory viruses. Sputum cultures generally are less appropriate because bacterial contamination and viscosity threaten tissue-culture cell viability. Aspirates of vesicular fluid are useful for isolation of HSV and VZV. Nasopharyngeal aspirates and stool specimens may be useful when the patient has fever and a rash and an enteroviral infection is suspected. Adenoviruses can be cultured from the urine of

1	HSV and VZV. Nasopharyngeal aspirates and stool specimens may be useful when the patient has fever and a rash and an enteroviral infection is suspected. Adenoviruses can be cultured from the urine of patients with hemorrhagic cystitis. CMV can frequently be isolated from cultures of urine or buffy coat. Biopsy material can be effectively cultured when viruses infect major organs, as in HSV encephalitis or adenovirus pneumonia.

1	The isolation of a virus does not necessarily establish disease causality. Viruses can persistently or intermittently colonize normal human mucosal surfaces. Saliva can be positive for herpesviruses, and normal urine samples can be positive for CMV. Isolations from blood, cerebrospinal fluid (CSF), or tissue are more often diagnostic of significant viral infection. Another method aimed at increasing the speed of viral diagnosis is direct testing for antigen or cytopathic effects. Virus-infected cells from the patient may be detected by staining with virus-specific monoclonal antibodies. For example, epithelial cells obtained by nasopharyngeal aspiration can be stained with a variety of specific monoclonal antibodies to identify the specific infecting respiratory virus. Antigen and serologic assays can be multiplexed to detect multiple analytes simultaneously by coupling of reagents to color-coded beads for each analyte and detection by flow cytometry.

1	Nucleic acid amplification techniques bring speed, sensitivity, and specificity to diagnostic virology. The ability to directly amplify minute amounts of viral nucleic acids in specimens means that detection no longer depends on viable virus and its replication. For example, amplification and detection of HSV nucleic acids in the CSF of patients with HSV encephalitis is a more sensitive detection method than culture of virus from CSF. The extreme sensitivity of these tests can be a problem, because subclinical infection or contamination can lead to false-positive results. Detection of viral nucleic acids does not necessarily indicate virus-induced disease.

1	Measurement of the amount of viral RNA or DNA in peripheral blood is an important means for determining whether a patient is at increased risk for virus-induced disease and for evaluating clinical responses to antiviral chemotherapy. Nucleic acid technologies for RNA quantification are routinely used in AIDS patients to evaluate responses to antiviral agents and to detect viral resistance or noncompliance with therapy. Virus-load measurements are also useful for evaluating the treatment of patients with HBV and HCV infections. Nucleic acid testing or direct staining with CMV-specific monoclonal antibodies to quantitate virus-infected cells in the peripheral blood (CMV antigenemia) is useful for identifying immunosuppressed patients who may be at risk for CMV-induced disease.

1	Multiple steps in the life cycles of viruses can be effectively targeted by antiviral drugs (Chaps. 215e and 216). Nucleoside and nonnucleoside reverse transcriptase inhibitors prevent HIV provirus synthesis, whereas protease inhibitors block maturation of the HIV and HCV polyprotein after infection of the cell. Enfuvirtide is a small peptide derived from HIV gp41 that acts before cell infection by preventing a conformational change required for initial fusion of the virus with the cell membrane. Raltegravir is an integrase inhibitor that is approved for use with other anti-HIV drugs. Amantadine and rimantadine inhibit the influenza M2 protein, preventing release of viral RNA early during infection, whereas zanamivir and oseltamivir inhibit the influenza neuraminidase, which is necessary for the efficient release of mature virions from infected cells.

1	Viral genomes can evolve resistance to drugs by mutation and selection, by recombination with a drug-resistant virus, or (in the case of influenza virus and other segmented RNA viral genomes) by reassortment. The emergence of drug-resistant strains can limit the efficacy of antiviral therapy. As in antibacterial therapy, excessive and inappropriate use of antiviral therapy can select for the emergence of drug-resistant strains. HIV genotyping is a rapid method for identifying drug-resistant viruses. Resistance to reverse transcriptase or protease inhibitors has been associated with specific mutations in the reverse transcriptase or protease genes. Identification of these mutations by polymerase chain reaction amplification and nucleic acid sequencing can be clinically useful for determining which antiviral agents may still be effective. Drug resistance also can arise in herpesviruses but is a less common clinical problem.

1	Viral vaccines are among the outstanding accomplishments of medical science. Smallpox has been eradicated except as a potential weapon of biological warfare or bioterrorism (Chap. 261e). Poliovirus eradication may soon follow. Measles can be contained or eliminated. Excess mortality due to influenza virus epidemics can be prevented, and the threat of influenza pandemics can be decreased by contemporary killed or live attenuated influenza vaccines. Mumps, rubella, and chickenpox are well controlled by childhood vaccination in the developed world. Reimmunization of mature adults can be used to control herpes zoster. New rotavirus vaccines can have a major impact on this leading cause of gastroenteritis and prominent cause of childhood death worldwide. Widespread HBV vaccination has dramatically lowered the frequency of acute and chronic hepatitis and is expected to lead to a dramatic decrease in the incidence of hepatocellular carcinoma. The HPV vaccine was the first vaccine

1	dramatically lowered the frequency of acute and chronic hepatitis and is expected to lead to a dramatic decrease in the incidence of hepatocellular carcinoma. The HPV vaccine was the first vaccine specifically licensed to prevent virus-induced cancer. Use of purified proteins, genetically engineered live-virus vaccines, and recombinant DNA–based strategies will make it possible to immunize against severe infections with other viruses. The development of effective HIV and HCV vaccines is complicated by the high mutation rate of viral RNA polymerase and reverse transcriptase, the population-based and individual divergence of HIV or HCV genomes, and repeated high-level exposure in some populations. Concerns about the use of smallpox and other viruses as weapons necessitate maintenance of immunity to agents that are not encountered naturally.

1	Viruses are being used experimentally to deliver biotherapeutic agents or novel vaccines. Foreign genes can be inserted into viral nucleic acids, and the recombinant virus vectors can be used to infect the patient or the patient’s cells ex vivo. Retrovirus integration into the cell genome has been used to functionally replace the abnormal gene in T cells of patients with severe combined immunodeficiency, thereby restoring immune function. Recombinant adenovirus, AAV, and retroviruses are being explored for use in diseases due to single-gene defects, such as cystic fibrosis and hemophilia. AAV carrying a lipoprotein lipase gene is now being used in Europe to treat a rare lipid-processing disease and is the first gene therapy approved for clinical use. Recombinant poxviruses, adenoviruses, and influenza viruses are also being used experimentally as vaccine vectors. Viral vectors are being tested experimentally for the expression of cytokines that can enhance immunity against tumor cells

1	influenza viruses are also being used experimentally as vaccine vectors. Viral vectors are being tested experimentally for the expression of cytokines that can enhance immunity against tumor cells or for the expression of proteins that can increase the sensitivity of tumor cells to chemotherapy. HSV deficient for replication in resting cells is being used to selectively kill proliferating glioblastoma cells after injections into CNS tumors. For improved safety, nonreplicating viruses are frequently used in clinical trials. Potential adverse events associated with virus-mediated gene transfer include the induction of inflammatory and antiviral immune responses. Instances of retrovirus-induced human malignances have raised concerns about the safety of retroviral gene therapy vectors.

1	Medical Virology Antiviral Chemotherapy, Excluding Antiretroviral Drugs Antiviral Chemotherapy, Excluding Antiretroviral Drugs Lindsey R. Baden, Raphael Dolin The field of antiviral therapy—both the number of antiviral drugs and our understanding of their optimal use—historically has lagged behind 215e that of antibacterial treatment, but significant progress has been made in recent years on new drugs for several viral infections. The development of antiviral drugs poses several challenges. Viruses replicate intracellularly and often use host cell enzymes, macromolecules, and organelles for synthesis of viral particles. Therefore, useful antiviral compounds must discriminate between host and viral functions with a high degree of specificity; agents without such selectivity are likely to be too toxic for clinical use.

1	Significant progress has also been made in the development of laboratory assays to assist clinicians in the appropriate use of antiviral drugs. Phenotypic and genotypic assays for resistance to antiviral drugs are becoming more widely available, and correlations of laboratory results with clinical outcomes are being better defined. Of particular note has been the development of highly sensitive and specific methods that measure the concentration of virus in blood (virus load) and permit direct assessment of the antiviral effect of a given drug regimen in that host site. Virus load measurements have been useful in recognizing the risk of disease progression in patients with viral infections and in identifying patients for whom antiviral chemotherapy might be of greatest benefit. As with any in vitro laboratory test, results are highly dependent on and likely vary with the laboratory techniques used.

1	Information regarding the pharmacodynamics of antiviral drugs, and particularly the relationship of concentration effects to efficacy, has been slow to develop but is also expanding. However, assays to measure concentrations of antiviral drugs, especially of their active moieties within cells, are still primarily research procedures not widely available to clinicians. Thus, there are limited guidelines for adjusting dosages of antiviral agents to maximize antiviral activity and minimize toxicity. Consequently, clinical use of antiviral drugs must be accompanied by particular vigilance for unanticipated adverse effects.

1	Like that of other infections, the course of viral infections is profoundly affected by interplay between the pathogen and a complex set of host defenses. The presence or absence of preexisting immunity, the ability to mount humoral and/or cell-mediated immune responses, and the stimulation of innate immunity are important determinants of the outcome of viral infections. The state of the host’s defenses needs to be considered when antiviral agents are used or evaluated.

1	As with any therapy, the optimal use of antiviral compounds requires a specific and timely diagnosis. For some viral infections, such as herpes zoster, the clinical manifestations are so characteristic that a diagnosis can be made on clinical grounds alone. For other viral infections, such as influenza A, epidemiologic information (e.g., the documentation of a community-wide influenza outbreak) can be used to make a presumptive diagnosis with a high degree of accuracy. However, for most of the remaining viral infections, including herpes simplex encephalitis, cytomegaloviral infections other than retinitis, and enteroviral infections, diagnosis on clinical grounds alone cannot be accomplished with certainty. For such infections, rapid viral diagnostic techniques are of great importance. Considerable progress has also been made in recent years in the development of such tests, which are now widely available for a number of viral infections.

1	Despite these complexities, the efficacy of a number of antiviral compounds has been clearly established in rigorously conducted and controlled studies. As summarized in Table 215e-1, this chapter reviews the antiviral drugs that are currently approved or are likely to be considered for approval in the near future for use against viral infections other than those caused by HIV. Antiretroviral drugs are reviewed in Chap. 226. (SEE ALSO CHAPS. 223 AND 224) ZANAMIVIR, OSELTAMIVIR, PERAMIVIR, AND LANINAMIVIR

1	Zanamivir and oseltamivir are inhibitors of the influenza viral neuraminidase enzyme, which is essential for release of virus from infected cells and for its subsequent spread throughout the respiratory tract of the infected host. The enzyme cleaves terminal sialic acid residues and thus destroys the cellular receptors to which the viral hemagglutinin attaches. Zanamivir and oseltamivir are sialic acid transition-state analogues and are highly active and specific inhibitors of the neuraminidases of both influenza A and B viruses. The antineuraminidase activity of the two drugs is similar, although zanamivir has somewhat greater in vitro activity against influenza B virus. Zanamivir may also be active against certain strains of influenza A virus that are resistant to oseltamivir. Both zanamivir and oseltamivir act through competitive and reversible inhibition of the active site of influenza A and B viral neuraminidases and have relatively little effect on mammalian cell enzymes.

1	Oseltamivir phosphate is an ethyl ester prodrug that is converted to oseltamivir carboxylate by esterases in the liver. Orally administered oseltamivir has a bioavailability of >60% and a plasma half-life of 7–9 h. The drug is excreted unmetabolized, primarily by the kidneys. Zanamivir has low oral bioavailability and is administered orally via a hand-held inhaler. By this route, ~15% of the dose is deposited in the lower respiratory tract, and low plasma levels of the drug are detected. The toxicities most frequently encountered with orally administered oseltamivir are nausea, gastrointestinal discomfort, and (less commonly) vomiting. Gastrointestinal discomfort is usually transient and is less likely if the drug is administered with food. Neuropsychiatric events (delirium, self-injury) have been reported in children who have been taking oseltamivir, primarily in Japan. Zanamivir is orally inhaled and is generally well tolerated, although exacerbations of asthma may occur. An IV

1	have been reported in children who have been taking oseltamivir, primarily in Japan. Zanamivir is orally inhaled and is generally well tolerated, although exacerbations of asthma may occur. An IV formulation of zanamivir is under development and is available from GlaxoSmithKline as part of clinical trials.

1	Inhaled zanamivir and orally administered oseltamivir have been effective in the treatment of naturally occurring, uncomplicated influenza A or B in otherwise healthy adults. In placebo-controlled studies, illness has been shortened by 1.0–1.5 days of therapy with either of these drugs when treatment is administered within 2 days of onset of symptoms. Pooled analyses of clinical studies of oseltamivir suggest that treatment may reduce the likelihood of hospitalizations and of certain respiratory tract complications associated with influenza, and observational studies suggest that oseltamivir may reduce mortality rates associated with influenza A outbreaks (Chap. 224). Once-daily inhaled zanamivir or once-daily orally administered oseltamivir can provide prophylaxis against laboratory-documented influenza A– and influenza B–associated illness.

1	Resistance to the neuraminidase inhibitors may develop by changes in the viral neuraminidase enzyme, by changes in the hemagglutinin that make it more resistant to the actions of the neuraminidase, or by both mechanisms. Isolates that are resistant to oseltamivir—most commonly through the H275Y mutation, which leads to a change from histidine to tyrosine at that residue in the neuraminidase—remain sensitive to zanamivir. Certain mutations impart resistance to both oseltamivir and zanamivir (e.g., I223R, which leads to a change from isoleucine to arginine). Because the mechanisms of action of the neuraminidase inhibitors differ from those of the adamantanes (see below), zanamivir and oseltamivir are active against strains of influenza A virus that are resistant to amantadine and rimantadine.

1	Appropriate use of antiviral agents against influenza viruses depends on a knowledge of the resistance patterns of circulating viruses. As of this writing, currently circulating influenza A/H1N1 and H3N2 viruses (2013–2014) were sensitive to zanamivir and oseltamivir, with a few exceptions for oseltamivir. Up-to-date information on patterns of resistance to antiviral drugs is available from the Centers for Disease Control and Prevention (CDC) at www.cdc.gov/flu. Influenza A and B: treatment Influenza A: treatment Influenza A and B: prophylaxis Influenza A: prophylaxis Varicella: immunocompetent host Varicella: immunocompromised host Oral IV IV IV Adults: 75 mg bid × 5 d Children 1–12 years: 30–75 mg bid, depending on weight,a × 5 d Adults and children ≥7 years: 10 mg bid × 5 d Adults: 100 mg qd or bid × 5–7 d Children 1–9 years: 5 mg/kg per day (maximum, 150 mg/d) × 5–7 d Adults: 75 mg/d Children ≥1 year: 30–75 mg/d, depending on weighta

1	Adults: 100 mg qd or bid × 5–7 d Children 1–9 years: 5 mg/kg per day (maximum, 150 mg/d) × 5–7 d Adults: 75 mg/d Children ≥1 year: 30–75 mg/d, depending on weighta Adults and children ≥5 years: 10 mg/d Adults: 200 mg/d Children 1–9 years: 5 mg/kg per day (maximum, 150 mg/d) 5 mg/kg bid × 14–21 d; then 5 mg/kg per day as maintenance dose 900 mg bid × 21 d; then 900 mg/d as maintenance dose 60 mg/kg q8h × 14–21 d; then 90–120 mg/kg per day as maintenance dose 5 mg/kg once weekly × 2 weeks, then once every other week; given with probenecid and hydration 330 mg on days 1 and 15 followed by 330 mg monthly as maintenance 20 mg/kg (maximum, 800 mg) 4 or 5 times daily × 5 d

1	Children 2–18 years: 20 mg/kg tid (not to exceed 1 g tid) × 5 d 20 mg/kg q8h × 14–21 d When started within 2 d of onset in uncomplicated disease, zanamivir and oseltamivir reduce symptom duration by 1.0–1.5 and 1.3 d, respectively. Their effectiveness in prevention or treatment of complications is unclear, although some analyses suggest that oseltamivir may reduce the frequency of respiratory tract complications and hospitalizations. Oseltamivir's side effects of nausea and vomiting can be reduced in frequency by drug administration with food. Zanamivir may exacerbate bronchospasm in patients with asthma. Amantadine and rimantadine are not recommended for routine use unless antiviral susceptibilities are known because of widespread resistance in A/H3N2 viruses since 2005–2006 and in pandemic A/H1N1 viruses in 2009–2010. Their efficacy in treatment of uncomplicated disease caused by sensitive viruses has been similar to that of neuraminidase inhibitors.

1	Prophylaxis must be continued for the duration of exposure and can be administered simultaneously with inactivated vaccine. Unless the sensitivity of isolates is known, neither amantadine nor rimantadine is currently recommended for prophylaxis or therapy. Use of ribavirin is to be considered for treatment of infants and young children hospitalized with RSV pneumonia and bronchiolitis, according to the American Academy of Pediatrics. Ganciclovir, valganciclovir, foscarnet, and cidofovir are approved for treatment of CMV retinitis in patients with AIDS. They are also used for colitis, pneumonia, or “wasting” syndrome associated with CMV and for prevention of CMV disease in transplant recipients. Valganciclovir has largely supplanted oral ganciclovir and is frequently used in place of IV ganciclovir. Foscarnet is not myelosuppressive and is active against acyclovirand ganciclovir-resistant herpesviruses.

1	Foscarnet is not myelosuppressive and is active against acyclovirand ganciclovir-resistant herpesviruses. Fomivirsen has reduced the rate of progression of CMV retinitis in patients in whom other regimens have failed or have not been well tolerated. The major form of toxicity is ocular inflammation. Treatment confers modest clinical benefit when administered within 24 h of rash onset. A change to oral valacyclovir can be considered once fever has subsided if there is no evidence of visceral involvement. Results are optimal when therapy is initiated early. Some authorities recommend treatment for 21 d to prevent relapses. Serious morbidity is common despite therapy. Prolonged oral administration after initial IV therapy has been suggested because of long-term sequelae associated with cutaneous recurrences of HSV infection. Genital herpes simplex, primary: treatment Genital herpes simplex, recurrent: treatment Genital herpes simplex, recurrent: suppression

1	Genital herpes simplex, primary: treatment Genital herpes simplex, recurrent: treatment Genital herpes simplex, recurrent: suppression Mucocutaneous herpes simplex in immunocompromised host: treatment Mucocutaneous herpes simplex in immunocompromised host: prevention of recurrence during intense immunosuppression Herpes simplex orolabialis, recurrente Herpes zoster: immunocompetent host Acyclovir 125 mg bid × 5 d, 1000 mg bid × 1 d, or 500 mg once, then 250 mg PO bid × 3 doses 800 mg bid 5 mg/kg q12h 500 mg to 1 g bid or tid 500 mg bidc 1.0% cream applied q2h during waking hours × 4 d 1 drop of 1% ophthalmic solution q2h while awake (maximum, 9 drops daily) 0.5-in. ribbon of 3% ophthalmic ointment 5 times daily 800 mg 5 times daily × 7–10 d The IV route is preferred for infections severe enough to warrant hospitalization or with neurologic complications.

1	The oral route is preferred for patients whose condition does not warrant hospitalization. Adequate hydration must be maintained. Topical use—largely supplemented by oral therapy—may obviate systemic administration to pregnant women. Systemic symptoms and untreated areas are not affected. Valacyclovir appears to be as effective as acyclovir but can be administered less frequently. Famciclovir appears to be similar in effectiveness to acyclovir. The clinical effect is modest and is enhanced if therapy is initiated early. Treatment does not affect recurrence rates. Suppressive therapy is recommended only for patients with at least 6–10 recurrences per year. “Breakthrough” occasionally takes place, and asymptomatic shedding of virus occurs. The need for suppressive therapy should be reevaluated after 1 year. Suppression with valacyclovir reduces transmission of genital HSV among virus-discordant couples.

1	The choice of the IV or oral route and the duration of therapy depend on the severity of infection and the patient's ability to take oral medication. Oral or IV treatment has supplanted topical therapy except for small, easily accessible lesions. Foscarnet is used for acyclovir-resistant viruses. Treatment is administered during periods when intense immunosuppression is expected— e.g., during antitumor chemotherapy or after transplantation—and is usually continued for 2–3 months. Treatment shortens healing time and symptom duration by 0.5–1.0 d (versus placebo). Therapy begun at earliest symptom reduces disease duration by 1 d. Therapy begun within 1 h of prodrome decreases time to healing by 1.8–2.2 d. Application at initial symptoms reduces healing time by 1 d. Therapy should be undertaken in consultation with an ophthalmologist.

1	Application at initial symptoms reduces healing time by 1 d. Therapy should be undertaken in consultation with an ophthalmologist. Valacyclovir may be more effective than acyclovir for pain relief; otherwise, it has a similar effect on cutaneous lesions and should be given within 72 h of rash onset. The duration of postherpetic neuralgia is shorter than with placebo. Famciclovir showed overall efficacy similar to that of acyclovir in a comparative trial. It should be given ≤72 h after rash onset. Acyclovir causes faster resolution of skin lesions than placebo and provides some relief of acute symptoms if given within 72 h of rash onset. Combined with tapering doses of prednisone, acyclovir improves quality-of-life outcomes. Antiviral Chemotherapy, Excluding Antiretroviral Drugs

1	Antiviral Chemotherapy, Excluding Antiretroviral Drugs Herpes zoster: immunocompromised host 1 million units per wart (maximum of 5) thrice weekly × 3 weeks 250,000 units per wart (maximum of 10) twice weekly × up to 8 weeks 100 mg/d × 12–18 months; 150 mg bid as part of therapy for HIV infection 0.5 mg/d × 48 weeks (1 mg/d if HBV is resistant to lamivudine) 1.5 μg weekly × 48 weeks 1.5 μg/kg weekly (IFN)/ 800–1400 mg daily (ribavirin) × 24–48 weeks 9–15 μg thrice weekly × 6–12 months Effectiveness in localized zoster is most marked when treatment is given early. Foscarnet may be used for acyclovir-resistant VZV infections. Treatment reduces ocular complications, including ocular keratitis and uveitis. Intralesional treatment frequently results in regression of warts, but lesions often recur. Parenteral administration may be useful if lesions are numerous. HBeAg and DNA are eliminated in 33–37% of cases. Histopathologic improvement is also seen.

1	HBeAg and DNA are eliminated in 33–37% of cases. Histopathologic improvement is also seen. ALT levels return to normal in 39% of patients, and histologic improvement occurs in 38%. Lamivudine monotherapy is well tolerated and effective in reduction of HBV DNA levels, normalization of ALT levels, and improvement in histopathology. However, resistance develops in 24% of recipients when lamivudine is used as monotherapy for 1 year. A return of ALT levels to normal is documented in 48–72% of recipients and improved liver histopathology in 53–64%. Adefovir is effective in lamivudine-resistant hepatitis B. Renal function should be monitored. Normalization of ALT is seen in 68–78% of recipients and loss of HBeAg in 21%. Entecavir is active against lamivudine-resistant HBV.

1	Normalization of ALT is seen in 68–78% of recipients and loss of HBeAg in 21%. Entecavir is active against lamivudine-resistant HBV. HBV DNA is reduced by >5 log10 copies/mL along with normalization of ALT levels in 74–77% of patients and improved histopathology in 65–67%. Resistance develops in 9–22% of patients after 2 years of therapy. Elevated CPK levels and myopathy may occur. ALT levels return to normal in 68–76% of patients, and liver histopathology improves in 72–74%. Resistance is uncommon with up to 2 years of therapy. SVRs are noted in 20–30% of patients. Normalization of ALT levels and improvements in liver histopathology are also seen. Combination therapy results in SVR in up to 40–50% of recipients.

1	SVRs are noted in 20–30% of patients. Normalization of ALT levels and improvements in liver histopathology are also seen. Combination therapy results in SVR in up to 40–50% of recipients. The slower clearance of pegylated IFNs than of standard IFNs permits once-weekly administration. Pegylated formulations appear to be superior to standard IFNs in efficacy, both as monotherapy and in combination with ribavirin, and have largely supplanted standard IFNs in treatment of hepatitis C. SVRs were seen in 42–51% of patients infected with HCV genotype 1 and in 76–82% of those infected with genotype 2 or 3. Doses of 9 and 15 μg are equivalent to IFN-α2a and IFN-α2b doses of 3 million units and 5 million units, respectively. HCV genotypes 1, 4, 5, and 6: 400 mg qd with daily weight-based ribavirin (1000 mg [<75 kg] to 1200 mg [>75 kg]) and weekly pegylated IFN for 12 weeks. Genotypes 2 and 3: 400 mg qd with daily weight-based ribavirin for 12 and 24 weeks, respectively

1	Alternative regimen for genotypes 1 and 4: 150 mg qd for 12 weeks plus daily ribavirin and weekly pegylated IFN for 24 weeks and for 24–48 weeks, respectively Sofosbuvir is generally well tolerated, and most common side effects have been attributable to concomitantly administered IFN and ribavirin. Sofosbuvir is recommended in triple combination with pegylated IFN and ribavirin as first-line therapy for genotypes 1, 4, 5, and 6, with SVRs in 89–97% of treatment-naïve patients, and in double combination with ribavirin for genotypes 2 and 3.

1	Simeprevir has supplanted the first-generation protease inhibitors boceprevir and telaprevir. Its metabolism by cytochrome CYP3A can result in interactions with other drugs. Photosensitivity and reversible hyperbilirubinemia are associated toxicities. Testing for the Q80K-resistant variant should be carried out since this variant is present in one-third of HCV genotype 1a infections. Triple combinations with pegylated IFN and ribavirin result in SVRs in 80% of genotype 1 infections without Q80K. Chronic hepatitis D IFN-α2a or IFN-α2b SC 9 million units thrice weekly × 12 The overall efficacy and the optimal regimen months and duration of therapy are not fully established. Pegylated IFN-α2b SC Sustained SVRs have been seen in 25–30% of 1.5 μg weekly × 48 weeks IFN-α.

1	IFN-α. aFor detailed weight recommendations and for children <1 year of age, see www.cdc.gov/flu/professionals/antivirals/summary-clinicians.htm. bAmantadine and rimantadine are not recommended for routine use because of widespread resistance in currently circulating A/H3N2 and pandemic A/H1N1 viruses. Their use may be considered if sensitivities become reestablished. cNot approved for this indication by the U.S. Food and Drug Administration (FDA). dApproved by the FDA for treatment of HIV-infected individuals. eAcyclovir suspension (15 mg/kg PO, to a maximum of 200 mg per dose) given for 7 d has been reported to be effective in treatment of primary herpetic gingivostomatitis in children. fActive ingredient: benzyl alcohol. Available without prescription. gConsult www.hcvguidelines.org for recommendations regarding treatment of null or partial responders to IFN regimens or of patients ineligible to receive IFN.

1	Abbreviations: ALT, alanine aminotransferase; CMV, cytomegalovirus; CPK, creatine phosphokinase; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; HSV, herpes simplex virus; IFN, interferon; RGT, response-guided therapy; RSV, respiratory syncytial virus; SVR, sustained virologic response; VZV, varicella-zoster virus. Zanamivir and oseltamivir have been approved by the U.S. Food and Drug Administration (FDA) for treatment of influenza in adults and in children (those ≥7 years old for zanamivir and those ≥1 year old for oseltamivir) who have been symptomatic for ≤2 days. Oseltamivir is approved for prophylaxis of influenza in individuals ≥1 year of age and zanamivir for those ≥5 years of age (Table 215e-1). Guidelines for the use of oseltamivir in children <1 year of age can be accessed through the CDC website, as noted in the footnote to Table 215e-1.

1	Peramivir is an investigational neuraminidase inhibitor that can be administered intravenously to patients for whom such an intervention is considered necessary. It has been approved in Japan, China, and South Korea but not in the United States, where it has been available in clinical trials through BioCryst Pharmaceuticals. Oseltamivir-resistant viruses generally exhibit reduced sensitivity to peramivir. Laninamivir octonoate is an investigational neuraminidase that has been approved in Japan. It is the prodrug of laninamivir, which is administered by oral inhalation and has a prolonged half-life of ~3 days. In limited studies, it has been investigated as single-dose therapy for influenza; its effects were similar to those obtained with multiple dosing of zanamivir or oseltamivir.

1	Amantadine and the closely related compound rimantadine are primary symmetric amines that have antiviral activity limited to influenza A viruses. Amantadine and rimantadine have a long history of efficacy in the prophylaxis and treatment of influenza A infections in humans. However, high frequencies of resistance to these drugs were noted among influenza A/H3N2 viruses in the 2005–2006 influenza season and continued to be seen in 2013–2014. The pandemic A/H1N1 viruses that circulated in 2009–2010 were also resistant to amantadine and rimantadine, and circulating influenza A/H1N1 viruses in the 2013–2014 season were largely resistant. Therefore, these agents are no longer recommended unless the sensitivity of the particular isolate of influenza A virus is known, in which case their use may be considered. Amantadine and rimantadine act through inhibition of the ion channel function of the influenza A M2 matrix protein, on which uncoating of the virus depends. A substitution of a single

1	be considered. Amantadine and rimantadine act through inhibition of the ion channel function of the influenza A M2 matrix protein, on which uncoating of the virus depends. A substitution of a single amino acid at critical sites in the M2 protein can result in a virus that is resistant to amantadine and rimantadine.

1	Amantadine and rimantadine have been shown to be effective in the prophylaxis of influenza A in large-scale studies of young adults and in less extensive studies of children and elderly persons. In such studies, efficacy rates of 55–80% in the prevention of influenza-like illness were noted, and even higher rates were reported when virus-specific attack rates were calculated. Amantadine and rimantadine have also been found to be effective in the treatment of influenza A infection in studies involving predominantly young adults and, to a lesser extent, children. Administration of these compounds within 24–72 h after the onset of illness has resulted in a reduction of the duration of signs and symptoms by ~50% compared with that in placebo recipients. The effect on signs and symptoms of illness is superior to that of commonly used antipyretic-analgesic agents. Only anecdotal reports are available concerning the efficacy of amantadine or rimantadine in the prevention or treatment of

1	illness is superior to that of commonly used antipyretic-analgesic agents. Only anecdotal reports are available concerning the efficacy of amantadine or rimantadine in the prevention or treatment of complications of influenza (e.g., pneumonia).

1	Amantadine and rimantadine are available only in oral formulations and are ordinarily administered to adults once or twice daily, with a dosage of 100–200 mg/d. Despite their structural similarities, the two compounds have different pharmacokinetics. Amantadine is not metabolized and is excreted almost entirely by the kidneys, with a half-life of 12–17 h and peak plasma concentrations of 0.4 μg/mL. In contrast, rimantadine is extensively metabolized to hydroxylated derivatives and has a half-life of 30 h. Only 30–40% of an orally administered dose of rimantadine is recovered in the urine. The peak plasma levels of rimantadine are approximately half those of amantadine,

1	Antiviral Chemotherapy, Excluding Antiretroviral Drugs but rimantadine is concentrated in respiratory secretions to a greater extent than amantadine. For prophylaxis, the compounds must be administered daily for the period at risk (i.e., duration of the exposure). For therapy, amantadine or rimantadine is generally administered for 5–7 days.

1	Although these compounds are generally well tolerated, 5–10% of amantadine recipients experience mild central nervous system side effects consisting primarily of dizziness, anxiety, insomnia, and difficulty in concentrating. These effects are rapidly reversible upon cessation of the drug’s administration. At a dose of 200 mg/d, rimantadine is better tolerated than amantadine; in a large-scale study of young adults, adverse effects were no more frequent among rimantadine recipients than among placebo recipients. Seizures and worsening of congestive heart failure have also been reported in patients treated with amantadine, although a causal relationship has not been established. The dosage of amantadine should be reduced to 100 mg/d in patients with renal insufficiency—i.e., a creatinine clearance rate (CrCl) of <50 mL/min—and in the elderly. A rimantadine dose of 100 mg/d should be used for patients with a CrCl of <10 mL/min and for the elderly.

1	Ribavirin is a synthetic nucleoside analogue that inhibits a wide range of RNA and DNA viruses. The mechanism of action of ribavirin is not completely defined and may be different for different groups of viruses. Ribavirin-5′-monophosphate blocks the conversion of inosine-5′-monophosphate to xanthosine-5′-monophosphate and interferes with the synthesis of guanine nucleotides as well as with that of both RNA and DNA. Ribavirin-5′-monophosphate also inhibits capping of virus-specific messenger RNA in certain viral systems.

1	Ribavirin administered as a small-particle aerosol to young children hospitalized with respiratory syncytial virus (RSV) infection has been clinically beneficial and has improved oxygenation in some studies (7 of 11). Although ribavirin has been approved for treatment of infants hospitalized with RSV infection, the American Academy of Pediatrics has recommended that it be considered on an individual basis rather than used routinely in that setting. Aerosolized ribavirin has also been administered to older children and adults (including immunosuppressed patients) with severe RSV and parainfluenza virus infections and to older children and adults with influenza A or B infection, but the benefit of this treatment, if any, is unclear. In RSV infections in immunosuppressed patients, ribavirin has been given in combination with anti-RSV immunoglobulins.

1	Orally administered ribavirin has not been effective in the treatment of influenza A virus infections. IV or oral ribavirin has reduced mortality rates among patients with Lassa fever; it has been particularly effective in this regard when given within the first 6 days of illness. IV ribavirin has been reported to be of clinical benefit in the treatment of hemorrhagic fever with renal syndrome caused by Hantaan virus and as therapy for Argentinean hemorrhagic fever. Oral ribavirin has also been recommended for the treatment and prophylaxis of Congo-Crimean hemorrhagic fever. Use of IV ribavirin in patients with hantavirus pulmonary syndrome in the United States has not been associated with clear-cut benefits.

1	Oral administration of ribavirin reduces serum aminotransferase levels in patients with chronic hepatitis C virus (HCV) infection; because it appears not to reduce serum HCV RNA levels, the mechanism of this effect is unclear. The drug provides added benefit when given by mouth in doses of 800–1200 mg/d in combination with interferon (IFN) α2b or α2a (see below), and the triple combination of ribavirin, IFN, and sofosbuvir or simeprevir has been approved for the treatment of patients with chronic HCV infection (see below). Recent data suggest that oral ribavirin may be beneficial in resolution of chronic hepatitis E infection associated with organ transplantation. Large oral doses of ribavirin (800–1000 mg/d) have been associated with reversible hematopoietic toxicity. This effect has not been observed with aerosolized ribavirin, apparently because little drug is absorbed systemically. Aerosolized administration of ribavirin is generally well tolerated but occasionally is associated

1	been observed with aerosolized ribavirin, apparently because little drug is absorbed systemically. Aerosolized administration of ribavirin is generally well tolerated but occasionally is associated with bronchospasm, rash, or conjunctival irritation. It should be administered under close supervision—particularly in the setting of mechanical ventilation, where precipitation of the drug is possible. Health care workers exposed to the drug have experienced minor toxicity, including eye and respiratory tract irritation. Because ribavirin is mutagenic, teratogenic, and embryotoxic, its use is generally contraindicated in pregnancy. Its administration as an aerosol poses a risk to pregnant health care workers. Because clearance of ribavirin is primarily renal, dose reduction is required in the setting of significant renal dysfunction.

1	DAS181 is an investigational antiviral agent with activity against influenza A and B and parainfluenza viruses. It is a sialidase linked to a respiratory epithelium–anchoring domain. This agent cleaves the terminal sialic acid residues on human respiratory cells, reducing the binding of the aforementioned respiratory viruses. DAS181 is administered by oral inhalation and is being evaluated in the treatment of parainfluenza type 3 infections in recipients of lung and stem cell transplants.

1	Acyclovir is a highly potent and selective inhibitor of the replication of certain herpesviruses, including herpes simplex virus (HSV) types 1 and 2, varicella-zoster virus (VZV), and Epstein-Barr virus (EBV). It is relatively ineffective in the treatment of human cytomegalovirus (CMV) infections; however, some studies have indicated effectiveness in the prevention of CMV-associated disease in immunosuppressed patients. Valacyclovir, the l-valyl ester of acyclovir, is converted almost entirely to acyclovir by intestinal and hepatic hydrolysis after oral administration. Valacyclovir offers pharmacokinetic advantages over orally administered acyclovir: it exhibits significantly greater oral bioavailability, results in higher blood levels, and can be given less frequently than acyclovir (two or three rather than five times daily).

1	The high degree of selectivity of acyclovir is related to its mechanism of action, which requires that the compound first be phosphorylated to acyclovir monophosphate. This phosphorylation occurs efficiently in herpesvirus-infected cells by means of a virus-coded thymidine kinase. In uninfected mammalian cells, little phosphorylation of acyclovir occurs, and the drug is therefore concentrated in herpesvirus-infected cells. Acyclovir monophosphate is subsequently converted by host cell kinases to a triphosphate that is a potent inhibitor of virus-induced DNA polymerase but has relatively little effect on host cell DNA polymerase. Acyclovir triphosphate can also be incorporated into viral DNA, with early chain termination.

1	Acyclovir is available in IV, oral, and topical forms, while valacyclovir is available in an oral formulation. IV acyclovir is effective in the treatment of mucocutaneous HSV infections in immunocompromised hosts, in whom it reduces time to healing, duration of pain, and virus shedding. When administered prophylactically during periods of intense immunosuppression (e.g., related to chemotherapy for leukemia or transplantation) and before the development of lesions, IV acyclovir reduces the frequency of HSV-associated disease. After prophylaxis is discontinued, HSV lesions recur. IV acyclovir is also effective in the treatment of HSV encephalitis.

1	Because VZV is generally less sensitive to acyclovir than is HSV, higher doses of acyclovir must be used to treat VZV infections. In immunocompromised patients with herpes zoster, IV acyclovir reduces the frequency of cutaneous dissemination and visceral complications and—in one comparative trial—was more effective than vidarabine. Acyclovir, administered at oral doses of 800 mg five times a day, had a modest beneficial effect on localized herpes zoster lesions in both immunocompromised and immunocompetent patients. Combination of acyclovir with a tapering regimen of prednisone appeared to be more effective than acyclovir alone in terms of quality-of-life outcomes in immunocompetent patients over age 50 with herpes zoster. A comparative study of acyclovir (800 mg PO five times daily) and valacyclovir (1 g PO three times daily) in immunocompetent patients with herpes zoster indicated that the latter drug may be more effective in eliciting the resolution of zoster-associated pain.

1	and valacyclovir (1 g PO three times daily) in immunocompetent patients with herpes zoster indicated that the latter drug may be more effective in eliciting the resolution of zoster-associated pain. Orally administered acyclovir (600 mg five times a day) reduced complications of herpes zoster ophthalmicus in a placebo-controlled trial.

1	In chickenpox, a modest overall clinical benefit is attained when oral acyclovir therapy is begun within 24 h of the onset of rash in otherwise healthy children (20 mg/kg, up to a maximum of 800 mg, four times a day) or adults (800 mg five times a day). IV acyclovir has also been reported to be effective in the treatment of immunocompromised children with chickenpox.

1	The most widespread use of acyclovir is in the treatment of genital HSV infections. IV or oral acyclovir or oral valacyclovir has shortened the duration of symptoms, reduced virus shedding, and accelerated healing when used for the treatment of primary genital HSV infections. Oral acyclovir and valacyclovir have also had a modest effect in treatment of recurrent genital HSV infections. However, the failure of treatment of either primary or recurrent disease to reduce the frequency of subsequent recurrences has indicated that acyclovir is ineffective in eliminating latent infection. Documented chronic oral administration of acyclovir for up to 6 years or of valacyclovir for up to 1 year has reduced the frequency of recurrences markedly during therapy; once the drug is discontinued, lesions recur. In one study, suppressive therapy with valacyclovir (500 mg once daily for 8 months) reduced transmission of HSV-2 genital infections among discordant couples by 50%. A modest effect on herpes

1	recur. In one study, suppressive therapy with valacyclovir (500 mg once daily for 8 months) reduced transmission of HSV-2 genital infections among discordant couples by 50%. A modest effect on herpes labialis (i.e., a reduction of disease duration by 1 day) was seen when valacyclovir was administered upon detection of the first symptom of a lesion at a dose of 2 g every 12 h for 1 day. In AIDS patients, chronic or intermittent administration of acyclovir has been associated with the development of HSV and VZV strains resistant to the action of the drug and with clinical failures. The most common mechanism of resistance is a deficiency of the virus-induced thymidine kinase. Patients with HSV or VZV infections resistant to acyclovir have frequently responded to foscarnet.

1	With the availability of the oral and IV forms, there are few indications for topical acyclovir, although treatment with this formulation has been modestly beneficial in primary genital HSV infections and in mucocutaneous HSV infections in immunocompromised hosts.

1	Overall, acyclovir is remarkably well tolerated and is generally free of toxicity. The most frequently encountered form of toxicity is renal dysfunction because of drug crystallization, particularly after rapid IV administration or with inadequate hydration. Central nervous system changes, including lethargy and tremors, are occasionally reported, primarily in immunosuppressed patients. However, whether these changes are related to acyclovir, to concurrent administration of other therapy, or to underlying infection remains unclear. Acyclovir is excreted primarily unmetabolized by the kidneys via both glomerular filtration and tubular secretion. Approximately 15% of a dose of acyclovir is metabolized to 9-[(carboxymethoxy)methyl]guanine or other minor metabolites. Reduction in dosage is indicated in patients with a CrCl of <50 mL/min. The half-life of acyclovir is ~3 h in normal adults, and the peak plasma concentration after a 1-h infusion of a dose of 5 mg/kg is 9.8 μg/mL.

1	is indicated in patients with a CrCl of <50 mL/min. The half-life of acyclovir is ~3 h in normal adults, and the peak plasma concentration after a 1-h infusion of a dose of 5 mg/kg is 9.8 μg/mL. Approximately 22% of an orally administered acyclovir dose is absorbed, and peak plasma concentrations of 0.3–0.9 μg/mL are attained after administration of a 200-mg dose. Acyclovir penetrates relatively well into the cerebrospinal fluid (CSF), with concentrations approaching half of those found in plasma.

1	Acyclovir causes chromosomal breakage at high doses, but its administration to pregnant women has not been associated with fetal abnormalities. Nonetheless, the potential risks and benefits of acyclovir should be carefully assessed before the drug is used in pregnancy.

1	Valacyclovir exhibits three to five times greater bioavailability than acyclovir. The concentration-time curve for valacyclovir, given as 1 g PO three times daily, is similar to that for acyclovir, given as 5 mg/kg IV every 8 h. The safety profiles of valacyclovir and acyclovir are similar, although thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome has been reported in immunocompromised patients who have received high doses of valacyclovir (8 g/d). Valacyclovir is approved for the treatment of herpes zoster, of initial and recurrent episodes of genital HSV infection, and of herpes labialis in immunocompetent adults as well as for suppressive treatment of genital herpes. Although it has not been extensively studied in other clinical settings involving HSV or VZV infections, many consultants use valacyclovir rather than oral acyclovir in settings where only the latter has been approved because of valacyclovir’s superior pharmacokinetics and more convenient dosing schedule.

1	Cidofovir is a phosphonate nucleotide analogue of cytosine. Its major use is in CMV infections, but it is active against a broad range of herpesviruses, including HSV, human herpesvirus (HHV) types 6A and 6B, HHV-8, and certain other DNA viruses such as polyomaviruses, papillomaviruses, adenoviruses, and poxviruses, including variola (smallpox) and vaccinia. Cidofovir does not require initial phosphorylation by virus-induced kinases; the drug is phosphorylated by host cell enzymes to cidofovir diphosphate, which is a competitive inhibitor of viral DNA polymerases and, to a lesser extent, of host cell DNA polymerases. Incorporation of cidofovir diphosphate slows or terminates nascent DNA chain elongation. Cidofovir is active against HSV isolates that are resistant to acyclovir because of absent or altered thymidine kinase and against CMV isolates that are resistant to ganciclovir because of UL97 phosphotransferase mutations. CMV isolates resistant to ganciclovir on the basis of UL54

1	absent or altered thymidine kinase and against CMV isolates that are resistant to ganciclovir because of UL97 phosphotransferase mutations. CMV isolates resistant to ganciclovir on the basis of UL54 mutations are usually resistant to cidofovir as well. Cidofovir is usually active against foscarnet-resistant CMV, although cross-resistance to foscarnet has been described.

1	Cidofovir has poor oral availability and is administered intravenously. It is excreted primarily by the kidney and has a plasma half-life of 2.6 h. Cidofovir diphosphate’s intracellular half-life of >48 h is the basis for the recommended dosing regimen of 5 mg/kg once a week for the initial 2 weeks and then 5 mg/kg every other week. The major toxic effect of cidofovir is proximal renal tubular injury, as manifested by elevated serum creatinine levels and proteinuria. The risk of nephrotoxicity can be reduced by vigorous saline hydration and by concomitant oral administration of probenecid. Neutropenia, rashes, and gastrointestinal tolerance may also occur.

1	IV cidofovir has been approved for the treatment of CMV retinitis in AIDS patients who are intolerant of ganciclovir or foscarnet or in whom those drugs have failed. In a controlled study, a maintenance dosage of 5 mg/kg per week administered to AIDS patients reduced the progression of CMV retinitis from that seen at 3 mg/kg. Intravitreal cidofovir has been used to treat CMV retinitis but has been associated with significant toxicity. IV cidofovir has been reported anecdotally to be effective for treatment of acyclovir-resistant mucocutaneous HSV infections. Likewise, topically administered cidofovir is reportedly beneficial against mucocutaneous HSV infections in HIV-infected patients. Anecdotal use of IV cidofovir has been described in disseminated adenoviral infections in immunosuppressed patients and in genitourinary infections with BK virus in renal transplant recipients; however, its efficacy, if any, in these circumstances is not established.

1	CMX-001 (brincidofovir) is an ester prodrug of cidofovir that can be administered orally and may be less nephrotoxic than IV cidofovir. It is being evaluated for prevention of CMV infection in stem cell transplant recipients and for treatment of BK nephropathy and adenovirus infections.

1	Fomivirsen is the first antisense oligonucleotide approved by the FDA for therapy in humans. This phosphorothioate oligonucleotide, 21 nucleotides in length, inhibits CMV replication through interaction with CMV messenger RNA. Fomivirsen is complementary to messenger transcripts of the major immediate early region 2 (IE2) of CMV, which codes for proteins regulating viral gene expression. In addition to its antisense mechanism of action, fomivirsen may exert activity against CMV through inhibition of viral adsorption to cells as well as direct inhibition of viral replication. Because of its different mechanism of action, fomivirsen is active against CMV isolates that are resistant to nucleoside or nucleotide analogues, such as ganciclovir, foscarnet, or cidofovir. Antiviral Chemotherapy, Excluding Antiretroviral Drugs

1	Antiviral Chemotherapy, Excluding Antiretroviral Drugs Fomivirsen has been approved for intravitreal administration in the treatment of CMV retinitis in AIDS patients who have failed to respond to other treatments or cannot tolerate them. Injections of 330 mg for two doses 2 weeks apart, followed by maintenance doses of 330 mg monthly, significantly reduce the rate of progression of CMV retinitis. The major toxicity is ocular inflammation, including vitritis and iritis, which usually responds to topically administered glucocorticoids.

1	An analogue of acyclovir, ganciclovir is active against HSV and VZV and is markedly more active than acyclovir against CMV. Ganciclovir triphosphate inhibits CMV DNA polymerase and can be incorporated into CMV DNA, whose elongation it eventually terminates. In HSV-and VZV-infected cells, ganciclovir is phosphorylated by virus-encoded thymidine kinases; in CMV-infected cells, it is phosphorylated by a viral kinase encoded by the UL97 gene. Ganciclovir triphosphate is present in tenfold higher concentrations in CMV-infected cells than in uninfected cells. Ganciclovir is approved for the treatment of CMV retinitis in immunosuppressed patients and for the prevention of CMV disease in transplant recipients. It is widely used for the treatment of other CMV-associated syndromes, including pneumonia, esophagogastrointestinal infections, hepatitis, and “wasting” illness.

1	Ganciclovir is available for IV or oral administration. Because its oral bioavailability is low (5–9%), relatively large doses (1 g three times daily) must be administered by this route. Oral ganciclovir has largely been supplanted by valganciclovir, which is the l-valyl ester of ganciclovir. Valganciclovir is well absorbed orally, with a bioavailability of 60%, and is rapidly hydrolyzed to ganciclovir in the intestine and liver. The area under the curve for a 900-mg dose of valganciclovir is equivalent to that for 5 mg/kg of IV ganciclovir, although peak serum concentrations are ~40% lower for valganciclovir. The serum half-life is 3.5 h after IV administration of ganciclovir and 4.0 h after PO administration of valganciclovir. Ganciclovir is excreted primarily by the kidneys in an unmetabolized form, and its dosage should be reduced in cases of renal failure. Ganciclovir therapy at the most commonly used initial IV dosage—i.e., 5 mg/kg every 12 h for 14–21 days—can be changed to

1	form, and its dosage should be reduced in cases of renal failure. Ganciclovir therapy at the most commonly used initial IV dosage—i.e., 5 mg/kg every 12 h for 14–21 days—can be changed to valganciclovir (900 mg PO twice daily) when the patient can tolerate oral therapy. The maintenance dose is 5 mg/kg IV daily or five times per week for ganciclovir and 900 mg by mouth once a day for valganciclovir. Dose adjustment in patients with renal dysfunction is required. Intraocular ganciclovir, given by either intravitreal injection or intraocular implantation, has also been used to treat CMV retinitis.

1	Ganciclovir is effective as prophylaxis against CMV-associated disease in organ and bone marrow transplant recipients. Oral ganciclovir administered prophylactically to AIDS patients with CD4+ T cell counts of <100/μL has provided protection against the development of CMV retinitis. However, the long-term benefits of this approach to prophylaxis in AIDS patients have not been established, and most experts do not recommend the use of oral ganciclovir for this purpose. As already mentioned, valganciclovir has supplanted oral ganciclovir in settings where oral prophylaxis or therapy is considered. The administration of ganciclovir has been associated with profound bone marrow suppression, particularly neutropenia, which significantly limits the drug’s use in many patients. Bone marrow toxicity is potentiated in the setting of renal dysfunction and when other bone marrow suppressants, such as zidovudine or mycophenolate mofetil, are used concomitantly.

1	Resistance has been noted in CMV isolates obtained after therapy with ganciclovir, especially those from patients with AIDS or from patients receiving prolonged ganciclovir therapy after organ transplantation. Such resistance may develop through a mutation in either the viral UL97 gene or the viral DNA polymerase. Ganciclovir-resistant isolates are usually sensitive to foscarnet (see below) or may be sensitive to cidofovir, depending on the mechanism of resistance (see above).

1	Famciclovir is the diacetyl 6-deoxyester of the guanosine analogue penciclovir. This agent is well absorbed orally, has a bioavailability of 77%, and is rapidly converted to penciclovir by deacetylation and oxidation in the intestine and liver. Penciclovir’s spectrum of activity and mechanism of action are similar to those of acyclovir. Thus, penciclovir usually is not active against acyclovir-resistant viruses. However, some acyclovir-resistant viruses with altered thymidine kinase or DNA polymerase substrate specificity may be sensitive to penciclovir. This drug is phosphorylated initially by a virus-encoded thymidine kinase and subsequently by cellular kinases to penciclovir triphosphate, which inhibits HSV-1, HSV-2, VZV, and EBV as well as hepatitis B virus (HBV). The serum half-life of penciclovir is 2 h, but the intracellular half-life of penciclovir triphosphate is 7–20 h—markedly longer than that of acyclovir triphosphate. The latter is the basis for the less frequent

1	of penciclovir is 2 h, but the intracellular half-life of penciclovir triphosphate is 7–20 h—markedly longer than that of acyclovir triphosphate. The latter is the basis for the less frequent (twice-daily) dosing schedule for famciclovir than for acyclovir. Penciclovir is eliminated primarily in the urine by both glomerular filtration and tubular secretion. The usually recommended dosage interval should be adjusted for renal insufficiency.

1	Clinical trials involving immunocompetent adults with herpes zoster showed that famciclovir was superior to placebo in eliciting the resolution of skin lesions and virus shedding and in shortening the duration of postherpetic neuralgia; moreover, administered at 500 mg every 8 h, famciclovir was at least as effective as acyclovir administered at an oral dose of 800 mg five times daily. Famciclovir was also effective in the treatment of herpes zoster in immunosuppressed patients. Clinical trials have demonstrated its effectiveness in the suppression of genital HSV infections for up to 1 year and in the treatment of initial and recurrent episodes of genital herpes. Famciclovir is effective as therapy for mucocutaneous HSV infections in HIV-infected patients. Application of a 1% penciclovir cream reduces the duration of signs and symptoms of herpes labialis in immunocompetent patients (by 0.5–1 day) and has been approved for that purpose by the FDA. Famciclovir is generally well

1	cream reduces the duration of signs and symptoms of herpes labialis in immunocompetent patients (by 0.5–1 day) and has been approved for that purpose by the FDA. Famciclovir is generally well tolerated, with occasional headache, nausea, and diarrhea reported in frequencies similar to those among placebo recipients. The administration of high doses of famciclovir for 2 years was associated with an increased incidence of mammary adenocarcinomas in female rats, but the clinical significance of this effect is unknown.

1	Foscarnet (phosphonoformic acid) is a pyrophosphate-containing compound that potently inhibits herpesviruses, including CMV. This drug inhibits DNA polymerases at the pyrophosphate binding site at concentrations that have relatively little effect on cellular polymerases. Foscarnet does not require phosphorylation to exert its antiviral activity and is therefore active against HSV and VZV isolates that are resistant to acyclovir because of deficiencies in thymidine kinase as well as against most ganciclovir-resistant strains of CMV. Foscarnet also inhibits the reverse transcriptase of HIV and is active against HIV in vivo.

1	Foscarnet is poorly soluble and must be administered intravenously via an infusion pump in a dilute solution over 1–2 h. The plasma half-life of foscarnet is 3–5 h and increases with decreasing renal function because the drug is eliminated primarily by the kidneys. It has been estimated that 10–28% of a dose may be deposited in bone, where it can persist for months. The most common initial dosage of foscarnet—60 mg/kg every 8 h for 14–21 days—is followed by a maintenance dose of 90–120 mg/kg once a day.

1	Foscarnet is approved for the treatment of CMV retinitis in patients with AIDS and of acyclovir-resistant mucocutaneous HSV infections. In a comparative clinical trial, the drug appeared to be about as efficacious as ganciclovir against CMV retinitis but was associated with a longer survival period, possibly because of its activity against HIV. Intraocular foscarnet has been used to treat CMV retinitis. In addition, foscarnet has been employed to treat acyclovir-resistant HSV and VZV infections as well as ganciclovir-resistant CMV infections, although resistance to foscarnet has been reported in CMV isolates obtained during therapy. Foscarnet has also been used to treat HHV-6 infections in immunosuppressed patients.

1	The major form of toxicity associated with foscarnet is renal impairment. Thus renal function should be monitored closely, particularly during the initial phase of therapy. Because foscarnet binds divalent metal ions, hypocalcemia, hypomagnesemia, hypokalemia, and hypoor hyperphosphatemia can develop. Saline hydration and slow infusion appear to protect the patient against nephrotoxicity and electrolyte disturbances. Although hematologic abnormalities have been documented (most commonly anemia), foscarnet is not generally myelosuppressive and can be administered concomitantly with myelosuppressive medications.

1	Trifluridine is a pyrimidine nucleoside active against HSV-1, HSV-2, and CMV. Trifluridine monophosphate irreversibly inhibits thymidylate synthetase, and trifluridine triphosphate inhibits viral and, to a lesser extent, cellular DNA polymerases. Because of systemic toxicity, trifluridine’s use is limited to topical therapy. Trifluridine is approved for treatment of HSV keratitis, against which trials have shown that it is more effective than topical idoxuridine but similar in efficacy to topical vidarabine. The drug has benefited some patients with HSV keratitis who have failed to respond to idoxuridine or vidarabine. Topical application of trifluridine to sites of acyclovir-resistant HSV mucocutaneous infection has also been beneficial in some cases.

1	Vidarabine is a purine nucleoside analogue with activity against HSV-1, HSV-2, VZV, and EBV. Vidarabine inhibits viral DNA synthesis through its 5′-triphosphorylated metabolite, although its precise molecular mechanisms of action are not completely understood. IV-administered vidarabine has been shown to be effective in the treatment of herpes simplex encephalitis, mucocutaneous HSV infections, herpes zoster in immunocompromised patients, and neonatal HSV infections. Its use has been supplanted by that of IV acyclovir, which is more effective and easier to administer. Production of the IV preparation has been discontinued by the manufacturer, but vidarabine is available as an ophthalmic ointment, which is effective in the treatment of HSV keratitis.

1	Maribavir is a benzimidazole that inhibits CMV and EBV. This drug inhibits the CMV UL97 kinase and does not require intracellular phosphorylation for its antiviral activity. Its mechanism of action involves blocking viral DNA synthesis and virion egress. Maribavir is orally administered and has been associated with taste disturbance and diarrhea. In phase 3 studies, it was not efficacious in the prevention of CMV infection in recipients of hematopoietic stem cell and adult liver transplants. However, when used at somewhat higher doses, it may be efficacious for the treatment of refractory or resistant CMV infections in transplant recipients.

1	Letermovir is an investigational drug with activity against CMV. It is a dihydroquinozoline that acts through inhibition of the viral terminase enzyme complex. This mechanism of action differs from that of ganciclovir, foscarnet, and cidofovir, which inhibit viral DNA polymerase; therefore, letermovir is active against CMV isolates that are resistant to those drugs. It is orally administered and is reportedly well tolerated. Letermovir is being evaluated as prophylaxis against CMV in hematopoietic stem cell recipients.

1	Inhibition of the helicase-primase heterotrimeric complex of HSV-1 and HSV-2 represents a novel mechanism of action of amenamevir and pritelivir. These drugs are being assessed for prevention and treatment of HSV genital infection. The efficacy of amenamevir, administered as a single oral dose of 1200 mg for recurrent genital herpes, was comparable to that of valacyclovir given for 3 days. Pritelivir has a long half-life (up to 80 h) and was studied in a placebo-controlled trial of suppression of genital HSV infections. Compared with placebo, pritelivir—a loading dose followed by either a daily oral dose of 75 mg for 4 weeks or a weekly dose of 400 mg for 4 weeks—reduced HSV shedding and days of genital lesions. Additional clinical studies of the helicase-primase inhibitors of HSV are planned.

1	Lamivudine is a pyrimidine nucleoside analogue that is used primarily in combination therapy against HIV infection (Chap. 226). Its activity against HBV is attributable to inhibition of the viral DNA polymerase. This drug has also been approved for the treatment of chronic HBV infection. At doses of 100 mg/d given for 1 year to patients positive for hepatitis B e antigen (HBeAg), lamivudine is well tolerated and results in suppression of HBV DNA levels, normalization of serum aminotransferase levels in 40–75% of patients, and reduction of hepatic inflammation and fibrosis in 50–60% of patients. Loss of HBeAg occurs in 30% of patients. Lamivudine also appears to be useful in the prevention or suppression of HBV infection associated with liver transplantation. Resistance to lamivudine develops in 24% of patients treated for 1 year and is associated with changes in the YMDD motif of HBV DNA polymerase. Because of the frequency of development of resistance, lamivudine has been largely

1	in 24% of patients treated for 1 year and is associated with changes in the YMDD motif of HBV DNA polymerase. Because of the frequency of development of resistance, lamivudine has been largely supplanted by less-resistanceprone drugs for the treatment of HBV infection.

1	Adefovir dipivoxil is the oral prodrug of adefovir, an acyclic nucleotide analogue of adenosine monophosphate that is active against HBV, HIV, HSV, CMV, and poxviruses. It is phosphorylated by cellular kinases to the active triphosphate moiety, which is a competitive inhibitor of HBV DNA polymerase and results in chain termination after incorporation into nascent viral DNA. Adefovir is administered orally and is eliminated primarily by the kidneys, with a plasma half-life of 5–7.5 h. In clinical studies, therapy with adefovir at a dose of 10 mg/d for 48 weeks resulted in normalization of serum alanine aminotransferase (ALT) levels in 48–72% of patients and improved liver histology in 53–64%; it also resulted in a 3.5to 3.9-log10 reduction in the number of HBV DNA copies per milliliter of plasma. Adefovir was effective in treatment-naïve patients as well as in those infected with lamivudineresistant HBV. Resistance to adefovir appears to develop less readily than that to lamivudine,

1	plasma. Adefovir was effective in treatment-naïve patients as well as in those infected with lamivudineresistant HBV. Resistance to adefovir appears to develop less readily than that to lamivudine, but adefovir resistance rates of 15–18% have been reported after 192 weeks of treatment and may reach 30% after 5 years. This agent is generally well tolerated. Significant nephrotoxicity attributable to adefovir is uncommon at the dose used in the treatment of HBV infections (10 mg/d) but is a treatment-limiting adverse effect at the higher doses used in therapy for HIV infections (30–120 mg/d). In any case, renal function should be monitored in patients taking adefovir, even at the lower dose. Adefovir is approved only for treatment of chronic HBV infection.

1	Tenofovir disoproxil fumarate is a prodrug of tenofovir, a nucleotide analogue of adenosine monophosphate with activity against both retroviruses and hepadnaviruses. In both immunocompetent and immunocompromised patients (including those co-infected with HIV and HBV), tenofovir given at a dose of 300 mg/d for 48 weeks reduced HBV replication by 4.6–6 log10, normalized ALT levels in 68–76% of patients, and improved liver histopathology in 72–74% of patients. Resistance develops uncommonly during ≥2 years of therapy, and tenofovir is active against lamivudine-resistant HBV. The safety profile of tenofovir is similar to that of adefovir, but nephrotoxicity has not been encountered at the dose used for HBV therapy. Tenofovir is approved for the treatment of HIV and chronic HBV infections. For a more detailed discussion of tenofovir, see Chap. 226.

1	Entecavir is a cyclopentyl 2′-deoxyguanosine analogue that inhibits HBV through interaction of entecavir triphosphate with several HBV DNA polymerase functions. At a dose of 0.5 mg/d given for 48 weeks, entecavir reduced HBV DNA copies by 5.0–6.9 log10, normalized serum aminotransferase levels in 68–78% of patients, and improved histopathology in 70–72% of patients. Entecavir inhibits lamivudineresistant viruses that have M550I or M550V/L526M mutations but only at serum concentrations 20or 30-fold higher than those obtained with the 0.5-mg/d dose. Thus, higher doses of entecavir (1 mg/d) are recommended for the treatment of patients infected with lamivudineresistant HBV. Development of resistance to entecavir is uncommon in treatment-naïve patients but does occur at unacceptably high rates

1	Antiviral Chemotherapy, Excluding Antiretroviral Drugs (43% after 4 years) in patients previously infected with lamivudineresistant virus. Entecavir-resistant strains appear to be sensitive to adefovir and tenofovir.

1	Entecavir is highly bioavailable but should be taken on an empty stomach because food interferes with its absorption. The drug is eliminated primarily in unchanged form by the kidneys, and its dosage should be adjusted for patients with CrCl values of <50 mL/min. Overall, entecavir is well tolerated, with a safety profile similar to that of lamivudine. As with other anti-HBV treatments, exacerbation of hepatitis may occur when entecavir therapy is stopped. Entecavir is approved for treatment of chronic hepatitis B, including infection with lamivudineresistant viruses, in adults. Entecavir has some activity against HIV-1 (median effective concentration, 0.026 to >10 μM) but should not be used as monotherapy in HIV-positive patients because of the potential for development of HIV resistance due to the M184V mutation.

1	Telbivudine is a β-l enantiomer of thymidine and is a potent, selective inhibitor of HBV. Its active form is telbivudine triphosphate, which inhibits HBV DNA polymerase and causes chain termination but has little or no activity against human DNA polymerase. Administration of telbivudine at an oral dose of 600 mg/d for 52 weeks to patients with chronic hepatitis B resulted in reduction of HBV DNA by 5.2–6.4 log10 copies/mL along with normalization of ALT levels in 74–77% of recipients and improved histopathology in 65–67% of patients. Telbivudineresistant HBV is generally cross-resistant with lamivudine-resistant virus but is usually susceptible to adefovir. After 2 years of therapy, resistance to telbivudine was noted in isolates from 22% of HBeAgpositive patients and in those from 9% of HBeAg-negative patients.

1	Orally administered telbivudine is rapidly absorbed; because it is eliminated primarily by the kidneys, its dosage should be reduced in patients with a CrCl value of <50 mL/min. Telbivudine is generally well tolerated, but increases in serum levels of creatinine kinases as well as fatigue and myalgias have been observed. As with other anti-HBV drugs, hepatitis may be exacerbated in patients who discontinue telbivudine therapy. Telbivudine has been approved for the treatment of adults with chronic hepatitis B who have evidence of viral replication and either persistently elevated serum aminotransferase levels or histopathologically active disease, but it has not been widely used because of the frequency of development of resistance, as noted above. IFNs are cytokines that exhibit a broad spectrum of antiviral activities as well as immunomodulating and antiproliferative properties. IFNs are not available for oral administration but must be given IM, SC, or

1	IV. Early studies with human leukocyte IFN demonstrated an effect in the prophylaxis of experimentally induced rhinovirus infections in humans and in the treatment of VZV infections in immunosuppressed patients. DNA recombinant technology has made available highly purified α, β, γ, and λ IFNs that have been evaluated in a variety of viral infections. Results of such trials have confirmed the effectiveness of intranasally administered IFN in the prophylaxis of rhinovirus infections, although its use has been associated with nasal mucosal irritation. Studies have also demonstrated a beneficial effect of intralesionally or systemically administered IFNs on genital warts. The effect of systemic administration consists primarily of a reduction in the size of the warts, and this mode of therapy may be useful in persons who have numerous warts that cannot easily be treated by individual intralesional injections. However, lesions frequently recur after either intralesional or systemic IFN

1	may be useful in persons who have numerous warts that cannot easily be treated by individual intralesional injections. However, lesions frequently recur after either intralesional or systemic IFN therapy is discontinued.

1	IFNs have undergone extensive study in the treatment of chronic HBV infection. The administration of standard IFN-α2b (5 million units daily or 10 million units three times a week for 16–24 weeks) to patients with stable chronic HBV infection resulted in loss of markers of HBV replication, such as HBeAg and HBV DNA, in 33–37% of cases; 8% of patients also became negative for hepatitis B surface antigen. In most patients who lose HBeAg and HBV DNA markers, serum aminotransferases return to normal levels, and both shortand long-term improvements in liver histopathology have been described. Predictors of a favorable response to standard IFN therapy include low pretherapy levels of HBV DNA, high pretherapy serum levels of ALT, a short duration of chronic HBV infection, and active inflammation in liver histopathology. Poor responses are seen in immunosuppressed patients, including those with HIV infection.

1	In pegylated IFNs, IFN alphas are linked to polyethylene glycol. This linkage results in slower absorption, decreased clearance, and more sustained serum concentrations, thereby permitting a more convenient, once-weekly dosing schedule; in many instances, pegylated IFN has supplanted standard IFN. After 48 weeks of treatment with 180 μg of pegylated IFN-α2a, HBV DNA was reduced by 4.1–4.5 log10 copies/mL, with normalization of serum ALT levels in 39% of patients and improved histology in 38%. Response rates were somewhat higher when lamivudine was administered with pegylated IFN-α2a. Adverse effects of IFN are common and include fever, chills, myalgia, fatigue, neurotoxicity (manifested primarily as somnolence, depression, anxiety, and confusion), and leukopenia. Autoantibodies (e.g., antithyroid antibodies) can also develop. IFN-α2b and pegylated IFN-α2a are approved for the treatment of patients with chronic hepatitis B. Data supporting the therapeutic efficacy of pegylated

1	antithyroid antibodies) can also develop. IFN-α2b and pegylated IFN-α2a are approved for the treatment of patients with chronic hepatitis B. Data supporting the therapeutic efficacy of pegylated interferon-α2b in HBV infection have been published; the drug has not been approved for this indication in the United States but has been approved for treatment of chronic HBV infection in other countries.

1	Several IFN preparations, including IFN-α2a, IFN-α2b, IFNalfacon-1, and IFN-αm1 (lymphoblastoid), have been studied as therapy for chronic HCV infections. A variety of monotherapy regimens have been studied, of which the most common for standard IFN is IFN-α2b or -α2a at 3 million units three times per week for 12–18 months. The addition of oral ribavirin to IFN-α2b—either as initial therapy or after failure of IFN therapy alone—results in significantly higher rates of sustained virologic and/or serum ALT responses (40–50%) than are obtained with monotherapy. Comparative studies indicate that pegylated IFN-α2b or -α2a therapy is more effective than standard IFN treatment against chronic HCV infection. The combination of SC pegylated IFN and oral ribavirin results in sustained virologic responses (SVRs) in 42–51% of patients with HCV genotype 1 infection and in 76–82% of patients with genotype 2 or 3 infection. Ribavirin appears to have a small antiviral effect in HCV infection but may

1	(SVRs) in 42–51% of patients with HCV genotype 1 infection and in 76–82% of patients with genotype 2 or 3 infection. Ribavirin appears to have a small antiviral effect in HCV infection but may also be working through an immunomodulatory effect in combination with IFN. Optimal results with ribavirin appear to be associated with weight-based dosing. Prognostic factors for a favorable response include an age of <40 years, a short duration of infection, low levels of HCV RNA, a lesser degree of liver histopathology, and infection with HCV genotypes other than 1. IFN-alfacon, a synthetic “consensus” α interferon, appears to produce response rates similar to those elicited by standard IFN-α2a or -α2b alone. In 2014, the approval of a polymerase inhibitor, sofosbuvir, and a second-generation protease inhibitor, simeprevir, led to revised recommendations for treatment of hepatitis C with triple combinations of pegylated IFN, ribavirin, and one of these two drugs, depending on the viral

1	protease inhibitor, simeprevir, led to revised recommendations for treatment of hepatitis C with triple combinations of pegylated IFN, ribavirin, and one of these two drugs, depending on the viral genotype (see below and Table 215e-1).

1	IFN-α and pegylated IFN-α are active against hepatitis D, but high doses are required (9 million units three times per week for 48 weeks). IFN-α elicited an SVR in 25–30% of patients, whereas pegylated IFN-α had a variable effect, evoking an SVR in 17–43% of patients. However, long-term biochemical and histologic improvements have been seen, even in the absence of sustained inhibition of viral replication. Sofosbuvir is the prodrug of a uridine nucleoside inhibitor of the HCV RNA NS5B polymerase. Its metabolism to the active uridine nucleoside triphosphate results in chain termination. Sofosbuvir is active against all HCV genotypes (1–6) and has a median effective concentration (EC50) of 0.7–2.6 μM against NS5B. Resistance to sofosbuvir is conferred by an S282T substitution in NS5B, but clinically expressed resistance to treatment has only rarely been encountered in patients who receive sofosbuvir.

1	Sofosbuvir is administered orally and is unaffected by food. After oral administration, plasma concentrations of sofosbuvir and of its active metabolite peak in 0.5–2 h and 2–4 h, respectively. Approximately 61–65% of sofosbuvir is bound in plasma proteins, but very little of the active metabolite is bound. Both sofosbuvir and its active metabolite are cleared renally, with t1/2 values of 0.4 h and 27 h, respectively. Sofosbuvir is relatively free from clinically significant drug interactions, although P‐glycoprotein inducers can reduce sofosbuvir concentrations. Sofosbuvir is generally well tolerated and has not been associated with significant toxicity. The most common side effects in recipients of sofosbuvir have been attributable to concomitant administration of IFN and ribavirin in combination clinical trials (see below).

1	Sofosbuvir has been studied in a variety of controlled and open‐label clinical trials. In late 2013, the results of these trials led to its recommendation—in triple combination with pegylated IFN and ribavirin—as first-line treatment for chronic hepatitis due to HCV genotypes 1, 4, 5, and 6, in which SVRs among treatment‐naïve patients were 89–97%. For HCV genotypes 2 and 3, IFN‐free regimens consisting of sofosbuvir and ribavirin have been recommended, with SVRs among treatment‐naïve patients of 93% for genotype 2 and 61% for genotype 3. BOCEPREVIR, TELAPREVIR

1	BOCEPREVIR, TELAPREVIR This drug class is specifically designed to inhibit the 3/4A (NS3/4A) HCV protease. These agents resemble the HCV polypeptide and, when processed by the viral protease, form a covalent bond with the catalytic NS3 serine residues, block further activity, and prevent proteolytic cleavage of the HCV polyprotein into NS4A, NS4B, NS5A, and NS5B proteins. Boceprevir and telaprevir are linear ketoamide compounds that are active against HCV genotype 1 (1b > 1a) and much less so against genotypes 2 and 3. These first-generation protease inhibitors received approval for combination therapy (with IFN and ribavirin) for genotype 1 infection. Neither boceprevir nor telaprevir is now recommended for the treatment of hepatitis C. These drugs have been supplanted by sofosbuvir and by simeprevir, a second‐generation protease inhibitor with improved pharmacokinetic properties, fewer drug–drug interactions, and less overall toxicity (see below).

1	Simeprevir is a second‐generation NS3/4A protease inhibitor with antiviral activity against genotype 1 (1b > 1a); the EC50 is 9.4 nM in an HCV genotype 1b replicon. The NS3 polymorphism Q80K, which is present in approximately one‐third of patients carrying HCV genotype 1b, increases the EC50 by elevenfold and results in clinical resistance to simeprevir. Thus, testing for Q80K should be carried out if treatment with simeprevir is being considered. Cross‐resistance occurs between simeprevir and the first‐generation protease inhibitors boceprevir and telaprevir.

1	Simeprevir is orally administered as a 150-mg capsule, and its bioavailability is increased by administration with food. The serum concentration peaks 4–6 h after oral administration. The drug’s elimination half-life is 10–13 h in healthy individuals and 41 h in patients with hepatitis C. Simeprevir is nearly entirely bound by plasma proteins and cleared by biliary excretion. Because there is no renal excretion, dose adjustments are not required in the presence of renal dysfunction. Simeprevir is metabolized by hepatic CYP3A and therefore should not be used in patients with decompensated liver function.

1	Because of its metabolism by cytochrome P450 3A (CYP3A), simeprevir interacts with drugs that induce or inhibit CYP3A, and these interactions may concomitantly increase or reduce plasma concentrations of simeprevir. Administration of simeprevir may also increase plasma concentrations of drugs that are substrates for hepatic organic anion‐transporting polypeptide 1B1 or 1B3 or for P glycoprotein transporters. Toxicity observed during clinical trials with simeprevir included photosensitivity (usually mild or moderate) in 28% of recipients and reversible hyperbilirubinemia (both conjugated and unconjugated), which was generally mild to moderate. Most of the other adverse effects that were seen in clinical trials with simeprevir were attributable to concomitant administration of IFN and ribavirin.

1	Simeprevir has been recommended as a component of alternative treatment—in combination with pegylated IFN and ribavirin—of chronic infection with HCV genotypes 1 and 4. Daily simeprevir, daily ribavirin, and weekly pegylated IFN for 12 weeks followed by another 12 weeks of pegylated IFN and ribavirin resulted in an SVR of 80% in the absence of the Q80K variant. In general, simeprevir‐based triple therapy appeared to be 10% less likely to yield an SVR than sofosbuvirbased therapy and more likely to cause adverse effects. However, for prior nonresponders or partial responders to pegylated IFN, the IFN‐free regimen of simeprevir, sofosbuvir, and ribavirin shows promise.

1	Next-generation direct-acting antivirals against HCV inhibitors are under active development. These agents include second-generation inhibitors of NS3/4, NS5B polymerase inhibitors, and inhibitors of NS5A (a membrane-associated phosphoprotein that is part of the HCV RNA replication complex). These investigational agents are making progress toward IFN-free regimens, shorter courses of therapy, improved tolerability, and reduction of resistance. For updated information, readers should consult http://www.hcvguidelines.org/. Antiviral Chemotherapy, Excluding Antiretroviral Drugs Herpes simplex virus Infections Lawrence Corey DEFINITION Herpes simplex viruses (HSV-1, HSV-2; Herpesvirus hominis) produce a variety of infections involving mucocutaneous surfaces, the central nervous system (CNS), and—on occasion—visceral organs. Prompt 216 sEC TIOn 12 InFECTIOns duE TO dnA vIRusEs recognition and treatment reduce the morbidity and mortality rates associated with HSV infections.

1	The genome of HSV is a linear, double-strand DNA molecule (molecular weight, ~100 × 106) that encodes >90 transcription units with 84 identified proteins. The genomic structures of the two HSV subtypes are similar. The overall genomic sequence homology between HSV-1 and HSV-2 is ~50%, whereas the proteome homology is >80%. The homologous sequences are distributed over the entire genome map, and most of the polypeptides specified by one viral type are antigenically related to polypeptides of the other viral type. Many type-specific regions unique to HSV-1 and HSV-2 proteins do exist, however, and a number of them appear to be important in host immunity. These type-specific regions have been used to develop serologic assays that distinguish between the two viral subtypes. Either restriction endonuclease analysis or sequencing of viral DNA can be used to distinguish between the two subtypes and among strains of each subtype. The variability of nucleotide sequences from clinical strains

1	endonuclease analysis or sequencing of viral DNA can be used to distinguish between the two subtypes and among strains of each subtype. The variability of nucleotide sequences from clinical strains of HSV-1 and HSV-2 is such that HSV isolates obtained from two individuals can be differentiated by restriction enzyme patterns or genomic sequences. Moreover, epidemiologically related sources, such as sexual partners, mother-infant pairs, or persons involved in a common-source outbreak, can be inferred from such patterns.

1	The viral genome is packaged in a regular icosahedral protein shell (capsid) composed of 162 capsomeres (see Fig. 214e-1). The outer covering of the virus is a lipid-containing membrane (envelope) acquired as the DNA-containing capsid buds through the inner nuclear membrane of the host cell. Between the capsid and lipid bilayer of the envelope is the tegument. Viral replication has both nuclear and cytoplasmic phases. Initial attachment to the cell membrane involves interactions of viral glycoproteins C and B with several cellular heparan sulfate–like surface receptors. Subsequently, viral glycoprotein D binds to cellular co-receptors that belong to the tumor necrosis factor receptor family of proteins, the immunoglobulin superfamily (nectin family), or both. The ubiquity of these receptors contributes to the wide host range of herpesviruses. HSV replication is highly regulated. After fusion and entry, the nucleocapsid enters the cytoplasm and several viral proteins are released from

1	contributes to the wide host range of herpesviruses. HSV replication is highly regulated. After fusion and entry, the nucleocapsid enters the cytoplasm and several viral proteins are released from the virion. Some of these viral proteins shut off host protein synthesis (by increasing cellular RNA degradation), whereas others “turn on” the transcription of early genes of HSV replication. These early gene products, designated α genes, are required for synthesis of the subsequent polypeptide group, the β polypeptides, many of which are regulatory proteins and enzymes required for DNA replication. Most current antiviral drugs interfere with β proteins, such as viral DNA polymerase. The third (γ) class of HSV genes requires viral DNA replication for expression and encodes most structural proteins specified by the virus.

1	After viral genome replication and structural protein synthesis, nucleocapsids are assembled in the cell’s nucleus. Envelopment occurs as the nucleocapsids bud through the inner nuclear membrane into the perinuclear space. In some cells, viral replication in the nucleus forms two types of inclusion bodies: type A basophilic Feulgen-positive bodies that contain viral DNA and eosinophilic inclusion bodies that are devoid of viral nucleic acid or protein and represent a “scar” of viral infection. Enveloped virions are then transported via the endoplasmic reticulum and the Golgi apparatus to the cell surface.

1	Viral genomes are maintained by some neuronal cells in a repressed state called latency. Latency, which is associated with transcription of only a limited number of virus-encoded RNAs, accounts for the presence of viral DNA and RNA in neural tissue at times when infectious virus cannot be isolated. Maintenance and growth of neural cells from latently infected ganglia in tissue culture result in production of infectious virions (explantation) and in subsequent permissive infection of susceptible cells (co-cultivation). Activation of the viral genome may then occur, resulting in reactivation—the normal pattern of regulated viral gene expression and replication and HSV release. The release of virions from the neuron follows a complex process of anterograde transport down the length of neuronal axons. In experimental animals, ultraviolet light, systemic and local immunosuppression, and trauma to the skin or ganglia are associated with reactivation.

1	Three noncoding RNA latency-associated transcripts (LATs) are found in the nuclei of latently infected neurons. Deletion mutants of the LAT region exhibit reduced efficiency in their later reactivation. Substitution of HSV-1 LATs for HSV-2 LATs induces an HSV-1 reactivation pattern. These data indicate that LATs apparently maintain— rather than establish—latency. HSV-1 LATs promote the survival of acutely infected neurons, perhaps by inhibiting apoptotic pathways. LAT transcript abundance and low genome-copy number correlate with subnuclear positioning of HSV genomes around the centromere. Indeed, chromatization of HSV DNA appears to play a vital role in silencing expression of lytic replication genes. Highly expressed during latency, LAT-derived micro-RNA appears to silence expression of the key neurovirulence factor infected-cell protein 34.5 (ICP34.5) and to bind in an antisense configuration to the immediate-early protein ICP0 messenger RNA to prevent expression, which is vital to

1	the key neurovirulence factor infected-cell protein 34.5 (ICP34.5) and to bind in an antisense configuration to the immediate-early protein ICP0 messenger RNA to prevent expression, which is vital to HSV reactivation. Although certain viral transcripts are known to be necessary for reactivation from latency, the molecular mechanisms of HSV latency are not fully understood, and strategies to interrupt or maintain latency in neurons are in developmental stages.

1	While latency is the predominant state of virus on a per-neuron basis, the high frequency of oral and genital tract reactivation for HSV-1 and HSV-2 suggests that the viruses are rarely quiescent within the entire biomass of ganglionic tissue. Recent data indicate that HSV-2 antigen is often shed: most persons infected with HSV-2 have frequent subclinical bursts of reactivation lasting 2–4 h, and the host mucosal immune system can contain viral reactivation in the mucosa before the development of clinical reactivation. Supporting this clinical observation, recent work using microdissection plus real-time polymerase chain reaction (PCR) of individual neurons from cadaveric trigeminal ganglia explants revealed that many more neurons (2–10%) harbor HSV than would be predicted by in situ hybridization studies for LATs. Viral copy number is highly variable between neurons, with extremely high levels in certain neurons, and HSV DNA copy numbers are similar in LAT-positive and LAT-negative

1	studies for LATs. Viral copy number is highly variable between neurons, with extremely high levels in certain neurons, and HSV DNA copy numbers are similar in LAT-positive and LAT-negative neurons; these findings add to the uncertainty about the role that LATs play in preventing reactivation.

1	Exposure to HSV at mucosal surfaces or abraded skin sites permits entry of the virus into cells of the epidermis and dermis and initiation of viral replication therein. HSV infections are usually acquired sub-clinically. Whether clinical or subclinical, HSV acquisition is associated with sufficient viral replication to permit infection of either sensory or autonomic nerve endings. On entry into the neuronal cell, the virus— or, more likely, the nucleocapsid—is transported intra-axonally to the nerve cell bodies in ganglia. In humans, the transit interval of spread to the ganglia after virus inoculation into peripheral tissue is unknown. During the initial phase of infection, viral replication occurs in ganglia 1176 and contiguous neural tissue. Virus then spreads to other mucocutaneous surfaces through centrifugal migration of infectious virions via peripheral sensory nerves. This mode of spread helps explain the large surface area involved, the high frequency of new lesions distant

1	through centrifugal migration of infectious virions via peripheral sensory nerves. This mode of spread helps explain the large surface area involved, the high frequency of new lesions distant from the initial crop of vesicles that is characteristic in patients with primary genital or oral-labial HSV infection, and the ability to recover virus from neural tissue distant from neurons innervating the inoculation site. Contiguous spread of locally inoculated virus also may take place and allow further mucosal extension of disease. Recent studies have demonstrated HSV viremia—another mechanism for extension of infection throughout the body—in ~30–40% of persons with primary HSV-2 infection. Latent infection with both viral subtypes in both sensory and autonomic ganglia has been demonstrated. For HSV-1 infection, trigeminal ganglia are most commonly infected, although extension to the inferior and superior cervical ganglia also occurs. With genital infection, sacral nerve root ganglia

1	For HSV-1 infection, trigeminal ganglia are most commonly infected, although extension to the inferior and superior cervical ganglia also occurs. With genital infection, sacral nerve root ganglia (S2–S5) are most commonly affected. After resolution of primary disease, infectious HSV can no longer be cultured from the ganglia; however, latent infection, as defined by the presence of viral DNA, persists in 2–11% of ganglionic cells in the anatomic region of the initial infection. The mechanism of reactivation from latency is unknown. Increasingly, studies indicate that host T cell responses at the ganglionic and peripheral mucosal levels influence the frequency and severity of HSV reactivation. HSV-specific T cells have been recovered from peripheral-nerve root ganglia. Many of these resident CD8+ T cells are juxtaposed with latently HSV-1-infected neurons in the trigeminal ganglia and can block reactivation with both interferon (IFN) γ release and granzyme B degradation of the

1	resident CD8+ T cells are juxtaposed with latently HSV-1-infected neurons in the trigeminal ganglia and can block reactivation with both interferon (IFN) γ release and granzyme B degradation of the immediate-early protein ICP4. In addition, there appears to be a latent viral load in the ganglia that correlates positively with the number of neurons infected and the rate of reactivation but inversely with the number of CD8+ cells present. It is not known whether reactivating stimuli transiently suppress these immune cells, independently upregulate transcription of lytic genes, or both. Moreover, host containment in the mucosa has been demonstrated. Once virus reaches the dermal-epidermal junction, there are three possible outcomes: rapid host containment of infection near the site of reactivation; spread of virus into the epidermis, with a micro-ulceration associated with low-titer subclinical shedding; and subsequent rapid (within hours) containment of virus with widespread replication

1	spread of virus into the epidermis, with a micro-ulceration associated with low-titer subclinical shedding; and subsequent rapid (within hours) containment of virus with widespread replication and necrosis of epithelial cells and subsequent clinical recurrence (the latter defined clinically by a skin blister and ulceration). Histologically, herpetic lesions involve a thin-walled vesicle or ulceration in the basal region, multinucleated cells that may include intranuclear inclusions, necrosis, and an acute inflammatory infection. Re-epithelialization occurs once viral replication is restricted, almost always in the absence of a scar. Analysis of the DNA from sequential isolates of HSV or from isolates from multiple infected ganglia in any one individual has revealed similar, if not identical, restriction endonuclease or DNA sequence patterns in most persons. As more sensitive genomic technologies are developed, evidence of multiple strains of the same subtype is increasingly being

1	restriction endonuclease or DNA sequence patterns in most persons. As more sensitive genomic technologies are developed, evidence of multiple strains of the same subtype is increasingly being reported. For example, infection of individual neurons with multiple strains of drug-susceptible and drug-resistant virus in severely immunosuppressed patients indicates that ganglia can be reseeded during chronic infection. Because exposure to mucosal shedding is relatively common during a person’s lifetime, current data suggest that exogenous infection with different strains of the same subtype, while possible, is uncommon.

1	Host responses influence the acquisition of HSV disease, the severity of infection, resistance to the development of latency, the maintenance of latency, and the frequency of recurrences. Both antibody-mediated and cell-mediated reactions are clinically important. Immunocompromised patients with defects in cell-mediated immunity experience more severe and more extensive HSV infections than those with deficits in humoral immunity, such as agammaglobulinemia. Experimental ablation of lymphocytes indicates that T cells play a major role in preventing lethal disseminated disease, although antibodies help reduce titers of virus in neural tissue. Some clinical manifestations of HSV appear to be related to the host immune response (e.g., stromal opacities associated with recurrent herpetic keratitis). The surface viral glycoproteins have been shown to be targets of antibodies that mediate neutralization and immune-mediated cytolysis (antibody-dependent cell-mediated cytotoxicity). Monoclonal

1	The surface viral glycoproteins have been shown to be targets of antibodies that mediate neutralization and immune-mediated cytolysis (antibody-dependent cell-mediated cytotoxicity). Monoclonal antibodies specific for each of the known viral glycoproteins have, in experimental infections, conferred protection against subsequent neurologic disease or ganglionic latency. In humans, however, subunit glycoprotein vaccines have been largely ineffective in reducing acquisition of infection. Multiple cell populations, including natural killer cells, macrophages, and a variety of T lymphocytes, play a role in host defenses against HSV infections, as do lymphokines generated by T lymphocytes. In animals, passive transfer of primed lymphocytes confers protection from subsequent HSV challenge. Maximal protection usually requires the activation of multiple T cell subpopulations, including cytotoxic T cells and T cells responsible for delayed hypersensitivity. The latter may confer protection by

1	protection usually requires the activation of multiple T cell subpopulations, including cytotoxic T cells and T cells responsible for delayed hypersensitivity. The latter may confer protection by the antigen-stimulated release of lymphokines (e.g., IFNs), which in turn have a direct antiviral effect and both activate and enhance a variety of specific and nonspecific effector cells. The HSV virion contains a variety of genes that are directed at the inhibition of host responses. These include gene no. 12 (US-12), which can bind to the cellular transporter-activating protein TAP-1 and reduce the ability of this protein to bind HSV peptides to human leukocyte antigen (HLA) class I, thereby reducing recognition of viral proteins by cytotoxic T cells of the host. This effect can be overcome by the addition of IFN-γ, but this reversal requires 24–48 h; thus, the virus has time to replicate and invade other host cells. Entry of infectious HSV-1 and HSV-2 inhibits several signaling pathways

1	the addition of IFN-γ, but this reversal requires 24–48 h; thus, the virus has time to replicate and invade other host cells. Entry of infectious HSV-1 and HSV-2 inhibits several signaling pathways of both CD4+ and CD8+ T cells, leading to their functional impairment in killing and influencing the spectrum of their cytokine secretion.

1	Increasing evidence suggests that HSV-specific CD8+ T cell responses are critical for clearance of virus from lesions. Immunosuppressed patients with frequent and prolonged HSV lesions have fewer functional CD8+ T cells directed at HSV. HSV-specific CD8+ T cells have been shown to persist in the genital skin at the dermal– epidermal junction contiguous to neuronal endings. Even during clinical quiescence, these CD8+ T cells make both antiviral and cytotoxic proteins indicative of immune surveillance. These resident memory CD8+ T cells appear to be “first responders” capable of controlling viral reactivation at the site of viral release into the dermis. This rapid “on and off” interplay between the virus and host helps explain the variability in clinical disease severity between episodes in any single individual. Differences of 30–60 min in host responses can result in 100to 1000-fold differences in viral levels and can determine whether an episode of disease is subclinical or

1	in any single individual. Differences of 30–60 min in host responses can result in 100to 1000-fold differences in viral levels and can determine whether an episode of disease is subclinical or clinical.

1	There is a strong association between the magnitude of the CD8+ T lymphocyte response and the clearance of virus from genital lesions. The location, effectiveness, and longevity of the T lymphocytes (and perhaps of other immune effector cells) may be important in the expression of disease and the likelihood of transmission over time. worldwide. The past 15 years have shown that the prevalence of HSV-2 is even higher in the developing than in the developed world. In sub-Saharan Africa, HSV-2 seroprevalence among pregnant women may approach 60%, and annual acquisition rates among teenage girls may verge on 20%. The global incidence has been estimated at ~23.6 million infections per year. As in the developed world, the rate of HSV-2 coital acquisition as well as the serologic prevalence is higher among women than among men. Most of this HSV-2 acquisition is preceded by acquisition of HSV-1; the frequency of genital HSV-1 in the developing world is low at present.

1	Infection with HSV-1 is acquired more frequently and earlier than infection with HSV-2. More than 90% of adults have antibodies to HSV-1 by the fifth decade of life. In populations of low socioeconomic status, most persons acquire HSV-1 infection before the third decade of life. Antibodies to HSV-2 are not detected routinely until puberty. Antibody prevalence rates correlate with past sexual activity and vary greatly among different population groups. There is evidence that the prevalence of HSV-2 has decreased slightly over the past decade in the United States. Serosurveys indicate that 15–20% of the U.S. population has antibodies to HSV-2. In most routine obstetric and family planning clinics, 25% of women have HSV-2 antibodies, although only 10% of those who are seropositive for HSV-2 report a history of genital lesions. As many as 50% of heterosexual adults attending sexually transmitted disease clinics have antibodies to HSV-2.

1	Many studies continue to show that both incident and—more important—prevalent HSV-2 infection enhances the acquisition rate of HIV-1. More specifically, HSV-2 infection is associated with a two-to fourfold increase in HIV-1 acquisition. This association has been amply demonstrated in heterosexual men and women in both the developed and the developing worlds. Epidemiologically, regions of the world with high HSV-2 prevalence and selected populations within such regions have a higher population-based incidence of HIV-1. One study indicated that approximately one-quarter of HIV infections in the high-prevalence city of Kisumu, Kenya, were directly attributable to HSV-2.

1	In addition, HSV-2 facilitates the spread of HIV into low-risk populations on a per-coital basis, and prevalent HSV-2 appears to increase the risk of HIV infection by sevento ninefold. Mathematical models suggest that ~33–50% of HIV-1 infections may be attributable to HSV-2 both in men who have sex with men (MSM) and in sub-Saharan Africa. In addition, HSV-2 is more frequently reactivated in and transmitted by persons co-infected with HIV-1 as opposed to persons not co-infected. Thus, most areas of the world with a high HIV-1 prevalence also have a high HSV-2 prevalence. A wide variety of serologic surveys have indicated a similar or even higher seroprevalence of HSV-2 in most parts of Central America, South America, and Africa. In Africa, HSV-2 seroprevalence has ranged from 40% to 70% in obstetric and other sexually experienced populations. Antibody prevalence rates average ~5–10% higher among women than among men.

1	Several studies suggest that many cases of “asymptomatic” genital HSV-2 infection are, in fact, simply unrecognized or confined to anatomic regions of the genital tract that are not easily visualized. Asymptomatic seropositive persons shed virus on mucosal surfaces almost as frequently as do those with symptomatic disease. This large reservoir of unidentified carriers of HSV-2 and the frequent asymptomatic reactivation of the virus from the genital tract have fostered the continued spread of genital herpes throughout the world. HSV-2 infection is an independent risk factor for the acquisition and transmission of infection with HIV-1. Among co-infected persons, HIV-1 virions can be shed from herpetic lesions of the genital region. This shedding may facilitate the spread of HIV through sexual contact. HSV-2 reactivation is associated with a localized persistent inflammatory response consisting of high concentrations of CCR5-enriched CD4+ T cells as well as inflammatory dendritic cells

1	contact. HSV-2 reactivation is associated with a localized persistent inflammatory response consisting of high concentrations of CCR5-enriched CD4+ T cells as well as inflammatory dendritic cells in the submucosa of the genital skin. These cells can support HIV infection and replication and hence are likely to account for the almost threefold increase in HIV acquisition among persons with genital herpes. Unfortunately, antiviral therapy does not reduce this subclinical postreactivation inflammation, probably because of the inability of current antiviral agents to prevent the release of small amounts of HSV antigen into the genital mucosa.

1	HSV infections occur throughout the year. Transmission can result from contact with persons who have active ulcerative lesions or with persons who have no clinical manifestations of infection but who are shedding HSV from mucocutaneous surfaces. HSV reactivation on genital skin and mucosal surfaces is common. The frequency of sampling influences the frequency of detection. Recent studies indicate that most HSV-1 and HSV-2 episodes last <4–6 h; thus, replication of virus and clearance by the host are rapid. Even with once-daily sampling, HSV DNA can be detected on 20–30% of days by PCR. Corresponding figures for HSV-1 in oral secretions are similar. Rates of shedding are highest during the initial years after acquisition, with viral shedding occurring on as many as 30–50% of days during this period. Immunosuppressed patients shed HSV from mucosal sites at an even higher frequency (20–80% of days). These high rates of mucocutane-1177 ous reactivation suggest that exposure to HSV from

1	this period. Immunosuppressed patients shed HSV from mucosal sites at an even higher frequency (20–80% of days). These high rates of mucocutane-1177 ous reactivation suggest that exposure to HSV from sexual or other close contact (kissing, sharing of glasses or silverware) is common and help explain the continuing spread and high seroprevalence of HSV infections worldwide. Reactivation rates vary widely among individuals. Among HIV-positive patients, a low CD4+ T cell count and a high HIV-1 load are associated with increased rates of HSV reactivation. Daily antiviral chemotherapy for HSV-2 infection can reduce shedding rates but does not eliminate shedding, as measured by PCR or culture.

1	HSV has been isolated from nearly all visceral and mucocutaneous sites. The clinical manifestations and course of HSV infection depend on the anatomic site involved, the age and immune status of the host, and the antigenic type of the virus. Primary HSV infections (i.e., first infections with either HSV-1 or HSV-2 in which the host lacks HSV antibodies in acute-phase serum) are frequently accompanied by systemic signs and symptoms. Compared with recurrent episodes, primary infections, which involve both mucosal and extramucosal sites, are characterized by a longer duration of symptoms and virus isolation from lesions. The incubation period ranges from 1 to 26 days (median, 6–8 days). Both viral subtypes can cause genital and oral-facial infections, and the infections caused by the two subtypes are clinically indistinguishable. However, the frequency of reactivation of infection is influenced by anatomic site and virus type. Genital HSV-2 infection is twice as likely to reactivate and

1	are clinically indistinguishable. However, the frequency of reactivation of infection is influenced by anatomic site and virus type. Genital HSV-2 infection is twice as likely to reactivate and recurs 8–10 times more frequently than genital HSV-1 infection. Conversely, oral-labial HSV-1 infection recurs more frequently than oral-labial HSV-2 infection. Asymptomatic shedding rates follow the same pattern.

1	Oral-Facial Infections Gingivostomatitis and pharyngitis are the most common clinical manifestations of first-episode HSV-1 infection, whereas recurrent herpes labialis is the most common clinical manifestation of reactivation HSV-1 infection. HSV pharyngitis and gingivostomatitis usually result from primary infection and are most common among children and young adults. Clinical symptoms and signs, which include fever, malaise, myalgias, inability to eat, irritabil ity, and cervical adenopathy, may last 3–14 days. Lesions may involve the hard and soft palate, gingiva, tongue, lip, and facial area. HSV-1 or HSV-2 infection of the pharynx usually results in exudative or ulcerative lesions of the posterior pharynx and/or tonsillar pillars. Lesions of the tongue, buccal mucosa, or gingiva may occur later in the course in one-third of cases. Fever lasting 2–7 days and cervical adenopathy are common. It can be difficult to differentiate HSV pharyngitis clinically from bacterial pharyngitis,

1	occur later in the course in one-third of cases. Fever lasting 2–7 days and cervical adenopathy are common. It can be difficult to differentiate HSV pharyngitis clinically from bacterial pharyngitis, Mycoplasma pneumoniae infections, and pharyngeal ulcerations of noninfectious etiologies (e.g., Stevens-Johnson syndrome). No substantial evidence suggests that reactivation of oral-labial HSV infection is associated with symptomatic recurrent pharyngitis.

1	Reactivation of HSV from the trigeminal ganglia may be associated with asymptomatic virus excretion in the saliva, development of intraoral mucosal ulcerations, or herpetic ulcerations on the vermilion border of the lip or external facial skin. About 50–70% of seropositive patients undergoing trigeminal nerve-root decompression and 10–15% of those undergoing dental extraction develop oral-labial HSV infection a median of 3 days after these procedures. Clinical differentiation of intraoral mucosal ulcerations due to HSV from aphthous, traumatic, or drug-induced ulcerations is difficult.

1	In immunosuppressed patients, HSV infection may extend into mucosal and deep cutaneous layers. Friability, necrosis, bleeding, severe pain, and inability to eat or drink may result. The lesions of HSV mucositis are clinically similar to mucosal lesions caused by cytotoxic drug therapy, trauma, or fungal or bacterial infections. Persistent ulcerative HSV infections are among the most common infections in patients with AIDS. HSV and Candida infections often occur concurrently. Systemic antiviral therapy speeds the rate of healing and relieves the pain of mucosal HSV infections in immunosuppressed patients. The frequency of HSV reactivation during the early phases of transplantation or induction chemotherapy is high (50–90%), and prophylactic systemic antiviral agents such as IV acyclovir and penciclovir or 1178 the oral congeners of these drugs are used to reduce reactivation rates. Patients with atopic eczema may also develop severe oral-facial HSV infections (eczema herpeticum), which

1	penciclovir or 1178 the oral congeners of these drugs are used to reduce reactivation rates. Patients with atopic eczema may also develop severe oral-facial HSV infections (eczema herpeticum), which may rapidly involve extensive areas of skin and occasionally disseminate to visceral organs. Extensive eczema herpeticum has resolved promptly with the administration of IV acyclovir. Erythema multiforme may also be associated with HSV infections (see Figs. 70-9 and 25e-25); some evidence suggests that HSV infection is the precipitating event in ~75% of cases of cutaneous erythema multiforme. HSV antigen has been demonstrated both in circulatory immune complexes and in skin lesion biopsy samples from these cases. Patients with severe HSV-associated erythema multiforme are candidates for chronic suppressive oral antiviral therapy. HSV-1 and varicella-zoster virus (VZV) have been implicated in the etiology of Bell’s palsy (flaccid paralysis of the mandibular portion of the facial nerve).

1	suppressive oral antiviral therapy. HSV-1 and varicella-zoster virus (VZV) have been implicated in the etiology of Bell’s palsy (flaccid paralysis of the mandibular portion of the facial nerve). Some but not all trials have documented quicker resolution of facial paralysis with the prompt initiation of antiviral therapy, with or without glucocorticoids. However, other trials have shown little benefit. Thus there is no consensus on the relative value of antiviral drugs alone, glucocorticoids alone, and the two modalities combined for the treatment of Bell’s palsy.

1	Genital Infections First-episode primary genital herpes is characterized by fever, headache, malaise, and myalgias. Pain, itching, dysuria, vaginal and urethral discharge, and tender inguinal lymphadenopathy are the predominant local symptoms. Widely spaced bilateral lesions of the external genitalia are characteristic (Fig. 216-1). Lesions may be present in varying stages, including vesicles, pustules, or painful erythematous ulcers. The cervix and urethra are involved in >80% of women with first-episode infections. First episodes of genital herpes in patients who have had prior HSV-1 infection are associated with systemic symptoms in a few patients and with faster healing than primary genital herpes. Subclinical DNAemia has been found in ~30% of cases of true primary genital herpes. The clinical courses of acute first-episode genital herpes are similar for HSV-1 and HSV-2 infection. However, the recurrence rates of genital disease differ with the viral subtype: the 12-month

1	The clinical courses of acute first-episode genital herpes are similar for HSV-1 and HSV-2 infection. However, the recurrence rates of genital disease differ with the viral subtype: the 12-month recurrence rates among patients with first-episode HSV-2 and HSV-1 infections are ~90% and ~55%, respectively (median number of recurrences, 4 and <1, respectively). Recurrence rates for genital HSV-2 infections vary greatly among individuals and over time within the same individual. HSV has been isolated from the urethra and urine of men and women without external genital lesions. A clear mucoid discharge and dysuria are characteristics of symptomatic HSV urethritis. HSV has been isolated from the urethra of 5% of women with the dysuria-frequency syndrome. Occasionally, HSV genital tract disease is manifested by endometritis and salpingitis in women and by prostatitis in men. About 15% of cases of HSV-2 acquisition are associated with nonlesional clinical syndromes, such as aseptic

1	disease is manifested by endometritis and salpingitis in women and by prostatitis in men. About 15% of cases of HSV-2 acquisition are associated with nonlesional clinical syndromes, such as aseptic meningitis, cervicitis, or urethritis. A more complete discussion of the differential diagnosis of genital herpes is presented in Chap. 163.

1	FIGuRE 216-1 Genital herpes: primary vulvar infection, with mul-tiple, extremely painful, punched-out, confluent, shallow ulcers on the edematous vulva and perineum. Micturition is often very painful. Associated inguinal lymphadenopathy is common. (Reprinted with permission from K Wolff et al: Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005.)

1	Both HSV-1 and HSV-2 can cause symptomatic or asymptomatic rectal and perianal infections. HSV proctitis is usually associated with rectal intercourse. However, subclinical perianal shedding of HSV is detected in women and men who report no rectal intercourse. This phenomenon is due to the establishment of latency in the sacral dermatome from prior genital tract infection, with subsequent reactivation in epithelial cells in the perianal region. Such reactivations are often subclinical. Symptoms of HSV proctitis include anorectal pain, anorectal discharge, tenesmus, and constipation. Sigmoidoscopy reveals ulcerative lesions of the distal 10 cm of the rectal mucosa. Rectal biopsies show mucosal ulceration, necrosis, polymorphonuclear and lymphocytic infiltration of the lamina propria, and (in occasional cases) multinucleated intranuclear inclusion-bearing cells. Perianal herpetic lesions are also found in immunosuppressed patients receiving cytotoxic therapy. Extensive perianal herpetic

1	occasional cases) multinucleated intranuclear inclusion-bearing cells. Perianal herpetic lesions are also found in immunosuppressed patients receiving cytotoxic therapy. Extensive perianal herpetic lesions and/or HSV proctitis is common among patients with HIV infection.

1	Herpetic Whitlow Herpetic whitlow—HSV infection of the finger— may occur as a complication of primary oral or genital herpes by inoculation of virus through a break in the epidermal surface or by direct introduction of virus into the hand through occupational or some other type of exposure. Clinical signs and symptoms include abrupt-onset edema, erythema, and localized tenderness of the infected finger. Vesicular or pustular lesions of the fingertip that are indistinguishable from lesions of pyogenic bacterial infection are seen. Fever, lymphadenitis, and epitrochlear and axillary lymphadenopathy are common. The infection may recur. Prompt diagnosis (to avoid unnecessary and potentially exacerbating surgical therapy and/or transmission) is essential. Antiviral chemotherapy is usually recommended (see below).

1	Herpes Gladiatorum HSV may infect almost any area of skin. Mucocutaneous HSV infections of the thorax, ears, face, and hands have been described among wrestlers. Transmission of these infections is facilitated by trauma to the skin sustained during wrestling. Several recent outbreaks have illustrated the importance of prompt diagnosis and therapy to contain the spread of this infection.

1	Eye Infections HSV infection of the eye is the most common cause of corneal blindness in the United States. HSV keratitis presents as an acute onset of pain, blurred vision, chemosis, conjunctivitis, and characteristic dendritic lesions of the cornea. Use of topical glucocorticoids may exacerbate symptoms and lead to involvement of deep structures of the eye. Debridement, topical antiviral treatment, and/or IFN therapy hastens healing. However, recurrences are common, and the deeper structures of the eye may sustain immunopathologic injury. Stromal keratitis due to HSV appears to be related to T cell–dependent destruction of deep corneal tissue. An HSV-1 epitope that is autoreactive with T cell–targeting corneal antigens has been postulated to be a factor in this infection. Chorioretinitis, usually a manifestation of disseminated HSV infection, may occur in neonates or in patients with HIV infection. HSV and VZV can cause acute necrotizing retinitis as an uncommon but severe

1	usually a manifestation of disseminated HSV infection, may occur in neonates or in patients with HIV infection. HSV and VZV can cause acute necrotizing retinitis as an uncommon but severe manifestation.

1	Central and Peripheral Nervous System Infections HSV accounts for 10–20% of all cases of sporadic viral encephalitis in the United States. The estimated incidence is ~2.3 cases per 1 million persons per year. Cases are distributed throughout the year, and the age distribution appears to be biphasic, with peaks at 5–30 and >50 years of age. HSV-1 causes >95% of cases.

1	The pathogenesis of HSV encephalitis varies. In children and young adults, primary HSV infection may result in encephalitis; presumably, exogenously acquired virus enters the CNS by neurotropic spread from the periphery via the olfactory bulb. However, most adults with HSV encephalitis have clinical or serologic evidence of mucocutaneous HSV-1 infection before the onset of CNS symptoms. In ~25% of the cases examined, the HSV-1 strains from the oropharynx and brain tissue of the same patient differ; thus some cases may result from reinfection with another strain of HSV-1 that reaches the CNS. Two theories have been proposed to explain the development of actively replicating HSV in localized areas of the CNS in persons whose ganglionic and CNS isolates are similar. Reactivation of latent HSV-1 infection in trigeminal or autonomic nerve roots may be associated with extension of virus into the CNS via nerves innervating the middle cranial fossa. HSV DNA has been demonstrated by DNA

1	HSV-1 infection in trigeminal or autonomic nerve roots may be associated with extension of virus into the CNS via nerves innervating the middle cranial fossa. HSV DNA has been demonstrated by DNA hybridization in brain tissue obtained at autopsy—even from healthy adults. Thus, reactivation of long-standing latent CNS infection may be another mechanism for the development of HSV encephalitis.

1	Recent studies have identified genetic polymorphisms in two separate genes among families with a high frequency of HSV encephalitis. Peripheral-blood mononuclear cells from these patients (predominantly children) appear to secrete reduced levels of IFN in response to HSV. These observations suggest that some cases of sporadic HSV encephalitis may be related to host genetic determinants. The clinical hallmark of HSV encephalitis has been the acute onset of fever and focal neurologic symptoms and signs, especially in the temporal lobe (Fig. 216-2). Clinical differentiation of HSV encephalitis from other viral encephalitides, focal infections, or noninfectious processes is difficult. Elevated cerebrospinal fluid (CSF) protein levels, leukocytosis (predominantly lymphocytes), and red blood cell counts due to hemorrhagic necrosis are common. While brain biopsy has been the gold standard for defining HSV encephalitis, a highly sensitive and specific PCR for detection of HSV DNA in CSF has

1	counts due to hemorrhagic necrosis are common. While brain biopsy has been the gold standard for defining HSV encephalitis, a highly sensitive and specific PCR for detection of HSV DNA in CSF has largely replaced biopsy for defining CNS infection. Although titers of antibody to HSV in CSF and serum increase in most cases of HSV encephalitis, they rarely do so earlier than 10 days into the illness and therefore, although useful in retrospect, generally are not helpful in establishing an early clinical diagnosis. In rare cases, demonstration of HSV antigen, HSV DNA, or HSV replication in brain tissue obtained by biopsy is highly sensitive; examination of such tissue also provides the opportunity to identify alternative, potentially treatable causes of encephalitis. Antiviral chemotherapy with acyclovir reduces the rate of death from HSV encephalitis. Most authorities recommend the administration of IV acyclovir to patients with presumed HSV encephalitis until the diagnosis is confirmed

1	acyclovir reduces the rate of death from HSV encephalitis. Most authorities recommend the administration of IV acyclovir to patients with presumed HSV encephalitis until the diagnosis is confirmed or an alternative diagnosis is made. All con-1179 firmed cases should be treated with IV acyclovir (30 mg/kg per day in three divided doses for 14–21 days). After the completion of therapy, the clinical recurrence of encephalitis requiring more treatment has been reported. For this reason, some authorities prefer to treat initially for 21 days, and many continue therapy until HSV DNA has been eliminated from the CSF. Even with therapy, neurologic sequelae are common, especially among persons >50 years of age.

1	HSV DNA has been detected in CSF from 3–15% of persons presenting to the hospital with aseptic meningitis. HSV meningitis, which is usually seen in association with primary genital HSV infection, is an acute, self-limited disease manifested by headache, fever, and mild photophobia and lasting 2–7 days. Lymphocytic pleocytosis in the CSF is characteristic. Neurologic sequelae of HSV meningitis are rare. HSV is the most commonly identified cause of recurrent lymphocytic meningitis (Mollaret’s meningitis). Demonstration of HSV antibodies in CSF or persistence of HSV DNA in CSF can establish the diagnosis. For persons with frequent recurrences of HSV meningitis, daily antiviral therapy has reduced the occurrence of such episodes.

1	Autonomic nervous system dysfunction, especially of the sacral region, has been reported in association with both HSV and VZV infections. Numbness, tingling of the buttocks or perineal areas, urinary retention, constipation, CSF pleocytosis, and (in males) impotence may occur. Symptoms appear to resolve slowly over days or weeks. Occasionally, hypoesthesia and/or weakness of the lower extremities persists for many months. Transitory hypoesthesia of the area of skin innervated by the trigeminal nerve and vestibular system dysfunction (as measured by electronystagmography) are the predominant signs of disease. Whether antiviral chemotherapy can abort these signs or reduce their frequency and severity is not yet known. Rarely, transverse myelitis, manifested by a rapidly progressive symmetric paralysis of the lower extremities or Guillain-Barré syndrome, follows HSV infection. Similarly, peripheral nervous system involvement (Bell’s palsy) or cranial polyneuritis may be related to

1	paralysis of the lower extremities or Guillain-Barré syndrome, follows HSV infection. Similarly, peripheral nervous system involvement (Bell’s palsy) or cranial polyneuritis may be related to reactivation of HSV-1 infection.

1	Visceral Infections HSV infection of visceral organs usually results from viremia, and multiple-organ involvement is common. Occasionally, however, the clinical manifestations of HSV infection involve only the esophagus, lung, or liver. HSV esophagitis may result from direct extension of oral-pharyngeal HSV infection into the esophagus or may occur de novo by reactivation and spread of HSV to the esophageal mucosa via the vagus nerve. The predominant symptoms of HSV esophagitis are odynophagia, dysphagia, substernal pain, and weight loss. Multiple oval ulcerations appear on an erythematous base with or without a patchy white pseudomembrane. The distal esophagus is most commonly involved. With extensive disease, diffuse friability may spread to the entire esophagus. Neither endoscopic nor barium examination can reliably differentiate HSV esophagitis from Candida esophagitis or from esophageal ulcerations due to thermal injury, radiation, or corrosives. Endoscopically obtained

1	nor barium examination can reliably differentiate HSV esophagitis from Candida esophagitis or from esophageal ulcerations due to thermal injury, radiation, or corrosives. Endoscopically obtained secretions for cytologic examination and culture or DNA detection by PCR provide the most useful material for diagnosis. Systemic antiviral chemotherapy usually reduces the severity and duration of symptoms and heals esophageal ulcerations. HSV pneumonitis is uncommon except in severely immunosuppressed patients and may result from extension of herpetic tracheobronchitis into lung parenchyma. Focal necrotizing pneumonitis usually ensues. Hematogenous dissemination of virus from sites of oral or genital mucocutaneous

1	FIGuRE 216-2 Computed tomography and diffusion-weighted magnetic resonance imag-ing scans of the brain of a patient with left-temporal-lobe herpes simplex virus encephalitis.

1	1180 disease may also occur, producing bilateral interstitial pneumonitis. Bacterial, fungal, and parasitic pathogens are commonly present in HSV pneumonitis. The mortality rate from untreated HSV pneumonia in immunosuppressed patients is high (>80%). HSV has also been isolated from the lower respiratory tract of persons with acute respiratory distress syndrome and prolonged intubation. Most authorities believe that the presence of HSV in tracheal aspirates in such settings is due to reactivation of HSV in the tracheal region and localized tracheitis in persons with long-term intubation. Such patients should be evaluated for extension of HSV infection into the lung parenchyma. Controlled trials assessing the role of antiviral agents used against HSV in morbidity and mortality associated with acute respiratory distress syndrome have not been conducted. The role of lower respiratory tract HSV infection in overall rates of morbidity and mortality associated with these conditions is

1	with acute respiratory distress syndrome have not been conducted. The role of lower respiratory tract HSV infection in overall rates of morbidity and mortality associated with these conditions is unclear. HSV is an uncommon cause of hepatitis in immunocompetent patients. HSV infection of the liver is associated with fever, abrupt elevations of bilirubin and serum aminotransferase levels, and leukopenia (<4000 white blood cells/μL). Disseminated intravascular coagulation may also develop. Other reported complications of HSV infection include monarticular arthritis, adrenal necrosis, idiopathic thrombocytopenia, and glomerulonephritis. Disseminated HSV infection in immunocompetent patients is rare. In immunocompromised patients, burn patients, or malnourished individuals, HSV occasionally disseminates to other visceral organs, such as the adrenal glands, pancreas, small and large intestines, and bone marrow. Rarely, primary HSV infection in pregnancy disseminates and may be associated

1	disseminates to other visceral organs, such as the adrenal glands, pancreas, small and large intestines, and bone marrow. Rarely, primary HSV infection in pregnancy disseminates and may be associated with the death of both mother and fetus. This uncommon event is usually related to the acquisition of primary infection in the third trimester. Disseminated HSV infection is best detected by the presence of HSV DNA in plasma or blood.

1	Neonatal HSV Infections Of all HSV-infected populations, neonates (infants younger than 6 weeks) have the highest frequency of visceral and/or CNS infection. Without therapy, the overall rate of death from neonatal herpes is 65%; <10% of neonates with CNS infection develop normally. Although skin lesions are the most commonly recognized features of disease, many infants do not develop lesions at all or do so only well into the course of disease. Neonatal infection is usually acquired perinatally from contact with infected genital secretions at delivery. Congenitally infected infants have been reported. Of neonatal HSV infections, 30–50% are due to HSV-1 and 50–70% to HSV-2. The risk of developing neonatal HSV infection is 10 times higher for an infant born to a mother who has recently acquired HSV than for other infants. Neonatal HSV-1 infections may also be acquired through postnatal contact with immediate family members who have symptomatic or asymptomatic oral-labial HSV-1

1	acquired HSV than for other infants. Neonatal HSV-1 infections may also be acquired through postnatal contact with immediate family members who have symptomatic or asymptomatic oral-labial HSV-1 infection or through nosocomial transmission within the hospital. All neonates with presumed herpes should be treated with IV acyclovir and then placed on maintenance oral antiviral therapy for the first 6–12 months of life. Antiviral chemotherapy with high-dose IV acyclovir (60 mg/kg per day) has reduced the mortality rate from neonatal herpes to ~15%. However, rates of morbidity, especially among infants with HSV-2 infection involving the CNS, are still very high.

1	HSV in Pregnancy In the United States, 22% of all pregnant women and 55% of non-Hispanic black pregnant women are seropositive for HSV-2. However, the risk of mother-to-child transmission of HSV in the perinatal period is highest when the infection is acquired near the time of labor—that is, in previously HSV-seronegative women. The clinical manifestations of recurrent genital herpes—including the frequency of subclinical versus clinical infection, duration of lesions, pain, and constitutional symptoms—are similar in pregnant and non-pregnant women. Recurrences increase in frequency over the course of pregnancy. However, when women are seropositive for HSV-2 at the outset of pregnancy, no effect on neonatal outcomes (including birth weight and gestational age) is seen. First-episode infections in pregnancy have more severe consequences for mother and infant. Maternal visceral dissemination during the third trimester occasionally occurs, as does premature birth or intrauterine growth

1	in pregnancy have more severe consequences for mother and infant. Maternal visceral dissemination during the third trimester occasionally occurs, as does premature birth or intrauterine growth retardation.

1	The acquisition of primary disease in pregnancy, whether related to HSV-1 or HSV-2, carries the risk of transplacental transmission of virus to the neonate and can result in spontaneous abortion, although this outcome is relatively uncommon. For newly acquired genital HSV infection during pregnancy, most authorities recommend treatment with acyclovir (400 mg three times daily) or valacyclovir (500–1000 mg twice daily) for 7–10 days. However, the impact of this intervention on transmission is unknown. The high HSV-2 prevalence rate in pregnancy and the low incidence of neonatal disease (1 case per 6000–20,000 live births) indicate that only a few infants are at risk of acquiring HSV. Therefore, cesarean section is not warranted for all women with recurrent genital disease. Because intrapartum transmission of infection accounts for the majority of cases, abdominal delivery need be considered only for women who are shedding HSV at delivery. Several studies have shown no correlation

1	transmission of infection accounts for the majority of cases, abdominal delivery need be considered only for women who are shedding HSV at delivery. Several studies have shown no correlation between recurrence of viral shedding before delivery and viral shedding at term. Hence, weekly virologic monitoring and amniocentesis are not recommended.

1	The frequency of transmission from mother to infant is markedly higher among women who acquire HSV near term (30–50%) than among those in whom HSV-2 infection is reactivated at delivery (<1%). Although maternal antibody to HSV-2 is protective, antibody to HSV-1 offers little or no protection against neonatal HSV-2 infection. Primary genital infection with HSV-1 leads to a particularly high risk of transmission during pregnancy and accounts for an increasing proportion of neonatal HSV cases. Moreover, during reactivation, HSV-1 appears more transmissible to the neonate than HSV-2. Only 2% of women who are seropositive for HSV-2 have HSV-2 isolated from cervical secretions at delivery, and only 1% of infants exposed in this manner develop infection, presumably because of the protective effects of maternally transferred antibodies and perhaps lower viral titers during reactivation. Despite the low frequency of transmission of HSV in this setting, 30–50% of infants with neonatal HSV are

1	of maternally transferred antibodies and perhaps lower viral titers during reactivation. Despite the low frequency of transmission of HSV in this setting, 30–50% of infants with neonatal HSV are born to mothers with established genital herpes.

1	Isolation of HSV by cervicovaginal swab at the time of delivery is the greatest risk factor for intrapartum HSV transmission (relative risk = 346); however, culture-negative, PCR-positive cases of intrapartum transmission are well described. New acquisition of HSV (odds ratio [OR] = 49), isolation of HSV-1 versus HSV-2 (OR = 35), cervical versus vulvar HSV detection (OR = 15), use of fetal scalp electrodes (OR = 3.5), and young age confer further risk of transmission, whereas abdominal delivery is protective (OR = 0.14). Physical examination poorly predicts the absence of shedding, and PCR far exceeds culture in terms of sensitivity and speed. Therefore, PCR detection at the onset of labor should be used to aid clinical decision-making for women with HSV-2 antibody. Because cesarean section appears to be an effective means of reducing maternal-fetal transmission, patients with recurrent genital herpes should be encouraged to come to the hospital early at the time of delivery for

1	section appears to be an effective means of reducing maternal-fetal transmission, patients with recurrent genital herpes should be encouraged to come to the hospital early at the time of delivery for careful examination of the external genitalia and cervix as well as collection of a swab sample for viral isolation. Women who have no evidence of lesions can have a vaginal delivery. The presence of active lesions on the cervix or external genitalia is an indication for cesarean delivery.

1	If first-episode exposure has occurred (e.g., if HSV serologies show that the mother is seronegative or if the mother is HSV-1-seropositive and the isolate at delivery is found to be HSV-2), many authorities would initiate antiviral therapy for the infant with IV acyclovir. At a minimum, samples for viral cultures and PCR should be obtained from the throat, nasopharynx, eyes, and rectum of these infants immediately and at 5to 10-day intervals. Lethargy, skin lesions, or fever should be evaluated promptly. All infants from whom HSV is isolated 24 h after delivery should be treated with IV acyclovir at recommended doses.

1	Both clinical and laboratory criteria are useful for diagnosing HSV infections. A clinical diagnosis can be made accurately when characteristic multiple vesicular lesions on an erythematous base are present. However, herpetic ulcerations may resemble skin ulcerations of other etiologies. Mucosal HSV infections may also present as urethritis or pharyngitis without cutaneous lesions. Thus, laboratory studies to confirm the diagnosis and to guide therapy are recommended. While staining of scrapings from the base of the lesions with Wright’s, Giemsa’s (Tzanck preparation), or Papanicolaou’s stain to detect giant cells or intranuclear inclusions of Herpesvirus infection is a well-described procedure, few clinicians are skilled in this technique, the sensitivity of staining is low (<30% for mucosal swabs), and these cytologic methods do not differentiate between HSV and VZV infections.

1	HSV infection is best confirmed in the laboratory by detection of virus, viral antigen, or viral DNA in scrapings from lesions. HSV DNA detection by PCR is the most sensitive laboratory technique for detecting mucosal or visceral HSV infections and should be used when available. HSV causes a discernible cytopathic effect in a variety of cell culture systems, and this effect can be identified within 48–96 h after inoculation. Spin-amplified culture with subsequent staining for HSV antigen has shortened the time needed to identify HSV to <24 h. The sensitivity of all detection methods depends on the stage of the lesions (with higher sensitivity for vesicular than for ulcerative lesions), on whether the patient has a first or a recurrent episode of the disease (with higher sensitivity in first than in recurrent episodes), and on whether the sample is from an immunosuppressed or an immunocompetent patient (with more antigen or DNA in immunosuppressed patients). Laboratory confirmation

1	first than in recurrent episodes), and on whether the sample is from an immunosuppressed or an immunocompetent patient (with more antigen or DNA in immunosuppressed patients). Laboratory confirmation permits subtyping of the virus; information on subtype may be useful epidemiologically and may help to predict the frequency of reactivation after first-episode oral-labial or genital HSV infection.

1	Serologic assays with whole-virus antigen preparations, such as complement fixation, neutralization, indirect immunofluorescence, passive hemagglutination, radioimmunoassay, and enzyme-linked immunosorbent assay, are useful for differentiating uninfected (seronegative) persons from those with past HSV-1 or HSV-2 infection, but they do not reliably distinguish between the two viral subtypes. Serologic assays that identify antibodies to type-specific surface proteins (epitopes) of the two viral subtypes have been developed and can distinguish reliably between the human antibody responses to HSV-1 and HSV-2. The most commonly used assays are those that measure antibodies to glycoprotein G of HSV-1 (gG1) and HSV-2 (gG2). A western blot assay that can detect several HSV type-specific proteins can also be used.

1	Acute-and convalescent-phase serum samples can be useful in demonstrating seroconversion during primary HSV-1 or HSV-2 infection. However, few available tests report titers, and increases in index values do not reflect first episodes in all patients. Serologic assays based on type-specific proteins should be used to identify asymptomatic carriers of HSV-1 or HSV-2. No reliable IgM method for defining acute HSV infection is available.

1	Several studies have shown that persons with previously unrecognized HSV-2 infection can be taught to identify symptomatic reactivations. Individuals seropositive for HSV-2 should be told about the high frequency of subclinical reactivation on mucosal surfaces that are not visible to the eye (e.g., cervix, urethra, perianal skin) or in microscopic ulcerations that may not be clinically symptomatic. Transmission of infection during such episodes is well established. HSV-2-seropositive persons should be educated about the high likelihood of subclinical shedding and the role condoms (male or female) may play in reducing transmission. Antiviral therapy with valacyclovir (500 mg once daily) has been shown to reduce the transmission of HSV-2 between sexual partners.

1	Many aspects of mucocutaneous and visceral HSV infections are amenable to antiviral chemotherapy. For mucocutaneous infections, acyclovir and its congeners famciclovir and valacyclovir have been the mainstays of therapy. Several antiviral agents are available for topical use in HSV eye infections: idoxuridine, trifluorothymidine, topical vidarabine, and cidofovir. For HSV encephalitis and neonatal herpes, IV acyclovir is the treatment of choice. All licensed antiviral agents for use against HSV inhibit the viral DNA polymerase. One class of drugs, typified by the drug acyclovir, is made up of substrates for the HSV enzyme thymidine kinase (TK).

1	Acyclovir, ganciclovir, famciclovir, and valacyclovir are all selectively 1181 phosphorylated to the monophosphate form in virus-infected cells. Cellular enzymes convert the monophosphate form of the drug to the triphosphate, which is then incorporated into the viral DNA chain. Acyclovir is the agent most frequently used for the treatment of HSV infections and is available in IV, oral, and topical formulations. Valacyclovir, the valyl ester of acyclovir, offers greater bioavailability than acyclovir and thus can be administered less frequently. Famciclovir, the oral formulation of penciclovir, is clinically effective in the treatment of a variety of HSV-1 and HSV-2 infections. Ganciclovir is active against both HSV-1 and HSV-2; however, it is more toxic than acyclovir, valacyclovir, and famciclovir and generally is not recommended for the treatment of HSV infections. Anecdotal case reports suggest that ganciclovir may also be less effective than acyclovir for treatment of HSV

1	and famciclovir and generally is not recommended for the treatment of HSV infections. Anecdotal case reports suggest that ganciclovir may also be less effective than acyclovir for treatment of HSV infections. All three recommended compounds—acyclovir, valacyclovir, and famciclovir—have proved effective in shortening the duration of symptoms and lesions of mucocutaneous HSV infections in both immunocompromised and immunocompetent patients (Table 216-1). IV and oral formulations prevent reactivation of HSV in seropositive immunocompromised patients during induction chemotherapy or in the period immediately after bone marrow or solid organ transplantation. Chronic daily suppressive therapy reduces the frequency of reactivation disease among patients with frequent genital or oral-labial herpes. Only valacyclovir has been subjected to clinical trials that demonstrated reduced transmission of HSV-2 infection between sexual partners. IV acyclovir (30 mg/kg per day, given as a 10-mg/kg infu

1	Only valacyclovir has been subjected to clinical trials that demonstrated reduced transmission of HSV-2 infection between sexual partners. IV acyclovir (30 mg/kg per day, given as a 10-mg/kg infu sion over 1 h at 8-h intervals) is effective in reducing rates of death and morbidity from HSV encephalitis. Early initiation of therapy is a critical factor in outcome. The major side effect associated with IV acyclovir is transient renal insufficiency, usually due to crystallization of the compound in the renal parenchyma. This adverse reaction can be avoided if the medication is given slowly over 1 h and the patient is well hydrated. Because CSF levels of acyclovir average only 30–50% of plasma levels, the dosage of acyclovir used for treatment of CNS infection (30 mg/kg per day) is double that used for treatment of mucocutaneous or visceral disease (15 mg/kg per day). Even higher doses of IV acyclovir are used for neonatal HSV infection (60 mg/kg per day in three divided doses).

1	Increasingly, shorter courses of therapy are being used for recurrent mucocutaneous infection with HSV-1 or HSV-2 in immunocompetent patients. One-day courses of famciclovir and valacyclovir are clinically effective, more convenient, and generally less costly than longer courses of therapy (Table 216-1). These short-course regimens should be reserved for immunocompetent hosts.

1	Recognition of the high frequency of subclinical reactivation provides a well-accepted rationale for the use of daily antiviral therapy to suppress reactivations of HSV, especially in persons with frequent clinical reactivations (e.g., those with recently acquired genital HSV infection). Immunosuppressed persons, including those with HIV infection, may also benefit from daily antiviral therapy. Recent studies have shown the efficacy of daily acyclovir and valacyclovir in reducing the frequency of HSV reactivations among HIV-positive persons. Regimens used include acyclovir (400–800 mg twice daily), famciclovir (500 mg twice daily), and valacyclovir (500 mg twice daily); valacyclovir at a dose of 4 g/d was associated with thrombotic thrombocytopenic purpura in one study of HIV-infected persons. In addition, daily treatment of HSV-2 reduces the titer of HIV RNA in plasma (0.5-log reduction) and in genital mucosa (0.33-log reduction).

1	Once-daily valacyclovir (500 mg) has been shown to reduce transmission of HSV-2 between sexual partners. Transmission rates are higher from males to females and among persons with frequent HSV-2 reactivation. Serologic screening can be used to identify at-risk couples. Daily valacyclovir appears to be more effective at reducing subclinical shedding than daily famciclovir. I. Mucocutaneous HSV infections A. Infections in immunosuppressed patients 1. Acute symptomatic first or recurrent episodes: IV acyclovir (5 mg/kg q8h) or oral acyclovir (400 mg qid), famciclovir (500 mg bid or tid), or valacyclovir (500 mg bid) is effective. Treatment duration may vary from 7 to 14 days. 2.

1	2. Suppression of reactivation disease (genital or oral-labial): IV acyclovir (5 mg/kg q8h) or oral valacyclovir (500 mg bid) or acyclovir (400–800 mg 3–5 times per day) prevents recurrences during the 30-day period immediately after transplantation. Longer-term HSV suppression is often used for persons with continued immunosuppression. In bone marrow and renal transplant recipients, oral valacyclovir (2 g/d) is also effective in reducing cytomegalovirus infection. Oral valacyclovir at a dose of 4 g/d has been associated with thrombotic thrombocytopenic purpura after extended use in HIV-positive persons. In HIV-infected persons, oral acyclovir (400–800 mg bid), valacyclovir (500 mg bid), or famciclovir (500 mg bid) is effective in reducing clinical and subclinical reactivations of HSV-1 and HSV-2. B. Infections in immunocompetent patients 1. Genital herpes a.

1	B. Infections in immunocompetent patients 1. Genital herpes a. First episodes: Oral acyclovir (200 mg 5 times per day or 400 mg tid), valacyclovir (1 g bid), or famciclovir (250 mg bid) for 7–14 days is effective. IV acyclovir (5 mg/kg q8h for 5 days) is given for severe disease or neurologic complications such as aseptic meningitis. b. Symptomatic recurrent genital herpes: Short-course (1to 3-day) regimens are preferred because of low cost, likelihood of adherence, and convenience. Oral acyclovir (800 mg tid for 2 days), valacyclovir (500 mg bid for 3 days), or famciclovir (750 or 1000 mg bid for 1 day, a 1500-mg single dose, or 500 mg stat followed by 250 mg q12h for 3 days) effectively shortens lesion duration. Other options include oral acyclovir (200 mg 5 times per day), valacyclovir (500 mg bid), and famciclovir (125 mg bid for 5 days). c.

1	c. Suppression of recurrent genital herpes: Oral acyclovir (400–800 mg bid) or valacyclovir (500 mg daily) is given. Patients with >9 episodes per year should take oral valacyclovir (1 g daily or 500 mg bid) or famciclovir (250 mg bid or 500 mg bid). 2. Oral-labial HSV infections a. First episode: Oral acyclovir is given (200 mg 5 times per day or 400 mg tid); an oral acyclovir suspension can be used (600 mg/m2 qid). Oral famciclovir (250 mg bid) or valacyclovir (1 g bid) has been used clinically. The duration of therapy is 5–10 days. b.

1	b. Recurrent episodes: If initiated at the onset of the prodrome, single-dose or 1-day therapy effectively reduces pain and speeds healing. Regimens include oral famciclovir (a 1500-mg single dose or 750 mg bid for 1 day) or valacyclovir (a 2-g single dose or 2 g bid for 1 day). Self-initiated therapy with 6-times-daily topical penciclovir cream effectively speeds healing of oral-labial HSV. Topical acyclovir cream has also been shown to speed healing. c. Suppression of reactivation of oral-labial HSV: If started before exposure and continued for the duration of exposure (usually 5–10 days), oral acyclovir (400 mg bid) prevents reactivation of recurrent oral-labial HSV infection associated with severe sun exposure. 3.

1	3. Surgical prophylaxis of oral or genital HSV infection: Several surgical procedures, such as laser skin resurfacing, trigeminal nerve-root decompression, and lumbar disk surgery, have been associated with HSV reactivation. IV acyclovir (3–5 mg/kg q8h) or oral acyclovir (800 mg bid), valacyclovir (500 mg bid), or famciclovir (250 mg bid) effectively reduces reactivation. Therapy should be initiated 48 h before surgery and continued for 3–7 days. 4. Herpetic whitlow: Oral acyclovir (200 mg) is given 5 times daily (alternative: 400 mg tid) for 7–10 days. 5. HSV proctitis: Oral acyclovir (400 mg 5 times per day) is useful in shortening the course of infection. In immunosuppressed patients or in patients with severe infection, IV acyclovir (5 mg/kg q8h) may be useful. 6.

1	6. Herpetic eye infections: In acute keratitis, topical trifluorothymidine, vidarabine, idoxuridine, acyclovir, penciclovir, and interferon are all beneficial. Debridement may be required. Topical steroids may worsen disease. II. Central nervous system HSV infections A. HSV encephalitis: IV acyclovir (10 mg/kg q8h; 30 mg/kg per day) is given for 10 days or until HSV DNA is no longer detected in cerebrospinal fluid. B. HSV aseptic meningitis: No studies of systemic antiviral chemotherapy exist. If therapy is to be given, IV acyclovir (15–30 mg/kg per day) should be used. C. Autonomic radiculopathy: No studies are available. Most authorities recommend a trial of IV acyclovir. III. Neonatal HSV infections: Oral acyclovir (60 mg/kg per day, divided into 3 doses) is given. The recommended duration of IV treatment is 21 days. Monitoring for relapse should be undertaken. Continued suppression with oral acyclovir suspension should be given for 3–4 months. IV. Visceral HSV infections

1	IV. Visceral HSV infections A. HSV esophagitis: IV acyclovir (15 mg/kg per day) is given. In some patients with milder forms of immunosuppression, oral therapy with valacyclovir or famciclovir is effective. B. HSV pneumonitis: No controlled studies exist. IV acyclovir (15 mg/kg per day) should be considered. V. Disseminated HSV infections: No controlled studies exist. IV acyclovir (5 mg/kg q8h) should be tried. Adjustments for renal insufficiency may be needed. No definite evidence indicates that therapy will decrease the risk of death. VI. Erythema multiforme associated with HSV: Anecdotal observations suggest that oral acyclovir (400 mg bid or tid) or valacyclovir (500 mg bid) will suppress erythema multiforme.

1	VI. Erythema multiforme associated with HSV: Anecdotal observations suggest that oral acyclovir (400 mg bid or tid) or valacyclovir (500 mg bid) will suppress erythema multiforme. VII. Infections due to acyclovir-resistant HSV: IV foscarnet (40 mg/kg IV q8h) should be given until lesions heal. The optimal duration of therapy and the usefulness of its continuation to suppress lesions are unclear. Some patients may benefit from cutaneous application of trifluorothymidine or 5% cidofovir gel.

1	Acyclovir-resistant strains of HSV have been identified. Most of these strains have an altered substrate specificity for phosphorylating acyclovir. Thus, cross-resistance to famciclovir and valacyclovir is usually found. Occasionally, an isolate with altered TK specificity arises and is sensitive to famciclovir but not to acyclovir. In some patients infected with TK-deficient virus, higher doses of acyclovir are associated with clearing of lesions. In others, clinical disease progresses despite high-dose therapy. Almost all clinically significant acyclovir resistance has been seen in immunocompromised patients, and HSV-2 isolates are more often resistant than HSV-1 strains. A study by the Centers for Disease Control and Prevention indicated that ~5% of HSV-2 isolates from HIV-positive persons exhibit some degree of in vitro resistance to acyclovir. Of HSV-2 isolates from immunocompetent patients attending sexually transmitted disease clinics, <0.5% show reduced in vitro sensitivity to

1	exhibit some degree of in vitro resistance to acyclovir. Of HSV-2 isolates from immunocompetent patients attending sexually transmitted disease clinics, <0.5% show reduced in vitro sensitivity to acyclovir. The lack of appreciable change in the frequency of detection of such isolates in the past 20 years probably reflects the reduced transmission of TK-deficient mutants. Isolation of HSV from lesions persisting despite adequate dosages and blood levels of acyclovir should raise the suspicion of acyclovir resistance. Therapy with the antiviral drug foscarnet is useful in acyclovir-resistant cases (Chap. 215e). Because of its toxicity and cost, this drug is usually reserved for patients with extensive mucocutaneous infections. Cidofovir is a nucleotide analogue and exists as a phosphonate or monophosphate form. Most TK-deficient strains of HSV are sensitive to cidofovir. Cidofovir ointment speeds healing of acyclovir-resistant lesions. No well-controlled trials of systemic cidofovir

1	or monophosphate form. Most TK-deficient strains of HSV are sensitive to cidofovir. Cidofovir ointment speeds healing of acyclovir-resistant lesions. No well-controlled trials of systemic cidofovir have been reported. True TK-negative variants of HSV appear to have a reduced capacity to spread because of altered neurovirulence—a feature important in the relatively infrequent presence of such strains in immunocompetent populations, even with increasing use of antiviral drugs.

1	Early studies of acyclovir-like drugs were performed solely in the developed world. Recent studies have shown that, although acyclovir-like drugs are effective in the developing world, their clinical and virologic benefits seem reduced from those in European and U.S. populations. The mechanism of this phenomenon is uncertain. Acyclovir therapy does not reduce the rate of HIV acquisition; however, HIV load among MSM in the United States decreased by 1.3 log10 in contrast to 0.9 log10 among Peruvian MSM and 0.5 log10 among African women.

1	The success of efforts to control HSV disease on a population basis through suppressive antiviral chemotherapy and/or educational programs will be limited. Barrier forms of contraception (especially condoms) decrease the likelihood of transmission of HSV infection, particularly during periods of asymptomatic viral excretion. When lesions are present, HSV infection may be transmitted by skin-to-skin contact despite the use of a condom. Nevertheless, the available data suggest that consistent condom use is an effective means of reducing the risk of genital HSV-2 transmission. Chronic daily antiviral therapy with valacyclovir can also be partially effective in reducing acquisition of HSV2, especially among susceptible women. There are no comparative efficacy studies of valacyclovir versus condom use. Most authorities suggest both approaches. The need for a vaccine to prevent acquisition of HSV infection is great, especially in light of the role HSV-2 plays in enhancing the acquisition

1	condom use. Most authorities suggest both approaches. The need for a vaccine to prevent acquisition of HSV infection is great, especially in light of the role HSV-2 plays in enhancing the acquisition and transmission of HIV-1.

1	A substantial portion of neonatal HSV cases could be prevented by reducing the acquisition of HSV by women in the third trimester of pregnancy. Neonatal HSV infection can result from either the acquisition of maternal infection near term or the reactivation of infection at delivery in the already-infected mother. Thus strategies for reducing neonatal HSV are complex. Some authorities have recommended that antiviral therapy with acyclovir or valacyclovir be given to HSV-2infected women in late pregnancy as a means of reducing reactivation of HSV-2 at term. Data are not available to support the efficacy of this approach. Moreover, the high treatment-to-prevention ratio makes this a dubious public health approach, even though it can reduce the frequency of HSV-associated cesarean delivery. varicella-zoster virus Infections Richard J. Whitley DEFINITION Varicella-zoster virus (VZV) causes two distinct clinical entities: varicella (chickenpox) and herpes zoster (shingles). Chickenpox, a

1	varicella-zoster virus Infections Richard J. Whitley DEFINITION Varicella-zoster virus (VZV) causes two distinct clinical entities: varicella (chickenpox) and herpes zoster (shingles). Chickenpox, a ubiquitous and extremely contagious infection, is usually a benign ill-217 ness of childhood characterized by an exanthematous vesicular rash. With reactivation of latent VZV (which is most common after the sixth decade of life), herpes zoster presents as a dermatomal vesicular rash, usually associated with severe pain.

1	Early in the twentieth century, similarities in the histopathologic features of skin lesions resulting from varicella and herpes zoster were demonstrated. Viral isolates from patients with chickenpox and herpes 1183 zoster produced similar alterations in tissue culture—specifically, the appearance of eosinophilic intranuclear inclusions and multinucleated giant cells. These results suggested that the viruses were biologically similar. Restriction endonuclease analyses of viral DNA from a patient with chickenpox who subsequently developed herpes zoster verified the molecular identity of the two viruses responsible for these different clinical presentations. VZV is a member of the family Herpesviridae, sharing with other members such structural characteristics as a lipid envelope surrounding a nucleocapsid with icosahedral symmetry, a total diameter of ~180–200 nm, and centrally located double-stranded DNA that is ~125,000 bp in length.

1	PATHOGENESIS AND PATHOLOGY Primary Infection Transmission occurs readily by the respiratory route; the subsequent localized replication of the virus at an undefined site (presumably the nasopharynx) leads to seeding of the lymphatic/reticuloendothelial system and ultimately to the development of viremia. Viremia in patients with chickenpox is reflected in the diffuse and scattered nature of the skin lesions and can be confirmed in selected cases by the recovery of VZV from the blood or routinely by the detection of viral DNA in either blood or lesions by polymerase chain reaction (PCR). Vesicles involve the corium and dermis, with degenerative changes characterized by ballooning, the presence of multinucleated giant cells, and eosinophilic intranuclear inclusions. Infection may involve localized blood vessels of the skin, resulting in necrosis and epidermal hemorrhage. With the evolution of disease, the vesicular fluid becomes cloudy because of the recruitment of polymorphonuclear

1	localized blood vessels of the skin, resulting in necrosis and epidermal hemorrhage. With the evolution of disease, the vesicular fluid becomes cloudy because of the recruitment of polymorphonuclear leukocytes and the presence of degenerated cells and fibrin. Ultimately, the vesicles either rupture and release their fluid (which includes infectious virus) or are gradually reabsorbed.

1	Recurrent Infection The mechanism of reactivation of VZV that results in herpes zoster is unknown. Presumably, the virus infects dorsal root ganglia during chickenpox, where it remains latent until reactivated. Histopathologic examination of representative dorsal root ganglia during active herpes zoster demonstrates hemorrhage, edema, and lymphocytic infiltration.

1	Active replication of VZV in other organs, such as the lung or the brain, can occur during either chickenpox or herpes zoster but is uncommon in the immunocompetent host. Pulmonary involvement is characterized by interstitial pneumonitis, multinucleated giant cell formation, intranuclear inclusions, and pulmonary hemorrhage. Central nervous system (CNS) infection leads to histopathologic evidence of perivascular cuffing similar to that encountered in measles and other viral encephalitides. Focal hemorrhagic necrosis of the brain, characteristic of herpes simplex virus (HSV) encephalitis, develops infrequently in VZV infection.

1	EPIDEMIOLOGY AND CLINICAL MANIFESTATIONS Chickenpox Humans are the only known reservoir for VZV. Chickenpox is highly contagious, with an attack rate of at least 90% among susceptible (seronegative) individuals. Persons of both sexes and all races are infected equally. The virus is endemic in the population at large; however, it becomes epidemic among susceptible individuals during seasonal peaks—namely, late winter and early spring in the temperate zone. Much of our knowledge of the disease’s natural history and incidence predates the licensure of the chickenpox vaccine in 1995. Historically, children 5–9 years old are most commonly affected and account for 50% of all cases. Most other cases involve children 1–4 and 10–14 years old. Approximately 10% of the population of the United States over the age of 15 is susceptible to infection. VZV vaccination during the second year of life has dramatically changed the epidemiology of infection, causing a significant decrease in the

1	States over the age of 15 is susceptible to infection. VZV vaccination during the second year of life has dramatically changed the epidemiology of infection, causing a significant decrease in the annualized incidence of chickenpox.

1	The incubation period of chickenpox ranges from 10 to 21 days but is usually 14–17 days. Secondary attack rates in susceptible siblings FIGuRE 217-1 Varicella lesions at various stages of evolution: vesicles on an erythematous base, umbilical vesicles, and crusts. within a household are 70–90%. Patients are infectious ~48 h before onset of the vesicular rash, during the period of vesicle formation (which generally lasts 4–5 days), and until all vesicles are crusted.

1	Clinically, chickenpox presents as a rash, low-grade fever, and malaise, although a few patients develop a prodrome 1–2 days before onset of the exanthem. In the immunocompetent patient, chickenpox is usually a benign illness associated with lassitude and with body temperatures of 37.8°–39.4°C (100°–103°F) of 3–5 days’ duration. The skin lesions—the hallmark of the infection—include maculopapules, vesicles, and scabs in various stages of evolution (Fig. 217-1). These lesions, which evolve from maculopapules to vesicles over hours to days, appear on the trunk and face and rapidly spread to involve other areas of the body. Most are small and have an erythematous base with a diameter of 5–10 mm. Successive crops appear over a 2to 4-day period. Lesions can also be found on the mucosa of the pharynx and/or the vagina. Their severity varies from one person to another. Some individuals have very few lesions, while others have as many as 2000. Younger children tend to have fewer vesicles than

1	and/or the vagina. Their severity varies from one person to another. Some individuals have very few lesions, while others have as many as 2000. Younger children tend to have fewer vesicles than older individuals. Secondary and tertiary cases within families are associated with a relatively large number of vesicles. Immunocompromised patients— both children and adults, particularly those with leukemia—have lesions (often with a hemorrhagic base) that are more numerous and take longer to heal than those of immunocompetent patients. Immunocompromised individuals are also at greater risk for visceral complications, which occur in 30–50% of cases and are fatal 15% of the time in the absence of antiviral therapy.

1	The most common infectious complication of varicella is secondary bacterial superinfection of the skin, which is usually caused by Streptococcus pyogenes or Staphylococcus aureus, including strains that are methicillin-resistant. Skin infection results from excoriation of lesions after scratching. Gram’s staining of skin lesions should help clarify the etiology of unusually erythematous and pustulated lesions.

1	The most common extracutaneous site of involvement in children is the CNS. The syndrome of acute cerebellar ataxia and meningeal inflammation generally appears ~21 days after onset of the rash and rarely develops in the pre-eruptive phase. The cerebrospinal fluid (CSF) contains lymphocytes and elevated levels of protein. CNS involvement is a benign complication of VZV infection in children and generally does not require hospitalization. Aseptic meningitis, encephalitis, transverse myelitis, and Guillain-Barré syndrome can also occur. Reye’s syndrome has been reported in children concomitantly treated with aspirin. Encephalitis is reported in 0.1–0.2% of children with chickenpox. Other than supportive care, no specific therapy (e.g., acyclovir administration) has proved efficacious for patients with CNS involvement.

1	Varicella pneumonia, the most serious complication following chickenpox, develops more often in adults (up to 20% of cases) than in children and is particularly severe in pregnant women. Pneumonia due to VZV usually has its onset 3–5 days into the illness and is associated with tachypnea, cough, dyspnea, and fever. Cyanosis, pleuritic chest pain, and hemoptysis are frequently noted. Roentgenographic evidence of disease consists of nodular infiltrates and interstitial pneumonitis. Resolution of pneumonitis parallels improvement of the skin rash; however, patients may have persistent fever and compromised pulmonary function for weeks.

1	Other complications of chickenpox include myocarditis, corneal lesions, nephritis, arthritis, bleeding diatheses, acute glomerulonephritis, and hepatitis. Hepatic involvement, distinct from Reye’s syndrome and usually asymptomatic, is common in chickenpox and is generally characterized by elevated levels of liver enzymes, particularly aspartate and alanine aminotransferases. Perinatal varicella is associated with mortality rates as high as 30% when maternal disease develops within 5 days before delivery or within 48 h thereafter. Illness in this setting is unusually severe because the newborn does not receive protective transplacental antibodies and has an immature immune system. Congenital varicella, with clinical manifestations of limb hypoplasia, cicatricial skin lesions, and microcephaly at birth, is extremely uncommon.

1	Herpes Zoster Herpes zoster (shingles) is a sporadic disease that results from reactivation of latent VZV from dorsal root ganglia. Most patients with shingles have no history of recent exposure to other individuals with VZV infection. Herpes zoster occurs at all ages, but its incidence is highest (5–10 cases per 1000 persons) among individuals in the sixth decade of life and beyond. Data suggest that 1.2 million cases occur annually in the United States. Recurrent herpes zoster is exceedingly rare except in immunocompromised hosts, especially those with AIDS.

1	Herpes zoster is characterized by a unilateral vesicular dermatomal eruption, often associated with severe pain. The dermatomes from T3 to L3 are most frequently involved. If the ophthalmic branch of the trigeminal nerve is involved, zoster ophthalmicus results. The factors responsible for the reactivation of VZV are not known. In children, reactivation is usually benign; in adults, it can be debilitating because of pain. The onset of disease is heralded by pain within the dermatome, which may precede lesions by 48–72 h; an erythematous maculopapular rash evolves rapidly into vesicular lesions (Fig. 217-2). In the normal host, these lesions may remain few in number and continue to form for only 3–5 days. The total duration of disease is generally 7–10 days; however, it may take as long as 2–4 weeks for the skin to return to normal. Patients with herpes zoster can transmit infection to seronegative individuals, with consequent chickenpox. In a few patients, characteristic localization

1	2–4 weeks for the skin to return to normal. Patients with herpes zoster can transmit infection to seronegative individuals, with consequent chickenpox. In a few patients, characteristic localization of pain to a dermatome with serologic evidence of herpes zoster has been reported in the absence of skin lesions, an entity known as zoster sine herpetica. When branches of the trigeminal nerve are involved, lesions may appear on the face, in the mouth, in the eye, or on the tongue. Zoster ophthalmicus is usually a debilitating condition that can result in blindness in the absence of antiviral therapy. In Ramsay Hunt syndrome, pain and vesicles appear in the external auditory canal, and patients lose their sense of taste in the anterior two-thirds of the tongue while developing ipsilateral facial palsy. The geniculate ganglion of the sensory branch of the facial nerve is involved.

1	FIGuRE 217-2 Close-up of lesions of disseminated zoster. Note lesions at different stages of evolution, including pustules and crusting. (Photo courtesy of Lindsey Baden; with permission.) In both normal and immunocompromised hosts, the most debilitating complication of herpes zoster is pain associated with acute neuritis and postherpetic neuralgia. Postherpetic neuralgia is uncommon in young individuals; however, at least 50% of zoster patients over age 50 report some degree of pain in the involved dermatome for months after the resolution of cutaneous disease. Changes in sensation in the dermatome, resulting in either hypoor hyperesthesia, are common.

1	CNS involvement may follow localized herpes zoster. Many patients without signs of meningeal irritation have CSF pleocytosis and moderately elevated levels of CSF protein. Symptomatic meningoencephalitis is characterized by headache, fever, photophobia, meningitis, and vomiting. A rare manifestation of CNS involvement is granulomatous angiitis with contralateral hemiplegia, which can be diagnosed by cerebral arteriography. Other neurologic manifestations include transverse myelitis with or without motor paralysis.

1	Like chickenpox, herpes zoster is more severe in immunocompromised than immunocompetent individuals. Lesions continue to form for >1 week, and scabbing is not complete in most cases until 3 weeks into the illness. Patients with Hodgkin’s disease and non-Hodgkin’s lymphoma are at greatest risk for progressive herpes zoster. Cutaneous dissemination (Fig. 217-3) develops in ~40% of these patients. Among patients with cutaneous dissemination, the risk of pneumonitis, meningoencephalitis, hepatitis, and other serious complications is increased by 5–10%. However, even in immunocompromised patients, disseminated zoster is rarely fatal. Recipients of hematopoietic stem cell transplants are at particularly high risk of VZV infection. Of all cases of posttransplantation VZV infection, 30% occur within 1 year (50% of these within 9 months); 45% of the patients involved have cutaneous or visceral dissemination. The mortality rate in this situation is 10%. Postherpetic neuralgia,

1	FIGuRE 217-3 Herpes zoster in an HIV-infected patient is seen as hemorrhagic vesicles and pustules on an erythematous base grouped in a dermatomal distribution. scarring, and bacterial superinfection are especially common in VZV 1185 infections occurring within 9 months of transplantation. Among infected patients, concomitant graft-versus-host disease increases the chance of dissemination and/or death.

1	(See also Chap. 25e) The diagnosis of chickenpox is not difficult. The characteristic rash and a history of recent exposure should lead to a prompt diagnosis. Other viral infections that can mimic chickenpox include disseminated HSV infection in patients with atopic dermatitis and the disseminated vesiculopapular lesions sometimes associated with coxsackievirus infection, echovirus infection, or atypical measles. However, these rashes are more commonly morbilliform with a hemorrhagic component rather than vesicular or vesiculopustular. Rickettsialpox (Chap. 211) is sometimes confused with chickenpox; however, rickettsialpox can be distinguished easily by detection of the “herald spot” at the site of the mite bite and the development of a more pronounced headache. Serologic testing is also useful in differentiating rickettsialpox from varicella and can confirm susceptibility in adults unsure of their chickenpox history. Concern about smallpox has recently increased because of the

1	also useful in differentiating rickettsialpox from varicella and can confirm susceptibility in adults unsure of their chickenpox history. Concern about smallpox has recently increased because of the threat of bioterrorism (Chap. 261e). The lesions of smallpox are larger than those of chickenpox and are all at the same stage of evolution at any given time.

1	Unilateral vesicular lesions in a dermatomal pattern should lead rapidly to the diagnosis of herpes zoster, although the occurrence of shingles without a rash has been reported. Both HSV and coxsackievirus infections can cause dermatomal vesicular lesions. Supportive diagnostic virology and fluorescent staining of skin scrapings with monoclonal antibodies are helpful in ensuring the proper diagnosis. In the prodromal stage of herpes zoster, the diagnosis can be exceedingly difficult and may be made only after lesions have appeared or by retrospective serologic assessment.

1	Unequivocal confirmation of the diagnosis is possible only through the isolation of VZV in susceptible tissue-culture cell lines, the demonstration of either seroconversion or a fourfold or greater rise in antibody titer between acute-phase and convalescent-phase serum specimens, or the detection of VZV DNA by PCR. A rapid impression can be obtained by a Tzanck smear, with scraping of the base of the lesions in an attempt to demonstrate multinucleated giant cells; however, the sensitivity of this method is low (~60%). PCR technology for the detection of viral DNA in vesicular fluid is available in a limited number of diagnostic laboratories. Direct immunofluorescent staining of cells from the lesion base or detection of viral antigens by other assays (such as the immunoperoxidase assay) is also useful, although these tests are not commercially available. The most frequently employed serologic tools for assessing host response are the immunofluorescent detection of antibodies to VZV

1	is also useful, although these tests are not commercially available. The most frequently employed serologic tools for assessing host response are the immunofluorescent detection of antibodies to VZV membrane antigens, the fluorescent antibody to membrane antigen (FAMA) test, immune adherence hemagglutination, and enzyme-linked immunosorbent assay (ELISA). The FAMA test and the ELISA appear to be most sensitive.

1	Medical management of chickenpox in the immunologically normal host is directed toward the prevention of avoidable complications. Obviously, good hygiene includes daily bathing and soaks. Secondary bacterial infection of the skin can be avoided by meticulous skin care, particularly with close cropping of fingernails. Pruritus can be decreased with topical dressings or the administration of antipruritic drugs. Tepid water baths and wet compresses are better than drying lotions for the relief of itching. Administration of aspirin to children with chickenpox should be avoided because of the association of aspirin derivatives with the development of Reye’s syndrome. Acyclovir (800 mg by mouth five times daily), valacyclovir (1 g three times daily), or famciclovir (250 mg three times daily) for 5–7 days is recommended for adolescents and adults with chickenpox of 1186 ≤24 h duration. (Valacyclovir is licensed for use in children and adolescents. Famciclovir is recommended but not licensed

1	5–7 days is recommended for adolescents and adults with chickenpox of 1186 ≤24 h duration. (Valacyclovir is licensed for use in children and adolescents. Famciclovir is recommended but not licensed for varicella.) Likewise, acyclovir therapy may be of benefit to children <12 years of age if initiated early in the disease (<24 h) at a dose of 20 mg/kg every 6 h. The advantages (i.e., pharmacokinetics) of the second-generation agents valacyclovir and famciclovir are described in Chap. 215e. Aluminum acetate soaks for the management of herpes zoster can be both soothing and cleansing. Patients with herpes zoster benefit from oral antiviral therapy, as evidenced by accelerated healing of lesions and resolution of zoster-associated pain with acyclovir, valacyclovir, or famciclovir. Acyclovir is administered at a dosage of 800 mg five times daily for 7–10 days. However, valacyclovir and famciclovir are superior in terms of pharmacokinetics and pharmacodynamics and should be used

1	Acyclovir is administered at a dosage of 800 mg five times daily for 7–10 days. However, valacyclovir and famciclovir are superior in terms of pharmacokinetics and pharmacodynamics and should be used preferentially. Famciclovir, the prodrug of penciclovir, is at least as effective as acyclovir and perhaps more so; the dose is 500 mg by mouth three times daily for 7 days. Valacyclovir, the prodrug of acyclovir, accelerates healing and resolution of zosterassociated pain more promptly than acyclovir. The dose is 1 g by mouth three times daily for 5–7 days. Compared with acyclovir, both famciclovir and valacyclovir offer the advantage of less frequent administration. All three of these drugs are now off patent. In severely immunocompromised hosts (e.g., transplant recipients, patients with lymphoproliferative malignancies), both chickenpox and herpes zoster (including disseminated disease) should be treated, at least at the outset, with IV acyclovir, which reduces the occurrence of

1	lymphoproliferative malignancies), both chickenpox and herpes zoster (including disseminated disease) should be treated, at least at the outset, with IV acyclovir, which reduces the occurrence of visceral complications but has no effect on healing of skin lesions or pain. The dose is 10 mg/kg every 8 h for 7 days. For low-risk immunocompromised hosts, oral therapy with valacyclovir or famciclovir appears beneficial. If medically feasible, it is desirable to decrease immunosuppressive treatment concomitant with the administration of IV acyclovir. Patients with varicella pneumonia often require ventilatory support. Persons with zoster ophthalmicus should be referred immediately to an ophthalmologist. Therapy for this condition consists of the administration of analgesics for severe pain and the use of atropine. Acyclovir, valacyclovir, and famciclovir all accelerate healing. Decisions about the use of glucocorticoids should be made by the ophthalmologist. The management of acute

1	and the use of atropine. Acyclovir, valacyclovir, and famciclovir all accelerate healing. Decisions about the use of glucocorticoids should be made by the ophthalmologist. The management of acute neuritis and/or postherpetic neuralgia can be particularly difficult. In addition to the judicious use of analgesics ranging from nonnarcotics to narcotic derivatives, drugs such as gabapentin, pregabalin, amitriptyline hydrochloride, lidocaine (patches), and fluphenazine hydrochloride are reportedly beneficial for pain relief. In one study, glucocorticoid therapy administered early in the course of localized herpes zoster significantly accelerated such quality-of-life improvements as a return to usual activity and termination of analgesic medications. The dose of prednisone administered orally was 60 mg/d on days 1–7, 30 mg/d on days 8–14, and 15 mg/d on days 15–21. This regimen is appropriate only for relatively healthy elderly persons with moderate or severe pain at presentation. Patients

1	60 mg/d on days 1–7, 30 mg/d on days 8–14, and 15 mg/d on days 15–21. This regimen is appropriate only for relatively healthy elderly persons with moderate or severe pain at presentation. Patients with osteoporosis, diabetes mellitus, glycosuria, or hypertension may not be appropriate candidates. Glucocorticoids should not be used without concomitant antiviral therapy.

1	Three methods are used for the prevention of VZV infections. First, a live attenuated varicella vaccine (Oka) is recommended for all children >1 year of age (up to 12 years of age) who have not had chickenpox and for adults known to be seronegative for VZV. Two doses are recommended for all children: the first at 12–15 months of age and the second at ~4–6 years of age. VZV-seronegative persons >13 years of age should receive two doses of vaccine at least 1 month apart. The vaccine is both safe and efficacious. Breakthrough cases are mild and may result in spread of the vaccine virus to susceptible contacts. The universal vaccination of children is resulting in a decreased incidence of chickenpox in sentinel communities. Furthermore, inactivation of the vaccine virus significantly decreases the occurrence of herpes zoster after hematopoietic stem-cell transplantation. 1. Exposure to a person with chickenpox or zoster a. Household: residence in the same household b.

1	1. Exposure to a person with chickenpox or zoster a. Household: residence in the same household b. Playmate: face-to-face indoor play c. Varicella: same 2to 4-bed room or adjacent beds in large ward, faceto-face contact with infectious staff member or patient, visit by a person deemed contagious Zoster: intimate contact (e.g., touching or hugging) with a person deemed contagious d. Newborn infant: onset of varicella in the mother ≤5 days before delivery or ≤48 h after delivery; VZIG not indicated if the mother has zoster 2. Patient should receive VZIG as soon as possible but not >96 h after exposure Candidates (Provided They Have Significant Exposure) Include: 1. Immunocompromised susceptible children without a history of varicella or varicella immunization 2. 3. Newborn infants whose mother had onset of chickenpox within 5 days before or within 48 h after delivery 4.

1	3. Newborn infants whose mother had onset of chickenpox within 5 days before or within 48 h after delivery 4. Hospitalized premature infant (≥28 weeks of gestation) whose mother lacks a reliable history of chickenpox or serologic evidence of protection against varicella 5. Hospitalized premature infant (<28 weeks of gestation or ≤1000-g birth weight), regardless of maternal history of varicella or VZV serologic status In individuals >50 years of age, a VZV vaccine with 18 times the viral content of the Oka vaccine decreased the incidence of shingles by 51%, the burden of illness by 61%, and the incidence of postherpetic neuralgia by 66%. The Advisory Committee on Immunization Practices has therefore recommended that persons in this age group be offered this vaccine in order to reduce the frequency of shingles and the severity of postherpetic neuralgia.

1	A second approach is to administer varicella-zoster immune globulin (VZIG) to individuals who are susceptible, are at high risk for developing complications of varicella, and have had a significant exposure. This product should be given within 96 h (preferably within 72 h) of the exposure. Indications for administration of VZIG appear in Table 217-1. Lastly, antiviral therapy can be given as prophylaxis to individuals at high risk who are ineligible for vaccine or who are beyond the 96-h window after direct contact. While the initial studies have used acyclovir, similar benefit can be anticipated with either valacyclovir or famciclovir. Therapy is instituted 7 days after intense exposure. At this time, the host is midway into the incubation period. This approach significantly decreases disease severity, if not totally preventing disease. Epstein-Barr virus Infections, 218 Including Infectious Mononucleosis Jeffrey I. Cohen

1	Epstein-Barr virus Infections, 218 Including Infectious Mononucleosis Jeffrey I. Cohen Epstein-Barr virus (EBV) is the cause of heterophile-positive infectious mononucleosis (IM), which is characterized by fever, sore throat, lymphadenopathy, and atypical lymphocytosis. EBV is also associated with several tumors, including nasopharyngeal and gastric carcinoma, Burkitt’s lymphoma, Hodgkin’s disease, and (in patients with immunodeficiencies) B cell lymphoma. The virus is a member of the family Herpesviridae. The two types of EBV that are widely prevalent in nature are not distinguishable by conventional serologic tests.

1	EBV infections occur worldwide. These infections are most common in early childhood, with a second peak during late adolescence. By adulthood, more than 90% of individuals have been infected and have antibodies to the virus. IM is usually a disease of young adults. In lower socioeconomic groups and in areas of the world with deficient standards of hygiene (e.g., developing regions), EBV tends to infect children at an early age, and IM is uncommon. In areas with higher standards of hygiene, infection with EBV is often delayed until adulthood, and IM is more prevalent.

1	EBV is spread by contact with oral secretions. The virus is frequently transmitted from asymptomatic adults to infants and among young adults by transfer of saliva during kissing. Transmission by less intimate contact is rare. EBV has been transmitted by blood transfusion and by bone marrow transplantation. More than 90% of asymptomatic seropositive individuals shed the virus in oropharyngeal secretions. Shedding is increased in immunocompromised patients and those with IM.

1	EBV is transmitted by salivary secretions. The virus infects the epithelium of the oropharynx and the salivary glands and is shed from these cells. While B cells may become infected after contact with epithelial cells, studies suggest that lymphocytes in the tonsillar crypts can be infected directly. The virus then spreads through the bloodstream. The proliferation and expansion of EBV-infected B cells along with reactive T cells during IM result in enlargement of lymphoid tissue. Polyclonal activation of B cells leads to the production of antibodies to host-cell and viral proteins. During the acute phase of IM, up to 1 in every 100 B cells in the peripheral blood is infected by EBV; after recovery, 1–50 in every 1 million B cells is infected. During IM, there is an inverted CD4+/CD8+ T cell ratio. The percentage of CD4+ T cells decreases, while there are large clonal expansions of CD8+ T cells; up to 40% of CD8+ T cells are directed against EBV antigens during acute infection. Memory

1	ratio. The percentage of CD4+ T cells decreases, while there are large clonal expansions of CD8+ T cells; up to 40% of CD8+ T cells are directed against EBV antigens during acute infection. Memory B cells, not epithelial cells, are the reservoir for EBV in the body. When patients are treated with acyclovir, shedding of EBV from the oropharynx stops but the virus persists in B cells.

1	The EBV receptor (CD21) on the surface of B cells is also the receptor for the C3d component of complement. EBV infection of epithelial cells results in viral replication and production of virions. When B cells are infected by EBV in vitro, they become transformed and can proliferate indefinitely. During latent infection of B cells, only the EBV nuclear antigens (EBNAs), latent membrane proteins (LMPs), and small EBV RNAs (EBERs) are expressed in vitro. EBV-transformed B cells secrete immunoglobulin; only a small fraction of these cells produce virus.

1	Cellular immunity is more important than humoral immunity in controlling EBV infection. In the initial phase of infection, suppressor T cells, natural killer cells, and nonspecific cytotoxic T cells are important in controlling the proliferation of EBV-infected B cells. Levels of markers of T cell activation and serum interferon γ are elevated. Later in infection, human leukocyte antigen–restricted cytotoxic T cells that recognize EBNAs and LMPs and destroy EBV-infected cells are generated. If T cell immunity is compromised, EBV-infected B cells may begin to proliferate. When EBV is associated with lymphoma in immunocompetent persons, virus-induced proliferation is but one step in a multi-step process of neoplastic transformation. In many EBV-containing tumors, LMP-1 mimics members of the tumor necrosis factor receptor family (e.g., CD40), transmitting growth-proliferating signals.

1	CLINICAL MANIFESTATIONS Signs and Symptoms Most EBV infections in infants and young children either are asymptomatic or present as mild pharyngitis with or without tonsillitis. In contrast, ~75% of infections in adolescents present as IM. IM in the elderly often presents with nonspecific symptoms, including prolonged fever, fatigue, myalgia, and malaise. In contrast, pharyngitis, lymphadenopathy, splenomegaly, and atypical lymphocytes are relatively rare in elderly patients. Median Percentage of Manifestation Patients (Range) Sore throat 75 (50–87) Malaise 47 (42–76) Headache 38 (22–67) Abdominal pain, nausea, or vomiting 17 (5–25) Chills 10 (9–11)

1	The incubation period for IM in young adults is ~4–6 weeks. A prodrome of fatigue, malaise, and myalgia may last for 1–2 weeks before the onset of fever, sore throat, and lymphadenopathy. Fever is usually low-grade and is most common in the first 2 weeks of the illness; however, it may persist for >1 month. Common signs and symptoms are listed along with their frequencies in Table 218-1. Lymphadenopathy and pharyngitis are most prominent during the first 2 weeks of the illness, while splenomegaly is more prominent during the second and third weeks. Lymphadenopathy most often affects the posterior cervical nodes but may be generalized. Enlarged lymph nodes are frequently tender and symmetric but are not fixed in place. Pharyngitis, often the most prominent sign, can be accompanied by enlargement of the tonsils with an exudate resembling that of streptococcal pharyngitis. A morbilliform or papular rash, usually on the arms or trunk, develops in ~5% of cases (Fig. 218-1). Many patients

1	of the tonsils with an exudate resembling that of streptococcal pharyngitis. A morbilliform or papular rash, usually on the arms or trunk, develops in ~5% of cases (Fig. 218-1). Many patients treated with ampicillin develop a macular rash; this rash is not predictive of future adverse reactions to penicillins. Erythema nodosum and erythema multiforme also have been described (Chap. 72). The severity of the disease correlates with the levels of CD8+ T cells and EBV DNA in the blood. Most patients have symptoms for 2–4 weeks, but nearly 10% have fatigue that persists for ≥6 months.

1	FIGuRE 218-1 Rash in a patient with infectious mononucleosis due to Epstein-Barr virus. (Courtesy of Maria Turner, MD; with permission.) Epstein-Barr Virus Infections, Including Infectious Mononucleosis FIGuRE 218-2 Atypical lymphocytes from a patient with infectious mononucleosis due to Epstein-Barr virus.

1	Epstein-Barr Virus Infections, Including Infectious Mononucleosis FIGuRE 218-2 Atypical lymphocytes from a patient with infectious mononucleosis due to Epstein-Barr virus. Laboratory Findings The white blood cell count is usually elevated and peaks at 10,000–20,000/μL during the second or third week of illness. Lymphocytosis is usually demonstrable, with >10% atypical lymphocytes. The latter cells are enlarged lymphocytes that have abundant cytoplasm, vacuoles, and indentations of the cell membrane (Fig. 218-2). CD8+ cells predominate among the atypical lymphocytes. Low-grade neutropenia and thrombocytopenia are common during the first month of illness. Liver function is abnormal in >90% of cases. Serum levels of aminotransferases and alkaline phosphatase are usually mildly elevated. The serum concentration of bilirubin is elevated in ~40% of cases.

1	Complications Most cases of IM are self-limited. Deaths are very rare and are most often due to central nervous system (CNS) complications, splenic rupture, upper airway obstruction, or bacterial superinfection.

1	When CNS complications develop, they usually do so during the first 2 weeks of EBV infection; in some patients, especially children, they are the only clinical manifestations of IM. Heterophile antibodies and atypical lymphocytes may be absent. Meningitis and encephalitis are the most common neurologic abnormalities, and patients may present with headache, meningismus, or cerebellar ataxia. Acute hemiplegia and psychosis also have been described. The cerebrospinal fluid contains mainly lymphocytes, with occasional atypical lymphocytes. Most cases resolve without neurologic sequelae. Acute EBV infection has also been associated with cranial nerve palsies (especially those involving cranial nerve VII), Guillain-Barré syndrome, acute transverse myelitis, and peripheral neuritis.

1	Autoimmune hemolytic anemia occurs in ~2% of cases during the first 2 weeks. In most cases, the anemia is Coombs-positive, with cold agglutinins directed against the red blood cell antigen. Most patients with hemolysis have mild anemia that lasts for 1–2 months, but some patients have severe disease with hemoglobinuria and jaundice. Nonspecific antibody responses may also include rheumatoid factor, antinuclear antibodies, anti–smooth muscle antibodies, antiplatelet antibodies, and cryoglobulins. IM has been associated with red-cell aplasia, severe granulocytopenia, thrombocytopenia, pancytopenia, and hemophagocytic lymphohistiocytosis. The spleen ruptures in <0.5% of cases. Splenic rupture is more common among male than female patients and may manifest as abdominal pain, referred shoulder pain, or hemodynamic compromise.

1	Hypertrophy of lymphoid tissue in the tonsils or adenoids can result in upper airway obstruction, as can inflammation and edema of the epiglottis, pharynx, or uvula. About 10% of patients with IM develop streptococcal pharyngitis after their initial sore throat resolves. Other rare complications associated with acute EBV infection include hepatitis (which can be fulminant), myocarditis or pericarditis, pneumonia with pleural effusion, interstitial nephritis, genital ulcerations, and vasculitis.

1	EBV-Associated Diseases Other Than IM EBV-associated lymphoproliferative disease has been described in patients with congenital or acquired immunodeficiency, including those with severe combined immunodeficiency, patients with AIDS, and recipients of bone marrow or organ transplants who are receiving immunosuppressive drugs (especially cyclosporine). Proliferating EBV-infected B cells infiltrate lymph nodes and multiple organs, and patients present with fever and lymphadenopathy or gastrointestinal symptoms. Pathologic studies show B cell hyperplasia or polyor monoclonal lymphoma.

1	X-linked lymphoproliferative disease is a recessive disorder of young boys who have a normal response to childhood infections but develop fatal lymphoproliferative disorders after infection with EBV. The protein associated with most cases of this syndrome (SAP) binds to a protein that mediates interactions of B and T cells. Most patients with this syndrome die of acute IM. Others develop hypogammaglobulinemia, malignant B cell lymphomas, aplastic anemia, or agranulocytosis. Disease resembling X-linked lymphoproliferative disease has also been associated with mutations in XIAP. Mutations in ITK, MagT1, or CD27 are associated with inability to control EBV and lymphoma. Moreover, IM has proved fatal to some patients with no obvious preexisting immune abnormality.

1	Oral hairy leukoplakia (Fig. 218-3) is an early manifestation of infection with HIV in adults (Chap. 226). Most patients present with raised, white corrugated lesions on the tongue (and occasionally on the buccal mucosa) that contain EBV DNA. Children infected with HIV can develop lymphoid interstitial pneumonitis; EBV DNA is often found in lung tissue from these patients.

1	Patients with chronic fatigue syndrome may have titers of antibody to EBV that are elevated but are not significantly different from those in healthy EBV-seropositive adults. While some patients have malaise and fatigue that persist for weeks or months after IM, persistent EBV infection is not a cause of chronic fatigue syndrome. Chronic active EBV infection is very rare and is distinct from chronic fatigue syndrome. The affected patients have an illness lasting >6 months, with elevated levels of EBV DNA in the blood, high titers of antibody to EBV, and evidence of organ involvement, including hepatosplenomegaly, lymphadenopathy, and pneumonitis, uveitis, or neurologic disease.

1	EBV is associated with several malignancies. About 15% of cases of Burkitt’s lymphoma in the United States and ~90% of those in Africa are associated with EBV (Chap. 134). African patients with Burkitt’s lymphoma have high levels of antibody to EBV, and their tumor tissue usually contains viral DNA. Malaria in African patients may impair cellular immunity to EBV and induce polyclonal B cell activation with an expansion of EBV-infected B cells. These changes may enhance the proliferation of B cells with elevated EBV DNA in the bloodstream, thereby increasing the likelihood of a c-myc translocation—the hallmark of Burkitt’s lymphoma. EBV-containing Burkitt’s lymphoma also occurs in patients with AIDS.

1	Anaplastic nasopharyngeal carcinoma is common in southern China and is uniformly associated with EBV; the affected tissues contain viral DNA and antigens. Patients with nasopharyngeal carcinoma often have elevated titers of antibody to EBV (Chap. 106). High levels of EBV plasma DNA before treatment or detectable levels of EBV DNA after radiation therapy correlate with lower rates of overall survival and relapse-free survival among patients with nasopharyngeal carcinoma. FIGuRE 218-3 Oral hairy leukoplakia often presents as white plaques on the lateral surface of the tongue and is associated with Epstein-Barr virus infection. Worldwide, the most common EBV-associated malignancy is gastric carcinoma. About 9% of these tumors are EBV-positive.

1	Worldwide, the most common EBV-associated malignancy is gastric carcinoma. About 9% of these tumors are EBV-positive. EBV has been associated with Hodgkin’s disease, especially the mixed-cellularity type (Chap. 134). Patients with Hodgkin’s disease often have elevated titers of antibody to EBV. In about half of cases in the United States, viral DNA and antigens are found in Reed-Sternberg cells. The risk of EBV-positive Hodgkin’s disease is significantly increased in young adults for several years after EBV-seropositive IM. About 50% of non-Hodgkin’s lymphomas in patients with AIDS are EBV-positive.

1	EBV is present in B cells of lesions from patients with lymphomatoid granulomatosis. In some cases, EBV DNA has been detected in tumors from immunocompetent patients with angiocentric nasal NK/T cell lymphoma, T cell lymphoma, and CNS lymphoma. Studies have demonstrated viral DNA in leiomyosarcomas from AIDS patients and in smooth-muscle tumors from organ transplant recipients. Virtually all CNS lymphomas in AIDS patients are associated with EBV. Studies have found that a history of IM and higher levels of antibodies to EBV before the onset of disease is more common in persons with multiple sclerosis than in the general population; additional research on a possible causal relationship is needed.

1	DIAGNOSIS Serologic Testing (Fig. 218-4) The heterophile test is used for the diagnosis of IM in children and adults. In the test for this antibody, human serum is absorbed with guinea pig kidney, and the heterophile titer is defined as the greatest serum dilution that agglutinates sheep, horse, or cow erythrocytes. The heterophile antibody does not interact with EBV proteins. A titer of ≥40 is diagnostic of acute EBV infection in a patient who has symptoms compatible with IM and atypical lymphocytes. Tests for heterophile antibodies are positive in 40% of patients with IM during the first week of illness and in 80–90% during the third week. Therefore, repeated testing may be necessary, especially if the initial test is performed early. Tests usually remain positive for 3 months after the onset of illness, but heterophile antibodies can persist for up to 1 year. These antibodies usually are not detectable in children <5 years of age, in the elderly, or in patients presenting with

1	the onset of illness, but heterophile antibodies can persist for up to 1 year. These antibodies usually are not detectable in children <5 years of age, in the elderly, or in patients presenting with symptoms not typical of IM. The commercially available monospot test for heterophile antibodies is somewhat more sensitive than the classic heterophile test. The monospot test is ~75% sensitive and ~90% specific compared with EBV-specific serologies (see below). False-positive monospot results are more common among persons with connective tissue disease, lymphoma, viral hepatitis, and malaria.

1	EBV-specific antibody testing is used for patients with suspected acute EBV infection who lack heterophile antibodies and for patients with atypical infections. Titers of IgM and IgG antibodies to viral capsid antigen (VCA) are elevated in the serum of more than 90% of patients at the onset of disease. IgM antibody to VCA is most useful for the diagnosis of acute IM because it is present at elevated titers only 1189 during the first 2–3 months of the disease; in contrast, IgG antibody to VCA is usually not useful for diagnosis of IM but is often used to assess past exposure to EBV because it persists for life. Seroconversion to EBNA positivity also is useful for the diagnosis of acute infection with EBV. Antibodies to EBNA become detectable relatively late (3–6 weeks after the onset of symptoms) in nearly all cases of acute EBV infection and persist for the lifetime of the patient. These antibodies may be lacking in immunodeficient patients and in those with chronic active EBV

1	of symptoms) in nearly all cases of acute EBV infection and persist for the lifetime of the patient. These antibodies may be lacking in immunodeficient patients and in those with chronic active EBV infection.

1	Titers of other antibodies also may be elevated in IM; however, these elevations are less useful for diagnosis. Antibodies to early antigens are detectable 3–4 weeks after the onset of symptoms in patients with IM. About 70% of individuals with IM have early antigen diffuse (EA-D) antibodies during the illness; the presence of EA-D antibodies is especially likely in patients with relatively severe disease. These antibodies usually persist for only 3–6 months. Levels of EA-D antibodies are also elevated in patients with nasopharyngeal carcinoma or chronic active EBV infection. Early antigen restricted (EA-R) antibodies are only occasionally detected in patients with IM but are often found at elevated titers in patients with African Burkitt’s lymphoma or chronic active EBV infection. IgA antibodies to EBV antigens have proved useful for the identification of patients with nasopharyngeal carcinoma and of persons at high risk for the disease.

1	Other Studies Detection of EBV DNA, RNA, or proteins has been valuable in demonstrating the association of the virus with various malignancies. The polymerase chain reaction has been used to detect EBV DNA in the cerebrospinal fluid of some AIDS patients with lymphomas and to monitor the amount of EBV DNA in the blood of patients with lymphoproliferative disease. Detection of high levels of EBV DNA in blood for a few days to several weeks after the onset of IM may be useful if serologic studies yield equivocal results. Culture of EBV from throat washings or blood is not helpful in the diagnosis of acute infection, since EBV persists in the oropharynx and in B cells for the lifetime of the infected individual.

1	Differential Diagnosis Whereas ~90% of cases of IM are due to EBV, 5–10% of cases are due to cytomegalovirus (CMV) (Chap. 219). CMV is the most common cause of heterophile-negative mononucleosis; less common causes of IM and differences from IM due to EBV are shown in Table 218-2. Therapy for IM consists of supportive measures, with rest and analgesia. Excessive physical activity during the first month should be avoided to reduce the possibility of splenic rupture, which often necessitates splenectomy. Glucocorticoid therapy is not indicated for uncomplicated IM and in fact may predispose to bacterial superinfection. Prednisone (40–60 mg/d for 2–3 days, with subsequent tapering of the dose over 1–2 weeks) has been used for the prevention of airway obstruction in patients with severe tonsillar hypertrophy, for Epstein-Barr Virus Infections, Including Infectious Mononucleosis Time of symptoms

1	Epstein-Barr Virus Infections, Including Infectious Mononucleosis Time of symptoms FIGuRE 218-4 Pattern of Epstein-Barr virus (EBV) serology during acute infection. EBNA, Epstein-Barr nuclear antigen; VCA, viral capsid antigen. (From JI Cohen, in NS Young et al [eds]: Clinical Hematology. Philadelphia, Mosby, 2006.) aMost commonly phenytoin, carbamazepine, sulfonamides, or minocycline. Abbreviations: CMV, cytomegalovirus; EBV, Epstein-Barr virus; HHV, human herpesvirus. autoimmune hemolytic anemia, for hemophagocytic lymphohistiocytosis, and for severe thrombocytopenia. Glucocorticoids have also been administered to rare patients with severe malaise and fever and to patients with severe CNS or cardiac disease. Acyclovir has had no significant clinical impact on IM in controlled trials. In one study, the combination of acyclovir and prednisolone had no significant effect on the duration of symptoms of IM.

1	Acyclovir, at a dosage of 400–800 mg five times daily, has been effective for the treatment of oral hairy leukoplakia (despite common relapses). The posttransplantation EBV lymphoproliferative syndrome (Chap. 169) generally does not respond to antiviral therapy. When possible, therapy should be directed toward reduction of immunosuppression. Antibody to CD20 (rituximab) has been effective in some cases. Infusions of donor lymphocytes are often effective for stem cell transplant recipients, although graftversus-host disease can occur. Infusions of EBV-specific cytotoxic T cells have been used to prevent EBV lymphoproliferative disease in high-risk settings as well as to treat the disease. Interferon α administration, cytotoxic chemotherapy, and radiation therapy (especially for CNS lesions) also have been used. Infusion of autologous EBV-specific cytotoxic T lymphocytes has shown promise in small studies of patients with nasopharyngeal carcinoma and Hodgkin’s disease. Treatment of

1	also have been used. Infusion of autologous EBV-specific cytotoxic T lymphocytes has shown promise in small studies of patients with nasopharyngeal carcinoma and Hodgkin’s disease. Treatment of several cases of X-linked lymphoproliferative disease with antibody to CD20 resulted in a successful outcome of what otherwise would probably have been fatal acute EBV infection.

1	The isolation of patients with IM is unnecessary. A vaccine directed against the major EBV glycoprotein reduced the frequency of IM but did not affect the rate of asymptomatic infection in a phase 2 trial. Cytomegalovirus and Human Herpesvirus Types 6, 7, and 8 Camille Nelson Kotton, Martin S. Hirsch CYTOMEGALOVIRuS DEFINITION 219

1	Cytomegalovirus (CMV), which was initially isolated from patients with congenital cytomegalic inclusion disease, is now recognized as an important pathogen in all age groups. In addition to inducing severe birth defects, CMV causes a wide spectrum of disorders in older chil-dren and adults, ranging from an asymptomatic subclinical infection + Older age at presentation, longer duration of fever ± Diffuse rash, oral/genital ulcers, aseptic meningitis ± Less splenomegaly, exposure to cats or raw meat − No splenomegaly, less fatigue ± Maculopapular rash, no splenomegaly + Fixed, nontender lymph nodes to a mononucleosis syndrome in healthy individuals to disseminated disease in immunocompromised patients. Human CMV is one of several related species-specific viruses that cause similar diseases in various animals. All are associated with the production of characteristic enlarged cells—hence the name cytomegalovirus.

1	CMV, a β-herpesvirus, has double-stranded DNA, four species of mRNA, a protein capsid, and a lipoprotein envelope. Like other herpesviruses, CMV demonstrates icosahedral symmetry, replicates in the cell nucleus, and can cause either a lytic and productive or a latent infection. CMV can be distinguished from other herpesviruses by certain biologic properties, such as host range and type of cytopathology. Viral replication is associated with the production of large intra-nuclear inclusions and smaller cytoplasmic inclusions. CMV appears to replicate in a variety of cell types in vivo; in tissue culture it grows preferentially in fibroblasts. Although there is little evidence that CMV is oncogenic in vivo, it does transform fibroblasts in rare instances, and genomic transforming fragments have been identified.

1	CMV has a worldwide distribution. In many regions of the world, the vast majority of adults are seropositive for CMV, whereas only half of adults in the United States and Canada are seropositive. In regions where the prevalence of CMV antibody is high, immunocompromised adults are more likely to undergo reactivation disease rather than primary infection. Data generated in specific regions should be considered in the context of local seropositivity rates, when appropriate.

1	Of newborns in the United States, ∼1% are infected with CMV; the percentages are higher in many less-developed countries. Communal living and poor personal hygiene facilitate spread. Perinatal and early childhood infections are common. CMV may be present in breast milk, saliva, feces, and urine. Transmission has occurred among young children in day-care centers and has been traced from infected toddler to pregnant mother to developing fetus. When an infected child introduces CMV into a household, 50% of susceptible family members seroconvert within 6 months.

1	CMV is not readily spread by casual contact but rather requires repeated or prolonged intimate exposure for transmission. In late adolescence and young adulthood, CMV is often transmitted sexually, and asymptomatic carriage in semen or cervical secretions is common. Antibody to CMV is present at detectable levels in a high proportion of sexually active men and women, who may harbor several strains simultaneously. Transfusion of blood products containing viable leukocytes may transmit CMV, with a frequency of 0.14–10% per unit transfused. Transfusion of leukocyte-reduced or CMV-seronegative blood significantly decreases the risk of CMV transmission.

1	Once infected, an individual generally carries CMV for life. The infection usually remains silent. CMV reactivation syndromes develop more frequently, however, when T lymphocyte–mediated immunity is compromised—for example, after organ transplantation, with lymphoid neoplasms and certain acquired immunodeficiencies (in particular, HIV infection; Chap. 226), or during critical illness in intensive care units. Most primary CMV infections in organ transplant recipients (Chap. 169) result from transmission via the graft. In CMV-seropositive transplant recipients, infection results from reactivation of latent virus or from infection by a new strain. CMV infection may also be associated with diseases as diverse as coronary artery stenosis and malignant gliomas, but these associations require further validation.

1	Congenital CMV infection can result from either primary or reactivation infection of the mother. However, clinical disease in the fetus or newborn is related almost exclusively to primary maternal infection (Table 219-1). The factors determining the severity of congenital infection are unknown; a deficient capacity to produce precipitating antibodies and to mount T cell responses to CMV is associated with relatively severe disease. Primary infection with CMV in late childhood or adulthood is often associated with a vigorous T lymphocyte response that may contribute to the development of a mononucleosis syndrome similar to that which follows infection with Epstein-Barr virus (Chap. 218). The hallmark of such infection is the appearance of atypical lymphocytes in the peripheral blood; these cells are predominantly activated CD8+ T lymphocytes. Polyclonal activation of B cells by CMV contributes to the development of rheumatoid factors and other autoantibodies during mononucleosis.

1	Once acquired, CMV persists indefinitely in host tissues. The sites of persistent infection probably include multiple cell types and various organs. Transmission via blood transfusion or organ transplantation is due primarily to latent infections in these tissues. If the host’s T cell responses become compromised by disease or by iatrogenic immunosuppression, latent virus can reactivate to cause a variety of syndromes. Chronic antigenic stimulation in the presence of immunosuppression (for example, after organ transplantation) appears to be an ideal setting for CMV activation and CMV disease. Certain particularly potent suppressants of T cell immunity (e.g., antithymocyte globulin, alemtuzumab) are associated with a high rate of clinical CMV syndromes. CMV may itself contribute to further T lymphocyte hyporesponsiveness, which often precedes superinfection with other opportunistic pathogens such as bacteria, molds, and Pneumocystis.

1	Cytomegalic cells in vivo (presumed to be infected epithelial cells) are two to four times larger than surrounding cells and often contain an 8to 10-μm intranuclear inclusion that is eccentrically placed and is surrounded by a clear halo, producing an “owl’s eye” appearance. Smaller granular cytoplasmic inclusions are demonstrated occasionally. Cytomegalic cells are found in a wide variety of organs, including the salivary gland, lung, liver, kidney, intestine, pancreas, adrenal gland, and central nervous system. The cellular inflammatory response to infection consists of plasma 1191 cells, lymphocytes, and monocyte-macrophages. Granulomatous reactions occasionally develop, particularly in the liver. Immunopathologic reactions may contribute to CMV disease. Immune complexes have been detected in infected infants, sometimes in association with CMV-related glomerulopathies. Immune-complex glomerulopathy has also been observed in some CMV-infected patients after renal transplantation.

1	CLINICAL MANIFESTATIONS Congenital CMV Infection Fetal infections range from subclinical to severe and disseminated. Cytomegalic inclusion disease develops in ∼5% of infected fetuses and is seen almost exclusively in infants born to mothers who develop primary infections during pregnancy. Petechiae, hepatosplenomegaly, and jaundice are the most common presenting features (60–80% of cases). Microcephaly with or without cerebral calcifications, intrauterine growth retardation, and prematurity are reported in 30–50% of cases. Inguinal hernias and chorioretinitis are less common. Laboratory abnormalities include elevated alanine aminotransferase levels in serum, thrombocytopenia, conjugated hyperbilirubinemia, hemolysis, and elevated protein levels in cerebrospinal fluid. The prognosis for severely infected infants is poor; the mortality rate is 20–30%, and few survivors escape intellectual or hearing difficulties later in childhood. The differential diagnosis of cytomegalic inclusion

1	severely infected infants is poor; the mortality rate is 20–30%, and few survivors escape intellectual or hearing difficulties later in childhood. The differential diagnosis of cytomegalic inclusion disease in infants includes syphilis, rubella, toxoplasmosis, infection with herpes simplex virus or enterovirus, and bacterial sepsis.

1	Most congenital CMV infections are clinically inapparent at birth. Of asymptomatically infected infants, 5–25% develop significant psychomotor, hearing, ocular, or dental abnormalities over the next several years. Perinatal CMV Infection The newborn may acquire CMV at delivery by passage through an infected birth canal or by postnatal contact with infected breast milk or other maternal secretions. Of infants who are breast-fed for >1 month by seropositive mothers, 40–60% become infected. Iatrogenic transmission can result from blood transfusion; use of leukocyte-reduced or CMV-seronegative blood products for transfusion into low-birth-weight seronegative infants or seronegative pregnant women decreases risk.

1	The great majority of infants infected at or after delivery remain asymptomatic. However, protracted interstitial pneumonitis has been associated with perinatally acquired CMV infection, particularly in premature infants, and occasionally has been accompanied by infection with Chlamydia trachomatis, Pneumocystis, or Ureaplasma urealyticum. Poor weight gain, adenopathy, rash, hepatitis, anemia, and atypical lymphocytosis may also be found, and CMV excretion often persists for months or years. CMV Mononucleosis The most common clinical manifestation of CMV infection in immunocompetent hosts beyond the neonatal period is a heterophile antibody–negative mononucleosis syndrome, which may develop spontaneously or follow transfusion of leukocyte-containing blood products. Although the syndrome occurs at all ages, it most Cytomegalovirus and Human Herpesvirus Types 6, 7, and 8

1	Person with AIDS <100 CD4+ T cells/μL; CMV seropositivity Retinitis; gastrointestinal Ganciclovir, valganciclovir, Oral valganciclovir disease; neurologic disease foscarnet, or cidofovir 1192 often involves sexually active young adults. With incubation periods of 20–60 days, the illness generally lasts for 2–6 weeks. Prolonged high fevers, sometimes with chills, profound fatigue, and malaise, characterize this disorder. Myalgias, headache, and splenomegaly are common, but in CMV (as opposed to Epstein-Barr virus) mononucleosis, exudative pharyngitis and cervical lymphadenopathy are rare. Occasional patients develop rubelliform rashes, often after exposure to ampicillin or certain other antibiotics. Less common are interstitial or segmental pneumonia, myocarditis, pleuritis, arthritis, and encephalitis. In rare cases, Guillain-Barré syndrome complicates CMV mononucleosis. The characteristic laboratory abnormality is relative lymphocytosis in peripheral blood, with >10% atypical

1	and encephalitis. In rare cases, Guillain-Barré syndrome complicates CMV mononucleosis. The characteristic laboratory abnormality is relative lymphocytosis in peripheral blood, with >10% atypical lymphocytes. Total leukocyte counts may be low, normal, or markedly elevated. Although significant jaundice is uncommon, serum aminotransferase and alkaline phosphatase levels are often moderately elevated. Heterophile antibodies are absent; however, transient immunologic abnormalities are common and may include the presence of cryoglobulins, rheumatoid factors, cold agglutinins, and antinuclear antibodies. Hemolytic anemia, thrombocytopenia, and granulocytopenia complicate recovery in rare instances. Most patients recover without sequelae, although postviral asthenia may persist for months. The excretion of CMV in urine, genital secretions, and/or saliva often continues for months or years. Rarely, CMV infection is fatal in immunocompetent hosts; survivors can have recurrent episodes of

1	The excretion of CMV in urine, genital secretions, and/or saliva often continues for months or years. Rarely, CMV infection is fatal in immunocompetent hosts; survivors can have recurrent episodes of fever and malaise, sometimes associated with autonomic nervous system dysfunction (e.g., attacks of sweating or flushing).

1	CMV Infection in the Immunocompromised Host (Table 219-1) CMV is the viral pathogen most commonly complicating organ transplantation (Chap. 169). In recipients of kidney, heart, lung, liver, pancreas, and vascularized composite (hand, face, other) transplants, CMV induces a variety of syndromes, including fever and leukopenia, hepatitis, colitis, pneumonitis, esophagitis, gastritis, and retinitis. CMV disease is an independent risk factor for both graft loss and death. Without prophylaxis, the period of maximal risk is between 1 and 4 months after transplantation. Disease likelihood and viral replication levels generally are greater after primary infection than after reactivation. Molecular studies indicate that seropositive transplant recipients are susceptible to infection with donor-derived, genotypically variant CMV, and such infection often results in disease. Reactivation infection, although common, is less likely than primary infection to be important clinically. The risk of

1	genotypically variant CMV, and such infection often results in disease. Reactivation infection, although common, is less likely than primary infection to be important clinically. The risk of clinical disease is related to various factors, such as degree of immunosuppression, use of antilymphocyte antibodies, lack of anti-CMV prophylaxis, and co-infection with other pathogens. The transplanted organ is particularly vulnerable as a target for CMV infection; thus there is a tendency for CMV hepatitis to follow liver transplantation and for CMV pneumonitis to follow lung transplantation.

1	CMV viremia occurs in roughly one-third of hematopoietic stem cell transplant recipients; the risk of severe disease may be reduced by prophylaxis or preemptive therapy with antiviral drugs. The risk is greatest 5–13 weeks after transplantation, and identified risk factors include certain types of immunosuppressive therapy, an allogeneic (rather than an autologous) graft, acute graft-versus-host disease, older age, and pretransplantation recipient seropositivity.

1	CMV is an important pathogen in patients with advanced HIV infection (Chap. 226), in whom it may cause retinitis or disseminated disease, particularly when peripheral-blood CD4+ T cell counts fall below 50–100/μL. As treatment for underlying HIV infection has improved, the incidence of serious CMV infections (e.g., retinitis) has decreased. However, during the first few weeks after institution of highly active antiretroviral therapy, acute flare-ups of CMV retinitis may occur secondary to an immune reconstitution inflammatory syndrome.

1	Syndromes produced by CMV in immunocompromised hosts often begin with prolonged fatigue, fever, malaise, anorexia, night sweats, and arthralgias or myalgias. Liver function abnormalities, leukopenia, thrombocytopenia, and atypical lymphocytosis may be observed during these episodes. The development of tachypnea, hypoxemia, and unproductive cough signals respiratory involvement. Radiologic examination of the lung often shows bilateral interstitial or reticulonodular infiltrates that begin in the periphery of the lower lobes and spread centrally and superiorly; localized segmental, nodular, or alveolar patterns are less common. The differential diagnosis includes Pneumocystis infection; other viral, bacterial, or fungal infections; pulmonary hemorrhage; and injury secondary to irradiation or to treatment with cytotoxic drugs.

1	Gastrointestinal CMV involvement may be localized or extensive and almost exclusively affects immunocompromised hosts. Colitis is the most common clinical manifestation in organ transplant recipients. Ulcers of the esophagus, stomach, small intestine, or colon may result in bleeding or perforation. CMV infection may lead to exacerbations of underlying ulcerative colitis. Hepatitis occurs frequently, particularly after liver transplantation. Acalculous cholecystitis and adrenalitis also have been described.

1	CMV rarely causes meningoencephalitis in otherwise healthy individuals. Two forms of CMV encephalitis are seen in patients with AIDS. One resembles HIV encephalitis and presents as progressive dementia; the other is a ventriculoencephalitis characterized by cranial-nerve deficits, nystagmus, disorientation, lethargy, and ventriculomegaly. In immunocompromised patients, CMV can also cause subacute progressive polyradiculopathy, which is often reversible if recognized and treated promptly. CMV retinitis is an important cause of blindness in immunocompromised patients, particularly patients with advanced AIDS (Chap. 226). Early lesions consist of small, opaque, white areas of granular retinal necrosis that spread in a centrifugal manner and are later accompanied by hemorrhages, vessel sheathing, and retinal edema (Fig. 219-1). CMV retinopathy must be distinguished from that due to other conditions, including toxoplasmosis, candidiasis, and herpes simplex virus infection.

1	Fatal CMV infections are often associated with persistent viremia and the involvement of multiple organ systems. Progressive pulmonary infiltrates, pancytopenia, hyperamylasemia, and hypotension are characteristic features that are frequently found in conjunction with a terminal bacterial, fungal, or protozoan superinfection. Extensive adrenal necrosis with CMV inclusions is often documented at autopsy, as is CMV involvement of many other organs.

1	CMV infection usually cannot be diagnosed reliably on clinical grounds alone. Isolation of CMV or detection of its antigens or DNA in appropriate clinical specimens is the preferred approach. The most common method of detection is quantitative nucleic acid testing (QNAT) for CMV by polymerase chain reaction (PCR) technology, for which blood or other specimens can be used; some centers use a CMV antigenemia test, an immunofluorescence assay that detects CMV antigens (pp65) in peripheral-blood leukocytes. Such assays may yield a positive result several days earlier than culture methods. QNAT may predict the risk for disease progression, particularly in immunocompromised hosts. CMV DNA in cerebrospinal fluid is useful in the diagnosis of CMV encephalitis or polyradiculopathy. Considerable variation exists among assays and laboratories; a recently introduced international testing standard should help reduce variation in PCR test results.

1	FIGuRE 219-1 Cytomegalovirus infection in a patient with AIDS may appear as an arcuate zone of retinitis with hemorrhages and optic disk swelling. Often CMV is confined to the retinal periphery, beyond view of the direct ophthalmoscope.

1	Virus excretion or viremia is readily detected by culture of appropriate specimens on human fibroblast monolayers. If CMV titers are high, as is common in congenital disseminated infection and in AIDS, characteristic cytopathic effects may be detected within a few days. However, in some situations (e.g., CMV mononucleosis), viral titers are low, and cytopathic effects may take several weeks to appear. Many laboratories expedite diagnosis with an overnight tissue-culture method (shell vial assay) involving centrifugation and an immunocytochemical detection technique employing monoclonal antibodies to an immediate-early CMV antigen. Isolation of virus from urine or saliva does not, by itself, constitute proof of acute infection, since excretion from these sites may continue for months or years after illness. Detection of viremia is a better predictor of acute infection.

1	A variety of serologic assays detect antibody to CMV. An increased level of IgG antibody to CMV may not be detectable for up to 4 weeks after primary infection. Detection of CMV-specific IgM is sometimes useful in the diagnosis of recent or active infection; however, circulating rheumatoid factors may result in occasional false-positive IgM tests. Serology is especially helpful when used to predict risk of CMV infection and disease in transplant recipients.

1	Prevention of CMV in organ and hematopoietic stem cell transplant recipients is usually based on one of two methods: universal prophylaxis or preemptive therapy. With universal prophylaxis, antiviral drugs are used for a defined period, often 3 or 6 months. One clinical trial demonstrated that, in CMV-seronegative recipients with seropositive donors, prophylaxis was more effective at prevention when given for 200 days rather than 100 days. With preemptive therapy, patients are monitored weekly for CMV viremia, and antiviral treatment is initiated once viremia is detected. Because of the bone marrow–suppressive effects of universal prophylaxis, preemptive therapy is more commonly employed in hematopoietic stem cell transplant recipients. For patients with advanced HIV infection (CD4+ T cell counts of <50/μL), some experts have advocated prophylaxis with valganciclovir (see below). However, side effects, lack of proven benefit, possible induction of viral resistance, and high cost have

1	counts of <50/μL), some experts have advocated prophylaxis with valganciclovir (see below). However, side effects, lack of proven benefit, possible induction of viral resistance, and high cost have precluded the wide acceptance of this practice. Preemptive therapy is under study in HIV-infected patients.

1	Several additional measures are useful for the prevention of CMV transmission to CMV-naïve, high-risk patients. The use of CMVseronegative or leukocyte-depleted blood greatly decreases the rate of transfusion-associated transmission. In a placebo-controlled trial, a CMV glycoprotein B vaccine reduced infection rates among 464 CMV-seronegative women; this outcome raises the possibility that this experimental vaccine will reduce rates of congenital infection, but further studies must validate this approach. A CMV glycoprotein B vaccine with MF59 adjuvant appeared effective in reducing the risk and duration of viremia in both seropositive and seronegative renal transplant recipients at risk for CMV infection. CMV immune globulin has been reported to prevent congenital CMV infection in infants of women with primary infection during pregnancy. Studies in hematopoietic stem cell transplant recipients have produced conflicting results.

1	Prophylactic acyclovir or valacyclovir may reduce rates of CMV infection and disease in renal transplant recipients, although neither drug is effective in the treatment of active CMV disease.

1	Ganciclovir is a guanosine derivative that has considerably more activity against CMV than its congener acyclovir. After intracellular conversion by a viral phosphotransferase encoded by CMV gene region UL97, ganciclovir triphosphate is a selective inhibitor of CMV DNA polymerase. Several clinical studies have indicated response rates of 70–90% among patients with AIDS who are given ganciclovir for the treatment of CMV retinitis or colitis. In severe infections (e.g., CMV pneumonia in hematopoietic stem cell transplant recipients), ganciclovir is often combined with CMV immune globulin. Prophylactic or suppressive ganciclovir may be useful in high-risk hematopoietic stem cell or organ transplant recipients (e.g., those who are CMV-seropositive before transplantation). In many patients with AIDS, persistently low CD4+ T cell counts, and CMV disease, clinical and virologic relapses occur promptly if treatment with ganciclovir is discontinued. Therefore, prolonged maintenance regimens

1	AIDS, persistently low CD4+ T cell counts, and CMV disease, clinical and virologic relapses occur promptly if treatment with ganciclovir is discontinued. Therefore, prolonged maintenance regimens are recommended for such patients. Resistance to ganciclovir is more common among patients treated for >3 months and is usually related to mutations in the CMV UL97 gene (or, less commonly, the UL54 gene).

1	Valganciclovir is an orally bioavailable prodrug that is rapidly metabolized to ganciclovir in intestinal tissues and the liver. Approximately 60–70% of an oral dose of valganciclovir is absorbed. An oral valganciclovir dose of 900 mg results in ganciclovir blood levels similar to those obtained with an IV ganciclovir dose of 5 mg/kg. Valganciclovir appears to be as effective as IV ganciclovir for both CMV induction (treatment) and maintenance regimens, while providing the ease of oral dosing. Furthermore, the adverse-event profiles and rates of resistance for the two drugs are similar.

1	Ganciclovir or valganciclovir therapy for CMV disease consists of a 14-to 21-day induction course (5 mg/kg IV twice daily for ganciclovir or 900 mg PO twice daily for valganciclovir), sometimes followed by maintenance therapy (e.g., valganciclovir, 900 mg/d). Peripheral-blood neutropenia develops in roughly one-quarter of treated patients but may be ameliorated by granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor. Whether to use maintenance therapy should depend on the overall level of immunocompromise and the risk of recurrent disease. Discontinuation of maintenance therapy should be considered in patients with AIDS who, while receiving antiretroviral therapy, have a sustained (3to 6-month) increase in CD4+ T cell counts to >100/μL.

1	For treatment of CMV retinitis, ganciclovir may also be administered via a slow-release pellet sutured into the eye. Although this intraocular device provides good local protection, contralateral eye disease and disseminated disease are not affected, and early retinal detachment is possible. A combination of intraocular and systemic therapy may be better than the intraocular implant alone.

1	Foscarnet (sodium phosphonoformate) inhibits CMV DNA polymerase. Because this agent does not require phosphorylation to be active, it is also effective against most ganciclovir-resistant isolates. Foscarnet is less well tolerated than ganciclovir and causes considerable toxicity, including renal dysfunction, hypomagnesemia, hypokalemia, hypocalcemia, genital ulcers, dysuria, nausea, and paresthesia. Moreover, foscarnet administration requires the use of an infusion pump and close clinical monitoring. With aggressive hydration and dose adjustments for renal dysfunction, the toxicity of foscarnet can be reduced. The use of foscarnet should be avoided when a saline load cannot be tolerated (e.g., in cardiomyopathy). The approved induction regimen is 60 mg/kg every 8 h for 2 weeks, although 90 mg/kg every 12 h is equally effective and no more toxic. Maintenance infusions should deliver 90–120 mg/ kg once daily. No oral preparation is available. Foscarnet-resistant virus may emerge during

1	mg/kg every 12 h is equally effective and no more toxic. Maintenance infusions should deliver 90–120 mg/ kg once daily. No oral preparation is available. Foscarnet-resistant virus may emerge during extended therapy. This drug is used more frequently after hematopoietic stem cell transplantation than in other situations to avoid the myelosuppressive effects of ganciclovir; in

1	Cytomegalovirus and Human Herpesvirus Types 6, 7, and 8 11 general, foscarnet is also the first choice for infections with ganciclovir-resistant CM Cidofovir is a nucleotide analogue with a long intracellular half-life that allows intermittent Iadministration. Induction regimens of 5 mgkg weekly for 2 weeks are followed by maintenance regimens of –5 mgkg every 2 weeks. Cidofovir can cause severe nephrotoxicity through dose-dependent proximal tubular cell injuryhowever, this adverse effect can be tempered somewhat by saline hydration and probenecid. Cidofovir is used primarily for ganciclovir-resistant virus. HUMAN HERPESVIRUS (HHV) TYPES 6, 7, ANd 8

1	HUMAN HERPESVIRUS (HHV) TYPES 6, 7, ANd 8 HHV-6 and -7 seropositivity rates are generally high throughout the world. HHV-6 was first isolated in 1986 from peripheral- blood leukocytes of six persons with various lymphoproliferative disorders. Two genetically distinct variants (HHV-6A and HHV-6B) are now recognized. HHV-6 appears to be transmitted by saliva and possibly by genital secretions.

1	Infection with HHV-6 frequently occurs during infancy as maternal antibody wanes. The peak age of acquisition is 9–21 months; by 24 months, seropositivity rates approach 80%. Older siblings appear to serve as a source of transmission. Congenital infection also may occur, and 1% of newborns are infected with HHV-6; placental infection with HHV-6 has been described. Most postnatally infected children develop symptoms (fever, fussiness, and diarrhea). A minority develop exanthem subitum (roseola infantum; see Fig. 25e-5), a common illness characterized by fever with subsequent rash. In addition, ~10–20% of febrile seizures without rash during infancy are caused by HHV-6. After initial infection, HHV-6 persists in peripheral-blood mononuclear cells as well as in the central nervous system, salivary glands, and female genital tract.

1	In older age groups, HHV-6 has been associated with mononucleosis syndromes; in immunocompromised hosts, encephalitis, pneumonitis, syncytial giant-cell hepatitis, and disseminated disease are seen. In transplant recipients, HHV-6 infection may also be associated with graft dysfunction. Acute HHV-6-associated limbic encephalitis has been reported in hematopoietic stem cell transplant recipients and is characterized by memory loss, confusion, seizures, hyponatremia, and abnormal electroencephalographic and MRI results. High plasma loads of HHV-6 DNA in hematopoietic stem cell transplant recipients are associated with allelic-mismatched donors, use of glucocorticoids, delayed monocyte and platelet engraftment, development of limbic encephalitis, and increased all-cause mortality rates. Like many other viruses, HHV-6 has been implicated in the pathogenesis of multiple sclerosis, although further study is needed to distinguish between association and etiology.

1	HHV-7 was isolated in 1990 from T lymphocytes from the peripheral blood of a healthy 26-year-old man. The virus is frequently acquired during childhood, albeit at a later age than HHV-6. HHV-7 is commonly present in saliva, which is presumed to be the principal source of infection; breast milk also can carry the virus. Viremia can be associated with either primary or reactivation infection. The most common clinical manifestations of childhood HHV-7 infections are fever and seizures. Some children present with respiratory or gastrointestinal signs and symptoms. An association has been made between HHV-7 and pityriasis rosea, but evidence is insufficient to indicate a causal relationship.

1	Clustering of HHV-6, HHV-7, and CMV infections in transplant recipients can make it difficult to sort out the roles of the various agents in individual clinical syndromes. HHV-6 and HHV-7 appear to be susceptible to ganciclovir and foscarnet, although definitive evidence of clinical response is lacking.

1	Unique herpesvirus-like DNA sequences were reported during 1994 and 1995 in tissues derived from Kaposi’s sarcoma (KS) and body cavity–based lymphoma occurring in patients with AIDS. The virus from which these sequences were derived is designated HHV-8 or Kaposi’s sarcoma–associated herpesvirus (KSHV). HHV-8, which infects B lymphocytes, macrophages, and both endothelial and epithelial cells, appears to be causally related not only to KS and a subgroup of AIDS-related B cell body cavity–based lymphomas (primary effusion lymphomas) but also to multicentric Castleman’s disease, a lymphoproliferative disorder of B cells. The association of HHV-8 with several other diseases has been reported but not confirmed.

1	HHV-8 seropositivity occurs worldwide, with areas of high endemicity influencing rates of disease. Unlike other herpes- virus infections, HHV-8 infection is much more common in some geographic areas (e.g., central and southern Africa) than in others (North America, Asia, northern Europe). In high-prevalence areas, infection occurs in childhood, seropositivity is associated with having a seropositive mother or (to a lesser extent) older sibling, and HHV-8 may be transmitted in saliva. In low-prevalence areas, infections typically occur in adults, probably with sexual transmission. Concurrent epidemics of HIV-1 and HHV-8 infections among certain populations (e.g., men who have sex with men) in the late 1970s and early 1980s appear to have resulted in the frequent association of AIDS and KS. Transmission of HHV-8 may also be associated with organ transplantation, injection drug use, and blood transfusion; however, transmission via blood transfusion in the United States appears to be

1	KS. Transmission of HHV-8 may also be associated with organ transplantation, injection drug use, and blood transfusion; however, transmission via blood transfusion in the United States appears to be quite rare.

1	Primary HHV-8 infection in immunocompetent children may manifest as fever and maculopapular rash. Among individuals with intact immunity, chronic asymptomatic infection is the rule, and neoplastic disorders generally develop only after subsequent immunocompromise. Immunocompromised persons with primary infection may present with fever, splenomegaly, lymphoid hyperplasia, pancytopenia, or rapid-onset KS. uantitative analysis of HHV-8 DNA suggests a predominance of latently infected cells in KS lesions and frequent lytic replication in multicentric Castleman’s disease.

1	Effective antiretroviral therapy for HIV-infected individuals has led to a marked reduction in rates of KS among persons dually infected with HHV-8 and HIV in resource-rich areas. HHV-8 itself is susceptible in vitro to ganciclovir, foscarnet, and cidofovir. A small, randomized, double-blind, placebo-controlled, crossover trial suggested that oral valganciclovir administered once daily reduced HHV-8 replication. However, clinical benefits of valganciclovir or other drugs in HHV-8 infection have not yet been demonstrated. Sirolimus has been shown to inhibit the progression of dermal KS in kidney transplant recipients while providing effective immunosuppression.

1	Molluscum Contagiosum, Monkeypox, and Other Poxvirus Infections Fred Wang The poxvirus family includes a large number of related DNA viruses 220e that infect various vertebrate hosts. The poxviruses responsible for infections in humans, the geographic locations in which these infections are found, the host reservoirs, and the main manifestations are listed in Table 220e-1. Infections with orthopoxviruses—e.g., smallpox (variola major) virus (Chap. 261e) or the zoonotic monkeypox virus—can result in systemic, potentially lethal human disease. Other poxvirus infections cause primarily localized skin disease in humans.

1	Molluscum contagiosum virus is an obligate human pathogen that causes distinctive proliferative skin lesions. These lesions measure 2–5 mm in diameter and are pearly, flesh-colored, and umbilicated, with a characteristic dimple at the center (Fig. 220e-1). A relative lack of inflammation and necrosis distinguishes these proliferative lesions from other poxvirus lesions. Lesions may be found—singly or in clusters—anywhere on the body except on the palms and soles and may be associated with an eczematous rash.

1	Molluscum contagiosum is highly prevalent among children and is the most common human disease resulting from poxvirus infection. Swimming pools are a common vector for transmission. Atopy and compromise of skin integrity increase the risk of infection. Genital lesions are more common in adults, to whom the virus may be transmitted by sexual contact. The incubation period ranges from 2 weeks to 6 months, with an average of 2–7 weeks. In most cases, the disease is self-limited and regresses spontaneously after 3–4 months in immunocompetent hosts. There are no systemic complications, but skin lesions may persist for 3–5 years. Molluscum contagiosum can be associated with immunosuppression and is frequently seen among HIV-infected patients (Chap. 226). The disease can be more generalized, severe, and persistent in AIDS patients than in other groups. Moreover, molluscum contagiosum can be exacerbated in the immune reconstitution inflammatory syndrome (IRIS) associated with the initiation

1	and persistent in AIDS patients than in other groups. Moreover, molluscum contagiosum can be exacerbated in the immune reconstitution inflammatory syndrome (IRIS) associated with the initiation of antiretroviral therapy.

1	The diagnosis of molluscum contagiosum is typically based on its clinical presentation and can be confirmed by histologic demonstration of the cytoplasmic eosinophilic inclusions (molluscum bodies) that are characteristic of poxvirus replication. Molluscum contagiosum FIGUrE 220e-1 Molluscum contagiosum is a cutaneous poxvirus infection characterized by multiple umbilicated flesh-colored or hypopigmented papules. virus cannot be propagated in vitro, but electron microscopy and molecular studies can be used for its identification. There is no specific systemic treatment for molluscum contagiosum, but a variety of techniques for physical ablation have been used. Cidofovir displays in vitro activity against many poxviruses, and case reports suggest that parenteral or topical cidofovir may have some efficacy in the treatment of recalcitrant molluscum contagiosum in immunosuppressed hosts.

1	Although monkeypox virus was named after the animal from which it was originally isolated, rodents are the primary viral reservoir. Human infections with monkeypox virus typically occur in Africa when humans come into direct contact with infected animals. Human-to-human propagation of monkeypox infection is rare. Human disease is characterized by a systemic illness and vesicular rash similar to those of variola. The clinical presentation of monkey-pox can be confused with that of the more common varicella-zoster virus infection (Chap. 217). Compared with the lesions of this herpes-virus infection, monkeypox lesions tend to be more uniform (i.e., in the same stage of development), diffuse, and peripheral in distribution. Lymphadenopathy is a prominent feature of monkeypox infection.

1	The first outbreak of human monkeypox infection in the Western Hemisphere occurred during 2003, when more than 70 cases were reported in the midwestern United States. The outbreak was linked to contact with pet prairie dogs that had become infected while being housed with rodents imported from Ghana. Patients presented most CHAPTER 220e Molluscum Contagiosum, Monkeypox, and Other Poxvirus Infections frequently with fever, rash, and lymphadenopathy ~12 days after exposure. Nine patients were hospitalized, but there were no deaths. Smallpox vaccination can provide cross-reactive immunity to monkeypox infection; studies of people exposed in the outbreak detected subclinical infection in a few vaccinated individuals—an observation suggesting the possibility of long-term vaccine protection. The risk of human disease from animal orthopoxvirus infections may increase as smallpox immunity wanes in the general population and the popularity of exotic animals as household pets grows.

1	Cowpox and buffalopox are rare zoonotic infections characterized by cutaneous poxlike lesions and mild systemic illness. Outbreaks of similar poxlike lesions among cattle and farm workers in Brazil have been due to Cantagalo and Araçatuba viruses, which are virtually identical to vaccinia virus and may have become established in cattle during smallpox vaccination programs. Parapoxviruses are widely scattered among animal species, but only a few are known to cause human disease via direct contact with infected animals. Parapoxviruses are antigenically distinct from orthopoxviruses and share no cross-immunity. Tanapox virus belongs to a separate, antigenically distinct genus and usually causes a single nodular lesion on the exposed area after contact with infected monkeys.

1	Parvovirus Infections Kevin E. Brown Parvoviruses, members of the family Parvoviridae, are small (diameter, ~22 nm), nonenveloped, icosahedral viruses with a linear single-strand DNA genome of ~5000 nucleotides. These viruses are dependent on either rapidly dividing host cells or helper viruses for replication. At 221 women, the estimated annual seroconversion rate is ~1%. Within 1195 households, secondary infection rates approach 50%. Detection of high-titer B19V in blood is not unusual (see “Pathogenesis,” below). Transmission can occur as a result of transfusion, most commonly of pooled components. To reduce the risk of transmission, plasma pools are screened by nucleic acid amplification technology, and high-titer pools are discarded. B19V is resistant to both heat and solvent-detergent inactivation.

1	least four groups of parvoviruses infect humans: parvovirus B19 (B19V), dependoparvoviruses (adeno-associated viruses; AAVs), PARV4/5 virus, and human bocaviruses (HBoVs). Human dependoparvoviruses are nonpathogenic and will not be considered further in this chapter. B19V is the type member of the genus Erythroparvovirus. On the basis of viral sequence, B19V is divided into three genotypes (designated 1, 2, and 3), but only a single B19V antigenic type has been described. Genotype 1 is predominant in most parts of the world; genotype 2 is rarely associated with active infection; and genotype 3 appears to predominate in parts of western Africa. B19V exclusively infects humans, and infection is endemic in virtually all parts of the world. Transmission occurs predominantly via the respiratory route and is followed by the onset of rash and arthralgia. By the age of 15 years, ~ 50% of children have detectable IgG; this figure rises to >90% among the elderly. In pregnant

1	B19V replicates primarily in erythroid progenitors. This specificity is due in part to the limited tissue distribution of the primary B19V receptor, blood group P antigen (globoside). Infection leads to high-titer viremia, with >1012 virus particles (or IU)/mL detectable in the blood at the apex (Fig. 221-1), and virus-induced cytotoxicity results in cessation of red cell production. In immunocompetent individuals, viremia and arrest of erythropoiesis are transient and resolve as the IgM and IgG antibody response is mounted. In individuals with normal erythropoiesis, there is only a minimal drop in hemoglobin levels; however, in those with increased erythropoiesis (especially with hemolytic anemia), this cessation of red cell production can induce a transient crisis with severe anemia (Fig. 221-1). Similarly, if an individual (or, after maternal infection, a fetus) does not mount a neutralizing antibody response and halt the lytic infection, erythroid production is compromised and

1	(Fig. 221-1). Similarly, if an individual (or, after maternal infection, a fetus) does not mount a neutralizing antibody response and halt the lytic infection, erythroid production is compromised and chronic anemia develops (Fig. 221-1).

1	The immune-mediated phase of illness, which begins 2–3 weeks after infection as the IgM response peaks, manifests as the rash of fifth disease together with arthralgia and/or frank arthritis. Low-level B19V DNA can be detected by polymerase chain reaction (PCR) in blood and tissues for months to years after acute infection. The B19V 1.0 0.2 manifestations Fever, chills, headache, myalgia Rash, arthralgia FIGuRE 221-1 Schematic of the time course of parvovirus B19V infection in (A) normals (erythema infectiosum), (B) transient aplastic crisis (TAC), and (C) chronic anemia/pure red-cell aplasia (PRCA). (Reprinted with permission from NS Young, KE Brown: N Engl J Med 350:586, 2004. © 2004 Massachusetts Medical Society. All rights reserved.) FIGuRE 221-2 Young child with erythema infectiosum, or fifth dis-ease, showing typical “slapped-cheek” appearance.

1	FIGuRE 221-2 Young child with erythema infectiosum, or fifth dis-ease, showing typical “slapped-cheek” appearance. receptor is found in a variety of other cells and tissues, including megakaryocytes, endothelial cells, placenta, myocardium, and liver. Infection of these tissues by B19V may be responsible for some of the more unusual presentations of the infection. Rare individuals who lack P antigen are naturally resistant to B19V infection.

1	CLINICAL MANIFESTATIONS Erythema Infectiosum Most B19V infections are asymptomatic or are associated with only a mild nonspecific illness. The main manifestation of symptomatic B19V infection is erythema infectiosum, also known as fifth disease or slapped-cheek disease (Fig. 221-2 and Fig. 25e-1A). Infection begins with a minor febrile prodrome ~7–10 days after exposure, and the classic facial rash develops several days later; after 2–3 days, the erythematous macular rash may spread to the extremities in a lacy reticular pattern. However, its intensity and distribution vary, and B19V-induced rash is difficult to distinguish from other viral exanthems. Adults typically do not exhibit the “slapped-cheek” phenomenon but present with arthralgia, with or without the macular rash.

1	Polyarthropathy Syndrome Although uncommon among children, arthropathy occurs in ~50% of adults and is more common among women than among men. The distribution of the affected joints is often symmetrical, with arthralgia affecting the small joints of the hands and occasionally the ankles, knees, and wrists. Resolution usually occurs within a few weeks, but recurring symptoms can continue for months. The illness may mimic rheumatoid arthritis, and rheumatoid factor can often be detected in serum. B19V infection may trigger rheumatoid disease in some patients and has been associated with juvenile idiopathic arthritis.

1	Transient Aplastic Crisis Asymptomatic transient reticulocytopenia occurs in most individuals with B19V infection. However, in patients who depend on continual rapid production of red cells, infection can cause transient aplastic crisis (TAC). Affected individuals include those with hemolytic disorders, hemoglobinopathies, red cell enzymopathies, and autoimmune hemolytic anemias. Patients present with symptoms of severe anemia (sometimes life-threatening) and a low reticulocyte count, and bone marrow examination reveals an absence of erythroid precursors and characteristic giant pronormoblasts. As its name indicates, the illness is transient, and anemia resolves with the cessation of cytopathic infection in the erythroid progenitors.

1	Pure Red-Cell Aplasia/Chronic Anemia Chronic B19V infection has been reported in a wide range of immunosuppressed patients, including those with congenital immunodeficiency, AIDS (Chap. 226), lymphoproliferative disorders (especially acute lymphocytic leukemia), and transplantation (Chap. 169). Patients have persistent anemia with reticulocytopenia, absent or low levels of B19V IgG, high titers of B19V DNA in serum, and—in many cases—scattered giant pronormoblasts in bone marrow. Rarely, nonerythroid hematologic lineages are also affected. Transient neutropenia, lymphopenia, and thrombocytopenia (including idiopathic thrombocytopenic purpura) have been observed. B19V occasionally causes a hemophagocytic syndrome.

1	Recent studies in Papua New Guinea and Ghana, where malaria is endemic, suggest that co-infection with Plasmodium and B19V plays a major role in the development of severe anemia in young children. Further studies must determine whether B19V infection contributes to severe anemia in other malarial regions. Hydrops Fetalis B19V infection during pregnancy can lead to hydrops fetalis and/or fetal loss. The risk of transplacental fetal infection is ~30%, and the risk of fetal loss (predominantly early in the second trimester) is ~9%. The risk of congenital infection is <1%. Although B19V does not appear to be teratogenic, anecdotal cases of eye damage and central nervous system (CNS) abnormalities have been reported. Cases of congenital anemia have also been described. B19V probably causes 10–20% of all cases of nonimmune hydrops.

1	unusual Manifestations B19V infection may rarely cause hepatitis, vasculitis, myocarditis, glomerulosclerosis, or meningitis. A variety of other cardiac manifestations, CNS diseases, and autoimmune infections have also been reported. However, B19V DNA can be detected by PCR for years in many tissues; this finding is of no known clinical significance, but its interpretation may cause confusion regarding B19V disease association. Diagnosis of B19V infection in immunocompetent individuals is generally based on detection of B19V IgM antibodies (Table 221-1). IgM can be detected at the time of rash in erythema infectiosum and Abbreviations: IU, international units (1 IU equals ~1 genome); n/a, not applicable; PCR, polymerase chain reaction.

1	Abbreviations: IU, international units (1 IU equals ~1 genome); n/a, not applicable; PCR, polymerase chain reaction. by the third day of TAC in patients with hematologic disorders; these antibodies remain detectable for ~3 months. B19V IgG is detectable by the seventh day of illness and persists throughout life. Quantitative detection of B19V DNA should be used for the diagnosis of early TAC or chronic anemia. Although B19V levels fall rapidly with the development of the immune response, DNA can be detectable by PCR for months or even years after infection, even in healthy individuals; therefore, quantitative PCR should be used. In acute infection at the height of viremia, >1012 B19V DNA IU/mL of serum can be detected; however, titers fall rapidly within 2 days. Patients with aplastic crisis or B19V-induced chronic anemia generally have >105 B19V DNA IU/mL.

1	No antiviral drug effective against B19V is available, and treatment of B19V infection often targets symptoms only. TAC precipitated by B19V infection frequently necessitates symptom-based treatment with blood transfusions. In patients receiving chemotherapy, temporary cessation of treatment may result in an immune response and resolution. If this approach is unsuccessful or not applicable, commercial immune globulin (IVIg; Gammagard, Sandoglobulin) from healthy blood donors can cure or ameliorate persistent B19V infection in immunosuppressed patients. Generally, the dose used is 400 mg/kg daily for 5–10 days. Like patients with TAC, immunosuppressed patients with persistent B19V infection should be considered infectious. Administration of IVIg is not beneficial for erythema infectiosum or B19V-associated polyarthropathy. Intrauterine blood transfusion can prevent fetal loss in some cases of fetal hydrops.

1	No vaccine has been approved for the prevention of B19V infection, although vaccines based on B19V virus-like particles expressed in insect cells are known to be highly immunogenic. Phase 1 trials of a putative vaccine were discontinued because of adverse side effects. The PARV4 viral sequence was initially detected in a patient with an acute viral syndrome. Similar sequences, including the related PARV5 sequence, have been detected in pooled plasma collections. The DNA sequence of PARV4/5 is distinctly different from that of all other parvoviruses, and this virus is now classified as a member of the newly described genus Tetraparvovirus.

1	PARV4 DNA is commonly found in plasma pools but at lower concentrations than levels of B19V DNA found before in plasma pools prior to screening. The higher levels of PARV4 DNA and IgG antibody in tissues (bone marrow and lymphoid tissue) and sera from IV drug users than in the corresponding specimens from control patients suggest that the virus is transmitted predominantly by parenteral means in the United States and Europe. Evidence for non-parenteral transmission in other parts of the world is limited. To date, PARV4/5 infection has been associated only with mild clinical disease (rash and/or transient aminotransferase elevation). Animal bocaviruses are associated with mild respiratory symptoms and enteritis in young animals. HBoV1 was originally identified in the respiratory tract of young children with lower respiratory tract infections. More recently, HBoV1 and the related viruses HBoV2, HBoV3, and HBoV4 have all been identified in human fecal samples.

1	Seroepidemiologic studies with HBoV virus-like particles suggest that human bocavirus infection is common. Worldwide, most individuals are infected before the age of 5 years. HBoV1 DNA is found in respiratory secretions from 2–20% of children with acute respiratory infection, often in the presence of other pathogens; in these circumstances, the role of HBoV1 in disease pathogenesis is unknown. Clinical disease due to HBoV1 is associated with evidence of primary infection (IgG seroconversion or the presence of IgM), HBoV1 DNA in serum, or high-titer HBoV1 DNA (>104 genome copies/mL) in respiratory secretions. Symptoms are not dissimilar from those of other viral respiratory infections, and cough and wheezing are commonly reported. There is no specific treatment for bocavirus infection. The role of human bocaviruses in childhood gastroenteritis remains to be established. Aaron C. Ermel, Darron R. Brown

1	Aaron C. Ermel, Darron R. Brown Investigation of human papillomavirus (HPV) infection began in earnest in the 1980s after Harold zur Hausen postulated that infection with these viruses was associated with cervical cancer. It is now recognized that HPV infection of the human genital tract is extremely common and causes clinical states ranging from asymptomatic infection to genital warts (condylomata acuminata); dysplastic lesions or invasive cancers of the anus, penis, vulva, vagina, and cervix; and a subset of oropharyngeal cancers. This chapter describes the epidemiology of HPV in general and as a pathogen, the natural history of HPV infections and associated cancers, strategies to prevent HPV infection and HPV-associated disease, and treatment modalities.

1	Molecular Overview HPV is an icosahedral, nonenveloped, 8000-base-pair, double-stranded DNA virus with a diameter of 55 nm. Like those of other papillomaviruses, HPV’s genome consists of an early (E) gene region, a late (L) gene region, and a noncoding region that contains regulatory elements. The E1, E2, E5, E6, and E7 proteins are expressed early in the growth cycle and are necessary for viral replication and cellular transformation. The E6 and E7 proteins cause malignant transformation by targeting the human cell cycle– regulatory molecules p53 and Rb (retinoblastoma protein), respectively, for degradation. Translation of the L1 and L2 transcripts and splicing of an E1^E4 transcript occur later. The L1 gene encodes the 54-kDa major capsid protein that makes up the majority of the virus shell; the 77-kDa L2 minor protein constitutes a smaller percentage of the capsid mass.

1	More than 125 HPV types have been identified and are numerically designated according to a unique L1 gene sequence. Approximately 40 HPV types are regularly found in the anogenital tract and are subdivided into high-risk and low-risk categories on the basis of the associated risk of cervical cancer. For example, HPV-6 and HPV-11 cause genital warts and ~10% of low-grade cervical lesions and are thus designated low risk. HPV-16 and HPV-18 cause dysplastic lesions and invasive cancers of the cervix and are considered high risk. HPV targets basal keratinocytes after microtrauma has exposed these cells to the virus. The HPV replication cycle is completed as keratinocytes undergo differentiation. Virions are assembled in the nuclei of differentiated keratinocytes and can be detected by electron microscopy. Infection is transmitted by contact with virus contained in these desquamated keratinocytes (or with free virus) from an infected individual.

1	1198 Immune Response to HPV Infection A cell-mediated immune response plays an important role in controlling the progression of natural HPV infection. Histologic examination of lesions in individuals who experience regression of genital warts demonstrates infiltration of T cells and macrophages. CD4+ T cell regulation is particularly important in controlling HPV infections, as evidenced by the higher rates of infection and disease among immunosuppressed individuals, particularly those who are infected with HIV. Specific T-cell responses may be measured against HPV proteins, the most important of which appear to be the E2 and E6 proteins. In women with HPV-16 cervical infection, a strong T-cell response to HPV-16-derived E2 protein is associated with a lack of progression of cervical disease. Natural HPV infection of the genital tract gives rise to a serum antibody response in only 60–70% of individuals because there is no viremic phase during infection. Significant, although

1	disease. Natural HPV infection of the genital tract gives rise to a serum antibody response in only 60–70% of individuals because there is no viremic phase during infection. Significant, although incomplete, protection against type-specific reinfection is associated with the presence of neutralizing antibodies. Serum antibodies likely reach the cervical epithelium and secretions by transudation and exudation. Therefore, protection against infection is related to the amount of neutralizing antibody at the site of infection and lasts as long as levels of neutralizing antibodies are sufficient.

1	EPIDEMIOLOGY AND NATuRAL HISTORY OF HPV-ASSOCIATED MALIGNANCY General Population HPV is transmitted by sexual intercourse, by oral sex, and possibly by touching of a partner’s genitalia. In cross-sectional and longitudinal studies, ~40% of young women have evidence of HPV infection, with peaks during the teens and early twenties—soon after first coitus. The number of lifetime sexual partners correlates with the likelihood of HPV infection and the subsequent risk of HPV-associated malignancy. HPV infection may develop in a monogamous person whose partner is infected. Most HPV infections become undetectable after 6–9 months. However, with prolonged follow-up and frequent sampling, the same HPV types may again be detected weeks or months after becoming undetectable. Whether such episodic detection indicates viral latency followed by reactivation or reinfection with an identical HPV type is still debated.

1	Although HPV is the causative agent of several cancers, most attention has focused on cervical cancer—the second most common cancer among women worldwide, which affects more than 500,000 women and kills more than 275,000 women annually. More than 85% of all cervical cancer cases and deaths occur in women living in low-income countries, especially in sub-Saharan Africa, Asia, and South and Central America. A quarter-century of evidence shows that HPV causes nearly 100% of cervical cancers. HPV infection is the most significant risk factor for cervical cancer; relative risks range from 10 to 20 and exceed 100 in prospective and case-control studies, respectively. The time from HPV infection to cervical cancer diagnosis may exceed 20 years. Cervical cancer peaks in the fifth and sixth decades of life among women living in developed countries but as much as a decade earlier among women living in resource-poor countries. Persistent carriers of oncogenic HPV types are at greatest risk for

1	of life among women living in developed countries but as much as a decade earlier among women living in resource-poor countries. Persistent carriers of oncogenic HPV types are at greatest risk for high-grade cervical dysplasia and cancer. Why only certain HPV infections eventually lead to malignancy is not clear. Biomarkers that can predict which women will develop cervical cancer are not available. Immunosuppression in general plays a significant role in re-detection/reactivation of HPV infections, while other factors such as smoking, hormonal changes, Chlamydia infection, and nutritional deficits promote viral persistence and cancer.

1	The International Agency for Research on Cancer concludes that HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 are carcinogenic in the uterine cervix. HPV-16 is particularly virulent and causes 50% of cervical cancers. Worldwide, HPV-16 and HPV-18 cause 70% of cervical squamous cell carcinomas and 85% of cervical adenocarcinomas. Oncogenic types other than HPV-16 and HPV-18 cause the remaining 30% of cervical cancers. HPV-16 and HPV-18 also cause nearly 90% of anal cancers worldwide. Although oncogenic HPV infection is necessary for the development of cervical malignancy, only ~3–5% of infected women will ever develop this cancer, even in the absence of cytologic screening.

1	In addition to cervical and anal cancer, other HPV-associated cancers include vulvar and vaginal cancer, which are associated with HPV in 50–70% of cases; penile cancer, which is caused by HPV in 50% of cases; and oropharyngeal squamous cell carcinoma (OPSCC). Over the past two decades, an epidemic of OPSCC related to oncogenic infection with HPV (primarily HPV-16) has developed. Annual rates of OPSCC among men in the United States have been increasing from a low of 0.27 case/100,000 in 1973 to 0.57 case/100,000 as of 2004; rates in women have remained relatively stable at ~0.17 case/100,000 per year. The increase in the incidence of OPSCC is greatest among white men 40–50 years of age. Nearly 14,000 new cases were diagnosed in the United States in 2013. Annual rates of OPSCCs of the base of the tongue and the tonsil have increased dramatically—i.e., by 1.3% and 0.6%, respectively. Fewer data are available from developing countries about OPSCCs.

1	Effects of HIV on HPV-Associated Disease HIV infection accelerates the natural progression of HPV infections. HIV-infected persons are more likely than other individuals to develop genital warts and to have lesions that are more recalcitrant to treatment. HIV infection has been consistently associated with precancerous cervical lesions, including low-grade cervical intraepithelial neoplasia (CIN) and CIN 3, the immediate precursor to cervical cancer. Women with HIV/AIDS have significantly higher rates of cervical cancer and of subsets of some vulvar, vaginal, and oropharyngeal tumors than women in the general population. Studies indicate a direct relation between low CD4+ T lymphocyte counts and the risk of cervical cancer. Some studies show a reduced likelihood of HPV infection and precancerous lesions of the cervix in HIV-infected women receiving antiretroviral therapy (ART). The incidence of cervical cancer among HIV-infected women has not changed significantly since ART was

1	precancerous lesions of the cervix in HIV-infected women receiving antiretroviral therapy (ART). The incidence of cervical cancer among HIV-infected women has not changed significantly since ART was introduced, possibly because of preexisting oncogenic HPV infections.

1	The burden of HPV-related cancers is expected to increase in HIV-infected patients, given the prolonged life expectancies made possible by ART. For women living in developing countries where cervical cancer screening is not widely available, this situation may have significant consequences. Thus, elucidating the interactions of HIV infection and cervical cancer with cofactors such as diet, other sexually transmitted infections, and environmental exposures is a research focus with potentially enormous implications for women living in lowand middle-income countries.

1	Similar to that of cervical cancer, the incidence of anal cancer is strongly influenced by HIV infection. HIV-infected men who have sex with men (MSM) and HIV-infected women have much higher rates of anal cancer than HIV-uninfected populations. Specifically, the incidence has been found to be as high as 130 cases/100,000 among HIV-positive MSM as opposed to only 5 cases/100,000 among HIV-negative MSM. The advent of ART has not impacted the incidence of anal cancer and high-grade anal intraepithelial neoplasia in the HIV-infected population. More information on screening, prevention, and treatment in the HIV-infected population can be found at the Department of Health and Human Services website (aidsinfo.nih.gov/guidelines).

1	HPV infects the female vulva, vagina, and cervix and the male urethra, penis, and scrotum. Perianal, anal, and oropharyngeal infections occur in both genders. Figures 222-1, 222-2, and 222-3 show vulvar, penile, and perianal warts, respectively. Genital warts are caused primarily by HPV-6 or HPV-11; their surface is either smooth or rough. Penile genital warts are usually 2–5 mm in diameter and often occur in groups. A second type of penile lesion, keratotic plaques, is slightly raised above the normal epithelium and has a rough, often pigmented surface. Vulvar warts are soft, whitish papules that either are sessile or have multiple fine, finger-like projections. These lesions are most often located in the introitus and on the labia. In nonmucosal areas, lesions are similar in appearance to those in men: dry and keratotic. Vulvar lesions can appear as smooth, sometimes pigmented papules that may coalesce. Vaginal lesions appear as multiple areas of elongated papillae. Biopsy of vulvar

1	those in men: dry and keratotic. Vulvar lesions can appear as smooth, sometimes pigmented papules that may coalesce. Vaginal lesions appear as multiple areas of elongated papillae. Biopsy of vulvar or vaginal lesions may reveal malignancy, which is not always reliably identified by clinical examination.

1	FIGuRE 222-1 Vulvar warts. (Downloaded from http://www2a.cdc .gov/stdtraining/ready-to-use/Manuals/HPV/hpv-slides-2013.pdf.) Subclinical cervical HPV infections are common, and the cervix may appear normal on examination. Cervical lesions often appear as papillary proliferations near the transformation zone. Irregular vascular loops are present beneath the surface epithelium. Patients who develop cervical cancer arising from HPV infection may present with a variety of symptoms. Early carcinomas appear eroded and bleed easily. More advanced carcinomas present as ulcerated lesions or as an exophytic cervical mass. Some cervical carcinomas are located in the cervical canal and may be difficult to see. Bleeding, symptoms of a mass lesion in late stages, and metastatic disease that may manifest as bowel or bladder obstruction due to direct extension of the tumor have also been described. FIGuRE 222-2 Condyloma acuminata of the shaft of the penis.

1	FIGuRE 222-2 Condyloma acuminata of the shaft of the penis. FIGuRE 222-3 Perianal warts. (Reprinted from K Wolff, RA Johnson, AP Saavedra: Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 7th ed. New York, McGraw-Hill, 2013.) Patients with squamous cell cancer of the anus have more variable presentations. The most common presentations include rectal bleeding and pain or a mass sensation. Of patients who are diagnosed with anal cancer, 20% may present with no specific symptoms at the time of diagnosis; rather, the lesion is found fortuitously. PREVENTION OF HPV INFECTION: HPV VACCINES

1	PREVENTION OF HPV INFECTION: HPV VACCINES Vaccines effective in preventing HPV infection and HPV-associated disease represent a major development in the last decade. The vaccines use virus-like particles (VLPs) that consist of the HPV L1 major capsid protein. The L1 protein self-assembles into VLPs when expressed in eukaryotic cells (i.e., yeast for the Merck vaccine or insect cells for the GlaxoSmithKline vaccine; see below). These VLPs contain the same epitopes as the HPV virion. However, they do not contain genetic material and cannot transmit infection. The immunogenicity of HPV vaccines relies on the development of conformational neutralizing antibodies to epitopes displayed on viral capsids.

1	Several large trials have demonstrated the high degree of safety and efficacy of HPV vaccines. The evidence to date has shown high and sustained efficacy against disease caused by those HPV types represented in the vaccines (HPV-6, -11, -16, and -18 in the Merck vaccine and HPV-16 and -18 in the GlaxoSmithKline vaccine). However, no therapeutic effect against active infection or disease has been found for either vaccine.

1	Bivalent Vaccine (Cervarix) A bivalent L1 VLP vaccine (HPV-16 and -18), marketed under the name Cervarix (GlaxoSmithKline), is administered by IM injection at months 0, 1, and 6. This vaccine was tested in 18,644 women 15–25 years of age who were residing in the United States, South America, Europe, and Asia. The primary endpoints of the study included vaccine efficacy against persistent infections with HPV16 and -18. Investigators also assessed the vaccine’s efficacy against CIN of grade 2 or higher due to HPV-16 and -18 in women who had no evidence of infection with these HPV types at baseline; in these women, vaccine efficacy was 94.9% (95% confidence interval [CI], 87.7 to 98.4) against CIN ≥2 related to HPV-16 or HPV-18, 91.7% (95% CI, 66.6 to 99.1) against CIN ≥3, and 100% (95% CI, –8.6 to 100) against adenocarcinoma in situ.

1	Adverse events were evaluated in phase 3 trials in a subset of 3077 women who received the bivalent vaccine and 3080 women (controls) who received hepatitis A vaccine. Injection-site adverse events (pain, redness, and swelling) and systemic adverse events (fatigue, headache, and myalgia) were reported more frequently in the HPV vaccine group than in the control group. Serious adverse events (mainly injection-site reactions), new-onset chronic disease, or medically significant conditions occurred in 3.5% of HPV vaccine recipients and in 3.5% of women receiving the control vaccine. 1200 The bivalent vaccine is approved in the United States for prevention of cervical cancer, CIN ≥2, adenocarcinoma in situ, and CIN 1 caused by HPV-16 and -18. This vaccine is approved for administration to girls and women 9–25 years of age.

1	Quadrivalent Vaccine (Gardasil) A quadrivalent L1 VLP (HPV-6, -11, -16, and -18) vaccine, marketed under the name Gardasil (Merck), is administered IM at months 0, 2, and 6. A combined efficacy analysis based on data from four randomized double-blind clinical studies including more than 20,000 participants demonstrated that the vaccine’s efficacy against external genital warts was 98.9% (95% CI, 93.7 to 100). Its efficacy was 95.2% (95% CI, 87.2 to 98.7) in protecting against CIN, 100% (95% CI, 92.9 to 100) against HPV-16or HPV-18-related CIN 2/3 or adenocarcinoma in situ, and 100% (95% CI, 55.5 to 100.0) against HPV-16or HPV-18-related vulvar intraepithelial neoplasia grades 2 and 3 (VIN 2/3) and vaginal intraepithelial neoplasia grades 2 and 3 (VaIN 2/3).

1	Safety data on the quadrivalent HPV vaccine are available from seven clinical trials including nearly 12,000 girls and women 9–26 years of age who received the vaccine and ~10,000 who received placebo. A larger proportion of young women reported injection-site adverse events in the vaccine groups than in the aluminum-containing or saline placebo groups. Systemic adverse events were reported by similar proportions of vaccine and placebo recipients and were described as mild or moderate by most participants. The types of serious adverse events reported were similar for the two groups. Ten persons who received the quadrivalent vaccine and seven persons who received placebo died during the course of the trials; no deaths were considered to be vaccine related.

1	During the course of the quadrivalent vaccine trials, surveillance data on the development of new medical conditions were collected for up to 4 years after vaccination. No statistically significant differences in the incidence of any medical conditions between vaccine and placebo recipients were found; this result indicated a very good safety profile. A recent safety review by the U.S. Food and Drug Administration and the Centers for Disease Control and Prevention (CDC) examined events related to Gardasil that had been reported to the Vaccine Adverse Events Reporting System. Adverse events were consistent with those seen in previous safety studies of the vaccine. It is noteworthy that rates of syncope and venous thrombotic events were higher with Gardasil than those that have usually been documented for other vaccines.

1	The quadrivalent vaccine is approved for (1) vaccination of girls and women 9–26 years of age to prevent genital warts and cervical cancer caused by HPV-6, -11, -16, and -18; (2) vaccination of the same population to prevent precancerous or dysplastic lesions, including cervical adenocarcinoma in situ, CIN 2/3, VIN 2/3, VaIN 2/3, and CIN 1; (3) vaccination of boys and men 9–26 years of age to prevent genital warts caused by HPV-6 and -11; and (4) vaccination of individuals 9–26 years of age to prevent anal cancer and associated precancerous lesions due to HPV-6, -11, -16, and -18.

1	Cross-Protection of HPV Vaccines Women vaccinated with either of the available vaccines produce neutralizing antibodies against types related to HPV-16 or -18. Analyses of data from clinical trials suggest that both vaccines may offer cross-protection against nonvaccine types. The bivalent vaccine appears more efficacious against HPV-31, -33, and -45 than the quadrivalent vaccine, but differences in study design make direct comparisons difficult. In addition, vaccine efficacy against persistent infections with HPV-31 and -45 appeared to wane over time in the bivalent vaccine trials, whereas efficacy against persistent infection with HPV-16 or -18 remained stable.

1	Second-Generation Vaccines While HPV-16 and -18 cause the majority of cervical cancers worldwide, global data have shown that HPV-31, -33, -35, -45, -52, and -58 are the next most frequently detected types in invasive cervical cancers. Second-generation vaccines that are in development incorporate VLPs of additional oncogenic HPV types (other than HPV-16 and -18), including HPV-31, -33, -45, -52, and -58; efficacy studies are ongoing. If vaccines with these five additional oncogenic types prove to be effective, mathematical models estimate that the level of protection could be raised to 90% of all squamous cell cervical cancers worldwide.

1	Recommendations for Vaccination The CDC’s Advisory Committee for Immunization Practice recommends administration of the quadrivalent HPV vaccine—with the schedule used in the vaccine trials—to all boys and girls 11–12 years of age as well as to boys/men and girls/ women 13–26 years of age who have not previously been vaccinated or who have not completed the full series. For women, Papanicolaou (Pap) smear testing and screening for HPV DNA are not recommended before vaccination. After vaccination, Pap testing is recommended to detect disease caused by other oncogenic HPV types.

1	After HPV infection occurs, prevention of HPV-associated disease relies on screening. Women residing in developing countries who lack access to cervical screening programs have a higher rate of cervical cancer and a lower rate of cancer-specific survival. Approximately 75% of women living in developed countries have been screened in the past 5 years, whereas the figure is only ~5% among women living in developing countries. Economic and logistic obstacles likely impede routine screening of these populations for cervical cancer.

1	The primary method used for cancer screening is cervical cytology via Pap smear. The guidelines of the American Society of Colposcopy and Cervical Pathology recommend initiation of cervical cancer screening at age 21, regardless of the age of sexual debut. Women 21–29 years old with a normal Pap smear should have the test repeated every 3 years. Although adolescent and young women often test positive for HPV DNA, they are at very low risk of cervical cancer. Co-testing, or testing for HPV DNA at the time of the Pap smear, is not recommended for women in this age group because the presence of HPV DNA does not correlate with the presence of high-grade squamous intraepithelial neoplasia. Women 30–65 years of age should have a Pap smear every 3 years if testing for HPV DNA is not performed. The screening interval for women in this age group can be extended to every 5 years if co-testing results are negative. HPV testing is not recommended for partners of women with HPV or for screening

1	The screening interval for women in this age group can be extended to every 5 years if co-testing results are negative. HPV testing is not recommended for partners of women with HPV or for screening for conditions other than cervical cancer.

1	Currently, there is no clear consensus regarding screening for anal cancer and its precursors, including high-grade anal intraepithelial lesions. This lack of clarity is due to an inadequate understanding of optimal treatment for lowor high-grade anal dysplasia found during cytologic screening. The current HIV treatment guidelines suggest that there may be a benefit to screening, but an effect on the associated morbidity and mortality of anal squamous cell cancer has not been consistently demonstrated.

1	A variety of treatment modalities are available for various HPV infections, but none has been proven to eliminate HPV from tissue adjacent to the destroyed and infected tissue. Treatment efficacies are limited by frequent recurrences (presumably due to reinfection acquired from an infected partner), reactivation of latent virus, or autoinoculation from nearby infected cells. The goals of treatment include prevention of virus transmission, eradication of premalignant lesions, and reduction of symptoms. Treatment is generally successful in eliminating visible lesions and grossly diseased tissue. Different therapies are indicated for genital warts, vaginal and cervical disease, and perianal and anal disease.

1	An optimal therapy for HPV-related genital tract disease that combines high efficacy, low toxicity, low cost, and low recurrence rate is not available. For genital warts of the penis or vulva, cryotherapy (see below) is safest, least expensive, and most effective. Guidelines for the treatment of genital warts can be found on the CDC website (www .cdc.gov/std/treatment/2010/genital-warts.htm). Women with vaginal lesions should be referred to a gynecologist experienced in colposcopy and treatment of these lesions. Treatment of cervical disease involves careful inspection, biopsy, and histopathologic grading to determine the severity and extent of disease. Women with evidence of cervical HPV infection should be referred to a gynecologist familiar with HPV and experienced in colposcopy. Optimal follow-up of these patients includes colposcopic examination of the cervix and vagina on a yearly basis. Guidelines from the American College of Gynecology and Obstetrics are available for the

1	follow-up of these patients includes colposcopic examination of the cervix and vagina on a yearly basis. Guidelines from the American College of Gynecology and Obstetrics are available for the treatment of cervical dysplasia and cancer.

1	For anal or perianal lesions, cryotherapy or surgical removal is safest and most effective. Anoscopy and/or sigmoidoscopy should be performed when patients have perianal lesions, and suspicious lesions should be biopsied to rule out malignancy. Tables 222-1 and 222-2 list the available patient-administered and physician-administered treatments, respectively. Podophyllotoxin Podophyllotoxin (0.05% solution or gel and 0.15% cream) induces necrosis of genital wart tissue that heals within a few days. It is relatively effective and can be self-administered. Podophyllotoxin is applied twice daily on three consecutive days of the week for a maximum of 4 weeks. Adverse effects are common and include pain, inflammation, erosion, and burning or itching. Podophyllotoxin should not be used to treat vaginal, cervical, or anal lesions. The safety of podophyllotoxin during pregnancy has not been established.

1	Sinecatechins Sinecatechins (15% ointment) is used to treat genital warts but should not be used to treat vaginal, cervical, or anal lesions. Sinecatechins causes an inflammatory response when applied topically three times a day for up to 4 months. Clearance rates approach 60% in some studies, and recurrence rates are 6–9%. Adverse effects (redness, burning, itching, and pain at the site of application) are generally mild. The safety of sinecatechins during pregnancy is unknown.

1	Imiquimod Imiquimod (5% or 3.75% cream) is a patient-applied topical immunomodulatory agent thought to activate immune cells by binding to a Toll-like receptor—an event that leads to an inflammatory response. Imiquimod 5% cream is applied to genital warts at bedtime three times per week for up to 16 weeks. Warts are cleared in ~56% of patients, more often in women than in men; recurrence rates approach 13%. Local inflammatory side effects are common. Rates of clearance of genital warts with the 3.75% formulation are not as high, but the duration of treatment is shorter (i.e., daily application for a maximum of 8 weeks), and fewer local and systemic adverse reactions occur. Imiquimod should not be used to treat vaginal, cervical, or anal lesions. The safety of imiquimod during pregnancy has not been established.

1	Cryotherapy Cryotherapy (liquid nitrogen) for HPV-associated lesions causes cellular death. Genital warts usually disappear after two or three weekly sessions but often recur. Cryotherapy, which Availability Cost Good Frequent Frequent, is nontoxic and is not associated with significant adverse reactions, can also be used for diseased cervical tissue. Local pain occurs frequently. Surgical Methods Exophytic lesions can be surgically removed after intradermal injection of 1% lidocaine. This treatment is well tolerated but can cause scarring and requires hemostasis. Genital warts can also be destroyed by electrocautery, in which no additional hemostasis is required.

1	Laser Therapy Laser treatment affords destruction of exophytic lesions and other HPV-infected tissue while preserving normal tissue. Local anesthetics are generally adequate. Efficacy for genital lesions is at least equal to that of other therapies (60–90%), with low recurrence rates (5–10%). Complications include local pain, vaginal discharge, periurethral swelling, and penile or vulvar swelling. Laser therapy has also been used successfully to treat cervical dysplasia and anal disease caused by HPV.

1	Interferon (IFN) Recombinant IFN-α is used for intralesional treatment of genital warts, including perianal lesions. The recommended dosage is 1.0 × 106 IU of IFN injected into each lesion three times weekly for 3 weeks. IFN therapy causes clearance of infected cells by immune-boosting effects. Adverse events include headache, nausea, vomiting, fatigue, and myalgia. IFN therapy is costly and should be reserved for severe cases that do not respond to cheaper treatments. IFN should not be used to treat vaginal, cervical, or anal lesions. Other Therapies Both trichloroacetic acid and bichloroacetic acid are caustic agents that destroy warts by coagulation of proteins. Neither of these agents is recommended for treatment.

1	Most sexually active adults will be infected with HPV during their lives. For all patients (vaccinated or unvaccinated), certain behavioral interventions can reduce the risk of acquiring HPV. Physicians can provide their patients with measures that can reduce this risk. The only way to avoid acquiring an HPV infection is to abstain from sexual activity, including intimate touching and oral sex. Practicing safe sex (partner reduction, condom use) may lower the likelihood of HPV transmission. Most HPV infections are controlled by the immune system and cause no symptoms or disease. Some infections lead to genital warts and cervical precancers. Genital warts can be treated for cosmetic reasons and to prevent spread of infection to others. Even after resolution of genital warts, latent virus can persist in normal-appearing skin or mucosa and thus theoretically can be transmitted to uninfected partners. Precancerous cervical lesions should be treated to prevent progression to cancer.

1	human diseases, accounting for one-half or more of all acute illnesses. The incidence of acute respiratory disease in the United States is 3–5.6 cases per person per year. The rates are highest among children <1 year old (6.1–8.3 cases per year) and remain high until age 6, when a progressive decrease begins. Adults have 3–4 cases per person per year. Morbidity from acute respiratory illnesses accounts for 30–50% of time lost from work by adults and for 60–80% of time lost from school by children. The use of antibacterial agents to treat viral respiratory infections represents a major source of abuse of that category of drugs.

1	It has been estimated that two-thirds to three-fourths of cases of acute respiratory illness are caused by viruses. More than 200 antigenically distinct viruses from 10 genera have been reported to cause acute respiratory illness, and it is likely that additional agents will be described in the future. The vast majority of these viral infections involve the upper respiratory tract, but lower respiratory tract disease can also develop, particularly in younger age groups, in the elderly, and in certain epidemiologic settings.

1	The illnesses caused by respiratory viruses traditionally have been divided into multiple distinct syndromes, such as the “common cold,” pharyngitis, croup (laryngotracheobronchitis), tracheitis, bronchiolitis, bronchitis, and pneumonia. Each of these general categories of illness has a certain epidemiologic and clinical profile; for example, croup occurs exclusively in very young children and has a characteristic clinical course. Some types of respiratory illness are more likely to be associated with certain viruses (e.g., the common cold with rhinoviruses), whereas others occupy characteristic epidemiologic niches (e.g., adenovirus infections in military recruits). The syndromes most commonly associated with infections with the major respiratory virus groups are summarized in Table 223-1. Most respiratory viruses clearly have the potential to cause more than one type of respiratory illness, and features of several types of illness may be found in the same patient. Moreover, the

1	223-1. Most respiratory viruses clearly have the potential to cause more than one type of respiratory illness, and features of several types of illness may be found in the same patient. Moreover, the clinical illnesses induced by these viruses are rarely sufficiently distinctive to permit an etiologic diagnosis on clinical grounds alone, although the epidemiologic setting increases the likelihood that one group of viruses rather than another is involved. In general, laboratory methods must be relied on to establish a specific viral diagnosis.

1	This chapter reviews viral infections caused by six of the major groups of respiratory viruses: rhinoviruses, coronaviruses, respiratory syncytial viruses, metapneumoviruses, parainfluenza viruses, and adenoviruses. The extraordinary outbreaks of lower respiratory tract disease associated with coronaviruses (severe acute respiratory syndrome [SARS] in 2002– 2003 and Middle East respiratory syndrome [MERS] in 2012–2013) are also discussed. Influenza viruses, which are a major cause of death as well as morbidity, are reviewed in Chap. 224. Herpesviruses, which occasionally cause pharyngitis and which also cause lower respiratory tract disease in immunosuppressed patients, are reviewed in Chap. 216. Enteroviruses, which account for occasional respiratory illnesses during the summer months, are reviewed in Chap. 228.

1	Rhinoviruses are members of the Picornaviridae family—small (15to 30-nm) nonenveloped viruses that contain a single-stranded RNA genome. Human rhinoviruses were first classified by immunologic serotype and are now divided into three genetic species: HRV-A, HRV-B, and HRV-C. The 102 serotypes described initially are encompassed by HRV-A and HRV-B species, whereas HRV-C encompasses more than 60 previously unrecognized serotypes. In contrast to other members of the picornavirus family, such as enteroviruses, rhinoviruses are acid-labile and are almost completely inactivated at pH ≤3. HRV-A and HRV-B species grow preferentially at 33°–34°C (the temperature of the human nasal passages) rather

1	Frequency of Respiratory Syndromes aSevere acute respiratory syndrome–associated coronavirus (SARS-CoV) caused epidemics of pneumonia from November 2002 to July 2003 (see text). bMiddle East respiratory syndrome coronavirus (MERS-CoV) has caused severe respiratory illnesses from 2012 to the time of this writing (2014); see text. cSerotypes 4 and 7 most commonly; also serotypes 14 and 21. dFever, cough, myalgia, malaise. eMay or may not have a respiratory component. than at 37°C (the temperature of the lower respiratory tract), whereas HRV-C viruses replicate well at either temperature. Of the 101 initially recognized serotypes of rhinovirus, 88 use intercellular adhesion molecule 1 (ICAM-1) as a cellular receptor and constitute the “major” receptor group, 12 use the low-density lipoprotein receptor (LDLR) and constitute the “minor” receptor group, and 1 uses decay-accelerating factor.

1	Rhinovirus infections are worldwide in distribution. They are a prominent cause of the common cold and have been detected in up to 50% of common cold–like illnesses by tissue culture and polymerase chain reaction (PCR) techniques. Overall rates of rhinovirus infection are higher among infants and young children and decrease with increasing age. Rhinovirus infections occur throughout the year, with seasonal peaks in early fall and spring in temperate climates. These infections are most often introduced into families by preschool or grade-school children <6 years old. Of initial illnesses in family settings, 25–70% are followed by secondary cases, with the highest attack rates among the youngest siblings at home. Attack rates also increase with family size.

1	Rhinoviruses appear to spread through direct contact with infected secretions, usually respiratory droplets. In some studies of volunteers, transmission was most efficient by hand-to-hand contact, with subsequent self-inoculation of the conjunctival or nasal mucosa. Other studies demonstrated transmission by largeor small-particle aerosol. Virus can be recovered from plastic surfaces inoculated 1–3 h previously; this observation suggests that environmental surfaces contribute to transmission. In studies of married couples in which neither partner had detectable serum antibody, transmission was associated with prolonged contact (≥122 h) during a 7-day period. Transmission was infrequent unless (1) virus was recoverable from the donor’s hands and nasal mucosa, (2) at least 1000 TCID50 (50% tissue culture infectious dose) of virus was present in nasal washes from the donor, and (3) the donor was at least moderately symptomatic with the “cold.” Despite anecdotal observations, exposure to

1	tissue culture infectious dose) of virus was present in nasal washes from the donor, and (3) the donor was at least moderately symptomatic with the “cold.” Despite anecdotal observations, exposure to cold temperatures, fatigue, and sleep deprivation have not been associated with increased rates of rhinovirus-induced illness in volunteers, although some studies have suggested that psychologically defined “stress” may contribute to development of symptoms.

1	By adulthood, nearly all individuals have neutralizing antibodies to multiple rhinovirus serotypes, although the prevalence of antibody to any one serotype varies widely. Multiple serotypes circulate simultaneously, and generally no single serotype or group of serotypes has been more prevalent than the others.

1	Rhinoviruses infect cells through attachment to specific cellular receptors; as mentioned above, most serotypes attach to ICAM-1, while a few use LDLR. Relatively limited information is available on the histopathology and pathogenesis of acute rhinovirus infections in humans. Examination of biopsy specimens obtained during experimentally induced and naturally occurring illness indicates that the nasal mucosa is edematous, is often hyperemic, and—during acute illness—is covered by a mucoid discharge. There is a mild infiltrate with inflammatory cells, including neutrophils, lymphocytes, plasma cells, and eosinophils. Mucus-secreting glands in the submucosa appear hyperactive; the nasal turbinates are engorged, a condition that may lead to obstruction of nearby openings of sinus cavities. Several mediators—e.g., bradykinin; lysylbradykinin; prostaglandins; histamine; interleukins 1β, 6, and 8; interferon γ–induced protein 10; and tumor necrosis factor α—have been linked to the

1	Several mediators—e.g., bradykinin; lysylbradykinin; prostaglandins; histamine; interleukins 1β, 6, and 8; interferon γ–induced protein 10; and tumor necrosis factor α—have been linked to the development of signs and symptoms in rhinovirus-induced colds.

1	The incubation period for rhinovirus illness is short, generally 1–2 days. Virus shedding coincides with the onset of illness or may begin shortly before symptoms develop. The mechanisms of immunity to rhinovirus infection are not well worked out. In some studies, the presence of homotypic antibody has been associated with significantly reduced rates of subsequent infection and illness, but data conflict regarding the relative importance of serum and local antibody in protection from rhinovirus infection.

1	The most common clinical manifestations of rhinovirus infections are those of the common cold. Illness usually begins with rhinorrhea and sneezing accompanied by nasal congestion. The throat is frequently sore, and in some cases sore throat is the initial complaint. Systemic signs and symptoms, such as malaise and headache, are mild or absent, and fever is unusual in adults but may occur in up to one-third of children. Illness generally lasts for 4–9 days and resolves spontaneously without sequelae. In children, bronchitis, bronchiolitis, and bronchopneumonia have been reported; nevertheless, it appears that rhinoviruses are not major causes of lower respiratory tract disease in children. Rhinoviruses may cause exacerbations of asthma and chronic pulmonary disease in adults. The vast majority of rhinovirus infections resolve without sequelae, but complications related to obstruction of the eustachian tubes or sinus ostia, including otitis media or acute sinusitis, can develop. In

1	majority of rhinovirus infections resolve without sequelae, but complications related to obstruction of the eustachian tubes or sinus ostia, including otitis media or acute sinusitis, can develop. In immunosuppressed patients, particularly bone marrow transplant recipients, severe and even fatal pneumonias have been associated with rhinovirus infections.

1	Although rhinoviruses are the most frequently recognized cause of the common cold, similar illnesses are caused by a variety of other viruses, and a specific viral etiologic diagnosis cannot be made on clinical grounds alone. Rather, rhinovirus infection is diagnosed by isolation of the virus from nasal washes or nasal secretions in tissue culture. In practice, this procedure is rarely undertaken because of the benign, self-limited nature of the illness. In most settings, detection of rhino-virus RNA is more sensitive and easier by PCR than by tissue culture. Accordingly, PCR has generally become the standard for detection of rhinoviruses in clinical specimens. Given the many serotypes of rhinovirus, diagnosis by serum antibody tests is currently impractical. Likewise, common laboratory tests, such as white blood cell count and erythrocyte sedimentation rate, are not helpful.

1	Because rhinovirus infections are generally mild and self-limited, treatment is not usually necessary. Therapy in the form of first-generation antihistamines and nonsteroidal anti-inflammatory drugs may be beneficial in patients with particularly pronounced symptoms, and an oral decongestant may be added if nasal obstruction is particularly troublesome. Reduction of activity is prudent in instances of significant discomfort or fatigability. Antibacterial agents should be used only if bacterial complications such as otitis media or sinusitis develop. Specific antiviral therapy is not available.

1	Intranasal application of interferon sprays has been effective in the prophylaxis of rhinovirus infections but is also associated with local irritation of the nasal mucosa. Studies of prevention of rhinovirus infection by blocking of ICAM-1 or by binding of drug (pleconaril) to parts of the viral capsid have yielded mixed results. Experimental vaccines to certain rhinovirus serotypes have been generated, but their usefulness is questionable because of the myriad serotypes involved and the uncertainty about mechanisms of immunity. Thorough hand washing, environmental decontamination, and protection against autoinoculation may help to reduce rates of transmission of infection.

1	Coronaviruses are pleomorphic, single-stranded RNA viruses that measure 100–160 nm in diameter. The name derives from the crown-like appearance produced by the club-shaped projections that stud the viral envelope. Coronaviruses infect a wide variety of animal species and have been divided into four genera. Coronaviruses that infect humans (HCoVs) fall into two genera: Alphacoronavirus 1204 and Betacoronavirus. Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are betacoronaviruses. In general, human coronaviruses have been difficult to cultivate in vitro, and some strains grow only in human tracheal organ cultures rather than in tissue culture. SARS-CoV and MERS-CoV are exceptions whose ready growth in African green monkey kidney (Vero E6) cells greatly facilitates their study.

1	Human coronavirus infections are present throughout the world. Seroprevalence studies of strains HCoV-229E and HCoV-OC43 have demonstrated that serum antibodies are acquired early in life and increase in prevalence with advancing age, so that >80% of adult populations have antibodies detectable by enzyme-linked immunosorbent assay (ELISA). Overall, coronaviruses account for 10–35% of common colds, depending on the season. Coronavirus infections appear to be particularly prevalent in late fall, winter, and early spring—times when rhinovirus infections are less common.

1	An extraordinary outbreak of the coronavirus-associated illness known as SARS occurred in 2002–2003. The outbreak apparently began in southern China and eventually resulted in 8096 recognized cases in 28 countries in Asia, Europe, and North and South America; ~90% of cases occurred in China and Hong Kong. The natural reservoir of SARS-CoV appeared to be the horseshoe bat, and the outbreak may have originated from human contact with infected semidomesticated animals such as the palm civet. In most cases, however, the infection was transmitted from human to human. Case–fatality rates varied among outbreaks, with an overall figure of ~9.5%. The disease appeared to be somewhat milder in cases in the United States and was clearly less severe among children. The outbreak ceased in 2003; 17 cases were detected in 2004, mostly in laboratory-associated settings, and no cases have been reported subsequently.

1	The mechanisms of transmission of SARS are incompletely understood. Clusters of cases suggest that spread may occur via both large-and small-droplet aerosols and perhaps via the fecal–oral route as well. The outbreak of illness in a large apartment complex in Hong Kong suggested that environmental sources, such as sewage or water, may also play a role in transmission. Some ill individuals (“super-spreaders”) appeared to be hyperinfectious and were capable of transmitting infection to 10–40 contacts, although most infections resulted in spread either to no one or to three or fewer individuals.

1	Since it began in June 2012, another extraordinary outbreak of serious respiratory illness, MERS, has been linked with a coronavirus (MERS-CoV). Through May 2014, a total of 536 cases and 145 deaths (27%) have been reported. All cases have been associated with contact or travel to six countries in or near the Arabian Peninsula: Jordan, Kuwait, Oman, Qatar, Saudi Arabia, and the United Arab Emirates. Cases have also been reported in France, Italy, Tunisia, Germany, Spain, and the United Kingdom. Person-to-person transmission has been documented, but sustained spread in communities has not. The source of MERS-CoV has not been established, but it is suspected that bats may be the animal reservoir and that camels serve as an intermediate host.

1	Coronaviruses that cause the common cold (e.g., strains HCoV-229E and HCoV-OC43) infect ciliated epithelial cells in the nasopharynx via the aminopeptidase N receptor (group 1) or a sialic acid receptor (group 2). Viral replication leads to damage of ciliated cells and induction of chemokines and interleukins, with consequent common-cold symptoms similar to those induced by rhinoviruses.

1	SARS-CoV infects cells of the respiratory tract via the angiotensinconverting enzyme 2 receptor. The result is a systemic illness in which virus is also found in the bloodstream, in the urine, and (for up to 2 months) in the stool. Virus persists in the respiratory tract for 2–3 weeks, and titers peak ~10 days after the onset of systemic illness. Pulmonary pathology consists of hyaline membrane formation, desquamation of pneumocytes in alveolar spaces, and an interstitial infiltrate made up of lymphocytes and mononuclear cells. Giant cells are frequently seen, and coronavirus particles have been detected in type II pneumocytes. Elevated levels of proinflammatory cytokines and chemokines have been detected in sera from patients with SARS. Because MERS-CoV was so recently detected, little is known at present about its pathogenesis. However, it may well be similar to that of SARS-CoV.

1	After an incubation period that generally lasts 2–7 days (range, 1–14 days), SARS usually begins as a systemic illness marked by the onset of fever, which is often accompanied by malaise, headache, and myalgias and is followed in 1–2 days by a nonproductive cough and dyspnea. Approximately 25% of patients have diarrhea. Chest x-rays can show a variety of infiltrates, including patchy areas of consolidation—most frequently in peripheral and lower lung fields—or interstitial infiltrates, which can progress to diffuse involvement. In severe cases, respiratory function may worsen during the second week of illness and progress to frank adult respiratory distress syndrome accompanied by multiorgan dysfunction. Risk factors for severe disease include an age of >50 years and comorbidities such as cardiovascular disease, diabetes, and hepatitis. Illness in pregnant women may be particularly severe, but SARS-CoV infection appears to be milder in children than in adults.

1	Information regarding the clinical manifestations of MERS-CoV is limited. The case–fatality rate has been high in the initial cases, but this may represent an ascertainment bias, and it is clear that mild cases occur as well. The median incubation period has been estimated to be 5.2 days, and a secondary case was estimated to have an incubation period of 9–12 days. Cases have been reported that begin with cough and fever and progress to acute respiratory distress and respiratory failure within a week. Other cases have manifested as mild upper respiratory symptoms only. Renal failure has been noted, and DPP-4, the host cell receptor for MERS-CoV, is expressed at high levels in the kidney; these findings suggest that direct viral infection of the kidney may lead to renal dysfunction. Diarrhea and vomiting are also common in MERS, and pericarditis has been reported.

1	The clinical features of common colds caused by human coronaviruses are similar to those of illness caused by rhinoviruses. In studies of volunteers, the mean incubation period of colds induced by corona-viruses (3 days) is somewhat longer than that of illness caused by rhinoviruses, and the duration of illness is somewhat shorter (mean, 6–7 days). In some studies, the amount of nasal discharge was greater in colds induced by coronaviruses than in those induced by rhinoviruses. Coronaviruses other than SARS-CoV have been recovered occasionally from infants with pneumonia and from military recruits with lower respiratory tract disease and have been associated with worsening of chronic bronchitis. Two novel coronaviruses, HCoV-NL63 and HCoVHKU1, have been isolated from patients hospitalized with acute respiratory illness. Their overall role as causes of human respiratory disease remains to be determined.

1	Laboratory abnormalities in SARS include lymphopenia, which is documented in ~50% of cases and which mostly affects CD4+ T cells but also involves CD8+ T cells and natural killer cells. Total white blood cell counts are normal or slightly low, and thrombocytopenia may develop as the illness progresses. Elevated serum levels of aminotransferases, creatine kinase, and lactate dehydrogenase have been reported.

1	A rapid diagnosis of SARS-CoV infection can be made by reverse-transcription PCR (RT-PCR) of respiratory tract samples and plasma early in the illness and of urine and stool later on. SARS-CoV can also be grown from respiratory tract samples by inoculation into Vero E6 tissue culture cells, in which a cytopathic effect is seen within days. RT-PCR appears to be more sensitive than tissue culture, but only around one-third of cases are positive by PCR at initial presentation. Serum antibodies can be detected by ELISA or immunofluorescence, and nearly all patients develop detectable serum antibodies within 28 days after the onset of illness.

1	Laboratory abnormalities in MERS-CoV infection include lymphopenia with or without neutropenia, thrombocytopenia, and elevated levels of lactate dehydrogenase. MERS-CoV can be isolated in tissue culture in Vero and LLC-MK2 cells, but PCR techniques are more sensitive and rapid and are the standard for laboratory diagnosis. Serologic tests using ELISA and immunofluorescence techniques have also been developed. Laboratory diagnosis of coronavirus-induced colds is rarely required. Coronaviruses that cause those illnesses are frequently difficult to cultivate in vitro but can be detected in clinical samples by ELISA or immunofluorescence assays or by RT-PCR for viral RNA. These research procedures can be used to detect coronaviruses in unusual clinical settings.

1	There is no specific therapy for SARS with established efficacy. Although ribavirin has frequently been used, it has little if any activity against SARS-CoV in vitro, and no beneficial effect on the course of illness has been demonstrated. Because of suggestions that immunopathology may contribute to the disease, glucocorticoids have also been widely used, but their benefit, if any, likewise remains to be established. Supportive care to maintain pulmonary and other organ-system functions remains the mainstay of therapy. Similarly, there is no established antiviral therapy for MERS. Interferon α2b and ribavirin have displayed activity against MERS-CoV in vitro and in a rhesus macaque model, but data are not available on its use in human cases of MERS. The approach to the treatment of common colds caused by coronaviruses is similar to that discussed above for rhinovirus-induced illnesses.

1	The recognition of SARS led to a worldwide mobilization of public health resources to apply infection control practices to contain the disease. Case definitions were established, travel advisories were proposed, and quarantines were imposed in certain locales. As of this writing, no additional cases of SARS have been reported since 2004. However, it remains unknown whether the disappearance of cases is a result of control measures, whether it is part of a seasonal or otherwise unexplained epidemiologic pattern of SARS, or when or whether SARS might reemerge. The U.S. Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) maintain recommendations for surveillance and assessment of potential cases of SARS (www.cdc.gov/sars/). The frequent transmission of the disease to health care workers makes it mandatory that strict infection-control practices be employed by health care facilities to prevent airborne, droplet, and contact transmission from any

1	of the disease to health care workers makes it mandatory that strict infection-control practices be employed by health care facilities to prevent airborne, droplet, and contact transmission from any suspected cases of SARS. Health care workers who enter areas in which patients with SARS may be present should don gowns, gloves, and eye and respiratory protective equipment (e.g., an N95 filtering facepiece respirator certified by the National Institute for Occupational Safety and Health).

1	Similarly, the WHO and the CDC have issued recommendations for identification, prevention, and control of MERS-CoV infections (www.cdc.gov/coronavirus/mers/index.html). Isolation precautions against airborne spread of infection should be instituted for patients hospitalized for suspected MERS, as described above for SARS. Vaccines have been developed against several animal coronaviruses but not against known human coronaviruses. The emergence of SARS-CoV and MERS-CoV has stimulated interest in the development of vaccines against such agents.

1	Human respiratory syncytial virus (HRSV) is a member of the Paramyxoviridae family (genus Pneumovirus). It is an enveloped virus ~150–350 nm in diameter and is so named because its replication in vitro leads to the fusion of neighboring cells into large multinucleated 1205 syncytia. The single-stranded RNA genome codes for 11 virus-specific proteins. Viral RNA is contained in a helical nucleocapsid surrounded by a lipid envelope bearing two glycoproteins: the G protein, by which the virus attaches to cells, and the F (fusion) protein, which facilitates entry of the virus into the cell by fusing host and viral membranes. HRSV is considered to be of a single antigenic type, but two distinct subgroups (A and B) and multiple subtypes within each subgroup have now been described. Antigenic diversity is reflected by differences in the G protein, whereas the F protein is relatively conserved. Both antigenic groups can circulate simultaneously in outbreaks, although there are typically

1	diversity is reflected by differences in the G protein, whereas the F protein is relatively conserved. Both antigenic groups can circulate simultaneously in outbreaks, although there are typically alternating patterns in which one subgroup predominates over 1to 2-year periods.

1	HRSV is a major respiratory pathogen of young children and the foremost cause of lower respiratory disease in infants. Infection with HRSV is seen throughout the world in annual epidemics that occur in late fall, winter, or spring and last up to 5 months. The virus is rarely encountered during the summer. Rates of illness are highest among infants 1–6 months of age, peaking at 2–3 months of age. The attack rates among susceptible infants and children are extraordinarily high, approaching 100% in settings such as daycare centers where large numbers of susceptible infants are present. By age 2, virtually all children will have been infected with HRSV. HRSV accounts for 20–25% of hospital admissions of young infants and children for pneumonia and for up to 75% of cases of bronchiolitis in this age group. It has been estimated that more than half of infants who are at risk will become infected during an HRSV epidemic.

1	In older children and adults, reinfection with HRSV is frequent, but disease is milder than in infancy. A common cold–like syndrome is the illness most commonly associated with HRSV infection in adults. It has been increasingly appreciated that severe lower respiratory tract disease with pneumonitis can occur in elderly (often institutionalized) adults, in individuals with cardiopulmonary disease, and in patients with immunocompromising disorders or treatment, including recipients of hematopoietic stem cell transplants (HSCTs) and solid-organ transplants (SOTs). HRSV is also an important nosocomial pathogen; during an outbreak, it can infect pediatric patients and up to 25–50% of the staff on pediatric wards. The spread of HRSV among families is efficient: up to 40% of siblings may become infected when the virus is introduced into the family setting.

1	HRSV is transmitted primarily by close contact with contaminated fingers or fomites and by self-inoculation of the conjunctiva or anterior nares. Virus may also be spread by coarse aerosols produced by coughing or sneezing, but it is inefficiently spread by fine-particle aerosols. The incubation period is ~4–6 days, and virus shedding may last for ≥2 weeks in children and for shorter periods in adults. In immunosuppressed patients, shedding can continue for weeks.

1	Little is known about the histopathology of minor HRSV infection. Severe bronchiolitis or pneumonia is characterized by necrosis of the bronchiolar epithelium and a peribronchiolar infiltrate of lymphocytes and mononuclear cells. Interalveolar thickening and filling of alveolar spaces with fluid can also be found. The correlates of protective immunity to HRSV are incompletely understood. Because reinfection occurs frequently and is often associated with illness, the immunity that develops after single episodes of infection clearly is not complete or long-lasting. However, the cumulative effect of multiple reinfections is to temper subsequent disease and to provide some temporary measure of protection against infection. Studies of experimentally induced disease in healthy volunteers indicate that the presence of nasal IgA neutralizing antibody correlates more closely with protection than does the presence of serum antibody. Studies in infants, however, suggest that maternally acquired

1	that the presence of nasal IgA neutralizing antibody correlates more closely with protection than does the presence of serum antibody. Studies in infants, however, suggest that maternally acquired antibody provides some protection from lower respiratory tract disease, although illness can be severe even in infants who have moderate levels of maternally derived serum antibody. The relatively severe disease observed in immunosuppressed 1206 patients and experimental animal models indicates that cell-mediated immunity is an important mechanism of host defense against HRSV. Evidence suggests that major histocompatibility class I–restricted cytotoxic T cells may be particularly important in this regard.

1	HRSV infection leads to a wide spectrum of respiratory illnesses. In infants, 25–40% of infections result in lower respiratory tract involvement, including pneumonia, bronchiolitis, and tracheobronchitis. In this age group, illness begins most frequently with rhinorrhea, low-grade fever, and mild systemic symptoms, often accompanied by cough and wheezing. Most patients recover gradually over 1–2 weeks. In more severe illness, tachypnea and dyspnea develop, and eventually frank hypoxia, cyanosis, and apnea can ensue. Physical examination may reveal diffuse wheezing, rhonchi, and rales. Chest radiography shows hyperexpansion, peribronchial thickening, and variable infiltrates ranging from diffuse interstitial infiltrates to segmental or lobar consolidation. Illness may be particularly severe in children born prematurely and in those with congenital cardiac disease, bronchopulmonary dysplasia, nephrotic syndrome, or immunosuppression. One study documented a 37% mortality rate among

1	severe in children born prematurely and in those with congenital cardiac disease, bronchopulmonary dysplasia, nephrotic syndrome, or immunosuppression. One study documented a 37% mortality rate among infants with HRSV pneumonia and congenital cardiac disease.

1	In adults, the most common symptoms of HRSV infection are those of the common cold, with rhinorrhea, sore throat, and cough. Illness is occasionally associated with moderate systemic symptoms such as malaise, headache, and fever. HRSV has also been reported to cause lower respiratory tract disease with fever in adults, including severe pneumonia in the elderly—particularly in nursing-home residents, among whom its impact can rival that of influenza. HRSV pneumonia can be a significant cause of morbidity and death among patients undergoing stem cell and solid organ transplantation, in whom case– fatality rates of 20–80% have been reported. Sinusitis, otitis media, and worsening of chronic obstructive and reactive airway disease have also been associated with HRSV infection.

1	The diagnosis of HRSV infection can be suspected on the basis of a suggestive epidemiologic setting—that is, severe illness among infants during an outbreak of HRSV in the community. Infections in older children and adults cannot be differentiated with certainty from those caused by other respiratory viruses. The specific diagnosis is established by detection of HRSV in respiratory secretions, such as sputum, throat swabs, or nasopharyngeal washes. Virus can be isolated in tissue culture, but this method has been largely supplanted by rapid viral diagnostic techniques consisting of immunofluorescence or ELISA of nasopharyngeal washes, aspirates, and (less satisfactorily) nasopharyngeal swabs. With specimens from children, these techniques have sensitivities and specificities of 80–95%; they are somewhat less sensitive with specimens from adults. RT-PCR detection techniques have shown even higher rates of sensitivity and specificity, particularly in adults. Serologic diagnosis may be

1	are somewhat less sensitive with specimens from adults. RT-PCR detection techniques have shown even higher rates of sensitivity and specificity, particularly in adults. Serologic diagnosis may be made by comparison of acuteand convalescent-phase serum specimens by ELISA or by neutralization or complement-fixation tests. These tests may be useful in older children and adults but are less sensitive in children <4 months of age.

1	Treatment of upper respiratory tract HRSV infection is aimed primarily at the alleviation of symptoms and is similar to that for other viral infections of the upper respiratory tract. For lower respiratory tract infections, respiratory therapy, including hydration, suctioning of secretions, and administration of humidified oxygen and antibronchospastic agents, is given as needed. In severe hypoxia, intubation and ventilatory assistance may be required. Studies of infants with HRSV infection who were given aerosolized ribavirin, a nucleoside analogue active in vitro against HRSV, demonstrated a modest beneficial effect on the resolution of lower respiratory tract illness, including alleviation of blood-gas abnormalities, in some studies. The American Academy of Pediatrics does not recommend routine use of ribavirin but states that treatment with aerosolized ribavirin “may be considered” for infants who are severely ill or who are at high risk for complications of HRSV infection;

1	routine use of ribavirin but states that treatment with aerosolized ribavirin “may be considered” for infants who are severely ill or who are at high risk for complications of HRSV infection; included are premature infants and those with bronchopulmonary dysplasia, congenital heart disease, or immunosuppression. The efficacy of ribavirin against HRSV pneumonia in older children and adults, including those with immunosuppression, has not been established. No benefit has been found in the treatment of HRSV pneumonia with standard immunoglobulin; immunoglobulin with high titers of antibody to HRSV (RSVIg), which is no longer available; or chimeric mouse–human monoclonal IgG antibody to HRSV (palivizumab). Combined therapy with aerosolized ribavirin and palivizumab is being evaluated in immunosuppressed patients with HRSV pneumonia.

1	Monthly administration of RSVIg (no longer available) or palivizumab has been approved as prophylaxis against HRSV for children <2 years of age who have bronchopulmonary dysplasia or cyanotic heart disease or who were born prematurely. Considerable interest exists in the development of vaccines against HRSV. Inactivated whole-virus vaccines have been ineffective; in one study, they actually potentiated disease in infants. Other approaches include immunization with purified F and G surface glycoproteins of HRSV or generation of stable live attenuated virus vaccines. In settings where rates of transmission are high (e.g., pediatric wards), barrier methods for the protection of hands and conjunctivae may be useful in reducing the spread of virus.

1	Human metapneumovirus (HMPV) is a viral respiratory pathogen that has been assigned to the Paramyxoviridae family (genus Metapneumovirus). Its morphology and genomic organization are similar to those of avian metapneumoviruses, which are recognized respiratory pathogens of turkeys. HMPV particles may be spherical, filamentous, or pleomorphic in shape and measure 150–600 nm in diameter. Particles contain 15-nm projections from the surface that are similar in appearance to those of other Paramyxoviridae. The single-stranded RNA genome codes for nine proteins that, except for the absence of nonstructural proteins, generally correspond to those of HRSV. HMPV is of only one antigenic type; two closely related genotypes (A and B), four subgroups, and two sublineages have been described.

1	HMPV infections are worldwide in distribution, are most frequent during the winter in temperate climates, and occur early in life, so that serum antibodies to the virus are present in 50% of children by age 2 and in nearly all children by age 5. HMPV infections have been detected in older age groups, including elderly adults, and in both immunocompetent and immunosuppressed hosts. This virus accounts for 1–5% of childhood upper respiratory tract infections and for 10–15% of respiratory tract illnesses requiring hospitalization of children. In addition, HMPV causes 2–4% of acute respiratory illnesses in ambulatory adults and elderly patients. HMPV has been detected in a few cases of SARS, but its role (if any) in these illnesses has not been established.

1	The spectrum of clinical illnesses associated with HMPV is similar to that associated with HRSV and includes both upper and lower respiratory tract illnesses, such as bronchiolitis, croup, and pneumonia. Reinfection with HMPV is common among older children and adults and has manifestations ranging from subclinical infections to common cold syndromes and occasionally pneumonia, which is seen primarily in elderly patients and those with cardiopulmonary diseases. Serious HMPV infections occur in immunocompromised patients, including those with neoplasia, recipients of HSCTs, and children with HIV infection. HMPV can be detected in nasal aspirates and respiratory secretions by immunofluorescence, by PCR (the most sensitive technique), or by growth in rhesus monkey kidney (LLC-MK2) tissue cultures. A serologic diagnosis can be made by ELISA, which uses HMPV-infected tissue culture lysates as sources of antigens.

1	Treatment for HMPV infections is primarily supportive and symptom-based. Ribavirin is active against HMPV in vitro, but its efficacy in vivo is unknown. Vaccines against HMPV are in the early stages of development. Parainfluenza viruses belong to the Paramyxoviridae family (genera Respirovirus and Rubulavirus). They are 150–200 nm in diameter, are enveloped, and contain a single-stranded RNA genome. The envelope is studded with two glycoproteins: one possesses both hemagglutinin and neuraminidase activity, and the other contains fusion activity. The viral RNA genome is enclosed in a helical nucleocapsid and codes for six structural and several accessory proteins. All types of parainfluenza virus (1, 2, 3, 4A, and 4B) share certain antigens with other members of the Paramyxoviridae family, including mumps and Newcastle disease viruses.

1	Parainfluenza viruses are distributed throughout the world; infection with serotypes 4A and 4B has been reported less widely, probably because these types are more difficult than the other three to grow in tissue culture. Infection is acquired in early childhood; by 5 years of age, most children have antibodies to serotypes 1, 2, and 3. Types 1 and 2 cause epidemics during the fall, often occurring in an alternate-year pattern. Type 3 infection has been detected during all seasons, but epidemics have occurred annually in the spring.

1	The contribution of parainfluenza infections to respiratory disease varies with both the location and the year. In studies conducted in the United States, parainfluenza virus infections have accounted for 4.3–22% of respiratory illnesses in children. The major importance of these viruses is as a cause of lower respiratory illness in young children, in whom they rank second only to HRSV in that regard. Parainfluenza virus type 1 is the most common cause of croup (laryngotracheobronchitis) in children, whereas serotype 2 causes similar, although generally less severe, disease. Type 3 is an important cause of bronchiolitis and pneumonia in infants, whereas illnesses associated with types 4A and 4B have generally been mild. Unlike types 1 and 2, type 3 frequently causes illness during the first month of life, when passively acquired maternal antibody is still present. Parainfluenza viruses are spread through infected respiratory secretions, primarily by person-to-person contact and/or by

1	month of life, when passively acquired maternal antibody is still present. Parainfluenza viruses are spread through infected respiratory secretions, primarily by person-to-person contact and/or by large droplets, and by contact with fomites contaminated with respiratory secretions. The incubation period has varied from 3 to 6 days in experimental infections but may be somewhat shorter for naturally occurring disease in children.

1	In adults, parainfluenza virus infections are generally mild and account for fewer than 10% of respiratory illnesses. The advent of contemporary laboratory methods for diagnosis has increased awareness of the impact of parainfluenza infections in adults. In a recent study, parainfluenza virus was the third most common viral isolate from patients 16–64 years old who required hospitalization (0.7 isolate/1000 population). In the 2009 influenza pandemic, parainfluenza virus type 3 was the second most common cause of illness after influenza virus.

1	Immunity to parainfluenza viruses is incompletely understood, but evidence suggests that immunity to infections with serotypes 1 and 2 is mediated by local IgA antibodies in the respiratory tract. Passively acquired serum neutralizing antibodies also confer some protection against infection with types 1, 2, and (to a lesser degree) 3. Studies in experimental animal models and in immunosuppressed patients suggest that T cell–mediated immunity may also be important in parainfluenza virus infections. Lack of cellular immune responses is associated with an increased risk of progressive and fatal disease in HSCT recipients.

1	Parainfluenza virus infections occur most frequently among children, in whom initial infection with serotype 1, 2, or 3 is associated with an acute febrile illness in 50–80% of cases. Children may present with coryza, sore throat, hoarseness, and cough that may or may not be croupy. In severe croup, fever persists, with worsening coryza and sore throat. A brassy or barking cough may progress to frank stridor. Most children recover over the next 1 or 2 days, although progressive airway obstruction and hypoxia ensue occasionally. If bronchiolitis or pneumonia develops, progressive cough accompanied by wheezing, tachypnea, and intercostal retractions may occur. In this setting, sputum production increases modestly. Physical examination documents nasopharyngeal discharge and oropharyngeal injection, along with rhonchi, wheezes, or coarse breath sounds. Chest x-rays can show air trapping and occasionally interstitial infiltrates.

1	In older children and adults, parainfluenza infections tend to be milder, presenting most frequently as a common cold or as hoarseness, with or without cough. Lower respiratory tract involvement in older children and adults is uncommon, although tracheobronchitis and community-acquired pneumonia have been reported in adults. Parainfluenza viruses, most frequently type 3, are important pathogens in immunosuppressed patients—particularly in HSCT recipients but also in SOT recipients (especially recipients of lung transplants). Patients receiving cancer chemotherapy are also at risk for severe parainfluenza infection. Severe, prolonged, and even fatal parainfluenzaassociated respiratory illnesses have been reported in children and adults with severe immunosuppression.

1	The clinical syndromes caused by parainfluenza viruses (with the possible exception of croup in young children) are not sufficiently distinctive to be diagnosed on clinical grounds alone. A specific diagnosis is established by detection of virus in respiratory tract secretions, throat swabs, or nasopharyngeal washings. Growth of the virus in tissue culture is detected either by hemagglutination or by a cytopathic effect. A rapid diagnosis may be made by identification of parainfluenza antigens in exfoliated cells from the respiratory tract with immunofluorescence or ELISA, although these techniques appear to be less sensitive than tissue culture. Highly specific and sensitive PCR assays have also been developed and have now become the standard for viral diagnosis. Serologic diagnosis can be established by hemagglutinationinhibition, complement-fixation, or neutralization testing of acuteand convalescent-phase specimens. However, because frequent heterotypic responses occur among the

1	be established by hemagglutinationinhibition, complement-fixation, or neutralization testing of acuteand convalescent-phase specimens. However, because frequent heterotypic responses occur among the parainfluenza serotypes, the serotype causing illness often cannot be identified by serologic techniques alone.

1	Acute epiglottitis caused by Haemophilus influenzae type b must be differentiated from viral croup. Influenza A virus is also a common cause of croup during epidemic periods.

1	For upper respiratory tract illness, symptoms can be treated as discussed for other viral respiratory tract illnesses. If complications such as sinusitis, otitis, or superimposed bacterial bronchitis develop, appropriate antibacterial drugs should be administered. Mild cases of croup should be treated with bed rest and moist air generated by vaporizers. More severe cases require hospitalization 1208 and close observation for the development of respiratory distress. If acute respiratory distress develops, humidified oxygen and intermittent racemic epinephrine are usually administered. Aerosolized or systemically administered glucocorticoids are beneficial; systemic administration has a more profound effect. No specific antiviral therapy has been established. Ribavirin is active against parainfluenza viruses in vitro, and anecdotal reports describe its use clinically, particularly in immunosuppressed patients, but its efficacy, if any, is unclear. DAS181, a sialidase with activity

1	parainfluenza viruses in vitro, and anecdotal reports describe its use clinically, particularly in immunosuppressed patients, but its efficacy, if any, is unclear. DAS181, a sialidase with activity against parainfluenza viruses, is undergoing evaluation in immunosuppressed patients.

1	Vaccines against parainfluenza viruses are under development.

1	Adenoviruses are complex DNA viruses that measure 70–80 nm in diameter. Human adenoviruses belong to the genus Mastadenovirus, which includes 51 serotypes. Adenoviruses have a characteristic morphology consisting of an icosahedral shell composed of 20 equilateral triangular faces and 12 vertices. The protein coat (capsid) consists of hexon subunits with group-specific and type-specific antigenic determinants and penton subunits at each vertex primarily containing group-specific antigens. A fiber with a knob at the end projects from each penton; this fiber contains type-specific and some group-specific antigens. Human adenoviruses have been divided into seven subgroups (A through G) on the basis of the homology of DNA genomes and other properties. Revised criteria for classifying human adenoviruses have been proposed; reflecting recent approaches to the characterization of novel adenoviruses, the revised criteria include genome sequence and computational analysis in addition to

1	adenoviruses have been proposed; reflecting recent approaches to the characterization of novel adenoviruses, the revised criteria include genome sequence and computational analysis in addition to traditional serologic criteria. The adenovirus genome is a linear double-stranded DNA that codes for structural and nonstructural polypeptides. The replicative cycle of adenovirus may result either in lytic infection of cells or in the establishment of a latent infection (primarily involving lymphoid cells). Some adenovirus types can induce oncogenic transformation, and tumor formation has been observed in rodents; however, despite intensive investigation, adenoviruses have not been associated with tumors in humans.

1	Adenovirus infections most frequently affect infants and children. Infections occur throughout the year but are most common from fall to spring. In the United States, adenoviruses account for ~10% of acute respiratory infections in children but for <2% of respiratory illnesses in civilian adults. Nearly 100% of adults have serum antibody to multiple serotypes—a finding indicating that infection is common in childhood. Types 1, 2, 3, and 5 are the most common isolates from children. Certain adenovirus serotypes—particularly 4 and 7 but also 3, 14, and 21—are associated with outbreaks of acute respiratory disease in military recruits. Clusters of particularly severe disease have been seen with adenovirus 14.

1	Adenovirus infection can be transmitted by inhalation of aerosolized virus, by inoculation of virus into conjunctival sacs, and probably by the fecal-oral route as well. Type-specific antibody generally develops after infection and is associated with protection—albeit incomplete—against infection with the same serotype.

1	In children, adenoviruses cause a variety of clinical syndromes. The most common is an acute upper respiratory tract infection, with prominent rhinitis. On occasion, lower respiratory tract disease, including bronchiolitis and pneumonia, also develops. Adenoviruses, particularly types 3 and 7, cause pharyngoconjunctival fever, a characteristic acute febrile illness of children that occurs in outbreaks, most often in summer camps. The syndrome is marked by bilateral conjunctivitis in which the bulbar and palpebral conjunctivae have a granular appearance. Low-grade fever is frequently present for the first 3–5 days, and rhinitis, sore throat, and cervical adenopathy develop. The illness generally lasts for 1–2 weeks and resolves spontaneously. Febrile pharyngitis without conjunctivitis has also been associated with adenovirus infection. Adenoviruses have been isolated from cases of whooping cough with or without Bordetella pertussis; the significance of adenovirus in that disease is

1	also been associated with adenovirus infection. Adenoviruses have been isolated from cases of whooping cough with or without Bordetella pertussis; the significance of adenovirus in that disease is unknown.

1	In adults, the most frequently reported illness has been acute respiratory disease caused by adenovirus types 4 and 7 in military recruits. This illness is marked by a prominent sore throat and the gradual onset of fever, which often reaches 39°C (102.2°F) on the second or third day of illness. Cough is almost always present, and coryza and regional lymphadenopathy are frequently seen. Physical examination may show pharyngeal edema, injection, and tonsillar enlargement with little or no exudate. If pneumonia has developed, auscultation and x-ray of the chest may indicate areas of patchy infiltration.

1	Adenoviruses have been associated with a number of non–respiratory tract diseases, including acute diarrheal illness caused by types 40 and 41 in young children and hemorrhagic cystitis caused by types 11 and 21. Epidemic keratoconjunctivitis, caused most frequently by types 8, 19, and 37, has been associated with contaminated common sources such as ophthalmic solutions and roller towels. Adenoviruses have also been implicated in disseminated disease and pneumonia in immunosuppressed patients, including recipients of SOTs or HSCTs. In HSCT recipients, adenovirus infections have manifested as pneumonia, hepatitis, nephritis, colitis, encephalitis, and hemorrhagic cystitis. In SOT recipients, adenovirus infection may involve the organ transplanted (e.g., hepatitis in liver transplants, nephritis in renal transplants) but can disseminate to other organs as well. In patients with AIDS, high-numbered and intermediate adenovirus serotypes have been isolated, usually in the setting of low

1	in renal transplants) but can disseminate to other organs as well. In patients with AIDS, high-numbered and intermediate adenovirus serotypes have been isolated, usually in the setting of low CD4+ T cell counts, but their isolation often has not been clearly linked to disease manifestations. Adenovirus nucleic acids have been detected in myocardial cells from patients with “idiopathic” myocardiopathies, and adenoviruses have been suggested as causative agents in some cases.

1	Adenovirus infection should be suspected in the epidemiologic setting of acute respiratory disease in military recruits and in certain clinical syndromes (such as pharyngoconjunctival fever or epidemic keratoconjunctivitis) in which outbreaks of characteristic illnesses occur. In most cases, however, illnesses caused by adenovirus infection cannot be differentiated from those caused by a number of other viral respiratory agents and Mycoplasma pneumoniae. A definitive diagnosis of adenovirus infection is established by detection of the virus in tissue culture (as evidenced by cytopathic changes) and by specific identification with immunofluorescence or other immunologic techniques. Rapid viral diagnosis can be established by immunofluorescence or ELISA of nasopharyngeal aspirates, conjunctival or respiratory secretions, urine, or stool. Highly sensitive and specific PCR assays and nucleic acid hybridization are available and have become the standard for diagnosis based on clinical

1	or respiratory secretions, urine, or stool. Highly sensitive and specific PCR assays and nucleic acid hybridization are available and have become the standard for diagnosis based on clinical specimens. Adenovirus types 40 and 41, which have been associated with diarrheal disease in children, require special tissue-culture cells for isolation, and these serotypes are most commonly detected by direct ELISA of stool or by PCR. Serum antibody rises can be demonstrated by complement-fixation or neutralization tests, ELISA, radioimmunoassay, or (for those adenoviruses that hemagglutinate red cells) hemagglutination-inhibition tests.

1	Only symptom-based treatment and supportive therapy are available for adenovirus infections, and clinically useful antiviral therapy has not been established. Ribavirin and cidofovir are active in vitro against certain adenoviruses. Retrospective studies and anecdotes describe the use of these agents in disseminated adenovirus infections, but definitive efficacy data from controlled studies are not available. An oral liposomal form of cidofovir (CMX001) is being evaluated for adenovirus infections in immunosuppressed patients.

1	Live vaccines have been developed against adenovirus types 4 and 7 and have been highly efficacious in control of acute respiratory disease among military recruits. These vaccines consist of live, unattenuated virus administered in enteric-coated capsules. Infection of the gastrointestinal tract with types 4 and 7 does not cause disease but stimulates local and systemic antibodies that are protective against subsequent acute respiratory disease due to those serotypes. These vaccines were not produced from 1999 to 2011 but are now available again and are being used effectively in military recruits. Adenoviruses are also being studied as live-virus vectors for the delivery of vaccine antigens and Yehuda Z. Cohen, Raphael Dolin

1	Yehuda Z. Cohen, Raphael Dolin Influenza is an acute respiratory illness caused by infection with influenza viruses. The illness affects the upper and/or lower respiratory tract and is often accompanied by systemic signs and symptoms such as fever, headache, myalgia, and weakness. Outbreaks of illness of variable extent and severity occur nearly every year. Such outbreaks result in significant morbidity rates in the general population and in increased mortality rates among certain high-risk patients, mainly as a result of pulmonary complications.

1	Influenza viruses are members of the Orthomyxoviridae family, of which influenza A, B, and C viruses constitute three separate genera. The designation of influenza viruses as type A, B, or C is based on antigenic characteristics of the nucleoprotein (NP) and matrix (M) protein antigens. Influenza A viruses are further subdivided (subtyped) on the basis of the surface hemagglutinin (H) and neuraminidase (N) antigens; individual strains are designated according to the site of origin, isolate number, year of isolation, and subtype—for example, influenza A/California/07/2009 (H1N1). Influenza A has 18 distinct H subtypes and 11 distinct N subtypes, of which only H1, H2, H3, N1, and N2 have been associated with epidemics of disease in humans. Avian influenza A viruses have been associated with small outbreaks and sporadic cases in humans (see below). Influenza B and C viruses are designated similarly to influenza A viruses, but H and N antigens from these viruses do not receive subtype

1	small outbreaks and sporadic cases in humans (see below). Influenza B and C viruses are designated similarly to influenza A viruses, but H and N antigens from these viruses do not receive subtype designations because intratypic variations in influenza B antigens are less extensive than those in influenza A viruses and may not occur with influenza C virus.

1	Influenza A and B viruses are major human pathogens and the most extensively studied of the Orthomyxoviridae. Type A and type B viruses are morphologically similar. The virions are irregularly shaped spherical particles, measure 80–120 nm in diameter, and have a lipid envelope from the surface of which the H and N glycoproteins project (Fig. 224-1). The hemagglutinin is the site by which the virus binds to sialic acid cell receptors, whereas the neuraminidase degrades the receptor and plays a role in the release of the virus from infected cells after replication has taken place. Influenza viruses enter cells by receptor-mediated endocytosis, forming a virus-containing endosome. The viral hemagglutinin mediates fusion of the endosomal membrane with the virus envelope, and viral nucleocapsids are subsequently released into the cytoplasm. Immune responses to the H antigen are the major determinants of protection against infection with influenza virus, whereas those to the N antigen limit

1	are subsequently released into the cytoplasm. Immune responses to the H antigen are the major determinants of protection against infection with influenza virus, whereas those to the N antigen limit viral spread and contribute to reduction of the infection. The lipid envelope of influenza A virus also contains the M proteins M1 and M2, which are involved in stabilization of the lipid envelope and in virus assembly. The virion also contains the NP antigen, which is associated with the viral genome, as well as three polymerase (P) proteins that are essential for transcription

1	FIGuRE 224-1 An electron micrograph of influenza A virus (×40,000). and synthesis of viral RNA. Two nonstructural proteins function as an interferon antagonist and posttranscriptional regulator (NS1) and a nuclear export factor (NS2 or NEP). The genomes of influenza A and B viruses consist of eight single-strand RNA segments, which code for the structural and nonstructural proteins. Because the genome is segmented, the opportunity for gene reassortment during infection is high; reassortment often takes place during infection of cells with more than one influenza A virus.

1	Influenza outbreaks occur virtually every year, although their extent and severity vary widely. Localized outbreaks take place at variable intervals, usually every 1–3 years. Global pandemics have occurred at variable intervals, but much less frequently than interpandemic outbreaks (Table 224-1). The most recent pandemic emerged in March of 2009 and was caused by an influenza A/H1N1 virus that rapidly spread worldwide over the next several months.

1	Influenza A Virus • antigenic variation and inflUenza oUtbreaks and pandemics The most extensive and severe outbreaks of influenza are caused by influenza A viruses, in part because of the remarkable propensity of the H and N antigens of these viruses to undergo periodic antigenic variation. Major antigenic variations, called antigenic shifts, are seen only with influenza A viruses and may be associated with pandemics. Minor variations are called antigenic drifts. Antigenic variation may involve the hemagglutinin alone or both the hemagglutinin and the neuraminidase. An example of an antigenic shift involving both the hemagglutinin and the neuraminidase is that of 1957, when the predominant influenza A virus subtype shifted from H1N1 to H2N2; this shift resulted in a severe pandemic, with an estimated 70,000 excess deaths (i.e., deaths in excess of the number expected without an influenza epidemic) in the United States alone. This excess mortality was significantly greater than that

1	an estimated 70,000 excess deaths (i.e., deaths in excess of the number expected without an influenza epidemic) in the United States alone. This excess mortality was significantly greater than that during interpandemic influenza seasons.

1	a As determined by retrospective serologic survey of individuals alive during those years (“seroarchaeology”). b Hemagglutinins formerly designated as Hsw and H0 are now classified as variants of H1. c From this time until 2008–2009, viruses of the H1N1 and H3N2 subtypes circulated either in alternating years or concurrently. d A novel influenza A/H1N1 virus emerged to cause this pandemic.

1	1210 In 1968, an antigenic shift involving only the hemagglutinin occurred (H2N2 to H3N2); the subsequent pandemic was less severe than that of 1957. In 1977, an H1N1 virus emerged and caused a pandemic that primarily affected younger individuals (i.e., those born after 1957). As shown in Table 224-1, H1N1 viruses circulated from 1918 to 1956; thus, individuals born prior to 1957 would be expected to have some degree of immunity to H1N1 viruses. The pandemic of 2009–2010 was caused by an A/H1N1 virus against which little immunity was present in the general population, although approximately one-third of individuals born before 1950 had some apparent immunity to related H1N1 strains. During most outbreaks of influenza A, a single subtype has circulated at a time. However, since 1977, H1N1 and H3N2 viruses have circulated simultaneously, resulting in outbreaks of varying severity. In some outbreaks, influenza B viruses have also circulated simultaneously with influenza A viruses. In

1	and H3N2 viruses have circulated simultaneously, resulting in outbreaks of varying severity. In some outbreaks, influenza B viruses have also circulated simultaneously with influenza A viruses. In 2009–2010, the pandemic A/H1N1 virus appeared to circulate nearly exclusively.

1	featUres of pandemic and interpandemic inflUenza a Pandemics provide the most dramatic evidence of the impact of influenza A. However, illnesses occurring between pandemics (interpandemic disease) also account for extensive mortality and morbidity, albeit over a longer period. In the United States, influenza was associated with an average of 23,000 excess deaths per season in 1976–2007 and with a maximum of 48,600 excess deaths during the 2003–2004 season.

1	Influenza A viruses that circulate between pandemics demonstrate antigenic drifts in the H antigen. These antigenic drifts result from point mutations in the RNA segment that codes for the hemagglutinin and occur most frequently in five hypervariable regions. Epidemiologically significant strains—that is, those with the potential to cause widespread outbreaks—exhibit changes in amino acids in at least two of the major antigenic sites in the hemagglutinin molecule. Because two point mutations are unlikely to occur simultaneously, it is believed that antigenic drifts result from point mutations occurring sequentially during the spread of virus from person to person. Antigenic drifts have been reported nearly annually since 1977 for H1N1 viruses and since 1968 for H3N2 viruses.

1	Interpandemic influenza A outbreaks usually begin abruptly, peak over a 2to 3-week period, generally last for 2–3 months, and often subside almost as rapidly as they began. In contrast, pandemic influenza may begin with rapid transmission at multiple locations, have high attack rates, and extend beyond the usual seasonality, with multiple waves of attack before or after the main outbreak. In interpandemic outbreaks, the first indication of influenza activity is an increase in the number of children with febrile respiratory illnesses who present for medical attention. This increase is followed by increases in rates of influenza-like illnesses among adults and eventually by an increase in hospital admissions for patients with pneumonia, worsening of congestive heart failure, and exacerbations of chronic pulmonary disease. Rates of absence from work and school also rise at this time. An increase in the number of deaths caused by pneumonia and influenza is generally a late observation in

1	of chronic pulmonary disease. Rates of absence from work and school also rise at this time. An increase in the number of deaths caused by pneumonia and influenza is generally a late observation in an outbreak. Attack rates have been highly variable from outbreak to outbreak in interpandemic influenza but most commonly are in the range of 10–20% of the general population.

1	Although pandemic influenza may occur throughout the year, interpandemic influenza occurs almost exclusively during the winter months in the temperate zones of the Northern and Southern hemispheres. In those locations, it is highly unusual to detect influenza A virus at other times, although rises in serum antibody titer or even outbreaks have been noted rarely during warm-weather months. In contrast, influenza virus infections occur throughout the year in the tropics. Where or how influenza A viruses persist between outbreaks in temperate zones is unknown. It is possible that the viruses are maintained in the human population on a worldwide basis by person-to-person transmission and that large population clusters support a low level of interepidemic transmission. Alternatively, human strains may persist in animal reservoirs. Convincing evidence to support either explanation is not available. In the modern era, rapid transportation may contribute to the transmission of viruses among

1	may persist in animal reservoirs. Convincing evidence to support either explanation is not available. In the modern era, rapid transportation may contribute to the transmission of viruses among widespread geographic locales.

1	The factors that result in the inception and termination of outbreaks of influenza A are incompletely understood. A major determinant of the extent and severity of an outbreak is the level of immunity in the population at risk. With the emergence of an antigenically novel influenza virus to which little or no immunity is present in a community, extensive outbreaks may occur. When the absence of immunity is worldwide, epidemic disease may spread around the globe, resulting in a pandemic. Such pandemic waves can continue for several years, until immunity in the population reaches a high level. In the years following pandemic influenza, antigenic drifts among influenza viruses result in outbreaks of variable severity in populations with high levels of immunity to the pandemic strain that circulated earlier. This situation persists until another antigenically novel pandemic strain emerges. On the other hand, outbreaks sometimes end despite the persistence of a large pool of susceptible

1	earlier. This situation persists until another antigenically novel pandemic strain emerges. On the other hand, outbreaks sometimes end despite the persistence of a large pool of susceptible individuals in the population. It has been suggested that certain influenza A viruses may be intrinsically less virulent and cause less severe disease than other variants, even in immunologically virgin subjects. If so, then other (undefined) factors besides the level of preexisting immunity must play a role in the epidemiology of influenza.

1	Avian and Swine Influenza Viruses Aquatic birds are the largest reservoir of influenza A viruses, harboring 16 hemagglutinin (H1–H16) and nine neuraminidase (N1–N9) subtypes. (In addition, H17N10 and H18N11 viruses are found in bats.) Influenza A pandemic strains in 1957 (A/H2N2) and in 1968 (A/H3N2) resulted from reassortment of gene segments between human and avian viruses. The influenza A/ H1N1 virus that caused the most severe pandemic of modern times (1918–1919) appears to have been an adaptation of an avian virus to human infection. Thus, there is concern that avian influenza viruses with novel hemagglutinin and neuraminidase antigens have the potential to emerge as pandemic strains.

1	Avian influenza A viruses have been reported to cause spo radic cases and small outbreaks in humans, usually after direct contact with birds (most commonly poultry). Sustained person-to-person transmission in the community has not been observed. Avian influenza A/H5N1 virus has been noted to cause illness in humans since 1997, with 648 cases reported to the World Health Organization as of January 2014. It is not clear whether the high observed case–fatality rate (59%) reflects preferential detection of severe cases. A/H7N7 infections have been noted in poultry industry workers; conjunctivitis was the most prominent feature, although a minority of individuals also had respiratory illness. More than 333 cases of avian A/H7N9 infection have been reported in China, with case–fatality rates of 36% among the infected patients admitted to the hospital. Most H7N9 isolates are sensitive to neuraminidase inhibitors, but a few isolates have exhibited high-level resistance to oseltamivir and

1	of 36% among the infected patients admitted to the hospital. Most H7N9 isolates are sensitive to neuraminidase inhibitors, but a few isolates have exhibited high-level resistance to oseltamivir and diminished sensitivity to zanamivir. Infections with avian H9N2 viruses have been reported primarily among children in Hong Kong and have consisted largely of mild respiratory illnesses. Mild cases of illness due to influenza H10N7 virus in Egypt and Australia have also been reported. In 2013, the first cases of human infection with avian A/ H10N8 and H6N1 viruses were described.

1	Influenza A viruses also circulate in swine but rarely infect humans. Whereas humans primarily have α-2,6-galactose receptors for hemagglutinins and birds primarily have α-2,3-galactose receptors, swine have both types of receptors. Thus, swine hosts efficiently sustain simultaneous infection with both human and avian viruses, thereby facilitating reassortment of genetic segments between viruses of both species. The pandemic A/H1N1 strain of 2009–2010 was a quadruple reassortant among swine, avian, and human influenza viruses. The influenza A virus subtypes that circulate most commonly in swine are H1N1, H1N2, and H3N2. When a predominantly swine virus causes infections in humans, it is designated a variant virus by the addition of “v” after the subtype. For example, influenza A/H3N2v virus was responsible for 321 cases of human infection reported in the United States in 2011 and 2012 and for 18 cases in 2013. Almost all of the affected patients had had close contact with swine. Only

1	was responsible for 321 cases of human infection reported in the United States in 2011 and 2012 and for 18 cases in 2013. Almost all of the affected patients had had close contact with swine. Only limited person-to-person transmission of swine influenza virus has been noted. Since 2005, 16 human cases caused by A/H1N1v virus and 5 caused by A/H1N2v virus have been detected in the United States.

1	Influenza B and C Viruses Influenza B virus causes outbreaks that are generally less extensive and are associated with less severe disease than those caused by influenza A virus, although the disease may occasionally be severe. The hemagglutinin and neuraminidase of influenza B viruses undergo less frequent and less extensive variation than those of influenza A viruses; this characteristic may account, in part, for the lesser severity of influenza B. Outbreaks of influenza B occur most frequently in schools and military camps, although outbreaks in institutions in which elderly individuals reside have also been noted on occasion. Since the 1980s, two antigenically distinct “lineages” of influenza B virus have circulated: Victoria and Yamagata.

1	In contrast to influenza A and B viruses, influenza C virus appears to be a relatively minor cause of disease in humans. It has been associated with common cold–like symptoms and occasionally with lower respiratory tract illness. The widespread prevalence of serum antibody to this virus indicates that asymptomatic infection may be common.

1	Influenza-Associated Morbidity and Mortality Rates Rates of morbidity and mortality caused by influenza outbreaks continue to be substantial. Most individuals who die in this setting have underlying diseases that place them at high risk for complications of influenza (Table 224-2). On average, there were 226,000 influenza-associated hospitalizations per year in the United States in 1979–2001. Recently, the moderately severe influenza season in 2012–2013 was associated with 381,500 hospitalizations (42 per 100,000 persons). Excess annual hospitalizations for groups of adults and children with high-risk medical conditions ranged from 40 to 1900 per 100,000 during outbreaks of influenza in 1973–2004. The most prominent high-risk conditions are chronic cardiac and pulmonary diseases and old age. Mortality rates among individuals with chronic metabolic or renal diseases or certain immunosuppressive diseases have also been elevated, although they remain lower than mortality rates among

1	age. Mortality rates among individuals with chronic metabolic or renal diseases or certain immunosuppressive diseases have also been elevated, although they remain lower than mortality rates among patients with chronic cardiopulmonary diseases. In the pandemic of 2009–2010, increased risk of severe disease was noted in children from birth to 4 years of age and in pregnant women. The morbidity rate attributable to influenza in the general population is considerable. It is estimated that interpandemic outbreaks of influenza currently incur annual economic costs of more than $87 billion in the United States. For pandemics, it is estimated that annual economic costs would range from $89.7 to $209.4 billion for attack rates of 15–35%.

1	The initial event in influenza is infection of the respiratory epithelium with influenza virus acquired from respiratory secretions of acutely infected individuals. In all likelihood, the virus is transmitted via aerosols generated by coughs and sneezes, although transmission through hand-to-hand contact, other personal contact, and even fomites may take place. Experimental evidence suggests that infection by a small-particle aerosol (particle diameter <10 μm) is more efficient than All children from birth to <5 years, especially <2 years All persons ≥50 years old Pregnant women Adults and children who have chronic pulmonary (including asthma) or car diovascular (except isolated hypertension), renal, hepatic, neurologic, hema tologic, or metabolic disorders (including diabetes mellitus) Persons who have immunosuppression (including that caused by medications or by HIV infection)

1	Children and adolescents (6 months to 18 years old) who are receiving longterm aspirin therapy and who might be at risk for Reye’s syndrome after influenza virus infection

1	Residents of nursing homes and other long-term care facilities Native Americans/Alaska Natives Persons who are morbidly obese (body mass index ≥40 kg/m2) that by larger droplets. Initially, viral infection involves the ciliated 1211 columnar epithelial cells, but it may also involve other respiratory tract cells, including alveolar cells, mucous gland cells, and macrophages. In infected cells, virus replicates within 4–6 h, after which infectious virus is released to infect adjacent or nearby cells. In this way, infection spreads from a few foci to a large number of respiratory cells over several hours. In experimentally induced infection, the incubation period of illness has ranged from 18 to 72 h, depending on the size of the viral inoculum. Histopathologic study reveals degenerative changes, including granulation, vacuolization, swelling, and pyknotic nuclei in infected ciliated cells. The cells eventually become necrotic and desquamate; in some areas, previously columnar

1	changes, including granulation, vacuolization, swelling, and pyknotic nuclei in infected ciliated cells. The cells eventually become necrotic and desquamate; in some areas, previously columnar epithelium is replaced by flattened and metaplastic epithelial cells. The severity of illness is correlated with the quantity of virus shed in secretions; thus, the degree of viral replication itself may be an important factor in pathogenesis. Despite the frequent development of systemic signs and symptoms such as fever, headache, and myalgias, influenza virus has only rarely been detected in extrapulmonary sites (including the bloodstream). Evidence suggests that the pathogenesis of systemic symptoms in influenza may be related to the induction of certain cytokines, particularly tumor necrosis factor α, interferon α, interleukin 6, and interleukin 8, in respiratory secretions and in the bloodstream.

1	The host response to influenza infections involves a complex interplay of humoral antibody, local antibody, cell-mediated immunity, interferon, and other host defenses. Serum antibody responses, which can be detected by the second week after primary infection, are measured by a variety of techniques: hemagglutination inhibition (HI), complement fixation (CF), neutralization, enzyme-linked immunosorbent assay (ELISA), and antineuraminidase antibody assay. Antibodies to the hemagglutinin appear to be the most important mediators of immunity; in several studies, HI titers of ≥40 have been associated with protection from infection. Secretory antibodies produced in the respiratory tract are predominantly of the IgA class, and secretory antibody neutralization titers of ≥4 have also been associated with protection. A variety of cell-mediated immune responses, both antigen-specific and antigen-nonspecific, can be detected early after infection and depend on the prior immune status of the

1	with protection. A variety of cell-mediated immune responses, both antigen-specific and antigen-nonspecific, can be detected early after infection and depend on the prior immune status of the host. These responses include T cell proliferative, T cell cytotoxic, and natural killer cell activity. In humans, CD8+ as well as CD4+ T lymphocytes are directed at conserved regions of internal proteins (NP, M, and P) as well as at the surface proteins H and N. Interferons can be detected in respiratory secretions shortly after the shedding of virus has begun, and rises in interferon titers coincide with decreases in virus shedding.

1	The host defense factors responsible for cessation of virus shedding and resolution of illness have not been defined specifically. Virus shedding generally stops within 2–5 days after symptoms first appear, at a time when serum and local antibody responses often are not detectable by conventional techniques, although antibody rises may be detected earlier by use of highly sensitive techniques, particularly in individuals with previous immunity to the virus. It has been suggested that interferon, cell-mediated immune responses, and/or nonspecific inflammatory responses all contribute to the resolution of illness. CD8+ cytotoxic T lymphocyte responses may be particularly important in this regard.

1	Influenza is most frequently described as a respiratory illness characterized by systemic symptoms, such as headache, feverishness, chills, myalgia, and malaise, as well as accompanying respiratory tract signs and symptoms, particularly cough and sore throat. In some cases, the onset is so abrupt that patients can recall the precise time they became ill. However, the spectrum of clinical presentations is wide, ranging from a mild, afebrile respiratory illness similar to the common cold (with either a gradual or an abrupt onset) to severe prostration with relatively few respiratory signs and symptoms. In most of the cases that come to a physician’s attention, the patient has a fever, with temperatures of 38°–41°C (100.4°–105.8°F). A rapid temperature rise within the first 24 h of illness is generally followed by gradual defervescence over 2–3 days, although, on occasion, fever may last as long as 1 week.

1	1212 Patients report a feverish feeling and chilliness, but true rigors are rare. Headache, either generalized or frontal, is often particularly troublesome. Myalgias may involve any part of the body but are most common in the legs and lumbosacral area. Arthralgias may also develop. Respiratory symptoms often become more prominent as systemic symptoms subside. Many patients have a sore throat or persistent cough, which may last for ≥1 week and which is often accompanied by substernal discomfort. Ocular signs and symptoms include pain on motion of the eyes, photophobia, and burning of the eyes. In the elderly, influenza may have a relatively subtle presentation. Typical features such as sore throat, myalgia, and even fever may be absent, and general symptoms such as anorexia, malaise, weakness, and dizziness may predominate. Physical findings are usually minimal in uncomplicated influenza. Early in the illness, the patient appears flushed, and the skin is hot and dry, although

1	weakness, and dizziness may predominate. Physical findings are usually minimal in uncomplicated influenza. Early in the illness, the patient appears flushed, and the skin is hot and dry, although diaphoresis and mottled extremities are sometimes evident, particularly in older patients. Examination of the pharynx may yield surprisingly unremarkable results despite a severe sore throat, but injection of the mucous membranes and postnasal discharge are apparent in some cases. Mild cervical lymphadenopathy may be noted, especially in younger individuals. The results of chest examination are largely negative in uncomplicated influenza, although rhonchi, wheezes, and scattered rales have been reported with variable frequency in different outbreaks. Frank dyspnea, hyperpnea, cyanosis, diffuse rales, and signs of consolidation are indicative of pulmonary complications. Patients with apparently uncomplicated influenza have been reported to have a variety of mild ventilatory defects and

1	rales, and signs of consolidation are indicative of pulmonary complications. Patients with apparently uncomplicated influenza have been reported to have a variety of mild ventilatory defects and increased alveolar-capillary diffusion gradients; thus, subclinical pulmonary involvement may be more common than is appreciated. In uncomplicated influenza, the acute illness generally resolves over 2–5 days, and most patients have largely recovered in 1 week, although cough may persist 1–2 weeks longer. In a significant minority (particularly the elderly), however, symptoms of weakness or lassitude (postinfluenza asthenia) may persist for several weeks and may prove troublesome for persons who wish to resume their full level of activity promptly. The pathogenetic basis for this asthenia is unknown, although pulmonary function abnormalities may persist for several weeks after uncomplicated influenza.

1	Complications of influenza occur most frequently in patients >65 years old and in those with certain chronic disorders, including cardiac or pulmonary diseases, diabetes mellitus, hemoglobinopathies, renal dysfunction, and immunosuppression. Pregnancy in the second or third trimester predisposes to complications with influenza. Children <5 years old (especially infants) are also at high risk for complications (Table 224-2). Pulmonary Complications • pneUmonia The most significant complication of influenza is pneumonia: “primary” influenza viral pneumonia, secondary bacterial pneumonia, or mixed viral and bacterial pneumonia (discussed below).

1	primary influenza viral pneumonia Primary influenza viral pneumonia is the least common but most severe of the pneumonic complications. It presents as acute influenza that does not resolve but instead progresses relentlessly, with persistent fever, dyspnea, and eventual cyanosis. Sputum production is generally scanty, but the sputum can contain blood. Few physical signs may be evident early in the illness. In more advanced cases, diffuse rales may be noted, and imaging findings consistent with diffuse interstitial infiltrates and/or acute respiratory distress syndrome may be present. In such cases, arterial blood-gas determinations show marked hypoxia. Viral cultures of respiratory secretions and lung parenchyma, especially if samples are taken early in illness, yield high titers of virus. In fatal cases of primary viral pneumonia, histopathologic examination reveals a marked inflammatory reaction in the alveolar septa, with edema and infiltration by lymphocytes, macrophages,

1	of virus. In fatal cases of primary viral pneumonia, histopathologic examination reveals a marked inflammatory reaction in the alveolar septa, with edema and infiltration by lymphocytes, macrophages, occasional plasma cells, and variable numbers of neutrophils. Fibrin thrombi in alveolar capillaries, along with necrosis and hemorrhage, have also been noted. Eosinophilic hyaline membranes can be found lining alveoli and alveolar ducts.

1	Primary influenza viral pneumonia has a predilection for individuals with cardiac disease, particularly those with mitral stenosis, but has also been reported in otherwise-healthy young adults as well as in older individuals with chronic pulmonary disorders. In some pandemics of influenza (notably those of 1918 and 1957), pregnancy increased the risk of primary influenza pneumonia. Subsequent epidemics of influenza have been associated with increased rates of hospitalization among pregnant women, which were also noted in the pandemic of 2009–2010.

1	secondary bacterial pneumonia Secondary bacterial pneumonia follows acute influenza. Improvement of the patient’s condition over 2–3 days is followed by a reappearance of fever along with clinical signs and symptoms of bacterial pneumonia, including cough, production of purulent sputum, and physical and x-ray signs of consolidation. The most common bacterial pathogens in this setting are Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae— organisms that can colonize the nasopharynx and that cause infection in the wake of changes in bronchopulmonary defenses. Secondary bacterial pneumonia occurs most frequently in high-risk individuals with chronic pulmonary and cardiac disease and in elderly individuals. Patients with secondary bacterial pneumonia often respond to appropriate antibiotic therapy when it is instituted promptly.

1	mixed viral and bacterial pneumonia Perhaps the most common pneumonic complications during outbreaks of influenza have mixed features of viral and bacterial pneumonia. Patients may experience a gradual progression of their acute illness or may show transient improvement followed by clinical exacerbation, with eventual manifestation of the clinical features of bacterial pneumonia. Sputum cultures may contain both influenza A virus and one of the bacterial pathogens described above. Patchy infiltrates or areas of consolidation may be detected by physical examination and chest x-ray. Patients with mixed viral and bacterial pneumonia generally have less widespread involvement of the lung than those with primary viral pneumonia, and their bacterial infections may respond to appropriate antibacterial drugs. Mixed viral and bacterial pneumonia occurs primarily in patients with chronic cardiovascular and pulmonary diseases.

1	otHer pUlmonary complications Other pulmonary complications associated with influenza include worsening of chronic obstructive pulmonary disease and exacerbation of chronic bronchitis and asthma. In children, influenza infection may present as croup. Sinusitis and otitis media (the latter occurring particularly often in children) may also be associated with influenza.

1	Extrapulmonary Complications Myositis, rhabdomyolysis, and myoglobinuria are occasional complications of influenza infection. Although myalgias are exceedingly common in influenza, true myositis is rare. Patients with acute myositis have exquisite tenderness of the affected muscles, most commonly in the legs, and may not be able to tolerate even the slightest pressure, such as the touch of bedsheets. In the most severe cases, there is frank swelling and bogginess of muscles. Serum levels of creatine phosphokinase and aldolase are markedly elevated, and an occasional patient develops renal failure from myoglobinuria. The pathogenesis of influenza-associated myositis is also unclear, although the presence of influenza virus in affected muscles has been reported.

1	Myocarditis and pericarditis were reported in association with influenza virus infection during the 1918–1919 pandemic; these reports were based largely on histopathologic findings, and these complications have been reported only infrequently since that time. Electrocardiographic changes during acute influenza are common among patients who have cardiac disease but have been ascribed most often to exacerbations of the underlying cardiac disease rather than to direct involvement of the myocardium with influenza virus. Epidemiologic data have shown an association between influenza outbreaks and increased cardiovascular-associated hospitalizations.

1	Central nervous system (CNS) complications such as encephalitis and transverse myelitis have been associated with influenza. Encephalitis is a rare but potentially serious complication that has been reported with influenza A and B virus infections. Children <5 years of age appear to be at greatest risk. The pathogenetic mechanisms by which influenza causes CNS disease are unclear. Guillain-Barré syndrome has been reported following influenza infection and, uncommonly, after influenza vaccination (see “Prophylaxis,” below). Toxic shock syndrome associated with S. aureus or group A streptococcal infection following acute influenza infection has been described (Chaps. 172 and 173).

1	Toxic shock syndrome associated with S. aureus or group A streptococcal infection following acute influenza infection has been described (Chaps. 172 and 173). Reye’s syndrome is a serious complication in children that is associated with influenza B and—to a lesser extent—influenza A virus infection as well as with varicella-zoster virus and other viral infections. An epidemiologic association between Reye’s syndrome and aspirin therapy for the antecedent viral infection has been noted; the syndrome’s incidence has decreased markedly with widespread warnings regarding aspirin use by children with acute viral respiratory infections.

1	In addition to complications involving the specific organ systems described above, influenza outbreaks include cases in which elderly and other high-risk individuals develop influenza and subsequently experience a gradual deterioration of underlying cardiovascular, pulmonary, or renal function—changes that occasionally are irreversible and lead to death. These deaths contribute to the overall excess mortality associated with influenza outbreaks.

1	During acute influenza, virus may be detected in throat swabs, nasopharyngeal swabs or washes, or sputum. Reverse-transcriptase polymerase chain reaction (RT-PCR) is the most sensitive and specific technique for detection of influenza viruses. RT-PCR can differentiate among influenza subtypes and is used for detection of avian influenza viruses. Rapid influenza diagnostic tests (RIDTs) detect influenza virus antigens by immunologic or enzymatic techniques. RIDTs yield results quickly, and some tests can distinguish between influenza A and B viruses. Although relatively specific, RIDTs vary in sensitivity with the technique and the virus to be detected.

1	Influenza virus may be isolated from tissue culture or chick embryos, but these labor-intensive procedures generally are no longer used for diagnostic purposes. Serologic methods for diagnosis require comparison of antibody titers in sera obtained during the acute illness with those in sera obtained 10–14 days after the onset of illness and are useful primarily in retrospect and for epidemiologic studies. Other laboratory tests generally are not helpful in the specific diagnosis of influenza virus infection. Leukocyte counts are variable, frequently being low early in illness and normal or slightly elevated later. Severe leukopenia has been described in overwhelming viral or bacterial infection, whereas leukocytosis with >15,000 cells/μL raises the suspicion of secondary bacterial infection.

1	During a community-wide outbreak, a clinical diagnosis of influenza can be made with a high degree of certainty in patients who present to a physician’s office with the typical febrile respiratory illness described above. In the absence of an outbreak (i.e., in sporadic or isolated cases), influenza may be difficult to differentiate on clinical grounds alone from an acute respiratory illness caused by any of a variety of respiratory viruses or by Mycoplasma pneumoniae. Severe streptococcal pharyngitis or early bacterial pneumonia may mimic acute influenza, although bacterial pneumonias generally do not run a self-limited course. Purulent sputum in which a bacterial pathogen can be detected by Gram’s staining is an important diagnostic feature in bacterial pneumonia.

1	(See also Chap. 215e) Specific antiviral therapy is available for influenza (Table 224-3): the neuraminidase inhibitors zanamivir and oseltamivir for both influenza A and influenza B and the adamantane agents amantadine and rimantadine for influenza A. The epidemiologic patterns of resistance to the influenza antiviral drugs are crucial elements in the selection of treatment. Up-to-date information on patterns of resistance to influenza antiviral drugs is available through www.cdc.gov/flu.

1	A 5-day course of oseltamivir or zanamivir reduces the duration of signs and symptoms of uncomplicated influenza by 1–1.5 days if treatment is started within 2 days of the onset of illness and may be effective if started up to 5 days after onset of symptoms. Zanamivir is administered via an oral inhalation device and may exacerbate bronchospasm in asthmatic patients. Oseltamivir has been associated with nausea and vomiting, whose frequency can be reduced by administration of the drug with food. Oseltamivir has also been associated with neuropsychiatric side effects in children. Peramivir, an investigational neuraminidase inhibitor that can be administered intravenously, is being evaluated in clinical trials, as is an intravenous form of zanamivir.

1	Amantadine and rimantadine are active only against influenza A, and widespread resistance exists among influenza A/H1N1 and A/ H3N2 viruses that are circulating currently; thus, the use of these drugs is not recommended unless influenza isolates are known to be sensitive. Amantadine or rimantadine treatment of illness caused by sensitive strains of influenza A virus reduces the duration of symptoms of uncomplicated influenza by ~50% if begun within 48 h after onset of illness—an effect similar to that of the neuraminidase inhibitors. Of amantadine recipients, 5–10% experience mild Treatment, influenza A Not approved 100 mg PO bid 100–200 mg/d

1	Treatment, influenza A Not approved 100 mg PO bid 100–200 mg/d Prophylaxis, influenza A Age 1–9, 5 mg/kg in 2 divided doses, Age ≥10, 100 mg PO bid 100–200 mg/d up to 150 mg/d a<15 kg: 30 mg bid; >15–23 kg: 45 mg bid; >23–40 kg: 60 mg bid; >40 kg: 75 mg bid. For children <1 year of age, see www.cdc.gov/h1n1flu/recommendations.htm. b<15 kg: 30 mg qd; >15–23 kg: 45 mg qd; >23–40 kg: 60 mg qd; >40 kg: 75 mg qd. For children <1 year of age, see www.cdc.gov/h1n1flu/recommendations.htm. cAmantadine and rimantadine are not currently recommended (2013–2014) because of widespread resistance in influenza A viruses. Their use may be reconsidered if viral susceptibility is reestablished.

1	1214 CNS side effects, primarily jitteriness, anxiety, insomnia, or difficulty concentrating. These side effects disappear promptly upon cessation of therapy. Rimantadine appears to be equally efficacious and is associated with less frequent CNS side effects than is amantadine. Ribavirin is a nucleoside analogue with activity against influenza A and B viruses in vitro. Its efficacy against influenza when administered as an aerosol is reportedly variable, and it is ineffective when administered orally. Its efficacy in the treatment of influenza A or B has not been established. The therapeutic efficacy of antiviral compounds in influenza has been demonstrated primarily in studies of young adults with uncomplicated disease. The effectiveness of these drugs in the treatment or prevention of complications of influenza is unclear. Pooled analyses of observational investigations and some efficacy studies have suggested that treatment with oseltamivir may reduce the frequency of lower

1	of complications of influenza is unclear. Pooled analyses of observational investigations and some efficacy studies have suggested that treatment with oseltamivir may reduce the frequency of lower respiratory complications and hospitalization. Therapy for primary influenza pneumonia is directed at maintaining oxygenation and is most appropriately undertaken in an intensive care unit, with aggressive respiratory and hemodynamic support as needed. Antibacterial drugs should be reserved for the treatment of bacterial complications of acute influenza, such as secondary bacterial pneumonia. The choice of antibiotics should be guided by Gram’s staining and culture of appropriate specimens of respiratory secretions, such as sputum. If the etiology of a case of bacterial pneumonia is unclear from an examination of respiratory secretions, empirical antibiotics effective against the most common bacterial pathogens in this setting (S. pneumoniae, S. aureus, and

1	H. influenzae) should be selected (Chaps. 171, 172, and 182). For uncomplicated influenza in individuals at low risk of complications, symptom-based rather than antiviral therapy may be considered. Acetaminophen or nonsteroidal anti-inflammatory agents can be used for relief of headache, myalgia, and fever, but salicylates should be avoided in children <18 years of age because of the possible association with Reye’s syndrome (see “Extrapulmonary Complications,” above). Because cough is ordinarily self-limited, treatment with cough suppressants generally is not indicated; codeine-containing compounds may be used if the cough is particularly troublesome. Patients should be advised to rest and maintain hydration during acute illness and to return to full activity only gradually after illness has resolved, especially if it has been severe.

1	The major public health measure for prevention of influenza is vaccination. Both inactivated (killed) and live attenuated vaccines are available and are generated from isolates of influenza A and B viruses that circulated in the previous influenza seasons and are anticipated to circulate in the upcoming season. For inactivated vaccines, 50–80% protection against influenza is expected if the vaccine virus and the currently circulating viruses are closely related. Available inactivated vaccines have been highly purified and are associated with few reactions. Up to 5% of individuals experience low-grade fever and mild systemic symptoms 8–24 h after vaccination, and up to one-third develop mild redness or tenderness at the vaccination site. Although the 1976 swine influenza vaccine appears to have been associated with an increased frequency of Guillain-Barré syndrome, influenza vaccines administered since 1976 generally have not been. Possible exceptions were noted during the 1992–1993

1	to have been associated with an increased frequency of Guillain-Barré syndrome, influenza vaccines administered since 1976 generally have not been. Possible exceptions were noted during the 1992–1993 and 1993–1994 influenza seasons, when there may have been an excess risk of this syndrome (slightly more than 1 case per 1 million vaccine recipients). Large-scale studies of vaccination with the 2009 pandemic H1N1 vaccine also suggested a possible increased risk of Guillain-Barré syndrome (1 case per 1 million vaccinees). However, the overall health risk following influenza substantially outweighs the potential risk associated with vaccination.

1	A live attenuated influenza vaccine administered by intranasal spray is available. The vaccine is generated by reassortment between currently circulating strains of influenza A and B viruses and a cold-adapted, attenuated master strain. The cold-adapted vaccine is well tolerated and highly efficacious (>90% protective) in young children; in one study, it provided protection against a circulating influenza virus that had drifted antigenically away from the vaccine strain. Live attenuated vaccine is approved for use in healthy nonpregnant persons 2–49 years of age.

1	Since 1975, influenza vaccines have been trivalent—i.e., they have contained two influenza A subtypes (H3N2 and H1N1) and one influenza B component. However, two antigenically distinct lineages of influenza B virus have circulated since the 1980s, and a quadrivalent vaccine that includes both B lineages is now available (2013–2014) as well. Quadrivalent vaccines are available in both inactivated and live-attenuated vaccine formulations. Inactivated influenza vaccines have been noted to be less immunogenic in the elderly. A higher-dose trivalent vaccine containing 60 μg of each antigen and a lower-dose, intradermally administered trivalent vaccine containing 9 μg of each antigen have been approved for use in individuals ≥65 years of age and individuals 18–64 years of age, respectively.

1	The influenza vaccines discussed above are manufactured in eggs and should not be administered to persons with true hypersensitivity to eggs. For use in this situation, an egg-free vaccine manufactured in cells through recombinant DNA techniques (Flublok®; Protein Sciences Corporation, Meriden, CT) has been approved. Active research is under way to develop vaccines with broad activity against antigenically distinct subtypes (“universal influenza vaccines”).

1	Historically, the U.S. Public Health Service has recommended influenza vaccination for certain groups at high risk for complications of influenza on the basis of age or underlying disease (Table 224-2) or for their close contacts. Although such individuals will continue to be the focus of vaccination programs, the recommendations have been progressively expanded, and immunization of the entire population above the age of 6 months has been recommended since 2010–2011. (Approved influenza vaccines are not available for infants <6 months of age.) This expanded recommendation reflects increased recognition of previously unappreciated risk factors (e.g., obesity, postpartum conditions, and racial or ethnic influences) as well as an appreciation that more widespread use of vaccine is required for influenza control. Inactivated vaccines may be administered safely to immunocompromised patients. Influenza vaccination is not associated with exacerbations of chronic nervous system diseases such

1	influenza control. Inactivated vaccines may be administered safely to immunocompromised patients. Influenza vaccination is not associated with exacerbations of chronic nervous system diseases such as multiple sclerosis. Vaccine should be administered early in the autumn before influenza outbreaks occur and should then be given annually to maintain immunity against the most current influenza virus strains.

1	Although antiviral drugs provide chemoprophylaxis against influenza, their use for that purpose has been limited because of concern about current and future development of resistance. Chemoprophylaxis with oseltamivir or zanamivir has been 84–89% efficacious against influenza A and B (Table 224-3). Chemoprophylaxis with amantadine or rimantadine is no longer recommended because of widespread resistance to these drugs. In earlier studies with sensitive viruses, prophylaxis with amantadine or rimantadine was 70–100% effective against illness associated with influenza A virus.

1	Chemoprophylaxis for healthy persons after community exposure generally is not recommended but may be considered for individuals at high risk of complications who have had close contact with an acutely ill person with influenza. During an outbreak, antiviral chemoprophylaxis can be administered simultaneously with inactivated vaccine because the drugs do not interfere with an immune response to the vaccine. However, concurrent administration of chemoprophylaxis and live attenuated vaccine may interfere with the immune response to the latter. Antiviral drugs should not be administered until at least 2 weeks after administration of live vaccine, and administration of live vaccine should not begin until at least 48 h after antiviral drug administration has been stopped. Chemoprophylaxis may also be considered to control nosocomial outbreaks of influenza. For that purpose, prophylaxis should be instituted promptly when influenza activity is detected and must be continued daily for the

1	also be considered to control nosocomial outbreaks of influenza. For that purpose, prophylaxis should be instituted promptly when influenza activity is detected and must be continued daily for the duration of the outbreak.

1	225e-1 The Human Retroviruses Dan L. Longo, Anthony S. Fauci The retroviruses, which make up a large family (Retroviridae), infect mainly vertebrates. These viruses have a unique replication cycle whereby their genetic information is encoded by RNA rather than DNA. Retroviruses contain an RNA-dependent DNA polymerase STRUCTURE AND LIFE CYCLE All retroviruses are similar in structure, genome organization, and mode of replication. Retroviruses are 70–130 nm in diameter and have a lipid-containing envelope surrounding an icosahedral capsid with a dense inner core. The core contains two identical copies of the single-strand RNA genome. The RNA molecules are 8–10 kb long and are complexed with reverse transcriptase and tRNA. Other viral proteins, such as integrase, are also components of the virion particle. The RNA has features usually found in mRNA: a cap site at the 5′ end of the mol-ecule, which is important in the initiation of mRNA translation, and 225e SECTion 14 infECTionS DuE To

1	virion particle. The RNA has features usually found in mRNA: a cap site at the 5′ end of the mol-ecule, which is important in the initiation of mRNA translation, and 225e SECTion 14 infECTionS DuE To HumAn immunoDEfiCiEnCy ViRuS AnD oTHER HumAn RETRoViRuSES (a reverse transcriptase) that directs the synthesis of a DNA form of the viral genome after infection of a host cell. The designation retrovirus denotes that information in the form of RNA is transcribed into DNA in the host cell—a sequence that overturned a central dogma of molecular biology: that information passes unidirectionally from DNA to RNA to protein. The observation that RNA was the source of genetic information in the causative agents of certain animal tumors led to a number of paradigm-shifting biologic insights regarding not only the direction of genetic information passage but also the viral etiology of certain cancers and the concept of oncogenes as normal host genes scavenged and altered by a viral vector.

1	The family Retroviridae includes seven subfamilies (Table 225e-1). Members of two of the families infect humans with pathologic consequences: the deltaretroviruses, of which human T cell lymphotropic virus (HTLV) type 1 is the most important in humans; and lentiviruses, of which HIV is the most important in humans.

1	The wide variety of interactions of a retrovirus with its host range from completely benign events (e.g., silent carriage of endogenous retroviral sequences in the germline genome of many animal species) to rapidly fatal infections (e.g., exogenous infection with an oncogenic virus such as Rous sarcoma virus in chickens). The ability of retroviruses to acquire and alter the structure and function of host cell sequences has revolutionized our understanding of molecular carcinogenesis. The viruses can insert into the germline genome of the host cell and behave as a transposable or movable genetic element. They can activate or inactivate genes near the site of integration into the genome. They can rapidly alter their own genome by recombination and mutation under selective environmental stimuli.

1	Most human viral diseases occur as a consequence of tissue destruction either directly by the virus itself or indirectly by the host’s response to the virus. Although these mechanisms are operative in retroviral infections, retroviruses have additional mechanisms of inducing disease, including the malignant transformation of an infected cell and the induction of an immunodeficiency state that renders the host susceptible to opportunistic diseases (infections and neoplasms; Chap. 226). a polyadenylation site at the 3′ end, which influences mRNA turnover (i.e., messages with shorter polyA tails turn over faster than messages with longer polyA tails). However, the retroviral RNA is not translated; instead it is transcribed into DNA. The DNA form of the retroviral genome is called a provirus.

1	The replication cycle of retroviruses proceeds in two phases (Fig. 225e-1). In the first phase, the virus enters the cytoplasm after binding to one or more specific cell-surface receptors; the viral RNA and reverse transcriptase synthesize a double-strand DNA version of the RNA template; and the provirus moves into the nucleus and integrates into the host cell genome. This proviral integration is permanent. Although some animal retroviruses integrate into a single specific site of the host genome in every infected cell, the human retroviruses integrate randomly. This first phase of replication depends entirely on gene products in the virus. The second phase includes the synthesis and processing of viral genomes, mRNAs, and proteins using host cell machinery, often under the influence of viral gene products. Virions are assembled and released from the cell by budding from the membrane; host cell membrane proteins are frequently incorporated into the envelope of the virus. Proviral

1	of viral gene products. Virions are assembled and released from the cell by budding from the membrane; host cell membrane proteins are frequently incorporated into the envelope of the virus. Proviral integration occurs during the S-phase of the cell cycle; thus, in general, nondividing cells are resistant to retroviral infection. Only the lentiviruses are able to infect nondividing cells. Once a host cell is infected, it is infected for the life of the cell. Retroviral genomes include both coding and noncoding sequences (Fig. 225e-2). In general, noncoding sequences are important recognition signals for DNA or RNA synthesis or processing events and are located in the 5′ and 3′ terminal regions of the genome. All retroviral genomes are terminally redundant, containing identical sequences called long terminal repeats (LTRs). The ends of the retroviral RNA genome differ slightly in sequence from the integrated retroviral DNA. In the latter, the LTR sequences are repeated in both the 5′

1	called long terminal repeats (LTRs). The ends of the retroviral RNA genome differ slightly in sequence from the integrated retroviral DNA. In the latter, the LTR sequences are repeated in both the 5′ and the 3′ terminus of the virus. The LTRs contain sequences involved in initiating the expression of the viral proteins, the integration of the provirus, and the polyadenylation of viral RNAs. The primer binding site, which is critical for the initiation of reverse transcription, and the viral packaging sequences are located outside the LTR sequences. The coding regions include the gag (group-specific antigen, core protein), pol (RNA-dependent DNA polymerase), and env (envelope) genes. The gag gene encodes a precursor polyprotein that is cleaved to form three to five capsid proteins; a fraction of the Gag precursor proteins also contain a protease responsible for cleaving the Gag and Pol polyproteins. A Gag-Pol polyprotein gives rise to the protease that is responsible for cleaving the

1	of the Gag precursor proteins also contain a protease responsible for cleaving the Gag and Pol polyproteins. A Gag-Pol polyprotein gives rise to the protease that is responsible for cleaving the Gag-Pol polyprotein. The pol gene encodes three proteins: the reverse transcriptase, the integrase, and the protease. The reverse transcriptase copies the viral RNA into the double-strand DNA provirus, which inserts itself into the host cell DNA via the action of integrase. The protease cleaves the Gag-Pol polyprotein into smaller protein products. The env gene encodes the envelope glycoproteins: one protein that binds to specific surface receptors and determines what cell types can be infected and a smaller transmembrane protein that anchors the complex to the envelope. Fig. 225e-3 shows how the retroviral gene products make up the virus structure.

1	CHAPTER 225e The Human Retroviruses Adsorption to specific receptor

1	FIgURE 225e-1 The life cycle of retroviruses. A. Overview of virus replication. The retrovirus enters a target cell by binding to a specific cell-surface receptor; once the virus is internalized, its RNA is released from the nucleocapsid and is reverse-transcribed into proviral DNA. The provirus is inserted into the genome and then transcribed into RNA; the RNA is translated; and virions assemble and are extruded from the cell membrane by budding. B. Overview of retroviral gene expression. The provirus is transcribed, capped, and polyadenylated. Viral RNA molecules then have one of three fates: they are exported to the cytoplasm, where they are packaged as the viral RNA in infectious viral particles; they are spliced to form the message for the envelope polyprotein; or they are translated into Gag and Pol proteins. Most of the messages for the Pol protein fail to initiate Pol translation because of a stop codon before its initiation; however, in a fraction of the messages, the stop

1	into Gag and Pol proteins. Most of the messages for the Pol protein fail to initiate Pol translation because of a stop codon before its initiation; however, in a fraction of the messages, the stop codon is missed and the Pol proteins are translated.

1	(Modified from JM Coffin, in BN Fields, DM Knipe [eds]: Fields Virology. New York, Raven, 1990; with permission.)

1	HTLVs have a region between env and the 3′ LTR that encodes several proteins and transcripts in overlapping reading frames (Fig. 225e-2). Tax is a 40-kDa protein that does not bind to DNA but induces the expression of host cell transcription factors that alter host cell gene expression and is capable of inducing cell transformation under certain circumstances. Rex is a 27-kDa protein that regulates the expression of viral mRNAs. Other transcripts from this region (p12, p13, p30) tend to restrict expression of viral genes and diminish the immunogenicity of infected cells. The protein of HBZ, a product of the complementary proviral DNA strand, interacts with many cellular transcription factors and signaling proteins. It stimulates proliferation of infected cells and is the only viral product universally expressed in HTLV-1-infected tumor cells. These proteins are produced from messages that are similar but that are spliced differently from overlapping but distinct exons.

1	The lentiviruses in general, and HIV-1 and -2 in particular, contain a larger genome than other pathogenic retroviruses. They contain an untranslated region between pol and env that encodes portions of several proteins, varying with the reading frame into which the mRNA is spliced. Tat is a 14-kDa protein that augments the expression of virus from the LTR. The Rev protein of HIV-1, similar to the Rex protein of HTLV, regulates RNA splicing and/or RNA transport. The Nef protein downregulates CD4, the cellular receptor for HIV; alters host T cell–activation pathways; and enhances viral infectivity. The Vif protein is necessary for the proper assembly of the HIV nucleoprotein core in many types of cells; without Vif, proviral DNA is not efficiently produced in these infected cells. In addition, the Vif protein targets APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide, a cytidine deaminase that mutates the viral sequence) for proteasomal degradation, thus blocking its

1	the Vif protein targets APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide, a cytidine deaminase that mutates the viral sequence) for proteasomal degradation, thus blocking its virus-suppressing effect. Vpr, Vpu (HIV-1 only), and Vpx (HIV-2 only) are viral proteins encoded by translation of the same message in different reading frames. As noted above, oncogenic retroviruses depend on cell proliferation for their replication; lentiviruses can infect nondividing cells, largely through effects mediated by Vpr. Vpr facilitates transport of the provirus into the nucleus and can induce other cellular changes, such as G2 growth arrest and differentiation of some target cells. Vpx is structurally related to Vpr, but its functions are not fully defined. Vpu promotes the degradation of CD4 in the endoplasmic reticulum and stimulates the release of virions from infected cells.

1	Retroviruses can be either exogenously acquired (by infection with an infected cell or a free virion capable of replication) or transmitted in the germline as endogenous virus. Endogenous retroviruses are often replication defective. The human genome contains endogenous retroviral sequences, but there are no known replication-competent endogenous retroviruses in humans.

1	In general, viruses that contain only the gag, pol, and env genes either are not pathogenic or take a long time to induce disease; these observations indicate the importance of the other regulatory genes in viral disease pathogenesis. The pathogenesis of neoplastic transformation by retroviruses relies on the chance integration of the provirus at a spot in the genome resulting in the expression of a cellular gene (protooncogene) that becomes transforming by virtue of its unregulated expression. For example, avian leukosis virus causes B cell leukemia by inducing the expression of myc. Some retroviruses possess captured and altered cellular genes near their integration site, and these viral oncogenes can transform the infected host cell. Viruses that have oncogenes often have lost a portion of their genome that is required for replication. Such viruses need helper viruses to reproduce, a feature that may explain why these acute transforming retroviruses are rare in nature. All human

1	of their genome that is required for replication. Such viruses need helper viruses to reproduce, a feature that may explain why these acute transforming retroviruses are rare in nature. All human retroviruses identified to date are exogenous and are not acutely transforming (i.e., they lack a transforming oncogene).

1	These remarkable properties of retroviruses have led to experimental efforts to use them as vectors to insert specific genes into particular cell types, a process known as gene therapy or gene transfer. The process could be used to repair a genetic defect or to introduce a new property that could be used therapeutically; for example, a gene (e.g., thymidine kinase) that would make a tumor cell susceptible to killing by a drug (e.g., ganciclovir) could be inserted. One source of concern about the use of retroviral vectors in humans is that replication-competent viruses might rescue endogenous retroviral replication, with unpredictable results. This concern is not merely hypothetical: the detection of proteins encoded by endogenous retroviral sequences on the surface of cancer cells implies that the genetic events leading to the cancer were able to activate the synthesis of these usually silent genes.

1	HTLV-1 was isolated in 1980 from a T cell lymphoma cell line from a patient originally thought to have cutaneous T cell lymphoma. Later it became clear that the patient had a distinct form of lymphoma (originally reported in Japan) called adult T cell leukemia/lymphoma (ATL). Serologic data have determined that HTLV-1 is the cause of at least two important diseases: ATL and tropical spastic paraparesis, also LTR GAG POL LTR LTR MA CA NC PR RT GP46 p21HTLV-I,II TAX, p40 REX, p27 p30 TAT, p14 PR POL REV, p19

1	FIgURE 225e-2 Genomic structure of retroviruses. The murine leukemia virus MuLV has the typical three structural genes: gag, pol, and env. The gag region gives rise to three proteins: matrix (MA), capsid (CA), and nucleic acid–binding (NC) proteins. The pol region encodes both a protease (PR) responsible for cleaving the viral polyproteins and a reverse transcriptase (RT ). In addition, HIV pol encodes an integrase (IN). The env region encodes a surface protein (SU) and a small transmembrane protein ( TM). The human retroviruses have additional gene products translated in each of the three possible reading frames. HTLV-1 and HTLV-2 have tax and rex genes with exons on either side of the env gene. HIV-1 and HIV-2 have six accessory gene products: tat, rev, vif, nef, vpr, and either vpu (in HIV-1) or vpx (in HIV-2). The genes for these proteins are located mainly between the pol and env genes. GP, glycoprotein; HBZ, HTLV-1 basic leucine zipper domain–containing protein; LTR, long termi

1	HIV-1) or vpx (in HIV-2). The genes for these proteins are located mainly between the pol and env genes. GP, glycoprotein; HBZ, HTLV-1 basic leucine zipper domain–containing protein; LTR, long termi nal repeat.

1	called HTLV-1-associated myelopathy (HAM). HTLV-1 may also play a role in infective dermatitis, arthritis, uveitis, and Sjögren’s syndrome. Two years after the isolation of HTLV-1, HTLV-2 was isolated from a patient with an unusual form of hairy cell leukemia that affected

1	Two years after the isolation of HTLV-1, HTLV-2 was isolated from a patient with an unusual form of hairy cell leukemia that affected FIgURE 225e-3 Schematic structure of human retroviruses. The surface glycoprotein (SU) is responsible for binding to receptors of host cells. The transmembrane protein (TM) anchors SU to the virus. NC is a nucleic acid–binding protein found in association with the viral RNA. A protease (PR) cleaves the polyproteins encoded by the gag, pol, and env genes into their functional components. RT is reverse transcriptase, and IN is an integrase present in some retroviruses (e.g., HIV-1) that facilitates insertion of the provirus into the host genome. The matrix protein (MA) is a Gag protein closely associated with the lipid of the envelope. The capsid protein (CA) forms the major internal structure of the virus, the core shell.

1	T cells. Epidemiologic studies of HTLV-2 failed to reveal a consistent disease association. Similarly, HTLV-3 and HTLV-4 have been identified but have no known disease association. Because the biology of HTLV-1 and that of HTLV-2 are similar, the following discussion will focus on HTLV-1.

1	Human glucose transporter protein 1 (GLUT-1) functions as a receptor for HTLV-1, probably acting together with neuropilin-1 (NRP1) and heparan sulfate proteoglycans. Generally, only T cells are productively infected, but infection of B cells and other cell types is occasionally detected. The most common outcome of HTLV-1 infection is latent carriage of randomly integrated provirus in CD4+ T cells. HTLV-1 does not contain an oncogene and does not insert into a unique site in the genome. Indeed, most infected cells express no viral gene products. The only viral gene product that is routinely expressed in tumor cells transformed by HTLV-1 in vivo is hbz. The tax gene is thought to be critical to the transformation process but is not expressed in the tumor cells of many ATL patients, possibly because of the immunogenicity of tax-expressing cells. Cells transformed in vitro, by contrast, actively transcribe HTLV-1 RNA and produce infectious virions. Most HTLV-1-transformed cell lines are

1	because of the immunogenicity of tax-expressing cells. Cells transformed in vitro, by contrast, actively transcribe HTLV-1 RNA and produce infectious virions. Most HTLV-1-transformed cell lines are the result of the infection of a normal host T cell in vitro. It is difficult to establish cell lines derived from authentic ATL cells.

1	Although tax does not itself bind to DNA, it does induce the expression of a wide range of host cell gene products, including transcription factors (especially c-rel/NF-κB, ets-1 and -2, and members of the fos/jun family), cytokines (e.g., interleukin [IL] 2, granulocytemacrophage colony-stimulating factor, and tumor necrosis factor), membrane proteins and receptors (major histocompatibility [MHC] molecules and IL-2 receptor α), and chromatin remodeling complexes. The genes activated by tax are generally controlled by transcription factors of the c-rel/NF-κB and cyclic AMP response element binding (CREB) protein families. It is unclear how this induction of host

1	CHAPTER225e The Human Retroviruses 225e-4 gene expression leads to neoplastic transformation; tax can interfere with G1 and mitotic cell-cycle checkpoints, block apoptosis, inhibit DNA repair, and promote antigen-independent T cell proliferation. Induction of a cytokine–autocrine loop has been proposed; however, IL-2 is not the crucial cytokine. The involvement of IL-4, IL-7, and IL-15 has been proposed. In light of the irregular expression of tax in ATL cells, it has been suggested that tax is important in the early phases of transformation but is not essential for the maintenance of the transformed state. The maintenance role is thought to be due to hbz expression. As is clear from the epidemiology of HTLV-1 infection, transformation of an infected cell is a rare event and may depend on heterogeneous second, third, or fourth genetic hits. No consistent chromosomal abnormalities have been described in ATL; however, aneuploidy is common and individual cases with p53 mutations and

1	on heterogeneous second, third, or fourth genetic hits. No consistent chromosomal abnormalities have been described in ATL; however, aneuploidy is common and individual cases with p53 mutations and translocations involving the T cell receptor genes on chromosome 14 have been reported. Tax may repress certain DNA repair enzymes, permitting the accumulation of genetic damage that would normally be repaired. However, the molecular pathogenesis of HTLV-1-induced neoplasia is not fully understood.

1	FEATURES OF HTLV-1 INFECTION Epidemiology HTLV-1 infection is transmitted in at least three ways: from mother to child, especially via breast milk; through sexual activity, more commonly from men to women; and through the blood—via contaminated transfusions or contaminated needles. The virus is most commonly transmitted perinatally. Compared with HIV, which can be transmitted in cell-free form, HTLV-1 is less infectious, and its transmission usually requires cell-to-cell contact.

1	HTLV-1 is endemic in southwestern Japan and Okinawa, where >1 million persons are infected. Antibodies to HTLV-1 are present in the serum of up to 35% of Okinawans, 10% of residents of the Japanese island of Kyushu, and <1% of persons in nonendemic regions of Japan. Despite this high prevalence of infection, only ~500 cases of ATL are diagnosed in this area each year. Clusters of infection have been noted in other areas of the Orient, such as Taiwan; in the Caribbean basin, including northeastern South America; in northwestern South America; in central and southern Africa; in Italy, Israel, Iran, and Papua New Guinea; in the Arctic; and in the southeastern part of the United States (Fig. 225e-4). An estimated 5–10 million persons have HTLV-1 infection worldwide.

1	Progressive spastic or ataxic myelopathy developing in an individual who is HTLV-1 positive (i.e., who has serum antibodies to HTLV-1) may be due to direct infection of the nervous system with the virus, but destruction of the pyramidal tracts appears to involve HTLV-1-infected CD4+ T cells; a similar disorder may result from infection with HIV or HTLV-2. In rare instances, patients with HAM are seronegative but have detectable antibody to HTLV-1 in cerebrospinal fluid (CSF).

1	The cumulative lifetime risk of developing ATL is 3% among HTLV-1-infected patients, with a threefold greater risk among men than among women; a similar cumulative risk is projected for HAM (4%), but with women more commonly affected than men. The distribution of these two diseases overlaps the distribution of HTLV-1, with >95% of affected patients showing serologic evidence of HTLV-1 infection. The latency period between infection and the emergence of disease is 20–30 years for ATL. For HAM, the median latency period is ~3.3 years (range, 4 months to 30 years). The development of ATL is rare among persons infected by blood products; however, ~20% of patients with HAM acquire HTLV-1 from contaminated blood. ATL is more common among perinatally infected individuals, whereas HAM is more common among persons infected via sexual transmission.

1	Associated Diseases • ATL Four clinical types of HTLV-1-induced neoplasia have been described: acute, lymphomatous, chronic, and smoldering. All of these tumors are monoclonal proliferations of CD4+ postthymic T cells with clonal proviral integrations and clonal T cell receptor gene rearrangements.

1	AcuTe ATL About 60% of patients who develop malignancy have classic acute ATL, which is characterized by a short clinical prodrome (~2 weeks between the first symptoms and the diagnosis) and an aggressive natural history (median survival period, 6 months). The clinical picture is dominated by rapidly progressive skin lesions, pulmonary involvement, hypercalcemia, and lymphocytosis with cells containing lobulated or “flower-shaped” nuclei (see Fig. 134-10). The malignant cells have monoclonal proviral integrations and express CD4, CD3, and CD25 (low-affinity IL-2 receptors) on their surface. Serum levels of CD25 can be used as a tumor marker. Anemia and thrombocytopenia are rare. The skin lesions may be difficult to distinguish from those in mycosis fungoides. Lytic bone lesions, which are common, do not contain tumor cells but rather are composed of osteolytic cells, usually without osteoblastic activity. Despite the leukemic picture, bone marrow involvement is patchy in most cases.

1	The hypercalcemia of ATL is multifactorial; the tumor cells produce osteoclast-activating factors (tumor necrosis factor α, IL-1, lymphotoxin) and can also produce a parathyroid hormone–like molecule. Affected patients have an underlying immunodeficiency that makes them susceptible to opportunistic infections similar to those seen in patients with AIDS (Chap. 226). The pathogenesis of the immunodeficiency is unclear. Pulmonary infiltrates in ATL patients reflect leukemic infiltration half the time and opportunistic infections with organisms such as Pneumocystis and other fungi the other half. Gastrointestinal symptoms are nearly always related to opportunistic infection. Strongyloides stercoralis is a gastrointestinal parasite that has a pattern of endemic distribution similar to that of HTLV-1. HTLV-1-infected persons also infected with this parasite may develop ATL more often or more rapidly than those without Strongyloides infections. Serum concentrations of lactate dehydrogenase

1	HTLV-1. HTLV-1-infected persons also infected with this parasite may develop ATL more often or more rapidly than those without Strongyloides infections. Serum concentrations of lactate dehydrogenase and alkaline

1	FIgURE 225e-4 Global distribution of HTLV-1 infection. Countries with a prevalence of HTLV-1 infection of 1–5% are shaded darkly. Note that the distribution of infected patients is not uniform in endemic countries. For example, the people of southwestern Japan and northeastern Brazil are more commonly affected than those in other regions of those countries. phosphatase are often elevated in ATL. About 10% of patients have leptomeningeal involvement leading to weakness, altered mental status, paresthesia, and/or headache. Unlike other forms of central nervous system (CNS) lymphoma, ATL may be accompanied by normal CSF protein levels. The diagnosis depends on finding ATL cells in the CSF (Chap. 134).

1	LymphomATous ATL The lymphomatous type of ATL occurs in ~20% of patients and is similar to the acute form in its natural history and clinical course, except that circulating abnormal cells are rare and lymphadenopathy is evident. The histology of the lymphoma is variable but does not influence the natural history. In general, the diagnosis is suspected on the basis of the patient’s birthplace (see “Epidemiology,” above) and the presence of skin lesions and hypercalcemia. The diagnosis is confirmed by the detection of antibodies to HTLV-1 in serum. chronic ATL Patients with the chronic form of ATL generally have normal serum levels of calcium and lactate dehydrogenase and no involvement of the CNS, bone, or gastrointestinal tract. The median duration of survival for these patients is 2 years. In some cases, chronic ATL progresses to the acute form of the disease.

1	smoLdering ATL Fewer than 5% of patients have the smoldering form of ATL. In this form, the malignant cells have monoclonal proviral integration; <5% of peripheral blood cells exhibit typical morphologic abnormalities; hypercalcemia, adenopathy, and hepatosplenomegaly do not develop; the CNS, the bones, and the gastrointestinal tract are not involved; and skin lesions and pulmonary lesions may be present. The median survival period for this small subset of patients appears to be ≥5 years.

1	hAm (TropicAL spAsTic pArApAresis) In contrast to ATL, in which there is a slight predominance of male patients, HAM affects female patients disproportionately. HAM resembles multiple sclerosis in certain ways (Chap. 458). The onset is insidious. Symptoms include weakness or stiffness in one or both legs, back pain, and urinary incontinence. Sensory changes are usually mild, but peripheral neuropathy may develop. The disease generally takes the form of slowly progressive and unremitting thoracic myelopathy; one-third of patients are bedridden within 10 years of diagnosis, and one-half are unable to walk unassisted by this point. Patients display spastic paraparesis or paraplegia with hyperreflexia, ankle clonus, and extensor plantar responses. Cognitive function is usually spared; cranial nerve abnormalities are unusual.

1	Magnetic resonance imaging (MRI) reveals lesions in both the white matter and the paraventricular regions of the brain as well as in the spinal cord. Pathologic examination of the spinal cord shows symmetric degeneration of the lateral columns, including the corticospinal tracts; some cases involve the posterior columns as well. The spinal meninges and cord parenchyma contain an inflammatory infiltrate with myelin destruction.

1	HTLV-1 is not usually found in cells of the CNS but may be detected in a small population of lymphocytes present in the CSF. In general, HTLV-1 replication is greater in HAM than in ATL, and patients with HAM have a stronger immune response to the virus. Antibodies to HTLV-1 are present in the serum and appear to be produced in the CSF of HAM patients, where titers are often higher than in the serum. The pathophysiology of HAM may involve the induction of autoimmune destruction of neural cells by T cells with specificity for viral components such as Tax or Env proteins. One theory is that susceptibility to HAM may be related to the presence of human leukocyte antigen (HLA) alleles capable of presenting viral antigens in a fashion that leads to autoimmunity. Insufficient data are available to confirm an HLA association. However, antibodies in the sera of HAM patients have been shown to bind a neuron-specific antigen (heteronuclear ribonuclear protein A1 [hnRNP A1]) and to interfere

1	to confirm an HLA association. However, antibodies in the sera of HAM patients have been shown to bind a neuron-specific antigen (heteronuclear ribonuclear protein A1 [hnRNP A1]) and to interfere with neurotransmission in vitro.

1	It is unclear what factors influence whether HTLV-1 infection will cause disease and, if it does, whether it will induce a neoplasm (ATL) or an autoimmune disorder (HAM). Differences in viral strains, the susceptibility of particular MHC haplotypes, the route of HTLV-1 infection, the viral load, and the nature of the HTLV-1-related immune 225e-5 response are putative factors, but few definitive data are available. oTher puTATive hTLv-1-reLATed diseAses Even in the absence of the full clinical picture of HAM, bladder dysfunction is common in HTLV-1-infected women. In areas where HTLV-1 is endemic, diverse inflammatory and autoimmune diseases have been attributed to the virus, including uveitis, dermatitis, pneumonitis, rheumatoid arthritis, and polymyositis. However, a causal relationship between HTLV-1 and these illnesses has not been established.

1	Prevention Women in endemic areas should not breast-feed their children, and blood donors should be screened for serum antibodies to HTLV-1. As in the prevention of HIV infection, the practice of safe sex and the avoidance of needle sharing are important.

1	For the small number of patients who develop HTLV-1-related disease, therapies are not curative. In patients with the acute and lymphomatous types of ATL, the disease progresses rapidly. Hypercalcemia is generally controlled by glucocorticoid administration and cytotoxic therapy directed against the neoplasm. The tumor is highly responsive to combination chemotherapy that is used against other forms of lymphoma; however, patients are susceptible to overwhelming bacterial and opportunistic infections, and ATL relapses within 4–10 months after remission in most cases. The combination of interferon α and zidovudine may extend survival. Because viral replication is not clearly associated with ATL progression, zidovudine is probably effective through its cytotoxic effects (as a chain-terminating thymidine analogue) rather than its antiviral effects. LSG15, a multidrug chemotherapy program developed in Japan, induces complete responses in about one-third of patients, about half of whom

1	thymidine analogue) rather than its antiviral effects. LSG15, a multidrug chemotherapy program developed in Japan, induces complete responses in about one-third of patients, about half of whom survive more than 2 years; however, but the median survival time is about 13 months. A pilot trial suggested that mogamulizumab, an antibody to CCR4 (a receptor for a number of chemokines, including RANTES and TARC), improved response rates when added to chemotherapy. An experimental approach using an yttrium 90–labeled or toxin-conjugated antibody to the IL-2 receptor appears promising but is not widely available. Patients with the chronic or smoldering form of ATL may be managed with an expectant approach: treat any infections, and watch and wait for signs of progression to acute disease.

1	Patients with HAM may obtain some benefit from the use of glucocorticoids to reduce inflammation. Antiretroviral regimens have not been effective. In one study, danazol (200 mg three times daily) produced significant neurologic improvement in five of six treated patients, with resolution of urinary incontinence in two cases, decreased spasticity in three, and restoration of the ability to walk after confinement to a wheelchair in two. Antibody to IL-15 receptor β chain has been tested with some promising clinical effects in small numbers of patients. Physical therapy and rehabilitation are important components of management. CHAPTER 225e The Human Retroviruses Epidemiology HTLV-2 is endemic in certain Native American tribes and in Africa. It is generally considered to be a New

1	CHAPTER 225e The Human Retroviruses Epidemiology HTLV-2 is endemic in certain Native American tribes and in Africa. It is generally considered to be a New World virus that was brought from Asia to the Americas 10,000–40,000 years ago during the migration of infected populations across the Bering land bridge. The mode of transmission of HTLV-2 is probably the same as that of HTLV-1 (see above). HTLV-2 may be less readily transmitted sexually than HTLV-1.

1	Studies of large cohorts of injection drug users with serologic assays that reliably distinguish HTLV-1 from HTLV-2 indicated that the vast majority of HTLV-positive cohort members were infected with HTLV-2. The seroprevalence of HTLV in a cohort of 7841 injection drug users from drug treatment centers in Baltimore, Chicago, Los Angeles, New Jersey (Asbury Park and Trenton), New York City (Brooklyn and Harlem), Philadelphia, and San Antonio was 20.9%, with >97% of cases due to HTLV-2. The seroprevalence of HTLV-2 was higher in the Southwest and the Midwest than in the Northeast. In contrast, the seroprevalence of HIV-1 was higher in the Northeast than in the Southwest or the Midwest. Approximately 3% of the cohort members were infected with both HTLV-2 and HIV-1. The seroprevalence of HTLV-2 increased linearly with age. Women were significantly more likely to be infected with HTLV-2 than were men; the virus is thought to be more efficiently transmitted from male to female than from

1	increased linearly with age. Women were significantly more likely to be infected with HTLV-2 than were men; the virus is thought to be more efficiently transmitted from male to female than from female to male.

1	Associated Diseases Although HTLV-2 was isolated from a patient with a T cell variant of hairy cell leukemia, this virus has not been consistently associated with a particular disease and in fact has been thought of as “a virus searching for a disease.” However, evidence is accumulating that HTLV-2 may play a role in certain neurologic, hematologic, and dermatologic diseases. These data require confirmation, particularly in light of the previous confusion regarding the relative prevalences of HTLV-1 and HTLV-2 among injection drug users. Prevention Avoidance of needle sharing, adherence to safe-sex practices, screening of blood (by assays for HTLV-1, which also detect HTLV-2), and avoidance of breast-feeding by infected women are important principles in the prevention of spread of HTLV-2.

1	HIV-1 and HIV-2 are members of the lentivirus subfamily of Retroviridae and are the only lentiviruses known to infect humans. The lentiviruses are slow-acting by comparison with viruses that cause acute infection (e.g., influenza virus) but not by comparison with other retroviruses. The features of acute primary infection with HIV resemble those of more classic acute infections. The characteristic chronicity of HIV disease is consistent with the designation lentivirus. For a detailed discussion of HIV, see Chap. 226. Human Immunodeficiency virus disease: AIds and Related disorders Anthony S. Fauci, H. Clifford Lane AIDS was first recognized in the United States in the summer of 1981, 226 when the U.S. Centers for Disease Control and Prevention (CDC) reported the unexplained occurrence of Pneumocystis jiroveci (formerly P. carinii) pneumonia in five previously healthy homosexual men in Los Angeles and of Kaposi’s sarcoma (KS) with or without

1	P. jiroveci pneumonia and other opportunistic infections in 26 previously healthy homosexual men in New York, San Francisco, and Los Angeles. The disease was soon recognized in male and female injection drug users; in hemophiliacs and blood transfusion recipients; among female sexual partners of men with AIDS; and among infants born to mothers with AIDS. In 1983, human immunodeficiency virus (HIV) was isolated from a patient with lymphadenopathy, and by 1984 it was demonstrated clearly to be the causative agent of AIDS. In 1985, a sensitive enzyme-linked immunosorbent assay (ELISA) was developed; this led to an appreciation of the scope and evolution of the HIV epidemic at first in the United States and other developed nations and ultimately among developing nations throughout the world (see “HIV Infection and AIDS Worldwide,” below). The staggering worldwide evolution of the HIV pandemic has been matched by an explosion of information in the areas of HIV virology, pathogenesis (both

1	“HIV Infection and AIDS Worldwide,” below). The staggering worldwide evolution of the HIV pandemic has been matched by an explosion of information in the areas of HIV virology, pathogenesis (both immunologic and virologic), treatment of HIV disease, treatment and prophylaxis of the opportunistic diseases associated with HIV infection, and prevention of HIV infection. The information flow related to HIV disease is enormous and continues to expand, and it has become almost impossible for the health care generalist to stay abreast of the literature.

1	The purpose of this chapter is to present the most current information available on the scope of the epidemic; on its pathogenesis, treatment, and prevention; and on prospects for vaccine development. Above all, the aim is to provide a solid scientific basis and practical clinical guidelines for a state-of-the-art approach to the HIV-infected patient.

1	The current U.S. CDC classification system for HIV infection and AIDS categorizes people on the basis of clinical conditions associated with HIV infection and CD4+ T lymphocyte measurement. A confirmed HIV case can be classified in one of five HIV infection stages (0, 1, 2, 3, or unknown). If there was a negative HIV test within 6 months of the first HIV infection diagnosis, the stage is 0, and remains 0 until 6 months after diagnosis. Advanced HIV disease (AIDS) is classified as stage 3 if one or more specific opportunistic illness has been diagnosed (Table 226-1). Otherwise, the stage is determined by CD4 test results and immunologic criteria (Table 226-2). If none of these criteria apply (e.g., because of missing information on CD4 test results), the stage is U (unknown). The definition and staging criteria of AIDS are complex and comprehensive and were established for surveillance purposes rather than for the practical care of patients. Thus, the clinician should not focus

1	Bacterial infections, multiple or recurrenta Candidiasis of bronchi, trachea, or lungs Candidiasis of esophagus Cervical cancer, invasiveb Coccidioidomycosis, disseminated or extrapulmonary Cryptococcosis, extrapulmonary Cryptosporidiosis, chronic intestinal (>1 month’s duration) Cytomegalovirus disease (other than liver, spleen, or nodes), onset at age >1 month Cytomegalovirus retinitis (with loss of vision) Encephalopathy attributed to HIV Herpes simplex: chronic ulcers (>1 month’s duration) or bronchitis, pneumo nitis, or esophagitis (onset at age >1 month) Histoplasmosis, disseminated or extrapulmonary Isosporiasis, chronic intestinal (>1 month’s duration) Kaposi’s sarcoma Lymphoma, Burkitt’s (or equivalent term) Lymphoma, immunoblastic (or equivalent term) Lymphoma, primary, of brain Mycobacterium avium complex or Mycobacterium kansasii, disseminated or

1	Mycobacterium tuberculosis of any site, pulmonary,b disseminated, or extrapulmonary Mycobacterium, other species or unidentified species, disseminated or extrapulmonary Pneumocystis jirovecii (previously known as Pneumocystis carinii) pneumonia Pneumonia, recurrentb Progressive multifocal leukoencephalopathy Salmonella septicemia, recurrent Toxoplasmosis of brain, onset at age >1 month Wasting syndrome attributed to HIV aOnly among children age <6 years. bOnly among adults, adolescents, and children age ≥6 years. Source: MMWR 63(No. RR-03), April 11, 2014.

1	Source: MMWR 63(No. RR-03), April 11, 2014. Human Immunodeficiency Virus Disease: AIDS and Related Disorders the transmembrane gp41. The HIV envelope exists as a trimeric heterodimer. The virion buds FIGuRE 226-1 A phylogenetic tree based on the complete genomes of primate immufrom the surface of the infected cell and incor-nodeficiency viruses. The scale (0.10) indicates a 10% difference at the nucleotide level. porates a variety of host proteins into its lipid (Prepared by Brian Foley, PhD, of the HIV Sequence Database, Theoretical Biology and Biophysics bilayer. The structure of HIV-1 is schematically Group, Los Alamos National Laboratory; additional information at www.hiv.lanl.gov/content/ diagrammed in Fig. 226-2B. sequence/HelpDocs/subtypes.html.)

1	Talapoin Dent’s DeBrazzas African green monkeys Red-capped mangabey Mandril-2 and drill Olive colobus Sun-tailed mandril-1 L’Hoest 0.10 Chimpanzee troglodytes HIV-2 and SIV from sooty mangabeys Mona, greater spot-nosed, and mustached monkeys Sykes Western red colobus HIV-1 O Group HIV-1 M Group HIV-1 N Group Chimpanzee troglodytes Chimpanzee schweinfurthii Gorilla HIV-1 P from chimpanzees and gorillas

1	FIGuRE 226-2 A. Electron micrograph of HIV. Figure illustrates a typical virion following budding from the surface of a CD4+ T lymphocyte, together with two additional incomplete virions in the process of budding from the cell membrane. B. Structure of HIV-1, including the gp120 envelope, gp41 transmembrane components of the envelope, genomic RNA, enzyme reverse transcriptase, p18(17) inner membrane (matrix), and p24 core protein (capsid). (Copyright by George V. Kelvin.) (Adapted from RC Gallo: Sci Am 256:46, 1987.)C. Scanning electron micrograph of HIV-1 virions infecting a human CD4+ T lymphocyte. The original photograph was imaged at 8000× magnification. (Courtesy of Elizabeth R. Fischer, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases; with permission.) the cytoplasm to reach the nucleus, the viral reverse transcriptase enzyme catalyzes the reverse transcription of the genomic RNA into DNA, resulting in the formation of double-stranded proviral

1	the cytoplasm to reach the nucleus, the viral reverse transcriptase enzyme catalyzes the reverse transcription of the genomic RNA into DNA, resulting in the formation of double-stranded proviral HIVDNA. At the preintegration steps of the replication cycle, the viral genome is vulnerable to cellular factors that can block the progression of infection. In particular, the cytoplasmic tripartite motif-containing protein 5-α (TRIM5-α) is a host restriction factor that interacts with retroviral capsids (Fig. 226-3). Although the exact mechanisms of action of TRIM5-α remain unclear, the HIV-1 capsid is not recognized by the human form of TRIM5-α. Thus this host factor is not effective in restricting HIV-1 replication in human cells. The apolipoprotein B mRNA editing enzyme (catalytic polypeptide-like 3 [APOBEC3]) family of cellular proteins also inhibits progression of virus infection after virus has entered the cell and prior to entering the nucleus (Fig. 226-3). APOBEC3 proteins, which

1	3 [APOBEC3]) family of cellular proteins also inhibits progression of virus infection after virus has entered the cell and prior to entering the nucleus (Fig. 226-3). APOBEC3 proteins, which are incorporated into virions and released into the cytoplasm of a newly infected cell, bind to the single minus-strand DNA intermediate and deaminate viral cytidine, causing hypermutation of retroviral genomes. HIV has evolved a powerful strategy to protect itself from APOBEC. The viral protein Vif targets APOBEC3 for proteasomal degradation.

1	With activation of the cell, the viral DNA accesses the nuclear pore and is exported from the cytoplasm to the nucleus, where it is integrated into the host cell chromosomes through the action of another virally encoded enzyme, integrase (Fig. 226-3). HIV provirus (DNA) integrates into the nuclear DNA preferentially within introns of active genes and regional hotspots. This provirus may remain transcriptionally inactive (latent) or it may manifest varying levels of gene expression, up to active production of virus.

1	Cellular activation plays an important role in the replication cycle of HIV and is critical to the pathogenesis of HIV disease (see “Pathogenesis and Pathophysiology,” below). Following initial binding, fusion, and internalization of the nucleic acid contents of virions into the target cell, incompletely reverse-transcribed DNA intermediates are labile in quiescent cells and do not integrate efficiently into the host cell genome unless cellular activation occurs shortly after infection. Furthermore, some degree of activation of the host cell is required for the initiation of transcription of the integrated proviral DNA into either genomic RNA or mRNA. This latter process may not necessarily be associated with the detectable expression of the classic cell-surface markers of activation. In this regard, activation of HIV expression from the latent state depends on the interaction of a number of cellular and viral factors. Following transcription, HIV mRNA is translated into proteins that

1	regard, activation of HIV expression from the latent state depends on the interaction of a number of cellular and viral factors. Following transcription, HIV mRNA is translated into proteins that undergo modification through glycosylation, myristoylation, phosphorylation,

1	CHAPTER 226 Human Immunodeficiency Virus Disease: AIDS and Related Disorders Genomic RNA Reverse transcriptase Cellular DNA Unintegrated linear DNA Fusion Uncoating CD4 gp120 Protein synthesis, processing, and assembly Budding Release TetherinTRIM5APOBEC3GmRNA Integrated proviral DNA Genomic RNA Mature HIV virion HIV FIGuRE 226-3 The replication cycle of HIV. See text for description. (Adapted from AS Fauci: Nature 384:529, 1996.) and cleavage. The viral particle is formed by the assembly of HIV with virion detachment, although the HIV accessory protein Vpu proteins, enzymes, and genomic RNA at the plasma membrane of counteracts the effect through direct interactions with tetherin. the cells. Budding of the progeny virion through the lipid bilayer of During or soon after budding, the virally encoded protease catalyzes the host cell membrane is the point at which the core acquires its the cleavage of the gag-pol precursor to yield the mature virion.

1	external envelope and where the host restriction factor tetherin can Progression through the virus replication cycle is profoundly influ inhibit the release of budding particles (Fig. 226-3). Tetherin is an enced by a variety of viral regulatory gene products. Likewise, each interferon (IFN)-induced type II transmembrane protein that interferes point in the replication cycle of HIV is a real or potential target for therapeutic intervention. Thus far, the reverse transcriptase, protease, and integrase enzymes as well as the process of virus–target cell binding and fusion have proved clinically to be susceptible to pharmacologic disruption.

1	Figure 226-5 illustrates schematically the arrangement of the HIV genome. Like other retroviruses, HIV-1 has genes that encode the structural proteins of the virus: gag encodes the proteins that form the core of the virion (including p24 antigen); pol encodes the enzymes responsible for protease processing of viral proteins, reverse transcription, and integration; and env encodes the envelope glycoproteins. However, HIV-1 is more complex than other retroviruses, particularly those of the nonprimate group, in that it also contains at least six other FIGuRE 226-4 Binding and fusion of HIV-1 with its target cell. HIV-1 binds to its target genes (tat, rev, nef, vif, vpr, and vpu), which code for cell via the CD4 molecule, leading to a conformational change in the gp120 molecule proteins involved in the modification of the host that allows it to bind to the co-receptor CCR5 (for R5-using viruses). The virus then firmly cell to enhance virus growth and the regulation of attaches to the host

1	in the modification of the host that allows it to bind to the co-receptor CCR5 (for R5-using viruses). The virus then firmly cell to enhance virus growth and the regulation of attaches to the host cell membrane in a coiled-spring fashion via the newly exposed viral gene expression. Several of these proteins are gp41 molecule. Virus-cell fusion occurs as the transitional intermediate of gp41 under-thought to play a role in the pathogenesis of HIV goes further changes to form a hairpin structure that draws the two membranes into disease; their various functions are listed in Fig. close proximity (see text for details). (Adapted from D Montefiori, JP Moore: Science 283:336, 226-5. Flanking these genes are the long terminal 1999; with permission.) repeats (LTRs), which contain regulatory elements

1	LTR Long terminal repeat Contains control regions that bind host transcription factors (NF-˜B, NFAT, Sp.1, TBP) Required for the initiation of transcription Contains RNA trans-acting response element (TAR) that binds Tat vifViral infectivity factor (p23) Overcomes inhibitory effects of APOBEC3, preventing hypermutation and viral DNA degradation vpuViral protein U Promotes CD4 degradation and influences virion release Overcomes inhibitory effects of tetherin envgp160 envelope protein Cleaved in endoplasmic reticulum to gp120 (SU) and gp41 (TM) gp120 mediates CD4 and chemokine receptor binding, while gp41 mediates fusion Contains RNA response element (RRE) that binds Rev gagPr55gag Polyprotein processed by PR MA, matrix (p17) Undergoes myristylation that helps target gag polyprotein to lipid rafts CA capsid (p24) polPolymerase Encodes a variety of viral enzymes, including PR (p10), RT and RNAase H (p66/51), and IN (p32) all processed by PR vprViral protein R (p15) Promotes G2 cell-cycle

1	rafts CA capsid (p24) polPolymerase Encodes a variety of viral enzymes, including PR (p10), RT and RNAase H (p66/51), and IN (p32) all processed by PR vprViral protein R (p15) Promotes G2 cell-cycle arrest Facilitates HIV infection of macrophages revRegulator of viral gene expression (p19) Binds RRE Inhibits viral RNA splicing and promotes nuclear export of incompletely spliced viral RNAs 5˛U3 RU5 U3 RU5

1	Binds cyclophilin A and CPSF6 Target of TRIM5° NC, nucleocapsid (p7) Zn finger, RNA-binding protein p6 Regulates the terminal steps in virion budding through interactions with TSG101 and ALIX1 Promotes down regulation of surface CD4 and MHC 1 expression Alters state of cellular activation Progression to disease slowed significantly in absence of Nef In presence of host cyclin T1 and CDK9 enhances RNA Pol II elongation on the viral DNA template FIGuRE 226-5 Organization of the genome of the HIV provirus together with a summary description of its 9 genes encoding 15 proteins. (Adapted from WC Greene, BM Peterlin: Nat Med 8:673, 2002.) involved in gene expression (Fig. 226-5). The major difference between the genomes of HIV-1 and HIV-2 is the fact that HIV-2 lacks the vpu gene and has a vpx gene not contained in HIV-1.

1	Molecular analyses of HIV isolates reveal varying levels of sequence diversity over all regions of the viral genome. For example, the degree of difference in the coding sequences of the viral envelope protein ranges from a few percent (very close, among isolates from the same infected individual) to more than 50% (extreme diversity, between isolates from the different groups of HIV 1: M, N, O, and P). The changes tend to cluster in hypervariable regions. HIV can evolve by several means, including simple base substitution, insertions and deletions, recombination, and gain and loss of glycosylation sites. HIV sequence diversity arises directly from the limited fidelity of the reverse transcriptase. The balance of immune pressure and functional constraints on proteins influences the regional level of variation within proteins. For example, Envelope, which is exposed on the surface of the virion and is under immune selective pressure from both antibodies and cytolytic T lymphocytes, is

1	level of variation within proteins. For example, Envelope, which is exposed on the surface of the virion and is under immune selective pressure from both antibodies and cytolytic T lymphocytes, is extremely variable, with clusters of mutations in hypervariable domains. In contrast, reverse transcriptase, with important enzymatic functions, is relatively conserved, particularly around the active site. The extraordinary variability of HIV-1 contrasts markedly with the relative stability of HTLV-1 and -2.

1	The four groups (M, N, O and P) of HIV-1 are the result of four separate chimpanzee-to-human (or possibly gorilla-to-human for groups O and P) transfers. Group M (major), which is responsible for most of the infections in the world, has diversified into subtypes and intersubtype recombinant forms, due to “sub-epidemics” within humans after one of those transfers.

1	Among primate lentiviruses, HIV-1 is most closely related to viruses isolated from chimpanzees and gorillas (Fig. 226-1). The chimpanzee subspecies Pan troglodytes troglodytes has been established to be the natural reservoir of the HIV-1 M and N groups. The rare viruses of the HIV-1 O and P groups are most closely related to viruses found in Cameroonian gorillas. The M group comprises nine subtypes, or clades, designated A, B, C, D, F, G, H, J, and K, as well as more than 60 known circulating recombinant forms (CRFs) and numerous unique recombinant forms. Intersubtype recombinants are generated by infection of an individual with two subtypes that then recombine and create a virus with a selective advantage. These CRFs range from highly prevalent forms such as CRF01_AE, common in southeast Asia, and CRF02_AG from west and central Africa, to a large number of CRFs that are relatively rare, either because they are of a more recent origin (newly recombined) or because they have not broken

1	Asia, and CRF02_AG from west and central Africa, to a large number of CRFs that are relatively rare, either because they are of a more recent origin (newly recombined) or because they have not broken out into a major population. The subtypes and CRFs create the major lineages of the M group of HIV-1. HIV-1 M group subtype C dominates the global pandemic, and there is much speculation that it is more transmissible than other subtypes, but solid data on transmissibility variations between subtypes are rare. Human population densities, access to prevention and treatment, prevalence of genital ulcers, iatrogenic transmissions, and other confounding host factors are all possible reasons why one subtype has spread more than another.

1	Figure 226-6 schematically diagrams the worldwide distribution of HIV-1 subtypes by region. Seven strains account for the vast majority of HIV infections globally: HIV-1 subtypes A, B, C, D, G and two of the CRFs, CRF01_AE and CRF02_AG. Subtype C viruses (of the M group) are by far the most common form worldwide, likely accounting for ~50% of prevalent infections worldwide. In sub-Saharan Africa, home to approximately two-thirds of all individuals living with HIV/AIDS, the majority of infections are caused by Human Immunodeficiency Virus Disease: AIDS and Related Disorders

1	FIGuRE 226-6 Global geographic distribution of HIV-1 subtypes and recombinant forms. Distributions derived from relative frequency of subtypes among >500,000 HIV genomic sequences in the Los Alamos National Laboratory HIV Sequence Database. (Additional information available at www.hiv.lanl.gov/components/sequence/HIV/geo/geo.comp.) subtype C, with smaller proportions of infections caused by subtype A, subtype G, CRF02_AG, and other subtypes and recombinants. In South Africa, the country with the largest number of prevalent infections (6.3 million in 2013), >97% of the HIV-1 isolates sequenced are of subtype C. In Asia, HIV-1 isolates of the CRF01_AE lineage and subtypes C and B predominate. CRF01_AE accounts for most infections in south and southeast Asia, while >95% of infections in India, home to an estimated 2.1 million HIV-infected individuals, are of subtype C (see “HIV Infection and AIDS Worldwide,” below). Subtype B viruses are the overwhelmingly predominant viruses seen in the

1	home to an estimated 2.1 million HIV-infected individuals, are of subtype C (see “HIV Infection and AIDS Worldwide,” below). Subtype B viruses are the overwhelmingly predominant viruses seen in the United States, Canada, certain countries in South America, western Europe, and Australia. It is thought that, purely by chance, subtype B was seeded into the United States and Europe in the late 1970s, thereby establishing an overwhelming founder effect. Many countries have co-circulating viral subtypes that are giving rise to new CRFs. Sequence analyses of HIV-1 isolates from infected individuals indicate that recombination among viruses of different clades likely occurs as a result of infection of an individual with viruses of more than one subtype, particularly in geographic areas where subtypes overlap, and more often in sub-epidemics driven by IV drug use than in those driven by sexual transmission.

1	The extraordinary diversity of HIV, reflected by the presence of multiple subtypes, circulating recombinant forms, and continuous viral evolution, has implications for possible differential rates of transmission, rates of disease progression, responses to therapy, and the development of resistance to antiretroviral drugs. This diversity is also a formidable obstacle to HIV vaccine development, as a broadly useful vaccine would need to induce protective responses against a wide range of viral strains.

1	HIV is transmitted primarily by sexual contact (both heterosexual and male to male); by blood and blood products; and by infected mothers to infants intrapartum, perinatally, or via breast milk. After more than 30 years of experience and observations regarding other potential modalities of transmission, there is no evidence that HIV is transmitted by casual contact or that the virus can be spread by insects, such as by a mosquito bite. Table 226-3 lists the estimated risk of HIV transmission for various types of exposures.

1	HIV infection is predominantly a sexually transmitted infection (STI) worldwide. By far the most common mode of infection, particularly in developing countries, is heterosexual transmission, although in many western countries a resurgence of male-to-male sexual transmission has occurred. Although a wide variety of factors including viral load and the presence of ulcerative genital diseases influence the efficiency of heterosexual transmission of HIV, such transmission is generally inefficient. A recent systemic review found a low per-act risk of heterosexual transmission in the absence of antiretrovirals: 0.04% for female-to-male transmission and 0.08% for male-to-female transmission during vaginal intercourse in the absence of antiretroviral therapy or condom use (Table 226-3).

1	HIV has been demonstrated in seminal fluid both within infected mononuclear cells and in cell-free material. The virus appears to concentrate in the seminal fluid, particularly in situations where there are increased numbers of lymphocytes and monocytes in the fluid, TABLE 226-3 EsTIMATEd PER-ACT PROBABILITy OF ACquIRIng HIv FROM An InFECTEd sOuRCE, By ExPOsuRE ACT Type of Exposure Risk per 10,000 Exposures aHIV transmission through these exposure routes is technically possible but unlikely and not well documented. Sources: CDC, www.cdc.gov/hiv/policies/law/risk.html; P Patel: AIDS 28:1509, 2014.

1	as in genital inflammatory states such as urethritis and epididymitis, conditions closely associated with other STIs. The virus has also been demonstrated in cervical smears and vaginal fluid. There is an elevated risk of HIV transmission associated with unprotected receptive anal intercourse (URAI) among both men and women compared to the risk associated with receptive vaginal intercourse. Although data are limited, the per-act risk for HIV transmission via URAI has been estimated to be ~1.4% (Table 226-3). The risk of HIV acquisition associated with URAI is probably higher than that seen in penile-vaginal intercourse because only a thin, fragile rectal mucosal membrane separates the deposited semen from potentially susceptible cells in and beneath the mucosa, and micro-trauma of the mucosal membrane may be associated with anal intercourse. Anal douching and sexual practices that traumatize the rectal mucosa also increase the likelihood of infection. It is likely that anal

1	of the mucosal membrane may be associated with anal intercourse. Anal douching and sexual practices that traumatize the rectal mucosa also increase the likelihood of infection. It is likely that anal intercourse provides at least two modalities of infection: (1) direct inoculation into blood in cases of traumatic tears in the mucosa; and (2) infection of susceptible target cells, such as Langerhans cells, in the mucosal layer in the absence of trauma. Insertive anal intercourse also confers an increased risk of HIV acquisition compared to insertive vaginal intercourse. Although the vaginal mucosa is several layers thicker than the rectal mucosa and less likely to be traumatized during intercourse, the virus can be transmitted to either partner through vaginal intercourse. As noted in Table 226-3, male-to-female HIV transmission is usually more efficient than female-to-male transmission. The differences in reported transmission rates between men and women may be due in part to the

1	Table 226-3, male-to-female HIV transmission is usually more efficient than female-to-male transmission. The differences in reported transmission rates between men and women may be due in part to the prolonged exposure to infected seminal fluid of the vaginal and cervical mucosa, as well as the endometrium (when semen enters through the cervical os). By comparison, the penis and urethral orifice are exposed relatively briefly to infected vaginal fluid. Among various cofactors examined in studies of heterosexual HIV transmission, the presence of other STIs has been strongly associated with HIV transmission. In this regard, there is a close association between genital ulcerations and transmission, owing to both susceptibility to infection and infectivity. Infections with microorganisms such as Treponema pallidum (Chap. 206), Haemophilus ducreyi (Chap. 182), and herpes simplex virus (HSV; Chap. 216) are important causes of genital ulcerations linked to transmission of HIV. In addition,

1	as Treponema pallidum (Chap. 206), Haemophilus ducreyi (Chap. 182), and herpes simplex virus (HSV; Chap. 216) are important causes of genital ulcerations linked to transmission of HIV. In addition, pathogens responsible for non-ulcerative inflammatory STIs such as those caused by Chlamydia trachomatis (Chap. 213), Neisseria gonorrhoeae (Chap. 181), and Trichomonas vaginalis (Chap. 254) also are associated with an increased risk of transmission of HIV infection. Bacterial vaginosis, an infection related to sexual behavior, but not strictly an STI, also may be linked to an increased risk of transmission of HIV infection. Several studies suggest that treating other STIs and genital tract syndromes may help prevent transmission of HIV. This effect is most prominent in populations in which the prevalence of HIV infection is relatively low. It is noteworthy that this principle may not apply to the treatment of HSV infections since it has been shown that even following anti-HSV therapy with

1	prevalence of HIV infection is relatively low. It is noteworthy that this principle may not apply to the treatment of HSV infections since it has been shown that even following anti-HSV therapy with resulting healing of HSV-related genital ulcers, HIV acquisition is not reduced. Biopsy studies revealed the likely explanation is that HIV receptor–positive inflammatory cells persisted in the genital tissue despite the healing of ulcers, and so HIV-susceptible targets remained at the site.

1	The quantity of HIV-1 in plasma is a primary determinant of the risk of HIV-1 transmission. In a cohort of heterosexual couples in Uganda discordant for HIV infection and not receiving antiretroviral therapy, the mean serum HIV RNA level was significantly higher among HIV-infected subjects whose partners seroconverted than among those whose partners did not seroconvert. In fact, transmission was rare when the infected partner had a plasma level of <1700 copies of HIV RNA per milliliter, even when genital ulcer disease was present (Fig. 226-7). The rate of HIV transmission per coital act was highest during the early stage of HIV infection when plasma HIV RNA levels were high and in advanced disease as the viral set point increased.

1	Antiretroviral therapy dramatically reduces plasma viremia in most HIV-infected individuals (see “Treatment,” below) and is associated with a reduction in risk of transmission. In a large study of serodiscordant couples, earlier treatment of the HIV-infected partner with among monogamous, heterosexual, HIV-serodiscordant couples in Uganda. (From RH Gray et al: Lancet 357:1149, 2001.) antiretroviral therapy rather than treatment delayed until the CD4+ T cells count fell below 250 cells per μL was associated with a 96% reduction in HIV transmission to the uninfected partner. This approach has been widely referred to as treatment as prevention or TasP. Several studies also have suggested a beneficial effect of antiretroviral treatment at the community level.

1	A number of studies including large, randomized, controlled trials clearly have indicated that male circumcision is associated with a lower risk of acquisition of HIV infection for heterosexual men. Studies are conflicting as to whether circumcision protects against HIV acquisition among men who have sex with men, but data suggest that circumcision is protective in those men who have sex with men who are insertive only. The benefit of circumcision may be due to increased susceptibility of uncircumcised men to ulcerative STIs, as well as to other factors such as microtrauma to the foreskin and glans penis. In addition, the highly vascularized inner foreskin tissue contains a high density of Langerhans cells as well as increased numbers of CD4+ T cells, macrophages, and other cellular targets for HIV. Finally, the moist environment under the foreskin may promote the presence or persistence of microbial flora that, via inflammatory changes, may lead to even higher concentrations of

1	targets for HIV. Finally, the moist environment under the foreskin may promote the presence or persistence of microbial flora that, via inflammatory changes, may lead to even higher concentrations of target cells for HIV in the foreskin. In addition, randomized trials have demonstrated that male circumcision also reduces hepatitis C virus (HCV) type 2, human papillomavirus virus (HPV), and genital ulcer disease in men as well as HPV, genital ulcer disease, bacterial vaginosis, and Trichomonas vaginalis infections among female partners of circumcised men. Thus, there may be an added benefit of diminution of risk for HIV acquisition to the female sexual partners of circumcised men.

1	In some studies the use of oral contraceptives was associated with an increase in incidence of HIV infection over and above that which might be expected by not using a condom for birth control. This phenomenon may be due to drug-induced changes in the cervical mucosa, rendering it more vulnerable to penetration by the virus. Adolescent girls might also be more susceptible to infection upon exposure due to the properties of an immature genital tract with increased cervical ectopy or exposed columnar epithelium. Oral sex is a much less efficient mode of transmission of HIV than is anal intercourse or vaginal intercourse (Table 226-3). A number of studies have reported that the incidence of transmission of infection by oral sex among couples discordant for HIV was extremely low. However, there have been well-documented reports of HIV transmission that likely resulted from fellatio or cunnilingus. Therefore, the assumption that oral sex is completely safe is not warranted.

1	The association of alcohol consumption and illicit drug use with unsafe sexual behavior, both homosexual and heterosexual, leads to an increased risk of sexual transmission of HIV. Methamphetamine and other so-called club drugs (e.g., ecstasy, ketamine, and gamma hydroxybutyrate), sometimes taken in conjunction with PDE-5 inhibitors such as sildenafil (Viagra), tadalafil (Cialis), or vardenafil (Levitra), have been associated with risky sexual practices and increased risk of HIV infection, particularly among men who have sex with men.

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1222 TRANSMISSION THROuGH INJECTION DRuG uSE HIV can be transmitted to injection drug users (IDUs) who are exposed to HIV while sharing injection paraphernalia such as needles, syringes, the water in which drugs are mixed, or the cotton through which drugs are filtered. Parenteral transmission of HIV during injection drug use does not require IV puncture; SC (“skin popping”) or IM (“muscling”) injections can transmit HIV as well, even though these behaviors are sometimes erroneously perceived as low-risk. Among IDUs, the risk of HIV infection increases with the duration of injection drug use; the frequency of needle sharing; the number of partners with whom paraphernalia are shared, particularly in the setting of “shooting galleries” where drugs are sold and large numbers of IDUs may share a limited number of “works”; comorbid psychiatric conditions such as antisocial personality disorder; the use of cocaine in

1	galleries” where drugs are sold and large numbers of IDUs may share a limited number of “works”; comorbid psychiatric conditions such as antisocial personality disorder; the use of cocaine in injectable form or smoked as “crack”; and the use of injection drugs in a geographic location with a high prevalence of HIV infection, such as certain inner-city areas in the United States. As noted in Table 226-3, the per-act risk of transmission from injection drug use with a contaminated needle has been estimated to be approximately 0.6%.

1	HIV can be transmitted to individuals who receive HIV-tainted blood transfusions, blood products, or transplanted tissue. The first cases of AIDS among transfusion recipients and individuals with hemophilia or other clotting disorders were reported in 1982. The vast majority of HIV infections acquired via contaminated blood transfusions, blood components, or transplanted tissue in resource-rich countries occurred prior to the spring of 1985, when mandatory testing of donated blood for HIV-1 was initiated. It is estimated that >90% of individuals exposed to HIV-contaminated blood products become infected (Table 226-3). Although blood screening for HIV is becoming more universal even in the developing world, unfortunately, in some resource-poor countries, HIV continues to be transmitted by blood, blood products, and tissues due to inadequate screening. Transfusions of whole blood, packed red blood cells, platelets, leukocytes, and plasma are all capable of transmitting HIV infection. In

1	blood products, and tissues due to inadequate screening. Transfusions of whole blood, packed red blood cells, platelets, leukocytes, and plasma are all capable of transmitting HIV infection. In contrast, hyperimmune gamma globulin, hepatitis B immune globulin, plasma-derived hepatitis B vaccine, and Rho immune globulin have not been associated with transmission of HIV infection. The procedures involved in processing these products either inactivate or remove the virus.

1	Currently, in the United States and in most developed countries, the following measures have made the risk of transmission of HIV infection by transfused blood or blood products extremely small: the screening of blood donations for antibodies to HIV-1 and HIV-2 and determination of the presence of HIV nucleic acid usually in mini-pools of several specimens; the careful selection of potential blood donors with health history questionnaires to exclude individuals with risk behavior; and opportunities for self-deferral and the screening out of HIV-negative individuals with serologic testing for infections that have shared risk factors with HIV, such as hepatitis B and C and syphilis. The chance of infection of a hemophiliac via clotting factor concentrates has essentially been eliminated because of the added layer of safety resulting from heat treatment of the concentrates. It is currently estimated that the risk of infection with HIV in the United States via transfused screened blood is

1	of the added layer of safety resulting from heat treatment of the concentrates. It is currently estimated that the risk of infection with HIV in the United States via transfused screened blood is approximately 1 in 2 million units. Therefore, since ~16 million donations are collected in the United States each year, despite the best efforts of science, one cannot completely eliminate the risk of transfusion-related transmission of HIV. In this regard, a case of transfusion-related transmission of HIV was reported in the United States in 2010, which was tracked to a blood donation in 2008; this was the first such reported case since 2002. Transmission of HIV (both HIV-1 and HIV-2) by blood or blood products is still an ongoing threat in certain developing countries, particularly in sub-Saharan Africa, where routine screening of blood is not universally practiced. In other countries, there have been reports of sporadic breakdowns in routinely available screening procedures in which

1	Africa, where routine screening of blood is not universally practiced. In other countries, there have been reports of sporadic breakdowns in routinely available screening procedures in which contaminated blood was allowed to be transfused, resulting in small clusters of patients becoming infected. For example, in China in the 1990s, a disturbingly large number of persons became infected by selling blood in situations where the collectors reused needles that were contaminated and, in some instances, mixed blood products from a number of individuals, separated the plasma, and reinfused mixed red blood cells back into the individual donors.

1	OCCuPATIONAL TRANSMISSION OF HIV: HEALTH CARE WORKERS, LABORATORY WORKERS, AND THE HEALTH CARE SETTING There is a small but definite occupational risk of HIV transmission to health care workers and laboratory personnel and potentially others who work with HIV-containing materials, particularly when sharp objects are used. An estimated 600,000 to 800,000 health care workers are stuck with needles or other sharp medical instruments in the United States each year. The global number of HIV infections among health care workers attributable to sharps injuries has been estimated to be 1000 cases (range, 200–5000) per year. As of 2010, there had been 57 documented cases of occupational HIV transmission to health care workers in the United States and 143 possible transmissions. There have been no confirmed cases reported since 1999.

1	Exposures that place a health care worker at potential risk of HIV infection are percutaneous injuries (e.g., a needle stick or cut with a sharp object) or contact of mucous membrane or nonintact skin (e.g., exposed skin that is chapped, abraded, or afflicted with dermatitis) with blood, tissue, or other potentially infectious body fluids. Large, multi-institutional studies have indicated that the risk of HIV transmission following skin puncture from a needle or a sharp object that was contaminated with blood from a person with documented HIV infection is ~0.3% and after a mucous membrane exposure it is 0.09% (see “HIV and the Health Care Worker,” below) if the injured and/or exposed person is not treated within 24 h with antiretroviral drugs. The risk of hepatitis B virus (HBV) infection following a similar type of exposure is ~6–30% in nonimmune individuals; if a susceptible worker is exposed to HBV, postexposure prophylaxis with hepatitis B immune globulin and initiation of HBV

1	following a similar type of exposure is ~6–30% in nonimmune individuals; if a susceptible worker is exposed to HBV, postexposure prophylaxis with hepatitis B immune globulin and initiation of HBV vaccine is >90% effective in preventing HBV infection. The risk of HCV infection following percutaneous injury is ~1.8% (Chap. 360).

1	Rare HIV transmission after nonintact skin exposure has been documented, but the average risk for transmission by this route has not been precisely determined; however, it is estimated to be less than the risk for mucous membrane exposure. Transmission of HIV through intact skin has not been documented. Currently in developed countries, virtually all puncture wounds and mucous membrane exposures in health care workers involving blood from a patient with documented HIV infection are treated prophylactically with combination antiretroviral therapy (cART). This practice, referred to as postexposure prophylaxis or PEP, has dramatically reduced the occurrence of puncture-related transmissions of HIV to health care workers.

1	In addition to blood and visibly bloody body fluids, semen and vaginal secretions also are considered potentially infectious; however, they have not been implicated in occupational transmission from patients to health care workers. The following fluids also are considered potentially infectious: cerebrospinal fluid, synovial fluid, pleural fluid, peritoneal fluid, pericardial fluid, and amniotic fluid. The risk for transmission after exposure to fluids or tissues other than HIV-infected blood also has not been quantified, but it is probably considerably lower than the risk after blood exposures. Feces, nasal secretions, saliva, sputum, sweat, tears, urine, and vomitus are not considered potentially infectious for HIV unless they are visibly bloody. Rare cases of HIV transmission via human bites have been reported, but not in the setting of occupational exposure.

1	An increased risk for HIV infection following percutaneous exposures to HIV-infected blood is associated with exposures involving a relatively large quantity of blood, as in the case of a device visibly contaminated with the patient’s blood, a procedure that involves a hollow-bore needle placed directly in a vein or artery, or a deep injury. Factors that might be associated with mucocutaneous transmission of HIV include exposure to an unusually large volume of blood and prolonged contact. In addition, the risk increases for exposures to blood from untreated patients with advanced-stage disease or those patients in the acute stage of HIV infection, owing to the higher levels of HIV in the blood under those circumstances. Since the beginning of the HIV epidemic, there have been rare instances where transmission of infection from a health care worker to patients seemed highly probable. Despite these small number of documented cases, the risk of HIV transmission involving health care

1	where transmission of infection from a health care worker to patients seemed highly probable. Despite these small number of documented cases, the risk of HIV transmission involving health care workers (infected or not) to patients is extremely low in developed countries—in fact, too low to be measured accurately. In this regard, several epidemiologic studies have been performed tracing thousands of patients of HIV-infected dentists, physicians, surgeons, obstetricians, and gynecologists, and no other cases of HIV transmission that could be linked to the health care providers were identified.

1	Breaches in infection control and the reuse of contaminated syringes, failure to properly sterilize surgical instruments, and/or hemodialysis equipment have also resulted rarely in the transmission of HIV from patient to patient in hospitals, nursing homes, and outpatient settings. Finally, these very rare occurrences of transmission of HIV as well as HBV and HCV to and from health care workers in the workplace underscore the importance of the use of universal precautions when caring for all patients (see below and Chap. 168).

1	HIV infection can be transmitted from an infected mother to her fetus during pregnancy, during delivery, or by breast-feeding. This remains an important form of transmission of HIV infection in certain developing countries, where the proportion of infected women to infected men is ~1:1. Virologic analyses of aborted fetuses indicate that HIV can be transmitted to the fetus during the first or second trimesters of pregnancy. However, maternal transmission to the fetus occurs most commonly in the perinatal period. Two studies performed in Rwanda and the Democratic Republic of Congo (then called Zaire) indicated that among all mother-to-child transmissions of HIV, the relative proportions were 23–30% before birth, 50–65% during birth, and 12–20% via breast-feeding.

1	In the absence of prophylactic antiretroviral therapy to the mother during pregnancy, labor, and delivery, and to the fetus following birth, the probability of transmission of HIV from mother to infant/ fetus ranges from 15% to 25% in industrialized countries and from 25% to 35% in developing countries. These differences may relate to the adequacy of prenatal care as well as to the stage of HIV disease and the general health of the mother during pregnancy. Higher rates of transmission have been reported to be associated with many factors—the best documented of which is the presence of high maternal levels of plasma viremia, with the risk increasing linearly with the level of maternal plasma viremia. It is very unlikely that mother-tochild transmission will occur if the mother’s level of plasma viremia is <1000 copies of HIV RNA/mL of blood and extremely unlikely if the level is undetectable (i.e., <50 copies/mL). However, there may not be a lower “threshold” below which transmission

1	plasma viremia is <1000 copies of HIV RNA/mL of blood and extremely unlikely if the level is undetectable (i.e., <50 copies/mL). However, there may not be a lower “threshold” below which transmission never occurs, since certain studies have reported rare transmission by women with viral RNA levels <50 copies/mL. Increased mother-to-child transmission is also correlated with closer human leukocyte antigen (HLA) match between mother and child. A prolonged interval between membrane rupture and delivery is another well-documented risk factor for transmission. Other conditions that are potential risk factors, but that have not been consistently demonstrated, are the presence of chorioamnionitis at delivery; STIs during pregnancy; illicit drug use during pregnancy; cigarette smoking; preterm delivery; and obstetric procedures such as amniocentesis, amnioscopy, fetal scalp electrodes, and episiotomy. In a seminal study conducted in the United States and France in the 1990s, zidovudine

1	delivery; and obstetric procedures such as amniocentesis, amnioscopy, fetal scalp electrodes, and episiotomy. In a seminal study conducted in the United States and France in the 1990s, zidovudine treatment of HIV-infected pregnant women from the beginning of the second trimester through delivery and of the infant for 6 weeks following birth dramatically decreased the rate of intrapartum and perinatal transmission of HIV infection from 22.6% in the untreated group to <5%. Today, the rate of motherto-child transmission has fallen to 1% or less in pregnant women who are receiving combination antiretroviral therapy (cART) for their HIV infection. Such treatment, combined with cesarean section delivery, has rendered mother-to-child transmission of HIV an unusual event in the United States and other developed nations. In this regard, both the United States Public Health Service and the World Health Organization guidelines recommend that all pregnant HIV-infected 1223 women should receive

1	other developed nations. In this regard, both the United States Public Health Service and the World Health Organization guidelines recommend that all pregnant HIV-infected 1223 women should receive cART for the health of the mother and to prevent perinatal transmission regardless of plasma HIV RNA copy number or CD4+ T cell counts.

1	Breast-feeding is an important modality of transmission of HIV infection in developing countries, particularly where mothers continue to breast-feed for prolonged periods. The risk factors for mother-tochild transmission of HIV via breast-feeding are not fully understood; factors that increase the likelihood of transmission include detectable levels of HIV in breast milk, the presence of mastitis, low maternal CD4+ T cell counts, and maternal vitamin A deficiency. The risk of HIV infection via breast-feeding is highest in the early months of breast-feeding. In addition, exclusive breast-feeding has been reported to carry a lower risk of HIV transmission than mixed feeding. In developed countries, breast feeding of babies by an HIV-infected mother is contraindicated since alternative forms of adequate nutrition, i.e., formulas, are readily available. In developing countries, where breast-feeding may be essential for the overall health of the infant, the continuation of cART in the

1	of adequate nutrition, i.e., formulas, are readily available. In developing countries, where breast-feeding may be essential for the overall health of the infant, the continuation of cART in the infected mother during the period of breastfeeding markedly diminishes the risk of transmission of HIV to the infant. In fact, once cART has been initiated in a pregnant woman, many experts recommend that therapy be continued for life.

1	Although HIV can be isolated typically in low titers from saliva of a small proportion of infected individuals, there is no convincing evidence that saliva can transmit HIV infection, either through kiss ing or through other exposures, such as occupationally to health care workers. Saliva contains endogenous antiviral factors; among these factors, HIV-specific immunoglobulins of IgA, IgG, and IgM isotypes are detected readily in salivary secretions of infected individuals. It has been suggested that large glycoproteins such as mucins and thrombospondin 1 sequester HIV into aggregates for clearance by the host. In addition, a number of soluble salivary factors inhibit HIV to various degrees in vitro, probably by targeting host cell receptors rather than the virus itself. Perhaps the best studied of these, secretory leukocyte protease inhibitor (SLPI), blocks HIV infection in several cell culture systems, and it is found in saliva at levels that approximate those required for inhibition

1	of these, secretory leukocyte protease inhibitor (SLPI), blocks HIV infection in several cell culture systems, and it is found in saliva at levels that approximate those required for inhibition of HIV in vitro. In this regard, higher salivary levels of SLPI in breast-fed infants were associated with a decreased risk of HIV transmission through breast milk. It has also been suggested that submandibular saliva reduces HIV infectivity by stripping gp120 from the surface of virions, and that saliva-mediated disruption and lysis of HIV-infected cells occurs because of the hypotonicity of oral secretions. There have been outlier cases of suspected transmission by saliva, but these have probably been blood-to-blood transmissions. Transmission of HIV by a human bite can occur but is a rare event. Although virus can be identified, if not isolated, from virtually any body fluid, there is no evidence that HIV transmission can occur as a result of exposure to tears, sweat, or urine. However,

1	Although virus can be identified, if not isolated, from virtually any body fluid, there is no evidence that HIV transmission can occur as a result of exposure to tears, sweat, or urine. However, there have been isolated cases of transmission of HIV infection by body fluids that may or may not have been contaminated with blood. Most of these situations occurred in the setting of a close relative providing intensive nursing care for an HIV-infected person without observing universal precautions, underscoring the importance of adhering to such precautions in the handling of body fluids and wastes from HIV-infected individuals.

1	HIV INFECTION AND AIDS WORLDWIDE HIV infection/AIDS is a global pandemic, with cases reported from virtually every country. At the end of 2013, an estimated 35.0 million individuals were living with HIV infection, according to the Joint United Nations Programme on HIV/AIDS (UNAIDS). An estimated 95% of people living with HIV/AIDS reside in lowand middle-income countries; ~50% are female, and 3.2 million are children <15 years. The distribution of these cases is illustrated in Fig. 226-8. The estimated number of people living with HIV—i.e., Human Immunodeficiency Virus Disease: AIDS and Related Disorders

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders FIGuRE 226-8 Estimated number of adults and children living with HIV infection as of December, 2013. Total: 35.0 (33.2 million–37.2 million). (From Joint United Nations Programme on HIV/AIDS [UNAIDS].) the global prevalence—has increased more than fourfold since 1990, reflecting the combined effects of continued high rates of new HIV infections and the life-prolonging impact of antiretroviral therapy (Fig. 226-9). In 2013, the global prevalence rate among persons age 15–49 years was 0.8%, with rates varying widely by country and region as illustrated in Fig. 226-10.

1	In 2013, an estimated 2.1 million new cases of HIV infection occurred worldwide, including 240,000 among children <15 years; about 40% of new infections were among persons under age 25. Between 2001 and 2013, the estimated number of new HIV infections globally fell by 38% (Fig. 226-9). Recent reductions in global HIV incidence likely reflect progress with HIV prevention efforts and the increased provision to HIV-infected people of antiretroviral therapy, which makes them much less likely to transmit the virus to sexual partners. In 2013, global AIDS deaths totaled 1.5 million (including 190,000 children <15 years), a 35% decrease since 2005 that coincides with a rapid expansion of access to antiretroviral therapy (Fig. 226-9). Since the beginning of the pandemic, an estimated 39 million people have died of an AIDS-related illness.

1	The HIV epidemic has occurred in “waves” in different regions of the world, each wave having somewhat different characteristics depending on the demographics of the country and region in question and the timing of the introduction of HIV into the population. Although the AIDS epidemic was first recognized in the United States and shortly thereafter in Western Europe, it very likely began in sub-Saharan Africa (see above), which has been particularly devastated by the epidemic. More than 70% of all people with HIV infection (~25 million), and nearly 90% of all HIV-infected children live in that region, even though sub-Saharan Africa is home to just 12% of the world’s population (Fig. 226-8). Within the region, southern Africa is worst-affected. In nine southern African countries, seroprevalence data indicate that >10% of the adult population age 15–49 is HIV-infected (Fig. 226-10). In addition, among high-risk individuals (e.g., commercial sex workers, patients attending STI clinics)

1	data indicate that >10% of the adult population age 15–49 is HIV-infected (Fig. 226-10). In addition, among high-risk individuals (e.g., commercial sex workers, patients attending STI clinics) who live in urban areas of sub-Saharan Africa, seroprevalence is now >50% in some places. Recent data offer promising signs of declining HIV incidence and prevalence in many countries in the region, although frequently at levels that remain high. Heterosexual exposure is

1	New HIV infections and deaths due to AIDS (millions) People living with HIV infection (millions) FIGuRE 226-9 Global estimates of HIV incidence and AIDS deaths (left) and, HIV prevalence (right), 1990–2013. (From UNAIDS.) 15.0% – 27.4% 5.0%– <15.0% 1.0% – <5.0% 0.5% – <1.0% Global Adult HIV Prevalence Rate = 0.8% 0.1% – <0.5% <0.1% No data FIGuRE 226-10 Global adult HIV prevalence rate, 2013. Data are estimates for adults aged 15-49 years. (From UNAIDS.) the primary mode of HIV transmission in sub-Saharan Africa, with women and girls disproportionately affected, accounting for ~60 percent of all HIV infections in that region. In 2013, an estimated 230,000 people were living with HIV in the Middle East/North Africa region. Cases are largely concentrated among IDUs, men who have sex with men, and sex workers and their clients.

1	In Asia and the Pacific, an estimated 4.8 million people were living with HIV at the end of 2013. In this region of the world, HIV prevalence is highest in southeast Asian countries, with wide variation in trends between different countries. Among countries in Asia, only Thailand has an adult seroprevalence rate of >1%. However, the populations of many Asian nations are so large (especially India and China) that even low infection and seroprevalence rates result in large numbers of people living with HIV. Although Asia’s epidemic has been concentrated for some time among specific populations—sex workers and their clients, men who have sex with men, and IDUs—it is expanding to the heterosexual partners of those most at risk.

1	The epidemic is expanding in Eastern Europe and Central Asia, where ~1.1 million people were living with HIV at the end of 2013. The Russian Federation and Ukraine account for the majority of HIV cases in the region. Driven initially by injection drug use and increasingly by heterosexual transmission, the number of new infections in this region has increased dramatically over the past decade. Approximately 1.9 million people are living with HIV/AIDS in Central and South America and the Caribbean. Brazil is home to the largest number of HIV-infected people in the region. However, the epidemic has been slowed in that country due to successful treatment and prevention efforts. Men who have sex with men account for the largest proportion of HIV infections in Central and South America. The Caribbean region has the highest regional adult seroprevalence rate after Africa. Heterosexual transmission, often tied to sex work, is the main driver of transmission in the region.

1	Approximately 2.3 million people are living with HIV/AIDS in North America and western and central Europe. The number of new infections among men who have sex with men has increased over the past decade in these mostly high-income areas, while rates of new infections among heterosexuals have stabilized and infections among women and IDUs have fallen. About 1.7 million people have been infected with HIV in the United States since the beginning of the epidemic, of whom >630,000 have died. Approximately 1.1 million individuals in the United States are living with HIV infection, ~16–18% of whom are unaware of their infection, according to recent estimates. As illustrated in Fig. 226-11, only a fraction of HIV-infected people are able to negotiate the steps in the HIV “care continuum,” from diagnosis, to entering into and staying in care, to receiving antiretroviral therapy, and ultimately to achieving a suppressed viral load (see “Treatment,” below).

1	More than 60% of those living with HIV/AIDS are Black/African-American or Hispanic/Latino, and more than half are men who have sex with men. The estimated HIV seroprevalence rate among all individuals age 13 years or older in the United States is ~0.5%. Approximately 2% of Black/African-American adults are HIV-infected in the United States, higher than any other group.

1	The number of new HIV infections in the United States, HIV incidence, peaked at about 130,000 per year in the late 1980s, followed by declines. For more than a decade, HIV incidence has remained stable at approximately 50,000 per year, with the proportion of new infections increasing in recent years among men who have sex with men and falling among women and IDUs. Among adults and adolescents newly diagnosed with HIV infection in 2011 (regardless of stage of infection), ~79% were males and ~21% were women. Of new HIV diagnoses among men, ~79% were attributed to male-to-male sexual contact, ~12% to heterosexual contact, ~6% to injection drug use, and ~4% to a combination of male-to-male sexual contact and injection drug use. Of new HIV diagnoses among women, ~86% were due to heterosexual contact and ~14% to injection drug use 82% 66% 37% 33% 25% 290,924 1,148,200 HIV-infected HIV-Linked to Retained in Prescribed Suppressed diagnosed HIV care HIV care ART viral load

1	HIV-infected HIV-Linked to Retained in Prescribed Suppressed diagnosed HIV care HIV care ART viral load FIGuRE 226-11 Estimated percentage of HIV-infected people engaged in selected stages of the continuum of HIV care in the United States. (Adapted from HI Hall et al: JAMA Intern Med 173:1337, 2013.) Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1226 Males Females n = 38,825 n = 10,257 FIGuRE 226-12 Transmission categories of adults and adolescents diagnosed with HIV infection (regardless of stage) in 2011, United States. (From CDC.) (Fig. 226-12). The estimated numbers of new HIV infections in 2011 in the United States for the 10 most affected subpopulations are shown in Fig. 226-13. Perinatal HIV transmission, from an HIV-infected mother to her baby, has declined significantly in the United States, largely due to Number of new HIV infections 2000 4000 6000 8000 10000 12000 Number of inf ections

1	Number of new HIV infections 2000 4000 6000 8000 10000 12000 Number of inf ections FIGuRE 226-13 Estimated number of new HIV infections in the United States, 2011, for the most affected subpopulations. FIGuRE 226-14 Estimated number of HIV-infected infants, United (From CDC.) States, 1990–2010. (From CDC.) the implementation of guidelines for the universal counseling and voluntary HIV testing of pregnant women and the use of antiretroviral therapy for pregnant women and newborn infants to prevent infection (Fig. 226-14). In 2011, fewer than 200 children were diagnosed with HIV infection in the United States.

1	HIV infection and AIDS have disproportionately affected minority populations in the United States. Among those diagnosed with HIV (regardless of stage of infection) in 2011, 47% percent were Blacks/ African Americans, a group that constitutes only 12% of the U.S. population (Fig. 226-15A). The estimated rate of new HIV diagnoses in 2011 by race/ethnicity per 100,000 population in the United States is shown in Fig. 226-15B.

1	The number of individuals diagnosed with AIDS and deaths among persons with AIDS in the United States rose steadily through the 1980s; AIDS cases peaked in 1993 and deaths in 1995 (Fig. 226-16). Since then, the annual numbers of AIDS-related deaths in the United States have fallen ~70%. This trend is due to several factors, including improved prophylaxis and treatment of opportunistic infections, growing experience among the health professions in caring for HIV-infected individuals, improved access to health care, and a decrease in new infections due to saturational effects and prevention efforts. n = 49,273 However, the most influential factor clearly has been 1227 the increased use of potent antiretroviral drugs, gen-Asian 2% American Indian/ erally administered in a combination of three or fourAlaska Native

1	American Indian/ erally administered in a combination of three or fourAlaska Native Native Hawaiian/ agents. Other Pacific Islander <1% Although the HIV/AIDS epidemic on the whole is <1% plateauing in the United States, it is spreading rapidly among certain populations, stabilizing in others, and decreasing in others. Similar to other STIs, HIV infection will not spread homogeneously throughout the population of the United States. However, it is clear that anyone who practices high-risk behavior is at risk for HIV infection. In addition, recent increases in infections and AIDS cases among young men who have sex with men as well as the spread in pockets of poverty in both urban and rural regions (particularly among underserved minority populations in the southern United States with inadequate access to health A care) testify that the epidemic of HIV infection in the United States remains a public health problem of major proportion.

1	The hallmark of HIV disease is a profound immunodeficiency resulting primarily from a progressive quantitative and qualitative deficiency of the subset of T lymphocytes referred to as helper T cells occurring in a setting of polyclonal immune activation. The helper subset of T cells is defined phenotypically by the presence on its surface of the CD4 molecule (Chap. 372e), which serves as the primary cellular receptor for HIV. A co-receptor must also be present together with CD4 for efficient binding, fusion, and entry of HIV-1 into its target cells (Figs. 226-3 and 226-4). HIV uses two major co-receptors, CCR5 and CXCR4, for fusion and entry; these co-receptors are also the primary receptors for certain chemoattractive cytokines termed chemo- kines and belong to the seven-transmembrane-domain G protein–coupled family of receptors. A number of mechanisms responsible for cellular depletion and/or B immune dysfunction of CD4+ T cells have been demonstrated in vitro; these include direct

1	G protein–coupled family of receptors. A number of mechanisms responsible for cellular depletion and/or B immune dysfunction of CD4+ T cells have been demonstrated in vitro; these include direct infection and FIGuRE 226-15 Race/ethnicity of persons (including children) diagnosed destruction of these cells by HIV, as well as indirect with HIV infection (regardless of stage) during 2011 in the United States. effects such as immune clearance of infected cells, cell

1	A. Estimated proportion of new infections by race/ethnicity. B. Estimated rate death associated with aberrant immune activation, and of new infections by race/ethnicity (per 100,000 population). (From CDC.) immune exhaustion due to aberrant cellular activation FIGuRE 226-16 Estimated number of AIDS cases and AIDS deaths, United States, 1985–2011. (From CDC.) 0 10 20 304050607080 Rate/100,000 population Human Immunodeficiency Virus Disease: AIDS and Related Disorders No. of cases and deaths (in thousands)

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders No. of cases and deaths (in thousands) FIGuRE 226-17 Typical course of an untreated HIV-infected individual. See text for detailed one other cell. Once infection is estab- lished, the virus replicates in lymphoid cells in the mucosa, the submucosa, and to some extent the lymphoreticular tissues that drain the gut tissues. For a108 variable period of time ranging from a few to several days, the virus cannot yet be detected in the plasma. This period is referred to as the “eclipse” phase of infection. As more virus is produced within 106 several days to weeks, it is disseminated, first to the draining lymph nodes and then to other lymphoid compartments 105 where it has easy access to dense concentrations of CD4+ T cell targets, allowing for a burst of high-level viremia that104 is readily detectable by currently avail- description. (From G Pantaleo et al: N Engl J Med 328:327, 1993. Copyright 1993 Massachusetts Medical

1	Society. All rights reserved.) with resulting cellular dysfunction. Patients with CD4+ T cell levels below certain thresholds are at high risk of developing a variety of opportunistic diseases, particularly the infections and neoplasms that are AIDS-defining illnesses. Some features of AIDS, such as Kaposi’s sarcoma and certain neurologic abnormalities, cannot be explained completely by the immunodeficiency caused by HIV infection, since these complications may occur prior to the development of severe immunologic impairment.

1	The combination of viral pathogenic and immunopathogenic events that occurs during the course of HIV disease from the moment of initial (primary) infection through the development of advanced-stage disease is complex and varied. It is important to appreciate that the pathogenic mechanisms of HIV disease are multifactorial and multiphasic and are different at different stages of the disease. Therefore, it is essential to consider the typical clinical course of an untreated HIV-infected individual in order to more fully appreciate these pathogenic events (Fig. 226-17). EARLY EVENTS IN HIV INFECTION: PRIMARY INFECTION AND INITIAL DISSEMINATION OF VIRuS

1	Using mucosal transmission as a model, the earliest events (within hours) that occur following exposure of HIV to the mucosal surface determine whether an infection will be established as well as the subsequent course of events following infection. Although the mucosal barrier is relatively effective in limiting access of HIV to susceptible targets in the lamina propria, the virus can cross the barrier by transport on Langerhans cells, an epidermal type of DC, just beneath the surface or through microscopic rents in the mucosa. Significant disruptions in the mucosal barrier as seen in ulcerative genital disease facilitate viral entry and increase the efficiency of infection. Virus then seeks susceptible targets, which are primarily CD4+ T cells that are spatially dispersed in the mucosa. This spatial dispersion of targets provides a significant obstacle to the establishment of infection. Such obstacles account for the low efficiency of sexual transmission of HIV (see “Sexual

1	mucosa. This spatial dispersion of targets provides a significant obstacle to the establishment of infection. Such obstacles account for the low efficiency of sexual transmission of HIV (see “Sexual Transmission,” above). Both “partially” resting CD4+ T cells and activated CD4+ T cells serve as early amplifiers of infection. Resting CD4+ T cells are more abundant; however, activated CD4+ T cells produce larger amounts of virus. In order for infection to become established, the basic reproductive rate (R0) must become equal to or greater than 1, i.e., each infected cell would infect at least able assays (Fig. 226-18). An important lymphoid organ, the gut-associated lymphoid tissue (GALT), is a major target of HIV infection and the location where large numbers of CD4+ T cells (usually memory cells) are infected and depleted, both by direct viral effects and by activation-associated apoptosis. Once virus virus is widely disseminated, infection is firmly established and the process is

1	cells) are infected and depleted, both by direct viral effects and by activation-associated apoptosis. Once virus virus is widely disseminated, infection is firmly established and the process is irreversible. It is important to point out that the initial infection of susceptible cells may vary somewhat with the route of infection. Virus that enters directly into the bloodstream via infected blood or blood products (i.e., transfusions, use of contaminated needles for injection drugs, sharp-object injuries, maternal-tofetal transmission either intrapartum or perinatally, or sexual intercourse where there is enough trauma to cause bleeding) is likely first cleared from the circulation to the spleen and other lymphoid organs, where primary focal infections begin, followed by wider dissemination throughout other lymphoid tissues as described above.

1	It has been demonstrated that sexual transmission of HIV is the result of a single infectious event and that a viral genetic bottleneck exists for transmission. In this regard, certain characteristics of the HIV envelope glycoprotein have a major influence on transmission, at least in subtype A and C viruses. Transmitting viruses, often referred to as “founder viruses,” are usually underrepresented in the circulating viremia of the transmitting partner and are less-diverged viruses with signature sequences including shorter V1–V2 loop sequences and fewer predicted N-linked glycosylation sites relative to the major circulating variants. These viruses are almost exclusively R5 strains and are usually sensitive to neutralization by antibody from the transmitting partner. Once replication proceeds in the newly infected partner, the founder virus diverges and accumulates glycosylation sites, becoming progressively more resistant to neutralization (Fig. 226-19).

1	The acute burst of viremia and wide dissemination of virus in primary HIV infection may be associated with an acute HIV syndrome, which occurs to varying degrees in ~50% of individuals with primary infection (see below). This syndrome is usually associated with high levels of viremia measured in millions of copies of HIV RNA per milliliter of plasma that last for several weeks. Acute mononucleosis-like symptoms are well correlated with the presence of viremia. Virtually all patients develop some degree of viremia during primary infection, which contributes to virus dissemination throughout the lymphoid tissue, even though they may remain asymptomatic or not recall experiencing symptoms. It appears that the initial level of plasma viremia in primary HIV infection does not necessarily determine the rate of disease progression; however, the set point of the level of steady-state plasma viremia after ~1 year does seem to correlate with the slope of disease progression in the untreated

1	the rate of disease progression; however, the set point of the level of steady-state plasma viremia after ~1 year does seem to correlate with the slope of disease progression in the untreated patient. The strikingly high levels of viremia observed in many patients with acute HIV infection is felt to be associated with a higher likelihood of transmission of the virus

1	FIGuRE 226-18 Summary of early events in HIV infection. See text for detailed description. CTLs, cytolytic T lymphocytes; HIV, human immunodeficiency virus. (Adapted from AT Haase: Nat Rev Immunol 5:783, 2005.) to others by a variety of routes including sexual transmission, shared needles and syringes, and mother-to-child transmission intrapartum, perinatally, or via breast milk.

1	ESTABLISHMENT OF CHRONIC AND PERSISTENT INFECTION Persistence of Virus Replication HIV infection is unique among human viral infections. Despite the robust cellular and humoral immune responses that are mounted following primary infection (see “Immune Response to HIV,” below), once infection has been established the virus succeeds in escaping complete immune-mediated clearance, paradoxically seems to thrive on immune activation, and is never eliminated completely from the body. Rather, a chronic infection develops and persists with varying degrees of continual virus replication in the untreated patient for a median of ~10 years before the patient becomes clinically ill (see “Advanced HIV Disease,” below). It is this establishment of a chronic, persistent infection that is the hallmark of HIV disease. Throughout the often protracted course of chronic infection, virus replication can invariably be detected in untreated patients by widely available assays that measure copies of HIV RNA

1	HIV disease. Throughout the often protracted course of chronic infection, virus replication can invariably be detected in untreated patients by widely available assays that measure copies of HIV RNA per milliliter of plasma. Levels of virus vary greatly in most untreated patients, ranging from several thousand to a few million copies of HIV RNA per milliliter of plasma. Studies using highly sensitive molecular techniques have demonstrated that even in certain

1	FIGuRE 226-19 As HIV diverges from founder to chronically replicating virus, it accumulates N-linked glycosylation sites. See text for detailed description. (Adapted from CA Derdeyn et al: Science 303:2019, 2004; B Chohan et al: J Virol 79:6528, 2005; and BF Keele et al: Proc Natl Acad Sci USA 105:7552, 2008.) patients in whom plasma viremia is suppressed to below detection (lower limit, 20–50 copies of HIV RNA/mL depending on manufacturer) by cART, there is a continual low level of virus replication. In other human viral infections, with very few exceptions, if the host survives, the virus is completely cleared from the body and a state of immunity against subsequent infection develops. HIV infection very rarely kills the host during primary infection. Certain viruses, such as HSV (Chap. 216), are not completely cleared from the body after infection, but instead enter a latent state; in these cases, clinical latency is accompanied by microbiologic latency. This is not the case with

1	216), are not completely cleared from the body after infection, but instead enter a latent state; in these cases, clinical latency is accompanied by microbiologic latency. This is not the case with HIV infection as described above. Chronicity associated with persistent virus replication can also be seen in certain cases of HBV and HCV infections (Chap. 362); however, in these infections the immune system is not a target of the virus.

1	Escape of HIV from Effective Immune System Control Inherent to the establishment of chronicity of HIV infection is the ability of the virus to evade adequate control and elimination by both the cellular and humoral limbs of the immune system. There are a number of mechanisms whereby the virus accomplishes this evasion. Paramount among these is the establishment of a sustained level of replication associated with the generation of viral diversity via mutation and recombination. The selection of mutants that escape control by CD8+ cytolytic T lymphocytes (CTLs) is critical to the propagation and progression of HIV infection. The high rate of virus replication associated with inevitable mutations also contributes to the inability of antibody to neutralize the autologous virus and thus to contain the virus quasispecies present in an individual at any given time. Extensive analyses of sequential HIV isolates and host responses have demonstrated that viral escape from B cell and CD8+ T cell

1	the virus quasispecies present in an individual at any given time. Extensive analyses of sequential HIV isolates and host responses have demonstrated that viral escape from B cell and CD8+ T cell epitopes occurs early after infection and allows the virus to stay one step ahead of effective immune responses. Virus-specific CD8+ CTLs expand greatly during primary HIV infection, and likely represent the high-affinity responses that would be expected to be most efficient in eliminating virus-infected cells; however, the restriction is generally incomplete as viral replication persists at relatively high levels in the majority of individuals. In addition to viral escape from CTLs through high rates of mutation, it is thought that the initially strong response becomes qualitatively dysfunctional owing to the overwhelming immune activation resulting from persistent viral replication, similar to the exhaustion of CD8+ CTLs that has been reported in the murine model of lymphocytic

1	dysfunctional owing to the overwhelming immune activation resulting from persistent viral replication, similar to the exhaustion of CD8+ CTLs that has been reported in the murine model of lymphocytic choriomeningitis virus (LCMV) infection. Several studies have indicated that exhaustion of HIV-specific CD8+ T cells during prolonged immune activation is associated with expression of inhibitory receptors, such

1	CHAPTER 226 Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1230 as programmed death (PD) 1 molecule (of the B7-CD28 family of molecules), as well as loss of polyreactivity and proliferative capacity. Another mechanism contributing to the evasion by HIV of immune system control is the downregulation of HLA class I molecules on the surface of HIV-infected cells by the viral proteins Nef, Tat, and Vpu, resulting in the lack of ability of the CD8+ CTL to recognize and kill the infected target cell. Although this downregulation of HLA class I molecules would favor elimination of HIV-infected cells by natural killer (NK) cells, this latter mechanism does not seem to remove HIV-infected cells effectively (see below). The principal targets of neutralizing antibodies against HIV are the envelope proteins gp120 and gp41. HIV employs at least three mechanisms to evade neutralizing responses: hypervariability in the primary sequence of the envelope, extensive glycosylation of

1	are the envelope proteins gp120 and gp41. HIV employs at least three mechanisms to evade neutralizing responses: hypervariability in the primary sequence of the envelope, extensive glycosylation of the envelope, and conformational masking of neutralizing epitopes. Several studies that have followed the humoral immune response to HIV from the earliest points after primary infection indicate that the virus continually mutates to escape the emerging antibody response such that the sequential antibodies that are induced do not neutralize the autologous virus. Broadly neutralizing antibodies capable of neutralizing a wide range of primary HIV isolates in vitro occur in only about 20% of HIV-infected individuals, and, when they do occur, it generally requires 2 to 3 years of infection with continual virus replication to drive the affinity maturation of the antibodies. Unfortunately, by the time these broadly neutralizing antibodies are formed, they are ineffective in containing the virus

1	virus replication to drive the affinity maturation of the antibodies. Unfortunately, by the time these broadly neutralizing antibodies are formed, they are ineffective in containing the virus replication in the patient. Persistent viremia also results in exhaustion of B cells similar to the exhaustion reported for CD4+ T cells, adding to the defects in the humoral response to HIV. CD4+ T cell help is essential for the integrity of antigen-specific immune responses, both humoral and cell-mediated. HIV preferentially infects activated CD4+ T cells including HIV-specific CD4+ T cells, and so this loss of viral-specific helper T cell responses has profound negative consequences for the immunologic control of HIV replication. Furthermore, this loss occurs early in the course of infection, and animal studies indicate that 40–70% of all memory CD4+ T cells in the GALT are eliminated during acute infection. Another potential means of escape of HIV-infected cells from elimination by CD8+ CTLs

1	animal studies indicate that 40–70% of all memory CD4+ T cells in the GALT are eliminated during acute infection. Another potential means of escape of HIV-infected cells from elimination by CD8+ CTLs is the sequestration of infected cells in immunologically privileged sites such as the central nervous system (CNS). Finally, the escape of HIV from immune-mediated elimination during primary infection allows the formation of a pool of latently infected cells that may not be recognized or completely eliminated by virus-specific CTLs or by ART (see below). Thus, despite a potent immune response and the marked downregulation of virus replication following primary HIV infection, HIV succeeds in establishing a state of chronic infection with a variable degree of persistent virus replication. During this period most patients make the clinical transition from acute primary infection to variable periods of clinical latency or smoldering disease activity (see below).

1	The HIV Reservoir: Obstacles to the Eradication of Virus A pool of latently infected, resting CD4+ T cells that serves as at least one component of the persistent reservoir of virus exists in virtually all HIV-infected individuals, including those who are receiving cART. Such cells carry an integrated form of HIV DNA in the genome of the host and can remain in this state until an activation signal drives the expression of HIV transcripts and ultimately replication-competent virus. This form of latency is to be distinguished from preintegration latency, in which HIV enters a resting CD4+ T cell and, in the absence of an activation signal, reverse transcription of the HIV genome occurs to a certain extent but the resulting proviral DNA fails to integrate into the host genome. This period of preintegration latency may last hours to days, and if no activation signal is delivered to the cell, the proviral DNA loses its capacity to initiate a productive infection. If these cells do become

1	preintegration latency may last hours to days, and if no activation signal is delivered to the cell, the proviral DNA loses its capacity to initiate a productive infection. If these cells do become activated prior to decay of the preintegration complex, reverse transcription proceeds to completion and the virus continues along its replication cycle (see above and Fig. 226-20). The pool of cells that are in the postintegration state of latency is established early during the

1	T cell activation (Ag, cytokines) Degradation of unintegrated HIV DNA T cell activation (Ag, cytokines) effect of Resting latently infected virus CD4+ memory T cells T cell activation (Ag, cytokines)

1	FIGuRE 226-20 Generation of latently infected, resting CD4+ T cells in HIV-infected individuals. See text for details. Ag, antigen; CTLs, cytolytic T lymphocytes. (Courtesy of TW Chun; with permission.) course of primary HIV infection. Despite the suppression of plasma viremia to <50 copies of HIV RNA per milliliter by potent regimens of cART administered over several years, this pool of latently infected cells persists and can give rise to replication-competent virus upon cellular activation. Modeling studies built on projections of decay curves have estimated that in such a setting of prolonged suppression, it would require a few to several years for the pool of latently infected cells to be completely eliminated. This has not been documented to occur spontaneously in any patients very likely because the latent viral reservoir is continually replenished by the low levels of persistent virus replication that may remain below the limits of detection of current assays (see below) (Fig.

1	because the latent viral reservoir is continually replenished by the low levels of persistent virus replication that may remain below the limits of detection of current assays (see below) (Fig. 226-20), even in patients who for the most part are treated successfully. Reservoirs of HIV-infected cells, latent or otherwise, can exist in a number of compartments including the lymphoid tissue, peripheral blood, and the CNS (likely in cells of the monocyte/ macrophage lineage) as well as in other unidentified locations. Over the past several years attempts have been made to eliminate HIV in the latent viral reservoir using agents that stimulate resting CD4+ T cells during the course of cART; however, such attempts have been unsuccessful. Thus, this persistent reservoir of infected cells and/or low levels of persistent virus replication are major obstacles to the goal of eradication of virus from infected individuals and hence a “cure,” despite the favorable clinical outcomes that have

1	low levels of persistent virus replication are major obstacles to the goal of eradication of virus from infected individuals and hence a “cure,” despite the favorable clinical outcomes that have resulted from cART.

1	Viral Dynamics The dynamics of viral production and turnover have been quantified using mathematical modeling in the setting of the administration of reverse transcriptase and protease inhibitors to HIV-infected individuals in clinical studies. Treatment with these drugs resulted in a precipitous decline in the level of plasma viremia, which typically fell by well over 90% within 2 weeks. The number of CD4+ T cells in the blood increased concurrently, which suggested that the Productively infected Latently infected per milliliter in the absence of therapy, there is virtually 1231 CD4+ lymphocytes CD4+ lymphocytes always some degree of ongoing virus replication. t1/2 = 1.0 day Uninfected, activated Long-lived

1	t1/2 = 1.0 day Uninfected, activated Long-lived In untreated patients or in patients in whom therapy has not adequately controlled virus replication, after a variable period, usually measured in years, the CD4+ T cell count falls below a critical level (<200/μL) and the patient becomes highly susceptible to opportunistic disease (Fig. 226-17). For this reason, the CDC case definition of AIDS includes all HIV-infected individuals over 5 years of age with CD4+ T cell counts below this level (Table 226-2). Patients may experience constitutional signs and symptoms or may develop an opportunistic disease abruptly without any prior symptoms, although the latter scenario is unusual. The depletion of CD4+ T cells continues to be progressive and unrelenting in this phase. It is not CD4+ lymphocytes infected with defective viruses

1	FIGuRE 226-21 Dynamics of HIV infection in vivo. See text for detailed description. (From AS Perelson et al: Science 271:1582, 1996.) killing of CD4+ T cells was linked directly to the levels of replicating virus. However, a significant component of the early rise in CD4+ T cell numbers following the initiation of therapy may be due to the redistribution of cells into the peripheral blood from other tissue compartments throughout the body as a consequence of therapy-related diminution in viremia-associated immune system activation. It was determined on the basis of modeling the kinetics of viral decline and the emergence of resistant mutants during therapy that 93–99% of the circulating virus originated from recently infected, rapidly turning over CD4+ T cells and that ~1–7% of circulating virus originated from longer-lived cells, likely monocytes/macrophages. A negligible amount of circulating virus originated from the pool of latently infected cells (Fig. 226-21). It was also

1	virus originated from longer-lived cells, likely monocytes/macrophages. A negligible amount of circulating virus originated from the pool of latently infected cells (Fig. 226-21). It was also determined that the half-life of a circulating virion was ~30–60 min and that of productively infected cells was 1 day. Given the relatively steady level of plasma viremia and of infected cells, it appears that extremely large amounts of virus (~1010–1011 virions) are produced and cleared from the circulation each day. In addition, data suggest that the minimal duration of the HIV-1 replication cycle in vivo is ~2 days. Other studies have demonstrated that the decrease in plasma viremia that results from cART correlates closely with a decrease in virus replication in lymph nodes, further confirming that lymphoid tissue is the main site of HIV replication and the main source of plasma viremia.

1	The level of steady-state viremia, called the viral set point, at ~1 year following acquisition of HIV infection has important uncommon for CD4+ T cell counts in the untreated patient to drop to as low as 10/μL or even to zero. In for opportunistic infections are readily accessible to such patients, survival is increased dramatically even in those patients with advanced HIV disease. In contrast, untreated patients who progress to this severest form of immunodeficiency usually succumb to opportunistic infections or neoplasms (see below).

1	It is important to distinguish between the terms long-term survivor and long-term nonprogressor. Long-term nonprogressors are by definition long-term survivors; however, the reverse is not always true. Predictions from one study that antedated the availability of effective cART estimated that ~13% of homosexual/bisexual men who were infected at an early age may remain free of clinical AIDS for >20 years. Many of these individuals may have progressed in their degree of immune deficiency; however, they certainly survived for a considerable period of time. With the advent of effective cART, the survival of HIV-infected individuals has dramatically increased. Early in the AIDS epidemic, prior to the availability of therapy, if a patient presented with a life-threatening opportunistic infection, the median survival was 26 weeks from the time of presentation. Currently, an HIV-infected 20-year-old individual in a high-income country who is appropriately treated with cART can expect to live

1	the median survival was 26 weeks from the time of presentation. Currently, an HIV-infected 20-year-old individual in a high-income country who is appropriately treated with cART can expect to live at least 50 years according to mathematical model projections. In the face of cART, long-term survival is becoming commonplace. Definitions of

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders prognostic implications for the progression of HIV disease 1 in the untreated patient. It has been demonstrated that as a group untreated HIV-infected individuals who have a low 0.9 HIV RNA copies/mL ˜9060 9,061 to 26,040 26,041 to 72,540 >72,540 set point at 6 months to 1 year following infection progress is very high at that time (Fig. 226-22). Clinical Latency versus Microbiologic Latency With the excep tion of long-term nonprogressors (see “Long-Term Survivors and Long-Term Nonprogressors,” below), the level of CD4+

1	Clinical Latency versus Microbiologic Latency With the excep tion of long-term nonprogressors (see “Long-Term Survivors and Long-Term Nonprogressors,” below), the level of CD4+ T cells in the blood decreases progressively in HIV-infected individuals in the absence of cART. The decline in CD4+ T cells may be gradual or abrupt, the latter usually reflecting a significant spike in the level of plasma viremia. Most patients 0.8 0.7 0.6 0.5 0.4 0.3 0.2 are relatively asymptomatic while this progressive decline is 0.1 taking place (see below) and are often described as being in a state of clinical latency. However, this term is misleading; it 0 does not mean disease latency, since progression, although slow in many cases, is generally relentless during this period. Time, years

1	Furthermore, clinical latency should not be confused with FIGuRE 226-22 Relationship between levels of virus and rates of disease microbiologic latency, since varying levels of virus replica-progression. Kaplan-Meier curves for AIDS-free survival stratified by basetion inevitably occur during this period of clinical latency. line HIV-1 RNA categories (copies per milliliter). (From JW Mellors et al: Science Even in those rare patients who have <50 copies of HIV RNA 272:1167, 1996.) 1232 long-term nonprogressors have varied considerably over the years, and so such individuals constitute a heterogeneous group. Long-term nonprogressors were first described in the 1990s. Originally, individuals were considered to be long-term nonprogressors if they had been infected with HIV for a long period (≥10 years), their CD4+ T cell counts were in the normal range, and they remained stable over years without receiving cART. Approximately 5–15% of HIV-infected individuals fell into this broader

1	(≥10 years), their CD4+ T cell counts were in the normal range, and they remained stable over years without receiving cART. Approximately 5–15% of HIV-infected individuals fell into this broader nonprogressor category. However, this group was rather heterogenous and over time a significant proportion of these individuals progressed and ultimately required therapy. From this broader group, a much smaller subgroup of “elite” controllers or nonprogressors was identified, and they constituted less than 1% of HIV-infected individuals. These elite controllers, by definition, have extremely low levels of plasma viremia and normal CD4+ T cell counts. It is noteworthy that certain of their HIV-specific immune responses are robust and clearly superior to those of HIV-infected progressors. In this group of elite controllers certain HLA class I haplotypes are overrepresented, particularly HLA-B57-01 and HLA-B27-05. Outside of the subgroup of elite controllers, a number of other genetic factors

1	group of elite controllers certain HLA class I haplotypes are overrepresented, particularly HLA-B57-01 and HLA-B27-05. Outside of the subgroup of elite controllers, a number of other genetic factors have been shown to be involved to a greater or lesser degree in the control of virus replication and thus in the rate of HIV disease progression (see “Genetic Factors in HIV-1 and AIDS Pathogenesis,” below).

1	Regardless of the portal of entry of HIV, lymphoid tissues are the major anatomic sites for the establishment and propagation of HIV infection. Despite the use of measurements of plasma viremia to determine the level of disease activity, virus replication occurs mainly in lymphoid tissue and not in blood; indeed, the level of plasma viremia directly reflects virus production in lymphoid tissue. Some patients experience progressive generalized lymphadenopathy early in the course of the infection; others experience varying degrees of transient lymphadenopathy. Lymphadenopathy reflects the cellular activation and immune response to the virus in the lymphoid tissue, which is generally characterized by follicular or germinal center hyperplasia. Lymphoid tissue involvement is a common denominator of virtually all patients with HIV infection, even those without easily detectable lymphadenopathy.

1	Simultaneous examinations of lymph tissue and peripheral blood in patients and monkeys during various stages of HIV and SIV infection, respectively, have led to substantial insight into the pathogenesis of HIV disease. In most of the original human studies, peripheral lymph nodes have been used predominantly as the source of lymphoid tissue. More recent studies in monkeys and humans have also focused on the GALT, where the earliest burst of virus replication occurs associated with marked depletion of CD4+ T cells. In detailed studies of peripheral lymph node tissue, using a combination of polymerase chain reaction (PCR) techniques for HIV DNA and HIV RNA in tissue and HIV RNA in plasma, in situ hybridization for HIV RNA, and light and electron microscopy, the following picture has emerged. During acute HIV infection resulting from mucosal transmission, virus replication progressively amplifies from scattered lymphoid cells in the lamina propria to draining lymphoid tissue, leading to

1	During acute HIV infection resulting from mucosal transmission, virus replication progressively amplifies from scattered lymphoid cells in the lamina propria to draining lymphoid tissue, leading to high levels of plasma viremia. The GALT plays a major role in the amplification of virus replication, and virus is disseminated from replication in the GALT to peripheral lymphoid tissue. A profound degree of cellular activation occurs (see below) and is reflected in follicular or germinal center hyperplasia. At this time copious amounts of extracellular virions (both infectious and defective) are trapped on the processes of the follicular dendritic cells (FDCs) in the germinal centers of the lymph nodes. Virions that have bound complement components on their surfaces attach to the surface of FDCs via interactions with complement receptors and likely via Fc receptors that bind to antibodies that are attached to the virions. In situ hybridization reveals expression of virus in individual

1	FDCs via interactions with complement receptors and likely via Fc receptors that bind to antibodies that are attached to the virions. In situ hybridization reveals expression of virus in individual cells of the paracortical area and, to a lesser extent, the germinal center (Fig. 226-23). The persistence of trapped virus after the transition from acute to chronic infection likely reflects a steady state whereby trapped virus turns over and is replaced by fresh virions that are continually produced. The trapped virus, either as whole virion or shed envelope, serves as a continual activator of CD4+ T cells, thus driving further virus replication.

1	FIGuRE 226-23 HIV in the lymph node of an HIV-infected indi-vidual. An individual cell infected with HIV shown expressing HIV RNA by in situ hybridization using a radiolabeled molecular probe. Original ×500. (Adapted from G Pantaleo, AS Fauci et al: Nature 362:355, 1993.)

1	During early and chronic/asymptomatic stages of HIV disease, the architecture of lymphoid tissues is generally preserved and may even be hyperplastic owing to an increased presence of B cells and specialized CD4+ T cells called follicular helper CD4+ T cells (TFH) in prominent germinal centers. Extracellular virions can be seen by electron microscopy attached to FDC processes. The trapping of antigen is a physiologically normal function for the FDCs, which present antigen to B cells and, along with the action of TFH cells, contribute to the generation of B cell memory. However, in the case of HIV, persistent cellular activation, resulting in the secretion of proinflammatory cytokines such as interleukin (IL) 1β, tumor necrosis factor (TNF) α, IFN-γ, and IL-6, which can induce viral replication (see below) and diminish the effectiveness of the immune response against the virus. In addition, the CD4+ TFH cells that are recruited into the germinal center to provide help to B cells in the

1	(see below) and diminish the effectiveness of the immune response against the virus. In addition, the CD4+ TFH cells that are recruited into the germinal center to provide help to B cells in the generation of an HIV-specific immune response are highly susceptible to infection by virus either trapped on FDC or propagated locally. Thus, in HIV infection, a normal physiologic function of the immune system that contributes to the clearance of virus, as well as to the generation of a specific immune response, can also have deleterious consequences.

1	As the disease progresses, the architecture of lymphoid tissues begins to show disruption. Confocal microscopy reveals destruction of the fibroblastic reticular cell (FRC) and FDC networks in the T cell zone and B cell follicles, respectively, and an incapacity to replenish naïve cells. The mechanisms of destruction are not completely understood, but they are thought to be associated with collagen deposition causing fibrosis and loss of production of cytokines such as IL-7 and lymphotoxin-α, which are critical to the maintenance of lymphoid tissues and their lymphocyte constituents. As the disease progresses to an advanced stage, there is complete disruption of the architecture of the lymphoid tissues, accompanied by dissolution of the FRC and FDC networks. At this point, the lymph nodes are “burnt out.” This destruction of lymphoid tissue compounds the immunodeficiency of HIV disease and contributes both to the inability to control HIV replication (leading usually to high levels of

1	are “burnt out.” This destruction of lymphoid tissue compounds the immunodeficiency of HIV disease and contributes both to the inability to control HIV replication (leading usually to high levels of plasma viremia in the untreated or inadequately treated patient) and to the inability to mount adequate immune responses against opportunistic pathogens. The events from primary infection to the ultimate destruction of the immune system are illustrated in Fig. 226-24. Recently, nonhuman primate studies and some human studies have examined GALT at various stages of HIV disease. Within the GALT, the basal level of activation combined with virus-mediated cellular activation results in the infection and elimination of an estimated 50–90% of CD4+ T cells in the gut. The extent of viremia are suppressed to below the level 1233 of detection by standard assays. From a virologic standpoint, although quiescent CD4+ T cells can be infected with Massive viremia

1	HIV, reverse transcription, integration, and virus spread are much more efficient in activated cells. Furthermore, cellular activation induces expression Wide dissemination to lymphoid organs of virus in cells latently infected with HIV. In essence, immune activation and Trapping of virus and inflammation provide the engine that establishment of chronic, drives HIV replication. In addition to endogenous factors such as cytokines, Partial immunologic a number of exogenous factors such control of virus replication as other microbes that are associated

1	Destruction of have important effects on HIV pathogen-envelope-mediated esis. Co-infection in vivo or in vitro with a range of viruses, such as HSV types 1 and 2, cytomegalovirus (CMV), human herpesvirus (HHV) 6, Epstein-Barr virus FIGuRE 226-24 Events that transpire from primary HIV infection through the establishment of (EBV), HBV, adenovirus, and HTLV-1 chronic persistent infection to the ultimate destruction of the immune system. See text for details. have been shown to upregulate HIV CTLs, cytolytic T lymphocytes; GALT, gut-associated lymphoid tissue. this early damage to GALT, which constitutes a major component of lymphoid tissue in the body, may play a role in determining the potential for immunologic recovery of the memory cell subset. IMMuNE ACTIVATION, INFLAMMATION, AND HIV PATHOGENESIS

1	IMMuNE ACTIVATION, INFLAMMATION, AND HIV PATHOGENESIS Activation of the immune system and variable degrees of inflammation are essential components of any appropriate immune response to a foreign antigen. However, immune activation and inflammation, which can be considered aberrant in HIV-infected individuals, play a critical role in the pathogenesis of HIV disease and other chronic conditions associated with HIV disease. Immune activation and inflammation in the HIV-infected individual contribute substantially to (1) the replication of HIV, (2) the induction of immune dysfunction, and (3) the increased incidence of chronic conditions associated with persistent immune activation and inflammation (Table 226-4).

1	indUction of Hiv replication by aberrant immUne activation The immune system is normally in a state of homeostasis, awaiting perturbation by foreign antigenic stimuli. Once the immune response deals with and clears the antigen, the system returns to relative quiescence (Chap. 372e). This is generally not the case in HIV infection where, in the untreated patient, virus replication is invariably persistent with very few exceptions and immune activation is persistent. HIV replicates most efficiently in activated CD4+ T cells; in HIV infection, chronic activation provides the cell substrates necessary for persistent virus replication throughout the course of HIV disease, particularly in the untreated patient and to variable degrees even in certain patients receiving cART whose levels of plasma expression. In addition, infestation with nematodes has been shown to be associ ated with a heightened state of immune activation that facilitates HIV replication; in certain studies deworming of

1	expression. In addition, infestation with nematodes has been shown to be associ ated with a heightened state of immune activation that facilitates HIV replication; in certain studies deworming of the infected host has resulted in a decrease in plasma viremia. Two diseases of extraordinary global health significance, malaria and tuberculosis (TB), have been shown to increase HIV viral load in dually infected individuals. Globally, Mycobacterium tuberculosis is the most common opportunistic infection in HIV-infected individuals (Chap. 202). In addition to the fact that HIV-infected individuals are more likely to develop active TB after exposure, it has been demonstrated that active TB can accelerate the course of HIV infection. It has also been shown that levels of plasma viremia are greatly elevated in HIV-infected individuals with active TB who are not on cART, compared with pre-TB levels and levels of viremia after successful treatment of the active TB. The situation is similar in

1	elevated in HIV-infected individuals with active TB who are not on cART, compared with pre-TB levels and levels of viremia after successful treatment of the active TB. The situation is similar in the interaction between HIV and malaria parasites (Chap. 248). Acute infection of HIV-infected individuals with Plasmodium falciparum increases HIV viral load, and the increased viral load is reversed by effective malaria treatment.

1	microbial translocation and persistent immUne activation One proposed mechanism of persistent immune activation involves the disruption of the mucosal barrier in the gut due to HIV replication in and disruption of submucosal lymphoid tissue. As a result of this disruption, there is an increase in the products, particularly lipopolysaccharide (LPS), of bacteria that translocate from the bowel lumen through the damaged mucosa to the circulation, leading to persistent systemic immune activation and inflammation. This effect can persist even after the HIV viral load is brought to <50 copies/mL by cART. Depletion in the GALT of IL-17–producing T cells, which are responsible for defense against extracellular bacteria and fungi, also is thought to contribute to HIV pathogenesis.

1	persistent immUne activation and inflammation indUce immUne dysfUnction The activated state in HIV infection is reflected by hyperactivation of B cells leading to hypergammaglobulinemia; increased lymphocyte turnover; activation of monocytes; expression of activation markers on CD4+ and CD8+ T cells; increased activation-associated cellular apoptosis; lymph node hyperplasia, particularly early in the course of disease; increased secretion of proinflammatory cytokines, particularly IL-6; elevated levels of high-sensitivity C-reactive protein, fibrinogen, d-dimer, neopterin, β2-microglobulin, acid-labile interferon, soluble (s) IL-2 receptors (R), sTNFR, sCD27, and sCD40L; and autoimmune phenomena (see “Autoimmune Phenomena,” below). Even in the absence of direct infection of a target cell, HIV envelope proteins can interact

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1234 with cellular receptors (CD4 molecules and chemokine receptors) to deliver potent activation signals resulting in calcium flux, the phosphorylation of certain proteins involved in signal transduction, co-localization of cytoplasmic proteins including those involved in cell trafficking, immune dysfunction, and, under certain circumstances, apoptosis. From an immunologic standpoint, chronic exposure of the immune system to a particular antigen over an extended period may ultimately lead to an inability to sustain an adequate immune response to the antigen in question. In many chronic viral infections, including HIV infection, persistent viremia is associated with “functional exhaustion” of virus-specific T cells, decreasing their capacity to proliferate and perform effector functions. It has been demonstrated that this phenomenon may be mediated, at least in part, by the upregulation of inhibitory receptors on

1	their capacity to proliferate and perform effector functions. It has been demonstrated that this phenomenon may be mediated, at least in part, by the upregulation of inhibitory receptors on HIV-specific T cells, such as PD-1 shared by both CD4+ and CD8+ T cells, as well as CTLA-4 on CD4+ and Tim-3, 2B4, and CD106 on CD8+ T cells. Furthermore, the ability of the immune system to respond to a broad spectrum of antigens may be compromised if immunocompetent cells are maintained in a state of chronic activation. The deleterious effects of chronic immune activation on the progression of HIV disease are well established. As in most conditions of persistent antigen exposure, the host must maintain sufficient activation of antigen (HIV)-specific responses but must also prevent excessive activation and potential immune-mediated damage to tissues. Certain studies suggest that normal immunosuppressive mechanisms that act to keep hyperimmune activation in check, particularly CD4+, FoxP3+, CD25+

1	and potential immune-mediated damage to tissues. Certain studies suggest that normal immunosuppressive mechanisms that act to keep hyperimmune activation in check, particularly CD4+, FoxP3+, CD25+ regulatory T cells (T-regs), may be dysfunctional or depleted in the context of advanced HIV disease.

1	medical conditions associated witH persistent immUne activation and inflammation in Hiv disease It has become clear, as the survival of HIV-infected individuals has increased, that a number of previously unrecognized medical complications are associated with HIV disease—and that these complications relate to chronic immune activation and inflammation (Table 226-4). These complications can appear even after patients have experienced years of adequate control of viral replication (plasma viremia below detectable levels) for several years. Of particular note are endothelial cell dysfunction and its relationship to cardiovascular disease. Other chronic conditions that have been reported include bone fragility, certain cancers, persistent immune dysfunction, diabetes, kidney and liver disease, and neurocognitive dysfunction, thus presenting an overall picture of accelerated aging.

1	Apoptosis Apoptosis is a form of programmed cell death that is a normal mechanism for the elimination of effete cells in organogenesis as well as in the cellular proliferation that occurs during a normal immune response (Chap. 372e). Apoptosis is largely dependent on cellular activation, and the aberrant cellular activation associated with HIV disease is correlated with a heightened state of apoptosis. HIV can trigger both Fas-dependent and Fas-independent pathways of apoptosis, the former of which is generally referred to as activation-induced cell death through an extrinsic pathway and involves the upregulation of the death receptor Fas and Fas ligand. Fas-independent pathways can be either extrinsic with different death receptors or intrinsic due to the downregulation of the antiapoptotic proteins such as Bcl-2. More recently, the phenomenon of pyroptosis, an inflammatory form of cell death involving the upregulation of the proinflammatory enzyme caspase-1 and release of the

1	proteins such as Bcl-2. More recently, the phenomenon of pyroptosis, an inflammatory form of cell death involving the upregulation of the proinflammatory enzyme caspase-1 and release of the proinflammatory cytokine IL-1 β, has been linked to a bystander effect of HIV replication on CD4+ T cells. Certain viral gene products have been associated with enhanced susceptibility to apoptosis; these include Env, Tat, and Vpr. In contrast, Nef has been shown to possess antiapoptotic properties. A number of studies, including those examining lymphoid tissue, have demonstrated that the rate of apoptosis is elevated in HIV infection and that apoptosis is seen in “bystander” cells such as CD8+ T cells and B cells as well as in uninfected CD4+ T cells. The intensity of apoptosis correlates with the general state of activation of the immune system and not with the stage of disease or with viral burden. It is likely that nonspecific apoptosis of immunocompetent cells related to immune activation

1	general state of activation of the immune system and not with the stage of disease or with viral burden. It is likely that nonspecific apoptosis of immunocompetent cells related to immune activation contributes to the immune abnormalities in HIV disease.

1	Autoimmune Phenomena The autoimmune phenomena that are common in HIV-infected individuals reflect, at least in part, chronic immune activation and the dysregulation of B and T cells. Although these phenomena usually occur in the absence of autoimmune disease, a wide spectrum of clinical manifestations that may be associated with autoimmunity have been described (see “Immunologic and Rheumatologic Diseases,” below). Autoimmune phenomena include antibodies against autoantigens expressed on intact lymphocytes and other cells, or against proteins released from dying cells. Antiplatelet antibodies have some clinical relevance in that they may contribute to the thrombocytopenia of HIV disease (see below). Antibodies to nuclear and cytoplasmic components of cells have been reported, as have antibodies to cardiolipin and phospholipids; CD4 molecules; CD43 molecules; C1q-A; variable regions of the T cell receptor α, β, and γ chains; Fas; denatured collagen; and IL-2. In addition,

1	as have antibodies to cardiolipin and phospholipids; CD4 molecules; CD43 molecules; C1q-A; variable regions of the T cell receptor α, β, and γ chains; Fas; denatured collagen; and IL-2. In addition, autoantibodies to a range of serum proteins, including albumin, immunoglobulin, and thyroglobulin, have been reported. Molecular mimicry, either from opportunistic pathogens or from HIV itself, is also a trigger or co-factor in autoimmunity. Antibodies against the HIV envelope proteins, especially gp41, often cross-react with host proteins; the best known examples are antibodies directed against the membrane-proximal external region of gp41 that also react with phospholipids and cardiolipin. The phenomenon of polyreactive HIV-specific antibodies may be beneficial to the host (see “Immune Response to HIV,” below).

1	The increased occurrence and/or exacerbation of certain autoimmune diseases have been reported in HIV infection; these diseases include psoriasis, idiopathic thrombocytopenic purpura, Graves’ disease, antiphospholipid syndrome, and primary biliary cirrhosis. The majority of these manifestations were described prior to the advent of cART and have decreased in frequency since its widespread use. However, with increasing availability of cART, an immune reconstitution inflammatory syndrome (IRIS) has become increasingly common in infected individuals, particularly those with low CD4+ T cell counts. IRIS is an autoimmune-like phenomenon characterized by a paradoxical deterioration of clinical condition, which is usually compartmentalized to a particular organ system, in individuals in whom cART has recently been initiated. It is associated with a decrease in viral load and at least partial recovery of immune competence, which is usually associated with increases in CD4+ T cell counts. The

1	has recently been initiated. It is associated with a decrease in viral load and at least partial recovery of immune competence, which is usually associated with increases in CD4+ T cell counts. The immunopathogenesis is felt to be related to an increase in immune response against the presence of residual antigens that are usually microbial and is commonly seen with underlying Mycobacterium tuberculosis and cryptococcosis. This syndrome is discussed in more detail below.

1	The immune system is homeostatically regulated by a complex network of immunoregulatory cytokines, which are pleiotropic and redundant and operate in an autocrine and paracrine manner. They are expressed continuously, even during periods of apparent quiescence of the immune system. On perturbation of the immune system by antigenic challenge, the expression of cytokines increases to varying degrees (Chap. 372e). Cytokines that are important components of this immunoregulatory network are thought to play major roles in HIV disease, during both the early and chronic phases of infection. A potent pro-inflammatory “cytokine storm” is induced during the acute phase of HIV infection, likely a response by inflammatory cells recruited to mucosal tissues where the virus initially replicates at very high levels. Cytokines that are induced during this early phase include IFN-α, IL-15, and the CXC chemokine IP-10 (CXCL10), followed by IL-6, IL-12, and TNF-α, and a delayed peak of the

1	at very high levels. Cytokines that are induced during this early phase include IFN-α, IL-15, and the CXC chemokine IP-10 (CXCL10), followed by IL-6, IL-12, and TNF-α, and a delayed peak of the anti-inflammatory cytokine IL-10. Soluble factors of innate immunity are also induced shortly after infection, including neopterin and β-microglobulin. Several of these early-expressed cytokines and factors are not down-regulated following the early phase of HIV infection, as seen in self-resolving viral infections, and persist during the chronic phase of infection and contribute to maintaining high levels of immune activation. Among the cytokines and factors associated with early innate immune responses, they are intended to contain viral replication, although most are potent inducers of HIV expression/replication because of their ability to induce immune activation that leads to enhanced viral production and an increase in readily available target cells for HIV (activated CD4+ T cells). The

1	because of their ability to induce immune activation that leads to enhanced viral production and an increase in readily available target cells for HIV (activated CD4+ T cells). The induction of IFN-α, one of the first cytokines induced during primary HIV infection, is thought to play a particularly important role in HIV pathogenesis by inducing a large number of IFN-associated genes that activate the immune system and alter the homeostasis of CD4+ T cells. Other cytokines that are elevated during the chronic phase of HIV infection and linked to immune activation include IFN-γ, the CC-chemokine RANTES (CCL5), macrophage inflammatory protein (MIP)-1β (CCL4), and IL-18.

1	Several specific cytokines and soluble factors have been associated with HIV pathogenesis at various stages of disease, in various tissues or organs, and in the regulation of HIV replication. Plasma levels of IP-10 are predictive of disease progression, whereas the proinflammatory cytokine IL-6, soluble CD14 (sCD14), and coagulation marker d-dimer are associated with increased risk of all-cause mortality in HIV-infected individuals. In particular, IL-6, sCD14, and d-dimer are associated with increased risk of cardiovascular disease and other causes of death, even in individuals receiving cART. IL-18 has also been shown to play a role in the development of the HIV-associated lipodystrophy syndrome, whereas increased levels of transforming growth factor (TGF)-β are associated with the induction of collagen deposition in lymph nodes (see above). Elevated levels of TNF-α and IL-6 have been demonstrated in plasma and cerebrospinal fluid (CSF), and increased expression of TNF-α, IL-1β,

1	of collagen deposition in lymph nodes (see above). Elevated levels of TNF-α and IL-6 have been demonstrated in plasma and cerebrospinal fluid (CSF), and increased expression of TNF-α, IL-1β, IFN-γ, and IL-6 has been demonstrated in the lymph nodes of HIV-infected individuals. RANTES, MIP-1α (CCL3), and MIP-1β (CCL4) (Chap. 372e) inhibit infection by and spread of R5 HIV-1 strains, while stromal cell–derived factor (SDF) 1 inhibits infection by and spread of X4 strains. The mechanisms whereby the CC-chemokines RANTES (CCL5), MIP-1α (CCL3), and MIP-1β (CCL4) inhibit infection of R5 strains of HIV, or SDF-1 blocks X4 strains of HIV, involve blocking of the binding of the virus to its co-receptors, the CC-chemokine receptor CCR5 and the CXC-chemokine receptor CXCR4, respectively. Other soluble factors that have not yet been fully characterized have also been shown to suppress HIV replication, independent of co-receptor usage.

1	The immune systems of patients with HIV infection are characterized by a profound increase in lymphocyte turnover that is immediately reduced with effective cART. Studies utilizing in vivo or in vitro labeling of lymphocytes in the S-phase of the cell cycle have demonstrated a tight correlation between the degree of lymphocyte turnover and plasma levels of HIV RNA. This increase in turnover is seen in CD4+ and CD8+ T lymphocytes as well as B lymphocytes and can be observed in peripheral blood and lymphoid tissue. Mathematical models derived from these data suggest that one can view the lymphoid pool as consisting of dynamically distinct subpopulations of cells that are differentially affected by HIV infection. A major consequence of HIV infection appears to be a shift in cells from a more quiescent pool to a pool with a higher turnover rate. It is likely that a consequence of a higher rate of turnover is a higher rate of cell death. The role of the thymus in adult human T cell

1	a more quiescent pool to a pool with a higher turnover rate. It is likely that a consequence of a higher rate of turnover is a higher rate of cell death. The role of the thymus in adult human T cell homeostasis and HIV pathogenesis is an area of controversy. While some data point to an important role for the thymus in maintaining T cell numbers and suggest that impairment of thymic function may be responsible for the declines in CD4+ T cells seen in the setting of HIV infection, other studies have concluded that the thymus plays a minor role in HIV pathogenesis. More recently, it has been suggested that the more rapid decline in CD4+ compared to CD8+ T cells may be linked to alterations in inflammatory and homeostatic cytokines that caused increased activation-induced death of CD4+ but not CD8+ T cells (see Table 226-5 for additional mechanisms of depletion).

1	As mentioned above, HIV-1 utilizes two major co-receptors along with CD4 to bind to, fuse with, and enter target cells; these co-receptors are CCR5 and CXCR4, which are also receptors for certain endogenous Loss of plasma membrane integrity Aberrant intracellular signaling due to viral budding events Accumulation of unintegrated viral Autoimmunity DNA Activation of DNA-dependent protein kinase during viral integration into host genome Interference with cellular RNA Innocent bystander killing of viral processing antigen–coated cells Intracellular gp120-CD4 autofusion Apoptosis, pyroptosis (caspase-1 events associated inflammation), autophagy Syncytia formation Inhibition of lymphopoiesis from

1	Intracellular gp120-CD4 autofusion Apoptosis, pyroptosis (caspase-1 events associated inflammation), autophagy Syncytia formation Inhibition of lymphopoiesis from Elimination of HIV-infected cells by virus-specific immune responses chemokines. Strains of HIV that utilize CCR5 as a co-receptor are referred to as R5 viruses. Strains of HIV that utilize CXCR4 are referred to as X4 viruses. Many virus strains are dual tropic in that they utilize both CCR5 and CXCR4; these are referred to as R5X4 viruses. The natural chemokine ligands for the major HIV co-receptors can readily block entry of HIV. For example, the CC-chemokines RANTES (CCL5), MIP-1α (CCL3), and MIP-1β (CCL4), which are the natural ligands for CCR5, block entry of R5 viruses, whereas SDF-1, the natural ligand for CXCR4, blocks entry of X4 viruses. The mechanism of inhibition of viral entry is a steric inhibition of binding that is not dependent on signal transduction (Fig. 226-25).

1	The transmitting virus is almost invariably an R5 virus that predominates during the early stages of HIV disease. In ~40% of HIV-infected individuals, there is a transition to a predominantly X4 virus that is associated with a relatively rapid progression of disease. However, at least 60% of infected individuals progress in their disease while maintaining predominance of an R5 virus. It should be pointed out that clade C viruses, unlike other subgroups, almost never switch from CCR5 tropism to CXCR4 tropism; the reason for this difference is unclear.

1	The basis for the tropism of different envelope glycoproteins for either CCR5 or CXCR4 relates to the ability of the HIV envelope, including the third variable region (V3 loop) of gp120, to interact with these co-receptors. In this regard, binding of gp120 to CD4 induces a conformational change in gp120 that increases its affinity for CCR5 (see above). Finally, R5 viruses are more efficient in infecting monocytes/macrophages and microglial cells of the brain (see “Neuropathogenesis in HIV Disease,” below).

1	tHe integrin α4β7 and mUcosal transmission of Hiv Several “accessory receptors” for HIV have been reported over the years, although only a few have withstood the test of time. These receptors are not necessary for virus binding and fusion to its target CD4+ T cell or for virus replication. However, the integrin α4β7 is an accessory receptor for HIV and it likely plays an important role in the transmission of HIV at mucosal surfaces such as the genital tract and gut. The integrin α4β7, which is the gut homing receptor for peripheral T cells, binds in its activated form to a specific tripeptide in the V2 loop of gp120, resulting in rapid activation of leukocyte function–associated antigen 1 (LFA-1), the central integrin in the establishment of virologic synapses, which facilitate efficient cell-to-cell spread of HIV. It has been demonstrated that α4β7high CD4+ T cells are more susceptible to productive infection than are α4β7low–neg CD4+ T cells because this cellular subset is enriched

1	cell-to-cell spread of HIV. It has been demonstrated that α4β7high CD4+ T cells are more susceptible to productive infection than are α4β7low–neg CD4+ T cells because this cellular subset is enriched with metabolically active CD4+ T cells that are CCR5high. These cells are present at the gut and genital tract mucosal surfaces. Importantly, it has been demonstrated that the virus that is transmitted during sexual exposure binds much more efficiently to α4β7 than does the virus that diversifies from the transmitting virus over time by mutation,

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders CC-Chemokine (RANTES, MIP-1˜, MIP-1°) ENV CD4 CXCR4 SDF-1 ENV CD4 CCR5 HIV HIV CD4+ Target Cell HIV HIV CD4+ Target Cell ABFIGuRE 226-25 Model for the role of co-receptors CXCR4 and CCR5 in the efficient binding and entry of X4 (A) and R5 (B) strains of HIV-1, respectively, into CD4+ target cells. Blocking of this initial event in the virus life cycle can be accomplished by inhibition of binding to the co-receptor by the normal ligand for the receptor in question. The ligand for CXCR4 is stromal cell–derived factor (SDF-1); the ligands for CCR5 are RANTES, MIP-1α, and MIP-1β. particularly involving the accumulation of glycosylation sites (see “Early Events in HIV Infection: Primary Infection and Initial Dissemination of Virus,” above).

1	particularly involving the accumulation of glycosylation sites (see “Early Events in HIV Infection: Primary Infection and Initial Dissemination of Virus,” above). Although the CD4+ T lymphocytes and to a lesser extent CD4+ cells of monocyte lineage are the principal targets of HIV, virtually any cell that expresses the CD4 molecule together with co-receptor molecules (see above and below) can potentially be infected with HIV. Circulating DCs have been reported to express low levels of CD4, and, depending on their stage of maturation, these cells can be infected with HIV. Epidermal Langerhans cells express CD4 and have been infected by HIV in vivo, although, as has been shown in vivo for DCs, FDCs, and B cells, these cells are more likely to bind and transfer virus to activated CD4+ T cells than to be productively infected themselves.

1	In vitro, HIV has been reported also to infect a wide range of cells and cell lines that express low levels of CD4, no detectable CD4, or only CD4 mRNA. However, since the only cells that have been shown unequivocally to be infected with HIV and to support replication of the virus are CD4+ T lymphocytes and cells of monocyte/macrophage lineage, the physiologic relevance of the in vitro infection of these other cell types is unclear.

1	Of potentially important clinical relevance is the demonstration that thymic precursor cells, which were assumed to be negative for CD3, CD4, and CD8 molecules, actually do express low levels of CD4 and can be infected with HIV in vitro. In addition, human thymic epithelial cells transplanted into an immunodeficient mouse can be infected with HIV by direct inoculation of virus into the thymus. Since these cells may play a role in the normal regeneration of CD4+ T cells, it is possible that their infection and depletion contribute, at least in part, to the impaired ability of the CD4+ T cell pool to completely reconstitute itself in certain infected individuals in whom cART has suppressed viral replication to <50 copies of HIV RNA per milliliter (see below). In addition, CD34+ monocyte precursor cells have been shown to be infected in vivo in patients with advanced HIV disease. It is likely that these cells express low levels of CD4, and therefore it is not essential to invoke

1	precursor cells have been shown to be infected in vivo in patients with advanced HIV disease. It is likely that these cells express low levels of CD4, and therefore it is not essential to invoke CD4-independent mechanisms to explain the infection.

1	ABNORMALITIES OF MONONuCLEAR CELLS CD4+ T Cells The primary immunopathogenic lesion in HIV infection involves CD4+ T cells, and the range of CD4+ T cell abnormalities in advanced HIV infection is broad. The defects are both quantitative and qualitative and ultimately impact virtually every limb of the immune system, indicating the critical dependence of the integrity of the immune system on the inducer/helper function of CD4+ T cells. In advanced HIV disease, most of the observed immune defects can ultimately be explained by the quantitative depletion of CD4+ T cells. However, T cell dysfunction can be demonstrated in patients early in the course of infection, even when the CD4+ T cell count is in the low-normal range. The degree and spectrum of dysfunctions increase as the disease progresses, reflecting the range of CD4+ T cell functional heterogeneity, especially in lymphoid tissues. One of the first sites of intense HIV replication is in the GALT where CD4+ TH17 cells reside; they

1	reflecting the range of CD4+ T cell functional heterogeneity, especially in lymphoid tissues. One of the first sites of intense HIV replication is in the GALT where CD4+ TH17 cells reside; they are important for host defense against extracellular pathogens in the intestinal mucosa and help maintain the integrity of the gut epithelium. In HIV infection, they are depleted by direct and indirect effects of viral replication and cause loss of gut homeostasis and integrity, as well as a shift to a more TH1 phenotype. Studies have shown that even after many years of cART, normalization of the CD4+ T cells in the GALT remains incomplete. In lymph nodes, HIV perturbs another important subset of the CD4+ helper T lineage, namely TFH cells (see “Lymphoid Organs and HIV Pathogenesis,” above). TFH cells, which are either derived directly from naïve CD4+ T cells or other TH precursors, migrate into B follicles during germinal center reactions and provide help to antigen-specific B cells through

1	cells, which are either derived directly from naïve CD4+ T cells or other TH precursors, migrate into B follicles during germinal center reactions and provide help to antigen-specific B cells through cell–cell interactions and secretion of cytokines to which B cells respond, the most important of which is IL-21. As with TH17 cells, TFH cells are highly susceptible to HIV infection. However, in contrast to TH17 and most other CD4+ T cell subsets, the number of TFH cells is increased in lymph nodes of HIV-infected individuals, especially those who are viremic. It is unclear whether this increase is helpful to responding B cells, although the likely outcome is that the increase in numbers is detrimental to the quality of the humoral immune response against HIV (see “Immune Response to HIV,” below). In addition, defects of central memory cells are a critical component of HIV immunopathogenesis. The progressive loss of antigen-specific CD4+ T cells has important implications for the

1	HIV,” below). In addition, defects of central memory cells are a critical component of HIV immunopathogenesis. The progressive loss of antigen-specific CD4+ T cells has important implications for the control of HIV infection. In this regard, there is a correlation between the maintenance of HIV-specific CD4+ T cell proliferative responses and improved control of infection. Essentially every T cell function has been reported to be abnormal at some stage of HIV infection. Loss of polyfunctional HIV-specific CD4+ T cells, especially those that produce IL-2, occurs early in disease, whereas IFN-producing CD4+ T cells are maintained longer and do not correlate with control of HIV viremia. Other abnormalities include impaired expression of IL-2 receptors, defective IL-2 production, reduced expression of the IL-7 receptor (CD127), and a decreased proportion of CD4+ T cells that express CD28, a major co-stimulatory molecule necessary for the normal activation of T cells, which is also

1	expression of the IL-7 receptor (CD127), and a decreased proportion of CD4+ T cells that express CD28, a major co-stimulatory molecule necessary for the normal activation of T cells, which is also depleted as a result of aging. Cells lacking expression of CD28 do not respond normally to activation signals and may express markers of terminal activation including HLA-DR, CD38, and CD45RO. As mentioned above (“Immune Activation, Inflammation, and HIV Pathogenesis”), a subset of CD4+ T cells referred to as T regulatory cells, or T-regs, may be involved in damping aberrant immune activation that propagates HIV replication. The presence of these T-reg cells correlates with lower viral loads and higher CD4+/CD8+ T cell ratios. A loss of this T-reg capability with advanced disease may be detrimental to the control of virus replication.

1	It is difficult to explain completely the profound immunodeficiency noted in HIV-infected individuals solely on the basis of direct infection and quantitative depletion of CD4+ T cells. This is particularly apparent during the early stages of HIV disease, when CD4+ T cell numbers may be only marginally decreased. In this regard, it is likely that CD4+ T cell dysfunction results from a combination of depletion of cells due to direct infection of the cell and a number of virus-related but indirect effects on the cell (Table 226-5). Several of these effects have been demonstrated ex vivo and/or by the analysis of cells isolated from the peripheral blood. However, as explained above, many of the defects are related to specialized CD4+ T cells that reside in lymphoid tissues. Furthermore, since the main targets of HIV infection are immunocompetent cells, these responses may contribute to immune cell depletion and immunologic dysfunction by eliminating both infected cells and “innocent

1	since the main targets of HIV infection are immunocompetent cells, these responses may contribute to immune cell depletion and immunologic dysfunction by eliminating both infected cells and “innocent bystander” cells. Soluble viral proteins, particularly gp120, can bind with high affinity to the CD4 molecules on uninfected T cells and monocytes; in addition, virus and/ or viral proteins can bind to DCs or FDCs. HIV-specific antibody can recognize these bound molecules and potentially collaborate in the elimination of the cells by ADCC. HIV envelope glycoproteins gp120 and gp160 manifest high-affinity binding to the CD4 molecule as well as to various chemokine receptors. Intracellular signals transduced by gp120 through both CD4 and CCR5/CXCR4 have been associated with a number of immunopathogenic processes including anergy, apoptosis, and abnormalities of cell trafficking. The molecular mechanisms responsible for these abnormalities include dysregulation of the T cell

1	of immunopathogenic processes including anergy, apoptosis, and abnormalities of cell trafficking. The molecular mechanisms responsible for these abnormalities include dysregulation of the T cell receptor–phosphoinositide pathway, p56lck activation, phosphorylation of focal adhesion kinase, activation of the MAP kinase and ras signaling pathways, and downregulation of the co-stimulatory molecules CD40 ligand and CD80.

1	The inexorable decline in CD4+ T cell counts that occurs in most HIV-infected individuals may result in part from the inability of the immune system to regenerate over an extended period of time the rapidly turning over CD4+ T cell pool efficiently enough to compensate for both HIV-mediated and naturally occurring attrition of cells. In this regard, the degree and duration of decline of CD4+ T cells at the time of initiation of therapy is an important predictor of the restoration of these cells. A person who maintains a very low CD4+ T cell count for a considerable period of time before the initiation of cART almost invariably has an incomplete reconstitution of such cells. At least two major mechanisms may contribute to the failure of the CD4+ T cell pool to reconstitute itself adequately over the course of HIV infection. The first is the destruction of lymphoid precursor cells, including thymic and bone marrow progenitor cells; the other is the gradual disruption of the lymphoid

1	over the course of HIV infection. The first is the destruction of lymphoid precursor cells, including thymic and bone marrow progenitor cells; the other is the gradual disruption of the lymphoid tissue microenvironment, which is essential for efficient regeneration of immunocompetent cells. Finally, during the advanced stages of CD4+ T lymphopenia, there are increased serum levels of the homeostatic cytokine IL-7. It was initially felt that this elevation was a homeostatic response to the lymphopenia; however, recent findings suggest that the increase in serum IL-7 was a result of reduced utilization of the cytokine related to the loss of cells expressing the IL-7 receptor, CD127, which serves as a normal physiologic regulator of IL-7 production.

1	CD8+ T Cells A relative CD8+ T lymphocytosis is generally associated with high levels of HIV plasma viremia and likely reflects an immune response to the virus as well as dysregulated homeostasis associated with generalized immune activation. During the late stages of HIV infection, there may be a significant reduction in the numbers of CD8+ T cells despite the presence of high levels of viremia. HIV-specific CD8+ CTLs have been demonstrated in HIV-infected individuals early in the course of disease, and their emergence often coincides with a decrease in plasma viremia—an observation that is a factor in 1237 the proposal that virus-specific CTLs can control HIV disease for a finite period of time in a certain percentage of infected individuals. However, emergence of HIV escape mutants that ultimately evade these HIV-specific CD8+ T cells has been described in the majority of HIV-infected individuals who are not receiving cART. In addition, as the disease progresses, the functional

1	that ultimately evade these HIV-specific CD8+ T cells has been described in the majority of HIV-infected individuals who are not receiving cART. In addition, as the disease progresses, the functional capability of these cells gradually decreases, at least in part due to the persistent nature of HIV infection that causes functional exhaustion via the upregulation of inhibitory receptors such as PD-1 on HIV-specific CD8+ T cells (see “Immune Activation, Inflammation, and HIV Pathogenesis,” above). As chronic immune activation persists, there are also systemic effects on CD8+ T cells, such that as a population they assume an abnormal phenotype characterized by expression of activation markers such as HLA-DR and CD38 with an absence of expression of the IL-2 receptor (CD25) and a reduced expression of the IL-7 receptor (CD127). In addition, CD8+ T cells lacking CD28 expression are increased in HIV disease, reflecting a skewed expansion of a less differentiated CD8+ T cell subset. This

1	of the IL-7 receptor (CD127). In addition, CD8+ T cells lacking CD28 expression are increased in HIV disease, reflecting a skewed expansion of a less differentiated CD8+ T cell subset. This skewing of subsets is also associated with diminished polyfunctionality, a qualitative difference that distinguishes nonprogressors from progressors. It has been reported that nonprogressors can also be distinguished from progressors by the maintenance in the former of a high proliferative capacity of their HIV-specific CD8+ T cells coupled to increases in perforin expression, characteristics that are markedly diminished in advanced HIV disease. It has been reported that the phenotype of CD8+ T cells in HIV-infected individuals may be of prognostic significance. Those individuals whose CD8+ T cells developed a phenotype of HLA-DR+/CD38– following seroconversion had stabilization of their CD4+ T cell counts, whereas those whose CD8+ T cells developed a phenotype of HLA-DR+/CD38+ had a more

1	cells developed a phenotype of HLA-DR+/CD38– following seroconversion had stabilization of their CD4+ T cell counts, whereas those whose CD8+ T cells developed a phenotype of HLA-DR+/CD38+ had a more aggressive course and a poorer prognosis. In addition to the defects in HIV-specific CD8+ CTLs, functional defects in other MHC-restricted CTLs, such as those directed against influenza and CMV, have been demonstrated. CD8+ T cells secrete a variety of soluble factors that inhibit HIV replication, including the CC-chemokines RANTES (CCL5), MIP-1α (CCL3), and MIP-1β (CCL4) as well as potentially a number of unidentified factors. The presence of high levels of HIV viremia in vivo as well as exposure of CD8+ T cells in vitro to HIV envelope, both of which are associated with aberrant immune activation, have been shown to be associated with a variety of cellular functional abnormalities. Furthermore, since the integrity of CD8+ T cell function depends in part on adequate inductive signals

1	have been shown to be associated with a variety of cellular functional abnormalities. Furthermore, since the integrity of CD8+ T cell function depends in part on adequate inductive signals from CD4+ T cells, the defect in CD8+ CTLs is likely compounded by the quantitative loss and qualitative dysfunction of CD4+ T cells.

1	B Cells The predominant defect in B cells from HIV-infected individuals is one of aberrant cellular activation, which is reflected by increased propensity to terminal differentiation and immunoglobulin secretion and increased expression of markers of activation and exhaustion. As a result of activation and differentiation in vivo, B cells from HIV viremic patients manifest a decreased capacity to mount a proliferative response to ligation of the B cell antigen receptor and other B cell stimuli in vitro. B cells from HIV-infected individuals manifest enhanced spontaneous secretion of immunoglobulins in vitro, a process that reflects their highly differentiated state in vivo. There is also an increased incidence of EBV-related B cell lymphomas in HIV-infected individuals that are likely due to combined effects of defective T cell immune surveillance and increased turnover that increases the risk of oncogenesis. Untransformed B cells cannot be infected with HIV, although HIV or its

1	due to combined effects of defective T cell immune surveillance and increased turnover that increases the risk of oncogenesis. Untransformed B cells cannot be infected with HIV, although HIV or its products can activate B cells directly. B cells from patients with high levels of viremia bind virions to their surface via the CD21 complement receptor. It is likely that in vivo activation of B cells by replication-competent or defective virus as well as viral products during the viremic state accounts at least in part for their activated phenotype. B cell subpopulations from HIV-infected individuals undergo a number of changes over the course of HIV disease, including the attrition of resting memory B cells and replacement with several aberrant memory and differentiated B cell subpopulations that collectively express reduced levels of CD21

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1238 and either increased expression of activation markers or inhibitory receptors associated with functional exhaustion. The more activated and differentiated B cells are also responsible for increased secretion of immunoglobulins and increased susceptibility to Fas-mediated apoptosis. In more advanced disease, there is also the appearance of immature B cells associated with CD4+ T cell lymphopenia. Cognate B cell–CD4+ T cell interactions are abnormal in viremic HIV-infected individuals in that B cells respond poorly to CD4+ T cell help and CD4+ T cells receive inadequate co-stimulatory signals from activated B cells. In vivo, the aberrant activated state of B cells manifests itself by hypergammaglobulinemia and by the presence of circulating immune complexes and autoantibodies. HIV-infected individuals respond poorly to primary and secondary immunizations with protein and polysaccharide antigens. Using immunization

1	of circulating immune complexes and autoantibodies. HIV-infected individuals respond poorly to primary and secondary immunizations with protein and polysaccharide antigens. Using immunization with influenza vaccine, it has been demonstrated that there is a memory B cell defect in HIV-infected individuals, particularly those with high levels of HIV viremia. There is also evidence that responses to HIV and non-HIV antigens in infected individuals, especially those who remain viremic, are enriched in abnormal subsets of B cells that either are highly prone to apoptosis or show signs of functional exhaustion. Taken together, these B cell defects are likely responsible in part for the inadequate response to HIV as well as to decreased response to vaccinations and the increase in certain bacterial infections seen in advanced HIV disease in adults, as well as for the important role of bacterial infections in the morbidity and mortality rates of HIV-infected children, who cannot mount an

1	infections seen in advanced HIV disease in adults, as well as for the important role of bacterial infections in the morbidity and mortality rates of HIV-infected children, who cannot mount an adequate humoral response to common bacterial pathogens. The absolute number of circulating B cells may be depressed in HIV infection; this phenomenon likely reflects increased activation-induced apoptosis as well as a redistribution of cells out of the circulation and into the lymphoid tissue—phenomena that are associated with ongoing viral replication.

1	Monocytes/Macrophages Circulating monocytes are generally normal in number in HIV-infected individuals; however, there is evidence of increased activation within this lineage. The increased level of sCD14 and other biomarkers (see above) reported in HIV-infected individuals is an indirect marker of monocyte activation in vivo. A number of other abnormalities of circulating monocytes have been reported in HIV-infected individuals, many of which may be related directly or indirectly to aberrant in vivo immune activation. In this regard, increased levels of lipopolysaccharide (LPS) are found in the sera of HIV-infected individuals due, at least in part, to translocation across the gut mucosal barrier (see above). LPS is a highly inflammatory bacterial product that preferentially binds to macrophages through CD14 and Toll-like receptors, resulting in cellular activation. Functional abnormalities of monocyte/macrophages in HIV disease include decreased secretion of IL-1 and IL-12;

1	to macrophages through CD14 and Toll-like receptors, resulting in cellular activation. Functional abnormalities of monocyte/macrophages in HIV disease include decreased secretion of IL-1 and IL-12; increased secretion of IL-10 and IL-18; defects in antigen presentation and induction of T cell responses due to decreased MHC class II expression; and abnormalities of Fc receptor function, C3 receptor–mediated clearance, oxidative burst responses, and certain cytotoxic functions such as ADCC, possibly related to low levels of expression of Fc and complement receptors. Monocytes express the CD4 molecule and several co-receptors for HIV on their surface, including CCR5, CXCR4, and CCR3, and thus are potential targets of HIV infection. The degree of cytopathicity of HIV for cells of the monocyte lineage is low, and HIV can replicate in cells of the monocyte lineage with relatively little cytopathic effect. Hence, monocyte-lineage cells may play a role in the dissemination of HIV in the body

1	lineage is low, and HIV can replicate in cells of the monocyte lineage with relatively little cytopathic effect. Hence, monocyte-lineage cells may play a role in the dissemination of HIV in the body and can serve as reservoirs of HIV infection, thus representing an obstacle to the eradication of HIV by antiretroviral drugs. In vivo infection of circulating monocytes is difficult to demonstrate; however, infection of tissue macrophages and macrophage-lineage cells in the brain (infiltrating macrophages or resident microglial cells) and lung (pulmonary alveolar macrophages) can be demonstrated easily. Tissue macrophages are an important source of HIV during the inflammatory response associated with opportunistic infections. Infection of monocyte precursors in the bone marrow may directly or indirectly be responsible for certain of the hematologic abnormalities in HIV-infected individuals. However, as with DCs, monocytes and macrophages express high levels of host restriction factors

1	or indirectly be responsible for certain of the hematologic abnormalities in HIV-infected individuals. However, as with DCs, monocytes and macrophages express high levels of host restriction factors that likely help explain the low contribution of myeloid cells to the overall viral burden in HIV-infected individuals.

1	Dendritic and Langerhans Cells DCs and Langerhans cells are thought to play an important role in the initiation of HIV infection by virtue of the ability of HIV to bind to cell-surface C-type lectin receptors, particularly DC-SIGN (see above) and Langerin. This allows efficient presentation of virus to CD4+ T cell targets that become infected; complexes of infected CD4+ T cells and DCs provide an optimal microenvironment for virus replication. There was once considerable disagreement regarding the HIV infectibility and hence the depletion as well as the dysfunction of DCs themselves. However, since the recognition of myeloid (mDC) and plasmacytoid (pDC) subsets, there has been a better appreciation of specific DC dysfunction in HIV disease. pDCs are an important component of the innate immune system and secrete large amounts of IFN-α in response to viral infections. The numbers of circulating pDCs are decreased in HIV infection through mechanisms that remain unclear, and there are

1	immune system and secrete large amounts of IFN-α in response to viral infections. The numbers of circulating pDCs are decreased in HIV infection through mechanisms that remain unclear, and there are conflicting reports regarding the frequency of pDCs in lymphoid tissues, with some studies suggesting that their increased tissue presence and secretion of inflammatory cytokines such as IFN-α contributes to lymphoid hyperplasia. The mDCs or conventional DCs are involved in the initiation of adaptive immunity in draining lymph nodes by presenting antigen to T cells and B cells, as well as by secreting cytokines such as IL-12, IL-15, and IL-18 that activate other immune cells. There are also indications that the relatively low infectibility of DCs may be associated with the expression of host restriction factors, including APOBEC3G (see above).

1	Natural Killer Cells The role of NK cells is to provide immunosurveillance against virus-infected cells, certain tumor cells, and allogeneic cells (Chap. 372e). There are no convincing data that HIV productively infects NK cells in vivo; however, functional abnormalities in NK cells have been observed throughout the course of HIV disease, and the severity of these abnormalities increases as disease progresses. NK cells are part of the innate immune system and act by direct killing of infected cells and secretion of antiviral cytokines. In early HIV infection there is an increase in the activation of NK cells, and the capacity to secrete IFN-γ is maintained, although they manifest reduced cytotoxic function. During chronic HIV infection, both NK cell cytotoxicity and cytokine secretion become impaired. Given that HIV infection of target cells downregulates HLA-A and -B, but not HLA-C and -D molecules, this may explain in part the relative inability of NK cells to kill HIV-infected

1	become impaired. Given that HIV infection of target cells downregulates HLA-A and -B, but not HLA-C and -D molecules, this may explain in part the relative inability of NK cells to kill HIV-infected target cells. However, the NK cell impairments, especially in patients with high levels of virus replication, are associated with an expansion of an “anergic” CD56–/CD16+ NK cell subset. This abnormal subset of NK cells manifests an increased expression of inhibitory NK cell receptors (iNKRs) and a substantial decrease in expression of natural cytotoxicity receptors (NCRs) and shows a markedly impaired lytic activity. The overrepresentation of this abnormal subset of NK cells may explain in part the observed defects in NK cell function in HIV-infected individuals and likely begins to occur during primary infection. NK cells also serve as important sources of HIV-inhibitory CC-chemokines. NK cells isolated from HIV-infected individuals constitutively produce high levels of MIP-1α (CCL3),

1	primary infection. NK cells also serve as important sources of HIV-inhibitory CC-chemokines. NK cells isolated from HIV-infected individuals constitutively produce high levels of MIP-1α (CCL3), MIP-1β (CCL4), and RANTES (CCL5), although the impact of these chemokines on HIV replication in vivo is unclear. Finally, NK cell–DC interactions are important for normal immune function. NK cells and DCs reciprocally modulate each other’s activation and maturation. These interactions are markedly impaired in HIV-infected individuals with high levels of plasma viremia.

1	pHenotypes of sUsceptibility and response to Hiv infection It is well known that individuals vary in their susceptibility to acquiring HIV infection and that there is wide variation in both the steady-state level of HIV that is established soon after infection (virologic setpoint) as well as the rate at which HIV-infected patients progress to AIDS. Some striking examples include sex workers who remain uninfected despite repeated exposure to HIV; HIV-infected individuals who spontaneously control viral replication in the absence of cART (HIV controllers); patients who resist disease progression for at least 8–10 years, despite viremia; and those progressing to AIDS within 3 years. Investigators have hypothesized that genetic differences may partly explain this interindividual variation in risk of acquiring HIV infection and disease progression rates. In addition to these phenotypes, it has been hypothesized that genetic variation may partly underpin the risk of developing specific

1	in risk of acquiring HIV infection and disease progression rates. In addition to these phenotypes, it has been hypothesized that genetic variation may partly underpin the risk of developing specific AIDS-defining illnesses (e.g., renal and neurologic diseases) and non-AIDS comorbidities (e.g., cardiovascular disease), as well as the variable recovery in CD4+ T cell counts observed while receiving cART.

1	Candidate gene approaches and genome-wide association studies (GWAS) have demonstrated associations between gene variations and the above-mentioned phenotypes. A list of some of these associations is shown in Table 226-6. While in vitro genome-wide functional scanning using RNA interference has identified hundreds of host factors that may be involved in the HIV life cycle, the association of these genes with HIV susceptibility and/or disease progression remains largely undefined. Below is a discussion of a few key genes with strong associations and their implications for improving clinical care.

1	associations witH ccr5 and translation of genetic findings to tHe clinic Possibly, the most dramatic example of the importance of genetic studies for identifying host factors that influence HIV-AIDS pathogenesis is from studies related to the gene that encodes for CC chemokine receptor 5 (CCR5). While in vitro studies established that CCR5 is the major HIV co-receptor for the cell entry of HIV-1 into the host, it was genetic studies that established the seminal in vivo role of this receptor for the initial entry of HIV and AIDS pathogenesis. Genetic analysis revealed that the in vitro resistance to CCR5-using R5 strains of HIV is in some instances due to carriage of two defective CCR5 alleles. This defect is a 32-bp deletion in the coding sequence (designated as the Δ32 allele). The CCR5 Δ32 allele encodes a truncated protein that is not expressed on the cell surface.

1	Approximately 1% of individuals of European ancestry are homozygous for the CCR5 Δ32 allele. Depending on the geographic region in Europe, up to 20% of individuals are heterozygous for the CCR5 Δ32 allele. The CCR5 Δ32 allele is either absent or extremely rare in other populations. The evolutionary pressure that resulted in the emergence of the CCR5 Δ32 allele in the European population remains unknown and has been speculated to be secondary to an ancestral pandemic such as the plague.

1	Individuals homozygous for the CCR5 Δ32 allele (Δ32/Δ32) lack CCR5 surface expression and are highly resistant to acquiring HIV infection. Heterozygosity for the CCR5 Δ32 allele is also associated with a reduced risk of acquiring HIV. Consequently, the frequency of the CCR5 Δ32 allele is enriched in individuals of European descent who remain uninfected despite exposure to the virus. Although the CCR5 Δ32/Δ32 genotype is associated with profound resistance to acquiring HIV, a few individuals with this genotype have become infected with the X4 HIV strain and, in some instances, experienced an accelerated disease course. In general, CCR5 Δ32 heterozygosity is associated with a slower HIV disease course.

1	Subsequent studies identified single nucleotide polymorphisms (SNPs) in the promoter (regulatory region) of CCR5 that influence its expression levels. Alleles bearing specific cassettes of linked polymorphisms (haplotypes) were identified and designated as human haplogroups A to G*2 (HHA to HHG*2). The CCR5 Δ32 is found on the HHG*2 haplotype. The CCR5 HHE haplotype was associated with higher CCR5 expression, and genetic association studies have shown that homozygosity for the CCR5 HHE haplotype is associated with an increased risk of acquiring HIV, progressing rapidly to AIDS, and reduced immune recovery on cART. The CCR2-64I-bearing HHF*2 haplotype is associated with a slower HIV disease course. The CCR5 HHA haplotype is the ancestral CCR5 haplotype and is associated with a lower CCR5 expression. The HHA haplotype was associated with slower disease progression in African populations and has been speculated to be a basis for why SIV-infected chimpanzees (who all carry the ancestral

1	expression. The HHA haplotype was associated with slower disease progression in African populations and has been speculated to be a basis for why SIV-infected chimpanzees (who all carry the ancestral CCR5 HHA haplotype) may resist disease progression. The CCR5 haplotypes also influence cell-mediated immunity and immune 1239 recovery on cART.

1	The association of variations in the CCR5 gene with HIV-AIDS phenotypes is also an example of how discoveries made in the laboratory (bench) have been translated to improve health outcomes (bedside). The discovery that the CCR5 Δ32/Δ32 genotype is associated with strong resistance to HIV infection, and that uninfected Caucasians bearing this genotype did not appear to have impaired immunity, led to the development of two kinds of therapies. First, it spurred the development of a new class of FDA-approved therapies, entry inhibitors (e.g., maraviroc), that block the interaction of CCR5 with the HIV envelope. Second, it led to the development of novel experimental cellular therapies. An HIV-infected patient with acute myelogenous leukemia was given an allogeneic stem-cell transplantation from an HLA-compatible person whose cells lacked expression of CCR5 due to the Δ32/Δ32 genotype. There has been no evidence of HIV-1 infection in the transplanted patient thus far (6 years). This

1	from an HLA-compatible person whose cells lacked expression of CCR5 due to the Δ32/Δ32 genotype. There has been no evidence of HIV-1 infection in the transplanted patient thus far (6 years). This observation spurred the hope of an HIV cure and led to the development of additional novel cellular therapies involving autologous transplantation of CD4+ T-cells in which the CCR5 gene is inactivated ex vivo using new gene editing procedures.

1	discovery of Hla class i alleles tHat associate witH virologic control of Hiv infection There is a strong association between variations within the HLA-B gene with protective (e.g., HLA-B*57 and -B*27 alleles) or detrimental (e.g., HLA-B*35 allele) outcomes during HIV infection. Carriage of the HLA-B*57 and/or HLA-B*27 alleles is associated with slower disease progression. The beneficial effects of these alleles may relate in part to their consistent associations with a lower virologic setpoint as well as to higher cell-mediated immunity. The protective effect of the HLA-B*57 and -B*27 alleles on HIV disease course is underscored by the finding that the prevalence of these alleles is higher among long-term nonprogressors and HIV elite controllers (see above). On the other hand, the HLA-B*35 allele has been associated with faster progression to AIDS and higher viral load. The prevalence of the HLA-B alleles differs between populations. HLA

1	B*57:01 in Europeans and HLA-B*57:03 in African Americans are the protective alleles. In some populations (e.g., Japanese) where the HLA-B*57/-B*27 alleles are absent, HLA-B*51 is associated with a protective phenotype. Possession of the protective HLA-B alleles is associated with broader and stronger CD8+ T cell responses to HIV epitopes. The mechanisms underlying the differential effects of the HLA-B alleles on HIV disease course may relate to differences in the ability of antigen-presenting cells to present immunodominant HIV epitopes to T helper or cytotoxic T lymphocytes in the context of MHC-encoded molecules. This may result in differential immune responses that influence viral replication. In this regard, the HLA-B alleles that impact HIV disease course differ in their amino acid residues in the HLA-B peptide-binding groove—and this may play a critical role in virologic control.

1	Investigators have also examined the influence of extended HLA haplotypes (linked alleles) on HIV disease course. The extended HLA ancestral haplotype (AH) 8.1 is defined by the presence of HLA-A1, HLA-B8, and HLA-DR3 alleles. AH 8.1 is the most common ancestral haplotype in Caucasians (present in 10%) and is associated with multiple autoimmune diseases in HIV-uninfected persons. These associations of AH 8.1 are thought to be due to a genetically determined hyperresponsiveness characterized by high TNF-α production and lack of complement C4A. Strong epidemiologic data indicate that carriage of AH 8.1 in HIV-infected persons is associated with a rapid decline in CD4+ T cells and faster progression to AIDS development. Gene–gene interactions between HLA alleles and other genes (e.g., killer cell immunoglobulin-like receptors) also may influence HIV disease progression rates.

1	polymorpHisms identified by gwas tHat associate witH virologic control Large-scale GWASs have been conducted for the phenotype of viral load, including in a large group of HIV controllers. GWAS in HIV-infected persons of European ancestry identified four SNPs in genes in the HLA class I loci that associated with virologic control. Human Immunodeficiency Virus Disease: AIDS and Related Disorders KIR+HLA KIR3DS1 + HLA-Bw4-80Ile Altered NK cell activity required to eliminate KIR3DS1 with HLA Bw4-80I +: delayed AIDS HIV-infected cells onset HLA-C1 + KIR2DL3, Reduction of inhibitory KIR likely results in HLA-C1+/KIR2DL3+: better immune recovery LILRB2+HLA LILRB2 + HLA class I Regulation of dendritic cells by LILRB2-HLA Control of HIV-1 engagement aRepresentative genes and polymorphisms and bpossible mechanisms are listed. cSome of the associations are population specific and may display cohort-specific effects.

1	Note: Apobec, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like; ApoE, apolipoprotein E; ART, antiretroviral therapy; CCL, CC ligand; CCL3L, CCL3-like; CCR5, CC chemokine receptor 5; CRP, C-reactive protein; CXCR6, chemokine (C-X-C motif ) receptor 6; DARC, Duffy antigen receptor for chemokines; HCP5, HLA class I histocompatibility antigen protein P5; HHE, human haplogroup E; HLA, human leukocyte antigen; IFN, interferon; IL, interleukin; LILRB2, leukocyte immunoglobulin-like receptor B2; KIR, killer cell immunoglobulin-like receptors; KS, Kaposi’s sarcoma; MBL, mannose-binding lectin; MHC, major histocompatibility complex; MICA: MHC class I polypeptide-related sequence A; ORF, open reading frame; PARD3B, par-3 family cell polarity regulator beta; PROX1, prospero homeobox 1; PSORS1C3, psoriasis susceptibility 1 candidate 3; SNP, single nucleotide polymorphism; rs#, SNP identification number in SNP database from NCBI; UTR, untranslated region; ZNRD1, zinc ribbon domain

1	1; PSORS1C3, psoriasis susceptibility 1 candidate 3; SNP, single nucleotide polymorphism; rs#, SNP identification number in SNP database from NCBI; UTR, untranslated region; ZNRD1, zinc ribbon domain containing 1; +, present, –, absent.

1	Sources: Sunil K Ahuja, MD, Weijing He, MD, and Gabriel Catano, MD. Reviews for additional information: P An et al: Trends Genet 26:119, 2010; J Fellay: Antivir Ther 14:731, 2009; RA Kaslow et al: J Infect Dis 191:S68, 2005; D van Manen et al: Retrovirology 9:70, 2012; MP Martin et al: Immunol Rev 254:245, 2013; S Limou et al: Front Immunol 4:118, 2013.

1	These SNPs are within or in the vicinity of HCP5, HLA-C, MICA, and PSORS1C3 genes. The protective effects of the SNPs in HCP5 and MICA may relate to their linkage with known protective HLA-B alleles. The protective HCP5 allele is in linkage disequilibrium with the HLAB*57:01, and the protective MICA allele tags with the HLA-B*57:01 and B*27:05 alleles. The protective HLA-C SNP is associated with higher HLA-C expression, and this effect is thought to be due to the altered binding of a microRNA to the HLA-C mRNA. Higher HLA-C expression has been associated with beneficial HIV phenotypes. The mechanism associated with the SNP in PSORS1C3 is unknown. GWAS in African Americans identified a SNP that tags the HLA-B*57:03 allele that is known to associate with lower virologic setpoint and slower disease course. Together these GWAS data underscore the importance of variations in HLA class I loci in control of viral replication.

1	GENETIC ASSOCIATIONS WITH SPECIFIC AIDS AND NON-AIDS CONDITIONS • carotid artery disease Many of the non-AIDS events in HIV-infected individuals resemble those related to immune senescence and those found in the HIV-uninfected aging population. A functional SNP in the ryanodine receptor 3 (RYR3) gene was found to be associated with an increased risk of common carotid intima–media thickness (cIMT), which is a surrogate for subclinical atherosclerosis. Functional studies on RYR3 and its isoforms demonstrate a major role of these receptors in modulating endothelial function and atherogenesis via calcium signaling pathways, providing a biologically plausible mechanism by which the SNP in RYR3 may associate with increased cIMT risk.

1	renal disease HIV-1-associated nephropathy (HIVAN) is a form of focal sclerosing glomerulonephritis caused by direct infection of kidney epithelial cells with HIV. HIVAN is more common in persons of African descent. There is evidence that polymorphisms in the MYH9 gene and in the neighboring APOL1 gene are a strong determinant of susceptibility to HIVAN in African Americans. The effect of carrying two APOL1 risk alleles explains nearly 35% of HIVAN. The mechanisms by which MYH9/APOL1 variants predispose to HIVAN are currently unknown.

1	The mechanisms by which MYH9/APOL1 variants predispose to HIVAN are currently unknown. Hiv-associated neurocognitive disorder HIV-associated neurocognitive disorder (HAND) comprises a spectrum of neurocognitive deficits due to HIV infection. Variations in the apolipoprotein E (ApoE) gene have strong associations with Alzheimer’s disease in the HIV-uninfected population. In HIV-infected persons, possession of the ApoE4 allele has been associated with several cognitive outcomes including dementia, peripheral neuropathy, and impairment in cognition and immediate and delayed verbal memory. Macrophage recruitment and activation plays a central role in the development of many of the HAND syndromes. Variations in chemokines that play an influential role in macrophage activation and recruitment, namely CCL2 (MCP-1) and CCL3 (MIP-1α), have been shown to alter the risk of developing HAND. Variations in mitochondrial genes also have been associated with risk of AIDS and HAND.

1	associations witH art-related adverse events Abacavir, an effective antiretroviral agent, is associated with significant risk of hypersensitivity reactions (2–9% of cases). Interestingly, while the HLA-B*57:01 allele is associated with a slower HIV disease course, possession of this allele is associated with a higher risk of abacavir-associated hypersensitivity. Pharmacogenetic screening for the HLA-B*57:01 allele is recommended before initiation of abacavir treatment.

1	While there has been a remarkable decrease in the incidence in the severe forms of HIV encephalopathy among those with access to treatment in the era of effective cART, HIV-infected individuals can still experience milder forms of neurocognitive impairment despite adequate cART. A variety of factors may contribute to the neurocognitive decline, which includes lack of complete control of HIV replication in the brain, production of HIV proteins that may be neurotoxic, low CD4+ T cell nadir, chronic immune activation, comorbidities such as

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1242 drug abuse, and the potential for neurotoxicity of certain of the antiretroviral drugs. HIV has been demonstrated in the brain and CSF of infected individuals with and without neuropsychiatric abnormalities. As opposed to lymphoid tissues, there are no resident lymphocytes in the brain. The main cell types that are infected in the brain in vivo are the perivascular macrophages and the microglial cells; low-level viral replication is also seen in perivascular astrocytes. Monocytes that have already been infected in the blood can migrate into the brain, where they then reside as macrophages, or macrophages can be directly infected within the brain. The precise mechanisms whereby HIV enters the brain are unclear; however, they are thought to relate, at least in part, to the ability of virus-infected and immune-activated macrophages to induce adhesion molecules such as E-selectin and vascular cell adhesion molecule 1

1	they are thought to relate, at least in part, to the ability of virus-infected and immune-activated macrophages to induce adhesion molecules such as E-selectin and vascular cell adhesion molecule 1 (VCAM-1) on brain endothelium. Other studies have demonstrated that HIV gp120 enhances the expression of intercellular adhesion molecule 1 (ICAM-1) in glial cells; this effect may facilitate entry of HIV-infected cells into the CNS. Virus isolates from the brain are preferentially R5 strains as opposed to X4 strains; in this regard, HIV-infected individuals who are heterozygous for CCR5-Δ32 appear to be relatively protected against the development of HIV encephalopathy. Once HIV enters the brain due to pressures of the local environment, it evolves to develop distinct sequences in the env, tat, and LTR genes. These unique sequences have been associated with neurocognitive dysfunction; however, it is unclear if they are causal (see below). HIV-infected individuals may manifest white matter

1	and LTR genes. These unique sequences have been associated with neurocognitive dysfunction; however, it is unclear if they are causal (see below). HIV-infected individuals may manifest white matter lesions as well as neuronal loss. The white matter lesions are due to axonal injury and a disruption of the blood-brain barrier and not due to demyelination. Given the absence of evidence of HIV infection of neurons either in vivo or in vitro, it is highly unlikely that direct infection of these cells accounts for their loss. Rather, the HIV-mediated effects on neurons are thought to involve indirect pathways whereby viral proteins, particularly gp120 and Tat, trigger the release of endogenous neurotoxins from macrophages and to a lesser extent from astrocytes. In addition, it has been demonstrated that both HIV-1 Nef and Tat can induce chemotaxis of leukocytes, including monocytes, into the CNS. Neurotoxins can be released from monocytes as a consequence of infection and/or immune

1	demonstrated that both HIV-1 Nef and Tat can induce chemotaxis of leukocytes, including monocytes, into the CNS. Neurotoxins can be released from monocytes as a consequence of infection and/or immune activation. Monocyte-derived neurotoxic factors have been reported to kill neurons via the N-methyld-aspartate (NMDA) receptor. In addition, HIV gp120 shed by virus-infected monocytes could cause neurotoxicity by antagonizing the function of vasoactive intestinal peptide (VIP), by elevating intracellular calcium levels, and by decreasing nerve growth factor levels in the cerebral cortex. A variety of monocyte-derived cytokines can contribute directly or indirectly to the neurotoxic effects in HIV infection; these include TNF-α, IL-1, IL-6, TGF-β, IFN-γ, platelet-activating factor, and endothelin. Furthermore, among the CC-chemokines, elevated levels of monocyte chemotactic protein-1 (MCP-1 or CCL-2) in the brain and CSF have been shown to correlate best with the presence and degree of HIV

1	Furthermore, among the CC-chemokines, elevated levels of monocyte chemotactic protein-1 (MCP-1 or CCL-2) in the brain and CSF have been shown to correlate best with the presence and degree of HIV encephalopathy. In addition, infection and/or activation of monocyte-lineage cells can result in increased production of eicosanoids, quinolinic acid, nitric oxide, excitatory amino acids such as l-cysteine and glutamate, arachidonic acid, platelet activating factor, free radicals, TNF-α, and TGF-β, which may contribute to neurotoxicity. Astrocytes may play diverse roles in HIV neuropathogenesis. Reactive gliosis or astrocytosis has been demonstrated in the brains of HIV-infected individuals, and TNF-α and IL-6 have been shown to induce astrocyte proliferation. In addition, astrocyte-derived IL-6 can induce HIV expression in infected cells in vitro. Furthermore, it has been suggested that astrocytes may downregulate macrophage-produced neurotoxins. Treatment with cART leads to improvement in

1	can induce HIV expression in infected cells in vitro. Furthermore, it has been suggested that astrocytes may downregulate macrophage-produced neurotoxins. Treatment with cART leads to improvement in neuropsychiatric manifestations and a decrease in these cytokine levels in CSF, suggesting that they are driven by the virus or by its products. However, even in patients on long-term cART, there may be evidence of persistently activated lymphocytes in the CSF. It is unclear if these lymphocytes may contribute to neuronal injury in the brain or are critical for controlling the CNS viral reservoir. The contribution of host genetic factors to development of neuropsychiatric manifestations of HIV infection has not been well studied. However, evidence supports the role of the E4 allele for apoE in an increased risk of HIV-associated neurocognitive disorders and peripheral neuropathy.

1	It has also been suggested that the CNS may serve as a relatively sequestered site for a reservoir of latently infected cells that might be a barrier for the eradication of virus by cART (see “Reservoirs of HIV-Infected Cells: Obstacles to the Eradication of Virus,” above).

1	There are at least four distinct epidemiologic forms of KS: (1) the classic form that occurs in older men of predominantly Mediterranean or eastern European Jewish backgrounds with no recognized contributing factors; (2) the equatorial African form that occurs in all ages, also without any recognized precipitating factors; (3) the form associated with organ transplantation and its attendant iatrogenic immunosuppressed state; and (4) the form associated with HIV-1 infection. In the latter two forms, KS is an opportunistic disease; in HIV-infected individuals, unlike typical opportunistic infections, its occurrence is not strictly related to the level of depression of CD4+ T cell counts. The pathogenesis of KS is complex; fundamentally, it is an angioproliferative disease that is not a true neoplastic sarcoma, at least not in its early stages. It is a manifestation of excessive proliferation of spindle cells that are believed to be of vascular origin and have features in common with

1	neoplastic sarcoma, at least not in its early stages. It is a manifestation of excessive proliferation of spindle cells that are believed to be of vascular origin and have features in common with endothelial and smooth-muscle cells. In HIV disease the development of KS is dependent on the interplay of a variety of factors including HIV-1 itself, human herpes virus 8 (HHV-8), immune activation, and cytokine secretion. A number of epidemiologic and virologic studies have clearly linked HHV-8, which is also referred to as Kaposi’s sarcoma–associated herpesvirus (KSHV), to KS not only in HIV-infected individuals but also in individuals with the other forms of KS. HHV-8 is a γ-herpesvirus related to EBV and herpesvirus saimiri. It encodes a homologue to human IL-6 and, in addition to KS, has been implicated in the pathogenesis of body cavity lymphoma, multiple myeloma, and monoclonal gammopathy of undetermined significance. Sequences of HHV-8 are found universally in the lesions of KS, and

1	implicated in the pathogenesis of body cavity lymphoma, multiple myeloma, and monoclonal gammopathy of undetermined significance. Sequences of HHV-8 are found universally in the lesions of KS, and patients with KS are virtually all seropositive for HHV-8. HHV-8 DNA sequences can be found in the B cells of 30–50% of patients with KS and 7% of patients with AIDS without clinically apparent KS.

1	Between 1 and 2% of eligible blood donors are positive for antibodies to HHV-8, while the prevalence of HHV-8 seropositivity in HIV-infected men is 30–35%. The prevalence of HHV-8 seropositivity in HIV-infected women is ~4%. This finding is reflective of the lower incidence of KS in women. It has been debated whether HHV-8 is actually the transforming agent in KS; the bulk of the cells in the tumor lesions of KS are not neoplastic cells. However, it has been demonstrated that endothelial cells can be transformed in vitro by HHV-8. In this regard, HHV-8 possesses a number of genes, including homologues of the IL-8 receptor, Bcl-2, and cyclin D, that can potentially transform the host cell. Despite the complexity of the pathogenic events associated with the development of KS in HIV-infected individuals, HHV-8 is the etiologic agent of this disease. The initiation and/or propagation of KS requires an activated state and is mediated, at least in part, by cytokines. A number of factors,

1	individuals, HHV-8 is the etiologic agent of this disease. The initiation and/or propagation of KS requires an activated state and is mediated, at least in part, by cytokines. A number of factors, including TNF-α, IL-1β, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), basic fibroblast growth factor, and oncostatin M, function in an autocrine and paracrine manner to sustain the growth and chemotaxis of the KS spindle cells. In this regard, KSHV-derived IL-6 has been demonstrated to induce proliferation of lymphoma cells and to inhibit the cytostatic effects of IFN-α on KSHV-infected lymphoma cells.

1	As detailed above and below, following the initial burst of viremia during primary infection, HIV-infected individuals mount robust immune responses that in most cases substantially curtail the levels of plasma viremia and likely contribute to delaying the ultimate development of clinically apparent disease for a median of 10 years in untreated individuals. This immune response contains elements of both humoral and cell-mediated immunity involving both innate and adaptive immune responses (Table 226-7; Fig. 226-26). It is directed against multiple antigenic determinants of the HIV virion as well as against viral proteins expressed on the surface of infected cells. 1243

1	Ironically, those CD4+ T cells with T cell receptors specific for HIV are theoretically those CD4+ T cells most likely to be activated—and thus to serve as early targets for productive HIV infection and the cell death or dysfunction associated with infection. Thus, an early consequence of HIV infection is interference with and decrease of the helper T cell Abbreviation: MHC, major histocompatibility complex. population needed to generate an effective immune response. Although a great deal of investigation has been directed toward delineating and better understanding the components of this immune response, it remains unclear which immunologic effector mechanisms are most important in delaying progression of infection and which, if any, play a role in the pathogenesis of HIV disease. This lack of knowledge has also hampered the ability to develop an effective vaccine for HIV disease.

1	Antibodies to HIV usually appear within 3–6 weeks and almost invariably within 12 weeks of primary infection (Fig. 226-27); rare exceptions are in individuals who have defects in the ability to produce HIV-specific antibodies. Detection of these antibodies forms the basis of most diagnostic screening tests for HIV infection. The appearance of HIV-binding antibodies detected by ELISA and Western blot assays occurs prior to the appearance of neutralizing antibodies; the latter generally appear following the initial decreases in plasma viremia and are more closely related to the appearance of HIV-specific CD8+ T lymphocytes. The first antibodies detected are those directed against the immunodominant region of the envelope gp41, followed by the appearance of antibodies to the structural or gag protein p24 and the gag precursor p55. Antibodies to p24 gag are followed by the appearance of antibodies to the outer envelope glycoprotein (gp120), the gag protein p17, and the products of the pol

1	protein p24 and the gag precursor p55. Antibodies to p24 gag are followed by the appearance of antibodies to the outer envelope glycoprotein (gp120), the gag protein p17, and the products of the pol gene (p31 and p66). In addition, one may see antibodies to the low-molecular-weight regulatory proteins encoded by the HIV genes vpr, vpu, vif, rev, tat, and nef. On rare occasion, levels of HIV-specific antibodies may decline during treatment of acute HIV infection.

1	While antibodies to multiple antigens of HIV are produced, the precise functional significance of these different antibodies is unclear. The only viral proteins that elicit neutralizing antibodies are the envelope proteins gp120 and gp41. Antibodies directed toward the envelope proteins of HIV have been characterized both as being protective and as possibly contributing to the pathogenesis of HIV disease. Among the protective antibodies are those that function to neutralize HIV directly and prevent the spread of infection to Human Immunodeficiency Virus Disease: AIDS and Related Disorders Activation, proliferation, cytokine and chemokine release additional cells, as well as those that participate in ADCC. The first neutralizing antibodies are directed against the autologous infecting 700 1:25,600 600 1:12,800 Levels of Anti-HIV antibody 1:6400 400 1:3200 300 1:1600 1:800200 100 1:400

1	Levels of Anti-HIV antibody 1:6400 400 1:3200 300 1:1600 1:800200 100 1:400 FIGuRE 226-27 Relationship between antigenemia and the devel-FIGuRE 226-26 Schematic representation of the different opment of antibodies to HIV. Levels of plasma HIV parallel those immunologic effector mechanisms thought to be active in the of p24 antigen. Antibodies to HIV proteins are generally seen 6–12 setting of HIV infection. Detailed descriptions are given in the weeks following infection and 3–6 weeks after the development of text. ADCC, antibody-dependent cellular cytotoxicity; MHC, major plasma viremia. Late in the course of illness, antibody levels to p24 histocompatibility complex; TCR, T cell receptor. decline, generally in association with a rising titer of p24 antigen.

1	1244 virus and appear after approximately 12 weeks of infection. Due to its high rate of mutation the virus is usually able to quickly escape these (and subsequent) neutralizing antibodies. One important mechanism of immune escape is the addition of N-linked glycosylation sites, forming a glycan shield that interferes with envelope recognition by these initial antibodies. A number of broad and potent HIV-neutralizing envelope-specific antibodies have been isolated from HIV-infected individuals in studies designed to better understand the host response to HIV infection. Approximately 20% of patients develop antibodies capable of neutralizing highly diverse strains. These studies have revealed at least five major sites within the HIV envelope that are able to elicit broadly-neutralizing antibodies. These sites include antibodies directed toward the CD4 binding site (CD4bs) of gp120, those binding glycandependent epitopes in the V1/V2 region of gp120, those near the base of the V3 region

1	These sites include antibodies directed toward the CD4 binding site (CD4bs) of gp120, those binding glycandependent epitopes in the V1/V2 region of gp120, those near the base of the V3 region of gp120, those binding to the gp120/gp41 bridge, and those binding to the membrane-proximal region of gp41 (Fig. 226-28). Several of these antibodies contain unique features including high levels of somatic hypermutation, selective germline gene usage (especially for CD4bs antibodies) and long heavy chain complementary determining regions (especially CDRH3). Of note, while these antibodies are broadly neutralizing in vitro, their precise in vivo significance is unclear and the patients from whom they were derived demonstrate evidence of ongoing viral replication unless treated with cART. The other major class of protective antibodies are those that participate in ADCC, a form of cell-mediated immunity (Chap. 372e) in which NK cells that bear Fc receptors are armed with specific anti-HIV

1	other major class of protective antibodies are those that participate in ADCC, a form of cell-mediated immunity (Chap. 372e) in which NK cells that bear Fc receptors are armed with specific anti-HIV antibodies that bind to the NK cells via their Fc portion. These armed NK cells then bind to and destroy cells expressing HIV antigens. The levels of anti-envelope antibodies capable of mediating ADCC are highest in the earlier stages of HIV infection. Antibodies to both gp120 and gp41 have been shown to participate in ADCC-mediated killing of HIV-infected cells. In vitro, IL-2 can augment ADCC-mediated killing. In addition to playing a role in host defense, HIV-specific antibodies have also been implicated in disease pathogenesis. Antibodies directed to gp41, when present in low titer, have been shown in vitro to be capable of facilitating infection of cells through an Fc receptor– mediated mechanism known as antibody enhancement. Thus, the same regions of the envelope protein of HIV that

1	shown in vitro to be capable of facilitating infection of cells through an Fc receptor– mediated mechanism known as antibody enhancement. Thus, the same regions of the envelope protein of HIV that give rise to antibodies capable of mediating ADCC can also elicit the production of antibodies that can facilitate infection of cells in vitro. In addition, it has been postulated that anti-gp120 antibodies that participate in the ADCC killing of HIV-infected cells might also kill uninfected CD4+ T cells if the uninfected cells had bound free gp120, a phenomenon referred to as bystander killing. One of the most primitive components of the humoral immune system is the complement system (Chap. 372e). This element of innate immunity consists of ~30 proteins that are found circulating in blood or associated with cell membranes. While HIV alone is capable of directly activating the complement cascade, the resulting lysis is

1	FIGuRE 226-28 Known targets of broadly neutralizing antibodies against HIV-1. (Adapted from PD Kwong, JR Mascola: Immunity 37:412, 2012.) weak due to the presence of host cell regulatory proteins captured in the virion envelope during budding. It is possible that complementopsonized HIV virions have increased infectivity in a manner analogous to antibody-mediated enhancement. Given that T cell–mediated immunity is known to play a major role in host defense against most viral infections (Chap. 372e), it is generally thought to be an important component of the host immune response to HIV. T cell immunity can be divided into two major categories: that mediated by helper/inducer CD4+ T cells and that mediated by cytotoxic/immunoregulatory CD8+ T cells.

1	HIV-specific CD4+ T cells can be detected in the majority of HIV-infected patients through the use of flow cytometry to measure intracellular cytokine production in response to MHC class II tetramers pulsed with HIV peptides or through lymphocyte proliferation assays utilizing HIV antigens such as p24. These cells likely play a critical role in the orchestration of the immune response to HIV by providing help to HIV-specific B cells and CD8+ T cells. They may also be capable of directly killing HIV-infected cells. HIV-specific CD4+ T cells may be preferential targets of HIV infection by HIV-infected antigen-presenting cells during the generation of an immune response to HIV (Fig. 226-26). However, they also are likely to undergo clonal expansions in response to HIV antigens and thus survive as a population of cells. No clear correlations exist between levels of HIV-specific CD4+ T lymphocytes and plasma HIV RNA levels; however, in the setting of high viral loads, CD4+ T cell responses

1	as a population of cells. No clear correlations exist between levels of HIV-specific CD4+ T lymphocytes and plasma HIV RNA levels; however, in the setting of high viral loads, CD4+ T cell responses to HIV antigens appear to shift from one of proliferation and IL-2 production to one of IFN-γ production. Thus, while a reverse correlation exists between the level of p24-specific proliferation and levels of plasma HIV viremia, the nature of the causal relationship between these parameters is unclear.

1	MHC class I–restricted, HIV-specific CD8+ T cells have been identified in the peripheral blood of patients with HIV-1 infection. These cells include CTLs that produce perforins and T cells that can be induced by HIV antigens to express an array of cytokines such as IFN-γ, IL-2, MIP-1β, and TNF-α. CTLs have been identified in the peripheral blood of patients within weeks of HIV infection and prior to the appearance of plasma virus. The selective pressure they exert on the evolution of the population of circulating viruses reflects their potential role in control of HIV infection. These CD8+ T lymphocytes, through their HIV-specific antigen receptors, bind to and cause the lytic destruction of target cells bearing autologous MHC class I molecules presenting HIV antigens. Two types of CTL activity can be demonstrated in the peripheral blood or lymph node mononuclear cells of HIV-infected individuals. The first type directly lyses appropriate target cells in culture without prior in vitro

1	can be demonstrated in the peripheral blood or lymph node mononuclear cells of HIV-infected individuals. The first type directly lyses appropriate target cells in culture without prior in vitro stimulation (spontaneous CTL activity). The other type of CTL activity reflects the precursor frequency of CTLs (CTLp); this type of CTL activity can be demonstrated by stimulation of CD8+ T cells in vitro with a mitogen such as phytohemagglutinin or anti-CD3 antibody.

1	In addition to CTLs, CD8+ T cells capable of being induced by HIV antigens to express cytokines such as IFN-γ also appear in the setting of HIV-1 infection. It is not clear whether these are the same or different effector pools compared with those cells mediating cytotoxicity; in addition, the relative roles of each in host defense against HIV are not fully understood. It does appear that these CD8+ T cells are driven to in vivo expansion by HIV antigen. There is a direct correlation between levels of CD8+ T cells capable of producing IFN-γ in response to HIV antigens and plasma levels of HIV-1 RNA. Thus, while these cells are clearly induced by HIV-1 infection, their overall ability to control infection remains unclear. Multiple HIV antigens, including Gag, Env, Pol, Tat, Rev, and Nef, can elicit CD8+ T cell responses. Among patients who control viral replication in the absence of antiretroviral drugs are a subset of patients referred to as elite nonprogressors (see “Long-Term

1	Nef, can elicit CD8+ T cell responses. Among patients who control viral replication in the absence of antiretroviral drugs are a subset of patients referred to as elite nonprogressors (see “Long-Term Survivors and Long-Term Nonprogressors,” above) whose peripheral blood contains a population of CD8+ T cells that undergo substantial proliferation and perforin expression in response to HIV antigens. It is possible that these cells play an important role in HIV-specific host defense.

1	At least three other forms of cell-mediated immunity to HIV have been described: non-cytolytic CD8+ T cell–mediated suppression of HIV replication, ADCC, and NK cell activity. Non-cytolytic CD8+ T cell–mediated suppression of HIV replication refers to the ability of CD8+ T cells from an HIV-infected patient to inhibit the replication of HIV in tissue culture without killing infected targets. There is no requirement for HLA compatibility between the CD8+ T cells and the HIV-infected cells. This effector mechanism is thus nonspecific and appears to be mediated by soluble factor(s) including the CC-chemokines RANTES (CCL5), MIP-1α (CCL3), and MIP-1β (CCL4). These CC-chemokines are potent suppressors of HIV replication and operate at least in part via blockade of the HIV co-receptor (CCR5) for R5 (macrophage-tropic) strains of HIV-1 (see above). ADCC, as described above in relation to humoral immunity, involves the killing of HIV-expressing cells by NK cells armed with specific antibodies

1	R5 (macrophage-tropic) strains of HIV-1 (see above). ADCC, as described above in relation to humoral immunity, involves the killing of HIV-expressing cells by NK cells armed with specific antibodies directed against HIV antigens. Finally, NK cells alone have been shown to be capable of killing HIV-infected target cells in tissue culture. This primitive cytotoxic mechanism of host defense is directed toward nonspecific surveillance for neoplastic transformation and viral infection through recognition of altered class I MHC molecules.

1	The establishment of HIV as the causative agent of AIDS and related syndromes early in 1984 was followed by the rapid development of sensitive screening tests for HIV infection. By March 1985, blood donors in the United States were routinely screened for antibodies to HIV. In 1996, blood banks in the United States added the p24 antigen capture assay to the screening process to help identify the rare infected individuals who were donating blood in the time (up to 3 months) between infection and the development of antibodies. In 2002, the ability to detect early infection with HIV was further enhanced by the licensure of nucleic acid testing (NAT) as a routine part of blood donor screening. These refinements decreased the interval between infection and detection (window period) from 22 days for antibody testing to 16 days with p24 antigen testing and subsequently to 12 days with NAT. The development of sensitive assays for monitoring levels of plasma viremia ushered in a new era of

1	days for antibody testing to 16 days with p24 antigen testing and subsequently to 12 days with NAT. The development of sensitive assays for monitoring levels of plasma viremia ushered in a new era of being able to monitor the progression of HIV disease more closely. Utilization of these tests, coupled with the measurement of levels of CD4+ T lymphocytes in peripheral blood, is essential in the management of patients with HIV infection.

1	The CDC has recommended that screening for HIV infection be performed as a matter of routine health care. The diagnosis of HIV infection depends on the demonstration of antibodies to HIV and/or the direct detection of HIV or one of its components. As noted above, antibodies to HIV generally appear in the circulation 3–12 weeks following infection.

1	The standard blood screening tests for HIV infection are based on the detection of antibodies to HIV. A common platform is the ELISA, also referred to as an enzyme immunoassay (EIA). This solid-phase assay is an extremely good screening test with a sensitivity of >99.5%. Most diagnostic laboratories use commercial kits that contain antigens from both HIV-1 and HIV-2 and thus are able to detect antibodies to either. These kits use both natural and recombinant antigens and are continuously updated to increase their sensitivity to newly discovered species, such as group O viruses (Fig. 226-1). The fourth-generation EIA tests combine detection of antibodies to HIV with detection of the p24 antigen of HIV. EIA tests are generally scored as positive (highly reactive), negative (nonreactive), or indeterminate (partially reactive). While the EIA is an extremely sensitive test, it is not optimal with regard to specificity. This is particularly true in studies of low-risk individuals, such as

1	indeterminate (partially reactive). While the EIA is an extremely sensitive test, it is not optimal with regard to specificity. This is particularly true in studies of low-risk individuals, such as volunteer blood donors. In this latter population, only 10% of EIA-positive individuals are subsequently confirmed to have HIV infection. Among the factors associated with false-positive EIA tests are antibodies to class II antigens (such as may be seen following pregnancy, blood transfusion, or transplantation), autoantibodies, hepatic disease, recent influenza vaccination, and acute viral infections. For these reasons, anyone suspected of having HIV infection based on a positive or inconclusive EIA result should 1245 ideally have the result confirmed with a more specific assay such as the Western blot. One can estimate whether an individual has a recent infection with HIV-1 by comparing the results on a standard EIA test that will score positive for all infected individuals with the

1	Western blot. One can estimate whether an individual has a recent infection with HIV-1 by comparing the results on a standard EIA test that will score positive for all infected individuals with the results on an assay modified to be less sensitive (“detuned assay”) that will score positive for individuals with established HIV infection and negative for individuals with recent infection. In rare instances, an HIV-infected individual treated early in the course of infection may revert to a negative EIA. This does not indicate clearing of infection; rather, it signifies levels of ongoing exposure to virus or viral proteins insufficient to maintain a measurable antibody response. When these individuals have discontinued therapy, viruses and antibodies have reappeared.

1	The most commonly used confirmatory test is the Western blot (Fig. 226-29). This assay takes advantage of the fact that multiple HIV antigens of different, well-characterized molecular weights elicit the production of specific antibodies. These antigens can be separated on the basis of molecular weight, and antibodies to each component can be detected as discrete bands on the Western blot. A negative Western blot is one in which no bands are present at molecular weights corresponding to HIV gene products. In a patient with a positive or indeterminate EIA and a negative Western blot, one can conclude with certainty that the EIA reactivity was a false positive. On the other hand, a Western blot demonstrating antibodies to products of all three of the major genes of HIV (gag, pol, and env) is conclusive evidence of infection with HIV. Criteria established by the U.S. Food and Drug Administration (FDA) in 1993 for a positive Western blot state that a result is considered positive if

1	is conclusive evidence of infection with HIV. Criteria established by the U.S. Food and Drug Administration (FDA) in 1993 for a positive Western blot state that a result is considered positive if antibodies exist to two of the three HIV proteins: p24, gp41, and gp120/160. Using these criteria, ~10% of all blood donors deemed positive for HIV-1 infection lacked an antibody band to the pol gene product p31. Some 50% of these blood donors were subsequently found to be false positives. Thus, the absence of the p31 band should increase the suspicion that one may be dealing with a false-positive test result. In this setting it is prudent to obtain additional confirmation with an RNA-based test for HIV-1 and/ or a follow-up Western blot. By definition, Western blot patterns of reactivity that do not fall into the positive or negative categories are considered “indeterminate.” There are two possible explanations for an indeterminate Western blot result. The most likely explanation in a

1	do not fall into the positive or negative categories are considered “indeterminate.” There are two possible explanations for an indeterminate Western blot result. The most likely explanation in a low-risk individual is that the patient being tested has antibodies that cross-react with one of the proteins of HIV. The most common patterns of cross-reactivity are antibodies that react with p24 and/or p55. The least likely explanation in this setting is that the individual is infected with HIV and is in the process of mounting a classic antibody response. In either instance, the Western blot should be repeated in 1 month to determine whether the indeterminate pattern is a pattern in evolution. In addition, one may attempt to confirm a diagnosis of HIV infection with the p24 antigen capture assay or one of the tests for HIV RNA (discussed below). While the Western blot is an excellent confirmatory test for HIV infection in patients with a positive or indeterminate EIA, it is a poor

1	assay or one of the tests for HIV RNA (discussed below). While the Western blot is an excellent confirmatory test for HIV infection in patients with a positive or indeterminate EIA, it is a poor screening test. Among individuals with a negative EIA and PCR for HIV, 20–30% may show one or more bands on Western blot. While these bands are usually faint and represent cross-reactivity, their presence creates a situation in which other diagnostic modalities (such as DNA PCR, RT-PCR, or p24 antigen capture) must be employed to ensure that the bands do not indicate early HIV infection.

1	A guideline for the use of these serologic tests in attempting to make a diagnosis of HIV infection is depicted in Fig. 226-30. In patients in whom HIV infection is suspected, the appropriate initial test is the EIA. If the result is negative, unless there is strong reason to suspect early HIV infection (as in a patient exposed within the previous 3 months), the diagnosis is ruled out and retesting should be performed only as clinically indicated. If the EIA is indeterminate or positive, the test should be repeated. If the repeat is negative on two occasions, one can assume that the initial positive reading was due to a technical error in the performance of the assay and that the patient is negative. If the repeat is indeterminate or positive, one should proceed to the HIV-1 Western blot. If the Western blot is positive, the diagnosis is HIV-1 Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1.

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1. Virus digested: digest separated into 1. Positive HIV-1 infection components by molecular weight 2. gp 160 immunization 2. Proteins transferred to filter paper: 3. Indeterminate (HIV-2 infection) reaction with test serum 4. Indeterminate (cross-reacting antibody to p24) 3. Enzyme-conjugated antihuman antibody added 5. Negative 4. FIGuRE 226-29 Western blot assay for detection of antibodies to HIV. A. Schematic representation of how a Western blot is performed. B. Examples of patterns of Western blot reactivity. In each instance the Western blot strip contains antigens to HIV-1. The serum from the patient immunized to the HIV-1 envelope gp160 contains only antibodies to the HIV-1 envelope proteins. The serum from the patient with HIV-2 infection cross-reacts with both reverse transcriptase and gag gene products of HIV-1.

1	infection. If the Western blot is negative, the EIA can be assumed to there is no progression in the Western blot, a diagnosis of HIV-1 is have been a false positive for HIV-1 and the diagnosis of HIV-1 infec-ruled out. If either the p24 or HIV-1 RNA assay is positive and/or the tion is ruled out. It would also be prudent at this point to perform spe-HIV-1 Western blot shows progression, a tentative diagnosis of HIV-1 cific serologic testing for HIV-2 following the same type of algorithm. infection can be made and later confirmed with a follow-up Western If the Western blot for HIV-1 is indeterminate, it should be repeated blot demonstrating a positive pattern. In addition to these standard in 4–6 weeks; in addition, one may proceed to a p24 antigen capture laboratory-based assays for detecting antibodies to HIV, an series of assay, HIV-1 RNA assay, or HIV-1 DNA PCR and specific serologic point-of-care tests can provide results in 1–60 min. Among the most testing for HIV-2. If the p24

1	antibodies to HIV, an series of assay, HIV-1 RNA assay, or HIV-1 DNA PCR and specific serologic point-of-care tests can provide results in 1–60 min. Among the most testing for HIV-2. If the p24 and HIV RNA assays are negative and popular of these is the OraQuick Rapid HIV-1 antibody test that can be run on blood, plasma, or saliva. The sensitivity and specificity of this test is ~99% when run on whole blood. Specificity remains the SEROLOGIC TESTS IN THE DIAGNOSIS OF HIV-1 OR same but sensitivity drops to 98% when the test is run on saliva. While

1	HIV-2 INFECTION negative results from this test are adequate to rule out a diagnosis of HIV infection, a positive finding should be considered preliminary and confirmed with standard serologic testing, as described above. Two rapid test kits are licensed for home use. They are the OraQuick In-Home HIV test and the Home Access HIV-1 test system.

1	A variety of laboratory tests are available for the direct detection of HIV or its components (Table 226-8). These tests may be of considerable help in making a diagnosis of HIV infection when the Western blot results are indeterminate. In addition, the tests detecting levels of HIV RNA can be used to determine prognosis and to assess the response to antiretroviral therapies. The simplest of the direct detection tests is the p24 antigen capture assay. This is an EIA-type assay in which the solid phase consists of antibodies to the p24 antigen of HIV. It detects the viral protein p24 in the blood of HIV-infected individuals where it exists either as free antigen or complexed to anti-p24 antibodies. Overall, ~30% of individuals with untreated HIV infection have detectable levels of free p24 antigen. This increases to ~50% when samples are treated with a weak acid to dissociate antigen-antibody FIGuRE 226-30 Algorithm for the use of serologic tests in the complexes. Throughout the course

1	antigen. This increases to ~50% when samples are treated with a weak acid to dissociate antigen-antibody FIGuRE 226-30 Algorithm for the use of serologic tests in the complexes. Throughout the course of HIV infection, an equilibrium diagnosis of HIV-1 or HIV-2 infection. *Stable indeterminate exists between p24 antigen and anti-p24 antibodies. During the first Western blot 4–6 weeks later makes HIV infection unlikely. However, few weeks of infection, before an immune response develops, there it should be repeated twice at 3-month intervals to rule out HIV infec-is a brisk rise in p24 antigen levels (Fig. 226-27). After the develoption. Alternatively, one may test for HIV-1 p24 antigen or HIV RNA. EIA, ment of anti-p24 antibodies, these levels decline. Late in the course of enzyme immunoassay. infection, when circulating levels of virus are high, p24 antigen levels aSensitivity figures refer to those approved by the U.S. Food and Drug Administration. bPrices may be lower in

1	immunoassay. infection, when circulating levels of virus are high, p24 antigen levels aSensitivity figures refer to those approved by the U.S. Food and Drug Administration. bPrices may be lower in large-volume settings. Abbreviations: bDNA, branched DNA; cDNA, complementary DNA; EIA, enzyme immunoassay; TMA, transcription-mediated amplification; NASBA, nucleic acid sequence–based amplifi cation; PCR, polymerase chain reaction.

1	also increase, particularly when detected by techniques involving dissociation of antigen-antibody complexes. The p24 antigen capture assay has its greatest use as a screening test for HIV infection in patients suspected of having the acute HIV syndrome, as high levels of p24 antigen are present prior to the development of antibodies. Its use as a stand-alone test for routine blood donor screening for HIV infection has been replaced by use of NAT or “fourth-generation” assays that combine antigen and antibody testing. The ability to measure and monitor levels of HIV RNA in the plasma of patients with HIV infection has been of extraordinary value in furthering our understanding of the pathogenesis of HIV infection, in monitoring the response to cART, and in providing a diagnostic tool in settings where measurements of anti-HIV antibodies may be misleading, such as in acute infection and neonatal infection. Four assays are predominantly used for this purpose. They are reverse

1	tool in settings where measurements of anti-HIV antibodies may be misleading, such as in acute infection and neonatal infection. Four assays are predominantly used for this purpose. They are reverse transcriptase PCR (RT-PCR; Amplicor); branched DNA (bDNA; VERSANT); transcription-mediated amplification (TMA; APTIMA); and nucleic acid sequence–based amplification (NASBA; NucliSENS). These tests are of value in making a diagnosis of HIV infection, in establishing initial prognosis, and in monitoring the effects of therapy. In addition to these four commercially available tests, the DNA PCR also is employed by research laboratories for making a diagnosis of HIV infection by amplifying HIV proviral DNA from peripheral blood mononuclear cells. The commercially available RNA detection tests have a sensitivity of 40–8100 copies of HIV RNA per milliliter of plasma. Research laboratory–based RNA assays can detect as few as one HIV RNA copy per milliliter, while the DNA PCR tests can detect

1	have a sensitivity of 40–8100 copies of HIV RNA per milliliter of plasma. Research laboratory–based RNA assays can detect as few as one HIV RNA copy per milliliter, while the DNA PCR tests can detect proviral DNA at a frequency of one copy per 10,000–100,000 cells. Thus, these tests are extremely sensitive. One frequent consequence of a high degree of sensitivity is some loss of specificity, and false-positive results have been reported with each of these techniques. For this reason, a positive EIA with a confirmatory Western blot remains the “gold standard” for a diagnosis of HIV infection, and the interpretation of other test results must be done with this in mind.

1	In the RT-PCR technique, following DNAse treatment, a cDNA copy is made of all RNA species present in plasma. Because HIV is an RNA virus, this will result in the production of DNA copies of the HIV genome in amounts proportional to the amount of HIV RNA present in plasma. This cDNA is then amplified and characterized using standard PCR techniques, employing primer pairs that can distinguish genomic cDNA from messenger cDNA. The bDNA assay involves the use of a solid-phase nucleic acid capture system and signal amplification through successive nucleic acid hybridizations to detect small quantities of HIV RNA. Both tests can achieve a tenfold increase in sensitivity to 40–50 copies of HIV RNA per milliliter with a preconcentration step in which plasma undergoes ultracentrifugation to pellet the viral particles. In the TMA assay, a cDNA copy of viral RNA is made using primers that contain a promoter sequence for T7 RNA polymerase. T7 polymerase is then added to produce multiple copies

1	the viral particles. In the TMA assay, a cDNA copy of viral RNA is made using primers that contain a promoter sequence for T7 RNA polymerase. T7 polymerase is then added to produce multiple copies of RNA amplicon from the DNA template. It is qualified at 100 copies/mL. The NASBA technique involves the isothermal amplification of a sequence within the gag region of HIV in the presence of internal standards and employs the production of multiple RNA copies through the action of T7-RNA polymerase. The resulting RNA species are quantitated through hybridization with a molecular beacon DNA probe that is quenched in the absence of hybridization. The lower limit of detection for the NucliSENS assay is 80 copies/mL.

1	In addition to being a diagnostic and prognostic tool, RT-PCR and DNA-PCR are also useful for amplifying defined areas of the HIV genome for sequence analysis and have become an important technique for studies of sequence diversity and microbial resistance to antiretroviral agents. In patients with a positive or indeterminate EIA test and an indeterminate Western blot, and in patients in whom serologic testing may be unreliable (such as patients with hypogammaglobulinemia or advanced HIV disease), these tests for quantitating HIV RNA in plasma or detecting proviral DNA in peripheral blood mononuclear cells are valuable tools for making a diagnosis of HIV infection; however, they should be used for diagnosis only when standard serologic testing has failed to provide a definitive result.

1	The epidemic of HIV infection and AIDS has provided the clinician with new challenges for integrating clinical and laboratory data to effect optimal patient management. The close relationship between clinical manifestations of HIV infection and CD4+ T cell count has made measurement of CD4+ T cell numbers a routine part of the evaluation of HIV-infected individuals. The discovery of HIV as the cause of AIDS led to the development of sensitive tests that allow one to monitor the levels of HIV in the blood. Determinations of peripheral blood CD4+ T cell counts and measurements of the plasma levels of HIV RNA provide a powerful set of tools for determining prognosis and monitoring response to therapy.

1	CD4+ T Cell Counts The CD4+ T cell count is the laboratory test generally accepted as the best indicator of the immediate state of immunologic competence of the patient with HIV infection. This measurement, which can be made directly or calculated as the product of the percentage of CD4+ T cells (determined by flow cytometry) and the total lymphocyte count (determined by the white blood cell count [WBC] multiplied by the lymphocyte differential percentage), has been shown to correlate very well with the level of immunologic competence. Patients with CD4+ T cell counts <200/μL are at high risk of disease from P. jiroveci, while patients with CD4+ T cell counts <50/ μL are at high risk of disease from CMV, mycobacteria of the M. avium complex (MAC), and/or T. gondii (Fig. 226-31). Once the CD4+ T cell count is <200/μL, patients should be placed on a regimen for P. jiroveci prophylaxis, and once the count is <50/μL, primary prophylaxis for MAC infection is indicated. As with any

1	the CD4+ T cell count is <200/μL, patients should be placed on a regimen for P. jiroveci prophylaxis, and once the count is <50/μL, primary prophylaxis for MAC infection is indicated. As with any laboratory measurement, one may wish to obtain two determinations prior to any significant changes in patient management based on CD4+ T cell count alone. Patients with HIV infection should have CD4+ T cell measurements performed at the time of diagnosis and every 3–6 months thereafter. More frequent measurements should be made if a declining trend is noted. For patients who have been on cART for at least 2 years

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders

1	FIGuRE 226-31 Relationship between CD4+ T cell counts and the development of opportunistic diseases. Boxplot of the median (line inside the box), first quartile (bottom of the box), third quartile (top of the box), and mean (asterisk) CD4+ lymphocyte count at the time of the development of opportunistic disease. Can, candidal esophagitis; CMV, cytomegalovirus infection; Crp, cryptosporidiosis; Cry, cryptococcal meningitis; DEM, AIDS dementia complex; HSV, herpes simplex virus infection; HZos, herpes zoster; KS, Kaposi’s sarcoma; MAC, Mycobacterium avium complex bacteremia; NHL, non-Hodgkin’s lymphoma; PCP, primary Pneumocystis jiroveci pneumonia; PCP2, secondary P. jiroveci untreated patient. Following the initiation of therapy or any change in therapy, plasma HIV RNA levels should be monitored approximately every 4 weeks until the effectiveness of the therapeutic regimen is determined by the development of a new steady-state level of HIV RNA. In most instances of effective

1	be monitored approximately every 4 weeks until the effectiveness of the therapeutic regimen is determined by the development of a new steady-state level of HIV RNA. In most instances of effective antiretroviral therapy the plasma level of HIV RNA will drop to <50 copies/mL within 6 months of the initiation of treatment. During therapy, levels of HIV RNA should be monitored every 3–6 months to evaluate the continuing effectiveness of therapy.

1	HIV Resistance Testing The availability of multiple antiretroviral drugs as treatment options has generated a great deal of interest in the potential for measuring the sensitivity of an individual’s HIV virus(es) to different antiretroviral agents. HIV resistance testing can be done through either genotypic pneumonia; PML, progressive multifocal leukoencephalopathy; Tox, Toxoplasma gondii encephalitis; or phenotypic measurements. In the WS, wasting syndrome. (From RD Moore, RE Chaisson: Ann Intern Med 124:633, 1996.) genotypic assays, sequence analyses with HIV RNA levels persistently <50 copies/mL, the monitoring of the CD4 count is felt by many to be optional. There are a handful of clinical situations in which the CD4+ T cell count may be misleading. Patients with HTLV-1/HIV co-infection may have elevated CD4+ T cell counts that do not accurately reflect their degree of immune competence. In patients with hypersplenism or those who have undergone splenectomy, and in patients

1	may have elevated CD4+ T cell counts that do not accurately reflect their degree of immune competence. In patients with hypersplenism or those who have undergone splenectomy, and in patients receiving medications that suppress the bone marrow such as IFN-α, the CD4+ T cell percentage may be a more reliable indication of immune function than the CD4+ T cell count. A CD4+ T cell percentage of 15 is comparable to a CD4+ T cell count of 200/μL.

1	HIV RNA Determinations Facilitated by highly sensitive techniques for the precise quantitation of small amounts of nucleic acids, the measurement of serum or plasma levels of HIV RNA has become an essential component in the monitoring of patients with HIV infection. As discussed in “Diagnosis of HIV Infection,” above, the most commonly used technique is the RT-PCR assay. This assay generates data in the form of number of copies of HIV RNA per milliliter of serum or plasma and can reliably detect as few as 40 copies of HIV RNA per milliliter of plasma. Research-based assays can detect down to one copy per milliliter. While it is common practice to describe levels of HIV RNA below these cut-offs as “undetectable,” this is a term that should be avoided as it is imprecise and leaves the false impression that the level of virus is 0. By utilizing more sensitive, nested PCR techniques and by studying tissue levels of virus as well as plasma levels, HIV RNA can be detected in virtually every

1	impression that the level of virus is 0. By utilizing more sensitive, nested PCR techniques and by studying tissue levels of virus as well as plasma levels, HIV RNA can be detected in virtually every patient with HIV infection. The one notable exception to this is a patient who underwent cytoreductive therapy followed by a bone marrow transplant from a CCR5Δ32 homozygous donor.

1	Measurements of changes in HIV RNA levels over time have been of great value in delineating the relationship between levels of virus and rates of disease progression (Fig. 226-22), the rates of viral turnover, the relationship between immune system activation and viral replication, and the time to development of drug resistance. HIV RNA measurements are greatly influenced by the state of activation of the immune system and may fluctuate greatly in the setting of secondary infections or immunization. For these reasons, decisions based on HIV RNA levels should never be made on a single determination. Measurements of plasma HIV RNA levels should be made at the time of HIV diagnosis and every 3–6 months thereafter in the of the HIV genomes obtained from patients are compared with sequences of viruses with known antiretroviral resistance profiles. In the phenotypic assays, the in vivo growth of viral isolates obtained from the patient is compared to the growth of reference strains of the

1	viruses with known antiretroviral resistance profiles. In the phenotypic assays, the in vivo growth of viral isolates obtained from the patient is compared to the growth of reference strains of the virus in the presence or absence of different antiretroviral drugs. A modification of this phenotypic approach utilizes a comparison of the enzymatic activities of the reverse transcriptase, protease, or integrase genes obtained by molecular cloning of patients’ isolates to the enzymatic activities of genes obtained from reference strains of HIV in the presence or absence of different drugs targeted to these genes. These tests are quite good in identifying those antiretroviral agents that have been utilized in the past and suggesting agents that may be of future value in a given patient. Resistance testing is recommended at the time of initial diagnosis and, if therapy is not initiated at that time, at the time of initiation of cART. Drug resistance testing is also indicated in the setting

1	testing is recommended at the time of initial diagnosis and, if therapy is not initiated at that time, at the time of initiation of cART. Drug resistance testing is also indicated in the setting of virologic failure and should be performed while the patient is still on the failing regimen because of the propensity for the pool of HIV quasispecies to rapidly revert to wild-type in the absence of the selective pressures of cART. In the hands of experts, resistance testing enhances the short-term ability to decrease viral load by ~0.5 log compared with changing drugs merely on the basis of drug history. In addition to the use of resistance testing to help in the selection of new drugs in patients with virologic failure, it may also be of value in selecting an initial regimen for treatment of therapy-naïve individuals. This is particularly true in geographic areas with a high level of background resistance. The patient needs to have an HIV-1 RNA level above 500– 1000 copies/mL for an

1	of therapy-naïve individuals. This is particularly true in geographic areas with a high level of background resistance. The patient needs to have an HIV-1 RNA level above 500– 1000 copies/mL for an accurate resistance determination. Resistance assays lose their consistency at lower levels of plasma viremia.

1	Co-Receptor Tropism Assays Following the licensure of maraviroc as the first CCR5 antagonist for the treatment of HIV infection (see below), it became necessary to be able to determine whether a patient’s virus was likely to respond to this treatment. Patients tend to have CCR5tropic virus early in the course of infection, with a trend toward CXCR4 viruses later in disease. The antiretroviral agent maraviroc is effective only against CCR5-tropic viruses. Because the genotypic determinants of cellular tropism are poorly defined, a phenotypic assay is necessary to determine this property of HIV. Two commercial assays, the Trofile assay (Monogram Biosciences) and the Phenoscript assay (VIRalliance), are available to make this determination. These assays clone the envelope regions of the patient’s virus into an indicator virus that is then used to infect target cells expressing either CCR5 or CXCR4 as their

1	TABLE 226-9 AssOCIATIOn BETwEEn HIgH-sEnsITIvITy CRP, IL-6, And D-dIMER wITH ALL-CAusE MORTALITy In PATIEnTs wITH HIv InFECTIOn IL-6 8.3 <.0001 11.8 <.0001 D-dimer 12.4 <.0001 26.5 <.0001 Abbreviations: Hs-CRP, high-sensitivity C-reactive protein; IL-6, interleukin 6. Source: From LH Kuller et al: PLoS Med 5:e203, 2008. co-receptor. These assays take weeks to perform and are expensive. Another, less costly option is to obtain a genotypic assay of the V3 region of HIV-1 and then employ a computer algorithm to predict viral tropism from the sequence. While this approach is less expensive than the classic phenotypic assay, there are fewer data to validate its predictive value.

1	Other Tests A variety of other laboratory tests have been studied as potential markers of HIV disease activity. Among these are quantitative culture of replication-competent HIV from plasma, peripheral blood mononuclear cells, or resting CD4+ T cells; circulating levels of β2-microglobulin, soluble IL-2 receptor, IgA, acid-labile endogenous IFN, or TNF-α; and the presence or absence of activation markers such as CD38, HLA-DR, and PD-1 on CD4+ or CD8+ T cells. Nonspecific serologic markers of inflammation and/or coagulation such as IL-6, d-dimer, and sCD14 have been shown to have a high correlation with all-cause mortality (Table 226-9). While these measurements have value as markers of disease activity and help to increase our understanding of the pathogenesis of HIV disease, they do not currently play a major role in the monitoring of patients with HIV infection.

1	The clinical consequences of HIV infection encompass a spectrum ranging from an acute syndrome associated with primary infection to a prolonged asymptomatic state to advanced disease. It is best to regard HIV disease as beginning at the time of primary infection and progressing through various stages. As mentioned above, active virus replication and progressive immunologic impairment occur throughout the course of HIV infection in most patients. With the exception of the rare, true, “elite” virus controllers or long-term nonprogressors (see “Long-Term Survivors and Long-Term Nonprogressors,” above), HIV disease in untreated patients inexorably progresses even during the clinically latent stage. Since the mid-1990s, cART has had a major impact on preventing and reversing the progression of disease over extended periods of time in a substantial proportion of adequately treated patients.

1	It is estimated that 50–70% of individuals with HIV infection experience an acute clinical syndrome ~3–6 weeks after primary infection (Fig. 226-32). Varying degrees of clinical severity have been reported, and although it has been suggested that symptomatic seroconversion leading to the seeking of medical attention indicates an increased risk for an accelerated course of disease, there does not appear to be a correlation between the level of the initial burst of viremia in acute HIV infection and the subsequent course of disease. The typical clinical findings in the acute HIV syndrome are listed in Table 226-10; they occur along with a burst of plasma viremia. It has been reported that several symptoms of the acute HIV syndrome (fever, skin rash, pharyngitis, and myalgia) occur less frequently in those infected by injection drug use compared with those infected by sexual contact. The syndrome is typical of an acute viral syndrome and has been likened to acute infectious

1	less frequently in those infected by injection drug use compared with those infected by sexual contact. The syndrome is typical of an acute viral syndrome and has been likened to acute infectious mononucleosis. Symptoms usually persist for one to several weeks and gradually subside as an immune response to HIV develops and the levels of plasma viremia decrease. Opportunistic infections have been reported during this stage of infection, reflecting the immunodeficiency that results from reduced numbers of CD4+

1	Plasma viremia (wide dissemination of virus) Acute syndrome Retrafficking of lymphocytes 1 week–3 months 1–2 weeks 3–6 weeks Immune response to HIV Curtailment of plasma viremia Primary Infection Establishment of chronic, persistent infection in lymphoid tissue Clinical latency FIGuRE 226-32 The acute HIV syndrome. See text for detailed description. (Adapted from G Pantaleo et al: N Engl J Med 328:327, 1993. Copyright 1993 Massachusetts Medical Society. All rights reserved.)

1	T cells and likely also from the dysfunction of CD4+ T cells owing to viral protein and endogenous cytokine-induced perturbations of cells (Table 226-5) associated with the extremely high levels of plasma viremia. A number of immunologic abnormalities accompany the acute HIV syndrome, including multiphasic perturbations of the numbers of circulating lymphocyte subsets. The number of total lymphocytes and T cell subsets (CD4+ and CD8+) are initially reduced. An inversion of the CD4+/CD8+ T cell ratio occurs later because of a rise in the number of CD8+ T cells. In fact, there may be a selective and transient expansion of CD8+ T cell subsets, as determined by T cell receptor analysis (see above). The total circulating CD8+ T cell count may remain elevated or return to normal; however, CD4+ T cell levels usually remain somewhat depressed, although there may be a slight rebound toward normal. Lymphadenopathy occurs in ~70% of individuals with primary HIV infection. Most patients recover

1	T cell levels usually remain somewhat depressed, although there may be a slight rebound toward normal. Lymphadenopathy occurs in ~70% of individuals with primary HIV infection. Most patients recover spontaneously from this syndrome and many are left with only a mildly depressed CD4+ T cell count that remains stable for a variable period before beginning its progressive decline; in some individuals, the CD4+ T cell count returns to the normal range. Approximately 10% of patients manifest a fulminant course of immunologic and clinical deterioration after primary infection, even after the disappearance of initial symptoms. In most patients, primary infection with or without the acute syndrome is followed by a prolonged period of clinical latency or smoldering low disease activity.

1	Although the length of time from initial infection to the development of clinical disease varies greatly, the median time for untreated patients is ~10 years. As emphasized above, HIV disease with active virus replication is ongoing and progressive during this asymptomatic period. The rate of disease progression is directly correlated with HIV Source: From B Tindall, DA Cooper: AIDS 5:1, 1991.

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1250 RNA levels. Patients with high levels of HIV RNA in plasma progress to symptomatic disease faster than do patients with low levels of HIV RNA (Fig. 226-22). Some patients referred to as long-term nonprogressors show little if any decline in CD4+ T cell counts over extended periods of time. These patients generally have extremely low levels of HIV RNA; a subset, referred to as elite nonprogressors, exhibits HIV RNA levels <50 copies/mL. Certain other patients remain entirely asymptomatic despite the fact that their CD4+ T cell counts show a steady progressive decline to extremely low levels. In these patients, the appearance of an opportunistic disease may be the first manifestation of HIV infection. During the asymptomatic period of HIV infection, the average rate of CD4+ T cell decline is ~50/μL per year. When the CD4+ T cell count falls to <200/μL, the resulting state of immunodeficiency is severe enough to place

1	period of HIV infection, the average rate of CD4+ T cell decline is ~50/μL per year. When the CD4+ T cell count falls to <200/μL, the resulting state of immunodeficiency is severe enough to place the patient at high risk for opportunistic infection and neoplasms and, hence, for clinically apparent disease.

1	Symptoms of HIV disease can appear at any time during the course of HIV infection. Generally speaking, the spectrum of illnesses that one observes changes as the CD4+ T cell count declines. The more severe and life-threatening complications of HIV infection occur in patients with CD4+ T cell counts <200/μL. A diagnosis of AIDS is made in individuals age 6 years and older with HIV infection and a CD4+ T cell count <200/μL (Stage 3, Table 226-2) and in anyone with HIV infection who develops one of the HIV-associated diseases considered to be indicative of a severe defect in cell-mediated immunity (Table 226-1). While the causative agents of the secondary infections are characteristically opportunistic organisms such as P. jiroveci, atypical mycobacteria, CMV, and other organisms that do not ordinarily cause disease in the absence of a compromised immune system, they also include common bacterial and mycobacterial pathogens. Following the widespread use of cART and implementation of

1	not ordinarily cause disease in the absence of a compromised immune system, they also include common bacterial and mycobacterial pathogens. Following the widespread use of cART and implementation of guidelines for the prevention of opportunistic infections (Table 226-11), the incidence of these secondary infections has decreased dramatically (Fig. 226-33). Overall, the clinical spectrum of HIV disease is constantly changing as patients live longer and new and better approaches to treatment and prophylaxis are developed. In addition to the classic AIDS-defining illnesses, patients with HIV infection also have an increase in serious non-AIDS illnesses, including non-AIDS related cancers and cardiovascular, renal, and hepatic disease. Non-AIDS events dominate the disease burden for patients with HIV infection receiving cART (Table 226-4). While AIDS-related illnesses are the leading cause of death in patients with HIV infection, they account for fewer than 50% of deaths.

1	for patients with HIV infection receiving cART (Table 226-4). While AIDS-related illnesses are the leading cause of death in patients with HIV infection, they account for fewer than 50% of deaths. Non-AIDS-defining malignancies, liver disease, and cardiovascular disease each account for 10–15% of deaths in patients with HIV infection. The physician providing care to a patient with HIV infection must be well versed in general internal medicine as well as HIV-related opportunistic diseases. In general, it should be stressed that a key element of treatment of symptomatic complications of HIV disease, whether they are primary or secondary, is achieving good control of HIV replication through the use of cART and instituting primary and secondary prophylaxis for opportunistic infections as indicated.

1	Diseases of the Respiratory System Acute bronchitis and sinusitis are prevalent during all stages of HIV infection. The most severe cases tend to occur in patients with lower CD4+ T cell counts. Sinusitis presents as fever, nasal congestion, and headache. The diagnosis is made by CT or MRI. The maxillary sinuses are most commonly involved; however, disease is also frequently seen in the ethmoid, sphenoid, and frontal sinuses. While some patients may improve without antibiotic therapy, radiographic improvement is quicker and more pronounced in patients who have received antimicrobial therapy. It is postulated that this high incidence of sinusitis results from an increased frequency of infection with encapsulated organisms such as H. influenzae and Streptococcus pneumoniae. In patients with low CD4+ T cell counts one may see mucormycosis infections of the sinuses. In contrast to the course of this infection in other patient populations, mucormycosis of the sinuses in patients with HIV

1	low CD4+ T cell counts one may see mucormycosis infections of the sinuses. In contrast to the course of this infection in other patient populations, mucormycosis of the sinuses in patients with HIV infection may progress more slowly. In this setting aggressive, frequent local debridement in addition to local and systemic amphotericin B may result in effective treatment.

1	Pulmonary disease is one of the most frequent complications of HIV infection. The most common manifestation of pulmonary disease is pneumonia. Three of the 10 most common AIDS-defining illnesses are recurrent bacterial pneumonia, tuberculosis, and pneumonia due to the unicellular fungus P. jiroveci. Other major causes of pulmonary infiltrates include other mycobacterial infections, other fungal infections, nonspecific interstitial pneumonitis, KS, and lymphoma.

1	Bacterial pneumonia is seen with an increased frequency in patients with HIV infection, with 0.8–2.0 cases per 100 person-years. Patients with HIV infection are particularly prone to infections with encapsulated organisms. S. pneumoniae (Chap. 171) and H. influenzae (Chap. 182) are responsible for most cases of bacterial pneumonia in patients with AIDS. This may be a consequence of altered B cell function and/or defects in neutrophil function that may be secondary to HIV disease (see above). Pneumonias due to S. aureus (Chap. 172) and P. aeruginosa (Chap. 189) also are reported to occur with an increased frequency in patients with HIV infection.

1	S. pneumoniae (pneumococcal) infection may be the earliest serious infection to occur in patients with HIV disease. This can present as pneumonia, sinusitis, and/or bacteremia. Patients with untreated HIV infection have a sixfold increase in the incidence of pneumococcal pneumonia and a 100-fold increase in the incidence of pneumococcal bacteremia. Pneumococcal disease may be seen in patients with relatively intact immune systems. In one study, the baseline CD4+ T cell count at the time of a first episode of pneumococcal pneumonia was ~300/μL. Of interest is the fact that the inflammatory response to pneumococcal infection appears proportional to the CD4+ T cell count. Due to this high risk of pneumococcal disease, immunization with the conjugated pneumococcal vaccine followed by booster immunization with the 23-valent pneumococcal polysaccharide vaccine is one of the generally recommended prophylactic measures for patients with HIV infection. This is likely most effective if given

1	immunization with the 23-valent pneumococcal polysaccharide vaccine is one of the generally recommended prophylactic measures for patients with HIV infection. This is likely most effective if given while the CD4+ T cell count is >200/μL and, if given to patients with lower CD4+ T cell counts, should be repeated once the count has been above 200 for 6 months. Although clear guidelines do not exist, it also makes sense to repeat immunization every 5 years. The incidence of bacterial pneumonia is cut in half when patients quit smoking.

1	Pneumocystis pneumonia (PCP), once the hallmark of AIDS, has dramatically declined in incidence following the development of effective prophylactic regimens and the widespread use of cART. It is, however, still the single most common cause of pneumonia in patients with HIV infection in the United States and can be identified as a likely etiologic agent in 25% of cases of pneumonia in patients with HIV infection, with an incidence in the range of 2–3 cases per 100 person-years. Approximately 50% of cases of HIV-associated PCP occur in patients who are unaware of their HIV status. The risk of PCP is greatest among those who have experienced a previous bout of PCP and those who have CD4+ T cell counts of <200/μL. Overall, 79% of patients with PCP have CD4+ T cell counts <100/μL and 95% of patients have CD4+ T cell counts <200/μL. Recurrent fever, night sweats, thrush, and unexplained weight loss also are associated with an increased incidence of PCP. For these reasons, it is strongly

1	patients have CD4+ T cell counts <200/μL. Recurrent fever, night sweats, thrush, and unexplained weight loss also are associated with an increased incidence of PCP. For these reasons, it is strongly recommended that all patients with CD4+ T cell counts <200/μL (or a CD4 percentage <15) receive some form of PCP prophylaxis. The incidence of PCP is approaching zero in patients with known HIV infection receiving appropriate cART and prophylaxis. In the United States, primary PCP is now occurring at a median CD4+ T cell count of 36/ μL, while secondary PCP is occurring at a median CD4+ T cell count of 10/μL. Patients with PCP generally present with fever and a cough that is usually nonproductive or productive of only scant amounts of white sputum. They may complain of a characteristic retrosternal chest pain that is worse on inspiration and is described as sharp or burning. HIV-associated PCP may have an indolent course characterized by weeks of vague symptoms and should be included in

1	chest pain that is worse on inspiration and is described as sharp or burning. HIV-associated PCP may have an indolent course characterized by weeks of vague symptoms and should be included in the differential diagnosis of fever, pulmonary complaints, or unexplained weight loss in any patient with HIV infection and <200 CD4+ T cells/μL. The most

1	Recommended as Standard of Care for Primary and Secondary Prophylaxis Prior bout of PCP Close contact with case of active pulmonary TB Drug resistant Same with high probability of exposure to drug-resistant TB Trimethoprim/sulfamethoxazole (TMP/SMX), 1 DS tablet qd PO TMP/SMX, 1 SS tablet qd PO Aerosolized pentamidine, 300 mg via Respirgard II nebulizer every month TMP/SMX, 1 SS PO daily Human Immunodeficiency Virus Disease: AIDS and Related Disorders All patients, preferably before CD4+ T cell count ≤200/μL Hepatitis B vaccine: 3 doses Hepatitis A vaccine: 2 doses (13) 0.5 mL IM × 1 followed in 8 weeks or more by pneumococcal polysaccharide vaccine (23) if CD4+ T cell count >200/μL Oseltamivir 75 mg PO qd Streptococcus pneumoniae Reimmunize patients initially immunized at a CD4+ T cell count <100/μL whose CD4+ T cell count then increases to >200/μL Human papillomavirus All patients 13–26 years of age HPV vaccine; 3 doses

1	Human papillomavirus All patients 13–26 years of age HPV vaccine; 3 doses Recommended for Prevention of Severe or Frequent Recurrences Abbreviations: ARV, antiretroviral; bid, twice daily; DS, double-strength; PCP, Pneumocystis jiroveci pneumonia; PO, by mouth; SS, single-strength; TB, tuberculosis.

1	common finding on chest x-ray is either a normal film, if the disease may demonstrate a patchy ground-glass appearance. Routine laborais suspected early, or a faint bilateral interstitial infiltrate. The classic tory evaluation is usually of little help in the differential diagnosis finding of a dense perihilar infiltrate is unusual in patients with AIDS. of PCP. A mild leukocytosis is common, although this may not be In patients with PCP who have been receiving aerosolized pentamidine obvious in patients with prior neutropenia. Elevation of lactate dehyfor prophylaxis, one may see an x-ray picture of upper lobe cavitary drogenase is common. Arterial blood-gases may indicate hypoxemia disease, reminiscent of TB. Other less common findings on chest with a decline in Pao and an increase in the arterial-alveolar (a–a) x-ray include lobar infiltrates and pleural effusions. Thin-section CT gradient. Arterial blood-gas measurements not only aid in making the diagnosis of PCP but also

1	in the arterial-alveolar (a–a) x-ray include lobar infiltrates and pleural effusions. Thin-section CT gradient. Arterial blood-gas measurements not only aid in making the diagnosis of PCP but also provide important information for staging the severity of the disease and directing treatment (see below). A definitive diagnosis of PCP requires demonstration of the organism in samples obtained from induced sputum, bronchoalveolar lavage, transbronchial biopsy, or open-lung biopsy. PCR has been used to10 detect specific DNA sequences for P. jiroveci in clinical specimens where histologic examinations have failed to make a diagnosis.

1	In addition to pneumonia, a number of other clinical problems have been reported in HIV-infected patients as a result of infection with P. jiroveci. Otic involvement may be seen as a primary infection, presenting as a polypoid mass involving the external auditory canal. PCP, one may see a variety of extrapulmonary manifestations of P. jiroveci. These include ophthalmic lesions of the choroid, a necrotiz ing vasculitis that resembles Burger’s disease, bone marrow hypopla sia, and intestinal obstruction. Other organs that have been involved include lymph nodes, spleen, liver, kidney, pancreas, pericardium, heart, thyroid, and adrenals. Organ infection may be associated with Human Immunodeficiency Virus Disease: AIDS and Related Disorders cystic lesions that may appear calcified on CT or ultrasound. The standard treatment for PCP or disseminated pneumocystosis

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders cystic lesions that may appear calcified on CT or ultrasound. The standard treatment for PCP or disseminated pneumocystosis No. of opportunistic infectionsper 100 person-years is trimethoprim/sulfamethoxazole (TMP/SMX). A high (20–85%) incidence of side effects, particularly skin rash and bone marrow suppression, is seen with TMP/SMX in patients with HIV infection.

1	12 Alternative treatments for mild to moderate PCP include dapsone/ trimethoprim, clindamycin/primaquine, and atovaquone. IV pentami dine is the treatment of choice for severe disease in the patient unable 8 to tolerate TMP/SMX. For patients with a Pao <70 mmHg or with an 6 a–a gradient >35 mmHg, adjunct glucocorticoid therapy should be used in addition to specific antimicrobials. Overall, treatment should be continued for 21 days and followed by secondary prophylaxis. 2 Prophylaxis for PCP is indicated for any HIV-infected individual who 0 has experienced a prior bout of PCP, any patient with a CD4+ T cell count of <200/μL or a CD4 percentage <15, any patient with unex-

1	FIGuRE 226-33 A. Decrease in the incidence of opportunistic infections and Kaposi’s sarcoma in HIV-infected individuals with CD4+ T cell counts <100/μL from 1992 through 1998. (Adapted and updated from FJ Palella et al: N Engl J Med 338:853, 1998, and JE Kaplan et al: Clin Infect Dis 30[S1]:S5, 2000, with permission.) B. Quarterly incidence rates of cytomegalovirus (CMV), Pneumocystis jiroveci pneumonia (PCP), and Mycobacterium avium complex (MAC) from 1995 to 2001. (From FJ Palella et al: AIDS 16:1617, 2002.) plained fever for >2 weeks, and any patient with a recent history of oropharyngeal candidiasis. The preferred regimen for prophylaxis is TMP/SMX, one double-strength tablet daily. This regimen also provides protection against toxoplasmosis and some bacterial respiratory pathogens. For patients who cannot tolerate TMP/SMX, alternatives for prophylaxis include dapsone plus pyrimethamine plus leucovorin, aerosolized pentamidine administered by the Respirgard II nebulizer, and

1	For patients who cannot tolerate TMP/SMX, alternatives for prophylaxis include dapsone plus pyrimethamine plus leucovorin, aerosolized pentamidine administered by the Respirgard II nebulizer, and atovaquone. Primary or secondary prophylaxis for PCP can be 1254 discontinued in those patients treated with cART who maintain good suppression of HIV (<50 copies/mL) and CD4+ T cell counts >200/μL for at least 3 months.

1	M. tuberculosis, once thought to be on its way to extinction in the United States, experienced a resurgence associated with the HIV epidemic (Chap. 202). Worldwide, approximately one-third of all AIDS-related deaths are associated with TB, and TB is the primary cause of death for 10–15% of patients with HIV infection. In the United States ~5% of AIDS patients have active TB. Patients with HIV infection are more likely to have active TB by a factor of 100 when compared with an HIV-negative population. For an asymptomatic HIV-negative person with a positive purified protein derivative (PPD) skin test, the risk of reactivation TB is around 1% per year. For the patient with untreated HIV infection, a positive PPD skin test, and no signs or symptoms of TB, the rate of reactivation TB is 7–10% per year. Untreated TB can accelerate the course of HIV infection. Levels of plasma HIV RNA increase in the setting of active TB and decline in the setting of successful TB treatment. Active TB is

1	per year. Untreated TB can accelerate the course of HIV infection. Levels of plasma HIV RNA increase in the setting of active TB and decline in the setting of successful TB treatment. Active TB is most common in patients 25–44 years of age, in African Americans and Hispanics, in patients in New York City and Miami, and in patients in developing countries. In these demographic groups, 20–70% of the new cases of active TB are in patients with HIV infection. The epidemic of TB embedded in the epidemic of HIV infection probably represents the greatest health risk to the general public and the health care profession associated with the HIV epidemic. In contrast to infection with atypical mycobacteria such as MAC, active TB often develops relatively early in the course of HIV infection and may be an early clinical sign of HIV disease. In one study, the median CD4+ T cell count at presentation of TB was 326/μL. The clinical manifestations of TB in HIV-infected patients are quite varied and

1	be an early clinical sign of HIV disease. In one study, the median CD4+ T cell count at presentation of TB was 326/μL. The clinical manifestations of TB in HIV-infected patients are quite varied and generally show different patterns as a function of the CD4+ T cell count. In patients with relatively high CD4+ T cell counts, the typical pattern of pulmonary reactivation occurs: patients present with fever, cough, dyspnea on exertion, weight loss, night sweats, and a chest x-ray revealing cavitary apical disease of the upper lobes. In patients with lower CD4+ T cell counts, disseminated disease is more common. In these patients the chest x-ray may reveal diffuse or lower-lobe bilateral reticulonodular infiltrates consistent with miliary spread, pleural effusions, and hilar and/or mediastinal adenopathy. Infection may be present in bone, brain, meninges, GI tract, lymph nodes (particularly cervical lymph nodes), and viscera. Some patients with advanced HIV infection and active TB may

1	adenopathy. Infection may be present in bone, brain, meninges, GI tract, lymph nodes (particularly cervical lymph nodes), and viscera. Some patients with advanced HIV infection and active TB may have no symptoms of illness, and thus screening for TB should be part of the initial evaluation of every patient with HIV infection. Approximately 60–80% of HIV-infected patients with TB have pulmonary disease, and 30–40% have extrapulmonary disease. Respiratory isolation and a negative-pressure room should be used for patients in whom a diagnosis of pulmonary TB is being considered. This approach is critical to limit nosocomial and community spread of infection. Culture of the organism from an involved site provides a definitive diagnosis. Blood cultures are positive in 15% of patients. This figure is higher in patients with lower CD4 +T cell counts. In the setting of fulminant disease one cannot rely on the accuracy of a negative PPD skin test to rule out a diagnosis of TB. In addition,

1	figure is higher in patients with lower CD4 +T cell counts. In the setting of fulminant disease one cannot rely on the accuracy of a negative PPD skin test to rule out a diagnosis of TB. In addition, IFN-γ release assays may be difficult to interpret due to high backgrounds as a consequence of HIV-associated immune activation. TB is one of the conditions associated with HIV infection for which cure is possible with appropriate therapy. Therapy for TB is generally the same in the HIV-infected patient as in the HIV-negative patient (Chap. 202). Due to the possibility of multidrug-resistant or extensively drug-resistant TB, drug susceptibility testing should be performed to guide therapy. Due to pharmacokinetic interactions, adjusted doses of rifabutin should be substituted for rifampin in patients receiving the HIV protease inhibitors or nonnucleoside reverse transcriptase inhibitors. Treatment is most effective in programs that involve directly observed therapy. Initiation of cART

1	in patients receiving the HIV protease inhibitors or nonnucleoside reverse transcriptase inhibitors. Treatment is most effective in programs that involve directly observed therapy. Initiation of cART and/or anti-TB therapy may be associated with clinical deterioration due to immune reconstitution inflammatory syndrome (IRIS) reactions. These are most common in patients initiating both treatments at the same time, may occur as early as 1 week after initiation of cART therapy, and are seen more frequently in patients with advanced HIV disease. For these reasons it is recommended that initiation of cART be delayed in antiretroviral-naïve patients with CD4 counts >50 cells/μL until 2–4 weeks following the initiation of treatment for TB. For patients with lower CD4 counts the benefits of more immediate cART outweigh the risks of IRIS, and cART should be started as soon as possible in those patients. Effective prevention of active TB can be a reality if the health care professional is

1	more immediate cART outweigh the risks of IRIS, and cART should be started as soon as possible in those patients. Effective prevention of active TB can be a reality if the health care professional is aggressive in looking for evidence of latent or active TB by making sure that all patients with HIV infection receive a PPD skin test or evaluation with an IFN-γ release assay. Anergy testing is not of value in this setting. Since these tests rely on the host mounting an immune response to

1	M. tuberculosis, patients with CD4+ T cell counts <200 cells/μL should be retested if their CD4+ T cell counts rise to persistently above 200. Patients at risk of continued exposure to TB should be tested annually. HIV-infected individuals with a skin-test reaction of >5 mm, those with a positive IFN-γ release assay, or those who are close household contacts of persons with active TB should receive treatment with 9 months of isoniazid and pyridoxine.

1	Atypical mycobacterial infections are also seen with an increased frequency in patients with HIV infection. Infections with at least 12 different mycobacteria have been reported, including M. bovis and representatives of all four Runyon groups. The most common atypical mycobacterial infection is with M. avium or M. intracellulare species— the Mycobacterium avium complex (MAC). Infections with MAC are seen mainly in patients in the United States and are rare in Africa. It has been suggested that prior infection with M. tuberculosis decreases the risk of MAC infection. MAC infections probably arise from organisms that are ubiquitous in the environment, including both soil and water. There is little evidence for person-to-person transmission of MAC infection. The presumed portals of entry are the respiratory and GI tracts. MAC infection is a late complication of HIV infection, occurring predominantly in patients with CD4+ T cell counts of <50/μL. The average CD4+ T cell count at the time

1	the respiratory and GI tracts. MAC infection is a late complication of HIV infection, occurring predominantly in patients with CD4+ T cell counts of <50/μL. The average CD4+ T cell count at the time of diagnosis is 10/μL. The most common presentation is disseminated disease with fever, weight loss, and night sweats. At least 85% of patients with MAC infection are mycobacteremic, and large numbers of organisms can often be demonstrated on bone marrow biopsy. The chest x-ray is abnormal in ~25% of patients, with the most common pattern being that of a bilateral, lower-lobe infiltrate suggestive of miliary spread. Alveolar or nodular infiltrates and hilar and/or mediastinal adenopathy can also occur. Other clinical findings include endobronchial lesions, abdominal pain, diarrhea, and lymphadenopathy. Anemia and elevated liver alkaline phosphatase are common. The diagnosis is made by the culture of blood or involved tissue. The finding of two consecutive sputum samples positive for MAC is

1	Anemia and elevated liver alkaline phosphatase are common. The diagnosis is made by the culture of blood or involved tissue. The finding of two consecutive sputum samples positive for MAC is highly suggestive of pulmonary infection. Cultures may take 2 weeks to turn positive. Therapy consists of a macrolide, usually clarithromycin, with ethambutol. Some physicians elect to add a third drug from among rifabutin, ciprofloxacin, or amikacin in patients with extensive disease. Therapy was generally for life; however, with the use of cART it is possible to discontinue therapy in patients with sustained suppression of HIV replication and CD4+ T cell counts >100/μL for 3–6 months. Primary prophylaxis for MAC is indicated in patients with HIV infection and CD4+ T cell counts <50/μL (Table 226-11). This may be discontinued in patients in whom cART induces a sustained suppression of viral replication and an increase in CD4+ T cell count to >100/μL for ≥6 months.

1	Rhodococcus equi is a gram-positive, pleomorphic, acid-fast, nonspore-forming bacillus that can cause pulmonary and/or disseminated infection in patients with advanced HIV infection. Fever and cough are the most common presenting signs. Radiographically one may see cavitary lesions and consolidation. Blood cultures are often positive. Treatment is based on antimicrobial sensitivity testing.

1	Fungal infections of the lung, in addition to PCP, can be seen in patients with AIDS. Patients with pulmonary cryptococcal disease present with fever, cough, dyspnea, and, in some cases, hemoptysis. A focal or diffuse interstitial infiltrate is seen on chest x-ray in >90% of patients. In addition, one may see lobar disease, cavitary disease, pleural effusions, and hilar or mediastinal adenopathy. More than half of patients are fungemic, and 90% of patients have concomitant CNS infection. Coccidioides immitis is a mold that is endemic in the southwest United States. It can cause a reactivation pulmonary syndrome in patients with HIV infection. Most patients with this condition will have CD4+ T cell counts <250/μL. Patients present with fever, weight loss, cough, and extensive, diffuse reticulonodular infiltrates on chest x-ray. One may also see nodules, cavities, pleural effusions, and hilar adenopathy. While serologic testing is of value in the immunocompetent host, serologies are

1	infiltrates on chest x-ray. One may also see nodules, cavities, pleural effusions, and hilar adenopathy. While serologic testing is of value in the immunocompetent host, serologies are negative in 25% of HIV-infected patients with coccidioidal infection. Invasive aspergillosis is not an AIDS-defining illness and is generally not seen in patients with AIDS in the absence of neutropenia or administration of glucocorticoids. When it does occur, Aspergillus infection may have an unusual presentation in the respiratory tract of patients with AIDS, where it gives the appearance of a pseudomembranous tracheobronchitis. Primary pulmonary infection of the lung may be seen with histoplasmosis. The most common pulmonary manifestation of histoplasmosis, however, is in the setting of disseminated disease, presumably due to reactivation. In this setting respiratory symptoms are usually minimal, with cough and dyspnea occurring in 10–30% of patients. The chest x-ray is abnormal in ~50% of patients,

1	presumably due to reactivation. In this setting respiratory symptoms are usually minimal, with cough and dyspnea occurring in 10–30% of patients. The chest x-ray is abnormal in ~50% of patients, showing either a diffuse interstitial infiltrate or diffuse small nodules, and the urine will often be positive for Histoplasma antigen.

1	Two forms of idiopathic interstitial pneumonia have been identified in patients with HIV infection: lymphoid interstitial pneumonitis (LIP) and nonspecific interstitial pneumonitis (NIP). LIP, a common finding in children, is seen in about 1% of adult patients with untreated HIV infection. This disorder is characterized by a benign infiltrate of the lung and is thought to be part of the polyclonal activation of lymphocytes seen in the context of HIV and EBV infections. Transbronchial biopsy is diagnostic in 50% of the cases, with an open-lung biopsy required for diagnosis in the remainder of cases. This condition is generally self-limited and no specific treatment is necessary. Severe cases have been managed with brief courses of glucocorticoids. Although rarely a clinical problem since the use of cART, evidence of NIP may be seen in up to half of all patients with untreated HIV infection. Histologically, interstitial infiltrates of lymphocytes and plasma cells in a perivascular and

1	the use of cART, evidence of NIP may be seen in up to half of all patients with untreated HIV infection. Histologically, interstitial infiltrates of lymphocytes and plasma cells in a perivascular and peribronchial distribution are present. When symptomatic, patients present with fever and nonproductive cough occasionally accompanied by mild chest discomfort. Chest x-ray is usually normal or may reveal a faint interstitial pattern. Similar to LIP, NIP is a self-limited process for which no therapy is indicated other than appropriate management of the underlying HIV infection. HIV-related pulmonary arterial hypertension (HIV-PAH) is seen in ~0.5% of HIV-infected individuals. Patients may present with an array of symptoms including shortness of breath, fatigue, syncope, chest pain, and signs of right-sided heart failure. Chest x-ray reveals dilated pulmonary vessels and right-sided cardiomegaly with right ventricular hypertrophy seen on electrocardiogram. cART does not appear to be of

1	of right-sided heart failure. Chest x-ray reveals dilated pulmonary vessels and right-sided cardiomegaly with right ventricular hypertrophy seen on electrocardiogram. cART does not appear to be of clear benefit, and the prognosis is quite poor with a median survival in the range of 2 years.

1	Neoplastic diseases of the lung including KS and lymphoma are discussed below in the section on neoplastic diseases.

1	Diseases of the Cardiovascular System Heart disease is a relatively common postmortem finding in HIV-infected patients (25–75% in autopsy series). The most common form of heart disease is coronary heart disease. In one large series the overall rate of myocardial infarction (MI) was 3.5/1000 patient-years, 28% of these events were fatal, and MI was responsible for 7% of all deaths in the cohort. In patients with HIV infection, cardiovascular disease may be associated with classic risk factors such as smoking, a direct consequence of HIV infection, or a complication of cART. Patients with HIV infection have higher levels of triglycerides, lower levels of high-density lipoprotein cholesterol, and a higher prevalence of smoking than cohorts of individuals without HIV infection. The finding that the rate of cardiovascular disease events was lower in patients on antiretroviral therapy than in those randomized to undergo a treatment interruption identified a clear association between HIV

1	that the rate of cardiovascular disease events was lower in patients on antiretroviral therapy than in those randomized to undergo a treatment interruption identified a clear association between HIV replication and risk of cardiovascular disease. In one study, a baseline CD4+ T cell count of <500/μL was found to be an independent risk factor for cardiovascular disease comparable in 1255 magnitude to that attributable to smoking. While the precise pathogenesis of this association remains unclear, it is likely related to the immune activation and increased propensity for coagulation seen as a consequence of HIV replication. Exposure to HIV protease inhibitors and certain reverse transcriptase inhibitors has been associated with increases in total cholesterol and/or risk of MI. Any increases in the risk of death from MI resulting from the use of certain antiretrovirals must be balanced against the marked increases in overall survival brought about by these drugs.

1	Another form of heart disease associated with HIV infection is a dilated cardiomyopathy associated with congestive heart failure (CHF) referred to as HIV-associated cardiomyopathy. This generally occurs as a late complication of HIV infection and, histologically, displays elements of myocarditis. For this reason some have advocated treatment with IV immunoglobulin (IVIg). HIV can be directly demonstrated in cardiac tissue in this setting, and there is debate over whether it plays a direct role in this condition. Patients present with typical findings of CHF including edema and shortness of breath. Patients with HIV infection may also develop cardiomyopathy as side effects of IFN-α or nucleoside analogue therapy. These are reversible once therapy is stopped. KS, cryptococcosis, Chagas’ disease, and toxoplasmosis can involve the myocardium, leading to cardiomyopathy. In one series, most patients with HIV infection and a treatable myocarditis were found to have myocarditis associated

1	disease, and toxoplasmosis can involve the myocardium, leading to cardiomyopathy. In one series, most patients with HIV infection and a treatable myocarditis were found to have myocarditis associated with toxoplasmosis. Most of these patients also had evidence of CNS toxoplasmosis. Thus, MRI or double-dose contrast CT scan of the brain should be included in the workup of any patient with advanced HIV infection and cardiomyopathy.

1	A variety of other cardiovascular problems are found in patients with HIV infection. Pericardial effusions may be seen in the setting of advanced HIV infection. Predisposing factors include TB, CHF, mycobacterial infection, cryptococcal infection, pulmonary infection, lymphoma, and KS. While pericarditis is quite rare, in one series 5% of patients with HIV disease had pericardial effusions that were considered to be moderate or severe. Tamponade and death have occurred in association with pericardial KS, presumably owing to acute hemorrhage. Nonbacterial thrombotic endocarditis has been reported and should be considered in patients with unexplained embolic phenomena. IV pentamidine, when given rapidly, can result in hypotension as a consequence of cardiovascular collapse.

1	Diseases of the Oropharynx and Gastrointestinal System Oropharyngeal and GI diseases are common features of HIV infection. They are most frequently due to secondary infections. In addition, oral and GI lesions may occur with KS and lymphoma.

1	Oral lesions, including thrush, hairy leukoplakia, and aphthous ulcers (Fig. 226-34), are particularly common in patients with untreated HIV infection. Thrush, due to Candida infection, and oral hairy leukoplakia, presumed due to EBV, are usually indicative of fairly advanced immunologic decline; they generally occur in patients with CD4+ T cell counts of <300/μL. In one study, 59% of patients with oral candidiasis went on to develop AIDS in the next year. Thrush appears as a white, cheesy exudate, often on an erythematous mucosa in the posterior oropharynx. While most commonly seen on the soft palate, early lesions are often found along the gingival border. The diagnosis is made by direct examination of a scraping for pseudohyphal elements. Culturing is of no diagnostic value, as patients with HIV infection may have a positive throat culture for Candida in the absence of thrush. Oral hairy leukoplakia presents as white, frondlike lesions, generally along the lateral borders of the

1	with HIV infection may have a positive throat culture for Candida in the absence of thrush. Oral hairy leukoplakia presents as white, frondlike lesions, generally along the lateral borders of the tongue and sometimes on the adjacent buccal mucosa (Fig. 226-34). Despite its name, oral hairy leukoplakia is not considered a premalignant condition. Lesions are associated with florid replication of EBV. While usually more disconcerting as a sign of HIV-associated immunodeficiency than a clinical problem in need of treatment, severe cases have been reported to respond to topical podophyllin or systemic therapy with anti-herpesvirus agents. Aphthous ulcers of the posterior oropharynx also are seen with regularity in patients with untreated HIV infection

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders FIGuRE 226-34 Various oral lesions in HIV-infected individuals. A. Thrush. B. Hairy leukoplakia. C. Aphthous ulcer. D. Kaposi’s sarcoma. (Fig. 226-34). These lesions are of unknown etiology and can be quite painful and interfere with swallowing. Topical anesthetics provide immediate symptomatic relief of short duration. The fact that thalidomide is an effective treatment for this condition suggests that the pathogenesis may involve the action of tissue-destructive cytokines. Palatal, glossal, or gingival ulcers may also result from cryptococcal disease or histoplasmosis.

1	Esophagitis (Fig. 226-35) may present with odynophagia and retrosternal pain. Upper endoscopy is generally required to make an accurate diagnosis. Esophagitis may be due to Candida, CMV, or HSV. While CMV tends to be associated with a single large ulcer, HSV infection is more often associated with multiple small ulcers. The esophagus may also be the site of KS and lymphoma. Like the oral mucosa, the esophageal mucosa may have large, painful ulcers of unclear etiology that may respond to thalidomide. While achlorhydria is a common problem in patients with HIV infection, other gastric problems are generally rare. Among the neoplastic conditions involving the stomach are KS and lymphoma. Infections of the small and large intestine leading to diarrhea, abdominal pain, and occasionally fever are among the most significant GI problems in HIV-infected patients. They include infections with bacteria, protozoa, and viruses.

1	Bacteria may be responsible for secondary infections of the GI tract. Infections with enteric pathogens such as Salmonella, Shigella, and Campylobacter are more common in men who have sex with men and are often more severe and more apt to relapse in patients with HIV infection. Patients with untreated HIV have approximately a 20-fold increased risk of infection with S. typhimurium. FIGuRE 226-35 Barium swallow of a patient with Candida esophagitis. The flow of barium along the mucosal surface is grossly irregular.

1	FIGuRE 226-35 Barium swallow of a patient with Candida esophagitis. The flow of barium along the mucosal surface is grossly irregular. They may present with a variety of nonspecific symptoms including fever, anorexia, fatigue, and malaise of several weeks’ duration. Diarrhea is common but may be absent. Diagnosis is made by culture of blood and stool. Long-term therapy with ciprofloxacin is the recommended treatment. HIV-infected patients also have an increased incidence of S. typhi infection in areas of the world where typhoid is a problem. Shigella spp., particularly S. flexneri, can cause severe intestinal disease in HIV-infected individuals. Up to 50% of patients will develop bacteremia. Campylobacter infections occur with an increased frequency in patients with HIV infection. While

1	C. jejuni is the strain most frequently isolated, infections with many other strains have been reported. Patients usually present with crampy abdominal pain, fever, and bloody diarrhea. Infection may also present as proctitis. Stool examination reveals the presence of fecal leukocytes. Systemic infection can occur, with up to 10% of infected patients exhibiting bacteremia. Most strains are sensitive to erythromycin. Abdominal pain and diarrhea may be seen with MAC infection. Fungal infections may also be a cause of diarrhea in patients with HIV infection. Histoplasmosis, coccidioidomycosis, and penicilliosis have all been identified as a cause of fever and diarrhea in patients with HIV infection. Peritonitis has been seen with C. immitis.

1	Cryptosporidia, microsporidia, and Isospora belli (Chap. 254) are the most common opportunistic protozoa that infect the GI tract and cause diarrhea in HIV-infected patients. Cryptosporidial infection may present in a variety of ways, ranging from a self-limited or intermittent diarrheal illness in patients in the early stages of HIV infection to a severe, life-threatening diarrhea in severely immunodeficient individuals. In patients with untreated HIV infection and CD4+ T cell counts of <300/μL, the incidence of cryptosporidiosis is ~1% per year. In 75% of cases the diarrhea is accompanied by crampy abdominal pain, and 25% of patients have nausea and/or vomiting. Cryptosporidia may also cause biliary tract disease in the HIV-infected patient, leading to cholecystitis with or without accompanying cholangitis and pancreatitis secondary to papillary stenosis. The diagnosis of cryptosporidial diarrhea is made by stool examination or biopsy of the small intestine. The diarrhea is

1	accompanying cholangitis and pancreatitis secondary to papillary stenosis. The diagnosis of cryptosporidial diarrhea is made by stool examination or biopsy of the small intestine. The diarrhea is noninflammatory, and the characteristic finding is the presence of oocysts that stain with acid-fast dyes. Therapy is predominantly supportive, and marked improvements have been reported in the setting of effective cART. Treatment with up to 2000 mg/d of nitazoxanide (NTZ) is associated with improvement in symptoms or a decrease in shedding of organisms in about half of patients. Its overall role in the management of this condition remains unclear. Patients can minimize their risk of developing cryptosporidiosis by avoiding contact with human and animal feces, by not drinking untreated water from lakes or rivers, and by not eating raw shellfish.

1	Microsporidia are small, unicellular, obligate intracellular parasites that reside in the cytoplasm of enteric cells (Chap. 254). The main species causing disease in humans is Enterocytozoon bieneusi. The clinical manifestations are similar to those described for cryptosporidia and include abdominal pain, malabsorption, diarrhea, and cholangitis. The small size of the organism may make it difficult to detect; however, with the use of chromotrope-based stains, organisms can be identified in stool samples by light microscopy. Definitive diagnosis generally depends on electron-microscopic examination of a stool specimen, intestinal aspirate, or intestinal biopsy specimen. In contrast to cryptosporidia, microsporidia have been noted in a variety of extraintestinal locations, including the eye, brain, sinuses, muscle, and liver, and they have been associated with conjunctivitis and hepatitis. The most effective way to deal with microsporidia in a patient with HIV infection is to restore

1	brain, sinuses, muscle, and liver, and they have been associated with conjunctivitis and hepatitis. The most effective way to deal with microsporidia in a patient with HIV infection is to restore the immune system by treating the HIV infection with cART. Albendazole, 400 mg bid, has been reported to be of benefit in some patients.

1	I. belli is a coccidian parasite (Chap. 254) most commonly found as a cause of diarrhea in patients from tropical and subtropical regions. Its cysts appear in the stool as large, acid-fast structures that can be differentiated from those of cryptosporidia on the basis of size, shape, and number of sporocysts. The clinical syndromes of Isospora infection are identical to those caused by cryptosporidia. The important distinction is that infection with Isospora is generally relatively easy to treat with 1257 TMP/SMX. While relapses are common, a thrice-weekly regimen of TMP/SMX appears adequate to prevent recurrence.

1	CMV colitis was once seen as a consequence of advanced immunodeficiency in 5–10% of patients with AIDS. It is much less common with the advent of cART. CMV colitis presents as diarrhea, abdominal pain, weight loss, and anorexia. The diarrhea is usually nonbloody, and the diagnosis is achieved through endoscopy and biopsy. Multiple mucosal ulcerations are seen at endoscopy, and biopsies reveal characteristic intranuclear and cytoplasmic inclusion bodies. Secondary bacteremias may result as a consequence of thinning of the bowel wall. Treatment is with either ganciclovir or foscarnet for 3–6 weeks. Relapses are common, and maintenance therapy is typically necessary in patients whose HIV infection is poorly controlled. Patients with CMV disease of the GI tract should be carefully monitored for evidence of CMV retinitis.

1	In addition to disease caused by specific secondary infections, patients with HIV infection may also experience a chronic diarrheal syndrome for which no etiologic agent other than HIV can be identified. This entity is referred to as AIDS enteropathy or HIV enteropathy. It is most likely a direct result of HIV infection in the GI tract. Histologic examination of the small bowel in these patients reveals low-grade mucosal atrophy with a decrease in mitotic figures, suggesting a hyporegenerative state. Patients often have decreased or absent small-bowel lactase and malabsorption with accompanying weight loss.

1	The initial evaluation of a patient with HIV infection and diarrhea should include a set of stool examinations, including culture, exami nation for ova and parasites, and examination for Clostridium difficile toxin. Approximately 50% of the time this workup will demonstrate infection with pathogenic bacteria, mycobacteria, or protozoa. If the initial stool examinations are negative, additional evaluation, including upper and/or lower endoscopy with biopsy, will yield a diagnosis of microsporidial or mycobacterial infection of the small intestine ~30% of the time. In patients for whom this diagnostic evaluation is nonrevealing, a presumptive diagnosis of HIV enteropathy can be made if the diarrhea has persisted for >1 month. An algorithm for the evalua tion of diarrhea in patients with HIV infection is given in Fig. 226-36. Rectal lesions are common in HIV-infected patients, particularly the perirectal ulcers and erosions due to the reactivation of HSV

1	FIGuRE 226-36 Algorithm for the evaluation of diarrhea in a patient with HIV infection. HIV-associated enteropathy is a diagnosis of exclusion and can be made only after other, generally treatable, forms of diarrheal illness have been ruled out. Human Immunodeficiency Virus Disease: AIDS and Related Disorders FIGuRE 226-37 Severe, erosive perirectal herpes simplex in a patient with AIDS. (Fig. 226-37). These lesions may appear quite atypical, as denuded skin without vesicles. They typically respond well to treatment with acyclovir, famciclovir, or foscarnet. Other rectal lesions encountered in patients with HIV infection include condylomata acuminata, KS, and intraepithelial neoplasia (see below).

1	Hepatobiliary Diseases Diseases of the hepatobiliary system are a major problem in patients with HIV infection. It has been estimated that approximately 15% of the deaths of patients with HIV infection are related to liver disease. While this is predominantly a reflection of the problems encountered in the setting of co-infection with hepatitis B or C, it is also a reflection of the hepatic injury, ranging from hepatic steatosis to hypersensitivity reactions to immune reconstitution, that can be seen in the context of cART.

1	The prevalence of co-infection with HIV and hepatitis viruses varies by geographic region. In the United States, ~90% of HIV-infected individuals have evidence of infection with HBV; 6–14% have chronic HBV infection; 5–50% of patients are co-infected with HCV; and co-infections with hepatitis D, E, and/or G viruses are common. Among IV drug users with HIV infection, rates of HCV infection range from 70% to 95%. HIV infection has a significant impact on the course of hepatitis virus infection. It is associated with approximately a threefold increase in the development of persistent hepatitis B surface antigenemia. Patients infected with both HBV and HIV have decreased evidence of inflammatory liver disease. The presumption that this is due to the immunosuppressive effects of HIV infection is supported by the observations that this situation can be reversed, and one may see the development of more severe hepatitis following the initiation of effective cART. In studies of the impact of

1	is supported by the observations that this situation can be reversed, and one may see the development of more severe hepatitis following the initiation of effective cART. In studies of the impact of HIV on HBV infection, fourto tenfold increases in liver-related mortality rates have been noted in patients with HIV and active HBV infection compared to rates in patients with either infection alone. There is, however, only a slight increase in overall mortality rate in HIV-infected individuals who are also hepatitis B surface antigen (HBsAg)–positive. IFN-α is less successful as treatment for HBV in patients with HIV co-infection. Lamivudine, emtricitabine, adefovir/tenofovir/ entecavir, and telbivudine alone or in combination are useful in the treatment of hepatitis B in patients with HIV infection. It is important to remember that all the above-mentioned drugs also have activity against HIV and should not be used alone in patients with HIV infection, in order to avoid the emergence of

1	It is important to remember that all the above-mentioned drugs also have activity against HIV and should not be used alone in patients with HIV infection, in order to avoid the emergence of quasispecies of HIV resistant to these drugs. For this reason, the need to treat hepatitis B infection in a patient with HIV infection is an indication to treat HIV infection in that same patient, regardless of CD4+ T cell count. HCV infection is more severe in the patient with HIV infection; it does not appear to affect overall mortality rates in HIV-infected individuals when other variables such as age, baseline CD4+ T cell count, and use of cART are taken into account. In the setting of HIV and HCV co-infection, levels of HCV are approximately tenfold higher than in the HIV-negative patient with HCV infection. There is a 50% higher overall mortality rate with a five-fold increased risk of death due to liver disease in patients chronically infected with both HCV and HIV. Use of directly acting

1	infection. There is a 50% higher overall mortality rate with a five-fold increased risk of death due to liver disease in patients chronically infected with both HCV and HIV. Use of directly acting agents for the treatment for HCV leads to cure rates approaching 100%, even in patients with HIV co-infection. Successful treatment of HCV in HIV-infected patients decreases mortality. Hepatitis A virus infection is not seen with an increased frequency in patients with HIV infection. It is recommended that all patients with HIV infection who have not experienced natural infection be immunized with hepatitis A and/or hepatitis B vaccines. Infection with hepatitis G virus, also known as GB virus C, is seen in ~50% of patients with HIV infection. For reasons that are currently unclear, there are data to suggest that patients with HIV infection co-infected with this virus have a decreased rate of progression to AIDS.

1	A variety of other infections also may involve the liver. Granulomatous hepatitis may be seen as a consequence of mycobacterial or fungal infections, particularly MAC infection. Hepatic masses may be seen in the context of TB, peliosis hepatis, or fungal infection. Among the fungal opportunistic infections, C. immitis and Histoplasma capsulatum are those most likely to involve the liver. Biliary tract disease in the form of papillary stenosis or sclerosing cholangitis has been reported in the context of cryptosporidiosis, CMV infection, and KS. When no diagnosis can be made, the term AIDS cholangiopathy is used. Hemophagocytic lymphohistiocytosis of the liver has been seen in the setting of Hodgkin’s disease.

1	Many of the drugs used to treat HIV infection are metabolized by the liver and can cause liver injury. Fatal hepatic reactions have been reported with a wide array of antiretrovirals including nucleoside analogues, nonnucleoside analogues, and protease inhibitors. Nucleoside analogues work by inhibiting DNA synthesis. This can result in toxicity to mitochondria, which can lead to disturbances in oxidative metabolism. This may manifest as hepatic steatosis and, in severe cases, lactic acidosis and fulminant liver failure. It is important to be aware of this condition and to watch for it in patients with HIV infection receiving nucleoside analogues. It is reversible if diagnosed early and the offending agent(s) discontinued. Nevirapine has been associated with at times fatal fulminant and cholestatic hepatitis, hepatic necrosis, and hepatic failure. Indinavir may cause mild to moderate elevations in serum bilirubin in 10–15% of patients in a syndrome similar to Gilbert’s syndrome. A

1	cholestatic hepatitis, hepatic necrosis, and hepatic failure. Indinavir may cause mild to moderate elevations in serum bilirubin in 10–15% of patients in a syndrome similar to Gilbert’s syndrome. A similar pattern of hepatic injury may be seen with atazanavir. In the patient receiving cART with an unexplained increase in hepatic transaminases, strong consideration should be given to drug toxicity.

1	Pancreatic injury is most commonly a consequence of drug toxicity, notably that secondary to pentamidine or dideoxynucleosides. While up to half of patients in some series have biochemical evidence of pancreatic injury, <5% of patients show any clinical evidence of pancreatitis that is not linked to a drug toxicity.

1	Diseases of the Kidney and Genitourinary Tract Diseases of the kidney or genitourinary tract may be a direct consequence of HIV infection, due to an opportunistic infection or neoplasm, or related to drug toxicity. Overall, microalbuminuria is seen in ~20% of untreated HIV-infected patients; significant proteinuria is seen in closer to 2%. The presence of microalbuminuria has been associated with an increase in all-cause mortality rate. HIV-associated nephropathy (HIVAN) was first described in IDUs and was initially thought to be IDU nephropathy in patients with HIV infection; it is now recognized as a true direct complication of HIV infection. Although the majority of patients with this condition have CD4+ T cell counts <200/μL, HIV-associated nephropathy can be an early manifestation of HIV infection and is also seen in children. Over 90% of reported cases have been in African-American or Hispanic individuals; the disease is not only more prevalent in these populations but also more

1	HIV infection and is also seen in children. Over 90% of reported cases have been in African-American or Hispanic individuals; the disease is not only more prevalent in these populations but also more severe and is the third leading cause of end-stage renal failure among African Americans age 20–64 in the United States. Proteinuria is the hallmark of this disorder. Edema and hypertension are rare. Ultrasound examination reveals enlarged, hyperechogenic kidneys. A definitive diagnosis is obtained through renal biopsy. Histologically, focal segmental glomerulosclerosis is present in 80%, and mesangial proliferation in 10–15% of cases. Prior to effective antiretroviral therapy, this disease was characterized by relatively rapid progression to end-stage renal disease. Patients with HIV-associated nephropathy should be treated for their HIV infection regardless of CD4+ T cell count. Treatment with angiotensin-converting enzyme (ACE) inhibitors and/or prednisone, 60 mg/d, also has been

1	nephropathy should be treated for their HIV infection regardless of CD4+ T cell count. Treatment with angiotensin-converting enzyme (ACE) inhibitors and/or prednisone, 60 mg/d, also has been reported to be of benefit in some cases. The incidence of this disease in patients receiving adequate cART has not been well defined; however, the impression is that it has decreased in frequency and severity. It is the leading cause of end-stage renal disease in patients with HIV infection.

1	Among the drugs commonly associated with renal damage in patients with HIV disease are pentamidine, amphotericin, adefovir, cidofovir, tenofovir, and foscarnet. TMP/SMX may compete for tubular secretion with creatinine and cause an increase in the serum creatinine level. Sulfadiazine may crystallize in the kidney and result in an easily reversible form of renal shutdown, while indinavir or atazanavir may form renal calculi. Adequate hydration is the mainstay of treatment and prevention for these latter two conditions.

1	Genitourinary tract infections are seen with a high frequency in patients with HIV infection; they present with skin lesions, dysuria, hematuria, and/or pyuria and are managed in the same fashion as in patients without HIV infection. Infections with HSV are covered below (“Dermatologic Diseases”). Infections with T. pallidum, the etiologic agent of syphilis, play an important role in the HIV epidemic. In HIV-negative individuals, genital syphilitic ulcers as well as the ulcers of chancroid are major predisposing factors for heterosexual transmission of HIV infection. While most HIV-infected individuals with syphilis have a typical presentation, a variety of formerly rare clinical problems may be encountered in the setting of dual infection. Among them are lues maligna, an ulcerating lesion of the skin due to a necrotizing vasculitis; unexplained fever; nephrotic syndrome; and neurosyphilis. The most common presentation of syphilis in the HIV-infected patient is that of condylomata

1	of the skin due to a necrotizing vasculitis; unexplained fever; nephrotic syndrome; and neurosyphilis. The most common presentation of syphilis in the HIV-infected patient is that of condylomata lata,a form of secondary syphilis. Neurosyphilis may be asymptomatic or may present as acute meningitis, neuroretinitis, deafness, or stroke. The rate of neurosyphilis may be as high as 1% in patients with HIV infection, and one should consider a lumbar puncture to look for neurosyphilis in all patients with HIV infection and secondary syphilis. As a consequence of the immunologic abnormalities seen in the setting of HIV infection, diagnosis of syphilis through standard serologic testing may be challenging. On the one hand, a significant number of patients have false-positive Venereal Disease Research Laboratory (VDRL) tests due to polyclonal B cell activation. On the other hand, the development of a new positive VDRL may be delayed in patients with new infections, and the anti–fluorescent

1	Laboratory (VDRL) tests due to polyclonal B cell activation. On the other hand, the development of a new positive VDRL may be delayed in patients with new infections, and the anti–fluorescent treponemal antibody (anti-FTA) test may be negative due to immunodeficiency. Thus, dark-field examination of appropriate specimens should be performed in any patient in whom syphilis is suspected, even if the patient has a negative VDRL. Similarly, any patient with a positive serum VDRL test, neurologic findings, and an abnormal spinal fluid examination should be considered to have neurosyphilis and treated accordingly, regardless of the CSF VDRL result. In any setting, patients treated for syphilis need to be carefully monitored to ensure adequate therapy. Approximately one-third of patients with HIV infection will experience a Jarisch-Herxheimer reaction upon initiation of therapy for syphilis.

1	Vulvovaginal candidiasis is a common problem in women with HIV infection. Symptoms include pruritus, discomfort, dyspareunia, and dysuria. Vulvar infection may present as a morbilliform rash that may extend to the thighs. Vaginal infection is usually associated with 1259 a white discharge, and plaques may be seen along an erythematous vaginal wall. Diagnosis is made by microscopic examination of the discharge for pseudohyphal elements in a 10% potassium hydroxide solution. Mild disease can be treated with topical therapy. More serious disease can be treated with fluconazole. Other causes of vaginitis include Trichomonas and mixed bacteria.

1	Diseases of the Endocrine System and Metabolic Disorders A variety of endocrine and metabolic disorders are seen in the context of HIV infection. These may be a direct consequence of HIV infection, secondary to opportunistic infections or neoplasms, or related to medication side effects. Between 33% and 75% of patients with HIV infection receiving thymidine analogues or protease inhibitors as a component of cART develop a syndrome often referred to as lipodystrophy, consisting of elevations in plasma triglycerides, total cholesterol, and apolipoprotein B, as well as hyperinsulinemia and hyperglycemia. Many of the patients have been noted to have a characteristic set of body habitus changes associated with fat redistribution, consisting of truncal obesity coupled with peripheral wasting (Fig. 226-38). Truncal obesity is apparent as an increase in abdominal girth related to increases in mesenteric fat, a dorsocervical fat pad (“buffalo hump”) reminiscent of patients with Cushing’s

1	(Fig. 226-38). Truncal obesity is apparent as an increase in abdominal girth related to increases in mesenteric fat, a dorsocervical fat pad (“buffalo hump”) reminiscent of patients with Cushing’s syndrome, and enlargement of the breasts. The peripheral wasting, or lipoatrophy, is particularly noticeable in the face and buttocks and by the prominence of the veins in the legs. These changes may develop at any time ranging from ~6 weeks to several years following the initiation of cART.

1	Approximately 20% of the patients with HIV-associated lipodystrophy meet the criteria for the metabolic syndrome as defined by The International Diabetes Federation or The U.S. National Cholesterol Education Program Adult Treatment Panel III. The lipodystrophy syndrome has been reported in association with regimens containing a variety of different drugs, and while initially reported in the setting of protease inhibitor therapy, it appears that similar changes can also be induced by protease-sparing regimens. It has been suggested that the lipoatrophy changes are particularly severe in patients receiv ing the thymidine analogues stavudine and zidovudine. National Cholesterol Education Program (NCEP) guidelines should be followed in the management of these lipid abnormalities (Chap. 291e), and consideration should be given to changing the components of cART with avoidance of thymidine analogues (azidothymidine and stavudine) and protease inhibitors. Due to concerns regarding drug

1	and consideration should be given to changing the components of cART with avoidance of thymidine analogues (azidothymidine and stavudine) and protease inhibitors. Due to concerns regarding drug interactions, the most commonly utilized lipid-lowering agents in this setting are gemfibrozil and atorvastatin. In addition, lactic acidosis is associated with cART. This is most commonly seen with nucleoside analogue reverse transcriptase inhibitors and can be fatal (see below).

1	Patients with advanced HIV disease may develop hyponatremia due to the syndrome of inappropriate antidiuretic hormone (vasopressin) secretion (SIADH) as a consequence of increased free-water intake and decreased free-water excretion. SIADH is usually seen in conjunction with pulmonary or CNS disease. Low serum sodium may also be due to adrenal insufficiency; a concomitant high serum potassium should alert one to this possibility. Hyperkalemia may be secondary to adrenal insufficiency; HIV nephropathy; or medications, particularly trimethoprim and pentamidine. Hypokalemia may be seen in the setting of tenofovir or amphotericin therapy. Adrenal gland disease may be due to mycobacterial infections, CMV disease, cryptococcal disease, histoplasmosis, or ketoconazole toxicity. Iatrogenic Cushing’s syndrome with suppression of the hypothalamic-pituitary-adrenal axis may be seen with the use of local glucocorticoids (injected or inhaled) in patients receiving ritonavir. This is due to

1	Cushing’s syndrome with suppression of the hypothalamic-pituitary-adrenal axis may be seen with the use of local glucocorticoids (injected or inhaled) in patients receiving ritonavir. This is due to inhibition of the hepatic enzyme CYP3A4 by ritonavir leading to prolongation of the glucocorticoid half-life.

1	Thyroid function may be altered in 10–15% of patients with HIV infection. Both hypoand hyperthyroidism may be seen. The predominant abnormality is subclinical hypothyroidism. In the setting of cART, up to 10% of patients have been noted to have elevated thyroid-stimulating hormone levels, suggesting that this may be

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1260 characterizes HIV infection and reflects the complex nature of the immune system and its regulatory mechanisms. Drug allergies are the most significant allergic reactions occurring in HIV-infected patients and appear to become more common as the disease progresses. They occur in up to 65% of patients who receive therapy with TMP/SMX for PCP. In general, these drug reactions are characterized by erythematous, morbilliform eruptions that are pruritic, tend to coalesce, and are often associated with fever. Nonetheless, ~33% of patients can be maintained on the offending therapy, and thus these reactions are not an immediate indication to stop the drug. Anaphylaxis is extremely rare in patients with HIV infection, and patients who have a cutaneous reaction during a single course of therapy can still be considered candidates for future treatment or prophylaxis with the same agent. The one exception to this is the

1	patients who have a cutaneous reaction during a single course of therapy can still be considered candidates for future treatment or prophylaxis with the same agent. The one exception to this is the nucleoside analogue abacavir, where fatal hypersensitivity reactions have been reported with rechallenge. This hypersensitivity is strongly associated with the HLA-B5701 haplotype, and a hypersensitivity reaction to abacavir is an absolute contraindication to future therapy. For other agents, including TMP/SMX, desensitization regimens are moderately successful. While the mechanisms underlying these allergic-type reactions remain unknown, patients with HIV infection have been noted to have elevated IgE levels that increase as the CD4+ T cell count declines. The numerous examples of patients with multiple drug reactions suggest that a common pathway is involved.

1	HIV infection shares many similarities with a variety of autoimmune diseases, including a substantial polyclonal B cell activation that is associated with a high incidence of antiphospho lipid antibodies, such as anticardiolipin antibodies, VDRL antibodies, and lupus-like anticoagulants. FIGuRE 226-38 Characteristics of lipodystrophy. A. Truncal obesity and buffalo hump. In addition, HIV-infected individuals have an B. Facial wasting. C. Accumulation of intraabdominal fat on CT scan.

1	increased incidence of antinuclear antibodies. Despite these serologic findings, there is no evidence that HIV-infected individuals have an a manifestation of immune reconstitution. Immune-reconstitution increase in two of the more common autoimmune diseases, i.e., Graves’ disease may occur as a late (9–48 months) complication of systemic lupus erythematosus and rheumatoid arthritis. In fact, itcART. In advanced HIV disease, infection of the thyroid gland may has been observed that these diseases may be somewhat ameliorated occur with opportunistic pathogens, including P. jiroveci, CMV, by the concomitant presence of HIV infection, suggesting that an mycobacteria, Toxoplasma gondii, and Cryptococcus neoformans. intact CD4+ T cell limb of the immune response plays an integralThese infections are generally associated with a nontender, diffuse role in the pathogenesis of these conditions. Similarly, there are anecenlargement of the thyroid gland. Thyroid function is usually normal. dotal

1	are generally associated with a nontender, diffuse role in the pathogenesis of these conditions. Similarly, there are anecenlargement of the thyroid gland. Thyroid function is usually normal. dotal reports of patients with common variable immunodeficiencyDiagnosis is made by fine-needle aspirate or open biopsy. (Chap. 374), characterized by hypogammaglobulinemia, who haveDepending on the severity of disease, HIV infection is associated had a normalization of Ig levels following the development of HIVwith hypogonadism in 20–50% of men. While this is generally a com-infection, suggesting a possible role for overactive CD4+ T cell plication of underlying illness, testicular dysfunction may also be a immunity in certain forms of that syndrome. The one autoimmuneside effect of ganciclovir therapy. In some surveys, up to two-thirds disease that may occur with an increased frequency in patientsof patients report decreased libido and one-third complain of erectile with HIV infection is a

1	therapy. In some surveys, up to two-thirds disease that may occur with an increased frequency in patientsof patients report decreased libido and one-third complain of erectile with HIV infection is a variant of primary Sjögren’s syndromedysfunction. Androgen-replacement therapy should be considered in (Chap. 383). Patients with HIV infection may develop a syndromepatients with symptomatic hypogonadism. HIV infection does not consisting of parotid gland enlargement, dry eyes, and dry mouthseem to have a significant effect on the menstrual cycle outside the that is associated with lymphocytic infiltrates of the salivary glandsetting of advanced disease. and lung. One also can see peripheral neuropathy, polymyositis, renal tubular acidosis, and hepatitis. In contrast to Sjögren’s syndrome, Immunologic and Rheumatologic Diseases Immunologic and rheumato-in which the lymphocytic infiltrates are composed predominantly logic disorders are common in patients with HIV infection and range of

1	Immunologic and Rheumatologic Diseases Immunologic and rheumato-in which the lymphocytic infiltrates are composed predominantly logic disorders are common in patients with HIV infection and range of CD4+ T cells, in patients with HIV infection the infiltrates are from excessive immediate-type hypersensitivity reactions (Chap. 376) composed predominantly of CD8+ T cells. In addition, while patients to an increase in the incidence of reactive arthritis (Chap. 384) to with Sjögren’s syndrome are mainly women who have autoantibodconditions characterized by a diffuse infiltrative lymphocytosis. The ies to Ro and La and who frequently have HLA-DR3 or -B8 MHC occurrence of these phenomena is an apparent paradox in the setting haplotypes, HIV-infected individuals with this syndrome are usually of the profound immunodeficiency and immunosuppression that African-American men who do not have anti-Ro or anti-La and who most often are HLA-DR5. This syndrome appears to be less common with the

1	of the profound immunodeficiency and immunosuppression that African-American men who do not have anti-Ro or anti-La and who most often are HLA-DR5. This syndrome appears to be less common with the increased use of effective cART. The term diffuse infiltrative lymphocytosis syndrome (DILS) is used to describe this entity and to distinguish it from Sjögren’s syndrome.

1	Approximately one-third of HIV-infected individuals experience arthralgias; furthermore, 5–10% are diagnosed as having some form of reactive arthritis, such as Reiter’s syndrome or psoriatic arthritis as well as undifferentiated spondyloarthropathy (Chap. 384). These syndromes occur with increasing frequency as the competency of the immune system declines. This association may be related to an increase in the number of infections with organisms that may trigger a reactive arthritis with progressive immunodeficiency or to a loss of important regulatory T cells. Reactive arthritides in HIV-infected individuals generally respond well to standard treatment; however, therapy with methotrexate has been associated with an increase in the incidence of opportunistic infections and should be used with caution and only in severe cases.

1	HIV-infected individuals also experience a variety of joint problems without obvious cause that are referred to generically as HIVor AIDS-associated arthropathy. This syndrome is characterized by sub-acute oligoarticular arthritis developing over a period of 1–6 weeks and lasting 6 weeks to 6 months. It generally involves the large joints, predominantly the knees and ankles, and is nonerosive with only a mild inflammatory response. X-rays are nonrevealing. Nonsteroidal anti-inflammatory drugs are only marginally helpful; however, relief has been noted with the use of intraarticular glucocorticoids. A second form of arthritis also thought to be secondary to HIV infection is called painful articular syndrome. This condition, reported as occurring in as many as 10% of AIDS patients, presents as an acute, severe, sharp pain in the affected joint. It affects primarily the knees, elbows, and shoulders; lasts 2–24 h; and may be severe enough to require narcotic analgesics. The cause of this

1	as an acute, severe, sharp pain in the affected joint. It affects primarily the knees, elbows, and shoulders; lasts 2–24 h; and may be severe enough to require narcotic analgesics. The cause of this arthropathy is unclear; however, it is thought to result from a direct effect of HIV on the joint. This condition is reminiscent of the fact that other lentiviruses, in particular the caprine arthritis-encephalitis virus, are capable of directly causing arthritis.

1	A variety of other immunologic or rheumatologic diseases have been reported in HIV-infected individuals, either de novo or in association with opportunistic infections or drugs. Using the criteria of widespread musculoskeletal pain of at least 3 months’ duration and the presence of at least 11 of 18 possible tender points by digital palpation, 11% of an HIV-infected cohort containing 55% IDUs were diagnosed as having fibromyalgia (Chap. 396). While the incidence of frank arthritis was less in this population than in other studied populations that consisted predominantly of men who have sex with men, these data support the concept that there are musculoskeletal problems that occur as a direct result of HIV infection. In addition there have been reports of leukocytoclastic vasculitis in the setting of zidovudine therapy. CNS angiitis and polymyositis also have been reported in HIV-infected individuals. Septic arthritis is surprisingly rare, especially given the increased incidence of

1	setting of zidovudine therapy. CNS angiitis and polymyositis also have been reported in HIV-infected individuals. Septic arthritis is surprisingly rare, especially given the increased incidence of staphylococcal bacteremias seen in this population. When septic arthritis has been reported, it has usually been due to Staphylococcus aureus, systemic fungal infection with

1	C. neoformans, Sporothrix schenckii, or H. capsulatum or to systemic mycobacterial infection with M. tuberculosis, M. haemophilum, M. avium, or M. kansasii.

1	Patients with HIV infection treated with cART have been found to have an increased incidence of osteonecrosis or avascular necrosis of the hip and shoulders. In a study of asymptomatic patients, 4.4% were found to have evidence of osteonecrosis on MRI. While precise causeand-effect relationships have been difficult to establish, this complication has been associated with the use of lipid-lowering agents, systemic glucocorticoids, and testosterone; bodybuilding exercise; alcohol consumption; and the presence of anticardiolipin antibodies. Osteoporosis has been reported in 7% of women with HIV infection, with 41% of women demonstrating some degree of osteopenia. Several studies have documented decreases in bone mineral density of 2–6% in the first 2 years following the initiation of cART. This may be particularly apparent with tenofovir-containing regimens.

1	worsening of an existing clinical condition or abrupt appearance of a new clinical finding (unmasking) is seen following the initiation of antiretroviral therapy weeks to months following the initiation of antiretroviral therapy most common in patients starting therapy with a CD4+ T cell count • Is frequently seen in the setting of tuberculosis; particularly when cART is starting soon after initiation of anti-TB therapy

1	Following the initiation of effective cART, a paradoxical worsening of preexisting, untreated, or partially treated opportunistic infections may be noted. One may also see exacerbations of pre-existing or the development of new autoimmune conditions following the initiation of antiretrovirals (Table 226-12). IRIS related to a known pre-existing infection or neoplasm is referred to as paradoxical IRIS, while IRIS associated with a previously undiagnosed condition is referred to as unmasking IRIS. The term immune reconstitution disease (IRD) is sometimes used to distinguish IRIS manifestations related to opportunistic diseases from IRIS manifestations related to autoimmune diseases. IRD is particularly common in patients with underlying untreated mycobacterial or fungal infections. IRIS is seen in 10–30% of patients, depending on the clinical setting, and is most common in patients starting therapy with CD4+ T cell counts <50 cells/μL who have a precipitous drop in HIV RNA levels

1	is seen in 10–30% of patients, depending on the clinical setting, and is most common in patients starting therapy with CD4+ T cell counts <50 cells/μL who have a precipitous drop in HIV RNA levels following the initiation of cART. Signs and symptoms may appear anywhere from 2 weeks to 2 years after the initiation of cART and can include localized lymphadenitis, prolonged fever, pulmonary infiltrates, hepatitis, increased intracranial pressure, uveitis, sarcoidosis, and Graves’ disease. The clinical course can be protracted, and severe cases can be fatal. The underlying mechanism appears to be related to a phenomenon similar to type IV hypersensitivity reactions and reflects the immediate improvements in immune function that occur as levels of HIV RNA drop and the immunosuppressive effects of HIV infection are controlled. In severe cases, the use of immunosuppressive drugs such as glucocorticoids may be required to blunt the inflammatory component of these reactions while specific

1	of HIV infection are controlled. In severe cases, the use of immunosuppressive drugs such as glucocorticoids may be required to blunt the inflammatory component of these reactions while specific antimicrobial therapy takes effect.

1	Diseases of the Hematopoietic System Disorders of the hematopoietic system including lymphadenopathy, anemia, leukopenia, and/or thrombocytopenia are common throughout the course of HIV infection and may be the direct result of HIV, manifestations of secondary infections and neoplasms, or side effects of therapy (Table 226-13). Direct histologic examination and culture of lymph node or bone marrow tissue are often diagnostic. A significant percentage of bone marrow aspirates from patients with HIV infection have been reported to contain lymphoid aggregates, the precise significance of which is unknown. Initiation of cART will lead to reversal of most hematologic complications that are the direct result of HIV infection.

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1262 Some patients, otherwise asymptomatic, may develop persistent generalized lymphadenopathy as an early clinical manifestation of HIV infection. This condition is defined as the presence of enlarged lymph nodes (>1 cm) in two or more extrainguinal sites for >3 months without an obvious cause. The lymphadenopathy is due to marked follicular hyperplasia in the node in response to HIV infection. The nodes are generally discrete and freely movable. This feature of HIV disease may be seen at any point in the spectrum of immune dysfunction and is not associated with an increased likelihood of developing AIDS. Paradoxically, a loss in lymphadenopathy or a decrease in lymph node size outside the setting of cART may be a prognostic marker of disease progression. In patients with CD4+ T cell counts >200/ μL, the differential diagnosis of lymphadenopathy includes KS, TB, Castleman’s disease, and lymphoma. In patients with more

1	marker of disease progression. In patients with CD4+ T cell counts >200/ μL, the differential diagnosis of lymphadenopathy includes KS, TB, Castleman’s disease, and lymphoma. In patients with more advanced disease, lymphadenopathy may also be due to atypical mycobacterial infection, toxoplasmosis, systemic fungal infection, or bacillary angiomatosis. While indicated in patients with CD4+ T cell counts <200/μL, lymph node biopsy is not indicated in patients with early-stage disease unless there are signs and symptoms of systemic illness, such as fever and weight loss, or unless the nodes begin to enlarge, become fixed, or coalesce. Monoclonal gammopathy of unknown significance (MGUS) (Chap. 136), defined as the presence of a serum monoclonal IgG, IgA, or IgM in the absence of a clear cause, has been reported in 3% of patients with HIV infection. The overall clinical significance of this finding in patients with HIV infection is unclear, although it has been associated with other viral

1	has been reported in 3% of patients with HIV infection. The overall clinical significance of this finding in patients with HIV infection is unclear, although it has been associated with other viral infections, non-Hodgkin’s lymphoma, and plasma cell malignancy. Anemia is the most common hematologic abnormality in HIV-infected patients and, in the absence of a specific treatable cause, is independently associated with a poor prognosis. While generally mild, anemia can be quite severe and require chronic blood transfusions. Among the specific reversible causes of anemia in the setting of HIV infection are drug toxicity, systemic fungal and mycobacterial infections, nutritional deficiencies, and parvovirus B19 infections. Zidovudine may block erythroid maturation prior to its effects on other marrow elements. A characteristic feature of zidovudine therapy is an elevated mean corpuscular volume (MCV). Another drug used in patients with HIV infection that has a selective effect on the

1	other marrow elements. A characteristic feature of zidovudine therapy is an elevated mean corpuscular volume (MCV). Another drug used in patients with HIV infection that has a selective effect on the erythroid series is dapsone. This drug can cause a serious hemolytic anemia in patients who are deficient in glucose-6-phosphate dehydrogenase and can create a functional anemia in others through induction of methemoglobinemia. Folate levels are usually normal in HIV-infected individuals; however, vitamin B12 levels may be depressed as a consequence of achlorhydria or malabsorption. True autoimmune hemolytic anemia is rare, although ~20% of patients with HIV infection may have a positive direct antiglobulin test as a consequence of polyclonal B cell activation. Infection with parvovirus B19 may also cause anemia. It is important to recognize this possibility given the fact that it responds well to treatment with IVIg. Erythropoietin levels in patients with HIV infection and anemia are

1	may also cause anemia. It is important to recognize this possibility given the fact that it responds well to treatment with IVIg. Erythropoietin levels in patients with HIV infection and anemia are generally lower than expected given the degree of anemia. Treatment with erythropoietin may result in an increase in hemoglobin levels. An exception to this is a subset of patients with zidovudine-associated anemia in whom erythropoietin levels may be quite high. During the course of HIV infection, neutropenia may be seen in approximately half of patients. In most instances it is mild; however, it can be severe and can put patients at risk of spontaneous bacterial infections. This is most frequently seen in patients with severely advanced HIV disease and in patients receiving any of a number of potentially myelosuppressive therapies. In the setting of neutropenia, diseases that are not commonly seen in HIV-infected patients, such as aspergillosis or mucormycosis, may occur. Both granulocyte

1	potentially myelosuppressive therapies. In the setting of neutropenia, diseases that are not commonly seen in HIV-infected patients, such as aspergillosis or mucormycosis, may occur. Both granulocyte colony-stimulating factor (G-CSF) and GM-CSF increase neutrophil counts in patients with HIV infection regardless of the cause of the neutropenia. Earlier concerns about the potential of these agents to also increase levels of HIV were not confirmed in controlled clinical trials. Thrombocytopenia may be an early consequence of HIV infection. Approximately 3% of patients with untreated HIV infection and CD4+ T cell counts ≥400/μL have platelet counts <150,000/μL. For untreated patients with CD4+ T cell counts <400/μL, this incidence increases to 10%. In patients receiving antiretrovirals, thrombocytopenia is associated with hepatitis C, cirrhosis, and ongoing high-level HIV replication. Thrombocytopenia is rarely a serious clinical problem in patients with HIV infection and generally

1	thrombocytopenia is associated with hepatitis C, cirrhosis, and ongoing high-level HIV replication. Thrombocytopenia is rarely a serious clinical problem in patients with HIV infection and generally responds well to successful cART. Clinically, it resembles the thrombocytopenia seen in patients with idiopathic thrombocytopenic purpura (Chap. 140). Immune complexes containing anti-gp120 antibodies and anti-anti-gp120 antibodies have been noted in the circulation and on the surface of platelets in patients with HIV infection. Patients with HIV infection have also been noted to have a platelet-specific antibody directed toward a 25-kDa component of the surface of the platelet. Other data suggest that the thrombocytopenia in patients with HIV infection may be due to a direct effect of HIV on megakaryocytes. Whatever the cause, it is very clear that the most effective medical approach to this problem has been the use of cART. For patients with platelet counts <20,000/μL, a more aggressive

1	megakaryocytes. Whatever the cause, it is very clear that the most effective medical approach to this problem has been the use of cART. For patients with platelet counts <20,000/μL, a more aggressive approach combining IVIg or anti-Rh Ig for an immediate response and cART for a more lasting response is appropriate. Rituximab has been used with some success in otherwise refractory cases. Splenectomy is a rarely needed option and is reserved for patients refractory to medical management. Because of the risk of serious infection with encapsulated organisms, all patients with HIV infection about to undergo splenectomy should be immunized with pneumococcal polysaccharide. It should be noted that, in addition to causing an increase in the platelet count, removal of the spleen will result in an increase in the peripheral blood lymphocyte count, making CD4+ T cell counts unreliable markers of immunocompetence. In this setting, the clinician should rely on the CD4+ T cell percentage for making

1	in the peripheral blood lymphocyte count, making CD4+ T cell counts unreliable markers of immunocompetence. In this setting, the clinician should rely on the CD4+ T cell percentage for making diagnostic decisions with respect to the likelihood of opportunistic infections. A CD4+ T cell percentage of 15 is approximately equivalent to a CD4+ T cell count of 200/μL. In patients with early HIV infection, thrombocytopenia has also been reported as a consequence of classic thrombotic thrombocytopenic purpura (Chap. 140). This clinical syndrome, consisting of fever, thrombocytopenia, hemolytic anemia, and neurologic and renal dysfunction, is a rare complication of early HIV infection. As in other settings, the appropriate management is the use of salicylates and plasma exchange. Other causes of thrombocytopenia include lymphoma, mycobacterial infections, and fungal infections.

1	The incidence of venous thromboembolic disease such as deep-vein thrombosis or pulmonary embolus is approximately 1% per year in patients with HIV infection. This is approximately 10 times higher than that seen in an age-matched population. Factors associated with an increased risk of clinical thrombosis include age over 45, history of an opportunistic infection, lower CD4 count, and estrogen use. Abnormalities of the coagulation cascade including decreased protein S activity, increases in factor VIII, anticardiolipin antibodies, or lupus-like anticoagulant have been reported in more than 50% of patients with HIV infection. The clinical significance of this increased propensity toward thromboembolic disease is likely reflected in the observation that elevations in d-dimer are strongly associated with all-cause mortality in patients with HIV infection (Table 226-9).

1	Dermatologic Diseases Dermatologic problems occur in >90% of patients with HIV infection. From the macular, roseola-like rash seen with the acute seroconversion syndrome to extensive end-stage KS, cutaneous manifestations of HIV disease can be seen throughout the course of HIV infection. Among the more common nonneoplastic problems are seborrheic dermatitis, folliculitis, and opportunistic infections. Extrapulmonary pneumocystosis may cause a necrotizing vasculitis. Neoplastic conditions are covered below. Seborrheic dermatitis occurs in 3% of the general population and in up to 50% of patients with HIV infection. Seborrheic dermatitis increases in prevalence and severity as the CD4+ T cell count declines. In HIV-infected patients, seborrheic dermatitis may be aggravated by concomitant infection with Pityrosporum, a yeastlike fungus; use of topical antifungal agents has been recommended in cases refractory to standard topical treatment.

1	Folliculitis is among the most prevalent dermatologic disorders in patients with HIV infection and is seen in ~20% of patients. It is more common in patients with CD4+ T cell counts <200 cells/μL. Pruritic papular eruption is one of the most common pruritic rashes in patients with HIV infection. It appears as multiple papules on the face, trunk, and extensor surfaces and may improve with cART. Eosinophilic pustular folliculitis is a rare form of folliculitis that is seen with increased frequency in patients with HIV infection. It presents as multiple, urticarial perifollicular papules that may coalesce into plaquelike lesions. Skin biopsy reveals an eosinophilic infiltrate of the hair follicle, which in certain cases has been associated with the presence of a mite. Patients typically have an elevated serum IgE level and may respond to treatment with topical anthelmintics. Pruritus is a common symptom in patients with HIV infection and can lead to prurigo nodularis. Patients with HIV

1	an elevated serum IgE level and may respond to treatment with topical anthelmintics. Pruritus is a common symptom in patients with HIV infection and can lead to prurigo nodularis. Patients with HIV infection have also been reported to develop a severe form of Norwegian scabies with hyperkeratotic psoriasiform lesions.

1	Both psoriasis and ichthyosis, although they are not reported to be increased in frequency, may be particularly severe when they occur in patients with HIV infection. Preexisting psoriasis may become guttate in appearance and more refractory to treatment in the setting of HIV infection.

1	Reactivation herpes zoster (shingles) is seen in 10–20% of patients with HIV infection. This reactivation syndrome of varicella-zoster virus indicates a modest decline in immune function and may be the first indication of clinical immunodeficiency. In one series, patients who developed shingles did so an average of 5 years after HIV infection. In a cohort of patients with HIV infection and localized zoster, the subsequent rate of the development of AIDS was 1% per month. In that study, AIDS was more likely to develop if the outbreak of zoster was associated with severe pain, extensive skin involvement, or involvement of cranial or cervical dermatomes. The clinical manifestations of reactivation zoster in HIV-infected patients, although indicative of immunologic compromise, are not as severe as those seen in other immunodeficient conditions. Thus, while lesions may extend over several dermatomes, involve the spinal cord, and/or be associated with frank cutaneous dissemination, visceral

1	as those seen in other immunodeficient conditions. Thus, while lesions may extend over several dermatomes, involve the spinal cord, and/or be associated with frank cutaneous dissemination, visceral involvement has not been reported. In contrast to patients without a known underlying immunodeficiency state, patients with HIV infection tend to have recurrences of zoster with a relapse rate of ~20%. Valacyclovir, acyclovir, or famciclovir is the treatment of choice. Foscarnet may be of value in patients with acyclovir-resistant virus.

1	Infection with herpes simplex virus in HIV-infected individuals is associated with recurrent orolabial, genital, and perianal lesions as part of recurrent reactivation syndromes (Chap. 216). As HIV disease progresses and the CD4+ T cell count declines, these infections become more frequent and severe. Lesions often appear as beefy red, are exquisitely painful, and have a tendency to occur high in the gluteal cleft (Fig. 226-37). Perirectal HSV may be associated with proctitis and anal fissures. HSV should be high in the differential diagnosis of any HIV-infected patient with a poorly healing, painful perirectal lesion. In addition to recurrent mucosal ulcers, recurrent HSV infection in the form of herpetic whitlow can be a problem in patients with HIV infection, presenting with painful vesicles or extensive cutaneous erosion. Valacyclovir, acyclovir or famciclovir is the treatment of choice in these settings. It is noteworthy that even subclinical reactivation of herpes simplex may be

1	or extensive cutaneous erosion. Valacyclovir, acyclovir or famciclovir is the treatment of choice in these settings. It is noteworthy that even subclinical reactivation of herpes simplex may be associated with increases in plasma HIV RNA levels.

1	Diffuse skin eruptions due to Molluscum contagiosum may be seen in patients with advanced HIV infection. These flesh-colored, umbilicated lesions may be treated with local therapy. They tend to regress with effective cART. Similarly, condyloma acuminatum lesions may be more severe and more widely distributed in patients with low CD4+ T cell counts. Imiquimod cream may be helpful in some cases. Atypical mycobacterial infections may present as erythematous cutaneous nodules, as may fungal infections, Bartonella, Acanthamoeba, and KS. Cutaneous infections with Aspergillus have been noted at the site of IV catheter placement.

1	The skin of patients with HIV infection is often a target organ for drug reactions (Chap. 74). Although most skin reactions are mild and not necessarily an indication to discontinue therapy, patients may have particularly severe cutaneous reactions, including erythroderma, 1263 Stevens-Johnson syndrome, and toxic epidermal necrolysis, as a reaction to drugs—particularly sulfa drugs, nonnucleoside reverse transcriptase inhibitors, abacavir, amprenavir, darunavir, fosamprenavir, and tipranavir. Similarly, patients with HIV infection are often quite photosensitive and burn easily following exposure to sunlight or as a side effect of radiation therapy (Chap. 75).

1	HIV infection and its treatment may be accompanied by cosmetic changes of the skin that are not of great clinical importance but may be troubling to patients. Yellowing of the nails and straightening of the hair, particularly in African-American patients, have been reported as a consequence of HIV infection. Zidovudine therapy has been associated with elongation of the eyelashes and the development of a bluish discoloration to the nails, again more common in African-American patients. Therapy with clofazimine may cause a yellow-orange discoloration of the skin and urine.

1	Neurologic Diseases Clinical disease of the nervous system accounts for a significant degree of morbidity in a high percentage of patients with HIV infection (Table 226-14). The neurologic problems that occur in HIV-infected individuals may be either primary to the pathogenic processes of HIV infection or secondary to opportunistic infections or neoplasms. Among the more frequent opportunistic diseases that involve the CNS are toxoplasmosis, cryptococcosis, progressive multifocal leukoencephalopathy, and primary CNS lymphoma. Other less common problems include mycobacterial infections; syphilis; and infection with CMV, HTLV-1, Trypanosoma cruzi, or Acanthamoeba. Overall, secondary diseases of the CNS have been reported to occur in approximately one-third of patients with AIDS. These data antedate the widespread use of cART, and this frequency is considerably lower in patients receiving effective antiretroviral drugs. Primary processes related to HIV infection of the nervous system are

1	the widespread use of cART, and this frequency is considerably lower in patients receiving effective antiretroviral drugs. Primary processes related to HIV infection of the nervous system are reminiscent of those seen with other lentiviruses, such as the Visna-Maedi virus of sheep.

1	Neurologic problems directly attributable to HIV occur throughout the course of infection and may be inflammatory, demyelinating, or degenerative in nature. The term HIV-associated neurocognitive disorders (HAND) is used to describe a spectrum of disorders that range from asymptomatic neurocognitive impairment (ANI) to minor neurocognitive disorder (MND) to clinically severe dementia. The most severe form, HIV-associated dementia (HAD), also referred to as the AIDS dementia complex, or HIV encephalopathy, is considered an AIDS-defining illness. Most HIV-infected patients have some neurologic problem during the course of their disease. Even in the setting of suppressive cART, approximately 50% of HIV-infected individuals can be shown to have mild to moderate neurocognitive impairment using sensitive neuropsychiatric testing. As noted in the section on Cryptococcosis HIV-associated neurocognitive disorders (HAND), including HIV encepha-

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1264 pathogenesis, damage to the CNS may be a direct result of viral infection of the CNS macrophages or glial cells or may be secondary to the release of neurotoxins and potentially toxic cytokines such as IL-1β, TNF-α, IL-6, and TGF-β. It has been reported that HIV-infected individuals with the E4 allele for apoE are at increased risk for AIDS encephalopathy and peripheral neuropathy. Virtually all patients with HIV infection have some degree of nervous system involvement with the virus. This is evidenced by the fact that CSF findings are abnormal in ~90% of patients, even during the asymptomatic phase of HIV infection. CSF abnormalities include pleocytosis (50–65% of patients), detection of viral RNA (~75%), elevated CSF protein (35%), and evidence of intrathecal synthesis of anti-HIV antibodies (90%). It is important to point out that evidence of infection of the CNS with HIV does not imply impairment of cognitive

1	protein (35%), and evidence of intrathecal synthesis of anti-HIV antibodies (90%). It is important to point out that evidence of infection of the CNS with HIV does not imply impairment of cognitive function. The neurologic function of an HIV-infected individual should be considered normal unless clinical signs and symptoms suggest otherwise. Aseptic meningitis may be seen in any but the very late stages of HIV infection. In the setting of acute primary infection, patients may experience a syndrome of headache, photophobia, and meningismus. Rarely, an acute encephalopathy due to encephalitis may occur. Cranial nerve involvement may be seen, predominantly cranial nerve VII but occasionally V and/or VIII. CSF findings include a lymphocytic pleocytosis, elevated protein level, and normal glucose level. This syndrome, which cannot be clinically differentiated from other viral meningitides (Chap. 165), usually resolves spontaneously within 2–4 weeks; however, in some patients, signs and

1	level. This syndrome, which cannot be clinically differentiated from other viral meningitides (Chap. 165), usually resolves spontaneously within 2–4 weeks; however, in some patients, signs and symptoms may become chronic. Aseptic meningitis may occur any time in the course of HIV infection; however, it is rare following the development of AIDS. This suggests that clinical aseptic meningitis in the context of HIV infection is an immune-mediated disease. Cryptococcus is the leading infectious cause of meningitis in patients with AIDS (Chap. 239). While the vast majority of these are due to C. neoformans, up to 12% may be due to C. gattii. Cryptococcal meningitis is the initial AIDS-defining illness in ~2% of patients and generally occurs in patients with CD4+ T cell counts <100/μL. Cryptococcal meningitis is particularly common in untreated patients with AIDS in Africa, occurring in ~5% of patients. Most patients present with a picture of subacute meningoencephalitis with fever, nausea,

1	meningitis is particularly common in untreated patients with AIDS in Africa, occurring in ~5% of patients. Most patients present with a picture of subacute meningoencephalitis with fever, nausea, vomiting, altered mental status, headache, and meningeal signs. The incidence of seizures and focal neurologic deficits is low. The CSF profile may be normal or may show only modest elevations in WBC or protein levels and decreases in glucose. The opening pressure in the CSF is usually elevated. In addition to meningitis, patients may develop cryptococcomas and cranial nerve involvement. Approximately one-third of patients also have pulmonary disease. Uncommon manifestations of cryptococcal infection include skin lesions that resemble molluscum contagiosum, lymphadenopathy, palatal and glossal ulcers, arthritis, gastroenteritis, myocarditis, and prostatitis. The prostate gland may serve as a reservoir for smoldering cryptococcal infection. The diagnosis of cryptococcal meningitis is made by

1	arthritis, gastroenteritis, myocarditis, and prostatitis. The prostate gland may serve as a reservoir for smoldering cryptococcal infection. The diagnosis of cryptococcal meningitis is made by identification of organisms in spinal fluid with india ink examination or by the detection of cryptococcal antigen. Blood cultures for fungus are often positive. A biopsy may be needed to make a diagnosis of CNS cryptococcoma. Treatment is with IV amphotericin B 0.7 mg/kg daily, or liposomal amphotericin 4–6 mg/kg daily, with flucytosine 25 mg/kg qid for at least 2 weeks if possible, continuing with amphotericin alone ideally until the CSF culture turns negative. Decreases in renal function in association with amphotericin can lead to increases in flucytosine levels and subsequent bone marrow suppression. Amphotericin is followed by fluconazole 400 mg/d PO for 8 weeks, and then fluconazole 200 mg/d until the CD4+ T cell count has increased to >200 cells/μL for 6 months in response to cART.

1	Amphotericin is followed by fluconazole 400 mg/d PO for 8 weeks, and then fluconazole 200 mg/d until the CD4+ T cell count has increased to >200 cells/μL for 6 months in response to cART. Repeated lumbar puncture may be required to manage increased intracranial pressure. Symptoms may recur with initiation of cART as an immune reconstitution syndrome (see above). Other fungi that may cause meningitis in patients with HIV infection are C. immitis and H. capsulatum. Meningoencephalitis has also been reported due to Acanthamoeba or Naegleria. HIV-associated dementia consists of a constellation of signs and symptoms of CNS disease. While this is generally a late complication of HIV infection that progresses slowly over months, it can be seen in patients with CD4+ T cell counts >350 cells/μL. A major feature of this entity is the development of dementia, defined as a decline in cognitive ability from a previous level. It may present as impaired ability to concentrate, increased

1	cells/μL. A major feature of this entity is the development of dementia, defined as a decline in cognitive ability from a previous level. It may present as impaired ability to concentrate, increased forgetfulness, difficulty reading, or increased difficulty performing complex tasks. Initially these symptoms may be indistinguishable from findings of situational depression or fatigue. In contrast to “cortical” dementia (such as Alzheimer’s disease), aphasia, apraxia, and agnosia are uncommon, leading some investigators to classify HIV encephalopathy as a “subcortical dementia” characterized by defects in short-term memory and executive function (see below). In addition to dementia, patients with HIV encephalopathy may also have motor and behavioral abnormalities. Among the motor problems are unsteady gait, poor balance, tremor, and difficulty with rapid alternating movements. Increased tone and deep tendon reflexes may be found in patients with spinal cord involvement. Late stages may

1	unsteady gait, poor balance, tremor, and difficulty with rapid alternating movements. Increased tone and deep tendon reflexes may be found in patients with spinal cord involvement. Late stages may be complicated by bowel and/or bladder incontinence. Behavioral problems include apathy, irritability, and lack of initiative, with progression to a vegetative state in some instances. Some patients develop a state of agitation or mild mania. These changes usually occur without significant changes in level of alertness. This is in contrast to the finding of somnolence in patients with dementia due to toxic/metabolic encephalopathies.

1	HIV-associated dementia is the initial AIDS-defining illness in ~3% of patients with HIV infection and thus only rarely precedes clinical evidence of immunodeficiency. Clinically significant encephalopathy eventually develops in ~25% of untreated patients with AIDS. As immunologic function declines, the risk and severity of HIV-associated dementia increases. Autopsy series suggest that 80–90% of patients with HIV infection have histologic evidence of CNS involvement. Several classification schemes have been developed for grading HIV encephalopathy; a commonly used clinical staging system is outlined in Table 226-15.

1	The precise cause of HIV-associated dementia remains unclear, although the condition is thought to be a result of a combination of direct effects of HIV on the CNS and associated immune activation. HIV has been found in the brains of patients with HIV encephalopathy by Southern blot, in situ hybridization, PCR, and electron microscopy. Multinucleated giant cells, macrophages, and microglial cells appear to be the main cell types harboring virus in the CNS. Histologically, the major changes are seen in the subcortical areas of the brain and include pallor and gliosis, multinucleated giant cell encephalitis, and vacuolar myelopathy. Less commonly, diffuse or focal spongiform changes occur in the white matter. Areas of the brain involved in motor function, language, and judgment are most severely affected.

1	There are no specific criteria for a diagnosis of HIV-associated dementia, and this syndrome must be differentiated from a number of other diseases that affect the CNS of HIV-infected patients (Table 226-14). The diagnosis of dementia depends on demonstrating a decline in Asymptomatic 1 SD below mean in No impairments in 2 cognitive domains activities of daily living Mild neurocognitive 1 SD below mean in Impairments in activities disorder 2 cognitive domains of daily living

1	Mild neurocognitive 1 SD below mean in Impairments in activities disorder 2 cognitive domains of daily living HIV-associated 2 SD below mean in Notable impairments in dementia 2 cognitive domains activities of daily living aNeurocognitive testing should include assessment of at least 5 domains, including attention-information processing, language, abstraction-executive, complex perceptual motor skills, memory (including learning and recall), simple motor skills, or sensory perceptual skills. Appropriate norms must be available to establish the number of domains in which performance is below 1 SD. bFunctional status is typically assessed by self-reporting but might be corroborated by a collateral source. No agreed measures exist for HIV-associated neurocognitive disorder criteria. Note that, for diagnosis of HIV-associated neurocognitive disorder, other causes of dementia must be ruled out and potential confounding effects of substance use or psychiatric illness should be considered.

1	Source: Adapted from A Antinori et al: Neurology 69:1789, 2007. FIGuRE 226-39 AIDS dementia complex. Postcontrast CT scan through the lateral ventricles of a 47-year-old man with AIDS, altered mental status, and dementia. The lateral and third ventricles and the cerebral sulci are abnormally prominent. Mild white matter hypodensity is seen adjacent to the frontal horns of the lateral ventricles.

1	cognitive function. This can be accomplished objectively with the use of a Mini-Mental Status Examination (MMSE) in patients for whom prior scores are available. For this reason, it is advisable for all patients with a diagnosis of HIV infection to have a baseline MMSE. However, changes in MMSE scores may be absent in patients with mild HIV encephalopathy. Imaging studies of the CNS, by either MRI or CT, often demonstrate evidence of cerebral atrophy (Fig. 226-39). MRI may also reveal small areas of increased density on T2-weighted images. Lumbar puncture is an important element of the evaluation of patients with HIV infection and neurologic abnormalities. It is generally most helpful in ruling out or making a diagnosis of opportunistic infections. In HIV encephalopathy, patients may have the nonspecific findings of an increase in CSF cells and protein level. While HIV RNA can often be detected in the spinal fluid and HIV can be cultured from the CSF, this finding is not specific for

1	the nonspecific findings of an increase in CSF cells and protein level. While HIV RNA can often be detected in the spinal fluid and HIV can be cultured from the CSF, this finding is not specific for HIV encephalopathy. There appears to be no correlation between the presence of HIV in the CSF and the presence of HIV encephalopathy. Elevated levels of macrophage chemoattractant protein (MCP-1), β2-microglobulin, neopterin, and quinolinic acid (a metabolite of tryptophan reported to cause CNS injury) have been noted in the CSF of patients with HIV encephalopathy. These findings suggest that these factors as well as inflammatory cytokines may be involved in the pathogenesis of this syndrome.

1	Combination antiretroviral therapy is of benefit in patients with HIV-associated dementia. Improvement in neuropsychiatric test scores has been noted for both adult and pediatric patients treated with antiretrovirals. The rapid improvement in cognitive function noted with the initiation of cART suggests that at least some component of this problem is quickly reversible, again supporting at least a partial role of soluble mediators in the pathogenesis. It should also be noted that these patients have an increased sensitivity to the side effects of neuroleptic drugs. The use of these drugs for symptomatic treatment is associated with an increased risk of extrapyramidal side effects; therefore, patients with HIV encephalopathy who receive these agents must be monitored carefully. It is felt by many physicians that the decrease in the prevalence of severe cases of HAND brought about by cART has resulted in an increase in the prevalence of milder forms of this disorder.

1	Seizures may be a consequence of opportunistic infections, neoplasms, or HIV encephalopathy (Table 226-16). The seizure threshold is often lower than normal in patients with advanced HIV infection due in part to the frequent presence of electrolyte abnormalities. Seizures are seen in 15–40% of patients with cerebral toxoplasmosis,

1	Overall Contribution Fraction of Patients Disease to First Seizure, % Who Have Seizures, % 15–35% of patients with primary CNS lymphoma, 8% of patients with cryptococcal meningitis, and 7–50% of patients with HIV encephalopathy. Seizures may also be seen in patients with CNS tuberculosis, aseptic meningitis, and progressive multifocal leukoencephalopathy. Seizures may be the presenting clinical symptom of HIV disease. In one study of 100 patients with HIV infection presenting with a first seizure, cerebral mass lesions were the most common cause, responsible for 32 of the 100 new-onset seizures. Of these 32 cases, 28 were due to toxoplasmosis and 4 to lymphoma. HIV encephalopathy accounted for an additional 24 new-onset seizures. Cryptococcal meningitis was the third most common diagnosis, responsible for 13 of the 100 seizures. In 23 cases, no cause could be found, and it is possible that these cases represent a subcategory of HIV encephalopathy. Of these 23 cases, 16 (70%) had 2 or

1	responsible for 13 of the 100 seizures. In 23 cases, no cause could be found, and it is possible that these cases represent a subcategory of HIV encephalopathy. Of these 23 cases, 16 (70%) had 2 or more seizures, suggesting that anticonvulsant therapy is indicated in all patients with HIV infection and seizures unless a rapidly correctable cause is found. While phenytoin remains the initial treatment of choice, hypersensitivity reactions to this drug have been reported in >10% of patients with AIDS, and therefore the use of phenobarbital or valproic acid should be considered as alternatives. Due to a variety of drug-drug interactions between antiseizure medications and antiretrovirals, drug levels need to be monitored carefully.

1	Patients with HIV infection may present with focal neurologic deficits from a variety of causes. The most common causes are toxoplasmosis, progressive multifocal leukoencephalopathy, and CNS lymphoma. Other causes include cryptococcal infections (discussed above; also Chap. 239), stroke, and reactivation of Chagas’ disease.

1	Toxoplasmosis has been one of the most common causes of secondary CNS infections in patients with AIDS, but its incidence is decreasing in the era of cART. It is most common in patients from the Caribbean and from France, where the seroprevalence of T. gondii is around 50%. This figure is closer to 15% in the United States. Toxoplasmosis is generally a late complication of HIV infection and usually occurs in patients with CD4+ T cell counts <200/μL. Cerebral toxoplasmosis is thought to represent a reactivation of latent tissue cysts. It is 10 times more common in patients with antibodies to the organism than in patients who are seronegative. Patients diagnosed with HIV infection should be screened for IgG antibodies to T. gondii during the time of their initial workup. Those who are seronegative should be counseled about ways to minimize the risk of primary infection including avoiding the consumption of undercooked meat and careful hand washing after contact with soil or changing the

1	should be counseled about ways to minimize the risk of primary infection including avoiding the consumption of undercooked meat and careful hand washing after contact with soil or changing the cat litter box. The most common clinical presentation of cerebral toxoplasmosis in patients with HIV infection is fever, headache, and focal neurologic deficits. Patients may present with seizure, hemiparesis, or aphasia as a manifestation of these focal deficits or with a picture more influenced by the accompanying cerebral edema and characterized by confusion, dementia, and lethargy, which can progress to coma. The diagnosis is usually suspected on the basis of MRI findings of multiple lesions in multiple locations, although in some cases only a single lesion is seen. Pathologically, these lesions generally exhibit inflammation and central necrosis and, as a result, demonstrate ring enhancement on contrast MRI (Fig. 226-40) or, if MRI is unavailable or contraindicated, on double-dose contrast

1	generally exhibit inflammation and central necrosis and, as a result, demonstrate ring enhancement on contrast MRI (Fig. 226-40) or, if MRI is unavailable or contraindicated, on double-dose contrast CT. There is usually evidence of surrounding edema. In addition to toxoplasmosis, the differential diagnosis of single or multiple enhancing mass lesions in

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders FIGuRE 226-40 Central nervous system toxoplasmosis. A coronal postcontrast T1-weighted MRI scan demonstrates a peripheral enhancing lesion in the left frontal lobe, associated with an eccentric nodular area of enhancement (arrow); this so-called eccentric target sign is typical of toxoplasmosis.

1	the HIV-infected patient includes primary CNS lymphoma and, less commonly, TB or fungal or bacterial abscesses. The definitive diagnostic procedure is brain biopsy. However, given the morbidity rate that can accompany this procedure, it is usually reserved for the patient who has failed 2–4 weeks of empiric therapy for toxoplasmosis. If the patient is seronegative for T. gondii, the likelihood that a mass lesion is due to toxoplasmosis is <10%. In that setting, one may choose to be more aggressive and perform a brain biopsy sooner. Standard treatment is sulfadiazine and pyrimethamine with leucovorin as needed for a minimum of 4–6 weeks. Alternative therapeutic regimens include clindamycin in combination with pyrimethamine; atovaquone plus pyrimethamine; and azithromycin plus pyrimethamine plus rifabutin. Relapses are common, and it is recommended that patients with a history of prior toxoplasmic encephalitis receive maintenance therapy with sulfadiazine, pyrimethamine, and leucovorin

1	plus rifabutin. Relapses are common, and it is recommended that patients with a history of prior toxoplasmic encephalitis receive maintenance therapy with sulfadiazine, pyrimethamine, and leucovorin as long as their CD4+ T cell counts remain <200 cells/μL. Patients with CD4+ T cell counts <100/μL and IgG antibody to Toxoplasma should receive primary prophylaxis for toxoplasmosis. Fortunately, the same daily regimen of a single double-strength tablet of TMP/SMX used for P. jiroveci prophylaxis provides adequate primary protection against toxoplasmosis. Secondary prophylaxis/maintenance therapy for toxoplasmosis may be discontinued in the setting of effective cART and increases in CD4+ T cell counts to >200/μL for 6 months.

1	JC virus, a human polyomavirus that is the etiologic agent of progressive multifocal leukoencephalopathy (PML), is an important opportunistic pathogen in patients with AIDS (Chap. 164). While ~80% of the general adult population has antibodies to JC virus, indicative of prior infection, <10% of healthy adults show any evidence of ongoing viral replication. PML is the only known clinical manifestation of JC virus infection. It is a late manifestation of AIDS and is seen in ~1–4% of patients with AIDS. The lesions of PML begin as small foci of demyelination in subcortical white matter that eventually coalesce. The cerebral hemispheres, cerebellum, and brainstem may all be involved. Patients typically have a protracted course with multifocal neurologic deficits, with or without changes in mental status. Approximately 20% of patients experience seizures. Ataxia, hemiparesis, visual field defects, aphasia, and sensory defects may occur. Headache, fever, nausea, and vomiting are rarely

1	mental status. Approximately 20% of patients experience seizures. Ataxia, hemiparesis, visual field defects, aphasia, and sensory defects may occur. Headache, fever, nausea, and vomiting are rarely seen. Their presence should suggest another diagnosis. MRI typically reveals multiple, nonenhancing white matter lesions that may coalesce and have a predilection for the occipital and parietal lobes. The lesions show signal hyperintensity on T2-weighted images and diminished signal on T1-weighted images. The measurement of JC virus DNA levels in CSF has a diagnostic sensitivity of 76% and a specificity of close to 100%. Prior to the availability of cART, the majority of patients with PML died within 3–6 months of the onset of symptoms. Paradoxical worsening of PML has been seen with initiation of cART as an immune reconstitution syndrome. There is no specific treatment for PML; however, a median survival of 2 years and survival of >15 years have been reported in patients with PML treated

1	of cART as an immune reconstitution syndrome. There is no specific treatment for PML; however, a median survival of 2 years and survival of >15 years have been reported in patients with PML treated with cART for their HIV disease. Despite having a significant impact on survival, only ~50% of patients with HIV infection and PML show neurologic improvement with cART. Studies with other antiviral agents such as cidofovir have failed to show clear benefit. Factors influencing a favorable prognosis for PML in the setting of HIV infection include a CD4+ T cell count >100/μL at baseline and the ability to maintain an HIV viral load of <500 copies/mL. Baseline HIV-1 viral load does not have independent predictive value of survival. PML is one of the few opportunistic infections that continues to occur with some frequency despite the widespread use of cART.

1	Reactivation American trypanosomiasis may present as acute meningoencephalitis with focal neurologic signs, fever, headache, vomiting, and seizures. Accompanying cardiac disease in the form of arrhythmias or heart failure should increase the index of suspicion. The presence of antibodies to T. cruzi supports the diagnosis. In South America, reactivation of Chagas’ disease is considered to be an AIDS-defining condition and may be the initial AIDS-defining condition. The majority of cases occur in patients with CD4+ T cell counts <200 cells/μL. Lesions appear radiographically as single or multiple hypodense areas, typically with ring enhancement and edema. They are found predominantly in the subcortical areas, a feature that differentiates them from the deeper lesions of toxoplasmosis. T. cruzi amastigotes, or trypanosomes, can be identified from biopsy specimens or CSF. Other CSF findings include elevated protein and a mild (<100 cells/μL) lymphocytic pleocytosis. Organisms can also be

1	amastigotes, or trypanosomes, can be identified from biopsy specimens or CSF. Other CSF findings include elevated protein and a mild (<100 cells/μL) lymphocytic pleocytosis. Organisms can also be identified by direct examination of the blood. Treatment consists of benzimidazole (2.5 mg/kg bid) or nifurtimox (2 mg/kg qid) for at least 60 days, followed by maintenance therapy for the duration of immunodeficiency with either drug at a dose of 5 mg/kg three times a week. As is the case with cerebral toxoplasmosis, successful therapy with antiretrovirals may allow discontinuation of therapy for Chagas’ disease.

1	Stroke may occur in patients with HIV infection. In contrast to the other causes of focal neurologic deficits in patients with HIV infection, the symptoms of a stroke are sudden in onset. Patients with HIV infection have an increased prevalence of many classic risk factors associated with stroke, including smoking and diabetes. It has been reported that HIV infection itself can lead to an increase in carotid artery stiffness. The relative increase in risk for stroke as a consequence of HIV infection is more pronounced in women and in individuals between the ages of 18 and 29. Among the secondary infectious diseases in patients with HIV infection that may be associated with stroke are vasculitis due to cerebral varicella zoster or neurosyphilis and septic embolism in association with fungal infection. Other elements of the differential diagnosis of stroke in the patient with HIV infection include atherosclerotic cerebral vascular disease, thrombotic thrombocytopenic purpura, and

1	fungal infection. Other elements of the differential diagnosis of stroke in the patient with HIV infection include atherosclerotic cerebral vascular disease, thrombotic thrombocytopenic purpura, and cocaine or amphetamine use.

1	Primary CNS lymphoma is discussed below in the section on neoplastic diseases.

1	Spinal cord disease, or myelopathy, is present in ~20% of patients with AIDS, often as part of HIV-associated neurocognitive disorder. In fact, 90% of the patients with HIV-associated myelopathy have some evidence of dementia, suggesting that similar pathologic processes may be responsible for both conditions. Three main types of spinal cord disease are seen in patients with AIDS. The first of these is a vacuolar myelopathy, as mentioned above. This condition is pathologically similar to subacute combined degeneration of the cord, such as that occurring with pernicious anemia. Although vitamin B12 deficiency can be seen in patients with AIDS as a primary complication of HIV infection, it does not appear to be responsible for the myelopathy seen in the majority of patients. Vacuolar myelopathy is characterized by a subacute onset and often presents with gait disturbances, predominantly ataxia and spasticity; it may progress to include bladder and bowel dysfunction. Physical findings

1	is characterized by a subacute onset and often presents with gait disturbances, predominantly ataxia and spasticity; it may progress to include bladder and bowel dysfunction. Physical findings include evidence of increased deep tendon reflexes and extensor plantar responses. The second form of spinal cord disease involves the dorsal columns and presents as a pure sensory ataxia. The third form is also sensory in nature and presents with paresthesias and dysesthesias of the lower extremities. In contrast to the cognitive problems seen in patients with HIV encephalopathy, these spinal cord syndromes do not respond well to antiretroviral drugs, and therapy is mainly supportive.

1	One important disease of the spinal cord that also involves the peripheral nerves is a myelopathy and polyradiculopathy seen in association with CMV infection. This entity is generally seen late in the course of HIV infection and is fulminant in onset, with lower extremity and sacral paresthesias, difficulty in walking, areflexia, ascending sensory loss, and urinary retention. The clinical course is rapidly progressive over a period of weeks. CSF examination reveals a predominantly neutrophilic pleocytosis, and CMV DNA can be detected by CSF PCR. Therapy with ganciclovir or foscarnet can lead to rapid improvement, and prompt initiation of foscarnet or ganciclovir therapy is important in minimizing the degree of permanent neurologic damage. Combination therapy with both drugs should be considered in patients who have been previously treated for CMV disease. Other diseases involving the spinal cord in patients with HIV infection include HTLV-1-associated myelopathy (HAM) (Chap. 225e),

1	in patients who have been previously treated for CMV disease. Other diseases involving the spinal cord in patients with HIV infection include HTLV-1-associated myelopathy (HAM) (Chap. 225e), neurosyphilis (Chap. 206), infection with herpes simplex (Chap. 216) or varicella-zoster (Chap. 217), TB (Chap. 202), and lymphoma (Chap. 134).

1	Peripheral neuropathies are common in patients with HIV infection. They occur at all stages of illness and take a variety of forms. Early in the course of HIV infection, an acute inflammatory demyelinating polyneuropathy resembling Guillain-Barré syndrome may occur (Chap. 460). In other patients, a progressive or relapsing-remitting inflammatory neuropathy resembling chronic inflammatory demyelinating polyneuropathy (CIDP) has been noted. Patients commonly present with progressive weakness, areflexia, and minimal sensory changes. CSF examination often reveals a mononuclear pleocytosis, and peripheral nerve biopsy demonstrates a perivascular infiltrate suggesting an autoimmune etiology. Plasma exchange or IVIg has been tried with variable success. Because of the immunosuppressive effects of glucocorticoids, they should be reserved for severe cases of CIDP refractory to other measures. Another autoimmune peripheral neuropathy seen in patients with AIDS is mononeuritis multiplex (Chaps.

1	glucocorticoids, they should be reserved for severe cases of CIDP refractory to other measures. Another autoimmune peripheral neuropathy seen in patients with AIDS is mononeuritis multiplex (Chaps. 460 and 385) due to a necrotizing arteritis of peripheral nerves. The most common peripheral neuropathy in patients with HIV infection is a distal sensory polyneuropathy (DSPN) also referred to as painful sensory neuropathy (HIV-SN), predominantly sensory neuropathy, or distal symmetric peripheral neuropathy. This condition may be a direct consequence of HIV infection or a side effect of dideoxynucleoside therapy. It is more common in taller individuals, older individuals, and those with lower CD4 counts. Two-thirds of patients with AIDS may be shown by electrophysiologic studies to have some evidence of peripheral nerve disease. Presenting symptoms are usually painful burning sensations in the feet and lower extremities. Findings on examination include a stocking-type sensory loss to

1	evidence of peripheral nerve disease. Presenting symptoms are usually painful burning sensations in the feet and lower extremities. Findings on examination include a stocking-type sensory loss to pinprick, temperature, and touch sensation and a loss of ankle reflexes. Motor changes are mild and are usually limited to weakness of the intrinsic foot muscles. Response of this condition to antiretrovirals has been variable, perhaps because antiretrovirals are responsible for the problem in some instances. When due to dideoxynucleoside therapy, patients with lower extremity peripheral neuropathy may complain of a sensation that they are walking on ice. Other entities in the differential diagnosis of peripheral neuropathy include diabetes mellitus, vitamin B12 deficiency, and side effects from metronidazole or dapsone. For distal symmetric polyneuropathy that fails to resolve following the discontinuation of dideoxynucleosides, therapy is symptomatic; gabapentin, carbamazepine, tricyclics,

1	metronidazole or dapsone. For distal symmetric polyneuropathy that fails to resolve following the discontinuation of dideoxynucleosides, therapy is symptomatic; gabapentin, carbamazepine, tricyclics, or analgesics may be effective for dysesthesias. Treatment-naïve patients may respond to cART.

1	Myopathy may complicate the course of HIV infection; causes include HIV infection itself, zidovudine, and the generalized wasting syndrome. HIV-associated myopathy may range in severity from an asymptomatic elevation in creatine kinase levels to a subacute syndrome characterized by proximal muscle weakness and myalgias. Quite pronounced elevations in creatine kinase may occur in asymptomatic patients, particularly after exercise. The clinical significance of this 1267 as an isolated laboratory finding is unclear. A variety of both inflammatory and noninflammatory pathologic processes have been noted in patients with more severe myopathy, including myofiber necrosis with inflammatory cells, nemaline rod bodies, cytoplasmic bodies, and mitochondrial abnormalities. Profound muscle wasting, often with muscle pain, may be seen after prolonged zidovudine therapy. This toxic side effect of the drug is dose-dependent and is related to its ability to interfere with the function of

1	wasting, often with muscle pain, may be seen after prolonged zidovudine therapy. This toxic side effect of the drug is dose-dependent and is related to its ability to interfere with the function of mitochondrial polymerases. It is reversible following discontinuation of the drug. Red ragged fibers are a histologic hallmark of zidovudine-induced myopathy.

1	Ophthalmologic Diseases Ophthalmologic problems occur in ~50% of patients with advanced HIV infection. The most common abnormal findings on funduscopic examination are cotton-wool spots. These are hard white spots that appear on the surface of the retina and often have an irregular edge. They represent areas of retinal ischemia secondary to microvascular disease. At times they are associated with small areas of hemorrhage and thus can be difficult to distinguish from CMV retinitis. In contrast to CMV retinitis, however, these lesions are not associated with visual loss and tend to remain stable or improve over time.

1	One of the most devastating consequences of HIV infection is CMV retinitis. Patients at high risk of CMV retinitis (CD4+ T cell count <100/μL) should undergo an ophthalmologic examination every 3–6 months. The majority of cases of CMV retinitis occur in patients with a CD4+ T cell count <50/μL. Prior to the availability of cART, this CMV reactivation syndrome was seen in 25–30% of patients with

1	AIDS. In the cART era this has dropped to close to 2%. CMV retinitis usually presents as a painless, progressive loss of vision. Patients may also complain of blurred vision, “floaters,” and scintillations. The disease is usually bilateral, although typically it affects one eye more than the other. The diagnosis is made on clinical grounds by an experienced ophthalmologist. The characteristic retinal appearance is that of perivascular hemorrhage and exudate. In situations where the diagnosis is in doubt due to an atypical presentation or an unexpected lack of response to therapy, vitreous or aqueous humor sampling with molecular diagnostic techniques may be of value. CMV infection of the retina results in a necrotic inflammatory process, and the visual loss that develops is irreversible. CMV retinitis may be complicated by rhegmatogenous retinal detachment as a consequence of retinal atrophy in areas of prior inflammation. Therapy for CMV retinitis consists of oral valganciclovir, IV

1	CMV retinitis may be complicated by rhegmatogenous retinal detachment as a consequence of retinal atrophy in areas of prior inflammation. Therapy for CMV retinitis consists of oral valganciclovir, IV ganciclovir, or IV foscarnet, with cidofovir as an alternative. Combination therapy with ganciclovir and foscarnet has been shown to be slightly more effective than either ganciclovir or foscarnet alone in the patient with relapsed CMV retinitis. A 3-week induction course is followed by maintenance therapy with oral valganciclovir. If CMV disease is limited to the eye, intravitreal injections of ganciclovir or foscarnet may be considered. Intravitreal injections of cidofovir are generally avoided due to the increased risk of uveitis and hypotony. Maintenance therapy is continued until the CD4+ T cell count remains >100 μL for >6 months. The majority of patients with HIV infection and CMV disease develop some degree of uveitis with the initiation of cART. The etiology of this is unknown;

1	T cell count remains >100 μL for >6 months. The majority of patients with HIV infection and CMV disease develop some degree of uveitis with the initiation of cART. The etiology of this is unknown; however, it has been suggested that this may be due to the generation of an enhanced immune response to CMV as an IRIS (see above). In some instances this has required the use of topical glucocorticoids.

1	Both HSV and varicella zoster virus can cause a rapidly progressing, bilateral necrotizing retinitis referred to as the acute retinal necrosis syndrome, or progressive outer retinal necrosis (PORN). This syndrome, in contrast to CMV retinitis, is associated with pain, keratitis, and iritis. It is often associated with orolabial HSV or trigeminal zoster. Ophthalmologic examination reveals widespread pale gray peripheral lesions. This condition is often complicated by retinal detachment. It is important to recognize and treat this condition with IV acyclovir as quickly as possible to minimize the loss of vision. Several other secondary infections may cause ocular problems in HIV-infected patients. P. jiroveci can cause a lesion of the choroid that may be detected as an incidental finding on ophthalmologic examination. These lesions are typically bilateral, are from half to twice the disc

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1268 diameter in size, and appear as slightly elevated yellow-white plaques. They are usually asymptomatic and may be confused with cotton-wool spots. Chorioretinitis due to toxoplasmosis can be seen alone or, more commonly, in association with CNS toxoplasmosis. KS may involve the eyelid or conjunctiva, while lymphoma may involve the retina. Syphilis may lead to a uveitis that is highly associated with the presence of neurosyphilis.

1	Additional Disseminated Infections and Wasting Syndrome Infections with species of the small, gram-negative, Rickettsia-like organism Bartonella (Chap. 197) are seen with increased frequency in patients with HIV infection. While it is not considered an AIDS-defining illness by the CDC, many experts view infection with Bartonella as indicative of a severe defect in cell-mediated immunity. It is usually seen in patients with CD4+ T cell counts <100/μL and is a significant cause of unexplained fever in patients with advanced HIV infection. Among the clinical manifestations of Bartonella infection are bacillary angiomatosis, cat-scratch disease, and trench fever. Bacillary angiomatosis is usually due to infection with B. henselae and is linked to exposure to flea-infested cats. It is characterized by a vascular proliferation that leads to a variety of skin lesions that have been confused with the skin lesions of KS. In contrast to the lesions of KS, the lesions of bacillary angiomatosis

1	by a vascular proliferation that leads to a variety of skin lesions that have been confused with the skin lesions of KS. In contrast to the lesions of KS, the lesions of bacillary angiomatosis generally blanch, are painful, and typically occur in the setting of systemic symptoms. Infection can extend to the lymph nodes, liver (peliosis hepatis), spleen, bone, heart, CNS, respiratory tract, and GI tract. Cat-scratch disease also is due to B. henselae and generally begins with a papule at the site of inoculation. This is followed several weeks later by the development of regional adenopathy and malaise. Infection with B. quintana is transmitted by lice and has been associated with case reports of trench fever, endocarditis, adenopathy, and bacillary angiomatosis. The organism is quite difficult to culture, and diagnosis often relies on identifying the organism in biopsy specimens using the Warthin-Starry or similar stains. Treatment is with either doxycycline or erythromycin for at

1	to culture, and diagnosis often relies on identifying the organism in biopsy specimens using the Warthin-Starry or similar stains. Treatment is with either doxycycline or erythromycin for at least 3 months.

1	Histoplasmosis is an opportunistic infection that is seen most frequently in patients in the Mississippi and Ohio River valleys, Puerto Rico, the Dominican Republic, and South America. These are all areas in which infection with H. capsulatum is endemic (Chap. 236). Because of this limited geographic distribution, the percentage of AIDS cases in the United States with histoplasmosis is only ~0.5. Histoplasmosis is generally a late manifestation of HIV infection; however, it may be the initial AIDS-defining condition. In one study, the median CD4+ T cell count for patients with histoplasmosis and AIDS was 33/μL. While disease due to H. capsulatum may present as a primary infection of the lung, disseminated disease, presumably due to reactivation, is the most common presentation in HIV-infected patients. Patients usually present with a 4to 8-week history of fever and weight loss. Hepatosplenomegaly and lymphadenopathy are each seen in about 25% of patients. CNS disease, either

1	HIV-infected patients. Patients usually present with a 4to 8-week history of fever and weight loss. Hepatosplenomegaly and lymphadenopathy are each seen in about 25% of patients. CNS disease, either meningitis or a mass lesion, is seen in 15% of patients. Bone marrow involvement is common, with thrombocytopenia, neutropenia, and anemia occurring in 33% of patients. Approximately 7% of patients have mucocutaneous lesions consisting of a maculopapular rash and skin or oral ulcers. Respiratory symptoms are usually mild, with chest x-ray showing a diffuse infiltrate or diffuse small nodules in ~50% of cases. The gastrointestinal tract may be involved. Diagnosis is made by silver staining of tissue, by culturing the organisms from blood, bone marrow, or tissue, or by detecting antigen in blood or urine. Treatment is typically with liposomal amphotericin B followed by maintenance therapy with oral itraconazole until the serum histoplasma antigen is <2 units, the patient has been on

1	in blood or urine. Treatment is typically with liposomal amphotericin B followed by maintenance therapy with oral itraconazole until the serum histoplasma antigen is <2 units, the patient has been on antiretrovirals for at least 6 months, and the CD4 count is >150 cells/μL. In the setting of mild infection, it may be appropriate to initiate therapy with itraconazole alone.

1	Following the spread of HIV infection to southeast Asia, disseminated infection with the fungus Penicillium marneffei was recognized as a complication of HIV infection and is considered an AIDS-defining condition in those parts of the world where it occurs. P. marneffei is the third most common AIDS-defining illness in Thailand, following TB and cryptococcosis. It is more frequently diagnosed in the rainy than the dry season. Clinical features include fever, generalized lymphadenopathy, hepatosplenomegaly, anemia, thrombocytopenia, and papular skin lesions with central umbilication. Treatment is with amphotericin B followed by itraconazole until the CD4+ T cell count is >100 cells/μL for at least 6 months.

1	Visceral leishmaniasis (Chap. 251) is recognized with increasing frequency in patients with HIV infection who live in or travel to areas endemic for this protozoal infection transmitted by sandflies. The clinical presentation is one of hepatosplenomegaly, fever, and hematologic abnormalities. Lymphadenopathy and other constitutional symptoms may be present. A chronic, relapsing course is seen in two-thirds of co-infected patients. Organisms can be isolated from cultures of bone marrow aspirates. Histologic stains may be negative, and antibody titers are of little help. Patients with HIV infection usually respond well initially to standard therapy with amphotericin B or pentavalent antimony compounds. Eradication of the organism is difficult, however, and relapses are common.

1	Patients with HIV infection are at a slightly increased risk of clinical malaria. This is particularly true for patients from nonendemic areas with presumed primary infection and in patients with lower CD4+ T cell counts. HIV-positive individuals with CD4+ T cell counts <300 cells/μL have a poorer response to malaria treatment than others. Co-infection with malaria is associated with a modest increase in HIV viral load. The risk of malaria may be decreased with TMP/SMX prophylaxis.

1	Generalized wasting is an AIDS-defining condition; it is defined as involuntary weight loss of >10% associated with intermittent or constant fever and chronic diarrhea or fatigue lasting >30 days in the absence of a defined cause other than HIV infection. Prior to the widespread use of cART it was the initial AIDS-defining condition in ~10% of patients with AIDS in the United States and is an indication for initiation of cART. Generalized wasting is rarely seen today with the earlier initiation of antiretrovirals. A constant feature of this syndrome is severe muscle wasting with scattered myofiber degeneration and occasional evidence of myositis. Glucocorticoids may be of some benefit; however, this approach must be carefully weighed against the risk of compounding the immunodeficiency of HIV infection. Androgenic steroids, growth hormone, and total parenteral nutrition have been used as therapeutic interventions with variable success.

1	Neoplastic Diseases The neoplastic diseases considered to be AIDS-defining conditions are Kaposi’s sarcoma, non-Hodgkin’s lymphoma, and invasive cervical carcinoma. In addition, there is also an increase in the incidence of a variety of non-AIDS-defining malignancies including Hodgkin’s disease; multiple myeloma; leukemia; melanoma; and cervical, brain, testicular, oral, lung, gastric, liver, renal, and anal cancers. Since the introduction of potent cART, there has been a marked reduction in the incidence of KS (Fig. 226-33) and CNS lymphoma, such that the non-AIDS-defining malignancies now account for more morbidity and mortality in patients with HIV infection than the AIDS-defining malignancies. Rates of non-Hodgkin’s lymphoma have declined as well; however, this decline has not been as dramatic as the decline in rates of KS. In contrast, cART has had little effect on human papillomavirus (HPV)-associated malignancies. As patients with HIV infection live longer, a wider array of

1	as dramatic as the decline in rates of KS. In contrast, cART has had little effect on human papillomavirus (HPV)-associated malignancies. As patients with HIV infection live longer, a wider array of cancers is seen in this population. While some may only reflect known risk factors (e.g., smoking, alcohol consumption, co-infection with other viruses such as hepatitis B) that are increased in patients with HIV infection, some may be a direct consequence of HIV and are clearly increased in patients with lower CD4+ T cell counts.

1	Kaposi’s sarcoma is a multicentric neoplasm consisting of multiple vascular nodules appearing in the skin, mucous membranes, and viscera. The course ranges from indolent, with only minor skin or lymph node involvement, to fulminant, with extensive cutaneous and visceral involvement. In the initial period of the AIDS epidemic, KS was a prominent clinical feature of the first cases of AIDS, occurring in 79% of the patients diagnosed in 1981. By 1989 it was seen in only 25% of cases, by 1992 the number had decreased to 9%, and by 1997 the number was <1%. HHV-8 (KSHV) has been strongly implicated as a viral cofactor in the pathogenesis of KS. Clinically, KS has varied presentations and may be seen at any stage of HIV infection, even in the presence of a normal CD4+ T cell FIGuRE 226-41 Kaposi’s sarcoma in three patients with AIDS demonstrating (A) periorbital edema and bruising; (B) classic truncal distribu-tion of lesions; and (C) upper extremity lesions.

1	count. The initial lesion may be a small, raised reddish-purple nodule on the skin (Fig. 226-41), a discoloration on the oral mucosa (Fig. 226-34D), or a swollen lymph node. Lesions often appear in sun-exposed areas, particularly the tip of the nose, and have a propensity to occur in areas of trauma (Koebner phenomenon). Because of the vascular nature of the tumors and the presence of extravasated red blood cells in the lesions, their colors range from reddish to purple to brown and often take the appearance of a bruise, with yellowish discoloration and tattooing. Lesions range in size from a few millimeters to several centimeters in diameter and may be either discrete or confluent. KS lesions most commonly appear as raised macules; however, they can also be papular, particularly in patients with higher CD4+ T cell counts. Confluent lesions may give rise to surrounding lymphedema and may be disfiguring when they involve the face and disabling when they involve the lower extremities or

1	with higher CD4+ T cell counts. Confluent lesions may give rise to surrounding lymphedema and may be disfiguring when they involve the face and disabling when they involve the lower extremities or the surfaces of joints. Apart from skin, the lymph nodes, GI tract, and lung are the organ systems most commonly affected by KS. Lesions have been reported in virtually every organ, including the heart and the CNS. In contrast to most malignancies, in which lymph node involvement implies metastatic spread and a poor prognosis, lymph node involvement may be seen very early in KS and is of no special clinical significance. In fact, some patients may present with disease limited to the lymph nodes. These are generally patients with relatively intact immune function and thus the patients with the best prognosis. Pulmonary involvement with KS generally presents with shortness of breath. Some 80% of patients with pulmonary KS also have cutaneous lesions. The chest x-ray characteristically shows

1	best prognosis. Pulmonary involvement with KS generally presents with shortness of breath. Some 80% of patients with pulmonary KS also have cutaneous lesions. The chest x-ray characteristically shows bilateral lower lobe infiltrates that obscure the margins of the mediastinum and diaphragm (Fig. 226-42). Pleural effusions are seen in 70% of cases of pulmonary KS, a fact that is often helpful in the differential diagnosis. GI involvement is seen in 50% of patients with KS and usually takes one of two forms: (1) mucosal involvement, which may lead to bleeding that can be severe; these patients sometimes also develop symptoms of GI obstruction if lesions become large; and (2) biliary tract involvement. KS lesions may infiltrate the gallbladder and biliary tree, leading to a clinical picture of obstructive jaundice similar to that seen with sclerosing cholangitis. Several staging systems have been proposed for KS. One in common use was developed by the National Institute of Allergy and

1	of obstructive jaundice similar to that seen with sclerosing cholangitis. Several staging systems have been proposed for KS. One in common use was developed by the National Institute of Allergy and Infectious Diseases AIDS Clinical Trials Group; it distinguishes patients on the basis of tumor extent, immunologic function, and presence or absence of systemic disease (Table 226-17).

1	FIGuRE 226-42 Chest x-ray of a patient with AIDS and pulmonary Kaposi’s sarcoma. The characteristic findings include dense bilateral lower lobe infiltrates obscuring the heart borders and pleural effusions. A diagnosis of KS is based on biopsy of a suspicious lesion. Histologically one sees a proliferation of spindle cells and endothelial cells, extravasation of red blood cells, hemosiderin-laden macrophages, and, in early cases, an inflammatory cell infiltrate. Included in the differential diagnosis are lymphoma (particularly for oral lesions), bacillary angiomatosis, and cutaneous mycobacterial infections.

1	Management of KS (Table 226-18) should be carried out in consultation with an expert since definitive treatment guidelines do not exist. In the majority of cases, effective cART will go a long way in achieving control. Antiretroviral therapy has been associated with the spontaneous regression of KS lesions. Paradoxically, it has also been associated with the initial appearance of KS as a form of IRIS. For patients in whom tumor persists or is compromising vital functions or in whom control of HIV replication is not possible, a variety of options exist. In some cases, lesions remain quite indolent, and many of these patients can be managed with no specific treatment. Fewer than 10% of AIDS patients with KS die as a consequence of their malignancy, Good Risk (Stage 0): Poor Risk (Stage 1): Parameter All of the Following Any of the Following

1	Good Risk (Stage 0): Poor Risk (Stage 1): Parameter All of the Following Any of the Following Human Immunodeficiency Virus Disease: AIDS and Related Disorders aDefined as unexplained fever, night sweats, >10% involuntary weight loss, or diarrhea persisting for more than 2 weeks. Observation and optimization of antiretroviral therapy Single or limited number of lesions

1	Combination chemotherapy with low-dose doxorubicin, bleomycin, and vinblastine (ABV) and death from secondary infections is considerably more common. Thus, whenever possible one should avoid treatment regimens that may further suppress the immune system and increase susceptibility to opportunistic infections. Treatment is indicated under two main circumstances. The first is when a single lesion or a limited number of lesions are causing significant discomfort or cosmetic problems, such as with prominent facial lesions, lesions overlying a joint, or lesions in the oropharynx that interfere with swallowing or breathing. Under these circumstances, treatment with localized radiation, intralesional vinblastine, topical 9-cis-retinoic acid, or cryotherapy may be helpful. It should be noted that patients with HIV infection are particularly sensitive to the side effects of radiation therapy. This is especially true with respect to the development of radiation-induced mucositis; doses of

1	that patients with HIV infection are particularly sensitive to the side effects of radiation therapy. This is especially true with respect to the development of radiation-induced mucositis; doses of radiation directed at mucosal surfaces, particularly in the head and neck region, should be adjusted accordingly. The use of systemic therapy, either IFN-α or chemotherapy, should be considered in patients with a large number of lesions or in patients with visceral involvement. The single most important determinant of response appears to be the CD4+ T cell count. This relationship between response rate and baseline CD4+ T cell count is particularly true for IFN-α. The response rate to IFN-α for patients with CD4+ T cell counts >600/μL is ~80%, while the response rate for patients with counts <150/μL is <10%. In contrast to the other systemic therapies, IFN-α provides an added advantage of having antiretroviral activity; thus, it may be the appropriate first choice for single-agent systemic

1	is <10%. In contrast to the other systemic therapies, IFN-α provides an added advantage of having antiretroviral activity; thus, it may be the appropriate first choice for single-agent systemic therapy for early patients with disseminated disease. A variety of chemotherapeutic agents also have been shown to have activity against KS. Four of them—liposomal daunorubicin, liposomal doxorubicin, vinblastine, and paclitaxel—have been approved by the FDA for this indication. Liposomal daunorubicin is approved as first-line therapy for patients with advanced KS. It has fewer side effects than conventional chemotherapy. In contrast, liposomal doxorubicin and paclitaxel are approved only for KS patients who have failed standard chemotherapy. Response rates vary from 23 to 88%, appear to be comparable to what had been achieved earlier with combination chemotherapy regimens, and are greatly influenced by CD4+ T cell count.

1	Lymphomas occur with an increased frequency in patients with congenital or acquired T cell immunodeficiencies (Chap. 374). AIDS is no exception; at least 6% of all patients with AIDS develop lymphoma at some time during the course of their illness. This is a 120-fold increase in incidence compared with the general population. In contrast to the situation with KS, primary CNS lymphoma, and most opportunistic infections, the incidence of AIDS-associated systemic lymphomas has not experienced a dramatic decrease as a consequence of the widespread use of effective cART. Lymphoma occurs in all risk groups, with the highest incidence in patients with hemophilia and the lowest incidence in patients from the Caribbean or Africa with heterosexually acquired infection. Lymphoma is a late manifestation of HIV infection, generally occurring in patients with CD4+ T cell counts <200/μL. As HIV disease progresses, the risk of lymphoma increases. The attack rate for lymphoma increases exponentially

1	of HIV infection, generally occurring in patients with CD4+ T cell counts <200/μL. As HIV disease progresses, the risk of lymphoma increases. The attack rate for lymphoma increases exponentially with increasing duration of HIV infection and decreasing level of immunologic function. At 3 years following a diagnosis of HIV infection, the risk of lymphoma is 0.8% per year; by 8 years after infection, it is 2.6% per year. As individuals with HIV infection live longer as a consequence of improved cART and better treatment and prophylaxis of opportunistic infections, it is anticipated that the incidence of lymphomas may increase.

1	Three main categories of lymphoma are seen in patients with HIV infection: grade III or IV immunoblastic lymphoma, Burkitt’s lymphoma, and primary CNS lymphoma. Approximately 90% of these lymphomas are B cell in phenotype; more than half contain EBV DNA. Some are associated with KSHV. These tumors may be either monoclonal or oligoclonal in nature and are probably in some way related to the pronounced polyclonal B cell activation seen in patients with AIDS.

1	Immunoblastic lymphomas account for ~60% of the cases of lymphoma in patients with AIDS. The majority of these are diffuse large B cell lymphomas (DLBCL). They are generally high grade and would have been classified as diffuse histiocytic lymphomas in earlier classification schemes. This tumor is more common in older patients, increasing in incidence from 0% in HIV-infected individuals <1 year old to >3% in those >50 years of age. Two variants of immunoblastic lymphoma that are seen primarily in HIV-infected patients are primary effusion lymphoma (PEL) and its solid variant, plasmacytic lymphoma of the oral cavity. PEL, also referred to as body cavity lymphoma, presents with lymphomatous pleural, pericardial, and/ or peritoneal effusions in the absence of discrete nodal or extranodal masses. The tumor cells do not express surface markers for B cells or T cells and are felt to represent a preplasmacytic stage of differentiation. While both HHV-8 and EBV DNA sequences have been found in

1	The tumor cells do not express surface markers for B cells or T cells and are felt to represent a preplasmacytic stage of differentiation. While both HHV-8 and EBV DNA sequences have been found in the genomes of the malignant cells from patients with body cavity lymphoma, KSHV is felt to be the driving force behind the oncogenesis (see above).

1	Small noncleaved cell lymphoma (Burkitt’s lymphoma) accounts for ~20% of the cases of lymphoma in patients with AIDS. It is most frequent in patients 10–19 years old and usually demonstrates characteristic c-myc translocations from chromosome 8 to chromosomes 14 or 22. Burkitt’s lymphoma is not commonly seen in the setting of immunodeficiency other than HIV-associated immunodeficiency, and the incidence of this particular tumor is more than 1000-fold higher in the setting of HIV infection than in the general population. In contrast to African Burkitt’s lymphoma, where 97% of the cases contain EBV genome, only 50% of HIV-associated Burkitt’s lymphomas are EBV-positive.

1	Primary CNS lymphoma accounts for ~20% of the cases of lymphoma in patients with HIV infection. In contrast to HIV-associated Burkitt’s lymphoma, primary CNS lymphomas are usually positive for EBV. In one study, the incidence of Epstein-Barr positivity was 100%. This malignancy does not have a predilection for any particular age group. The median CD4+ T cell count at the time of diagnosis is ~50/ μL. Thus, CNS lymphoma generally presents at a later stage of HIV infection than does systemic lymphoma. This may explain, at least in part, the poorer prognosis for this subset of patients.

1	The clinical presentation of lymphoma in patients with HIV infection is quite varied, ranging from focal seizures to rapidly growing mass lesions in the oral mucosa (Fig. 226-43) to persistent unexplained fever. At least 80% of patients present with extranodal disease, and a similar percentage have B-type symptoms of fever, night sweats, or weight loss. Virtually any site in the body may be involved. The most common extranodal site is the CNS, which is involved in approximately one-third of all patients with lymphoma. Approximately 60% of these cases are primary CNS lymphoma. Primary CNS lymphoma generally presents with focal neurologic deficits, including cranial nerve findings, headaches, and/or seizures. MRI or CT generally reveals a limited number (one to three) of 3to 5-cm lesions (Fig. 226-44). The lesions often show ring enhancement on contrast administration and may occur in any location. Contrast enhancement is usually less pronounced than that seen with toxoplasmosis.

1	FIGuRE 226-43 Immunoblastic lymphoma involving the hard palate of a patient with AIDS. Locations that are most commonly involved with CNS lymphoma are deep in the white matter. The main diseases in the differential diagnosis are cerebral toxoplasmosis and cerebral Chagas’ disease. In addition to the 20% of lymphomas in HIV-infected individuals that are primary CNS lymphomas, CNS disease is also seen in HIV-infected patients with systemic lymphoma. Approximately 20% of patients with systemic lymphoma have CNS disease in the form of leptomeningeal involvement. This fact underscores the importance of lumbar puncture in the staging evaluation of patients with systemic lymphoma.

1	Systemic lymphoma is seen at earlier stages of HIV infection than primary CNS lymphoma. In one series the mean CD4+ T cell count was 226/μL. In addition to lymph node involvement, systemic lymphoma may commonly involve the GI tract, bone marrow, liver, and lung. GI tract involvement is seen in ~25% of patients. Any site in the GI tract may be involved, and patients may complain of difficulty swallowing or abdominal pain. The diagnosis is usually suspected on the basis of CT or MRI of the abdomen. Bone marrow involvement is seen in ~20% of patients and may lead to pancytopenia. Liver and lung involvement are each seen in ~10% of patients. Pulmonary disease may present as a mass lesion, multiple nodules, or an interstitial infiltrate.

1	FIGuRE 226-44 Central nervous system lymphoma. Postcontrast T1-weighted MRI scan in a patient with AIDS, an altered mental status, and hemiparesis. Multiple enhancing lesions, some ring-enhancing, are present. The left sylvian lesion shows gyral and subcortical enhancement, and the lesions in the caudate and splenium (arrowheads) show enhancement of adjacent ependymal surfaces.

1	Both conventional and unconventional approaches have been 1271 employed in an attempt to treat HIV-related lymphomas. Systemic lymphoma is generally treated by the oncologist with combination chemotherapy. Earlier disappointing figures are being replaced with more optimistic results for the treatment of systemic lymphoma following the availability of more effective cART and the use of rituximab in CD20+ tumors. While there is some controversy regarding the use of antiretrovirals during chemotherapy, there is no question that their use overall in patients with HIV lymphoma has improved survival. Concerns regarding synergistic bone marrow toxicities with chemotherapy and cART are mitigated with the use of cART regimens that avoid bone marrow–toxic antiretrovirals. As in most situations in patients with HIV disease, those with higher CD4+ T cell counts tend to fare better. Response rates as high as 72% with a median survival of 33 months and disease-free intervals up to 9 years have been

1	with HIV disease, those with higher CD4+ T cell counts tend to fare better. Response rates as high as 72% with a median survival of 33 months and disease-free intervals up to 9 years have been reported. Treatment of primary CNS lymphoma remains a significant challenge. Treatment is complicated by the fact that this illness usually occurs in patients with advanced HIV disease. Palliative measures such as radiation therapy provide some relief. The prognosis remains poor in this group, with a 2-year survival of 29%.

1	Multicentric Castleman’s disease is a KSHV-associated lymphoproliferative disorder that is seen with an increased frequency in patients with HIV infection. While not a true malignancy, it shares many features with lymphoma including generalized lymphadenopathy, hepatosplenomegaly, and systemic symptoms of fever, fatigue, and weight loss. Pulmonary symptoms may be seen in ~50% of patients. KS is present in 75–82% of cases. Lymph node biopsies reveal a predomi nance of interfollicular plasma cells and/or germinal centers with vascularization and an “onionskin” (hyaline vascular) appearance. Prior to the availability of cART, HIV-infected patients with multicentric Castleman’s disease had a 15-fold increased risk of developing nonHodgkin’s lymphoma compared with HIV-infected patients in general. Treatment typically involves chemotherapy. Anecdotal reports of success with rituximab suggest that more specific treatment may be successful, although, in one series treatment with rituximab was

1	Treatment typically involves chemotherapy. Anecdotal reports of success with rituximab suggest that more specific treatment may be successful, although, in one series treatment with rituximab was associated with worsening of coexisting KS. The median survival of patients with treated multicentric Castleman’s disease pre-cART was initially reported as 14 months. This has increased to a 2-year survival of more than 90% in the era of cART.

1	Evidence of infection with human papillomavirus (HPV), associated with intraepithelial dysplasia of the cervix or anus, is approximately twice as common in HIV-infected individuals as in the general population and can lead to intraepithelial neoplasia and eventually invasive cancer. In a series of studies, HIV-infected men were examined for evidence of anal dysplasia, and Papanicolaou (Pap) smears were found to be abnormal in 20–80%. These changes tend to persist and are generally not affected by cART, raising the possibility of a subsequent transition to a more malignant condition. While the incidence of an abnormal Pap smear of the cervix is ~5% in otherwise healthy women, the incidence of abnormal cervical smears in women with HIV infection is 30–60%, and invasive cervical cancer is included as an AIDS-defining condition. While only small increases in the absolute numbers of cervical or anal cancers have been seen as a consequence of HIV infection, the relative risk of these

1	is included as an AIDS-defining condition. While only small increases in the absolute numbers of cervical or anal cancers have been seen as a consequence of HIV infection, the relative risk of these conditions when one compares HIV-infected to -noninfected men and women is on the order of 10to 100-fold. Given the high rates of dysplasia and relative risks for cervical and anal cancer, a comprehensive gynecologic and rectal examination, including Pap smear, is indicated at the initial evaluation and 6 months later for all patients with HIV infection. If these examinations are negative at both time points, the patient should be followed with yearly evaluations. If an initial or repeat Pap smear shows evidence of severe inflammation with reactive squamous changes, the next Pap smear should be performed at 3 months. If, at any time, a Pap smear shows evidence of squamous intraepithelial lesions, colposcopic examination with biopsies as indicated should be performed. The 2-year survival

1	be performed at 3 months. If, at any time, a Pap smear shows evidence of squamous intraepithelial lesions, colposcopic examination with biopsies as indicated should be performed. The 2-year survival rate for HIV-infected patients with invasive cervical cancer is 64% compared with 79% in non-HIV-infected patients. In addition to rectal and cervical lesions, HPV can also lead to head and

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1272 neck cancers. In one study of men who have sex with men, 25% were found to have oral HPV; high-risk HPV genotypes were three times more common in the HIV-infected men. The most common HPV genotypes in the general population and the genotypes upon which current HPV vaccines are based are 6, 11, 16, and 18. This is not the case in the HIV-infected population, where other genotypes such as 58 and 53 also are prominent. This raises concerns about the level of effectiveness of the current HPV vaccines for HIV-infected patients. Despite this, it is recommended that patients with HIV infection be vaccinated against HPV.

1	A syndrome was recognized in 1992 characterized by an absolute CD4+ T cell count of <300/μL or <20% of total T cells on a minimum of two occasions at least 6 weeks apart; no evidence of HIV-1, HIV-2, HTLV-1, or HTLV-2 on testing; and the absence of any defined immunodeficiency or therapy associated with decreased levels of CD4+ T cells. By mid-1993, ~100 patients had been described. After extensive multicenter investigations, a series of reports were published in early 1993, which together allowed a number of conclusions. Idiopathic CD4+ lymphocytopenia (ICL) is a very rare syndrome, as determined by studies of blood donors and cohorts of HIV-seronegative men who have sex with men. Cases were clearly identified as early as 1983 and were remarkably similar to the clinical features of ICL that had been identified decades earlier. The definition of ICL based on CD4+ T cell counts coincided with the ready availability of testing for CD4+ T cells in patients suspected of being

1	of ICL that had been identified decades earlier. The definition of ICL based on CD4+ T cell counts coincided with the ready availability of testing for CD4+ T cells in patients suspected of being immunodeficient. Although, as a result of immune deficiency, certain patients with ICL develop some of the opportunistic diseases (particularly cryptococcosis, nontuberculous mycobacterial infections, and cervical dysplasia) seen in HIV-infected patients, the syndrome is demographically, clinically, and immunologically unlike HIV infection and AIDS. Fewer than half of the reported ICL patients had risk factors for HIV infection, and there were wide geographic and age distributions. The fact that a significant proportion of patients did have risk factors probably reflects a selection bias, in that physicians who take care of HIV-infected patients are more likely to monitor CD4+ T cells. Approximately half of the patients are women, compared with approximately one-third among HIV-infected

1	that physicians who take care of HIV-infected patients are more likely to monitor CD4+ T cells. Approximately half of the patients are women, compared with approximately one-third among HIV-infected individuals in the United States. Many patients with ICL remained clinically stable, and their condition did not deteriorate progressively as is common with seriously immunodeficient HIV-infected patients. Approximately 15% of patients with ICL experience spontaneous reversal of the CD4+ T lymphocytopenia. Immunologic abnormalities in ICL are somewhat different from those of HIV infection. ICL patients often have increases in CD4+ T cell activation with decreases in CD8+ T cells and B cells. Furthermore, immunoglobulin levels are either normal or, more commonly, decreased in patients with ICL, compared with the usual hypergammaglobulinemia of HIV-infected individuals. Virologic studies of these patients have revealed no evidence of HIV-1, HIV2, HTLV-1, or HTLV-2 or of any other mononuclear

1	compared with the usual hypergammaglobulinemia of HIV-infected individuals. Virologic studies of these patients have revealed no evidence of HIV-1, HIV2, HTLV-1, or HTLV-2 or of any other mononuclear cell–tropic virus. Furthermore, there has been no epidemiologic evidence to suggest that a transmissible microbe was involved. The cases of ICL have been widely dispersed, with no clustering. Close contacts and sexual partners who were studied were clinically well and were serologically, immunologically, and virologically negative for HIV. ICL is a heterogeneous syndrome, and it is highly likely that there is no common cause; however, there may be common causes among subgroups of patients that are currently unrecognized.

1	Patients who present with laboratory data consistent with ICL should be worked up for underlying diseases that could be responsible for the immune deficiency. If no underlying cause is detected, no specific therapy should be initiated. However, if opportunistic diseases occur, they should be treated appropriately (see above). Depending on the level of the CD4+ T cell count, patients should receive prophylaxis for the commonly encountered opportunistic infections.

1	The CDC guidelines call for the testing for HIV infection to be a part of routine medical care. It is recommended that the patient be informed of the intention to test, as is the case with other routine laboratory determinations, and be given the opportunity to “opt out.” Such an approach is critical to the goal of identifying as many infected individuals as possible since 16–18% of the >1.1 million individuals in the United States who are HIV-infected are not aware of their status. Under these circumstances of routine testing, although it is desirable, pretest counseling may not always be built into the testing process. However, no matter how well prepared a patient is for adversity, the discovery of a diagnosis of HIV infection is a devastating event. Thus, physicians should be sensitive to this fact and, where possible, execute some degree of pretest counseling to at least partially prepare the patient should the results demonstrate the presence of HIV infection. Following a

1	to this fact and, where possible, execute some degree of pretest counseling to at least partially prepare the patient should the results demonstrate the presence of HIV infection. Following a diagnosis of HIV infection, the health care provider should be prepared to immediately activate support systems for the newly diagnosed patient. These should include an experienced social worker or nurse who can spend time talking to the person and ensuring that he or she is emotionally stable. Most communities have HIV support centers that can be of great help in these difficult situations.

1	The treatment of patients with HIV infection requires not only a comprehensive knowledge of the possible disease processes that may occur and up-to-date knowledge of and experience with cART, but also the ability to deal with the problems of a chronic, potentially life-threatening illness. A comprehensive knowledge of internal medicine is required to deal with the changing spectrum of illnesses associated with HIV infection, many of which are similar to a state of accelerated aging. Great advances have been made in the treatment of patients with HIV infection. The appropriate use of potent cART and other treatment and prophylactic interventions are of critical importance in providing each patient with the best opportunity to live a long and healthy life despite the presence of HIV infection. In contrast to the earlier days of this epidemic, a diagnosis of HIV infection need no longer be equated with having an inevitably fatal disease. In addition to medical interventions, the health

1	In contrast to the earlier days of this epidemic, a diagnosis of HIV infection need no longer be equated with having an inevitably fatal disease. In addition to medical interventions, the health care provider has a responsibility to provide each patient with appropriate counseling and education concerning their disease as part of a comprehensive care plan. Patients must be educated about the potential transmissibility of their infection and about the fact that while health care providers may refer to levels of the virus as “undetectable,” this is more a reflection of the sensitivity of the assay being used to measure the virus than a comment on the presence or absence of the virus. It is important for patients to be aware that the virus is still present and capable of being transmitted at all stages of HIV disease. Thus, there must be frank discussions concerning sexual practices and the sharing of syringes and other paraphernalia used in illicit drug use. The treating physician not

1	all stages of HIV disease. Thus, there must be frank discussions concerning sexual practices and the sharing of syringes and other paraphernalia used in illicit drug use. The treating physician not only must be aware of the latest medications available for patients with HIV infection but also must educate patients concerning the natural history of their illness and listen and be sensitive to their fears and concerns. As with other diseases, therapeutic decisions should be made in consultation with the patient, when possible, and with the patient’s proxy if the patient is incapable of making decisions. In this regard, it is recommended that all patients with HIV infection, and in particular those with CD4+ T cell counts <200/μL, designate a trusted individual with durable power of attorney to make medical decisions on their behalf, if necessary.

1	Following a diagnosis of HIV infection, there are several examinations and laboratory studies that should be performed to help determine the extent of disease and provide baseline standards for future reference (Table 226-19). In addition to routine chemistry, fasting lipid profile, aspartate aminotransferase, alanine aminotransferase, total and direct bilirubin, fasting glucose and hematology screening panels, Pap smear, urinalysis, and chest x-ray, one should also obtain a CD4+ T cell count, two separate plasma HIV

1	History and physical examination Routine chemistry and hematology AST, ALT, direct and indirect bilirubin Lipid profile and fasting glucose CD4+ T lymphocyte count Two plasma HIV RNA levels HIV resistance testing HLA-B5701 screening RPR or VDRL test Anti-Toxoplasma antibody titer PPD skin test or IFN-γ release assay Mini-Mental Status Examination Serologies for hepatitis A, hepatitis B, and hepatitis C Immunization with pneumococcal polysaccharide; influenza as indicated Immunization with hepatitis A and hepatitis B if seronegative Counseling regarding natural history and transmission Help contacting others who might be infected Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; PPD, purified protein derivative; RPR, rapid plasma reagin; VDRL, Venereal Disease Research Laboratory.

1	RNA levels, an HIV resistance test, a rapid plasma reagin or VDRL test, an anti-Toxoplasma antibody titer, and serologies for hepatitis A, B, and C. A PPD test or IFN-γ release assay should be done and an MMSE performed and recorded. A pregnancy test should be done in women in whom the drug efavirenz is being considered, and HLA-B5701 testing should be done in all patients in whom the drug abacavir is being considered. Patients should be immunized with pneumococcal polysaccharide, with annual influenza shots, and, if seronegative for these viruses, with HPV, hepatitis A, and hepatitis B vaccines. The status of hepatitis C infection should be determined. In addition, patients should be counseled with regard to sexual practices and needle sharing, and counseling should be offered to those whom the patient knows or suspects may also be infected. Once these baseline activities are performed, shortand long-term medical management strategies should be developed based on the most recent

1	whom the patient knows or suspects may also be infected. Once these baseline activities are performed, shortand long-term medical management strategies should be developed based on the most recent information available and modified as new information becomes available. The field of HIV medicine is changing rapidly, and it is difficult to remain fully up to date. Fortunately there are a series of excellent sites on the Internet that are frequently updated, and they provide the most recent information on a variety of topics, including consensus panel reports on treatment (Table 226-20).

1	Combination antiretroviral therapy (cART), also referred to as highly active antiretroviral therapy (HAART), is the cornerstone of management of patients with HIV infection. Following the initiation of widespread use of cART in the United States in 1995–1996, marked declines were noted in the incidence of most AIDS-defining conditions (Fig. 226-33). Suppression of HIV replication is an important component in prolonging life as well as in improving the quality of life in patients with HIV infection. Adequate suppression requires www.aidsinfo.nih.gov AIDSinfo, a service of the U.S. Department of Health and Human Services, posts federally approved treatment guidelines for HIV and AIDS; provides information on federally funded and privately funded clinical trials and CDC publications and data www.cdcnpin.org Updates on epidemiologic data and prevention information from the CDC Abbreviation: CDC, Centers for Disease Control and Prevention.

1	strict adherence to prescribed regimens of antiretroviral drugs. This 1273 has been facilitated by the coformulations of antiretrovirals and the development of once-daily regimens. Unfortunately, many of the most important questions related to the treatment of HIV disease currently lack definitive answers. Among them are the questions of when therapy should be started, what the best initial regimen is, when a given regimen should be changed, and what it should be changed to when a change is made. Notwithstanding these uncertainties, the physician and patient must come to a mutually agreeable plan based on the best available data. In an effort to facilitate this process, the U.S. Department of Health and Human Services makes available on the Internet (www.aidsinfo.nih.gov) a series of periodically updated guidelines, including “Guidelines for the Use of Antiretroviral Agents in HIV-Infected Adults and Adolescents” and “Guidelines for the Prevention of Opportunistic Infections in

1	periodically updated guidelines, including “Guidelines for the Use of Antiretroviral Agents in HIV-Infected Adults and Adolescents” and “Guidelines for the Prevention of Opportunistic Infections in Persons Infected with Human Immunodeficiency Virus.” At present, an extensive clinical trials network, involving both clinical investigators and patient advocates, is in place attempting to develop improved approaches to therapy. Consortia comprising representatives of academia, industry, independent foundations, and the federal government are involved in the process of drug development, including a wide-ranging series of clinical trials. As a result, new therapies and new therapeutic strategies are continually emerging. New drugs are often available through expanded-access programs prior to official licensure. Given the complexity of this field, decisions regarding cART are best made in consultation with experts.

1	Currently available drugs for the treatment of HIV infection as part of a combination regimen fall into four categories: those that inhibit the viral reverse transcriptase enzyme (nucleoside and nucleotide reverse transcriptase inhibitors; nonnucleoside reverse transcriptase inhibitors), those that inhibit the viral protease enzyme (protease inhibitors), those that inhibit the viral integrase enzyme (integrase inhibitors), and those that interfere with viral entry (fusion inhibitors; CCR5 antagonists) (Table 226-21; Fig. 226-45).

1	The FDA-approved reverse transcriptase inhibitors include the nucleoside analogues zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, and emtricitabine; the nucleotide analogue tenofovir; and the nonnucleoside reverse transcriptase inhibitors nevirapine, delavirdine, efavirenz, and etravirine (Table 226-21; Fig. 226-45). These represent the first class of drugs licensed for the treatment of HIV infection. They are indicated for this use as part of combination regimens. It should be stressed that none of these drugs should be used as monotherapy for HIV infection due to the relative ease with which drug resistance may develop under such circumstances. Thus, when lamivudine, emtricitabine, or tenofovir is used to treat hepatitis B infection in the setting of HIV infection, one should ensure that the patient is also on additional antiretroviral medication. The reverse transcriptase inhibitors block the HIV replication cycle at the point of RNA-dependent DNA synthesis,

1	one should ensure that the patient is also on additional antiretroviral medication. The reverse transcriptase inhibitors block the HIV replication cycle at the point of RNA-dependent DNA synthesis, the reverse transcription step. While the nonnucleoside reverse transcriptase inhibitors are quite selective for the HIV-1 reverse transcriptase, the nucleoside and nucleotide analogues inhibit a variety of DNA polymerases in addition to those of the HIV-1 reverse transcriptase. For this reason, serious side effects are more varied with the nucleoside analogues and include mitochondrial damage that can lead to hepatic steatosis and lactic acidosis as well as peripheral neuropathy and pancreatitis. The use of either of the thymidine analogues zidovudine and stavudine has been associated with a syndrome of hyperlipidemia, glucose intolerance/insulin resistance, and fat redistribution often referred to as lipodystrophy syndrome (discussed in “Diseases of the Endocrine System and Metabolic

1	a syndrome of hyperlipidemia, glucose intolerance/insulin resistance, and fat redistribution often referred to as lipodystrophy syndrome (discussed in “Diseases of the Endocrine System and Metabolic Disorders,” above). The reverse transcriptase inhibitors preferred for use according to the DHHS Panel on the use of antiretroviral drugs are lamivudine, emtricitabine, abacavir, tenofovir, and rilpivirine.

1	Lamivudine (3TC; 2’,3’-dideoxy-3’-thiacytidine) is the fifth of the nucleoside analogues to be licensed in the United States. It is the negative enantiomer of a dideoxy analogue of cytidine. In actual practice, lamivudine or the closely related drug emtricitabine (see Human Immunodeficiency Virus Disease: AIDS and Related Disorders Etravirine (Intelence) Licensed In combination with other antiretroviral agents in treatment-experienced patients whose HIV is resistant to nonnucleoside reverse transcriptase inhibitors and other antiretroviral medications but the discontinuation rate in the indinavir group was unexpectedly high, accounting for most treatment “failures”); CD4 cell increase (~140/μL in each group) at 24 weeks 200 mg bid Higher rates of HIV RNA suppression to <50 copies/mL (56% vs 39%); greater increases in CD4+ T cell count (89 vs 64 cells) compared to placebo when given in combination with an optimized background regimen Rash, nausea, hypersensitivity reactions

1	Enfuvirtide (Fuzeon) Licensed In combination 90 mg SC bid In treatment of experienced with other agents in patients, superior to placebo when treatment-experienced added to new optimized back-patients with evidence ground (37% vs 16% with <400 HIV of HIV-1 replication Maraviroc (Selzentry) Licensed In combination with 150–600 mg bid At 24 weeks, among 635 patients other antiretroviral depending on con-with CCR5-tropic virus and HIV-1 agents in adults infected comitant medica-RNA >5000 copies/mL despite at with only CCR5-tropic tions (see text) least 6 months of prior therapy HIV-1 with at least 1 agent from 3 of the 4 antiretroviral drug classes, 61% of patients randomized to maraviroc achieved HIV RNA levels <400 copies/mL compared with 28% of patients randomized to placebo Local injection reactions, hypersensitivity reactions, increased rate of bacterial pneumonia Hepatotoxicity, nasopharyngitis, fever, cough, rash, abdominal pain, dizziness, musculoskeletal symptoms

1	Local injection reactions, hypersensitivity reactions, increased rate of bacterial pneumonia Hepatotoxicity, nasopharyngitis, fever, cough, rash, abdominal pain, dizziness, musculoskeletal symptoms Human Immunodeficiency Virus Disease: AIDS and Related Disorders Raltegravir (Isentress) Licensed In combination with 400 mg bid other antiretroviral agents (Available only in combination with cobicistat, tenofovir, and emtricitabine [Stribild]) Dolutegravir Licensed In combination with 50 mg daily for (Tivicay) 50 mg twice daily for treatment-experienced patients or those also receiving efavirenz or rifampin At 24 weeks, among 436 patients with 3-class drug resistance, 76% of patients randomized to receive raltegravir achieved HIV RNA levels <400 copies/mL compared with 41% of patients randomized to receive placebo Noninferior to raltegravir or atazanavir/ritonavir in treatment-experienced patients.

1	Noninferior to raltegravir or atazanavir/ritonavir in treatment-experienced patients. Noninferior to raltegravir, superior to efavirenz or darunavir/ritonavir Nausea, headache, diarrhea, CPK elevation, muscle weakness, rhabdomyolysis Diarrhea, nausea, upper respiratory infections, headache Insomnia, headache, hypersensitivity reactions, hepatotoxicity Abbreviations: ARC, AIDS-related complex; NRTIs, nonnucleoside reverse transcriptase inhibitors.

1	Insomnia, headache, hypersensitivity reactions, hepatotoxicity Abbreviations: ARC, AIDS-related complex; NRTIs, nonnucleoside reverse transcriptase inhibitors. 1276 below) is a frequent element of many different combination regi-on hepatitis B, direct effects on HIV, and immune reconstitution mens currently in use. These two drugs and the nucleotide reverse (see above). To prevent the development of resistant strains of HIV, transcriptase inhibitor tenofovir (see below) also have activity these drugs should never be used on their own for the treatment against hepatitis B virus. For this reason flares of hepatitis may be of hepatitis B in the patient with HIV infection. Lamivudine is avail-seen in co-infected patients starting and/or or stopping any of able either alone or in coformulations including zidovudine and/or these three agents due to the confounding issues of direct effects abacavir (Table 226-22). One reason behind the excellent synergy

1	FIGuRE 226-45 Molecular structures of antiretroviral agents. Human Immunodeficiency Virus Disease: AIDS and Related Disorders seen between lamivudine and the other nucleoside analogues may be that strains of HIV resistant to lamivudine (M184V substitution) appear to have enhanced sensitivity to other nucleosides, and thus development of dual resistance is more difficult. In addition, there is a suggestion that 3TC-resistant strains of HIV may be less virulent and are less able to generate new mutants than are strains of HIV that are 3TC-sensitive. Lamivudine is among the best tolerated and least toxic of the nucleoside analogues. aNot licensed in the United States.

1	aNot licensed in the United States. Emtricitabine (FTC; 5-fluoro-1-(2R,5S)-[2-(hydroxymethyl)-1,3oxathiolan-5-yl]cytosine) is the negative enantiomer of a thio analogue of cytidine with a fluorine in the 5 position. It is licensed for use in combination with other antiretroviral agents for treatment of HIV-1 infection in adults. Compared with lamivudine, it is similar in activity and has a longer half-life. It is available either alone or coformulated with tenofovir or tenofovir and efavirenz (Table 226-22). As with lamivudine, resistance to emtricitabine is associated with the M184V mutation in reverse transcriptase. Viruses showing the K65R mutation in reverse transcriptase may have reduced susceptibility to emtricitabine.

1	Abacavir {(1S,cis)-4-[2-amino-6-(cyclopropylamino)-9H-purin9-yl]-2-cyclopentene-1-methanol sulfate (salt)(2:1)} is a synthetic carbocyclic analogue of the nucleoside guanosine. It is licensed to be used in combination with other antiretroviral agents for the treatment of HIV-1 infection. Hypersensitivity reactions that may occur with initial therapy or rechallenge have been reported in ~4% of patients treated with this drug, and patients developing signs or symptoms of hypersensitivity such as fever, skin rash, fatigue, and GI symptoms should discontinue the drug and not restart it. Fatal hypersensitivity reactions have been reported with rechallenge. Abacavir hypersensitivity occurs with a higher frequency in patients who are HLA-B5701-positive. It is recommended that patients be screened for HLA-B5701 prior to initiation of abacavir and that 1278 abacavir only be used as a last resort and with close monitoring in patients who are HLA-B5701-positive. Abacavir-resistant strains of HIV

1	for HLA-B5701 prior to initiation of abacavir and that 1278 abacavir only be used as a last resort and with close monitoring in patients who are HLA-B5701-positive. Abacavir-resistant strains of HIV are typically also resistant to lamivudine, emtricitabine, didanosine, and zalcitabine. In randomized trials abacavir was found to be inferior to tenofovir in patients with baseline HIV RNA levels >100,000 copies/mL. Abacavir is formulated alone as well as in combination with lamivudine, with zidovudine and lamivudine or with lamivudine and dolutegravir. Tenofovir disoproxil fumarate (9-[(R)-2-[[bis[[(isopropoxycarbonyl) oxy]methoxy]phosphinyl]methoxy]propyl]adenine fumarate [1:1]) is an acyclic nucleoside phosphonate diester analogue of adenosine monophosphate. It undergoes diester hydrolysis to form the nucleoside monophosphate (nucleotide) tenofovir and is the first nucleotide analogue to be licensed for treatment of HIV infection. It is indicated in combination with other

1	hydrolysis to form the nucleoside monophosphate (nucleotide) tenofovir and is the first nucleotide analogue to be licensed for treatment of HIV infection. It is indicated in combination with other antiretroviral agents for the treatment of HIV-1 infection and in combination with emtricitabine for pre-exposure prophylaxis for HIV-1 prevention in populations at high risk of HIV infection. HIV isolates with increased resistance typically express a K65R mutation in reverse transcriptase and a threeto fourfold reduction in sensitivity to tenofovir. Tenofovir is primarily eliminated by the kidneys, and renal impairment including a Fanconi-like syndrome with hypophosphatemia may occur. Tenofovir is contraindicated in patients with renal impairment. An investigational prodrug analogue with less renal toxicity, tenofovir alafenamide fumarate is currently in clinical trials. Small but statistically significant decreases in bone mineral density have been noted in patients receiving tenofovir.

1	renal toxicity, tenofovir alafenamide fumarate is currently in clinical trials. Small but statistically significant decreases in bone mineral density have been noted in patients receiving tenofovir. Coadministration of tenofovir with didanosine leads to a 60% increase in didanosine levels, and thus doses of didanosine need to be adjusted and patients monitored carefully if these two drugs are used in combination. In addition, CD4+ T cell increases may be blunted in patients on this combination. Coadministration of tenofovir with atazanavir leads to a decrease in atazanavir levels, and thus low-dose ritonavir (see below) needs to be added when these drugs are used in combination. Tenofovir is available alone and coformulated with emtricitabine, emtricitabine and efavirenz, emtricitabine and rilpivirine, or emtricitabine, elvitegravir and cobicistat. Nevirapine, delavirdine, efavirenz, etravirine, and rilpivirine are nonnucleoside inhibitors of the HIV-1 reverse transcriptase and are

1	and rilpivirine, or emtricitabine, elvitegravir and cobicistat. Nevirapine, delavirdine, efavirenz, etravirine, and rilpivirine are nonnucleoside inhibitors of the HIV-1 reverse transcriptase and are licensed for use in combination with nucleoside analogues for the treatment of HIV-infected adults. Coformulations that include efavirenz or nevirapine are available (Table 226-22). These agents inhibit reverse transcriptase by binding to regions of the enzyme outside the active site and causing conformational changes in the enzyme that render it inactive. Although these agents are active in the nanomolar range, they are also very selective for the reverse transcriptase of HIV-1, have no activity against HIV-2, and, when used as mono-therapy, are associated with the rapid emergence of drug-resistant mutants (Table 226-21; Fig. 226-46). Efavirenz and rilpivirine are administered once a day, nevirapine and etravirine twice a day, and delavirdine three times a day. All are associated with

1	mutants (Table 226-21; Fig. 226-46). Efavirenz and rilpivirine are administered once a day, nevirapine and etravirine twice a day, and delavirdine three times a day. All are associated with the development of a maculopapular rash, generally seen within the first few weeks of therapy. While it is possible to treat through this rash, it is important to be sure that one is not dealing with a more severe eruption such as Stevens-Johnson syndrome by looking carefully for signs of mucosal involvement, significant fever, or painful lesions with desquamation. Severe, life-threatening, and in some cases fatal hepatotoxicity, including fulminant and cholestatic hepatitis, hepatic necrosis, and hepatic failure, have been reported in patients treated with nevirapine. There is a suggestion that this is more common in women with higher CD4+ T cell counts. Many patients treated with efavirenz note a feeling of light-headedness, dizziness, or out of sorts following the initiation of therapy. Some

1	is more common in women with higher CD4+ T cell counts. Many patients treated with efavirenz note a feeling of light-headedness, dizziness, or out of sorts following the initiation of therapy. Some complain of vivid dreams. These symptoms tend to disappear after several weeks of therapy. Aside from difficulties with dreams, taking efavirenz at bedtime may minimize the side effects. Efavirenz may cause fetal harm when administered during the first trimester to a pregnant woman. Women of childbearing potential should undergo pregnancy testing prior to initiation of efavirenz. Efavirenz is commonly used in combination with two nucleoside analogues as part of initial treatment regimens. Etravirine is a diarylpyrimidine derivative currently licensed for treatment of HIV infection in combination with other agents. In contrast to the other nonnucleoside reverse transcriptase inhibitors, which all exhibit cross-resistance, etravirine may be active against strains of HIV that are resistant to

1	with other agents. In contrast to the other nonnucleoside reverse transcriptase inhibitors, which all exhibit cross-resistance, etravirine may be active against strains of HIV that are resistant to other nonnucleoside reverse transcriptase inhibitors. Among its side effects are rash, headache, nausea, and diarrhea. Rilpivirine is effective across a broad range of NNRTI-resistant viruses and shares cross-resistance with etravirine. It is better tolerated and a has higher rate of virologic failure than efavirenz, particularly in those with HIV RNA >100,000. It is only available as part of a combination regimen with tenofovir and emtricitabine.

1	The HIV-1 protease inhibitors (saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, fosamprenavir, lopinavir/ritonavir, atazanavir, tipranavir, and darunavir) are a major part of the therapeutic armamentarium of antiretrovirals. When used as part of initial regimens in combination with reverse transcriptase inhibitors, these agents have been shown to be capable of suppressing levels of HIV replication to under 50 copies/mL in the majority of patients for a minimum of 5 years. As in the case of reverse transcriptase inhibitors, resistance to protease inhibitors can develop rapidly in the setting of monotherapy, and thus these agents should be used only as part of combination therapeutic regimens. A summary of known resistance mutations for protease inhibitors is shown in Fig. 226-46. The protease inhibitors preferred for use according to the DHHS Panel on the use of antiretroviral drugs are ritonavir (only as a pharmacokinetic enhancer), atazanavir, and darunavir.

1	Ritonavir was the first protease inhibitor for which clinical efficacy was demonstrated. In a study of 1090 patients with CD4+ T cell counts <100/μL who were randomized to receive either placebo or ritonavir in addition to any other licensed medications, patients receiving ritonavir had a reduction in the cumulative incidence of clinical progression or death from 34% to 17%. Mortality decreased from 10.1% to 5.8%. At full doses, ritonavir is poorly tolerated. Among the main side effects are nausea, diarrhea, abdominal pain, hyperlipidemia, and circumoral paresthesia. Ritonavir has a high affinity for several isoforms of cytochrome P450 (3A4, 2D6), and its use can result in large increases in the plasma concentrations of drugs metabolized by these pathways. Among the agents affected in this manner are most other protease inhibitors, macrolide antibiotics, R-warfarin, ondansetron, rifabutin, most calcium channel blockers, glucocorticoids, and some of the chemotherapeutic agents used to

1	manner are most other protease inhibitors, macrolide antibiotics, R-warfarin, ondansetron, rifabutin, most calcium channel blockers, glucocorticoids, and some of the chemotherapeutic agents used to treat KS and/or lymphomas. In addition, ritonavir may increase the activity of glucuronyltransferases, thus decreasing the levels of drugs metabolized by this pathway. Overall, great care must be taken when prescribing additional drugs to patients taking protease inhibitors in general and ritonavir in particular. As mentioned above, the pharmacodynamic boosting property of ritonavir, seen with doses as low as 100–200 mg once or twice a day, is often used in the setting of cART for HIV infection to derive more convenient regimens. For example, when given with low-dose ritonavir, saquinavir and indinavir can be given on twice-a-day schedules and taken with food.

1	Atazanavir is an azapeptide inhibitor of the HIV-1 protease that was licensed in 2003. An advantage of atazanavir is that total cholesterol and triglyceride levels do not increase as much with atazanavir as with other protease inhibitors. This coupled with the fact that it can be given on a once-daily schedule made atazanavir a popular component of initial treatment regimens following its licensure. Atazanavir is associated with increases in serum bilirubin, renal stones, and prolongations of the ECG PR interval. Atazanavirresistant isolates emerging in previously treatment-naïve individuals frequently harbor an I50L substitution. This mutation in some instances is associated with increased sensitivity to other protease inhibitors. Atazanavir requires an acidic gastric pH for absorption, and its use in combination with a proton pump inhibitor is contraindicated due to concerns about absorption. Atazanavir is an inhibitor of cytochrome P3A, and its use may be associated with

1	Mutations in the Reverse Transcriptase Gene Associated with Resistance to Reverse Transcriptase Inhibitors 1279 Multi-nRTI Resistance: 69 Insertion Complex (affects all nRTIs currently approved by the US FDA) Multi-nRTI Resistance: 151 Complex (affects all nRTIs currently approved by the US FDA except tenofovir) A VF FQ Multi-nRTI Resistance: Thymidine Analogue-Associated Mutations (TAMs; affect all nRTIs currently approved by the US FDA) M DK LTK L Amino acid, wild-type Amino acid position 65 100 Amino acid substitution

1	FIGuRE 226-46 Amino acid substitutions conferring resistance to antiretroviral drugs. For each amino acid residue, the letter above the bar indicates the amino acid associated with wild-type virus and the letter(s) below indicate the substitution(s) that confer viral resistance. The number shows the position of the mutation in the protein. Mutations selected by protease inhibitors in Gag cleavage sites are not listed. HR1 indicates first heptad repeat; NAMs indicates nRTI-associated mutations; nRTI indicates nucleoside reverse transcriptase inhibitor; NNRTI indicates nonnucleoside reverse transcriptase inhibitor; PI indicates protease inhibitor. Amino acid abbreviations: A, alanine; C, cysteine; D, aspartate; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine. (Reprinted with permission from the

1	K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine. (Reprinted with permission from the International Antiviral Society—USA. AM Wensing, V Calvez, HR Günthard et al: 2014 Update of the Drug Resistance Mutations in HIV-1. Top Antivir Med. 2014; 22(3):642–650. Updated information [and thorough explanatory notes] is available at www.iasusa.org.)

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1280 Mutations in the Protease Gene Associated with Resistance to Protease Inhibitors Mutations in the Envelope Gene Associated with Resistance to Entry Inhibitors Maraviroc Activity limited to patients with R5 viruses

1	Mutations in the Integrase Gene Associated with Resistance to Integrase Strand Transfer Inhibitors increased levels of calcium channel blockers, macrolide antibiotics, main reasons for discontinuation were bilirubin elevations and HMG-CoA reductase inhibitors, and sildenafil. Levels of atazana-gastrointestinal side effects. vir are lower in the presence of tenofovir or efavirenz. In these Darunavir is a nonpeptidic HIV protease inhibitor initially licensed settings, levels of atazanavir should be boosted with the use of in 2006. It is indicated for coadministration with 100 mg of ritonavir low-dose ritonavir. In a head-to-head comparison, more patients and other antiretroviral agents for the treatment of HIV infecdiscontinued atazanavir than either darunavir or raltegravir. The tion. In initial studies in treatment-experienced subjects, 46% of patients achieved a reduction in HIV RNA viral loads to <50 copies/ mL. Studies in treatment-naïve patients demonstrated efficacy superior to

1	initial studies in treatment-experienced subjects, 46% of patients achieved a reduction in HIV RNA viral loads to <50 copies/ mL. Studies in treatment-naïve patients demonstrated efficacy superior to lopinavir/ritonavir-containing regimens but inferior to dolutegravir. Skin rash, which may be severe, is seen in 7% of patients and may be related to the sulfonamide moiety contained in the molecule. GI intolerance and headache are the other most frequent side effects.

1	Entry inhibitors act by interfering with the binding of HIV to its receptor or co-receptor or by interfering with the process of fusion (see above). The first drug in this class to be licensed was the fusion inhibitor enfuvirtide, or T-20, followed by the CCR5 antagonist maraviroc. A variety of additional small molecules that bind to HIV-1 co-receptors are currently in clinical trials.

1	Enfuvirtide is a linear 36-amino-acid synthetic peptide with the N terminus acetylated and the C terminus a carboxamide. It is composed of naturally occurring L-amino acid residues and interferes with the fusion of the viral and cellular membranes by binding to the HR1 region in the gp41 subunit of the HIV-1 envelope. This binding interferes with the coil-coil interaction required to approximate the viral envelope and the host cell membrane during the process of viral fusion. Enfuvirtide was licensed in 2003 for treatment of HIV-1 infection in combination with other antiretroviral agents in treatment-experienced patients with ongoing viral replication despite antiretroviral therapy. Enfuvirtide is not active against HIV-2. Enfuvirtide-resistant isolates of HIV exhibit amino acid changes in positions 36–45 of gp41. In two independent studies, patients who had persistent viremia despite prior treatment with agents from all three available classes of drugs were randomized to receive an

1	in positions 36–45 of gp41. In two independent studies, patients who had persistent viremia despite prior treatment with agents from all three available classes of drugs were randomized to receive an individualized regimen (based on prior treatment history and resistance profile) with or without enfuvirtide. The change in plasma HIV-1 RNA from baseline was ~1 log greater (–1.53 vs –0.68) in patients randomized to receive enfuvirtide. Among the drawbacks of this agent are the requirement for twice-a-day injection, the occurrence of injection-site reactions in close to 100% of patients, and an increase in bacterial pneumonia in the enfuvirtide-treated patients compared with the control patients (4.68 vs 0.61 events per 100 patient-years) in the phase III studies.

1	Maraviroc is a CCR5 antagonist that interferes with HIV binding at the stage of co-receptor engagement. It was licensed in 2007 for treatment of HIV infection in combination with other agents in treatment-experienced patients infected with only CCR5-tropic (R5) virus resistant to multiple agents. The license was extended in 2009 to include treatment-naïve patients with R5 virus. A co-receptor tropism assay should be performed if one is considering the use of maraviroc to ensure that the potential patient is only harboring R5 viruses. In phase III trials of treatment-experienced patients randomized to receive optimal therapy plus maraviroc or placebo, 61% of patients randomized to maraviroc achieved HIV RNA levels <400 copies/mL compared with 28% of patients randomized to placebo. An allergic reaction–associated hepatotoxicity has been reported with maraviroc. Among the most common side effects of maraviroc are dizziness due to postural hypotension, cough, fever, colds, rash, muscle

1	reaction–associated hepatotoxicity has been reported with maraviroc. Among the most common side effects of maraviroc are dizziness due to postural hypotension, cough, fever, colds, rash, muscle and joint pain, and stomach pain. Maraviroc is a substrate of CYP3A and Pgp, and the recommend dose varies depending on concomitant medications. In combination with nucleoside analogues, tipranavir/ritonavir, enfuvirtide, and/or nevirapine, the dose is 300 mg twice daily. In the presence of CYP3A inhibitors, such as most protease inhibitors, the dose is 150 mg twice daily. In the presence of CYP3A inducers such as efavirenz, the dose is 600 mg twice daily.

1	Integrase inhibitors act by blocking the action of the HIV integrase enzyme and thus preventing integration of the HIV provirus into the host cell genome. They are among the most potent and safest of the antiretroviral drugs and frequently part of initial combination regimens. The three licensed integrase inhibitors are raltegravir, elvitegravir and dolutegravir.

1	Raltegravir is an inhibitor of the viral enzyme integrase and the first of this class to be approved. It acts by interfering with the binding of the preintegration complex to host DNA and as such is referred to as an integrase strand transfer inhibitor (INSTI). Raltegravir 1281 was approved in 2007 for treatment of HIV infection in combination with other agents in treatment-experienced patients, and the approval was extended in 2009 to include treatment-naïve patients. Raltegravir exhibits a wide range of activity against HIV-1 and HIV-2, including viruses with multiple resistance mutations to other classes of drugs. As with several other compounds, resistance to raltegravir comes at the expense of replicative fitness. In two phase III studies in which 436 patients with 3-class antiretroviral drug resistance were randomized to an optimized background regimen with raltegravir or placebo, 76% of patients receiving raltegravir achieved HIV RNA levels <400 copies/mL compared with 41% of

1	drug resistance were randomized to an optimized background regimen with raltegravir or placebo, 76% of patients receiving raltegravir achieved HIV RNA levels <400 copies/mL compared with 41% of patients randomized to the placebo arm. In contrast to many other antiretroviral drugs the side-effect profile of raltegravir is minimal, with similar side-effect profiles noted for the raltegravir and placebo groups.

1	Elvitegravir is an integrase inhibitor that was approved in 2012 as part of a fixed-dose combination tablet also containing tenofovir, emtricitabine, and cobicistat (Stribild). The cobicistat acts much in the same way as low-dose ritonavir to boost the concentrations of elvitegravir by inhibiting CYP3A such that once-a-day dosing of Stribild is sufficient. Elvitegravir demonstrates cross-resistance with raltegravir. In two randomized, controlled trials, elvitegravir was found to be noninferior to efavirenz in one study and noninferior to atazanavir/ritonavir in the other. The most common side effects experienced with elvitegravir are diarrhea, nausea, upper respiratory infection, and headache. The cobicistat component of the fixed-dose tablet inhibits tubular secretion of creatinine, resulting in increases in serum creatinine, and is not recommended for patients with estimated creatinine clearances <70 mL/min.

1	Dolutegravir was approved in 2013 for use as part of a combination regimen in either treatment-naïve or -experienced patients. It comes as a 50-mg tablet and is given once daily in treatment-naïve patients and twice daily in treatment-experienced patients. Isolates of HIV that have developed resistance to raltegravir or elvitegravir may still be sensitive to dolutegravir. Its main side effects are insomnia and headache. In two randomized, controlled trials it has been shown to be superior to either efavirenz (n = 833) or darunavir/ ritonavir (n = 484) in combination with nucleos(t)ide analogues due to lower rates of discontinuation. In a third trial of 822 patients it was shown to be noninferior to raltegravir.

1	The principles of therapy for HIV infection have been articulated by a panel sponsored by the U.S. Department of Health and Human Services as a working group of the NIH Office of AIDS Research Advisory Council. These principles are summarized in Table 226-23. As noted in these guidelines, cART of HIV infection does not lead to eradication or cure of HIV. The single possible exception to this is an individual with HIV infection who received an allogeneic stem cell transplant for treatment of acute myelogenous leukemia. His conditioning regimen included cytotoxic chemotherapy, total-body irradiation, and antithymocyte immunoglobulin. The donor cells were homozygous for the CCR5Δ32 mutation (see above) and thus resistant to HIV infection. Despite cART being stopped the day of the transplant, the patient has exhibited no signs of active HIV infection for more than 8 years.

1	Treatment decisions must take into account the fact that one is dealing with a chronic infection that requires daily therapy. While early therapy is generally the rule in infectious diseases, immediate treatment of every HIV-infected individual upon diagnosis may not be prudent, and therapeutic decisions must take into account the balance between risks and benefits. Patients initiating antiretroviral therapy must be willing to commit to life-long treatment and understand the importance of adherence to their prescribed regimen. The importance of adherence is illustrated by the observation that treatment interruption is associated with rapid increases in HIV RNA levels, rapid declines in CD4+ T cell counts, and an increased risk of clinical progression. While it seems reasonable to assume that the complications associated with cART could be minimized by Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1.

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1. Ongoing HIV replication leads to immune system damage, progression to AIDS, and systemic immune activation. 2. Plasma HIV RNA levels indicate the magnitude of HIV replication and the rate of CD4+ T cell destruction. CD4+ T cell counts indicate the current level of competence of the immune system. 3. Maximal suppression of viral replication is a goal of therapy; the greater the suppression the less likely the appearance of drug-resistant quasispecies. 4. The most effective therapeutic strategies involve the simultaneous initiation of combinations of effective anti-HIV drugs with which the patient has not been previously treated and that are not cross-resistant with antiretroviral agents that the patient has already received. 5. The antiretroviral drugs used in combination regimens should be used according to optimum schedules and dosages. 6.

1	5. The antiretroviral drugs used in combination regimens should be used according to optimum schedules and dosages. 6. The number of available drugs is limited. Any decisions on antiretroviral therapy have a long-term impact on future options for the patient. 7. Women should receive optimal antiretroviral therapy regardless of pregnancy status. 8. The same principles apply to children and adults. The treatment of HIV-infected children involves unique pharmacologic, virologic, and immunologic considerations. 9. Compliance is an important part of ensuring maximal effect from a given regimen. The simpler the regimen, the easier it is for the patient to be compliant. Source: Modified from Principles of Therapy of HIV Infection, USPHS, and the Henry J. Kaiser Family Foundation.

1	Source: Modified from Principles of Therapy of HIV Infection, USPHS, and the Henry J. Kaiser Family Foundation. intermittent treatment regimens designed to minimize exposure to the drugs in question, all efforts to do so have paradoxically been associated with an increase in serious adverse events in the patients randomized to intermittent therapy, suggesting that some “nonAIDS-associated” serious adverse events such as heart attack and stroke may be linked to HIV replication. Thus, unless contraindicated for reasons of toxicity, patients started on cART should remain on cART.

1	At present, the U.S. Department of Health and Human Services Guidelines panel recommends that everyone with HIV infection be treated with cART. The evidence for this is strongest for patients with CD4+ T cell counts <350/μL. Clinical trials are underway to more carefully determine the benefit of initiating therapy in patients with CD4+ T cell counts ≥350/μL. In addition, one may wish to administer a 6-week course of therapy to uninfected individuals immediately following a high-risk exposure to HIV. The combination of tenofovir and emtricitabine is also indicated for pre-exposure prophylaxis in individuals at high risk of HIV infection. For patients diagnosed with an opportunistic infection and HIV infection at the same time, one may consider a 2to 4-week delay in the initiation of antiretroviral therapy during which time treatment is focused on the opportunistic infection. This delay may decrease the severity of any subsequent immune reconstitution inflammatory syndrome by lowering

1	therapy during which time treatment is focused on the opportunistic infection. This delay may decrease the severity of any subsequent immune reconstitution inflammatory syndrome by lowering the antigenic burden of the opportunistic infection. For patients with advanced HIV infection (CD4+ <50 cells/μL), however, cART should be initiated as soon as possible.

1	Once the decision has been made to initiate therapy, the health care provider must decide which drugs to use as the first regimen. The decision regarding choice of drugs not only will affect the immediate response to therapy but also will have implications regarding options for future therapeutic regimens. The initial regimen is usually the most effective insofar as the virus has yet to develop significant resistance. HIV is capable of rapidly developing resistance to any single agent, and therapy must be given as a multidrug combination. Given that patients can be infected with viruses that harbor drug resistance mutations, it is recommended that a viral genotype be done prior to the initiation of therapy to optimize the selection of antiretroviral agents. The combination regimens currently recommended for initial therapy in any treatment-naïve patient are listed in Table 226-24. Other regimens containing abacavir and rilpivirine may be appropriate for patients with HIV RNA levels

1	recommended for initial therapy in any treatment-naïve patient are listed in Table 226-24. Other regimens containing abacavir and rilpivirine may be appropriate for patients with HIV RNA levels <100,000 copies/ mL. It is currently unclear whether treatment-naïve individuals with

1	I. Non-Nucleoside Reverse Transcriptase Inhibitor Based: II. Protease Inhibitor Based: III. Integrase Inhibitor Based: Source: Guidelines for the Use of Antiretroviral Agents in HIV-Infected Adults and Adolescents, USPHS.

1	<50 copies/mL of HIV RNA benefit from cART. Following the initiation of therapy one should expect a rapid, at least 1-log (tenfold) reduction in plasma HIV RNA levels within 1–2 months and then a slower decline in plasma HIV RNA levels to <50 copies/mL within 6 months. During this same time there should be a rise in the CD4+ T cell count of 100–150/μL that is also particularly brisk during the first month of therapy. Subsequently, one should anticipate a CD4+ T cell count increase of 50–100 cells/year until numbers approach normal. Many clinicians feel that failure to achieve these endpoints is an indication for a change in therapy. Other reasons for a change in therapy include a persistently declining CD4+ T cell count, a consistent increase in HIV RNA levels to >200 copies/mL, clinical deterioration, or drug toxicity (Table 226-25). As in the case of initiating therapy, changing therapy may have a lasting impact on future therapeutic options. When changing therapy because of

1	deterioration, or drug toxicity (Table 226-25). As in the case of initiating therapy, changing therapy may have a lasting impact on future therapeutic options. When changing therapy because of treatment failure (clinical progression or worsening laboratory parameters), it is important to attempt to provide a regimen with at least two new active drugs. This decision can be guided by resistance testing (see below). In the patient in whom a change is made for reasons of drug toxicity, a simple replacement of one drug is reasonable. It should be stressed that in attempting to sort out a drug toxicity it may be advisable to hold all therapy for a period of time to distinguish between drug toxicity and disease progression. Drug toxicity will usually begin to show signs of reversal within 1–2 weeks. Prior to changing a treatment regimen because of drug failure, it is important to ensure that the patient has been adherent to the prescribed regimen. As in the case of initial therapy, the

1	weeks. Prior to changing a treatment regimen because of drug failure, it is important to ensure that the patient has been adherent to the prescribed regimen. As in the case of initial therapy, the simpler the new therapeutic regimen, the easier it is for the patient to be compliant. Plasma HIV RNA levels should be monitored every 3–6 months during therapy and more frequently if one is contemplating a change in regimen due to an increase in viral load or immediately following a change in regimen.

1	In order to determine an optimal therapeutic regimen for initial therapy or for a patient on a failing regimen, one may attempt to measure antiretroviral drug susceptibility through genotyping or phenotyping of HIV quasispecies and to determine adequacy of dosing through measurement of drug levels. Genotyping may be done through cDNA sequencing. Phenotypic assays typically measure the Less than a 1-log drop in plasma HIV RNA by 4 weeks following the initiation of therapy A reproducible significant increase (defined as threefold or greater) from the nadir of plasma HIV RNA level not attributable to intercurrent infection, vaccination, or test methodology aGenerally speaking, a change should involve the initiation of at least two drugs felt to be effective in the given patient. The exception to this is when change is being made to manage toxicity, in which case a single substitution is reasonable.

1	Source: Guidelines for the Use of Antiretroviral Agents in HIV-Infected Adults and Adolescents, USPHS.

1	enzymatic activity of viral enzymes in the presence or absence of different concentrations of different drugs and have also been used to determine co-receptor tropism. These assays will generally detect quasispecies present at a frequency of ≥10%. NextGen sequencing may allow detection of quasispecies at frequencies down to 1%. It is generally recommended that resistance testing be used in selecting initial therapy in settings where the risk of transmission of resistant virus is high (such as the United States and Europe) and in determining new regimens for patients experiencing virologic failure while on therapy. Resistance testing may be of particular value in distinguishing drug-resistant virus from poor patient compliance. Due to the rapid rate at which drug-resistant viruses revert to wild-type, it is recommended that resistance testing performed in the setting of drug failure be carried out while the patient is still on the failing regimen. Measurement of plasma drug levels can

1	wild-type, it is recommended that resistance testing performed in the setting of drug failure be carried out while the patient is still on the failing regimen. Measurement of plasma drug levels can also be used to tailor an individual treatment. The inhibitory quotient, defined as the trough blood level/IC50 of the patient’s virus, is used by some to determine the adequacy of dosing of a given treatment regimen. Despite the best of efforts there will still be patients with ongoing high levels of HIV replication while receiving the best available therapy. These patients will receive benefit from remaining on antiretroviral therapy even though it is not fully suppressive.

1	In addition to the licensed medications discussed above, a large number of experimental agents are being evaluated as possible therapies for HIV infection. Therapeutic strategies are being developed to interfere with virtually every step of the replication cycle of the virus (Fig. 226-3). In addition, as more is discovered about the role of the immune system in controlling viral replication, additional strategies, generically referred to as “immune-based therapies,” are being developed as a complement to antiviral therapy. Among the antiviral agents in early clinical trials are additional nucleoside and nucleotide analogues, protease inhibitors, fusion inhibitors, receptor and co-receptor antagonists, and integrase inhibitors as well as new antiviral strategies including antisense nucleic acids and maturation inhibitors. Among the immune-based therapies being evaluated are IFN-α, bone marrow transplantation, adoptive transfer of lymphocytes genetically modified to resist infection or

1	acids and maturation inhibitors. Among the immune-based therapies being evaluated are IFN-α, bone marrow transplantation, adoptive transfer of lymphocytes genetically modified to resist infection or enhance HIV-specific immunity, active immunotherapy with inactivated HIV or its components, IL-7, and IL-15.

1	Health care workers, especially those who deal with large numbers of HIV-infected patients, have a small but definite risk of becoming infected with HIV as a result of professional activities (see “Occupational Transmission of HIV: Health Care Workers, Laboratory Workers, and the Health Care Setting,” above). The first case of HIV transmission from a patient to health care worker was reported in 1984. Occupational transmission of HIV has been reported in most countries; as noted above, the global number of HIV infections among health care workers attributable to punctures/cuts has been estimated to be 1000 cases (range, 200–5000) per year.

1	In the United States 57 health care workers for whom case investigations were completed as of 2010 had documented seroconversions to HIV following occupational exposures. The routes of exposure resulting in infection were as follows: 48 percutaneous (puncture/cut injury); 5 mucocutaneous (mucous membrane and/or skin); 2 both percutaneous and mucocutaneous; and 2 of unknown route. Of the 57 health care personnel, 49 were exposed to HIV-infected blood; 3 to concentrated virus in a laboratory; 1 to visibly bloody fluid; and 4 to an unspecified fluid. The individuals with documented seroconversions included 19 laboratory workers (16 of whom were clinical laboratory workers), 24 nurses, 6 physicians, 2 surgical technicians, 1 dialysis technician, 1 respiratory therapist, 1 health aide, 1 embalmer/ morgue technician, and 2 housekeeper/maintenance workers. In addition, more than 140 possible cases of occupationally acquired HIV infection have been reported among health care personnel in the

1	morgue technician, and 2 housekeeper/maintenance workers. In addition, more than 140 possible cases of occupationally acquired HIV infection have been reported among health care personnel in the United States. The number of these workers who actually acquired their infection through occupational exposures is not known. Taken together, data from several large studies suggest that the risk of HIV 1283 infection following a percutaneous exposure to HIV-contaminated blood is ~0.3%, and after a mucous membrane exposure, ~0.09%. Although episodes of HIV transmission after nonintact skin exposure have been documented, the average risk for transmission by this route has not been precisely quantified but is estimated to be less than the risk for mucous membrane exposures. The risk for transmission after exposure to fluids or tissues other than HIV-infected blood also has not been quantified but is probably considerably lower than for blood exposures. A seroprevalence survey of 3420 orthopedic

1	after exposure to fluids or tissues other than HIV-infected blood also has not been quantified but is probably considerably lower than for blood exposures. A seroprevalence survey of 3420 orthopedic surgeons, 75% of whom practiced in an area with a relatively high prevalence of HIV infection and 39% of whom reported percutaneous exposure to patient blood, usually through an accident involving a suture needle, failed to reveal any cases of possible occupational infection, suggesting that the risk of infection with a suture needle may be considerably less than that with a blood-drawing (hollow-bore) needle.

1	Most cases of health care worker seroconversion occur as a result of needle-stick injuries. When one considers the circumstances that result in needle-stick injuries, it is immediately obvious that adhering to the standard guidelines for dealing with sharp objects would result in a significant decrease in this type of accident. In one study, 27% of needle-stick injuries resulted from improper disposal of the needle (over half of these were due to recapping the needle), 23% occurred during attempts to start an IV line, 22% occurred during blood drawing, 16% were associated with an IM or SC injection, and 12% were associated with giving an IV infusion. Clinicians should consider potential occupational exposures to

1	HIV as urgent medical concerns to ensure timely postexposure management and possible administration of postexposure antiretroviral prophylaxis (PEP). Recommendations regarding PEP must take into account that a variety of circumstances determine the risk of transmission of HIV following occupational exposure. In this regard, several factors have been associated with an increased risk for occupational transmission of HIV infection, including deep injury, the presence of visible blood on the instrument causing the exposure, injury with a device that had been placed in the vein or artery of the source patient, terminal illness in the source patient, and lack of postexposure cART in the exposed health care worker. Other important considerations when considering PEP in the health care worker include known or suspected pregnancy or breast-feeding, the possibility of exposure to drug-resistant virus, and toxicities of PEP regimens. Regardless of the decision to use PEP, the wound should be

1	include known or suspected pregnancy or breast-feeding, the possibility of exposure to drug-resistant virus, and toxicities of PEP regimens. Regardless of the decision to use PEP, the wound should be cleansed immediately and antiseptic applied. If a decision is made to offer PEP, U.S. Public Health Service guidelines recommend (1) a combination of two nucleoside analogue reverse transcriptase inhibitors given for 4 weeks for less severe exposures, or (2) a combination of two nucleoside analogue reverse transcriptase inhibitors plus a third drug given for 4 weeks for more severe exposures. Most clinicians administer the latter regimen in all cases in which a decision is made to treat. Detailed guidelines are available from the Updated U.S. Public Health Service Guidelines for the Management of Occupational Exposures to HIV and Recommendations for Postexposure Prophylaxis (CDC, 2005). The report emphasizes the importance of adherence to PEP when it is indicated, follow-up of exposed

1	of Occupational Exposures to HIV and Recommendations for Postexposure Prophylaxis (CDC, 2005). The report emphasizes the importance of adherence to PEP when it is indicated, follow-up of exposed workers to improve PEP adherences, monitoring for adverse events (including seroconversion), and expert consultation in the management of exposures.

1	For consultation on the treatment of occupational exposures to HIV and other bloodborne pathogens, the clinician managing the exposed patient can call the National Clinicians’ Post-Exposure Prophylaxis Hotline (PEPline) at 888-448-4911. This service is available 24 hours a day at no charge. (Additional information on the Internet is available at www.nccc.ucsf.edu.) PEPline support may be especially useful in challenging situations, such as when drug-resistant HIV strains are suspected or the health care worker is pregnant. Health care workers can minimize their risk of occupational HIV infection by following the CDC guidelines of July 1991, which include adherence to universal precautions, refraining from direct patient care if one has exudative lesions or weeping dermatitis, and disinfecting and sterilizing reusable devices employed in invasive procedures.

1	Human Immunodeficiency Virus Disease: AIDS and Related Disorders 1284 The premise of universal precautions is that every specimen should be handled as if it came from someone infected with a bloodborne pathogen. All samples should be double-bagged, gloves should be worn when drawing blood, and spills should be immediately disinfected with bleach. In attempting to put this small but definite risk to the health care worker in perspective, it is important to point out that ~200 health care workers die each year as a result of occupationally acquired hepatitis B infection. The tragedy in this instance is that these infections and deaths due to HBV could be greatly decreased by more extended use of the HBV vaccine. The risk of HBV infection following a needle-stick injury from a hepatitis antigen–positive patient is much higher than the risk of HIV infection (see “Transmission,” above). There are multiple examples of needle-stick injuries where the patient was positive for both HBV and HIV

1	patient is much higher than the risk of HIV infection (see “Transmission,” above). There are multiple examples of needle-stick injuries where the patient was positive for both HBV and HIV and the health care worker became infected only with HBV. For these reasons, it is advisable, given the high prevalence of HBV infection in HIV-infected individuals, that all health care workers dealing with HIV-infected patients be immunized with the HBV vaccine. TB is another infection common to HIV-infected patients that can be transmitted to the health care worker. For this reason, all health care workers should know their PPD status, have it checked yearly, and receive 6 months of isoniazid treatment if their skin test converts to positive. In addition, all patients in whom a diagnosis of TB is being entertained should be placed immediately in respiratory isolation, pending results of the diagnostic evaluation. The emergence of drug-resistant organisms, including the extensively drug-resistant

1	entertained should be placed immediately in respiratory isolation, pending results of the diagnostic evaluation. The emergence of drug-resistant organisms, including the extensively drug-resistant TB strains that have been identified in Africa, has made TB an increasing problem for health care workers. This is particularly true for the health care worker with preexisting HIV infection. One of the most charged issues ever to come between health care workers and patients is that of transmission of infection from HIV-infected health care workers to their patients. This is discussed in “Occupational Transmission of HIV: Health Care Workers, Laboratory Workers, and the Health Care Setting,” above. Theoretically, the same universal precautions that are used to protect the health care worker from the HIV-infected patient will also protect the patient from the HIV-infected health care worker.

1	Given that human behavior, especially human sexual behavior, is extremely difficult to change, a critical modality for preventing the spread of HIV infection is the development of a safe and effective vaccine. Historically, vaccines have provided a safe, cost-effective, and efficient means of preventing illness, disability, and death from infectious diseases. Successful vaccines for the most part are predicated on the assumptions that the body can mount an adequate immune response to the microbe or virus in question during natural infection and that the vaccine will mimic the natural response to infection. Even with serious diseases, such as smallpox, poliomyelitis, measles, and influenza among others, the body in the vast majority of cases clears the infectious agent and provides protection, which is usually life-long against future exposure. Unfortunately, this is not the case with HIV infection since the natural immune response to HIV infection is unable to clear the virus from the

1	which is usually life-long against future exposure. Unfortunately, this is not the case with HIV infection since the natural immune response to HIV infection is unable to clear the virus from the body and cases of superinfection are not uncommon. Some of the factors that contribute to the problematic nature of development of a preventive HIV vaccine are the high mutability of the virus, the fact that the infection can be transmitted by cell-free or cell-associated virus, the fact that the HIV provirus integrates itself into the genome of the target cell and may remain in a latent form unexposed to the immune system, the likely need for the development of effective mucosal immunity, and the fact that it has been difficult to establish the precise correlates of protective immunity to HIV infection. A fraction of a percent of HIV-infected individuals are “elite controllers” in that they maintain extremely low and even undetectable levels of viremia in the absence of cART, and a number of

1	A fraction of a percent of HIV-infected individuals are “elite controllers” in that they maintain extremely low and even undetectable levels of viremia in the absence of cART, and a number of individuals have been exposed to HIV multiple times but remain uninfected; these facts suggest that there are elements of host defense or an HIV-specific immune response that have the potential to be protective. Early attempts to develop a vaccine with the envelope protein gp120 aimed at inducing neutralizing antibodies in humans were unsuccessful in that the elicited antisera failed to neutralize primary isolates of HIV cultured and tested in fresh peripheral blood mononuclear cells. In this regard, two phase 3 trials were undertaken in the United States and Thailand using soluble gp120, and the vaccines failed to protect human volunteers from HIV infection. In addition, two separate vaccine trials aimed at eliciting CD8+ T cell responses to prevent infection and, if unsuccessful in preventing

1	failed to protect human volunteers from HIV infection. In addition, two separate vaccine trials aimed at eliciting CD8+ T cell responses to prevent infection and, if unsuccessful in preventing infection to control postinfection viremia, also failed at both goals. Recently, a vaccine using a poxvirus vector prime expressing various viral proteins followed by an envelope protein boost was tested in a 16,000-person clinical trial (RV144) conducted in Thailand among predominantly low-prevalence heterosexuals. The vaccine provided the first positive, albeit very modest, signal ever reported in an HIV vaccine trial, showing 31% protection against acquisition of infection. Such a result is certainly not sufficient justification for clinical use of the vaccine, but it served as an important first step in the direction of the development of a safe and effective vaccine against HIV infection. Follow-up studies of RV144 indicate that nonneutralizing or weakly neutralizing antibody responses

1	step in the direction of the development of a safe and effective vaccine against HIV infection. Follow-up studies of RV144 indicate that nonneutralizing or weakly neutralizing antibody responses against certain constant epitopes in the otherwise highly variable V1-V2 region of the HIV envelope may be associated with the modest degree of protection observed in that clinical trial. Additional studies are planned in attempts to improve on the results of RV144 by a variety of approaches, including increasing the number of vaccine boosts with envelope protein.

1	An area of HIV vaccine research that is currently being actively pursued is the attempt to induce broadly neutralizing antibodies by developing as immunogens for vaccination certain epitopes on the HIV envelope that are the targets of naturally occurring broadly neutralizing antibodies during HIV infection. It is curious that only about 20% of HIV-infected individuals develop broadly neutralizing antibodies in response to natural infection and they do so only after 2 to 3 years of ongoing infection. By the time these antibodies appear, they can neutralize a broad range of primary HIV isolates, but they appear to be ineffective against the autologous virus in the infected subject. Upon close examination, these broadly neutralizing antibodies manifest a high degree of somatic mutations that were accumulated over time and are responsible for their affinity maturation and broadly neutralizing capacity. The goal of current efforts is to develop the conformationally correct HIV envelope

1	were accumulated over time and are responsible for their affinity maturation and broadly neutralizing capacity. The goal of current efforts is to develop the conformationally correct HIV envelope epitopes that, when used as immunogens, would direct the immune response of an uninfected individual to the production of broadly neutralizing antibodies over a reasonable time frame by sequential immunizations. It remains to be seen whether this approach will be feasible.

1	Education, counseling, and behavior modification are the cornerstones of any HIV prevention strategy. A major problem in the United States and elsewhere is that many infections are passed on by those who do not know that they are infected. Of the ~1.1 million persons in the United States who are HIV-infected, it is estimated that ~16–18% do not know their HIV status and approximately 49% of all new infections are transmitted by those people who are not aware that they are infected. In this regard, the CDC has recommended that HIV testing become part of routine medical care and that all individuals between the ages of 13 and 64 years be tested at least one time. These individuals should be informed of the testing and be tested without the need for written informed consent. Each individual could “opt out” of testing, but testing would otherwise be routinely administered. Individuals who are practicing high-risk behavior should be tested more often. In addition to identifying individuals

1	“opt out” of testing, but testing would otherwise be routinely administered. Individuals who are practicing high-risk behavior should be tested more often. In addition to identifying individuals who might benefit from cART, information gathered from such an approach should serve as the basis for behavior-modification programs, both for infected individuals who may be unaware of their HIV status and who could infect others and for uninfected individuals practicing high-risk behavior. The practice of “safer sex” is the most effective way for sexually active uninfected individuals to avoid contracting HIV infection and for infected individuals to avoid spreading infection. Abstinence from sexual relations is the only absolute way to prevent sexual transmission of HIV infection. However, for many individuals this may not be feasible, and there are a number of relatively safe practices that can markedly decrease the chances of transmission of HIV infection. Partners engaged in monogamous

1	many individuals this may not be feasible, and there are a number of relatively safe practices that can markedly decrease the chances of transmission of HIV infection. Partners engaged in monogamous sexual relationships who wish to be assured of safety should both be tested for HIV antibody. If both are negative, it must be understood that any divergence from monogamy puts both partners at risk; open discussion of the importance of honesty in such relationships should be encouraged. When the HIV status of either partner is not known, or when one partner is positive, there are a number of options. Use of condoms can markedly decrease the chance of HIV transmission. It should be remembered that condoms are not 100% effective in preventing transmission of HIV infection, and there is a ~10% failure rate of condoms used for contraceptive purposes. Most condom failures result from breakage or improper usage, such as not wearing the condom for the entire period of intercourse. Latex condoms

1	rate of condoms used for contraceptive purposes. Most condom failures result from breakage or improper usage, such as not wearing the condom for the entire period of intercourse. Latex condoms are preferable, since virus has been shown to leak through natural skin condoms. Petroleum-based gels should never be used for lubrication of the condom, since they increase the likelihood of condom rupture. Some men who have sex with men practice fellatio as a “minimal risk” activity compared with anal intercourse. It should be emphasized that receptive fellatio is definitely not safe sex, and although the incidence of transmission via fellatio is considerably less than that of rectal or vaginal intercourse, there has been documentation of transmission of HIV where receptive fellatio was the only sexual act performed (see “Transmission,” above). Topical microbicides composed of gels containing antiretroviral drugs have been shown to be efficacious in preventing acquisition of HIV infection in

1	sexual act performed (see “Transmission,” above). Topical microbicides composed of gels containing antiretroviral drugs have been shown to be efficacious in preventing acquisition of HIV infection in women engaging in vaginal intercourse. However, there has been a considerable degree of variability in efficacy related to the variable adherence of participants to the use of the intervention. In general, it is felt that microbicides can be quite efficacious; however, adherence is a major stumbling block to 1285 their broad effectiveness. Pre-exposure prophylaxis (PreP) using oral antiretroviral drugs on a daily basis in uninfected men who have sex with men and transgender women has been shown to be efficacious in preventing acquisition of HIV infection. The degree of efficacy can be very high (>90%) if subjects adhere strictly to the regimen. However, adherence has proven to be a problem in maximizing the overall effectiveness of this approach.

1	Adult male circumcision has been shown to result in a 50% to 65% reduction in HIV acquisition in the circumcised subject. Clearly, this approach has considerable potential as a preventive strategy for HIV infection and is currently being pursued, particularly in developing nations, as a component of HIV prevention. The most effective way to prevent transmission of HIV infection among IDUs is to stop the use of injectable drugs. Unfortunately, that is extremely difficult to accomplish unless the individual enters a treatment program. For those who will not or cannot participate in a drug treatment program and who will continue to inject drugs, the avoidance of sharing of needles and other paraphernalia (“works”) is the next best way to avoid transmission of infection. However, the cultural and social factors that contribute to the sharing of paraphernalia are complex and difficult to overcome. In addition, needles and syringes may be in short supply. Under these circumstances,

1	cultural and social factors that contribute to the sharing of paraphernalia are complex and difficult to overcome. In addition, needles and syringes may be in short supply. Under these circumstances, paraphernalia should be cleaned after each usage with a virucidal solution, such as undiluted sodium hypochlorite (household bleach). Programs that provide sterile needles to addicts in exchange for used needles have resulted in a marked decrease in HIV transmission without increasing the use of injection drugs. It is important for IDUs to be tested for HIV infection and counseled to avoid transmission to their sexual partners. Oral PreP also is effective in preventing acquisition of HIV infections among IDUs. Prevention of transmission through blood or blood products and prevention of mother-to-child transmission are discussed in “Transmission,” above.

1	Umesh D. Parashar, Roger I. Glass Acute infectious gastroenteritis is a common illness that affects persons of all ages worldwide. It is a leading cause of mortality among children in developing countries, accounting for an estimated 0.7 million deaths each year, and is responsible for up to 10–12% of all hospitalizations among children in industrialized countries, including the United States. Elderly persons, especially those with debilitating health conditions, also are at risk of severe complications and death from acute gastroenteritis. Among healthy young adults, acute gastroenteritis is rarely fatal but incurs substantial medical and social costs, including those of time lost from work.

1	Several enteric viruses have been recognized as important etiologic agents of acute infectious gastroenteritis (Table 227-1, Fig. 227-1). Although most viral gastroenteritis is caused by RNA viruses, the DNA viruses that are occasionally involved (e.g., adenovirus types 40 and 41) are included in this chapter. Illness caused by these viruses is characterized by the acute onset of vomiting and/or diarrhea, which may be accompanied by fever, nausea, abdominal cramps, anorexia, and malaise. As shown in Table 227-2, several features can help distinguish gastroenteritis caused by viruses from that caused by bacterial agents. However, the distinction based on clinical and epidemiologic parameters alone is often difficult, and laboratory tests are required to confirm the diagnosis.

1	HuMAN CALICIVIRuSES Etiologic Agent The Norwalk virus is the prototype strain of a group of small (27–40 nm), nonenveloped, round, icosahedral viruses with relatively amorphous surface features on visualization by electron microscopy. These viruses have been difficult to classify because they have not been adapted to growth in cell culture and no animal models are available. Molecular cloning and characterization have demonstrated that the viruses have a single, positive-strand RNA genome ~7.5 kb in length and possess a single virion-associated protein—similar to that of typical caliciviruses—with a molecular mass of 60 kDa. On the basis of these molecular characteristics, these viruses are presently classified in two genera belonging to the family Caliciviridae: the noroviruses and the sapoviruses (previously called Norwalk-like viruses and Sapporo-like viruses, respectively).

1	Epidemiology Infections with the Norwalk and related human caliciviruses are common worldwide, and most adults have antibodies to these viruses. Antibody is acquired at an earlier age in developing countries—a pattern consistent with the presumed fecal-oral mode of transmission. Infections occur year-round, although, in temperate climates, a distinct increase has been noted in cold-weather months. Noroviruses may be the most common infectious agents of mild gastroenteritis in the community and affect all age groups, whereas sapoviruses primarily cause gastroenteritis in children. Noroviruses also cause traveler’s diarrhea, and outbreaks have occurred among military personnel deployed to various parts of the world. The limited data available indicate that norovirus may be the second most common viral agent (after rotavirus) among young Norovirus Caliciviridae Positive-sense All ages + + EM, RT-PCR single-strand RNA

1	Norovirus Caliciviridae Positive-sense All ages + + EM, RT-PCR single-strand RNA Sapovirus Caliciviridae Positive-sense Children <5 years + EM, RT-PCR single-strand RNA Astrovirus Astroviridae Positive-sense Children <5 years + EM, EIA, RT-PCR single-strand RNA Adenovirus (mainly types Adenoviridae Double-strand DNA Children <5 years +/+ + EM, EIA (commercial), PCR 40 and 41) Abbreviations: EIA, enzyme immunoassay; EM, electron microscopy; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RT-PCR, reverse-transcription PCR.

1	Abbreviations: EIA, enzyme immunoassay; EM, electron microscopy; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RT-PCR, reverse-transcription PCR. children and the most common agent among older children and adults. In the United States, with the decline in severe rotavirus disease following implementation of rotavirus vaccines, norovirus has become the leading cause of medically attended gastroenteritis in young children. Noroviruses are also recognized as the major cause of epidemics of gastroenteritis worldwide. In the United States, >90% of outbreaks of nonbacterial gastroenteritis are caused by noroviruses.

1	Virus is transmitted predominantly by the fecal-oral route but is also present in vomitus. Because an inoculum with very few viruses can be infectious, transmission can occur by aerosolization, by contact with contaminated fomites, and by person-to-person contact. Viral shedding and infectivity are greatest during the acute illness, but challenge studies with Norwalk virus in volunteers indicate that viral antigen may be shed by asymptomatically infected persons and also by symptomatic persons before the onset of symptoms and for several weeks after the resolution of illness. Viral shedding can be prolonged in immunocompromised individuals.

1	Pathogenesis The exact sites and cellular receptors for attachment of viral particles have not been determined. Data suggest that carbohydrates that are similar to human histo-blood group antigens and are present on the gastroduodenal epithelium of individuals with the secretor phenotype may serve as ligands for the attachment of Norwalk virus. Additional studies must more fully elucidate norovirus-carbohydrate interactions, including potential strain-specific variations. After the infection of volunteers, reversible lesions are noted in the upper jejunum, with broadening and blunting of the villi, shortening of the microvilli, vacuolization of the lining epithelium, crypt hyperplasia, and infiltration of the lamina propria by polymorphonuclear neutrophils and lymphocytes. The lesions persist for at least 4 days after the resolution of symptoms and are associated with malabsorption of carbohydrates and fats and a decreased level of brush-border enzymes.

1	FIGuRE 227-1 Viral agents of gastroenteritis. NV, norovirus; SV, sapovirus. Adenylate cyclase activity is not altered. No histopathologic changes are seen in the stomach or colon, but gastric motor function is delayed, and this alteration is believed to contribute to the nausea and vomiting that are typical of this illness.

1	Clinical Manifestations Gastroenteritis caused by Norwalk and related human caliciviruses has a sudden onset following an average incubation period of 24 h (range, 12–72 h). The illness generally lasts 12–60 h and is characterized by one or more of the following symptoms: nausea, vomiting, abdominal cramps, and diarrhea. Vomiting is more prevalent among children, whereas a greater proportion of adults develop diarrhea. Constitutional symptoms are common, including headache, fever, chills, and myalgias. The stools are characteristically loose and watery, without blood, mucus, or leukocytes. White cell counts are generally normal; rarely, leukocytosis with relative lymphopenia may be observed. Death is a rare outcome and usually results from severe dehydration in vulnerable persons (e.g., elderly patients with debilitating health conditions).

1	Immunity Approximately 50% of persons challenged with Norwalk virus become ill and acquire short-term immunity against the infecting strain. Immunity to Norwalk virus appears to correlate inversely with level of antibody; i.e., persons with higher levels of preexisting antibody to Norwalk virus are more susceptible to illness. This observation suggests that some individuals have a genetic predisposition to illness. Specific ABO, Lewis, and secretor blood group phenotypes may influence susceptibility to norovirus infection.

1	Diagnosis Cloning and sequencing of the genomes of Norwalk and several other human caliciviruses have allowed the development of assays based on polymerase chain reaction (PCR) for detection of virus in stool and vomitus. Virus-like particles produced by expression of capsid proteins in a recombinant baculovirus vector have been used to develop enzyme immunoassays (EIAs) for detection of virus in stool or a serologic response to a specific viral antigen. These newer diagnostic techniques are considerably more sensitive than previous detection methods, such as electron microscopy, immune electron microscopy, and EIAs based on reagents derived from humans. However, no currently available single assay can detect all human caliciviruses because of their great genetic and antigenic diversity. In addition, the assays are still cumbersome and are available primarily in research laboratories, although they are increasingly being adopted by public health laboratories for routine screening of

1	In addition, the assays are still cumbersome and are available primarily in research laboratories, although they are increasingly being adopted by public health laboratories for routine screening of fecal specimens from patients affected by outbreaks of gastroenteritis. Commercial EIA kits have limited sensitivity and usefulness in clinical practice and are of greatest utility in outbreaks, in which many specimens are tested and only a few need be positive to identify norovirus as the cause.

1	The disease is self-limited, and oral rehydration therapy is generally adequate. If severe dehydration develops, IV fluid therapy is indicated. No specific antiviral therapy is available. Prevention Epidemic prevention relies on situation-specific measures, such as control of contamination of food and water, exclusion of ill food handlers, and reduction of person-to-person spread through good personal hygiene and disinfection of contaminated fomites. The role of immunoprophylaxis is not clear, given the lack of long-term immunity from natural disease, but efforts to develop norovirus vaccines are ongoing. In a clinical study, a candidate virus-like particle norovirus vaccine was shown to protect against homologous viral challenge.

1	ROTAVIRuS Etiologic Agent Rotaviruses are members of the family Reoviridae. The viral genome consists of 11 segments of double-strand RNA that are enclosed in a triple-layered, nonenveloped, icosahedral capsid 75 nm in diameter. Viral protein 6 (VP6), the major structural protein, is the target of commercial immunoassays and determines the group specificity of rotaviruses. There are seven major groups of rotavirus (A through G); human illness is caused primarily by group A and, to a much lesser extent, by groups B and C. Two outer-capsid proteins, VP7 (G-protein) and VP4 (P-protein), determine serotype specificity, induce neutralizing antibodies, and form the basis for binary classification of rotaviruses (G and P types). The segmented genome of rotavirus allows genetic reassortment (i.e., exchange of genome segments between viruses) during co-infection—a property that may play a role in viral evolution and that has been utilized in the development of reassortant animal-human

1	(i.e., exchange of genome segments between viruses) during co-infection—a property that may play a role in viral evolution and that has been utilized in the development of reassortant animal-human rotavirus–based vaccines.

1	Epidemiology Worldwide, nearly all children are infected with rotavirus by 3–5 years of age. Neonatal infections are common but are often asymptomatic or mild, presumably because of protection by maternal antibody or breast milk. Compared with rotavirus disease in industrialized countries, disease in developing countries occurs at a younger age, is less seasonal, and is more frequently caused by uncommon rotavirus strains. Moreover, because of suboptimal access to hydration therapy, rotavirus is a leading cause of diarrheal death among children in the developing world, with the highest mortality rates among children in sub-Saharan Africa and South Asia (Fig. 227-2).

1	First infections after 3 months of age are likely to be symptomatic, and the incidence of disease peaks among children 4–23 months of age. Reinfections are common, but the severity of disease decreases with each repeat infection. Therefore, severe rotavirus infections are less common among older children and adults than among younger individuals. Nevertheless, rotavirus can cause illness in parents and caretakers of children with rotavirus diarrhea, immunocompromised persons, travelers, and elderly individuals and should be considered in the differential diagnosis of gastroenteritis among adults.

1	In tropical settings, rotavirus disease occurs year-round, with less pronounced seasonal peaks than in temperate settings, where rota-virus disease occurs predominantly during the cooler fall and winter months. Before the introduction of rotavirus vaccine in the United States, the rotavirus season each year began in the Southwest during the autumn and early winter (October through December) and migrated across the continent, peaking in the Northeast during late winter and spring (March through May). The reasons for this characteristic pattern are not clear but may be correlated with state-specific differences in birth rates, which could influence the rate of accumulation of susceptible infants after each rotavirus season. After the implementation of routine vaccination of U.S. infants against rotavirus in 2006, the characteristic prevaccine geotemporal pattern of U.S. rota-virus was dramatically altered, and these changes were accompanied by substantial declines in rotavirus

1	against rotavirus in 2006, the characteristic prevaccine geotemporal pattern of U.S. rota-virus was dramatically altered, and these changes were accompanied by substantial declines in rotavirus detections by a national network of sentinel laboratories (Fig. 227-3). During the latest two seasons with available data (spanning 2010–2012), the number of rotavirus detections declined by 74–90% from the prevaccine baseline, and the common. While human rotavirus strains that possess a high degree of genetic homology with animal strains have been identified, animal-to-human transmission appears to be uncommon. Group B rotaviruses have been associated with several large epidemics of severe gastroenteritis among adults in China since 1982 and have also been identified in India. Group C rotaviruses have been associated with a small proportion of pediatric gastroenteritis cases in several countries worldwide.

1	<10 deaths per 100,000 10 to 50 deaths per 100,000 and ultimately destroy mature enterocytes in the villous epithe 50 to 100 deaths per 100,000 100 to 500 deaths per 100,000 lium of the proximal small intes-FIGuRE 227-2 Rotavirus mortality rates by country, per 100,000 children <5 years of age. (Reproduced tine. The loss of absorptive villous with permission from UD Parashar et al: J Infect Dis 200:S9, 2009.) epithelium, coupled with the pro liferation of secretory crypt cells, results in secretory diarrhea. Brush-annual proportion of rotavirus tests that were positive was below 10% border enzymes characteristic of differentiated cells are reduced, in both seasons (compared with a prevaccine baseline median of 26%). and this change leads to the accumulation of unmetabolized disac-A pattern of biennial increases in rotavirus activity has emerged during charides and consequent osmotic diarrhea. Studies in mice indithe five postvaccine seasons (2007–2012), but activity has remained cate

1	of biennial increases in rotavirus activity has emerged during charides and consequent osmotic diarrhea. Studies in mice indithe five postvaccine seasons (2007–2012), but activity has remained cate that a nonstructural rotavirus protein, NSP4, functions as an substantially below prevaccine levels in each season. enterotoxin and contributes to secretory diarrhea by altering epi-

1	During episodes of rotavirus-associated diarrhea, virus is shed in thelial cell function and permeability. In addition, rotavirus may large quantities in stool (107–1012/g). Viral shedding detectable by EIA evoke fluid secretion through activation of the enteric nervous usually subsides within 1 week but may persist for >30 days in immu-system in the intestinal wall. Data indicate that rotavirus antigennocompromised individuals; it may be detected for longer periods by emia and viremia are common among children with acute rotavirus sensitive molecular assays, such as PCR. The virus is transmitted pre-infection, although the antigen and RNA levels in serum are substandominantly through the fecal-oral route. Spread through respiratory tially lower than those in stool. secretions, person-to-person contact, or contaminated environmental surfaces has been postulated to explain the rapid acquisition of anti-Clinical Manifestations The clinical spectrum of rotavirus infection body in the

1	contact, or contaminated environmental surfaces has been postulated to explain the rapid acquisition of anti-Clinical Manifestations The clinical spectrum of rotavirus infection body in the first 3 years of life, regardless of sanitary conditions. ranges from subclinical infection to severe gastroenteritis leading to

1	At least 10 different G serotypes of group A rotavirus have been life-threatening dehydration. After an incubation period of 1–3 days, identified in humans, but only 5 types (G1 through G4 and G9) are the illness has an abrupt onset, with vomiting frequently preceding the onset of diarrhea. Up to one-third of patients may have a temperature of >39°C. The stools are characteristically loose and watery and only infrequently contain red or white cells. Gastrointestinal symptoms generally resolve60 in 3–7 days. Respiratory and neurologic features in children with rotavirus infection have been reported, but causal associations have not been proven. Moreover, rotavirus infection has been associated with a variety of other clinical conditions (e.g., sudden infant death syndrome, necrotizing enterocolitis, intussusception, Kawasaki’s disease, and type 1 diabetes), but no causal relationship has been confirmed with any of these syndromes.

1	Kawasaki’s disease, and type 1 diabetes), but no causal relationship has been confirmed with any of these syndromes. Rotavirus does not appear to be a major opportunistic pathogen in children with HIV infection. In severely immunodeficient chil dren, rotavirus can cause protracted diarrhea with prolonged viral excretion and, in rare instances, can disseminate systemically. Persons who are immunosuppressed for bone marrow transplantation also are at risk for severe or even fatal rotavirus disease.

1	FIGuRE 227-3 Percentage of rotavirus tests with positive results, by week of year, July– June, 2000–2012. The maximal or minimal percentage of rotavirus-positive tests for 2000– Immunity Protection against rotavirus disease 2006 may have occurred during any of the six baseline seasons. Data are from the National is correlated with the presence of virus-specific Respiratory and Enteric Virus Surveillance System. (Adapted from Centers for Disease Control and secretory IgA antibodies in the intestine Prevention, 2012.) and, to some extent, the serum. Because

1	Percent of tests rotavirus positive virus-specific IgA production at the intestinal surface is short lived, complete protection against disease is only temporary. However, each infection and subsequent reinfection confers progressively greater immunity; thus severe disease is most common among young children with first or second infections. Immunologic memory is believed to be important in the attenuation of disease severity upon reinfection. Diagnosis Illness caused by rotavirus is difficult to distinguish clinically from that caused by other enteric viruses. Because large quantities of virus are shed in feces, the diagnosis can usually be confirmed by a wide variety of commercially available EIAs or by techniques for detecting viral RNA, such as gel electrophoresis, probe hybridization, or PCR.

1	Rotavirus gastroenteritis can lead to severe dehydration. Thus appropriate treatment should be instituted early. Standard oral rehydration therapy is successful for most children who can take fluids by mouth, but IV fluid replacement may be required for patients who are severely dehydrated or are unable to tolerate oral therapy because of frequent vomiting. The therapeutic roles of probiotics, bismuth subsalicylate, enkephalinase inhibitors, and nitazoxanide have been evaluated in clinical studies but are not clearly defined. Antibiotics and antimotility agents should be avoided. In immunocompromised children with chronic symptomatic rotavirus disease, orally administered immunoglobulins or colostrum may result in the resolution of symptoms, but the best choices regarding agents and their doses have not been well studied, and treatment decisions are often empirical.

1	Prevention Efforts to develop rotavirus vaccines were pur sued because it was apparent—given the similar rates in less developed and industrialized nations—that improvements in hygiene and sanitation were unlikely to reduce disease incidence. The first rotavirus vaccine licensed in the United States in 1998 was withdrawn from the market within 1 year because it was linked with a low incidence of intussusception, a severe bowel obstruction.

1	In 2006, promising safety and efficacy results for two new rotavirus vaccines were reported from large clinical trials conducted in North America, Europe, and Latin America. Both vaccines are now recommended for routine immunization of all U.S. infants, and their use has rapidly led to a >70–80% decline in rotavirus hospitalizations and emergency department visits at hospitals across the United States. Indirect benefits from vaccination (i.e., herd immunity) have also been documented in many settings. In April 2009, the World Health Organization recommended the use of rotavirus vaccines in all countries worldwide. As of May 2013, a total of 42 countries, including 5 low-income countries in Africa and Asia, have incorporated rota-virus vaccine into their national childhood immunization programs. In Mexico and in Brazil, a decline in deaths from childhood diarrhea following introduction of rotavirus vaccines has been documented. Postmarketing surveillance has identified a low risk of

1	In Mexico and in Brazil, a decline in deaths from childhood diarrhea following introduction of rotavirus vaccines has been documented. Postmarketing surveillance has identified a low risk of intussusception in some countries; however, the benefits of vaccination exceed the risks, and no changes in vaccine administration policy have been implemented. The different epidemiology of rotavirus disease and the greater prevalence of co-infection with other enteric pathogens, of comorbidities, and of malnutrition in developing countries may adversely affect the performance of oral rotavirus vaccines, as is the case with oral vaccines against poliomyelitis, cholera, and typhoid in these regions. Therefore, evaluation of the efficacy of rotavirus vaccines in resource-poor settings of Africa and Asia was specifically recommended, and these trials have now been completed. As anticipated, the efficacy of rotavirus vaccines was moderate (50– 65%) in these settings when compared with that in

1	and Asia was specifically recommended, and these trials have now been completed. As anticipated, the efficacy of rotavirus vaccines was moderate (50– 65%) in these settings when compared with that in industrialized countries. Nevertheless, even a moderately efficacious rotavirus vaccine would be likely to have substantial public health benefits in these areas with a high disease burden.

1	Enteric adenoviruses of serotypes 40 and 41 belonging to subgroup F are 70to 80-nm viruses with double-strand DNA that cause ~2–12% of all diarrhea episodes in young children. Unlike adenoviruses that cause respiratory illness, enteric adenoviruses are difficult to cultivate in cell lines, but they can be detected with commercially available EIAs. Adenovirus types 31 and 42–49 have been linked to diarrhea in HIV-infected and other immunocompromised persons.

1	Astroviruses are 28to 30-nm viruses with a characteristic icosahedral structure and a positive-sense, single-strand RNA. At least seven serotypes have been identified, of which serotype 1 is most common. Astroviruses are primarily pediatric pathogens, causing ~2–10% of cases of mild to moderate gastroenteritis in children. The availability of simple immunoassays to detect virus in fecal specimens and of molecular methods to confirm and characterize strains will permit more comprehensive assessment of the etiologic role of these agents. Toroviruses are 100to 140-nm, enveloped, positive-strand RNA viruses that are recognized as causes of gastroenteritis in horses (Berne virus) and cattle (Breda virus). Their role as a cause of diarrhea in humans is still unclear, but studies from Canada have demonstrated associations between torovirus excretion and both nosocomial gastroenteritis and necrotizing enterocolitis in neonates. These associations require further evaluation.

1	Picobirnaviruses are small, bisegmented, double-strand RNA viruses that cause gastroenteritis in a variety of animals. Their role as primary causes of gastroenteritis in humans remains unclear, but several studies have found an association between picobirnaviruses and gastroen teritis in HIV-infected adults. Several other viruses (e.g., enteroviruses, reoviruses, pestiviruses, and parvovirus B) have been identified in the feces of patients with diarrhea, but their etiologic role in gastroenteritis has not been proven. Diarrhea has also been noted as a manifestation of infection with recently recognized viruses that primarily cause severe respiratory illness: the severe acute respiratory syndrome–associated coronavirus (SARS-CoV), influenza A/H5N1 virus, and the current pandemic strain of influenza A/H1N1 virus. Enterovirus, Parechovirus, and Jeffrey I. Cohen

1	Enteroviruses, members of the family Picornaviridae, are so designated because of their ability to multiply in the gastrointestinal tract. Despite their name, these viruses are not a prominent cause of gastroenteritis. Enteroviruses encompass more than 100 human serotypes: 3 serotypes of poliovirus, 21 serotypes of coxsackievirus A, 6 serotypes of coxsackievirus B, 28 serotypes of echovirus, enteroviruses 68–71, and multiple new enteroviruses (beginning with enterovirus 73) that have been identified by molecular techniques. Human enteroviruses have been reclassified into four species designated A–D. Echoviruses 22 and 23 have been reclassified as parechoviruses 1 and 2 on the basis of low nucleotide homology and differences in viral proteins. Enterovirus surveillance conducted in the United States by the Centers for Disease Control and Prevention (CDC) in 2007–2008 showed that the most common enterovirus serotype, coxsackievirus B1, was followed in frequency by echoviruses 18, 9, and

1	States by the Centers for Disease Control and Prevention (CDC) in 2007–2008 showed that the most common enterovirus serotype, coxsackievirus B1, was followed in frequency by echoviruses 18, 9, and 6; together, these four viruses accounted for 52% of all isolates.

1	Human enteroviruses contain a single-stranded RNA genome surrounded by an icosahedral capsid comprising four viral proteins. These viruses have no lipid envelope and are stable in acidic environments, Enterovirus, Parechovirus, and Reovirus Infections 1290 including the stomach. They are susceptible to chlorine-containing cleansers but resistant to inactivation by standard disinfectants (e.g., alcohol, detergents) and can persist for days at room temperature.

1	Much of what is known about the pathogenesis of enteroviruses has been derived from studies of poliovirus infection. After ingestion, poliovirus is thought to infect epithelial cells in the mucosa of the gastrointestinal tract and then to spread to and replicate in the submucosal lymphoid tissue of the tonsils and Peyer’s patches. The virus next spreads to the regional lymph nodes, a viremic phase ensues, and the virus replicates in organs of the reticuloendothelial system. In some cases, a second episode of viremia occurs and the virus replicates further in various tissues, sometimes causing symptomatic disease.

1	It is uncertain whether poliovirus reaches the central nervous system (CNS) during viremia or whether it also spreads via peripheral nerves. Since viremia precedes the onset of neurologic disease in humans, it has been assumed that the virus enters the CNS via the bloodstream. The poliovirus receptor is a member of the immunoglobulin super-family. Poliovirus infection is limited to primates, largely because their cells express the viral receptor. Studies demonstrating the poliovirus receptor in the end-plate region of muscle at the neuromuscular junction suggest that, if the virus enters the muscle during viremia, it could travel across the neuromuscular junction up the axon to the anterior horn cells. Studies of monkeys and of transgenic mice expressing the poliovirus receptor show that, after IM injection, poliovirus does not reach the spinal cord if the sciatic nerve is cut. Taken together, these findings suggest that poliovirus can spread directly from muscle to the CNS by neural

1	after IM injection, poliovirus does not reach the spinal cord if the sciatic nerve is cut. Taken together, these findings suggest that poliovirus can spread directly from muscle to the CNS by neural pathways.

1	Poliovirus can usually be cultured from the blood 3–5 days after infection, before the development of neutralizing antibodies. While viral replication at secondary sites begins to slow 1 week after infection, it continues in the gastrointestinal tract. Poliovirus is shed from the oropharynx for up to 3 weeks after infection and from the gastrointestinal tract for as long as 12 weeks; hypogammaglobulinemic patients can shed poliovirus for >20 years. During replication in the gastrointestinal tract, attenuated oral poliovirus can mutate, reverting to a more neurovirulent phenotype within a few days; however, additional mutations are probably required for full neurovirulence. One patient with hypogammaglobulinemia who had been infected 12 years earlier and was receiving IV immune globulin suddenly developed quadriplegia and respiratory muscle paralysis and died; analysis showed that the virus had reverted to a more wild-type sequence.

1	Humoral and secretory immunity in the gastrointestinal tract is important for the control of enterovirus infections. Enteroviruses induce specific IgM, which usually persists for <6 months, and specific IgG, which persists for life. Capsid protein VP1 is the predominant target of neutralizing antibody, which generally confers lifelong protection against subsequent disease caused by the same serotype but does not prevent infection or virus shedding. Enteroviruses also induce cellular immunity whose significance is uncertain. Patients with impaired cellular immunity are not known to develop unusually severe disease when infected with enteroviruses. In contrast, the severe infections in patients with agammaglobulinemia emphasize the importance of humoral immunity in controlling enterovirus infections. Disseminated enterovirus infections have occurred in hematopoietic cell transplant recipients. IgA antibodies are instrumental in reducing poliovirus replication in and shedding from the

1	infections. Disseminated enterovirus infections have occurred in hematopoietic cell transplant recipients. IgA antibodies are instrumental in reducing poliovirus replication in and shedding from the gastrointestinal tract. Breast milk contains IgA specific for enteroviruses and can protect humans from infection.

1	Enteroviruses have a worldwide distribution. More than 50% of nonpoliovirus enterovirus infections and more than 90% of poliovirus infections are subclinical. When symptoms do develop, they are usually nonspecific and occur in conjunction with fever; only a minority of infections are associated with specific clinical syndromes. The incubation period for most enterovirus infections ranges from 2 to 14 days but usually is <1 week.

1	Enterovirus infection is more common in socioeconomically disadvantaged areas, especially in those where conditions are crowded and in tropical areas where hygiene is poor. Infection is most common among infants and young children; serious illness develops most often during the first few days of life and in older children and adults. In developing countries, where children are infected at an early age, poliovirus infection has less often been associated with paralysis; in countries with better hygiene, older children and adults are more likely to be seronegative, become infected, and develop paralysis. Passively acquired maternal antibody reduces the risk of symptomatic infection in neonates. Young children are the most frequent shedders of enteroviruses and are usually the index cases in family outbreaks. In temperate climates, enterovirus infections occur most often in the summer and fall; no seasonal pattern is apparent in the tropics.

1	Most enteroviruses are transmitted primarily by the fecal-oral or oral-oral route. Patients are most infectious shortly before and after the onset of symptomatic disease, when virus is present in the stool and throat. The ingestion of virus-contaminated food or water also can cause disease. Certain enteroviruses (such as enterovirus 70, which causes acute hemorrhagic conjunctivitis) can be transmitted by direct inoculation from the fingers to the eye. Airborne transmission is important for some viruses that cause respiratory tract disease, such as coxsackievirus A21. Enteroviruses can be transmitted across the placenta from mother to fetus, causing severe disease in the newborn. The transmission of enteroviruses through blood transfusions or insect bites has not been documented. Nosocomial spread of coxsackievirus and echovirus has taken place in hospital nurseries.

1	CLINICAL FEATuRES Poliovirus Infection Most infections with poliovirus are asymptomatic. After an incubation period of 3–6 days, ~5% of patients present with a minor illness (abortive poliomyelitis) manifested by fever, malaise, sore throat, anorexia, myalgias, and headache. This condition usually resolves in 3 days. About 1% of patients present with aseptic meningitis (nonparalytic poliomyelitis). Examination of cerebrospinal fluid (CSF) reveals lymphocytic pleocytosis, a normal glucose level, and a normal or slightly elevated protein level; CSF polymorphonuclear leukocytes may be present early. In some patients, especially children, malaise and fever precede the onset of aseptic meningitis.

1	paralytic poliomyelitis The least common presentation is that of paralytic disease. After one or several days, signs of aseptic meningitis are followed by severe back, neck, and muscle pain and by the rapid or gradual development of motor weakness. In some cases the disease appears to be biphasic, with aseptic meningitis followed first by apparent recovery but then (1–2 days later) by the return of fever and the development of paralysis; this form is more common among children than among adults. Weakness is generally asymmetric, is proximal more than distal, and may involve the legs (most commonly); the arms; or the abdominal, thoracic, or bulbar muscles. Paralysis develops during the febrile phase of the illness and usually does not progress after defervescence. Urinary retention may also occur. Examination reveals weakness, fasciculations, decreased muscle tone, and reduced or absent reflexes in affected areas. Transient hyperreflexia sometimes precedes the loss of reflexes.

1	may also occur. Examination reveals weakness, fasciculations, decreased muscle tone, and reduced or absent reflexes in affected areas. Transient hyperreflexia sometimes precedes the loss of reflexes. Patients frequently report sensory symptoms, but objective sensory testing usually yields normal results. Bulbar paralysis may lead to dysphagia, difficulty in handling secretions, or dysphonia. Respiratory insufficiency due to aspiration, involvement of the respiratory center in the medulla, or paralysis of the phrenic or intercostal nerves may develop, and severe medullary involvement may lead to circulatory collapse. Most patients with paralysis recover some function weeks to months after infection. About two-thirds of patients have residual neurologic sequelae.

1	Paralytic disease is more common among older individuals, pregnant women, and persons exercising strenuously or undergoing trauma at the time of CNS symptoms. Tonsillectomy predisposes to bulbar poliomyelitis, and IM injections increase the risk of paralysis in the involved limb(s).

1	vaccine-associated poliomyelitis The risk of developing poliomyelitis after oral vaccination is estimated at 1 case per 2.5 million doses. The risk is ~2000 times higher among immunodeficient persons, especially in persons with hypoor agammaglobulinemia. Before 1997, an average of eight cases of vaccine-associated poliomyelitis occurred—in both vaccinees and their contacts—in the United States each year. With the change in recommendations first to a sequential regimen of inactivated poliovirus vaccine (IPV) and oral poliovirus vaccine (OPV) in 1997 and then to an all-IPV regimen in 2000, the number of cases of vaccine-associated polio declined. From 1997 to 1999, six such cases were reported in the United States; no cases have been reported since 1999.

1	postpolio syndrome The postpolio syndrome presents as a new onset of weakness, fatigue, fasciculations, and pain with additional atrophy of the muscle group involved during the initial paralytic disease 20–40 years earlier. The syndrome is more common among women and with increasing time after acute disease. The onset is usually insidious, and weakness occasionally extends to muscles that were not involved during the initial illness. The prognosis is generally good; progression to further weakness is usually slow, with plateau periods of 1–10 years. The postpolio syndrome is thought to be due to progressive dysfunction and loss of motor neurons that compensated for the neurons lost during the original infection and not to persistent or reactivated poliovirus infection.

1	Other Enteroviruses An estimated 5–10 million cases of symptomatic disease due to enteroviruses other than poliovirus occur in the United States each year. Among neonates, enteroviruses are the most common cause of aseptic meningitis and nonspecific febrile illnesses. Certain clinical syndromes are more likely to be caused by certain serotypes (Table 228-1). nonspecific febrile illness (sUmmer grippe) The most common clinical manifestation of enterovirus infection is a nonspecific febrile illness. After an incubation period of 3–6 days, patients present with an acute onset of fever, malaise, and headache. Occasional cases are associated with upper respiratory symptoms, and some cases include nausea and vomiting. Symptoms often last for 3–4 days, and most cases resolve in a week. While infections with other respiratory viruses occur more often from late fall to early spring, febrile illness due to enteroviruses frequently occurs in the summer and early fall.

1	Serotype(s) of Indicated Virus generalized disease of tHe newborn Most serious enterovirus infections 1291 in infants develop during the first week of life, although severe disease can occur up to 3 months of age. Neonates often present with an illness resembling bacterial sepsis, with fever, irritability, and lethargy. Laboratory abnormalities include leukocytosis with a left shift, thrombocytopenia, elevated values in liver function tests, and CSF pleocytosis. The illness can be complicated by myocarditis and hypotension, fulminant hepatitis and disseminated intravascular coagulation, meningitis or meningoencephalitis, or pneumonia. It may be difficult to distinguish neonatal enterovirus infection from bacterial sepsis, although a history of a recent virus-like illness in the mother provides a clue.

1	aseptic meningitis and encepHalitis In children and young adults, enteroviruses are the cause of up to 90% of cases of aseptic meningitis in which an etiologic agent can be identified. Patients with aseptic meningitis typically present with an acute onset of fever, chills, headache, photophobia, and pain on eye movement. Nausea and vomiting also are common. Examination reveals meningismus without localizing neurologic signs; drowsiness or irritability may also be apparent. In some cases, a febrile illness may be reported that remits but returns several days later in conjunction with signs of meningitis. Other systemic manifestations may provide clues to an enteroviral cause, including diarrhea, myalgias, rash, pleurodynia, myocarditis, and herpangina. Examination of the CSF invariably reveals pleocytosis; the CSF cell count shows a shift from neutrophil to lymphocyte predominance within 1 day of presentation, and the total cell count does not exceed 1000/μL. The CSF glucose level is

1	pleocytosis; the CSF cell count shows a shift from neutrophil to lymphocyte predominance within 1 day of presentation, and the total cell count does not exceed 1000/μL. The CSF glucose level is usually normal (in contrast to the low CSF glucose level in mumps), with a normal or slightly elevated protein concentration. Partially treated bacterial meningitis may be particularly difficult to exclude in some instances. Enteroviral meningitis is more common in summer and fall in temperate climates, while viral meningitis of other etiologies is more common in winter and spring. Symptoms ordinarily resolve within a week, although CSF abnormalities can persist for several weeks. Enteroviral meningitis is often more severe in adults than in children. Neurologic sequelae are rare, and most patients have an excellent prognosis.

1	Enteroviral encephalitis is much less common than enteroviral aseptic meningitis. Occasional highly inflammatory cases of enteroviral meningitis may be complicated by a mild form of encephalitis that is recognized on the basis of progressive lethargy, disorientation, and sometimes seizures. Less commonly, severe primary encephalitis may develop. An estimated 10–35% of cases of viral encephalitis are due to enteroviruses. Immunocompetent patients generally have a good prognosis. Patients with hypogammaglobulinemia, agammaglobulinemia, or severe combined immunodeficiency may develop chronic meningitis or encephalitis; about half of these patients have a dermatomyositis-like syndrome, with peripheral edema, rash, and myositis. They may also have chronic hepatitis. Patients may develop neurologic disease while receiving immunoglobulin replacement therapy. Echoviruses (especially echovirus 11) are the most common pathogens in this situation.

1	Paralytic disease due to enteroviruses other than poliovirus occurs sporadically and is usually less severe than poliomyelitis. Most cases are due to enterovirus 70 or 71 or to coxsackievirus A7 or A9. Guillain-Barré syndrome is also associated with enterovirus infection. While earlier studies suggested a link between enteroviruses and chronic fatigue syndrome, most recent studies have not demonstrated such an association.

1	pleUrodynia (bornHolm disease) Patients with pleurodynia present with an acute onset of fever and spasms of pleuritic chest or upper abdominal pain. Chest pain is more common in adults, and abdominal pain is more common in children. Paroxysms of severe, knifelike pain usually last 15–30 min and are associated with diaphoresis and tachypnea. Fever peaks within an hour after the onset of paroxysms and subsides when pain resolves. The involved muscles are tender to palpation, and a pleural rub may be detected. The white blood cell count and chest x-ray results are usually normal. Most cases are due to coxsackievirus B and occur during epidemics. Symptoms resolve in a few days, and recurrences are rare. Treatment includes the administration of nonsteroidal anti-inflammatory agents or the application of heat to the affected muscles.

1	Enterovirus, Parechovirus, and Reovirus Infections 1292 myocarditis and pericarditis Enteroviruses are estimated to cause up to one-third of cases of acute myocarditis. Coxsackievirus B and its RNA have been detected in pericardial fluid and myocardial tissue in some cases of acute myocarditis and pericarditis. Most cases of enteroviral myocarditis or pericarditis occur in newborns, adolescents, or young adults. More than two-thirds of patients are male. Patients often present with an upper respiratory tract infection that is followed by fever, chest pain, dyspnea, arrhythmias, and occasionally heart failure. A pericardial friction rub is documented in half of cases, and the electrocardiogram shows ST-segment elevations or STand T-wave abnormalities. Serum levels of myocardial enzymes are often elevated. Neonates commonly have severe disease, while most older children and adults recover completely. Up to 10% of cases progress to chronic dilated cardiomyopathy. Chronic constrictive

1	are often elevated. Neonates commonly have severe disease, while most older children and adults recover completely. Up to 10% of cases progress to chronic dilated cardiomyopathy. Chronic constrictive pericarditis may also be a sequela.

1	eXantHems Enterovirus infection is the leading cause of exanthems in children in the summer and fall. While exanthems are associated with many enteroviruses, certain types have been linked to specific syndromes. Echoviruses 9 and 16 have frequently been associated with exanthem and fever. Rashes may be discrete or confluent, beginning on the face and spreading to the trunk and extremities. Echovirus 9 is the most common cause of a rubelliform (discrete) rash. Unlike the rash of rubella, the enteroviral rash occurs in the summer and is not associated with lymphadenopathy. Roseola-like rashes develop after defervescence, with macules and papules on the face and trunk. The Boston exanthem, caused by echovirus 16, is a roseola-like rash. A variety of other rashes have been associated with enteroviruses, including erythema multiforme (see Fig. 25e-25) and vesicular, urticarial, petechial, or purpuric lesions. Enanthems also occur, including lesions that resemble the Koplik’s spots seen

1	enteroviruses, including erythema multiforme (see Fig. 25e-25) and vesicular, urticarial, petechial, or purpuric lesions. Enanthems also occur, including lesions that resemble the Koplik’s spots seen with measles (see Fig. 25e-2).

1	Hand-foot-and-moUtH disease (Fig. 228-1) After an incubation period of 4–6 days, patients with hand-foot-and-mouth disease present with fever, anorexia, and malaise; these manifestations are followed by the development of sore throat and vesicles (see Fig. 25e-23) on the buccal mucosa and often on the tongue and then by the appearance of tender vesicular lesions on the dorsum of the hands, sometimes with involvement of the palms. The vesicles may form bullae and quickly ulcerate. About one-third of patients also have lesions on the palate, uvula, or tonsillar pillars, and one-third have a rash on the feet (including the soles) or on the buttocks. The disease is highly infectious, with attack rates of close to 100% among young children. The lesions usually resolve in 1 week. Most cases are due to coxsackievirus A16 or enterovirus 71.

1	An epidemic of enterovirus 71 infection in Taiwan in 1998 resulted in thousands of cases of hand-foot-and-mouth dis ease or herpangina (see below). Severe complications included CNS disease, myocarditis, and pulmonary hemorrhage. About 90% of those who died were children ≤5 years old, and death was associated with pulmonary edema or pulmonary hemorrhage. CNS disease included aseptic meningitis, flaccid paralysis (similar to that seen in poliomyelitis), and rhombencephalitis with myoclonus and tremor or ataxia. The mean age of patients with CNS complications was 2.5 years, and MRI in cases with encephalitis usually showed brain-stem lesions. Follow-up of children at 6 months showed persistent dysphagia, cranial nerve palsies, hypoventilation, limb weakness, and atrophy; at 3 years, persistent neurologic sequelae were documented, with delayed development and impaired cognitive function.

1	Another epidemic of enterovirus 71 infection occurred in China in 2008–2010, with nearly 500,000 infections and 126 deaths. Infections were associated with fever, rash, brain-stem encephalitis with myoclonic jerks, and limb trembling; some cases progressed to seizures and coma. Lung findings included pulmonary edema and hemorrhage; while the level of creatine kinase MB was sometimes elevated, myocardial necrosis was generally not found. Cyclic epidemics occur every 2–3 years in other Asian countries. However, the virus circulates at lower rates in the United States, Europe, and Africa. In the United States, hand-foot-and-mouth disease is most commonly associated with coxsackievirus A16. Between November 2011 and February 2012, outbreaks of hand-foot-and-mouth disease

1	FIGuRE 228-1 Vesicular eruptions of the hand (A), foot (B), and mouth (C) of a 6-year-old boy with coxsackievirus A6 infection. (Images reprinted courtesy of Centers for Disease Control and Prevention/Emerging Infectious Diseases.) due to coxsackievirus A6 occurred in several U.S. states, and 19% of the affected persons were hospitalized. Herpangina Herpangina is usually caused by coxsackievirus A and presents as acute-onset fever, sore throat, odynophagia, and grayish-white papulovesicular lesions on an erythematous base that ulcerate. The lesions can persist for weeks; are present on the soft palate, anterior pillars of the tonsils, and uvula; and are concentrated in the posterior portion of the mouth. In contrast to herpes stomatitis, enteroviral herpangina is not associated with gingivitis. Acute lymphonodular pharyngitis associated with coxsackievirus A10 presents as white or yellow nodules surrounded by erythema in the posterior oropharynx. The lesions do not ulcerate.

1	acUte HemorrHagic conjUnctivitis Patients with acute hemorrhagic conjunctivitis present with an acute onset of severe eye pain, blurred vision, photophobia, and watery discharge from the eye. Examination reveals edema, chemosis, and subconjunctival hemorrhage and often shows punctate keratitis and conjunctival follicles as well (Fig. 228-2). Preauricular adenopathy is often found. Epidemics and nosocomial spread have been associated with enterovirus 70 and coxsackievirus A24. Systemic symptoms, including headache and fever, develop in 20% of cases, and recovery is usually complete in 10 days. The sudden onset and short duration of the illness help to distinguish acute hemorrhagic conjunctivitis from other ocular infections, such as those due to adenovirus and Chlamydia trachomatis. Paralysis has been associated with some cases of acute hemorrhagic conjunctivitis due to enterovirus 70 during epidemics.

1	otHer manifestations Enteroviruses are an infrequent cause of childhood pneumonia and the common cold. In the fall of 2014, enterovirus D68 infection was confirmed in more than 500 persons with mild to severe respiratory illnesses in 43 U.S. states. Nearly all reported cases were in children, many of whom had asthma. Enterovirus D68 was detected in upper respiratory tract specimens from some patients with unexplained acute neurologic disease during outbreaks of infection with this virus; however, the virus was not detected in the CSF, and at present the link between the virus and neurologic disease is uncertain. Coxsackievirus B has been isolated at autopsy from the pancreas of a few children presenting with type 1 diabetes mellitus; however, most attempts to isolate the virus have been unsuccessful. Other diseases that have been associated with enterovirus infection include parotitis, bronchitis, bronchiolitis, croup, infectious lymphocytosis, polymyositis, acute arthritis, and acute

1	Other diseases that have been associated with enterovirus infection include parotitis, bronchitis, bronchiolitis, croup, infectious lymphocytosis, polymyositis, acute arthritis, and acute nephritis.

1	Isolation of enterovirus in cell culture is the traditional diagnostic procedure. While cultures of stool, nasopharyngeal, or throat samples from patients with enterovirus diseases are often positive, isolation of the virus from these sites does not prove that it is directly associated with disease because these sites are frequently colonized for weeks in

1	FIGuRE 228-2 Acute hemorrhagic conjunctivitis due to enterovirus 70. (Image reprinted with permission from Red Book 2012: Committee on Infectious Diseases, 29th ed. Used with permission of the American Academy of Pediatrics.) patients with subclinical infections. Isolation of virus from the throat is 1293 more likely to be associated with disease than is isolation from the stool since virus is shed for shorter periods from the throat. Cultures of CSF, serum, fluid from body cavities, or tissues are positive less frequently, but a positive result is indicative of disease caused by enterovirus. In some cases, the virus is isolated only from the blood or only from the CSF; therefore, it is important to culture multiple sites. Cultures are more likely to be positive earlier than later in the course of infection. Most human enteroviruses can be detected within a week after inoculation of cell cultures. Cultures may be negative because of the presence of neutralizing antibody, lack of

1	the course of infection. Most human enteroviruses can be detected within a week after inoculation of cell cultures. Cultures may be negative because of the presence of neutralizing antibody, lack of susceptibility of the cells used, or inappropriate handling of the specimen. Coxsackievirus A may require inoculation into special cell-culture lines or into suckling mice.

1	Identification of the enterovirus serotype is useful primarily for epidemiologic studies and, with a few exceptions, has little clinical utility. It is important to identify serious infections with enterovirus during epidemics and to distinguish the vaccine strain of poliovirus from the other enteroviruses in the throat or in the feces. Stool and throat samples for culture as well as acute-and convalescent-phase serum specimens should be obtained from all patients with suspected poliomyelitis. In the absence of a positive CSF culture, a positive culture of stool obtained within the first 2 weeks after the onset of symptoms is most often used to confirm the diagnosis of poliomyelitis. If polio-virus infection is suspected, two or more fecal and throat swab samples should be obtained at least 1 day apart and cultured for enterovirus as soon as possible. If poliovirus is isolated, it should be sent to the CDC for identification as either wild-type or vaccine virus.

1	Reverse-transcriptase polymerase chain reaction (PCR) has been used to amplify viral nucleic acid from CSF, serum, urine, stool, conjunctiva, throat swabs, and tissues. A pan-enterovirus PCR assay can detect all human enteroviruses. With the proper controls, PCR of the CSF is highly sensitive (70–100%) and specific (>80%) and is more rapid than culture. PCR of the CSF is less likely to be positive when patients present ≥3 days after the onset of meningitis or with enterovirus 71 infection; in these cases, PCR of throat or rectal swabs— although less specific than PCR of CSF—should be considered. PCR of serum is also highly sensitive and specific in the diagnosis of disseminated disease. PCR may be particularly helpful for the diagnosis and follow-up of enterovirus disease in immunodeficient patients receiving immunoglobulin therapy, whose viral cultures may be negative. Antigen detection is less sensitive than PCR.

1	Serologic diagnosis of enterovirus infection is limited by the large number of serotypes and the lack of a common antigen. Demonstration of seroconversion may be useful in rare cases for confirmation of culture results, but serologic testing is usually limited to epidemiologic studies. Serum should be collected and frozen soon after the onset of disease and again ~4 weeks later. Measurement of neutralizing titers is the most accurate method for antibody determination; measurement of complement-fixation titers is usually less sensitive. Titers of virus-specific IgM are elevated in both acute and chronic infection.

1	Most enterovirus infections are mild and resolve spontaneously; however, intensive supportive care may be needed for cardiac, hepatic, or CNS disease. IV, intrathecal, or intraventricular immunoglobulin has been used with apparent success in some cases for the treatment of chronic enterovirus meningoencephalitis and dermatomyositis in patients with hypogammaglobulinemia or agammaglobulinemia. The disease may stabilize or resolve during therapy; however, some patients decline inexorably despite therapy. IV immunoglobulin often prevents severe enterovirus disease in these patients. IV administration of immunoglobulin with high titers of antibody to the infecting virus has been used in some cases of life-threatening infection in neonates, who may not have maternally acquired antibody. In one trial involving neonates with enterovirus infections, immunoglobulin containing very high titers of antibody to the infecting virus reduced rates of viremia; however, the study was too small to show

1	trial involving neonates with enterovirus infections, immunoglobulin containing very high titers of antibody to the infecting virus reduced rates of viremia; however, the study was too small to show a substantial clinical benefit. The level of

1	Enterovirus, Parechovirus, and Reovirus Infections 1294 enteroviral antibodies varies with the immunoglobulin preparation. A phase 2 trial of pleconaril for severe neonatal enterovirus disease has been completed; however, as of this writing, the results have not been reported and the drug is not available on a compassionate-use basis. Glucocorticoids are contraindicated. Good hand-washing practices and the use of gowns and gloves are important in limiting nosocomial transmission of enteroviruses during epidemics. Enteric precautions are indicated for 7 days after the onset of enterovirus infections. Enterovirus 71 vaccine candidates are under development.

1	(See also Chap. 148) After a peak of 57,879 cases of poliomy elitis in the United States in 1952, the introduction of IPV in 1955 and of OPV in 1961 ultimately eradicated disease due to wild-type poliovirus in the Western Hemisphere. Such disease has not been documented in the United States since 1979, when cases occurred among religious groups who had declined immunization. In the Western Hemisphere, paralysis due to wild-type poliovirus was last documented in 1991.

1	In 1988, the World Health Organization adopted a resolution to eradicate poliomyelitis by the year 2000. From 1988 to 2001, the number of cases worldwide decreased by >99%, with only 496 confirmed cases reported in 2001. Wild-type poliovirus type 2 has not been detected in the world since 1999. The Americas were certified free of indigenous wild-type poliovirus transmission in 1994, the Western Pacific Region in 2000, and the European Region in 2002. However, in 2002, there were 1922 cases of polio, with 1600 cases reported in India. In fact, after the nadir of 496 cases in 2001, 21 countries that had previously been free of polio reported cases imported from 6 polio-endemic countries in 2002–2005. By 2006, polio transmission had been reduced in most of these 21 countries. In 2012, 293 cases of polio were reported (the lowest number ever in a 1-year period); 85% were from Nigeria, Pakistan, and Afghanistan, the only countries where polio remains endemic (Table 228-2). As of November

1	of polio were reported (the lowest number ever in a 1-year period); 85% were from Nigeria, Pakistan, and Afghanistan, the only countries where polio remains endemic (Table 228-2). As of November 2013, there had been 390 cases of polio in 2013 compared with 293 cases in 2012. The increase was associated with a marked rise in imported cases, including more than 180 cases in Somalia, more than 10 cases each in Kenya and Syria, and cases in Cameroon and Ethiopia. Also in 2013, wild-type poliovirus was detected in sewage in Israel, prompting a massive vaccination campaign with OPV. As of November 2013, India had not reported a case of polio since January 2011. Polio is a source of concern for unimmunized or partially immunized travelers. Importation of polio-virus accounted for ~50% of cases in 2013. Clearly, global eradication of polio is necessary to eliminate the risk of importation of wild-type virus. Outbreaks are thought to have been facilitated by suboptimal rates of vaccination,

1	in 2013. Clearly, global eradication of polio is necessary to eliminate the risk of importation of wild-type virus. Outbreaks are thought to have been facilitated by suboptimal rates of vaccination, isolated pockets of unvaccinated children, poor sanitation and crowding, improper vaccine-storage conditions, and a reduced level of response to one of the serotypes in the vaccine.

1	Country Type of Transmission No. of Cases aOf these cases, 8 were vaccine-derived. bOf these cases, 16 were vaccine-derived. cOf these cases, 9 were vaccine-derived. dOf these cases, 12 were vaccine-derived. Source: World Health Organization. While the global eradication campaign has markedly reduced the number of cases of endemic polio, doubts have been raised as to whether eradication is a realistic goal, given the large number of asymptomatic infections and the political instability in developing countries.

1	The occurrence of outbreaks of poliomyelitis due to circulating vaccine-derived poliovirus of all three types has been increasing, especially in areas with low vaccination rates. In Egypt, 32 cases of vaccine-derived polio occurred in 1983–1993; in the Dominican Republic and Haiti, 21 cases occurred in 2000–2001; in Indonesia, 46 cases were reported in 2005; in Nigeria, 385 cases occurred in 2005–2012; in the Democratic Republic of the Congo, 64 cases were reported in 2008–2012; in Pakistan, 16 cases occurred in 2012, and at least 30 cases occurred in 2013. These OPV-derived viruses reverted to a more neurovirulent phenotype after undetected circulation (probably for >2 years). The epidemic in Hispaniola was rapidly terminated after intensive vaccination with OPV. In 2005, a case of vaccine-derived polio occurred in an unvaccinated U.S. woman returning from a visit to Central and South America. In the same year, an unvaccinated immunocompromised infant in Minnesota was found to be

1	polio occurred in an unvaccinated U.S. woman returning from a visit to Central and South America. In the same year, an unvaccinated immunocompromised infant in Minnesota was found to be shedding vaccine-derived poliovirus; further investigation identified 4 of 22 infants in the same community who were shedding the virus. All 5 infants were asymptomatic. These outbreaks emphasize the need for maintaining high levels of vaccine coverage and continued surveillance for circulating virus.

1	IPV is used in most industrialized countries and OPV in most developing countries, including those in which polio still is or recently was endemic. While IM injections of other vaccines (live or attenuated) can be given concurrently with OPV, unnecessary IM injections should be avoided during the first month after OPV vaccination because they increase the risk of vaccine-associated paralysis. Since 1988, an enhanced-potency inactivated poliovirus vaccine has been available in the United States.

1	After several doses of OPV alone, the seropositivity rate for individual poliovirus serotypes may still be suboptimal for children in developing countries; one or more supplemental doses of IPV can increase the rate of seropositivity for these serotypes. Against a given serotype, monovalent OPV containing only that serotype is more immunogenic than trivalent vaccine because of a lack of interference from other serotypes. With eradication of wild-type poliovirus type 2, bivalent OPV (types 1 and 3), which was shown to be superior to trivalent OPV, has been the vaccine of choice to eliminate polio and has markedly reduced rates of polio in Nigeria. As the frequency of wild-type polio declines and reports of polio associated with circulating vaccine-derived viruses increase, the World Health Organization is investigating whether IPV can be produced from OPV strains that require less biocontainment, ultimately replacing OPV.

1	OPV and IPV induce antibodies that persist for at least 5 years. Both vaccines induce IgG and IgA antibodies. Compared with recipients of IPV, recipients of OPV shed less virus and less frequently develop reinfection with wild-type virus after exposure to poliovirus. Although IPV is safe and efficacious, OPV offers the advantages of ease of administration, lower cost, and induction of intestinal immunity resulting in a reduction in the risk of community transmission of wild-type virus. Because of progress toward global eradication of polio and the continued occurrence of cases of vaccine-associated polio, an all-IPV regimen was recommended in 2000 for childhood poliovirus vaccination in the United States, with vaccine administration at 2, 4, and 6–18 months and 4–6 years of age. The risk of vaccine-associated polio should be discussed before OPV is administered. Recommendations for vaccination of adults are listed in Table 228-3.

1	There are concerns about discontinuing vaccination in the event that endemic spread of poliovirus is eliminated. Among the reasons for these concerns are that poliovirus is shed from some immunocompromised persons for >10 years, that vaccine-derived poliovirus can circulate and cause disease, and that wild-type poliovirus is present in research laboratories. Human parechoviruses (HPeVs), like enteroviruses, are members of the family Picornaviridae. The 16 serotypes of HPeV commonly cause 1. Most adults in the United States have been vaccinated during childhood and are at little risk of exposure to wild-type virus in the United States. Immunization is recommended for those with a higher risk of exposure than the general population, including: a. travelers to areas where poliovirus is or may be epidemic or endemic; b. members of communities or population groups with disease caused by wild-type polioviruses; c. d.

1	travelers to areas where poliovirus is or may be epidemic or endemic; b. members of communities or population groups with disease caused by wild-type polioviruses; c. d. health care workers in close contact with patients who may be excreting wild-type polioviruses. 2. Three doses of IPV are recommended for adults who need to be immunized. The second dose should be given 1–2 months after the first dose; the third dose should be given 6–12 months after the second dose. 3. Adults who are at increased risk of exposure to wild-type poliovirus and who have previously completed primary immunization should receive a single dose of IPV. Adults who did not complete primary immunization should receive the remaining required doses of IPV. Abbreviation: IPV, inactivated poliovirus vaccine. Source: Modified from Pickering LK, ed. Red Book 2012: Committee on Infectious Diseases, 29th ed.

1	Source: Modified from Pickering LK, ed. Red Book 2012: Committee on Infectious Diseases, 29th ed. infections in early childhood. HPeV-1 infections occur throughout the year, while other parechovirus infections occur more commonly in summer and fall. Infections with HPeVs present similarly to those due to enteroviruses and may cause generalized disease of the newborn, aseptic meningitis, encephalitis, transient paralysis, exanthems, respiratory tract disease, and gastroenteritis. While HPeV-1 is the most common serotype and generally causes mild disease, deaths of infants in the United States have been associated with HPeV-1, HPeV-3, and HPeV-6. HPeVs can be isolated from the same sites as enteroviruses, including the nasopharynx, stool, and respiratory tract secretions. PCR using pan-enterovirus primers does not detect HPeVs, and while PCR assays are performed by the CDC and research laboratories, many commercial laboratories do not perform the test.

1	Reoviruses are double-stranded RNA viruses encompassing three serotypes. Serologic studies indicate that most humans are infected with reoviruses during childhood. Most infections either are asymptomatic or cause mild upper respiratory tract symptoms. Reovirus is considered a rare cause of mild gastroenteritis or meningitis in infants and children. Speculation regarding an association of reovirus type 3 with idiopathic neonatal hepatitis and extrahepatic biliary atresia is based on an elevated prevalence of antibody to reovirus in some affected patients and the detection of viral RNA by PCR in hepatobiliary tissues in some studies. New orthoreoviruses have been associated with human disease—e.g., Melaka and Kampar viruses with fever and acute respiratory disease in Malaysia, and Nelson Bay virus with acute respiratory disease in a traveler from Bali.

1	Measles (Rubeola) Kaitlin Rainwater-Lovett, William J. Moss DEFINITION Measles is a highly contagious viral disease that is characterized by a prodromal illness of fever, cough, coryza, and conjunctivitis followed by the appearance of a generalized maculopapular rash. 229 Before the widespread use of measles vaccines, it was estimated that measles caused between 5 million and 8 million deaths worldwide each year.

1	Before the widespread use of measles vaccines, it was estimated that measles caused between 5 million and 8 million deaths worldwide each year. tion. In the Americas, intensive vaccination and surveillance efforts—based in part on the successful Pan American Health Organization strategy of periodic nationwide measles vaccination campaigns (supplementary immunization activities, or SIAs)—and high levels of routine measles vaccine coverage interrupted endemic transmission of measles virus. In the United States, high-level coverage with two doses of measles vaccine eliminated endemic measles virus transmission in 2000. More recently, progress has been made in reducing measles incidence and mortality rates in sub-Saharan Africa and Asia as a consequence of increasing routine measles vaccine coverage and provision of a second dose of measles vaccine through mass measles vaccination campaigns and childhood immunization programs.

1	In 2003, the World Health Assembly endorsed a resolution urging member countries to reduce the number of deaths attributed to measles by 50% (compared with 1999 estimates) by the end of 2005. This target was met. Global measles mortality rates were further reduced in 2008; during that year, there were an estimated 164,000 deaths due to measles (uncertainty bounds: 115,000 and 222,000 deaths). These achievements attest to the enormous public-health significance of measles vaccination. However, recent large outbreaks of measles in Europe and Africa illustrate the challenges faced in sustaining measles control: in these outbreaks, measles was imported into countries that had eliminated indigenous transmission of measles virus.

1	The Measles and Rubella Initiative, a partnership led by the American Red Cross, the United Nations Foundation, UNICEF, the U.S. Centers for Disease Control and Prevention (CDC), and the World Health Organization (WHO), is playing an important role in reducing global measles incidence and mortality rates. Since its inception in 2001, the Initiative has provided governments and communities in more than 80 countries with technical and financial support for routine immunization activities, mass vaccination campaigns, and disease surveillance systems. Through its 2012–2020 Global Measles and Rubella Strategic Plan, the Initiative aims to reduce measles deaths by 95% (compared with year 2000 estimates) by 2015 and to eliminate measles from at least five of the six WHO regions by 2020. As regional goals for measles elimination are set, global measles eradication is likely to become a public health goal in the near future.

1	Measles virus is a spherical, nonsegmented, single-stranded, negative-sense RNA virus and a member of the Morbillivirus genus in the family Paramyxoviridae. Measles was originally a zoonotic infection, arising from animal-to-human transmission of an ancestral morbillivirus ~10,000 years ago, when human populations had attained sufficient size to sustain virus transmission. Although RNA viruses typically have high mutation rates, measles virus is considered to be an antigenically monotypic virus; i.e., the surface proteins responsible for inducing protective immunity have retained their antigenic structure across time and distance. The public health significance of this stability is that measles vaccines developed decades ago from a single strain of measles virus remain protective worldwide. Measles virus is killed by ultraviolet light and heat, and attenuated measles vaccine viruses retain these characteristics, necessitating a cold chain for vaccine transport and storage.

1	Measles virus is one of the most highly contagious directly transmitted pathogens. Outbreaks can occur in populations in which <10% of persons are susceptible. Chains of transmission are common among household contacts, school-age children, and health care workers. There are no latent or persistent measles virus infections that result in prolonged contagiousness, nor are there animal reservoirs for the virus. Thus, measles virus can be maintained in human populations only by an unbroken chain of acute infections, which requires 1296 a continuous supply of susceptible individuals. Newborns become susceptible to measles virus infection when passively acquired maternal antibody is lost; when not vaccinated, these infants account for the bulk of new susceptible individuals. Endemic measles has a typical temporal pattern characterized by yearly seasonal epidemics superimposed on longer epidemic cycles of 2–5 years or more. In temperate climates, annual measles outbreaks typically occur in

1	a typical temporal pattern characterized by yearly seasonal epidemics superimposed on longer epidemic cycles of 2–5 years or more. In temperate climates, annual measles outbreaks typically occur in the late winter and early spring. These annual outbreaks are probably attributable to social networks facilitating transmission (e.g., congregation of children at school) and environmental factors favoring the viability and transmission of measles virus. Measles cases continue to occur during interepidemic periods in large populations, but at low incidence. The longer epidemic cycles occurring every several years result from the accumulation of susceptible persons over successive birth cohorts and the subsequent decline in the number of susceptibles following an outbreak. Secondary attack rates among susceptible household and institutional contacts generally exceed 90%. The average age at which measles occurs depends on rates of contact with infected persons, protective maternal antibody

1	among susceptible household and institutional contacts generally exceed 90%. The average age at which measles occurs depends on rates of contact with infected persons, protective maternal antibody decline, and vaccine coverage. In densely populated urban settings with low-level vaccination coverage, measles is a disease of infants and young children. The cumulative distribution can reach 50% by 1 year of age, with a significant proportion of chil dren acquiring measles before 9 months—the age of routine vaccina tion in many countries, in line with the schedule recommended by the

1	WHO’s Expanded Programme on Immunization. As measles vaccine coverage increases or population density decreases, the age distribu tion shifts toward older children. In such situations, measles cases predominate in school-age children. Infants and young children, although susceptible if not protected by vaccination, are not exposed transmitted by direct contact with infected secretions but does not survive for long on fomites.

1	The incubation period for measles is ~10 days to fever onset and 14 days to rash onset. This period may be shorter in infants and longer (up to 3 weeks) in adults. Infection is initiated when measles virus is deposited on epithelial cells in the respiratory tract, oropharynx, or conjunctivae (Fig. 229-1A). During the first 2–4 days after infection, measles virus proliferates locally in the respiratory mucosa and spreads to draining lymph nodes. Virus then enters the bloodstream in infected leukocytes (primarily monocytes), producing the primary viremia that disseminates infection throughout the reticuloendothelial system. Further replication results in secondary viremia that begins 5–7 days after infection and disseminates measles virus throughout the body. Replication of measles virus in these target organs, together with the host’s immune response, is responsible for the signs and symptoms of measles that occur 8–12 days after infection and mark the end of the incubation period

1	in these target organs, together with the host’s immune response, is responsible for the signs and symptoms of measles that occur 8–12 days after infection and mark the end of the incubation period (Fig. 229-1B).

1	to measles virus at a rate sufficient to cause a large disease burden in this age group. As vaccination coverage increases further, the age distribution of cases may be shifted into adolescence and adulthood; this distribution is seen in measles outbreaks in the United States and age groups. Persons with measles are infectious for several days before and after the onset of rash, when levels of measles virus in blood and body fluids are highest and when cough, coryza, and sneezing, which facilitate virus spread, are most severe. The contagiousness of measles before the onset of recognizable disease hinders the effectiveness of quarantine measures. Viral shedding by children with impaired cell-mediated immunity can be prolonged.

1	Medical settings are well-recognized sites of measles virus transmission. Children may present to health care facilities during the prodrome, when the diagnosis is not obvious although the child is Days after infection infectious and is likely to infect susceptible contacts. Health care workers can acquire measles from infected children and transmit measles virus to others. Nosocomial transmission can be reduced by maintenance of a high index of clinical suspicion, use of appropriate isolation precautions when measles is suspected, administration of measles vaccine to susceptible children and health care workers, and documentation of health care workers’ immunity to measles (i.e., proof of receipt of two doses of measles vaccine or detection of antibodies to measles virus).

1	As efforts at measles control are increasingly successful, public perceptions of the risk of measles as a disease diminish and are replaced by concerns about possible adverse events associated with measles vaccine. As a consequence, numerous measles outbreaks have occurred because of opposition to vaccination on religious or philosophical grounds or unfounded fears of serious adverse events (see “Active Immunization,” below). Measles virus is transmitted primarily by respiratory droplets over short distances and, less commonly, by small-particle aerosols that remain suspended in the air for long periods. Airborne transmission appears to be important in certain settings, including schools, physicians’ offices, hospitals, and enclosed public places. The virus can be FIGuRE 229-1 Measles virus infection: pathogenesis, clinical features, and immune responses. A. Spread of measles virus, from initial infection of the respiratory tract through dissemination to the skin.

1	B. Appearance of clinical signs and symptoms, including Koplik’s spots and rash. C. Antibody and T cell responses to measles virus. The signs and symptoms of measles arise coincident with the host immune response. (Source: Modified from WJ Moss, DE Griffin: Nat Rev Microbiol 4:900, 2006.)

1	Host immune responses to measles virus are essential for viral clearance, clinical recovery, and the establishment of long-term immunity (Fig. 229-1C). Early nonspecific (innate) immune responses during the prodromal phase include activation of natural killer cells and increased production of antiviral proteins. The adaptive immune responses consist of measles virus–specific antibody and cellular responses. The protective efficacy of antibodies to measles virus is illustrated by the immunity conferred to infants from passively acquired maternal antibodies and the protection of exposed, susceptible individuals after administration of anti–measles virus immunoglobulin. The first measles virus–specific antibodies produced after infection are of the IgM subtype, with a subsequent switch to predominantly IgG1 and IgG4 isotypes. The IgM antibody response is typically absent following reexposure or revaccination and serves as a marker of primary infection.

1	The importance of cellular immunity to measles virus is demonstrated by the ability of children with agammaglobulinemia (congenital inability to produce antibodies) to recover fully from measles and the contrasting picture for children with severe defects in T lymphocyte function, who often develop severe or fatal disease (Chap. 374). The initial predominant TH1 response (characterized by interferon γ) is essential for viral clearance, and the later TH2 response (characterized by interleukin 4) promotes the development of measles virus–specific antibodies that are critical for protection against reinfection. The duration of protective immunity following wild-type measles virus infection is generally thought to be lifelong. Immunologic memory to measles virus includes both continued production of measles virus–specific antibodies and circulation of measles virus–specific CD4+ and CD8+ T lymphocytes.

1	However, the intense immune responses induced by measles virus infection are paradoxically associated with depressed responses to unrelated (non–measles virus) antigens, which persist for several weeks to months beyond resolution of the acute illness. This state of immune suppression enhances susceptibility to secondary infections with bacteria and viruses that cause pneumonia and diarrhea and is responsible for a substantial proportion of measles-related morbidity and deaths. Delayed-type hypersensitivity responses to recall antigens, such as tuberculin, are suppressed, and cellular and humoral responses to new antigens are impaired. Reactivation of tuberculosis and remission of autoimmune diseases after measles have been described and are attributed to this period of immune suppression. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: Clinicians should consider measles in persons presenting with fever and generalized erythematous rash, particularly when measles virus is known to be circulating or the patient has a history of travel to endemic areas. Appropriate precautions must be taken to prevent nosocomial transmission. The diagnosis requires laboratory confirmation except during large outbreaks in which an epidemiologic link to a confirmed case can be established. Care is largely supportive and consists of the administration of vitamin A and antibiotics (see “Treatment,” below). Complications of measles, including secondary bacterial infections and encephalitis, may occur after acute illness and require careful monitoring, particularly in immunocompromised persons.

1	In most persons, the signs and symptoms of measles are highly characteristic (Fig. 229-1B). Fever and malaise beginning ~10 days after exposure are followed by cough, coryza, and conjunctivitis. These signs and symptoms increase in severity over 4 days. Koplik’s spots (see Fig. 25e-2) develop on the buccal mucosa ~2 days before the rash appears. The characteristic rash of measles (see Fig. 25e-3) begins 2 weeks after infection, when the clinical manifestations are most severe, and signal the host’s immune response to the replicating virus. 1297 Headache, abdominal pain, vomiting, diarrhea, and myalgia may be present. Koplik’s spots (see Fig. 25e-2) are pathognomonic of measles and consist of bluish white dots ~1 mm in diameter surrounded by erythema. The lesions appear first on the buccal mucosa opposite the lower molars but rapidly increase in number to involve the entire buccal mucosa. They fade with the onset of rash.

1	The rash of measles begins as erythematous macules behind the ears and on the neck and hairline. The rash progresses to involve the face, trunk, and arms (see Fig. 25e-3), with involvement of the legs and feet by the end of the second day. Areas of confluent rash appear on the trunk and extremities, and petechiae may be present. The rash fades slowly in the same order of progression as it appeared, usually beginning on the third or fourth day after onset. Resolution of the rash may be followed by desquamation, particularly in undernourished children.

1	Because the characteristic rash of measles is a consequence of the cellular immune response, it may not develop in persons with impaired cellular immunity (e.g., those with AIDS; Chap. 226). These persons have a high case-fatality rate and frequently develop giant-cell pneumonitis caused by measles virus. T lymphocyte defects due to causes other than HIV-1 infection (e.g., cancer chemotherapy) also are associated with increased severity of measles. A severe atypical measles syndrome was observed in recipients of a formalin-inactivated measles vaccine (used in the United States from 1963 to 1967 and in Canada until 1970) who were subsequently exposed to wild-type measles virus. The atypical rash began on the palms and soles and spread centripetally to the proximal extremities and trunk, sparing the face. The rash was initially erythematous and maculopapular but frequently progressed to vesicular, petechial, or purpuric lesions (see Fig. 25e-22).

1	The differential diagnosis of measles includes other causes of fever, rash, and conjunctivitis, including rubella, Kawasaki disease, infectious mononucleosis, roseola, scarlet fever, Rocky Mountain spotted fever, entero virus or adenovirus infection, and drug sensitivity. Rubella is a milder illness without cough and with distinctive lymphadenopathy. The rash of roseola (exanthem subitum) (see Fig. 25e-5) appears after fever has subsided. The atypical lymphocytosis in infectious mononucleosis contrasts with the leukopenia commonly observed in children with measles.

1	Measles is readily diagnosed on clinical grounds by clinicians familiar with the disease, particularly during outbreaks. Koplik’s spots (see Fig. 25e-2) are especially helpful because they appear early and are pathognomonic. Clinical diagnosis is more difficult (1) during the prodromal illness; (2) when the rash is attenuated by passively acquired antibodies or prior immunization; (3) when the rash is absent or delayed in immunocompromised children or severely undernourished children with impaired cellular immunity; and (4) in regions where the incidence of measles is low and other pathogens are responsible for the majority of illnesses with fever and rash. The CDC case definition for measles requires (1) a generalized maculopapular rash of at least 3 days’ duration; (2) fever of at least 38.3°C (101°F); and (3) cough, coryza, or conjunctivitis.

1	Serology is the most common method of laboratory diagnosis. The detection of measles virus–specific IgM in a single specimen of serum or oral fluid is considered diagnostic of acute infection, as is a fourfold or greater increase in measles virus–specific IgG antibody levels between acuteand convalescent-phase serum specimens. Primary infection in the immunocompetent host results in antibodies that are detectable within 1–3 days of rash onset and reach peak levels in 2–4 weeks. Measles virus–specific IgM antibodies may not be detectable until 4–5 days or more after rash onset and usually fall to undetectable levels within 4–8 weeks of rash onset.

1	Several methods for measurement of antibodies to measles virus are available. Neutralization tests are sensitive and specific, and the results are highly correlated with protective immunity; however, these tests 1298 require propagation of measles virus in cell culture and thus are expensive and laborious. Commercially available enzyme immunoassays are most frequently used. Measles can also be diagnosed by isolation of the virus in cell culture from respiratory secretions, nasopharyngeal or conjunctival swabs, blood, or urine. Direct detection of giant cells in respiratory secretions, urine, or tissue obtained by biopsy provides another method of diagnosis. For detection of measles virus RNA by reverse-transcriptase polymerase chain reaction amplification of RNA extracted from clinical specimens, primers targeted to highly conserved regions of measles virus genes are used. Extremely sensitive and specific, this assay may also permit identification and characterization of measles virus

1	primers targeted to highly conserved regions of measles virus genes are used. Extremely sensitive and specific, this assay may also permit identification and characterization of measles virus genotypes for molecular epidemiologic studies and can distinguish wild-type from vaccine virus strains.

1	There is no specific antiviral therapy for measles. Treatment consists of general supportive measures, such as hydration and administration of antipyretic agents. Because secondary bacterial infections are a major cause of morbidity and death attributable to measles, effective case management involves prompt antibiotic treatment for patients who have clinical evidence of bacterial infection, including pneumonia and otitis media. Streptococcus pneumoniae and Haemophilus influenzae type b are common causes of bacterial pneumonia following measles; vaccines against these pathogens probably lower the incidence of secondary bacterial infections following measles.

1	Vitamin A is effective for the treatment of measles and can markedly reduce rates of morbidity and mortality. The WHO recommends administration of once-daily doses of 200,000 IU of vitamin A for 2 consecutive days to all children with measles who are ≥12 months of age. Lower doses are recommended for younger children: 100,000 IU per day for children 6–12 months of age and 50,000 IU per day for children <6 months old. A third dose is recommended 2–4 weeks later for children with evidence of vitamin A deficiency. While such deficiency is not a widely recognized problem in the United States, many American children with measles do, in fact, have low serum levels of vitamin A, and these children experience increased measles-associated morbidity. The Committee on Infectious Diseases of the American Academy of Pediatrics recommends that the administration of two consecutive daily doses of vitamin A be considered for children who are hospitalized with measles and its complications as well as

1	Academy of Pediatrics recommends that the administration of two consecutive daily doses of vitamin A be considered for children who are hospitalized with measles and its complications as well as for children with measles who are immunodeficient; who have ophthalmologic evidence of vitamin A deficiency, impaired intestinal absorption, or moderate to severe malnutrition; or who have recently immigrated from areas with high measles mortality rates. Parenteral and oral formulations of vitamin A are available.

1	Anecdotal reports have described the recovery of previously healthy pregnant and immunocompromised patients with measles pneumonia and of immunocompromised patients with measles encephalitis after treatment with aerosolized and IV ribavirin. However, the clinical benefits of ribavirin in measles have not been conclusively demonstrated in clinical trials. Most complications of measles involve the respiratory tract and include the effects of measles virus replication itself and secondary bacterial infections. Acute laryngotracheobronchitis (croup) can occur during measles and may result in airway obstruction, particularly in young children. Giant-cell pneumonitis due to replication of measles virus in the lungs can develop in immunocompromised children, including those with HIV-1 infection. Many children with measles develop diarrhea, which contributes to undernutrition.

1	Most complications of measles result from secondary bacterial infections of the respiratory tract that are attributable to a state of immune suppression lasting for several weeks to months after acute measles. Otitis media and bronchopneumonia are most common and may be caused by S. pneumoniae, H. influenzae type b, or staphylococci. Recurrence of fever or failure of fever to subside with the rash suggests secondary bacterial infection.

1	Rare but serious complications of measles involve the central nervous system (CNS). Postmeasles encephalomyelitis complicates ~1 in 1000 cases, affecting mainly older children and adults. Encephalomyelitis occurs within 2 weeks of rash onset and is characterized by fever, seizures, and a variety of neurologic abnormalities. The finding of periventricular demyelination, the induction of immune responses to myelin basic protein, and the absence of measles virus in the brain suggest that postmeasles encephalomyelitis is an autoimmune disorder triggered by measles virus infection. Other CNS complications that occur months to years after acute infection are measles inclusion body encephalitis (MIBE) and subacute sclerosing panencephalitis (SSPE). In contrast to postmeasles encephalomyelitis, MIBE and SSPE are caused by persistent measles virus infection. MIBE is a rare but fatal complication that affects individuals with defective cellular immunity and typically occurs months after

1	MIBE and SSPE are caused by persistent measles virus infection. MIBE is a rare but fatal complication that affects individuals with defective cellular immunity and typically occurs months after infection. SSPE is a slowly progressive disease characterized by seizures and progressive deterioration of cognitive and motor functions, with death occurring 5–15 years after measles virus infection. SSPE most often develops in persons infected with measles virus at <2 years of age.

1	Most persons with measles recover and develop long-term protective immunity to reinfection. Measles case-fatality proportions vary with the average age of infection, the nutritional and immunologic status of the population, measles vaccine coverage, and access to health care. Among previously vaccinated persons who do become infected, disease is less severe and mortality rates are significantly lower. In developed countries, <1 in 1000 children with measles die. In endemic areas of sub-Saharan Africa, the measles case-fatality proportion may be 5–10% or even higher. Measles is a major cause of childhood deaths in refugee camps and in internally displaced populations, where case-fatality proportions have been as high as 20–30%.

1	PREVENTION Passive Immunization Human immunoglobulin given shortly after exposure can attenuate the clinical course of measles. In immunocompetent persons, administration of immunoglobulin within 72 h of exposure usually prevents measles virus infection and almost always prevents clinical measles. Administered up to 6 days after exposure, immunoglobulin will still prevent or modify the disease. Prophylaxis with immunoglobulin is recommended for susceptible household and nosocomial contacts who are at risk of developing severe measles, particularly children <1 year of age, immunocompromised persons (including HIV-infected persons previously immunized with live attenuated measles vaccine), and pregnant women. Except for premature infants, children <6 months of age usually will be partially or completely protected by passively acquired maternal antibody. If measles is diagnosed in a household member, all unimmunized children in the household should receive immunoglobulin. The recommended

1	completely protected by passively acquired maternal antibody. If measles is diagnosed in a household member, all unimmunized children in the household should receive immunoglobulin. The recommended dose is 0.25 mL/kg given intramuscularly. Immunocompromised persons should receive 0.5 mL/ kg. The maximum total dose is 15 mL. IV immunoglobulin contains antibodies to measles virus; the usual dose of 100–400 mg/kg generally provides adequate prophylaxis for measles exposures occurring as long as 3 weeks or more after IV immunoglobulin administration.

1	Active Immunization The first live attenuated measles vaccine was developed by passage of the Edmonston strain in chick embryo fibroblasts to produce the Edmonston B virus, which was licensed in 1963 in the United States. Further passage of Edmonston B virus produced the more attenuated Schwarz vaccine that currently serves as the standard in much of the world. The Moraten (“more attenuated Enders”) strain, which was licensed in 1968 and is used in the United States, is genetically closely related to the Schwarz strain.

1	Lyophilized measles vaccines are relatively stable, but reconstituted vaccine rapidly loses potency. Live attenuated measles vaccines are inactivated by light and heat and lose about half their potency at 20°C and almost all their potency at 37 within 1 h after reconstitution. 12 Therefore, a cold chain must be maintained before and after reconstitution. Antibodies first appear 12–15 days after vaccination, and titers peak at 1–3 months. Measles vaccines are often combined with other live attenuated virus vaccines, such as those for mumps and rubella (MMR) and for mumps, rubella, and varicella (MMR-V).

1	The recommended age of first vaccination varies from 6 to 15 months and represents a balance between the optimal age for seroconversion and the probability of acquiring measles before that age. The proportions of children who develop protective levels of antibody after measles vaccination approximate 85% at 9 months of age and 95% at 12 months. Common childhood illnesses concomitant with vaccination may reduce the level of immune response, but such illness is not a valid reason to withhold vaccination. Measles vaccines have been well tolerated and immunogenic in HIV-1-infected children and adults, although antibody levels may wane. Because of the potential severity of wild-type measles virus infection in HIV-1-infected children, routine measles vaccination is recommended except for those who are severely immunocompromised. Measles vaccination is contraindicated in individuals with other severe deficiencies of cellular immunity because of the possibility of disease due to progressive

1	who are severely immunocompromised. Measles vaccination is contraindicated in individuals with other severe deficiencies of cellular immunity because of the possibility of disease due to progressive pulmonary or CNS infection with the vaccine virus.

1	The duration of vaccine-induced immunity is at least several decades if not longer. Rates of secondary vaccine failure 10–15 years after immunization have been estimated at ~5% but are probably lower when vaccination takes place after 12 months of age. Decreasing antibody concentrations do not necessarily imply a complete loss of protective immunity: a secondary immune response usually develops after reexposure to measles virus, with a rapid rise in antibody titers in the absence of overt clinical disease. Standard doses of currently licensed measles vaccines are safe for immunocompetent children and adults. Fever to 39.4 (103) occurs in ~5% of seronegative vaccine recipients, and 2% of vaccine recipients develop a transient rash. Mild transient thrombocytopenia has been reported, with an incidence of ~1 case per 40,000 doses of MMR vaccine.

1	Since the publication of a report in 1998 hypothesizing that MMR vaccine may cause a syndrome of autism and intestinal inflammation, much public attention has focused on this purported association. The events that followed publication of this report led to diminished vaccine coverage in the United Kingdom and provide important lessons in the misinterpretation of epidemiologic evidence and the communication of scientific results to the public. The publication that incited the concern was a case series describing 12 children with a regressive developmental disorder and chronic enterocolitis; 9 of these children had autism. In 8 of the 12 cases, the parents associated onset of the developmental delay with MMR vaccination. This simple temporal association was misinterpreted and misrepresented as a possible causal relationship, first by the lead author of the study and then by elements of the media and the public. Subsequently, several comprehensive reviews and additional epidemiologic

1	as a possible causal relationship, first by the lead author of the study and then by elements of the media and the public. Subsequently, several comprehensive reviews and additional epidemiologic studies refuted evidence of a causal relationship between MMR vaccination and autism.

1	Progress in global measles control has renewed discussion of measles eradication. In contrast to poliovirus eradication, the eradication of measles virus will not entail challenges posed by prolonged shedding of potentially virulent vaccine viruses and environmental viral reservoirs. However, in comparison with smallpox eradication, higher levels of population immunity will be necessary to interrupt measles virus transmission, more highly skilled health care workers will be required to administer measles vaccines, and containment through case detection and ring vaccination will be more difficult for measles virus because of infectivity before rash onset. New tools, such as aerosol administration of measles vaccines, will facilitate mass vaccination campaigns. Despite enormous progress, measles remains a leading vaccine-preventable cause of childhood mortality worldwide and continues to cause outbreaks in communities with low vaccination coverage rates in industrialized nations.

1	Laura A. Zimmerman, Susan E. Reef Rubella was historically viewed as a variant of measles or scarlet fever. Not until 1962 was a separate viral agent for rubella isolated. After an epidemic of rubella in Australia in the early 1940s, the ophthalmologist Norman Gregg noticed the occurrence of congenital cataracts among infants whose mothers had reported rubella infection during early pregnancy, and congenital rubella syndrome (CRS; see “Clinical Manifestations,” below) was first described. Rubella virus is a member of the Togaviridae family and the only member of the genus Rubivirus. This single-stranded RNA enveloped virus measures 50–70 nm in diameter. Its core protein is surrounded by a single-layer lipoprotein envelope with spike-like projections containing two glycoproteins, E1 and E2. There is only one antigenic type of rubella virus, and humans are its only known reservoir.

1	Although the pathogenesis of postnatal (acquired) rubella has been well documented, data on pathology are limited because of the mildness of the disease. Rubella virus is spread from person to person via respiratory droplets. Primary implantation and replication in the nasopharynx are followed by spread to the lymph nodes. Subsequent viremia occurs, which in pregnant women often results in infection of the placenta. Placental virus replication may lead to infection of fetal organs. The pathology of CRS in the infected fetus is well defined, with almost all organs found to be infected; however, the pathogenesis of CRS is only poorly delineated. In tissue, infections with rubella virus have diverse effects, ranging from no obvious impact to cell destruction. The hallmark of fetal infection is chronicity, with persistence throughout fetal development in utero and for up to 1 year after birth.

1	Individuals with acquired rubella may shed virus from 7 days before rash onset to ~5–7 days thereafter. Both clinical and subclinical infections are considered contagious. Infants with CRS may shed large quantities of virus from bodily secretions, particularly from the throat and in the urine, up to 1 year of age. Outbreaks of rubella, including some in nosocomial settings, have originated with index cases of CRS. Thus only individuals immune to rubella should have contact with infants who have CRS or who are congenitally infected with rubella virus but are not showing signs of CRS.

1	The largest recent rubella epidemic in the United States took place in 1964–1965, when an estimated 12.5 million cases occurred, resulting in ~20,000 cases of CRS. Since the introduction of the routine rubella vaccination program in the United States in 1969, the number of rubella cases reported each year has dropped by >99%; the rate of vaccination coverage with rubella-containing vaccine has been >90% among children 19–35 months old since 1995 and >95% for kindergarten and first-grade entrants since 1980. In 1989 a goal for the elimination of rubella and CRS in the United States was set, and in 2004 a panel of experts agreed unanimously that rubella was no longer an endemic disease in this country. The criteria used to document lack of endemic transmission included low disease incidence, high nationwide rubella antibody seroprevalence, outbreaks that were few and contained (i.e., small numbers of cases), and lack of endemic virus transmission (as assessed by genetic sequencing). In

1	high nationwide rubella antibody seroprevalence, outbreaks that were few and contained (i.e., small numbers of cases), and lack of endemic virus transmission (as assessed by genetic sequencing). In the United States, interruption of endemic transmission of rubella virus has been sustained since 2001; in 2012, however, three cases of CRS were reported in infants whose mothers had acquired rubella infection abroad. Thus health care providers should remain vigilant, considering the possibility of rubella infection in patients emigrating or returning from countries without rubella control programs and the accompanying potential for CRS among their infants.

1	FIGURE 230e-1 Mild maculopapular rash of rubella in a child. Although rubella and CRS are no longer endemic in the United States, they remain important public health problems globally. The number of rubella cases reported worldwide in 1999 was ~900,000; this figure declined steadily to 94,030 in 2012. However, numbers of rubella cases are substantially underestimated because cases in many countries are identified through measles surveillance systems that are not specific for rubella. In 2010, it was estimated that 103,000 cases of CRS occur globally.

1	CLINICAL FEATURES Acquired Rubella Acquired rubella commonly presents with a generalized maculopapular rash that usually lasts for up to 3 days (Fig. 230e-1), although as many as 50% of cases may be subclinical or without rash. When it occurs, the rash is usually mild and may be difficult to detect in persons with darker skin. In children, rash is usually the first sign of illness. However, in older children and adults, a 1to 5-day prodrome often precedes the rash and may include low-grade fever, malaise, and upper respiratory symptoms. The incubation period is 14 days (range, 12–23 days). Lymphadenopathy, particularly occipital and postauricular, may be noted during the second week after exposure. Although acquired rubella is usually thought of as a benign disease, arthralgia and arthritis are common in infected adults, particularly women. Thrombocytopenia and encephalitis are less common complications.

1	Congenital Rubella Syndrome The most serious consequence of rubella virus infection can develop when a woman becomes infected during pregnancy, particularly during the first trimester. The resulting complications may include miscarriage, fetal death, premature delivery, or live birth with congenital defects. Infants infected with rubella virus in utero may have myriad physical defects (Table 230e-1), which most commonly relate to the eyes, ears, and heart. This constellation of severe birth defects is known as congenital rubella syndrome. In addition to permanent manifestations, there are a host of transient physical manifestations, including thrombocytopenia with purpura/petechiae (e.g., dermal erythropoiesis, “blueberry muffin syndrome”). Some infants may be born with congenital rubella virus infection but have no apparent signs or symptoms of CRS and are referred to as “infants with congenital rubella infection only.”

1	DIAGNOSIS Acquired Rubella Clinical diagnosis of acquired rubella is difficult because of the mimicry of many illnesses with rashes, the varied Interstitial pneumonitis Congenital heart defects (patent ductus arteriosus, pulmonary arterial

1	Interstitial pneumonitis Congenital heart defects (patent ductus arteriosus, pulmonary arterial Thrombocytopenia with purpura/ stenosis) petechiae (e.g., dermal erythropoiesis, or “blueberry muffin syndrome”) Eye defects (cataracts, cloudy cornea, microphthalmos, pigmentary reti-Hemolytic anemia nopathy, congenital glaucoma) Bony radiolucencies Microcephaly Intrauterine growth retardation Central nervous system sequelae Adenopathy (mental and motor delay, autism) Meningoencephalitis clinical presentations, and the high rates of subclinical and mild disease. Illnesses that may be similar to rubella in presentation include scarlet fever, roseola, toxoplasmosis, fifth disease, measles, and illnesses with suboccipital and postauricular lymphadenopathy. Thus laboratory documentation of rubella virus infection is considered the only reliable way to confirm acute disease.

1	Laboratory assessment of rubella infection is conducted by serologic and virologic methods. For acquired rubella, serologic diagnosis is most common and depends on the demonstration of IgM antibodies in an acute-phase serum specimen or a fourfold rise in IgG antibody titer between acuteand convalescent-phase specimens. The enzyme-linked immunosorbent assay IgM capture technique is considered most accurate for serologic diagnosis, but the indirect IgM assay also is acceptable. After rubella virus infection, IgM antibody may be detectable for up to 6 weeks. In case of a negative result for IgM in specimens taken earlier than day 5 after rash onset, serologic testing should be repeated. Although uncommon, reinfection with rubella virus is possible, and IgM antibodies may be present. To detect a rise in IgG antibody titer indicative of acute disease, the acute-phase serum specimen should be collected within 7–10 days after onset of illness and the convalescent-phase specimen ~14–21 days

1	a rise in IgG antibody titer indicative of acute disease, the acute-phase serum specimen should be collected within 7–10 days after onset of illness and the convalescent-phase specimen ~14–21 days after the first specimen.

1	IgG avidity testing is used in conjunction with IgG testing. Low-avidity antibodies indicate recent infection. Mature (high-avidity) IgG antibodies most likely indicate an infection occurring at least 2 months previously. This test helps distinguish primary infection from reinfection. Avidity testing may be particularly useful in diagnosing rubella in pregnant women and assessing the risk of CRS. Rubella virus can be isolated from the blood and nasopharynx during the prodromal period and for as long as 2 weeks after rash onset. However, as the secretion of virus in individuals with acquired rubella is maximal just before or up to 4 days after rash onset, this is the optimal time frame for collecting specimens for viral cultures. Rubella can also be diagnosed by viral RNA detection in a reverse-transcriptase polymerase chain reaction (RT-PCR) assay.

1	Congenital Rubella Syndrome A clinical diagnosis of CRS is reasonable when an infant presents with a combination of cataracts, hearing impairment, and heart defects; this pattern is seen in ~10% of infants with CRS. Infants may present with different combinations of defects depending on when infection occurs during gestation. Hearing impairment is the most common single defect of CRS. However, as with acquired rubella, laboratory diagnosis of congenital infection is highly recommended, particularly because most features of the clinical presentation are nonspecific and may be associated with other intrauterine infections. Early diagnosis of CRS facilitates appropriate medical intervention for specific disabilities and prompts implementation of infection control measures.

1	Diagnostic tests used to confirm CRS include serologic assays and virus detection. In an infant with congenital infection, serum IgM antibodies are normally present for up to 6 months but may be detectable for up to 1 year after birth. In some instances, IgM may not be detectable until 1 month of age; thus infants who have symptoms consistent with CRS but who test negative shortly after birth should be retested at 1 month. A rubella serum IgG titer persisting beyond the time expected after passive transfer of maternal IgG antibody (i.e., a rubella titer that does not decline at the expected rate of a twofold dilution per month) is another serologic criterion used to confirm CRS.

1	In congenital infection, rubella virus is isolated most commonly from throat swabs and less commonly from urine and cerebrospinal fluid. Infants with congenital rubella may excrete virus for up to 1 year, but specimens for virus isolation are most likely to be positive if obtained within the first 6 months after birth. Rubella virus in infants with CRS can also be detected by RT-PCR. Rubella Diagnosis in Pregnant Women In the United States, screening for rubella IgG antibodies is recommended as part of routine prenatal care. Pregnant women with a positive IgG antibody serologic test are considered immune. Susceptible pregnant women should be vaccinated postpartum.

1	A susceptible pregnant woman exposed to rubella virus should be tested for IgM antibodies and/or a fourfold rise in IgG antibody titer between acuteand convalescent-phase serum specimens to determine whether she was infected during pregnancy. Pregnant women with evidence of acute infection must be clinically monitored, and gestational age at the time of maternal infection must be determined to assess the possibility of risk to the fetus. Of women infected with rubella virus during the first 11 weeks of gestation, up to 90% deliver an infant with CRS; for maternal infection during the first 20 weeks of pregnancy, the CRS rate is 20%.

1	No specific therapy is available for rubella virus infection. Symptom-based treatment for various manifestations, such as fever and arthralgia, is appropriate. Immunoglobulin does not prevent rubella virus infection after exposure and therefore is not recommended as routine postexposure prophylaxis. Although immunoglobulin may modify or suppress symptoms, it can create an unwarranted sense of security: infants with congenital rubella have been born to women who received immunoglobulin shortly after exposure. Administration of immunoglobulin should be considered only if a pregnant woman who has been exposed to rubella will not consider termination of the pregnancy under any circumstances. In such cases, IM administration of 20 mL of immunoglobulin within 72 h of rubella exposure may reduce—but does not eliminate—the risk of rubella.

1	After the isolation of rubella virus in the early 1960s and the occurrence of a devastating pandemic, a vaccine for rubella was developed and licensed in 1969. Currently, the majority of rubella-containing vaccines (RCVs) used worldwide are combined measles and rubella (MR) or measles, mumps, and rubella (MMR) formulations. A tetravalent measles, mumps, rubella, and varicella (MMRV) vaccine is available but is not widely used. The public health burden of rubella infection is measured primarily through the resulting CRS cases. The 1964–1965 rubella epidemic in the United States encompassed >30,000 infections during pregnancy. CRS occurred in ~20,000 infants born alive, including >11,000 infants who were deaf, >3500 infants who were blind, and almost 2000 infants who were mentally retarded. The cost of this epidemic exceeded $1.5 billion. In 1983, the cost per child with CRS was estimated at $200,000.

1	In most countries, there is little documented evidence to illuminate the epidemiology of CRS. Clusters of CRS cases have been reported in developing countries. Before the introduction of an immunization program, the incidence of CRS is 0.1–0.2 per 1000 live births during endemic periods and 1–4 per 1000 live births during epidemic periods. Where rubella virus is circulating and women of childbearing age are susceptible, CRS cases will continue to occur. The most effective method of preventing acquired rubella and CRS is through vaccination with an RCV. One dose induces seroconversion in ≥95% of persons ≥1 year of age. Immunity is considered long-term and 230e-3 3,500 Kilometers No (60 countries or 31%) Yes (134 countries or 69%) 0 875 1,750

1	FIGURE 230e-2 Countries using rubella vaccine in their national immunization schedule, 2012. (From the World Health Organization.) is probably lifelong. The most commonly used vaccine globally is the RA27/3 virus strain. The current recommendation for routine rubella vaccination in the United States is a first dose of MMR vaccine at 12–15 months of age and a second dose at 4–6 years. Target groups for rubella vaccine include children ≥1 year of age, adolescents and adults without documented evidence of immunity, individuals in congregate settings (e.g., college students, military personnel, child care and health care workers), and susceptible women before and after pregnancy.

1	Because of the theoretical risk of transmission of live attenuated rubella vaccine virus to the developing fetus, women known to be pregnant should not receive an RCV. In addition, pregnancy should be avoided for 28 days after receipt of an RCV. In follow-up studies of 680 unknowingly pregnant women who received rubella vaccine, no infant was born with CRS. Receipt of an RCV during pregnancy is not ordinarily a reason to consider termination of the pregnancy. As of 2012, 134 (69%) of the 194 member countries of the World Health Organization recommended inclusion of an RCV in the routine childhood vaccination schedule (Fig. 230e-2). Goals for control or elimination of rubella and CRS have been established in the American Region, the European Region, the South-East Asia Region, and the Western Pacific Region. The other two regions (Eastern Mediterranean and African) have not yet set such goals.

1	Mumps Steven A. Rubin, Kathryn M. Carbone DEFINITION Mumps is an illness characterized by acute-onset unilateral or bilateral tender, self-limited swelling of the parotid or other salivary gland(s) that lasts at least 2 days and has no other apparent cause. 231e

1	Mumps is caused by a paramyxovirus with a negative-strand, nonsegmented RNA genome of 15,384 bases encoding at least 8 proteins: the nucleo(N), phospho(P), V, matrix (M), fusion (F), small hydrophobic (SH), hemagglutinin-neuraminidase (HN), and large (L) proteins. The N, P, and L proteins together provide the polymerase activity responsible for genome transcription and replication. The viral genome is surrounded by a host cell–derived lipid bilayer envelope containing the M, F, SH, and HN proteins. The M protein is involved in viral assembly, whereas the HN and F proteins are responsible for cell attachment and entry and are the major targets of virus-neutralizing antibody. The V and SH proteins are accessory proteins, acting as antagonists of the host antiviral response; the former interferes with the interferon response and the latter with the tumor necrosis factor α (TNF-α)–mediated apoptotic signaling pathway. Because of the hypervariability of the SH gene, its nucleotide sequence

1	with the interferon response and the latter with the tumor necrosis factor α (TNF-α)–mediated apoptotic signaling pathway. Because of the hypervariability of the SH gene, its nucleotide sequence is used to “genotype” the virus for molecular epidemiologic purposes. Thus far, 12 mumps virus genotypes have been assigned by SH gene sequence and are designated A–N (with the exclusion of E and M, which have been merged with genotypes C and K, respectively).

1	Nucleotide sequencing of clinical isolates shows that virus genotypes D and G circulate predominantly in the Western Hemisphere; genotypes F, C, and I in the Asia–Pacific region; and genotypes B, H, J, and K in the Southern Hemisphere (Fig. 231e-1). Although numerous mumps virus genotypes have been identified and some vary antigenically from others, only one serotype exists, and there is no evidence to suggest that certain circulating virus strains are more virulent or contagious than others.

1	Mumps is endemic worldwide, with epidemics every 3–5 years in unvaccinated populations. These epidemics typically occur in locations where children and young adults congregate, such as schools, military barracks, and other institutions. In countries without national mumps vaccination programs, the estimated annual global incidence is 100–1000 cases per 100,000 population. After the introduction of mumps vaccine in the United States in 1967, the number of reported cases declined dramatically. By 2001, fewer than 300 cases were reported, representing a 99.8% reduction from prevaccineera levels. Mumps incidence remained at historic lows in the United States until 2006, when 6584 cases were reported—the largest outbreak since 1987. At the time of the 2006 outbreak, the disease was resurging globally, even in populations with high-level vaccination coverage. The number of reported U.S. cases declined precipitously in the 2 years that followed but then spiked in 2009–2010, with focal

1	globally, even in populations with high-level vaccination coverage. The number of reported U.S. cases declined precipitously in the 2 years that followed but then spiked in 2009–2010, with focal outbreaks in New York and New Jersey, and again in 2011, with a focal outbreak in California. A recent study by the Centers for Disease Control and Prevention (CDC) showed that two-dose coverage with measles– mumps–rubella (MMR) vaccine in major U.S. cities (94.8%) remains at or very near the level needed to contain these childhood infections; however, focal areas with inadequate vaccination coverage still leave some children at risk. Sporadic, large-scale mumps outbreaks continue to be reported worldwide, sometimes in countries where the disease was once under control.

1	Although historically a disease of unvaccinated children, with the largest proportion of cases occurring in children 5–9 years of age during the prevaccine era, mumps now frequently occurs in older age groups—primarily college students, most of whom were vaccinated in early childhood. This shift in age distribution and the occurrence of mumps in vaccinated populations are probably the result of several coincident circumstances, including (1) situations promoting the spread of respiratory viruses among young adults (e.g., residence in college dormitories), (2) waning of vaccine immunity with time, (3) lack of endemically circulating wild-type virus to periodically boost vaccine-induced immune responses, and (4) continuing global epidemics (due to either lack of mumps vaccination programs or, where such programs do exist, low rates of mumps vaccination). The notable

1	FIGurE 231e-1 Distribution of reported mumps genotypes, 2005–2011 (data as of April 20, 2012). Pie-slice size is proportional to the number of years each genotype was reported. (Figure courtesy of WHO, with permission; http://www.who.int/immunization_monitoring/diseases/ mumps/en/index.html; accessed September 11, 2012.) decline in mumps vaccination–induced immunity with time may be due to both declining titers and decreasing avidity of antibodies. The waning of mumps immunity over time is supported by studies suggesting that a third dose of MMR vaccine significantly reduces the mumps attack rate; however, these studies were not adequately controlled to rule out the possibility that the observed declines in mumps incidence were unrelated to the intervention. Therefore, the effectiveness of a third dose of MMR vaccine remains to be demonstrated.

1	Humans are the only natural hosts for mumps virus infection. The incubation period of mumps is ~19 days (range, 7–23 days). The virus is transmitted by the respiratory route via droplets, saliva, and fomites. Mumps virus is typically shed from 1 week before to 1 week after symptom onset, although this window appears to be narrower in vaccinated individuals. Persons are most contagious 1–2 days before onset of clinical symptoms. Inference from related respiratory diseases and animal studies indicates that primary replication likely occurs in the nasal mucosa or upper respiratory mucosal epithelium. Mononuclear cells and cells within regional lymph nodes can become infected; such infection facilitates the development of viremia and poses a risk for a wide array of acute inflammatory reactions. Classic sites of mumps virus replication include the salivary glands, testes, pancreas, ovaries, mammary glands, and central nervous system (CNS).

1	Little is known about the pathology of mumps since the disease is rarely fatal. The virus replicates well in glandular epithelium, but classic parotitis is not a necessary component of mumps infection. Affected glands contain perivascular and interstitial mononuclear cell infiltrates and exhibit hemorrhage with prominent edema. Necrosis of acinar and epithelial duct cells is evident in the salivary glands and in the germinal epithelium of the seminiferous tubules of the testes. The virus probably enters cerebrospinal fluid (CSF) through the choroid plexus or via transiting mononuclear cells during plasma viremia. Although relevant data are limited, typical mumps encephalitis appears to be secondary to respiratory spread and is probably a parainfectious process, as suggested by perivenous demyelination, perivascular mononuclear cell inflammation, and relative sparing of neurons. Although rare, presumed primary encephalitis has been associated with mumps virus isolation from brain

1	demyelination, perivascular mononuclear cell inflammation, and relative sparing of neurons. Although rare, presumed primary encephalitis has been associated with mumps virus isolation from brain tissue. Evidence of placental and intrauterine spread in pregnancy has been found in both early and late gestation.

1	Up to half of mumps virus infections are asymptomatic or lead to nonspecific respiratory symptoms. Inapparent infections are more common in adults than in children. The prodrome of mumps consists of low-grade fever, malaise, myalgia, headache, and anorexia. Mumps parotitis—acute-onset unilateral or bilateral swelling of the parotid or other salivary glands that lasts >2 days and has no other apparent cause—develops in 70–90% of symptomatic infections, usually within 24 h of prodromal symptoms but sometimes as long as 1 week thereafter. Parotitis is generally bilateral, although the two sides may not be involved synchronously. Unilateral involvement is documented in about one-third of cases. Swelling of the parotid is accompanied by tenderness and obliteration of the space between the earlobe and the angle of the mandible (Figs. 231e-2 and 231e-3). The patient frequently reports an earache and finds it difficult to eat, swallow, or talk. The orifice of the parotid duct is commonly red

1	and the angle of the mandible (Figs. 231e-2 and 231e-3). The patient frequently reports an earache and finds it difficult to eat, swallow, or talk. The orifice of the parotid duct is commonly red and swollen. The submaxillary and sublingual glands are involved less often than the parotid gland and are almost never involved alone. Glandular swelling increases for a few days and then gradually subsides, disappearing within 1 week. Recurrent sialadenitis is a rare sequela of mumps parotitis. In ~6% of mumps cases, obstruction of lymphatic drainage secondary to bilateral salivary gland swelling may lead to presternal pitting edema, associated often with submandibular adenitis and rarely with the more life-threatening supraglottic edema.

1	FIGurE 231e-2 A comparison of a person before acquiring mumps (A) and on day 3 (B) of acute bilateral parotitis. (Courtesy of patient C.M. From Shanley JD. The resurgence of mumps in young adults and adolescents. Cleve Clin J Med 2007; 74:42-48. Reprinted with permission. Copyright © 2007 Cleveland Clinic Foundation. All rights reserved.)

1	Epididymo-orchitis is the next most common manifestation of mumps, developing in 15–30% of cases in postpubertal males, with bilateral involvement in 10–30% of those cases. Orchitis, accompanied by fever, typically occurs during the first week of parotitis but can develop up to 6 weeks after parotitis or in its absence. The testis is painful and tender and can be enlarged to several times its normal size; this condition usually resolves within 1 week. Testicular atrophy develops in one-half of affected men. Sterility after mumps is rare, although subfertility is estimated to occur in 13% of cases of unilateral orchitis and in 30–87% of cases of bilateral orchitis. Oophoritis occurs in ~5% of women with mumps and may be associated with lower abdominal pain and vomiting but has only rarely been associated with sterility or premature menopause. Mumps infection in postpubertal women may also present with mastitis.

1	Documented CSF pleocytosis indicates that mumps virus invades the CNS in ~50% of cases; however, symptomatic CNS disease, typically in the form of aseptic meningitis, occurs in <10% of cases, with a male predominance. CNS symptoms of aseptic meningitis (e.g., stiff neck, headache, and drowsiness) appear ~5 days after parotitis and also occur often in the absence of parotid involvement. Within the first 24 h polymorphonuclear leukocytes may predominate in CSF (1000–2000 cells/μL), but by the second day nearly all the cells are lymphocytes. The glucose level in CSF may be low and the protein concentration high, a pattern reminiscent of bacterial meningitis. Mumps meningitis is a self-limited manifestation without significant risk of death or long-term sequelae. Cranial nerve palsies have occasionally led to permanent sequelae, particularly deafness. The reported incidence of mumps-associated hearing loss varies between 1 in 1000 and 1 in 100,000. In ~0.1% of infections, mumps virus may

1	led to permanent sequelae, particularly deafness. The reported incidence of mumps-associated hearing loss varies between 1 in 1000 and 1 in 100,000. In ~0.1% of infections, mumps virus may cause encephalitis, which presents as high fever with marked changes in the level of consciousness, seizures, and focal neurologic symptoms. Electroencephalographic abnormalities may be seen. Permanent sequelae are sometimes identified in survivors, and adult infections more commonly have poor outcomes than do pediatric infections. The mortality rate associated with mumps encephalitis is ~1.5%. Other CNS problems occasionally associated with mumps include cerebellar ataxia, facial palsy, transverse myelitis, hydrocephalus, Guillain-Barré syndrome, flaccid paralysis, and behavioral changes.

1	FIGurE 231e-3 Schematic drawing of a parotid gland infected with mumps virus (right) compared with a normal gland (left). An enlarged cervical lymph node is usually posterior to the imaginary line. (Reprinted with permission from Gershon A et al: Mumps, in Krugman’s Infectious Diseases of Children, 11th ed. Philadelphia, Elsevier, 2004, p 392.)

1	Mumps pancreatitis, which may present as abdominal pain, occurs in ~4% of infections but is difficult to diagnose because an elevated serum amylase level can be associated with either parotitis or pancreatitis. An etiologic association of mumps virus and juvenile diabetes mellitus remains controversial. Myocarditis and endocardial fibroelastosis are rare and self-limited but may represent severe complications of mumps infection; however, mumps-associated electrocardiographic abnormalities have been reported in up to 15% of cases. Other unusual complications include thyroiditis, nephritis, arthritis, hepatic disease, keratouveitis, and thrombocytopenic purpura. Abnormal renal function is common, but severe, life-threatening nephritis is rare. It remains at issue whether an excessive number of spontaneous abortions are associated with gestational mumps. Mumps in pregnancy does not appear to lead to premature birth, low birth weight, or fetal malformations.

1	During a mumps outbreak, the diagnosis is made easily in patients with parotitis and a history of recent exposure; however, when disease incidence is low, other causes of parotitis should be considered and laboratory testing is required for case confirmation. Infectious causes of parotitis include other viruses (e.g., HIV, coxsackievirus, parainfluenza virus type 3, influenza A virus, Epstein-Barr virus, adenovirus, parvovirus B19, lymphocytic choriomeningitis virus, human herpes-virus 6), gram-positive bacteria, atypical mycobacteria, and Bartonella species. Rarely, other gram-negative or anaerobic bacteria are associated with parotitis. Parotitis can also develop in the setting of sarcoidosis, Sjögren’s syndrome, Mikulicz’s syndrome, Parinaud’s syndrome, uremia, diabetes mellitus, laundry starch ingestion, malnutrition, cirrhosis, and some drug treatments. Unilateral parotitis can be caused by ductal obstruction, cysts, and tumors. In the absence of parotitis or other salivary gland

1	starch ingestion, malnutrition, cirrhosis, and some drug treatments. Unilateral parotitis can be caused by ductal obstruction, cysts, and tumors. In the absence of parotitis or other salivary gland enlargement, symptoms of other visceral organ and/or CNS involvement may predominate, and a laboratory diagnosis is required. Other entities should be considered when manifestations consistent with mumps appear in organs other than the parotid. Testicular torsion may produce a painful scrotal mass resembling that seen in mumps orchitis. Other viruses (e.g., enteroviruses) may cause aseptic meningitis that is clinically indistinguishable from that due to mumps virus.

1	Laboratory diagnosis is primarily based on detection of viral RNA by reverse-transcriptase polymerase chain reaction (RT-PCR) or on serology. Detection of viral antigens (e.g., via mumps virus–specific immunofluorescent staining of cultured clinical specimens) is comparatively inefficient and is no longer commonly performed.

1	For RT-PCR-based testing, viral RNA can be extracted either directly from clinical samples or from cell cultures incubated with clinical samples. Buccal swabs appear to be the best specimens for virus detection, particularly when obtained within 2 days of clinical onset; however, mumps virus can also be detected readily in throat swabs and saliva and, in cases of meningitis, in CSF. Despite the apparent high frequency of viremia during mumps, mumps virus has rarely been detected in blood. The ability to detect viral RNA in clinical samples rapidly diminishes beyond the first week after symptom onset, and 231e-3 in several studies rates of virus detection were substantially lower in recipients of two vaccine doses than in unvaccinated persons or recipients of one dose. The rate of false-negative RT-PCR findings can be quite high, approaching 70% in some studies.

1	A serologic diagnosis of mumps is typically made by enzyme-linked immunosorbent assay (ELISA). The data must be interpreted with caution. In vaccinated persons with mumps, IgM is typically absent; thus, a negative IgM result in a vaccinated person does not rule out mumps. In addition, regardless of vaccination status, IgM may not be detectable if serum is assayed too early (prior to day 3 of symptom onset) or too late (beyond 6 weeks after symptom onset) in the course of disease. Reliance on a rise in IgG titer in paired acuteand convalescent-phase sera also is problematic: IgG titers in convalescent-phase sera may be only nominally greater than those in acute-phase sera. Thus, at present, the capacity of RNA or viral antigen detection to confirm cases is much greater than that of serologic testing. Traditional and labor-intensive serologic tests such as complement fixation, hemagglutination inhibition, and virus neutralization are now performed only rarely. The main downside to

1	testing. Traditional and labor-intensive serologic tests such as complement fixation, hemagglutination inhibition, and virus neutralization are now performed only rarely. The main downside to replacement of these functional serologic assays with the more rapid ELISA method is the latter’s detection of all virus-specific antibodies, including those that are nonneutralizing (i.e., nonprotective). Thus, an individual who is seropositive by ELISA may lack protective levels of antibody. While there is a strong association between the presence of mumps virus neutralizing antibody and protection from disease, an absolute antibody titer predictive of serologic protection is lacking; in this respect, mumps differs from other respiratory infections, such as measles.

1	Mumps is generally a benign, self-resolving illness. Therapy for parotitis and other clinical manifestations is symptom based and supportive. The administration of analgesics and the application of warm or cold compresses to the parotid area may be helpful. Testicular pain may be minimized by the local application of cold compresses and gentle support for the scrotum. Anesthetic blocks also may be used. Neither the administration of glucocorticoids nor incision of the tunica albuginea is of proven value in severe orchitis. Anecdotal information on a small number of patients with orchitis suggests that SC administration of interferon α2b may help preserve the organ and fertility. Lumbar puncture is occasionally performed to relieve headache associated with meningitis. Mumps immune globulin has not been consistently shown to be effective in preventing mumps and is not recommended for treatment or postexposure prophylaxis.

1	Vaccination is the only practical control measure. Nearly all developed countries use mumps-containing vaccines, but in many countries mumps is not a notifiable disease and vaccination is often voluntary. However, where used, mumps vaccination has had a tremendous impact, with reductions in incidence and morbidity typically exceeding 90%. Despite the tremendous success of mumps vaccination programs, large mumps outbreaks continue to occur globally, even in settings of high-level two-dose vaccine coverage. Whereas outbreaks historically involved young (often unvaccinated) children in primary and secondary schools, more recent outbreaks have predominantly involved young adults, particularly on college and university campuses. While primary and secondary (waning-immunity) vaccine failures have been hypothesized to be factors in mumps outbreaks in several countries, in some countries other factors may have played a role, such as lack of compliance with the recommended vaccine schedule,

1	have been hypothesized to be factors in mumps outbreaks in several countries, in some countries other factors may have played a role, such as lack of compliance with the recommended vaccine schedule, changes to vaccination schedules resulting in missed cohorts, or changes in population demographics, such as large-scale immigration. In the United States, the benefit-cost ratios for mumps vaccination alone are >13 for direct costs (e.g., medical expenses) and >24 for societal costs (including productivity losses for patients and caregivers).

1	Several mumps virus vaccines are used throughout the world; in the United States, only the live attenuated Jeryl Lynn strain is used. Current recommendations are that mumps vaccine be administered as part of the combined trivalent measles-mumps-rubella vaccine (M-M-R® II) or the quadrivalent measles-mumps-rubella-varicella vaccine (ProQuad®). Monovalent vaccine is no longer produced for the U.S. market but is available in other countries. Before administering mumps-containing vaccine, physicians should always consult the latest recommendations from the Advisory Committee on Immunization Practices (ACIP). Current recommendations for children specify two doses of mumps-containing vaccine: the first dose given on or after the first birthday and the second dose administered no earlier than 28 days after the first. In the United States, children often receive the second dose between the ages of 4 and 6 years.

1	In 2009, the ACIP revised its recommendations for evidence of mumps immunity in health care personnel to include (1) documented administration of two doses of a preparation containing live mumps vaccine, (2) laboratory evidence of immunity or laboratory confirmation of disease, or (3) birth date before 1957. For unvaccinated health care personnel born before 1957 who lack laboratory evidence of mumps immunity or laboratory confirmation of mumps, health care facilities should consider two doses of MMR vaccine separated by the appropriate interval; during a mumps outbreak, vaccination of these individuals is recommended.

1	Mumps vaccine contains live attenuated virus. It is not recommended for pregnant women, for individuals who have had a life-threatening allergic reaction to components of the vaccine, or for people in settings of clinically significant primary or secondary immunosuppression. (For details, see the ACIP guidelines on the CDC’s website [www.cdc.gov/vaccines/pubs/acip-list.htm].) Occasionally, febrile reactions and parotitis have been reported soon after mumps vaccination. Allergic reactions after vaccination (e.g., rash and pruritus) are uncommon and are usually mild and self-limited. More serious complications, such as aseptic meningitis, have been causally associated with certain vaccine strains but not with the Jeryl Lynn strain.

1	Immunity to mumps is associated with the development of neutralizing antibody, although a specific correlate of protection has not been established. Seroconversion occurs in ~95% of recipients of the Jeryl Lynn strain; however, vaccine efficacy is ~80% for one dose and 90% for two doses. Recent data indicate declining seropositivity rates with time since vaccination. Studies are under way to assess the value of including a third dose in the immunization schedule. Although it is generally accepted that mumps virus is serologically monotypic, antigenic differences between virus isolates have been detected. It is unclear whether such differences can lead to immune escape. The role of the cellular arm of the immune response is unclear, but there is evidence that it may help limit virus spread and complications. The authors thank and acknowledge Dr. Anne Gershon, the author of this chapter in earlier editions.

1	The authors thank and acknowledge Dr. Anne Gershon, the author of this chapter in earlier editions. Rabies and Other Rhabdovirus InfectionsCHAPTER 232Rabies and Other Rhabdovirus Infections Alan C. Jackson RABIES Rabies is a rapidly progressive, acute infectious disease of the central 232 nervous system (CNS) in humans and animals that is caused by infection with rabies virus. The infection is normally transmitted from animal vectors. Rabies has encephalitic and paralytic forms that progress to death.

1	Rabies virus is a member of the family Rhabdoviridae. Two genera in this family, Lyssavirus and Vesiculovirus, contain species that cause human disease. Rabies virus is a lyssavirus that infects a broad range of animals and causes serious neurologic disease when transmitted to humans. This single-strand RNA virus has a nonsegmented, negative-sense (antisense) genome that consists of 11,932 nucleotides and encodes 5 proteins: nucleocapsid protein, phosphoprotein, matrix protein, glycoprotein, and a large polymerase protein. Rabies virus variants, which can be characterized by distinctive nucleotide sequences, are associated with specific animal reservoirs. Six other non–rabies virus species in the Lyssavirus genus have been reported to cause a clinical picture similar to rabies. Vesicular stomatitis virus, a vesiculovirus, causes vesiculation and ulceration in cattle, horses, and other animals and causes a self-limited, mild, systemic illness in humans (see “Other Rhabdoviruses,”

1	stomatitis virus, a vesiculovirus, causes vesiculation and ulceration in cattle, horses, and other animals and causes a self-limited, mild, systemic illness in humans (see “Other Rhabdoviruses,” below).

1	1300 EPIDEMIOLOGY Rabies is a zoonotic infection that occurs in a variety of mammals throughout the world except in Antarctica and on some islands. Rabies virus is usually transmitted to humans by the bite of an infected animal. Worldwide, endemic canine rabies is estimated to cause 55,000 human deaths annually. Most of these deaths occur in Asia and Africa, with rural populations and children most frequently affected. Thus, in many resource-poor and resource-limited countries, canine rabies continues to be a threat to humans. However, in Latin America, rabies control efforts in dogs have been quite successful in recent years. Endemic canine rabies has been eliminated from the United States and most other resource-rich countries. Rabies is endemic in wildlife species, and a variety of animal reservoirs have been identified in different countries. Surveillance data from 2012 identified 6162 confirmed animal cases of rabies in the United States (including Puerto Rico). Only 8% of these

1	reservoirs have been identified in different countries. Surveillance data from 2012 identified 6162 confirmed animal cases of rabies in the United States (including Puerto Rico). Only 8% of these cases were in domestic animals, including 257 cases in cats, 84 in dogs, and 115 in cattle. In North American wildlife reservoirs, including bats, raccoons, skunks, and foxes, the infection is endemic, with involvement of one or more rabies virus variants in each reservoir species (Fig. 232-1). “Spillover” of rabies to other wildlife species and to domestic animals occurs. Bat rabies virus variants are present in every state except Hawaii and are responsible for most indigenously acquired human rabies cases in the United States. Raccoon rabies is endemic along the entire eastern coast of the United States. Skunk rabies is present in the midwestern states, with another focus in California. Rabies in foxes occurs in Texas, New Mexico, Arizona, and Alaska. In Canada and Europe, epizootics of

1	States. Skunk rabies is present in the midwestern states, with another focus in California. Rabies in foxes occurs in Texas, New Mexico, Arizona, and Alaska. In Canada and Europe, epizootics of rabies in red foxes have been well controlled with the use of baits containing rabies vaccine. A similar approach is used in Canada to control raccoon rabies. Rabies virus variants isolated from humans or other mammalian species can be identified by reverse-transcription polymerase chain reaction (RT-PCR) amplification and sequencing or by characterization with monoclonal antibodies. These techniques are helpful in human cases with no known history of exposure. Worldwide, most human rabies is transmitted from dogs in countries with endemic canine rabies and dog-to-dog transmission, and human cases can be imported by travelers returning from these regions. In North America, human disease is usually associated with transmission from bats; there may be no known history of bat bite or other bat

1	can be imported by travelers returning from these regions. In North America, human disease is usually associated with transmission from bats; there may be no known history of bat bite or other bat exposure in these cases. Most human cases are due to a bat rabies virus variant associated with silver-haired and tricolored bats. These are small bats whose bite may not be recognized, and the virus has adapted for replication at skin temperature and in cell types that are present in the skin.

1	Transmission from nonbite exposures is relatively uncommon. Aerosols generated in the laboratory or in caves containing millions of Brazilian free-tail bats have rarely caused human rabies. Transmission has resulted from corneal transplantation and also from solid organ transplantation and a vascular conduit (for a liver transplant) from undiagnosed donors with rabies in Texas, Florida, and Germany. Human-to-human transmission is extremely rare, although hypothetical concern about transmission to health care workers has prompted the implementation of barrier techniques to prevent exposures.

1	The incubation period of rabies (defined as the interval between exposure and the onset of clinical disease) is usually 20–90 days but in rare cases is as short as a few days or >1 year. During most of the incubation period, rabies virus is thought to be present at or close to the site of inoculation (Fig. 232-2). In muscles, the virus is known to bind to nicotinic acetylcholine receptors on postsynaptic membranes at neuromuscular junctions, but the exact details of viral entry into the skin and SC tissues have not yet been clarified. Rabies virus spreads centripetally along peripheral nerves toward the CNS at a rate of up to ~250 mm/d via retrograde fast axonal transport to the spinal cord or brainstem. Once the virus enters the CNS, it rapidly disseminates to other regions of the CNS via fast axonal transport along neuroanatomic connections. Neurons are prominently infected in rabies; infection of astrocytes is unusual. After CNS infection becomes established, there is centrifugal

1	fast axonal transport along neuroanatomic connections. Neurons are prominently infected in rabies; infection of astrocytes is unusual. After CNS infection becomes established, there is centrifugal spread along sensory and autonomic nerves to other tissues, including the salivary glands, heart, adrenal glands, and skin. Rabies virus replicates in acinar cells of the salivary glands and is secreted in the saliva of rabid animals that serve as vectors of

1	FIGuRE 232-1 Distribution of the major rabies virus variants among wild terrestrial reservoirs in the United States and Puerto Rico, 2008–2012. *Potential host-shift event. (From JL Dyer et al: J Am Vet Med Assoc 243:805, 2013.)

1	Eye Salivary glands Dorsal root ganglion Spinal cord Brain Infection of brain neurons with neuronal dysfunction 6 Centrifugal spread along nerves to salivary glands, skin, cornea, and other organs 7 Virus binds to nicotinic acetylcholine receptors at neuromuscular junction 3 Replication in motor neurons of the spinal cord and local dorsal root ganglia and rapid ascent to brain 5 Virus inoculated 1 Viral replication in muscle 2 Virus travels within axons in peripheral nerves via retrograde fast axonal transport 4 Sensory nerves to skin Skeletal muscle FIGuRE 232-2 Schematic representation of the pathogenetic events following periph-eral inoculation of rabies virus by an animal bite. (Adapted from Jackson AC: Human disease, in Rabies: scientific basis of the disease and its management, 3rd ed., AC Jackson [ed], Oxford, UK, Elsevier Academic Press, 2013, pp 269–298; with permission.)

1	In rabies, the emphasis must be on postexposure prophylaxis (PEP) initiated before any symptoms or signs develop. Rabies should usually be suspected on the basis of the clinical presentation. The disease usually presents as atypical encephalitis with relative preservation of consciousness. Rabies may be difficult to recognize late in the clinical course when progression to coma has occurred. A minority of patients present with acute flaccid paralysis. There are prodromal, acute neurologic, and comatose phases that usually progress to death despite aggressive therapy (Table 232-1).

1	Prodromal Features The clinical features of rabies begin with nonspecific prodromal manifestations, including fever, malaise, headache, nausea, and vomiting. Anxiety or agitation may also occur. The earliest specific neurologic symptoms of rabies include paresthesias, pain, or pruritus near the site of the exposure, one or more of which occur in 50–80% of patients and strongly suggest rabies. The wound has usually healed by this point, and these symptoms probably reflect infection with associated inflammatory changes in local dorsal root or cranial sensory ganglia.

1	Encephalitic Rabies Two acute neurologic forms of rabies are seen in humans: encephalitic (furious) in 80% and paralytic in 20%. Some of the manifestations of encephalitic rabies, including fever, confusion, hallucinations, combativeness, and seizures, may be seen in other viral encephalitides as well. Autonomic dysfunction is common and may result in hypersalivation, gooseflesh, cardiac arrhythmia, and priapism. In encephalitic rabies, episodes of hyperexcitability are typically followed by periods of complete lucidity that become shorter as the disease progresses. Rabies encephalitis is distinguished by early brainstem involvement, which results in the classic features of hydrophobia (involuntary, painful contraction of the diaphragm and accessory respiratory, laryngeal, and pharyngeal muscles in response to swallowing liquids) the disease. There is no well-documented evidence for hematogenous spread of rabies virus.

1	Pathologic studies show mild inflammatory changes in the CNS in rabies, with mononuclear inflammatory infiltration in the leptomeninges, perivascular regions, and parenchyma, including microglial nodules called Babes nodules. Degenerative neuronal changes usually are not prominent, and there is little evidence of neuronal death; neuronophagia is observed occasionally. The pathologic changes are surprisingly mild in light of the clinical severity and fatal outcome of the disease. The most characteristic pathologic finding in rabies is the Negri body (Fig. 232-3). Negri bodies are eosinophilic cytoplasmic inclusions in brain neurons that are composed of rabies virus proteins and viral RNA. These inclusions occur in a minority of infected neurons, are commonly observed in Purkinje cells of the cerebellum and in pyramidal neurons of the hippocampus, and are less frequently seen in cortical and brainstem neurons. Negri bodies are not observed in all cases of rabies. The lack of prominent

1	the cerebellum and in pyramidal neurons of the hippocampus, and are less frequently seen in cortical and brainstem neurons. Negri bodies are not observed in all cases of rabies. The lack of prominent degenerative neuronal changes has led to the concept that neuronal dysfunction—rather than neuronal death—is responsible for clinical disease in rabies. The basis for behavioral changes, including the aggressive behavior of rabid animals, is not well understood.

1	and aerophobia (the same features caused by stimulation from a draft of air). These symptoms are probably due to dysfunction of infected brainstem neurons that normally inhibit inspiratory neurons near the nucleus ambiguus, resulting in exaggerated defense reflexes that protect the respiratory tract. The combination of hypersalivation and pharyngeal dysfunction is also responsible for the classic appearance of “foaming at the mouth” (Fig. 232-4). Brainstem dysfunction progresses rapidly, and coma—followed within days by death—is the rule unless the course is prolonged by supportive measures. With such measures, late complications can include cardiac and/or respiratory failure, disturbances of water balance (syndrome of inappropriate antidiuretic hormone secretion or diabetes insipidus), noncardiogenic pulmonary edema, and gastrointestinal hemorrhage. Cardiac arrhythmias may be due to dysfunction affecting vital centers in the brainstem or to myocarditis. Multiple-organ failure is

1	noncardiogenic pulmonary edema, and gastrointestinal hemorrhage. Cardiac arrhythmias may be due to dysfunction affecting vital centers in the brainstem or to myocarditis. Multiple-organ failure is common in patients treated aggressively in critical care units.

1	Paralytic Rabies About 20% of patients have paralytic rabies in which muscle weakness predominates and cardinal features of encephalitic rabies (hyperexcitability, hydrophobia, and aerophobia) are lacking. There is early and prominent flaccid muscle weakness, often beginning in the bitten extremity and spreading to produce quadriparesis and facial FIGuRE 232-3 Three large Negri bodies in the cytoplasm of a cerebellar Purkinje cell from an 8-year-old boy who died of rabies after being bitten by a rabid dog in Mexico. (From AC Jackson, E Lopez-Corella: N Engl J Med 335:568, 1996. © Massachusetts Medical Society.) weakness. Sphincter involvement is common, sensory involvement is usually mild, and these cases are commonly misdiagnosed as Guillain-Barré syndrome. Patients with paralytic rabies generally survive a few days longer than those with encephalitic rabies, but multiple-organ failure nevertheless ensues.

1	Most routine laboratory tests in rabies yield normal results or show nonspecific abnormalities. Complete blood counts are usually normal. Examination of cerebrospinal fluid (CSF) often reveals mild mononuclear cell pleocytosis with a mildly elevated protein level. Severe pleocytosis (>1000 white cells/μL) is unusual and should prompt a search for an alternative diagnosis. CT head scans are usually normal in rabies. MRI brain scans may show signal abnormalities in the brainstem or other gray-matter areas, but these findings are variable and nonspecific. Electroencephalograms show only nonspecific abnormalities. Of course, important tests in suspected cases of rabies include those that may identify an alternative, potentially treatable diagnosis (see “Differential Diagnosis,” below). Source: MAW Hattwick: Rabies virus, in Principles and Practice of Infectious Diseases, GL Mandell et al (eds). New York, Wiley, 1979, pp 1217–1228. Adapted with permission from Elsevier.

1	Source: MAW Hattwick: Rabies virus, in Principles and Practice of Infectious Diseases, GL Mandell et al (eds). New York, Wiley, 1979, pp 1217–1228. Adapted with permission from Elsevier. FIGuRE 232-4 Hydrophobic spasm of inspiratory muscles associ-ated with terror in a patient with encephalitic (furious) rabies who is attempting to swallow water. (Copyright DA Warrell, Oxford, UK; with permission.)

1	In North America, a diagnosis of rabies often is not considered until relatively late in the clinical course, even with a typical clinical presentation. This diagnosis should be considered in patients presenting with acute atypical encephalitis or acute flaccid paralysis, including those in whom Guillain-Barré syndrome is suspected. The absence of an animal-bite history is common in North America. The lack of hydrophobia is not unusual in rabies. Once rabies is suspected, rabies-specific laboratory tests should be performed to confirm the diagnosis. Diagnostically useful specimens include serum, CSF, fresh saliva, skin biopsy samples from the neck, and brain tissue (rarely obtained before death). Because skin biopsy relies on the demonstration of rabies virus antigen in cutaneous nerves at the base of hair follicles, samples are usually taken from hairy skin at the nape of the neck. Corneal impression smears are of low diagnostic yield and are generally not performed. Negative

1	at the base of hair follicles, samples are usually taken from hairy skin at the nape of the neck. Corneal impression smears are of low diagnostic yield and are generally not performed. Negative antemortem rabies-specific laboratory tests never exclude a diagnosis of rabies, and tests may need to be repeated after an interval for diagnostic confirmation.

1	Rabies Virus–Specific Antibodies In a previously unimmunized patient, serum neutralizing antibodies to rabies virus are diagnostic. However, because rabies virus infects immunologically privileged neuronal tissues, serum antibodies may not develop until late in the disease. Antibodies may be detected within a few days after the onset of symptoms, but some patients die without detectable antibodies. The presence of rabies virus–specific antibodies in the CSF suggests rabies encephalitis, regardless of immunization status. A diagnosis of rabies is questionable in patients who recover from rabies without developing serum neutralizing antibodies to rabies virus.

1	RT-PCR Amplification Detection of rabies virus RNA by RT-PCR is highly sensitive and specific. This technique can detect virus in fresh saliva samples, skin, CSF, and brain tissues. In addition, RT-PCR with genetic sequencing can distinguish among rabies virus variants, permitting identification of the probable source of an infection. Direct Fluorescent Antibody Testing Direct fluorescent antibody (DFA) testing with rabies virus antibodies conjugated to fluorescent dyes is highly sensitive and specific and can be performed quickly and applied to skin biopsies and brain tissue. In skin biopsies, rabies virus antigen may be detected in cutaneous nerves at the base of hair follicles.

1	The diagnosis of rabies may be difficult without a history of animal exposure, and no exposure to an animal (e.g., a bat) may be recalled. The presentation of rabies is usually quite different from that of acute viral encephalitis due to most other causes, including herpes simplex encephalitis and arboviral (e.g., West Nile) encephalitis. Early neurologic symptoms may occur at the site of the bite, and there may be early features of brainstem involvement with preservation of consciousness. Anti-N-methyl-d-aspartate receptor (anti-NMDA) encephalitis occurs in young patients (especially females) and is characterized by behavioral changes, autonomic instability, hypoventilation, and seizures. Postinfectious (immune-mediated) encephalomyelitis may follow influenza, measles, mumps, and other infections; it may also occur as a sequela of immunization with rabies vaccine derived from neural tissues, which are used only in resource-limited and resource-poor countries. Rabies may present with

1	it may also occur as a sequela of immunization with rabies vaccine derived from neural tissues, which are used only in resource-limited and resource-poor countries. Rabies may present with unusual neuropsychiatric symptoms and may be misdiagnosed as a psychiatric disorder. Rabies hysteria may occur as a psychological response to the fear of rabies and is often characterized by a shorter incubation period than rabies, aggressive behavior, inability to communicate, and a long course with recovery.

1	As previously mentioned, paralytic rabies may mimic Guillain-Barré syndrome. In these cases, fever, bladder dysfunction, a normal sensory examination, and CSF pleocytosis favor a diagnosis of rabies. Conversely, Guillain-Barré syndrome may occur as a complication of rabies vaccination with a neural tissue–derived product (e.g., suckling mouse brain vaccine) and may be mistaken for paralytic rabies (i.e., vaccine failure). There is no established treatment for rabies. There have been many recent treatment failures with the combination of antiviral drugs, ketamine, and therapeutic (induced) coma—measures that were used in a healthy survivor in whom antibodies to rabies virus were detected at presentation. Expert opinion should be sought before a course of experimental therapy is embarked upon. A palliative approach may be appropriate for some patients.

1	Rabies is an almost uniformly fatal disease but is nearly always preventable with appropriate postexposure therapy during the early incubation period (see below). There are seven well-documented cases of survival from rabies. All but one of these patients had received rabies vaccine before disease onset. The single survivor who had not received vaccine had neutralizing antibodies to rabies virus in serum and CSF at clinical presentation. Most patients with rabies die within several days of the onset of illness, despite aggressive care in a critical care unit.

1	PREVENTION Postexposure Prophylaxis Since there is no effective therapy for rabies, it is extremely important to prevent the disease after an animal exposure. Figure 232-5 shows the steps involved in making decisions about PEP. On the basis of the history of the exposure and local epidemiologic information, the physician must decide whether initiation of PEP is warranted. Healthy dogs, cats, or ferrets may be confined and observed for 10 days. PEP is not necessary if the animal remains healthy. If the animal develops signs of rabies during the observation period, it should be euthanized immediately; the head should be transported to the laboratory under refrigeration, rabies virus should be sought by DFA testing, and viral isolation should be attempted by cell culture and/or mouse inoculation. Any animal other than a dog, cat, or ferret should be euthanized immediately and the head submitted for laboratory examination. In high-risk exposures and in areas where canine rabies is

1	inoculation. Any animal other than a dog, cat, or ferret should be euthanized immediately and the head submitted for laboratory examination. In high-risk exposures and in areas where canine rabies is endemic, rabies prophylaxis should be initiated without waiting for laboratory results. If the laboratory results prove to be negative, it may safely be concluded that the animal’s saliva did not

1	Did the animal bite the patient or did saliva contaminate a scratch, abrasion, open wound, or mucous membrane? Is rabies known or suspected to be present in the species and the geographic area? Was the animal captured? Does laboratory examination of the brain by fluorescent antibody staining confirm rabies? Was the animal a normally behaving dog, cat, or ferret? Does the animal become ill under observation over the next 10 days?

1	Was the animal a normally behaving dog, cat, or ferret? Does the animal become ill under observation over the next 10 days? FIGuRE 232-5 Algorithm for rabies postexposure prophylaxis. RIG, rabies immune globulin. (From L Corey, in Harrison’s Principles of Internal Medicine, 15th ed. E Braunwald et al [eds]: New York, McGraw-Hill, 2001; adapted with permission.) contain rabies virus, and immunization should be discontinued. If an animal escapes after an exposure, it must be considered rabid, and PEP must be initiated unless information from public health officials indicates otherwise (i.e., there is no endemic rabies in the area). Although controversial, the use of PEP may be warranted when a person (e.g., a small child or a sleeping adult) has been present in the same space as a bat and an unrecognized bite cannot be reliably excluded.

1	PEP includes local wound care and both active and passive immunization. Local wound care is essential and may greatly decrease the risk of rabies virus infection. Wound care should not be delayed, even if the initiation of immunization is postponed pending the results of the 10-day observation period. All bite wounds and scratches should be washed thoroughly with soap and water. Devitalized tissues should be debrided, tetanus prophylaxis given, and antibiotic treatment initiated whenever indicated.

1	All previously unvaccinated persons (but not those who have previously been immunized) should be passively immunized with rabies immune globulin (RIG). If RIG is not immediately available, it should be administered no later than 7 days after the first vaccine dose. After day 7, endogenous antibodies are being produced, and passive immunization may actually be counterproductive. If anatomically feasible, the entire dose of RIG (20 IU/kg) should be infiltrated at the site of the bite; otherwise, any RIG remaining after infiltration of the bite site should be administered IM at a distant site. With multiple or large wounds, the RIG preparation may need to be diluted in order to obtain a sufficient volume for adequate infiltration of all wound sites. If the exposure involves a mucous membrane, the entire dose should be administered IM. Rabies vaccine and RIG should never be administered at the same site or with the same syringe. Commercially available RIG in the United States is purified

1	the entire dose should be administered IM. Rabies vaccine and RIG should never be administered at the same site or with the same syringe. Commercially available RIG in the United States is purified from the serum of hyper-immunized human donors. These human RIG preparations are much better tolerated than are the equine-derived preparations still in use in 1304 some countries (see below). Serious adverse effects of human RIG are uncommon. Local pain and low-grade fever may occur. Two purified inactivated rabies vaccines are available for rabies PEP in the United States. They are highly immunogenic and remarkably safe compared with earlier vaccines. Four 1-mL doses of rabies vaccine should be given IM in the deltoid area. (The anterolateral aspect of the thigh also is acceptable in children.) Gluteal injections, which may not always reach muscle, should not be given and have been associated with rare vaccine failures. Ideally, the first dose should be given as soon as possible after

1	Gluteal injections, which may not always reach muscle, should not be given and have been associated with rare vaccine failures. Ideally, the first dose should be given as soon as possible after exposure; failing that, it should be given without further delay. The three additional doses should be given on days 3, 7, and 14; a fifth dose on day 28 is no longer recommended. Pregnancy is not a contraindication for immunization. Glucocorticoids and other immunosuppressive medications may interfere with the development of active immunity and should not be administered during PEP unless they are essential. Routine measurement of serum neutralizing antibody titers is not required, but titers should be measured 2–4 weeks after immunization in immunocompromised persons. Local reactions (pain, erythema, edema, and pruritus) and mild systemic reactions (fever, myalgias, headache, and nausea) are common; anti-inflammatory and antipyretic medications may be used, but immunization should not be

1	edema, and pruritus) and mild systemic reactions (fever, myalgias, headache, and nausea) are common; anti-inflammatory and antipyretic medications may be used, but immunization should not be discontinued. Systemic allergic reactions are uncommon, but anaphylaxis does occur rarely and can be treated with epinephrine and antihistamines. The risk of rabies development should be carefully considered before the decision is made to discontinue vaccination because of an adverse reaction. Most of the burden of rabies PEP is borne by persons with the fewest resources. In addition to the rabies vaccines discussed above, vaccines grown in either primary cell lines (hamster or dog kidney) or continuous cell lines (Vero cells) are satisfactory and are available in many countries outside the United States. Less expensive vaccines derived from neural tissues are still used in a diminishing number of developing countries; however, these vaccines are associated with serious neuroparalytic

1	United States. Less expensive vaccines derived from neural tissues are still used in a diminishing number of developing countries; however, these vaccines are associated with serious neuroparalytic complications, including postinfectious encephalomyelitis and Guillain-Barré syndrome. The use of these vaccines should be discontinued as soon as possible, and progress has been made in this regard. Worldwide, >10 million individuals receive postexposure rabies vaccine each year. If human RIG is unavailable, purified equine RIG can be used in the same manner at a dose of 40 IU/kg. Before the administration of equine RIG, hypersensitivity should be assessed by intradermal testing with a 1:10 dilution. The incidence of anaphylactic reactions and serum sickness has been low with recent equine RIG products.

1	Preexposure Rabies Vaccination Preexposure rabies prophylaxis should be considered for people with an occupational or recreational risk of rabies exposures, including certain travelers to rabies-endemic areas. The primary schedule consists of three doses of rabies vaccine given on days 0, 7, and 21 or 28. Serum neutralizing antibody tests help determine the need for subsequent booster doses. When a previously immunized individual is exposed to rabies, two booster doses of vaccine should be administered on days 0 and 3. Wound care remains essential. As stated above, RIG should not be administered to previously vaccinated persons. A growing number of lyssaviruses other than rabies virus have been discovered to infect bat populations in Europe, Africa,

1	A growing number of lyssaviruses other than rabies virus have been discovered to infect bat populations in Europe, Africa, Asia, and Australia. Six of these viruses have produced a very small number of cases of a human disease indistinguishable from rabies: European bat lyssaviruses 1 and 2, Australian bat lyssavirus, Irkut virus, and Duvenhage virus. Mokola virus, a lyssavirus that has been isolated from shrews with an unknown reservoir species in Africa, may also produce human disease indistinguishable from rabies.

1	Vesicular stomatitis is a viral disease of cattle, horses, pigs, and some wild mammals. Vesicular stomatitis virus is a member of the genus Vesiculovirus in the family Rhabdoviridae. Outbreaks of vesicular stomatitis in horses and cattle occur sporadically in the southwestern United States. The animal infection is associated with severe vesiculation and ulceration of oral tissues, teats, and feet and may be clinically indistinguishable from the more dangerous foot-and-mouth disease. Epidemics are usually seasonal, typically beginning in the late spring, and are probably due to arthropod vectors. Direct animal-to-animal spread can also occur, although the virus cannot penetrate intact skin. Transmission to humans usually results from direct contact with infected animals (particularly cattle) and occasionally follows laboratory exposure. In human disease, early conjunctivitis is followed by an acute influenza-like illness with fever, chills, nausea, vomiting, headache, retrobulbar pain,

1	and occasionally follows laboratory exposure. In human disease, early conjunctivitis is followed by an acute influenza-like illness with fever, chills, nausea, vomiting, headache, retrobulbar pain, myalgias, substernal pain, malaise, pharyngitis, and lymphadenitis. Small vesicular lesions may be present on the buccal mucosa or on the fingers. Encephalitis is very rare. The illness usually lasts 3–6 days, with complete recovery. Subclinical infections are common. A serologic diagnosis can be made on the basis of a rise in titer of complement-fixing or neutralizing antibodies. Therapy is symptom-based.

1	Jens H. Kuhn, Clarence J. Peters

1	This chapter summarizes the major features of selected arthropod-borne and rodent-borne viruses. Numerous viruses of this category are transmitted in nature among animals without ever infecting humans. Other viruses incidentally infect humans, but only a proportion of these viruses induce human disease. In addition, certain viral agents are regularly introduced into human populations or spread among humans by certain arthropods (specifically, insects and ticks) or by chronically infected rodents. These zoonotic viruses are taxonomically diverse and therefore differ fundamentally from one another in terms of virion morphology, replication strategies, genomic organization, and genome sequence. While a virus’s classification in a taxon is enlightening with regard to natural maintenance strategies, sensitivity to antiviral agents, and particular aspects of pathogenesis, the classification does not necessarily predict which clinical signs and symptoms (if any) the virus will cause in

1	strategies, sensitivity to antiviral agents, and particular aspects of pathogenesis, the classification does not necessarily predict which clinical signs and symptoms (if any) the virus will cause in humans. Zoonotic viruses are evolving, and “new” zoonotic viruses are regularly discovered. The epizootiology and epidemiology of zoonotic viruses continue to change as a result of environmental alterations affecting vectors, reservoirs, wildlife, livestock, and humans. Zoonotic viruses are most numerous in the tropics but are also found in temperate and even frigid climates. The distribution and seasonal activity of a zoonotic virus may vary, and the rate at which it changes is likely to depend largely on ecologic conditions (e.g., rainfall and temperature), which can affect the density of virus vectors and reservoirs and the development of infection.

1	Arthropod-borne viruses (arboviruses) infect their vectors after ingestion of a blood meal from a viremic, usually nonhuman vertebrate; some arthropods may also become infected by saliva-activated transmission. The arthropod vectors then develop chronic, systemic infection as the viruses penetrate the gut and spread throughout the body to the salivary glands; such virus dissemination, referred to as extrinsic incubation, typically lasts 1–3 weeks in mosquitoes. At this point, if the salivary glands become involved, the arthropod vector is competent to continue the chain of transmission by infecting a vertebrate during a subsequent blood meal. An alternative mechanism for virus maintenance in its arthropod vector is transovarial transmission. The arthropod generally is unharmed by the infection, and the natural vertebrate partner usually has only transient viremia with no overt disease.

1	Rodent-borne viruses are maintained in nature by transmission between rodents, which become chronically infected. Usually a high degree of rodent–virus specificity is observed, and overt disease in the reservoir host is rare. Arthropod-borne and rodent-borne zoonotic viruses belong to at least seven families: Arenaviridae, Bunyaviridae, Flaviviridae, Orthomyxoviridae, Reoviridae, Rhabdoviridae, and Togaviridae (Table 233-1).

1	The members of the family Arenaviridae that infect humans are all assigned to the genus Arenavirus. The members of this genus are divided into two main phylogenetic branches: Old World viruses (the Lassa–lymphocytic choriomeningitis serocomplex) and New World viruses (the Tacaribe serocomplex). Human arenaviruses form spherical, oval, or pleomorphic enveloped and spiked virions (~50–300 nm in diameter) that bud from the infected cell’s plasma membrane. The particles contain two genomic single-stranded RNAs (S, ~3.5 kb; and L, ~7.5 kb) encoding structural proteins in an ambisense orientation. Most arenaviruses persist in nature by chronically infecting rodents. The Old World viruses are maintained by murid rodents that often are persistently viremic and commonly transmit viruses vertically and horizontally. New World viruses are found in cricetid rodents; horizontal transmission is typical, vertical infection may occur, and persistent viremia may be observed. Strikingly, each

1	and horizontally. New World viruses are found in cricetid rodents; horizontal transmission is typical, vertical infection may occur, and persistent viremia may be observed. Strikingly, each arenavirus is predominantly adapted to one particular type of rodent. Humans usually become infected through inhalation of or direct contact with infected rodent excreta or secreta (e.g., aerosols of rodents in harvesting machines; aerosolized dried rodent urine or feces in barns or houses; direct contact with rodents in traps). Person-to-person transmission of arenaviruses is uncommon.

1	The family Bunyaviridae includes four medically significant genera: Hantavirus, Nairovirus, Orthobunyavirus, and Phlebovirus. The members of all these genera form spherical to pleomorphic enveloped virions containing three genomic single-stranded RNAs (S, ~1–2 kb; M, 3.6–5.3 kb; and L, 6.4–12.3 kb) of negative (hantaviruses, nairoviruses, orthobunyaviruses) or ambisense (nairoviruses) polarity. Bunyaviruses mature into particles ~80–120 nm in diameter in the Golgi complex of infected cells and exit these cells by exocytosis.

1	Hantaviruses are unique among the bunyaviruses in that they are not transmitted by arthropods but instead are maintained in nature by rodents that chronically shed virions. Old World hantaviruses are harbored by murid and cricetid rodents, and New World hantaviruses are maintained by cricetid rodents. As with arenaviruses, individual hantaviruses usually are specifically adapted to a particular type of rodent. However, hantaviruses do not cause chronic viremia in their rodent hosts and are transmitted only horizontally from rodent to rodent. Similar to arenaviruses, hantaviruses infect humans primarily through inhalation of or direct contact with rodent excreta or secreta, and person-to-person transmission is not a common event (with the notable exception of Andes virus). Although there is overlap, the human Old World hantaviruses usually are the etiologic agents of hemorrhagic fever with renal syndrome, whereas the New World viruses usually cause hantavirus pulmonary syndrome.

1	Nairoviruses are maintained by ixodid ticks, which vertically (transovarially and transstadially) transmit these viruses to progeny tick generations and horizontally spread them through viremic vertebrate hosts. Humans are usually infected via a tick bite or during handling of infected vertebrates. Orthobunyaviruses are largely mosquito-borne and rarely midge-borne and have viremic vertebrate intermediate hosts. Many orthobunyaviruses are also transovarially transmitted in their mosquito host. Numerous orthobunyaviruses have been associated with human infection and disease. They have been considered to be members of ~19 serogroups based on antigenic cross-reactions, but this grouping is currently undergoing revision with the accumulation of new genomic 1305 data and phylogenetic analyses. Human viruses are found in at least nine serogroups.

1	Phleboviruses are transmitted vertically (transovarially) in their arthropod hosts and horizontally through viremic vertebrate hosts. Phleboviruses are divided into two groups: the phlebotomus group viruses are transmitted by sandflies and the Uukuniemi group viruses by ticks. Phleboviruses are assigned to at least 10 serocomplexes; human pathogens are found in at least four of these serocomplexes.

1	The family Flaviviridae currently includes four genera, one of which (Flavivirus) comprises arthropod-borne human viruses. Flaviviruses sensu stricto have single-stranded positive-sense RNA genomes (~11 kb) and form spherical enveloped particles 40–60 nm in diameter. The flaviviruses discussed here belong to two phylogenetically and antigenically distinct groups and are transmitted among vertebrates by mosquitoes and ixodid ticks, respectively. Vectors are usually infected when they feed on viremic hosts; as in the case of most other viruses discussed here, humans are accidental hosts who usually are infected by arthropod bites. Arthropods maintain flavivirus infections horizontally, although transovarial transmission has been documented. Under certain circumstances, flaviviruses can also be transmitted by aerosols or via contaminated food products; in particular, raw milk can transmit tick-borne encephalitis virus.

1	The family Orthomyxoviridae includes two genera of medically relevant arthropod-borne viruses: Quaranjavirus and Thogotovirus. Quaranjaviruses are transmitted among birds by ixodid ticks, whereas thogotoviruses have a predilection for mammalian host reservoirs and can be transmitted by both ixodid ticks and mosquitoes.

1	The family Reoviridae contains viruses with linear, multisegmented, double-stranded RNA genomes (~16–29 kb in total). These viruses produce particles that have icosahedral symmetry and are 60–80 nm in diameter. In contrast to all other virions discussed here, reovirions are not enveloped and thus are insensitive to detergent inactivation. Fifteen genera of reoviruses are currently recognized. Human arthropod-borne viruses are found among the genera Coltivirus (subfamily Spinareovirinae), Orbivirus, and Seadornavirus (subfamily Sedoreovirinae). Arthropod-borne coltiviruses possess 12 genome segments. Coltiviruses are transmitted by numerous tick types transstadially but not transovarially. Overall maintenance of the transmission cycle, therefore, involves viremic mammalian hosts infected by tick bites. Arthropod-borne orbiviruses have 10 genome segments and are transmitted by mosquitoes or ixodid ticks, whereas relevant seadornaviruses have 12 genome segments and are transmitted

1	by tick bites. Arthropod-borne orbiviruses have 10 genome segments and are transmitted by mosquitoes or ixodid ticks, whereas relevant seadornaviruses have 12 genome segments and are transmitted exclusively by mosquitoes.

1	The family Rhabdoviridae is included in the order Mononegavirales. Viruses of the nine rhabdovirus genera have linear, nonsegmented, single-stranded RNA genomes of negative polarity (~11–15 kb) and form bullet-shaped to pleomorphic enveloped particles (100–430 nm long and 45–100 nm wide). Only the genus Vesiculovirus includes human arthropod-borne viruses, all of which are transmitted by insects (biting midges, mosquitoes, and sandflies). The general properties of rhabdoviruses are discussed in more detail in Chap. 232. The members of the family Togaviridae have linear, singleand positive-stranded RNA genomes (~9.7–11.8 kb) and form enveloped icosahedral virions (~60–70 nm in diameter) that bud from the plasma membrane of the infected cell. The togaviruses discussed here are all members of the genus Alphavirus and are transmitted among vertebrates by mosquitoes. Unknown Unknown Eulipotyphla, least weasles (Mustela nivalis), rodents Unknown Unknown Birds, cattle, rodents

1	Unknown Unknown Eulipotyphla, least weasles (Mustela nivalis), rodents Unknown Unknown Birds, cattle, rodents Birds Cattle, pigs Bats, camels, horses Camels, cattle Gray four-eyed opossums (Philander opossum) Cattle, horses, pigs Cattle, horses, pigs Unknown Sandflies (Phlebotomus papatasi, P. perfiliewi, P. perniciosus) Sandflies (Phlebotomus spp.) Sandflies (Phlebotomus papatasi, P. perfiliewi) Ixodid ticks (Ixodes spp.) Mosquitoes (Aedes, Anopheles, Culiseta spp.) Mosquitoes (Aedes, Anopheles, Culex spp.), ixodid ticks (Dermacentor, Hyalomma, Ornithodoros spp.) Ixodid ticks (Amblyomma, Hyalomma, Rhipicephalus spp.) Mosquitoes (Aedes aegypti), sandflies (Phlebotomus, Sergentomyia spp.) Mosquitoes (Aedes, Culex, Toxorhynchites spp.) Sandflies (Lutzomyia spp.) Biting midges (Culicoides spp.), chloropid flies, mosquitoes (Culex, Mansonia spp.), muscoid flies (Musca spp.), simuliid flies F/M F/M F/M F/M E, F/M F/M E, F/M

1	E, F/M E, F/M F/M aAbbreviations refer to the syndrome most commonly associated with the virus: A/R, arthritis/rash; E, encephalitis; F/M, fever/myalgia; P, pulmonary; VHF, viral hemorrhagic fever. Abbreviations are placed in parentheses when cases are either extremely rare or controversial. bIn the older literature, chikungunya virus often is also listed as a causative agent of VHF. However, later studies revealed that, in most cases, people with “chikungunya hemorrhagic fever” were co-infected with one or more dengue viruses, an observation suggesting that the VHF was actually severe dengue. cWhitewater Arroyo virus is often listed as a causative agent of VHF in the literature, but convincing data associating this virus with VHF have not been published. dAlso includes Kunjin virus. eIncludes the recently described Alkhurma/Alkhumra variant of Kyasanur Forest disease virus. fAlso known as Ganjam virus. gAlso known as Brezová virus, Cvilín virus, Kharagysh virus, Koliba virus, or

1	eIncludes the recently described Alkhurma/Alkhumra variant of Kyasanur Forest disease virus. fAlso known as Ganjam virus. gAlso known as Brezová virus, Cvilín virus, Kharagysh virus, Koliba virus, or Lipovník virus. hAlso known as Čalovo virus or Chittoor virus. iAlso known as Palma virus. j The final virus name has not yet been decided. Alternatives used in the literature are Huaiyangshan virus (HYSV) and Henan fever virus (HNFV). kAlso known as Astra virus and Batken virus.

1	The distributions of arthropod-borne and rodent-borne viruses are restricted by the areas inhabited by their reservoir hosts and/or vectors. Consequently, a patient’s geographic origin or travel history can provide important clues in the differential diagnosis. Table 233-2 lists the approximate geographic distribution of most arthropod-borne and rodent-borne infections. Many of these diseases can be acquired in either rural or urban settings; these diseases include yellow fever, dengue (previously called dengue fever), severe dengue (previously called dengue hemorrhagic fever and dengue shock syndrome), chikungunya virus disease, hemorrhagic fever with renal syndrome caused by Seoul virus, sandfly fever caused by sandfly fever Naples and Sicilian viruses, and Oropouche virus disease.

1	In patients with suspected viral infection, a recognized history of mosquito bite has little diagnostic significance, but a history of tick bite is more diagnostically useful. Exposure to rodents is sometimes reported by persons infected with arenaviruses or hantaviruses. Laboratory diagnosis is required in all cases, although epidemics occasionally provide enough clinical and epidemiologic clues for a presumptive etiologic diagnosis. For most arthropod-borne and rodent-borne viruses, acute-phase serum samples (collected within 3 or 4 days of onset) have yielded isolates. Paired serum samples have been used to demonstrate rising antibody titers. Intensive efforts to develop rapid tests for viral hemorrhagic fevers have resulted in reliable antigen-detection enzyme-linked immunosorbent assays (ELISAs), IgM-capture ELISAs, and multiplex polymerase chain reaction (PCR) assays. These tests can provide a diagnosis based on a single serum sample within a few hours and are particularly

1	assays (ELISAs), IgM-capture ELISAs, and multiplex polymerase chain reaction (PCR) assays. These tests can provide a diagnosis based on a single serum sample within a few hours and are particularly useful in patients with severe disease. More sensitive reverse-transcription PCR (RT-PCR) assays may yield diagnoses based on samples without detectable antigen and may also provide useful genetic information about the etiologic agent.

1	Hantavirus infections differ from other viral infections discussed here in that severe acute disease is immunopathologic; patients present with serum IgM that serves as the basis for a sensitive and specific test. At diagnosis, patients with encephalitides generally are no longer viremic or antigenemic and usually do not have virions in cerebrospinal fluid (CSF). In this situation, the value of serologic methods for IgM determination and RT-PCR is high. IgM-capture ELISA is increasingly being used for the simultaneous testing of serum and CSF. IgG ELISA or classic serology is useful in the evaluation of past exposure to viruses, many of which circulate in areas with minimal medical infrastructures and sometimes cause only mild or subclinical infection.

1	The spectrum of possible human responses to infection with arthropod-or rodent-borne viruses is wide, and knowledge of the outcome of most of these infections is limited. People infected with these viruses may not develop signs of illness. If viral disease is recognized, it can usually be grouped into one of five broad categories: arthritis and rash, encephalitis, fever and myalgia, pulmonary disease, or viral hemorrhagic fever (VHF) (Table 233-3). These categories often overlap. For example, infections with West Nile and Venezuelan equine Type of Diseasea Type of Diseasea

1	Type of Diseasea Type of Diseasea Europe Lymphocytic (Avalon) and Bhanja virus Dengue/severe Dhori and Eyach, — Chikungunya choriomeningitis/ infections; California dengue; tick-Thogoto virus Kemerovo, infections hemorrhagic fever with renal rhagic fever; syndrome; Inkoo virus infec-(Usutu virus infection; sandfly fever/Pappataci tion); West Nile fever/phlebotomus fever; virus infection Uukuniemi virus infection aQuotation marks indicate common usage in the absence of International Classification of Disease version 10 (ICD-10) recognition. Diseases not acknowledged by the ICD-10 are designated as “virus infection.” Disease names are placed in parentheses when cases are either extremely rare or controversial. aVirus names are placed in parentheses when human infections are either extremely rare or controversial.

1	encephalitis viruses are discussed here as encephalitides, but during epidemics many patients present with much milder febrile syndromes. Similarly, Rift Valley fever virus is best known as a cause of VHF, but the attack rates for febrile disease are far higher, and encephalitis and blindness occasionally occur as well. Lymphocytic choriomeningitis virus is classified here as a cause of fever and myalgia because this syndrome is the most common disease manifestation; even when central nervous system (CNS) disease evolves during infection with this virus, neural manifestations are usually mild and are preceded by fever and myalgia. Infection with any dengue virus type (1, 2, 3, or 4) is considered as a cause of fever and myalgia because this syndrome is by far the most common manifestation worldwide. However, severe dengue is a VHF with a complicated pathogenesis that is of tremendous importance in pediatric practice in certain areas of the world. Unfortunately, most of the known

1	worldwide. However, severe dengue is a VHF with a complicated pathogenesis that is of tremendous importance in pediatric practice in certain areas of the world. Unfortunately, most of the known arthropodor rodent-borne viral diseases have not been studied in detail with modern medical approaches; thus available data may be incomplete or biased. The reader must be aware that data on geographic distribution are often fuzzy: the literature frequently is not clear as to whether the data pertain to the distribution of a particular virus or the areas where human disease has been observed. In addition, the designations for viruses and viral diseases have changed multiple times over decades. Here, virus and taxon names are in line with the latest reports of the International Committee on Taxonomy of Viruses, and disease names are largely in accordance with the World Health Organization’s International Classification of Disease version 10 (ICD-10) and more recent updates.

1	Arthritides are common accompaniments of several viral diseases, such as hepatitis B, parvovirus B19 infection, and rubella, and occasionally accompany infection due to adenoviruses, enteroviruses, herpesviruses, and mumps virus. Two ungrouped bunyaviruses, Gan Gan virus and Trubanaman virus, and the flavivirus Kokobera virus have been associated with single cases of polyarthritic disease. Arthropod-borne alphaviruses are also common causes of arthritides—usually acute febrile diseases accompanied by the development of a maculopapular rash. Rheumatic involvement includes arthralgia alone, periarticular swelling, and (less commonly) joint effusions. Most alphavirus infections are less severe and have fewer articular manifestations in children than in adults. In temperate climates, these ailments are summer diseases. No specific therapies or licensed vaccines exist. The most important alphavirus arthritides are Barmah Forest virus infection, chikungunya virus disease, Ross River

1	ailments are summer diseases. No specific therapies or licensed vaccines exist. The most important alphavirus arthritides are Barmah Forest virus infection, chikungunya virus disease, Ross River disease, and Sindbis virus infection. A large (>2 million cases), albeit isolated, epidemic was caused by o’nyong nyong virus in 1959–1961 (o’nyong nyong fever). Mayaro, Semliki Forest, and Una viruses have caused isolated cases or limited and infrequent epidemics (30 to several hundred cases per year) in the past. Signs and symptoms of infections with these viruses often are similar to those observed with chikungunya virus disease.

1	Chikungunya Virus Disease Disease caused by chikungunya virus is endemic in rural areas of Africa. Intermittent epidemics take place in towns and cities of both Africa and Asia. Aedes aegypti mosquitoes are the usual vectors for the disease in urban areas. In 2004, a massive epidemic began in the Indian Ocean region (in particular on the islands of Réunion and Mauritius) and was most likely spread by travelers; Aedes albopictus was identified as the major vector of chikungunya virus during that epidemic. Between 2013 and 2014, several thousand chikungunya virus infections were reported (and several tens of thousands of cases were suspected) from Caribbean islands. The virus was imported to Italy, France, and the United States by travelers from the Caribbean. Chikungunya virus poses a threat to the continental United States as suitable vector mosquitoes are present in the southern states. The disease is most common among adults, in whom the clinical presentation may be dramatic. The

1	to the continental United States as suitable vector mosquitoes are present in the southern states. The disease is most common among adults, in whom the clinical presentation may be dramatic. The abrupt onset of chikungunya virus disease follows an incubation period of 2–10 days. Fever (often severe) with a saddleback pattern and severe arthralgia are accompanied by chills and constitutional symptoms and signs, such as abdominal pain, anorexia, conjunctival injection, headache, nausea, and photophobia. Migratory polyarthritis mainly affects the small 1313 joints of the ankles, feet, hands, and wrists, but the larger joints are not necessarily spared. Rash may appear at the outset or several days into the illness; its development often coincides with defervescence, which occurs around day 2 or 3 of the disease. The rash is most intense on the trunk and limbs and may desquamate. Young children develop less prominent signs and are therefore less frequently hospitalized. Children also

1	2 or 3 of the disease. The rash is most intense on the trunk and limbs and may desquamate. Young children develop less prominent signs and are therefore less frequently hospitalized. Children also often develop a bullous rather than a maculopapular/ petechial rash. Maternal–fetal transmission has been reported and in some cases has led to fetal death. Recovery may require weeks, and some elderly patients may continue to experience joint pain, recurrent effusions, or stiffness for several years. This persistence of signs and symptoms may be especially common in HLA-B27-positive patients. In addition to arthritis, petechiae are occasionally seen and epistaxis is not uncommon, but chikungunya virus should not be considered a VHF agent. A few patients develop leukopenia. Elevated concentrations of aspartate aminotransferase (AST) and C-reactive protein have been described, as have mildly decreased platelet counts. Treatment of chikungunya virus disease relies on nonsteroidal

1	concentrations of aspartate aminotransferase (AST) and C-reactive protein have been described, as have mildly decreased platelet counts. Treatment of chikungunya virus disease relies on nonsteroidal anti-inflammatory drugs and sometimes chloroquine for refractory arthritis.

1	Barmah Forest Virus Infection and Ross River Disease Barmah Forest virus and Ross River virus cause diseases that are indistinguishable on clinical grounds alone (hence the previously common disease designation epidemic polyarthritis for both infections). Ross River virus has caused epidemics in Australia, Papua New Guinea, and the South Pacific since the beginning of the twentieth century and continues to be responsible for ~4800 cases of disease in rural and suburban areas annually. In 1979–1980, the virus swept through the Pacific Islands, causing more than 500,000 infections. Ross River virus is predominantly transmitted by Aedes normanensis, Aedes vigilax, and Culex annulirostris. Wallabies and rodents are probably the main vertebrate hosts. Barmah Forest virus infections have been on the rise in recent years. In 2005– 2006, roughly 2000 cases were recorded in Australia. Barmah Forest virus is transmitted by both Aedes and Culex mosquitoes and has been isolated from biting

1	been on the rise in recent years. In 2005– 2006, roughly 2000 cases were recorded in Australia. Barmah Forest virus is transmitted by both Aedes and Culex mosquitoes and has been isolated from biting midges. The vertebrate hosts remain to be deter mined, but serologic studies implicate horses and possums.

1	Of the human Barmah Forest and Ross River virus infections surveyed, 55–75% were asymptomatic; however, these viral diseases can be debilitating. The incubation period is 7–9 days; the onset of illness is sudden, and disease is usually ushered in by disabling symmetrical joint pain. A nonitchy, diffuse, maculopapular rash (more common in Barmah Forest virus infection) generally develops coincidentally or follows shortly, but in some patients it can precede joint pains by several days. Constitutional symptoms such as low-grade fever, asthenia, headache, myalgia, and nausea are not prominent or are absent in many patients. Most patients are incapacitated for considerable periods (≥6 months) by joint involvement, which interferes with grasping, sleeping, and walking. Ankle, interphalangeal, knee, metacarpophalangeal, and wrist joints are most often involved, although elbows, shoulders, and toes may also be affected. Periarticular swelling and tenosynovitis are common, and one-third of

1	knee, metacarpophalangeal, and wrist joints are most often involved, although elbows, shoulders, and toes may also be affected. Periarticular swelling and tenosynovitis are common, and one-third of patients have true arthritis (more common in Ross River disease). Myalgia and nuchal stiffness may accompany joint pains. Only half of all patients with arthritis can resume normal activities within 4 weeks, and 10% still must limit their activity after 3 months. Occasional patients are symptomatic for 1–3 years but without progressive arthropathy.

1	In the diagnosis of either infection, clinical laboratory values are normal or variable. Tests for rheumatoid factor and antinuclear antibodies are negative, and the erythrocyte sedimentation rate is acutely elevated. Joint fluid contains 1000–60,000 mononuclear cells/μL, and viral antigen can usually be detected in macrophages. IgM antibodies are valuable in the diagnosis of this infection, although such antibodies occasionally persist for years. Isolation of the virus from blood after mosquito inoculation or growth of the virus in cell culture is possible early in the illness. Because of the great economic impact of annual epidemics in Australia, an inactivated Ross River virus vaccine is under development. Nonsteroidal anti-inflammatory drugs such as naproxen or acetylsalicylic acid are effective for treatment.

1	1314 Sindbis Virus Infection Sindbis virus is transmitted among birds by infected mosquitoes. Infections with northern European or southern African variants are particularly likely in rural environments. After an incubation period of <1 week, Sindbis virus infection begins with rash and arthralgia. Constitutional clinical signs are not marked, and fever is modest or lacking altogether. The rash, which lasts ~1 week, begins on the trunk, spreads to the extremities, and evolves from macules to papules that often vesiculate. The arthritis is multiarticular, migratory, and incapacitating, with resolution of the acute phase in a few days; the ankles, elbows, knees, phalangeal joints, wrists, and—to a much lesser extent—proximal and axial joints are involved. Persistence of joint pain and occasionally of arthritis is a major problem and may continue for months or even years despite lack of deformities.

1	Zika Virus Infection Zika virus is an emerging pathogen that is transmitted among nonhuman primates and humans by Aedes mosquitoes. Human infections are usually benign and are most likely misdiagnosed as dengue or influenza. Zika virus infection is characterized by influenza-like clinical signs, including fever, headaches, and malaise. A maculopapular rash, conjunctivitis, myalgia, and arthralgia usually accompany or follow those manifestations. Zika virus infection was first documented in Africa in 1947 and was later recognized in southeastern and southern Asia. In recent years, the number of Zika virus infections reported from Micronesia and Polynesia has increased steadily.

1	The major encephalitis viruses are found in the families Bunyaviridae, Flaviviridae, Rhabdoviridae, and Togaviridae. However, individual agents of other families, including Dhori virus and thogotovirus (Orthomyxoviridae) as well as Banna virus (Reoviridae), have been known to cause isolated cases of encephalitis as well. Arboviral encephalitides are seasonal diseases, commonly occurring in the warmer months. Their incidence varies markedly with time and place, depending on ecologic factors. The causative viruses differ substantially in terms of case–infection ratio (i.e., the ratio of clinical to sub-clinical infections), lethality rate, and residual disease. Humans are not important amplifiers of these viruses.

1	All the viral encephalitides discussed in this section have a similar pathogenesis. An infected arthropod ingests blood from a human and thereby initiates infection. The initial viremia is thought to originate from the lymphoid system. Viremia leads to multifocal entry into the CNS, presumably through infection of olfactory neuroepithelium, with passage through the cribriform plate; “Trojan horse” entry with infected macrophages; or infection of brain capillaries. During the viremic phase, there may be little or no recognizable disease except in tick-borne flavivirus encephalitides, which may manifest with clearly delineated phases of fever and systemic illness.

1	CNS lesions arise partly from direct neuronal infection and subsequent damage and partly from edema, inflammation, and other indirect effects. The usual pathologic features of arboviral encephalitides are focal necroses of neurons, inflammatory glial nodules, and perivascular lymphoid cuffing. Involved areas display the “luxury perfusion” phenomenon, with normal or increased total blood flow and low oxygen extraction. The typical patient presents with a prodrome of nonspecific constitutional signs and symptoms, including fever, abdominal pain, sore throat, and respiratory signs. Headache, meningeal signs, photophobia, and vomiting follow quickly. The severity of human infection varies from an absence of signs/symptoms to febrile headache, aseptic meningitis, and full-blown encephalitis. The proportions and severity of these manifestations vary with the infecting virus. Involvement of deeper brain structures in less severe cases may be signaled by lethargy, somnolence, and intellectual

1	proportions and severity of these manifestations vary with the infecting virus. Involvement of deeper brain structures in less severe cases may be signaled by lethargy, somnolence, and intellectual deficit (as disclosed by the mental status examination). More severely affected patients are obviously disoriented and may become comatose. Tremors, loss of abdominal reflexes, cranial nerve palsies, hemiparesis, monoparesis, difficulty swallowing, limb-girdle syndrome, and frontal lobe signs are all common. Spinal and motor neuron diseases are documented after West Nile and Japanese encephalitis virus infections. Seizures and focal signs may be evident early or may appear during the course of the disease. Some patients present with an abrupt onset of fever, convulsions, and other signs of CNS involvement. The acute encephalitis usually lasts from a few days to as long as 2–3 weeks. The infections may be fatal, or recovery may be slow, with weeks or months required for the return of maximal

1	The acute encephalitis usually lasts from a few days to as long as 2–3 weeks. The infections may be fatal, or recovery may be slow, with weeks or months required for the return of maximal recoupable function, or incomplete, with persisting long-term deficits. Difficulty concentrating, fatigability, tremors, and personality changes are common during recovery.

1	The diagnosis of arboviral encephalitides depends on the careful evaluation of a febrile patient with CNS disease and the performance of laboratory studies to determine etiology. Clinicians should (1) consider empirical acyclovir treatment for herpesvirus meningoencephalitis and antibiotic treatment for bacterial meningitis until test results are received; (2) exclude intoxination and metabolic or oncologic causes, including paraneoplastic syndromes, hyperammonemia, liver failure, and anti-NMDA receptor encephalitis; and (3) rule out a brain abscess or a stroke. Leptospirosis, neurosyphilis, Lyme disease, cat-scratch disease, and more recently described viral encephalitides (e.g., Nipah virus infection) should be considered if epidemiologically relevant. CSF examination usually shows a modest increase in leukocyte counts—in the tens or hundreds or perhaps a few thousand. Early in the process, a significant proportion of these leukocytes may be polymorphonuclear, but mononuclear cells

1	increase in leukocyte counts—in the tens or hundreds or perhaps a few thousand. Early in the process, a significant proportion of these leukocytes may be polymorphonuclear, but mononuclear cells are usually predominant later. CSF glucose concentrations are generally normal. There are exceptions to this pattern of findings: in eastern equine encephalitis, for example, polymorphonuclear leukocytes may predominate during the first 72 h of disease and hypoglycorrhachia may be detected. In lymphocytic choriomeningitis/meningoencephalitis, lymphocyte counts may be in the thousands, and the glucose concentration may be diminished. A humoral immune response is usually detectable at or near the onset of disease. Both serum (acuteor convalescent-phase) and CSF should be examined for IgM antibodies and viruses by plaque-reduction neutralization assay and/or (RT)-PCR. Virus generally cannot be isolated from blood or CSF, although Japanese encephalitis virus has been recovered from CSF of patients

1	viruses by plaque-reduction neutralization assay and/or (RT)-PCR. Virus generally cannot be isolated from blood or CSF, although Japanese encephalitis virus has been recovered from CSF of patients with severe disease. RT-PCR analysis of CSF may yield positive results. Viral antigen is present in brain tissue, although its distribution may be focal. Electroencephalography usually shows diffuse abnormalities and is not directly helpful.

1	Experience with medical imaging is still evolving. Both computed tomography (CT) and magnetic resonance imaging (MRI) scans may be normal except for evidence of preexisting conditions or occasional diffuse edema. Imaging is generally nonspecific in that most patients do not present with pathognomonic lesions, but it can be used to rule out other suspected causes of disease. It is important to remember that imaging may yield negative results if done early in the disease course but later may detect lesions. For example, eastern equine encephalitis (focal abnormalities) and severe Japanese encephalitis (hemorrhagic bilateral thalamic lesions) have caused lesions detectable by medical imaging.

1	Comatose patients may require management of intracranial pressure elevations, inappropriate secretion of antidiuretic hormone, respiratory failure, or seizures. Specific therapies for these viral encephalitides are not available. The only practical preventive measures are vector management and personal protection against the arthropod transmitting the virus. For Japanese encephalitis or tick-borne viral encephalitis, vaccination should be considered in certain circumstances (see relevant sections below).

1	Bunyaviruses: California (Meningo)encephalitis The isolation of California encephalitis virus established California serogroup orthobunyaviruses as causes of encephalitides. However, California encephalitis virus has been implicated in only a very few cases of encephalitis, whereas its close relative, La Crosse virus, is the major cause of encephalitis in this serogroup (~70 cases per year in the United States). California (meningo)encephalitis due to La Crosse virus infection is most commonly reported from the upper Midwest of the United States but is also found in other areas of the central and eastern parts of the country, most often in West Virginia, Tennessee, North Carolina, and Georgia. The serogroup includes 13 other viruses, some of which (e.g., Inkoo, Jamestown Canyon, Lumbo, snowshoe hare, and Tahyña viruses) also cause human disease. Transovarial transmission is a strong component of transmission of the California serogroup viruses in Aedes and Ochlerotatus mosquitoes. The

1	hare, and Tahyña viruses) also cause human disease. Transovarial transmission is a strong component of transmission of the California serogroup viruses in Aedes and Ochlerotatus mosquitoes. The mosquito vector of La Crosse virus is Ochlerotatus triseriatus. In addition to transovarial transmission, acquisition through feeding on viremic chipmunks and other mammals as well as venereal transmission can result in infection of this mosquito. O. triseriatus breeds in sites such as tree holes and abandoned tires and bites during daylight hours. The habits of this mosquito correlate with the risk factors for human cases: recreation in forested areas, residence at a forest’s edge, and the presence of water-containing abandoned tires around the home. Intensive environmental modification based on these findings has reduced the incidence of disease in a highly endemic area in the U.S. Midwest.

1	Most humans are infected from July through September. The Asian tiger mosquito (A. albopictus) efficiently transmits La Crosse virus to mice and also transmits the agent transovarially in the laboratory; this aggressive anthropophilic mosquito has the capacity to urbanize, and its possible impact on transmission of virus to humans is of concern. The prevalence of antibody to La Crosse virus in humans is ≥20% in endemic areas, a figure indicating that infection is common but often asymptomatic. CNS disease has been recognized primarily in children <15 years of age.

1	The illness from La Crosse virus varies from aseptic meningitis accompanied by confusion to severe and occasionally fatal encephalitis (lethality rate, <0.5%). The incubation period is ~3–7 days. Although there may be prodromal symptoms/signs, the onset of CNS disease is sudden, with fever, headache, and lethargy often joined by nausea and vomiting, convulsions (in one-half of patients), and coma (in one-third of patients). Focal seizures, hemiparesis, tremor, aphasia, chorea, Babinski signs, and other evidence of significant neurologic dysfunction are common, but residual disease is not. Approximately 10% of patients have recurrent seizures in the succeeding months. Other serious sequelae of La Crosse virus infection are rare, although a decrease in scholastic standing in children has been reported and mild personality change has occasionally been suggested.

1	The blood leukocyte count is commonly elevated in patients with La Crosse virus infection, sometimes reaching 20,000/μL, and is usually accompanied by a left shift. CSF leukocyte counts are typically 30–500/μL with a mononuclear cell predominance (although 25–90% of cells are polymorphonuclear in some patients). The blood protein concentration is normal or slightly increased, and the glucose concentration is normal. Specific virologic diagnosis based on IgM-capture assays of serum and CSF is efficient. The only human anatomic site from which virus has been isolated is the brain. Treatment is supportive over a 1to 2-week acute phase during which status epilepticus, cerebral edema, and inappropriate secretion of antidiuretic hormone are important concerns. A phase 2B clinical trial of IV ribavirin in children with La Crosse virus infection was discontinued during dose escalation because of adverse effects.

1	Jamestown Canyon virus has been implicated in several cases of encephalitis in adults, usually with a significant respiratory illness at onset. Human infection with this virus has been documented in New York, Wisconsin, Ohio, Michigan, Ontario, and other areas of North America where the vector mosquito, Aedes stimulans, feeds on its main host, the white-tailed deer. Tahyña virus can be found in central Europe, Russia, China, and Africa. The virus is a prominent cause of febrile disease but can also cause pharyngitis, pulmonary syndromes, aseptic meningitis, or meningoencephalitis.

1	Flaviviruses The most important flavivirus encephalitides are Japanese encephalitis, St. Louis encephalitis, tick-borne encephalitis, and West Nile virus infection. Australian encephalitis (Murray Valley encephalitis) and Rocio virus infection resemble Japanese encephalitis but are documented only occasionally in Australia and Brazil, respectively. Powassan virus has caused ~50 cases of often-severe disease (lethality rate, ~10%), frequently occurring among children in eastern Canada and the United States. Usutu virus has caused only individual cases of human infection, but such infections may be underdiagnosed.

1	japanese encepHalitis Japanese encephalitis is the most important viral encephalitis in Asia. Each year 35,000–50,000 cases and more than 15,000 deaths are reported. Japanese encephalitis virus is found 1315 throughout Asia, including far eastern Russia, Japan, China, India, Pakistan, and southeastern Asia, and causes occasional epidemics on western Pacific islands. The virus has been detected in the Torres Strait islands, and five human encephalitis cases have been identified on the nearby Australian mainland. The virus is particularly common in areas where irrigated rice fields attract the natural avian vertebrate hosts and provide abundant breeding sites for mosquitoes such as Culex tritaeniorhynchus, which transmit the virus to humans. Additional amplification by pigs, which suffer abortion, and horses, which develop encephalitis, may be significant as well. Vaccination of these additional amplifying hosts may reduce the transmission of the virus.

1	Clinical signs of Japanese encephalitis emerge after an incubation period of 5–15 days and range from an unspecific febrile presentation (nausea, vomiting, diarrhea, cough) to aseptic meningitis, meningoencephalitis, acute flaccid paralysis, and severe encephalitis. Common findings are cerebellar signs, cranial nerve palsies, and cognitive and speech impairments. A Parkinsonian presentation and seizures are typical in severe cases. Effective vaccines are available. Vaccination is indicated for summer travelers to rural Asia, where the risk of acquiring Japanese encephalitis is considered to be about 1 per 5000 to 1 per 20,000 travelers per week if travel duration exceeds 3 weeks. Usually two intramuscular doses of the vaccine are given 28 days apart, with the second dose administered at least 1 week prior to travel.

1	st. loUis encepHalitis St. Louis encephalitis virus is transmitted between mosquitoes and birds. This virus causes a low-level endemic infection among rural residents of the western and central United States, where Culex tarsalis is the vector (see “Western Equine Encephalitis,” below). The more urbanized mosquitoes Culex pipiens and Culex quinquefasciatus have been responsible for epidemics resulting in hundreds or even thousands of cases in cities of the central and eastern United States. Most cases occur in June through October. The urban mosquitoes breed in accumulations of stagnant water and sewage with high organic content and readily feed on humans in and around houses at dusk. The elimination of open sewers and trash-filled drainage systems is expensive and may not be possible, but screening of houses and implementation of personal protective measures may be an effective approach to the prevention of infection. The rural mosquito vector is most active at dusk and outdoors; its

1	screening of houses and implementation of personal protective measures may be an effective approach to the prevention of infection. The rural mosquito vector is most active at dusk and outdoors; its bites can be avoided by modification of activities and use of repellents.

1	Disease severity increases with age. St. Louis encephalitis virus infections that result in aseptic meningitis or mild encephalitis are concentrated among children and young adults, while severe and fatal cases primarily affect the elderly. Infection rates are similar in all age groups; thus the greater susceptibility of older persons to disease is a biologic consequence of aging. St. Louis encephalitis has an abrupt onset after an incubation period of 4–21 days, sometimes following a prodrome, and begins with fever, lethargy, confusion, and headache. In addition, nuchal rigidity, hypotonia, hyperreflexia, myoclonus, and tremors are common. Severe cases can include cranial nerve palsies, hemiparesis, and seizures. Patients often report dysuria and may have viral antigen in urine as well as pyuria. The overall rate of lethality is generally ~7% but may reach 20% among patients >60 years of age. Recovery is slow. Emotional lability, difficulties with concentration and memory, asthenia,

1	pyuria. The overall rate of lethality is generally ~7% but may reach 20% among patients >60 years of age. Recovery is slow. Emotional lability, difficulties with concentration and memory, asthenia, and tremors are commonly prolonged in older convalescent patients. The CSF of patients with St. Louis encephalitis usually contains tens to hundreds of leukocytes, with a lymphocytic predominance and a left shift. The CSF glucose concentration is normal in these patients.

1	tick-borne viral encepHalitis Tick-borne encephalitis viruses are currently subdivided into four groups: the western/European subtype (previously called central European encephalitis virus), the (Ural-) Siberian subtype (previously called Russian spring-summer encephalitis virus), the Far Eastern subtype, and the louping ill subtype (previously called louping ill virus or, in Japan, Negishi virus). Small mammals and grouse, deer, and sheep are the vertebrate amplifiers for these viruses, which are transmitted by ticks. The risk of infection varies by geographic area and can be highly localized within a given area; human 1316 infections usually follow either outdoor activities resulting in tick bites or consumption of raw (unpasteurized) milk from infected goats or, less commonly, from other infected animals (cows, sheep). Milk seems to represent the main transmission route for louping ill viruses, which cause disease only very rarely. The western/European subtype viruses are

1	from other infected animals (cows, sheep). Milk seems to represent the main transmission route for louping ill viruses, which cause disease only very rarely. The western/European subtype viruses are transmitted mainly by Ixodes ricinus from Scandinavia to the Ural Mountains. (Ural-)Siberian viruses are transmitted predominantly by Ixodes persulcatus from Europe across the Ural Mountains to the Pacific Ocean; louping ill viruses seem to be confined primarily to Great Britain. Several thousand infections with tick-borne encephalitis virus are recorded each year among people of all ages. Human tick-borne viral encephalitis occurs between April and October, with a peak in June and July. Western/European viruses classically caused bimodal disease. After an incubation period of 7–14 days, the illness begins with a fever– myalgia phase (arthralgia, fever, headaches, myalgia, nausea) that lasts for 2–4 days and is thought to correlate with viremia. A subsequent remission for several days is

1	illness begins with a fever– myalgia phase (arthralgia, fever, headaches, myalgia, nausea) that lasts for 2–4 days and is thought to correlate with viremia. A subsequent remission for several days is followed by the recurrence of fever and the onset of meningeal signs. The CNS phase (7–10 days before onset of improvement) varies from mild aseptic meningitis, which is more common among younger patients, to severe (meningo-)encephalitis with coma, seizures, tremors, and motor signs. Spinal and medullary involvement can lead to typical limb-girdle paralysis and respiratory paralysis. Most patients with western/European virus infections recover (lethality rate, 1%), and only a minority of patients have significant deficits. However, the lethality rate from (Ural-)Siberian virus infections reaches 7–8%. Infections with Far Eastern viruses generally run a more abrupt course. The encephalitic syndrome caused by these viruses sometimes begins without a remission from the fever–myalgia phase

1	7–8%. Infections with Far Eastern viruses generally run a more abrupt course. The encephalitic syndrome caused by these viruses sometimes begins without a remission from the fever–myalgia phase and has more severe manifestations than the western/European syndrome. The lethality rate is high (20–40%), and major sequelae—most notably, lower motor neuron paralyses of the proximal muscles of the extremities, trunk, and neck—are common, developing in approximately one-half of patients. Thrombocytopenia sometimes develops during the initial febrile illness, resembling the early hemorrhagic phase of some other tick-borne flavivirus infections, such as Kyasanur Forest disease. In the early stage of the illness, virus may be isolated from the blood. In the CNS phase, IgM antibodies are detectable in serum and/or CSF. Diagnosis of tick-borne viral encephalitis primarily relies on serology and detection of viral genomes by RT-PCR. There is no specific therapy for infection. However, effective

1	in serum and/or CSF. Diagnosis of tick-borne viral encephalitis primarily relies on serology and detection of viral genomes by RT-PCR. There is no specific therapy for infection. However, effective alum-adjuvanted, formalin-inactivated virus vaccines are produced in Austria, Germany, and Russia in chicken embryo cells (FSME-Immun® and Encepur®). Two doses of the Austrian vaccine separated by an interval of 1–3 months appear to be effective in the field, and antibody responses are similar when vaccine is given on days 0 and 14. Because rare cases of postvaccination Guillain-Barré syndrome have been reported, vaccination should be reserved for persons likely to experience rural exposure in an endemic area during the season of transmission. Cross-neutralization for the western/European and Far Eastern variants has been established, but there are no published field studies on cross-protection among formalin-inactivated vaccines. Because 0.2–4% of ticks in endemic areas may be infected,

1	Far Eastern variants has been established, but there are no published field studies on cross-protection among formalin-inactivated vaccines. Because 0.2–4% of ticks in endemic areas may be infected, the use of immunoglobulin prophylaxis of tick-borne viral encephalitis has been raised. Prompt administration of high-titered specific antibody preparations should probably be undertaken, although no controlled data are available to prove the efficacy of this measure. Immunoglobulins should be considered because of the risk of antibody-mediated enhancement of infection or antigen–antibody complex deposition in tissues.

1	west nile virUs infection West Nile virus is now the primary cause of arboviral encephalitis in the United States. In 2012, 2873 cases of neuroinvasive disease (e.g., meningitis, encephalitis, acute flaccid paralysis), with 270 deaths, and 2801 cases of non-neuroinvasive infection were reported. West Nile virus was initially described as being transmitted among wild birds by Culex mosquitoes in Africa, Asia, and southern Europe. In addition, the virus has been implicated in severe and fatal hepatic necrosis in Africa. West Nile virus was introduced into New York City in 1999 and subsequently spread to other areas of the northeastern United States, causing die-offs among crows, exotic zoo birds, and other birds. The virus has continued to spread and is now found in almost all states as well as in Canada, Mexico, South America, and the Caribbean islands. C. pipiens remains the major vector in the northeastern United States, but several other Culex species and A. albopictus are also

1	well as in Canada, Mexico, South America, and the Caribbean islands. C. pipiens remains the major vector in the northeastern United States, but several other Culex species and A. albopictus are also involved. Jays compete with crows and other corvids as amplifiers and lethal targets in other areas of the country.

1	West Nile virus is a common cause of febrile disease without CNS involvement (incubation period, 3–14 days), but it occasionally causes aseptic meningitis and severe encephalitis, particularly among the elderly. The fever–myalgia syndrome caused by West Nile virus differs from that caused by other viruses in terms of the frequent—rather than occasional—appearance of a maculopapular rash concentrated on the trunk (especially in children) and the development of lymphadenopathy. Back pain, fatigue, headache, myalgia, retroorbital pain, sore throat, nausea and vomiting, and arthralgia (but not arthritis) are common accompaniments that may persist for several weeks. Encephalitis, sequelae, and death are all more common among elderly, diabetic, and hypertensive patients and among patients with previous CNS insults. In addition to the more severe motor and cognitive sequelae, milder findings may include tremor, slight abnormalities in motor skills, and loss of executive functions. Intense

1	previous CNS insults. In addition to the more severe motor and cognitive sequelae, milder findings may include tremor, slight abnormalities in motor skills, and loss of executive functions. Intense clinical interest and the availability of laboratory diagnostic methods have made it possible to define a number of unusual clinical features. Such features include chorioretinitis, flaccid paralysis with histologic lesions resembling poliomyelitis, and initial presentation with fever and focal neurologic deficits in the absence of diffuse encephalitis. Immunosuppressed patients may have fulminant courses or develop persistent CNS infection. Virus transmission through both transplantation and blood transfusion has necessitated screening of blood and organ donors by nucleic acid–based tests. Occasionally, pregnant women infect their fetuses with West Nile virus.

1	Rhabdoviruses: Chandipura Virus Infection Chandipura virus seems to be an emerging and increasingly important human virus in India, where it is transmitted among hedgehogs by mosquitoes and sandflies. In humans, the disease begins as an influenza-like illness, with fever, headache, abdominal pain, nausea, and vomiting; these manifestations are followed by neurologic impairment and infection-related or autoimmune-mediated encephalitis. Chandipura virus infection is characterized by high lethality in children. Several hundred cases of infection are recorded in India every year. Infections with other arthropod-borne rhabdoviruses (Isfahan, Piry, vesicular stomatitis Indiana, vesicular stomatitis New Jersey) may imitate the early febrile stage of Chandipura virus infection.

1	Togaviruses • eastern eQUine encepHalitis This disease is encountered primarily in swampy foci along the eastern coast of the United States, with a few inland foci as far removed as Michigan. Infected humans present for medical care from June through October. During this period, the bird–Culiseta mosquito cycle spills over into other mosquitoes such as Aedes sollicitans or Aedes vexans, which are more likely to feed on mammals. There is concern over the potential role of the introduced anthropophilic mosquito species A. albopictus, which has been found to be infected with eastern equine encephalitis virus and is an effective experimental vector in the laboratory. Horses are a common target for the virus. Contact with unvaccinated horses may be associated with human disease, but horses probably do not play a significant role in amplification of the virus.

1	Eastern equine encephalitis is one of the most destructive of the arboviral diseases, with a sudden onset after an incubation period of ~5–10 days, rapid progression, 50–75% lethality, and frequent sequelae in survivors. This severity is reflected in the extensive necrotic lesions and polymorphonuclear infiltrates found at postmortem examination of the brain. Acute polymorphonuclear CSF pleocytosis, often occurring during the first 1–3 days of disease, is another indication of severity. In addition, leukocytosis with a left shift is a common feature. A formalin-inactivated vaccine has been used to protect laboratory workers but is not generally available or applicable.

1	venezUelan eQUine encepHalitis Venezuelan equine encephalitis viruses are separated into epizootic viruses (subtypes IA/B and IC) and enzootic viruses (subtypes ID, IE, and IF). Closely related enzootic viruses are Everglades virus, Mucambo virus, and Tonate virus. Enzootic viruses are found primarily in humid tropical-forest habitats and are maintained between culicoid mosquitoes and rodents. These viruses cause human disease but are not pathogenic for horses and do not cause epizootics. Enzootic viruses are common causes of acute febrile disease. Everglades virus has caused encephalitis in humans in Florida. Extrapolation from the rate of genetic change suggests that Everglades virus may have been introduced into Florida <200 years ago. Everglades virus is most closely related to the ID subtype viruses that appear to have given evolutionary rise to the epizootic variants active in South America.

1	Epizootic viruses have an unknown natural cycle but periodically cause extensive epizootics/epidemics in equids and humans in the Americas. These epizootics/epidemics are the result of high-level viremia in horses and mules, which transmit the infection to several types of mosquitoes. Infected mosquitoes in turn infect humans and perpetuate virus transmission. Humans also have high-level viremia, but their role in virus transmission is unclear. Epizootics of Venezuelan equine fever occurred repeatedly in South America at intervals of ≤10 years from the 1930s until 1969, when a massive epizootic spread throughout Central America and Mexico, reaching southern Texas in 1971. Genetic sequencing suggested that the virus from that outbreak originated from residual “un-inactivated” IA/B subtype virus in veterinary vaccines. The outbreak was terminated in Texas with a live attenuated vaccine (TC-83) originally developed for human use by the

1	U.S. Army; the epizootic virus was then used for further production of inactivated veterinary vaccines. No further epizootic disease was identified until 1995, when additional epizootics took place in Colombia, Venezuela, and Mexico. The viruses involved in these epizootics as well as previously epizootic IC viruses are close phylogenetic relatives of known enzootic ID viruses. This finding suggests that active evolution and selection of epizootic viruses are under way in South America.

1	During epizootics, extensive human infection is the rule, with clinical disease in 10–60% of infected individuals. Most infections result in notable acute febrile disease, while relatively few infections (5–15%) result in neurologic disease. A low rate of CNS invasion is supported by the absence of encephalitis among the many infections resulting from exposure to aerosols in the laboratory setting or from vaccination accidents. The most recent large epizootic of Venezuelan equine fever occurred in Colombia and Venezuela in 1995; of the more than 85,000 clinical cases, 4% (with a higher proportion among children than adults) included neurologic symptoms/signs, and 300 cases ended in death.

1	The prevention of epizootic Venezuelan equine fever depends on vaccination of horses with the attenuated TC-83 vaccine or with an inactivated vaccine prepared from that variant. Enzootic viruses are genetically and antigenically different from epizootic viruses, and protection against the former with vaccines prepared from the latter is relatively ineffective. Humans can be protected by immunization with similar vaccines prepared from Everglades virus, Mucambo virus, and Venezuelan equine encephalitis virus, but the use of the vaccines is restricted to laboratory personnel because of reactogenicity, possible fetal pathogenicity, and limited availability.

1	western eQUine encepHalitis The primary maintenance cycle of western equine encephalitis virus in the United States is between C. tarsalis and birds, principally sparrows and finches. Equids and humans become infected, and both suffer encephalitis without amplifying the virus in nature. St. Louis encephalitis virus is transmitted in a similar cycle in the same regions harboring western equine encephalitis virus; disease caused by the former occurs about a month earlier than that caused by the latter (July through October). Large epidemics of western equine encephalitis occurred in the western and central United States and Canada during the 1930s through 1950s, but in recent years the disease has been uncommon. From 1964 through 2010, only 640 cases were reported in the United States. This decline in incidence may reflect in part the integrated approach to mosquito management that has been employed in irrigation projects and the increasing use of 1317 agricultural pesticides. The

1	This decline in incidence may reflect in part the integrated approach to mosquito management that has been employed in irrigation projects and the increasing use of 1317 agricultural pesticides. The decreased incidence of western equine encephalitis almost certainly reflects the increased tendency for humans to be indoors behind closed windows at dusk—the peak biting period by the major vector.

1	After an incubation period of ~5–10 days, western equine encephalitis virus causes a typical diffuse viral encephalitis, with an increased attack rate and increased morbidity among the young, particularly children <2 years old. In addition, the lethality rate is high among the young and the very elderly (3–7% overall). One-third of individuals who have convulsions during the acute illness have subsequent seizure activity. Infants <1 year old—particularly those in the first months of life—are at serious risk of motor and intellectual damage. Twice as many males as females develop clinical encephalitis after 5–9 years of age. This difference in incidence may be related to greater outdoor exposure of boys to the vector but may also be due in part to biologic differences. A formalin-inactivated vaccine has been used to protect laboratory workers but is not generally available.

1	The fever and myalgia syndrome is most commonly associated with zoonotic virus infection. Many of the numerous viruses listed in Table 233-1 probably cause at least a few cases of this syndrome, but only some of these viruses have prominent associations with the syndrome and are of biomedical importance. The fever and myalgia syndrome typically begins with the abrupt onset of fever, chills, intense myalgia, and malaise. Patients may also report joint or muscle pains, but true arthritis is not found. Anorexia is characteristic and may be accompanied by nausea or even vomiting. Headache is common and may be severe, with photophobia and retroorbital pain. Physical findings are minimal and are usually confined to conjunctival injection with pain on palpation of muscles or the epigastrium. The duration of symptoms/signs is quite variable (generally 2–5 days), with a biphasic course in some instances. The spectrum of disease varies from sub-clinical to temporarily incapacitating. Less

1	The duration of symptoms/signs is quite variable (generally 2–5 days), with a biphasic course in some instances. The spectrum of disease varies from sub-clinical to temporarily incapacitating. Less constant findings include a nonpruritic maculopapular rash. Epistaxis may occur but does not necessarily indicate a bleeding diathesis. A minority of the patients may develop aseptic meningitis. This diagnosis is difficult to make in remote areas, given patients’ photophobia and myalgia as well as the lack of opportunity to examine the CSF. Although pharyngitis or radiographic evidence of pulmonary infiltrates is found in some patients, the agents causing this syndrome are not primary respiratory pathogens.

1	The differential diagnosis includes anicteric leptospirosis, rickettsial diseases, and the early stages of other syndromes discussed in this chapter. The fever and myalgia syndrome is often described as “influenza-like,” but the usual absence of cough and coryza makes influenza an unlikely confounder except at the earliest stages. Treatment is supportive, but acetylsalicylic acid is avoided because of the potential for exacerbated bleeding or Reye’s syndrome. Complete recovery is the general outcome for people with this syndrome, although prolonged asthenia and nonspecific symptoms have been described in some patients, particularly after infection with lymphocytic choriomeningitis virus or dengue virus types 1–4.

1	Efforts at prevention of viral infection are best based on vector control, which, however, may be expensive or impossible. For mosquito control, destruction of breeding sites is generally the most economically and environmentally sound approach. Emerging containment technologies include the release of genetically modified mosquitoes and the spread of Wolbachia bacteria to limit mosquito multiplication rates. Depending on the vector and its habits, other possible approaches include the use of screens or other barriers (e.g., permethrin-impregnated bed nets) to prevent the vector from entering dwellings, judicious application of arthropod repellents such as N,N,-diethyltoluamide (DEET) to the skin, wearing of long-sleeved and ideally permethrin-impregnated clothing, and avoidance of the vectors’ habitats and times of peak activity.

1	Arenaviruses Lymphocytic choriomeningitis/meningoencephalitis is the only human arenavirus infection resulting predominantly in fever 1318 and myalgia. Lymphocytic choriomeningitis virus is transmitted to humans from the common house mouse (Mus musculus) by aerosols of excreta and secreta. The virus is maintained in the mouse mainly by vertical transmission from infected dams. The vertically infected mouse remains viremic and sheds virus for life, with high concentrations of virus in all tissues. Infected colonies of pet hamsters also can serve as a link to humans. Infections among scientists and animal caretakers can occur because the virus is widely used in immunology laboratories as a model of T cell function and can silently infect cell cultures and passaged tumor lines. In addition, patients may have a history of residence in rodent-infested housing or other exposure to rodents. An antibody prevalence of ~5–10% has been reported among adults from Argentina, Germany, and the

1	patients may have a history of residence in rodent-infested housing or other exposure to rodents. An antibody prevalence of ~5–10% has been reported among adults from Argentina, Germany, and the United States. Lymphocytic choriomeningitis/meningoencephalitis differs from the general syndrome of fever and myalgia in that the onset is gradual. Conditions occasionally associated with the disease are orchitis, transient alopecia, arthritis, pharyngitis, cough, and maculopapular rash. An estimated one-fourth of patients (or fewer) experience a febrile phase of 3–6 days. After a brief remission, many develop renewed fever accompanied by severe headache, nausea and vomiting, and meningeal signs lasting for ~1 week (the CNS phase). These patients virtually always recover fully, as do the rare patients with clear-cut signs of encephalitis. Recovery may be delayed by transient hydrocephalus. During the initial febrile phase, leukopenia and thrombocytopenia are common, and virus can usually be

1	with clear-cut signs of encephalitis. Recovery may be delayed by transient hydrocephalus. During the initial febrile phase, leukopenia and thrombocytopenia are common, and virus can usually be isolated from blood. During the CNS phase, the virus may be found in the CSF, and antibodies are present in the blood. The pathogenesis of lymphocytic choriomeningitis/meningoencephalitis is thought to resemble that following direct intracranial inoculation of the virus into adult mice. The onset of the immune response leads to T cell–mediated immunopathologic meningitis. During the meningeal phase, CSF mononuclear-cell counts range from the hundreds to the low thousands per microliter, and hypoglycorrhachia is found in one-third of patients. IgM-capture ELISA, immunochemistry, and RT-PCR are used in the diagnosis of lymphocytic choriomeningitis/meningoencephalitis. IgM-capture ELISA of serum and CSF usually yields positive results; RT-PCR assays have been developed for probing CSF. Because

1	in the diagnosis of lymphocytic choriomeningitis/meningoencephalitis. IgM-capture ELISA of serum and CSF usually yields positive results; RT-PCR assays have been developed for probing CSF. Because patients who have fulminant infections transmitted by recent organ transplantation do not mount an immune response, immunohistochemistry or RT-PCR is required for diagnosis. Infection should be suspected in acutely ill febrile patients with marked leukopenia and thrombocytopenia. In patients with aseptic meningitis, any of the following suggests lymphocytic choriomeningitis/meningoencephalitis: a well-marked febrile prodrome, adult age, occurrence in the autumn, low CSF glucose levels, or CSF mononuclear-cell counts of >1000/μL. In pregnant women, infection may lead to fetal invasion with consequent congenital hydrocephalus and chorioretinitis. Because the maternal infection may be mild, causing only a short febrile illness, antibodies to the virus should be sought in both the mother and the

1	congenital hydrocephalus and chorioretinitis. Because the maternal infection may be mild, causing only a short febrile illness, antibodies to the virus should be sought in both the mother and the fetus under suspicious circumstances, particularly in TORCH (toxoplasmosis, rubella, cytomegalovirus, herpes simplex, and HIV)–negative neonatal hydrocephalus.

1	Bunyaviruses Numerous bunyaviruses cause fever and myalgia. Many of these viruses cause individual infections and usually do not result in epidemics—e.g., the viruses of the orthobunyavirus Anopheles A serogroup (e.g., Tacaiuma virus), Bwamba serogroup (Bwamba virus, Pongola virus), Guama serogroup (Catu virus, Guama virus), Nyando serogroup (Nyando virus), and Wyeomyia serogroup (Wyeomyia virus); the unclassified bunyavirus Tataguine virus; the phlebovirus Bhanja complex (Bhanja virus, Heartland virus) and Candiru complex (Alenquer, Candiru, Escharate, Maldonado, Morumbi, and Serra Norte viruses); the hantavirus Choclo virus; and the Dugbe and Nairobi sheep disease nairoviruses. In the relevant orthobunyaviral Bunyamwera serogroup (Bunyamwera, Batai, Cache Valley, Fort Sherman, Germiston, Guaroa, Ilesha, Ngari, Shokwe, and Xingu viruses), Ngari virus recently has been implicated in a large epidemic in Africa.

1	ortHobUnyavirUs groUp c serogroUp Apeú, Caraparú, Itaquí, Madrid, Marituba, Murutucú, Nepuyo, Oriboca, Ossa, Restan, and Zungarococha viruses are among the most common causes of arboviral infection in humans entering South American jungles. These viruses cause acute febrile disease and are transmitted by mosquitoes in neotropical forests. ortHobUnyavirUs simbU serogroUp Oropouche virus is transmitted in Central and South America by a biting midge, Culicoides paraensis, which often breeds to high density in cacao husks and other vegetable detritus found in towns and cities. Explosive epidemics involving thousands of patients have been reported from several towns in Brazil and Peru. Rash and aseptic meningitis have been detected in a number of patients. Iquitos virus, a recently discovered reassortant and close relative of Oropouche virus, causes disease that is easily mistaken for Oropouche virus disease; its overall epidemiologic significance remains to be determined.

1	pHlebovirUs sandfly fever serogroUp A previous designation for sandfly fever, “3-day fever,” instructively describes the brief debilitating course associated with this essentially benign infection. There is neither a rash nor CNS involvement, and complete recovery is the rule. Sandfly fever is caused by at least six distinct phleboviruses of the phlebovirus sandfly fever serocomplex (Chagres virus, sandfly fever Cyprus virus, sandfly fever Naples virus, sandfly fever Sicilian virus, sandfly fever Turkey virus, and Toscana virus). Sandfly fever Naples virus, sandfly fever Sicilian virus, and Toscana viruses are the most important human pathogens of this group. Phlebotomus sandflies transmit the viruses, probably among small mammals, and infect humans by bites. Female sandflies may be infected by the oral route as they take a blood meal and may transmit the virus to offspring when they lay their eggs after a second blood meal. This prominent transovarial transmission confounds virus

1	by the oral route as they take a blood meal and may transmit the virus to offspring when they lay their eggs after a second blood meal. This prominent transovarial transmission confounds virus control.

1	Sandfly fever is found in the circum-Mediterranean area, extending to the east through the Balkans into parts of China as well as into western Asia. Chagres virus is endemic in Panama. Sandflies are found in both rural and urban settings and are known for their short flight ranges and their small sizes; the latter enables them to penetrate standard mosquito screens and netting. Epidemics have been described in the wake of natural disasters and wars. After World War II, extensive spraying in parts of Europe to control malaria greatly reduced sandfly populations and sandfly fever Naples virus transmission; the incidence of sandfly fever continues to be low.

1	A common pattern of disease in endemic areas consists of high attack rates among travelers and military personnel and little or no disease in the local population, who are protected after childhood infection. Toscana virus infection is common during the summer among rural residents and vacationers, particularly in Italy, Spain, and Portugal; a number of cases have been identified in travelers returning to Germany and Scandinavia. The disease may manifest as an uncomplicated febrile illness but is often associated with aseptic meningitis, with virus isolated from the CSF.

1	Punta Toro virus is a phlebovirus that is not part of the sandfly fever serocomplex but that, like the members of this complex, is transmitted by sandflies. Punta Toro virus causes a sandfly fever–like disease in the Latin American tropical forest, where the vectors rest on tree buttresses. Epidemics have not been reported, but antibody prevalence among inhabitants of villages in endemic areas indicates a cumulative lifetime exposure rate of >50%.

1	Flaviviruses The most clinically important flaviviruses that cause the fever and myalgia syndrome are dengue viruses 1–4. In fact, dengue is probably the most important arthropod-borne viral disease worldwide, with 50–100 million infections occurring per year. Year-round transmission of dengue viruses 1–4 occurs between latitudes of 25°N and 25°S, but seasonal forays of the viruses into the United States and Europe have been documented. All four viruses have A. aegypti as their principal vector. Through increasing spread of mosquitoes throughout the tropics and subtropics and international travel of infected humans, large areas of the world have become vulnerable to the introduction of dengue viruses. Thus, dengue and severe dengue (see “Viral Hemorrhagic Fevers,” below) are becoming increasingly common. For instance, conditions favorable to dengue virus 1–4 transmission via A. aegypti exist in Hawaii and the southern United States. The range of a lesser dengue virus vector, A.

1	increasingly common. For instance, conditions favorable to dengue virus 1–4 transmission via A. aegypti exist in Hawaii and the southern United States. The range of a lesser dengue virus vector, A. albopictus, now extends from Asia to the United States, the Indian Ocean, parts of Europe, and Hawaii.

1	A. aegypti typically breeds near human habitation, using relatively fresh water from sources such as water jars, vases, discarded containers, coconut husks, and old tires. The mosquito usually inhabits dwellings and bites during the day. Bursts of dengue cases are to be expected in the southern United States, particularly along the Mexican border, where containers of water may be infested with A. aegypti. Closed habitations with air-conditioning may inhibit transmission of many arboviruses, including dengue viruses 1–4.

1	Dengue begins after an incubation period averaging 4–7 days, when the typical patient experiences the sudden onset of fever, frontal headache, retroorbital pain, and back pain along with severe myalgias. These symptoms gave rise to the colloquial designation of dengue as “break-bone fever.” Often a transient macular rash appears on the first day, as do adenopathy, palatal vesicles, and scleral injection. The illness may last a week, with additional symptoms and clinical signs usually including anorexia, nausea or vomiting, and marked cutaneous hypersensitivity. Near the time of defervescence on days 3–5, a maculopapular rash begins on the trunk and spreads to the extremities and the face. Epistaxis and scattered petechiae are often noted in uncomplicated dengue, and preexisting gastrointestinal lesions may bleed during the acute illness.

1	Laboratory findings of dengue include leukopenia, thrombocytopenia, and, in many cases, elevations of serum aminotransferase concentrations. The diagnosis is made by IgM ELISA or paired serology during recovery or by antigen-detection ELISA or RT-PCR during the acute phase. Virus is readily isolated from blood in the acute phase if mosquito inoculation or mosquito cell culture is used.

1	Reoviruses Several orbiviruses (Lebombo, Kemerovo, Orungo, and Tribeč viruses) and coltiviruses (Colorado tick fever, Eyach, and Salmon River viruses) can cause fever and myalgia in humans. With the exception of Lebombo and Orungo viruses, all of these viruses are transmitted by ticks. The most important reoviral arthropod-borne disease is Colorado tick fever. Several hundred patients with this disease are reported annually in the United States. The infection is acquired between March and November through the bite of an infected ixodid tick, the Rocky Mountain wood tick (Dermacentor andersoni), in mountainous western regions at altitudes of 1200–3000 m. Small mammals serve as amplifying hosts. The most common presentation is fever and myalgia; meningoencephalitis is not uncommon, and hemorrhagic disease, pericarditis, myocarditis, orchitis, and pulmonary presentations have also been reported. Rash develops in a minority of patients. Leukopenia and thrombocytopenia are also noted. The

1	disease, pericarditis, myocarditis, orchitis, and pulmonary presentations have also been reported. Rash develops in a minority of patients. Leukopenia and thrombocytopenia are also noted. The disease usually lasts 7–10 days and is often biphasic. The most important differential diagnostic considerations since the beginning of the twentieth century have been Rocky Mountain spotted fever (although Colorado tick fever is much more common in Colorado) and tularemia. Colorado tick fever virus replicates for several weeks in erythropoietic cells and can be found in erythrocytes. This feature, detected in erythroid smears stained by immunofluorescence, can be diagnostically helpful and is important during screening of blood donors.

1	Hantavirus pulmonary syndrome (HPS) was first described in 1993, but retrospective identification of cases by immunohistochemistry (1978) and serology (1959) support the idea that HPS is a recently discovered rather than a truly new disease. The causative agents are hantaviruses of a distinct phylogenetic lineage that is associated with the cricetid rodent subfamily Sigmodontinae. Sin Nombre virus, which chronically infects North American deer mice (Peromyscus maniculatus), is the most important agent of HPS in the United States. Several other related viruses (Anajatuba, Andes, Araraquara, Araucária, Bayou, Bermejo, Black Creek Canal, Blue River, Castelo dos Sonhos, El Moro Canyon, Juquitiba, Laguna Negra, Lechiguanas, Maciel, Monongahela, Muleshoe, New York, Orán, Paranoá, Pergamino, Río Mamoré, and Tunari) cause the disease in North and South America, 1319 but Andes virus is unusual in that it has been implicated in human-tohuman transmission. HPS particularly affects rural

1	Río Mamoré, and Tunari) cause the disease in North and South America, 1319 but Andes virus is unusual in that it has been implicated in human-tohuman transmission. HPS particularly affects rural residents living in dwellings permeable to rodent entry or working in occupations that pose a risk of rodent exposure. Each type of rodent has its own particular habits; in the case of deer mice, these behaviors include living in and around human habitation.

1	HPS begins with a prodrome of ~3–4 days (range, 1–11 days) comprising fever, malaise, myalgia, and—in many cases—gastrointestinal disturbances such as abdominal pain, nausea, and vomiting. Dizziness is common and vertigo occasional. Severe prodromal symptoms/ signs may bring some patients to medical attention, but most cases are recognized as the pulmonary phase begins. Typical signs are slightly lowered blood pressure, tachycardia, tachypnea, mild hypoxemia, thrombocytopenia, and early radiographic signs of pulmonary edema. Physical findings in the chest are often surprisingly scant. The conjunctival and cutaneous signs of vascular involvement seen in hantavirus VHFs (see below) are uncommon. During the next few hours, decompensation may progress rapidly to severe hypoxemia and respiratory failure.

1	The HPS differential diagnosis includes abdominal surgical conditions and pyelonephritis as well as rickettsial disease, sepsis, meningococcemia, plague, tularemia, influenza, and relapsing fever. A specific diagnosis is best made by IgM antibody testing of acute-phase serum, which has yielded positive results even in the prodrome. Tests using a Sin Nombre virus antigen detect antibodies to the related HPS-causing hantaviruses. Occasionally, heterotypic viruses will react only in the IgG ELISA, but such a finding is highly suspicious given the very low seroprevalence of these viruses in normal populations. RT-PCR is usually positive when used to test blood clots obtained in the first 7–9 days of illness and when used to test tissues; this assay is useful in identifying the infecting virus in areas outside the home range of deer mice and in atypical cases.

1	During the prodrome, the differential diagnosis of HPS is difficult, but by the time of presentation or within 24 h thereafter, a number of diagnostically helpful clinical features become apparent. Cough usually is not present at the outset. Interstitial edema is evident on a chest x-ray. Later, bilateral alveolar edema with a central distribution develops in the setting of a normal-sized heart; occasionally, the edema is initially unilateral. Pleural effusions are often seen. Thrombocytopenia, circulating atypical lymphocytes, and a left shift (often with leukocytosis) are almost always evident; thrombocytopenia is a particularly important early clue. Hemoconcentration, hypoalbuminemia, and proteinuria should also be sought for diagnosis. Although thrombocytopenia virtually always develops and prolongation of the partial thromboplastin time is the rule, clinical evidence for coagulopathy or laboratory indications of disseminated intravascular coagulation (DIC) are found in only a

1	and prolongation of the partial thromboplastin time is the rule, clinical evidence for coagulopathy or laboratory indications of disseminated intravascular coagulation (DIC) are found in only a minority of severely ill patients. Patients with severe illness also have acidosis and elevated serum lactate concentrations. Mildly increased values in renal function tests are common, but patients with severe HPS often have markedly elevated serum creatinine concentrations. Some New World hantaviruses other than Sin Nombre virus (e.g., Andes virus) have been associated with more kidney involvement, but few such cases have been studied.

1	Management of HPS during the first few hours after presentation is critical. The goal is to prevent severe hypoxemia by oxygen therapy, with intubation and intensive respiratory management if needed. During this period, hypotension and shock with increasing hematocrit invite aggressive fluid administration, but this intervention should be undertaken with great caution. Because of low cardiac output with myocardial depression and increased pulmonary vascular permeability, shock should be managed expectantly with pressors and modest infusion of fluid guided by pulmonary capillary wedge pressure. Mild cases can be managed by frequent monitoring and oxygen administration without intubation. Many patients require intubation to manage hypoxemia and developing shock. Extracorporeal membrane oxygenation is instituted in severe cases, ideally before the onset of shock. The procedure is indicated in patients who have a cardiac index of 2.3 L/min/m2 or an arterial oxygen tension/fractional

1	oxygenation is instituted in severe cases, ideally before the onset of shock. The procedure is indicated in patients who have a cardiac index of 2.3 L/min/m2 or an arterial oxygen tension/fractional inspired oxygen (PaO2/FiO2) ratio of 1320 <50 and who are unresponsive to conventional support. Lethality rates remain at ~30–40% even with good management, but most patients surviving the first 48 h of hospitalization are extubated and discharged within a few days with no apparent long-term residua. The antiviral drug ribavirin inhibits hantaviruses in vitro but did not have a marked effect on patients treated in an open-label study.

1	VHF is a constellation of findings based on vascular instability and decreased vascular integrity. An assault, direct or indirect, on the microvasculature leads to increased permeability and (particularly when platelet function is decreased) to actual disruption and local hemorrhage (a positive tourniquet sign). Blood pressure is decreased, and in severe cases shock supervenes. Cutaneous flushing and conjunctival suffusion are examples of common, observable abnormalities in the control of local circulation. Hemorrhage occurs infrequently. In most patients, hemorrhage is an indication of widespread vascular damage rather than a life-threatening loss of blood volume. In some VHFs, specific organs may be particularly impaired. For instance, the kidneys are primary targets in hemorrhagic fever with renal syndrome (HFRS), and the liver is a primary target in yellow fever and filovirus diseases. However, in all of these diseases, generalized circulatory disturbance is critically important.

1	with renal syndrome (HFRS), and the liver is a primary target in yellow fever and filovirus diseases. However, in all of these diseases, generalized circulatory disturbance is critically important. The pathogenesis of VHF is poorly understood and varies among the viruses regularly implicated in the syndrome. In some viral infections, direct damage to the vascular system or even to parenchymal cells of target organs is an important factor; in other viral infections, soluble mediators are thought to play a major role in the development of hemorrhage or fluid redistribution.

1	The acute phase in most cases of VHF is associated with ongoing virus replication and viremia. VHFs begin with fever and myalgia, usually of abrupt onset. (Arenavirus infections are the exceptions as they often develop gradually.) Within a few days, the patient presents for medical attention because of increasing prostration that is often accompanied by abdominal or chest pain, anorexia, dizziness, severe headache, hyperesthesia, photophobia, and nausea or vomiting and other gastrointestinal disturbances. Initial examination often reveals only an acutely ill patient with conjunctival suffusion, tenderness to palpation of muscles or abdomen, and borderline hypotension or postural hypotension, perhaps with tachycardia. Petechiae (often best visualized in the axillae), flushing of the head and thorax, periorbital edema, and proteinuria are common. AST concentrations are usually elevated at presentation or within a day or two thereafter. Hemoconcentration from vascular leakage, which is

1	and thorax, periorbital edema, and proteinuria are common. AST concentrations are usually elevated at presentation or within a day or two thereafter. Hemoconcentration from vascular leakage, which is usually evident, is most marked in HFRS and in severe dengue. The seriously ill patient progresses to more severe clinical signs and develops shock and other findings typical of the causative virus. Shock, multifocal bleeding, and CNS involvement (encephalopathy, coma, seizures) are all poor prognostic signs.

1	One of the major diagnostic clues to VHF is travel to an endemic area within the incubation period for a given syndrome. Except in infections with Seoul, dengue, and yellow fever viruses, which have urban hosts/vectors, travel to a rural setting is especially suggestive of a diagnosis of VHF. In addition, several diseases considered in the differential diagnosis—falciparum malaria, shigellosis, typhoid fever, leptospirosis, relapsing fever, and rickettsial diseases—are treatable and potentially lethal.

1	Early recognition of VHF is important because of the need for virus-specific therapy and supportive measures. Such measures include prompt, atraumatic hospitalization; judicious fluid therapy that takes into account the patient’s increased capillary permeability; administration of cardiotonic drugs; use of pressors to maintain blood pressure at levels that will support renal perfusion; treatment of the relatively common secondary bacterial (and the more rare fungal) infections; replacement of clotting factors and platelets as indicated; and the usual precautionary measures used in the treatment of patients with hemorrhagic diatheses. DIC should be treated only if clear laboratory evidence of its existence is found and if laboratory monitoring of therapy is feasible; there is no proven benefit of such therapy. The available evidence suggests that VHF patients have decreased cardiac output and will respond poorly to fluid loading as it is often practiced in the treatment of shock

1	benefit of such therapy. The available evidence suggests that VHF patients have decreased cardiac output and will respond poorly to fluid loading as it is often practiced in the treatment of shock associated with bacterial sepsis. Specific therapy is available for several of the VHFs. Strict barrier nursing and other precautions against infection of medical staff and visitors are indicated when VHFs are encountered except when the illness is due to dengue viruses, hantaviruses, Rift Valley fever virus, or yellow fever virus.

1	Novel VHF-causing agents are still being discovered. Besides the viruses listed below, the latest addition may be the unclassified rhabdovirus Bas-Congo virus, which has been associated with three cases of VHF in the Democratic Republic of the Congo. However, Koch’s postulates have not yet been fulfilled to prove cause and effect. Arenaviruses The most important arenaviruses causing VHF are Junín virus, Lassa virus, and Machupo virus. Chapare, Guanarito, Lujo, and Sabiá viruses have caused limited and/or infrequent outbreaks or individual cases.

1	jUnín/argentinian and macHUpo/bolivian HemorrHagic fevers These severe diseases (with fetal lethality rates reaching 15–30%) are caused by Junín virus and Machupo virus, respectively. Their clinical presentations are similar, but their epidemiology differs because of the distribution and behavior of the viruses’ rodent reservoirs. Junín/ Argentinian hemorrhagic fever has thus far been recorded only in rural areas of Argentina, whereas Machupo/Bolivian hemorrhagic fever seems to be confined to rural Bolivia. Infection with the causative agents almost always results in disease, and all ages and both sexes are affected. Person-to-person or nosocomial transmission is rare but has occurred. The transmission of Junín/Argentinian hemorrhagic fever from convalescing men to their wives suggests the need for counseling of patients with arenavirus hemorrhagic fever concerning the avoidance of intimate contacts for several weeks after recovery. Compared with the pattern in Lassa fever (see

1	the need for counseling of patients with arenavirus hemorrhagic fever concerning the avoidance of intimate contacts for several weeks after recovery. Compared with the pattern in Lassa fever (see below), thrombocytopenia—often marked—is the rule, hemorrhage is common, and CNS dysfunction (e.g., marked confusion, tremors of the upper extremities and tongue, and cerebellar signs) is much more common in disease caused by Junín virus and Machupo virus. Some cases follow a predominantly neurologic course, with a poor prognosis.

1	The clinical laboratory is helpful in diagnosis since thrombocytopenia, leukopenia, and proteinuria are typical findings. Junín/ Argentinian hemorrhagic fever is readily treated with convalescent-phase plasma given within the first 8 days of illness. In the absence of passive antibody therapy, IV ribavirin in the dose recommended for Lassa fever is likely to be effective in all the South American VHFs caused by arenaviruses. A safe, effective, live attenuated vaccine exists for Junín/Argentinian hemorrhagic fever. After vaccination of more than 250,000 high-risk persons in the endemic area, the incidence of this VHF decreased markedly. In experimental animals, this vaccine is cross-protective against Machupo/Bolivian hemorrhagic fever.

1	lassa fever Lassa virus is known to cause endemic and epidemic disease in Nigeria, Sierra Leone, Guinea, and Liberia, although it is probably more widely distributed in western Africa. In countries where Lassa virus is endemic, Lassa fever can be a prominent cause of febrile disease. For example, in one hospital in Sierra Leone, laboratory-confirmed Lassa fever is consistently responsible for one-fifth of admissions to the medical wards. In western Africa alone, probably tens of thousands of Lassa virus infections occur annually. Lassa virus can be transmitted by close person-to-person contact. The virus is often present in urine during convalescence and is suspected to be present in seminal fluid early in recovery. Nosocomial spread has occurred but is uncommon if proper sterile parenteral techniques are used. All ages and both sexes are affected; the incidence of disease is highest in the dry season, but transmission takes place year-round.

1	Among the VHF agents, only arenaviruses are typically associated with a gradual onset of illness, which begins after an incubation period of 5–16 days. Hemorrhage is seen in only ~15–30% of Lassa fever patients; a maculopapular rash is often noted in light-skinned patients. Effusions are common, and male-dominant pericarditis may develop late. Maternal lethality is higher than the usual 15–30% and is especially increased during the last trimester. The fetal death rate reaches 90%. Excavation of the uterus may increase survival rates of pregnant women, but data on Lassa fever and pregnancy are still sparse. These figures suggest that interruption of the pregnancy of Lassa virus–infected women should be considered. White blood cell counts are normal or slightly elevated, and platelet counts are normal or somewhat low. Deafness coincides with clinical improvement in ~20% of patients and is permanent and bilateral in some patients. Reinfection may occur but has not been associated with

1	are normal or somewhat low. Deafness coincides with clinical improvement in ~20% of patients and is permanent and bilateral in some patients. Reinfection may occur but has not been associated with severe disease.

1	High-level viremia or a high serum AST concentration statistically predicts a fatal outcome. Thus, patients with an AST concentration of >150 IU/mL should be treated with IV ribavirin. This antiviral nucleoside analogue appears to be effective in reducing case–fatality rates from those documented among retrospective controls. However, possible side effects, such as reversible anemia (which usually does not require transfusion), dependent hemolytic anemia, and bone marrow suppression, need to be kept in mind. Ribavirin should be given by slow IV infusion in a dose of 32 mg/kg; this dose should be followed by 16 mg/kg every 6 h for 4 days and then by 8 mg/kg every 8 h for 6 days. Inactivated Lassa virus vaccines failed in preclinical studies.

1	Bunyaviruses The most important VHF-causing bunyaviruses are Crimean-Congo hemorrhagic fever virus, hantaviruses, Rift Valley fever virus, and “severe fever with thrombocytopenia syndrome virus.” Other bunyaviruses—e.g., the Garissa variant of Ngari virus and Ilesha virus—have caused sporadic VHF outbreaks in Africa.

1	crimean-congo HemorrHagic fever (ccHf) This severe VHF has a wide geographic distribution, potentially emerging wherever virus-bearing ticks occur. Because of the propensity of CCHF virus–transmitting ticks to feed on domestic livestock and certain wild mammals, veterinary serosurveys are the most effective mechanism for the monitoring of virus circulation in a particular region. Human infections are acquired via tick bites or during the crushing of infected ticks. Domestic animals do not become ill but do develop viremia. Thus, there is risk of acquiring CCHF during sheep shearing, slaughter, and contact with infected hides or carcasses from recently slaughtered infected animals. Nosocomial epidemics are common and are usually related to extensive blood exposure or needlesticks.

1	Although generally similar to other VHFs, CCHF causes extensive liver damage, resulting in jaundice in some patients. Clinical laboratory values indicate DIC and show elevations in concentrations of AST, creatine phosphokinase, and bilirubin. Patients who do not survive generally have more distinct changes than survivors in the concentrations of these markers, even in the early days of illness, and also develop leukocytosis rather than leukopenia. In addition, thrombocytopenia is more marked and develops earlier in patients who do not survive than in survivors. The benefit of IV ribavirin for treatment remains hotly debated, but clinical experience and retrospective comparison of patients with ominous clinical laboratory values suggest that ribavirin may be efficacious. No human or veterinary vaccines are recommended.

1	HemorrHagic fever witH renal syndrome HFRS is the most important VHF today, with more than 100,000 cases of severe disease in Asia annually and milder infections numbering in the thousands in Europe. The disease is widely distributed in Eurasia. The major causative viruses are Puumala virus (Europe), Dobrava-Belgrade virus (the Balkans), and Hantaan virus (eastern Asia). Amur/Soochong, Gou, Kurkino, Muju, Saaremaa, Sochi, and Tula viruses also cause HFRS but much less frequently and in more geographically confined areas determined by the distribution of reservoir hosts. Seoul virus is exceptional in that it is associated with brown rats (Rattus norvegicus); therefore, the virus has a worldwide distribution because of the migration of these rodents on ships. Despite the wide distribution of Seoul virus, only mild or moderate HFRS occurs in Asia, and human disease has been difficult to identify in many areas of the world. Most cases of HFRS occur in rural residents or vacationers; the

1	of Seoul virus, only mild or moderate HFRS occurs in Asia, and human disease has been difficult to identify in many areas of the world. Most cases of HFRS occur in rural residents or vacationers; the exception is Seoul virus infection, which may be acquired in an urban or rural setting or from contaminated laboratory-rat colonies. Classic Hantaan virus infection in Korea and in rural China is most common in the spring and fall and 1321 is related to rodent density and agricultural practices. Human infection is acquired primarily through aerosols of rodent urine, although virus is also present in rodent saliva and feces. Patients with HFRS are not infectious.

1	Severe cases of HFRS evolve in four identifiable stages. The febrile stage lasts 3 or 4 days and is identified by the abrupt onset of fever, headache, severe myalgia, thirst, anorexia, and often nausea and vomiting. Photophobia, retroorbital pain, and pain on ocular movement are common, and the vision may become blurred with ciliary body inflammation. Flushing over the face, the V area of the neck, and the back is characteristic, as are pharyngeal injection, periorbital edema, and conjunctival suffusion. Petechiae often develop in areas of pressure, the conjunctivae, and the axillae. Back pain and tenderness to percussion at the costovertebral angle reflect massive retroperitoneal edema. Laboratory evidence of mild to moderate DIC is present. Other laboratory findings of HFRS include proteinuria and active urinary sediment. The hypotensive stage lasts from a few hours to 48 h and begins with falling blood pressure and sometimes shock. The relative bradycardia typical of the febrile

1	and active urinary sediment. The hypotensive stage lasts from a few hours to 48 h and begins with falling blood pressure and sometimes shock. The relative bradycardia typical of the febrile phase is replaced by tachycardia. Kinin activation is marked. The rising hematocrit reflects increasing vascular leakage. Leukocytosis with a left shift develops, and thrombocytopenia continues. Atypical lymphocytes—which in fact are activated CD8+ and, to a lesser extent, CD4+ T cells—circulate. Proteinuria is marked, and the urine’s specific gravity falls to 1.010. Renal circulation is congested and compromised from local and systemic circulatory changes resulting in necrosis of tubules, particularly at the corticomedullary junction, and oliguria. During the oliguric stage, hemorrhagic tendencies continue, probably in large part because of uremic bleeding defects. Oliguria persists for 3–10 days before the return of renal function marks the onset of the polyuric stage (diuresis and

1	tendencies continue, probably in large part because of uremic bleeding defects. Oliguria persists for 3–10 days before the return of renal function marks the onset of the polyuric stage (diuresis and hyposthenuria), which carries the danger of dehydration and electrolyte abnormalities.

1	Mild cases of HFRS may be much less stereotypical. The presentation may include only fever, gastrointestinal abnormalities, and transient oliguria followed by hyposthenuria. Infections with Puumala virus, the most common cause of HFRS in Europe (nephropathia epidemica), result in a much-attenuated picture but the same general presentation. Bleeding manifestations are found in only 10% of patients, hypotension rather than shock is usually documented, and oliguria is present in only about half of patients. The dominant features may be fever, abdominal pain, proteinuria, mild oliguria, and sometimes blurred vision or glaucoma followed by polyuria and hyposthenuria in recovery. The lethality rate is <1%.

1	HFRS should be suspected in patients with rural exposure in an endemic area. Prompt recognition of the disease permits rapid hospitalization and expectant management of shock and renal failure. Useful clinical laboratory parameters include leukocytosis, which may be leukemoid and is associated with a left shift; thrombocytopenia; and proteinuria. HFRS is readily diagnosed by an IgM-capture ELISA that is positive at admission or within 24–48 h thereafter. The isolation of hantaviruses is difficult, but RT-PCR of a blood clot collected early in the clinical course or of tissues obtained postmortem should give positive results. Such testing is usually undertaken if definitive identification of the infecting virus is required.

1	Mainstays of therapy are management of shock, reliance on vasopressors, modest crystalloid infusion, IV human serum albumin administration, and treatment of renal failure with prompt dialysis to prevent overhydration that may result in pulmonary edema and to control hypertension that increases the possibility of intracranial hemorrhage. Use of IV ribavirin has reduced lethality and morbidity in severe cases, provided treatment is begun within the first 4 days of illness. Lethality may be as high as 15% but with proper therapy should be <5%. Sequelae have not been definitively established.

1	rift valley fever The natural range of Rift Valley fever virus was previously confined to sub-Saharan Africa, with circulation of the virus markedly enhanced by substantial rainfall. The El Niño Southern Oscillation phenomenon of 1997 facilitated subsequent spread of Rift 1322 Valley fever to the Arabian Peninsula, with epidemic disease in 2000. The virus has also been found in Madagascar and has been introduced into Egypt, where it caused major epidemics in 1977–1979, 1993, and thereafter. Rift Valley fever virus is maintained in nature by transovarial transmission in floodwater Aedes mosquitoes and presumably also has a vertebrate amplifier. Increased transmission during particularly heavy rains leads to epizootics characterized by high-level viremia in cattle, goats, or sheep. Numerous types of mosquitoes then feed on these animals and become infected, thereby increasing the possibility of human infections. Remote sensing via satellite can detect the ecologic changes associated

1	types of mosquitoes then feed on these animals and become infected, thereby increasing the possibility of human infections. Remote sensing via satellite can detect the ecologic changes associated with high rainfall that predict the likelihood of Rift Valley fever virus transmission. High-resolution satellites can also detect the special depressions in floodwaters from which the mosquitoes emerge. In addition, the virus can be transmitted by contact with blood or aerosols from domestic animals. Transmission risk is therefore high during birthing, and both abortuses and placentas need to be handled with caution. Slaughtered animals are not infectious because anaerobic glycolysis in postmortem tissues results in an acidic environment that rapidly inactivates bunyaviruses. Neither person-to-person nor nosocomial transmission of Rift Valley fever has been documented. Rift Valley fever virus is unusual in that it causes several clinical syndromes. Most infections are manifested as the

1	nor nosocomial transmission of Rift Valley fever has been documented. Rift Valley fever virus is unusual in that it causes several clinical syndromes. Most infections are manifested as the fever–myalgia syndrome. A small proportion of infections result in VHF with especially prominent liver involvement. Renal failure and DIC are also common features. Perhaps 10% of otherwise mild infections lead to retinal vasculitis, and some patients have permanently impaired vision. Funduscopic examination reveals edema, hemorrhages, and infarction of the retina as well as optic nerve degeneration. In a small proportion of patients (<1 in 200), retinal vasculitis is followed by viral encephalitis. No proven therapy exists for Rift Valley fever. Both retinal disease and encephalitis occur after the acute febrile syndrome has resolved and serum neutralizing antibody has developed—events suggesting that only supportive care need be given. Epidemic disease is best prevented by vaccination of livestock.

1	febrile syndrome has resolved and serum neutralizing antibody has developed—events suggesting that only supportive care need be given. Epidemic disease is best prevented by vaccination of livestock. The ability of this virus to propagate after introduction into Egypt suggests that other potentially receptive areas, including the United States, should develop response plans. Rift Valley fever, like Venezuelan equine encephalitis, is likely to be controlled only with adequate stocks of an effective live attenuated vaccine, but such global stocks are unavailable. A formalin-inactivated vaccine confers immunity in humans, but quantities are limited and three injections are required; this vaccine is recommended for potentially exposed laboratory workers and for veterinarians working in sub-Saharan Africa. A new live attenuated vaccine, MP-12, is being tested in humans and may soon become available for general use. The vaccine is safe and licensed for use in sheep and cattle.

1	severe fever witH tHrombocytopenia syndrome This is a recently described tick-borne disease caused by a previously unknown and still-unclassified phlebovirus. Numerous human infections have been reported during the past few years from China, and several cases have also been detected in Japan and South Korea. The clinical presentation ranges from mild nonspecific fever to severe VHF with a high (>12%) lethality rate.

1	Flaviviruses The most important flaviviruses that cause VHF are the mosquito-borne dengue viruses 1–4 and yellow fever viruses. These viruses are widely distributed and cause tens to hundreds of thousands of infections each year. Kyasanur Forest disease virus and Omsk hemorrhagic fever virus are geographically very restricted but important tick-borne flaviviruses that cause VHF, sometimes with subsequent viral encephalitis. Tick-borne encephalitis virus has caused VHF in a few patients. There is currently no therapy for these VHFs, but an inactivated vaccine has been used in India to prevent Kyasanur Forest disease.

1	severe dengUe Several weeks after convalescence from infection with dengue virus 1, 2, 3, or 4, the transient protection conferred by that infection against reinfection with a heterotypic dengue virus usually wanes. Heterotypic reinfection may result in classic dengue or, less commonly, in severe dengue. In the past 20 years, A. aegypti has progressively reinvaded Latin America and other areas, and frequent travel by infected individuals has introduced multiple variants of dengue viruses 1–4 from many geographic areas. Thus the pattern of hyperendemic transmission of multiple dengue virus serotypes established in the Americas and the Caribbean has led to the emergence of severe dengue as a major problem. Among the millions of dengue virus 1–4 infections, ~500,000 cases of severe dengue occur annually, with a lethality rate of ~2.5%. The induction of vascular permeability and shock depends on multiple factors, such as the presence or absence of enhancing and nonneutralizing antibodies,

1	annually, with a lethality rate of ~2.5%. The induction of vascular permeability and shock depends on multiple factors, such as the presence or absence of enhancing and nonneutralizing antibodies, age (susceptibility to severe dengue drops considerably after 12 years of age), sex (females are more often affected than males), race (whites are more often affected than blacks), nutritional status (malnutrition is protective), or sequence of infections (e.g., dengue virus 1 infection followed by dengue virus 2 infection seems to be more dangerous than dengue virus 4 infection followed by dengue virus 2 infection). In addition, considerable heterogeneity exists among each dengue virus population. For instance, Southeast Asian dengue virus 2 variants have more potential to cause severe dengue than do other variants.

1	Severe dengue is identified by the detection of bleeding tendencies (tourniquet test, petechiae) or overt bleeding in the absence of underlying causes, such as preexisting gastrointestinal lesions. Shock may result from increased vascular permeability. In milder cases of severe dengue, restlessness, lethargy, thrombocytopenia (<100,000/μL), and hemoconcentration are detected 2–5 days after the onset of typical dengue, usually at the time of defervescence. The maculopapular rash that often develops in dengue may also appear in severe dengue. In more severe cases, frank shock is apparent, with low pulse pressure, cyanosis, hepatomegaly, pleural effusions, and ascites; in some patients, severe ecchymoses and gastrointestinal bleeding develop. The period of shock lasts only 1 or 2 days.

1	A virologic diagnosis of severe dengue can be made by the usual means. However, multiple flavivirus infections result in broad immune responses to several members of the genus, and this situation may result in a lack of virus specificity of the IgM and IgG immune responses. A secondary antibody response can be sought with tests against several flavivirus antigens to demonstrate the characteristic wide spectrum of reactivity. Most patients with shock respond promptly to close monitoring, oxygen administration, and infusion of crystalloid or—in severe cases— colloid. The case–fatality rates reported vary greatly with case ascertainment and quality of treatment; however, most patients with severe dengue respond well to supportive therapy, and the overall lethality rate at an experienced center in the tropics is probably as low as 1%.

1	The key to control of both dengue and severe dengue is the control of A. aegypti, which also reduces the risk of urban yellow fever and chikungunya virus circulation. Control efforts have been handicapped by the presence of nondegradable tires and long-lived plastic containers in trash repositories (perfect mosquito breeding grounds when filled with water during rainfall) and by insecticide resistance. Urban poverty and an inability of the public health community to mobilize the populace to respond to the need to eliminate mosquito breeding sites are also factors in lack of mosquito control. A tetravalent live attenuated dengue vaccine based on the attenuated yellow fever virus 17D platform is currently being evaluated in phase 3 clinical trials in Latin America, Asia, and Australia. At least two other live attenuated candidate vaccines based on modified recombinant dengue viruses have been evaluated in phase 1 clinical studies, but the results have not been promising.

1	yellow fever Yellow fever virus had caused major epidemics in Africa and Europe before its transmission by A. aegypti mosquitoes was discovered in 1900. Urban yellow fever became established in the New World as a result of colonization with A. aegypti, originally an African mosquito. Subsequently, different types of mosquitoes and nonhuman primates were found to maintain yellow fever virus in Africa and also in Central and South American jungles. Transmission to humans

1	Ebolavirus and Marburgvirus Infections Jens H. Kuhn Several viruses of the family Filoviridae cause severe and frequently fatal viral hemorrhagic fevers in humans. Introduction of filoviruses 234 is incidental, occurring via bites from mosquitoes that have fed on viremic monkeys. After the identification of A. aegypti mosquitoes as vectors of yellow fever, containment strategies were aimed at increased mosquito control. Today, urban yellow fever transmission occurs only in some African cities, but the threat exists in the great cities of South America, where reinfestation by A. aegypti has taken place and dengue virus 1–4 transmission by the same mosquito is common. Despite the existence of a highly effective and safe vaccine, several hundred jungle yellow fever cases occur annually in South America, and thousands of jungle and urban cases occur each year in Africa (29,000–60,000 estimated for 2013).

1	Yellow fever is a typical VHF accompanied by prominent hepatic necrosis. A period of viremia, typically lasting 3 or 4 days, is followed by a period of “intoxication.” During the latter phase in severe cases, characteristic jaundice, hemorrhages, black vomit, anuria, and terminal delirium occur, perhaps related in part to extensive hepatic involvement. Blood leukocyte counts may be normal or reduced and are often high in terminal stages. Albuminuria is usually noted and may be marked. As renal function fails in terminal or severe cases, the concentration of blood urea nitrogen rises proportionately. Abnormalities detected in liver function tests range from modest elevations of AST concentrations in mild cases to severe derangement.

1	Urban yellow fever can be prevented by the control of A. aegypti. The continuing sylvatic cycles require vaccination of all visitors to areas of potential transmission with live attenuated variant 17D vaccine virus, which cannot be transmitted by mosquitoes. With few exceptions, reactions to the vaccine are minimal; immunity is provided within 10 days and lasts for at least 25–35 years. An egg allergy mandates caution in vaccine administration. Although there are no documented harmful effects of the vaccine on fetuses, pregnant women should be immunized only if they are definitely at risk of exposure to yellow fever virus. Because vaccination has been associated with several cases of encephalitis in children <6 months of age, it is contraindicated in this age group, nor is it recommended for infants 6–8 months of age unless the risk of exposure is very high. Rare, serious, multisystemic adverse reactions (occasionally fatal) have been reported, particularly affecting the elderly, and

1	for infants 6–8 months of age unless the risk of exposure is very high. Rare, serious, multisystemic adverse reactions (occasionally fatal) have been reported, particularly affecting the elderly, and risk-to-benefit should be weighed prior to vaccine administration to individuals ≥60 years of age. Nevertheless, the number of deaths of unvaccinated travelers with yellow fever exceeds the number of deaths from vaccination, and a liberal vaccination policy for travelers to involved areas should be pursued. Timely information on changes in yellow fever distribution and yellow fever vaccine requirements can be obtained from the

1	U.S. Centers for Disease Control and Prevention (http://www.cdc.gov/ vaccines/vpd-vac/yf/default.htm). into human populations is an extremely rare event that most likely occurs by direct or indirect contact with healthy mammalian filovirus hosts or by contact with infected, sick, or deceased nonhuman primates. Filoviruses are highly infectious but not very contagious. Natural human-to-human transmission takes place through direct person-to-person (usually skin-to-skin) contact or exposure to infected bodily fluids and tissues; there is no evidence of such transmission by aerosol or respiratory droplets. Infections progress rapidly from influenza-like to hemorrhagic manifestations and typically culminate in multiple-organ dysfunction syndrome and shock. Treatment of filovirus infections is of necessity entirely supportive because no spe-1323 cific efficacious antiviral agents or vaccines are yet available.

1	Filoviruses are categorized as World Health Organization (WHO) Risk Group 4 Pathogens. Consequently, all work with material suspected of containing filoviruses should be conducted only in maximal containment (biosafety level 4) laboratories. Experienced personnel handling these viruses must wear appropriate personal protective gear (see “Prevention,” below) and follow rigorous standard operating procedures. The proper authorities and WHO reference laboratories should be contacted immediately when filovirus infections are suspected.

1	The family Filoviridae includes three genera: Cuevavirus, Ebolavirus, and Marburgvirus (Table 234-1 and Fig. 234-1). The available data suggest that the only known cuevavirus, Lloviu virus (LLOV), and one ebolavirus, Reston virus (RESTV), are not pathogenic for humans. The remaining four ebolaviruses—Bundibugyo virus (BDBV), Ebola virus (EBOV), Sudan virus (SUDV), and Taï Forest virus (TAFV)—cause Ebola virus disease (EVD; International Classification of Disease, Tenth Revision [ICD-10], code A98.4). The two marburgviruses, Marburg virus (MARV) and Ravn virus (RAVV), are the etiologic agents of Marburg virus disease (MVD; ICD-10 code A98.3).

1	Filoviruses have linear, nonsegmented, single-stranded, nega tive-sense RNA genomes that are ~19 kb in length. These genomes contain six or seven genes that encode the following seven structural proteins: nucleoprotein, polymerase cofactor (VP35), matrix protein (VP40), glycoprotein (GP1,2), transcriptional cofactor (VP30), secondary matrix protein (VP24), and RNA-dependent RNA polymerase (L protein). Cuevaviruses and ebolaviruses, but not marburgviruses, also encode three nonstructural proteins of unknown function (sGP, ssGP, and Δ-peptide). Filovirions are unique among human virus particles in that they are predominantly pleomorphic filaments but also assume torusor 6-like shapes (width, ~80 nm; average length, ≥790 nm). These enveloped virions contain helical ribonucleocapsids and are covered with GP1,2 spikes (Fig. 234-2). Virus 1: Marburg virus Virus: Lake Victoria (MARV) marburgvirus (MARV) Virus 2: Ravn virus (RAVV)

1	Virus 1: Marburg virus Virus: Lake Victoria (MARV) marburgvirus (MARV) Virus 2: Ravn virus (RAVV) Genus Ebolavirus Genus Ebolavirus Species Taï Forest ebolavirus Species Cote d’Ivoire ebolavirus [sica] Virus: Taï Forest virus Virus: Cote d’Ivoire ebolavirus (TAFV) [sic] (CIEBOV) Species Reston ebolavirus Species Reston ebolavirus Virus: Reston virus (RESTV) Virus: Reston ebolavirus (REBOV) Species Sudan ebolavirus Species Sudan ebolavirus Virus: Sudan virus (SUDV) Virus: Sudan ebolavirus (SEBOV) Species Zaire ebolavirus Species Zaire ebolavirus Virus: Ebola virus (EBOV) Virus: Zaire ebolavirus (ZEBOV) Species Bundibugyo ebolavirus Virus: Bundibugyo virus (BDBV) Genus Cuevavirus Species Lloviu cuevavirus Virus: Lloviu virus (LLOV) aThe correct spelling of the country for which this virus is named is Côte d’Ivoire. The lack of a circumflex in “Cote” in the virus designation produced a false country name. This fact is denoted by “[sic].” DQ447652 marburgviruses 1,100 DQ447650

1	DQ447652 marburgviruses 1,100 DQ447650 DQ447653 filovirus 10,436 7,583 3,210 2,128 ebolaviruses 4,364 3,807 0.2 1 FIGuRE 234-1 Filovirus phylogeny/evolution. Bayesian coalescent analysis of representative variants of all known filovirus clades (represented by underlined GenBank accession numbers). The maximal clade credibility tree is shown with the most recent common ancestor (MRCA) at each node. Posterior probability values are shown beneath MRCA estimates in years. Scale is in substitutions/site based on an analysis performed by Dr. Serena Carroll, Centers for Disease Control and Prevention. BDBV, Bundibugyo virus; EBOV, Ebola virus; LLOV, Lloviu virus; MARV, Marburg virus; RAVV, Ravn virus; RESTV, Reston virus; SUDV, Sudan virus; TAFV, Taï Forest virus.

1	To date (i.e., as of December 3, 2014), a total of 20,012 human filovirus infections and 8058 fatalities have been recorded (Fig. 234-3). These numbers emphasize both the high degree of lethality (number of deaths per number of sick people; 40.3%) and the overall low mortality (impact on healthy population) of filovirus infections. At least for the moment, natural filovirus infections do not FIGuRE 234-2 Ebola virus particle: the first transmission electron micrograph of an Ebola virion in a culture of Vero cells inoculated with a blood sample from a patient from the 1976 Zaire outbreak of Ebola virus disease. Shown is the typical and unique filamentous and pleomorphic structure of filovirions. (PHIL ID#1833, taken by Dr. Fredrick

1	A. Murphy, Centers for Disease Control and Prevention.) pose a global threat. Filoviruses pathogenic for humans appear to be exclusively endemic to Equatorial Africa, although this distribution may change if natural or artificial environmental alterations lead to filovirus host migration and increased contacts between nonhuman hosts and humans (Fig. 234-4). The majority of recorded EVD and MVD outbreaks can be traced back to single index cases who transmitted the infection to others. These chains of contacts suggest that only around 50 natural host-to-human spillover events have occurred since the discovery of filoviruses in 1967. Outbreak frequency, case numbers, and overall lethality probably depend on the particular etiologic agent, the geographic location and socioeconomic conditions of the affected country, and local customs. In particular, the availability of personal protective gear and reusable medical equipment, such as syringes and needles, has affected overall case numbers

1	of the affected country, and local customs. In particular, the availability of personal protective gear and reusable medical equipment, such as syringes and needles, has affected overall case numbers in the past, and outbreaks have been contained when local burial practices, such as ritual washing, have been either prevented or altered by the use of gloves. The incidence of EVD and MVD may have increased over the past two decades (Figs. 234-3 and 234-4), but researchers debate whether the observed change is due to increased filovirus activity, more frequent contact between filovirus hosts and humans, or continuous improvement in surveillance capabilities.

1	EVD and MVD outbreaks are associated with distinct meteorologic and geographic conditions and are probably associated with distinct hosts or reservoirs. The four ebolaviruses that cause disease in humans are endemic in humid rainforests. EVD outbreaks have often been linked to hunting or contact with bush meat (i.e., meat from apes, other nonhuman primates, duikers, or bush pigs) in forests. Ecologic studies indicate that EBOV may be the etiologic agent of extensive and frequently fatal epizootics among wild chimpanzee and gorilla populations. However, replicating isolates of ebolaviruses from wild nonhuman primates have thus far been obtained only in the case of TAFV, which was isolated from a succumbed western chimpanzee in Côte d’Ivoire in 1994. The marburgviruses MARV and RAVV, on the other hand, seem to infect hosts inhabiting arid woodlands. MVD outbreaks have almost always been epidemiologically linked to visits to or work in natural or artificial caves or mines. A pteropid

1	the other hand, seem to infect hosts inhabiting arid woodlands. MVD outbreaks have almost always been epidemiologically linked to visits to or work in natural or artificial caves or mines. A pteropid (fruit) bat, the cave-dwelling Egyptian rousette (Rousettus aegyptiacus), serves as a natural and subclinically infected reservoir for both MARV and RAVV. Although bats are suspected to be the hosts for ebolaviruses as

1	FIGuRE 234-3 Characteristics of outbreaks of human filovirus disease. Six of eight known filoviruses have caused disease in humans in the past. Outbreaks are listed by virus in chronological order. Laboratory infections are shaded gray and italicized. Arrows indicate international case exportation. Total number of cases and total number of lethal cases are summarized in the middle column (2014 EBOV infections as of December 3). The lethality/case–fatality rate (black dots) for each outbreak is plotted on a 0–100% scale along with 99% confidence intervals (black horizontal lines). The overall case–fatality rate for disease caused by a particular virus is delineated by vertical bold-colored lines, with vertical bold-colored dashed lines indicating the corresponding 99% confidence intervals; the overall case–fatality rate for all ebolavirus infections, all marburgvirus infections, and all filovirus infections are shown by vertical gray bars. BDBV, Bundibugyo virus; COD, Democratic

1	the overall case–fatality rate for all ebolavirus infections, all marburgvirus infections, and all filovirus infections are shown by vertical gray bars. BDBV, Bundibugyo virus; COD, Democratic Republic of the Congo (formerly Zaire); COG, Republic of the Congo; EBOV, Ebola virus; MARV, Marburg virus; RAVV, Ravn virus; SUDV, Sudan virus; TAFV, Taï Forest virus; UK, United Kingdom; USSR, Union of Soviet Socialist Republics (today Russia).

1	FIGuRE 234-4 Geographic distribution of human filovirus disease outbreaks and years of occurrence. Arrows indicate international case exportation. BDBV, Bundibugyo virus; COD, Democratic Republic of the Congo (formerly Zaire); COG, Republic of the Congo; EBOV, Ebola virus; MARV, Marburg virus; RAVV, Ravn virus; SUDV, Sudan virus; TAFV, Taï Forest virus. well, definitive proof is still lacking. In fact, thus far, only EBOV and RESTV have been loosely connected to frugivorous and insectivorous bats by means of antibody or genome fragment detection, whereas the hosts of BDBV, SUDV, and TAFV remain unclear.

1	Human infections typically occur through direct exposure of skin lesions or mucosal surfaces to contaminated bodily fluids or material or by parenteral inoculation (e.g., via accidental needlesticks or reuse of needles in poorly equipped hospitals). Numerous studies, both in vitro and in vivo (in several animal models of human disease), have shed light on key pathogenetic events that evolve subsequent to filovirion exposure. The GP1,2 spikes on the surface of filovirions determine their cell and tissue tropism by engaging yet-unidentified cell-surface molecules and the intracellular receptor Niemann-Pick C1. One of the pathogenetic hallmarks of filovirus infection is a pronounced suppression of the immune system. The first targets of filovirions are local macrophages, monocytes, and dendritic cells.

1	Several structural proteins of filovirions, in particular VP35, VP40, and VP24, then suppress cellular innate immune responses by, for instance, inhibiting the interferon pathway and thereby enabling a productive filovirus infection. The result is the secretion of copious numbers of progeny virions, as evidenced by high titers in the bloodstream (>106 plaque-forming units [pfu]/mL of serum in humans) and the lymphatics, and dissemination to most tissues. Filovirions then infect additional phagocytic cells, such as other macrophages (alveolar, peritoneal, pleural), Kupffer cells in the liver, and microglia, as well as other targets, such as adrenal cortical cells, fibroblasts, hepatocytes, endothelial cells, and a variety of epithelial cells. Infection leads to the secretion of soluble signaling molecules (varying with the cell type) that most likely are crucial factors in immune response modulation and development of multiorgan dysfunction syndrome. For instance, infected macrophages

1	molecules (varying with the cell type) that most likely are crucial factors in immune response modulation and development of multiorgan dysfunction syndrome. For instance, infected macrophages react by secreting proinflammatory cytokines, a response that leads to further recruitment of macrophages to the site of infection. In contrast, infected dendritic cells are not activated to secrete cytokines, and expression of major histocompatibility class II antigens is partially suppressed. Immunosuppression occurs in part by massive lymphoid depletion in lymph nodes, spleen, and thymus in the absence of reactive inflammatory cellular responses. Results from animal studies suggest that depletion is a direct consequence of considerable bystander apoptosis of lymphocytes; this explanation would also account for the severe lymphopenia that develops in patients. The consequence of these events is not only florid filovirus dissemination but also a proclivity of the patient for secondary bacterial

1	account for the severe lymphopenia that develops in patients. The consequence of these events is not only florid filovirus dissemination but also a proclivity of the patient for secondary bacterial and fungal infections.

1	Other pathogenetic hallmarks of filovirus infections are a severe disturbance of the clotting system and the impairment of vascular integrity. Disseminated intravascular coagulation is the cause of the severe imbalance in the clotting system of filovirus-infected patients. Thrombocytopenia, increased concentrations of tissue factor, consumption of clotting factors, increased concentrations of fibrin degradation products (d-dimers), and declining concentrations of protein C are typical features of infection. Consequently, the occlusion of small vessels by widely distributed microthrombi leads to extensive necroses/hypoxic infarcts in target tissues (particularly the gonads, kidneys, liver, and spleen) in the absence of marked inflammatory 1327 responses. In addition, petechiae, ecchymoses, extensive visceral effusions, and other hemorrhagic signs are observed in internal organs, mucous membranes, and skin. Actual severe blood loss, however, is a rare event. Aberrance in cytokines or

1	extensive visceral effusions, and other hemorrhagic signs are observed in internal organs, mucous membranes, and skin. Actual severe blood loss, however, is a rare event. Aberrance in cytokines or other factors such as nitric oxide and direct infection and activation of endothelial cells most likely are responsible for upregulated permeability of the endothelia of blood vessels. This upregulation leads to fluid redistribution (third spacing); interstitial and myocardial edema and hypovolemic shock are common developments. Clinical improvement is rare and is usually characterized by falling viral titers during the development of a virus-specific immune response.

1	MVD and EVD cannot be differentiated by mere observation of clinical manifestations. The incidence of clinical signs does not differ significantly among infections caused by disparate filoviruses (Table 234-2). The incubation period ranges from 3 to 25 days, after which infected people develop a biphasic syndrome with a 1to 2-day Abbreviations: BDBV, Bundibugyo virus; EBOV, Ebola virus; MARV, Marburg virus; NR, not reported.

1	1328 relative remission separating the two phases. The first phase (disease onset until around day 5–7) resembles influenza and is characterized by sudden onset of fever and chills, severe headaches, cough, myalgia, pharyngitis, arthralgia of the larger joints, development of a maculopapular rash, and other signs/symptoms (Table 234-2). The second phase (approximately 5–7 days after disease onset and thereafter) involves the gastrointestinal tract (abdominal pain with vomiting and/ or diarrhea), respiratory tract (chest pain, cough), vascular system (postural hypotension, edema), and central nervous system (confusion, coma, headache). Hemorrhagic manifestations such as subconjunctival injection, nosebleeds, hematemesis, hematuria, and melena are typical (Table 234-2). Typical laboratory findings are leukopenia (with cell counts as low as 1000/μL) with a left shift prior to leukocytosis, thrombocytopenia (with counts as low as 50,000/μL), increased concentrations of liver and

1	findings are leukopenia (with cell counts as low as 1000/μL) with a left shift prior to leukocytosis, thrombocytopenia (with counts as low as 50,000/μL), increased concentrations of liver and pancreatic enzymes (aspartate aminotransferase > alanine aminotransferase, γ-glutamyltransferase, serum amylase), hypokalemia, hypoproteinemia, increased creatinine and urea concentrations with proteinuria, and prolonged prothrombin and partial thromboplastin times. Patients usually succumb to disease 4–14 days after infection. Patients who survive experience prolonged and sometimes incapacitating sequelae such as arthralgia, asthenia, iridocyclitis, hearing loss, myalgia, orchitis, parotitis, psychosis, recurrent hepatitis, transverse myelitis, or uveitis. Temporary hair loss and desquamation of skin areas previously affected by a typical maculopapular rash are visible consequences of the disease. Rarely, filoviruses can persist in the liver, eyes, or testicles of survivors and may cause

1	of skin areas previously affected by a typical maculopapular rash are visible consequences of the disease. Rarely, filoviruses can persist in the liver, eyes, or testicles of survivors and may cause recurrent disease months after convalescence.

1	Filovirus infections cannot be diagnosed on the basis of clinical presentation alone. Numerous diseases typical for Equatorial Africa need to be considered in the differential diagnosis of a febrile patient. Almost all of these diseases occur at a much higher incidence than filovirus infections and are therefore the more likely candidates during differential diagnostic deliberations. The most important of the infectious diseases that closely mimic EVD and MVD are falciparum malaria and typhoid fever; also important are enterohemorrhagic Escherichia coli enteritis, gram-negative septicemia (including shigellosis), meningococcal septicemia, rickettsial infections, fulminant viral hepatitis, leptospirosis, measles, and all other viral hemorrhagic fevers (in particular, yellow fever). Other ailments, such as venomous snakebites, warfarin intoxication, and the many transient or inherited platelet and vascular disorders, also must be considered. Visits to caves or mines and direct contact

1	ailments, such as venomous snakebites, warfarin intoxication, and the many transient or inherited platelet and vascular disorders, also must be considered. Visits to caves or mines and direct contact with bats, nonhuman primates (especially apes), or bush meat should raise suspicion of filovirus infection, as should admission to or treatment in rural hospitals or direct contact with severely ill local residents.

1	If EVD or MVD is suspected on the basis of epidemiologic history, exposure history, and/or clinical manifestations, infectious disease specialists and the proper public health authorities, including the WHO, should be notified immediately. Laboratory diagnosis of EVD and MVD is relatively straightforward but requires maximal containment (biosafety level 4), which usually is not available in filovirus-endemic countries, or the involvement of on-site personnel trained in the use of diagnostic assays adapted for field use. Consequently, diagnostic samples should be collected with great caution and with use of proper personal protective equipment and strict barrier nursing techniques. With adherence to established biosafety precautionary measures, samples should be sent in suitable transport media to national or international WHO reference laboratories. Acute-phase blood/serum is the preferred diagnostic specimen because it usually contains high titers of filovirions and

1	transport media to national or international WHO reference laboratories. Acute-phase blood/serum is the preferred diagnostic specimen because it usually contains high titers of filovirions and filovirion-specific antibodies.

1	The current methods of choice for the diagnosis of filovirus infection are reverse-transcription polymerase chain reaction (detection limit, 1000–2000 virus genome copies per milliliter of serum) and antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of filovirus genomes and filovirion components, respectively. Direct IgM and IgG or IgM capture ELISA is used for the detection of filovirion-targeting antibodies from patients in later stages of disease—i.e., those who have been able to mount a detectable immune response, including survivors. All these assays can be conducted on samples treated with guanidinium isothiocyanate (for polymerase chain reaction) or cobalt-60 irradiation (for ELISA) or subjected to other effective measures that render filoviruses noninfectious. Virus isolation in cell culture and plaque assays for quantification or diagnostic confirmation is relatively easy but must be performed in maximal-containment laboratories. If available,

1	Virus isolation in cell culture and plaque assays for quantification or diagnostic confirmation is relatively easy but must be performed in maximal-containment laboratories. If available, electron microscopic examination of properly inactivated samples or cultures can confirm the diagnosis because filovirions have unique filamentous shapes (Fig. 234-2). Formalin-fixed skin biopsies can be useful for safe postmortem diagnoses.

1	Any treatment of patients with suspected or confirmed filovirus infection must be administered under increased safety precautions by experienced specialists using appropriate personal protective equipment (see “Prevention,” below). Treatment of EVD and MVD is entirely supportive because no accepted/approved, efficacious, specific antiviral agents or vaccines are yet available. The one exception is hyperimmune equine immunoglobulin, which has been approved in Russia—in the absence of convincing efficacy data—for emergency treatment of laboratory infections. Given the extraordinarily high lethality of filoviruses, special protocols may be established by ad hoc expert groups to outline treatment of exposed individuals with one of several regimens that have shown promise in experimental nonhuman primates. Current options include postexposure vaccination with filovirus GP1,2-expressing recombinant replicating vesicular stomatitis Indiana virus; administration of specific filovirus genomeor

1	primates. Current options include postexposure vaccination with filovirus GP1,2-expressing recombinant replicating vesicular stomatitis Indiana virus; administration of specific filovirus genomeor transcript-targeting small interfering RNAs or phosphorodiamidate morpholino oligomers; administration of filovirusspecific antibodies or antibody cocktails (convalescent sera have not yet been proven effective); and use of a synthetic adenosine analog (BCX4430) that acts as a non-obligate RNA chain terminator. In the absence of these candidate treatments, measures to stabilize patients include those generally recommended for severe septicemia/sepsis/shock. Countermeasures should address hypotension and hypoperfusion, vascular leakage in the systemic and pulmonary circulatory system, disseminated intravascular coagulation and overt hemorrhaging, acute kidney failure, and electrolyte (especially potassium) imbalances. Pain management and administration of antipyretics and antiemetics should

1	intravascular coagulation and overt hemorrhaging, acute kidney failure, and electrolyte (especially potassium) imbalances. Pain management and administration of antipyretics and antiemetics should always be considered.

1	Given the severe immunosuppression induced by filovirus infection, secondary infections should be kept in mind and appropriately treated as early as possible. Pregnancy and labor cause severe and frequently fatal complications in filovirus infections due to clotting factor consumption, fetal loss, and/or severe blood loss during birth. The prognosis of filovirus infections is generally poor, although outcome probably depends somewhat on which particular virus causes the infection (Fig. 234-3). Convalescence may take months, with skin peeling, alopecia, prostration, weight loss, orchitis, amnesia, confusion, and anxiety as typical sequelae. Rarely, filoviruses persist in apparently healthy survivors and are either reactivated by unknown means at a later point or transmitted sexually. Condom use or abstinence from sexual activity for at least 3 months after disappearance of clinical signs is therefore recommended for survivors.

1	Currently, filovirus vaccines are not available. Prevention of filovirus infection in nature is difficult because the ecology of the viruses is not completely understood. As stated above, frugivorous cave-dwelling diagnosis and Treatment of Fungal Infections John E. Edwards, Jr. TERMINOLOGY AND MICROBIOLOGY Traditionally, fungal infections have been classified into specific cat-235 sECTIOn 16 FungAL InFECTIOns

1	Diagnosis and Treatment of Fungal Infections pteropid bats (Egyptian rousettes) have been identified as healthy carriers of MARV and RAVV. Avoidance of direct or indirect contact with these bats is therefore useful advice to people entering or living in areas where the animals can be found. Prevention seems to be more difficult in the case of ebolaviruses, for which definite reservoirs have not yet been pinpointed. EVD outbreaks have been associated not with bats but rather with hunting or consumption of nonhuman primates. The mechanism of introduction of ebolaviruses into nonhuman primate populations is unclear. Therefore, the best advice to locals and travelers is to avoid contact with bush meat, nonhuman primates, and bats.

1	Relatively simple barrier nursing techniques, vigilant use of proper personal protective equipment, and quarantine measures usually suffice to terminate or at least contain filovirus disease outbreaks. Isolation of filovirus-infected people and avoidance of direct person-to-person contact without proper personal protective equipment usually suffice to prevent further spread as the pathogens are not 1329 transmitted through droplets or aerosols under natural conditions. Typical protective gear sufficient to prevent filovirus infections consists of disposable gloves, gowns, and shoe covers and a face shield and/or goggles. If available, N-95/N-100 respirators may be used to further limit infection risk. Positive air pressure respirators should be considered for high-risk medical procedures such as intubation or suctioning. Medical equipment used in the care of a filovirus-infected patient, such as gloves or syringes, should never be reused unless safety-tested sterilization or

1	such as intubation or suctioning. Medical equipment used in the care of a filovirus-infected patient, such as gloves or syringes, should never be reused unless safety-tested sterilization or disinfection methods are properly applied. Because filovirions are enveloped, disinfection with detergents, such as 1% sodium deoxycholate, diethyl ether, or phenolic compounds, is relatively straightforward. Bleach solutions of 1:100 and 1:10 are recommended for surface disinfection and application to excreta/corpses, respectively. Whenever possible, potentially contaminated materials should be autoclaved, irradiated, or destroyed.

1	egories based on both anatomic location and epidemiology. The most common general anatomic categories are mucocutaneous and deep organ infection; the most common general epidemiologic categories are endemic and opportunistic infection. Although mucocutaneous infections can cause serious morbidity, they are rarely fatal. Deep organ infections also cause severe illness in many cases and, in contrast to mucocutaneous infections, are often fatal. The endemic mycoses (e.g., coccidioidomycosis) are caused by fungal organisms that are not part of the normal human microbiota but rather are acquired from environmental sources. In contrast, opportunistic mycoses are caused by organisms (e.g., Candida and Aspergillus) that commonly are components of the normal human microbiota and whose ubiquity in nature renders them easily acquired by the immunocompromised host (Table 235-1). Opportunistic fungi cause serious infections when the immunologic response of the host becomes ineffective, allowing

1	nature renders them easily acquired by the immunocompromised host (Table 235-1). Opportunistic fungi cause serious infections when the immunologic response of the host becomes ineffective, allowing the organisms to transition from harmless commensals to invasive pathogens. Frequently, the diminished effectiveness of the immune system is a result of advanced modern therapies that coincidentally either cause an imbalance in the host’s microbiota or directly interfere with immunologic responses. Endemic mycoses cause more severe

1	Pneumocystosis aThe endemic mycoses can also occur as opportunistic infections. illness in immunocompromised patients than in immunocompetent individuals.

1	illness in immunocompromised patients than in immunocompetent individuals. Patients acquire deep organ infection with endemic fungi almost exclusively by inhalation. Cutaneous infections result either from hematogenous dissemination or, more often, from direct contact with soil—the natural reservoir for the vast majority of endemic mycoses. The dermatophytic fungi may be acquired by human-to-human transmission, but the majority of infections result from environmental contact. In contrast, the opportunistic fungus Candida invades the host from normal sites of colonization, usually the mucous membranes of the gastrointestinal tract. In general, innate immunity is the primary defense mechanism against fungi. Although antibodies are formed during many fungal infections (and even during commensalism), they generally do not constitute the primary mode of host defense. Nevertheless, in selected infections, as discussed below, measurement of antibody titers may be a useful diagnostic test.

1	Three other terms frequently used in clinical discussions of fungal infections are yeast, mold, and dimorphic fungus. Yeasts are seen as rounded single cells or as budding organisms. Candida and Cryptococcus are traditionally classified as yeasts. Molds grow as filamentous forms called hyphae both at room temperature and in invaded tissue. Aspergillus, Rhizopus (the genus that causes mucormycosis, also known as zygomycosis), and fungi commonly infecting the skin to cause ringworm and related cutaneous conditions are classified as molds. Variations occur within this classification of yeasts and molds. For instance, when Candida infects tissue, both yeasts and filamentous forms may be present (except with C. glabrata, which forms only yeasts in tissue); in contrast, Cryptococcus exists only in yeast form. Dimorphic is the term used to describe fungi that grow as yeasts or large spherical structures in tissue but as filamentous forms at room temperature in the environment. Classified in

1	in yeast form. Dimorphic is the term used to describe fungi that grow as yeasts or large spherical structures in tissue but as filamentous forms at room temperature in the environment. Classified in this group are the organisms causing blastomycosis, paracoccidioidomycosis, coccidioidomycosis, histoplasmosis, and sporotrichosis.

1	The incidence of nearly all fungal infections has risen substantially. Opportunistic infections have increased in frequency as a consequence of intentional immunosuppression in organ and stem cell transplantation and other disorders, the administration of cytotoxic chemotherapy for cancers, the liberal use of antibacterial agents, and, more recently, the increasing use of monoclonal antibodies. Within a global context, the incidence of endemic mycoses has stantial population growth. When advances in medical care (e.g., more aggressive treatment of cancer or organ transplantation) are introduced into a given area, the opportunistic mycoses increase in incidence.

1	1330 DIAGNOSIS The definitive diagnosis of any fungal infection requires histopathologic identification of the fungus invading tissue and accompanying evidence of an inflammatory response. The identification of an inflammatory response has been especially important with regard to Aspergillus infection. Aspergillus is ubiquitous and can float in the air onto biopsy material. Therefore, in rare but important instances, this fungus is an ex vivo contaminant during processing of a specimen for microscopy, with a consequent incorrect diagnosis. The stains most commonly used to identify fungi are periodic acid–Schiff and Gomori methenamine silver. Candida, unlike other fungi, is visible on gram-stained tissue smears. Hematoxylin and eosin stain is not sufficient to identify Candida in tissue specimens. When positive, an india ink preparation of cerebrospinal fluid (CSF) is diagnostic for cryptococcosis. Most laboratories now use calcofluor white staining coupled with fluorescent microscopy

1	When positive, an india ink preparation of cerebrospinal fluid (CSF) is diagnostic for cryptococcosis. Most laboratories now use calcofluor white staining coupled with fluorescent microscopy to identify fungi in fluid specimens. Extensive investigations of the diagnosis of deep organ fungal infections have yielded a variety of tests with different degrees of specificity and sensitivity. The most reliable tests are the detection of antibody to Coccidioides immitis in serum and CSF; of Histoplasma capsulatum antigen in urine, serum, and CSF; and of cryptococcal polysaccharide antigen in serum and CSF. These tests have a general sensitivity and specificity of 90%; however, because of variability among laboratories, testing on multiple occasions is advisable. The test for galactomannan has been used extensively in Europe and is now approved in the United States for diagnosis of aspergillosis. Sources of concern regarding galactomannan are the incidence of false-negative results and the

1	been used extensively in Europe and is now approved in the United States for diagnosis of aspergillosis. Sources of concern regarding galactomannan are the incidence of false-negative results and the need for multiple serial tests to reduce this incidence. The β-glucan test for Candida is also under evaluation but, like the galactomannan test, still requires additional validation; this test has a negative predictive value of ~90%. Both of these tests are being used with increasing frequency, especially for guiding the timing of initiation and duration of therapy. The galactomannan test is being evaluated in both serum and bronchoalveolar lavage fluid. Numerous polymerase chain reaction assays to detect antigens are in the developmental stages, as are nucleic acid hybridization techniques; currently, these tests are not widely available. Of the fungal organisms, Candida is by far the most frequently recovered from blood. Although Candida species can be detected with any of the

1	currently, these tests are not widely available. Of the fungal organisms, Candida is by far the most frequently recovered from blood. Although Candida species can be detected with any of the automated blood culture systems widely used at present, the lysis-centrifugation technique increases the sensitivity of blood cultures for Candida and for less common organisms (e.g., H. capsulatum). Lysis-centrifugation should be used when disseminated fungal infection is suspected. Except in the cases of coccidioidomycosis, cryptococcosis, and histoplasmosis, there are no fully validated and widely used tests for serodiagnosis of disseminated fungal infection. Skin tests for the endemic mycoses are no longer available.

1	This discussion is intended as a brief overview of general strategies for the use of antifungal agents in the treatment of fungal infections. Regimens, schedules, and strategies are detailed in the chapters on specific mycoses that follow in this section. The doses cited here are standard doses for adults with invasive infection. Since fungal organisms are eukaryotic cells that contain most of the same organelles (with many of the same physiologic functions) as human cells, the identification of drugs that selectively kill or inhibit fungi but are not toxic to human cells has been highly problematic. Far fewer antifungal than antibacterial agents have been introduced into clinical medicine.

1	The introduction of amphotericin B (AmB) in the late 1950s revolutionized the treatment of fungal infections in deep organs. Before AmB became available, cryptococcal meningitis and other disseminated fungal infections were nearly always fatal. For nearly a decade after AmB was introduced, it was the only effective agent for the treatment of life-threatening fungal infections. AmB remains the broadest-spectrum antifungal agent but carries several disadvantages, including significant nephrotoxicity, lack of an oral preparation, and unpleasant side effects (fever, chills, and nausea) during treatment. To circumvent nephrotoxicity and infusion side effects, lipid formulations of AmB were developed and have virtually replaced the original colloidal deoxycholate formulation in clinical use (although the older formulation is still available). The lipid formulations include liposomal AmB (L-AmB; 3–5 mg/kg per day) and AmB lipid complex (ABLC; 5 mg/kg per day). A third preparation, AmB

1	(although the older formulation is still available). The lipid formulations include liposomal AmB (L-AmB; 3–5 mg/kg per day) and AmB lipid complex (ABLC; 5 mg/kg per day). A third preparation, AmB colloidal dispersion (ABCD; 3–4 mg/kg per day), is rarely used because of the high incidence of side effects associated with infusion.

1	The lipid formulations of AmB have the disadvantage of being considerably more expensive than the deoxycholate formulation. Experience is still accumulating on the comparative efficacy, toxicity, and advantages of the different formulations for specific clinical fungal infections, including central nervous system (CNS) infection. Whether there is a clinically significant difference in these drugs with respect to CNS penetration or nephrotoxicity remains controversial. Despite these issues and despite the expense, the lipid formulations are now much more commonly used than AmB deoxycholate in developed countries. In developing countries, AmB deoxycholate is still preferred because of the expense of the lipid formulations.

1	This class of antifungal drugs offers important advantages over AmB: the azoles cause little or no nephrotoxicity and are available in oral formulations. Early azoles included ketoconazole and miconazole, which have been replaced by newer agents for the treatment of deep organ fungal infections. The azoles’ mechanism of action is inhibition of ergosterol synthesis in the fungal cell wall. Unlike AmB, these drugs are considered fungistatic, not fungicidal.

1	Fluconazole Since its introduction, fluconazole has played an extremely important role in the treatment of a wide variety of serious fungal infections. Its major advantages are the availability of both oral and IV formulations, a long half-life, satisfactory penetration of most body fluids (including ocular fluid and CSF), and minimal toxicity (especially relative to that of AmB). Its disadvantages include (usually reversible) hepatotoxicity and—at high doses—alopecia, muscle weakness, and dry mouth with a metallic taste. Fluconazole is not effective for the treatment of aspergillosis, mucormycosis, or Scedosporium apiospermum infections. It is less effective than the newer azoles against Candida glabrata and Candida krusei.

1	Fluconazole has become the agent of choice for the treatment of coccidioidal meningitis, although relapses have followed therapy with this drug. In addition, fluconazole is useful as both consolidation and maintenance therapy for cryptococcal meningitis. This agent has been shown to be as efficacious as AmB in the treatment of candidemia. The effectiveness of fluconazole in candidemia and the drug’s relatively minimal toxicity, in conjunction with the inadequacy of diagnostic tests for widespread hematogenously disseminated candidiasis, have led to a change in the paradigm for candidemia management. The standard of care is now to treat all candidemic patients with an antifungal agent and to change all their intravascular lines, if feasible, rather than merely removing a singular suspect intravascular line and then observing the patient. The usual fluconazole regimen for treatment of candidemia is 400 mg/d given until 2 weeks after the last positive blood culture.

1	Fluconazole is considered effective as fungal prophylaxis in bone marrow transplant recipients and high-risk liver transplant patients. Its general use for prophylaxis in patients with leukemia, in AIDS patients with low CD4+ T cell counts, and in patients on surgical intensive care units remains controversial. Because of concerns about the possibility of infection due to resistant Candida species and of infection with Aspergillus species, many clinicians are initiating therapy with an echinocandin, which is then replaced by fluconazole once a susceptible Candida species is recovered and concern about Aspergillus is diminished.

1	Voriconazole Voriconazole, which is available in both oral and IV formulations, has a broader spectrum than fluconazole against Candida species (including C. glabrata and C. krusei) and is active against Aspergillus, Scedosporium, and Fusarium. It is generally considered the first-line drug of choice for treatment of aspergillosis. A few case reports have shown voriconazole to be effective in individual patients with coccidioidomycosis, blastomycosis, and histoplasmosis, but because of limited data this agent is not recommended for primary treatment of the endemic mycoses. Among the disadvantages of voriconazole (compared with fluconazole) are its more numerous interactions with many of the drugs used in patients predisposed to fungal infections. Hepatotoxicity, skin rashes (including photosensitivity), and visual disturbances are relatively common. Skin cancer surveillance is now recommended for patients taking voriconazole. In addition, voriconazole is considerably more expensive

1	and visual disturbances are relatively common. Skin cancer surveillance is now recommended for patients taking voriconazole. In addition, voriconazole is considerably more expensive than fluconazole. Moreover, it is advisable to monitor voriconazole levels in certain patients since (1) this drug is completely metabolized in the liver by CYP2C9, CYP3A4, and CYP2C19; and (2) human genetic variability in CYP2C19 activity exists. Dosages should be reduced accordingly in patients with liver failure. Dose adjustments for renal insufficiency are not necessary; however, because the IV formulation is prepared in cyclodextrin, it should not be given to patients with severe renal insufficiency.

1	Itraconazole Itraconazole is available in IV and oral (capsule and suspension) formulations. Varying blood levels among patients taking oral itraconazole reflect a disadvantage compared with the other azoles. Itraconazole is the drug of choice for mild to moderate histoplasmosis and blastomycosis and has often been used for chronic mucocutaneous candidiasis. It has been approved by the

1	U.S. Food and Drug Administration (FDA) for use in febrile neutropenic patients. Itraconazole has also proved useful for the treatment of chronic coccidioidomycosis, sporotrichosis, and S. apiospermum infection. The mucocutaneous and cutaneous fungal infections that have been treated successfully with itraconazole include oropharyngeal candidiasis (especially in AIDS patients), tinea versicolor, tinea capitis, and onychomycosis. Disadvantages of itraconazole include its poor penetration into CSF, the use of cyclodextrin in both the oral suspension and the IV formulation, the variable absorption of the drug in capsule form, and the need for monitoring of blood levels in patients taking capsules for disseminated mycoses. Reported cases of severe congestive heart failure in patients taking itraconazole have been a source of concern. Like the other azoles, itraconazole can cause hepatic toxicity.

1	Posaconazole Posaconazole is approved by the FDA for prophylaxis of aspergillosis and candidiasis in patients at high risk for developing these infections because of severe immunocompromise. It has also been approved for the treatment of oropharyngeal candidiasis and has been evaluated as therapy for zygomycosis, fusariosis, aspergillosis, cryptococcosis, and various other forms of candidal infection. The relevant studies of posaconazole in zygomycosis, fusariosis, and aspergillosis have examined salvage therapy. A study of more than 90 patients whose zygomycosis was refractory to other therapy yielded encouraging results. No trials of posaconazole for the treatment of candidemia have yet been reported. Case reports have described the drug’s efficacy in coccidioidomycosis and histoplasmosis. Controlled trials have shown its effectiveness as a prophylactic agent in patients with acute leukemia and in bone marrow transplant recipients. In addition, posaconazole has been found to be

1	Controlled trials have shown its effectiveness as a prophylactic agent in patients with acute leukemia and in bone marrow transplant recipients. In addition, posaconazole has been found to be effective against fluconazole-resistant Candida species. The results of a large-scale study of the use of posaconazole as salvage therapy for aspergillosis indicated that it is an alternative to other agents for salvage therapy; however, that study predated the use of voriconazole and the echinocandins.

1	The echinocandins, including the FDA-approved drugs caspofungin, anidulafungin, and micafungin, have added considerably to the antifungal armamentarium. All three of these agents inhibit β-1,3-glucan synthase, which is necessary for cell wall synthesis in 1331 fungi and is not a component of human cells. None of these agents is currently available in an oral formulation. The echinocandins are considered fungicidal for Candida and fungistatic for Aspergillus. Their greatest use to date is against candidal infections. They offer two advantages: broad-spectrum activity against all Candida species and relatively low toxicity. The minimal inhibitory concentrations (MICs) of all the echinocandins are highest against Candida parapsilosis; it is not clear whether these higher MIC values represent less clinical effectiveness against this species. The echinocandins are among the safest antifungal agents.

1	In controlled trials, caspofungin has been at least as efficacious as AmB for the treatment of candidemia and invasive candidiasis and as efficacious as fluconazole for the treatment of candidal esophagi-tis. In addition, caspofungin has been efficacious as salvage therapy for aspergillosis. Anidulafungin has been approved by the FDA as therapy for candidemia in nonneutropenic patients and for Candida esophagitis, intraabdominal infection, and peritonitis. In controlled trials, anidulafungin has been shown to be noninferior and possibly superior to fluconazole against candidemia and invasive candidiasis. It is as efficacious as fluconazole against candidal esophagitis. When anidulafungin is used with cyclosporine, tacrolimus, or voriconazole, no dosage adjustment is required for either drug in the combination. Micafungin has been approved for the treatment of esophageal candidiasis and candidemia and for prophylaxis in patients receiving stem cell transplants. In a head-to-head trial,

1	in the combination. Micafungin has been approved for the treatment of esophageal candidiasis and candidemia and for prophylaxis in patients receiving stem cell transplants. In a head-to-head trial, micafungin was noninferior to caspofungin for the treatment of candidemia. Studies thus far have shown that coadministration of micafungin and cyclosporine does not require dose adjustments for either drug. When micafungin is given with sirolimus, the area under the plasma drug concentration–time curve rises for sirolimus, usually necessitating a reduction in its dose. In open-label trials, favorable results have been obtained with micafungin for the treatment of deep-seated Aspergillus and Candida infections.

1	The use of flucytosine has diminished as newer antifungal drugs have been developed. This agent is now used most commonly in combination with AmB (deoxycholate or lipid formulations) for the initial treatment of cryptococcal meningitis. Flucytosine has a unique mechanism of action based on intrafungal conversion to 5-fluorouracil, which is toxic to the fungal cell. Development of resistance to the compound has limited its use as a single agent. Flucytosine is nearly always used in combination with AmB. Its good penetration into the CSF makes it attractive for use with AmB for treatment of cryptococcal meningitis. Flucytosine has also been recommended for the treatment of candidal meningitis in combination with AmB; comparative trials with AmB alone have not been done. Significant and frequent bone marrow depression is seen with flucytosine when this drug is used with AmB.

1	Historically, griseofulvin has been useful primarily for ringworm infection. This agent is usually given for relatively long periods. Terbinafine has been used primarily for onychomycosis but also for ringworm. In comparative studies, terbinafine has been as effective as itraconazole and more effective than griseofulvin for both conditions.

1	A detailed discussion of the agents used for the treatment of cutaneous fungal infections and onychomycosis is beyond the scope of this chapter; the reader is referred to the dermatology literature. Many classes of compounds have been used to treat the common fungal infections of the skin. Among the azoles used are clotrimazole, econazole, miconazole, oxiconazole, sulconazole, ketoconazole, tioconazole, butoconazole, and terconazole. In general, topical treatment of vaginal candidiasis has been successful. Since little difference is thought to exist in the efficacy of the various vaginal preparations, the choice of agent is made by the physician and/or

1	Diagnosis and Treatment of Fungal Infections 1332 the patient on the basis of preference and availability. Fluconazole given orally at 150 mg has the advantage of not requiring repeated intravaginal application. Nystatin is a polyene that has been used for both oropharyngeal thrush and vaginal candidiasis. Useful agents in other classes include ciclopirox olamine, haloprogin, terbinafine, naftifine, tolnaftate, and undecylenic acid. Histoplasmosis Chadi A. Hage, L. Joseph Wheat ETIOLOGY Histoplasma capsulatum, a thermal dimorphic fungus, is the etiologic agent of histoplasmosis. In most endemic areas, H. capsulatum var. capsulatum is the causative agent. In 236 Africa, H. capsulatum var. duboisii also is found; var. duboisii can be differentiated from var. capsulatum as the duboisii yeasts are larger. In Central and South America, histoplasmosis is common and is caused by clades of H. capsulatum var. capsulatum that differ genetically from those involved elsewhere.

1	Mycelia—the naturally infectious form of Histoplasma—have a characteristic appearance, with microconidial and macroconidial forms. Microconidia are oval and are small enough (2–4 μm) to reach the terminal bronchioles and alveoli. Shortly after infecting the host, mycelia transform into the yeasts that are found inside macrophages and other phagocytes. The yeast forms are characteristically small (2–5 μm), with occasional narrow budding. In the laboratory, mycelia are best grown at room temperature, whereas yeasts are grown at 37°C on enriched media. Histoplasmosis is the most prevalent endemic mycosis in

1	North America. Although this fungal disease has been reported throughout the world, its endemicity is particularly notable in certain parts of North, Central, and South America; Africa; and Asia. In Europe, histoplasmosis is diagnosed fairly often, mostly in emigrants from or travelers to endemic areas on other continents. In the United States, the endemic areas spread over the Ohio and Mississippi river valleys. This pattern is related to the humid and acidic nature of the soil in these areas. Soil enriched with bird or bat droppings promotes the growth and sporulation of Histoplasma. Disruption of soil containing the organism leads to aerosolization of the microconidia and exposure of humans nearby. Activities associated with high-level exposure include spelunking, excavation, cleaning of chicken coops, demolition and remodeling of old buildings, and cutting of dead trees. Most cases seen outside of highly endemic areas represent imported disease—e.g., cases reported in Europe after

1	chicken coops, demolition and remodeling of old buildings, and cutting of dead trees. Most cases seen outside of highly endemic areas represent imported disease—e.g., cases reported in Europe after travel to the Americas, Africa, or Asia.

1	Infection follows inhalation of microconidia (Fig. 236-1). Once they reach the alveolar spaces, microconidia are rapidly recognized and engulfed by alveolar macrophages. At this point, the microconidia transform into budding yeasts (Fig. 236-2), a process that is integral to the pathogenesis of histoplasmosis and is dependent on the availability of calcium and iron inside the phagocytes. The yeasts are capable of growing and multiplying inside resting macrophages. Neutrophils and then lymphocytes are attracted to the site of infection. Before the development of cellular immunity, yeasts use the phagosomes as a vehicle for translocation to local draining lymph nodes, whence they spread hematogenously throughout the reticuloendothelial system. Adequate cellular immunity develops ~2 weeks after infection. T cells produce interferon γ to assist the macrophages in killing the organism and controlling the progression of disease. Interleukin 12 and tumor necrosis factor α (TNF-α) play an

1	after infection. T cells produce interferon γ to assist the macrophages in killing the organism and controlling the progression of disease. Interleukin 12 and tumor necrosis factor α (TNF-α) play an essential role in cellular immunity to H. capsulatum. In the immunocompetent host, macrophages, lymphocytes, and epithelial cells eventually organize and form granulomas that contain the organisms. These granulomas typically fibrose and calcify; calcified mediastinal lymph nodes and hepatosplenic calcifications are frequently found in healthy individuals from endemic areas. In immunocompetent hosts, infection with H. capsulatum confers some immunity to reinfection. In patients with impaired cellular immunity, the infection is not contained and can disseminate. Progressive disseminated histoplasmosis (PDH) can involve multiple organs, most commonly the bone marrow, spleen, liver (Fig. 236-3), adrenal glands, and mucocutaneous membranes. Unlike latent tuberculosis, latent histoplasmosis

1	(PDH) can involve multiple organs, most commonly the bone marrow, spleen, liver (Fig. 236-3), adrenal glands, and mucocutaneous membranes. Unlike latent tuberculosis, latent histoplasmosis rarely reactivates.

1	FIGuRE 236-1 Spiked spherical conidia of H. capsulatum (lacto- phenol cotton blue stain). Structural lung disease (e.g., emphysema) impairs the clearance of pulmonary histoplasmosis, and chronic pulmonary disease can result. This chronic process is characterized by progressive inflammation, tissue necrosis, and fibrosis mimicking cavitary tuberculosis. FIGuRE 236-2 Small (2–5 μm) narrow budding yeasts of H. capsulatum from bronchoalveolar lavage fluid (Grocott’s methena-mine silver stain). FIGuRE 236-3 Intracellular yeasts (arrows) of H. capsulatum in a liver biopsy specimen (hematoxylin and eosin stain). The clinical spectrum of histoplasmosis ranges from asymptomatic infection to life-threatening illness. The attack rate and the extent and severity of the disease depend on the intensity of exposure, the immune status of the exposed individual, and the underlying lung architecture of the host.

1	In immunocompetent individuals with low-level exposure, most Histoplasma infections are either asymptomatic or mild and self-limited. Of adults residing in endemic areas, 50–80% have skin-test and/or radiographic evidence of previous infection without clinical manifestations. When symptoms do develop, they usually appear 1–4 weeks after exposure. Heavy exposure leads to a flulike illness with fever, chills, sweats, headache, myalgia, anorexia, cough, dyspnea, and chest pain. Chest radiographs usually show signs of pneumonitis with prominent hilar or mediastinal adenopathy. Pulmonary infiltrates may be focal with light exposure or diffuse with heavy exposure. Rheumatologic symptoms of arthralgia or arthritis, often associated with erythema nodosum, occur in 5–10% of patients with acute histoplasmosis. Pericarditis may also develop. These manifestations represent inflammatory responses to the acute infection rather than its direct effects. Hilar or mediastinal lymph nodes may undergo

1	Pericarditis may also develop. These manifestations represent inflammatory responses to the acute infection rather than its direct effects. Hilar or mediastinal lymph nodes may undergo necrosis and coalesce to form large mediastinal masses that can cause compression of great vessels, proximal airways, and the esophagus. These necrotic lymph nodes may also rupture and create fistulas between mediastinal structures (e.g., bronchoesophageal fistulas).

1	PDH is typically seen in immunocompromised individuals, who account for ~70% of cases. Common risk factors include AIDS (CD4+ T cell count, <200/μL), extremes of age, immunosuppressive medications administered for prevention or treatment of rejection following transplantation (e.g., prednisone, mycophenolate, calcineurin inhibitors, and biologic response modifiers), and methotrexate, anti-TNF-α agents, or other biologic response modifiers given for inflammatory arthritis or Crohn’s disease.

1	The spectrum of PDH ranges from an acute, rapidly fatal course— with diffuse interstitial or reticulonodular lung infiltrates causing respiratory failure, shock, coagulopathy, and multiorgan failure—to a more subacute course with a focal organ distribution. Common manifestations include fever and weight loss. Hepatosplenomegaly also is common. Other findings may include meningitis or focal brain lesions, ulcerations of the oral mucosa, gastrointestinal ulcerations, and adrenal insufficiency. Prompt recognition of this devastating illness is of paramount importance in patients with more severe manifestations or with underlying immunosuppression, especially AIDS (Chap. 226).

1	Chronic cavitary histoplasmosis is seen in smokers who have structural lung disease (e.g., bullous emphysema). This chronic illness is characterized by productive cough, dyspnea, low-grade fever, night sweats, and weight loss. Chest radiographs usually show upper-lobe infiltrates, cavitation, and pleural thickening—findings resembling those of tuber-1333 culosis. Without treatment, the course is slowly progressive. Fibrosing mediastinitis is an uncommon and serious complication of histoplasmosis. In certain patients, acute infection is followed for unknown reasons by progressive fibrosis around the hilar and mediastinal lymph nodes. Involvement may be unilateral or bilateral; bilateral involvement carries a worse prognosis. Major manifestations include superior vena cava syndrome, obstruction of pulmonary vessels, and airway obstruction. Patients may experience recurrent pneumonia, hemoptysis, or respiratory failure. Fibrosing mediastinitis is fatal in up to one-third of cases.

1	In healed histoplasmosis, calcified mediastinal nodes or lung parenchyma may erode through the walls of the airways and cause hemoptysis. This condition is called broncholithiasis. The clinical features and management of histoplasmosis caused by the genetically different clades in Central and South America are similar to those of the disease in North America. African histoplasmosis caused by var. duboisii is clinically distinct and is characterized by frequent skin and bone involvement.

1	America are similar to those of the disease in North America. African histoplasmosis caused by var. duboisii is clinically distinct and is characterized by frequent skin and bone involvement. Fungal culture remains the gold standard diagnostic test for histoplasmosis. However, culture results may not be known for up to 1 month, and cultures are often negative in less severe cases. Cultures are positive in ~75% of cases of PDH and chronic pulmonary histoplasmosis. Cultures of bronchoalveolar lavage (BAL) fluid are positive in about half of cases that include acute pulmonary histoplasmosis causing diffuse infiltrates with hypoxemia. In PDH, the culture yield is highest for BAL fluid, bone marrow aspirate, and blood. Cultures of sputum or bronchial washings are usually positive in chronic pulmonary histoplasmosis. Cultures are typically negative, however, in other forms of histoplasmosis.

1	Fungal stains of cytopathology or biopsy materials showing structures resembling Histoplasma yeasts are helpful in the diagnosis of PDH, yielding positive results in about half of cases. Yeasts can be seen in BAL fluid (Fig. 236-2) from patients with diffuse pulmonary infiltrates, in bone marrow biopsy samples, and in biopsy specimens of other involved organs (e.g., the adrenal glands). Occasionally, yeasts are seen in blood smears from patients with severe PDH. However, artifacts and other fungal elements sometimes stain positive and may be misidentified as Histoplasma yeasts.

1	The detection of Histoplasma antigen in body fluids is extremely useful in the diagnosis of PDH and acute diffuse pulmonary histoplasmosis. The sensitivity of this technique is >95% in patients with PDH and >80% in patients with acute pulmonary histoplasmosis if both urine and serum are tested. Antigen level correlates with severity of illness in PDH and can be used to follow disease progression as levels predictably decrease with effective therapy. An increase in antigen levels also predicts relapse. Antigen can be detected in cerebrospinal fluid from patients with meningitis and in BAL fluid from those with pneumonia. Cross-reactivity occurs with African histoplasmosis, blastomycosis, coccidioidomycosis, paracoccidioidomycosis, and Penicillium marneffei infection.

1	Serologic tests, including immunodiffusion and complement fixation, are useful for the diagnosis of histoplasmosis in immunocompetent patients. Serum antibody titers may rise fourfold in patients with acute histoplasmosis. Serologic tests are especially useful for the diagnosis of chronic pulmonary histoplasmosis. The limitations of serology, however, include insensitivity early in the course of infection (at least 1 month is required for the production of antibodies), insensitivity in immunosuppressed patients, and persistence of detectable antibody for several years after infection. Positive results from past infection may lead to a misdiagnosis of active histoplasmosis in a patient with another disease process. Treatment recommendations for histoplasmosis are summarized in Table 236-1. Treatment is indicated for all patients with PDH or chronic pulmonary histoplasmosis as well as for symptomatic Type of Treatment Histoplasmosis Recommendations Comments

1	Type of Treatment Histoplasmosis Recommendations Comments Abbreviation: AmB, amphotericin B. patients with acute pulmonary histoplasmosis causing diffuse infiltrates, especially with hypoxemia. In most cases of pulmonary histoplasmosis, treatment is not recommended because the degree of exposure is not heavy; the infection is asymptomatic or symptoms are mild, subacute, and not progressive; and the illness resolves without therapy. The preferred treatments for histoplasmosis include the lipid formulations of amphotericin B (AmB) in more severe cases and itraconazole in others. Liposomal AmB has been more effective than the deoxycholate formulation for treatment of PDH in patients with AIDS. The deoxycholate formulation is an alternative to lipid formulations for patients at low risk for nephrotoxicity. Posaconazole, voriconazole, and fluconazole are alternatives for patients who cannot take itraconazole.

1	In severe cases requiring hospitalization, a lipid formulation of AmB is followed by itraconazole. In patients with meningitis, a lipid formulation of AmB should be given for 4–6 weeks before the switch to itraconazole. In immunosuppressed patients, the degree of immunosuppression should be reduced if possible, although immune reconstitution inflammatory syndrome (IRIS) may ensue. Antiretroviral treatment improves the outcome of PDH in patients with AIDS and is recommended; however, whether antiretroviral treatment should be delayed to avoid IRIS is unknown.

1	Blood levels of itraconazole should be monitored to ensure adequate drug exposure, with target concentrations of the parent drug and its hydroxy metabolites of 1–5 μg/mL as measured by high-performance liquid chromatography and 2–10 μg/mL as measured by microbiologic assay. Drug interactions should be carefully assessed: itraconazole not only is cleared by cytochrome P450 metabolism but also inhibits cytochrome P450. This profile causes interactions with many other medications. The duration of treatment for acute pulmonary histoplasmosis is 6–12 weeks, while that for PDH and chronic pulmonary histoplasmosis is ≥1 year. Antigen levels in urine and serum should be monitored during and for at least 1 year after therapy for PDH. Stable or rising antigen levels suggest treatment failure or relapse.

1	Previously, lifelong itraconazole maintenance therapy was recommended for patients with AIDS once histoplasmosis was diagnosed. Today, however, maintenance therapy is not required for patients who respond well to antiretroviral therapy, with CD4+ T cell counts of at least 150/μL (preferably >250/μL); who complete at least 1 year of itraconazole therapy; and who exhibit neither clinical evidence of active histoplasmosis nor an antigenuria level of >4 ng/ mL. Maintenance therapy also appears to be unnecessary in patients receiving immunosuppressive treatment if the degree of immunosuppression can be reduced through an approach similar to that used for patients with AIDS.

1	Fibrosing mediastinitis, which represents a chronic fibrotic reaction to past mediastinal histoplasmosis rather than an active infection, does not respond to antifungal therapy. While treatment is often prescribed for patients with pulmonary histoplasmosis who have not recovered within 1 month and for those with persistent mediastinal lymphadenopathy, the effectiveness of antifungal therapy in these situations is unknown. Neil M. Ampel Coccidioidomycosis, commonly known as Valley fever (see “Epidemiology,” below), is caused by dimorphic soil-dwelling fungi of the genus Coccidioides. Genetic analysis has demonstrated the existence of two species, C. immitis and C. posadasii. These species are indistinguishable with regard to the clinical disease they cause and their appearance on routine laboratory media. Thus, the organisms will be referred to simply as Coccidioides for the remainder of this chapter.

1	Coccidioidomycosis is confined to the Western Hemisphere between the latitudes of 40°N and 40°S. In the United States, areas of high endemicity include the southern portion of the San Joaquin Valley of California and the south-central region of Arizona. However, infection may be acquired in other areas of the southwestern United States, including the southern coastal counties in California, southern Nevada, southwestern Utah, southern New Mexico, and western Texas, including the Rio Grande Valley. Outside the United States, coccidioidomycosis is endemic to northern Mexico as well as to localized regions of Central America. In South America, there are endemic foci in Colombia, Venezuela, northeastern Brazil, Paraguay, Bolivia, and north-central Argentina.

1	The risk of infection is increased by direct exposure to soil harboring Coccidioides. Because of difficulty in isolating Coccidioides from the soil, the precise characteristics of potentially infectious soil are not known. In the United States, several outbreaks of coccidioidomycosis have been associated with soil from archaeologic excavations of Amerindian sites both within and outside of the recognized endemic region. These cases often involved alluvial soils in regions of relative aridity with moderate temperature ranges. Coccidioides was isolated at depths of 2–20 cm below the surface. The recent identification of three cases of coccidioidomycosis in eastern Washington State may suggest that the endemic region is expanding.

1	In endemic areas, many cases of Coccidioides infection occur without obvious soil or dust exposure. Climatic factors appear to increase the infection rate in these regions. In particular, periods of aridity following rainy seasons have been associated with marked increases in the number of symptomatic cases. Overall, the incidence within the United States has increased substantially over the past decade, with nearly 43 cases per 100,000 residents of the endemic region in 2011. Most of that increase has occurred in south-central Arizona, where most of that state’s population resides, and in the southern San Joaquin Valley of California, a much less populated region. The factors causing this increase have not been fully elucidated; however, an influx of older individuals without prior coccidioidal infection appears to be involved. Other variables, such as climate change, construction activity, and increased awareness and reporting, may also be factors. Health care providers should

1	coccidioidal infection appears to be involved. Other variables, such as climate change, construction activity, and increased awareness and reporting, may also be factors. Health care providers should consider coccidioidomycosis when evaluating persons with pneumonia who live in or have traveled to endemic areas.

1	PATHOGENESIS, PATHOLOGY, AND IMMuNE RESPONSE On agar media and in the soil, Coccidioides organisms exist as filamentous molds. Within this mycelial structure, individual filaments (hyphae) elongate and branch, some growing upward. Alternating cells within the hyphae degenerate, leaving barrel-shaped viable elements called arthroconidia. Measuring ∼2 by 5 μm, arthroconidia may become airborne for extended periods. Their small size allows them to evade initial mechanical mucosal defenses and reach deep into the bronchial tree, where infection is initiated in the nonimmune host.

1	Once in a susceptible host, the arthroconidia enlarge, become rounded, and develop internal septations. The resulting structures, called spherules(Fig. 237-1), may attain sizes of 200 μm and are unique to Coccidioides. The septations encompass uninuclear elements called endospores. Spherules may rupture and release packets of endospores that can themselves develop into spherules, thus propagating infection locally. If returned to artificial media or the soil, the fungus reverts to its mycelial stage. Clinical observations and data from studies of animals strongly support the critical role of a robust cellular immune response in the host’s control of coccidioidomycosis. Necrotizing granulomas containing

1	FIGuRE 237-1 Life cycle of Coccidioides.(From TN Kirkland, J Fierer: Emerg Infect Dis 2:192, 1996.) spherules are typically identified in patients with resolved pulmonary 1335 infection. In disseminated disease, granulomas are generally poorly formed or do not develop at all, and a polymorphonuclear leukocyte response occurs frequently. In patients who are asymptomatic or in whom the initial pulmonary infection resolves, delayed-type hypersensitivity to coccidioidal antigens has been routinely documented. Of infected individuals, 60% are completely asymptomatic, and the remaining 40% have symptoms that are related principally to pulmonary infection, including fever, cough, and pleuritic chest pain. The risk of symptomatic illness increases with age. Coccidioidomycosis is commonly misdiagnosed as community-acquired bacterial pneumonia.

1	There are several cutaneous manifestations of primary pulmonary coccidioidomycosis. Toxic erythema consisting of a maculopapular rash has been noted in some cases. Erythema nodosum (see Fig. 25e-40)— typically over the lower extremities—or erythema multiforme (see Fig. 25e-25)—usually in a necklace distribution—may occur; these manifestations are seen particularly often in women. Arthralgias and arthritis may develop. The diagnosis of primary pulmonary coccidioidomycosis is suggested by a history of night sweats or profound fatigue as well as by peripheral-blood eosinophilia and hilar or mediastinal lymphadenopathy on chest radiography. While pleuritic chest pain is common, pleural effusions occur in fewer than 10% of cases. Such effusions are invariably associated with a pulmonary infiltrate on the same side. The cellular content of these effusions is mononuclear in nature; Coccidioides is rarely grown from effusions.

1	In most patients, primary pulmonary coccidioidomycosis usually resolves without sequelae in weeks. However, several pneumonic complications may arise. Pulmonary nodules are residua of primary pneumonia. Generally single, frequently located in the upper lobes, and ≤4 cm in diameter, nodules are often discovered on a routine chest radiograph in an asymptomatic patient. Calcification is uncommon. Coccidioidal pulmonary nodules can be difficult to distinguish radiographically from pulmonary malignancies. Like malignancies, coccidioidal nodules often enhance on positron emission tomography. However, routine CT often demonstrates multiple nodules in coccidioidomycosis. Biopsy is often required to distinguish between these two conditions.

1	Pulmonary cavities occur when a nodule extrudes its contents into the bronchus, resulting in a thin-walled shell. These cavities can be associated with persistent cough, hemoptysis, and pleuritic chest pain. Rarely, a cavity may rupture into the pleural space, causing pyopneumothorax. In such cases, patients present with acute dyspnea, and the chest radiograph reveals a collapsed lung with a pleural air-fluid level. Chronic or persistent pulmonary coccidioidomycosis manifests with prolonged symptoms of fever, cough, and weight loss and is radio-graphically associated with pulmonary scarring, fibrosis, and cavities. It occurs most commonly in patients who already have chronic lung disease due to other etiologies.

1	In some cases, primary pneumonia presents as a diffuse reticulonodular pulmonary process (detected by plain chest radiography) in association with dyspnea and fever. Primary diffuse coccidioidal pneumonia may occur in settings of intense environmental exposure or profoundly suppressed cellular immunity (e.g., in patients with AIDS), with unrestrained fungal growth that is frequently associated with fungemia.

1	Clinical dissemination outside the thoracic cavity occurs in fewer than 1% of infected individuals. Dissemination is more likely to occur in male patients, particularly those of African-American or Filipino ancestry, and in persons with depressed cellular immunity, including patients with HIV infection and peripheral-blood CD4+ T cell counts of <250/μL; those receiving chronic glucocorticoid therapy; those with allogeneic solid-organ transplants; and those being treated with tumor necrosis factor α antagonists. Women who acquire infection during the second or third trimester of pregnancy also are at risk for disseminated disease. Common sites for dissemination include the skin, bone, joints, soft tissues, and meninges. Dissemination may follow symptomatic or asymptomatic pulmonary infection and may involve 1336 only one site or multiple anatomic foci. When it occurs, clinical dissemination is usually evident within the first few months after primary pulmonary infection. Coccidioidal

1	and may involve 1336 only one site or multiple anatomic foci. When it occurs, clinical dissemination is usually evident within the first few months after primary pulmonary infection. Coccidioidal meningitis, if untreated, is uniformly fatal. Patients usually present with a persistent headache, which is sometimes accompanied by lethargy and confusion. Nuchal rigidity, if present, is not severe. Examination of cerebrospinal fluid (CSF) demonstrates lymphocytic pleocytosis with profound hypoglycorrhachia and elevated protein levels. CSF eosinophilia is occasionally documented. With or without appropriate therapy, patients may develop hydrocephalus, which presents clinically as a marked decline in mental status, often with gait disturbances.

1	As mentioned above, coccidioidomycosis is often misdiagnosed as community-acquired bacterial pneumonia. Clues that suggest a diagnosis of coccidioidomycosis include peripheral-blood eosinophilia, hilar or mediastinal adenopathy on radiographic imaging, marked fatigue, and failure to improve with antibiotic therapy.

1	Serology plays an important role in establishing a diagnosis of coccidioidomycosis. Several techniques are available, including the traditional tube-precipitin (TP) and complement-fixation (CF) assays, immunodiffusion (IDTP and IDCF), and enzyme immunoassay (EIA) to detect IgM and IgG antibodies. TP and IgM antibodies are found in serum soon after infection and persist for weeks. They are not useful for gauging disease progression and are not found in the CSF. The CF and IgG antibodies occur later in the course of the disease and persist longer than TP and IgM antibodies. Rising CF titers are associated with clinical progression, and the presence of CF antibody in CSF is indicative of coccidioidal meningitis. Antibodies disappear over time in persons whose clinical illness resolves.

1	Because of its commercial availability, the coccidioidal EIA is frequently used as a screening tool for coccidioidal serology. There has been concern that the IgM EIA is occasionally falsely positive, particularly in asymptomatic individuals. In addition, while the sensitivity and specificity of the IgG EIA appear to be higher than those of the CF and IDCF assays, the optical density obtained in the EIA does not correlate with the serologic titer of either of the latter tests.

1	Coccidioides grows within 3–7 days at 37°C on a variety of artificial media, including blood agar. Therefore, it is always useful to obtain samples of sputum or other respiratory fluids and tissues for culture in suspected cases of coccidioidomycosis. The clinical laboratory should be alerted to the possibility of this diagnosis, since Coccidioides poses a significant laboratory hazard if it is inadvertently inhaled. The organism can also be identified directly. While treatment of samples with potassium hydroxide is rarely fruitful in establishing the diagnosis, examination of sputum or other respiratory fluids after Papanicolaou or Gomori methenamine silver staining reveals spherules in a significant proportion of patients with pulmonary coccidioidomycosis. For fixed tissues (e.g., those obtained from biopsy specimens), spherules with surrounding inflammation can be demonstrated with hematoxylineosin or Gomori methenamine silver staining.

1	A commercially available test for coccidioidal antigenuria and antigenemia has been developed and appears to be particularly useful in immunosuppressed patients with severe or disseminated disease. False-positive results may occur in cases of histoplasmosis or blastomycosis. Some laboratories offer genomic detection by polymerase chain reaction. Currently, two main classes of antifungal agents are useful for the treatment of coccidioidomycosis (Table 237-1). While once prescribed routinely, amphotericin B in all its formulations is now reserved for only the most severe cases of dissemination and for intrathecal or intraventricular administration to patients with coccidioidal meningitis in whom triazole antifungal therapy has failed. The original formulation of amphotericin B, which is dispersed with deoxycholate, is usually administered intravenously in doses

1	TABLE 237-1 CLInICAL PREsEnTATIOns OF COCCIdIOIdOMyCOsIs, THEIR FREquEnCy, And RECOMMEndEd InITIAL THERAPy FOR THE IMMunOCOMPETEnT HOsT Clinical Presentation Frequency, % Recommended Therapy Primary pneumonia 40 In most cases, nonea (focal) Cavity — In most cases, noneb Skin, bone, joint, soft — Prolonged triazole therapyc aTreatment is indicated for hosts with depressed cellular immunity as well as for those with prolonged symptoms and signs of increased severity, including night sweats for >3 weeks, weight loss of >10%, a complement-fixation titer of >1:16, and extensive pulmonary involvement on chest radiography. bTreatment (usually with the oral triazoles fluconazole and itraconazole) is recommended for persistent symptoms. cIn severe cases, some clinicians would use amphotericin B as initial therapy. dIntraventricular or intrathecal amphotericin B is recommended in cases of triazole failure. Hydrocephalus may occur, requiring a CSF shunt.

1	Note: See text for dosages and durations. of 0.7–1.0 mg/kg either daily or three times per week. The newer lipid-based formulations—amphotericin B lipid complex (ABLC), amphotericin B colloidal dispersion (ABCD), and amphotericin B liposomal complex (L-AmB)—are associated with less renal toxicity. The lipid dispersions are administered intravenously at doses of 5 mg/kg daily or three times per week.

1	Triazole antifungals are the principal drugs now used to treat most cases of coccidioidomycosis. Clinical trials have demonstrated the usefulness of both fluconazole and itraconazole. Evidence indicates that itraconazole may be more efficacious against bone and joint disease. Because of its demonstrated penetration into CSF, fluconazole is the azole of choice for the treatment of coccidioidal meningitis, but itraconazole also is effective. For both drugs, a minimal oral adult dosage of 400 mg/d should be used. The maximal dose of itraconazole is 200 mg three times daily, but higher doses of fluconazole may be given. Two newer triazole antifungals, posaconazole and voriconazole, are now available. Data suggest that both drugs may be useful against infections, including meningitis, in which prior fluconazole therapy has failed. High-dose triazole therapy may be teratogenic during the first trimester of pregnancy; thus, amphotericin B should be considered as therapy for

1	in which prior fluconazole therapy has failed. High-dose triazole therapy may be teratogenic during the first trimester of pregnancy; thus, amphotericin B should be considered as therapy for coccidioidomycosis in pregnant women during this period.

1	Most patients with focal primary pulmonary coccidioidomycosis require no therapy. Patients for whom antifungal therapy should be considered include those with underlying cellular immunodeficiencies and those with prolonged symptoms and signs of extensive disease. Specific criteria include symptoms persisting for ≥2 months, night sweats occurring for >3 weeks, weight loss of >10%, a serum CF antibody titer of >1:16, and extensive pulmonary involvement apparent on chest radiography. Diffuse pulmonary coccidioidomycosis represents a special situation. Because most patients with this form of disease are profoundly hypoxemic and critically ill, many clinicians favor beginning therapy with amphotericin B and switching to an oral triazole antifungal once clinical improvement occurs.

1	The nodules that may follow primary pulmonary coccidioidomycosis do not require treatment. As noted above, these nodules are not easily distinguished from pulmonary malignancies by means of radiographic imaging. Close clinical follow-up and biopsy may be required to distinguish between these two entities. Most pulmonary cavities do not require therapy. Antifungal treatment should be considered in patients with persistent cough, pleuritic chest pain, and hemoptysis. Occasionally, pulmonary coccidioidal cavities become secondarily infected. This development is usually manifested by an air-fluid level within the cavity. Bacterial flora or Aspergillus species are commonly involved, and therapy directed at these organisms should be considered. Surgery is rarely required except in cases of persistent hemoptysis or pyopneumothorax. For chronic pulmonary coccidioidomycosis, prolonged antifungal therapy—lasting for at least 1 year—is usually required, with monitoring of symptoms, radiographic

1	hemoptysis or pyopneumothorax. For chronic pulmonary coccidioidomycosis, prolonged antifungal therapy—lasting for at least 1 year—is usually required, with monitoring of symptoms, radiographic changes, sputum cultures, and serologic titers.

1	Most cases of disseminated coccidioidomycosis require prolonged antifungal therapy. Duration of treatment is based on resolution of the signs and symptoms of the lesions in conjunction with a significant decline in serum CF antibody titer. Such therapy routinely is continued for at least several years. Relapse occurs in 15–30% of individuals once therapy is discontinued.

1	Coccidioidal meningitis poses a special challenge. While most patients with this form of disease respond to treatment with oral triazoles, 80% experience relapse when therapy is stopped. Thus, lifelong therapy is recommended. In cases of triazole failure, intrathecal or intraventricular amphotericin B may be used. Installation requires considerable expertise and should be undertaken only by an experienced health care provider. Shunting of CSF in addition to appropriate antifungal therapy is required in cases of meningitis complicated by hydrocephalus. It is prudent to obtain expert consultation in all cases of coccidioidal meningitis.

1	There are no proven methods to reduce the risk of acquiring coccidioidomycosis among residents of an endemic region, but avoidance of direct contact with uncultivated soil or with visible dust containing soil is reasonable. For individuals with suppressed cellular immunity, the risk of developing symptomatic coccidioidomycosis is greater than that in the general population. Among those about to undergo allogeneic solid-organ transplantation, antifungal therapy is appropriate when there is evidence of active or recent coccidioidomycosis. Several cases of donor-transmitted coccidioidomycosis have occurred during transplantation. If possible, donors from an endemic region should be screened for coccidioidomycosis before transplantation. Data on the use of antifungal agents for prophylaxis in other situations are limited. The administration of an antifungal drug to prevent symptomatic coccidioidomycosis is not recommended for HIV-1-infected patients who live in an endemic region. Most

1	in other situations are limited. The administration of an antifungal drug to prevent symptomatic coccidioidomycosis is not recommended for HIV-1-infected patients who live in an endemic region. Most experts would administer a triazole to patients with a history of active coccidioidomycosis or a positive coccidioidal serology in whom therapy with tumor necrosis factor α antagonists is being initiated.

1	Blastomycosis Donna C. Sullivan, Rathel L. Nolan, III Blastomycosis is a systemic pyogranulomatous infection, involving pri-marily the lungs, that follows inhalation of the conidia of Blastomyces dermatitidis. Pulmonary blastomycosis varies from an asymptomatic infection to acute or chronic pneumonia. Hematogenous dissemina-238 tion to skin, bones, and the genitourinary system is common; however, almost any organ can be involved. B. dermatitidis is the asexual state of Ajellomyces dermatitidis. Two serotypes have been identified on the basis of the presence or absence of the A antigen. Distinct genotypic groups have been differentiated by rDNA polymerase chain reaction restriction fragment length FIGuRE 238-1 Blastomyces dermatitidis broad-based budding yeast in the aspirate of a chest wall abscess. Note the presence of multiple nuclei, the thickened cell wall, and the broad-based bud.

1	FIGuRE 238-1 Blastomyces dermatitidis broad-based budding yeast in the aspirate of a chest wall abscess. Note the presence of multiple nuclei, the thickened cell wall, and the broad-based bud. polymorphisms and microsatellite markers. B. dermatitidis exhibits thermal dimorphism, growing as the mycelial phase at room temperature and as the yeast phase at 37°C. Primary isolation in the laboratory is most dependable for the mycelial phase incubated at 30°C. Definitive identification usually requires conversion to the yeast phase at 37°C or— now more commonly—the use of nucleic acid amplification techniques that detect mycelial-phase growth. Under the microscope, the yeast cells are usually 8–15 μm in diameter, have thick refractile cell walls, are multinucleate, and exhibit a single, large, broad-based bud (Fig. 238-1).

1	Most cases of blastomycosis have been reported in North America. Endemic areas include the southeastern and south-central states bordering the Mississippi and Ohio river basins, the midwestern states, and the Canadian provinces bordering the Great Lakes. A small endemic area exists in New York and Canada along the St. Lawrence River. Acute blastomycosis is typically found only in North America, and the clinical presentation of blastomycosis in nonendemic areas is as a chronic disease. Outside North America, blastomycosis occurs sporadically in Nigeria, Zimbabwe, Tunisia, Saudi Arabia, Israel, Lebanon, and India. The disease has been reported most frequently in Africa.

1	Outside North America, blastomycosis occurs sporadically in Nigeria, Zimbabwe, Tunisia, Saudi Arabia, Israel, Lebanon, and India. The disease has been reported most frequently in Africa. Early studies indicated that middle-aged men with outdoor occupations were at greatest risk. Reported outbreaks, however, do not suggest a predilection according to sex, age, race, occupation, or season. The specific niche in nature in which the organism resides remains uncertain; B. dermatitidis probably grows as microfoci in the warm, moist soil of wooded areas rich in organic debris. Inhalation of conidia following exposure to soil, whether related to work or recreation, appears to be the common factor associated with infection. Outbreaks of human disease may be preceded by the occurrence of disease in simultaneously exposed dogs. Zoonotic transmission is rare but has been reported in association with dog bites, pet kinkajou bites, cat scratches, and animal necropsies.

1	Alveolar macrophages and polymorphonuclear leukocytes are critical for phagocytosis and killing of the inhaled conidia of B. dermatitidis. The interaction of these mediators of the innate immune response with local host factors, such as lung surfactant, plays a significant role in inhibiting conversion to the pathogenic yeast form. This inhibition prevents the establishment of symptomatic disease and may account for the high frequency of asymptomatic infections in outbreaks. Once conversion to the thick-walled yeast form has occurred, phagocytosis and killing are much more difficult, and the development of clinically 1338 apparent infection is much more likely. Ultimately, the T lymphocyte response—specifically, a TH1 response—is the primary factor in limiting infection and dissemination. Moreover, yeast-phase conversion results in the expression of yeast phase–specific proteins such as the 120-kDa glycoprotein adhesin BAD-1 and the Blastomyces yeast phase–specific protein 1 (BYS1).

1	Moreover, yeast-phase conversion results in the expression of yeast phase–specific proteins such as the 120-kDa glycoprotein adhesin BAD-1 and the Blastomyces yeast phase–specific protein 1 (BYS1). BAD-1 has been well characterized as a virulence factor and is the major epitope for humoral and cellular immunity. The role of BYS1, putatively identified as a signal peptide, has not been determined.

1	APPROACH TO THE PATIENT:

1	Blastomycosis most commonly presents as acute or chronic pneumonia that has been refractory to therapy with antibacterial drugs. Whether acute or chronic, blastomycosis may mimic many other disease processes. For example, acute pulmonary blastomycosis may present with signs and symptoms indistinguishable from those of bacterial pneumonia or influenza, and chronic pulmonary blastomycosis may mimic malignancy or tuberculosis. Skin lesions are often misdiagnosed as basal cell or squamous cell carcinoma, pyoderma gangrenosum, or keratoacanthoma. Laryngeal lesions are frequently mistaken for squamous cell carcinoma. Thus, the clinician must maintain a high index of suspicion and ensure that secretions or biopsy materials from patients who live in or have visited regions endemic for blastomycosis are subjected to careful histologic evaluation. This diligence is especially important in caring for individuals with pneumonia who fail to respond to treatment with antibacterial agents.

1	Acute pulmonary infection is often diagnosed in association with point-source outbreaks. Typical symptoms include the abrupt onset of fever, chills, pleuritic chest pain, arthralgias, and myalgias. Cough is initially nonproductive but frequently becomes purulent as disease progresses. Chest radiographs usually reveal alveolar infiltrates with consolidation. Pleural effusions and hilar adenopathy are uncommon. Most patients diagnosed with pulmonary blastomycosis have chronic indolent pneumonia with signs and symptoms of fever, weight loss, productive cough, and hemoptysis. The most common radiologic findings are alveolar infiltrates with or without cavitation, mass lesions that mimic bronchogenic carcinoma, and fibronodular infiltrates. Hematogenous dissemination to the skin, bones, and genitourinary tract occurs most often in association with chronic pulmonary disease. Although blastomycosis is not considered an opportunistic infection, immunosuppression has been recognized as a risk

1	tract occurs most often in association with chronic pulmonary disease. Although blastomycosis is not considered an opportunistic infection, immunosuppression has been recognized as a risk factor for more serious pulmonary involvement, including respiratory failure (adult respiratory distress syndrome) associated with miliary disease or diffuse pulmonary infiltrates. In the late stages of AIDS, mortality rates of ≥50% have been documented. Most deaths occur within the first few days of therapy. Solid-organ transplant recipients with endemic fungal infections, including both histoplasmosis and blastomycosis, frequently have more severe pulmonary disease as well as dissemination. Blastomycosis has been associated with a mortality rate of 36% in these patients.

1	In Africa, pulmonary cases typically include bony involvement (frequently of the vertebrae), with subcutaneous abscesses of the chest wall or legs. All of the manifestations seen in African patients fall within the spectrum of blastomycosis observed in North America. The increased prevalence of chronic and disseminated bone disease in these patients may reflect a delay in diagnosis in regions where spinal disease is often treated empirically as tuberculosis. Skin disease is the most common extrapulmonary manifestation of blastomycosis. Two types of skin lesions occur: verrucous (more common) and ulcerative. Osteomyelitis occurs in as many as one-fourth of B. dermatitidis infections. The vertebrae, pelvis, sacrum, skull, ribs, and long bones are most frequently involved. Patients with

1	B. dermatitidis osteomyelitis often present with contiguous soft-tissue abscesses or chronic draining sinuses. In men, blastomycosis may involve the prostate and epididymis. Central nervous system (CNS) disease occurs in fewer than 5% of immunocompetent patients with blastomycosis. A recent multicenter review identified 22 patients with CNS disease, of whom 12 (54%) met at least one criterion for immunosuppression; although most cases of CNS blastomycosis are associated with infection at other sites, 22.7% of the reviewed cases had only CNS involvement. CNS disease, usually presenting as a brain abscess, has been reported in ~40% of cases in patients with AIDS. Less common forms of CNS disease are cranial or spinal epidural abscess and meningitis.

1	Definitive diagnosis of blastomycosis requires growth of the organism from sputum, bronchial washings, pus, or biopsy material. Specimens should be inoculated onto a fungal medium such as Sabouraud dextrose agar, with or without chloramphenicol. B. dermatitidis is generally visible in 5–10 days but may require incubation for up to 30 days if only a few organisms are present in the specimen. A presumptive diagnosis may be based on demonstration of the characteristic broad-based budding yeast by microscopic examination of wet preps of sputum in pneumonia or of skin-lesion scrapings. Serologic testing for antibodies to B. dermatitidis by complement fixation, immunodiffusion, or enzyme immunoassay is of little value for diagnosis because of limited sensitivity and specificity as well as cross-reactivity with other fungal antigens.

1	A Blastomyces antigen assay that detects antigen in urine and serum is commercially available and is reasonably sensitive and specific (MiraVista Diagnostics, Indianapolis, IN). Antigen detection appears to be more sensitive in urine than in serum. This antigen test may be useful for monitoring of patients during therapy or for early detection of relapse. Chemiluminescent DNA probes (AccuProbe; GenProbe Inc., San Diego, CA) are commonly used to confirm identification of B. dermatitidis once growth has been detected in culture. Repetitive sequence–based PCR is available (DiversiLab System; bioMérieux, Durham, NC). Molecular identification techniques are currently used only to supplement traditional diagnostic methods.

1	The Infectious Diseases Society of America has published guidelines for the treatment of blastomycosis. Selection of an appropriate therapeutic regimen must be based on the clinical form and severity of the disease, the immune status of the patient, and the toxicity of the antifungal agent (Table 238-1). Although spontaneous cures of acute pulmonary infection are well documented, there are no criteria by which to distinguish patients whose disease will progress or resolve without treatment. Thus all patients with blastomycosis should be treated.

1	Itraconazole is the agent of choice for immunocompetent patients with mild to moderate pulmonary or non-CNS extrapulmonary disease. Therapy is continued for 6–12 months. Amphotericin B (AmB) is the preferred initial treatment for patients who are severely immunocompromised, who have life-threatening disease or CNS disease, or whose disease progresses during treatment with itraconazole. Although not rigorously studied, lipid formulations of AmB provide an alternative for patients who cannot tolerate AmB deoxycholate. Most patients with non-CNS disease whose clinical condition improves after an initial course of AmB (usually 2 weeks in duration) can be switched to itraconazole to complete 6–12 months of therapy. Fluconazole, because of its excellent penetration of the CNS, is useful in the treatment of patients with brain abscess or meningitis after an initial course of AmB.

1	Voriconazole has been used successfully to treat refractory blastomycosis, blastomycosis in immunosuppressed patients, and—given 1339TABLE 238-1 TREATMEnT OF BLAsTOMyCOsIsa Disease Primary Therapy Alternative Therapy Immunocompetent Patient/Life-Threatening Disease Pulmonary Lipid AmB, 3–5 mg/ kg qd, or AmB deoxycholate, 0.7–1.0 mg/kg qd (total dose: 1.5–2.5 g) Itraconazole, 200–400 mg/d (once patient's condition has stabilized) Cryptococcosis Arturo Casadevall DEFINITION AND ETIOLOGY Cryptococcus, a genus of yeast-like fungi, is the etiologic agent of cryptococcosis. Both species, C. neoformans and C. gattii, can cause cryptococcosis in humans. The two varieties of C. neoformans—grubii 239 dose: 1.5–2.5 g) aTherapy is generally given for 6–12 months. For bone and joint disease, a 12-month course is usually necessary. bSuppressive therapy with itraconazole may be considered for patients whose immunocompromised state continues. Fluconazole (800 mg/d) may be useful for patients who have

1	course is usually necessary. bSuppressive therapy with itraconazole may be considered for patients whose immunocompromised state continues. Fluconazole (800 mg/d) may be useful for patients who have CNS disease or cannot tolerate itraconazole.

1	Abbreviations: AmB, amphotericin B; CNS, central nervous system. its good penetration of cerebrospinal fluid—CNS disease. Posaconazole has also been used for refractory pulmonary disease. The echinocandins have variable activity against B. dermatitidis and therefore are not used in the treatment of blastomycosis. Cure rates are 90–95% among compliant immunocompetent patients given itraconazole for mild to moderate pulmonary and extrapulmonary disease without CNS involvement. Bone and joint disease usually requires 12 months of therapy. The fewer than 5% of infections that relapse after an initial course of itraconazole usually respond well to a second treatment course. The authors thank Dr. Stanley W. Chapman, Professor Emeritus, University of Mississippi, for his continued help and support and for his contributions to this chapter in an earlier edition. and neoformans—correlate with serotypes A and D, respectively.

1	and neoformans—correlate with serotypes A and D, respectively. C. gattii, although not divided into varieties, also is antigenically diverse, encompassing serotypes B and C. Most clinical microbiology laboratories do not routinely distinguish between C. neoformans and C. gattii, or among varieties, but rather identify and report all isolates simply as C. neoformans.

1	Cryptococcosis was first described in the 1890s but remained relatively rare until the mid-twentieth century, when advances in diagnosis and increases in the number of immunosuppressed individuals markedly raised its reported prevalence. Although serologic evidence of cryptococcal infection is common among immunocompetent individuals, cryptococcal disease (cryptococcosis) is relatively rare in the absence of impaired immunity. Individuals at high risk for disease due to C. neoformans include patients with hematologic malignancies, recipients of solid organ transplants who require ongoing immunosuppressive therapy, persons whose medical conditions necessitate glucocorticoid therapy, and patients with advanced HIV infection and CD4+ T lymphocyte counts of <200/μL. In contrast, C. gattii–related disease is not associated with specific immune deficits and often occurs in immunocompetent individuals. Cryptococcal infection is acquired from the environment.

1	Cryptococcal infection is acquired from the environment. C. neoformans and C. gattii inhabit different ecologic niches. C. neoformans is frequently found in soils contaminated with avian excreta and can easily be recovered from shaded and humid soils contaminated with pigeon droppings. In contrast, C. gattii is not found in bird feces. Instead, it inhabits a variety of arboreal species, including several types of eucalyptus tree. C. neoformans strains are found throughout the world; however, var. grubii (serotype A) strains are far more common than var. neoformans (serotype D) strains among both clinical and environmental isolates. The geographic distribution of C. gattii was thought to be largely limited to tropical regions until an outbreak of cryptococcosis caused by a new serotype B strain began in Vancouver in 1999. This outbreak has extended into the United States, and C. gattii infections are being encountered increasingly in several states in the Pacific Northwest.

1	The global burden of cryptococcosis was recently estimated at ~1 million cases, with >600,000 deaths annually. Thus cryptococci are important human pathogens. Since the onset of the HIV pandemic in the early 1980s, the overwhelming majority of cryptococcosis cases have occurred in patients with AIDS (Chap. 226). To comprehend the impact of HIV infection on the epidemiology of cryptococcosis, it is instructive to note that in the early 1990s there were >1000 cases of cryptococcal meningitis each year in New York City—a figure far exceeding that for all cases of bacterial meningitis. With the advent of effective antiretroviral therapy, the incidence of AIDS-related cryptococcosis has been sharply reduced among treated individuals. Thus most cases of cryptococcosis now occur in resource-limited regions of the world. The disease remains distressingly common in regions where antiretroviral therapy is not readily available (e.g., parts of Africa and Asia); in these regions, up to one-third

1	regions of the world. The disease remains distressingly common in regions where antiretroviral therapy is not readily available (e.g., parts of Africa and Asia); in these regions, up to one-third of patients with AIDS have cryptococcosis. Among HIV-infected persons, those with a decreased percentage of memory B cells expressing IgM may be at greater risk for cryptococcosis.

1	1340 PATHOGENESIS Cryptococcal infection is acquired by inhalation of aerosolized infectious particles. The exact nature of these particles is not known; the two leading candidate forms are small desiccated yeast cells and basidiospores. Little is known about the pathogenesis of initial infection. Serologic studies have shown that cryptococcal infection is acquired in childhood, but it is not known whether the initial infection is symptomatic. Given that cryptococcal infection is common while disease is rare, the consensus is that pulmonary defense mechanisms in immunologically intact individuals are highly effective at containing this fungus. It is not clear whether initial infection leads to a state of immunity or whether most individuals are subject throughout life to frequent and recurrent infections that resolve without clinical disease. However, evidence indicates that some human cryptococcal infections lead to a state of latency in which viable organisms are harbored for

1	recurrent infections that resolve without clinical disease. However, evidence indicates that some human cryptococcal infections lead to a state of latency in which viable organisms are harbored for prolonged periods, possibly in granulomas. Thus the inhalation of cryptococcal cells and/or spores can be followed by either clearance or establishment of the latent state. The consequences of prolonged harboring of cryptococcal cells in the lung are not known, but evidence from animal studies indicates that the organisms’ prolonged presence could alter the immunologic milieu in the lung and predispose to allergic airway disease. Cryptococcosis usually presents clinically as chronic meningoencephalitis. The mechanisms by which the fungus undergoes extrapulmonary dissemination and enters the central nervous system (CNS) remain poorly understood. The mechanism by which cryptococcal cells cross the blood–brain barrier is a subject of intensive study. Current evidence suggests that both direct

1	nervous system (CNS) remain poorly understood. The mechanism by which cryptococcal cells cross the blood–brain barrier is a subject of intensive study. Current evidence suggests that both direct fungal-cell migration across the endothelium and fungal-cell carriage inside macrophages as “Trojan horse” invaders can occur. Cryptococcus species have well-defined virulence factors that include the expression of the polysaccharide capsule, the ability to make melanin, and the elaboration of enzymes (e.g., phospholipase and urease) that enhance the survival of fungal cells in tissue. Among these virulence factors, the capsule and melanin production have been most extensively studied. The cryptococcal capsule is antiphagocytic, and the capsular polysaccharide has been associated with numerous deleterious effects on host immune function. Cryptococcal infections can elicit little or no tissue inflammatory response. The immune dysfunction seen in cryptococcosis has been attributed to the release

1	effects on host immune function. Cryptococcal infections can elicit little or no tissue inflammatory response. The immune dysfunction seen in cryptococcosis has been attributed to the release of copious amounts of capsular polysaccharide into tissues, where it probably interferes with local immune responses (Fig. 239-1). In clinical practice, the capsular polysaccharide is the antigen that is measured as a diagnostic marker of cryptococcal infection.

1	FIGuRE 239-1 Cryptococcal antigen in human brain tissue, as revealed by immunohistochemical staining. Brown areas show polysaccharide deposits in the midbrain of a patient who died of cryptococcal meningitis. (Reprinted with permission from SC Lee et al: Hum Pathol 27:839, 1996.) APPROACH TO THE PATIENT: Cryptococcosis should be included in the differential diagnosis when any patient presents with findings suggestive of chronic meningitis. Concern about cryptococcosis is heightened by a history of headache and neurologic symptoms in a patient with an underlying immunosuppressive disorder or state that is associated with an increased incidence of cryptococcosis, such as advanced HIV infection or solid organ transplantation.

1	The clinical manifestations of cryptococcosis reflect the site of fungal infection. The spectrum of disease caused by Cryptococcus species consists predominantly of meningoencephalitis and pneumonia, but skin and soft tissue infections also occur; in fact, cryptococcosis can affect any tissue or organ. CNS involvement usually presents as signs and symptoms of chronic meningitis, such as headache, fever, lethargy, sensory deficits, memory deficits, cranial nerve paresis, vision deficits, and meningismus. Cryptococcal meningitis differs from bacterial meningitis in that many Cryptococcus-infected patients present with symptoms of several weeks’ duration. In addition, classic characteristics of meningeal irritation, such as meningismus, may be absent in cryptococcal meningitis. Indolent cases can present as subacute dementia. Meningeal cryptococcosis can lead to sudden catastrophic vision loss.

1	Pulmonary cryptococcosis usually presents as cough, increased sputum production, and chest pain. Patients infected with C. gattii can present with granulomatous pulmonary masses known as cryptococcomas. Fever develops in a minority of cases. Like CNS disease, pulmonary cryptococcosis can follow an indolent course, and the majority of cases probably do not come to clinical attention. In fact, many cases are discovered incidentally during the workup of an abnormal chest radio-graph obtained for other diagnostic purposes. Pulmonary cryptococcosis can be associated with antecedent diseases such as malignancy, diabetes, and tuberculosis.

1	Skin lesions are common in patients with disseminated cryptococcosis and can be highly variable, including papules, plaques, purpura, vesicles, tumor-like lesions, and rashes. The spectrum of cryptococcosis in HIV-infected patients is so varied and has changed so much since the advent of antiretroviral therapy that a distinction between HIV-related and HIV-unrelated cryptococcosis is no longer pertinent. In patients with AIDS and solid organ transplant recipients, the lesions of cutaneous cryptococcosis often resemble those of molluscum contagiosum (Fig. 239-2; Chap. 220e).

1	A diagnosis of cryptococcosis requires the demonstration of yeast cells in normally sterile tissues. Visualization of the capsule of fungal cells in cerebrospinal fluid (CSF) mixed with India ink is a useful rapid diagnostic technique. Cryptococcal cells in India ink have a distinctive appearance because their capsules exclude ink particles. However, the CSF India ink examination may yield negative results in patients with a low fungal burden. This examination should be performed by a trained individual, since leukocytes and fat globules can sometimes be mistaken for fungal cells. Cultures of CSF and blood that are positive for cryptococcal cells are diagnostic for cryptococcosis. In cryptococcal meningitis, CSF examination usually reveals evidence of chronic meningitis with mononuclear cell pleocytosis and increased protein levels. A particularly useful test is cryptococcal antigen (CRAg) detection in CSF and blood. The assay is based on serologic detection of cryptococcal

1	cell pleocytosis and increased protein levels. A particularly useful test is cryptococcal antigen (CRAg) detection in CSF and blood. The assay is based on serologic detection of cryptococcal polysaccharide and is both sensitive and specific. A positive CRAg test provides strong presumptive evidence for cryptococcosis; however, because the result is often negative in pulmonary cryptococcosis, the test is less useful in the diagnosis of pulmonary disease and is of only limited usefulness in monitoring the response to therapy.

1	FIGuRE 239-2 Disseminated fungal infection. A liver transplant recipient developed six cutaneous lesions similar to the one shown. Biopsy and serum antigen testing demonstrated Cryptococcus. Important features of the lesion include a benign-appearing fleshy papule with central umbilication resembling molluscum contagio-sum. (Photo courtesy of Dr. Lindsey Baden; with permission.) In areas of Africa where there is a high prevalence of HIV infection, routine screening of blood for CRAg in HIV-infected patients with low CD4+ T lymphocyte counts may identify individuals at high risk of cryptococcal disease who are candidates for antifungal therapy. Similarly, CRAg screening has shown that a significant proportion of HIV-infected patients hospitalized with pneumonia in Thailand harbor cryptococcal infection. Inexpensive point-of-care CRAg tests that are under development could be of great diagnostic benefit in resource-limited regions.

1	Both the site of infection and the immune status of the host must be considered in the selection of therapy for cryptococcosis. The disease has two general patterns of manifestation: (1) pulmonary cryptococcosis, with no evidence of extrapulmonary dissemination; and (2) extrapulmonary (systemic) cryptococcosis, with or without meningoencephalitis. Pulmonary cryptococcosis in an immunocompetent host sometimes resolves without therapy. However, given the propensity of Cryptococcus species to disseminate from the lung, the inability to gauge the host’s immune status precisely, and the availability of low-toxicity therapy in the form of fluconazole, the current recommendation is for pulmonary cryptococcosis in an immunocompetent individual to be treated with fluconazole (200– 400 mg/d for 3–6 months). Extrapulmonary cryptococcosis without CNS involvement in an immunocompetent host can be treated with the same regimen, although amphotericin B (AmB; 0.5–1 mg/ kg daily for 4–6 weeks) may be

1	months). Extrapulmonary cryptococcosis without CNS involvement in an immunocompetent host can be treated with the same regimen, although amphotericin B (AmB; 0.5–1 mg/ kg daily for 4–6 weeks) may be required for more severe cases. In general, extrapulmonary cryptococcosis without CNS involvement requires less intensive therapy, with the caveat that morbidity and death in cryptococcosis are associated with meningeal involvement. Thus the decision to categorize cryptococcosis as “extrapulmonary without CNS involvement” should be made only after careful evaluation of the CSF reveals no evidence of cryptococcal infection. For CNS involvement in a host without AIDS or obvious immune impairment, most authorities recommend initial therapy with AmB (0.5–1 mg/kg daily) during an induction phase, which is followed by prolonged therapy with fluconazole (400 mg/d) during a consolidation phase. For cryptococcal meningoencephalitis without a concomitant immunosuppressive condition, the recommended

1	is followed by prolonged therapy with fluconazole (400 mg/d) during a consolidation phase. For cryptococcal meningoencephalitis without a concomitant immunosuppressive condition, the recommended regimen is AmB (0.5–1 mg/kg) plus flucytosine (100 mg/kg) daily for 6–10 weeks. Alternatively, patients can be treated with AmB (0.5–1 1341 mg/kg) plus flucytosine (100 mg/kg) daily for 2 weeks and then with fluconazole (400 mg/d) for at least 10 weeks. Patients with immunosuppression are treated with the same initial regimens except that consolidation therapy with fluconazole is given for a prolonged period to prevent relapse.

1	Cryptococcosis in patients with HIV infection always requires aggressive therapy and is considered incurable unless immune function improves. Consequently, therapy for cryptococcosis in the setting of AIDS has two phases: induction therapy (intended to reduce the fungal burden and alleviate symptoms) and lifelong maintenance therapy (to prevent a symptomatic clinical relapse). Pulmonary and extrapulmonary cryptococcosis without evidence of CNS involvement can be treated with fluconazole (200–400 mg/d). In patients who have more extensive disease, flucytosine (100 mg/kg per day) may be added to the fluconazole regimen for 10 weeks, with lifelong fluconazole maintenance therapy thereafter. For HIV-infected patients with evidence of CNS involvement, most authorities recommend induction therapy with AmB. An acceptable regimen is AmB (0.7–1 mg/kg) plus flucytosine (100 mg/kg) daily for 2 weeks followed by fluconazole (400 mg/d) for at least 10 weeks and then by lifelong maintenance therapy

1	with AmB. An acceptable regimen is AmB (0.7–1 mg/kg) plus flucytosine (100 mg/kg) daily for 2 weeks followed by fluconazole (400 mg/d) for at least 10 weeks and then by lifelong maintenance therapy with fluconazole (200 mg/d). Fluconazole (400–800 mg/d) plus flucytosine (100 mg/kg per day) for 6–10 weeks followed by fluconazole (200 mg/d) as maintenance therapy is an alternative. Newer triazoles like voriconazole and posaconazole are highly active against cryptococcal strains and appear effective clinically, but clinical experience with these agents in the treatment of cryptococcosis is limited. Lipid formulations of AmB can be substituted for AmB deoxycholate in patients with renal impairment. Neither caspofungin nor micafungin is effective against Cryptococcus species; consequently, neither drug has a role in the treatment of cryptococcosis. Cryptococcal meningoencephalitis is often associated with increased intracranial pressure, which is believed to be responsible for damage to

1	drug has a role in the treatment of cryptococcosis. Cryptococcal meningoencephalitis is often associated with increased intracranial pressure, which is believed to be responsible for damage to the brain and cranial nerves. Appropriate management of CNS cryptococcosis requires careful attention to the management of intracranial pressure, including the reduction of pressure by repeated therapeutic lumbar puncture and the placement of shunts. Recent studies suggest that the addition of a short course of interferon γ to antifungal therapy in patients with HIV infection increases clearance of cryptococci from the CSF.

1	In HIV-infected patients with previously treated cryptococcosis who are receiving fluconazole maintenance therapy, it may be possible to discontinue antifungal drug treatment if antiretroviral therapy results in immunologic improvement. However, certain recipients of maintenance therapy who have a history of successfully treated cryptococcosis can develop a troublesome immune reconstitution syndrome when antiretroviral therapy produces a rebound in immunologic function.

1	Even with antifungal therapy, cryptococcosis is associated with high rates of morbidity and death. For the majority of patients with cryptococcosis, the most important prognostic factors are the extent and the duration of the underlying immunologic deficits that predisposed them to develop the disease. Therefore, cryptococcosis is often curable with antifungal therapy in individuals with no apparent immunologic dysfunction, but, in patients with severe immunosuppression (e.g., those with AIDS), the best that can be hoped for is that antifungal therapy will induce remission, which can then be maintained with lifelong suppressive therapy. Before the advent of antiretroviral therapy, the median overall survival period for AIDS patients with cryptococcosis was <1 year. Cryptococcosis in patients with underlying neoplastic disease has a particularly poor prognosis. For CNS cryptococcosis, poor prognostic markers are a CSF assay positive for yeast cells on initial India ink examination

1	with underlying neoplastic disease has a particularly poor prognosis. For CNS cryptococcosis, poor prognostic markers are a CSF assay positive for yeast cells on initial India ink examination (evidence of a heavy fungal burden), high CSF pressure, low CSF glucose levels, low CSF pleocytosis (<2/μL), recovery of yeast cells from extraneural sites, absence of antibody to capsular 1342 polysaccharide, a CSF or serum cryptococcal antigen level of ≥1:32, and concomitant glucocorticoid therapy or hematologic malignancy. A response to treatment does not guarantee cure since relapse of cryptococcosis is common even among patients with relatively intact immune systems. Complications of CNS cryptococcosis include cranial nerve deficits, vision loss, and cognitive impairment.

1	No vaccine is available for cryptococcosis. In patients at high risk (e.g., those with advanced HIV infection and CD4+ T lymphocyte counts of <200/μL), primary prophylaxis with fluconazole (200 mg/d) is effective in reducing the prevalence of disease. Since antiretroviral therapy raises the CD4+ T lymphocyte count, it constitutes an immunologic form of prophylaxis. However, cryptococcosis in the setting of immune reconstitution has been reported in patients with HIV infection and in recipients of solid organ transplants. Candidiasis John E. Edwards, Jr. The genus Candida encompasses more than 150 species, only a few of which cause disease in humans. With rare exceptions (although the exceptions are increasing in number), the human pathogens are C. albicans, C. guilliermondii, C. krusei, C. parapsilosis, C. tropicalis, 240

1	C. kefyr, C. lusitaniae, C. dubliniensis, and C. glabrata. Ubiquitous in nature, they inhabit the gastrointestinal tract (including the mouth and oropharynx), the female genital tract, and the skin. Although cases of candidiasis have been described since antiquity in debilitated patients, the advent of Candida species as common human pathogens dates to the introduction of modern therapeutic approaches that suppress normal host defense mechanisms. Of these relatively recent advances, the most important is the use of antibacterial agents that alter the normal human microbiota and allow nonbacterial species to become more prevalent in the commensal flora. With the introduction of anti-fungal agents, the causes of Candida infections shifted from an almost complete dominance of C. albicans to the common involvement of

1	C. glabrata and the other species listed above. The non-albicans species now account for approximately half of all cases of candidemia and hematogenously disseminated candidiasis. Recognition of this change is clinically important, since the various species differ in susceptibility to the newer antifungal agents. In developed countries, where medical therapeutics are commonly used, Candida species are now among the most common nosocomial pathogens. Candida is a small, thin-walled, ovoid yeast that measures 4–6 μm in diameter and reproduces by budding. Organisms of this genus occur in three forms in tissue: blastospores, pseudohyphae, and hyphae. Candida grows readily on simple medium; lysis centrifugation enhances its recovery from blood. Species are identified by biochemical testing (currently with automated devices) or on special agar (e.g., CHROMagar).

1	Candida organisms are ubiquitous in nature; worldwide, these fungi are present in humans as commensals, in animals, in foods, and on inanimate objects. In developed countries, where advanced medical therapeutics are commonly used (see “Treatment,” below), Candida species are now among the most common health care–associated pathogens. In the United States, these species are the fourth most common isolates from the blood of hospitalized patients. In countries where advanced medical care is rarely available, mucocutaneous Candida infections, such as thrush, are more common than deep organ infections, which rarely occur; however, the incidence of deep organ candidiasis increases steadily as advances in health care—such as therapy with broad-spectrum antibiotics, more aggressive treatment of cancer, and the use of immunosuppression for sustaining organ transplants—are introduced and implemented. In recent decades, as a result of the HIV epidemic, the incidence of thrush and Candida

1	of cancer, and the use of immunosuppression for sustaining organ transplants—are introduced and implemented. In recent decades, as a result of the HIV epidemic, the incidence of thrush and Candida esophagitis has increased substantially. In aggregate, the global incidence of infections due to Candida species has risen steadily over the past few decades.

1	In the most serious form of Candida infection, the organisms disseminate hematogenously and form microabscesses and small macroabscesses in major organs. Although the exact mechanism is not known, Candida probably enters the bloodstream from mucosal surfaces after growing to large numbers as a consequence of bacterial suppression by antibacterial drugs; alternatively, in some instances, the organism may enter from the skin. A change from the blastospore stage to the pseudohyphal and hyphal stages is generally considered integral to the organism’s penetration into tissue. However, C. glabrata can cause extensive infection even though it does not transform into pseudohyphae or hyphae. Adherence to both epithelial and endothelial cells, thought to be the first step in invasion and infection, has been studied extensively, and several adhesins have been identified. Biofilm formation also is considered important in pathogenesis. Numerous reviews of cases of hematogenously disseminated

1	has been studied extensively, and several adhesins have been identified. Biofilm formation also is considered important in pathogenesis. Numerous reviews of cases of hematogenously disseminated candidiasis have identified the predisposing factors or conditions associated with disseminated disease (Table 240-1). Women who receive antibacterial agents may develop vaginal candidiasis.

1	Innate immunity is the most important defense mechanism against hematogenously disseminated candidiasis, and the neutrophil is the most important component of this defense. Macrophages also play an important defensive role. STAT1, Dectin-1, CARD9, and TH1 and TH17 lymphocytes contribute significantly to innate defense (see “Clinical Manifestations,” below). Although many immunocompetent individuals have antibodies to Candida, the role of these antibodies in defense against the organism is not clear. Multiple genetic polymorphisms that predispose to disseminated candidiasis will most likely be identified in future studies.

1	CLINICAL MANIFESTATIONS Mucocutaneous Candidiasis Thrush is characterized by white, adherent, painless, discrete or confluent patches in the mouth, on the tongue, or in the esophagus, occasionally with fissuring at the corners of the mouth. This form of disease caused by Candida can also occur at points of contact with dentures. Organisms are identifiable in gram-stained scrapings from lesions. The occurrence of thrush in a young, otherwise healthy-appearing person should prompt an investigation for underlying HIV infection. More commonly, thrush is seen as a nonspecific manifestation of severe debilitating illness. Vulvovaginal candidiasis is accompanied by pruritus, pain, and vaginal discharge which is usually thin but may contain whitish “curds” in severe cases. A subset of patients with recurrent vulvovaginitis have a deficiency in the surface expression of Dectin-1, a major recognition factor for β-glucan on Candida. This deficiency leads to suboptimal functioning of the CARD9

1	with recurrent vulvovaginitis have a deficiency in the surface expression of Dectin-1, a major recognition factor for β-glucan on Candida. This deficiency leads to suboptimal functioning of the CARD9 pathway, which ultimately increases the propensity for recurrent vaginal infections.

1	FIGuRE 240-1 Macronodular skin lesions associated with hematogenously disseminated candidiasis. Candida organisms are usually but not always visible on histopathologic examination. The fungi grow when a portion of the biopsied specimen is cultured. Therefore, for optimal identification, both histopathology and culture should be performed. (Image courtesy of Dr. Noah Craft and the Victor Newcomer collection at UCLA, archived by Logical Images, Inc.; with permission.)

1	Other Candida skin infections include paronychia, a painful swelling at the nail-skin interface; onychomycosis, a fungal nail infection rarely caused by this genus; intertrigo, an erythematous irritation with redness and pustules in the skin folds; balanitis, an erythematouspustular infection of the glans penis; erosio interdigitalis blastomycetica, an infection between the digits of the hands or toes; folliculitis, with pustules developing most frequently in the area of the beard; perianal candidiasis, a pruritic, erythematous, pustular infection surrounding the anus; and diaper rash, a common erythematous-pustular perineal infection in infants. Generalized disseminated cutaneous candidiasis, another form of infection that occurs primarily in infants, is characterized by widespread eruptions over the trunk, thorax, and extremities. The diagnostic macronodular lesions of hematogenously disseminated candidiasis (Fig. 240-1) indicate a high probability of dissemination to multiple

1	eruptions over the trunk, thorax, and extremities. The diagnostic macronodular lesions of hematogenously disseminated candidiasis (Fig. 240-1) indicate a high probability of dissemination to multiple organs as well as the skin. While the lesions are seen predominantly in immunocompromised patients treated with cytotoxic drugs, they may also develop in patients without neutropenia.

1	Chronic mucocutaneous candidiasis is a heterogeneous infection of the hair, nails, skin, and mucous membranes that persists despite intermittent therapy. The onset of disease usually comes in infancy or within the first two decades of life but in rare cases comes in later life. The condition may be mild and limited to a specific area of the skin or nails, or it may take a severely disfiguring form (Candida granuloma) characterized by exophytic outgrowths on the skin. Chronic mucocutaneous candidiasis is usually associated with specific immunologic dysfunction; most frequently reported is a failure of T lymphocytes to proliferate or to excrete cytokines in response to stimulation by Candida antigens in vitro. A subset of the affected patients have mutations in the STAT1 gene resulting in an insufficiency of interferon γ, interleukin 17, and interleukin 22.

1	Approximately half of patients with chronic mucocutaneous candidiasis have associated endocrine abnormalities that together are designated the autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) syndrome. This syndrome is due to mutations in the autoimmune regulator (AIRE) gene and is most prevalent among Finns, Iranian Jews, Sardinians, northern Italians, and Swedes. Conditions that usually follow the onset of the disease include hypoparathyroidism, adrenal insufficiency, autoimmune thyroiditis, Graves’ disease, chronic active hepatitis, alopecia, juvenile-onset pernicious anemia, malabsorption, and primary hypogonadism. In addition, dental enamel dysplasia, vitiligo, pitted nail dystrophy, and calcification of the tympanic membranes may occur. Patients with chronic mucocutaneous candidiasis rarely develop hematogenously 1343 disseminated candidiasis, probably because their neutrophil function remains intact.

1	Deeply Invasive Candidiasis Deeply invasive Candida infections may or may not be due to hematogenous seeding. Deep esophageal infection may result from penetration by organisms from superficial esophageal erosions; joint or deep wound infection from contiguous spread of organisms from the skin; kidney infection from catheter-initiated spread of organisms through the urinary tract; infection of intraabdominal organs and the peritoneum from perforation of the gastrointestinal tract; and gallbladder infection from retrograde migration of organisms from the gastrointestinal tract into the biliary drainage system.

1	However, far more commonly, deeply invasive candidiasis results from hematogenous seeding of various organs as a complication of candidemia. Once the organism gains access to the intravascular compartment (either from the gastrointestinal tract or, less often, from the skin through the site of an indwelling intravascular catheter), it may spread hematogenously to a variety of deep organs. The brain, chorioretina (Fig. 240-2), heart, and kidneys are most commonly infected and the liver and spleen less commonly so (most often in neutropenic patients). In fact, nearly any organ can become involved, including the endocrine glands, pancreas, heart valves (native or prosthetic), skeletal muscle, joints (native or prosthetic), bone, and meninges. Candida organisms can also spread hematogenously to the skin and cause classic macronodular lesions (Fig. 240-1). Frequently, painful muscular involvement also is evident beneath the area of affected skin. Chorioretinal involvement and skin

1	to the skin and cause classic macronodular lesions (Fig. 240-1). Frequently, painful muscular involvement also is evident beneath the area of affected skin. Chorioretinal involvement and skin involvement are highly significant, since both findings are associated with a very high probability of abscess formation in multiple deep organs as a result of generalized hematogenous seeding. Ocular involvement (Fig. 240-2) may require specific treatment (e.g., partial vitrectomy or intraocular injection of antifungal agents) to prevent permanent blindness. An ocular examination is indicated for all patients with candidemia, whether or not they have ocular manifestations.

1	The diagnosis of Candida infection is established by visualization of pseudohyphae or hyphae on wet mount (saline and 10% KOH), tissue Gram’s stain, periodic acid–Schiff stain, or methenamine silver stain in the presence of inflammation. Absence of organisms on hematoxylineosin staining does not reliably exclude Candida infection. The most

1	FIGuRE 240-2 Hematogenous Candida endophthalmitis. A classic off-white lesion projecting from the chorioretina into the vitreous causes the surrounding haze. The lesion is composed primarily of inflammatory cells rather than organisms. Lesions of this type may progress to cause extensive vitreal inflammation and eventual loss of the eye. Partial vitrectomy, combined with IV and possibly intravitreal antifungal therapy, may be helpful in controlling the lesions. (Image courtesy of Dr. Gary Holland; with permission.) challenging aspect of diagnosis is determining which patients with Candida isolates have hematogenously disseminated candidiasis. For instance, recovery of Candida from sputum, urine, or peritoneal catheters may indicate mere colonization rather than deep-seated infection, and Candida isolation from the blood of patients with indwelling intravascular catheters may reflect inconsequential seeding of the blood from or growth of the organisms on the catheter. Despite extensive

1	isolation from the blood of patients with indwelling intravascular catheters may reflect inconsequential seeding of the blood from or growth of the organisms on the catheter. Despite extensive research into both antigen and antibody detection systems, there is currently no widely available and validated diagnostic test to distinguish patients with inconsequential seeding of the blood from those whose positive blood cultures represent hematogenous dissemination to multiple organs. Many studies are under way to establish the utility of the β-glucan test; at present, its greatest utility is its negative predictive value (∼90%). Meanwhile, the presence of ocular or macronodular skin lesions is highly suggestive of widespread infection of multiple deep organs. Although extensive research is being conducted on other tests for infection, such as PCR, none of these tests is fully validated or widely available at present.

1	The treatment of mucocutaneous candidiasis is summarized in Table 240-2. All patients with candidemia are treated with a systemic antifungal agent. A certain percentage of patients, including many of those who have candidemia associated with an indwelling intravascular catheter, probably have “benign” candidemia rather than deep-organ seeding. However, because there is no reliable way to distinguish benign candidemia from deep-organ infection, and because antifungal drugs less toxic than amphotericin B are available, anti-fungal treatment for candidemia—with or without clinical evidence of deep-organ involvement—has become the standard of practice. In addition, if an indwelling intravascular catheter is present, it is best to remove or replace the device whenever feasible.

1	The drugs used for the treatment of candidemia and suspected disseminated candidiasis are listed in Table 240-3. Various lipid formulations of amphotericin B, three echinocandins, and the azoles fluconazole and voriconazole are used; no agent within a given class has been clearly identified as superior to the others. Most institutions choose an agent from each class on the basis of their own specific microbial epidemiology, strategies to minimize toxicities, and cost considerations. Unless azole resistance is considered likely, fluconazole is the agent of choice for the treatment of candidemia and suspected disseminated candidiasis in nonneutropenic, hemodynamically stable patients. Initial treatment in the context of likely azole resistance depends, as mentioned above, on the epidemiology of the individual hospital. For example, certain hospitals have a high rate of recovery of C. glabrata, while others do not. At institutions where non-albicans Candida species are frequently

1	of the individual hospital. For example, certain hospitals have a high rate of recovery of C. glabrata, while others do not. At institutions where non-albicans Candida species are frequently recovered, therapy with an echinocandin is typically started while the results of sensitivity testing are awaited. For hemodynamically unstable or neutropenic patients, initial treatment with broader-spectrum agents is desirable; these drugs include polyenes, echinocandins, or later-generation azoles such as voriconazole. Once the clinical response has been assessed and the pathogen specifically identified, the regimen can be altered accordingly. At present, the vast majority of C. albicans isolates are sensitive to fluconazole. Isolates of C. glabrata and C. krusei are less sensitive to fluconazole and more sensitive to polyenes and echinocandins. C. parapsilosis is less sensitive to echinocandins in vitro; however, this lesser sensitivity is considered nonsignificant.

1	Some generalizations exist regarding the management of specific Candida infections. Recovery of Candida from sputum is almost never indicative of underlying pulmonary candidiasis and does not by itself warrant antifungal treatment. Similarly, Candida in the urine of a patient with an indwelling bladder catheter may represent colonization only rather than bladder or kidney infection; however, the threshold for systemic treatment is lower in severely ill patients in

1	Agent Route of Administration Dosea Comment aFor loading doses and adjustments in renal failure, see Pappas PG et al: Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis 48:503, 2009. The recommended duration of therapy is 2 weeks beyond the last positive blood culture and the resolution of signs and symptoms of infection. bAlthough ketoconazole is approved for the treatment of disseminated candidiasis, it has been replaced by the newer agents listed in this table. Posaconazole has been approved for prophylaxis in neutropenic patients and for oropharyngeal candidiasis. FDA, U.S. Food and Drug Administration.

1	this category since it is impossible to distinguish colonization from 1345 lower or upper urinary tract infection. If the isolate is C. albicans, Aspergillosis most clinicians use oral fluconazole rather than a bladder washout with amphotericin B, which was more commonly used in the past. David W. Denning Caspofungin has been used with success; although echinocandins are poorly excreted into the urine, they may be an option, especially for non-albicans isolates. The doses and duration are the same as for disseminated candidiasis. The significance of the recovery of Candida from abdominal drains in postoperative patients is unclear, but again the threshold for treatment is generally low because most of the affected patients have been subjected to factors predisposing to disseminated candidiasis.

1	Removal of the infected valve and long-term antifungal therapy constitute appropriate treatment for Candida endocarditis. Although definitive studies are not available, patients usually are treated for weeks with a systemic antifungal agent (Table 240-2) and then given chronic suppressive therapy for months or years (sometimes indefinitely) with an oral azole (usually fluconazole at 400–800 mg/d).

1	Hematogenous Candida endophthalmitis is a special problem requiring ophthalmologic consultation. When lesions are expanding or are threatening the macula, an IV polyene combined with flucytosine (25 mg/kg four times daily) has been the regimen of choice, although comparative studies with other regimens have not yet been reported. As more data on the azoles and echinocandins become available, new strategies involving these agents are developing. Of paramount importance is the decision to perform a partial vitrectomy. This procedure debulks the infection and can preserve sight, which may otherwise be lost as a result of vitreal scarring. All patients with candidemia should undergo ophthalmologic examination because of the relatively high frequency of this ocular complication. Not only can this examination detect a developing eye lesion early in its course; in addition, identification of a lesion signifies a probability of ~90% of deep-organ abscesses and may prompt prolongation of

1	can this examination detect a developing eye lesion early in its course; in addition, identification of a lesion signifies a probability of ~90% of deep-organ abscesses and may prompt prolongation of therapy for candidemia beyond the recommended 2 weeks after the last positive blood culture. Although the basis for the consensus is a very small data set, the recommended treatment for Candida meningitis is a polyene (Table 240-3) plus flucytosine (25 mg/kg four times daily). Successful treatment of Candida-infected prosthetic material (e.g., an artificial joint) nearly always requires removal of the infected material followed by long-term administration of an antifungal agent selected on the basis of the isolate’s sensitivity and the logistics of administration.

1	The use of antifungal agents to prevent Candida infections has been controversial, but some general principles have emerged. Most centers administer prophylactic fluconazole (400 mg/d) to recipients of allogeneic stem cell transplants. High-risk liver transplant recipients also are given fluconazole prophylaxis in most centers. The use of prophylaxis for neutropenic patients has varied considerably from center to center; many centers that elect to give prophylaxis to this population use either fluconazole (200–400 mg/d) or a lipid formulation of amphotericin B (AmBiSome, 1–2 mg/d). Caspofungin (50 mg/d) also has been recommended. Some centers have used itraconazole suspension (200 mg/d). Posaconazole (200 mg three times daily) also has been approved by the FDA for prophylaxis in neutropenic patients and is gaining in popularity.

1	Prophylaxis is sometimes given to surgical patients at very high risk. The widespread use of prophylaxis for nearly all patients in general surgical or medical intensive care units is not—and should not be—a common practice for three reasons: (1) the incidence of disseminated candidiasis is relatively low, (2) the cost-benefit ratio is suboptimal, and (3) increased resistance with widespread prophylaxis is a valid concern. Prophylaxis for oropharyngeal or esophageal candidiasis in HIV-infected patients is not recommended unless there are frequent recurrences.

1	Aspergillosis is the collective term used to describe all disease entities caused by any one of ~50 pathogenic and allergenic species of Aspergillus. Only those species that grow at 37°C can cause invasive infection, although some species without this ability can cause allergic syndromes. A. fumigatus is responsible for most cases of invasive aspergillosis, almost all cases of chronic aspergillosis, and most allergic syndromes. A. flavus is more prevalent in some hospitals and causes a higher proportion of cases of sinus infections, cutaneous infections, and keratitis than A. fumigatus. A. niger can cause invasive infection but more commonly colonizes the respiratory tract and causes external otitis. A. terreus causes only invasive disease, usually with a poor prognosis. A. nidulans occasionally causes invasive infection, primarily in patients with chronic granulomatous disease.

1	Aspergillus has a worldwide distribution, most commonly growing in decomposing plant materials (i.e., compost) and in bedding. This hyaline (nonpigmented), septate, branching mold produces vast numbers of conidia (spores) on stalks above the surface of mycelial growth. Aspergilli are found in indoor and outdoor air, on surfaces, and in water from surface reservoirs. Daily exposures vary from a few to many millions of conidia; the latter high numbers of conidia are encountered in hay barns and other very dusty environments. The required size of the infecting inoculum is uncertain; however, only intense exposures (e.g., during construction work, handling of moldy bark or hay, or composting) are sufficient to cause disease in healthy immunocompetent individuals. Allergic syndromes may be exacerbated by continuous antigenic exposure arising from sinus or airway colonization or from nail infection. High-efficiency particulate air (HEPA) filtration is often protective against infection;

1	exacerbated by continuous antigenic exposure arising from sinus or airway colonization or from nail infection. High-efficiency particulate air (HEPA) filtration is often protective against infection; thus HEPA filters should be installed and monitored for efficiency in operating rooms and in areas of the hospital that house high-risk patients.

1	The incubation period of invasive aspergillosis after exposure is highly variable, extending in documented cases from 2 to 90 days. Thus community acquisition of an infecting strain frequently manifests as invasive infection during hospitalization, although nosocomial acquisition is also common. Outbreaks usually are directly related to a contaminated air source in the hospital.

1	mated (Table 241-1). However, given the inadequate diagnos tic capability in almost all lowand middle-income countries, the accuracy of these estimates is uncertain. The frequency of different manifestations of aspergillosis varies considerably with geographic location; most notably, chronic granulomatous sinusitis is rare outside the Middle East and India, and fungal keratitis is particularly common in Nepal, Myanmar, Bhutan, and India (800 and 113 cases/100,000 population, respectively). The potential effects of chronic pulmonary aspergillosis after pulmonary tuberculosis have only recently been appreciated and include life-threatening hemoptysis, misdiagnosis of smear-negative tuberculosis, and general exacerbation of posttuberculosis morbidity.

1	The primary risk factors for invasive aspergillosis are profound neutropenia and glucocorticoid use; risk increases with longer duration of these conditions. Higher doses of glucocorticoids increase the risk of both acquisition of invasive aspergillosis and death from the infection. Neutrophil and/or phagocyte dysfunction also is an important risk factor, as evidenced by aspergillosis in chronic granulomatous disease, advanced HIV infection, and relapsed leukemia. An increasing incidence of invasive aspergillosis in medical intensive care units suggests that, in patients who are not immunocompromised, temporary abrogation of protective responses as a result of glucocorticoid use or a general anti-inflammatory state is a significant risk factor. Many Type of Disease Culture ✓ ✓✓ Microscopy ✓ ?? Antigen ✓✓✓ ? ✓✓✓ Real-time PCR ✓✓✓ ✓✓✓ ✓✓✓ β-D-glucan ✓✓ ✓ ? Real-time PCR ✓✓ ??

1	Type of Disease Culture ✓ ✓✓ Microscopy ✓ ?? Antigen ✓✓✓ ? ✓✓✓ Real-time PCR ✓✓✓ ✓✓✓ ✓✓✓ β-D-glucan ✓✓ ✓ ? Real-time PCR ✓✓ ?? aIncidence and prevalence figures are for Europe. From www.ecdc.europa.eu/en/publications/ publications/risk-assessment-impact-environmental-usage-of-triazoles-on-aspergillus-sppresistance-to-medical-triazoles.pdf. bPeople are not born with allergic fungal disease; the annual frequency with which it occurs is not known. cAllergic bronchopulmonary aspergillosis and severe asthma with fungal sensitization. dGD Brown et al: Human fungal infections: the hidden killers. Sci Transl Med 2012:4:165rv13. eKey for sensitivity: 1 check = limited (as the text indicates, 10–30% for culture); 2 checks = higher; 3 checks = >80%; and 4 checks = ~95%. Abbreviation: PCR, polymerase chain reaction.

1	Abbreviation: PCR, polymerase chain reaction. patients have some evidence of prior pulmonary disease—typically, a history of pneumonia or chronic obstructive pulmonary disease. Therapy with infliximab, adalimumab, alemtuzumab, daclizumab, rituximab, and possibly bevacizumab therapy also carries an increased risk of invasive aspergillosis, as do severe liver disease and high levels of stored iron in bone marrow. Patients with chronic pulmonary aspergillosis have a wide spectrum of underlying pulmonary disease, including tuberculosis, prior pneumothorax, or chronic obstructive pulmonary disease. These patients are immunocompetent except for some cytokine regulation defects, most of which are consistent with an inability to mount an inflammatory immune (TH1-like) response or to control it adequately. Glucocorticoids accelerate disease progression.

1	Allergic bronchopulmonary aspergillosis (ABPA) is associated with polymorphisms of interleukin (IL) 4Ra, IL-10, and SPA2 genes (and others) and with heterozygosity of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. These associations suggest a strong genetic basis for the development of a TH2-like and “allergic” response to A. fumigatus. CD4+CD25+ T (Treg) cells also appear to be pivotal in determining disease phenotype. Remarkably, high-dose glucocorticoid treatment for exacerbations of ABPA almost never leads to invasive aspergillosis.

1	Invasive Pulmonary Aspergillosis Both the frequency of invasive disease and the pace of its progression increase with greater degrees of immunocompromise. Invasive aspergillosis is arbitrarily classified as acute and subacute, with courses of ≤1 month and 1–3 months, respectively. More than 80% of cases of invasive aspergillosis involve the lungs. The most common clinical features are no symptoms at all, fever, cough (sometimes productive), nondescript chest discomfort, trivial hemoptysis, and shortness of breath. Although the fever often responds to glucocorticoids, the disease progresses. The keys to early diagnosis in at-risk patients are a high index of suspicion, screening for circulating antigen (in leukemia), and urgent CT of the thorax. Invasive aspergillosis is one of the most common diagnostic errors revealed at autopsy.

1	Invasive Sinusitis The sinuses are involved in 5–10% of cases of invasive aspergillosis, especially affecting patients with leukemia and recipients of hematopoietic stem cell transplants. In addition to fever, the most common features are nasal or facial discomfort, blocked nose, and nasal discharge (sometimes bloody). Endoscopic examination of the nose reveals pale, dusky or necrotic-looking tissue in any location. CT or MRI of the sinuses is essential but does not distinguish invasive Aspergillus sinusitis from preexisting allergic or bacterial sinusitis early in the disease process.

1	Tracheobronchitis Occasionally, only the airways are infected by Aspergillus. The resulting manifestations range from acute or chronic bronchitis to ulcerative or pseudomembranous tracheobronchitis. These entities are particularly common among lung transplant recipients. Obstruction with mucous plugs occurs in normal individuals; in persons with ABPA, cystic fibrosis, and/or bronchiectasis; and in immunocompromised patients.

1	Disseminated Aspergillosis In the most severely immunocompromised patients, Aspergillus disseminates from the lungs to multiple organs— most often to the brain but also to the skin, thyroid, bone, kidney, liver, gastrointestinal tract, eye (endophthalmitis), and heart valve. Aside from cutaneous lesions, the most common features are gradual clinical deterioration over 1–3 days, with low-grade fever and features of mild sepsis, and nonspecific abnormalities in laboratory tests. In most cases, at least one localization becomes apparent before death. Blood cultures are almost always negative. Cerebral Aspergillosis Hematogenous dissemination to the brain is a devastating complication of invasive aspergillosis. Single or multiple lesions may develop. In acute disease, hemorrhagic infarction is most

1	Type of Disease typical, and cerebral abscess is common. Rarer manifestations include meningitis, mycotic aneurysm, and cerebral granuloma (mimicking a brain tumor). Local spread from cranial sinuses also occurs. Postoperative infection develops rarely and is exacerbated by glucocorticoids, often given after neurosurgery. The presentation can be either acute or subacute, with mood changes, focal signs, seizures, and decline in mental status. MRI is the most useful immediate investigation; unenhanced CT of the brain is usually nonspecific, and contrast is often contraindicated because of poor renal function.

1	Endocarditis Most cases of Aspergillus endocarditis are prosthetic valve infections resulting from contamination during surgery. Native valve disease is reported, especially as a feature of disseminated infection and in persons using illicit IV drugs. Culture-negative endocarditis with large vegetations is the most common presentation; embolectomy occasionally reveals the diagnosis. Cutaneous Aspergillosis Dissemination of Aspergillus occasionally results in cutaneous features, usually an erythematous or purplish nontender area that progresses to a necrotic eschar. Direct invasion of the skin occurs in neutropenic patients at the site of IV catheter insertion and in burn patients; such invasion may also follow trauma. Wounds may become infected with Aspergillus (especially A. flavus) after surgery.

1	Chronic Pulmonary Aspergillosis The hallmark of chronic cavitary pulmonary aspergillosis (also called semi-invasive aspergillosis, chronic necrotizing aspergillosis, or complex aspergilloma) (Fig. 241-1) is one or more pulmonary cavities expanding over a period of months or years in association with pulmonary symptoms and systemic manifestations such as fatigue and weight loss. (Pulmonary aspergillosis developing over <3 months is better classified as subacute invasive aspergillosis.) Often mistaken initially for tuberculosis, almost all cases occur in patients with prior pulmonary disease (e.g., tuberculosis, atypical mycobacterial infection, sarcoidosis, rheumatoid lung disease, pneumothorax, bullae) or lung surgery. The onset is insidious, and systemic features may be more prominent than pulmonary symptoms. Cavities may have a fluid level or a well-formed fungal ball, but pericavitary infiltrates and multiple cavities—with or without pleural thickening—are typical. An irregular

1	than pulmonary symptoms. Cavities may have a fluid level or a well-formed fungal ball, but pericavitary infiltrates and multiple cavities—with or without pleural thickening—are typical. An irregular internal cavity surface and thickened cavity walls are indicative of disease activity.

1	FIGuRE 241-1 CT scan image of the chest in a patient with longstanding bilateral chronic cavitary pulmonary aspergillosis. This patient had a history of several bilateral pneumothoraces and had required bilateral pleurodesis in 1990. CT scan then demonstrated multiple bullae, and sputum cultures grew A. fumigatus. The patient had initially weakly and later strongly positive serum Aspergillus antibody tests (precipitins). This scan (2003) shows a mixture of thickand thin-walled cavities in both lungs (each marked with C), with a probable fungal ball (black arrow) protruding into the large cavity on the patient's right side (R). There is also considerable pleural thickening bilaterally.

1	IgG antibodies (usually precipitating) to Aspergillus are almost always 1347 detectable in blood, and levels fall slowly with successful therapy. Some patients have concurrent infections—even without a fungal ball—with atypical mycobacteria and/or other bacterial pathogens. One or more Aspergillus nodules that resemble early lung carcinoma and may cavitate have been recognized. If untreated, chronic pulmonary aspergillosis typically progresses (sometimes relatively rapidly) to unilateral or upper-lobe fibrosis. This end-stage entity is termed chronic fibrosing pulmonary aspergillosis.

1	Aspergilloma Aspergilloma (fungal ball) occurs in up to 20% of residual pulmonary cavities ≥2.5 cm in diameter. Signs and symptoms associated with single (simple) aspergillomas are minor, including cough (sometimes productive), hemoptysis, wheezing, and mild fatigue. More significant signs and symptoms are associated with chronic cavitary pulmonary aspergillosis and should be treated as such. The vast majority of fungal balls are caused by A. fumigatus, but A. niger has been implicated, particularly in diabetic patients; aspergillomas due to A. niger can lead to oxalosis with renal dysfunction. The most significant complication of aspergilloma is life-threatening hemoptysis, which may be the presenting manifestation. Some fungal balls resolve spontaneously, but the cavity may still be infected.

1	Chronic Aspergillus Sinusitis Three entities are subsumed under this broad designation: fungal ball of the sinus, chronic invasive sinusitis, and chronic granulomatous sinusitis. Fungal ball of the sinus is limited to the maxillary sinus (except in rare cases involving the sphenoid sinus) and consists of a chronic saprophytic entity in which the sinus cavity is filled with a fungal ball. Maxillary disease is associated with prior upper-jaw root canal work and chronic (bacterial) sinusitis. About 90% of CT scans show focal hyperattenuation related to concretions; on MRI scans, the T2-weighted signal is decreased, whereas it is increased in bacterial sinusitis. Removal of the fungal ball is curative. No tissue invasion is demonstrable histologically or radiologically.

1	In contrast, chronic invasive sinusitis is a slowly destructive process that most commonly affects the ethmoid and sphenoid sinuses but can involve any sinus. Patients are usually but not always immunocompromised to some degree (e.g., as a result of diabetes or HIV infection). Imaging of the cranial sinuses shows opacification of one or more sinuses, local bone destruction, and invasion of local structures. The differential diagnosis is wide, including infections caused by numerous other fungi; sphenoid sinusitis is often caused by bacteria. Apart from a history of chronic nasal discharge and blockage, loss of the sense of smell, and persistent headache, the usual presenting features are related to local involvement of critical structures. The orbital apex syndrome (blindness and proptosis) is characteristic. Facial swelling, cavernous sinus thrombosis, carotid artery occlusion, pituitary fossa, and brain and skull base invasion have been described.

1	Chronic granulomatous sinusitis due to Aspergillus is most commonly seen in the Middle East and India and is often caused by A. flavus. It typically presents late, with facial swelling and unilateral proptosis. The prominent granulomatous reaction histologically distinguishes this disease from chronic invasive sinusitis, in which tissue necrosis with a low-grade mixed-cell infiltrate is typical. IgG antibodies to A. flavus are usually detectable.

1	Allergic Bronchopulmonary Aspergillosis In almost all cases, ABPA represents a hypersensitivity reaction to A. fumigatus; rare cases are due to other aspergilli and other fungi. ABPA occurs in ~2.5% of patients with asthma who are referred to secondary care and in up to 15% of adults with cystic fibrosis. Episodes of bronchial obstruction with mucous plugs leading to coughing fits, “pneumonia,” consolidation, and breathlessness are typical. Many patients report coughing up thick sputum casts. Eosinophilia commonly develops before systemic glucocorticoids are given. The cardinal diagnostic tests include an elevated serum level of total IgE (usually >1000 IU/mL), a positive skin-prick test in response to A. fumigatus extract, or detection of Aspergillus-specific IgE and IgG antibodies. The presence of hyperattenuated mucus in airways is highly specific. Central bronchiectasis is characteristic, and some patients develop chronic cavitary pulmonary aspergillosis.

1	1348 Severe Asthma with Fungal Sensitization Many adults with severe asthma do not fulfill the criteria for ABPA and yet are allergic to fungi. Although A. fumigatus is a common allergen, numerous other fungi (e.g., Cladosporium and Alternaria species) are implicated by skin-prick testing and/or specific IgE radioallergosorbent testing. Serum total IgE concentrations are <1000 IU/mL, and bronchiectasis is moderately common.

1	Allergic Sinusitis Like the lungs, the sinuses manifest allergic responses to Aspergillus and other fungi. The affected patients present with chronic (i.e., perennial) sinusitis that is relatively unresponsive to antibiotics. Many of these patients have nasal polyps, and all have congested nasal mucosae and sinuses full of mucoid material. The histologic hallmarks of allergic fungal sinusitis are local eosinophilia and Charcot-Leyden crystals. Removal of abnormal mucus and polyps, with local and occasionally systemic administration of glucocorticoids, usually leads to resolution. Persistent or recurrent signs and symptoms may require more extensive surgery (ethmoidectomy) and possibly local antifungal therapy. Recurrence is common, often after another bacterial or viral infection.

1	Superficial Aspergillosis Aspergillus can cause keratitis and otitis externa. The former may be difficult to diagnose early enough to save the patient’s sight. Treatment requires local surgical debridement as well as intensive topical antifungal therapy. Otitis externa usually resolves with debridement and local application of antifungal agents.

1	Several techniques are required to establish the diagnosis of any form of aspergillosis with confidence (Table 241-1). Patients with acute invasive aspergillosis have a relatively heavy load of fungus in the affected organ; thus culture, molecular diagnosis, antigen detection, and histopathology usually confirm the diagnosis. However, the pace of progression leaves only a narrow window for making the diagnosis without losing the patient, and some invasive procedures are not possible because of coagulopathy, respiratory compromise, and other factors. Currently, ~40% of cases of invasive aspergillosis are missed clinically and are diagnosed only at autopsy. Histologic examination of affected tissue reveals either infarction, with invasion of blood vessels by many fungal hyphae, or acute necrosis, with limited inflammation and fewer hyphae. Aspergillus hyphae are hyaline, narrow, and septate, with branching at 45°; no yeast forms are present in infected tissue. Hyphae can be seen in

1	necrosis, with limited inflammation and fewer hyphae. Aspergillus hyphae are hyaline, narrow, and septate, with branching at 45°; no yeast forms are present in infected tissue. Hyphae can be seen in cytology or microscopy preparations, which therefore provide a rapid means of presumptive diagnosis.

1	Culture is important in confirming the diagnosis, given that multiple other (rarer) fungi can mimic Aspergillus species histologically. Bacterial agar is less sensitive than fungal media for culture. Thus, if physicians do not request fungal culture, the diagnosis may be missed. Culture may be falsely positive (e.g., in patients whose airways are colonized by Aspergillus) or falsely negative. Only 10–30% of patients with invasive aspergillosis have a positive culture at any time. Both antigen detection and real-time polymerase chain reaction (PCR) are faster and much more sensitive than culture of respiratory samples and blood. The Aspergillus antigen test relies on detection of galactomannan release from Aspergillus organisms during growth. Positive serum antigen results usually precede clinical or radiologic features by several days. The sensitivity of antigen detection is reduced by antifungal prophylaxis and empirical therapy.

1	Definitive confirmation of the diagnosis requires (1) a positive culture of a sample taken directly from an ordinarily sterile site (e.g., a brain abscess) or (2) positive results of both histologic testing and culture of a sample taken from an affected organ (e.g., sinuses or skin). Most diagnoses of invasive aspergillosis are inferred from fewer data, including the presence of the halo sign on a high-resolution thoracic CT scan, in which a localized ground-glass appearance representing hemorrhagic infarction surrounds a nodule. While a halo sign may be produced by other fungi, Aspergillus species are by far the most common cause. Halo signs are present for ~7 days early in the course of infection in neutropenic patients and are a good prognostic feature, reflecting an early diagnosis. Thick CT sections can give the false appearance of a halo sign, as can other technical factors. Other common radiologic features of invasive pulmonary aspergillosis include nodules and pleural-based

1	Thick CT sections can give the false appearance of a halo sign, as can other technical factors. Other common radiologic features of invasive pulmonary aspergillosis include nodules and pleural-based infarction or cavitation, with pleural fluid apparent in 10% of patients.

1	For chronic invasive aspergillosis, Aspergillus antibody testing is invaluable although relatively imprecise. Biopsy of new nodules reveals hyphae surrounded by cells of chronic inflammation and sometimes granulomas. Antibody titers fall with successful therapy. Cultures are infrequently positive but are important in checking for azole resistance. Real-time PCR of sputum is often strongly positive. Some patients with chronic pulmonary aspergillosis also have elevated titers of total serum IgE and Aspergillus-specific IgE. ABPA and severe asthma with fungal sensitization are diagnosed serologically with elevated total and specific serum IgE levels and with skin-prick tests. Allergic Aspergillus sinusitis is usually diagnosed histologically, although precipitating antibodies in blood also may be useful.

1	Antifungal drugs active against Aspergillus include voriconazole, itraconazole, posaconazole, caspofungin, micafungin, and amphotericin B (AmB). Drug interactions with azoles must be considered before these agents are prescribed. In addition, plasma azole concentrations vary substantially from one patient to another, and many authorities recommend monitoring to ensure that drug concentrations are adequate but not excessive. Initial IV administration is preferred for acute invasive aspergillosis and oral administration for all other disease that requires antifungal therapy. Current recommendations are shown in Table 241-3.

1	Voriconazole is the preferred agent for invasive aspergillosis; caspofungin, posaconazole, and lipid-associated AmB are second-line agents. AmB is not active against A. terreus or A. nidulans. An infectious disease consultation is advised for patients with invasive disease, given the complexity of management. Combination therapy (voriconazole plus an echinocandin) for acute invasive aspergillosis may be beneficial for non-neutropenic patients. Immune reconstitution can complicate recovery. The duration of therapy for invasive aspergillosis varies from ~3 months to several years, depending on the patient’s immune status and response to therapy. Relapse occurs if the response is suboptimal and immune reconstitution is not complete.

1	Itraconazole is currently the preferred oral agent for chronic and allergic forms of aspergillosis. Voriconazole or posaconazole can be substituted when failure, emergence of resistance, or adverse events occur. An itraconazole dose of 200 mg twice daily is recommended, with monitoring of drug concentrations in the blood. Chronic cavitary pulmonary aspergillosis probably requires lifelong therapy, whereas the duration of treatment for other forms of chronic and allergic aspergillosis requires case-by-case evaluation.

1	Resistance to one or more azoles, although uncommon, is present in isolates from the environment in many regions, including northern Europe, India, China, and North America. Resistance may be derived from azole fungicide use for crops. In addition, resistance arising from multiple mechanisms may develop during long-term treatment, and a positive culture during antifungal therapy is an indication for susceptibility testing. Combined resistance to itraconazole and voriconazole is the most common type of cross-resistance. Glucocorticoids should be used in chronic cavitary pulmonary aspergillosis only if covered by adequate antifungal therapy. Surgical treatment is important in several forms of aspergillosis, including fungal ball of the sinus and single aspergillomas, in which surgery is curative; invasive aspergillosis involving bone, Prophylaxis Posaconazole, itracon-AI azole solution Chronic pulmonaryc Itraconazole, voricon-BII azole

1	Prophylaxis Posaconazole, itracon-AI azole solution Chronic pulmonaryc Itraconazole, voricon-BII azole ABPA/SAFS Itraconazole AI Drug interactions (especially with rifampin), renal failure (IV only) Diarrhea and vomiting with itraconazole, vincristine interaction Multicavity disease: poor outcome of surgery, medical therapy preferable Poor absorption of itraconazole capsules with proton pump inhibitors or H2 blockers Some glucocorticoid interactions, including with inhaled formulations AmB, caspofungin, posaconazole, micafungin Micafungin, aerosolized AmB Itraconazole, voriconazole, intracavity AmB Posaconazole, IV AmB, IV micafungin Voriconazole, posaconazole As primary therapy, voriconazole carries 20% more responses than AmB. Consider initial combination therapy with an echinocandin in nonneutropenic patients. Some centers monitor plasma levels of itraconazole and posaconazole. Single large cavities with an aspergilloma are best resected.

1	Some centers monitor plasma levels of itraconazole and posaconazole. Single large cavities with an aspergilloma are best resected. Resistance may emerge during treatment, especially if plasma drug levels are subtherapeutic. Long-term therapy is helpful in most cases. No evidence indicates whether therapy modifies progression to bronchiectasis/fibrosis. aFor information on duration of therapy, see text. bEvidence levels are those used in treatment guidelines (TJ Walsh et al: Treatment of aspergillosis: Clinical practice guidelines of the Infectious Diseases Society of America [IDSA]. Clin Infect Dis 46:327, 2008). cAn infectious disease consultation is appropriate for these patients.

1	Note: The oral dose is usually 200 mg bid for voriconazole and itraconazole and 400 mg bid for posaconazole suspension. The IV dose of voriconazole for adults is 6 mg/kg twice at 12-h intervals (loading doses) followed by 4 mg/kg q12h; a larger dose is required for children and teenagers. Plasma monitoring is helpful in optimizing the dosage. Caspofungin is given as a single loading dose of 70 mg and then at 50 mg/d; some authorities use 70 mg/d for patients weighing >80 kg, and lower doses are required with hepatic dysfunction. Micafungin is given as 50 mg/d for prophylaxis and as at least 150 mg/d for treatment; this drug has not yet been approved by the U.S. Food and Drug Administration (FDA) for this indication. AmB deoxycholate is given at a daily dose of 1 mg/kg if tolerated. Several strategies are available for minimizing renal dysfunction. Lipid-associated AmB is given at 3 mg/kg (AmBisome) or 5 mg/kg (Abelcet). Different regimens are available for aerosolized AmB, but none is

1	strategies are available for minimizing renal dysfunction. Lipid-associated AmB is given at 3 mg/kg (AmBisome) or 5 mg/kg (Abelcet). Different regimens are available for aerosolized AmB, but none is FDA approved. Other considerations that may alter dose selection or route include age; concomitant medications; renal, hepatic, or intestinal dysfunction; and drug tolerability.

1	Abbreviations: AmB, amphotericin B; ABPA, allergic bronchopulmonary aspergillosis; SAFS, severe asthma with fungal sensitization. heart valve, sinuses, and proximal areas of the lung; brain abscess; keratitis; and endophthalmitis. In allergic fungal sinusitis, removal of abnormal mucus and polyps, with local and occasionally systemic glucocorticoid treatment, usually leads to resolution. Persistent or recurrent signs and symptoms may require more extensive surgery (ethmoidectomy) and possibly local antifungal therapy. Surgery is problematic in chronic pulmonary aspergillosis, usually resulting in serious complications. Bronchial artery embolization is preferred for problematic hemoptysis.

1	In situations in which moderate or high risk is predicted (e.g., after induction therapy for acute myeloid leukemia), the need for antifungal prophylaxis for superficial and systemic candidiasis and for invasive aspergillosis is generally accepted. Fluconazole is commonly used in these situations but has no activity against Aspergillus species. Itraconazole capsules are ineffective, and itraconazole solution offers only modest efficacy. Posaconazole solution is more effective. Some data support the use of IV micafungin. No prophylactic regimen is completely successful.

1	Invasive aspergillosis is curable if immune reconstitution occurs, whereas allergic and chronic forms are not. The mortality rate for invasive aspergillosis is ~50% if the infection is treated but is 100% if the diagnosis is missed. Infection with a voriconazole-resistant strain carries a mortality rate of 88%. Cerebral aspergillosis, Aspergillus endocarditis, and bilateral extensive invasive pulmonary aspergillosis have very poor outcomes, as does invasive infection in persons with late-stage AIDS or relapsed uncontrolled leukemia and in recipients of allogeneic hematopoietic stem cell transplants. Deterioration with no therapy over 12 months Natural history FIGuRE 241-2 Comparison of the impact of itraconazole therapy (400 mg/d) and standard care on chronic cavitary pulmonary aspergillosis at 6 and 12 months. (After R Agarwal et al: Itraconazole in chronic cavitary pulmonary aspergillosis: a randomised controlled trial and systematic review of literature. Mycoses 56:559, 2013.)

1	The mortality rate for chronic cavitary pulmonary aspergillosis is ~30% 6 months after presentation, falling to ~15% annually thereafter. After 12 months with no antifungal therapy, 70% of patients have deteriorated and 30% are stable (Fig. 241-2). Therapy fails in ~30% of recipients of antifungal therapy and still more often if azole resistance is present. Both ABPA and SAFS patients respond to antifungal therapy; ~60% respond to itraconazole and ~80% to voriconazole and posaconazole (if tolerated). If the severity of asthma declines, the inhaled glucocorticoid dose can be reduced and oral glucocorticoids can be stopped. Brad Spellberg, Ashraf S. Ibrahim

1	Brad Spellberg, Ashraf S. Ibrahim Mucormycosis represents a group of life-threatening infections caused by fungi of the order Mucorales of the subphylum Mucoromycotina (formerly known as the class Zygomycetes). Infection caused by the Mucorales is most accurately referred to as mucormycosis, although the term zygomycosis may still be used by some sources. Mucormycosis is highly invasive and relentlessly progressive, resulting in higher rates of morbidity and mortality than many other infections. However, recent studies have suggested that mortality rates from mucormycosis have declined with newer therapies. A high index of suspicion is critical for diagnosis, and early initiation of therapy—often before confirmation of the diagnosis—is necessary to optimize outcomes. Fungi of the order Mucorales belong to seven medically relevant families (Table 242-1), all of which can cause mucormycosis.

1	Among the Mucorales, Rhizopus oryzae (in the family Mucoraceae) is by far the most common cause of infection in the Western Hemisphere. Less frequently isolated species of the Mucoraceae that cause a similar spectrum of infections include Rhizopus microsporus, Rhizomucor pusillus, Lichtheimia corymbifera (formerly Absidia corymbifera), Apophysomyces elegans, and Mucor species (which, despite its name, only rarely causes mucormycosis). Increasing numbers of cases of mucormycosis due to infection with Cunninghamella species (family Cunninghamellaceae) have also been reported, particularly in highly immunocompromised patients. Rare case reports have demonstrated the ability of fungi in the remaining families of the Mucorales to cause mucormycosis, although other Mucorales can be the major cause of disease in certain geographic areas (e.g., A. elegans in India and Mucor irregularis in China).

1	The Mucorales are ubiquitous environmental fungi to which humans are constantly exposed. These fungi cause infection primarily in patients with diabetes or defects in phagocytic function (e.g., those associated with neutropenia or glucocorticoid treatment). Patients with elevated levels of free iron, which supports fungal growth in serum and tissues, are likewise at increased risk for mucormycosis. In iron-overloaded patients with end-stage renal failure, treatment with deferoxamine predisposes to the development of rapidly fatal disseminated mucormycosis; this agent, an iron chelator for the human host, serves as a fungal siderophore, directly delivering iron to the Mucorales. Furthermore, patients with diabetic ketoacidosis (DKA) are at high risk of developing rhinocerebral mucormycosis. The acidosis causes dissociation of iron from sequestering proteins in serum, TABLE 242-1 TAxOnOMy OF FungI CAusIng MuCORMyCOsIs (suBPHyLuM MuCOROMyCOTInA, ORdER MuCORALEs)

1	TABLE 242-1 TAxOnOMy OF FungI CAusIng MuCORMyCOsIs (suBPHyLuM MuCOROMyCOTInA, ORdER MuCORALEs) Lichtheimiaceae Lichtheimia (formerly Mycocladus, formerly Absidia) Cunninghamellaceae Cunninghamella Thamnidiaceae Cokeromyces Mortierellaceae Mortierella Saksenaceae Saksenaea Apophysomyces Syncephalastraceae Syncephalastrum resulting in enhanced fungal survival and virulence. Nevertheless, the majority of diabetic patients who present with mucormycosis are not acidotic, and, even absent acidosis, hyperglycemia directly contributes to the risk of mucormycosis by at least three likely mechanisms: (1) hyperglycation of iron-sequestering proteins, disrupting normal iron sequestration; (2) upregulation of a mammalian cell receptor (GRP78) that binds to Mucorales, enabling tissue penetration (due to both a direct effect of hyperglycemia and increasing levels of free iron, which independently enhances GRP78 expression); and (3) induction of poorly characterized defects in phagocytic function.

1	Mucormycosis typically occurs in patients with diabetes mellitus, solid organ or hematopoietic stem cell transplantation (HSCT), prolonged neutropenia, or malignancy. The majority of diabetic patients are not acidotic on presentation with mucormycosis. Furthermore, patients often have no previously recognized history of diabetes mellitus when they present with mucormycosis. In these instances, presentation for mucormycosis may result in the first clinical recognition of hyperglycemia, which may have been unmasked by recent glucocorticoid use. Thus a high index of suspicion of mucormycosis must be maintained, even in the absence of a known history of diabetes, if hyperglycemia is present. In patients undergoing HSCT, mucormycosis develops at least as commonly during nonneutropenic as during neutropenic periods, probably because of glucocorticoid treatment of graft-versus-host disease. Mucormycosis can occur as isolated cutaneous or subcutaneous infection in immunologically normal

1	during neutropenic periods, probably because of glucocorticoid treatment of graft-versus-host disease. Mucormycosis can occur as isolated cutaneous or subcutaneous infection in immunologically normal individuals after traumatic implantation of soil or vegetation (e.g., due to natural disasters or motor vehicle accidents) or in nosocomial settings via direct access through IV catheters, SC injections, or maceration of the skin by a moist dressing.

1	Patients receiving antifungal prophylaxis with either itraconazole or voriconazole may be at increased risk of mucormycosis. These patients typically present with disseminated mucormycosis, the most lethal form of disease. Breakthrough mucormycosis also has been described in patients receiving posaconazole or echinocandin prophylaxis. Mucormycosis can be divided into at least six clinical categories based on clinical presentation and the involvement of a particular anatomic site: rhino-orbital-cerebral, pulmonary, cutaneous, gastrointestinal, disseminated, and miscellaneous. These categories of invasive mucormycosis tend to affect patients with specific defects in host defense. For example, patients with DKA typically develop the rhino-orbital-cerebral form and much more rarely develop pulmonary or disseminated disease. In contrast, pulmonary mucormycosis occurs most commonly in leukemic patients who are receiving chemotherapy and in patients undergoing HSCT.

1	Rhino-Orbital-Cerebral Disease Rhino-orbital-cerebral mucormycosis continues to be the most common form of the disease. Most cases occur in patients with diabetes, although such cases (probably due to glucocorticoid use) are increasingly being described in the transplantation setting, often along with glucocorticoid-induced diabetes mellitus. The initial symptoms of rhino-orbital-cerebral mucormycosis are nonspecific and include eye or facial pain and facial numbness followed by the onset of conjunctival suffusion and blurry vision. Fever may be absent in up to half of cases. White blood cell counts are typically elevated as long as the patient has functioning bone marrow. If untreated, infection usually spreads from the ethmoid sinus to the orbit, resulting in compromise of extraocular muscle function and proptosis, typically with chemosis. Onset of signs and symptoms in the contralateral eye, with resulting bilateral proptosis, chemosis, vision loss, and ophthalmoplegia, is ominous,

1	function and proptosis, typically with chemosis. Onset of signs and symptoms in the contralateral eye, with resulting bilateral proptosis, chemosis, vision loss, and ophthalmoplegia, is ominous, suggesting the development of cavernous sinus thrombosis.

1	Upon visual inspection, infected tissue may appear to be normal during the earliest stages of fungal spread, then progressing through an erythematous phase, with or without edema, before the onset of a violaceous appearance and finally the development of a black necrotic eschar. Infection can sometimes extend from the sinuses into the mouth and produce painful necrotic ulcerations of the hard palate, but this is a late finding that suggests extensive, well-established infection.

1	Pulmonary Disease Pulmonary mucormycosis is the second most common manifestation. Symptoms include dyspnea, cough, and chest pain; fever is often but not invariably present. Angioinvasion results in necrosis, cavitation, and/or hemoptysis. Lobar consolidation, isolated masses, nodular disease, cavities, or wedge-shaped infarcts may be seen on chest radiography. High-resolution chest CT is the best method for determining the extent of pulmonary mucormycosis and may demonstrate evidence of infection before it is seen on chest x-ray. In the setting of cancer, where mucormycosis may be difficult to differentiate from aspergillosis, the presence of ≥10 pulmonary nodules, pleural effusion, or concomitant sinusitis makes mucormycosis more likely. It is critical to distinguish mucormycosis from aspergillosis as rapidly as possible because treatments for these infections differ. Indeed, voriconazole— the first-line treatment for aspergillosis—exacerbates mucormycosis in mouse and fly models of

1	as rapidly as possible because treatments for these infections differ. Indeed, voriconazole— the first-line treatment for aspergillosis—exacerbates mucormycosis in mouse and fly models of infection.

1	Cutaneous Disease Cutaneous mucormycosis may result from external implantation of the fungus or conversely from hematogenous dissemination. External implantation–related infection has been described in the setting of soil exposure from trauma (e.g., in a motor vehicle accident or natural disaster), penetrating injury with plant material (e.g., a thorn), injections of medications (e.g., insulin), catheter insertion, contamination of surgical dressings, and use of tape to secure endotracheal tubes. Cutaneous disease can be highly invasive, penetrating into muscle, fascia, and even bone. In mucormycosis, necrotizing fasciitis carries a mortality rate approaching 80%. Necrotic cutaneous lesions in the setting of hematogenous dissemination also are associated with an extremely high mortality rate. However, with prompt, aggressive surgical debridement, isolated cutaneous mucormycosis has a favorable prognosis and a low mortality rate.

1	Gastrointestinal Disease In the past, gastrointestinal mucormycosis occurred primarily in premature neonates in association with disseminated disease and necrotizing enterocolitis. However, there has been a marked increase in case reports describing adults with neutropenia or other immunocompromising conditions. In addition, gastrointestinal disease has been reported as a nosocomial process following administration of medications mixed with contaminated wooden applicator sticks. Nonspecific abdominal pain and distention associated with nausea and vomiting are the most common symptoms. Gastrointestinal bleeding is common, and fungating masses may be seen in the stomach at endoscopy. The disease may progress to visceral perforation, with extremely high mortality rates.

1	Disseminated and Miscellaneous Forms of Disease Hematogenously disseminated mucormycosis may originate from any primary site of infection. The most common site of dissemination is the brain, but metastatic lesions may also be found in any other organ. The mortality rate associated with dissemination to the brain approaches 100%. Even without central nervous system (CNS) involvement, mortality rates for disseminated mucormycosis exceed 90%. Miscellaneous forms of mucormycosis may affect any body site, including bones, mediastinum, trachea, kidneys, and (in association with dialysis) peritoneum.

1	A high index of suspicion is required for diagnosis of mucormycosis. Unfortunately, autopsy series have shown that up to half of cases are diagnosed only post-mortem. Because the Mucorales are environmental isolates, definitive diagnosis requires a positive culture from a sterile site (e.g., a needle aspirate, a tissue biopsy specimen, or pleural fluid) or histopathologic evidence of invasive mucormycosis. A probable diagnosis of mucormycosis can be established by culture FIGuRE 242-1 Histopathology sections of Rhizopus oryzae in infected brain. A. Broad, ribbon-like, nonseptate hyphae in the parenchyma (arrows) and a thrombosed blood vessel with extensive intravascular hyphae (arrowhead) (hematoxylin and eosin). B. Extensive, broad, ribbon-like hyphae invading the parenchyma (Gomori methenamine silver).

1	from a nonsterile site (e.g., sputum or bronchoalveolar lavage) when a patient has appropriate risk factors as well as clinical and radiographic evidence of disease. However, given the urgency of administering therapy early, the patient should be treated while confirmation of the diagnosis is awaited.

1	Biopsy with histopathologic examination remains the most sensitive and specific modality for definitive diagnosis (Fig. 242-1). Biopsy reveals characteristic wide (≥6to 30-μm), thick-walled, ribbon-like, aseptate hyphal elements that branch at right angles. Other fungi, including Aspergillus, Fusarium, and Scedosporium species, have septa, are thinner, and branch at acute angles. Because artificial septa may result from folding of tissue during processing (which may also alter the appearance of the angle of branching), the width and the ribbon-like form of the fungus are the most reliable features distinguishing mucormycosis. The Mucorales are visualized most effectively with periodic acid–Schiff or methenamine silver stain or, if the organism burden is high, with hematoxylin and eosin. While histopathology can identify the Mucorales, specific species can be identified only by culture. Polymerase chain reaction (PCR) is being investigated as a diagnostic tool for mucormycosis but is

1	While histopathology can identify the Mucorales, specific species can be identified only by culture. Polymerase chain reaction (PCR) is being investigated as a diagnostic tool for mucormycosis but is not yet approved by the U.S. Food and Drug Administration (FDA) for this purpose and is not generally available.

1	Unfortunately, cultures are positive in fewer than half of cases of mucormycosis. Nevertheless, the Mucorales are not fastidious organisms and tend to grow quickly (i.e., within 48 h) on culture media. The likely explanation for the low sensitivity of culture is that the Mucorales form long filamentous structures that are killed by tissue homogenization—the standard method for preparing tissue cultures in the clinical microbiology laboratory. Thus the laboratory should be advised when a diagnosis of mucormycosis is suspected, and the tissue should be cut into sections and placed in the center of culture dishes rather than homogenized. There is also substantial variability among isolates in optimal growth temperature, so growth at both room temperature and 37°C is advisable.

1	Imaging techniques often yield subtle findings that underestimate the extent of disease. For example, the most common finding on CT or MRI of the head or sinuses of a patient with rhino-orbital mucormycosis is sinusitis that is indistinguishable from bacterial sinusitis. It is also common to detect no abnormalities in sinus bones despite clinical evidence of progressive disease. MRI is more sensitive (~80%) for detecting orbital and CNS disease than is CT. High-risk patients should always undergo endoscopy and/or surgical exploration, with biopsy of the areas of suspected infection. If mucormycosis is suspected, initial empirical therapy with a polyene antifungal agent should be initiated while the diagnosis is being confirmed.

1	Other mold infections, including aspergillosis, scedosporiosis, fusariosis, and infections caused by the dematiaceous fungi (brown-pigmented 1352 soil organisms), can cause clinical syndromes identical to mucormycosis. Histopathologic examination usually allows distinction of the Mucorales from these other organisms, and a positive culture permits definitive species identification. As stated above, it is important to distinguish the Mucorales from these other fungi, as the preferred antifungal treatments differ (i.e., polyenes for the Mucorales vs. expanded-spectrum triazoles for most septate molds). The entomophthoromycoses caused by Basidiobolus and Conidiobolus also can cause identical clinical syndromes. These fungi may appear similar to the Mucorales on histopathology and can be reliably distinguished from the latter only by culture. In a patient with sinusitis and proptosis, orbital cellulitis and cavernous sinus thrombosis caused by bacterial pathogens (most commonly

1	be reliably distinguished from the latter only by culture. In a patient with sinusitis and proptosis, orbital cellulitis and cavernous sinus thrombosis caused by bacterial pathogens (most commonly Staphylococcus aureus, but also streptococcal and gram-negative species) must be excluded. Klebsiella rhinoscleromatis is a rare cause of an indolent facial rhinoscleroma syndrome that may appear similar to mucormycosis. Finally, the Tolosa-Hunt syndrome causes painful ophthalmoplegia, ptosis, headache, and cavernous sinus inflammation; biopsies and clinical follow-up may be needed to distinguish the Tolosa-Hunt syndrome from mucormycosis by the lack of progression of the former entity.

1	The successful treatment of mucormycosis requires four steps: (1) early diagnosis; (2) reversal of underlying predisposing risk factors, if possible; (3) surgical debridement; and (4) prompt anti-fungal therapy. Early diagnosis of mucormycosis is critical, since early initiation of therapy is associated with improved survival rates. It is also crucial to reverse (or prevent) underlying defects in host defense during treatment (e.g., by stopping or reducing the dosage of immunosuppressive medications or by rapidly restoring euglycemia and normal acid-base status). Finally, iron administration to patients with active mucormycosis should be avoided, as iron exacerbates infection in animal models. Blood transfusion typically results in some liberation of free iron due to hemolysis, so a conservative approach to red blood cell transfusions is advisable.

1	Blood vessel thrombosis and resulting tissue necrosis during mucormycosis can result in poor penetration of antifungal agents to the site of infection. Therefore, debridement of all necrotic tissues is critical for eradication of disease. Surgery has been found (by logistic regression and in multiple case series) to be an independent variable for favorable outcome in patients with mucormycosis. Limited data from a retrospective study support the use of intraoperative frozen sections to delineate the margins of infected tissues, with sparing of tissues lacking evidence of infection. A multidisciplinary team, including an internist, an infectious disease specialist, and surgical specialists whose expertise is relevant to the sites of infection, is typically required for the management of mucormycosis.

1	Primary therapy for mucormycosis should be based on a polyene antifungal agent (Table 242-2), except perhaps for mild localized infection (e.g., isolated suprafascial cutaneous infection) that has been eradicated surgically in an immunocompetent patient. Amphotericin B (AmB) deoxycholate remains the only licensed antifungal agent for the treatment of mucormycosis. However, lipid formulations of AmB are significantly less nephrotoxic, can be administered at higher doses, and are probably more effective than AmB deoxycholate for this purpose. Liposomal amphotericin B (LAmB) is preferred to amphotericin B lipid complex (ABLC) for management of CNS infection on the basis of retrospective survival data and superior brain penetration; there is no clear advantage of either agent for non-CNS infections.

1	aPrimary therapy should generally include a polyene. Non-polyene-based regimens may be appropriate for patients who refuse or are intolerant of polyene therapy or for relatively immunocompetent patients with mild disease (e.g., isolated suprafascial cutaneous infection) that can be surgically eradicated. bProspective randomized trials are necessary to confirm the suggested benefit (from animal and small retrospective human studies) of combination therapy for mucormycosis. Dose escalation of any echinocandin is not recommended because of a paradoxical loss of benefit of combination therapy at echinocandin doses of ≥3 mg/kg qd. Abbreviations: ABLC, AmB lipid complex; AmB, amphotericin B; CNS, central nervous system; LAmB, liposomal AmB. Source: Modified from B Spellberg et al: Clin Infect Dis 48:1743, 2009.

1	Abbreviations: ABLC, AmB lipid complex; AmB, amphotericin B; CNS, central nervous system; LAmB, liposomal AmB. Source: Modified from B Spellberg et al: Clin Infect Dis 48:1743, 2009. The optimal dosages for antifungal treatment of mucormycosis are not known. Starting dosages of 1 mg/kg per day for AmB deoxycholate and 5 mg/kg per day for LAmB and ABLC are commonly given to adults and children. Dose escalation of LAmB to 7.5 or 10 mg/kg per day for CNS mucormycosis may be considered in light of the limited penetration of polyenes into the brain. Because of auto-induction of metabolism, which results in paradoxically lower drug levels, there is no advantage to escalating the LAmB dose above 10 mg/kg per day, and doses of 5 mg/kg per day are probably adequate for non-CNS infections. ABLC dose escalation above 5 mg/ kg per day is not advisable given the lack of relevant data and the drug’s potential toxicity.

1	Echinocandin–lipid polyene combinations improved survival rates among mice with disseminated mucormycosis (including CNS disease) and were associated with significantly better outcomes than polyene monotherapy in a small retrospective clinical study involving patients with rhino-orbital-cerebral mucormycosis. Although combination therapy may be considered on the basis of these limited data sets, definitive clinical trials are needed to establish whether it offers any real advantage over monotherapy for mucormycosis. Echinocandins should be administered at standard, FDA-approved doses, since dose escalation has resulted in paradoxical loss of efficacy in preclinical models.

1	In contrast to deferoxamine, the iron chelator deferasirox is fungicidal against clinical isolates of the Mucorales. In mice with DKA and disseminated mucormycosis, combination deferasirox-LAmB therapy resulted in synergistic improvement of survival rates and reduced the fungal burden in brain. Unfortunately, a randomized, double-blind, phase 2 safety clinical trial of adjunctive therapy with deferasirox (plus LAmB) documented excess mortality in the patients treated with deferasirox. It is noteworthy that the study population included primarily patients with active malignancy, and few patients in the study had diabetes mellitus as their only risk factor. Deferasirox is therefore contraindicated as therapy in patients with active malignancy, but its role in patients who have diabetes mellitus without malignancy (the setting in which its preclinical efficacy was optimal) remains uncertain.

1	Posaconazole is the only FDA-approved azole with in vitro activity against the Mucorales. However, pharmacokinetic/pharmacodynamic data raise concerns about the reliability with which adequate in vivo levels of orally administered posaconazole are attained. Furthermore, posaconazole is inferior in efficacy to AmB for the treatment of murine mucormycosis and is not superior to placebo for treatment of murine infection with R. oryzae. Moreover, posaconazole-polyene combination therapy is not superior to polyene monotherapy for mucormycosis in mice, and no comparative data are available for combination therapy in humans. The roles of recombinant cytokines and neutrophil transfusions in the primary treatment of mucormycosis are not clear, although it is intuitive that earlier recovery of neutrophil counts should improve survival rates. Limited data indicate that hyperbaric oxygen may be useful in centers with the appropriate technical expertise and facilities.

1	In general, antifungal therapy for mucormycosis should be continued until resolution of clinical signs and symptoms of infection and resolution of underlying immunosuppression. For patients with mucormycosis who are receiving immunosuppressive medications, secondary antifungal prophylaxis is typically continued for as long as the immunosuppressive regimen is administered. The role of radiographic follow-up in determining prognosis and therapeutic duration is being studied. Analysis of data from the phase 2 DEFEAT Mucor study indicated that early radiographic progression (within the first 2 weeks) did not predict long-term mortality risk, nor did early radiographic stability/regression predict long-term survival. Therefore, caution should be used in reacting to short-term, serial radiographic results, and greater emphasis should be placed on clinical response, particularly within the first 2–4 weeks after initiation of therapy. Carol A. Kauffman

1	Carol A. Kauffman Dimorphic fungi exist in discrete environmental niches as molds that produce conidia, which are their infectious form. In tissues and at temperatures of >35°C, the mold converts to the yeast form. Other endemic mycoses—histoplasmosis, coccidioidomycosis, and blastomycosis—are discussed in Chaps. 236, 237, and 238, respectively.

1	SPOROTRICHOSIS Etiologic Agent Sporothrix schenckii is a thermally dimorphic fungus that is found worldwide in sphagnum moss, decaying vegetation, and soil. Epidemiology and Pathogenesis Sporotrichosis most commonly infects persons who participate in outdoor activities such as landscaping, gardening, and tree farming. Infected animals can transmit S. schenckii to humans. An outbreak of sporotrichosis in Rio de Janeiro that began in 1998 and that has involved >2000 people has been traced to cats, which are highly susceptible to this infection. Sporotrichosis is primarily a localized infection of skin and subcutaneous tissues that follows traumatic inoculation of conidia. Osteoarticular sporotrichosis is uncommon, occurring most often in middle-aged men who abuse alcohol, and pulmonary sporotrichosis occurs almost exclusively in persons with chronic obstructive pulmonary disease who have inhaled the organism from the environment. Dissemination occurs rarely, almost always in markedly

1	occurs almost exclusively in persons with chronic obstructive pulmonary disease who have inhaled the organism from the environment. Dissemination occurs rarely, almost always in markedly immunocompromised patients, especially those with AIDS.

1	Clinical Manifestations Days or weeks after inoculation, a papule develops at the site and then usually ulcerates but is not very painful. Similar lesions develop sequentially along the lymphatic channels proximal to the original lesion (Fig. 243-1). Some patients develop a fixed cutaneous lesion that can be verrucous or ulcerative and that remains localized without lymphatic extension. The differential diagnosis of lymphocutaneous sporotrichosis includes nocardiosis, tularemia, nontuberculous mycobacterial infection (especially that due to Mycobacterium marinum), and leishmaniasis. Osteoarticular sporotrichosis can present as chronic synovitis or septic arthritis. Pulmonary sporotrichosis must be differentiated from tuberculosis and from other fungal pneumonias.

1	FIGuRE 243-1 Several nodular lesions that developed after a young boy pricked his index finger with a thorn. A culture yielded S. schenckii. (Courtesy of Dr. Angela Restrepo.) 1354 Numerous ulcerated skin lesions, with or without spread to visceral organs (including the central nervous system [CNS]), are characteristic of disseminated sporotrichosis. Diagnosis S. schenckii usually grows readily as a mold when material from a cutaneous lesion is incubated at room temperature. Histopathologic examination of biopsy material shows a mixed granulomatous and pyogenic reaction, and tiny oval or cigar-shaped yeasts are sometimes visualized with special stains. Serologic testing is not useful.

1	Treatment and Prognosis Guidelines for the management of the various forms of sporotrichosis have been published by the Infectious Diseases Society of America (Table 243-1). Itraconazole is the drug of choice for lymphocutaneous sporotrichosis. Fluconazole is less effective; voriconazole and posaconazole have not been used for sporotrichosis. Saturated solution of potassium iodide (SSKI) also is effective for lymphocutaneous infection and costs much less than itraconazole. However, SSKI is poorly tolerated because of adverse reactions, including metallic taste, salivary gland swelling, rash, and fever. Terbinafine appears to be effective but has been used in few patients. Treatment for lymphocutaneous sporotrichosis is continued for 2–4 weeks after all lesions have resolved, usually for a total of 3–6 months. Pulmonary and osteoarticular forms of sporotrichosis are treated with itraconazole for at least 1 year. Severe pulmonary infection and disseminated sporotrichosis, including that

1	total of 3–6 months. Pulmonary and osteoarticular forms of sporotrichosis are treated with itraconazole for at least 1 year. Severe pulmonary infection and disseminated sporotrichosis, including that involving the CNS, are treated initially with amphotericin B (AmB), which is followed by itraconazole after improvement has been noted. Lifelong suppressive therapy with itraconazole is required for AIDS patients. The success rate for treatment of lymphocutaneous sporotrichosis is 90–100%, but other forms of the disease respond poorly to antifungal therapy.

1	aThe starting dosage is 5–10 drops tid in water or juice. The dosage is increased weekly by 10 drops per dose, as tolerated, up to 40–50 drops tid. bThe dosage of lipid AmB is 3–5 mg/kg daily; the higher dosage should be used when the central nervous system is involved. cThe dosage of AmB deoxycholate is 0.6–1.0 mg/kg daily. Abbreviations: AmB, amphotericin B; SSKI, saturated solution of potassium iodide; TMPSMX, trimethoprim-sulfamethoxazole.

1	Abbreviations: AmB, amphotericin B; SSKI, saturated solution of potassium iodide; TMPSMX, trimethoprim-sulfamethoxazole. Etiologic Agent, Epidemiology, and Pathogenesis Paracoccidioides brasiliensis is a thermally dimorphic fungus that is endemic in humid areas of Central and South America, especially in Brazil. A striking male-to-female ratio varies from 14:1 to as high as 70:1 (in rural Brazil). Most patients are middle-aged or elderly men from rural areas. Paracoccidioidomycosis develops after the inhalation of aerosolized conidia encountered in the environment. For most patients, disease rarely develops at the time of the initial infection but appears years later, presumably after reactivation of a latent infection.

1	Clinical Manifestations Two major syndromes are associated with paracoccidioidomycosis: the acute or juvenile form and the chronic or adult form. The acute form is uncommon, occurs mostly in persons <30 years old, and manifests as disseminated infection of the reticuloendothelial system. Immunocompromised individuals also manifest this type of rapidly progressive disease. The chronic form of paracoccidioidomycosis accounts for ∼90% of cases and predominantly affects older men. The primary manifestation is progressive pulmonary disease, primarily in the lower lobes, with fibrosis. Ulcerative and nodular mucocutaneous lesions in the nares and mouth—another common manifestation of chronic paracoccidioidomycosis—must be differentiated from leishmaniasis (Chap. 251) and squamous cell carcinoma (Chap. 105).

1	Diagnosis The diagnosis is established by growth of the organism in culture. A presumptive diagnosis can be made by detection of the distinctive thick-walled yeast, with multiple narrow-necked buds attached circumferentially, in purulent material or tissue biopsies. Treatment and Prognosis Itraconazole is the treatment of choice for paracoccidioidomycosis (Table 243-1). Ketoconazole is also effective but more toxic; voriconazole and posaconazole have been used with success in a few cases. Sulfonamides also are effective and are the least costly agents, but the response is slower and the relapse rate higher. Seriously ill patients should be treated with AmB initially. Patients with paracoccidioidomycosis have an excellent response to therapy, but pulmonary fibrosis is often progressive in those with chronic disease.

1	Etiologic Agent, Epidemiology, and Pathogenesis Penicillium marneffei is a thermally dimorphic fungus that is endemic in the soil in certain areas of Vietnam, Thailand, and several other southeastern Asian countries. The epidemiology of penicilliosis is linked to bamboo rats, which are infected with the fungus but rarely manifest disease. The disease occurs most often among persons living in rural areas in which the rats are found, but there is no evidence for transmission of the infection directly from rats to humans. Infection is rare in immunocompetent hosts, and most cases are reported in persons who have advanced AIDS. Infection results from the inhalation of conidia from the environment. The organism converts to the yeast phase in the lungs and then spreads hematogenously to the reticuloendothelial system.

1	Clinical Manifestations The clinical manifestations of penicilliosis mimic those of disseminated histoplasmosis and include fever, fatigue, weight loss, dyspnea, diarrhea (in some cases), lymphadenopathy, hepatosplenomegaly, and skin lesions, which appear as papules that often umbilicate and resemble molluscum contagiosum (Chap. 220e). Diagnosis Penicilliosis is diagnosed by culture of P. marneffei from blood or from biopsy samples of skin, bone marrow, or lymph node. The organism usually grows within 1 week as a mold that produces a distinctive red pigment. Histopathologic examination of tissues and smears of blood or material from skin lesions shows oval or elliptical yeast-like organisms with central septation and can quickly establish a presumptive diagnosis.

1	Treatment and Prognosis Patients who have severe disease should be treated initially with AmB until their condition improves; therapy can then be changed to itraconazole (Table 243-1). Patients who have mild symptoms can be treated from the start with itraconazole. For patients with AIDS, suppressive therapy with itraconazole is recommended until immune reconstitution (related to successful therapy for HIV infection with antiretroviral drugs) is evident. Disseminated penicilliosis is usually fatal if not treated. With treatment, the mortality rate is ∼10%.

1	In these common soil organisms (also called dematiaceous fungi), melanin causes the hyphae and/or conidia to be darkly pigmented. The term phaeohyphomycosis is used to describe any infection with a pigmented mold. This definition encompasses two specific syndromes—eumycetoma and chromoblastomycosis—as well as all other types of infections caused by these organisms. It is important to note that eumycetomas can be caused by hyaline molds as well as brown-black molds and that only about half of all mycetomas are due to fungi. Actinomycetes cause the remainder (Chap. 199). Most of the involved fungi cause localized subcutaneous infections after direct inoculation, but disseminated infection and serious focal visceral infections also occur, especially in immunocompromised patients.

1	Etiologic Agents A large number of pigmented molds can cause human infection. All are found in the soil or on plants, and some cause economically important plant diseases. The most common cause of eumycetoma is Madurella species. Fonsecaea and Cladophialophora species are responsible for most cases of chromoblastomycosis. Disseminated infection and focal visceral infections are caused by a variety of dematiaceous fungi; Alternaria, Exophiala, Curvularia, and Wangiella species are among the more common molds reported to cause human infection. Recently, Exserohilum species have caused a large outbreak of severe, sometimes fatal CNS and osteoarticular infections following the injection of methylprednisolone contaminated with this fungus.

1	Epidemiology and Pathogenesis Eumycetoma and chromoblastomycosis are acquired by inoculation through the skin. These two syndromes are seen almost entirely in tropical and subtropical areas and occur mostly in rural laborers who are frequently exposed to the organisms. Other infections with dematiaceous molds are acquired by inhalation, by traumatic inoculation into the eye or through the skin, or by injection of contaminated medication. Melanin is a virulence factor for all the pigmented molds. Several organisms, specifically Cladophialophora bantiana and Rhinocladiella mackenziei, are neurotropic and likely to cause CNS infection. In an immunocompromised patient or when a pigmented mold is injected directly into a deep structure, these organisms become opportunists, invading blood vessels and mimicking better-known opportunistic infections, such as aspergillosis.

1	Clinical Manifestations Eumycetoma is a chronic subcutaneous and cutaneous infection that usually occurs on the lower extremities and that is characterized by swelling, development of sinus tracts, and the appearance of grains that are actually colonies of fungi discharged from the sinus tract. As the infection progresses, adjacent fascia and bony structures become involved. The disease is indolent and disfiguring, progressing slowly over years. Complications include fractures of infected bone and bacterial superinfection. Chromoblastomycosis is an indolent subcutaneous infection characterized by nodular, verrucous, or plaque-like painless lesions that occur predominantly on the lower extremities and grow slowly over months to years. There is hardly ever extension to adjacent structures, as is seen with eumycetoma. Long-term consequences include bacterial superinfection, chronic lymphedema, and (rarely) the development of squamous cell carcinoma.

1	Dematiaceous molds are the most common cause of allergic fungal sinusitis and a less common cause of invasive fungal sinusitis. Keratitis occurs with traumatic corneal inoculation. Even in many immunocompromised patients, inoculation through the skin generally produces localized cyst-like, nodular lesions at the entry site. However, other immunocompromised patients develop pneumo-1355 nia, brain abscess, or disseminated infection. Epidural injection of Exserohilum-contaminated steroids has led to meningitis, basilar stroke, epidural abscess or phlegmon, vertebral osteomyelitis, and arachnoiditis.

1	Diagnosis The specific diagnosis of infection with a pigmented mold is established by growth of the organism in culture. However, in eumycetoma, a tentative clinical diagnosis can be made when a patient presents with a lesion characterized by swelling, sinus tracts, and grains. Histopathologic examination and culture are necessary to confirm that the etiologic agent is a mold and not an actinomycete. In chromoblastomycosis, the diagnosis rests on the histologic demonstration of sclerotic bodies (dark brown, thick-walled, septate fungal forms that resemble large yeasts) in the tissues; culture establishes which pigmented mold is causing the infection. For other infections, growth of the organism is essential to differentiate infection with a hyaline mold (e.g., Aspergillus or Fusarium) from that due to a pigmented mold. No serologic assays for pigmented molds are available. Polymerase chain reaction (PCR) assays are increasingly used in the diagnosis of infection due to pigmented molds

1	that due to a pigmented mold. No serologic assays for pigmented molds are available. Polymerase chain reaction (PCR) assays are increasingly used in the diagnosis of infection due to pigmented molds but are available only through fungal reference laboratories.

1	Treatment and Prognosis Treatment of eumycetoma and chromoblastomycosis involves both surgical extirpation of the lesion and use of antifungal agents. Surgical removal of the lesions of both eumycetoma and chromoblastomycosis is most effective if performed before extensive spread has occurred. In chromoblastomycosis, cryosurgery and laser therapy have been used with variable success. The antifungal agents of choice are itraconazole, voriconazole, and posaconazole. The most experience has accrued with itraconazole; less experience has been gained with the newer azoles, which are active in vitro and have been reported to be effective in a few patients. Flucytosine and terbinafine also have been used to treat chromoblastomycosis. Chromoblastomycosis and eumycetoma are chronic indolent infections that are difficult to cure but are not life-threatening.

1	Disseminated and focal visceral infections are treated with the appropriate antifungal agent; the choice of agent is based on the location and extent of the infection, in vitro testing, and clinical experience with the specific infecting organism. AmB is not effective against many of these organisms but has been used successfully against others. The most experience has accrued with itraconazole in the treatment of localized infections. Voriconazole is increasingly used when infections are disseminated or involve the CNS because this drug reaches adequate concentrations within the CNS and because both IV and well-absorbed oral formulations are available. The role of posaconazole has not been established but will likely expand. Disseminated and focal visceral infections, especially those involving the CNS, are associated with high mortality rates.

1	Two genera of hyaline (nonpigmented) molds, Fusarium and Scedosporium, and one yeast-like genus, Trichosporon, have become prominent pathogens among immunocompromised patients. Infections caused by Fusarium and Scedosporium species overlap with invasive aspergillosis in their clinical manifestations, and, when seen in tissues, these organisms appear similar to Aspergillus. In the immunocompetent host, these fungi cause localized infections of skin, skin structures, and subcutaneous tissues, but their role as causes of infection in immunocompromised patients will be emphasized in this section.

1	Etiologic Agent, Epidemiology, and Pathogenesis Fusarium spe cies, which are found worldwide in soil and on plants, have emerged as major opportunists in markedly immunocompromised patients. Most human infections follow inhalation of conidia, but ingestion and direct inoculation also can lead to disease. An outbreak of severe Fusarium keratitis among soft contact lens wearers was traced back to a particular brand of contact lens solution and 1356 individual contact lens cases that had been contaminated. Disseminated infection is reported most often in patients who have a hematologic malignancy, are neutropenic, have received a stem cell or solid organ transplant, or have a severe burn.

1	Clinical Manifestations In immunocompetent persons, Fusarium species cause localized infections of various organs. These organisms commonly cause fungal keratitis, which can extend into the anterior chamber of the eye; cause loss of vision; and require corneal transplantation. Onychomycosis due to Fusarium species, while basically an annoyance in immunocompetent patients, is a source of subsequent hematogenous dissemination and should be aggressively sought and treated in neutropenic patients. In profoundly immunocompromised patients, fusariosis is angioinvasive, and clinical manifestations mimic those of aspergillosis. Pulmonary infection is characterized by multiple nodular lesions. Sinus infection is likely to lead to invasion of adjacent structures. Disseminated fusariosis occurs primarily in neutropenic patients with hematologic malignancies and in allogeneic stem cell transplant recipients, especially those with graft-versus-host disease. Disseminated fusariosis differs from

1	in neutropenic patients with hematologic malignancies and in allogeneic stem cell transplant recipients, especially those with graft-versus-host disease. Disseminated fusariosis differs from disseminated aspergillosis in that skin lesions are extremely common with fusariosis; the lesions are nodular or necrotic, are usually painful, and appear over time in different locations (Fig. 243-2).

1	Diagnosis The diagnostic approach usually includes both documentation of the growth of Fusarium species from involved tissue and demonstration of invasion by histopathologic techniques that show septate hyphae in tissues. The organism is difficult to differentiate from Aspergillus species in tissues; thus, identification with culture is imperative. An extremely helpful diagnostic clue is growth in blood cultures, which are positive in as many as 50% of patients with disseminated fusariosis. There are no serologic assays for Fusarium. PCR techniques have proved useful but are available only through fungal reference laboratories.

1	Treatment and Prognosis Fusarium species are resistant to many anti-fungal agents. A lipid formulation of AmB (at least 5 mg/kg daily), voriconazole (200–400 mg twice daily), or posaconazole (300 mg daily) is recommended. Many physicians use both a lipid formulation of AmB and either voriconazole or posaconazole because susceptibility information is not available when therapy must be initiated. Serum drug levels should be monitored with either azole to ensure that absorption is adequate and with voriconazole to avoid toxicity. Mortality rates for disseminated fusariosis have been as high as 85%. With the improved antifungal therapy now available, mortality rates have fallen to ∼50%. However, if neutropenia persists, the mortality rate approaches 100%. FIGuRE 243-2 Painful necrotic foot lesion that developed over a week in a woman who had acute leukemia and who had been neu-tropenic for 2 months. Fusarium species were grown from a punch biopsy. (Courtesy of Dr. Nessrine Ktaich.)

1	SCEDOSPORIOSIS Etiologic Agent The genus Scedosporium includes several pathogens. The major causes of human infections are Scedosporium apiospermum, which in its sexual state is termed Pseudallescheria boydii, and S. prolificans. The S. apiospermum complex encompasses several species but will be referred to here simply as S. apiospermum. Epidemiology and Pathogenesis S. apiospermum is found worldwide in temperate climates in tidal flats, swamps, ponds, manure, and soil. This organism is known as a cause of pneumonia, disseminated infection, and brain abscess in near-drowning victims. S. prolificans is also found in soil but is more geographically restricted. Infection occurs predominantly through inhalation of conidia, but direct inoculation through the skin or into the eye also can occur.

1	Clinical Manifestations Among immunocompetent persons, Scedosporium species are a prominent cause of eumycetoma. Keratitis as a result of accidental corneal inoculation is a sight-threatening infection. In patients who have hematologic malignancies (especially acute leukemia with neutropenia), recipients of solid organ or stem cell transplants, and patients receiving glucocorticoids, Scedosporium species are angioinvasive, causing pneumonia and widespread dissemination with abscesses. Pulmonary infection mimics aspergillosis; nodules, cavities, and lobar infiltrates are common. Disseminated infection involves the skin, heart, brain, and many other organs. Skin lesions are not as common or as painful as those of fusariosis.

1	Diagnosis Diagnosis depends on the growth of Scedosporium species from involved tissue and the demonstration of invasion by histopathologic techniques that show septate hyphae in tissues. Culture evidence is essential because Scedosporium species are difficult to differentiate from Aspergillus in tissues. Demonstration of tissue invasion is essential because these ubiquitous environmental molds can be mere contaminants or colonizers. S. prolificans can grow in blood cultures, but S. apiospermum usually does not. There are no serologic assays for Scedosporium. PCR techniques have proved useful but are available only through fungal reference laboratories.

1	Treatment and Prognosis Scedosporium species are resistant to AmB, echinocandins, and some azoles. Voriconazole is the agent of choice for S. apiospermum, and posaconazole also has been used for this infection. S. prolificans is resistant in vitro to almost every available antifungal agent; the addition of agents such as terbinafine to a voriconazole regimen has been attempted because in vitro data suggest possible synergy against some strains of S. prolificans. Mortality rates for invasive S. apiospermum infection are ∼50%, but those for invasive S. prolificans infection remain as high as 85–100%. TRICHOSPORONOSIS Etiologic Agent The genus Trichosporon contains many species, some of which cause localized infection of hair and nails. The major pathogen responsible for invasive infection is Trichosporon asahii. Trichosporon species grow as yeast-like colonies in vitro; in vivo, however, hyphae, pseudohyphae, and arthroconidia can also be seen.

1	Epidemiology and Pathogenesis These yeasts are commonly found in soil, sewage, and water and in rare instances can colonize human skin and the human gastrointestinal tract. Most infections follow fungal inhalation or entry via central venous catheters. Systemic infection occurs almost exclusively in immunocompromised hosts, including those who have hematologic malignancies, are neutropenic, have received a solid organ transplant, or are receiving glucocorticoids. Clinical Manifestations Disseminated trichosporonosis resembles invasive candidiasis, and fungemia is often the initial manifestation of infection. Pneumonia, skin lesions, and sepsis syndrome are common. The skin lesions begin as papules or nodules surrounded by erythema and progress to central necrosis. A chronic form of infection mimics hepatosplenic candidiasis (chronic disseminated candidiasis).

1	Diagnosis The diagnosis of systemic Trichosporon infection is established by growth of the organism from involved tissues or from blood. Histopathologic examination of a skin lesion showing a mixture of yeast forms, arthroconidia, and hyphae can lead to an early presumptive diagnosis of trichosporonosis. The serum cryptococcal antigen latex agglutination test may be positive in patients with disseminated trichosporonosis because T. asahii and Cryptococcus neoformans share polysaccharide antigens. Treatment and Prognosis Rates of response to AmB have been disappointing, and many Trichosporon isolates are resistant in vitro. Voriconazole appears to be the antifungal agent of choice and is used at a dosage of 200–400 mg twice daily. The mortality rates for disseminated Trichosporon infection have been as high as 70% but are decreasing with the use of newer azoles, such as voriconazole; however, patients who remain neutropenic are likely to succumb to this infection.

1	Fungal infections of the skin and skin structures are caused by molds and yeasts that do not invade deeper tissues but rather cause disease merely by inhabiting the superficial layers of skin, hair follicles, and nails. These agents are the most common cause of fungal infections of humans but only rarely cause serious infections. YEAST INFECTIONS Etiologic Agents The lipophilic yeast Malassezia is dimorphic in that it lives on the skin in the yeast phase but transforms to the mold phase as it causes disease. Most species require exogenous lipids for growth. Epidemiology and Pathogenesis Malassezia species are part of the indigenous human flora found in the stratum corneum of the back, chest, scalp, and face—areas rich in sebaceous glands. Disease is more common in humid areas. The organisms do not invade below the stratum corneum and generally elicit little if any inflammatory response.

1	Clinical Manifestations Malassezia species cause tinea versicolor (also called pityriasis versicolor), folliculitis, and seborrheic dermatitis. Tinea versicolor presents as flat round scaly patches of hypoor hyperpigmented skin on the neck, chest, or upper arms. The lesions are usually asymptomatic but can be pruritic. They can be mistaken for vitiligo, but the latter is not scaly. Folliculitis occurs over the back and chest and mimics bacterial folliculitis. Seborrheic dermatitis manifests as erythematous pruritic scaly lesions in the eyebrows, moustache, nasolabial folds, and scalp. The scalp lesions are termed cradle cap in babies and dandruff in adults. Seborrheic dermatitis can be severe in patients with advanced AIDS. Fungemia and disseminated infection occur rarely with Malassezia species—almost always in premature neonates receiving parenteral lipid preparations through a central venous catheter.

1	Diagnosis Malassezia infections are diagnosed clinically in most cases. If scrapings are collected on a microscope slide on which a drop of potassium hydroxide has been placed, a mixture of budding yeasts and short septate hyphae is seen. In order to culture M. furfur from those patients in whom disseminated infection is suspected, sterile olive oil must be added to the medium.

1	Treatment and Prognosis Topical creams and lotions, including selenium sulfide shampoo, ketoconazole shampoo or cream, terbinafine cream, and ciclopirox cream, are effective in treating Malassezia infections and are usually given for 2 weeks. Mild topical steroid creams are sometimes used to treat seborrheic dermatitis. For extensive disease, itraconazole (200 mg/d) or fluconazole (200 mg/d) can be used for 5–7 days. The rare cases of fungemia caused by Malassezia species are treated with AmB or fluconazole, prompt removal of the catheter, and discontinuance of parenteral lipid infusions. Malassezia skin infections are benign and self-limited, although recurrences are the rule. The outcome of systemic infection depends on the host’s underlying conditions, but most infected infants do well. DERMATOPHYTE (MOLD) INFECTIONS Etiologic Agents The molds that cause skin infections in humans include the genera Trichophyton, Microsporum, and Epidermophyton.

1	DERMATOPHYTE (MOLD) INFECTIONS Etiologic Agents The molds that cause skin infections in humans include the genera Trichophyton, Microsporum, and Epidermophyton. These organisms, which are not components of the normal skin flora, 1357 can live within the keratinized structures of the skin—hence the term dermatophytes.

1	wide, and infections with these organisms are extremely com mon. Some organisms cause disease only in humans and can be transmitted by person-to-person contact and by fomites, such as hairbrushes or wet floors, that have been contaminated by infected individuals. Several species cause infections in cats and dogs and can readily be transmitted from these animals to humans. Finally, some dermatophytes are spread from contact with soil. The characteristic ring shape of cutaneous lesions is the result of the organisms’ outward growth in a centrifugal pattern in the stratum corneum. Fungal invasion of the nail usually occurs through the lateral or superficial nail plates and then spreads throughout the nail; when hair shafts are invaded, the organisms can be found either within the shaft or surrounding it. Symptoms are caused by the inflammatory reaction elicited by fungal antigens and not by tissue invasion. Dermatophyte infections occur more commonly in male than in female patients, and

1	it. Symptoms are caused by the inflammatory reaction elicited by fungal antigens and not by tissue invasion. Dermatophyte infections occur more commonly in male than in female patients, and progesterone has been shown to inhibit dermatophyte growth.

1	Clinical Manifestations Dermatophyte infection of the skin is often called ringworm. This term is confusing because worms are not involved. Tinea, the Latin word for worm, describes the serpentine nature of the skin lesions and is a less confusing designation that is used in conjunction with the name of the body part affected—e.g., tinea capitis (head), tinea pedis (feet), tinea corporis (body), tinea cruris (crotch), and tinea unguium (nails, although infection at this site is more often termed onychomycosis).

1	Tinea capitis occurs most commonly in children 3–7 years old. Children with tinea capitis usually present with well-demarcated scaly patches in which hair shafts are broken off right above the skin; alopecia can result. Tinea corporis is manifested by well-demarcated, annular, pruritic, scaly lesions that undergo central clearing. Usually one or several small lesions are present. In some cases, tinea corporis can involve much of the trunk or manifest as folliculitis with pustule formation. The rash should be differentiated from contact dermatitis, eczema, and psoriasis. Tinea cruris is seen almost exclusively in men. The perineal rash is erythematous and pustular, has a discrete scaly border, is without satellite lesions, and is usually pruritic. The rash must be differentiated from intertriginous candidiasis, erythrasma, and psoriasis.

1	Tinea pedis also is more common among men than among women. It usually starts in the web spaces of the toes; peeling, maceration, and pruritus are followed by development of a scaly pruritic rash along the lateral and plantar surfaces of the feet. Hyperkeratosis of the soles of the feet often ensues. Tinea pedis has been implicated in lower-extremity cellulitis, as streptococci and staphylococci can gain entrance to the tissues through fissures between the toes. Onychomycosis affects toenails more often than fingernails and is most common among persons who have tinea pedis. The nail becomes thickened and discolored and may crumble; onycholysis almost always occurs. Onychomycosis is more common in older adults and in persons with vascular disease, diabetes mellitus, and trauma to the nails. Fungal infection must be differentiated from psoriasis, which can mimic onychomycosis but usually has associated skin lesions.

1	Diagnosis Many dermatophyte infections are diagnosed by their clinical appearance. If the diagnosis is in doubt, as is often the case in children with tinea capitis, scrapings should be taken from the edge of a lesion with a scalpel blade, transferred to a slide to which a drop of potassium hydroxide is added, and examined under a microscope for the presence of hyphae. Cultures are indicated if an outbreak is suspected or the patient does not respond to therapy. Culture of the nail is especially useful as an aid to decisions about both diagnosis and treatment. Treatment and Prognosis Dermatophyte infections usually respond to topical therapy. Lotions or sprays are easier than creams to apply to large or hairy areas. Particularly for tinea cruris, the affected Terbinafine 250 mg/d for 1–2 weeks Adverse reactions minimal with short treatment period

1	Terbinafine 250 mg/d for 1–2 weeks Adverse reactions minimal with short treatment period Itraconazolea 200 mg/d for 1–2 weeks Adverse reactions minimal with short treatment period except for drug interactions aItraconazole capsules require food and gastric acid for absorption, whereas itraconazole solution is taken on an empty stomach.

1	area should be kept as dry as possible. When patients have extensive skin lesions, oral itraconazole or terbinafine can hasten resolution (Table 243-2). Terbinafine interacts with fewer drugs than itraconazole and is generally the first-line agent. Onychomycosis does not respond to topical therapy, although ciclopirox nail lacquer applied daily for a year is occasionally beneficial. Itraconazole and terbinafine both accumulate in the nail plate and can be used to treat onychomycosis (Table 243-2). These agents are more effective and better tolerated than griseofulvin and ketoconazole. The major decision to be made with regard to therapy is whether the extent of nail involvement justifies the use of systemic antifungal agents that have adverse effects, may interact with other drugs, and are costly. Treating for cosmetic reasons alone is discouraged. Relapses of tinea cruris and tinea pedis are common and should be treated early with topical creams to avoid development of more extensive

1	costly. Treating for cosmetic reasons alone is discouraged. Relapses of tinea cruris and tinea pedis are common and should be treated early with topical creams to avoid development of more extensive disease. Relapses of onychomycosis follow treatment in 25–30% of cases.

1	Henry Masur, Alison Morris Pneumocystis is an opportunistic pathogen that is an important cause of pneumonia in immunocompromised hosts, particularly those with HIV infection (Chap. 226), and in individuals with organ transplants, those with hematologic malignancies, and those receiving immunosuppressive therapy. The organism was discovered in rodents in 1906 and was initially believed to be a protozoan. Because Pneumocystis cannot be cultured, our understanding of its biology has been limited, but molecular techniques have demonstrated that the organism is actually a fungus. Formerly known as Pneumocystis carinii, the species infecting humans has been renamed Pneumocystis jirovecii.

1	P. jirovecii pneumonia (PCP) came to medical attention when cases were reported in malnourished orphans in Europe during World War II. The disease was later recognized in other immunosuppressed populations but was rare in the era before HIV/AIDS and before intensive immunosuppressive therapy for organ transplantation and autoimmune disorders. In 1981, PCP was first reported in men who had sex with men and in IV drug users who had no obvious cause of immunosuppression. These cases were subsequently recognized as the first cases of what came to be known as the acquired immunodeficiency syndrome (AIDS) (Chap. 226). The incidence of PCP increased dramatically as the AIDS epidemic grew: without chemoprophylaxis or antiretroviral therapy (ART), 80–90% of patients with HIV/AIDS in North America and Western Europe ultimately develop one or more episodes of PCP. While its incidence declined with the introduction of anti-Pneumocystis prophylaxis and combination ART, PCP has continued to be a

1	and Western Europe ultimately develop one or more episodes of PCP. While its incidence declined with the introduction of anti-Pneumocystis prophylaxis and combination ART, PCP has continued to be a leading cause of AIDS-associated morbidity in the United States and Western Europe, particularly in individuals who do not know they are infected with HIV until they are profoundly immunosuppressed and in HIV-infected patients who are not receiving ART or PCP prophylaxis.

1	PCP also develops in HIV-uninfected patients who are immunocompromised secondary to hematologic or malignant neoplasms, stem cell or solid organ transplantation, and immunosuppressive medications. The incidence of PCP depends on the degree of immunosuppression. PCP is increasingly reported among individuals receiving tumor necrosis factor α inhibitors and antilymphocyte monoclonal antibodies for rheumatologic or other diseases. While clinical PCP in immunocompetent hosts has not been clearly documented, studies have shown that Pneumocystis organisms can colonize the airways of children and adults who are not overtly immunocompromised. The relevance of these organisms to acute or chronic syndromes, such as chronic obstructive pulmonary disease (COPD), in immunocompetent patients is being investigated. In some developing countries, the incidence of PCP among

1	In some developing countries, the incidence of PCP among HIV-infected individuals has been found to be lower than that in industrialized countries. This lower incidence may be due to competing mortality from infectious diseases such as tuberculosis and bacterial pneumonia, which typically occur before patients become immunosuppressed enough to develop PCP. Geographic variations in Pneumocystis exposure and underdiagnosis also may explain the apparent lower frequency of PCP in some countries.

1	PATHOGENESIS AND PATHOLOGY Life Cycle and Transmission The life cycle of Pneumocystis involves both sexual and asexual reproduction, and the organism exists as a trophic form, a cyst, and a precyst at various points. Serologic and molecular studies have demonstrated that most humans are exposed to Pneumocystis early in life. It was historically thought that Pneumocystis developed from reactivation of latent infection, but de novo infections from environmental sources and person-to-person transmission occur as well. Outbreaks of PCP suggest that nosocomial transmission can take place, and studies with rodents show that immunocompetent animals can serve as reservoirs for transmission of P. carinii (the infecting species in rodents) to immunocompetent and immunosuppressed animals. However, Pneumocystis organisms are species specific, and thus humans are infected only by other humans who transmit P. jirovecii; humans cannot be infected with animal species of Pneumocystis such as P. murina

1	organisms are species specific, and thus humans are infected only by other humans who transmit P. jirovecii; humans cannot be infected with animal species of Pneumocystis such as P. murina (mice) or P. oryctolagi (rabbits). The utility of respiratory isolation in preventing transmission from patients with PCP to other immunosuppressed individuals has been debated; no clear evidence exists, although it seems prudent to isolate patients with active PCP from other immunosuppressed patients.

1	Role of Immunity Defects in cellular and/or humoral immunity predispose to development of PCP. CD4+ T cells are critical in host defense against Pneumocystis. For HIV-infected patients, the incidence is inversely related to the CD4+ T cell count: at least 80% of cases occur at counts of <200 cells/μL, and most of these cases develop at counts of <100 cells/μL. CD4+ T cell counts are less specific and thus less useful in predicting the risk of PCP in HIV-uninfected, immunosuppressed patients. Lung Pathology Pneumocystis has a unique tropism for the lung. It is presumably inhaled into the alveolar space. Clinically apparent pneumonia occurs only if an individual is immunocompromised. Pneumocystis proliferates in the lung, provoking a mononuclear cell

1	FIGuRE 244-1 Direct microscopy of Pneumocystis pneumonia. A. Transbronchial lung biopsy stained with hematoxylin and eosin shows eosinophilic alveolar filling. B. Methenamine silver–stained bronchoalveolar lavage (BAL) fluid. C. Giemsa-stained BAL fluid. D. Immunofluorescent stain of BAL fluid. response. The alveoli become filled with proteinaceous material, and alveolar damage results in increased alveolar-capillary injury and surfactant abnormalities. Stained lung sections typically show foamy, vacuolated alveolar exudates composed largely of viable and nonviable organisms (Fig. 244-1A). Interstitial edema and fibrosis may develop, and organisms can be seen in the alveolar space with silver or other stains. Moreover, the organisms can be seen when tissue is subjected to colorimetric or immunofluorescent staining (Fig. 244-1B–D).

1	CLINICAL FEATuRES Clinical Presentation PCP presents as acute or subacute pneumonia that may initially be characterized by a vague sense of dyspnea alone but that subsequently manifests as fever and nonproductive cough with progressive shortness of breath ultimately resulting in respiratory failure and death. Extrapulmonary manifestations of PCP are rare but can include involvement of almost any organ, most notably lymph nodes, spleen, and liver. Physical Examination The physical examination findings in PCP are nonspecific. Patients have decreased oxygen saturation—at rest or with exertion—that, without treatment, progresses to severe hypoxemia. Patients may initially have a normal chest examination and no adventitious sounds but later, without treatment, develop diffuse rales and signs of consolidation. Oral thrush in a patient with HIV infection indicates an increased risk for PCP.

1	Laboratory Findings The results of routine laboratory tests are nonspecific in PCP. Serum levels of lactate dehydrogenase (LDH) are often elevated due to pulmonary damage; however, a normal LDH level does not rule out PCP, nor is an elevated LDH value specific for PCP. The peripheral white blood cell count may be elevated, but the increase is usually modest. Hepatic and renal function are typically normal.

1	Radiographic Findings Although the initial chest radiograph may be normal when patients have mild symptoms, the classic radiographic appearance of PCP consists of diffuse bilateral interstitial infiltrates that are perihilar and symmetric (Fig. 244-2A)—yet another finding that is not specific for PCP. The interstitial infiltrates can progress to alveolar filling (Fig. 244-2B). High-resolution chest CT shows diffuse ground-glass opacities in virtually all patients with PCP (Fig. 244-2C). A normal chest CT essentially rules out the diagnosis of PCP. Cysts and pneumothoraces are common chest radiographic findings (Fig. 244-2D). A wide variety of atypical radiographic findings have been described, including asymmetric patterns, upper lobe infiltrates, mediastinal adenopathy, nodules, cavities, and effusions.

1	The optimal sample for diagnostic examination depends on how ill the patient is and what resources are available. Before the 1990s, diagnoses of PCP were usually established by open lung biopsy; later, transbronchial lung biopsy was employed. Hematoxylin and eosin staining of pulmonary FIGuRE 244-2 Radiographs in Pneumocystis pneumonia. A. Posterior-anterior chest radiograph showing symmetric interstitial infiltrates. B. Posterior-anterior chest radiograph showing symmetric alveolar infiltrates (courtesy of Alison Morris). C. CT image demonstrating symmetric interstitial infiltrates and ground-glass opacities. D. CT image showing symmetric interstitial infiltrates, ground-glass opacities, and pneumatoceles.

1	tissue demonstrates a foamy alveolar infiltrate and a mononuclear interstitial infiltrate (Fig. 244-2A). This appearance is pathognomonic for PCP even though the organisms cannot be specifically identified with this stain. The diagnosis is typically established in lung tissue or pulmonary secretions by highly specific staining of the cyst—e.g., with methenamine silver (Fig. 244-2B), toluidine blue O, or Giemsa (Fig. 244-2C)—or by staining with a specific immunofluorescent antibody (Fig. 244-2D).

1	The demonstration of organisms in bronchoalveolar lavage fluid is almost 100% sensitive and specific for PCP in patients with either HIV infection or immunosuppression of other etiologies. The organisms are identified with the specific stains indicated above for lung biopsy. While expectorated sputum or throat swabs have very low sensitivity, an induced sputum sample obtained and interpreted by an experienced provider can be highly sensitive and specific. The reported sensitivity of induced sputum for PCP is widely variable (55–90%), however, and is dependent on both the characteristics of the patient and the experience of the center conducting the test.

1	Recently, many laboratories have offered polymerase chain reaction (PCR) testing of respiratory specimens for Pneumocystis. However, these PCR tests are so sensitive that it is difficult to distinguish patients with colonization (i.e., those whose acute lung disease is due to some other process but who have low levels of Pneumocystis DNA in the lungs) from those with acute pneumonia due to Pneumocystis. Such PCR tests on appropriate samples may be more useful for ruling out a diagnosis of PCP if they are negative than for definitively attributing the disease to Pneumocystis. There has been considerable interest in serologic tests such as assays for (1→3)-β-D-glucan, levels of which are frequently elevated in patients with PCP. However, no serologic assays developed to date offer substantial sensitivity or specificity.

1	Untreated, PCP is invariably fatal. Patients with HIV infection often have an indolent course that presents as mild exercise intolerance or chest tightness without fever or cough and a normal or nearly normal posterior-anterior chest radiograph, with progression over days, weeks, or even a few months to fever, cough, diffuse alveolar infiltrates, and profound hypoxemia. Some patients with HIV infection and most patients with other types of immunosuppression have more acute disease that progresses over a few days to respiratory failure. Rare patients also develop distributive shock. A few unusual patients present with extrapulmonary manifestations in the skin or soft tissue, retina, brain, liver, kidney, or spleen that are nonspecific in presentation and can be diagnosed only by histology.

1	Factors that influence mortality risk include the patient’s age and degree of immunosuppression as well as the presence of preexisting lung disease, a low serum albumin level, the need for mechanical ventilation, and the development of pneumothorax. With advances in supportive critical care, the prognosis for patients with PCP who require intubation and respiratory support has improved and now depends to a large extent on comorbidities and the prognosis of the underlying disease. Since patients typically do not respond to therapy for 4–8 days, supportive care for a minimum of 10 days is a reasonable consideration if such support is compatible with the patient’s wishes and the prognosis of comorbidities. Patients whose condition continues to deteriorate after 3 or 4 days or has not improved after 7–10 days should be reevaluated to determine whether other infectious processes are present (either having been missed on initial evaluation or having developed during treatment) or whether

1	after 7–10 days should be reevaluated to determine whether other infectious processes are present (either having been missed on initial evaluation or having developed during treatment) or whether noninfectious processes (e.g., congestive heart failure, pulmonary emboli, pulmonary hypertension, drug toxicity, or a neoplastic process) are causing pulmonary dysfunction.

1	TREATMEnT p. jirovecii Pneumonia

1	The treatment of choice for PCP is trimethoprim-sulfamethoxazole (TMP-SMX), given either IV or PO for 14–21 days (Table 244-1). TMP-SMX, which interferes with the organism’s folate metabolism, is at least as effective as alternative agents and is better tolerated. However, TMP-SMX can cause leukopenia, hepatitis, rash, and fever as well as anaphylactic and anaphylactoid reactions, and patients with HIV infection have an unusually high incidence of hypersensitivity to TMP-SMX. Monitoring of serum drug levels is useful if renal function or toxicities are issues. Maintenance of a 2-h post-dose sulfamethoxazole level of 100–150 μg/mL has been associated with a successful outcome. Resistance to TMP-SMX cannot be measured by organism growth inhibition in the laboratory because Pneumocystis cannot be cultured. However, mutations in the target gene for sulfamethoxazole that confer in vitro sulfa resistance to other organisms have been found in Pneumocystis. The clinical relevance of these

1	be cultured. However, mutations in the target gene for sulfamethoxazole that confer in vitro sulfa resistance to other organisms have been found in Pneumocystis. The clinical relevance of these mutations for the response to therapy is unknown. Sulfadiazine plus pyrimethamine, an oral regimen more often used

1	Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; TMP-SMX, trimethoprimsulfamethoxazole. for treatment of toxoplasmosis, also is highly effective. Dapsone 1361 plus pyrimethamine or dapsone plus trimethoprim also can be used. Intravenous pentamidine or the combination of clindamycin plus primaquine is an option for patients who cannot tolerate TMP-SMX and for patients in whose treatment TMP-SMX appears to be failing. Pentamidine must be given IV over at least 60 min to avoid potentially lethal hypotension. Adverse effects can be severe and irreversible and include renal dysfunction, dysglycemia (life-threatening hypoglycemia that can occur days or weeks after initial infusion and be followed by hyperglycemia), neutropenia, and torsades des pointes. Clindamycin plus primaquine is effective, but primaquine can be given only by the oral route—a disadvantage for patients who cannot ingest or absorb oral drugs.

1	A major advance in therapy for PCP was the recognition that glucocorticoids could improve survival rates among HIV-infected patients with moderate to severe disease (room air PO2, <70 mmHg; or alveolar-arterial oxygen gradient, ≥35 mmHg). Glucocorticoids appear to reduce the pulmonary inflammation that occurs after specific therapy is started and organisms begin to die, eliciting inflammation. Therapy with glucocorticoids should be the standard of care for patients with HIV infection and probably is also effective for patients with other immunodeficiencies. This treatment should be started for moderate or severe disease when therapy for PCP is initiated, even if the diagnosis has not yet been confirmed. If HIV-infected or HIV-uninfected patients are receiving high-dose glucocorticoids when they develop PCP, there are theoretical advantages to increasing or decreasing the steroid dose, but there is no convincing evidence on which to base any specific strategy.

1	No definitive trials have defined the best therapeutic algorithm for patients in whom TMP-SMX treatment for PCP is failing. If no other treatable infectious or noninfectious processes are detected and pulmonary dysfunction appears to be due to PCP alone, many authorities would switch from TMP-SMX to either IV pentamidine or IV clindamycin plus oral primaquine. Some authorities would add the second drug or drug combination to TMP-SMX rather than switching regimens. If patients are not already receiving them, glucocorticoids should be added to the regimen; the dosage and regimen, which are usually chosen empirically, depend on what glucocorticoid regimen (if any) the patient was receiving when PCP therapy was begun.

1	For patients with HIV infection who present with PCP before the initiation of ART, ART should be started within the first 2 weeks of therapy for PCP in most cases. Immune reconstitution inflammatory syndrome (IRIS) can occur, however, and the decision to initiate ART thus requires considerable expertise in terms of optimal timing relative to PCP recovery as well as in the other factors that are relevant when ART is initiated in any patient.

1	The most effective method for preventing PCP is to eliminate the cause of immunosuppression by withdrawing immunosuppressive therapy or treating the underlying cause, e.g., HIV infection. Patients who are susceptible to PCP benefit from chemoprophylaxis during the period of susceptibility. For patients with HIV infection, CD4+ T cell counts are a reliable marker of susceptibility, and counts below 200 cells/μL are an indication to start prophylaxis (Table 244-2). For patients with HIV infection who are not receiving ART, oral candidiasis or prior PCP also is an indication for chemoprophylaxis, regardless of CD4+ T cell count. For such patients not receiving ART, any prior episode of an AIDS-defining illness or pneumonia should encourage the use of chemoprophylaxis. However, patients who are not adherent to ART are not likely to take PCP chemoprophylaxis.

1	For patients without HIV infection, there is no laboratory parameter, including the CD4+ T cell count, that predicts susceptibility to PCP with adequate positive and negative accuracy. The period of susceptibility is usually estimated on the basis of experience with the Drug(s) Dose, Route Comments TMP-SMX 1 tablet (doubleor Incidence of hypersensisingle-strength) qd PO tivity is high. Rechallenge for nonlife-threatening hypersensitivity; consider dose-escalation protocol. optimal absorption. Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; TMP-SMX, trimethoprimsulfamethoxazole.

1	optimal absorption. Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; TMP-SMX, trimethoprimsulfamethoxazole. underlying disease and immunosuppressive regimen. Patients receiving a prolonged course of high-dose glucocorticoids appear to be particularly susceptible to PCP. The glucocorticoid exposure threshold that warrants chemoprophylaxis is controversial, but such preventive therapy should be strongly considered for any patient receiving more than the equivalent of 20 mg of prednisone daily for 30 days. TMP-SMX is the most effective prophylactic drug: few patients experience a PCP breakthrough when they are reliably taking a recommended TMP-SMX chemoprophylactic regimen. Several TMPSMX regimens have been used successfully. One double-strength tablet daily is the regimen with which there is the most experience, but either one single-strength tablet daily or one double-strength tablet two or three times weekly also has been recommended for various populations of patients.

1	For patients who cannot tolerate TMP-SMX (usually because of hypersensitivity or bone marrow suppression), alternative drugs include daily dapsone, weekly dapsone-pyrimethamine, and monthly aerosol pentamidine. Patients who develop hypersensitivity to TMP-SMX can sometimes tolerate the drug if a gradual dose-escalation protocol is used. Dapsone cross-reacts with sulfonamides in a substantial fraction of patients and therefore is rarely useful in patients with a history of life-threatening reactions to TMP-SMX. Aerosolized pentamidine is highly effective, but it is not as effective as TMP-SMX and may not provide protection in areas of the lung that are not well ventilated. Atovaquone is also effective and well tolerated; however, this drug is available only as an oral preparation, and gastrointestinal absorption is unpredictable in patients with abnormal gastrointestinal motility or function.

1	245e-1 Laboratory Diagnosis of Parasitic Infections Sharon L. Reed, Charles E. Davis The cornerstone for the diagnosis of parasitic infections is a thorough history of the patient’s illness. Epidemiologic aspects of the illness are especially important because the risks of acquiring many parasites T. saginata are morphologically indistinguishable from those of Taenia solium (the cause of cysticercosis), motility is an important distinguishing characteristic. Microscopic examination of feces is not complete until direct wet mounts have been evaluated and concentration techniques as well as permanent stains have been applied. Before accepting a report of negativity for ova and parasites as final, the physician should insist that the laboratory undertake each of these procedures. Some intestinal parasites are more readily detected in material other than feces. For example, examination of duodenal contents is sometimes necessary to 245e SECTIon 17 PRoTozoAL AnD HELmInTHIC InfECTIonS:

1	parasites are more readily detected in material other than feces. For example, examination of duodenal contents is sometimes necessary to 245e SECTIon 17 PRoTozoAL AnD HELmInTHIC InfECTIonS: GEnERAL ConSIDERATIonS are closely related to occupation, recreation, or travel to areas of high endemicity. Without a basic knowledge of the epidemiology and life cycles of the major parasites, it is difficult to approach the diagnosis of parasitic infections systematically. Accordingly, the medical classification of important human parasites in this chapter emphasizes their geographic distribution, their transmission, and the anatomic location and stages of their life cycle in humans. The text and tables are intended to serve as a guide to the correct diagnostic procedures for the major parasitic infections; in addition, the reader is referred to other chapters that contain more comprehensive information about each infection (Chaps. 247–260). Tables 245e-1, 245e-2, and 245e-3 summarize the

1	infections; in addition, the reader is referred to other chapters that contain more comprehensive information about each infection (Chaps. 247–260). Tables 245e-1, 245e-2, and 245e-3 summarize the geographic distributions, the anatomic locations, and the methods employed for the diagnosis of flatworm, roundworm, and protozoal infections, respectively.

1	In addition to selecting the correct diagnostic procedures, physicians must counsel their patients to ensure that specimens are collected properly and arrive at the laboratory promptly. For example, the diagnosis of bancroftian filariasis is unlikely to be confirmed by the laboratory unless blood is drawn near midnight, when the nocturnal microfilariae are active. Laboratory personnel and surgical pathologists should be notified in advance when a parasitic infection is suspected. Continuing interaction with the laboratory staff and the surgical pathologists increases the likelihood that parasites in body fluids or biopsy specimens will be examined carefully by the most capable individuals.

1	Most helminths and protozoa exit the body in the fecal stream. Many laboratories now use a stool collection kit with instructions for patients to transfer portions of the sample directly into bacterial carrier medium and fixative, which increases yield. If kits are not available, the patient should be instructed to collect feces in a clean waxed or cardboard container and to record the time of collection on the container. Refrigeration will preserve trophozoites for a few hours and protozoal cysts and helminthic ova for several days. Contamination with water (which could contain free-living protozoa) or with urine (which can damage trophozoites) should be avoided. Fecal samples should be collected before ingestion of barium or other contrast agents for radiologic procedures and before treatment with antidiarrheal agents and antacids; these substances change the consistency of the feces and interfere with microscopic detection of parasites. Because of the cyclic shedding of most

1	treatment with antidiarrheal agents and antacids; these substances change the consistency of the feces and interfere with microscopic detection of parasites. Because of the cyclic shedding of most parasites in the feces, a minimum of three samples collected on alternate days should be examined. Examination of a single sample can be up to 50% less sensitive.

1	Analysis of fecal samples entails both macroscopic and microscopic examination. Watery or loose stools are more likely to contain protozoal trophozoites, but protozoal cysts and all stages of helminths may be found in formed feces. If adult worms or tapeworm segments are observed, they should be transported promptly to the laboratory or washed and preserved in fixative for later examination. The only tapeworm with motile segments is Taenia saginata, the beef tapeworm, which patients sometimes bring to the physician. Because the ova of detect Giardia lamblia, Cryptosporidium, and Strongyloides larvae. Use of the “cellophane tape” technique to detect pinworm ova on the perianal skin sometimes also reveals ova of T. saginata deposited perianally when the motile segments disintegrate (Table 245e-4).

1	Two routine solutions are used to make wet mounts for identification of the various life stages of helminths and protozoa: physiologic saline for trophozoites, cysts, ova, and larvae and dilute iodine solution for protozoal cysts and ova. Iodine solution must never be used to examine specimens for trophozoites because it kills the parasites and thus eliminates their characteristic motility. The two most common concentration procedures for detecting small numbers of cysts and ova are formalin-ether sedimentation and zinc sulfate flotation. The formalin-ether technique is preferable because all parasites sediment but not all float. Slides permanently stained for trophozoites should be prepared before concentration. Additional slides stained for cysts and ova may be made from the concentrate.

1	In many instances, especially in the differentiation of Entamoeba histolytica from other amebas, identification of parasites from wet mounts or concentrates must be considered tentative. Permanently stained smears allow study of the cellular detail necessary for definitive identification. The iron-hematoxylin stain is excellent for critical work, but trichrome staining, which can be completed in 1 h, is a satisfactory alternative that also reveals parasites in specimens preserved in polyvinyl alcohol fixative. Modified acid-fast staining and fluorescent auramine microscopy are useful adjuncts for detection and identification of several intestinal protozoa, including Cryptosporidium and Cyclospora, while direct fluorescent antibody testing is in common use for Cryptosporidium and Giardia. Microsporidia, which cause chronic diarrhea in HIV-infected patients, may be missed unless a special modified trichrome stain or fluorescent screening with calcofluor white is requested (Table

1	Microsporidia, which cause chronic diarrhea in HIV-infected patients, may be missed unless a special modified trichrome stain or fluorescent screening with calcofluor white is requested (Table 245e-3).

1	Invasion of tissue by protozoa and helminths renders the choice of diagnostic techniques more difficult. For example, physicians must understand that aspiration of an amebic liver abscess rarely reveals

1	E. histolytica because the trophozoites are located primarily in the abscess wall. They must remember that the urine sediment offers the best opportunity to detect Schistosoma haematobium in the young Ethiopian immigrant or the American traveler who returns from Africa with hematuria. Tables 245e-1, 245e-2, and 245e-3, which offer a quick guide to the geographic distribution and anatomic locations of the major tissue parasites, should help the physician to select the appropriate body fluid or biopsy site for microscopic examination. Tables 245e-5 and 245e-6 provide additional information about the identification of parasites in samples from specific anatomic locations. The laboratory procedures for detection of parasites in other body fluids are similar to those used in the examination of feces. The physician should insist on wet mounts, concentration techniques, and permanent stains for all body fluids. The trichrome or iron-hematoxylin stain is satisfactory for all tissue helminths

1	feces. The physician should insist on wet mounts, concentration techniques, and permanent stains for all body fluids. The trichrome or iron-hematoxylin stain is satisfactory for all tissue helminths in body fluids other than blood,

1	CHAPTER 245e Laboratory Diagnosis of Parasitic Infections aLarvae also can mature in intestinal villi of humans and mice. bWhen there are two intermediate hosts, the first is separated from the second by a dash. Definitive hosts are infected by the second intermediate host. cT. solium can cause either intestinal infections or cysticercosis. Its ova are identical to those of T. saginata; scolices and segments of the two species differ. dOva seldom reach the fecal stream during acute disease. Note: CNS, central nervous system; EIA, enzyme immunoassay; WB, western blot. Serologic tests listed in Tables 245e-1, 245e-2, and 245e-3 are available commercially or from the Centers for Disease Control and Prevention, Atlanta, GA. but microfilarial worms and blood protozoa are more easily visualized a polycarbonate filter (pore size, 3–5 μm) facilitates the detection of with Giemsa or Wright’s staining. microfilariae. The intracellular amastigote forms of Leishmania species

1	The parasites most commonly detected in Giemsa-stained blood and T. cruzi can sometimes be visualized in stained smears of periphsmears are the plasmodia, microfilariae, and African trypanosomes eral blood, but aspirates of the bone marrow, liver, and spleen are the (Table 245e-5). Most patients with Chagas’ disease present in the best sources for microscopic detection and culture of Leishmania in chronic phase, when Trypanosoma cruzi is no longer microscopically kala-azar and of T. cruzi in chronic Chagas’ disease. The diagnosis of detectable in blood smears. Wet mounts are sometimes more sensitive malaria and the critical distinction among the various Plasmodium than stained smears for the detection of microfilariae and African try-species are made by microscopic examination of stained thick and thin panosomes because these active parasites cause noticeable movement blood films (Chap. 248). Because the lab infrastructure and technical of the erythrocytes in the microscopic field.

1	thick and thin panosomes because these active parasites cause noticeable movement blood films (Chap. 248). Because the lab infrastructure and technical of the erythrocytes in the microscopic field. Filtration of blood through expertise may not be available in many rural areas with high levels of

1	Trichuris trichiura Temperate and Soil, fecal-oral Humans Ova Feces — Rectal prolapse (whipworm) tropical zones Ascaris lumbricoi-Temperate and Soil, fecal-oral Humans Ova Feces — Sx of pulmonary des (roundworm tropical zones migration of humans) Ancylostoma duo-Eurasia, Africa, Soil to skin Humans Ova/larvae Feces — Sx of pulmonary denale (Old World Pacific migration, anemia hookworm) Necator america-U.S., Africa, Soil to skin Humans Ova/larvae Feces — Sx of pulmonary nus (New World worldwide migration, anemia hookworm) EIA, RAPID, PCRb Nocturnal periodicitya EIA, RAPID, PCRb Nocturnal LIPSb, PCRb May be visible in eye, diurnal LIPSb, PCRb Examine nodules or skin snips Ancylostoma bra-Tropical and tem-Soil to skin Dogs/cats, Larvae Skin — Dog and cat ziliense (creeping perate zones

1	Ancylostoma bra-Tropical and tem-Soil to skin Dogs/cats, Larvae Skin — Dog and cat ziliense (creeping perate zones Toxocara canis Tropical and tem-Soil, fecal-oral Dogs/cats, Larvae Viscera, CNS, eye EIA Also caused by and T. cati (visceral perate zones raccoons, roundworms of larva migrans), aExcept for infection acquired in the South Pacific, blood should be drawn at midnight. bLIPS (the luciferase immunoprecipitation system) for serology and PCR (polymerase chain reaction) are available from the Laboratory of Parasitic Diseases at the National Institutes of Health: 301-496-5398. Abbreviations: CNS, central nervous system; CSF, cerebrospinal fluid; EIA, enzyme immunoassay; RAPID, rapid immunographic assay (available internationally); Sx, signs/symptoms.

1	CHAPTER 245e Laboratory Diagnosis of Parasitic Infections malaria, rapid detection tests (RDTs) are increasingly being used to fill this gap. These are immunochromatographic capture assays with monoclonal antibodies to species-specific antigens (histidine-rich protein 2 [PfHRP2] or aldolase of Plasmodium falciparum) or conserved Plasmodium antigens (lactate dehydrogenase). The World Health Organization sponsored a major testing program evaluating the different RDTs. Lower performance rates have been reported in a variety of field sites, especially in areas where deletions of the pfhrp2 gene have been detected. Subpatent infection and identification of Plasmodium species can also be confirmed by polymerase chain reaction (PCR), but for this purpose PCR is primarily a research tool. P. knowlesi, a simian parasite, has been identified as the cause of an increasing number of infections in Malaysian Borneo and other areas of Southeast Asia; PCR or another molecular method is required to

1	a simian parasite, has been identified as the cause of an increasing number of infections in Malaysian Borneo and other areas of Southeast Asia; PCR or another molecular method is required to differentiate P. knowlesi from P. malariae.

1	Although most tissue parasites stain with the traditional hematoxylin and eosin, appropriate special stains should also be applied to surgical biopsy specimens. The surgical pathologist who is accustomed to applying silver stains for Pneumocystis to induced sputum and trans-bronchial biopsies may need to be reminded to examine wet mounts and iron-hematoxylin–stained preparations of pulmonary specimens for helminthic ova and E. histolytica. The clinician should also be able to advise the surgeon and pathologist about optimal techniques for the identification of parasites in specimens obtained by certain specialized minor procedures (Table 245e-6). For example, the excision of skin snips for the diagnosis of onchocerciasis, the collection of rectal snips aAcid-fastness is best demonstrated by auramine fluorescence or modified acid-fast stain. bContact the CDC at 404-718-4100. cCard agglutination is provided to endemic countries by the World Health Organization. dLimited specificity;

1	by auramine fluorescence or modified acid-fast stain. bContact the CDC at 404-718-4100. cCard agglutination is provided to endemic countries by the World Health Organization. dLimited specificity; most sensitive for L. donovani.

1	Abbreviations: CNS, central nervous system; CSF, cerebrospinal fluid; DFA, direct fluorescent antibody; EIA, enzyme immunoassay; IFA, indirect fluorescent antibody; IIF, indirect immunofluorescence; PCR, polymerase chain reaction; RDT, rapid detection test; RE, reticuloendothelial; troph, trophozoite; tryp, trypomastigote form. for the diagnosis of schistosomiasis, and punch biopsy of skin lesions Intestinal helminths provoke eosinophilia only during pulmonary for the identification and culture of cutaneous and mucocutaneous migration of the larval stages. Eosinophilia is not a manifestation species of Leishmania are simple procedures, but the diagnosis can be of protozoal infections. Parasitic causes of eosinophilia in cerebromissed if the specimens are improperly obtained or processed. spinal fluid include nematodes (e.g., Angiostrongylus, Gnathostoma, Toxocara, and Baylisascaris species) as well as flatworms (e.g., T. solium and Schistosoma species).

1	T. solium and Schistosoma species). Like the hypochromic microcytic anemia of heavy hookworm Eosinophilia (>500/μL) commonly accompanies infections with most infections, other nonspecific laboratory abnormalities may suggest of the tissue helminths; absolute numbers of eosinophils may be high parasitic infection in patients with appropriate geographic and/or in trichinellosis and the migratory phases of filariasis (Table 245e-7). environmental exposures. Biochemical evidence of cirrhosis or an T. solium ova and segments Serology; brain biopsy for neurocysticercosis Clonorchis (Opisthorchis) sinensis ova Examination of bile for ova and adults in cholangitis Fasciola hepatica ova Examination of bile for ova and adults in cholangitis Paragonimus ova Serology; sputum; biopsy of lung or brain for larvae Schistosoma ova Serology for all; rectal snips (especially for S. mansoni), urine (S. haematobium), liver biopsy and liver ultrasound

1	Schistosoma ova Serology for all; rectal snips (especially for S. mansoni), urine (S. haematobium), liver biopsy and liver ultrasound Ascaris lumbricoides ova and adults Examination of sputum for larvae in lung disease Hookworm ova and occasional larvae Examination of sputum for larvae in lung disease Microsporidial spores Duodenal aspirate or jejunal biopsy aStains and concentration techniques are discussed in the text. bIsospora and Cryptosporidium are acid-fast. Body Fluid, Parasite Enrichment/Stain Culture Technique Plasmodium spp. Thick and thin smears/ Not useful for diagnosis Giemsa or Wright’s Leishmania spp. Buffy coat/Giemsa Media available from CDC

1	Plasmodium spp. Thick and thin smears/ Not useful for diagnosis Giemsa or Wright’s Leishmania spp. Buffy coat/Giemsa Media available from CDC African trypanosomesa Buffy coat, anion Mouse or rat column/wet mount and inoculationb Giemsa aTrypanosoma rhodesiense and T. gambiense. bInject mice intraperitoneally with 0.2 mL of whole heparinized blood (0.5 mL for rats). After 5 days, check tail blood daily for trypanosomes as described above. Call the CDC (770-488-7775) for information on diagnosis and treatment. cDetectable in blood by conventional techniques only during acute disease. Xenodiagnosis is successful in ∼50% of patients with chronic Chagas’ disease. dDaytime (1000–1400 h) and nighttime (2200–0200 h) blood samples should be drawn to maximize the chance of detecting Wuchereria (nocturnal except for Pacific strains), Brugia (nocturnal), and Loa loa (diurnal). Loa loa adults and O. volvulus adults and microfilariae Schistosoma ova of all species, but especially S. mansoni

1	Loa loa adults and O. volvulus adults and microfilariae Schistosoma ova of all species, but especially S. mansoni T. rhodesiense trypomastigotes Acanthamoeba spp. trophozoites or cysts Cutaneous and mucocutaneous Leishmania spp. Skin snips: Lift skin with a needle and excise ∼1 mg to a depth of 0.5 mm from several sites. Weigh each sample, place it in 0.5 mL of saline for 4 h, and examine wet mounts and Giemsa stains of the saline either directly or after filtration. Count microfilariae.a Biopsies of subcutaneous nodules: Stain routine histopathologic sections and impression smears with Giemsa. Muscle biopsies: Excise ∼1.0 g of deltoid or gastrocnemius muscle and squash between two glass slides for direct microscopic examination. Rectal snips: From four areas of mucosa, take 2-mg snips, tease onto a glass slide, and flatten with a second slide before examining directly at 10×. Preparations may be fixed in alcohol or stained.

1	Rectal snips: From four areas of mucosa, take 2-mg snips, tease onto a glass slide, and flatten with a second slide before examining directly at 10×. Preparations may be fixed in alcohol or stained. Aspirate of chancre or lymph node:b Aspirate center with an 18-gauge needle, place a drop on a slide, and examine for motile forms. An otherwise insufficient volume of material may be stained with Giemsa. Corneal scrapings: Have an ophthalmologist obtain a sample for immediate Giemsa staining and culture on nutrient agar overlaid with Escherichia coli. Swabs, aspirates, or punch biopsies of skin lesions: Obtain a specimen from the margin of a lesion for Giemsa staining of impression smears; section and culture on special media from the CDC. CHAPTER 245e Laboratory Diagnosis of Parasitic Infections aCounts of >100/mg are associated with a significant risk of complications. bLymph node aspiration is contraindicated in some infections and should be used judiciously.

1	Taenia solium During muscle encystation and in cerebrospinal fluid with neurocysticercosis Paragonimus spp. Uniformly high in acute stage Fasciola hepatica May be high in acute stage Clonorchis (Opisthorchis) sinensis Variable Schistosoma mansoni 50% of infected travelers S. haematobium 25% of infected travelers S. japonicum Up to 6000/μL in acute infection aVirtually every helminth has been associated with eosinophilia. This table includes both common and uncommon parasites that frequently elicit eosinophilia during infection. bWuchereria bancrofti, Brugia spp., Loa loa, and Onchocerca volvulus.

1	abnormal urine sediment in an African immigrant certainly raises the possibility of schistosomiasis, and anemia and thrombocytopenia in a febrile traveler or immigrant are among the hallmarks of malaria. CT and MRI also contribute to the diagnosis of infections with many tissue parasites and have become invaluable adjuncts in the diagnosis of neurocysticercosis and cerebral toxoplasmosis. Useful antibody assays for many of the important tissue parasites are available; most of those listed in Table 245e-8 can be obtained from the Centers for Disease Control and Prevention (CDC) in Atlanta. The results of serologic tests not listed in the tables should be interpreted with caution.

1	The value of antibody assays is limited by several factors. For example, the preparation of thick and thin blood smears remains the procedure of choice for the diagnosis of malaria in individual patients because diagnostic titers to plasmodia develop slowly and do not differentiate species—a critical step in patient management. Filarial antigens cross-react with those from other nematodes; as in assays for antibody to most parasites, the presence of antibody in the filarial assay fails to distinguish between past and current infection. Despite these specific limitations, the restricted geographic distribution of many tropical parasites increases the diagnostic usefulness of both the presence and the absence of antibody in travelers from industrialized countries. In contrast, a large proportion of the world’s population has been exposed to Toxoplasma gondii, and the presence of IgG antibody to T. gondii does not constitute proof of active disease.

1	Fewer antibody assays are available for the diagnosis of infection with intestinal parasites. E. histolytica is the major exception. Sensitive, Amebiasis EIA EIA,b RAPID,b PCR Giardiasis EIA,b RAPID,b DFA, PCR Cryptosporidiosis EIA,b DFA, RAPID,b PCR Malaria (all species) IIFd RAPID, PCR Babesiosis IIF PCR Chagas’ disease IIF, EIA PCR Leishmaniasis IIF, EIA PCRb Toxoplasmosis IIF, EIA (IgM)e PCRb Microsporidiosis DFA, PCR Naegleriasis DFA, PCR Balamuthiasis aUnless otherwise noted, all tests are available at the CDC. bResearch or commercial laboratories only. cAvailable at the NIH (301-496-5398) and commercially. dOf limited use for management of acute disease. eDetermination of infection within the last 3 months may require additional tests by a research laboratory.

1	Note: DFA, direct fluorescent antibody; EIA, enzyme immunoassay; IIF, indirect immunofluorescence; PCR, polymerase chain reaction; RAPID, rapid immunographic assay; WB, western blot. Most antigen and antibody parasite detection kits are available commercially. Most PCRs listed are now available at the CDC and in commercial or research laboratories. Contact the CDC (404-718-4100). specific serologic tests are invaluable in the diagnosis of amebiasis. Commercial kits for the detection of antigen by enzyme-linked immunosorbent assay or of whole organisms by fluorescent antibody assay are now available for several protozoan parasites (Table 245e-8).

1	DNA hybridization with probes that are repeated many times in the genome of a specific parasite and amplification of a specific DNA fragment by PCR have now been established as useful techniques for the diagnosis of several protozoan infections (Table 245e-8). Although PCR is very sensitive, it is an adjunct to conventional techniques for parasite detection and should be requested only when microscopic and immunodiagnostic procedures fail to establish the probable diagnosis. For example, only multiple negative blood smears or the failure to identify the infecting species justifies PCR for the diagnosis or proper management of malaria. In addition to PCR of anticoagulated blood, the CDC (www.cdc.gov/dpdx/) and several commercial laboratories now perform PCRs for detection of certain specific parasites in stool samples, biopsy specimens, and bronchoalveolar lavage fluid (Table 245e-8). Although PCRs are now used primarily for the detection of protozoans, active research efforts are

1	parasites in stool samples, biopsy specimens, and bronchoalveolar lavage fluid (Table 245e-8). Although PCRs are now used primarily for the detection of protozoans, active research efforts are likely to establish their feasibility for the detection of several helminths.

1	Agents Used to Treat Parasitic Infections Thomas A. Moore Parasitic infections afflict more than half of the world’s population and impose a substantial health burden, particularly in underdeveloped 246e

1	CHAPTER 246e Agents Used to Treat Parasitic Infections nations, where they are most prevalent. The reach of some parasitic diseases, including malaria, has expanded over the past few decades as a result of factors such as deforestation, population shifts, global warming, and other climatic events. Despite major efforts at vaccine development and vector control, chemotherapy remains the single most effective means of controlling parasitic infections. Efforts to combat the spread of some diseases are hindered by the development and spread of drug resistance, the limited introduction of new antiparasitic agents, and the proliferation of counterfeit medications. However, there are good reasons to be optimistic. Ambitious global initiatives aimed at controlling or eliminating threats such as AIDS, tuberculosis, and malaria have demonstrated some early successes. Recognition of the substantial burden imposed by the “neglected” tropical diseases has generated multinational partnerships to

1	AIDS, tuberculosis, and malaria have demonstrated some early successes. Recognition of the substantial burden imposed by the “neglected” tropical diseases has generated multinational partnerships to develop and deploy effective antiparasitic agents. Vaccines against several tropical diseases are being developed, and clinical trials for vaccines against parasites continue.

1	This chapter deals exclusively with the agents used to treat infections due to parasites. Specific treatment recommendations for the parasitic diseases of humans are listed in subsequent chapters. Many of the agents discussed herein are approved by the U.S. Food and Drug Administration (FDA) but are considered investigational for the treatment of certain infections. Drugs marked in the text with an asterisk (*) are available through the Centers for Disease Control and Prevention (CDC) Drug Service (telephone: 404-639-3670 or 404-639-2888; www.cdc.gov/ncpdcid/dsr/). Drugs marked with a dagger (†) are available only through their manufacturers; contact information for these manufacturers may be available from the CDC. Table 246e-1 presents a brief overview of each agent (including some drugs that are covered in other chapters), along with major toxicities, spectrum of activity, and safety for use during pregnancy 246e-1 and lactation.

1	Albendazole Like all benzimidazoles, albendazole acts by selectively binding to free β-tubulin in nematodes, inhibiting the polymerization of tubulin and the microtubule-dependent uptake of glucose. Irreversible damage occurs in gastrointestinal (GI) cells of the nematodes, resulting in starvation, death, and expulsion by the host. This fundamental disruption of cellular metabolism offers treatment for a wide range of parasitic diseases.

1	Albendazole is poorly absorbed from the GI tract. Administration with a fatty meal increases its absorption by twoto sixfold. Poor absorption may be advantageous for the treatment of intestinal helminths, but successful treatment of tissue helminth infections (e.g., hydatid disease and neurocysticercosis) requires that a sufficient amount of active drug reach the site of infection. The metabolite albendazole sulfoxide is responsible for the drug’s therapeutic effect outside the gut lumen. Albendazole sulfoxide crosses the blood-brain barrier, reaching a level significantly higher than that achieved in plasma. The high concentrations of albendazole sulfoxide attained in cerebrospinal fluid (CSF) may explain the efficacy of albendazole in the treatment of neurocysticercosis.

1	Albendazole is extensively metabolized in the liver, but there are few data regarding the drug’s use in patients with hepatic disease. Single-dose albendazole therapy in humans is largely without side effects (overall frequency, ≤1%). More prolonged courses (e.g., as administered for cystic and alveolar echinococcal disease) have been associated with liver function abnormalities and bone marrow toxicity. Thus, when prolonged use is anticipated, the drug should be administered in treatment cycles of 28 days interrupted by 14-day intervals off therapy. Prolonged therapy with full-dose albendazole (800 mg/d) should be approached cautiously in patients also receiving drugs with known effects on the cytochrome P450 system. Amodiaquine Amodiaquine has been widely used in the treatment of malaria for >40 years. Like chloroquine (the other major 4-aminoquinoline), amodiaquine is now of limited use because of the spread of resistance. Amodiaquine interferes with hemozoin formation

1	Amodiaquine Malariab Agranulocytosis, hepatotoxicity Chloroquine Malariab Occasional: pruritus, nausea, vomiting, headache, hair depigmentation, exfoliative dermatitis, reversible corneal opacity. Rare: irreversible retinal injury, nail discoloration, blood dyscrasias Primaquine Malariab Frequent: hemolysis in patients with G6PD deficiency Occasional: methemoglobinemia, GI disturbances. Rare: CNS symptoms Tafenoquine Malariab Frequent: hemolysis in patients with G6PD deficiency, mild GI upset. Occasional: methemoglobinemia, headaches No information Not No assigned information Antacids and kaolin: reduced Not Yes absorption of chloroquine assignedc Ampicillin: bioavailability reduced by chloroquine Cimetidine: increased serum levels of chloroquine Cyclosporine: serum levels increased by chloroquine Quinacrine: potentiated Contraindicated No information toxicity of primaquine

1	Cimetidine: increased serum levels of chloroquine Cyclosporine: serum levels increased by chloroquine Quinacrine: potentiated Contraindicated No information toxicity of primaquine Paromomycin Amebiasis,b infection with Dientamoeba fragilis, giardiasis, cryptosporidiosis, leishmaniasis Amphotericin B Leishmaniasis,d amebic meningoencephalitis Amphotericin B lipid complex, ABLC (Abelcet) Amphotericin B, liposomal (AmBisome) Atovaquone Malaria,b babesiosis Frequent: abdominal pain, diarrhea. Occasional: ECG disturbances (dose-related prolongation of QTc and PR interval), nausea, pruritus. Contraindicated in persons who have cardiac disease or who have taken mefloquine in the preceding 3 weeks Occasional: nausea, vomiting, diarrhea, abdominal pain, anorexia, headache, dizziness Frequent: GI disturbances (oral dosing only). Occasional: nephrotoxicity, ototoxicity, vestibular toxicity (parenteral dosing only)

1	Frequent: GI disturbances (oral dosing only). Occasional: nephrotoxicity, ototoxicity, vestibular toxicity (parenteral dosing only) Frequent: fever, chills, hypokalemia, hypomagnesemia, nephrotoxicity. Occasional: vomiting, dyspnea, hypotension Frequent: arthralgias/myalgias, pancreatitis, ECG changes (QT prolongation, T wave flattening or inversion) Frequent: arthralgias/myalgias, pancreatitis, ECG changes (QT prolongation, T wave flattening or inversion) Occasional: neurotoxicity (ataxia, convulsions), nausea, vomiting, anorexia, contact dermatitis Frequent: nausea, vomiting. Occasional: abdominal pain, headache Concomitant use of agents that prolong QTc interval contraindicated Antineoplastic agents: renal toxicity, bronchospasm, hypotension Glucocorticoids, ACTH, digitalis: hypokalemia Zidovudine: increased myeloand nephrotoxicity Antiarrhythmics and tricyclic antidepressants: increased risk of cardiotoxicity

1	Glucocorticoids, ACTH, digitalis: hypokalemia Zidovudine: increased myeloand nephrotoxicity Antiarrhythmics and tricyclic antidepressants: increased risk of cardiotoxicity Mefloquine: levels decreased and clearance accelerated by artesunate Mefloquine: increased absorption Plasma levels decreased by rifampin, tetracycline; bioavailability decreased by metoclopramide Albendazole Ascariasis, capillariasis, clonorchiasis, cutaneous larva migrans, cysticercosis,b echinococcosis,b enterobiasis, eosinophilic enterocolitis, gnathostomiasis, hookworm, lymphatic filariasis, microsporidiosis, strongyloidiasis, trichinellosis, trichostrongyliasis, trichuriasis, visceral larva migrans Mebendazole Ascariasis,b capillariasis, eosinophilic enterocolitis, enterobiasis,b hookworm,b trichinellosis, trichostrongyliasis, trichuriasis,b visceral larva migrans Thiabendazole Strongyloidiasis,b cutaneous larva migrans,b visceral larva migransb Triclabendazole Fascioliasis, paragonimiasis

1	Thiabendazole Strongyloidiasis,b cutaneous larva migrans,b visceral larva migransb Triclabendazole Fascioliasis, paragonimiasis Clindamycin Babesiosis, malaria, toxoplasmosis Eflornithineh (difluo-Trypanosomiasis romethylornithine, DFMO) Occasional: nausea, vomiting, abdominal pain, headache, reversible alopecia, elevated aminotransferases. Rare: leukopenia, rash Occasional: diarrhea, abdominal pain, elevated aminotransferases. Rare: agranulocytosis, thrombocytopenia, alopecia Frequent: anorexia, nausea, vomiting, diarrhea, headache, dizziness, asparagus-like urine odor. Occasional: drowsiness, giddiness, crystalluria, elevated aminotransferases, psychosis. Rare: hepatitis, seizures, angioneurotic edema, Stevens-Johnson syndrome, tinnitus Occasional: abdominal cramps, diarrhea, biliary colic, transient headache Frequent: rash, pruritus, nausea, leukopenia, paresthesias

1	Occasional: abdominal cramps, diarrhea, biliary colic, transient headache Frequent: rash, pruritus, nausea, leukopenia, paresthesias Occasional: pseudomembranous colitis, abdominal pain, diarrhea, nausea/vomiting. Rare: pruritus, skin rashes Frequent: flatulence. Occasional: nausea, vomiting, diarrhea. Rare: pruritus Frequent: pancytopenia. Occasional: diarrhea, seizures. Rare: transient hearing loss vinca alkaloids, pimozide, alprazolam, diazepam, midazolam, triazolam, verapamil, atorvastatin, cerivastatin, lovastatin, simvastatin, tacrolimus, sirolimus, indinavir, ritonavir, saquinavir, alfentanil, buspirone, methylprednisolone, trimetrexate: plasma levels increased by azoles Carbamazepine, phenobarbital, phenytoin, isoniazid, rifabutin, rifampin, antacids, H2-receptor antagonists, proton pump inhibitors, nevirapine: decreased plasma levels of azoles Clarithromycin, erythromycin, indinavir, ritonavir: increased plasma levels of azoles

1	Clarithromycin, erythromycin, indinavir, ritonavir: increased plasma levels of azoles Dexamethasone, praziquantel: plasma level of albendazole sulfoxide increased by ~50% Cimetidine: inhibited mebendazole metabolism Theophylline: serum levels increased by thiabendazole CHAPTER 246e Agents Used to Treat Parasitic Infections diarrhea, abdominal pain. Rare: angioedema, cholestatic jaundice levels increased by azithromycin Nelfinavir: increased levels of azithromycin Miltefosine Leishmaniasisb, primary amebic meningoencephalitis Nitazoxanide Cryptosporidiosis,b giardiasisb Metronidazole Amebiasis,b balantidiasis, dracunculiasis, giardiasis, trichomoniasis,b D. fragilis infection Tinidazole Amebiasis,b giardiasis, trichomoniasis Paromomycin Amebiasis,b D. fragilis infection, giardiasis, cryptosporidiosis, leishmaniasis Occasional: GI disturbances, transient skin eruptions. Rare: thrombocytopenia, QT prolongation in an infant, cholestatic hepatitis

1	Frequent: lightheadedness, nausea, headache. Occasional: confusion; nightmares; insomnia; visual disturbance; transient and clinically silent ECG abnormalities, including sinus bradycardia, sinus arrhythmia, first-degree AV block, prolongation of QTc interval, and abnormal T waves. Rare: psychosis, convulsions, hypotension Frequent: myocardial injury, encephalopathy, peripheral neuropathy, hypertension. Occasional: G6PD-induced hemolysis, erythema nodosum leprosum. Rare: hypotension Frequent: abdominal pain, nausea, vomiting, diarrhea, headache, vertigo, bronchospasm. Rare: cholinergic symptoms Frequent: mild and transient (1–2 days) GI disturbances within first 2 weeks of therapy (resolve after treatment completion); motion sickness. Occasional: reversible elevations of creatinine and aminotransferases Occasional: nausea, vomiting, dizziness, pruritus

1	Occasional: nausea, vomiting, dizziness, pruritus Frequent: nausea, vomiting, abdominal pain, insomnia, paresthesias, weakness, tremors. Rare: seizures (all reversible and dose-related) Occasional: abdominal pain, diarrhea. Rare: vomiting, headache Frequent: nausea, headache, anorexia, metallic aftertaste. Occasional: vomiting, insomnia, vertigo, paresthesias, disulfiram-like effects. Rare: seizures, peripheral neuropathy Occasional: nausea, vomiting, metallic taste Occasional: dizziness, drowsiness, headache, orange urine, elevated aminotransferases. Rare: seizures Frequent: GI disturbances (oral dosing only). Occasional: nephrotoxicity, ototoxicity, vestibular toxicity (parenteral dosing only) No major interactions Not assignedc Yesg Administration of halofan-C Yes trine <3 weeks after mefloquine use may produce fatal QTc prolongation. Mefloquine may lower plasma levels of anticonvulsants. Levels are decreased and clearance is accelerated by artesunate.

1	Warfarin: effect enhanced by B Yes metronidazole Disulfiram: psychotic reaction Phenobarbital, phenytoin: accelerate elimination of metronidazole Lithium: serum levels elevated by metronidazole Cimetidine: prolonged half-life of metronidazole See metronidazole. C Yes No major interactions Oral: B No information Parenteral: not assignedc CHAPTER 246e Agents Used to Treat Parasitic Infections Pentamidine isethionate Leishmaniasis, trypanosomiasis Piperazine Ascariasis, enterobiasis Diethylcarbamazinee Lymphatic filariasis, loiasis, tropical pulmonary eosinophilia Praziquantel Clonorchiasis,b cysticercosis, diphyllobothriasis, hymenolepiasis, taeniasis, opisthorchiasis, intestinal trematodes, paragonimiasis, schistosomiasisb Pyrantel pamoate Ascariasis, eosinophilic enterocolitis, enterobiasis,b hookworm, trichostrongyliasis Quinine and quinidine Malaria, babesiosis Ciprofloxacin Cyclosporiasis, isosporiasis

1	Pyrantel pamoate Ascariasis, eosinophilic enterocolitis, enterobiasis,b hookworm, trichostrongyliasis Quinine and quinidine Malaria, babesiosis Ciprofloxacin Cyclosporiasis, isosporiasis Frequent: hypotension, hypoglycemia, pancreatitis, sterile abscesses at IM injection sites, GI disturbances, reversible renal failure. Occasional: hepatotoxicity, cardiotoxicity, delirium. Rare: anaphylaxis Occasional: nausea, vomiting, diarrhea, abdominal pain, headache. Rare: neurotoxicity, seizures Frequent: dose-related nausea, vomiting. Rare: fever, chills, arthralgias, headaches Frequent: abdominal pain, diarrhea, dizziness, headache, malaise. Occasional: fever, nausea. Rare: pruritus, singultus Occasional: GI disturbances, headache, dizziness, elevated aminotransferases Occasional: headache, nausea

1	Occasional: GI disturbances, headache, dizziness, elevated aminotransferases Occasional: headache, nausea Frequent: headache, nausea, vomiting, bitter taste. Occasional: yellow-orange discoloration of skin, sclerae, urine; begins after 1 week of treatment and lasts up to 4 months after drug discontinuation. Rare: psychosis, exfoliative dermatitis, retinopathy, G6PD-induced hemolysis, exacerbation of psoriasis, disulfiram-like effects Frequent: cinchonism (tinnitus, high-tone deafness, headache, dysphoria, nausea, vomiting, abdominal pain, visual disturbances, postural hypotension), hyperinsulinemia resulting in life-threatening hypoglycemia. Occasional: deafness, hemolytic anemia, arrhythmias, hypotension due to rapid IV infusion Occasional: nausea, diarrhea, vomiting, abdominal pain/ discomfort, headache, restlessness, rash. Rare: myalgias/ arthralgias, tendon rupture, CNS symptoms (nervousness, agitation, insomnia, anxiety, nightmares or paranoia); convulsions

1	None reported to date B Yes Primaquine: toxicity potenti-C No information ated by quinacrine Carbonic anhydrase X Yesg inhibitors, thiazide diuretics: reduced renal elimination of quinidine Amiodarone, cimetidine: increased quinidine levels Nifedipine: decreased quinidine levels; quinidine slows metabolism of nifedipine Phenobarbital, phenytoin, rifampin: accelerated hepatic elimination of quinidine Verapamil: reduced hepatic clearance of quinidine Diltiazem: decreased clearance of quinidine Probenecid: increased serum C Yes levels of ciprofloxacin Theophylline, warfarin: serum levels increased by ciprofloxacin Tetracyclines Balantidiasis, D. fragilis infection, malaria; lymphatic filariasis (doxycycline) Frequent: immediate: fever, urticaria, nausea, vomiting, hypotension; delayed (up to 24 h): exfoliative dermatitis, stomatitis, paresthesias, photophobia, renal dysfunction. Occasional: nephrotoxicity, adrenal toxicity, optic atrophy, anaphylaxis

1	Frequent: GI disturbances. Occasional: photosensitivity dermatitis. Rare: exfoliative dermatitis, esophagitis, hepatotoxicity No major interactions Not assigned No information Warfarin: effect prolonged D Yes by tetracyclines aBased on U.S. Food and Drug Administration pregnancy categories of A–D, X. bApproved by the FDA for this indication. cUse in pregnancy is recommended by international organizations outside the United States. dOnly AmBisome has been approved by the FDA for this indication. eAvailable through the CDC. f Only artemether (in combination with lumefantrine) and artesunate have been approved by the FDA for this indication. gNot believed to be harmful. hAvailable through the manufacturer. Abbreviations: ACTH, adrenocorticotropic hormone; AV, atrioventricular; CNS, central nervous system; ECG, electrocardiogram; G6PD, glucose 6-phosphate dehydrogenase; GI, gastroin testinal; MAO, monoamine oxidase.

1	through complexation with heme. It is rapidly absorbed and acts as a prodrug after oral administration; the principal plasma metabolite monodesethylamodiaquine is the predominant antimalarial agent. Amodiaquine and its metabolites are all excreted in urine, but there are no recommendations concerning dosage adjustment in patients with impaired renal function. Agranulocytosis and hepatotoxicity can develop with repeated use; therefore, this drug should not be used for prophylaxis. Despite widespread resistance, amodiaquine has been shown to be effective in some areas when combined with other antimalarial drugs (e.g., artesunate, sulfadoxine-pyrimethamine), particularly in children. Although licensed, amodiaquine is not yet available in the United States. Amphotericin B See Table 246e-1 and Chap. 235.

1	Antimonials* Despite associated adverse reactions and the need for prolonged parenteral treatment, the pentavalent antimonial compounds (designated Sbv) have remained the first-line therapy for all forms of leishmaniasis throughout the world, primarily because they are affordable and effective and have survived the test of time. Pentavalent antimonials are active only after bioreduction to the trivalent Sb(III) form, which inhibits trypanothione reductase, a critical enzyme involved in the oxidative stress management of Leishmania species. The fact that Leishmania species use trypanothione rather than glutathione (which is used by mammalian cells) may explain the parasite-specific activity of antimonials. The drugs are taken up by the reticuloendothelial system, and their activity against Leishmania species may be enhanced by this localization. Sodium stibogluconate is the only pentavalent antimonial available in the United States; meglumine antimoniate is used principally in

1	against Leishmania species may be enhanced by this localization. Sodium stibogluconate is the only pentavalent antimonial available in the United States; meglumine antimoniate is used principally in francophone countries.

1	Resistance is a major problem in some areas. Although low-level unresponsiveness to Sbv was identified in India in the 1970s, incremental increases in both the recommended daily dosage (to 20 mg/kg) and the duration of treatment (to 28 days) satisfactorily compensated for the growing resistance until around 1990. There has since been steady erosion in the capacity of Sbv to induce long-term cure in patients with kala-azar who live in eastern India. Co-infection with HIV impairs the treatment response. Sodium stibogluconate is available in aqueous solution and is administered parenterally. Antimony appears to have two elimination phases. When the drug is administered IV, the mean half-life of the first phase is <2 h; the mean half-life of the terminal elimination phase is nearly 36 h. This slower phase may be due to conversion of pentavalent antimony to a trivalent form that is the likely cause of the side effects often seen with prolonged therapy.

1	Artemisinin Derivatives* Artesunate, artemether, arteether, and the parent compound artemisinin are sesquiterpene lactones derived from the wormwood plant Artemisia annua. These agents are at least tenfold more potent in vivo than other antimalarial drugs and presently show no cross-resistance with known antimalarial drugs; thus, they have become first-line agents for the treatment of severe falciparum malaria. The artemisinin compounds are rapidly effective against the asexual blood forms of Plasmodium species but are not active against intrahepatic forms. Artemisinin and its derivatives are highly lipid soluble and readily cross both host and parasite cell membranes. One factor that explains the drugs’ highly selective toxicity against malaria is that parasitized erythrocytes concentrate artemisinin and its derivatives to concentrations 100-fold higher than those in uninfected erythrocytes. The antimalarial effect of these agents results primarily from dihydroartemisinin, a compound

1	artemisinin and its derivatives to concentrations 100-fold higher than those in uninfected erythrocytes. The antimalarial effect of these agents results primarily from dihydroartemisinin, a compound to which artemether and artesunate are both converted. In the presence of heme or molecular iron, the endoperoxide moiety of dihydroartemisinin decomposes, generating free radicals and other metabolites that damage parasite proteins. The compounds are available for oral, rectal, IV, or IM administration, depending on the derivative. In the United States, IV artesunate is available for the treatment of severe, quinidine-unresponsive malaria through the CDC malaria hot-line (770-488-7788, M–F, 0800–1630 EST; 770-488-7100 after hours). Artemisinin and its derivatives are cleared rapidly from the circulation. Their short half-lives limit their value for prophylaxis and monotherapy. These agents should be used only in combination with another, longer-acting agent (e.g., artesunate-mefloquine,

1	circulation. Their short half-lives limit their value for prophylaxis and monotherapy. These agents should be used only in combination with another, longer-acting agent (e.g., artesunate-mefloquine, dihydroartemisininpiperaquine). A combined formulation of artemether and lumefantrine is available for the treatment of acute uncomplicated falciparum malaria acquired in areas where Plasmodium falciparum is resistant to chloroquine and antifolates.

1	Atovaquone Atovaquone is a hydroxynaphthoquinone that exerts broad-spectrum antiprotozoal activity via selective inhibition of parasite mitochondrial electron transport. This agent exhibits potent activity against toxoplasmosis and babesiosis when used with pyrimethamine and azithromycin, respectively. Atovaquone possesses a novel mode of action against Plasmodium species, inhibiting the electron transport system at the level of the cytochrome bc1 complex. The drug is active against both the erythrocytic and the exoerythrocytic stages of Plasmodium species; however, because it does not eradicate hypnozoites from the liver, patients with Plasmodium vivax or Plasmodium ovale infections must be given radical prophylaxis.

1	Malarone® is a fixed-dose combination of atovaquone and proguanil used for malaria prophylaxis as well as for the treatment of acute, uncomplicated P. falciparum malaria. Malarone has been shown to be effective in regions with multidrug-resistant P. falciparum. Resistance to atovaquone has yet to be reported. The bioavailability of atovaquone varies considerably. Absorption after a single oral dose is slow, increases twoto threefold with a fatty CHAPTER 246e Agents Used to Treat Parasitic Infections meal, and is dose-limited above 750 mg. The elimination half-life is increased in patients with moderate hepatic impairment. Because of the potential for drug accumulation, the use of atovaquone is generally contraindicated in persons with a creatinine clearance rate <30 mL/min. No dosage adjustments are needed in patients with mild to moderate renal impairment. Azithromycin See Table 246e-1 and Chap. 170. Azoles See Table 246e-1 and Chap. 235.

1	Azithromycin See Table 246e-1 and Chap. 170. Azoles See Table 246e-1 and Chap. 235. Benznidazole* This oral nitroimidazole derivative is used to treat Chagas’ disease, with cure rates of 80–90% recorded in acute infections. Benznidazole is believed to exert its trypanocidal effects by generating oxygen radicals to which the parasites are more sensitive than mammalian cells because of a relative deficiency in antioxidant enzymes. Benznidazole also appears to alter the balance between proand anti-inflammatory mediators by downregulating the synthesis of nitrite, interleukin (IL) 6, and IL-10 in macrophages. Benznidazole is highly lipophilic and readily absorbed. The drug is extensively metabolized; only 5% of the dose is excreted unchanged in the urine. Benznidazole is well tolerated; adverse effects are rare and usually manifest as GI upset or pruritic rash.

1	Chloroquine This 4-aminoquinoline has marked, rapid schizonticidal and gametocidal activity against blood forms of P. ovale and Plasmodium malariae and against susceptible strains of P. vivax and

1	P. falciparum. It is not active against intrahepatic forms (P. vivax and P. ovale). Parasitized erythrocytes accumulate chloroquine in significantly greater concentrations than do normal erythrocytes. Chloroquine, a weak base, concentrates in the food vacuoles of intraerythrocytic parasites because of a relative pH gradient between the extracellular space and the acidic food vacuole. Once it enters the acidic food vacuole, chloroquine is rapidly converted to a membrane-impermeable protonated form and is trapped. Continued accumulation of chloroquine in the parasite’s acidic food vacuoles results in drug levels that are 600-fold higher at this site than in plasma. The high accumulation of chloroquine results in an increase in pH within the food vacuole to a level above that required for the acid proteases’ optimal activity, inhibiting parasite heme polymerase; as a result, the parasite is effectively killed with its own metabolic waste. Compared with susceptible strains,

1	for the acid proteases’ optimal activity, inhibiting parasite heme polymerase; as a result, the parasite is effectively killed with its own metabolic waste. Compared with susceptible strains, chloroquine-resistant plasmodia transport chloroquine out of intraparasitic compartments more rapidly and maintain lower chloroquine concentrations in their acid vesicles. Hydroxychloroquine, a congener of chloroquine, is equivalent to chloroquine in its antimalarial efficacy but is preferred to chloroquine for the treatment of autoimmune disorders because it produces less ocular toxicity when used in high doses.

1	Chloroquine is well absorbed. However, because it exhibits extensive tissue binding, a loading dose is required to yield effective plasma concentrations. A therapeutic drug level in plasma is reached 2–3 h after oral administration (the preferred route). Chloroquine can be administered IV, but excessively rapid parenteral administration can result in seizures and death from cardiovascular collapse. The mean half-life of chloroquine is 4 days, but the rate of excretion decreases as plasma levels decline, making once-weekly administration possible for prophylaxis in areas with sensitive strains. About one-half of the parent drug is excreted in urine, but the dose should not be reduced for persons with acute malaria and renal insufficiency. Ciprofloxacin See Table 246e-1 and Chap. 170. Clindamycin See Table 246e-1 and Chap. 170. Dapsone See Table 246e-1 and Chap. 205e.

1	Ciprofloxacin See Table 246e-1 and Chap. 170. Clindamycin See Table 246e-1 and Chap. 170. Dapsone See Table 246e-1 and Chap. 205e. Dehydroemetine Emetine is an alkaloid derived from ipecac; dehydroemetine is synthetically derived from emetine and is considered less toxic. Both agents are active against Entamoeba histolytica and appear to work by blocking peptide elongation and thus inhibiting protein synthesis. Emetine is rapidly absorbed after parenteral administration, rapidly distributed throughout the body, and slowly excreted in the urine in unchanged form. Both agents are contraindicated in patients with renal disease.

1	Diethylcarbamazine* A derivative of the antihelminthic agent piperazine with a long history of successful use, diethylcarbamazine (DEC) remains the treatment of choice for lymphatic filariasis and loiasis and has also been used for visceral larva migrans. Although piperazine itself has no antifilarial activity, the piperazine ring of DEC is essential for the drug’s activity. DEC’s mechanism of action remains to be fully defined. Proposed mechanisms include immobilization due to inhibition of parasite cholinergic muscle receptors, disruption of microtubule formation, and alteration of helminthic surface membranes resulting in enhanced killing by the host’s immune system. DEC enhances adherence properties of eosinophils. The development of resistance under drug pressure (i.e., a progressive decrease in efficacy when the drug is used widely in human populations) has not been observed, although DEC has variable effects when administered to persons with filariasis. Monthly administration

1	decrease in efficacy when the drug is used widely in human populations) has not been observed, although DEC has variable effects when administered to persons with filariasis. Monthly administration provides effective prophylaxis against both bancroftian filariasis and loiasis.

1	DEC is well absorbed after oral administration, with peak plasma concentrations reached within 1–2 h. No parenteral form is available. The drug is eliminated largely by renal excretion, with <5% found in feces. If more than one dose is to be administered to an individual with renal dysfunction, the dose should be reduced commensurate with the reduction in creatinine clearance rate. Alkalinization of the urine prevents renal excretion and increases the half-life of DEC. Use in patients with onchocerciasis can precipitate a Mazzotti reaction, with pruritus, fever, and arthralgias. Like other piperazines, DEC is active against Ascaris species. Patients co-infected with this nematode may expel live worms after treatment.

1	Diloxanide Furoate Diloxanide furoate, a substituted acetanilide, is a luminally active agent used to eradicate the cysts of E. histolytica. After ingestion, diloxanide furoate is hydrolyzed by enzymes in the lumen or mucosa of the intestine, releasing furoic acid and the ester diloxanide; the latter acts directly as an amebicide. Diloxanide furoate is given alone to asymptomatic cyst passers. For patients with active amebic infections, diloxanide is generally administered in combination with a 5-nitroimidazole such as metronidazole or tinidazole. Diloxanide furoate is rapidly absorbed after oral administration. When coadministered with a 5-nitroimidazole, only diloxanide appears in the systemic circulation; levels peak within 1 h and disappear within 6 h. About 90% of an oral dose is excreted in the urine within 48 h, chiefly as the glucuronide metabolite. Diloxanide furoate is contraindicated in pregnant and breast-feeding women and in children <2 years of age.

1	Eflornithine* Eflornithine (difluoromethylornithine, or DFMO) is a fluorinated analogue of the amino acid ornithine. Although originally designed as an antineoplastic agent, eflornithine has proven effective against some trypanosomatids. At one point, the production of this effective agent ceased despite the increasing incidence of human African trypanosomiasis; however, production resumed after eflornithine was discovered to be an effective cosmetic depilatory agent.

1	Eflornithine has specific activity against all stages of infection with Trypanosoma brucei gambiense; however, it is inactive against Trypanosoma brucei rhodesiense. The drug acts as an irreversible suicide inhibitor of ornithine decarboxylase, the first enzyme in the biosynthesis of the polyamines putrescine and spermidine. Polyamines are essential for the synthesis of trypanothione, an enzyme required for the maintenance of intracellular thiols in the correct redox state and for the removal of reactive oxygen metabolites. However, polyamines are also essential for cell division in eukaryotes, and ornithine decarboxylase is similar in trypanosomes and mammals. The selective antiparasitic activity of eflornithine is partly explained by the structure of the trypanosomal enzyme, which lacks a 36-amino-acid C-terminal sequence found on mammalian ornithine decarboxylase. This difference results in a lower turnover of ornithine decarboxylase and a more rapid decrease of polyamines in

1	lacks a 36-amino-acid C-terminal sequence found on mammalian ornithine decarboxylase. This difference results in a lower turnover of ornithine decarboxylase and a more rapid decrease of polyamines in trypanosomes than in the mammalian host.

1	The diminished effectiveness of eflornithine against T. b. rhodesiense appears to be due to the parasite’s ability to replace the inhibited enzyme more rapidly than T. b. gambiense. Eflornithine is less toxic but more costly than conventional therapy. It can be administered IV or PO. The dose should be reduced in renal failure. Eflornithine readily crosses the blood-brain barrier; CSF levels are highest in persons with the most severe central nervous system (CNS) involvement.

1	Fumagillin† Fumagillin is a water-insoluble antibiotic that is derived from the fungus Aspergillus fumigatus and is active against microsporidia. This drug is effective when used topically to treat ocular infections due to Encephalitozoon species. When given systemically, fumagillin was effective but caused thrombocytopenia in all recipients in the second week of treatment; this side effect was readily reversed when administration of the drug was stopped. The mechanisms by which fumagillin inhibits microsporidial replication are poorly understood, although the drug may inhibit methionine aminopeptidase 2 by irreversibly blocking the active site.

1	Furazolidone This nitrofuran derivative is an effective alternative agent for the treatment of giardiasis and also exhibits activity against Isospora belli. Because it is the only agent active against Giardia that is available in liquid form, it is most often used to treat young children. Furazolidone undergoes reductive activation in Giardia lamblia trophozoites—an event that, unlike the reductive activation of metronidazole, involves an NADH oxidase. The killing effect correlates with the toxicity of reduced products, which damage important cellular components, including DNA. Although furazolidone had been thought to be largely unabsorbed when administered orally, the occurrence of systemic adverse reactions indicates that this is not the case. More than 65% of the drug dose can be recovered from the urine as colored metabolites. Omeprazole reduces the oral bioavailability of furazolidone.

1	Furazolidone is a monoamine oxidase (MAO) inhibitor; thus, caution should be used in its concomitant administration with other drugs (especially indirectly acting sympathomimetic amines) and in the consumption of food and drink containing tyramine during treatment. However, hypertensive crises have not been reported in patients receiving furazolidone, and it has been suggested that—because furazolidone inhibits MAOs gradually over several days—the risks are small if treatment is limited to a 5-day course. Because hemolytic anemia can occur in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency and glutathione instability, furazolidone treatment is contraindicated in mothers who are breast-feeding and in neonates.

1	Halofantrine This 9-phenanthrenemethanol is one of three classes of arylaminoalcohols first identified as potential antimalarial agents by the World War II Malaria Chemotherapy Program. Its activity is believed to be similar to that of chloroquine, although it is an oral alternative for the treatment of malaria due to chloroquine-resistant P. falciparum. Although the mechanism of action is poorly understood, halofantrine is thought to share mechanism(s) with the 4-aminoquinolines, forming a complex with ferriprotoporphyrin IX and interfering with the degradation of hemoglobin.

1	Halofantrine exhibits erratic bioavailability, but its absorption is significantly enhanced when it is taken with a fatty meal. The elimination half-life of halofantrine is 1–2 days; it is excreted mainly in feces. Halofantrine is metabolized into N-debutyl-halofantrine by the cytochrome P450 enzyme CYP3A4. Grapefruit juice should be avoided during treatment because it increases both halofantrine’s bioavailability and halofantrine-induced QT interval prolongation by inhibiting CYP3A4 at the enterocyte level.

1	Iodoquinol Iodoquinol (diiodohydroxyquin), a hydroxyquinoline, is an effective luminal agent for the treatment of amebiasis, balantidiasis, and infection with Dientamoeba fragilis. Its mechanism of action is unknown. It is poorly absorbed. Because the drug contains 64% organically bound iodine, it should be used with caution in patients with thyroid disease. Iodine dermatitis occurs occasionally during 246e-9 iodoquinol treatment. Protein-bound serum iodine levels may be increased during treatment and can interfere with certain tests of thyroid function. These effects may persist for as long as 6 months after discontinuation of therapy. Iodoquinol is contraindicated in patients with liver disease. Most serious are the reactions related to prolonged high-dose therapy (optic neuritis, peripheral neuropathy), which should not occur if the recommended dosage regimens are followed.

1	Ivermectin Ivermectin (22,23-dihydroavermectin) is a derivative of the macrocyclic lactone avermectin produced by the soil-dwelling actinomycete Streptomyces avermitilis. Ivermectin is active at low doses against a wide range of helminths and ectoparasites. It is the drug of choice for the treatment of onchocerciasis, strongyloidiasis, cutaneous larva migrans, and scabies. Ivermectin is highly active against microfilariae of the lymphatic filariases but has no macrofilaricidal activity. When ivermectin is used in combination with other agents such as DEC or albendazole for treatment of lymphatic filariasis, synergistic activity is seen. Although active against the intestinal helminths Ascaris lumbricoides and Enterobius vermicularis, ivermectin is only variably effective in trichuriasis and is ineffective against hookworms. Widespread use of ivermectin for treatment of intestinal nematode infections in sheep and goats has led to the emergence of drug resistance in veterinary practice;

1	is ineffective against hookworms. Widespread use of ivermectin for treatment of intestinal nematode infections in sheep and goats has led to the emergence of drug resistance in veterinary practice; this development may portend problems in human medical use.

1	Data suggest that ivermectin acts by opening the neuromuscular membrane-associated, glutamate-dependent chloride channels. The influx of chloride ions results in hyperpolarization and muscle paralysis—particularly of the nematode pharynx, with consequent blockage of the oral ingestion of nutrients. As these chloride channels are present only in invertebrates, paralysis is seen only in the parasite. Ivermectin is available for administration to humans only as an oral formulation. The drug is highly protein bound; it is almost completely excreted in feces. Both food and beer increase the bioavailability of ivermectin significantly. Ivermectin is distributed widely throughout the body; animal studies indicate that it accumulates at the highest concentration in adipose tissue and liver, with little accumulation in the brain. Few data exist to guide therapy in hosts with conditions that may influence drug pharmacokinetics.

1	Ivermectin is generally administered as a single dose of 150–200 μg/kg. In the absence of parasitic infection, the adverse effects of ivermectin in therapeutic doses are minimal. Adverse effects in patients with filarial infections include fever, myalgia, malaise, lightheadedness, and (occasionally) postural hypotension. The severity of such side effects is related to the intensity of parasite infection, with more symptoms in individuals with a heavy parasite burden. In onchocerciasis, skin edema, pruritus, and mild eye irritation may also occur. The adverse effects are generally self-limiting and only occasionally require symptom-based treatment with antipyretics or antihistamines. More severe complications of ivermectin therapy for onchocerciasis include encephalopathy in patients heavily infected with Loa loa.

1	Lumefantrine Lumefantrine (benflumetol), a fluorene arylaminoalcohol derivative synthesized in the 1970s by the Chinese Academy of Military Medical Sciences (Beijing), has marked blood schizonticidal activity against a wide range of plasmodia. This agent conforms structurally and in mode of action to other arylaminoalcohols (quinine, mefloquine, and halofantrine). Lumefantrine exerts its antimalarial effect as a consequence of its interaction with heme, a degradation product of hemoglobin metabolism. Although its antimalarial activity is slower than that of the artemisinin-based drugs, the recrudescence rate with the recommended lumefantrine regimen is lower. The pharmacokinetic properties of lumefantrine are reminiscent of those of halofantrine, with variable oral bioavailability, considerable augmentation of oral bioavailability by concomitant fat intake, and a terminal elimination half-life of ~4–5 days in patients with malaria.

1	Artemether and lumefantrine have synergistic activity, and the combined formulation of artemether and lumefantrine is effective for the treatment of falciparum malaria in areas where P. falciparum is resistant to chloroquine and antifolates. CHAPTER 246e Agents Used to Treat Parasitic Infections Mebendazole This benzimidazole is a broad-spectrum antiparasitic agent widely used to treat intestinal helminthiases. Its mechanism of action is similar to that of albendazole; however, it is a more potent inhibitor of parasite malic dehydrogenase and exhibits a more specific and selective effect against intestinal nematodes than the other benzimidazoles.

1	Mebendazole is available only in oral form but is poorly absorbed from the GI tract; only 5–10% of a standard dose is measurable in plasma. The proportion absorbed from the GI tract is extensively metabolized in the liver. Metabolites appear in the urine and bile; impaired liver or biliary function results in higher plasma mebendazole levels in treated patients. No dose reduction is warranted in patients with renal function impairment. Because mebendazole is poorly absorbed, its incidence of side effects is low. Transient abdominal pain and diarrhea sometimes occur, usually in persons with massive parasite burdens.

1	Mefloquine Mefloquine is the preferred drug for prophylaxis of chloroquine-resistant malaria; high doses can be used for treatment. Despite the development of drug-resistant strains of P. falciparum in parts of Africa and Southeast Asia, mefloquine is an effective drug throughout most of the world. Cross-resistance of mefloquine with halofantrine and with quinine has been documented in limited areas. Like quinine and chloroquine, this quinoline is active only against the asexual erythrocytic stages of malarial parasites. Unlike quinine, however, mefloquine has a relatively poor affinity for DNA and, as a result, does not inhibit the synthesis of parasitic nucleic acids and proteins. Although both mefloquine and chloroquine inhibit hemozoin formation and heme degradation, mefloquine differs in that it forms a complex with heme that may be toxic to the parasite.

1	Mefloquine HCl is poorly water soluble and intensely irritating when given parenterally; thus it is available only in tablet form. Its absorption is adversely affected by vomiting and diarrhea but is significantly enhanced when the drug is administered with or after food. About 98% of the drug binds to protein. Mefloquine is excreted mainly in the bile and feces; therefore, no dose adjustment is needed in persons with renal insufficiency. The drug and its main metabolite are not appreciably removed by hemodialysis. No special chemoprophylactic dosage adjustments are indicated for the achievement of plasma concentrations in dialysis patients that are similar to those in healthy persons. Pharmacokinetic differences have been detected among various ethnic populations. In practice, however, these distinctions are of minor importance compared with host immune status and parasite sensitivity. In patients with impaired liver function, the elimination of mefloquine may be prolonged, leading

1	these distinctions are of minor importance compared with host immune status and parasite sensitivity. In patients with impaired liver function, the elimination of mefloquine may be prolonged, leading to higher plasma levels.

1	Mefloquine should be used with caution by individuals participating in activities requiring alertness and fine-motor coordination. A recent FDA review found that dizziness, vertigo, or tinnitus can persist or become permanent as a result of treatment with the drug; thus, a boxed warning was mandated. If the drug is to be administered for a prolonged period, periodic evaluations are recommended, including liver function tests and ophthalmic examinations. Sleep abnormalities (insomnia, abnormal dreams) have occasionally been reported. Psychosis and seizures occur rarely; mefloquine should not be prescribed to patients with neuropsychiatric conditions, including depression, generalized anxiety disorder, psychosis, schizophrenia, and seizure disorder. If acute anxiety, depression, restlessness, or confusion develops during prophylaxis, these psychiatric symptoms may be considered prodromal to a more serious event, and the drug should be discontinued.

1	Concomitant use of quinine, quinidine, or drugs producing β-adrenergic blockade may cause significant electrocardiographic abnormalities or cardiac arrest. Halofantrine must not be given simultaneously with or <3 weeks after mefloquine because a potentially fatal prolongation of the QTc interval on electrocardiography may occur. No data exist on mefloquine use after halofantrine use. Administration of mefloquine with quinine or chloroquine may increase the risk of convulsions. Mefloquine may lower plasma levels of anticonvulsants. Caution should be exercised with regard to concomitant antiretroviral therapy, since mefloquine has been shown to exert variable effects on ritonavir pharmacokinetics that are not explained by hepatic CYP3A4 activity or ritonavir protein binding. Vaccinations with attenuated live bacteria should be completed at least 3 days before the first dose of mefloquine.

1	Women of childbearing age who are traveling to areas where malaria is endemic should be warned against becoming pregnant and encouraged to practice contraception during malaria prophylaxis with mefloquine and for up to 3 months thereafter. However, in the case of unplanned pregnancy, use of mefloquine is not considered an indication for pregnancy termination. Analysis of prospectively monitored cases demonstrates a prevalence of birth defects and fetal loss comparable to background rates.

1	Melarsoprol* Melarsoprol has been used since 1949 for the treatment of human African trypanosomiasis. This trivalent arsenical compound is indicated for the treatment of African trypanosomiasis with neurologic involvement and for the treatment of early disease that is resistant to suramin or pentamidine. Melarsoprol, like other drugs containing heavy metals, interacts with thiol groups of several different proteins; however, its antiparasitic effects appear to be more specific. Trypanothione reductase is a key enzyme involved in the oxidative stress management of both Trypanosoma and Leishmania species, helping to maintain an intracellular reducing environment by reduction of disulfide trypanothione to its dithiol derivative dihydrotrypanothione. Melarsoprol sequesters dihydrotrypanothione, depriving the parasite of its main sulfhydryl antioxidant, and inhibits trypanothione reductase, depriving the parasite of the essential enzyme system that is responsible for keeping trypanothione

1	depriving the parasite of its main sulfhydryl antioxidant, and inhibits trypanothione reductase, depriving the parasite of the essential enzyme system that is responsible for keeping trypanothione reduced. These effects are synergistic. The selectivity of arsenical action against trypanosomes is due at least in part to the greater melarsoprol affinity of reduced trypanothione than of other monothiols (e.g., cysteine) on which the mammalian host depends for maintenance of high thiol levels. Melarsoprol enters the parasite via an adenosine transporter; drug-resistant strains lack this transport system.

1	Melarsoprol is always administered IV. A small but therapeutically significant amount of the drug enters the CSF. The compound is excreted rapidly, with ~80% of the arsenic found in feces. Melarsoprol is highly toxic. The most serious adverse reaction is reactive encephalopathy, which affects 6% of treated individuals and usually develops within 4 days of the start of therapy, with an average case-fatality rate of 50%. Glucocorticoids are administered with melarsoprol to prevent this development. Because melarsoprol is intensely irritating, care must be taken to avoid infiltration of the drug.

1	Metrifonate Metrifonate has selective activity against Schistosoma haematobium. This organophosphorous compound is a prodrug that is converted nonenzymatically to dichlorvos (2,2-dichlorovinyl dimethylphosphate, DDVP), a highly active chemical that irreversibly inhibits the acetylcholinesterase enzyme. Schistosomal cholinesterase is more susceptible to dichlorvos than is the corresponding human enzyme. The exact mechanism of action of metrifonate is uncertain, but the drug is believed to inhibit tegumental acetylcholine receptors that mediate glucose transport.

1	Metrifonate is administered in a series of three doses at 2-week intervals. After a single oral dose, metrifonate produces a 95% decrease in plasma cholinesterase activity within 6 h, with a fairly rapid return to normal. However, 2.5 months are required for erythrocyte cholinesterase levels to return to normal. Treated persons should not be exposed to neuromuscular blocking agents or organophosphate insecticides for at least 48 h after treatment. Metronidazole and Other Nitroimidazoles See Table 246e-1 and Chap. 170.

1	Metronidazole and Other Nitroimidazoles See Table 246e-1 and Chap. 170. Miltefosine In the early 1990s, miltefosine (hexadecylphosphocholine), originally developed as an antineoplastic agent, was discovered to have significant antiproliferative activity against Leishmania species, Trypanosoma cruzi, and T. brucei parasites in vitro and in experimental animal models. Miltefosine is the first oral drug that has proved to be highly effective and comparable to amphotericin B against visceral leishmaniasis in India, where antimonial-resistant cases are prevalent. Miltefosine is also effective in previously untreated visceral infections. Cure rates in cutaneous leishmaniasis are comparable to those obtained with antimony. Miltefosine has also been shown to be effective against the free-living ameba Naegleria fowleri.

1	The activity of miltefosine is attributed to interaction with cell signal transduction pathways and inhibition of phospholipid and sterol biosynthesis. Resistance to miltefosine has not been observed clinically. The drug is readily absorbed from the GI tract, is widely distributed, and accumulates in several tissues. The efficacy of a 28-day treatment course in Indian visceral leishmaniasis is equivalent to that of amphotericin B therapy; however, it appears that a shortened course of 21 days may be equally efficacious. General recommendations for the use of miltefosine are limited by the exclusion of specific groups from the published clinical trials: persons <12 or >65 years of age, persons with the most advanced disease, breast-feeding women, HIV-infected patients, and individuals with significant renal or hepatic insufficiency.

1	Niclosamide† Niclosamide is active against a wide variety of adult tapeworms but not against tissue cestodes. It is also a molluscicide and is used in snail-control programs. The drug uncouples oxidative phosphorylation in parasite mitochondria, thereby blocking the uptake of glucose by the intestinal tapeworm and resulting in the parasite’s death. Niclosamide rapidly causes spastic paralysis of intestinal cestodes in vitro. Its use is limited by its side effects, the necessarily long duration of therapy, the recommended use of purgatives, and—most important—limited availability (i.e., on a named-patient basis from the manufacturer).

1	Niclosamide is poorly absorbed. Tablets are given on an empty stomach in the morning after a liquid meal the night before, and this dose is followed by another 1 h later. For treatment of hymenolepiasis, the drug is administered for 7 days. A second course is often prescribed. The scolex and proximal segments of the tapeworms are killed on contact with niclosamide and may be digested in the gut. However, disintegration of the adult tapeworm results in the release of viable ova, which theoretically can result in autoinfection. Although fears of the development of cysticercosis in patients with Taenia solium infections have proved unfounded, it is still recommended that a brisk purgative be given 2 h after the first dose.

1	Nifurtimox* This nitrofuran compound is an inexpensive and effective oral agent for the treatment of acute Chagas’ disease. Trypanosomes lack catalase and have very low levels of peroxidase; as a result, they are very vulnerable to by-products of oxygen reduction. When nifurtimox is reduced in the trypanosome, a nitro anion radical is formed and undergoes autooxidation, resulting in the generation of the superoxide anion O2–, hydrogen peroxide (H2O2), hydroperoxyl radical (HO2), and other highly reactive and cytotoxic molecules. Despite the abundance of catalases, peroxidases, and superoxide dismutases that neutralize these destructive radicals in mammalian cells, nifurtimox has a poor therapeutic index. Prolonged use is required, but the course may have to be interrupted because of drug toxicity, which develops in 40–70% of recipients. Nifurtimox is well absorbed and undergoes rapid and extensive biotransformation; <0.5% of the original drug is excreted in urine.

1	Nitazoxanide Nitazoxanide is a 5-nitrothiazole compound used for the treatment of cryptosporidiosis and giardiasis; it is active against other intestinal protozoa as well. The drug is approved for use in children 1–11 years of age. The antiprotozoal activity of nitazoxanide is believed to be due to interference with the pyruvate-ferredoxin oxidoreductase (PFOR) enzyme–dependent electron transfer reaction that is essential to anaerobic energy metabolism. Studies have shown that the PFOR enzyme from G. lamblia directly reduces nitazoxanide by transfer of electrons in the absence of ferredoxin. The DNA-derived PFOR protein sequence of Cryptosporidium parvum appears to be similar to that of G. lamblia. Interference with the PFOR enzyme–dependent electron transfer reaction may not be the only pathway by which nitazoxanide 246e-11 exerts antiprotozoal activity.

1	After oral administration, nitazoxanide is rapidly hydrolyzed to an active metabolite, tizoxanide (desacetyl-nitazoxanide). Tizoxanide then undergoes conjugation, primarily by glucuronidation. It is recommended that nitazoxanide be taken with food; however, no studies have been conducted to determine whether the pharmacokinetics of tizoxanide and tizoxanide glucuronide differ in fasted versus fed subjects. Tizoxanide is excreted in urine, bile, and feces, and tizoxanide glucuronide is excreted in urine and bile. The pharmacokinetics of nitazoxanide in patients with impaired hepatic and/or renal function have not been studied. Tizoxanide is highly bound to plasma protein (>99.9%). Therefore, caution should be used when administering this agent concurrently with other highly plasma protein–bound drugs with narrow therapeutic indices, as competition for binding sites may occur.

1	Oxamniquine This tetrahydroquinoline derivative is an effective alternative agent for the treatment of Schistosoma mansoni, although susceptibility to this drug exhibits regional variation. Oxamniquine exhibits anticholinergic properties, but its primary mode of action seems to rely on ATP-dependent enzymatic drug activation generating an intermediate that alkylates essential macromolecules, including DNA. In treated adult schistosomes, oxamniquine produces marked tegumental alterations that are similar to those seen with praziquantel but that develop less rapidly, becoming evident 4–8 days after treatment.

1	Oxamniquine is administered orally as a single dose and is well absorbed. Food retards absorption and reduces bioavailability. About 70% of an administered dose is excreted in urine as a mixture of pharmacologically inactive metabolites. Patients should be warned that their urine might have an intense orange-red color. Side effects are uncommon and usually mild, although hallucinations and seizures have been reported. Paromomycin (Aminosidine) First isolated in 1956, this aminoglycoside is an effective oral agent for the treatment of infections due to intestinal protozoa. Parenteral paromomycin appears to be effective against visceral leishmaniasis in India.

1	Paromomycin inhibits protozoan protein synthesis by binding to the 30S ribosomal RNA in the aminoacyl-tRNA site, causing misreading of mRNA codons. Paromomycin is less active against G. lamblia than standard agents; however, like other aminoglycosides, paromomycin is poorly absorbed from the intestinal lumen, and the high levels of drug in the gut compensate for this relatively weak activity. If absorbed or administered systemically, paromomycin can cause ototoxicity and nephrotoxicity. However, systemic absorption is very limited, and toxicity should not be a concern in persons with normal kidneys. Topical formulations are not generally available.

1	Pentamidine Isethionate This diamidine is an effective alternative agent for some forms of leishmaniasis and trypanosomiasis. It is available for parenteral and aerosolized administration. Although its mechanism of action remains undefined, it is known to exert a wide range of effects, including interaction with trypanosomal kinetoplast DNA; interference with polyamine synthesis by a decrease in the activity of ornithine decarboxylase; and inhibition of RNA polymerase, ribosomal function, and the synthesis of nucleic acids and proteins.

1	Pentamidine isethionate is well absorbed, is highly tissue bound, and is excreted slowly over several weeks, with an elimination half-life of 12 days. No steady-state plasma concentration is attained in persons given daily injections; the result is extensive accumulation of pentamidine in tissues, primarily the liver, kidney, adrenal, and spleen. Pentamidine does not penetrate well into the CNS. Pulmonary concentrations of pentamidine are increased when the drug is delivered in aerosolized form. Piperazine The antihelminthic activity of piperazine is confined to ascariasis and enterobiasis. Piperazine acts as an agonist at extrasynaptic γ-aminobutyric acid (GABA) receptors, causing an influx of chloride ions in the nematode somatic musculature. Although the initial result is hyperpolarization of the muscle fibers, the ultimate effect is flaccid

1	CHAPTER 246e Agents Used to Treat Parasitic Infections paralysis, leading to the expulsion of live worms. Patients should be warned, as this occurrence can be unsettling. Praziquantel This heterocyclic pyrazinoisoquinoline derivative is highly active against a broad spectrum of trematodes and cestodes. It is the mainstay of treatment for schistosomiasis and is a critical part of community-based control programs.

1	All of the effects of praziquantel can be attributed either directly or indirectly to an alteration of intracellular calcium concentrations. Although the exact mechanism of action remains unclear, the major mechanism is disruption of the parasite tegument, causing tetanic contractures with loss of adherence to host tissues and, ultimately, disintegration or expulsion. Praziquantel induces changes in the antigenicity of the parasite by causing the exposure of concealed antigens. Praziquantel also produces alterations in schistosomal glucose metabolism, including decreases in glucose uptake, lactate release, glycogen content, and ATP levels.

1	Praziquantel exerts its parasitic effects directly and does not need to be metabolized to be effective. It is well absorbed but undergoes extensive first-pass hepatic clearance. Levels of the drug are increased when it is taken with food, particularly carbohydrates, or with cimetidine. Serum levels are reduced by glucocorticoids, chloroquine, carbamazepine, and phenytoin. Praziquantel is completely metabolized in humans, with 80% of the dose recovered as metabolites in urine within 4 days. It is not known to what extent praziquantel crosses the placenta, but retrospective studies suggest that it is safe in pregnancy. Patients with schistosomiasis who have heavy parasite burdens may develop abdominal discomfort, nausea, headache, dizziness, and drowsiness. Symptoms begin 30 min after ingestion, may require spasmolytics for relief, and usually disappear spontaneously after a few hours.

1	Primaquine Phosphate Primaquine, an 8-aminoquinoline, has a broad spectrum of activity against all stages of plasmodial development in humans but has been used most effectively for eradication of the hepatic stage of these parasites. Despite its toxicity, it remains the drug of choice for radical cure of P. vivax infections. Primaquine must be metabolized by the host to be effective. It is, in fact, rapidly metabolized; only a small fraction of the dose of the parent drug is excreted unchanged. Although the parasiticidal activity of the three oxidative metabolites remains unclear, they are believed to affect both pyrimidine synthesis and the mitochondrial electron transport chain. The metabolites appear to have significantly less antimalarial activity than primaquine; however, their hemolytic activity is greater than that of the parent drug.

1	Primaquine causes marked hypotension after parenteral administration and therefore is given only by the oral route. It is rapidly and almost completely absorbed from the GI tract. Patients should be tested for G6PD deficiency before they receive primaquine. The drug may induce the oxidation of hemoglobin into methemoglobin, irrespective of the G6PD status of the patient. Primaquine is otherwise well tolerated. Proguanil (Chloroguanide) Proguanil inhibits plasmodial dihydrofolate reductase and is used with atovaquone for oral treatment of uncomplicated malaria or with chloroquine for malaria prophylaxis in parts of Africa without widespread chloroquine-resistant P. falciparum.

1	Proguanil exerts its effect primarily by means of the metabolite cycloguanil, whose inhibition of dihydrofolate reductase in the parasite disrupts deoxythymidylate synthesis, thus interfering with a key pathway involved in the biosynthesis of pyrimidines required for nucleic acid replication. There are no clinical data indicating that folate supplementation diminishes drug efficacy; women of childbearing age for whom atovaquone/proguanil is prescribed should continue taking folate supplements to prevent neural tube birth defects. Proguanil is extensively absorbed regardless of food intake. The drug is 75% protein bound. The main routes of elimination are hepatic biotransformation and renal excretion; 40–60% of the proguanil dose is excreted by the kidneys. Drug levels are increased and elimination is impaired in patients with hepatic insufficiency.

1	Pyrantel Pamoate Pyrantel is a tetrahydropyrimidine formulated as pamoate. This safe, well-tolerated, inexpensive drug is used to treat a variety of intestinal nematode infections but is ineffective in trichuriasis. Pyrantel pamoate is usually effective in a single dose. Its target is the nicotinic acetylcholine receptor on the surface of nematode somatic muscle. Pyrantel depolarizes the neuromuscular junction of the nematode, resulting in its irreversible paralysis and allowing the natural expulsion of the worm. Pyrantel pamoate is poorly absorbed from the intestine; >85% of the dose is passed unaltered in feces. The absorbed portion is metabolized and excreted in urine. Piperazine is antagonistic to pyrantel pamoate and should not be used concomitantly. Pyrantel pamoate has minimal toxicity at the oral doses used to treat intestinal helminthic infection. It is not recommended for pregnant women or for children <12 months old.

1	Pyrantel pamoate has minimal toxicity at the oral doses used to treat intestinal helminthic infection. It is not recommended for pregnant women or for children <12 months old. Pyrimethamine When combined with short-acting sulfonamides, this diaminopyrimidine is effective in malaria, toxoplasmosis, and isosporiasis. Unlike mammalian cells, the parasites that cause these infections cannot use preformed pyrimidines obtained through salvage pathways but rather rely completely on de novo pyrimidine synthesis, for which folate derivatives are essential cofactors. The efficacy of pyrimethamine is increasingly limited by the development of resistant strains of P. falciparum and P. vivax. Single amino acid substitutions to parasite dihydrofolate reductase confer resistance to pyrimethamine by decreasing the enzyme’s binding affinity for the drug.

1	Pyrimethamine is well absorbed; the drug is 87% bound to human plasma proteins. In healthy volunteers, drug concentrations remain at therapeutic levels for up to 2 weeks; drug levels are lower in patients with malaria. At the usual dosage, pyrimethamine alone causes little toxicity except for occasional skin rashes and, more rarely, blood dyscrasias. Bone marrow suppression sometimes occurs at the higher doses used for toxoplasmosis; at these doses, the drug should be administered with folinic acid.

1	Pyronaridine This potent antimalarial is a benzonaphthyridine derivative first synthesized by Chinese researchers in 1970. Like chloroquine, pyronaridine targets hematin formation, inhibiting the production of β-hematin by forming complexes with it, with consequent enhancement of hematin-induced hemolysis. However, this drug is more potent than chloroquine: for complete lysis, pyronaridine is required at only 1/100th of the concentration needed with chloroquine. It also inhibits glutathione-dependent heme degradation. Despite its similar mode of action, pyronaridine remains effective against chloroquine-resistant strains. When combined with artesunate, it has been shown to be effective for the treatment of acute, uncomplicated infection caused by P. falciparum or P. vivax in areas of low transmission with evidence of artemisinin resistance.

1	Pyronaridine is readily absorbed, widely distributed throughout the body, metabolized by the liver, and excreted in urine and stool. Its use is contraindicated in patients with severe liver or kidney impairment. Pyronaridine has been shown in vitro to be an inhibitor of both CYP2D6 and P-glycoprotein, and these effects may have clinical relevance for patients taking medications for cardiac disease (e.g., metoprolol and digoxin). Quinacrine* Quinacrine is the only drug approved by the FDA for the treatment of giardiasis. Although its production was discontinued in 1992, quinacrine can be obtained from alternative sources through the CDC Drug Service. The antiprotozoal mechanism of quinacrine has not been fully elucidated. The drug inhibits NADH oxidase—the same enzyme that activates furazolidone. The differing relative quinacrine uptake rate between human cells and G. lamblia may explain the selective toxicity of the drug. Resistance correlates with decreased drug uptake.

1	Quinacrine is rapidly absorbed from the intestinal tract and is widely distributed in body tissues. Alcohol is best avoided due to a disulfiram-like effect. Quinine and Quinidine When combined with another agent, the cinchona alkaloid quinine is effective for the oral treatment of both uncomplicated, chloroquine-resistant malaria and babesiosis. Quinine acts rapidly against the asexual blood stages of all forms of the human malaria parasites. For severe malaria, only quinidine (the dextroisomer of quinine) is available in the United States. Quinine concentrates in the acidic food vacuoles of Plasmodium species. The drug inhibits the nonenzymatic polymerization of the highly reactive, toxic heme molecule into the nontoxic polymer pigment hemozoin.

1	Quinine is readily absorbed when given orally. In patients with malaria, the elimination half-life of quinine increases according to the severity of the infection. However, toxicity is avoided by an increase in the concentration of plasma glycoproteins. The cinchona alkaloids are extensively metabolized, particularly by CYP3A4; only 20% of the dose is excreted unchanged in urine. The drug’s metabolites are also excreted in urine and may be responsible for toxicity in patients with renal failure. Renal excretion of quinine is decreased when cimetidine is taken and increased when the urine is acidic. The drug readily crosses the placenta. Quinidine is both more potent as an antimalarial and more toxic than quinine. Its use requires cardiac monitoring. Dose reduction is necessary in persons with severe renal impairment.

1	Quinidine is both more potent as an antimalarial and more toxic than quinine. Its use requires cardiac monitoring. Dose reduction is necessary in persons with severe renal impairment. Spiramycin† This macrolide is used to treat acute toxoplasmosis in pregnancy and congenital toxoplasmosis. While the mechanism of action is similar to that of other macrolides, the efficacy of spiramycin in toxoplasmosis appears to stem from its rapid and extensive intracellular penetration, which results in macrophage drug concentrations 10–20 times greater than serum concentrations. Spiramycin is rapidly and widely distributed throughout the body and reaches concentrations in the placenta up to five times those in serum. This agent is excreted mainly in bile. Indeed, in humans, the urinary excretion of active compounds represents only 20% of the administered dose.

1	Serious reactions to spiramycin are rare. Of the available macrolides, spiramycin appears to have the lowest risk of drug interactions. Complications of treatment are rare but, in neonates, can include life-threatening ventricular arrhythmias that disappear with drug discontinuation. Sulfonamides See Table 246e-1 and Chap. 170. Suramin* This derivative of urea is the drug of choice for the early stage of African trypanosomiasis. The drug is polyanionic and acts by forming stable complexes with proteins, thus inhibiting multiple enzymes essential to parasite energy metabolism. Suramin appears to inhibit all trypanosome glycolytic enzymes more effectively than it inhibits the corresponding host enzymes. Suramin is parenterally administered. It binds to plasma proteins and persists at low levels for several weeks after infusion. Its metabolism is negligible. This drug does not penetrate the CNS.

1	Suramin is parenterally administered. It binds to plasma proteins and persists at low levels for several weeks after infusion. Its metabolism is negligible. This drug does not penetrate the CNS. Tafenoquine Tafenoquine is an 8-aminoquinoline with causal prophylactic activity. Its prolonged half-life (2–3 weeks) allows longer dosing intervals when the drug is used for prophylaxis. Tafenoquine has been well tolerated in clinical trials. When tafenoquine is taken with food, its absorption is increased by 50% and the most commonly reported adverse event—mild GI upset—is diminished. Like primaquine, tafenoquine is a potent oxidizing agent, causing hemolysis in patients with G6PD deficiency as well as methemoglobinemia. Tetracyclines See Table 246e-1 and Chap. 170.

1	Tetracyclines See Table 246e-1 and Chap. 170. Thiabendazole Discovered in 1961, thiabendazole remains one of the most potent of the numerous benzimidazole derivatives. However, its use has declined significantly because of a higher frequency of adverse 246e-13 effects than is seen with other, equally effective agents.

1	Thiabendazole is active against most intestinal nematodes that infect humans. Although the exact mechanism of its antihelminthic activity has not been fully elucidated, it is likely to be similar to that of other benzimidazole drugs: namely, inhibition of polymerization of parasite β-tubulin. The drug also inhibits the helminth-specific enzyme fumarate reductase. In animals, thiabendazole has anti-inflammatory, antipyretic, and analgesic effects, which may explain its usefulness in dracunculiasis and trichinellosis. Thiabendazole also suppresses egg and/or larval production by some nematodes and may inhibit the subsequent development of eggs or larvae passed in feces. Despite the emergence and global spread of thiabendazole-resistant trichostrongyliasis among sheep, there have been no reports of drug resistance in humans.

1	Thiabendazole is available in tablet form and as an oral suspension. The drug is rapidly absorbed from the GI tract but can also be absorbed through the skin. Thiabendazole should be taken after meals. This agent is extensively metabolized in the liver before ultimately being excreted; most of the dose is excreted within the first 24 h. The usual dose of thiabendazole is determined by the patient’s weight, but some treatment regimens are parasite specific. No particular adjustments are recommended in patients with renal or hepatic failure; only cautious use is advised. Coadministration of thiabendazole to patients taking theophylline can result in an increase in theophylline levels by >50%. Therefore, serum levels of theophylline should be monitored closely in this situation.

1	Tinidazole This nitroimidazole is effective for the treatment of amebiasis, giardiasis, and trichomoniasis. Like metronidazole, tinidazole must undergo reductive activation by the parasite’s metabolic system before it can act on protozoal targets. Tinidazole inhibits the synthesis of new DNA in the parasite and causes degradation of existing DNA. The reduced free-radical derivatives alkylate DNA, with consequent cytotoxic damage to the parasite. This damage appears to be produced by short-lived reduction intermediates, resulting in helix destabilization and strain breakage of DNA. The mechanism of action and side effects of tinidazole are similar to those of metronidazole, but adverse events appear to be less frequent and severe with tinidazole. In addition, the significantly longer half-life of tinidazole (>12 h) offers potential cure with a single dose.

1	Triclabendazole While most benzimidazoles have broad-spectrum antihelminthic activity, they exhibit minimal or no activity against F. hepatica. In contrast, the antihelminthic activity of triclabendazole is highly specific for Fasciola and Paragonimus species, with little activity against nematodes, cestodes, and other trematodes. Triclabendazole is effective against all stages of Fasciola species. The active sulfoxide metabolite of triclabendazole binds to fluke tubulin by assuming a unique nonplanar configuration and disrupts microtubule-based processes. Resistance to triclabendazole in veterinary use has been reported in Australia and Europe; however, no resistance has been documented in humans.

1	Triclabendazole is rapidly absorbed after oral ingestion; administration with food enhances its absorption and shortens the elimination half-life of the active metabolite. Both the sulfoxide and the sulfone metabolites are highly protein bound (>99%). Treatment with triclabendazole is typically given in one or two doses. No clinical data are available regarding dose adjustment in renal or hepatic insufficiency; however, given the short course of therapy and extensive hepatic metabolism of triclabendazole, dose adjustment is unlikely to be necessary. No information exists on drug interactions. Trimethoprim-Sulfamethoxazole See Table 246e-1 and Chap. 170. CHAPTER 246e Agents Used to Treat Parasitic Infections Amebiasis and Infection with Free-Living Amebas Rosa M. Andrade, Sharon L. Reed AMEBIASIS DEFINITION 247 sEC TIOn 18 Amebiasis and Infection with Free-Living Amebas

1	Amebiasis and Infection with Free-Living Amebas Rosa M. Andrade, Sharon L. Reed AMEBIASIS DEFINITION 247 sEC TIOn 18 Amebiasis and Infection with Free-Living Amebas Amebiasis is an infection with the intestinal protozoan Entamoeba histolytica. About 90% of infections are asymptomatic, and the remaining 10% produce a spectrum of clinical syndromes ranging from dysentery to abscesses of the liver or other organs.

1	E. histolytica is acquired by ingestion of viable cysts from fecally contaminated water, food, or hands. Food-borne exposure is most prevalent and is particularly likely when food handlers are shedding cysts or food is being grown with feces-contaminated soil, fertilizer, or water. Besides the drinking of contaminated water, less common means of transmission include oral and anal sexual practices and—in rare instances—direct rectal inoculation through colonic irrigation devices. Motile trophozoites are released from cysts in the small intestine and, in most patients, remain as harmless commensals in the large bowel. After encystation, infectious cysts are shed in the stool and can survive for several weeks in a moist environment. In some patients, the trophozoites invade either the bowel mucosa, causing symptomatic colitis, or the bloodstream, causing distant abscesses of the liver, lungs, or brain. The trophozoites may not encyst in patients with active dysentery, and motile

1	bowel mucosa, causing symptomatic colitis, or the bloodstream, causing distant abscesses of the liver, lungs, or brain. The trophozoites may not encyst in patients with active dysentery, and motile hematophagous trophozoites are frequently present in fresh stools. Trophozoites are rapidly killed by exposure to air or stomach acid, however, and therefore cannot transmit infection.

1	About 10% of the world’s population is infected with Entamoeba, the majority with noninvasive Entamoeba dispar. Amebiasis results from infection with E. histolytica and is the third most common cause of death from parasitic disease (after schistosomiasis and malaria). Invasive colitis and liver abscesses are sevenfold more common among men than among women; this difference has been attributed to a disparity in complement-mediated killing. The wide spectrum of clinical disease caused by Entamoeba is due in part to the differences between these two infecting species. E. histolytica has unique isoenzymes, surface antigens, DNA markers, and virulence properties that distinguish it from other genetically related and morphologically identical species, such as E. dispar and E. moshkovskii.

1	Most asymptomatic carriers, including men who have sex with men (MSM) and patients with AIDS, harbor E. dispar and have self-limited infections. In this respect, E. dispar is dissimilar to other enteric pathogens such as Cryptosporidium and Cystoisospora belli, which can cause self-limited illnesses in immunocompetent hosts but devastating diarrhea in patients with AIDS. These observations indicate that E. dispar is incapable of causing invasive disease. Unlike E. dispar, E. histolytica can cause invasive disease, as demonstrated in recent reports from Korea, China, and India that suggest higher prevalences of amebic seroconversion, invasive amebiasis, and amebic liver abscesses among HIV-positive than HIV-negative patients. In another study, 10% of asymptomatic patients who were colonized with E. histolytica went on to develop amebic colitis, while the rest remained asymptomatic and cleared the infection within 1 year.

1	The potential of E. moshkovskii to cause diarrhea, weight loss, and colitis was recently demonstrated in a mouse model of cecal infection. However, the pathogenic potential of this species is not clear. A prospective evaluation of children from the Mirpur community of Dhaka, Bangladesh, found that most children who had diarrheal diseases associated with E. moshkovskii were simultaneously infected with at least one other enteric pathogen.

1	Areas of highest incidence of Entamoeba infection (due to inadequate sanitation and crowding) include most developing countries in the tropics, particularly Mexico, India, and nations of Central and South America, tropical Asia, and Africa. In a 4-year follow-up study of preschool children in a highly endemic area of Bangladesh, 80% of children had at least one episode of E. histolytica infection and 53% had more than one episode. Naturally acquired immunity did develop but was usually short-lived and correlated with the presence in the stool of secretory IgA antibody to the major adherence lectin galactose N-acetylgalactosamine (Gal/GalNAc). The main groups at risk for amebiasis in developed countries are returned travelers, recent immigrants, MSM, military personnel, and inmates of institutions. Data from the GeoSentinel Surveillance Network, which come from tropical medicine clinics on six continents, showed that, among long-term travelers (trip duration, >6 months), diarrhea due

1	Data from the GeoSentinel Surveillance Network, which come from tropical medicine clinics on six continents, showed that, among long-term travelers (trip duration, >6 months), diarrhea due to E. histolytica was among the most common diagnoses.

1	1364 PATHOGENESIS AND PATHOLOGY Both trophozoites (Fig. 247-1) and cysts (Fig. 247-2) are found in the intestinal lumen, but only trophozoites of E. histolytica invade tissue. The trophozoite is 20–60 μm in diameter and contains vacuoles and a nucleus with a characteristic central nucleolus. In animals, depletion of intestinal mucus, diffuse inflammation, and disruption of the epithelial barrier precede trophozoite contact with the colonic mucosa. Trophozoites attach to colonic mucus and epithelial cells by their Gal/ GalNAc lectin. The earliest intestinal lesions are microulcerations of the mucosa of the cecum, sigmoid colon, or rectum that release erythrocytes, inflammatory cells, and epithelial cells. Proctoscopy reveals small ulcers with heaped-up margins and normal intervening mucosa (Fig. 247-3A). Submucosal extension of ulcerations under viable-appearing surface mucosa causes the classic “flask-shaped” ulcer containing trophozoites at the margins of dead and viable tissues.

1	(Fig. 247-3A). Submucosal extension of ulcerations under viable-appearing surface mucosa causes the classic “flask-shaped” ulcer containing trophozoites at the margins of dead and viable tissues. Although neutrophilic infiltrates may accompany the early lesions in animals, human intestinal infection is marked by a paucity of inflammatory cells, probably in part because of the killing of neutrophils by trophozoites (Fig. 247-3B). Treated ulcers characteristically heal with little or no scarring. Occasionally, however, full-thickness necrosis and perforation occur. Rarely, intestinal infection results in the formation of a mass lesion, or ameboma, in the bowel lumen. The overlying mucosa is usually thin and ulcerated, while other layers of the wall are thickened, edematous, and hemorrhagic; this condition results in exuberant formation of granulation tissue with little fibrous-tissue response. A number of virulence factors have been linked to the ability of E. histolytica to invade

1	this condition results in exuberant formation of granulation tissue with little fibrous-tissue response. A number of virulence factors have been linked to the ability of E. histolytica to invade through the interglandular epithelium. One factor consists of the extracellular cysteine proteinases that degrade collagen, elastin, IgA, IgG, and the anaphylatoxins C3a and C5a. Other enzymes may disrupt glycoprotein bonds between mucosal epithelial cells in the gut. Amebas can lyse neutrophils, monocytes, lymphocytes, and cells of colonic and hepatic lines. The cytolytic effect of amebas appears to require direct contact with target cells and may be linked to the release of phospholipase A and pore-forming peptides. E. histolytica trophozoites also cause apoptosis of human cells. Phagocytosis is a virulence factor that leads to defective parasite proliferation if inhibited. This process is potentially modulated by calmodulin-like calcium-binding protein 3, which pairs with actin and myosin

1	a virulence factor that leads to defective parasite proliferation if inhibited. This process is potentially modulated by calmodulin-like calcium-binding protein 3, which pairs with actin and myosin during initiation and formation of phagosomes. Another virulence factor is the ability to resist reactive oxygen species, reactive nitrogen species such as nitric oxide, or S-nitrosothiols such as S-nitrosoglutathione (GSNO) and S-nitrosocysteine (CySNO). E. histolytica trophozoites are constantly exposed to reactive oxygen and nitrogen species from their own metabolism and host defenses during tissue invasion. Overexpression of hydrogen peroxide regulatory motif–binding protein appears to increase E. histolytica cytotoxicity. Since E. histolytica lacks glutathione and glutathione reductase, it relies on its thioredoxin/thioredoxin reductase system to prevent, regulate, and repair the damage caused by oxidative stress. This antioxidant system is versatile in that it can reduce

1	FIGuRE 247-1 Trophozoite of E. histolytica. A single nucleus with a central, dot-like nucleolus is seen (trichrome stain). FIGuRE 247-2 Cyst of E. histolytica. Three of the four nuclei are vis-ible (trichrome stain). FIGuRE 247-3 Endoscopic and histopathologic features of intestinal amebiasis. A. Appearance of ulcers on colonoscopy (arrows). B. Inflammatory infiltrate and E. histolytica trophozoites (arrow) in invasive amebic colitis (hematoxylin and eosin). (Courtesy of the Department of Pathology and Gastroenterology, VA San Diego Medical Center.) reactive nitrogen species and use an alternative electron donor such as the reduced form of nicotinamide adenine dinucleotide. Metronidazole, the current standard of therapy for amebiasis, seems to exert its anti-parasitic effect through the inhibition of this antioxidant system. Newer therapeutic candidates targeting this system, such as auranofin, also have demonstrated in vitro and in vivo efficacy against this parasite.

1	Liver abscesses are always preceded by intestinal colonization, which may be asymptomatic. Blood vessels may be compromised early by wall lysis and thrombus formation. Trophozoites invade veins to reach the liver through the portal venous system. E. histolytica is resistant to complement-mediated lysis—a property critical to survival in the bloodstream. In contrast, E. dispar is rapidly lysed by complement and is thus restricted to the bowel lumen. Inoculation of amebas into the portal system of hamsters results in an acute cellular infiltrate consisting predominantly of neutrophils. Later, the neutrophils are lysed by contact with amebas, and the release of neutrophil toxins may contribute to necrosis of hepatocytes. The liver parenchyma is replaced by necrotic material that is surrounded by a thin rim of congested liver tissue. The necrotic contents of a liver abscess are classically described as “anchovy paste,” although the fluid is variable in color and is composed of

1	surrounded by a thin rim of congested liver tissue. The necrotic contents of a liver abscess are classically described as “anchovy paste,” although the fluid is variable in color and is composed of bacteriologically sterile granular debris with few or no cells. Amebas, if seen, tend to be found near the capsule of the abscess.

1	Host innate and adaptive immunity are important factors that determine susceptibility to invasive disease and its clinical outcome. While neutrophils were thought to contribute to tissue damage in intestinal and liver amebiasis due to their cytotoxic effects on host epithelial cells, a recent report suggests that they may exert a protective effect in susceptible mice. Neutropenia, induced with an antibody to Gr-1 (i.e., to peripheral neutrophils), led to death in C3H/HeJ mice and to severe disease in CBA mice (both of which are relatively susceptible to E. histolytica infection), while it had no effect on C57BL/6 mice, which are known for their intrinsic resistance to infection with this parasite.

1	E. histolytica infection), while it had no effect on C57BL/6 mice, which are known for their intrinsic resistance to infection with this parasite. Antimicrobial peptides, such as cathelicidins, are an important part of innate immunity and are induced by E. histolytica upon intestinal invasion in a mouse model. In this model, cecal cathelicidin-related antimicrobial peptide (CRAMP) mRNA increased more than fourfold by 3 days and more than 100-fold at 7 days. However, E. histolytica remained resistant to cathelicidin-mediated killing, probably because the antimicrobial peptide was digested by amebic cysteine proteinases. IgA plays a critical role in acquired immunity to E. histolytica. A study in Bangladeshi schoolchildren revealed that an intestinal IgA response to Gal/GalNAc reduced the risk of new

1	IgA plays a critical role in acquired immunity to E. histolytica. A study in Bangladeshi schoolchildren revealed that an intestinal IgA response to Gal/GalNAc reduced the risk of new E. histolytica infection by 64%. Serum IgG antibody is not protective; titers correlate with the duration of illness rather than with the severity of disease. Indeed, Bangladeshi children with a serum IgG response were more likely than those without such a response to develop new E. histolytica infection. In infants from this same Bangladeshi community, passive immunity conferred by maternal parasite-specific IgA via breastfeeding resulted in a 39% reduction in risk of infection and a 64% reduction in risk of diarrheal disease from E. histolytica during the first year of life. A link between nutrition and immunity is demonstrated by the elevated rate of infections due to protozoan parasites, including

1	A link between nutrition and immunity is demonstrated by the elevated rate of infections due to protozoan parasites, including E. histolytica, among undernourished children in developing countries. Resistance to amebiasis is associated with a polymorphism in the receptor for the adipocytokine leptin. Children in a Bangladeshi cohort with a mutant R223 leptin receptor allele were nearly four times more likely to be infected with E. histolytica than those carrying the ancestral Q223 allele. This mutant allele is overrepresented in many geographic areas with a high prevalence of amebiasis, such as Bangladesh and India. CLINICAL SYNDROMES Intestinal Amebiasis The most common type of amebic infection is asymptomatic cyst passage. Even in highly endemic areas, most patients harbor E. dispar.

1	CLINICAL SYNDROMES Intestinal Amebiasis The most common type of amebic infection is asymptomatic cyst passage. Even in highly endemic areas, most patients harbor E. dispar. Symptomatic amebic colitis develops 2–6 weeks after the ingestion of infectious E. histolytica cysts. A gradual onset of lower abdominal pain and mild diarrhea is followed by malaise, weight loss, and diffuse lower abdominal or back pain. Cecal involvement may mimic acute 1365 appendicitis. Patients with full-blown dysentery may pass 10–12 stools per day. The stools contain little fecal material and consist mainly of blood and mucus. In contrast to those with bacterial diarrhea, fewer than 40% of patients with amebic dysentery are febrile. Virtually all patients have heme-positive stools.

1	More fulminant intestinal infection, with severe abdominal pain, high fever, and profuse diarrhea, is rare and occurs predominantly in children. Patients may develop toxic megacolon, in which there is severe bowel dilation with intramural air. Patients receiving glucocorticoids are at risk for severe amebiasis. Uncommonly, patients develop a chronic form of amebic colitis, which can be confused with inflammatory bowel disease. The association between severe amebiasis complications and glucocorticoid therapy emphasizes the importance of excluding amebiasis when inflammatory bowel disease is suspected. An occasional patient presents with only an asymptomatic or tender abdominal mass caused by an ameboma, which is easily confused with cancer on barium studies. A positive serologic test or biopsy can prevent unnecessary surgery in this setting. The syndrome of post–amebic colitis—i.e., persistent diarrhea following documented cure of amebic colitis—is controversial; no evidence of

1	or biopsy can prevent unnecessary surgery in this setting. The syndrome of post–amebic colitis—i.e., persistent diarrhea following documented cure of amebic colitis—is controversial; no evidence of recurrent amebic infection can be found, and re-treatment usually has no effect.

1	Amebic Liver Abscess Extraintestinal infection by E. histolytica most often involves the liver. Of travelers who develop an amebic liver abscess after leaving an endemic area, 95% do so within 5 months. Young patients with an amebic liver abscess are more likely than older patients to present in the acute phase with prominent symptoms of <10 days’ duration. Most patients are febrile and have right-upperquadrant pain, which may be dull or pleuritic in nature and may radiate to the shoulder. Point tenderness over the liver and right-sided pleural effusion are common. Jaundice is rare. Although the initial site of infection is the colon, fewer than one-third of patients with an amebic abscess have active diarrhea. Older patients from endemic areas are more likely to have a subacute course lasting 6 months, with weight loss and hepatomegaly. About one-third of patients with chronic pre sentations are febrile. Thus, the clinical diagnosis of an amebic liver abscess may be difficult to

1	lasting 6 months, with weight loss and hepatomegaly. About one-third of patients with chronic pre sentations are febrile. Thus, the clinical diagnosis of an amebic liver abscess may be difficult to establish because the symptoms and signs are often nonspecific. Since 10–15% of patients present only with fever, amebic liver abscess must be considered in the differential diagnosis of fever of unknown origin (Chap. 26).

1	Complications of Amebic Liver Abscess Pleuropulmonary involvement, which is reported in 20–30% of patients, is the most frequent complication of amebic liver abscess. Manifestations include sterile effusions, contiguous spread from the liver, and rupture into the pleural space. Sterile effusions and contiguous spread usually resolve with medical therapy, but frank rupture into the pleural space requires drainage. A hepatobronchial fistula may cause cough productive of large amounts of necrotic material that may contain amebas. This dramatic complication carries a good prognosis. Abscesses that rupture into the peritoneum may present as an indolent leak or an acute abdomen and require both percutaneous catheter drainage and medical therapy. Rupture into the pericardium, usually from abscesses of the left lobe of the liver, carries the gravest prognosis; it can occur during medical therapy and requires surgical drainage.

1	Other Extraintestinal Sites The genitourinary tract may become involved by direct extension of amebiasis from the colon or by hematogenous spread of the infection. Painful genital ulcers, characterized by a punched-out appearance and profuse discharge, may develop secondary to extension from either the intestine or the liver. Both of these conditions respond well to medical therapy. Cerebral involvement has been reported in fewer than 0.1% of patients in large clinical series. Symptoms and prognosis depend on the size and location of the lesion. DIAGNOSTIC TESTS Laboratory Diagnosis Stool examinations, serologic tests, and noninvasive imaging of the liver are the most important procedures in the diagnosis of amebiasis. Fecal findings suggestive of amebic colitis

1	Amebiasis and Infection with Free-Living Amebas 1366 include a positive test for heme, a paucity of neutrophils, and amebic cysts or trophozoites. The definitive diagnosis of amebic colitis is made by the demonstration of hematophagous trophozoites of E. histolytica (Fig. 247-1). Because trophozoites are killed rapidly by water, drying, or barium, it is important to examine at least three fresh stool specimens. Examination of a combination of wet mounts, iodine-stained concentrates, and trichrome-stained preparations of fresh stool and concentrates for cysts (Fig. 247-2) or trophozoites (Fig. 247-1) confirms the diagnosis in 75–95% of cases. Culture of amebas is more sensitive, but this diagnostic method is not routinely available. If stool examinations are negative, sigmoidoscopy with biopsy of the edge of ulcers may increase the yield, but this procedure is dangerous during fulminant colitis because of the risk of perforation. Trophozoites in a biopsy specimen from a colonic mass

1	biopsy of the edge of ulcers may increase the yield, but this procedure is dangerous during fulminant colitis because of the risk of perforation. Trophozoites in a biopsy specimen from a colonic mass confirm the diagnosis of ameboma, but trophozoites are rare in liver aspirates because they are found in the abscess capsule and not in the readily aspirated necrotic center. Accurate diagnosis requires experience, since the trophozoites may be confused with neutrophils and the cysts must be differentiated morphologically from those of Entamoeba hartmanni, Entamoeba coli, and Endolimax nana, which do not cause clinical disease and do not warrant therapy. Unfortunately, the cysts of E. histolytica cannot be distinguished microscopically from those of E. dispar or

1	E. moshkovskii. Therefore, the microscopic diagnosis of E. histolytica can be made only by the detection of Entamoeba trophozoites that have ingested erythrocytes. In terms of sensitivity, stool diagnostic tests based on the detection of the Gal/GalNAc lectin of E. histolytica compare favorably with the polymerase chain reaction and with isolation in culture followed by isoenzyme analysis.

1	Serology is an important addition to the methods used for parasitologic diagnosis of invasive amebiasis. Enzyme-linked immunosorbent assays and agar gel diffusion assays are positive in more than 90% of patients with colitis, amebomas, or liver abscess. Positive results in conjunction with the appropriate clinical syndrome suggest active disease because serologic findings usually revert to negative within 6–12 months. Even in highly endemic areas such as South Africa, fewer than 10% of asymptomatic individuals have a positive amebic serology. The interpretation of the indirect hemagglutination test is more difficult because titers may remain positive for as long as 10 years. Up to 10% of patients with acute amebic liver abscess may have negative serologic findings; in suspected cases with an initially negative result, testing should be repeated in 1 week. In contrast to carriers of

1	E. dispar, most asymptomatic carriers of E. histolytica develop antibodies. Thus, serologic tests are helpful in assessing the risk of invasive amebiasis in asymptomatic, cyst-passing individuals in nonendemic areas. Serologic tests also should be performed in patients with ulcerative colitis before the institution of glucocorticoid therapy to prevent the development of severe colitis or toxic megacolon owing to unsuspected amebiasis.

1	Routine hematology and chemistry tests usually are not very helpful in the diagnosis of invasive amebiasis. About three-fourths of patients with an amebic liver abscess have leukocytosis (>10,000 cells/μL); this condition is particularly likely if symptoms are acute or complications have developed. Invasive amebiasis does not elicit eosinophilia. Anemia, if present, is usually multifactorial. Even with large liver abscesses, liver enzyme levels are normal or minimally elevated. The alkaline phosphatase level is most often elevated and may remain so for months. Aminotransferase elevations suggest acute disease or a complication. Radiographic Studies Radiographic barium studies are potentially dangerous in acute amebic colitis. Amebomas are usually identified first by a barium enema, but biopsy is necessary for differentiation from carcinoma.

1	Radiographic techniques such as ultrasonography, CT, and MRI are all useful for detection of the round or oval hypoechoic cyst of an amebic liver abscess. More than 80% of patients who have had symptoms for >10 days have a single abscess of the right lobe of the liver (Fig. 247-4). Approximately 50% of patients who have had symptoms for <10 days have multiple abscesses. Findings associated with complications include large abscesses (>10 cm) in the superior part of the right lobe, which may rupture into the pleural space; multiple lesions, which must be differentiated from pyogenic abscesses; and lesions of the left lobe, which may rupture into the pericardium. Because abscesses resolve slowly and may increase in size in patients who are responding clinically to therapy, frequent follow-up ultrasonography may prove confusing. Complete resolution of a liver abscess within 6 months can be anticipated in two-thirds of patients, but 10% may have persistent abnormalities for a year.

1	FIGuRE 247-4 Abdominal CT scan of a large amebic abscess of the right lobe of the liver. (Courtesy of the Department of Radiology, UCSD Medical Center, San Diego; with permission.) Differential Diagnosis The differential diagnosis of intestinal amebiasis includes bacterial diarrheas (Chap. 160) caused by Campylobacter (Chap. 192); enteroinvasive Escherichia coli (Chap. 186); and species of Shigella (Chap. 191), Salmonella (Chap. 190), and Vibrio (Chap. 193). Although the typical patient with amebic colitis has less prominent fever than in these other conditions as well as heme-positive stools with few neutrophils, correct diagnosis requires bacterial cultures, microscopic examination of stools, and amebic serologic testing. As has already been mentioned, amebiasis must be ruled out in any patient thought to have inflammatory bowel disease.

1	Because of the variety of presenting signs and symptoms, amebic liver abscess can easily be confused with pulmonary or gallbladder disease or with any febrile illness with few localizing signs, such as malaria (Chap. 248) or typhoid fever (Chap. 190). The diagnosis should be considered in members of high-risk groups who have recently traveled outside the United States (Chap. 149) and in inmates of institutions. Once radiographic studies have identified an abscess in the liver, the most important differential diagnosis is between amebic and pyogenic abscess. Patients with pyogenic abscess typically are older and have a history of underlying bowel disease or recent surgery. Amebic serology is helpful, but aspiration of the abscess, with Gram’s staining and culture of the material, may be required for differentiation of the two diseases.

1	The drugs used to treat amebiasis can be classified according to their primary site of action (Table 247-1). Luminal amebicides are poorly absorbed; they reach high concentrations in the bowel, but their activity is limited to cysts and trophozoites close to the mucosa. Only two luminal drugs are available in the United States: iodoquinol and paromomycin. Indications for the use of luminal agents include eradication of cysts in patients with colitis or a liver abscess and treatment of asymptomatic carriers. The majority of Asymptomatic carriage Luminal agent: iodoquinol (650-mg tablets), (250-mg tablets), 500 mg tid for 10 days Acute colitis Metronidazole (250or 500-mg tablets), tinidazole, 2 g/d PO for 3 days Luminal agent as above Amebic liver abscess Metronidazole, 750 mg PO or IV for 5–10 days; or tinidazole, 2 g PO once; or ornidazole,a 2 g Luminal agent as above aNot available in the United States.

1	Amebic liver abscess Metronidazole, 750 mg PO or IV for 5–10 days; or tinidazole, 2 g PO once; or ornidazole,a 2 g Luminal agent as above aNot available in the United States. asymptomatic individuals who pass cysts are colonized with E. dis-par, which does not warrant specific therapy. However, it is prudent to treat asymptomatic individuals who pass cysts unless E. dispar colonization can be definitively demonstrated by specific antigen-detection tests.

1	Tissue amebicides reach high concentrations in the blood and tissue after oral or parenteral administration. The development of nitroimidazole compounds, especially metronidazole, was a major advance in the treatment of invasive amebiasis. Patients with amebic colitis should be treated with IV or oral metronidazole. Side effects include nausea, vomiting, abdominal discomfort, and a disulfiram-like reaction. Another longer-acting imidazole compound, tinidazole, is also effective and available in the United States. All patients should also receive a full course of therapy with a luminal agent, since metronidazole does not eradicate cysts. Resistance to metronidazole has been selected in the laboratory but has not been found in clinical isolates. Relapses are not uncommon and probably represent reinfection or failure to eradicate amebas from the bowel because of an inadequate dosage or duration of therapy.

1	Metronidazole is the drug of choice for amebic liver abscess. Longer-acting nitroimidazoles (tinidazole and ornidazole) have been effective as single-dose therapy in developing countries. With early diagnosis and therapy, mortality rates from uncomplicated amebic liver abscess are <1%. There is no evidence that combined therapy with two drugs is more effective than the single-drug regimen. Studies of South Africans with liver abscesses demonstrated that 72% of patients without intestinal symptoms had bowel infection with E. histolytica; thus, all treatment regimens should include a luminal agent to eradicate cysts and prevent further transmission. Amebic liver abscess recurs rarely.

1	E. histolytica; thus, all treatment regimens should include a luminal agent to eradicate cysts and prevent further transmission. Amebic liver abscess recurs rarely. More than 90% of patients respond dramatically to metronidazole therapy with decreases in both pain and fever within 72 h. Indications for aspiration of liver abscesses are (1) the need to rule out a pyogenic abscess, particularly in patients with multiple lesions; (2) the lack of a clinical response in 3–5 days; (3) the threat of imminent rupture; and (4) the need to prevent rupture of left-lobe abscesses into the pericardium. There is no evidence that aspiration, even of large abscesses (up to 10 cm), accelerates healing. Percutaneous drainage may be successful even if the liver abscess has already ruptured. Surgery should be reserved for instances of bowel perforation and rupture into the pericardium.

1	Amebic infection is spread by ingestion of food or water contaminated with cysts. Since an asymptomatic carrier may excrete up to 15 million cysts per day, prevention of infection requires adequate sanitation and eradication of cyst carriage. In high-risk areas, infection can be minimized by the avoidance of unpeeled fruits and vegetables and the use of bottled water. Because cysts are resistant to readily 1367 attainable levels of chlorine, disinfection by iodination (tetraglycine hydroperiodide) is recommended. There is no effective prophylaxis.

1	Free-living amebas of the genera Acanthamoeba and Naegleria are distributed throughout the world and have been isolated from a wide variety of fresh and brackish water, including that from lakes, taps, hot springs, swimming pools, and heating and air-conditioning units, and even from the nasal passages of healthy children. Encystation may protect the protozoa from desiccation and food deprivation. The persistence of Legionella pneumophila in water supplies may be attributable in part to chronic infection of free-living amebas, particularly Naegleria. Free-living amebas of the genus Balamuthia have been isolated from soil samples, including a sample from a flowerpot linked to a fatal infection in a child.

1	Primary amebic meningoencephalitis caused by Naegleria fowleri follows the aspiration of water contaminated with trophozoites or cysts or the inhalation of contaminated dust, leading to invasion of the olfactory neuroepithelium. Infection is most common among otherwise healthy children or young adults, who often report recent swimming in lakes or heated swimming pools. Rarely, some cases occur when contaminated water is used for nasal irrigation. After an incubation period of 2–15 days, severe headache, high fever, nausea, vomiting, and meningismus develop. Photophobia and palsies of the third, fourth, and sixth cranial nerves are common. Rapid progression to seizures and coma may follow. The prognosis is uniformly poor: most patients die within a week. Recently, two surviving children were treated with miltefosine, an investigational drug that is available through the Centers for Disease Control and Prevention (CDC) for the treatment of Naegleria infections.

1	The diagnosis of Naegleria infection should be considered in any patient who has purulent meningitis without evidence of bacteria on Gram’s staining, antigen detection assay, and culture. Other laboratory findings resemble those for fulminant bacterial meningitis, with elevated intracranial pressure, high white blood cell counts (up to 20,000/μL), and elevated protein concentrations and low glucose levels in cerebrospinal fluid (CSF). Diagnosis depends on the detection of motile trophozoites in wet mounts of fresh spinal fluid. Antibodies to Naegleria species have been detected in healthy adults; serologic testing is not useful in the diagnosis of acute infection.

1	ACANTHAMOEBA INFECTIONS Granulomatous Amebic Encephalitis Infection with Acanthamoeba species follows a more indolent course and typically occurs in chronically ill or debilitated patients. Risk factors include lymphoproliferative disorders, chemotherapy, glucocorticoid therapy, lupus erythematosus, and AIDS. Infection usually reaches the central nervous system hematogenously from a primary focus in the sinuses, skin, or lungs. In the central nervous system, the onset is insidious, and the syndrome often mimics a space-occupying lesion. Altered mental status, headache, and stiff neck may be accompanied by focal findings such as cranial nerve palsies, ataxia, and hemiparesis. Cutaneous ulcers or hard nodules containing amebas are frequently detected in AIDS patients with disseminated Acanthamoeba infection and can be an important diagnostic site.

1	Examination of the CSF for trophozoites may be diagnostically helpful, but lumbar puncture may be contraindicated because of increased intracerebral pressure. CT frequently reveals cortical and subcortical lesions of decreased density consistent with embolic infarcts. In other patients, multiple enhancing lesions with edema may mimic the computed tomographic appearance of toxoplasmosis (Chap. 253). Demonstration of the trophozoites and cysts of Acanthamoeba on wet mounts or in biopsy specimens establishes the diagnosis. Culture on nonnutrient agar plates seeded with Escherichia coli also may be helpful. Fluorescein-labeled antiserum is available Amebiasis and Infection with Free-Living Amebas

1	Amebiasis and Infection with Free-Living Amebas FIGuRE 247-5 Double-walled cyst of Acanthamoeba castellanii, as seen by phase-contrast microscopy. (From DJ Krogstad et al, in A Balows et al [eds]: Manual of Clinical Microbiology, 5th ed. Washington, DC, American Society for Microbiology, 1991.) from the CDC for the detection of protozoa in biopsy specimens. Granulomatous amebic encephalitis in patients with AIDS may have an accelerated course (with survival for only 3–40 days) because of poor granuloma formation in these individuals. Various antimicrobial agents have been used to treat Acanthamoeba infection, but the infection is almost uniformly fatal. The CDC has now made miltefosine available because of improved survival rates when the drug is included in treatment regimens.

1	Keratitis The incidence of keratitis caused by Acanthamoeba has increased in the past 30 years, in part as a result of improved diagnosis. Earlier infections were associated with trauma to the eye and exposure to contaminated water. At present, most infections are linked to extended-wear contact lenses, and rare cases are associated with laser-assisted in situ keratomileusis (LASIK). Risk factors include the use of homemade saline, the wearing of lenses while swimming, and inadequate disinfection. Since contact lenses presumably cause microscopic trauma, the early corneal findings may be nonspecific. The first symptoms usually include tearing and the painful sensation of a foreign body. Once infection is established, progression is rapid; the characteristic clinical sign is an annular, paracentral corneal ring representing a corneal abscess. Deeper corneal invasion and loss of vision may follow.

1	The differential diagnosis includes bacterial, mycobacterial, and herpetic infection. The irregular polygonal cysts of Acanthamoeba (Fig. 247-5) may be identified in corneal scrapings or biopsy material, and trophozoites can be grown on special media. Cysts are resistant to available drugs, and the results of medical therapy have been disappointing. Some reports have suggested partial responses to propamidine isethionate eyedrops. Severe infections usually require keratoplasty.

1	Balamuthia mandrillaris, a free-living ameba previously referred to as a leptomyxid ameba, is an important etiologic agent of amebic meningoencephalitis in immunocompetent hosts. The course is typically sub-acute, with focal neurologic signs, fever, seizures, and headaches leading to death within 1 week to several months after onset. Examination of CSF reveals mononuclear or neutrophilic pleocytosis, elevated protein levels, and normal to low glucose concentrations. Multiple hypodense lesions are usually detected with imaging studies (Fig. 247-6). This mixed picture of space-occupying lesions with CSF pleocytosis is suggestive of Balamuthia. Fluorescent antibody is available from the CDC for brain biopsy specimens. The variety of drugs used to treat the few surviving patients (i.e., fewer than five reported in the United States) includes pentamidine, flucytosine, sulfadiazine, and macrolides. The CDC recommends that miltefosine now be included, as for the other free-living amebas. The

1	than five reported in the United States) includes pentamidine, flucytosine, sulfadiazine, and macrolides. The CDC recommends that miltefosine now be included, as for the other free-living amebas. The differential diagnosis includes tuberculomas (Chap. 202) and neurocysticercosis (Chap. 260).

1	FIGuRE 247-6 Brain MRI of amebic meningoencephalitis due to Balamuthia mandrillaris. A large lesion in the parieto-occipital lobe and other smaller lesions are seen. (Courtesy of the Department of Radiology, UCSD Medical Center, San Diego.) Nicholas J. White, Joel G. Breman Humanity has but three great enemies: Fever, famine, and war; of these by far the greatest, by far the most terrible, is fever. —William Osler

1	Malaria is a protozoan disease transmitted by the bite of infected Anopheles mosquitoes. The most important of the parasitic diseases of humans, it is transmitted in 106 countries containing 3 billion people and causes approximately 2000 deaths each day; mortality rates are decreasing as a result of highly effective control programs in several countries. Malaria has been eliminated from the United States, Canada, Europe, and Russia; in the late twentieth and early twenty-first centuries, however, its prevalence rose in many parts of the tropics. Increases in the drug resistance of the parasite, the insecticide resistance of its vectors, and human travel and migration have contributed to this resurgence. Occasional local transmission after importation of malaria has occurred in several southern and eastern areas of the United States and in Europe, indicating the continual danger to non-malarious countries. Although there are many successful new control initiatives as well as promising

1	and eastern areas of the United States and in Europe, indicating the continual danger to non-malarious countries. Although there are many successful new control initiatives as well as promising research initiatives, malaria remains today, as it has been for centuries, a heavy burden on tropical communities, a threat to nonendemic countries, and a danger to travelers.

1	Six species of the genus Plasmodium cause nearly all malarial infections in humans. These are P. falciparum, P. vivax, two morphologically identical sympatric species of P. ovale (as suggested by recent evidence), P. malariae, and—in Southeast Asia—the monkey malaria parasite P. knowlesi (Table 248-1). While almost all deaths are caused aIn Southeast Asia, the monkey malaria parasite P. knowlesi also causes disease in humans. Young ring forms resemble those of P. falciparum, while older trophozoites resemble those of P. malariae. Reliable identification requires molecular genotyping. bParasitemias of >2% are suggestive of P. falciparum infection. Antibodies to sporozoites block invasion of hepatocytes Antibodies to merozoites block invasion of RBCs Antibodies to malaria "toxins" Antibodies to parasite antigens on infected RBCs block cytoadherence to endothelium

1	Antibodies block fertilization, development, and invasion by falciparum malaria, P. knowlesi and occasionally P. vivax also can cause severe illness. Human infection begins when a female anopheline mosquito inoculates plasmodial sporozoites from its salivary gland during a blood meal (Fig. 248-1). These microscopic motile forms of the malaria parasite are carried rapidly via the bloodstream to the liver, where they invade hepatic parenchymal cells and begin a period of asexual reproduction. By this amplification process (known as intrahepatic or preerythrocytic schizogony or merogony), a single sporozoite eventually may produce from 10,000 to >30,000 daughter merozoites. The swollen infected liver cells eventually burst, discharging motile merozoites into the bloodstream. These merozoites then invade the red blood cells (RBCs) and multiply sixto twentyfold every 48 h (P. knowlesi, 24 h; P. malariae, 72 h). When the parasites reach densities of ~50/μL of blood (~100 million parasites

1	then invade the red blood cells (RBCs) and multiply sixto twentyfold every 48 h (P. knowlesi, 24 h; P. malariae, 72 h). When the parasites reach densities of ~50/μL of blood (~100 million parasites in the blood of an adult), the symptomatic stage of the infection begins. In P. vivax and P. ovale infections, a proportion of the intrahepatic forms do not divide immediately but remain inert for a period ranging from 3 weeks to ≥1 year before reproduction begins. These dormant forms, or hypnozoites, are the cause of the relapses that characterize infection with these two species.

1	After entry into the bloodstream, merozoites rapidly invade erythrocytes and become trophozoites. Attachment is mediated via a specific erythrocyte surface receptor. For P. falciparum, the reticulocyte-binding protein homologue 5 (PfRh5) is indispensable for erythrocyte invasion. Basigin (CD147, EMMPRIN) is the erythrocyte receptor of PfRh5. In the case of P. vivax, this receptor is related to the Duffy blood-group antigen Fya or Fyb. Most West Africans and people with origins in that region carry the Duffy-negative FyFy phenotype and are therefore resistant to P. vivax malaria. During the early stage of intraerythrocytic development, the small “ring forms” of the different parasitic species appear similar under light microscopy. As the trophozoites enlarge, species-specific characteristics become evident, pigment becomes visible, and the parasite assumes an irregular or ameboid shape. By the end of the intraerythrocytic life cycle, the parasite has consumed two-thirds of the RBC’s

1	become evident, pigment becomes visible, and the parasite assumes an irregular or ameboid shape. By the end of the intraerythrocytic life cycle, the parasite has consumed two-thirds of the RBC’s hemoglobin and has grown to occupy most of the cell. It is now called a schizont. Multiple nuclear divisions have taken place (schizogony or merogony). The RBC then ruptures to release 6–30 daughter merozoites, each potentially capable of invading a new RBC and repeating the cycle. The disease in human beings is caused by the direct effects of the asexual parasite—RBC invasion and destruction—and by the host’s reaction. After release from the liver (P. vivax, P. ovale, P. malariae, P. knowlesi), some of the blood-stage parasites develop into morphologically distinct,

1	FIGuRE 248-1 The malaria transmission cycle from mosquito to human and targets of immunity. RBC, red blood cell. 1370 longer-lived sexual forms (gametocytes) that can transmit malaria. In falciparum malaria, a delay of several asexual cycles precedes this switch to gametocytogenesis. After being ingested in the blood meal of a biting female anopheline mosquito, the male and female gametocytes form a zygote in the insect’s midgut. This zygote matures into an ookinete, which penetrates and encysts in the mosquito’s gut wall. The resulting oocyst expands by asexual division until it bursts to liberate myriad motile sporozoites, which then migrate in the hemolymph to the salivary gland of the mosquito to await inoculation into another human at the next feeding. Senegal The Gambia Guinea-

1	Senegal The Gambia Guinea- Malaria occurs throughout most of the tropical regions of the world (Fig. 248-2). P. falciparum predominates in Africa, New Guinea, and Hispaniola (i.e., the Dominican Republic and Haiti); P. vivax is more common in Central America. The prevalence of these two species is approximately equal in South America, the Indian subcontinent, eastern Asia, and Oceania. P. malariae is found in most endemic areas, especially throughout sub-Saharan Africa, but is much less common. P. ovale is relatively unusual outside of Africa and, where it is found, comprises <1% of isolates. Patients infected with P. knowlesi have been identified on the island of Borneo and, to a lesser extent, elsewhere in Southeast Asia, where the main hosts, long-tailed and pig-tailed macaques, are found.

1	MexicoArgentinaBoliviaColombiaVenezuelaPeruBrazilFrenchGuianaSurinameGuyanaChileEcuadorGalapagosIslandsParaguayUruguayFalklandIslandsDominican Republic HaitiBelize Guatemala El Salvador Nicaragua Costa Rica Panama Honduras Mongolia China Japan North Korea South Korea Philippines Taiwan Australia New Zealand New Caledonia Vanuatu Solomon Islands Papua New Guinea Pacific Islands (Palau) Fiji India IranIraq Jordan ThailandBangladesh Laos Bhutan Nepal Sri Lanka Madagascar Pakistan Afghanistan Turkmenistan Uzbekistan Kyrgyzstan Azerbaijan Armenia Georgia Saudi Arabia South Africa Egypt Turkey Malaysia Vietnam Macau Indonesia Algeria Libya Morocco Namibia Nigeria Cameroon Togo Benin Ghana Niger Chad CAR Sudan Mali Burkina Faso Western Sahara Ethiopia Botswana Angola Zambia DROC Rwanda Uganda Tanzania Kenya Burundi Somalia Syria CyprusIsrael Tunisia Greece Yemen Eritrea Oman Singapore Portugal Spain France Myanmar Gabon Congo Equatorial Guinea Guinea Sierra Leone Liberia Cote d'IvoireLesotho

1	Kenya Burundi Somalia Syria CyprusIsrael Tunisia Greece Yemen Eritrea Oman Singapore Portugal Spain France Myanmar Gabon Congo Equatorial Guinea Guinea Sierra Leone Liberia Cote d'IvoireLesotho Swaziland Mozambique Zimbabwe Malawi UAE Kuwait Qatar Chloroquine-resistant Chloroquine-sensitive None Malaria-Endemic Areas Djibouti FIGuRE 248-2 Malaria-endemic countries in the Americas (bottom) and in Africa, the Middle East, Asia, and the South Pacific (top), 2007. CAR, Central African Republic; DROC, Democratic Republic of the Congo; UAE, United Arab Emirates. Several countries in the Americas, the Middle East, and North Africa are close to eliminating malaria.

1	The epidemiology of malaria is complex and may vary considerably even within relatively small geographic areas. Endemicity traditionally has been defined in terms of parasitemia rates or palpable-spleen rates in children 2–9 years of age and classified as hypoendemic (<10%), mesoendemic (11–50%), hyperendemic (51–75%), and holoendemic (>75%). Until recently, it was uncommon to use these indices for planning control programs; however, many countries are now conducting national surveys to assess program progress. In holoand hyperendemic areas (e.g., certain regions of tropical Africa or coastal New Guinea) where there is intense P. falciparum transmission, people may sustain more than one infectious mosquito bite per day and are infected repeatedly throughout their lives. In such settings, rates of morbidity and mortality due to malaria are considerable during early childhood. Immunity against disease is hard won in these areas, and the burden of disease in young children is high; by

1	rates of morbidity and mortality due to malaria are considerable during early childhood. Immunity against disease is hard won in these areas, and the burden of disease in young children is high; by adulthood, however, most malarial infections are asymptomatic. As control measures progress and urbanization expands, environmental conditions become less conducive to transmission, and all age groups may lose protective immunity and become susceptible to illness. Constant, frequent, year-round infection is termed stable transmission. In areas where transmission is low, erratic, or focal, full protective immunity is not acquired, and symptomatic disease may occur at all ages. This situation usually exists in hypoendemic areas and is termed unstable transmission. Even in stable-transmission areas, there is often an increased incidence of symptomatic malaria coinciding with increased mosquito breeding and transmission during the rainy season. Malaria can behave like an epidemic disease in

1	there is often an increased incidence of symptomatic malaria coinciding with increased mosquito breeding and transmission during the rainy season. Malaria can behave like an epidemic disease in some areas, particularly those with unstable malaria, such as northern India (the Punjab region), the horn of Africa, Rwanda, Burundi, southern Africa, and Madagascar. An epidemic can develop when there are changes in environmental, economic, or social conditions, such as heavy rains following drought or migrations (usually of refugees or workers) from a nonmalarious region to an area of high transmission, along with failure to invest in national programs; a breakdown in malaria control and prevention services caused by war or civil disorder can intensify epidemic conditions. This situation usually results in considerable mortality among all age groups.

1	The principal determinants of the epidemiology of malaria are the number (density), the human-biting habits, and the longevity of the anopheline mosquito vectors. More than 100 of the >400 anopheline species can transmit malaria, but the ~40 species that do so commonly vary considerably in their efficiency as malaria vectors. More specifically, the transmission of malaria is directly proportional to the density of the vector, the square of the number of human bites per day per mosquito, and the tenth power of the probability of the mosquito’s surviving for 1 day. Mosquito longevity is particularly important because the portion of the parasite’s life cycle that takes place within the mosquito—from gametocyte ingestion to subsequent inoculation (sporogony)—lasts 8–30 days, depending on ambient temperature; thus, to transmit malaria, the mosquito must survive for >7 days. Sporogony is not completed at cooler temperatures—i.e., <16°C (60.8°F) for P. vivax and <21°C (69.8°F) for P.

1	on ambient temperature; thus, to transmit malaria, the mosquito must survive for >7 days. Sporogony is not completed at cooler temperatures—i.e., <16°C (60.8°F) for P. vivax and <21°C (69.8°F) for P. falciparum; thus transmission does not occur below these temperatures or at high altitudes, although malaria outbreaks and transmission have occurred in the highlands (>1500 m) of eastern Africa, which were previously free of vectors. The most effective mosquito vectors of malaria are those, such as Anopheles gambiae in Africa, that are long-lived, occur in high densities in tropical climates, breed readily, and bite humans in preference to other animals. The entomologic inoculation rate (i.e., the number of sporozoitepositive mosquito bites per person per year) is the most common measure of malaria transmission and varies from <1 in some parts of Latin America and Southeast Asia to >300 in parts of tropical Africa.

1	After invading an erythrocyte, the growing malarial parasite progressively consumes and degrades intracellular proteins, principally hemoglobin. The potentially toxic heme is detoxified by lipid-mediated 1371 crystallization to biologically inert hemozoin (malaria pigment). The parasite also alters the RBC membrane by changing its transport properties, exposing cryptic surface antigens, and inserting new parasite-derived proteins. The RBC becomes more irregular in shape, more antigenic, and less deformable.

1	In P. falciparum infections, membrane protuberances appear on the erythrocyte’s surface 12–15 h after the cell’s invasion. These “knobs” extrude a high-molecular-weight, antigenically variant, strain-specific erythrocyte membrane adhesive protein (PfEMP1) that mediates attachment to receptors on venular and capillary endothelium—an event termed cytoadherence. Several vascular receptors have been identified, of which intercellular adhesion molecule 1 is probably the most important in the brain, chondroitin sulfate B in the placenta, and CD36 in most other organs. Thus, the infected erythrocytes stick inside and eventually block capillaries and venules. At the same stage, these

1	P. falciparum–infected RBCs may also adhere to uninfected RBCs (to form rosettes) and to other parasitized erythrocytes (agglutination). The processes of cytoadherence, rosetting, and agglutination are central to the pathogenesis of falciparum malaria. They result in the sequestration of RBCs containing mature forms of the parasite in vital organs (particularly the brain), where they interfere with microcirculatory flow and metabolism. Sequestered parasites continue to develop out of reach of the principal host defense mechanism: splenic processing and filtration. As a consequence, only the younger ring forms of the asexual parasites are seen circulating in the peripheral blood in falciparum malaria, and the level of peripheral parasitemia underestimates the true number of parasites within the body. Severe malaria is also associated with reduced deformability of the uninfected erythrocytes, which compromises their passage through the partially obstructed capillaries and venules and

1	the body. Severe malaria is also associated with reduced deformability of the uninfected erythrocytes, which compromises their passage through the partially obstructed capillaries and venules and shortens RBC survival.

1	In the other human malarias, sequestration does not occur, and all stages of the parasite’s development are evident on peripheral-blood smears. Whereas P. vivax, P. ovale, and P. malariae show a marked predilection for either young RBCs (P. vivax, P. ovale) or old cells (P. malariae) and produce a level of parasitemia that is seldom >2%, P. falciparum can invade erythrocytes of all ages and may be associated with very high levels of parasitemia.

1	Initially, the host responds to plasmodial infection by activating nonspecific defense mechanisms. Splenic immunologic and filtrative clearance functions are augmented in malaria, and the removal of both parasitized and uninfected erythrocytes is accelerated. The spleen is able to remove damaged ring-form parasites and return the once-infected erythrocytes to the circulation, where their survival period is shortened. The parasitized cells escaping splenic removal are destroyed when the schizont ruptures. The material released induces the activation of macrophages and the release of proinflammatory cytokines, which cause fever and exert other pathologic effects. Temperatures of ≥40°C (104°F) damage mature parasites; in untreated infections, the effect of such temperatures is to further synchronize the parasitic cycle, with eventual production of the regular fever spikes and rigors that originally served to characterize the different malarias. These regular fever patterns (quotidian,

1	the parasitic cycle, with eventual production of the regular fever spikes and rigors that originally served to characterize the different malarias. These regular fever patterns (quotidian, daily; tertian, every 2 days; quartan, every 3 days) are seldom seen today in patients who receive prompt and effective antimalarial treatment.

1	The geographic distributions of sickle cell disease, hemoglobins C and E, hereditary ovalocytosis, the thalassemias, and glucose-6-phosphate dehydrogenase (G6PD) deficiency closely resemble that of falciparum malaria before the introduction of control measures. This similarity suggests that these genetic disorders confer protection against death from falciparum malaria. For example, HbA/S heterozygotes (sickle cell trait) have a sixfold reduction in the risk of dying from severe falciparum malaria. Hemoglobin S–containing RBCs impair parasite growth at low oxygen tensions, and P. falciparum–infected RBCs containing hemoglobins S and C exhibit reduced cytoadherence because of reduced surface presentation of the adhesin PfEMP1. Parasite multiplication in HbA/E heterozygotes is reduced at high 1372 parasite densities. In Melanesia, children with α-thalassemia appear to have more frequent malaria (both vivax and falciparum) in the early years of life, and this pattern of infection appears

1	1372 parasite densities. In Melanesia, children with α-thalassemia appear to have more frequent malaria (both vivax and falciparum) in the early years of life, and this pattern of infection appears to protect them against severe disease. In Melanesian ovalocytosis, rigid erythrocytes resist merozoite invasion, and the intraerythrocytic milieu is hostile. Nonspecific host defense mechanisms stop the infection’s expansion, and the subsequent strain-specific immune response then controls the infection. Eventually, exposure to sufficient strains confers protection from high-level parasitemia and disease but not from infection. As a result of this state of infection without illness (premunition), asymptomatic parasitemia is common among adults and older children living in regions with stable and intense transmission (i.e., holoor hyperendemic areas) and also in parts of low-transmission areas. Immunity is mainly specific for both the species and the strain of infecting malarial parasite.

1	and intense transmission (i.e., holoor hyperendemic areas) and also in parts of low-transmission areas. Immunity is mainly specific for both the species and the strain of infecting malarial parasite. Both humoral immunity and cellular immunity are necessary for protection, but the mechanisms of each are incompletely understood (Fig. 248-1). Immune individuals have a polyclonal increase in serum levels of IgM, IgG, and IgA, although much of this antibody is unrelated to protection. Antibodies to a variety of parasitic antigens presumably act in concert to limit in vivo replication of the parasite. In the case of falciparum malaria, the most important of these antigens is the surface adhesin— the variant protein PfEMP1. Passively transferred IgG from immune adults has been shown to reduce levels of parasitemia in children. Passive transfer of maternal antibody contributes to the relative (but not complete) protection of infants from severe malaria in the first months of life. This

1	levels of parasitemia in children. Passive transfer of maternal antibody contributes to the relative (but not complete) protection of infants from severe malaria in the first months of life. This complex immunity to disease declines when a person lives outside an endemic area for several months or longer. Several factors retard the development of cellular immunity to malaria. These factors include the absence of major histocompatibility antigens on the surface of infected RBCs, which precludes direct T cell recognition; malaria antigen–specific immune unresponsiveness; and the enormous strain diversity of malarial parasites, along with the ability of the parasites to express variant immunodominant antigens on the erythrocyte surface that change during the course of infection. Parasites may persist in the blood for months or years (or, in the case of P. malariae, for decades) if treatment is not given. The complexity of the immune response in malaria, the sophistication of the

1	may persist in the blood for months or years (or, in the case of P. malariae, for decades) if treatment is not given. The complexity of the immune response in malaria, the sophistication of the parasites’ evasion mechanisms, and the lack of a good in vitro correlate with clinical immunity have all slowed progress toward an effective vaccine.

1	Malaria is a very common cause of fever in tropical countries. The first symptoms of malaria are nonspecific; the lack of a sense of wellbeing, headache, fatigue, abdominal discomfort, and muscle aches followed by fever are all similar to the symptoms of a minor viral illness. In some instances, a prominence of headache, chest pain, abdominal pain, cough, arthralgia, myalgia, or diarrhea may suggest another diagnosis. Although headache may be severe in malaria, the neck stiffness and photophobia seen in meningitis do not occur. While myalgia may be prominent, it is not usually as severe as in dengue fever, and the muscles are not tender as in leptospirosis or typhus. Nausea, vomiting, and orthostatic hypotension are common. The classic malarial paroxysms, in which fever spikes, chills, and rigors occur at regular intervals, are relatively unusual and suggest infection with P. vivax or P. ovale. The fever is usually irregular at first (that of falciparum malaria may never become

1	and rigors occur at regular intervals, are relatively unusual and suggest infection with P. vivax or P. ovale. The fever is usually irregular at first (that of falciparum malaria may never become regular); the temperature of nonimmune individuals and children often rises above 40°C (104°F) in conjunction with tachycardia and sometimes delirium. Although childhood febrile convulsions may occur with any of the malarias, generalized seizures are specifically associated with falciparum malaria and may herald the development of encephalopathy (cerebral malaria). Many clinical abnormalities have been described in acute malaria, but most patients with uncomplicated infections have few abnormal physical findings other than fever, malaise, mild anemia, and (in some cases) a palpable spleen. Anemia is common among young children living in areas with stable transmission, particularly where resistance has compromised the efficacy of antimalarial drugs. In nonimmune individuals with acute malaria,

1	is common among young children living in areas with stable transmission, particularly where resistance has compromised the efficacy of antimalarial drugs. In nonimmune individuals with acute malaria, the spleen takes several days to become palpable, but splenic enlargement is found in a high proportion of otherwise healthy individuals in malaria-endemic areas and reflects repeated infections. Slight enlargement of the liver is also common, particularly among young children. Mild jaundice is common among adults; it may develop in patients with otherwise uncomplicated malaria and usually resolves over 1–3 weeks. Malaria is not associated with a rash like those seen in meningococcal septicemia, typhus, enteric fever, viral exanthems, and drug reactions. Petechial hemorrhages in the skin or mucous membranes—features of viral hemorrhagic fevers and leptospirosis— develop only very rarely in severe falciparum malaria.

1	Appropriately and promptly treated, uncomplicated falciparum malaria (i.e., the patient can swallow medicines and food) carries a mortality rate of <0.1%. However, once vital-organ dysfunction occurs or the total proportion of erythrocytes infected increases to >2% (a level corresponding to >1012 parasites in an adult), mortality risk rises steeply. The major manifestations of severe falciparum malaria are shown in Table 248-2, and features indicating a poor prognosis are listed in Table 248-3. Unarousable coma/ Failure to localize or respond appropriately to Acidemia/acidosis Arterial pH of <7.25 or plasma bicarbonate level of <15 mmol/L; venous lactate level of >5 mmol/L; manifests as labored deep breathing, often termed “respiratory distress” Severe normochromic, Hematocrit of <15% or hemoglobin level of normocytic anemia <50 g/L (<5 g/dL) with parasitemia <10,000/μL

1	Severe normochromic, Hematocrit of <15% or hemoglobin level of normocytic anemia <50 g/L (<5 g/dL) with parasitemia <10,000/μL Renal failure Serum or plasma creatinine level of >265 μmol/L (>3 mg/dL); urine output (24 h) of <400 mL in adults or <12 mL/kg in children; no improvement with rehydration Pulmonary edema/adult Noncardiogenic pulmonary edema, often respiratory distress aggravated by overhydration syndrome Hypoglycemia Plasma glucose level of <2.2 mmol/L (<40 mg/dL) Hypotension/shock Systolic blood pressure of <50 mmHg in children 1–5 years or <80 mmHg in adults; core/ skin temperature difference of >10°C; capillary refill >2 s intravascular coagulation the gums, nose, and gastrointestinal tract and/or evidence of disseminated intravascular coagulation

1	Convulsions More than two generalized seizures in 24 h; signs of continued seizure activity, sometimes subtle (e.g., tonic-clonic eye movements without limb or face movement) aHemoglobinuria may also occur in uncomplicated malaria and in patients with G6PD deficiency who take primaquine. bIn children who are normally able to sit. Abbreviation: G6PD, glucose-6-phosphate dehydrogenase. Marked agitation Hyperventilation (respiratory distress) Hypothermia (<36.5°C; <97.7°F) Bleeding Deep coma Repeated convulsions Anuria Shock Hypoglycemia (<2.2 mmol/L) Acidosis (arterial pH <7.3, serum HCO3 <15 mmol/L) Elevated liver enzymes (AST/ALT 3 times upper limit of normal) Elevated muscle enzymes (CPK ↑, myoglobin ↑) Leukocytosis (>12,000/μL) Decreased platelet count (<50,000/μL) Increased mortality at >100,000/μL

1	Elevated muscle enzymes (CPK ↑, myoglobin ↑) Leukocytosis (>12,000/μL) Decreased platelet count (<50,000/μL) Increased mortality at >100,000/μL High mortality at >500,000/μL >20% of parasites identified as pigment-containing trophozoites and >5% of neutrophils with visible pigment Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CPK, creatine phosphokinase; PCV, packed cell volume. Cerebral Malaria Coma is a characteristic and ominous feature of falciparum malaria and, despite treatment, is associated with death rates of ~20% among adults and 15% among children. Any obtundation, delirium, or abnormal behavior should be taken very seriously. The onset may be gradual or sudden following a convulsion.

1	Cerebral malaria manifests as diffuse symmetric encephalopathy; focal neurologic signs are unusual. Although some passive resistance to head flexion may be detected, signs of meningeal irritation are absent. The eyes may be divergent and a pout reflex is common, but other primitive reflexes are usually absent. The corneal reflexes are preserved, except in deep coma. Muscle tone may be either increased or decreased. The tendon reflexes are variable, and the plantar reflexes may be flexor or extensor; the abdominal and cremasteric reflexes are absent. Flexor or extensor posturing may be seen. On routine funduscopy, ~15% of patients have retinal hemorrhages; with pupillary dilation and indirect ophthalmoscopy, this figure increases to 30–40%. Other funduscopic abnormalities (Fig. 248-3) include discrete spots of retinal opacification (30–60%), papilledema (8% among children, rare among adults), cotton wool spots (<5%), and decolorization of a retinal vessel or segment of vessel

1	include discrete spots of retinal opacification (30–60%), papilledema (8% among children, rare among adults), cotton wool spots (<5%), and decolorization of a retinal vessel or segment of vessel (occasional cases). Convulsions, usually generalized and often repeated, occur in ~10% of adults and up to 50% of children with cerebral malaria. More covert seizure activity also is common, particularly among children, and may manifest as repetitive tonic-clonic eye movements or even hypersalivation. Whereas adults rarely (i.e., in <3% of cases) suffer neurologic sequelae, ~10% of children surviving cerebral malaria—especially those with hypoglycemia, severe anemia, repeated seizures, and deep coma—have residual neurologic deficits when they regain consciousness; hemiplegia, cerebral palsy, cortical blindness, deafness, and impaired cognition have been reported. The majority of these deficits improve markedly or resolve completely within 6 months. However, the prevalence of some other

1	cortical blindness, deafness, and impaired cognition have been reported. The majority of these deficits improve markedly or resolve completely within 6 months. However, the prevalence of some other deficits increases over time; ~10% of children surviving cerebral malaria have a persistent language deficit. There may also be deficits in learning, planning and executive functions, attention, memory, and nonverbal functioning. The incidence of epilepsy is increased and life expectancy decreased among these children.

1	FIGuRE 248-3 The eye in cerebral malaria: perimacular whitening and pale-centered retinal hemorrhages. (Courtesy of N. Beare, T. Taylor, S. Harding, S. Lewallen, and M. Molyneux; with permission.)

1	S. Harding, S. Lewallen, and M. Molyneux; with permission.) Hypoglycemia Hypoglycemia, an important and common complication of severe malaria, is associated with a poor prognosis and is particularly problematic in children and pregnant women. Hypoglycemia in malaria results from a failure of hepatic gluconeogenesis and an increase in the consumption of glucose by both the host and, to a much lesser extent, the malaria parasites. To compound the situation, quinine, which is still widely used for the treatment of both severe and uncomplicated falciparum malaria, is a powerful stimulant of pancreatic insulin secretion. Hyperinsulinemic hypoglycemia is especially troublesome in pregnant women receiving quinine treatment. In severe disease, the clinical diagnosis of hypoglycemia is difficult: the usual physical signs (sweating, gooseflesh, tachycardia) are absent, and the neurologic impairment caused by hypoglycemia cannot be distinguished from that caused by malaria.

1	Acidosis Acidosis, an important cause of death from severe malaria, results from accumulation of organic acids. Hyperlactatemia commonly coexists with hypoglycemia. In adults, coexisting renal impairment often compounds the acidosis; in children, ketoacidosis also may contribute. Other, still-unidentified organic acids are major contributors to acidosis. Acidotic breathing, sometimes called “respiratory distress,” is a sign of poor prognosis. It is followed often by circulatory failure refractory to volume expansion or inotropic drug treatment and ultimately by respiratory arrest. The plasma concentrations of bicarbonate or lactate are the best biochemical prognosticators in severe malaria. Hypovolemia is not a major contributor to acidosis. Lactic acidosis is caused by the combination of anaerobic glycolysis in tissues where sequestered parasites interfere with microcirculatory flow, 1374 lactate production by the parasites, and a failure of hepatic and renal lactate clearance. The

1	anaerobic glycolysis in tissues where sequestered parasites interfere with microcirculatory flow, 1374 lactate production by the parasites, and a failure of hepatic and renal lactate clearance. The prognosis of severe acidosis is poor.

1	Noncardiogenic Pulmonary Edema Adults with severe falciparum malaria may develop noncardiogenic pulmonary edema even after several days of antimalarial therapy. The pathogenesis of this variant of the adult respiratory distress syndrome is unclear. The mortality rate is >80%. This condition can be aggravated by overly vigorous administration of IV fluid. Noncardiogenic pulmonary edema can also develop in otherwise uncomplicated vivax malaria, where recovery is usual.

1	Renal Impairment Acute kidney injury is common in severe falciparum malaria, but oliguric renal failure is rare among children. The pathogenesis of renal failure is unclear but may be related to erythrocyte sequestration and agglutination interfering with renal micro-circulatory flow and metabolism. Clinically and pathologically, this syndrome manifests as acute tubular necrosis. Renal cortical necrosis never develops. Acute renal failure may occur simultaneously with other vital-organ dysfunction (in which case the mortality risk is high) or may progress as other disease manifestations resolve. In survivors, urine flow resumes in a median of 4 days, and serum creatinine levels return to normal in a mean of 17 days (Chap. 334). Early dialysis or hemofiltration considerably enhances the likelihood of a patient’s survival, particularly in acute hypercatabolic renal failure.

1	Hematologic Abnormalities Anemia results from accelerated RBC removal by the spleen, obligatory RBC destruction at parasite schizogony, and ineffective erythropoiesis. In severe malaria, both infected and uninfected RBCs show reduced deformability, which correlates with prognosis and development of anemia. Splenic clearance of all RBCs is increased. In nonimmune individuals and in areas with unstable transmission, anemia can develop rapidly and transfusion is often required. As a consequence of repeated malarial infections, children in many areas of Africa and on the island of New Guinea may develop severe anemia resulting from both shortened survival of uninfected RBCs and marked dyserythropoiesis. Anemia is a common consequence of antimalarial drug resistance, which results in repeated or continued infection.

1	Slight coagulation abnormalities are common in falciparum malaria, and mild thrombocytopenia is usual (a normal platelet count should raise questions about the diagnosis of malaria). Of patients with severe malaria, <5% have significant bleeding with evidence of disseminated intravascular coagulation. Hematemesis from stress ulceration or acute gastric erosions also may occur rarely.

1	Liver Dysfunction Mild hemolytic jaundice is common in malaria. Severe jaundice is associated with P. falciparum infections; is more common among adults than among children; and results from hemolysis, hepatocyte injury, and cholestasis. When accompanied by other vital-organ dysfunction (often renal impairment), liver dysfunction carries a poor prognosis. Hepatic dysfunction contributes to hypoglycemia, lactic acidosis, and impaired drug metabolism. Occasional patients with falciparum malaria may develop deep jaundice (with hemolytic, hepatic, and cholestatic components) without evidence of other vital-organ dysfunction, in which case the prognosis is good.

1	Other Complications HIV/AIDS and malnutrition predispose to more severe malaria in nonimmune individuals; malaria anemia is worsened by concurrent infections with intestinal helminths, hookworm in particular. Septicemia may complicate severe malaria, particularly in children. Differentiating severe malaria from sepsis with incidental parasitemia in childhood is very difficult. In endemic areas, Salmonella bacteremia has been associated specifically with P. falciparum infections. Chest infections and catheter-induced urinary tract infections are common among patients who are unconscious for >3 days. Aspiration pneumonia may follow generalized convulsions. The frequencies of complications of severe falciparum malaria are summarized in Table 248-4. Malaria in early pregnancy causes abortion. In areas of high malaria transmission, falciparum malaria in primiand secundigravid women Note: –, rare; +, infrequent; ++, frequent; +++, very frequent.

1	Malaria in early pregnancy causes abortion. In areas of high malaria transmission, falciparum malaria in primiand secundigravid women Note: –, rare; +, infrequent; ++, frequent; +++, very frequent. is associated with low birth weight (average reduction, ~170 g) and consequently increased infant mortality rates. In general, infected mothers in areas of stable transmission remain asymptomatic despite intense accumulation of parasitized erythrocytes in the placental microcirculation. Maternal HIV infection predisposes pregnant women to more frequent and higher-density malaria infections, predisposes their newborns to congenital malarial infection, and exacerbates the reduction in birth weight associated with malaria.

1	In areas with unstable transmission of malaria, pregnant women are prone to severe infections and are particularly vulnerable to high parasitemias with anemia, hypoglycemia, and acute pulmonary edema. Fetal distress, premature labor, and stillbirth or low birth weight are common results. Fetal death is usual in severe malaria. Congenital malaria occurs in <5% of newborns whose mothers are infected; its frequency and the level of parasitemia are related directly to the parasite density in maternal blood and in the placenta. P. vivax malaria in pregnancy is also associated with a reduction in birth weight (average, 110 g), but, in contrast to the situation in falciparum malaria, this effect is more pronounced in multigravid than in primigravid women. About 350,000 women die in childbirth yearly, with most deaths occurring in low-income countries; maternal death from hemorrhage at childbirth is correlated with malaria-induced anemia.

1	Most of the 660,000 persons who die of falciparum malaria each year are young African children. Convulsions, coma, hypoglycemia, metabolic acidosis, and severe anemia are relatively common among children with severe malaria, whereas deep jaundice, oliguric acute kidney injury, and acute pulmonary edema are unusual. Severely anemic children may present with labored deep breathing, which in the past has been attributed incorrectly to “anemic congestive cardiac failure” but in fact is usually caused by metabolic acidosis, often compounded by hypovolemia. In general, children tolerate antimalarial drugs well and respond rapidly to treatment.

1	Malaria can be transmitted by blood transfusion, needle-stick injury, sharing of needles by infected injection drug users, or organ transplantation. The incubation period in these settings is often short because there is no preerythrocytic stage of development. The clinical features and management of these cases are the same as for naturally acquired infections. Radical chemotherapy with primaquine is unnecessary for transfusion-transmitted P. vivax and P. ovale infections.

1	Chronic or repeated malarial infections produce hypergammaglobulinemia; normochromic, normocytic anemia; and, in certain situations, splenomegaly. Some residents of malaria-endemic areas in tropical Africa and Asia exhibit an abnormal immunologic response to repeated infections that is characterized by massive splenomegaly, hepatomegaly, marked elevations in serum titers of IgM and malarial antibody, hepatic sinusoidal lymphocytosis, and (in Africa) peripheral B cell lymphocytosis. This syndrome has been associated with the production of cytotoxic IgM antibodies to CD8+ T lymphocytes, antibodies to CD5+ T lymphocytes, and an increase in the ratio of CD4+ to CD8+ T cells. These events may lead to uninhibited B cell production of IgM and the formation of cryoglobulins (IgM aggregates and immune complexes). This immunologic process stimulates reticuloendothelial hyperplasia and clearance activity and eventually produces splenomegaly. Patients with hyperreactive malarial splenomegaly

1	immune complexes). This immunologic process stimulates reticuloendothelial hyperplasia and clearance activity and eventually produces splenomegaly. Patients with hyperreactive malarial splenomegaly present with an abdominal mass or a dragging sensation in the abdomen and occasional sharp abdominal pains suggesting perisplenitis. Anemia and some degree of pancytopenia are usually evident, and in some cases malarial parasites cannot be found in peripheral-blood smears. Vulnerability to respiratory and skin infections is increased; many patients die of overwhelming sepsis. Persons with hyperreactive malarial splenomegaly who are living in endemic areas should receive antimalarial chemoprophylaxis; the results are usually good. In nonendemic areas, antimalarial treatment is advised. In some cases refractory to therapy, clonal lymphoproliferation may develop and can then evolve into a malignant lymphoproliferative disorder.

1	Chronic or repeated infections with P. malariae (and possibly with other malarial species) may cause soluble immune complex injury to the renal glomeruli, resulting in the nephrotic syndrome. Other unidentified factors must contribute to this process since only a very small proportion of infected patients develop renal disease. The histologic appearance is that of focal or segmental glomerulonephritis with splitting of the capillary basement membrane. Subendothelial dense deposits are seen on electron microscopy, and immunofluorescence reveals deposits of complement and immunoglobulins; in samples of renal tissue from children, P. malariae antigens are often visible. A coarse-granular pattern of basement membrane immunofluorescent deposits (predominantly IgG3) with selective proteinuria carries a better prognosis than a fine-granular, predominantly IgG2 pattern with nonselective proteinuria. Quartan nephropathy usually responds poorly to treatment with either antimalarial agents or

1	carries a better prognosis than a fine-granular, predominantly IgG2 pattern with nonselective proteinuria. Quartan nephropathy usually responds poorly to treatment with either antimalarial agents or glucocorticoids and cytotoxic drugs.

1	It is possible that malaria-related immune dysregulation provokes infection with lymphoma viruses. Burkitt’s lymphoma is strongly associated with Epstein-Barr virus. The prevalence of this childhood tumor is high in malarious areas of Africa.

1	The diagnosis of malaria rests on the demonstration of asexual forms of the parasite in stained peripheral-blood smears. After a negative blood smear, repeat smears should be made if there is a high degree of suspicion. Of the Romanowsky stains, Giemsa at pH 7.2 is preferred; Field’s, Wright’s, or Leishman’s stain can also be used. Both thin (Figs. 248-4 and 248-5; see also Figs. 250e-3 and 250e-4) and thick (Figs. 248-6, 248-7, 248-8, and 248-9) blood smears should be examined. The thin blood smear should be rapidly air-dried, fixed in anhydrous methanol, and stained; the RBCs in the tail of the film should then be examined under oil immersion (×1000 magnification). The level of parasitemia is expressed as the number of parasitized erythrocytes per 1000 RBCs. The thick blood film should be of uneven thickness. The smear should be dried thoroughly and stained without fixing. As many layers of erythrocytes overlie one another and are lysed during the staining procedure, the thick film

1	be of uneven thickness. The smear should be dried thoroughly and stained without fixing. As many layers of erythrocytes overlie one another and are lysed during the staining procedure, the thick film has the advantage of concentrating the parasites (by 40to 100-fold compared with a thin blood film) and thus increasing diagnostic sensitivity. Both parasites and white blood cells (WBCs) are counted, and the number of parasites per unit volume is calculated from the total leukocyte count. Alternatively, a WBC count of 8000/μL is assumed. This figure is converted to the number of parasitized erythrocytes per microliter. A minimum of 200 WBCs should be counted under oil immersion. Interpretation of blood smear films requires some experience because artifacts are common. Before a thick smear is judged to be negative, 100–200 fields should be examined under oil immersion. In high-transmission areas, the presence of up to 10,000 parasites/μL of blood may be tolerated without symptoms or signs

1	judged to be negative, 100–200 fields should be examined under oil immersion. In high-transmission areas, the presence of up to 10,000 parasites/μL of blood may be tolerated without symptoms or signs in partially immune individuals. Thus in these areas the detection of malaria parasites is sensitive but has low specificity in identifying

1	FIGuRE 248-4 Thin blood films of Plasmodium falciparum. A. Young trophozoites. B. Old trophozoites. C. Pigment in polymorphonuclear cells and trophozoites. D. Mature schizonts. E. Female gametocytes. F. Male gametocytes. (Reproduced from Bench Aids for the Diagnosis of Malaria Infections, 2nd ed, with the permission of the World Health Organization.) FIGuRE 248-5 Thin blood films of Plasmodium vivax. A. Young trophozoites. B. Old trophozoites. C. Mature schizonts. D. Female gametocytes. E. Male gametocytes. (Reproduced from Bench Aids for the Diagnosis of Malaria Infections, 2nd ed, with the permission of the World Health Organization.) FIGuRE 248-6 Thick blood films of Plasmodium falciparum. A. Trophozoites. B. Gametocytes. (Reproduced from Bench Aids for the Diagnosis of Malaria Infections, 2nd ed, with the permission of the World Health Organization.) malaria as the cause of illness. Low-density parasitemia is common in other conditions causing fever.

1	Rapid, simple, sensitive, and specific antibody-based diagnostic stick or card tests that detect P. falciparum–specific, histidine-rich protein 2 (PfHRP2), lactate dehydrogenase, or aldolase antigens in finger-prick blood samples are now being used widely in control programs (Table 248-5). Some of these rapid diagnostic tests carry a second antibody, which allows falciparum malaria to be distinguished from the less dangerous malarias. PfHRP2-based tests may remain positive for several weeks after acute infection. This feature is a disadvantage in high-transmission areas where infections are frequent, but it is of value in the diagnosis of severe malaria in patients who have taken antimalarial drugs and cleared peripheral parasitemia (but in whom the PfHRP2 test remains strongly positive). Rapid diagnostic tests are replacing microscopy in many areas because of their simplicity and speed. Their disadvantage is that they do not quantify parasitemia.

1	The relationship between parasitemia and prognosis is complex; in general, patients with >105 parasites/μL are at increased risk of dying, but nonimmune patients may die with much lower counts, and partially immune persons may tolerate parasitemia levels many times higher with only minor symptoms. In severe malaria, a poor prognosis is indicated by a predominance of more mature P. falciparum FIGuRE 248-7 Thick blood films of Plasmodium vivax. A. Trophozoites. B. Schizonts. C. Gametocytes. (Reproduced from Bench Aids for the Diagnosis of Malaria Infections, 2nd ed, with the permission of the World Health Organization.) FIGuRE 248-8 Thick blood films of Plasmodium ovale. A. Trophozoites. B. Schizonts. C. Gametocytes. (Reproduced from Bench Aids for the Diagnosis of Malaria Infections, 2nd ed, with the permission of the World Health Organization.)

1	FIGuRE 248-9 Thick blood films of Plasmodium malariae. A. Trophozoites. B. Schizonts. C. Gametocytes. (Reproduced from Bench Aids for the Diagnosis of Malaria Infections, 2nd ed, with the permission of the World Health Organization.) Plasmodium LDH dipstick or A drop of blood is placed on the stick or card, which Rapid; sensitivity similar to or Slightly more difficult preparation card test is then immersed in washing solutions. Monoclonal slightly lower than that of thick than PfHRP2 tests; may miss low-level antibody capture of parasitic antigens reads out as two films for P. falciparum (~0.001% parasitemia with P. vivax, P. ovale, and colored bands. One band is genus specific (all malarias), parasitemia) P. malariae and may not speciate and the other is specific for P. falciparum.

1	P. falciparum parasitemia aMalaria cannot be diagnosed clinically with accuracy, but treatment should be started on clinical grounds if laboratory confirmation is likely to be delayed. In areas of the world where malaria is endemic and transmission is high, low-level asymptomatic parasitemia is common in otherwise healthy people. Thus malaria may not be the cause of a fever, although in this context the presence of >10,000 parasites/μL (~0.2% parasitemia) does indicate that malaria is the cause. Antibody and polymerase chain reaction tests have no role in the diagnosis of malaria except that PCR is increasingly used for genotyping and speciation in mixed infections and for detection of low-level parasitemias in asymptomatic residents of endemic areas. bAsexual parasites/200 WBCs × 40 = parasite count/μL (assumes a WBC count of 8000/μL). See Figs. 248-6 through 248-9. cP. falciparum gametocytemia may persist for days or weeks after clearance of asexual parasites. Gametocytemia without

1	count/μL (assumes a WBC count of 8000/μL). See Figs. 248-6 through 248-9. cP. falciparum gametocytemia may persist for days or weeks after clearance of asexual parasites. Gametocytemia without asexual parasitemia does not indicate active infection. dParasitized RBCs (%) × hematocrit × 1256 = parasite count/μL. See Figs. 248-4 and 248-5. eThe presence of >100,000 parasites/μL (~2% parasitemia) is associated with an increased risk of severe malaria, but some patients have severe malaria with lower counts. At any level of parasitemia, the finding that >50% of parasites are tiny rings (cytoplasm thickness less than half of nucleus width) carries a relatively good prognosis. The presence of visible pigment in >20% of parasites or of phagocytosed pigment in >5% of polymorphonuclear leukocytes (indicating massive recent schizogony) carries a worse prognosis. fPersistence of PfHRP2 is a disadvantage in high-transmission settings, where many asymptomatic people have positive tests, but can be

1	massive recent schizogony) carries a worse prognosis. fPersistence of PfHRP2 is a disadvantage in high-transmission settings, where many asymptomatic people have positive tests, but can be used to diagnostic advantage in low-transmission settings when a sick patient has previously received unknown treatment (which, in endemic areas, often consists of antimalarial drugs). A positive PfHRP2 test indicates that the illness is falciparum malaria, even if the blood smear is negative.

1	Abbreviations: LDH, lactate dehydrogenase; PfHRP2, P. falciparum histidine-rich protein 2; RBCs, red blood cells; WBCs, white blood cells.

1	1378 parasites (i.e., >20% of parasites with visible pigment) in the peripheral-blood film or by the presence of phagocytosed malarial pigment in >5% of neutrophils. In P. falciparum infections, gametocytemia peaks 1 week after the peak of asexual parasites. Because the mature gametocytes of P. falciparum (unlike those of other plasmodia) are not affected by most antimalarial drugs, their persistence does not constitute evidence of drug resistance. Phagocytosed malarial pigment is sometimes seen inside peripheral-blood monocytes or polymorphonuclear leukocytes and may provide a clue to recent infection if malaria parasites are not detectable. After the clearance of the parasites, this intraphagocytic malarial pigment is often evident for several days in the peripheral blood films or for longer in bone marrow aspirates or smears of fluid expressed after intradermal puncture. Staining of parasites with the fluorescent dye acridine orange allows more rapid diagnosis of malaria (but not

1	longer in bone marrow aspirates or smears of fluid expressed after intradermal puncture. Staining of parasites with the fluorescent dye acridine orange allows more rapid diagnosis of malaria (but not speciation of the infection) in patients with low-level parasitemia. Molecular diagnosis by polymerase chain reaction (PCR) amplification of parasite nucleic acid is more sensitive than microscopy or rapid diagnostic tests for detecting malaria parasites and defining malarial species. While currently impractical in the standard clinical setting, PCR is used in reference centers in endemic areas. In epidemiologic surveys, sensitive PCR detection may prove very useful in identifying asymptomatic infections as control and eradication programs drive parasite prevalence down to very low levels. Serologic diagnosis with either indirect fluorescent antibody or enzyme-linked immunosorbent assays may prove useful as measures of transmission intensity in future epidemiologic studies. Serology has

1	diagnosis with either indirect fluorescent antibody or enzyme-linked immunosorbent assays may prove useful as measures of transmission intensity in future epidemiologic studies. Serology has no place in the diagnosis of acute illness.

1	Normochromic, normocytic anemia is usual. The leukocyte count is generally normal, although it may be raised in very severe infections. There is slight monocytosis, lymphopenia, and eosinopenia, with reactive lymphocytosis and eosinophilia in the weeks after the acute infection. The erythrocyte sedimentation rate, plasma viscosity, and levels of C-reactive protein and other acute-phase proteins are high. The platelet count is usually reduced to ~105/μL. Severe infections may be accompanied by prolonged prothrombin and partial thromboplastin times and by more severe thrombocytopenia. Levels of antithrombin III are reduced even in mild infection. In uncomplicated malaria, plasma concentrations of electrolytes, blood urea nitrogen (BUN), and creatinine are usually normal. Findings in severe malaria may include metabolic acidosis, with low plasma concentrations of glucose, sodium, bicarbonate, calcium, phosphate, and albumin together with elevations in lactate, BUN, creatinine, urate,

1	malaria may include metabolic acidosis, with low plasma concentrations of glucose, sodium, bicarbonate, calcium, phosphate, and albumin together with elevations in lactate, BUN, creatinine, urate, muscle and liver enzymes, and conjugated and unconjugated bilirubin. Hypergammaglobulinemia is usual in immune and semi-immune subjects. Urinalysis generally gives normal results. In adults and children with cerebral malaria, the mean cerebrospinal fluid (CSF) opening pressure at lumbar puncture is ~160 mm; usually the CSF content is normal or there is a slight elevation of total protein level (<1.0 g/L [<100 mg/dL]) and cell count (<20/μL).

1	(Table 248-6) When a patient in or from a malarious area presents with fever, thick and thin blood smears should be prepared and examined immediately to confirm the diagnosis and identify the species of infecting parasite (Figs. 248-4 through 248-9). Repeat blood smears should be performed at least every 12–24 h for 2 days if the first smears are negative and malaria is strongly suspected. Alternatively, a rapid antigen detection card or stick test should be performed. Patients with severe malaria or those unable to take oral drugs should receive parenteral antimalarial therapy. If there is any doubt about the resistance status of the infecting organism, it should be considered resistant. Antimalarial drug susceptibility testing can be performed but is rarely available, has poor predictive value in an individual case, and yields results too slowly to influence the choice of treatment. Several drugs are available for oral treatment. The choice of drug depends on the likely sensitivity

1	value in an individual case, and yields results too slowly to influence the choice of treatment. Several drugs are available for oral treatment. The choice of drug depends on the likely sensitivity of

1	Type of Disease or Treatment Regimen(s) Known chloroquine-Chloroquine (10 mg of base/kg stat followed by 5 mg/ sensitive strains of kg at 12, 24, and 36 h or by 10 mg/kg at 24 h and Plasmodium vivax, 5 mg/kg at 48 h) P. malariae, P. ovale, or P. knowlesi, Amodiaquine (10–12 mg of base/kg qd for 3 days) P. falciparumb Radical treatment In addition to chloroquine or amodiaquine as detailed for P. vivax or P. ovale above, primaquine (0.5 mg of base/kg qd in tropical infection regions and 0.25 mg/kg for temperate-origin P. vivax) should be given for 14 days to prevent relapse. In mild G6PD deficiency, 0.75 mg of base/kg should be given once weekly for 8 weeks. Primaquine should not be given in severe G6PD deficiency. P. falciparum (25 mg/kg)/pyrimethamine (1.25 mg/kg) as a single malariac dose Artesunated (4 mg/kg qd for 3 days) plus amodiaquine (10 mg of base/kg qd for 3 days)e Multidrug-resistant Either artemether-lumefantrined (1.5/9 mg/kg bid for

1	Artesunated (4 mg/kg qd for 3 days) plus amodiaquine (10 mg of base/kg qd for 3 days)e Multidrug-resistant Either artemether-lumefantrined (1.5/9 mg/kg bid for P. falciparum malaria 3 days with food) Artesunated (4 mg/kg qd for 3 days) plus mefloquine (24–25 mg of base/kg—either 8 mg/kg qd for 3 days or 15 mg/kg on day 2 and then 10 mg/kg on day 3)e Dihydroartemisinin-piperaquined (2.5/20 mg/kg qd for 3 days) Second-line treat-Either artesunated (2 mg/kg qd for 7 days) or quinine ment/treatment of (10 mg of salt/kg tid for 7 days) plus 1 of the following 3: imported malaria 1. 2. 3. Atovaquone-proguanil (20/8 mg/kg qd for 3 days with food) Artesunated (2.4 mg/kg stat IV followed by 2.4 mg/kg at 12 and 24 h and then daily if necessary)h or, if unavailable, Artemetherd (3.2 mg/kg stat IM followed by 1.6 mg/ kg qd) or, if unavailable, Quinine dihydrochloride (20 mg of salt/kgi infused over 4 h, followed by 10 mg of salt/kg infused over 2–8 h q8hj) or, if unavailable,

1	Quinidine (10 mg of base/kgi infused over 1–2 h, followed by 1.2 mg of base/kg per hourj with electrocardiographic monitoring) aIn endemic areas, except in pregnant women and infants, a single dose of primaquine (0.25 mg of base/kg) should be added as a gametocytocide to all falciparum malaria treatments to prevent transmission. This addition is considered safe even in G6PD deficiency. bVery few areas now have chloroquine-sensitive P. falciparum malaria (Fig. 248-2). cIn areas where the partner drug to artesunate is known to be effective. dArtemisinin derivatives are not readily available in some temperate countries. eFixed-dose coformulated combinations are available. The World Health Organization now recommends artemisinin combination regimens as first-line therapy for falciparum malaria in all tropical countries and advocates use of fixed-dose combinations. fTetracycline and doxycycline should not be given to pregnant women or to children <8 years of age. gOral treatment should be

1	in all tropical countries and advocates use of fixed-dose combinations. fTetracycline and doxycycline should not be given to pregnant women or to children <8 years of age. gOral treatment should be substituted as soon as the patient recovers sufficiently to take fluids by mouth. hArtesunate is the drug of choice when available. The doses in children weighing <20 kg should be 3 mg/kg. The data from large studies in Southeast Asia showed a 35% lower mortality rate than with quinine, and very large studies in Africa showed a 22.5% reduction in mortality rate compared with quinine. iA loading dose should not be given if therapeutic doses of quinine or quinidine have definitely been administered in the previous 24 h. Some authorities recommend a lower dose of quinidine. jInfusions can be given in 0.9% saline and 5–10% dextrose in water. Infusion rates for quinine and quinidine should be carefully controlled.

1	Abbreviation: G6PD, glucose-6-phosphate dehydrogenase. the infecting parasites. Despite increasing evidence of chloroquine resistance in P. vivax (from parts of Indonesia, Oceania, eastern and southern Asia, and Central and South America), chloroquine remains a first-line treatment for the non-falciparum malarias (P. vivax, P. ovale, P. malariae, P. knowlesi) except in Indonesia and Papua New Guinea, where high levels of resistance in P. vivax are prevalent.

1	The treatment of falciparum malaria has changed radically in recent years. In all endemic areas, the World Health Organization (WHO) now recommends artemisinin-based combinations (ACTs) as first-line treatment for uncomplicated falciparum malaria. These combinations are also highly effective for the other malarias. These rapidly and reliably effective drugs are sometimes unavailable in temperate countries, where treatment recommendations are limited by the registered available drugs. Fake or substandard antimalarials are commonly sold in many Asian and African countries. Thus, careful attention is required at the time of purchase and later, especially if the patient fails to respond as expected. Characteristics of antimalarial drugs are shown in Table 248-7.

1	In large studies, parenteral artesunate, a water-soluble artemisinin derivative, has reduced mortality rates in severe falciparum malaria among Asian adults and children by 35% and among African children by 22.5% compared with mortality rates with quinine treatment. Artesunate has therefore become the drug of choice for all patients with severe malaria everywhere. Artesunate is given by IV injection but can also be given by IM injection. Artemether and the closely related drug artemotil (arteether) are oil-based formulations given by IM injection; they are erratically absorbed and do not confer the same survival benefit as artesunate. A rectal formulation of artesunate has been developed as a community-based pre-referral treatment for patients in the rural tropics who cannot take oral medications. Pre-referral administration of rectal artesunate has been shown to decrease mortality risk among severely ill children in communities without access to immediate parenteral treatment.

1	medications. Pre-referral administration of rectal artesunate has been shown to decrease mortality risk among severely ill children in communities without access to immediate parenteral treatment. Although the artemisinin compounds are safer than quinine and considerably safer than quinidine, only one formulation is available in the United States. IV artesunate has been approved by the U.S. Food and Drug Administration for emergency use against severe malaria and can be obtained through the Centers for Disease Control and Prevention (CDC) Drug Service (see end of chapter for contact information). The antiarrhythmic quinidine gluconate is as effective as quinine and, as it was more readily available, replaced quinine for the treatment of malaria in the United States. The administration of quinidine must be closely monitored if dysrhythmias and hypotension are to be avoided. If total plasma levels exceed 8 μg/mL or the QTc interval exceeds 0.6 s or the QRS complex widens by more than

1	quinidine must be closely monitored if dysrhythmias and hypotension are to be avoided. If total plasma levels exceed 8 μg/mL or the QTc interval exceeds 0.6 s or the QRS complex widens by more than 25% of baseline, then infusion rates should be slowed or infusion stopped temporarily. If arrhythmia or saline-unresponsive hypotension develops, treatment with this drug should be discontinued. Quinine is safer than quinidine; cardiovascular monitoring is not required except when the recipient has cardiac disease.

1	Severe falciparum malaria constitutes a medical emergency requiring intensive nursing care and careful management. The patient should be weighed and, if comatose, placed on his or her side. Frequent evaluation of the patient’s condition is essential. Adjunctive treatments such as high-dose glucocorticoids, urea, heparin, dextran, desferrioxamine, antibody to tumor necrosis factor α, high-dose phenobarbital (20 mg/kg), mannitol, or large-volume fluid or albumin boluses have proved either ineffective or harmful in clinical trials and should not be used. In acute renal failure or severe metabolic acidosis, hemofiltration or hemodialysis should be started as early as possible.

1	In severe malaria, parenteral antimalarial treatment should be started immediately. Artesunate, given by either IV or IM injection, is the agent of choice; it is simple to administer, safe, and rapidly effective. It does not require dose adjustments in liver dysfunction or renal failure, and it should be used in pregnant women with severe malaria. If artesunate is unavailable and artemether, quinine, or quinidine is used, an initial loading dose must be given so that 1379 therapeutic concentrations are reached as soon as possible. Both quinine and quinidine will cause dangerous hypotension if injected rapidly; when given IV, they must be administered carefully by rate-controlled infusion only. If this approach is not possible, quinine may be given by deep IM injections into the anterior thigh. The optimal therapeutic range for quinine and quinidine in severe malaria is not known with certainty, but total plasma concentrations of 8–15 mg/L for quinine and 3.5–8.0 mg/L for quinidine are

1	The optimal therapeutic range for quinine and quinidine in severe malaria is not known with certainty, but total plasma concentrations of 8–15 mg/L for quinine and 3.5–8.0 mg/L for quinidine are effective and do not cause serious toxicity. The systemic clearance and apparent volume of distribution of these alkaloids are markedly reduced and plasma protein binding is increased in severe malaria, so that the blood concentrations attained with a given dose are higher. If the patient remains seriously ill or in acute renal failure for >2 days, maintenance doses of quinine or quinidine should be reduced by 30–50% to prevent toxic accumulation of the drug. The initial doses should never be reduced. If safe and feasible, exchange transfusion may be considered for patients with severe malaria, although the precise indications for this procedure have not been agreed upon and there is no clear evidence that this measure is beneficial, particularly with artesunate treatment. Convulsions should

1	the precise indications for this procedure have not been agreed upon and there is no clear evidence that this measure is beneficial, particularly with artesunate treatment. Convulsions should be treated promptly with IV (or rectal) benzodiazepines. The role of prophylactic anticonvulsants in children is uncertain. If respiratory support is not available, then a full loading dose of phenobarbital (20 mg/kg) to prevent convulsions should not be given as it may cause respiratory arrest.

1	When the patient is unconscious, the blood glucose level should be measured every 4–6 h. All patients should receive a continuous infusion of dextrose, and blood concentrations ideally should be maintained above 4 mmol/L. Hypoglycemia (<2.2 mmol/L or 40 mg/ dL) should be treated immediately with bolus glucose. The parasite count and hematocrit level should be measured every 6–12 h. Anemia develops rapidly; if the hematocrit falls to <20%, then whole blood (preferably fresh) or packed cells should be transfused slowly, with careful attention to circulatory status. Renal function should be checked daily. Children presenting with severe anemia and acidotic breathing require immediate blood transfusion. Accurate assessment is vital. Management of fluid balance is difficult in severe malaria, particularly in adults, because of the thin dividing line between overhydration (leading to pulmonary edema) and underhydration (contributing to renal impairment). As soon as the patient can take

1	particularly in adults, because of the thin dividing line between overhydration (leading to pulmonary edema) and underhydration (contributing to renal impairment). As soon as the patient can take fluids, oral therapy should be substituted for parenteral treatment.

1	Infections due to sensitive strains of P. vivax, P. knowlesi, P. malariae, and P. ovale should be treated with oral chloroquine (total dose, 25 mg of base/kg) or with an ATC known to be efficacious. In much of the tropics, drug-resistant P. falciparum has been increasing in distribution, frequency, and intensity. It is now accepted that, to prevent resistance, falciparum malaria should be treated with drug combinations and not with single drugs in endemic areas; the same rationale has been applied successfully to the treatment of tuberculosis, HIV/AIDS, and cancers. This combination strategy is based on simultaneous use of two or more drugs with different modes of action. ACT regimens are now recommended as first-line treatment for falciparum malaria throughout the malaria-affected world. These regimens are safe and effective in adults, children, and after the first trimester of pregnancy (uncertainty regarding safety currently precludes their use in the first trimester). The rapidly

1	These regimens are safe and effective in adults, children, and after the first trimester of pregnancy (uncertainty regarding safety currently precludes their use in the first trimester). The rapidly eliminated artemisinin component is usually an artemisinin derivative (artesunate, artemether, or dihydroartemisinin) given for 3 days, and the partner drug is usually a more slowly eliminated antimalarial to which

1	P. falciparum is sensitive. Five ACT regimens are currently recommended by the WHO. In areas with multidrug-resistant falciparum malaria (parts of Asia and South America, including those with mefloquine-resistant parasites; Fig. 248-10), artemether-lumefantrine, artesunate-mefloquine, or dihydroartemisinin-piperaquine should be used; these regimens provide cure rates of >90%. In areas with sensitive parasites, the aforementioned combinations, artesunatesulfadoxine-pyrimethamine, or artesunate-amodiaquine also may be aHalofantrine should not be used by patients with long ECG QTc intervals or known conduction disturbances or by those taking drugs that may affect ventricular repolarization—e.g., quinidine, quinine, mefloquine, chloroquine, neuroleptics, antiarrhythmics, tricyclic antidepressants, and some antihistamines. bTetracycline and doxycycline should not be given to pregnant women or to children <8 years of age.

1	Abbreviations: Cl, systemic clearance; ECG, electrocardiogram; G6PD, glucose-6-phosphate dehydrogenase; Vd , total apparent volume of distribution. Andaman Sea Gulf of Thailand ChinaMyanmarThailandCambodiaBhutanIndiaBangladeshVietnamLaosMalaysiaFIGuRE 248-10 Mefloquine and artemisinin resistance in Plasmodium falciparum in Southeast Asia: high-level mefloquine resistance (dark red), low-level mefloquine resistance (pink), and mefloquine sensitivity (failure rate, <20%; green). There is insufficient information for other areas. Artemisinin resistance is now prevalent in areas where mefloquine resistance has been reported (pink areas).

1	used. Pyronaridine-artesunate is still under evaluation. Atovaquoneproguanil is highly effective everywhere, although it is seldom used in endemic areas because of its high cost and the propensity for rapid emergence of resistance. Of great concern is the emergence of artemisinin-resistant P. falciparum in western Cambodia and eastern Myanmar. Infections with these parasites are cleared slowly from the blood, with clearance times typically exceeding 3 days, and cure rates with ACTs are reduced.

1	The 3-day ACT regimens are all well tolerated, although mefloquine is associated with increased rates of vomiting and dizziness. As second-line treatments for recrudescence following first-line therapy, a different ACT regimen may be given; another alternative is a 7-day course of either artesunate or quinine plus tetracycline, doxycycline, or clindamycin. Tetracycline and doxycycline cannot be given to pregnant women or to children <8 years of age. Oral quinine is extremely bitter and regularly produces cinchonism comprising tinnitus, high-tone deafness, nausea, vomiting, and dysphoria. Adherence is poor with the required 7-day regimens of quinine.

1	Patients should be monitored for vomiting for 1 h after the administration of any oral antimalarial drug. If there is vomiting, the dose should be repeated. Symptom-based treatment, with tepid sponging and acetaminophen administration, lowers fever and thereby reduces the patient’s propensity to vomit these drugs. Minor central nervous system reactions (nausea, dizziness, sleep disturbances) are common. The incidence of serious adverse neuropsychiatric reactions to mefloquine treatment is ~1 in 1000 in Asia but may be as high as 1 in 200 among Africans and Caucasians. All the antimalarial quinolines (chloroquine, mefloquine, and quinine) exacerbate the orthostatic hypotension associated with malaria, and all are tolerated better by children than by adults. Pregnant women, young children, patients unable to tolerate oral therapy, and nonimmune individuals (e.g., travelers) with suspected malaria should be evaluated carefully and hospitalization considered. If there is any doubt as to

1	patients unable to tolerate oral therapy, and nonimmune individuals (e.g., travelers) with suspected malaria should be evaluated carefully and hospitalization considered. If there is any doubt as to the identity of the infecting malarial species, treatment for falciparum malaria should be given. A negative blood smear makes malaria unlikely but does not rule it out completely; thick blood films should be checked again 1 and 2 1381 days later to exclude the diagnosis. Nonimmune patients receiving treatment for malaria should have daily parasite counts performed until the thick films are negative. If the level of parasitemia does not fall below 25% of the admission value in 48 h or if parasitemia has not cleared by 7 days (and adherence is assured), drug resistance is likely and the regimen should be changed.

1	To eradicate persistent liver stages and prevent relapse (radical treatment), primaquine (0.5 mg of base/kg or, in infections acquired in temperate areas, 0.25 mg/kg) should be given daily for 14 days to patients with P. vivax or P. ovale infections after laboratory tests for G6PD deficiency have proved negative. If the patient has a mild variant of G6PD deficiency, primaquine can be given in a dose of 0.75 mg of base/kg (45 mg maximum) once weekly for 8 weeks. Pregnant women with vivax or ovale malaria should not be given primaquine but should receive suppressive prophylaxis with chloroquine (5 mg of base/kg per week) until delivery, after which radical treatment can be given.

1	COMPLICATIONS Acute Renal Failure If the plasma level of BUN or creatinine rises despite adequate rehydration, fluid administration should be restricted to prevent volume overload. As in other forms of hypercatabolic acute renal failure, renal replacement therapy is best performed early (Chap. 334). Hemofiltration and hemodialysis are more effective than peritoneal dialysis and are associated with lower mortality risk. Some patients with renal impairment pass small volumes of urine sufficient to allow control of fluid balance; these cases can be managed conservatively if other indications for dialysis do not arise. Renal function usually improves within days, but full recovery may take weeks.

1	Acute Pulmonary Edema (Acute Respiratory Distress Syndrome) Patients should be positioned with the head of the bed at a 45° elevation and given oxygen and IV diuretics. Pulmonary artery occlusion pressures may be normal, indicating increased pulmonary capillary perme ability. Positive-pressure ventilation should be started early if the immediate measures fail (Chap. 326). Hypoglycemia An initial slow injection of 50% dextrose (0.5 g/kg) should be followed by an infusion of 10% dextrose (0.10 g/kg per hour). The blood glucose level should be checked regularly thereafter as recurrent hypoglycemia is common, particularly among patients receiving quinine or quinidine. In severely ill patients, hypoglycemia commonly occurs together with metabolic (lactic) acidosis and carries a poor prognosis.

1	Other Complications Patients who develop spontaneous bleeding should be given fresh blood and IV vitamin K. Convulsions should be treated with IV or rectal benzodiazepines and, if necessary, respiratory support. Aspiration pneumonia should be suspected in any unconscious patient with convulsions, particularly with persistent hyperventilation; IV antimicrobial agents and oxygen should be administered, and pulmonary toilet should be undertaken. Hypoglycemia or gram-negative septicemia should be suspected when the condition of any patient suddenly deteriorates for no obvious reason during antimalarial treatment. In malaria-endemic areas where a high proportion of children are parasitemic, it is usually impossible to distinguish severe malaria from bacterial sepsis with confidence. These children should be treated with both antimalarials and broad-spectrum antibiotics from the outset. Because nontyphoidal Salmonella infections are particularly common, empirical antibiotics should be

1	children should be treated with both antimalarials and broad-spectrum antibiotics from the outset. Because nontyphoidal Salmonella infections are particularly common, empirical antibiotics should be selected to cover these organisms. Antibiotics should be considered for severely ill patients of any age who are not responding to antimalarial treatment.

1	In recent years, considerable progress has been made in malaria prevention, control, and research. Distribution of insecticide-treated 1382 bed-nets (ITNs) has been shown to reduce all-cause mortality in African children by 20%. New drugs have been discovered and developed, and one vaccine candidate (the RTS,S vaccine) will soon be considered for registration. Highly effective drugs, long-lasting ITNs, and insecticides for spraying dwellings are being purchased for endemic countries by the Global Fund to Fight AIDS, Tuberculosis, and Malaria; the President’s Malaria Initiative (funded by the U.S. Agency for International Development and managed by the CDC in partnership with endemic countries); UNICEF; and other organizations. Malaria research and control are being strongly supported by the National Institute of Allergy and Infectious Diseases, the CDC, the Wellcome Trust, the Bill & Melinda Gates Foundation, the Multilateral Initiative on Malaria, the Roll Back Malaria Partnership,

1	the National Institute of Allergy and Infectious Diseases, the CDC, the Wellcome Trust, the Bill & Melinda Gates Foundation, the Multilateral Initiative on Malaria, the Roll Back Malaria Partnership, and the WHO among others. While a laudable goal, the global eradication of malaria is not feasible in the immediate future because of the widespread distribution of Anopheles breeding sites; the great number of infected persons; the continued use of ineffective antimalarial drugs; and inadequacies in human and material resources, infrastructure, and control programs. The call for and commitment to ultimate eradication of malaria by the Gates Foundation in 2007— seconded by Margaret Chan, Director General of the WHO—added great impetus to all malaria initiatives, especially those aimed at discovery and implementation of new interventions. Malaria may be contained by judicious use of insecticides to kill the mosquito vector, rapid diagnosis, patient management, and—where effective and

1	discovery and implementation of new interventions. Malaria may be contained by judicious use of insecticides to kill the mosquito vector, rapid diagnosis, patient management, and—where effective and feasible—administration of intermittent preventive treatment, seasonal malaria chemoprevention, or chemoprophylaxis to high-risk groups such as pregnant women, young children, and travelers from nonendemic regions. Malaria researchers are intensifying their efforts to gain a better understanding of parasite-human-mosquito interactions and to develop more effective control and prevention interventions. Despite the enormous investment in efforts to develop a malaria vaccine and the 30–60% efficacy in African children of a recombinant protein sporozoite-targeted adjuvanted vaccine (RTS,S) in field trials, no safe, highly effective, long-lasting vaccine is likely to be available for general use in the near future (Chap. 148). Indeed, protection from RTS,S in the very youngest recipients

1	field trials, no safe, highly effective, long-lasting vaccine is likely to be available for general use in the near future (Chap. 148). Indeed, protection from RTS,S in the very youngest recipients dropped to 16% only 4 years after vaccination. While there is great promise for one or several malaria vaccines on the more distant horizon, prevention and control measures continue to rely on antivector and drug-use strategies. Furthermore, recent gains are threatened by increasing insecticide resistance and behavioral changes (to avoid ITN contact) in anopheline mosquito vectors and by spreading artemisinin resistance in P. falciparum.

1	Simple measures to reduce the frequency of infected-mosquito bites in malarious areas are very important. These measures include the avoidance of exposure to mosquitoes at their peak feeding times (usually dusk to dawn) and the use of insect repellents containing 10–35% DEET (or, if DEET is unacceptable, 7% picaridin), suitable clothing, and ITNs or other insecticide-impregnated materials. Widespread use of bed nets treated with residual pyrethroids reduces the incidence of malaria in areas where vectors bite indoors at night. (Table 248-8; wwwnc.cdc.gov/travel/yellowbook/2014/chapter-3infectious-diseases-related-to-travel/malaria) Recommendations for prophylaxis depend on knowledge of local patterns of Plasmodium species drug sensitivity and the likelihood of acquiring malarial infection. When there is uncertainty, drugs effective against resistant

1	P. falciparum should be used (atovaquone-proguanil [Malarone], doxycycline, or mefloquine). Chemoprophylaxis is never entirely reliable, and malaria should always be considered in the differential diagnosis of fever in patients who have traveled to endemic areas, even if they are taking prophylactic antimalarial drugs.

1	Pregnant women traveling to malarious areas should be warned about the potential risks. All pregnant women at risk in endemic areas should be encouraged to attend regular antenatal clinics. Mefloquine is the only drug advised for pregnant women traveling to areas with drug-resistant malaria; this drug is generally considered safe in the second and third trimesters of pregnancy, and the data on first-trimester exposure, although limited, are reassuring. Chloroquine and proguanil are regarded as safe. The safety of other prophylactic antimalarial agents in pregnancy has not been established. Antimalarial prophylaxis has been shown to reduce mortality rates among children between the ages of 3 months and 4 years in malaria-endemic areas; however, it is not a logistically or economically feasible option in many countries. The alternative—to give intermittent preventive treatment or seasonal malaria chemoprevention—shows promise for more widespread use in infants, young children, and

1	feasible option in many countries. The alternative—to give intermittent preventive treatment or seasonal malaria chemoprevention—shows promise for more widespread use in infants, young children, and pregnant women. Children born to nonimmune mothers in endemic areas (usually expatriates moving to malaria-endemic areas) should receive prophylaxis from birth.

1	Travelers should start taking antimalarial drugs 2 days to 2 weeks before departure so that any untoward reactions can be detected and so that therapeutic antimalarial blood concentrations will be present when needed (Table 248-8). Antimalarial prophylaxis should continue for 4 weeks after the traveler has left the endemic area, except if atovaquone-proguanil or primaquine has been taken; these drugs have significant activities against the liver stage of the infection (causal prophylaxis) and can be discontinued 1 week after departure from the endemic area. If suspected malaria develops while a traveler is abroad, obtaining a reliable diagnosis and antimalarial treatment locally is a top priority. Presumptive self-treatment for malaria with atovaquoneproguanil (for 3 consecutive days) or another drug can be considered under special circumstances; medical advice on self-treatment should be sought before departure for malarious areas and as soon as possible after illness begins. Every

1	another drug can be considered under special circumstances; medical advice on self-treatment should be sought before departure for malarious areas and as soon as possible after illness begins. Every effort should be made to confirm the diagnosis by parasitologic studies.

1	Atovaquone-proguanil (Malarone; 3.75/1.5 mg/kg or 250/100 mg, daily adult dose) is a fixed-combination, once-daily prophylactic agent that is very well tolerated by adults and children, with fewer adverse gastrointestinal effects than chloroquine-proguanil and fewer adverse central nervous system effects than mefloquine. It is proguanil itself, rather than the antifolate metabolite cycloguanil, that acts synergistically with atovaquone. This combination is effective against all types of malaria, including multidrug-resistant falciparum malaria. Atovaquone-proguanil is best taken with food or a milky drink to optimize absorption. There are insufficient data on the safety of this regimen in pregnancy.

1	Mefloquine (250 mg of salt weekly, adult dose) has been widely used for malarial prophylaxis because it is usually effective against multidrug-resistant falciparum malaria and is reasonably well tolerated. The drug has been associated with rare episodes of psychosis and seizures at prophylactic doses; these reactions are more frequent at the higher doses used for treatment. More common side effects with prophylactic doses of mefloquine include mild nausea, dizziness, fuzzy thinking, disturbed sleep patterns, vivid dreams, and malaise. The drug is contraindicated for use by travelers with known hypersensitivity to mefloquine or related compounds (e.g., quinine, quinidine) and by persons with active or recent depression, anxiety disorder, psychosis, schizophrenia, another major psychiatric disorder, or seizures; mefloquine is not recommended for persons with cardiac conduction abnormalities although the evidence that it is cardiotoxic is very weak. Confidence is increasing with regard

1	disorder, or seizures; mefloquine is not recommended for persons with cardiac conduction abnormalities although the evidence that it is cardiotoxic is very weak. Confidence is increasing with regard to the safety of mefloquine prophylaxis during pregnancy; in studies in Africa, mefloquine prophylaxis was found to be effective and safe during pregnancy. However, in one study from Thailand, treatment of malaria with mefloquine was associated with an increased risk of stillbirth; this effect was not seen subsequently.

1	Daily administration of doxycycline (100 mg daily, adult dose) is an effective alternative to atovaquone-proguanil or mefloquine. Doxycycline is generally well tolerated but may cause vulvovaginal thrush, diarrhea, and photosensitivity and cannot be used by children <8 years old or by pregnant women. Chloroquine can no longer be relied upon to prevent P. falciparum infections in most areas but is used to prevent and treat malaria due to the other human Plasmodium species and for P. falciparum malaria in Central American countries west and north of the Panama Primaquine For prevention of malaria 30 mg of base 0.5 mg of base/kg (0.8 mg of Begin 1–2 days before travel to malarious areas. in areas with mainly (52.6 mg of salt) salt/kg) PO qd, up to adult dose; Take daily at the same time each day while in

1	P. vivax PO qd should be taken with food the malarious areas and for 7 days after leaving such areas. Primaquine is contraindicated in persons with G6PD deficiency. It is also contraindicated during pregnancy and in lactation unless the infant being breast-fed has a documented normal G6PD level. Primaquine Used for presumptive 30 mg of base 0.5 mg of base/kg (0.8 mg of This therapy is indicated for persons who have antirelapse therapy (52.6 mg of salt) PO salt/kg), up to adult dose, PO qd had prolonged exposure to P. vivax and/or (terminal prophylaxis) to qd for 14 days after for 14 days after departure from P. ovale. It is contraindicated in persons with G6PD decrease risk of relapses departure from the the malarious area deficiency as well as during pregnancy and in of P. vivax and P. ovale malarious area lactation unless the infant being breast-fed has a documented normal G6PD level.

1	aAn adult tablet contains 250 mg of atovaquone and 100 mg of proguanil hydrochloride. bA pediatric tablet contains 62.5 mg of atovaquone and 25 mg of proguanil hydrochloride. cVery few areas now have chloroquine-sensitive malaria (Fig. 248-2). Source: CDC: www.cdc.gov/malaria/travelers/drugs.html.

1	Canal, Caribbean countries, and some countries in the Middle East. potential problem with protracted prophylactic use; such myopathy is Chloroquine-resistant P. vivax has been reported from parts of eastern more likely to occur at the high doses used in the treatment of rheuma-Asia, Oceania, and Central and South America. This drug is gener-toid arthritis. Neuropsychiatric reactions and skin rashes are unusual. ally well tolerated, although some patients cannot take it because of When used continuously, amodiaquine, a related aminoquinoline, is malaise, headache, visual symptoms (due to reversible keratopathy), associated with a high risk of agranulocytosis (~1 person in 2000) and gastrointestinal intolerance, or pruritus. Chloroquine is considered hepatotoxicity (~1 person in 16,000); thus this agent should not be safe in pregnancy. With chronic administration for >5 years, a char-used for prophylaxis. acteristic dose-related retinopathy may develop, but this condition is Primaquine

1	this agent should not be safe in pregnancy. With chronic administration for >5 years, a char-used for prophylaxis. acteristic dose-related retinopathy may develop, but this condition is Primaquine (daily adult dose, 0.5 mg of base/kg or 30 mg taken rare at the doses used for antimalarial prophylaxis. Idiosyncratic or with food), an 8-aminoquinoline compound, has proved safe and allergic reactions are also rare. Skeletal and/or cardiac myopathy is a effective in the prevention of drug-resistant falciparum and vivax 1384 malaria in adults. This drug can be considered for persons who are traveling to areas with or without drug-resistant P. falciparum and who are intolerant to other recommended drugs. Abdominal pain and oxidant hemolysis—the principal adverse effects—are not common as long as the drug is taken with food and is not given to G6PDdeficient persons, in whom it can cause serious hemolysis. Travelers must be tested for G6PD deficiency and be shown to have a level in the normal

1	the drug is taken with food and is not given to G6PDdeficient persons, in whom it can cause serious hemolysis. Travelers must be tested for G6PD deficiency and be shown to have a level in the normal range before receiving primaquine. Primaquine should not be given to pregnant women or neonates. Primaquine, given in a single dose of 0.25 mg/kg as a gametocytocide, together with an ACT is recommended in falciparum malaria treatment regimens in malaria elimination programs. In the past, the dihydrofolate reductase inhibitors pyrimethamine and proguanil (chloroguanide) were administered widely, but the rapid selection of resistance in both P. falciparum and P. vivax has limited their use. Whereas antimalarial quinolines such as chloroquine (a 4-aminoquinoline) act on the erythrocyte stage of parasitic development, the dihydrofolate reductase inhibitors also inhibit preerythrocytic growth in the liver (causal prophylaxis) and development in the mosquito (sporontocidal activity). Proguanil

1	parasitic development, the dihydrofolate reductase inhibitors also inhibit preerythrocytic growth in the liver (causal prophylaxis) and development in the mosquito (sporontocidal activity). Proguanil is safe and well tolerated, although mouth ulceration occurs in ~8% of persons using this drug; it is considered safe for antimalarial prophylaxis in pregnancy. The prophylactic use of the combination of pyrimethamine and sulfadoxine is not recommended because of an unacceptable incidence of severe toxicity, principally exfoliative dermatitis and other skin rashes, agranulocytosis, hepatitis, and pulmonary eosinophilia (incidence, 1:7000; fatal reactions, 1:18,000). The combination of pyrimethamine with dapsone (0.2/1.5 mg/kg weekly; 12.5/100 mg, adult dose) has been used in some countries. Dapsone may cause methemoglobinemia and allergic reactions and (at higher doses) may pose a significant risk of agranulocytosis. Proguanil and the pyrimethamine-dapsone combination are not available in

1	may cause methemoglobinemia and allergic reactions and (at higher doses) may pose a significant risk of agranulocytosis. Proguanil and the pyrimethamine-dapsone combination are not available in the United States. Because of the increasing spread and intensity of antimalarial drug resistance (Figs. 248-2 and 248-10), the CDC recommends that travelers and their providers consider their destination, type of travel, and current medications and health risks when choosing antimalarial chemoprophylaxis. There is an increasingly appreciated problem of counterfeit and substandard antimalarial drugs (and other medicines) on the shelves of pharmacies in Southeast Asia and sub-Saharan Africa; hence, travelers should purchase their preventive drugs from a reputable source before going to a malarious country. Consultation for the evaluation of prophylaxis failures or treatment of malaria can be obtained from state and local health departments and the CDC Malaria Hotline (770-488-7788) or the CDC

1	country. Consultation for the evaluation of prophylaxis failures or treatment of malaria can be obtained from state and local health departments and the CDC Malaria Hotline (770-488-7788) or the CDC Emergency Operations Center (770-488-7100).

1	Edouard G. Vannier, Peter J. Krause Babesiosis is an emerging tick-borne infectious disease caused by protozoan parasites of the genus Babesia that invade and eventually lyse red blood cells (RBCs). Most cases are due to Babesia microti and occur in the United States, particularly in the Northeast and upper Midwest. The infection typically is mild in young and otherwise healthy individuals but can be severe and sometimes fatal in persons >50 years of age and in immunocompromised patients. Sporadic cases have been reported in Europe and the rest of the world.

1	ETIOLOGY AND EPIDEMIOLOGY Geographic Distribution More than 100 Babesia species are found in wild and domestic animals; a few of these species cause infection in humans (Fig. 249-1). B. microti, a parasite of small rodents, is the most common etiologic agent of human babesiosis and is endemic in the northeastern and upper midwestern United States. Seven states in these two regions (Connecticut, Massachusetts, Minnesota, New Jersey, New York, Rhode Island, and Wisconsin) account for >95% of the reported cases. Other etiologic agents include Babesia duncani and B. duncani–type organisms on the West Coast and Babesia divergens–like organisms in Kentucky, Missouri, and Washington State. The primary causative agent of human babesiosis in Europe is

1	The primary causative agent of human babesiosis in Europe is B. divergens, but Babesia venatorum and B. microti also have been reported. In Asia, cases due to B. microti–like organisms have been documented in Japan, Taiwan, and the People’s Republic of China. A case caused by B. venatorum also has been reported from the People’s Republic of China. A case of B. microti infection was described in Australia. Sporadic cases due to uncharacterized species have been reported in Colombia, Egypt, India, Mozambique, and South Africa.

1	Incidence More than 1100 cases were reported in the United States in 2011, the year the disease became nationally notifiable. This figure represents a fivefold increase in incidence over the past decade. The incidence of babesiosis is markedly underestimated because symptoms are nonspecific and because young healthy individuals typically experience a mild or asymptomatic infection and may not seek medical attention. Fewer than 50 cases of B. divergens, B. divergens–like, and B. venatorum infections have been reported. Babesiosis caused by B. duncani and B. duncani–type organisms has also been sporadic, with fewer than 10 reported cases.

1	B. duncani and B. duncani–type organisms has also been sporadic, with fewer than 10 reported cases. Modes of Transmission In the United States, B. microti is transmitted to humans primarily by the nymphal stage of the deer tick (Ixodes scapularis), the same tick that transmits the causative agents of Lyme disease (Chap. 210) and human granulocytotropic anaplasmosis (Chap. 211). Transmission generally occurs from May through October, with three-fourths of cases presenting in July and August. The vectors for transmission of B. duncani and B. divergens– like organisms are thought to be Ixodes pacificus and Ixodes dentatus, respectively. In Europe, Ixodes ricinus is the vector for B. divergens and B. venatorum. In Japan, B. microti–like organisms have been found in Ixodes ovatus ticks.

1	Babesiosis occasionally is acquired through transfusion of blood or blood products. B. microti is the most common transfusion-transmitted pathogen reported to the U.S. Food and Drug Administration, and more than 170 such cases have been identified. Three transfusion-transmitted cases caused by B. duncani have been documented. Transfusion-transmitted cases occur year-round, although most cases occur from June through November. More than 80% of cases occur in endemic areas. Transfusion-transmitted babesiosis occurs in nonendemic areas when unrecognized Babesia-contaminated blood products are imported from endemic areas: asymptomatically infected residents of endemic areas donate blood in nonendemic areas, or residents of nonendemic areas travel to endemic areas, become infected, and donate blood after they return home. Approximately three-quarters of the transfusion-transmitted babesiosis cases reported between 1979 and 2009 occurred in the last decade of this period, and about

1	donate blood after they return home. Approximately three-quarters of the transfusion-transmitted babesiosis cases reported between 1979 and 2009 occurred in the last decade of this period, and about one-fifth of patients died.

1	Seven cases of probable or confirmed congenital B. microti infection have been described. Other cases of neonatal babesiosis have been acquired by transfusion or tick bite. CLINICAL MANIFESTATIONS Asymptomatic B. microti Infection At least 20% of adults and 40% of children do not experience symptoms following B. microti infection. Asymptomatic infection, whether treated or not, may persist for >1 year after acute babesial illness. There is no evidence of long-term complications following asymptomatic infection; however, people who are asymptomatically infected may transmit the infection when they donate blood. Mild to Moderate B. microti Illness Symptoms typically develop following an incubation period of 1–4 weeks after tick bite and 1–9 weeks (but as long as 6 months) after transfusion of blood products. Patients experience a gradual onset of malaise, fatigue, and weakness. Fever I. persulcatus B. duncani I. scapularis B. microti B. divergens I. ricinus I. ovatus

1	FIGuRE 249-1 Worldwide distribution of human babesiosis. Dark colors designate areas where human babesiosis is either endemic or sporadic (as defined by more than three tick-borne cases reported in a country or state). Isolated cases of babesiosis are denoted by circles. Colors designate causative Babesia species: Babesia microti and B. microti–like organisms in red, Babesia duncani and B. duncani–type organisms in orange, Babesia divergens and B. divergens–like organisms in blue, Babesia venatorum in purple, KO1 in black, and unspeciated Babesia organisms in white. Due to space constraints, the 10 cases reported from Montenegro are denoted by a single white circle, and those from Australia, Mozambique, and South Africa are not shown. Light colors denote areas that are enzootic for Ixodes tick species known to transmit one or several Babesia species but where human babesiosis has yet to be documented. (Adapted from E Vannier, PJ Krause: N Engl J Med 366:2397, 2012.) can reach 40.9°C

1	tick species known to transmit one or several Babesia species but where human babesiosis has yet to be documented. (Adapted from E Vannier, PJ Krause: N Engl J Med 366:2397, 2012.) can reach 40.9°C (105.6°F) and is accompanied by one or more of the following: chills, sweats, headache, myalgia, arthralgia, nausea, anorexia, and dry cough. Less common symptoms include sore throat, photo-phobia, abdominal pain, vomiting, weight loss, shortness of breath, and depression. On physical examination, fever is the salient feature. Ecchymoses and petechiae have been reported. An erythema migrans rash signifies concurrent Lyme disease (Chap. 210). Splenomegaly and hepatomegaly occasionally are noted. Lymphadenopathy is absent. Jaundice, slight pharyngeal erythema, and retinopathy with splinter hemorrhages and retinal infarcts rarely occur. Symptoms typically last 1–2 weeks, but fatigue may persist for several months.

1	Severe B. microti Illness Severe babesiosis requires hospital admission and typically occurs in patients with one or more of the following: age of >50 years, neonatal prematurity, male gender, asplenia, HIV/AIDS, malignancy, hemoglobinopathy, and immunosuppressive therapy. More than one-third of hospitalized patients develop complications, including acute respiratory distress syndrome, disseminated intra-vascular coagulation, congestive heart failure, renal failure, splenic infarcts, and splenic rupture. Patients who develop complications tend to have severe anemia (hemoglobin, ≤10 g/L). Laboratory prognostic factors for severe outcome—defined by hospitalization for >14 days, an intensive care unit stay of >2 days, or death—include an elevated alkaline phosphatase level (>125 U/L) and parasitemia of >4%. The fatality rate is 5–9% among hospitalized patients but is ~20% among immunocompromised patients and patients with transfusion-transmitted babesiosis.

1	Other Babesia Infections Cases of B. duncani infection range in severity from asymptomatic to fatal. Clinical manifestations are similar to those reported for B. microti infection. All three patients infected with B. divergens–like organisms in the United States required hospitalization; one died. Most cases of B. divergens infection in Europe have occurred in people lacking a spleen. The incubation period is 1–3 weeks. Symptoms appear suddenly and consist of fever (>41°C or 105.8°F), shaking chills, drenching sweats, headache, myalgia, and lumbar and abdominal pain. Hemoglobinuria and jaundice are commonly noted, and mild hepatomegaly may occur. If the infection is not treated, patients often develop pulmonary edema and renal failure. All four patients infected with B. venatorum in Europe had been splenectomized; their illness ranged from mild to severe, and none died. A child in China who developed B. venatorum illness had an intact spleen and survived the infection.

1	Anemia is a key feature of the pathogenesis of babesiosis. Hemolytic anemia caused by rupture of infected RBCs generates cell debris that may accumulate in the kidney and cause renal failure. Anemia also results from the clearance of intact RBCs as they pass through the splenic red pulp and encounter resident macrophages. Babesia antigens expressed at the RBC membrane promote opsonization and facilitate uptake by splenic macrophages. In addition, RBCs are poorly deformable as a result of oxidation generated by the parasite and the host immune response and are filtered out as they attempt to squeeze across the venous vasculature. Bone marrow suppression due to cytokine production may also contribute to anemia.

1	An appropriate immune response is necessary for the control and clearance of Babesia. However, several lines of evidence suggest that an excessive response contributes to pathogenesis. Studies using laboratory mice have clearly established that CD4+ T cells are critical for resistance to and resolution of B. microti infection. CD4+ T cells are a major source of interferon γ (IFN-γ), and lack of this cytokine causes resistant mice to become highly susceptible to B. microti. IFN-γ is central to host resistance in B. duncani infection, but natural killer cells are its main source. B. duncani infection is more severe than B. microti infection in rodents and is characterized by pulmonary inflammation. Tumor necrosis factor α is expressed around alveolar septa, whereas IFN-γ is detected around pulmonary vessels. Blockade of either cytokine promotes the survival of mice infected with B. duncani.

1	A diagnosis of babesiosis should be considered for any patient who lives or travels in a Babesia-endemic area and presents with a febrile illness in the late spring, summer, or early autumn or within 6 months after a blood transfusion. Co-infection with Babesia should be considered in cases of Lyme disease or human granulocytotropic anaplasmosis when symptoms are more severe or prolonged than usual. Screening laboratory tests can help support the diagnosis of babesiosis. A complete blood count often shows anemia and thrombocytopenia. Low hematocrit, hemoglobin, and haptoglobin levels and elevated reticulocyte counts and lactate dehydrogenase levels are consistent with hemolytic anemia. Liver enzyme tests often reveal elevated levels of alkaline phosphatase, aspartate and alanine aminotransferases, and bilirubin. Urinalysis may show hemoglobinuria, excess urobilinogen, and proteinuria. Elevated levels of blood urea nitrogen and serum creatinine indicate renal compromise.

1	A specific diagnosis usually is established by microscopic examination of Giemsa-stained thin blood smears (Fig. 249-2). Babesia trophozoites appear round, pear-shaped, or ameboid. The ring form is most common and lacks the central brownish deposit (hemozoin) typical of Plasmodium falciparum trophozoites (see Fig. 250e-1C). Other distinguishing features are the absence of schizonts and gametocytes and the occasional presence of tetrads (“Maltese cross”). FIGuRE 249-2 Giemsa-stained thin blood films showing Babesia microti parasites. B. microti are obligate parasites of erythrocytes. Trophozoites may appear as ring forms (A) or as ameboid forms (B). Merozoites can be arranged in tetrads and are pathognomonic (C). Extracellular parasites can be noted, particularly when parasitemia is high (D). (Adapted from E Vannier, PJ Krause: N Engl J Med 366:2397, 2012.)

1	Tetrads are characteristic of B. microti, B. duncani, and B. divergens–like organisms in human erythrocytes. Because the number of parasitized RBCs may be low, particularly at the onset of symptoms, identification of the parasite may require multiple blood smears over several days. Parasitemia levels can range from 1% to 20% in immunocompetent hosts and can be as high as 85% in immunocompromised patients. If parasites cannot be identified by microscopy and the disease is still suspected, amplification of the babesial 18S rRNA gene by polymerase chain reaction (PCR) is recommended. Quantitative PCR has greatly lowered the threshold for detection of B. microti DNA.

1	Serology can suggest or confirm the diagnosis of babesiosis. An indirect immunofluorescent antibody test for B. microti is most commonly used. IgM titers of ≥1:64 and IgG titers of ≥1:1024 suggest active or recent infection. Titers typically decline over 6–12 months. Antibodies to B. microti do not cross-react with B. duncani or B. divergens antigen. In B. divergens infection, serology is of limited use because symptoms often appear before antibodies can be detected. Sera from patients infected with B. divergens–like organisms or B. venatorum are reactive against B. divergens antigen. ASYMPTOMATIC B. MICROTI INFECTION

1	B. venatorum are reactive against B. divergens antigen. ASYMPTOMATIC B. MICROTI INFECTION People who experience asymptomatic B. microti infection often are not diagnosed and treated. Current guidelines recommend antibiotic therapy for asymptomatic carriers only if parasitemia persists for >3 months. Laboratory-based tests are being developed for the purpose of screening the blood supply and will result in the identification of a greater number of asymptomatic B. microti carriers, raising the question of whether they should be treated. MILD TO MODERATE B. MICROTI ILLNESS Atovaquone plus azithromycin, given orally for 7–10 days, is the recommended antibiotic treatment combination for mild to moderate B. microti Infection (Mild to Moderate Illnessa,b) Atovaquone (750 mg q12h PO) Atovaquone (20 mg/kg q12h PO; maximum, 750 mg/dose)

1	B. microti Infection (Mild to Moderate Illnessa,b) Atovaquone (750 mg q12h PO) Atovaquone (20 mg/kg q12h PO; maximum, 750 mg/dose) Azithromycin (500 mg/d PO on day 1, 250 mg/d PO thereafter) Azithromycin (10 mg/kg qd PO on day 1 [maximum, 500 mg/dose], 5 mg/kg qd PO thereafter [maximum, 250 mg/dose]) B. microti Infection (Severe Illnessc,d) B. divergens Infection Clindamycin (7–10 mg/kg q6–8h IV or 7–10 mg/kg q6–8h PO; maximum, 600 mg/dose) Quinine (8 mg/kg q8h PO; maximum, 650 mg/dose) Clindamycin (600 mg q6–8h IV) Clindamycin (7–10 mg/kg q6–8h IV; maximum, 600 mg/dose)

1	Quinine (8 mg/kg q8h PO; maximum, 650 mg/dose) Clindamycin (600 mg q6–8h IV) Clindamycin (7–10 mg/kg q6–8h IV; maximum, 600 mg/dose) Quinine (650 mg q8h PO) Quinine (8 mg/kg q8h PO; maximum, aTreatment duration, 7–10 days. bA high dose of azithromycin (600–1000 mg) combined with atovaquone has been recommended for immunocompromised hosts. cTreatment typically is given for 7–10 days, but its duration may vary. In severely immunocompromised patients, therapy should be continued for at least 6 weeks, including 2 weeks after parasites are no longer detected on blood smear. dSeveral alternative regimens have been used in a limited number of cases of B. microti infection, and their efficacy is uncertain. These regimens include atovaquone, azithromycin, and clindamycin; atovaquone, azithromycin, and doxycycline; atovaquone, clindamycin, and doxycycline; atovaquone, doxycycline, and artemisinin; atovaquone-proguanil; azithromycin and quinine; and azithromycin, clindamycin, and doxycycline.

1	Sources: (1) ME Falagas, MS Klempner: Clin Infect Dis 22:809, 1996. (2) PJ Krause et al: N Engl J Med 343:1454, 2000. (3) PJ Krause et al: Clin Infect Dis 46:370, 2008. (4) CM Shih, CC Wang: Am J Trop Med Hyg 59:509, 1998. (5) CP Stowell et al: N Engl J Med 356:2313, 2007. (6) JM Vyas et al: Clin Infect Dis 45:1588, 2007. (7) GP Wormser et al: Clin Infect Dis 50:381, 2010.

1	babesiosis (Table 249-1). Clindamycin plus quinine is a second choice. Symptoms usually begin to resolve within 48 h of therapy initiation, but complete resolution may take weeks to months. An atypical or poor response to therapy should raise concern about the possibility of concurrent Lyme disease (Chap. 210) or human granulocytotropic anaplasmosis (Chap. 211). In the first prospective trial of antibabesial therapy, the combination of atovaquone plus azithromycin was compared with clindamycin plus quinine in adults. These two drug combinations were equally effective in resolving symptoms and clearing parasitemia. Adverse effects were reported in 15% of trial participants who received atovaquone plus azithromycin but in 72% of those who received clindamycin plus quinine. Adverse reactions were so severe that treatment had to be stopped in about one-third of participants taking clindamycin plus quinine but in only 2% of those taking atovaquone plus azithromycin.

1	SEVERE B. MICROTI ILLNESS Clindamycin given intravenously plus quinine given orally for 7–10 days constitute the recommended treatment for severe babesiosis. Intravenous quinidine may be used instead of oral quinine but requires cardiac monitoring because of the risk of QT prolongation and polymorphic ventricular tachycardia.

1	Standard antimicrobial therapy is sometimes insufficient to resolve symptoms and clear parasitemia, especially in patients with marked immunosuppression due to splenectomy, HIAIDS, malig-13 nancy, andor immunosuppressive therapy (including rituximab for cell lymphomas). In such patients, antimicrobial therapy should be administered for at least weeks, including 2 weeks after parasites are no longer observed on blood smear. High-dose azithromycin (00–1000 mgd) plus atovauone have been successfully used in immunocompromised patients. esistance to atovauone plus azithromycin has occurred in a few cases. In patients who are unresponsive to atovauone plus azithromycin or who do not tolerate clindamycin plus uinine, alternative regimens have been used (Table 21).

1	Partial or complete C exchange transfusion is recommended in patients with high-grade parasitemia (0, severe anemia (hemoglobin, 0 gdL), or pulmonary, hepatic, or renal compromise. Parasitemia and hematocrit should be monitored daily until symptoms recede and the parasitemia level is The regimen for B. duncani infections typically consists of intravenous clindamycin (00 mg tidid or 1200 mg bid) plus oral uinine (00–50 mg tid) for 7–10 days. A regimen often used for B. divergens–like infections is intravenous clindamycin (00 mg tidid, 0 mg tid, or 1200 mg bid) plus oral uinine or uinidine (50 mg tid). In Europe, B. divergens infection is considered a medical emergency. The recommended treatment is immediate complete blood exchange transfusion and therapy with intravenous clindamycin plus either oral uinine or intravenous uinidine. Some cases have been cured with exchange transfusion and clindamycin monotherapy. Anemia may persist for month and reuire additional transfusion.

1	No vaccine is available for human use. There is no role for antibiotic prophylaxis. Individuals who reside in endemic areas, especially those at risk for severe babesiosis, should wear clothing that covers the lower part of the body, apply tick repellents (such as DEET) to clothing, and limit outdoor activities where ticks may abound from May through October. The skin should be thoroughly examined after outdoor activities, and ticks should be removed with tweezers. Individuals with a history of symptomatic babesiosis or with positive Babesia serology are indefinitely deferred from donating blood.

1	Atlas of Blood Smears of Malaria and Babesiosis Nicholas J. White, Joel G. Breman Six species of blood protozoan parasites cause human malaria (Chap. 248): the potentially lethal and often drug-resistant Plasmodium falci-parum; the relapsing parasites Plasmodium vivax and Plasmodium ovale 250e (with what appear to be two morphologically identical sympatric species of P. ovale); Plasmodium malariae, which can persist at low densities for years; and, in infections in individuals living in or close to tropical forests in Southeast Asia, Plasmodium knowlesi, a monkey parasite that microscopically resembles P. falciparum (young forms) and P. malariae (older forms) but is identified definitively by molecular methods.

1	The malaria parasites are readily seen under the microscope (×1000 250e-1 magnification) in thick and thin blood smears stained with supravital dyes (e.g., Giemsa’s, Field’s, Wright’s, Leishman’s). The morphologic characteristics of the parasites are summarized in Table 250e-1. In the thick film, lysis of red blood cells by water leaves the stained white cells and parasites, allowing detection of densities as low as 50 parasites/μL. This degree of sensitivity is up to 100 times greater than that of the thin film, in which the cells are fixed and the malaria parasites are seen inside the red cells. The thin film is better for speciation and provides useful prognostic information in severe falciparum malaria. Several findings are associated with increased mortality risk: high parasite counts, more mature parasites (>20% containing visible malaria pig ment), and phagocytosed malaria pigment in >5% of neutrophils. Babesia microti (Chap. 249) appears as a small ring form resembling

1	P. falciparum. Unlike Plasmodium, Babesia does not cause the production of pigment in parasites, nor are schizonts or gametocytes formed. aThe early trophozoites of Plasmodium knowlesi resemble those of P. falciparum. The late and mature trophozoites and schizonts of P. knowlesi appear very similar to those of P. malariae. The differences are that (1) P. knowlesi trophozoites may have double chromatin dots, with two or three parasites per RBC, and may cause higher-level parasitemia; and (2) P. knowlesi mature schizonts have 16 merozoites rather than the 8–10 found with P. malariae. bTwo morphologically identical sympatric species, according to recent evidence. Abbreviation: RBC, red blood cell. CHAPTER 250e Atlas of Blood Smears of Malaria and Babesiosis

1	Abbreviation: RBC, red blood cell. CHAPTER 250e Atlas of Blood Smears of Malaria and Babesiosis Figure 250e-1 Thin blood films of Plasmodium falciparum. A. Young trophozoites. B. Old trophozoites. C. Pigment in polymorphonuclear cells and trophozoites. D. Mature schizonts. E. Female gametocytes. F. Male gametocytes. (Reproduced from Bench Aids for the Diagnosis of Malaria Infections, 2nd ed, with the permission of the World Health Organization.) Figure 250e-2 Thin blood films of Plasmodium vivax. A. Young trophozoites. B. Old trophozoites. C. Mature schizonts. D. Female gametocytes. E. Male gametocytes. (Reproduced from Bench Aids for the Diagnosis of Malaria Infections, 2nd ed, with the permission of the World Health Organization.)

1	Figure 250e-3 Thin blood films of Plasmodium ovale. A. Old trophozoites. B. Mature schizonts. C. Male gametocytes. D. Female gametocytes. (Reproduced from Bench Aids for the Diagnosis of Malaria Infections, 2nd ed, with the permission of the World Health Organization.) Figure 250e-4 Thin blood films of Plasmodium malariae. A. Old trophozoites. B. Mature schizonts. C. Male gametocytes. D. Female gametocytes. (Reproduced from Bench Aids for the Diagnosis of Malaria Infections, 2nd ed, with the permission of the World Health Organization.) Figure 250e-5 Thick blood films of Plasmodium falciparum. A. Trophozoites. B. Gametocytes. (Reproduced from Bench Aids for the Diagnosis of Malaria Infections, 2nd ed, with the permission of the World Health Organization.) CHAPTER 250e Atlas of Blood Smears of Malaria and Babesiosis

1	CHAPTER 250e Atlas of Blood Smears of Malaria and Babesiosis Figure 250e-6 Thick blood films of Plasmodium vivax. A. Trophozoites. B. Schizonts. C. Gametocytes. (Reproduced from Bench Aids for the Diagnosis of Malaria Infections, 2nd ed, with the permission of the World Health Organization.) Figure 250e-7 Thick blood films of Plasmodium ovale. A. Trophozoites. B. Schizonts. C. Gametocytes. (Reproduced from Bench Aids for the Diagnosis of Malaria Infections, 2nd ed, with the permission of the World Health Organization.) Figure 250e-8 Thick blood films of Plasmodium malariae. A. Trophozoites. B. Schizonts. C. Gametocytes. (Reproduced from Bench Aids for the Diagnosis of Malaria Infections, 2nd ed, with the permission of the World Health Organization.)

1	Figure 250e-9 Thin blood film showing trophozoites of Babesia. (Reproduced from Bench Aids for the Diagnosis of Malaria Infections, 2nd ed, with the permission of the World Health Organization.) 1387 Leishmaniasis Shyam Sundar DEFINITION Encompassing a complex group of disorders, leishmaniasis is caused by unicellular eukaryotic obligatory intracellular protozoa of the genus Leishmania and primarily affects the host’s reticuloendothelial system. 251 Leishmania species produce widely varying clinical syndromes ranging from self-healing cutaneous ulcers to fatal visceral disease. These syndromes fall into three broad categories: visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), and mucosal leishmaniasis (ML).

1	Leishmaniasis is caused by ~20 species of the genus Leishmania in the order Kinetoplastida and the family Trypanosomatidae (Table 251-1). Several clinically important species are of the subspecies Viannia. The organisms are transmitted by phlebotomine sandflies of the genus Phlebotomus in the “Old World” (Asia, Africa, and Europe) and the genus Lutzomyia in the “New World” (the Americas). Transmission may be anthroponotic (i.e., the vector transmits the infection from infected humans to healthy humans) or zoonotic (i.e., the vector transmits the infection from an animal reservoir to humans). Human-tohuman transmission via shared infected needles has been documented in IV drug users in the Mediterranean region. In utero transmission to the fetus occurs rarely.

1	Leishmania organisms occur in two forms: extracellular, flagellate promastigotes (length, 10–20 μm) in the sandfly vector and intracellular, nonflagellate amastigotes (length, 2–4 μm; Fig. 251-1) in vertebrate hosts, including humans. Promastigotes are introduced through the proboscis of the female sandfly into the skin of the vertebrate host. Neutrophils predominate among the host cells that first encounter and take up promastigotes at the site of parasite delivery. The infected neutrophils may undergo apoptosis and release viable parasites that are taken up by macrophages, or the apoptotic cells may themselves be taken up by macrophages and dendritic cells. The parasites multiply as amastigotes inside macrophages, causing cell rupture with subsequent invasion of other macrophages. While feeding on infected hosts, sand-flies pick up amastigotes, which transform into the flagellate form in the flies’ posterior midgut and multiply by binary fission; the promastigotes then migrate to

1	feeding on infected hosts, sand-flies pick up amastigotes, which transform into the flagellate form in the flies’ posterior midgut and multiply by binary fission; the promastigotes then migrate to the anterior midgut and can infect a new host when flies take another blood meal.

1	Leishmaniasis occurs in 98 countries—most of them developing—in tropical and temperate regions (Fig. 251-2). More than 1.5 million cases occur annually, of which 0.7–1.2 million are CL (and its variations) and 200,000–400,000 are VL. More than 350 million people are at risk, with an overall prevalence of 12 million. Although the distribution of Leishmania is limited by the distribution of sandfly vectors, human leishmaniasis is on the increase worldwide. VL (also known as kala-azar, a Hindi term meaning “black fever”) is caused by the Leishmania donovani complex, which includes

1	VL (also known as kala-azar, a Hindi term meaning “black fever”) is caused by the Leishmania donovani complex, which includes L. donovani and Leishmania infantum (the latter designated Leishmania chagasi in the New World); these species are responsible for anthroponotic and zoonotic transmission, respectively. India and neighboring Bangladesh, Sudan, South Sudan, Ethiopia, and Brazil are the four largest foci of VL and account for 90% of the world’s VL burden, with India being the worst affected. Zoonotic VL is reported from all countries in the Middle East, Pakistan, and other countries from western Asia to China. Endemic foci also exist in the independent states of the former Soviet Union, mainly Georgia and Azerbaijan. In the Horn of Africa, Sudan, South Sudan, Ethiopia, Kenya, Uganda, and Somalia report VL. In Sudan and South Sudan, large outbreaks are thought to be anthroponotic, although zoonotic transmission also occurs. VL is rare in West and sub-Saharan Africa.

1	Organism, Clinical Endemic Region Syndrome Species Vector Reservoir Transmission Setting aL. infantum is designated L. chagasi in the New World. Abbreviations: CL, cutaneous leishmaniasis; DCL, diffuse cutaneous leishmaniasis; ML, mucosal leishmaniasis; PKDL, post–kala-azar dermal leishmaniasis; VL, visceral leishmaniasis. Mediterranean VL, long an established endemic disease due to on the immune system. IV drug users are at particular risk. Other L. infantum, has a large canine reservoir and was seen primarily in forms of immunosuppression (e.g., that associated with organ trans-infants before the advent of HIV infection. In Mediterranean Europe, plantation) also predispose to VL. In the Americas, disease caused by 70% of adult VL cases are associated with HIV co-infection. The com-L. infantum is endemic from Mexico to Argentina, but 90% of cases in bination is deadly because of the impact of the two infections together the New World are reported from northeastern Brazil.

1	FIGuRE 251-1 A macrophage with numerous intracellular amasti-gotes (2–4 μm) in a Giemsa-stained splenic smear from a patient with visceral leishmaniasis. Each amastigote contains a nucleus and a char-acteristic kinetoplast consisting of multiple copies of mitochondrial DNA. A few extracellular parasites are also visible. Immunopathogenesis The majority of individuals infected by

1	L. donovani or L. infantum mount a successful immune response and control the infection, never developing symptomatic disease. Forty-eight hours after intradermal injection of killed promastigotes, these individuals exhibit delayed-type hypersensitivity (DTH) to leishmanial antigens in the leishmanin skin test (also called the Montenegro skin test). Results in mouse models indicate that the development of acquired resistance to leishmanial infection is controlled by the production of interleukin (IL) 12 by antigen-presenting cells and the subsequent secretion of interferon (IFN) γ, tumor necrosis factor (TNF) α, and other proinflammatory cytokines by the T helper 1 (TH1) subset of T lymphocytes. The immune response in patients developing active VL is complex; in addition to increased production of multiple proinflammatory cytokines and chemokines, patients with active disease have markedly elevated levels of IL-10 in serum as well as enhanced IL-10 mRNA expression in lesional tissues.

1	of multiple proinflammatory cytokines and chemokines, patients with active disease have markedly elevated levels of IL-10 in serum as well as enhanced IL-10 mRNA expression in lesional tissues. The main disease-promoting activity of IL-10 in VL may be to condition host macrophages for enhanced survival and growth of the parasite. IL-10 1389 can render macrophages unresponsive to activation signals and inhibit killing of amastigotes by downregulating the production of TNF-α and nitric oxide. Multiple antigen-presentation functions of dendritic cells and macrophages are also suppressed by IL-10. Patients with such suppression do not have positive leishmanin skin tests, nor do their peripheral-blood mononuclear cells respond to leishmanial antigens in vitro. Organs of the reticuloendothelial system are predominantly affected, with remarkable enlargement of the spleen, liver, and lymph nodes in some regions. The tonsils and intestinal submucosa are also heavily infiltrated with parasites.

1	system are predominantly affected, with remarkable enlargement of the spleen, liver, and lymph nodes in some regions. The tonsils and intestinal submucosa are also heavily infiltrated with parasites. Bone marrow dysfunction results in pancytopenia.

1	Clinical Features On the Indian subcontinent and in the Horn of Africa, persons of all ages are affected by VL. In endemic areas of the Americas and the Mediterranean basin, immunocompetent infants and small children as well as immunodeficient adults are affected especially often. The most common presentation of VL is an abrupt onset of moderateto high-grade fever associated with rigor and chills. Fever may continue for several weeks with decreasing intensity, and the patient may become afebrile for a short period before experiencing another bout of fever. The spleen may be palpable by the second week of illness and, depending on the duration of illness, may become hugely enlarged (Fig. 251-3). Hepatomegaly (usually moderate in degree) soon follows. Lymphadenopathy is common in most endemic regions of the world except the Indian subcontinent, where it is rare. Patients lose weight and feel weak, and the skin gradually develops dark discoloration due to hyperpigmentation that is most

1	regions of the world except the Indian subcontinent, where it is rare. Patients lose weight and feel weak, and the skin gradually develops dark discoloration due to hyperpigmentation that is most easily seen in brown-skinned individuals. In advanced illness, hypoalbuminemia may manifest as pedal edema and ascites. Anemia appears early and may become severe enough to cause congestive heart failure. Epistaxis, retinal hemorrhages, and gastrointestinal bleeding are associated with thrombocytopenia. Secondary infections such as measles, pneumonia, tuberculosis, bacillary or amebic dysentery, and gastroenteritis are common. Herpes zoster, chickenpox, boils in the skin, and scabies may also occur. Untreated, the disease is fatal in most patients, including 100% of those with HIV co-infection.

1	Leukopenia and anemia occur early and are followed by thrombocytopenia. There is a marked polyclonal increase in serum immunoglobulins. Serum levels of hepatic aminotransferases are raised in a significant proportion of patients, and serum bilirubin levels are elevated occasionally. Renal dysfunction is uncommon. Laboratory Diagnosis Demonstration of amastigotes in smears of tissue aspirates is the gold standard for the diagnosis of VL (Fig. 251-1). The FIGuRE 251-2 Worldwide distribution of human leishmaniasis. CL, cutaneous leishmaniasis; VL, visceral leishmaniasis. FIGuRE 251-3 A patient with visceral leishmaniasis has a hugely enlarged spleen visible through the surface of the abdomen. Splenomegaly is the most important feature of visceral leishmaniasis.

1	FIGuRE 251-3 A patient with visceral leishmaniasis has a hugely enlarged spleen visible through the surface of the abdomen. Splenomegaly is the most important feature of visceral leishmaniasis. sensitivity of splenic smears is >95%, whereas smears of bone marrow (60–85%) and lymph node aspirates (50%) are less sensitive. Culture of tissue aspirates increases sensitivity. Splenic aspiration is invasive and may be dangerous in untrained hands. Several serologic techniques are currently used to detect antibodies to Leishmania. An enzyme-linked immunosorbent assay (ELISA) and the indirect immunofluorescent antibody test (IFAT) are used in sophisticated laboratories.

1	In the field, however, a rapid immunochromatographic test based on the detection of antibodies to a recombinant antigen (rK39) consisting of 39 amino acids conserved in the kinesin region of L. infantum is used worldwide. The test requires only a drop of fingerprick blood or serum, and the result can be read within 15 min. Except in East Africa (where both its sensitivity and its specificity are lower), the sensitivity of the rK39 rapid diagnostic test (RDT) in immunocompetent individuals is ~98% and its specificity is 90%. In Sudan, an RDT based on a new synthetic polyprotein, rK28, was more sensitive (96.8%) and specific (96.2%) than rK39-based RDTs. Qualitative detection of leishmanial nucleic acid by polymerase chain reaction (PCR) and quantitative detection by real-time PCR are confined to specialized laboratories and have yet to be used for routine diagnosis of VL in endemic areas. PCR can distinguish among the major species of Leishmania infecting humans.

1	Differential Diagnosis VL is easily mistaken for malaria. Other febrile illnesses that may mimic VL include typhoid fever, tuberculosis, brucellosis, schistosomiasis, and histoplasmosis. Splenomegaly due to portal hypertension, chronic myeloid leukemia, tropical splenomegaly syndrome, and (in Africa) schistosomiasis may also be confused with VL. Fever with neutropenia or pancytopenia in patients from an endemic region strongly suggests a diagnosis of VL; hypergammaglobulinemia in patients with long-standing illness strengthens the diagnosis. In nonendemic countries, a careful travel history is essential when any patient presents with fever.

1	Severe anemia should be corrected by blood transfusion, and other comorbid conditions should be managed promptly. Treatment of VL is complex because the optimal drug, dosage, and duration vary with the endemic region. Despite completing recommended treatment, some patients experience relapse (most often within 6 months), and prolonged follow-up is recommended. A pentavalent antimonial is the drug of choice in most endemic regions of the world, but there is widespread resistance to antimony in the Indian state of Bihar, where either amphotericin B (AmB) deoxycholate or miltefosine is preferred. Dose requirements for AmB are lower in India than in the Americas, Africa, or the Mediterranean region. In Mediterranean countries, where cost is seldom an issue, liposomal AmB is the drug of choice. In immunocompetent patients, relapses are uncommon with AmB in its deoxycholate and lipid formulations. Antileishmanial therapy has recently evolved as new drugs and delivery systems have become

1	In immunocompetent patients, relapses are uncommon with AmB in its deoxycholate and lipid formulations. Antileishmanial therapy has recently evolved as new drugs and delivery systems have become available and resistance to antimonial compounds has emerged.

1	Except for AmB (deoxycholate and lipid formulations), antileishmanial drugs are available in the United States only from the Centers for Disease Control and Prevention.

1	Two pentavalent antimonial (SbV) preparations are available: sodium stibogluconate (100 mg of SbV/mL) and meglumine antimoniate (85 mg of SbV/mL). The daily dose is 20 mg/kg by IV infusion or IM injection, and therapy continues for 28–30 days. Cure rates exceed 90% in Africa, the Americas, and most of the Old World but are <50% in Bihar, India, as a result of resistance. Adverse reactions to SbV treatment are common and include arthralgia, myalgia, and elevated serum levels of aminotransferases. Electrocardiographic changes are common. Concave ST-segment elevation is not significant, but prolongation of QTc to >0.5 s may herald ventricular arrhythmia and sudden death. Chemical pancreatitis is common but usually does not require discontinuation of treatment; severe clinical pancreatitis occurs in immunosuppressed patients.

1	AmB is currently used as a first-line drug in Bihar, India. In other parts of the world, it is used when initial antimonial treatment fails. Conventional AmB deoxycholate is administered in doses of 0.75–1.0 mg/kg on alternate days for a total of 15 infusions. Fever with chills is an almost universal adverse reaction to AmB infusions. Nausea and vomiting are also common, as is thrombophlebitis in the infused veins. Acute toxicities can be minimized by administration of antihistamines like chlorpheniramine and antipyretic agents like acetaminophen before each infusion. AmB can cause renal dysfunction and hypokalemia and, in rare instances, elicits hypersensitivity reactions, bone marrow suppression, and myocarditis, all of which can be fatal.

1	The several lipid formulations of AmB developed to replace the deoxycholate formulation are preferentially taken up by reticuloendothelial tissues. Because very little free drug is available to cause toxicity, a large amount of drug can be delivered over a short period. Liposomal AmB has been used extensively to treat VL in all parts of the world. With a terminal half-life of ~150 h, liposomal AmB can be detected in the liver and spleen of animals for several weeks after a single dose. This is the only drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of VL; the regimen is 3 mg/kg daily on days 1–5, 14, and 21 (total dose, 21 mg/kg). However, the total-dose requirement for different regions of the world varies widely. In Asia, it is 10–15 mg/kg; in Africa, ~18 mg/kg; and in Mediterranean/American regions, ≥20 mg/kg. The daily dose is flexible (1–10 mg/kg). In a study in India, a single dose of 10 mg/kg cured infection in 96% of patients. Adverse effects of

1	mg/kg; and in Mediterranean/American regions, ≥20 mg/kg. The daily dose is flexible (1–10 mg/kg). In a study in India, a single dose of 10 mg/kg cured infection in 96% of patients. Adverse effects of liposomal AmB are usually mild and include infusion reactions, backache, and occasional reversible nephrotoxicity.

1	Paromomycin (aminosidine) is an aminocyclitol-aminoglycoside antibiotic with antileishmanial activity. Its mechanism of action against Leishmania has yet to be established. Paromomycin is approved in India for the treatment of VL at an IM dose of 11 mg of base/kg daily for 21 days; this regimen produces a cure rate of 95%. However, the optimal dose has not been established in other endemic regions. Paromomycin is a relatively safe drug, but some patients develop hepatotoxicity, reversible ototoxicity, and (in rare instances) nephrotoxicity and tetany.

1	Miltefosine, an alkylphosphocholine, is the first oral compound approved for the treatment of leishmaniasis. This drug has a long half-life (150–200 h); its mechanism of action is not clearly understood. The recommended therapeutic regimens for patients on the Indian subcontinent are a daily dose of 50 mg for 28 days for patients weighing <25 kg, a twice-daily dose of 50 mg for 28 days for patients weighing ≥25 kg, and 2.5 mg/kg for 28 days for children 2–11 years of age. These regimens have resulted in a cure rate of 94% in India. However, recent studies from the Indian subcontinent indicate a decline in the cure rate. Doses in other regions remain to be established. Because of its long half-life, miltefosine is prone to induce resistance in Leishmania. Its adverse effects include mild to moderate vomiting and diarrhea in 40% and 20% of patients, respectively; these reactions usually clear spontaneously after a few days. Rare cases of severe allergic dermatitis, hepatotoxicity, and

1	to moderate vomiting and diarrhea in 40% and 20% of patients, respectively; these reactions usually clear spontaneously after a few days. Rare cases of severe allergic dermatitis, hepatotoxicity, and nephrotoxicity have been reported. Because miltefosine is expensive and is associated with significant adverse events, it is best administered as directly observed therapy to ensure completion of treatment and to minimize the risk of resistance induction. Because miltefosine is teratogenic in rats, its use is contraindicated during pregnancy and (unless contraceptive measures are strictly adhered to for at least 3 months after treatment) in women of childbearing age.

1	Multidrug therapy for leishmaniasis is likely to be preferred in the future. Its potential advantages in VL include (1) better compliance and lower costs associated with shorter treatment courses and decreased hospitalization, (2) less toxicity due to lower drug doses and/or shorter duration of treatment, and (3) a reduced likelihood that resistance to either agent will develop. In a study from India, one dose of liposomal AmB (5 mg/kg) followed by miltefosine for 7 days, paromomycin for 10 days, or both miltefosine and paromomycin simultaneously for 10 days (in their usual daily doses) produced a cure rate of >97% (all three combinations). In Africa, a combination of SbV and paromomycin given for 17 days was as effective and safe as SbV given for 30 days.

1	Recovery from VL is quick. Within a week after the start of treatment, defervescence, regression of splenomegaly, weight gain, and recovery of hematologic parameters are evident. With effective treatment, no parasites are recovered from tissue aspirates at the posttreatment evaluation. Continued clinical improvement over 6–12 months is suggestive of cure. A small percentage of patients (with the exact figure depending on the regimen used) relapse but respond well to treatment with AmB deoxycholate or lipid formulations.

1	HIV/VL co-infection has been reported from 35 countries. Where both infections are endemic, VL behaves as an opportunistic infection in HIV-1-infected patients. HIV infection can increase the risk of developing VL by severalfold in endemic areas. Co-infected patients usually show the classic signs of VL, but they may present with atypical features due to loss of immunity and involvement of unusual anatomic locations, with, for example, infiltration of the skin, oral mucosa, gastrointestinal tract, lungs, and other organs. Serodiagnostic tests are commonly negative. Parasites can be recovered from unusual sites such as bronchoalveolar lavage fluid and buffy coat. Liposomal AmB is the drug of choice for HIV/VL co-infection—both for primary treatment and for treatment of relapses. A total dose of 40 mg/kg, administered as 4 mg/kg on days 1–5, 10, 17, 24, 31, and 38, is considered optimal and is approved by the FDA, but most patients experience a relapse within 1 year. Pentavalent

1	dose of 40 mg/kg, administered as 4 mg/kg on days 1–5, 10, 17, 24, 31, and 38, is considered optimal and is approved by the FDA, but most patients experience a relapse within 1 year. Pentavalent antimonials and AmB deoxycholate can also be used where liposomal AmB is not accessible. Reconstitution of patients’ immunity by antiretroviral therapy has led to a dramatic decline in the incidence of co-infection in the Mediterranean basin. In contrast, HIV/VL co-infection is on the rise in African and Asian countries. Ethiopia is worst affected: up to 30% of VL patients are also infected with HIV. Because restoration of the CD4+ T cell count to >200/μL does decrease the frequency of relapse, antiretroviral therapy (in addition to antileishmanial therapy) is a cornerstone for the management of HIV/VL co-infection. Secondary prophylaxis with lipo somal AmB has been shown to delay relapses, but no regimen has been established as optimal.

1	On the Indian subcontinent and in Sudan and other East African countries, 2–50% of patients develop skin lesions concurrent with or after the cure of VL. Most common are hypopigmented macules, papules, and/or nodules or diffuse infiltration of the skin and sometimes of the oral mucosa. The African and Indian diseases differ in several respects; important features of post–kala-azar dermal leishmaniasis (PKDL) in these two regions are listed in Table 251-2, and disease in an Indian patient is depicted in Fig. 251-4. In PKDL, parasites are scanty in hypopigmented macules but may be seen and cultured more easily from nodular lesions. Cellular infiltrates are heavier in nodules than in macules. Lymphocytes are the dominant cells; next most common are histiocytes and plasma TABLE 251-2 CLInICAL, EPIdEMIOLOgIC, And THERAPEuTIC FEATuREs OF POsT–KALA-AzAR dERMAL LEIsHMAnIAsIs: EAsT AFRICA And THE IndIAn suBCOnTInEnT

1	TABLE 251-2 CLInICAL, EPIdEMIOLOgIC, And THERAPEuTIC FEATuREs OF POsT–KALA-AzAR dERMAL LEIsHMAnIAsIs: EAsT AFRICA And THE IndIAn suBCOnTInEnT FIGuRE 251-4 Post–kala-azar dermal leishmaniasis in an Indian patient. Note nodules of varying size involving the entire face. The face is erythematous, and the surface of some of the large nodules is discolored.

1	cells. In about half of cases, epithelioid cells—scattered individually or forming compact granulomas—are seen. The diagnosis is based on history and clinical findings, but rK39 and other serologic tests are positive in most cases. Indian PKDL is treated with pentavalent antimonials for 60–120 days. This prolonged course frequently leads to noncompliance. The alternative—several courses of AmB spread over several months—is expensive and unacceptable for most patients. Oral miltefosine for 12 weeks, in the usual daily doses, cures most patients with Indian PKDL. In East Africa, a majority of patients experience spontaneous healing. In those with persistent lesions, the response to 60 days of treatment with a pentavalent antimonial is good.

1	CL can be broadly divided into Old World and New World forms. Old World CL caused by Leishmania tropica is anthroponotic and is confined to urban or suburban areas throughout its range. Zoonotic CL is most commonly due to Leishmania major, which naturally parasitizes several species of desert rodents that act as reservoirs over wide areas of the Middle East, Africa, and central Asia. Local outbreaks of human disease are common. Major outbreaks currently affect Afghanistan and Pakistan in association with refugees and population movement. CL is increasingly seen in tourists and military personnel on mission in CL-endemic regions of countries like Afghanistan and as a co-infection in HIV-infected patients. Leishmania aethiopica is restricted to the highlands of Ethiopia, Kenya, and Uganda, where it is a natural parasite of hyraxes. New World CL is mainly zoonotic and is most often caused by Leishmania mexicana, Leishmania (Viannia) panamensis, and Leishmania amazonensis. A wide range of

1	it is a natural parasite of hyraxes. New World CL is mainly zoonotic and is most often caused by Leishmania mexicana, Leishmania (Viannia) panamensis, and Leishmania amazonensis. A wide range of forest animals act as reservoirs, and human infections with these species are predominantly rural. As a result of extensive urbanization and deforestation, Leishmania (Viannia) braziliensis has adapted to peridomestic and urban animals, and CL due to this organism is increasingly becoming an urban disease. In the United States, a few cases of CL have been acquired indigenously in Texas.

1	Immunopathogenesis As in VL, the proinflammatory (TH1) response in CL may result in either asymptomatic or subclinical infection. However, in some individuals, the immune response causes ulcerative skin lesions, the majority of which heal spontaneously, leaving a scar. Healing is usually followed by immunity to reinfection with that species of parasite.

1	Clinical Features A few days or weeks after the bite of a sandfly, a papule develops and grows into a nodule that ulcerates over some weeks or months. The base of the ulcer, which is usually painless, consists of necrotic tissue and crusted serum, but secondary bacterial infection sometimes occurs. The margins of the ulcer are raised and indurated. Lesions may be single or multiple and vary in size from 0.5 to >3 cm (Fig. 251-5). Lymphatic spread and lymph gland involvement may be palpable and may precede the appearance of the skin lesion. There may be satellite lesions, especially in L. major and L. tropica infections. The lesions usually heal spontaneously after 2–15 months. Lesions due to L. major and L. mexicana tend to heal rapidly, whereas those due to

1	L. major and L. mexicana tend to heal rapidly, whereas those due to L. tropica and parasites of subspecies Viannia heal more slowly. In CL caused by L. tropica, new lesions—usually scaly, erythematous papules and nodules—develop in the center or periphery of a healed sore, a condition known as leishmaniasis recidivans. Lesions of L. mexicana and Leishmania (Viannia) peruviana closely resemble those seen in the Old World; however, lesions on the pinna of the ear are common, chronic, and destructive in the former infections. L. mexicana is responsible for chiclero’s ulcer, the so-called self-healing sore of Mexico. CL lesions on exposed body parts (e.g., the face and hands), permanent scar formation, and social stigmatization may cause anxiety and depression and may affect the quality of life of CL patients.

1	Differential Diagnosis A typical history (an insect bite followed by the events leading to ulceration) in a resident of or a traveler to an endemic focus strongly suggests CL. Cutaneous tuberculosis, fungal infections, leprosy, sarcoidosis, and malignant ulcers are sometimes mistaken for CL. Laboratory Diagnosis Demonstration of amastigotes in material obtained from a lesion remains the diagnostic gold standard. Microscopic examination of slit skin smears, aspirates, or biopsies of the lesion is used for detection of parasites. Culture of smear or biopsy material may yield Leishmania. PCR is more sensitive than microscopy and culture and allows identification of Leishmania to the species level. This information is important in decisions about therapy because responses to treatment can vary with the species. Isoenzyme profiling is used to determine species for research purposes.

1	FIGuRE 251-5 Cutaneous leishmaniasis in a Bolivian child. There are multiple ulcers resulting from several sandfly bites. The edges of the ulcers are raised. (Courtesy of P. Desjeux, Retired Medical Officer, World Health Organization, Geneva, Switzerland.) Although lesions heal spontaneously in the majority of cases, their spread or persistence indicates that treatment may be needed. One or a few small lesions due to “self-healing species” can be treated with topical agents. Systemic treatment is required for lesions over the face, hands, or joints; multiple lesions; large ulcers; lymphatic spread; New World CL with the potential for development of ML; and CL in HIV co-infected patients.

1	A pentavalent antimonial is the first-line drug for all forms of CL and is used in a dose of 20 mg/kg for 20 days, as for VL. The exceptions to this rule are CL caused by Leishmania (Viannia) guyanensis, for which pentamidine isethionate is the drug of choice (two injections of 4 mg of salt/kg separated by a 48-h interval), and CL due to

1	L. aethiopica, which responds to paromomycin (16 mg/kg daily) but not to antimonials. Relapses usually respond to a second course of treatment. In Peru, topical imiquimod (5–7.5%) plus parenteral antimonials have been shown to cure CL more rapidly than antimonials alone. Azoles and triazoles have been used with mixed responses in both Old and New World CL but have not been adequately assessed for this indication in clinical trials. In L. major infection, oral fluconazole (200 mg/d for 6 weeks) resulted in a higher rate of cure than placebo (79% vs 34%) and also cured infection faster. Adverse effects include gastrointestinal symptoms and hepatotoxicity. Ketoconazole (600 mg/d for 28 days) is 76–90% effective in CL due to L. (V.) panamensis and L. mexicana in Panama and Guatemala. Miltefosine has been used in CL in doses of 2.5 mg/kg for 28 days. This agent is effective against L. major infections. In Colombia, where CL is due to L. (V.) panamensis, miltefosine was also effective, with

1	has been used in CL in doses of 2.5 mg/kg for 28 days. This agent is effective against L. major infections. In Colombia, where CL is due to L. (V.) panamensis, miltefosine was also effective, with a cure rate of 91%. For L. (V.) braziliensis infections, however, the results with miltefosine are less consistent. In Brazil, miltefosine cured 71% of patients with L. (V.) guyanensis infection. Other drugs, such as dapsone, allopurinol, rifampin, azithromycin, and pentoxifylline, have been used either alone or in combinations, but most of the relevant studies have had design limitations that preclude meaningful conclusions.

1	Small lesions (≤3 cm in diameter) may conveniently be treated weekly until cure with an intralesional injection of a pentavalent antimonial at a dose adequate to blanch the lesion (0.2–2.0 mL). An ointment containing 15% paromomycin sulfate, either alone or with 0.5% gentamicin or 12% methylbenzonium chloride, cured 70–82% of lesions due to L. major in 20 days and may be suitable for lesions caused by other species. Heat therapy with an FDA-approved radio-frequency generator and cryotherapy with liquid nitrogen have also been used successfully.

1	Diffuse Cutaneous Leishmaniasis (DCL) DCL is a rare form of leishmaniasis caused by L. amazonensis and L. mexicana in South and Central America and by L. aethiopica in Ethiopia and Kenya. DCL is characterized by the lack of a cell-mediated immune response to the parasite, the uncontrolled multiplication of which thus continues unabated. The DTH response does not develop, and lymphocytes do not respond to leishmanial antigens in vitro. DCL patients have a polarized immune response with high levels of immunosuppressive cytokines, including IL-10, transforming growth factor (TGF) β, and IL-4, and low concentrations of IFN-γ. Profound immunosuppression leads to widespread cutaneous disease. Lesions may initially be confined to the face or a limb but spread over months or years to other areas of the skin. They may be symmetrically or asymmetrically distributed and include papules, nodules, plaques, and areas of diffuse infiltration. These lesions do not ulcerate. The overlying skin is

1	of the skin. They may be symmetrically or asymmetrically distributed and include papules, nodules, plaques, and areas of diffuse infiltration. These lesions do not ulcerate. The overlying skin is usually erythematous in pale-skinned patients. The lesions are teeming with parasites, which are therefore easy to recover. DCL does not heal spontaneously and is difficult to treat. If relapse and drug resistance are to be prevented, treatment should be continued for some time after lesions have healed and parasites can no longer be isolated. In the New World, repeated 20-day courses of pentavalent antimonials are given, with an intervening drug-free period of 10 days. Miltefosine has been used for several months with a good initial response. Combinations should be tried. 1393 In Ethiopia, a combination of paromomycin (14 mg/kg per day) and sodium stibogluconate (10 mg/kg per day) is effective.

1	The subgenus Viannia is widespread from the Amazon basin to Paraguay and Costa Rica and is responsible for deep sores and for ML (Table 251-1). In L. (V.) braziliensis infections, cutaneous lesions may be simultaneously accompanied by mucosal spread of the disease or followed by spread years later. ML is typically caused by L. (V.) braziliensis and rarely by L. amazonensis, L. (V.) guyanensis, and L. (V.) panamensis. Young men with chronic lesions of CL are at particular risk. Overall, ~3% of infected persons develop ML. Not every patient with ML has a history of prior CL. ML is almost entirely confined to the Americas. In rare cases, ML may also be caused by Old World species like L. major, L. infantum (L. chagasi), or L. donovani.

1	Immunopathogenesis and Clinical Features The immune response is polarized toward a TH1 response, with marked increases of IFN-γ and TNF-α and varying levels of TH2 cytokines (IL-10 and TGF-β). Patients have a stronger DTH response with ML than with CL, and their peripheral-blood mononuclear cells respond strongly to leishmanial antigens. The parasite spreads via the lymphatics or the bloodstream to mucosal tissues of the upper respiratory tract. Intense inflammation leads to destruction, and severe disability ensues. Lesions in or around the nose or mouth (espundia; Fig. 251-6) are the typical presentation of ML. Patients usually provide a history of self-healed CL preceding ML by 1–5 years. Typically, ML presents as nasal stuffiness and bleed ing followed by destruction of nasal cartilage, perforation of the nasal septum, and collapse of the nasal bridge. Subsequent involvement of the pharynx and larynx leads to difficulty in swallowing and phonation.

1	FIGuRE 251-6 Mucosal leishmaniasis in a Brazilian patient. There is extensive inflammation around the nose and mouth, destruction of the nasal mucosa, ulceration of the upper lip and nose, and destruction of the nasal septum. (Courtesy of R. Dietz, Universidade Federal do Espírito Santo, Vitória, Brazil.) in the New World. Chagas disease and African Trypanosomiasis Louis V. Kirchhoff, Anis Rassi Jr. Although the genus Trypanosoma contains many species of protozo-ans, only T. cruzi, T. brucei gambiense, and T. brucei rhodesiense cause 252 1394 The lips, cheeks, and soft palate may also be affected. Secondary bacterial infection is common, and aspiration pneumonia may be fatal. Despite the high degree of TH1 immunity and the strong DTH response, ML does not heal spontaneously.

1	Laboratory Diagnosis Tissue biopsy is essential for identification of parasites, but the rate of detection is poor unless PCR techniques are used. The strongly positive DTH response fails to distinguish between past and present infection.

1	The regimen of choice is a pentavalent antimonial agent administered at a dose of 20 mg of SbV/kg for 30 days. Patients with ML require long-term follow-up with repeated oropharyngeal and nasal examination. With failure of therapy or relapse, patients may receive another course of an antimonial but then become unresponsive, presumably because of resistance in the parasite. In this situation, AmB should be used. An AmB deoxycholate dose totaling 25–45 mg/kg is appropriate. There are no controlled trials of liposomal AmB, but administration of 2–3 mg/kg for 20 days is considered adequate. Miltefosine (2.5 mg/kg for 28 days) cured 71% of ML patients in Bolivia. The more extensive the disease, the worse the prognosis; thus prompt, effective treatment and regular follow-up are essential.

1	No vaccine is available for any form of leishmaniasis. Inoculation with live L. major (“leishmanization”) is practiced in Iran. Anthroponotic leishmaniasis is controlled by case finding, treatment, and vector control with insecticide-impregnated bed nets and curtains and residual insecticide spraying. Control of zoonotic leishmaniasis is more difficult. Use of insecticide-impregnated collars for dogs, treatment of infected domestic dogs, and culling of street dogs are measures that have been used with uncertain efficacy to prevent transmission of

1	L. infantum. In Brazil, a canine vaccine has been found to promote a decrease in the human and canine incidence of zoonotic VL. Two vaccines, Leishmune and Leish-Tec®, are licensed in Brazil; Leishmune provides significant protection to vaccinated dogs. CaniLeish® is the first licensed canine vaccine developed in Europe. Personal prophylaxis with bed nets and repellants may reduce the risk of CL infections disease in humans. T. cruzi is the etiologic agent of Chagas disease in the Americas; T. b. gambiense and T. b. rhodesiense cause African trypanosomiasis.

1	Chagas disease, or American trypanosomiasis, is a zoonosis caused by the protozoan parasite T. cruzi. Acute Chagas disease is usually a mild febrile illness that results from initial infection with the organism. After spontaneous resolution of the acute illness, most infected persons remain for life in the indeterminate phase of chronic Chagas disease, which is characterized by subpatent parasitemia, easily detectable IgG antibodies to T. cruzi, and an absence of associated signs and symptoms. In 10–30% of chronically infected patients, cardiac and/or gastrointestinal symptoms develop that can lead to serious morbidity and even death.

1	In 10–30% of chronically infected patients, cardiac and/or gastrointestinal symptoms develop that can lead to serious morbidity and even death. T. cruzi is transmitted among its mammalian hosts by hematophagous triatomine insects, often called reduviid bugs. The insects become infected by sucking blood from animals or humans with circulating parasites. Ingested organisms multiply in the gut of the triatomines, and infective forms are discharged with the feces at the time of subsequent blood meals. Transmission to a second vertebrate host occurs when breaks in the skin, mucous membranes, or conjunctivae become contaminated with bug feces that contain infective parasites. T. cruzi can also be transmitted by transfusion of blood donated by infected persons, by organ transplantation, from mother to unborn child, by ingestion of contaminated food or drink, and in laboratory accidents.

1	Initial infection at the site of parasite entry is characterized by local histologic changes that include the presence of parasites within leukocytes and cells of subcutaneous tissues and the development of interstitial edema, lymphocytic infiltration, and reactive hyperplasia of adjacent lymph nodes. After dissemination of the organisms through the lymphatics and the bloodstream, primarily muscles (including the myocardium) (Fig. 252-1) and ganglion cells may become heavily parasitized. The characteristic pseudocysts present in sections of infected tissues are intracellular aggregates of multiplying parasites.

1	In persons with chronic T. cruzi infections who develop related clinical manifestations, the heart is the organ most commonly affected. Changes include thinning of the ventricular walls, biventricular enlargement, apical aneurysms, and mural thrombi. Widespread lymphocytic infiltration, diffuse interstitial fibrosis, and atrophy of myocardial cells are often apparent. Although parasites are difficult to find in myocardial tissue by conventional histologic methods, more sensitive techniques of parasite detection, such as immunohistochemistry and polymerase chain reaction (PCR), have more frequently demonstrated T. cruzi antigens and parasite DNA in chronic lesions. Conduction-system abnormalities often affect the right branch and the left anterior branch of the bundle of His. In chronic Chagas disease of the gastrointestinal tract (megadisease), the esophagus and colon may exhibit varying degrees of dilation. On microscopic examination, focal inflammatory lesions with lymphocytic

1	Chagas disease of the gastrointestinal tract (megadisease), the esophagus and colon may exhibit varying degrees of dilation. On microscopic examination, focal inflammatory lesions with lymphocytic infiltration are seen, and the number of neurons in the myenteric plexus may be markedly reduced. Accumulating evidence implicates the persistence of parasites and the accompanying chronic inflammation—rather than autoimmune mechanisms—as the basis for the pathology in patients with chronic

1	T. cruzi infection. FIGuRE 252-1 Trypanosoma cruzi in the heart muscle of a child who died of acute Chagas myocarditis. An infected myocyte containing several dozen T. cruzi amastigotes is in the center of the field (hematoxylin and eosin, 900 ×). T. cruzi is found only in the Americas. Wild and domestic mammals harboring T. cruzi and infected triatomines are found in spotty distributions from the southern United States to southern Argentina. Humans become involved in the cycle of transmission when infected vectors take up residence in the primitive wood, adobe, and stone houses common in much of Latin America. Thus human T. cruzi infection is a health problem primarily among the poor in rural areas of Mexico and Central and South America. Most new

1	T. cruzi infections in rural settings occur in children, but the incidence is unknown because most cases go undiagnosed. Historically, transfusion-associated transmission of T. cruzi was a serious public health problem in many endemic countries. Transmission by this route has been largely eliminated, however, as effective programs for serologic screening of donated blood have been implemented. Several dozen patients with HIV and chronic T. cruzi infections who underwent acute recrudescence of the latter have been described. These patients generally presented with T. cruzi brain abscesses, a manifestation of the illness that does not occur in immunocompetent persons. Currently, it is estimated that 8 million people are chronically infected with T. cruzi and that 14,000 deaths due to the illness occur each year. The resulting morbidity and mortality make Chagas disease the most important parasitic disease burden in Latin America.

1	In recent years, the rate of T. cruzi transmission has decreased markedly in several endemic countries as a result of successful programs involving vector control, screening of donated blood, and education of at-risk populations. A major program, which began in 1991 in the “southern cone” nations of South America (Uruguay, Paraguay, Bolivia, Brazil, Chile, and Argentina), has provided the framework for much of this progress. Uruguay and Chile were certified free of transmission by the main domiciliary vector species (Triatoma infestans) in the late 1990s, and Brazil was declared transmission-free in 2006. Transmission has been reduced markedly in Argentina as well. Similar control programs have been initiated in the countries of northern South America and in the Central American nations.

1	Acute Chagas disease is rare in the United States, where 22 cases of autochthonous transmission and seven instances of transmission by blood transfusion have been reported. Moreover, T. cruzi was transmitted to five recipients of organs from three T. cruzi–infected donors, two of whom became infected through cardiac transplants. Acute Chagas disease has been reported in only one tourist returning to the United States from Latin America, although three such instances have been reported in Europe as well as one in Canada. In contrast, the prevalence of chronic T. cruzi infections in the United States has increased considerably in recent years. An estimated 23 million immigrants from Chagas-endemic countries currently live in the United States, ~17 million of whom are Mexicans. The total number of

1	T. cruzi–infected persons living in the United States is estimated to be 300,000. Screening of the U.S. blood supply for T. cruzi infection began in January 2007. The overall prevalence of T. cruzi infection among donors is ~1 in 13,300, and to date nearly 3000 infected donors have been identified and deferred permanently (see “Diagnosis,” below).

1	The first signs of acute Chagas disease develop at least 1 week after invasion by the parasites. When the organisms enter through a break in the skin, an indurated area of erythema and swelling (the chagoma), accompanied by local lymphadenopathy, may appear. Romaña sign— the classic finding in acute Chagas disease, which consists of unilateral painless edema of the palpebrae and periocular tissues—can result when the conjunctiva is the portal of entry. These initial local signs may be followed by malaise, fever, anorexia, and edema of the face and lower extremities. Generalized lymphadenopathy and hepatosplenomegaly may develop. Severe myocarditis develops rarely; most deaths in acute Chagas disease are due to heart failure. Neurologic signs are not common, but meningoencephalitis occurs occasionally, especially in children <2 years old. Usually within 4–8 weeks, acute signs and symptoms resolve spontaneously in virtually all patients, with commencement of the asymptomatic or

1	occurs occasionally, especially in children <2 years old. Usually within 4–8 weeks, acute signs and symptoms resolve spontaneously in virtually all patients, with commencement of the asymptomatic or indeterminate form of chronic T. cruzi infection.

1	Symptomatic chronic Chagas disease becomes apparent years 1395 or even decades after the initial infection. The heart is commonly involved, and symptoms are caused by rhythm disturbances, segmental or dilated cardiomyopathy, and thromboembolism. Right bundle branch block is a common electrocardiographic abnormality, but other types of intraventricular and atrioventricular blocks, premature ventricular contractions, and tachyand bradyarrhythmias occur frequently. Cardiomyopathy often results in biventricular heart failure, with a predominance of right-sided failure at advanced stages. Embolization of mural thrombi to the brain or other areas may take place. Sudden death is the main cause of death in Chagas heart disease; congestive heart failure and stroke are next most common. Patients with megaesophagus suffer from dysphagia, odynophagia, chest pain, and regurgitation. Aspiration can occur (especially during sleep) in patients with severe esophageal dysfunction, and repeated episodes

1	megaesophagus suffer from dysphagia, odynophagia, chest pain, and regurgitation. Aspiration can occur (especially during sleep) in patients with severe esophageal dysfunction, and repeated episodes of aspiration pneumonitis are common. Weight loss, cachexia, and pulmonary infection can result in death. Patients with megacolon are plagued by abdominal pain and chronic constipation, which predisposes to fecaloma formation. Advanced megacolon can cause obstruction, volvulus, septicemia, and death.

1	The diagnosis of acute Chagas disease requires the detection of parasites. Microscopic examination of fresh anticoagulated blood or the buffy coat is the simplest way to see the motile organisms. Parasites also can be seen in Giemsa-stained thin and thick blood smears. Microhematocrit tubes containing acridine orange as a stain can be used for the same purpose. When used repeatedly by experienced personnel, all of these methods yield positive results in a high proportion of cases of acute Chagas disease. Serologic testing does not play a major role in diagnosing acute Chagas disease. PCR assays often give positive results in infected patients in whom traditional parasitologic tests are negative, including infants with congenital Chagas disease.

1	Chronic Chagas disease is diagnosed by the detection of specific IgG antibodies that bind to T. cruzi antigens. Demonstration of the parasite is not of primary importance. In Latin America, ~30 assays are commercially available, including several based on recombinant antigens. Although these tests usually show good sensitivity and reasonable specificity, false-positive reactions may occur—typically with samples from patients who have other infectious and parasitic diseases or autoimmune disorders. In addition, confirmatory testing has presented a persistent challenge. For these reasons, the World Health Organization recommends that specimens be tested in at least two assays and that well-characterized positive and negative comparison samples be included in each run. The Chagas radioimmune precipitation assay (RIPA), a highly sensitive and specific confirmatory method for detecting antibodies to T. cruzi, is approved under the Clinical Laboratory Improvement Amendment and available in

1	precipitation assay (RIPA), a highly sensitive and specific confirmatory method for detecting antibodies to T. cruzi, is approved under the Clinical Laboratory Improvement Amendment and available in the laboratory of one of the authors (L.V.K.). In 2006, the U.S. Food and Drug Administration (FDA) approved a test to screen blood and organ donors for T. cruzi infection (Ortho T. cruzi ELISA Test System; Ortho-Clinical Diagnostics, Raritan, NJ). Since January 2007, the vast majority of U.S. blood donors have been screened, and positive units have undergone confirmatory testing with the Chagas RIPA. A second test for donor screening was approved by the FDA in 2010 (Abbott PRISM® Chagas Assay; Abbott Laboratories, Abbott Park, IL), as was an enzyme strip assay (Abbott ESA Chagas) in 2011. The use of PCR assays to detect T. cruzi DNA in chronically infected persons has been studied extensively; unfortunately, the sensitivity of this approach has not been shown to be reliably greater than

1	use of PCR assays to detect T. cruzi DNA in chronically infected persons has been studied extensively; unfortunately, the sensitivity of this approach has not been shown to be reliably greater than that of serology.

1	Therapy for Chagas disease is still unsatisfactory. For many years now, only two drugs—nifurtimox and benznidazole—have been available for this purpose. Regrettably, both drugs lack efficacy and may cause bothersome side effects. In acute Chagas disease, nifurtimox markedly reduces the duration of symptoms and parasitemia and decreases the mortality rate.

1	1396 Nevertheless, limited studies have shown that only ~70% of acute infections are cured by a full course of treatment. Common adverse effects of nifurtimox include anorexia, nausea, vomiting, weight loss, and abdominal pain. Neurologic reactions to the drug may include restlessness, disorientation, insomnia, twitching, paresthesia, polyneuritis, and seizures. These symptoms usually disappear when the dosage is reduced or treatment is discontinued. The recommended daily dosage is 8–10 mg/kg for adults, 12.5–15 mg/kg for adolescents, and 15–20 mg/kg for children 1–10 years of age. The drug should be given orally in four divided doses each day, and therapy should be continued for 90–120 days. Nifurtimox is available from the Drug Service of the Centers for Disease Control and Prevention (CDC) in Atlanta (telephone number, 404-639-3670). The efficacy of benznidazole is similar or even superior to that of nifurtimox. A cure rate of >90% among congenitally infected infants treated before

1	in Atlanta (telephone number, 404-639-3670). The efficacy of benznidazole is similar or even superior to that of nifurtimox. A cure rate of >90% among congenitally infected infants treated before their first birthday has been reported. Adverse effects include rash, peripheral neuropathy, and rare instances of granulocytopenia. The recommended oral dosage is 5 mg/kg per day for 60 days for adults and 5–10 mg/kg per day for 60 days for children, with administration of two or three divided doses. Benznidazole is generally considered the drug of choice in Latin America. The question of whether adults in the indeterminate or chronic symptomatic phase of Chagas disease should be treated with nifurtimox or benznidazole has been debated for years. The fact that cure rates in persons with long-established chronic infection are notably inferior to those in patients with acute or recent chronic infection is central to this controversy. No convincing evidence from randomized controlled trials

1	chronic infection are notably inferior to those in patients with acute or recent chronic infection is central to this controversy. No convincing evidence from randomized controlled trials indicates that nifurtimox or benznidazole treatment of adults in the indeterminate or chronic symptomatic phase reduces either the appearance or progression of symptoms or mortality rates. On the basis of results of some observational studies, a panel of experts convened by the CDC in 2006 recommended that adults <50 years old with presumably long-standing indeterminate T. cruzi infections— or even with mild to moderate disease—be offered treatment. A large randomized clinical trial (the BENEFIT multicenter trial) designed to assess the parasitologic and clinical efficacy of benznidazole in 2856 adults (18–75 years old) with chronic Chagas heart disease (without advanced lesions) is being performed in Brazil, Argentina, Colombia, Bolivia, and El Salvador, but results will not be available until 2015.

1	years old) with chronic Chagas heart disease (without advanced lesions) is being performed in Brazil, Argentina, Colombia, Bolivia, and El Salvador, but results will not be available until 2015. In contrast, randomized studies have shown that treatment of children is useful, and the current consensus of Latin American authorities and the CDC panel of experts is that all

1	T. cruzi–infected persons up to 18 years old and all adults known to have become infected recently should be given benznidazole or nifurtimox. The usefulness of antifungal azoles for the treatment of Chagas disease has been studied in laboratory animals and to a lesser extent in humans. To date, none of these drugs has exhibited a level of anti–T. cruzi activity that would justify its use in humans. Several newer drugs in this class have shown promise in animal studies and are currently being evaluated in phase 2 clinical trials.

1	Patients who develop cardiac and/or gastrointestinal disease in association with T. cruzi infection should be referred to appropriate subspecialists for further evaluation and treatment. Pacemakers can be useful in patients with ominous arrhythmias. The usefulness of implantable cardioverter defibrillators in persons with Chagas heart disease has not been established and currently is being studied in a prospective randomized trial. Cardiac transplantation is an option for patients with end-stage chagasic cardiomyopathy; more than 150 such transplantations have been done in Brazil and the United States. The survival rate among Chagas disease cardiac transplant recipients seems to be higher than that among persons receiving cardiac transplants for other reasons. This better outcome may be due to the fact that lesions are limited to the heart in most patients with symptomatic chronic Chagas disease.

1	Because drug therapy has limitations and vaccines are not available, the control of T. cruzi transmission in endemic countries depends on the reduction of domiciliary vector populations by spraying of insecticides, improvements in housing, and education of at-risk persons. As noted above, these measures, coupled with serologic screening of blood donors, have markedly reduced transmission of the parasite in many endemic countries. Tourists would be wise to avoid sleeping in dilapidated houses in rural areas of endemic countries. Mosquito nets and insect repellent can provide additional protection.

1	In view of the possibly serious consequences of chronic T. cruzi infection, it would be prudent for all immigrants from endemic regions who are living in the United States to be tested for evidence of infection. Identification of persons harboring the parasite would permit periodic electrocardiographic monitoring, which is important to detect incipient heart disease and guide further diagnostic studies and treatment. The possibility of congenital transmission is yet another justification for screening. T. cruzi is classified as a Risk Group 2 agent in the United States and a Risk Group 3 agent in some European countries. Laboratory staff should work with the parasite or infected vectors and mammals at containment levels consistent with the risk group designation in their areas.

1	Sleeping sickness, or human African trypanosomiasis (HAT), is caused by flagellated protozoan parasites that belong to the T. brucei complex and are transmitted to humans by tsetse flies. In untreated patients, the trypanosomes first cause a febrile illness that is followed months or years later by progressive neurologic impairment and death.

1	The East African (rhodesiense) and the West African (gambiense) forms of sleeping sickness are caused, respectively, by two trypanosome subspecies: T. b. rhodesiense and T. b. gambiense. These subspecies are morphologically indistinguishable but cause illnesses that are epidemiologically and clinically distinct (Table 252-1). The parasites are transmitted by blood-sucking tsetse flies of the genus Glossina. The insects acquire the infection when they ingest blood from infected mammalian hosts. After many cycles of multiplication in the midgut of the vector, the parasites migrate to the salivary glands. Their transmission takes place when they are inoculated into a mammalian host during a subsequent blood meal. The injected trypanosomes multiply in the blood (Fig. 252-2) and other extracellular spaces and evade immune destruction for long periods by undergoing antigenic variation, a process driven by gene switching in which the antigenic structure of the organisms’ surface coat of

1	spaces and evade immune destruction for long periods by undergoing antigenic variation, a process driven by gene switching in which the antigenic structure of the organisms’ surface coat of glycoproteins changes periodically.

1	West African East African Point of Comparison (gambiense) (rhodesiense) Abbreviation: CNS, central nervous system. Source: Reprinted with permission from LV Kirchhoff, in GL Mandell et al (eds): Principles and Practice of Infectious Diseases, 7th ed. Philadelphia, Elsevier Churchill Livingstone, 2010. FIGuRE 252-2 Trypanosoma brucei rhodesiense parasites in rat blood. The slender parasite is thought to be the form that multiplies in mammalian hosts, whereas the stumpy forms are nondividing and are capable of infecting insect vectors (Giemsa, 1200 × ). (Courtesy of Dr. G. A. Cook, Madison, WI; with permission.)

1	A self-limited inflammatory lesion (trypanosomal chancre) may appear a week or so after the bite of an infected tsetse fly. A systemic febrile illness then evolves as the parasites are disseminated through the lymphatics and bloodstream. Systemic HAT without central nervous system (CNS) involvement is generally referred to as stage 1 disease. In this stage, widespread lymphadenopathy and splenomegaly reflect marked lymphocytic and histiocytic proliferation and invasion of morular cells, which are plasmacytes that may be involved in the production of IgM. Endarteritis, with perivascular infiltration of both parasites and lymphocytes, may develop in lymph nodes and the spleen. Myocarditis develops frequently in patients with stage 1 disease and is especially common in T. b. rhodesiense infections.

1	Hematologic manifestations that accompany stage 1 HAT include moderate leukocytosis, thrombocytopenia, and anemia. High levels of immunoglobulins, consisting primarily of polyclonal IgM, are a constant feature, and heterophile antibodies, antibodies to DNA, and rheumatoid factor are often detected. High levels of antigen–antibody complexes may play a role in the tissue damage and increased vascular permeability that facilitate dissemination of the parasites. Stage 2 disease involves invasion of the CNS. The presence of trypanosomes in perivascular areas is accompanied by intense infiltration of mononuclear cells. Abnormalities in cerebrospinal fluid (CSF) include increased pressure, elevated total protein concentration, and pleocytosis. In addition, trypanosomes are frequently found in CSF.

1	The trypanosomes that cause sleeping sickness are found only in sub-Saharan Africa. After its near-eradication in the mid 1960s, sleeping sickness underwent a resurgence in the 1990s, primarily in Uganda, Sudan, the Central African Republic, the Democratic Republic of the Congo, and Angola. A subsequent increase in control activities reduced the incidence in many endemic areas, however, and in 2009 fewer than 10,000 cases were reported to the World Health Organization. Although underreporting is a persistent problem, the level of control achieved to date was the basis for convening a panel of experts in 2009 to develop a vision for eradication of HAT.

1	Humans are the only reservoir of T. b. gambiense, which occurs in widely distributed foci in tropical rain forests of Central and West Africa. Gambiense trypanosomiasis is primarily a problem in rural populations; tourists rarely become infected. Trypanotolerant antelope species in savanna and woodland areas of Central and East Africa are the principal reservoir of T. b. rhodesiense. Cattle can also be infected with this and other trypanosome species but generally succumb to the infection. Because risk results from contact with tsetse flies that feed on wild animals, humans acquire T. b. rhodesiense infection only inciden-1397 tally, usually while visiting or working in areas where infected game and vectors are present. Roughly one or two imported cases of HAT acquired in East African parks are reported to the CDC each year.

1	A painful trypanosomal chancre appears in some patients at the site of inoculation of the parasite. Hematogenous and lymphatic dissemination (stage 1 disease) is marked by the onset of fever. Typically, bouts of high temperatures lasting several days are separated by afebrile periods. Lymphadenopathy is prominent in T. b. gambiense trypanosomiasis. The nodes are discrete, movable, rubbery, and nontender. Cervical nodes are often visible, and enlargement of the nodes of the posterior cervical triangle, or Winterbottom’s sign, is a classic finding. Pruritus and maculopapular rashes are common. Inconstant findings include malaise, headache, arthralgias, weight loss, edema, hepatosplenomegaly, and tachycardia. The differential diagnosis of stage 1 HAT includes many diseases that are common in the tropics and are associated with fevers. HIV infection, malaria, and typhoid fever are common in populations at risk for HAT and need to be considered.

1	CNS invasion (stage 2 disease) is characterized by the insidious development of protean neurologic manifestations that are accompanied by progressive abnormalities in the CSF. A picture of progressive indifference and daytime somnolence develops (hence the designation “sleeping sickness”), sometimes alternating with restlessness and insomnia at night. A listless gaze accompanies a loss of spontaneity, and speech may become halting and indistinct. Extrapyramidal signs may include choreiform movements, tremors, and fasciculations. Ataxia is frequent, and the patient may appear to have Parkinson’s disease, with a shuffling gait, hypertonia, and tremors. In the final phase, progressive neurologic impairment ends in coma and death.

1	The most striking difference between the gambiense and rhodesiense forms of HAT is that the latter illness tends to follow a more acute course. Typically, in tourists with T. b. rhodesiense disease, systemic signs of infection, such as fever, malaise, and headache, appear before the end of the trip or shortly after the return home. Persistent tachycar dia unrelated to fever is common early in the course of T. b. rhodesiense trypanosomiasis, and death may result from arrhythmias and congestive heart failure before CNS disease develops. In general, untreated T. b. rhodesiense trypanosomiasis leads to death in a matter of weeks to months, often without a clear distinction between the hemolymphatic and CNS stages. In contrast, T. b. gambiense disease can smolder for many months or even for years.

1	A definitive diagnosis of HAT requires detection of the parasite. If a chancre is present, fluid should be expressed and examined directly by light microscopy for the highly motile trypanosomes. The fluid also should be fixed and stained with Giemsa. Material obtained by needle aspiration of lymph nodes early in the illness should be examined similarly. Examination of wet preparations and Giemsa-stained thin and thick films of serial blood samples is also useful. If parasites are not seen initially in blood, efforts should be made to concentrate the organisms, which can be done in microhematocrit tubes containing acridine orange. Alternatively, the buffy coat from 10–15 mL of anti-coagulated blood can be examined directly under a microscope. The likelihood of finding parasites in blood is higher in stage 1 than in stage 2 disease and in patients infected with T. b. rhodesiense rather than T. b. gambiense. Trypanosomes may also be seen in material aspirated from the bone marrow; the

1	is higher in stage 1 than in stage 2 disease and in patients infected with T. b. rhodesiense rather than T. b. gambiense. Trypanosomes may also be seen in material aspirated from the bone marrow; the aspirate can be inoculated into liquid culture medium, as can blood, buffy coat, lymph node aspirates, and CSF. It is essential to examine CSF from all patients in whom HAT is suspected. Abnormalities in the CSF that may be associated with stage 2 disease include an increase in the CSF cell count as well as increases in opening pressure and in levels of total protein and IgM. Trypanosomes may be seen in the sediment of centrifuged CSF. Any CSF abnormality in a patient in whom trypanosomes have been found at other sites must be viewed as pathognomonic for CNS involvement and thus must prompt specific treatment for CNS disease. In patients 1398 with CSF pleocytosis in whom parasites are not found, tuberculous meningitis and HIV-associated CNS infections such as cryptococcosis should be

1	specific treatment for CNS disease. In patients 1398 with CSF pleocytosis in whom parasites are not found, tuberculous meningitis and HIV-associated CNS infections such as cryptococcosis should be considered in the differential diagnosis. A number of serologic assays, such as the card agglutination test for trypanosomes (CATT) for T. b. gambiense, are available to aid in the diagnosis of HAT. Their ease of use makes them valuable for epidemiologic surveys, but their variable sensitivity and specificity mandate that decisions about treatment be based on demonstration of the parasite. Accurate PCR assays for detecting African trypanosomes in humans have been developed, but the lack of the necessary technical and human resources in most endemic areas stands in the way of their widespread use.

1	The drugs used for treatment of HAT are suramin, pentamidine, eflornithine, and the organic arsenical melarsoprol. In the United States, these drugs can be obtained from the CDC. Therapy for HAT must be individualized on the basis of the infecting subspecies, the presence or absence of CNS disease, adverse reactions, and occasionally drug resistance. The choices of drugs for the treatment of HAT are summarized in Table 252-2.

1	Suramin is highly effective against stage 1 rhodesiense HAT. However, it can cause serious adverse effects and must be administered under the close supervision of a physician. A 100to 200-mg IV test dose should be given to detect hypersensitivity. The dosage for adults is 20 mg/kg on days 1, 5, 12, 18, and 26. The drug is given by slow IV infusion of a freshly prepared 10% aqueous solution. Approximately 1 patient in 20,000 has an immediate, severe, and potentially fatal reaction to the drug, developing nausea, vomiting, shock, and seizures. Less severe reactions include fever, photo-phobia, pruritus, arthralgias, and skin eruptions. Renal damage is the most common important adverse effect of suramin. Transient proteinuria often appears during treatment. A urinalysis should be done before each dose, and treatment should be discontinued if proteinuria increases or if casts and red cells appear in the sediment. Suramin should not be given to patients with renal insufficiency.

1	Pentamidine is the first-line drug for treatment of stage 1 gambiense HAT. The dose for both adults and children is 4 mg/kg per day, given IM or IV for 7–10 days. Frequent, immediate adverse reactions include nausea, vomiting, tachycardia, and hypotension. These reactions are usually transient and do not warrant cessation of therapy. Other adverse reactions include nephrotoxicity, abnormal liver function tests, neutropenia, rashes, hypoglycemia, and sterile abscesses. Suramin is an alternative agent for stage 1 T. b. gambiense disease.

1	Eflornithine is highly effective for treatment of both stages of gambiense sleeping sickness. In the trials on which the FDA based its approval, this agent cured >90% of 600 patients with stage 2 disease. The recommended treatment schedule is 400 mg/kg per day, given IV in four divided doses, for 2 weeks. Adverse reactions include diarrhea, anemia, thrombocytopenia, seizures, and hearing loss. The high dosage and duration of therapy required are disadvantages that make widespread use of eflornithine difficult. A randomized trial Abbreviations: CSF, cerebrospinal fluid; NECT, nifurtimox-eflornithine combination therapy.

1	Abbreviations: CSF, cerebrospinal fluid; NECT, nifurtimox-eflornithine combination therapy. comparing the standard eflornithine regimen (400 mg/kg per day infused over 6 h for 14 days) with nifurtimox-eflornithine combination therapy (NECT; oral nifurtimox, 15 mg/kg per day in three divided doses, plus IV eflornithine, 200 mg/kg per day in two divided doses, both for 7 days) in adults with stage 2 gambiense HAT showed improved efficacy and reduced adverse effects with combination therapy, making this drug suitable for first-line use.

1	The arsenical melarsoprol is the drug of choice for the treatment of rhodesiense HAT with CNS involvement and is an alternative agent for stage 2 gambiense disease. The “short course” of melarsoprol that is currently recommended has been shown to be noninferior to the decades-old treatment course for T. b. rhodesiense, which was administered over several weeks and was much more toxic. The short-course regimen consists of 10 daily doses of 2.2 mg/kg IV, each given with prednisolone (1 mg/kg).

1	Melarsoprol is highly toxic and should be administered with great care. As noted, all patients receiving melarsoprol should be given prednisolone to reduce the likelihood of drug-induced encephalopathy. Without prednisolone prophylaxis, the incidence of reactive encephalopathy has been as high as 18% in some series. Clinical manifestations of reactive encephalopathy include high fever, headache, tremor, impaired speech, seizures, and even coma and death. Treatment with melarsoprol should be discontinued at the first sign of encephalopathy but may be restarted cautiously at lower doses a few days after signs have resolved. Extravasation of the drug results in intense local reactions. Vomiting, abdominal pain, nephrotoxicity, and myocardial damage can occur.

1	HAT poses complex public-health and epizootic problems in Africa. Considerable progress has been made in many areas through control programs that focus on eradication of vectors and drug treatment of infected humans. People can reduce their risk of acquiring trypanosomiasis by avoiding areas known to harbor infected insects, by wearing protective clothing, and by using insect repellent. Chemoprophylaxis is not recommended, and no vaccine is available to prevent transmission of the parasites. Kami Kim, Lloyd H. Kasper

1	Kami Kim, Lloyd H. Kasper Toxoplasmosis is caused by infection with the obligate intracellular parasite Toxoplasma gondii. Acute infection acquired after birth may be asymptomatic but is thought to result in the lifelong chronic persistence of cysts in the host’s tissues. In both acute and chronic toxoplasmosis, the parasite is responsible for clinically evident disease, including lymphadenopathy, encephalitis, myocarditis, and pneumonitis. Congenital toxoplasmosis is an infection of newborns that results from the transplacental passage of parasites from an infected mother to the fetus. These infants may be asymptomatic at birth, but most later manifest a wide range of signs and symptoms, including chorioretinitis, strabismus, epilepsy, and psychomotor retardation. In immunocompetent individuals, toxoplasmosis can also present as acute disease (typically chorioretinitis) associated with foodor waterborne sources.

1	T. gondii is an intracellular coccidian that infects both birds and mammals. There are two distinct stages in the life cycle of T. gondii that yield transmissible forms of the parasite (Fig. 253-1). In the asexual stages, tissue cysts that contain bradyzoites or sporulated oocysts that contain sporozoites are ingested by an intermediate host (e.g., a human, mouse, sheep, pig, or bird). The cyst is rapidly digested by the Intermediate host: birds, mammals, humans Bradyzoites encyst within the CNS and muscle of the infected host. Oocysts are excreted in cat feces. Contaminated soil is ingested by birds, mammals, and humans.

1	Oocysts are excreted in cat feces. Contaminated soil is ingested by birds, mammals, and humans. T. gondii infects a wide range of mammals and birds. Its seroprevalence depends on the locale and the age of the population. Generally, hot arid climatic conditions are associated with a low prevalence of infection. In the United States and most European countries, the seroprevalence increases with age and exposure. For example, in the United States, 5–30% of individuals 10–19 years old and 10–67% of those >50 years old have serologic evidence of expo- the host, replicate, and cause tissue sure. In Central America, France, Turkey, damage. and Brazil, the seroprevalence is higher. Because of increased awareness of food-borne infections, the prevalence of seropositivity has decreased worldwide.

1	TRANSMISSION Toxoplasmic Oral Transmission Most cases of human encephalitis Toxoplasma infection are thought to be acquired by the oral route. Transmission can be attributable to ingestion of FIGuRE 253-1 Life cycle ofToxoplasma gondii. The cat is the definitive host in which the sexual sporulated oocysts from contaminated phase of the cycle is completed. Oocysts shed in cat feces can infect a wide range of animals, soil, food, or water. During acute feline including birds, rodents, grazing domestic animals, and humans. The bradyzoites found in the infection, a cat may excrete as many as muscle of food animals may infect humans who eat insufficiently cooked meat products, particularly 100 million parasites per day. These very lamb and pork. Although human disease can take many forms, congenital infection and encepha-stable sporozoite-containing oocysts are litis from reactivation of latent infection in the brains of immunosuppressed persons are the most highly infectious and may

1	congenital infection and encepha-stable sporozoite-containing oocysts are litis from reactivation of latent infection in the brains of immunosuppressed persons are the most highly infectious and may remain viable important manifestations. CNS, central nervous system. (Courtesy of Dominique Buzoni-Gatel, Institut for many years in soil or water. Humans

1	Pasteur, Paris; with permission.) acidic-pH gastric secretions. Bradyzoites or sporozoites are released, enter the small-intestinal epithelium, and transform into rapidly dividing tachyzoites. The tachyzoites can infect and replicate in all mammalian cells except red blood cells. The parasite actively penetrates the cell and forms a parasitophorous vacuole. Parasite replication continues within the vacuole. After the parasites reach a critical mass, intracellular signaling within the host and the parasite, including calcium fluxes, result in parasite egress from the vacuole. The host cell is destroyed, and the released tachyzoites infect adjoining cells. The tachyzoite replication cycle within an infected organ causes cytopathology. Most tachyzoites are eliminated by the host’s humoral and cell-mediated immune responses. Tissue cysts containing many bradyzoites develop 7–10 days after systemic tachyzoite infection. These tissue cysts occur in various host organs but persist

1	humoral and cell-mediated immune responses. Tissue cysts containing many bradyzoites develop 7–10 days after systemic tachyzoite infection. These tissue cysts occur in various host organs but persist principally within the central nervous system (CNS) and muscle. The development of this chronic stage completes the asexual portion of the life cycle. Active infection in the immunocompromised host is most likely to be due to the spontaneous release of encysted parasites that undergo rapid transformation into tachyzoites within the CNS and are not contained by the immune system.

1	The sexual stage in the life cycle takes place in the cat (the definitive host). The parasite’s sexual phase is defined by the formation of oocysts within the feline host. This enteroepithelial cycle begins with the ingestion of the bradyzoite tissue cysts and, after several intermediate stages, culminates in the production of gametes. Gamete fusion produces a zygote, which envelops itself in a rigid wall and is secreted in the feces as an unsporulated oocyst. After 2–3 days of exposure to air at ambient temperature, the noninfectious oocyst sporulates to produce eight sporozoite progeny. The sporulated oocyst can be ingested by an intermediate host, such as a person emptying a cat’s litter box or a pig rummaging in a barnyard. It is in the intermediate host that

1	T. gondii completes its life cycle. Sporulated oocysts are environmentally hardy and very infectious; they are thought to be sources of waterborne outbreaks such as those reported in Victoria (British Columbia, Canada) and in South America. ies to the oocyst/sporozoite. Children and adults also can acquire infection from tissue cysts containing bradyzoites. The ingestion of a single cyst is all that is required for human infection. Undercooking or insufficient freezing of meat is an important source of infection in the developed world. In the United States, lamb products and pork products may yield evidence of cysts that contain bradyzoites, but the overall prevalence of T. gondii has been gradually decreasing. The incidence in beef is much lower— perhaps as low as 1%. Direct ingestion of bradyzoite cysts in these various meat products leads to acute infection.

1	Transmission via Blood or Organs In addition to being transmitted orally, T. gondii can be transmitted directly from a seropositive donor to a seronegative recipient in a transplanted heart, heart-lung, kidney, liver, or pancreas. Viable parasites can be cultured from refrigerated anticoagulated blood, which may be a source of infection in individuals receiving blood transfusions. T. gondii reactivation has been reported in bone marrow, hematopoietic stem cell, and liver transplant recipients as well as in individuals with AIDS. Although antibody titers generally are not useful in monitoring T. gondii infection, individuals with higher antibody titers may be at relatively high risk for reactivation after hematopoietic stem cell transplantation; thus routine polymerase chain reaction (PCR) screening of blood from these patients may be in order. Finally, laboratory personnel can be infected after contact with contaminated needles or glassware or with infected tissue.

1	Transplacental Transmission On average, about one-third of all women who acquire infection with T. gondii during pregnancy transmit the parasite to the fetus; the remainder give birth to normal, uninfected babies. Of the various factors that influence fetal outcome, gestational age at the time of infection is the most critical (see below). Few data support a role for recrudescent maternal infection as the source of congenital disease, although rare cases of transmission by immunocompromised women (e.g., those infected with HIV or those receiving high-dose glucocorticoids) have been reported. Thus, women who are seropositive before pregnancy usually are protected against acute infection and do not give birth to congenitally infected neonates.

1	1400 The following general guidelines can be used to evaluate congenital infection. There is essentially no risk if the mother becomes infected ≥6 months before conception. If infection is acquired <6 months before conception, the likelihood of transplacental infection increases as the interval between infection and conception decreases. Women with documented acute toxoplasmosis should be counseled to use appropriate measures to prevent pregnancy for 6 months after infection. In pregnancy, if the mother becomes infected during the first trimester, the incidence of transplacental infection is lowest (~15%), but the disease in the neonate is most severe. If maternal infection occurs during the third trimester, the incidence of transplacental infection is greatest (65%), but the infant is usually asymptomatic at birth. Infected infants who are normal at birth may have a higher incidence of learning disabilities and chronic neurologic sequelae than uninfected children. Only a small

1	is usually asymptomatic at birth. Infected infants who are normal at birth may have a higher incidence of learning disabilities and chronic neurologic sequelae than uninfected children. Only a small proportion (20%) of women infected with T. gondii develop clinical signs of infection. Often the diagnosis is first appreciated when routine postconception serologic tests show evidence of specific antibody.

1	Upon the host’s ingestion of either tissue cysts containing bradyzoites or oocysts containing sporozoites, the parasites are released from the cysts by the digestive process. Bradyzoites are resistant to the effect of pepsin and invade the host’s gastrointestinal tract. Within enterocytes (or other gut-associated cells), the parasites undergo morphologic transformation, giving rise to invasive tachyzoites. These tachyzoites induce a parasite-specific secretory IgA response. From the gastrointestinal tract, parasites disseminate to a variety of organs, particularly lymphatic tissue, skeletal muscle, myocardium, retina, placenta, and the CNS. At these sites, the parasite infects host cells, replicates, and invades the adjoining cells. In this fashion, the hallmarks of the infection develop: cell death and focal necrosis surrounded by an acute inflammatory response.

1	In the immunocompetent host, both the humoral and the cellular immune responses control infection; parasite virulence and tissue tropism may be strain specific. Tachyzoites are sequestered by a variety of immune mechanisms, including induction of parasiticidal antibody, activation of macrophages with radical intermediates, production of interferon γ (IFN-γ), and stimulation of CD8+ cytotoxic T lymphocytes. These antigen-specific lymphocytes are capable of killing both extracellular parasites and target cells infected with parasites. As tachyzoites are cleared from the acutely infected host, tissue cysts containing bradyzoites begin to appear, usually within the CNS and the retina. Studies indicate that Toxoplasma secretes signaling molecules into infected host cells and that these molecules modulate host gene expression, host metabolism, and host immune response. While it was initially thought that cysts with bradyzoites are not eliminated by the immune system, recent studies in the

1	modulate host gene expression, host metabolism, and host immune response. While it was initially thought that cysts with bradyzoites are not eliminated by the immune system, recent studies in the murine model indicate that both CD8+ T cells and alternatively activated macrophages are able to kill cysts in vivo; some cysts persist, however, and the ability to eliminate cysts may depend on the genetic background of the infected host.

1	In the immunocompromised or fetal host, the immune factors necessary to control the spread of tachyzoite infection are lacking. This altered immune state allows the persistence of tachyzoites and gives rise to progressive focal destruction that results in organ failure (i.e., necrotizing encephalitis, pneumonia, and myocarditis).

1	It is thought that all infected individuals have persistent infection with cysts containing bradyzoites, but this lifelong infection usually remains subclinical. Although bradyzoites are in a slow metabolic phase, cysts do degenerate and rupture within the CNS. This degenerative process, with the development of new bradyzoite-containing cysts, is the most probable source of recrudescent infection in immunocompromised individuals and the most likely stimulus for the persistence of antibody titers in the immunocompetent host. Although the concept is controversial, the persistence of toxoplasmosis has been hypothesized to be a contributing factor to a variety of neuropsychiatric conditions, including schizophrenia and bipolar disease. In rodents, infection clearly has significant effects on behavior, increasing predation.

1	Cell death and focal necrosis due to replicating tachyzoites induce an intense mononuclear inflammatory response in any tissue or cell type infected. Tachyzoites rarely can be visualized by routine histopathologic staining of these inflammatory lesions. However, immunofluorescent staining with parasitic antigen–specific antibodies can reveal either the organism itself or evidence of antigen. In contrast to this inflammatory process caused by tachyzoites, bradyzoite-containing cysts cause inflammation only at the early stages of development, and even this inflammation may be a response to the presence of tachyzoite antigens. Once the cysts reach maturity, the inflammatory process can no longer be detected, and the cysts remain immunologically quiescent within the brain matrix until they rupture.

1	Lymph Nodes During acute infection, lymph node biopsy demonstrates characteristic findings, including follicular hyperplasia and irregular clusters of tissue macrophages with eosinophilic cytoplasm. Granulomas rarely are evident in these specimens. Although tachyzoites are not usually visible, they can be sought either by subinoculation of infected tissue into mice, with resultant disease, or by PCR. PCR amplification of DNA fragments of Toxoplasma genes is effective and sensitive in establishing lymph node infection by tachyzoites. Eyes In the eye, infiltrates of monocytes, lymphocytes, and plasma cells may produce unior multifocal lesions. Granulomatous lesions and chorioretinitis can be observed in the posterior chamber after acute necrotizing retinitis. Other ocular complications include iridocyclitis, cataracts, and glaucoma.

1	Central Nervous System During CNS involvement, both focal and diffuse meningoencephalitis can be documented, with evidence of necrosis and microglial nodules. Necrotizing encephalitis in patients without AIDS is characterized by small diffuse lesions with perivascular cuffing in contiguous areas. In the AIDS population, polymorphonuclear leukocytes may be present in addition to monocytes, lymphocytes, and plasma cells. Cysts containing bradyzoites frequently are found contiguous with the necrotic tissue border. As a consequence of combined antiretroviral therapy (cART) for AIDS, the incidence of toxoplasmosis has decreased in the developed world. Its incidence in under-resourced settings is not known.

1	Lungs and Heart Among patients with AIDS who die of toxoplasmosis, 40–70% have involvement of the lungs and heart. Interstitial pneumonitis can develop in neonates and immunocompromised patients. Thickened and edematous alveolar septa infiltrated with mononuclear and plasma cells are apparent. This inflammation may extend to the endothelial walls. Tachyzoites and bradyzoite-containing cysts have been observed within the alveolar membrane. Superimposed bronchopneumonia can be caused by other microbial agents. Cysts and aggregates of parasites in cardiac muscle tissue are evident in patients with AIDS who die of toxoplasmosis. Focal necrosis surrounded by inflammatory cells is associated with hyaline necrosis and disrupted myocardial cells. Pericarditis is associated with toxoplasmosis in some patients.

1	Gastrointestinal Tract Rare cases of human gastrointestinal tract infection with T. gondii have presented as ulcerations in the mucosa. Acute infection in certain strains of inbred mice (C57BL/6) results in lethal ileitis within 7–9 days. This inflammatory bowel disease has been recognized in several other mammalian species, including pigs and nonhuman primates. Although the association between human inflammatory bowel disease and either acute or recurrent Toxoplasma infection has not been established, studies have demonstrated recognition of the infection by human intestinal epithelial cells, as evidenced by mitogen-activated protein kinase phosphorylation, nuclear factor κB translocation, and interleukin (IL) 8 secretion.

1	Other Sites Pathologic changes during disseminated infection are similar to those described for the lymph nodes, eyes, and CNS. In patients with AIDS, the skeletal muscle, pancreas, stomach, and kidneys can be involved, with necrosis, invasion by inflammatory cells, and (rarely) tachyzoites detectable by routine staining. Large necrotic lesions may cause direct tissue destruction. In addition, secondary effects from acute infection of these various organs, including pancreatitis, myositis, and glomerulonephritis, have been reported.

1	Acute Toxoplasma infection evokes a cascade of protective immune responses in the immunocompetent host. Toxoplasma enters the host at the gut mucosal level and evokes a mucosal immune response that includes the production of antigen-specific secretory IgA. Titers of serum IgA antibody directed at p30 (SAG1) are a useful marker for congenital and acute toxoplasmosis. Milk-whey IgA from acutely infected mothers contains a high titer of antibody to T. gondii and can block infection of enterocytes in vitro. In mice, IgA intestinal secretions directed at the parasite are abundant and are associated with the induction of mucosal T cells.

1	Within the host, T. gondii rapidly induces detectable levels of both IgM and IgG serum antibodies. Monoclonal gammopathy of the IgG class can occur in congenitally infected infants. IgM levels may be increased in newborns with congenital infection. The polyclonal IgG antibodies evoked by infection are parasiticidal in vitro in the presence of serum complement and are the basis for the Sabin-Feldman dye test. However, cell-mediated immunity is the major protective response evoked by the parasite during host infection. Macrophages are activated after phagocytosis of antibody-opsonized parasites. This activation can lead to death of the parasite by either an oxygen-dependent or an oxygen-independent process. If the parasite is not phagocytosed and enters the macrophage by active penetration, it continues to replicate, and this replication may represent the mechanism for transport and dissemination to distant organs. Toxoplasma stimulates a robust IL-12 response by human dendritic cells.

1	it continues to replicate, and this replication may represent the mechanism for transport and dissemination to distant organs. Toxoplasma stimulates a robust IL-12 response by human dendritic cells. The requirement for costimulation via CD40/154 has been established. The CD4+ and CD8+ T cell responses are antigen-specific and further stimulate the production of a variety of important lymphokines that expand the T cell and natural killer cell repertoire. T. gondii is a potent inducer of a TH1 phenotype, with IL-12 and IFN-γ playing an essential role in the control of the parasites’ growth in the host. Regulation of the inflammatory response is at least partially under the control of a TH2 response that includes the production of IL-4 and IL-10 in seropositive individuals. Both asymptomatic patients and those with active infection may have a depressed CD4+-to-CD8+ ratio. This shift may be correlated with a disease syndrome but is not necessarily correlated with disease outcome. Human T

1	patients and those with active infection may have a depressed CD4+-to-CD8+ ratio. This shift may be correlated with a disease syndrome but is not necessarily correlated with disease outcome. Human T cell clones of both the CD4+ and the CD8+ phenotypes are cytolytic against parasite-infected macrophages. These T cell clones produce cytokines that are “microbistatic.” IL-18, IL-7, and IL-15 upregulate the production of IFN-γ and may be important during acute and chronic infection. The effect of IFN-γ may be paradoxical, with stimulation of a host down-regulatory response as well.

1	Although T. gondii infection is believed to be recrudescent in patients with AIDS or other immunocompromised states, antibody titers are not useful in establishing reactivation or in following the activity of infection. An absence of positive serologies suggests an alternative diagnosis, although AIDS patients may have borderline positive or low serologies. T cells from AIDS patients with reactivation of toxoplasmosis fail to secrete both IFN-γ and IL-2. This alteration in the production of these critical immune cytokines contributes to the persistence of infection. Toxoplasma infection frequently develops late in the course of AIDS, when the loss of T cell–dependent protective mechanisms, particularly CD8+ T cells, becomes most pronounced.

1	In persons whose immune systems are intact, acute toxoplasmosis is usually asymptomatic and self-limited. This condition can go unrecognized in 80–90% of adults and children with acquired infection. The asymptomatic nature of this infection makes diagnosis difficult in mothers infected during pregnancy. In contrast, the wide range of clinical manifestations in congenitally infected children includes severe neurologic complications such as hydrocephalus, microcephaly, mental retardation, and chorioretinitis. If prenatal infection is severe, multiorgan failure and subsequent intrauterine fetal death can occur. In children and adults, chronic infection can persist throughout life, 1401 with little consequence to the immunocompetent host.

1	Toxoplasmosis in Immunocompetent Patients The most common manifestation of acute toxoplasmosis is cervical lymphadenopathy. The nodes may be single or multiple, are usually nontender, are discrete, and vary in firmness. Lymphadenopathy also may be found in suboccipital, supraclavicular, inguinal, and mediastinal areas. Generalized lymphadenopathy occurs in 20–30% of symptomatic patients. Between 20% and 40% of patients with lymphadenopathy also have headache, malaise, fatigue, and fever (usually with a temperature of <40°C [<104°F]). A smaller proportion of symptomatic individuals have myalgia, sore throat, abdominal pain, maculopapular rash, meningoencephalitis, and confusion. Rare complications associated with infection in the normal immune host include pneumonia, myocarditis, encephalopathy, pericarditis, and polymyositis. Signs and symptoms associated with acute infection usually resolve within several weeks, although the lymphadenopathy may persist for some months. In one

1	pericarditis, and polymyositis. Signs and symptoms associated with acute infection usually resolve within several weeks, although the lymphadenopathy may persist for some months. In one epidemic, toxoplasmosis was diagnosed correctly in only 3 of the 25 patients who consulted physicians. If toxoplasmosis is considered in the differential diagnosis, routine laboratory and serologic screening should precede node biopsy.

1	It is now appreciated that genotypes of T. gondii prevalent in in North America or Europe. These genotypes may be associated with acute or recurrent ocular disease in immunocompetent individuals and have also been associated with pneumonitis and a fulminant sepsis picture in immunologically normal individuals. Thus a detailed history is critical for establishing a diagnosis.

1	The results of routine laboratory studies are usually unremarkable except for minimal lymphocytosis, an elevated erythrocyte sedimentation rate, and a nominal increase in serum aminotransferase levels. Evaluation of cerebrospinal fluid (CSF) in cases with evidence of encephalopathy or meningoencephalitis shows an elevation of intra-cranial pressure, mononuclear pleocytosis (10–50 cells/mL), a slight increase in protein concentration, and (occasionally) an increase in the gamma globulin level. PCR amplification of the Toxoplasma DNA target sequence in CSF may be beneficial. The CSF of chronically infected individuals is normal.

1	Infection of Immunocompromised Patients Patients with AIDS and those receiving immunosuppressive therapy for lymphoproliferative disorders are at greatest risk for developing acute toxoplasmosis. Toxoplasmosis has also been reported after treatment with antibodies to tumor necrosis factor. The infection may be due either to reactivation of latent infection or to acquisition of parasites from exogenous sources such as blood or transplanted organs. In individuals with AIDS, >95% of cases of Toxoplasma encephalitis (TE) are believed to be due to recrudescent infection. In most of these cases, encephalitis develops when the CD4+ T cell count falls below 100/µL. In immunocompromised hosts, the disease may be rapidly fatal if untreated. Thus, accurate diagnosis and initiation of appropriate therapy are necessary to prevent fulminant infection.

1	Toxoplasmosis is a principal opportunistic infection of the CNS in persons with AIDS. Although geographic origin may be related to frequency of infection, it has no correlation with the severity of disease in immunocompromised hosts. Individuals with AIDS who are seropositive for T. gondii are at high risk for encephalitis. Before the advent of current cART, about one-third of the 15–40% of adult AIDS patients in the United States who were latently infected with T. gondii developed TE. TE may still be a presenting infection in individuals who are unaware of their positive HIV status.

1	The signs and symptoms of acute toxoplasmosis in immunocompromised patients principally involve the CNS (Fig. 253-2). More than 50% of patients with clinical manifestations have intracerebral involvement. Clinical findings at presentation range from nonfocal to focal dysfunction. CNS findings include encephalopathy, meningoencephalitis, and mass lesions. Patients may present with altered mental status (75%), fever (10–72%), seizures (33%), headaches (56%), and focal neurologic findings (60%), including motor deficits, cranial nerve

1	FIGuRE 253-2 Toxoplasmic encephalitis in a 36-year-old patient with AIDS. The mul-tiple lesions are demonstrated by MRI scanning (T1-weighted with gadolinium enhance-ment). (Courtesy of Clifford Eskey, Dartmouth Hitchcock Medical Center, Hanover, NH; with permission.) palsies, movement disorders, dysmetria, visual-field loss, and aphasia. Patients who present with evidence of diffuse cortical dysfunction develop evidence of focal neurologic disease as infection progresses. This altered condition is due not only to the necrotizing encephalitis caused by direct invasion by the parasite but also to secondary effects, including vasculitis, edema, and hemorrhage. The onset of infection can range from an insidious process over several weeks to an acute presentation with fulminant focal deficits, including hemiparesis, hemiplegia, visual-field defects, localized headache, and focal seizures.

1	Although lesions can occur anywhere in the CNS, the areas most often involved appear to be the brainstem, basal ganglia, pituitary gland, and corticomedullary junction. Brainstem involvement gives rise to a variety of neurologic dysfunctions, including cranial nerve palsy, dysmetria, and ataxia. With basal ganglionic infection, patients may develop hydrocephalus, choreiform movements, and choreoathetosis. Toxoplasma usually causes encephalitis, and meningeal involvement is uncommon. CSF findings may be unremarkable or may include a modest increase in cell count and in protein—but not glucose—concentration. Nonetheless, the parasite may be detected by PCR in CSF from many patients with TE.

1	Cerebral toxoplasmosis must be differentiated from other opportunistic infections or tumors in the CNS of AIDS patients. The differential diagnosis includes herpes simplex encephalitis, cryptococcal meningitis, progressive multifocal leukoencephalopathy, and primary CNS lymphoma. Involvement of the pituitary gland can give rise to panhypopituitarism and hyponatremia from inappropriate secretion of vasopressin (antidiuretic hormone). HIV-associated neurocognitive disorder (HAND) may present as cognitive impairment, attention loss, and altered memory. Brain biopsy in patients who have been treated for TE but who continue to exhibit neurologic dysfunction often fails to identify organisms.

1	Autopsies of Toxoplasma-infected patients have demonstrated the involvement of multiple organs, including the lungs, gastrointestinal tract, pancreas, skin, eyes, heart, and liver. Toxoplasma pneumonia can be confused with Pneumocystis pneumonia (PcP). Respiratory involvement usually presents as dyspnea, fever, and a nonproductive cough and may rapidly progress to acute respiratory failure with hemoptysis, metabolic acidosis, hypotension, and (occasionally) disseminated intravascular coagulation. Histopathologic studies demonstrate necrosis and a mixed cellular infiltrate. The presence of organisms is a helpful diagnostic indicator, but organisms can also be found in healthy tissue. Infection of the heart is usually asymptomatic but can be associated with cardiac tamponade or biventricular failure. Infections of the gastrointestinal tract and the liver have been documented.

1	Congenital Toxoplasmosis Between 400 and 4000 infants born each year in the United States are affected by congenital toxoplasmosis. Acute infection in mothers acquiring T. gondii during pregnancy is usually asymptomatic; most such women are diagnosed via prenatal serologic screening. Infection of the placenta leads to hematogenous infection of the fetus. As gestation proceeds, the proportion of fetuses that become infected increases, but the clinical severity of the infection declines. Although infected children may initially be asymptomatic, the persistence of

1	T. gondii can result in reactivation and clinical disease—most frequently chorioretinitis—decades later. Factors associated with relatively severe disabilities include delays in diagnosis and in initiation of therapy, neonatal hypoxia and hypoglycemia, profound visual impairment (see “Ocular Infection,” below), uncorrected hydrocephalus, and increased intracranial pressure. If treated appropriately, upwards of 70% of children have normal developmental, neurologic, and ophthalmologic findings at follow-up evaluations. Treatment for 1 year with pyrimethamine, a sulfonamide, and folinic acid is tolerated with minimal toxicity (see “Treatment,” below). Ocular Infection Infection with T. gondii is estimated to cause 35% of all cases of chorioretinitis in the United States and

1	Europe. It was formerly thought that the majority of cases of ocular disease were due to congenital infection. New ocular toxoplasmosis in immunocompetent individuals occurs more commonly than was previously appreciated and has been associated with outbreaks in Victoria (British Columbia) and in South America. A variety of ocular manifestations are documented, including blurred vision, scotoma, photophobia, and eye pain. Macular involvement occurs, with loss of central vision, and nystagmus is secondary to poor fixation. Involvement of the extraocular muscles may lead to disorders of convergence and to strabismus. Ophthalmologic examination should be undertaken in newborns with suspected congenital infection. As the inflammation resolves, vision improves, but episodic flare-ups of chorioretinitis, which progressively destroy retinal tissue and lead to glaucoma, are common. The ophthalmologic examination reveals yellow-white, cotton-like patches with indistinct margins of hyperemia. As

1	which progressively destroy retinal tissue and lead to glaucoma, are common. The ophthalmologic examination reveals yellow-white, cotton-like patches with indistinct margins of hyperemia. As the lesions age, white plaques with distinct borders and black spots within the retinal pigment become more apparent. Lesions usually are located near the posterior pole of the retina; they may be single but are more commonly multiple. Congenital lesions may be unilateral or bilateral and show evidence of massive chorioretinal degeneration with extensive fibrosis. Surrounding these areas of involvement are a normal retina and vasculature. In patients with AIDS, retinal lesions are often large, with diffuse retinal necrosis, and include both free tachyzoites and cysts containing bradyzoites. Toxoplasmic chorioretinitis may be a prodrome to the development of encephalitis.

1	DIAGNOSIS Tissue and Body Fluids The differential diagnosis of acute toxoplasmosis can be made by appropriate culture, serologic testing, and PCR (Table 253-1). Although available only at specialized laboratories, the isolation of T. gondii from blood or other body fluids can be accomplished after subinoculation of the sample into the peritoneal cavity of mice. If no parasites are found in the mouse’s peritoneal fluid 6–10 days after inoculation, its anti-Toxoplasma serum titer can be evaluated 4–6 weeks after inoculation. Isolation of T. gondii from the patient’s body fluids reflects acute infection, whereas isolation from biopsied tissue is an indication only of the presence of tissue cysts and should not be misinterpreted as evidence of acute toxoplasmosis. Persistent parasitemia in patients with latent, asymptomatic infection is rare. Histologic examination of lymph nodes may suggest the characteristic changes described above. Demonstration of tachyzoites in lymph nodes

1	in patients with latent, asymptomatic infection is rare. Histologic examination of lymph nodes may suggest the characteristic changes described above. Demonstration of tachyzoites in lymph nodes establishes the diagnosis of acute toxoplasmosis. Like subinoculation into mice, histologic demonstration of cysts containing bradyzoites confirms prior infection with T. gondii but is nondiagnostic for acute infection.

1	Serology The procedures mentioned above have great diagnostic value but are limited by difficulties encountered either in the growth Abbreviations: CNS, central nervous system; PCR, polymerase chain reaction. Source: Adapted from JD Schwartzman: Toxoplasmosis, in Principles and Practice of Clinical Parasitology. Hoboken, Wiley, 2001. of parasites in vivo or in the identification of tachyzoites by histochemical methods. Serologic testing has become the routine method of diagnosis.

1	Diagnosis of acute infection with T. gondii can be established by detection of the simultaneous presence of IgG and IgM antibodies to Toxoplasma in serum. The presence of circulating IgA favors the diagnosis of an acute infection. The Sabin-Feldman dye test, the indirect fluorescent antibody test, and the enzyme-linked immunosorbent assay (ELISA) all satisfactorily measure circulating IgG antibody to Toxoplasma. Positive IgG titers (>1:10) can be detected as early as 2–3 weeks after infection. These titers usually peak at 6–8 weeks and decline slowly to a new baseline level that persists for life. Antibody avidity increases with time and can be useful in difficult cases during pregnancy for establishing when infection may have occurred. The serum IgM titer should be measured in concert with the IgG titer to better establish the time of infection; either the double-sandwich IgM-ELISA or the IgM-immunosorbent assay (IgM-ISAGA) should be used. Both assays are specific and sensitive, with

1	the IgG titer to better establish the time of infection; either the double-sandwich IgM-ELISA or the IgM-immunosorbent assay (IgM-ISAGA) should be used. Both assays are specific and sensitive, with fewer false-positive results than other commercial tests. The double-sandwich IgA-ELISA is more sensitive than the IgM-ELISA for detecting congenital infection in the fetus and newborn. Although a negative IgM result with a positive IgG titer indicates distant infection, IgM can persist for >1 year and should not necessarily be considered a reflection of acute disease. If acute toxoplasmosis is suspected, a more extensive panel of serologic tests can be performed. In the United States, testing is available at the Toxoplasma Serology Laboratory at Palo Alto Medical 1403 Foundation (http://www.pamf.org/serology/clinicianguide.html).

1	T. gondii in biologic samples independent of the serologic response. Results obtained with PCR have suggested high sensitivity, specificity, and clinical utility in the diagnosis of TE, and PCR technology may be becoming more readily available in resource-poor settings. Real-time PCR is a promising technique that can provide quantitative results. Isolates can be genotyped and polymorphic sequences can be obtained, with consequent identification of the precise strain. Molecular epidemiologic studies with polymorphic markers have been useful in correlating clinical signs and symptoms of disease with different T. gondii genotypes.

1	The Immunocompetent Adult or Child For the patient who presents with lymphadenopathy only, a positive IgM titer is an indication of acute infection—and an indication for therapy, if clinically warranted (see “Treatment,” below). The serum IgM titer should be determined again in 3 weeks. An elevation in the IgG titer without an increase in the IgM titer suggests that infection is present but is not acute. If there is a borderline increase in either IgG or IgM, the titers should be reassessed in 3–4 weeks.

1	The Immunocompromised Host A presumptive clinical diagnosis of TE in patients with AIDS is based on clinical presentation, history of exposure (as evidenced by positive serology), and radiologic evaluation. To detect latent infection with T. gondii, HIV-infected persons should be tested for IgG antibody to Toxoplasma soon after HIV infection is diagnosed. When these criteria are used, the predictive value is as high as 80%. More than 97% of patients with AIDS and toxoplasmosis have IgG antibody to T. gondii in serum. IgM serum antibody usually is not detectable. Although IgG titers do not correlate with active infection, serologic evidence of infection virtually always precedes the development of TE. It is therefore important to determine the Toxoplasma antibody status of all patients infected with HIV. Antibody titers may range from negative to 1:1024 in patients with AIDS and TE. Fewer than 3% of patients have no demonstrable antibody to Toxoplasma at diagnosis of TE.

1	Patients with TE have focal or multifocal abnormalities demonstrable by CT or MRI. Neuroradiologic evaluation should include double-dose contrast CT of the head. By this test, single and frequently multiple contrast-enhancing lesions (<2 cm) may be identified. MRI usually demonstrates multiple lesions located in both hemispheres, with the basal ganglia and corticomedullary junction most commonly involved; MRI provides a more sensitive evaluation of the efficacy of therapy than does CT (Fig. 253-2). These findings are not pathognomonic of Toxoplasma infection, because 40% of CNS lymphomas are multifocal and 50% are ring-enhancing. For both MRI and CT scans, the rate of false-negative results is ~10%. The finding of a single lesion on an MRI scan increases the likelihood of primary CNS lymphoma (in which solitary lesions are four times more likely than in TE) and strengthens the argument for the performance of a brain biopsy. A therapeutic trial of anti-Toxoplasma medications is

1	lymphoma (in which solitary lesions are four times more likely than in TE) and strengthens the argument for the performance of a brain biopsy. A therapeutic trial of anti-Toxoplasma medications is frequently used to assess the diagnosis. Treatment of presumptive TE with pyrimethamine plus sulfadiazine or clindamycin results in quantifiable clinical improvement in >50% of patients by day 3. Leucovorin is administered to prevent bone marrow toxicity. By day 7, >90% of treated patients show evidence of improvement. In contrast, if patients fail to respond or have lymphoma, clinical signs and symptoms worsen by day 7. Patients in this category require brain biopsy with or without a change in therapy. This procedure can now be performed by a stereotactic CT-guided method that reduces the potential for complications. Brain biopsy for T. gondii identifies organisms in 50–75% of cases. PCR amplification of CSF may also confirm toxoplasmosis or suggest alternative diagnoses (Table 253-1), such

1	for complications. Brain biopsy for T. gondii identifies organisms in 50–75% of cases. PCR amplification of CSF may also confirm toxoplasmosis or suggest alternative diagnoses (Table 253-1), such as progressive multifocal leukoencephalopathy (JC virus positive) or primary CNS lymphoma (Epstein-Barr virus positive).

1	CT and MRI with contrast are currently the standard diagnostic imaging tests for TE. As in other conditions, the radiologic response may lag behind the clinical response. Resolution of lesions may take 1404 from 3 weeks to 6 months. Some patients show clinical improvement despite worsening radiographic findings. Congenital Infection The issue of concern when a pregnant woman has evidence of recent T. gondii infection is whether the fetus is infected. PCR analysis of the amniotic fluid for the B1 gene of

1	Congenital Infection The issue of concern when a pregnant woman has evidence of recent T. gondii infection is whether the fetus is infected. PCR analysis of the amniotic fluid for the B1 gene of T. gondii has replaced fetal blood sampling. Serologic diagnosis is based on the persistence of IgG antibody or a positive IgM titer after the first week of life (a time frame that excludes placental leak). The IgG determination should be repeated every 2 months. An increase in IgM beyond the first week of life is indicative of acute infection. Up to 25% of infected newborns may be seronegative and have normal routine physical examinations. Thus assessment of the eye and the brain, with ophthalmologic testing, CSF evaluation, and radiologic studies, is important in establishing the diagnosis.

1	Ocular Toxoplasmosis The serum antibody titer may not correlate with the presence of active lesions in the fundus, particularly in cases of congenital toxoplasmosis. In general, a positive IgG titer (measured in undiluted serum if necessary) in conjunction with typical lesions establishes the diagnosis. Antibody production in ocular fluids, expressed in terms of the Goldmann-Witmer coefficient, has been described for diagnosis of ocular disease but does not always correlate with PCR results. If lesions are atypical and the serum antibody titer is in the low-positive range, the diagnosis is presumptive. The parasitic antigen–specific polyclonal IgG assay as well as parasite-specific PCR may facilitate the diagnosis. Accordingly, the clinical diagnosis of ocular toxoplasmosis can be supported in 60–90% of cases by laboratory tests, depending on the time of anterior chamber puncture and the panel of antibody analyses used. In the remaining cases, the possibility of a falsely negative

1	in 60–90% of cases by laboratory tests, depending on the time of anterior chamber puncture and the panel of antibody analyses used. In the remaining cases, the possibility of a falsely negative laboratory diagnosis or of an incorrect clinical diagnosis cannot be clarified further.

1	Congenitally infected neonates are treated with daily oral pyrimethamine (1 mg/kg) and sulfadiazine (100 mg/kg) with folinic acid for 1 year. Depending on the signs and symptoms, prednisone (1 mg/kg per day) may be used for congenital infection. Some U.S. states and some countries routinely screen pregnant women (France, Austria) and/or newborns (Denmark, Massachusetts). Management and treatment regimens vary with the country and the treatment center. Most experts use spiramycin to treat pregnant women who have acute toxoplasmosis early in pregnancy and use pyrimethamine/ sulfadiazine/folinic acid to treat women who seroconvert after 18 weeks of pregnancy or in cases of documented fetal infection. This treatment is somewhat controversial: clinical studies, which have included few untreated women, have not proven the efficacy of such therapy in preventing congenital toxoplasmosis. However, studies do suggest that treatment during pregnancy decreases the severity of infection. Many

1	women, have not proven the efficacy of such therapy in preventing congenital toxoplasmosis. However, studies do suggest that treatment during pregnancy decreases the severity of infection. Many women who are infected in the first trimester elect termination of pregnancy. Those who do not terminate pregnancy are offered prenatal antibiotic therapy to reduce the frequency and severity of Toxoplasma infection in the infant. The optimal duration of treatment for a child with asymptomatic congenital toxoplasmosis is not clear, although most clinicians in the United States would treat the child for 1 year in light of cohort investigations conducted by the National Collaborative Chicago-Based, Congenital Toxoplasmosis Study.

1	Immunologically competent adults and older children who have only lymphadenopathy do not require specific therapy unless they have persistent, severe symptoms. Patients with ocular toxoplasmosis are usually treated for 1 month with pyrimethamine plus either sulfadiazine or clindamycin and sometimes with prednisone. Treatment should be supervised by an ophthalmologist familiar with Toxoplasma disease. Ocular disease can be self-limited without treatment, but therapy is typically considered for lesions that are severe or close to the fovea or optic disc.

1	INFECTION IN IMMuNOCOMPROMISED PATIENTS Primary Prophylaxis Patients with AIDS should be treated for acute toxoplasmosis; in immunocompromised patients, toxoplasmosis is rapidly fatal if untreated. Before the introduction of cART, the median survival time was >1 year for patients who could tolerate treatment for TE. Despite their toxicity, the drugs used to treat TE were required for survival prior to cART. The incidence of TE has declined as the survival of patients with HIV infection has increased through the use of cART.

1	In Africa, many patients are diagnosed with HIV infection only after developing opportunistic infections. Hence, the optimal management of these opportunistic infections is important if the benefits of subsequent cART are to be realized. The incidence of TE in under-resourced settings is not clear because of a lack of facilities for serologic testing and imaging. AIDS patients who are seropositive for T. gondii and who have a CD4+ T lymphocyte count of <100/µL should receive prophylaxis against TE.

1	Of the currently available agents, trimethoprim-sulfamethoxazole (TMP-SMX) appears to be an effective alternative for treatment of TE in resource-poor settings where the preferred combination of pyrimethamine plus sulfadiazine is not available. The daily dose of TMP-SMX (one double-strength tablet) that is recommended as the preferred regimen for prophylaxis of PcP is effective against TE. If patients cannot tolerate TMP-SMX, the recommended alternative is dapsone-pyrimethamine, which likewise is effective against PcP. Atovaquone with or without pyrimethamine also can be considered. Prophylactic monotherapy with dapsone, pyrimethamine, azithromycin, clarithromycin, or aerosolized pentamidine is probably insufficient. AIDS patients who are seronegative for Toxoplasma and are not receiving prophylaxis for PcP should be retested for IgG antibody to Toxoplasma if their CD4+ T cell count drops to <100/µL. If seroconversion has taken place, then the patient should be given prophylaxis as

1	prophylaxis for PcP should be retested for IgG antibody to Toxoplasma if their CD4+ T cell count drops to <100/µL. If seroconversion has taken place, then the patient should be given prophylaxis as described above.

1	Discontinuing Primary Prophylaxis Current studies indicate that prophylaxis against TE can be discontinued in patients who have responded to cART and whose CD4+ T lymphocyte count has been >200/μL for 3 months. Although patients with CD4+ T lymphocyte counts of <100/μL are at greatest risk for developing TE, the risk that this condition will develop when the count has increased to 100–200/μL has not been established. Thus, prophylaxis should be discontinued when the count has increased to >200/μL. Discontinuation of therapy reduces the pill burden; the potential for drug toxicity, drug interaction, or selection of drug-resistant pathogens; and cost. Prophylaxis should be recommenced if the CD4+ T lymphocyte count again decreases to <100–200/μL.

1	Individuals who have completed initial therapy for TE should receive treatment indefinitely unless immune reconstitution, with a CD4+ T cell count of >200/μL, occurs as a consequence of cART. Combination therapy with pyrimethamine plus sulfadiazine plus leucovorin is effective for this purpose. An alternative to sulfadiazine in this regimen is clindamycin. Patients receiving secondary prophylaxis for TE are at low risk for recurrence when they have completed initial therapy for TE, remain asymptomatic, and have evidence of restored immune function. Individuals with HIV infection should have a CD4+ T lymphocyte count of >200/μL for at least 6 months after cART. This recommendation is consistent with more extensive data indicating the safety of discontinuing secondary prophylaxis for other opportunistic infections during advanced HIV disease. A repeat MRI brain scan is recommended. Secondary prophylaxis should be reintroduced if the CD4+ T lymphocyte count decreases to <200/μL.

1	All HIV-infected persons should be counseled regarding sources of Toxoplasma infection. The chances of primary infection with Toxoplasma can be reduced by not eating undercooked meat and Causes: Asymptomatic infection, acute diarrhea, or chronic diarrhea and malabsorption. Small bowel may demonstrate villous blunting, crypt hypertrophy, and mucosal inflammation. Excystation follows exposure to stomach acid and intestinal proteases, releasing trophozoite forms that multiply by binary fission and reside in the upper small bowel adherent to enterocytes.

1	Excystation follows exposure to stomach acid and intestinal proteases, releasing trophozoite forms that multiply by binary fission and reside in the upper small bowel adherent to enterocytes. Cysts and trophozoites are passed in the stool into the environment. Cysts are ingested (10-25 cysts) in contaminated water or food or by direct fecal-oral transmission (as in day-care centers). Encystation occurs under conditions of bile salt concentration changes and alkaline pH. Smooth-walled cysts can contain two trophozoites. Cysts can survive in the environment (up to several weeks in cold water). They may also infect nonhuman mammalian species. FIGuRE 254-1 Life cycle of Giardia. (Reprinted with permission from RL Guerrant et al [eds]: Tropical Infectious Diseases: Principles, Pathogens and Practice, 2nd ed, p 987. © 2006, with permission from Elsevier Science.)

1	Surface water, ranging from mountain streams to large municipal reservoirs, can become contaminated with fecally derived Giardia cysts. The efficacy of water as a means of transmission is enhanced by the small infectious inoculum of Giardia, the prolonged survival of cysts by avoiding oocyst-contaminated material (i.e., a cat’s litter box). Specifically, lamb, beef, and pork should be cooked to an internal temperature of 165°–170°F; from a more practical perspective, meat cooked until it is no longer pink inside usually satisfies this requirement. Hands should be washed thoroughly after work in the garden, and all fruits and vegetables should be washed. Ingestion of raw shellfish is a risk factor for toxoplasmosis, given that the filter-feeding mechanism of clams and mussels concentrates oocysts. FIGuRE 254-2 Flagellated, binucleate Giardia trophozoites. Protozoal Intestinal Infections and Trichomoniasis Peter F. Weller PROTOZOAL INFECTIONS GIARDIASIS 254

1	FIGuRE 254-2 Flagellated, binucleate Giardia trophozoites. Protozoal Intestinal Infections and Trichomoniasis Peter F. Weller PROTOZOAL INFECTIONS GIARDIASIS 254 If the patient owns a cat, the litter box should be cleaned or changed daily, preferably by an HIV-negative, nonpregnant person; alternatively, patients should wash their hands thoroughly after changing the litter box. Litter boxes should be changed daily if possible, as freshly excreted oocysts will not have sporulated and will not be infectious. Patients should be encouraged to keep their cats inside and not to adopt or handle stray cats. Cats should be fed only canned or dried commercial food or well-cooked table food, not raw or undercooked meats. Patients need not be advised to part with their cats or to have their cats tested for toxoplasmosis. Blood intended for transfusion into Toxoplasma-seronegative immunocompromised individuals should be screened for antibody to

1	T. gondii. Although such serologic screening is not routinely performed, seronegative women should be screened for evidence of infection several times during pregnancy if they are exposed to environmental conditions that put them at risk for infection with T. gondii. HIV-positive individuals should adhere closely to these preventive measures. Giardia intestinalis (also known as G. lamblia or G. duodena lis) is a cosmopolitan protozoal parasite that inhabits the small intestines of humans and other mammals. Giardiasis is one of the most common parasitic diseases in both developed and developing countries worldwide, causing both endemic and epidemic intestinal disease and diarrhea.

1	Life Cycle and Epidemiology (Fig. 254-1) Infection follows the ingestion of environmentally hardy cysts, which excyst in the small intestine, releasing flagellated trophozoites (Fig. 254-2) that multiply by binary fission. Giardia remains a pathogen of the proximal small bowel and does not disseminate hematogenously. Trophozoites remain free in the lumen or attach to the mucosal epithelium by means of a ventral sucking disk. As a trophozoite encounters altered conditions, it forms a morphologically distinct cyst, which is the stage of the parasite usually found in the feces. Trophozoites may be present and even predominate in loose or watery stools, but it is the resistant cyst that survives outside the body and is responsible for transmission. Cysts do not tolerate heating or desiccation, but they do remain viable for months in cold fresh water. The number of cysts excreted varies widely but can approach 107 per gram of stool.

1	Ingestion of as few as 10 cysts is sufficient to cause infection in humans. Because cysts are infectious when excreted, person-to-person transmission occurs where fecal hygiene is poor. Giardiasis is especially prevalent in day-care centers; person-to-person spread also takes place in other institutional settings with poor fecal hygiene and during anal-oral contact. If food is contaminated with Giardia cysts after cooking or preparation, food-borne transmission can occur. Waterborne transmission accounts for episodic infections (e.g., in campers and travelers) and for major epidemics in metropolitan areas. 1406 in cold water, and the resistance of cysts to killing by routine chlorination methods that are adequate for controlling bacteria. Viable cysts or filtration. In the United States, Giardia (like

1	In the United States, Giardia (like Cryptosporidium; see below) is a com-Isospora -+ + mon cause of waterborne epidemics of Cyclospora -+ + gastroenteritis. Giardia is common in develop-Microsporidia ing countries, and infections may be acquired stains, tissue by travelers.

1	There are several recognized genotypes or aO+P, ova and parasites. bNucleic acid amplification tests. assemblages of G. intestinalis. Human infections are due to assemblages A and B, whereas other assemblages are more common in other animals, including cats the stools, and other signs and symptoms of colitis are uncommon and and dogs. Like beavers from reservoirs implicated in epidemics, dogs suggest a different diagnosis or a concomitant illness. Symptoms tend and cats have been found to be infected with assemblages A and B, an to be intermittent yet recurring and gradually debilitating, in contrast observation suggesting that these animals might be sources of human with the acute disabling symptoms associated with many enteric bacteinfection. rial infections. Because of the less severe illness and the propensity for

1	Giardiasis, like cryptosporidiosis, creates a significant economic chronic infections, patients may seek medical advice late in the course burden because of the costs incurred in the installation of water of the illness; however, disease can be severe, resulting in malabsorpfiltration systems required to prevent waterborne epidemics, in the tion, weight loss, growth retardation, and dehydration. A number of management of epidemics that involve large communities, and in the extraintestinal manifestations have been described, such as urticaria, evaluation and treatment of endemic infections. anterior uveitis, and arthritis; whether these are caused by giardiasis or concomitant processes is unclear.

1	Pathophysiology The reasons that some, but not all, infected patients Giardiasis can be severe in patients with hypogammaglobulinemiadevelop clinical manifestations and the mechanisms by which Giardia and can complicate other preexisting intestinal diseases, such as thatcauses alterations in small-bowel function are largely unknown. occurring in cystic fibrosis. In patients with AIDS, Giardia can causeAlthough trophozoites adhere to the epithelium, they are not invasive enteric illness that is refractory to treatment.

1	but may elicit apoptosis of enterocytes, epithelial barrier dysfunction, and epithelial cell malabsorption and secretion. Consequent lactose Diagnosis (Table 254-1) Giardiasis is diagnosed by detection of intolerance and, in a minority of infected adults and children, signifi-parasite antigens in the feces, by identification of cysts in the feces cant malabsorption are clinical signs of the loss of brush-border enzyme or of trophozoites in the feces or small intestines, or by nucleic acid activities. In most infections, the morphology of the bowel is unaltered; amplification tests (NAATs). Cysts are oval, measure 8–12 μm × however, in chronically infected, symptomatic patients, the histopatho-7–10 μm, and characteristically contain four nuclei. Trophozoites are logic findings (including flattened villi) and the clinical manifestations pear-shaped, dorsally convex, flattened parasites with two nuclei and at times resemble those of tropical sprue and gluten-sensitive enteropa-four pairs

1	villi) and the clinical manifestations pear-shaped, dorsally convex, flattened parasites with two nuclei and at times resemble those of tropical sprue and gluten-sensitive enteropa-four pairs of flagella (Fig. 254-2). The diagnosis is sometimes difficult thy. The pathogenesis of diarrhea in giardiasis is not known. to establish. Direct examination of fresh or properly preserved stools as

1	The natural history of Giardia infection varies markedly. Infections well as concentration methods should be used. Because cyst excretion may be aborted, transient, recurrent, or chronic. G. intestinalis is variable and may be undetectable at times, repeated examination parasites vary genotypically, and such variations might contribute to of stool, sampling of duodenal fluid, and biopsy of the small intestine different courses of infection. Parasite as well as host factors may be may be required to detect the parasite. Tests for parasitic antigens in important in determining the course of infection and disease. Both stool are at least as sensitive and specific as good microscopic examinacellular and humoral responses develop in human infections, but their tions and are easier to perform. Newer NAATs are highly sensitive but precise roles in disease pathogenesis and/or control of infection are are not always available for clinical use at present. unknown. Because patients with

1	Newer NAATs are highly sensitive but precise roles in disease pathogenesis and/or control of infection are are not always available for clinical use at present. unknown. Because patients with hypogammaglobulinemia suffer from prolonged, severe infections that are poorly responsive to treatment, humoral immune responses appear to be important. The greater sus-

1	Cure rates with metronidazole (250 mg thrice daily for 5 days) are ceptibilities of the young than of the old and of newly exposed persons than of chronically exposed populations suggest that at least partial usually >90%. Tinidazole (2 g once by mouth) may be more effective than metronidazole. Albendazole (400 mg daily for 5–10 days) is as protective immunity may develop. effective as metronidazole and is associated with fewer side effects.

1	effective as metronidazole and is associated with fewer side effects. Clinical Manifestations Disease manifestations of giardiasis range from Nitazoxanide (500 mg twice daily for 3 days) is an alternative agent asymptomatic carriage to fulminant diarrhea and malabsorption. Most for treatment of giardiasis. Paromomycin, an oral aminoglycoside infected persons are asymptomatic, but in epidemics the proportion of that is not well absorbed, can be given to symptomatic pregnantsymptomatic cases may be higher. Symptoms may develop suddenly patients, although information is limited on how effectively thisor gradually. In persons with acute giardiasis, symptoms develop after agent eradicates infection.

1	an incubation period that lasts at least 5–6 days and usually 1–3 weeks. Almost all patients respond to therapy and are cured, although Prominent early symptoms include diarrhea, abdominal pain, bloating, some with chronic giardiasis experience delayed resolution of sympbelching, flatus, nausea, and vomiting. Although diarrhea is common, toms after eradication of Giardia. For many of the latter patients,upper intestinal manifestations such as nausea, vomiting, bloating, and residual symptoms probably reflect delayed regeneration of intesabdominal pain may predominate. The duration of acute giardiasis is tinal brush-border enzymes. Continued infection should be docuusually >1 week, although diarrhea often subsides. Individuals with mented by stool examinations before treatment is repeated. Patientschronic giardiasis may present with or without having experienced who remain infected after repeated treatments should be evaluated an antecedent acute symptomatic episode. Diarrhea is not

1	Patientschronic giardiasis may present with or without having experienced who remain infected after repeated treatments should be evaluated an antecedent acute symptomatic episode. Diarrhea is not necessarily for reinfection through family members, close personal contacts,prominent, but increased flatus, loose stools, sulfurous belching, and and environmental sources as well as for hypogammaglobulinemia.

1	(in some instances) weight loss occur. Symptoms may be continual or In cases refractory to multiple treatment courses, prolonged therapyepisodic and may persist for years. Some persons who have relatively with metronidazole (750 mg thrice daily for 21 days) or therapy withmild symptoms for long periods recognize the extent of their discom-varied combinations of multiple agents has been successful.fort only in retrospect. Fever, the presence of blood and/or mucus in Prevention Giardiasis can be prevented by consumption of uncontaminated food and water and by personal hygiene during the provision of care for infected children. Boiling or filtering potentially contaminated water prevents infection.

1	The coccidian parasite Cryptosporidium causes diarrheal dis ease that is self-limited in immunocompetent human hosts but can be severe in persons with AIDS or other forms of immunodeficiency. Two species of Cryptosporidium, C. hominis (especially in the United States, sub-Saharan Africa, and Asia) and C. parvum (in Europe), cause most human infections.

1	Life Cycle and Epidemiology Cryptosporidium species are widely distributed in the world. Cryptosporidiosis is acquired by the consumption of oocysts (50% infectious dose: ~132 C. parvum oocysts in nonimmune individuals), which excyst to liberate sporozoites that in turn enter and infect intestinal epithelial cells. The parasite’s further development involves both asexual and sexual cycles, which produce forms capable of infecting other epithelial cells and of generating oocysts that are passed in the feces. Cryptosporidium species infect a number of animals, and C. parvum can spread from infected animals to humans. Since oocysts are immediately infectious when passed in feces, person-to-person transmission takes place in day-care centers and among household contacts and medical providers. Waterborne transmission (especially that of C. hominis) accounts for infections in travelers and for common-source epidemics. Oocysts are quite hardy and resist killing by routine chlorination. Both

1	Waterborne transmission (especially that of C. hominis) accounts for infections in travelers and for common-source epidemics. Oocysts are quite hardy and resist killing by routine chlorination. Both drinking water and recreational water (e.g., pools, waterslides) have been increasingly recognized as sources of infection.

1	Pathophysiology Although intestinal epithelial cells harbor cryptosporidia in an intracellular vacuole, the means by which secretory diarrhea is elicited remain uncertain. No characteristic pathologic changes are found by biopsy. The distribution of infection can be spotty within the principal site of infection, the small bowel. Cryptosporidia are found in the pharynx, stomach, and large bowel of some patients and at times in the respiratory tract. Especially in patients with AIDS, involvement of the biliary tract can cause papillary stenosis, sclerosing cholangitis, or cholecystitis.

1	Clinical Manifestations Asymptomatic infections can occur in both immunocompetent and immunocompromised hosts. In immunocompetent persons, symptoms develop after an incubation period of ~1 week and consist principally of watery nonbloody diarrhea, sometimes in conjunction with abdominal pain, nausea, anorexia, fever, and/or weight loss. In these hosts, the illness usually subsides after 1–2 weeks. In contrast, in immunocompromised hosts (especially those with AIDS and CD4+ T cell counts <100/μL), diarrhea can be chronic, persistent, and remarkably profuse, causing clinically significant fluid and electrolyte depletion. Stool volumes may range from 1 to 25 L/d. Weight loss, wasting, and abdominal pain may be severe. Biliary tract involvement can manifest as mid-epigastric or right-upper-quadrant pain.

1	Diagnosis (Table 254-1) Evaluation starts with fecal examination for small oocysts, which are smaller (4–5 μm in diameter) than the fecal stages of most other parasites. Because conventional stool examination for ova and parasites (O+P) does not detect Cryptosporidium, specific testing must be requested. Detection is enhanced by evaluation of stools (obtained on multiple days) by several techniques, including modified acid-fast and direct immunofluorescent stains and enzyme immunoassays. Newer NAATs are being employed. Cryptosporidia can also be identified by light and electron microscopy at the apical surfaces of intestinal epithelium from biopsy specimens of the small bowel and, less frequently, the large bowel.

1	Nitazoxanide, approved by the U.S. Food and Drug Administration (FDA) for the treatment of cryptosporidiosis, is available in tablet form for adults (500 mg twice daily for 3 days) and as an elixir for children. To date, however, this agent has not been effective for 1407 the treatment of HIV-infected patients, in whom improved immune status due to antiretroviral therapy can lead to amelioration of cryptosporidiosis. Otherwise, treatment includes supportive care with replacement of fluids and electrolytes and administration of antidiarrheal agents. Biliary tract obstruction may require papillotomy or T-tube placement. Prevention requires minimizing exposure to infectious oocysts in human or animal feces. Use of submicron water filters may minimize acquisition of infection from drinking water.

1	The coccidian parasite Cystoisospora belli causes human intestinal disease. Infection is acquired by the consumption of oocysts, after which the parasite invades intestinal epithelial cells and undergoes both sexual and asexual cycles of development. Oocysts excreted in stool are not immediately infectious but must undergo further maturation.

1	Although C. belli infects many animals, little is known about the epidemiology or prevalence of this parasite in humans. It is most common in tropical and subtropical countries. Acute infections can begin abruptly with fever, abdominal pain, and watery nonbloody diarrhea and can last for weeks or months. In patients who have AIDS or are immunocompromised for other reasons, infections often are not self-limited but rather resemble cryptosporidiosis, with chronic, profuse watery diarrhea. Eosinophilia, which is not found in other enteric protozoan infections, may be detectable. The diagnosis (Table 254-1) is usually made by detection of the large (~25-μm) oocysts in stool by modified acid-fast staining. Oocyst excretion may be low-level and intermittent; if repeated stool examinations are unrevealing, sampling of duodenal contents by aspiration or small-bowel biopsy (often with electron microscopic examination) may be necessary. NAATs are promising newer diagnostic tools.

1	Trimethoprim-sulfamethoxazole (TMP-SMX, 160/800 mg four times daily for 10 days; and for HIV-infected patients, then continuing three times daily for 3 weeks) is effective. For patients intolerant of sulfonamides, pyrimethamine (50–75 mg/d) can be used. Relapses can occur in persons with AIDS and necessitate maintenance therapy with TMP-SMX (160/800 mg three times per week).

1	Cyclospora cayetanensis, a cause of diarrheal illness, is globally distributed: illness due to C. cayetanensis has been reported in the United States, Asia, Africa, Latin America, and Europe. The epidemiology of this parasite has not yet been fully defined, but waterborne transmission and food-borne transmission (e.g., by basil, sweet peas, and imported raspberries) have been recognized. The full spectrum of illness attributable to Cyclospora has not been delineated. Some infected patients may be without symptoms, but many have diarrhea, flulike symptoms, and flatulence and belching. The illness can be self-limited, can wax and wane, or, in many cases, can involve prolonged diarrhea, anorexia, and upper gastrointestinal symptoms, with sustained fatigue and weight loss in some instances. Diarrheal illness may persist for >1 month. Cyclospora can cause enteric illness in patients infected with HIV.

1	The parasite is detectable in epithelial cells of small-bowel biopsy samples and elicits secretory diarrhea by unknown means. The absence of fecal blood and leukocytes indicates that disease due to Cyclospora is not caused by destruction of the small-bowel mucosa. The diagnosis (Table 254-1) can be made by detection of spherical 8to 10-μm oocysts in the stool, although routine stool O+P examinations are not sufficient. Specific fecal examinations must be requested to detect the oocysts, which are variably acid-fast and are fluorescent when viewed with ultraviolet light microscopy. Newer NAATs are proving to be sensitive. Cyclosporiasis should be considered in the differential diagnosis of prolonged diarrhea, with or without a history of travel by the patient to other countries. rare among immunocompetent hosts. In patients with AIDS, intestinal infections with Enterocytozoon bieneusi and Encephalitozoon (formerly

1	rare among immunocompetent hosts. In patients with AIDS, intestinal infections with Enterocytozoon bieneusi and Encephalitozoon (formerly Cyclosporiasis is treated with TMP-SMX (160/800 mg twice daily for 7–10 days). HIV-infected patients may experience relapses after such treatment and thus may require longer-term suppressive maintenance therapy.

1	Microsporidia are obligate intracellular spore-forming protozoa that infect many animals and cause disease in humans, especially as opportunistic pathogens in AIDS. Microsporidia are members of a distinct phylum, Microspora, which contains dozens of genera and hundreds of species. The various microsporidia are differentiated by their developmental life cycles, ultrastructural features, and molecular taxonomy based on ribosomal RNA. The complex life cycles of the organisms result in the production of infectious spores (Fig. 254-3). Currently, eight genera of microsporidia—Encephalitozoon, Pleistophora, Nosema, Vittaforma, Trachipleistophora, Anncalia, Microsporidium, and Enterocytozoon—are recognized as causes of human disease. Although some microsporidia are probably prevalent causes of self-limited or asymptomatic infections in immunocompetent patients, little is known about how microsporidiosis is acquired.

1	Microsporidiosis is most common among patients with AIDS, less common among patients with other types of immunocompromise, and Septata) intestinalis are recognized to contribute to chronic diarrhea and wasting; these infections had been found in 10–40% of patients with chronic diarrhea. Both organisms have been found in the biliary tracts of patients with cholecystitis. E. intestinalis may also disseminate to cause fever, diarrhea, sinusitis, cholangitis, and bronchiolitis. In patients with AIDS, Encephalitozoon hellem has caused superficial keratoconjunctivitis as well as sinusitis, respiratory tract disease, and disseminated infection. Myositis due to Pleistophora has been documented. Nosema, Vittaforma, and Microsporidium have caused stromal keratitis associated with trauma in immunocompetent patients.

1	Microsporidia are small gram-positive organisms with mature spores measuring 0.5–2 μm × 1–4 μm. Diagnosis of microsporidial infections in tissue often requires electron microscopy, although intracellular spores can be visualized by light microscopy with hematoxylin and eosin, Giemsa, or tissue Gram’s stain. For the diagnosis of intestinal microsporidiosis, modified trichrome or chromotrope 2R-based staining and Uvitex 2B or calcofluor fluorescent staining reveal spores in smears of feces or duodenal aspirates. Definitive therapies for microsporidial infections remain to be established. For superficial keratoconjunctivitis due to E. hellem, topical therapy with fumagillin suspension has shown promise (Chap. 246e). For enteric infections with E. bieneusi and E. intestinalis in HIV-infected patients, therapy with albendazole may be efficacious (Chap. 246e). Microsporidia Enterocytozoon bieneusi, Encephalitozoon spp., et al.

1	Microsporidia Enterocytozoon bieneusi, Encephalitozoon spp., et al. Intracellular multiplication via merogony and sporogony Encephalitozoon intestinalis in epithelial cells, endothelial cells, or macrophages E. bieneusi in epithelial cell Polar tubule pierces host epithelial cell, injects sporoplasm Presumed ingestion or respiratory acquisition of spores Person-to-person, zoonotic, water-borne, or Spore-laden host epithelial cells sloughed into lumina of gastrointestinal, respiratory, or genitourinary tract While E. bieneusi is primarily in the gastrointestinal tract, other species may invade the lung or eye or disseminate to cause: Chronic diarrhea Cholangitis Sinusitis Bronchitis Nephritis Cystitis/prostatitis Keratoconjunctivitis Encephalitis food-borne transmission? Sloughed cells Diagnostic spores present in stool, urine, respiratory fluids, cerebrospinal fluid, or various tissue specimens

1	Diagnostic spores present in stool, urine, respiratory fluids, cerebrospinal fluid, or various tissue specimens FIGuRE 254-3 Life cycle of microsporidia. (Reprinted with permission from RL Guerrant et al [eds]: Tropical Infectious Diseases: Principles, Pathogens and Practice, 2nd ed, p 1128. © 2006, with permission from Elsevier Science.) Balantidiasis Balantidium coli is a large ciliated protozoal parasite that can produce a spectrum of large-intestinal dis ease analogous to amebiasis. The parasite is widely distributed in the world. Since it infects pigs, cases in humans are more common where pigs are raised. Infective cysts can be transmitted from person to person and through water, but many cases are due to the ingestion of cysts derived from porcine feces in association with slaughtering, with use of pig feces for fertilizer, or with contamination of water supplies by pig feces.

1	Ingested cysts liberate trophozoites, which reside and replicate in the large bowel. Many patients remain asymptomatic, but some have persisting intermittent diarrhea, and a few develop more fulminant dysentery. In symptomatic individuals, the pathology in the bowel— both gross and microscopic—is similar to that seen in amebiasis, with varying degrees of mucosal invasion, focal necrosis, and ulceration. Balantidiasis, unlike amebiasis, only rarely spreads hematogenously to other organs. The diagnosis is made by detection of the trophozoite stage in stool or sampled colonic tissue. Tetracycline (500 mg four times daily for 10 days) is an effective therapeutic agent.

1	Blastocystosis Blastocystis hominis remains an organism of uncertain pathogenicity. Some patients who pass B. hominis in their stools are asymptomatic, whereas others have diarrhea and associated intestinal symptoms. Diligent evaluation reveals other potential bacterial, viral, or protozoal causes of diarrhea in some but not all patients with symptoms. Because the pathogenicity of B. hominis is uncertain and because therapy for Blastocystis infection is neither specific nor uniformly effective, patients with prominent intestinal symptoms should be fully evaluated for other infectious causes of diarrhea. If diarrheal symptoms associated with Blastocystis are prominent, either metronidazole (750 mg thrice daily for 10 days) or TMP-SMX (160 mg/800 mg twice daily for 7 days) can be used.

1	dientamoebiasis Dientamoeba fragilis is unique among intestinal protozoa in that it has a trophozoite stage but not a cyst stage. How trophozoites survive to transmit infection is not known. When symptoms develop in patients with D. fragilis infection, they are generally mild and include intermittent diarrhea, abdominal pain, and anorexia. The diagnosis is made by the detection of trophozoites in stool; the lability of these forms accounts for the greater yield when fecal samples are preserved immediately after collection. Since fecal excretion rates vary, examination of several samples obtained on alternate days increases the rate of detection. Iodoquinol (650 mg three times daily for 20 days) or paromomycin (25–35 mg/kg per day in three doses for 7 days) is appropriate for treatment. Various species of trichomonads can be found in the mouth (in association with periodontitis) and occasionally in the gastrointestinal tract.

1	Various species of trichomonads can be found in the mouth (in association with periodontitis) and occasionally in the gastrointestinal tract. Trichomonas vaginalis—one of the most prevalent protozoal parasites 140in the United States—is a pathogen of the genitourinary tract and a major cause of symptomatic vaginitis (Chap. 163). Life Cycle and Epidemiology T. vaginalis is a pear-shaped, actively motile organism that measures about 10 × 7 om, replicates by binary fission, and inhabits the lower genital tract of females and the urethra and prostate of males. In the United States, it accounts for ~3 million infections per year in women. While the organism can survive for a few hours in moist environments and could be acquired by direct contact, person-to-person venereal transmission accounts for virtually all cases of trichomoniasis. Its prevalence is greatest among persons with multiple sexual partners and among those with other sexually transmitted diseases (Chap. 163).

1	Clinical Manifestations Many men infected with T. vaginalis are asymptomatic, although some develop urethritis and a few have epididymitis or prostatitis. In contrast, infection in women, which has an incubation period of 5–28 days, is usually symptomatic and manifests with malodorous vaginal discharge (often yellow), vulvar erythema and itching, dysuria or urinary frequency (in 30–50% of patients), and dyspareunia. These manifestations, however, do not clearly distinguish trichomoniasis from other types of infectious vaginitis.

1	diagnosis Detection of motile trichomonads by microscopic examination of wet mounts of vaginal or prostatic secretions has been the conventional means of diagnosis. Although this approach provides an immediate diagnosis, its sensitivity for the detection of T. vaginalis is only ~50–60% in routine evaluations of vaginal secretions. Direct immunofluorescent antibody staining is more sensitive (70–90%) than wet-mount examinations. T. vaginalis can be recovered from the urethra of both males and females and is detectable in males after prostatic massage. A new NAAT, APTIMA, is FDA approved and is highly sensitive and specific for urine and for endocervical and vaginal swabs from women.

1	Metronidazole (either a single 2-g dose or 500-mg doses twice daily for 7 days) or tinidazole (a single 2-g dose) is effective. All sexual partners must be treated concurrently to prevent reinfection, especially from asymptomatic males. In males with persistent symptomatic urethritis after therapy for nongonococcal urethritis, metronidazole therapy should be considered for possible trichomoniasis. Alternatives to metronidazole for treatment during pregnancy are not readily available. einfection often accounts for apparent treatment failures, but strains of T. vaginalis exhibiting high-level resistance to metronidazole have been encountered. Treatment of these resistant infections with higher oral doses, parenteral doses, or concurrent oral and vaginal doses of metronidazole or with tinidazole has been successful. Introduction to Helminthic Infections Peter F. Weller

1	Introduction to Helminthic Infections Peter F. Weller The word helminth is derived from the Greek helmins (“parasitic worm”). Helminthic worms are highly prevalent and, depending on the species, may exist as free-living organisms or as parasites of plant or animal hosts. The parasitic helminths have co-evolved with specific mammalian and other host species. Accordingly, most helminthic infections are restricted to nonhuman hosts, and only rarely do these zoonotic helminths accidentally cause human infections.

1	Helminthic parasites of humans belong to two phyla: Nemathelminthes, which includes nematodes (roundworms), and Platyhelminthes, which includes cestodes (tapeworms) and trematodes (flukes). Helminthic parasites of humans reside within the human body and hence are the cause of true infections. In contrast, parasites of other genera that reside only on mucocutaneous surfaces of humans (e.g., the parasites causing myiasis and scabies) are considered to represent infestations rather than infections.

1	Helminthic parasites differ substantially from protozoan parasites in several respects. First, protozoan parasites are unicellular organisms, whereas helminthic parasites are multicellular worms that possess differentiated organ systems. Second, helminthic parasites have complex life cycles that require sequential stages of development outside the human host. Thus, most helminths do not complete their replication within the human host; rather, they develop to a certain stage within the mammalian host and, as part of their obligatory life cycle, must mature further outside that host. During the “extra-human” stages of their life cycle, helminths exist either as free-living organisms or as parasites within another host species and thereafter mature into new developmental stages capable of infecting humans. Thus, with only two exceptions (Strongyloides stercoralis and Capillaria philippinensis, which are capable of internal reinfection), increases in the number of adult helminths (i.e.,

1	humans. Thus, with only two exceptions (Strongyloides stercoralis and Capillaria philippinensis, which are capable of internal reinfection), increases in the number of adult helminths (i.e., the “worm burden”) within the human host require repeated exogenous reinfections. In the case of protozoan parasites, a brief, even singular exposure (e.g., a single mosquito bite transmitting malaria) may lead rapidly to intense parasite loads and overwhelming infections; in contrast, for all but the two helminths noted above, increases in worm burden require multiple and usually ongoing exposures to infectious forms, such as ingestion of eggs of intestinal helminths or waterborne exposures to infectious cercariae of Schistosoma mansoni. This requirement is germane both to the consideration of helminthic infections in individuals and to ongoing global efforts to interrupt and/or minimize the acquisition of helminthic infections by humans.

1	Third, helminthic infections have a predilection toward stimulation of host immune responses that elicit eosinophilia within human tissues and blood. The many protozoan infections characteristically do not elicit eosinophilia in infected humans, with only three exceptions (two intestinal protozoan parasites, Cystoisospora belli and Dientamoeba fragilis, and tissue-borne Sarcocystis species). The magnitude of helminth-elicited eosinophilia tends to correlate with the extent of tissue invasion by larvae or adult helminths. For example, in several helminthic infections, including acute schistosomiasis (Katayama syndrome), paragonimiasis, and hookworm and Ascaris infections, eosinophilia is most pronounced during the early phases of infection, when migrations of infecting larvae and progression of subsequent developmental stages through the tissues are greatest. In established infections, local eosinophilia is often present around helminths in tissues, but blood eosinophilia may be

1	of subsequent developmental stages through the tissues are greatest. In established infections, local eosinophilia is often present around helminths in tissues, but blood eosinophilia may be intermittent, mild, or absent. In helminthic infections in which parasites are well contained within tissues (e.g., echinococcal cysts) or confined within the lumen of the intestinal tract (e.g., adult Ascaris or tapeworms), eosinophilia is usually absent.

1	Nematodes are nonsegmented roundworms. Species of nematodes are remarkably diverse and abundant in nature. Among the many thousands of nematode species, few are parasites of humans. Most nematodes are free-living, and these species have variably evolved to survive in diverse ecologic niches, including saltwater, freshwater, or soil. The well-studied organism Caenorhabditis elegans is a free-living nematode. Nematodes can be either beneficial or deleterious parasites of plants. Parasitic nematodes have co-evolved with specific mammalian hosts and have no capacity to live their full life cycles in other hosts. Uncommonly, humans are exposed to infectious stages of nonhuman nematode parasites, and the resultant zoonotic nematode infections can elicit inflammatory and immune responses as larval forms migrate and die in the unsuitable human host. Examples include pulmonary coin lesions due to mosquito-transmitted infections with the dog heartworm Dirofilaria immitis; eosinophilic

1	as larval forms migrate and die in the unsuitable human host. Examples include pulmonary coin lesions due to mosquito-transmitted infections with the dog heartworm Dirofilaria immitis; eosinophilic meningoencephalitis due to ingested eggs of the raccoon ascarid Baylisascaris procyonis; and eosinophilic meningitis due to ingestion of larvae of the rat lungworm Angiostrongylus cantonensis.

1	Nematode parasites of humans include worms that reside in the intestinal tract or localize in extraintestinal vascular or tissue sites. Roundworms are bisexual, with separate male and female forms (except for S. stercoralis, whose adult females are hermaphroditic in the human intestinal tract). Depending on the species, fertilized females release either larvae or eggs containing larvae. Nematodes have five developmental stages: an adult stage and four sequential larval stages. These parasites characteristically are surrounded by a durable outer cuticular layer. Nematodes have a nervous system; a muscular system, including muscle cells under the cuticle; and a developed intestinal tract, including an oral cavity and an elongated gut that ends in an anal pore. Adults may range in size from minute to >1 meter in length (with Dracunculus medinensis, for example, at the long end of this spectrum).

1	Humans acquire infections with nematode parasites by various routes, depending on the parasitic species. Ingestion of eggs passed in human feces is a major global health problem with many of the intestinal helminths (e.g., Ascaris lumbricoides). In other species, infecting larvae penetrate skin exposed to fecally contaminated soil (e.g., S. stercoralis) or traverse the skin after the bite of infected insect vectors (e.g., filariae). Some nematode infections are acquired by consumption of specific animal-derived foods (e.g., trichinellosis from raw or undercooked pork or wild carnivorous mammals). As noted above, only two nematodes, S. stercoralis and C. philippinensis, can internally reinfect humans.; thus, for all other nematodes, any increases in worm burden must be due to continued exogenous reinfections.

1	Tapeworms are the cestode parasites of humans. Adult tapeworms are elongated, segmented, hermaphroditic flatworms that reside in the intestinal lumen or, in their larval forms, may live in extraintestinal tissues. Tapeworms include a head (scolex) and a number of attached segments (proglottids). The worms attach to the intestinal tract via their scolices, which may possess suckers, hooks, or grooves. The scolex is the site of formation of new proglottids. Tapeworms do not have a functional gut tract; rather, each tapeworm segment passively and actively obtains nutrients through its specialized surface tegument. Mature proglottids possess both male and female sex organs, but insemination usually occurs between adjacent proglottids. Fertilized proglottids release eggs that are passed in the feces. When ingested by an intermediate host, an egg releases an oncosphere that penetrates the

1	CHAPTER 255e Introduction to Helminthic Infections gut and develops further in tissues as a cysticercus. Humans acquire infection by ingesting animal tissues that contain cysticerci, and the resultant tapeworms develop and reside in the proximal small bowel (e.g., Taenia solium, T. saginata). Alternatively, if humans ingest eggs of these cestodes that have been passed in human or animal feces, oncospheres develop and can cause space-occupying extraintestinal cystic lesions in tissues; examples include cysticercosis due to T. solium and hydatid disease due to species of Echinococcus.

1	Trematodes of medical importance include blood flukes, intestinal flukes, and tissue flukes. Adult flukes are often leaf-shaped flatworms. Oral and/or ventral suckers help adult flukes maintain their positions in situ. Flukes have an oral cavity but no distal anal pore. Nutrients are obtained both through their integument and by ingestion into the blind intestinal tract. Flukes are hermaphroditic except for blood flukes (schistosomes), which are bisexual. Eggs are passed in human feces (Fasciola, Fasciolopsis, Clonorchis, Schistosoma japonicum, S. mansoni), urine (S. haematobium), or sputum and feces (Paragonimus). Expelled eggs release miracidia—usually in water—that infect specific snail species. Within snails, parasites multiply and cercariae are released. Depending on the species, cercariae can penetrate the skin (schistosomes) or can develop into metacercariae that can be ingested with plants (e.g., watercress for Fasciola) or with fish (Clonorchis) or crabs (Paragonimus).

1	Many of the so-called neglected tropical diseases are due to helminthic infections. The health impacts of many helminthic infections are varied and are based on the frequent need for repeated exposures to increase the worm burdens in infected humans. In global regions where exposures to specific helminths occur even in childhood (e.g., fecally derived intestinal nematodes, mosquito-transmitted filariae, or waterborne snail-transmitted schistosome infections), the morbidities in infected individuals can include nutritional, developmental, cognitive, and functional impairments. Ongoing global mass-treatment programs are currently aimed at diminishing the local prevalences of specific helminths and their consequent impacts on the health of local populations.

1	1410 ~1 week, female worms release newborn larvae that migrate via the circulation to striated muscle. The larvae of all species except T. pseu- dospiralis, T. papuae, and T. zimbabwensis then encyst by inducing a nematode Infections radical transformation in the muscle cell architecture. Although host Peter F. Weller Nematodes are elongated, symmetric roundworms. Parasitic nematodes of medical significance may be broadly classified as either predominantly intestinal or tissue nematodes. This chapter covers the tissue nematodes that cause trichinellosis, visceral and ocular larva migrans, cutaneous larva migrans, cerebral angiostrongyliasis, and gnathostomiasis. All of these zoonotic infections result from incidental exposure to infectious nematodes. The clinical symptoms of these infections are due largely to invasive larval stages that (except in the case of Trichinella) do not reach maturity in humans.

1	Trichinellosis develops after the ingestion of meat containing cysts of Trichinella (e.g., pork or other meat from a carnivore). Although most infections are mild and asymptomatic, heavy infections can cause severe enteritis, periorbital edema, myositis, and (infrequently) death. Life Cycle and Epidemiology Eight species of Trichinella are recognized as causes of infection in humans. Two species are distributed worldwide: T. spiralis, which is found in a great variety of carnivorous and omnivorous animals, and T. pseudospiralis, which is found in mammals and birds. T. nativa is present in Arctic regions and infects bears; T. nelsoni is found in equatorial eastern Africa, where it is common among felid predators and scavengers such as hyenas and bush pigs; and T. britovi is found in Europe, western Africa, and western Asia among carnivores but not among domestic swine. T. murrelli is present in North American game animals.

1	After human consumption of trichinous meat, encysted larvae are liberated by digestive acid and proteases (Fig. 256-1). The larvae invade the small-bowel mucosa and mature into adult worms. After Larvae migrate, penetrate striated muscle, reside in "nurse-cells," and encyst,* causing: Muscle pain, fever, periorbital edema, Larvae are released eosinophilia, occasional in the stomach and mature CNS or cardiac damage in the small bowel, causing: immune responses may help to expel intestinal adult worms, they have few deleterious effects on muscle-dwelling larvae.

1	Human trichinellosis is often caused by the ingestion of infected pork products and thus can occur in almost any location where the meat of domestic or wild swine is eaten. Human trichinellosis may also be acquired from the meat of other animals, including dogs (in parts of Asia and Africa), horses (in Italy and France), and bears and walruses (in northern regions). Although cattle (being herbivores) are not natural hosts of Trichinella, beef has been implicated in outbreaks when contaminated or adulterated with trichinous pork. Laws that prohibit the feeding of uncooked garbage to pigs have greatly reduced the transmission of trichinellosis in the United States. About 12 cases of trichinellosis are reported annually in this country, but most mild cases probably remain undiagnosed. Recent U.S. and Canadian outbreaks have been attributable to consumption of wild game (especially bear meat) and, less frequently, of pork.

1	Pathogenesis and Clinical Features Clinical symptoms of trichinellosis arise from the successive phases of parasite enteric invasion, larval migration, and muscle encystment (Fig. 256-1). Most light infections (those with <10 larvae per gram of muscle) are asymptomatic, whereas heavy infections (which can involve >50 larvae per gram of muscle) can be life-threatening. Invasion of the gut by large numbers of parasites occasionally provokes diarrhea during the first week after infection. Abdominal pain, constipation, nausea, or vomiting also may be prominent.

1	Symptoms due to larval migration and muscle invasion begin to appear in the second week after infection. The migrating Trichinella larvae provoke a marked local and systemic hypersensitivity reaction, with fever and hypereosinophilia. Periorbital and facial edema is common, as are hemorrhages in the subconjunctivae, retina, and nail beds (“splinter” hemorrhages). A maculopapular rash, headache, cough, dyspnea, or dysphagia sometimes develops. Myocarditis with tachyarrhythmias or heart failure—and, less commonly, encephalitis or pneumonitis—may develop and accounts for most deaths of patients with trichinellosis.

1	Upon onset of larval encystment in muscle 2–3 weeks after infection, symptoms of myositis with myalgias, muscle edema, and weakness develop, usually overlapping with the inflammatory reactions to migrating larvae. The most commonly involved muscle groups include the extraocular muscles; the biceps; and the muscles of the jaw, neck, lower back, and diaphragm. Peaking ~3 weeks after infection, symptoms subside only gradually during a prolonged convalescence. Uncommon infections with T. pseudospiralis, whose larvae do not encapsulate in muscles, elicit prolonged polymyositis-like illness. Laboratory Findings and Diagnosis Blood eosinophilia develops in >90% of patients with symptomatic trichinellosis and may peak at a level of >50% 2–4 weeks after infection. Serum levels of muscle enzymes, including creatine phosphoki-

1	Encysted larvae ingested Similar cycle (as humans) in undercooked pork, in swine or other carnivores nase, are elevated in most symptomatic patients. boar, horse, or bear (rats, bears, foxes, dogs, or horses) Patients should be questioned thoroughly about their consumption of pork or wild animal meat *T. papuae, T. zimbabwensis, and T. pseudospiralis do not encyst.

1	and about illness in other individuals who ate FIGuRE 256-1 Life cycle ofTrichinella spiralis (cosmopolitan); nelsoni (equatorial Africa); the same meat. A presumptive clinical diagnosis britovi (Europe, western Africa, western Asia); nativa (Arctic); murrelli (North America); pap-can be based on fevers, eosinophilia, periorbital uae (Papua New Guinea); zimbabwensis (Tanzania); and pseudospiralis (cosmopolitan). CNS, edema, and myalgias after a suspect meal. A rise central nervous system. (Reprinted from RL Guerrant et al [eds]: Tropical Infectious Diseases: in the titer of parasite-specific antibody, which Principles, Pathogens and Practice, 2nd ed, p 1218. © 2006, with permission from Elsevier Science.) usually does not occur until after the third week of infection, confirms the diagnosis. Alternatively, a definitive diagnosis requires surgical biopsy of at least 1 g of involved muscle; the yields are highest near tendon insertions. The fresh muscle tissue should be compressed

1	Alternatively, a definitive diagnosis requires surgical biopsy of at least 1 g of involved muscle; the yields are highest near tendon insertions. The fresh muscle tissue should be compressed between glass slides and examined microscopically (Fig. 256-2), because larvae may be missed by examination of routine histopathologic sections alone.

1	FIGuRE 256-2 Trichinella larva encysted in a characteristic hyalinized capsule in striated muscle tissue. (Photo/Wadsworth Center, New York State Department of Health. Reprinted from MMWR 53:606, 2004; public domain.) Most lightly infected patients recover uneventfully with bed rest, antipyretics, and analgesics. Glucocorticoids like prednisone (Table 256-1) are beneficial for severe myositis and myocarditis. Mebendazole and albendazole are active against enteric stages of the parasite, but their efficacy against encysted larvae has not been conclusively demonstrated. Prevention Larvae may be killed by cooking pork until it is no longer pink or by freezing it at -15°C for 3 weeks. However, Arctic T. nativa

1	Prevention Larvae may be killed by cooking pork until it is no longer pink or by freezing it at -15°C for 3 weeks. However, Arctic T. nativa Trichinellosis Mild Supportive Moderate Albendazole (400 mg bid × 8–14 days) or Mebendazole (200–400 mg tid × 3 days, then 400 mg tid × 8–14 days) Severe Add glucocorticoids (e.g., prednisone, 1 mg/kg qd × 5 days) Visceral larva Mild to Supportive for adults, 400 mg bid for children) with glucocorticoids × 5–20 days has been effective Ivermectin (single dose, 200 μg/kg) or migrans Angiostrongyliasis Mild to Supportive moderate Severe Glucocorticoids (as above) larvae in walrus or bear meat are relatively resistant and may remain 1411 viable despite freezing.

1	Angiostrongyliasis Mild to Supportive moderate Severe Glucocorticoids (as above) larvae in walrus or bear meat are relatively resistant and may remain 1411 viable despite freezing. Visceral larva migrans is a syndrome caused by nematodes that are normally parasitic for nonhuman host species. In humans, these nematode larvae do not develop into adult worms but instead migrate through host tissues and elicit eosinophilic inflammation. The most common form of visceral larva migrans is toxocariasis due to larvae of the canine ascarid Toxocara canis; the syndrome is due less commonly to the feline ascarid T. cati and even less commonly to the pig ascarid Ascaris suum. Rare cases with eosinophilic meningoencephalitis have been caused by the raccoon ascarid Baylisascaris procyonis.

1	Life Cycle and Epidemiology The canine roundworm T. canis is distributed among dogs worldwide. Ingestion of infective eggs by dogs is followed by liberation of Toxocara larvae, which penetrate the gut wall and migrate intravascularly into canine tissues, where most remain in a developmentally arrested state. During pregnancy, some larvae resume migration in bitches and infect puppies prenatally (through transplacental transmission) or after birth (through suckling). Thus, in lactating bitches and puppies, larvae return to the intestinal tract and develop into adult worms, which produce eggs that are released in the feces. Eggs must undergo embryonation over several weeks to become infectious. Humans acquire toxocariasis mainly by eating soil contaminated by puppy feces that contains infective T. canis eggs. Visceral larva migrans is most common among children who habitually eat dirt.

1	Pathogenesis and Clinical Features Clinical disease most commonly afflicts preschool children. After humans ingest Toxocara eggs, the larvae hatch and penetrate the intestinal mucosa, from which they are carried by the circulation to a wide variety of organs and tissues. The larvae invade the liver, lungs, central nervous system (CNS), and other sites, provoking intense local eosinophilic granulomatous responses. The degree of clinical illness depends on larval number and tissue distribution, reinfection, and host immune responses. Most light infections are asymptomatic and may be manifest only by blood eosinophilia. Characteristic symptoms of visceral larva migrans include fever, malaise, anorexia and weight loss, cough, wheezing, and rashes. Hepatosplenomegaly is common. These features may be accompanied by extraordinary peripheral eosinophilia, which may approach 90%. Uncommonly, seizures or behavioral disorders develop. Rare deaths are due to severe neurologic, pneumonic, or

1	may be accompanied by extraordinary peripheral eosinophilia, which may approach 90%. Uncommonly, seizures or behavioral disorders develop. Rare deaths are due to severe neurologic, pneumonic, or myocardial involvement.

1	The ocular form of the larva migrans syndrome occurs when Toxocara larvae invade the eye. An eosinophilic granulomatous mass, most commonly in the posterior pole of the retina, develops around the entrapped larva. The retinal lesion can mimic retinoblastoma in appearance, and mistaken diagnosis of the latter condition can lead to unnecessary enucleation. The spectrum of eye involvement also includes endophthalmitis, uveitis, and chorioretinitis. Unilateral visual disturbances, strabismus, and eye pain are the most common presenting symptoms. In contrast to visceral larva migrans, ocular toxocariasis usually develops in older children or young adults with no history of pica; these patients seldom have eosinophilia or visceral manifestations.

1	Diagnosis In addition to eosinophilia, leukocytosis and hypergammaglobulinemia may be evident. Transient pulmonary infiltrates are apparent on chest x-rays of about one-half of patients with symptoms of pneumonitis. The clinical diagnosis can be confirmed by an enzyme-linked immunosorbent assay for toxocaral antibodies. Stool examination for parasite eggs is worthless in toxocariasis, since the larvae do not develop into egg-producing adults in humans.

1	The vast majority of Toxocara infections are self-limited and resolve without specific therapy. In patients with severe myocardial, CNS, or pulmonary involvement, glucocorticoids may be employed to reduce inflammatory complications. Available anthelmintic drugs, 1412 including mebendazole and albendazole, have not been shown conclusively to alter the course of larva migrans. Control measures include prohibiting dog excreta in public parks and playgrounds, deworming dogs, and preventing pica in children. Treatment of ocular disease is not fully defined, but the administration of albendazole in conjunction with glucocorticoids has been effective (Table 256-1).

1	Cutaneous larva migrans (“creeping eruption”) is a serpiginous skin eruption caused by burrowing larvae of animal hookworms, usually the dog and cat hookworm Ancylostoma braziliense. The larvae hatch from eggs passed in dog and cat feces and mature in the soil. Humans become infected after skin contact with soil in areas frequented by dogs and cats, such as areas underneath house porches. Cutaneous larva migrans is prevalent among children and travelers in regions with warm humid climates, including the southeastern United States.

1	After larvae penetrate the skin, erythematous lesions form along the tortuous tracks of their migration through the dermal-epidermal junction; the larvae advance several centimeters in a day. The intensely pruritic lesions may occur anywhere on the body and can be numerous if the patient has lain on the ground. Vesicles and bullae may form later. The animal hookworm larvae do not mature in humans and, without treatment, will die after an interval ranging from weeks to a couple of months, with resolution of skin lesions. The diagnosis is made on clinical grounds. Skin biopsies only rarely detect diagnostic larvae. Symptoms can be alleviated by ivermectin or albendazole (Table 256-1). Angiostrongylus cantonensis, the rat lungworm, is the most common cause of human eosinophilic meningitis (Fig. 256-3).

1	Angiostrongylus cantonensis, the rat lungworm, is the most common cause of human eosinophilic meningitis (Fig. 256-3). Southeast Asia and the Pacific Basin but has spread to other areas of the world, including the Caribbean islands, countries in Central and South America, and the southern United States. A. cantonensis larvae produced by adult worms in the rat lung migrate to the gastrointestinal tract and are expelled with the feces. They develop into infective larvae in land snails and slugs. Humans acquire the infection by ingesting raw infected mollusks; vegetables contaminated by mollusk slime; or crabs, freshwater shrimp, and certain marine fish that have themselves eaten infected mollusks. The larvae then migrate to the brain.

1	Eosinophilic meningitis 2 weeks 3rd-stage larvae (consumed in snail or slime) penetrate gut, go to CNS (then lung in rat) Larvae consumed by land snail/slug (Achatina fulica) viable in fresh water Adult in pulmonary artery produces fertile eggs; larvae hatch, penetrate arterioles, migrate up bronchi, and are coughed up, swallowed, and passed in feces FIGuRE 256-3 Life cycle of Angiostrongylus cantonensis (rat lung worm), found in Southeast Asia, Pacific Islands, Cuba, Australia, Japan, China, Mauritius, and U.S. ports. CNS, central nervous system. (Reprinted from RL Guerrant et al [eds]: Tropical Infectious Diseases: Principles, Pathogens and Practice, 2nd ed, p 1225. © 2006, with permission from Elsevier Science.)

1	Pathogenesis and Clinical Features The parasites eventually die in the CNS, but not before initiating pathologic consequences that, in heavy infections, can result in permanent neurologic sequelae or death. Migrating larvae cause marked local eosinophilic inflammation and hemorrhage, with subsequent necrosis and granuloma formation around dying worms. Clinical symptoms develop 2–35 days after the ingestion of larvae. Patients usually present with an insidious or abrupt excruciating frontal, occipital, or bitemporal headache. Neck stiffness, nausea and vomiting, and paresthesias are also common. Fever, cranial and extraocular nerve palsies, seizures, paralysis, and lethargy are uncommon.

1	Laboratory Findings Examination of cerebrospinal fluid (CSF) is mandatory in suspected cases and usually reveals an elevated opening pressure, a white blood cell count of 150–2000/μL, and an eosinophilic pleocytosis of >20%. The protein concentration is usually elevated and the glucose level normal. The larvae of A. cantonensis are only rarely seen in CSF. Peripheral-blood eosinophilia may be mild. The diagnosis is generally based on the clinical presentation of eosinophilic meningitis together with a compatible epidemiologic history.

1	Specific chemotherapy is not of benefit in angiostrongyliasis; larvicidal agents may exacerbate inflammatory brain lesions. Management consists of supportive measures, including the administration of analgesics, sedatives, and—in severe cases—glucocorticoids (Table 256-1). Repeated lumbar punctures with removal of CSF can relieve symptoms. In most patients, cerebral angiostrongyliasis has a self-limited course, and recovery is complete. The infection may be prevented by adequately cooking snails, crabs, and prawns and inspecting vegetables for mollusk infestation. Other parasitic or fungal causes of eosinophilic meningitis in endemic areas may include gnathostomiasis (see below), paragonimiasis (Chap. 259), schistosomiasis (Chap. 259), neurocysticercosis (Chap. 260), and coccidioidomycosis (Chap. 237).

1	Infection of human tissues with larvae of Gnathostoma spinigerum can cause eosinophilic meningoencephalitis, migratory cutaneous swellings, or invasive masses of the eye and visceral organs.

1	miasis occurs in many countries and is notably endemic in Southeast Asia and parts of China and Japan. In nature, the mature adult worms parasitize the gastrointestinal tract of dogs and cats. First-stage larvae hatch from eggs passed into water and are ingested by Cyclops species (water fleas). Infective third-stage larvae develop in the flesh of many animal species (including fish, frogs, eels, snakes, chickens, and ducks) that have eaten either infected Cyclops or another infected second intermediate host. Humans typically acquire the infection by eating raw or undercooked fish or poultry. Raw fish dishes, such as som fak in Thailand and sashimi in Japan, account for many cases of human gnathostomiasis. Some cases in Thailand result from the local practice of applying frog or snake flesh as a poultice.

1	Pathogenesis and Clinical Features Clinical symptoms are due to the aberrant migration of a single larva into cutaneous, visceral, neural, or ocular tissues. After invasion, larval migration may cause local inflammation, with pain, cough, or hematuria accompanied by fever and eosinophilia. Painful, itchy, migratory swellings may develop in the skin, particularly in the distal extremities or periorbital area. Cutaneous swellings usually last ~1 week but often recur intermittently over many years. Larval invasion of the eye can provoke a sight-threatening inflammatory response. Invasion of the CNS results in eosinophilic meningitis with myeloencephalitis, a serious complication due to ascending larval migration along a large nerve track. Patients characteristically present with agonizing radicular pain and paresthesias in the trunk or a limb, which are followed shortly by paraplegia. Cerebral involvement, with focal hemorrhages and tissue destruction, is often fatal.

1	Diagnosis and Treatment Cutaneous migratory swellings with marked peripheral eosinophilia, supported by an appropriate geographic and dietary history, generally constitute an adequate basis for a clinical diagnosis of gnathostomiasis. However, patients may present with ocular or cerebrospinal involvement without antecedent cutaneous swellings. In the latter case, eosinophilic pleocytosis is demonstrable (usually along with hemorrhagic or xanthochromic CSF), but worms are almost never recovered from CSF. Surgical removal of the parasite from subcutaneous or ocular tissue, though rarely feasible, is both diagnostic and therapeutic. Albendazole or ivermectin may be helpful (Table 256-1). At present, cerebrospinal involvement is managed with supportive measures and generally with a course of glucocorticoids. Gnathostomiasis can be prevented by adequate cooking of fish and poultry in endemic areas. Peter F. Weller, Thomas B. Nutman

1	Peter F. Weller, Thomas B. Nutman More than a billion persons worldwide are infected with one or more species of intestinal nematodes. Table 257-1 summarizes biologic and clinical features of infections due to the major intestinal parasitic nematodes. These parasites are most common in regions with poor fecal sanitation, particularly in resource-poor countries in the tropics and subtropics, but they have also been seen with increasing frequency among immigrants and refugees to resource-rich countries. Although nematode infections are not usually fatal, they contribute to malnutrition and diminished work capacity. It is interesting that these helminth infections may protect some individuals from allergic disease. Humans may on occasion be infected with nematode parasites that ordinarily infect animals; these zoonotic infections produce diseases such as trichostrongyliasis, anisakiasis, capillariasis, and abdominal angiostrongyliasis.

1	Intestinal nematodes are roundworms; they range in length from 1 mm to many centimeters when mature (Table 257-1). Their life cycles are complex and highly varied; some species, including Strongyloides stercoralis and Enterobius vermicularis, can be transmitted directly from person to person, while others, such as Ascaris lumbricoides, Necator americanus, and Ancylostoma duodenale, require a soil phase for development. Because most helminth parasites do not self-replicate, the acquisition of a heavy burden of adult worms requires repeated exposure to the parasite in its infectious stage, whether larva or egg. Hence, clinical disease, as opposed to asymptomatic infection, generally develops only with prolonged residence in an endemic area and is typically related to infection intensity. In persons with marginal nutrition, intestinal helminth infections may impair growth and development. Eosinophilia and elevated serum IgE levels are features of many helminth infections and, when

1	In persons with marginal nutrition, intestinal helminth infections may impair growth and development. Eosinophilia and elevated serum IgE levels are features of many helminth infections and, when unexplained, should always prompt a search for intestinal helminths. Significant protective immunity to intestinal nematodes appears not to develop in humans, although mechanisms of parasite immune evasion and host immune responses to these infections have not been elucidated in detail.

1	A. lumbricoides is the largest intestinal nematode parasite of humans, reaching up to 40 cm in length. Most infected individuals have low worm burdens and are asymptomatic. Clinical disease arises from 1413 larval migration in the lungs or effects of the adult worms in the intestines.

1	Life Cycle Adult worms live in the lumen of the small intestine. Mature female Ascaris worms are extraordinarily fecund, each producing up to 240,000 eggs a day, which pass with the feces. Ascarid eggs, which are remarkably resistant to environmental stresses, become infective after several weeks of maturation in the soil and can remain infective for years. After infective eggs are swallowed, larvae hatched in the intestine invade the mucosa, migrate through the circulation to the lungs, break into the alveoli, ascend the bronchial tree, and return— through swallowing—to the small intestine, where they develop into adult worms. Between 2 and 3 months elapse between initial infection and egg production. Adult worms live for 1–2 years.

1	Epidemiology Ascaris is widely distributed in tropical and subtropical regions as well as in other humid areas, including the rural southeastern United States. Transmission typically occurs through fecally contaminated soil and is due either to a lack of sanitary facilities or to the use of human feces as fertilizer. With their propensity for hand-to-mouth fecal carriage, younger children are most affected. Infection outside endemic areas, though uncommon, can occur when eggs on transported vegetables are ingested.

1	Clinical Features During the lung phase of larval migration, ~9–12 days after egg ingestion, patients may develop an irritating nonproductive cough and burning substernal discomfort that is aggravated by coughing or deep inspiration. Dyspnea and blood-tinged sputum are less common. Fever is usually reported. Eosinophilia develops during this symptomatic phase and subsides slowly over weeks. Chest x-rays may reveal evidence of eosinophilic pneumonitis (Löffler’s syndrome), with rounded infiltrates a few millimeters to several centimeters in size. These infiltrates may be transient and intermittent, clearing after several weeks. Where there is seasonal transmission of the parasite, seasonal pneumonitis with eosinophilia may develop in previously infected and sensitized hosts.

1	In established infections, adult worms in the small intestine usually cause no symptoms. In heavy infections, particularly in children, a large bolus of entangled worms can cause pain and small-bowel obstruction, sometimes complicated by perforation, intussusception, or volvulus. Single worms may cause disease when they migrate into aberrant sites. A large worm can enter and occlude the biliary tree, causing biliary colic, cholecystitis, cholangitis, pancreatitis, or (rarely) intrahepatic abscesses. Migration of an adult worm up the esophagus can provoke coughing and oral expulsion of the worm. In highly endemic areas, intestinal and biliary ascariasis can rival acute appendicitis and gallstones as causes of surgical acute abdomen.

1	Laboratory Findings Most cases of ascariasis can be diagnosed by microscopic detection of characteristic Ascaris eggs (65 by 45 μm) in fecal samples. Occasionally, patients present after passing an adult worm— identifiable by its large size and smooth cream-colored surface—in the stool or, much less commonly, through the mouth or nose. During the early transpulmonary migratory phase, when eosinophilic pneumonitis occurs, larvae can be found in sputum or gastric aspirates before diagnostic eggs appear in the stool. The eosinophilia that is prominent during this early stage usually decreases to minimal levels in established infection. Adult worms may be visualized, occasionally serendipitously, on contrast studies of the gastrointestinal tract. A plain abdominal film may reveal masses of worms in gas-filled loops of bowel in patients with intestinal obstruction. Pancreaticobiliary worms can be detected by ultrasound and endoscopic retrograde cholangiopancreatography; the latter method

1	in gas-filled loops of bowel in patients with intestinal obstruction. Pancreaticobiliary worms can be detected by ultrasound and endoscopic retrograde cholangiopancreatography; the latter method also has been used to extract biliary Ascaris worms.

1	Ascariasis should always be treated to prevent potentially serious complications. Albendazole (400 mg once), mebendazole (100 g twice daily for 3 days or 500 mg once), or ivermectin (150–200 μg/kg aTime from infection to egg production by mature female worm. once) is effective. These medications are contraindicated in pregnancy, however. Mild diarrhea and abdominal pain are uncommon side effects of these agents. Partial intestinal obstruction should be managed with nasogastric suction, IV fluid administration, and instillation of piperazine through the nasogastric tube, but complete obstruction and its severe complications require immediate surgical intervention.

1	Two hookworm species (A. duodenale and N. americanus) are responsible for human infections. Most infected individuals are asymptomatic. Hookworm disease develops from a combination of factors—a heavy worm burden, a prolonged duration of infection, and an inadequate iron intake—and results in iron-deficiency anemia and, on occasion, hypoproteinemia.

1	Life Cycle Adult hookworms, which are ~1 cm long, use buccal teeth (Ancylostoma) or cutting plates (Necator) to attach to the small-bowel mucosa and suck blood (0.2 mL/d per Ancylostoma adult) and interstitial fluid. The adult hookworms produce thousands of eggs daily. The eggs are deposited with feces in soil, where rhabditiform larvae hatch and develop over a 1-week period into infectious filariform larvae. Infective larvae penetrate the skin and reach the lungs by way of the bloodstream. There they invade alveoli and ascend the airways before being swallowed and reaching the small intestine. The prepatent period from skin invasion to appearance of eggs in the feces is ~6–8 weeks, but it may be longer with A. duodenale. Larvae of A. duodenale, if swallowed, can survive and develop directly in the intestinal mucosa. Adult hookworms may survive over a decade but usually live ~6–8 years for A. duodenale and 2–5 years for N. americanus. Hot, humid regions Worldwide Worldwide

1	Hot, humid regions Worldwide Worldwide Small-bowel mucosa Cecum, colonic Cecum, appendix mucosa Decades (owing to 5 years 2 months autoinfection) 5000–10,000 3000–7000 2000 Epidemiology A. duodenale is prevalent in southern Europe, North Africa, and northern Asia, and N. americanus is the predominant species in the Western Hemisphere and equatorial Africa. The two species overlap in many tropical regions, particularly Southeast Asia. In most areas, older children have the highest incidence and greatest intensity of hookworm infection. In rural areas where fields are fertilized with human feces, older working adults also may be heavily infected.

1	Clinical Features Most hookworm infections are asymptomatic. Infective larvae may provoke pruritic maculopapular dermatitis (“ground itch”) at the site of skin penetration as well as serpiginous tracks of subcutaneous migration (similar to those of cutaneous larva migrans; Chap. 256) in previously sensitized hosts. Larvae migrating through the lungs occasionally cause mild transient pneumonitis, but this condition develops less frequently in hookworm infection than in ascariasis. In the early intestinal phase, infected persons may develop epigastric pain (often with postprandial accentuation), inflammatory diarrhea, or other abdominal symptoms accompanied by eosinophilia. The major consequence of chronic hookworm infection is iron deficiency. Symptoms are minimal if iron intake is adequate, but marginally nourished individuals develop symptoms of progressive iron-deficiency anemia and hypoproteinemia, including weakness and shortness of breath.

1	Laboratory Findings The diagnosis is established by the finding of characteristic 40by 60-μm oval hookworm eggs in the feces. Stool-concentration procedures may be required to detect light infections. Eggs of the two species are indistinguishable by light microscopy. In a stool sample that is not fresh, the eggs may have hatched to release rhabditiform larvae, which need to be differentiated from those of S. stercoralis. Hypochromic microcytic anemia, occasionally with eosinophilia or hypoalbuminemia, is characteristic of hookworm disease.

1	Hookworm infection can be eradicated with several safe and highly effective anthelmintic drugs, including albendazole (400 mg once) and mebendazole (500 mg once). Mild iron-deficiency anemia can often be treated with oral iron alone. Severe hookworm disease with protein loss and malabsorption necessitates nutritional support and oral iron replacement along with deworming. There is some concern that the benzimidazoles (mebendazole and albendazole) are becoming less effective against human hookworms.

1	Ancylostoma caninum and Ancylostoma braziliense A. caninum, the canine hookworm, has been identified as a cause of human eosinophilic enteritis, especially in northeastern Australia. In this zoonotic infection, adult hookworms attach to the small intestine (where they may be visualized by endoscopy) and elicit abdominal pain and intense local eosinophilia. Treatment with mebendazole (100 mg twice daily for 3 days) or albendazole (400 mg once) or endoscopic removal is effective. Both of these animal hookworm species can cause cutaneous larva migrans (“creeping eruption”; Chap. 256).

1	S. stercoralis is distinguished by its ability—unique among helminths (except for Capillaria; see below)—to replicate in the human host. This capacity permits ongoing cycles of autoinfection as infective larvae are internally produced. Strongyloidiasis can thus persist for decades without further exposure of the host to exogenous infective larvae. In immunocompromised hosts, large numbers of invasive Strongyloides larvae can disseminate widely and can be fatal.

1	Life Cycle In addition to a parasitic cycle of development, Strongyloides can undergo a free-living cycle of development in the soil (Fig. 257-1). This adaptability facilitates the parasite’s survival in the absence of mammalian hosts. Rhabditiform larvae passed in feces can transform into infectious filariform larvae either directly or after a free-living phase of development. Humans acquire strongyloidiasis when filariform larvae in fecally contaminated soil penetrate the skin or mucous membranes. The larvae then travel through the bloodstream to the lungs, where they break into the alveolar spaces, ascend the bronchial tree, are swallowed, and thereby reach the small intestine. There the larvae mature into adult worms that penetrate the mucosa of the proximal small bowel. The minute (2-mm-long) parasitic adult female worms reproduce by parthenogenesis; adult males do not exist. Eggs hatch in the intestinal mucosa, releasing rhabditiform larvae that migrate to the lumen and pass

1	(2-mm-long) parasitic adult female worms reproduce by parthenogenesis; adult males do not exist. Eggs hatch in the intestinal mucosa, releasing rhabditiform larvae that migrate to the lumen and pass with the feces into soil. Alternatively, rhabditiform larvae in the bowel can develop directly into filariform larvae that penetrate the colonic wall or perianal skin and enter the circulation to repeat the migration that establishes ongoing internal reinfection. This autoinfection cycle allows strongyloidiasis to persist for decades.

1	Epidemiology S. stercoralis is spottily dis tributed in tropical areas and other hot, cutaneous and/or abdominal symptoms. Recurrent urticaria, often 1415 involving the buttocks and wrists, is the most common cutaneous manifestation. Migrating larvae can elicit a pathognomonic serpiginous eruption, larva currens (“running larva”). This pruritic, raised, erythematous lesion advances as rapidly as 10 cm/h along the course of larval migration. Adult parasites burrow into the duodenojejunal mucosa and can cause abdominal (usually midepigastric) pain, which resembles peptic ulcer pain except that it is aggravated by food ingestion. Nausea, diarrhea, gastrointestinal bleeding, mild chronic colitis, and weight loss can occur. Small-bowel obstruction may develop with early, heavy infection. Pulmonary symptoms are rare in uncomplicated strongyloidiasis. Eosinophilia is common, with levels fluctuating over time.

1	The ongoing autoinfection cycle of strongyloidiasis is normally constrained by unknown factors of the host’s immune system. Abrogation of host immunity, especially with glucocorticoid therapy and much less commonly with other immunosuppressive medications, leads to hyperinfection, with the generation of large numbers of filariform larvae. Colitis, enteritis, or malabsorption may develop. In disseminated strongyloidiasis, larvae may invade not only gastrointestinal tissues and the lungs but also the central nervous system, peritoneum, liver, and kidneys. Moreover, bacteremia may develop because of the passage of enteric flora through disrupted mucosal barriers. Gram-negative sepsis, pneumonia, or meningitis may complicate or dominate the clinical course. Eosinophilia is often absent in severely infected patients. Disseminated strongyloidiasis, particularly in patients with unsuspected infection who are given glucocorticoids, can be fatal. Strongyloidiasis is a frequent complication of

1	infected patients. Disseminated strongyloidiasis, particularly in patients with unsuspected infection who are given glucocorticoids, can be fatal. Strongyloidiasis is a frequent complication of infection with human

1	Intestinal Nematode InfectionsCHAPTER 2572-mm hermaphroditic adult s penetrate small-bowel mucosa and release eggs, which hatch to rhabditiform larvae. Larvae shed in stool Lung or intestinal stage may cause: Eosinophilia and intermittent epigastric pain Autoinfection: Transform within the intestine into filariform larvae, which penetrate perianal skin or bowel mucosa, causing: Pruritic larva currens Eosinophilia Hyperinfection: With immunosuppression, larger Larvae migrate via bloodstream or lymphatics to lungs, ascend airway to trachea and pharynx, and are swallowed. T cell lymphotropic virus type 1, but disseminated strongyloidiasis is not common among patients infected with HIV-1.

1	T cell lymphotropic virus type 1, but disseminated strongyloidiasis is not common among patients infected with HIV-1. Free-living 1-mm adults in soil Eggs in soil Indirect development (heterogonic) (can multiply outside host for several generations) in soil Direct development Rhabditiform larvae in soil numbers of filariform larvae develop, penetrate bowel, and disseminate, causing: Colitis, polymicrobial sepsis, pneumonitis, or meningitis humid regions and is particularly common in Southeast Asia, sub-Saharan Africa, and Brazil. In the United States, the parasite is endemic in parts of the Southeast and is found in immigrants, refugees, travelers, and military personnel who have lived in endemic areas.

1	FIGuRE 257-1 Life cycle of Strongyloides stercoralis. (Adapted from Guerrant RL et al Clinical Features In uncomplicated strongyloidia-[eds]: Tropical Infectious Diseases: Principles, Pathogens and Practice, 2nd ed, p 1276. © 2006, sis, many patients are asymptomatic or have mild with permission from Elsevier Science.) 1416 Diagnosis In uncomplicated strongyloidiasis, the finding of rhabditiform larvae in feces is diagnostic. Rhabditiform larvae are ~250 μm long, with a short buccal cavity that distinguishes them from hookworm larvae. In uncomplicated infections, few larvae are passed and single stool examinations detect only about one-third of cases. Serial examinations and the use of the agar plate detection method improve the sensitivity of stool diagnosis. In uncomplicated strongyloidiasis (but not in hyperinfection), stool examinations may be repeatedly negative. Strongyloides larvae may also be found by sampling of the duodenojejunal contents by aspiration or biopsy. An

1	(but not in hyperinfection), stool examinations may be repeatedly negative. Strongyloides larvae may also be found by sampling of the duodenojejunal contents by aspiration or biopsy. An enzyme-linked immunosorbent assay for serum antibodies to antigens of Strongyloides is a sensitive method for diagnosing uncomplicated infections. Such serologic testing should be performed for patients whose geographic histories indicate potential exposure, especially those who exhibit eosinophilia and/or are candidates for glucocorticoid treatment of other conditions. In disseminated strongyloidiasis, filariform larvae should be sought in stool as well as in samples obtained from sites of potential larval migration, including sputum, bronchoalveolar lavage fluid, or surgical drainage fluid.

1	Even in the asymptomatic state, strongyloidiasis must be treated because of the potential for subsequent dissemination and fatal hyperinfection. Ivermectin (200 μg/kg daily for 2 days) is consistently more effective than albendazole (400 mg daily for 3 days). For disseminated strongyloidiasis, treatment with ivermectin should be extended for at least 5–7 days or until the parasites have been eradicated. In immunocompromised hosts, the course of ivermectin should be repeated 2 weeks after initial treatment. Most infections with Trichuris trichiura are asymptomatic, but heavy infections may cause gastrointestinal symptoms. Like the other soil-transmitted helminths, whipworm is distributed globally in the tropics and subtropics and is most common among poor children from resource-poor regions of the world.

1	Life Cycle Adult Trichuris worms reside in the colon and cecum, the anterior portions threaded into the superficial mucosa. Thousands of eggs laid daily by adult female worms pass with the feces and mature in the soil. After ingestion, infective eggs hatch in the duodenum, releasing larvae that mature before migrating to the large bowel. The entire cycle takes ~3 months, and adult worms may live for several years. Clinical Features Tissue reactions to Trichuris are mild. Most infected individuals have no symptoms or eosinophilia. Heavy infections may result in anemia, abdominal pain, anorexia, and bloody or mucoid diarrhea resembling inflammatory bowel disease. Rectal prolapse can result from massive infections in children, who often suffer from malnourishment and other diarrheal illnesses. Moderately heavy Trichuris burdens also contribute to growth retardation.

1	Diagnosis and Treatment The characteristic 50by 20-μm lemon-shaped Trichuris eggs are readily detected on stool examination. Adult worms, which are 3–5 cm long, are occasionally seen on proctoscopy. Mebendazole (500 mg once) or albendazole (400 mg daily for 3 doses) is safe and moderately effective for treatment, with cure rates of 70–90%. Ivermectin (200 μg/kg daily for 3 doses) is also safe but is not quite as efficacious as the benzimidazoles. E. vermicularis is more common in temperate countries than in the tropics. In the United States, ~40 million persons are infected with pinworms, with a disproportionate number of cases among children.

1	Life Cycle and Epidemiology Enterobius adult worms are ~1 cm long and dwell in the cecum. Gravid female worms migrate nocturnally into the perianal region and release up to 2000 immature eggs each. The eggs become infective within hours and are transmitted by hand-to-mouth passage. From ingested eggs, larvae hatch and mature into adults. This life cycle takes ~1 month, and adult worms survive for ~2 months. Self-infection results from perianal scratching and transport of infective eggs on the hands or under the nails to the mouth. Because of the ease of person-to-person spread, pinworm infections are common among family members.

1	Clinical Features Most pinworm infections are asymptomatic. Perianal pruritus is the cardinal symptom. The itching, which is often worse at night as a result of the nocturnal migration of the female worms, may lead to excoriation and bacterial superinfection. Heavy infections have been alleged to cause abdominal pain and weight loss. On rare occasions, pinworms invade the female genital tract, causing vulvovaginitis and pelvic or peritoneal granulomas. Eosinophilia is uncommon. Diagnosis Since pinworm eggs are not released in feces, the diagnosis cannot be made by conventional fecal ova and parasite tests. Instead, eggs are detected by the application of clear cellulose acetate tape to the perianal region in the morning. After the tape is transferred to a slide, microscopic examination will detect pinworm eggs, which are oval, measure 55 by 25 μm, and are flattened along one side.

1	Infected children and adults should be treated with mebendazole (100 mg once) or albendazole (400 mg once), with the same treatment repeated after 2 weeks. Treatment of household members is advocated to eliminate asymptomatic reservoirs of potential reinfection. Trichostrongylus species, which are normally parasites of herbivo rous animals, occasionally infect humans, particularly in Asia and Africa. Humans acquire the infection by accidentally ingesting Trichostrongylus larvae on contaminated leafy vegetables. The larvae do not migrate in humans but mature directly into adult worms in the small bowel. These worms ingest far less blood than hookworms; most infected persons are asymptomatic, but heavy infections may give rise to mild anemia and eosinophilia. In stool examinations, Trichostrongylus eggs resemble hookworm eggs but are larger (85 by 115 μm). Treatment consists of mebendazole or albendazole (Chap. 246e).

1	Anisakiasis is a gastrointestinal infection caused by the accidental ingestion in uncooked saltwater fish of nematode larvae belonging to the family Anisakidae. The incidence of anisakiasis in the United States has increased as a result of the growing popularity of raw fish dishes. Most cases occur in Japan, the Netherlands, and Chile, where raw fish—sashimi, pickled green herring, and ceviche, respectively—are national culinary staples. Anisakid nematodes parasitize large sea mammals such as whales, dolphins, and seals. As part of a complex parasitic life cycle involving marine food chains, infectious larvae migrate to the musculature of a variety of fish. Both Anisakis simplex and Pseudoterranova decipiens have been implicated in human anisakiasis, but an identical gastric syndrome may be caused by the red larvae of eustrongylid parasites of fish-eating birds. When humans consume infected raw fish, live larvae may be coughed up within 48 h. Alternatively, larvae may immediately

1	may be caused by the red larvae of eustrongylid parasites of fish-eating birds. When humans consume infected raw fish, live larvae may be coughed up within 48 h. Alternatively, larvae may immediately penetrate the mucosa of the stomach. Within hours, violent upper abdominal pain accompanied by nausea and occasionally vomiting ensues, mimicking an acute abdomen. The diagnosis can be established by direct visualization on upper endoscopy, outlining of the worm by contrast radiographic studies, or histopathologic examination of extracted tissue. Extraction of the burrowing larvae during endoscopy is curative. In addition, larvae may pass to the small bowel, where they penetrate the mucosa and provoke a vigorous eosinophilic granulomatous response. Symptoms may appear 1–2 weeks after the infective meal, with intermittent abdominal pain, diarrhea, nausea, and fever resembling the manifestations of Crohn’s disease. The diagnosis may be suggested by barium studies and confirmed by curative

1	meal, with intermittent abdominal pain, diarrhea, nausea, and fever resembling the manifestations of Crohn’s disease. The diagnosis may be suggested by barium studies and confirmed by curative surgical resection of a granuloma in which the worm is embedded. Anisakid eggs are not found in the stool, since the larvae do not mature in humans. Serologic tests have been developed but are not widely available.

1	Anisakid larvae in saltwater fish are killed by cooking to 60°C, freezing at -20°C for 3 days, or commercial blast freezing, but usually not by salting, marinating, or cold smoking. No medical treatment is available; surgical or endoscopic removal should be undertaken.

1	Intestinal capillariasis is caused by ingestion of raw fish infected with Capillaria philippinensis. Subsequent autoinfec tion can lead to a severe wasting syndrome. The disease occurs in the Philippines and Thailand and, on occasion, elsewhere in Asia. The natural cycle of C. philippinensis involves fish from fresh and brackish water. When humans eat infected raw fish, the larvae mature in the intestine into adult worms, which produce invasive larvae that cause intestinal inflammation and villus loss. Capillariasis has an insidious onset with nonspecific abdominal pain and watery diarrhea. If untreated, progressive autoinfection can lead to protein-losing enteropathy, severe malabsorption, and ultimately death from cachexia, cardiac failure, or superinfection. The diagnosis is established by identification of the characteristic peanut-shaped (20by 40-μm) eggs on stool examination. Severely ill patients require hospitalization and supportive therapy in addition to prolonged

1	established by identification of the characteristic peanut-shaped (20by 40-μm) eggs on stool examination. Severely ill patients require hospitalization and supportive therapy in addition to prolonged anthelmintic treatment with albendazole (200 mg twice daily for 10 days; Chap. 246e).

1	Abdominal angiostrongyliasis is found in Latin America and Africa. The zoonotic parasite Angiostrongylus costaricensis causes eosinophilic ileocolitis after the ingestion of contaminated vegetation. A. costaricensis normally parasitizes the cotton rat and other rodents, with slugs and snails serving as intermediate hosts. Humans become infected by accidentally ingesting infective larvae in mollusk slime deposited on fruits and vegetables; children are at highest risk. The larvae penetrate the gut wall and migrate to the mesenteric artery, where they develop into adult worms. Eggs deposited in the gut wall provoke an intense eosinophilic granulomatous reaction, and adult worms may cause mesenteric arteritis, thrombosis, or frank bowel infarction. Symptoms may mimic those of appendicitis, including abdominal pain and tenderness, fever, vomiting, and a palpable mass in the right iliac fossa. Leukocytosis and eosinophilia are prominent. CT with contrast medium typically shows inflamed

1	including abdominal pain and tenderness, fever, vomiting, and a palpable mass in the right iliac fossa. Leukocytosis and eosinophilia are prominent. CT with contrast medium typically shows inflamed bowel, often with concomitant obstruction, but a definitive diagnosis is usually made surgically with partial bowel resection. Pathologic study reveals a thickened bowel wall with eosinophilic granulomas surrounding the Angiostrongylus eggs. In nonsurgical cases, the diagnosis rests solely on clinical grounds because larvae and eggs cannot be detected in the stool. Medical therapy for abdominal angiostrongyliasis is of uncertain efficacy. Careful observation and surgical resection for severe symptoms are the mainstays of treatment.

1	Thomas B. Nutman, Peter F. Weller

1	Filarial worms are nematodes that dwell in the subcutaneous tissues and the lymphatics. Eight filarial species infect humans (Table 258-1); of these, four—Wuchereria bancrofti, Brugia malayi, Onchocerca volvulus, and Loa loa—are responsible for most serious filarial infections. Filarial parasites, which infect an estimated 170 million persons worldwide, are transmitted by specific species of mosquitoes or other arthropods and have a complex life cycle, including infective larval stages carried by insects and adult worms that reside in either lymphatic or subcutaneous tissues of humans. The offspring of adults are microfi-1417 lariae, which, depending on their species, are 200–250 μm long and 5–7 μm wide, may or may not be enveloped in a loose sheath, and either circulate in the blood or migrate through the skin (Table 258-1). To complete the life cycle, microfilariae are ingested by the arthropod vector and develop over 1–2 weeks into new infective larvae. Adult worms live for many

1	migrate through the skin (Table 258-1). To complete the life cycle, microfilariae are ingested by the arthropod vector and develop over 1–2 weeks into new infective larvae. Adult worms live for many years, whereas microfilariae survive for 3–36 months. The bacterial endosymbiont Wolbachia has been found intracellularly in all stages of Brugia, Wuchereria, Mansonella, and Onchocerca species and has become a target for antifilarial chemotherapy.

1	Usually, infection is established only with repeated, prolonged exposures to infective larvae. Since the clinical manifestations of filarial diseases develop relatively slowly, these infections should be considered to induce chronic infections with possible long-term debilitating effects. In terms of the nature, severity, and timing of clinical manifestations, patients with filarial infections who are native to endemic areas and have lifelong exposure may differ significantly from those who are travelers or who have recently moved to these areas. Characteristically, filarial disease is more acute and intense in newly exposed individuals than in natives of endemic areas. Lymphatic filariasis is caused by W. bancrofti, B. malayi, or B. timori. The threadlike adult parasites reside in lymphatic channels or lymph nodes, where they may remain viable for more than two decades.

1	W. bancrofti, the most widely distributed filarial parasite of humans, affects an estimated 110 million people and is found throughout the tropics and subtropics, including Asia and the Pacific Islands, Africa, areas of South America, and the Caribbean basin. Humans are the only definitive host for the parasite. Generally, the subperiodic form is found only in the Pacific Islands; elsewhere, W. bancrofti is nocturnally periodic. Nocturnally periodic forms of microfilariae are scarce in peripheral blood by day and increase at night, whereas subperiodic forms are present in peripheral blood at all times and reach maximal levels in the afternoon. Natural vectors for W. bancrofti are Culex fatigans mosquitoes in urban settings and Anopheles or Aedes mosquitoes in rural areas.

1	W. bancrofti are Culex fatigans mosquitoes in urban settings and Anopheles or Aedes mosquitoes in rural areas. Brugian filariasis due to B. malayi occurs primarily in eastern India, Indonesia, Malaysia, and the Philippines. B. malayi also has two forms distinguished by the periodicity of microfilaremia. The more common nocturnal form is transmitted in areas of coastal rice fields, while the subperiodic form is found in forests. B. malayi naturally infects cats as well as humans. The distribution of B. timori is limited to the islands of southeastern Indonesia.

1	The principal pathologic changes result from inflammatory damage to the lymphatics, which is typically caused by adult worms and not by microfilariae. Adult worms live in afferent lymphatics or sinuses of lymph nodes and cause lymphatic dilation and thickening of the vessel walls. The infiltration of plasma cells, eosinophils, and macrophages in and around the infected vessels, along with endothelial and connective tissue proliferation, leads to tortuosity of the lymphatics and damaged or incompetent lymph valves. Lymphedema and chronic stasis changes with hard or brawny edema develop in the overlying skin. These consequences of filarial infection are due both to the direct effects of the worms and to the host’s inflammatory response to the parasite. Inflammatory responses are believed to cause the granulomatous and proliferative processes that precede total lymphatic obstruction. It is thought that the lymphatic vessel remains patent as long as the worm remains viable and that the

1	to cause the granulomatous and proliferative processes that precede total lymphatic obstruction. It is thought that the lymphatic vessel remains patent as long as the worm remains viable and that the death of the worm leads to enhanced granulomatous reactions and fibrosis. Lymphatic obstruction results, and, despite collateralization, lymphatic function is compromised.

1	The most common presentations of the lymphatic filariases are asymptomatic (or subclinical) microfilaremia, hydrocele (Fig. 258-1), acute adenolymphangitis (ADL), and chronic lymphatic disease. In areas where W. bancrofti or B. malayi is endemic, the overwhelming majority of infected individuals have few overt clinical manifestations of filarial infection despite large numbers of circulating microfilariae in the peripheral blood. Although they may be clinically asymptomatic, virtually all persons with W. bancrofti or B. malayi microfilaremia have some degree of subclinical disease that includes microscopic hematuria and/or proteinuria, dilated (and tortuous) lymphatics (visualized by imaging), and—in men with W. bancrofti infection—scrotal lymphangiectasia (detectable by ultrasound). Despite these findings, the majority of individuals appear to remain clinically asymptomatic for years; in relatively few does the infection progress to either acute or chronic disease.

1	ADL is characterized by high fever, lymphatic inflammation (lymphangitis and lymphadenitis), and transient local edema. The lymphangitis is retrograde, extending peripherally from the lymph node draining the area where the adult parasites reside. Regional lymph nodes are often enlarged, and the entire lymphatic channel can become indurated and inflamed. Concomitant local thrombophlebitis can occur as well. In brugian filariasis, a single local abscess may form along the involved lymphatic tract and subsequently rupture to the surface. The lymphadenitis and lymphangitis can involve both the upper and lower extremities in both bancroftian and brugian filariasis, but involvement of the genital lymphatics occurs almost exclusively with W. bancrofti infection. This genital involvement can be manifested by funiculitis, epididymitis, and scrotal pain and tenderness. In endemic areas, another type of acute disease—dermatolymphangioadenitis (DLA)—is recognized as a syndrome that includes high

1	by funiculitis, epididymitis, and scrotal pain and tenderness. In endemic areas, another type of acute disease—dermatolymphangioadenitis (DLA)—is recognized as a syndrome that includes high fever, chills, myalgias, and headache. Edematous inflammatory plaques clearly demarcated from normal skin are seen. Vesicles, ulcers, and hyperpigmentation may also be noted. There is often a history of trauma, burns, irradiation, insect bites, punctiform lesions, or chemical injury. Entry lesions, especially in the interdigital area, are common. DLA is often diagnosed as cellulitis.

1	FIGuRE 258-1 Hydrocele associated with Wuchereria bancrofti infection. If lymphatic damage progresses, transient lymphedema can develop into lymphatic obstruction and the permanent changes associated with elephantiasis (Fig. 258-2). Brawny edema follows early pitting edema, the subcutaneous tissues thicken, and hyperkeratosis occurs. Fissuring of the skin develops, as do hyperplastic changes. Superinfection of these poorly vascularized tissues becomes a problem. In bancroftian filariasis, in which genital involvement is common, hydroceles may develop (Fig. 258-1); in advanced stages, this condition may evolve into scrotal lymphedema and scrotal elephantiasis. Furthermore, if there is obstruction of the retroperitoneal lymphatics, increased renal lymphatic pressure leads to rupture of the renal lymphatics and the development of chyluria, which is usually intermittent and most prominent in the morning.

1	The clinical manifestations of filarial infections in travelers or trans-migrants who have recently entered an endemic region are distinctive. Given a sufficient number of bites by infected vectors, usually over a 3to 6-month period, recently exposed patients can develop acute lymphatic or scrotal inflammation with or without urticaria and localized angioedema. Lymphadenitis of epitrochlear, axillary, femoral, or inguinal lymph nodes is often followed by retrogradely evolving lymphangitis. Acute attacks are short-lived and are not usually accompanied by fever. With prolonged exposure to infected mosquitoes, these attacks, if untreated, become more severe and lead to permanent lymphatic inflammation and obstruction.

1	A definitive diagnosis can be made only by detection of the parasites and hence can be difficult. Adult worms localized in lymphatic vessels or nodes are largely inaccessible. Microfilariae can be found in blood, in hydrocele fluid, or (occasionally) in other body fluids. Such fluids can be examined microscopically, either directly or—for greater sensitivity—after concentration of the parasites by the passage of fluid through a polycarbonate cylindrical-pore filter (pore size, 3 μm) or by the centrifugation of fluid fixed in 2% formalin (Knott’s concentration technique). The timing of blood collection is critical and should be based on the periodicity of the microfilariae in the endemic region involved. Many infected individuals do not have microfilaremia, and definitive diagnosis in such cases can be difficult. Assays for circulating antigens of W. bancrofti permit the diagnosis of microfilaremic and cryptic (amicrofilaremic) infection. Two tests are commercially available: an

1	such cases can be difficult. Assays for circulating antigens of W. bancrofti permit the diagnosis of microfilaremic and cryptic (amicrofilaremic) infection. Two tests are commercially available: an enzyme-linked immunosorbent assay (ELISA) and a rapid-format immunochromatographic card test. Both assays have sensitivities of 93–100% and specificities approaching 100%. There are currently no tests for circulating antigens in brugian filariasis.

1	FIGuRE 258-2 Elephantiasis of the lower extremity associated with Wuchereria bancrofti infection. Polymerase chain reaction (PCR)–based assays for DNA of W. bancrofti and B. malayi in blood have been developed. A number of studies indicate that the sensitivity of this diagnostic method is equivalent to or greater than that of parasitologic methods.

1	In cases of suspected lymphatic filariasis, examination of the scrotum, the lymph nodes, or (in female patients) the breast by means of high-frequency ultrasound in conjunction with Doppler techniques may result in the identification of motile adult worms within dilated lymphatics. Worms may be visualized in the lymphatics of the spermatic cord in up to 80% of men infected with W. bancrofti. Live adult worms have a distinctive pattern of movement within the lymphatic vessels (termed the filarial dance sign). Radionuclide lymphoscintigraphic imaging of the limbs reliably demonstrates widespread lymphatic abnormalities in both subclinical microfilaremic persons and those with clinical manifestations of lymphatic pathology. Although of potential utility in the delineation of anatomic changes associated with infection, lymphoscintigraphy is unlikely to assume primacy in the diagnostic evaluation of individuals with suspected infection; it is principally a research tool, although it has

1	associated with infection, lymphoscintigraphy is unlikely to assume primacy in the diagnostic evaluation of individuals with suspected infection; it is principally a research tool, although it has been used more widely for assessment of lymphedema of any cause. Eosinophilia and elevated serum concentrations of IgE and antifilarial antibody support the diagnosis of lymphatic filariasis. There is, however, exten-1419 sive cross-reactivity between filarial antigens and antigens of other helminths, including the common intestinal roundworms; thus, interpretations of serologic findings can be difficult. In addition, residents of endemic areas can become sensitized to filarial antigens (and thus be serologically positive) through exposure to infected mosquitoes without having patent filarial infections.

1	The ADL associated with lymphatic filariasis must be distinguished from thrombophlebitis, infection, and trauma. Retrograde evolution is a characteristic feature that helps distinguish filarial lymphangitis from ascending bacterial lymphangitis. Chronic filarial lymphedema must also be distinguished from the lymphedema of malignancy, postoperative scarring, trauma, chronic edematous states, and congenital lymphatic system abnormalities. With newer definitions of clinical syndromes in lymphatic filariasis and new tools to assess clinical status (e.g., ultrasound, lymphoscintigraphy, circulating filarial antigen assays, PCR), approaches to treatment based on infection status can be considered.

1	Orally administered diethylcarbamazine (DEC; 6 mg/kg daily for 12 days), which has both macroand microfilaricidal properties, remains the drug of choice for the treatment of active lymphatic filariasis (defined by microfilaremia, antigen positivity, or adult worms on ultrasound), although albendazole (400 mg twice daily by mouth for 21 days) has also demonstrated macrofilaricidal efficacy. A 4to 6-week course of oral doxycycline (targeting the intracellular Wolbachia) also has significant macrofilaricidal activity, as does DEC/ albendazole used daily for 7 days. The addition of DEC to a 3-week course of doxycycline is efficacious in lymphatic filariasis.

1	Regimens that combine single doses of albendazole (400 mg) with either DEC (6 mg/kg) or ivermectin (200 μg/kg) all have a sustained microfilaricidal effect and are the mainstay of programs for the eradication of lymphatic filariasis in Africa (albendazole/ ivermectin) and elsewhere (albendazole/DEC) (see “Prevention and Control,” below).

1	As has already been mentioned, a growing body of evidence indicates that, although they may be asymptomatic, virtually all persons with W. bancrofti or B. malayi microfilaremia have some degree of subclinical disease (hematuria, proteinuria, abnormalities on lymphoscintigraphy). Thus, early treatment of asymptomatic persons who have microfilaremia is recommended to prevent further lymphatic damage. For ADL, supportive treatment (including the administration of antipyretics and analgesics) is recommended, as is antibiotic therapy if secondary bacterial infection is likely. Similarly, because lymphatic disease is associated with the presence of adult worms, treatment with DEC is recommended for microfilaria-negative carriers of adult worms.

1	In persons with chronic manifestations of lymphatic filariasis, treatment regimens that emphasize hygiene, prevention of secondary bacterial infections, and physiotherapy have gained wide acceptance for morbidity control. These regimens are similar to those recommended for lymphedema of most nonfilarial causes and are known by a variety of names, including complex decongestive physiotherapy and complex lymphedema therapy. Hydroceles (Fig. 258-1) can be managed surgically. With chronic manifestations of lymphatic filariasis, drug treatment should be reserved for individuals who have evidence of active infection; however, a 6-week course of doxycycline has been shown to provide improvement in filarial lymphedema irrespective of disease activity.

1	Side effects of DEC treatment include fever, chills, arthralgias, headaches, nausea, and vomiting. Both the development and the severity of these reactions are directly related to the number of microfilariae circulating in the bloodstream. The adverse reactions may represent either an acute hypersensitivity reaction to the antigens being released by dead and dying parasites or an inflammatory reaction induced by the intracellular Wolbachia endosymbionts freed from their intracellular niche. 1420 Ivermectin has a side effect profile similar to that of DEC when used in lymphatic filariasis. In patients infected with L. loa who have high levels of microfilaremia, DEC—like ivermectin (see “Loiasis,” below)—can elicit severe encephalopathic complications. When used in single-dose regimens for the treatment of lymphatic filariasis, albendazole is associated with relatively few side effects.

1	To protect themselves against filarial infection, individuals must avoid contact with infected mosquitoes by using personal protective measures, including bed nets, particularly those impregnated with insecticides such as permethrin. Community-based intervention is the current approach to elimination of lymphatic filariasis as a public health problem. The underlying tenet of this approach is that mass annual distribution of antimicrofilarial chemotherapy—albendazole with either DEC (for all areas except those where onchocerciasis is coendemic; see section on onchocerciasis treatment, below) or ivermectin—will profoundly suppress microfilaremia. If the suppression is sustained, then transmission can be interrupted. Created by the World Health Organization in 1997, the Global

1	Created by the World Health Organization in 1997, the Global Programme to Eliminate Lymphatic Filariasis is based on mass administration of single annual doses of DEC plus albendazole in non-African regions and of albendazole plus ivermectin in Africa. Available information from late 2013 indicated that more than 792 million persons in 53 countries had thus far participated. Not only has lymphatic filariasis been eliminated in some defined areas, but collateral benefits—avoidance of disability and treatment of intestinal helminths and other conditions (e.g., scabies and louse infestation)— have also been noted. The strategy of the global program is being refined, and attempts are being made to integrate this effort with other mass-treatment strategies (e.g., deworming programs, malaria control, and trachoma control) in an integrated control strategy.

1	Tropical pulmonary eosinophilia (TPE) is a distinct syndrome that develops in some individuals infected with the lymphatic- dwelling filarial species. This syndrome affects males and females in a ratio of 4:1, often during the third decade of life. The majority of cases have been reported from India, Pakistan, Sri Lanka, Brazil, Guyana, and Southeast Asia.

1	Clinical Features The main features include a history of residence in filarial-endemic regions, paroxysmal cough and wheezing (usually nocturnal and probably related to the nocturnal periodicity of microfilariae), weight loss, low-grade fever, lymphadenopathy, and pronounced blood eosinophilia (>3000 eosinophils/μL). Chest x-rays or CT scans may be normal but generally show increased bronchovascular markings. Diffuse miliary lesions or mottled opacities may be present in the middle and lower lung fields. Tests of pulmonary function show restrictive abnormalities in most cases and obstructive defects in half. Characteristically, total serum IgE levels (4–40 KIU/ mL) and antifilarial antibody titers are markedly elevated.

1	Pathology In TPE, microfilariae and parasite antigens are rapidly cleared from the bloodstream by the lungs. The clinical symptoms result from allergic and inflammatory reactions elicited by the cleared parasites. In some patients, trapping of microfilariae in other reticuloendothelial organs can cause hepatomegaly, splenomegaly, or lymphadenopathy. A prominent, eosinophil-enriched, intraalveolar infiltrate is often reported, and with it comes the release of cytotoxic proinflammatory eosinophil granule proteins that may mediate some of the pathology seen in TPE. In the absence of successful treatment, interstitial fibrosis can lead to progressive pulmonary damage.

1	Differential Diagnosis TPE must be distinguished from asthma, Löffler’s syndrome, allergic bronchopulmonary aspergillosis, allergic granulomatosis with angiitis (Churg-Strauss syndrome), the systemic vasculitides (most notably, periarteritis nodosa and granulomatosis with polyangiitis), chronic eosinophilic pneumonia, and the idiopathic hypereosinophilic syndrome. DEC is used at a daily dosage of 4–6 mg/kg for 14 days. Symptoms usually resolve within 3–7 days after the initiation of therapy. Relapse, which occurs in ~12–25% of cases (sometimes after an interval of years), requires re-treatment.

1	Onchocerciasis (“river blindness”) is caused by the filarial nematode O. volvulus, which infects an estimated 37 million individuals in 35 countries worldwide. The majority of individuals infected with O. volvulus live in the equatorial region of Africa extending from the Atlantic coast to the Red Sea. In the Americas, isolated foci were identified in Mexico, Guatemala, Colombia, Ecuador, Venezuela, and Brazil. The infection is also found in Yemen.

1	Infection in humans begins with the deposition of infective larvae on the skin by the bite of an infected blackfly. The larvae develop into adults, which are typically found in subcutaneous nodules. About 7 months to 3 years after infection, the gravid female releases microfilariae that migrate out of the nodule and throughout the tissues, concentrating in the dermis. Infection is transmitted to other persons when a female fly ingests microfilariae from the host’s skin and these microfilariae then develop into infective larvae. Adult O. volvulus females and males are ~40–60 cm and ~3–6 cm in length, respectively. The life span of adults can be as long as 18 years, with an average of ~9 years. Because the blackfly vector breeds along free-flowing rivers and streams (particularly in rapids) and generally restricts its flight to an area within several kilometers of these breeding sites, both biting and disease transmission are most intense in these locations.

1	Onchocerciasis primarily affects the skin, eyes, and lymph nodes. In contrast to the pathology in lymphatic filariasis, the damage in onchocerciasis is elicited by microfilariae and not by adult parasites. In the skin, there are mild but chronic inflammatory changes that can result in loss of elastic fibers, atrophy, and fibrosis. The subcutaneous nodules (onchocercomata) consist primarily of fibrous tissues surrounding the adult worm, often with a peripheral ring of inflammatory cells (characterized as lymphatic in origin) surrounded by an endothelial layer. In the eye, neovascularization and corneal scarring lead to corneal opacities and blindness. Inflammation in the anterior and posterior chambers frequently results in anterior uveitis, chorioretinitis, and optic atrophy. Although punctate opacities are due to an inflammatory reaction surrounding dead or dying microfilariae, the pathogenesis of most manifestations of onchocerciasis is still unclear.

1	CLINICAL FEATuRES Skin Pruritus and rash are the most common manifestations of onchocerciasis. The pruritus can be incapacitating; the rash is typically a papular eruption (Fig. 258-3) that is generalized rather than localized to a particular region of the body. Long-term infection results in exaggerated and premature wrinkling of the skin, loss of elastic fibers, and epidermal atrophy that can lead to loose, redundant skin and hypoor hyperpigmentation. Localized eczematoid dermatitis can cause hyperkeratosis, scaling, and pigmentary changes. In an immunologically hyperreactive form of onchodermatitis (commonly termed sowdah or localized onchodermatitis), the affected skin darkens as a consequence of the profound inflammation that occurs as microfilariae in the skin are cleared.

1	Onchocercomata These subcutaneous nodules, which can be palpable and/or visible, contain the adult worm. In African patients, they are common over the coccyx and sacrum, the trochanter of the femur, the lateral anterior crest, and other bony prominences; in patients from South and Central America, nodules tend to develop preferentially in the upper part of the body, particularly on the head, neck, and shoulders. Nodules vary in size and characteristically are firm and not tender. It has been estimated that, for every palpable nodule, there are four deeper nonpalpable ones. FIGuRE 258-3 Papular eruption as a consequence of onchocerciasis.

1	FIGuRE 258-3 Papular eruption as a consequence of onchocerciasis. Ocular Tissue Visual impairment is the most serious complication of onchocerciasis and usually affects only those persons with moderate or heavy infections. Lesions may develop in all parts of the eye. The most common early finding is conjunctivitis with photophobia. Punctate keratitis—acute inflammatory reactions surrounding dying microfilariae and manifested as “snowflake” opacities—is common among younger patients and resolves without apparent complications.

1	Sclerosing keratitis occurs in 1–5% of infected persons and is the leading cause of onchocercal blindness in Africa. Anterior uveitis and iridocyclitis develop in ~5% of infected persons in Africa. In Latin America, complications of the anterior uveal tract (pupillary deformity) may cause secondary glaucoma. Characteristic chorioretinal lesions develop as a result of atrophy and hyperpigmentation of the retinal pigment epithelium. Constriction of the visual fields and overt optic atrophy may occur. Lymph Nodes Mild to moderate lymphadenopathy is common, particularly in the inguinal and femoral areas, where the enlarged nodes may hang down in response to gravity (“hanging groin”), sometimes predisposing to inguinal and femoral hernias. Systemic Manifestations Some heavily infected individuals develop cachexia with loss of adipose tissue and muscle mass. Among adults who become blind, there is a threeto fourfold increase in the mortality rate.

1	Definitive diagnosis depends on the detection of an adult worm in an excised nodule or, more commonly, of microfilariae in a skin snip. Skin snips are obtained with a corneal-scleral punch, which collects a blood-free skin biopsy sample extending to just below the epidermis, or by lifting of the skin with the tip of a needle and excision of a small (1to 3-mm) piece with a sterile scalpel blade. The biopsy tissue is incubated in tissue culture medium or in saline on a glass slide or flat-bottomed microtiter plate. After incubation for 2–4 h (or occasionally overnight in light infections), microfilariae emergent from the skin can be seen by low-power microscopy. Eosinophilia and elevated serum IgE levels are common but, because they occur in many parasitic infections, are not diagnostic in themselves. Assays to detect specific antibodies to Onchocerca and 1421 PCR to detect onchocercal DNA in skin snips are used in specialized laboratories and are highly sensitive and specific.

1	The main goals of therapy are to prevent the development of irreversible lesions and to alleviate symptoms. Surgical excision is recommended when nodules are located on the head (because of the proximity of microfilaria-producing adult worms to the eye), but chemotherapy is the mainstay of management. Ivermectin, a semisynthetic macrocyclic lactone active against microfilariae, is the first-line agent for the treatment of onchocerciasis. It is given orally in a single dose of 150 μg/kg, either yearly or semiannually. More frequent ivermectin administration (every 3 months) has been suggested to ameliorate pruritus and skin disease After treatment, most individuals have few or no reactions.

1	After treatment, most individuals have few or no reactions. Pruritus, cutaneous edema, and/or maculopapular rash occurs in ~1–10% of treated individuals. In areas of Africa coendemic for O. volvulus and L. loa, however, ivermectin is contraindicated (as it is for pregnant or breast-feeding women) because of severe posttreatment encephalopathy, especially in patients who are heavily microfilaremic for L. loa (>8000 microfilariae/mL). Although ivermectin treatment results in a marked drop in microfilarial density, its effect can be short-lived (<3 months in some cases). Thus, it is occasionally necessary to give ivermectin more frequently for persistent symptoms. A 6-week course of doxycycline is macrofilaristatic, rendering female adult worms sterile for long periods.

1	A 6-week course of doxycycline is macrofilaristatic, rendering female adult worms sterile for long periods. Vector control has been beneficial in highly endemic areas in which breeding sites are vulnerable to insecticide spraying, but most areas endemic for onchocerciasis are not suited to this type of control. Community-based administration of ivermectin every 6–12 months is being used to interrupt transmission in endemic areas. This measure, in conjunction with vector control, has already helped eliminate the infection in most of Latin America and has reduced the prevalence of disease in many endemic foci in Africa. No drug has proved useful for prophylaxis of O. volvulus infection.

1	Loiasis is caused by L. loa (the African eye worm), which is present in the rainforests of West and Central Africa. Adult parasites (females, 50–70 mm long and 0.5 mm wide; males, 25–35 mm long and 0.25 mm wide) live in subcutaneous tissues. Microfilariae circulate in the blood with a diurnal periodicity that peaks between 12:00 noon and 2:00 p.m.

1	Manifestations of loiasis in natives of endemic areas may differ from those in temporary residents or visitors. Among the indigenous population, loiasis is often an asymptomatic infection with microfilaremia. Infection may be recognized only after subconjunctival migration of an adult worm (Fig. 258-4) or may be manifested by episodic Calabar swellings—evanescent localized areas of angioedema and erythema developing on the extremities and less frequently at other sites. Nephropathy, encephalopathy, and cardiomyopathy can occur but are rare. In patients who are not residents of endemic areas, allergic symptoms predominate, episodes of Calabar swelling tend to be more frequent and debilitating, microfilaremia is less common, and eosinophilia and increased levels of antifilarial antibodies are characteristic. The pathogenesis of the manifestations of loiasis is poorly understood. Calabar swellings are thought to result from a hypersensitivity reaction to adult worm antigens.

1	The pathogenesis of the manifestations of loiasis is poorly understood. Calabar swellings are thought to result from a hypersensitivity reaction to adult worm antigens. FIGuRE 258-4 Adult Loa loa worm being surgically removed after its subconjunctival migration.

1	FIGuRE 258-4 Adult Loa loa worm being surgically removed after its subconjunctival migration. Definitive diagnosis of loiasis requires the detection of microfilariae in the peripheral blood or the isolation of the adult worm from the eye (Fig. 258-4) or from a subcutaneous biopsy specimen collected from a site of swelling developing after treatment. PCR-based assays for the detection of L. loa DNA in blood are available in specialized laboratories and are highly sensitive and specific, as are some newer recombinant antigen–based serologic techniques. In practice, the diagnosis must often be based on a characteristic history and clinical presentation, blood eosinophilia, and elevated levels of antifilarial antibodies, particularly in travelers to an endemic region, who are usually amicrofilaremic. Other clinical findings in travelers include hypergammaglobulinemia, elevated levels of serum IgE, and elevated leukocyte and eosinophil counts.

1	DEC (8–10 mg/kg per day administered orally for 21 days) is effective against both the adult and the microfilarial forms of L. loa, but multiple courses are frequently necessary before loiasis resolves completely. In cases of heavy microfilaremia, allergic or other inflammatory reactions can take place during treatment, including central nervous system involvement with coma and encephalitis. Heavy infections can be treated initially with apheresis to remove the microfilariae and with glucocorticoids (40–60 mg of prednisone per day) followed by doses of DEC (0.5 mg/kg per day). If antifilarial treatment has no adverse effects, the prednisone dose can be rapidly tapered and the dose of DEC gradually increased to 8–10 mg/ kg per day.

1	Albendazole or ivermectin is effective in reducing microfilarial loads, although neither is approved for this purpose by the U.S. Food and Drug Administration. Moreover, ivermectin is contraindicated in patients with >8000 microfilariae/mL because this drug has been associated with severe adverse events (including encephalopathy and death) in heavily infected patients with loiasis in West and Central Africa. DEC (300 mg weekly) is an effective prophylactic regimen for loiasis.

1	Mansonella streptocerca, found mainly in the tropical forest belt of Africa from Ghana to the Democratic Republic of the Congo, is transmitted by biting midges. The major clinical manifestations involve the skin and include pruritus, papular rashes, and pigmentation changes. Many infected individuals have inguinal adenopathy, although most are asymptomatic. The diagnosis is made by detection of the characteristic microfilariae in skin snips. Ivermectin at a single dose of 150 μg/kg leads to sustained suppression of microfilariae in the skin and is probably the treatment of choice for streptocerciasis.

1	M. perstans, distributed across the center of Africa and in northeastern South America, is transmitted by midges. Adult worms reside in serous cavities—pericardial, pleural, and peri toneal—as well as in the mesentery and the perirenal and retroperitoneal tissues. Microfilariae circulate in the blood without periodicity. The clinical and pathologic features of the infection are poorly defined. Most patients appear to be asymptomatic, but manifestations may include transient angioedema and pruritus of the arms, face, or other parts of the body (analogous to the Calabar swellings of loiasis); fever; headache; arthralgias; and right-upper-quadrant pain. Occasionally, pericarditis and hepatitis occur. The diagnosis is based on the demonstration of microfilariae in blood or serosal effusions. Perstans filariasis is often associated with peripheral-blood eosinophilia and antifilarial antibody elevations.

1	With the identification of a Wolbachia endosymbiont in M. perstans, doxycycline (200 mg twice a day) for 6 weeks has been established as the first effective treatment for this infection. The distribution of M. ozzardi is restricted to Central and South America and certain Caribbean islands. Adult worms are rarely recovered from humans. Microfilariae circulate in the blood without periodicity. Although this organism has often been considered nonpathogenic, headache, articular pain, fever, pulmonary symptoms, adenopathy, hepatomegaly, pruritus, and eosinophilia have been ascribed to M. ozzardi infection. The diagnosis is made by detection of microfilariae in peripheral blood. Ivermectin is effective in treating this infection.

1	The incidence of dracunculiasis, caused by Dracunculus medi nensis, has declined dramatically because of global eradication efforts. In 2012, only 542 cases worldwide had been identified. The infection is currently endemic only in Chad, Ethiopia, Mali, and South Sudan. Humans acquire D. medinensis when they ingest water containing infective larvae derived from Cyclops, a crustacean that is the intermediate host. Larvae penetrate the stomach or intestinal wall, mate, and mature. The adult male probably dies; the female worm develops over a year and migrates to subcutaneous tissues, usually in the lower extremity. As the thin female worm, ranging in length from 30 cm to 1 m, approaches the skin, a blister forms that, over days, breaks down and forms an ulcer. When the blister opens, large numbers of motile, rhabditiform larvae can be released into stagnant water; ingestion by Cyclops completes the life cycle.

1	Few or no clinical manifestations of dracunculiasis are evident until just before the blister forms, when there is an onset of fever and generalized allergic symptoms, including periorbital edema, wheezing, and urticaria. The emergence of the worm is associated with local pain and swelling. When the blister ruptures (usually as a result of immersion in water) and the adult worm releases larva-rich fluid, symptoms are relieved. The shallow ulcer surrounding the emerging adult worm heals over weeks to months. Such ulcers, however, can become secondarily infected, the result being cellulitis, local inflammation, abscess formation, or (uncommonly) tetanus. Occasionally, the adult worm does not emerge but becomes encapsulated and calcified. The diagnosis is based on the findings developing with the emergence of the adult worm, as described above.

1	The diagnosis is based on the findings developing with the emergence of the adult worm, as described above. schistosomiasis and Other Trematode Infections Charles H. King, Adel A. F. Mahmoud Trematodes, or flatworms, are a group of morphologically and biologically heterogeneous organisms that belong to the phylum 259 Gradual extraction of the worm by winding of a few centimeters on a stick each day remains the common and effective practice. Worms may be excised surgically. No drug is effective in treating dracunculiasis. Prevention, which remains the only real control measure, depends on the provision of safe drinking water.

1	Prevention, which remains the only real control measure, depends on the provision of safe drinking water. Dirofilariae that affect primarily dogs, cats, and raccoons occasionally infect humans incidentally, as do Brugia and Onchocerca parasites that affect small mammals. Because humans are an abnormal host, the parasites never develop fully. Pulmonary dirofilarial infection caused by the canine heartworm Dirofilaria immitis generally presents in humans as a solitary pulmonary nodule. Chest pain, hemoptysis, and cough are uncommon. Infections with D. repens (from dogs) or D. tenuis (from raccoons) can cause local subcutaneous nodules in humans. Zoonotic Brugia infection can produce isolated lymph node enlargement, whereas zoonotic Onchocerca can cause subconjunctival masses. Eosinophilia levels and antifilarial antibody titers are not commonly elevated. Excisional biopsy is both diagnostic and curative. These infections usually do not respond to chemotherapy.

1	Platyhelminthes. Human infection with trematodes occurs in many geographic areas and can cause considerable morbidity and mortality. The dependence on one drug—praziquantel—for treatment of most infections caused by trematodes raises the specter of developing resistance in these worms; several instances of reduced drug efficacy have already been reported. The widespread use of oxamniquine in the 1970s to reduce the impact of schistosomiasis resulted in the development of significant resistance. Recently, a single quantitative trait locus on schistosomal chromosome 6 was identified as the genetic basis for resistance.

1	For clinical purposes, significant trematode infections of humans may be divided according to the tissues invaded by the adult stage of the fluke, whether bloodstream, biliary tree, intestines, or lungs (Table 259-1). Trematodes share some common morphologic features, including macroscopic size (from one to several centimeters); dorsoventral, flattened, bilaterally symmetric bodies (adult worms); and the prominence of two suckers. Except for schistosomes, all human parasitic trematodes are hermaphroditic. Their life cycles involve a definitive host (mammalian/human), in which adult worms initiate sexual reproduction, and an intermediate host (snail), in which asexual multiplication of larvae occurs. More than one intermediate host may be necessary for some species of trematodes. Human infection is initiated either by direct penetration of intact skin or by ingestion. Upon maturation within humans, adult flukes initiate sexual reproduction and egg production. Helminth ova leave the

1	infection is initiated either by direct penetration of intact skin or by ingestion. Upon maturation within humans, adult flukes initiate sexual reproduction and egg production. Helminth ova leave the definitive host in excreta or sputum and, upon reaching suitable environmental

1	Schistosoma mansoni Skin penetration by Africa, South America, cercariae released from Middle East snails S. japonicum Skin penetration by China, Philippines, cercariae released from Indonesia snails S. intercalatum Skin penetration by West Africa cercariae released from snails S. mekongi Skin penetration by Southeast Asia cercariae released from snails S. haematobium Skin penetration by Africa, Middle East cercariae released from snails Clonorchis sinensis Ingestion of metacercar-Eastern Asia iae in freshwater fish Opisthorchis viverrini Ingestion of metacercar-Eastern Asia, Thailand iae in freshwater fish O. felineus Ingestion of metacercar-Eastern Asia, Europe iae in freshwater fish Fasciola hepatica Ingestion of metacer-Worldwide cariae on aquatic plants or in water F. gigantica Ingestion of metacer-Sporadic, Africa cariae on aquatic plants or in water Fasciolopsis buski Ingestion of metacercar-Southeast Asia iae on aquatic plants

1	F. gigantica Ingestion of metacer-Sporadic, Africa cariae on aquatic plants or in water Fasciolopsis buski Ingestion of metacercar-Southeast Asia iae on aquatic plants Heterophyes heterophyes Ingestion of metacer-Eastern Asia, North cariae in freshwater or Africa brackish-water fish Paragonimus westermani Ingestion of metacercar-Global except North and related species iae in crayfish or crabs America and Europe conditions, they hatch, releasing free-living miracidia that seek specific snail intermediate hosts. After asexual reproduction, cercariae are released from infected snails. In certain species, these organisms infect humans; in others, they find a second intermediate host to allow encystment into metacercariae—the infective stage for humans.

1	The host-parasite relationship in trematode infections is a product of certain biologic features of these organisms: they are multicellular, undergo several developmental changes within the host, and usually result in chronic infections. In general, the distribution of worm infections in human populations is overdispersed; i.e., it follows a negative binomial statistical distribution in which most infected individuals harbor low worm burdens while a small percentage are heavily infected. It is the heavily infected minority who are particularly prone to disease sequelae and who constitute an epidemiologically significant reservoir of infection in endemic areas. Recent evidence indicates that the prevalence of morbidity in infected populations is greater than was previously thought. Morbidity and death due to trematode infections reflect a multifactorial process that results from the tipping of a delicate balance between intensity of infection and host reactions, which initiate and

1	and death due to trematode infections reflect a multifactorial process that results from the tipping of a delicate balance between intensity of infection and host reactions, which initiate and modulate immunologic and pathologic outcome. Furthermore, the genetics of the parasite and of the human host contribute to the outcome of infection and disease. Infections with trematodes that migrate through or reside in host tissues are associated with a moderate to high degree of peripheral-blood eosinophilia; this association is of significance in protective and immunopathologic sequelae and is a useful clinical indicator of infection.

1	APPROACH TO THE PATIENT: The approach to individuals with suspected trematode infection begins with a question: Where have you been? Details of geographic history, exposure to freshwater bodies, and indulgence in local eating habits (without ensuring safety of food and drink) are all essential elements in eliciting the history of the present illness. The workup plan must include a detailed physical examination and tests appropriate for suspected infection. Diagnosis is based either on detection of the relevant stage of the parasite in excreta, sputum, or (rarely) tissue samples or on sensitive and specific serologic tests. Consultation with physicians familiar with these infections or with the U.S. Centers for Disease Control and Prevention (CDC) is helpful in guiding diagnosis and selecting therapy. GLOBAL CONSIDERATIONS: EPIDEMIOLOGY OF TREMATODE INFECTIONS

1	GLOBAL CONSIDERATIONS: EPIDEMIOLOGY OF TREMATODE INFECTIONS Except among international travelers, trematode infections are quite rare in high-income countries because good sanitation and hygiene block trematode transmission and because transmission is tied to the distribution of the specific snail species that serve as intermediate hosts during the parasites’ life cycle. In contrast, parasitic fluke infections are quite common in underdeveloped areas of Africa, Asia, and South America, with an estimated 440 million people affected by past or present Schistosoma infection and another 60 million people affected by the other foodborne trematodes. These infections are not benign; they result in multiyear chronic inflammatory disorders that significantly affect performance status and health-related quality of life. Global disease burden estimates indicate that at least 5 million years of healthy life are lost each year in the more than 90 endemic countries around the world.

1	BLOOD FLuKES: SCHISTOSOMIASIS Human schistosomiasis is caused by five species of the parasitic trematode genus Schistosoma: S. mansoni, S. japonicum, S. mekongi, and S. intercalatum cause intestinal and hepatic schistosomiasis, and S. haematobium causes urogenital schistosomiasis. Infection may cause considerable morbidity in the intestines, liver, or urinary tract, and a small proportion of affected individuals die. Other schistosomes (e.g., avian species) may invade human skin but then die in subcutaneous tissue, producing only self-limiting cutaneous manifestations.

1	Human infection is initiated by penetration of intact skin with infective cercariae. These organisms, which are released from infected snails in freshwater bodies, measure ~2 mm in length and possess an anterior and a ventral sucker that attach to the skin and facilitate penetration. Once in subcutaneous tissue, cercariae transform into schistosomula, with morphologic, membrane, and immunologic changes. The cercarial outer membrane changes from a trilaminar to a heptalaminar structure that is then maintained throughout the organism’s life span in humans. This transformation is thought to be the schistosome’s main adaptive mechanism for survival in humans. Schistosomula begin their migration within 2–4 days via venous or lymphatic vessels, reaching the lungs and finally the liver parenchyma. Sexually mature worms descend into the venous system at specific anatomic locations: intestinal veins (S. mansoni, S. japonicum, S. mekongi, and S. intercalatum) and vesical and other pelvic veins

1	Sexually mature worms descend into the venous system at specific anatomic locations: intestinal veins (S. mansoni, S. japonicum, S. mekongi, and S. intercalatum) and vesical and other pelvic veins (S. haematobium). After mating, adult gravid females travel against venous blood flow to small tributaries, where they deposit their ova intravascularly. Schistosome ova (Fig. 259-1) have specific morphologic features that vary with the species. Aided by enzymatic secretions through minipores in eggshells, ova move through the venous wall, traversing host tissues to reach the lumen of the intestinal or urinary tract, and are voided with stools or urine. Approximately 50% of ova are retained in host tissues locally (intestines or urinary tract) or are carried by venous blood flow to the liver and other organs. Schistosome ova that reach freshwater bodies hatch, releasing free-living miracidia that seek the snail intermediate host and undergo several cycles of asexual multiplication. Finally,

1	other organs. Schistosome ova that reach freshwater bodies hatch, releasing free-living miracidia that seek the snail intermediate host and undergo several cycles of asexual multiplication. Finally, infective cercariae are shed from snails to complete the transmission cycle.

1	Adult schistosomes are ~1–2 cm long. Males are slightly shorter than females, with flattened bodies and anteriorly curved edges forming the gynecophoral canal, in which mature adult females are usually held. Females are longer, slender, and rounded in cross-section. The precise nature of biochemical and reproductive exchanges between the two sexes is unknown, as are the regulatory mechanisms for pairing. Adult schistosomes parasitize specific sites in the host venous system. What guides adult intestinal schistosomes to branches of the superior or inferior mesenteric veins or adult S. haematobium worms to the vesical plexus is unknown. In addition, adult worms inhibit the coagulation cascade and evade the effector arms of the host immune responses by still-undetermined mechanisms. The genome of schistosomes is relatively large (~270 Mb) and is arrayed on seven pairs of autosomes and one pair of sex chromosomes. Sequencing of the S. japonicum,

1	S. mansoni, and S. haematobium genomes has provided insight into the worms’ genomic and proteomic features, offering an opportunity to FIGuRE 259-1 Morphology of schistosome eggs, the diagnostic stage of the parasite’s life cycle. A. Schistosoma haematobium egg (in a urine sample) is large (~140 mm long), with a terminal spine. B. S. mansoni egg (in a fecal sample) is large (~150 mm long), with a thin shell and lateral spine. C. S. japonicum egg (fecal) is smaller than that of S. mansoni (~90 mm long), with a small spine or hooklike structure. D. S. mekongi egg (fecal) is similar to that of S. japonicum but smaller (~65 mm long). E. S. intercalatum egg (fecal) is larger than that of S. haematobium (~190 mm long), with a longer, sharply pointed spine. (From LR Ash, TC Orihel: Atlas of Human Parasitology, 3rd ed. Chicago, ASCP Press, 1990; with permission.)

1	FIGuRE 259-2 Global distribution of schistosomiasis. A. Schistosoma mansoni infection (dark blue) is endemic in Africa, the Middle East, South America, and a few Caribbean countries. S. intercalatum infection (green) is endemic in sporadic foci in West and Central Africa. B. S. haematobium infection (purple) is endemic in Africa and the Middle East. The major endemic countries for S. japonicum infection (green) are China, the Philippines, and Indonesia. S. mekongi infection (red) is endemic in sporadic foci in Southeast Asia. discover new drug targets and to understand the molecular basis of pathogenesis.

1	discover new drug targets and to understand the molecular basis of pathogenesis. The global distribution of schistosome infection in human populations (Fig. 259-2) is dependent on both parasite and host factors. Information on prevalence and global distribution is inexact. At present, the five Schistosoma species are estimated to infect 200–300 million individuals (mostly children and young adults) in South America, the Caribbean, Africa, the Middle East, and Southeast Asia. Notably, parasite-related disease persists after active infection resolves, leaving a substantial health burden among adult populations. Thus, the overall number of humans likely to be affected by Schistosoma-related disease is now ~440 million. The total population living under conditions favoring transmission risk numbers ~700 million—a fact reflecting the global public health significance of schistosomiasis.

1	In endemic areas, the rate of yearly onset of new infection (incidence) is generally low. Prevalence, on the other hand, starts to be appreciable by the age of 3–4 years and builds to a maximum that varies by endemic region (up to 100%) in the 12to 20-year age group. Prevalence then stabilizes or decreases slightly in older age groups (>40 years). Intensity of infection (as measured by fecal or urinary egg counts, which correlate with adult worm burdens in most circumstances) follows the increase in prevalence up to the age of 12–20 years and then declines markedly in older age groups. This decline may reflect acquisition of resistance or may be due to changes in water contact patterns, since older people have less exposure. Infection with schistosomes in human populations has a peculiar pattern. Most infected individuals harbor low worm burdens, and only a small proportion suffer from high-intensity infection. This pattern may be due to differences in worm infectivity or to a

1	pattern. Most infected individuals harbor low worm burdens, and only a small proportion suffer from high-intensity infection. This pattern may be due to differences in worm infectivity or to a spectrum of genetic susceptibilities in human populations.

1	Disease due to schistosome infection is the consequence of parasitologic, host, and associated viral infections and of nutritional and environmental factors. Most disease syndromes relate to the presence of one or more of the parasite stages in humans. Disease manifestations in the populations of endemic areas correlate, in general, with intensity and duration of infection as well as with age and genetic susceptibility of the host. Overall, severe Schistosoma-specific disease manifestations are relatively rare among persons infected with any of the intestinal schistosomes. In contrast, symptoms of urogenital schistosomiasis manifest clinically in most S. haematobium–infected individuals. In addition, all forms of Schistosoma infection are associated with subclinical systemic morbidities that can significantly affect physical and cognitive performance, causing, for example, growth stunting, undernutrition, and anemia of chronic inflammation. New estimates of total morbidity due to

1	that can significantly affect physical and cognitive performance, causing, for example, growth stunting, undernutrition, and anemia of chronic inflammation. New estimates of total morbidity due to chronic schistosomiasis indicate a significantly greater burden than was previously appreciated.

1	Schistosomiasis appears to be a cofactor in the spread and progression of HIV/AIDS in areas where both diseases are endemic. Increased emphasis should be placed on the treatment of schistosome infections in persons at risk of HIV/AIDS.

1	Cercarial invasion is associated with dermatitis arising from dermal and subdermal inflammatory responses, both humoral and cell-mediated. As the parasites approach sexual maturity in the liver of infected individuals and as oviposition commences, acute schistosomiasis or Katayama syndrome (a serum sickness–like illness; see “Clinical Features,” below) may occur. The associated antigen excess results in formation of soluble immune complexes, which may be deposited in several tissues, initiating multiple pathologic events. In chronic schistosomiasis, most disease manifestations are due to eggs retained in host tissues. The granulomatous response around these ova is cell-mediated and is regulated both positively and negatively by a cascade of cytokine, cellular, and humoral responses. Granuloma formation begins with recruitment of a host of inflammatory cells in response to antigens secreted by the living organism within the ova. Cells recruited initially include phagocytes,

1	Granuloma formation begins with recruitment of a host of inflammatory cells in response to antigens secreted by the living organism within the ova. Cells recruited initially include phagocytes, antigen-specific T cells, and eosinophils. Fibroblasts, giant cells, and B lymphocytes predominate later. Over time, these cumulative lesions reach a size many times that of parasite eggs, thus inducing organomegaly and obstruction. Immunomodulation or downregulation of host responses to schistosome eggs plays a significant role in limiting the extent of the granulomatous lesions—and consequently disease—in chronically infected experimental animals or humans. The underlying mechanisms involve another cascade of regulatory cytokines and idiotypic antibodies. Subsequent to the granulomatous response, fibrosis sets in, resulting in more permanent disease sequelae. Because schistosomiasis is also a

1	FIGuRE 259-3 Chronic hepatosplenomegaly caused by schistosomiasis mansoni. Liver and spleen enlargement, ascites, and wasting are characteristically seen in patients with chronic Schistosoma mansoni infection. chronic infection, the accumulation of antigen–antibody complexes results in deposits in renal glomeruli and may cause significant kidney disease. The better-studied pathologic sequelae in schistosomiasis are those observed in liver disease. Ova that are carried by portal blood embolize to the liver. Because of their size (~150 × 60 μm in the case of

1	S. mansoni), they lodge at presinusoidal sites, where granulomas are formed. These granulomas contribute to the hepatomegaly observed in infected individuals (Fig. 259-3). Schistosomal liver enlargement is also associated with certain class I and class II human leukocyte antigen (HLA) haplotypes and markers; its genetic basis appears to be polygenic. Presinusoidal portal blockage causes several hemodynamic changes, including portal hypertension and associated development of portosystemic collaterals at the esophagogastric junction and other sites. Esophageal varices are most likely to break and cause repeated episodes of hematemesis. Because changes in hepatic portal blood flow occur slowly, compensatory arterialization of the blood flow through the liver is established. Although this compensatory mechanism may be associated with certain metabolic side effects, retention of hepatocyte perfusion permits maintenance of normal liver function for several years.

1	The second most significant pathologic change in the liver relates to fibrosis. It is characteristically periportal (Symmers’ clay pipe–stem fibrosis) but may be diffuse. Fibrosis, when diffuse, may be seen in areas of egg deposition and granuloma formation but is also seen in distant locations such as portal tracts. Schistosomiasis results in pure fibrotic lesions in the liver; cirrhosis occurs only when other toxic factors or infectious agents (e.g., hepatitis B or C virus) are involved. Deposition of fibrotic tissue in the extracellular matrix results from the interaction of T lymphocytes with cells of the fibroblast series; several cytokines, such as interleukin (IL) 2, IL-4, IL-1, and transforming growth factor β, are known to stimulate fibrogenesis. The process may be dependent on the genetic constitution of the host. Furthermore, regulatory cytokines that can suppress T cell responses and fibrogenesis, such as IL-10, interferon γ, or IL-12, may play a role in modulating the

1	the genetic constitution of the host. Furthermore, regulatory cytokines that can suppress T cell responses and fibrogenesis, such as IL-10, interferon γ, or IL-12, may play a role in modulating the response.

1	Although the above description focuses on granuloma formation and fibrosis of the liver, similar processes occur in urogenital schistosomiasis. Granuloma formation at the lower end of the ureters obstructs urinary flow, with subsequent development of hydroureter and hydronephrosis. Similar lesions in the urinary bladder cause the protrusion of papillomatous structures into its cavity; these may ulcerate and/or bleed. The chronic stage of infection is associated with scarring and deposition of calcium in the bladder wall. Among women, involvement of the birth canal can cause cervical or vaginal wall polyps and friability leading to contact bleeding, with an apparently increased risk of HIV transmission. Secondary infertility or subfecundity can also result from female genital schistosomiasis involving the uterus, fallopian tubes, or ovaries. Among men, S. haematobium infection can result in prostatic and testicular lesions with hematospermia. Superficial cutaneous lesions of the

1	involving the uterus, fallopian tubes, or ovaries. Among men, S. haematobium infection can result in prostatic and testicular lesions with hematospermia. Superficial cutaneous lesions of the perineum can occur in both sexes.

1	Studies on immunity to schistosomiasis, whether innate or adaptive, have expanded our knowledge of the components of these responses and target antigens. The critical question, however, is whether humans acquire immunity to schistosomes. Epidemiologic data suggest the onset of acquired immunity during the course of infection in young adults. Curative treatment of infected populations in endemic areas is followed by differentiation in the pattern of reinfection. Some (susceptible) individuals acquire reinfection rapidly, whereas other (resistant) individuals are reinfected slowly. This difference may be explained by differences in transmission, immunologic response, or genetic susceptibility. The mechanism of acquired immunity involves antibodies, complement, and several effector cells, particularly eosinophils. Furthermore, the intensity of schistosome infection has been correlated with a region in chromosome 5. In several studies, a few protective schistosome antigens have been

1	particularly eosinophils. Furthermore, the intensity of schistosome infection has been correlated with a region in chromosome 5. In several studies, a few protective schistosome antigens have been identified as vaccine candidates, but none has been fully evaluated in human populations to date.

1	In general, disease manifestations of schistosomiasis occur in three stages, which vary not only by species but also by intensity of infection and other host factors, such as age and genetics of the human host. During the phase of cercarial invasion, a form of dermatitis may be observed. This so-called swimmers’ itch occurs most often with S. mansoni and S. japonicum infections, manifesting 2 or 3 days after invasion as an itchy maculopapular rash on the affected areas of the skin. The condition is particularly severe when humans are exposed to avian schistosomes. This form of cercarial dermatitis is also seen around freshwater lakes in the northern United States, particularly in the spring and summer months. Cercarial dermatitis is a self-limiting clinical entity. During worm maturation and at the beginning of oviposition (i.e., 4–8 weeks after skin invasion), acute schistosomiasis or Katayama syndrome—a serum sickness–like illness with fever, generalized lymphadenopathy, and

1	and at the beginning of oviposition (i.e., 4–8 weeks after skin invasion), acute schistosomiasis or Katayama syndrome—a serum sickness–like illness with fever, generalized lymphadenopathy, and hepatosplenomegaly—may develop. Individuals with acute schistosomiasis have a high degree of peripheral-blood eosinophilia. Parasite-specific antibodies may be detected before schistosome eggs are identified in excreta.

1	Acute schistosomiasis has become an important clinical entity worldwide because of increased travel to endemic areas. Travelers are exposed to parasites while swimming or wading in freshwater bodies and upon their return present with acute manifestations. The course of acute schistosomiasis is generally benign, but central nervous system (CNS) schistosomiasis and even deaths are occasionally reported in association with heavy exposure to schistosomes among travelers and migrants. The main clinical manifestations of chronic schistosomiasis are species-dependent. Intestinal species (S. mansoni, S. japonicum,

1	The main clinical manifestations of chronic schistosomiasis are species-dependent. Intestinal species (S. mansoni, S. japonicum, S. mekongi, and S. intercalatum) cause intestinal and hepatosplenic disease as well as several manifestations associated with portal hypertension. During the intestinal phase, which may begin a few months after infection and may last for years, symptomatic patients characteristically have colicky abdominal pain, bloody diarrhea, and anemia. Patients may also report fatigue and an inability to perform daily routine functions and may show evidence of growth retardation and anemia. This more subtle form of schistosomiasis morbidity is generally underappreciated. The severity of intestinal schistosomiasis is often related to the intensity of the worm burden. The disease runs a chronic course and may result in colonic polyposis, which has been reported from some endemic areas, such as Egypt and Uganda.

1	The hepatosplenic phase of disease manifests early (during the first year of infection, particularly in children) with liver enlargement due to parasite-induced granulomatous lesions. Hepatomegaly is seen in ~15–20% of infected individuals; it correlates roughly with intensity of infection, occurs more often in children, and may be related to specific HLA haplotypes. In subsequent phases of infection, presinusoidal blockage of blood flow leads to portal hypertension and splenomegaly (Fig. 259-3). Moreover, portal hypertension may lead to varices at the lower end of the esophagus and at other sites. Patients with schistosomal liver disease may have right-upper-quadrant “dragging” pain during the hepatomegaly phase, and this pain may move to the left upper quadrant as splenomegaly progresses. Bleeding from esophageal varices may, however, be the first clinical manifestation of this phase. Patients may experience repeated bleeding but seem to tolerate its impact, because an adequate

1	Bleeding from esophageal varices may, however, be the first clinical manifestation of this phase. Patients may experience repeated bleeding but seem to tolerate its impact, because an adequate total hepatic blood flow permits normal liver function for a considerable period. In late-stage disease, typical fibrotic changes occur along with liver function deterioration and the onset of ascites, hypoalbuminemia, and defects in coagulation. Intercurrent viral infections of the liver (especially hepatitis B and C), toxic insults (excessive ethanol ingestion or exposure to organic poisons or aflatoxin), or nutritional deficiencies may well accelerate or exacerbate the deterioration of hepatic function.

1	The extent and severity of intestinal and hepatic disease in schistosomiasis mansoni and japonica have been well described. Although it was originally thought that S. japonicum might induce more severe disease manifestations because the adult worms can produce 10 times more eggs than S. mansoni, subsequent field studies have not supported this claim. Clinical observations of individuals infected with S. mekongi or S. intercalatum have been less detailed, partly because of the limited geographic distribution of these organisms.

1	The clinical manifestations of S. haematobium infection occur relatively early and involve a high percentage of infected individuals. Up to 80% of children infected with S. haematobium have dysuria, frequency, and hematuria. Hematuria may sometimes occur only at the end of voiding. Urine examination reveals blood and albumin as well as an unusually high frequency of bacterial urinary tract infections and urinary sediment cellular metaplasia. These manifestations correlate with the intensity of infection, the presence of urinary bladder granulomas, and subsequent ulceration. Along with local effects of granuloma formation in the urinary bladder, obstruction of the lower end of the ureters results in hydroureter and hydronephrosis, which may be seen in 25–50% of infected children. As infection progresses, bladder granulomas undergo fibrosis, which results in typical sandy patches visible on cystoscopy. In many endemic areas, an association between squamous cell carcinoma of the bladder

1	progresses, bladder granulomas undergo fibrosis, which results in typical sandy patches visible on cystoscopy. In many endemic areas, an association between squamous cell carcinoma of the bladder and S. haematobium infection has been observed. Such malignancy is detected in a younger age group than is transitional cell carcinoma. In fact, S. haematobium has now been classified as a human carcinogen. Genital schistosomiasis (described in the previous section) is a common presenting symptom among adults of both sexes.

1	Significant disease may occur in other organs during chronic schistosomiasis. Lung and CNS disease have been documented; other sites, such as the skin and the genital organs, are less frequently affected. In pulmonary schistosomiasis, embolized eggs lodge in small arterioles, producing acute necrotizing arteriolitis and granuloma formation. During S. mansoni and S. japonicum infection, schistosome eggs reach the lungs after the development of portosystemic collateral circulation; in S. haematobium infection, ova may reach the lungs directly via connections between the vesical and systemic circulation. Subsequent fibrous tissue deposition leads to endarteritis obliterans, pulmonary hypertension, and cor pulmonale. The most common symptoms are cough, fever, and dyspnea. Cor pulmonale may be diagnosed radiologically on the basis of prominence of the right side of the heart and dilation of the pulmonary artery. Frank evidence of right-sided heart failure may be seen in late cases.

1	Although less common than pulmonary manifestations, CNS 1427 schistosomiasis is important, characteristically occurring in association with S. japonicum infection. Migratory worms deposit eggs in the brain and induce a granulomatous response. The frequency of this manifestation among infected individuals in some endemic areas (e.g., the Philippines) is calculated at 2–4%. Jacksonian epilepsy due to

1	S. japonicum infection is the second most common cause of epilepsy in these areas. S. mansoni and S. haematobium infections have been associated with transverse myelitis. This syndrome is thought to be due to eggs traveling to the venous plexus around the spinal cord. In schistosomiasis mansoni, transverse myelitis is usually seen in the chronic stage after the development of portal hypertension and portosystemic shunts, which allow ova to travel to the spinal cord veins. This proposed sequence of events has been challenged because of a few reports of transverse myelitis occurring early in the course of S. mansoni infection. More information is needed to confirm these observations. During schistosomiasis caused by Schistosoma haematobium, ova may travel through communication between vesical and systemic veins, resulting in spinal cord disease that may be detected at any stage of infection. Pathologic study of lesions in schistosomal transverse myelitis may reveal eggs along with

1	vesical and systemic veins, resulting in spinal cord disease that may be detected at any stage of infection. Pathologic study of lesions in schistosomal transverse myelitis may reveal eggs along with necrotic or granulomatous lesions. Patients usually present with acute or rapidly progressing lower-leg weakness accompanied by sphincter dysfunction.

1	Physicians in areas not endemic for schistosomiasis face considerable diagnostic challenges. In the most common clinical presentation, a traveler returns with symptoms and signs of acute syndromes of schistosomiasis—namely, cercarial dermatitis or Katayama syndrome. Central to a correct diagnosis is a thorough inquiry into the patient’s history of travel and exposure to freshwater bodies—whether slowor fast-running—in an endemic area. Differential diagnosis of fever in returned travelers includes a spectrum of infections whose etiologies are viral (e.g., dengue fever), bacterial (e.g., enteric fever, leptospirosis), rickettsial, or protozoal (e.g., malaria). In cases of Katayama syndrome, prompt diagnosis is essential and is based on clinical presentation, high-level peripheral-blood eosinophilia, and a positive serologic assay for schistosomal antibodies. Two tests are available at the CDC: the Falcon assay screening test/enzyme-linked immunosorbent assay (FAST-ELISA) and the

1	eosinophilia, and a positive serologic assay for schistosomal antibodies. Two tests are available at the CDC: the Falcon assay screening test/enzyme-linked immunosorbent assay (FAST-ELISA) and the confirmatory enzyme-linked immunoelectrotransfer blot (EITB). Both tests are highly sensitive and ~96% specific. In some instances, examination of stool or urine for ova may yield positive results.

1	Individuals with established infection are diagnosed by a combination of geographic history, characteristic clinical presentation, and presence of schistosome ova in excreta. The diagnosis may also be established with the serologic assays mentioned above or with those that detect circulating schistosome antigens. These assays can be applied to blood, urine, or other body fluids (e.g., cerebrospinal fluid). For suspected schistosome infection, stool examination by the Kato thick smear or any other concentration method generally identifies most patients with heavy infection but does not identify all lightly infected individuals. For the latter patients, a point-of-care test to detect parasite circulating cathodic antigen in urine may prove very useful in establishing the presence of active S. mansoni infection and in monitoring the clearance of infection after treatment. For S. haematobium, urine may be examined by microscopy of sediment or by filtration of a known volume through

1	S. mansoni infection and in monitoring the clearance of infection after treatment. For S. haematobium, urine may be examined by microscopy of sediment or by filtration of a known volume through Nuclepore filters. Sensitivity can be further improved by testing for parasite DNA in urine sediment. The Kato thick smear and Nuclepore filtration provide quantitative data on the intensity of infection, which is of value in assessing the degree of tissue damage and in monitoring the effect of chemotherapy. Schistosome infection may also be diagnosed by examination of tissue specimens, typically rectal biopsy samples; except in rare circumstances, other biopsy procedures (e.g., liver biopsy) are not needed.

1	The differential diagnosis of schistosomal hepatomegaly must include viral hepatitis of all etiologies, miliary tuberculosis, malaria, visceral leishmaniasis, ethanol abuse, and causes of hepatic and portal vein obstruction. The differential diagnosis of hematuria in S. haematobium 1428 infection includes bacterial cystitis, tuberculosis, urinary stones, and follow-up visit with a health care provider is strongly recommended. malignancy. Prevention of infection in inhabitants of endemic areas is a significant challenge. Residents of these regions use freshwater bodies for sanitary, domestic, recreational, and agricultural purposes. Several control measures have been used, including application of molluscicides, provision

1	Treatment of schistosomiasis depends on the stage of infection and the clinical presentation. Other than topical dermatologic applications for relief of itching, no specific treatment is indicated for cercarial dermatitis caused by avian schistosomes. Therapy for acute schistosomiasis or Katayama syndrome needs to be adjusted appropriately for each case. Although antischistosomal chemotherapy may be used, it does not have a significant impact on maturing worms. In severe acute schistosomiasis, management in an acute-care setting is necessary, with supportive measures and consideration of glucocorticoid treatment to reduce inflammation. Once the acute critical phase is over, specific chemotherapy is indicated for parasite elimination. For all individuals with established infection, treatment to eradicate the parasite should be administered. The drug of choice is praziquantel, which—depending on the infecting species (Table 259-2)—is administered PO as a total of 40 or 60 mg/kg in two

1	to eradicate the parasite should be administered. The drug of choice is praziquantel, which—depending on the infecting species (Table 259-2)—is administered PO as a total of 40 or 60 mg/kg in two or three doses over a single day. Praziquantel treatment results in parasitologic cure in ~85% of cases and reduces egg counts by >90%. Efficacy rates among children <5 years old have been reported to be lower. These children are more likely to need re-treatment to effect a cure. Few side effects have been encountered, and those that do develop usually do not interfere with completion of treatment. Dependence on a single chemotherapeutic agent has raised the possibility of development of resistance in schistosomes; to date, such resistance does not seem to be clinically significant. The effect of antischistosomal treatment on disease manifestations varies by stage. Early hepatomegaly and bladder lesions are known to resolve after chemotherapy, but the late established manifestations, such as

1	antischistosomal treatment on disease manifestations varies by stage. Early hepatomegaly and bladder lesions are known to resolve after chemotherapy, but the late established manifestations, such as fibrosis, do not recede. Additional management modalities are needed for individuals with other manifestations, such as hepatocellular failure or recurrent hematemesis. The use of these interventions is guided by general medical and surgical principles.

1	Transmission of schistosomiasis is dependent on human behavior. Because the geographic distribution of infections in endemic regions of the world is not clearly demarcated, it is prudent for travelers to endemic areas to avoid contact with all freshwater bodies, irrespective of the speed of water flow or unsubstantiated claims of safety. Some topical agents, when applied to the skin, may inhibit cercarial penetration, but none is currently available. If exposure occurs, a Paragonimus westermani Praziquantela 25 mg/kg, 3 doses per day for 2 days aNot approved by the U.S. Food and Drug Administration for this indication.

1	Paragonimus westermani Praziquantela 25 mg/kg, 3 doses per day for 2 days aNot approved by the U.S. Food and Drug Administration for this indication. of sanitary water and sewage disposal, chemotherapy, and health education to effect behavioral change in terms of water-contact activities. Current recommendations to countries endemic for schistosomiasis emphasize the use of multiple approaches. With the advent of an oral, safe, and effective broad-spectrum antischistosomal agent (praziquantel), chemotherapy has been most successful in reducing the intensity of infection and reversing disease. The duration of this positive impact depends on the transmission dynamics of the parasite in any specific endemic region. The ultimate goal of research on prevention and control is the development of a vaccine. Although there are a few promising leads, this goal probably is not within reach during the next decade.

1	Several species of biliary fluke infecting humans are particu larly common in Southeast Asia and Russia. Other species are transmitted in Europe, Africa, and the Americas. On the basis of their migratory pathway in humans, these infections may be divided into the Clonorchis and Fasciola groups (Table 259-1). Infection with Clonorchis sinensis, the Chinese or oriental fluke, is endemic among fish-eating mammals in Southeast Asia. Humans are an incidental host; the prevalence of human infection is highest in China, Vietnam, and Korea. Infection with Opisthorchis viverrini and O. felineus is zoonotic in cats and dogs. Transmission to humans occurs occasionally, particularly in Thailand (O. viverrini) and in Southeast Asia and eastern Europe (O. felineus). Data on the exact geographic distribution of these infectious agents in human populations are rudimentary.

1	Infection with any of these three species is established by ingestion of raw or inadequately cooked freshwater fish harboring metacercariae. These organisms excyst in the duodenum, releasing larvae that travel through the ampulla of Vater and mature into adult worms in bile canaliculi. Mature flukes are flat and elongated, measuring 1–2 cm in length. The hermaphroditic worms reproduce by releasing small operculated eggs, which pass with bile into the intestines and are voided with stools. The life cycle is completed in the environment in specific freshwater snails (the first intermediate host) along with later encystment of snail-derived cercariae as infectious metacercariae in freshwater fish.

1	Except for late sequelae, the exact clinical syndromes caused by clonorchiasis and opisthorchiasis are not well defined. Because most infected individuals harbor a low worm burden, many are minimally symptomatic. Moderate to heavy infection may be associated with vague right-upper-quadrant pain. In contrast, chronic or repeated infection is associated with manifestations such as cholangitis, cholangiohepatitis, and biliary obstruction. Cholangiocarcinoma is epidemiologically related to C. sinensis infection in China and to O. viverrini infection in northeastern Thailand. This association has resulted in classification of these infectious agents as human carcinogens.

1	Infections with Fasciola hepatica and F. gigantica are world wide zoonoses that are particularly endemic in sheep-raising countries. Human cases have been reported in South America, Europe, Africa, and Australia. Recent estimates indicate a worldwide prevalence of 17 million cases. High endemicity has been reported in certain areas of Peru and Bolivia. In most endemic areas the predominant species is F. hepatica, but in Asia and Africa a varying degree of overlap with F. gigantica has been observed.

1	Humans acquire fascioliasis by ingestion of metacercariae attached to certain aquatic plants, such as watercress, water caltrop, and water chestnuts. Infection may also be acquired by consumption of contaminated water or ingestion of food items washed with such water. Acquisition of human infection through consumption of freshly prepared raw liver containing immature flukes has been reported. Infection is initiated when metacercariae excyst, penetrate the gut wall, and travel through the peritoneal cavity to invade the liver capsule. Adult worms migrate through the liver parenchyma and finally reach bile ducts, where they produce large operculated eggs that are voided in bile through the gastrointestinal tract to the outside environment. The flukes’ life cycle is completed in specific snails (the first intermediate host) followed by encystment on aquatic plants.

1	Clinical features of fascioliasis relate to the stage and intensity of infection. Acute disease develops during parasite migration (1–2 weeks after infection) and includes fever, right-upper-quadrant pain, hepatomegaly, and eosinophilia. Computed tomography (CT) of the liver may show multiple parenchymal holes/or migratory tracks. Symptoms and signs usually subside as the parasites reach their final habitat. In individuals with chronic infection, bile duct obstruction and biliary cirrhosis are infrequently demonstrated. No relation to hepatic malignancy has been ascribed to fascioliasis. Diagnosis of infection with any of the biliary flukes depends on a high degree of suspicion, elicitation of an appropriate geographic history, and stool examination for characteristically shaped parasite ova. Additional evidence may be obtained by documenting peripheral-blood eosinophilia or imaging the liver. Serologic testing is helpful, particularly in lightly infected individuals.

1	Drug therapy (praziquantel or triclabendazole) is summarized in Table 259-2. Patients with anatomic lesions in the biliary tract or malignancy are managed according to general medical guidelines. Two species of intestinal flukes cause human infection in defined geographic areas worldwide (Table 259-1). The large

1	Two species of intestinal flukes cause human infection in defined geographic areas worldwide (Table 259-1). The large Fasciolopsis buski (adults measure 2 × 7 cm) is endemic in Southeast Asia, whereas the smaller Heterophyes heterophyes is found in the Nile Delta of Egypt. Infection is initiated by ingestion of metacercariae attached to aquatic plants (F. buski) or encysted in freshwater or brackish-water fish (H. heterophyes). Flukes mature in human intestines, and eggs are passed with stools. Most individuals infected with intestinal flukes are asymptomatic. In heavy F. buski infection, diarrhea, abdominal pain, and malabsorption may be encountered. Heavy infection with H. heterophyes may be associated with abdominal pain and mucous diarrhea. The diagnosis is established by detection of characteristically shaped ova in stool samples. The drug of choice for treatment is praziquantel (Table 259-2).

1	Infection with the lung fluke Paragonimus westermani (Table 259-1) and related species (e.g., P. africanus) is endemic in many parts of the world, excluding North America and Europe. Endemicity is particularly noticeable in West Africa, Central and South America, and Asia. In nature, the reservoir hosts of P. westermani are wild and domestic felines. In Africa, P. africanus has been found in other species, such as dogs. Adult lung flukes, which are 7–12 mm in length, are found encapsulated in the lungs of infected persons. In rare circumstances, flukes are found encysted in the CNS (cerebral paragonimiasis) or the abdominal cavity. Humans acquire lung fluke infection by ingesting infective metacercariae encysted in the muscles and viscera of crayfish and freshwater crabs. In endemic areas, these 1429 crustaceans are consumed raw, marinated, or pickled. Once the organisms reach the duodenum, they excyst, penetrate the gut wall, and travel through the peritoneal cavity, diaphragm, and

1	these 1429 crustaceans are consumed raw, marinated, or pickled. Once the organisms reach the duodenum, they excyst, penetrate the gut wall, and travel through the peritoneal cavity, diaphragm, and pleural space to reach the lungs. Mature flukes are found in the bronchioles surrounded by cystic lesions. Parasite eggs are either expectorated with sputum or swallowed and passed to the outside environment with feces. The life cycle is completed in snails and freshwater crustaceans.

1	When maturing flukes lodge in lung tissues, they cause hemorrhage and necrosis, resulting in cyst formation. The adjacent lung parenchyma shows evidence of inflammatory infiltration, predominantly by eosinophils. Cysts usually measure 1–2 cm in diameter and may contain one or two worms each. With the onset of oviposition, cysts usually rupture in adjacent bronchioles—an event allowing ova to exit the human host. Older cysts develop thickened walls, which may undergo calcification. During the active phase of paragonimiasis, lung tissues surrounding parasite cysts may show evidence of pneumonia, bronchitis, bronchiectasis, and fibrosis.

1	Pulmonary paragonimiasis is particularly symptomatic in persons with moderate to heavy infection. Productive cough with brownish sputum or frank hemoptysis associated with peripheral-blood eosinophilia is usually the presenting feature. Chest examination may reveal signs of pleurisy. In chronic cases, bronchitis or bronchiectasis may predominate, but these conditions rarely proceed to lung abscess. Imaging of the lungs demonstrates characteristic features, including patchy densities, cavities, pleural effusion, and ring shadows. Cerebral paragonimiasis presents as either space-occupying lesions or epilepsy. Pulmonary paragonimiasis is diagnosed by detection of parasite ova in sputum and/or stools. Serology is of considerable help in egg-negative cases and in cerebral paragonimiasis. The differential diagnosis includes active tuberculosis, bacterial lung abscess, and lung carcinoma.

1	The drug of choice for treatment is praziquantel (Table 259-2). Other medical or surgical management may be needed for pulmonary or cerebral lesions.

1	For residents of nonendemic areas who are visiting an endemic region, the only effective preventive measure is to avoid ingestion of local plants, fish, or crustaceans; if their ingestion is necessary, these items should be washed and cooked thoroughly. Instruction on water and food preparation and consumption should be included in physicians’ advice to travelers (Chap. 149). Interruption of transmission among residents of endemic areas depends on avoiding ingestion of infective stages and disposing of feces and sputum appropriately to prevent hatching of eggs in the environment. These two approaches rely greatly on socioeconomic development, health education, and significant behavioral change. In countries where economic progress has resulted in financial and social improvements, transmission has decreased. The third approach to control in endemic communities entails selective use of chemotherapy for individuals posing the highest risk of transmission (i.e., those with heavy

1	transmission has decreased. The third approach to control in endemic communities entails selective use of chemotherapy for individuals posing the highest risk of transmission (i.e., those with heavy infections). The availability of praziquantel—a broad-spectrum, safe, and effective anthelmintic agent—provides a means for reducing the reservoirs of infection in human populations. However, the existence of most of these helminthic infections as zoonoses in several animal species complicates control efforts.

1	A. Clinton White, Jr., Peter F. Weller Cestodes, or tapeworms, are segmented worms. The adults reside in the gastrointestinal tract, but the larvae can be found in almost any organ. Human tapeworm infections can be divided into two major clinical groups. In one group, humans are the definitive hosts, with the adult tapeworms living in the gastrointestinal tract (Taenia saginata, Diphyllobothrium, Hymenolepis, and Dipylidium caninum). In the other, humans are intermediate hosts, with larval-stage parasites present in the tissues; diseases in this category include echinococcosis, sparganosis, and coenurosis. Humans may be either the definitive or the intermediate hosts for Taenia solium. Both stages of Hymenolepis nana are found simultaneously in the human intestines.

1	The ribbon-shaped tapeworm attaches to the intestinal mucosa by means of sucking cups or hooks located on the scolex. Behind the scolex is a short, narrow neck from which proglottids (segments) form. As each proglottid matures, it is displaced further back from the neck by the formation of new, less mature segments. The progressively elongating chain of attached proglottids, called the strobila, constitutes the bulk of the tapeworm. The length varies among species. In some, the tapeworm may consist of more than 1000 proglottids and may be several meters long. The mature proglottids are hermaphroditic and produce eggs, which are subsequently released. Because eggs of the different Taenia species are morphologically identical, differences in the morphology of the scolex or proglottids provide the basis for diagnostic identification to the species level.

1	Most human tapeworms require at least one intermediate host for complete larval development. After ingestion of the eggs or proglottids by an intermediate host, the larval oncospheres are activated, escape the egg, and penetrate the intestinal mucosa. The oncosphere migrates to tissues and develops into an encysted form known as a cysticercus (single scolex), a coenurus (multiple scolices), or a hydatid (cyst with daughter cysts, each containing several protoscolices). The definitive host’s ingestion of tissues containing a cyst enables a scolex to develop into a tapeworm. The beef tapeworm T. saginata occurs in all countries where raw or undercooked beef is eaten. It is most prevalent in sub- Saharan African and Middle Eastern countries. T. asiatica is closely related to T. saginata and is found in Asia, with pigs as intermediate hosts. The clinical manifestations and morphology of these two species are very similar and are therefore discussed together.

1	Etiology and Pathogenesis Humans are the only definitive host for the adult stage of T. saginata and T. asiatica. The tapeworms, which can reach 8 m in length with 1000–2000 proglottids, inhabit the upper jejunum. The scolex of T. saginata has four prominent suckers, whereas

1	T. asiatica has an unarmed rostellum. Each gravid segment has 15–30 uterine branches (in contrast to 8–12 for T. solium). The eggs are indistinguishable from those of T. solium; they measure 30–40 μm, contain the oncosphere, and have a thick brown striated shell. Eggs deposited on vegetation can live for months or years until they are ingested by cattle or other herbivores (T. saginata) or pigs (T. asiatica). The embryo released after ingestion invades the intestinal wall and is carried to striated muscle or viscera, where it transforms into the cysticercus. When ingested in raw or undercooked meat, this form can infect humans. After the cysticercus is ingested, it takes ~2 months for the mature adult worm to develop.

1	Clinical Manifestations Patients become aware of the infection most commonly by noting passage of proglottids in their feces. The proglottids are often motile, and patients may experience perianal discomfort when proglottids are discharged. Mild abdominal pain or discomfort, nausea, change in appetite, weakness, and weight loss can occur. Diagnosis The diagnosis is made by the detection of eggs or proglottids in the stool. Eggs may also be present in the perianal area; thus, if proglottids or eggs are not found in the stool, the perianal region should be examined with use of a cellophane-tape swab (as in pinworm infection; Chap. 257). Distinguishing T. saginata or T. asiatica from T. solium requires examination of mature proglottids. All three species can be distinguished by examining the scolex. Available serologic tests are not helpful diagnostically. Eosinophilia and elevated levels of serum IgE may be detected. A single dose of praziquantel (10 mg/kg) is highly effective.

1	A single dose of praziquantel (10 mg/kg) is highly effective. Prevention The major method of preventing infection is the adequate cooking of beef or pork viscera; exposure to temperatures as low as 56°C for 5 min will destroy cysticerci. Refrigeration or salting for long periods or freezing at −10°C for 9 days also kills cysticerci in beef. General preventive measures include inspection of beef and proper disposal of human feces. The pork tapeworm T. solium can cause two distinct forms of infection in humans: adult tapeworms in the intestine or larval forms in the tissues (cysticercosis). Humans are the only definitive hosts for T. solium; pigs are the usual intermediate hosts, although other animals may harbor the larval forms. T. solium is found worldwide in areas where pigs are raised and have access to human feces. However, it is most prevalent in Latin America, sub-Saharan Africa, China, India, and

1	T. solium is found worldwide in areas where pigs are raised and have access to human feces. However, it is most prevalent in Latin America, sub-Saharan Africa, China, India, and Southeast Asia. Cysticercosis occurs in industrialized nations largely as a result of the immigration of infected persons from endemic areas.

1	Etiology and Pathogenesis The adult tapeworm generally resides in the upper jejunum. The scolex attaches by both sucking disks and two rows of hooklets. The adult worm usually lives for a few years. The tapeworm, usually ~3 m in length, may have as many as 1000 proglottids, each of which produces up to 50,000 eggs. Proglottids are released and excreted into the feces, and the eggs in these proglottids are infective for both humans and animals. The eggs may survive in the environment for several months. After ingestion of eggs by the pig intermediate host, the larvae are activated, escape the egg, penetrate the intestinal wall, and are carried to many tissues; they are most frequently identified in striated muscle of the neck, tongue, and trunk. Within 60–90 days, the encysted larval stage develops. These cysticerci can survive for months to years. By ingesting undercooked pork containing cysticerci, humans acquire infections that lead to intestinal tapeworms. Infections that cause

1	develops. These cysticerci can survive for months to years. By ingesting undercooked pork containing cysticerci, humans acquire infections that lead to intestinal tapeworms. Infections that cause human cysticercosis follow the ingestion of T. solium eggs, usually from close contact with a tapeworm carrier. Autoinfection may occur if an individual with an egg-producing tapeworm ingests eggs derived from his or her own feces.

1	Clinical Manifestations Intestinal infections with T. solium may be asymptomatic. Fecal passage of proglottids may be noted by patients. Other symptoms are infrequent.

1	In cysticercosis, the clinical manifestations are variable. Cysticerci can be found anywhere in the body but are most commonly detected in the brain, cerebrospinal fluid (CSF), skeletal muscle, subcutaneous tissue, or eye. The clinical presentation of cysticercosis depends on the number and location of cysticerci as well as on the extent of associated inflammatory responses or scarring. Neurologic manifestations are the most common (Fig. 260-1). Seizures are associated with inflammation surrounding cysticerci in the brain parenchyma. These seizures may be generalized, focal, or Jacksonian. Hydrocephalus results from CSF flow obstruction by cysticerci and accompanying inflammation or by CSF outflow obstruction from arachnoiditis. Symptoms of increased intra-cranial pressure, including headache, nausea, vomiting, changes in this problem can be overcome by using 1431 the more specific immunoblot assay. An immunoblot assay using lentil lectin purified glycoproteins is >99% specific and

1	nausea, vomiting, changes in this problem can be overcome by using 1431 the more specific immunoblot assay. An immunoblot assay using lentil lectin purified glycoproteins is >99% specific and highly sensitive. However, patients with single intracranial lesions or with calcifications may be seronegative. With this assay, serum samples provide greater diagnostic sensitivity than CSF. All of the diagnostic antigens have been cloned, and assays using recombinant antigens are being developed. Antigen detection assays using monoclonal antibodies to detect parasite antigen in the blood or CSF may also facilitate diagnosis

1	FIGuRE 260-1 Neurocysticercosis is caused by Taenia solium. Neurologic infection can be and patient follow-up. These assays are onlyclassified on the basis of the location and viability of the parasites. When the parasites are in the now becoming available for patient care.ventricles, they often cause obstructive hydrocephalus. Left: Magnetic resonance imaging show-Studies have demonstrated that cliniing a cysticercus in the lateral ventricle, with resultant hydrocephalus. The arrow points to the cal criteria can aid in diagnosis in selected scolex within the cystic parasite. Center: CT showing a parenchymal cysticercus, with enhance-cases. In patients from endemic areas who ment of the cyst wall and an internal scolex (arrow). Right: Multiple cysticerci, including calcified had single enhancing lesions presentinglesions from prior infection (arrowheads), viable cysticerci in the basilar cisterns (white arrow), with seizures, a normal physical examinaand a large degenerating

1	single enhancing lesions presentinglesions from prior infection (arrowheads), viable cysticerci in the basilar cisterns (white arrow), with seizures, a normal physical examinaand a large degenerating cysticercus in the Sylvian fissure (black arrow). (Modified with permission tion, and no evidence of systemic disease from JC Bandres et al: Clin Infect Dis 15:799, 1992. © The University of Chicago Press.) (e.g., no fever, adenopathy, or chest radio- vision, dizziness, ataxia, or confusion, are often evident. Patients with hydrocephalus may develop papilledema or display altered mental status. When cysticerci develop at the base of the brain or in the sub-arachnoid space, they may cause chronic meningitis or arachnoiditis, communicating hydrocephalus, hemorrhages, or strokes.

1	Diagnosis The diagnosis of intestinal T. solium infection is made by the detection of eggs or proglottids, as described for T. saginata. More sensitive methods, including antigen-capture enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and serology for tapeworm stage-specific antigens, are currently available only as research techniques. In cysticercosis, diagnosis can be difficult. A consensus conference has delineated absolute, major, minor, and epidemiologic criteria for diagnosis (Table 260-1). Diagnostic certainty is possible only with definite demonstration of the parasite (absolute criteria). This task can be accomplished by histologic observation of the parasite in excised tissue, by funduscopic visualization of the parasite in the eye (in the anterior chamber, vitreous, or subretinal spaces), or by neuroimaging studies demonstrating cystic lesions containing a characteristic scolex (Fig. 260-1). With improving resolution of neuroimaging studies, the

1	vitreous, or subretinal spaces), or by neuroimaging studies demonstrating cystic lesions containing a characteristic scolex (Fig. 260-1). With improving resolution of neuroimaging studies, the scolex can now be identified in many cases. In other instances, a clinical diagnosis is based on a combination of clinical presentation, radiographic studies, serologic tests, and exposure history.

1	Neuroimaging findings suggestive of neurocysticercosis constitute the primary major diagnostic criterion (Fig. 260-1). These findings include cystic lesions with or without enhancement (e.g., ring enhancement), one or more nodular calcifications (which may also have associated enhancement), or focal enhancing lesions. Cysticerci in the brain parenchyma are usually 5–20 mm in diameter and rounded. Cystic lesions in the subarachnoid space or fissures may enlarge up to 6 cm in diameter and may be lobulated. For cysticerci within the subarachnoid space or ventricles, the walls may be very thin and the cyst fluid is often isodense with CSF. Thus, obstructive hydrocephalus or enhancement of the basilar meninges may be the only finding on CT in extraparenchymal neurocysticercosis. Cysticerci in the ventricles or subarachnoid space are usually visible to an experienced neuroradiologist on MRI or on CT with intraventricular contrast injection. CT is more sensitive than MRI in identifying

1	in the ventricles or subarachnoid space are usually visible to an experienced neuroradiologist on MRI or on CT with intraventricular contrast injection. CT is more sensitive than MRI in identifying calcified lesions, whereas MRI is better for identifying cystic lesions, scolices, and enhancement.

1	The second major diagnostic criterion is detection of specific antibodies to cysticerci. Although most tests using unfractionated antigen have high rates of false-positive and false-negative results, graphic abnormalities), the constellation of with no midline shift was almost always caused by neurocysticercosis. Finally, spontaneous resolution or resolution after therapy with albendazole alone is consistent with neurocysticercosis. Minor diagnostic criteria include neuroimaging findings consistent with but less characteristic of cysticercosis, clinical manifestations suggestive of neurocysticercosis (e.g., seizures, hydrocephalus, or altered mental status), evidence of cysticercosis outside the central nervous system (CNS) (e.g., cigar-shaped soft-tissue calcifications), or detection of antibody in CSF by ELISA. Epidemiologic criteria include 1. Absolute criteria a. Demonstration of cysticerci by histologic or microscopic examination of biopsy material b.

1	Demonstration of cysticerci by histologic or microscopic examination of biopsy material b. Visualization of the parasite in the eye by funduscopy c. Neuroradiologic demonstration of cystic lesions containing a characteristic scolex 2. Major criteria a. Neuroradiologic lesions suggestive of neurocysticercosis b. Demonstration of antibodies to cysticerci in serum by enzyme-linked immunoelectrotransfer blot c. Resolution of intracranial cystic lesions spontaneously or after therapy with albendazole or praziquantel alone 3. Minor criteria a. Lesions compatible with neurocysticercosis detected by neuroimaging studies b. Clinical manifestations suggestive of neurocysticercosis c. Demonstration of antibodies to cysticerci or cysticercal antigen in cerebrospinal fluid by enzyme-linked immunosorbent assay d. Evidence of cysticercosis outside the central nervous system (e.g., cigar-shaped soft-tissue calcifications) 4. Epidemiologic criteria a. b.

1	Evidence of cysticercosis outside the central nervous system (e.g., cigar-shaped soft-tissue calcifications) 4. Epidemiologic criteria a. b. Frequent travel to a cysticercosis-endemic area c. Household contact with an individual infected with Taenia solium aDiagnosis is confirmed by either one absolute criterion or a combination of two major criteria, one minor criterion, and one epidemiologic criterion. A probable diagnosis is supported by the fulfillment of (1) one major criterion plus two minor criteria; (2) one major criterion plus one minor criterion and one epidemiologic criterion; or (3) three minor criteria plus one epidemiologic criterion. Source: Modified from OH Del Brutto et al: Neurology 57:177, 2001. 1432 exposure to a tapeworm carrier or household member infected with T. solium, current or prior residence in an endemic area, and frequent travel to an endemic area.

1	1432 exposure to a tapeworm carrier or household member infected with T. solium, current or prior residence in an endemic area, and frequent travel to an endemic area. The diagnosis is confirmed in patients with either one absolute criterion or a combination of two major criteria, one minor criterion, and one epidemiologic criterion (Table 260-1). A probable diagnosis is supported by the fulfillment of (1) one major criterion plus two minor criteria; (2) one major criterion plus one minor criterion and one epidemiologic criterion; or (3) three minor criteria plus one epidemiologic criterion. Although the CSF is usually abnormal in neurocysticercosis, CSF abnormalities are not pathognomonic. Patients may have CSF pleocytosis with a predominance of lymphocytes, neutrophils, or eosinophils. The protein level in CSF may be elevated; the glucose concentration is usually normal but may be depressed.

1	Intestinal T. solium infection is treated with a single dose of praziquantel (10 mg/kg). However, praziquantel occasionally evokes an inflammatory response in the CNS if concomitant cryptic cysticercosis is present. Niclosamide (2 g) is also effective but is not widely available.

1	The initial management of neurocysticercosis should focus on symptom-based treatment of seizures or hydrocephalus. Seizures can usually be controlled with antiepileptic treatment. If parenchymal lesions resolve without development of calcifications and patients remain free of seizures, antiepileptic therapy can usually be discontinued after 1–2 years. Placebo-controlled trials are clarifying the clinical advantage of antiparasitic drugs for parenchymal neurocysticercosis. Trends toward faster resolution of neuroradiologic abnormalities have been observed in most studies. The clinical benefits are less dramatic and consist mainly of shortening the period during which recurrent seizures occur and decreasing the number of patients who have many recurrent seizures. For the treatment of patients with brain parenchymal cysticerci, most authorities favor antiparasitic drugs, including albendazole (15 mg/kg per day for 8–28 days) or praziquantel (50–100 mg/kg daily in three divided doses for

1	with brain parenchymal cysticerci, most authorities favor antiparasitic drugs, including albendazole (15 mg/kg per day for 8–28 days) or praziquantel (50–100 mg/kg daily in three divided doses for 15–30 days). A combination of albendazole and praziquantel (50 mg/kg per day) may be more effective in patients with multiple lesions. A longer course or combination therapy is often needed in patients with multiple subarachnoid cysticerci. Both agents may exacerbate the inflammatory response around the dying parasite, thereby exacerbating seizures or hydrocephalus as well. Thus, patients receiving these drugs should be carefully monitored. High-dose glucocorticoids should be used during treatment. Because glucocorticoids induce first-pass metabolism of praziquantel and may decrease its antiparasitic effect, cimetidine should be co-administered to inhibit praziquantel metabolism.

1	For patients with hydrocephalus, the emergent reduction of intra-cranial pressure is the mainstay of therapy. In the case of obstructive hydrocephalus, the preferred approach is removal of the cysticercus via endoscopic surgery. However, this intervention is not always possible. An alternative approach is initially to perform a diverting procedure, such as ventriculoperitoneal shunting. Historically, shunts have usually failed, but low failure rates have been attained with administration of antiparasitic drugs and glucocorticoids. Open craniotomy to remove cysticerci is now required only infrequently but is an alternative for fourth-ventricular cysticerci. For patients with subarachnoid cysts or giant cysticerci, anti-inflammatory medications such as glucocorticoids are needed to reduce arachnoiditis and accompanying vasculitis. Most authorities recommend prolonged courses of antiparasitic drugs as well as shunting when hydrocephalus is present. Methotrexate should be used as a

1	arachnoiditis and accompanying vasculitis. Most authorities recommend prolonged courses of antiparasitic drugs as well as shunting when hydrocephalus is present. Methotrexate should be used as a steroid-sparing agent in patients requiring prolonged therapy. In patients with diffuse cerebral edema and elevated intracranial pressure due to multiple inflamed lesions, glucocorticoids are the mainstay of therapy, and antiparasitic drugs should be avoided. For ocular and spinal medullary lesions, drug-induced inflammation may cause irreversible damage. Ocular disease should be managed surgically. Recent data suggest that either medical or surgical therapy can be used for spinal disease.

1	Prevention Measures for the prevention of intestinal T. solium infection consist of the application to pork of precautions similar to those described above for beef with regard to T. saginata infection. The prevention of cysticercosis involves minimizing the opportunities for ingestion of fecally derived eggs by means of good personal hygiene, effective fecal disposal, and treatment and prevention of human intestinal infections. Mass chemotherapy has been administered to human and porcine populations in efforts at disease eradication. Finally, vaccines to prevent porcine cysticercosis have shown promise in studies and are under development.

1	Echinococcosis is an infection caused in humans by the larval stage of the Echinococcus granulosus complex, E. multilocu laris, or E. vogeli. E. granulosus complex parasites produce cystic hydatid disease, with unilocular cystic lesions. These infections are prevalent in most areas where livestock is raised in association with dogs. Molecular evidence suggests that E. granulosus strains may actually belong to more than one species; specifically, strains from sheep, cattle, pigs, horses, and camels probably represent separate species. These parasites are found on all continents, with areas of high prevalence in China, central Asia, the Middle East, the Mediterranean region, eastern Africa, and parts of South America. E. multilocularis, which causes multilocular alveolar lesions that are locally invasive, is found in Alpine, sub-Arctic, or Arctic regions, including Canada, the United States, and central and northern Europe; China; and central Asia. E. vogeli causes polycystic hydatid

1	locally invasive, is found in Alpine, sub-Arctic, or Arctic regions, including Canada, the United States, and central and northern Europe; China; and central Asia. E. vogeli causes polycystic hydatid disease and is found only in Central and South America.

1	Like other cestodes, echinococcal species have both intermediate and definitive hosts. The definitive hosts are canines that pass eggs in their feces. After the ingestion of eggs, cysts develop in the intermediate hosts—sheep, cattle, humans, goats, camels, and horses for the E. granulosus complex and mice and other rodents for E. multilocularis. When a dog (E. granulosus) or fox (E. multilocularis) ingests infected meat containing cysts, the life cycle is completed.

1	Etiology The small (5-mm-long) adult E. granulosus complex worms, which live for 5–20 months in the jejunum of dogs, have only three proglottids: one immature, one mature, and one gravid. The gravid segment splits to release eggs that are morphologically similar to Taenia eggs and are extremely hardy. After humans ingest the eggs, embryos escape from the eggs, penetrate the intestinal mucosa, enter the portal circulation, and are carried to various organs, most commonly the liver and lungs. Larvae develop into fluid-filled unilocular hydatid cysts that consist of an external membrane and an inner germinal layer. Daughter cysts develop from the inner aspect of the germinal layer, as do germinating cystic structures called brood capsules. New larvae, called protoscolices, develop in large numbers within the brood capsule. The cysts expand slowly over a period of years.

1	The life cycle of E. multilocularis is similar except that wild canines, such as foxes, serve as the definitive hosts and small rodents serve as the intermediate hosts. The larval form of E. multilocularis, however, is quite different in that it remains in the proliferative phase, the parasite is always multilocular, and vesicles without brood capsule or protoscolices progressively invade the host tissue by peripheral extension of processes from the germinal layer.

1	Clinical Manifestations Slowly enlarging echinococcal cysts generally remain asymptomatic until their expanding size or their space-occupying effect in an involved organ elicits symptoms. The liver and the lungs are the most common sites of these cysts. The liver is involved in about two-thirds of E. granulosus infections and in nearly all E. multilocularis infections. Because a period of years elapses before cysts enlarge sufficiently to cause symptoms, they may be discovered incidentally on a routine x-ray or ultrasound study.

1	Patients with hepatic echinococcosis who are symptomatic most often present with abdominal pain or a palpable mass in the right upper quadrant. Compression of a bile duct or leakage of cyst fluid into the biliary tree may mimic recurrent cholelithiasis, and biliary obstruction can result in jaundice. Rupture of or episodic leakage from a hydatid cyst may produce fever, pruritus, urticaria, eosinophilia, or anaphylaxis. Pulmonary hydatid cysts may rupture into the bronchial tree or pleural cavity and produce cough, salty phlegm, dyspnea, chest pain, or hemoptysis. Rupture of hydatid cysts, which can occur spontaneously or at surgery, may lead to multifocal dissemination of protoscolices, which can form additional cysts. Other presentations are due to the involvement of bone (invasion of the medullary cavity with slow bone erosion producing pathologic fractures), the CNS (spaceoccupying lesions), the heart (conduction defects, pericarditis), and the pelvis (pelvic mass).

1	The larval forms of E. multilocularis characteristically present as a slowly growing hepatic tumor, with progressive destruction of the liver and extension into vital structures. Patients commonly report upper-quadrant and epigastric pain. Liver enlargement and obstructive jaundice may be apparent. The lesions may infiltrate adjoining organs (e.g., diaphragm, kidneys, or lungs) or may metastasize to the spleen, lungs, or brain.

1	Diagnosis Radiographic and related imaging studies are important in detecting and evaluating echinococcal cysts. Plain x-rays will define pulmonary cysts of E. granulosus—usually as rounded masses of uniform density—but may miss cysts in other organs unless there is cyst wall calcification (as occurs in the liver). MRI, CT, and ultrasound reveal well-defined cysts with thick or thin walls. When older cysts contain a layer of hydatid sand that is rich in accumulated protoscolices, these imaging methods may detect this fluid layer of different density. However, the most pathognomonic finding, if demonstrable, is that of daughter cysts within the larger cyst. This finding, like eggshell or mural calcification on CT, is indicative of E. granulosus infection and helps to distinguish the cyst from carcinomas, bacterial or amebic liver abscesses, or hemangiomas. In contrast, ultrasound or CT of alveolar hydatid cysts reveals indistinct solid masses with central necrosis and plaquelike

1	cyst from carcinomas, bacterial or amebic liver abscesses, or hemangiomas. In contrast, ultrasound or CT of alveolar hydatid cysts reveals indistinct solid masses with central necrosis and plaquelike calcifications.

1	A specific diagnosis of E. granulosus infection can be made by the examination of aspirated fluids for protoscolices or hooklets, but diagnostic aspiration is not usually recommended because of the risk of fluid leakage resulting in either dissemination of infection or anaphy-1433 lactic reactions. Serodiagnostic assays can be useful, although a negative test does not exclude the diagnosis of echinococcosis. Cysts in the liver elicit positive antibody responses in ~90% of cases, whereas up to 50% of individuals with cysts in the lungs are seronegative. Detection of antibody to specific echinococcal antigens by immunoblotting has the highest degree of specificity.

1	Therapy for cystic echinococcosis is based on considerations of the size, location, and manifestations of cysts and the overall health of the patient. Surgery has traditionally been the principal definitive method of treatment. Currently, ultrasound staging is recommended for E. granulosus infections (Fig. 260-2). Small CL, CE1, and CE3 lesions may respond to chemotherapy with albendazole. For CE1 lesions and uncomplicated CE3 lesions, PAIR (percutaneous aspiration, infusion of scolicidal agents, and reaspiration) is now recommended instead of surgery. PAIR is contraindicated for superficially located cysts (because of the risk of rupture), for cysts with multiple thick internal septal divisions (honeycombing pattern), and for cysts communicating with the biliary tree. For prophylaxis of secondary peritoneal echinococcosis due to inadvertent spillage of fluid during PAIR, the administration of albendazole (15 mg/kg daily in two divided doses) should be initiated at least 2 days before

1	peritoneal echinococcosis due to inadvertent spillage of fluid during PAIR, the administration of albendazole (15 mg/kg daily in two divided doses) should be initiated at least 2 days before the procedure and continued for at least 4 weeks afterward. Ultrasound-or CT-guided aspiration allows confirmation of the diagnosis by demonstration of protoscolices in the aspirate. After aspiration, contrast material should be injected to detect occult communications with the biliary tract. Alternatively, the fluid should be checked for bile staining visually and by dipstick. If no bile is found and no communication is visualized, the contrast material is reaspirated, with subsequent infusion of scolicidal agents (usually 95% ethanol; alternatively, hypertonic saline). This approach, when implemented by a skilled practitioner, yields rates of cure and relapse equivalent to those following surgery, with less perioperative morbidity and shorter hospitalization. In experienced hands, some CE2

1	by a skilled practitioner, yields rates of cure and relapse equivalent to those following surgery, with less perioperative morbidity and shorter hospitalization. In experienced hands, some CE2 lesions can be treated by aspiration with a trocar. Daughter cysts within the primary cyst may need to be punctured separately, and catheter drainage may be required.

1	FIGuRE 260-2 Management of cystic hydatid disease caused by Echinococcus granulosus should be based on viability of the parasite, which can be estimated from radiographic appearance. The ultrasound appearance includes lesions classified as active, transitional, and inactive. Active cysts include types CL (with a cystic lesion and no visible cyst wall), CE1 (with a visible cyst wall and internal echoes [snowflake sign]), and CE2 (with a visible cyst wall and internal septation). Transitional cysts (CE3) may have detached laminar membranes or may be partially collapsed. Inactive cysts include types CE4 (a nonhomogeneous mass) and CE5 (a cyst with a thick calcified wall). (Adapted from RL Guerrant et al [eds]: Tropical Infectious Diseases: Principles, Pathogens and Practice, 2nd ed, p 1312. © 2005, with permission from Elsevier Science.) 1434 Surgery remains the treatment of choice for complicated

1	E. granulosus cysts (e.g., those communicating with the biliary tract), for most thoracic and intracranial cysts, and for areas where PAIR is not possible. For E. granulosus of the liver, the preferred surgical approach is pericystectomy, in which the entire cyst and the surrounding fibrous tissue are removed. The risks posed by leakage of fluid during surgery or PAIR include anaphylaxis and dissemination of infectious protoscolices. The latter complication has been minimized by careful attention to the prevention of spillage of the cyst and by soaking of the drapes with hypertonic saline. Infusion of scolicidal agents is no longer recommended because of problems with hypernatremia, intoxication, or sclerosing cholangitis. Albendazole, which is active against Echinococcus, should be administered adjunctively, beginning several days before resection of the liver and continuing for several weeks for E. granulosus. Praziquantel (50 mg/kg daily for 2 weeks) may hasten the death of the

1	adjunctively, beginning several days before resection of the liver and continuing for several weeks for E. granulosus. Praziquantel (50 mg/kg daily for 2 weeks) may hasten the death of the protoscolices. Medical therapy with albendazole alone for 12 weeks to 6 months results in cure in ~30% of cases and in improvement in another 50%. In many instances of treatment failure, E. granulosus infections are subsequently treated successfully with PAIR or additional courses of medical therapy. Response to treatment is best assessed by serial imaging studies, with attention to cyst size and consistency. Some cysts may not demonstrate complete radiologic resolution even though no viable protoscolices are present. Some of these cysts with partial radiologic resolution (e.g., CE4 or CE5) can be managed with observation only.

1	Surgical resection remains the treatment of choice for E. multilocularis infection. Complete removal of the parasite continues to offer the best chance for cure. Ongoing therapy with albendazole for at least 2 years after presumptively curative surgery is recommended. Positron emission tomography can be used to follow disease activity. Most cases are diagnosed at a stage at which complete resection is not possible; in these cases, albendazole treatment should be continued indefinitely, with careful monitoring. In some cases, liver transplantation has been used because of the size of the necessary liver resection. However, continuous immunosuppression favors the proliferation of E. multilocularis larvae and reinfection of the transplant. Thus, indefinite treatment with albendazole is required.

1	Prevention In endemic areas, echinococcosis can be pre vented by administering praziquantel to infected dogs, by denying dogs access to infected animals, or by vaccinating sheep. Limitation of the number of stray dogs is helpful in reducing the prevalence of infection among humans. In Europe, E. multilocularis infection has been associated with gardening; gloves should be used when working with soil. Infection with Hymenolepis nana, the dwarf tapeworm, is the most common of all the cestode infections. H. nana is endemic in both temperate and tropical regions of the world. Infection is spread by fecal/oral contamination and is common among institutionalized children.

1	Etiology and Pathogenesis H. nana is the only cestode of humans that does not require an intermediate host. Both the larval and adult phases of the life cycle take place in the human. The adult—the smallest tapeworm parasitizing humans—is ~2 cm long and dwells in the proximal ileum. Proglottids, which are small and rarely seen in the stool, release spherical eggs 30–44 μm in diameter, each of which contains an oncosphere with six hooklets. The eggs are immediately infective and are unable to survive for >10 days in the external environment. When the egg is ingested by a new host, the oncosphere is freed and penetrates the intestinal villi, becoming a cysticercoid larva. Larvae migrate back into the intestinal lumen, attach to the mucosa, and mature into adult worms over 10–12 days. Eggs may also hatch before passing into the stool, causing internal autoinfection with increasing numbers of intestinal worms. Although the life span of adult H. nana worms is only ~4–10 weeks, the

1	Eggs may also hatch before passing into the stool, causing internal autoinfection with increasing numbers of intestinal worms. Although the life span of adult H. nana worms is only ~4–10 weeks, the autoinfection cycle perpetuates the infection.

1	Clinical Manifestations H. nana infection, even with many intestinal worms, is usually asymptomatic. When infection is intense, anorexia, abdominal pain, and diarrhea develop. Diagnosis Infection is diagnosed by the finding of eggs in the stool. Praziquantel (25 mg/kg once) is the treatment of choice, because it acts against both the adult worms and the cysticercoids in the intestinal villi. Nitazoxanide (500 mg bid for 3 days) may be used as an alternative. Prevention Good personal hygiene and improved sanitation can eradicate the disease. Epidemics have been controlled by mass chemotherapy coupled with improved hygiene. Hymenolepis diminuta, a cestode of rodents, occasionally infects small children, who ingest the larvae in uncooked cereal foods contaminated by fleas and other insects in which larvae develop. Infection is usually asymptomatic and is diagnosed by the detection of eggs in the stool. Treatment with praziquantel results in cure in most cases.

1	Diphyllobothrium latum and other Diphyllobothrium species are found in the lakes, rivers, and deltas of the Northern Hemisphere, central Africa, and South America. Etiology and Pathogenesis The adult worm—the longest tapeworm (up to 25 m)—attaches to the ileal and occasionally to the jejunal mucosa by its suckers, which are located on its elongated scolex. The adult worm has 3000–4000 proglottids, which release ~1 million eggs daily into the feces. If an egg reaches water, it hatches and releases a free-swimming embryo that can be eaten by small freshwater crustaceans (Cyclops or Diaptomus species). After an infected crustacean containing a developed procercoid is swallowed by a fish, the larva migrates into the fish’s flesh and grows into a plerocercoid, or sparganum larva. Humans acquire the infection by ingesting infected raw or smoked fish. Within 3–5 weeks, the tapeworm matures into an adult in the human intestine.

1	Clinical Manifestations Most D. latum infections are asymptomatic, although manifestations may include transient abdominal discomfort, diarrhea, vomiting, weakness, and weight loss. Occasionally, infection can cause acute abdominal pain and intestinal obstruction; in rare cases, cholangitis or cholecystitis may be produced by migrating proglottids. Because the tapeworm absorbs large quantities of vitamin B12 and interferes with ileal B12 absorption, vitamin B12 deficiency can develop, but this effect has been noted only in Scandinavia, where up to 2% of infected patients, especially the elderly, have megaloblastic anemia resembling pernicious anemia and may exhibit neurologic sequelae of B12 deficiency. Diagnosis The diagnosis is made readily by the detection of the characteristic eggs in the stool. The eggs possess a single shell with an operculum at one end and a knob at the other. Mild to moderate eosinophilia may be detected.

1	Praziquantel (5–10 mg/kg once) is highly effective. Parenteral vitamin B12 should be given if B12 deficiency is manifest. Prevention Infection can be prevented by heating fish to 54°C for 5 min or by freezing it at −18°C for 24 h. Placing fish in brine with a high salt concentration for long periods kills the eggs. Dipylidium caninum, a common tapeworm of dogs and cats, may accidentally infect humans. Dogs, cats, and occasionally humans become infected by ingesting fleas harboring cysticercoids. Children are more likely to become infected than adults. Most infections are asymptomatic, but abdominal pain, diarrhea, anal pruritus, urticaria, eosinophilia, or passage of segments in the stool may occur. The diagnosis is made by the detection of proglottids or ova in the stool. As in D. latum infection, therapy consists of praziquantel. Prevention requires anthelmintic treatment and flea control for pet dogs or cats.

1	Humans can be infected by the sparganum, or plerocercoid larva, of a diphyllobothrid tapeworm of the genus Spirometra. Infection can be acquired by the consumption of water containing infected Cyclops; 1435 by the ingestion of infected snakes, birds, or mammals; or by the application of infected flesh as poultices. The worm migrates slowly in tissues, and infection commonly presents as a subcutaneous swelling. Periorbital tissues can be involved, and ocular sparganosis may destroy the eye. Surgical excision is used to treat localized sparganosis. This rare infection of humans by the larval stage (coenurus) of the dog tapeworm Taenia multiceps or T. serialis results in a space-occupying cystic lesion. As in cysticercosis, involvement of the CNS and subcutaneous tissue is most common. Both definitive diagnosis and treatment require surgical excision of the lesion. Chemotherapeutic agents generally are not effective.

1	the genetic modification of microbes for the purposes of antimicrobial resistance or evasion by the immune system, creation of fine-particle aerosols, chemical treatment to stabilize and prolong infectivity, and H. Clifford Lane, Anthony S. Fauci alteration of host range through changes in surface proteins. Certain

1	Descriptions of the use of microbial pathogens as potential weapons of war or terrorism date from ancient times. Among the most frequently cited of such episodes are the poisoning of water supplies in the sixth century b.c. with the fungus Claviceps purpurea (rye ergot) by the Assyrians, the hurling of the dead bodies of plague victims over the walls of the city of Kaffa by the Tartar army in 1346, and the efforts by the British to spread smallpox to the Native American population loyal to the French via contaminated blankets in 1763. The tragic attacks on the World Trade Center and the Pentagon on September 11, 2001, followed closely by the mailing of letters containing anthrax spores to media and congressional offices through the U.S. Postal Service, dramatically changed the mindset of the American public regarding both our vulnerability to microbial bioterrorist attacks and the seriousness and intent of the federal government to protect its citizens against future attacks. Modern

1	the American public regarding both our vulnerability to microbial bioterrorist attacks and the seriousness and intent of the federal government to protect its citizens against future attacks. Modern science has revealed methods of deliberately spreading or enhancing disease in ways not appreciated by our ancestors. The combination of basic research, good medical practice, and constant vigilance will be needed to defend against such attacks.

1	Although the potential impact of a bioterrorist attack could be enormous, leading to thousands of deaths and high morbidity rates, acts of bioterrorism would be expected to produce their greatest impact through the fear and terror they generate. In contrast to biowarfare, where the primary goal is destruction of the enemy through mass casualties, an important goal of bioterrorism is to destroy the morale of a society through fear and uncertainty. Although the actual biologic impact of a single act may be small, the degree of disruption created by the realization that such an attack is possible may be enormous. This was readily apparent with the impact on the U.S. Postal Service and the functional interruption of the activities of the legislative branch of the U.S. government following the anthrax attacks noted above. Thus, the key to the defense against these attacks is a highly functioning system of public health surveillance and education so that attacks can be quickly recognized

1	anthrax attacks noted above. Thus, the key to the defense against these attacks is a highly functioning system of public health surveillance and education so that attacks can be quickly recognized and effectively contained. This is complemented by the availability of appropriate countermeasures in the form of diagnostics, therapeutics, and vaccines, both in response to and in anticipation of bioterrorist attacks.

1	The Working Group for Civilian Biodefense created a list of key features that characterize the elements of biologic agents that make them particularly effective as weapons (Table 261e-1). Included among these are the ease of spread and transmission of the agent and the presence of an adequate database to allow newcomers to the field to quickly apply the good science of others to bad intentions of their own. Agents of bioterrorism may be used in their naturally occurring forms, or they can be deliberately modified to deliver greater impact. Among the approaches to maximizing the deleterious effects of biologic agents are Key Features oF BioLogiC agents used as Bioweapons 1. 2. 3. 4. Lack of rapid diagnostic capability 5. Lack of universally available effective vaccine 6. Potential to cause anxiety 7. Availability of pathogen and feasibility of production 8. 9. Database of prior research and development 10. Potential to be “weaponized”

1	Potential to cause anxiety 7. Availability of pathogen and feasibility of production 8. 9. Database of prior research and development 10. Potential to be “weaponized” Source: From L Borio et al: JAMA 287:2391, 2002; with permission. of these approaches fall under the category of weaponization, which is a term generally used to describe the processing of microbes or toxins in a manner that would ensure a devastating effect following release. For example, weaponization of anthrax by the Soviets involved the production of vast numbers of spores of appropriate size to reach the lower respiratory tract easily in a form that maintained aerosolization for prolonged periods of time and that could be delivered in a massive release, such as via widely dispersed bomblets.

1	The U.S. Centers for Disease Control and Prevention (CDC) classifies potential biologic threats into three categories: A, B, and C (Table 261e-2). Category A agents are the highest-priority pathogens. They pose the greatest risk to national security because they (1) can be easily disseminated or transmitted from person to person, (2) result in high mortality rates and have the potential for major public health impact, (3) might cause public panic and social disruption, and (4) require special action for public health preparedness. Category B agents are the second highest priority pathogens and include those that are moderately easy to disseminate, result in moderate morbidity rates and low mortality rates, and require specifically enhanced diagnostic capacity. Category C agents are the third highest priority. These include certain emerging pathogens to which the general population lacks immunity; that could be engineered for mass dissemination in the future because of availability,

1	third highest priority. These include certain emerging pathogens to which the general population lacks immunity; that could be engineered for mass dissemination in the future because of availability, ease of production, and ease of dissemination; and that have a major public health impact and the potential for high morbidity and mortality rates. It should be pointed out, however, that these A, taBLe 261e-2 CdC Category a, B, and C agents

1	Arenaviruses: Lassa, New World (Machupo, Junin, Guanarito, and Sabia) Bunyaviridae: Crimean-Congo, Rift Valley Filoviridae: Ebola, Marburg Brucellosis (Brucella spp.) Epsilon toxin of Clostridium perfringens Food safety threats (e.g., Salmonella spp., Escherichia coli 0157:H7, Shigella) Glanders (Burkholderia mallei) Melioidosis (Burkholderia pseudomallei) Psittacosis (Chlamydophila psittaci) Q fever (Coxiella burnetii) Ricin toxin from Ricinus communis (castor beans) Staphylococcal enterotoxin B Typhus fever (Rickettsia prowazekii) Viral encephalitis (alphaviruses [e.g., Venezuelan, eastern, and western equine encephalitis]) Water safety threats (e.g., Vibrio cholerae, Cryptosporidium parvum) Emerging infectious diseases threats such as Nipah, hantavirus, SARS or MERS coronavirus, and pandemic influenza Abbreviations: MERS, Middle East respiratory syndrome; SARS, severe acute respiratory syndrome.

1	Abbreviations: MERS, Middle East respiratory syndrome; SARS, severe acute respiratory syndrome. Source: Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases. B, and C designations are empirical and, depending on evolving circumstances such as intelligence-based threat assessments, the priority rating of any given microbe or toxin could change. The CDC classification system also largely reflects the severity of illness produced by a given agent, rather than its accessibility to potential terrorists. See also Chap. 175.

1	Bacillus anthracis as a Bioweapon Anthrax may be the prototypic disease of bioterrorism. Although rarely, if ever, spread from person to person, the illness embodies the other major features of a disease introduced through terrorism, as outlined in Table 261e-1. U.S. and British government scientists studied anthrax as a potential biologic weapon beginning approximately at the time of World War II (WWII). Offensive bioweapons activity including bioweapons research on microbes and toxins in the United States ceased in 1969 as a result of two executive orders by President Richard M. Nixon. Although the 1972 Biological and Toxin Weapons Convention Treaty outlawed research of this type worldwide, the Soviet Union produced and stored tons of anthrax spores for potential use as a bioweapon until at least the late 1980s. At present, there is suspicion that research on anthrax as an agent of bioterrorism is ongoing by several nations and extremist groups. One example of this was the release

1	at least the late 1980s. At present, there is suspicion that research on anthrax as an agent of bioterrorism is ongoing by several nations and extremist groups. One example of this was the release of anthrax spores by the Aum Shinrikyo cult in Tokyo in 1993. Fortunately, there were no casualties associated with that episode because of the inadvertent use of a nonpathogenic strain of anthrax by the terrorists.

1	The potential impact of anthrax spores as a bioweapon was clearly demonstrated in 1979 following the accidental release of spores into the atmosphere from a Soviet Union bioweapons facility in Sverdlovsk, Russia. Although total figures are not known, at least 77 cases of anthrax were diagnosed with certainty, of which 66 were fatal. These victims were exposed in an area within 4 km downwind of the facility, and deaths due to anthrax were also noted in livestock up to 50 km further downwind. Based on recorded wind patterns, the interval between the time of exposure and development of clinical illness ranged from 2 to 43 days. The majority of cases were within the first 2 weeks. Death typically occurred within 1–4 days following the onset of symptoms. It is likely that the widespread use of postexposure penicillin prophylaxis limited the total number of cases. The extended period of time between exposure and disease in some individuals supports the data from nonhuman primate studies,

1	of postexposure penicillin prophylaxis limited the total number of cases. The extended period of time between exposure and disease in some individuals supports the data from nonhuman primate studies, suggesting that anthrax spores can lie dormant in the respiratory tract for at least 4–6 weeks without evoking an immune response. This extended period of microbiologic latency following exposure poses a significant challenge for management of victims in the postexposure period.

1	In September 2001, the American public was exposed to anthrax spores as a bioweapon delivered through the U.S. Postal Service by an employee of the United States Army Medical Research Institute for Infectious Diseases (USAMRIID) who had access to such materials and who committed suicide prior to being indicted for this crime. The CDC identified 22 confirmed or suspected cases of anthrax as a consequence of this attack. These included 11 patients with inhalational anthrax, of whom 5 died, and 11 patients with cutaneous anthrax (7 confirmed), all of whom survived (Fig. 261e-1). Cases occurred in individuals who opened contaminated letters as well as in postal workers involved in the processing of mail. A minimum of five letters mailed from Trenton, NJ, served as the vehicles for these attacks. One of these letters was reported to contain 2 g of material, equivalent to 100 billion to 1 trillion weapon-grade spores. Based on studies performed in the 1950s using monkeys exposed to

1	attacks. One of these letters was reported to contain 2 g of material, equivalent to 100 billion to 1 trillion weapon-grade spores. Based on studies performed in the 1950s using monkeys exposed to aerosolized anthrax demonstrating that ~10,000 spores were required to produce lethal disease in 50% of animals exposed to this dose (the LD50), the contents of one letter had the theoretical potential, under optimal conditions, of causing illness or death in up to 50 million individuals. The strain used in this attack was the Ames strain. Although it was noted to have an inducible β-lactamase and to constitutively express a cephalosporinase, it was susceptible to all antibiotics standard for B. anthracis.

1	Envelopes mailed to news media companies, Sept. 18 Envelopes mailed to government leaders, Oct. 9 Anthrax first confirmed 0 2Numberof cases4 6 Symptom onset dates, September–November 2001 Dist. of Columbia* = inhalation anthrax cases. * = Metropolitan District of Columbia Area, cases were residents of Maryland (3) and Virginia (2). Figure 261e-1 Confirmed anthrax cases associated with bioterrorism: United States, 2001. A. Geographic location, clinical manifestation, and outcome of the 11 cases of confirmed inhalational and 11 cases of confirmed cutaneous anthrax. B. Epidemic curve for 22 cases of anthrax. (From DB Jernigan et al: Emerg Infect Dis 8:1019, 2002; with permission.)

1	Microbiology and Clinical Features Anthrax is caused by B. anthracis, a gram-positive, nonmotile, spore-forming rod that is found in soil and predominantly causes disease in herbivores such as cattle, goats, and sheep. Anthrax spores can remain viable for decades. The remarkable stability of these spores makes them an ideal bioweapon, and their destruction in decontamination activities can be a challenge. Naturally occurring human infection is generally the result of contact with anthrax-infected animals or animal products such as goat hair in textile mills or animal skins used in making drums. While an LD50 of 10,000 spores is a generally accepted number, it has also been suggested that as few as one to three spores may be adequate to cause disease in some settings. Advanced technology is likely to be necessary to generate a bioweapon containing spores of the optimal size (1–5 μm) to travel to the alveolar spaces.

1	The three major clinical forms of anthrax are gastrointestinal, cutaneous, and inhalational. Gastrointestinal anthrax typically results from the ingestion of contaminated meat; the condition is rarely seen and is unlikely to be the result of a bioterrorism event. The lesion of cutaneous anthrax typically begins as a papule following the introduction of spores through an opening in the skin. This papule then evolves to a painless vesicle followed by the development of a coal-black, necrotic eschar (Fig. 261e-2). It is the Greek word for coal (anthrax) that gives the organism and the disease its name. Cutaneous anthrax was ~20% fatal prior to the availability of antibiotics. Inhalational anthrax is the Figure 261e-2 Clinical manifestations of a pediatric case of cutaneous anthrax associated with the bioterrorism attack of 2001. The lesion progresses from vesicular on day 5 (A) to necrotic with the classic black eschar on day 12 (B) to a healed scar 2 months later (C).

1	(Photographs provided by Dr. Mary Wu Chang. Part A reproduced with permission from KJ Roche et al: N Engl J Med 345:1611, 2001 and Parts B and C reproduced with permission from A Freedman et al: JAMA 287:869, 2002.) form most likely to be responsible for death in the setting of a bioterrorist attack. It occurs following the inhalation of spores that become deposited in the alveolar spaces. These spores are phagocytized by macrophages and transported to the mediastinal and peribronchial lymph nodes where they germinate, leading to active bacterial growth and elaboration of the bacterial products edema toxin and lethal toxin. Subsequent hematogenous spread of bacteria is accompanied by cardiovascular collapse and death. The earliest symptoms are typically a viral-like prodrome with fever, malaise, and abdominal and/or chest symptoms that progress over the course of a few days to a moribund state. Characteristic findings on chest x-ray include mediastinal widening and pleural effusions

1	malaise, and abdominal and/or chest symptoms that progress over the course of a few days to a moribund state. Characteristic findings on chest x-ray include mediastinal widening and pleural effusions (Fig. 261e-3). Although initially thought to be 100% fatal, the experiences at Sverdlovsk in 1979 and in the United States in 2001 (see below) indicate that with prompt initiation of antibiotic therapy, survival is possible. The characteristics of the 11 cases of inhalational anthrax diagnosed in the United States in 2001 following exposure to contaminated letters postmarked September 18 or October 9, 2001, followed the classic pattern established for this illness, with patients presenting with a rapidly progressive course characterized by fever, fatigue or malaise, nausea or vomiting, cough, and shortness of breath. At presentation, the total white blood cell counts were ~10,000 cells/μL; transaminases tended to be elevated, and all 11 had abnormal findings on chest x-ray and computed

1	and shortness of breath. At presentation, the total white blood cell counts were ~10,000 cells/μL; transaminases tended to be elevated, and all 11 had abnormal findings on chest x-ray and computed tomography (CT). Radiologic findings included infiltrates, mediastinal widening, and hemorrhagic pleural effusions. For cases in which the dates of exposure were known, symptoms appeared within 4–6 days. Death occurred within 7 days of diagnosis in the five fatal cases (overall mortality rate 55%). Rapid diagnosis and prompt initiation of antibiotic therapy were key to survival.

1	Anthrax can be successfully treated if the disease is promptly recognized and appropriate therapy with antibiotics and antitoxin is initiated early. Although penicillin, ciprofloxacin, and doxycycline are the currently licensed antibiotics for this indication, clindamycin and rifampin also have in vitro activity against the organism and have been used as part of treatment regimens. Until sensitivity results are known, suspected cases are best managed with a combination of broadly active agents (Table 261e-3). The B. anthracis toxins lethal factor and edema factor share a component known as protective antigen. Raxibacumab, a monoclonal antibody directed toward protective antigen, was licensed in 2012 under the animal rule (see below) for treatment of adult and pediatric patients with inhalational anthrax in combination with appropriate antibacterial drugs. Patients with inhalational anthrax are not contagious and do not require special isolation procedures.

1	Vaccination and Prevention The first successful vaccine for anthrax was developed for animals by Louis Pasteur in 1881. At present, the single vaccine licensed for human use is a product produced from the cell-free culture supernatant of an attenuated, nonencapsulated strain of B. anthracis (Stern strain), referred to as anthrax vaccine adsorbed (AVA). Clinical trials for safety in humans and efficacy in animals are currently under way to evaluate the role of recombinant protective antigen as an alternative to AVA. In a postexposure setting in nonhuman primates, a 2-week course of AVA plus ciprofloxacin was found to be superior to ciprofloxacin alone in preventing the development of clinical disease and death. Although the current recommendation for postexposure prophylaxis is 60 days of antibiotics, it would seem prudent to include immunization with anthrax vaccine if available. Given the potential for

1	B. anthracis to be engineered to express penicillin resistance, the empirical regimen of choice in this setting is either ciprofloxacin or doxycycline. In settings where these approaches are not available or appropriate, one can administer the antitoxin monoclonal antibody raxibacumab. See also Chap. 196.

1	Yersinia pestis as a Bioweapon Although it lacks the environmental stability of anthrax, the highly contagious nature and high mortality rate of plague make it a close to ideal agent of bioterrorism, particularly if delivered in a weaponized form. Occupying a unique place in history, plague has been alleged to have been used as a biologic weapon for centuries. The catapulting of plague-infected corpses into besieged fortresses is a practice that was first noted in 1346 during the assault of the Crimean city of Kaffa by the Mongolian Tartars. Although unlikely to have resulted in disease transmission, some believe that this event may have played a role in the start of the Black Death pandemic of the fourteenth and fifteenth centuries in Europe. Given that plague was already moving across Asia toward Europe at this time, it is unclear whether such an allegation is accurate. During WWII, the infamous Unit 731 of the Japanese army was reported to have repeatedly dropped plague-infested

1	Asia toward Europe at this time, it is unclear whether such an allegation is accurate. During WWII, the infamous Unit 731 of the Japanese army was reported to have repeatedly dropped plague-infested fleas over parts of China, including Manchuria. These drops were associated with subsequent outbreaks of plague in the targeted areas. Following WWII, the United States and the Soviet Union conducted programs of research on how to create aerosolized Y. pestis that could be used as a bioweapon to cause primary pneumonic plague. As mentioned above, plague was thought to be an excellent bioweapon due to the fact that in addition to causing infection in those inhaling the aerosol, significant numbers of secondary cases of primary pneumonic plague would also likely occur due to the contagious nature of the disease and person-to-person transmission via respiratory aerosol. Secondary reports of research conducted during that time suggest that organisms remain viable for up to 1 h and can be

1	nature of the disease and person-to-person transmission via respiratory aerosol. Secondary reports of research conducted during that time suggest that organisms remain viable for up to 1 h and can be dispersed for

1	Figure 261e-3 Progression of chest x-ray findings in a patient with inhalational anthrax. Findings evolved from subtle hilar prominence and right perihilar infiltrate to a progressively widened mediastinum, marked perihilar infiltrates, peribronchial cuffing, and air bronchograms.

1	(From L Borio et al: JAMA 286:2554, 2001; with permission.) distances up to 10 km. Although the offensive bioweapons program in the United States was terminated prior to production of sufficient quantities of plague organisms for use as a weapon, it is believed that Soviet scientists did manufacture quantities sufficient for such a purpose. It has also been reported that more than 10 Soviet institutes and >1000 scientists were working with plague as a biologic weapon. Of concern is the fact that in 1995 a microbiologist in Ohio was arrested for having obtained Y. pestis in the mail from the American Type Culture Collection, using a credit card and a false letterhead. In the wake of this incident, the U.S. Congress passed a law in 1997 requiring that anyone intending to send or receive any of 42 different agents that could potentially be used as bioweapons first register with the CDC.

1	Microbiology and Clinical Features Plague is caused by Y. pestis, a non-motile, gram-negative bacillus that exhibits bipolar, or “safety pin,” staining with Wright, Giemsa, or Wayson stains. It has had a major impact on the course of history, thus adding to the element of fear evoked by its mention. The earliest reported plague epidemic was in 224 b.c. in China. The most infamous pandemic began in Europe in the fourteenth century, during which time one-third to one-half of the entire population of Europe was killed. During a plague outbreak in India in 1994, even though the number of confirmed cases was relatively small, it is estimated that 500,000 individuals fled their homes in fear of this disease. In the first decade of the twenty-first century, 21,725 cases of plague were reported to the World Health Organization (WHO). Over 90% of these cases were from Africa, and the overall case fatality rate was 7.4%.

1	The clinical syndromes of plague generally reflect the mode of infection. Bubonic plague is the consequence of an insect bite; primary pneumonic plague arises through the inhalation of bacteria. Most of the plague seen in the world today is bubonic plague and is the result of a bite by a plague-infected flea. In part as a consequence of past pandemics, plague infection of rodents exists widely in nature, including in the southwestern United States, and each year thousands of cases of plague occur worldwide through contact with infected animals or fleas. Following inoculation of regurgitated bacteria into the skin by a flea bite, organisms travel through the lymphatics to regional lymph nodes, where they are phagocytized but not destroyed. Inside the cell, they multiply rapidly leading to inflammation, painful lymphadenopathy with necrosis, fever, bacteremia, septicemia, and death. The characteristic enlarged, inflamed lymph nodes, or buboes, give this form of plague its name. In some

1	inflammation, painful lymphadenopathy with necrosis, fever, bacteremia, septicemia, and death. The characteristic enlarged, inflamed lymph nodes, or buboes, give this form of plague its name. In some instances, patients may develop bacteremia without lymphadenopathy following infection, a condition referred to as primary septicemic plague. Extensive ecchymoses may develop due 261e-5 taBLe 261e-3 CLiniCaL syndroMes, prevention, and treatMent strategies For diseases Caused By Category a agents

1	Cutaneous lesion: Papule to eschar Inhalational disease: Fever, malaise, chest and abdominal discomfort Pleural effusion, widened mediastinum on chest x-ray Fever, cough, dyspnea, hemoptysis Fever, malaise, headache, backache, emesis Maculopapular to vesicular to pustular skin lesions Fever, chills, malaise, myalgia, chest discomfort, dyspnea, headache, skin rash, pharyngitis, conjunctivitis Fever, myalgia, rash, encephalitis, prostration Dry mouth, blurred vision, ptosis, weakness, dysarthria, dysphagia, dizziness, respiratory failure, progressive paralysis, dilated pupils 1–12 days Culture, Gram stain, PCR, Wright stain of peripheral smear 1–60 days Postexposure: Ciprofloxacin, 500 mg, PO bid × 60 d or Doxycycline, 100 mg PO bid × 60 d or Amoxicillin, 500 mg PO q8h × 60 d, likely to be effective if strain is penicillin sensitive Active disease: Ciprofloxacin, 400 mg IV q12h or doxycycline, 100 mg IV q12h plus

1	Amoxicillin, 500 mg PO q8h × 60 d, likely to be effective if strain is penicillin sensitive Active disease: Ciprofloxacin, 400 mg IV q12h or doxycycline, 100 mg IV q12h plus Clindamycin, 900 mg IV q8h and/or rifampin, 300 mg IV q12h; switch to PO when stable × 60 d total plus Raxibacumab, 40 mg/kg IV over 2.25 h; diphenhydramine to reduce reaction Gentamicin, 2.0 mg/kg IV loading then 1.7 mg/kg q8h IV or Streptomycin, 1.0 g q12h IM or IV Alternatives include doxycycline, 100 mg bid PO or IV; chloramphenicol, 500 mg qid PO or IV Supportive measures; consideration for cidofovir, tecovirimat, antivaccinia immunoglobulin Streptomycin, 1 g IM bid or Gentamicin, 5 mg/kg per day div q8h IV for 14 days or Doxycycline, 100 mg IV bid or Chloramphenicol, 15 mg/kg up to 1 g IV qid or Ciprofloxacin, 400 mg IV bid Ribavirin 30 mg/kg up to 2 g × 1, followed by 16 mg/kg IV up to 1 g q6h for 4 days, followed by 8 mg/ kg IV up to 0.5 g q8h × 6 days

1	Ciprofloxacin, 400 mg IV bid Ribavirin 30 mg/kg up to 2 g × 1, followed by 16 mg/kg IV up to 1 g q6h for 4 days, followed by 8 mg/ kg IV up to 0.5 g q8h × 6 days Supportive measures including ventilation, HBAT equine antitoxin from the CDC Emergency Operations Center, 770-488-7100 Anthrax vaccine adsorbed Recombinant protective antigen vaccines are under study Raxibacumab when alternative therapies are not available or appropriate Doxycycline, 100 mg PO bid or Levofloxacin, 500 mg PO daily Doxycycline, 100 mg PO bid × 14 days or Ciprofloxacin, 500 mg PO bid × 14 days Administration of antitoxin Abbreviations: CDC, U.S. Centers for Disease Control and Prevention; FDA, U.S. Food and Drug Administration; HBAT, heptavalent botulinum antitoxin; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase polymerase chain reaction.

1	to disseminated intravascular coagulation, and gangrene of the digits infiltrates and consolidation on chest x-ray. In the absence of antibiotand/or nose may develop in patients with advanced septicemic plague. ics, the mortality rate of this form of plague is on the order of 85%, and It is thought that this appearance of some patients gave rise to the term death usually occurs within 2–6 days. Black Death in reference to the plague epidemic of the fourteenth and fifteenth centuries. Some patients may develop pneumonia (secondary pneumonic plague) as a complication of bubonic or septicemic plague. These patients may then transmit the agent to others via the respira-Streptomycin, tetracycline, doxycycline, and levofloxacin are tory route, causing cases of primary pneumonic plague. Primary pneu-licensed by the U.S. Food and Drug Administration (FDA) for the monic plague is the manifestation most likely to occur as the result of a treatment of plague. Levofloxacin was approved for this

1	pneu-licensed by the U.S. Food and Drug Administration (FDA) for the monic plague is the manifestation most likely to occur as the result of a treatment of plague. Levofloxacin was approved for this indication bioterrorist attack, with an aerosol of bacteria spread over a wide area in 2012 via the animal rule (see below). Multiple additional antibiotor a particular environment that is densely populated. In this setting, ics licensed for other infections are commonly used and are likely patients would be expected to develop fever, cough with hemoptysis, effective. Among these are aminoglycosides such as gentamicin, dyspnea, and gastrointestinal symptoms 1–6 days following exposure. cephalosporins, trimethoprim/sulfamethoxazole, chloramphenicol, Clinical features of pneumonia would be accompanied by pulmonary and ciprofloxacin (Table 261e-3). A multidrug-resistant strain of

1	Y. pestis was identified in 1995 from a patient with bubonic plague in Madagascar. Although this organism was resistant to streptomycin, ampicillin, chloramphenicol, sulfonamides, and tetracycline, it retained its susceptibility to other aminoglycosides and cephalosporins. Given the subsequent identification of a similar organism in 1997 coupled with the fact that this resistance is plasmid-mediated, it seems likely that genetically modifying Y. pestis to a multidrugresistant form is possible. Unlike patients with inhalational anthrax (see above), patients with pulmonary plague should be cared for under conditions of strict respiratory isolation comparable to that used for multidrug-resistant tuberculosis.

1	Vaccination and Prevention A formalin-fixed, whole-organism vaccine was licensed by the FDA for the prevention of plague. That vaccine is no longer being manufactured, but its potential value as a current countermeasure against bioterrorism would likely have been modest at best as it was ineffective against animal models of primary pneumonic plague. Efforts are under way to develop a second generation of vaccines that will protect against aerosol challenge. Among the candidates being tested are recombinant forms of the fraction 1 capsular (F1) antigen and the virulence component of the type III secretion apparatus (V) antigen of Y. pestis. It is likely that doxycycline or levofloxacin would provide coverage in a chemoprophylaxis setting. Unlike the case with anthrax, in which one has to be concerned about the persistence of ungerminated spores in the respiratory tract, the duration of prophylaxis against plague need only extend to 7 days following exposure. See also Chap. 220e.

1	Variola Virus as a Bioweapon Given that most of the world’s population was once vaccinated against smallpox, variola virus would not have been considered a good candidate as a bioweapon 30 years ago. However, with the cessation of immunization programs in the United States in 1972 and throughout the world in 1980 due to the successful global eradication of smallpox, close to 50% of the U.S. population is fully susceptible to smallpox today. Given its infectious nature and the 10–30% mortality rate in unimmunized individuals, the deliberate spread of this virus could have a devastating effect on our society and unleash a previously conquered deadly disease. It is estimated that an initial infection of 50–100 persons in a first generation of cases could expand by a factor of 10–20 with each succeeding generation in the absence of any effective containment measures. Although the likely implementation of an effective public health response makes this scenario unlikely, it does illustrate

1	succeeding generation in the absence of any effective containment measures. Although the likely implementation of an effective public health response makes this scenario unlikely, it does illustrate the potential damage and disruption that can result from a smallpox outbreak.

1	In 1980, the WHO recommended that all immunization programs be terminated; that representative samples of variola virus be transferred to two locations, one at the CDC in Atlanta, GA, in the United States and the other at the Institute of Virus Preparations in the Soviet Union; and that all other stocks of smallpox be destroyed. Several years later, it was recommended that these two authorized collections be destroyed. However, these latter recommendations were placed on hold in the wake of increased concerns on the use of variola virus as a biologic weapon and thus the need to maintain an active program of defensive research. Many of these concerns were based on allegations made by former Soviet officials that extensive programs had been in place in that country for the production and weaponization of large quantities of smallpox virus. The dismantling of these programs with the fall of the Soviet Union and the subsequent weakening of security measures led to fears that stocks of

1	weaponization of large quantities of smallpox virus. The dismantling of these programs with the fall of the Soviet Union and the subsequent weakening of security measures led to fears that stocks of Variola major may have made their way to other countries or terrorist organizations. In addition, accounts that efforts had been taken to produce recombinant strains of Variola that would be more virulent and more contagious than the wild-type virus have led to an increase in the need to be vigilant for the reemergence of this often fatal infectious disease.

1	Microbiology and Clinical Features Smallpox is caused by one of two variants of variola virus, V. major and V. minor. Variola is a double-strand DNA virus and member of the Orthopoxvirus genus of the Poxviridae family. Infections with V. minor are generally less severe than those of V. major, with milder constitutional symptoms and lower mortality rates; thus V. major is the only one considered to be a viable bioweapon. Infection with V. major typically occurs following contact with an infected person. Patients are infectious from the time that a maculopapular rash appears on the skin and oropharynx through the resolution and scabbing of the pustular lesions. Infection occurs principally during close contact, through the inhalation of saliva droplets containing virus from the oropharyngeal exanthem. Aerosolized material from contaminated clothing or linen can also spread infection. Several days after exposure, a primary viremia is believed to occur that results in dissemination of

1	exanthem. Aerosolized material from contaminated clothing or linen can also spread infection. Several days after exposure, a primary viremia is believed to occur that results in dissemination of virus to lymphoid tissues. A secondary viremia occurs ~4 days later that leads to localization of infection in the dermis. Approximately 12–14 days following the initial exposure, the patient develops high fever, malaise, vomiting, headache, backache, and a maculopapular rash that begins on the face and extremities and spreads to the trunk (centripetal) with lesions in the same developmental stage in any given location. This is in contrast to the rash of varicella (chickenpox) that begins on the trunk and face and spreads to the extremities (centrifugal) with lesions at all stages of development. The lesions are initially maculopapular and evolve to vesicles that eventually become pustules and then scabs. The oral mucosa also develops maculopapular lesions that evolve to ulcers. The lesions

1	The lesions are initially maculopapular and evolve to vesicles that eventually become pustules and then scabs. The oral mucosa also develops maculopapular lesions that evolve to ulcers. The lesions appear over a period of 1–2 days and evolve at the same rate. Although virus can be isolated from the scabs on the skin, the conventional thinking is that once the scabs have formed the patient is no longer contagious. Smallpox is associated with 10–30% mortality rates, with patients typically dying of severe systemic illness during the second week of symptoms. Historically, ~5–10% of naturally occurring smallpox cases take either of two highly virulent atypical forms, classified as hemorrhagic and malignant. These are difficult to diagnose because of their atypical presentations. The hemorrhagic form is uniformly fatal and begins with the relatively abrupt onset of a severely prostrating illness characterized by high fevers and severe headache and back and abdominal pain. This form of the

1	form is uniformly fatal and begins with the relatively abrupt onset of a severely prostrating illness characterized by high fevers and severe headache and back and abdominal pain. This form of the illness resembles a severe systemic inflammatory syndrome, in which patients have a high viremia but die without developing the characteristic rash. Cutaneous erythema develops accompanied by petechiae and hemorrhages into the skin and mucous membranes. Death usually occurs within 5–6 days. The malignant, or “flat,” form of smallpox is frequently fatal and has an onset similar to the hemorrhagic form, but with confluent skin lesions developing more slowly and never progressing to the pustular stage.

1	Given the infectious nature of smallpox and the extreme vulnerability of contemporary society, patients who are suspected cases should be handled with strict isolation procedures. Although laboratory confirmation of a suspected case by culture, polymerase chain reaction (PCR), and electron microscopy is essential, it is equally important that appropriate precautions be used when obtaining samples for culture and laboratory testing. All health care and laboratory workers caring for patients should have been recently immunized with vaccinia, and all samples should be transported in doubly sealed containers. Patients should be cared for in negative-pressure rooms with strict isolation precautions.

1	There is no licensed specific therapy for smallpox, and historic treatments have focused solely on supportive care. Although several antiviral agents, including cidofovir, that are licensed for other diseases have in vitro activity against V. major, they have never been tested in the setting of human disease. For this reason, it is difficult to predict whether or not they would be effective in cases of smallpox and, if effective, whether or not they would be of value in patients with advanced disease. Agents currently being studied as possible antiviral compounds against V.�major include a viral egress inhibitor (tecovirimat, ST-246, or Arestvyr) and a lipid-conjugated form of cidofovir (brincidofovir, CMX001). preparations were made for mass production of 261e-7

1	preparations were made for mass production of 261e-7 CoMpLiCations FroM 438,134 adMinistrations oF vaCCinia during the u.s. departMent oF deFense (dod) sMaLLpox iMMunization CaMpaign initiated in F. tularensis by the United States, but no stockdeCeMBer 2002 piling of any agent took place. Stocks of F. tula rensis were reportedly generated by the Soviet DoD Rate per Million Number of Vaccinees (95% Historic Rate Per Union in the mid-1950s. It has also been sug-Complication Cases Confidence Interval) Million Vaccinees gested that the Soviet program extended into the era of molecular biology and that some strains were engineered to be resistant to common anti biotics. F. tularensis is an extremely infectious606a organism, and human infections have occurred streaked with colonies. Given these facts, it is 2.6–8.7a reasonable to conclude that this organism might 100b be used as a bioweapon through either an aero2–35a sol or contamination of food or drinking water.

1	1–7a Microbiology and Clinical Features Although 1–2a similar in many ways to anthrax and plague, aBased on adolescent and adult smallpox vaccinations from 1968 studies, both primary and revaccinations. bBased on tularemia, also referred to as rabbit fever or deer case series in Finnish military recruits given the Finnish strain of smallpox vaccine. cPotentially attributable to vaccina-fly fever, is neither as lethal nor as fulminant astion; after lupus-like illness. either of these other two category A bacterial Source: From JD Grabenstein and W Winkenwerder: http://www.smallpox.mil/event/SPSafetySum.asp. infections. It is, however, extremely infectious,

1	Vaccination and Prevention In 1796, Edward Jenner demonstrated that deliberate infection with cowpox virus could prevent illness on subsequent exposure to smallpox. Today, smallpox is a preventable disease following immunization with vaccinia. The current dilemma facing our society regarding assessment of the risk and benefit of smallpox vaccination is that the degree of risk that someone will deliberately and effectively release smallpox into our society is unknown. Given that there are well-described risks associated with vaccination, the degree of risk/benefit for the general population does not favor immunization. As a prudent first step in preparedness for a smallpox attack, however, members of the U.S. armed services received primary or booster immunizations with vaccinia before 1990 and after 2002. In addition, a number of civilian health care workers who comprise smallpox-response teams at the state and local public health level have been vaccinated.

1	Initial fears regarding the immunization of a segment of the American population with vaccinia when there are more individuals receiving immunosuppressive drugs and other immunocompromised patients than ever before were dispelled by the data generated from the military and civilian immunization campaigns of 2002–2004. Adverse event rates for the first 450,000 immunizations were similar to and, in certain categories of adverse events, even lower than those from prior historic data, in which most severe sequelae of vaccination occurred in young infants (Table 261e-4). In addition, 11 patients with early-stage HIV infection were inadvertently immunized without problem. One significant concern during that immunization campaign, however, was the description of a syndrome of myopericarditis, which had not been appreciated during prior immunization campaigns with vaccinia. In an effort to provide a safer vaccine to protect against smallpox, ACAM 2000, a cloned virus propagated in tissue

1	which had not been appreciated during prior immunization campaigns with vaccinia. In an effort to provide a safer vaccine to protect against smallpox, ACAM 2000, a cloned virus propagated in tissue culture, was developed and became the first second-generation smallpox vaccine to be licensed. This vaccine is now the only vaccinia product currently licensed in the United States and has been used by the U.S. military since 2008. It is part of the U.S. government stockpile. Research continues on attenuated forms of vaccinia such as modified vaccinia Ankara (MVA). Vaccinia immune globulin is available to treat those who experience a severe reaction to immunization with vaccinia.

1	See also Chap. 195.

1	Francisella tularensis as a Bioweapon Tularemia has been studied as an agent of bioterrorism since the mid-twentieth century. It has been speculated by some that the outbreak of tularemia among German and Soviet soldiers during fighting on the Eastern Front during WWII was the consequence of a deliberate release. Unit 731 of the Japanese army studied the use of tularemia as a bioweapon during WWII. Large and as few as 10 organisms can lead to establishment of infection. Despite this fact, it is not spread from person to person. Tularemia is caused by F. tularensis, a small, nonmotile, gram-negative coccobacillus. Although it is not a spore-forming organism, it is a hardy bacterium that can survive for weeks in the environment. Infection typically comes from insect bites or contact with organisms in the environment. Infections have occurred in laboratory workers studying the agent. Large waterborne outbreaks have been recorded. It is most likely that the outbreak among German and

1	organisms in the environment. Infections have occurred in laboratory workers studying the agent. Large waterborne outbreaks have been recorded. It is most likely that the outbreak among German and Russian soldiers and Russian civilians noted above during WWII represented a large waterborne tularemia outbreak in a Tularensis-enzootic area devastated by warfare.

1	Humans can become infected through a variety of environmental sources. Infection is most common in rural areas where a variety of small mammals may serve as reservoirs. Human infections in the summer are often the result of insect bites from ticks, flies, or mosquitoes that have bitten infected animals. In colder months, infections are most likely the result of direct contact with infected mammals and are most common in hunters. In these settings, infection typically presents as a systemic illness with an area of inflammation and necrosis at the site of tissue entry. Drinking of contaminated water may lead to an oropharyngeal form of tularemia characterized by pharyngitis with cervical and/or retropharyngeal lymphadenopathy (Chap. 195). The most likely mode of dissemination of tularemia as a biologic weapon would be as an aerosol, as has occurred in a number of natural outbreaks in rural areas, including Martha’s Vineyard in the United States. Approximately 1–14 days following

1	as a biologic weapon would be as an aerosol, as has occurred in a number of natural outbreaks in rural areas, including Martha’s Vineyard in the United States. Approximately 1–14 days following exposure by this route, one would expect to see inflammation of the airways with pharyngitis, pleuritis, and bronchopneumonia. Typical symptoms would include the abrupt onset of fever, fatigue, chills, headache, and malaise (Table 261e-3). Some patients might experience conjunctivitis with ulceration, pharyngitis, and/or cutaneous exanthems. A pulse-temperature dissociation might be present. Approximately 50% of patients would show a pulmonary infiltrate on chest x-ray. Hilar adenopathy might also be present, and a small percentage of patients could have adenopathy without infiltrates. The highly variable presentation makes acute recognition of aerosol-disseminated tularemia very difficult. The diagnosis would likely be made by immunohistochemistry, molecular techniques, or culture of infected

1	presentation makes acute recognition of aerosol-disseminated tularemia very difficult. The diagnosis would likely be made by immunohistochemistry, molecular techniques, or culture of infected tissues or blood. Untreated, mortality rates range from 5 to 15% for cutaneous routes of infection and from 30 to 60% for infection by inhalation. Since the advent of antibiotic therapy, these rates have dropped to <2%.

1	Both streptomycin and doxycycline are licensed for treatment of tularemia. Other agents likely to be effective include gentamicin, chloramphenicol, and ciprofloxacin (Table 261e-3). Given the potential for genetic modification of this organism to yield antibiotic-resistant strains, broad-spectrum coverage should be the rule until sensitivities have been determined. As mentioned above, special isolation procedures for patients are not required. Vaccination and Prevention There are no vaccines currently licensed for the prevention of tularemia. Although a live, attenuated strain of the organism has been used in the past with some reported success, there are inadequate data to support its widespread use at this time. Development of a vaccine for this agent is an important part of the current biodefense research agenda. In the absence of an effective vaccine, postexposure chemoprophylaxis with either doxycycline or ciprofloxacin appears to be a reasonable approach (Table 261e-3).

1	See also Chaps. 233 and 234. Hemorrhagic Fever Viruses as Bioweapons Several of the hemorrhagic fever viruses have been reported to have been weaponized by the Soviet Union and the United States. Nonhuman primate studies indicate that infection can be established with very few virions and that infectious aerosol preparations can be produced. Under the guise of wanting to aid victims of an Ebola outbreak, members of the Aum Shinrikyo cult in Japan were reported to have traveled to central Africa in 1992 in an attempt to obtain Ebola virus for use in a bioterrorist attack. Thus, although there has been no evidence that these agents have ever been used in a biologic attack, there is clear interest in their potential for this purpose.

1	Microbiology and Clinical Features The viral hemorrhagic fevers are a group of illnesses caused by any one of a number of similar viruses (Table 261e-2). These viruses are all enveloped, single-strand RNA viruses that are thought to depend on a host reservoir for long-term survival. Although rodents or insects have been identified as the hosts for some of these viruses, for others the hosts are unknown. These viruses tend to be geographically restricted according to the migration patterns of their hosts. Great apes are not a natural reservoir for Ebola virus, but large numbers of these animals in sub-Saharan Africa have died from Ebola infection over the past decade. Humans can become infected with hemorrhagic fever viruses if they come into contact with an infected host or other infected animals. Person-to-person transmission, largely through direct contact with virus-containing body fluids, has been documented for Ebola, Marburg, and Lassa viruses and rarely for the New World

1	animals. Person-to-person transmission, largely through direct contact with virus-containing body fluids, has been documented for Ebola, Marburg, and Lassa viruses and rarely for the New World arenaviruses. Although there is no clear evidence of respiratory spread among humans, these viruses have been shown in animal models to be highly infectious by the aerosol route. This, coupled with mortality rates as high as 90%, makes them excellent candidate agents of bioterrorism.

1	The clinical features of the viral hemorrhagic fevers vary depending on the particular agent (Table 261e-3). Initial signs and symptoms typically include fever, myalgia, prostration, and disseminated intravascular coagulation with thrombocytopenia and capillary hemorrhage. These findings are consistent with a cytokine-mediated systemic inflammatory syndrome. A variety of different maculopapular or erythematous rashes may be seen. Leukopenia, temperature-pulse dissociation, renal failure, and seizures may also be part of the clinical presentation.

1	Outbreaks of most of these diseases are sporadic and unpredictable. As a consequence, most studies of pathogenesis have been performed using laboratory animals. The diagnosis should be suspected in anyone with temperature >38.3°C for <3 weeks who also exhibits at least two of the following: hemorrhagic or purpuric rash, epistaxis, hematemesis, hemoptysis, or hematochezia in the absence of any other identifiable cause. In this setting, samples of blood should be sent after consultation to the CDC or the USAMRIID for serologic testing for antigen and antibody as well as reverse transcriptase polymerase chain reaction (RT-PCR) testing for hemorrhagic fever viruses. All samples should be handled with double-bagging. Given how little is known regarding the human-to-human transmission of these viruses, appropriate isolation measures would include full barrier precautions with negative-pressure rooms and use of powered air-purifying respirators (PAPRs).

1	Unprotected skin contact with cadavers has been implicated in the transmission of certain hemorrhagic fever viruses such as Ebola, so it is recommended that autopsies of suspected cases be performed using the strictest measures for protection and that burial or cremation be performed promptly without embalming. There are no approved and effective antiviral therapies for this class of viruses (Table 261e-3). Although there are anecdotal reports of the efficacy of ribavirin, interferon-α, or hyperimmune immunoglobulin, definitive data are lacking. The best data for ribavirin are in arenavirus (Lassa and New World) infections. In some in vitro systems, specific immunoglobulin has been reported to enhance infectivity, and thus these potential treatments must be approached with caution.

1	Vaccination and Prevention There are no licensed and effective vaccines for these agents. Studies are currently under way examining the potential role of DNA, recombinant viruses, and attenuated viruses as vaccines for several of these infections. Among the most promising at present are vaccines for Argentine, Ebola, Rift Valley, and Kyasanur Forest viruses. A series of monoclonal antibodies directed against the envelope glycoproteins of Ebola have demonstrated protection against infection in a postexposure setting in nonhuman primates and are being further developed for human use. See also Chap. 178.

1	Botulinum Toxin as a Bioweapon In a bioterrorist attack, botulinum toxin would likely be dispersed as an aerosol or as contamination of a food supply. Although contamination of a water supply is possible, it is likely that any toxin would be rapidly inactivated by the chlorine used to purify drinking water. Similarly, toxin can be inactivated by heating any food to >85°C for >5 min. Without external facilitation, the environmental decay rate is estimated at 1% per minute, and thus the time interval between weapon release and ingestion or inhalation needs to be rather short. The Japanese biologic warfare group, Unit 731, is reported to have conducted experiments on botulism poisoning in prisoners in the 1930s. The United States and the Soviet Union both acknowledged producing botulinum toxin, and there is some evidence that the Soviet Union attempted to create recombinant bacteria containing the gene for botulinum toxin. In records submitted to the United Nations, Iraq admitted to

1	toxin, and there is some evidence that the Soviet Union attempted to create recombinant bacteria containing the gene for botulinum toxin. In records submitted to the United Nations, Iraq admitted to having produced 19,000 L of concentrated toxin—enough toxin to kill the entire population of the world three times over. By many accounts, botulinum toxin was the primary focus of the pre-1991 Iraqi bioweapons program. In addition to these examples of state-supported research into the use of botulinum toxin as a bioweapon, the Aum Shinrikyo cult unsuccessfully attempted on at least three occasions to disperse botulinum toxin into the civilian population of Tokyo.

1	Microbiology and Clinical Features Unique among the category A agents for not being a live microorganism, botulinum toxin is one of the most potent toxins ever described and is thought by some to be the most poisonous substance in existence. It is estimated that 1 g of botulinum toxin would be sufficient to kill 1 million individuals if adequately dispersed. Botulinum toxin is produced by the gram-positive, spore-forming anaerobe C. botulinum (Chap. 178). Its natural habitat is soil. There are seven antigenically distinct forms of botulinum toxin, designated A–G. The majority of naturally occurring human cases are of types A, B, and E. Antitoxin directed toward one of these will have little to no activity against the others. The toxin is a 150-kDa zinc-containing protease that prevents the intracellular fusion of acetylcholine vesicles with the motor neuron membrane, thus preventing the release of acetylcholine. In the absence of acetylcholine-dependent triggering of muscle fibers, a

1	intracellular fusion of acetylcholine vesicles with the motor neuron membrane, thus preventing the release of acetylcholine. In the absence of acetylcholine-dependent triggering of muscle fibers, a flaccid paralysis develops. Although botulism does not spread from person to person, the ease of production of the toxin coupled with its high morbidity and 60–100% mortality make it a close to ideal bioweapon.

1	Botulism can result from the growth of C. botulinum infection in a wound or the intestine, the ingestion of contaminated food, or the inhalation of aerosolized toxin. The latter two forms are the most likely modes of transmission for bioterrorism. Once toxin is absorbed into the bloodstream, it binds to the neuronal cell membrane, enters the cell, and cleaves one of the proteins required for the intracellular binding of the synaptic vesicle to the cell membrane, thus preventing release of the neurotransmitter to the membrane of the adjacent muscle cell. Patients initially develop multiple cranial nerve palsies that are followed by a descending flaccid paralysis. The extent of the neuromuscular compromise is dependent on the level of toxemia. The majority of patients experience diplopia, dysphagia, dysarthria, dry mouth, ptosis, dilated pupils, fatigue, and extremity weakness. There are minimal true central nervous system effects, and patients rarely show significant alterations in

1	dysphagia, dysarthria, dry mouth, ptosis, dilated pupils, fatigue, and extremity weakness. There are minimal true central nervous system effects, and patients rarely show significant alterations in mental status. Severe cases can involve complete muscular collapse, loss of the gag reflex, and respiratory failure. Recovery requires the regeneration of new motor neuron synapses with the muscle cell, a process that can take weeks to months. In the absence of secondary infections, which may be common during the protracted recovery phase of this illness, patients remain afebrile. The diagnosis is suspected on clinical grounds and confirmed by a mouse bioassay or toxin immunoassay.

1	Treatment for botulism is mainly supportive and may require intubation, mechanical ventilation, and parenteral nutrition (Table 261e-3). If diagnosed early enough, administration of equine antitoxin may reduce the extent of nerve injury and decrease the severity of disease. At present, a heptavalent botulinum antitoxin (HBAT) is available through the CDC as an investigational agent for treatment of naturally occurring noninfant botulism. HBAT contains horse serum–derived antibody fragments to all seven known botulinum toxins (A–G). It is composed of <2% intact immunoglobulin and ≥90% Fab and F(ab′)2 immunoglobulin fragments. A single dose of antitoxin is usually adequate to neutralize any circulating toxin. Repeat dosing may be needed in a setting of continued toxin exposure. Given that this product is derived from horse serum, one needs to be vigilant for hypersensitivity reactions, including serum sickness and anaphylaxis following its administration. Once the damage to the nerve

1	this product is derived from horse serum, one needs to be vigilant for hypersensitivity reactions, including serum sickness and anaphylaxis following its administration. Once the damage to the nerve axon has been done, however, there is little possible in the way of specific therapy. At this point, vigilance for secondary complications such as infections during the protracted recovery phase is of the utmost importance. Due to their ability to worsen neuromuscular blockade, aminoglycosides and clindamycin should be avoided in the treatment of these infections.

1	Vaccination and Prevention A botulinum toxoid preparation has been used as a vaccine for laboratory workers at high risk of exposure and in certain military situations; however, it is not currently available in quantities that could be used for the general population. At present, early recognition of the clinical syndrome and use of appropriate equine antitoxin is the mainstay of prevention of full-blown disease in exposed individuals. The development of human monoclonal antibodies as a replacement for equine antitoxin antibodies is an area of active research interest.

1	The category B agents include those that are easy or moderately easy to disseminate and result in moderate morbidity and low mortality rates. A listing of the current category B agents is provided in Table 261e-2. As can be seen, it includes a wide array of microorganisms and products of microorganisms. Several of these agents have been used in bioterrorist attacks, although never with the impact of the category A agents described above. Among the more notorious of these was 261e-9 the contamination of salad bars in Oregon in 1984 with Salmonella typhimurium by the religious cult Rajneeshee. In this outbreak, which many consider to be the first bioterrorist attack against U.S. citizens, >750 individuals were poisoned and 40 were hospitalized in an effort to influence a local election. The intentional nature of this outbreak went unrecognized for more than a decade.

1	Category C agents are the third highest priority agents in the biodefense agenda. These agents include emerging pathogens to which little or no immunity exists in the general population, such as the severe acute respiratory syndrome (SARS) or Middle East respiratory syndrome (MERS) coronavirus or pandemic-potential strains of influenza that could be obtained from nature and deliberately disseminated. These agents are characterized as being relatively easy to produce and disseminate, having high morbidity and mortality rates, and having a significant public health impact. There is no running list of category C agents at the present time.

1	As noted above, a large number and diverse array of agents have the potential to be used in a bioterrorist attack. In contrast to the military situation with biowarfare, where the primary objective is to inflict mass casualties on a healthy and prepared militia, the objectives of bioterrorism are to harm civilians as well as to create fear and disruption among the civilian population. Although the military need only to prepare their troops to deal with the limited number of agents that pose a legitimate threat of biowarfare, the public health system needs to prepare the entire civilian population to deal with the multitude of agents and settings that could be used in a bioterrorism attack. This includes anticipating issues specific to the very young and the very old, the pregnant patient, and the immunocompromised individual. The challenges in this regard are enormous and immediate. Whereas military preparedness emphasizes vaccines toward a limited number of agents, civilian

1	patient, and the immunocompromised individual. The challenges in this regard are enormous and immediate. Whereas military preparedness emphasizes vaccines toward a limited number of agents, civilian preparedness needs to rely on rapid diagnosis and treatment of a wide array of conditions.

1	The medical profession must maintain a high index of suspicion that unusual clinical presentations or the clustering of cases of a rare disease may not be a chance occurrence but rather the first sign of a bioterrorist event. This is particularly true when such diseases occur in traditionally healthy populations, when surprisingly large numbers of rare conditions occur, and when diseases commonly seen in rural settings appear in urban populations. Given the importance of rapid diagnosis and early treatment for many of these conditions, it is essential that the medical care team report any suspected cases of bioterrorism immediately to local and state health authorities and/or to the CDC (888-246-2675). Enhancements made to the U.S. public health surveillance network after the anthrax attacks of 2001 have greatly facilitated the rapid sharing of information among public health agencies.

1	A series of efforts are in place to ensure the biomedical security of the civilian population of the United States. The Public Health Service has a more highly trained, fully deployable force. The Strategic National Stockpile (SNS) maintained by the CDC provides rapid access to quantities of pharmaceuticals, antidotes, vaccines, and other medical supplies that may be of value in the event of biologic or chemical terrorism. The SNS has two basic components. The first of these consists of “push packages” that can be deployed anywhere in the United States within 12 h. These push packages are a preassembled set of supplies, pharmaceuticals, and medical equipment ready for immediate delivery to the field. Given that an actual threat may not have been precisely identified at the time of stockpile deployment, they provide treatment for a variety of conditions. The contents of the push packs are constantly updated to ensure that they reflect current needs as determined by national security

1	deployment, they provide treatment for a variety of conditions. The contents of the push packs are constantly updated to ensure that they reflect current needs as determined by national security risk assessments; they include antibiotics for treatment of anthrax, plague, and tularemia as well as a cache of vaccine to deal with a smallpox threat. The second component of the SNS comprises inventories managed by specific vendors and consists of the provision of additional pharmaceuticals, supplies, and/ or products tailored to the specific attack.

1	The number of FDA-approved and licensed drugs and vaccines for category A and B agents is currently limited and not reflective of the pharmacy of today. In an effort to speed the licensure of additional drugs and vaccines for these diseases, the FDA has a rule for the licensure of such countermeasures against agents of bioterrorism when adequate and well-controlled clinical efficacy studies cannot be ethically conducted in humans. This is commonly referred to as the “Animal Rule.” Thus, for indications in which field trials of prophylaxis or therapy for a naturally occurring disease are not feasible, the FDA will rely on evidence solely from laboratory animal studies. For this rule to apply, it must be shown that (1) there are reasonably well-understood pathophysiologic mechanisms for the condition and its treatment; (2) the effect of the intervention is independently substantiated in at least two animal species, including species expected to react with a response predictive for

1	the condition and its treatment; (2) the effect of the intervention is independently substantiated in at least two animal species, including species expected to react with a response predictive for humans; (3) the animal study endpoint is clearly related to the desired benefit in humans; and (4) the data in animals allow selection of an effective dose in humans. As noted above, levofloxacin for treatment of plague and raxibacumab for treatment of inhalational anthrax have been licensed via this mechanism.

1	Finally, the Biomedical Advanced Research and Development Authority (BARDA) was established in 2006 within the U.S. Department of Health and Human Services to provide an integrated, systematic approach to the development and purchase of the necessary vaccines, drugs, therapies, and diagnostic tools for public health medical emergencies. As authorized by the All-Hazards Preparedness Reauthorization Act of 2013 in conjunction with the Project Bioshield Act of 2006 and the Pandemic and All Hazards Act of 2006, BARDA manages a series of initiatives designed to facilitate biodefense research within the federal government, create a stable source of funding for the purchase of countermeasures against agents of bioterrorism, and create a category of “emergency use authorization” to allow the FDA to approve the use of unlicensed countermeasures during times of extraordinary unmet needs, as might be present in the context of a bioterrorist attack.

1	Although the prospect of a deliberate attack on civilians with disease-producing agents may seem to be an act of incomprehensible evil, history shows us that it is something that has been done in the past and will likely be done again in the future. It is the responsibility of health care providers to be aware of this possibility, to be able to recognize early signs of a potential bioterrorist attack and alert the public health system, and to respond quickly to provide care to the individual patient. Among the websites with current information on microbial bioterrorism are www.bt.cdc.gov, www.niaid.nih.gov, and www.cidrap.umn.edu.

1	Chemical terrorism Charles G. Hurst, Jonathan Newmark, James A. Romano, Jr. The use of chemical warfare agents (CWAs) in modern warfare dates back to World War I (WWI). Sulfur mustard and nerve agents were used by Iraq against the Iranian military and Kurdish civilians. Most 262e recently the nerve agent Sarin, GB, was used by the Syrian military against their civilian population. Since the Japanese sarin attacks in 1994–1995 and the terrorist strikes of September 11, 2001, the alltoo-real possibility of chemical or biological terrorism against civilian populations anywhere in the world has attracted increased attention.

1	Military planners consider the WWI blistering agent sulfur mustard and the organophosphorus nerve agents as the most likely agents to be used on the battlefield. In a civilian or terrorist scenario, the choice widens considerably. For example, many of the CWAs of WWI, including chlorine, phosgene, and cyanide, are used today in large amounts in industry. They are produced in chemical plants, are stockpiled in large tanks, and travel up and down highways and railways in large tanker cars. The rupture of any of these stores by accident or on purpose could cause many injuries and deaths. In three attacks in February 2007, for example, insurgents in Iraq used chlorine gas released from tankers after explosions as a crude form of chemical weaponry; these attacks killed 12 people and intoxicated more than 140 others. Countless hazardous materials (HAZMATs) that are not used on the battlefield can be used as terrorist weapons. Some of them, including insecticides and ammonia, could wreak as

1	more than 140 others. Countless hazardous materials (HAZMATs) that are not used on the battlefield can be used as terrorist weapons. Some of them, including insecticides and ammonia, could wreak as much damage and injury as the weaponized chemical agents.

1	Many mistakenly believe that chemical attacks will always be so severe that little can be done except to bury the dead. History proves the opposite. Even in WWI, when IV fluids, endotracheal tubes, and antibiotics were unavailable, the mortality rate among U.S. forces on the battlefield from CWAs—chiefly sulfur mustard and the pulmonary intoxicants—was only 1.9%. That figure was far lower than the 7% mortality rate from conventional wounds. In the 1995 Tokyo subway sarin incident, among the 5500 patients who sought medical attention at hospitals, 80% were not actually symptomatic and only 12 died. Recent events should prompt not a fatalistic attitude but a realistic wish to understand the pathophysiology of the syndromes these agents cause, with a view to treating expeditiously all patients who present for care and an expectation of saving the vast majority. As we prepare to defend our civilian population from the effects of chemical terrorism, we also must consider the fact that

1	who present for care and an expectation of saving the vast majority. As we prepare to defend our civilian population from the effects of chemical terrorism, we also must consider the fact that terrorism itself can produce sequelae such as physiologic or neurologic effects that may resemble the effects of nonlethal exposures to CWAs. These effects are due to a general fear of chemicals, fear of decontamination, fear of protective ensembles, or other phobic reactions. The increased difficulty in differentiating between stress reactions and nerve agent–induced organic brain syndromes has been pointed out. Knowledge of the behavioral effects of CWAs and their medical countermeasures is imperative to ensure that military and civilian medical and mental health organizations can deal with possible incidents involving weapons of mass destruction.

1	For the reader’s benefit, the CWAs, their two-letter North Atlantic Treaty Organization (NATO) codes (which were established by a NATO international convention and convey no clinical implications), their unique physical features, and their initial effects are listed in Table 262e-1. Table 262e-2 provides guidelines for immediate treatment. The focus of this chapter is on the blister and nerve CWAs, which have been employed in battle and against civilians and have had a significant public health impact. VESICANTS: SULFUR MUSTARD Sulfur mustard has been a military threat since it first appeared on the battlefield in Belgium during WWI. In modern times, it remains a threat on the battlefield as well as a potential chemical terrorism aLetters in parentheses indicate NATO codes for designated agents. bFrostbite may occur from skin contact with liquid arsine, cyanogen chloride, or phosgene.

1	Source: State of New York, Department of Health, as modified by the Chemical Casualty Care Division, U.S. Army Medical Research Institute of Chemical Defense. weapon because of the simplicity of its manufacture and its extreme effectiveness. Sulfur mustard accounted for 70% of the 1.3 million chemical casualties in WWI. Occasional cases of sulfur mustard intoxication continue to occur in the United States among people exposed to WWIand WWII-era munitions. Mechanism Sulfur mustard constitutes both a vapor and a liquid threat to all exposed epithelial surfaces. The effects are delayed, aFor frostbite areas, DO NOT remove any adhering clothing. Wash area with plenty of warm water to release clothing. Source: State of New York, Department of Health.

1	appearing hours after exposure. The organs most commonly affected blister formation. Mustard also has mild cholinergic activity, which are the skin (with erythema and vesicles), the eyes (with manifestations may be responsible for effects such as early gastrointestinal and CNS ranging from mild conjunctivitis to severe eye damage), and the air-symptoms. Mustard reacts with tissue within minutes of entering the ways (with effects ranging from mild upper airway irritation to severe body. Its circulating half-life in unaltered form is extremely brief. bronchiolar damage). After exposure to large quantities of mustard, precursor cells of the bone marrow are damaged, with consequent pan-Clinical Features Topical effects of mustard occur in the skin, airways, cytopenia and secondary infection. The gastrointestinal mucosa may and eyes; the eyes are most sensitive and the airways next most sensibe damaged, and there are sometimes central nervous system (CNS) tive. Absorbed mustard may produce

1	gastrointestinal mucosa may and eyes; the eyes are most sensitive and the airways next most sensibe damaged, and there are sometimes central nervous system (CNS) tive. Absorbed mustard may produce effects in the bone marrow, gas-signs of unknown mechanism. No specific antidotes exist; manage-trointestinal tract, and CNS. Direct injury to the gastrointestinal tract ment is entirely supportive. Immediate decontamination of the liquid also may occur after ingestion of the compound through contaminais the only way to reduce damage. Complete decontamination in 2 min tion of water or food. stops clinical injury; decontamination at 5 min reduces skin injury by Erythema is the mildest and earliest form of mustard skin injury. It ~50%. Table 262e-2 lists approaches to decontamination after expo-resembles sunburn and is associated with pruritus, burning, or sting-sure to mustard and other CWAs. ing pain. Erythema begins to appear within 2 h to 2 days after vapor

1	Mustard dissolves slowly in aqueous media such as sweat, but, once exposure. Time of onset depends on severity of exposure, ambient dissolved, it rapidly forms cyclic ethylene sulfonium ions that are temperature and humidity, and type of skin. The most sensitive sites extremely reactive with cell proteins, cell membranes, and especially are warm moist locations and areas of thin delicate skin, such as the DNA in rapidly dividing cells. The ability of mustard to react with and perineum, external genitalia, axillae, antecubital fossae, and neck. alkylate DNA gives rise to the effects by which it has been character-Within the erythematous areas, small vesicles can develop, which ized as “radiomimeti”—i.e., similar to radiation injury. Mustard has may later coalesce to form bullae (Fig. 262e-1). The typical bulla is many biologic actions, but its actual mechanism of action is largely large, dome-shaped, flaccid, thin-walled, translucent, and surrounded unknown. Much of the biologic damage

1	The typical bulla is many biologic actions, but its actual mechanism of action is largely large, dome-shaped, flaccid, thin-walled, translucent, and surrounded unknown. Much of the biologic damage from mustard results from by erythema. The blister fluid, a transudate, is clear to straw-colored DNA alkylation and cross-linking in rapidly dividing cells: corneal and becomes yellow, tending to coagulate. The fluid does not contain epithelium, basal keratinocytes, bronchial mucosal epithelium, gastro-mustard and is not itself a vesicant. Lesions from high-dose liquid intestinal mucosal epithelium, and bone marrow precursor cells. This exposure may develop a central zone of coagulation necrosis with blisdamage may lead to cellular death and inflammatory reactions. In the ter formation at the periphery. These lesions take longer to heal and are skin, proteolytic digestion of anchoring filaments at the epidermal-more prone to secondary infection than are the uncomplicated lesions dermal

1	periphery. These lesions take longer to heal and are skin, proteolytic digestion of anchoring filaments at the epidermal-more prone to secondary infection than are the uncomplicated lesions dermal junction may be the major mechanism of action resulting in seen at lower exposure levels. Severe lesions may require skin grafting.

1	FIgURE 262e-1 Large bulla formation from mustard burn in a patient. Although the blisters in this case involved only 7% of the body surface area, the patient still required hospitalization in a burn intensive care unit. The primary airway lesion is necrosis of the mucosa with possible damage to underlying smooth muscle. The damage begins in the upper airways and descends to the lower airways in a dose-dependent manner. Usually the terminal airways and alveoli are affected only as a terminal event. Pulmonary edema is not usually present unless the damage is very severe, and then it becomes hemorrhagic.

1	The earliest effects of mustard—and perhaps the only effects of a low concentration—involve the nose, sinuses, and pharynx. There may be irritation or burning of the nares, epistaxis, sinus pain, and pharyngeal pain. As the concentration increases, laryngitis, voice changes, and nonproductive cough develop. Damage to the trachea and upper bronchi leads to a productive cough. Lower airway involvement causes dyspnea, severe cough, and increasing quantities of sputum. Terminally, there may be necrosis of the smaller airways with hemorrhagic edema into surrounding alveoli. Hemorrhagic pulmonary edema is rare. Necrosis of airway mucosa causes “pseudomembrane” formation. These membranes may obstruct the bronchi. During WWI, high-dose mustard exposure caused acute death via this mechanism in a small minority of cases (Fig. 262e-2).

1	The eyes are the organs most sensitive to mustard vapor injury. The latent period is shorter for eye injury than for skin injury and is also dependent on exposure concentration. After low-dose vapor exposure, irritation evidenced by reddening of the eyes may be the only effect. As the dose increases, the injury includes progressively severe conjunctivitis, photophobia, blepharospasm, pain, and corneal damage FIgURE 262e-2 Schematic diagram of pseudomembrane forma-tion as is seen in high-dose sulfur mustard vapor inhalation exposure. In World War I, severe inhalation exposure often caused death via obstruction of large airways. FIgURE 262e-3 World War I photograph of troops exposed to sulfur mustard vapor. The vast majority of these troops survived with no long-term damage to the eyes; however, they were rendered effectively blind for days or weeks.

1	(Fig. 262e-3). About 90% of eye injuries related to mustard heal in 2 weeks to 2 months without sequelae. Scarring between the iris and the lens may follow severe effects; this scarring may restrict pupillary movements and predispose victims to glaucoma. The most severe damage is caused by liquid mustard. Extensive eye exposure can be followed by severe corneal damage with possible perforation of the cornea and loss of the eye. In some individuals, latent chronic keratitis, sometimes associated with corneal ulcerations, has been described as early as 8 months and as late as 20 years after initial exposure.

1	The mucosa of the gastrointestinal tract is susceptible to mustard damage from either systemic absorption or ingestion of the agent. Mustard exposure in small amounts will cause nausea and vomiting lasting up to 24 h. The mechanism of the nausea and vomiting is not understood, but mustard does have a cholinergic-like effect. The CNS effects of mustard also remain poorly defined. Exposure to large amounts can cause seizures in animals. Reports from WWI and from the Iran–Iraq war described people exposed to small amounts of mustard acting sluggish, apathetic, and lethargic. These reports suggest that minor psychological problems could linger for ≥1 year.

1	The cause of death in the majority of mustard poisoning cases is sepsis and respiratory failure. Mechanical obstruction via pseudomembrane formation and agent-induced laryngospasm is important in the first 24 h, but only in cases of severe exposure. From the third through the fifth day after exposure, secondary pneumonia due to bacterial invasion of denuded necrotic mucosa can be expected. The third wave of death is caused by agent-induced bone marrow suppression, which peaks 7–21 days after exposure and causes death via sepsis. A patient severely ill from mustard poisoning requires the general supportive care provided for any severely ill patient as well as the specific care given to a burn patient. Liberal use of systemic analgesics, maintenance of fluid and electrolyte balance, provision of nutrition, administration of appropriate antibiotics, and other supportive measures are necessary (Table 262e-2).

1	The management of a patient exposed to mustard may range from simple (as in the provision of symptom-based care for a sunburn-like erythema) to complex (as in total management of a severely ill patient with burns, immunosuppression, and multi-system involvement). Before raw denuded areas of skin develop, especially with less severe exposures, topical cortisone creams or lotions may be of benefit. Some very basic research data point to the early use of anti-inflammatory preparations. Small blisters (<1–2 cm) should be left intact. Because larger bullae eventually will break, they should be unroofed carefully. Denuded areas should be irrigated three or four times daily with saline, other sterile solutions, or soapy water and then liberally covered with the topical antibiotic of choice, such as silver sulfadiazine, mafenide acetate, or triple antibiotic ointment to a thickness of 1–2 mm. Some physicians advocate sterile needle drainage of large blisters, with collapsing of the blister

1	as silver sulfadiazine, mafenide acetate, or triple antibiotic ointment to a thickness of 1–2 mm. Some physicians advocate sterile needle drainage of large blisters, with collapsing of the blister roof to form a sterile dressing. Mustard blister fluid does not contain sulfur mustard but rather consists only of sterile tissue fluid. Health care staff should not fear possible contamination. If an antibiotic cream is not available, sterile petrolatum will be useful. Modified Dakin’s solution (sodium hypochlorite, 0.5%) was used for field-expedient irrigation and antisepsis both in WWI and for Iranian casualties in the Iran–Iraq war (1984–1987). Large areas of vesication require hospitalization, IV therapy, and whirlpool bath irrigation.

1	Systemic analgesics should be used liberally, particularly before manipulation of the patient. Monitoring of fluids and electrolytes is important in any sick patient, but it must be recognized that fluid loss is not of the magnitude seen with thermal burns. Overly rigorous hydration seems to have precipitated pulmonary edema in a few Iranian casualties sent to European hospitals.

1	Conjunctival irritation from a low-vapor exposure responds to any of a number of available ophthalmic solutions after the eyes are irrigated thoroughly. A topical antibiotic applied several times a day reduces the incidence and severity of infection. Animal laboratory data reflect remarkable results with early application of commercially available topical antibiotic/glucocorticoid ophthalmologic ointments. An ophthalmologist should be consulted. Topical glucocorticoids are not of proven value, but their use during the first few hours or days may significantly reduce inflammation and subsequent damage. Further use should be relegated to an ophthalmologist. Petroleum jelly or a similar substance should be applied regularly to the edges of the eyelids to prevent them from sticking together. Topical analgesics, although of limited value, may be useful initially if blepharospasm is too severe to permit an adequate examination.

1	A productive cough and dyspnea accompanied by fever and leukocytosis occurring within 12–24 h are indicative of chemical pneumonitis. The clinician must resist the urge to use prophylactic antibiotics for this process. Infection often occurs on the third to fifth day and is signaled by increased fever, a pulmonary infiltrate, and increased sputum production with a change in color. Appropriate antibiotic therapy should await confirmation by Gram’s stain and, later, by culture and sensitivity assessment. Intubation may be necessary if laryngeal spasm or edema makes breathing difficult or becomes life-threatening. Intubation permits better ventilation and facilitates suctioning of necrotic and inflammatory debris. Early use of positive end-expiratory pressure (PEEP) or continuous positive airway pressure (CPAP) may be beneficial. Pseudomembrane formation may require fiberoptic bronchoscopy for suctioning of necrotic debris.

1	Bronchodilators are of benefit for bronchospasm. If additional relief of bronchospasm is needed, glucocorticoids should be used. There is little evidence that the routine use of glucocorticoids is beneficial except for additional relief of bronchospasm.

1	Leukopenia begins around day 3 with major systemic absorption. Marrow suppression peaks at 7–14 days. In the Iran–Iraq war, a white blood cell count of ≤200/μL usually resulted in death of the patient. Sterilization of the gut by nonabsorbable antibiotics should be considered to reduce the possibility of sepsis from enteric organisms. Cellular replacement (bone marrow transplants or transfusions) may be successful. Granulocyte colony-stimulating factor produced a 50% reduction in the time required for bone marrow recovery in nonhuman primates exposed to sulfur mustard. Medication for nausea and vomiting may be necessary for gastrointestinal side effects. Lymphopenia precedes general leukopenia by a day or more and may be a useful clinical tip-off to impending leukopenia.

1	Excellent experimental assessments of the contributions of DNA alkylation, inflammation, activation of proteolytic enzymes, or lipid peroxidation to the mustard injury have been developed in the past 15–20 years. Some examples include (1) the demonstration of a reduction by up to 75% of inflammation and tissue damage in the mouse ear swelling test by vanilloid compounds and (2) the demonstration of 50–60% protection by N-acetylcysteine in the generation of free radicals within guinea pig lung exposed to mustard. In many cases, the demonstration of protection is dependent on the availability of sufficient amounts of drugs with adequate half-lives. Strategies to enhance bioavailability include attachment of polyethylene glycol to the antioxidant drug/enzyme and/or delivery of the drug/enzyme in a liposome.

1	The organophosphorus nerve agents are the deadliest of the CWAs. They work by inhibition of tissue synaptic acetylcholinesterase, creating an acute cholinergic crisis. Death ensues because of respiratory depression and can occur within seconds to minutes. The nerve agents tabun and sarin were first used on the battlefield by Iraq against Iran during the first Persian Gulf War (1984–1987). Estimates of casualties from these agents range from 20,000–100,000. In 1994 and 1995, the Japanese cult Aum Shinrikyo used sarin in two terrorist attacks in Matsumoto and Tokyo. Two U.S. soldiers were exposed to sarin while rendering safe an improvised explosive device in Iraq in 2004.

1	The “classic” nerve agents include tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), and VX; VR, similar to VX, was manufactured in the former Soviet Union (Table 262e-1). All the nerve agents are organophosphorus compounds, which are liquid at standard temperature and pressure. The “G” agents evaporate at about the rate of water, except for cyclosarin, which is oily and thus probably will have evaporated within 24 h after deposition on the ground. Their high volatility thus makes a spill of any amount a serious vapor hazard. In the Tokyo subway attack in which sarin was used, 100% of the symptomatic patients inhaled sarin vapor that spilled out on the floor of the subway cars. The low vapor pressure of VX, an oily liquid, makes it much less of a vapor hazard but potentially a greater environmental hazard because it persists in the environment far longer.

1	Mechanism Acetylcholinesterase inhibition accounts for the major life-threatening effects of nerve agent poisoning. The efficacy of antidotal therapy in the reversal of this inhibition proves that this is the primary toxic action of these poisons. At cholinergic synapses, acetylcholinesterase, bound to the postsynaptic membrane, functions as a turn-off switch to regulate cholinergic transmission. Inhibition of acetylcholinesterases causes the released neurotransmitter, acetylcholine, to accumulate abnormally. End-organ overstimulation, which is recognized by clinicians as a cholinergic crisis, ensues (Fig. 262e-4). Clinical Features Clinical effects of nerve agent exposure are identical for vapor and liquid exposure routes if the dose is sufficiently large. The speed and order of symptom onset will differ (Table 262e-2).

1	Exposure of a patient to nerve agent vapor, by far the more likely route of exposure in both battlefield and terrorist scenarios, will cause cholinergic symptoms in the order in which the toxin encounters cholinergic synapses. The most exposed synapses on the human integument are in the pupillary muscles. Nerve agent vapor easily crosses the cornea, interacts with these synapses, and produces miosis, described by Tokyo subway victims as “the world going black.” Rarely, FIgURE 262e-4 Schematic diagram of the pathophysiology of nerve agent exposure. Nerve agent ( ) binds to the active site of acetylcholinesterase (AChE), which is shown as floating free in space but is in reality a postsynaptic membrane-bound enzyme. As a result, acetylcholine ( ), which normally is released from presynaptic membrane but then is degraded, accumulates, and this leads ( ) to organ overstimulation and cholinergic crisis.

1	this vapor also can cause eye pain and nausea. Exocrine glands in the nose, mouth, and pharynx are next exposed to the vapor, and cholinergic overload here causes increased secretions, rhinorrhea, excess salivation, and drooling. Toxin then interacts with exocrine glands in the upper airway, causing bronchorrhea, and with bronchial smooth muscle, causing bronchospasm. This combination of events can result in hypoxia. Once the victim has inhaled, vapor can passively cross the alveolar-capillary membrane, enter the bloodstream, and incidentally and asymptomatically inhibit circulating cholinesterases, particularly free butyrylcholinesterase and erythrocyte acetylcholinesterase, both of which can be assayed. Unfortunately, the results of this assay may not be easily interpretable without a baseline, since cholinesterase levels vary enormously between individuals and over time in an individual, healthy patient.

1	Usually the first organ system to become symptomatic from bloodborne nerve agent exposure is the gastrointestinal tract, where cholinergic overload causes abdominal cramping and pain, nausea, vomiting, and diarrhea. After the gastrointestinal tract becomes involved, nerve agents will affect the heart, distant exocrine glands, muscles, and brain. Because there are cholinergic synapses on both the vagal (parasympathetic) and sympathetic sides of the autonomic input to the heart, one cannot predict how heart rate and blood pressure will change once intoxication has occurred. Remote exocrine activity will include oversecretion in the salivary, nasal, respiratory, and sweat glands—the patient will be “wet all over.” Bloodborne nerve agents will overstimulate neuromuscular junctions in skeletal muscles, causing fasciculations followed by frank twitching. If the process goes on long enough, ATP in muscles will eventually be depleted and flaccid paralysis will ensue.

1	In the brain, since the cholinergic system is so widely distributed, bloodborne nerve agents will, in sufficient doses, cause rapid loss of consciousness, seizures, and central apnea leading to death within minutes. If respiration is supported, status epilepticus that does not respond to usual anticonvulsants may ensue (Chap. 445). If status epilepticus persists, neuronal death and permanent brain dysfunction may occur. Even in mild nerve-agent intoxication, patients may recover but may experience weeks of irritability, sleep disturbance, and nonspecific neurobehavioral manifestations. The time from exposure to development of the full-blown cholinergic crisis after nerve agent vapor inhalation can be minutes or even seconds, yet there is no depot effect. Since nerve agents have a short circulating half-life, if the patient is supported and, ideally, treated 262e-5 with antidotes, improvement should be rapid, without subsequent deterioration.

1	Liquid exposure to nerve agents results in different speeds and orders of symptom onset. A nerve agent on intact skin will partially evaporate and partially begin to travel through the skin, causing localized sweating and then, when it encounters neuromuscular junctions, localized fasciculations. Once in muscle, the agent will cross into the circulation and cause gastrointestinal discomfort, respiratory distress, heart rate changes, generalized fasciculations and twitching, loss of consciousness, seizures, and central apnea. The time course will be much longer than with vapor inhalation; even a large, lethal droplet can take up to 30 min to exert an effect, and a small, sublethal dose could progressively take effect over 18 h. Clinical worsening that occurs hours after treatment has started is far more likely with liquid than with vapor exposure. In addition, miosis, which is practically unavoidable with vapor exposure, is not always present with liquid exposure and may be the last

1	is far more likely with liquid than with vapor exposure. In addition, miosis, which is practically unavoidable with vapor exposure, is not always present with liquid exposure and may be the last manifestation to develop in this situation; such a delay is due to the relative insulation of the pupillary muscle from the systemic circulation.

1	Unless the cholinesterase is reactivated by specific therapy (oximes), its binding to the enzyme is essentially irreversible. Erythrocyte acetylcholinesterase activity recovers at ~1% per day. Plasma butyrylcholinesterase recovers more quickly and is a better guide to recovery of tissue enzyme activity. Acute nerve agent poisoning is treated by decontamination, respiratory support, and three antidotes: an anticholinergic, an oxime, and an anticonvulsant (Table 262e-3). In acute cases, all these forms of therapy may be given simultaneously.

1	Decontamination of a vapor is formally unnecessary; however, in the Tokyo subway attack, sarin vapor trapped in patients’ clothing caused miosis in 10% of emergency personnel. Removal of clothing would have prevented most of these instances. Expedient decontamination methods for CWAs are available. For soap and water decontamination, the skin surface and hair are washed in warm or tepid water at least three times, or the exposed individual showers for 2 min, washing with soap and rinsing. The rapid physical removal Elderly, frail Atropine (1 mg IM) and 2-PAM Cl (10 mg/kg IM or 5–10 mg/kg IV slowly) Atropine (2–4 mg IM) and 2-PAM Cl (25 mg/kg IM or 5–10 mg/kg IV slowly) Assisted ventilation after antidotes for severe Repeat atropine (2 mg IM, or 1 mg IM for infants) at 5to 10-min intervals until secretions have diminished and breathing is comfortable or airway resistance has returned to nearly normal.

1	Repeat atropine (2 mg IM, or 1 mg IM for infants) at 5to 10-min intervals until secretions have diminished and breathing is comfortable or airway resistance has returned to nearly normal. Diazepam for convulsions (0.2–0.5 mg IV for infants <5 years; 1 mg IV for children >5 years; 5 mg IV for adults). aMild/moderate effects include localized sweating, muscle fasciculations, nausea, vomiting, weakness, and dyspnea. bSevere effects include unconsciousness, convulsions, apnea, and flaccid paralysis. cIf the calculated dose exceeds the adult IM dose, adjust accordingly. Abbreviation: 2-PAM Cl, 2-pralidoxime (or Protopam®) chloride. Source: State of New York, Department of Health.

1	of a chemical agent is essential. Scrubbing of exposed skin with a stiff brush or bristles is discouraged, because skin damage may occur and may increase absorption of agent. “Gentle” liquid dish soap and copious amounts of water should be used, with mild to moderate friction applied with a single-use sponge or washcloth in the first and second washes. The third wash should be a rinse with copious amounts of warm or tepid water. Shampoo can be used to wash the hair. If only cold water is available, it should be used; decontamination should not be delayed while warm water is sought. Spot (local) decontamination with reactive skin decontamination lotion (RSDL), followed by a soap and water wash/shower, is the method preferred by the Department of Defense. RSDL is available for purchase by civilians and has been shown to be superior across a broad spectrum of nerve agents as well as sulfur mustard. RSDL is the only product approved by the U.S. Food and Drug Administration (FDA) for

1	by civilians and has been shown to be superior across a broad spectrum of nerve agents as well as sulfur mustard. RSDL is the only product approved by the U.S. Food and Drug Administration (FDA) for initial spot decontamination. An important caveat is that RSDL and 0.5% sodium hypochlorite (dilute bleach military field expedient) should not be used concurrently because of a potential exothermic reaction. In any event, decontamination must be accomplished before the patient enters the medical facility to avoid contaminating the facility and its staff. In patients with contaminated wounds, potentially contaminated clothing and other foreign material that may serve as a depot for the liquid agent should be extracted.

1	Death from nerve agent poisoning is almost always attributable to respiratory causes. Ventilation will be complicated by increased resistance and secretions. Atropine should be given before ventilation or as it begins, since it will make ventilation far easier.

1	ANTIDOTAL THERAPY Atropine In theory, any anticholinergic agent could be used to treat nerve agent poisoning. Worldwide, however, the choice is invariably atropine because of its wide temperature stability and rapid effectiveness when administered either IM or IV and because inadvertent administration of this drug usually causes little CNS dysfunction (Table 262e-3). Atropine rapidly reverses cholinergic overload at muscarinic synapses but has little effect at nicotinic synapses. The practical implication is that atropine can quickly treat the life-threatening respiratory effects of nerve agents but probably will not help with neuromuscular (and possibly sympathetic) effects. In the field, military personnel are given a combined autoinjector containing both 2.1 mg of atropine and oxime (2-pralidoxime chloride [2-PAM Cl])—a product licensed by the FDA under the trade name Duodote . Its miltary designation is the Antidote Treatment Nerve Agent Autoinjector (ATNAA) (Fig. 262e-5). Only

1	chloride [2-PAM Cl])—a product licensed by the FDA under the trade name Duodote . Its miltary designation is the Antidote Treatment Nerve Agent Autoinjector (ATNAA) (Fig. 262e-5). Only full—and not divided—autoinjector doses can be administered. The field loading dose is 2, 4, or 6 mg, with re-treatment every 5–10 min until the patient’s breathing improves and secretions diminish. The Iranian military initially used larger doses during the Iran–Iraq war, in which oximes were in short supply. When the patient reaches a level of medical care at which drugs can be given IV, this is the preferred route. In small children, the IV route may be the initial avenue for atropine therapy; however, pediatric autoinjectors of 0.5 mg and 1 mg are manufactured. There is no upper limit to atropine therapy (whether IM or IV), but the total average dose for a severely afflicted adult is usually 20–30 mg.

1	In a mildly afflicted patient with miosis and no other systemic symptoms, atropine or homatropine eyedrops may suffice for therapy. This treatment will result in ~24 h of mydriasis. Frank miosis or imperfect accommodation may persist for weeks or even months after all other signs and symptoms have resolved.

1	Oximes Oximes are nucleophiles that reactivate the cholinesterase whose active site has been occupied and bound to nerve agent (Table 262e-3). Therapy with oximes therefore restores normal enzyme function. Oxime therapy is limited by a second side reaction, called “aging,” in which a side chain on nerve agents falls off the complex at a characteristic rate. “Aged” complexes are negatively charged, and oximes cannot reactivate negatively charged complexes. The practical effect of this limitation differs from one nerve agent to another since each ages at a characteristic rate. For example, sarin ages in 3–5 h, tabun ages over a longer period (12–13 h), and VX ages much less rapidly (>48 h). All these intervals are so much longer than the patient’s expected life span and expected treatment time after acute nerve-agent toxicity that they are irrelevant. Soman, in contrast, ages in 2 min; thus, only a few minutes after exposure, oximes are useless in treating soman poisoning. The oxime

1	time after acute nerve-agent toxicity that they are irrelevant. Soman, in contrast, ages in 2 min; thus, only a few minutes after exposure, oximes are useless in treating soman poisoning. The oxime used varies by country; the United States has approved and fielded 2-PAM Cl. MARK 1 kits and Duodotes® (Fig. 262e-5A) both contain autoinjectors holding 600 mg of 2-PAM Cl. Initial field loading doses are 600, 1200, and 1800 mg. Since blood pressure may become elevated after administration of 45 mg/kg in adults, field use of 2-PAM Cl is restricted to 1800 mg/h IM. During the time when more oxime cannot be given, atropine alone is recommended. In the hospital setting, 2.5–25 mg/kg of 2-PAM Cl by the IV route has been found to reactivate 50% of inhibited cholinesterase. The usual recommendation is 1000 mg by slow IV drip over 20–30 min, with ≤2500 mg over a period of 1–1.5 h. Active research aims to field a more effective and broader-spectrum oxime than 2-PAM Cl.

1	Anticonvulsants Nerve agent–induced seizures do not respond to the usual anticonvulsants used for status epilepticus, including phenytoin, phenobarbital, carbamazepine, valproic acid, and lamotrigine (Chap. 445). The only anticonvulsants that have been shown to stop this form of status are the benzodiazepines. Diazepam is the only benzodiazepine approved for seizures in humans, although other FDA-approved benzodiazepines (notably midazolam) work well against nerve agent–induced seizures in animal models. Diazepam FIgURE 262e-5 Antidotes to nerve agents. A. The Antidote Treatment Nerve Agent Autoinjector (ATNAA) replaces the MARK I Kit. It is easier to self-administer and allows prompt distribution of the antidotes atropine and 2-pralidoxime chloride (2-PAM Cl). B. Diazepam 10-mg autoinjectors are carried by all U.S. military forces in a potential chemical battlefield and are being stockpiled by civilian first responders.

1	B. Diazepam 10-mg autoinjectors are carried by all U.S. military forces in a potential chemical battlefield and are being stockpiled by civilian first responders. therefore is manufactured in 10-mg injectors for IM use and given to

1	U.S. forces for this purpose (Fig. 262e-5B). Civilian agencies are stockpiling this field product (convulsive antidote for nerve agent, CANA), which generally has not been used in hospital practice. Extrapolation from animal studies indicates that adults will probably require 30–40 mg of diazepam given IM to stop nerve agent–induced status epilepticus. In the hospital or in a small child unable to receive the autoinjector, IV diazepam may be used at similar doses. The clinician may confuse seizures with the neuromuscular signs of nerve agent poisoning. In the hospital, early electroencephalography is advised to distinguish among nonconvulsive status epilepticus, actual seizures, and postictal paralysis. Animal studies have shown that the most effective benzodiazepine in this situation is midazolam, which is not FDA-approved for seizures. At the time of this writing, a new drug application for use of midazolam against seizures has been submitted to the FDA. The superiority of IM

1	is midazolam, which is not FDA-approved for seizures. At the time of this writing, a new drug application for use of midazolam against seizures has been submitted to the FDA. The superiority of IM midazolam to IV lorazepam in a large community trial of status epilepticus suggests that emergency personnel will soon incorporate autoinjectors into routine clinical practice and that these field products will thus become integrated into clinical medicine.

1	Peripheral neuropathy and the so-called intermediate syndrome, which are prominent long-term effects of insecticide poisoning, are not described in nerve agent survivors. Recent research has explored approaches leading to transient “immunity” and eventually to biologic products that will be protective against lethal nerve agents yet be devoid of side effects. A novel approach is to use enzymes to scavenge these highly toxic nerve agents before they attack their intended targets. The accumulated work has shown that if a scavenger is present at the time of nerve agent exposure, toxicant levels are rapidly reduced. This reduction is so rapid and profound that the need to administer a host of pharmacologically active drugs as antidotes is, according to laboratory studies, eliminated.

1	Cyanide (CN–) has become an agent of particular interest in terrorist scenarios because of its applicability to indoor targets. In recent years, for example, attacks with this agent have targeted the water supply of the U.S. Embassy in Italy. The 1993 World Trade Center bombing in New York may have been intended as a cyanide release as well.

1	Hydrogen cyanide and cyanogen chloride, the major forms of cyanide, are either true gases or liquids very close to their boiling points at standard room temperature. Hydrogen cyanide gas is lighter than air and does not remain concentrated outdoors for long; thus it is a poor military weapon but an effective weapon in an indoor space such as a train station or a sports arena. Cyanide is also water-soluble and poses a threat to the food and water supply from either accidents or malign intent. It is well absorbed from the gastrointestinal tract, through the skin, or via inhalation—the preferred terrorist route. Cyanide smells like bitter almonds, but 50% of persons lack the ability to smell it.

1	Unique among CWAs, cyanide is a normal constituent of the environment and actually is a required cofactor in many compounds important in metabolism, including vitamin B12. Cyanide is present in many plants, including tobacco; therefore, smokers, for instance, chronically carry cyanide at three times the usual level. Humans have evolved a detoxification mechanism for cyanide. Cyanide poisoning results if a large challenge of CN– overwhelms this mechanism, while treatment of cyanide poisoning exploits it.

1	Mechanism Cyanide directly poisons the last step in the mitochondrial electron transport chain, cytochrome a3, which results in a shutdown of cellular energy production. Tissues are poisoned in direct proportion to their metabolic rate, with the carotid baroreceptors and the brain—the most metabolically active tissues in the body—affected fastest and most severely. This poisoning results from cyanide’s high affinity for certain metals, notably Co and Fe+++. Cytochrome a3 contains Fe+++, to which CN− binds. Cyanide-poisoned tissues cannot extract oxygen from the blood; even though pulmonary oxygen exchange and cardiac function are preserved, cells die of hypoxia—i.e., 262e-7 of histotoxic rather than cardiopulmonary cause.

1	Clinical Features Hyperpnea occurs ~15 s after inhalation of a high concentration of cyanide and is followed within 15–30 s by the onset of convulsions and electrical status epilepticus. Respiratory activity stops 2–3 min later, and cardiac activity ceases several minutes after that. Exposure, especially via inhalation, to a large challenge of CN– can cause death in as little as 8 min. Smaller challenges will cause symptom spread over a longer period; very low doses may produce no effects at all because of the body’s ability to detoxify small amounts. Cyanogen chloride additionally produces mucous membrane irritation. Many but not all patients have a cherry-red appearance because their venous blood remains oxygenated.

1	Differential Diagnosis In a mass casualty incident caused by a chemical agent, the primary differential diagnosis of cyanide poisoning will be nerve agent poisoning. Cyanide-poisoned patients lack the prominent cholinergic signs seen in nerve agent poisoning, such as miosis and increased secretions. The cherry-red appearance often seen in cyanide poisoning is never seen in nerve agent poisoning. Cyanosis, confusingly, is not a prominent early sign in cyanide poisoning. Treatment of cyanide poisoning may require simply evacuation of the patient from the source of contamination. Decontamination of a true gas, other than clothing removal to avoid gas trapped in clothing air cells, is probably not a major concern. Oxygen, supplied via mask, nasal cannula, or endotracheal tube, has been shown to benefit patients, although the benefit is not explained by the known mechanism of action of CN–.

1	Oxygen, supplied via mask, nasal cannula, or endotracheal tube, has been shown to benefit patients, although the benefit is not explained by the known mechanism of action of CN–. Cyanide antidotes exploit the body’s innate detoxification mechanism, the hepatic enzyme rhodanese. They also exploit cyanide’s affinity for certain metal ions. Antidote recommendations are summarized in Table 262e-4.

1	The classic two-step cyanide antidote kit includes two IV solutions: sodium nitrite and sodium thiosulfate. It may also include amyl nitrite perles for inhalation. Nitrites are methemoglobin formers; when administered to a patient, nitrite converts a fraction of the body’s hemoglobin into methemoglobin by converting heme iron from Fe++ to Fe+++. CN– has a greater affinity for methemoglobin Fe+++ than for cytochrome a3. As a result, administration of nitrite creates a “sink” of cyanmethemoglobin; formation of methemoglobin pulls CN– off mitochondrial cytochrome a3, allowing cellular respiration to resume. Recent work suggests that nitrites may also work via a second mechanism involving the neurotransmitter nitrous oxide; if so, this mechanism may explain why cyanide-poisoned patients improve after nitrite administration faster than is explained by the known rate of methemoglobin formation.

1	Nitrite administration may save the patient acutely but creates an unstable pool of cyanmethemoglobin, the elimination of which requires a sulfur donor: sodium thiosulfate. Sodium thiosulfate donates sulfur to the reaction catalyzed by rhodanese; cyanide is converted to thiocyanate, a compound the body eliminates harmlessly in urine. Sodium thiosulfate alone may be administered to fire victims, whose oxygen-carrying capacity is already reduced and in whom the administration of nitrite may form so much methemoglobin as to render the blood unable to carry oxygen at all.

1	Hydroxocobalamin, or vitamin B12a, has recently been approved for use as an alternative cyanide antidote. Unlike sodium nitrite and sodium thiosulfate, it must be reconstituted at the scene. Hydroxocobalamin lacks the propensity of nitrites for hypotension, but many cases in which it has been beneficial have also required the use of sodium thiosulfate. Hydroxocobalamin causes an orange discoloration of the skin that is of no functional significance.

1	All of these antidotes require the placement of an IV line. Amyl nitrite is currently the only non-intravenous cyanide antidote available and has never been formally approved by the FDA. Amyl nitrite aVictims whose clothing or skin is contaminated with hydrogen cyanide liquid or solution can secondarily contaminate response personnel by direct contact or through off-gassing vapors. Dermal contact with cyanide-contaminated victims or with the gastric contents of victims who may have ingested cyanide-containing materials should be avoided. Victims exposed only to hydrogen cyanide gas do not pose contamination risks to rescuers. If the patient is a victim of recent smoke inhalation (and thus may have high carboxyhemoglobin levels), only sodium thiosulfate should be administered. bIf sodium nitrite is unavailable, administer amyl nitrite by inhalation from crushable ampoules. cAvailable in cyanide antidote kits, which can be purchased from various manufacturers.

1	Source: State of New York, Department of Health. perles in cyanide antidote kits can be crushed and inhaled by a Prognosis Cyanide casualties tend to recover much more quickly than patient who is still breathing. Amyl nitrite can also be given through casualties exposed to other chemical agents. Many patients with indusa respirator. trial cases have returned to work within the same shift. If a patient None of the cyanide antidotes is specifically approved for pedi-receives a large challenge and dies, death usually takes place within atric use. minutes of exposure.

1	None of the cyanide antidotes is specifically approved for pedi-receives a large challenge and dies, death usually takes place within atric use. minutes of exposure. radiation terrorism Christine E. Hill-Kayser, Eli Glatstein, Zelig A. Tochner The threat of a terror attack employing nuclear or radiation-related devices is unequivocal in the twenty-first century. Such an attack would certainly have the potential to cause unique and devastating 263e medical and psychological effects that would require prompt action by members of the medical community. This chapter outlines the most probable scenarios for an attack involving radiation as well as the medical principles for handling such threats.

1	Potential terrorist incidents with radiologic consequences may be considered in two major categories. The first is the use of radiologic dispersal devices. Such devices disseminate radioactive material purposefully and without nuclear detonation. An attack with a goal of radiologic dispersal could take place through use of conventional explosives with incorporated radionuclides (“dirty bombs”), one or more fixed nuclear facilities, or nuclear-powered surface vessels or submarines. Other means could include detonation of malfunctioning nuclear weapons with no nuclear yield (nuclear “duds”) and installation of radionuclides in food or water. The second and less probable scenario is the actual use of nuclear weapons. Each scenario poses its own specific medical threats, including “conventional” blast or thermal injury, introduction to a radiation field, and exposure to either external or internal contamination from a radioactive explosion.

1	Atomic isotopes with uneven numbers of protons and/or neutrons are typically unstable; such isotopes discharge particles or energy to matter as they move to stability, a process that is defined as radiation. Mass-containing particles, including alpha particles, electrons, and/ or neutrons, may be transferred during this process (alpha radiation, beta radiation, and neutron radiation, respectively); alternatively, the transfer may consist only of energy in the form of a gamma ray. Alpha (α) radiation consists of heavy, positively charged particles, each of which contains two protons and two neutrons. Alpha particles usually are emitted from isotopes that have an atomic number of ≥82, such as uranium and plutonium. Due to their large size, alpha particles have limited penetrating power. Fine obstacles such as cloth and human skin usually can stop these particles from penetrating into the body. Thus they represent a small risk from external exposure. If they are somehow internalized,

1	Fine obstacles such as cloth and human skin usually can stop these particles from penetrating into the body. Thus they represent a small risk from external exposure. If they are somehow internalized, however, alpha particles can cause significant damage to human cells that are in their immediate proximity.

1	Beta (β) radiation consists of electrons, which are small, light, negatively charged particles (about 1/2000 the mass of a neutron or proton). Electrons can travel only a short, finite distance in tissue, with the precise distance depending on their energy. Exposure to beta particles is common in many radiation accidents. Radioactive iodine released in nuclear plant accidents is the best-known form of beta radiation. Plastic layers and clothing can stop most beta particles, and their penetration is generally measured at a few millimeters. A large quantum of energy delivered via beta particles to the basal stratum of the skin can cause a burn that is similar to a thermal burn and is treated as such.

1	Gamma (γ) rays and x-rays (both photons) are similar. Gamma rays are uncharged electromagnetic radiation discharged from a nucleus as a wave of energy. X-rays are the product of abrupt mechanical deceleration of electrons striking a heavy target such as tungsten. Although they are generated by different sources, gamma rays and x-rays have similar properties; that is, they have no charge and no mass, just energy. They travel easily through matter and thus are sometimes referred to as penetrating radiation. Gamma rays and x-rays are the principal types of radiation that cause dangerous total-body exposure. Gamma rays and x-rays of the same energy will cause the same biologic effects, and these effects will require the same treatment.

1	Neutron (η) particles are heavy and uncharged and are often emitted during nuclear detonation. They possess a wide energy range; their ability to penetrate tissues is variable, depending on their energy. They 263e-1 are less likely to be present in most scenarios of radiation bioterrorism than are the other forms of radiation discussed above.

1	Radiation interactions with atoms can result in ionization and the formation of free radicals that damage tissue by disrupting chemical bonds and molecular structures in the cell, including DNA. Protons, electrons, and gamma rays cause cellular damage through ionization of DNA. Depending on energy and other factors, some fraction of this damage will be caused by a direct strike to the DNA molecule (direct ionization). The remainder will be caused by ionization of water molecules to create free radicals that, in turn, damage DNA (indirect ionization). Ionization of DNA resulting from neutrons is exclusively indirect. Radiation damage can lead to cell death; the cells that recover may be mutated and at higher risk for subsequent cancer evolution. Cell sensitivity increases as the replication rate increases and cell differentiation decreases.

1	The commonly used units of radiation are the rad and the gray (Gy). The rad (radiation absorbed dose) is energy deposited within living matter and is equal to 100 ergs/g of tissue. The traditional rad has been replaced by the Système Internationale (SI) unit of the gray; 100 rad = 1 Gy, while 1 Gy is equal to 1 joule/kg. The sievert (Sv) is the SI unit that refers to the equivalent radiation dose in biologic tissues. While 1 Sv is equal to 1 joule/kg, Sv and Gy are not interchangeable units: Sv refers to the biologic effect of the radiation, while Gy refers to the physical energy being transferred.

1	Whole-body exposure occurs when radiation energy is deposited throughout the entire body. During a whole-body exposure, alpha and beta particles have limited penetration and do not cause significant noncutaneous injury unless emission results from an internalized source. Whole-body exposure from gamma rays, x-rays, or neutrons, which can penetrate through the body (the degree of which depends on their energy), can result in damage to multiple tissues and organs. The damage is proportional to the radiation exposure of the specific organ or tissue.

1	External contamination is a result of fallout of radioactive particles that land on the body surface, clothing, skin, and hair. This is the dominant element to consider in the mass-casualty situation resulting from a radioactive terrorist strike. The common contaminants primarily emit alpha and beta radiation. Alpha particles do not penetrate beyond the skin and thus have minimal systemic effects. Beta emitters can cause significant cutaneous burns and scarring. Due to their ability to penetrate tissue, gamma emitters can cause not only local damage but also whole-body radiation exposures and injury. Medical treatment primarily entails decontamination of the body, including wounds and burns, to prevent internalization of radioactive contaminants. Removal of contaminated clothing reduces levels of contamination significantly and is a first step in the decontamination process. Generally, patients do not constitute a significant radiation hazard to health care providers, and lifesaving

1	levels of contamination significantly and is a first step in the decontamination process. Generally, patients do not constitute a significant radiation hazard to health care providers, and lifesaving treatment should not be delayed for fear of secondary contamination of the medical team. Although risk is relatively low, any damage to health care personnel will depend directly on the duration of exposure and will be inversely proportional to the square of the distance from any radioactive source. Gowns that can be easily removed offer protection.

1	Internal contamination occurs when radioactive material is inhaled or ingested or enters the body through open wounds or burns or via skin absorption. In principle, any externally contaminated casualty should be evaluated for internal contamination. Because of their chemical properties, some isotopes may exert toxic effects on specific target organs in addition to causing radiologic injury. The respiratory system is the main portal of entry for internal contamination, and the lung is the organ at greatest risk. Aerosol particles <5 μm in diameter can reach the alveoli, whereas larger particles will remain in the proximal airways. The tiny particles can be absorbed by the lymphatic system or the bloodstream. Bronchial lavage is often a helpful treatment in this situation. Radioactive material entering the gastrointestinal tract is absorbed according to its chemical structure and solubility.

1	The insoluble radionuclides may affect the lower gastrointestinal tract. Intact skin is normally an effective barrier to most radionuclides. Penetration through the skin usually takes place when wounds or burns have compromised the skin barrier. Therefore, any skin erosion should be cleaned and decontaminated promptly. Absorbed radioactive materials travel throughout the body. Liver, kidney, adipose tissue, thyroid, and bone and bone marrow tend to bind and retain radioactive material more than other tissues do. Medical treatment thus includes the prevention of absorption, the reduction of incorporation, and the enhancement of elimination (see below).

1	Localized exposure refers to close contact between a highly radioactive source and a part of the body, with consequent discrete damage to the skin and deeper tissues that resembles a thermal burn. Later signs include epilation, erythema, moist desquamation, ulceration, blistering, and necrosis in proportion to exposure. Alopecia, transient or permanent, is dose related and starts at cutaneous doses of >3 Gy. Overt tissue damage can take weeks or even months to develop; the healing process can also be very slow, lasting for months. Long-term cutaneous changes, including keratosis, fibrosis, and telangiectasis, may appear years after the exposure. Treatment is based on analgesia and infection prophylaxis. Nevertheless, severe burns often require grafting or even amputation. Long-term radiation effects are characterized by cell loss, cell death, and tissue atrophy.

1	Radiologic dispersal incidents are generally of two types, resulting from small, usually localized sources or from wide dispersals over large areas. The methods that could be utilized to formulate an attack using dispersal of radiation are incredibly diverse. Radioactive materials can take the form of solid state, aerosol, gas, or liquid. They can be put into food or water, released from vehicles, or spread by explosion. The principal route of exposure is usually direct contact between the victim’s skin and the radioactive particles, although internal contamination can occur if the material is inhaled or ingested. The radiation field is also a potential source of whole-body exposure. The psychosocial effects that accompany such an event are significant and are beyond the scope of this chapter. A list of radioactive materials, including information on their major properties and medical treatment, is given in Table 263e-1.

1	In a localized event, the amount and spread of the radioactive materials are usually limited and can be treated like a spill of hazardous material. Protective clothing prevents or minimizes the contamination of emergency responders. The use of explosives coupled with a large amount of radioactive materials can result in wide dispersion of radiation, which is of far greater concern. Other potential sources of radiation are nuclear reactors, spent nuclear fuel, and transport vehicles. Less probable but still possible is the use of a large source of penetrating radiation without explosion. It is expected that most exposures would be low-level and that the principal health and psychosocial effects would be similar to those in the former scenario but on a larger scale. Whenever an explosion is involved, conventional lifesaving treatment should be given first priority. Only then should decontamination and specific treatment be given for the radiation exposure.

1	Silent exposure represents a scenario in which a powerful radiologic source, often called a radiologic exposure device, could be hidden in a crowded place and spread radioactive materials without being recognized or reported. Recognition of the event and the source of exposure might take a long time. A major clue in this situation is the appearance of unusual clinical manifestations in many individuals; such manifestations are often nonspecific and include symptoms of acute radiation sickness (see “Acute Radiation Syndrome,” below) such as headache, fatigue, malaise, and opportunistic infections. Gastrointestinal phenomena such as diarrhea, nausea, vomiting, and anorexia may occur. Dermatologic symptoms (e.g., burns, ulceration, and epilation) and hematopoietic manifestations (e.g., bleeding tendency, thrombocytopenia, purpura, lymphopenia, and neutropenia) are also possible and are dose-related. Careful epidemiologic studies may be necessary to identify the source of such exposure.

1	The most likely scenario in a nuclear terror attack is the detonation of a single low-yield device. The estimated yield of such a device is anywhere between 0.01 and 10 kilotons of 2,4,6-trinitrotoluene (TNT). The expected effects of such an explosion are a combination of several components: ground shock, air blast, thermal radiation, initial nuclear radiation, crater formation, and radioactive fallout.

1	A nuclear detonation, like a conventional explosion, produces a shock wave that can further damage structures and cause many casualties. In addition, the detonation can produce an extremely hot fireball that can ignite materials and cause severe burns. The detonation releases an intense pulse of ionizing radiation consisting mainly of gamma rays and neutrons. The radiation produced in the first minute is termed initial radiation, whereas the ongoing radiation due to fallout is termed residual radiation. Both types of radiation can cause acute radiation sickness, and winds can carry fallout and contaminate large areas. The LD50/30 (i.e., the dose that causes a 50% mortality rate at 30 days) is ~4 Gy for whole-body exposure without medical support; with medical support, the LD ranges between 8 and 10 Gy. On top of its immediate effects, a massive blast forms a crater in the soil and usually produces ground shock that compounds the physical damage and the number of casualties. Inhalation

1	8 and 10 Gy. On top of its immediate effects, a massive blast forms a crater in the soil and usually produces ground shock that compounds the physical damage and the number of casualties. Inhalation of large amounts of radioactive dust causes pneumonitis that can lead to pulmonary fibrosis. Use of a mask covering the mouth and nose can result in effective prevention. The intense flash of infrared and visible light can cause either temporary or permanent blindness. Cataracts can develop months to years later among survivors.

1	Acute radiation syndrome (ARS) refers to multisystem symptomatology resulting hours to weeks after radiation exposure. As discussed earlier, cell sensitivity to radiation damage increases as the cell replication rate increases and as cell differentiation decreases. Bone marrow and mucosal surfaces of the gastrointestinal tract, which have vast mitotic activity, are significantly more sensitive to radiation than are slowly dividing tissues such as bones and muscles. After exposure of all or most of the human body to ionizing radiation, ARS can develop. The clinical manifestations of ARS reflect the dose and type of radiation as well as the parts of the body exposed.

1	ARS manifests as three major groups of signs and symptoms: hematopoietic, gastrointestinal, and neurovascular. In addition, ARS exists in four stages: prodrome, latent phase, clinical illness, and recovery or death. The higher the radiation doses, the shorter and more severe each stage. The prodrome appears within minutes to 4 days after exposure, lasts from a few hours to a few days, and can include nausea, vomiting, anorexia, and diarrhea. At the end of the prodrome, ARS progresses to the latent phase. Minimal or no symptoms are present during the latent phase, which commonly lasts up to 2.5 weeks but can last up to 6 weeks. The duration depends on the radiation dose, the prior health of the patient, and coexisting illness or injury. After the latent phase, the exposed person manifests illness that may end in recovery or lead to death.

1	With exposure to low doses of <1 Gy, ARS is generally mild. At this dose, symptoms can be minimal or nonexistent, even if the entire body is exposed to penetrating radiation. The main feature of the clinical picture is transient depression of bone marrow (lymphopenia) that lasts up to 2–3 weeks and then improves. ARS is significantly more acute and severe with exposure to very high radiation doses (>30 Gy). At these doses, the prodrome appears in minutes and is followed by 5–6 hours of latency before cardiovascular collapse occurs secondary to irreversible damage to the microcirculation.

1	Exposure to intermediate radiation doses may result in variable ARS courses. The type and dose of radiation and the part of the body exposed determine not only the timing of the different stages of ARS but also the dominant clinical picture. At low radiation doses of 0.7–4 Gy, hematopoietic depression due to bone marrow suppression is the main constituent of illness. The patient may develop infection and bleeding secondary to low leukocyte and platelet counts, respectively. The bone marrow eventually recovers in almost all patients if Mode of Accumulation Isotope Name Symbol Common Usage t1/2 Biologic (days) Exposure Type Contamination in Body Treatment Manganese Mn-56 Reactors, research β, γ; 2.6 h; 5.7 External, internal N/A Liver N/A laboratories Cobalt Co-60 Medical radio-β, γ; 5.26 y; 9.5 External, internal Lungs Liver Gastric lavage, therapy devices, Strontium Sr-90 Fission product of β; 28 y; 18,000 Internal GI tract Bones (similar to Strontium, calcium, uranium

1	Strontium Sr-90 Fission product of β; 28 y; 18,000 Internal GI tract Bones (similar to Strontium, calcium, uranium Molybdenum Mo-99 Hospitals: scans β, γ; 66.7 h; 3 External, internal N/A Kidneys N/A Technetium Tc-99m Hospitals: scans β, γ; 6.049 h; 1 External, internal IV administration Kidneys, total Potassium per-body chlorate to reduce thyroid dose Cesium Cs-137 Medical radiother-β, γ; 30 y; 70 External, internal Lungs, GI tract, Renal excretion Ion-exchange resapy devices ins, Prussian blue potassium Gadolinium Gd-153 Hospitals β, γ; 242 d; 1000 External, internal N/A N/A N/A Iridium Ir-192 Commercial β, γ; 74 d; 50 External, internal N/A Spleen N/A radiography Radium Ra-226 Instrument illumina-α, β, γ; 1602 y; External, internal GI tract Bones MgSO4 lavage, tion, industrial appli-16,400 ammonium cations, old medical chloride, calcium equipment, former

1	Radium Ra-226 Instrument illumina-α, β, γ; 1602 y; External, internal GI tract Bones MgSO4 lavage, tion, industrial appli-16,400 ammonium cations, old medical chloride, calcium equipment, former Tritium H-3 Luminescent β; 12.5 y; 12 Internal Inhalation, GI Total body Dilution with gunsights, muzzle- tract, wounds controlled water velocity detectors, intake, diuretics nuclear weapons Iodine-131 Reactors, thyroid β, γ; 8.1 d; 138 Internal Inhalation, GI Thyroid Potassium/sodium ablators tract, wounds iodide, propylthiouracil, methimazole Uranium U-235 Depleted uranium, α, (α, β, γ); 7.1 × Internal GI tract Kidneys, bones NaHCO3, chelation natural uranium, 108 y; 15 with EDTA fuel rods, weapons-grade material Plutonium Pu-239 Produced from α; 2.2 × 104 y; 73,000 Internal Limited lung Lungs, bones, Chelation with uranium in reactors, absorption, high bone marrow, DTPA or EDTA nuclear weapons retention liver, gonads

1	Americium Am-241 Smoke detectors, α; 458 y; 73,000 Internal Inhalation, skin Lungs, liver, Chelation with nuclear weapons wounds bones, bone DTPA or EDTA (in form of fallout) Polonium Po-210 Calibration source α; 138.4 d; 60 Internal Inhalation, Spleen, kidneys Lavage, wounds Thorium Th-232 Calibration source α; 1.41 × 1010 y; Internal N/A N/A N/A 73,000 Phosphorus P-32 Research labo-β; 14.3 d; 1155 Internal Inhalation, GI Bones, bone mar-Lavage, aluminum ratories, medical tract, wounds row, rapidly repli-hydroxide, phosfacilities Abbreviations: DTPA, diethylenetriamine pentaacetic acid; EDTA, ethylenediamine tetraacetic acid; GI, gastrointestinal; h, hours; N/A, not available; y, years.

1	they are supported with transfusions and fluids; antibiotics are often simply do not recover. In addition to the gastrointestinal syndrome needed in addition. Patients with isolated hematopoietic manifesta-associated with very high-level exposures, patients may develop a neutions of ARS can almost always survive with proper supportive care. rovascular syndrome that includes vascular collapse, seizures, and con-After exposure to 6–8 Gy, a significantly more complicated clinical fusion; death occurs within a few days. The neurovascular syndrome picture may ensue. At these doses, the bone marrow does not always dominates after whole-body exposure to >20 Gy. In this variant, the recover and death may occur as a result. A gastrointestinal syndrome prodrome and latent phase both last only a few hours. may accompany the hematopoietic manifestations and further worsen the patient’s condition. Gastrointestinal injury due to compromise of the absorptive layer of the gut alters absorption of

1	few hours. may accompany the hematopoietic manifestations and further worsen the patient’s condition. Gastrointestinal injury due to compromise of the absorptive layer of the gut alters absorption of fluids, electrolytes, and nutrients. Such injury can lead to vomiting, diarrhea, gastrointestinal bleeding, sepsis, and electrolyte and fluid imbalance. Generally, The treatment of ARS is focused on maintaining homeostasis, thus these symptoms are also accompanied by a severe hematopoietic giving damaged organs a chance to recover. Aggressive support syndrome, with only a slim chance of bone marrow recovery. These is provided for every damaged system. Treatment for the hemafactors in constellation often lead to death. Whole-body exposure to topoietic system targets mainly neutropenia and infection, with >9–10 Gy is almost always fatal. Crucial elements of the bone marrow measures that may include transfusion of leukoreduced irradiated blood as needed and administration of hematopoietic

1	with >9–10 Gy is almost always fatal. Crucial elements of the bone marrow measures that may include transfusion of leukoreduced irradiated blood as needed and administration of hematopoietic growth factors. The value of bone marrow transplantation in this situation is questionable. None of the bone marrow transplantations that were performed among the victims of the nuclear reactor accident in Chernobyl proved successful. Bone marrow transplantation could be considered for casualties with whole-body exposure to 6–10 Gy when the hematopoietic syndrome is dominant and the bone marrow is less likely to recover with time, although the efficacy of this treatment has not been proved. Another major component of the treatment of ARS is the provision of partial or total parenteral nutrition to bypass the damaged gastrointestinal system. For blast and thermal injuries, standard therapy for trauma is given. Psychological support is essential in many cases. A treatment algorithm is outlined in

1	the damaged gastrointestinal system. For blast and thermal injuries, standard therapy for trauma is given. Psychological support is essential in many cases. A treatment algorithm is outlined in Fig. 263e-1.

1	Victims of radiation bioterrorism can suffer from conventional thermal or blast injuries, exposure to radiation, and contamination by radioactive materials. Many will have combinations of the above, which can cause higher morbidity and mortality rates than each exposure would alone. The number of casualties will be a major factor in determining the response of the medical system to an act of radiation bioterrorism. If only a few persons are affected, no significant changes and adaptations of the system are needed to treat the victims. If a terror attack results in dozens of casualties or more, however, an organized disaster plan at the local and state levels must be invoked to deal with the crisis properly. Useful U.S. planning documents that include many universal planning concepts can be found at http://www.remm .nlm.gov/remm_Preplanning.htm. Ideally, medical personnel will have had a prior assignment and training and be prepared to function in a scenario with which they are

1	can be found at http://www.remm .nlm.gov/remm_Preplanning.htm. Ideally, medical personnel will have had a prior assignment and training and be prepared to function in a scenario with which they are familiar. Stockpiles of specific equipment and medications should be obtained ahead of time and stored safely (see the Centers for Disease Control and Prevention Web site at http:// www.bt.cdc.gov/stockpile/). One of the goals of terrorist attackers is to overwhelm medical facilities and minimize the salvage of casualties. Initial management consists of primary triage and transportation of the wounded to medical facilities for treatment. The rationale behind triage is to sort patients into classes according to the severity of injury for the purpose of expediting clinical care and maximizing the use of the available clinical services and facilities. Triage requires determination of the level of emergency care needed. The higher the number and broader the range of casualties, the more complex

1	use of the available clinical services and facilities. Triage requires determination of the level of emergency care needed. The higher the number and broader the range of casualties, the more complex and difficult triage becomes. The mildly wounded and victims of contamination only can be sent to evacuation, registration (with disaster response teams), and decontamination/treatment centers. Figure 263e-2 illustrates an evacuation scheme after a radiologic event causing multiple casualties. The goal of such an algorithm is to treat all possible victims of exposure and minor injury outside of the hospital setting. This approach prevents hospitals from being directly overwhelmed and enhances treatment for persons who are severely wounded. Emergency treatment should be administered initially for conventional injuries such as wounds, trauma, and thermal or chemical burns. Individuals with such injuries should be stabilized, if possible, and immediately transported to a medical facility.

1	for conventional injuries such as wounds, trauma, and thermal or chemical burns. Individuals with such injuries should be stabilized, if possible, and immediately transported to a medical facility. Removing clothing and wrapping the victim in clean blankets or nylon sheets reduce both the exposure of the patient and the contamination risk to the staff. Less severely injured victims should undergo preliminary decontamination before or during evacuation to a hospital.

1	FIGURE 263e-1 General guidelines for treatment of radiation casualties. CBC, complete blood count. One must remember that radionuclide contamination of the skin commonly is not an acute life-threatening situation for the patient or for the personnel who care for the patient. Only powerful gamma emitters are likely to cause real damage from contamination. It is important to emphasize that exposure to a radiation field alone does not necessarily create any contamination. The exposed person, if not contaminated, is not radioactive and does not directly emit any radiation.

1	To protect the staff, protective gear (gowns, gloves, masks, and caps) should be used. Protective masks with filters and chemically protective overgarments provide excellent protection from contamination. Waterproof shoe covers are also important. Remaining in the contaminated area and dealing with lifesaving procedures should take place according to the “ALARA” principle: as low as reasonably achievable. It is better to send many people to do the job for short exposure times than to send a few people for longer periods.

1	Decontamination of victims should take place in the field before their arrival at medical facilities, but radiologic decontamination should never interfere with medical care. Removal of outer clothing and shoes usually reduces a patient’s contamination by 80–90%. Contaminated clothes should be carefully removed by rolling them over themselves, placed in marked plastic bags, and removed to a predefined area for contaminated clothes and equipment. A radiation detector should then be used to check for the presence of any residual radiologic contamination on the patient’s body. To prevent internalization of the radioactive materials, one should cover open wounds before decontamination. Showering or washing of the entire skin and hair is very important and should be done as soon as possible. The skin should then be dried and reassessed for residual contamination until no radiation is found. FIGURE 263e-2 Algorithm for evacuation in a multicasualty radiologic event.

1	FIGURE 263e-2 Algorithm for evacuation in a multicasualty radiologic event. Contamination-removing chemical agents are more than sufficient to remove radiologic contamination. Wound decontamination should be as conservative as possible. The main goal is to prevent both extensive local damage and internal contamination through lacerated skin. The bandages should be removed and the wounds flushed. The wound should then be dried and assessed for radiation. This procedure can be repeated again and again until contamination is undetectable. Excision of contaminated wounds should be attempted only when surgically necessary. Radioactive shrapnel that can penetrate through the skin should be removed.

1	In the hospital, staff can wear normal hospital barrier clothing, including two pairs of gloves, a gown, shoe covers, a head cover, and a face mask. Eye protection is recommended. Decontamination of medical personnel is obligatory after emergency treatment and decontamination of the patient. After use, all protective clothing should be placed in a designated container for contaminated clothing.

1	Radiation intensity decays rapidly with the square of the distance from the source; thus increasing the distance from the source and decreasing the time spent near it are basic principles of radiation safety. Shielding with lead can be used as protection from small radioactive gamma sources. Geiger counters can detect gamma and beta radiation. Pocket chambers and dosimeters, film badges, and thermoluminescent dosimeters can measure accumulated exposure to gamma radiation. All these detectors are in common use in medical facilities and should be employed to help define the level of contamination. Alpha radiation is harder to detect due to its poor penetration. An alpha scintillation counter, which is capable of detecting alpha radiation, is not commonly used in medical facilities.

1	Figure 263e-3 shows a model for hospital arrangement for triage. Persons contaminated either externally or internally should be identified, externally decontaminated, and, if need be, treated immediately and specifically for internal contamination as detailed below. In all other cases, the need for treatment of radiation injuries does not constitute a medical emergency. Early actions—e.g., blood sampling both to assess the severity of the exposure and to perform blood typing and cross-matching for possible transfusion—need to be taken promptly if ARS is evident or if whole-body exposure is suspected. Contaminated O.R.

1	In the hospital entrance, a distinct decontamination area should be set up promptly. Separation between clean and contaminated areas is essential. Medical personnel in contaminated areas should wear protective gear, as noted above, and should be rotated in their assignments every 1–2 h to ensure minimal exposure to radiation. If patients are critically wounded and require either surgery or resuscitation, they need to be transported directly to “contaminated” operating rooms or resuscitation sites for lifesaving procedures. Once the condition of such patients is stable, they should be decontaminated. It is important to obtain details concerning the exposure, to look for prodromal signs of radiation sickness, and to conduct a physical examination. One of the simplest ways to estimate exposure clinically is to measure the time of prodromal appearance. The earlier the prodromal signs and symptoms appear, the higher is the dose of radiation exposure. A few laboratory tests need to be done

1	clinically is to measure the time of prodromal appearance. The earlier the prodromal signs and symptoms appear, the higher is the dose of radiation exposure. A few laboratory tests need to be done routinely, such as complete blood count and urinalysis. If internal contamination is suspected, specific treatment should be given as outlined below.

1	Table 263e-2 summarizes the common treatment regimens for internal radionuclide contamination. Treatment for internal radionuclide contamination, also referred to as decorporation, should be started as soon as possible after suspected or known exposure. The approximate upper limit of radionuclide contamination that can reasonably be ignored from a radiation safety point of view is not well defined. These are judgments that will depend on the circumstances of the event and the resources available. The Clinical Decision Guide within the National Council on Radiation Protection and Measurements (NCRP) Report 161 is a decision tool for determining the need for treatment of a contaminated person. Purchase of these volumes by major triage centers (available at http://www .ncrppublications.org/Reports/161_I) may be a prudent investment that would help health care workers in a critical situation to determine which patients should undergo decorporation.

1	The goal is to leave the smallest amount of radionuclide possible in the body. Treatment is given to reduce absorption and enhance elimination and excretion. Some decorporation agents are not approved by the U.S. Food and Drug Administration (FDA) for these indications, and few clinical data support the efficacy of their use. The gastrointestinal tract may be cleared by stomach lavage, with emetics (such as apomorphine, 5–10 mg; or ipecac, 1to 2-g capsules or 15 mL in syrup), or by use of purgatives, laxatives, ion exchangers, and aluminum antacids. Prussian blue (1 g tid for a minimum of 3 weeks) is an ion exchanger used to treat cesium-137 internal contamination. Aluminum antacids (such as aluminum phosphate gel) may reduce strontium uptake in the gut if given immediately after exposure. Aluminum hydroxide is less effective.

1	Radionuclide interaction with tissues can be prevented or reversed through use of agents that block absorption; dilute, mobilize, or release radionucleotides from tissues; or chelate radio-nucleotides.

1	Blocking agents prevent the entrance of radioactive materials. The best-recognized effective blocking agent is potassium iodide (KI), which blocks the uptake of radioactive iodine (131I) by the thyroid. KI is most effective if taken within the first hour after exposure and is still effective 6 h after exposure. Its effectiveness subsequently declines until 24 h after exposure; however, it is recommended that KI be taken up to 48 h after exposure. The KI dose is based on age, predicted thyroid exposure, and pregnancy and lactation status. Adults between the ages of 18 and 40 should receive 130 mg/d for 7–14 days if exposed to ≥10 cGy of radioactive iodine. Other thyroid-blocking agents include propylthiouracil (100 mg tid for 8 days) and methimazole (10 mg tid for 2 days followed by 5 mg tid for 6 days). These agents are somewhat less effective than KI.

1	Diluting agents decrease the absorption of the radionuclide; for example, water may be used as a diluting agent in the treatment for tritium (3H) contamination. The recommended amount is 3–4 L/d for at least 3 weeks. Mobilizing agents are most effective when given immediately; however, they may be effective for up to 2 weeks after exposure. These agents include antithyroid drugs, parathyroid extract, glucocorticoids, ammonium chloride, diuretics, expectorants, and inhalants. All of the latter agents should induce the release of radionuclides from tissues.

1	Chelating agents can bind many radioactive materials, after which the complexes are excreted from the body. In this regard, diethylenetriaminepentaacetic acid (DTPA)—as either Ca-DTPA or Zn-DTPA—is superior to ethylenediaminetetraacetic acid (EDTA); DTPA has been approved by the FDA to treat internal contamination with plutonium, americium, and curium, but it also chelates berkelium, californium, and any other material with an atomic number >92. Ca-DTPA is more effective than Zn-DTPA during the first 24 h after internal contamination, and the two drugs are equally effective after the initial 24 h. If both drugs are available, Ca-DTPA should be given as the first dose. If additional treatment is needed, treatment should be switched to Zn-DTPA. The dose is 1 g of Ca-DTPA or Zn-DTPA, dissolved in 250 mL of normal saline or 5% glucose and given intravenously over 1 h daily. The duration of chelation treatment depends on the amount of internal contamination and the individual response to

1	in 250 mL of normal saline or 5% glucose and given intravenously over 1 h daily. The duration of chelation treatment depends on the amount of internal contamination and the individual response to treatment. DTPA also can be administrated by nebulized inhalation; 1 g is given in a 1:1 dilution with water or saline over 15–20 min. Nebulized Zn-DTPA is recommended if inhalation was the only route of internal contamination. The IV route is recommended and should be used if the route of internal contamination is not known or if multiple routes of internal contamination are likely. DTPA penta-ethyl ester is a prodrug that has a favorable oral-absorption profile and whose therapeutic effects have been demonstrated in initial efficacy studies. Because it can be given orally, this prodrug may ultimately prove more useful in the setting of mass casualties than IV or nebulized forms of the drug. Treating uranium contamination with DTPA is contraindicated due to synergistic damage to the kidneys.

1	Lung lavage can reduce radiation-induced pneumonitis and is indicated only when a large amount of radionuclide enters the lungs and has the potential to cause acute radiation injury. The procedure requires anesthesia.

1	One of the major difficulties in treating victims exposed to radiation is determination of the amount of exposure. Immediately after a terrorist event, when victims are being triaged, information regarding source, dose, and exposure time is unlikely to be available. Clinical assessment of the patient is the best approach and includes history, physical examination, and observation for onset of the ARS prodrome. An early prodrome indicates high-level exposure to radiation. Victims who arrive at the hospital reporting severe weakness, nausea, vomiting, diarrhea, or seizures probably will not survive despite supportive measures. A very limited number of tests can be performed to estimate 263e-7 radiation exposure and contamination. The Biodosimetry Assessment Tool (BAT) facilitates treatment decisions during radiation exposure incidents. Developed by the U.S. Armed Forces Radiobiology Research Institute (AFRRI), the BAT provides a method of estimating radiation exposure on the basis of a

1	decisions during radiation exposure incidents. Developed by the U.S. Armed Forces Radiobiology Research Institute (AFRRI), the BAT provides a method of estimating radiation exposure on the basis of a single lymphocyte count, the lymphocyte depletion rate, and the time from exposure to onset of emesis. The BAT algorithms are based on large datasets from human radiation exposure and are available at http://www.usuhs.mil/afrri/outreach/request.htm. The patient should be observed for clinical symptoms, and the severity and time of onset of nausea, vomiting, headache, anorexia, fever, hypotension, tachycardia, weakness, cognitive changes, skin desquamation, diarrhea, and bloody stools should be recorded. The AFRRI Biodosimetry Worksheet (http://www.usuhs.mil/afrri/outreach/pdf/ afrriform331.pdf ) is a useful resource for detailed recording. Baseline tests should include a complete blood count with differential and platelet count, renal evaluation, and determination of electrolytes, serum

1	) is a useful resource for detailed recording. Baseline tests should include a complete blood count with differential and platelet count, renal evaluation, and determination of electrolytes, serum amylase, and serum C-reactive protein. Urine and stool samples should be obtained if internal contamination is suspected. Nasal swabs taken from each nostril within the first 1–2 h after the exposure may be useful for determination of radionuclide inhalation. After exhalation, each swab is labeled, sealed in a plastic bag, and sent for analysis to appropriate laboratories. Patients exposed to 0.7–4 Gy develop pancytopenia from as early as 10 days to as late as 8 weeks after exposure. Lymphocytes show the most rapid decline, whereas counts of other leukocytes and platelets decline less rapidly. Erythrocytes are the least vulnerable blood elements.

1	Absolute lymphocyte counts should be repeated every 4–6 h for 5–6 days; they are the most valuable early indicator because they constitute a sensitive marker for radiation damage and correlate with both the exposure and the prognosis. A 50% drop in absolute lymphocyte count within the first 24 h indicates a significant injury. HLA typing is necessary whenever irreversible bone marrow damage is suspected. Lymphocyte chromosomal analysis can detect exposure to as little as 0.03–0.06 Gy, and 15 mL of blood for this purpose should be drawn as early as possible in a heparinized collection tube and kept cool. Radiation-induced chromosomal aberrations visible in peripheral-blood lymphocytes include dicentric chromosomes and ring forms that last for a few weeks. Calibration of a dose-response curve makes it possible to assess the radiation dose on the basis of the presence of these aberrations. Dicentric quantification requires multiple days to perform and is available only in select centers.

1	Another method for estimating exposure is the in vitro cytokinesis– block micronucleus assay. Micronuclei can be the result of small acentric chromosome fragments that arise during exposure to radiation. The technique to score the micronuclei in peripheral-blood lymphocytes has been standardized in the last few years. It can be a useful tool in small-scale exposures but is not feasible in a mass casualty setting.

1	It is desirable to continue follow-up over the long termin some circumstances. In general, only persons who are exposed to <8–10 Gy of whole-body irradiation have a chance to survive in the long term, and they are at risk of developing cataracts, sterility, and cancers as well as lung, kidney, and bone marrow problems. In light of their age, their gender, and the amount and type of exposure, they should be followed for many years. A major public health issue is the risk of secondary malignancy in individuals and populations that have been exposed to low doses of radiation. Leukemia and breast, brain, thyroid, and lung cancer develop most commonly, but the exposed population is at increased risk for many other cancers as well. Appropriate follow-up protocols should be based on the type of exposure and the exposed population. In cases of internal contamination, long-term follow-up should be focused on the organ at risk. Substantial psychosocial support will likely be needed for a

1	of exposure and the exposed population. In cases of internal contamination, long-term follow-up should be focused on the organ at risk. Substantial psychosocial support will likely be needed for a community in the years after an attack including radiologic agents.

1	Approach to the Patient with Possible Cardiovascular Disease Joseph Loscalzo THE MAGNITUDE OF THE PROBLEM Cardiovascular diseases comprise the most prevalent serious disorders in industrialized nations and are a rapidly growing problem in devel-oping nations (Chap. 266e). Age-adjusted death rates for coronary heart disease have declined by two-thirds in the last four decades in the United States, reflecting the identification and reduction of risk factors as well as improved treatments and interventions for the man-agement of coronary artery disease, arrhythmias, and heart failure. Nonetheless, cardiovascular diseases remain the most common causes of death, responsible for 35% of all deaths, almost 1 million deaths each year. Approximately one-fourth of these deaths are sudden. In addition, cardiovascular diseases are highly prevalent, diagnosed in 80 million adults, or ~35% of the adult population. The growing preva-lence of obesity (Chap. 416), type 2 diabetes mellitus (Chap. 417),

1	cardiovascular diseases are highly prevalent, diagnosed in 80 million adults, or ~35% of the adult population. The growing preva-lence of obesity (Chap. 416), type 2 diabetes mellitus (Chap. 417), and metabolic syndrome (Chap. 422), which are important risk factors for atherosclerosis, now threatens to reverse the progress that has been made in the age-adjusted reduction in the mortality rate of coronary heart disease. For many years cardiovascular disease was considered to be more common in men than in women. In fact, the percentage of all deaths secondary to cardiovascular disease is higher among women (43%) than among men (37%) (Chap. 6e). In addition, although the absolute number of deaths secondary to cardiovascular disease has declined over the past decades in men, this number has actually risen in women. Inflammation, obesity, type 2 diabetes mellitus, and the metabolic syndrome appear to play more prominent roles in the development of coronary atherosclerosis in women than in

1	risen in women. Inflammation, obesity, type 2 diabetes mellitus, and the metabolic syndrome appear to play more prominent roles in the development of coronary atherosclerosis in women than in men. Coronary artery disease (CAD) is more frequently associated with dysfunction of the coronary microcirculation in women than in men. Exercise electro-cardiography has a lower diagnostic accuracy in the prediction of epicardial obstruction in women than in men. NATURAL HISTORY Cardiovascular disorders often present acutely, as in a previously asymptomatic person who develops an acute myocardial infarction (Chap. 295), or a previously asymptomatic patient with hypertrophic cardiomyopathy (Chap. 287), or with a prolonged QT interval (Chap. 277) whose first clinical manifestation is syncope or even sudden death. However, the alert physician may recognize the patient at risk for these complications long before they occur and often can take measures to prevent their occurrence. For example, a

1	sudden death. However, the alert physician may recognize the patient at risk for these complications long before they occur and often can take measures to prevent their occurrence. For example, a patient with acute myocardial infarction will often have had risk factors for athero-sclerosis for many years. Had these risk factors been recognized, their elimination or reduction might have delayed or even prevented the infarction. Similarly, a patient with hypertrophic cardiomyopathy may have had a heart murmur for years and a family history of this disor-der. These findings could have led to an echocardiographic examina-tion, recognition of the condition, and appropriate therapy long before the occurrence of a serious acute manifestation. Patients with valvular heart disease or idiopathic dilated cardiomy-opathy, by contrast, may have a prolonged course of gradually increas-ing dyspnea and other manifestations of chronic heart failure that is punctuated by episodes of acute deterioration

1	cardiomy-opathy, by contrast, may have a prolonged course of gradually increas-ing dyspnea and other manifestations of chronic heart failure that is punctuated by episodes of acute deterioration only late in the course of the disease. Understanding the natural history of various cardiac 264 SEC Tion 1 inTRoDuC Tion To CARDiovASCulAR DiSoRDERS PART 10: Disorders of the Cardiovascular System disorders is essential for applying appropriate diagnostic and therapeutic measures to each stage of the condition, as well as for providing the patient and family with the likely prognosis.

1	The symptoms caused by heart disease result most commonly from myocardial ischemia, disturbance of the contraction and/or relaxation of the myocardium, obstruction to blood flow, or an abnormal cardiac rhythm or rate. Ischemia, which is caused by an imbalance between the heart’s oxygen supply and demand, is manifest most frequently as chest discomfort (Chap. 19), whereas reduction of the pumping ability of the heart commonly leads to fatigue and elevated intravascular pressure upstream of the failing ventricle. The latter results in abnormal fluid accumulation, with peripheral edema (Chap. 50) or pulmonary congestion and dyspnea (Chap. 47e). Obstruction to blood flow, as occurs in valvular stenosis, can cause symptoms resembling those of myocardial failure (Chap. 279). Cardiac arrhythmias often develop suddenly, and the resulting symptoms and signs—palpitations (Chap. 52), dyspnea, hypotension, and syncope (Chap. 27)—generally occur abruptly and may disappear as rapidly as they

1	often develop suddenly, and the resulting symptoms and signs—palpitations (Chap. 52), dyspnea, hypotension, and syncope (Chap. 27)—generally occur abruptly and may disappear as rapidly as they develop.

1	Although dyspnea, chest discomfort, edema, and syncope are cardinal manifestations of cardiac disease, they occur in other conditions as well. Thus, dyspnea is observed in disorders as diverse as pulmonary disease, marked obesity, and anxiety (Chap. 47e). Similarly, chest discomfort may result from a variety of noncardiac and cardiac causes other than myocardial ischemia (Chap. 19). Edema, an important finding in untreated or inadequately treated heart failure, also may occur with primary renal disease and in hepatic cirrhosis (Chap. 50). Syncope occurs not only with serious cardiac arrhythmias but in a number of neurologic conditions as well (Chap. 27). Whether heart disease is responsible for these symptoms frequently can be determined by carrying out a careful clinical examination (Chap. 267), supplemented by noninvasive testing using electrocardiography at rest and during exercise (Chap. 268), echocardiography, roentgenography, and other forms of myocardial imaging (Chap. 270e).

1	Myocardial or coronary function that may be adequate at rest may be insufficient during exertion. Thus, dyspnea and/or chest discomfort that appear during activity are characteristic of patients with heart disease, whereas the opposite pattern, i.e., the appearance of these symptoms at rest and their remission during exertion, is rarely observed in such patients. It is important, therefore, to question the patient carefully about the relation of symptoms to exertion.

1	Many patients with cardiovascular disease may be asymptomatic both at rest and during exertion but may present with an abnormal physical finding such as a heart murmur, elevated arterial pressure, or an abnormality of the electrocardiogram (ECG) or imaging test. It is important to assess the global risk of CAD in asymptomatic individuals, using a combination of clinical assessment and measurement of cholesterol and its fractions, as well as other biomarkers, such as C-reactive protein, in some patients (Chap. 291e). Since the first clinical manifestation of CAD may be catastrophic—sudden cardiac death, acute myocardial infarction, or stroke in previous asymptomatic persons—it is mandatory to identify those at high risk of such events and institute further testing and preventive measures.

1	As outlined by the New York Heart Association (NYHA), the elements of a complete cardiac diagnosis include the systematic consideration of the following: 1. The underlying etiology. Is the disease congenital, hypertensive, ischemic, or inflammatory in origin? Approach to the Patient with Possible Cardiovascular Disease No limitation of physical activity Marked limitation of physical activity No symptoms with ordinary Less than ordinary activity causes exertion symptoms Slight limitation of physical activity Class IV Ordinary activity causes symptoms Inability to carry out any physical activity without discomfort Source: Modified from The Criteria Committee of the New York Heart Association. 2. The anatomic abnormalities. Which chambers are involved? Are they hypertrophied, dilated, or both? Which valves are affected? Are they regurgitant and/or stenotic? Is there pericardial involvement? Has there been a myocardial infarction? 3.

1	3. The physiologic disturbances. Is an arrhythmia present? Is there evidence of congestive heart failure or myocardial ischemia? 4. Functional disability. How strenuous is the physical activity required to elicit symptoms? The classification provided by the NYHA has been found to be useful in describing functional disability (Table 264-1).

1	One example may serve to illustrate the importance of establishing a complete diagnosis. In a patient who presents with exertional chest discomfort, the identification of myocardial ischemia as the etiology is of great clinical importance. However, the simple recognition of ischemia is insufficient to formulate a therapeutic strategy or prognosis until the underlying anatomic abnormalities responsible for the myocardial ischemia, e.g., coronary atherosclerosis or aortic stenosis, are identified and a judgment is made about whether other physiologic disturbances that cause an imbalance between myocardial oxygen supply and demand, such as severe anemia, thyrotoxicosis, or supra-ventricular tachycardia, play contributory roles. Finally, the severity of the disability should govern the extent and tempo of the workup and strongly influence the therapeutic strategy that is selected.

1	The establishment of a correct and complete cardiac diagnosis usually commences with the history and physical examination (Chap. 267). Indeed, the clinical examination remains the basis for the diagnosis of a wide variety of disorders. The clinical examination may then be supplemented by five types of laboratory tests: (1) ECG (Chap. 268), (2) noninvasive imaging examinations (chest roentgenogram, echocardiogram, radionuclide imaging, computed tomographic imaging, positron emission tomography, and magnetic resonance imaging) (Chap. 270e), (3) blood tests to assess risk (e.g., lipid determinations, C-reactive protein [Chap. 291e]) or cardiac function (e.g., brain natriuretic peptide [BNP] [Chap. 279]), (4) occasionally specialized invasive examinations (i.e., cardiac catheterization and coronary arteriography [Chap. 272]), and (5) genetic tests to identify monogenic cardiac diseases (e.g., hypertrophic cardiomyopathy [Chap. 287], Marfan’s syndrome [Chap. 427], and abnormalities of

1	coronary arteriography [Chap. 272]), and (5) genetic tests to identify monogenic cardiac diseases (e.g., hypertrophic cardiomyopathy [Chap. 287], Marfan’s syndrome [Chap. 427], and abnormalities of cardiac ion channels that lead to prolongation of the QT interval and an increase in the risk of sudden death [Chap. 276]). These tests are becoming more widely available.

1	In eliciting the history of a patient with known or suspected cardiovascular disease, particular attention should be directed to the family history. Familial clustering is common in many forms of heart disease. Mendelian transmission of single-gene defects may occur, as in hyper-trophic cardiomyopathy (Chap. 287), Marfan’s syndrome (Chap. 427), and sudden death associated with a prolonged QT syndrome (Chap. 277). Premature coronary disease and essential hypertension, type 2 diabetes mellitus, and hyperlipidemia (the most important risk factors for CAD) are usually polygenic disorders. Although familial transmission may be less obvious than in the monogenic disorders, it is helpful in assessing risk and prognosis in polygenic disorders, as well. Familial clustering of cardiovascular diseases not only may occur on a genetic basis but also may be related to familial dietary or behavior patterns, such as excessive ingestion of salt or calories and cigarette smoking.

1	When an attempt is made to determine the severity of functional impairment in a patient with heart disease, it is helpful to ascertain the level of activity and the rate at which it is performed before symptoms develop. Thus, it is not sufficient to state that the patient complains of dyspnea. The breathlessness that occurs after running up two long flights of stairs denotes far less functional impairment than do similar symptoms that occur after taking a few steps on level ground. Also, the degree of customary physical activity at work and during recreation should be considered. The development of two-flight dyspnea in a well-conditioned marathon runner may be far more significant than the development of one-flight dyspnea in a previously sedentary person. The history should include a detailed consideration of the patient’s therapeutic regimen. For example, the persistence or development of edema, breathlessness, and other manifestations of heart failure in a patient who is receiving

1	consideration of the patient’s therapeutic regimen. For example, the persistence or development of edema, breathlessness, and other manifestations of heart failure in a patient who is receiving optimal doses of diuretics and other therapies for heart failure (Chap. 279) is far graver than are similar manifestations in the absence of treatment. Similarly, the presence of angina pectoris despite treatment with optimal doses of multiple antianginal drugs (Chap. 293) is more serious than it is in a patient on no therapy. In an effort to determine the progression of symptoms, and thus the severity of the underlying illness, it may be useful to ascertain what, if any, specific tasks the patient could have carried out 6 months or 1 year earlier that he or she cannot carry out at present.

1	(See also Chap. 268) Although an ECG usually should be recorded in patients with known or suspected heart disease, with the exception of the identification of arrhythmias, conduction abnormalities, ventricular hypertrophy, and acute myocardial infarction, it generally does not establish a specific diagnosis. The range of normal electrocardiographic findings is wide, and the tracing can be affected significantly by many noncardiac factors, such as age, body habitus, and serum electrolyte concentrations. In general, electrocardiographic changes should be interpreted in the context of other abnormal cardiovascular findings. (Fig. 264-1) The cause of a heart murmur can often be readily elucidated from a systematic evaluation of its major attributes: timing, duration, intensity, quality, frequency, configuration, location, and radiation when considered in the light of the history, general physical examination, and other features of the cardiac examination, as described in Chap. 267.

1	The majority of heart murmurs are midsystolic and soft (grades I–II/VI). When such a murmur occurs in an asymptomatic child or young adult without other evidence of heart disease on clinical examination, it is usually benign and echocardiography generally is not required. By contrast, two-dimensional and Doppler echocardiography (Chap. 270e) are indicated in patients with loud systolic murmurs (grades ≥III/VI), especially those that are holosystolic or late systolic, and in most patients with diastolic or continuous murmurs.

1	Increasing subspecialization in internal medicine and the perfection of advanced diagnostic techniques in cardiology can lead to several undesirable consequences. Examples include the following: 1. Failure by the noncardiologist to recognize important cardiac manifestations of systemic illnesses. For example, the presence of mitral stenosis, patent foramen ovale, and/or transient atrial arrhythmia should be considered in a patient with stroke, or the presence of pulmonary hypertension and cor pulmonale should be considered in a patient with scleroderma or Raynaud’s syndrome. A cardiovascular examination should be carried out to identify and estimate

1	PRESENCE OF CARDIAC MURMUR Systolic Murmur Diastolic or Continuous Murmur Grade I + II and midsystolic Grade III or >, holosystolic, or late systolic Other signs or symptoms of cardiac disease No further workup Normal ECG and chest X-ray Abnormal ECG or chest X-ray Asymptomatic and no associated findings Echocardiography Cardiac consult if appropriate FIgURE 264-1 Approach to the evaluation of a heart murmur. ECG, electrocardiogram. (From RA O’Rourke, in Primary Cardiology, 2nd ed, E Braunwald, L Goldman [eds]. Philadelphia, Saunders, 2003.) the severity of the cardiovascular involvement that accompanies many noncardiac disorders. 2.

1	2. Failure by the cardiologist to recognize underlying systemic disorders in patients with heart disease. For example, hyperthyroidism should be considered in an elderly patient with atrial fibrillation and unexplained heart failure, and Lyme disease should be considered in a patient with unexplained fluctuating atrioventricular block. A cardiovascular abnormality may provide the clue critical to the recognition of some systemic disorders. For example, an unexplained pericardial effusion may provide an early clue to the diagnosis of tuberculosis or a neoplasm. 3. Overreliance on and overutilization of laboratory tests, particularly invasive techniques, for the evaluation of the cardiovascular system. Cardiac catheterization and coronary arteriography (Chap.

1	272) provide precise diagnostic information that may be crucial in developing a therapeutic plan in patients with known or suspected CAD. Although a great deal of attention has been directed to these examinations, it is important to recognize that they serve to supplement, not supplant, a careful examination carried out with clinical and noninvasive techniques. A coronary arteriogram should not be performed in lieu of a careful history in patients with chest pain suspected of having ischemic heart disease. Although coronary arteriography may establish whether the coronary arteries are obstructed and to what extent, the results of the procedure by themselves often do not provide a definitive answer to the question of whether a patient’s complaint of chest discomfort is attributable to coronary atherosclerosis and whether or not revascularization is indicated.

1	Despite the value of invasive tests in certain circumstances, they entail some small risk to the patient, involve discomfort and substantial cost, and place a strain on medical facilities. Therefore, they should be carried out only if the results can be expected to modify the patient’s management. The prevention of heart disease, especially of CAD, is one of the most important tasks of primary care health givers as well as cardiologists. Prevention begins with risk assessment, followed by attention to lifestyle, such as achieving optimal weight, physical activity, and smoking cessation, and then aggressive treatment of all abnormal risk factors, such as hypertension, hyperlipidemia, and diabetes mellitus (Chap. 417). After a complete diagnosis has been established in patients with known heart disease, a number of management options are usually available. Several examples may be used to demonstrate some of the 1441 principles of cardiovascular therapeutics: 1.

1	In the absence of evidence of heart disease, the patient should be clearly informed of this assessment and not be asked to return at intervals for repeated examinations. If there is no evidence of disease, such continued attention may lead to the patient’s developing inappropriate concern about the possibility of heart disease. 2. If there is no evidence of cardiovascular disease but the patient has one or more risk factors for the development of ischemic heart disease (Chap. 293), a plan for their reduction should be developed and the patient should be retested at intervals to assess compliance and efficacy in risk reduction. 3.

1	3. Asymptomatic or mildly symptomatic patients with valvular heart disease that is anatomically severe should be evaluated periodically, every 6 to 12 months, by clinical and noninvasive examinations. Early signs of deterioration of ventricular function may signify the need for surgical treatment before the development of disabling symptoms, irreversible myocardial damage, and excessive risk of surgical treatment (Chap. 283). 4.

1	4. In patients with CAD (Chap. 293), available practice guidelines should be considered in the decision on the form of treatment (medical, percutaneous coronary intervention, or surgical revascularization). Mechanical revascularization may be employed too frequently in the United States and too infrequently in Eastern Europe and developing nations. The mere presence of angina pectoris and/or the demonstration of critical coronary arterial narrowing at angiography should not reflexively evoke a decision to treat the patient by revascularization. Instead, these interventions should be limited to patients with CAD whose angina has not responded adequately to medical treatment or in whom revascularization has been shown to improve the natural history (e.g., acute coronary syndrome or multivessel CAD with left ventricular dysfunction). Basic Biology of the Cardiovascular System

1	Basic Biology of the Cardiovascular System Basic Biology of the Cardiovascular System Joseph Loscalzo, Peter Libby, Jonathan A. Epstein THE BLOOD VESSEL VASCULAR ULTRASTRUCTURE 265e

1	Blood vessels participate in homeostasis on a moment-to-moment basis and contribute to the pathophysiology of diseases of virtually every organ system. Hence, an understanding of the fundamentals of vascular biology furnishes a foundation for understanding the normal function of all organ systems and many diseases. The smallest blood vessels—capillaries—consist of a monolayer of endothelial cells apposed to a basement membrane, adjacent to occasional smooth-muscle-like cells known as pericytes (Fig. 265e-1A). Unlike larger vessels, pericytes do not invest the entire microvessel to form a continuous sheath. Arteries typically have a trilaminar structure (Fig. 265e-1B–E). The intima consists of a monolayer of endothelial cells continuous with those of the capillaries. The middle layer, or tunica media, consists of layers of smooth-muscle cells; in veins, the media can contain just a few layers of smooth-muscle cells (Fig. 265e-1B). The outer layer, the adventitia, consists of looser

1	tunica media, consists of layers of smooth-muscle cells; in veins, the media can contain just a few layers of smooth-muscle cells (Fig. 265e-1B). The outer layer, the adventitia, consists of looser extracellular matrix with occasional fibroblasts, mast cells, and nerve terminals. Larger arteries have their own vasculature, the vasa vasorum, which nourishes the outer aspects of the tunica media. The adventitia of many veins surpasses the intima in thickness.

1	The tone of muscular arterioles regulates blood pressure and flow through various arterial beds. These smaller arteries have a relatively thick tunica media in relation to the adventitia (Fig. 265e-1C). Medium-size muscular arteries similarly contain a prominent tunica media (Fig. 265e-1D); atherosclerosis commonly affects this type of muscular artery. The larger elastic arteries have a much more structured tunica media consisting of concentric bands of smooth-muscle cells, interspersed with strata of elastin-rich extracellular matrix A. Capillary B. Vein sandwiched between layers of smooth-muscle cells (Fig. 265e-1E). 265e-1 Larger arteries have a clearly demarcated internal elastic lamina that forms the barrier between the intima and the media. An external elastic lamina demarcates the media of arteries from the surrounding adventitia.

1	The intima in human arteries often contains occasional resident smooth-muscle cells beneath the monolayer of vascular endothelial cells. The embryonic origin of smooth-muscle cells in various types of artery differs. Some upper-body arterial smooth-muscle cells derive from the neural crest, whereas lower-body arteries generally recruit smooth-muscle cells from neighboring mesodermal structures during development. Derivatives of the proepicardial organ, which gives rise to the epicardial layer of the heart, contribute to the vascular smooth-muscle cells of the coronary arteries. Bone marrow–derived endothelial progenitors may aid repair of damaged or aging arteries. In addition, multipotent vascular stem cells resident in vessel walls may give rise to the smooth-muscle cells that accumulate in injured or atheromatous arteries (Chaps. 88, 89e, and 90e).

1	VASCULAR CELL BIOLOGY Endothelial Cell The key cell of the vascular intima, the endothelial cell, has manifold functions in health and disease. The endothelium forms the interface between tissues and the blood compartment. It therefore must regulate the entry of molecules and cells into tissues in a selective manner. The ability of endothelial cells to serve as a selectively permeable barrier fails in many vascular disorders, including atherosclerosis, hypertension, and renal disease. This dysregulation of permeability also occurs in pulmonary edema and other situations of “capillary leak.” The endothelium also participates in the local regulation of blood flow and vascular caliber. Endogenous substances produced by endothelial cells such as prostacyclin, endothelium-derived hyperpolarizing factor, nitric oxide (NO), and hydrogen peroxide (H2O2) provide tonic vasodilatory stimuli under physiologic conditions in vivo (Table 265e-1). Impaired production or excess catabolism of NO

1	C. Small muscular artery CHAPTER 265e Basic Biology of the Cardiovascular System D. Large muscular artery Vascular smooth-muscle cell E. Large elastic artery Internal elastic lamina External elastic lamina Adventitia Pericyte Endothelial cell FIGURE 265e-1 Schematics of the structures of various types of blood vessels. A. Capillaries consist of an endothelial tube in contact with a discontinuous population of pericytes. B. Veins typically have thin medias and thicker adventitias. C. A small muscular artery features a prominent tunica media. D. Larger muscular arteries have a prominent media with smooth-muscle cells embedded in a complex extracellular matrix. E. Larger elastic arteries have cylindrical layers of elastic tissue alternating with concentric rings of smooth-muscle cells. Optimize balance between Impaired dilation, vasoconstriction vasodilation and vasoconstriction

1	Optimize balance between Impaired dilation, vasoconstriction vasodilation and vasoconstriction Antithrombotic, profibrinolytic Prothrombotic, antifibrinolytic impairs this endothelium-dependent vasodilator function and may contribute to excessive vasoconstriction in various pathologic situations. Measurement of flow-mediated dilatation can assess endothelial vasodilator function in humans (Fig. 265e-2). By contrast, endothelial cells also produce potent vasoconstrictor substances such as endothelin in a regulated fashion. Excessive production of reactive oxygen species, such as superoxide anion (O2−), by endothelial or smooth-muscle cells under pathologic conditions (e.g., excessive exposure to angiotensin II), can promote local oxidative stress and inactivate NO.

1	The endothelial monolayer contributes critically to inflammatory processes involved in normal host defenses and pathologic states. The normal endothelium resists prolonged contact with blood leukocytes; however, when activated by bacterial products such as endotoxin or by proinflammatory cytokines released during infection or injury, endothelial cells express an array of leukocyte adhesion molecules that bind various classes of leukocytes. The endothelial cells appear to recruit selectively different classes of leukocytes in different pathologic conditions. The gamut of adhesion molecules and chemokines generated during acute bacterial infection tends to recruit granulocytes. In chronic inflammatory diseases such as tuberculosis and atherosclerosis, endothelial cells express adhesion molecules that favor the recruitment of mononuclear leukocytes that characteristically accumulate in these conditions.

1	The endothelium also dynamically regulates thrombosis and hemostasis. NO, in addition to its vasodilatory properties, can limit platelet activation and aggregation. Like NO, prostacyclin produced by endothelial cells under normal conditions not only provides a vasodilatory stimulus but also antagonizes platelet activation and aggregation. Thrombomodulin expressed on the surface of endothelial cells binds thrombin at low concentrations and inhibits coagulation through activation of the protein C pathway, inactivating clotting factors Va and VIIIa and thus combating thrombus formation. The surface of endothelial cells contains heparan sulfate glycosaminoglycans that furnish an endogenous antithrombotic coating to the vasculature. Endothelial cells also participate actively in fibrinolysis and its regulation. They express receptors for plasminogen and plasminogen activators and produce tissue-type plasminogen activator. Through local generation of plasmin, the normal endothelial

1	and its regulation. They express receptors for plasminogen and plasminogen activators and produce tissue-type plasminogen activator. Through local generation of plasmin, the normal endothelial monolayer can promote the lysis of nascent thrombi.

1	When activated by inflammatory cytokines, bacterial endotoxin, or angiotensin II, for example, endothelial cells can produce substantial quantities of the major inhibitor of fibrinolysis, plasminogen activator inhibitor 1 (PAI-1). Thus, in pathologic circumstances, the endothelial cell may promote local thrombus accumulation rather than combat it. Inflammatory stimuli also induce the expression of the potent pro-coagulant tissue factor, a contributor to disseminated intravascular coagulation in sepsis.

1	Endothelial cells also participate in the pathophysiology of a number of immune-mediated diseases. Lysis of endothelial cells mediated by complement provides an example of immunologically mediated tissue injury. The presentation of foreign histocompatibility complex antigens by endothelial cells in solid-organ allografts can promote allograft arteriopathy. In addition, immune-mediated endothelial injury may contribute in some patients with thrombotic thrombocytopenic purpura and patients with hemolytic-uremic syndrome. Thus, in addition to the involvement of innate immune responses, endothelial cells participate actively in both humoral and cellular limbs of the immune response. PART 10 Disorders of the Cardiovascular System

1	PART 10 Disorders of the Cardiovascular System FIGURE 265e-2 Assessment of endothelial function in vivo using blood pressure cuff occlusion and release. Upon deflation of the cuff, an ultrasound probe monitors changes in diameter (A) and blood flow (B) of the brachial artery (C). (Reproduced with permission of J. Vita, MD.) Endothelial cells regulate growth of subjacent smooth-muscle cells. Heparan sulfate glycosaminoglycans elaborated by endothelial cells can inhibit smooth-muscle proliferation. In contrast, when exposed to various injurious stimuli, endothelial cells can elaborate growth factors and chemoattractants, such as platelet-derived growth factor, that can promote the migration and proliferation of vascular smooth-muscle cells. Dysregulated elaboration of these growth-stimulatory molecules may promote smooth-muscle accumulation in atherosclerotic lesions.

1	Vascular Smooth-Muscle Cell The vascular smooth-muscle cell, the major cell type of the media layer of blood vessels, also contributes actively to vascular pathobiology. Contraction and relaxation of smooth-muscle cells at the level of the muscular arteries controls blood pressure and, hence, regional blood flow and the afterload experienced by the left ventricle (see below). The vasomotor tone of veins, which is governed by smooth-muscle cell tone, regulates the capacitance of the venous tree and influences the preload experienced by both ventricles. Smooth-muscle cells in the adult vessel seldom replicate in the absence of arterial injury or inflammatory activation. Proliferation and migration of arterial smooth-muscle cells, associated with functional modulation characterized by lower content of contractile proteins and greater production of extracellular matrix macromolecules, can contribute to the development of arterial stenoses in atherosclerosis, arteriolar remodeling that can

1	of contractile proteins and greater production of extracellular matrix macromolecules, can contribute to the development of arterial stenoses in atherosclerosis, arteriolar remodeling that can sustain and propagate hypertension, and the hyperplastic response of arteries injured by percutaneous intervention. In the pulmonary circulation, smooth-muscle migration and proliferation contribute decisively to the pulmonary vascular disease that gradually occurs in response to sustained high-flow states such as left-to-right shunts. Such pulmonary vascular disease provides a major obstacle to the management of many patients with adult congenital heart disease. Among other mediators, microRNAs have emerged as powerful regulators of this transition, offering new targets for intervention.

1	Smooth-muscle cells secrete the bulk of vascular extracellular matrix. Excessive production of collagen and glycosaminoglycans contributes to the remodeling and altered functions and biomechanics of arteries affected by hypertension or atherosclerosis. In larger elastic arteries, the elastin synthesized by smooth-muscle cells serves to maintain not only normal arterial structure, but also hemodynamic 265e-3 function. The ability of the larger arteries, such as the aorta, to store the kinetic energy of systole promotes tissue perfusion during diastole. Arterial stiffness associated with aging or disease, as manifested by a widening pulse pressure, increases left ventricular afterload and portends a poor outcome.

1	Like endothelial cells, vascular smooth-muscle cells do not merely respond to vasomotor or inflammatory stimuli elaborated by other cell types but can themselves serve as a source of such stimuli. For example, when exposed to bacterial endotoxin or other proinflammatory stimuli, smooth-muscle cells can elaborate cytokines and other inflammatory mediators. Like endothelial cells, upon inflammatory activation, arterial smooth-muscle cells can produce prothrombotic mediators such as tissue factor, the antifibrinolytic protein PAI-1, and other molecules that modulate thrombosis and fibrinolysis. Smooth-muscle cells also elaborate autocrine growth factors that can amplify hyperplastic responses to arterial injury.

1	Vascular Smooth-Muscle Cell Function Vascular smooth-muscle cells govern vessel tone. Those cells contract when stimulated by a rise in intracellular calcium concentration by calcium influx through the plasma membrane and by calcium release from intracellular stores (Fig. 265e-3). In vascular smooth-muscle cells, voltage-dependent L-type calcium channels open with membrane depolarization, which is regulated by energy-dependent ion pumps such as the Na+,K+-ATPase pump and ion channels such as the Ca2+-sensitive K+ channel. Local changes in intracellular calcium concentration, termed calcium sparks, result from the influx of calcium through the voltage-dependent calcium channel and are caused by the coordinated activation of a CHAPTER 265e Basic Biology of the Cardiovascular System NE, ET-1, Ang II PLC ANPNO pGC AC RhoA Rho Kinase IP3 G G SR Calcium MLCK MLCP IP3R RyrR Plb ATPase cGMP GTP ATP cAMP VDCC K+ Ch Na-K ATPase sGC Beta-Agonist PKG PKA

1	NE, ET-1, Ang II PLC ANPNO pGC AC RhoA Rho Kinase IP3 G G SR Calcium MLCK MLCP IP3R RyrR Plb ATPase cGMP GTP ATP cAMP VDCC K+ Ch Na-K ATPase sGC Beta-Agonist PKG PKA FIGURE 265e-3 Regulation of vascular smooth-muscle cell calcium concentration and actomyosin ATPase-dependent contraction. AC, adenylyl cyclase; Ang II, angiotensin II; ANP, atrial natriuretic peptide; DAG, diacylglycerol; ET-1, endothelin-1; G, G protein; IP3, inositol 1,4,5trisphosphate; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; NE, norepinephrine; NO, nitric oxide; pGC, particular guanylyl cyclase; PIP2, phosphatidylinositol 4,5-bisphosphate; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; PLC, phospholipase C; sGC, soluble guanylyl cyclase; SR, sarcoplasmic reticulum; VDCC, voltage-dependent calcium channel. (Modified from B Berk, in Vascular Medicine, 3rd ed. Philadelphia, Saunders, Elsevier, 2006, p. 23; with permission.)

1	PART 10 Disorders of the Cardiovascular System 265e-4 cluster of ryanodine-sensitive calcium release channels in the sarcoplasmic reticulum (see below). Calcium sparks directly augment intracellular calcium concentration and indirectly increase intracellular calcium concentration by activating chloride channels. In addition, calcium sparks reduce smooth-muscle contractility by activating large-conductance calcium-sensitive K+ channels, hyperpolarizing the cell membrane and thereby limiting further voltage-dependent increases in intracellular calcium. Biochemical agonists also increase intracellular calcium concentration, in this case by receptor-dependent activation of phospholipase C with hydrolysis of phosphatidylinositol 4,5-bisphosphate, resulting in the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). These membrane lipid derivatives in turn activate protein kinase C and increase intracellular calcium concentration. In addition, IP3 binds to specific

1	(DAG) and inositol 1,4,5-trisphosphate (IP3). These membrane lipid derivatives in turn activate protein kinase C and increase intracellular calcium concentration. In addition, IP3 binds to specific receptors on the sarcoplasmic reticulum membrane to increase calcium efflux from this calcium storage pool into the cytoplasm. Vascular smooth-muscle cell contraction depends principally on the phosphorylation of myosin light chain, which in the steady state, reflects the balance between the actions of myosin light chain kinase and myosin light chain phosphatase. Calcium activates myosin light chain kinase through the formation of a calciumcalmodulin complex. Phosphorylation of myosin light chain by this kinase augments myosin ATPase activity and enhances contraction. Myosin light chain phosphatase dephosphorylates myosin light chain, reducing myosin ATPase activity and contractile force. Phosphorylation of the myosin-binding subunit (thr695) of myosin light chain phosphatase by Rho kinase

1	dephosphorylates myosin light chain, reducing myosin ATPase activity and contractile force. Phosphorylation of the myosin-binding subunit (thr695) of myosin light chain phosphatase by Rho kinase inhibits phosphatase activity and induces calcium sensitization of the contractile apparatus. Rho kinase is itself activated by the small GTPase RhoA, which is stimulated by guanosine exchange factors and inhibited by GTPase-activating proteins. Both cyclic AMP and cyclic GMP relax vascular smooth-muscle cells through complex mechanisms. β agonists, acting through their G-protein-coupled receptors activate adenylyl cyclase to convert ATP to cyclic AMP; NO and atrial natriuretic peptide acting directly and via a G-protein-coupled receptor, respectively, activate guanylyl cyclase to convert GTP to cyclic GMP. These agents in turn activate protein kinase A and protein kinase G, respectively, which inactivate myosin light chain kinase and decrease vascular smooth-muscle cell tone. In addition,

1	cyclic GMP. These agents in turn activate protein kinase A and protein kinase G, respectively, which inactivate myosin light chain kinase and decrease vascular smooth-muscle cell tone. In addition, protein kinase G can interact directly with the myosinbinding substrate subunit of myosin light chain phosphatase, increasing phosphatase activity and decreasing vascular tone. Finally, several mechanisms drive NO-dependent, protein kinase G–mediated reductions in vascular smooth-muscle cell calcium concentration, including phosphorylation-dependent inactivation of RhoA; decreased IP3 formation; phosphorylation of the IP3 receptor–associated cyclic GMP kinase substrate, with subsequent inhibition of IP3 receptor function; phosphorylation of phospholamban, which increases calcium ATPase activity and sequestration of calcium in the sarcoplasmic reticulum; and protein kinase G–dependent stimulation of plasma membrane calcium ATPase activity, perhaps by activation of the Na+,K+-ATPase pump or

1	and sequestration of calcium in the sarcoplasmic reticulum; and protein kinase G–dependent stimulation of plasma membrane calcium ATPase activity, perhaps by activation of the Na+,K+-ATPase pump or hyperpolarization of the cell membrane by activation of calcium-dependent K+ channels.

1	Control of Vascular Smooth-Muscle Cell Tone The autonomic nervous system and endothelial cells modulate vascular smooth-muscle cells in a tightly regulated manner. Autonomic neurons enter the blood vessel medial layer from the adventitia and modulate vascular smooth-muscle cell tone in response to baroreceptors and chemoreceptors within the aortic arch and carotid bodies and in response to thermoreceptors in the skin. These regulatory components include rapidly acting reflex arcs modulated by central inputs that respond to sensory inputs (olfactory, visual, auditory, and tactile) as well as emotional stimuli. Three classes of nerves mediate autonomic regulation of vascular tone: sympathetic, whose principal neurotransmitters are epinephrine and norepinephrine; parasympathetic, whose principal neurotransmitter is acetylcholine; and nonadrenergic/noncholinergic, which include two subgroups—nitrergic, whose principal neurotransmitter is NO, and peptidergic, whose principal

1	whose principal neurotransmitter is acetylcholine; and nonadrenergic/noncholinergic, which include two subgroups—nitrergic, whose principal neurotransmitter is NO, and peptidergic, whose principal neurotransmitters are substance P, vasoactive intestinal peptide, calcitonin gene-related peptide, and ATP.

1	Each of these neurotransmitters acts through specific receptors on the vascular smooth-muscle cell to modulate intracellular calcium and, consequently, contractile tone. Norepinephrine activates α receptors, and epinephrine activates α and β receptors (adrenergic receptors); in most blood vessels, norepinephrine activates postjunctional α1 receptors in large arteries and α2 receptors in small arteries and arterioles, leading to vasoconstriction. Most blood vessels express β2-adrenergic receptors on their vascular smooth-muscle cells and respond to β agonists by cyclic AMP–dependent relaxation. Acetylcholine released from parasympathetic neurons binds to muscarinic receptors (of which there are five subtypes, M1–5) on vascular smooth-muscle cells to yield vasorelaxation. In addition, NO stimulates presynaptic neurons to release acetylcholine, which can stimulate the release of NO from the endothelium. Nitrergic neurons release NO produced by neuronal NO synthase, which causes vascular

1	presynaptic neurons to release acetylcholine, which can stimulate the release of NO from the endothelium. Nitrergic neurons release NO produced by neuronal NO synthase, which causes vascular smooth-muscle cell relaxation via the cyclic GMP–dependent and –independent mechanisms described above. The peptidergic neurotransmitters all potently vasodilate, acting either directly or through endothelium-dependent NO release to decrease vascular smooth-muscle cell tone. For the detailed molecular physiology of the autonomic nervous system, see Chap. 454.

1	The endothelium modulates vascular smooth-muscle tone by the direct release of several effectors, including NO, prostacyclin, hydrogen sulfide, and endothelium-derived hyperpolarizing factor, all of which cause vasorelaxation, and endothelin, which causes vasoconstriction. The release of these endothelial effectors of vascular smooth-muscle cell tone is stimulated by mechanical (shear stress, cyclic strain, etc.) and biochemical mediators (purinergic agonists, muscarinic agonists, peptidergic agonists), with the biochemical mediators acting through endothelial receptors specific to each class. In addition to these local paracrine modulators of vascular smooth-muscle cell tone, circulating mediators can affect tone, including norepinephrine and epinephrine, vasopressin, angiotensin II, bradykinin, and the natriuretic peptides (atrial natriuretic peptide [ANP], brain natriuretic peptide [BNP], C-type natriuretic peptide [CNP], and dendroaspis natriuretic peptide [DNP]), as discussed

1	bradykinin, and the natriuretic peptides (atrial natriuretic peptide [ANP], brain natriuretic peptide [BNP], C-type natriuretic peptide [CNP], and dendroaspis natriuretic peptide [DNP]), as discussed above.

1	Growth of new blood vessels can occur in response to conditions such as chronic hypoxemia and tissue ischemia. Growth factors, including vascular endothelial growth factor (VEGF) and forms of fibroblast growth factor (FGF), activate a signaling cascade that stimulates endothelial proliferation and tube formation, defined as angiogenesis. Guidance molecules, including members of the semaphorin family of secreted peptides, direct blood vessel patterning by attracting or repelling nascent endothelial tubes. The development of collateral vascular networks in the ischemic myocardium, an example of angiogenesis, can result from selective activation of endothelial progenitor cells, which may reside in the blood vessel wall or home to the ischemic tissue from the bone marrow. True arteriogenesis, or the development of a new blood vessel that includes all three cell layers, normally does not occur in the cardiovascular system of adult mammals. The molecular mechanisms and progenitor cells that

1	or the development of a new blood vessel that includes all three cell layers, normally does not occur in the cardiovascular system of adult mammals. The molecular mechanisms and progenitor cells that can recapitulate blood vessel development de novo are under rapidly advancing study (Chaps. 88, 89e, and 90e).

1	The last decade has witnessed considerable progress in efforts to define the genetic differences underlying individual variations in vascular pharmacologic responses. Many investigators have focused on receptors and enzymes associated with neurohumoral modulation of vascular function as well as hepatic enzymes that metabolize drugs that affect vascular tone. The genetic polymorphisms thus far associated with differences in vascular response often (but not invariably) relate to functional differences in the activity or expression of the receptor or enzyme of interest. Some of these CHAPTER 265e Basic Biology of the Cardiovascular System polymorphisms appear to have different allele frequencies in specific ethnic groups.

1	CHAPTER 265e Basic Biology of the Cardiovascular System polymorphisms appear to have different allele frequencies in specific ethnic groups. About three-fourths of the ventricular mass is composed of cardiomyocytes, normally 60–140 μm in length and 17–25 μm in diameter (Fig. 265e-4A). Each cell contains multiple, rodlike cross-banded strands (myofibrils) that run the length of the cell and are composed of serially repeating structures, the sarcomeres. The cytoplasm between the myofibrils contains other cell constituents, including the single

1	FIGURE 265e-4 A shows the branching myocytes making up the cardiac myofibers. B illustrates the critical role played by the changing [Ca2+] in the myocardial cytosol. Ca2+ ions are schematically shown as entering through the calcium channel that opens in response to the wave of depolarization that travels along the sarcolemma. These Ca2+ ions “trigger” the release of more calcium from the sarcoplasmic reticulum (SR) and thereby initiate a contraction-relaxation cycle. Eventually the small quantity of Ca2+ that has entered the cell leaves predominantly through an Na+/Ca2+ exchanger, with a lesser role for the sarcolemmal Ca2+ pump. The varying actin-myosin overlap is shown for (B) systole, when [Ca2+] is maximal, and (C) diastole, when [Ca2+] is minimal. D. The myosin heads, attached to the thick filaments, interact with the thin actin filaments. (From LH Opie: Heart Physiology: From Cell to Circulation, 4th ed. Philadelphia, Lippincott, Williams & Wilkins, 2004. Reprinted with

1	to the thick filaments, interact with the thin actin filaments. (From LH Opie: Heart Physiology: From Cell to Circulation, 4th ed. Philadelphia, Lippincott, Williams & Wilkins, 2004. Reprinted with permission. Copyright LH Opie, 2004.) centrally located nucleus, numerous mitochondria, and the intracel-265e-5 lular membrane system, the sarcoplasmic reticulum.

1	The sarcomere, the structural and functional unit of contraction, lies between adjacent Z lines, which are dark repeating bands that are apparent on transmission electron microscopy. The distance between Z lines varies with the degree of contraction or stretch of the muscle and ranges between 1.6 and 2.2 μm. Within the confines of the sarcomere are alternating light and dark bands, giving the myocardial fibers their striated appearance under the light microscope. At the center of the sarcomere is a dark band of constant length (1.5 μm), the A band, which is flanked by two lighter bands, the I bands, which are of variable length. The sarcomere of heart muscle, like that of skeletal muscle, consists of two sets of interdigitating myofilaments. Thicker filaments, composed principally of the protein myosin, traverse the A band; theyare about 10 nm (100 Å) in diameter, with tapered ends. Thinner filaments, composed primarily of actin, course from the Z lines through the I band into the A

1	protein myosin, traverse the A band; theyare about 10 nm (100 Å) in diameter, with tapered ends. Thinner filaments, composed primarily of actin, course from the Z lines through the I band into the A band; they are approximately 5 nm (50Å) in diameter and 1.0 μm in length. Thus, thick and thin filaments overlap only within the (dark) A band, whereas the (light) I band contains only thin filaments. On electron-microscopic examination, bridges may be seen to extend between the thick and thin filaments within the A band; these are myosin heads (see below) bound to actin filaments.

1	The sliding filament model for muscle contraction rests on the fundamental observation that both the thick and the thin filaments are constant in overall length during both contraction and relaxation. With activation, the actin filaments are propelled farther into the A band. In the process, the A band remains constant in length, whereas the I band shortens and the Z lines move toward one another.

1	The myosin molecule is a complex, asymmetric fibrous protein with a molecular mass of about 500,000 Da; it has a rodlike portion that is about150 nm (1500 Å) in length with a globular portion (head) at its end. These globular portions of myosin form the bridges between the myosin and actin molecules and are the site of ATPase activity. In forming the thick myofilament, which is composed of ~300 longitudinally stacked myosin molecules, the rodlike segments of the myosin molecules are laid down in an orderly, polarized manner, leaving the globular portions projecting outward so that they can interact with actin to generate force and shortening (Fig. 265e-4B).

1	Actin has a molecular mass of about 47,000 Da. The thin filament consists of a double helix of two chains of actin molecules wound about each other on a larger molecule, tropomyosin. A group of regulatory proteins—troponins C, I, and T—are spaced at regular intervals on this filament (Fig. 265e-5). In contrast to myosin, actin lacks intrinsic enzymatic activity but does combine reversibly with myosin in the presence of ATP and Ca2+. The calcium ion activates the myosin ATPase, which in turn breaks down ATP, the energy source for contraction (Fig. 265e-5). The activity of myosin ATPase determines the rate of forming and breaking of the actomyosin cross-bridges and ultimately the velocity of muscle contraction. In relaxed muscle, tropomyosin inhibits this interaction. Titin (Fig. 265e-4D) is a large, flexible, myofibrillar protein that connects myosin to the Z line; its stretching contributes to the elasticity of the heart.

1	1. ATP hydrolysis 2.Formation of 4.Dissociation of 3.Product dissociation ADP Relaxed, energized Pi PART 10 Disorders of the Cardiovascular System

1	FIGURE 265e-5 Four steps in cardiac muscle contraction and relaxation. In relaxed muscle (upper left), ATP bound to the myosin cross-bridge dissociates the thick and thin filaments. Step 1: Hydrolysis of myosin-bound ATP by the ATPase site on the myosin head transfers the chemical energy of the nucleotide to the activated cross-bridge (upper right). When cytosolic Ca2+ concentration is low, as in relaxed muscle, the reaction cannot proceed because tropomyosin and the troponin complex on the thin filament do not allow the active sites on actin to interact with the cross-bridges. Therefore, even though the cross-bridges are energized, they cannot interact with actin. Step 2: When Ca2+ binding to troponin C has exposed active sites on the thin filament, actin interacts with the myosin cross-bridges to form an active complex (lower right) in which the energy derived from ATP is retained in the actin-bound cross-bridge, whose orientation has not yet shifted. Step 3: The muscle contracts

1	to form an active complex (lower right) in which the energy derived from ATP is retained in the actin-bound cross-bridge, whose orientation has not yet shifted. Step 3: The muscle contracts when ADP dissociates from the cross-bridge. This step leads to the formation of the low-energy rigor complex (lower left) in which the chemical energy derived from ATP hydrolysis has been expended to perform mechanical work (the “rowing” motion of the cross-bridge). Step 4: The muscle returns to its resting state, and the cycle ends when a new molecule of ATP binds to the rigor complex and dissociates the cross-bridge from the thin filament. This cycle continues until calcium is dissociated from troponin C in the thin filament, which causes the contractile proteins to return to the resting state with the cross-bridge in the energized state. ADP, adenosine diphosphate; ATP, adenosine triphosphate; ATPase, adenosine triphosphatase. (From AM Katz: Heart failure: Cardiac function and dysfunction, in

1	the cross-bridge in the energized state. ADP, adenosine diphosphate; ATP, adenosine triphosphate; ATPase, adenosine triphosphatase. (From AM Katz: Heart failure: Cardiac function and dysfunction, in Atlas of Heart Diseases, 3rd ed, WS Colucci [ed]. Philadelphia, Current Medicine, 2002. Reprinted with permission.)

1	Dystrophin is a long cytoskeletal protein that has an amino-terminal actin-binding domain and a carboxy-terminal domain that binds to the dystroglycan complex at adherens junctions on the cell membrane, thus tethering the sarcomere to the cell membrane at regions tightly coupled to adjacent contracting myocytes. Mutations in components of the dystrophin complex lead to muscular dystrophy and associated cardiomyopathy.

1	During activation of the cardiac myocyte, Ca2+ becomes attached to one of three components of the heterotrimer troponin C, which results in a conformational change in the regulatory protein tropomyosin; the latter, in turn, exposes the actin cross-bridge interaction sites (Fig. 265e-5). Repetitive interaction between myosin heads and actin filaments is termed cross-bridge cycling, which results in sliding of the actin along the myosin filaments, ultimately causing muscle shortening and/or the development of tension. The splitting of ATP then dissociates the myosin cross-bridge from actin. In the presence of ATP (Fig. 265e-5), linkages between actin and myosin filaments are made and broken cyclically as long as sufficient Ca2+ is present; these linkages cease when [Ca2+] falls below a critical level, and the troponintropomyosin complex once more prevents interactions between the myosin cross-bridges and actin filaments (Fig. 265e-6).

1	Intracytoplasmic Ca2+ is a principal determinant of the inotropic state of the heart. Most agents that stimulate myocardial contractility (positive inotropic stimuli), including the digitalis glycosides and β-adrenergic agonists, increase the [Ca2+] in the vicinity of the myofilaments, which in turn triggers cross-bridge cycling. Increased impulse traffic in the cardiac adrenergic nerves stimulates myocardial contractility as a consequence of the release of norepinephrine from cardiac adrenergic nerve endings. Norepinephrine activates myocardial β receptors and, through the Gs-stimulated guanine nucleotide-binding protein, activates the enzyme adenylyl cyclase, which leads to the formation of the intracellular second messenger cyclic AMP from ATP (Fig. 265e-6). Cyclic AMP in turn activates protein kinase A (PKA), which phosphorylates the Ca2+ channel in the myocardial sarcolemma, thereby enhancing the influx of Ca2+ into the myocyte. Other functions of PKA are discussed below.

1	The sarcoplasmic reticulum (SR) (Fig. 265e-7), a complex network of anastomosing intracellular channels, invests the myofibrils. Its longitudinally disposed tubules closely invest the surfaces of individual sarcomeres but have no direct continuity with the outside of the cell. However, closely related to the SR, both structurally and functionally, are the transverse tubules, or T system, formed by tubelike invaginations of the sarcolemma that extend into the myocardial fiber along the Z lines, i.e., the ends of the sarcomeres.

1	In the inactive state, the cardiac cell is electrically polarized; i.e., the interior has a negative charge relative to the outside of the cell, with a transmembrane potential of –80 to –100 mV (Chap. 273e). The sarcolemma, which in the resting state is largely impermeable to Na+, has a Na+and K+-stimulating pump energized by ATP that extrudes Na+ from the cell; this pump plays a critical role in establishing the resting potential. Thus, intracellular [K+] is relatively high and [Na+] is far lower; conversely, extracellular [Na+] is high and [K+] is low. At the same time, in the resting state, extracellular [Ca2+] greatly exceeds free intracellular [Ca2+].

1	The action potential has four phases (see Fig. 273e-1B). During the plateau of the action potential (phase 2), there is a slow inward current through L-type Ca2+ channels in the sarcolemma (Fig. 265e-7). The depolarizing current not only extends across the surface of the cell but penetrates deeply into the cell by way of the ramifying T tubular system. The absolute quantity of Ca2+ that crosses the sarcolemma and the T system is relatively small and by itself appears to be insufficient to bring about full activation of the contractile apparatus. However, this Ca2+ current triggers the release of much larger quantities of Ca2+ from the SR, a process termed Ca2+-induced Ca2+ release. The latter is a major determinant of intracytoplasmic [Ca2+] and therefore of myocardial contractility.

1	Ca2+ is released from the SR through a Ca2+ release channel, a cardiac isoform of the ryanodine receptor (RyR2), which controls intra-cytoplasmic [Ca2+] and, as in vascular smooth-muscle cells, leads to the local changes in intracellular [Ca2+] called calcium sparks. A number of regulatory proteins, including calstabin 2, inhibit RyR2 and thereby the release of Ca2+ from the SR. PKA dissociates calstabin from the RyR2, enhancing Ca2+ release and thereby myocardial contractility. Excessive plasma catecholamine levels and cardiac sympathetic neuronal release of norepinephrine cause hyperphosphorylation of PKA, leading to calstabin 2–depleted RyR2. The latter depletes SR Ca2+ stores and thereby impairs cardiac contraction, leading to heart failure, and also triggers ventricular arrhythmias.

1	1.rate of contraction ˜ 2. peak force 3.rate of relaxation there is an exchange of Ca2+ for Na+ at the sarcolemma (Fig. 265e-7), reducing the cytoplasmic [Ca2+]. Cyclic AMP–dependent PKA phosphorylates the SR protein phospholamban; the latter, in turn, permits activation of the Ca2+ pump, thereby increasing the uptake of Ca2+ by the SR, accelerating the rate of relaxation, and providing larger quantities of Ca2+ in the SR for release by subsequent depolarization, thereby stimulating contraction. Thus, the combination of the cell membrane, transverse tubules, and SR, with their ability to transmit the action potential and release and then reaccumulate Ca2+, plays a fundamental role in the rhythmic contraction and relaxation of heart muscle. Genetic or pharmacologic alterations of any component, whatever its etiology, can disturb these functions.

1	The extent of shortening of heart muscle and, therefore, the stroke volume of the ventricle in the intact heart depend on three major influences: (1) the length of the muscle at the onset of contraction, i.e., the preload; (2) the tension that the muscle is called on to develop during contraction, i.e., the afterload; and (3) the contractility of the muscle, i.e., the extent and velocity of shortening at any given preload and afterload. The major determinants of preload, afterload, and contractility are shown in Table 265e-2.

1	The preload determines the length of the sarcomeres at the onset of contraction. The length of the sarcomeres associated with the most forceful contraction is ~2.2 μm. This length provides the optimum configuration for the interaction between the two sets of myofilaments. The length of the sarcomere also regulates the extent of activation of the contractile system, i.e., its sensitivity to Ca2+. According to this concept, termed length-dependent activation, myofilament sensitivity to Ca2+ is also maximal at the optimal sarcomere length. The relation between the initial length of the muscle fibers and the developed force has prime importance for the function of heart muscle. This relationship forms the basis of Starling’s law of the heart, which states that within limits, the force of ventricular contraction depends on the end-diastolic length of the cardiac muscle; in the intact heart, the latter relates closely to the ventricular end-diastolic volume.

1	The ventricular end-diastolic or “filling” pressure sometimes is used as a surrogate for the end-diastolic volume. In isolated heart and heart-lung preparations, the stroke volume varies directly with the end-diastolic fiber length (preload) and inversely with the arterial resistance (afterload), and as the heart fails—i.e., as its contractility declines—it deliv ers a progressively smaller stroke volume from a normal or even elevated end-diastolic volume. The relation between the ventricular end-diastolic pressure and the stroke work of the ventricle (the ventricular function curve) provides a useful definition of the level of contractility of the heart in the intact organism. An increase in contractility is accompanied by a shift of the ventricular function curve upward and to the left (greater stroke work at any level of ventricular end-diastolic pressure, or lower end-diastolic volume at any level of stroke work), whereas a shift downward and to the right characterizes depression

1	(greater stroke work at any level of ventricular end-diastolic pressure, or lower end-diastolic volume at any level of stroke work), whereas a shift downward and to the right characterizes depression of contractility (Fig. 265e-8).

1	CHAPTER 265e Basic Biology of the Cardiovascular System Pattern of contraction

1	FIGURE 265e-6 Signal systems involved in positive inotropic and lusitropic (enhanced relaxation) effects of β-adrenergic stimulation. When the β-adrenergic agonist interacts with the β receptor, a series of G protein–mediated changes leads to activation of adenylyl cyclase and the formation of cyclic adenosine monophosphate (cAMP). The latter acts via protein kinase A to stimulate metabolism (left) and phosphorylate the Ca2+ channel protein (right). The result is an enhanced opening probability of the Ca2+ channel, thereby increasing the inward movement of Ca2+ ions through the sarcolemma (SL) of the T tubule. These Ca2+ ions release more calcium from the sarcoplasmic reticulum (SR) to increase cytosolic Ca2+ and activate troponin C. Ca2+ ions also increase the rate of breakdown of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and inorganic phosphate (Pi). Enhanced myosin ATPase activity explains the increased rate of contraction, with increased activation of troponin C

1	triphosphate (ATP) to adenosine diphosphate (ADP) and inorganic phosphate (Pi). Enhanced myosin ATPase activity explains the increased rate of contraction, with increased activation of troponin C explaining increased peak force development. An increased rate of relaxation results from the ability of cAMP to activate as well the protein phospholamban, situated on the membrane of the SR, that controls the rate of uptake of calcium into the SR. The latter effect explains enhanced relaxation (lusitropic effect). P, phosphorylation; PL, phospholamban; TnI, troponin I. (Modified from LH Opie: Heart Physiology: From Cell to Circulation, 4th ed. Philadelphia, Lippincott, Williams & Wilkins, 2004. Reprinted with permission. Copyright LH Opie, 2004.)

1	The Ca2+ released from the SR then diffuses toward the myofibrils, where, as already described, it combines with troponin C (Fig. 265e-6). By repressing this inhibitor of contraction, Ca2+ activates the myofilaments to shorten. During repolarization, the activity of the Ca2+ pump in the SR, the SR Ca2+ ATPase (SERCA2A), reaccumulates Ca2+ against a concentration gradient, and the Ca2+ is stored in the SR by its attachment to a protein, calsequestrin. This reaccumulation of Ca2+ is an energy (ATP)-requiring process that lowers the cytoplasmic [Ca2+] to a level that inhibits the actomyosin interaction responsible for contraction, and in this manner leads to myocardial relaxation. Also,

1	PART 10 Disorders of the Cardiovascular System 265e-8 Na+ Na+/Ca2+ Plasma membrane ventricle. Conversely, at the same aortic pressure pump exchanger Ca2+ pump and ventricular diastolic volume, the afterload on B1 B2 a hypertrophied ventricle is lower than of a normal chamber. The aortic pressure in turn depends on the peripheral vascular resistance, the physical characteristics of the arterial tree, and the volume Intracellular of blood it contains at the onset of ejection. Plasma membrane Cisterna (cytosol) Ventricular afterload critically regulates cardioCa2+ vascular performance (Fig. 265e-9). As already channel noted, elevations in both preload and contractil- Ca2+-ity increase myocardial fiber shortening, whereas increases in afterload reduce it. The extent of determine stroke volume. An increase in arterial G pressure induced by vasoconstriction, for example, Calsequestrin augments afterload, which opposes myocardial Mitochondria fiber shortening, reducing stroke volume.

1	G pressure induced by vasoconstriction, for example, Calsequestrin augments afterload, which opposes myocardial Mitochondria fiber shortening, reducing stroke volume. When myocardial contractility becomes CD impaired and the ventricle dilates, afterload rises (Laplace’s law) and limits cardiac output. Increased afterload also may result from neural and humoral stimuli that occur in response to a fall in cardiac output. This increased afterload may reduce cardiac output further, thereby increasing ventricular EF volume and initiating a vicious circle, especially in patients with ischemic heart disease and limited myocardial O2 supply. Treatment with vasodilators has the opposite effect; when afterload is reduced, cardiac output rises (Chap. 279). Under normal circumstances, the various influ above interact in a complex fashion to maintain

1	FIGURE 265e-7 The Ca2+ fluxes and key structures involved in cardiac excitation- cardiac output at a level appropriate to the require- contraction coupling. The arrows denote the direction of Ca2+ fluxes. The thickness ments of the metabolizing tissues (Fig. 265e-9); of each arrow indicates the magnitude of the calcium flux. Two Ca2+ cycles regulate interference with a single mechanism may not excitation-contraction coupling and relaxation. The larger cycle is entirely intracellular influence the cardiac output. For example, a mod- and involves Ca2+ fluxes into and out of the sarcoplasmic reticulum, as well as Ca2+ erate reduction of blood volume or the loss of the binding to and release from troponin C. The smaller extracellular Ca2+ cycle occurs atrial contribution to ventricular contraction ordi when this cation moves into and out of the cell. The action potential opens plasma membrane Ca2+ channels to allow passive entry of Ca2+ into the cell from the extra- cardiac output at

1	ordi when this cation moves into and out of the cell. The action potential opens plasma membrane Ca2+ channels to allow passive entry of Ca2+ into the cell from the extra- cardiac output at rest. Under these circumstances, cellular fluid (arrow A). Only a small portion of the Ca2+ that enters the cell directly other factors, such as increases in the frequency activates the contractile proteins (arrow A ). The extracellular cycle is completed when 1 of adrenergic nerve impulses to the heart, heart

1	Ca2+ is actively transported back out to the extracellular fluid by way of two plasma rate, and venous tone, will serve as compensatory membrane fluxes mediated by the sodium-calcium exchanger (arrow B ) and the ). In the intracellular Ca2+ cycle, passive 2 individual. Ca2+ release occurs through channels in the cisternae (arrow C) and initiates contraction; active Ca2+ uptake by the Ca2+ pump of the sarcotubular network (arrow D) relaxes the heart. Diffusion of Ca2+ within the sarcoplasmic reticulum (arrow G) returns

1	The integrated response to exercise illustrates this activator cation to the cisternae, where it is stored in a complex with calseques the interactions among the three determinants of trin and other calcium-binding proteins. Ca2+ released from the sarcoplasmic stroke volume: preload, afterload, and contractil reticulum initiates systole when it binds to troponin C (arrow E). Lowering of cytosolic ity (Fig. 265e-8). Hyperventilation, the pumping [Ca2+] by the sarcoplasmic reticulum (SR) causes this ion to dissociate from action of the exercising muscles, and venocon troponin (arrow F) and relaxes the heart. Ca2+ also may move between mitochondria and cytoplasm (H). (Adapted from AM Katz: Physiology of the Heart, 4th ed. Philadelphia, Lippincott, Williams & Wilkins, 2005, with permission.) (Table 265e-2). Simultaneously, the increase in the adrenergic nerve impulse traffic to the myocardium, the increased concentration of circu-

1	VENTRICULAR AFTERLOAD lating catecholamines, and the tachycardia that In the intact heart, as in isolated cardiac muscle, the extent and veloc-occur during exercise combine to augment the contractility of the ity of shortening of ventricular muscle fibers at any level of preload myocardium (Fig. 265e-8, curves 1 and 2) and together elevate and of myocardial contractility relate inversely to the afterload, i.e., stroke volume and stroke work, without a change in or even a reducthe load that opposes shortening. In the intact heart, the afterload tion of end-diastolic pressure and volume (Fig. 265e-8, points A may be defined as the tension developed in the ventricular wall dur-and B). Vasodilation occurs in the exercising muscles, thus tending ing ejection. Afterload is determined by the aortic pressure as well as to limit the increase in arterial pressure that otherwise would occur by the volume and thickness of the ventricular cavity. Laplace’s law as cardiac output rises to levels as

1	pressure as well as to limit the increase in arterial pressure that otherwise would occur by the volume and thickness of the ventricular cavity. Laplace’s law as cardiac output rises to levels as high as five times greater than states that the tension of the myocardial fiber is the product of the basal levels during maximal exercise. This vasodilation ultimately intracavitary ventricular pressure and ventricular radius divided by allows the achievement of a greatly elevated cardiac output during wall thickness. Therefore, at any particular level of aortic pressure, exercise at an arterial pressure only moderately higher than in the the afterload on a dilated left ventricle exceeds that on a normal-sized resting state.

1	I. Ventricular Preload A. Blood volume B. Distribution of blood volume 1. 2. 3. 4. 5. Pumping action of skeletal muscles C. Atrial contraction II. Ventricular Afterload A. Systemic vascular resistance B. Elasticity of arterial tree C. Arterial blood volume D. Ventricular wall tension 1. 2. III. Myocardial Contractilitya A. Intramyocardial [Ca2+] ↑↓ B. Cardiac adrenergic nerve activity ↑↓b C. Circulating catecholamines ↑↓b D. Cardiac rate ↑↓b E. Exogenous inotropic agents ↑ F. Myocardial ischemia ↓ G. Myocardial cell death (necrosis, apoptosis, autophagy) ↓ H. Alterations of sarcomeric and cytoskeletal proteins ↓ 1. 2. I. Myocardial fibrosis ↓ J. Chronic overexpression of neurohormones ↓ K. Ventricular remodeling ↓ L. Chronic and/or excessive myocardial hypertrophy ↓ aArrows indicate directional effects of determinants of contractility. bContractility rises initially but later becomes depressed.

1	L. Chronic and/or excessive myocardial hypertrophy ↓ aArrows indicate directional effects of determinants of contractility. bContractility rises initially but later becomes depressed. Several techniques can define impaired cardiac function in clinical practice. The cardiac output and stroke volume may be depressed in the presence of heart failure, but not uncommonly, these variables are within normal limits in this condition. A somewhat more sensitive index of cardiac function is the ejection fraction, i.e., the ratio of stroke volume to end-diastolic volume (normal value = 67 ± 8%), which is frequently depressed in systolic heart failure even when the stroke volume itself is normal. Alternatively, abnormally elevated ventricular end-diastolic volume (normal value = 75 ± 20 mL/m2) or end-systolic volume (normal value = 25 ± 7 mL/m2) signifies impairment of left ventricular systolic function.

1	Noninvasive techniques, particularly echocardiography as well as radionuclide scintigraphy and cardiac magnetic resonance imaging (MRI) (Chap. 270e), have great value in the clinical assessment of myocardial function. They provide measurements of end-diastolic and end-systolic volumes, ejection fraction, and systolic shortening rate, and they allow assessment of ventricular filling (see below) as well as regional contraction and relaxation. The latter measurements are particularly important in ischemic heart disease, as myocardial infarction causes regional myocardial damage.

1	A limitation of measurements of cardiac output, ejection fraction, and ventricular volumes in assessing cardiac function is that ventricular loading conditions strongly influence these variables. Thus, a depressed ejection fraction and lowered cardiac output may occur in patients with normal ventricular function but reduced preload, as occurs in hypovolemia, or with increased afterload, as occurs in acutely elevated arterial pressure. Stretching of myocardium

1	Stretching of myocardium FIGURE 265e-8 The interrelations among influences on ventricular end-diastolic volume (EDV) through stretching of the myocardium and the contractile state of the myocardium. Levels of ventricular EDV associated with filling pressures that result in dyspnea and pulmonary edema are shown on the abscissa. Levels of ventricular performance required when the subject is at rest, while walking, and during maximal activity are designated on the ordinate. The broken lines are the descending limbs of the ventricular-performance curves, which are rarely seen during life but show the level of ventricular performance if end-diastolic volume could be elevated to very high levels. For further explanation, see text. (Modified from WS Colucci and E Braunwald: Pathophysiology of heart failure, in Braunwald’s Heart Disease, 7th ed, DP Zipes et al [eds]. Philadelphia: Elsevier, 2005, pp 509–538.) CHAPTER 265e Basic Biology of the Cardiovascular System

1	CHAPTER 265e Basic Biology of the Cardiovascular System FIGURE 265e-9 Interactions in the intact circulation of preload, contractility, and afterload in producing stroke volume. Stroke volume combined with heart rate determines cardiac output, which, when combined with peripheral vascular resistance, determines arterial pressure for tissue perfusion. The characteristics of the arterial system also contribute to afterload, an increase that reduces stroke volume. The interaction of these components with carotid and aortic arch baroreceptors provides a feedback mechanism to higher medullary and vasomotor cardiac centers and to higher levels in the central nervous system to effect a modulating influence on heart rate, peripheral vascular resistance, venous return, and contractility.

1	(From MR Starling: Physiology of myocardial contraction, in Atlas of Heart Failure: Cardiac Function and Dysfunction, 3rd ed, WS Colucci and E Braunwald [eds]. Philadelphia: Current Medicine, 2002, pp 19–35.) 265e-10 Normal as from the cell’s breakdown of its glycogen stores Disorders of the Cardiovascular System

1	Disorders of the Cardiovascular System FIGURE 265e-10 The responses of the left ventricle to increased afterload, (glycogenolysis). These two principal sources of acetyl coenzyme A in cardiac muscle vary reciprocally. Glucose is broken down in the cytoplasm into a three-carbon product, pyruvate, which passes into the mitochondria, where it is metabolized to the two-carbon fragment, acetyl-CoA, and undergoes oxidation. FFAs are converted to acyl-CoA in the cytoplasm and acetyl-CoA in the mitochondria. Acetyl-CoA enters the citric acid (Krebs) cycle to produce ATP by oxidative phosphorylation within the mitochondria; ATP then enters the cytoplasm from the mitochondrial compartment. Intracellular ADP, resulting from the breakdown of ATP, enhances mitochondrial ATP production.

1	In the fasted, resting state, circulating FFA concentrations and their myocardial uptake are high, and they furnish most of the heart’s acetyl-CoA (~70%). In the fed state, with elevations of blood increased preload, and increased and reduced contractility are shown in the glucose and insulin, glucose oxidation increases and pressure-volume plane. Left. Effects of increases in preload and afterload on the

1	FFA oxidation subsides. Increased cardiac work, the pressure-volume loop. Because there has been no change in contractility, the administration of inotropic agents, hypoxia, and mild end-systolic pressure-volume relationship (ESPVR) is unchanged. With an increase in ischemia all enhance myocardial glucose uptake, glu afterload, stroke volume falls (1 → 2); with an increase in preload, stroke volume rises cose production resulting from glycogenolysis, and (1 → 3). Right. With increased myocardial contractility and constant left ventricular glucose metabolism to pyruvate (glycolysis). By con end-diastolic volume, the ESPVR moves to the left of the normal line (lower end trast, β-adrenergic stimulation, as occurs during stress, systolic volume at any end-systolic pressure) and stroke volume rises (1 → 3). With raises the circulating levels and metabolism of FFAs reduced myocardial contractility, the ESPVR moves to the right; end-systolic volume is in favor of glucose. Severe ischemia

1	rises (1 → 3). With raises the circulating levels and metabolism of FFAs reduced myocardial contractility, the ESPVR moves to the right; end-systolic volume is in favor of glucose. Severe ischemia inhibits the cyto increased, and stroke volume falls (1 → 2).

1	plasmic enzyme pyruvate dehydrogenase, and despite The end-systolic left ventricular pressure-volume relationship is a both glycogen and glucose breakdown, glucose is metabolized only to lactic acid (anaerobic glycoly particularly useful index of ventricular performance because it does sis), which does not enter the citric acid cycle. Anaerobic glycolysis not depend on preload and afterload (Fig. 265e-10). At any level of produces much less ATP than does aerobic glucose metabolism, in myocardial contractility, left ventricular end-systolic volume varies which glucose is metabolized to pyruvate and subsequently oxidized to inversely with end-systolic pressure; as contractility declines, endCO2. High concentrations of circulating FFAs, which can occur when adrenergic stimulation is superimposed on severe ischemia, reduce systolic volume (at any level of end-systolic pressure) rises.

1	Ventricular filling is influenced by the extent and speed of myocardial relaxation, which in turn depends on the rate of uptake of Ca2+ by the SR; the latter may be enhanced by adrenergic activation and reduced by ischemia, which reduces the ATP available for pumping Ca2+ into the SR (see above). The stiffness of the ventricular wall also may impede filling. Ventricular stiffness increases with hypertrophy and conditions that infiltrate the ventricle, such as amyloid, or is caused by an extrinsic constraint (e.g., pericardial compression) (Fig. 265e-11).

1	Ventricular filling can be assessed by continuously measuring the velocity of flow across the mitral valve using Doppler ultrasound. Normally, the velocity of inflow is more rapid in early diastole than during atrial systole; with mild to moderately impaired relaxation, the rate of early diastolic filling declines, whereas the rate of presystolic filling rises. With further impairment of filling, the pattern is “pseudonormalized,” and early ventricular filling becomes more rapid as left atrial pressure upstream to the stiff left ventricle rises.

1	The heart requires a continuous supply of energy (in the form of ATP) not only to perform its mechanical pumping functions, but also to regulate intracellular and transsarcolemmal ionic movements and concentration gradients. Among its pumping functions, the development of tension, the frequency of contraction, and the level of myocardial oxidative phosphorylation and also cause ATP wastage; the myocardial content of ATP declines and impairs myocardial contraction. In addition, products of FFA breakdown can exert toxic effects on cardiac cell membranes and may be arrhythmogenic. contractility are the principal determinants of the heart’s substantial FIGURE 265e-11 Mechanisms that cause diastolic dysfunction energy needs, making its O2 requirements approximately 15% of that reflected in the pressure-volume relation. The bottom half of the of the entire organism. pressure-volume loop is depicted. Solid lines represent normal sub-

1	Most ATP production depends on the oxidation of substrate (glu-jects; broken lines represent patients with diastolic dysfunction. (From cose and free fatty acids [FFAs]). Myocardial FFAs are derived from JD Carroll et al: The differential effects of positive inotropic and vasodilator circulating FFAs, which result principally from lipolysis in adipose therapy on diastolic properties in patients with congestive cardiomyopatissue, whereas the myocyte’s glucose derives from plasma as well thy. Circulation 74:815, 1986; with permission.) 265e-11 development, such as NKX2-5 and GATA4. Mutations in these genes are responsible for some forms of inherited congenital heart disease. Cardiac precursors coalesce to form a midline heart tube composed of a single cell layer of endocardium surrounded by a single layer of myocardial precursors. The caudal, inflow region of the heart tube, which is destined to adopt a more rostral final position, represents the atrial anlagen, whereas the rostral,

1	by a single layer of myocardial precursors. The caudal, inflow region of the heart tube, which is destined to adopt a more rostral final position, represents the atrial anlagen, whereas the rostral, outflow portion of the tube forms the truncus arteriosus, which divides to produce the aorta and the proximal pulmonary artery. Between these extremes lie the structural precursors of the ventricles. The linear heart tube undergoes an asymmetric looping process (the first gross evidence of left-right asymmetry in the developing embryo), which positions the portion of the heart tube destined to become the left ventricle to the left of the more rostral precursors of the right ven-tricle and outflow tract. Looping is coordinated with chamber specifi-cation and ballooning of various regions of the heart tube to produce the presumptive atria and ventricles. Relatively recent work has demonstrated that significant portions of the right ventricle are formed by cells that are added to the

1	of the heart tube to produce the presumptive atria and ventricles. Relatively recent work has demonstrated that significant portions of the right ventricle are formed by cells that are added to the developing heart after looping has occurred. These cells, which are derived from what is called the second heart field, migrate to the heart from the ventral pharynx and express markers that allow for their identification, including Islet-1. Different embryologic origins of cells within the right and left ventricles may help explain why some forms of congenital and adult heart diseases affect these regions of the heart to varying degrees. After looping and chamber formation, a series of septation events divide the left and right sides of the heart, separate the atria from the ventricles, and form the aorta and pulmonary artery from the truncus arteriosus. Cardiac valves form between the atria and the ventricles and between the ventricles and the out-flow vessels. Early in development, the

1	the aorta and pulmonary artery from the truncus arteriosus. Cardiac valves form between the atria and the ventricles and between the ventricles and the out-flow vessels. Early in development, the single layer of myocardial cells secretes an extracellular matrix rich in hyaluronic acid. This extracellular matrix, termed “cardiac jelly,” accumulates within the endocardial cushions, precursors of the cardiac valves. Signals from overlying myocardial cells, including members of the transforming growth factor β family, trigger migration, invasion, and pheno-typic changes of underlying endocar-dial cells, which undergo an epithelial-mesenchymal transformation and invade the cardiac jelly to cellularize the endocardial cushions. Mesenchymal components proliferate and remodel to form the mature valve leaflets. The great vessels form as a series of bilaterally symmetric aortic arch arteries that undergo asymmetric remodel-ing events to form the mature vascula-ture. The immigration of neural

1	valve leaflets. The great vessels form as a series of bilaterally symmetric aortic arch arteries that undergo asymmetric remodel-ing events to form the mature vascula-ture. The immigration of neural crest regions Second heart field RA RV RV LVLV LA First heart field coelom Forming heart ABCDE

1	Myocardial energy is stored as creatine phosphate (CP), which is in equilibrium with ATP, the immediate source of energy. In states of reduced energy availability, the CP stores decline first. Cardiac hypertrophy, fibrosis, tachycardia, increased wall tension resulting from ventricular dilation, and increased intracytoplasmic [Ca2+] all contribute to increased myocardial energy needs. When coupled with reduced coronary flow reserve, as occurs with obstruction of coronary arteries or abnormalities of the coronary microcirculation, an imbalance in myocardial ATP production relative to demand may occur, and the resulting ischemia can worsen or cause heart failure.

1	Developmental Biology of the Cardiovascular System The heart is the first organ to form during embryogenesis (Fig. 265e-12) and must accomplish the simultaneous challenges of circulating blood, nutrients, and oxygen to the other forming organs while continuing to grow and undergo complex morphogenetic changes. Early progenitors of the heart arise within very early crescent-shaped fields of lateral splanchnic mesoderm under the influence of multiple signals, including those derived from neural ectoderm long before neural tube closure. Early cardiac precursors express genes encoding regulatory transcription factors that play reiterated roles in cardiac CHAPTER 265e Basic Biology of the Cardiovascular System

1	FIGURE 265e-12 A. Schematic depiction of a transverse section through an early embryo depicts cells that arise in the dorsal neural tube the bilateral regions where early heart tubes form. B. The bilateral heart tubes subsequently orchestrates this process. These cells are migrate to the midline and fuse to form the linear heart tube. C. At the early cardiac crescent stage required for aortic arch remodeling and of embryonic development, cardiac precursors include a primary heart field fated to form the linear septation of the truncus arteriosus. They heart tube and a second heart field fated to add myocardium to the inflow and outflow poles of develop into smooth-muscle cells within the heart. D. Second heart field cells populate the pharyngeal region before subsequently migrat-the tunica media of the aortic arch, the ing to the maturing heart. E. Large portions of the right ventricle and outflow tract and some cells ductus arteriosus, and the carotid arterwithin the atria derive

1	media of the aortic arch, the ing to the maturing heart. E. Large portions of the right ventricle and outflow tract and some cells ductus arteriosus, and the carotid arterwithin the atria derive from the second heart field. F. The aortic arch arteries form as symmetric ies. Smooth-muscle cells within the sets of vessels that then remodel under the influence of the neural crest to form the asymmetric descending aorta arise from a differ-mature vasculature. LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. ent embryologic source, the lateral plate 265e-12 mesoderm, and smooth muscle of the proximal outflow tract arises from the second heart field. Neural crest cells are sensitive to both vitamin A and folic acid, and congenital heart disease involving abnormal remodeling of the aortic arch arteries has been associated with maternal deficiencies of these vitamins. Congenital heart disease involving the outflow tract can be associated with other defects of neural

1	of the aortic arch arteries has been associated with maternal deficiencies of these vitamins. Congenital heart disease involving the outflow tract can be associated with other defects of neural crest, such as cleft palate or craniofacial abnormalities. Coronary artery formation requires yet another cell population that initiates extrinsic to the embryonic heart fields. Epicardial cells arise in the proepicardial organ, a derivative of the septum transversum, which also contributes to the fibrous portion of the diaphragm and to the liver. Proepicardial cells contribute to the smooth-muscle cells of the coronary arteries and are required for their proper patterning. Other cell types within the heart, including fibroblasts and potentially some myocardial and endocardial cells, also can arise from the proepicardium. The cardiac conduction system, which functions both to generate and to propagate electrical impulses, develops primarily from multi-potential cardiac precursors, which also

1	from the proepicardium. The cardiac conduction system, which functions both to generate and to propagate electrical impulses, develops primarily from multi-potential cardiac precursors, which also give rise to cardiac muscle. The conduction system is composed of slow-conducting (proximal) components, such as the sinoatrial (SA) and atrioventricular (AV) nodes, as well as fast-conducting (distal) components, including the His bundle, bundle branches, and Purkinje fibers. The AV node primarily serves to delay the electrical impulse between atria and ventricles (manifesting decremental conduction), whereas the distal conduction system rapidly delivers the impulse throughout the ventricles. Significant recent attention has been focused on the embryo-logic origins of various components of the specialized conduction

1	PART 10 Disorders of the Cardiovascular System network. Precursors within the sinus venosus give rise to the SA node, whereas those within the AV canal mature into heterogeneous cell types that compose the AV node. Myocardial cells transdifferentiate into Purkinje fibers to form the distal conduction system. Fast and slow conducting cell types within the nodes and bundles are characterized by expression of distinct gap junction proteins, including connexins, and ion channels that characterize unique cell fates and electrical properties of the tissues. Developmental defects in conduction system morphogenesis and lineage determination can lead to various electrophysiologic disorders, including congenital heart block and preexcitation syndromes such as the Wolff-Parkinson-White syndrome (Chap. 276).

1	Studies of cardiac stem and progenitor cells suggest that progressive lineage restriction results in the gradual and stepwise determination of mature cell fates within the heart, with early precursors capable of adopting endothelial, smooth-muscle, or cardiac phenotypes, and subsequent further specialization into atrial, ventricular, and specialized conduction cell types. Until very recently, adult mammalian myocardial cells were viewed as fully differentiated and without regenerative potential. Evidence currently supports the existence of limited regenerative potential of the mature heart. Considerable current effort is being devoted to evaluating the utility of various putative stem cell populations and regenerative approaches to enhance cardiac repair after injury. The success of such approaches would offer the exciting possibility of reconstructing an infarcted or failing ventricle (Chaps. 88 and 90e).

1	Epidemiology of Cardiovascular Disease Thomas A. Gaziano, J. Michael Gaziano Cardiovascular disease (CVD) is now the most common cause of death worldwide. Before 1900, infectious diseases and malnutrition were the 266e Stage Description Deaths Related to CVD, % Predominant CVD Type most common causes, and CVD was responsible for less than 10% of all deaths. In 2010, CVD accounted for approximately 16 million deaths worldwide (30%), including nearly 40% of deaths in high-income countries and about 28% in lowand middle-income countries.

1	The global rise in CVD is the result of an unprecedented transformation in the causes of morbidity and mortality dur ing the twentieth century. Known as the epidemiologic transition, this shift is driven by industrialization, urbanization, and associated lifestyle changes and is taking place in every part of the world among all races, ethnic groups, and cultures. The transition is divided into four basic stages: pestilence and famine, receding pandemics, degenerative and man-made diseases, and delayed degenerative diseases. A fifth stage, characterized by an epidemic of inactivity and obesity, is emerging in some countries (Table 266e-1).

1	The age of pestilence and famine is marked by malnutrition, infectious diseases, and high infant and child mortality that are offset by high fertility. Tuberculosis, dysentery, cholera, and influenza are often fatal, resulting in a mean life expectancy of about 30 years. CVD, which accounts for less than 10% of deaths, takes the form of rheumatic heart disease and cardiomyopathies due to infection and malnutrition. Approximately 10% of the world’s population remains in the age of pestilence and famine.

1	Per capita income and life expectancy increase during the age of receding pandemics as the emergence of public health systems, cleaner water supplies, and improved nutrition combine to drive down deaths from infectious disease and malnutrition. Infant and childhood mortality also decline, but deaths due to CVD increase to between 10 and 35% of all deaths. Rheumatic valvular disease, hypertension, coronary heart disease (CHD), and stroke are the predominant forms of CVD. Almost 40% of the world’s population is currently in this stage.

1	The age of degenerative and man-made diseases is distinguished 266e-1 by mortality from noncommunicable diseases—primarily CVD— surpassing mortality from malnutrition and infectious diseases. Caloric intake, particularly from animal fat, increases. CHD and stroke are prevalent, and between 35 and 65% of all deaths can be traced to CVD. Typically, the rate of CHD deaths exceeds that of stroke by a ratio of 2:1 to 3:1. During this period, average life expectancy surpasses the age of 50. Roughly 35% of the world’s population falls into this category.

1	In the age of delayed degenerative diseases, CVD and cancer remain the major causes of morbidity and mortality, with CVD accounting for 40% of all deaths. However, age-adjusted CVD mortality declines, aided by preventive strategies (for example, smoking cessation programs and effective blood pressure control), acute hospital management, and technologic advances, such as the availability of bypass surgery. CHD, stroke, and congestive heart failure are the primary forms of CVD. About 15% of the world’s population is now in the age of delayed degenerative diseases or is exiting this age and moving into the fifth stage of the epidemiologic transition.

1	In the industrialized world, physical activity continues to decline while total caloric intake increases. The resulting epidemic of overweight and obesity may signal the start of the age of inactivity and obesity. Rates of type 2 diabetes mellitus, hypertension, and lipid abnormalities are on the rise, trends that are particularly evident in children. If these risk factor trends continue, age-adjusted CVD mortality rates could increase in the coming years.

1	Unique regional features have modified aspects of the transition in various parts of the world. High-income countries experienced declines in CVD death rates by as much as 50–60% over the last 60 years, whereas CVD death rates increased by 15% over the past 20 years in the lowand middle-income range. However, given the large amount of available data, the United States serves as a useful reference point for comparisons. The age of pestilence and famine occurred before 1900, with a largely agrarian economy and population. Infectious diseases accounted for more deaths than any other cause. By the 1930s, the country proceeded through the age of receding pandemics. The establishment of public health infrastructures resulted in dramatic declines in infectious disease mortality rates. Lifestyle changes due to rapid urbanization resulted in a simultaneous increase in CVD mortality rates, reaching approximately 390 per 100,000. Between 1930 and 1965, the country entered the age of degenerative

1	CHAPTER 266e Epidemiology of Cardiovascular Disease Pestilence and famine Predominance of malnutrition and infectious diseases <10 as causes of death; high rates of infant and child mortality; low mean life expectancy Receding pandemics Improvements in nutrition and public health lead to 10–35 decrease in rates of deaths related to malnutrition and infection; precipitous decline in infant and child mortality rates man-made diseases cal activity lead to emergence of hypertension and atherosclerosis; with increase in life expectancy, mortality from chronic, noncommunicable diseases exceeds mortality from malnutrition and infectious disease Delayed degenerative CVD and cancer are the major causes of morbidity and 40–50 diseases mortality; better treatment and prevention efforts help avoid deaths among those with disease and delay primary events; age-adjusted CVD morality declines; CVD affecting older and older individuals

1	Inactivity and obesity Overweight and obesity increase at alarming rate; 33 diabetes and hypertension increase; decline in smoking rates levels off; a minority of the population meets physical activity recommendations Rheumatic heart disease, cardiomyopathies caused by infection and malnutrition Rheumatic valvular disease, hypertension, CHD, and stroke (predominantly hemorrhagic) CHD, stroke, and congestive heart failure CHD, stroke, and congestive heart failure, peripheral vascular disease Abbreviations: CHD, coronary heart disease; CVD, cardiovascular disease. Source: Adapted from AR Omran: The epidemiologic transition: A theory of the epidemiology of population change. Milbank Mem Fund Q 49:509, 1971; and SJ Olshansky, AB Ault: The fourth stage of the epidemiologic transition: The age of delayed degenerative diseases. Milbank Q 64:355, 1986.

1	PART 10 Disorders of the Cardiovascular System 266e-2 and man-made diseases. Infectious disease mortality rates fell to fewer than 50 per 100,000 per year, whereas CVD mortality rates reached peak levels with increasing urbanization and lifestyle changes in diet, physical activity, and tobacco consumption. The age of delayed degenerative diseases took place between 1965 and 2000. New therapeutic approaches, preventive measures, and exposure to public health campaigns promoting lifestyle modifications led to substantial declines in age-adjusted mortality rates and a steadily rising age at which a first CVD event occurs. Currently, the United States is entering what appears to be a fifth phase. The decline in the age-adjusted CVD death rate of 3% per year through the 1970s and 1980s has tapered off in the 1990s to 2%. However, CVD death rates have declined by 3–5% per year during the first decade of the new millennium. Competing trends appear to be at play. One the one hand, an increase

1	off in the 1990s to 2%. However, CVD death rates have declined by 3–5% per year during the first decade of the new millennium. Competing trends appear to be at play. One the one hand, an increase in the prevalence of diabetes and obesity, a slowing in the rate of decline in smoking, and a leveling off in the rate of detection and treatment for hypertension are in the negative column. On the other hand, cholesterol levels continue to decline in the face of increased statin use. Many high-income countries (HICs)—which together account for 15% of the population—have proceeded through four stages of the epidemiologic transition in roughly the same pattern as the United States. CHD is the dominant form of CVD in these countries, with rates that tend to be twoto five-fold higher than stroke rates. However, variations exist. Whereas North America, Australia, and central northwestern European HICs experienced significant increases then rapid declines in CVD rates, southern and central

1	rates. However, variations exist. Whereas North America, Australia, and central northwestern European HICs experienced significant increases then rapid declines in CVD rates, southern and central European countries experienced a more gradual rise and fall in rates. More specifically, central European countries (i.e., Austria, Belgium, and Germany) declined at slower rates compared to their northern counterparts (i.e., Finland, Sweden, Denmark, and Norway). Countries such as Portugal, Spain, and Japan never reached the high mortality rates that the United States and other countries did, with CHD mortality rates at 200 per 100,000, or less. The countries of Western Europe also exhibit a clear north/south gradient in absolute rates of CVD, with rates highest in northern countries (i.e., Finland, Ireland, and Scotland) and lowest in Mediterranean countries (i.e., France, Spain, and Italy). Japan is unique among the HICs, most likely due to the unique dietary patterns of its population.

1	Ireland, and Scotland) and lowest in Mediterranean countries (i.e., France, Spain, and Italy). Japan is unique among the HICs, most likely due to the unique dietary patterns of its population. Although stroke rates increased dramatically, CHD rates did not rise as sharply in Japan. However, Japanese dietary habits are undergoing substantial changes, reflected in an increase in cholesterol levels.

1	Global deaths by cause, 2010 Patterns in lowand middle-income countries (LMICs; gross national income per capita less than U.S. $12,615) depend, in part, on cultural differences, secular trends, and responses at the country level, with regard to both public health and treatment infrastructure. Although communicable diseases continue to be a major cause of death, CVD has emerged as a significant health concern in LMICs. With 85% of the world’s population, LMICs are driving the rates of change in the global burden of CVD (Fig. 266e-1). In most LMICs, an urban/rural gradient has emerged for CHD, stroke, and hypertension, with higher rates in urban centers.

1	However, although CVD rates are rapidly rising, vast differences exist among the regions and countries, and even within the countries themselves (Fig. 266e-2). The East Asia and Pacific regions appear to be straddling the second and third phases of the epidemiologic transition. CVD is a major cause of death in China, but like Japan, stroke causes more deaths than CHD in a ratio of about three to one. Vietnam and Cambodia, on the other hand, are just emerging from the pestilence and famine transition. The Middle East and North Africa regions also appear to be entering the third phase of the epidemiologic transition, with increasing life expectancy and CVD death rates just below those of HICs. In general, Latin America appears to be in the third phase of the transition, although there is vast regional heterogeneity with some areas in the second phase of the transition and some in the fourth. The Eastern Europe and Central Asia regions, however, are firmly in the peak of the third phase,

1	regional heterogeneity with some areas in the second phase of the transition and some in the fourth. The Eastern Europe and Central Asia regions, however, are firmly in the peak of the third phase, with the highest death rates due to CVD (~66%) in the world. Importantly, deaths due to CHD are not limited to the elderly in this region and have a significant effect on working-age populations. South Asia—and more specifically, India, which accounts for the greatest proportion of the region’s population—is experiencing an alarming increase in heart disease. The transition appears to be in the Western style, with CHD as the dominant form of CVD. However, rheumatic heart disease continues to be a major cause of morbidity and mortality. As in South Asia, rheumatic heart disease is also an important cause of CVD morbidity and mortality in sub-Saharan Africa, which largely remains in the first phase of the epidemiologic transition.

1	Many factors contribute to this heterogeneity among LMICs. First, the regions are in various stages of the epidemiologic transition. Second, vast differences in lifestyle and behavioral risk factors exist. Third, racial and ethnic differences may lead to altered susceptibilities to various forms of CVD. In addition, it should be noted that for most countries in these regions, accurate country-wide data on cause-specific mortality are not complete. FIGuRE 266-1 Global deaths by cause, 2010. CMNN, communicable, maternal, neonatal, and nutritional disorders; CVD, cardiovascular diseases; INJ, injuries; ONC, other noncommunicable diseases. (Based on data from Global Burden of Disease Study 2010: Global Burden of Disease Study 2010 Mortality Results 1970–2010. Seattle, Institute for Health Metrics and Evaluation, 2012.) Latin American and the Caribbean Middle East and North Africa Europe and Central Asia 266e-3 28.8% 42.3% 58.2% (601 million) (422 million) (404 million)

1	Latin American and the Caribbean Middle East and North Africa Europe and Central Asia 266e-3 28.8% 42.3% 58.2% (601 million) (422 million) (404 million) High-income 35.8% (970 million) South Asia 20.4% (1,609 million) Sub-Saharan Africa East Asia and Pacific 8.8% 35.7%(823 million) (1,991 million) FIGuRE 266-2 Cardiovascular disease deaths as a percentage of total deaths and total population in seven economic regions of the world defined by the World Bank.(Based on data from Global Burden of Disease Study 2010: Global Burden of Disease Study 2010 Mortality Results CHAPTER 266e Epidemiology of Cardiovascular Disease 1970–2010. Seattle, Institute for Health Metrics and Evaluation, 2012.)

1	CHAPTER 266e Epidemiology of Cardiovascular Disease 1970–2010. Seattle, Institute for Health Metrics and Evaluation, 2012.) CVD accounts for nearly 30% of deaths worldwide, a number that is expected to increase. In 2010, CHD accounted for 13.3% of all deaths globally and the largest portion of global years of life lost (YLLs) and disability-adjusted life-years (DALYs). The second largest cause of death was stroke (11.1% of all deaths), which was also the third largest contributor to global YLLs and DALYs (Table 266e-2). Together, CHD and stroke accounted for nearly a quarter of all deaths worldwide. The burden of stroke is of growing concern among LMICs. The impact of stroke on DALYs and mortality rates is more than three times greater in LMICs as compared to HICs. By 2030, the number of deaths due to stroke is projected to increase by more than 30%, the majority of which will occur in LMICs.

1	With nearly 85% of the world’s population, LMICs largely drive global CVD rates and trends. Ten million CVD deaths occurred in TAblE 266e-2 moRbiDiTy RElATED To HEART DiSEASE: 2010–2030 Abbreviations: CHD, coronary heart disease; CVD, cardiovascular disease. Source: Adapted from Global Burden of Disease Study 2010: Global Burden of Disease Study 2010 Mortality Results 1970-2010. Seattle, Institute for Health Metrics and Evaluation, 2012; J Mackay, G Mensah: Atlas of Heart Disease and Stroke. Geneva, World Health Organization, 2004. LMICs in 2010, compared to 5.6 million in HICs. Globally, there is evidence of significant delays in age of occurrence and/or improvements in case fatality rates; between 1990 and 2010, the number of CVD deaths increased by 31%, but age-adjusted death rates decreased by 21.2% in the same period.

1	Although HIC population growth will be fueled by emigration from LMICs, the populations of HICs will shrink as a proportion of the world’s population. The modest decline in CVD death rates that began in the HICs in the latter third of the twentieth century will continue, but the rate of decline appears to be slowing. However, these countries are expected to see an increase in the prevalence of CVD, as well as the absolute number of deaths as the population ages. Significant portions of the population living in LMICs have entered the third phase of the epidemiologic transition, and some are entering the fourth stage. Changing demographics play a significant role in future predictions for CVD throughout the world. For example, the population growth rate in Eastern Europe and Central Asia was 0.7% in 2012, whereas it was 1.3% in South Asia.

1	CVD rates will also have an economic impact. Even assuming no increase in CVD risk factors, most countries, but especially India and South Africa, will see a large number of people between 35 and 64 die of CVD over the next 30 years, as well as an increasing level of morbidity among middle-aged people related to heart disease and stroke. In China, it is estimated that there will be 9 million deaths from CVD in 2030—up from 2.4 million in 2002—with half occurring in individuals between 35 and 64 years old. The global variation in CVD rates is related to temporal and regional variations in known risk behaviors and factors. Ecological analyses of major CVD risk factors and mortality demonstrate high correlations between expected and observed mortality rates for the three main risk factors—smoking, serum cholesterol, and hypertension—and suggest

1	PART 10 Disorders of the Cardiovascular System 266e-4 that many of the regional variations are based on differences in conventional risk factors.

1	Behavioral Risk Factors • TOBACCO Over 1.3 billion people use tobacco worldwide, a number that is projected to increase to 1.6 billion by 2030. Tobacco use currently causes about 5 million deaths annually (9% of all deaths), approximately 1.6 million of which are CVD-related. If current smoking patterns continue, the global burden of disease attributable to tobacco will reach 10 million deaths by 2030. Although tobacco use has been greatest in HICs historically, consumption has shifted dramatically to LMICs in recent decades. Some of the highest tobacco use now occurs in the East Asia and Pacific region. A unique feature of LMICs is easy access to smoking during the early stages of the epidemiologic transition due to the availability of relatively inexpensive tobacco products. In South Asia, the prominence of other locally produced forms of tobacco besides manufactured cigarettes makes control of consumption more challenging. Secondhand smoke is another well-established cause of CHD,

1	the prominence of other locally produced forms of tobacco besides manufactured cigarettes makes control of consumption more challenging. Secondhand smoke is another well-established cause of CHD, responsible for 600,000 deaths of nonsmokers in 2011. Although smoking bans have both immediate and long-term benefits, implementation varies greatly between countries.

1	DIET Total caloric intake per capita increases as countries develop. With regard to CVD, a key element of dietary change is an increase in intake of saturated animal fats and hydrogenated vegetable fats, which contain atherogenic trans fatty acids, along with a decrease in intake of plant-based foods and an increase in simple carbohydrates. Fat contributes less than 20% of calories in rural China and India, less than 30% in Japan, and well above 30% in the United States. Caloric contributions from fat appear to be falling in the HICs. In the United States, between 1971 and 2010, the percentage of calories derived from saturated fat decreased from 13% to 11%.

1	PHYSICAL INACTIVITY The increased mechanization that accompanies the economic transition leads to a shift from physically demanding, agriculture-based work to largely sedentary industryand office-based work. In the United States, approximately one-quarter of the population does not participate in any leisure-time physical activity, and only 51.6% of adults report engaging in physical activity three or more times a week. Physical inactivity is similarly high in other regions of the world and is increasing in countries that are rapidly urbanizing as part of their economic transition. In urban China, for example, the proportion of adults who participate in moderateor high-level activity has decreased significantly, whereas those who participate in low-level activity has increased.

1	Examination of trends in metabolic risk factors provides insight into changes in the CVD burden globally. Here we describe four metabolic risk factors—lipid levels, hypertension, obesity, and diabetes mellitus—using data from the Global Burden of Disease, Injuries, and Risk Factors Study (GBD 2010). The GBD project identified and compiled mortality and morbidity data from 187 countries from 1980 to 2010.

1	Lipid Levels Worldwide, high cholesterol levels are estimated to play a role in 56% of ischemic heart disease events and 18% of strokes, amounting to 4.4 million deaths annually. Although mean population plasma cholesterol levels tend to rise as countries move through the epidemiologic transition, mean serum total cholesterol levels have decreased globally between 1980 and 2008 by 0.08 mmol/L per decade in men and 0.07 mmol/L per decade in women. In 2008, age-standardized mean total cholesterol was 4.64 mmol/L (179.4 mg/dL) in men and 4.76 mmol/L (184.2 mg/dL) in women. Large declines occurred in Australasia, North America, and Western Europe (0.19–0.21 mmol/L). Countries in the East Asia and Pacific region experienced increases of greater than 0.08 mmol/L in both men and women. Social and individual changes that accompany urbanization clearly play a role because plasma cholesterol levels tend to be higher among urban residents than among rural residents. This shift is largely driven

1	individual changes that accompany urbanization clearly play a role because plasma cholesterol levels tend to be higher among urban residents than among rural residents. This shift is largely driven by greater consumption of dietary fats—primarily from animal products and processed vegetable oils—and decreased physical activity. In HICs, in general, mean population cholesterol levels are falling, whereas wide variation is seen in the LMICs.

1	Hypertension Elevated blood pressure is an early indicator of the epidemiologic transition. Worldwide, approximately 62% of strokes and 49% of CHD are attributable to suboptimal (>115 mmHg systolic) blood pressure, which is believed to account for more than 7 million deaths annually. Remarkably, nearly half of this burden occurs among those with systolic blood pressure less than 140 mmHg, even as this level is used at the arbitrary threshold for defining hypertension in many national guidelines. Between 1980 and 2008, the age-standardized prevalence of uncontrolled prevalence has decreased even as the number of people with uncontrolled hypertension has increased. This trend results largely from population growth and aging. Rising mean population blood pressure also occurs as populations industrialize and move from rural to urban settings. For example, the prevalence of hypertension in urban India is 25%, but varies between 10% and 15% in rural communities. One major concern in LMICs

1	industrialize and move from rural to urban settings. For example, the prevalence of hypertension in urban India is 25%, but varies between 10% and 15% in rural communities. One major concern in LMICs is the high rate of undetected, and therefore untreated, hypertension. This may explain, at least in part, the higher stroke rates in these countries in relation to CHD rates during the early stages of the transition. The high rates of hypertension throughout Asia, especially undiagnosed hypertension, likely contribute to the high prevalence of hemorrhagic stroke in the region. Globally, however, mean systolic blood pressure has decreased among both genders (0.8 mmHg per decade among men; 1.0 mmHg per decade among women).

1	Obesity Although clearly associated with increased risk of CHD, much of the risk posed by obesity may be mediated by other CVD risk factors, including hypertension, diabetes mellitus, and lipid profile imbalances. According to the latest GBD data, nearly 1.46 billion adults were overweight (body mass index ≥25 kg/m2) in 2008, and approximately 508 million were obese (BMI ≥30 kg/m2). Obesity is increasing throughout the world, particularly in developing countries, where the trajectories are steeper than those experienced by the developed countries. In many of the LMICs, obesity appears to coexist with undernutrition and malnutrition. Adolescents are at particular risk. Currently, 1 in 10 children are estimated to be overweight, a number that is increasing worldwide. Women are also more affected than men, with the number of overweight women generally exceeding underweight women based on data from 36 LMICs.

1	Diabetes Mellitus As a consequence of, or in addition to, increasing body mass index and decreasing levels of physical activity, worldwide rates of diabetes—predominantly type 2 diabetes—are on the rise. According to the most recent data from the GBD project, mean fasting plasma glucose levels have increased globally between 1980 and 2008. An estimated 346 million people worldwide have diabetes. The International Diabetes Foundation predicts that this number will reach 522 million by 2030, a yearly rate of growth that is higher than that of the world’s adult population. Nearly 50% of people with diabetes are undiagnosed, and 80% live in LMICs. The highest regional prevalence for diabetes occurs in the Middle East and North Africa, where an estimated 12.5% of the adult population has diabetes. Future growth will also largely occur in this region, along with other LMICs in South Asia and sub-Saharan Africa. There appear to be clear genetic susceptibilities to diabetes mellitus of

1	diabetes. Future growth will also largely occur in this region, along with other LMICs in South Asia and sub-Saharan Africa. There appear to be clear genetic susceptibilities to diabetes mellitus of various racial and ethnic groups. For example, migration studies suggest that South Asians and Indians tend to be at higher risk than those of European extraction.

1	Although CVD rates are declining in the HICs, they are increasing in virtually every other region of the world. The consequences of this preventable epidemic will be substantial on many levels, including individual mortality and morbidity, family suffering, and staggering economic costs. Three complementary strategies can be used to lessen the impact. First, the overall burden of CVD risk factors can be lowered through population-wide public health measures, such as national campaigns CHAPTER 266e Epidemiology of Cardiovascular Disease

1	Disorders of the Cardiovascular SystemPhysical Examination of the Cardiovascular System Patrick T. O’Gara, Joseph Loscalzo The approach to a patient with known or suspected cardiovascular disease begins with the time-honored traditions of a directed history and a targeted physical examination. The scope of these activities depends on the clinical context at the time of presentation, rang-ing from an elective ambulatory follow-up visit to a more focused emergency department encounter. There has been a gradual decline in physical examination skills over the last two decades at every level, from student to faculty specialist, a development of great concern to both clinicians and medical educators. Classic cardiac findings are recognized by only a minority of internal medicine and family practice residents. Despite popular perceptions, clinical performance does not improve predictably as a function of experience; instead, the acquisi-tion of new examination skills may become more

1	practice residents. Despite popular perceptions, clinical performance does not improve predictably as a function of experience; instead, the acquisi-tion of new examination skills may become more difficult for a busy individual practitioner. Less time is now devoted to mentored cardio-vascular examinations during the training of students and residents. One widely recognized outcome of these trends is the progressive overutilization of noninvasive imaging studies to establish the pres-ence and severity of cardiovascular disease even when the examina-tion findings imply a low pretest probability of significant pathology. Educational techniques to improve bedside skills include repetition, patient-centered teaching conferences, and visual display feedback of auscultatory events with Doppler echocardiographic imaging. The evidence base that links the findings from the history and physi-cal examination to the presence, severity, and prognosis of cardiovas-cular disease has been established

1	imaging. The evidence base that links the findings from the history and physi-cal examination to the presence, severity, and prognosis of cardiovas-cular disease has been established most rigorously for coronary artery disease, heart failure, and valvular heart disease. For example, obser-vations regarding heart rate, blood pressure, signs of pulmonary congestion, and the presence of mitral regurgitation (MR) contribute importantly to bedside risk assessment in patients with acute coronary syndromes. Observations from the physical examination in this set-ting can inform clinical decision making before the results of cardiac biomarkers testing are known. The prognosis of patients with systolic heart failure can be predicted on the basis of the jugular venous pressure (JVP) and the presence or absence of a third heart sound (S3). Accurate characterization of cardiac murmurs provides important insight into the natural history of many valvular and congenital heart lesions. Finally, the

1	or absence of a third heart sound (S3). Accurate characterization of cardiac murmurs provides important insight into the natural history of many valvular and congenital heart lesions. Finally, the important role played by the physical examination in enhancing the clinician-patient relationship cannot be overestimated. THE GENERAL PHYSICAL EXAMINATION Any examination begins with an assessment of the general appear-ance of the patient, with notation of age, posture, demeanor, and 267 SEC Tion 2 DiAgnoSiS oF CARDiovASCulAR DiSoRDERS overall health status. Is the patient in pain or resting quietly, dyspneic or diaphoretic? Does the patient choose to avoid certain body positions to reduce or eliminate pain, as might be the case with suspected acute pericarditis? Are there clues indicating that dyspnea may have a pulmonary cause, such as a barrel chest deformity with an increased anterior-posterior diameter, tachypnea, and pursed-lip breathing? Skin pallor, cyanosis, and jaundice can be

1	dyspnea may have a pulmonary cause, such as a barrel chest deformity with an increased anterior-posterior diameter, tachypnea, and pursed-lip breathing? Skin pallor, cyanosis, and jaundice can be appreciated readily and provide additional clues. A chronically ill-appearing emaciated patient may suggest the presence of long-standing heart failure or another systemic disorder, such as a malignancy. Various genetic syndromes, often with cardiovascular involvement, can also be recognized easily, such as trisomy 21, Marfan’s syndrome, and Holt-Oram syndrome. Height and weight should be measured routinely, and both body mass index and body surface area should be calculated. Knowledge of the waist circumference and the waist-to-hip ratio can be used to predict long-term cardiovascular risk. Mental status, level of alertness, and mood should be assessed continuously during the interview and examination.

1	Skin Central cyanosis occurs with significant right-to-left shunting at the level of the heart or lungs, allowing deoxygenated blood to reach the systemic circulation. Peripheral cyanosis or acrocyanosis, in contrast, is usually related to reduced extremity blood flow due to small vessel constriction, as seen in patients with severe heart failure, shock, or peripheral vascular disease; it can be aggravated by the use of β-adrenergic blockers with unopposed α-mediated constriction. Differential cyanosis refers to isolated cyanosis affecting the lower but not the upper extremities in a patient with a large patent ductus arteriosus (PDA) and secondary pulmonary hypertension with right-to-left to shunting at the great vessel level. Hereditary telangiectasias on the lips, tongue, and mucous membranes, as part of the Osler-Weber-Rendu syndrome (hereditary hemorrhagic telangiectasia), resemble spider nevi and can be a source of right-to-left shunting when also present in the lung. Malar

1	membranes, as part of the Osler-Weber-Rendu syndrome (hereditary hemorrhagic telangiectasia), resemble spider nevi and can be a source of right-to-left shunting when also present in the lung. Malar telangiectasias also are seen in patients with advanced mitral stenosis and scleroderma. An unusually tan or bronze discoloration of the skin may suggest hemochromatosis as the cause of the associated systolic heart failure. Jaundice, which may be visible first in the sclerae, has a broad differential diagnosis but, in the appropriate setting, can be consistent with advanced right heart failure and congestive hepatomegaly or late-term “cardiac cirrhosis.” Cutaneous ecchymoses are seen frequently among patients taking vitamin K antagonists or antiplatelet agents such as aspirin and thienopyridines. Various lipid disorders sometimes are associated with subcutaneous xanthomas, particularly along the tendon sheaths or over the extensor surfaces of the extremities. Severe hypertriglyceridemia

1	Various lipid disorders sometimes are associated with subcutaneous xanthomas, particularly along the tendon sheaths or over the extensor surfaces of the extremities. Severe hypertriglyceridemia can be associated with eruptive xanthomatosis and lipemia retinalis. Palmar crease xanthomas are specific for type III hyperlipoproteinemia. Pseudoxanthoma elasticum, a disease associated with premature atherosclerosis, is manifested by a leathery, cobblestoned appearance of the skin in the axilla and neck creases and by angioid streaks on funduscopic examination. Extensive lentiginoses have been described in a variety of development delay–cardiovascular syndromes, including Carney’s syndrome, which includes multiple atrial myxomas. Cutaneous manifestations of sarcoidosis such as lupus pernio and erythema nodosum may suggest this disease as a cause of an associated dilated cardiomyopathy, especially with heart block, intraventricular conduction delay, or ventricular tachycardia.

1	Head and Neck Dentition and oral hygiene should be assessed in every patient both as a source of potential infection and as an index of general health. A high-arched palate is a feature of Marfan’s syndrome and other connective tissue disease syndromes. Bifid uvula has been described in patients with Loeys-Dietz syndrome, and orange tonsils are characteristic of Tangier disease. The ocular manifestations of hyperthyroidism have been well described. Many patients with congenital heart disease have associated hypertelorism, low-set ears, or micrognathia. Blue sclerae are a feature of osteogenesis imperfecta. An arcus senilis pattern lacks specificity as an index of coronary heart disease risk. The funduscopic examination is an often underused method by which to assess the microvasculature, especially among patients with established atherosclerosis, hypertension, or diabetes mellitus. A mydriatic agent may be necessary for optimal visualization. A funduscopic examination should be

1	especially among patients with established atherosclerosis, hypertension, or diabetes mellitus. A mydriatic agent may be necessary for optimal visualization. A funduscopic examination should be performed routinely in the assessment of patients with suspected endocarditis and those with a history of acute visual change. Branch retinal artery occlusion or visualization of a Hollenhorst plaque can narrow the differential diagnosis rapidly in the appropriate setting. Relapsing polychondritis may manifest as an inflamed pinna or, in its later stages, as a saddle-nose deformity because of destruction of nasal cartilage; granulomatosis with polyangiitis (Wegener’s) can also lead to a saddle-nose deformity.

1	Chest Midline sternotomy, left posterolateral thoracotomy, or infraclavicular scars at the site of pacemaker/defibrillator generator implantation should not be overlooked and may provide the first clue regarding an underlying cardiovascular disorder in patients unable to provide a relevant history. A prominent venous collateral pattern may suggest subclavian or vena caval obstruction. If the head and neck appear dusky and slightly cyanotic and the venous pressure is grossly elevated without visible pulsations, a diagnosis of superior vena cava syndrome should be entertained. Thoracic cage abnormalities have been well described among patients with connective tissue disease syndromes. They include pectus carinatum (“pigeon chest”) and pectus excavatum (“funnel chest”). Obstructive lung disease is suggested by a barrel chest deformity, especially with tachypnea, pursed-lip breathing, and use of accessory muscles. The characteristically severe kyphosis and compensatory lumbar, pelvic, and

1	is suggested by a barrel chest deformity, especially with tachypnea, pursed-lip breathing, and use of accessory muscles. The characteristically severe kyphosis and compensatory lumbar, pelvic, and knee flexion of ankylosing spondylitis should prompt careful auscultation for a murmur of aortic regurgitation (AR). Straight back syndrome refers to the loss of the normal kyphosis of the thoracic spine and has been described in patients with mitral valve prolapse (MVP) and its variants. In some patients with cyanotic congenital heart disease, the chest wall appears to be asymmetric, with anterior displacement of the left hemithorax. The respiratory rate and pattern should be noted during spontaneous breathing, with additional attention to depth, audible wheezing, and stridor. Lung examination can reveal adventitious sounds indicative of pulmonary edema, pneumonia, or pleuritis.

1	Abdomen In some patients with advanced obstructive lung disease, the point of maximal cardiac impulse may be in the epigastrium. The liver is frequently enlarged and tender in patients with chronic heart failure. Systolic pulsations over the liver signify severe tricuspid regurgitation (TR). Splenomegaly may be a feature of infective endocarditis, particularly when symptoms have persisted for weeks or months. Ascites is a nonspecific finding but may be present with advanced chronic right heart failure, constrictive pericarditis, hepatic cirrhosis, or an intraperitoneal malignancy. The finding of an elevated JVP implies a cardiovascular etiology. In nonobese patients, the aorta typically is palpated between the epigastrium and the umbilicus. The sensitivity of palpation for the detection of an abdominal aortic aneurysm (pulsatile and expansile mass) decreases as a function of body size. Because palpation alone is not sufficiently accurate to establish this diagnosis, a screening

1	of an abdominal aortic aneurysm (pulsatile and expansile mass) decreases as a function of body size. Because palpation alone is not sufficiently accurate to establish this diagnosis, a screening ultrasound examination is advised. The 1443 presence of an arterial bruit over the abdomen suggests high-grade atherosclerotic disease, although precise localization is difficult.

1	Extremities The temperature and color of the extremities, the presence of clubbing, arachnodactyly, and pertinent nail findings can be surmised quickly during the examination. Clubbing implies the presence of central right-to-left shunting, although it has also been described in patients with endocarditis. Its appearance can range from cyanosis and softening of the root of the nail bed, to the classic loss of the normal angle between the base of the nail and the skin, to the skeletal and periosteal bony changes of hypertrophic osteoarthropathy, which is seen rarely in patients with advanced lung or liver disease. Patients with the Holt-Oram syndrome have an unopposable, “fingerized” thumb, whereas patients with Marfan’s syndrome may have arachnodactyly and a positive “wrist” (overlapping of the thumb and fifth finger around the wrist) or “thumb” (protrusion of the thumb beyond the ulnar aspect of the hand when the fingers are clenched over the thumb in a fist) sign. The Janeway

1	of the thumb and fifth finger around the wrist) or “thumb” (protrusion of the thumb beyond the ulnar aspect of the hand when the fingers are clenched over the thumb in a fist) sign. The Janeway lesions of endocarditis are non- tender, slightly raised hemorrhages on the palms and soles, whereas Osler’s nodes are tender, raised nodules on the pads of the fingers or toes. Splinter hemorrhages are classically identified as linear petechiae in the midposition of the nail bed and should be distinguished from the more common traumatic petechiae, which are seen closer to the distal edge.

1	Lower extremity or presacral edema in the setting of an elevated JVP defines volume overload and may be a feature of chronic heart failure or constrictive pericarditis. Lower extremity edema in the absence of jugular venous hypertension may be due to lymphatic or venous obstruction or, more commonly, to venous insufficiency, as further suggested by the appearance of varicosities, venous ulcers (typically medial in location), and brownish cutaneous discoloration from hemosiderin deposition (eburnation). Pitting edema can also be seen in patients who use dihydropyridine calcium channel blockers. A Homan’s sign (posterior calf pain on active dorsiflexion of the foot against resistance) is neither specific nor sensitive for deep venous thrombosis. Muscular atrophy or the absence of hair along an extremity is consistent with severe arterial insufficiency or a primary neuromuscular disorder.

1	CARDIOVASCULAR EXAMINATION Jugular Venous Pressure and Waveform JVP is the single most important bedside measurement from which to estimate the volume status. The internal jugular vein is preferred because the external jugular vein is valved and not directly in line with the superior vena cava and right atrium. Nevertheless, the external jugular vein has been used to discriminate between high and low central venous pressure (CVP) when tested among medical students, residents, and attending physicians. Precise estimation of the central venous or right atrial pressure from bedside assessment of the jugular venous waveform has proved difficult. Venous pressure traditionally has been measured as the vertical distance between the top of the jugular venous pulsation and the sternal inflection point (angle of Louis). A distance >4.5 cm at 30° elevation is considered abnormal. However, the actual distance between the mid-right atrium and the angle of Louis varies considerably as a function of

1	(angle of Louis). A distance >4.5 cm at 30° elevation is considered abnormal. However, the actual distance between the mid-right atrium and the angle of Louis varies considerably as a function of both body size and the patient angle at which the assessment is made (30°, 45°, or 60°). The use of the sternal angle as a reference point leads to systematic underestimation of CVP, and this method should be used less for semiquantification than to distinguish a normal from an abnormally elevated CVP. The use of the clavicle may provide an easier reference for standardization. Venous pulsations above this level in the sitting position are clearly abnormal, as the distance between the clavicle and the right atrium is at least 10 cm. The patient should always be placed in the sitting position, with the legs dangling below the bedside, when an elevated pressure is suspected in the semisupine position. It should also be noted that bedside estimates of CVP are made in centimeters of water but

1	with the legs dangling below the bedside, when an elevated pressure is suspected in the semisupine position. It should also be noted that bedside estimates of CVP are made in centimeters of water but must be converted to millimeters of mercury to provide correlation with accepted hemodynamic norms (1.36 cmH2O = 1.0 mmHg).

1	Physical Examination of the Cardiovascular System 1444 The venous waveform sometimes can be difficult to distinguish from the carotid pulse, especially during casual inspection. Nevertheless, the venous waveform has several characteristic features, and its individual components can be appreciated in most patients (Fig. 267-1). The arterial pulsation is not easily obliterated with palpation; the venous waveform in patients with sinus rhythm is usually biphasic, while the carotid pulse is monophasic; and the jugular venous pulsation should change with changes in posture or inspiration (unless the venous pressure is quite elevated). The venous waveform is divided into several distinct peaks. The a wave reflects right atrial presystolic contraction and occurs just after the electrocardiographic P wave, preceding the first heart sound (S1). A prominent a wave is seen in patients with reduced right ventricular compliance; a cannon a wave occurs with atrioventricular (AV) dissociation and

1	P wave, preceding the first heart sound (S1). A prominent a wave is seen in patients with reduced right ventricular compliance; a cannon a wave occurs with atrioventricular (AV) dissociation and right atrial contraction against a closed tricuspid valve. In a patient with a wide complex tachycardia, the appreciation of cannon a waves in the jugular venous waveform identifies the rhythm as ventricular in origin. The a wave is not present with atrial fibrillation. The x descent defines the fall in right atrial pressure after inscription of the a wave. The c wave interrupts this x descent and is followed by a further descent. The v wave represents atrial filling (atrial diastole) and occurs during ventricular systole. The height of the v wave is determined by right atrial compliance as well as the volume of blood returning to the right atrium either antegrade from the cavae or retrograde through an incompetent tricuspid valve. In patients with TR, the v wave is accentuated and the

1	well as the volume of blood returning to the right atrium either antegrade from the cavae or retrograde through an incompetent tricuspid valve. In patients with TR, the v wave is accentuated and the subsequent fall in pressure (y descent) is rapid. With progressive degrees of TR, the v wave merges with the c wave, and the right atrial and jugular vein waveforms become “ventricularized.” The y descent, which follows the peak of the v wave, can become prolonged or blunted with obstruction to right ventricular inflow, as may occur with tricuspid stenosis or pericardial tamponade. Normally, the venous pressure should fall by at least 3 mmHg with inspiration. Kussmaul’s sign is defined by either a rise or a lack of fall of the JVP with inspiration and is classically associated with constrictive pericarditis, although it has been reported in patients with restrictive cardiomyopathy, massive pulmonary embolism, right ventricular infarction, and advanced left ventricular systolic heart

1	pericarditis, although it has been reported in patients with restrictive cardiomyopathy, massive pulmonary embolism, right ventricular infarction, and advanced left ventricular systolic heart failure. It is also a common, isolated finding in patients after cardiac surgery without other hemodynamic abnormalities. Venous hypertension sometimes can be elicited by performance of the abdominojugular reflex or with passive leg elevation. When these signs are positive, a volume-overloaded state with limited compliance of an overly distended or constricted venous system is present. The abdominojugular reflex is elicited with firm and consistent pressure over the upper portion of the abdomen, preferably over the right upper quadrant, for at least 10 s. A positive response is defined by a sustained rise of more than 3 cm in JVP for at least 15 s after release of the hand. Patients must be coached to refrain from breath holding or a Valsalva-like maneuver during the procedure. The

1	by a sustained rise of more than 3 cm in JVP for at least 15 s after release of the hand. Patients must be coached to refrain from breath holding or a Valsalva-like maneuver during the procedure. The abdominojugular reflex is useful in predicting a pulmonary artery wedge pressure in excess of 15 mmHg in patients with heart failure. Although the JVP estimates right ventricular filling pressure, it has a predictable relationship with the pulmonary artery wedge pressure. In a large study of patients with advanced heart failure, the presence of a right atrial pressure >10 mmHg (as predicted on bedside examination) had a positive value of 88% for the prediction of a pulmonary artery wedge pressure of >22 mmHg. In addition, an elevated JVP has prognostic significance in patients with both symptomatic heart failure and asymptomatic left ventricular systolic dysfunction. The presence of an elevated JVP is associated with a higher risk of subsequent hospitalization for heart failure, death

1	heart failure and asymptomatic left ventricular systolic dysfunction. The presence of an elevated JVP is associated with a higher risk of subsequent hospitalization for heart failure, death from heart failure, or both.

1	Assessment of Blood Pressure Measurement of blood pressure usually is delegated to a medical assistant but should be repeated by the clinician. Accurate measurement depends on body position, arm size, time of measurement, place of measurement, device, device size, technique, and examiner. In general, physician-recorded blood pressures are higher than both nurse-recorded pressures and self-recorded pressures at home. Blood pressure is best measured in the seated position with

1	FIGURE 267-1 A. Jugular venous pulse wave tracing (top) with heart sounds (bottom). The A wave represents right atrial presystolic contraction and occurs just after the electrocardiographic P wave and just before the first heart sound (I). In this example, the A wave is accentuated and larger than normal due to decreased right ventricular compliance, as also suggested by the right-sided S4 (IV). The C wave may reflect the carotid pulsation in the neck and/or an early systolic increase in right atrial pressure as the right ventricle pushes the closed tricuspid valve into the right atrium. The x descent follows the A wave just as atrial pressure continues to fall. The V wave represents atrial filling during ventricular systole and peaks at the second heart sound (II). The y descent corresponds to the fall in right atrial pressure after tricuspid valve opening. B. Jugular venous wave forms in mild (middle) and severe (top) tricuspid regurgitation, compared with normal, with

1	corresponds to the fall in right atrial pressure after tricuspid valve opening. B. Jugular venous wave forms in mild (middle) and severe (top) tricuspid regurgitation, compared with normal, with phonocardiographic representation of the corresponding heart sounds below. With increasing degrees of tricuspid regurgitation, the waveform becomes “ventricularized.” C. Electrocardiogram (ECG) (top), jugular venous waveform (JVP) (middle), and heart sounds (bottom) in pericardial constriction. Note the prominent and rapid y descent, corresponding in timing to the pericardial knock (K). (From J Abrams: Synopsis of Cardiac Physical Diagnosis, 2nd ed. Boston, Butterworth Heinemann, 2001, pp 25–35.) the arm at the level of the heart, using an appropriately sized cuff, after 5–10 min of relaxation. When it is measured in the supine position, the arm should be raised to bring it to the level of the mid-right atrium. The length and width of the blood pressure cuff bladder should be 80% and 40% of

1	When it is measured in the supine position, the arm should be raised to bring it to the level of the mid-right atrium. The length and width of the blood pressure cuff bladder should be 80% and 40% of the arm’s circumference, respectively. A common source of error in practice is to use an inappropriately small cuff, resulting in marked overestimation of true blood pressure, or an inappropriately large cuff, resulting in underestimation of true blood pressure. The cuff should be inflated to 30 mmHg above the expected systolic pressure and the pressure released at a rate of 2–3 mmHg/s. Systolic and diastolic pressures are defined by the first and fifth Korotkoff sounds, respectively. Very low (even 0 mmHg) diastolic blood pressures may be recorded in patients with chronic, severe AR or a large arteriovenous fistula because of enhanced diastolic “run-off.” In these instances, both the phase IV and phase V Korotkoff sounds should be recorded. Blood pressure is best assessed at the brachial

1	arteriovenous fistula because of enhanced diastolic “run-off.” In these instances, both the phase IV and phase V Korotkoff sounds should be recorded. Blood pressure is best assessed at the brachial artery level, though it can be measured at the radial, popliteal, or pedal pulse level. In general, systolic pressure increases and diastolic pressure decreases when measured in more distal arteries. Blood pressure should be measured in both arms, and the difference should be less than 10 mmHg. A blood pressure differential that exceeds this threshold may be associated with atherosclerotic or inflammatory subclavian artery disease, supravalvular aortic stenosis, aortic coarctation, or aortic dissection. Systolic leg pressures are usually as much as 20 mmHg higher than systolic arm pressures. Greater leg–arm pressure differences are seen in patients with chronic severe AR as well as patients with extensive and calcified lower extremity peripheral arterial disease. The ankle-brachial index

1	Greater leg–arm pressure differences are seen in patients with chronic severe AR as well as patients with extensive and calcified lower extremity peripheral arterial disease. The ankle-brachial index (lower pressure in the dorsalis pedis or posterior tibial artery divided by the higher of the two brachial artery pressures) is a powerful predictor of long-term cardiovascular mortality.

1	The blood pressure measured in an office or hospital setting may not accurately reflect the pressure in other venues. “White coat hypertension” is defined by at least three separate clinic-based measurements >140/90 mmHg and at least two non-clinic-based measurements <140/90 mmHg in the absence of any evidence of target organ damage. Individuals with white coat hypertension may not benefit from drug therapy, although they may be more likely to develop sustained hypertension over time. Masked hypertension should be suspected when normal or even low blood pressures are recorded in patients with advanced atherosclerotic disease, especially when evidence of target organ damage is present or bruits are audible.

1	Orthostatic hypotension is defined by a fall in systolic pressure >20 mmHg or in diastolic pressure >10 mmHg in response to assumption of the upright posture from a supine position within 3 min. There may also be a lack of a compensatory tachycardia, an abnormal response that suggests autonomic insufficiency, as may be seen in patients with diabetes or Parkinson’s disease. Orthostatic hypotension is a common cause of postural lightheadedness/syncope and should be assessed routinely in patients for whom this diagnosis might pertain. It can be exacerbated by advanced age, dehydration, certain medications, food, deconditioning, and ambient temperature.

1	Arterial Pulse The carotid artery pulse occurs just after the ascending aortic pulse. The aortic pulse is best appreciated in the epigastrium, just above the level of the umbilicus. Peripheral arterial pulses that should be assessed routinely include the subclavian, brachial, radial, ulnar, femoral, popliteal, dorsalis pedis, and posterior tibial. In patients in whom the diagnosis of either temporal arteritis or polymyalgia rheumatica is suspected, the temporal arteries also should be examined. Although one of the two pedal pulses may not be palpable in up to 10% of normal subjects, the pair should be symmetric. The integrity of the arcuate system of the hand is assessed by Allen’s test, which is performed routinely before instrumentation of the radial artery. The pulses should be examined for their symmetry, volume, timing, contour, amplitude, and duration. If necessary, simultaneous auscultation of the heart can help identify a delay in the arrival of an arterial pulse. Simultaneous

1	for their symmetry, volume, timing, contour, amplitude, and duration. If necessary, simultaneous auscultation of the heart can help identify a delay in the arrival of an arterial pulse. Simultaneous palpation of the radial and femoral pulses may reveal a femoral delay in a patient with hypertension and suspected aortic coarctation. The carotid upstrokes should never be examined simultaneously or before listening for a bruit. Light pressure should always be used to avoid precipitation of carotid hypersensitivity 1445 syndrome and syncope in a susceptible elderly individual. The arterial pulse usually becomes more rapid and spiking as a function of its distance from the heart, a phenomenon that reflects the muscular status of the more peripheral arteries and the summation of the incident and reflected waves. In general, the character and contour of the arterial pulse depend on the stroke volume, ejection velocity, vascular compliance, and systemic vascular resistance. The pulse

1	and reflected waves. In general, the character and contour of the arterial pulse depend on the stroke volume, ejection velocity, vascular compliance, and systemic vascular resistance. The pulse examination can be misleading in patients with reduced cardiac output and in those with stiffened arteries from aging, chronic hypertension, or peripheral arterial disease.

1	The character of the pulse is best appreciated at the carotid level (Fig. 267-2). A weak and delayed pulse (pulsus parvus et tardus) defines severe aortic stenosis (AS). Some patients with AS may also have a slow, notched, or interrupted upstroke (anacrotic pulse) with a thrill or shudder. With chronic severe AR, by contrast, the carotid upstroke has a sharp rise and rapid fall-off (Corrigan’s or water-hammer pulse). Some patients with advanced AR may have a bifid or bisferiens pulse, in which two systolic peaks can be appreciated. A bifid pulse is also described in patients with hypertrophic obstructive cardiomyopathy (HOCM), with inscription of percussion and tidal waves. A bifid pulse is easily appreciated in patients on intraaortic balloon counterpulsation (IABP), in whom the second pulse is diastolic in timing.

1	Pulsus paradoxus refers to a fall in systolic pressure >10 mmHg with inspiration that is seen in patients with pericardial tamponade but also is described in those with massive pulmonary embolism, hemorrhagic shock, severe obstructive lung disease, and tension pneumothorax.

1	FIGURE 267-2 Schematic diagrams of the configurational changes in carotid pulse and their differential diagnoses. Heart sounds are also illustrated. A. Normal. S4, fourth heart sound; S1, first heart sound; A2 aortic component of second heart sound; P2 pulmonic component of second heart sound. B. Aortic stenosis. Anacrotic pulse with slow upstroke to a reduced peak. C. Bisferiens pulse with two peaks in systole. This pulse is rarely appreciated in patients with severe aortic regurgitation. D. Bisferiens pulse in hypertrophic obstructive cardiomyopathy. There is a rapid upstroke to the first peak (percussion wave) and a slower rise to the second peak (tidal wave). E. Dicrotic pulse with peaks in systole and diastole. This waveform may be seen in patients with sepsis or during intraaortic balloon counterpulsation with inflation just after the dicrotic notch. (From K Chatterjee, W Parmley [eds]: Cardiology: An Illustrated Text/Reference. Philadelphia, Gower Medical Publishers, 1991.)

1	Physical Examination of the Cardiovascular System 1446 Pulsus paradoxus is measured by noting the difference between the patient with calf claudication, a decrease in pulse amplitude between systolic pressure at which the Korotkoff sounds are first heard (dur-the common femoral and popliteal arteries will localize the obstrucing expiration) and the systolic pressure at which the Korotkoff tion to the level of the superficial femoral artery, although inflow sounds are heard with each heartbeat, independent of the respiratory obstruction above the level of the common femoral artery may coexist. phase. Between these two pressures, the Korotkoff sounds are heard Auscultation for carotid, subclavian, abdominal aortic, and femoral only intermittently and during expiration. The cuff pressure must be artery bruits should be routine. However, the correlation between the decreased slowly to appreciate the finding. It can be difficult to mea-presence of a bruit and the degree of vascular

1	must be artery bruits should be routine. However, the correlation between the decreased slowly to appreciate the finding. It can be difficult to mea-presence of a bruit and the degree of vascular obstruction is poor. A sure pulsus paradoxus in patients with tachycardia, atrial fibrillation, cervical bruit is a weak indicator of the degree of carotid artery steno-or tachypnea. A pulsus paradoxus may be palpable at the brachial sis; the absence of a bruit does not exclude the presence of significant artery or femoral artery level when the pressure difference exceeds luminal obstruction. If a bruit extends into diastole or if a thrill is pres15 mmHg. This inspiratory fall in systolic pressure is an exaggerated ent, the obstruction is usually severe. Another cause of an arterial bruit consequence of interventricular dependence. is an arteriovenous fistula with enhanced flow.

1	Pulsus alternans, in contrast, is defined by beat-to-beat variability of The likelihood of significant lower extremity peripheral arterial pulse amplitude. It is present only when every other phase I Korotkoff disease increases with typical symptoms of claudication, cool skin, sound is audible as the cuff pressure is lowered slowly, typically in a abnormalities on pulse examination, or the presence of a vascular patient with a regular heart rhythm and independent of the respiratory bruit. Abnormal pulse oximetry (a >2% difference between finger and cycle. Pulsus alternans is seen in patients with severe left ventricular toe oxygen saturation) can be used to detect lower extremity peripheral systolic dysfunction and is thought to be due to cyclic changes in intra-arterial disease and is comparable in its performance characteristics to cellular calcium and action potential duration. When pulsus alternans the ankle-brachial index. is associated with electrocardiographic T-wave alternans,

1	in its performance characteristics to cellular calcium and action potential duration. When pulsus alternans the ankle-brachial index. is associated with electrocardiographic T-wave alternans, the risk for an arrhythmic event appears to be increased. Inspection and Palpation of the Heart The left ventricular apex beat

1	Ascending aortic aneurysms can rarely be appreciated as a pulsa-may be visible in the midclavicular line at the fifth intercostal space tile mass in the right parasternal area. Appreciation of a prominent in thin-chested adults. Visible pulsations anywhere other than this abdominal aortic pulse should prompt noninvasive imaging for better expected location are abnormal. The left anterior chest wall may heave characterization. Femoral and/or popliteal artery aneurysms should be in patients with an enlarged or hyperdynamic left or right ventricle. sought in patients with abdominal aortic aneurysm disease. As noted previously, a visible right upper parasternal pulsation may be The level of a claudication-producing arterial obstruction can often suggestive of ascending aortic aneurysm disease. In thin, tall patients be identified on physical examination (Fig. 267-3). For example, in a and patients with advanced obstructive lung disease and flattened

1	Anterior superior Posterior tibial a. External iliac a. Deep femoral a. Palpatation of Blood pressure popliteal artery pulse cuff Femoral a. Popliteal a. Dorsalis pedis a. Dorsalis pedis a. A Major arteries of the lower limb B Measurement of ankle systolic pressure FIGURE 267-3 A. Anatomy of the major arteries of the leg. B. Measurement of the ankle systolic pressure. (From NA Khan et al: JAMA 295:536, 2006.) diaphragms, the cardiac impulse may be visible in the epigastrium and should be distinguished from a pulsatile liver edge. Palpation of the heart begins with the patient in the supine position at 30° and can be enhanced by placing the patient in the left lateral decubitus position. The normal left ventricular impulse is less than 2 cm in diameter and moves quickly away from the fingers; it is better appreciated at end expiration, with the heart closer to the anterior chest wall. Characteristics such as size, amplitude, and rate of force development should be noted.

1	Enlargement of the left ventricular cavity is manifested by a leftward and downward displacement of an enlarged apex beat. A sustained apex beat is a sign of pressure overload, such as that which may be present in patients with AS or chronic hypertension. A palpable presystolic impulse corresponds to the fourth heart sound (S4) and is indicative of reduced left ventricular compliance and the forceful contribution of atrial contraction to ventricular filling. A palpable third sound (S3), which is indicative of a rapid early filling wave in patients with heart failure, may be present even when the gallop itself is not audible. A large left ventricular aneurysm may sometimes be palpable as an ectopic impulse, discrete from the apex beat. HOCM may very rarely cause a triple cadence beat at the apex with contributions from a palpable S4 and the two components of the bisferiens systolic pulse.

1	Right ventricular pressure or volume overload may create a sternal lift. Signs of either TR (cv waves in the jugular venous pulse) and/or pulmonary arterial hypertension (a loud single or palpable P2) would be confirmatory. The right ventricle can enlarge to the extent that left-sided events cannot be appreciated. A zone of retraction between the right and left ventricular impulses sometimes can be appreciated in patients with right ventricle pressure or volume overload when they are placed in the left lateral decubitus position. Systolic and diastolic thrills signify turbulent and high-velocity blood flow. Their locations help identify the origin of heart murmurs.

1	CARDIAC AUSCULTATION Heart Sounds Ventricular systole is defined by the interval between the first (S1) and second (S2) heart sounds (Fig. 267-4). The first heart sound (S1) includes mitral and tricuspid valve closure. Normal splitting can be appreciated in young patients and those with right bundle branch block, in whom tricuspid valve closure is relatively delayed. The intensity of S1 is determined by the distance over which the anterior leaflet of the mitral valve must travel to return to its annular plane, leaflet mobility, left ventricular contractility, and the PR interval. S1 is classically loud in the early phases of rheumatic mitral stenosis (MS) and in patients with hyperkinetic circulatory states or short PR intervals. S1 becomes softer in the later stages of MS when the leaflets are rigid and calcified, after exposure to β-adrenergic receptor blockers, with long PR intervals, and with left ventricular contractile dysfunction. The intensity of heart sounds, however, can be

1	are rigid and calcified, after exposure to β-adrenergic receptor blockers, with long PR intervals, and with left ventricular contractile dysfunction. The intensity of heart sounds, however, can be reduced by any process that increases the distance between the stethoscope and the responsible cardiac event, including mechanical ventilation, obstructive lung disease, obesity, pneumothorax, and a pericardial effusion.

1	Aortic and pulmonic valve closure constitutes the second heart sound (S2). With normal or physiologic splitting, the A2–P2 interval increases with inspiration and narrows during expiration. This physiologic interval will widen with right bundle branch block because of the further delay in pulmonic valve closure and in patients with severe MR because of the premature closure of the aortic valve. An unusually narrowly split or even a singular S2 is a feature of pulmonary arterial hypertension. Fixed splitting of S2, in which the A2–P2 interval is wide and does not change during the respiratory cycle, occurs in patients with a secundum atrial septal defect. Reversed or paradoxical splitting refers to a pathologic delay in aortic valve closure, such as that which occurs in patients with left bundle branch block, right ventricular pacing, severe AS, HOCM, and acute myocardial ischemia. With reversed or paradoxical splitting, the individual components of S2 are audible at end expiration,

1	bundle branch block, right ventricular pacing, severe AS, HOCM, and acute myocardial ischemia. With reversed or paradoxical splitting, the individual components of S2 are audible at end expiration, and their interval narrows with inspiration, the opposite of what would be expected under normal physiologic conditions. P2 is considered loud when its intensity exceeds that of A2 at the base, when it can be palpated in the area of the proximal main pulmonary

1	C Expiratory splitting with inspiratory S1 S2S1 S2 increase (RBBB, idiopathic dilatation PA) P2 (LBBB, aortic stenosis) S1 S2 S1 S2 FIGURE 267-4 Heart sounds. A. Normal. S1, first heart sound; S2, second heart sound; A2, aortic component of the second heart sound; P2, pulmonic component of the second heart sound. B. Atrial septal defect with fixed splitting of S2. C. Physiologic but wide splitting of S2 with right bundle branch block (RBBB). PA, pulmonary artery. D. Reversed or paradoxical splitting of S2 with left bundle branch block (LBBB). E. Narrow splitting of S2 with pulmonary hypertension. (From NO Fowler: Diagnosis of Heart Disease. New York, Springer-Verlag, 1991, p 31.) artery (second left interspace), or when both components of S2 can be appreciated at the lower left sternal border or apex. The intensity of A2 and P2 decreases with aortic and pulmonic stenosis, respectively. In these conditions, a single S2 may result.

1	Systolic Sounds An ejection sound is a high-pitched early systolic sound that corresponds in timing to the upstroke of the carotid pulse. It usually is associated with congenital bicuspid aortic or pulmonic valve disease; however, ejection sounds are also sometimes audible in patients with isolated aortic or pulmonary root dilation and normal semilunar valves. The ejection sound that accompanies bicuspid aortic valve disease becomes softer and then inaudible as the valve calcifies and becomes more rigid. The ejection sound that accompanies pulmonic stenosis (PS) moves closer to the first heart sound as the severity of the stenosis increases. In addition, the pulmonic ejection sound is the only right-sided acoustic event that decreases in intensity with inspiration. Ejection sounds are often heard more easily at the lower left sternal border than they are at the base. Nonejection sounds (clicks), which occur after the onset of the carotid upstroke, are related to MVP and may be single

1	heard more easily at the lower left sternal border than they are at the base. Nonejection sounds (clicks), which occur after the onset of the carotid upstroke, are related to MVP and may be single or multiple. The nonejection click may introduce a murmur. This click-murmur complex will move away from the first heart sound with maneuvers that increase ventricular preload, such as squatting. On standing, the click and murmur move closer to S1.

1	Diastolic Sounds The high-pitched opening snap (OS) of MS occurs after a very short interval after the second heart sound. The A2–OS interval is inversely proportional to the height of the left atrial–left ventricular diastolic pressure gradient. The intensity of both S1 and the OS of MS decreases with progressive calcification and rigidity of the anterior mitral leaflets. The pericardial knock (PK) is also high-pitched

1	Physical Examination of the Cardiovascular System 1448 and occurs slightly later than the OS, corresponding in timing to the abrupt cessation of ventricular expansion after tricuspid valve opening and to an exaggerated y descent seen in the jugular venous waveform in patients with constrictive pericarditis. A tumor plop is a lower-pitched sound that rarely can be heard in patients with atrial myxoma. It may be appreciated only in certain positions and arises from the diastolic prolapse of the tumor across the mitral valve. The third heart sound (S3) occurs during the rapid filling phase of ventricular diastole. It can be a normal finding in children, adolescents, and young adults; however, in older patients, it signifies heart failure. A left-sided S3 is a low-pitched sound best heard over the left ventricular (LV) apex. A right-sided S3 is usually better heard over the lower left sternal border and becomes louder with inspiration. A left-sided S3 in patients with chronic heart

1	over the left ventricular (LV) apex. A right-sided S3 is usually better heard over the lower left sternal border and becomes louder with inspiration. A left-sided S3 in patients with chronic heart failure is predictive of cardiovascular morbidity and mortality. Interestingly, an S3 is equally prevalent among heart failure patients with and without LV systolic dysfunction. The fourth heart sound (S4) occurs during the atrial filling phase of ventricular diastole and indicates LV presystolic expansion. An S4 is more common among patients who derive significant benefit from the atrial contribution to ventricular filling, such as those with chronic LV hypertrophy or active myocardial ischemia. An S4 is not present with atrial fibrillation.

1	Cardiac Murmurs Heart murmurs result from audible vibrations that are caused by increased turbulence and are defined by their timing within the cardiac cycle. Not all murmurs are indicative of structural heart disease, and the accurate identification of a benign or functional systolic murmur often can obviate the need for additional testing in healthy subjects. The duration, frequency, configuration, and intensity of a heart murmur are dictated by the magnitude, variability, and duration of the responsible pressure difference between two cardiac chambers, the two ventricles, or the ventricles and their respective great arteries. The intensity of a heart murmur is graded on a scale of 1 to 6; a thrill is present with murmurs of grade 4 or greater intensity. Other attributes of the murmur that aid in its accurate identification include its location, radiation, and response to bedside maneuvers. Although clinicians can detect and correctly identify heart murmurs with only fair

1	murmur that aid in its accurate identification include its location, radiation, and response to bedside maneuvers. Although clinicians can detect and correctly identify heart murmurs with only fair reliability, a careful and complete bedside examination usually can identify individuals with valvular heart disease for whom transthoracic echocardiography and clinical follow-up are indicated and exclude subjects for whom no further evaluation is necessary.

1	Systolic murmurs can be early, mid, late, or holosystolic in timing (Fig. 267-5). Acute severe MR results in a decrescendo early systolic murmur, the characteristics of which are related to the progressive attenuation of the left ventricular to left atrial pressure gradient during systole because of the steep and rapid rise in left atrial pressure in this context. Severe MR associated with posterior leaflet prolapse or flail radiates anteriorly and to the base, where it can be confused with the murmur of AS. MR that is due to anterior leaflet involvement radiates posteriorly and to the axilla. With acute TR in patients with normal pulmonary artery pressures, an early systolic murmur that may increase in intensity with inspiration may be heard at the left lower sternal border, with regurgitant cv waves visible in the jugular venous pulse.

1	A midsystolic murmur begins after S1 and ends before S2; it is typically crescendo-decrescendo in configuration. AS is the most common cause of a midsystolic murmur in an adult. It is often difficult to estimate the severity of the valve lesion on the basis of the physical examination findings, especially in older hypertensive patients with stiffened carotid arteries or patients with low cardiac output in whom the intensity of the systolic heart murmur is misleadingly soft. Examination findings consistent with severe AS would include parvus et tardus carotid upstrokes, a late-peaking grade 3 or greater midsystolic murmur, a soft A2, a sustained LV apical impulse, and an S4. It is sometimes difficult to distinguish aortic sclerosis from more advanced degrees of valve stenosis. The former is defined by focal thickening and calcification of the aortic valve leaflets that is not severe enough to result in obstruction. These valve changes are associated with a Doppler jet velocity across

1	is defined by focal thickening and calcification of the aortic valve leaflets that is not severe enough to result in obstruction. These valve changes are associated with a Doppler jet velocity across the aortic valve of 2.5 m/s or less. Patients

1	FIGURE 267-5 A. Top. Graphic representation of the systolic pressure difference (green shaded area) between left ventricle and left atrium with phonocardiographic recording of a holosystolic murmur (HSM) indicative of mitral regurgitation. ECG, electrocardiogram; LAP, left atrial pressure; LVP, left ventricular pressure; S1, first heart sound; S2 second heart sound. Bottom. Graphic representation of the systolic pressure gradient (green shaded area) between left ventricle and aorta in patient with aortic stenosis. A midsystolic murmur (MSM) with a crescendo-decrescendo configuration is recorded. AOP, aortic pressure. B. Top. Graphic representation of the diastolic pressure difference between the aorta and left ventricle (blue shaded area) in a patient with aortic regurgitation, resulting in a decrescendo, early diastolic murmur (EDM) beginning with A2. Bottom. Graphic representation of the diastolic left atrial–left ventricular gradient (blue areas) in a patient with mitral stenosis

1	in a decrescendo, early diastolic murmur (EDM) beginning with A2. Bottom. Graphic representation of the diastolic left atrial–left ventricular gradient (blue areas) in a patient with mitral stenosis with a mid-diastolic murmur (MDM) and late presystolic murmurs (PSM).

1	with aortic sclerosis can have grade 2 or 3 midsystolic murmurs identical in their acoustic characteristics to the murmurs heard in patients with more advanced degrees of AS. Other causes of a midsystolic heart murmur include pulmonic valve stenosis (with or without an ejection sound), HOCM, increased pulmonary blood flow in patients with a large atrial septal defect and left-to-right shunting, and several states associated with accelerated blood flow in the absence of structural heart disease, such as fever, thyrotoxicosis, pregnancy, anemia, and normal childhood/adolescence.

1	The murmur of HOCM has features of both obstruction to LV outflow and MR, as would be expected from knowledge of the pathophysiology of this condition. The systolic murmur of HOCM usually can be distinguished from other causes on the basis of its response to bedside maneuvers, including Valsalva, passive leg raising, and standing/ squatting. In general, maneuvers that decrease LV preload (or increase LV contractility) will cause the murmur to intensify, whereas maneuvers that increase LV preload or afterload will cause a decrease in the intensity of the murmur. Accordingly, the systolic murmur of HOCM becomes louder during the strain phase of the Valsalva maneuver and after standing quickly from a squatting position. The murmur becomes softer with passive leg raising and when squatting. The murmur of AS is typically loudest in the second right interspace with radiation into the carotids, whereas the murmur of HOCM is best heard between the lower left sternal border and the apex. The

1	The murmur of AS is typically loudest in the second right interspace with radiation into the carotids, whereas the murmur of HOCM is best heard between the lower left sternal border and the apex. The murmur of PS is best heard in the second left interspace. The midsystolic murmur associated with enhanced pulmonic blood flow in the setting of a large atrial septal defect (ASD) is usually loudest at the mid-left sternal border.

1	is low-pitched or rumbling, and is introduced by an OS in the early stages of the rheumatic disease process. Presystolic accentuation refers to an increase in the intensity of the murmur just before the first heart sound and occurs in patients with sinus rhythm. It is absent FIGURE 267-6 Behavior of the click (C) and murmur (M) of mitral valve prolapse with in patients with atrial fibrillation. The aus changes in loading (volume, impedance) and contractility. S1, first heart sound; S2, sec cultatory findings in patients with rheumatic ond heart sound. With standing (left side of figure), volume and impedance decrease, as a tricuspid stenosis typically are obscured by result of which the click and murmur move closer to S . With squatting (right), the click and 1 left-sided events, although they are similar in murmur move away from S due to the increases in left ventricular volume and impedance 1 nature to those described in patients with MS.

1	(afterload). Ao, aorta; LV, left ventricle. (Adapted from RA O’Rourke, MH Crawford: Curr Prob Physical Examination of the Cardiovascular System Cardiol 1:9, 1976.) A late systolic murmur, heard best at the apex, indicates MVP. As previously noted, the murmur may or may not be introduced by a nonejection click. Differential radiation of the murmur, as previously described, may help identify the specific leaflet involved by the myxomatous process. The click-murmur complex behaves in a manner directionally similar to that demonstrated by the murmur of HOCM during the Valsalva and stand/squat maneuvers (Fig. 267-6). The murmur of MVP can be identified by the accompanying nonejection click.

1	Holosystolic murmurs are plateau in configuration and reflect a continuous and wide pressure gradient between the left ventricle and left atrium with chronic MR, the left ventricle and right ventricle with a ventricular septal defect (VSD), and the right ventricle and right atrium with TR. In contrast to acute MR, in chronic MR the left atrium is enlarged and its compliance is normal or increased to the extent that there is little if any further increase in left atrial pressure from any increase in regurgitant volume. The murmur of MR is best heard over the cardiac apex. The intensity of the murmur increases with maneuvers that increase LV afterload, such as sustained hand grip. The murmur of a VSD (without significant pulmonary hypertension) is holosystolic and loudest at the mid-left sternal border, where a thrill is usually present. The murmur of TR is loudest at the lower left sternal border, increases in intensity with inspiration (Carvallo’s sign), and is accompanied by visible

1	border, where a thrill is usually present. The murmur of TR is loudest at the lower left sternal border, increases in intensity with inspiration (Carvallo’s sign), and is accompanied by visible cv waves in the jugular venous wave form and, on occasion, by pulsatile hepatomegaly.

1	Diastolic Murmurs In contrast to some systolic murmurs, diastolic heart murmurs always signify structural heart disease (Fig. 267-5). The murmur associated with acute, severe AR is relatively soft and of short duration because of the rapid rise in LV diastolic pressure and the progressive diminution of the aortic-LV diastolic pressure gradient. In contrast, the murmur of chronic severe AR is classically heard as a decrescendo, blowing diastolic murmur along the left sternal border in patients with primary valve pathology and sometimes along the right sternal border in patients with primary aortic root pathology.

1	With chronic AR, the pulse pressure is wide 1449 and the arterial pulses are bounding in character. These signs of significant diastolic run-off are absent in the acute phase. The murmur of pulmonic regurgitation is also heard along the left sternal border. It is most commonly due to pulmonary hypertension and enlargement of the annulus of the pulmonic valve. S2 is single and loud and may be palpable. There is a right ventricular/parasternal lift that is indicative of chronic right ventricular pressure overload. A less impressive murmur of PR is present after repair of tetralogy of Fallot or pulmonic valve atresia. In this postoperative setting, the murmur is softer and lower-pitched, and the severity of the accompanying pulmonic regurgitation can be underestimated significantly.

1	MS is the classic cause of a midto late diastolic murmur, which is best heard over the apex in the left lateral decubitus position, to the generation of mid-diastolic murmurs that are created by increased and accelerated transvalvular diastolic flow, even in the absence of valvular obstruction, in the setting of severe MR, severe TR, or a large ASD with left-to-right shunting. The Austin Flint murmur of chronic severe AR is a low-pitched midto late apical diastolic murmur that sometimes can be confused with MS. The Austin Flint murmur typically decreases in intensity after exposure to vasodilators, whereas the murmur of MS may be accompanied by an opening snap and also may increase in intensity after vasodilators because of the associated increase in cardiac output. Unusual causes of a mid-diastolic murmur include atrial myxoma, complete heart block, and acute rheumatic mitral valvulitis.

1	Continuous Murmur A continuous murmur is predicated on a pressure gradient that persists between two cardiac chambers or blood vessels across systole and diastole. The murmurs typically begin in systole, envelop the second heart sound (S2), and continue through some portion of diastole. They can often be difficult to distinguish from individual systolic and diastolic murmurs in patients with mixed valvular heart disease. The classic example of a continuous murmur is that associated with a PDA, which usually is heard in the second or third interspace at a slight distance from the sternal border. Other causes of a continuous murmur include a ruptured sinus of Valsalva aneurysm with creation of an aortic–right atrial or right ventricular fistula, a coronary or great vessel arteriovenous fistula, and an arteriovenous fistula constructed to provide dialysis access. There are two types of benign continuous murmurs. The cervical venous hum is heard in children or adolescents in the

1	fistula, and an arteriovenous fistula constructed to provide dialysis access. There are two types of benign continuous murmurs. The cervical venous hum is heard in children or adolescents in the supraclavicular fossa. It can be obliterated with firm pressure applied to the diaphragm of the stethoscope, especially when the subject turns his or her head toward the examiner. The mammary soufflé of pregnancy relates to enhanced arterial blood flow through engorged breasts. The diastolic component of the murmur can be obliterated with firm pressure over the stethoscope.

1	Dynamic Auscultation Diagnostic accuracy can be enhanced by the performance of simple bedside maneuvers to identify heart murmurs and characterize their significance (Table 267-1). Except for the pulmonic Respiration Right-sided murmurs and sounds generally increase with inspiration, except for the PES. Left-sided murmurs and sounds are usually louder during expiration. Valsalva maneuver Most murmurs decrease in length and intensity. Two exceptions are the systolic murmur of HOCM, which usually becomes much louder, and that of MVP, which becomes longer and often louder. After release of the Valsalva maneuver, right-sided murmurs tend to return to control intensity earlier than do left-sided murmurs.

1	After VPB or AF Murmurs originating at normal or stenotic semilunar valves increase in the cardiac cycle after a VPB or in the cycle after a long cycle length in AF. By contrast, systolic murmurs due to AV valve regurgitation do not change, diminish (papillary muscle dysfunction), or become shorter (MVP). Positional changes With standing, most murmurs diminish, with two exceptions being the murmur of HOCM, which becomes louder, and that of MVP, which lengthens and often is intensified. With squatting, most murmurs become louder, but those of HOCM and MVP usually soften and may disappear. Passive leg raising usually produces the same results.

1	Exercise Murmurs due to blood flow across normal or obstructed valves (e.g., PS, MS) become louder with both isotonic and submaximal isometric (hand grip) exercise. Murmurs of MR, VSD, and AR also increase with hand grip exercise. However, the murmur of HOCM often decreases with nearly maximum hand grip exercise. Left-sided S4 and S3 sounds are often accentuated by exercise, particularly when due to ischemic heart disease. Abbreviations: AF, atrial fibrillation; AR, aortic regurgitation; HOCM, hypertrophic obstructive cardiomyopathy; MR, mitral regurgitation; MS, mitral stenosis; MVP, mitral valve prolapse; PES, pulmonic ejection sound; PR, pulmonic regurgitation; PS, pulmonic stenosis; TR, tricuspid regurgitation; TS, tricuspid stenosis; VPB, ventricular premature beat; VSD, ventricular septal defect.

1	ejection sound, right-sided events increase in intensity with inspiration and decrease with expiration; left-sided events behave oppositely (100% sensitivity, 88% specificity). As previously noted, the intensity of the murmurs associated with MR, VSD, and AR will increase in response to maneuvers that increase LV afterload, such as hand grip and vasopressors. The intensity of these murmurs will decrease after exposure to vasodilating agents. Squatting is associated with an abrupt increase in LV preload and afterload, whereas rapid standing results in a sudden decrease in preload. In patients with MVP, the click and murmur move away from the first heart sound with squatting because of the delay in onset of leaflet prolapse at higher ventricular volumes. With rapid standing, however, the click and murmur move closer to the first heart sound as prolapse occurs earlier in systole at a smaller chamber dimension. The murmur of HOCM behaves similarly, becoming softer and shorter with

1	the click and murmur move closer to the first heart sound as prolapse occurs earlier in systole at a smaller chamber dimension. The murmur of HOCM behaves similarly, becoming softer and shorter with squatting (95% sensitivity, 85% specificity) and longer and louder on rapid standing (95% sensitivity, 84% specificity). A change in the intensity of a systolic murmur in the first beat after a premature beat or in the beat after a long cycle length in patients with atrial fibrillation suggests valvular AS rather than MR, particularly in an older patient in whom the murmur of the AS may be well transmitted to the apex (Gallavardin effect). Of note, however, the systolic murmur of HOCM also increases in intensity in the beat after a premature beat. This increase in intensity of any LV outflow murmur in the beat after a premature beat relates to the combined effects of enhanced LV filling (from the longer diastolic period) and postextrasystolic potentiation of LV contractile function. In

1	murmur in the beat after a premature beat relates to the combined effects of enhanced LV filling (from the longer diastolic period) and postextrasystolic potentiation of LV contractile function. In either instance, forward flow will accelerate, causing an increase in the gradient across the LV outflow tract (dynamic or fixed) and a louder systolic murmur. In contrast, the intensity of the murmur of MR does not change in a postpremature beat, because there is relatively little change in the nearly constant LV to left atrial pressure gradient or further alteration in mitral valve flow. Bedside exercise can sometimes be performed to increase cardiac output and, secondarily, the intensity of both systolic and diastolic heart murmurs. Most left-sided heart murmurs decrease in intensity and duration during the strain phase of the Valsalva maneuver. The murmurs associated with MVP and HOCM are the two notable exceptions. The Valsalva maneuver also can be used to assess the integrity of the

1	during the strain phase of the Valsalva maneuver. The murmurs associated with MVP and HOCM are the two notable exceptions. The Valsalva maneuver also can be used to assess the integrity of the heart and vasculature in the setting of advanced heart failure.

1	Prosthetic Heart Valves The first clue that prosthetic valve dysfunction may contribute to recurrent symptoms is frequently a change in the quality of the heart sounds or the appearance of a new murmur. The heart sounds with a bioprosthetic valve resemble those generated by native valves. A mitral bioprosthesis usually is associated with a grade 2 or 3 midsystolic murmur along the left sternal border (created by turbulence across the valve struts as they project into the LV outflow tract) as well as by a soft mid-diastolic murmur that occurs with normal LV filling. This diastolic murmur often can be heard only in the left lateral decubitus position and after exercise. A high pitched or holosystolic apical murmur is indicative of pathologic MR due to a paravalvular leak and/or intra-annular bioprosthetic regurgitation from leaflet degeneration, for which additional imaging is usually indicated. Clinical deterioration can occur rapidly after the first expression of mitral bioprosthetic

1	bioprosthetic regurgitation from leaflet degeneration, for which additional imaging is usually indicated. Clinical deterioration can occur rapidly after the first expression of mitral bioprosthetic failure. A tissue valve in the aortic position is always associated with a grade 2 to 3 midsystolic murmur at the base or just below the suprasternal notch. A diastolic murmur of AR is abnormal in any circumstance. Mechanical valve dysfunction may first be suggested by a decrease in the intensity of either the opening or the closing sound. A high-pitched apical systolic murmur in patients with a mechanical mitral prosthesis and a diastolic decrescendo murmur in patients with a mechanical aortic prosthesis indicate paravalvular regurgitation. Patients with prosthetic valve thrombosis may present clinically with signs of shock, muffled heart sounds, and soft murmurs.

1	Pericardial Disease A pericardial friction rub is nearly 100% specific for the diagnosis of acute pericarditis, although the sensitivity of this finding is not nearly as high, because the rub may come and go over the course of an acute illness or be very difficult to elicit. The rub is heard as a leathery or scratchy three-component or two-component sound, although it may be monophasic. Classically, the three components are ventricular systole, rapid early diastolic filling, and late presystolic filling after atrial contraction in patients in sinus rhythm. It is necessary to listen to the heart in several positions. Additional clues may be present from the history and 12-lead electrocardiogram. The rub typically disappears as the volume of any pericardial effusion increases. Pericardial tamponade can be diagnosed with a sensitivity of 98%, a specificity of 83%, and a positive likelihood ratio of 5.9 (95% confidence interval 2.4–14) by a pulsus paradoxus that exceeds 12 mmHg in a

1	tamponade can be diagnosed with a sensitivity of 98%, a specificity of 83%, and a positive likelihood ratio of 5.9 (95% confidence interval 2.4–14) by a pulsus paradoxus that exceeds 12 mmHg in a patient with a large pericardial effusion.

1	The findings on physical examination are integrated with the symptoms previously elicited with a careful history to construct an appropriate differential diagnosis and proceed with indicated imaging and laboratory assessment. The physical examination is an irreplaceable component of the diagnostic algorithm and in selected patients can inform prognosis. Educational efforts to improve clinician competence eventually may result in cost saving, particularly if the indications for imaging can be influenced by the examination findings. Ary L. Goldberger An electrocardiogram (ECG or EKG) is a graphic recording of electric potentials generated by the heart. The signals are detected by means of metal electrodes attached to the extremities and chest wall and then are amplified and recorded by the electrocardiograph. ECG leads actually display the instantaneous differences in potential between the electrodes.

1	The clinical utility of the ECG derives from its immediate availability as a noninvasive, inexpensive, and highly versatile test. In addition to its use in detecting arrhythmias, conduction disturbances, and myocardial ischemia, electrocardiography may reveal findings related to life-threatening metabolic disturbances (e.g., hyperkalemia) or increased susceptibility to sudden cardiac death (e.g., QT prolongation syndromes). (See also Chaps. 274 and 276) Depolarization of the heart is the initiating event for cardiac contraction. The electric currents that spread through the heart are produced by three components: cardiac pacemaker cells, specialized conduction tissue, and the heart muscle itself. The ECG, however, records only the depolarization (stimulation) and repolarization (recovery) potentials generated by the “working” atrial and ventricular myocardium.

1	The depolarization stimulus for the normal heartbeat originates in the sinoatrial (SA) node (Fig. 268-1), or sinus node, a collection of pacemaker cells. These cells fire spontaneously; that is, they exhibit automaticity. The first phase of cardiac electrical activation is the spread of the depolarization wave through the right and left atria, followed by atrial contraction. Next, the impulse stimulates pacemaker and specialized conduction tissues in the atrioventricular (AV) nodal and His-bundle areas; together, these two regions constitute the AV junction. The bundle of His bifurcates into two main branches, the right and left bundles, which rapidly transmit depolarization wavefronts to the right and left ventricular myocardium by way of Purkinje fibers. The main left bundle bifurcates into two primary subdivisions: a left anterior fascicle and a left posterior fascicle. The depolarization wavefronts then spread through the ventricular wall, from endocardium to epicardium,

1	into two primary subdivisions: a left anterior fascicle and a left posterior fascicle. The depolarization wavefronts then spread through the ventricular wall, from endocardium to epicardium, triggering ventricular contraction.

1	Since the cardiac depolarization and repolarization waves have direction and magnitude, they can be represented by vectors. Vector analysis illustrates a central concept of electrocardiography: The ECG records the complex spatial and temporal summation of electrical potentials from multiple myocardial fibers conducted to the surface of the body. This principle accounts for inherent limitations in both ECG sensitivity (activity from certain cardiac regions may be canceled out or may be too weak to be recorded) and specificity (the same vectorial sum can result from either a selective gain or a loss of forces in opposite directions).

1	The ECG waveforms are labeled alphabetically, beginning with the P wave, which represents atrial depolarization (Fig. 268-2). The QRS complex represents ventricular depolarization, and the ST-T-U complex (ST segment, T wave, and U wave) represents ventricular repolarization. The J point is the junction between the end of the QRS complex and the beginning of the ST segment. Atrial repolarization (STa and T a) is usually too low in amplitude to be detected, but it may become apparent in conditions such as acute pericarditis and atrial infarction. The QRS-T waveforms of the surface ECG correspond in a general way with the different phases of simultaneously obtained ventricular action potentials, the intracellular recordings from single myocardial FIGURE 268-1 Schematic of the cardiac conduction system. FIGURE 268-2 Basic ECG waveforms and intervals. Not shown is the RR interval, the time between consecutive QRS complexes.

1	FIGURE 268-1 Schematic of the cardiac conduction system. FIGURE 268-2 Basic ECG waveforms and intervals. Not shown is the RR interval, the time between consecutive QRS complexes. fibers (Chap. 274). The rapid upstroke (phase 0) of the action potential corresponds to the onset of QRS. The plateau (phase 2) corresponds to the isoelectric ST segment, and active repolarization (phase 3) corresponds to the inscription of the T wave. Factors that decrease the slope of phase 0 by impairing the influx of Na+ (e.g., hyperkalemia and drugs such as flecainide) tend to increase QRS duration. Conditions that prolong phase 2 (amiodarone, hypocalcemia) increase the QT interval. In contrast, shortening of ventricular repolarization (phase 2), such as by digitalis administration or hypercalcemia, abbreviates the ST segment.

1	The ECG ordinarily is recorded on special graph paper that is divided into 1-mm2 gridlike boxes. Since the usual ECG paper speed is 25 mm/s, the smallest (1 mm) horizontal divisions correspond to 0.04 (40 ms), with heavier lines at intervals of 0.20 s (200 ms). Vertically, the ECG graph measures the amplitude of a specific wave or deflection (1 mV = 10 mm with standard calibration; the voltage criteria for hypertrophy mentioned below are given in millimeters). There are four major ECG intervals: RR, PR, QRS, and QT (Fig. 268-2). The heart rate (beats per minute) can be computed readily from the interbeat (RR) interval by dividing the number of large (0.20 s) time units between consecutive R waves into 300 or the number of small (0.04 s) units into 1500. The PR interval measures the time (normally 120–200 ms) between atrial and ventricular depolarization, which includes the physiologic delay imposed by stimulation of cells in the AV junction area. The QRS interval (normally 100–110 ms

1	120–200 ms) between atrial and ventricular depolarization, which includes the physiologic delay imposed by stimulation of cells in the AV junction area. The QRS interval (normally 100–110 ms or less) reflects the duration of ventricular depolarization. The QT interval includes both ventricular depolarization and repolarization times and varies inversely with the heart rate. A rate-related (“corrected”) QT interval, QTc, can be calculated as QT/√RR and normally is ≤0.44 s. (Some references give QTc upper normal limits as 0.43 s in men and 0.45 s in women. Also, a number of different formulas have been proposed, without consensus, for calculating the QTc.)

1	The QRS complex is subdivided into specific deflections or waves. If the initial QRS deflection in a particular lead is negative, it is termed a Q wave; the first positive deflection is termed an R wave. A negative deflection after an R wave is an S wave. Subsequent positive or negative waves are labeled R′ and S′, respectively. Lowercase letters (qrs) are used for waves of relatively small amplitude. An entirely negative QRS complex is termed a QS wave. The 12 conventional ECG leads record the difference in potential between electrodes placed on the surface of the body. These leads are divided into two groups: six limb (extremity) leads and six chest (precordial) leads. The limb leads record potentials transmitted onto the frontal plane (Fig. 268-3A), and the chest leads record potentials transmitted onto the horizontal plane (Fig. 268-3B). + V4 deflection will be recorded.

1	+ V4 deflection will be recorded. FIGURE 268-3 The six frontal plane (A) and six horizontal plane (B) leads provide a P WAVE ambulatory ECG (Holter) recordings usually employ only one or two modified leads. Intracardiac electrocardiography and electrophysiologic testing are discussed in Chaps. 274 and 276. The ECG leads are configured so that a positive (upright) deflection is recorded in a lead if a wave of depolarization spreads toward the positive pole of that + lead, and a negative deflection is recorded if the wave spreads toward the negative pole. If the mean orientation + of the depolarization vector is at right angles to a particu lar lead axis, a biphasic (equally positive and negative) three-dimensional representation of cardiac electrical activity.

1	The spatial orientation and polarity of the six frontal plane leads is represented on the hexaxial diagram (Fig. 268-4). The six chest leads (Fig. 268-5) are unipolar recordings obtained by electrodes in the following positions: lead V1, fourth intercostal space, just to the right of the sternum; lead V2, fourth intercostal space, just to the left of the sternum; lead V3, midway between V2 and V4; lead V4, midclavicular line, fifth intercostal space; lead V5, anterior axillary line, same level as V4; and lead V6, midaxillary line, same level as V4 and V5. Additional posterior leads are sometimes placed on the same horizontal plane as V4 to facilitate detection of acute posterolateral infarction (V7, midaxillary line; V8 posterior axillary line; and V9, posterior scapular line).

1	Together, the frontal and horizontal plane electrodes provide a three-dimensional representation of cardiac electrical activity. Each lead can be likened to a different video camera angle “looking” at the same events—atrial and ventricular depolarization and repolarization— from different spatial orientations. The conventional 12-lead ECG can be supplemented with additional leads in special circumstances. For example, right precordial leads V3R, V4R, etc., are useful in detecting evidence of acute right ventricular ischemia. Bedside monitors and The normal atrial depolarization vector is oriented down

1	Rightaxisdeviation Normalaxis Leftaxisdeviation –90° –aVF –60° –III –30° +aVL 0° +I +30° –aVR +60° +II +90° +aVF +120° +III +150° – aVL 180° –I –150° +aVR –120° –II Extremeaxisdeviation FIGURE 268-4 The frontal plane (limb or extremity) leads are rep-resented on a hexaxial diagram. Each ECG lead has a specific spatial orientation and polarity. The positive pole of each lead axis (solid line) and the negative pole (hatched line) are designated by their angular position relative to the positive pole of lead I (0°). The mean electrical axis of the QRS complex is measured with respect to this display.

1	ward and toward the subject’s left, reflecting the spread of depolarization from the sinus node to the right and then the left atrial myocardium. Since this vector points toward the positive pole of lead II and toward the negative pole of lead aVR, the normal P wave will be positive in lead II and negative in lead aVR. By contrast, activation of the atria from an ectopic pacemaker in the lower part of either atrium or in the AV junction region may produce retrograde P waves (negative in lead II, positive in lead aVR). The normal P wave in lead V1 may be biphasic with a positive component reflecting right atrial depolarization, followed by a small (<1 mm2) negative component reflecting left atrial depolarization.

1	Normal ventricular depolarization proceeds as a rapid, continuous spread of activation wave fronts. This complex process can be divided into two major sequential phases, and each phase can be represented by a mean vector (Fig. 268-6). The first phase is depolarization of the interventricular septum from the left to the right and anteriorly (vector 1). The second results from the simultaneous depolarization of the right and left ventricles; it normally is dominated by the more massive left ventricle, so that vector 2 points leftward and posteriorly. Therefore, a right precordial lead (V1) will record this biphasic depolarization process with a small positive deflection (septal r wave) followed by a larger negative deflection (S wave). A left precordial lead, e.g., V6, will record the same sequence with a small negative deflection (septal q wave) followed by a relatively tall positive deflection (R wave). Intermediate leads show a relative increase in R-wave amplitude (normal R-wave

1	sequence with a small negative deflection (septal q wave) followed by a relatively tall positive deflection (R wave). Intermediate leads show a relative increase in R-wave amplitude (normal R-wave progression) and a decrease in S-wave amplitude progressing across the chest from right to left. The precordial lead where

1	V1V3RV4RV2V3V4V5V6FIGURE 268-5 The horizontal plane (chest or precordial) leads are obtained with electrodes in the locations shown. major phases, each represented by a vector. A. The first phase (arrow 1) denotes depolarization of the ventricular septum, beginning on the left side and spreading to the right. This process is represented by a small “septal” r wave in lead V1 and a small septal q wave in lead V6. B. Simultaneous depolarization of the left and right ventricles (LV and RV) constitutes the second phase. Vector 2 is oriented to the left and posteriorly, reflecting the electrical predominance of the LV. C. Vectors (arrows) representing these two phases are shown in reference to the horizontal plane leads. (After AL Goldberger et al: Goldberger’s Clinical Electrocardiography: A Simplified Approach, 8th ed. Philadelphia, Elsevier/Saunders, 2013.) the R and S waves are of approximately equal amplitude is referred to as the transition zone (usually V3 or V4) (Fig. 268-7).

1	The QRS pattern in the extremity leads may vary considerably from one normal subject to another depending on the electrical axis of the QRS, which describes the mean orientation of the QRS vector with reference to the six frontal plane leads. Normally, the QRS axis ranges from –30° to +100° (Fig. 268-4). An axis more negative than –30° is referred to as left axis deviation, and an axis more positive than +100° is referred to as right axis deviation. Left axis deviation may occur as a normal variant but is more commonly associated with left ventricular hypertrophy, also may occur as a normal variant (particularly in children and young 1453 adults), as a spurious finding due to reversal of the left and right arm electrodes, or in conditions such as right ventricular overload (acute or chronic), infarction of the lateral wall of the left ventricle, dextrocardia, left pneumothorax, and left posterior fascicular block.

1	Normally, the mean T-wave vector is oriented roughly concordant with the mean QRS vector (within about 45° in the frontal plane). Since depolarization and repolarization are electrically opposite processes, this normal QRS–T-wave vector concordance indicates that repolarization normally must proceed in the reverse direction from depolarization (i.e., from ventricular epicardium to endocardium). The normal U wave is a small, rounded deflection (≤1 mm) that follows the T wave and usually has the same polarity as the T wave. An abnormal increase in U-wave amplitude is most commonly due to drugs (e.g., dofetilide, amiodarone, sotalol, quinidine) or to hypokalemia. Very prominent U waves are a marker of increased susceptibility to the torsades de pointes type of ventricular tachycardia (Chap. 276). Inversion of the U wave in the precordial leads is abnormal and may be a subtle sign of ischemia.

1	Right atrial overload (acute or chronic) may lead to an increase in P-wave amplitude (≥2.5 mm) (Fig. 268-8), sometimes referred to as “P-pulmonale.” Left atrial overload typically produces a biphasic P wave in V1 with a broad negative component or a broad (≥120 ms), often notched P wave in one or more limb leads (Fig. 268-8). This pattern, previously referred to as “P-mitrale,” may also occur with left atrial conduction delays in the absence of actual atrial enlargement, leading to the more general designation of left atrial abnormality.

1	Right ventricular hypertrophy due to a sustained, severe pressure load (e.g., due to tight pulmonic valve stenosis or certain pulmonary artery hypertension syndromes) is characterized by a relatively tall R wave in lead V1 (R ≥ S wave), usually with right axis deviation (Fig. 268-9); alternatively, there may be a qR pattern in V1 or V3R. ST depression and T-wave inversion in the right-to-midprecordial leads are also often present. This pattern, formerly called right ventricular “strain,” is attributed to repolarization abnormalities in acutely or chronically overloaded muscle. Prominent S waves may occur in the left lateral precordial leads. Right ventricular hypertrophy due to ostium secundum–type atrial septal defects, with the accompanying a block in the anterior fascicle of the left FIGURE 268-7 Normal electrocardiogram from a healthy subject. Sinus rhythm is present with bundle system (left anterior fascicular a heart rate of 75 beats per minute. PR interval is 0.16 s; QRS

1	left FIGURE 268-7 Normal electrocardiogram from a healthy subject. Sinus rhythm is present with bundle system (left anterior fascicular a heart rate of 75 beats per minute. PR interval is 0.16 s; QRS interval (duration) is 0.08 s; QT interval block or hemiblock), or inferior myo-is 0.36 s; QTc is 0.40 s; the mean QRS axis is about +70°. The precordial leads show normal R-wave cardial infarction. Right axis deviation progression with the transition zone (R wave = S wave) in lead V3.

1	FIGURE 268-8 Right atrial (RA) overload may cause tall, peaked P waves in the limb or precordial leads. Left atrial (LA) abnormality may cause broad, often notched P waves in the limb leads and a biphasic P wave in lead V1 with a prominent negative component representing delayed depolarization of the LA. (After MK Park, WG Guntheroth: How to Read Pediatric ECGs, 4th ed. St. Louis, Mosby/ Elsevier, 2006.) FIGURE 268-9 Left ventricular hypertrophy (LVH) increases the amplitude of electrical forces directed to the left and posteriorly. In addition, repolarization abnormalities may cause ST-segment depression and T-wave inversion in leads with a prominent R wave. Right ventricular hypertrophy (RVH) may shift the QRS vector to the right; this effect usually is associated with an R, RS, or qR complex in lead V1. T-wave inversions may be present in right precordial leads.

1	right ventricular volume overload, is commonly associated with an incomplete or complete right bundle branch block pattern with a rightward QRS axis. Acute cor pulmonale due to pulmonary embolism (Chap. 300), for example, may be associated with a normal ECG or a variety of abnormalities. Sinus tachycardia is the most common arrhythmia, although other tachyarrhythmias, such as atrial fibrillation or flutter, may occur. The QRS axis may shift to the right, sometimes in concert with the so-called S1Q3T3 pattern (prominence of the S wave in lead I and the Q wave in lead III, with T-wave inversion in lead III). Acute right ventricular dilation also may be associated with slow R-wave progression and ST-T abnormalities in V1 to V4 simulating acute anterior infarction. A right ventricular conduction disturbance may appear.

1	Chronic cor pulmonale due to obstructive lung disease (Chap. 279) usually does not produce the classic ECG patterns of right ventricular hypertrophy noted above. Instead of tall right precordial R waves, chronic lung disease more typically is associated with small R waves in right-to-midprecordial leads (slow R-wave progression) due in part to downward displacement of the diaphragm and the heart. Low-voltage complexes are commonly present, owing to hyperaeration of the lungs.

1	A number of different voltage criteria for left ventricular hypertrophy (Fig. 268-9) have been proposed on the basis of the presence of tall left precordial R waves and deep right precordial S waves (e.g., SV1 + [RV5 or RV6] >35 mm). Repolarization abnormalities (ST depression with T-wave inversions, formerly called the left ventricular “strain” pattern) also may appear in leads with prominent R waves. However, prominent precordial voltages may occur as a normal variant, especially in athletic or young individuals. Left ventricular hypertrophy may increase limb lead voltage with or without increased precordial voltage (e.g., RaVL + SV3 >20 mm in women and >28 mm in men). The presence of left atrial abnormality increases the likelihood of underlying left ventricular hypertrophy in cases with borderline voltage criteria. Left ventricular hypertrophy often progresses to incomplete or complete left bundle branch block. The sensitivity of conventional voltage criteria for left ventricular

1	borderline voltage criteria. Left ventricular hypertrophy often progresses to incomplete or complete left bundle branch block. The sensitivity of conventional voltage criteria for left ventricular hypertrophy is decreased in obese persons and smokers. ECG evidence for left ventricular hypertrophy is a major noninvasive marker of increased risk of cardiovascular morbidity and mortality rates, including sudden cardiac death. However, because of false-positive and false-negative diagnoses, the ECG is of limited utility in diagnosing atrial or ventricular enlargement. More definitive information is provided by echocardiography (Chap. 270e).

1	Intrinsic impairment of conduction in either the right or the left bundle system (intraventricular conduction disturbances) leads to prolongation of the QRS interval. With complete bundle branch blocks, the QRS interval is ≥120 ms in duration; with incomplete blocks, the QRS interval is between 100 and 120 ms. The QRS vector usually is oriented in the direction of the myocardial region where depolarization is delayed (Fig. 268-10). Thus, with right bundle branch block, the terminal QRS vector is oriented to the right and anteriorly (rSR′ in V1 and qRS in V6, typically). Left bundle branch block alters both early and later phases of ventricular depolarization. The major QRS vector is directed to the left and posteriorly. In addition, the normal early left-to-right pattern of septal activation is disrupted such that septal depolarization proceeds from right to left as well. As a result, left bundle branch block generates wide, predominantly negative (QS) complexes in lead V1 and

1	activation is disrupted such that septal depolarization proceeds from right to left as well. As a result, left bundle branch block generates wide, predominantly negative (QS) complexes in lead V1 and entirely positive (R) complexes in lead V6. A pattern identical to that of left bundle branch block, preceded by a sharp spike, is seen in most cases of electronic right ventricular pacing because of the relative delay in left ventricular activation.

1	Bundle branch block may occur in a variety of conditions. In subjects without structural heart disease, right bundle branch block is seen more commonly than left bundle branch block. Right bundle branch block also occurs with heart disease, both congenital (e.g., atrial septal defect) and acquired (e.g., valvular, ischemic). Left bundle branch block is often a marker of one of four underlying conditions associated with increased risk of cardiovascular morbidity and mortality rates: coronary heart disease (frequently with impaired left ventricular FIGURE 268-10 Comparison of typical QRS-T patterns in right bundle branch block (RBBB) and left bundle branch block (LBBB) with the normal pattern in leads V1 and V6. Note the secondary T-wave inversions (arrows) in leads with an rSR′ complex with RBBB and in leads with a wide R wave with LBBB.

1	function), hypertensive heart disease, aortic valve disease, and cardiomyopathy. Bundle branch blocks may be chronic or intermittent. A bundle branch block may be rate-related; for example, it often occurs when the heart rate exceeds some critical value.

1	Bundle branch blocks and depolarization abnormalities secondary to artificial pacemakers not only affect ventricular depolarization (QRS) but also are characteristically associated with secondary repolarization (ST-T) abnormalities. With bundle branch blocks, the T wave is typically opposite in polarity to the last deflection of the QRS (Fig. 268-10). This discordance of the QRS–T-wave vectors is caused by the altered sequence of repolarization that occurs secondary to altered depolarization. In contrast, primary repolarization abnormalities are independent of QRS changes and are related instead to actual alterations in the electrical properties of the myocardial fibers themselves (e.g., in the resting membrane potential or action potential duration), not just to changes in the sequence of repolarization. Ischemia, electrolyte imbalance, and drugs such as digitalis all cause such primary ST–T-wave changes. Primary and secondary T-wave changes may coexist. For example, T-wave

1	of repolarization. Ischemia, electrolyte imbalance, and drugs such as digitalis all cause such primary ST–T-wave changes. Primary and secondary T-wave changes may coexist. For example, T-wave inversions in the right precordial leads with left bundle branch block or in the left precordial leads with right bundle branch block may be important markers of underlying ischemia or other abnormalities. A distinctive abnormality simulating right bundle branch block with ST-segment elevarightward than +110–120°) is extremely rare as an isolated finding 1455 and requires exclusion of other factors causing right axis deviation mentioned earlier.

1	More complex combinations of fascicular and bundle branch blocks may occur that involve the left and right bundle system. Examples of bifascicular block include right bundle branch block and left posterior fascicular block, right bundle branch block with left anterior fascicular block, and complete left bundle branch block. Chronic bifascicular block in an asymptomatic individual is associated with a relatively low risk of progression to high-degree AV heart block. In contrast, new bifascicular block with acute anterior myocardial infarction carries a much greater risk of complete heart block. Alternation of right and left bundle branch block is a sign of trifascicular disease. However, the presence of a prolonged PR interval and bifascicular block does not necessarily indicate trifascicular involvement, since this combination may arise with AV node disease and bifascicular block. Intraventricular conduction delays also can be caused by extrinsic (toxic) factors that slow ventricular

1	involvement, since this combination may arise with AV node disease and bifascicular block. Intraventricular conduction delays also can be caused by extrinsic (toxic) factors that slow ventricular conduction, particularly hyperkalemia or drugs (e.g., class 1 antiarrhythmic agents, tricyclic antidepressants, phenothiazines).

1	Prolongation of QRS duration does not necessarily indicate a conduction delay but may be due to preexcitation of the ventricles via a bypass tract, as in Wolff-Parkinson-White (WPW) patterns (Chap. 276) and related variants. The diagnostic triad of WPW consists of a wide QRS complex associated with a relatively short PR interval and slurring of the initial part of the QRS (delta wave), with the latter effect being due to aberrant activation of ventricular myocardium. The presence of a bypass tract predisposes to reentrant supraventricular tachyarrhythmias.

1	(See also Chap. 295) The ECG is a cornerstone in the diagnosis of acute and chronic ischemic heart disease. The findings depend on several key factors: the nature of the process (reversible [i.e., ischemia] versus irreversible [i.e., infarction]), the duration (acute versus chronic), the extent (transmural versus subendocardial), and localization (anterior versus inferoposterior), as well as the presence of other underlying abnormalities (ventricular hypertrophy, conduction defects).

1	Ischemia exerts complex time-dependent effects on the electrical properties of myocardial cells. Severe, acute ischemia lowers the resting membrane potential and shortens the duration of the action potential. Such changes cause a voltage gradient between normal and ischemic zones. As a consequence, current flows between those regions. These currents of injury are represented on the surface ECG by deviation of the ST segment (Fig. 268-11). When the acute ischemia is transmural, the ST vector usually is shifted in the direction of the outer (epicardial) layers, producing ST elevations and sometimes, in the earliest stages of ischemia, tall, positive so-called hyperacute T waves over the ischemic zone. With ischemia confined primarily to the subendocardium, the ST vector typically shifts toward the subendocardium and ventricular cavity, so that overlying (e.g., anterior precordial) leads show ST-segment depression (with ST elevation in lead aVR). Multiple factors affect the amplitude of

1	the subendocardium and ventricular cavity, so that overlying (e.g., anterior precordial) leads show ST-segment depression (with ST elevation in lead aVR). Multiple factors affect the amplitude of acute ischemic ST deviations. Profound ST elevation or depression in multiple leads usually indicates very severe ischemia.

1	tions in the right chest leads is seen with the A

1	Brugada pattern (Chap. 276). Partial blocks (fascicular or “hemiblocks”) in the left bundle system (left anterior or posterior fascicular blocks) generally do not prolong the QRS duration substantially but instead are associated with shifts in the frontal plane QRS axis (leftward or rightward, ST respectively). Left anterior fascicular block FIGURE 268-11 Acute ischemia causes a current of injury. With predominant subendocar(QRS axis more negative than –45°) is prob-dial ischemia (A), the resultant ST vector will be directed toward the inner layer of the affected ably the most common cause of marked ventricle and the ventricular cavity. Overlying leads therefore will record ST depression. With left axis deviation in adults. In contrast, left ischemia involving the outer ventricular layer (B) (transmural or epicardial injury), the ST vector posterior fascicular block (QRS axis more will be directed outward. Overlying leads will record ST elevation.

1	1456 From a clinical viewpoint, the division of V1 V2 V3 V4 V5 V6 acute myocardial infarction into ST-segment elevation and non-ST elevation types is useful since the efficacy of acute reperfusion therapy is limited to the former group. The ECG leads are usually more helpful in localizing regions of ST elevation than non-ST elevation ischemia. For example, acute transmural anterior (including apical and FIGURE 268-12 Severe anterior wall ischemia (with or without infarction) may cause promilateral) wall ischemia is reflected by ST eleva-nent T-wave inversions in the precordial leads. This pattern (sometimes referred to as Wellens tions or increased T-wave positivity in one T waves) is usually associated with a high-grade stenosis of the left anterior descending or more of the precordial leads (V1–V6) and coronary artery. leads I and aVL. Inferior wall ischemia produces changes in leads II, III, and aVF. “Posterior” wall ischemia (usu-tissue may lead to decreased R-wave amplitude or

1	leads (V1–V6) and coronary artery. leads I and aVL. Inferior wall ischemia produces changes in leads II, III, and aVF. “Posterior” wall ischemia (usu-tissue may lead to decreased R-wave amplitude or abnormal Q waves ally associated with lateral or inferior involvement) may be indirectly (even in the absence of transmurality) in the anterior or inferior leads recognized by reciprocal ST depressions in leads V1 to V3 (thus consti-(Fig. 268-13). Previously, abnormal Q waves were considered marktuting an ST elevation “equivalent” acute coronary syndrome). Right ers of transmural myocardial infarction, whereas subendocardial ventricular ischemia usually produces ST elevations in right-sided infarcts were thought not to produce Q waves. However, careful ECG-chest leads (Fig. 268-5). When ischemic ST elevations occur as the ear-pathology correlative studies have indicated that transmural infarcts liest sign of acute infarction, they typically are followed within a period may occur without Q

1	ST elevations occur as the ear-pathology correlative studies have indicated that transmural infarcts liest sign of acute infarction, they typically are followed within a period may occur without Q waves and that subendocardial (nontransmural) ranging from hours to days by evolving T-wave inversions and often infarcts sometimes may be associated with Q waves. Therefore, infarcts by Q waves occurring in the same lead distribution. Reversible trans-are more appropriately classified as “Q-wave” or “non-Q-wave.” The mural ischemia, for example, due to coronary vasospasm (Prinzmetal’s major acute ECG changes in syndromes of ischemic heart disease variant angina and possibly the Tako-tsubo “stress” cardiomyopathy are summarized schematically in Fig. 268-14. Loss of depolarizasyndrome), may cause transient ST-segment elevations without devel-tion forces due to posterior or lateral infarction may cause reciprocal opment of Q waves, as may very early reperfusion in acute coronary increases in

1	transient ST-segment elevations without devel-tion forces due to posterior or lateral infarction may cause reciprocal opment of Q waves, as may very early reperfusion in acute coronary increases in R-wave amplitude in leads V1 and V2 without diagnostic Q syndromes. Depending on the severity and duration of ischemia, the waves in any of the conventional leads. Atrial infarction may be asso-ST elevations may resolve completely in minutes or be followed by ciated with PR-segment deviations due to an atrial current of injury, T-wave inversions that persist for hours or even days. Patients with changes in P-wave morphology, or atrial arrhythmias. In the weeks ischemic chest pain who present with deep T-wave inversions in mul-and months after infarction, these ECG changes may persist or begin tiple precordial leads (e.g., V1–V4,, I, and aVL) with or without cardiac to resolve. Complete normalization of the ECG after Q-wave infarcenzyme elevations typically have severe obstruction in the

1	tiple precordial leads (e.g., V1–V4,, I, and aVL) with or without cardiac to resolve. Complete normalization of the ECG after Q-wave infarcenzyme elevations typically have severe obstruction in the left anterior tion is uncommon but may occur, particularly with smaller infarcts. In descending coronary artery system (Fig. 268-12). In contrast, patients contrast, ST-segment elevations that persist for several weeks or more whose baseline ECG already shows abnormal T-wave inversions may after a Q-wave infarct usually correlate with a severe underlying wall develop T-wave normalization (pseudonormalization) during episodes motion disorder (akinetic or dyskinetic zone), although not necessarof acute transmural ischemia. ily a frank ventricular aneurysm. ECG changes due to ischemia may

1	With infarction, depolarization (QRS) changes often accompany occur spontaneously or may be provoked by various exercise protocols repolarization (ST-T) abnormalities. Necrosis of sufficient myocardial (stress electrocardiography; Chap. 293). A ECG sequence with anterior Q-wave infarction FIGURE 268-13 Sequence of depolarization and repolarization changes with (A) acute anterior and (B) acute inferior wall Q-wave infarc-tions. With anterior infarcts, ST elevation in leads I and aVL and the precordial leads may be accompanied by reciprocal ST depressions in leads II, III, and aVF. Conversely, acute inferior (or posterolateral) infarcts may be associated with reciprocal ST depressions in leads V1 to V3. (After AL Goldberger et al: Goldberger’s Clinical Electrocardiography: A Simplified Approach, 8th ed. Philadelphia, Elsevier/Saunders, 2013.)

1	Noninfarction subendocardial ischemia (classic angina) Transient ST depressions Noninfarction transmural ischemia Transient ST elevations or paradoxical T wave normalization, sometimes followed by T wave inversions Non-Q wave (Non-ST elevation) infarction ST depressions or T wave inversions without Q waves MYOCARDIAL ISCHEMIA ST elevation/ Q wave infarction New Q waves preceded by hyperacute T waves/ST elevations and followed by T wave inversions FIGURE 268-14 Variability of ECG patterns with acute myocardial ischemia. The ECG also may be normal or nonspecifically abnormal. Furthermore, these categorizations are not mutually exclusive. (After AL Goldberger et al: Goldberger’s Clinical Electrocardiography: A Simplified Approach, 8th ed. Philadelphia, Elsevier/Saunders, 2013.)

1	The ECG has important limitations in both sensitivity and specificity in the diagnosis of ischemic heart disease. Although a single normal ECG does not exclude ischemia or even acute infarction, a normal ECG throughout the course of an acute infarct is distinctly uncommon. Prolonged chest pain without diagnostic ECG changes therefore should always prompt a careful search for other noncoronary causes of chest pain (Chap. 19). Furthermore, the diagnostic changes of acute or evolving ischemia are often masked by the presence of left bundle branch block, electronic ventricular pacemaker patterns, and Wolff-Parkinson-White preexcitation. However, clinicians continue to over-diagnose ischemia or infarction based on the presence of ST-segment elevations or depressions; T-wave inversions; tall, positive T waves; or Q waves not related to ischemic heart disease (pseudoinfarct patterns). For example, ST-segment elevations simulating ischemia may occur with acute pericarditis or myocarditis, as

1	T waves; or Q waves not related to ischemic heart disease (pseudoinfarct patterns). For example, ST-segment elevations simulating ischemia may occur with acute pericarditis or myocarditis, as a normal variant (including the typical “early repolarization” pattern), or in a variety of other conditions (Table 268-1). Similarly, tall, positive T waves do not invariably represent hyperacute ischemic changes but may also be caused by normal variants, hyperkalemia, cerebrovascular injury, and left ventricular volume overload due to mitral or aortic regurgitation, among other causes.

1	ST-segment elevations and tall, positive T waves are common findings in leads V1 and V2 in left bundle branch block or left ventricular hypertrophy in the absence of ischemia. The differential diagnosis of Q waves includes physiologic or positional variants, ventricular hypertrophy, acute or chronic noncoronary myocardial injury, hypertrophic cardiomyopathy, and ventricular conduction disorders. Digoxin, ventricular hypertrophy, hypokalemia, and a variety of other factors may cause ST-segment depression mimicking subendocardial ischemia. Prominent T-wave inversion may occur with ventricular hypertrophy, cardiomyopathies, myocarditis, and cerebrovascular injury (particularly intracranial bleeds), among many other conditions.

1	A variety of metabolic and pharmacologic agents alter the ECG and, in particular, cause changes in repolarization (ST-T-U) and sometimes QRS prolongation. Certain life-threatening electrolyte disturbances may be diagnosed initially and monitored from the ECG. Hyperkalemia produces a sequence of changes (Fig. 268-15), usually beginning with narrowing and peaking (tenting) of the T waves. Further elevation of extracellular K+ leads to AV conduction disturbances, diminution in P-wave amplitude, and widening of the QRS interval. Severe hyperkalemia Noninfarction, transmural ischemia (Prinzmetal’s angina, and probably Tako-tsubo syndrome, which may also exactly simulate classical acute infarction) Brugada patterns (right bundle branch block–like pattern with ST elevations in right precordial leads)a Trauma to ventricles aUsually localized to V1–V2 or V3.

1	Brugada patterns (right bundle branch block–like pattern with ST elevations in right precordial leads)a Trauma to ventricles aUsually localized to V1–V2 or V3. Source: Modified from AL Goldberger et al: Goldberger’s Clinical Electrocardiography: A Simplified Approach, 8th ed. Philadelphia, Elsevier/Saunders, 2013.

1	eventually causes cardiac arrest with a slow sinusoidal type of mechanism (“sine-wave” pattern) followed by asystole. Hypokalemia (Fig. 268-16) prolongs ventricular repolarization, often with prominent U waves. Prolongation of the QT interval is also seen with drugs that increase the duration of the ventricular action potential: class 1A antiarrhythmic agents and related drugs (e.g., quinidine, disopyramide, procainamide, tricyclic antidepressants, phenothiazines) and class III agents (e.g., amiodarone [Fig. 268-16], dofetilide, dronedarone, sotalol, ibutilide). Marked QT prolongation, sometimes with deep, wide T-wave inversions, may occur with intracranial bleeds, particularly subarachnoid hemorrhage (“CVA T-wave” pattern) (Fig. 268-16). Systemic hypothermia also prolongs repolarization, usually with a distinctive convex elevation of the J point (Osborn wave). Hypocalcemia typically prolongs the QT interval (ST portion), whereas hypercalcemia shortens it (Fig. 268-17). Digitalis

1	usually with a distinctive convex elevation of the J point (Osborn wave). Hypocalcemia typically prolongs the QT interval (ST portion), whereas hypercalcemia shortens it (Fig. 268-17). Digitalis glycosides also shorten the QT interval, often with a characteristic “scooping” of the ST–T-wave complex (digitalis effect).

1	Many other factors are associated with ECG changes, particularly alterations in ventricular repolarization. T-wave flattening, minimal T-wave inversions, or slight ST-segment depression (“nonspecific ST–T-wave changes”) may occur with a variety of electrolyte and acid-base disturbances, a number of infectious processes, central nervous system disorders, endocrine abnormalities, many drugs, ischemia, hypoxia, and virtually any type of cardiopulmonary abnormality. Although subtle ST–T-wave changes may be markers of ischemia, transient nonspecific repolarization changes may also occur after a meal or with postural (orthostatic) change, hyperventilation, or exercise in healthy individuals.

1	Low QRS voltage is arbitrarily defined as peak-to-trough QRS amplitudes of ≤5 mm in the six limb leads and/or ≤10 mm in the chest leads. Multiple factors may be responsible. Among the most serious include pericardial (Fig. 268-18) or pleural effusions, chronic obstructive pulmonary disease, infiltrative cardiomyopathies, and anasarca. Electrical alternans—a beat-to-beat alternation in one or more components of the ECG signal—is a common type of nonlinear cardiovascular response to a variety of hemodynamic and electrophysiologic

1	FIGURE 268-15 The earliest ECG change with hyperkalemia is usually peaking (“tenting”) of the T waves. With further increases in the serum potassium concentration, the QRS complexes widen, the P waves decrease in amplitude and may disappear, and finally a sine-wave pattern leads to asystole unless emergency therapy is given. (After AL Goldberger et al: Goldberger’s Clinical Electrocardiography: A Simplified Approach, 8th ed. Philadelphia, Elsevier/Saunders, 2013.) perturbations. Total electrical alternans (P-QRS-T) with sinus tachy-Hypocalcemia Normal Hypercalcemia cardia is a relatively specific sign of pericardial effusion, usually with cardiac tamponade (Fig. 268-18). The mechanism relates to a periodic II swinging motion of the heart in the effusion at a frequency exactly one-half the heart rate. In contrast, pure repolarization (ST-T or U wave) alternans is a sign of electrical instability and may precede ventricular tachyarrhythmias.

1	Accurate analysis of ECGs requires thoroughness and care. The patient’s age, gender, and clinical status should always be taken into account. Many mistakes in ECG interpretation are errors of omission. Therefore, a systematic approach is essential. The following 14 QT 0.48 s QT 0.36 s QT 0.26 s points should be analyzed carefully in every ECG: (1) standardization QT 0.52 QT 0.41 QT 0.36 (calibration) and technical features (including lead placement and artifacts), (2) rhythm, (3) heart rate, (4) PR interval/AV conduction, FIGURE 268-17 Prolongation of the Q-T interval (ST-segment QRS interval, (6) QT/QTc intervals, (7) mean QRS electrical axis, portion) is typical of hypocalcemia. Hypercalcemia may cause abbre P waves, (9) QRS voltages, (10) precordial R-wave progression, viation of the ST segment and shortening of the QT interval.

1	P waves, (9) QRS voltages, (10) precordial R-wave progression, viation of the ST segment and shortening of the QT interval. FIGURE 268-16 A variety of metabolic derangements, drug effects, and other factors may prolong ventricular repolarization with QT prolongation or prominent U waves. Prominent repolarization prolongation, particularly if due to hypokalemia, inherited “channelopathies,” or certain pharmacologic agents, indicates increased susceptibility to torsades des pointes–type ventricular tachycardia (Chap. 277). Marked systemic hypothermia is associated with a distinctive convex “hump” at the J point (Osborn wave, arrow) due to altered ventricular action potential characteristics. Note QRS and QT prolongation along with sinus tachycardia in the case of tricyclic antidepressant overdose.

1	FIgURE 268-18 Classic triad of findings for pericardial effusion with cardiac tamponade: (1) sinus tachycardia; (2) low QRS voltages; and (3) electrical alternans (best seen in leads V3 and V4 in this case; arrows). This triad is highly specific for pericardial effusion, usually with tamponade physiology, but of limited sensitivity. (Adapted from LA Nathanson et al: ECG Wave-Maven. http://ecg.bidmc.harvard.edu.) (11) abnormal Q waves, (12) ST segments, (13) T waves, and (14) U waves. Comparison with any previous ECGs is invaluable. The diagnosis and management of specific cardiac arrhythmias and conduction disturbances are discussed in Chaps. 274 and 276.

1	Computerized ECG systems are widely used for immediate retrieval of thousands of ECG records. Computer interpretation of ECGs still has major limitations. Incomplete or inaccurate readings are most likely with arrhythmias and complex abnormalities. Therefore, computerized interpretation (including measurements of basic ECG intervals) should not be accepted without careful clinician review. Atlas of Electrocardiography Ary L. Goldberger The electrocardiograms (ECGs) in this atlas supplement those illustrated in Chap. 268. The interpretations emphasize findings of specific teaching value. All of the figures are from ECG Wave-Maven, Copyright 2003, Beth Israel Deaconess Medical Center, http://ecg.bidmc.harvard.edu. The abbreviations used in this chapter are as follows: CHAPTER 269e Atlas of Electrocardiography FIguRE 269e-1 Anterior wall ischemia (deep T-wave inversions and ST-segment depressions in I, aVL, V3–V6) in a patient with LVH (increased voltage in V2–V5).

1	FIguRE 269e-1 Anterior wall ischemia (deep T-wave inversions and ST-segment depressions in I, aVL, V3–V6) in a patient with LVH (increased voltage in V2–V5). PART 10 Disorders of the Cardiovascular System FIguRE 269e-2 Acute anterolateral wall ischemia with ST elevations in V4–V6. Probable prior inferior MI with Q waves in leads II, III, and aVF. FIguRE 269e-3 Acute lateral ischemia with ST elevations in I and aVL with probable reciprocal ST depressions inferiorly (II, III, and aVF). Ischemic ST depressions also in V3 and V4. Left atrial abnormality. FIguRE 269e-4 Sinus tachycardia. Marked ischemic ST-segment elevations in inferior limb leads (II, III, aVF) and laterally (V6) suggestive of acute inferolateral MI, and prominent ST-segment depressions with upright T waves in V1–V4 are consistent with associated acute posterior MI.

1	FIguRE 269e-5 Acute, extensive anterior MI with marked ST elevations in I, aVL, V1–V6 and low amplitude pathologic Q waves in V3–V6. Marked reciprocal ST-segment depressions in III and aVF. CHAPTER 269e Atlas of Electrocardiography PART 10 Disorders of the Cardiovascular System FIguRE 269e-6 Acute anterior wall MI with ST elevations and Q waves in V1–V4 and aVL and reciprocal inferior ST depressions. FIguRE 269e-7 SR with premature atrial complexes. RBBB; pathologic Q waves and ST elevation due to acute anterior/septal MI in V1–V3. FIguRE 269e-8 Acute anteroseptal MI (Q waves and ST elevations in V1–V4) with RBBB (note terminal R waves in V1). CHAPTER 269e Atlas of Electrocardiography FIguRE 269e-9 Extensive prior MI involving inferior-posterior-lateral wall (Q waves in leads II, III, aVF, tall R waves in V1, V2, and Q waves in V5, V6). T-wave abnormalities in leads I and aVL, V5, and V6. PART 10 Disorders of the Cardiovascular System

1	PART 10 Disorders of the Cardiovascular System FIguRE 269e-10 SR with PR prolongation (“first-degree AV block”), left atrial abnormality, LVH, and RBBB. Pathologic Q waves in V1–V5 and aVL with ST elevations (a chronic finding in this patient). Findings compatible with prior anterolateral MI and left ventricular aneurysm. FIguRE 269e-11 Prior inferior-posterior MI. Wide (0.04 s) Q waves in the inferior leads (II, III, aVF); broad R wave in V1 (a Q wave “equivalent” here). Absence of right-axis deviation and the presence of upright T waves in V1–V2 are also against RVH. FIguRE 269e-12 SR with RBBB (broad terminal R wave in V1) and left anterior fascicular block (hemiblock) and pathologic anterior Q waves in V1–V3. Patient had severe multivessel coronary artery disease, with echocardiogram showing septal dyskinesis and apical akinesis. CHAPTER 269e Atlas of Electrocardiography

1	CHAPTER 269e Atlas of Electrocardiography FIguRE 269e-13 Acute pericarditis with diffuse ST elevations in I, II, III, aVF, V3–V6, without T-wave inversions. Also note concomitant PR-segment elevation in aVR and PR depression in the inferolateral leads. PART 10 Disorders of the Cardiovascular System FIguRE 269e-14 SR; diffuse ST elevations (I, II, aVL, aVF, V2–V6) with associated PR deviations (elevated PR in aVR; depressed in V4–V6); borderline low voltage. Q-wave and T-wave inversions in II, III, and aVF. Diagnosis: acute pericarditis with inferior Q-wave MI. FIguRE 269e-15 SR, prominent left atrial abnormality (see I, II, V1), right-axis deviation, and RVH (tall, relatively narrow R wave in V1) in a patient with mitral stenosis.

1	FIguRE 269e-15 SR, prominent left atrial abnormality (see I, II, V1), right-axis deviation, and RVH (tall, relatively narrow R wave in V1) in a patient with mitral stenosis. FIguRE 269e-16 SR, left atrial abnormality, and LVH by voltage criteria with borderline right-axis deviation in a patient with mixed mitral stenosis (left atrial abnormality and right-axis deviation) and mitral regurgitation (LVH). Prominent precordial T-wave inversions and QT prolongation also present. CHAPTER 269e Atlas of Electrocardiography FIguRE 269e-17 Coarse AF, tall R in V2 with vertical QRS axis (positive R in aVF) indicating RVH. Tall R in V4 may be due to concomitant LVH. Patient had severe mitral stenosis with moderate mitral regurgitation. PART 10 Disorders of the Cardiovascular System

1	PART 10 Disorders of the Cardiovascular System FIguRE 269e-18 SR; first-degree AV “block” (PR prolongation); LVH (tall R in aVL); RBBB (wide multiphasic R wave in V1) and left anterior fascicular block in a patient with HCM. Deep Q waves in I and aVL are consistent with septal hypertrophy. FIguRE 269e-19 LVH with deep T-wave inversions in limb leads and precordial leads. Striking T-wave inversions in mid-precordial leads suggest apical HCM (Yamaguchi’s syndrome). FIguRE 269e-20 Sinus tachycardia with S1Q3T3 pattern (T-wave inversion in III), incomplete RBBB, and right precordial T-wave inversions consistent with acute RV overload in a patient with pulmonary emboli. CHAPTER 269e Atlas of Electrocardiography FIguRE 269e-21 Sinus tachycardia, right-axis deviation, RVH with tall R in V1 and deep S in V6, and inverted T waves in II, III, aVF, and V1–V5 in a patient with atrial septal defect and severe pulmonary hypertension. PART 10 Disorders of the Cardiovascular System

1	PART 10 Disorders of the Cardiovascular System FIguRE 269e-22 Signs of right atrial/RV overload in a patient with chronic obstructive lung disease: (1) peaked P waves in II; (2) QR in V1 with narrow QRS; (3) delayed precordial transition, with terminal S waves in V /V ; (4) superior axis deviation with an S -S -S pattern. FIguRE 269e-23 (1) Low voltage; (2) incomplete RBBB (rsr′ in V1–V3); (3) borderline peaked P waves in lead II with vertical P-wave axis (probable right atrial overload); (4) slow R-wave progression in V1–V3; (5) prominent S waves in V6; and (6) atrial premature beats. This combination is seen typically in severe chronic obstructive lung disease. FIguRE 269e-24 Prominent U waves (II, III, and V4–V6) with ventricular repolarization prolongation in a patient with severe hypokalemia. CHAPTER 269e Atlas of Electrocardiography

1	FIguRE 269e-24 Prominent U waves (II, III, and V4–V6) with ventricular repolarization prolongation in a patient with severe hypokalemia. CHAPTER 269e Atlas of Electrocardiography FIguRE 269e-25 Abbreviated ST segment such that the T wave looks like it takes off directly from QRS in some leads (I, V4, aVL, and V5) in a patient with severe hypercalcemia. Note also high takeoff of ST segment in V2/V3 simulating acute ischemia. PART 10 Disorders of the Cardiovascular System FIguRE 269e-26 SR with LVH, left atrial abnormality, and tall peaked T waves in the precordial leads with inferolateral ST depressions (II, III, aVF, and V6); left anterior fascicular block and borderline prolonged QT interval in a patient with renal failure, hypertension, and hyperkalemia; prolonged QT is secondary to associated hypocalcemia.

1	FIguRE 269e-27 Normal ECG in an 11-year-old male. T-wave inversions in V1–V2. Vertical QRS axis (+90°) and early precordial transition between V2 and V3 are normal findings in children. FIguRE 269e-28 Left atrial abnormality and LVH in a patient with long-standing hypertension. FIguRE 269e-29 Normal variant ST-segment elevations in a healthy 21-year-old male (commonly referred to asbenign early repolarization pattern). ST elevations exhibit upward concavity and are most apparent in V3 and V4, and less than 1 mm in the limb leads. Precordial QRS voltages are prominent, but within normal limits for a young adult. No evidence of left atrial abnormality or ST depression/T-wave inversions to go along with LVH. CHAPTER 269e Atlas of Electrocardiography PART 10 Disorders of the Cardiovascular System FIguRE 269e-30 SR with first-degree AV “block” (PR interval = 0.24 s) and complete LBBB.

1	CHAPTER 269e Atlas of Electrocardiography PART 10 Disorders of the Cardiovascular System FIguRE 269e-30 SR with first-degree AV “block” (PR interval = 0.24 s) and complete LBBB. FIguRE 269e-31 Dextrocardia with: (1) inverted P waves in I and aVL; (2) negative QRS complex and T wave in I; and (3) progressively decreasing voltage across the precordium. FIguRE 269e-32 Sinus tachycardia; intraventricular conduction delay (IVCD) with a rightward QRS axis. QT interval is prolonged for the rate. The triad of sinus tachycardia, a wide QRS complex, and a long QT in appropriate clinical context suggests tricyclic antidepressant overdose. Terminal S wave (rS) in I and terminal R wave (qR) in aVR are also noted as part of this IVCD variant. CHAPTER 269e Atlas of Electrocardiography ECG Wave-Maven http://ecg.bidmc.harvard.edu Copyright, 2007 Beth Israel Deaconess Med Ctr

1	CHAPTER 269e Atlas of Electrocardiography ECG Wave-Maven http://ecg.bidmc.harvard.edu Copyright, 2007 Beth Israel Deaconess Med Ctr FIguRE 269e-33 Borderline sinus bradycardia (59 beats/min), prolonged PR interval (250 ms), and RBBB are present with marked right-axis deviation (RAD), the latter consistent with left posterior fascicular block (LPFB). LPFB is a diagnosis of exclusion, which requires ruling out lead reversal, normal variant, RV overload syndromes, or lateral MI, in particular, as causes of the RAD. This ECG also shows nondiagnostic Q waves in the inferior leads. In concert with RBBB, the LPFB indicates bifascicular block. (From LA Nathanson et al: ECG Wave-Maven. http://ecg.bidmc.harvard.edu.)

1	The ability to image the heart and blood vessels noninvasively has been one of the greatest advances in cardiovascular medicine since the development of the electrocardiogram. Cardiac imaging complements history taking and physical examination, blood and laboratory testing, and exercise testing in the diagnosis and management of most diseases of the cardiovascular system. Modern cardiovascular imaging consists of echocardiography (cardiac ultrasound), nuclear scintigraphy including positron emission tomography (PET) imaging, magnetic resonance imaging (MRI), and computed tomography (CT). These studies, often used in conjunction with exercise testing, can be used independently or in concert depending on the specific diagnostic needs. In this chapter, we review the principles of each of these modalities and the utility and relative benefits of each for the most common cardiovascular diseases.

1	Echocardiography uses high-frequency sound waves (ultrasound) to penetrate the body, reflect from relevant structures, and generate an image. The basic physical principles of echocardiography are identical to other types of ultrasound imaging, although the hardware and software are optimized for evaluation of cardiac structure and function. Early echocardiography machines displayed “M-mode” echo-cardiograms in which a single ultrasound beam was displayed over time on a moving sheet of paper (Fig. 270e-1, left panel). Modern Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging

1	Marcelo F. Di Carli, Raymond Y. Kwong, Scott D. Solomon echocardiographic machinery uses phased array transducers that 270e-1 contain up to 512 elements and emit ultrasound in sequence. The reflected ultrasound is then sensed by the receiving elements. A “scan converter” uses information about the timing and magnitude of the reflected ultrasound to generate an image (Fig. 270e-1, right panel). This sequence happens repeatedly in “real time” to generate moving images with frame rates that are typically greater than 30 frames per second, but can exceed 100 frames per second. The gray scale of the image features indicates the intensity of the reflected ultrasound; fluid or blood appears black, and highly reflective structures, such as calcifications on cardiac valves or the pericardium, appear white. Tissues such as myocardium appear more gray, and tissues such as muscle display a unique speckle pattern. Although M-mode echocardiography has largely been supplanted by two-dimensional

1	appear white. Tissues such as myocardium appear more gray, and tissues such as muscle display a unique speckle pattern. Although M-mode echocardiography has largely been supplanted by two-dimensional echocardiography, it is still used because of its high temporal resolution and accuracy for making linear measurements.

1	The spatial resolution of ultrasound is dependent on the wavelength: the smaller the wavelength and the higher the frequency of the ultrasound beam, the greater are the spatial resolution and ability to discern small structures. Increasing the frequency of ultrasound will increase resolution but at the expense of reduced penetration. Higher frequencies can be used in pediatric imaging or transesophageal echocardiography where the transducer can be much closer to the structures being interrogated, and this is a rationale for using trans-esophageal echocardiography to obtain higher quality images. Three-dimensional ultrasound transducers use a waffle-like matrix array transducer and receive a pyramidal data sector. Three-dimensional echocardiography is being increasingly used for assessment of congenital heart disease and valves, although current image quality lags behind two-dimensional ultrasound (Fig. 270e-2).

1	In addition to the generation of two-dimensional images that provide information about cardiac structure and function, echocardiography can be used to interrogate blood flow within the heart and blood vessels by using the Doppler principle to ascertain the velocity of blood flow. When ultrasound emitted from a transducer reflects off red blood cells that are moving toward the transducer, the reflected ultrasound will return at a slightly higher frequency than emitted; the opposite is true when flow is away from the transducer. That frequency difference, termed the Doppler shift, is directly related to the velocity CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging

1	CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging FIGURE 270e-1 Principle of image generation in two-dimensional (2D) echocardiography. An electronically steerable phased-array transducer emits ultrasound from piezoelectric elements, and returning echoes are used to generate a 2D image (right) using a scan converter. Early echocardiography machines used a single ultrasound beam to generate an “M-mode” echocardiogram (see text), although modern equipment generates M-mode echocardiograms digitally from the 2D data. LV, left ventricle. PART 10 Disorders of the Cardiovascular System FIGURE 270e-2 Three-dimensional (3D) probe and 3D image.

1	PART 10 Disorders of the Cardiovascular System FIGURE 270e-2 Three-dimensional (3D) probe and 3D image. of the flow of the red blood cells. The velocity of blood flow between two chambers will be directly related to the pressure gradient between those chambers. A modified form of the Bernoulli equation, where p = the pressure gradient and v = the velocity of blood flow in meters per second, can be used to calculate this pressure gradient in the majority of clinical circumstances. This principle can be used to determine the pressure gradient between chambers and across valves and has become central to the quantitative assessment of valvular heart disease.

1	There are three types of Doppler ultrasound that are typically used in standard echocardiographic examinations: spectral Doppler, which consists of both pulsed wave Doppler and continuous wave Doppler, and color flow Doppler. Both types of spectral Doppler will display a waveform representing the velocity of blood flow, with time on the horizontal axis and velocity on the vertical axis. Pulsed wave Doppler is used to interrogate relatively low velocity flow and has the ability to determine blood flow velocity at a particular location within the heart. Continuous wave Doppler is used to assess high-velocity flow but can only identify the highest velocity in a particular direction and cannot interrogate the velocity at a specific depth location. Both of these techniques can only accurately assess velocities that are in the direction of the ultrasound scan lines, and velocities that are at an angle to the direction of the ultrasound beam will be underestimated. Color flow Doppler is a

1	assess velocities that are in the direction of the ultrasound scan lines, and velocities that are at an angle to the direction of the ultrasound beam will be underestimated. Color flow Doppler is a form of pulsed wave Doppler in which the velocity of blood flow is color encoded according to a scale and superimposed on a two-dimensional grayscale image in real time, giving the appearance of real-time flow within the heart. The Doppler principle can also be used to assess the velocity of myocardial motion, which is a sensitive way to assess myocardial function (Fig. 270e-3). A standard full transthoracic echocardiographic examination consists of a series of two-dimensional views made up of different imaging planes from various scanning locations and spectral and color flow Doppler assessment.

1	Transesophageal echocardiography is a form of echocardiography in which the transducer is located on the tip of an endoscope that can be inserted into the esophagus. This procedure allows closer, less obstructed views of cardiac structures, without having to penetrate through chest wall, muscle, and ribs. Because less penetration is needed, a higher frequency probe can be used, and image quality and spatial resolution are generally higher than with standard trans-thoracic imaging, particularly for structures that are more posterior. Transesophageal echocardiography has become the test of choice for assessment of small lesions in the heart such as valvular vegetations, especially in the setting of a prosthetic valve disease, and intracardiac thrombi, including assessment of the left atrial appendage, which is difficult to visualize with standard transthoracic imaging, and for assessment of congenital abnormalities. Transesophageal echocardiography requires both topical and systemic

1	appendage, which is difficult to visualize with standard transthoracic imaging, and for assessment of congenital abnormalities. Transesophageal echocardiography requires both topical and systemic anesthesia, generally conscious sedation, and carries additional risks such as potential damage to the esophagus, including the rare possibility of perforation, aspiration, and anesthesia-related complications. Patients generally need to give consent for transesophageal echocardiography and be monitored during and subsequent to the procedure. Transesophageal echocardiography can be carried out in intubated patients and is routinely used for intraoperative monitoring during cardiac surgery.

1	Stress echocardiography is routinely used to assess cardiac function during exercise and can be used to identify myocardial ischemia or to assess valvular function under exercise conditions. Stress echocardiography is typically performed in conjunction with treadmill or bicycle exercise testing, but can also be performed using pharmacologic stress most typically with an intravenous infusion of dobutamine (see section on stress imaging below).

1	Whereas typical echocardiographic equipment is large, bulky, and expensive, small hand-held ultrasound equipment developed over the last decade now offers diagnostic quality imaging in a package small enough to be carried on rounds (Fig. 270e-4). These relatively inexpensive point-of-care devices currently lack full diagnostic capabilities but represent an excellent screening tool if used by an experienced operator. As these units become even smaller and less expensive, they are being increasingly used not just by cardiologists, but also by emergency medicine physicians, intensivists, anesthesiologists, and internists. Radionuclide imaging techniques are commonly used for the evaluation of patients with known or suspected coronary artery disease (CAD),

1	Radionuclide imaging techniques are commonly used for the evaluation of patients with known or suspected coronary artery disease (CAD), FIGURE 270e-3 Three types of Doppler ultrasound. A and B. Pulsed and continuous wave Doppler waveforms with time on horizontal axis and velocity of blood flow on vertical axis. C. Color flow Doppler, where velocities are encoded by colors according to scale on right side of screen and superimposed on a two-dimensional grayscale image.

1	including for initial diagnosis and risk stratification as well as the assessment of myocardial viability. These techniques use small amounts of radiopharmaceuticals (Table 270e-1), which are injected intravenously and trapped in the heart and/or vascular cells. Radioactivity within the heart and vasculature decays by emitting gamma rays. The interaction between these gamma rays and the detectors in specialized scanners (single-photon emission computed tomography [SPECT] and PET) creates a scintillation event or light output, which can be captured by digital recording equipment to form an image of the heart and vasculature. Like CT and MRI, radionuclide images also generate tomographic (three-dimensional) views of the heart and vasculature. Radiopharmaceuticals Used in Clinical Imaging Table 270e-1 summarizes the most commonly used radiopharmaceuticals in clinical SPECT and PET imaging.

1	Protocols for Stress Myocardial Perfusion Imaging Both exercise and pharmacologic stress can be used for myocardial perfusion imaging. Exercise stress is generally preferred because it is physiologic and provides additional clinically important information (i.e., clinical and hemodynamic responses, ST-segment changes, exercise duration, and functional status). However, submaximal effort will lower the sensitivity of the test and should be avoided, especially if the test is requested for initial diagnosis of CAD. In patients who are unable to exercise or who exercise submaximally, pharmacologic stress offers an adequate alternative to exercise stress testing. Pharmacologic stress can be accomplished either with coronary vasodilators, such as adenosine, dipyridamole, or regadenoson, or β1-receptor agonists, such as dobutamine. For patients unable to exercise, vasodilators are the most commonly used stressors in combination with myocardial perfusion imaging. Dobutamine is a potent

1	β1-receptor agonists, such as dobutamine. For patients unable to exercise, vasodilators are the most commonly used stressors in combination with myocardial perfusion imaging. Dobutamine is a potent β1-receptor agonist that increases myocardial oxygen demand by augmenting contractility, heart rate, and blood pressure similar to exercise. It is generally used as an alternative to vasodilator stress in patients with chronic pulmonary disease, in whom vasodilators may be contraindicated. Dobutamine is also commonly used as a pharmacologic alternative to stress 270e-3 testing in stress echocardiography.

1	FIGURE 270e-4 Two examples of hand-held ultrasound equipment: V-Scan (General Electric, left) and Sonosite (right). Imaging protocols are tailored to the individual patient based on the clinical question, patient’s risk, ability to exercise, body mass index, and other factors.

1	For SPECT imaging, technetium-99m (99mTc)-labeled tracers are the most commonly used imaging agents because they are associated with the best image quality and the lowest radiation dose to the patient (Fig. 270e-5). Selection of the protocol (stress-only, single-day, or 2-day) depends on the patient and clinical question. After intravenous injection, myocardial uptake of 99mTc-labeled tracers is rapid (1–2 min). After uptake, these tracers become trapped intracellularly in mitochondria and show minimal change over time. This is why 99mTc tracers can be helpful in patients with chest pain of unclear etiology occurring at rest, because patients can be injected while having chest pain and imaged some time later after symptoms subside. Because the radiotracer is trapped at the time of injection, the images provide a snapshot of myocardial perfusion at the time of injection, even if the acquisition is delayed. Indeed, a normal myocardial perfusion study following a rest injection in a

1	the images provide a snapshot of myocardial perfusion at the time of injection, even if the acquisition is delayed. Indeed, a normal myocardial perfusion study following a rest injection in a patient with active chest pain effectively excludes myocardial ischemia as the cause of chest pain (high negative predictive value). While used commonly in the past for perfusion imaging, thallium-201 protocols are now rarely used because they are typically associated with a higher radiation dose to the patient.

1	PET myocardial perfusion imaging is an alternative to SPECT and is associated with improved diagnostic accuracy and lower radiation dose to patients due to the fact that radiotracers are typically short lived (Table 270e-1). The ultra-short half-life of some PET radiopharmaceuticals in clinical use (e.g., rubidium-82) is the primary reason why imaging is generally combined with pharmacologic stress, as opposed to exercise, because this allows for faster imaging of these rapidly decaying radiopharmaceuticals. However, exercise is possible for relatively longer lived radiotracers (e.g., 13N-ammonia). PET imaging protocols are typically faster than SPECT, but more expensive. For myocardial perfusion imaging, rubidium-82 does not require an on-site medical cyclotron (it is available from a strontium-82/rubidium-82 generator) and, thus, is the most commonly used radiopharmaceutical. 13N-ammonia has better flow characteristics (higher myocardial extraction) and imaging properties than

1	a strontium-82/rubidium-82 generator) and, thus, is the most commonly used radiopharmaceutical. 13N-ammonia has better flow characteristics (higher myocardial extraction) and imaging properties than rubidium-82, but it does require an on-site medical cyclotron. In comparison to SPECT, PET has improved spatial and contrast resolution and provides absolute measures of myocardial perfusion (in mL/min per gram of tissue), thereby providing the patients’ regional and global coronary flow reserve. The latter helps improve diagnostic accuracy and risk stratification, especially in obese patients, women, and higher risk individuals (e.g., diabetes mellitus) (Fig. 270e-6). Contemporary PET and SPECT scanners are combined with a CT scanner (so-called hybrid PET/CT and SPECT/CT). CT is used primarily to guide patient positioning in the field of view and for correcting inhomogeneities in radiotracer distribution due to attenuation by soft tissues (so-called attenuation

1	CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging Abbreviations: PET, positron emission tomography; SPECT, single-photon emission computed tomography. PART 10 Disorders of the Cardiovascular System Perfusion Defect blackout map 506.0 100.0% 0.0 0.0% Perfusion Defect blackout map 145.0 100.0% 0.0 0.0% Perfusion Defect blackout map 50.0% 100.0% 0.0% 0.0%

1	Perfusion Defect blackout map 506.0 100.0% 0.0 0.0% Perfusion Defect blackout map 145.0 100.0% 0.0 0.0% Perfusion Defect blackout map 50.0% 100.0% 0.0% 0.0% FIGURE 270e-5 Tomographic stress (top of each pair) and rest myocardial perfusion images with technetium-99m sestamibi single-photon emission computed tomography imaging demonstrating a large perfusion defect throughout the anterior and anteroseptal walls. The right panel demonstrates the quantitative extent of the perfusion abnormality at stress (top bull’s-eye), at rest (middle bull’s-eye), and the extent of defect reversibility (lower bull’s-eye). The lower left panel demonstrates electrocardiogram-gated myocardial perfusion images from which one can determine the presence of regional wall motion abnormalities and calculate left ventricular volumes and ejection fraction.

1	correction). However, it can also be used to obtain diagnostic data including coronary artery calcium score and/or CT coronary angiography (discussed below). For the evaluation of myocardial viability in patients with ischemic cardiomyopathy, myocardial perfusion imaging (with SPECT or PET) is usually combined with metabolic imaging (i.e., fluorodeoxyglucose [FDG] PET). In hospital settings lacking access to PET scanning, thallium-201 SPECT imaging is an excellent alternative.

1	CT acquires images by passing a thin x-ray beam through the body at many angles to generate cross-sectional images. The x-ray transmission measurements are collected by a detector array and digitized into pixels that form an image. The grayscale information in individual pixels is determined by the attenuation of the x-ray beam along its path by tissues of different densities, referenced to the value for water in units known as Hounsfield units. In the resulting CT images, bone appears bright white, air is black, and blood and muscle show varying shades of gray. However, due to the limited contrast between cardiac chambers and vascular structures, iodinated contrast agents are necessary for most cardiovascular indications. Cardiac CT produces tomographic images of the heart and surrounding structures. With modern CT scanners, a three-dimensional dataset of the heart can be acquired in 5–15 s with submillimeter spatial resolution.

1	CT Calcium Scoring CT calcium scoring is the simplest application of cardiac CT and does not require administration of iodinated contrast. The presence of coronary artery calcification has been associated with increased burden of atherosclerosis and cardiovascular mortality. Coronary calcium is then quantified (e.g., Agatston score) and categorized as minimal (0–10), mild (10–100), moderate (100–400), or severe (>400) (Fig. 270e-7). Coronary artery calcium (CAC) scores are then normalized by age and gender and reported as percentile scores. Population-based studies in asymptomatic cohorts have reported high cardiac prognostic value of CT calcium score. With appropriate techniques, the radiation dose associated with CAC scanning is very low (~1–2 mSv).

1	CT Coronary Angiography Coronary CT angiography (CTA) is emerging as a viable alternative to coronary angiography in selected patients. Imaging of the coronary arteries by CT is challenging because of their small luminal size and because of cardiac and respiratory motion. Respiratory motion can be reduced by breath-holding, and cardiac motion is best reduced by slowing the patient’s heart rate, ideally to under 60 beats/min, using intravenous or oral beta blockade or other rate-lowering drugs. When performing a coronary CTA, image quality is further enhanced using sublingual nitroglycerin to enlarge the coronary lumen just prior to contrast injection. Imaging the whole-heart volume is synchronized to the administration of weight-based and appropriately timed intravenous iodinated contrast. Image acquisition is linked to the timing of the cardiac cycle through electrocardiogram (ECG) triggering. Prospective ECG triggering, whereby the x-ray beam is turned on during a specific part of

1	Image acquisition is linked to the timing of the cardiac cycle through electrocardiogram (ECG) triggering. Prospective ECG triggering, whereby the x-ray beam is turned on during a specific part of the cardiac cycle (e.g., end systole, combined end-systolic and end-diastolic timing, or mid-diastole), is generally used to limit the radiation exposure to the patient by acquiring data only through that portion of the cardiac cycle with least motion. Dose modulation is another method that should be routinely used to reduce radiation when performing CTA. It delivers a maximal amount of x-ray during the portion of the cardiac cycle of interest, but reduces x-ray delivery throughout the remaining portion of the cardiac cycle. The resulting images are then postprocessed using a three-dimensional workstation, which facilitates interpretation of the coronary anatomy and estimation of the severity of atherosclerosis (Fig. 270e-7).

1	Cardiac magnetic resonance (CMR) imaging is based on imaging of protons in hydrogen. Hydrogen is abundant because 80% of the human body consists of water. When put inside the MRI scanner, Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging MixedNoncalcifiedRCAAoAoAoLADABCDLADCalcifiedPA LAD 2.32 1.09 2.12 LCX 2.29 1.08 2.12 RCA 2.30 0.99 2.33 200 TOT 2.30 1.06 2.18 100

1	LAD 2.32 1.09 2.12 LCX 2.29 1.08 2.12 RCA 2.30 0.99 2.33 200 TOT 2.30 1.06 2.18 100 FIGURE 270e-6 Multidimensional cardiac imaging protocol with positron emission tomography. The left upper panel demonstrates stress and rest short-axis images of the left and right ventricles demonstrating normal regional myocardial perfusion. The middle panel demonstrates the quantitative bull’s-eye display to evaluate the extent and severity of perfusion defects. The lower right panel illustrates the time-activity curves for quantification of myocardial blood flow. The right upper panel demonstrates electrocardiogram-gated myocardial perfusion images from which one can determine the presence of regional wall motion abnormalities and calculate left ventricular volumes and ejection fraction. LAD, left anterior descending artery; LCX, left circumflex artery; RCA, right coronary artery; TOT, total left ventricle.

1	FIGURE 270e-7 Examples of non-contrastand contrast-enhanced coronary imaging with computed tomography (CT). A. Calcified coronary plaques in the distal left main and proximal left anterior descending coronary artery (LAD) in a noncontrast cardiac CT scan. Calcium deposits are dense and present as bright white structures on CT, even without contrast enhancement. B, C, and D. Different types of atherosclerotic plaques on contrast-enhanced CT scans. Importantly, noncalcified plaques are evident only on contrast-enhanced CT scans. AO, aorta; PA, pulmonary artery; RCA, right coronary artery.

1	the magnetic field causes the protons (spins) to spin around their axis (a process known as precession) at specific frequencies. Spins within water have a different frequency than spins within more complex macromolecules such as fat or protein. Inside the MRI, a set of gradient coils slightly modifies the magnetic field in each of the three orthogonal directions. As a result, this additional process alters the frequencies of spins, and now the spins can be spatially located inside the MRI bore. This system allows the MRI to selectively deposit radio-frequency energy (in the form or radiofrequency pulse) into specific locations of the body for the purpose of imaging those locations. Once the radiofrequency pulses stop, the energy absorbed by the body will quickly be released back. Using the proper arrangement of surface phased-array coils, this released energy can be read, and important information such as spin locations and frequencies can be digitally recorded in a data matrix known

1	proper arrangement of surface phased-array coils, this released energy can be read, and important information such as spin locations and frequencies can be digitally recorded in a data matrix known as the K-space, before reconstructed into a magnetic resonance image. Radiofrequency energy deposition into the patient’s body can be arranged in many complex ways, known as pulse sequences, that allow extraction of different types of information from the body regions of interests. In CMR, these types of information in general are categorized under T1, T2, or T2∗ weighting, each of them containing a different combination of diagnostic information regarding cardiac structure, tissue characteristics, blood flow, or other physiologic properties of the heart.

1	In clinical CMR, most pulse sequences are T1-weighted sequences, which characterize cardiac structure and function, blood flow, and myocardial perfusion, whereas T2-weighted and T2∗-weighted pulse sequences characterize myocardial edema and myocardial iron infiltration, respectively. A combination of more than one weighting is possible in some pulse sequences. ECG-triggered cine CMR is the modality that serves as a reference standard for quantifying ventricular volumes and function. Respiratory motion during CMR imaging is suppressed most commonly using repetitive patient breath-holding, but more advanced algorithms such as motion averaging or gating diaphragmatic motion (known as navigator guidance) are also used in clinical CMR. A list of common pulse sequence used in CMR is shown in Table 270e-2.

1	Echocardiography, CMR, and cardiac CT are all capable of assessing cardiac structure and function, although echocardiography is generally considered the primary imaging method for these assessments. Radionuclide imaging can also be used to assess left ventricular PART 10 Disorders of the Cardiovascular System regional and global systolic function. Echocardiography is most often used to assess the size of all four chambers and thickness of ventricular walls, which are affected by both cardiac and systemic diseases.

1	The structure of the left ventricle is generally assessed by determining its volume and mass. Left ventricular volumes can be easily estimated from two-dimensional echocardiography by using one of several validated methods. The accuracy of these methods by echocardiography is limited by the fact that, as a nontomographic technique, foreshortening of the imaging plane can lead to underestimation of volumes. Moreover, virtually all of these methods require accurate identification of the endocardial border, which is dependent on image quality. In this regard, high-resolution tomographic techniques such as CMR or cardiac CT are considered generally more accurate for volumetric assessment. Three-dimensional echocardiography has several advantages over two-dimensional echocardiography by not requiring any geometric assumptions about the left ventricle for quantification of volumes and ejection fraction. However, acquisition of three-dimensional echocardiographic images requires substantial

1	any geometric assumptions about the left ventricle for quantification of volumes and ejection fraction. However, acquisition of three-dimensional echocardiographic images requires substantial expertise, and these techniques are not widely used in practice.

1	Left ventricular dilatation is common to a number of cardiac diseases. For example, regional dysfunction secondary to myocardial infarction can ultimately lead to progressive ventricular dilatation or remodeling. Although dilatation often begins in the region affected by the infarction, subsequent compensatory dilatation can occur in remote myocardial regions as well. The presence of regional wall motion abnormalities associated with ventricular thinning (reflecting scar) in a coronary distribution is strongly suggestive of an ischemic etiology. Direct assessment of infarcted myocardium is possible with both CMR (evident as areas of late gadolinium enhancement [LGE]) and radionuclide imaging (as assessed by regional perfusion or metabolic defects at rest). CMR can be particularly useful in determining etiology of ventricular dilatation and dysfunction, with LGE in coronary distributions being nearly pathognomonic for infarction (Video 270e-1).

1	More global ventricular dilatation is seen in cardiomyopathy and dilatation due to valvular heart disease. Idiopathic, nonischemic cardiomyopathies will typically result in global ventricular dilatation and dysfunction, with thinning of the walls. Patients with substantial ventricular dyssynchrony due to conduction abnormalities will have a typical pattern of contraction (e.g., delay of contraction of the lateral wall with left bundle branch block). Although various methods for determining ventricular dyssynchrony have been proposed as ways to identify patients who would benefit from cardiac resynchronization therapy, it is not yet clear that they are superior to ECG assessment of QRS duration and morphology. As discussed later in this chapter, regurgitant lesions of either the mitral or aortic valves can lead to substantial ventricular dilatation, and assessment of ventricular size is integral in the evaluation and timing of surgical correction. Because changes in ventricular size

1	aortic valves can lead to substantial ventricular dilatation, and assessment of ventricular size is integral in the evaluation and timing of surgical correction. Because changes in ventricular size are used clinically to determine which patients should undergo valve surgery, accurate assessment of changes in ventricular size is essential. Although serial echocardiography can provide these data, serial assessment by CMR may be more accurate when appreciation of subtle changes over time is important.

1	Left ventricular wall thickness and mass are also important measures of cardiac and systemic disease. The left ventricle will hypertrophy under any condition in which its afterload is increased, including conditions that obstruct outflow, such as aortic stenosis, hypertrophic cardiomyopathy, and subaortic membranes; in postcardiac aortic obstruction seen in coarctation; or in systemic conditions characterized by increased afterload, such as hypertension. The pattern of ventricular hypertrophy can change depending on the etiology. Aortic stenosis and hypertension are typically characterized by concentric hypertrophy, in which the ventricular walls thicken “concentrically” and cavity size is usually small. In volume overload conditions such as mitral or aortic regurgitation, there may be minimal increase in ventricular wall thickness, but substantial ventricular dilatation leads to marked increases in left ventricular mass.

1	Ventricular wall thickness can be measured and ventricular mass can be calculated by either echocardiography or CMR. Although radionuclide imaging and cardiac CT can also provide measures of left ventricular mass, they are not generally used for this purpose. Although measurement of wall thickness with echocardiography is relatively straightforward and accurate, determining left ventricular mass by echocardiography requires using one of several formulas that takes into account both wall thickness and ventricular cavity dimensions. Assessment of left ventricular mass by CMR has the advantage of not requiring geometric assumptions and is thus more accurate than echocardiography.

1	Assessment of ejection fraction, or the percentage of blood ejected with each beat, has been the primary method to assess systolic function and is generally calculated by subtracting end-systolic volume from end-diastolic volume and dividing by end-diastolic volume. All cardiac imaging modalities can provide direct measurements of left ventricular ejection fraction (LVEF). As discussed above, tomographic techniques (e.g., CMR, CT, and radionuclide imaging [SPECT and PET]) are generally more accurate and reproducible than echocardiography because there are no geometric assumptions. A LVEF of 55% or greater is generally considered normal, and an LVEF of 50–55% is considered in the low-normal range.

1	Newer methods to assess systolic function, such as myocardial strain or deformation imaging using speckle-tracking methods on echocardiography or myocardial tagging on CMR, can provide a more sensitive approach to detection of systolic dysfunction. Additional assessments based on these novel methods include assessment of myocardial twist and torsion. Although these techniques are not used routinely, they may be especially useful in certain conditions such as valvular heart disease and early detection of cardiotoxicity following chemotherapy and/ or radiation therapy. In addition to estimation or calculation of ejection fraction, stroke volume can be assessed by virtually all cardiac imaging methods, generally by subtracting the end-systolic volume from the end-diastolic volume, or by Doppler methods (only on echocardiography), and offers another measure of systolic function that provides independent information from ejection fraction.

1	Echocardiography remains the primary method for clinical assessment of diastolic function. Recent advances in Doppler tissue imaging allow for accurate assessment of the velocity of myocardial wall motion by assessing the excursion of the mitral annulus in diastole. Mitral annular relaxation velocity, or E′, is inversely related to the time constant of relaxation, tau, and has been shown to have prognostic significance. Dividing the standard mitral inflow maximal velocity, E, by the mitral annular relaxation velocity yields E/E′, which has been shown to correlate with left ventricular filling pressures. The utility of standard E and A wave ratios for assessment of diastolic function has been questioned. Mitral deceleration time can be a useful measure if very short (<150 ms), suggesting restrictive physiology and severe diastolic dysfunction. Several grading methods for diastolic function have been proposed that take into account a number of diastolic parameters, including Doppler

1	restrictive physiology and severe diastolic dysfunction. Several grading methods for diastolic function have been proposed that take into account a number of diastolic parameters, including Doppler tissue-based relaxation velocities, pulmonary venous Doppler, and left atrial size (Fig. 270e-8). Diastolic function worsens with aging, and most diastolic parameters need to be adjusted for age.

1	Right ventricular size and function have been shown to be prognostically important in a variety of conditions. Right ventricular size and function can be assessed by echocardiography, CMR, CT, or radionuclide imaging methods. CMR is considered the most accurate noninvasive technique to evaluate the structure and right ventricular ejection fraction of the right ventricle (Video 270e-2). Although first-pass imaging by radionuclide angiography can provide accurate and reproducible measurements of right ventricular volumes and ejection fraction, it is not commonly used. Assessment of the right ventricle by echocardiography has generally been qualitative, owing in part to the unusual geometry of the right ventricle. However, several quantitative methods are available for assessment of right ventricular function, including fractional area change (FAC = [diastolic area – systolic area]/ 270e-7 diastolic area), which has been shown to correlate with outcomes in heart failure and after

1	ventricular function, including fractional area change (FAC = [diastolic area – systolic area]/ 270e-7 diastolic area), which has been shown to correlate with outcomes in heart failure and after myocardial infarction. Excursion of the tricuspid annulus (tricuspid annular plane systolic excursion) is another widely used method to assess right ventricular function, although it is mostly used in research settings.

1	Abnormalities of right ventricular size and function are generally secondary to either diseases that affect the right ventricle intrinsically or disease in which the right ventricle responds to abnormalities elsewhere in the heart or pulmonary vasculature. Intrinsic diseases that affect the RV include congenital abnormalities, including hypoplastic right ventricle and arrhythmogenic right ventricular dysplasia, and acquired diseases, such as right ventricular infarction and infiltrative diseases that affect the right ventricle. Long-standing pulmonary hypertension or pulmonary outflow tract obstruction leads to right ventricular hypertrophy and ultimately dilatation. Although right ventricular dilatation can occur due to both chronic and acute processes, chronic right ventricular dilatation is usually secondary to long-standing increases in pulmonary pressures and can thus be distinguished from the acute processes that cause right ventricular dilatation. One such acute process that

1	is usually secondary to long-standing increases in pulmonary pressures and can thus be distinguished from the acute processes that cause right ventricular dilatation. One such acute process that can cause profound right ventricular dilatation and dysfunction is acute pulmonary embolism. In the setting of acute occlusion of a pulmonary artery or branch, an acute rise in pulmonary vascular resistance causes a previously normal right ventricle to dilate and fail due to the increased afterload. In acute pulmonary embolism, right ventricular dilatation and dysfunction are signs of substantial hemodynamic compromise and are associated with a marked increase risk in likelihood of death. In addition to right ventricular dilatation, acute pulmonary embolism is often associated with a specific pattern of regional right ventricular dysfunction, commonly referred to as the McConnell sign, characterized by preservation of right ventricular wall motion in the basal and apical regions and dyskinesis

1	of regional right ventricular dysfunction, commonly referred to as the McConnell sign, characterized by preservation of right ventricular wall motion in the basal and apical regions and dyskinesis in the region of the mid right ventricular free wall. This abnormality is highly specific for acute pulmonary embolism and is likely secondary to acute increases in right ventricular load.

1	Any disease that causes increased pulmonary vascular resistance can lead to right ventricular dilatation and dysfunction. Long-standing chronic obstructive pulmonary disease results in cor pulmonale in which right ventricular pressures become elevated as the right ventricle hypertrophies in response to the increased pulmonary vascular resistance. Acute pneumonia can cause findings that are similar to acute pulmonary embolism. In patients with right ventricular dilatation without obvious pulmonary disease, intracardiac shunts should be considered. The increased flow through the pulmonary vasculature as a result of an atrial septal or ventricular septal defect can, over time, result in elevation in pulmonary resistance with subsequent dilatation and hypertrophy of the right ventricle. Right ventricular dilatation and dysfunction also have prognostic significance in left-sided heart disease and have been shown to be important predictors of outcome in patients with heart failure or acute

1	ventricular dilatation and dysfunction also have prognostic significance in left-sided heart disease and have been shown to be important predictors of outcome in patients with heart failure or acute myocardial infarction.

1	In addition to assessment of left ventricular structure, assessment of the other cardiac chambers also provides important clues to intracardiac and systemic diseases. Enlargement of the left atrium is common in patients with hypertension and is also suggestive of increased left ventricular filling pressures; indeed, left atrial size is often termed the “hemoglobin A1c” of diastolic function, because left atrial enlargement reflects long-standing increase in left-sided filling pressures. Right atrial dilatation and dilatation of the inferior vena cava are common in conditions in which central venous pressure is elevated. CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging

1	CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging Both cardiac CT and radionuclide imaging expose patients to ionizing radiation. Several recent publications have raised concern regarding the potential harmful effects of ionizing radiation associated with cardiac imaging. The effective dose is a measure used to estimate the biologic effects of radiation and is expressed in millisieverts (mSv). However, measuring the radiation effective dose associated with diagnostic imaging is complex and imprecise and often results in varying estimates,

1	PART 10 Disorders of the Cardiovascular System Normal diastolic function Mitral inflow Mitral inflow at peak valsalva maneuver Doppler tissue imaging of mitral annular motion Pulmonary venous flow Flow propagation velocity (Vp) on color M-mode Left ventricular relaxation Left ventricular compiance Atrial pressure Normal Normal Normal Impaired Normal to Normal Impaired Impaired Impaired Mild diastolic dysfunction Impaired relaxation Moderate diastolic dysfunction Pseudonormal Reversible restrictive Fixed restrictive Severe diastolic dysfunction 0.75< E/A<1.5 DT >140 ms Adur 0 Velocity, m/s 2.0 A E 0.75 <E/A<1.5 DT>140 ms E/A˜1.5 DT<140 ms E/A>1.5 DT<140 ms E/A°0.75 ˛E/A<0.5 0 Velocity, m/s 2.0 AE E/e’<10 Velocity, m/s0 0.15 e’ a’ ˛E/A˜0.5 ˛E/A˜0.5 ˛E/A˜0.5˛E/A<0.5 S˜D ARdur<Adur Vp >50 cm/s E/Vp <1.5 >45 cm/s E/Vp <1.5 >45 cm/s E/Vp >2.5 >45 cm/s E/Vp >2.5 >45 cm/s E/Vp <2.5 ARdur Time, ms Time, ms Time, ms Time, ms Time, ms 0 Velocity, m/s 2.0 S<D or ARdur>Adur+30 ms S<D or

1	Vp >50 cm/s E/Vp <1.5 >45 cm/s E/Vp <1.5 >45 cm/s E/Vp >2.5 >45 cm/s E/Vp >2.5 >45 cm/s E/Vp <2.5 ARdur Time, ms Time, ms Time, ms Time, ms Time, ms 0 Velocity, m/s 2.0 S<D or ARdur>Adur+30 ms S<D or ARdur>Adur+30 ms S<D or ARdur>Adur+30 ms S>D ARdur<Adur E/e’<10 E/e’˜10 E/e’˜10 E/e’˜10 FIGURE 270e-8 Stages of diastolic function based on various parameters, including mitral inflow (with and without Valsalva maneuver), Doppler tissue imaging, pulmonary venous flow, and flow propagation. (Adapted with permission from MM Redfield et al: JAMA 289:194, 2003.) even among experts. The effective dose from a typical myocardial perfusion SPECT scan ranges between ~4 and 11 mSv, depending on the protocol and type of scanner used. The effective dose from a typical myocardial perfusion PET scan is lower, ~2.5–4 mSv. Radiation exposure associated with cardiac CT is variable and, as with radionuclide imaging, also depends on the imaging protocol and scanner used. Although historic radiation doses

1	~2.5–4 mSv. Radiation exposure associated with cardiac CT is variable and, as with radionuclide imaging, also depends on the imaging protocol and scanner used. Although historic radiation doses with cardiac CT have been quite high, the introduction of newer technologies described above (e.g., x-ray tube modulation, prospective ECG gating) has resulted in a significant dose reduction. The current average radiation dose for a coronary CTA ranges from 5–15 mSv and, in selected cases, can be as low as 1 mSv. Imaging laboratories follow the ALARA (as low as reasonably achievable) principle when balancing the clinical need and imaging approach. By comparison, the average dose for invasive coronary angiography is ~7 mSv, whereas exposure to radiation from natural sources in the United States amounts to ~3 mSv annually.

1	The risk of a fatal malignancy from medical imaging–related radiation is difficult to estimate precisely but is likely small and difficult to discern from the background risk of natural malignancies. The small but potential radiation risks from imaging mandate an assessment of the risk-versus-benefit ratio in the individual patient. In this context, one must not fail to take into account the risks of missing important diagnostic information by not performing a test (which could potentially influence near-term management and outcomes) for a theoretical concern of a small long-term risk of malignancy. Before ordering any test, especially one associated with ionizing radiation, we must ensure the appropriateness of the study and that the potential benefits outweigh the risks. The likelihood that the study being considered will affect clinical management of the patient should be addressed before testing is performed. It is also important that “routine” follow-up scans in asymptomatic

1	that the study being considered will affect clinical management of the patient should be addressed before testing is performed. It is also important that “routine” follow-up scans in asymptomatic individuals be avoided.

1	Contrast agents are commonly used in cardiac CT, CMR, and echocardiography. Although their use significantly enhances the diagnostic information of each of these tests, there are also potential risks from the administration of contrast agents that should be considered.

1	The risk of adverse reactions from iodinated contrast agents used in cardiac CT is well established. The precise pathogenesis of contrast reactions following intravascular administration of iodinated contrast media is not known. The overall incidence of contrast reactions is 0.4–3% with nonionic formulations and higher for ionic formulations. Most contrast adverse reactions are mild and self-limiting. The risk of contrast-induced nephropathy (CIN) in patients with relatively normal renal function (glomerular filtration rate [GFR] >60 mL/min) is low. In most patients, CIN is self-limited, and renal function usually returns to baseline within 7–10 days, without progressing to chronic renal failure. However, this risk increases in patients with GFR <60 mL/min, especially older diabetic subjects. In such patients, appropriate screening and preand postscan hydration are necessary.

1	The use of gadolinium-based contrast agents (GBCAs) in CMR imaging enhances the versatility of this technique. Although there are several commercially available GBCAs in the United States, their use in cardiac imaging is considered off-label. Mild reactions from GBCAs occur in ~1% of patients, but severe or anaphylactic reactions are very rare. All GBCAs are chelated to make the compounds nontoxic and to allow renal excretion. Exposure to the nonchelated component of GBCA (Gd3+) has been associated with a rare condition known as nephrogenic systemic fibrosis (NSF), which is an interstitial inflammatory reaction that leads to severe skin induration, contracture of the extremities, fibrosis of internal organs, and even death. Risk factors to developing NSF include high-dose (>0.1 mmol/ kg) GBCA use in presence of severe renal dysfunction (estimated GFR [eGFR] <30 mL/min per 1.73 m2), need for hemodialysis, an eGFR <15 mL/min per 1.73 m2, use of gadodiamine contrast agent, acute renal

1	GBCA use in presence of severe renal dysfunction (estimated GFR [eGFR] <30 mL/min per 1.73 m2), need for hemodialysis, an eGFR <15 mL/min per 1.73 m2, use of gadodiamine contrast agent, acute renal failure, acute systemic illness, and presence of concurrent pro-inflammatory events. With the use of weight-based dosing and pretest screening, recent data suggest that NSF is extremely rare. Previously, an incidence of 0.02% in 83,121 patients exposed to GBCA over 10 years was noted; however, with current eGFR screening guidelines that have been widely practiced since 2006, a near-zero incidence of NSF has been reported.

1	Contrast agents can also be used in echocardiography. Injected agitated saline is used routinely to assess cardiac shunts, because these “bubbles” are too large to traverse the pulmonary circulation. After saline injection, the presence of bubbles in the left side of the heart is indicative of shunt, although the location can sometimes be difficult to determine. Dedicated echocardiographic contrast agents have been developed for opacification of left-sided structures and perfusion, although these are only currently U.S. Food and Drug Administration (FDA) approved for perfusion. These agents are either albuminor lipid-based microspheres 270e-9 filled with inert gases, typically perfluorocarbons. They are considered extremely safe, although they have, in extremely rare instances, been associated with allergic reactions and neurologic events.

1	The risks of performing CMR in the presence of a pacemaker include generation of electrical current from the metallic hardware (especially if wire loops exist), device movement induced by the magnetic field, inappropriate pacing and sensing, and heating as a result of the “antenna’s effect.” While the presence of a permanent pacemaker remains a contraindication to CMR, highly experienced centers had reported success in performing MRI in these patients in a carefully monitored clinical setting. In general, patients need to be not pacemaker-dependent, the setting of the pacemaker needs to be modified to aysnchronous mode, and the pulse sequence needs to be modified to reduce the amount of radiofrequency energy deposition. Pacemakers implantated for less than 6 weeks and the presence of epicardial, abandoned, or nonfixation leads are considered unsafe. Collectively, evidence from combined reports of >250 patients with pacemaker models manufactured after year 2000 suggests that CMR at 1.5

1	abandoned, or nonfixation leads are considered unsafe. Collectively, evidence from combined reports of >250 patients with pacemaker models manufactured after year 2000 suggests that CMR at 1.5 T or less can be performed without significant risk for the patient and with minor nonpermanent alteration of pacemaker settings and function. Similar safety data exists for automatic implantable cardioverter-defibrillators (AICDs), but they are based only on small numbers of patients. In 2011, the first CMR-compatible FDA-approved permanent pacemaker became available commercially. Currently, no AICD has achieved FDA clearance for MRI compatibility.

1	The basis for the diagnostic application of imaging tests in patients with known or suspected CAD should be viewed in light of the pretest probability of disease as well as the specific characteristics of imaging tests (i.e., sensitivity and specificity). In symptomatic patients, the prevalence or pretest probability of CAD differs based on the type of symptom (typical angina, atypical angina, noncardiac chest pain), as well as on age, gender, and coronary risk factors. In an individual patient, the results of the initial test informs the posttest likelihood of CAD. In patients undergoing sequential testing (e.g., ECG treadmill testing followed by stress imaging), the posttest probability of disease after the first test becomes the pretest likelihood of disease for the second test. Regardless of the sequence, the expectation is that a test will provide sufficient information to confirm or exclude the diagnosis of CAD and that such information will allow accurate risk stratification to

1	of the sequence, the expectation is that a test will provide sufficient information to confirm or exclude the diagnosis of CAD and that such information will allow accurate risk stratification to be able to guide management decisions.

1	Table 270e-3 summarizes the relative diagnostic accuracies of cardiac imaging modalities for the diagnosis of CAD. It is important to highlight that the vast majority of studies included in meta-analyses of the diagnostic accuracy of cardiac imaging modalities for the diagnosis of CAD were retrospective, small, single-center studies, comprising predominantly male patients with a high prevalence CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging SPECT MPI 113 studies (n = 11,212 patients) 88% 76% Coronary CTA 18 studies (n = 1286 patients) 99% 89% Note: In these studies, the diagnosis of coronary artery disease was based on the presence of a >50% or >70% stenosis on invasive coronary angiography. Abbreviations: CMR, cardiac magnetic resonance; CTA, computed tomography angiography; MPI, myocardial perfusion imaging; PET, positron emission tomography; SPECT, single-photon emission computed tomography.

1	PART 10 Disorders of the Cardiovascular System 270e-10 of CAD (>50–60%). Multicenter studies assessing the performance of individual modalities or comparing different modalities have consistently resulted in more modest diagnostic accuracies, tracking more closely Stress with how these tests perform in practice.

1	Stress Echocardiography The hallmark of myocardial ischemia during stress echocardiography is the development of new regional wall motion abnormalities and reduced systolic wall thickening (Video 270e-3). Stress Stress echocardiography can be performed in conjunction with exercise or dobutamine stress. Stress echocardiography is best at identifying inducible wall motion Rest abnormalities in previously normally contracting segments. In a patient with wall motion abnormalities at rest, the specificity of stress echocardiography is Stress reduced, and worsening regional function of a previously abnormal segment might reflect worsening contractile function in the setting of increased wall stress Rest rather than new evidence of inducible ischemia.

1	The advantages of stress echocardiography over other stress imaging techniques include its relatively FIGURE 270e-9 Selected technetium-99m sestamibi myocardial perfusion good diagnostic accuracy, widespread availability, no single-photon emission computed tomography images of two different use of ionizing radiation, and relatively low cost. patients demonstrating a reversible perfusion defect involving the anterior and Limitations of stress echocardiography include (1) the septal left ventricular wall, reflecting ischemia in the left anterior descending corotechnical challenges associated with image acquisition nary territory (arrows in left panel ), and a fixed perfusion defect involving the inferior at peak exercise because of exertional hyperpnoea and and inferolateral walls consistent with myocardial scar in the right coronary territory cardiac excursion, (2) the fact that rapid recovery of (arrow in right panel ).

1	wall motion abnormalities can be seen with mild ischemia (especially with one-vessel disease, which limits sensitivity), (3) difficulty detecting residual ischemia within an infarcted territory because of resting wall motion abnormality, (4) high operator dependence for acquisition of echocardiographic data and analysis of images, and (5) the fact that good-quality complete images viewing all myocardial segments occurs in only 85% of patients. Newer techniques including second harmonic imaging and the use of intravenous contrast agents improve image quality, but their effect on diagnostic accuracy has not been well documented. The use of IV contrast agents may also allow assessment of myocardial perfusion, although this is not approved or generally reimbursed, and data concerning the utility of contrast perfusion echocardiography are limited.

1	As with nuclear perfusion imaging, stress echocardiography is often used for risk stratification in patients with suspected or known CAD. A negative stress echocardiogram is associated with an excellent prognosis, allowing identification of patients at low risk. Conversely, the risk of adverse events increases with the extent and severity of wall motion abnormalities on stress echocardiography. Stress Radionuclide Imaging SPECT myocardial perfusion imaging is the most common form of stress imaging tests for CAD evaluation. The presence of a reversible myocardial perfusion defect is indicative of ischemia (Fig. 270e-9, left panel), whereas a fixed perfusion defect generally reflects prior myocardial infarction (Fig. 270e-9, right panel). As discussed above, PET has advantages compared to SPECT, but it is not widely available and is more expensive and, thus, considered an emerging technology in clinical practice.

1	Nuclear perfusion imaging is another robust approach to diagnose obstructive CAD, quantify the magnitude of inducible myocardial ischemia, assess the extent of tissue viability, and guide therapeutic management (i.e., selection of patients for revascularization). One of the most valuable clinical applications of radionuclide perfusion imaging is for risk stratification. It is well established that patients with a normal SPECT or PET study exhibit a median rate of major adverse cardiac events of <1% annually. Importantly, the risks of death and myocardial infarction increase linearly with increasing magnitude of perfusion abnormalities, reflecting the extent and severity of CAD.

1	Despite the widespread use and clinical acceptance of radionuclide imaging in CAD evaluation, a recognized limitation of this approach is that it often uncovers only coronary territories supplied by the most severe stenoses. Consequently, it is relatively insensitive to accurately delineate the extent of obstructive angiographic CAD, especially in the setting of multivessel disease. The use of quantitative myocardial blood flow and coronary flow reserve with PET can help mitigate this limitation. In patients with so-called “balanced” ischemia or diffuse CAD, measurements of coronary flow reserve uncover areas of myocardium at risk that would generally be missed by performing only relative assessments of myocardial perfusion (Fig. 270e-10). Conversely, a normal coronary flow reserve is associated with a very high negative predictive value for excluding high-risk angiographic CAD. These measurements of coronary flow reserve also contribute to risk stratification across the spectrum of

1	with a very high negative predictive value for excluding high-risk angiographic CAD. These measurements of coronary flow reserve also contribute to risk stratification across the spectrum of ischemic changes, including patients with visually normal myocardial perfusion.

1	Hybrid CT and nuClear perfusion imaging Because many of the newer generation nuclear medicine scanners integrate CT and a gamma camera in the same acquisition gantry, it is now possible to acquire and quantify myocardial scar and ischemia and CAC scoring from a single dual-modality study (SPECT/CT or PET/CT) (Fig. 270e-11). The rationale for this integrated approach is predicated on the fact that the perfusion imaging approach is designed to uncover only obstructive atherosclerosis. Conversely, CAC scoring (or CT coronary angiography) provides a quantitative measure of the anatomic extent of atherosclerosis. This provides an opportunity to improve the conventional models for risk assessment using nuclear imaging alone, especially in patients without known CAD.

1	Cardiac CT Voluminous plaques are more prone to calcification, and stenotic lesions frequently contain large amounts of calcium. Indeed, there is evidence that high CAC scores are generally predictive of a higher likelihood of obstructive CAD, and the available data support the concept of a threshold phenomenon governing this relationship (i.e., Agatston score >400). However, given the fact that CAC scores are not specific markers of obstructive CAD, one should be cautious in using this information as the basis for referral of patients to coronary angiography, especially in symptomatic patients with low-risk stress tests. Conversely, CAC scores <400, especially in symptomatic patients with intermediate-high likelihood of CAD, as in those with typical angina, may be less effective in excluding CAD, especially in young symptomatic men and women who may have primarily noncalcified atherosclerosis (Fig. 270e-12).

1	FIGURE 270e-10 Coronary angiographic (left panel) and rubidium-82 myocardial perfusion positron emission tomograph images (right panel) in an 85-year-old female with diabetes presenting with chest pain. The coronary angiogram demonstrates significant stenoses of the left main and circumflex coronary arteries. However, the perfusion images demonstrate only a reversible lateral wall defect. Quantification of stress and rest myocardial blood flow demonstrated a significant, global reduction on coronary flow reserve (estimated at 1.2, normal value >2.0), reflecting extensive myocardium risk that was underestimated by the semiquantitative estimates of myocardial perfusion. LAD, left ante-rior descending artery; LCX, left circumflex artery; LM, left main artery; RCA, right coronary artery. CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging

1	CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging FIGURE 270e-11 Stress and rest rubidium-82 myocardial perfusion positron emission tomography (PET) images (left) and noncontrast gated computed tomography (CT) images (right) delineating the extent and severity of coronary artery calcifications obtained with integrated PET/CT imaging. The images demonstrate extensive atherosclerosis (Agatston coronary calcium score = 1330) without flow-limiting disease based on the normal perfusion study. aAo, ascending aorta; dAo, descending aorta; PA, pulmonary artery. PART 10 Disorders of the Cardiovascular System

1	PART 10 Disorders of the Cardiovascular System FIGURE 270e-12 Stress and rest rubidium-82 myocardial perfusion positron emission tomography images (top), noncontrast gated computed tomography images (lower right), and selected coronary angiographic images obtained on a 59-year-old male patient with atypical angina. Despite the absence of significant coronary calcifications (Agatston calcium score = 0), the perfusion images demonstrated a dense and reversible perfusion defect involving the anterior and anteroseptal walls (arrows), reflecting significant obstructive disease in the left anterior descending coronary artery (LAD), confirmed on angiography. LM, left main artery.

1	As discussed above, the improved temporal and spatial resolution of modern multidetector CT scanners offer a unique noninvasive approach to delineate the extent and severity of coronary atherosclerosis. The extremely high sensitivity of this approach offers a very effective means for excluding the presence of CAD (high negative predictive value) (Table 270e-3). In the setting of high coronary calcium scores (e.g., >400), however, specificity is reduced because the blooming artifact of calcium does not allow one to evaluate the vessel lumen accurately. Given the high negative predictive value of CTA, a normal scan result effectively excludes obstructive CAD and abolishes the need for further investigation. As discussed below, this may be quite useful in patients with low-intermediate clinical risk presenting to the emergency room for chest pain. However, the limited capability of this technique to determine the severity of stenosis and to predict which obstructions are flow limiting

1	risk presenting to the emergency room for chest pain. However, the limited capability of this technique to determine the severity of stenosis and to predict which obstructions are flow limiting can make abnormal scan results more difficult to interpret, especially in terms of the possible need of coronary revascularization. There are emerging data suggesting that by adding a stress myocardial perfusion CT evaluation (similar to stress perfusion CMR) (Fig. 270e-13, top panel) or an estimated fractional flow reserve (so-called FFRCT) (Fig. 270e-13, lower panel), one can define the hemodynamic significance of anatomic stenosis. However, these are not in routine clinical use and remain emerging technologies.

1	As with invasive coronary angiography, assessments of the extent of CAD by CTA can also provide useful prognostic information. A low 1-year cardiac event rate has been reported for patients without obstructive CAD on CTA. For patients with obstructive CAD, the risk of adverse cardiac events increases proportionally with the extent of angiographically obstructive CAD. Although CTA can be helpful in assessing patency of bypass grafts, the assessment of stents is somewhat more challenging because the limited spatial resolution of CT and stent diameter (<3 mm being associated with the highest number of partial lumen visualization and nondiagnostic scans) both contribute to limited clinical results.

1	CMR Imaging The two approaches used with CMR to evaluate known or suspected CAD include the assessment of regional myocardial perfusion or wall motion at rest and during stress, the latter being analogous to dobutamine echocardiography. Although treadmill or bicycle exercise stress CMR is practiced in a small number of specialized centers, the logistics for stress MRI studies currently require the use of pharmacologic stress agents including vasodilators or dobutamine. Myocardial perfusion is evaluated by injecting a bolus of a GBCA followed by continuous data acquisition as the contrast passes through the cardiac chambers and into the myocardium. Relative perfusion deficits are recognized as regions of low signal intensity (black) within the myocardium (Video 270e-4). In addition, LGE imaging allows detection of bright areas of myocardial scar (white), which further enhances the utility of this approach for diagnosis of CAD (Fig. 270e-14).

1	The major advantage of dobutamine CMR over dobutamine echocardiography is better image quality and sharper definition of endocardial borders from the blood pool. Consequently, dobutamine CMR appears to have better diagnostic accuracy than dobutamine echocardiography for detection of CAD, especially in patients with poor acoustic window (Table 270e-3). A limitation of high-dose dobutamine stress CMR is that it bears the potential risk of severe side effects, such as hypotension and severe ventricular arrhythmias in the inhospitable environment of the magnetic resonance 270e-13 scanner. It occurs rarely (~5%), and most cases can be prevented with proper monitoring of vital signs and regional cine function. The advantage of stress perfusion CMR over SPECT is its clearly higher spatial resolution, allowing detection of subendocardial defects that may be missed by SPECT. The addition of the information from LGE imaging allows differentiation of hypoperfused (potentially ischemic) from

1	allowing detection of subendocardial defects that may be missed by SPECT. The addition of the information from LGE imaging allows differentiation of hypoperfused (potentially ischemic) from infarcted myocardium and characterizes the extent of myocardial ischemia.

1	FFRCT0.64 LAD FFRCT FFR 0.57 0.90.80.7 As with other imaging modalities, there is evidence that ischemia measurements derived from stress CMR studies also have prognostic value. In line with the nuclear and echocardiography literature, a normal CMR study is associated with a good prognosis. Conversely, the presence of new wall motion abnormali- FIGURE 270e-13 Examples of novel approaches to the assessment of flow-limiting coronary ties, regional perfusion defects, the com- artery disease (CAD) with cardiac computed tomography (CT). In the top panel, representative bination of wall motion abnormalities views of a coronary CT angiogram (CTA; left), coronary angiogram (middle), and stress myocardial and perfusion defects, and the presence of perfusion CT (right) images in a patient with CAD and prior stenting of the left anterior descending LGE are all predictors of adverse events.

1	coronary artery (LAD) are presented. On the CTA, the stent (arrows) is totally occluded as evidenced by the loss of contrast enhancement distal to the stent. The coronary angiogram demonstrates a concordant total occlusion of the LAD. On the perfusion CT images, there is a black rim (arrows) without Known CAD As discussed above, involving the anterior and anterolateral walls, indicating the lack of contrast opacification during there are many options for the evaluation stress consistent with myocardial ischemia. (Images courtesy of CORE 320 investigators.) The lower of a patient with suspected CAD present- panel illustrates an example of fractional flow reserve (FFR) estimates with coronary CTA (left) com ing with chest pain symptoms. The criti pared to the reference standard of invasive FFR. The FFR reflects the pressure differential between cal questions to be answered by a testing a coronary segment distal to a stenosis and the aorta. In normal coronary arteries, there is no gradi

1	FFR. The FFR reflects the pressure differential between cal questions to be answered by a testing a coronary segment distal to a stenosis and the aorta. In normal coronary arteries, there is no gradi strategy include the following: (1) Does ent, and FFR is 1. An FFR <0.80 is consistent with a hemodynamically significant stenosis. (Images the chest pain reflect obstructive CAD?

1	courtesy of Dr. James Min, Cornell University, New York.) (2) What are the shortand long-term risks? (3) Does the patient need to be considered for revascularization? For symptomatic patients without a prior history of CAD and a normal or nearly normal resting ECG who are able to exercise, the American Colloge of Cardiology/American Heart Association guidelines recommend standard exercise treadmill testing (ETT) as the initial testing strategy. The guidelines further suggest that patients who are categorized as low risk by ETT (e.g., those achieving >10 metabolic equivalents [METS] without chest pain or ECG changes) be treated initially with medical therapy, and those with high-risk ETT findings (i.e., typical angina with >2 mm ST-segment depression in multiple leads, ST elevation during exercise, drop in blood pressure, or sustained ventricular arrhtyhmias) be referred for coronary angiography.

1	The use of exercise testing in women presents difficulties that are not seen in men, reflecting the differences in the lower prevalence of obstructive CAD in women and the different accuracy of exercise testing in men and women. Compared with men, the lower pretest probability of disease in women means that more test results are false positive. In some of these patients, a positive ETT may reflect true myocardial ischemia caused my microvascular coronary artery dysfunction (so-called microvascular disease). In addition, the inability of many women to exercise to maximum aerobic capacity, the greater prevalence of mitral valve prolapse and microvascular disease in women, and possibly other reasons may contribute to the differences with men as well. The difficulties of using exercise testing for diagnosing obstructive CAD in women have led to speculation that stress imaging may be preferred over standard stress testing. However, recent data from the WOMEN study suggests that in

1	testing for diagnosing obstructive CAD in women have led to speculation that stress imaging may be preferred over standard stress testing. However, recent data from the WOMEN study suggests that in symptomatic, low-risk women who are able to exercise, standard ETT is a very effective initial diagnostic strategy as compared to stress radionuclide imaging. Women included in the study were randomized to standard ETT or exercise radionuclide perfusion imaging. The primary endpoint was the 2-year incidence of major adverse cardiac

1	FIGURE 270e-14 The image shows the late gadolinium enhance-ment image of a mid short-axis view. There is no evidence of infarction in the anterior wall, which would be seen as bright white areas, indicating that the stress perfusion defect primarily represents myocardial ischemia. This patient had a significant stenosis of the left anterior descending coronary artery. CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging 8.9 9.1 7.8 6.7 6.4 5.7 6 3.6 3.6 2.9 2.7 1.8 1.7 1.5 0.7 0.3 0.4 0 FIGURE 270e-15 Incremental risk stratification of stress imaging over Duke treadmill score in patients with suspected coronary artery disease. Stress imaging is most valuable in the intermediate-risk group. SPECT, single-photon emission computed tomography; VD, vessel disease. (Reproduced with permission from R Hachamovitch et al: Circulation 93:905, 1996; and TH Marwick et al: Circulation 103:2566, 2001.)

1	PART 10 Disorders of the Cardiovascular System events, defined as CAD death or hospitalization for an acute coronary syndrome or heart failure. At 2 years, there was no difference in major adverse cardiac events. As expected, ETT resulted in 48% lower costs compared to exercise radionuclide imaging.

1	Patients with intermediate-high risk after ETT (e.g., low exercise duration, chest pain, and/or ST-segment depression without high-risk features) will often require additional testing, either stress imaging or noninvasive CT coronary angiography, to more accurately characterize clinical risk. Most common stress imaging strategies in intermediate-risk patients include stress echocardiography and radionuclide imaging. In such patients, stress imaging with either SPECT or echocardiography has been shown to accurately reclassify patients who are initially classified as intermediate risk by ETT as low or high risk (Fig. 270e-15). Following this staged strategy of applying the low-cost ETT first and reserving more expensive imaging to refine risk stratification to patients initially classified as intermediate risk by ETT is more cost effective than applying stress or anatomic imaging as the initial test routinely.

1	A stress imaging strategy is the recommended first step for patients who are unable to exercise to an adequate workload and/or those with abnormal resting ECGs (e.g., left ventricular hypertrophy with strain, left bundle branch block). Importantly, the most recent documents regarding appropriate use of radionuclide and echocardiography imaging also considered that an imaging strategy may be an appropriate first step in patients with intermediate-high likelihood of CAD (e.g., diabetics, renal impairment) due to increased overall sensitivity for diagnosis of CAD and improved risk stratification.

1	In considering the potential clinical application of imaging modalities, the evidence supporting the role of assessment of ischemia versus anatomy must be considered. From the discussion above, a normal CTA is helpful because it effectively excludes the presence of obstructive CAD and the need for further testing, defines a low clinical risk, and makes management decisions regarding referral to coronary angiography straightforward. Because of its limited accuracy to define stenosis severity and predict ischemia, however, abnormal CTA results are more problematic to interpret and to use as the basis for defining the potential need of invasive coronary angiography and revascularization. In such patients, a follow-up stress test is usually required to determine the possible need of revascularization (Fig. 270e-16).

1	The justification of stress imaging in testing strategies has hinged on the identification of which patients may avoidance of excess catheterizations with their associated cost and risk and the potential for intervening unnecessarily. The acceptable diagnostic accuracy of stress imaging approaches, along with their robust risk stratification, and the ability of ischemia information to identify patients who would benefit from revascularization suggest a potential role as a first imaging strategy in patients with intermediate-high likelihood of CAD. Although the available data suggest similar diagnostic accuracy for SPECT, PET, echocardiography, and CMR, the choice of strategy depends on availability and local expertise.

1	Selecting a Testing Strategy in Patients with Known CAD Use and selection of testing strategies in symptomatic patients with established CAD (i.e., prior angiography, prior myocardial infarction, prior revascularization) differ from those in patients without prior CAD. Although standard benefit from a revascularization strategy by means of FIGURE 270e-16 Selected views from coronary computed tomography angiononinvasive estimates of jeopardized myocardium rather graphic (CTA) images (top panel) and stress and rest rubidium-82 myocardial than angiography-derived anatomic stenoses. Indeed, perfusion positron emission tomography images (lower panel) obtained on there is evidence that only the presence of moder-a 64-year-old male patient with atypical angina. The CTA images demonstrate ate-severe ischemia identifies patients with apparent dense focal calcifications in the left main (LM) and left anterior descending (LAD) improved survival with revascularization. Patients with coronary

1	ischemia identifies patients with apparent dense focal calcifications in the left main (LM) and left anterior descending (LAD) improved survival with revascularization. Patients with coronary arteries and a significant noncalcified plaque in the mid right coronary mild or no ischemia are better candidates for optimal artery (RCA; arrow). The myocardial perfusion images demonstrated no evidence of medical therapy. The advantages of this approach include flow-limiting stenosis. LCx, left circumflex artery; OM, obtuse marginal branch.

1	ETT may help distinguish cardiac from noncardiac chest pain, exercise ECG has a number of limitations following myocardial infarction and revascularization (especially coronary artery bypass grafting). These patients frequently have rest ECG abnormalities. In addition, there is a clinical need to document both the magnitude and localization of ischemia to be able to direct therapy, especially the potential need for targeted revascularization. Consequently, imaging tests are preferred for evaluating patients with known CAD.

1	There are also important differences in the effectiveness of imaging tests in these patients. As discussed above, coronary CTA is limited in patients with prior revascularization. Patients with prior coronary artery bypass grafting are a particularly heterogeneous group with respect to the anatomic basis of ischemia and its implications for subsequent morbidity and mortality. In addition to graft attrition, progression of disease in the native coronary arteries is not uncommon in symptomatic patients. While CTA provides excellent visualization of the bypass grafts, the native circulation tends to get heavily calcified and is generally not a good target for imaging with CTA. Likewise, blooming artifacts from metallic stents also limit the application of coronary CTA in patients with prior percutaneous coronary intervention. Although newer stent material may change the potential role of CTA in the future, it is probably not the first line of testing in these patients. If an anatomic

1	percutaneous coronary intervention. Although newer stent material may change the potential role of CTA in the future, it is probably not the first line of testing in these patients. If an anatomic strategy is indicated, direct referral to invasive angiography is preferred.

1	Stress imaging approaches are especially useful and preferred in symptomatic patients with established CAD. As in patients without prior CAD, normal imaging studies in symptomatic patients with established CAD also identify a low-risk cohort. In those with abnormal stress imaging studies, the degree of abnormality relates to posttest risk. In addition, stress imaging approaches can localize and quantify the magnitude of ischemia (especially with perfusion imaging), thereby assisting in planning targeted revascularization procedures. As in patients without prior CAD, the choice of stress imaging strategy depends on availability and local expertise.

1	Testing Strategy Considerations in Patients Presenting with Chest Pain to the Emergency Department Although acute chest pain is a frequent reason for patient visits to the emergency department (ED), only a small minority of those presentations represent an acute coronary syndrome (ACS). Strategies used in the evaluation of these patients include novel cardiac biomarkers (e.g., serum troponins), conventional stress testing (ETT), and noninvasive cardiac imaging. It is generally accepted that the primary goal of this evaluation is exclusion of ACS and other serious conditions rather than detection of CAD.

1	The routine evaluation of acute chest pain in most centers in the United States includes admission to a chest pain unit to rule out ACS with the use of serial ECGs and cardiac biomarkers. In selected patients, stress testing with or without imaging may be used for further risk stratification. Stress echocardiography and radionuclide imaging are among the most frequently used imaging approaches in these patients. The relative strengths and weaknesses of these testing options have been discussed above. Both approaches have been shown to be effective for identifying low-risk patients who can be safely discharged from the ED. Multiparametric CMR imaging has also been used successfully in patients with acute chest pain. In addition to the combined assessment of regional and global left ventricular function, myocardial perfusion, and tissue viability, it is also possible to evaluate the presence of myocardial edema to characterize the myocardium at risk secondary to reduced coronary flow

1	function, myocardial perfusion, and tissue viability, it is also possible to evaluate the presence of myocardial edema to characterize the myocardium at risk secondary to reduced coronary flow (Video 270e-5). Due to its ability to probe multiple aspects of myocardial physiology, cardiac anatomy, and tissue characterization with LGE imaging, CMR is also useful in diagnosing conditions that mimic ACS (e.g., acute myocarditis, takotsubo cardiomyopathy, pericarditis) (Fig. 270e-17). Thus, CMR imaging offers unique information of myocardial pathophysiology in the spectrum of ACS and is, perhaps, the most versatile of all noninvasive imaging techniques. Unfortunately, it is not widely available even at specialized centers and is not a first-line testing strategy. The main disadvantages of the “functional” testing strategy are that it is time consuming and is generally associated with a prolonged length of stay and, thus, is more costly.

1	FIGURE 270e-17 A four-chamber long-axis late gadolinium enhancement (LGE) image of a patient with acute myocarditis. Note that the LGE primarily involved the epicardial aspect of the myocardium (arrows), sparing the endocardium, which is a feature that distinguishes myocarditis from myocardial infarction, which affects the endocardium. Also note the multiple foci of LGE in this case affecting the lateral wall of the left ventricle. Viral myocarditis often presents with this pattern.

1	As discussed above, coronary CTA is a rapid and accurate imaging technique to exclude the presence of CAD and is well suited for the evaluation of patients with acute chest pain (Fig. 270e-18). Several single-center and, more recently, multicenter studies have demonstrated the feasibility, safety, and accuracy of coronary CTA in the ED. There have been four randomized controlled trials evaluating the efficacy of coronary CTA as the initial testing strategy as compared to usual care (which typically includes stress imaging). Patients in these trials had a very low clinical risk. Overall, there were no deaths and very few myocardial infarctions without differences between the groups. Likewise, there were no differences in postdischarge ED visits or rehospitalizations. These studies showed decreased length of stay with coronary CTA, and most but not all reported cost savings. An observation from a recent meta-analysis was that, compared to usual care, more patients assigned to coronary

1	decreased length of stay with coronary CTA, and most but not all reported cost savings. An observation from a recent meta-analysis was that, compared to usual care, more patients assigned to coronary CTA underwent cardiac catheterization (6.3% vs 8.4%, respectively) and revascularization (2.6% vs 4.6%, respectively). The relative increased frequency in the referral to cardiac catheterization and revascularization after coronary CTA compared to stress imaging testing strategies has also been observed in patients with stable chest pain syndromes.

1	Taken together, the available data clearly suggest that not all patients presenting with acute chest pain require specialized imaging testing. Patients with very low clinical risk and negative biomarkers (especially high-sensitivity troponin assays) can be safely triaged. The use of imaging tests in patients with low-intermediate risk should be carefully considered, especially given the trade-offs discussed above. Abnormalities of any of the four valvular structures in the heart can lead to significant cardiac dysfunction, heart failure, or even death. Echocardiography, CMR, and cardiac CT can be used for the evaluation of valvular heart disease, although echocardiography has generally been considered the first imaging test of choice for the assessment of valvular heart disease. In addition, echocardiography is the most cost-effective screening method for valvular heart disease. In some cases,

1	CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging PART 10 Disorders of the Cardiovascular System FIGURE 270e-18 Representative coronary computed tomography angiographic (CTA) images of two patients presenting to the emergency department with chest pain and negative biomarkers. The patient in A had angiographically normal coronary arteries; the panel shows a representative view of the right coronary artery (RCA). B and C show a corresponding significant stenosis in the mid portion of the RCA on both the CTA (B) and invasive angiographic view (C). (Images courtesy of Dr. Quynh Truong, Massachusetts General Hospital, Boston, MA.) CMR can complement echocardiography when echocardiographic acoustic window is inadequate, quantifying blood flow data more precisely, or providing complimentary assessment of adjacent vascular structures relevant to the valvular condition.

1	Echocardiography can be used to assess both regurgitant and stenotic lesions of any of the cardiac valves. Typical indications for echocardiography to assess valvular heart disease include cardiac murmurs identified on physical examination, symptoms of breathlessness that may represent valvular heart disease, syncope or presyncope, and preoperative exams in patients undergoing bypass surgery. A standard echocardiographic examination should include qualitative and quantitative assessment of all valves regardless of indication and should serve as an adequate screening test for significant valvular disease.

1	General Principles of Valvular Assessment • direCT visualizaTion of valvular sTruCTures Direct visualization of valve structures by two-dimensional echocardiography represents the first step in valvular evaluation. The morphology of valvular structures provides useful information regarding the etiology and severity of valvular disease. For example, two-dimensional imaging assessment of the aortic valve can identify the number of leaflets, determine whether the valve is bicuspid or tricuspid, and determine the severity of calcification and degree of leaflet excursion. Similarly, the classic appearance of a rheumatic mitral valve is extremely useful in determining the etiology of mitral stenosis, and mitral valve prolapse can be instantly identified without even the need for Doppler-based quantification.

1	evaluaTion of sTenoTiC valves As described earlier in the chapter, evaluation of stenotic valves generally includes estimation of the pressure gradient across the stenosis and determination of the valve area. Both of these measures have diagnostic and prognostic value. For example, when Doppler echocardiography is used to assess the maximal velocity across a stenotic aortic valve, this calculation will provide an accurate measure of the instantaneous gradient across the valve. This gradient will be higher than the mean gradient, as well as higher than that peakto-peak gradient obtained at cardiac catheterization. This gradient is dependent on both the degree of stenosis and the contractile function of the left ventricle. Patients with significant left ventricular dysfunction may have severe aortic stenosis but will be unable to generate a high gradient across the valve because generated pressure within the left ventricle will be diminished.

1	Assessment of stenotic valves generally requires estimation of both the pressure gradient across the valve and the valve area. Pressure gradient is estimated through direct application of the Bernoulli principle, and the formula p = 4v2 is usually sufficient to estimate the gradient across the valve. Several methods can be used to estimate valve areas, including the continuity principle based on the principle of conservation of mass. By this method, flow is estimated in two places. For example, for assessment of the aortic valve area, we measure the flow in the region of the left ventricular outflow tract and the cross-sectional area in this region, the product of which should be equal to the flow across the stenotic aortic valve and its cross-sectional area. Estimation of the mitral valve area in patients with suspected mitral stenosis can also be performed in a number of ways, including planimetry of the valve directly, estimation with continuity methods, or the most commonly used

1	area in patients with suspected mitral stenosis can also be performed in a number of ways, including planimetry of the valve directly, estimation with continuity methods, or the most commonly used pressure half-time method, in which the stenosis severity is estimated by the time it takes for the pressure—estimated from velocity by the Bernoulli equation—to reach half of its original value during mitral inflow.

1	evaluaTion of regurgiTanT lesions Regurgitant lesions are generally assessed by both visual assessment of the valve morphology and a variety of Doppler-based methods to assess the severity of regurgitation. The etiology of regurgitation can often be inferred from visual inspection. For example, prolapse of the mitral valve leaflets—and to a lesser extent, the aortic valve leaflets—can be easily visualized with two-dimensional echocardiography. In general, valvular regurgitation can be caused by abnormalities of the valve leaflets themselves or abnormalities of the annulus and supporting structures, and these can usually be distinguished visually on transthoracic echocardiography (see discussion below).

1	Quantification of valvular regurgitation is more difficult with echocardiography than quantification of valvular stenoses. Doppler-based methods are best suited to assess blood velocities rather than volumetric flow. The most widely used technique for assessing the severity of valvular regurgitation is color flow Doppler estimation, which is qualitative. More quantitative methods such as the proximal isovelocity surface area (PISA) method (see below) allow for more accurate assessment of regurgitation and provide estimation of the regurgitant fraction and effective regurgitant orifice area but are less widely used. Assessment of regurgitant lesions with CMR also has a number of advantages (see below).

1	Assessment of Aortic Stenosis Aortic stenosis, one of the most common forms of valvular heart disease, most often occurs because of gradual progression of valvular calcification in both normal and congenitally abnormal valves. Assessment of aortic stenosis is most commonly performed with echocardiography, although techniques for quantitative assessment of aortic stenosis with CMR have been developed and increasingly used over the past decade. Echocardiographic assessment generally begins with visual inspection of the valve, usually in the parasternal long-axis and short-axis views. This allows for assessment of valvular morphology, whether it is tricuspid, bicuspid, or some variant; degree of leaflet calcification; and leaflet excursion.

1	The normal aortic valve consists of three leaflets or cusps: the right coronary, the left coronary, and the noncoronary cusps. Abnormalities of cusp development are some of the most common congenital heart anomalies, the most common of which is bicuspid aortic valve, with two opening leaflets rather than three (Fig. 270e-19). The aortic valve can be visualized on echocardiography, although sometimes it can be difficult to distinguish true bicuspid aortic valve from variants, including the presence of a vestigial commissure (raphe). Bicuspid aortic estimating both the pressure gradient across the valve and the valve area. Patients with moderate aortic stenosis or higher generally have peak instantaneous velocities of 3.0 m/s and higher, and often higher than 4.0 m/s, corresponding to pressure gradients of 36 and 64 mmHg, respectively. Because pressure gradients across the aortic valve can be underestimated in patients with severe left ventricular dysfunction, estimation of valve area

1	gradients of 36 and 64 mmHg, respectively. Because pressure gradients across the aortic valve can be underestimated in patients with severe left ventricular dysfunction, estimation of valve area by the continuity principle is the most accurate technique for assessing the severity of the stenosis. However, evaluation of the patient with so-called low-flow or low-gradient aortic stenosis can be challenging and can sometimes require provocative testing such as dobutamine echocardiography. In these cases, it is important to distinguish whether the valve is indeed capable of opening further or simply behaving like a stenotic valve because of the low-pressure gradient.

1	270e-17 CHAPTER 270e valve, one of the most common congenital anomalies, predisposes to both aortic stenosis and aortic insufficiency. As discussed above, the degree of aortic stenosis is assessed by ABCFIGURE 270e-19 Normal aortic valve in the parasternal long-axis view (A) and short-axis view (B), and bicuspid aortic valve showing typical 10 o’clock to 4 o’clock leaflet orientation (C). aortic insufficiency, paravalvular leak resulting from patient–prosthe-sis mismatch). Echocardiography is the imaging modality of choice for long-term surveillance. Aortic valve areas less than 1.0 cm2 are generally considered severe, and valve areas less than 0.6 cm2 are considered critical. Because patients with good left ventricular function can often tolerate severe aortic stenosis for a considerable period of time, valve areas or gradients alone should not be used to determine whether an individual patient should undergo aortic valve surgery, as this remains a clinical decision.

1	Some patients with apparent aortic stenosis actually have subvalvular or even supravalvular obstruction. Hypertrophic cardiomyopathy represents the classic form of subvalvular aortic stenosis, but this is usually easily distinguished from aortic stenosis on echocardiography as the valve leaflets can be seen opening during systole. Subaortic membranes can behave very similarly to leaflet aortic stenosis, and the membranes themselves can be very thin and difficult to visualize, although the presence of a murmur, a gradient across the valve with aortic leaflets that appear to open normally, is highly suggestive of a membrane. Supravalvular aortic stenosis, although exceedingly rare, also occurs.

1	The emergence of transcatheter aortic valve intervention as a therapeutic option for patients with severe aortic stenosis who are not optimal candidates for surgical replacement has resulted in a very important clinical role for multimodality imaging. Imaging plays a critical role in preprocedural planning, intraprocedural implantation optimization, and follow-up of these patients. CT plays an important role in defining the eligibility of the proposed access site (CTA of the aorta and iliac arteries) and in defining the anatomic relationships between the aortic valve and aortic root, left ventricle, and coronary ostia. Cardiac CT and transesophageal echocardiography are also used to define the device size. Transesophageal echocardiography is used during the device implantation to ensure the best prosthesis–patient match, to assess pros-Assessment of Aortic Regurgitation Assessment of aortic regurgitation requires qualitative assessment of the aortic valve structure. Aortic

1	to ensure the best prosthesis–patient match, to assess pros-Assessment of Aortic Regurgitation Assessment of aortic regurgitation requires qualitative assessment of the aortic valve structure. Aortic regurgitation is common with congenital abnormalities of the aortic valve, the most common of which is bicuspid aortic valve. Aortic regurgitation often coexists with aortic stenosis, and it is not uncommon for patients to have both severe aortic stenosis and regurgitation. Congenital abnormalities of the aortic leaflets, such as bicuspid aortic valve, are common causes of aortic insufficiency. Dilatation of the aortic root, as occurs in patients with hypertension and other disorders in which aortic dilatation can occur, can also lead to aortic regurgitation even when the valve leaflets are intrinsically normal due to malcoaptation of the leaflets. Aortic root dilatation is common in patients with aortic regurgitation, both as a cause or coexisting lesion, and the aortic root and

1	are intrinsically normal due to malcoaptation of the leaflets. Aortic root dilatation is common in patients with aortic regurgitation, both as a cause or coexisting lesion, and the aortic root and ascending aorta should be measured and followed in these patients (Fig. 270e-20).

1	Because aortic regurgitation can result in dilatation of the left ventricle over time with ultimate reduction in ventricular function, caring for the patient with aortic regurgitation requires serial assessment of ventricular size and function. Patients whose ventricles dilate beyond an end-systolic diameter of 5.5 cm or whose LVEF declines below normal are at significantly higher risk of death or heart failure, and these measures are often used to decide the need for valve surgery. Quantitation of regurgitation itself can be performed using a number of methods. Semiquantitative visual assessment of aortic regurgitant jet width and depth by color flow Doppler remains the most used. The jet diameter as a ratio of the left ventricular outflow tract diameter proximal to the valve represents one of the most reliable indices of severity and correlates well with angiographic assessment. Similarly, the vena contracta, which represents the smallest diameter of the regurgitant flow at the

1	one of the most reliable indices of severity and correlates well with angiographic assessment. Similarly, the vena contracta, which represents the smallest diameter of the regurgitant flow at the level of the valve, can be used to assess the severity of aortic regurgitation. Other Doppler-based methods include assessing the pressure half-time, or rate of decline of the pressure gradient between the aorta and left ventricle, a measure of acuity of aortic regurgitation, and assessing aortic flow reversal in the descending aorta. The regurgitant volume can be calculated by comparing the

1	Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging thesis position and function after deployment, FIGURE 270e-20 Aortic regurgitation visualized by color flow Doppler in the parasternal and to identify immediate complications (e.g., long-axis view (A) and the parasternal short-axis view (B). FIGURE 270e-21 The resultant flow curve generated from phase contrast imaging demonstrates a forward flow of 123 mL and a regurgitant volume of 67 mL, yielding a regurgitant fraction of 54% indicating severe aortic regurgitation. flow across the aortic and pulmonic valves, assuming the pulmonic valve is competent.

1	flow across the aortic and pulmonic valves, assuming the pulmonic valve is competent. CMR offers a number of advantages over echocardiography in the assessment of aortic regurgitation. CMR can be more accurate than echocardiography for assessing small changes in cardiac size or function that can occur over time in patients with aortic insufficiency. In addition, CMR techniques can very accurately quantify regurgitant volume in patients with aortic insufficiency, a known limitation of echocardiography. CMR can also capture three-dimensional imaging PART 10 Disorders of the Cardiovascular System of the aortic size that in some cases can be helpful in determining the etiology of the aortic regurgitation or in monitoring the patient (Fig. 270e-21 and Video 270e-6).

1	Assessment of Mitral Regurgitation The normal mitral valve consists of an anterior and posterior leaflet in a saddle shape configuration (Fig. 270e-22). The leaflets are attached to the papillary muscles via chordae tendineae that insert on the ventricular side of the leaflets. Mitral regurgitation can occur due to abnormalities of the leaflets, the chordal structures, or the ventricle, or any combination of these (Fig. 270e-23).

1	Mitral valve prolapse, in which one leaflet moves behind the plane of the other leaflet, can be due to myxomatous degeneration of the valves and leaflet redundancy, disruption of chordal structures secondary to degenerative disease, or papillary muscle rupture or dysfunction following myocardial infarction. Regurgitant jets can be visualized using color flow Doppler. The velocity of regurgitant jets is driven by the pressure gradient between the two chambers. This velocity tends to be quite high for left-sided regurgitant lesions, including mitral regurgitation and aortic regurgitation, resulting in turbulent jets on color flow Doppler (Fig. 270e-23). Visual estimation of color flow Doppler is generally sufficient for qualitative assessment of regurgitant severity but can dramatically underor overestimate regurgitation severity, particularly when regurgitant jets are quite eccentric. For this reason, quantitative assessment is generally recommended, especially when making clinical

1	underor overestimate regurgitation severity, particularly when regurgitant jets are quite eccentric. For this reason, quantitative assessment is generally recommended, especially when making clinical decisions about surgical intervention. The PISA method is generally used for quantitative assessment of severity of mitral regurgitation. This method relies on estimation of the velocity of flow acceleration at a specific distance proximal to the valve with the assumption that the flow accelerates in concentric hemispheres.

1	FIGURE 270e-22 Normal mitral valve in two-dimensional views (left) and with three-dimensional imaging (right). FIGURE 270e-23 A. Mitral valve prolapse with posterior leaflet visualized prolapsing behind the plane of the anterior leaflet (arrow). B. Color flow Doppler showing mitral regurgitation in a patient with mitral valve prolapse. C. Severe functional mitral regurgitation in a patient with a dilated left ventricle.

1	As with aortic insufficiency, assessment of ventricular structure and function is also integral in the evaluation of mitral regurgitation. Although some patients have mitral regurgitation due to intrinsic abnormalities of the valve itself, in others, the valve can be relatively normal but the mitral regurgitation can be secondary to dilatation and remodeling of the left ventricle. So-called functional mitral regurgitation is generally secondary to apical displacement of the papillary muscles in a dilated ventricle, resulting in the leaflets of the mitral valve being pulled toward the apex of the heart, resulting in poor coaptation during systole and resultant relatively central mitral regurgitation. This type of mitral regurgitation can generally be distinguished from intrinsic mitral valve disease, and the surgical or procedural treatment of these conditions can be different. Knowledge of the etiology of mitral regurgitation can be important for a surgeon planning mitral valve

1	valve disease, and the surgical or procedural treatment of these conditions can be different. Knowledge of the etiology of mitral regurgitation can be important for a surgeon planning mitral valve surgery. Moreover, new procedural approaches to mitral valve disease may be different depending on the etiology.

1	Ventricular dilatation is an important predictor of outcome in patients with mitral regurgitation of any cause. It is important to realize that in a patient with significant mitral regurgitation, a large portion of the blood being ejected from the left ventricle with every beat is regurgitant, thus artificially increasing the ejection fraction. Thus, an ejection fraction of 55% in a patient with severe mitral regurgitation may actually represent substantial reduction in myocardial systolic function. CMR can be helpful in evaluating mitral regurgitation in a subset of patients when echocardiographic assessment is inadequate. CMR can directly quantify regurgitant volume of the mitral regurgitant jet or indirectly quantify regurgitant volume by measuring the difference of left ventricular stroke volume and aortic forward flow.

1	Assessment of Mitral Stenosis Rheumatic mitral disease remains the most common cause of mitral stenosis, although mitral stenosis can also result from severe calcification of the mitral leaflets. Rheumatic mitral stenosis has a distinct appearance characterized by tethering at the leaflet tips and relative pliability of the leaflets themselves, resulting in a hockey stick–type deformation particularly of the anterior leaflet (Fig. 270e-24). Narrowing of the mitral orifice impedes flow from the left atrium to the left ventricle, resulting in increased pressures in the left atrium, which are then transmitted backward into the pulmonary vasculature and the right side of the heart. When mitral stenosis is suspected, echocardiography can be useful for determining etiology (specifically whether it is rheumatic or not), estimating the valve areas and gradients across the valve, assessing the left atrium, and assessing right ventricular size and function. Assessment of left atrial size and

1	it is rheumatic or not), estimating the valve areas and gradients across the valve, assessing the left atrium, and assessing right ventricular size and function. Assessment of left atrial size and right ventricular size and function is particularly useful in helping determine the severity of the mitral stenosis.

1	MYOCARDIAL INFARCTION AND HEART FAILURE Role of Imaging after Myocardial Infarction Imaging can be particularly useful in the immediate and long-term follow-up of patients with myocardial infarction. As discussed earlier in the chapter, CMR is the best technique for direct assessment of infarcted myocardium. LGE imaging by CMR provides accurate delineation of infarct size and morphology. In a recent multicenter study, LGE imaging by CMR identified infarct location accurately and detected acute and chronic infarcts with a sensitivity of 99% and 94%, respectively. With an in-plane spatial resolution of 1.5–2 mm and a high contrast-to-noise ratio, LGE by CMR has excellent sensitivity in detecting small areas of myocardial scar. In addition, regions of microvascular obstruction (no-reflow) can be seen as dense hypoenhanced areas within the core of a bright region of infarction. Both the presence of LGE and microvascular obstruction are markers of increased clinical risk.

1	While echocardiography is often used to assess myocardial function immediately after myocardial infarction, myocardial stunning is common in the early post–myocardial infarction period, especially in patients who undergo reperfusion therapy. In these patients, either partial or complete recovery of ventricular function is common within several days, so that early estimation of ejection fraction may be misleading. In patients with uncomplicated myocardial infarction, imaging can generally be deferred for several days so that a more accurate assessment of cardiac function, including regional wall motion, can be assessed (Fig. 270e-25).

1	Echocardiography is the best method for assessment of patients with suspected mechanical complications after myocardial infarction. These include mitral regurgitation secondary to either papillary muscle dysfunction or rupture of papillary muscle head, ventricular septal defect, or even cardiac rupture. A new severe systolic murmur should raise suspicions for either severe mitral regurgitation or ventricular septal defect. While cardiac rupture is often catastrophic, contained ruptures, also known as pseudoaneurysms, can occur, and early diagnosis and surgical treatment are the best way to maximize survival. The presence of thrombus within the pericardial space following myocardial infarction should immediately raise suspicion of myocardial rupture and represents a surgical emergency. CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging

1	CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging FIGURE 270e-24 A. Rheumatic mitral stenosis showing pliable leaflets tethered at the tips (arrow). Note the characteristically enlarged left atrium. B. Mitral stenosis visualized from a three-dimensional echocardiogram. FIGURE 270e-25 Acute left anterior descending artery distribution myocardial infarction at end systole showing akinetic region (arrows). PART 10 Disorders of the Cardiovascular System

1	FIGURE 270e-25 Acute left anterior descending artery distribution myocardial infarction at end systole showing akinetic region (arrows). PART 10 Disorders of the Cardiovascular System Some patients demonstrate progressive left ventricular dilatation and dysfunction, known as cardiac remodeling, after myocardial infarction. Assessment of cardiac function and regional wall motion is useful in the follow-up period, generally between 1 and 6 months following infarction. The persistence of left ventricular systolic dysfunction following infarction is used to determine the type of therapy (e.g., angiotensin-converting enzyme inhibitors or angiotensin receptor blockers are typically used in patients with systolic dysfunction following myocardial infarction).

1	In patients with acute or subacute myocardial infarction, investigation of residual ischemia and/or viability is occasionally an important clinical question, especially among those with recurrent symptoms after myocardial infarction (Fig. 270e-26). All cardiac imaging techniques can provide information regarding myocardial viability and ischemia. In the absence of definitive trials offering head-to-head comparisons between techniques in large series of patients, uncertainty persists concerning the relative accuracies of each method for predicting functional and prognostic benefit after revascularization. Thus, one should exercise caution in the interpretation of the relative diagnostic accuracy of each imaging technology. Nevertheless, the available data suggest that radionuclide imaging, especially PET, is highly sensitive, with higher negative predictive value than dobutamine echocardiography. In contrast, dobutamine echocardiography tends to be associated with higher specificity

1	especially PET, is highly sensitive, with higher negative predictive value than dobutamine echocardiography. In contrast, dobutamine echocardiography tends to be associated with higher specificity and positive predictive accuracy than the radionuclide imaging methods. The experience with CMR suggests that it offers similar predictive accuracies as those seen with dobutamine echocardiography.

1	Role of Imaging in New-Onset Heart Failure Echocardiography is usually a first-line test in patients presenting with new-onset heart failure. As discussed above, this test provides a direct assessment of ventricular function and can help distinguish patients with reduced ejection fraction from those with preserved ejection fraction. In addition, it provides additional structural information including an assessment of valves, myocardium, and pericardium. Although coronary angiography is commonly performed in patients with reduced ejection fraction, the determination of heart failure etiology in an individual patient may be difficult even if angiographically obstructive CAD is present. Indeed, patients with heart failure and no angiographic CAD may have typical angina or regional wall motion abnormalities on noninvasive imaging, whereas patients with angiographically obstructive CAD may have no symptoms of angina

1	FIGURE 270e-26 Examples of myocardial viability patterns obtained with cardiac magnetic resonance imaging (MRI) and positron emission tomography (PET) in three different patients with coronary artery disease. The top panel demonstrates extensive late gadolinium enhancement (bright white areas) involving the anterior, anteroseptal, and apical left ventricular walls (arrows), consistent with myocardial scar and nonviable myocardium. The lower left panel demonstrates rubidium-82 myocardial perfusion and 18F-fluorodeoxyglucose (FDG) images showing a large and severe perfusion defect in the anterior, anterolateral, and apical walls, indicating preserved glucose metabolism (so-called perfusion-metabolic mismatch) consistent with viable myocardium. The right lower panel shows similar PET images demonstrating concordant reduction in perfusion and metabolism (so called perfusion-metabolic match) in the lateral wall, consistent with nonviable myocardium.

1	FIGURE 270e-27 A case of cardiac amyloidosis. Note on this late gadolinium enhancement image that there were multiple foci of gadolinium accumulation in the left ventricle (LV) myocardium (red arrows), as well as the left atrial (LA) walls (blue arrows). The LV walls were markedly increased in thickness, and both atria were dilated, consistent with a restrictive cardiac morphology. The blood pool signal was diminished after contrast injection, which was consistent with high burden of amyloid disease in other organs that causes gadolinium concentration in the blood to rapidly go down. RA, right atrium; RV, right ventricle.

1	or history of myocardial infarction. Thus, the appropriate classification for any given patient is not always clear, and it often requires the complementary information of coronary angiography and noninvasive imaging. Stress radionuclide imaging and echocardiography can be helpful in delineating the extent and severity of inducible myocardial ischemia and viability. Multiparametric CMR can be quite helpful in the differential diagnosis of heart failure etiologies. Apart from quantifying left and right ventricular volumes and function, CMR can provide information about myocardial ischemia and scar. The pattern of LGE helps differentiate infarction (typically starting in the subendocardium and involving a coronary territory) from other forms of infiltrative or inflammatory cardiomyopathies (typically involving the midor subepicardial layers without following a coronary distribution) (Fig. 270e-27). In addition, it can assess the presence of myocardial edema (e.g., myocarditis)

1	(typically involving the midor subepicardial layers without following a coronary distribution) (Fig. 270e-27). In addition, it can assess the presence of myocardial edema (e.g., myocarditis) cardiomyopathy has variable degree of increased ventricular thickness, 270e-21 and often is seen to have outflow obstruction and intense LGE in regions with marked hypertrophy (Fig. 270e-28). CMR also can quantify myocardial iron content in patients at risk of iron-overload cardiomyopathy (Video 270e-7).

1	PET metabolic imaging has a complementary role in the evaluation of inflammatory cardiomyopathies, especially sarcoidosis. In patients with suspected cardiac sarcoidosis, the presence of focal and/or diffuse glucose uptake can help identify areas of active sarcoidosis. In addition, for patients undergoing immunosuppressive therapy, PET is frequently used to monitor therapeutic response (Fig. 270e-29). In patients with ischemic cardiomyopathy, radionuclide imaging in general and PET in particular are frequently used to quantify the presence and extent of myocardial ischemia and viability to assist with clinical decision making related to myocardial revascularization (Fig. 270e-26).

1	Therapies used to treat cancer can adversely affect the cardiovascular system. As the efficacy of cancer treatment and survival improve, many patients are presenting with late adverse consequences from chemotherapy and/or radiation therapy on cardiovascular function. Thus, the morbidity and mortality from late cardiovascular complications threaten to offset the early gains in cancer survival, especially among children and young adults. Early recognition and treatment of cardiomyocyte injury are critical for successful application of preventative therapies, but difficult because the adverse effects on cardiac function are a relatively late manifestation after exposure to anticancer therapy.

1	The accepted standard for clinical diagnosis of cardiotoxicity is defined as a >5% reduction in LVEF to <55% with symptoms of heart failure, or a >10% drop in LVEF to <55% in patients who are asymptomatic. Thus, noninvasive imaging plays a major role in diagnosing and monitoring for cardiac toxicity in patients undergoing cancer treatment. Radionuclide angiography has been the technique of choice

1	CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging and quantify myocardial iron deposi-FIGURE 270e-28 This figure demonstrates three pulse sequence techniques by cardiac magtion that can potentially lead to cardiac netic resonance that are often used to assess patients with hypertrophic cardiomyopathy, all toxicity. Infiltrative cardiomyopathy such displayed in the mid short-axis scan plane. The center panel demonstrates that the left ventricle as amyloidosis typically has a restrictive (LV) was markedly thickened in its wall thickness especially in the LV septum (red arrows). This findcardiomyopathy pattern (bilateral atrial ing was matched by marked regions of late gadolinium enhancement (LGE), which was consisenlargement and biventricular increased tent with fibrosis in these segments (right panel, white arrows). The left panel was cine myocardial wall thickness). CMR of patients with car-tagging in the

1	and biventricular increased tent with fibrosis in these segments (right panel, white arrows). The left panel was cine myocardial wall thickness). CMR of patients with car-tagging in the same slice plane. Myocardial tagging is used to assess the normal intramyocardial diac amyloidosis often also demonstrates strain by assessing distortion of the myocardial grids during systole. In this case, despite normal-a characteristic pattern of diffuse endo-appearing systolic radial wall thickening, the myocardial strain as assessed by the distortion of cardial infiltration of the left ventricle grids was markedly reduced (left panel, white arrows). This finding is consistent with substantial and the atria (Fig. 270e-27). Hypertrophic myofibril disarray in the anterior and anteroseptal segments in this patient. RV, right ventricle.

1	PART 10 Disorders of the Cardiovascular System FIGURE 270e-29 Representative cardiac magnetic resonance (CMR; top panel) and positron emission tomography (PET; lower panel) images from a 45-year-old male presenting with complete heart block. The CMR images demonstrate extensive late gadolinium enhance-ment in the subepicardial left ventricular (LV) anterior and anteroseptal walls and also in the right ventricular (RV) free wall (arrows). The PET images demonstrate extensive fluorodeoxyglucose uptake in the same areas, most consistent with active inflammation due to sarcoidosis. for quite some time. However, this is rapidly changing, and echocardiography now plays a major role in this application.

1	for quite some time. However, this is rapidly changing, and echocardiography now plays a major role in this application. Recently, more novel imaging approaches have been advocated, including deformation imaging with echocardiography and fibrosis imaging with CMR. These techniques have shown promising results in experimental animal models and in humans. In addition, there are also proof-of-concept studies in animal models using molecular imaging approaches targeting the mechanisms of cardiac toxicity (e.g., apoptosis and oxidant stress), which can presumably provide the earliest signs of the off-target effects of these therapies. However, all of these techniques are currently considered experimental.

1	The fibroelastic pericardial sac surrounding the heart consists of a visceral, or epicardial, layer and a parietal layer, with a generally small amount of pericardial fluid in between layers. The pericardium is generally quite pliable and moves easily with the heart during contraction and relaxation. Abnormalities of the pericardium can affect cardiac function primarily by impairing the heart’s ability to fill. Inflammation of the pericardium can lead to an accumulation of fluid between the two layers, or pericardial effusion, which can be visualized by echocardiography, CMR, or CT. Other reasons for accumulation of pericardial fluid include infection, malignancy, and bleeding into the pericardium. The latter can be the result of catastrophic processes such as trauma, cardiac rupture, perforation in the setting of a cardiac procedure, cardiac surgery, or dissection of the aorta with extension in the pericardium.

1	Echocardiography remains the initial test of choice for assessing pericardial disease, especially effusions (Fig. 270e-30). Moreover, echocardiography can be useful in evaluating for pericardial constrictive physiology, in which a thick noncompliant pericardium impairs cardiac filling. The location, size, and physiologic consequences of accumulated pericardial effusion can generally easily be determined by echocardiography. Pericardial tamponade occurs when enough pericardial fluid accumulates so that the intrapericardial pressure exceeds filling pressures of the heart, generally the right ventricle. The balance between intrapericardial pressure and ventricular pressure is more important than the extent of fluid accumulation. Conditions in which pericardial effusions accumulate over a long period of time, as can be the case in the setting of malignant effusions, can lead to large pericardial fluid accumulations without the classic hemodynamic findings associated with pericardial

1	a long period of time, as can be the case in the setting of malignant effusions, can lead to large pericardial fluid accumulations without the classic hemodynamic findings associated with pericardial tamponade. In contrast, rapid accumulations of pericardial fluid, such as those that occur due to cardiac rupture or perforation, can lead to tamponade physiology without very large effusions. In patients with suspected pericardial effusion or tamponade, echocardiography can usually be performed rapidly, at the bedside, and even by operators with limited skill. The distance from the parietal to the visceral pericardial layer can be measured, and when this exceeds approximately 1 cm, an effusion is considered significant. Echocardiographic features suggestive of tamponade include diastolic collapse of the right ventricular free wall, suggestive of pericardial pressures that exceed right ventricular filling pressures, and Doppler evidence of respiratory flow variation, which is the Doppler

1	of the right ventricular free wall, suggestive of pericardial pressures that exceed right ventricular filling pressures, and Doppler evidence of respiratory flow variation, which is the Doppler equivalent of pulsus paradoxus. Despite the benefits of echocardiography in suspected pericardial tamponade, the diagnosis of tamponade remains a clinical diagnosis, and other important features, such as patient’s blood pressure in the presence of pulsus paradoxus, needs to be taken into account when considering therapeutic options.

1	FIGURE 270e-30 Pericardial effusion with tamponade physiology. The right ventricle (arrow) is small and collapsing in end diastole due to increased pericardial pressure.

1	Chronic inflammation of the pericardium can lead to thickening and potentially calcification of the parietal pericardium, resulting in pericardial constriction in which diastolic filling can be severely impaired. In these cases, filling of the ventricles comes to an abrupt halt when the volume of ventricular filling is impaired by the constricting pericardium. Assessment of pericardial thickness in these patients is important, but it is just as important to note that approximately one in five patients with severe pericardial constriction have no significant pericardial thickening by imaging or at surgery. Thus, a lack of thickened pericardium does not rule out pericardial constriction, and patients’ signs and symptomatology and physiologic evidence of constriction should be assessed independently. Pericardial constriction typically demonstrates marked respiratory changes in diastolic flow on Doppler echocardiography, in contrast to restrictive cardiomyopathy, but substantial overlap

1	Pericardial constriction typically demonstrates marked respiratory changes in diastolic flow on Doppler echocardiography, in contrast to restrictive cardiomyopathy, but substantial overlap exists. CT and CMR offer tomographic, whole-heart assessment of pericardial thickening and other anatomy abnormalities in pericardial constriction (enlarged atria, vena cavas, pleural and pericardial effusions) (Fig. 270e-31 and Video 270e-8). CMR offers the additional information of pericardial fibrosis and inflammation by LGE imaging, as well as evidence of constrictive physiology (e.g., regional relaxation concordance due to myocardial adhesions, paradoxical septal motion at rest or during Valsalva maneuver).

1	Echocardiography is usually the modality that first detects the presence of a cardiac mass. Differential diagnoses of an intracardiac mass most often include thrombus, tumor, or vegetation. Given their unrestricted tomographic views and multiplanar three-dimensional imaging, CMR and CT can complement echocardiography by further characterizing the physical features of the cardiac mass. Compared to CT, CMR has the advantage of higher tissue contrast differentiation, more robust cine imaging, and the use of multifaceted techniques within the same imaging session to determine the physiologic characteristics of the mass. Gadolinium contrast enhancement patterns of increased capillary perfusion can help to determine the presence and extent of FIGURE 270e-31 A female patient developed pericardial constriction and right heart failure, secondary to radiation therapy for breast cancer. Note the multiple pericardial adhesions (red arrows).

1	FIGURE 270e-31 A female patient developed pericardial constriction and right heart failure, secondary to radiation therapy for breast cancer. Note the multiple pericardial adhesions (red arrows). FIGURE 270e-32 Cardiac thrombus (arrow) in an apical aneurysmal region following acute myocardial infarction.

1	vascularity within the mass, relevant for differentiating tumor from thrombus. Structures that are known to mimic a cardiac mass include (1) anatomic variants, such as the Eustachian valve, Chiari network, crista sagittalis or terminalis, and the right ventricular moderator band, and (2) “pseudotumors,” such as interatrial septal aneurysm, coronary or aortic aneurysm, lipomatous hypertrophy of interatrial septum, hiatal hernia, or a catheter/pacemaker lead. A number of coexisting conditions should raise the likelihood of a cardiac thrombus (Fig. 270e-32), including regional wall motion abnormality from infarction or ventricular aneurysm, atrial fibrillation leading to slow flow in the left atrial appendage, or presence of venous catheters or recent endovascular injury. CMR has the advantage of being able to assess regional wall motion and infarction or ventricular aneurysm in matching scan planes, adjacent to the cardiac thrombus, using cine and LGE imaging, respectively. For

1	advantage of being able to assess regional wall motion and infarction or ventricular aneurysm in matching scan planes, adjacent to the cardiac thrombus, using cine and LGE imaging, respectively. For ventricular thrombus, gadolinium-enhanced LGE imaging can detect thrombus at a higher sensitivity than echocardiography by depicting high-contrast difference between the dark thrombus and its adjacent structures and by imaging in three dimensions. In addition, mural thrombus does not enhance on first-pass perfusion and often has a characteristic “etched” appearance (black border surrounding a bright center) on LGE imaging, thus providing higher diagnostic specificity than anatomic information alone (Fig. 270e-33). Comparing the signal intensities of a mass before and after contrast injection may confirm the lack of tissue vascularity (i.e., thrombus)

1	CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging FIGURE 270e-33 Late gadolinium enhancement image of a massive anterior infarction complicated by a dyskinetic left ventricular LV aneurysm and intracavitary thrombus (red asterisk). Disorders of the Cardiovascular System FIGURE 270e-34 A case of a cardiac fibroma. A patient presented with shortness of breath and was found to have a large myocardial mass on echocardiography. Cine cardiac magnetic resonance imaging confirmed the large myocardial mass involving the anterolateral wall. Shortly after gadolinium contrast was injected, the myocardial mass demonstrated intense accumulation of contrast on LGE imaging (right panel, asterisk). This is a case of cardiac fibroma. The patient also has gingival hyperplasia and bifid thoracic ribs, a part of the rare Gorlin’s syndrome.

1	by the lack of signal enhancement after contrast administration. Like intracardiac thrombus, regions of microvascular obstruction also appear dark, but microvascular obstruction is confined within the myocardium and surrounded by infarction and thus can be differentiated from intracardiac thrombus. CMR imaging for small thrombus in the left atrial appendage is difficult due to slow flow in the atrium and rhythm irregularity from atrial fibrillation, but it may be helpful in cases where transesophageal echocardiography is suboptimal or not feasible.

1	The majority of cardiac malignancy is metastatic, and metastatic cardiac malignancy is far more common than primary cardiac malignancies; these metastatic involvements of the heart are the result of direct invasion (e.g., lung and breast), lymphatic spread (e.g., lymphomas and melanomas), or hematogenous spread (e.g., renal cell carcinoma). Primary benign cardiac tumors are seen mostly in children and young adults and include atrial myxoma, rhabdomyoma, fibroma, and endocardial fibroelastoma (Fig. 270e-34). Atrial myxomas are often seen as a round or multilobar mass in the left atrium (75%), right atrium (20%), or ventricles or mixed chambers (5%). They typically have inhomogeneous brightness in the center on cine steady-state free precession imaging due to their gelatinous contents and may have a pedunculated attachment to the fossa ovalis. Primary malignant cardiac tumors are extremely rare and include angiosarcoma, fibrosarcoma, rhabdomyosarcoma, and liposarcoma.

1	Patients with suspected endocarditis often undergo echocardiography for the purpose of identifying vegetations or intramyocardial abscesses. Vegetations are generally highly mobile structures that most typically are attached to valves or present in areas of the heart with turbulent flow. The absence of a vegetation on echocardiography does not rule out endocarditis, because small vegetations below the resolution of the imaging techniques can be present. Echocardiography remains the best technique for assessment of vegetations because its high temporal resolution allows visualization of the typical oscillating motion, although large vegetations can be visualized with other techniques (Fig. 270e-35). The size and location of a vegetation do not necessarily provide any specific information about the type of infection. Abscesses, particularly around the aortic and mitral annuli, are particularly concerning in patients with endocarditis and should be suspected in patients with prolongation

1	the type of infection. Abscesses, particularly around the aortic and mitral annuli, are particularly concerning in patients with endocarditis and should be suspected in patients with prolongation of cardiac intervals in the setting of endocarditis. Visualization of both vegetations and possible abscesses is best done with transesophageal echocardiography, particularly in patients with prosthetic valves. Indeed, transesophageal echocardiography is the first test of choice in a patient with a mechanical mitral or aortic valve and suspected endocarditis (Fig. 270e-35). Vegetations should be measured because their size has prognostic importance and can be used to decide whether a patient should be taken to surgery.

1	PET metabolic imaging is emerging as a potentially useful imaging technique to identify the source of infection in patients with prosthetic valves, vascular grafts, and implantable pacemakers/defibrillators, especially in patients in whom echocardiography and/or blood cultures are negative. There is an emerging literature documenting the potential value of macrophage-targeted metabolic imaging with 18F-FDG and PET (Fig. 270e-36). Likewise, FDG PET is also useful to identify vascular inflammation and monitor the response to immunosuppressive therapy (Fig. 270e-37). While a discussion of complex congenital heart disease is beyond the scope of this chapter, a number of common congenital abnormalities are present in adults, and cardiac imaging is essential to diagnosing and managing these conditions. Abnormalities of the interatrial

1	FIGURE 270e-35 Vegetation on native mitral valve (left panel, arrow). Left atrium (LA) and left ventricle (LV) are indicated. Middle panel shows a vegetation on a mechanical prosthesis (St. Jude) indicated by an arrow; right panel shows vegetation on prosthesis after excision.

1	FIGURE 270e-36 Representative cross-sectional computed tomography (CT; left), fluorodeoxyglucose (FDG) positron emission tomography (PET; middle), and fused CT and PET (right) images before and after antibiotic treatment in a patient with fever and suspected infection of the stent placed in the descending portion of the aortic arch (arrow) for treatment of aortic coarctation. The FDG images before treatment demonstrate intense glucose uptake within the stent, consistent with inflammation/infection. The lower panel demonstrates significant attenuation of the FDG signal after treatment. (Images courtesy of Dr. Sharmila Dorbala, Brigham and Women’s Hospital.) septum probably represent the most common adult congenital cardiac abnormalities. Patent foramen ovale (PFO) can be identified in almost 25% of patients. In patients with PFO, a one-way flap in the region of the fossa ovalis is normally kept close by the left atrial pressure, which is generally higher than right atrial pressure for

1	25% of patients. In patients with PFO, a one-way flap in the region of the fossa ovalis is normally kept close by the left atrial pressure, which is generally higher than right atrial pressure for the majority of the cardiac cycle. However, right-to-left flow through a PFO can occur any time the right atrial pressure exceeds the left atrial pressure, including with maneuvers or conditions in which intrathoracic pressure is increased. The presence of a PFO can increase the likelihood of the paradoxical embolus, and thus the presence of a PFO should be determined in patients with stroke or systemic embolus of unknown etiology. Because the one-way flap of the PFO will be closed during much of the cardiac cycle, color flow Doppler will usually not reveal a PFO. Instead, agitated saline (bubble study) is the best way to assess for PFO or atrial septal defect. Saline is agitated and injected peripherally and then enters the right atrium. If no shunt is present, only the right side of the

1	study) is the best way to assess for PFO or atrial septal defect. Saline is agitated and injected peripherally and then enters the right atrium. If no shunt is present, only the right side of the heart will be pacified because the air bubbles will be too small to traverse the lungs. Because PFO is a one-way flap, maneuvers should be used to temporarily increase right atrial pressure. Either a Valsalva maneuver or sniff maneuver can be effective.

1	Atrial septal defects occur most commonly in the region of the fossa extremely high to reflect the pressure gradient between the left and ovalis, referred to as secundum-type defects (Fig. 270e-38). Additional right ventricles. Defects can occur in both the muscular and membraatrial septal defects include defects of the sinus venosus and atrium nous portions of the ventricular septum. primum. Color flow Doppler echocardiography is usually sufficient for In patients with either atrial or ventricular septal defects, estimation of diagnosis of a secundum-type atrial septal defect, but agitated saline is the severity of the left-to-right shunt is essential and can be an important generally needed for the diagnosis of other types of atrial septal defects. determinant in management decisions. Shunts are generally assessed

1	Ventricular septal defects can generally be visualized by color by echocardiography by assessing the relationship between pulmonary flow Doppler as turbulent high-velocity jets from the left to the right flow and aortic flow, the Qp/Qs ratio. Shunts and cardiac anatomy of ventricle. In cases where the jet origin is unclear, continuous wave most congenital heart diseases can also be accurately evaluated by CMR Doppler can estimate the velocities. These would be expected to be (Fig. 270e-39). noted in the subcostal view with color flow Doppler showing flow through the defect (right). CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging

1	CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging FIGURE 270e-37 Representative coronal computed tomography (CT) angiographic (CTA; left panel), fluorodeoxyglucose (FDG) positron emission tomography (PET; middle panel), and fused CT and PET (right panel) images in a patient with suspected aortitis. The CTA images demonstrate thickening of the ascending aorta (Ao), which correlates with intense, focal FDG uptake consistent with active inflammation. LV, left ventricle.

1	FIGURE 270e-39 A and B are phase contrast images that display blood flow (phase images on A) and anatomy (structural images on B) of the aorta (red) and pulmonary artery (green). C demonstrates the flow curves of the aorta (red) and the pulmonary artery (green). Note that the total flow (area under the curve) was substantially higher in the pulmonary artery than the aorta, indicative of a marked elevated pulmonary-tosystemic shunt ratio, as a result of the partial anomalous pulmonary venous return that drained into the superior vena cava.

1	VIDEO 270e-1 Cine steady-state free precession (SSFP) imaging (left) in short axis in a patient who had a large anterior myocardial infarction. Only one cut of a stack of short axis is shown. This method allows quantification of left ventricular (LV) and right ventricular (RV) volumes in diastole and systole and calculation of the LV ejection fraction, stroke volumes, and cardiac output (a product of LV stroke volume and heart rate). Note that in this case there is anterior and anteroseptal akinesia (lack of systolic wall thickening, as shown by the left cine movie, red arrows) matching by a near-transmural myocardial infarction as seen by the matching late gadolinium enhancement (LGE) image (right picture, white arrows).

1	VIDEO 270e-2 This is cine cardiac magnetic resonance (CMR) imaging of a patient in the long-axis four-chamber view. Note that the basal aspect of the right ventricular (RV) free wall is thickened, aneurysmal, and akinetic (red arrows). The global RV systolic function is mildly reduced, and the RV is dilated. CMR can image the RV using tomographic views and can quantify the RV volumes and ejection fraction volumetrically. This is a patient who presented with syncopal spells and inducible ventricular tachycardia on subsequent workup. He was diagnosed to have arrhythmogenic right ventricular dysplasia. VIDEO 270e-3 Exercise echocardiogram showing rest images on left and poststress images on right, with parasternal long-axis, upper panel, and apical four-chamber, lower panel, end-systolic frames. Following exercise, the distal septal/apical region becomes akinetic. A = upper left (UL); B = upper right (UR); C = lower left (LL); D = lower right (LR).

1	VIDEO 270e-4 The video shows cardiac magnetic resonance (CMR) myocardial perfusion imaging during vasodilating stress, in three parallel-short-axis views. An bolus of gadolinium contrast was injected intravenously while rapid imaging acquisition occurred. The contrast enhances the right ventricle first, then travels through the pulmonary circulation, enters the left ventricle (LV), and then perfuses the LV myocardium. Myocardial perfusion defects with this technique show as black subendocardial rims, reflecting lack of contrast accumulation due to ischemia and/or scar. In this case, the anterior wall has a severe perfusion defect (red arrow). Figure 270-14 shows the late gadolinium enhancement (LGE) image of a mid short-axis view. There is no evidence of infarction in the anterior wall, which would be seen as bright white areas, indicating that the stress perfusion defect primarily represents myocardial ischemia. This patient had a significant stenosis of the left anterior descending

1	would be seen as bright white areas, indicating that the stress perfusion defect primarily represents myocardial ischemia. This patient had a significant stenosis of the left anterior descending coronary artery.

1	VIDEO 270e-5 A 60-year-old female presented with intermittent chest pain of 3 days in duration but was pain free at the time of assessment in the emergency room. Admission electrocardiogram (ECG) demonstrated T-wave inversion in the anterior precordial lead, but cardiac enzymes were normal. A resting cardiac magnetic resonance (CMR) study reviewed a large area of anteroseptal hypokinesia (left picture, region of hypokinesia shown by the red arrows), matching with a large resting perfusion defect (middle picture, perfusion defect shown by the blue arrows). Late gadolinium enhancement (LGE) imaging (right picture), however, did not show any enhancement to indicate any infarction in the anteroseptal wall, suggesting that the hypocontractile and hypoperfused anteroseptal wall was viable. Urgent coronary angiography demonstrated an acute thrombus in the mid left anterior descending coronary artery, which required coronary stenting. This case represents an example of acute coronary syndrome

1	coronary angiography demonstrated an acute thrombus in the mid left anterior descending coronary artery, which required coronary stenting. This case represents an example of acute coronary syndrome with hibernating but viable myocardium in the anteroseptal wall. The anteroseptal wall recovered contractile function when reassessed 6 months later.

1	VIDEO 270e-6 A patient with severe aortic regurgitation quantified by cardiac magnetic resonance (CMR). Notice the dark flow jet during diastolic across the aortic valve. For quantitation of the aortic regurgitation severity, a cross-sectional cut was made just below the aortic valve, perpendicular to the aortic regurgitation jet, using phase contrast flow imaging. Apart from aortic regurgitation fraction and 270e-27 volume, CMR also can volumetrically quantify ventricular sizes and dimensions of the aorta, which are useful in monitoring patients with aortic valve diseases. VIDEO 270e-7 These are T2* images of the heart (left panel) and the liver (right panel) of a patient who has hemochromatosis.

1	VIDEO 270e-7 These are T2* images of the heart (left panel) and the liver (right panel) of a patient who has hemochromatosis. Note that iron and the liver are markedly darkened in these movies, indicating high load of iron in the heart muscle and liver. The rate of signal reduction (decay) in the myocardium and liver can be calculated as T2∗ value expressed in milliseconds. In this case, the T2∗ was at 10 ms. T2∗ <20 ms in patients with cardiomyopathy has been shown to indicate iron toxicity as the etiology of the cardiomyopathy, and it carries prognostic valve for such patients at risk of cardiac iron toxicity. VIDEO 270e-8 This video shows the heart in long and short axis. Note the large atria, thickened pericardium, and extensive pericardial adhesions. Given the extensive pericardial adhesions, there is little shearing motion of the ventricles against the parietal pericardium.

1	CHAPTER 270e Noninvasive Cardiac Imaging: Echocardiography, Nuclear Cardiology, and Magnetic Resonance/Computed Tomography Imaging Atlas of Noninvasive Imaging Marcelo F. Di Carli, Raymond Y. Kwong, Scott D. Solomon This chapter provides “movie” image clips as they are viewed in clini-cal practice, as well as additional static images. Noninvasive cardiac imaging is essential to the diagnosis and management of patients with 271e known or suspected cardiovascular disease. This atlas supplements Chap. 270e, which describes the principles and clinical applications of these important techniques. 1.4 2.8 1.4 2.8

1	1.4 2.8 1.4 2.8 Figure 271e-1 A 48-year-old man with new-onset substernal chest pain. Echocardiography shows evidence of acute anterior myocardial infarction involving the interventricular septum and apex secondary to an occlusion of the left anterior descending coronary artery seen from the parasternal long axis view (left) and the apical four-chamber view (right). LV, left ventricle; RV, right ventricle. (See Videos 271e-1 and 271e-2.) CHAPTER 271e Atlas of Noninvasive Imaging

1	CHAPTER 271e Atlas of Noninvasive Imaging Figure 271e-2 A 55-year-old man with exertional chest discomfort and dyspnea. He exercised for 12 min on a standard Bruce protocol, experiencing typical chest pain and ST-segment depression in V2–V5. End-systolic frame of a stress echocardiogram shows apical four-chamber view at rest (left) and after exercise (right). After exercise, there is a clear regional wall motion abnormality in the distal septum through the apex, consistent with a stenosis in the left anterior descending artery distribution (arrows). LV, left ventricle. (See Videos 271e-3 and 271e-4.)

1	Figure 271e-3 Exercise single-photon emission computed tomography (SPECT) myocardial perfusion technetium-99m (99mTc) sestamibi scan in a 54-year-old male with a history of coronary artery disease and a prior coronary stent. The stress images (left and middle) show a large defect involving the apex, all apical segments, mid-inferior, mid-inferoseptum, and mid-anteroseptum (arrowheads), which is completely reversible at rest (right), reflecting a large area of exercise-induced myocardial ischemia throughout the left anterior descending coronary territory. The bull's eye displays on the right panel depict the semiquantitative extent of ischemia (light yellow and blue areas represent the extent PART 10 Disorders of the Cardiovascular System and severity of ischemia).

1	PART 10 Disorders of the Cardiovascular System and severity of ischemia). Figure 271e-4 Coronary computed tomography angiography (CTA). Curved multiplanar reformations demonstrating coronary artery disease severity, defined as normal (no plaque or stenosis), mild (<40%), moderate (40–69%), and severe (>70%) luminal narrowing. By guidelines for CTA reporting, alternative classification provides for stenosis grading as normal, minimal (1–24%), mild (25–49%), moderate (50–69%), severe (70–99%), and occluded (100%). (From GL Raff et al: SCCT guidelines for the interpretation and reporting of coronary computed tomographic angiography. J Cardiovasc Comput Tomogr 3:122, 2009; with permission.) -CAC score 271e = 96th percentile for age, race and ethnicity1 10-year hard CHD risk is 6% (observed age) vs 30% (arterial age)2#

1	Figure 271e-5 Coronary artery calcium (CAC) scan on a 51-yearold white male without clinical cardiovascular disease or treated diabetes, referred for CAC for risk stratification to guide preventive therapies. A. Gated, noncontrast cardiac computed tomography (CT; 3-mm slice thickness), axial view, demonstrating calcified left anterior descending (LAD) artery atherosclerosis. B. Whole-heart three-dimensional image reconstruction, inverted maximum-intensity projection, demonstrating overall burden of CAC with predominant LAD distribution (arrow). Top right. CAC scores for each coronary artery with calcified plaque involvement, scored by Agatston method and total volume. #For white male with observed age 51 years, total cholesterol 220 mg/dL, high-density lipoprotein 45 mg/dL, nonsmoker, no hypertension, and systolic blood pressure 120 mmHg. Calculated arterial age is 81 years. CHD, coronary heart disease; CX, left circumflex artery; LM, left main artery. 1Data from RL McClelland et al:

1	hypertension, and systolic blood pressure 120 mmHg. Calculated arterial age is 81 years. CHD, coronary heart disease; CX, left circumflex artery; LM, left main artery. 1Data from RL McClelland et al: Circulation 113:30-37, 2006. 2Data from RL McClelland et al: Am J Cardiol 103: 59-63, 2009.

1	Figure 271e-6 Cardiac magnetic resonance (CMR) stress myocardial perfusion images in a 60-year-old patient with atypical chest pain. Cine movie short-axis image (left upper panel) shows normal left ventricle size and global and regional function at rest. During vasodilator stress, there is marked reduction of lateral wall perfusion (white arrow, right upper panel) as well as a mild defect in the septal wall. This region is confirmed to be viable by matching late gadolinium enhancement imaging (left lower panel), which demonstrated no evidence of infarction in the lateral wall. These findings are consistent with a severe coronary stenosis in the left circumflex artery. On angiography performed subsequently, there is a tight lesion in the left circumflex artery (red arrow, right lower panel). (See Videos 271e-5 and 271e-6.) LAD 79% 75% 1/8 1.05 1.40 LCX 65% 59% 2.22 0.89 2.49 RCA 76% 68% 1.99 0.96 2.08 TOT 74% 69% 1.75 0.98 1.79

1	Figure 271e-7 Adenosine positron emission tomography (PET) myocardial perfusion 13N-ammonia scan in a 60-year-old female with atypical chest pain. The stress images (left) show a large defect involving the apex, all apical segments, mid-inferior, mid-inferoseptum, and midanteroseptum (arrowheads), which is completely reversible at rest (right). This is consistent with a medium-sized area of stress-induced ischemia in the mid portion of the left anterior descending (LAD) coronary artery. The right panel illustrates the time-activity curves used for quantification of myocardial blood flow (in mL/min per g of tissue) at peak stress (upper panel) and at rest (lower panel). Coronary flow reserve is then calculated as the ratio of stress/rest myocardial blood flow. The coronary flow reserve is abnormal in the LAD territory, and normal in the left circumflex (LCX) and right coronary artery (RCA) territories (i.e., >2.0). TOT, total left ventricle.

1	Figure 271e-8 Coronary computed tomography angiography (CTA) obtained on a 35-year-old female presenting to an outpatient clinic with a history of unexplained syncope and a 6-month complaint of intermittent, atypical chest pain occurring primarily during rest. Physical examination is normal. An exercise treadmill test is performed demonstrating good exercise capacity with no exertional chest pain or ischemic ECG changes. For persistent, unexplained symptoms, coronary CTA is obtained. A. Three-dimensional cardiac CT image reconstruction demonstrating anomalous right coronary artery (RCA) origin from the left coronary cusp with an acute angle takeoff (arrow) and an intraarterial course between the aorta (Ao) and main pulmonary artery (PA). B, C. Contrast-enhanced CTA in two-dimensional axial (B) and coronal oblique views (C) demonstrating proximal RCA intraarterial course between the Ao and main PA. PART 10 Disorders of the Cardiovascular System

1	PART 10 Disorders of the Cardiovascular System Figure 271e-9 Coronary computed tomography angiography (CTA) obtained from a 13-year-old boy with a history of Kawasaki disease who presented with limited exercise capacity and occasional, atypical chest pain. A, B. Three-dimensional cardiac CT image reconstruction demonstrating large three-vessel coronary artery diffuse aneurysms with proximal, nondominant left circumflex (LCX) artery occlusion. C. Two-dimensional contrast-enhanced coronary CTA demonstrating mid-RCA and mid-LAD thrombi that are nonocclusive layered and near circumferential thrombi, respectively, and proximal LCX occlusion. Ao, aorta; CT, computed tomography; LAD, left anterior descending; RCA, right coronary artery.

1	Figure 271e-10 A case of viability assessment in a patient with inferior myocardial infarction. The cine movie in the upper panel shows an area of inferior akinesis (green arrows). Magnetic resonance image demonstrates transmural contrast enhancement of the inferior wall (red arrows) and the right ventricle (white arrows), which is consistent with infarction. Imaging the heart 10–15 min after injection of gadolinium allows for the accumulation of gadolinium in infarcted tissue (red arrows), which identifies nonviable infarcted myocardium as bright. Viability assessment, as in this case, can provide guidance for any benefits of invasive coronary intervention. In this case, the inferior wall is nonviable. Apart from the inferior wall infarction (red arrows), there is extensive right ventricular infarction (white arrows). (See Video 271e-7.)

1	Figure 271e-11 Rest myocardial perfusion and metabolism positron emission tomography (PET) scan obtained with 13N-ammonia (perfusion) and 18F-fluorodeoxyglucose (FDG; glucose metabolism) in a 48-year-old male with a prior myocardial infarction. The rest perfusion Atlas of Noninvasive Imaging images show a large defect involving the apex, apical segments, and mid-anteroseptal and anterior segments (arrowheads), which has associ-ated increase in glucose uptake (perfusion-metabolic mismatch), reflecting viable but hibernating myocardium throughout the left anterior descending coronary territory.

1	Figure 271e-12 A 70-year-old patient with known cardiac murmur and progressive shortness of breath and a recent episode of syncope. Echocardiography shows severe calcific aortic stenosis. A heavily calcified aortic valve (arrow) is shown in the parasternal long-axis views (top panels) and short-axis view (bottom left). Doppler interrogation shows a peak transaortic velocity of 5.2 m/s consistent with a peak instantaneous gradient of 109 mmHg and a mean gradient of 66 mmHg, and a corresponding aortic valve area of <0.6 cm2 (lower right). Ao, aorta; LA, left atrium; LV, left ventricle; RV, right ventricle. (See Videos 271e-8, 271e-9, and 271e-10.)

1	Figure 271e-13 A 66-year-old patient with multiple myeloma and progressive shortness of breath. Echocardiography shows features typical of cardiac amyloidosis, including thickened myocardium with a “sparkly” appearance and left atrial enlargement. Systolic function is mildly reduced, and diastolic function is severely reduced. LA, left atrium; LV, left ventricle; RV, right ventricle. (See Videos 271e-11 and 271e-12.)

1	Figure 271e-14 Magnetic resonance image with contrast enhancement in magnitude (A), and phase-sensitive reconstructed images (B) 5–10 min after injection of gadolinium in a patient with transthyretin (TTR)-mediated amyloidosis. The phase-sensitive reconstruction (B) enhances the region of abnormal collection of gadolinium, making gadolinium enhancement in the ventricle (red arrows) and the atrium (green arrows) more prominent. Amyloidosis causes accumulation of abnormal interstitial proteins, which results in late gadolinium enhancement in a diffuse subendocardial pattern (red arrows). Blood pool signal is characteristically dark (asterisk) owing to sequestration of gadolinium into other organs.

1	Figure 271e-15 A 34-year-old woman with known cardiac murmur and syncope with a family history of sudden cardiac death. Echocardiogram shows classic findings of hypertrophic cardiomyopathy, including marked left ventricular wall thickness, particularly in the interventricular septum, notable in the parasternal long-axis view (upper left) and apical view (upper right). Note reverse septal curvature in the apical view (upper left). There is substantial flow acceleration through the left ventricular outflow tract (lower left) with evidence of a late peaking systolic gradient (arrow, lower right) caused by outflow tract obstruction. Ao, aorta; IVS, interventricular septum; LA, left atrium; LV, left ventricle; PW, posterior wall; RV, right ventricle. (See Videos 271e-13, 271e-14, and 271e-15.)

1	Figure 271e-16 Magnetic resonance image with contrast enhancement in a patient with hypertrophic cardiomyopathy. Note the marked thickened anteroseptal wall (black arrows, left panel) consistent with asymmetric septal hypertrophy. After contrast is injected, this region demonstrated heterogeneous foci of contrast enhancement (right panel, red arrows) consistent with myocardial fibrosis due to myofibril disarray in this condition. This typical enhancement pattern of hypertrophic cardiomyopathy is found in the areas of maximum wall thickness, Atlas of Noninvasive Imaging typically at the anteroseptum as in this case. (See Video 271e-16.) Nonischemic cardiomyopathy sarcoid (with both MRI and PET) Apex Mid Base Perfusion FDG Short-axis MRI Late enhancement Perfusion FDG Horizontal Long-axis Vertical Long-axis LVRV LVRV

1	Figure 271e-17 Late gadolinium enhancement cardiac magnetic resonance imaging (MRI) (left panel) and rest myocardial perfusion and fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) studies (middle and right panels) in a 48-year-old male with a complete heart block. The MRI images demonstrate a linear area of mid-wall late gadolinium enhancement involving the inferior and inferolateral walls (arrows). The myocardial perfusion images were normal. However, the FDG images demonstrated a focal area of intense glucose uptake in the inferolateral wall, corresponding to the area of late enhancement on MRI. This is consistent with focal active sarcoidosis. LV, left ventricle; RV, right ventricle.

1	Figure 271e-18 A 46-year-old patient with malignant melanoma who presents with acute shortness of breath. Echocardiogram reveals a large pericardial effusion (arrow, upper left) with evidence of cardiac tamponade. M-mode echocardiography (upper right) shows evidence of collapse of the right ventricular free wall during diastole (arrow). Doppler echocardiography (lower panel) shows evidence of respiratory flow variation, consistent with a pulsus paradoxus. LA, left atrium; LV, left ventricle; RV, right ventricle. (See Video 271e-17.) PART 10 Disorders of the Cardiovascular System

1	PART 10 Disorders of the Cardiovascular System Figure 271e-19 Diffuse pericardial thickening (left; red arrows) and circumferential effusion (right; white arrows) associated with effusive-constrictive pericarditis. Effusive-constrictive pericarditis is a progressive condition that has varying degrees of hemodynamic consequences due initially to the collection of pericardial fluid and ultimately to pericardial constriction. It is typically suspected in cases where pericardiocentesis fails to normalize intracardiac pressures. In this example, pericardial fluid analysis resulted in a sterile exudate of leukocytes and erythrocytes. LV, left ventricle; RV, right ventricle.

1	Figure 271e-20 A 48-year-old woman with severe idiopathic pulmonary hypertension. Echocardiography reveals evidence of marked right ventricular volume and pressure overload as evidenced by enlarged right ventricle (upper left and right), small left ventricle (upper left and upper right), and flattening of the interventricular septum (D-shaped septum) in systole and diastole (upper right). Tricuspid regurgitation velocity, which reflects the pressure gradient between the right ventricle and the left ventricle, is markedly elevated at 5 m/s, consistent with a right ventricle to right atrial pressure gradient of 100 mmHg, which is consistent with systemic right-sided pressures. LA, left atrium; LV, left ventricle; RV, right ventricle. (See Videos 271e-18 and 271e-19.) CHAPTER 271e Atlas of Noninvasive Imaging

1	CHAPTER 271e Atlas of Noninvasive Imaging Figure 271e-21 Metastatic cardiac tumor diagnosed by cardiac magnetic resonance (MR) in a patient who presented with chest pain and inferior ST elevation. Left heart catheterization was normal. Cardiac MR demonstrates extensive myocardial edema (A, white arrows) with marked reduction in first perfusion (B) and accumulation of gadolinium within the cardiac mass 10–15 min after injection of gadolinium (C, red arrows). D. Positron emission tomography scan showed increased fluorodeoxyglucose uptake in a lung mass as well as in the cardiac mass, consistent with cardiac metastasis. Biopsy of the lung mass revealed adenosquamous carcinoma of the lung. LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. (See Videos 271e-20 and 271e-21.) PART 10 Disorders of the Cardiovascular System

1	PART 10 Disorders of the Cardiovascular System Figure 271e-22 Fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) in a 52-year-old male with a prior aortic valve replacement who presented with fever and was found to have Haemophilus parainfluenzae bacteremia. The multiplanar reformatted fused PET/CT images demonstrate intense FDG uptake surrounding the aortic valve prosthesis (arrowheads), compatible with a paravalvular abscess. The patient was found to have purulent fluid around the valve during surgery, and he underwent an aortic valve replacement. Ao, aorta; AV, aortic valve; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle.

1	Figure 271e-23 Cardiac computed tomography (CT) pulmonary vein mapping in a 62-year-old male with symptomatic, paroxysmal atrial fibrillation referred for cardiac CT for pulmonary vein mapping prior to planned pulmonary vein isolation. Three-dimensional image reconstruction, demonstrating (A) normal pulmonary vein anatomy and (B) common variant with presence of separate right middle pulmonary vein (RMPV) ostium. LLPV, left lower pulmonary vein; LUPV, left upper pulmonary vein; RLPV, right lower pulmonary vein; RUPV, right upper pulmonary vein.

1	Disorders of the Cardiovascular SystemDiagnostic Cardiac Catheterization and Coronary Angiography Jane A. Leopold, David P. Faxon Diagnostic cardiac catheterization and coronary angiography are 272 considered the gold standard in the assessment of the anatomy and physiology of the heart and its associated vasculature. In 1929, Forssmann demonstrated the feasibility of cardiac catheterization in humans when he passed a urological catheter from a vein in his arm to his right atrium and documented the catheter’s position in the heart by x-ray. In the 1940s, Cournand and Richards applied this technique to patients with cardiovascular disease to evaluate cardiac function. These three physicians were awarded the Nobel Prize in 1956. In 1958, Sones inadvertently performed the first selective coronary angiography when a catheter in the left ventricle slipped back across the aortic valve, engaged the right coronary artery, and power-injected 40 mL of contrast down the vessel. The resulting

1	coronary angiography when a catheter in the left ventricle slipped back across the aortic valve, engaged the right coronary artery, and power-injected 40 mL of contrast down the vessel. The resulting angiogram provided superb anatomic detail of the artery, and the patient suffered no adverse effects. Sones went on to develop selective coronary catheters, which were modified further by Judkins, who developed preformed catheters and allowed coronary artery angiography to gain widespread use as a diagnostic tool. In the United States, cardiac catheterization is the second most common operative procedure, with more than one million procedures performed annually.

1	INDICATIONS, RISKS, AND PREPROCEDURE MANAGEMENT Cardiac catheterization and coronary angiography are indicated to evaluate the extent and severity of cardiac disease in symptomatic patients and to determine if medical, surgical, or catheter-based interventions are warranted (Table 272-1). They are also used to exclude severe disease in symptomatic patients with equivocal findings on noninvasive studies and in patients with chest-pain syndromes of unclear etiology for whom a definitive diagnosis is necessary for management. Cardiac catheterization is not mandatory prior to cardiac surgery in some younger patients who have congenital or valvular heart disease that is well defined by noninvasive imaging and who do not have symptoms or risk factors that suggest concomitant coronary artery disease.

1	The risks associated with elective cardiac catheterization are relatively low, with a reported risk of 0.05% for myocardial infarction, 0.07% for stroke, and 0.08–0.14% for death. These risks increase substantially if the catheterization is performed emergently, during acute myocardial infarction or in hemodynamically unstable patients. Canadian Cardiology Society Class II, III, or IV stable angina on medical therapy Chest-pain syndrome of unclear etiology and equivocal findings on noninvasive tests Reperfusion with primary percutaneous coronary intervention Persistent or recurrent ischemia Pulmonary edema and/or reduced ejection fraction Cardiogenic shock or hemodynamic instability Risk stratification or positive stress test after acute myocardial infarction Mechanical complications—mitral regurgitation, ventricular septal defect Suspected severe valve disease in symptomatic patients—dyspnea, angina, heart failure, syncope Infective endocarditis with need for cardiac surgery

1	Suspected severe valve disease in symptomatic patients—dyspnea, angina, heart failure, syncope Infective endocarditis with need for cardiac surgery Asymptomatic patients with aortic regurgitation and cardiac enlargement or Prior to cardiac surgery in patients with suspected coronary artery disease New onset with angina or suspected undiagnosed coronary artery disease New-onset cardiomyopathy of uncertain cause or suspected to be due to coronary artery disease Prior to surgical correction, when symptoms or noninvasive testing suggests coronary disease Symptomatic patients with suspected cardiac tamponade or constrictive pericarditis Hypertrophic cardiomyopathy with angina Diseases of the aorta when knowledge of coronary artery involvement is necessary for management

1	Hypertrophic cardiomyopathy with angina Diseases of the aorta when knowledge of coronary artery involvement is necessary for management Additional risks of the procedure include tachyor bradyarrhythmias that require countershock or pharmacologic therapy, acute renal failure leading to transient or permanent dialysis, vascular complications that necessitate surgical repair, and significant access-site bleeding. Of these risks, vascular access-site bleeding is the most common complication, occurring in 1.5–2.0% of patients, with major bleeding events associated with a worse shortand long-term outcome.

1	In patients who understand and accept the risks associated with cardiac catheterization, there are no absolute contraindications when the procedure is performed in anticipation of a life-saving intervention. Relative contraindications do, however, exist; these include decompensated congestive heart failure; acute renal failure; severe chronic renal insufficiency, unless dialysis is planned; bacteremia; acute stroke; active gastrointestinal bleeding; severe, uncorrected electrolyte abnormalities; a history of an anaphylactic/anaphylactoid reaction to iodinated contrast agents; and a history of allergy/bronchospasm to aspirin in patients for whom progression to a percutaneous coronary intervention is likely and aspirin desensitization has not been performed.

1	Contrast allergy and contrast-induced acute kidney injury merit further consideration, because these adverse events may occur in otherwise healthy individuals and prophylactic measures exist to reduce risk. Allergic reactions to contrast agents occur in <5% of cases, with severe anaphylactoid (clinically indistinguishable from anaphylaxis, but not mediated by an IgE mechanism) reactions occurring in 0.1%–0.2% of patients. Mild reactions manifest as nausea, vomiting, and urticaria, while severe anaphylactoid reactions lead to hypotensive shock, pulmonary edema, and cardiorespiratory arrest. Patients with a history of significant contrast allergy should be premedicated with corticosteroids and antihistamines (H1and H2-blockers) and studies performed with nonionic, low-osmolar contrast agents that have a lower reported rate of allergic reactions.

1	Contrast-induced acute kidney injury, defined as an increase in creatinine >0.5 mg/dL or 25% above baseline that occurs 48–72 hours after contrast administration, occurs in ~2–7% of patients with rates of 20–30% reported in high-risk patients, including those with diabetes mellitus, congestive heart failure, chronic kidney disease, anemia, and older age. Dialysis is required in 0.3–0.7% of patients and is associated with a fivefold increase in in-hospital mortality. For all patients, adequate intravascular volume expansion with intravenous 0.9% saline (1.0–1.5 mL/kg per hour) for 3–12 hours before and continued 6–24 hours after the procedure limits the risk of contrast-induced acute kidney injury. Pretreatment with N-acetylcysteine (Mucomyst) has not reduced the risk of contrast-induced acute kidney injury consistently and, therefore, is no longer recommended routinely. Diabetic patients treated with metformin should stop the drug 48 hours prior to the procedure to limit the

1	acute kidney injury consistently and, therefore, is no longer recommended routinely. Diabetic patients treated with metformin should stop the drug 48 hours prior to the procedure to limit the associated risk of lactic acidosis. Other strategies to decrease risk include the administration of sodium bicarbonate (3 mL/kg per hour) 1 hour before and 6 hours after the procedure; use of lowor iso-osmolar contrast agents; and limiting the volume of contrast to <100 mL per procedure.

1	Cardiac catheterization is performed after the patient has fasted for 6 hours and has received intravenous conscious sedation to remain awake but sedated during the procedure. All patients with suspected coronary artery disease are pretreated with 325 mg aspirin. In patients in whom the procedure is likely to progress to a percutaneous coronary intervention, an additional antiplatelet agent should be started: clopidogrel (600-mg loading dose and 75 mg daily) or prasugrel (60-mg loading dose and 10 mg daily), or ticagrelor (180-mg loading and 90 mg twice daily). Prasugrel should not be selected for individuals with prior stroke or transient ischemic attack. Warfarin is held starting 2–3 days prior to the catheterization to allow the international normalized ratio (INR) to fall to <1.7 and limit access-site bleeding complications. Cardiac catheterization is a sterile procedure, so antibiotic prophylaxis is not required.

1	Cardiac catheterization and coronary angiography provide a detailed hemodynamic and anatomic assessment of the heart and coronary arteries. The selection of procedures is dependent on the patient’s symptoms and clinical condition, with some direction provided by noninvasive studies.

1	Vascular Access Cardiac catheterization procedures are performed using a percutaneous technique to enter the femoral artery and vein as the preferred access sites for left and right heart catheterization, respectively. A flexible sheath is inserted into the vessel over a guidewire, allowing diagnostic catheters to be introduced into the vessel and advanced toward the heart using fluoroscopic guidance. The radial artery (or brachial artery) may also be used as an arterial access site in patients, particularly those with peripheral arterial disease that involves the abdominal aorta, iliac, or femoral vessels; severe iliac artery tortuosity; morbid obesity; or preference for early postprocedure ambulation. Use of radial-artery access is gaining popularity due to a lower rate of access-site bleeding complications. A normal Allen’s test confirming dual blood supply to the hand from the radial and ulnar arteries is recommended prior to access at this site. The internal jugular or

1	bleeding complications. A normal Allen’s test confirming dual blood supply to the hand from the radial and ulnar arteries is recommended prior to access at this site. The internal jugular or antecubital veins serve as alternate access sites to the right heart when the patient has an inferior vena cava filter in place or 1461 requires prolonged hemodynamic monitoring.

1	Right Heart Catheterization This procedure measures pressures in the right heart. Right heart catheterization is no longer a routine part of diagnostic cardiac catheterization, but it is reasonable in patients with unexplained dyspnea, valvular heart disease, pericardial disease, right and/or left ventricular dysfunction, congenital heart disease, and suspected intracardiac shunts. Right heart catheterization uses a balloon-tipped flotation catheter that is advanced sequentially to the right atrium, right ventricle, pulmonary artery, and pulmonary wedge position (as a surrogate for left atrial pressure) using fluoroscopic guidance; in each cardiac chamber, pressure is measured and blood samples are obtained for oxygen saturation analysis to screen for intracardiac shunts.

1	Left Heart Catheterization This procedure measures pressures in the left heart as a determinant of left ventricular performance. With the aid of fluoroscopy, a catheter is guided to the ascending aorta and across the aortic valve into the left ventricle to provide a direct measure of left ventricular pressure. In patients with a tilting-disc prosthetic aortic valve, crossing the valve with a catheter is contraindicated, and the left heart may be accessed via a transseptal technique from the right atrium using a needle-tipped catheter to puncture the atrial septum at the fossa ovalis. Once the catheter crosses from the right to the left atrium, it can be advanced across the mitral valve to the left ventricle. This technique is also used for mitral valvuloplasty. Heparin is given for prolonged procedures to limit the risk of stroke from embolism of clots that may form on the catheter. For patients with heparin-induced thrombocytopenia, the direct thrombin inhibitors bivalirudin (0.75

1	procedures to limit the risk of stroke from embolism of clots that may form on the catheter. For patients with heparin-induced thrombocytopenia, the direct thrombin inhibitors bivalirudin (0.75 mg/kg bolus, 1.75 mg/kg per hour for the duration of the procedure) or argatroban (350 μg/kg bolus, 15 μg/kg per minute for the duration of the procedure) may be used.

1	A comprehensive hemodynamic assessment involves obtaining pressure measurements in the right and left heart and peripheral arterial system and determining the cardiac output (Table 272-2). The shape Arteriovenous oxygen difference (vol %) 3.5–4.8 Cardiac index ([L-min]/m2) 2.8–4.2

1	Hemodynamic measurements are also used to differentiate between cardiac tamponade, constrictive pericarditis, and restrictive cardiomyopathy (Table 272-3). In cardiac tamponade, right atrial pressure is increased with a decreased or absent “y” descent, indicative of impaired right atrial emptying in diastole, and there is diastolic equalization of pressures in all cardiac chambers. In constrictive pericarditis, right atrial pressure is elevated with a prominent “y” descent, indicating rapid filling of the right ventricle during early diastole. A diastolic dip and plateau or “square root sign,” in the ventricular waveforms due to an abrupt halt in ventricular filling during diastole; right ventricular and pulmonary artery pressures are elevated; and discordant pressure changes in the right and left ventricles with inspiration (right ventricular systolic pressure increases while left ventricular systolic pressure decreases) are observed. The latter hemodynamic phenomenon is the most

1	and left ventricles with inspiration (right ventricular systolic pressure increases while left ventricular systolic pressure decreases) are observed. The latter hemodynamic phenomenon is the most specific for constriction. Restrictive cardiomyopathy may be distinguished from constrictive pericarditis by a marked increase in right ventricular and pulmonary artery systolic pressures (usually >60 mmHg), a separation of the left and right ventricular diastolic pressures by and magnitude of the pressure waveforms provide important diag->5 mmHg (at baseline or with acute volume loading), and concordant nostic information; an example of normal pressure tracings is shown changes in left and right ventricular diastolic filling pressures with in Fig. 272-1. In the absence of valvular heart disease, the atria and inspiration (both increase). ventricles are “one chamber” during diastole when the tricuspid and mitral valves are open while in systole, when the pulmonary and aortic Cardiac Output

1	the atria and inspiration (both increase). ventricles are “one chamber” during diastole when the tricuspid and mitral valves are open while in systole, when the pulmonary and aortic Cardiac Output Cardiac output is measured by the Fick method or the valves are open, the ventricles and their respective outflow tracts are thermodilution technique. Typically, the Fick method and thermodiconsidered “one chamber.” These concepts form the basis by which lution technique are both performed during cardiac catheterization, hemodynamic measurements are used to assess valvular stenosis. although the Fick method is considered more reliable in the presence When aortic stenosis is present, there is a systolic pressure gradient of tricuspid regurgitation and in low-output states. The Fick method

1	FIGURE 272-1 Normal hemodynamic waveforms recorded during right heart catheteriza-tion. Atrial pressure tracings have a characteristic “a” wave that reflects atrial contraction and a “v” wave that reflects pressure changes in the atrium during ventricular systole. Ventricular pres-sure tracings have a low-pressure diastolic filling period and a sharp rise in pressure that occurs during ventricular systole. d, diastole; PA, pulmonary artery; PCWP, pulmonary capillary wedge pressure; RA, right atrium; RV, right ventricle; s, systole.

1	between the left ventricle and the aorta; when mitral stenosis is present, there is a diastolic pressure gradient between the pulmonary capillary wedge (left atrial) pressure and the left ventricle (Fig. 272-2). Hemodynamic measurements also discriminate between aortic stenosis and hypertrophic obstructive cardiomyopathy where the asymmetrically hypertrophied septum creates a dynamic intraventricular pressure gradient during ven-200 tricular systole. The magnitude of this obstruction is measured using an end-hole catheter positioned at the left ventricular apex that is pulled back while recording pressure; once the catheter has passed the septal obstruction and is positioned in the apex of the left ventricle, a gradient can be measured between the left ventricular apex and the aorta. Hypertrophic obstructive cardiomy-100 opathy is confirmed by the Brockenbrough-Braunwald sign: following a premature ventricular contraction, there is an increase in the left ventricular–aorta pressure

1	obstructive cardiomy-100 opathy is confirmed by the Brockenbrough-Braunwald sign: following a premature ventricular contraction, there is an increase in the left ventricular–aorta pressure gradient with a simultaneous decrease in the aortic pulse pressure. These findings are absent in aortic stenosis.

1	Regurgitant valvular lesions increase volume (and pressure) in the “receiving” cardiac chamber. In severe 0 mmHg mmHg mitral and tricuspid regurgitation, the increase in blood flow to the atria takes place during ventricular systole, FIGURE 272-2 Severe aortic and mitral stenosis. Simultaneous recording of left leading to an increase in the v wave (two times greater ventricular (LV) and aortic (Ao) pressure tracings demonstrates a 62-mmHg mean than the mean pressure). Severe aortic regurgitation systolic gradient (shaded area) that corresponds to an aortic valve area of 0.6 cm2 leads to a decrease in aortic diastolic pressure with a (left). Simultaneous recording of LV and pulmonary capillary wedge (PCW) pressure concomitant rise in left ventricular end-diastolic pres-tracings reveals a 14-mmHg mean diastolic gradient (shaded area) that is consistent sure, resulting in equalization of pressures between the with critical mitral stenosis (mitral valve area = 0.5 cm2). d, diastole; e,

1	14-mmHg mean diastolic gradient (shaded area) that is consistent sure, resulting in equalization of pressures between the with critical mitral stenosis (mitral valve area = 0.5 cm2). d, diastole; e, end diastole; s, two chambers at end-diastole. systole.

1	1463TABlE 272-3 HEMoDYnAMiC FinDingS in TAMPonADE, ConSTRiCTivE PERiCARDiTiS, AnD RESTRiCTivE CARDioMYoPATHY Right atrium pressure ↑↑↑ (Fails to decrease by 50% or to <10 mmHg after pericardiocentesis) uses oxygen as the indicator substance and is based on the principle that the amount of a substance taken up or released by an organ (oxygen consumption) is equal to the product of its blood flow (cardiac output) and the difference in the concentration of the substance in the arterial and venous circulation (arterial-venous oxygen difference). Thus, the formula for calculating the Fick cardiac output is:

1	Oxygen consumption is estimated as 125 mL oxygen/minute × body surface area, and the arterial-venous oxygen difference is determined by first calculating the oxygen carrying capacity of blood (hemoglobin [g/100 mL] × 1.36 [mL oxygen/g hemoglobin] × 10) and multiplying this product by the fractional oxygen saturation. The thermodilution method measures a substance that is injected into and adequately mixes with blood. In contemporary practice, thermodilution cardiac outputs are measured using temperature as the indicator. Measurements are made with a thermistor-tipped catheter that detects temperature deviations in the pulmonary artery after the injection of 10 mL of room-temperature normal saline into the right atrium.

1	Vascular Resistance Resistance across the systemic and pulmonary circulations is calculated by extrapolating from Ohm’s law of electrical resistance and is equal to the mean pressure gradient divided by the mean flow (cardiac output). Therefore, systemic vascular resistance is ([mean aortic pressure − mean right atrial pressure]/cardiac output) multiplied by 80 to convert the resistance from Wood units to dyns-cm−5. Similarly, the pulmonary vascular resistance is ([mean pulmonary artery − mean pulmonary capillary wedge pressure]/cardiac output) × 80. Pulmonary vascular resistance is lowered by oxygen, nitroprusside, calcium channel blockers, prostacyclin infusions, and inhaled nitric oxide; these therapies may be administered during catheterization to determine if increased pulmonary vascular resistance is fixed or reversible.

1	Valve Area Hemodynamic data may also be used to calculate the valve area using the Gorlin formula that equates the area to the flow across the valve divided by the pressure gradient between the cardiac chambers surrounding the valve. The formula for the assessment of valve area is: Area = (cardiac output [cm3/min]/[systolic ejection period or diastolic filling period][heart rate])/44.3 C × square root of the pressure gradient, where C = 1 for aortic valve and 0.85 for the mitral valve. A valve area of <1.0 cm2 and a mean gradient of greater than 40 mmHg indicate severe aortic stenosis, while a valve area of <1.5 cm2 and a mean gradient >5–10 mmHg is consistent with moderate-to-severe mitral stenosis; in symptomatic patients with a mitral valve area >1.5 cm2, a mean gradient >15 mmHg, pulmonary artery pressure >60 mmHg, or a pulmonary artery wedge pressure >25 mmHg after exercise is also considered significant and may warrant intervention. The modified Hakki formula has also been used

1	artery pressure >60 mmHg, or a pulmonary artery wedge pressure >25 mmHg after exercise is also considered significant and may warrant intervention. The modified Hakki formula has also been used to estimate aortic valve area. This formula calculates the valve area as the cardiac output (L/min) divided by the square root of the pressure gradient. Aortic valve area calculations based on the Gorlin formula are flow-dependent and, therefore, for patients with low cardiac outputs, it is imperative to determine if a decreased valve area actually reflects a fixed stenosis or is overestimated by a low cardiac output and stroke volume that is insufficient to open the valve leaflets fully. In these instances, cautious hemodynamic manipulation using dobutamine to increase the cardiac output and recalculation of the aortic valve area may be necessary.

1	Intracardiac Shunts In patients with congenital heart disease, detection, localization, and quantification of the intracardiac shunt should be evaluated. A shunt should be suspected when there is unexplained arterial desaturation or increased oxygen saturation of venous blood. A “step up” or increase in oxygen content indicates the presence of a left-to-right shunt while a “step down” indicates a right-to-left shunt. The shunt is localized by detecting a difference in oxygen saturation levels of 5–7% between adjacent cardiac chambers. The severity of the shunt is determined by the ratio of pulmonary blood flow (Qp) to the systemic blood flow (Qs), or Qp/Qs = ([systemic arterial oxygen content − mixed venous oxygen content]/pulmonary vein oxygen content − pulmonary artery oxygen content). For an atrial septal defect, a shunt ratio of 1.5 is considered significant and factored with other clinical variables to determine the need for intervention. When a congenital ventricular septal

1	For an atrial septal defect, a shunt ratio of 1.5 is considered significant and factored with other clinical variables to determine the need for intervention. When a congenital ventricular septal defect is present, a shunt ratio of ≥2.0 with evidence of left ventricular volume overload is a strong indication for surgical correction.

1	Ventriculography to assess left ventricular function may be performed during cardiac catheterization. A pigtail catheter is advanced retrograde across the aortic valve into the left ventricle and 30–45 mL of contrast is power-injected to visualize the left ventricular chamber during the cardiac cycle. The ventriculogram is usually performed in the right anterior oblique projection to examine wall motion and mitral valve function. Normal wall motion is observed as symmetric contraction of all segments; hypokinetic segments have decreased contraction, akinetic segments do not contract, and dyskinetic segments appear to bulge paradoxically during systole (Fig. 272-3). Ventriculography may also reveal a left ventricular aneurysm, pseudoaneurysm, or diverticulum and can be used to assess mitral valve prolapse and the severity of mitral regurgitation. The degree of mitral regurgitation is estimated

1	FIGURE 272-3 Left ventriculogram at end diastole (left) and end systole (right). In patients with normal left ventricular function, the ventriculogram reveals symmetric contraction of all walls (top). Patients with coronary artery disease may have wall motion abnormalities on ventriculography as seen in this 60-year-old male following a large anterior myocardial infarction. In systole, the anterior, apical, and inferior walls are akinetic (white arrows) (bottom).

1	by comparing the density of contrast opacification of the left atrium with that of the left ventricle. Minimal contrast reflux into the left atrium is considered 1+ mitral regurgitation, while contrast density in the left atrium that is greater than that in the left ventricle with reflux of contrast into the pulmonary veins within three beats defines 4+ mitral regurgitation. Ventriculography performed in the left anterior oblique projection can be used to identify a ventricular septal defect. Calculation of the ventricular volumes in systole and diastole allows calculation of stroke volume and cardiac output.

1	Aortography in the cardiac catheterization laboratory visualizes abnormalities of the ascending aorta, including aneurysmal dilation and involvement of the great vessels, as well as dissection with compression of the true lumen by an intimal flap that separates the true and false arteries arising from the lumina. Aortography can also be used to identify patent saphenous vein grafts that elude selective cannulation, identify shunts that involve the aorta such as a patent ductus arteriosus, and provide a qualitative assessment of aortic regurgitation using a 1+−4+ scale similar to that used for mitral regurgitation.

1	Selective coronary angiography is catheterization and is used to define the coronary anatomy and determine the extent of epicardial coronary artery ease. Specially shaped coronary cath eters are used to engage the left and right coronary ostia. Hand injection of coronary “luminogram” that is recorded phy). Because the coronary arteries are three-dimensional objects that are in motion with the cardiac cycle, angio grams of the vessels using several differ ent orthogonal projections are taken to best visualize the vessels without overlap or foreshortening. The normal coronary anatomy is highly variable between individuals, but, in general, there are two coronary ostia and three major coronary vessels—the left anterior descending, the left circumflex, and the right coronary arteries with the left anterior descending and left circumflex left main coronary artery (Fig. 272-4).

1	When the right coronary artery is the origin of the atrioventricular nodal branch, the posterior descending artery, and the posterior lateral vessels, the circulation is defined as right dominant; this is found in ~85% of individuals. When these branches arise from the left circumflex artery as occurs in ~5% of individuals, the circulation is defined as left dominant. The remaining ~10% of patients have a codominant circulation with vessels arising from both the right and left coronary circulation. In some patients, a ramus intermedius branch arises directly from the left main coronary artery; this finding is a normal variant. Coronary artery anomalies occur in 1–2% of patients, with separate ostia for the left anterior descending and left circumflex arteries being the most common (0.41%).

1	FIGURE 272-4 Normal coronary artery anatomy. A. Coronary angiogram showing the left circumflex (LCx) artery and its obtuse marginal (OM) branches. The left anterior descending artery (LAD) is also seen but may be foreshortened in this view. B. The LAD and its diagonal (D) branches are best seen in cranial views. In this angiogram, the left main (LM) coronary artery is also seen. C. The right coronary artery (RCA) gives off the posterior descending artery (PDA), so this is a right dominant circulation. FIGURE 272-5 Coronary stenoses on cine angiogram and intra-vascular ultrasound. Significant stenoses in the coronary artery are seen as narrowings (black arrows) of the vessel. Intravascular ultrasound shows a normal segment of artery (A), areas with eccentric plaque (B, C), and near total obliteration of the lumen at the site of the significant stenosis (D). Note that the intravascular ultrasound catheter is present in the images as a black circle.

1	Coronary angiography visualizes coronary artery stenoses as luminal narrowings on the cine angiogram. The degree of narrowing is referred to as the percent stenosis and is determined visually by comparing the most severely diseased segment with a proximal or distal “normal segment”; a stenosis >50% is considered significant (Fig. 272-5). Online quantitative coronary angiography can provide a more accurate assessment of the percent stenosis and lessen the tendency to overestimate lesion severity visually. The presence of a myocardial bridge, which most commonly involves the left anterior descending artery, may be mistaken for a significant stenosis; this occurs when a portion of the vessel dips below the epicardial surface into the myocardium and is subject to compressive forces during ventricular systole. The key to differentiating a myocardial bridge from a fixed stenosis is that the “stenosed” part of the vessel returns to normal during diastole. Coronary calcification is also seen

1	systole. The key to differentiating a myocardial bridge from a fixed stenosis is that the “stenosed” part of the vessel returns to normal during diastole. Coronary calcification is also seen during angiography prior to the injection of contrast agents. Collateral blood vessels may be seen traversing from one ves(Fig. 272-5). Intravascular ultrasound (IVUS) is performed using 1465 a small flexible catheter with a 40-mHz transducer at its tip that is advanced into the coronary artery over a guidewire. Data from intra-vascular ultrasound studies may be used to image atherosclerotic plaque precisely, determine luminal cross-sectional area, and measure vessel size; it is also used during or following percutaneous coronary intervention to assess the stenosis and determine the adequacy of stent placement. Optical coherence tomography (OCT) is a catheter-based imaging technique that uses near-infrared light to generate images with better spatial resolution than IVUS; however, the depth of

1	placement. Optical coherence tomography (OCT) is a catheter-based imaging technique that uses near-infrared light to generate images with better spatial resolution than IVUS; however, the depth of field is smaller. The advantage of OCT imaging over IVUS lies in its ability to image characteristics of the atherosclerotic plaque (lipid, fibrous cap) with high definition and to assess coronary stent placement, apposition, and patency (Fig. 272-6).

1	Measurement of the fractional flow reserve provides a functional assessment of the stenosis and is more accurate in predicting longterm clinical outcome than imaging techniques. The fractional flow reserve is the ratio of the pressure in the coronary artery distal to the stenosis divided by the pressure in the artery proximal to the stenosis at maximal vasodilation. Fractional flow reserve is measured using a coronary pressure–sensor guidewire at rest and at maximal hyperemia following the injection of adenosine. A fractional flow reserve of <0.80 indicates a hemodynamically significant stenosis that would benefit from intervention.

1	Once the procedure is completed, vascular access sheaths are removed. If the femoral approach is used, direct manual compression or vascular closure devices that immediately close the arteriotomy site with a staple/clip, collagen plug, or sutures are used to achieve hemostasis. These devices decrease the length of supine bed rest (from 6 hours to 2–4 hours) and improve patient satisfaction but have not been shown definitively to be superior to manual compression with respect to access-site complications. With radial-artery access, bed rest is needed for only 2 hours. When cardiac catheterization is performed as an elective outpatient procedure, the patient completes postprocedure bed rest in a monitored setting and is discharged home with instructions to liberalize fluids because contrast agents promote an osmotic diuresis, to avoid strenuous activity, and to observe the vascular access site for signs of complications. Overnight hospitalization may be required for high-risk patients

1	agents promote an osmotic diuresis, to avoid strenuous activity, and to observe the vascular access site for signs of complications. Overnight hospitalization may be required for high-risk patients with significant comorbidities, patients with complications occurring during the catheterization, or patients who have undergone a percutaneous coronary intervention. Hypotension early after the procedure may be due to inadequate fluid replacement or retroperitoneal bleeding from the access site. Patients who received >2 Gy of radiation during the procedure should be examined for signs of erythema. For patients who received higher doses (>5 Gy), clinical follow-up within 1 month to assess for skin injury is recommended.

1	sel to the distal vasculature of a severely stenosed or totally occluded vessel. Thrombolysis in myocardial infarction (TIMI) flow grade, a measure of the relative duration of time that it takes for contrast to opacify the coronary artery fully, may provide an additional clue to the degree of lesion severity, and the presence of TIMI grade 1 (minimal filling) or 2 ABCDE (delayed filling) suggests that a significant coronary FIGURE 272-6 Optical coherence tomography imaging. A. The optical coherence artery stenosis is present.

1	tomography (OCT) catheter (*) in the lumen of a coronary artery with limited neointima INTRAVASCULAR ULTRASOUND, OPTICAL COHERENCE formation. The intima is seen with high definition, but unlike intravascular ultrasound TOMOGRAPHY, AND FRACTIONAL FLOW RESERVE imaging, the vessel media and adventitia are not well visualized. B. A fibrous plaque During coronary angiography, intermediate steno-(arrow) is characterized by a bright signal. C. A large, eccentric, lipid-rich plaque obscures ses (40–70%), indeterminate findings, or anatomic part of the vessel lumen. Because lipid in the plaque absorbs light, the lipid-rich plaque findings that are incongruous with the patient’s appears as a dark area with irregular borders (arrow). The plaque is covered by a thin symptoms may require further interrogation. In fibrous cap (arrowhead) typical of a vulnerable plaque. D. A thrombus (arrow) adherthese cases, intravascular ultrasound provides a ent to a ruptured plaque that is protruding into the

1	In fibrous cap (arrowhead) typical of a vulnerable plaque. D. A thrombus (arrow) adherthese cases, intravascular ultrasound provides a ent to a ruptured plaque that is protruding into the vessel lumen. E. A coronary stent is more accurate anatomic assessment of the coronary that is well opposed to the vessel wall. The stent struts appear as short bright lines with artery and the degree of coronary atherosclerosis dropout behind the struts (arrow).

1	Principles of Electrophysiology David D. Spragg, Gordon F. Tomaselli HISTORY AND INTRODUCTION The field of cardiac electrophysiology was ushered in with the devel-opment of the electrocardiogram (ECG) by Einthoven at the turn of 273e SECTion 3 DiSoRDERS of RHy THm

1	FIGURE 273e-1 A. Cellular atrial and ventricular action potentials. Phases 0–4 are the rapid upstroke, early repolarization, plateau, late repolarization, and diastole, respectively. The ionic currents and their respective genes are shown above and below the action potentials. The currents that underlie the action potentials vary in atrial and ventricular myocytes. B. A ventricular action potential with a schematic of the ionic currents flowing during the phases of the action potential. Potassium current (IK1) is the principal current during phase 4 and determines the resting membrane potential of the myocyte. Sodium current generates the upstroke of the action potential (phase 0); activation of Ito with inactivation of the Na current inscribes early repolarization (phase 1). The plateau (phase 2) is generated by a balance of repolarizing potassium currents and depolarizing calcium current. Inactivation of the calcium current with persistent activation of potassium currents

1	plateau (phase 2) is generated by a balance of repolarizing potassium currents and depolarizing calcium current. Inactivation of the calcium current with persistent activation of potassium currents (predominantly IKr and IKs) causes phase 3 repolarization.

1	the twentieth century. Subsequent recording of cellular membrane currents demonstrated that the body surface ECG is the timed sum of the cellular action potentials in the atria and ventricles. In the late 1960s, the development of intracavitary recording, in particular, His bundle electrograms, marked the beginning of contemporary clinical electrophysiology. Adoption of radiofrequency technology to ablate cardiac tissue in the early 1990s heralded the birth of interventional cardiac electrophysiology.

1	The clinical problem of sudden death caused by ventricular arrhythmias, most commonly in the setting of coronary artery obstruction, was recognized as early as the late nineteenth century. The problem was vexing and led to the development of pharmacologic and nonpharmacologic therapies, including transthoracic defibrillators, cardiac massage, and, most recently, implantable defibrillators. Over time the limitations of antiarrhythmic drug therapy have been highlighted repeatedly in clinical trials, and now ablation and devices are first-line therapy for a number of cardiac arrhythmias. In the last two decades, the genetic basis of a number of heritable arrhythmias has been elucidated, revealing important insights into the mechanisms not only of these rare arrhythmias but also of similar rhythm disturbances observed in more common forms of heart disease. SCN5A (Nav1.5) CACNA1C (Cav1.2) ICa-L SLC8A1 (NCX1.1) KCNJ2 (Kir2.1) IK1 KCND3/KCNIP2 (Kv4.3/KChIP2) to

1	SCN5A (Nav1.5) CACNA1C (Cav1.2) ICa-L SLC8A1 (NCX1.1) KCNJ2 (Kir2.1) IK1 KCND3/KCNIP2 (Kv4.3/KChIP2) to KCNH2/KCNE2 (HERG/MiRP-1) IKr KCNQ1/KCNE1 (KVLQT1/minK) IKs KCNA5 (Kv1.5)

1	The normal cardiac impulse is generated by pacemaker cells in the sinoatrial node situated at the junction of the right atrium and the superior vena cava (see Fig. 268-1). This impulse is transmitted slowly through nodal tissue to the anatomically complex atria, where it is conducted more rapidly to the atrioventricular node (AVN), inscribing the P wave of the ECG (see Fig. 268-2). There is a perceptible delay in conduction through the anatomically and functionally heterogeneous AVN. The time needed for activation of the atria and the AVN delay is represented as the PR interval of the ECG. The AVN is the only electrical connection between the atria and the ventricles in the normal heart. The electrical impulse emerges from the AVN and is transmitted to the His-Purkinje system, specifically the common bundle of His, then the left and right bundle branches, and then to the Purkinje network, facilitating activation of ventricular muscle. In normal circumstances, the ventricles are

1	the common bundle of His, then the left and right bundle branches, and then to the Purkinje network, facilitating activation of ventricular muscle. In normal circumstances, the ventricles are activated rapidly in a well-defined fashion that is determined by the course of the Purkinje network, and this inscribes the QRS complex (see Fig. 268-2). Recovery of electrical excitability occurs more slowly and is governed by the time of activation and duration of regional action potentials. The relative brevity of epicardial action potentials in the ventricle results in repolarization that occurs first on the epicardial surface and then proceeds to the endocardium, which inscribes a T wave normally of the same polarity as the QRS complex. The duration of ventricular activation and recovery is determined by the action potential duration and is represented on the body surface ECG by the QT interval (see Fig. 268-2).

1	Cardiac myocytes exhibit a characteristically long action potential (200–400 ms) compared with neurons and skeletal muscle cells (1–5 ms). The action potential profile is sculpted by the orchestrated activity of multiple distinctive timeand voltage-dependent ionic currents (Fig. 273e-1A). The currents are carried by transmembrane CHAPTER 273e Principles of Electrophysiology

1	PART 10 Disorders of the Cardiovascular System 273e-2 proteins that passively conduct ions down their electrochemical gradients through selective pores (ion channels), actively transport ions against their electrochemical gradient (pumps, transporters), or electrogenically exchange ionic species (exchangers). Action potentials in the heart are regionally distinct. The regional variability in cardiac action potentials is a result of differences in the number and types of ion channel proteins expressed by different cell types in the heart. Further, unique sets of ionic currents are active in pacemaking and muscle cells, and the relative contributions of these currents may vary in the same cell type in different regions of the heart (Fig. 273e-1A). Ion channels are complex, multisubunit transmembrane glycoproteins that open and close in response to a number of biologic stimuli, including a change in membrane voltage, ligand binding (directly to the channel or to a G protein–coupled

1	transmembrane glycoproteins that open and close in response to a number of biologic stimuli, including a change in membrane voltage, ligand binding (directly to the channel or to a G protein–coupled receptor), and mechanical deformation (Fig. 273e-2). Other ion motive exchangers and transporters contribute importantly to cellular excitability in the heart. Ion pumps establish and maintain the ionic gradients across the cell membrane that serve as the driving force for current flow through ion channels. Transporters or exchangers that do not move ions in an electrically neutral manner (e.g., the sodium-calcium exchanger transports three Na+ for one Ca2+) are termed electrogenic and contribute directly to the action potential profile. The most abundant superfamily of ion channels expressed in the heart is voltage gated. Several structural themes are common to all voltage-dependent ion channels. First, the architecture is modular, consisting either of four homologous subunits (e.g., K

1	in the heart is voltage gated. Several structural themes are common to all voltage-dependent ion channels. First, the architecture is modular, consisting either of four homologous subunits (e.g., K channels) or of four internally homologous domains (e.g., Na and Ca channels). Second, the proteins fold around a central pore lined by amino acids that exhibit exquisite conservation within a given channel family of like selectivity (e.g., all Na channels have very similar P segments). Third, the general strategy for activation gating (opening and closing in response to changes in membrane voltage) is highly conserved: the fourth trans-membrane segment (S4), studded with positively charged residues, lies within the membrane field and moves in response to depolarization, opening the channel. Fourth, most ion channel complexes include not only the pore-forming proteins (α subunits) but also auxiliary subunits (e.g., β subunits) that modify channel function (Fig. 273e-2). Na and Ca channels

1	most ion channel complexes include not only the pore-forming proteins (α subunits) but also auxiliary subunits (e.g., β subunits) that modify channel function (Fig. 273e-2). Na and Ca channels are the primary carriers of depolarizing current in both the atria and the ventricles; inactivation of these currents and activation of repolarizing K currents hyperpolarize the heart cells, reestablishing the negative resting membrane potential (Fig. 273e-1B). The plateau phase is a time when little current is flowing, and relatively minor changes in depolarizing or repolarizing currents can have profound effects on the shape and duration of the action profile. Mutations in subunits of these channel proteins produce arrhythmogenic alterations in the action potentials that cause long and short QT syndrome, Brugada syndrome, idiopathic ventricular fibrillation, familial atrial fibrillation, and some forms of conduction system disease.

1	Cardiac arrhythmias result from abnormalities of electrical impulse generation, conduction, or both. Bradyarrhythmias typically arise from disturbances in impulse formation at the level of the sinoatrial node or from disturbances in impulse propagation at any level, including exit block from the sinus node, conduction block in the AVN, and impaired conduction in the His-Purkinje system. Tachyarrhythmias can be classified according to mechanism, including enhanced automaticity (spontaneous depolarization of atrial, junctional, or ventricular pacemakers), triggered arrhythmias (initiated by afterdepolarizations occurring during or immediately after cardiac repolarization, during phase 3 or 4 of the action potential), or reentry (circus propagation of a depolarizing wavefront). A variety of mapping and pacing maneuvers typically performed during invasive electrophysiologic testing can often determine the underlying mechanism of a tachyarrhythmia (Table 273e-1).

1	Alterations in Impulse Initiation: Automaticity Spontaneous (phase 4) diastolic depolarization underlies the property of automaticity characteristic of pacemaking cells in the sinoatrial (SA) and atrioventricular

1	FIGURE 273e-2 Topology and subunit composition of the voltage-dependent ion channels. Potassium channels are formed by the tetramerization of α or pore-forming subunits and one or more β subunits; only single β subunits are shown for clarity. Sodium and calcium channels are composed of α subunits with four homologous domains and one or more ancillary subunits. In all channel types, the loop of protein between the fifth and sixth membrane-spanning repeat in each subunit or domain forms the ion-selective pore. In the case of the sodium channel, the channel is a target for phosphorylation, the linker between the third and fourth homologous domain is critical to inactivation, and the sixth membrane-spanning repeat in the fourth domain is important in local anesthetic antiarrhythmic drug binding. The Ca channel is a muiltisubunit protein complex with the α1 subunit containing the pore and major drug binding domain.

1	Normal or enhanced automaticity of subsidiary latent pacemakers 273e-3 produces escape rhythms in the setting of failure of more dominant pacemakers. Suppression of a pacemaker cell by a faster rhythm leads to an increased intracellular Na+ load ([Na+]i), and extrusion of Na+ from the cell by Na, K-ATPase produces an increased background repolarizing current that slows phase 4 diastolic depolarization. At slower rates, [Na+]i is decreased, as is the activity of the Na, K-ATPase, warm-up of the tachycardia rate. Overdrive suppression and warm-up are characteristic of, but may not be observed in, all automatic tachy cardias. Abnormal conduction into tissue with enhanced automaticity (entrance block) may blunt or eliminate the phenomena of overdrive CHAPTER 273e Principles of Electrophysiology suppression and warm-up of automatic tissue.

1	CHAPTER 273e Principles of Electrophysiology suppression and warm-up of automatic tissue. Abnormal automaticity may produce atrial tachycardia, acceler ated idioventricular rhythms, and ventricular tachycardia, particularly associated with ischemia and reperfusion. It has also been suggested that injury currents at the borders of ischemic myocardium may depolarize adjacent nonischemic tissue, predisposing to automatic ventricular tachycardia. Abbreviations: AP, action potential; AV, atrioventricular; DADs, delayed afterdepolarizations; EADs, early afterdepolarizations; HF, heart failure; LVH, left ventricular hypertrophy; VF, ventricular fibrillation; VT, ventricular tachyarrhythmia.

1	(AV) nodes, His-Purkinje system, coronary sinus, and pulmonary veins. Phase 4 depolarization results from the concerted action of a number of ionic currents, including K+ currents, Ca2+ currents, electrogenic Na, K-ATPase, the Na-Ca exchanger, and the so-called funny, or pacemaker, current (If); however, the relative importance of these currents remains controversial.

1	The rate of phase 4 depolarization and, therefore, the firing rates of pacemaker cells are dynamically regulated. Prominent among the factors that modulate phase 4 is autonomic nervous system tone. The negative chronotropic effect of activation of the parasympathetic nervous system is a result of the release of acetylcholine that binds to muscarinic receptors, releasing G protein βγ subunits that activate a potassium current (IKACh) in nodal and atrial cells. The resulting increase in K+ conductance opposes membrane depolarization, slowing the rate Afterdepolarizations and Triggered Automaticity Triggered automaticity or activity refers to impulse initiation that is dependent on afterdepolarizations (Fig. 273e-3). Afterdepolarizations are membrane voltage oscillations that occur during (early afterdepolarizations, EADs) or after (delayed afterdepolarizations, DADs) an action potential.

1	The cellular feature common to the induction of DADs is the presence of an increased Ca2+ load in the cytosol and sarcoplasmic reticulum. Digitalis glycoside toxicity, catecholamines, and ischemia all can enhance Ca2+ loading sufficiently to produce DADs. Accumulation of lysophospholipids in ischemic myocardium with consequent Na+ and Ca2+ overload has been suggested as a mechanism for DADs and triggered automaticity. Cells from damaged areas or cells that survive a myocardial infarction may display spontaneous release of calcium from the sarcoplasmic reticulum, and this may generate “waves” of intracellular calcium elevation and arrhythmias.

1	EADs occur during the action potential and interrupt the orderly repolarization of the myocyte. Traditionally, EADs have been thought to arise from action potential prolongation and reactivation of depolarizing currents, but more recent experimental evidence suggests a previously unappreciated interrelationship between intracellular calcium loading and EADs. Cytosolic calcium may increase when action potentials are prolonged. This, in turn, appears to enhance L-type Ca current, further prolonging action potential duration as well as providing the inward current driving EADs. Intracellular calcium loading by action potential prolongation may also enhance the likelihood of DADs. The interrelationship among intracellular [Ca2+], EADs, and DADs may be one explanation for the susceptibility of hearts that are of rise of phase 4 of the action potential. Conversely, augmentation of 0 mV sympathetic nervous system tone increases myocardial catecholamine concentrations, which activate both

1	of hearts that are of rise of phase 4 of the action potential. Conversely, augmentation of 0 mV sympathetic nervous system tone increases myocardial catecholamine concentrations, which activate both α-and β-adrenergic receptors. The effect of β1-adrenergic stimulation predominates in pacemaking cells, augmenting both L-type Ca current (I ) and I , thus increasing the slope of phase 4. Enhanced sympathetic nervous system activity can dramatically increase the rate of firing of SA nodal cells, producing sinus tachycardia with rates >200 beats/min. By contrast, the increased rate of firing of Purkinje cells is more limited, rarely producing ven tricular tachyarrhythmias >120 beats/min.

1	Normal automaticity may be affected by a number of other factors 0.5 s associated with heart disease. Hypokalemia and ischemia may reduce the activity of Na, K-ATPase, thereby reducing the background repolar-FIGURE 273e-3 Schematic action potentials with early after-izing current and enhancing phase 4 diastolic depolarization. The end depolarizations (EADs) and delayed afterdepolarizations (DADs). result would be an increase in the spontaneous firing rate of pacemaking Afterdepolarizations are spontaneous depolarizations in cardiac cells. Modest increases in extracellular potassium may render the maxi-myocytes. EADs occur before the end of the action potential (phases mum diastolic potential more positive, thereby also increasing the firing 2 and 3), interrupting repolarization. DADs occur during phase 4 of rate of pacemaking cells. A more significant increase in [K+]o, however, the action potential after completion of repolarization. The cellular renders the heart inexcitable by

1	occur during phase 4 of rate of pacemaking cells. A more significant increase in [K+]o, however, the action potential after completion of repolarization. The cellular renders the heart inexcitable by depolarizing the membrane potential. mechanisms of EADs and DADs differ (see text).

1	PART 10 Disorders of the Cardiovascular System 273e-4 calcium loaded (e.g., in ischemia or congestive heart failure) to develop arrhythmias, particularly on exposure to action potential–prolonging drugs. EAD-triggered arrhythmias exhibit rate dependence. In general, the amplitude of an EAD is augmented at slow rates when action potentials are longer. Indeed, a fundamental condition that underlies the development of EADs is action potential and QT prolongation. Hypokalemia, hypomagnesemia, bradycardia, and, most commonly, drugs can predispose to the generation of EADs, invariably in the context of prolonging the action potential. Antiarrhythmics with class IA and III action (see below) produce action potential and QT prolongation intended to be therapeutic but frequently causing arrhythmias. Noncardiac drugs such as phenothiazines, nonsedating antihistamines, and some antibiotics can also prolong the action potential duration and predispose to EAD-mediated triggered arrhythmias.

1	Noncardiac drugs such as phenothiazines, nonsedating antihistamines, and some antibiotics can also prolong the action potential duration and predispose to EAD-mediated triggered arrhythmias. Decreased [K+]o paradoxically may decrease membrane potassium currents (particularly the delayed rectifier current, IKr) in the ventricular myocyte, explaining why hypokalemia causes action potential prolongation and EADs. In fact, potassium infusions in patients with the congenital long QT syndrome (LQTS) and in those with drug-induced acquired QT prolongation shorten the QT interval. EAD-mediated triggered activity probably underlies initiation of the characteristic polymorphic ventricular tachycardia, torsades des pointes, seen in patients with congenital and acquired forms of LQTS. Structural heart disease, such as cardiac hypertrophy and heart failure, may also delay ventricular repolarization (so-called electrical remodeling) and predispose to arrhythmias related to abnormalities of

1	heart disease, such as cardiac hypertrophy and heart failure, may also delay ventricular repolarization (so-called electrical remodeling) and predispose to arrhythmias related to abnormalities of repolarization. The abnormalities of repolarization in hypertrophy and heart failure are often magnified by concomitant drug therapy or electrolyte disturbances.

1	Abnormal Impulse Conduction: Reentry The most common arrhythmia mechanism is reentry resulting from abnormal electrical impulse conduction and is defined as the circulation of an activation wave around an inexcitable obstacle. The requirements for reentry are two electrophysiologically dissimilar pathways for impulse propagation around an inexcitable region (Fig. 273e-4). Reentry can occur around a fixed anatomic structure (e.g., myocardial scar), with a stable pattern of cardiac depolarization moving in series over the anterograde and retrograde limbs of the circuit. This form of reentry, referred to as anatomic reentry or excitable gap reentry (see below), is initiated when a depolarizing wavefront encounters an area of unidirectional conduction block in the retrograde limb of the circuit. Conduction across the anterograde limb occurs with a delay that, if of sufficient duration, allows for recovery of conduction in the retrograde limb with reentry of the depolarization wave into

1	Conduction across the anterograde limb occurs with a delay that, if of sufficient duration, allows for recovery of conduction in the retrograde limb with reentry of the depolarization wave into the retrograde limb of

1	FIGURE 272-4 Schematic diagram of reentry. A. The circuit contains two limbs, one with slow conduction. B. A premature impulse blocks in the fast pathway and conducts over the slow pathway, allowing the fast pathway to recover so that the activation wave can reenter the fast pathway from the retrograde direction. C. During sustained reentry utilizing such a circuit, a gap (excitable gap) exists between the activating head of the wave and the recovering tail. D. One mechanism of termination of reentry occurs when the conduction and recovery characteristics of the circuit change and the activating head of the wave collides with the tail, extinguishing the tachycardia.

1	the circuit. Sustained reentry requires that the functional dimension of depolarized tissue or the tachycardia wavelength (λ = conduction velocity × refractory period) fits within the total anatomic length of the circuit, referred to as the path length. When the path length of the circuit exceeds the λ of the tachycardia, the region between the head of the activation wave and the refractory tail is referred to as the excitable gap. Anatomically determined, excitable gap reentry can explain several clinically important tachycardias, such as AV reentry, atrial flutter, bundle branch reentry ventricular tachycardia, and ventricular tachycardia in scarred myocardium.

1	Reentrant arrhythmias may exist in the heart in the absence of an excitable gap and with a tachycardia wavelength nearly the same size as the path length. In this case, the wavefront propagates through partially refractory tissue without a fixed anatomic obstacle and no fully excitable gap; this is referred to as leading circle reentry, a form of functional reentry (reentry that depends on functional properties of the tissue). Unlike excitable gap reentry, there is no fixed anatomic circuit in leading circle reentry, and it may, therefore, not be possible to disrupt the tachycardia with pacing or destruction of a part of the circuit. Furthermore, the circuit in leading circle reentry tends to be less stable than that in excitable gap reentrant arrhythmias, with large variations in cycle length and a predilection to termination. There is strong evidence to suggest that less organized arrhythmias, such as atrial and ventricular fibrillation, are associated with more complex activation

1	length and a predilection to termination. There is strong evidence to suggest that less organized arrhythmias, such as atrial and ventricular fibrillation, are associated with more complex activation of the heart and are due to functional reentry.

1	Catheter-based and pharmacologic therapies for reentrant arrhythmias are designed to disrupt the anatomic circuit or alter the relationship between the wavelength and path length of the arrhythmia circuit, eliminating pathologic conduction. For example, antiarrhythmic drugs that prolong the action potential (Class III) are effective if they sufficiently prolong the λ such that it can no longer fit within the anatomic circuit. Catheter ablation is often undertaken with the goal of identifying and destroying a critical limb of the reentrant circuit (i.e., ablation of the cavotricuspid isthmus in the treatment of typical, right atrial flutter). Due to the less defined pathways of myocardial activation seen in functional reentry, ablation of these rhythms tends to target initiating triggers (e.g., pulmonary vein potentials in catheter ablation of atrial fibrillation) rather than the anatomic circuit.

1	Structural heart disease is associated with changes in conduction and refractoriness that increase the risk of reentrant arrhythmias. Chronically ischemic myocardium exhibits a downregulation of the gap junction channel protein (connexin 43) that carries intercellular ionic current. The border zones of infarcted and failing ventricular myocardium exhibit not only functional alterations of ionic currents but also remodeling of tissue and altered distribution of gap junctions. The changes in gap junction channel expression and distribution, in combination with macroscopic tissue alterations, support a role for slowed conduction in reentrant arrhythmias that complicate chronic coronary artery disease (CAD). Aged human atrial myocardium exhibits altered conduction, manifest as highly fractionated atrial electrograms, producing an ideal substrate for the reentry that may underlie the very common development of atrial fibrillation in the elderly. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: The evaluation of patients with suspected cardiac arrhythmias is highly individualized; however, two key features—the history and ECG—are pivotal in directing the diagnostic workup and therapy. Patients with cardiac arrhythmias exhibit a wide spectrum of clinical presentations that range from asymptomatic ECG abnormalities to survival from cardiac arrest. In general, the more severe the presenting symptoms are, the more aggressive the evaluation and treatment are. Loss of consciousness that is believed to be of cardiac origin typically mandates an exhaustive search for the etiology and often requires invasive, device-based therapy. The presence of structural heart disease and prior myocardial infarction dictates a change in the approach to the management of syncope or ventricular arrhythmias. The presence of a family history of serious ventricular arrhythmias or premature sudden death will influence the evaluation of presumed heritable arrhythmias.

1	The physical examination is focused on determining whether there is cardiopulmonary disease that is associated with specific cardiac arrhythmias. The absence of significant cardiopulmonary disease often, but not always, suggests benignity of the rhythm disturbance. In contrast, palpitations, syncope, or near syncope in the setting of significant heart or lung disease have more ominous implications. In addition, the physical examination may reveal the presence of a persistent arrhythmia such as atrial fibrillation.

1	The judicious use of noninvasive diagnostic tests is an important element in the evaluation of patients with arrhythmias, and there is no test more important than the ECG, particularly if recorded at the time of symptoms. Uncommon but diagnostically important signatures of electrophysiologic disturbances may be unearthed on the resting ECG, such as delta waves in Wolff-Parkinson-White (WPW) syndrome, prolongation or shortening of the QT interval, right precordial ST-segment abnormalities in Brugada syndrome, and epsilon waves in arrhythmogenic right ventricular dysplasia. Variants of body surface ECG recording can provide important information about arrhythmia substrates and triggers. Holter monitoring and event recording, either continuous or intermittent, record the body surface ECG over longer periods, enhancing the possibility of observing the cardiac rhythm during symptoms. Holter monitoring is particularly useful in assessing daily symptoms thought to be attributable to

1	ECG over longer periods, enhancing the possibility of observing the cardiac rhythm during symptoms. Holter monitoring is particularly useful in assessing daily symptoms thought to be attributable to arrhythmia or for quantifying a particular arrhythmia phenomenon (e.g., premature ventricular complex burden). Ambulatory event monitors are indicated when symptoms thought to be due to arrhythmia occur less frequently (i.e., several episodes per month), and, because the monitors are typically patient-activated, they are optimal for correlating symptoms with rhythm disturbances. Implantable long-term monitors permit prolonged telemetric monitoring both for diagnosis and to assess the efficacy of therapy. Implantable monitors are typically used for the evaluation of malignant symptoms that occur quite infrequently and that cannot be provoked at diagnostic electro-physiology study.

1	Exercise electrocardiography is important in determining the presence of myocardial demand ischemia; more recently, analysis of the morphology of the QT interval with exercise has been used to assess the risk of serious ventricular arrhythmias. The exercise ECG may be particularly useful in patients with symptoms that occur during activity. Cardiac imaging plays an important role in the detection and characterization of myocardial structural abnormalities that may render the heart more susceptible to arrhythmia. Ventricular tachyarrhythmias, for instance, occur more frequently in patients with ventricular systolic dysfunction and chamber dilation, in hypertrophic cardiomyopathy, and in the setting of infiltrative diseases such as sarcoidosis. Supraventricular arrhythmias may be associated with particular congenital conditions, including AV reentry in the setting of Ebstein’s anomaly. Echocardiography is a frequently employed imaging technique to screen for disorders of cardiac

1	with particular congenital conditions, including AV reentry in the setting of Ebstein’s anomaly. Echocardiography is a frequently employed imaging technique to screen for disorders of cardiac structure and function. Increasingly, magnetic resonance imaging of the myocardium is being used to screen for scar burden, fibrofatty infiltration of the myocardium as seen in arrhythmogenic right ventricular cardiomyopathy, and other structural changes that affect arrhythmia susceptibility.

1	Head-up tilt (HUT) testing is useful in the evaluation of patients with syncope in whom there is a suspicion that exaggerated vagal tone or vasodepression may play a causal role. The physiologic response to HUT is incompletely understood; however, redistribution of blood volume and increased ventricular contractility occur consistently. Exaggerated activation of a central reflex in response to HUT produces a stereotypic response of an initial increase in heart rate, then a drop in blood pressure followed by a reduction in heart rate characteristic of neurally mediated hypotension. Other responses to HUT may be observed in patients with orthostatic hypotension and autonomic insufficiency. HUT is used most often in patients with recurrent syncope, although it may be useful in patients with single syncopal episodes with associated injury, particularly in the absence of structural heart disease. In patients with structural heart disease, HUT may be indicated in those with syncope, in whom

1	syncopal episodes with associated injury, particularly in the absence of structural heart disease. In patients with structural heart disease, HUT may be indicated in those with syncope, in whom other causes (e.g., asystole, ventricular tachyarrhythmias) have been excluded. HUT has been suggested as a useful tool in the diagnosis of and therapy for recurrent idiopathic vertigo, chronic fatigue syndrome, recurrent transient ischemic attacks, and repeated falls of unknown etiology in the elderly. Importantly, HUT is relatively contraindicated in the presence of severe CAD with proximal coronary stenoses, known severe cerebrovascular disease, severe mitral stenosis, and obstruction to left ventricular outflow (e.g., aortic stenosis).

1	Electrophysiologic testing is central to the understanding and treatment of many cardiac arrhythmias. Indeed, most frequently, electrophysiologic testing is interventional, providing both diagnosis and therapy. The indications for electrophysiologic testing fall into several categories: to define the mechanism of an arrhythmia; to deliver catheter-based ablative treatment; and to determine the etiology of symptoms that may be caused by an arrhythmia (e.g., syncope, palpitations). The components of the electrophysiologic test are baseline measurements of conduction under resting and stressed (rate or pharmacologic) conditions and maneuvers, both pacing and pharmacologic, to induce arrhythmias. A number of sophisticated electrical mapping and catheter-guidance techniques have been developed to facilitate catheter-based therapeutics in the electrophysiology laboratory.

1	The interaction of antiarrhythmic drugs with cardiac tissues and the resulting electrophysiologic changes are complex. An incomplete understanding of the effects of these drugs has produced serious missteps that have had adverse effects on patient outcomes and the development of newer pharmacologic agents. Currently, antiarrhythmic drugs have been relegated to an ancillary role in the treatment of most cardiac arrhythmias.

1	There are several explanations for the complexity of antiarrhythmic drug action: the structural similarity of target ion channels; regional differences in the levels of expression of channels and transporters, which change with disease; time and voltage dependence of drug action; and the effect of these drugs on targets other than ion channels. Because of the limitations of any scheme to classify antiarrhythmic agents, a shorthand that is useful in describing the major mechanisms of action is of some utility. Such a classification scheme was proposed in 1970 by Vaughan-Williams and later modified by Singh and Harrison. The classes of antiarrhythmic action are class I, local anesthetic effect due to blockade of Na+ current; class II, interference with the action of catecholamines at the β-adrenergic receptor; class III, delay of repolarization due to inhibition of K+ current or activation of depolarizing current; and class IV, interference with calcium conductance (Table 273e-2). Class

1	receptor; class III, delay of repolarization due to inhibition of K+ current or activation of depolarizing current; and class IV, interference with calcium conductance (Table 273e-2). Class I antiarrhythmics have been further subdivided based on the kinetics and potency of Na+ channel binding; class Ia agents (quinidine, procainamide) are those with moderate potency and intermediate kinetics; class Ib agents (lidocaine, mexiletine) are those with low potency and rapid kinetics; and class Ic drugs (flecainide, propafenone) are those with high potency and the slowest kinetics. The limitations of the Vaughan-Williams classification scheme include multiple actions of most drugs, overwhelming consideration of antagonism as a mechanism of action, and the fact that several agents have none of the four classes of action in the scheme.

1	The use of catheter ablation is based on the principle that there is a critical anatomic region of impulse generation or propagation that CHAPTER 273e Principles of Electrophysiology is required for the initiation and maintenance of cardiac arrhythmias. Destruction of such a critical region results in the elimination of the arrhythmia. The use of radiofrequency (RF) energy in clinical medicine is nearly a century old. The first catheter ablation using a DC energy source was performed in the early 1980s by Scheinman and colleagues. By the early 1990s, RF had been adapted for use in catheter-based ablation in the heart (Fig. 273e-5).

1	The RF band (300–30,000 kHz) is used to generate energy for several biomedical applications, including coagulation and cauterization of tissues. Energy of this frequency will not stimulate skeletal muscle or the heart and heats tissue by a resistive mechanism, with the intensity of heating and tissue destruction being proportional to the delivered power. Alternative, less frequently used energy sources for catheter ablation of cardiac arrhythmias include microwaves (915 MHz or 2450 MHz), lasers, ultrasound, and freezing (cryoablation). Of these alternative ablation techniques, cryoablation is being used clinically with the most frequency, especially ablation in the region of the AVN. At temperatures just below 32°C, membrane ion transport is disrupted, producing depolarization of cells, decreased action potential amplitude and duration, and slowed conduction velocity (resulting in local conduction block)—all of which are reversible if the tissue is rewarmed in a timely fashion.

1	FIGURE 273e-5 Catheter ablation of cardiac arrhythmias. A. A schematic of the catheter system and generator in a patient undergoing radiofrequency catheter ablation (RFCA); the circuit involves the catheter in the heart and a dispersive patch placed on the body surface (usually the back). The inset shows a diagram of the heart with a catheter located at the AV valve ring for ablation of an accessory pathway.

1	B. A right anterior oblique fluoroscopic image of the catheter position for ablation of a left-sided accessory pathway. A catheter is placed in the atrial side of the mitral valve ring (abl) via a transseptal puncture. Other catheters are placed in the coronary sinus (CS), in the right atrium (RA), and in the right ventricular (RV) apex to record local electrical activation. C. Body surface electrocardiogram recordings (I, II, V1) and endocardial electrograms (HRA, high right atrium; HISp, proximal His bundle electrogram; CS 7, 8, recordings from poles 7 and 8 of a decapolar catheter placed in the coronary sinus) during RFCA of a left-sided accessory pathway in a patient with Wolff-Parkinson-White syndrome. The QRS narrows at the fourth complex; the arrow shows the His bundle electrogram, which becomes apparent with elimination of ventricular preexcitation over the accessory pathway.

1	Tissue cooling can be used for mapping and ablation. Cryomapping can be used to confirm the location of a desired ablation target, such as an accessory pathway in WPW syndrome, or can be used to determine the safety of ablation around the AVN by monitoring AV conduction during cooling. Another advantage of cryoablation is that once the catheter tip cools below freezing, it adheres to the tissue, increasing catheter stability independent of the rhythm or pacing. Bradyarrhythmias due either to primary sinus node dysfunction or to atrioventricular conduction defects are readily treated through implantation of a permanent pacemaker. Clinical indications for pacemaker implantation often depend on the presence either of symptomatic bradycardia or of an unreliable endogenous escape rhythm and are more fully reviewed in Chaps. 274 and 275.

1	Ventricular tachyarrhythmias, particularly those occurring in the 273e-7 context of progressive structural heart diseases such as ischemic cardiomyopathy or arrhythmogenic right ventricular cardiomyopathy, may recur despite therapy with antiarrhythmic drugs or catheter ablation. In appropriate candidates, implantation of an internal cardioverter-defibrillator (ICD) may reduce mortality rates from sudden cardiac death. In a subset of patients with congestive heart failure (CHF) and ventricular mechanical dyssynchrony, ICD or pacemaker platforms can be used to provide cardiac resynchronization therapy, typically through implantation of a left ventricular pacing lead. In patients with dyssynchronous CHF, such therapy has been shown to improve both morbidity and mortality rates. CHAPTER 273e Principles of Electrophysiology

1	The Bradyarrhythmias: Disorders of the Sinoatrial node David D. Spragg, Gordon F. Tomaselli Electrical activation of the heart normally originates in the sinoatrial (SA) node, the predominant pacemaker. Other subsidiary 274 120 negative resting membrane potential compared with atrial or ventricular myocytes. Electrical diastole in nodal cells is characterized by slow diastolic depolarization (phase 4), which generates an action potential as the membrane voltage reaches threshold. The action potential upstrokes (phase 0) are slow compared with atrial or ventricular myocytes, being mediated by calcium rather than sodium current. Cells with properties of SA and AV nodal tissue are electrically connected to the remainder of the myocardium by cells with an electrophysiologic phenotype between that of nodal cells and that of atrial or ventricular myocytes. Cells in the SA node exhibit the most rapid phase 4 depolarization and thus are the dominant pacemakers in a normal heart.

1	Bradycardia results from a failure of either impulse initiation or impulse conduction. Failure of impulse initiation may be caused by depressed automaticity resulting from a slowing or failure of phase 4 diastolic depolarization (Fig. 274-2), which may result from disease or exposure to drugs. Prominently, the autonomic nervous system modulates the rate of phase 4 diastolic depolarization and thus the firing rate of both primary (SA node) and subsidiary pacemakers. Failure of conduction of an impulse from nodal tissue to atrial or ventricular myocardium may produce bradycardia as a result of exit block. Conditions that alter the activation and connectivity of cells (e.g., fibrosis) in the heart may result in failure of impulse conduction.

1	SA node dysfunction and AV conduction block are the most common causes of pathologic bradycardia. SA node dysfunction may be difficult to distinguish from physiologic sinus bradycardia, particularly in the young. SA node dysfunction increases in frequency between the fifth and sixth decades of life and should be considered in patients with fatigue, exercise intolerance, or syncope and sinus bradycardia. Permanent pacemaking is the only reliable therapy for symptomatic bradycardia in the absence of extrinsic and reversible etiologies such as increased vagal tone, hypoxia, hypothermia, and drugs (Table 274-1). Approximately 50% of the 150,000 permanent pacemakers implanted in the United States and 20–30% of the 150,000 of those in Europe were implanted for SA node disease.

1	The SA node is composed of a cluster of small fusiform cells in the sulcus terminalis on the epicardial surface of the heart at the right atrial– superior vena caval junction, where they envelop the SA nodal artery. The SA node is structurally heterogeneous, but the central prototypic nodal cells have fewer distinct myofibrils than does the surrounding atrial myocardium, no intercalated disks visible on light microscopy, a poorly developed sarcoplasmic reticulum, and no T-tubules. Cells in the peripheral regions of the SA node are transitional in both structure and function. The SA nodal artery arises from the right coronary artery in 55–60% and the left circumflex artery in 40–45% of persons. The SA node is richly innervated by sympathetic and parasympathetic nerves and ganglia.

1	pacemakers in the atrioventricular (AV) node, specialized conducting system, and muscle may initiate elec- trical activation if the SA node is dysfunctional or sup- pressed. Typically, subsidiary pacemakers discharge at a slower rate and, in the absence of an appropriate increase in stroke volume, may result in tissue hypoperfusion. Spontaneous activation and contraction of the heart are a consequence of the specialized pacemaking tissue

1	Spontaneous activation and contraction of the heart are a consequence of the specialized pacemaking tissue Voltage, mV in these anatomic locales. As described in Chap. 273e, –100 action potentials in the heart are regionally heterogeneous. The action potentials in cells isolated from nodal FIGURE 274-1 Action potential profiles recorded in cells isolated from sinoatrial tissue are distinct from those recorded from atrial and or atrioventricular nodal tissue compared with those of cells from atrial or ventricuventricular myocytes (Fig. 274-1). The complement lar myocardium. Nodal cell action potentials exhibit more depolarized resting memof ionic currents present in nodal cells results in a less brane potentials, slower phase 0 upstrokes, and phase 4 diastolic depolarization.

1	FIGURE 274-2 Schematics of nodal action potentials and the currents that contribute to phase 4 depolarization. Relative increases in depolarizing L(I ) and T(I ) type calcium and pacemaker cur rents (If) along with a reduction in repolarizing inward rectifier (IK1) and delayed rectifier (IK) potassium currents result in depolarization. Activation of ACh-gated (IKACh) potassium current and beta blockade slow the rate of phase 4 and decrease the pacing rate. (Modified from J Jalife et al: Basic Cardiac Electrophysiology for the Clinician, Blackwell Publishing, 1999.)

1	Irregular and slow propagation of impulses from the SA node can be explained by the electrophysiology of nodal cells and the structure of the SA node itself. The action potentials of SA nodal cells are characterized by a relatively depolarized membrane potential (Fig. 274-1) of −40 to −60 mV, slow phase 0 upstroke, and relatively rapid phase 4 diastolic depolarization compared with the action potentials recorded in cardiac muscle cells. The relative absence of inward rectifier

1	Vasovagal (cardioinhibitory) stimulation Inflammatory Drugs Pericarditis Beta blockers Myocarditis (including viral) Calcium channel blockers Rheumatic heart disease Digoxin Collagen vascular diseases Ivabradine Lyme disease Antiarrhythmics (class I and III) Senile amyloidosis Adenosine Congenital heart disease Clonidine (other sympatholytics) TGA/Mustard and Fontan repairs Lithium carbonate Iatrogenic Cimetidine Radiation therapy Amitriptyline Postsurgical Phenothiazines Chest trauma Narcotics (methadone) Familial Pentamidine SSS2, AD, OMIM #163800 (15q24-25) Hypothyroidism SSS1, AR OMIM #608567 (3p21) Sleep apnea SSS3, AD, OMIM #614090 (14q11.2) Hypoxia SA node disease with myopia, OMIM #182190 Endotracheal suctioning (vagal maneuvers) Kearns-Sayre syndrome, OMIM #530000 Increased intracranial pressure Type 1, OMIM #160900 (19q13.2-13.3) Type 2, OMIM #602668 (3q13.3-q24) Friedreich’s ataxia, OMIM #229300 (9q13, 9p23-p11)

1	Increased intracranial pressure Type 1, OMIM #160900 (19q13.2-13.3) Type 2, OMIM #602668 (3q13.3-q24) Friedreich’s ataxia, OMIM #229300 (9q13, 9p23-p11) Abbreviations: AD, autosomal dominant; AR, autosomal recessive; MI, myocardial infarction; OMIM, Online Mendelian Inheritance in Man (database); TGA, transposition of the great arteries.

1	potassium current (IK1) accounts for the depolarized membrane poten-1467 tial; the slow upstroke of phase 0 results from the absence of available fast sodium current (INa) and is mediated by L-type calcium current ); and phase 4 depolarization is a result of the aggregate activity (ICa-L of a number of ionic currents. Prominently, both Land T-type (ICa-T) calcium currents, the pacemaker current (so-called funny current, or If) formed by hyperpolarization-activated cyclic nucleotide-gated channels, and the electrogenic sodium-calcium exchanger provide depolarizing current that is antagonized by delayed rectifier (IKr) and acetylcholine-gated (IKACh) potassium currents. ICa-L , and If are modulated by , ICa-T β-adrenergic stimulation and IKACh by vagal stimulation, explaining the exquisite sensitivity of diastolic depolarization to autonomic nervous system activity. The slow conduction within the SA node is explained by the absence of INa and poor electrical coupling of cells in the

1	sensitivity of diastolic depolarization to autonomic nervous system activity. The slow conduction within the SA node is explained by the absence of INa and poor electrical coupling of cells in the node, resulting from sizable amounts of interstitial tissue and a low abundance of gap junctions. The poor coupling allows for graded electrophysiologic properties within the node, with the peripheral transitional cells being silenced by electrotonic coupling to atrial myocardium.

1	SA nodal dysfunction has been classified as intrinsic or extrinsic. The distinction is important because extrinsic dysfunction is often reversible and generally should be corrected before pacemaker therapy is considered (Table 274-1). The most common causes of extrinsic SA node dysfunction are drugs and autonomic nervous system influences that suppress automaticity and/or compromise conduction. Other extrinsic causes include hypothyroidism, sleep apnea, and conditions likely to occur in critically ill patients such as hypothermia, hypoxia, increased intracranial pressure (Cushing’s response), and endotracheal suctioning via activation of the vagus nerve.

1	Intrinsic sinus node dysfunction is degenerative and often is characterized pathologically by fibrous replacement of the SA node or its connections to the atrium. Acute and chronic coronary artery disease (CAD) may be associated with SA node dysfunction, although in the setting of acute myocardial infarction (MI; typically inferior), the abnormalities are transient. Inflammatory processes may alter SA node function, ultimately producing replacement fibrosis. Pericarditis, myocarditis, and rheumatic heart disease have been associated with SA nodal disease with sinus bradycardia, sinus arrest, and exit block. Carditis associated with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and mixed connective tissue disorders (MCTDs) may also affect SA node structure and function. Senile amyloidosis is an infiltrative disorder in patients typically in the ninth decade of life; deposition of amyloid protein in the atrial myocardium can impair SA node function. Some SA node disease

1	amyloidosis is an infiltrative disorder in patients typically in the ninth decade of life; deposition of amyloid protein in the atrial myocardium can impair SA node function. Some SA node disease is iatrogenic and results from direct injury to the SA node during cardiothoracic surgery.

1	Rare heritable forms of sinus node disease have been described, and several have been characterized genetically. Autosomal dominant sinus node dysfunction in conjunction with supraventricular tachycardia (i.e., tachycardia-bradycardia variant of sick-sinus syndrome [SSS2]) has been linked to mutations in the pacemaker current (If) subunit gene HCN4 on chromosome 15. An autosomal recessive form of SSS1 with the prominent feature of atrial inexcitability and absence of P waves on the electrocardiogram (ECG) is caused by mutations in the cardiac sodium channel gene, SCN5A, on chromosome 3. Variants in myosin heavy chain 6 (MYH6) increase the susceptibility to SSS (SSS3). SA node dysfunction associated with myopia has been described but not genetically characterized. There are several neuromuscular diseases, including Kearns-Sayre syndrome (ophthalmoplegia, pigmentary degeneration of the retina, and cardiomyopathy) and myotonic dystrophy, that have a predilection for the conducting system

1	diseases, including Kearns-Sayre syndrome (ophthalmoplegia, pigmentary degeneration of the retina, and cardiomyopathy) and myotonic dystrophy, that have a predilection for the conducting system and SA node.

1	SSS in both the young and the elderly is associated with an increase in fibrous tissue in the SA node. The onset of SSS may be hastened by coexisting disease, such as CAD, diabetes mellitus, hypertension, and valvular diseases and cardiomyopathies. SA node dysfunction may be completely asymptomatic and manifest as an ECG anomaly such as sinus bradycardia; sinus arrest and

1	The Bradyarrhythmias: Disorders of the Sinoatrial Node 1468 exit block; or alternating supraventricular tachycardia, usually atrial fibrillation, and bradycardia. Symptoms associated with SA node dysfunction, in particular tachycardia-bradycardia syndrome, may be related to both slow and fast heart rates. For example, tachycardia may be associated with palpitations, angina pectoris, and heart failure, and bradycardia may be associated with hypotension, syncope, presyncope, fatigue, and weakness. In the setting of SSS, overdrive suppression of the SA node may result in prolonged pauses and syncope upon termination of the tachycardia. In many cases, symptoms associated with SA node dysfunction result from concomitant cardiovascular disease. A significant minority of patients with SSS develop signs and symptoms of heart failure that may be related to slow or fast heart rates.

1	One-third to one-half of patients with SA node dysfunction develop supraventricular tachycardia, usually atrial fibrillation or atrial flutter. The incidence of persistent atrial fibrillation in patients with SA node dysfunction increases with advanced age, hypertension, diabetes mellitus, left ventricular dilation, valvular heart disease, and ventricular pacing. Remarkably, some symptomatic patients may experience an improvement in symptoms with the development of atrial fibrillation, presumably from an increase in their average heart rate. Patients with the tachycardia-bradycardia variant of SSS, similar to patients with atrial fibrillation, are at risk for thromboembolism, and those at greatest risk, including patients ≥65 years and patients with a prior history of stroke, valvular heart disease, left ventricular dysfunction, or atrial enlargement, should be treated with anticoagulants. Up to one-quarter of patients with SA node disease will have concurrent AV conduction disease,

1	disease, left ventricular dysfunction, or atrial enlargement, should be treated with anticoagulants. Up to one-quarter of patients with SA node disease will have concurrent AV conduction disease, although only a minority will require specific therapy for high-grade AV block.

1	The natural history of SA node dysfunction is one of varying intensity of symptoms even in patients who present with syncope. Symptoms related to SA node dysfunction may be significant, but overall mortality usually is not compromised in the absence of other significant comorbid conditions. These features of the natural history need to be taken into account in considering therapy for these patients.

1	The electrocardiographic manifestations of SA node dysfunction include sinus bradycardia, sinus pauses, sinus arrest, sinus exit block, tachycardia (in SSS), and chronotropic incompetence. It is often difficult to distinguish pathologic from physiologic sinus bradycardia. By definition, sinus bradycardia is a rhythm driven by the SA node with a rate of <60 beats/min; sinus bradycardia is very common and typically benign. Resting heart rates <60 beats/min are very common in young healthy individuals and physically conditioned subjects. A sinus rate of <40 beats/min in the awake state in the absence of physical conditioning generally is considered abnormal. Sinus pauses and sinus arrest result from failure of the SA node to discharge, producing a pause without P waves visible on the ECG (Fig. 274-3). Sinus pauses of up to 3 s are common in awake athletes, and pauses of this duration or longer may be observed in asymptomatic elderly subjects. Intermittent failure of conduction from the

1	274-3). Sinus pauses of up to 3 s are common in awake athletes, and pauses of this duration or longer may be observed in asymptomatic elderly subjects. Intermittent failure of conduction from the SA node produces sinus exit block. The severity of sinus exit block may vary in a manner similar to that of AV block (Chap. 275). Prolongation of conduction from the sinus node will not be apparent on the ECG; second-degree SA block will produce intermittent conduction from the SA node and a regularly irregular atrial rhythm.

1	FIGURE 274-3 Sinus slowing and pauses on the electrocardiogram (ECG). The ECG is recorded during sleep in a young patient without heart disease. The heart rate before the pause is slow, and the PR interval is prolonged, consistent with an increase in vagal tone. The P waves have a morphology consistent with sinus rhythm. The recording is from a two-lead telemetry system in which the tracing labeled II mimics frontal lead II and V represents Modified Central Lead 1, which mimics lead V1 of the standard 12-lead ECG.

1	Type I second-degree SA block results from progressive prolongation of SA node conduction with intermittent failure of the impulses originating in the sinus node to conduct to the surrounding atrial tissue. Second-degree SA block appears on the ECG as an intermittent absence of P waves (Fig. 274-4). In type II second-degree SA block, there is no change in SA node conduction before the pause. Complete or third-degree SA block results in no P waves on the ECG. Tachycardia-bradycardia syndrome is manifest as alternating sinus bradycardia and atrial tachyarrhythmias. Although atrial tachycardia, atrial flutter, and atrial fibrillation may be observed, the latter is the most common tachycardia. Chronotropic incompetence is the inability to increase the heart rate in response to exercise or other stress appropriately and is defined in greater detail below.

1	SA node dysfunction is most commonly a clinical or electrocardiographic diagnosis. Sinus bradycardia or pauses on the resting ECG are rarely sufficient to diagnose SA node disease, and longer-term recording and symptom correlation generally are required. Symptoms in the absence of sinus bradyarrhythmias may be sufficient to exclude a diagnosis of SA node dysfunction. Electrocardiographic recording plays a central role in the diagnosis and management of SA node dysfunction. Despite the limitations of the resting ECG, longer-term recording employing Holter or event monitors may permit correlation of symptoms with the cardiac rhythm. Many contemporary event monitors may be automatically triggered to record the ECG when certain programmed heart rate criteria are met. Implantable ECG monitors permit long-term recording (12–18 months) in particularly challenging patients.

1	Failure to increase the heart rate with exercise is referred to as chronotropic incompetence. This is alternatively defined as failure to reach 85% of predicted maximal heart rate at peak exercise or failure to achieve a heart rate >100 beats/min with exercise or a maximal heart rate with exercise less than two standard deviations below that of an age-matched control population. Exercise testing may be useful in dis-criminating chronotropic incompetence from resting bradycardia and may aid in the identification of the mechanism of exercise intolerance.

1	FIGURE 274-4 Mobitz type I SA nodal exit block. A theoretical SA node electrogram (SAN EG) is shown. Note that there is grouped beating producing a regularly irregular heart rhythm. The SA node EG rate is constant with progressive delay in exit from the node and activation of the atria, inscribing the P wave. This produces subtly decreasing P-P intervals before the pause, and the pause is less than twice the cycle length of the last sinus interval. Autonomic nervous system testing is useful in diagnosing carotid sinus hypersensitivity; pauses >3 s are consistent with the diagnosis but may be present in asymptomatic elderly subjects. Determining the intrinsic heart rate (IHR) may distinguish SA node dysfunction from slow heart rates that result from high vagal tone. The normal IHR after administration of 0.2 mg/kg propranolol and 0.04 mg/kg atropine is 117.2 − (0.53 × age) in beats/min; a low IHR is indicative of SA disease.

1	Electrophysiologic testing may play a role in the assessment of patients with presumed SA node dysfunction and in the evaluation of syncope, particularly in the setting of structural heart disease. In this circumstance, electrophysiologic testing is used to rule out more malignant etiologies of syncope, such as ventricular tachyarrhythmias and AV conduction block. There are several ways to assess SA node function invasively. They include the sinus node recovery time (SNRT), defined as the longest pause after cessation of overdrive pacing of the right atrium near the SA node (normal: <1500 ms or, corrected for sinus cycle length, <550 ms), and the sinoatrial conduction time (SACT), defined as one-half the difference between the intrinsic sinus cycle length and a noncompensatory pause after a premature atrial stimulus (normal <125 ms). The combination of an abnormal SNRT, an abnormal SACT, and a low IHR is a sensitive and specific indicator of intrinsic SA node disease.

1	Since SA node dysfunction is not associated with increased mortality rates, the aim of therapy is alleviation of symptoms. Exclusion of extrinsic causes of SA node dysfunction and correlation of the cardiac rhythm with symptoms is an essential part of patient management. Pacemaker implantation is the primary therapeutic intervention in patients with symptomatic SA node dysfunction. Pharmacologic considerations are important in the evaluation and management of patients with SA nodal disease. A number of drugs modulate SA node function and are extrinsic causes of dysfunction (Table 274-1). Beta blockers and calcium channel blockers increase SNRT in patients with SA node dysfunction, and antiarrhythmic drugs with class I and III action may promote SA node exit block. In general, such agents should be discontinued before decisions regarding the need for permanent pacing in patients with SA node disease are made. Chronic pharmacologic therapy for sinus bradyarrhythmias is limited. Some

1	should be discontinued before decisions regarding the need for permanent pacing in patients with SA node disease are made. Chronic pharmacologic therapy for sinus bradyarrhythmias is limited. Some pharmacologic agents may improve SA node function; digitalis, for example, has been shown to shorten SNRT in patients with SA node dysfunction. Isoproterenol or atropine administered IV may increase the sinus rate acutely. Theophylline has been used both acutely and chronically to increase heart rate but has liabilities when used in patients with tachycardiabradycardia syndrome, increasing the frequency of supraventricular tachyarrhythmias, and in patients with structural heart disease, increasing the risk of potentially serious ventricular arrhythmias. Currently, there is only a single randomized study of therapy for SA node dysfunction. In patients with resting heart rates <50 and >30 beats/min on a Holter monitor, patients who received dual-chamber pacemakers experienced significantly

1	study of therapy for SA node dysfunction. In patients with resting heart rates <50 and >30 beats/min on a Holter monitor, patients who received dual-chamber pacemakers experienced significantly fewer syncopal episodes and had symptomatic improvement compared with patients randomized to theophylline or no treatment.

1	In certain circumstances, sinus bradycardia requires no specific treatment or only temporary rate support. Sinus bradycardia is common in patients with acute inferior or posterior MI and can be exacerbated by vagal activation induced by pain or the use of drugs such as morphine. Ischemia of the SA nodal artery probably occurs in acute coronary syndromes more typically with involvement with the right coronary artery, and even with infarction, the effect on SA node function most often is transient.

1	Sinus bradycardia is a prominent feature of carotid sinus hypersensitivity and neurally mediated hypotension associated with vasovagal syncope that responds to pacemaker therapy. Carotid hypersensitivity with recurrent syncope or presyncope associated 1469 with a predominant cardioinhibitory component responds to pacemaker implantation. Several randomized trials have investigated the efficacy of permanent pacing in patients with drug-refractory vasovagal syncope, with mixed results. Although initial trials suggested that patients undergoing pacemaker implantation have fewer recurrences and a longer time to recurrence of symptoms, at least one follow-up study did not confirm these results.

1	Nomenclature and Complications The main therapeutic intervention in SA node dysfunction is permanent pacing. Since the first implementation of permanent pacing in the 1950s, many advances in technology have resulted in miniaturization, increased longevity of pulse generators, improvement in leads, and increased functionality. To better understand pacemaker therapy for bradycardias, it is important to be familiar with the fundamentals of pacemaking. Pacemaker modes and function are named using a five-letter code.

1	The first letter indicates the chamber(s) that is paced (O, none; A, atrium; V, ventricle; D, dual; S, single), the second is the chamber(s) in which sensing occurs (O, none; A, atrium; V, ventricle; D, dual; S, single), the third is the response to a sensed event (O, none; I, inhibition; T, triggered; D, inhibition + triggered), the fourth refers to the programmability or rate response (R, rate responsive), and the fifth refers to the existence of antitachycardia functions if present (O, none; P, antitachycardia pacing; S, shock; D, pace + shock). Almost all modern pacemakers are multiprogrammable and have the capability for rate responsiveness using one of several rate sensors: activity or motion, minute ventilation, or QT interval. The most commonly programmed modes of implanted singleand dual-chamber pacemakers are VVIR and DDDR, respectively, although multiple modes can be programmed in modern pacemakers.

1	Although pacemakers are highly reliable, they are subject to a number of complications related to implantation and electronic function. In adults, permanent pacemakers are most commonly implanted with access to the heart by way of the subclavian–superior vena cava venous system. Rare, but possible, acute complications of transvenous pacemaker implantation include infection, hematoma, pneumothorax, cardiac perforation, diaphragmatic/phrenic nerve stimulation, and lead dislodgment. Limitations of chronic pacemaker therapy include infection, erosion, lead failure, and abnormalities resulting from inappropriate programming or interaction with the patient’s native electrical cardiac function. Rotation of the pacemaker pulse generator in its subcutaneous pocket, either intentionally or inadvertently, often referred to as “twiddler’s syndrome,” can wrap the leads around the generator and produce dislodgment with failure to sense or pace the heart. The small size and light weight of

1	inadvertently, often referred to as “twiddler’s syndrome,” can wrap the leads around the generator and produce dislodgment with failure to sense or pace the heart. The small size and light weight of contemporary pacemakers make this a rare complication.

1	Complications stemming from chronic cardiac pacing also result from disturbances in atrioventricular synchrony and/or left ventricular mechanical synchrony. Pacing modes that interrupt or fail to restore atrioventricular synchrony may lead to a constellation of signs and symptoms, collectively referred to as pacemaker syndrome, that include neck pulsation, fatigue, palpitations, cough, confusion, exertional dyspnea, dizziness, syncope, elevation in jugular venous pressure, canon A waves, and stigmata of congestive heart failure, including edema, rales, and a third heart sound. Right ventricular apical pacing can induce dyssynchronous activation of the left ventricle, leading to compromised left ventricular systolic function, mitral valve regurgitation, and the previously mentioned stigmata of congestive heart failure. Maintenance of AV synchrony can minimize the sequelae of pacemaker syndrome. Selection of pacing modes that minimize unnecessary ventricular pacing or implantation of a

1	of congestive heart failure. Maintenance of AV synchrony can minimize the sequelae of pacemaker syndrome. Selection of pacing modes that minimize unnecessary ventricular pacing or implantation of a device capable of right and left ventricular pacing (biventricular pacing) can help minimize the deleterious consequences of pacing-induced mechanical dyssynchrony at the ventricular level.

1	The Bradyarrhythmias: Disorders of the Sinoatrial Node 1470 Pacemaker Therapy in SA Node Dysfunction Pacing in SA nodal disease is indicated to alleviate symptoms of bradycardia. Consensus guidelines published by the American Heart Association (AHA)/American College of Cardiology/Heart Rhythm Society (ACC/HRS) outline the indications for the use of pacemakers and categorize them by class based on levels of evidence. Class I conditions are those for which there is evidence or consensus of opinion that therapy is useful and effective. In class II conditions, there is conflicting evidence or a divergence of opinion about the efficacy of a procedure or treatment; in class IIa conditions, the weight of evidence or opinion favors treatment; and in class IIb conditions, efficacy is less well established by the evidence or opinion of experts. In class III conditions, the evidence or weight of opinion indicates that the therapy is not efficacious or useful and may be harmful. Class I

1	well established by the evidence or opinion of experts. In class III conditions, the evidence or weight of opinion indicates that the therapy is not efficacious or useful and may be harmful. Class I indications for pacing in SA node dysfunction include documented symptomatic bradycardia, sinus node dysfunction– associated long-term drug therapy for which there is no alternative, and symptomatic chronotropic incompetence. Class IIa indications include those outlined previously in which sinus node dysfunction is suspected but not documented and for syncope of unexplained origin in the presence of major abnormalities of SA node dysfunction. Mildly symptomatic individuals with heart rates consistently <40 beats/min constitute a class IIb indication for pacing. Pacing is not indicated in patients with SA node dysfunction who do not have symptoms and in those in whom bradycardia is associated with the use of nonessential drugs (Table 274-2). There is some controversy about the mode of

1	patients with SA node dysfunction who do not have symptoms and in those in whom bradycardia is associated with the use of nonessential drugs (Table 274-2). There is some controversy about the mode of pacing that should be employed in SA node disease. A number of randomized, single-blind trials of pacing mode have been performed. There are no trials that demonstrate an improvement in mortality rate with AV synchronous pacing compared with single-chamber pacing in SA node disease. In some of these studies, the incidence of atrial fibrillation and thromboembolic events was reduced with AV synchronous pacing. In trials of patients with dual-chamber pacemakers designed to compare single-chamber with dual-chamber pacing by crossover design, the need for AV synchronous pacing due to pacemaker syndrome was common. Pacing modes that preserve AV synchrony appear to be associated with a reduction in the incidence of atrial fibrillation and improved quality of life. Because of the low but finite

1	was common. Pacing modes that preserve AV synchrony appear to be associated with a reduction in the incidence of atrial fibrillation and improved quality of life. Because of the low but finite 1.

1	SA node dysfunction with symptomatic bradycardia or sinus pause 2. Symptomatic SA node dysfunction as a result of essential long-term drug therapy with no acceptable alternatives 3. 4. Atrial fibrillation with bradycardia and pauses >5 s 1. SA node dysfunction with heart rates <40 beats/min without a clear and consistent relationship between bradycardia and symptoms 2. SA node dysfunction with heart rates <40 beats/min on an essential longterm drug therapy with no acceptable alternatives, without a clear and consistent relationship between bradycardia and symptoms 3. Syncope of unknown origin when major abnormalities of SA node dysfunction are discovered or provoked by electrophysiologic testing 1. Mildly symptomatic patients with waking chronic heart rates <40 beats/min 1. SA node dysfunction in asymptomatic patients, even those with heart rates <40 beats/min 2. SA node dysfunction in which symptoms suggestive of bradycardia are not associated with a slow heart rate 3.

1	SA node dysfunction in which symptoms suggestive of bradycardia are not associated with a slow heart rate 3. SA node dysfunction with symptomatic bradycardia due to nonessential drug therapy Source: Modified from AE Epstein et al: J Am Coll Cardiol 51:e1, 2008 and CM Tracy et al: J Am Coll Cardiol 61:e6, 2013. incidence of AV conduction disease, patients with SA node dysfunction usually undergo dual-chamber pacemaker implantation.

1	Pacemaker Therapy in Carotid Sinus Hypersensitivity and Vasovagal Syncope Carotid sinus hypersensitivity, if accompanied by a significant cardioinhibitory component, responds well to pacing. In this circumstance, pacing is required only intermittently and single-chamber ventricular pacing is often sufficient. The mechanism of vasovagal syncope is incompletely understood but appears to involve activation of cardiac mechanoreceptors with consequent activation of neural centers that mediate vagal activation and withdrawal of sympathetic nervous system tone. Several randomized clinical trials have been performed in patients with drug-refractory vasovagal syncope, with some studies suggesting reduction in the frequency and the time to recurrent syncope in patients who were paced compared with those who were not. A recent follow-up study to one of those initial trials, however, found less convincing results, casting some doubt on the utility of pacing for vagally mediated syncope.

1	The Bradyarrhythmias: Disorders of the Atrioventricular node David D. Spragg, Gordon F. Tomaselli Impulses generated in the sinoatrial (SA) node or in ectopic atrial loci are conducted to the ventricles through the electrically and anatomically complex atrioventricular (AV) node. As described in Chap. 274, the electrophysiologic properties of nodal tissue are distinct from atrial and ventricular myocardium. Cells located in the AV node sit at a relatively higher resting membrane potential than surrounding atrial and ventricular myocytes, exhibit spontaneous depolarization during phase 4 of the action potential, and have slower phase 0 depolarization (mediated by calcium influx in nodal tissue) than that seen in ventricular tissue (mediated by sodium influx).

1	Bradycardia may occur when conduction across the AV node is compromised, resulting in ineffective ventricular rates, with the possibility of attendant symptoms, including fatigue, syncope, and (if subsidiary pacemaker activity is insufficient) even death. It is important to recognize that in the setting of disturbed AV conduction, SA activation and atrial systole may occur at normal or even accelerated rates, while ventricular activation is either slowed or nonexistent. Transient AV conduction block is common in the young and is most likely the result of high vagal tone found in up to 10% of young adults. Acquired and persistent failure of AV conduction is decidedly rare in healthy adult populations, with an estimated incidence of 200 per million population per year. In the setting of myocardial ischemia, aging and fibrosis, or cardiac infiltrative diseases, however, persistent AV block is much more common.

1	As with symptomatic bradycardia arising from SA node dysfunction, permanent pacing is the only reliable therapy for symptoms arising from AV conduction block. Approximately 50% of the 150,000 permanent pacemakers implanted in the United States and 70–80% of those in Europe are implanted for disorders of AV conduction.

1	The AV conduction axis is structurally complex, involving the atria and ventricles as well as the AV node. Unlike the SA node, the AV node is a subendocardial structure originating in the transitional zone, which is composed of aggregates of cells in the posterior-inferior right atrium. Superior, medial, and posterior transitional atrionodal bundles converge on the compact AV node. The compact AV node (~1 × 3 × 5 mm) is situated at the apex of the triangle of Koch, which is defined by the coronary sinus ostium posteriorly, the septal tricuspid valve annulus anteriorly, and the tendon of Todaro superiorly. The compact AV node continues as the penetrating AV bundle where it immediately traverses the central fibrous body and is in close proximity to the aortic, mitral, and tricuspid valve annuli; thus, it is subject to injury in the setting of valvular heart disease or its surgical treatment. The penetrating AV bundle continues through the annulus fibrosis and emerges along the

1	valve annuli; thus, it is subject to injury in the setting of valvular heart disease or its surgical treatment. The penetrating AV bundle continues through the annulus fibrosis and emerges along the ventricular septum adjacent to the membranous septum as the bundle of His. The right bundle branch (RBB) emerges from the distal AV bundle in a band that traverses the right ventricle (moderator band). In contrast, the left bundle branch (LBB) is a broad subendocardial sheet of tissue on the septal left ventricle. The Purkinje fiber network emerges from the RBB and LBB and extensively ramifies on the endocardial surfaces of the right and left ventricles, respectively.

1	The blood supply to the penetrating AV bundle is from the AV nodal artery and first septal perforator of the left anterior descending coronary artery. The bundle branches also have a dual blood supply from the septal perforators of the left anterior descending coronary artery and branches of the posterior descending coronary artery. The AV node is highly innervated with postganglionic sympathetic and parasympathetic nerves. The bundle of His and distal conducting system are minimally influenced by autonomic tone.

1	The cells that constitute the AV node complex are heterogeneous with a range of action potential profiles. In the transitional zones, the cells have an electrical phenotype between those of atrial myocytes and cells of the compact node (see Fig. 274-1). Atrionodal transitional connections may exhibit decremental conduction, defined as slowing of conduction with increasingly rapid rates of stimulation. Fast and slow AV nodal pathways have been described, but it is controversial whether these two types of pathway are anatomically distinct or represent functional heterogeneities in different regions of the AV nodal complex. Myocytes that constitute the compact node are depolarized (resting membrane potential ~–60 mV) and exhibit action potentials with low amplitudes, slow upstrokes of phase 0 (<10 V/s), and phase 4 diastolic depolarization; high-input resistance; and relative insensitivity to external [K+]. The action potential phenotype is explained by the complement of ionic currents

1	0 (<10 V/s), and phase 4 diastolic depolarization; high-input resistance; and relative insensitivity to external [K+]. The action potential phenotype is explained by the complement of ionic currents expressed. AV nodal cells lack a robust inward rectifier potassium current (IK1) and fast sodium current (INa); L-type calcium current (ICa-L) is responsible for phase 0; and phase 4 depolarization reflects the composite activity of the depolarizing currents—funny current (I ), I , T-type calcium current (I ), and sodium calcium exchanger current (INCX)—and the repolarizing currents—delayed rectifier (I ) and acetylcholine-gated (I ) potassium currents. Electrical coupling between cells in the AV node is tenuous due to the relatively sparse expression of gap junction channels (predominantly connexin-40) and increased extracellular volume.

1	The His bundle and the bundle branches are insulated from ventricular myocardium. The most rapid conduction in the heart is observed in these tissues. The action potentials exhibit very rapid upstrokes (phase 0), prolonged plateaus (phase 2), and modest automaticity (phase 4 depolarization). Gap junctions, composed largely of connexin-40, are abundant, but bundles are poorly connected transversely to ventricular myocardium.

1	Conduction block from the atrium to the ventricle can occur for a variety of reasons in a number of clinical situations, and AV conduction block may be classified in a number of ways. The etiologies may be functional or structural, in part analogous to extrinsic and intrinsic causes of SA nodal dysfunction. The block may be classified by its severity from first to third degree or complete AV block or by the location of block within the AV conduction system. Table 275-1 summarizes the etiologies of AV conduction block. Those that are functional (autonomic, metabolic/endocrine, and drug-related) tend to be reversible. Most other etiologies produce structural changes, typically fibrosis, in segments of the AV conduction axis that are generally permanent. Heightened vagal tone during sleep or in well-conditioned individuals can be associated with all grades of AV block. Carotid sinus hypersensitivity, vasovagal syncope, and cough and micturition syncope may be associated with SA node

1	or in well-conditioned individuals can be associated with all grades of AV block. Carotid sinus hypersensitivity, vasovagal syncope, and cough and micturition syncope may be associated with SA node slowing and AV conduction block. Transient metabolic and endocrinologic disturbances as well as a number of pharmacologic agents also may produce reversible AV conduction block.

1	Congenital heart disease Facioscapulohumeral MD, OMIM #158900 (4q35) Maternal SLE Emery-Dreifuss MD, OMIM Kearns-Sayre syndrome, OMIM #310300 (Xq28) #530000 Progressive familial heart block, Myotonic dystrophy type IA OMIM #113900 (3p21) Type 1, OMIM #160900 Progressive familial heart block, (19q13.2-13.3) type IB, OMIM #604559 (19q13.32) Type 2, OMIM #602668 Progressive familial heart block, (3q13.3-q24) type II, OMIM %140400 Abbreviations: MCTD, mixed connective tissue disease; MI, myocardial infarction; OMIM, Online Mendelian Inheritance in Man (database; designations: #, phenotypic description, molecular basis known; %, phenotypic description); SLE, systemic lupus erythematosus.

1	Several infectious diseases have a predilection for the conducting system. Lyme disease may involve the heart in up to 50% of cases; 10% of patients with Lyme carditis develop AV conduction block, which is generally reversible but may require temporary pacing support. Chagas’ disease, which is common in Latin America, and syphilis may produce more persistent AV conduction disturbances. Some autoimmune and infiltrative diseases may produce AV conduction block, including systemic lupus erythematosus (SLE), rheumatoid arthritis, mixed connective tissue disease, scleroderma, amyloidosis (primary and secondary), sarcoidosis, and hemochromatosis; rare malignancies also may impair AV conduction.

1	Idiopathic progressive fibrosis of the conduction system is one of the more common and degenerative causes of AV conduction block. Aging is associated with degenerative changes in the summit of the ventricular septum, central fibrous body, and aortic and mitral annuli and has been described as “sclerosis of the left cardiac skeleton.” The process typically begins in the fourth decade of life and may be accelerated by atherosclerosis, hypertension, and diabetes mellitus. Accelerated forms The Bradyarrhythmias: Disorders of the Atrioventricular Node

1	The Bradyarrhythmias: Disorders of the Atrioventricular Node FIGURE 275-1 First-degree AV block with slowing of conduction in the AV node as indicated by the prolonged atrial-to-His bundle electro-gram (AH) interval, in this case 157 ms. The His bundle-to-earliest ventricular activation on the surface ECG (HV) interval is normal. The normal HV interval suggests normal conduction below the AV node to the ventricle. I and V1 are surface ECG leads, and HIS is the recording of the endocavitary electrogram at the His bundle position. A, H, and V are labels for the atrial, His bundle, and right ventricular electrograms, respec tively. of progressive familial heart block have been identified in families with mutations in the cardiac sodium channel gene (SCN5A) and other loci that have been mapped to chromosomes 1 and 19.

1	AV conduction block has been associated with heritable neuromuscular diseases, including the nucleotide repeat disease myotonic dystrophy, the mitochondrial myopathy Kearns-Sayre syndrome (Chap. 462e), and several of the monogenic muscular dystrophies. Congenital AV block may be observed in complex congenital cardiac anomalies (Chap. 282), such as transposition of the great arteries, ostium primum atrial septal defects (ASDs), ventricular septal defects (VSDs), endocardial cushion defects, and some single-ventricle defects. Congenital AV block in the setting of a structurally normal heart has been seen in children born to mothers with SLE. Iatrogenic AV block may occur during mitral or aortic valve surgery, rarely in the setting of thoracic radiation, and as a consequence of catheter ablation. AV block is a decidedly rare complication of the surgical repair of VSDs or ASDs but may complicate repairs of transposition of the great arteries.

1	Coronary artery disease may produce transient or persistent AV block. In the setting of coronary spasm, ischemia, particularly in the right coronary artery distribution, may produce transient AV block. In acute myocardial infarction (MI), AV block transiently develops in 10–25% of patients; most commonly, this is first-or second-degree AV block, but complete heart block (CHB) may also occur. Second-degree and higher-grade AV block tends to occur more often in inferior than in anterior acute MI; however, the level of block in inferior MI tends to be in the AV node with more stable, narrow escape rhythms. In contrast, acute anterior MI is associated with block in the distal AV nodal complex, His bundle, or bundle branches and results in wide complex, unstable escape rhythms and a worse prognosis with high mortality rates.

1	AV conduction block typically is diagnosed electrocardiographically, which characterizes the severity of the conduction disturbance and allows one to draw inferences about the location of the block. AV conduction block manifests as slow conduction in its mildest forms and failure to conduct, either intermittent or persistently, in more severe varieties. First-degree AV block (PR interval >200 ms) is a slowing of conduction through the AV junction (Fig. 275-1). The site of delay is typically in the AV node but may be in the atria, bundle of His, or His-Purkinje system. A wide QRS is suggestive of delay in the distal conduction system, whereas a narrow QRS suggests delay in the AV node proper or, less commonly, in the bundle of His. In second-degree AV block there is an intermittent failure of electrical impulse conduction from atrium to ventricle. Second-degree AV block is subclassified as Mobitz type I (Wenckebach) or Mobitz type II. The periodic failure of conduction in Mobitz type I

1	of electrical impulse conduction from atrium to ventricle. Second-degree AV block is subclassified as Mobitz type I (Wenckebach) or Mobitz type II. The periodic failure of conduction in Mobitz type I block is characterized by a progressively lengthening PR interval, shortening of the RR interval, and a pause that is less than two times the immediately preceding RR interval on the electrocardiogram (ECG). The ECG complex after the pause exhibits a shorter PR interval than that immediately preceding the pause (Fig. 275-2). This ECG pattern most often arises because of decremental conduction of electrical impulses in the AV node.

1	0.40 FIGURE 275-2 Mobitz type I second-degree AV block. The PR interval prolongs before the pause, as shown in the ladder diagram. The ECG pattern results from slowing of conduction in the AV node. FIGURE 275-3 Paroxysmal AV block. Multiple nonconducted P waves after a period of sinus bradycardia with a normal PR interval. This implies significant conduction system disease, requiring permanent pacemaker implantation.

1	It is important to distinguish type I from type II second-degree AV nodal block because the latter has more serious prognostic implications. Type II second-degree AV block is characterized by intermittent failure of conduction of the P wave without changes in the preceding PR or RR intervals. When AV block is 2:1, it may be difficult to distinguish type I from type II block. Type II second-degree AV block typically occurs in the distal or infra-His conduction system, is often associated with intraventricular conduction delays (e.g., bundle branch block), and is more likely to proceed to higher grades of AV block than is type I second-degree AV block. Second-degree AV block (particularly type II) may be associated with a series of nonconducted P waves, referred to as paroxysmal AV block(Fig. 275-3), and implies significant conduction system disease and is an indication for permanent pacing. Complete failure of conduction from atrium to ventricle is referred to as complete or

1	block(Fig. 275-3), and implies significant conduction system disease and is an indication for permanent pacing. Complete failure of conduction from atrium to ventricle is referred to as complete or third-degree AV block. AV block that is intermediate between second degree and third degree is referred to as high-grade AV block and, as with CHB, implies advanced AV conduction system disease. In both cases, the block is most often distal to the AV node, and the duration of the QRS complex can be helpful in determining the level of the block. In the absence of a preexisting bundle branch block, a wide QRS escape rhythm (Fig. 275-4B) implies a block in the distal His or bundle branches; in contrast, a narrow QRS rhythm implies a block in the AV node or proximal His and an escape rhythm originating in the AV junction (Fig. 275-4A). Narrow QRS escape rhythms are typically faster and more stable than wide QRS escape rhythms and originate more proximally in the AV conduction system.

1	Diagnostic testing in the evaluation of AV block is aimed at determining the level of conduction block, particularly in asymptomatic patients, since the prognosis and therapy depend on whether the block is in or below the AV node. Vagal maneuvers, carotid sinus massage, exercise, and administration of drugs such as atropine and isoproterenol may be diagnostically informative. Owing to the differences in the innervation of the AV node and infranodal conduction system, vagal stimulation and carotid sinus massage slow conduction in the AV node but have less of an effect on infranodal tissue and may even improve conduction due to a reduced rate of activation of distal tissues. Conversely, atropine, isoproterenol, and exercise improve conduction through the AV node and impair infranodal conduction. In patients with congenital CHB and a narrow QRS complex, exercise typically increases heart rate; by contrast, those with acquired CHB, particularly with wide QRS, do not respond to exercise

1	In patients with congenital CHB and a narrow QRS complex, exercise typically increases heart rate; by contrast, those with acquired CHB, particularly with wide QRS, do not respond to exercise with an increase in heart rate.

1	Additional diagnostic evaluation, including electrophysiologic testing, may be indicated in patients with syncope and suspected high-grade AV block. This is particularly relevant if noninvasive testing does not reveal the cause of syncope or if the patient has structural heart disease with ventricular tachyarrhythmias as a cause of symptoms. Electrophysiologic testing provides more precise information regarding the location of AV conduction block and permits studies of AV conduction under conditions of pharmacologic stress and exercise. Recording of the His bundle electrogram by a catheter positioned at the superior margin of the tricuspid valve annulus provides information about conduction at all levels of the AV conduction axis. A properly recorded His bundle electrogram reveals local atrial activity, the His electrogram, and local ventricular activation; when it is monitored simultaneously with recorded body surface electrocardiographic traces, intraatrial, AV nodal, and infranodal

1	activity, the His electrogram, and local ventricular activation; when it is monitored simultaneously with recorded body surface electrocardiographic traces, intraatrial, AV nodal, and infranodal conduction times can be assessed (Fig. 275-1). The time from the most rapid deflection of the

1	CHAPTER 275 The Bradyarrhythmias: Disorders of the Atrioventricular Node FIGURE 275-4 High-grade AV block.A. Multiple nonconducted P waves with a regular narrow complex QRS escape rhythm probably emanating from the AV junction. B. A wide complex QRS escape and a single premature ventricular contraction. In both cases, there is no consistent temporal relationship between the P waves and QRS complexes.

1	FIGURE 275-5 High-grade AV block below the His. The AH interval is normal and is not changing before the block. Atrial and His bundle electrograms are recorded consistent with block below the distal AV junction. I, II, III, and V1 are surface ECG leads. HISp, HISd, and RVA are the proximal HIS, distal HIS, and right ventricular apical electrical recordings, respectively. A, H, and V represent the atrial, His, and ventricular electro-grams on the His bundle recording, respectively. (Tracing courtesy of Dr. Joseph Marine; with permission.) atrial electrogram in the His bundle recording to the His electrogram (AH interval) represents conduction through the AV node and is normally <130 ms. The time from the His electrogram to the earliest onset of the QRS on the surface ECG (HV interval) represents the conduction time through the His-Purkinje system and is normally ≤55 ms.

1	Rate stress produced by pacing can unveil abnormal AV conduction. Mobitz I second-degree AV block at short atrial paced cycle lengths is a normal response. However, when it occurs at atrial cycle lengths >500 ms (<120 beats/min) in the absence of high vagal tone, it is abnormal. Typically, type I second-degree AV block is associated with prolongation of the AH interval, representing conduction slowing and block in the AV node. AH prolongation occasionally is due to the effect of drugs (beta blockers, calcium channel blockers, digitalis) or increased vagal tone. Atropine can be used to reverse high vagal tone; however, if AH prolongation and AV block at long pacing cycle lengths persists, intrinsic AV node disease is likely. Type II second-degree block is typically infranodal, often in the His-Purkinje system. Block below the node with prolongation of the HV interval or a His bundle electrogram with no ventricular activation (Fig. 275-5) is abnormal unless it is elicited at fast pacing

1	His-Purkinje system. Block below the node with prolongation of the HV interval or a His bundle electrogram with no ventricular activation (Fig. 275-5) is abnormal unless it is elicited at fast pacing rates or short coupling intervals with extra stimulation. It is often difficult to determine the type of second-degree AV block when 2:1 conduction is present; however, the finding of a His bundle electrogram after every atrial electrogram indicates that block is occurring in the distal conduction system.

1	Intracardiac recording at electrophysiologic study that reveals prolongation of conduction through the His-Purkinje system (i.e., long HV interval) is associated with an increased risk of progression to higher grades of block and is generally an indication for pacing. In the setting of bundle branch block, the HV interval may reveal the condition of the unblocked bundle and the prognosis for developing more advanced AV conduction block. Prolongation of the HV interval in patients with asymptomatic bundle branch block is associated with an increased risk of developing higher-grade AV block. The risk increases with greater prolongation of the HV interval such that in patients with an HV interval >100 ms, the annual incidence of complete AV block approaches 10%, indicating a need for pacing. In patients with acquired CHB, even if intermittent, there is little role for electrophysiologic testing, and pacemaker implantation is almost always indicated.

1	TREATMEnT ManageMent of aV conduction Block

1	Temporary or permanent artificial pacing is the most reliable treatment for patients with symptomatic AV conduction system disease. However, exclusion of reversible causes of AV block and the need for temporary heart rate support based on the hemodynamic condition of the patient are essential considerations in each patient. Correction of electrolyte derangements and ischemia, inhibition of excessive vagal tone, and withholding of drugs with AV nodal blocking properties may increase the heart rate. Adjunctive pharmacologic treatment with atropine or isoproterenol may be useful if the block is in the AV node. Since most pharmacologic treatment may take some time to initiate and become effective, temporary pacing may be necessary. The most expeditious technique is the use of transcutaneous pacing, where pacing patches are placed anteriorly over the cardiac apex (cathode) and posteriorly between the spine and the scapula or above the right nipple (anode). Acutely, transcutaneous pacing is

1	where pacing patches are placed anteriorly over the cardiac apex (cathode) and posteriorly between the spine and the scapula or above the right nipple (anode). Acutely, transcutaneous pacing is highly effective, but its duration is limited by patient discomfort and longer-term failure to capture the ventricle owing to changes in lead impedance. If a patient requires more 1. Third-degree or high-grade AV block at any anatomic level associated with: a.

1	b. c. Periods of asystole >3 s or any escape rate <40 beats/min while awake, or an escape rhythm originating below the AV node d. Postoperative AV block not expected to resolve e. Catheter ablation of the AV junction f. Neuromuscular diseases such as myotonic dystrophy, Kearns-Sayre syndrome, Erb dystrophy, and peroneal muscular atrophy, regardless of the presence of symptoms 2. Second-degree AV block with symptomatic bradycardia 3. Type II second-degree AV block with a wide QRS complex with or without symptoms 4. Exercise-induced second-or third-degree AV block in the absence of ischemia 5. Atrial fibrillation with bradycardia and pauses >5 s 1. Asymptomatic third-degree AV block regardless of level 2. Asymptomatic type II second-degree AV block with a narrow QRS complex 3. Asymptomatic type II second-degree AV block with block within or below the His at electrophysiologic study 4. Firstor second-degree AV block with symptoms similar to pacemaker syndrome 1.

1	Asymptomatic type II second-degree AV block with block within or below the His at electrophysiologic study 4. Firstor second-degree AV block with symptoms similar to pacemaker syndrome 1. AV block in the setting of drug use/toxicity, when the block is expected to recur even with drug discontinuation 2. Neuromuscular diseases such as myotonic dystrophy, Kearns-Sayre syndrome, Erb dystrophy, and peroneal muscular atrophy with any degree of AV block regardless of the presence of symptoms 1. 2. Asymptomatic type I second-degree AV block at the AV node level 3. AV block that is expected to resolve or is unlikely to recur (Lyme disease, drug toxicity) Source: Modified from AE Epstein et al: J Am Coll Cardiol 51:e1, 2008.

1	AV block that is expected to resolve or is unlikely to recur (Lyme disease, drug toxicity) Source: Modified from AE Epstein et al: J Am Coll Cardiol 51:e1, 2008. The Bradyarrhythmias: Disorders of the Atrioventricular Node than a few minutes of pacemaker support, transvenous temporary pacing should be instituted. Temporary pacing leads can be placed from the jugular or subclavian venous system and advanced to the right ventricle, permitting stable temporary pacing for many days, if necessary. In most circumstances, in the absence of prompt resolution, conduction block distal to the AV node requires permanent pacemaking.

1	There are no randomized trials that evaluate the efficacy of pacing in patients with AV block, as there are no reliable therapeutic alternatives for AV block and untreated high-grade AV block is potentially lethal. The consensus guidelines for pacing in acquired AV conduction block in adults provide a general outline for situations in which pacing is indicated (Table 275-2). Pacemaker implantation should be performed in any patient with symptomatic bradycardia and irreversible second-or third-degree AV block, regardless of the cause or level of block in the conducting system. Symptoms may include those directly related to bradycardia and low cardiac output or to worsening heart failure, angina, or intolerance to an essential medication. Pacing in patients with asymptomatic AV block should be individualized; situations in which pacing should be considered are patients with acquired CHB, particularly in the setting of cardiac enlargement; left ventricular dysfunction; and waking heart

1	be individualized; situations in which pacing should be considered are patients with acquired CHB, particularly in the setting of cardiac enlargement; left ventricular dysfunction; and waking heart rates ≤40 beats/min. Patients who have asymptomatic second-degree AV block of either type should be considered for pacing if the block is demonstrated to be intraor infra-His or is associated with a wide QRS complex. Pacing may be indicated in asymptomatic patients in special circumstances, in patients with profound first-degree AV block and left ventricular dysfunction in whom a shorter AV interval produces hemodynamic improvement, and in the setting of milder forms of AV conduction delay (first-degree AV block, intraventricular conduction delay) in patients with neuromuscular diseases that have a predilection for the conduction system, such as myotonic dystrophy and other muscular dystrophies, and Kearns-Sayre syndrome.

1	AV block in acute MI is often transient, particularly in inferior infarction. The circumstances in which pacing is indicated in acute MI are persistent second-or third-degree AV block, particularly if symptomatic, and transient second-or third-degree AV block associated with bundle branch block (Table 275-3). Pacing is generally not indicated in the setting of transient AV block in the absence of intra-ventricular conduction delays or in the presence of fascicular block 1. Persistent second-degree AV block in the His-Purkinje system with bilateral bundle branch block or third-degree block within or below the His after AMI 2. Transient advanced (second-or third-degree) infranodal AV block and associated bundle branch block. If the site of block is uncertain, an electrophysiologic study may be necessary 3. 1. Persistent secondor third-degree AV block at the AV node level 1. Transient AV block in the absence of intraventricular conduction defects 2.

1	1. Persistent secondor third-degree AV block at the AV node level 1. Transient AV block in the absence of intraventricular conduction defects 2. Transient AV block in the presence of isolated left anterior fascicular block 3. Acquired left anterior fascicular block in the absence of AV block 4. Persistent first-degree AV block in the presence of bundle branch block that is old or age-indeterminate Source: Modified from AE Epstein et al: J Am Coll Cardiol 51:e1, 2008. 1. 2. 3. 1. Syncope not demonstrated to be due to AV block when other likely causes (e.g., ventricular tachycardia) have been excluded 2. Incidental finding at electrophysiologic study of a markedly prolonged HV interval (>100 ms) in asymptomatic patients 3.

1	Incidental finding at electrophysiologic study of a markedly prolonged HV interval (>100 ms) in asymptomatic patients 3. Incidental finding at electrophysiologic study of pacing-induced infra-His block that is not physiologic 1. Neuromuscular diseases such as myotonic dystrophy, Kearns-Sayre syndrome, Erb dystrophy, and peroneal muscular atrophy with any degree of fascicular block regardless of the presence of symptoms, because there may be unpredictable progression of AV conduction disease 1. 2. Fascicular block with first-degree AV block without symptoms Source: Modified from AE Epstein et al: J Am Coll Cardiol 51:e1, 2008. or first-degree AV block that develops in the setting of preexisting bundle branch block. Fascicular blocks that develop in acute MI in the absence of other forms of AV block also do not require pacing (Table 275-3 and Table 275-4).

1	Distal forms of AV conduction block may require pacemaker implantation in certain clinical settings. Patients with bifascicular or trifascicular block and symptoms, particularly syncope that is not attributable to other causes, should undergo pacemaker implantation. Pacemaking is indicated in asymptomatic patients with bifascicular or trifascicular block who experience intermittent third-degree, type II second-degree AV block or alternating bundle branch block. In patients with fascicular block who are undergoing electro-physiologic study, a markedly prolonged HV interval or block below the His at long cycle lengths also may constitute an indication for permanent pacing. Patients with fascicular block and the neuromuscular diseases previously described should also undergo pacemaker implantation (Table 275-4).

1	In general, a pacing mode that maintains AV synchrony reduces complications of pacing such as pacemaker syndrome and pacemaker-mediated tachycardia. This is particularly true in younger patients; the importance of dual-chamber pacing in the elderly, however, is not well established. Several studies have failed to demonstrate a difference in mortality rate in older patients with AV block treated with a single-(VVI) compared with a dual-(DDD) chamber pacing mode. In some of the studies that randomized pacing mode, the risk of chronic atrial fibrillation and stroke risk decreased with physiologic pacing. In patients with sinus rhythm and AV block, the very modest increase in risk with dual-chamber pacemaker implantation appears to be justified to avoid the possible complications of single-chamber pacing. Gregory F. Michaud, William G. Stevenson

1	Gregory F. Michaud, William G. Stevenson Supraventricular tachyarrhythmias originate from or are dependent on conduction through the atrium or atrioventricular (AV) node to the ventricles. Most produce narrow QRS-complex tachycardia (QRS duration <120 ms) characteristic of ventricular activation over the Purkinje system. Conduction block in the left or right bundle branch or activation of the ventricles from an accessory pathway produces a wide QRS complex during supraventricular tachycardia that must be distinguished from ventricular tachycardia (Chap. 277). Supraventricular tachyarrhythmia may be divided into physiologic sinus tachycardia and pathologic tachycardia (Table 276-1). The prognosis and treatment vary considerably depending on the mechanism and underlying heart disease.

1	Supraventricular tachycardia can be of brief duration, termed nonsustained, or can be sustained such that an intervention, such as cardioversion or drug administration, is required for termination. Episodes that occur with sudden onset and termination are referred to as paroxysmal. Paroxysmal supraventricular tachycardia (PSVT) refers to a family of tachycardias including AV node reentry, AV reentry using an accessory pathway, and atrial tachycardia. Symptoms of supraventricular arrhythmia vary depending on the rate, duration, associated heart disease, and comorbidities and include palpitations, chest pain, dyspnea, diminished exertional capacity, and occasionally syncope. Rarely, a supraventricular arrhythmia precipitates cardiac arrest in patients with the Wolff-Parkinson-White syndrome or severe heart disease, such as hypertrophic cardiomyopathy.

1	Diagnosis requires obtaining an electrocardiogram (ECG) at the time of symptoms. For transient arrhythmia, ambulatory ECG recording is warranted (see Table 277-1). Exercise testing is useful for assessing exercise-related symptoms. Occasionally an invasive electro-physiology study is warranted to provoke the arrhythmia with pacing, confirm the mechanism, and often, perform catheter ablation.

1	Physiologic Sinus Tachycardia The sinus node is comprised of a group of cells dispersed within the superior aspect of the thick ridge of muscle known as the crista terminalis where the posterior smooth atrial wall derived from the sinus venosus meets the trabeculated anterior portion of the right atrium (Fig. 276-1). Sinus p waves are characterized by a frontal plane axis directed inferiorly and leftward, with positive p waves in leads II, III, and aVF; a negative p wave in aVR; and an initially positive biphasic p wave in V1. Normal sinus rhythm has a rate of 60–100 beats/min. Sinus tachycardia (>100 beats/min) typically occurs in response to sympathetic stimulation and vagal withdrawal, whereby the rate of spontaneous depolarization of the sinus node increases and the focus of earliest activation within the node typically shifts more leftward and closer to the superior septal aspect of the crista terminalis, thus producing taller p waves in the inferior limb leads when compared to

1	activation within the node typically shifts more leftward and closer to the superior septal aspect of the crista terminalis, thus producing taller p waves in the inferior limb leads when compared to normal sinus rhythm.

1	Sinus tachycardia is considered physiologic when it is an appropriate response to exercise, stress, or illness. Sinus tachycardia can be difficult to distinguish from focal atrial tachycardia (see below) that originates from a focus near the sinus node. A causative factor (such as exertion) and a gradual increase and decrease in rate favors sinus tachycardia, whereas an abrupt onset and offset favor atrial tachycardia. The distinction can be difficult and occasionally requires extended ECG monitoring or even invasive electrophysiology study. Treatment for physiologic sinus tachycardia is aimed at the underlying condition (Table 276-2). Nonphysiologic Sinus Tachycardia Inappropriate sinus tachycardia is an uncommon condition in which the sinus rate increases spontaneously at rest or out of proportion to physiologic stress or exertion. I. Physiologic sinus tachycardia Defining feature: normal sinus mechanism precipitated by exertion, stress, concurrent illness (Table 276-2) II. A.

1	I. Physiologic sinus tachycardia Defining feature: normal sinus mechanism precipitated by exertion, stress, concurrent illness (Table 276-2) II. A. Tachycardia originating from the atrium Defining feature: tachycardia may continue despite beats that fail to conduct to the ventricles, indicating that the AV node is not participating in the tachycardia circuit 1. Inappropriate sinus tachycardia Defining feature: tachycardia from the normal sinus node area that occurs without an identifiable precipitating factor as a result of dysfunctional autonomic regulation 2. Focal atrial tachycardia Defining feature: Regular atrial tachycardia with defined p wave; may be sustained, nonsustained, paroxysmal, or incessant. Frequent sites of origin occur along the valve annuli of left or right atrium, pulmonary veins, coronary sinus musculature, superior vena cava 3. Atrial flutter – macroreentrant atrial tachycardia

1	Defining feature: organized reentry creates organized atrial activity, commonly seen as sawtooth flutter waves at rates typically faster than 200 beats/min a. i. Right atrial reentry parallel to the tricuspid annulus and dependent on conduction through the isthmus between the inferior vena cava and tricuspid annulus 1. Counterclockwise (as viewed from the ventricular aspect) 2. b. i. Usually due to reentry in left or right atrium associated with scars usually from prior surgery or catheter ablation for atrial fibrillation, but may be idiopathic 4. Atrial fibrillation Defining feature: chaotic rapid atrial electrical activity with variable ventricular rate; the most common sustained cardiac arrhythmia in older adults 5. Multifocal atrial tachycardia Defining feature: multiple discrete p waves often seen in patients with pulmonary disease during acute exacerbations of pulmonary insufficiency B. AV nodal reentry tachycardia

1	Defining feature: multiple discrete p waves often seen in patients with pulmonary disease during acute exacerbations of pulmonary insufficiency B. AV nodal reentry tachycardia Defining feature: paroxsymal regular tachycardia with P waves visible at the end of the QRS complex or not visible at all; the most common paroxysmal sustained tachycardia in healthy young adults; more common in women C. Tachycardias associated with accessory atrioventricular pathways a. Orthodromic AV reentry tachycardia Defining feature: paroxysmal sustained tachycardia similar to AV nodal reentry; during sinus rhythm, evidence of ventricular preexcitation may be present (Wolff-Parkinson-White syndrome) or absent (concealed accessory pathway) b. Preexcited tachycardia Defining feature: wide QRS tachycardia with QRS morphology similar to VT 1. 2.

1	2. Atrial fibrillation with preexcitation – irregular wide complex, or intermittently wide complex tachycardia, some with dangerously rapid rates faster than 250/min 3. Atrial tachycardia or flutter with preexcitation Abbreviations: AV, atrioventricular; VT, ventricular tachycardia.

1	Affected individuals are often women in the third or fourth decade of life. Fatigue, dizziness, and even syncope may accompany palpitations, which can be disabling. Additional symptoms of chest pain, headaches, and gastrointestinal upset are common. It must be distinguished from appropriate sinus tachycardia and from focal atrial tachycardia, as discussed above. Misdiagnosis of physiologic sinus tachycardia with an anxiety disorder is common. Therapy is often ineffective or poorly tolerated. Careful titration of beta blockers and/ or calcium channel blockers may reduce symptoms. Clonidine and 1477 serotonin reuptake inhibitors have also been used. Ivabradine, a drug that blocks the If current causing sinus node depolarization, is promising but is not approved for use in the United States. Catheter ablation of the sinus node has been used, but long-term control of symptoms is usually poor, and it often leaves young individuals with a permanent pacemaker.

1	When symptomatic sinus tachycardia occurs with postural hypo-tension, the syndrome is called postural orthostatic tachycardia syndrome (POTS). Symptoms are often similar to those in patients with inappropriate sinus tachycardia. POTS is sometimes due to autonomic dysfunction following a viral illness and may resolve spontaneously over 3–12 months. Volume expansion with salt supplementation, oral fludrocortisone, compression stockings, and the α-agonist midodrine, often in combination, can be helpful. Exercise training has also been purported to improve symptoms.

1	Focal Atrial Tachycardia Focal atrial tachycardia (AT) can be due to abnormal automaticity, triggered automaticity, or a small reentry circuit confined to the atrium or atrial tissue extending into a pulmonary vein, the coronary sinus, or vena cava. It can be sustained, nonsustained, paroxysmal, or incessant. Focal AT accounts for approximately 10% of PSVT referred for catheter ablation. Nonsustained AT is commonly observed on 24-h ambulatory ECG recordings, and the prevalence increases with age. Tachycardia can occur in the absence of structural heart disease or can be associated with any form of heart disease that affects the atrium. Sympathetic stimulation is a promoting factor such that AT can be a sign of underlying illness. AT with AV block can occur in digitalis toxicity. Symptoms are similar to other supraventricular tachycardias (SVTs). Incessant AT can cause tachycardia-induced cardiomyopathy.

1	AT typically presents as an SVT either with 1:1 AV conduction or with AV block that can be Wenckebach type conduction or fixed (e.g., 2:1 or 3:1) block. Because it is not dependent on AV nodal conduction, AT will not terminate with AV block, and the atrial rate will not be affected, which distinguishes AT from most AV nodal–dependent SVTs, such as AV nodal reentry and AV reentry using an accessory pathway (see below). An accelerated warm-up phase after initiation or cool-down phase prior to termination also favors AT rather than AV nodal–dependent SVT. P waves are often discrete, with an intervening isoelectric segment, in contrast to atrial flutter and macroreentrant AT (see below). When 1:1 conduction to the ventricles is present, the arrhythmia can resemble sinus tachycardia typically with a P-R interval shorter than the R-P interval (Fig. 276-2). It can be distinguished from sinus tachycardia by the p-wave morphology, which usually differs from sinus p waves depending on the

1	with a P-R interval shorter than the R-P interval (Fig. 276-2). It can be distinguished from sinus tachycardia by the p-wave morphology, which usually differs from sinus p waves depending on the location of the focus. Focal AT tends to originate in areas of complex atrial anatomy, such as the crista terminalis, valve annuli, atrial septum, and atrial muscle extending along cardiac thoracic veins (superior vena cava, coronary sinus, and pulmonary veins) (Fig. 276-3), and the location can often be estimated by the P-wave morphology. AT from the right atrium has a positive P-wave morphology in lead I and biphasic P-wave morphology in lead V1. AT from the atrial septum will frequently have a narrower P-wave duration than sinus rhythm. AT from the left atrium will usually have a monophasic, positive P wave in lead V1. AT that originates from superior atrial locations, such as the superior vena cava or superior pulmonary veins, will be positive in the inferior limb leads II, III, and aVF,

1	P wave in lead V1. AT that originates from superior atrial locations, such as the superior vena cava or superior pulmonary veins, will be positive in the inferior limb leads II, III, and aVF, whereas AT from a more inferior location, such as the ostium of the coronary sinus, will inscribe negative P waves in these same leads. When the focus is in the superior aspect of the crista terminalis, close to the sinus node, however, the p wave will resemble that of sinus tachycardia. Abrupt onset and offset then favor AT rather than sinus tachycardia. Depending on the atrial rate, the P wave may fall on top of the t wave or, during 2:1 conduction, may fall coincident with the QRS. Maneuvers that increase AV block, such as carotid sinus massage, Valsalva maneuver, or administration of AV nodal–blocking agents, such as adenosine, are useful to create AV block that will expose the p wave (Fig. 276-4).

1	II, III, aVF FIGURE 276-1 Right atrial anatomy pertinent to normal sinus rhythm and supraventricular tachycardia. A. Typical P-wave morphology during normal sinus rhythm based on standard 12-lead electrocardiogram. There is a positive P wave in leads II, III, and aVF; biphasic, initially positive P wave in V1; and negative P wave in aVR. B. Right atrial anatomy seen from a right lateral perspective with the lateral wall opened to view the septum. AVN, atrioventricular node; CS Os, coronary sinus ostium; FO, fossa ovalis; IVC, inferior vena cava; SVC, superior vena cava; TVA, FIGURE 276-3 Location of focal atrial tachycardia focus estimated by P-wave morphology. LAA, left atrial appendage; LIV, left inferior pulmonary vein; LSV, left superior pulmonary vein; RAA, right atrial appendage; RIV, right inferior pulmonary vein; RSV, right superior pulmonary vein; SVC, superior vena cava.

1	FIGURE 276-2 Common mechanisms underlying paroxysmal supraventricular tachycardia along with typical R-P relationships. A. Schematic showing a four-chamber view of the heart with atrioventricular node in green and an accessory pathway between the left atrium and left ventricle in yellow. Atrial tachycardia (AT; red circuit) is confined completely to atrial tissue. Atrioventricular nodal reentry tachycardia (AVNRT; blue circuit) uses atrioventricular (AV) nodal and perinodal atrial tissue. Atrioventricular reentry tachycardia (AVRT; black circuit) uses atrial and ventricular tissue, accessory pathway, AV node, and specialized conduction fibers (His-Purkinje) as part of the reentry circuit. B. Typical relation of the p wave to QRS, commonly described as the R-P to P-R relationships for the different tachycardia mechanisms. tricuspid valve annulus. 1. 2. Acute illness with fever, infection, pain 3. Hypovolemia, anemia 4. 5. 6.

1	tricuspid valve annulus. 1. 2. Acute illness with fever, infection, pain 3. Hypovolemia, anemia 4. 5. 6. Drugs that have sympathomimetic, vagolytic, or vasodilator properties, e.g., albuterol, theophylline, tricyclic antidepressants, nifedipine, hydralazine 7. II, III, aVF FIGURE 276-4 Atrial tachycardia (AT) with 1:1 and 2:1 atrioventricular (AV) conduction. Arrows indicate p waves. A. AT with 1:1 AV relationship and R-P > P-R. B. Same AT with 2:1 AV relationship after AV nodal–blocking agent administered. (Adapted from F Marchlinski: The tachyarrhythmias. In Longo DL et al [eds]: Harrison’s Principles of Internal Medicine, 18th ed. New York, McGraw-Hill, 2012, pp 1878–1900.)

1	(Adapted from F Marchlinski: The tachyarrhythmias. In Longo DL et al [eds]: Harrison’s Principles of Internal Medicine, 18th ed. New York, McGraw-Hill, 2012, pp 1878–1900.) Acute management of sudden-onset, sustained AT is the same as for PSVT (see below), but the response to pharmacologic therapy is variable, likely depending on the mechanism. For AT due to reentry, administration of adenosine or vagal maneuvers may transiently increase AV block without terminating tachycardia. Some ATs terminate with a sufficient dose of adenosine, consistent with triggered activity as the mechanism. Cardioversion can be effective in some, but fails in others, suggesting automaticity as the mechanism. Beta blockers and calcium channel blockers may slow the ventricular rate by increasing AV block, which can improve tolerance of the arrhythmias. Potential precipitating factors and intercurrent illness should be sought and corrected. Underlying heart disease should be considered and excluded.

1	For patients with recurrent episodes, beta blockers, the calcium channel blockers diltiazem or verapamil, and the antiarrhythmic drugs flecainide, propafenone, disopyramide, sotalol, and amiodarone can be effective, but potential toxicities and adverse effects often warrant avoiding these agents (Tables 276-3, 276-4, and 276-5). Catheter ablation targeting the AT focus is effective in more than 80% of patients and is recommended for 1479 recurrent symptomatic AT when drugs fail or are not desired or for incessant AT causing tachycardia-induced cardiomyopathy.

1	Atrioventricular Nodal Reentry Tachycardia AV nodal reentry tachycardia (AVNRT) is the most common form of PSVT, representing approximately 60% of cases referred for catheter ablation. It most commonly manifests in the second to fourth decades of life, often in women. It is often well tolerated, but rapid tachycardia, particularly in the elderly, may cause angina, pulmonary edema, hypotension, or syncope. It is not usually associated with structural heart disease.

1	The mechanism is reentry involving the AV node and likely the perinodal atrium, made possible by the existence of multiple pathways for conduction from the atrium into the AV node (Fig. 276-5). In the most common form, a slowly conducting AV nodal pathway extends from the compact AV node near the bundle of His, inferiorly along the tricuspid annulus, adjacent to the coronary sinus os. The reentry wavefront propagates up this slow pathway to the compact AV node and then exits from the fast pathway at the top of the AV node. The path back to the slow pathway to complete the circuit is not defined. The conduction time from the compact AV node region to the atrium is similar to that from the compact node to the His bundle and ventricles, such that atrial activation occurs at about the same time as ventricular activation. The p wave is therefore inscribed during, slightly before, or slightly after the QRS and can be difficult to discern. Often the P wave is seen at the end of the QRS

1	time as ventricular activation. The p wave is therefore inscribed during, slightly before, or slightly after the QRS and can be difficult to discern. Often the P wave is seen at the end of the QRS complex as a pseudo-r′ in lead V1 and pseudo-S waves in leads II, III, and aVF (Fig. 276-5A). The rate can vary with sympathetic tone. Simultaneous atrial and ventricular contraction results in atrial contraction against a closed tricuspid valve that produces cannon a waves visible in the jugular venous pulse and that the patient often perceives as a fluttering sensation in the neck. Elevated venous pressures may also lead to release of natriuretic peptides that cause posttachycardia diuresis. Less frequently, the AV nodal reentry circuit revolves in the opposite direction and gives rise to a tachycardia with an R-P interval longer than the P-R interval, similar to AT. The p wave will have the morphology noted above, and in contrast to ATs, maneuvers or medications that produce AV block

1	a tachycardia with an R-P interval longer than the P-R interval, similar to AT. The p wave will have the morphology noted above, and in contrast to ATs, maneuvers or medications that produce AV block terminate the arrhythmia.

1	Acute treatment is the same as for PSVT (discussed below). Whether ongoing therapy is warranted depends on the severity of symptoms and frequency of episodes. Reassurance and instruction as to performance of the Valsalva maneuver to terminate episodes are sufficient for many patients. Administration of an oral beta blocker, verapamil, or diltiazem at the onset of an episode has been used to

1	Adenosine 6–18 mg (rapid bolus) N/A Terminate reentrant SVT — involving AV node Amiodarone 15 mg/min for 10 min, 1 mg/ 0.5–1 mg/min AF, AFL, SVT, VT/VF III min for 6 h Digoxin 0.25 mg q2h until 1 mg total 0.125–0.25 mg/d AF/AFL rate control — Diltiazem 0.25 mg/kg over 3–5 min 5–15 mg/h SVT, AF/AFL rate control IV (max 20 mg) Esmolol 500 μg/kg over 1 min 50 μg/kg per min AF/AFL rate control II Ibutilide 1 mg over 10 min if over 60 kg N/A Terminate AF/AFL III Lidocaine 1–3 mg/kg at 20–50 mg/min 1–4 mg/min VT IB Metoprolol 5 mg over 3–5 min × 3 doses 1.25–5 mg q6h SVT, AF rate control; exercise-II induced VT; long QT Procainamide 15 mg/kg over 60 min 1–4 mg/min Convert/prevent AF/VT IA Quinidine 6–10 mg/kg at 0.3–0.5 mg/kg N/A Convert/prevent AF/VT IA per min Verapamil 5–10 mg over 3–5 min 2.5–10 mg/h SVT, AF rate control IV aClassification of antiarrhythmic drugs: class I—agents that primarily block inward sodium current; class IA agents also prolong action potential duration; class

1	2.5–10 mg/h SVT, AF rate control IV aClassification of antiarrhythmic drugs: class I—agents that primarily block inward sodium current; class IA agents also prolong action potential duration; class II—antisympathetic agents; class III—agents that primarily prolong action potential duration; class IV—calcium channel–blocking agents.

1	Abbreviations: AF, atrial fibrillation; AFL, atrial flutter; AV, atrioventricular; SVT, supraventricular tachycardia; VF, ventricular fibrillation; VT, ventricular tachycardia. Digoxin 0.125–0.25 qd 38–48 Renal AF rate control — Diltiazem 30–60 q6h 3–4.5 Hepatic AF rate control/SVT IV Disopyramide 100–300 q6–8h 4–10 Renal 50%/hepatic AF/SVT prevention Ia Dofetilide 0.125–0.5 q12h 10 Renal AF prevention Nadolol 40–240 per d 10–24 Renal Same as metoprolol II Verapamil 80–120 q6–8h 4.5–12 Hepatic/renal AF rate control/RVOT VT IV Idiopathic LV VT aClassification of antiarrhythmic drugs: class I—agents that primarily block inward sodium current; class II—antisympathetic agents; class III—agents that primarily prolong action potential duration; class IV—calcium channel-blocking agents. bAmiodarone and dronedarone both are grouped in class III, but both also have class I, II, and IV properties.

1	Abbreviations: AF, atrial fibrillation; LV, left ventricular; RVOT, right ventricular outflow tract; SVT, supraventricular tachycardia; VT, ventricular tachycardia.

1	facilitate termination. Chronic therapy with these medications or frequently encountered as an incessant tachycardia in children, often flecainide is an option if prophylactic therapy is needed. Catheter abla-in the perioperative period of surgery for congenital heart disease. tion of the slow AV nodal pathway is recommended for patients with It presents as a narrow QRS tachycardia, often with ventriculoatrial recurrent or severe episodes or when drug therapy is ineffective, not (VA) block, such that AV dissociation is present. JET can occur as tolerated, or not desired by the patient. Catheter ablation is curative in a manifestation of increased adrenergic tone and may be seen after over 95% of patients. The major risk is heart block requiring perma-administration of isoproterenol. It may also occur for a short period of nent pacemaker implantation, which occurs in less than 1% of patients. time after ablation for AVNRT.

1	Accelerated junctional rhythm is a junctional automatic rhythm Junctional Tachycardia Junctional ectopic tachycardia (JET) is due between 50 and 100 beats/min. Initiation may occur with gradual to automaticity within the AV node. It is rare in adults and more acceleration in rate, suggesting an automatic focus, or after a premature Sotalol Long QT and torsades des pointes Hypotension, bronchospasm from β-blocking effect Abbreviations: AF, atrial fibrillation; AV, atrioventricular; VT, ventricular tachycardia. extension: Compact AV node: Fast pathway FIGURE 276-5 Atrioventricular (AV) node reentry. A. Leads II and V1 are shown. P waves are visible at the end of the QRS complex and are negative in lead II, and may give the impression of S waves in the inferior limb leads II, III, and aVF and an R’ in lead V1. B. Stylized version of the AV nodal reentry circuit within the triangle of Koch (Fig. 276-1) that involves AV node and its extensions along with perinodal atrial tissue.

1	ventricular contraction, suggesting a focus of triggered automaticity. VA conduction is usually present, with p-wave morphology and timing such that it resembles slow AVNRT. Accessory pathways (APs) occur in 1 in 1500–2000 people and are associated with a variety of arrhythmias including narrow-complex PSVT, wide-complex tachycardias, and, rarely, sudden death. Most patients have structurally normal hearts, but APs are associated with Ebstein’s anomaly of the tricuspid valve and forms of hypertrophic cardiomyopathy including PRKAG2 mutations, Danon’s disease, and Fabry’s disease.

1	APs are abnormal connections that allow conduction between the atrium and ventricles across the AV ring (Fig. 276-6). They are present from birth and are due to failure of complete partitioning of atrium and ventricle by the fibrous AV rings. They occur across either an AV valve annulus or the septum, most frequently between the left atrium and free wall of the left ventricle, followed by posteroseptal, right free wall, and anteroseptal locations. If the AP conducts from atrium to ventricle (antegrade) with a shorter conduction time than the AV node and His bundle, then the ventricles are preexcited during sinus rhythm, and the ECG shows a short P-R interval (<0.12 s), slurred initial portion of the QRS (delta wave), and prolonged QRS duration produced by slow conduction through direct activation of ventricular myocardium over the AP (Fig. 276-6A). The morphology of the QRS and delta wave is determined by the AP location (Fig. 276-7) and the degree of fusion between the excitation

1	activation of ventricular myocardium over the AP (Fig. 276-6A). The morphology of the QRS and delta wave is determined by the AP location (Fig. 276-7) and the degree of fusion between the excitation wavefronts from conduction over the AV node and conduction over the AP. Right-sided pathways preexcite the right ventricle, producing a left bundle branch block–like configuration in lead V1, and often show marked preexcitation because of relatively close proximity of the AP to the sinus node (Fig. 276-7). Left-sided pathways preexcite the left ventricle and may produce a right bundle branch–like configuration in lead V1 and a negative delta wave in aVL, indicating initial depolarization of the lateral portion of the left ventricle that can mimic q waves of lateral wall infarction (Fig. 276-7). Preexcitation due to an AP at the diaphragmatic surface of the heart, typically in the paraseptal region, produces delta waves that are negative in leads III and aVF, mimicking the q waves of

1	276-7). Preexcitation due to an AP at the diaphragmatic surface of the heart, typically in the paraseptal region, produces delta waves that are negative in leads III and aVF, mimicking the q waves of inferior wall infarction (Fig. 276-7). Preexcitation can be intermittent and disappear during exercise as conduction over the AV node accelerates and takes over ventricular activation completely.

1	Wolff-Parkinson-White (WPW) syndrome is defined as a preexcited QRS during sinus rhythm and episodes of PSVT. There are a number of variations of APs, which may not cause preexcitation and/ or arrhythmias. Concealed APs allow only retrograde conduction, from ventricle to atrium, so no preexcitation is present during sinus rhythm, but SVT can occur. Fasciculoventricular connections between the His bundle and ventricular septum produce preexcitation but do not cause arrhythmia, nor do fibers such as atrio-Hisian connections, probably because the circuit is too short to promote reentry. Atriofascicular pathways, also known as Mahaim fibers, probably represent a duplicate AV node and His-Purkinje system that connect the right atrium to fascicles of the right bundle branch and conduct slowly only in the anterograde direction.

1	AV Reentry Tachycardia The most common tachycardia caused by an AP is the PSVT designated orthodromic AV reentry. The circulating reentry wavefront propagates from the atrium anterogradely over the AV node and His-Purkinje system to the ventricles and then reenters the atria via retrograde conduction over the AP (Fig. 276-6B). The QRS is narrow or may have typical right or left bundle branch block, but without preexcitation during tachycardia. Because excitation through the normal AV conduction system and AP are necessary, AV or VA block results in tachycardia termination. During sinus rhythm, preexcitation is seen if the pathway also allows anterograde conduction (Fig. 276-6A). Most commonly, during tachycardia the R-P interval is shorter than the P-R interval and can resemble AVNRT (Fig. 276-1). Unlike typical AVNRT, P-wave timing is never simultaneous with a narrow QRS complex because the ventricles must be activated before the reentry wavefront reaches the AP and conducts back to

1	276-1). Unlike typical AVNRT, P-wave timing is never simultaneous with a narrow QRS complex because the ventricles must be activated before the reentry wavefront reaches the AP and conducts back to the atrium. The morphology of the P wave is determined by the pathway location, but can be difficult to assess because it is usually inscribed during the ST segment. The p wave in posteroseptal APs is negative in leads II, III, and aVF, similar to that of AV nodal reentry, but P-wave morphology differs from AV nodal reentry for pathways in other locations (Fig. 276-7).

1	Occasionally, an AP conducts extremely slowly in the retrograde direction, which results in tachycardia with a long R-P interval, similar to most ATs. These pathways are usually located in the septal region and have negative p waves in leads II, III, and aVF. Slow conduction facilitates reentry, often leading to nearly incessant tachycardia, known as paroxysmal junctional reciprocating tachycardia (PJRT). Tachycardia-induced cardiomyopathy can occur. Without an invasive electrophysiology study, it may be difficult to distinguish this form of orthodromic AV reentry from atypical AV nodal reentry or AT. FIGURE 276-6 Wolff-Parkinson-White (WPW) syndrome. A. A 12-lead electrocardiogram in sinus rhythm (SR) of a patient with WPW demonstrating short P-R interval, delta waves, and widened QRS complex. This patient had an anteroseptal location of the AP.

1	B. Orthodromic AV reentry in a patient with WPW syndrome using a posteroseptal AP. Note the P waves in the ST segment (arrows) seen in lead III and normal appearance of QRS complex. C. Three most common rhythms associated with WPW syndrome: sinus rhythm demonstrating antegrade conduction over the AP and AV node; orthodromic AVRT using retrograde conduction over the AP and antegrade conduction over the AV node; and antidromic AVRT using retrograde conduction over the AV node and antegrade conduction over the AP. AP, accessory pathway; AV, atrioventricular; AVRT, atrioventricular reentry tachycardia; WPW, Wolff-Parkinson-White.

1	Preexcited Tachycardias Preexcitated tachycardia occurs when the ventricles are activated by antegrade conduction over the AP (Fig. 276-6C). The most common is antidromic AV reentry in which activation propagates from atrium to ventricle via the AP and then conducts retrogradely to the atria via the His-Purkinje system and the AV node (or rarely a second AP). The wide QRS complex is produced entirely via ventricular excitation over the AP because there is no contribution of ventricular activation over more rapidly conducting specialized His-Purkinje fibers. This tachycardia is often indistinguishable from monomorphic ventricular tachycardia. The presence of preexcitation in sinus rhythm suggests the diagnosis.

1	FIGURE 276-7 Potential locations for accessory pathways in patients with Wolff-Parkinson-White Syndrome and typical QRS appearance of delta waves that can mimic underlying structural heart disease such as myocardial infraction of bundle branch block. AV, aor-tic valve; MV, mitral valve; PV, pulmonary valve; TV, tricuspid valve.

1	Preexcitated tachycardia also occurs if an AP allows antegrade conduction to the ventricles during AT, atrial flutter, atrial fibrillation (Fig. 276-8), or AV nodal reentry. Atrial fibrillation and atrial flutter are potentially life threatening if the AP allows very rapid repetitive conduction. Approximately 25% of APs causing preexcitation allow minimum R-to-R intervals of less than 250 ms during atrial fibrillation are therefore associated with a risk of inducing ventricular fibrillation and sudden death. Preexcited atrial fibrillation presents as a wide-complex, very irregular rhythm. During atrial fibrillation, the ventricular rate is determined by the conduction properties of the AP and AV node. The QRS complex can appear quite bizarre and change on a beat-to-beat basis due to the variability in the degree of fusion from activation over the AV node and AP, or all beats may be due to conduction over the AP (Fig. 276-8). Ventricular activation from the Purkinje system may

1	to the variability in the degree of fusion from activation over the AV node and AP, or all beats may be due to conduction over the AP (Fig. 276-8). Ventricular activation from the Purkinje system may depolarize the ventricular end of the AP and prevent 1:1 atrial wavefront conduction over the AP. Slowing AV nodal conduction can thereby facilitate AP conduction and dangerously accelerate the ventricular rate. Administration of AV nodal–blocking agents including oral or intravenous verapamil, diltiazem, beta blockers, intravenous adenosine, and intravenous amiodarone are contraindicated. Preexcited tachycardias should be treated with electrical cardioversion or intravenous procainamide or ibutilide, which may terminate or slow the ventricular rate.

1	Management of Patients with Accessory Pathways Acute management of orthodromic AV reentry is discussed below for PSVT. Patients with WPW syndrome may have wide-complex tachycardia due to antidromic AV reentry, orthodromic AV with bundle branch block, or a preexcited tachycardia, and treatment depends on the underlying rhythm. Initial patient evaluation should include assessment for aggravating factors, including intercurrent illness and factors that increase sympathetic tone. Examination should focus on excluding underlying heart disease. An echocardiogram is reasonable to exclude Ebstein’s anomaly and hypertrophic cardiomyopathy.

1	Patients with preexcitation who have symptoms of arrhythmia are at risk for developing atrial fibrillation and sudden death if they have an AP with high-risk properties. The risk of cardiac arrest is in the range of 2 per 1000 patients in adults but is likely greater in children. An invasive electrophysiology study is warranted to determine if the AP is high enough risk to warrant potentially curative catheter ablation. For patients with concealed APs or known low-risk APs causing orthodromic AV reentry, chronic therapy is guided by symptoms and frequency of events. Vagal maneuvers may terminate episodes, as may a dose of beta blocker, verapamil, or diltiazem taken at the onset of an episode. Chronic therapy with these agents or flecainide can reduce the frequency of episodes in some patients. Catheter ablation is warranted for recurrent arrhythmias when drugs are ineffective, not tolerated, or not desired by the patient or if the AP is considered high risk (Fig. 276-8). Efficacy is

1	Catheter ablation is warranted for recurrent arrhythmias when drugs are ineffective, not tolerated, or not desired by the patient or if the AP is considered high risk (Fig. 276-8). Efficacy is in the range of 95% depending on the location of the AP. Serious complications occur in fewer than 3% of patients, but can include AV block, cardiac tamponade, thromboemboli, coronary artery injury, and vascular access complications. Mortality occurs in less than 1 in 1000 patients.

1	Adults who have preexcitation but no arrhythmia symptoms have a risk of sudden death estimated to be 1 per 1000 patient-years. Electrophysiology study is usually advised for people in occupations for which an arrhythmia occurrence would place them or others at risk, such as police, military, and pilots, or for individuals who desire evaluation for risk. Routine follow-up without therapy is reasonable in others. Children are at greater risk of sudden death, approximately 2 per 1000 patient-years. FIGURE 276-8 Preexcited atrial fibrillation (AF) due to conduction over a left free wall accessory pathway (AP). The electrocardiogram shows rapid irregular QRS complexes that represent fusion between conduction over the atrioventricular node and left free wall AP. Shortest R-R intervals between preexcited QRS complexes of less than 250 ms, as in this case, indicate a risk of sudden death with this arrhythmia.

1	Acute management of narrow QRS PSVT is guided by the clinical presentation. Continuous ECG monitoring should be implemented and a 12-lead ECG should always be obtained when possible. In the presence of hypotension with unconsciousness or respiratory distress, QRS-synchronous direct current cardioversion is warranted, but this is rarely needed, because intravenous adenosine works promptly in most situations (see below). For stable individuals, initial therapy takes advantage of the fact that most PSVTs are dependent on AV nodal conduction (AV nodal reentry or orthodromic AV reentry) and therefore likely to respond to sympatholytic and vagotonic maneuvers and drugs (Fig. 276-9). As these are administered, the ECG should be continuously recorded, because the response can Hemodynamically stable regular, Narrow QRS tachyca Hemodynamically stable regular, narrow QRS tachycardia

1	Hemodynamically stable regular, Narrow QRS tachyca Hemodynamically stable regular, narrow QRS tachycardia Vagal maneuversi.v. adenosine i.v. verapamil/diltiazemNo terminationVagal maneuvers IV adenosine IV verapamil/diltiazem No termination i.v. Ibutilide + AV nodal blocking agenti.v. procainamide + AV nodal blocking agent cardioversion IV ibutilide + AV nodal–blocking agent IV procainamide + AV nodal–blocking agent Cardioversion FIGURE 276-9 Treatment algorithm for patients presenting with hemodynamically stable paroxysmal supraventricular tachycardia. AV, atrioventricular.

1	1484 establish the diagnosis. AV block with only transient slowing of tachycardia may expose ongoing p waves, indicating AT or atrial flutter as the mechanism. Carotid sinus massage is reasonable provided the risk of carotid vascular disease is low, as indicated by absence of carotid bruits and no prior history of stroke. A Valsalva maneuver should be attempted in cooperative individuals, and if effective, the patient can be taught to perform this maneuver as needed. If vagal maneuvers fail or cannot be performed, intravenous adenosine will terminate the vast majority of PSVT by transiently blocking conduction in the AV node. Adenosine may produce transient chest pain, dyspnea, and anxiety. It is contraindicated in patients with prior cardiac transplantation due to potential hypersensitivity. It can theoretically aggravate bronchospasm. Adenosine precipitates atrial fibrillation, which is usually brief, in up to 15% of patients, so it should be used cautiously in patients with WPW

1	It can theoretically aggravate bronchospasm. Adenosine precipitates atrial fibrillation, which is usually brief, in up to 15% of patients, so it should be used cautiously in patients with WPW syndrome in whom AF may produce hemodynamic instability. Intravenous beta blockers and calcium channel blockers (verapamil or diltiazem) are also effective but may cause hypotension before and after arrhythmia termination and have a longer duration of action. These agents can also be given orally and can be taken by the patient on an as-needed basis to slow ventricular rate and facilitate termination by Valsalva maneuver. The differential diagnosis of wide-complex tachycardia includes ventricular tachycardia (Chap. 277), PSVT with bundle branch block aberrancy, and preexcited tachycardia (see above). In general, these should be managed as ventricular tachycardia until proven otherwise. If the tachycardia is regular and the patient is stable, a trial of intravenous adenosine is reasonable. Very

1	In general, these should be managed as ventricular tachycardia until proven otherwise. If the tachycardia is regular and the patient is stable, a trial of intravenous adenosine is reasonable. Very irregular wide-complex tachycardia should be managed with cardioversion, intravenous procainamide, or ibutilide, which presumes preexcited atrial fibrillation or flutter (see above). If the diagnosis of PSVT with aberrancy is unequivocal, as may be the case in patients with prior episodes, treatment for PSVT is reasonable. In all cases, continuous ECG monitoring should be implemented, and emergency cardioversion and defibrillation should be available.

1	Macrorrentrant atrial tachycardia is due to a large reentry circuit, often associated with areas of scar in the atria. Common or typical right atrial flutter is due to a circuit that revolves around the tricuspid valve annulus, bounded anteriorly by the annulus and posteriorly by functional conduction block in the crista terminalis. The wavefront passes through an isthmus between the inferior vena cava and the tricuspid valve annulus, known as the sub-Eustachian or cavotricuspid isthmus, where it is susceptible to interruption by catheter ablation. Thus, common atrial flutter is cavotricuspid isthmus-dependent atrial flutter. This circuit most commonly revolves in a counterclockwise direction (as viewed looking toward the tricuspid annulus from the ventricular aspect), which produces the characteristic negative sawtooth flutter waves in leads II, III, and aVF and positive P waves in lead V1 (Fig. 276-10). When the direction is reversed, clockwise rotation produces the opposite P-wave

1	characteristic negative sawtooth flutter waves in leads II, III, and aVF and positive P waves in lead V1 (Fig. 276-10). When the direction is reversed, clockwise rotation produces the opposite P-wave vector in those leads. The atrial rate is typically 240–300 beats/min but may be slower in the presence of atrial disease or antiarrhythmic drugs. It often conducts to the ventricles with 2:1 AV block, creating a regular tachycardia at 150 beats/min, with p waves that may be difficult to discern. Maneuvers that increase AV nodal block will typically expose flutter waves, allowing diagnosis.

1	Common right atrial flutter often occurs in association with atrial fibrillation and with atrial scar from senescence or prior cardiac surgery. Some patients with atrial fibrillation that is treated with an antiarrhythmic drug, particularly flecainide, propafenone, or amiodarone, will present with atrial flutter rather than fibrillation. Macroreentrant ATs that are not dependent on conduction through the cavotricuspid isthmus are referred to as atypical atrial flutters. They can occur in either atrium and are usually associated with areas of scar. Left atrial flutter and perimitral left atrial flutter are commonly seen after extensive left atrial ablation for atrial fibrillation or atrial surgery. The clinical presentation is similar to common atrial flutter, but with different P-wave morphologies. They can be difficult to distinguish from focal AT, and in most cases, the mechanism can only be confirmed by an electrophysiology study.

1	FIGURE 276-10 A. Common right atrial flutter, also known as cavotricuspid isthmus flutter, showing positive P waves in lead V1 and negative “sawtooth” pattern in lead II typical of counterclockwise rotation relative to the tricuspid valve annulus. (Adapted from F Marchlinski: The tachyarrhythmias. In Longo DL et al [eds]: Harrison's Principles of Internal Medicine, 18th ed. New York, McGraw-Hill, 2012, pp 1878–1900.) B. A right atrial map of common counterclockwise flutter is shown. Colors indicate activation time, progressing from red to yellow to green, blue, and purple. The reentry path parallels the tricuspid annulus.

1	Initial management of atrial flutter is similar to that for atrial fibrillation, discussed in more detail below. Electrical cardioversion is warranted for hemodynamic instability or severe symptoms. Otherwise, rate control can be achieved with administration of AV nodal–blocking agents, but this is often more difficult than for atrial fibrillation. The risk of thromboembolic events is felt to be similar to that associated with atrial fibrillation. Anticoagulation is warranted prior to conversion for episodes more than 48 h in duration and chronically for patients at increased risk of thromboembolic stroke based on the CHA2DS2-VASc scoring system (Table 276-6).

1	For a first episode of atrial flutter, conversion to sinus rhythm with no antiarrhythmic drug therapy is reasonable. For recurrent episodes, antiarrhythmic drug therapy with sotalol, dofetilide, disopyramide, and amiodarone may be considered, but more than 70% of patients experience recurrences. For recurrent episodes of common atrial flutter, catheter ablation of the cavotricuspid isthmus abolishes the arrhythmia in over 90% of patients with a low risk of complications that are largely related to vascular access and infrequent heart block. Approximately 50% of patients presenting with atrial flutter develop atrial fibrillation within the next 5 years.

1	Multifocal AT (MAT) is characterized by at least three distinct P-wave morphologies with rates typically between 100 and 150 beats/min. Unlike atrial fibrillation, there are clear isoelectric intervals between P waves (Fig. 276-11). The mechanism is likely triggered automaticity from multiple atrial foci. It is usually encountered in patients with chronic pulmonary disease and acute illness. Therapy for MAT is directed at treating the underlying disease and correcting any metabolic abnormalities. Electrical cardioversion has no effect. The calcium channel blockers verapamil or diltiazem may slow the atrial and ventricular rate. Patients with severe pulmonary disease often do not tolerate beta blocker therapy. MAT may respond to amiodarone, but long-term therapy with this agent is usually avoided due to its toxicities, particularly pulmonary fibrosis.

1	Atrial fibrillation (AF) is characterized by disorganized, rapid, and irregular atrial activation with loss of atrial contraction and with an irregular ventricular rate that is determined by AV nodal conduction (Fig. 276-12). In an untreated patient, the ventricular rate also tends aModified from GY Lip et al: Lancet 379:648, 2012. bU.S. Food and Drug Administration recommended dosing; other regimens are available outside the United States. Abbreviations: CCr, creatinine clearance; Cr, creatinine; INR, international normalized ratio; TIA, transient ischemic attack. FIGURE 276-11 Multifocal atrial tachycardia. Rhythm strip obtained from a patient with severe pulmonary disease during an acute illness. Arrows note three distinct P-wave morphologies. to be rapid and variable, between 120 and 160 beats/min, but in some patients, it may exceed 200 beats/min. Patients with high vagal tone or AV nodal conduction disease may have slow rates.

1	AF is the most common sustained arrhythmia and is a major public health problem. Prevalence increases with age, and more than 95% of AF patients are older than 60 years of age. The prevalence by age 80 is approximately 10%. The lifetime risk of developing AF for individuals 40 years old is approximately 25%. AF is slightly more common in men than women and more common in whites than blacks. Risk factors for developing AF in addition to age include hypertension, diabetes mellitus, cardiac disease, and sleep apnea. AF is a marker for heart disease, the severity of heart disease, and age, and it is therefore difficult to determine the extent to which AF itself contributes to associated increased mortality and morbidity. AF is associated with increased risk of developing heart failure. AF increases the risk of stroke by fivefold and is estimated to be the cause of 25% of strokes. It also increases the risk of dementia.

1	AF is occasionally associated with an acute precipitating factor such as hyperthyroidism, acute alcohol intoxication, or an acute illness including myocardial infarction or pulmonary embolism. AF occurs in up to 30% of patients recovering from cardiac surgery, associated with inflammatory pericarditis.

1	The clinical type of AF suggests the underlying pathophysiology (Fig. 276-12). Paroxysmal AF is defined as episodes that start and stop spontaneously. It is often initiated by small reentrant or rapidly firing foci in sleeves of atrial muscle along the pulmonary veins. Catheter ablation that isolates these foci usually abolishes the AF. Persistent AF has a longer duration, exceeding 7 days, and, in many cases, will continue unless cardioversion is performed. Cardioversion can be followed by prolonged periods of sinus rhythm. Episodes may be initiated by rapidly firing foci, but persistence of the arrhythmia is likely due to single or multiple areas of reentry facilitated by structural and electrophysiologic atrial abnormalities. In patients with long-standing persistent AF (>1 year), significant structural changes are present in the atrium that support reentry and automaticity, making it difficult to restore and maintain sinus rhythm. Some patients progress over years from paroxysmal

1	structural changes are present in the atrium that support reentry and automaticity, making it difficult to restore and maintain sinus rhythm. Some patients progress over years from paroxysmal to persistent AF. Fibrosis that develops with aging and atrial hypertrophy in response to hypertension and other cardiac disease may be an important promoting factor, although electrophysiologic changes to conduction and refractoriness occur as well in response to chronic tachycardia in the atrium.

1	Types of AF Paroxysmal AF Persistent AF requires cardioversion Long-standing persistent or permanent AF Ectopic foci Triggers Electrophysiologic remodeling fibrosis Chronic substrate fibrosis FIGURE 276-12 A rhythm strip of atrial fibrillation (AF) showing no distinct P-wave morphology and irregular ventricular response. Diagram depicts atrial fibrillation types. Paroxysmal AF is initiated by premature beats, as shown in the rhythm strip (arrow) after two sinus beats. Triggering foci are often an important cause of this arrhythmia. Persistent AF is associated with atrial structural and electrophysiologic remod-eling, as well as with triggering foci in many patients. Long-standing persistent AF is associated with greater structural remodeling with atrial fibrosis and electrophysiologic remodeling.

1	Clinical consequences are related to rapid ventricular rates, loss of atrial contribution to ventricular filling, and predisposition to thrombus formation in the left atrial appendage with potential embolization. Presentations vary with the ventricular rate and underlying heart disease and comorbidities. Many patients are asymptomatic. Rapid rates may cause hemodynamic collapse or heart failure exacerbations particularly in patients with impaired cardiac function, hypertrophic cardiomyopathy, and heart failure with preserved systolic function. Exercise intolerance and easy fatigability are common. Occasionally, dizziness or syncope occurs due to pauses when AF terminates to sinus rhythm (Fig. 276-13).

1	Treatment for AF is primarily guided by patients’ symptoms, the hemodynamic effect of AF, the duration of AF if there are persistent risk factors for stroke, and underlying heart disease. Oral anticoagulation in high-risk patients with AF includes vitamin K antagonists or the newer anticoagulants such as thrombin inhibitors (dabigatran) or factor Xa inhibitors (rivaroxaban, apixaban), but not antiplatelet agents (aspirin and clopidogrel), which have substantially less effect.

1	New-onset AF that produces severe hypotension, pulmonary edema, or angina should be electrically cardioverted starting with a QRS synchronous shock of 200 J, ideally after sedation or anesthesia is achieved. Greater shock energy and different electrode placements may be tried if the shock fails to terminate AF. If AF terminates and reinitiates, administration of an antiarrhythmic drug, such as ibutilide, and repeat cardioversion may be considered. If the patient is stable, immediate management involves rate control to alleviate or prevent symptoms, anticoagulation if appropriate, and cardioversion to restore 1487 sinus rhythm if AF is persistent. Anticoagulation strategies for new-onset AF are debated. In the absence of contraindications, it is usually appropriate to initiate systemic anticoagulation with heparin immediately, while evaluation and other therapies are implemented.

1	Cardioversion within 48 h of the onset of AF is common practice in patients who have not been anticoagulated, provided that they are not at high risk for stroke due to a prior history of embolic events, rheumatic mitral stenosis, or hypertrophic cardiomyopathy with marked left atrial enlargement. These patients are usually at risk of recurrence, such that initiation of anticoagulation is considered based on the patient’s individual risk for stroke, commonly assessed from the CHA2DS2-VASc score.

1	If the duration of AF exceeds 48 h or is unknown, there is greater concern for thromboembolism with cardioversion, even in patients considered low risk for stroke. There are two approaches to mitigate the risk related to cardioversion. One option is to anticoagulate continuously for 3 weeks before and a minimum of 4 weeks after cardioversion. A second approach is to start anticoagulation and perform a transesophageal echocardiogram to determine if thrombus is present in the left atrial appendage. If thrombus is absent, cardioversion can be performed and anticoagulation continued for a minimum of 4 weeks because recovery of atrial mechanical function after electrical or pharmacologic cardioversion may be delayed and thrombus can form and embolize days after cardioversion. Some patients may merit ongoing anticoagulation after cardioversion, depending on stroke risk profile.

1	Acute rate control can be achieved with beta blockers and/or the calcium channel blockers verapamil and diltiazem administered either intravenously or orally, as warranted by the urgency of the clinical situation. Digoxin may be added, particularly in heart failure Recorded: 02/24/2013 @ 12:44 AM (CT) 25 mm/sec, 32 mm/mV Continues-> Recorded: 02/24/2013 @ 12:44 AM (CT) 25 mm/sec, 32 mm/mV Continues-> Recorded: 02/24/2013 @ 12:44 AM (CT) 25 mm/sec, 32 mm/mV Continues-> Post trigger 7.2 seconds FIGURE 276-13 A continuous rhythm strip is shown. Atrial fibrillation is present at the top and abruptly terminates in the second tracing, with atrial and ventricular standstill for 7.2 s until resumption of sinus rhythm. The patient experienced syncope.

1	1488 patients, because it does not have negative inotropic effects, particularly if use of AV nodal–blocking agents is limited by poor tolerance or is contraindicated. Its effect is modest but synergistic with the other AV nodal–blocking agents, but it is particularly limited when sympathetic tone is elevated. Typically, the goal of acute rate control is to reduce the ventricular rate to less than 100/min, but the goal must be guided by the clinical situation.

1	For patients who remain in AF chronically, the goal of rate control is to alleviate and prevent symptoms and prevent deterioration of ventricular function from excessive rates. β-Adrenergic blockers, calcium channel blockers, and digoxin are used, sometimes in combination. Rate should be assessed with exertion and medications adjusted accordingly. Exertion-related symptoms are often an indication of inadequate rate control. The initial goal is a resting heart rate of less than 80 beats/min that increases to less than 100 beats/min with light exertion, such as walking. If it is difficult to slow the ventricular rate to that degree, allowing a resting rate of up to 110 beats/min is acceptable provided it does not cause symptoms and ventricular function remains normal. Periodic assessment of ventricular function is warranted because some patients develop tachycardia-induced cardiomyopathy.

1	If adequate rate control in AF is difficult to achieve, further consideration should be given to restoring sinus rhythm. Catheter ablation of the AV junction to create heart block and implantation of a permanent pacemaker reliably achieve rate control without the need for AV nodal agents, but implement life-long permanent pacing. Right ventricular apical pacing induces dyssynchronous ventricular activation that can be symptomatic or depress ventricular function in some patients. Biventricular pacing may be used to minimize the degree of ventricular dyssynchrony.

1	The majority of patients warrant chronic anticoagulation, but selection of therapy should be individualized based on patient profile and risks and benefits of individual agents. Anticoagulation with a vitamin K antagonist is warranted for all patients with AF who have rheumatic mitral stenosis or mechanical heart valves for whom the newer anticoagulants have not been tested. Anticoagulation with a vitamin K antagonist (warfarin) or the newer oral anticoagulants is warranted for patients who have had more than 48 h of AF and are undergoing cardioversion, for patients who have a prior history of stroke, or for patients with a CHA2DS2-VASc score of ≥2, but it may be considered in patients with a risk score of 1. The approach to patients with paroxysmal AF is the same as for persistent AF. It is recognized that many patients who appear to have infrequent AF episodes often have asymptomatic episodes that put them at risk. Absence of AF during periodic monitoring is not sufficient to

1	AF. It is recognized that many patients who appear to have infrequent AF episodes often have asymptomatic episodes that put them at risk. Absence of AF during periodic monitoring is not sufficient to indicate low risk. The role of continuous monitoring with implanted recorders or pacemakers is not yet clear as a guide for anticoagulation in patients with a borderline risk profile. Bleeding is the major risk of anticoagulation. Major bleeding requiring transfusion or in a critical area (e.g., intracranial) occurs in approximately 1% of patients per year. Risk factors for bleeding include age >65–75 years, heart failure, history of anemia, and excessive alcohol or nonsteroidal anti-inflammatory drug use. Patients with coronary stents who require antiplatelet therapy with aspirin and a thienopyridine are at particularly high risk of bleeding.

1	Warfarin reduces the annual risk of stroke by 64% compared to placebo and by 37% compared to antiplatelet therapy. The newer anticoagulants, dabigatran, rivaroxaban, and apixaban, have been found to be noninferior to warfarin in individual trials, and analysis of pooled data suggests superiority to warfarin by small absolute margins of 0.4–0.7% in reduction of mortality, stroke, major bleeding, and intracranial hemorrhage. Warfarin is an inconvenient agent that requires several days to achieve a therapeutic effect (prothrombin time [PT]/international normalized ratio [INR] >2), requires monitoring of PT/INR to adjust dose, and has many drug and food interactions, thus limiting patient compliance. The newer agents are easier to use and achieve reliable anticoagulation promptly without requiring dosage adjustment based on blood tests. Dabigatran, rivaroxaban, and apixaban have renal excretion, cannot be used with severe renal insufficiency, and require dose adjustment for modest renal

1	dosage adjustment based on blood tests. Dabigatran, rivaroxaban, and apixaban have renal excretion, cannot be used with severe renal insufficiency, and require dose adjustment for modest renal impairment, which is of particular concern in the elderly, who are at increased bleeding risk. Excretion can also be influenced by P-glycoprotein inducers and inhibitors. Warfarin anticoagulation can be reversed by administration of fresh frozen plasma and vitamin K. Reversing agents for the newer anticoagulants are lacking (but in development), and bleeding must be managed with supportive care, with the expectation that clotting will improve over 12 h as the anticoagulant is excreted.

1	The antiplatelet agents aspirin and clopidogrel are inferior to warfarin for stroke prevention in AF and do not reduce the risk of bleeding. Clopidogrel combined with aspirin is better than aspirin alone but inferior to warfarin and has greater bleeding risk than aspirin alone. Chronic anticoagulation is contraindicated in some patients due to bleeding risks. Because most atrial thrombi are felt to originate in the left atrial appendage, surgical removal of the appendage, combined with atrial maze surgery, may be considered for patients undergoing surgery, although removal of the appendage has not been unequivocally shown to reduce the risk of thromboembolism. Percutaneous devices that occlude or ligate the left atrial appendage are being studied for safety and efficacy.

1	The decision to administer antiarrhythmic drugs or perform catheter ablation to attempt maintenance of sinus rhythm (commonly referred to as the “rhythm control strategy”) is mainly guided by patient symptoms and preferences regarding the benefits and risks of therapies. In general, patients who maintain sinus rhythm have better survival than those who continue to have AF. This is likely because continued AF is a marker of disease severity. In randomized trials, administration of antiarrhythmic medications to maintain sinus rhythm did not improve survival or symptoms compared to a rate control strategy, and the drug therapy group had more hospitalizations. Disappointing efficacy and toxicities of available antiarrhythmic drugs and patient selection bias may be factors that influenced the results of these trials. The impact of catheter ablation on mortality is not known. A rhythm control strategy is usually selected for patients with symptomatic paroxysmal AF, a first episode of

1	the results of these trials. The impact of catheter ablation on mortality is not known. A rhythm control strategy is usually selected for patients with symptomatic paroxysmal AF, a first episode of symptomatic persistent AF, AF with difficult rate control, and AF that has resulted in depressed ventricular function or that aggravates heart failure. A rhythm control strategy is more likely to be favored in younger patients than in sedentary or elderly patients in whom rate control is usually easily achieved. Even if sinus rhythm is apparently maintained, anticoagulation is recommended according to the CHA2DS2-VASc stroke risk profile because asymptomatic episodes of AF are common. Following a first episode of persistent AF, a strategy using AV nodal–blocking agents, cardioversion, and anticoagulation is reasonable, in addition to addressing possible aggravating factors, including hypertension, heart failure, and sleep apnea. If recurrences are infrequent, periodic cardioversion is

1	anticoagulation is reasonable, in addition to addressing possible aggravating factors, including hypertension, heart failure, and sleep apnea. If recurrences are infrequent, periodic cardioversion is reasonable.

1	Pharmacologic Therapy for Maintaining Sinus Rhythm The goal of pharmacologic therapy is to maintain sinus rhythm or reduce episodes of AF. Drug therapy can be instituted once sinus rhythm has been established or in anticipation of cardioversion. β-Adrenergic blockers and calcium channel blockers help control ventricular rate, improve symptoms, and possess a low-risk profile, but have low efficacy for preventing AF episodes. Risks and side effects of antiarrhythmic drugs are a major consideration in selecting therapy. Class I sodium channel–blocking agents (e.g., flecainide, 1489 propafenone, disopyramide) are options for subjects without sig-ventricular Arrhythmias nificant structural heart disease, but they have negative inotropic

1	Roy M. John, William G. Stevenson and proarrhythmic effects that warrant avoidance in patients with coronary artery disease or heart failure. The class III agents sotalol and dofetilide can be administered to patients with coronary artery disease or structural heart disease but have approximately a 3% risk of inducing excessive QT prolongation and torsades des pointes. Dofetilide should be initiated only in a hospital with ECG monitoring, and many physicians take this approach with sotalol as well. Dronedarone increases mortality in patients with heart failure. All of these agents have modest efficacy in patients with paroxysmal AF, of whom approximately 30–50% will benefit. Amiodarone is more effective, maintaining sinus rhythm in approximately two-thirds of patients. It can be administered to patients with heart failure and coronary artery disease. Over 20% of patients experience toxicities during long-term therapy.

1	Catheter ablation avoids antiarrhythmic drug toxicities but has procedural risks and requires an experienced center. For patients with previously untreated but recurrent paroxysmal AF, catheter ablation has similar efficacy to antiarrhythmic drug therapy and is superior to antiarrhythmic drugs for patients who have recurrent AF despite drug treatment. The procedure involves cardiac catheterization, transatrial septal puncture, and radiofrequency ablation or cryoablation to electrically isolate the regions around the pulmonary veins, abolishing the effect of triggering foci to interact with the left atrial AF substrate. Extensive areas of ablation are required, and gaps in healed ablation areas necessitate a repeat procedure in 20–50% of patients. Sinus rhythm is maintained for more than 1 year after one procedure in approximately 60% of patients and in 70–80% of patients after multiple procedures. Some patients become more responsive to antiarrhythmic drugs.

1	There is a 2–7% risk of major complications, including stroke (0.5–1%), cardiac tamponade (1%), phrenic nerve paralysis, bleeding from femoral access sites, and fluid overload with heart failure, that can emerge 1–3 days after the procedure. It is important to recognize the potential for delayed presentation of some complications. Ablation within the pulmonary veins can lead to pulmonary vein stenosis, presenting weeks to months after the procedure with dyspnea or hemoptysis. Esophageal ulcers can form immediately after the procedure and may rarely lead to a fistula between the left atrium and esophagus (estimated incidence of 0.1%) that presents as endocarditis and stroke 10 days to 3 weeks after the procedure.

1	Catheter ablation is less effective for persistent AF. More extensive ablation is often required, including areas that likely support reentry in regions outside the pulmonary venous antra, but individual strategies are debated. More than one ablation procedure is often required to maintain sinus rhythm. Surgical ablation of AF is typically performed concomitant with cardiac valve or coronary artery surgery and less commonly as a stand-alone procedure; however, for patients with persistent AF, surgical or hybrid procedures may have higher single-procedure efficacy. Risks include sinus node injury requiring pacemaker implantation. Surgical removal of the left atrial appendage may reduce stroke risk, although thrombus can form in the remnant of the appendage or if the appendage is not completely ligated. Portions of this chapter were retained from the work of the previous author, Francis Marchlinski.

1	Portions of this chapter were retained from the work of the previous author, Francis Marchlinski. Arrhythmias that originate in the ventricular myocardium or His-Purkinje system include premature ventricular beats, ventricular tachycardias that can be sustained or nonsustained, and ventricular fibrillation. Arrhythmia may emerge from a focus of myocardial or Purkinje cells capable of automaticity, or triggered automaticity, or from reentry through areas of scar or a diseased Purkinje system. Ventricular arrhythmias are often associated with structural heart disease and are an important cause of sudden death (Chap. 327). They also occur in some structurally normal hearts, in which case they are usually benign. Evaluation and management are guided by the risk of arrhythmic death, which is assessed based on symptoms, type of arrhythmia, and associated underlying heart disease.

1	Ventricular arrhythmias are characterized by their electrocardiographic appearance and duration. Conduction away from the ventricular focus through the ventricular myocardium is slower than activation of the ventricles over the Purkinje system. Hence, the QRS complex during ventricular arrhythmias will be wide, typically >0.12 s. Premature ventricular beats (also referred to as premature ventricular contractions [PVCs]) are single ventricular beats that fall earlier than the next anticipated supraventricular beat (Fig. 277-1). PVCs that originate from the same focus will have the same QRS morphology and are referred to as unifocal (Fig. 277-1A). PVCs that originate from different ventricular sites have different QRS morphologies and are referred to as multifocal (Fig. 277-1B). Two consecutive ventricular beats are ventricular couplets.

1	Ventricular tachycardia (VT) is three or more consecutive beats at a rate faster than 100 beats/min. Three or more consecutive beats at slower rates are designated an idioventricular rhythm (Fig. 277-1C). VT that terminates spontaneously within 30 s is designated nonsustained (Fig. 277-2), whereas sustained VT persists longer than 30 s or is terminated by an active intervention, such as administration of an intravenous medication, external cardioversion, or pacing or a shock from an implanted cardioverter-defibrillator.

1	Monomorphic VT has the same QRS complex from beat to beat, indicating that the activation sequence is the same from beat to beat and that each beat likely originates from the same source (Fig. 277-3A). The initial site of ventricular activation largely determines the sequence of ventricular activation. Therefore, the QRS morphology of PVCs and monomorphic VT provides an indication of the site of origin within the ventricles (Fig. 277-4). The likely origin often suggests whether an arrhythmia is idiopathic or associated with structural disease. Arrhythmias that originate from the right ventricle or septum result in late activation of much of the left ventricle, thereby producing a prominent S wave in V1 referred to as a left bundle branch block–like configuration. Arrhythmias that originate from the free wall of the left ventricle have a prominent positive deflection in V1, thereby producing a right bundle branch block–like morphology in V1. The frontal plane axis of the QRS is also

1	from the free wall of the left ventricle have a prominent positive deflection in V1, thereby producing a right bundle branch block–like morphology in V1. The frontal plane axis of the QRS is also useful. An axis that is directed inferiorly, as indicated by dominant R waves in leads II, III, and AVF, suggests initial activation of the cranial portion of the ventricle, whereas a frontal plane axis that is directed superiorly (dominant S waves in II, III, and AVF) suggests initial activation at the inferior wall.

1	Very rapid monomorphic VT has a sinusoidal appearance, also called ventricular flutter, because it is not possible to distinguish the QRS complex from the T wave (Fig. 277-3B). Relatively slow sinusoidal VTs have a wide QRS indicative of slowed ventricular conduction (Fig. 277-3C). Hyperkalemia, toxicity from excessive effects of drugs that blocks sodium channels (e.g., flecainide, propafenone, or Art. Pr. 1000 ms I

1	Art. Pr. 1000 ms I FIGURE 277-1 Examples of types of premature ventricular contractions (PVCs). A. Unifocal PVCs follow every sinus beat in a bigeminal frequency. Trace shows electrocardiogram lead 1 and arterial pressure (Art. Pr.). Sinus rhythm beats are followed by normal arterial waveform. The arterial pressure following premature beats is attenuated (arrows) and imperceptible to palpation. The pulse in this patient is registered at half the heart rate. B. Multifocal PVCs. The two PVCs shown have different morphologies. C. Example of accelerated idioventricular rhythm. The second QRS is a normally conducted beat. All other QRS complexes on this rhythm strip are ventricular due to accelerated idioventricular rhythm. tricyclic antidepressants), and severe global myocardial ischemia are causes.

1	tricyclic antidepressants), and severe global myocardial ischemia are causes. Polymorphic VT has a continually changing QRS morphology indicating a changing ventricular activation sequence. Polymorphic VT that occurs in the context of congenital or acquired prolongation of the QT interval often has a waxing and waning QRS amplitude creating a “twisting about the points” appearance referred to as Torsade de Pointes (Fig. 277-3D). Ventricular fibrillation (VF) has continuous irregular activation with no discrete QRS complexes (Fig. 277-3E). Monomorphic or polymorphic VT may transition to VF in susceptible patients. FIGURE 277-3 Examples of types of ventricular tachycardia (VT). A. Monomorphic VT with dissociated P waves (short arrows). B. Ventricular flutter. C. Sinusoidal VT due to electrolyte disturbance or drug effects. D. Polymorphic VT resulting from prolongation of QT interval (torsade de pointes VT). E. Ventricular fibrillation.

1	Common symptoms of ventricular arrhythmias include palpitations, dizziness, exercise intolerance, episodes of lightheadedness, syncope, or sudden death. These arrhythmias can be asymptomatic and encountered unexpectedly as an irregular pulse or heart sounds on examination, or seen on a routine electrocardiogram (ECG), exercise test, or cardiac ECG monitoring. Syncope is a concerning symptom that can be due to an episode of VT with hypotension. Syncope due to a ventricular arrhythmia often indicates that there is a significant risk for subsequent cardiac arrest and sudden death with arrhythmia recurrence. Although benign causes of syncope, such as reflex-mediated neurocardiogenic (vasovagal) syncope and orthostatic hypotension, are generally more common, it is important to consider the possibility of heart disease or a genetic syndrome causing VT. When these are suspected, hospitalization for further evaluation and monitoring is often appropriate.

1	Sustained VT may present with cardiac arrest, often with degeneration of the VT to VF. Occasionally a sustained VT will be hemo dynamically tolerated and present with diminished exercise capacity or exacerbation of heart failure. Many patients who are at risk for VT have known heart disease and may have an implantable cardioverterdefibrillator (ICD). In patients with an ICD, spontaneous episodes of VT may elicit an episode of transient lightheadedness, palpitations, or syncope that may be followed by a shock from the ICD (see below).

1	The diagnosis of ventricular arrhythmias is established by recording of the arrhythmia on an ECG or, in some cases, initiation of the arrhythmia during an electrophysiologic study (Table 277-1). A 12-lead ECG of the arrhythmia should be obtained when possible and often provides clues to the potential site of origin and possible presence of underlying heart disease (see above). When the arrhythmia is intermittent with days to weeks between symptoms, prolonged ambulatory monitoring to capture the ECG at the time of symptoms is required to make the diagnosis. Continuous ambulatory monitoring or looping event recording monitors are options. Exercise testing should be considered in patients with exercise-induced symptoms. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: Initial assessment focuses on hemodynamic stability and evaluation for underlying heart disease. A family history of sudden death or cardiomyopathy suggests the possibility of a genetic basis for the arrhythmia and greater risk. The electrocardiogram can provide important clues. Patients with benign idiopathic arrhythmias usually have a completely normal ECG during sinus rhythm. Cardiac imaging is warranted to assess ventricular function and look for evidence of depressed ventricular function indicative of a cardiomyopathy or ventricular hypertrophy that may indicate hypertrophic cardiomyopathy. Cardiac magnetic resonance imaging (MRI) with late gadolinium enhancement can detect areas of ventricular scar, which are usually present in patients who are at risk for sustained monomorphic VT (Fig. 277-5). Evaluation to exclude atherosclerotic coronary artery disease should be performed in patients at risk, guided by age and other risk factors.

1	SPECIFIC ARRHYTHMIAS PVCs and Nonsustained VT Ventricular extrasystoles (Fig. 277-1A) can be due to automaticity or reentry (Chap. 278e). PVCs can be a sign of increased sympathetic tone; myocardial ischemia; hypoxia; electrolyte abnormalities, particularly hypokalemia; or underlying heart disease. During myocardial ischemia or in association with other heart disease, PVCs can be a harbinger of sustained VT or II III III II LVRVV1 = LBBB Septal or RV origin V1 = RBBB LV origin V1 II, III AVF = Inferior axis superior origin II, III AVF = Superior axis inferior origin FIGURE 277-4 Site of VT origin based on QRS morphology. LBBB, left bundle branch block; LV, left ventricle; RBBB, right bundle branch block; RV, right ventricle. I. 12-Lead ECG A. Should be obtained for PVCs, nonsustained VT, and monomorphic VT when possible

1	I. 12-Lead ECG A. Should be obtained for PVCs, nonsustained VT, and monomorphic VT when possible B. QRS morphology suggests ventricular region of origin V1 – dominant S = septum or RV V1 – dominant R = LV Superior axis = inferior wall origin Inferior axis = outflow region or anterior wall II. A. 24to 48-h continuous Holter monitor Useful for evaluation of daily symptoms to quantitate PVCs B. Event recorder: can be used for weeks at a time Useful for evaluation of infrequent symptoms Some require patient activation and will miss asymptomatic III. A. B. QT interval response to exercise may be abnormal in long QT syndrome IV. A. Can establish definitive diagnosis of VT versus supraventricular tachycardia with aberrancy or ventricular preexcitation B. Can provoke some arrhythmias that are otherwise infrequent C. D. Procedural risks determined by vascular access, whether ablation is performed, and the location of the arrhythmia substrate

1	B. Can provoke some arrhythmias that are otherwise infrequent C. D. Procedural risks determined by vascular access, whether ablation is performed, and the location of the arrhythmia substrate Abbreviation: RV, right ventricle. See text for other abbreviations. VF. In patients with heart disease, a higher frequency of ectopy and complexity (couplets and nonsustained VT) are associated with more severe disease and, in those with heart failure, with increased mortality. However, suppression of these arrhythmias with antiarrhythmic drugs does not improve survival. In the absence of cardiac disease, PVCs and nonsustained VT generally have a benign prognosis. PVCs that occur at a bigeminal frequency may not generate sufficient cardiac output for a radial pulse and hence may register at rates half that of the heart rate (Fig. 277-1A). Very frequent PVCs can depress ventricular function (see below).

1	Evaluation and managEmEnt When encountered during acute illness or as a new finding, evaluation should focus on detection and correction of potential aggravating factors and causes, specifically myocardial ischemia, ventricular dysfunction, and electrolyte abnormalities, most commonly hypokalemia. Underlying heart disease should be defined.

1	The ECG characteristics of the arrhythmia are often suggestive of whether structural heart disease is present. PVCs with smooth uninterrupted contours and sharp QRS deflections suggest an ectopic focus in relatively normal myocardium, whereas broad notching and slurred QRS deflections suggest a diseased myocardial substrate. The most frequent site of origin for idiopathic ventricular arrhythmias is the right ventricular outflow tract, giving rise to PVCs or VT that have a left bundle branch block configuration, with an inferiorly directed frontal plane axis as discussed below (Fig. 277-2). However, QRS morphology alone is not reliable as an indicator of disease or subsequent risk. Nonsustained VT is usually monomorphic with rates less than 200 beats/min and typically lasts less than 8 beats (Fig. 277-2). Nonsustained VT that is very rapid, polymorphic, or with a first beat that occurs prior to the peak of the T wave (“short-coupled”) is uncommon and should prompt careful evaluation

1	(Fig. 277-2). Nonsustained VT that is very rapid, polymorphic, or with a first beat that occurs prior to the peak of the T wave (“short-coupled”) is uncommon and should prompt careful evaluation for underlying disease or genetic syndromes associated with sudden death.

1	A family history of sudden death should prompt evaluation for genetic syndromes associated with sudden death, including cardiomyopathy, long QT syndrome, and arrhythmogenic right ventricular cardiomyopathy (see below). Any abnormality on the 12-lead ECG warrants further evaluation (Fig. 277-6). Repolarization abnormalities are seen in a number of genetically determined syndromes associated with sudden death, including the long QT syndrome, Brugada syndrome, arrhythmogenic right ventricular cardiomyopathy (ARVC), and hypertrophic cardiomyopathy. An echocardiogram is often necessary to assess ventricular function, wall motion abnormalities, and valvular heart disease. Cardiac magnetic resonance (CMR) imaging is also useful for this purpose and for the detection of ventricular scarring that is the substrate for sustained VT (Fig. 277-5). Exercise stress testing should be performed in patients with effort-related symptoms and in those at risk for coronary artery disease.

1	idiopathic pvcs and nonsustainEd vt For PVC and nonsustained VT in the absence of structural heart disease or a genetic sudden death syndrome, no specific therapy is needed unless the patient has significant symptoms or evidence that frequent PVCs are depressing ventricular function (see below). Reassurance that the arrhythmia is benign is often sufficient to allow the patient to cope with the symptoms, which will often wax and wane in frequency over years. Avoiding stimulants, such as caffeine, is helpful in some patients. If symptoms require treatment, β-adrenergic blockers and nondihydropyridine calcium channel blockers (verapamil and diltiazem) are sometimes helpful (see Table 276-3). If these fail, more potent antiarrhythmic drugs or catheter

1	CHAPTER 2771493 FIGURE 277-5 Imaging studies of the left ventricle (LV) used to assist ablation for ventricle tachycardia (VT). Left panel is a magnetic resonance image of a longitudinal section demonstrating thinning of the anterior wall and late gadolinium enhancement in a subendocardial scar (white arrows). The middle panel shows a two-dimensional image of the LV in long axis corresponding to the sector through the mid LV (arrow, right panel) obtained by an intracardiac echo probe positioned in the right ventricle. An electroanatomic three-dimensional map of the LV in the left anterior oblique projection is displayed in the right panel. The purple color depicts areas of normal voltage (>1.5 mV). Blue, green, and yellow represent progressively lower voltages with the red areas indicating scar (<0.5 mV). Channels of viable myocardium with slow conduction within the scar are identified with the light blue dots. Areas of ablation delivered to regions involved in reentrant VT are

1	scar (<0.5 mV). Channels of viable myocardium with slow conduction within the scar are identified with the light blue dots. Areas of ablation delivered to regions involved in reentrant VT are indicated by maroon dots.

1	ablation can be considered. The antiarrhythmic agents flecainide, propafenone, mexiletine, and amiodarone can be effective, but the potential for side effects warrants careful consideration. Catheter ablation can be effective if the arrhythmia occurs with sufficient frequency or is readily provoked such that its origin can be identified for ablation in a similar manner to that for idiopathic monomorphic VT as discussed below. Benefit must be carefully weighed against the procedure-related risks (see below). pvcs and nonsustainEd vt associatEd with acutE coronary syndromEs

1	pvcs and nonsustainEd vt associatEd with acutE coronary syndromEs During and early after acute myocardial infarction (MI), PVCs and nonsustained VT are common and can be an early manifestation of ischemia and a harbinger of subsequent VF. Treatment with β-adrenergic blockers and correction of hypokalemia and hypomagnesemia reduce the risk of VF. Routine administration of the antiarrhythmic drugs such as lidocaine has not been shown to reduce mortality and is not indicated for suppression of PVCs or asymptomatic nonsustained VT.

1	Following recovery from acute MI, frequent PVCs (typically >10 PVCs per hour), repetitive PVCs with couplets, and nonsustained VT are markers for depressed ventricular function and increased mortality, but routine antiarrhythmic drug therapy to suppress these arrhythmias is not warranted. Treatment with the sodium channel blocker flecainide increased mortality. Amiodarone therapy reduces sudden death, but does not improve total mortality. Therefore, amiodarone is an option for treatment of symptomatic arrhythmias in this population when the potential benefit outweighs its potential toxicities. β-Adrenergic blockers reduce sudden death but have limited effect on spontaneous arrhythmias.

1	For survivors of an acute MI, an ICD reduces mortality in certain high-risk groups: patients who have survived >40 days after the acute MI and have a left ventricular (LV) ejection fraction of ≤0.30 or who have an ejection fraction <0.35 and have symptomatic heart failure (functional class II or III); and patients >5 days after MI who have a reduced LV ejection fraction, nonsustained VT, and inducible sustained VT or VF on electrophysiologic testing. ICDs do not reduce mortality when routinely implanted soon after MI or in patients after recent coronary artery revascularization surgery.

1	pvcs and nonsustainEd vt associatEd with dEprEssEd vEntricular function and hEart failurE PVCs and nonsustained VT are common in patients with depressed ventricular function and heart failure and are markers for disease severity and increased mortality, but antiarrhythmic drug therapy to suppress these arrhythmias has not been shown to improve survival. Antiarrhythmic drugs whose major action is blockade of the cardiac sodium channel (flecainide, propafenone, mexiletine, quinidine, and disopyramide) are avoided in patients with structural heart disease because of a

1	FIGURE 277-6 Precordial chest leads V1–V3 showing typical abnormalities of arrhyth-risk of proarrhythmia, negative inotropic effects, mogenic right ventricular cardiomyopathy (ARVC) (A) and Brugada syndrome (B). and increased mortality. Therapy with the potas-In ARVC, there is T inversion and delayed ventricular activation manifest as epsilon waves sium channel blockers, e.g., dofetilide, does not (arrows). Panel B shows ST elevation in V1 and V2 typical of the Brugada syndrome. (Figures reduce mortality. Amiodarone suppresses venreproduced from F Marchlinski: The tachyarrhythmias. In Longo DL et al [eds]: Harrison’s tricular ectopy and reduces sudden death but does Principles of Internal Medicine, 18th edition. New York, McGraw-Hill, 2012, pp 1878–1900.) not improve overall survival. ICDs are the major 1494 therapy to protect against sudden death in patients at high risk and are recommended for those with LV ejection fraction <0.35 and New York Heart Association class II and III

1	ICDs are the major 1494 therapy to protect against sudden death in patients at high risk and are recommended for those with LV ejection fraction <0.35 and New York Heart Association class II and III heart failure, in whom they reduce mortality by 20%, from 36% to 29%, over 5 years.

1	othEr cardiac disEasEs Ventricular ectopy is associated with increased mortality in patients with hypertrophic cardiomyopathy (Chap. 287) or with congenital heart disease (Chap. 282) associated with right ventricular or LV dysfunction. In these patients, management is similar to that for patients with ventricular dysfunction. Pharmacologic suppression of the arrhythmia has not been shown to improve mortality. ICDs are indicated for patients considered at high risk for sudden cardiac death.

1	pvc-inducEd vEntricular dysfunction Very frequent ventricular ectopy and repetitive nonsustained VT (Fig. 277-2) can depress ventricular function, possibly through an effect similar to chronic tachycardia or by inducing ventricular dyssynchrony. Depression of ventricular function rarely occurs unless PVCs account for more than 10–20% of total beats over a 24-h period. Often the PVCs are idiopathic and unifocal, most commonly originating from the LV papillary muscles or outflow tract regions where they can be targeted for ablation. The distinction between PVC-induced ventricular dysfunction as compared to a cardiomyopathic process causing ventricular dysfunction and arrhythmia is difficult and in some cases can be made only retrospectively by observing an improvement in ventricular function after the arrhythmia is suppressed with an antiarrhythmic drug, such as amiodarone, or by catheter ablation.

1	Idioventricular Rhythms Three or more ventricular beats at a rate slower than 100 beats/min are termed idioventricular rhythm (Fig. 277-1C). Automaticity is the likely mechanism. Idioventricular rhythms are common during acute MI (Chap. 295) and may emerge during sinus bradycardia. Atropine may be administered to increase the sinus rates if the loss of atrioventricular synchrony leads to hemodynamic compromise. This rhythm is also common in patients with cardiomyopathies or sleep apnea. It can also be idiopathic, often emerging when the sinus rate slows during sleep. Therapy should target any underlying cause and correction of bradycardia. Specific therapy for asymptomatic idioventricular rhythm is not necessary.

1	Sustained Monomorphic VT Sustained monomorphic VT presents as a wide QRS tachycardia that has the same QRS configuration from beat to beat, indicating an identical sequence of ventricular depolarization for each beat (Fig. 277-3A). VT originates from a stable focus or reentry circuit. In structural heart disease, the substrate is often an area of patchy replacement fibrosis due to infarction, inflammation, or prior cardiac surgery that creates anatomical or functional reentry pathways (Fig. 277-5). Less commonly, VT is related to reentry or automaticity in a diseased Purkinje system. In the absence of structural heart disease, idiopathic VT can present as sustained monomorphic VTs that are due to focal automaticity or reentry involving a portion of the Purkinje system.

1	The clinical presentation can vary depending on the rate of the arrhythmia, underlying cardiac function, and autonomic adaptation in response to the arrhythmia. Whereas patients with normal cardiac function might tolerate rapid VTs, those with severe LV dysfunction often experience symptoms of hypotension, even if VT is not particularly fast. Monomorphic VT may deteriorate to VF, which may be the initial cardiac rhythm recorded at the time of resuscitation.

1	diagnosis Sustained monomorphic VT has to be distinguished from other causes of uniform wide QRS tachycardia. These include supra-ventricular tachycardia with left or right bundle branch block aberrant conduction, supraventricular tachycardias conducted to the ventricles over an accessory pathway (Chap. 276), and rapid cardiac pacing in a patient with a pacemaker or defibrillator. In the presence of known heart disease, VT is the most likely diagnosis of a wide QRS tachycardia. Hemodynamic stability during the arrhythmia does not exclude VT. A number of ECG criteria have been evaluated. The presence of AV dissociation is usually a reliable marker for VT (Fig. 277-7), but P waves can be difficult to define. A P wave following each QRS does not VT versus Supraventricular Tachycardia with Aberrancy VT any of V1 toV6 Possible SVT with aberrancy VT still possible

1	VT versus Supraventricular Tachycardia with Aberrancy VT any of V1 toV6 Possible SVT with aberrancy VT still possible FIGURE 277-7 Algorithm for differentiation of ventricular tachycardia (VT) from supraventricular tachycardia (SVT) with aberration. AV, atrioventricular.

1	FIGURE 277-7 Algorithm for differentiation of ventricular tachycardia (VT) from supraventricular tachycardia (SVT) with aberration. AV, atrioventricular. exclude VT because 1:1 conduction from ventricle to atrium can occur. A monophasic R wave or Rs complex in AVR or concordance from V1 to V6 of monophasic R or S waves is also relatively specific for VT (Fig. 277-7). Other QRS morphology criteria have also been described, but all have limitations and are not very reliable in patients with severe heart disease. In patients with known bundle branch block, the same QRS morphology during tachycardia as during sinus rhythm suggests supraventricular tachycardia rather than VT, but is not absolutely reliable. An electrophysiologic study is sometimes required for definitive diagnosis. Rarely, noise and movement artifacts on telemetry recordings can simulate VT; prompt recognition can avoid unnecessary tests and interventions.

1	When LV function is depressed or there is evidence of structural myocardial disease, scar-related reentry is the most likely diagnosis. Scars are suggested by pathologic Q waves on the ECG, segmental left or right ventricular wall motion abnormalities on echocardiogram or nuclear imaging, and areas of delayed gadolinium enhancement during MRI (Fig. 277-5).

1	trEatmEnt and prognosis Initial management follows Advanced Cardiac Life Support (ACLS) guidelines. If hypotension, impaired consciousness, or pulmonary edema is present, QRS synchronous electrical cardioversion should be performed, ideally after sedation if the patient is conscious. For stable tachycardia, a trial of adenosine is reasonable, as this may clarify a supraventricular tachycardia with aberrancy (Chap. 276). Intravenous amiodarone is the drug of choice if heart disease is present. Following restoration of sinus rhythm, hospitalization and evaluation to define underlying heart disease are required. Assessment of cardiac biomarkers for evidence of MI is appropriate, but acute MI is rarely a cause of sustained monomorphic VT, and elevations in troponin or creatine kinase (CK)-MB are more likely to indicate myocardial damage that is secondary to hypotension and ischemia from the VT. Subsequent management is determined by the underlying heart disease and frequency of VT. If VT

1	are more likely to indicate myocardial damage that is secondary to hypotension and ischemia from the VT. Subsequent management is determined by the underlying heart disease and frequency of VT. If VT recurs frequently or is incessant, administration of antiarrhythmic medications or catheter ablation may be required to restore stability. More commonly, sustained monomorphic VT occurs as an isolated episode, but with a risk of recurrence. ICDs are usually considered for VT associated with structural heart disease.

1	Patients who present with sustained VT associated with coronary artery disease typically have a history of prior large MI and present years after the acute infarct with a remodeled ventricle and markedly depressed LV function. Even when there is biomarker evidence of acute MI, a preexisting scar from previous MI should be suspected as the cause of the VT. Infarct scars provide a durable substrate for sustained VT, and up to 70% of patients have a recurrence of the arrhythmia within 2 years. Scar-related reentry is not dependent on recurrent acute myocardial ischemia, so coronary revascularization cannot be anticipated to prevent recurrent VT, even when it may be appropriate for other indications. Depressed ventricular function, which is a risk factor for sudden death, is usually present. Implantation of an ICD is warranted for most patients provided that there is a reasonable expectation of survival with acceptable functional status for the next year after recovery from the VT

1	Implantation of an ICD is warranted for most patients provided that there is a reasonable expectation of survival with acceptable functional status for the next year after recovery from the VT episode. ICDs reduce annual mortality from 12.3% to 8.8% and lower arrhythmic deaths by 50% in patients with hemodynamically significant sustained VT or a history of cardiac arrest compared with pharmacologic therapy. Chronic amiodarone therapy may be considered for patients who are not candidates for or who decline ICD placement.

1	Following ICD implantation, patients remain at risk for heart failure, recurrent ischemic events, and recurrent VT, with a 5-year mortality that exceeds 30%. Attention to therapies with survival benefit, including β-adrenergic blocking agents, angiotensin-converting enzyme inhibitors, and statins, is important. Patients with frequent symptomatic recurrences of VT require antiarrhythmic drug therapy or catheter ablation.

1	nonischEmic dilatEd cardiomyopathy Sustained monomorphic VT associated with nonischemic cardiomyopathy is usually due to scar-related reentry. The etiology of scar is often unclear, but progressive replacement fibrosis is the likely cause. On cardiac MRI, scars are detectable as areas of delayed gadolinium enhancement and are more often intramural or subepicardial in location as compared with patients with prior MI. Scars that cause VT are often located adjacent to a valve annulus and can occur in either ventricle. Any cardiomyopathic process can cause scars and VT, but cardiac sarcoidosis (Chap. 390) and Chagas’ disease (Chap. 252) are particularly associated with monomorphic VT (Table 277-2). An ICD is usually indicated with additional drugs or ablation for control of recurrent VT.

1	monomorphic vt in arvc ARVC (Chap. 287) is a rare genetic disorder most commonly due to mutations in genes encoding for cardiac desmosomal proteins. Approximately 50% have a familial transmission with autosomal dominant inheritance. A less common, autosomal recessive form is associated with cardiocutaneous syndromes that include Naxos disease and Carvajal syndrome. Patients typically present between the second and fifth decade with palpitations, syncope, or cardiac arrest owing to sustained monomorphic VT, although polymorphic VT can also occur. Fibrosis and fibro-fatty replacement most commonly involve the right ventricular myocardium and provide the substrate for reentrant VT that usually has a left bundle branch block–like configuration, consistent with the right ventricular origin. The sinus rhythm ECG suggests the disease in more than 85% of patients, most often showing T-wave inversions in V1–V3 (Fig. 277-6). Delayed activation of the right ventricle may cause a widened QRS

1	The sinus rhythm ECG suggests the disease in more than 85% of patients, most often showing T-wave inversions in V1–V3 (Fig. 277-6). Delayed activation of the right ventricle may cause a widened QRS (≥110 ms) in the right precordial leads and a prolonged S-wave upstroke in those leads, and occasionally a deflection at the end of the QRS known as an epsilon wave (Fig. 277-6). Cardiac imaging may show right ventricular enlargement or areas of abnormal motion or reveal areas of scar on CMR imaging with gadolinium. The monomorphic VT of early ARVC can sometimes be difficult to differentiate from idiopathic right ventricular outflow tract VT.

1	LV involvement can occur and occasionally precede manifest right ventricular disease. Heart failure is rare except in late stages, and survival to advanced age can be anticipated provided that VT can be controlled. An ICD is recommended. When VT is exercise-induced, it may respond to β-adrenergic blockers and limiting exercise. Sotalol, amiodarone, and catheter ablation have been used to reduce recurrences. Ablation targets are often located in the subepicardium of the RV. tEtralogy of fallot VT occurs in 3–14% of patients late after repair of tetralogy of Fallot (Chap. 282) and contributes to a 2% per decade risk of I. Idiopathic VT without structural heart disease A. Outflow tract origin 1. RV outflow tract: left bundle branch block pattern with inferior axis (tall QRS in inferior leads) and late transition in the precordial leads 2. LV outflow tract: prominent R in V1 with inferior axis

1	LV outflow tract: prominent R in V1 with inferior axis B. Left posterior fascicular VT 1. Right bundle branch block pattern with left axis deviation (most common) II. A. Monomorphic VT is common with prior large myocardial infarction B. III. A. Polymorphic VT and VF more common but fibrotic scars can cause monomorphic VT especially with sarcoidosis and Chagas’ disease IV. A. Monomorphic VT usually of right ventricular origin (left bundle branch morphology) B. Polymorphic VT and VF can occur independently or through degeneration of monomorphic VT V. Repaired tetralogy of Fallot A. Monomorphic VT of right ventricular origin (usually left bundle branch morphology) VI. A. B. Less commonly, monomorphic VT associated with myocardial scars VIII. A. Long QT syndrome: torsade de pointes VT B. Brugada syndrome: VF C. Catecholaminergic polymorphic VT: polymorphic VT or bidirectional VT D. Short QT syndrome: ventricular fibrillation E.

1	A. Long QT syndrome: torsade de pointes VT B. Brugada syndrome: VF C. Catecholaminergic polymorphic VT: polymorphic VT or bidirectional VT D. Short QT syndrome: ventricular fibrillation E. Early repolarization syndrome: polymorphic VT or VF Abbreviation: RV, right ventricle. See text for other abbreviations. sudden death. Monomorphic VT is due to reentry around areas of surgically created scar in the RV (Table 277-2). Factors associated with VT risk include age >5 years at the time of repair, high-grade ventricular ectopy, inducible VT on an electrophysiologic study, abnormal right ventricular hemodynamics, and sinus rhythm QRS duration >180 ms. An ICD is usually warranted for patients who have a spontaneous episode of VT, but criteria for a prophylactic ICD in other patients have not been established. Catheter ablation is used to control recurrent episodes.

1	BundlE Branch rEEntry vt Reentry through the Purkinje system occurs in approximately 5% of patients with monomorphic VT in the presence of structural heart disease. The reentry circuit typically revolves retrograde via the left bundle and anterograde down the right bundle, thereby producing VT that has a left bundle branch block configuration. Catheter ablation of the right bundle branch abolishes this VT. Bundle branch reentry is usually associated with severe underlying heart disease. Other scar-related VTs are often present and often require additional therapy or ICD implantation.

1	idiopathic monomorphic vt Idiopathic VT in patients without structural heart disease usually presents with palpitations, lightheadedness, and occasionally syncope, often provoked by sympathetic stimulation during exercise or emotional upset. The QRS morphology of the arrhythmia suggests the diagnosis (see below). The sinus rhythm ECG is normal. Cardiac imaging shows normal ventricular function and no evidence of ventricular scar. Occasionally a patient with structural heart disease is found to have concomitant idiopathic VT, unrelated to the structural disease. Sudden death is rare. Outflow tract VTs originate from a focus, usually with features consistent with triggered automaticity. The arrhythmia may present with 1. Congenital long QT syndromes (see text for details) Long QT syndrome type 1: Reduced repolarizing current IKs due to mutation in KCNQ1 gene Long QT syndrome type 2: Reduced repolarizing current IKr due to mutation in KCNH2 gene

1	Long QT syndrome type 1: Reduced repolarizing current IKs due to mutation in KCNQ1 gene Long QT syndrome type 2: Reduced repolarizing current IKr due to mutation in KCNH2 gene Long QT syndrome type 3: Delayed inactivation of the INa due to mutations in SCN5A gene Others: Several other types of long QT syndromes have been described; long QT types 1, 2, and 3 account for 80–90% of cases 2. Acquired prolongation of QT interval Class IA: Quinidine, disopyramide, procainamide Class III: Sotalol, amiodarone (QT prolongation common but torsade ventricular tachycardia is rare), ibutilide, dofetilide, almokalant Macrolides: Erythromycin, clarithromycin, azithromycin Fluoroquinolones: Levofloxacin, moxifloxacin, gatifloxacin Antifungals: Ketoconazole, itraconazole Antivirals: Amantadine

1	Haloperidol, phenothiazines, thioridazine, trifluoperazine, sertindole, zimelidine, ziprasidone Tricyclic and tetracyclic antidepressants sustained VT, nonsustained VT, or PVCs often provoked by exercise or emotional upset. Repeated bursts of nonsustained VTs, which may occur incessantly, are known as repetitive monomorphic VTs and can cause tachycardia-induced cardiomyopathy with depressed ventricular function that recovers after suppression of the arrhythmia (Fig. 277-2). Most originate in the right ventricular outflow tract, which gives rise to VT that has a left bundle branch block configuration in V1 and an axis that is directed inferiorly, with tall R waves in leads II, III, and AVF (Fig. 277-2). Idiopathic VT can also arise in the LV outflow tract or in sleeves of myocardium that extend along the aortic root. LV origin is suspected when lead V1 or V2 has prominent R waves. Although this typical outflow tract QRS morphology favors idiopathic VT, some cardiomyopathies, notably

1	extend along the aortic root. LV origin is suspected when lead V1 or V2 has prominent R waves. Although this typical outflow tract QRS morphology favors idiopathic VT, some cardiomyopathies, notably ARVC, can cause PVCs or VT from this region. Excluding these diseases is an initial focus of evaluation.

1	LV intrafascicular VT presents with sustained VT that has a right bundle branch block–like configuration. It is often exercise-induced and occurs more often in men than women. The mechanism is reentry in or near the septal ramifications of the LV Purkinje system. This VT can be terminated by intravenous administration of verapamil. managEmEnt of idiopathic vt Treatment is required for symptoms or when frequent or incessant arrhythmias depress ventricular function. β-Adrenergic blockers are first-line therapy. Nondihydropyridine calcium channel blockers (diltiazem and verapamil) are sometimes effective. Catheter ablation is warranted for severe symptoms or when beta blockers or calcium channel blockers are not effective or not desired. Efficacy and risks of catheter ablation vary with the specific site of origin of the VT, being most favorable for arrhythmias originating in the right ventricular outflow tract.

1	LV fascicular VT can be terminated by intravenous administration of verapamil, although chronic therapy with oral verapamil is not Terfenadine, astemizole, diphenhydramine, hydroxyzine Cholinergic antagonists: Cisapride, organophosphates Citrate (massive blood transfusions) Cocaine Methadone Fluoxetine (in conjunction with other drugs that prolong QT) Cardiac conditions always effective. Catheter ablation is recommended if β-adrenergic blockers or calcium channel blockers are ineffective or not desired.

1	Polymorphic VT Sustained polymorphic VT can be seen with any form of structural heart disease (Table 277-2). However, unlike sustained monomorphic VT, polymorphic VT does not always indicate a structural abnormality or focus of automaticity. Reentry with continually changing reentrant paths, spiral wave reentry, and multiple automatic foci are potential mechanisms (Chap. 278e). Sustained polymorphic VT usually degenerates into VF. Polymorphic VT is typically seen in association with acute MI or myocardial ischemia, ventricular hypertrophy, and a number of genetic mutations that affect cardiac ion channels (Table 277-3).

1	polymorphic vt associatEd with acutE mi/myocardial ischEmia Acute MI or ischemia is a common cause of polymorphic VT and should be the initial consideration in management. Approximately 10% of patients with acute MI develop VT that degenerates to VF, related to reentry through the infarct border zone. The risk is greatest in the first hour of acute MI. Following resuscitation as per the ACLS guidelines, management is as for acute MI (Chap. 295). β-Adrenergic blockers, correction of electrolyte abnormalities, and prompt myocardial reperfusion are required. Repeated episodes of polymorphic VT suggest ongoing myocardial ischemia and warrant assessment of adequacy of myocardial reperfusion. Polymorphic VT and VF that occur within the first 48 h of acute MI are associated with greater in-hospital mortality, but those who survive past hospital discharge are not at increased risk for arrhythmic sudden death. Long-term therapy for postinfarct ventricular arrhythmia is determined by residual

1	mortality, but those who survive past hospital discharge are not at increased risk for arrhythmic sudden death. Long-term therapy for postinfarct ventricular arrhythmia is determined by residual LV function, with an ICD being indicated for persistent severe LV dysfunction (LV ejection fraction <0.35).

1	congenital long Qt syndrome The con-1497 genital long QT syndrome (LQTS) is caused by mutations in genes coding for cardiac ion channels responsible for ventricular repolarization. The corrected QT (QTc) is typically prolonged to greater than 440 ms in men and 460 ms in women. Symptoms are due to Torsade de Pointes VT (Fig. 277-8). Several forms of congenital LQTS have been identified, but three groups of mutations that lead to LQTS type 1 (LQTS-1), LQTS type 2 (LQTS-2), or LQTS type 3 (LQTS-3) account for A 90% of cases. The most frequently encountered mutations, LQTS1 and LQTS2, are due to abnormalities of potassium channels, but mutations affecting the sodium channel (LQTS3) and calcium channels have also been described (Table 277-3).

1	Patients often present with syncope or cardiac arrest, usually during childhood. In LQTS-1, episodes tend to occur during exertion, particularly swimming. In LQTS-2, sudden auditory stimuli or emotional upset predispose to events. In LQTS-3, sudden death during sleep is a notable feature. FIGURE 277-8 Electrocardiogram (ECG) of a patient with prolonged QT and episodes of torsade de pointes ventricular tachycardia (VT). A. Twelve-lead ECG showing a heart rate of 54, anterior ered in the course of family screening wall T inversion, and QT interval of 600 ms. The corrected QT interval (QTc) is 585 ms. B. Telemetry or on a routine ECG. Genotyping can

1	ECG tracing with digital pulse waveform demonstrating bursts of torsade de pointes VT. The initiating sequence of the VT is characteristic, with a PVC inducing a pause followed by a sinus beat that had a to provide reassurance regarding the longer QT and interruption of the T wave by a PVC that is the first beat of VT. The VT is self-terminat diagnosis. Correlations of genotype ing in this case.

1	RepolaRization abnoRmalities and genetic aRRhythmia syndRomes • acquired long Qt Abnormal prolongation of the QT interval is associated with the polymorphic VT Torsade de Pointes (Fig. 277-8). The VT often has a characteristic initiation sequence of a premature ventricular beat that induces a pause, followed by a sinus beat that has a longer QT interval and interruption of the T wave by the PVC that is the first beat of the polymorphic VT. This characteristic initiation is termed “pause-dependent” (Fig. 277-8). Causes of QT prolongation include electrolyte abnormalities, bradycardia, and a number of medications that block repolarizing potassium currents, notably the antiarrhythmic drugs sotalol, dofetilide, and ibutilide, but also a number of other medications used for noncardiac diseases, including erythromycin, pentamidine, haloperidol, phenothiazines, and methadone (Table 2773). Individual susceptibility may be related to genetic polymorphisms or mutations that influence

1	diseases, including erythromycin, pentamidine, haloperidol, phenothiazines, and methadone (Table 2773). Individual susceptibility may be related to genetic polymorphisms or mutations that influence repolarization.

1	Patients typically present with near-syncope, syncope, or cardiac arrest. Sustained episodes degenerate to VF requiring defibrillation. PVCs and nonsustained VT often precede episodes of sustained VT. Intravenous administration of 1–2 g of magnesium sulphate usually suppresses recurrent episodes. If magnesium alone is ineffective, increasing heart rate with isoproterenol infusion or pacing, to a rate of 100–120 beats/min as required to suppress PVCs, usually suppresses VT recurrences. These maneuvers allow time for correction of associated electrolyte disturbance (hypokalemia and hypocalcemia) and bradycardia and removal of any causative drugs (Table 277-3). Drug interactions that elevate levels of the offending agent are often a precipitating factor. Patients who experience a polymorphic VT induced by QT prolongation should be considered to have a susceptibility to the arrhythmia and should avoid all future exposure to medications known to prolong the QT interval.

1	with risk and response to therapy are beginning to emerge. In most patients with LQTS-1 or LQTS-2, adequate doses of beta blocker therapy (the non-selective agents nadolol or propranolol) are sufficient protection from arrhythmia episodes. Markers of increased risk include QTc interval exceeding 0.5 s, female gender, and a history of syncope or cardiac arrest. Recurrent syncope despite beta blocker therapy or a high-risk profile merits consideration of an ICD. Avoidance of QT-prolonging drugs is critical for all patients with LQTS, including those who are genotype positive but have normal QT intervals. short Qt syndrome Short QT syndrome is very rare compared to LQTS. The QTc is shorter than 0.36 s, and usually less than 0.3 s. The genetic abnormality causes a gain of function of the potassium channel (I Kr) or reduced inward depolarizing currents. The abnormality is associated with atrial fibrillation, polymorphic VT, and sudden death.

1	Brugada syndrome Brugada syndrome is a rare syndrome characterized by >0.2 mV of ST-segment elevation with a coved ST segment and negative T wave in more than one anterior precordial lead (V1–V3) (Fig. 277-6) and episodes of syncope or cardiac arrest due to polymorphic VT in the absence of structural heart disease. Cardiac arrest may occur during sleep or be provoked by febrile illness. Males are more commonly affected than females. Mutations involving cardiac sodium channels are identified in approximately 25% of cases. Distinction from patients with similar ST elevation owing to LV hypertrophy, pericarditis, myocardial ischemia or MI hyperkalemia, hypothermia, right bundle branch block, and ARVC is often difficult. Furthermore, the characteristic ST-segment elevation can wax and wane over time and may become pronounced during acute illness and fever. Administration of the sodium channel blocking drug flecainide, ajmaline, or procainamide can augment or unmask ST elevation in

1	wane over time and may become pronounced during acute illness and fever. Administration of the sodium channel blocking drug flecainide, ajmaline, or procainamide can augment or unmask ST elevation in affected individuals. An ICD is indicated for individuals who have had unexplained syncope or been resuscitated from cardiac arrest.

1	1498 Quinidine has been used successfully to suppress frequent episodes of VT. Early repolarization syndrome Patients resuscitated from VF who have no structural heart disease or other identified abnormality have a higher prevalence of J-point elevation with notching in the terminal QRS. A family history of sudden death is present in some patients, suggesting a potential genetic basis. J-point elevation is also seen in some patients with the Brugada syndrome and associated with a higher risk of arrhythmias. An ICD is recommended for those who have had prior cardiac arrest. It should be noted that J-point elevation is commonly seen as a normal variant, and in the absence of specific symptoms, the clinical relevance is not known.

1	catecholaminergic polymorphic vt This rare familial syndrome is due to mutations in the cardiac ryanodine receptor and, less commonly, the sarcoplasmic calcium binding protein, calsequestin 2. These mutations result in abnormal sarcoplasmic calcium handling and polymorphic ventricular arrhythmias that resemble those seen with digitalis toxicity. The VT is polymorphic or has a characteristic alternating QRS morphology termed bidirectional VT. Patients usually present during childhood with exerciseor emotion-induced palpitations, syncope, or cardiac arrest. β-Adrenergic blockers (e.g., nadolol and propranolol) and an ICD are recommended. Verapamil, flecainide, or surgical left cardiac sympathetic denervation reduces or prevents recurrent VT in some patients.

1	hypertrophic cardiomyopathy (hcm) HCM is the most common genetic cardiovascular disorder, occurring in 1 in 500 individuals, and is a prominent cause of sudden death before the age of 35 years (Chap. 287). Sudden death can be due to polymorphic VT/VF. Rarely, sustained monomorphic VT occurs related to areas of ventricular scar. Risk factors include young age, nonsustained VT, failure of blood pressure to increase during exercise, recent (within 6 months) syncope, ventricular wall thickness >3 cm, and possibly the severity of LV outflow obstruction. An ICD is generally indicated for high-risk patients, but the specific risk profile warranting an ICD continues to be debated. Surgical myectomy, performed to relieve outflow obstruction, has been associated with a sudden death rate of less than 1% per year. The reported annual rate of sustained VT or sudden death after transcoronary ethanol septal ablation done to relieve outflow obstruction has been reported to range between 1 and 5%.

1	genetic dilated cardiomyopathies Genetic dilated cardiomyopathies account for 30–40% of cases of nonischemic dilated cardiomyopathies. Some are associated with muscular dystrophy. Autosomal dominant, recessive, X-linked, and mitochondrial inheritance patterns are recognized. Mutations in genes coding for structural proteins of the nuclear lamina (lamin A and C) and the SCN5A gene are particularly associated with conduction system disease and ventricular arrhythmias. Patients can experience polymorphic VT and cardiac arrest or develop areas of scar causing sustained monomorphic VT. ICDs are recommended for those who have had a sustained VT or are at high risk due to significantly depressed ventricular function (LV ejection fraction of ≤0.35 and associated with heart failure) or a malignant family history of sudden death.

1	Ventricular Fibrillation VF is characterized by disordered electrical ventricular activation without identifiable QRS complexes (Fig. 2773E). Spiral wave reentry and multiple circulating reentry wavefronts are possible mechanisms. Sustained polymorphic or monomorphic VT that degenerates to VF is a common cause of out-of-hospital cardiac arrest. Treatment follows ACLS guidelines with defibrillation to restore sinus rhythm. If resuscitation is successful, further evaluation is performed to identify and treat underlying heart disease and potential causes of the arrhythmia, including the possibility that monomorphic or polymorphic VT could have initiated VF. If a transient reversible cause such as acute MI is not identified, therapy to reduce the risk of sudden death with an ICD is often warranted. Chronic amiodarone therapy may be considered for individuals who are not ICD candidates.

1	Incessant VT and Electrical Storm VT is incessant when it continues to recur shortly after electrical, pharmacologic, or spontaneous conversion to sinus rhythm. “VT storm” or “electrical storm” refers to three or more separate episodes of VT within 24 h, most commonly encountered in patients with ICDs. Slow incessant VT is sometimes asymptomatic, but can cause heart failure or tachycardia-induced cardiomyopathy. More commonly, these presentations are life-threatening and require emergent therapy. Measures to reduce sympathetic tone, including β-adrenergic blockade, sedation, and general anesthesia, have been used effectively. Intravenous administration of amiodarone and lidocaine can be effective for suppression. Urgent catheter ablation can be lifesaving.

1	Use of antiarrhythmic drugs is based on consideration of the risks and potential benefit for the individual patient. The potential to increase the frequency of VT or cause a new VT, an undesirable effect known as “proarrhythmia,” is a potential risk. Many drugs have multiple effects, often blocking more than one channel. Drug doses, metabolism, and adverse effects are summarized in Chap. 277. β-Adrenergic Blockers Many ventricular arrhythmias are sensitive to sympathetic stimulation, and β-adrenergic stimulation also diminishes the electrophysiologic effects of many antiarrhythmic drugs. The safety of β-blocking agents makes them the first choice of therapy for most ventricular arrhythmias. They are particularly useful for exercise-induced arrhythmias and idiopathic arrhythmias, but have limited efficacy for most arrhythmias associated with heart disease. Bradyarrhythmias are the major cardiac toxicity.

1	Calcium Channel Blockers The nondihydropyridine calcium channel blockers diltiazem and verapamil can be effective for some idiopathic VTs. The risk of proarrhythmia is low, but they have negative inotropic and vasodilatory effects that can aggravate hypotension.

1	Sodium Channel-Blocking Agents Drugs whose major effect is mediated through sodium channel blockade include mexiletine, quinidine, disopyramide, flecainide, and propafenone, which are available for chronic oral therapy (Table 277-3). Lidocaine, quinidine, and procainamide are available as intravenous formulations. Quinidine, disopyramide, and procainamide also have potassium channel-blocking effects that prolong the QT interval. These agents have potential proarrhythmic effects and, with the possible exception of quinidine, also have negative inotropic effects that may contribute to increased mortality observed in patients with prior MI. Long-term therapy is generally avoided in patients with structural heart disease but may be used to reduce symptomatic arrhythmias in patients with ICDs.

1	Potassium Channel-Blocking Agents Sotalol and dofetilide block the delayed rectifier potassium channel IKr, thereby prolonging the QT interval. Sotalol also has nonselective β-adrenergic blocking activity. It has a modest effect on reducing ICD shocks due to ventricular and atrial arrhythmias. Proarrhythmia with Torsade de Pointes due to QT prolongation occurs in 3–5% of patients. Both sotalol and dofetilide are excreted via the kidneys, necessitating dose adjustment or avoidance in renal insufficiency. These drugs must be avoided in patients with other risk factors for Torsade de Pointes, including QT prolongation, hypokalemia, and significant bradycardia.

1	Amiodarone and Dronedarone Amiodarone, which blocks multiple cardiac ionic currents and has sympatholytic activity, suppresses a variety of ventricular arrhythmias. It is administered intravenously for life-threatening arrhythmias. During chronic oral therapy, electrophysiologic effects develop over several days. It is more effective than sotalol in reducing ICD shocks and is the preferred drug for ventricular arrhythmias in patients with heart disease who are not candidates for an ICD. Bradyarrhythmias are the major cardiac adverse effect. Ventricular proarrhythmia can occur, but Torsade de Pointes VT is rare. Noncardiac toxicities are a major problem and contribute to drug discontinuation in approximately a third of patients during long-term therapy. Pneumonitis or pulmonary fibrosis occurs in approximately 1% of patients. Photosensitivity is common, and neuropathy and ocular toxicity can occur. Systematic monitoring is recommended during chronic therapy, including assessment for

1	occurs in approximately 1% of patients. Photosensitivity is common, and neuropathy and ocular toxicity can occur. Systematic monitoring is recommended during chronic therapy, including assessment for thyroid and liver toxicity every 6 months and lung toxicity with a chest radiograph and/or determination of lung diffusing capacity annually. Dronedarone has structural similarities to amiodarone but without the iodine moiety. Efficacy for ventricular arrhythmias is poor, and it increases mortality in patients with heart failure.

1	ICDs are highly effective for termination of VT and VF and also provide bradycardia pacing. ICDs decrease mortality in patients at risk for sudden death due to structural heart diseases. In all cases, ICDs are recommended only if there is also expectation for survival of at least a year with acceptable functional capacity. The exception is in cases of patients with end-stage heart disease who are awaiting cardiac transplantation outside the hospital, or who have left bundle branch block QRS prolongation such that they are likely to have improvement in ventricular function with cardiac resynchronization therapy from a biventricular ICD.

1	ICDs can often terminate monomorphic VT by a burst of rapid pacing faster than the VT, known as antitachycardia pacing (ATP) (Fig. 277-9A). If ATP fails or is not a programmed treatment, as is often the case for rapid VT or VF, a shock is delivered (Fig. 277-9B). Shocks are painful if the patient is conscious. The most common ICD complication is the delivery of unnecessary therapy (either ATP or shocks) in response to a rapid supraventricular tachycardia or electrical noise as a result of an ICD lead fracture. Interrogation of 1499 the ICD, which can be performed remotely and communicated via Internet, is critical after an ICD shock to determine the arrhythmia diagnosis and exclude an unnecessary therapy. Device infection occurs in approximately 1% of patients.

1	Despite prompt termination of VT or VF by an ICD, the occurrence of these arrhythmias predicts subsequent increased mortality and risk of heart failure. Occurrence of VT or VF should therefore prompt assessment for potential causes including worsening heart failure, electrolyte abnormalities, and ischemia. Repeated shocks, even if appropriate, often induce posttraumatic stress disorder. Antiarrhythmic drugs mostly in the form of amiodarone or catheter ablation are often required for suppression of recurrent arrhythmias. Antiarrhythmic drug therapy can alter the VT rate and the energy required for defibrillation, thereby necessitating programming changes in the ICD’s algorithms for detection and therapy.

1	Catheter ablation is performed by guiding an electrode catheter to the arrhythmia origin and producing a thermal injury with radiofrequency current. The size and location of the arrhythmia substrate determine the ease and likely effectiveness of the procedure, as well as potential complications. The most common complications, which occur in <5% of patients, are related to vascular access, including bleeding, femoral hematomas, arteriovenous fistulae, and pseudoaneurysms. Catheter ablation is a reasonable first-line therapy for many patients with symptomatic idiopathic VTs. Success rates for those originating from a focus in the right ventricular outflow tract are in the range of 80–90% but lower for idiopathic VTs arising in less common locations such as from the LV outflow tract or aortic root, along the atrioventricular valve annuli, and from the papillary muscles. Failure of ablation is often due to inability to induce the

1	FIGURE 277-9 Implantable cardioverter-defibrillator (ICD) and therapies for ventricular arrhythmias. A. A monomorphic ventricular tachycardia (VT) is terminated by a burst of pacing impulses at a rate faster than VT (antitachycardia pacing). B. A rapid VT is converted with a high-voltage shock (arrow). The chest x-ray in panel C shows the components of an ICD capable of biventricular pacing. ICD generator in the subcutaneous tissue of the left upper chest, pacing leads in the right atrium and the left ventricular (LV) branch of the coronary sinus (LV lead), and a pacing/defibrillating lead in the right ventricle (RV lead) are shown.

1	1500 arrhythmia for precise localization or because the origin of the VT is from a site that is inaccessible or in close proximity to a coronary artery. Complications are infrequent but can include perforation with cardiac tamponade, atrioventricular block due to injury to the conduction system, and coronary artery injury for foci in proximity to a coronary vessel. In patients with scar-related VT due to prior infarction or cardiomyopathy, ablation targets abnormal regions in the scar. Because these scars often contain multiple reentry circuits over relatively large regions, extensive areas of ablation are required, and these areas are often identified as regions of low voltage displayed on anatomic reconstructions of the ventricle (Fig. 277-5). If the circuits are not confined to the subendocardial scar, epicardial mapping and ablation can be performed via a subxiphoid pericardial puncture, similar to a pericardiocentesis. Epicardial mapping and ablation are often required for VTs

1	scar, epicardial mapping and ablation can be performed via a subxiphoid pericardial puncture, similar to a pericardiocentesis. Epicardial mapping and ablation are often required for VTs due to nonischemic cardiomyopathy, but also have potentially greater risks of bleeding, coronary injury, and post-procedure pericarditis, which is usually transient. For drug-refractory VT due to prior MI, ablation abolishes VT in approximately half of patients and reduces the frequency of VT in an additional 20%. More than one procedure is necessary in up to 30% of patients. Ablation can be lifesaving for patients with very frequent or incessant VT. Procedure-related mortality is in the range of 3%, with most mortality due to continued uncontrollable VT when the procedure fails. In nonischemic heart disease, the arrhythmia substrate locations are more variable and outcomes are less well defined. Catheter ablation can also be lifesaving for rare patients with recurrent polymorphic VT and VF that is

1	the arrhythmia substrate locations are more variable and outcomes are less well defined. Catheter ablation can also be lifesaving for rare patients with recurrent polymorphic VT and VF that is repeatedly initiated by a uniform PVC. The initiating ectopic beat often originates from the Purkinje system or the right ventricular outflow tract and can be targeted for ablation. When antiarrhythmic drug therapy and catheter ablation fail or are not options, surgical cryoablation, often combined with aneurysmectomy, can be effective therapy for recurrent VT due to prior MI and has also been used successfully in a few patients with nonischemic heart disease. Few centers now maintain the expertise for this therapy. Injection of absolute ethanol into the coronary arterial blood supply of the arrhythmia substrate has also been used for ablation in a small number of patients who have failed catheter ablation and drugs.

1	Patients with ventricular arrhythmias fall into three general groups. The first are those who have associated structural heart disease that must be detected. The risk of life-threatening arrhythmias causing sudden death is indicated by the nature of the arrhythmia—sustained (or causing cardiac arrest) or nonsustained, in which case the risk of life-threatening arrhythmias is assessed from the severity of the heart disease, usually the severity of ventricular dysfunction. ICDs provide the most protection from sudden arrhythmic death. The second group comprises those who do not have recognizable structural heart disease, but have a genetic syndrome associated with increased risk of sudden death. A family history of sudden death and abnormal electrocardiogram most frequently suggest the diagnosis. The third group includes individuals with benign idiopathic arrhythmias who may require therapy to control symptoms, but who are not at significant risk for life-threatening arrhythmias. The

1	The third group includes individuals with benign idiopathic arrhythmias who may require therapy to control symptoms, but who are not at significant risk for life-threatening arrhythmias. The appropriate recognition of these patients is facilitated by thoughtful application of ECG and cardiac imaging. High-risk individuals benefit from specialized care for consideration of ICDs, catheter ablation, and antiarrhythmic drug therapy.

1	Atlas of Cardiac Arrhythmias Ary L. Goldberger The electrocardiograms in this atlas supplement those illustrated in Chaps. 274 and 276. The interpretations emphasize findings of specific teaching value. All of the figures are adapted from cases in ECG Wave-Maven, Copyright 2003, Beth Israel Deaconess Medical Center, http://ecg .bidmc.harvard.edu. The abbreviations used in this chapter are as follows: CHAPTER 278e Atlas of Cardiac Arrhythmias Figure 278e-1 Respiratory sinus arrhythmia, a physiologic finding in a healthy young adult. The rate of the sinus pacemaker is relatively slow at the beginning of the strip during expiration, then accelerates during inspiration and slows again with expiration. Changes are due to cardiac vagal tone modulation with breathing. PART 10 Disorders of the Cardiovascular System

1	PART 10 Disorders of the Cardiovascular System Figure 278e-2 Sinus tachycardia (110/min) with first-degree AV “block” (conduction delay) with PR interval = 0.28 s. The P wave is visible after the ST-T wave in V1−V3 and superimposed on the T wave in other leads. Atrial (nonsinus) tachycardias may produce a similar pattern, but the rate is usually faster. Figure 278e-3 Sinus rhythm (P wave rate about 60/min) with 2:1 AV (second-degree) block causing marked bradycardia (ventricular rate of about 30/min). LVH is also present, along with left atrial abnormality. Figure 278e-4 Sinus rhythm (P wave rate about 60/min) with 2:1 (second-degree) AV block yielding a ventricular (pulse) rate of about 30/min. Left atrial abnormality. RBBB with left anterior fascicular block. Possible inferior MI. CHAPTER 278e Atlas of Cardiac Arrhythmias

1	CHAPTER 278e Atlas of Cardiac Arrhythmias Figure 278e-5 Marked junctional bradycardia (25 beats/min). Rate is regular with a flat baseline between narrow QRS complexes, without evident P waves. Patient was on atenolol, with possible underlying sick sinus syndrome. The serum potassium was slightly elevated at 5.5 mEq/L. PART 10 Disorders of the Cardiovascular System Figure 278e-6 Sinus rhythm at a rate of 64/min (P wave rate) with third-degree (complete) AV block yielding an effective heart (pulse) rate of 40/min. The slow, narrow QRS complexes indicate an AV junctional escape pacemaker. Left atrial abnormality. Figure 278e-7 Sinus rhythm at a rate of 90/min with advanced second-degree AV block and possible transient complete heart block with Lyme carditis.

1	Figure 278e-7 Sinus rhythm at a rate of 90/min with advanced second-degree AV block and possible transient complete heart block with Lyme carditis. Figure 278e-8 Multifocal atrial tachycardia with varying P-wave morphologies and P-P intervals; right atrial overload with peaked P waves in II, III, and aVF (with vertical P-wave axis); superior QRS axis; slow R-wave progression with delayed transition in precordial leads in patient with severe chronic obstructive lung disease. Figure 278e-9 NSR in a patient with Parkinson’s disease. Tremor artifact, best seen in limb leads. This tremor artifact may sometimes be confused with atrial flutter/fibrillation. Borderline voltage criteria for LVH are present. CHAPTER 278e Atlas of Cardiac Arrhythmias PART 10 Disorders of the Cardiovascular System

1	CHAPTER 278e Atlas of Cardiac Arrhythmias PART 10 Disorders of the Cardiovascular System Figure 278e-10 Atrial tachycardia with atrial rate of about 200/min (note lead V1), 2:1 AV block (conduction), and one premature ventricular complex. Also present: LVH with intraventricular conduction delay and slow precordial R-wave progression (cannot rule out prior anterior MI). Figure 278e-11 Atrial tachycardia with 2:1 block. P-wave rate is about 150/min, with ventricular (QRS) rate of about 75/min. The nonconducted (“extra”) P waves just after the QRS complex are best seen in lead V1. Also, note incomplete RBBB and borderline QT prolongation. Figure 278e-12 Atrial tachycardia (180/min with 2:1 AV block [see lead V1]). LVH by precordial voltage and nonspecific ST-T changes. Slow R-wave progression (V1−V4) raises consideration of prior anterior MI.

1	Figure 278e-13 AV nodal reentrant tachycardia (AVNRT) at a rate of 150/min. Note subtle “pseudo” R waves in lead aVR due to retrograde atrial activation, which usually occurs nearly simultaneous with ventricles in AVNRT. Left-axis deviation consistent with left anterior fascicular block (hemiblock) is also present. CHAPTER 278e Atlas of Cardiac Arrhythmias PART 10 Disorders of the Cardiovascular System Figure 278e-14 Atrial flutter with 2:1 AV conduction. Note typical atrial flutter waves, partly hidden in the early ST segment, seen, for example, in leads II and V1. Figure 278e-15 Atrial flutter with atrial rate 300/min and variable (predominant 2:1 and 3:1) AV conduction. Typical flutter waves are best seen in lead II.

1	Figure 278e-15 Atrial flutter with atrial rate 300/min and variable (predominant 2:1 and 3:1) AV conduction. Typical flutter waves are best seen in lead II. Figure 278e-16 Wide complex tachycardia. Atrial flutter with 2:1 AV conduction (block) and LBBB, not to be mistaken for VT. Typical atrial flutter activity is clearly present in lead II, at a cycle rate of about 320/min, yielding an effective ventricular rate of about 160/min. Figure 278e-17 AF with LBBB. The ventricular rhythm is erratically irregular. Coarse fibrillatory waves are best seen in lead V1, with a typical LBBB pattern. CHAPTER 278e Atlas of Cardiac Arrhythmias PART 10 Disorders of the Cardiovascular System Figure 278e-18 AF with complete heart block and a junctional escape mechanism causing a slow regular ventricular response (45/ min). The QRS complexes show an intraventricular conduction delay with left-axis deviation and LVH. Q-T (U) prolongation is also present.

1	Figure 278e-19 AF with right-axis deviation and LVH. Tracing suggests biventricular hypertrophy in a patient with mitral stenosis and aortic valve disease. Figure 278e-20 WPW preexcitation pattern, with triad of short PR, wide QRS, and delta waves. Polarity of the delta waves (slightly positive in leads V1 and V2 and most positive in lead II and lateral chest leads) is consistent with a right-sided bypass tract. Figure 278e-21 AF in patient with the WPW syndrome and antegrade conduction down the bypass tract leading to a wide complex tachycardia. Rhythm is “irregularly irregular,” and rate is extremely rapid (about 230/min). Not all beats are preexcited. CHAPTER 278e Atlas of Cardiac Arrhythmias PART 10 Disorders of the Cardiovascular System Figure 278e-22 Accelerated idioventricular rhythm (AIVR) originating from the LV and accounting for RBBB morphology. ST elevations in the precordial leads from underlying acute MI.

1	Figure 278e-22 Accelerated idioventricular rhythm (AIVR) originating from the LV and accounting for RBBB morphology. ST elevations in the precordial leads from underlying acute MI. Figure 278e-23 Prolonged (0.60 s) QT interval in a patient with a hereditary long QT syndrome. CHAPTER 278e Atlas of Cardiac Arrhythmias Figure 278e-24 Monomorphic VT at rate of 170/min. The RBBB morphology in V1 and the R:S ratio <1 in V6 are both suggestive of VT. The morphology of the VT is suggestive of origin from the left side of the heart, near the base (RBBB with inferior/rightward axis). Baseline artifact is present in leads V1−V3. Heart Failure: Pathophysiology and Diagnosis Douglas L. Mann, Murali Chakinala HEART FAILURE DEFINITION 279 SEC Tion 4

1	Heart Failure: Pathophysiology and Diagnosis Douglas L. Mann, Murali Chakinala HEART FAILURE DEFINITION 279 SEC Tion 4 Despite repeated attempts to develop a mechanistic definition that encompasses the heterogeneity and complexity of heart failure (HF), no single conceptual paradigm has withstood the test of time. The current American College of Cardiology Foundation (ACCF)/American Heart Association (AHA) guidelines define HF as a complex clinical syndrome that results from structural or functional impairment of ventricular filling or ejection of blood, which in turn leads to the cardinal clinical symptoms of dyspnea and fatigue and signs of HF, namely edema and rales. Because many patients present without signs or symptoms of volume overload, the term “heart failure” is preferred over the older term “congestive heart failure.”

1	HF is a burgeoning problem worldwide, with more than 20 million people affected. The overall prevalence of HF in the adult population in developed countries is 2%. HF prevalence follows an exponential pattern, rising with age, and affects 6–10% of people over age 65. Although the relative incidence of HF is lower in women than in men, women constitute at least one-half the cases of HF because of their longer life expectancy. In North America and Europe, the lifetime risk of developing HF is approximately one in five for a 40-year-old. The overall prevalence of HF is thought to be increasing, in part because current therapies for cardiac disorders, such as myocardial infarction (MI), valvular heart disease, and arrhythmias, are allowing patients to survive longer. Very little is known about the prevalence or risk of developing HF in emerging nations because of the lack of population-based studies in those countries. HF was once thought to arise primarily in the setting of a depressed

1	the prevalence or risk of developing HF in emerging nations because of the lack of population-based studies in those countries. HF was once thought to arise primarily in the setting of a depressed left ventricular (LV) ejection fraction (EF); however, epidemiologic studies have shown that approximately one-half of patients who develop HF have a normal or preserved EF (EF ≥50%). Accordingly, the historical terms “systolic” and “diastolic” HF have been abandoned, and HF patients are now broadly categorized into HF with a reduced EF (HFrEF; formerly systolic failure) or HF with a preserved EF (HRpEF; formerly diastolic failure).

1	As shown in Table 279-1, any condition that leads to an alteration in LV structure or function can predispose a patient to developing HF. Although the etiology of HF in patients with a preserved EF differs from that of patients with depressed EF, there is considerable overlap between the etiologies of these two conditions. In industrialized countries, coronary artery disease (CAD) has become the predominant cause in men and women and is responsible for 60–75% of cases of HF. Hypertension contributes to the development of HF in 75% of patients, including most patients with CAD. Both CAD and hypertension interact to augment the risk of HF, as does diabetes mellitus.

1	In 20–30% of the cases of HF with a depressed EF, the exact etiologic basis is not known. These patients are referred to as having nonischemic, dilated, or idiopathic cardiomyopathy if the cause is unknown (Chap. 287). Prior viral infection or toxin exposure (e.g., alcoholic or chemotherapeutic) also may lead to a dilated cardiomyopathy. Moreover, it is becoming increasingly clear that a large number of cases of dilated cardiomyopathy are secondary to specific genetic defects, most notably those in the cytoskeleton. Most forms of familial dilated cardiomyopathy are inherited in an autosomal dominant fashion. Mutations of genes that encode cytoskeletal proteins (desmin, cardiac myosin, vinculin) and nuclear membrane proteins (laminin) have been identified thus far. Dilated cardiomyopathy also is associated with Duchenne’s, Becker’s, and limb-girdle muscular dystrophies. Conditions that lead to a high cardiac output (e.g., arteriovenous fistula, anemia) are seldom responsible for the

1	also is associated with Duchenne’s, Becker’s, and limb-girdle muscular dystrophies. Conditions that lead to a high cardiac output (e.g., arteriovenous fistula, anemia) are seldom responsible for the development of HF in a normal heart; however, in the presence of underlying structural heart disease, these conditions can lead to overt HF.

1	Chronic volume overload Chagas’ disease Regurgitant valvular disease Disorders of rate and rhythm Intracardiac (left-to-right) shunting Chronic bradyarrhythmias Extracardiac shunting Chronic tachyarrhythmias Primary (hypertrophic cardiomy-Infiltrative disorders (amyloidosis, opathies) sarcoidosis) Metabolic disorders Excessive blood flow requirements Thyrotoxicosis Systemic arteriovenous shunting Nutritional disorders (beriberi) Chronic anemia aIndicates conditions that can also lead to heart failure with a preserved ejection fraction. Class I Patients with cardiac disease but without resulting limitation of physical activity. Ordinary physical activity does not cause undue fatigue, palpitations, dyspnea, or anginal pain. Class II Patients with cardiac disease resulting in slight limitation of physical activity. They are comfortable at rest. Ordinary physical activity results in fatigue, palpitation, dyspnea, or anginal pain.

1	Class III Patients with cardiac disease resulting in marked limitation of physical activity. They are comfortable at rest. Less than ordinary activity causes fatigue, palpitation, dyspnea, or anginal pain. Class IV Patients with cardiac disease resulting in inability to carry on any physical activity without discomfort. Symptoms of heart failure or the anginal syndrome may be present even at rest. If any physical activity is undertaken, discomfort is increased. Source: Adapted from New York Heart Association, Inc., Diseases of the Heart and Blood Vessels: Nomenclature and Criteria for Diagnosis, 6th ed. Boston, Little Brown, 1964, p. 114.

1	Source: Adapted from New York Heart Association, Inc., Diseases of the Heart and Blood Vessels: Nomenclature and Criteria for Diagnosis, 6th ed. Boston, Little Brown, 1964, p. 114. Rheumatic heart disease remains a major cause of HF in Africa and Asia, especially in the young. Hypertension is an important cause of HF in the African and African-American populations. Chagas’ disease is still a major cause of HF in South America. Not surprisingly, anemia is a frequent concomitant factor in HF in many developing nations. As developing nations undergo socioeconomic development, the epidemiology of HF is becoming similar to that of Western Europe and North America, with CAD emerging as the single most common cause of HF. Although the contribution of diabetes mellitus to HF is not well understood, diabetes accelerates atherosclerosis and often is associated with hypertension.

1	Despite many recent advances in the evaluation and management of HF, the development of symptomatic HF still carries a poor prognosis. Community-based studies indicate that 30–40% of patients die within 1 year of diagnosis and 60–70% die within 5 years, mainly from worsening HF or as a sudden event (probably because of a ventricular arrhythmia). Although it is difficult to predict prognosis in an individual, patients with symptoms at rest (New York Heart Association [NYHA] class IV) have a 30–70% annual mortality rate, whereas patients with symptoms with moderate activity (NYHA class II) have an annual mortality rate of 5–10%. Thus, functional status is an important predictor of patient outcome (Table 279-2).

1	Figure 279-1 provides a general conceptual framework for considering the development and progression of HFrEF. As shown, HF may be viewed as a progressive disorder that is initiated after an index event either damages the heart muscle, with a resultant loss of functioning cardiac myocytes, or, alternatively, disrupts the ability of the myocardium to generate force, thereby preventing the heart from contracting normally. This index event may have an abrupt onset, as in the case of an MI; it may have a gradual or insidious onset, as in the case of hemodynamic pressure or volume overloading; or it may be hereditary, as in the case of many of the genetic cardiomyopathies. Regardless of the nature of the inciting event, the feature that is common to each of these index events is that they all in some manner produce a decline in the pumping capacity of the heart. In most instances, patients remain asymptomatic or minimally symptomatic after the initial decline in pumping capacity of the

1	in some manner produce a decline in the pumping capacity of the heart. In most instances, patients remain asymptomatic or minimally symptomatic after the initial decline in pumping capacity of the heart or develop symptoms only after the dysfunction has been present for some time.

1	Although the precise reasons why patients with LV dysfunction may remain asymptomatic is not certain, one potential explanation is that a number of compensatory mechanisms become activated in the presence of cardiac injury and/or LV dysfunction allowing patients Heart Failure: Pathophysiology and Diagnosis Time, years

1	FIGURE 279-1 Pathogenesis of heart failure with a depressed ejection fraction. Heart failure begins after an index event produces an initial decline in the heart's pumping capacity. After this initial decline in pumping capacity, a variety of compensatory mechanisms are activated, including the adrenergic nervous system, the reninangiotensin-aldosterone system, and the cytokine system. In the short term, these systems are able to restore cardiovascular function to a normal homeostatic range with the result that the patient remains asymptomatic. However, with time, the sustained activation of these systems can lead to secondary end-organ damage within the ventricle, with worsening left ventricular remodeling and subsequent cardiac decompensation. (From D Mann: Circulation 100:999, 1999.) to sustain and modulate LV function for a period of months to years. The compensatory mechanisms that have been described thus far include (1) activation of the renin-angiotensin-aldosterone (RAA) and

1	sustain and modulate LV function for a period of months to years. The compensatory mechanisms that have been described thus far include (1) activation of the renin-angiotensin-aldosterone (RAA) and adrenergic nervous systems, which are responsible, respectively, for maintaining cardiac output through increased retention of salt and water (Fig. 279-2), and (2) increased myocardial contractility. In addition, there is activation of a family of countervailing vasodilatory molecules, including the atrial and brain natriuretic peptides (ANP and BNP), prostaglandins (PGE2 and PGI2), and nitric oxide (NO), that offsets the excessive peripheral vascular vasoconstriction. Genetic background, sex, age, or environment may influence these compensatory mechanisms, which are able to modulate LV function within a physiologic/homeostatic range so that the functional capacity of the patient is preserved or is depressed only minimally. Thus, patients may remain asymptomatic or minimally symptomatic for

1	within a physiologic/homeostatic range so that the functional capacity of the patient is preserved or is depressed only minimally. Thus, patients may remain asymptomatic or minimally symptomatic for a period of years; however, at some point patients become overtly symptomatic, with a resultant striking increase in morbidity and mortality rates. Although the exact mechanisms that are responsible for this transition are not known, as will be discussed below, the transition to symptomatic HF is accompanied by increasing activation of neurohormonal, adrenergic, and cytokine systems that lead to a series of adaptive changes within the myocardium collectively referred to as LV remodeling.

1	In contrast to our understanding of the pathogenesis of HF with a depressed EF, our understanding of the mechanisms that contribute to the development of HF with a preserved EF is still evolving. That is, although diastolic dysfunction (see below) was thought to be the only mechanism responsible for the development of HF with a preserved EF, community-based studies suggest that additional extracardiac mechanisms may be important, such as increased vascular stiffness and impaired renal function. BASIC MECHANISMS OF HEART FAILURE Heart Failure with a Reduced Ejection Fraction LV remodeling develops in response to a series of complex events that occur at the cellular and molecular levels (Table 279-3). These changes include (1) myocyte hypertrophy; (2) alterations in the contractile properties of the myocyte; (3) progressive loss of myocytes through necrosis,

1	FIGURE 279-2 Activation of neurohormonal systems in heart failure. The decreased cardiac output in heart failure (HF) patients results in an "unloading" of high-pressure baroreceptors (circles) in the left ventricle, carotid sinus, and aortic arch. This unloading of the peripheral baroreceptors leads to a loss of inhibitory parasympathetic tone to the central nervous system (CNS), with a resultant generalized increase in efferent sympathetic tone, and nonosmotic release of arginine vasopressin (AVP) from the pituitary. AVP (or antidiuretic hormone [ADH]) is a powerful vasoconstrictor that increases the permeability of the renal collecting ducts, leading to the reabsorption of free water. These afferent signals to the CNS also activate efferent sympathetic nervous system pathways that innervate the heart, kidney, peripheral vasculature, and skeletal muscles.

1	Sympathetic stimulation of the kidney leads to the release of renin, with a resultant increase in the circulating levels of angiotensin II and aldosterone. The activation of the renin-angiotensin-aldosterone system promotes salt and water retention and leads to vasoconstriction of the peripheral vasculature, myocyte hypertrophy, myocyte cell death, and myocardial fibrosis. Although these neurohormonal mechanisms facilitate short-term adaptation by maintaining blood pressure, and hence perfusion to vital organs, the same neurohormonal mechanisms are believed to contribute to end-organ changes in the heart and the circulation and to the excessive salt and water retention in advanced HF. (Modified from A Nohria et al: Neurohormonal, renal and vascular adjustments, in Atlas of Heart Failure: Cardiac Function and Dysfunction, 4th ed, WS Colucci [ed]. Philadelphia, Current Medicine Group 2002, p. 104.) apoptosis, and autophagic cell death; (4) β-adrenergic desensitization; (5) abnormal

1	Cardiac Function and Dysfunction, 4th ed, WS Colucci [ed]. Philadelphia, Current Medicine Group 2002, p. 104.) apoptosis, and autophagic cell death; (4) β-adrenergic desensitization; (5) abnormal myocardial energetics and metabolism; and (6) reorganization of the extracellular matrix with dissolution of the organized structural collagen weave surrounding myocytes and subsequent replacement by an interstitial collagen matrix that does not

1	Source: Adapted from D. Mann: Pathophysiology of heart failure, in Braunwald's Heart Disease, 8th ed, PL Libby et al (eds). Philadelphia, Elsevier, 2008, p. 550. provide structural support to the myocytes. The biologic stimuli for these profound changes include mechanical stretch of the myocyte, circulating neurohormones (e.g., norepinephrine, angiotensin II), inflammatory cytokines (e.g., tumor necrosis factor [TNF]), other peptides and growth factors (e.g., endothelin), and reactive oxygen species (e.g., superoxide). The sustained overexpression of these biologically active molecules is believed to contribute to the progression of HF by virtue of the deleterious effects they exert on the heart and the circulation. Indeed, this insight forms the clinical rationale for using pharmacologic agents that antagonize these systems (e.g., angiotensin-converting enzyme [ACE] inhibitors and beta blockers) in treating patients with HF (Chap. 280).

1	To understand how the changes that occur in the failing cardiac myocyte contribute to depressed LV systolic function in HF, it is instructive first to review the biology of the cardiac muscle cell (Chap. 265e). Sustained neurohormonal activation and mechanical overload result in transcriptional and posttranscriptional changes in the genes and proteins that regulate excitation-contraction coupling and cross-bridge interaction (see Figs. 265e-6 and 265e-7). The changes that regulate excitation-contraction include decreased function of sarcoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA2A), resulting in decreased calcium uptake into the sarcoplasmic reticulum (SR), and hyperphosphorylation of the ryanodine receptor, leading to calcium leakage from the SR. The changes that occur in the cross-bridges include decreased expression of α-myosin heavy chain and increased expression of β-myosin heavy chain, myocytolysis, and disruption of the cytoskeletal links between the sarcomeres and

1	cross-bridges include decreased expression of α-myosin heavy chain and increased expression of β-myosin heavy chain, myocytolysis, and disruption of the cytoskeletal links between the sarcomeres and the extracellular matrix. Collectively, these changes impair the ability of the myocyte to contract and therefore contribute to the depressed LV systolic function observed in patients with HF.

1	Myocardial relaxation is an adenosine triphosphate (ATP)dependent process that is regulated by uptake of cytoplasmic calcium into the SR by SERCA2A and extrusion of calcium by sarcolemmal pumps (see Fig. 265e-7). Accordingly, reductions in ATP concentration, as occurs in ischemia, may interfere with these processes and lead to slowed myocardial relaxation. Alternatively, if LV filling is delayed because LV compliance is reduced (e.g., from hypertrophy or fibrosis), LV filling pressures will similarly remain elevated at end diastole (see Fig. 265e-11). An increase in heart rate disproportionately shortens the time for diastolic filling, which may lead to elevated LV filling pressures, particularly in noncompliant ventricles. Elevated LV end-1503 diastolic filling pressures result in increases in pulmonary capillary pressures, which can contribute to the dyspnea experienced by patients with diastolic dysfunction. In addition to impaired myocardial relaxation, increased myocardial

1	increases in pulmonary capillary pressures, which can contribute to the dyspnea experienced by patients with diastolic dysfunction. In addition to impaired myocardial relaxation, increased myocardial stiffness secondary to cardiac hypertrophy and increased myocardial collagen content may contribute to diastolic failure. Importantly, diastolic dysfunction can occur alone or in combination with systolic dysfunction in patients with HF.

1	Left Ventricular Remodeling Ventricular remodeling refers to the changes in LV mass, volume, and shape and the composition of the heart that occur after cardiac injury and/or abnormal hemodynamic loading conditions. LV remodeling may contribute independently to the progression of HF by virtue of the mechanical burdens that are engendered by the changes in the geometry of the remodeled LV. In addition to the increase in LV end-diastolic volume, LV wall thinning occurs as the left ventricle begins to dilate. The increase in wall thinning, along with the increase in afterload created by LV dilation, leads to a functional afterload mismatch that may contribute further to a decrease in stroke volume. Moreover, the high end-diastolic wall stress might be expected to lead to (1) hypoperfusion of the subendocardium, with resultant worsening of LV function; (2) increased oxidative stress, with the resultant activation of families of genes that are sensitive to free radical generation (e.g.,

1	of the subendocardium, with resultant worsening of LV function; (2) increased oxidative stress, with the resultant activation of families of genes that are sensitive to free radical generation (e.g., TNF and interleukin 1β); and (3) sustained expression of stretch-activated genes (angiotensin II, endothelin, and TNF) and/or stretch activation of hypertrophic signaling pathways. Increasing LV dilation also results in tethering of the papillary muscles with resulting incompetence of the mitral valve apparatus and functional mitral regurgitation, which in turn leads to further hemodynamic overloading of the ventricle. Taken together, the mechanical burdens that are engendered by LV remodeling contribute to the progression of HF. Recent studies have shown that LV remodeling can be reversed following medical and device therapy and that reverse LV remodeling is associated with improved clinical outcomes in patients with HFrEF. Indeed, one of the goals of therapy for HF is to prevent and/or

1	medical and device therapy and that reverse LV remodeling is associated with improved clinical outcomes in patients with HFrEF. Indeed, one of the goals of therapy for HF is to prevent and/or reverse LV remodeling.

1	CLINICAL MANIFESTATIONS Symptoms The cardinal symptoms of HF are fatigue and shortness of breath. Although fatigue traditionally has been ascribed to the low cardiac output in HF, it is likely that skeletal-muscle abnormalities and other noncardiac comorbidities (e.g., anemia) also contribute to this symptom. In the early stages of HF, dyspnea is observed only during exertion; however, as the disease progresses, dyspnea occurs with less strenuous activity, and it ultimately may occur even at rest. The origin of dyspnea in HF is probably multifactorial (Chap. 47e). The most important mechanism is pulmonary congestion with accumulation of interstitial or intra-alveolar fluid, which activates juxtacapillary J receptors, which in turn stimulate the rapid, shallow breathing characteristic of cardiac dyspnea. Other factors that contribute to dyspnea on exertion include reductions in pulmonary compliance, increased airway resistance, respiratory muscle and/or diaphragm fatigue, and anemia.

1	cardiac dyspnea. Other factors that contribute to dyspnea on exertion include reductions in pulmonary compliance, increased airway resistance, respiratory muscle and/or diaphragm fatigue, and anemia. Dyspnea may become less frequent with the onset of right ventricular (RV) failure and tricuspid regurgitation.

1	orthopnEa Orthopnea, which is defined as dyspnea occurring in the recumbent position, is usually a later manifestation of HF than is exertional dyspnea. It results from redistribution of fluid from the splanchnic circulation and lower extremities into the central circulation during recumbency, with a resultant increase in pulmonary capillary pressure. Nocturnal cough is a common manifestation of this process and a frequently overlooked symptom of HF. Orthopnea generally is relieved by sitting upright or sleeping with additional pillows. Although orthopnea is a relatively specific symptom of HF, it may occur in patients with abdominal obesity or ascites and patients with pulmonary disease whose lung mechanics favor an upright posture. paroxysmal nocturnal dyspnEa (pnd) This term refers to acute episodes of severe shortness of breath and coughing that generally occur at night and awaken the patient from sleep, usually 1–3 h after the

1	Heart Failure: Pathophysiology and Diagnosis 1504 patient retires. PND may manifest as coughing or wheezing, possibly because of increased pressure in the bronchial arteries leading to airway compression, along with interstitial pulmonary edema that leads to increased airway resistance. Whereas orthopnea may be relieved by sitting upright at the side of the bed with the legs in a dependent position, patients with PND often have persistent coughing and wheezing even after they have assumed the upright position. Cardiac asthma is closely related to PND, is characterized by wheezing secondary to bronchospasm, and must be differentiated from primary asthma and pulmonary causes of wheezing.

1	chEynE-stokEs rEspiration Also referred to as periodic respiration or cyclic respiration, Cheyne-Stokes respiration is present in 40% of patients with advanced HF and usually is associated with low cardiac output. Cheyne-Stokes respiration is caused by an increased sensitivity of the respiratory center to arterial PCO2. There is an apneic phase, during which arterial PO2 falls and arterial PCO2 rises. These changes in the arterial blood gas content stimulate the respiratory center, resulting in hyperventilation and hypocapnia, followed by recurrence of apnea. Cheyne-Stokes respirations may be perceived by the patient or the patient’s family as severe dyspnea or as a transient cessation of breathing. acutE pulmonary EdEma See Chap. 326

1	acutE pulmonary EdEma See Chap. 326 Other Symptoms Patients with HF also may present with gastrointestinal symptoms. Anorexia, nausea, and early satiety associated with abdominal pain and fullness are common complaints and may be related to edema of the bowel wall and/or a congested liver. Congestion of the liver and stretching of its capsule may lead to right upper-quadrant pain. Cerebral symptoms such as confusion, disorientation, and sleep and mood disturbances may be observed in patients with severe HF, particularly elderly patients with cerebral arteriosclerosis and reduced cerebral perfusion. Nocturia is common in HF and may contribute to insomnia.

1	A careful physical examination is always warranted in the evaluation of patients with HF. The purpose of the examination is to help determine the cause of HF as well as to assess the severity of the syndrome. Obtaining additional information about the hemodynamic profile and the response to therapy and determining the prognosis are important additional goals of the physical examination.

1	General Appearance and Vital Signs In mild or moderately severe HF, the patient appears to be in no distress at rest except for feeling uncomfortable when lying flat for more than a few minutes. In more severe HF, the patient must sit upright, may have labored breathing, and may not be able to finish a sentence because of shortness of breath. Systolic blood pressure may be normal or high in early HF, but it generally is reduced in advanced HF because of severe LV dysfunction. The pulse pressure may be diminished, reflecting a reduction in stroke volume. Sinus tachycardia is a nonspecific sign caused by increased adrenergic activity. Peripheral vasoconstriction leading to cool peripheral extremities and cyanosis of the lips and nail beds is also caused by excessive adrenergic activity.

1	Jugular Veins (See also Chap. 267) Examination of the jugular veins provides an estimation of right atrial pressure. The jugular venous pressure is best appreciated with the patient lying recumbent, with the head tilted at 45°. The jugular venous pressure should be quantified in centimeters of water (normal ≤8 cm) by estimating the height of the venous column of blood above the sternal angle in centimeters and then adding 5 cm. In the early stages of HF, the venous pressure may be normal at rest but may become abnormally elevated with sustained (~15 seconds) pressure on the abdomen (positive abdominojugular reflux). Giant v waves indicate the presence of tricuspid regurgitation.

1	Pulmonary Examination Pulmonary crackles (rales or crepitations) result from the transudation of fluid from the intravascular space into the alveoli. In patients with pulmonary edema, rales may be heard widely over both lung fields and may be accompanied by expiratory wheezing (cardiac asthma). When present in patients without concomitant lung disease, rales are specific for HF. Importantly, rales are frequently absent in patients with chronic HF, even when LV filling pressures are elevated, because of increased lymphatic drainage of alveolar fluid. Pleural effusions result from the elevation of pleural capillary pressure and the resulting transudation of fluid into the pleural cavities. Since the pleural veins drain into both the systemic and the pulmonary veins, pleural effusions occur most commonly with biventricular failure. Although pleural effusions are often bilateral in HF, when they are unilateral, they occur more frequently in the right pleural space.

1	Cardiac Examination Examination of the heart, although essential, frequently does not provide useful information about the severity of HF. If cardiomegaly is present, the point of maximal impulse (PMI) usually is displaced below the fifth intercostal space and/or lateral to the midclavicular line, and the impulse is palpable over two interspaces. Severe LV hypertrophy leads to a sustained PMI. In some patients, a third heart sound (S3) is audible and palpable at the apex. Patients with enlarged or hypertrophied right ventricles may have a sustained and prolonged left parasternal impulse extending throughout systole. An S3 (or protodiastolic gallop) is most commonly present in patients with volume overload who have tachycardia and tachypnea, and it often signifies severe hemodynamic compromise. A fourth heart sound (S4) is not a specific indicator of HF but is usually present in patients with diastolic dysfunction. The murmurs of mitral and tricuspid regurgitation are frequently

1	compromise. A fourth heart sound (S4) is not a specific indicator of HF but is usually present in patients with diastolic dysfunction. The murmurs of mitral and tricuspid regurgitation are frequently present in patients with advanced HF.

1	Abdomen and Extremities Hepatomegaly is an important sign in patients with HF. When it is present, the enlarged liver is frequently tender and may pulsate during systole if tricuspid regurgitation is present. Ascites, a late sign, occurs as a consequence of increased pressure in the hepatic veins and the veins draining the peritoneum. Jaundice, also a late finding in HF, results from impairment of hepatic function secondary to hepatic congestion and hepatocellular hypoxemia and is associated with elevations of both direct and indirect bilirubin.

1	Peripheral edema is a cardinal manifestation of HF, but it is nonspecific and usually is absent in patients who have been treated adequately with diuretics. Peripheral edema is usually symmetric and dependent in HF and occurs predominantly in the ankles and the pretibial region in ambulatory patients. In bedridden patients, edema may be found in the sacral area (presacral edema) and the scrotum. Long-standing edema may be associated with indurated and pigmented skin. Cardiac Cachexia With severe chronic HF, there may be marked weight loss and cachexia. Although the mechanism of cachexia is not entirely understood, it is probably multifactorial and includes elevation of the resting metabolic rate; anorexia, nausea, and vomiting due to congestive hepatomegaly and abdominal fullness; elevation of circulating concentrations of cytokines such as TNF; and impairment of intestinal absorption due to congestion of the intestinal veins. When present, cachexia augurs a poor overall prognosis.

1	The diagnosis of HF is relatively straightforward when the patient presents with classic signs and symptoms of HF; however, the signs and symptoms of HF are neither specific nor sensitive. Accordingly, the key to making the diagnosis is to have a high index of suspicion, particularly for high-risk patients. When these patients present with signs or symptoms of HF, additional laboratory testing should be performed. Routine Laboratory Testing Patients with new-onset HF and those with chronic HF and acute decompensation should have a complete blood count, a panel of electrolytes, blood urea nitrogen, serum creatinine, hepatic enzymes, and a urinalysis. Selected patients should have assessment for diabetes mellitus (fasting serum glucose or oral glucose tolerance test), dyslipidemia (fasting lipid panel), and thyroid abnormalities (thyroid-stimulating hormone level).

1	Electrocardiogram (ECG) A routine 12-lead ECG is recommended. The major importance of the ECG is to assess cardiac rhythm and determine the presence of LV hypertrophy or a prior MI (presence or absence of Q waves) as well as to determine QRS width to ascertain whether the patient may benefit from resynchronization therapy (see below). A normal ECG virtually excludes LV systolic dysfunction. Chest X-Ray A chest x-ray provides useful information about cardiac size and shape, as well as the state of the pulmonary vasculature, and may identify noncardiac causes of the patient’s symptoms. Although patients with acute HF have evidence of pulmonary hypertension, interstitial edema, and/or pulmonary edema, the majority of patients with chronic HF do not. The absence of these findings in patients with chronic HF reflects the increased capacity of the lymphatics to remove interstitial and/or pulmonary fluid.

1	Assessment of LV Function Noninvasive cardiac imaging (Chap. 270e) is essential for the diagnosis, evaluation, and management of HF. The most useful test is the two-dimensional (2-D) echocardiogram/ Doppler, which can provide a semiquantitative assessment of LV size and function as well as the presence or absence of valvular and/ or regional wall motion abnormalities (indicative of a prior MI). The presence of left atrial dilation and LV hypertrophy, together with abnormalities of LV diastolic filling provided by pulse-wave and tissue Doppler, is useful for the assessment of HF with a preserved EF. The 2-D echocardiogram/Doppler is also invaluable in assessing RV size and pulmonary pressures, which are critical in the evaluation and management of cor pulmonale (see below). Magnetic resonance imaging (MRI) also provides a comprehensive analysis of cardiac anatomy and function and is now the gold standard for assessing LV mass and volumes. MRI also is emerging as a useful and accurate

1	imaging (MRI) also provides a comprehensive analysis of cardiac anatomy and function and is now the gold standard for assessing LV mass and volumes. MRI also is emerging as a useful and accurate imaging modality for evaluating patients with HF, both in terms of assessing LV structure and for determining the cause of HF (e.g., amyloidosis, ischemic cardiomyopathy, hemochromatosis).

1	The most useful index of LV function is the EF (stroke volume divided by end-diastolic volume). Because the EF is easy to measure by noninvasive testing and easy to conceptualize, it has gained wide acceptance among clinicians. Unfortunately, the EF has a number of limitations as a true measure of contractility, since it is influenced by alterations in afterload and/or preload. Nonetheless, with the exceptions indicated above, when the EF is normal (≥50%), systolic function is usually adequate, and when the EF is significantly depressed (<30–40%), contractility is usually depressed.

1	Biomarkers Circulating levels of natriuretic peptides are useful and important adjunctive tools in the diagnosis of patients with HF. Both B-type natriuretic peptide (BNP) and N-terminal pro-BNP (NT-proBNP), which are released from the failing heart, are relatively sensitive markers for the presence of HF with depressed EF; they also are elevated in HF patients with a preserved EF, albeit to a lesser degree. In ambulatory patients with dyspnea, the measurement of BNP or NT-proBNP is useful to support clinical decision making regarding the diagnosis of HF, especially in the setting of clinical uncertainty. Moreover, the measurement of BNP or NT-proBNP is useful for establishing prognosis or disease severity in chronic HF and can be useful to achieve optimal dosing of medical therapy in select clinically euvolemic patients. However, it is important to recognize that natriuretic peptide levels increase with age and renal impairment, are more elevated in women, and can be elevated in

1	select clinically euvolemic patients. However, it is important to recognize that natriuretic peptide levels increase with age and renal impairment, are more elevated in women, and can be elevated in right HF from any cause. Levels can be falsely low in obese patients. Other biomarkers, such as soluble ST-2 and galectin-3, are newer biomarkers that can be used for determining the prognosis of HF patients.

1	Exercise Testing Treadmill or bicycle exercise testing is not routinely advocated for patients with HF, but either is useful for assessing the need for cardiac transplantation in patients with advanced HF (Chap. 281). A peak oxygen uptake (vo2) <14 mL/kg per min is asso-1505 ciated with a relatively poor prognosis. Patients with a vo2 <14 mL/ kg per min have been shown, in general, to have better survival when transplanted than when treated medically.

1	HF resembles but should be distinguished from (1) conditions in which there is circulatory congestion secondary to abnormal salt and water retention but in which there is no disturbance of cardiac structure or function (e.g., renal failure), and (2) noncardiac causes of pulmonary edema (e.g., acute respiratory distress syndrome). In most patients who present with classic signs and symptoms of HF, the diagnosis is relatively straightforward. However, even experienced clinicians have difficulty differentiating the dyspnea that arises from cardiac and pulmonary causes (Chap. 47e). In this regard, noninvasive cardiac imaging, biomarkers, pulmonary function testing, and chest x-ray may be useful. A very low BNP or NT-proBNP may be helpful in excluding a cardiac cause of dyspnea in this setting. Ankle edema may arise secondary to varicose veins, obesity, renal disease, or gravitational effects. When HF develops in patients with a preserved EF, it may be difficult to determine the relative

1	Ankle edema may arise secondary to varicose veins, obesity, renal disease, or gravitational effects. When HF develops in patients with a preserved EF, it may be difficult to determine the relative contribution of HF to the dyspnea that occurs in chronic lung disease and/or obesity.

1	Cor pulmonale, often referred to as pulmonary heart disease, can be defined as altered RV structure and/or function in the context of chronic lung disease and is triggered by the onset of pulmonary hypertension. Although RV dysfunction is also an important sequela of HFpEF and HFrEF, this is not considered as cor pulmonale. Cor pulmonale develops in response to acute or chronic changes in the pulmonary vasculature and/or the lung parenchyma that are sufficient to cause pulmonary hypertension. The true prevalence of cor pulmonale is difficult to ascertain. First, not all patients with chronic lung disease will develop cor pulmonale, which may be subclinical in compensated individuals. Second, our ability to diagnose pulmonary hypertension and cor pulmonale by routine physical examination and laboratory testing is relatively insensitive. However, advances in 2-D echo/Doppler imaging and biomarkers (BNP) can make it easier to identify cor pulmonale.

1	Once patients with chronic pulmonary or pulmonary vascular disease develop cor pulmonale, the prognosis worsens. Although chronic obstructive pulmonary disease (COPD) and chronic bronchitis are responsible for approximately 50% of the cases of cor pulmonale in North America (Chap. 314), any disease that affects the pulmonary vasculature (Chap. 304) or parenchyma can lead to cor pulmonale (Table 279-4). Primary pulmonary vascular disorders are relatively rare causes of cor pulmonale, but cor pulmonale is extremely common with these conditions, given the magnitude of pulmonary hypertension present.

1	Although many conditions can lead to cor pulmonale, the common pathophysiologic mechanism is pulmonary hypertension that is sufficient to alter RV structure (i.e., dilation with or without hypertrophy) and function. Normally, pulmonary artery pressures are only ~15 mmHg and do not increase even with multiples of resting cardiac output, because of vasodilation and blood vessel recruitment of the pulmonary circulatory bed. But, in the setting of parenchymal lung diseases, primary pulmonary vascular disorders, or chronic (alveolar) hypoxia, the circulatory bed undergoes varying degrees of vascular remodeling, vasoconstriction, and destruction. As a result, pulmonary artery pressures and RV afterload increase, setting the stage for cor pulmonale (Table 279-4). The systemic consequences of cor pulmonale relate to alterations in cardiac output as well as salt and Heart Failure: Pathophysiology and Diagnosis Diseases of the Lung Parenchyma Disorders of Chronic (Alveolar) Hypoxia

1	Heart Failure: Pathophysiology and Diagnosis Diseases of the Lung Parenchyma Disorders of Chronic (Alveolar) Hypoxia Kyphoscoliosis Living at high altitude Diseases of the Pulmonary Vasculature water homeostasis. Anatomically, the RV is a thin-walled, compliant chamber that is better suited to handle volume overload than pressure overload. Thus, the sustained pressure overload imposed by pulmonary hypertension and increased pulmonary vascular resistance eventually causes the RV to fail.

1	The response of the RV to pulmonary hypertension depends on the acuteness and severity of the pressure overload. Acute cor pulmonale occurs after a sudden and severe stimulus (e.g., massive pulmonary embolus), with RV dilatation and failure but no RV hypertrophy (Chap. 300). Chronic cor pulmonale, however, is associated with a more slowly evolving and progressive pulmonary hypertension that leads to initial modest RV hypertrophy and subsequent RV dilation. Acute decompensation of previously compensated chronic cor pulmonale is a common clinical occurrence. Triggers include worsening hypoxia from any cause (e.g., pneumonia), acidemia (e.g., exacerbation of COPD), acute pulmonary embolus, atrial tachyarrhythmia, hypervolemia, and mechanical ventilation that leads to compressive forces on alveolar blood vessels.

1	CLINICAL MANIFESTATIONS Symptoms The symptoms of chronic cor pulmonale generally are related to the underlying pulmonary disorder. Dyspnea, the most common symptom, is usually the result of the increased work of breathing secondary to changes in elastic recoil of the lung (fibrosing lung diseases), altered respiratory mechanics (e.g., overinflation with COPD), or inefficient ventilation (e.g., primary pulmonary vascular disease). Orthopnea and PND are rarely symptoms of isolated right HF and usually point toward concurrent left heart dysfunction. Rarely, these symptoms reflect increased work of breathing in the supine position resulting from compromised diaphragmatic excursion. Abdominal pain and ascites that occur with cor pulmonale are similar to the right HF that ensues in chronic HF. Lower-extremity edema may occur secondary to neurohormonal activation, elevated RV filling pressures, or increased levels of carbon dioxide and hypoxemia, which can lead to peripheral vasodilation and

1	edema may occur secondary to neurohormonal activation, elevated RV filling pressures, or increased levels of carbon dioxide and hypoxemia, which can lead to peripheral vasodilation and edema formation.

1	Signs Many of the signs encountered in cor pulmonale are also present in HF patients with a depressed EF, including tachypnea, elevated jugular venous pressures, hepatomegaly, and lower-extremity edema. Patients may have prominent v waves in the jugular venous pulse as a result of tricuspid regurgitation. Other cardiovascular signs include an RV heave palpable along the left sternal border or in the epigastrium. The increase in intensity of the holosystolic murmur of tricuspid regurgitation with inspiration (“Carvallo’s sign”) may be lost eventually as RV failure worsens. Cyanosis is a late finding in cor pulmonale and is secondary to a low cardiac output with systemic vasoconstriction and ventilation-perfusion mismatches in the lung.

1	The most common cause of right HF is not pulmonary parenchymal or vascular disease but left HF. Therefore, it is important to evaluate the patient for LV systolic and diastolic dysfunction. The ECG in severe pulmonary hypertension shows P pulmonale, right axis deviation, and RV hypertrophy. Radiographic examination of the chest may show enlargement of the main central pulmonary arteries and hilar vessels. Spirometry and lung volumes can identify obstructive and/ or restrictive defects indicative of parenchymal lung diseases; arterial blood gases can demonstrate hypoxemia and/or hypercapnia. Spiral computed tomography (CT) scans of the chest are useful in diagnosing acute thromboembolic disease; however, ventilation-perfusion lung scanning remains best suited for diagnosing chronic thromboembolic disease (Chap. 300). A high-resolution CT scan of the chest can identify interstitial lung disease.

1	Two-dimensional echocardiography is useful for measuring RV thickness and chamber dimensions. Location of the RV behind the sternum and its crescent shape challenge assessment of RV function by echocardiography, especially when parenchymal lung disease is present. Calculated measures of RV function (e.g., tricuspid annular plane systolic excursion [TAPSE] or the Tei Index) supplement more subjective assessments of RV function. The interventricular septum may move paradoxically during systole in the presence of pulmonary hypertension. As noted, Doppler echocardiography can be used to assess pulmonary artery pressures. MRI is also useful for assessing RV structure and function, particularly in patients who are difficult to image with 2-D echocardiography because of severe lung disease. Right-heart catheterization is useful for confirming the diagnosis of pulmonary hypertension and for excluding elevated left-heart pressures (measured as the pulmonary capillary wedge pressure) as a cause

1	catheterization is useful for confirming the diagnosis of pulmonary hypertension and for excluding elevated left-heart pressures (measured as the pulmonary capillary wedge pressure) as a cause for right HF. BNP and N-terminal BNP levels are elevated in patients with cor pulmonale secondary to RV myocardial stretch and may be dramatically elevated in acute pulmonary embolism.

1	class II or III symptoms, who received sildenafil at 20 mg three times 1507 Heart Failure: Management daily for 3 months, followed by 60 mg three times daily for another 3 months, compared with a placebo. There was no improvement in Mandeep R. Mehra functional capacity, quality of life, or other clinical and surrogate

1	Distinctive phenotypes of presentation with diverse management targets exemplify the vast syndrome of heart failure. These range from chronic heart failure with reduced ejection fraction (HFrEF) or heart failure with preserved ejection fraction (HFpEF), acute decompensated heart failure (ADHF), and advanced heart failure. Early management evolved from symptom control to disease-modifying therapy in HFrEF with the advent of renin-angiotensin-aldosterone system (RAAS)– directed therapy, beta receptor antagonists, mineralocorticoid receptor antagonists, cardiac resynchronization therapy, and implantable cardio-defibrillators. However, similar advances have been elusive in the syndromes of HFpEF and ADHF, which have remained devoid of convincing therapeutic advances to alter their natural history. In advanced heart failure, a stage of disease typically encountered in HFrEF, the patient remains markedly symptomatic with demonstrated refractoriness or inability to tolerate full-dose

1	history. In advanced heart failure, a stage of disease typically encountered in HFrEF, the patient remains markedly symptomatic with demonstrated refractoriness or inability to tolerate full-dose neurohormonal antagonism, often requires escalating doses of diuretics, and exhibits persistent hyponatremia and renal insufficiency with frequent episodes of heart failure decompensation requiring recurrent hospitalizations. Such individuals are at the highest risk of sudden or progressive pump failure–related deaths (Chap. 281). In contrast, early-stage asymptomatic left ventricular dysfunction is amenable to preventive care, and its natural history is modifiable by neurohormonal antagonism (not further discussed).

1	Therapeutic targets in HFpEF include control of congestion, stabilization of heart rate and blood pressure, and efforts at improving exercise tolerance. Addressing surrogate targets, such as regression of ventricular hypertrophy in hypertensive heart disease, and use of lusitropic agents, such as calcium channel blockers and beta receptor antagonists, have been disappointing. Experience has demonstrated that lowering blood pressure alleviates symptoms more effectively than targeted therapy with specific agents.

1	The Candesartan in Heart Failure—Assessment of Mortality and Morbidity (CHARM) Preserved study showed a statistically significant reduction in hospitalizations but no difference in all-cause mortality in patients with HFpEF who were treated with the angiotensin receptor blocker (ARB), candesartan. Similarly, the Irbesartan in Heart Failure with Preserved Systolic Function (I-PRESERVE) trial demonstrated no differences in meaningful endpoints in such patients treated with irbesartan. An earlier analysis of a subset of the Digitalis Investigation Group (DIG) trial found no role for digoxin in the treatment of HFpEF. In the Study of the Effects of Nebivolol Intervention on Outcomes and Rehospitalization in Seniors with Heart Failure (SENIORS) trial of nebivolol, a vasodilating beta blocker, the subgroup of elderly patients with prior hospitalization and HFpEF did not appear to benefit in terms of all-cause or cardiovascular mortality. Much smaller mechanistic studies in the elderly with

1	the subgroup of elderly patients with prior hospitalization and HFpEF did not appear to benefit in terms of all-cause or cardiovascular mortality. Much smaller mechanistic studies in the elderly with the angiotensin-converting enzyme inhibitor (ACEI) enalapril showed no effect on peak exercise oxygen consumption, 6-minute walk distance, aortic distensibility, left ventricular mass, or peripheral neurohormone expression.

1	A small trial demonstrated that the phosphodiesterase-5 inhibitor sildenafil improved filling pressures and right ventricular function in a cohort of HFpEF patients with pulmonary venous hypertension. This finding led to the phase II trial, Phosphodiesterase-5 Inhibition to Improve Clinical Status and Exercise Capacity in Diastolic Heart Failure (RELAX), in HFpEF patients (left ventricular ejection fraction [LVEF] >50%) with New York Heart Association (NYHA) functional parameters. Conceptually targeting myocardial fibrosis in HFpEF, the large-scale Aldosterone Antagonist Therapy in Adults with Preserved Ejection Fraction Congestive Heart Failure (TOPCAT) trial has been completed. This trial demonstrated no improvement in the primary composite end-point, but did show a secondary signal of benefit on HF hospitalizations, counterbalanced, however, by an increase in adverse effects, particularly hyperkalemia. However, pessimism has been generated by the negative outcome of the Aldosterone

1	on HF hospitalizations, counterbalanced, however, by an increase in adverse effects, particularly hyperkalemia. However, pessimism has been generated by the negative outcome of the Aldosterone Receptor Blockade in Diastolic Heart Failure (ALDO-DHF) study wherein spironolactone improved echocardiographic indices of diastolic dysfunction but failed to improve exercise capacity, symptoms, or quality-of-life measures. A unique molecule that hybridizes an ARB with an endopeptidase inhibitor, LCZ696, increases the generation of myocardial cyclic guanosine 3′,5′-monophosphate, enhances myocardial relaxation, and reduces ventricular hypertrophy. This dual blocker has been shown to reduce circulating natriuretic peptides and reduce left atrial size to a significantly greater extent than valsartan alone in patients with HFpEF.

1	Even as efforts to control hypertension in HFpEF are critical, evaluation for and correction of underlying ischemia may be beneficial. Appropriate identification and treatment of sleep-disordered breathing should be strongly considered. Excessive decrease in preload with vasodilators may lead to underfilling the ventricle and subsequent hypotension and syncope. Some investigators have suggested that the exercise intolerance in HFpEF is a manifestation of chronotropic insufficiency and that such aberrations could be corrected with use of rate responsive pacemakers, but this remains an inadequately investigated contention (Fig. 280-1).

1	ADHF is a heterogeneous clinical syndrome most often resulting in need for hospitalization due to confluence of interrelated abnormalities of decreased cardiac performance, renal dysfunction, and alterations in vascular compliance. Admission with a diagnosis of ADHF is associated with excessive morbidity and mortality, with nearly half of these patients readmitted for management within 6 months, and a high short-term (5–8% in-hospital) and long-term mortality (20% at 1 year). Importantly, long-term aggregate outcomes remain poor, with a combined incidence of cardiovascular deaths, heart failure hospitalizations, myocardial infarction, strokes, or sudden death reaching 50% at 12 months after hospitalization. The management of these patients has remained difficult and principally revolves around volume control and decrease of vascular impedance while maintaining attention to end-organ perfusion (coronary and renal).

1	The first principle of management of these patients is to identify and tackle known precipitants of decompensation. Identification and management of medication nonadherence and use of prescribed medicines such as nonsteroidal anti-inflammatory drugs, cold and flu preparations with cardiac stimulants, and herbal preparations, including licorice, ginseng, and ma huang (an herbal form of ephedrine now banned in most places), are required. Active infection and overt or covert pulmonary thromboembolism should be sought, identified, and treated when clinical clues suggest such direction. When possible, arrhythmias should be corrected by controlling heart rate or restoring sinus rhythm in patients with poorly tolerated rapid atrial fibrillation and by correcting ongoing ischemia with coronary revascularization or by correcting offenders such as ongoing bleeding in demand-related ischemia. A parallel step in management involves stabilization of hemodynamics in those with instability. The

1	revascularization or by correcting offenders such as ongoing bleeding in demand-related ischemia. A parallel step in management involves stabilization of hemodynamics in those with instability. The routine use of a pulmonary artery catheter is not recommended and should be restricted to those who respond poorly to diuresis or experience hypotension or signs and symptoms suggestive of a low cardiac output where therapeutic targets are unclear. Analysis of in-hospital registries has identified several

1	Heart Failure: Management Heart Failure with Preserved Ejection Fraction: Management • Reduce the congestive state – Caution to not reduce preload excessively – Efforts to maintain sinus rhythm in atrial fibrilation may be beneficial – May mimic HF as an “angina equivalent” – Common comorbidity causing systemic hypertension, pulmonary hypertension, and right heart dysfunction – ? Targeted pacing (unproven) FIGURE 280-1 Pathophysiologic correlations, general therapeutic principles, and results of specific “directed” therapy in heart failure (HF) with preserved ejection fraction. ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker.

1	parameters associated with worse outcomes: a blood urea nitrogen level greater than 43 mg/dL (to convert to mmol/L, multiply by 0.357), systolic blood pressure less than 115 mmHg, a serum creatinine level greater than 2.75 mg/dL (to convert to μmol/L, multiply by 88.4), and an elevated troponin I level. A useful clinical schema to identify treatment targets for the various phenotypic presentations and management goals in ADHF is depicted in Fig. 280-2.

1	VOLUME MANAGEMENT Intravenous Diuretic Agents Intravenous diuretic agents rapidly and effectively relieve symptoms of congestion and are essential when oral drug absorption is impaired. When high doses of diuretic agents are required or when the effect is suboptimal, a continuous infusion may be needed to reduce toxicity and maintain stable serum drug levels. Randomized clinical trials of high-versus low-dose or bolus versus continuous infusion diuresis have not provided clear justification for the best diuretic strategy in ADHF, and as such, the use of diuretic regimens remains an art rather than science. Addition of a thiazide diuretic agent such as metolazone in combination provides a synergistic effect and is often required in patients receiving long-term therapy with loop diuretic agents. Change in weight is often used as a surrogate for adequate diuresis, but this objective measure of volume status may be surprisingly difficult to interpret, and weight loss during

1	diuretic agents. Change in weight is often used as a surrogate for adequate diuresis, but this objective measure of volume status may be surprisingly difficult to interpret, and weight loss during hospitalization does not necessarily correlate closely with outcomes. It is generally advisable to continue diuresis until euvolemia has been achieved. Physical examination findings, specifically the jugular venous pressure coupled with biomarker trends, are useful in timing discharge planning.

1	The Cardiorenal Syndrome The cardiorenal syndrome is being recognized increasingly as a complication of ADHF. Multiple definitions have been proposed for the cardiorenal syndrome, but at its simplest, it can be thought to reflect the interplay between abnormalities of heart and kidney function, with deteriorating function of one organ while therapy is administered to preserve the other. Approximately 30% of patients hospitalized with ADHF exhibit abnormal renal function at baseline, and this is associated with longer hospitalizations and increased mortality. However, mechanistic studies have been largely unable to find correlation between deterioration in renal function, cardiac output, left-sided filling pressures, and reduced renal perfusion; most patients with cardiorenal syndrome demonstrate a preserved cardiac output. It is hypothesized that in patients with established heart failure, this syndrome represents a complex interplay of neurohormonal factors, potentially exacerbated

1	a preserved cardiac output. It is hypothesized that in patients with established heart failure, this syndrome represents a complex interplay of neurohormonal factors, potentially exacerbated by “backward failure” resulting from increased intra-abdominal pressure and impairment in return of renal venous blood flow. Continued use of diuretic therapy may be associated with a reduction in glomerular filtration rate and a worsening of the cardiorenal syndrome when right-sided filling pressures remain elevated. In patients in the late stages of disease characterized by profound low cardiac output state, inotropic therapy or mechanical circulatory support has been shown to preserve or improve renal function in selected individuals in the short term until more definitive therapy such as assisted circulation or cardiac transplantation is implemented.

1	Ultrafiltration Ultrafiltration (UF) is an invasive fluid removal technique that may supplement the need for diuretic therapy. Proposed benefits of UF include controlled rates of fluid removal, neutral effects on serum electrolytes, and decreased neurohormonal activity. This technique has also been referred to as aquapheresis in recognition of its electrolyte depletion–sparing effects. Current UF systems function with two large-bore, peripherally inserted venous lines. In a pivotal study evaluating UF versus conventional therapy, fluid removal was improved and subsequent heart failure hospitalizations and urgent clinic visits were reduced with UF; however, no improvement in renal function and no subjective differences in dyspnea scores or adverse outcomes were noted. More recently, in the Cardiorenal Rescue Study in Acute Decompensated Heart Failure (CARRESS-HF) trial, 188 patients with ADHF and worsening renal failure were randomized to stepped pharmacologic care or UF. The primary

1	Cardiorenal Rescue Study in Acute Decompensated Heart Failure (CARRESS-HF) trial, 188 patients with ADHF and worsening renal failure were randomized to stepped pharmacologic care or UF. The primary endpoint was a change in serum creatinine and change in weight (reflecting fluid removal) at 96 hours. Although similar weight loss occurred in both groups (approximately 5.5 kg), there was worsening in creatinine in the UF group. Deaths and hospitalizations for heart failure were no different between groups, but there were more severe adverse events in the UF group, mainly due to kidney failure, bleeding complications, and intravenous catheter-related complications. This investigation argues against using UF as a primary strategy in patients with ADHF who are nonetheless responsive to diuretics. Whether UF is useful in states of diuretic unresponsiveness remains an open question, and this strategy continues to be employed judiciously in such situations.

1	Heart Failure: ManagementExtreme distress Pulmonary congestion Renal failure Low pulse pressure Cool extremities Cardio-renal syndrome Hepatic congestion New-onset arrhythmia Valvular heart disease Inflammatory heart disease Myocardial ischemia CNS injury Drug toxicity Renal insufficiency Biomarkers of injury Acute coronary syndrome, arrhythmia, hypoxia, pulmonary embolism, infection High-Risk Features Hypertensive Heterogeneity of ADHF: Management Principles Severe Pulmonary Congestion with Hypoxia Hypoperfusion with End-Organ Dysfunction Hypotension, Low Cardiac Output, and End-Organ Failure Acute Decompensation “Typical” Acute Decompensation “Pulmonary edema” Acute Decompensation “Low output” Acute Decompensation “Cardiogenic shock” Normotensive (usually volume overloaded) (usually not volume overloaded) Hemodynamic monitoring (suboptimal initial therapeutic response) Inotropic therapy (usually catecholamines) Mechanical circulatory support (IABP, percutaneous VAD, ultrafiltration)

1	volume overloaded) Hemodynamic monitoring (suboptimal initial therapeutic response) Inotropic therapy (usually catecholamines) Mechanical circulatory support (IABP, percutaneous VAD, ultrafiltration) Inotropic therapy (if low blood pressure or diuretic refractoriness) Vasodilators Vasodilators Vasodilators Opiates Diuretics O2 and noninvasive ventilation Diuretics FIGURE 280-2 The distinctive phenotypes of acute decompensated heart failure (ADHF), their presentations, and suggested therapeu-tic routes. (Unique causes of ADHF, such as isolated right heart failure and pericardial disease, and rare causes, such as aortic and coronary dis-section or ruptured valve structures or sinuses of Valsalva, are not delineated and are covered elsewhere.) IABP, intraaortic balloon pump; VAD, ventricular assist device.

1	Vasodilators including intravenous nitrates, nitroprusside, and nesiritide (a recombinant brain-type natriuretic peptide) have been advocated for upstream therapy in an effort to stabilize ADHF. The latter agent was introduced in a fixed dose for therapy after a comparison with intravenous nitrates suggested more rapid and greater reduction in pulmonary capillary wedge pressure. Enthusiasm for nesiritide waned due to concerns within the pivotal trials for development of renal insufficiency and an increase in mortality. To address these concerns, a large-scale morbidity and mortality trial, the Acute Study of Clinical Effectiveness of Nesiritide in Decompensated Heart Failure (ASCEND-HF) study was completed in 2011 and randomly enrolled 7141 patients with ADHF to nesiritide or placebo for 24 to 168 hours in addition to standard care. Nesiritide was not associated with an increase or a decrease in the rates of death and rehospitalization and had a clinically insignificant benefit on

1	24 to 168 hours in addition to standard care. Nesiritide was not associated with an increase or a decrease in the rates of death and rehospitalization and had a clinically insignificant benefit on dyspnea. Renal function did not worsen, but increased rates of hypotension were noted. Although this trial established the safety for this drug, the routine use cannot be advocated due to lack of significant efficacy. Recombinant human relaxin-2, or serelaxin, is a peptide upregulated in pregnancy and examined in ADHF patients with a normal or elevated blood pressure. In the Relaxin in Acute Heart Failure (RELAX-AHF) trial, serelaxin or placebo was added to a regimen of standard therapy in 1161 patients hospitalized with ADHF, evidence of congestion, and systolic pressure >125 mmHg. Serelaxin improved dyspnea, reduced signs and symptoms of congestion, and was associated with less early worsening of HF. Exploratory endpoints of hard outcomes at 6 months suggested positive signals in favor of

1	dyspnea, reduced signs and symptoms of congestion, and was associated with less early worsening of HF. Exploratory endpoints of hard outcomes at 6 months suggested positive signals in favor of mortality reduction. This agent is being tested in a large, more confirmatory trial setting.

1	Impairment of myocardial contractility often accompanies ADHF, and pharmacologic agents that increase intracellular concentration of cyclic adenosine monophosphate via direct or indirect pathways, such as sympathomimetic amines (dobutamine) and phosphodiesterase-3 inhibitors (milrinone), respectively, serve as positive inotropic agents. Their activity leads to an increase in cytoplasmic calcium. Inotropic therapy in those with a low-output state augments cardiac output, improves perfusion, and relieves congestion acutely. Although milrinone and dobutamine have similar hemodynamic profiles, milrinone is slower acting and is renally excreted and thus requires dose adjustments in the setting of kidney dysfunction. Since milrinone acts downstream from the β1-adrenergic receptor, it may provide an advantage in patients receiving beta blockers when admitted to the hospital. Studies are in universal agreement that long-term inotropic therapy increases mortality. However, the short-term use

1	an advantage in patients receiving beta blockers when admitted to the hospital. Studies are in universal agreement that long-term inotropic therapy increases mortality. However, the short-term use of inotropic agents in ADHF 1510 is also associated with increased arrhythmia, hypotension, and no beneficial effects on hard outcomes. Inotropic agents are currently indicated as bridge therapy (to either left ventricular assist device support or to transplant) or as selectively applied palliation in end-stage heart failure. Novel inotropic agents that leverage the concept of myofilament calcium sensitization rather than increasing intracellular calcium levels have been introduced. Levosimendan is a calcium sensitizer that provides inotropic activity, but also possesses phosphodiesterase-3 inhibition properties that are vasodilators in action. This makes the drug unsuitable in states of low output in the setting of hypotension. Two trials, the second Randomized Multicenter Evaluation of

1	properties that are vasodilators in action. This makes the drug unsuitable in states of low output in the setting of hypotension. Two trials, the second Randomized Multicenter Evaluation of Intravenous Levosimendan Efficacy (REVIVE II) and Survival of Patients with Acute Heart Failure in Need of Intravenous Inotropic Support (SURVIVE), have tested this agent in ADHF. SURVIVE compared levosimendan with dobutamine, and despite an initial reduction in circulating B-type natriuretic peptide levels in the levosimendan group compared with patients in the dobutamine group, this drug did not reduce all-cause mortality at 180 days or affect any secondary clinical outcomes. The second trial compared levosimendan against traditional noninotropic therapy and found a modest improvement in symptoms with worsened short-term mortality and ventricular arrhythmias. Another drug that functions as a selective myosin activator, omecamtiv mecarbil, prolongs the ejection period and increases fractional

1	with worsened short-term mortality and ventricular arrhythmias. Another drug that functions as a selective myosin activator, omecamtiv mecarbil, prolongs the ejection period and increases fractional shortening. Distinctively, the force of contraction is not increased, and as such, this agent does not increase myocardial oxygen demand. In a 600-patient trial called ATOMIC-HF (A Trial of Omecamtiv Mecarbil to Increase Contractility in Acute Heart Failure), this agent showed improvement in dyspnea scores in the highest dose cohort, but not across all enrolled patients. How this agent performs in broader populations remains uncertain. Other inotropic agents that increase myocardial calcium sensitivity through mechanisms that reduce cTnI phosphorylation or inhibit protein kinase A are being developed. (Table 280-1 depicts typical inotropic, vasodilator, and diuretic drugs used in ADHF.)

1	Other trials testing unique agents have yielded disappointing results in the situation of ADHF. The Placebo-Controlled Randomized Study of the Selective A1 Adenosine Receptor Antagonist Rolofylline for Patients Hospitalized with Acute Decompensated Heart Failure and Volume Overload to Assess Treatment Effect on Congestion and Renal Function (PROTECT) trial of selective adenosine antagonism and the Efficacy of Vasopressin Antagonism in Heart Failure Outcome Study with Tolvaptan (EVEREST) trial of an oral selective vasopressin-2 antagonist in ADHF were both negative with respect to hard outcomes. In patients who fail to respond adequately to medical therapy, mechanical assist devices may be required. This is covered in more detail in Chap. 281. The past 50 years have witnessed great strides in the management of HFrEF. The treatment of symptomatic heart failure that evolved from a renocentric (diuretics) and hemodynamic therapy model

1	The past 50 years have witnessed great strides in the management of HFrEF. The treatment of symptomatic heart failure that evolved from a renocentric (diuretics) and hemodynamic therapy model FIGURE 280-3 Progressive decline in mortality with angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs), beta blockers, mineralocorticoid receptor antagonists, and balanced vasodilators (∗selected populations such as African Americans); further stack-on neurohormonal therapy is ineffective or results in worse outcome; management of comorbidity is of unclear efficacy. EPO, erythropoietin; HF, heart failure; HFrEF, heart failure with reduced ejection fraction; PUFA, polyunsaturated fatty acid; SSRI, selective serotonin reuptake Heart Failure: Management inhibitor.

1	Heart Failure: Management inhibitor. (digoxin, inotropic therapy) ushered in the era of disease-modifying therapy with neurohormonal antagonism. In this regard, ACEIs and beta blockers form the cornerstone of pharmacotherapy and lead to attenuation of decline and improvement in cardiac structure and function with consequent reduction in symptoms, improvement in quality of life, decreased burden of hospitalizations, and a decline in mortality from both pump failure and arrhythmic deaths (Fig. 280-3).

1	Meta-analyses suggest a 23% reduction in mortality and a 35% reduction in the combination endpoint of mortality and hospitalizations for heart failure in patients treated with ACEIs. Patients treated with beta blockers provide a further 35% reduction in mortality on top of the benefit provided by ACEIs alone. Increased experience with both agents in a broad range of patients with HFrEF has demonstrated the safety of ACEIs in treating patients with mild renal insufficiency and the tolerability of beta blockers in patients with moderately controlled diabetes, asthma, and obstructive lung disease. The benefits of ACEIs and beta blockers extend to advanced symptoms of disease (NYHA class IIIb–IV). However, a substantial number of patients with advanced heart failure may not be able to achieve optimal doses of neurohormonal inhibitors and require cautious reduction in dose exposure to maintain clinical stability. Such individuals with lower exposure to ACEIs and beta blockers represent a

1	optimal doses of neurohormonal inhibitors and require cautious reduction in dose exposure to maintain clinical stability. Such individuals with lower exposure to ACEIs and beta blockers represent a high-risk cohort with poor prognosis.

1	Class Effect and Sequence of Administration ACEIs exert their beneficial effects in HFrEF as a class; however, the beneficial effects of beta blockers are thought to be limited to specific drugs. Beta blockers with intrinsic sympathomimetic activity (xamoterol) and other agents, including bucindolol, have not demonstrated a survival benefit. On the basis of investigations, beta blocker use in HFrEF should be restricted to carvedilol, bisoprolol, and metoprolol succinate—agents tested and proven to improve survival in clinical trials. Whether beta blockers or ACEIs should be started first was answered by the Cardiac Insufficiency Bisoprolol Study (CIBIS) III, in which outcomes did not vary when either agent was initiated first. Thus, it matters little which agent is initiated first; what does matter is that optimally titrated doses of both ACEIs and beta blockers be established in a timely manner. Limits of Pharmacologic Therapy in HFrEF 1511

1	Limits of Pharmacologic Therapy in HFrEF 1511 Dose and Outcome A trial has indicated that higher tolerated doses of ACEIs achieve greater reduction in hospitalizations without materially improving survival. Beta blockers demonstrate a dose-dependent improvement in cardiac function and reductions in mortality and hospitalizations. Clinical experience suggests that, in the absence of symptoms to suggest hypotension (fatigue and dizziness), pharmacotherapy may be up-titrated every 2 weeks in hemodynamically stable and euvolemic ambulatory patients as tolerated.

1	Aldosterone antagonism is associated with a reduction in mortality in all stages of symptomatic NYHA class II to IV HFrEF. Elevated aldosterone levels in HFrEF promote sodium retention, electrolyte imbalance, and endothelial dysfunction and may directly contribute to myocardial fibrosis. The selective agent eplerenone (tested in NYHA class II and post–myocardial infarction heart failure) and the nonselective antagonist spironolactone (tested in NYHA class III and IV heart failure) reduce mortality and hospitalizations, with significant reductions in sudden cardiac death (SCD). Hyperkalemia and worsening renal function are concerns, especially in patients with underlying chronic kidney disease, and renal function and serum potassium levels must be closely monitored.

1	Neurohormonal “escape” has been witnessed in patients with HFrEF by the finding that circulating levels of angiotensin II return to pretreatment levels with long-term ACEI therapy. ARBs blunt this phenomenon by binding competitively to the AT1 receptor. A large meta-analysis of 24 randomized trials showed the superiority of ARBs to placebo in patients with intolerable adverse effects with ACEIs and their noninferiority in all-cause mortality or hospitalizations when compared with ACEIs. The Valsartan Heart Failure Trial (Val-HeFT) suggested that addition of valsartan in patients already receiving treatment with ACEIs and beta blockers was associated with a trend toward worse outcomes. Similarly, adding valsartan to captopril in patients with heart failure after myocardial infarction who were receiving background beta blocker therapy was associated with an increase in adverse events without any added benefit compared with 1512 monotherapy for either group. Thus, the initial clinical

1	were receiving background beta blocker therapy was associated with an increase in adverse events without any added benefit compared with 1512 monotherapy for either group. Thus, the initial clinical strategy should be to use a two-drug combination first (ACEI and beta blocker; if beta blocker intolerant, then ACEI and ARB; if ACEI intolerant, then ARB and beta blocker). In symptomatic patients (NYHA class II–IV), an aldosterone antagonist should be strongly considered, but four-drug therapy should be avoided. A recent trial called the Aliskiren Trial on Acute Heart Failure Outcomes (ASTRONAUT) tested a direct renin inhibitor, aliskiren, in addition to other heart failure medications, within a week after discharge from a hospitalization for decompensated HFrEF. No significant difference in cardiovascular death or hospitalization at 6 or 12 months was noted. Aliskiren was associated with a reduction in circulating natriuretic peptides, but any disease-modifying effect was overcome by

1	in cardiovascular death or hospitalization at 6 or 12 months was noted. Aliskiren was associated with a reduction in circulating natriuretic peptides, but any disease-modifying effect was overcome by excessive adverse events including hyperkalemia, hypo-tension, and renal dysfunction.

1	The combination of hydralazine and nitrates has been demonstrated to improve survival in HFrEF. Hydralazine reduces systemic vascular resistance and induces arterial vasodilatation by affecting intracellular calcium kinetics; nitrates are transformed in smooth muscle cells into nitric oxide, which stimulates cyclic guanosine monophosphate production and consequent arterial-venous vasodilation. This combination improves survival, but not to the magnitude evidenced by ACEIs or ARBs. However, in individuals with HFrEF unable to tolerate renin-angiotensin-aldosterone–based therapy for reasons such as renal insufficiency or hyperkalemia, this combination is preferred as a disease-modifying approach. A trial conducted in self-identified African Americans, the African-American Heart Failure Trial (A-Heft), studied a fixed dose of isosorbide dinitrate with hydralazine in patients with advanced symptoms of HFrEF who were receiving standard background therapy. The study demonstrated benefit in

1	(A-Heft), studied a fixed dose of isosorbide dinitrate with hydralazine in patients with advanced symptoms of HFrEF who were receiving standard background therapy. The study demonstrated benefit in survival and hospitalization recidivism in the treatment group. Adherence to this regimen is limited by the thrice-daily dosing schedule. Table 280-2 lists the common neurohormonal and vasodilator regimens for HFrEF.

1	Ivabradine, an inhibitor of the If current in the sinoatrial node, slows the heart rate without a negative inotropic effect. The Systolic Heart Failure Treatment with Ivabradine Compared with Placebo Trial (SHIFT) was conducted in patients with class II or III HFrEF, a heart rate >70 beats/min, and history of hospitalization for heart failure during the previous year. Ivabradine reduced hospitalizations and the combined endpoint of cardiovascular-related death and heart failure hospitalization. The study population was not necessarily representative of North American patients with HFrEF since, with a few exceptions, most did not receive internal cardioverter-defibrillation or cardiac resynchronization therapy and 40% did not receive a mineralocorticoid receptor antagonist. Although 90% received beta blockers, only a quarter were on full doses. Whether this agent, now available outside the United States, would have been effective in patients receiving robust, guideline-recommended

1	received beta blockers, only a quarter were on full doses. Whether this agent, now available outside the United States, would have been effective in patients receiving robust, guideline-recommended therapy for heart failure remains enigmatic. In the 2012 European Society of Cardiology guidelines for the treatment of heart failure, ivabradine was suggested as second-line therapy before digoxin is considered in patients who remain symptomatic after guideline-based ACEIs, beta blockers, and mineralocorticoid receptor antagonists and with residual heart rate >70 beats/min. Another group in whom potential benefit may be expected includes those unable to tolerate beta blockers.

1	Digitalis glycosides exert a mild inotropic effect, attenuate carotid sinus baroreceptor activity, and are sympathoinhibitory. These effects decrease serum norepinephrine levels, plasma renin levels, and possibly aldosterone levels. The DIG trial demonstrated a reduction in heart failure hospitalizations in the treatment group but no reduction in mortality or improvement in quality of life. Importantly, treatment with digoxin resulted in a higher mortality rate in women than men. Furthermore, the effects of digoxin in reducing hospitalizations were lower in women than in men. It should be noted that low doses of digoxin are sufficient to achieve any potentially beneficial outcomes, and higher doses breach the therapeutic safety index. Although digoxin levels should be checked to minimize toxicity and although dose reductions are indicated for higher levels, no adjustment is made for low levels. Generally, digoxin is now relegated as therapy for patients who remain profoundly

1	minimize toxicity and although dose reductions are indicated for higher levels, no adjustment is made for low levels. Generally, digoxin is now relegated as therapy for patients who remain profoundly symptomatic despite optimal neurohormonal blockade and adequate volume control.

1	Neurohormonal activation results in avid salt and water retention. Loop diuretic agents are often required because of their increased potency, Metoprolol succinate CR/XL 159 12.5–25 qd 200 qd Carvedilol 37 3.125 bid 25–50 bid and frequent dose adjustments may be necessary because of variable oral absorption and fluctuations in renal function. Importantly, clinical trial data confirming efficacy are limited, and no data suggest that these agents improve survival. Thus, diuretic agents should ideally be used in tailored dosing schedules to avoid excessive exposure. Indeed, diuretics are essential at the outset to achieve volume control before neurohormonal therapy is likely to be well tolerated or titrated.

1	Amlodipine and felodipine, second-generation calcium channel– blocking agents, safely and effectively reduce blood pressure in HFrEF but do not affect morbidity, mortality, or quality of life. The first-generation agents, including verapamil and diltiazem, may exert negative inotropic effects and destabilize previously asymptomatic patients. Their use should be discouraged.

1	Despite an abundance of animal and clinical data demonstrating deleterious effects of activated neurohormonal pathways beyond the RAAS and sympathetic nervous system, targeting such pathways with incremental blockade has been largely unsuccessful. As an example, the endothelin antagonist bosentan is associated with worsening heart failure in HFrEF despite demonstrating benefits in right-sided heart failure due to pulmonary arterial hypertension. Similarly, the centrally acting sympatholytic agent moxonidine worsens outcomes in left heart failure. The combined drug omapatrilat hybridizes an ACEI with a neutral endopeptidase inhibitor, and this agent was tested in the Omapatrilat Versus Enalapril Randomized Trial of Utility in Reducing Events (OVERTURE) trial. This drug did not favorably influence the primary outcome measure of the combined risk of death or hospitalization for heart failure requiring intravenous treatment. The risk of angioedema was notably higher with omapatrilat than

1	the primary outcome measure of the combined risk of death or hospitalization for heart failure requiring intravenous treatment. The risk of angioedema was notably higher with omapatrilat than ACEIs alone. LCZ696 and ARB with an endopeptidase inhibitor have shown benefit in a large trial versus ARB alone.

1	Targeting inflammatory cytokines such as tumor necrosis factor α (TNF-α) by using anticytokine agents such as infliximab and etanercept has been unsuccessful and associated with worsening heart failure. Nonspecific immunomodulation has been tested in the large Advanced Chronic Heart Failure Clinical Assessment of Immune Modulation Therapy (ACCLAIM-HF) trial of 2426 HFrEF patients with NYHA functional class II to IV symptoms. Ex vivo exposure of a blood sample to controlled oxidative stress initiates apoptosis of leukocytes soon after intramuscular gluteal injection of the treated sample. The physiologic response to apoptotic cells results in a reduction in inflammatory cytokine production and upregulation of anti-inflammatory cytokines. This promising hypothesis was not proven, although certain subgroups (those with no history of previous myocardial infarction and those with mild heart failure) showed signals in favor of immunomodulation. Use of intravenous immunoglobulin therapy in

1	certain subgroups (those with no history of previous myocardial infarction and those with mild heart failure) showed signals in favor of immunomodulation. Use of intravenous immunoglobulin therapy in nonischemic etiology of heart failure has not been shown to result in beneficial outcomes.

1	Potent lipid-altering and pleiotropic effects of statins reduce major cardiovascular events and improve survival in non–heart failure populations. Once heart failure is well established, this therapy may not be as beneficial and theoretically could even be detrimental by depleting ubiquinone in the electron transport chain. Two trials, Controlled Rosuvastatin Multinational Trial in Heart Failure (CORONA) and Gruppo Italiano per lo Studio della Sopravvivenza nell’Insufficienza Cardiac (GISSI-HF), have tested low-dose rosuvastatin in patients with HFrEF and demonstrated no improvement in aggregate clinical outcomes. If statins are required to treat progressive coronary artery disease in the background setting of heart failure, then they should be employed. However, no rationale appears to exist for routine statin therapy in nonischemic heart failure.

1	HFrEF is accompanied by a hypercoagulable state and therefore a high risk of thromboembolic events, including stroke, pulmonary embolism, and peripheral arterial embolism. Although long-term oral anticoagulation is established in certain groups, including patients with atrial fibrillation, the data are insufficient to support the use of warfarin in patients in normal sinus rhythm without a history of thromboembolic events or echocardiographic evidence of left ventricular thrombus. In the large Warfarin versus Aspirin in Reduced Cardiac Ejection Fraction (WARCEF) trial, 2305 patients with HFrEF were randomly allocated to either full-dose aspirin or international normalized ratio– controlled warfarin with follow-up for 6 years. Among patients with reduced LVEF who were in sinus rhythm, there was no significant overall difference in the primary outcome between treatment with warfarin and treatment with aspirin. A reduced risk of ischemic stroke with warfarin was offset by an increased

1	was no significant overall difference in the primary outcome between treatment with warfarin and treatment with aspirin. A reduced risk of ischemic stroke with warfarin was offset by an increased risk of major hemorrhage. Aspirin blunts ACEI-mediated prostaglandin synthesis, but the clinical importance of this finding remains unclear. Current guidelines support the use of aspirin in patients with ischemic cardiomyopathy.

1	Treatment with long-chain omega-3 polyunsaturated fatty acids (ω-3 PUFAs) has been shown to be associated with modestly improved clinical outcomes in patients with HFrEF. This observation from the GISSI-HF trial was extended to measurements of ω-3 PUFAs in plasma phospholipids at baseline and after 3 months. Three-month treatment with ω-3 PUFAs enriched circulating eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Low EPA levels are inversely related to total mortality in patients with HFrEF.

1	A growing body of evidence suggests an association between heart failure and micronutrient status. Reversible heart failure has been described as a consequence of severe thiamine and selenium deficiency. Thiamine deficiency has received attention in heart failure due to the fact that malnutrition and diuretics are prime risk factors for thiamine loss. Small exploratory randomized studies have suggested a benefit of supplementation of thiamine in HFrEF with evidence of improved cardiac function. This finding is restricted to chronic heart failure states and does not appear to be beneficial in the ADHF phenotype. Due to the preliminary nature of the evidence, no recommendations for routine supplementation or testing for thiamine deficiency can be made.

1	Peripheral lower extremity therapy using graded external pneumatic compression at high pressure is administered in 1-hour sessions for 35 treatments (7 weeks) and has been proposed to reduce angina symptoms and extend time to exercise-induced ischemia in patients with coronary artery disease. The Prospective Evaluation of Enhanced External Counterpulsation in Congestive Heart Failure (PEECH) study assessed the benefits of enhanced external counterpulsation in the treatment of patients with mild-to-moderate heart failure. This randomized trial improved exercise tolerance, quality of life, and NYHA functional classification but without an accompanying increase in peak oxygen consumption. A placebo effect due to the nature of the intervention simply cannot be excluded.

1	The Heart Failure: A Controlled Trial Investigating Outcomes of Exercise Training (HF-ACTION) study investigated short-term (3-month) and long-term (12-month) effects of a supervised exercise training program in patients with moderate HFrEF. Exercise was safe, improved patients’ sense of well-being, and correlated with a trend toward mortality reduction. Maximal changes in 6-minute walk distance were evident at 3 months with significant improvements in cardiopulmonary exercise time and peak oxygen consumption persisting at 12 months. Therefore, exercise training is recommended as an adjunctive treatment in patients with heart failure. Heart Failure: Management

1	Sleep-disordered breathing is common in HF and particularly in HFrEF. A range of presentations exemplified by obstructive sleep apnea, central sleep apnea, and its extreme form of Cheyne-Stokes breathing are noted. Frequent periods of hypoxia and repeated micro-and macro-arousals trigger adrenergic surges, which can worsen hypertension and impair systolic and diastolic function. A high index of suspicion is required, especially in patients with difficult-to-control hypertension or with predominant symptoms of fatigue despite reverse remodeling in response to optimal medical therapy. Worsening of right heart function with improvement of left ventricular function noted on medical therapy should immediately trigger a search for underlying sleep-disordered breathing or pulmonary complications such as occult embolism or pulmonary hypertension. Treatment with nocturnal positive airway pressure improves oxygenation, LVEF, and 6-minute walk distance. However, no conclusive data exist to

1	such as occult embolism or pulmonary hypertension. Treatment with nocturnal positive airway pressure improves oxygenation, LVEF, and 6-minute walk distance. However, no conclusive data exist to support this therapy as a disease-modifying approach with reduction in mortality.

1	Anemia is common in heart failure patients, reduces functional status and quality of life, and is associated with increased proclivity for hospital admissions and mortality. Anemia in heart failure is more common in the elderly, in those with advanced stages of HFrEF, in the presence of renal insufficiency, and in women and African Americans. The mechanisms include iron deficiency, dysregulation of iron metabolism, and occult gastrointestinal bleeding. Intravenous iron using either iron sucrose or carboxymaltose (Ferric Carboxymaltose Assessment in Patients with Iron Deficiency and Chronic Heart Failure [FAIR-HF] trial) has been shown to correct anemia and improve functional capacity. Erythropoiesis-regulating agents such as erythropoietin analogues have been studied with disappointing results. The Reduction of Events by Darbepoetin Alfa in Heart Failure (RED-HF) trial evaluated 2278 mild-to-moderate anemia patients with HFrEF and demonstrated that treatment with darbepoetin alfa did

1	The Reduction of Events by Darbepoetin Alfa in Heart Failure (RED-HF) trial evaluated 2278 mild-to-moderate anemia patients with HFrEF and demonstrated that treatment with darbepoetin alfa did not improve clinical outcomes in patients with systolic heart failure.

1	Depression is common in HFrEF, with a reported prevalence of one in five patients, and is associated with a poor quality of life, limited functional status, and increased risk of morbidity and mortality in this population. Antidepressants may improve depression, promote vascular health, and decrease systemic inflammation in HFrEF. However, the largest randomized study of depression in HFrEF, the Sertraline Against Depression and Heart Disease in Chronic Heart Failure (SADHART-CHF) trial, showed that sertraline was safe, but did not provide greater reduction in depression or improve cardiovascular status among patients with heart failure and depression compared with nurse-driven multidisciplinary management.

1	Atrial arrhythmias, especially atrial fibrillation, are common and serve as a harbinger of worse prognosis in patients with heart failure. When rate control is inadequate or symptoms persist, pursuing a rhythm control strategy is reasonable. Rhythm control may be achieved via pharmacotherapy or by percutaneous or surgical techniques, and referral to practitioners or centers experienced in these modalities is recommended. Antiarrhythmic drug therapy should be restricted to amiodarone and dofetilide, both of which have been shown to be safe and effective but do not alter the natural history of the underlying disease. The Antiarrhythmic Trial with Dronedarone in Moderate-to-Severe Congestive Heart Failure Evaluating Morbidity Decrease (ANDROMEDA) studied the effects of the novel antiarrhythmic agent dronedarone and found an increased mortality due to worsening heart failure. Catheter ablation and pulmonary vein isolation appear to be safe and effective in this high-risk cohort and

1	agent dronedarone and found an increased mortality due to worsening heart failure. Catheter ablation and pulmonary vein isolation appear to be safe and effective in this high-risk cohort and compare favorably with the more established practice of atrioventricular node ablation and biventricular pacing.

1	Nonsynchronous contraction between the walls of the left ventricle (intraventricular) or between the ventricular chambers (interventricular) impairs systolic function, decreases mechanical efficiency of contraction, and adversely affects ventricular filling. Mechanical dyssynchrony results in an increase in wall stress and worsens functional mitral regurgitation. The single most important association of extent of dyssynchrony is a widened QRS interval on the surface electrocardiogram, particularly in the presence of a left bundle branch block pattern. With placement of a pacing lead via the coronary sinus to the lateral wall of the ventricle, cardiac resynchronization therapy (CRT) enables a more synchronous ventricular contraction by aligning the timing of activation of the opposing walls. Early studies showed improved exercise capacity, reduction in symptoms, and evidence of reverse remodeling. The Cardiac Resynchronization in Heart Failure Study (CARE-HF) trial was the first study

1	Early studies showed improved exercise capacity, reduction in symptoms, and evidence of reverse remodeling. The Cardiac Resynchronization in Heart Failure Study (CARE-HF) trial was the first study to demonstrate a reduction in all-cause mortality with CRT placement in patients with HFrEF on optimal therapy with continued moderate-to-severe residual symptoms of NYHA class III or IV heart failure. More recent clinical trials have demonstrated disease-modifying properties of CRT in even minimally symptomatic patients with HFrEF, including the Resynchronization–Defibrillation for Ambulatory Heart Failure Trial (RAFT) and Multicenter Automatic Defibrillator Implantation Trial with Cardiac Resynchronization Therapy (MADIT-CRT), both of which sought to use CRT in combination with an implantable defibrillator. Most benefit in mildly symptomatic HFrEF patients accrues from applying this therapy in those with a QRS width of >149 ms and a left bundle branch block pattern. Attempts to further

1	defibrillator. Most benefit in mildly symptomatic HFrEF patients accrues from applying this therapy in those with a QRS width of >149 ms and a left bundle branch block pattern. Attempts to further optimize risk stratification and expand indications for CRT using modalities other than electrocardiography have proven disappointing. In particular, echocardiographically derived measures of dyssynchrony vary tremendously, and narrow QRS dyssynchrony has not proven to be a good target for treatment. Uncertainty surrounds the benefits of CRT in those with ADHF, a predominant right bundle branch block pattern, atrial fibrillation, and evidence of scar in the lateral wall, which is the precise location where the CRT lead is positioned.

1	SCD due to ventricular arrhythmias is the mode of death in approximately half of patients with heart failure and is particularly proportionally prevalent in HFrEF patients with early stages of the disease. Patients who survive an episode of SCD are considered to be at very high risk and qualify for placement of an implantable cardioverterdefibrillator (ICD). Although primary prevention is challenging, the degree of residual left ventricular dysfunction despite optimal medical therapy (≤35%) to allow for adequate remodeling and the underlying etiology (post–myocardial infarction or ischemic cardiomyopathy) are the two single most important risk markers for stratification of need and benefit. Currently, patients with NYHA class II or III symptoms of heart failure and an LVEF <35%, irrespective of etiology of heart failure, are appropriate candidates for ICD prophylactic therapy. In patients with a myocardial infarction and optimal medical therapy with residual LVEF ≤30% (even when

1	of etiology of heart failure, are appropriate candidates for ICD prophylactic therapy. In patients with a myocardial infarction and optimal medical therapy with residual LVEF ≤30% (even when asymptomatic), placement of an ICD is appropriate. In patients with a terminal illness and a predicted life span of less than 6 months or in those with NYHA class IV symptoms who are refractory to medications and who are not candidates for transplant, the risks of multiple ICD shocks must be carefully weighed against the survival benefits. If a patient meets the QRS criteria for CRT, combined CRT with ICD is often employed (Table 280-3).

1	Coronary artery bypass grafting (CABG) is considered in patients with ischemic cardiomyopathy with multivessel coronary artery disease. The recognition that hibernating myocardium, defined as myocardial tissue with abnormal function but maintained cellular function, could recover after revascularization led to the notion that revascularization with CABG would be useful in those with living myocardium. Revascularization is most robustly supported in individuals with ongoing angina and left ventricular failure. Revascularizing those with left ventricular failure in the absence of angina remains controversial. The Surgical Treatment for Ischemic Heart Failure (STICH) trial enrolled 1212 patients with an ejection fraction of 35% or less and coronary artery disease amenable to CABG and randomly assigned them to mismatch patients is generally due to progressive LVD, not a focal arrhythmia substrate (except in patients with post-MI HF)

1	Diminishing returns with Intervention at early stages of HF advanced disease most successful since sudden death diminishes as cause of death with advanced HF Timing of benefits LVEF should be evaluated on optimal medical therapy or after revascularization before ICD therapy is employed; no benefit to ICD implant within 40 days of an MI (unless for secondary prevention) Estimation of benefits and Patients and clinicians often over- prognosis estimate benefits of ICDs; an ICD discharge is not equivalent to an episode of sudden death (some ventricular arrhythmias terminate spontaneously); appropriate ICD discharges are associated with a worse near-term prognosis Abbreviations: HF, heart failure; ICD, implantable cardioverter-defibrillator; LVD, left ventricular disease; LVEF, left ventricular ejection fraction; MI, myocardial infarction.

1	Abbreviations: HF, heart failure; ICD, implantable cardioverter-defibrillator; LVD, left ventricular disease; LVEF, left ventricular ejection fraction; MI, myocardial infarction. medical therapy alone or medical therapy plus CABG. There was no significant difference between groups with respect to the primary endpoint of death from any cause. Patients assigned to CABG had lower rates of death from cardiovascular causes and of death from any cause or hospitalization for cardiovascular causes. An ancillary study of this trial also determined that the detection of hibernation pre-revascularization did not materially influence the efficacy of this approach, nor did it help to define a population unlikely to benefit if hibernation was not detected.

1	Surgical ventricular restoration (SVR), a technique characterized by infarct exclusion to remodel the left ventricle by reshaping it surgically in patients with ischemic cardiomyopathy and dominant anterior left ventricular dysfunction, has been proposed. However, in a 1000-patient trial in patients with HFrEF who underwent CABG alone or CABG plus SVR, the addition of SVR to CABG had no disease-modifying effect. Cardiac symptoms and exercise tolerance improved from baseline to a similar degree in both study groups. SVR resulted in lower left ventricular volumes at 4 months after operation. However, left ventricular aneurysm surgery is still advocated in those with refractory heart failure, ventricular arrhythmias, or thromboembolism arising from an akinetic aneurysmal segment of the ventricle. Other remodeling procedures, such as use of an external mesh-like net attached around the heart to limit further enlargement, have not been shown to provide hard clinical benefits, although

1	Other remodeling procedures, such as use of an external mesh-like net attached around the heart to limit further enlargement, have not been shown to provide hard clinical benefits, although favorable cardiac remodeling was noted.

1	Mitral regurgitation (MR) occurs with varying degrees in patients with HFrEF and dilated ventricles. Annular dilatation and leaflet noncoaptation in the setting of anatomically normal papillary muscles, chordal structures, and valve leaflets characterize functional MR. In patients who are not candidates for surgical coronary revascularization, mitral valve repair remains controversial. Ischemic MR (or infarct-related MR) is typically associated with leaflet tethering and displacement related to abnormal left ventricular wall motion and geometry. No evidence to support the use of surgical or percutaneous valve correction for functional MR exists as disease-modifying therapy even though MR can be corrected.

1	The cardiomyocyte is no longer considered a terminally differentiated cell and possesses regenerative capacity. Such renewal is accelerated under conditions of stress and injury, such as an ischemic event or 1515 heart failure. Investigations that use either bone marrow–derived precursor cells or autologous cardiac-derived cells have gained traction. A number of smalland moderate-scale trials of such therapy have focused on post–myocardial infarction patients and have used autologous bone marrow–derived progenitor or stem cells. These trials have had variable results, with most demonstrating modest improvements in parameters of cardiac structure and remodeling. More promising, however, are cardiac-derived stem cells. Two preliminary pilot trials delivering cells via an intracoronary approach have been reported. In one, autologous c-kit–positive cells isolated from the atria obtained from patients undergoing CABG were cultured and rein-fused. In another, cardiosphere-derived cells

1	have been reported. In one, autologous c-kit–positive cells isolated from the atria obtained from patients undergoing CABG were cultured and rein-fused. In another, cardiosphere-derived cells grown from endomyocardial biopsy specimens were used. These small trials demonstrated improvements in left ventricular function but require far more work to usher in a clinical therapeutic success. The appropriate route of administration, the quantity of cells to achieve a minimal therapeutic threshold, the constitution of these cells (single source or mixed), the mechanism by which benefit accrues, and shortand long-term safety remain to be elucidated.

1	Targeting molecular aberrations using gene transfer therapy, mostly with an adenoviral vector, is emerging in HFrEF. Several methods of gene delivery have been developed, including direct intramyocardial injection, coronary artery or venous infusion, and injection into the pericardial space. Cellular targets under consideration include β2-adrenergic receptors and calcium cycling proteins such as inhibitors of phospholamban. SERCA2a is deficient in patients with HFrEF and is primarily responsible for reincorporating calcium into the sarcoplasmic reticulum during diastole. A phase II randomized, double-blind, placebo-controlled trial called CUPID (Efficacy and Safety Study of Genetically Targeted Enzyme Replacement Therapy for Advanced Heart Failure) was completed. This study used coronary arterial infusion of adeno-associated virus type 1 carrying the gene for SERCA2a and demonstrated that natriuretic peptides were decreased, reverse remodeling was noted, and symptomatic improvements

1	arterial infusion of adeno-associated virus type 1 carrying the gene for SERCA2a and demonstrated that natriuretic peptides were decreased, reverse remodeling was noted, and symptomatic improvements were forthcoming. Stromal-derived factor 1 enhances myocardial repair and facilitates “homing” of stem cells to the site of tissue injury. Strategies using intramyocardial injections to deploy this gene at sites of injury are being studied.

1	More advanced therapies for late-stage heart failure such as left ventricular assist devices and cardiac transplantation are covered in detail in Chap. 281.

1	Despite stellar outcomes with medical therapy, admission rates following heart failure hospitalization remain high, with nearly half of all patients readmitted to hospital within 6 months of discharge. Recurrent heart failure and related cardiovascular conditions account for only half of readmissions in patients with heart failure, whereas other comorbidity-related conditions account for the rest. The key to achieving enhanced outcomes must begin with the attention to transitional care at the index hospitalization with facilitated discharge through comprehensive discharge planning, patient and caregiver education, appropriate use of visiting nurses, and planned follow-up. Early postdischarge follow-up, whether by telephone or clinic-based, may be critical to ensuring stability because most heart failure–related readmissions tend to occur within the first 2 weeks after discharge. Although routinely advocated, intensive surveillance of weight and vital signs with use of telemonitoring

1	heart failure–related readmissions tend to occur within the first 2 weeks after discharge. Although routinely advocated, intensive surveillance of weight and vital signs with use of telemonitoring has not decreased hospitalizations. Intrathoracic impedance measurements have been advocated for the identification of early rise in filling pressure and worsened hemodynamics so that preemptive management may be employed. However, this has not been successful and may worsen outcomes in the short term. Implantable pressure monitoring systems do tend to provide signals for early decompensation, and in patients with moderately advanced symptoms, such systems have been shown to provide information that can allow implementation of therapy to avoid hospitalizations by as much as 39% (in the CardioMEMS Heart Sensor Allows Monitoring of Pressure to Improve Outcomes in NYHA Class III Heart

1	Heart Failure: Management

1	Cardiac Transplantation and Prolonged Assisted Circulation Sharon A. Hunt, Hari R. Mallidi Advanced or end-stage heart failure is an increasingly frequent sequela of many types of heart disease, as progressively more effective pallia-281 1516 Failure Patients [CHAMPION] trial). Once heart failure becomes advanced, regularly scheduled review of the disease course and options with the patient and family is recommended including discussions surrounding end-of-life preferences when patients are comfortable in an outpatient setting. As the disease state advances further, integrating care with social workers, pharmacists, and community-based nursing may be critical in improving patient satisfaction with the therapy, enhancing quality of life, and avoiding heart failure hospitalizations. Equally important is attention to seasonal influenza vaccinations and periodic pneumococcal vaccines that may obviate non–heart failure hospitalizations in these ill patients. When nearing end of life,

1	important is attention to seasonal influenza vaccinations and periodic pneumococcal vaccines that may obviate non–heart failure hospitalizations in these ill patients. When nearing end of life, facilitating a shift in priorities to outpatient and hospice palliation is key, as are discussions around advanced therapeutics and continued use of ICD prophylaxis, which may worsen quality of life and prolong death.

1	Substantial differences exist in the practice of heart failure therapeutics and outcomes by geographic location. International guidelines produced by the American College of Cardiology/American Heart Association, European Society of Cardiology, and National Institute for Health and Clinical Excellence (United Kingdom) differ in their approach to evaluation of evidence and prioritization of therapy. The penetrance of CRT and ICD is higher in the United States than in Europe. Conversely, therapy unavailable in the United States, such as ivabradine and levosimendan, is designated as useful in Europe. Although ACEIs appear to be similarly effective across populations, variation in the benefits of beta blockers based on world region remains an area of controversy. In oral pharmacologic therapy trials of HFrEF, patients from southwest Europe have a lower incidence of ischemic cardiomyopathy and those in North America tend to have more diabetes and prior coronary revascularization. There is

1	trials of HFrEF, patients from southwest Europe have a lower incidence of ischemic cardiomyopathy and those in North America tend to have more diabetes and prior coronary revascularization. There is also regional variation in medication use even after accounting for indication. In trials of ADHF, patients in Eastern Europe tend to be younger, with higher ejection fractions and lower natriuretic peptide levels. Patients from South America tend to have the lowest rates of comorbidities, revascularization, and device use. In contrast, patients from North America have the highest comorbidity burden with high revascularization and device use rates. Given geographic differences in baseline characteristics and clinical outcomes, the generalizability of therapeutic outcomes in patients in the United States and Western Europe may require verification.

1	tion for the earlier stages of heart disease and prevention of sudden death associated with heart disease become more widely recognized and employed (Chap. 279). When patients with end-stage or refractory heart failure are identified, the physician is faced with the decision of advising compassionate end-of-life care or choosing to recommend extraordinary life-extending measures. For the occasional patient who is relatively young and without serious comorbidities, the latter may represent a reasonable option. Current therapeutic options are limited to cardiac transplantation (with the option of mechanical cardiac assistance as a “bridge” to transplantation) or permanent mechanical assistance of the circulation. In the future, it is possible that genetic modulation of ventricular function or cell-based cardiac repair will be options for such patients. Currently, both of the latter approaches are considered to be experimental.

1	Surgical techniques for orthotopic transplantation of the heart were devised in the 1960s and taken into the clinical arena in 1967. The procedures did not gain widespread clinical acceptance until the introduction of “modern” and more effective immunosuppression in the early 1980s. By the 1990s, the demand for transplantable hearts met, and then exceeded, the available donor supply and leveled off at about 4000 heart transplantations annually worldwide, according to data from the Registry of the International Society for Heart and Lung Transplantation (ISHLT). Subsequently, heart transplantation activity in the United States has remained stable at ~2200 per year, but worldwide activity reported to this registry has decreased somewhat. This apparent decline in numbers may be a result of the fact that reporting is legally mandated in the United States but not elsewhere, and several countries have started their own databases.

1	Donor and recipient hearts are excised in virtually identical operations with incisions made across the atria and atrial septum at the mid-atrial level (with the posterior walls of the atria left in place) and across the great vessels just above the semilunar valves. The donor heart is generally “harvested” by a separate surgical team, transported from the donor hospital in a bag of iced saline solution, and reanastomosed into the waiting recipient in the orthotopic or normal anatomic position. The only change in surgical technique since this method was first described has been a movement in recent years to move the right atrial anastomosis back to the level of the superior and inferior venae cavae to better preserve right atrial geometry and prevent atrial arrhythmias. Both methods of implantation leave the recipient with a surgically denervated heart that does not respond to any direct sympathetic or parasympathetic stimuli but does respond to circulating catecholamines. The

1	of implantation leave the recipient with a surgically denervated heart that does not respond to any direct sympathetic or parasympathetic stimuli but does respond to circulating catecholamines. The physiologic responses of the denervated heart to the demands of exercise are atypical but quite adequate for continuation of normal physical activity.

1	In the United States, the allocation of donor organs is accomplished under the supervision of the United Network for Organ Sharing, a private organization under contract to the federal government. The United States is divided geographically into eleven regions for donor heart allocation. Allocation of donor hearts within a region is decided according to a system of priority that takes into account (1) the severity of illness, (2) the geographic distance from the donor, and (3) the patient’s time on the waiting list. A physiologic limit of ~3 h of “ischemic” (out-of-body) time for hearts precludes a national sharing of hearts. This allocation system design is reissued annually and is responsive to input from a variety of constituencies, including both donor families and transplantation professionals.

1	At the current time, the highest priority according to severity of illness is assigned to patients requiring hospitalization at the transplantation center for IV inotropic support, with a pulmonary artery catheter in place for hemodynamic monitoring, or to patients requiring mechanical circulatory support—i.e., use of an intra-aortic balloon pump or a right or left ventricular assist device (RVAD, LVAD), extracorporeal membrane oxygenation, or mechanical ventilation. The second highest priority is given to patients requiring ongoing inotropic support, but without a pulmonary artery catheter in place. All other patients are assigned a priority according to time accrued on the waiting list, and matching generally is based only on compatibility in terms of ABO blood group and gross body size.

1	While HLA matching of donor and recipient would be ideal, the relatively small numbers of patients as well as the time constraints involved make such matching impractical. However, some patients who are “presensitized” and have preexisting antibodies to human leukocyte antigens (HLAs) undergo prospective cross-matching with the HEART TRANSPLANTS 1517 Kaplan-Meier Survival (Transplants: January 1982–June 2010)

1	HEART TRANSPLANTS 1517 Kaplan-Meier Survival (Transplants: January 1982–June 2010) FIGURE 281-1 Global survival rates after heart transplantation since 1982. Rates were calculated by the Kaplan-Meier method, which incorporates information from all transplant recipients for whom any follow-up has been provided. Because many patients are still alive and some patients have been lost to follow-up, the survival rates are estimates rather than exact figures because the time of death is not known for all patients. Therefore, 95% confidence limits are provided. (From J Stehlik et al: J Heart Lung Transplant 31:1052, 2012.) donor; these patients are commonly multiparous women or patients who have received multiple transfusions.

1	Heart failure is an increasingly common cause of death, particularly in the elderly. Most patients who reach what has recently been categorized as stage D, or refractory end-stage heart failure, are appropriately treated with compassionate end-of-life care. A subset of such patients who are younger and without significant comorbidities can be considered as candidates for heart transplantation. Exact criteria vary in different centers but generally take into consideration the patient’s physiologic age and the existence of comorbidities such as peripheral or cerebrovascular disease, obesity, diabetes, cancer, or chronic infection. A registry organized by the ISHLT has tracked worldwide and

1	A registry organized by the ISHLT has tracked worldwide and U.S. survival rates after heart transplantation since 1982. The most recent update reveals survival rates of 83% and 76% 1 and 3 years after transplantation, respectively, or a posttransplantation “half-life” of 10.00 years (Fig. 281-1). The quality of life of survivors is generally excellent, with well over 90% of patients in the registry returning to normal and unrestricted function after transplantation.

1	Medical regimens employed to suppress the normal immune response to a solid organ allograft vary from center to center and are in a constant state of evolution, as more effective agents with improved side-effect profiles and less toxicity are introduced. All currently used regimens are nonspecific, providing general hyporeactivity to foreign antigens rather than donor-specific hyporeactivity and also causing the attendant, and unwanted, susceptibility to infections and malignancy. Most cardiac transplantation programs currently use a three-drug regimen that includes a calcineurin inhibitor (cyclosporine or tacrolimus), an inhibitor of T cell proliferation or differentiation (azathioprine, mycophenolate mofetil, or sirolimus), and at least a short initial course of glucocorticoids. Many programs also include an initial “induction” course of polyclonal or monoclonal antibodies to T cells in the perioperative period to decrease the frequency or severity of early posttransplantation

1	programs also include an initial “induction” course of polyclonal or monoclonal antibodies to T cells in the perioperative period to decrease the frequency or severity of early posttransplantation rejection. Most recently introduced have been monoclonal antibodies (daclizumab and basiliximab) that block the interleukin 2 receptor and may prevent allograft rejection without additional global immunosuppression.

1	Cardiac allograft rejection is usually diagnosed by endomyocardial biopsy conducted either on a surveillance basis or in response to clinical deterioration. Biopsy surveillance is performed on a regular basis in most programs for the first year postoperatively (or the first 5 years in many programs). Therapy consists of augmentation of immunosuppression, the intensity and duration of which are dictated by the severity of rejection. Increasing numbers of heart transplant recipients are surviving for years following transplantation and constitute a population of patients with a number of long-term management issues.

1	Allograft Coronary Artery Disease Despite usually having young donor hearts, cardiac allograft recipients are prone to develop coronary artery disease (CAD). This CAD is generally a diffuse, concentric, and longitudinal process that is quite different from “ordinary” atherosclerotic CAD, which is more focal and often eccentric. The underlying etiology most likely is primarily immunologic injury of the vascular endothelium, but a variety of risk factors influence the existence and progression of CAD, including nonimmunologic factors such as dyslipidemia, diabetes mellitus, and cytomegalovirus (CMV) infection. It is hoped that newer and improved immunosuppressive modalities will reduce the incidence and impact of these devastating complications, which currently account for the majority of late posttransplantation deaths. Thus far, the immunosuppressive agents mycophenolate mofetil and the mammalian target of the rapamycin (mTOR) inhibitors sirolimus and everolimus have been shown to be

1	posttransplantation deaths. Thus far, the immunosuppressive agents mycophenolate mofetil and the mammalian target of the rapamycin (mTOR) inhibitors sirolimus and everolimus have been shown to be associated with short-term lower incidence and extent of coronary intimal thickening; in anecdotal reports, institution of sirolimus was associated with some reversal of CAD. The use of statins also is associated with a reduced incidence of this vasculopathy, and these drugs are now used almost universally in transplant recipients unless contraindicated. Palliation of CAD with percutaneous interventions is probably safe and effective in the short term, although the disease often advances relentlessly. Because of the denervated status of the organ, patients rarely experience angina pectoris, even in advanced stages of disease.

1	Retransplantation is the only definitive form of therapy for advanced allograft CAD. However, the scarcity of donor hearts makes the 1518 decision to pursue retransplantation in an individual patient difficult and ethically complex.

1	Malignancy An increased incidence of malignancy is a well-recognized sequela of any program of chronic immunosuppression, and organ transplantation is no exception. Lymphoproliferative disorders are among the most frequent posttransplantation complications and, in most cases, seem to be driven by Epstein-Barr virus. Effective therapy includes reduction of immunosuppression (a clear “double-edged sword” in the setting of a life-sustaining organ), administration of antiviral agents, and traditional chemoand radiotherapy. Most recently, specific antilymphocyte (CD20) therapy has shown great promise. Cutaneous malignancies (both basal cell and squamous cell carcinomas) also occur with increased frequency among transplant recipients and can follow aggressive courses. The role of decreasing immunosuppression in the treatment of these cancers is far less clear.

1	Infections The use of currently available nonspecific immunosuppressive modalities to prevent allograft rejection naturally results in increased susceptibility to infectious complications in transplant recipients. Although the incidence has decreased since the introduction of cyclosporine, infections with unusual and opportunistic organisms are still the major cause of death during the first postoperative year and remain a threat to the chronically immunosuppressed patient throughout life. Effective therapy depends on careful surveillance for early signs and symptoms of opportunistic infection, an extremely aggressive approach to obtaining a specific diagnosis, and expertise in recognizing the more common clinical presentations of infections caused by CMV, Aspergillus, and other opportunistic agents.

1	The modern era of mechanical circulatory support can be traced back to 1953, when cardiopulmonary bypass was first used in a clinical setting and ushered in the possibility of brief periods of circulatory support to permit open-heart surgery. Subsequently, a variety of extracorporeal pumps to provide circulatory support for brief periods have been developed. The use of a mechanical device to support the circulation for more than a few hours initially progressed slowly, with the implantation of a total artificial heart in 1969 in Texas by Cooley. This patient survived for 60 h until a donor organ became available, at which point he underwent transplantation. Unfortunately, the patient died of pulmonary complications after transplantation. The entire field of mechanical replacement of the heart then took a decade-long hiatus until the 1980s, when total artificial hearts were reintroduced with much publicity; however, they failed to produce the hoped-for treatment of end-stage heart

1	heart then took a decade-long hiatus until the 1980s, when total artificial hearts were reintroduced with much publicity; however, they failed to produce the hoped-for treatment of end-stage heart disease. Starting in the 1970s, in parallel with the development of the total artificial heart, intense research had addressed the development of ventricular assist devices, which provide mechanical assistance for (rather than replacing) the failing ventricle.

1	Although conceived of initially as alternatives to biologic replacement of the heart, LVADs were introduced—and are still employed primarily—as temporary “bridges” to heart transplantation in candidates in whom medical therapy begins to fail before a donor heart becomes available. Several devices are approved by the U.S. Food and Drug Administration (FDA) and are in widespread use (see later). Those that are implantable within the body are compatible with hospital discharge and offer the patient a chance for life at home during a wait for a donor heart. However successful such “bridging” is for the individual patient, it does nothing to alleviate the scarcity of donor hearts; the ultimate goal in the field remains that of providing a reasonable alternative to biologic replacement of the heart—one that is widely and easily available and cost-effective.

1	Currently, there are two major indications for ventricular assistance. First, patients at risk of imminent death from cardiogenic shock are eligible for mechanical support. These patients are generally managed with temporary cardiac assist devices. Second, if patients have a left ventricular ejection fraction <25% or a peak VO2 <14 mL/kg per min or are dependent on inotropic therapy or support with intra-aortic balloon counterpulsation, they may be eligible for mechanical support. If they are eligible for heart transplantation, the mechanical circulatory assistance is termed the “bridge to transplantation.” By contrast, if the patient has a contraindication to heart transplantation, the use of the device is deemed to constitute “destination” left ventricular assistance therapy.

1	BASIC CONCEPTS Pulsatile vs. Nonpulsatile Devices Pulsatile devices are ventricular assist devices whose mechanism of action mandates the alternating filling and emptying of a volume chamber within the device that mimics the mechanism of action of the natural heart. Nonpulsatile devices have a mechanism of action that results in continuous blood flow through the device, eliminating the need for pulsatility. The pulsatile devices are larger, bulkier, and associated with greater energy requirements and higher rates of complications than the nonpulsatile devices. However, pulsatile devices provide greater degrees of support and may even be capable of replacing the function of the heart entirely in the form of a total artificial heart. Because of the bulkiness of these devices, many patients are too small to be supported with intracorporeal pulsatile pumps. However, paracorporeal versions are available. These devices are versatile and can be used for right, left, or biventricular

1	are too small to be supported with intracorporeal pulsatile pumps. However, paracorporeal versions are available. These devices are versatile and can be used for right, left, or biventricular assistance/replacement.

1	Continuous-flow (nonpulsatile) devices are further categorized on the basis of impeller design and mechanism. The older designs have tended to be axial-flow pumps, which operate on the Archimedes screw principle. These devices have an impeller that is in line with the direction of blood flow, and the inlet direction of blood is the same as the outlet direction. Continuous-flow devices have been dependent on the presence of blood-washed bearings within the pump housing and may be associated with an increased risk of blood and platelet activation. The newer devices are centrifugal in design; the blood flow takes a 90° turn between the inlet section of the pump and the outlet section. Another major difference in the newer devices is the absence of blood-washed bearings (with most devices having magnetically levitated impellers). This design allows the construction of smaller pumps with less blood-element activation than the axial-flow designs.

1	Available Devices In the United States, there are currently four FDA-approved devices that are used as bridges to transplantation in adults. Of these four devices, one is also approved for use as destination therapy or as long-term mechanical support of the heart. A number of other devices are approved only for short-term support in post–cardiac surgery shock or in cardiogenic shock secondary to acute myocardial infarction or fulminant myocarditis; these will not be considered here. So far, no long-term device is totally implantable, and, because of the need for transcutaneous connections, all share a common problem with infectious complications. Likewise, all share some tendency to thromboembolic complications and are subject to the possibility of mechanical failure common to any machine.

1	The total artificial heart (TAH) (Syncardia, Tucson, AZ) is a pneumatic, biventricular, orthotopically implanted ventricular assist device with an externalized driveline connecting it to its console. The TAH is currently the only FDA-approved device for use in patients who have severe biventricular failure. The Thoratec LVAD (Thoratec Corp., Pleasanton, CA) is an extra-corporeal pump that takes blood from a large cannula placed in the left ventricular apex and propels it forward through an outflow cannula inserted into the ascending aorta. The extracorporeal nature of this pump allows its use in small adults for whom intracorporeal pumps would be too large. This device provides not only left but also right ventricular assistance and can be utilized for biventricular support within the same patient (BiVentricular Assist Device).

1	The HeartMate II LVAD (Thoratec) similarly uses a drainage cannula in the left ventricular apex to drain blood into a small chamber, where the blood is driven by an electrically powered motor that spins a rotor, accelerating blood outflow into the ascending aorta (Fig. 281-2). This device is currently the only FDA-approved axial-flow pump that can be used both as a bridge to transplantation and as destination therapy. FIGURE 281-2 Diagram of HeartMate II left ventricular assist device (LVAD). (Reprinted with permission from Thoratec Corp., Pleasanton, CA.) The HeartWare Ventricular Assist System with the HVAD pump (HeartWare Inc., Framingham, MA) is the first third-generation device to be granted FDA approval for use in patients as a bridge to transplantation. The device is a centrifugal pump that is housed completely within the patient’s pericardial cavity and provides adequate support for many patients.

1	The use of these devices in the United States is limited mainly to patients with post–cardiac surgery shock and to those who are bridged to transplantation. The results of bridging to transplantation with the available devices are quite good, with nearly 75% of younger patients receiving a transplant by 1 year and having excellent posttransplantation survival rates.

1	Publication of the REMATCH (Randomized Evaluation of Mechanical Assistance in the Treatment of Heart Failure) trial in 2001 documented a somewhat improved survival rate in patients who had end-stage heart disease, were not candidates for transplantation, and were randomized to a pulsatile LVAD (albeit with a high rate of complications, especially neurologic issues) as opposed to continued medical therapy. This result led to renewed interest in use of the devices for nonbiologic permanent replacement of heart function as well. Subsequently, this device was supplanted by the HeartMate II axial-flow device, which has dramatically improved the survival of patients with severe end-stage heart disease in whom medical therapy has failed. The patients who had this device implanted had a 2-year survival rate of 58%, whereas the survival rate for patients in the medically treated arm of the original REMATCH trial was only 8%. More recent experience has shown that the mean survival period of

1	survival rate of 58%, whereas the survival rate for patients in the medically treated arm of the original REMATCH trial was only 8%. More recent experience has shown that the mean survival period of patients with a continuous-flow LVAD for destination therapy is approaching 5 years.

1	Several studies have evaluated the benefit of LVAD therapy as a bridge to transplantation. The most recent data come from a series of 140 patients who underwent implantation of a HeartWare HVAD. Of these patients, 94% achieved the principal outcome (defined as survival to transplantation, recovery of heart function, or ongoing device support) at 180 days. With increased experience and improved outcomes using LVADs as a bridge to transplantation, the ability to maintain end-organ function and limit the progression of pulmonary hypertension—or even to decrease pulmonary vascular resistance—makes mechanical unloading a more attractive option than continued inotropic support. The early bridge-to-transplantation experience demonstrated reduced posttransplantation survival compared with medical management; however, more recent experience has shown equivalent outcomes following transplantation. This result is likely secondary to a trend toward earlier device implantation—i.e., prior to the

1	management; however, more recent experience has shown equivalent outcomes following transplantation. This result is likely secondary to a trend toward earlier device implantation—i.e., prior to the onset of irreversible end-organ damage.

1	in the Adult Jamil A. Aboulhosn, John S. Child Over a hundred years ago, Sir William Osler, in his classic textbook The Principles and Practice of Medicine (New York, Appleton & Co, 1892, pp 659–663), devoted only five pages to “Congenital Affections of the Heart,” with the first sentence declaring that “[t]hese [disorders] have only limited clinical interest, as in a large proportion of cases the anomaly is not compatible with life, and in others nothing can be done to remedy the defect or even to relieve symptoms.” Fortunately, in the intervening century, considerable progress has been made in understanding the basis for these disorders and their effective treatment.

1	The most common birth defects are cardiovascular in origin. These malformations are due to complex multifactorial genetic and environmental causes. Recognized chromosomal aberrations and mutations of single genes account for <10% of all cardiac malformations. Congenital heart disease (CHD) complicates ~1% of all live births in the general population—about 40,000 births/year—but occurs more frequently in the offspring (about 4–10%, depending on maternal CHD type) of women with CHD. Owing to the remarkable surgical advances over the last 60 years, >90% of afflicted neonates and children now reach adulthood; women with CHD may now frequently successfully bear children after competent repairs. As such, the population with CHD is steadily increasing. Women with CHD are at increased risk for periand postpartum complications, but maternal CHD is generally not considered an absolute contraindication to pregnancy unless the mother has certain high-risk features (e.g., cyanosis, pulmonary

1	periand postpartum complications, but maternal CHD is generally not considered an absolute contraindication to pregnancy unless the mother has certain high-risk features (e.g., cyanosis, pulmonary hypertension, decompensated heart failure, arrhythmias, aortic aneurysm, among others). Consultation with an adult CHD expert is warranted for all females with CHD who desire to become pregnant.

1	Nearly one and a half million adults with operated or unoperated CHD live in the United States today; there are now more adults than children with CHD in the United States. Because true surgical cures are rare, and all repairs—be they palliative or corrective—may leave residua, sequelae, or complications, most require some degree of lifetime expert surveillance. The anatomic and physiologic changes in the heart and circulation due to any specific CHD lesion are not static but, rather, progress from prenatal life to adulthood. Malformations that are benign or escape detection in childhood may become clinically significant in the adult. Unfortunately, the growing number of adults with CHD has not been paralleled by an adequate increase in the number of specialists and

1	Congenital Heart Disease in the Adult 1520 specialty centers that are trained and equipped to manage this challenging population. Ongoing efforts to increase awareness, resources, and advocacy are essential for the necessary growth of this specialty.

1	(See also Chap. 265e) CHD is generally the result of aberrant embryonic development of a normal structure or failure of such a structure to progress beyond an early stage of embryonic or fetal development. This brief section serves to introduce the reader to normal development so that defects may be better understood; by necessity, it is not exhaustive. Cardiogenesis is a finely tuned process with transcriptional control of a complex group of regulatory proteins that activate or inhibit their gene targets in a locationand time-dependent manner. At about 3 weeks of embryonic development, two cardiac cords form and become canalized; at that point, the primordial cardiac tube develops from two sources (cardiac crescent or the first heart field, pharyngeal mesoderm or the second heart field); by 21 days, these fuse into a single cardiac tube beginning at the cranial end. The cardiac tube then elongates and develops discrete constrictions with the following segments from caudal to cranial

1	by 21 days, these fuse into a single cardiac tube beginning at the cranial end. The cardiac tube then elongates and develops discrete constrictions with the following segments from caudal to cranial location: sinus venosus receives the umbilical, vitelline, and common cardinal veins: atrium, ventricle, bulbus cordis, truncus arteriosus, aortic sac, and the aortic arches. The cardiac tube is fixed at the sinus venosus and arterial ends.

1	Subsequently, in the next few weeks, differential growth of cells causes the tube to elongate and loop as an “S” with the bulboventricular portion moving rightward and the atrium and sinus venosus moving posterior to the ventricle. The primitive atrium and ventricle communicate via the atrioventricular canal from which the endocardial cushion develops into two parts (ventrally and dorsally). The cushions fuse and divide the atrioventricular canal into two atrioventricular inlets and also migrate to help form the ventricular septum. The primitive atrium is divided first by a septum primum membrane, which grows down from the superior wall to the cushions; as this fusion occurs, the mid-portion resorbs in the center forming the ostium secundum. Rightward of the septum primum, a second septum secundum membrane grows down from the ventral-cranial wall toward—but not reaching—the cushions, and covering most, but not all, of the ostium secundum, resulting in a flap of the foramen ovale. The

1	secundum membrane grows down from the ventral-cranial wall toward—but not reaching—the cushions, and covering most, but not all, of the ostium secundum, resulting in a flap of the foramen ovale. The primitive ventricle is partitioned by a finely tuned set of events. The interventricular septum grows up toward the cushions, and the cushions form an upper inlet septum; between the two portions is a hole called the interventricular foramen. The left and right ventricles begin to develop side by side, and the atria and their respective inlet valves align over their ventricles. Finally, these two parts of the septum fuse with the bulboventricular ridges, which, once having septated the truncus arteriosus, extend into the ventricle. The bulbocordis divides into a subaortic portion as the muscular conus resorbs, whereas the subpulmonary section has elongation of its muscular conus. Spiral division of the common truncus arteriosus rotates and aligns the pulmonary artery and aortic portions

1	conus resorbs, whereas the subpulmonary section has elongation of its muscular conus. Spiral division of the common truncus arteriosus rotates and aligns the pulmonary artery and aortic portions over their respective outflow tracts, the aortic valve moving posterior over the left ventricle (LV) outflow tract and the pulmonary valve moving anterior over the right ventricle (RV) outflow tract, with a wraparound relationship of the two great arteries.

1	Early on, the venous systems are bilateral and symmetric and enter two horns of the sinus venosus. Ultimately, except for the coronary sinus, most of the left-sided portions and the left sinus–venosus horn regress, and the systemic venous system empties into the right horn via the inferior and superior vena cavae. The pulmonary venous system, initially connecting to the systemic venous system, develops as buds from the developing lungs, which fuse together in the pulmonary venous confluence, at which point the connection to the systemic system regresses. Simultaneously, a projection from the back wall of the left atrium (the common pulmonary vein) grows posteriorly to merge with the confluence, which then becomes a part of the posterior left atrial wall.

1	The truncus arteriosus and aortic sac initially develop six paired symmetric arches, which curve posteriorly and become the paired dorsal aortae. The detailed description of the selective regression of some of the arches is not presented in this chapter. In brief summary, Mild congenital mitral valve disease (e.g., except parachute valve, cleft this process results in the development of arch 3 as the internal carotid arteries, left arch 4 as the aortic arch and right subclavian artery, and part of arch 6 as the patent ductus arteriosus. The two dorsal thoracic aortae fuse in the abdomen with persistence of the left dorsal aorta.

1	Tables 282-1, 282-2, and 282-3 list CHD malformations as simple, intermediate, or complex. Simple defects generally are single lesions with a shunt or a valvular malformation. Intermediate defects may have two or more simple defects. Complex defects generally have components of an intermediate defect plus more complex cardiac and vascular anatomy, often with cyanosis, and frequently with transposition complexes. The goal of these tables is to suggest when cardiology consultation or advanced CHD specialty care is needed. Patients with complex CHD (which includes most “named” surgeries that usually involve complex CHD) should virtually always be managed in conjunction with an experienced specialty adult CHD center. Patients with intermediate lesions should have an initial consultation and subsequent occasional intermittent follow-up with an adult CHD specialist. Patients with simple lesions often may be managed by a well-informed internist or general cardiologist, although consultation

1	occasional intermittent follow-up with an adult CHD specialist. Patients with simple lesions often may be managed by a well-informed internist or general cardiologist, although consultation with a specifically trained adult congenital cardiologist is occasionally advisable.

1	Atrial septal defect (ASD) is a common cardiac anomaly that may be first encountered in the adult and occurs more frequently in females. Sinus venosus ASD occurs high in the atrial septum near the entry of the superior vena cava into the right atrium and is associated frequently with anomalous pulmonary venous connection from the right lung to the superior vena cava or right atrium (Fig. 282-1). Ostium primum ASDs lie adjacent to the atrioventricular valves, either of which may be deformed and regurgitant. Ostium primum ASDs are common in Down’s syndrome, often as part of complex atrioventricular septal defects with a common atrioventricular valve and a posterior defect of the basal portion of the interventricular septum. The most common ostium secundum ASD involves the fossa ovalis and is midseptal in location; this should not be confused with a patent foramen Anomalous pulmonary venous drainage, partial or total

1	Anomalous pulmonary venous drainage, partial or total Ventricular septal defect, complicated (e.g., absent or abnormal valves or with associated obstructive lesions, aortic regurgitation) Coarctation of the aorta Pulmonic valve stenosis (moderate to severe) Infundibular right ventricular outflow obstruction of significance Pulmonary valve regurgitation (moderate to severe) Patent ductus arteriosus (nonclosed)—moderate to large Sinus of Valsalva fistula/aneurysm Tetralogy of Fallot or pulmonary atresia (all forms)

1	Transposition of the great arteries ovale. Anatomic obliteration of the foramen ovale ordinarily follows its functional closure soon after birth, but residual “probe patency” is a common normal variant; ASD denotes a true deficiency of the atrial septum and implies functional and anatomic patency. The magnitude of the left-to-right shunt depends on the ASD size, ventricular diastolic properties, and the relative impedance in the pulmonary and systemic circulations. The left-to-right shunt causes diastolic overloading of the RV and increased pulmonary blood flow. Patients with ASD are usually asymptomatic in early life, although there may be some physical underdevelopment and an increased tendency for respiratory infections; cardiorespiratory symptoms occur in many older patients. Beyond the fourth decade, a significant number of patients develop atrial arrhythmias, pulmonary arterial hypertension, and right heart failure. Patients exposed to the chronic environmental hypoxemia of high

1	fourth decade, a significant number of patients develop atrial arrhythmias, pulmonary arterial hypertension, and right heart failure. Patients exposed to the chronic environmental hypoxemia of high altitude tend to develop pulmonary hypertension at younger ages. In older patients, left-to-right shunting across the ASD increases as progressive systemic hypertension and/or coronary artery disease (CAD) result in reduced compliance of the LV.

1	Physical Examination Examination usually reveals a prominent RV impulse and palpable pulmonary artery pulsation. The first heart sound is normal or split, with accentuation of the tricuspid valve closure sound. Increased flow across the pulmonic valve is responsible for a midsystolic pulmonary outflow murmur. The second heart sound is widely split and is fixed in relation to respiration. A mid-diastolic rumbling murmur, loudest at the fourth intercostal space and along the left sternal border, reflects increased flow across the tricuspid valve. In ostium primum ASD, an apical holosystolic murmur indicates associated mitral or tricuspid regurgitation or a ventricular septal defect (VSD). FIGURE 282-1 Types and locations of congenital cardiac defects. ASD, atrial septal defect; PDA, patent ductus arteriosus; RMPV, right middle pulmonary vein; RUPV, right upper pulmonary vein; VSD, ventricular septal defect.

1	These findings are altered when increased pulmonary vascular 1521 resistance causes diminution of the left-to-right shunt. Both the pulmonary outflow and tricuspid inflow murmurs decrease in intensity, the pulmonic component of the second heart sound and a systolic ejection sound are accentuated, the two components of the second heart sound may fuse, and a diastolic murmur of pulmonic regurgitation appears. Cyanosis and clubbing accompany the development of a right-to-left shunt (see “Ventricular Septal Defect” later in this chapter). In adults with an ASD and atrial fibrillation, the physical findings may be confused with mitral stenosis with pulmonary hypertension because the tricuspid diastolic flow murmur and widely split second heart sound may be mistakenly thought to represent the diastolic murmur of mitral stenosis and the mitral “opening snap,” respectively.

1	Electrocardiogram In ostium secundum ASD, electrocardiogram (ECG) usually shows right-axis deviation and an rSr´ pattern in the right precordial leads representing enlargement of the RV outflow tract. An ectopic atrial pacemaker or first-degree heart block may occur with the sinus venous ASD. In ostium primum ASD, the RV conduction defect is accompanied by left superior axis deviation and counterclockwise rotation of the frontal plane QRS loop. Varying degrees of RV and right atrial (RA) enlargement or hypertrophy may occur with each type of defect, depending on the presence and degree of pulmonary hypertension. Chest x-ray shows an enlarged RA and RV, and pulmonary artery and its branches; increased pulmonary vascular markings of left-to-right shunt vascularity will diminish if pulmonary vascular disease develops.

1	Echocardiogram Echocardiography reveals pulmonary arterial and RV and RA dilatation with abnormal (paradoxical) ventricular septal motion in the presence of a significant right heart volume overload. The ASD may be visualized directly by two-dimensional imaging, color-flow imaging, or echocontrast. Echocardiography and Doppler examination have supplanted cardiac catheterization. Transesophageal echocardiography is indicated if the transthoracic echocardiogram is ambiguous, which is often the case with sinus venosus defects, or for guiding catheter device closure (Fig. 282-2). Cardiac catheterization is performed if inconsistencies exist in the clinical data, if significant pulmonary hypertension or associated malformations are suspected, if CAD is a possibility, or when attempting transcatheter closure of the ASD.

1	Operative repair, usually with a patch of pericardium or of prosthetic material or percutaneous transcatheter device closure, if the ASD is of an appropriate size and shape, should be advised for all patients with uncomplicated secundum ASD with significant left-to-right shunting, i.e., pulmonary-to-systemic flow ratios ≥1.5:1. Excellent results may be anticipated, at low risk, even in patients >40 years, in the absence of severe pulmonary hypertension. In ostium primum ASD, cleft mitral valves may require repair in addition to patch closure of the ASD. Closure is not usually carried out in patients with small defects and trivial left-to-right shunts or in those with severe pulmonary vascular disease without a significant left-to-right shunt. However, the use of pulmonary vasodilators with resultant reduction in pulmonary artery pressure and resistance may allow closure of ASD in patients with pulmonary vascular disease.

1	Patients with sinus venosus or ostium secundum ASDs rarely die before the fifth decade. During the fifth and sixth decades, the incidence of progressive symptoms, often leading to severe disability, increases substantially. Medical management should include prompt treatment of respiratory tract infections; antiarrhythmic medications for atrial fibrillation or supraventricular tachycardia; and the usual measures for hypertension, coronary disease, or heart failure (Chap. 279), if these complications occur. The risk of infective endocarditis is low, and antibiotic prophylaxis is not recommended (Chap. 155). Congenital Heart Disease in the Adult

1	Congenital Heart Disease in the Adult FIGURE 282-2 A. Transesophageal echocardiogram demonstrating a secundum-type atrial septal defect (ASD) with shunting from the left atrium (LA) to the right atrium (RA). The right pulmonary artery (RPA) and superior vena cava (SVC) are labeled. B. Transcatheter balloon sizing of the ASD. C. Atrial septal occluder placement with a small manually created “fenestration” within the device that continues to allow a small amount of flow from the LA to the RA; this is used as a means of preventing left atrial hypertension after ASD closure. Left atrial hypertension may occur in older patients with decreased left ventricular compliance. D. Three-dimensional image of the septal occluder en-face; note the fenestration in the LA disc. The mitral valve (MV) and right inferior pulmonary vein (RIPV) are labeled.

1	VSD is one of the most common of all cardiac birth defects, either as an isolated defect or as a component of a combination of anomalies (Fig. 282-1). The VSD is usually single and situated in the membranous or midmuscular portion of the septum. The functional disturbance depends on its size and on the status of the pulmonary vascular bed. Only smallor moderate-size VSDs are seen initially in adulthood, as most patients with an isolated large VSD come to medical or surgical attention early in life.

1	A wide spectrum exists in the natural history of VSD, ranging from spontaneous closure to congestive cardiac failure and death in infancy. Included within this spectrum is the possible development of pulmonary vascular obstruction, RV outflow tract obstruction, aortic regurgitation, or infective endocarditis. Spontaneous closure is more common in patients born with a small VSD, which occurs in early childhood in most. The pulmonary vascular bed is often a principal determinant of the clinical manifestations and course of a given VSD and feasibility of surgical repair. Increased pulmonary arterial pressure results from increased pulmonary blood flow and/or resistance, the latter usually the result of obstructive, obliterative structural changes within the pulmonary vascular bed. It is important to quantitate and compare pulmonary-to-systemic flows and resistances in patients with severe pulmonary hypertension. The term Eisenmenger’s syndrome is applied to patients with a large

1	is important to quantitate and compare pulmonary-to-systemic flows and resistances in patients with severe pulmonary hypertension. The term Eisenmenger’s syndrome is applied to patients with a large communication between the two circulations at the aortopulmonary, ventricular, or atrial levels and bidirectional or predominantly right-to-left shunts because of high resistance and obstructive pulmonary hypertension.

1	Patients with large VSDs and pulmonary hypertension are at greatest risk for developing pulmonary vascular disease. Large VSDs should be corrected early in life when pulmonary vascular disease is not severely elevated. In patients with Eisenmenger’s syndrome, symptoms in adult life consist of exertional dyspnea, chest pain, syncope, and hemoptysis. The right-to-left shunt leads to cyanosis, clubbing, and erythrocytosis (see below). The degree to which pulmonary vascular resistance is elevated before operation is a critical factor determining prognosis. If the pulmonary vascular resistance is one-third or less of the systemic value, progression of pulmonary vascular disease after operation is unusual; however, if a moderate to severe increase in pulmonary vascular resistance exists preoperatively, either no change or a progression of pulmonary vascular disease is common postoperatively. Pregnancy is contraindicated in Eisenmenger’s syndrome. The mother’s health is most at risk if she

1	either no change or a progression of pulmonary vascular disease is common postoperatively. Pregnancy is contraindicated in Eisenmenger’s syndrome. The mother’s health is most at risk if she has a cardiovascular lesion associated with pulmonary vascular disease and pulmonary hypertension (e.g., Eisenmenger’s physiology or mitral stenosis) or severe LV outflow tract obstruction (e.g., aortic stenosis), but she is also at risk of death with any malformation that may cause heart failure or a hemodynamically important arrhythmia. The fetus is most at risk with maternal cyanosis, heart failure, or pulmonary hypertension.

1	RV outflow tract obstruction develops in ~5–10% of patients who present in infancy with a moderate to large left-to-right shunt. With time, as subvalvular RV outflow tract obstruction progresses, the findings in these patients whose VSD remains sizable begin to resemble more closely those of the cyanotic tetralogy of Fallot. In ~5% of patients, aortic valve regurgitation results from insufficient cusp tissue or prolapse of the cusp through the interventricular defect; the aortic regurgitation then complicates and dominates the clinical course. Echocardiography with spectral and color Doppler examination defines the number and location of defects in the ventricular septum and associated anomalies and the hemodynamic physiology of the defect(s). Hemodynamic and angiographic study may be occasionally required to assess the status of the pulmonary vascular bed and clarify details of the altered anatomy. Cross-sectional imaging modalities such as computed tomography (CT) or magnetic

1	be occasionally required to assess the status of the pulmonary vascular bed and clarify details of the altered anatomy. Cross-sectional imaging modalities such as computed tomography (CT) or magnetic resonance imaging (MRI) are useful in delineating complex anatomy and assessing extra-cardiac structures.

1	Closure is not recommended for patients with normal pulmonary arterial pressures with small shunts (pulmonary-to-systemic flow ratios of <1.5:1). Operative correction or transcatheter closure is indicated when there is a moderate to large left-to-right shunt with a pulmonary-to-systemic flow ratio >1.5:1, in the absence of prohibitively high levels of pulmonary vascular resistance (pulmonary arterial resistance is less than two-thirds of systemic arterial resistance).

1	In patients with Eisenmenger’s VSD, pulmonary arterial vasodilators and both singleor double-lung transplantation with intracardiac defect repair or heart/lung transplantation show promise for improvement in symptoms (Chaps. 281 and 320e). Chronic hypoxemia in cyanotic CHD results in secondary erythrocytosis due to increased erythropoietin production (Chap. 49). The term polycythemia is a misnomer; white cell counts are normal, and platelet counts are normal to decreased. Compensated erythrocytosis with iron-replete equilibrium hematocrits rarely results in symptoms of hyperviscosity at hematocrits <65% and occasionally not even with hematocrits ≥70%. For this reason, therapeutic phlebotomy is rarely required in compensated erythrocytosis. In contrast, patients with decompensated erythrocytosis fail to establish equilibrium with unstable, rising hematocrits and recurrent hyperviscosity symptoms. Therapeutic phlebotomy, a two-edged sword, allows temporary relief of symptoms but limits

1	fail to establish equilibrium with unstable, rising hematocrits and recurrent hyperviscosity symptoms. Therapeutic phlebotomy, a two-edged sword, allows temporary relief of symptoms but limits oxygen delivery, begets instability of the hematocrit, and compounds the problem by iron depletion. Iron-deficiency symptoms are usually indistinguishable from those of hyperviscosity; progressive symptoms after recurrent phlebotomy are usually due to iron depletion with hypochromic microcytosis. Iron depletion results in a larger number of smaller (microcytic) hypochromic red cells that are less capable of carrying oxygen and less deformable in the microcirculation; with more of them relative to plasma volume, viscosity is greater than for an equivalent hematocrit with fewer, larger, iron-replete, deformable cells. As such, iron-depleted erythrocytosis results in increasing symptoms due to decreased oxygen delivery to the tissues.

1	Hemostasis is abnormal in cyanotic CHD, due, in part, to the increased blood volume and engorged capillaries, abnormalities in platelet function, and sensitivity to aspirin or nonsteroidal anti-inflammatory agents, as well as abnormalities of the extrinsic and intrinsic coagulation system. Oral contraceptives are often contraindicated for cyanotic women because of the enhanced risk of vascular thrombosis. Symptoms of hyperviscosity can be produced in any cyanotic patient with erythrocytosis if dehydration reduces plasma volume. Phlebotomy for symptoms of hyperviscosity not due to dehydration or iron deficiency is a simple outpatient removal of 500 mL of blood over 45 min with isovolumetric replacement with isotonic saline. Acute phlebotomy without volume replacement is contraindicated. Iron repletion in decompensated iron-depleted erythrocytosis reduces iron-deficiency symptoms, but must be done gradually to avoid an excessive rise in hematocrit and resulting hyperviscosity.

1	The ductus arteriosus is a vessel leading from the bifurcation of the pulmonary artery to the aorta just distal to the left subclavian artery (Fig. 282-1). Normally, the vascular channel is open in the fetus but closes immediately after birth. The flow across the ductus is determined by the pressure and resistance relationships between the systemic and pulmonary circulations and by the cross-sectional area and length of the ductus. In most adults with this anomaly, pulmonary pressures are normal, and a gradient and shunt from aorta to pulmonary artery persist throughout the cardiac cycle, resulting in a characteristic thrill 1523 and a continuous “machinery” murmur with late systolic accentuation at the upper left sternal edge. In adults who were born with a large left-to-right shunt through the ductus arteriosus, pulmonary vascular obstruction (Eisenmenger’s syndrome) with pulmonary hypertension, right-to-left shunting, and cyanosis have usually developed. Severe pulmonary vascular

1	the ductus arteriosus, pulmonary vascular obstruction (Eisenmenger’s syndrome) with pulmonary hypertension, right-to-left shunting, and cyanosis have usually developed. Severe pulmonary vascular disease results in reversal of flow through the ductus; unoxygenated blood is shunted to the descending aorta; and the toes—but not the fingers—become cyanotic and clubbed, a finding termed differential cyanosis (Fig. 282-3). The leading causes of death in adults with patent ductus arteriosus are cardiac failure and infective endocarditis; occasionally, severe pulmonary vascular obstruction may cause aneurysmal dilatation, calcification, and rupture of the ductus.

1	In the absence of severe pulmonary vascular disease and predominant left-to-right shunting of blood, the patent ductus should be surgically ligated or divided. Transcatheter closure has become common for appropriately shaped defects. Operation should be deferred for several months in patients treated successfully for infective endocarditis because the ductus may remain somewhat edematous and friable.

1	The three most common causes of aortic root–to–right-heart shunts are congenital aneurysm of an aortic sinus of Valsalva with fistula, coronary arteriovenous fistula, and anomalous origin of the left coronary artery from the pulmonary trunk. Aneurysm of an aortic sinus of Valsalva consists of a separation or lack of fusion between the media of the aorta and the annulus of the aortic valve. Rupture usually occurs in the third or fourth decade of life; most often, the aorticocardiac fistula is between the right coronary cusp and the RV; but occasionally, when the noncoronary cusp is involved, the fistula drains into the RA. Abrupt rupture causes chest pain, bounding pulses, a continuous murmur accentuated in diastole, and volume overload of the heart. Diagnosis is confirmed by two-dimensional and Doppler echocardiographic studies; cardiac catheterization quantitates the left-to-right shunt, and thoracic aortography visualizes the fistula. Medical management is directed at cardiac

1	and Doppler echocardiographic studies; cardiac catheterization quantitates the left-to-right shunt, and thoracic aortography visualizes the fistula. Medical management is directed at cardiac failure, arrhythmias, or endocarditis. At operation, the aneurysm is closed and amputated, and the aortic wall is reunited with the heart, either by direct suture or with a patch or prosthesis. Transcatheter device closure is a less invasive and effective alternative to surgery.

1	Coronary arteriovenous fistula, an unusual anomaly, consists of a communication between a coronary artery and another cardiac chamber, usually the coronary sinus, RA, or RV. The shunt is usually of small magnitude, and myocardial blood flow is not usually compromised; if the shunt is large, there may be a coronary “steal” syndrome with myocardial ischemia and possible angina or ventricular arrhythmias. Potential complications include infective endocarditis; thrombus formation with occlusion or distal embolization with myocardial infarction; rupture of an aneurysmal fistula; and, rarely, pulmonary hypertension and congestive failure. A loud, superficial, continuous murmur at the lower or midsternal border usually prompts a further evaluation of asymptomatic patients. Doppler echocardiography demonstrates the site of drainage; if the site of origin is proximal, it may be detectable by two-dimensional echocardiography. Angiography (classic catheterization, CT, or magnetic resonance

1	demonstrates the site of drainage; if the site of origin is proximal, it may be detectable by two-dimensional echocardiography. Angiography (classic catheterization, CT, or magnetic resonance angiography) permits identification of the size and anatomic features of the fistulous tract, which may be closed by suture or transcatheter obliteration.

1	The third anomaly causing a shunt from the aortic root to the right heart is anomalous origin of the left coronary artery from the pulmonary artery. In this condition, oxygenated blood from the aortic root flows via a dilated right coronary artery and collaterals to the left coronary artery and retrograde to the lower pressure pulmonary artery circulation via the anomalous left main coronary artery (which emerges from the pulmonary artery). Myocardial infarction and Congenital Heart Disease in the Adult

1	Congenital Heart Disease in the Adult FIGURE 282-3 A. Patent ductus arteriosus (PDA) in a patient with severe pulmonary hypertension (Eisenmenger’s syndrome). Due to the suprasystemic pulmonary arterial resistance, deoxygenated (cyanotic) blood from the right ventricle (RV) and pulmonary artery (PA) is shunted across the PDA to the aorta (Ao). The left atrium (LA) and left ventricle (LV) are labeled. B. Differential clubbing and cyanosis of the toes due to lower extremity perfusion by the deoxygenated blood crossing the PDA. C. Angiogram in a dilated main pulmonary artery (MPA) with shunting noted across the PDA to the descending aorta (dAo). The left pulmonary artery (LPA) is labeled. D. Direct pressure recordings in the Ao and PA demonstrating suprasystemic PA systolic pressure.

1	fibrosis commonly lead to death within the first year, although up to 20% of patients survive to adolescence and beyond without surgical correction. The diagnosis is supported by the ECG findings of an anterolateral myocardial infarction and left ventricular hypertrophy (LVH). Operative management of adults consists of coronary artery reimplantation, coronary artery bypass with an internal mammary artery graft, or saphenous vein–coronary artery graft.

1	Malformations that cause obstruction to LV outflow include congenital valvular aortic stenosis, discrete subaortic stenosis, or supravalvular aortic stenosis. Bicuspid aortic valves are more common in males than in females. The congenital bicuspid aortic valve, which may initially be functionally normal, is one of the most common congenital malformations of the heart and may go undetected in early life. Because bicuspid valves may develop stenosis or regurgitation with time or be the site of infective endocarditis, the lesion may be difficult to distinguish in older adults from acquired rheumatic or degenerative calcific aortic valve disease. The dynamics of blood flow associated with a congenitally deformed, rigid aortic valve commonly lead to thickening of the cusps and, in later life, to calcification. Hemodynamically significant obstruction causes concentric hypertrophy of the LV wall. The ascending aorta is often dilated, misnamed “poststenotic” dilatation; this is due to

1	life, to calcification. Hemodynamically significant obstruction causes concentric hypertrophy of the LV wall. The ascending aorta is often dilated, misnamed “poststenotic” dilatation; this is due to histologic abnormalities of the aortic media and may result in aortic dissection. Diagnosis is made by echocardiography, which reveals the morphology of the aortic valve and aortic root and quantitates severity of stenosis or regurgitation. The clinical manifestations and hemodynamic abnormalities are discussed in Chap. 283.

1	In patients with diminished cardiac reserve, medical management includes the administration of digoxin and diuretics and sodium restriction while awaiting operation. A dilated aortic root may require beta blockers, angiotensin receptor blockers, or angiotensin-converting enzyme inhibitors. Aortic valve replacement is indicated in adults with critical obstruction, i.e., with an aortic valve area <0.45 cm2/m2, with symptoms secondary to LV dysfunction or myocardial ischemia, or with hemodynamic evidence of LV dysfunction. In asymptomatic children or adolescents or young adults with critical aortic stenosis without valvular calcification or these features, aortic balloon valvuloplasty is often useful (Chap. 296e). If surgery is contraindicated in older patients because of a complicating medical problem such as malignancy or renal or hepatic failure, balloon valvuloplasty may provide short-term improvement. This procedure may serve as a bridge to aortic valve replacement in patients with

1	problem such as malignancy or renal or hepatic failure, balloon valvuloplasty may provide short-term improvement. This procedure may serve as a bridge to aortic valve replacement in patients with severe heart failure. Transcatheter aortic valve replacement is a potential alternative to surgery.

1	suBaortic stEnosis The discrete form of subaortic stenosis consists of a membranous diaphragm or fibromuscular ring encircling the LV outflow tract just beneath the base of the aortic valve. The jet impact from the subaortic stenotic jet on the underside of the aortic valve often begets progressive aortic valve fibrosis and valvular regurgitation. Echocardiography demonstrates the anatomy of the subaortic obstruction; Doppler studies show turbulence proximal to the aortic valve and can quantitate the pressure gradient and severity of aortic regurgitation. Treatment consists of complete excision of the membrane or fibromuscular ring.

1	supravalvular aortic stEnosis This is a localized or diffuse narrowing of the ascending aorta originating just above the level of the coronary arteries at the superior margin of the sinuses of Valsalva. In contrast to other forms of aortic stenosis, the coronary arteries are subjected to elevated systolic pressures from the LV, are often dilated and tortuous, and are susceptible to premature atherosclerosis. The coronary ostia may also become obstructed by the aortic valve leaflets. In most patients, a genetic defect for the anomaly is located in the same chromosomal region as elastin on chromosome 7. Supravalvular aortic stenosis is the most commonly associated cardiac defect in Williams-Beuren syndrome, typically comprising the following: “elfin” facies, low nasal bridge, cheerful demeanor, mental retardation with retained language skills and love of music, supravalvular aortic stenosis, and transient hypercalcemia.

1	Narrowing or constriction of the lumen of the aorta may occur anywhere along its length but is most common distal to the origin of the left subclavian artery near the insertion of the ligamentum arteriosum. Coarctation occurs in ~7% of patients with CHD, is more common in males than females, and is particularly frequent in patients with gonadal dysgenesis (e.g., Turner’s syndrome). Clinical manifestations depend on the site and extent of obstruction and the presence of associated cardiac anomalies, most commonly a bicuspid aortic valve. Circle of Willis aneurysms may occur in up to 10%.

1	Most children and young adults with isolated, discrete coarctation are asymptomatic. Headache, epistaxis, chest pressure, and claudication with exercise may occur, and attention is usually directed to the cardiovascular system when a heart murmur or hypertension in the upper extremities and absence, marked diminution, or delayed pulsations in the femoral arteries are detected on physical examination. Enlarged and pulsatile collateral vessels may be palpated in the intercostal spaces anteriorly, in the axillae, or posteriorly in the inter-scapular area. The upper extremities and thorax may be more developed than the lower extremities. A midsystolic murmur over the left interscapular space may become continuous if the lumen is narrowed sufficiently to result in a high-velocity jet across the lesion throughout the cardiac cycle. Additional systolic and continuous murmurs over the lateral thoracic wall may reflect increased flow through dilated and tortuous collateral vessels. The ECG

1	the lesion throughout the cardiac cycle. Additional systolic and continuous murmurs over the lateral thoracic wall may reflect increased flow through dilated and tortuous collateral vessels. The ECG usually reveals LV hypertrophy. Chest x-ray may show a dilated left subclavian artery high on the left mediastinal border and a dilated ascending aorta. Indentation of the aorta at the site of coarctation and preand poststenotic dilatation (the “3” sign) along the left paramediastinal shadow are essentially pathognomonic. Notching of the third to ninth ribs, an important radiographic sign, is due to inferior rib erosion by dilated collateral vessels.

1	Two-dimensional echocardiography from suprasternal windows iden-1525 tifies the site of coarctation; Doppler quantitates the pressure gradient. Transesophageal echocardiography and MRI or CT allow visualization of the length and severity of the obstruction and associated collateral arteries. In adults, cardiac catheterization is indicated primarily to evaluate the coronary arteries or to perform catheter-based intervention (angioplasty and stent of the coarctation). The chief hazards of proximal aortic severe hypertension include cerebral aneurysms and hemorrhage, aortic dissection and rupture, premature coronary arteriosclerosis, aortic valve failure, and LV failure; infective endarteritis may occur on the coarctation site or endocarditis may settle on an associated bicuspid aortic valve, which is estimated to be present in 50% of patients.

1	Treatment is surgical or involves percutaneous catheter balloon dilatation with stent placement; the details of selection of therapy are beyond this review; however, the use of transcatheter treatment techniques has increased dramatically, and many previously “surgical” cases are treated via percutaneous or hybrid techniques. Late postoperative systemic hypertension in the absence of residual coarctation is related partly to the duration of preoperative hypertension. Follow-up of rest and exercise blood pressures is important; many have systolic hypertension only during exercise, in part due to a diffuse vasculopathy and to noncompliance of the stented or surgically reconstructed region. All operated or stented coarctation patients deserve a high-quality MRI or CT procedure in follow-up.

1	Obstruction to RV outflow may be localized to the supravalvular, valvular, or subvalvular levels or occur at a combination of these sites. Multiple sites of narrowing of the peripheral pulmonary arteries are a feature of rubella embryopathy and may occur with both the familial and sporadic forms of supravalvular aortic stenosis. Valvular pulmonic stenosis (PS) is the most common form of isolated RV obstruction.

1	The severity of the obstructing lesion, rather than the site of narrowing, is the most important determinant of the clinical course. In the presence of a normal cardiac output, a peak systolic pressure gradient <30 mmHg indicates mild PS and >50 mmHg indicates severe PS; pressures between these limits are considered to indicate moderate stenosis. Patients with mild PS are generally asymptomatic and demonstrate little or no progression in the severity of obstruction with age. In patients with more significant stenosis, the severity may increase with time. Symptoms vary with the degree of obstruction. Fatigue, dyspnea, RV failure, and syncope may limit the activity of older patients, in whom moderate or severe obstruction may prevent an augmentation of cardiac output with exercise. In patients with severe obstruction, the systolic pressure in the RV may exceed that in the LV, because the ventricular septum is intact. RV ejection is prolonged with moderate or severe stenosis, and the

1	with severe obstruction, the systolic pressure in the RV may exceed that in the LV, because the ventricular septum is intact. RV ejection is prolonged with moderate or severe stenosis, and the sound of pulmonary valve closure is delayed and soft. RV hypertrophy reduces the compliance of that chamber, and a forceful RA contraction is necessary to augment RV filling. A fourth heart sound; prominent a waves in the jugular venous pulse; and, occasionally, presystolic pulsations of the liver reflect vigorous atrial contraction. The clinical diagnosis is supported by a left parasternal lift and harsh systolic crescendo-decrescendo murmur and thrill at the upper left sternal border, typically preceded by a systolic ejection sound if the obstruction is due to a mobile nondysplastic pulmonary valve. The holosystolic murmur of tricuspid regurgitation may accompany severe PS, especially in the presence of congestive heart failure. Cyanosis usually reflects right-to-left shunting through a patent

1	The holosystolic murmur of tricuspid regurgitation may accompany severe PS, especially in the presence of congestive heart failure. Cyanosis usually reflects right-to-left shunting through a patent foramen ovale or ASD. In patients with supravalvular or peripheral pulmonary arterial stenosis, the murmur is systolic or continuous and is best heard over the area of narrowing, with radiation to the peripheral lung fields.

1	Congenital Heart Disease in the Adult FIGURE 282-4 A. Transesophageal echocardiogram of a patient with severe pulmonary stenosis due to a mobile and doming pulmonary valve (PV). The pulmonary artery (PA) and the right ventricle (RV) are labeled. B. Following balloon valvuloplasty, the pulmonary valve orifice is larger. C. Simultaneous RV and PA pressure tracings before balloon valvuloplasty; the peak-to-peak gradient across the pulmonary valve is ~70 mmHg. D. After balloon valvuloplasty, the peak-to-peak gradient is reduced to ~25 mmHg.

1	D. After balloon valvuloplasty, the peak-to-peak gradient is reduced to ~25 mmHg. In mild cases, the ECG is normal, whereas moderate and severe stenoses are associated with RV hypertrophy. The chest x-ray with mild or moderate PS shows a heart of normal size with normal lung vascularity. In pulmonary valvular stenosis, dilatation of the main and left pulmonary arteries occurs in part due to the direction of the PS jet and in part due to intrinsic tissue weakness. With severe obstruction, RV hypertrophy is generally evident. The pulmonary vascularity may be reduced with severe stenosis, RV failure, and/or a right-to-left shunt at the atrial level. Twoand three-dimensional echocardiography visualizes pulmonary valve morphology; the outflow tract pressure gradient is quantitated by Doppler echocardiography (Fig. 282-4). The cardiac catheter technique of balloon valvuloplasty (Chap.

1	The cardiac catheter technique of balloon valvuloplasty (Chap. 272) is usually effective, and the surgery is rarely necessary. Multiple stenoses of the peripheral pulmonary arteries are effectively treated with transcatheter angioplasty or stenting. The four components of the tetralogy of Fallot are malaligned VSD, obstruction to RV outflow, aortic override of the VSD, and RV hypertrophy due to the RV’s response to aortic pressure via the large VSD. The severity of RV outflow obstruction determines the clinical presentation. The severity of hypoplasia of the RV outflow tract varies from mild to complete (pulmonary atresia). Pulmonary valve stenosis and supravalvular and peripheral pulmonary arterial obstruction may coexist; rarely, there is unilateral absence of a pulmonary artery (usually the left). A right-sided aortic arch and descending thoracic aorta occur in ~25%.

1	The relationship between the resistance of blood flow from the ventricles into the aorta and into the pulmonary artery plays a major role in determining the hemodynamic and clinical picture. When the RV outflow obstruction is severe, pulmonary blood flow is reduced markedly, and a large volume of desaturated systemic venous blood shunts right-to-left across the VSD. Severe cyanosis and erythrocytosis occur, and symptoms of systemic hypoxemia are prominent. In many infants and children, the obstruction is mild but progressive.

1	The ECG shows RV hypertrophy. Chest x-ray shows a normal-sized, boot-shaped heart (coeur en sabot) with a prominent RV and a concavity in the region of the pulmonary conus. Pulmonary vascular markings are typically diminished, and the aortic arch and knob may be on the right side. Echocardiography demonstrates the malaligned VSD with the overriding aorta and the site and severity of PS, which may be subpulmonic (fixed or dynamic), at the pulmonary valve or in the main or branch pulmonary arteries. Classic contrast angiography may provide details regarding the RV outflow tract, pulmonary valve and annulus, and caliber of the main branches of the pulmonary artery, as well as about possible associated aortopulmonary collaterals. Coronary arteriography identifies the anatomy and course of the coronary arteries, which may be anomalous. Cardiac MRI and CT complement echocardiography and provide much of the information gathered by angiography as well as additional functional information. MRI

1	coronary arteries, which may be anomalous. Cardiac MRI and CT complement echocardiography and provide much of the information gathered by angiography as well as additional functional information. MRI is considered the clinical gold standard for quantification of RV volume and function as well as quantification of the pulmonary regurgitation severity.

1	For a variety of reasons, only a few adults with tetralogy of Fallot have not had some form of previous surgical intervention. Reoperation in adults is most commonly for severe pulmonary regurgitation or pulmonary stenosis. Long-term concerns about ventricular function persist. Ventricular and atrial arrhythmias occur, respectively, in 15% and 25% of adults and may require medical treatment, electrophysiologic study and ablation, defibrillator placement, or transcatheter or surgical intervention, usually including pulmonary valve replacement. Transcatheter pulmonary valve replacement is widely used in patients meeting anatomic criteria. The aortic root has a medial tissue defect; it is commonly enlarged and associated with aortic regurgitation. Endocarditis remains a risk despite surgical repair.

1	This condition is commonly called dextro-or D-transposition of the great arteries. The aorta arises rightward anteriorly from the RV, and the pulmonary artery emerges leftward and posteriorly from the LV, which results in two separate parallel circulations; some communication between them must exist after birth to sustain life. Most patients have an interatrial communication, two-thirds have a patent ductus arteriosus, and about one-third have an associated VSD. Transposition is more common in males and accounts for ~10% of cyanotic heart disease. The course is determined by the degree of tissue hypoxemia, the ability of each ventricle to sustain an increased workload in the presence of reduced coronary arterial oxygenation, the nature of the associated cardiovascular anomalies, and the status of the pulmonary vascular bed. Patients who do not undergo surgical palliation generally do not survive to reach adulthood. The long-term outcomes in those that have undergone surgery are in

1	status of the pulmonary vascular bed. Patients who do not undergo surgical palliation generally do not survive to reach adulthood. The long-term outcomes in those that have undergone surgery are in large part determined by the type of surgery performed. By the third decade of life, ~30% of patients with “atrial switch” operations will have developed decreased RV function and progressive tricuspid regurgitation, which may lead to congestive heart failure. Pulmonary vascular obstruction develops by 1–2 years of age in patients with an associated large VSD or large patent ductus arteriosus in the absence of obstruction to LV outflow.

1	The balloon or blade catheter or surgical creation or enlargement of an interatrial communication in the neonate is the simplest procedure for providing increased intracardiac mixing of systemic and pulmonary venous blood. Systemic pulmonary artery anastomosis may be indicated in the patient with severe obstruction to LV outflow and diminished pulmonary blood flow. Intracardiac repair may be accomplished by rearranging the venous returns (intraatrial switch, i.e., Mustard or Senning operation) so that the 1527 systemic venous blood is directed to the mitral valve and, thence, to the LV and pulmonary artery, while the pulmonary venous blood is diverted through the tricuspid valve and RV to the aorta. The late survival after these repairs is good, but arrhythmias (e.g., atrial flutter) or conduction defects (e.g., sick sinus syndrome) occur in ~50% of such patients by 30 years after the intraatrial switch surgery. Progressive dysfunction of the systemic subaortic RV, tricuspid

1	or conduction defects (e.g., sick sinus syndrome) occur in ~50% of such patients by 30 years after the intraatrial switch surgery. Progressive dysfunction of the systemic subaortic RV, tricuspid regurgitation, ventricular arrhythmias, cardiac arrest, and late sudden death are worrisome features. Preferably, this malformation is corrected in infancy by transposing both coronary arteries to the posterior artery and transecting, contraposing, and anastomosing the aorta and pulmonary arteries (arterial-switch operation). For patients with a VSD in whom it is necessary to bypass a severely obstructed LV outflow tract, corrective operation employs an intra-cardiac ventricular baffle and extracardiac prosthetic conduit to replace the pulmonary artery (Rastelli procedure).

1	This is a family of complex lesions with both atrioventricular valves or a common atrioventricular valve opening to a single ventricular chamber. Associated anomalies include abnormal great artery positional relationships, pulmonic valvular or subvalvular stenosis, and subaortic stenosis. Survival to adulthood depends on a relatively normal pulmonary blood flow, yet normal pulmonary resistance and good ventricular function. Modifications of the Fontan approach are generally applied to carefully selected patients with creation of a pathway(s) from the systemic veins to the pulmonary arteries. This malformation is characterized by atresia of the tricuspid valve; an interatrial communication; and, frequently, hypoplasia of the RV and pulmonary artery. The clinical picture is usually dominated by severe cyanosis due to obligatory admixture of systemic and pulmonary venous blood in the LV. The ECG characteristically shows RA enlargement, left-axis deviation, and LV hypertrophy.

1	Atrial septostomy and palliative operations to increase pulmonary blood flow, often by anastomosis of a systemic artery or vein to a pulmonary artery, may allow survival to the second or third decade. A Fontan atriopulmonary or total cavopulmonary connection may then allow functional correction in patients with normal or low pulmonary arterial resistance pressure and good LV function. There are a number of important long-term considerations with the Fontan circulation, including the development of arrhythmias, progressive liver dysfunction, thromboembolic complications, and potential long-term need for heart or heart and liver transplantation.

1	Characterized by a downward displacement of the tricuspid valve into the RV, due to anomalous attachment of the tricuspid leaflets, the Ebstein tricuspid valve tissue is dysplastic and results in tricuspid regurgitation. The abnormally situated tricuspid orifice produces an “atrialized” portion of the RV lying between the atrioventricular ring and the origin of the valve, which is continuous with the RA chamber. Often, the RV is hypoplastic. Although the clinical manifestations are variable, some patients come to initial attention because of either (1) progressive cyanosis from right-to-left atrial shunting, (2) symptoms due to tricuspid regurgitation and RV dysfunction, or (3) paroxysmal atrial tachyarrhythmias with or without atrioventricular bypass tracts (Wolff-Parkinson-White [WPW] syndrome). Diagnostic findings by two-dimensional echocardiography include the abnormal positional relation between the tricuspid and mitral valves with abnormally increased apical displacement of the

1	Diagnostic findings by two-dimensional echocardiography include the abnormal positional relation between the tricuspid and mitral valves with abnormally increased apical displacement of the septal tricuspid leaflet. Tricuspid regurgitation is quantitated by Doppler examination. Surgical approaches include prosthetic replacement of the tricuspid valve when the leaflets are tethered or repair of the native valve.

1	Congenital Heart Disease in the Adult 1528 CONGENITALLY CORRECTED TRANSPOSITION The two fundamental anatomic abnormalities in this malformation are transposition of the ascending aorta and pulmonary trunk and inversion of the ventricles. This arrangement results in desaturated systemic venous blood passing from the RA through the mitral valve to the LV and into the pulmonary trunk, whereas oxygenated pulmonary venous blood flows from the left atrium through the tricuspid valve to the RV and into the aorta. Thus, the circulation is corrected functionally. The clinical presentation, course, and prognosis of patients with congenitally corrected transposition vary depending on the nature and severity of any complicating intracardiac anomalies and of development of dysfunction of the systemic subaortic RV. Progressive RV dysfunction and tricuspid regurgitation may also develop in one-third of patients by age 30; Ebstein-type anomalies of the left-side tricuspid atrioventricular valve are

1	subaortic RV. Progressive RV dysfunction and tricuspid regurgitation may also develop in one-third of patients by age 30; Ebstein-type anomalies of the left-side tricuspid atrioventricular valve are common. VSD or PS due to obstruction to outflow from the right-sided subpulmonary (anatomic left) ventricle may coexist. Complete heart block occurs at a rate of 2–10% per decade. The diagnosis of the malformation and associated lesions can be established by comprehensive two-dimensional echocardiography and Doppler examination.

1	Positional anomalies refer to conditions in which the cardiac apex is in the right side of the chest (dextrocardia) or at the midline (mesocardia), or in which there is a normal location of the heart in the left side of the chest but abnormal position of the viscera (isolated levocardia). Knowledge of the position of the abdominal organs and of the branching pattern of the main stem bronchi is important in categorizing these malpositions. When dextrocardia occurs without situs inversus, when the visceral situs is indeterminate, or if isolated levocardia is present, associated, often complex, multiple cardiac anomalies are usually present. In contrast, mirror-image dextrocardia is usually observed with complete situs inversus, which occurs most frequently in individuals whose hearts are otherwise normal.

1	Owing to the enormous strides in cardiovascular surgical techniques that have occurred in the past 70 years, a large number of long-term survivors of palliative or corrective operations in infancy and childhood have reached adulthood. These patients are often challenging because of the diversity of anatomic, hemodynamic, and electrophysiologic residua and sequelae of cardiac operations.

1	The proper care of the survivor of an operation for CHD requires that the clinician understand the details of the malformation before operation; pay meticulous attention to the details of the operative procedure; and recognize the postoperative residua (conditions left totally or partially uncorrected), the sequelae (conditions caused by surgery), and the complications that may have resulted from the operation. Except for ligation of an uncomplicated patent ductus arteriosus, almost every other surgical repair leaves behind or causes some abnormality of the heart and circulation that may range from trivial to serious. Thus, even with results that are considered clinically to be good to excellent, continued long-term postoperative follow-up is advisable.

1	Cardiac operations involving the atria, such as closure of ASD, repair of total or partial anomalous pulmonary venous return, or venous switch corrections of complete transposition of the great arteries (the Mustard or Senning operations), may be followed years later by sinus node or atrioventricular node dysfunction and/or by atrial arrhythmias (especially atrial flutter). Intraventricular surgery may also result in electrophysiologic consequences, including complete heart block necessitating pacemaker insertion to avoid sudden death. Valvular problems may arise late after initial cardiac operation. An example is the progressive stenosis of an initially nonobstructive bicuspid aortic valve in the patient who underwent aortic coarctation repair. Such aortic valves may also be the site of infective endocarditis. After repair of the ostium primum ASD, the cleft mitral valve may become progressively regurgitant. Tricuspid regurgitation may also be progressive in the postoperative patient

1	endocarditis. After repair of the ostium primum ASD, the cleft mitral valve may become progressively regurgitant. Tricuspid regurgitation may also be progressive in the postoperative patient with tetralogy of Fallot if RV outflow tract obstruction was not relieved adequately at initial surgery. In many patients with surgically modified CHD, inadequate relief of an obstructive lesion, a residual regurgitant lesion, or a residual shunt will cause or hasten the onset of clinical signs and symptoms of myocardial dysfunction. Despite a good hemodynamic repair, many patients with a subaortic RV develop RV decompensation and signs of left heart failure. In many patients, particularly those who were cyanotic for many years before operation, a preexisting compromise in ventricular performance is due to the original underlying malformation.

1	A final category of postoperative problems involves the use of prosthetic valves, patches, or conduits in the operative repair. The special risks include infective endocarditis, thrombus formation, and premature degeneration and calcification of the prosthetic materials. There are many patients in whom extracardiac conduits are required to correct the circulation functionally and often to carry blood to the lungs from the RA or RV. These conduits may develop intraluminal obstruction, and if they include a prosthetic valve, it may show progressive calcification and thickening. Many such patients face reintervention (interventional cardiac catheterization or surgical reoperation) one or more times in their lives. Such care should be directed to centers specializing in adults with complex congenital cardiovascular malformations. The effect of pregnancy in postoperative patients depends on the outcome of the repair, including the presence and severity of residua, sequelae, or

1	complex congenital cardiovascular malformations. The effect of pregnancy in postoperative patients depends on the outcome of the repair, including the presence and severity of residua, sequelae, or complications. Contraception is an important topic with such patients. Tubal ligation should be considered in those in whom pregnancy is strictly contraindicated.

1	Endocarditis Prophylaxis Two major predisposing causes of infective endocarditis are a susceptible cardiovascular substrate and a source of bacteremia. The clinical and bacteriologic profile of infective endocarditis in patients with CHD has changed with the advent of intracardiac surgery and of prosthetic devices. Prophylaxis includes both antimicrobial and hygienic measures. Meticulous dental and skin care are required. Routine antimicrobial prophylaxis is recommended for bacteremic dental procedures or instrumentation through an infected site in most patients with operated CHD, particularly if foreign material, such as a prosthetic valve, conduit, or surgically constructed shunt, is in place. In the case of patches, in the absence of a high-pressure patch leak, or transcatheter devices, prophylaxis is usually recommended for 6 months until there is endothelialization. Individuals with unrepaired cyanotic heart disease are also generally recommended to receive prophylaxis (Chap.

1	prophylaxis is usually recommended for 6 months until there is endothelialization. Individuals with unrepaired cyanotic heart disease are also generally recommended to receive prophylaxis (Chap. 155).

1	Patrick T. O’Gara, Joseph Loscalzo

1	Primary valvular heart disease ranks well below coronary heart disease, stroke, hypertension, obesity, and diabetes as a major threat to the public health. Nevertheless, it is the source of significant morbidity and mortality rates. Rheumatic fever (Chap. 381) is the dominant cause of valvular heart disease in developing and low-income countries. Its prevalence has been estimated to range from as low as 1 per 100,000 school-age children in Costa Rica to as high as 150 per 100,000 in China. Rheumatic heart disease accounts for 12–65% of hospital admissions related to cardiovascular disease and 2–10% of hospital discharges in some developing countries. Prevalence and mortality rates vary among communities even within the same country as a function of overcrowding and the availability of medical resources and population-wide programs for detection and treatment of group A streptococcal pharyngitis. In economically deprived areas, tropical and subtropical climates (particularly on the

1	medical resources and population-wide programs for detection and treatment of group A streptococcal pharyngitis. In economically deprived areas, tropical and subtropical climates (particularly on the Indian subcontinent), Central

1	America, and the Middle East, rheumatic valvular disease progresses more rapidly than in more-developed nations and frequently causes serious symptoms in patients younger than 20 years of age. This accelerated natural history may be due to repeated infections with more virulent strains of rheumatogenic streptococci. Approximately 15 million to 20 million people live with rheumatic heart disease worldwide, an estimated prevalence characterized by 300,000 new cases and 233,000 case fatalities per year, with the highest mortality rates reported from Southeast Asia (~7.6 per 100,000).

1	Although there have been recent reports of isolated outbreaks of streptococcal infection in North America, valve disease in high-income countries is dominated by degenerative or inflammatory processes that lead to valve thickening, calcification, and dysfunction. The prevalence of valvular heart disease increases with age for both men and women. Important left-sided valve disease may affect as many as 12–13% of adults older than the age of 75. In the United States, there were 85,000 hospital discharges with valvular heart disease in 2010, and the vast majority of these were related to surgical procedures for heart valve disease (mostly involving the aortic and mitral valves).

1	The incidence of infective endocarditis (Chap. 155) has increased with the aging of the population, the more widespread prevalence of vascular grafts and intracardiac devices, the emergence of more virulent multidrug-resistant microorganisms, and the growing epidemic of diabetes. The more restricted use of antibiotic prophylaxis since 2007 has thus far not been associated with an increase in incidence rates. Infective endocarditis has become a relatively more frequent cause of acute valvular regurgitation. Bicuspid aortic valve disease affects as many as 0.5–1.4% of the general population, with an associated incidence of aortopathy involving root or ascending aortic aneurysm disease or coarctation. An increasing number of childhood survivors of congenital heart disease present later in life with valvular dysfunction. The global burden of valvular heart disease is expected to progress.

1	As is true for many other chronic health conditions, disparities in access to and quality of care for patients with valvular heart disease have been well documented. Management decisions and outcome differences based on age, gender, race, and geography require educational efforts across all levels of providers. The role of the physical examination in the evaluation of patients with valvular heart disease is also considered in Chaps. 51e and 267; of electrocardiography (ECG) in Chap. 268; of echocardiography and other noninvasive imaging techniques in Chap. 270e; and of cardiac catheterization and angiography in Chap. 272. Aortic stenosis (AS) occurs in about one-fourth of all patients with chronic valvular heart disease; approximately 80% of adult patients with symptomatic, valvular AS are male.

1	(Table 283-1) AS in adults is due to degenerative calcification of the aortic cusps and occurs most commonly on a substrate of congenital disease (bicuspid aortic valve), chronic (trileaflet) deterioration, or previous rheumatic inflammation. A pathologic study of specimens removed at the time of aortic valve replacement for AS showed that 53% were bicuspid and 4% unicuspid. The process of aortic valve deterioration and calcification is not a passive one, but rather one that shares many features with vascular atherosclerosis, including endothelial dysfunction, lipid accumulation, inflammatory cell activation, cytokine release, and upregulation of several signaling pathways (Fig. 283-1). Eventually, valvular myofibroblasts differentiate phenotypically into osteoblasts and actively produce bone matrix proteins that allow for the deposition of calcium hydroxyapatite crystals. Genetic polymorphisms involving the vitamin D receptor, the estrogen receptor in postmenopausal women,

1	produce bone matrix proteins that allow for the deposition of calcium hydroxyapatite crystals. Genetic polymorphisms involving the vitamin D receptor, the estrogen receptor in postmenopausal women, interleukin 10, and apolipoprotein E4 have been linked to the development of calcific AS, and a strong familial clustering of cases has been reported from western France. Several traditional atherosclerotic risk factors have also been associated with the development and progression of calcific

1	Aortic stenosis Congenital (bicuspid, unicuspid) AS, including low-density lipoprotein (LDL) cholesterol, lipoprotein a (Lp[a]), diabetes mellitus, smoking, chronic kidney disease, and the metabolic syndrome. The presence of aortic valve sclerosis (focal thickening and calcification of the leaflets not severe enough to cause obstruction) is associated with an excess risk of cardiovascular death and myocardial infarction (MI) among persons older than age 65. Approximately 30% of persons older than 65 years exhibit aortic valve sclerosis, whereas 2% exhibit frank stenosis.

1	Rheumatic disease of the aortic leaflets produces commissural fusion, sometimes resulting in a bicuspid-appearing valve. This condition, in turn, makes the leaflets more susceptible to trauma and ultimately leads to fibrosis, calcification, and further narrowing. By the time the obstruction to left ventricular (LV) outflow causes serious clinical disability, the valve is usually a rigid calcified mass, and careful examination may make it difficult or even impossible to determine the etiology of the underlying process. Rheumatic AS is almost always associated with involvement of the mitral valve and with aortic regurgitation. Mediastinal radiation can also result in late scarring, fibrosis, and calcification of the leaflets with AS.

1	A bicuspid aortic valve (BAV) is the most common congenital heart valve defect and occurs in 0.5–1.4% of the population with a 2–4:1 male-to-female predominance. The inheritance pattern appears to be autosomal dominant with incomplete penetrance, although some have questioned an X-linked component as suggested by the prevalence of BAV disease among patients with Turner’s syndrome. The prevalence of BAV disease among first-degree relatives of an affected individual is approximately 10%. A single gene defect to explain the majority of cases has not been identified, although a mutation in the NOTCH1 gene has been described in some families. Abnormalities in endothelial nitric oxide synthase and NKX2.5 have been implicated as well. Medial degeneration with ascending aortic aneurysm formation occurs commonly among patients with BAV disease; aortic coarctation is less frequently encountered. Patients with BAV disease have larger aortas than patients with comparable tricuspid aortic valve

1	occurs commonly among patients with BAV disease; aortic coarctation is less frequently encountered. Patients with BAV disease have larger aortas than patients with comparable tricuspid aortic valve disease. The aortopathy develops independent of the hemodynamic severity of the valve lesion and is a risk factor for aneurysm formation and/or dissection. A BAV can be a component of more complex congenital heart disease with or without other left heart obstructing lesions, as seen in Shone’s complex.

1	Foam cell with ApoB) FIGURE 283-1 Pathogenesis of calcific aortic stenosis. Inflammatory cells infiltrate across the endothelial barrier and release cytokines that act on fibroblasts to promote cellular proliferation and matrix remodeling. LDL is oxidatively modified and taken up by macrophage scavengers to become foam cells. Angiotensin-converting enzyme colocalizes with ApoB. A subset of myofibroblasts differentiates into an osteoblast phenotype capable of promoting bone formation. ACE, angiotensin-converting enzyme; ApoB, apolipoprotein B; LDL, low-density lipoprotein; IL, interleukin; MMP, matrix metalloproteinase; TGF, transforming growth factor. (From RV Freeman, CM Otto: Circulation 111:3316, 2005; with permission.)

1	In addition to valvular AS, three other lesions may be responsible for obstruction to LV outflow: hypertrophic obstructive cardiomyopathy (Chap. 287), discrete fibromuscular/membranous subaortic stenosis, and supravalvular AS (Chap. 282). The causes of LV outflow obstruction can be differentiated on the basis of the cardiac examination and Doppler echocardiographic findings.

1	The obstruction to LV outflow produces a systolic pressure gradient between the LV and aorta. When severe obstruction is suddenly produced experimentally, the LV responds by dilation and reduction of stroke volume. However, in some patients, the obstruction may be present at birth and/or increase gradually over the course of many years, and LV contractile performance is maintained by the presence of concentric LV hypertrophy. Initially, this serves as an adaptive mechanism because it reduces toward normal the systolic stress developed by the myocardium, as predicted by the Laplace relation (S = Pr/h, where S = systolic wall stress, P = pressure, r = radius, and h = wall thickness). A large transaortic valve pressure gradient may exist for many years without a reduction in cardiac output (CO) or LV dilation; ultimately, however, excessive hypertrophy becomes maladaptive, LV systolic function declines because of afterload mismatch, abnormalities of diastolic function progress, and

1	(CO) or LV dilation; ultimately, however, excessive hypertrophy becomes maladaptive, LV systolic function declines because of afterload mismatch, abnormalities of diastolic function progress, and irreversible myocardial fibrosis develops.

1	A mean systolic pressure gradient >40 mmHg with a normal CO or an effective aortic orifice area of approximately <1 cm2 (or approximately <0.6 cm2/m2 body surface area in a normal-sized adult)—i.e., less than approximately one-third of the normal orifice area—is generally considered to represent severe obstruction to LV outflow. The elevated LV end-diastolic pressure observed in many patients with severe AS and preserved ejection fraction (EF) signifies the presence of diminished compliance of the hypertrophied LV. Although the CO at rest is within normal limits in most patients with severe AS, it usually fails to rise normally during exercise. Loss of an appropriately timed, vigorous atrial contraction, as occurs in atrial fibrillation (AF) or atrioventricular dissociation, may cause rapid progression of symptoms. Late in the course, contractile function deteriorates because of afterload excess, the CO and LV–aortic pressure gradient decline, and the mean left atrial (LA), pulmonary

1	progression of symptoms. Late in the course, contractile function deteriorates because of afterload excess, the CO and LV–aortic pressure gradient decline, and the mean left atrial (LA), pulmonary artery (PA), and right ventricular (RV) pressures rise. LV performance can be further compromised by superimposed coronary artery disease (CAD). Stroke volume (and thus CO) can also be reduced in patients with significant hypertrophy and a small LV cavity despite a normal EF. Low-flow, low-gradient AS (with either reduced or normal LV systolic function) is both a diagnostic and therapeutic challenge.

1	The hypertrophied LV causes an increase in myocardial oxygen requirements. In addition, even in the absence of obstructive CAD, coronary blood flow is impaired to the extent that ischemia can be precipitated under conditions of excess demand. Capillary density is reduced relative to wall thickness, compressive forces are increased, and the elevated LV end-diastolic pressure reduces the coronary driving pressure. The subendocardium is especially vulnerable to ischemia by this mechanism. AS is rarely of clinical importance until the valve orifice has narrowed to approximately 1 cm2. Even severe AS may exist for many years without producing any symptoms because of the ability of the hypertrophied LV to generate the elevated intraventricular pressures required to maintain a normal stroke volume. Once symptoms occur, valve replacement is indicated.

1	Most patients with pure or predominant AS have gradually increasing obstruction over years but do not become symptomatic until the sixth to eighth decades. Adult patients with BAV disease, however, develop significant valve dysfunction and symptoms one to two decades sooner. Exertional dyspnea, angina pectoris, and syncope are the three cardinal symptoms. Often, there is a history of insidious progression of fatigue and dyspnea associated with gradual curtailment of activities and reduced effort tolerance. Dyspnea results primarily from elevation of the pulmonary capillary pressure caused by elevations of LV diastolic pressures secondary to impaired relaxation and reduced LV compliance. Angina pectoris usually develops somewhat later and reflects an imbalance between the augmented myocardial oxygen requirements and reduced oxygen availability. CAD may or may not be present, although its coexistence is common among AS patients older than age 65. Exertional syncope may result from a

1	oxygen requirements and reduced oxygen availability. CAD may or may not be present, although its coexistence is common among AS patients older than age 65. Exertional syncope may result from a decline in arterial pressure caused by vasodilation in the exercising muscles and inadequate vasoconstriction in nonexercising muscles in the face of a fixed CO, or from a sudden fall in CO produced by an arrhythmia.

1	Because the CO at rest is usually well maintained until late in the course, marked fatigability, weakness, peripheral cyanosis, cachexia, and other clinical manifestations of a low CO are usually not prominent until this stage is reached. Orthopnea, paroxysmal nocturnal dyspnea, and pulmonary edema, i.e., symptoms of LV failure, also occur only in the advanced stages of the disease. Severe pulmonary hypertension leading to RV failure and systemic venous hypertension, hepatomegaly, AF, and tricuspid regurgitation (TR) are usually late findings in patients with isolated severe AS. When AS and mitral stenosis (MS) coexist, the reduction in flow (CO) induced by MS lowers the pressure gradient across the aortic valve and, thereby, masks many of the clinical findings produced by AS. The transaortic pressure gradient can be increased in patients with concomitant aortic regurgitation (AR) due to higher aortic valve flow rates.

1	The rhythm is generally regular until late in the course; at other times, AF should suggest the possibility of associated mitral valve disease. The systemic arterial pressure is usually within normal limits. In the late stages, however, when stroke volume declines, the systolic pressure may fall and the pulse pressure narrow. The carotid arterial pulse rises slowly to a delayed peak (pulsus parvus et tardus). A thrill or anacrotic “shudder” may be palpable over the carotid arteries, more commonly the left. In the elderly, the stiffening of the arterial wall may mask this important physical sign. In many patients, the a wave in the jugular venous pulse is accentuated. This results from the diminished distensibility of the RV cavity caused by the bulging, hypertrophied interventricular septum.

1	The LV impulse is sometimes displaced laterally in the later stages of the disease. A double apical impulse (with a palpable S4) may be recognized, particularly with the patient in the left lateral recumbent position. A systolic thrill may be present at the base of the heart to the right of the sternum when leaning forward or in the suprasternal notch.

1	Auscultation An early systolic ejection sound is frequently audible in children, adolescents, and young adults with congenital BAV disease. This sound usually disappears when the valve becomes calcified and rigid. As AS increases in severity, LV systole may become prolonged so that the aortic valve closure sound no longer precedes the pulmonic valve closure sound, and the two components may become synchronous, or aortic valve closure may even follow pulmonic valve closure, causing paradoxical splitting of S2 (Chap. 267). The sound of aortic valve closure can be heard most frequently in patients with AS who have pliable valves, and calcification diminishes the intensity of this sound. Frequently, an S4 is audible at the apex and reflects the presence of LV hypertrophy and an elevated LV end-diastolic pressure; an S3 generally occurs late in the course, when the LV dilates and its systolic function becomes severely compromised.

1	The murmur of AS is characteristically an ejection (mid) systolic murmur that commences shortly after the S1, increases in intensity to reach a peak toward the middle of ejection, and ends just before aortic valve closure. It is characteristically low-pitched, rough and rasping in character, and loudest at the base of the heart, most commonly in the second right intercostal space. It is transmitted upward along the carotid arteries. Occasionally it is transmitted downward and to the apex, where it may be confused with the systolic murmur of mitral regurgitation (MR) (Gallavardin effect). In almost all patients with severe obstruction and preserved CO, the murmur is at least grade III/VI. In patients with mild degrees of obstruction or in those with severe stenosis with heart failure and low CO in whom the stroke volume and, therefore, the transvalvular flow rate are reduced, the murmur may be relatively soft and brief.

1	LABORATORY EXAMINATION 1531 ECG In most patients with severe AS, there is LV hypertrophy. In advanced cases, ST-segment depression and T-wave inversion (LV “strain”) in standard leads I and aVL and in the left precordial leads are evident. However, there is no close correlation between the ECG and the hemodynamic severity of obstruction, and the absence of ECG signs of LV hypertrophy does not exclude severe obstruction. Many patients with AS have systemic hypertension, which can also contribute to the development of hypertrophy.

1	Echocardiogram The key findings on TTE are thickening, calcification, and reduced systolic opening of the valve leaflets and LV hypertrophy. Eccentric closure of the aortic valve cusps is characteristic of congenitally bicuspid valves. TEE imaging can display the obstructed orifice extremely well, but it is not routinely required for accurate characterization of AS. The valve gradient and aortic valve area can be estimated by Doppler measurement of the transaortic velocity. Severe AS is defined by a valve area <1 cm2, whereas moderate AS is defined by a valve area of 1–1.5 cm2 and mild AS by a valve area of 1.5–2 cm2.

1	Aortic valve sclerosis, conversely, is accompanied by a jet velocity of less than 2.5 meters/s (peak gradient <25 mmHg). LV dilation and reduced systolic shortening reflect impairment of LV function. There is increasing experience with the use of longitudinal strain and strain rate to characterize earlier changes in LV systolic function, well before a decline in EF can be appreciated. Doppler indices of impaired diastolic function are frequently seen.

1	Echocardiography is useful for identifying coexisting valvular abnormalities; for differentiating valvular AS from other forms of LV outflow obstruction; and for measurement of the aortic root and proximal ascending aortic dimensions. These aortic measurements are particularly important for patients with BAV disease. Dobutamine stress echocardiography is useful for the evaluation of patients with AS and severe LV systolic dysfunction (low-flow, low-gradient, severe AS with reduced EF), in whom the severity of the AS can often be difficult to judge. Patients with severe AS (i.e., valve area <1 cm2) with a relatively low mean gradient (<40 mmHg) despite a normal EF (low-flow, low-gradient, severe AS with normal EF) are often hypertensive, and efforts to control their systemic blood pressure should be optimized before Doppler echocardiography is repeated. The use of dobutamine stress echocardiography in this setting is under investigation. When there is continued uncertainty regarding

1	should be optimized before Doppler echocardiography is repeated. The use of dobutamine stress echocardiography in this setting is under investigation. When there is continued uncertainty regarding the severity of AS in patients with reduced CO, quantitative analysis of the amount of aortic valve calcium with chest computed tomography (CT) may be helpful.

1	Chest X-Ray The chest x-ray may show no or little overall cardiac enlargement for many years. Hypertrophy without dilation may produce some rounding of the cardiac apex in the frontal projection and slight backward displacement in the lateral view. A dilated proximal ascending aorta may be seen along the upper right heart border in the frontal view. Aortic valve calcification may be discernible in the lateral view, but is usually readily apparent on fluoroscopic examination or by echocardiography; the absence of valvular calcification on fluoroscopy in an adult suggests that severe valvular AS is not present. In later stages of the disease, as the LV dilates, there is increasing roentgenographic evidence of LV enlargement, pulmonary congestion, and enlargement of the LA, PA, and right heart chambers.

1	Catheterization Right and left heart catheterization for invasive assessment of AS is performed infrequently but can be useful when there is a discrepancy between the clinical and noninvasive findings. Concern has been raised that attempts to cross the aortic valve for measurement of LV pressures are associated with a risk of cerebral embolization. Catheterization is also useful in three distinct categories of patients: (1) patients with multivalvular disease, in whom the role played by each valvular deformity should be defined to aid in the planning of operative treatment; (2) young, asymptomatic patients with noncalcific congenital AS, to define the severity of obstruction to LV outflow, because operation or percutaneous aortic balloon valvuloplasty (PABV) may be indicated in these patients if severe AS is 1532 present, even in the absence of symptoms; and (3) patients in whom it is suspected that the obstruction to LV outflow may not be at the level of the aortic valve but rather

1	if severe AS is 1532 present, even in the absence of symptoms; and (3) patients in whom it is suspected that the obstruction to LV outflow may not be at the level of the aortic valve but rather at the subor supravalvular level. Coronary angiography is indicated to screen for CAD in appropriate patients with severe AS who are being considered for surgery. The incidence of significant CAD for which bypass grafting is indicated at the time of aortic valve replacement (AVR) exceeds 50% among adult patients.

1	Death in patients with severe AS occurs most commonly in the seventh and eighth decades. Based on data obtained at postmortem examination in patients before surgical treatment became widely available, the average time to death after the onset of various symptoms was as follows: angina pectoris, 3 years; syncope, 3 years; dyspnea, 2 years; congestive heart failure, 1.5–2 years. Moreover, in >80% of patients who died with AS, symptoms had existed for <4 years. Among adults dying with valvular AS, sudden death, which presumably resulted from an arrhythmia, occurred in 10–20%; however, most sudden deaths occurred in patients who had previously been symptomatic. Sudden death as the first manifestation of severe AS is very uncommon (<1% per year) in asymptomatic adult patients. Calcific AS is a progressive disease, with an annual reduction in valve area averaging 0.1 cm2 and annual increases in the peak jet velocity and mean valve gradient averaging 0.3 meters/s and 7 mmHg, respectively

1	a progressive disease, with an annual reduction in valve area averaging 0.1 cm2 and annual increases in the peak jet velocity and mean valve gradient averaging 0.3 meters/s and 7 mmHg, respectively (Table 283-2).

1	TREATMEnT aortic StenoSiS (fig. 283-2)

1	In patients with severe AS (valve area <1 cm2), strenuous physical activity and competitive sports should be avoided, even in the asymptomatic stage. Care must be taken to avoid dehydration and hypovolemia to protect against a significant reduction in CO. Medications used for the treatment of hypertension or CAD, including beta blockers and angiotensin-converting enzyme (ACE) inhibitors, are generally safe for asymptomatic patients with preserved LV systolic function. Nitroglycerin is helpful in relieving angina pectoris in patients with CAD. Retrospective studies have shown that patients with degenerative calcific AS who receive HMG-CoA reductase inhibitors (“statins”) exhibit slower progression of leaflet calcification and aortic valve area reduction than those who do not. However, randomized prospective studies with either high-dose atorvastatin or combination simvastatin/ezetimibe have failed to show a measurable effect on valve-related outcomes. The use of statin medications

1	randomized prospective studies with either high-dose atorvastatin or combination simvastatin/ezetimibe have failed to show a measurable effect on valve-related outcomes. The use of statin medications should continue to be driven by considerations regarding primary and secondary prevention of atherosclerotic cardiovascular disease (ASCVD) events. ACE inhibitors have not been studied prospectively for AS-related outcomes. The need for endocarditis prophylaxis is restricted to AS patients with a prior history of endocarditis.

1	Asymptomatic patients with calcific AS and severe obstruction should be followed carefully for the development of symptoms and by serial echocardiograms for evidence of deteriorating LV function. Operation is indicated in patients with severe AS (valve aData are for the first two quarters of calendar year 2013, during which 1004 sites reported a total of 135,666 procedures. Data are available from the Society of Thoracic Surgeons at http://www.sts.org/sites/default/files/documents/2013_3rdHarvestExecutiveSummary.pdf. Abbreviations: AVR, aortic valve replacement; CAB, coronary artery bypass; MVR, mitral valve replacement.

1	area <1 cm2 or 0.6 cm2/m2 body surface area) who are symptomatic, those who exhibit LV systolic dysfunction (EF <50%), and those with BAV disease and an aneurysmal root or ascending aorta (maximal dimension >5.5 cm). Operation for aneurysm disease is recommended at smaller aortic diameters (4.5–5.0 cm) for patients with a family history of an aortic catastrophe and for patients who exhibit rapid aneurysm growth (>0.5 cm/year). Patients with asymptomatic moderate or severe AS who are referred for coronary artery bypass grafting surgery should also have AVR. In patients without heart failure, the operative risk of AVR (including patients with AS or AR) is approximately 2% (Table 283-2) but increases as a function of age and the need for concomitant aortic surgery or coronary revascularization with bypass grafting. The indications for AVR in the asymptomatic patient have been the subject of intense debate over the past 5 years, as surgical outcomes in selected patients have continued to

1	with bypass grafting. The indications for AVR in the asymptomatic patient have been the subject of intense debate over the past 5 years, as surgical outcomes in selected patients have continued to improve. Relative indications for which surgery can be considered include an abnormal response to treadmill exercise; rapid progression of AS, especially when urgent access to medical care might be compromised; very severe AS, defined by an aortic valve jet velocity >5 meters/s or mean gradient >60 mmHg and low operative risk; and excessive LV hypertrophy in the absence of systemic hypertension. Exercise testing can be safely performed in the asymptomatic patient, as many as one-third of whom will show signs of functional impairment.

1	Operation should be carried out promptly after symptom onset. In patients with low-flow, low-gradient severe AS with reduced LVEF, the perioperative mortality risk is high (15–20%), and evidence of myocardial disease may persist even when the operation is technically successful. Long-term postoperative survival correlates with preoperative LV function. Nonetheless, in view of the even worse prognosis of such patients when they are treated medically, there is usually little choice but to advise valve replacement, especially in patients in whom contractile reserve can be demonstrated by dobutamine stress echocardiography (defined by a ≥20% increase in stroke volume after dobutamine challenge). Patients in this high surgical risk group may benefit from transcatheter aortic valve replacement (TAVR, see below). The treatment of patients with low-flow, low-gradient severe AS with normal LVEF is also difficult. Outcomes appear to be better with surgery compared with conservative medical care

1	see below). The treatment of patients with low-flow, low-gradient severe AS with normal LVEF is also difficult. Outcomes appear to be better with surgery compared with conservative medical care for symptomatic patients with this type of “paradoxical” low-flow AS, but more research is needed to guide therapeutic decision-making. In patients in whom severe AS and CAD coexist, relief of the AS and revascularization may sometimes result in striking clinical and hemodynamic improvement (Table 283-2).

1	Because many patients with calcific AS are elderly, particular attention must be directed to the adequacy of hepatic, renal, and pulmonary function before AVR is recommended. Age alone is not a contraindication to AVR for AS. The perioperative mortality rate depends to a substantial extent on the patient’s preoperative clinical and hemodynamic state. Treatment decisions for AS patients who are not at low operative risk should be made by a multidisciplinary heart team with representation from general cardiology, interventional cardiology, imaging, cardiac surgery, and other allied specialties as needed, including geriatrics. The 10-year survival rate of older adult patients with AVR is approximately 60%. Approximately 30% of bioprosthetic valves evidence primary valve failure in 10 years, requiring re-replacement, and an approximately equal percentage of patients with mechanical prostheses develop significant hemorrhagic complications as a consequence of treatment with vitamin K

1	requiring re-replacement, and an approximately equal percentage of patients with mechanical prostheses develop significant hemorrhagic complications as a consequence of treatment with vitamin K antagonists. Homograft AVR is usually reserved for patients with aortic valve endocarditis.

1	The Ross procedure involves replacement of the diseased aortic valve with the autologous pulmonic valve and implantation of a homograft in the native pulmonic position. Its use has declined considerably in the United States because of the technical complexity of the procedure and the incidence of late postoperative aortic root dilation and autograft failure with AR. There is also a low incidence of pulmonary homograft stenosis. Vmax 3 m/s –3.9 m/s ˛Pmean 20–39 mmHg Severe AS Vmax °4 m/s ˛Pmean °40 mmHg AVR (I) AVR (IIa) AVR (IIb) Vmax °5 m/s ˛Pmean °60 mmHg low surgical risk ˛Vmax >0.3 m/s/y low surgical risk Abnormal ETT Asymptomatic (stage C) LVEF <50% (stage C2) Other cardiac surgery Symptomatic (stage D1)

1	FIGURE 283-2 Management strategy for patients with aortic stenosis. Preoperative coronary angiography should be performed routinely as determined by age, symptoms, and coronary risk factors. Cardiac catheterization and angiography may also be helpful when there is a discrepancy between clinical and noninvasive findings. Patients who do not meet criteria for intervention should be monitored periodically with clinical and echocardiographic follow-up. The class designations refer to the American College of Cardiology/American Heart Association methodology for treatment recommendations. Class I recommendations should be performed or are indicated; Class IIa recommendations are considered reasonable to perform; Class IIb recommendations may be considered. The stages refer to the stages of progression of the disease. At disease stage A, risk factors are present for the development of valve dysfunction; stage B refers to progressive, mild-moderate, asymptomatic valve disease; stage C disease

1	of the disease. At disease stage A, risk factors are present for the development of valve dysfunction; stage B refers to progressive, mild-moderate, asymptomatic valve disease; stage C disease is severe in nature but clinically asymptomatic; stage C1 characterizes asymptomatic patients with severe valve disease but compensated ventricular function; stage C2 refers to asymptomatic, severe disease with ventricular decompensation; stage D refers to severe, symptomatic valve disease. With aortic stenosis, stage D1 refers to symptomatic patients with severe aortic stenosis and a high valve gradient (>40 mmHg mean gradient); stage D2 comprises patients with symptomatic, severe, low-flow, low-gradient aortic stenosis and low left ventricular ejection fraction; and stage D3 characterizes patients with symptomatic, severe, low-flow, low-gradient aortic stenosis and preserved left ventricular ejection fraction (paradoxical, low-flow, low-gradient severe aortic stenosis). AS, aortic stenosis;

1	with symptomatic, severe, low-flow, low-gradient aortic stenosis and preserved left ventricular ejection fraction (paradoxical, low-flow, low-gradient severe aortic stenosis). AS, aortic stenosis; AVA; aortic valve area; AVR, aortic valve replacement by either surgical or transcatheter approach; BP, blood pressure; DSE, dobutamine stress echocardiography; ETT, exercise treadmill test; LVEF, left ventricular ejection fraction; ΔP , mean pressure gradient; and V , maximum velocity. (Adapted from RA Nishimura et al: 2014 AHA/ACC Guideline for the Management of Patients with Valvular Heart Disease. J Am Coll Cardiol doi: 10.1016/j.jacc.2014.02.536, 2014, with permission.)

1	This procedure is preferable to operation in many children and young adults with congenital, noncalcific AS (Chap. 282). It is not commonly used as definitive therapy in adults with severe calcific AS because of a very high restenosis rate (80% within 1 year) and the risk of procedural complications, but on occasion, it has been used successfully as a “bridge to operation” in patients with severe LV dysfunction and shock who are too ill to tolerate surgery. It is performed routinely as part of the TAVR procedure (see below). TAVR for treatment of AS has been performed in more than 50,000 prohibitiveor high-surgical-risk adult patients worldwide using one of two available systems, a balloon-expandable valve and a self-expanding valve, both of which incorporate a pericardial prosthesis (Fig. 283-3). More than 250 U.S. centers now offer this procedure.

1	TAVR is most frequently performed via the transfemoral route, although trans-LV apical, subclavian, carotid, and ascending aortic routes have been used. Aortic balloon valvuloplasty under rapid RV pacing is performed as a first step to create an orifice of sufficient size for the prosthesis. Procedural success rates exceed 90%. Among elderly patients with severe AS who are considered inoperable (i.e., prohibitive surgical risk), 1and 2-year survival rates are significantly higher with TAVR compared with medical therapy (including PABV) (Fig. 283-4). Oneand 2-year survival rates are essentially equal for high-surgical-risk patients treated with TAVR or surgical AVR (SAVR) (Fig. 283-5). TAVR is associated with an early hazard for stroke and a higher incidence of postprocedural, paravalvular AR, a risk factor for mortality over the next 2 years. Postprocedural heart block requiring permanent pacemaker therapy is observed significantly more frequently with the self-expanding valve. Valve

1	AR, a risk factor for mortality over the next 2 years. Postprocedural heart block requiring permanent pacemaker therapy is observed significantly more frequently with the self-expanding valve. Valve performance characteristics are excellent. Overall outcomes with this transformative technology have been very favorable and have allowed the extension of AVR to groups of patients previously considered at high or prohibitive risk for conventional surgery. Nevertheless, some patients are not candidates for this procedure because their comorbidity profile, including an assessment of frailty, would make its undertaking inappropriate. The heart team is specifically charged with making challenging decisions of this nature. The use of these devices for the treatment of patients at intermediate operative risk and for those with structural deterioration of bioprosthetic aortic and mitral valves (“valve-in-valve”), as an alternative to reoperative valve replacement, is under active study.

1	FIGURE 283-3 Balloon-expandable (A) and self-expanding (B) valves for transcatheter aortic valve replacement (TAVR). B, inflated balloon; N, nose cone; V, valve. (Part A, courtesy of Edwards Lifesciences, Irvine, CA; with permission. NovaFlex+ is a trademark of Edwards Lifesciences Corporation. Part B, © Medtronic, Inc. 2015. Medtronic CoreValve Transcatheter Aortic Valve. CoreValve is a registered trademark of Medtronic, Inc.) Hazard ratio, 0.56 (95% CI, 0.43–0.73) P<0.001 (Table 283-1) AR may be caused by primary valve disease or by primary aortic root disease. Primary Valve Disease Rheumatic disease results in thickening, deformity, and shortening of the individual aortic valve cusps, changes that prevent their proper opening during systole and closure during diastole. A rheumatic origin is much less common in patients with isolated AR who do not have associated rheumatic mitral valve disease. Patients with congenital BAV disease may develop predominant AR,

1	Death from any Cause, Intention-to-Treat Population 68.0 Standard therapy 43.3 TAVR 0 10 20 30Death from any cause (%)40 50 60 Surgery TAVR Hazard ratio, 0.90 (95% CI, 0.71–1.15) P = 0.41 No. at Risk

1	No. at Risk FIGURE 283-4 Twenty-four-month outcomes following transcatheter aortic valve replacement (TAVR) for inoperable patients in the PARTNER I trial (cohort B). CI, confidence interval. (Adapted from RR Makkar et al: N Engl J Med 366:1696, 2012; with permission.) and approximately 20% of patients will require aortic valve surgery between 10 and 40 years of age. Congenital fenestrations of the aortic valve occasionally produce mild AR. Membranous subaortic stenosis often leads to thickening and scarring of the aortic valve leaflets with secondary AR. Prolapse of an aortic cusp, resulting in progressive chronic AR, occurs in approximately 15% of patients with ventricular septal defect (Chap. 282) but may also occur as an isolated phenomenon or as a consequence of myxomatous degeneration sometimes associated with mitral and/or tricuspid valve involvement.

1	AR may result from infective endocarditis, which can develop on a valve previously affected by rheumatic disease, a congenitally deformed valve, or on a normal aortic valve, and may lead to perforation or erosion of one or more leaflets. The aortic valve leaflets may become scarred and retracted during the course of syphilis or ankylosing spondylitis and contribute further to the AR that derives primarily from the associated root disease. Although traumatic rupture or avulsion of an aortic cusp is an uncommon cause of acute AR, it represents the most frequent serious lesion in patients surviving nonpenetrating cardiac injuries. The coexistence of hemodynamically significant AS with AR usually excludes all the rarer forms of AR because it occurs almost exclusively in patients with rheumatic or congenital AR. In patients with AR due to primary valvular disease, dilation of the aortic annulus may occur secondarily and lead to worsening regurgitation.

1	Primary Aortic Root Disease AR also may be due entirely to marked aortic annular dilation, i.e., aortic root disease, without primary involvement of the valve leaflets; widening of the aortic annulus and separation of the aortic leaflets are responsible for the AR (Chap. 301). Medial degeneration of the ascending aorta, which may or may not be associated with other manifestations of Marfan’s syndrome; idiopathic dilation of the aorta; annuloaortic ectasia; osteogenesis imperfecta; and severe, chronic hypertension may all widen the aortic annulus and lead to progressive AR. Occasionally AR is caused by retrograde dissection of the aorta involving the aortic annulus. Syphilis and ankylosing spondylitis, both of which may affect the aortic leaflets, may also be associated with cellular infiltration and scarring of the media of the thoracic aorta, leading to aortic dilation, aneurysm formation, and severe regurgitation. In syphilis of the aorta (Chap. 206), now a very rare condition, the

1	and scarring of the media of the thoracic aorta, leading to aortic dilation, aneurysm formation, and severe regurgitation. In syphilis of the aorta (Chap. 206), now a very rare condition, the involvement of the intima may narrow the coronary ostia, which in turn may be responsible for myocardial ischemia.

1	The total stroke volume ejected by the LV (i.e., the sum of the effective forward stroke volume and the volume of blood that regurgitates back into the LV) is increased in patients with AR. In patients with severe AR, the volume of regurgitant flow may equal the effective forward stroke volume. In contrast to MR, in which a portion of the LV stroke volume is delivered into the low-pressure LA, in AR the entire LV stroke volume is ejected into a high-pressure zone, the aorta. An increase in the LV end-diastolic volume (increased preload) constitutes the major hemodynamic compensation for AR. The dilation and eccentric hypertrophy of the LV allow this chamber to eject a larger stroke volume without requiring any increase in the relative shortening of each myofibril. Therefore, severe AR may occur with a normal effective forward stroke volume and a normal LVEF (total [forward plus regurgitant] stroke volume/end-diastolic volume), together with an elevated LV end-diastolic pressure and

1	occur with a normal effective forward stroke volume and a normal LVEF (total [forward plus regurgitant] stroke volume/end-diastolic volume), together with an elevated LV end-diastolic pressure and volume. However, through the operation of Laplace’s law, LV dilation increases the LV systolic tension required to develop any given level of systolic pressure. Chronic AR is, thus, a state in which LV preload and afterload are both increased. Ultimately, these adaptive measures fail. As LV function deteriorates, the end-diastolic volume rises further and the forward stroke volume and EF decline. Deterioration of LV function often precedes the development of symptoms. Considerable thickening of the LV wall also occurs with chronic AR, and at autopsy, the hearts of these patients may be among the largest encountered, sometimes weighing >1000 g.

1	The reverse pressure gradient from aorta to LV, which drives the AR flow, falls progressively during diastole, accounting for the decrescendo nature of the diastolic murmur. Equilibration between aortic and LV 1535 pressures may occur toward the end of diastole in patients with chronic severe AR, particularly when the heart rate is slow. In patients with acute severe AR, the LV is unprepared for the regurgitant volume load. LV compliance is normal or reduced, and LV diastolic pressures rise rapidly, occasionally to levels >40 mmHg. The LV pressure may exceed the LA pressure toward the end of diastole, and this reversed pressure gradient closes the mitral valve prematurely.

1	In patients with chronic severe AR, the effective forward CO usually is normal or only slightly reduced at rest, but often it fails to rise nor mally during exertion. An early sign of LV dysfunction is a reduction in the EF. In advanced stages, there may be considerable elevation of the LA, PA wedge, PA, and RV pressures and lowering of the forward CO at rest. Myocardial ischemia may occur in patients with AR because myocardial oxygen requirements are elevated by LV dilation, hypertrophy, and elevated LV systolic tension, and coronary blood flow may be compromised. A large fraction of coronary blood flow occurs during diastole, when arterial pressure is low, thereby reducing coronary perfusion or driving pressure. This combination of increased oxygen demand and reduced supply may cause myocardial ischemia, particularly of the subendocardium, even in the absence of epicardial CAD.

1	Approximately three-fourths of patients with pure or predominant valvular AR are men; women predominate among patients with primary valvular AR who have associated rheumatic mitral valve disease. A history compatible with infective endocarditis may sometimes be elicited from patients with rheumatic or congenital involvement of the aortic valve, and the infection often precipitates or seriously aggravates preexisting symptoms. In patients with acute severe AR, as may occur in infective endocarditis, aortic dissection, or trauma, the LV cannot dilate sufficiently to maintain stroke volume, and LV diastolic pressure rises rapidly with associated marked elevations of LA and PA wedge pressures. Pulmonary edema and/or cardiogenic shock may develop rapidly.

1	Chronic severe AR may have a long latent period, and patients may remain relatively asymptomatic for as long as 10–15 years. However, uncomfortable awareness of the heartbeat, especially on lying down, may be an early complaint. Sinus tachycardia, during exertion or with emotion, or premature ventricular contractions may produce particularly uncomfortable palpitations as well as head pounding. These complaints may persist for many years before the development of exertional dyspnea, usually the first symptom of diminished cardiac reserve. The dyspnea is followed by orthopnea, paroxysmal nocturnal dyspnea, and excessive diaphoresis. Anginal chest pain even in the absence of CAD may occur in patients with severe AR, even in younger patients. Anginal pain may develop at rest as well as during exertion. Nocturnal angina may be a particularly troublesome symptom, and it may be accompanied by marked diaphoresis. The anginal episodes can be prolonged and often do not respond satisfactorily to

1	exertion. Nocturnal angina may be a particularly troublesome symptom, and it may be accompanied by marked diaphoresis. The anginal episodes can be prolonged and often do not respond satisfactorily to sublingual nitroglycerin. Systemic fluid accumulation, including congestive hepatomegaly and ankle edema, may develop late in the course of the disease.

1	In chronic severe AR, the jarring of the entire body and the bobbing motion of the head with each systole can be appreciated, and the abrupt distention and collapse of the larger arteries are easily visible. The examination should be directed toward the detection of conditions predisposing to AR, such as bicuspid valve, endocarditis, Marfan’s syndrome, and ankylosing spondylitis.

1	Arterial Pulse A rapidly rising “water-hammer” pulse, which collapses suddenly as arterial pressure falls rapidly during late systole and diastole (Corrigan’s pulse), and capillary pulsations, an alternate flushing and paling of the skin at the root of the nail while pressure is applied to the tip of the nail (Quincke’s pulse), are characteristic 1536 of chronic severe AR. A booming “pistol-shot” sound can be heard over the femoral arteries (Traube’s sign), and a to-and-fro murmur (Duroziez’s sign) is audible if the femoral artery is lightly compressed with a stethoscope. The arterial pulse pressure is widened as a result of both systolic hypertension and a lowering of the diastolic pressure. The measurement of arterial diastolic pressure with a sphygmomanometer may be complicated by the fact that systolic sounds are frequently heard with the cuff completely deflated. However, the level of cuff pressure at the time of muffling of the Korotkoff sounds (phase IV) generally corresponds

1	fact that systolic sounds are frequently heard with the cuff completely deflated. However, the level of cuff pressure at the time of muffling of the Korotkoff sounds (phase IV) generally corresponds fairly closely to the true intraarterial diastolic pressure. As the disease progresses and the LV end-diastolic pressure rises, the arterial diastolic pressure may actually rise as well, because the aortic diastolic pressure cannot fall below the LV end-diastolic pressure. For the same reason, acute severe AR may also be accompanied by only a slight widening of the pulse pressure. Such patients are invariably tachycardic as the heart rate increases in an attempt to preserve the CO.

1	Palpation In patients with chronic severe AR, the LV impulse is heaving and displaced laterally and inferiorly. The systolic expansion and diastolic retraction of the apex are prominent. A diastolic thrill may be palpable along the left sternal border in thin-chested individuals, and a prominent systolic thrill may be palpable in the suprasternal notch and transmitted upward along the carotid arteries. This systolic thrill and the accompanying murmur do not necessarily signify the coexistence of AS. In some patients with AR or with combined AS and AR, the carotid arterial pulse may be bisferiens, i.e., with two systolic waves separated by a trough (see Fig. 267-2D).

1	Auscultation In patients with severe AR, the aortic valve closure sound (A2) is usually absent. A systolic ejection sound is audible in patients with BAV disease, and occasionally an S4 also may be heard. The murmur of chronic AR is typically a high-pitched, blowing, decrescendo diastolic murmur, heard best in the third intercostal space along the left sternal border (see Fig. 267-5B). In patients with mild AR, this murmur is brief, but as the severity increases, it generally becomes louder and longer, indeed holodiastolic. When the murmur is soft, it can be heard best with the diaphragm of the stethoscope and with the patient sitting up, leaning forward, and with the breath held in forced expiration. In patients in whom the AR is caused by primary valvular disease, the diastolic murmur is usually louder along the left than the right sternal border. However, when the murmur is heard best along the right sternal border, it suggests that the AR is caused by aneurysmal dilation of the

1	is usually louder along the left than the right sternal border. However, when the murmur is heard best along the right sternal border, it suggests that the AR is caused by aneurysmal dilation of the aortic root. “Cooing” or musical diastolic murmurs suggest eversion of an aortic cusp vibrating in the regurgitant stream.

1	A mid-systolic ejection murmur is frequently audible in isolated AR. It is generally heard best at the base of the heart and is transmitted along the carotid arteries. This murmur may be quite loud without signifying aortic obstruction. A third murmur sometimes heard in patients with severe AR is the Austin Flint murmur, a soft, low-pitched, rumbling mid-to-late diastolic murmur. It is probably produced by the diastolic displacement of the anterior leaflet of the mitral valve by the AR stream and is not associated with hemodynamically significant mitral obstruction. The auscultatory features of AR are intensified by strenuous and sustained handgrip, which augments systemic vascular resistance. In acute severe AR, the elevation of LV end-diastolic pressure may lead to early closure of the mitral valve, a soft S1, a pulse pressure that is not particularly wide, and a soft, short, early diastolic murmur of AR.

1	LABORATORY EXAMINATION ECG In patients with chronic severe AR, the ECG signs of LV hypertrophy become manifest (Chap. 268). In addition, these patients frequently exhibit ST-segment depression and T-wave inversion in leads I, aVL, V5, and V6 (“LV strain”). Left-axis deviation and/or QRS prolongation denote diffuse myocardial disease, generally associated with patchy fibrosis, and usually signify a poor prognosis.

1	Echocardiogram LV size is increased in chronic AR and systolic function is normal or even supernormal until myocardial contractility declines, as signaled by a decrease in EF or increase in the end-systolic dimension. A rapid, high-frequency diastolic fluttering of the anterior mitral leaflet produced by the impact of the regurgitant jet is a characteristic finding. The echocardiogram is also useful in determining the cause of AR, by detecting dilation of the aortic annulus and root, aortic dissection (see Fig. 270e-5), or primary leaflet pathology. With severe AR, the central jet width assessed by color flow Doppler imaging exceeds 65% of the LV outflow tract, the regurgitant volume is ≥60 mL/beat, the regurgitant fraction is ≥50%, and there is diastolic flow reversal in the proximal descending thoracic aorta. The continuous-wave Doppler profile of the AR jet shows a rapid deceleration time in patients with acute severe AR, due to the rapid increase in LV diastolic pressure.

1	descending thoracic aorta. The continuous-wave Doppler profile of the AR jet shows a rapid deceleration time in patients with acute severe AR, due to the rapid increase in LV diastolic pressure. Surveillance transthoracic echocardiography forms the cornerstone of longitudinal follow-up and allows for the early detection of changes in LV size and/or function. For patients in whom transthoracic echocardiography (TTE) is limited by poor acoustical windows or inadequate semiquantitative assessment of LV function or the severity of the regurgitation, cardiac magnetic resonance imaging (MRI) can be performed. This modality also allows for accurate assessment of aortic size and contour. Transesophageal echocardiography (TEE) can also provide detailed anatomic assessment of the valve, root, and portions of the aorta.

1	Chest X-Ray In chronic severe AR, the apex is displaced downward and to the left in the frontal projection. In the left anterior oblique and lateral projections, the LV is displaced posteriorly and encroaches on the spine. When AR is caused by primary disease of the aortic root, aneurysmal dilation of the aorta may be noted, and the aorta may fill the retrosternal space in the lateral view. Echocardiography, cardiac MRI, and chest CT angiography are more sensitive than the chest x-ray for the detection of root and ascending aortic enlargement. Cardiac Catheterization and Angiography When needed, right and left heart catheterization with contrast aortography can provide confirmation of the magnitude of regurgitation and the status of LV function. Coronary angiography is performed routinely in appropriate patients prior to surgery. ACUTE AORTIC REGURGITATION (FIG. 283-6)

1	ACUTE AORTIC REGURGITATION (FIG. 283-6) Patients with acute severe AR may respond to intravenous diuretics and vasodilators (such as sodium nitroprusside), but stabilization is usually short-lived and operation is indicated urgently. Intraaortic balloon counterpulsation is contraindicated. Beta blockers are also best avoided so as not to reduce the CO further or slow the heart rate, thus allowing more time for diastolic filling of the LV. Surgery is the treatment of choice and is usually necessary within 24 h of diagnosis.

1	Early symptoms of dyspnea and effort intolerance respond to treatment with diuretics; vasodilators (ACE inhibitors, dihydropyridine calcium channel blockers, or hydralazine) may be useful as well. Surgery can then be performed in a more controlled setting. The use of vasodilators to extend the compensated phase of chronic severe AR before the onset of symptoms or the development of LV dysfunction is more controversial and less well established. Systolic blood pressure should be controlled (goal <140 mmHg) in patients with chronic AR, and vasodilators are an excellent first choice as antihypertensive agents. It is often difficult to achieve adequate control because of the increased stroke volume that accompanies severe AR. Cardiac arrhythmias and systemic infections are poorly tolerated in patients with severe AR and must be treated promptly and vigorously. Although nitroglycerin and long-acting nitrates are not as helpful in relieving anginal pain as they are in patients with ischemic

1	patients with severe AR and must be treated promptly and vigorously. Although nitroglycerin and long-acting nitrates are not as helpful in relieving anginal pain as they are in patients with ischemic heart disease, they are worth a trial. Patients with

1	Vena contracta >0.6 cm (stage B) Holodiastolic aortic flow reversal Vena contracta °0.6 cm vena contracta >0.6 cm ERO ˜0.3 cm2 ERO <0.3 cm2 LV dilation O ˜0.3 cm

1	FIGURE 283-6 Management of patients with aortic regurgitation. See legend for Fig. 283-2 for explanation of treatment recommendations (Class I, IIa, and IIb) and disease stages (B, C1, C2, D). Preoperative coronary angiography should be performed routinely as determined by age, symptoms, and coronary risk factors. Cardiac catheterization and angiography may also be helpful when there is a discrepancy between clinical and noninvasive findings. Patients who do not meet criteria for intervention should be monitored periodically with clinical and echocardiographic follow-up. AR, aortic regurgitation; AVR, aortic valve replacement (valve repair may be appropriate in selected patients); ERO, effective regurgitant orifice; LV, left ventricular; LVEDD, left ventricular end-diastolic dimension; LVEF, left ventricular ejection fraction; LVESD, left ventricular end-systolic dimension; RF, regurgitant fraction; RVol, regurgitant volume. (Adapted from RA Nishimura et al: 2014 AHA/ACC Guideline for

1	left ventricular ejection fraction; LVESD, left ventricular end-systolic dimension; RF, regurgitant fraction; RVol, regurgitant volume. (Adapted from RA Nishimura et al: 2014 AHA/ACC Guideline for the Management of Patients with Valvular Heart Disease. J Am Coll Cardiol doi: 10.1016/j.jacc.2014.02.536, 2014, with permission.) syphilitic aortitis should receive a full course of penicillin therapy (Chap. 206). Beta blockers and the angiotensin receptor blocker losartan may be useful to retard the rate of aortic root enlargement in young patients with Marfan’s syndrome and aortic root dilation. Early reports of the efficacy of losartan in patients with Marfan’s syndrome have led to its use in other populations of patients including those with BAV disease and aortopathy. The use of beta blockers in patients with valvular AR was previously felt to be relatively contraindicated due to concerns that the resulting slowing of the heart rate would allow more time for diastolic regurgitation.

1	in patients with valvular AR was previously felt to be relatively contraindicated due to concerns that the resulting slowing of the heart rate would allow more time for diastolic regurgitation. More recent observational reports, however, suggest that beta blockers may provide functional benefit in patients with chronic AR. Beta blockers can sometimes provide incremental blood pressure lowering in patients with chronic AR and hypertension. Patients with severe AR, particularly those with an associated aortopathy, should avoid isometric exercises.

1	In deciding on the advisability and proper timing of surgical treatment, two points should be kept in mind: (1) patients with chronic severe AR usually do not become symptomatic until after the development of myocardial dysfunction; and (2) when delayed too long (defined as >1 year from onset of symptoms or LV dysfunction), surgical treatment often does not restore normal LV function. Therefore, in patients with chronic severe AR, careful clinical follow-up and noninvasive testing with echocardiography at approximately 6-to 12-month intervals are necessary if operation is to be undertaken at the optimal time, i.e., after the onset of LV dysfunction but prior to the development of severe symptoms. Exercise testing may be helpful to assess effort tolerance more objectively. Operation can be deferred as long as the patient both remains asymptomatic and retains normal LV function without severe chamber dilation.

1	AVR is indicated for the treatment of severe AR in symptomatic patients irrespective of LV function. In general, the operation should be carried out in asymptomatic patients with severe AR and progressive LV dysfunction defined by an LVEF <50%, an LV end-systolic dimension >50 mm, or an LV diastolic dimension >65 mm. Smaller dimensions may be appropriate thresholds in individuals of smaller stature. Patients with severe AR without indications for operation should be followed by clinical and echocardiographic examination every 6–12 months. FIGURE 283-7 Valve-sparing aortic root reconstruction (David procedure). (From P Steltzer et al [eds]: Valvular Heart Disease: A Companion to Braunwald’s Heart Disease, 3rd ed, Fig 12-27, p. 200.)

1	Surgical options for management of aortic valve and root disease have expanded considerably over the past decade. AVR with a suitable mechanical or tissue prosthesis is generally necessary in patients with rheumatic AR and in many patients with other forms of regurgitation. Rarely, when a leaflet has been perforated during infective endocarditis or torn from its attachments to the aortic annulus by thoracic trauma, primary surgical repair may be possible. When AR is due to aneurysmal dilation of the root or proximal ascending aorta rather than to primary valve involvement, it may be possible to reduce or eliminate the regurgitation by narrowing the annulus or by excising a portion of the aortic root without replacing the valve. Elective, valve-sparing aortic root reconstruction generally involves reimplantation of the valve in a contoured graft with reattachment of the coronary artery buttons into the side of the graft and is best undertaken in specialized surgical centers (Fig.

1	involves reimplantation of the valve in a contoured graft with reattachment of the coronary artery buttons into the side of the graft and is best undertaken in specialized surgical centers (Fig. 283-7). Resuspension of the native aortic valve leaflets is possible in approximately 50% of patients with acute AR in the setting of type A aortic dissection. In other conditions, however, regurgitation can be effectively eliminated only by replacing the aortic valve, the dilated or aneurysmal ascending aorta responsible for the regurgitation, and implanting a composite valve-graft conduit. This formidable procedure entails a higher risk than isolated AVR.

1	As in patients with other valvular abnormalities, both the operative risk and the late mortality rate are largely dependent on the stage of the disease and myocardial function at the time of operation. The overall operative mortality rate for isolated AVR (performed for either or both AS or AR) is approximately 2% (Table 283-2). However, patients with AR, marked cardiac enlargement, and prolonged LV dysfunction experience an operative mortality rate of approximately 10% and a late mortality rate of approximately 5% per year due to LV failure despite a technically satisfactory operation. Nonetheless, because of the very poor prognosis with medical management, even patients with LV systolic failure should be considered for operation. Patients with acute severe AR require prompt surgical treatment, which may be lifesaving.

1	Patients with acute severe AR require prompt surgical treatment, which may be lifesaving. Mitral valve Disease Patrick T. O’Gara, Joseph Loscalzo The role of the physical examination in the evaluation of patients with valvular heart disease is also considered in Chaps. 51e and 267; of electrocardiography (ECG) in Chap. 268; of echocardiography and other noninvasive imaging techniques in Chap. 270e; and of cardiac 284 catheterization and angiography in Chap. 272.

1	Rheumatic fever is the leading cause of mitral stenosis (MS) (Table 284-1). Other less common etiologies of obstruction to left ventricular inflow include congenital mitral valve stenosis, cor triatriatum, mitral annular calcification with extension onto the leaflets, systemic lupus erythematosus, rheumatoid arthritis, left atrial myxoma, and infective endocarditis with large vegetations. Pure or predominant MS occurs in approximately 40% of all patients with rheumatic heart disease and a history of rheumatic fever (Chap. 381). In other patients with rheumatic heart disease, lesser degrees of MS may accompany mitral regurgitation (MR) and aortic valve disease. With reductions in the incidence of acute rheumatic fever, particularly in temperate climates and developed countries, the incidence of MS has declined considerably over the past several decades. However, it remains a major problem in developing nations, especially in tropical and semitropical climates.

1	In rheumatic MS, chronic inflammation leads to diffuse thickening of the valve leaflets with formation of fibrous tissue and/or calcific deposits. The mitral commissures fuse, the chordae tendineae fuse and shorten, the valvular cusps become rigid, and these changes, in turn, lead to narrowing at the apex of the funnel-shaped (“fish-mouth”) valve. Although the initial insult to the mitral valve is rheumatic, later changes may be exacerbated by a nonspecific process resulting from trauma to the valve due to altered flow patterns. Calcification of the Mitral stenosis Rheumatic fever Congenital Severe mitral annular calcification SLE, RA Mitral regurgitation Acute Endocarditis Papillary muscle rupture (post-MI) Trauma Chordal rupture/leaflet flail (MVP, IE) Chronic Myxomatous (MVP) Rheumatic fever Endocarditis (healed) Mitral annular calcification Congenital (cleft, AV canal) HOCM with SAM Ischemic (LV remodeling) Dilated cardiomyopathy Radiation

1	Chronic Myxomatous (MVP) Rheumatic fever Endocarditis (healed) Mitral annular calcification Congenital (cleft, AV canal) HOCM with SAM Ischemic (LV remodeling) Dilated cardiomyopathy Radiation Abbreviations: AV, atrioventricular; IE, infective endocarditis; HOCM, hypertrophic obstructive cardiomyopathy; LV, left ventricular; MI, myocardial infarction; MVP, mitral valve prolapse; RA, rheumatoid arthritis; SAM, systolic anterior motion; SLE, systemic lupus erythematosus. stenotic mitral valve immobilizes the leaflets and narrows the orifice 1539 further. Thrombus formation and arterial embolization may arise from the calcific valve itself, but in patients with atrial fibrillation (AF), thrombi arise more frequently from the dilated left atrium (LA), particularly from within the LA appendage.

1	In normal adults, the area of the mitral valve orifice is 4–6 cm2. In the presence of significant obstruction, i.e., when the orifice area is reduced to < ~2 cm2, blood can flow from the LA to the left ventricle (LV) only if propelled by an abnormally elevated left atrioventricular pressure gradient, the hemodynamic hallmark of MS. When the mitral valve opening is reduced to <1.5 cm2, referred to as “severe” MS, an LA pressure of ~25 mmHg is required to maintain a normal cardiac output (CO). The elevated pulmonary venous and pulmonary arterial (PA) wedge pressures reduce pulmonary compliance, contributing to exertional dyspnea. The first bouts of dyspnea are usually precipitated by clinical events that increase the rate of blood flow across the mitral orifice, resulting in further elevation of the LA pressure (see below).

1	To assess the severity of obstruction hemodynamically, both the transvalvular pressure gradient and the flow rate must be measured (Chap. 272). The latter depends not only on the CO but on the heart rate, as well. An increase in heart rate shortens diastole proportionately more than systole and diminishes the time available for flow across the mitral valve. Therefore, at any given level of CO, tachycardia, including that associated with rapid AF, augments the transvalvular pressure gradient and elevates further the LA pressure. Similar considerations apply to the pathophysiology of tricuspid stenosis.

1	The LV diastolic pressure and ejection fraction (EF) are normal in isolated MS. In MS and sinus rhythm, the elevated LA and PA wedge pressures exhibit a prominent atrial contraction pattern (a wave) and a gradual pressure decline after the v wave and mitral valve opening (y descent). In severe MS and whenever pulmonary vascular resistance is significantly increased, the PA pressure (PAP) is elevated at rest and rises further during exercise, often causing secondary elevations of right ventricular (RV) end-diastolic pressure and volume. Cardiac Output In patients with severe MS (mitral valve orifice 1–1.5 cm2), the CO is normal or almost so at rest, but rises subnormally during exertion. In patients with very severe MS (valve area <1 cm2), particularly those in whom pulmonary vascular resistance is markedly elevated, the CO is subnormal at rest and may fail to rise or may even decline during activity.

1	Pulmonary Hypertension The clinical and hemodynamic features of MS are influenced importantly by the level of the PAP. Pulmonary hypertension results from: (1) passive backward transmission of the elevated LA pressure; (2) pulmonary arteriolar constriction (the so-called “second stenosis”), which presumably is triggered by LA and pulmonary venous hypertension (reactive pulmonary hypertension); interstitial edema in the walls of the small pulmonary vessels; and at end stage, organic obliterative changes in the pulmonary vascular bed. Severe pulmonary hypertension results in RV enlargement, secondary tricuspid regurgitation (TR), and pulmonic regurgitation (PR), as well as right-sided heart failure.

1	In temperate climates, the latent period between the initial attack of rheumatic carditis (in the increasingly rare circumstances in which a history of one can be elicited) and the development of symptoms due to MS is generally about two decades; most patients begin to experience disability in the fourth decade of life. Studies carried out before the development of mitral valvotomy revealed that once a patient with MS became seriously symptomatic, the disease progressed inexorably to death within 2–5 years.

1	In patients whose mitral orifices are large enough to accommodate a normal blood flow with only mild elevations of LA pressure, marked elevations of this pressure leading to dyspnea and cough may be precipitated by sudden changes in the heart rate, volume status, or CO, as, for example, with severe exertion, excitement, fever, severe anemia, paroxysmal AF 1540 and other tachycardias, sexual intercourse, pregnancy, and thyrotoxicosis. As MS progresses, lesser degrees of stress precipitate dyspnea, the patient becomes limited in daily activities, and orthopnea and paroxysmal nocturnal dyspnea develop. The development of persistent AF often marks a turning point in the patient’s course and is generally associated with acceleration of the rate at which symptoms progress. Hemoptysis (Chap. 48) results from rupture of pulmonary-bronchial venous connections secondary to pulmonary venous hypertension. It occurs most frequently in patients who have elevated LA pressures without markedly

1	48) results from rupture of pulmonary-bronchial venous connections secondary to pulmonary venous hypertension. It occurs most frequently in patients who have elevated LA pressures without markedly elevated pulmonary vascular resistances and is rarely fatal. Recurrent pulmonary emboli (Chap. 300), sometimes with infarction, are an important cause of morbidity and mortality late in the course of MS. Pulmonary infections, i.e., bronchitis, bronchopneumonia, and lobar pneumonia, commonly complicate untreated MS, especially during the winter months.

1	Pulmonary Changes In addition to the aforementioned changes in the pulmonary vascular bed, fibrous thickening of the walls of the alveoli and pulmonary capillaries occurs commonly in MS. The vital capacity, total lung capacity, maximal breathing capacity, and oxygen uptake per unit of ventilation are reduced (Chap. 306e). Pulmonary compliance falls further as pulmonary capillary pressure rises during exercise. Thrombi and Emboli Thrombi may form in the left atria, particularly within the enlarged atrial appendages of patients with MS. Systemic embolization, the incidence of which is 10–20%, occurs more frequently in patients with AF, in patients >65 years of age, and in those with a reduced CO. However, systemic embolization may be the presenting feature in otherwise asymptomatic patients with only mild MS. (See also Chaps. 51e and 267)

1	(See also Chaps. 51e and 267) Inspection and Palpation In patients with severe MS, there may be a malar flush with pinched and blue facies. In patients with sinus rhythm and severe pulmonary hypertension or associated tricuspid stenosis (TS), the jugular venous pulse reveals prominent a waves due to vigorous right atrial systole. The systemic arterial pressure is usually normal or slightly low. An RV tap along the left sternal border signifies an enlarged RV. A diastolic thrill may rarely be present at the cardiac apex, with the patient in the left lateral recumbent position.

1	Auscultation The first heart sound (S1) is usually accentuated in the early stages of the disease and slightly delayed. The pulmonic component of the second heart sound (P2) also is often accentuated with elevated PA pressures, and the two components of the second heart sound (S2) are closely split. The opening snap (OS) of the mitral valve is most readily audible in expiration at, or just medial to, the cardiac apex. This sound generally follows the sound of aortic valve closure (A2) by 0.05–0.12 s. The time interval between A2 and OS varies inversely with the severity of the MS. The OS is followed by a low-pitched, rumbling, diastolic murmur, heard best at the apex with the patient in the left lateral recumbent position (see Fig. 267-5); it is accentuated by mild exercise (e.g., a few rapid sit-ups) carried out just before auscultation. In general, the duration of this murmur correlates with the severity of the stenosis in patients with preserved CO. In patients with sinus rhythm,

1	rapid sit-ups) carried out just before auscultation. In general, the duration of this murmur correlates with the severity of the stenosis in patients with preserved CO. In patients with sinus rhythm, the murmur often reappears or becomes louder during atrial systole (presystolic accentuation). Soft, grade I or II/VI systolic murmurs are commonly heard at the apex or along the left sternal border in patients with pure MS and do not necessarily signify the presence of MR. Hepatomegaly, ankle edema, ascites, and pleural effusion, particularly in the right pleural cavity, may occur in patients with MS and RV failure.

1	Associated Lesions With severe pulmonary hypertension, a pansystolic murmur produced by functional TR may be audible along the left sternal border. This murmur is usually louder during inspiration and diminishes during forced expiration (Carvallo’s sign). When the CO is markedly reduced in MS, the typical auscultatory findings, including the diastolic rumbling murmur, may not be detectable (silent MS), but they may reappear as compensation is restored. The Graham Steell murmur of PR, a high-pitched, diastolic, decrescendo blowing murmur along the left sternal border, results from dilation of the pulmonary valve ring and occurs in patients with mitral valve disease and severe pulmonary hypertension. This murmur may be indistinguishable from the more common murmur produced by aortic regurgitation (AR), although it may increase in intensity with inspiration and is accompanied by a loud and often palpable P2.

1	LABORATORY EXAMINATION ECG In MS and sinus rhythm, the P wave usually suggests LA enlargement (see Fig. 268-8). It may become tall and peaked in lead II and upright in lead V1 when severe pulmonary hypertension or TS complicates MS and right atrial (RA) enlargement occurs. The QRS complex is usually normal. However, with severe pulmonary hypertension, right axis deviation and RV hypertrophy are often present.

1	Echocardiogram (See also Chap. 270e) Transthoracic echocardiography (TTE) with color flow and spectral Doppler imaging provides critical information, including measurements of mitral inflow velocity during early (E wave) and late (A wave in patients in sinus rhythm) diastolic filling, estimates of the transvalvular peak and mean gradients and of the mitral orifice area, the presence and severity of any associated MR, the extent of leaflet calcification and restriction, the degree of distortion of the subvalvular apparatus, and the anatomic suitability for percutaneous mitral balloon valvotomy (percutaneous mitral balloon valvuloplasty [PMBV]; see below). In addition, TTE provides an assessment of LV and RV function, chamber sizes, an estimation of the PAP based on the tricuspid regurgitant jet velocity, and an indication of the presence and severity of any associated valvular lesions, such as aortic stenosis and/or regurgitation. Transesophageal echocardiography (TEE) provides

1	jet velocity, and an indication of the presence and severity of any associated valvular lesions, such as aortic stenosis and/or regurgitation. Transesophageal echocardiography (TEE) provides superior images and should be used when TTE is inadequate for guiding management decisions. TEE is especially indicated to exclude the presence of LA thrombus prior to PMBV. The performance of TTE with exercise to evaluate the mean mitral diastolic gradient and PA pressures can be very helpful in the evaluation of patients with MS when there is a discrepancy between the clinical findings and the resting hemodynamics.

1	Chest X-Ray The earliest changes are straightening of the upper left border of the cardiac silhouette, prominence of the main PAs, dilation of the upper lobe pulmonary veins, and posterior displacement of the esophagus by an enlarged LA. Kerley B lines are fine, dense, opaque, horizontal lines that are most prominent in the lower and mid-lung fields and that result from distention of interlobular septae and lymphatics with edema when the resting mean LA pressure exceeds approximately 20 mmHg.

1	Like MS, significant MR may also be associated with a prominent diastolic murmur at the apex due to increased antegrade transmitral flow, but in patients with isolated MR, this diastolic murmur commences slightly later than in patients with MS, and there is often clear-cut evidence of LV enlargement. An OS and increased P2 are absent, and S1 is soft or absent. An apical pansystolic murmur of at least grade III/VI intensity as well as an S3 suggest significant MR. Similarly, the apical mid-diastolic murmur associated with severe AR (Austin Flint murmur) may be mistaken for MS but can be differentiated from it because it is not intensified in presystole and becomes softer with administration of amyl nitrite or other arterial vasodilators. TS, which occurs rarely in the absence of MS, may mask many of the clinical features of MS or be clinically silent; when present, the diastolic murmur of TS increases with inspiration and the y descent in the jugular venous pulse is delayed.

1	Atrial septal defect (Chap. 282) may be mistaken for MS; in both conditions, there is often clinical, ECG, and chest x-ray evidence of RV enlargement and accentuation of pulmonary vascularity. However, the absence of LA enlargement and of Kerley B lines and the demonstration of fixed splitting of S2 with a grade II or III mid-systolic murmur at the mid to upper left sternal border all favor atrial septal defect over MS. Atrial septal defects with large left-to-right shunts may result in functional TS because of the enhanced diastolic flow.

1	Left atrial myxoma (Chap. 289e) may obstruct LA emptying, causing dyspnea, a diastolic murmur, and hemodynamic changes resembling those of MS. However, patients with an LA myxoma often have features suggestive of a systemic disease, such as weight loss, fever, anemia, systemic emboli, and elevated serum IgG and interleukin 6 (IL-6) concentrations. The auscultatory findings may change markedly with body position. The diagnosis can be established by the demonstration of a characteristic echo-producing mass in the LA with TTE.

1	Left and right heart catheterization can be useful when there is a discrepancy between the clinical and noninvasive findings, including those from TEE and exercise echocardiographic testing as appropriate. Catheterization is helpful in assessing associated lesions, such as aortic stenosis (AS) and AR. Catheterization and coronary angiography are not usually necessary to aid in decision-making about surgery in patients younger than 65 years of age with typical findings of severe mitral obstruction on physical examination and TTE. In men older than 40 years of age, women older than 45 years of age, and younger patients with coronary risk factors, especially those with positive noninvasive stress tests for myocardial ischemia, coronary angiography is advisable preoperatively to identify patients with critical coronary obstructions that should be bypassed at the time of operation. Computed tomographic coronary angiography (CTCA) (Chap. 270e) is now often used to screen preoperatively for

1	with critical coronary obstructions that should be bypassed at the time of operation. Computed tomographic coronary angiography (CTCA) (Chap. 270e) is now often used to screen preoperatively for the presence of coronary artery disease (CAD) in patients with valvular heart 1541 disease and low pretest likelihood of CAD. Catheterization and left ventriculography may be useful in patients who have undergone PMBV or previous mitral valve surgery for MS, and who have redeveloped limiting symptoms, especially if questions regarding the severity of the valve lesion(s) remain after noninvasive study.

1	(Fig. 284-1) Penicillin prophylaxis of group A β-hemolytic streptococcal infections (Chap. 381) for secondary prevention of rheumatic fever is important for at-risk patients with rheumatic MS. Recommendations for infective endocarditis prophylaxis are similar to those for other valve lesions and are restricted to patients at high risk for complications from infection, including patients with a history of endocarditis. In symptomatic patients, some improvement usually occurs with restriction of sodium intake and small doses of oral diuretics. Beta blockers, nondihydropyridine calcium channel blockers (e.g., verapamil or diltiazem), and digitalis glycosides are useful in slowing the ventricular rate of patients with AF. Warfarin therapy targeted to an international normalized ratio (INR) of 2–3 should be administered indefinitely to patients with MS who have AF or a history of thromboembolism. The routine use of warfarin in patients in sinus rhythm with LA enlargement (maximal dimension

1	Rheumatic MS Very severe MS MVA ˜1 cm2 T½ °220 ms Severe MS MVA ˜1.5 cm2 T½ °150 ms Progressive MS MVA >1.5 cm2 T½ <150 ms Asymptomatic (stage C) Asymptomatic (stage C) Symptomatic (stage D) Symptomatic with no other cause Class I Class IIa Class IIb

1	FIGURE 284-1 Management of rheumatic mitral stenosis. See legend for Fig. 283-2 for explanation of treatment recommendations (class I, IIa, IIb) and disease stages (C, D). Preoperative coronary angiography should be performed routinely as determined by age, symptoms, and coronary risk factors. Cardiac catheterization and angiography may also be helpful when there is a discrepancy between clinical and noninvasive findings. AF, atrial fibrillation; LA, left atrial; MR, mitral regurgitation; MS, mitral stenosis; MVA, mitral valve area; MVR, mitral valve surgery (repair or replacement); NYHA, New York Heart Association; PCWP, pulmonary capillary wedge pressure; PMBC, percutaneous mitral balloon commissurotomy; and T ½, pressure half-time. (Adapted from RA Nishimura et al: 2014 AHA/ACC Guideline for the Management of Patients with Valvular Heart Disease. J Am Coll Cardiol doi: 10.1016/j.jacc.2014.02.536, 2014, with permission.) 1542 >5.5 cm) with or without spontaneous echo contrast is

1	for the Management of Patients with Valvular Heart Disease. J Am Coll Cardiol doi: 10.1016/j.jacc.2014.02.536, 2014, with permission.) 1542 >5.5 cm) with or without spontaneous echo contrast is more controversial. The novel oral anticoagulants are not approved for use in patients with significant valvular heart disease. If AF is of relatively recent onset in a patient whose MS is not severe enough to warrant PMBV or surgical commissurotomy, reversion to sinus rhythm pharmacologically or by means of electrical countershock is indicated. Usually, cardioversion should be undertaken after the patient has had at least 3 consecutive weeks of anticoagulant treatment to a therapeutic INR. If cardioversion is indicated more urgently, then intravenous heparin should be provided and TEE performed to exclude the presence of LA thrombus before the procedure. Conversion to sinus rhythm is rarely successful or sustained in patients with severe MS, particularly those in whom the LA is especially

1	to exclude the presence of LA thrombus before the procedure. Conversion to sinus rhythm is rarely successful or sustained in patients with severe MS, particularly those in whom the LA is especially enlarged or in whom AF has been present for more than 1 year.

1	Unless there is a contraindication, mitral valvotomy is indicated in symptomatic (New York Heart Association [NYHA] Functional Class II–IV) patients with isolated severe MS, whose effective orifice (valve area) is < ~1 cm2/m2 body surface area, or <1.5 cm2 in normal-sized adults. Mitral valvotomy can be carried out by two techniques: PMBV and surgical valvotomy. In PMBV (Figs. 284-2 and 284-3), a catheter is directed into the LA after transseptal puncture, and a single balloon is directed across the valve and inflated in the valvular orifice. Ideal patients have relatively pliable leaflets with little or no commissural calcium. In addition, the subvalvular structures should not be significantly scarred or thickened, and there should be no LA thrombus. The shortand long-term results of this procedure in appropriate patients are similar to those of surgical valvotomy, but with less morbidity and a lower periprocedural mortality rate. Event-free

1	FIGURE 284-2 Inoue balloon technique for percutaneous mitral balloon valvotomy. A. After transseptal puncture, the deflated balloon catheter is advanced across the interatrial septum, then across the mitral valve and into the left ventricle. B–D. The balloon is inflated stepwise within the mitral orifice. Mean mitral gradient 3 mmHg Cardiac output 3.8 L/min Mitral valve area 1.8 cm2 LV LA Mean mitral gradient 15 mmHg Cardiac output 3 L/min Mitral valve area 0.6 cm2 40 20 0 LV LA FIGURE 284-3 Simultaneous left atrial (LA) and left ventricular (LV) pressure before and after percutaneous mitral balloon valvuloplasty (PMBV) in a patient with severe mitral stenosis. ECG, electrocardiogram.

1	(Courtesy of Raymond G. McKay, MD; with permission.) survival in younger (<45 years) patients with pliable valves is excellent, with rates as high as 80–90% over 3–7 years. Therefore, PMBV has become the procedure of choice for such patients when it can be performed by a skilled operator in a high-volume center. TTE is helpful in identifying patients for the percutaneous procedure, and TEE is performed routinely to exclude LA thrombus and to assess the degree of MR at the time of the scheduled procedure. An “echo score” has been developed to help guide decision-making. The score accounts for the degree of leaflet thickening, calcification, and mobility, and for the extent of subvalvular thickening. A lower score predicts a higher likelihood of successful PMBV.

1	In patients in whom PMBV is not possible or unsuccessful, or in many patients with restenosis after previous surgery, an “open” valvotomy using cardiopulmonary bypass is necessary. In addition to opening the valve commissures, it is important to loosen any subvalvular fusion of papillary muscles and chordae tendineae; to remove large deposits of calcium, thereby improving valvular function; and to remove atrial thrombi. The perioperative mortality rate is ~2%.

1	Successful valvotomy is defined by a 50% reduction in the mean mitral valve gradient and a doubling of the mitral valve area. Successful valvotomy, whether balloon or surgical, usually results in striking symptomatic and hemodynamic improvement and prolongs survival. However, there is no evidence that the procedure improves the prognosis of patients with slight or no functional impairment. Therefore, unless recurrent systemic embolization or severe pulmonary hypertension has occurred (PA systolic pressures >50 mmHg at rest or >60 mmHg with exercise), valvotomy is not recommended for patients who are entirely asymptomatic and/or who have mild or moderate stenosis (mitral valve area >1.5 cm2). When there is little symptomatic improvement after valvotomy, it is likely that the procedure was ineffective, that it induced MR, or that associated valvular or myocardial disease was present. About half of all patients undergoing surgical mitral valvotomy require reoperation by 10 years. In the

1	ineffective, that it induced MR, or that associated valvular or myocardial disease was present. About half of all patients undergoing surgical mitral valvotomy require reoperation by 10 years. In the pregnant patient with MS, valvotomy should be carried out if pulmonary congestion occurs despite intensive medical treatment. PMBV is the preferred strategy in this setting and is performed with TEE and no or minimal x-ray exposure.

1	Mitral valve replacement (MVR) is necessary in patients with MS and significant associated MR, those in whom the valve has been severely distorted by previous transcatheter or operative aData are for the first two quarters of calendar year 2013, during which 1004 sites reported a total of 135,666 procedures. Data are available from the Society of Thoracic Surgeons at http://www.sts.org/sites/default/files/documents/2013_3rdHarvestExecutiveSummary.pdf. Abbreviations: CAB, coronary artery bypass; MVR, mitral valve replacement; MVRp, mitral valve repair.

1	manipulation, or those in whom the surgeon does not find it possible to improve valve function significantly with valvotomy. MVR is now routinely performed with preservation of the chordal attachments to optimize LV functional recovery. Perioperative mortality rates with MVR vary with age, LV function, the presence of CAD, and associated comorbidities. They average 5% overall but are lower in young patients and may be twice as high in patients >65 years of age with significant comorbidities (Table 284-2). Because there are also long-term complications of valve replacement, patients in whom preoperative evaluation suggests the possibility that MVR may be required should be operated on only if they have severe MS—i.e., an orifice area ≤1.5 cm2—and are in NYHA Class III, i.e., symptomatic with ordinary activity despite optimal medical therapy. The overall 10-year survival of surgical survivors is ~70%. Long-term prognosis is worse in patients >65 years of age and those with marked

1	with ordinary activity despite optimal medical therapy. The overall 10-year survival of surgical survivors is ~70%. Long-term prognosis is worse in patients >65 years of age and those with marked disability and marked depression of the CO preoperatively. Pulmonary hypertension and RV dysfunction are additional risk factors for poor outcome.

1	MR may result from an abnormality or disease process that affects any one or more of the five functional components of the mitral valve apparatus (leaflets, annulus, chordae tendineae, papillary muscles, and subjacent myocardium) (Table 284-1). Acute MR can occur in the setting of acute myocardial infarction (MI) with papillary muscle rupture (Chap. 295), following blunt chest wall trauma, or during the course of infective endocarditis. With acute MI, the posteromedial papillary muscle is involved much more frequently than the anterolateral papillary muscle because of its singular blood supply. Transient, acute MR can occur during periods of active ischemia and bouts of angina pectoris. Rupture of chordae tendineae can result in “acute-on-chronic MR” in patients with myxomatous degeneration of the valve apparatus.

1	Chronic MR can result from rheumatic disease, mitral valve prolapse (MVP), extensive mitral annular calcification, congenital valve defects, hypertrophic obstructive cardiomyopathy (HOCM), and dilated cardiomyopathy (Chap. 287). Distinction also should be drawn between primary (degenerative, organic) MR, in which the leaflets and/or chordae tendineae are primarily responsible for abnormal valve function, and functional (secondary) MR, in which the leaflets and chordae tendineae are structurally normal but the regurgitation is caused by annular enlargement, papillary muscle displacement, leaflet tethering, or their combination. The rheumatic process produces rigidity, deformity, and retraction of the valve cusps and commissural fusion, as well as shortening, contraction, and fusion of the chordae tendineae. The MR associated with both MVP and HOCM is usually dynamic in nature. MR in HOCM occurs as a consequence of anterior papillary muscle displacement and systolic anterior motion of

1	chordae tendineae. The MR associated with both MVP and HOCM is usually dynamic in nature. MR in HOCM occurs as a consequence of anterior papillary muscle displacement and systolic anterior motion of the anterior mitral valve leaflet into the narrowed LV outflow tract. Annular calcification is especially prevalent among patients with advanced renal disease and is commonly observed in women >65 years of age with hypertension and diabetes. MR may occur as a congenital anomaly (Chap. 282), most commonly as a defect of the endocardial 1543 cushions (atrioventricular cushion defects). A cleft anterior mitral valve leaflet accompanies primum atrial septal defect. Chronic MR is frequently secondary to ischemia and may occur as a consequence of ventricular remodeling, papillary muscle displacement, and leaflet tethering, or with fibrosis of a papillary muscle, in patients with healed MI(s) and ischemic cardiomyopathy. Similar mechanisms of annular dilation and ventricular remodeling contribute

1	and leaflet tethering, or with fibrosis of a papillary muscle, in patients with healed MI(s) and ischemic cardiomyopathy. Similar mechanisms of annular dilation and ventricular remodeling contribute to the MR that occurs among patients with nonischemic forms of dilated cardiomyopathy once the LV end-diastolic dimension reaches 6 cm.

1	Irrespective of cause, chronic severe MR is often progressive, because enlargement of the LA places tension on the posterior mitral leaflet, pulling it away from the mitral orifice and thereby aggravating the valvular dysfunction. Similarly, LV dilation increases the regurgitation, which, in turn, enlarges the LA and LV further, resulting in a vicious circle; hence the aphorism, “mitral regurgitation begets mitral regurgitation.”

1	The resistance to LV emptying (LV afterload) is reduced in patients with MR. As a consequence, the LV is decompressed into the LA during ejection, and with the reduction in LV size during systole, there is a rapid decline in LV tension. The initial compensation to MR is more complete LV emptying. However, LV volume increases progressively with time as the severity of the regurgitation increases and as LV contractile function deteriorates. This increase in LV volume is often accompanied by a reduced forward CO. LV compliance is often increased, and thus, LV diastolic pressure does not increase until late in the course. The regurgitant volume varies directly with the LV systolic pressure and the size of the regurgitant orifice; the latter, in turn, is influenced by the extent of LV and mitral annular dilation. Because EF rises in severe MR in the presence of normal LV function, even a modest reduction in this parameter (<60%) reflects significant dysfunction.

1	During early diastole, as the distended LA empties, there is a particularly rapid y descent in the absence of accompanying MS. A brief, early diastolic LA-LV pressure gradient (often generating a rapid filling sound [S3] and mid-diastolic murmur masquerading as MS) may occur in patients with pure, severe MR as a result of the very rapid flow of blood across a normal-sized mitral orifice.

1	Semiquantitative estimates of LV ejection fraction (LVEF), CO, PA systolic pressure, regurgitant volume, regurgitant fraction (RF), and the effective regurgitant orifice area can be obtained during a careful Doppler echocardiographic examination. These measurements can also be obtained accurately with cardiac magnetic resonance (CMR) imaging, although this technology is not widely available. Left and right heart catheterization with contrast ventriculography is used less frequently. Severe, nonischemic MR is defined by a regurgitant volume ≥60 mL/beat, RF ≥50%, and effective regurgitant orifice area ≥0.40 cm2. Severe ischemic MR, however, is usually associated with an effective regurgitant orifice area of >0.2 cm2. In the latter instance, lesser degrees of MR carry relatively greater prognostic weight.

1	LA Compliance In acute severe MR, the regurgitant volume is delivered into a normal-sized LA having normal or reduced compliance. As a result, LA pressures rise markedly for any increase in LA volume. The v wave in the LA pressure pulse is usually prominent, LA and pulmonary venous pressures are markedly elevated, and pulmonary edema is common. Because of the rapid rise in LA pressures during ventricular systole, the murmur of acute MR is early in timing and decrescendo in configuration ending well before S2, as a reflection of the progressive diminution in the LV-LA pressure gradient. LV systolic function in acute MR may be normal, hyperdynamic, or reduced, depending on the clinical context.

1	Patients with chronic severe MR, on the other hand, develop marked LA enlargement and increased LA compliance with little if any increase in LA and pulmonary venous pressures for any increase in LA volume. The LA v wave is relatively less prominent. The murmur of chronic MR is classically holosystolic in timing and plateau in configuration, as a reflection of the near-constant LV-LA pressure gradient. These patients usually complain of severe fatigue and exhaustion secondary 1544 to a low forward CO, whereas symptoms resulting from pulmonary congestion are less prominent initially; AF is almost invariably present once the LA dilates significantly.

1	Patients with chronic mild-to-moderate, isolated MR are usually asymptomatic. This form of LV volume overload is well tolerated. Fatigue, exertional dyspnea, and orthopnea are the most prominent complaints in patients with chronic severe MR. Palpitations are common and may signify the onset of AF. Right-sided heart failure, with painful hepatic congestion, ankle edema, distended neck veins, ascites, and secondary TR, occurs in patients with MR who have associated pulmonary vascular disease and pulmonary hypertension. Acute pulmonary edema is common in patients with acute severe MR. In patients with chronic severe MR, the arterial pressure is usually normal, although the carotid arterial pulse may show a sharp, low-volume upstroke owing to the reduced forward CO. A systolic thrill is often palpable at the cardiac apex, the LV is hyperdynamic with a brisk systolic impulse and a palpable rapid-filling wave (S3), and the apex beat is often displaced laterally.

1	In patients with acute severe MR, the arterial pressure may be reduced with a narrow pulse pressure, the jugular venous pressure and wave forms may be normal or increased and exaggerated, the apical impulse is not displaced, and signs of pulmonary congestion are prominent.

1	Auscultation S1 is generally absent, soft, or buried in the holosystolic murmur of chronic, severe MR. In patients with severe MR, the aortic valve may close prematurely, resulting in wide but physiologic splitting of S2. A low-pitched S3 occurring 0.12–0.17 s after the aortic valve closure sound, i.e., at the completion of the rapid-filling phase of the LV, is believed to be caused by the sudden tensing of the papillary muscles, chordae tendineae, and valve leaflets. It may be followed by a short, rumbling, mid-diastolic murmur, even in the absence of structural MS. A fourth heart sound is often audible in patients with acute severe MR who are in sinus rhythm. A presystolic murmur is not ordinarily heard with isolated MR.

1	A systolic murmur of at least grade III/VI intensity is the most characteristic auscultatory finding in chronic severe MR. It is usually holosystolic (see Fig. 267-5A), but as previously noted, it is decrescendo and ceases in mid to late systole in patients with acute severe MR. The systolic murmur of chronic MR is usually most prominent at the apex and radiates to the axilla. However, in patients with ruptured chordae tendineae or primary involvement of the posterior mitral leaflet with prolapse or flail, the regurgitant jet is eccentric, directed anteriorly, and strikes the LA wall adjacent to the aortic root. In this situation, the systolic murmur is transmitted to the base of the heart and, therefore, may be confused with the murmur of AS. In patients with ruptured chordae tendineae, the systolic murmur may have a cooing or “seagull” quality, whereas a flail leaflet may produce a murmur with a musical quality. The systolic murmur of chronic MR not due to MVP is intensified by

1	the systolic murmur may have a cooing or “seagull” quality, whereas a flail leaflet may produce a murmur with a musical quality. The systolic murmur of chronic MR not due to MVP is intensified by isometric exercise (handgrip) but is reduced during the strain phase of the Valsalva maneuver because of the associated decrease in LV preload.

1	LABORATORY EXAMINATION ECG In patients with sinus rhythm, there is evidence of LA enlargement, but RA enlargement also may be present when pulmonary hypertension is significant and affects RV function. Chronic severe MR is frequently associated with AF. In many patients, there is no clear-cut ECG evidence of enlargement of either ventricle. In others, the signs of eccentric LV hypertrophy are present.

1	Echocardiogram TTE is indicated to assess the mechanism of the MR and its hemodynamic severity. LV function can be assessed from LV end-diastolic and end-systolic volumes and EF. Observations can be made regarding leaflet structure and function, chordal integrity, LA and LV size, annular calcification, and regional and global LV systolic function. Doppler imaging should demonstrate the width or area of the color flow MR jet within the LA, the duration and intensity of the continuous wave Doppler signal, the pulmonary venous flow contour, the early peak mitral inflow velocity, and quantitative measures of regurgitant volume, RF, and effective regurgitant orifice area. In addition, the PAPs can be estimated from the TR jet velocity. TTE is also indicated to follow the course of patients with chronic MR and to provide rapid assessment for any clinical change. The echocardiogram in patients with MVP is described in the next section. TEE provides greater anatomic detail than TTE (see Fig.

1	chronic MR and to provide rapid assessment for any clinical change. The echocardiogram in patients with MVP is described in the next section. TEE provides greater anatomic detail than TTE (see Fig. 270e-5). Exercise testing with TTE can be useful to assess exercise capacity as well as any dynamic change in MR severity, PA systolic pressures, and biventricular function, for patients in whom there is a discrepancy between clinical findings and the results of functional testing performed at rest.

1	Chest X-Ray The LA and LV are the dominant chambers in chronic MR. Late in the course of the disease, the LA may be massively enlarged and forms the right border of the cardiac silhouette. Pulmonary venous congestion, interstitial edema, and Kerley B lines are sometimes noted. Marked calcification of the mitral leaflets occurs commonly in patients with long-standing, combined rheumatic MR and MS. Calcification of the mitral annulus may be visualized, particularly on the lateral view of the chest. Patients with acute severe MR may have asymmetric pulmonary edema if the regurgitant jet is directed predominantly to the orifice of an upper lobe pulmonary vein. MEDICAL TREATMENT (FIG. 284-4)

1	The management of chronic severe MR depends to some degree on its cause. Warfarin should be provided once AF intervenes with a target INR of 2–3. Novel oral anticoagulants are not approved for this indication. Cardioversion should be considered depending on the clinical context and LA size. In contrast to the acute setting, there are no large, long-term prospective studies to substantiate the use of vasodilators for the treatment of chronic, isolated severe MR with preserved LV systolic function in the absence of systemic hypertension. The severity of MR in the setting of an ischemic or nonischemic dilated cardiomyopathy may diminish with aggressive guideline-directed treatment of heart failure including the use of diuretics, beta blockers, angiotensin-converting enzyme (ACE) inhibitors, digitalis, and biventricular pacing (cardiac resynchronization therapy [CRT]) when otherwise indicated. Asymptomatic patients with severe MR in sinus rhythm with normal LV size and systolic function

1	digitalis, and biventricular pacing (cardiac resynchronization therapy [CRT]) when otherwise indicated. Asymptomatic patients with severe MR in sinus rhythm with normal LV size and systolic function should avoid isometric forms of exercise.

1	Patients with acute severe MR require urgent stabilization and preparation for surgery. Diuretics, intravenous vasodilators (particularly sodium nitroprusside), and even intraaortic balloon counterpulsation may be needed for patients with post-MI papillary muscle rupture or other forms of acute severe MR.

1	In the selection of patients with chronic, nonischemic, primary or organic, severe MR for surgical treatment, the often slowly progressive nature of the condition must be balanced against the immediate and long-term risks associated with operation. These risks are significantly lower for primary valve repair than for valve replacement (Table 287-2). Repair usually consists of valve reconstruction using a variety of valvuloplasty techniques and insertion of an annuloplasty ring. Repair spares the patient the long-term adverse consequences of valve replacement, including thromboembolic and hemorrhagic complications in the case of mechanical prostheses and late valve failure necessitating repeat valve replacement in the case of bioprostheses. In addition, by preserving the integrity of the papillary muscles, subvalvular apparatus, and chordae tendineae, mitral repair and valvuloplasty maintain LV function to a relatively greater degree.

1	Vena contracta ˜0.7 cm RVol ˜60% cc RF ˜50% ERO ˜0.4 cm2 LV dilation (stage B) Vena contracta ˜0.7 cm RVol <60 cc RF <50% ERO <0.4 cm2

1	FIGURE 284-4 Management of mitral regurgitation. See legend for Fig. 283-2 for explanation of treatment recommendations (class I, IIa, IIb) and disease stages (B, C1, C2, D). Preoperative coronary angiography should be performed routinely as determined by age, symptoms, and coronary risk factors. Cardiac catheterization and angiography may also be helpful when there is a discrepancy between clinical and noninvasive findings. AF, atrial fibrillation; CAD, coronary artery disease; CRT, cardiac resynchronization therapy; ERO, effective regurgitant orifice; HF, heart failure; LV, left ventricular; LVEF, left ventricular ejection fraction; LVESD, left ventricular end-systolic dimension; MR, mitral regurgitation, MV, mitral valve; MVR, mitral valve replacement; NYHA, New York Heart Association; PASP, pulmonary artery systolic pressure; RF, regurgitant fraction; RVol, regurgitant volume; and Rx, therapy. ∗Mitral valve repair preferred over MVR when possible. (Adapted from RA Nishimura et al:

1	PASP, pulmonary artery systolic pressure; RF, regurgitant fraction; RVol, regurgitant volume; and Rx, therapy. ∗Mitral valve repair preferred over MVR when possible. (Adapted from RA Nishimura et al: 2014 AHA/ACC Guideline for the Management of Patients with Valvular Heart Disease. J Am Coll Cardiol doi: 10.1016/j.jacc.2014.02.536, 2014, with permission.)

1	Surgery for chronic nonischemic severe MR is indicated once symptoms occur, especially if valve repair is feasible (Fig. 284-4). Other indications for early consideration of mitral valve repair include recent-onset AF and pulmonary hypertension defined as a systolic PA pressure ≥50 mmHg at rest or ≥60 mmHg with exercise. Surgical treatment of chronic nonischemic severe MR is indicated for asymptomatic patients when LV dysfunction is progressive with the LVEF falling below 60% and/or end-systolic dimension increasing beyond 40 mm. These aggressive recommendations for surgery are predicated on the outstanding results achieved with mitral valve repair particularly when applied to patients with myxomatous disease such as that associated with prolapse or flail leaflet. Indeed primary valvuloplasty repair of patients younger than 75 years with normal LV systolic function and no CAD can now be performed by experienced surgeons with <1% perioperative mortality risk. The risk of stroke,

1	repair of patients younger than 75 years with normal LV systolic function and no CAD can now be performed by experienced surgeons with <1% perioperative mortality risk. The risk of stroke, however, is also approximately 1%. Repair is feasible in up to 95% of patients with myxomatous disease operated on by a high-volume surgeon in a referral center of excellence. Long-term durability is excellent; the incidence of reoperative surgery for failed primary repair is ~1% per year for the first 10 years after surgery. For patients with AF, left or biatrial maze surgery, or radiofrequency, isolation of the pulmonary veins is often performed to reduce the risk of recurrent postoperative AF.

1	The surgical management of patients with functional, ischemic MR is more complicated and most often involves simultaneous coronary artery revascularization. Current surgical practice includes annuloplasty repair with an undersized, rigid ring or chord-sparing valve replacement for patients with moderate or greater degrees of MR. Valve repair for ischemic MR is associated with lower perioperative mortality rates but higher rates of recurrent MR over time. In patients with ischemic MR and significantly impaired LV systolic function (EF <30%), the risk of surgery is higher, recovery of LV performance is incomplete, and long-term survival is reduced. Referral for surgery must be individualized and made only after aggressive attempts with guideline-directed medical therapy and CRT, when indicated. The routine performance of valve repair in patients with significant MR in the setting of severe, functional, nonischemic dilated cardiomyopathy has not been shown to improve long-term survival

1	The routine performance of valve repair in patients with significant MR in the setting of severe, functional, nonischemic dilated cardiomyopathy has not been shown to improve long-term survival compared with optimal medical therapy. Patients with acute severe MR can often be stabilized temporarily with appropriate medical therapy, but surgical correction will be necessary emergently in the case of papillary muscle rupture and within days to weeks in most other settings.

1	When surgical treatment is contemplated, left and right heart catheterization and left ventriculography may be helpful in confirming the presence of severe MR in patients in whom there is a discrepancy between the clinical and TTE findings that cannot be resolved with TEE or CMR. Coronary angiography identifies patients who require concomitant coronary revascularization. FIGURE 284-5 Clip used to grasp the free edges of the anterior and posterior leaflets in their midsections during transcatheter repair of selected patients with mitral regurgitation. (Courtesy of Abbott Vascular. © 2014 Abbott Laboratories. All rights reserved.)

1	A transcatheter approach to the treatment of either organic or functional MR may be feasible in selected patients with appropriate anatomy. The proper role of currently available techniques remains under active investigation. One approach involves the deployment of a clip delivered via transseptal puncture that grasps the leading edges of the mitral leaflets in their mid-portion (anterior scallop to posterior scallop or A2-P2; Fig. 284-5). The length and width of the gap between these leading edges dictate patient eligibility. The device is commercially available for the treatment of prohibitive surgical risk patients with severe, degenerative (organic) MR and is undergoing study in the United States for treatment of patients with symptomatic heart failure, reduced LVEF, and severe, functional MR despite guideline-directed medical therapy. A second approach involves the deployment of a device within the coronary sinus that can be adjusted to reduce its circumference, thus secondarily

1	MR despite guideline-directed medical therapy. A second approach involves the deployment of a device within the coronary sinus that can be adjusted to reduce its circumference, thus secondarily decreasing the circumference of the mitral annulus and the effective orifice area of the valve much like a surgically implanted ring. Variations in the anatomic relationship of the coronary sinus to the mitral annulus and circumflex coronary artery have limited the applicability of this technique. Attempts to reduce the septal-lateral dimension of a dilated annulus using adjustable cords placed across the LV in a subvalvular location have also been investigated.

1	MVP, also variously termed the systolic click-murmur syndrome, Barlow’s syndrome, floppy-valve syndrome, and billowing mitral leaflet syndrome, is a relatively common but highly variable clinical syndrome resulting from diverse pathologic mechanisms of the mitral valve apparatus. Among these are excessive or redundant mitral leaflet tissue, which is commonly associated with myxomatous degeneration and greatly increased concentrations of certain glycosaminoglycans. In most patients with MVP, the cause is unknown, but in some, it appears to be genetically determined. A reduction in the production of type III collagen has been incriminated, and electron microscopy has revealed fragmentation of collagen fibrils.

1	MVP is a frequent finding in patients with heritable disorders of connective tissue, including Marfan’s syndrome (Chap. 427), osteogenesis imperfecta, and Ehlers-Danlos syndrome. MVP may be associated with thoracic skeletal deformities similar to but not as severe as those in Marfan’s syndrome, such as a high-arched palate and alterations of the chest and thoracic spine, including the so-called straight back syndrome. In most patients with MVP, myxomatous degeneration is confined to the mitral valve, although the tricuspid and aortic valves may also be affected. The posterior mitral leaflet is usually more affected than the anterior, and the mitral valve annulus is often dilated. In many patients, elongated, redundant, or ruptured chordae tendineae cause or contribute to the regurgitation.

1	MVP also may occur rarely as a sequel to acute rheumatic fever, in ischemic heart disease, and in various cardiomyopathies, as well as in 20% of patients with ostium secundum atrial septal defect. MVP may lead to excessive stress on the papillary muscles, which, in turn, leads to dysfunction and ischemia of the papillary muscles and the subjacent ventricular myocardium. Rupture of chordae tendineae and progressive annular dilation and calcification contribute to valvular regurgitation, which then places more stress on the diseased mitral valve apparatus, thereby creating a vicious circle. ECG changes (see below) and ventricular arrhythmias described in some patients with MVP appear to result from regional ventricular dysfunction related to the increased stress placed on the papillary muscles.

1	MVP is more common in women and occurs most frequently between the ages of 15 and 30 years; the clinical course is most often benign. MVP may also be observed in older (>50 years) patients, often men, in whom MR is often more severe and requires surgical treatment. There is an increased familial incidence for some patients, suggesting an autosomal dominant form of inheritance with incomplete penetrance. MVP varies in its clinical expression, ranging from only a systolic click and murmur with mild prolapse of the posterior leaflet to severe MR due to chordal rupture and leaflet flail. The degree of myxomatous change of the leaflets can also vary widely. In many patients, the condition progresses over years or decades; in others, it worsens rapidly as a result of chordal rupture or endocarditis.

1	Most patients are asymptomatic and remain so for their entire lives. However, in North America, MVP is now the most common cause of isolated severe MR requiring surgical treatment. Arrhythmias, most commonly ventricular premature contractions and paroxysmal supraventricular and ventricular tachycardia, as well as AF, have been reported and may cause palpitations, light-headedness, and syncope. Sudden death is a very rare complication and occurs most often in patients with severe MR and depressed LV systolic function. There may be an excess risk of sudden death among patients with a flail leaflet. Many patients have chest pain that is difficult to evaluate; it is often substernal, prolonged, and not related to exertion, but may rarely resemble angina pectoris. Transient cerebral ischemic attacks secondary to emboli from the mitral valve due to endothelial disruption have been reported. Infective endocarditis may occur in patients with MR and/or leaflet thickening.

1	Auscultation A frequent finding is the mid or late (nonejection) systolic click, which occurs 0.14 s or more after S1 and is thought to be generated by the sudden tensing of slack, elongated chordae tendineae or by the prolapsing mitral leaflet when it reaches its maximal excursion. Systolic clicks may be multiple and may be followed by a high-pitched, mid-late systolic crescendo-decrescendo murmur, which occasionally is “whooping” or “honking” and is heard best at the apex. The click and murmur occur earlier with standing, during the strain phase of the Valsalva maneuver, and with any intervention that decreases LV volume, exaggerating the propensity of mitral leaflet prolapse. Conversely, squatting and isometric exercises, which increase LV volume, diminish MVP; the click-murmur complex is delayed, moves away from S1, and may even disappear. Some patients have a mid-systolic click without a murmur; others have a murmur without a click. Still others have both sounds at different

1	is delayed, moves away from S1, and may even disappear. Some patients have a mid-systolic click without a murmur; others have a murmur without a click. Still others have both sounds at different times.

1	The ECG most commonly is normal but may show biphasic or inverted T waves in leads II, III, and aVF, and occasionally supraventricular or ventricular premature beats. TTE is particularly effective in identifying the abnormal position and prolapse of the mitral valve leaflets. A useful echocardiographic definition of MVP is systolic displacement (in the parasternal long axis view) of the mitral valve leaflets by at least 2 mm into the LA superior to the plane of the mitral annulus. Color flow and continuous wave Doppler imaging is helpful to evaluate the associated MR and provide semiquantitative estimates of severity. The jet lesion of MR due to MVP is most often eccentric, and assessment of RF and effective regurgitant orifice area can be difficult. TEE is indicated when more accurate information is required and is performed routinely for intraoperative guidance for valve repair. Invasive left ventriculography is rarely necessary but can also show prolapse of the posterior and

1	information is required and is performed routinely for intraoperative guidance for valve repair. Invasive left ventriculography is rarely necessary but can also show prolapse of the posterior and sometimes of both mitral valve leaflets.

1	Infective endocarditis prophylaxis is indicated only for patients with a prior history of endocarditis. Beta blockers sometimes relieve chest pain and control palpitations. If the patient is symptomatic from severe MR, mitral valve repair (or rarely, chord-sparing replacement) is indicated (Fig. 284-4). Antiplatelet agents, such as aspirin, should be given to patients with transient ischemic attacks, and if these are not effective, warfarin should be considered. Warfarin is also indicated once AF intervenes.

1	Tricuspid and Pulmonic valve Disease Patrick T. O’Gara, Joseph Loscalzo TRICUSPID STENOSIS Tricuspid stenosis (TS), which is much less prevalent than mitral 285 stenosis (MS) in North America and Western Europe, is generally rheumatic in origin, and is more common in women than men (Table 285-1). It does not occur as an isolated lesion and is usually associated with MS. Hemodynamically significant TS occurs in 5–10% of patients with severe MS; rheumatic TS is commonly associated with some degree of tricuspid regurgitation (TR). Nonrheumatic causes of TS are rare.

1	A diastolic pressure gradient between the right atrium (RA) and right ventricle (RV) defines TS. It is augmented when the transvalvular blood flow increases during inspiration and declines during expiration. A mean diastolic pressure gradient of 4 mmHg is usually sufficient to elevate the mean RA pressure to levels that result in systemic venous congestion. Unless sodium intake has been restricted and diuretics administered, this venous congestion is associated with hepatomegaly, ascites, and edema, sometimes severe. In patients with sinus rhythm, the RA a wave may be extremely tall and may even approach the level of the RV systolic pressure. The y descent is prolonged. The cardiac output (CO) at rest is usually depressed, and it fails to rise during exercise. The low CO is responsible for the normal or only slightly elevated left atrial (LA), pulmonary artery (PA), and RV systolic pressures despite the presence of MS. Thus, the presence of TS can mask the hemodynamic and clinical

1	for the normal or only slightly elevated left atrial (LA), pulmonary artery (PA), and RV systolic pressures despite the presence of MS. Thus, the presence of TS can mask the hemodynamic and clinical features of any associated MS.

1	Because the development of MS generally precedes that of TS, many patients initially have symptoms of pulmonary congestion and fatigue. Characteristically, patients with severe TS complain of relatively little dyspnea for the degree of hepatomegaly, ascites, and edema that they have. However, fatigue secondary to a low CO and discomfort due to refractory edema, ascites, and marked hepatomegaly are common in patients with advanced TS and/or TR. In some patients, TS may be Secondary (functional) RV and tricuspid annular dilatation due to multiple causes of RV enlargement (e.g., long-standing pulmonary HTN, remodeling post-RV MI) Chronic RV apical pacing Abbreviations: HTN, hypertension; MI, myocardial infarction; RV, right ventricular; TVP, tricuspid valve prolapse. suspected for the first time when symptoms of right-sided failure persist after an adequate mitral valvotomy.

1	suspected for the first time when symptoms of right-sided failure persist after an adequate mitral valvotomy. Because TS usually occurs in the presence of other obvious valvular disease, the diagnosis may be missed unless it is considered. Severe TS is associated with marked hepatic congestion, often resulting in cirrhosis, jaundice, serious malnutrition, anasarca, and ascites. Congestive hepatomegaly and, in cases of severe tricuspid valve disease, splenomegaly are present. The jugular veins are distended, and in patients with sinus rhythm, there may be giant a waves. The v waves are less conspicuous, and because tricuspid obstruction impedes RA emptying during diastole, there is a slow y descent. In patients with sinus rhythm, there may be prominent presystolic pulsations of the enlarged liver as well.

1	On auscultation, an opening snap (OS) of the tricuspid valve may rarely be heard approximately 0.06 s after pulmonic valve closure. The diastolic murmur of TS has many of the qualities of the diastolic murmur of MS, and because TS almost always occurs in the presence of MS, it may be missed. However, the tricuspid murmur is generally heard best along the left lower sternal border and over the xiphoid process, and is most prominent during presystole in patients with sinus rhythm. The murmur of TS is augmented during inspiration, and it is reduced during expiration and particularly during the strain phase of the Valsalva maneuver, when tricuspid transvalvular flow is reduced.

1	The electrocardiogram (ECG) features of RA enlargement (see Fig. 268-8) include tall, peaked P waves in lead II, as well as prominent, upright P waves in lead V1. The absence of ECG evidence of RV 1548 hypertrophy (RVH) in a patient with right-sided heart failure who is believed to have MS should suggest associated tricuspid valve disease. The chest x-ray in patients with combined TS and MS shows particular prominence of the RA and superior vena cava without much enlargement of the PA and with less evidence of pulmonary vascular congestion than occurs in patients with isolated MS. On echocardiographic examination, the tricuspid valve is usually thickened and domes in diastole; the transvalvular gradient can be estimated by continuous wave Doppler echocardiography. Severe TS is characterized by a valve area ≤1 cm2 or pressure half-time of ≥190 ms. The RA and inferior vena cava (IVC) are enlarged. Transthoracic echocardiography (TTE) provides additional information regarding the

1	by a valve area ≤1 cm2 or pressure half-time of ≥190 ms. The RA and inferior vena cava (IVC) are enlarged. Transthoracic echocardiography (TTE) provides additional information regarding the severity of any associated TR, mitral valve structure and function, left ventricle (LV) and RV size and function, and PA pressure. Cardiac catheterization is not routinely necessary for assessment of TS.

1	Patients with TS generally exhibit marked systemic venous congestion; salt restriction, bed rest, and diuretic therapy are required during the preoperative period. Such a preparatory period may diminish hepatic congestion and thereby improve hepatic function sufficiently so that the risks of operation, particularly bleeding, are diminished. Surgical relief of the TS should be carried out, preferably at the time of surgical mitral valvotomy or mitral valve replacement (MVR) for mitral valve disease, in patients with moderate or severe TS who have mean diastolic pressure gradients exceeding ~4 mmHg and tricuspid orifice areas <1.5–2 cm2. TS is almost always accompanied by significant TR. Operative repair may permit substantial improvement of tricuspid valve function. If repair cannot be accomplished, the tricuspid valve may have to be replaced. Meta-analysis has shown no difference in overall survival between mechanical and tissue valve replacement. Mechanical valves in the tricuspid

1	the tricuspid valve may have to be replaced. Meta-analysis has shown no difference in overall survival between mechanical and tissue valve replacement. Mechanical valves in the tricuspid position are more prone to thromboembolic complications than in other positions. Percutaneous tricuspid balloon valvuloplasty for isolated severe TS without significant TR is very rarely performed.

1	In at least 80% of cases, TR is secondary to marked dilation of the tricuspid annulus from RV enlargement due to PA hypertension (Table 285-1). Functional TR may complicate RV enlargement of any cause, however, including an inferior myocardial infarction (MI) that involves the RV. It is commonly seen in the late stages of heart failure due to rheumatic or congenital heart disease with severe PA hypertension (PA systolic pressure >55 mmHg), as well as in ischemic and idiopathic dilated cardiomyopathies. It is reversible in part if PA hypertension can be relieved. Functional TR can also develop from chronic RV apical pacing. Rheumatic fever may produce primary (organic) TR, often associated with TS. Infarction of RV papillary muscles, tricuspid valve prolapse, carcinoid heart disease, endomyocardial fibrosis, radiation, infective endocarditis, and leaflet trauma all may produce TR. Less commonly, TR results from congenitally deformed tricuspid valves, and it occurs with defects of the

1	fibrosis, radiation, infective endocarditis, and leaflet trauma all may produce TR. Less commonly, TR results from congenitally deformed tricuspid valves, and it occurs with defects of the atrioventricular canal, as well as with Ebstein’s malformation of the tricuspid valve (Chap. 282).

1	The incompetent tricuspid valve allows blood to flow backward from the RV into the RA, the volume of which is dependent on the driving pressure (i.e., RV systolic pressure) and the size of the regurgitant orifice. The severity and physical signs of TR can vary as a function of PA systolic pressure (in the absence of RV outflow tract stenosis), the dimension of the tricuspid valve annulus, the respiratory cycle– dependent changes in RV preload, and RA compliance. RV filling is increased during inspiration. Forward CO is reduced and does not augment with exercise. Significant degrees of TR will lead to RA enlargement and elevation of the RA and jugular venous pressures with prominent c-v waves in the pulse tracings. Progressively severe TR can lead to “ventricularization” of the RA wave form (see Fig. 267-1B). Severe TR is also characterized by RV dilation (RV volume overload) and eventual systolic dysfunction, the rate of which can be accelerated by a concomitant pressure load from PA

1	(see Fig. 267-1B). Severe TR is also characterized by RV dilation (RV volume overload) and eventual systolic dysfunction, the rate of which can be accelerated by a concomitant pressure load from PA hypertension or by myocardial fibrosis from previous injury.

1	Mild or moderate degrees of TR are usually well tolerated in the absence of other hemodynamic disturbances. Because TR most often coexists with left-sided valve lesions, LV dysfunction, and/or PA hypertension, symptoms related to these lesions may dominate the clinical picture. Fatigue and exertional dyspnea owing to reduced forward CO are early symptoms of isolated, severe TR. As the disease progresses and RV function declines, patients may report cervical pulsations, abdominal full-ness/bloating, diminished appetite, and muscle wasting, although with progressive weight gain and painful swelling of the lower extremities.

1	The neck veins in patients with severe TR are distended with prominent c-v waves and rapid y descents (in the absence of TS). TR is more often diagnosed by examination of the neck veins than by auscultation of the heart sounds. Other findings may include marked hepatomegaly with systolic pulsations, ascites, pleural effusions, edema, and a positive hepatojugular reflex. A prominent RV pulsation along the left parasternal region and a blowing holosystolic murmur along the lower left sternal margin, which may be intensified during inspiration (Carvallo’s sign) and reduced during expiration or the strain phase of the Valsalva maneuver, are characteristic findings. The murmur of TR may sometimes be confused with that of MR unless attention is paid to its variation during the respiratory cycle and the extent of RV enlargement is appreciated. Atrial fibrillation (AF) is usually present in the chronic phase of the disease.

1	The ECG may show changes characteristic of the lesion responsible for the TR, e.g., an inferior Q-wave MI suggestive of a prior RV MI, RVH, or a bizarre right bundle branch block type pattern with preexcitation in patients with Ebstein’s anomaly. ECG signs of RA enlargement may be present in patients with sinus rhythm; AF is frequently noted. The chest x-ray may show RA and RV enlargement, depending on the chronicity and severity of TR. TTE is usually definitive with demonstration of RA dilation and RV volume overload and prolapsing, flail, scarred, or displaced/tethered tricuspid leaflets; the diagnosis and assessment of TR can be made by color flow Doppler imaging (see Fig. 270e-8). Severe TR is accompanied by hepatic vein systolic flow reversal. Continuous wave Doppler of the TR velocity profile is useful in estimating PA systolic pressure. Accurate assessment of TR severity, PA pressures, and RV size and systolic function with TTE can be quite challenging in many patients.

1	velocity profile is useful in estimating PA systolic pressure. Accurate assessment of TR severity, PA pressures, and RV size and systolic function with TTE can be quite challenging in many patients. Real-time three-dimensional echocardiography and cardiac magnetic resonance (CMR) imaging provide alternative imaging modalities, although they are not widely available. In patients with severe TR, the CO is usually markedly reduced, and the RA pressure pulse may exhibit no x descent during early systole but a prominent c-v wave with a rapid y descent. The mean RA and RV end-diastolic pressures are often elevated. Exercise testing can be used to assess functional capacity in patients with asymptomatic severe TR. The prognostic significance of exercise-induced changes in TR severity and RV function has not been well studied.

1	TREATMEnT tricuSPid regurgitation (fig. 285-1) Diuretics can be useful for patients with severe TR and signs of right heart failure. An aldosterone antagonist may be particularly helpful because many patients have secondary hyperaldosteronism from marked hepatic congestion. Therapies to reduce elevated PA pressures and/or pulmonary vascular resistance, including those targeted at left-sided heart disease, can also be considered for patients with PA hypertension and severe functional TR. Tricuspid valve surgery is recommended for patients with severe TR who are undergoing

1	FIGURE 285-1 Management of tricuspid regurgitation. See legend for Fig. 283-2 for explanation of treatment recommendations (Class I, IIa, IIb) and disease stages (B, C, D). Preoperative coronary angiography should be performed routinely as determined by age, symptoms, and coronary risk factors. Cardiac catheterization and angiography may also be helpful when there is a discrepancy between clinical and noninvasive findings. PHTN, pulmonary hypertension; RV, right ventricular; TA, tricuspid annular; TTE, transthoracic echocardiogram; TR, tricuspid regurgitation; TV, tricuspid valve; TVR, tricuspid valve replacement. ∗ TA dilation is defined by >40 mm on TTE (>21 mm/m2) or >70 mm on direct intraoperative measurement. (Adapted from RA Nishimura et al: 2014 AHA/ACC Guideline for the Management of Patients with Valvular Heart Disease. J Am Coll Cardiol doi: 10.1016/j.jacc.2014.02.536, 2014, with permission.) left-sided valve surgery and is also undertaken frequently for treatment of even

1	of Patients with Valvular Heart Disease. J Am Coll Cardiol doi: 10.1016/j.jacc.2014.02.536, 2014, with permission.) left-sided valve surgery and is also undertaken frequently for treatment of even moderate TR in patients undergoing left-sided valve surgery who have tricuspid annular dilation (>40 mm), a history of right heart failure, or PA hypertension. Operation most often comprises repair rather than replacement in these settings and has become routine in most major surgical centers. Surgery may also infrequently be required for treatment of severe, primary TR with right heart failure not responsive to standard medical therapy or because of progressively declining RV systolic function. Reported perioperative mortality rates for isolated tricuspid valve surgery (repair and replacement) are high (~8-9%) and likely are influenced by the hazards encountered during reoperation on patients who have undergone previous left-sided valve surgery and have reduced RV function. Indwelling

1	are high (~8-9%) and likely are influenced by the hazards encountered during reoperation on patients who have undergone previous left-sided valve surgery and have reduced RV function. Indwelling pacemaker or defibrillator leads can also pose technical challenges.

1	Pulmonic valve stenosis (PS) is essentially a congenital disorder (Table 285-1). With isolated PS, the valve is typically domed. Dysplastic pulmonic valves are seen as part of the Noonan’s syndrome (Chap. 302), which maps to chromosome 12. Much less common etiologies include carcinoid and obstructing tumors or bulky vegetations. The pulmonic valve is only very rarely affected by the rheumatic process.

1	PS is defined hemodynamically by a systolic pressure gradient between the RV and main PA. RV hypertrophy develops as a consequence of sustained obstruction to RV outflow, and systolic ejection is prolonged. Compared with the ability of the LV to compensate for the pressure overload imposed by aortic stenosis (AS), RV dysfunction from afterload mismatch occurs earlier in the course of PS and at lower peak systolic pressures, because the RV adapts less well to this type of hemodynamic burden. With normal systolic function and CO, severe PS is defined by a peak systolic gradient across the pulmonic valve of >50 mmHg; moderate PS correlates with a peak gradient of 30–50 mmHg. PS rarely progresses in patients with peak gradients less than 30 mmHg, but may worsen in those with moderate disease due to valve thickening and calcification with age. The RA a wave elevates in relation to the higher pressures needed to fill a noncompliant, hypertrophied RV. A prominent RA v wave signifies

1	disease due to valve thickening and calcification with age. The RA a wave elevates in relation to the higher pressures needed to fill a noncompliant, hypertrophied RV. A prominent RA v wave signifies functional TR from RV and annular dilation. The CO is maintained until late in the course of the disease.

1	Patients with mild or even moderate PS are usually asymptomatic and first come to medical attention because of a heart murmur that leads to echocardiography. With severe PS, patients may report exertional dyspnea or early-onset fatigue. Anginal chest pain from RV oxygen supply-demand mismatch and syncope may occur with very severe forms of obstruction, particularly in the presence of a destabilizing trigger such as atrial fibrillation, fever, infection, or anemia.

1	The murmur of mild or moderate PS is mid-systolic in timing, crescendo-decrescendo in configuration, heard best in the left second interspace, and usually introduced by an ejection sound (click) in younger adults whose valves are still pliable. The ejection sound is the only right-sided acoustic event that decreases in intensity with inspiration. This phenomenon reflects premature opening of the pulmonic valve by the elevated RV end-diastolic (postatrial a wave) pressure. The systolic murmur increases in intensity during inspiration. With progressively severe PS, the ejection sound moves closer to the first heart sound and eventually becomes inaudible. A right-sided fourth heart sound may emerge. The systolic murmur peaks later and may persist through the aortic component of the second heart sound (A2). Pulmonic valve 1550 closure is delayed, and the pulmonic component of the second heart sound (P2) is reduced or absent. A prominent a wave, indicative of the higher atrial pressure

1	heart sound (A2). Pulmonic valve 1550 closure is delayed, and the pulmonic component of the second heart sound (P2) is reduced or absent. A prominent a wave, indicative of the higher atrial pressure necessary to fill the noncompliant RV, may be seen in the jugular venous pulse. A parasternal or RV lift can be felt with significant pressure overload. Signs of right heart failure, such as hepatomegaly, ascites, and edema, are uncommon but may appear very late in the disease.

1	The ECG will show right axis deviation, RVH, and RA enlargement in adult patients with severe PS. Chest x-ray findings include poststenotic dilation of the main PA in the frontal plane projection and filling of the retrosternal airspace due to RV enlargement on the lateral film. In some patients with RVH, the cardiac apex appears to be lifted off the left hemidiaphragm. The RA may also be enlarged. TTE allows definitive diagnosis and characterization in most cases, with depiction of the valve and assessment of the gradient, RV function, PA pressures (which should be low), and any associated cardiac lesions. TEE may be useful in some patients for improved delineation of the RV outflow tract (RVOT) and assessment of infundibular hypertrophy. Cardiac catheterization is not usually necessary, but if performed, pressures should be obtained from just below and above the pulmonic valve with attention to the possibility that a dynamic component to the gradient may exist. The correlation

1	but if performed, pressures should be obtained from just below and above the pulmonic valve with attention to the possibility that a dynamic component to the gradient may exist. The correlation between Doppler assessment of peak instantaneous gradient and catheterization-measured peak-to-peak gradient is weak. The latter may correlate better with the Doppler mean gradient.

1	Diuretics can be used to treat symptoms and signs of right heart failure. Provided there is less than moderate pulmonic regurgitation, pulmonic balloon valvotomy is recommended for symptomatic patients with a domed valve and a peak gradient >50 mmHg (or mean gradient >30 mmHg) and for asymptomatic patients with a peak gradient >60 mmHg (or mean gradient >40 mmHg). Surgery may be required when the valve is dysplastic (as seen in patients with Noonan’s syndrome and other disorders). A multidisciplinary heart team is best positioned to make treatment decisions of this nature.

1	Pulmonic regurgitation (PR) may develop as a consequence of primary valve pathology, annular enlargement, or their combination; after surgical treatment of RVOT obstruction in children with such disorders as tetralogy of Fallot; or after pulmonic balloon valvotomy (Table 285-1). Carcinoid usually causes mixed pulmonic valve disease with PR and PS. Long-standing severe PA hypertension from any cause can result in dilation of the pulmonic valve ring and PR.

1	Severe PR results in RV chamber enlargement and eccentric hypertrophy. As is the case for aortic regurgitation (AR), PR is a state of increased preload and afterload. The reverse pressure gradient from the PA to the RV, which drives the PR, progressively decreases throughout diastole and accounts for the decrescendo nature of the diastolic murmur. As RV diastolic pressure increases, the murmur becomes shorter in duration. The forward CO is preserved during the early stages of the disease, but may not increase normally with exercise and declines over time. A reduction in RV ejection fraction may be an early indicator of hemodynamic compromise. In advanced stages, there is significant enlargement of the RV and RA with marked elevation of the jugular venous pressure.

1	Mild or moderate degrees of PR do not, by themselves, result in symptoms. Other problems, such as PA hypertension, may dominant the clinical picture. With progressively severe PR and RV dysfunction, fatigue, exertional dyspnea, abdominal fullness/bloating, and lower extremity swelling may be reported.

1	The physical examination hallmark of PR is a high-pitched, decrescendo diastolic murmur (Graham Steell murmur) heard along the left sternal border that can be difficult to distinguish from the more frequently appreciated murmur of aortic regurgitation. The Graham Steell murmur may become louder with inspiration and is usually associated with a loud and sometimes palpable P2 and an RV lift, as would be expected in patients with significant PA hypertension of any cause. Survivors of childhood surgery for tetralogy of Fallot or PS/pulmonary atresia may have an RV-PA conduit that is freely regurgitant because it does not contain a valve. PA pressures in these individuals are not elevated and the diastolic murmur can be misleadingly low pitched and of short duration despite significant degrees of PR and RV volume overload.

1	Depending on both the etiology and severity of PR, the ECG may show findings of RVH and RA enlargement. On chest x-ray, the RV and RA may be enlarged. Pulmonic valve morphology and function can be assessed with transthoracic Doppler echocardiography. PA pressures can be estimated from the tricuspid valve systolic jet velocity. CMR provides greater anatomic detail, particularly in patients with repaired congenital heart disease, and more precise assessment of RV volumes. Cardiac catheterization is not routinely necessary but would be performed as part of a planned transcatheter procedure.

1	In patients with functional PR due to PA hypertension and annular dilation, efforts to reduce PA vascular resistance and pressure should be optimized. Such efforts may include pharmacologic/ vasodilator and/or surgical/interventional strategies, depending on the cause of the PA hypertension. Diuretics can be used to treat the manifestations of right heart failure. Surgical valve replacement for primary, severe, pulmonic valve disease, such as carcinoid or endocarditis, is rarely undertaken. Transcatheter pulmonic valve replacement has been successfully performed in many patients with severe PR after childhood repair of tetralogy of Fallot or pulmonic valve stenosis or atresia. This procedure was introduced clinically prior to transcatheter aortic valve replacement. Patrick T. O’Gara, Joseph Loscalzo

1	Many acquired and congenital cardiac lesions may result in stenosis and/or regurgitation of one or more heart valves. For example, rheumatic heart disease can involve the mitral (mitral stenosis [MS], mitral regurgitation [MR], or MS and MR), aortic (aortic stenosis [AS], aortic regurgitation [AR], or AS and AR), and/or tricuspid (tricuspid stenosis [TS], tricuspid regurgitation [TR], or TS and TR) valve, alone or in combination. The common association of functional TR with significant mitral valve disease is discussed in Chap. 285. Severe mitral annular calcification can result in regurgitation (due to decreased annular shortening during systole) and mild stenosis (caused by extension of the calcification onto the leaflets resulting in restricted valve opening). Patients with severe AS may develop functional MR that may not improve after isolated aortic valve replacement (AVR). Chordal rupture has been described infrequently in patients with severe AS. Aortic valve infective

1	AS may develop functional MR that may not improve after isolated aortic valve replacement (AVR). Chordal rupture has been described infrequently in patients with severe AS. Aortic valve infective endocarditis may secondarily involve the mitral apparatus either by abscess formation and contiguous spread via the inter-valvular fibrosa or by “drop metastases” from the aortic leaflets onto the anterior leaflet of the mitral valve. Mediastinal radiation may result in aortic, mitral, and even tricuspid valve disease, most often with mixed stenosis and regurgitation. Carcinoid heart disease may cause mixed lesions of either or both the tricuspid and pulmonic valves. Ergotamines, and the previously used combination of fenfluramine and phentermine, can rarely result in mixed lesions of the aortic and/ or mitral valve. Patients with Marfan’s syndrome may have both AR from aortic root dilation and MR due to mitral valve prolapse (MVP). Myxomatous degeneration causing prolapse of multiple valves

1	and/ or mitral valve. Patients with Marfan’s syndrome may have both AR from aortic root dilation and MR due to mitral valve prolapse (MVP). Myxomatous degeneration causing prolapse of multiple valves (mitral, aortic, tricuspid) can also occur in the absence of an identifiable connective tissue disorder. Bicuspid aortic or pulmonic valve disease can result in mixed stenosis and regurgitation.

1	In patients with multivalvular heart disease, the pathophysiologic derangements associated with the more proximal valve disease can mask the full expression of the attributes of the more distal valve lesion. For example, in patients with rheumatic mitral and aortic valve disease, the reduction in cardiac output (CO) imposed by the mitral valve disease will decrease the magnitude of the hemodynamic derangements related to the severity of the aortic valve lesion (stenotic, regurgitant, or both). Alternatively, the development of atrial fibrillation (AF) during the course of MS can lead to sudden worsening in a patient whose aortic valve disease was not previously felt to be significant. The development of reactive pulmonary vascular disease, sometimes referred to as a “secondary obstructive lesion in series,” can impose an additional challenge in these settings. As CO falls with progressive tricuspid valve disease, the severity of any associated mitral or aortic disease can be

1	lesion in series,” can impose an additional challenge in these settings. As CO falls with progressive tricuspid valve disease, the severity of any associated mitral or aortic disease can be underestimated.

1	One of the most common examples of multivalve disease is that of functional TR in the setting of significant mitral valve disease. Functional TR occurs as a consequence of right ventricular and annular dilation; pulmonary artery (PA) hypertension is often present. The tricuspid leaflets are morphologically normal. Progressive degrees of TR lead to right ventricular volume overload and continued chamber and annular dilation. The TR is usually central in origin; reflux into the right atrium (RA) is expressed as large, systolic c-v waves in the RA pressure pulse. The height of the c-v wave is dependent on RA compliance and the volume of regurgitant flow. The RA wave form may become “ventricularized” in advanced stages of chronic, severe TR with PA hypertension. CO falls and the severity of the associated mitral valve disease may become more difficult to appreciate. Primary rheumatic tricuspid valve disease may occur with rheumatic mitral disease and cause hemodynamic changes reflective

1	the associated mitral valve disease may become more difficult to appreciate. Primary rheumatic tricuspid valve disease may occur with rheumatic mitral disease and cause hemodynamic changes reflective of TR, TS, or their combination. With TS, the y descent in the RA pressure pulse is prolonged.

1	Another example of rheumatic, multivalve disease involves the combination of mitral and aortic valve pathology, frequently characterized by MS and AR. In isolated MS, left ventricular (LV) preload and diastolic pressure are reduced as a function of the severity of inflow obstruction. With concomitant AR, however, LV filling is enhanced and diastolic pressure may rise depending on the compliance characteristics of the chamber. Because the CO falls with progressive degrees of MS, transaortic valve flows will decline, masking the potential severity of the aortic valve lesion (AR, AS, or its combination). As noted above, onset of AF in such patients can be especially deleterious.

1	Functional MR may complicate the course of some patients with severe AS. The mitral valve leaflets and chordae tendineae are usually normal. Incompetence is related to changes in LV geometry (remodeling) and abnormal systolic tethering of the leaflets in the context of markedly elevated LV systolic pressures. Relief of the excess afterload with surgical or transcatheter AVR often, but not always, results in reduction or elimination of the MR. Persistence of significant MR following AVR is associated with impaired functional outcomes and reduced survival. Identification of patients who would benefit from concomitant treatment of their functional MR at time of AVR is quite challenging. Most surgeons advocate for repair of moderate-to-severe 1551 or severe functional MR at time of surgical AVR.

1	In patients with mixed AS and AR, assessment of valve stenosis can be influenced by the magnitude of the regurgitant valve flow. Because transvalvular systolic flow velocities are augmented in patients with AR and preserved LV systolic function, the LV-aortic Doppler-derived pressure gradient and the intensity of the systolic murmur will be elevated to values higher than expected for the true systolic valve orifice size as delineated by planimetry. Uncorrected, the Gorlin formula, which relies on forward CO (systolic transvalvular flow) and the mean pressure gradient for calculation of valve area, is not accurate in the setting of mixed aortic valve disease. Similar considerations apply to patients with mixed mitral valve disease. The peak mitral valve Doppler E wave velocity (v0) is increased in the setting of severe MR because of enhanced early diastolic flow and may not accurately reflect the contribution to left atrial (LA) hypertension from any associated MS. When either AR or MR

1	in the setting of severe MR because of enhanced early diastolic flow and may not accurately reflect the contribution to left atrial (LA) hypertension from any associated MS. When either AR or MR is the dominant lesion in patients with mixed aortic or mitral valve disease, respectively, the LV is dilated. When AS or MS predominates, LV chamber size will be normal or small. It can sometimes be difficult to ascertain whether stenosis or regurgitation is the dominant lesion in patients with mixed valve disease, although an integrated clinical and noninvasive assessment can usually provide clarification for purposes of patient management and follow-up.

1	Patients with significant AS, a nondilated LV chamber, and concentric hypertrophy will poorly tolerate the abrupt development of aortic regurgitation, as may occur, for example, with infective endocarditis or after surgical or transcatheter AVR complicated by paravalvular leakage. The noncompliant LV is not prepared to accommodate the sudden volume load, and as a result, LV diastolic pressure rises rapidly and severe heart failure develops. Indeed, paravalvular regurgitation is a significant risk factor for shortto intermediate-term death following transcatheter AVR. Conditions in which the LV may not be able to dilate in response to chronic AR (or MR) include radiation heart disease and, in some patients, the cardiomyopathy associated with obesity and diabetes. Noncompliant ventricles of small chamber size predispose to earlier onset diastolic dysfunction and heart failure in response to any further perturbation in valve function.

1	Compared with patients with isolated, single-lesion valve disease, patients with multiple or mixed valve disease may develop symptoms at a relatively earlier stage in the natural history of their disease. Symptoms such as exertional dyspnea and fatigue are usually related to elevated filling pressures, reduced CO, or their combination. Palpitations may signify AF and identify mitral valve disease as an important component of the clinical presentation, even when not previously suspected. Chest pain compatible with angina could reflect left or right ventricular oxygen supply/demand mismatch on a substrate of hypertrophy and pressure/volume overload with or without superimposed coronary artery disease. Symptoms related to right heart failure (abdominal fullness/bloating, edema) are late manifestations of advanced disease.

1	Mixed disease of a single valve is most often manifested by systolic and diastolic murmurs, each with the attributes expected for the valve in question. Thus, patients with AS and AR will have characteristic mid-systolic, crescendo-decrescendo and blowing, decrescendo diastolic murmurs at the base of the heart in the second right interspace and along the left sternal edge, respectively. Many patients with significant AR have mid-systolic outflow murmurs even in the absence of valve sclerosis/stenosis, and other findings of AS must be sought. The separate murmurs of AS and AR can occasionally be difficult to distinguish from the continuous murmurs associated with either a patent ductus arteriosus (PDA) or ruptured sinus of Valsalva aneurysm. With mixed aortic valve disease, the systolic murmur should end before, and not envelope or extend through, the second heart sound (S2). The murmur associated with a PDA is heard best to the left of the upper sternum. The continuous murmur heard

1	murmur should end before, and not envelope or extend through, the second heart sound (S2). The murmur associated with a PDA is heard best to the left of the upper sternum. The continuous murmur heard with a ruptured sinus of Valsalva 1552 aneurysm is often first appreciated after an episode of acute chest pain. An early ejection click, which usually defines bicuspid aortic valve dis- ease in young adults, is often not present in patients with congenital, mixed AS and AR. As noted above, both the intensity and duration of these separate murmurs can be influenced by a reduction in CO and transvalvular flow due to coexistent mitral valve disease. In patients with isolated MS and MR, expected findings would include a blowing, holosystolic murmur and a mid-diastolic rumble (with or without an opening snap) best heard at the cardiac apex. An irregularly irregular heart rhythm in such patients would likely signify AF. Findings with TS and TR would mimic those of left-sided MS and MR, save

1	opening snap) best heard at the cardiac apex. An irregularly irregular heart rhythm in such patients would likely signify AF. Findings with TS and TR would mimic those of left-sided MS and MR, save for the expected changes in the murmurs with respiration. The murmurs of pulmonic stenosis and regurgitation behave in a fashion directionally similar to AS and AR; dynamic changes during respiration should be noted. Specific attributes of these cardiac murmurs are reviewed in Chap. 285.

1	The electrocardiogram (ECG) may show evidence of ventricular hypertrophy and/or atrial enlargement. ECG signs indicative of right-sided cardiac abnormalities in patients with left-sided valve lesions should prompt additional assessment for PA hypertension and/or right-sided valve disease. The presence of AF in patients with aortic valve disease may be a clue to the presence of previously unsuspected mitral valve disease in the appropriate context. The chest x-ray can be reviewed for evidence of cardiac chamber enlargement, valve and/or annular calcification, and any abnormalities in the appearance of the pulmonary vasculature. The latter could include enlargement of the main and proximal pulmonary arteries with PA hypertension and pulmonary venous redistribution/engorgement or Kerley B lines with increasing degrees of LA hypertension. An enlarged azygos vein in the frontal projection indicates RA hypertension. Roentgenographic findings not expected based on a single or mixed valve

1	lines with increasing degrees of LA hypertension. An enlarged azygos vein in the frontal projection indicates RA hypertension. Roentgenographic findings not expected based on a single or mixed valve lesion may reflect other valve disease.

1	Transthoracic echocardiography (TTE) is the most commonly used imaging modality for the diagnosis and characterization of multiple and/or mixed valvular heart disease and may often demonstrate findings not clinically suspected. Transesophageal echocardiography (TEE) may sometimes be required for more accurate assessment of valve anatomy (specifically, the mitral valve) and when infective endocarditis (IE) is considered responsible for the clinical presentation. TTE findings of particular interest include those related to valve morphology and function, calcification, chamber size, ventricular wall thickness, estimated PA systolic pressure, and the dimensions of the great vessels, including the root and ascending aorta, PA, and inferior vena cava. Exercise testing (with or without echocardiography) can be useful when the degree of functional limitation reported by the patient is not adequately explained by the findings on TTE performed at rest. An integrated assessment of the clinical

1	can be useful when the degree of functional limitation reported by the patient is not adequately explained by the findings on TTE performed at rest. An integrated assessment of the clinical and TTE findings is needed to help determine the dominant valve lesion(s) and establish an appropriate plan for treatment and follow-up. Natural history is usually influenced to a relatively greater degree by the dominant lesion. Exercise testing (with or without echocardiography) can

1	Cardiac magnetic resonance (CMR) can be used to provide additional anatomic and physiologic information when echocardiography proves suboptimal, but is less well suited to the evaluation of valve morphology. Cardiac computed tomography (CT) has been used to assess intracardiac structures in patients with complicated IE. Coronary CT angiography provides a noninvasive alternative for the assessment of coronary artery anatomy prior to surgery.

1	Invasive hemodynamic evaluation with right and left heart catheterization may be required to characterize more completely the individual contributions of each lesion in patients with either multiple or mixed valvular heart disease. Measurement of PA pressures and calculation of pulmonary vascular resistance (PVR) can help inform clinical decision-making in certain patient subsets, such as those with advanced mitral and tricuspid valve disease. Attention to the accurate assessment of CO is essential. Coronary angiography (if indicated) can be performed as part of the procedure. Contrast ventriculography and great vessel angiography are performed infrequently.

1	Management of patients with multiple or mixed valve disease can be challenging. As noted above, it is helpful to determine the dominant valve lesion and proceed according to the treatment and follow-up recommendations for it (Chaps. 283 to 285), being mindful of deviations from the expected course because of problems related to another valve disorder. For example, AF that emerges in the course of moderate mitral valve disease may precipitate heart failure in patients with concomitant, severe aortic valve disease.

1	Medical therapies are limited and include diuretics when indicated for relief of congestion and vitamin K antagonists for anticoagulation to prevent stroke and thromboembolism in patients with AF. The novel oral anticoagulants are not approved for use in the setting of significant valvular heart disease. Blood pressure–lowering medications may be needed to treat systemic hypertension, which may aggravate left-sided regurgitant valve lesions, but should be initiated and titrated carefully. Pulmonary vasodilators to lower PVR are not generally effective in this context.

1	There is a paucity of evidence to inform practice guidelines for surgical and/or transcatheter valve intervention in patients with multiple or mixed valve disease. When there is a clear, dominant lesion, as for example in a patient with severe AS and mild AR, indications for intervention are straightforward and follow those recommended for patients with AS (Chap. 283). In other patients, however, there is less clarity, and decisions regarding intervention should be based on several considerations, including those related to lesion severity, ventricular remodeling, functional capacity, and PA pressures. In this regard, it is important to realize that patients with multiple and/or mixed valve disease may develop limiting symptoms or signs of physiologic impairment even with moderate valve lesions.

1	Concomitant aortic and mitral valve replacement surgery is associated with a significantly higher perioperative mortality risk than replacement of either valve alone (see Tables 283-2 and 284-2), and operation should be carefully considered. Double valve replacement surgery is usually performed for treatment of severe (unrepairable) valve disease at both locations and for the combination of severe disease at one location with moderate disease at the other, so as to avoid the hazards of reoperation in the intermediate to late term for progressive disease of the unoperated valve. In addition, the presence of a prosthesis in the aortic position significantly restricts surgical exposure of the native mitral valve. The need for double valve replacement may also impact the decision regarding the type of prosthesis (i.e., mechanical vs tissue).

1	Tricuspid valve repair for moderate or severe functional TR at the time of left-sided valve surgery is now commonplace, particularly if there is dilation of the tricuspid annulus (>40 mm). The addition of tricuspid valve repair, consisting usually of insertion of an annuloplasty ring, adds little time or complexity to the procedure and is well tolerated. Reoperation for repair (or replacement) of progressive TR years after initial surgery for left-sided valve disease, on the other hand, is associated with a relatively high perioperative mortality risk. Repair of moderate or severe functional MR at time of AVR for AS can usually be undertaken with acceptable risk for perioperative death or major complication.

1	The presence of moderate or severe MR in patients with rheumatic MS is a contraindication to percutaneous mitral balloon valvotomy (PMBV). Likewise, the presence of significant AR in patients with AS disqualifies them from percutaneous aortic balloon valvotomy (PABV). The presence of severe, coexistent AR was an exclusion criterion for enrollment in the initial PARTNER trials of transcatheter AVR (TAVR) in prohibitiveand high-surgical-risk patients with severe, calcific AS. Transcatheter management of both severe AS (with TAVR) and functional MR (with deployment of a MitralClip) has been reported. Further advances in transcatheter treatments for multiple and mixed valve disease are anticipated.

1	Cardiomyopathy and Myocarditis Neal K. Lakdawala, Lynne Warner Stevenson, Joseph Loscalzo DEFINITION AND CLASSIFICATION Cardiomyopathy is disease of the heart muscle. It is estimated 287 that cardiomyopathy accounts for 5–10% of the heart failure in the 5–6 million patients carrying that diagnosis in the United States. This term is intended to exclude cardiac dysfunction that results from other structural heart disease, such as coronary artery disease, primary valve disease, or severe hypertension; however, in general usage, the phrase ischemic cardiomyopathy is sometimes applied to describe diffuse dysfunction attributed to multivessel coronary artery disease, and nonischemic cardiomyopathy to describe cardiomyopathy from other causes. As of 2006, cardiomyopathies are defined as “a heterogeneous group of diseases of the myocardium associated with mechanical and/or electrical dysfunction that usually (but not invariably) exhibit inappropriate ventricular hypertrophy or dilatation and

1	group of diseases of the myocardium associated with mechanical and/or electrical dysfunction that usually (but not invariably) exhibit inappropriate ventricular hypertrophy or dilatation and are due to a variety of causes that frequently are genetic.”1

1	The traditional classification of cardiomyopathies into a triad of dilated, restrictive, and hypertrophic was based initially on autopsy specimens and later on echocardiographic findings. Dilated and hypertrophic cardiomyopathies can be distinguished on the basis of left ventricular wall thickness and cavity dimension; however, restrictive cardiomyopathy can have variably increased wall thickness and chamber dimensions that range from reduced to slightly increased, with prominent atrial enlargement. Restrictive cardiomyopathy is now defined more on the basis of abnormal diastolic function, which is also present but initially less prominent in dilated and hypertrophic cardiomyopathy. Restrictive cardiomyopathy can overlap in presentation, gross morphology, and etiology with both hypertrophic and dilated cardiomyopathies (Table 287-1).

1	Expanding information renders this classification triad based on phenotype increasingly inadequate to define disease or therapy. Identification of more genetic determinants of cardiomyopathy has suggested a four-way classification scheme of etiology as primary (affecting primarily the heart) and secondary to other systemic disease. The primary causes are then divided into genetic, mixed genetic and acquired, and acquired; however, genetic information is often unavailable at the time of initial presentation, the phenotypic expression of a given mutation varies widely, and genetic predisposition influences the clinical phenotype of acquired cardiomyopathies, as well. Although the proposed genetic classification does not yet guide many current clinical strategies, it will likely become increasingly relevant as classification of disease moves beyond individual organ pathology to more integrated systems approaches.

1	For all cardiomyopathies, the early symptoms often relate to exertional intolerance with breathlessness or fatigue, usually from inadequate cardiac reserve during exercise. These symptoms may initially go unnoticed or be attributed to other causes, commonly lung disease or age-dependent exercise limitation. As fluid retention leads to elevation of resting filling pressures, shortness of breath may occur during routine daily activity such as dressing and may manifest as dyspnea or cough when lying down at night. Although often considered the hallmark of congestion, peripheral edema may be absent despite severe fluid retention, particularly in younger patients in whom ascites and abdominal discomfort may dominate. The nonspecific term congestive heart failure describes only the resulting syndrome of fluid retention, which is common to all three types of cardiomyopathy and also to cardiac structural diseases associated 1From BJ Maron et al: Circulation 113:1807, 2006.

1	with elevated filling pressures. All three types of cardiomyopathy 1553 can be associated with atrioventricular valve regurgitation, typical and atypical chest pain, atrial and ventricular tachyarrhythmias, and embolic events (Table 287-1). Initial evaluation begins with a detailed clinical history and examination, looking for clues to cardiac, extracardiac, and familial disease (Table 287-2).

1	Estimates for the prevalence of genetic etiology for cardiomyopa thy continue to rise, with increasing attention paid to the family history and the availability of genetic testing. Well-recognized in hypertrophic cardiomyopathy, heritability is also present in at least 30% of dilated cardiomyopathy without other clear etiology. Careful family history should elicit not only known cardiomyopathy and heart failure, but also family members who have had sudden death, often incorrectly attributed to “a massive heart attack,” who have had atrial fibrillation or pacemaker implantation by middle age, or who have muscular dystrophy.

1	Most familial cardiomyopathies are inherited in an autosomal dominant pattern, with occasional autosomal recessive and X-linked inheritance (Table 287-3). Missense mutations with amino acid substitutions are the most common in cardiomyopathy. Expressed mutant proteins may interfere with function of the normal allele through a dominant negative mechanism. Mutations introducing a premature stop codon (nonsense) or shift in the reading frame (frameshift) may create a truncated or unstable protein the lack of which causes cardiomyopathy (haploinsufficiency). Deletions or duplications of an entire exon or gene are uncommon causes of cardiomyopathy, except for the dystrophinopathies.

1	Many different genes have been implicated in human cardiomyopathy (locus heterogeneity), and many mutations within those genes have been associated with disease (allelic heterogeneity). Although most identified mutations are “private” to individual families, several specific mutations are found repeatedly, either due to a founder effect or recurrent mutations at a common residue.

1	Genetic cardiomyopathy is characterized by age dependence and incomplete penetrance. The defining phenotype of cardiomyopathy is rarely present at birth and, in some individuals, may never manifest. Related individuals who carry the same mutation may differ in the severity of cardiomyopathy and associated consequences of rhythm disorders and need for transplantation, indicating the important role of other genetic, epigenetic, and environmental modifiers in disease expression. Sex appears to play a role, as penetrance and clinical severity may be greater in men for most cardiomyopathies. Clinical disease expression is generally more severe in the 3–5% of individuals who harbor two or more mutations linked to cardiomyopathy. However, the clinical course of a patient usually cannot be predicted based on which mutation is present; thus, current therapy is based on the phenotype rather than the genetic defect. Currently, the greatest utility of genetic testing for cardiomyopathy is to

1	based on which mutation is present; thus, current therapy is based on the phenotype rather than the genetic defect. Currently, the greatest utility of genetic testing for cardiomyopathy is to inform family evaluations. However, genetic testing occasionally enables the detection of a disease for which specific therapy is indicated, such as the replacements for defective metabolic enzymes in Fabry’s disease and Gaucher disease.

1	Mutations in sarcomeric genes, encoding the thick and thin myofilament proteins, are the best characterized. While the majority are associated with hypertrophic cardiomyopathy, an increasing number of sarcomeric mutations have now been implicated in dilated cardiomyopathy, and some in left ventricular noncompaction. Few mutations have been identified in excitation-contraction coupling proteins, perhaps because they are too crucial for survival to allow variation. The most commonly recognized genetic causes of dilated cardiomyopathy are structural mutations of the giant protein titin, encoded TTN, which maintains sarcomere structure and acts as a key signaling molecule. As cytoskeletal proteins play crucial roles in the structure, connection, and stability of the myocyte, multiple defects in these proteins can lead to cardiomyopathy, usually with a dilated phenotype (Fig. 287-1). For example, desmin forms intermediate filaments that connect the nuclear and plasma membranes, Z-lines,

1	Normal or decreased Increased, may also be primarily affected Related to annular dilation; mitral appears earlier during decompensation; tricuspid regurgitation with right ventricular dysfunction Left before right, except right prominent in young adults Ventricular tachyarrhythmia; conduction block in Chagas’ disease, and some families. Atrial fibrillation. Normal or increased Markedly increased Increased; may be massive Increased; related to elevated filling pressures Related to endocardial involvement; Related to valve-septum frequent mitral and tricuspid interaction; mitral regurgitation regurgitation, rarely severe Exertional intolerance, fluid retention Exertional intolerance; may early, may have dominant right-sided have chest pain symptoms Right often dominates Left-sided congestion at rest may develop late Ventricular uncommon except in Ventricular tachyarrhythmias; sarcoidosis, conduction block in atrial fibrillation sarcoidosis and amyloidosis. Atrial fibrillation.

1	aLeft-sided symptoms of pulmonary congestion: dyspnea on exertion, orthopnea, paroxysmal nocturnal dyspnea. Right-sided symptoms of systemic venous congestion: hepatic and abdominal distention, discomfort on bending, peripheral edema. Thorough history and physical examination to identify cardiac and noncardiac disordersa Detailed family history of heart failure, cardiomyopathy, skeletal myopathy, conduction disorders, tachyarrhythmias, and sudden death History of alcohol, illicit drugs, chemotherapy or radiation therapya Assessment of ability to perform routine and desired activitiesa Assessment of volume status, orthostatic blood pressure, body mass indexa Chest radiographa Two-dimensional and Doppler echocardiograma Magnetic resonance imaging for evidence of myocardial inflammation and

1	Chest radiographa Two-dimensional and Doppler echocardiograma Magnetic resonance imaging for evidence of myocardial inflammation and Chemistry: Serum sodium,a potassium,a calcium, a magnesiuma Fasting glucose (glycohemoglobin in diabetes mellitus) Creatinine,a blood urea nitrogena Albumin,a total protein,a liver function testsa Lipid profile Thyroid-stimulating hormonea Serum iron, transferrin saturation Urinalysis Creatine kinase isoforms Cardiac troponin levels Hematology: Hemoglobin/hematocrita White blood cell count with differential,a including eosinophils Erythrocyte sedimentation rate Titers for infection in the setting of clinical suspicion: Acute viral (coxsackie, echovirus, influenza) Human immunodeficiency virus Chagas’ (Trypanosoma cruzi), Lyme (Borrelia burgdorferi), toxoplasmosis Catheterization with coronary angiography in patients with angina who are candidates for interventiona

1	Catheterization with coronary angiography in patients with angina who are candidates for interventiona Serologies for active rheumatologic disease Endomyocardial biopsy including sample for electron microscopy when suspecting specific diagnosis with therapeutic implications aLevel I recommendations from ACC/AHA Practice Guidelines for Chronic Heart Failure in the Adult. and the intercalated disks between muscle cells. Desmin mutations impair the transmission of force and signaling for both cardiac and skeletal muscle and may cause combined cardiac and skeletal myopathy.

1	Sarcolemmal membrane protein defects are associated with dilated cardiomyopathy. The best known is dystrophin, encoded by the X chromosome gene DMD, abnormalities of which cause Duchenne’s and Becker’s muscle dystrophy. (Interestingly, abnormal dystrophin can be acquired when the coxsackie virus cleaves dystrophin during viral myocarditis.) This protein provides a network that supports the sarcolemma and also connects to the sarcomere. The progressive functional defect in both cardiac and skeletal muscle reflects vulnerability to mechanical stress. Dystrophin is associated at the membrane with a complex of other proteins, such as metavinculin, abnormalities of which also cause dilated cardiomyopathy. Defects in the sarcolemmal channel proteins (channelopathies) are generally associated with primary arrhythmias, but mutations in SCN5A, distinct from those that cause the Brugada or long-QT syndromes, have been implicated in dilated cardiomyopathy with conduction disease.

1	Nuclear membrane protein defects in cardiac and skeletal muscle occur in either autosomal (lamin A/C) or X-linked (emerin) patterns. These defects are associated with a high prevalence of atrial arrhythmias and conduction system disease, which can occur in some family members without or before detectable cardiomyopathy. Intercalated disks contribute to intracellular connections, allowing mechanical and electrical coupling between cells and also connections to desmin filaments within the cell. Mutations in proteins of the desmosomal complex compromise attachment of the myocytes, which can become disconnected and die, to be replaced by fat and fibrous tissue. These areas are highly arrhythmogenic and may dilate to form aneurysms. Although more often noted in the right ventricle (arrhythmogenic right ventricular dysplasia), this condition can affect both ventricles and has also been termed “arrhythmogenic cardiomyopathy.”

1	Owing to the conservation of signaling pathways in multiple systems, we may expect to discover more extracardiac manifestations of genetic abnormalities initially considered to manifest exclusively in the heart. In contrast, the monogenic disorders of metabolism that affect the heart are already clearly recognized to affect multiple organ systems. Currently, it is most important to diagnose defective enzymes for which specific enzyme replacement therapy can now ameliorate the course of disease, such as with alpha-galactosidase A deficiency (Fabry’s disease). Abnormalities of mitochondrial DNA (maternally transmitted) impair energy production with multiple clinical manifestations, including impaired cognitive function and skeletal myopathy. The phenotypic expression is highly variable depending on the

1	Abbreviations: AD, autosomal dominant; AR, autosomal recessive; ARVC, arrhythmogenic right ventricular cardiomyopathy; CDDC, conduction disease with dilated cardiomyopathy; DCM, dilated cardiomyopathy; HCM, hypertrophic cardiomyopathy; HCM+, HCM with preexcitation; HCMc, HCM with conduction disease; LVNC, left ventricular noncompaction; MELAS, (mitochondrial) myopathy, encephalopathy, lactic acidosis, and strokelike episodes syndrome; MERRF, myoclonic epilepsy with ragged red fibers; RCM, restrictive cardiomyopathy. distribution of the maternal mitochondria during embryonic development. Heritable systemic diseases, such as familial amyloidosis and hemochromatosis, can affect the heart without mutation of genes expressed in the heart.

1	For any patient with suspected or proven genetic disease, family members should be considered and evaluated in a longitudinal fashion. Screening includes an echocardiogram and electrocardiogram (ECG). The indications and implications for confirmatory specific genetic testing vary depending on the specific mutation. The profound questions raised by families about diseases shared and passed down merit serious and sensitive discussion, ideally provided by a trained genetic counselor.

1	An enlarged left ventricle with decreased systolic function as measured by left ventricular ejection fraction characterizes dilated cardiomyopathy (Figs. 287-2, 287-3, and 287-4). Systolic failure is more marked than diastolic dysfunction. Although the syndrome of dilated cardiomyopathy has multiple etiologies (Table 287-4), there appear to be common pathways of secondary response and disease progression. When myocardial injury is acquired, some myocytes may die initially, whereas others survive only to have later programmed cell death (apoptosis), and remaining myocytes hypertrophy in response to increased wall stress. Local and circulating factors stimulate deleterious secondary responses that contribute to progression of disease. Dynamic remodeling of the interstitial scaffolding affects diastolic function and the amount of ventricular dilation. Mitral regurgitation commonly develops as the valvular apparatus is distorted and is usually substantial by the time heart failure is

1	diastolic function and the amount of ventricular dilation. Mitral regurgitation commonly develops as the valvular apparatus is distorted and is usually substantial by the time heart failure is severe. Many cases that present “acutely” have progressed silently through these stages over months to years. Dilation and decreased function of the right ventricle may result from the initial injury and occasionally dominate, but more commonly appear later in relation to mechanical interactions with the failing left ventricle and the elevated afterload presented by secondary pulmonary hypertension.

1	FIGURE 287-1 Drawing of myocyte indicating multiple sites of abnormal gene products associated with cardiomyopathy. Major func-tional groups include the sarcomeric proteins (actin, myosin, tropomyosin, and the associated regulatory proteins), the dystrophin complex stabilizing and connecting the cell membrane to intracellular structures, the desmosome complexes associated with cell-cell connections and stability, and multiple cytoskeletal proteins that integrate and stabilize the myocyte. ATP, adenosine triphosphate. (Figure adapted from Jeffrey A. Towbin, MD, University of Cincinnati, with permission.)

1	Regardless of the nature and degree of direct cell injury, the resulting functional impairment often includes some contribution from secondary responses that may be modifiable or reversible. Almost half of all patients with new-onset cardiomyopathy demonstrate substantial spontaneous recovery. Even with long-standing disease, some patients have dramatic improvement to near-normal ejection fractions during pharmacologic therapy, particularly notable with the β-adrenergic antagonists coupled with renin-angiotensin system inhibition. For patients in whom left bundle branch block precedes clinical heart failure by many years, cardiac resynchronization pacing may be particularly likely to improve ejection fraction and decrease ventricular size. Interest in the potential for recovery of cardiomyopathy has been

1	FIGURE 287-2 Dilated cardiomyopathy. This gross specimen of a heart removed at the time of transplantation shows massive left ventricular dilation and moderate right ventricular dilation. Although the left ventricular wall in particular appears thinned, there is significant hypertrophy of this heart, which weighs more than 800 g (upper limit of normal = 360 g). A defibrillator lead is seen traversing the tricuspid valve into the right ventricular apex. (Image courtesy of Robert Padera, MD, PhD, Department of Pathology, Brigham and Women’s Hospital, Boston.) further stimulated by occasional “recovery” of left ventricular function after prolonged mechanical circulatory support. The diagnosis and therapy for dilated cardiomyopathy are generally dictated by the stage of heart failure (Chap. 279), with specific aspects discussed for relevant etiologies below.

1	Myocarditis (inflammation of the heart) can result from multiple causes but is most commonly attributed to infective agents that can injure the myocardium through direct invasion, production of cardiotoxic substances, or chronic inflammation with or without persistent infection. Myocarditis cannot be assumed from a presentation of decreased systolic function in the setting of an acute infection, as any severe infection causing systemic cytokine release can depress cardiac function transiently. Infectious myocarditis has been reported with almost all types of infective agents but is most commonly associated with viruses and the protozoan Trypanosoma cruzi.

1	The pathogenesis of viral myocarditis has been extensively studied in murine models. After viruses gain entry through the respiratory or gastrointestinal tract, they can infect organs possessing specific receptors, such as the coxsackie-adenovirus receptor on the heart. Viral infection and replication can cause myocardial injury and lysis. For example, the enteroviral protease 2A facilitates viral replication and infection through degradation of the myocyte protein dystrophin, which is crucial for myocyte stability. Activation of viral receptor proteins can also activate host tyrosine kinases, which modify the cytoskeleton to facilitate further viral entry.

1	The first host response to infection is the nonspecific innate immune response, heavily dependent on Toll-like receptors that recognize common antigenic patterns. Cytokine release is rapid, followed by triggered activation and expansion of specific Tand B-cell populations. This initial response appears to be crucial, as early immunosuppression

1	FIGURE 287-3 Dilated cardiomyopathy. This echocardiogram of a young man with dilated cardiomyopathy shows massive global dilation and thinning of the walls of the left ventricle (LV). The left atrium (LA) is also enlarged compared to normal. Note that the echocardiographic and pathologic images are vertically opposite, such that the LV is by convention on the top right in the echocardiographic image and bottom right in the pathologic images. RA, right atrium; RV, right ventricle. (Image courtesy of Justina Wu, MD, Brigham and Women’s Hospital, Boston.) in animal models can increase viral replication and worsen cardiac injury. However, successful recovery from viral infection depends not only on the efficacy of the immune response to limit viral infection, but also on timely downregulation to prevent overreaction and autoimmune injury to the host.

1	FIGURE 287-4 Dilated cardiomyopathy. Microscopic specimen of a dilated cardiomyopathy showing the nonspecific changes of interstitial fibrosis and myocyte hypertrophy characterized by increased myocyte size and enlarged, irregular nuclei. Hematoxylin and eosin– stained section, 100× original magnification. (Image courtesy of Robert Padera, MD, PhD, Department of Pathology, Brigham and Women’s Hospital, Boston.) The secondary acquired immune response is more specifically addressed against the viral proteins and can include both T-cell infiltration and antibodies to viral proteins. If unchecked, the acquired immune response can perpetuate secondary cardiac damage. Ongoing Viral (coxsackie,a adenovirus,a HIV, hepatitis C) Parasitic (T. cruzi—Chagas’ disease, trypanosomiasis, toxoplasmosis) Bacterial (diphtheria) Spirochetal (Borrelia burgdorferi—Lyme disease) Rickettsial (Q fever) Fungal (with systemic infection)

1	Eosinophilic myocarditis Polymyositis, dermatomyositis Collagen vascular disease Peripartum cardiomyopathy Transplant rejection Alcohol Catecholamines: amphetamines, cocaine Chemotherapeutic agents (anthracyclines, trastuzumab) Interferon Other therapeutic agents (hydroxychloroquine, chloroquine) Drugs of misuse (emetine, anabolic steroids) Heavy metals: lead, mercury Occupational exposure: hydrocarbons, arsenicals Nutritional deficiencies: thiamine, selenium, carnitine Electrolyte deficiencies: calcium, phosphate, magnesium Endocrinopathy Skeletal and cardiac myopathy Dystrophin-related dystrophy (Duchenne’s, Becker’s) Mitochondrial myopathies (e.g., Kearns-Sayre syndrome) Arrhythmogenic ventricular dysplasia Hemochromatosis Associated with other systemic diseases Susceptibility to immune-mediated myocarditis Overlap with Nondilated Cardiomyopathy Miscellaneous (Shared Elements of Above Etiologies)

1	Overlap with Nondilated Cardiomyopathy Miscellaneous (Shared Elements of Above Etiologies) Supraventricular arrhythmias with uncontrolled rate Very frequent nonsustained ventricular tachycardia or high premature ventricular complex burden Left bundle branch block (LBBB) has been implicated as a cause of dilated cardiomyopathy appearing late after idiopathic LBBB and responding with near-normal left ventricle size and function after cardiac resynchronization therapy. aSome specific cases can be linked now to specific genetic mutation in a familial cardiomyopathy; others with similar phenotypes that appear to be acquired or idiopathic may represent genetic factors not yet identified.

1	cytokine release activates matrix metalloproteinases that can disrupt the collagen and elastin scaffolding of the heart, potentiating ventricular dilation. Stimulation of profibrotic factors leads to pathologic interstitial fibrosis. Some of the antibodies triggered through co-stimulation or molecular mimicry also recognize targets within the host myocyte, such as the β-adrenergic receptor, troponin, and Na+/K+ ATPase, but it remains unclear whether these antibodies contribute actively to cardiac dysfunction in humans or merely serve as markers of cardiac injury.

1	It is not known how long the viruses persist in the human heart, whether late persistence of the viral genome continues to be deleterious, or how often a dormant virus can again become pathogenic. Genomes of common viruses have frequently been detected in patients with clinical diagnoses of myocarditis or dilated cardiomyopathy, but there is little information on how often these are present in patients without cardiac disease (see below). Further information is needed to understand the relative timing and contribution of infection, immune responses, and secondary adaptations in the progression of heart failure after viral myocarditis (Fig 287-5).

1	Clinical Presentation of Viral Myocarditis Acute viral myocarditis often presents with symptoms and signs of heart failure. Some patients present with chest pain suggestive of pericarditis or acute myocardial infarction. Occasionally, the presentation is dominated by atrial or ventricular tachyarrhythmias, or by pulmonary or systemic emboli from intracardiac thrombi. Electrocardiographic or echocardiographic abnormalities may also be detected incidentally during evaluation for other diagnoses. The typical patient with presumed viral myocarditis is a young to middle-aged adult who develops progressive dyspnea and weakness within a few days to weeks after a viral syndrome that was accompanied by fever and myalgias.

1	A small number of patients present with fulminant myocarditis, with rapid progression from a severe febrile respiratory syndrome to cardiogenic shock that may involve multiple organ systems, leading to renal failure, hepatic failure, and coagulopathy. These patients are typically young adults who have recently been dismissed from urgent care settings with antibiotics for bronchitis or oseltamivir for viral syndromes, only to return within a few days in rapidly progressive cardiogenic shock. Prompt triage is vital to provide aggressive support with high-dose intravenous catecholamine therapy and sometimes with temporary mechanical circulatory support. Recognition of patients with this fulminant presentation is potentially life-saving as more than half can survive, with marked improvement demonstrable within the first few weeks. The ejection fraction function of these patients often recovers to near-normal, although residual diastolic dysfunction may limit vigorous exercise for some

1	demonstrable within the first few weeks. The ejection fraction function of these patients often recovers to near-normal, although residual diastolic dysfunction may limit vigorous exercise for some survivors.

1	Chronic viral myocarditis is often invoked, but rarely proven, as a diagnosis when no other cause of dilated cardiomyopathy can be identified. However, some cases of otherwise unexplained cardiomyopathy will later be recognized to have a genetic basis, or ultimately found to have resulted from excess alcohol consumption or illicit drugs. There are likely many other causes that cannot yet be identified. The prevalence of previous or persistent viral infection as the cause for chronic dilated cardiomyopathy remains highly controversial. Laboratory evaluation for myocarditis The initial evaluation for suspected myocarditis includes an ECG, an echocardiogram, and serum levels of troponin and creatine phosphokinase fractions. Magnetic resonance imaging is increasingly used for the diagnosis of myocarditis, which is supported by evidence of increased tissue edema and gadolinium enhancement (Fig. 287-6), particularly in the mid-wall (as distinct from usual coronary artery territories).

1	Endomyocardial biopsy is not often indicated for the initial evaluation of suspected viral myocarditis unless ventricular tachyarrhythmias suggest possible etiologies of sarcoidosis or giant cell myocarditis. The indications and benefit of endomyocardial biopsy for evaluation of myocarditis or new-onset cardiomyopathy remain controversial. FIGURE 287-5 Schematic diagram demonstrating the possible progression from infection through direct, secondary, and autoimmune responses to dilated cardiomyopathy. Most of the supporting evidence for this sequence is derived from animal models. It is not known to what degree persistent infection and/or ongoing immune responses contribute to ongoing myocardial injury in the chronic phase.

1	The Dallas Criteria for myocarditis on endomyocardial biopsy include lymphocytic infiltrate with evidence of myocyte necrosis (Fig. 287-7) and are negative in 80–90% of patients with clinical myocarditis. Negative Dallas Criteria can reflect sampling error or early resolution of lymphocytic infiltrates, but also the insensitivity of the test when inflammation results from cytokines and antibody-mediated injury. Routine histologic examination of endomyocardial biopsy rarely reveals a specific infective etiology, such as toxoplasmosis or Cytomegalovirus. Immunohistochemistry of myocardial biopsy samples is commonly used to identify active lymphocyte subtypes and may also detect upregulation of HLA antigens and the presence of complement components attributed to inflammation, but the specificity and significance of these findings are uncertain.

1	An increase in circulating viral titers between acute and convalescent blood samples supports a diagnosis of acute viral myocarditis with potential spontaneous improvement. There is no established role for measuring circulating anti-heart antibodies, which may be the result, rather than a cause, of myocardial injury and have been found also in patients with coronary artery disease and genetic cardiomyopathy. FIGURE 287-6 Magnetic resonance image of myocarditis showing the typical mid-wall location (arrow) for late gadolinium enhance-ment from cardiac inflammation and scarring. (Image courtesy of Ron Blankstein, MD, and Marcelo Di Carli, MD, Division of Nuclear Medicine, Brigham and Women’s Hospital, Boston.) Patients with recent or ongoing viral syndromes can be classified into three levels of diagnosis: 1. Possible subclinical acute myocarditis is diagnosed when a patient has a typical viral syndrome but no cardiac symptoms, with one or more of the following:

1	Elevated biomarkers of cardiac injury (troponin or CK-MB) ECG findings suggestive of acute injury Abnormality on cardiac imaging, usually echocardiography FIGURE 287-7 Acute myocarditis. Microscopic image of an endomyocardial biopsy showing massive infiltration with mononuclear cells and occasional eosinophils associated with clear myocyte damage. The myocyte nuclei are enlarged and reactive. Such extensive involvement of the myocardium would lead to extensive replacement fibrosis even if the inflammatory response could be suppressed. Hematoxylin and eosin–stained section, 200× original magnification.

1	(Image courtesy of Robert Padera, MD, PhD, Department of Pathology, Brigham and Women’s Hospital, Boston.) 1560 2. Probable acute myocarditis is diagnosed when the above criteria are met and accompanied also by cardiac symptoms, such as shortness of breath or chest pain, which can result from pericarditis or myocarditis. When clinical findings of pericarditis (pleuritic chest pain, ECG abnormalities, pericardial rub or effusion) are accompanied by elevated troponin or CK-MB or abnormal cardiac wall motion, the terms perimyocarditis or myopericarditis are sometimes used. 3. Definite myocarditis is diagnosed when there is histologic or immunohistologic evidence of inflammation on endomyocardial biopsy (see below) and does not require any other laboratory or clinical criteria.

1	In humans, viruses are often suspected but rarely proven to be the direct cause of clinical myocarditis. First implicated was the picornavirus family of RNA viruses, principally the enteroviruses, coxsackie virus, echovirus, and poliovirus. Influenza, another RNA virus, is implicated with varying frequency every winter and spring as epitopes change. Of the DNA viruses, adenovirus, vaccinia (smallpox vaccine), and the herpesviruses (varicella zoster, cytomegalovirus, Epstein-Barr virus, and human herpesvirus 6 [HHV6]) are well-recognized to cause myocarditis but also occur commonly in the healthy population. Polymerase chain reaction (PCR) detects viral genomes in the majority of patients with dilated cardiomyopathy, but also in normal “control” hearts. Most often detected are parvovirus B19 and HHV6, which may affect the cardiovascular system, in part, through infection of vascular endothelial cells. However, their contribution to chronic cardiomyopathy is uncertain, as serologic

1	B19 and HHV6, which may affect the cardiovascular system, in part, through infection of vascular endothelial cells. However, their contribution to chronic cardiomyopathy is uncertain, as serologic evidence of exposure is present in many children and most adults.

1	Human immunodeficiency virus (HIV) was associated with an incidence of dilated cardiomyopathy of 1–2%; however, with the advent of highly active antiretroviral therapy (HAART), HIV has been associated with a significantly lower incidence of cardiac disease. Cardiomyopathy in HIV may result from cardiac involvement with other associated viruses, such as cytomegalovirus and hepatitis C, as well as by HIV directly. Antiviral drugs to treat chronic HIV can cause cardiomyopathy, both directly and through drug hypersensitivity. The clinical picture may be complicated by pericardial effusions and pulmonary hypertension. There is a high frequency of lymphocytic myocarditis found at autopsy, and viral particles have been demonstrated in the myocardium in some cases, consistent with direct causation.

1	Hepatitis C has been repeatedly implicated in cardiomyopathy, particularly in Germany and Asia. Cardiac dysfunction may improve after interferon therapy. As this cytokine itself often depresses cardiac function transiently, careful coordination of administration and ongoing clinical evaluation are critical. Involvement of the heart with hepatitis B is uncommon, but can be seen when associated with systemic vasculitis (polyarteritis nodosa). Additional viruses implicated specifically in myocarditis include mumps, respiratory syncytial virus, the arboviruses (dengue fever and yellow fever), and arenaviruses (Lassa fever). However, for any serious infection, the systemic inflammatory response can cause nonspecific depression of cardiac function, which is generally reversible if the patient survives.

1	There is currently no specific therapy recommended during any stage of viral myocarditis. During acute infection, therapy with anti-inflammatory or immunosuppressive medications is avoided, as their use has been shown to increase viral replication and myocardial injury in animal models. Therapy with specific antiviral agents (such as oseltamivir) has not been studied in relation to cardiac involvement. There is ongoing investigation into the impact of antiviral therapy to treat chronic viral persistence identified from endomyocardial biopsy. Large trials of immunosuppressive therapy for Dallas Criteria–positive myocarditis have been negative. There are some initial encouraging results and ongoing investigations with immunosuppressive therapy for immune-mediated myocarditis defined by immunohistologic criteria on biopsy or circulating anti-heart antibodies in the absence of myocardial viral genomes. However, neither antiviral nor anti-inflammatory therapies are currently recommended.

1	criteria on biopsy or circulating anti-heart antibodies in the absence of myocardial viral genomes. However, neither antiviral nor anti-inflammatory therapies are currently recommended. Until we have a better understanding of the different phases of viral myocarditis and its sequelae and the effects of timed or targeted therapies, treatment will continue to be directed to the clinical cardiovascular stage of the disease, for dilated cardiomyopathy in general.

1	Parasitic Myocarditis Chagas’ disease is the third most common parasitic infection in the world and the most common infective cause of cardiomyopathy. The protozoan T. cruzi is transmitted by the bite of the reduviid bug, endemic in the rural areas of South and Central America. Transmission can also occur through blood transfusion, organ donation, from mother to fetus, and occasionally orally. While programs to eradicate the insect vector have decreased the prevalence from about 16 million to less than 10 million in South America, cases are increasingly recognized in Western developed countries. Approximately 100,000 affected individuals are currently living in the United States, most of whom contracted the disease in endemic areas.

1	Multiple pathogenic mechanisms are implicated. The parasite itself can cause myocyte lysis and primary neuronal damage, and specific immune responses may recognize the parasites or related antigens and lead to chronic immune activation in the absence of detectable parasites. Molecular techniques have revealed persistent parasite DNA fragments in infected individuals. Further evidence for persistent infection is the eruption of parasitic skin lesions during immunosuppression after cardiac transplantation. As with viral myocarditis, the relative roles of persistent infection and of secondary autoimmune injury have not been resolved (Fig. 287-5). An additional factor in the progression of Chagas’ disease is the autonomic dysfunction and microvascular damage that may contribute to cardiac and gastrointestinal disease.

1	The acute phase of Chagas’ disease with parasitemia is usually unrecognized, but in fewer than 5% of cases, it presents clinically within a few weeks of infection, with nonspecific symptoms or occasionally with acute myocarditis and meningoencephalitis. In the absence of antiparasitic therapy, the silent stage progresses slowly over 10–30 years in almost half of patients to manifest in the cardiac and gastrointestinal systems in the chronic stages. Features typical of Chagas’ disease are conduction system abnormalities, particularly sinus node and atrioventricular (AV) node dysfunction and right bundle branch block. Atrial fibrillation and ventricular tachyarrhythmias also occur. Small ventricular aneurysms are common, particularly at the ventricular apex. These dilated ventricles are particularly thrombogenic, giving rise to pulmonary and systemic emboli. Xenodiagnosis, detection of the parasite itself, is rarely performed. The serologic tests for specific IgG antibodies against the

1	thrombogenic, giving rise to pulmonary and systemic emboli. Xenodiagnosis, detection of the parasite itself, is rarely performed. The serologic tests for specific IgG antibodies against the trypanosome lack sufficient specificity and sensitivity, thereby requiring two separate positive tests required to make a diagnosis.

1	Treatment of the advanced stages focuses on clinical manifestations of the disease and includes heart failure medications, pacemakerdefibrillators, and anticoagulation. Increasing attention is directed to antiparasitic therapy even in chronic disease without obvious active infection. The most common effective antiparasitic therapies are benznidazole and nifurtimox, both associated with multiple severe reactions, including dermatitis, gastrointestinal distress, and neuropathy. Survival is less than 30% at 5 years after the onset of overt clinical heart failure. Patients without major extracardiac disease have occasionally undergone transplantation, after which they may require lifelong therapy to suppress reactivation of infection.

1	African trypanosomiasis infection results from the tsetse fly bite and can occur in travelers exposed during trips to Africa. The West African form is caused by Trypanosoma brucei gambiense and progresses silently over years. The East African form caused by T. brucei rhodesiense can progress rapidly through perivascular infiltration to myocarditis and heart failure, with frequent arrhythmias. The diagnosis is made by identification of trypanosomes in blood, lymph nodes, or other affected sites. Antiparasitic therapy has limited efficacy and is determined by the specific type and the stage of infection (hemolymphatic or neurologic).

1	Toxoplasmosis is contracted through undercooked infected beef or pork, transmission from feline feces, organ transplantation, transfusion, or maternal-fetal transmission. Immunocompromised hosts are most likely to experience reactivation of latent infection from cysts. The cysts have been found in up to 40% of autopsies of patients dying from HIV infection. Toxoplasmosis may present with encephalitis or chorioretinitis and, in the heart, can cause myocarditis, pericardial effusion, constrictive pericarditis, and heart failure. The diagnosis in an immunocompetent patient is made when the IgM is positive and the IgG becomes positive later. Active toxoplasmosis may be suspected in an immunocompromised patient with myocarditis and a positive IgG titer for toxoplasmosis, particularly when avidity testing identifies high specificity of the antibody. Fortuitous sampling occasionally reveals the cysts in the myocardium. Combination therapy can include pyrimethamine and sulfadiazine or

1	avidity testing identifies high specificity of the antibody. Fortuitous sampling occasionally reveals the cysts in the myocardium. Combination therapy can include pyrimethamine and sulfadiazine or clindamycin.

1	Trichinellosis is caused by Trichinella spiralis larva ingested with undercooked meat. Larvae migrating into skeletal muscles cause myalgias, weakness, and fever. Periorbital and facial edema and conjunctival and retinal hemorrhage may also be seen. Although the larva may occasionally invade the myocardium, clinical heart failure is rare and, when observed, attributed to the eosinophilic inflammatory response. The diagnosis is made from the specific serum antibody and is further supported by the presence of eosinophilia. Treatment includes antihelminthic drugs (albendazole, mebendazole) and glucocorticoids if inflammation is severe. Cardiac involvement with Echinococcus is rare, but cysts can form and rupture in the myocardium and pericardium.

1	Bacterial Infections Most bacterial infections can involve the heart occasionally through direct invasion and abscess formation, but do so rarely. More commonly, systemic inflammatory responses depress contractility in severe infection and sepsis. Diphtheria specifically affects the heart in almost one-half of cases, and cardiac involvement is the most common cause of death in patients with this infection. The prevalence of vaccines has shifted the incidence of diphtheria from children worldwide to countries without routine immunization and to older populations who have lost their immunity. The bacillus releases a toxin that impairs protein synthesis and may particularly affect the conduction system. The specific antitoxin should be administered as soon as possible, with higher priority than antibiotic therapy. Other systemic bacterial infections that can involve the heart include brucellosis, chlamydophila, legionella, meningococcus, mycoplasma, psittacosis, and salmonellosis, for

1	than antibiotic therapy. Other systemic bacterial infections that can involve the heart include brucellosis, chlamydophila, legionella, meningococcus, mycoplasma, psittacosis, and salmonellosis, for which specific treatment is directed at the systemic infection.

1	Clostridial infections cause myocardial damage from the released toxin. Gas bubbles can be detected in the myocardium, and occasionally abscesses can form in the myocardium and pericardium. Streptococcal infection with β-hemolytic streptococci is most commonly associated with acute rheumatic fever and is characterized by inflammation and fibrosis of cardiac valves and systemic connective tissue, but it can also lead to a myocarditis with focal or diffuse infiltrates of mononuclear cells. Tuberculosis can involve the myocardium directly as well as through tuberculous pericarditis, but rarely does so when the disease is treated with antibiotics. Whipple’s disease is caused by Tropheryma whipplei. The usual manifestations are in the gastrointestinal tract, but pericarditis, coronary arteritis, valvular lesions, and occasionally clinical heart failure may also occur. Multidrug antituberculous regimens are effective, but the disease tends to relapse even with appropriate treatment.

1	Other Infections Spirochetal myocarditis has been diagnosed from myocardial biopsies containing Borrelia burgdorferi that causes Lyme disease. Lyme carditis most often presents with arthritis and conduction system disease that resolves within 1–2 weeks of antibiotic treatment, only rarely implicated in chronic heart failure. Fungal myocarditis can occur due to hematogenous or direct spread of infection from other sites, as has been described for aspergillosis, actinomycosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, and mucormycosis. However, cardiac involvement is rarely the dominant clinical feature of these infections. The rickettsial infections, Q fever, Rocky Mountain spotted fever, 1561 and scrub typhus are frequently accompanied by ECG changes, but most clinical manifestations relate to systemic vascular involvement.

1	Myocardial inflammation can occur without apparent preceding infection. The paradigm of noninfective inflammatory myocarditis is cardiac transplant rejection, from which we have learned that myocardial depression can develop and reverse quickly, that noncellular mediators such as antibodies and cytokines play a major role in addition to lymphocytes, and that myocardial antigens are exposed by prior physical injury and viral infection.

1	The most commonly diagnosed noninfective inflammation is granulomatous myocarditis, including both sarcoidosis and giant cell myocarditis. Sarcoidosis, as discussed in Chap. 390, is a multisystem disease most commonly affecting the lungs. Although classically presenting with higher prevalence in young African-American men, the epidemiology appears to be changing, with increasing recogni tion of sarcoidosis in Caucasian patients in nonurban areas. Patients with pulmonary sarcoid are at high risk for cardiac involvement, but cardiac sarcoidosis also occurs without clinical lung disease. Regional clustering of the disease supports the suspicion that the granulomatous reaction is triggered by an infectious or environmental allergen not yet identified.

1	The sites and density of cardiac granulomata, the time course, and the degree of extracardiac involvement are remarkably variable. Patients may present with rapid-onset heart failure and ventricular tachyarrhythmias, conduction block, chest pain syndromes, or minor cardiac findings in the setting of ocular involvement, an infiltrative skin rash, or a nonspecific febrile illness. They may also present less acutely after months to years of fluctuating cardiac symptoms. When ventricular tachycardia or conduction block dominates the initial presentation of heart failure without coronary artery disease, suspicion should be high for these granulomatous myocarditides.

1	Depending on the time course, the ventricles may appear restrictive or dilated. There is often right ventricular predominance of both dilation and ventricular arrhythmias, sometimes initially attributed to arrhythmogenic right ventricular dysplasia. Small ventricular aneurysms are common. Computed tomography of the chest often reveals pulmonary lymphadenopathy even in the absence of clinical lung disease. Metabolic imaging (positron emission tomography (PET]) of the whole chest can highlight active sarcoid lesions that are avid for glucose. Magnetic resonance imaging (MRI) of the heart can identify areas likely to be inflammatory. To rule out chronic infections, such as tuberculosis or histoplasmosis as the cause of adenopathy, the diagnosis usually requires pathologic confirmation. Biopsy of enlarged mediastinal nodes may provide the highest yield. The scattered granulomata of sarcoidosis can easily be missed on cardiac biopsy (Fig. 287-8).

1	Immunosuppressive treatment for sarcoidosis is initiated with high-dose glucocorticoids, which are often more effective for arrhythmias than for the heart failure. Patients with sarcoid lesions that persist or recur during tapering of corticosteroids are considered candidates for other immunosuppressive therapies, frequently with agents also used for cardiac transplantation. Pacemakers and implantable defibrillators are generally indicated to prevent life-threatening heart block or ventricular tachycardia, respectively. Because the inflammation often resolves into extensive fibrosis that impairs cardiac function and provides pathways for reentrant arrhythmias, the prognosis for improvement is best when the granulomata are not extensive and the ejection fraction is not severely reduced.

1	Giant cell myocarditis is less common than sarcoidosis, but accounts for 10–20% of biopsy-positive cases of myocarditis. Giant cell myocarditis typically presents with rapidly progressive heart failure and tachyarrhythmias. Diffuse granulomatous lesions are surrounded by extensive inflammatory infiltrate unlikely to be missed on endomyocardial biopsy, often with extensive eosinophilic infiltration. Associated conditions are thymomas, thyroiditis, pernicious anemia, other autoimmune diseases, and occasionally recent infections. Glucocorticoid therapy is less effective than for sarcoidosis and is

1	FIGURE 287-8 Sarcoidosis. Microscopic image of an endomyocardial biopsy showing a noncaseating granuloma and associated interstitial fibrosis typical of sarcoidosis. No microorganisms were present on special stains, and no foreign material was identified. Hematoxylin and eosin–stained section, 200× original magnification. (Image courtesy of Robert Padera, MD, PhD, Department of Pathology, Brigham and Women’s Hospital, Boston.) sometimes combined with other immunosuppressive agents. The course is generally of rapid deterioration requiring urgent transplantation. Although the severity of presentation and myocardial histology are more fulminant than with sarcoidosis, the occasional finding of giant cell myocarditis after sarcoidosis suggests that they may in some cases represent different stages of the same disease spectrum.

1	Eosinophilic myocarditis can be an important manifestation of the hypereosinophilic syndrome, which in Western countries is often idiopathic, although in Mediterranean and African countries, it is likely a consequence of antecedent infection. It may also be seen with systemic eosinophilic syndromes such as Churg-Strauss syndrome or malignancies. Hypersensitivity myocarditis is often an unexpected diagnosis, made when the biopsy reveals infiltration with lymphocytes and mononuclear cells with a high proportion of eosinophils. Most commonly, the reaction is attributed to antibiotics, particularly those taken chronically, but thiazides, anticonvulsants, indomethacin, and methyldopa have also been implicated. Occasional associations with the smallpox vaccine have been reported. Although the circulating eosinophil count may be slightly elevated in hypersensitivity myocarditis, it does not reach the high levels of the hypereosinophilic syndrome. High-dose glucocorticoids and discontinuation

1	eosinophil count may be slightly elevated in hypersensitivity myocarditis, it does not reach the high levels of the hypereosinophilic syndrome. High-dose glucocorticoids and discontinuation of the trigger agent can be curative for hypersensitivity myocarditis.

1	Myocarditis is often associated with systemic inflammatory diseases, such as polymyositis and dermatomyositis, which affect skeletal and cardiac muscle. Although noninfective inflammatory myocarditis is sometimes included in the differential diagnosis of cardiac findings in patients with connective tissue disease such as systemic lupus erythematosus, pericarditis, vasculitis, pulmonary hypertension, and accelerated coronary artery disease are more common cardiac manifestations of connective tissue disease.

1	Peripartum cardiomyopathy (PPCM) develops during the last trimester or within the first 6 months after pregnancy, with a frequency between 1:2000 and 1:15,000 deliveries. Risk factors are increased maternal age, increased parity, twin pregnancy, malnutrition, use of tocolytic therapy for premature labor, and preeclampsia or toxemia of pregnancy. Heart failure early after delivery was previously common in Nigeria, when the custom for new mothers included salt ingestion while reclining on a warm bed, which likely impaired mobilization of the excess circulating volume after delivery. In the Western world, lymphocytic myocarditis has sometimes been found on myocardial biopsy. This inflammation has been hypothesized to reflect increased susceptibility to viral myocarditis or an autoimmune myocarditis due to cross-reactivity of anti-uterine antibodies against cardiac muscle. Another proposed mechanism invokes an abnormal prolactin cleavage fragment, which is induced by oxidative stress and

1	due to cross-reactivity of anti-uterine antibodies against cardiac muscle. Another proposed mechanism invokes an abnormal prolactin cleavage fragment, which is induced by oxidative stress and may trigger myocardial apoptosis; this observation has led to preliminary investigation of bromocriptine as possible therapy.

1	Very recently, PPCM has been found to be associated with increased antiangiogenic signaling, a process that is exacerbated by preeclampsia. In animal models of this disease, proangiogenic therapies have proven curative. As the increased circulatory demand of pregnancy can aggravate other cardiac disease that was clinically unrecognized, it is crucial to the diagnosis of PPCM that there be no evidence for a preexisting cardiac disorder. By contrast, heart failure presenting earlier in pregnancy has been termed pregnancy-associated cardiomyopathy (PACM). Both PPCM and PACM have been found in some families with other presentations of dilated cardiomyopathy, in some cases with known sarcomeric protein mutations. Pregnancy may, thus, represent an environmental trigger for accelerated phenotypic expression of genetic cardiomyopathy.

1	Cardiotoxicity has been reported with multiple environmental and pharmacologic agents. Often these associations are seen only with very high levels of exposure or acute overdoses, in which acute electrocardiographic and hemodynamic abnormalities may reflect both direct drug effect and systemic toxicity.

1	Alcohol is the most common toxin implicated in chronic dilated cardiomyopathy. Excess consumption may contribute to more than 10% of cases of heart failure, including exacerbation of cases with other primary etiologies such as valvular disease or previous infarction. Toxicity is attributed both to alcohol and to its primary metabolite, acetaldehyde. Polymorphisms of the genes encoding alcohol dehydrogenase and the angiotensin-converting enzyme increase the likelihood of alcoholic cardiomyopathy in an individual with excess consumption. Superimposed vitamin deficiencies and toxic alcohol additives are rarely implicated. The alcohol consumption necessary to produce cardiomyopathy in an otherwise normal heart has been estimated to be five to six drinks (about 4 ounces of pure ethanol) daily for 5–10 years, but frequent binge drinking may also be sufficient. Many patients with alcoholic cardiomyopathy are fully functional in their daily lives without apparent stigmata of alcoholism. The

1	for 5–10 years, but frequent binge drinking may also be sufficient. Many patients with alcoholic cardiomyopathy are fully functional in their daily lives without apparent stigmata of alcoholism. The cardiac impairment in severe alcoholic cardiomyopathy is the sum of both permanent damage and a substantial component that is reversible after cessation of alcohol consumption. Atrial fibrillation occurs commonly both early in the disease (“holiday heart”) and in advanced stages. Medical therapy includes neurohormonal antagonists and diuretics as needed for fluid management. Withdrawal should be supervised to avoid exacerbations of heart failure or arrhythmias, and ongoing support arranged. Even with severe disease, marked improvement can occur within 3–6 months of abstinence. Implantable defibrillators are generally deferred until an adequate period of abstinence, after which they may not be necessary if the ejection fraction has improved. With continued consumption, the prognosis is

1	defibrillators are generally deferred until an adequate period of abstinence, after which they may not be necessary if the ejection fraction has improved. With continued consumption, the prognosis is grim.

1	Cocaine, amphetamines, and related catecholaminergic stimulants can produce chronic cardiomyopathy as well as acute ischemia and tachyarrhythmias. Pathology reveals microinfarcts consistent with small vessel ischemia, similar to those seen with pheochromocytoma. Chemotherapy agents are the most common drugs implicated in toxic cardiomyopathy. Judicious use of these drugs requires balancing the risks of the malignancy and the risks of cardiotoxicity, as many cancers have a chronic course with better prognosis than heart failure.

1	Anthracyclines cause characteristic histologic changes of vacuolar degeneration and myofibrillar loss. Generation of reactive oxygen species involving heme compounds is currently the favored explanation for myocyte injury and fibrosis. Disruption of the large titin protein may contribute to loss of sarcomere organization. Risk for cardiotoxicity increases with higher doses, preexisting cardiac disease, and concomitant chest irradiation. There are three different presentations of anthracycline-induced cardiomyopathy. (1) Heart failure can develop acutely during administration of a single dose, but may clinically resolve in a few weeks. (2) Early-onset doxorubicin cardiotoxicity develops in about 3% of patients during or shortly after a chronic course, relating closely to total dose. It may be rapidly progressive, but may also resolve to good, but not normal, ventricular function. (3) The chronic presentation differs according to whether therapy was given before or after puberty.

1	It may be rapidly progressive, but may also resolve to good, but not normal, ventricular function. (3) The chronic presentation differs according to whether therapy was given before or after puberty. Patients who received doxorubicin while still growing may have impaired development of the heart, which leads to clinical heart failure by the time the patient reaches the early twenties. Late after adult exposure, patients may develop the gradual onset of symptoms or an acute onset precipitated by a reversible second insult, such as influenza or atrial fibrillation. Doxorubicin cardiotoxicity leads to a relatively nondilated ventricle, perhaps due to the accompanying fibrosis. Thus, the stroke volume may be severely reduced with an ejection fraction of 30–40%, which would be well tolerated in a patient with a larger ventricle typical of other cardiomyopathies with systolic dysfunction. Therapy includes angiotensin-converting enzyme inhibitors and β-adrenergic blocking agents used for

1	in a patient with a larger ventricle typical of other cardiomyopathies with systolic dysfunction. Therapy includes angiotensin-converting enzyme inhibitors and β-adrenergic blocking agents used for other causes of heart failure, with careful suppression of “inappropriate” sinus tachycardia, and attention to postural hypotension that can occur in these patients. Once thought to have an inexorable downward course, some patients with doxorubicin cardiotoxicity improve under careful management to near-normal clinical function for many years.

1	Trastuzumab (Herceptin) is a monoclonal antibody that interferes with cell surface receptors crucial for some tumor growth and for cardiac adaptation. The incidence of cardiotoxicity is lower than for anthracyclines but enhanced by coadministration with them. Although considered to be more often reversible, trastuzumab cardiotoxicity does not always resolve, and some patients progress to clinical heart failure and death. As with anthracycline cardiotoxicity, therapy is as usual for heart failure, but it is not clear whether the spontaneous rate of improvement is enhanced by neurohormonal antagonists.

1	Cardiotoxicity with cyclophosphamide and ifosfamide generally occurs acutely and with very high doses. 5-Fluorouracil, cisplatin, and some other alkylating agents can cause recurrent coronary spasm that occasionally leads to depressed contractility. Acute administration of interferon-α can cause hypotension and arrhythmias. Clinical heart failure occurring during repeated chronic administration usually resolves after discontinuation.

1	Many small-molecule tyrosine kinase inhibitors are under development for different malignancies. Although these agents are “targeted” at specific tumor receptors or pathways, the biologic conservation of signaling pathways can cause these inhibitors to have “off-target” effects that include the heart and vasculature. Recognition of cardiotoxicity during therapy with these agents is complicated because they occasionally cause peripheral fluid accumulation (ankle edema, periorbital swelling, pleural effusions) due to local factors rather than elevated central venous pressures. Therapeutic approaches include withdrawal of the tyrosine kinase inhibitor (when possible) and substitution with a congener (when available), as well as conventional treatment for heart failure. Prophylactic treatment with beta blockers and angiotensin-converting enzyme inhibitors prior to and during chemotherapy is a topic of ongoing investigation.

1	Other therapeutic drugs that can cause cardiotoxicity during chronic use include hydroxychloroquine, chloroquine, emetine, and antiretroviral therapies. Toxic exposures can cause arrhythmias or respiratory injury acutely during accidents. Chronic exposures implicated in cardiotoxicity include hydrocarbons, fluorocarbons, arsenicals, lead, and mercury.

1	Endocrine disorders affect multiple organ systems, including the heart. Hyperthyroidism and hypothyroidism do not often cause clinical heart failure in an otherwise normal heart, but commonly exacerbate heart failure. Clinical signs of thyroid disease may be masked, so tests of thyroid function are part of the routine evaluation of cardiomyopathy. Hyperthyroidism should always be considered with new-onset atrial fibrillation or ventricular tachycardia or atrial fibrillation in which the rapid ventricular response is difficult to control. The most common current reason for thyroid abnormalities in the cardiac population is the treatment of tachyarrhythmias with amiodarone, 1563 a drug with substantial iodine content. Hypothyroidism should be treated with very slow escalation of thyroid supplements to avoid exacerbating tachyarrhythmias and heart failure. Hyperthyroidism and heart failure are a dangerous combination that merits very close supervision, often hospitalization, during

1	supplements to avoid exacerbating tachyarrhythmias and heart failure. Hyperthyroidism and heart failure are a dangerous combination that merits very close supervision, often hospitalization, during titration of antithyroid medications, during which decompensation of heart failure may occur precipitously and fatally.

1	Pheochromocytoma is rare, but should be considered when a patient has heart failure and very labile blood pressure and heart rate, sometimes with episodic palpitations (Chap. 407). Patients with pheochromocytoma often have postural hypotension. In addition to α-adrenergic receptor antagonists, definitive therapy requires surgical extirpation. Very high renin states, such as those caused by renal artery stenosis, can lead to modest depression in ejection fraction with little or no ventricular dilation and markedly labile symptoms with flash pulmonary edema, related to sudden shifts in vascular tone and intravascular volume.

1	Controversies remain regarding whether diabetes and obesity are sufficient to cause cardiomyopathy. Most heart failure in diabetes results from epicardial coronary disease, with further increase in coronary artery risk due to accompanying hypertension and renal dysfunction. Cardiomyopathy may result in part from insulin resistance and increased advanced-glycosylation end products, which impair both systolic and diastolic function. However, much of the dysfunction can be attributed to scattered focal ischemia resulting from distal coronary artery tapering and limited microvascular perfusion even without proximal focal stenoses. Diabetes is a typical factor in heart failure with “preserved” ejection fraction, along with hypertension, advanced age, and female gender.

1	The existence of a cardiomyopathy due to obesity is generally accepted. In addition to cardiac involvement from associated diabetes, hypertension, and vascular inflammation of the metabolic syndrome, obesity alone is associated with impaired excretion of excess volume load, which, over time, can lead to increased wall stress and secondary adaptive neurohumoral responses. Fluid retention may be aggravated by large fluid intake and the rapid clearance of natriuretic peptides by adipose tissue. In the absence of another obvious cause of cardiomyopathy in an obese patient with systolic dysfunction without marked ventricular dilation, effective weight reduction is often associated with major improvement in ejection fraction and clinical function. Improvement in cardiac function has been described after successful bariatric surgery, although all major surgical therapy poses increased risk for patients with heart failure. Postoperative malabsorption and nutritional deficiencies, such as

1	after successful bariatric surgery, although all major surgical therapy poses increased risk for patients with heart failure. Postoperative malabsorption and nutritional deficiencies, such as calcium and phosphate deficiencies, may be particularly deleterious for patients with cardiomyopathy.

1	Nutritional deficiencies can occasionally cause dilated cardiomyopathy but are not commonly implicated in developed Western countries. Beri-beri heart disease due to thiamine deficiency can result from poor nutrition in undernourished populations and in patients deriving most of their calories from alcohol, and has been reported in teenagers subsisting only on highly processed foods. This disease is initially a vasodilated state with very high output heart failure that can later progress to a low output state; thiamine repletion can lead to prompt recovery of cardiovascular function. Abnormalities in carnitine metabolism can cause dilated or restrictive cardiomyopathies, usually in children. Deficiency of trace elements such as selenium can cause cardiomyopathy (Keshan’s disease).

1	Calcium is essential for excitation-contraction coupling. Chronic deficiencies of calcium, such as can occur with hypoparathyroidism (particularly postsurgical) or intestinal dysfunction (from diarrheal syndromes and following extensive resection), can cause severe chronic heart failure that responds over days or weeks to vigorous calcium repletion. Phosphate is a component of high-energy compounds needed for efficient energy transfer and multiple signaling pathways. Hypophosphatemia can develop during starvation and early refeeding following a prolonged fast, and occasionally during hyperalimentation. Magnesium is a cofactor for thiamine-dependent reactions and for the sodium-potassium adenosine triphosphatase (ATPase), but 1564 The most recognizable familial cardiomyopathy syndromes with extra-cardiac manifestations are the muscular dystrophies. Both Duchenne’s and the milder Becker’s dystrophy result from abnormalities in the X-linked dystrophin gene of the sarcolemmal membrane.

1	extra-cardiac manifestations are the muscular dystrophies. Both Duchenne’s and the milder Becker’s dystrophy result from abnormalities in the X-linked dystrophin gene of the sarcolemmal membrane. Skeletal myopathy is present in multiple other genetic cardiomyopathies (Table 287-3), some of which are associated with creatine kinase elevations. Families with a history of atrial arrhythmias, conduction system disease, and cardiomyopathy may have abnormalities of the nuclear membrane lamin proteins. While all dilated cardiomyopathies carry a risk of sudden death, a family history of cardiomyopathy with sudden death raises suspicion for a particularly arrhythmogenic mutation; affected family members may be considered for implantable defibrillators even before meeting the reduced ejection fraction threshold for primary prevention of sudden death. A prominent family history of sudden death or ventricular tachycardia before clinical cardiomyopathy suggests genetic defects in the desmosomal

1	threshold for primary prevention of sudden death. A prominent family history of sudden death or ventricular tachycardia before clinical cardiomyopathy suggests genetic defects in the desmosomal proteins (Fig. 287-10). Originally described as affecting the right ventricle (arrhythmogenic right ventricular dysplasia [ARVD]), this disorder (arrhythmogenic ventricular dysplasia) can affect either or both ventricles. Patients often present first with ventricular tachycardia. Genetic defects in proteins of the desmosomal complex disrupt myocyte junctions and adhesions, leading to replacement of myocardium by deposits of fat. Thin ventricular walls may be recognized on echocardiography but are better visualized on MRI. Because desmosomes are also important for elasticity hypomagnesemia rarely becomes sufficiently profound to cause clini-of hair and skin, some of the defective desmosomal proteins are cal cardiomyopathy. associated with striking “woolly hair” and thickened skin on the

1	becomes sufficiently profound to cause clini-of hair and skin, some of the defective desmosomal proteins are cal cardiomyopathy. associated with striking “woolly hair” and thickened skin on the Hemochromatosis is variably classified as a metabolic or storage palms and soles. Implantable defibrillators are usually indicated to disease (Chap. 428). It is included among the causes of restrictive prevent sudden death. There is variable progression to right, left, or cardiomyopathy, but the clinical presentation is often that of a dilated biventricular failure. cardiomyopathy. The autosomal recessive form is related to the HFE Left ventricular noncompaction is a condition of unknown prevagene. With up to 10% of the population heterozygous for one muta-lence that is increasingly revealed with the refinement of imaging tion, the clinical prevalence might be as high as 1 in 500. The lower techniques. The diagnostic criteria include the presence of multiple observed rates highlight the limited

1	refinement of imaging tion, the clinical prevalence might be as high as 1 in 500. The lower techniques. The diagnostic criteria include the presence of multiple observed rates highlight the limited penetrance of the disease, suggest-trabeculations in the left ventricle distal to the papillary muscles, creing the role of additional genetic and environmental factors for clinical ating a “spongy” appearance of the apex. Noncompaction has been expression. Hemochromatosis can also be acquired from iron overload associated with multiple genetic variants in the sarcomeric and other due to hemolytic anemia and transfusions. Excess iron is deposited genes, such as TAZ (encoding tafazzin). The diagnosis may be made in the perinuclear compartment of cardiomyocytes, with resulting incidentally or in patients previously diagnosed with cardiomyopathy, disruption of intracellular architecture and mitochondrial function. in whom the criteria for noncompaction may appear and resolve with Diagnosis is

1	patients previously diagnosed with cardiomyopathy, disruption of intracellular architecture and mitochondrial function. in whom the criteria for noncompaction may appear and resolve with Diagnosis is easily made from measurement of serum iron and trans-changing left ventricular size and function. The three cardinal clinical ferrin saturation, with a threshold of >60% for men and >45–50% for features are ventricular arrhythmias, embolic events, and heart failure. women. MRI can help to quantitate iron stores in the liver and heart, Treatment generally includes anticoagulation and early consideration and endomyocardial biopsy tissue can be stained for iron (Fig. 287-9), for an implantable defibrillator, in addition to neurohormonal antagowhich is particularly important if the patient has another cause for nists as indicated by stage of disease. cardiomyopathy. If diagnosed early, hemochromatosis can often be Some families inherit a susceptibility to viral-induced myocarditis. managed by

1	cause for nists as indicated by stage of disease. cardiomyopathy. If diagnosed early, hemochromatosis can often be Some families inherit a susceptibility to viral-induced myocarditis. managed by repeated phlebotomy to remove iron. For more severe This propensity may relate to abnormalities in cell surface receptors, iron overload, iron chelation therapy with desferrioxamine (deferox-such as the coxsackie-adenovirus receptor, that bind viral proteins. amine) or deferasirox can help to improve cardiac function if myocyte loss and replacement fibrosis are not too severe. Inborn disorders of metabolism occasionally present with dilated cardiomyopathy, although they are most often associated with restrictive cardiomyopathy (Table 287-4).

1	The genetic basis for cardiomyopathy is discussed in the section, “Genetic Etiologies of Cardiomyopathy.” The recognized frequency of familial involvement in dilated cardiomyopathy has increased to over 30%. Mutations in TTN, encoding the giant sarcomeric protein titin, are the most common cause of dilated cardiomyopa-AB thy, accounting for up to 25% of familial disease. FIGURE 287-10 Arrhythmogenic right ventricular dysplasia. A. Cross-sectional slice of On average, men with TTN mutations develop a pathology specimen removed at transplantation, showing severe dysplasia of the right cardiomyopathy a decade before women, without ventricle (RV) with extensive fatty replacement of right ventricular myocardium. B. The distinctive clinical features. Mutations in thick and remarkably thin right ventricular free wall is revealed by transillumination. LV, left ventricle. thin filament genes account for ~8% of dilated car-(Images courtesy of Gayle Winters, MD, and Richard Mitchell, MD, PhD,

1	ventricular free wall is revealed by transillumination. LV, left ventricle. thin filament genes account for ~8% of dilated car-(Images courtesy of Gayle Winters, MD, and Richard Mitchell, MD, PhD, Division of Pathology, diomyopathy and may manifest in early childhood. Brigham and Women’s Hospital, Boston.)

1	FIGURE 287-9 Hemochromatosis. Microscopic image of an endo-myocardial biopsy showing extensive iron deposition within the cardiac myocytes with the Prussian blue stain (400× original mag-nification). (Image courtesy of Robert Padera, MD, PhD, Department of Pathology, Brigham and Women’s Hospital, Boston.) Some may have partial homology with viral proteins such that an autoimmune response is triggered against the myocardium.

1	Some may have partial homology with viral proteins such that an autoimmune response is triggered against the myocardium. Prognosis and therapy of familial dilated cardiomyopathy are dictated primarily by the stage of clinical disease and the risk for sudden death. In some cases, the familial etiology facilitates prognostic decisions, particularly regarding the likelihood of recovery after a new diagnosis, which is unlikely for familial disease. The rate of progression of disease, once manifest, is, to some extent, heritable, although marked variation can be seen. However, there have been cases of remarkable clinical remission after acute presentation, likely after a reversible additional insult, such as prolonged tachycardia or infective myocarditis.

1	The apical ballooning syndrome, or stress-induced cardiomyopathy, occurs typically in older women after sudden intense emotional or physical stress. The ventricle shows global ventricular dilation with basal contraction, forming the shape of the narrow-necked jar (takotsubo) used in Japan to trap octopi. Originally described in Japan, it is increasingly recognized elsewhere during emergency cardiac catheterization and intensive care unit admissions for noncardiac conditions. Presentations include pulmonary edema, hypotension, and chest pain with ECG changes mimicking an acute infarction. The left ventricular dysfunction extends beyond a specific coronary artery distribution and generally resolves within days to weeks. Animal models and ventricular biopsies suggest that this acute cardiomyopathy may result from intense sympathetic activation with heterogeneity of myocardial autonomic innervation, diffuse microvascular spasm, and/or direct catecholamine toxicity. Coronary angiography

1	may result from intense sympathetic activation with heterogeneity of myocardial autonomic innervation, diffuse microvascular spasm, and/or direct catecholamine toxicity. Coronary angiography may be required to rule out acute coronary occlusion. No therapies have been proven beneficial, but reasonable strategies include nitrates for pulmonary edema, intraaortic balloon pump if needed for low output, combined alpha and beta blockers rather than selective beta blockade if hemodynamically stable, and magnesium for arrhythmias related to QT prolongation. Anticoagulation is generally withheld due to the occasional occurrence of ventricular rupture. While the prognosis is generally good, recurrences have been described in up to 10% of patients.

1	Idiopathic dilated cardiomyopathy is a diagnosis of exclusion, when all other known factors have been excluded. Approximately two-thirds of dilated cardiomyopathies are still labeled as idiopathic; however, a substantial proportion of these may reflect unrecognized genetic disease. Continued reconsideration of etiology during chronic heart failure management often reveals specific causes later in a patient’s course.

1	The limitations of our phenotypic classification are revealed through the multiple overlaps between the etiologies and presentations of the three types. Cardiomyopathy with reduced systolic function but without severe dilation can represent early dilated cardiomyopathy, “minimally dilated cardiomyopathy,” or restrictive diseases without marked increases in ventricular wall thickness. For example, sarcoidosis and hemochromatosis can present as dilated or restrictive disease. Early stages of amyloidosis are often mistaken for hypertrophic cardiomyopathy. Progression of hypertrophic cardiomyopathy into a “burned-out” phase occurs occasionally, with decreased contractility and modest ventricular dilation. Overlaps are particularly common with the inherited metabolic disorders, which can present as any of the three major phenotypes (Fig. 287-4).

1	Multiple genetic disorders of metabolic pathways can cause myocardial disease, due to infiltration of abnormal products or cells containing them between the myocytes, and storage disease, due to their accumulation within cells (see HPIM 18e, Table 238-4, and 287-5). The restrictive phenotype is most common, but mildly dilated cardiomyopathy may occur. Hypertrophic cardiomyopathy may be mimicked by the myocardium thickened with these abnormal products causing “pseudohypertrophy.” Most of these diseases are diagnosed during childhood. Inherited metabolic defectsa Fabry’s disease Glycogen storage disease (II, III) Drugs: e.g., serotonin, ergotamine Overlap with Other Cardiomyopathies aCan be familial.

1	Fabry’s disease results from a deficiency of the lysosomal enzyme alpha-galactosidase A caused by one of more than 160 mutations. This disorder of glycosphingolipid metabolism is an X-linked recessive disorder that may also cause clinical disease in female carriers. Glycolipid accumulation may be limited to the cardiac tissues or may also involve the skin and kidney. Electron microscopy of endomyocardial biopsy tissue shows diagnostic vesicles containing concentric lamellar figures (Fig. 287-11). Diagnosis is crucial because enzyme replacement can reduce abnormal deposits and improve cardiac and clinical function. The magnitude of clinical impact has not been well-established for this therapy, which requires frequent infusions of the enzyme at a cost of over $100,000 a year. Enzyme replacement can also improve the course of Gaucher’s disease, in which cerebroside-rich cells accumulate in multiple organs due to a deficiency of beta-glucosidase. Cerebrosiderich cells infiltrate the

1	can also improve the course of Gaucher’s disease, in which cerebroside-rich cells accumulate in multiple organs due to a deficiency of beta-glucosidase. Cerebrosiderich cells infiltrate the heart, which can also lead to a hemorrhagic pericardial effusion and valvular disease.

1	Glycogen storage diseases lead to accumulation of lysosomal storage products and intracellular glycogen accumulation, particularly with glycogen storage disease type III, due to a defective debranching enzyme. There are more than 10 types of mucopolysaccharidoses, in which autosomal dominant or X-linked deficiencies of lysosomal enzymes lead to the accumulation of glycosaminoglycans in the skeleton, nervous system, and occasionally the heart. With characteristic facies, short stature, and frequent cognitive impairment, most individuals are diagnosed early in childhood and die before adulthood.

1	Carnitine is an essential cofactor in long-chain fatty acid metabolism. Multiple defects have been described that lead to carnitine deficiency, causing intracellular lipid inclusions and restrictive or dilated cardiomyopathy, often presenting in children. Fatty acid oxidation requires many metabolic steps with specific enzymes that can be deficient, with complex interactions with carnitine. Depending on the defect, cardiac and skeletal myopathy can be ameliorated with replacement of fatty acid intermediates and carnitine. FIGURE 287-11 Fabry’s disease. Transmission electron micrograph of a right ventricular endomyocardial biopsy specimen at high magnification showing the characteristic concentric lamellar inclusions of glycosphingolipids accumulating as a result of deficiency of the lysosomal enzyme alpha-galactosidase A. Image taken at 15,000× original magnification. (Image courtesy of Robert Padera, MD, PhD, Department of Pathology, Brigham and Women’s Hospital, Boston.)

1	Two monogenic metabolic cardiomyopathies have recently been described as causes of increased ventricular wall thickness without an increase of muscle subunits or an increase in contractility. Mutations in the gamma-2 regulatory subunit of the adenosine monophosphate (AMP)-activated protein kinase important for glucose metabolism (PRKAG2) have been associated with a high prevalence of conduction abnormalities, such as AV block and ventricular preexcitation (WolffParkinson-White syndrome). Several defects have been reported in an X-linked lysosome-associated membrane protein (LAMP2). This defect can be maternally transmitted or sporadic and has occasionally been isolated to the heart, although it often leads to a syndrome of skeletal myopathy, mental retardation, and hepatic dysfunction referred to as Danon’s disease. Extreme left ventricular hypertrophy appears early, often in childhood, and can progress rapidly to end-stage heart failure with low ejection fraction. Electron microscopy

1	to as Danon’s disease. Extreme left ventricular hypertrophy appears early, often in childhood, and can progress rapidly to end-stage heart failure with low ejection fraction. Electron microscopy of these metabolic disorders shows that the myocytes are enlarged by multiple intracellular vacuoles of metabolic by-products.

1	The least common of the physiologic triad of cardiomyopathies is restrictive cardiomyopathy, which is dominated by abnormal diastolic function, often with mildly decreased contractility and ejection fraction (usually >30–50%). Both atria are enlarged, sometimes massively. Modest left ventricular dilation can be present, usually with an end-diastolic dimension <6 cm. End-diastolic pressures are elevated in both ventricles, with preservation of cardiac output until late in the disease. Subtle exercise intolerance is usually the first symptom but is often not recognized until after clinical presentation with congestive symptoms. The restrictive diseases often present with relatively more right-sided symptoms, such as edema, abdominal discomfort, and ascites, although filling pressures are elevated in both ventricles. The cardiac impulse is less displaced than in dilated cardiomyopathy and less dynamic than in hypertrophic cardiomyopathy. A fourth heart sound is more common than a third

1	in both ventricles. The cardiac impulse is less displaced than in dilated cardiomyopathy and less dynamic than in hypertrophic cardiomyopathy. A fourth heart sound is more common than a third heart sound in sinus rhythm, but atrial fibrillation is common. Jugular venous pressures often show rapid Y descents and may increase during inspiration (positive Kussmaul’s sign). Most restrictive cardiomyopathies are due to infiltration of abnormal substances between myocytes, storage of abnormal metabolic products within myocytes, or fibrotic injury (Table 287-5). The differential diagnosis should include constrictive pericardial disease, which may also be dominated by right-sided heart failure.

1	Amyloidosis is the major cause of restrictive cardiomyopathy (Figs. 287-12, 287-13, and 287-14). Several proteins can self-assemble to form the beta-sheets of amyloid proteins, which deposit with different consequences depending on the type of protein. The systemic amyloidoses are discussed in Chap. 137. In addition to cardiac infiltration, neurologic involvement occurs commonly with primary amyloidosis (immunoglobulin light chains) and with familial amyloidosis (genetic abnormalities of transthyretin). There are over 100 identified mutations in transthyretin on chromosome 13, among which the V122I transthyretin mutation has been identified in about 4% of African Americans and in 10% of African Americans with heart failure and may contribute importantly to heart failure in general in the elderly African-American population. Organ dysfunction was previously attributed solely to physical disruption from the infiltrating amyloid fibrils, but newer information suggests additional direct

1	elderly African-American population. Organ dysfunction was previously attributed solely to physical disruption from the infiltrating amyloid fibrils, but newer information suggests additional direct toxicity from the immunoglobulin light chain and abnormal transthyretin protein aggregates themselves. In senile amyloidosis, there is abnormal accumulation of normal transthyretin or natriuretic peptide folding, detected in 10% of people over 80 years and half of those over 90 years but often without apparent clinical disease. Men show a greater burden of amyloid deposition and 20-fold greater likelihood of clinical disease with senile amyloidosis. The aging of the population will soon render senile amyloidosis the most common of the amyloidoses.

1	Cardiac amyloid is classically suspected from thickened ventricular walls with an ECG that shows low voltage. However, low voltage is not always present and is less common in familial or senile amyloidosis than in primary AL amyloidosis. A characteristic refractile brightness in the septum on echocardiography is suggestive of the diagnosis, but neither sensitive nor specific. Both atria are dilated, often dramatically, and diastolic dysfunction may be more obvious than in left ventricular hypertrophy from other causes. Amyloid infiltration can also be detected with gadolinium enhancement in MRI.

1	FIGURE 287-12 Restrictive cardiomyopathy—amyloidosis. Gross specimen of a heart with amyloidosis. The heart is firm and rubbery with a waxy cut surface. The atria are markedly dilated, and the left atrial endocardium, normally smooth, has yellow-brown amyloid deposits that give texture to the surface. (Image courtesy of Robert Padera, MD, PhD, Department of Pathology, Brigham and Women’s Hospital, Boston.) LV LA Septum RA RV Pacing lead in RV Lateral wall of LV Pericardial effusion FIGURE 287-13 Restrictive cardiomyopathy—amyloidosis.

1	FIGURE 287-13 Restrictive cardiomyopathy—amyloidosis. Echocardiogram showing thickened walls of both ventricles without major chamber dilation. The atria are markedly dilated, consistent with chronically elevated ventricular filling pressures. In this example, there is a characteristic hyperrefractile “glittering” of the myocardium typical of amyloid infiltration, which is often absent (especially with more recent echocardiographic systems of better resolution). The mitral and tricuspid valves are thickened. A pacing lead is visible in the right ventricle (RV), and a pericardial effusion is evident. Note that the echocardiographic and pathologic images are vertically opposite, such that the left ventricle (LV) is by convention on the top right in the echocardiographic image and bottom right in the pathologic images. LA, left atrium; RA, right atrium. (Image courtesy of Justina Wu, MD, Brigham and Women’s Hospital, Boston.)

1	The diagnosis of primary or familial amyloidosis can sometimes be made from biopsies of an abdominal fat pad or the rectum, but cardiac amyloidosis is most reliably identified from a biopsy of the heart, in which amyloid fibrils infiltrate the myocardium diffusely, particularly around the conduction system and coronary vessels (Fig. 287-14). Diagnosis of the type of amyloid protein requires immunohistochemistry of biopsied tissue rather than serum or urine electrophoresis, which can lead to incorrect classification.

1	Therapy for all types of amyloid is predominantly for symptoms 1567 of fluid retention, which often requires high doses of loop diuretics. Digoxin bound to the amyloid fibrils can reach toxic levels, and should therefore be used only in very low doses, if at all. There is no evidence regarding use of neurohormonal antagonists in amyloid heart disease, where the possible theoretical benefit has to be balanced against the possibility of aggravating postural hypotension and diminishing the crucial heart rate reserve. The risk of intracardiac thrombi may warrant chronic anticoagulation.

1	The prognosis is worst for primary amyloid, with a median sur vival of 6–12 months after symptoms of heart failure. If present, multiple myeloma is treated with chemotherapy, the extent of which is often limited by the potential of worsening cardiac dysfunction. Immunoglobulin-associated amyloid has occasionally been treated with sequential heart transplantation and delayed bone marrow transplant, with frequent recurrence of amyloid in the transplanted heart. Abnormal transthyretin-associated cardiac amyloid has a somewhat better prognosis and can be treated in selected patients with heart and liver transplantation. Senile cardiac amyloid has the slowest progres sion and best overall prognosis.

1	Progressive fibrosis can cause restrictive myocardial disease without ventricular dilation. Thoracic radiation, common for breast and lung cancer or mediastinal lymphoma, can produce early or late restrictive cardiomyopathy. Patients with radiation cardiomyopathy may present with a possible diagnosis of constrictive pericarditis, as the two conditions often coexist. Careful hemodynamic evaluation and, often, endomyocardial biopsy should be performed if considering pericardial stripping surgery, which is unlikely to be successful in the presence of underlying restrictive cardiomyopathy.

1	Scleroderma causes small vessel spasm and ischemia that can lead to a small, stiff heart with reduced ejection fraction without dilation. The pulmonary hypertension associated with scleroderma may lead to more clinical right heart failure because of concomitant fibrotic disease of the right ventricle. Doxorubicin causes direct myocyte injury usually leading to dilated cardiomyopathy, but the limited degree of dilation may result from increased fibrosis, which restricts remodeling.

1	The physiologic picture of elevated filling pressures with atrial enlargement and preserved ventricular contractility with normal or reduced ventricular volumes can result from extensive fibrosis of the endocardium, without transmural myocardial disease. For patients who have not lived in the equatorial regions, this picture is rare, and when seen is often associated with a history of chronic hypereosinophilic syndrome (Löffler’s endocarditis), which is more common in men than women. In this disease, persistent hypereosinophilia of >1500 eos/mm3 for at least 6 months can cause an acute phase of eosinophilic injury in the endocardium (see earlier discussion of eosinophilic myocarditis), with systemic illness and injury to other organs. There is usually no obvious cause, but the hypereosinophilia can occasionally be explained by allergic, parasitic, or malignant disease. It is postulated to be followed by a period in which cardiac inflammation is replaced by evidence of fibrosis with

1	can occasionally be explained by allergic, parasitic, or malignant disease. It is postulated to be followed by a period in which cardiac inflammation is replaced by evidence of fibrosis with superimposed thrombosis. In severe disease, the dense fibrotic layer can obliterate the ventricular apices and extend to thicken and tether the AV

1	FIGURE 287-14 Amyloidosis—microscopic images of amyloid involving the myocar-valve leaflets. The clinical disease may present with dium. The left panel (hematoxylin and eosin stain) shows glassy, grey-pink amorphous heart failure, embolic events, and atrial arrhythmaterial infiltrating between cardiomyocytes, which stain a darker pink. The right panel mias. While plausible, the sequence of transition shows a sulfated blue stain that highlights the amyloid green and stains the cardiac from eosinophilic myocarditis or Löffler’s endocarmyocytes yellow. (The Congo red stain can also be used to highlight amyloid; under ditis to endomyocardial fibrosis has not been clearly polarized light, amyloid will have an apple-green birefringence when stained with Congo demonstrated. red.) Images at 100× original magnification. (Image courtesy of Robert Padera, MD, PhD, In tropical countries, up to one-quarter of heart Department of Pathology, Brigham and Women’s Hospital, Boston.) failure may be due

1	original magnification. (Image courtesy of Robert Padera, MD, PhD, In tropical countries, up to one-quarter of heart Department of Pathology, Brigham and Women’s Hospital, Boston.) failure may be due to endomyocardial fibrosis, 1568 affecting either or both ventricles. This condition shares with the previous condition the partial obliteration of the ventricular apex with fibrosis extending into the valvular inflow tract and leaflets; however, it is not clear that the etiologies are the same for all cases. Pericardial effusions frequently accompany endomyocardial fibrosis but are not common in Löffler’s endocarditis. For endomyocardial fibrosis, there is no gender difference, but a higher prevalence in African-American populations. While tropical endomyocardial fibrosis could represent the end-stage of previous hypereosinophilic disease triggered by endemic parasites, neither prior parasitic infection nor hypereosinophilia is usually documented. Geographic nutritional deficiencies have

1	end-stage of previous hypereosinophilic disease triggered by endemic parasites, neither prior parasitic infection nor hypereosinophilia is usually documented. Geographic nutritional deficiencies have also been proposed as an etiology. Medical treatment focuses on glucocorticoids and chemotherapy to suppress hypereosinophilia when present. Fluid retention may become increasingly resistant to diuretic therapy. Anticoagulation is recommended. Atrial fibrillation is associated with worse symptoms and prognosis, but may be difficult to suppress. Surgical resection of the apices and replacement of the fibrotic valves can improve symptoms, but surgical morbidity and mortality and later recurrence rates are high. The serotonin secreted by carcinoid tumors can produce fibrous plaques in the endocardium and right-sided cardiac valves, occasionally affecting left-sided valves, as well. Valvular lesions may be stenotic or regurgitant. Systemic symptoms include flushing and diarrhea. Liver disease

1	and right-sided cardiac valves, occasionally affecting left-sided valves, as well. Valvular lesions may be stenotic or regurgitant. Systemic symptoms include flushing and diarrhea. Liver disease from hepatic metastases may play a role by limiting hepatic function and thereby allowing more serotonin to reach the venous circulation.

1	Hypertrophic cardiomyopathy is defined as left ventricular hypertrophy that develops in the absence of causative hemodynamic factors, such as hypertension, aortic valve disease, or systemic infiltrative or storage diseases (Figs. 287-15 and 287-16). It has previously been termed hypertrophic obstructive cardiomyopathy (HOCM), asymmetric septal hypertrophy (ASH), and idiopathic hypertrophic subaortic stenosis (IHSS). However, the accepted terminology is now hypertrophic

1	FIGURE 287-15 Hypertrophic cardiomyopathy. Gross specimen of a heart with hypertrophic cardiomyopathy removed at the time of transplantation, showing asymmetric septal hypertrophy (septum much thicker than left ventricular free wall) with the septum bulging into the left ventricular outflow tract causing obstruction. The forceps are retracting the anterior leaflet of the mitral valve, demonstrating the characteristic plaque of systolic anterior motion, manifest as endocardial fibrosis on the interventricular septum in a mirror-image pattern to the valve leaflet. There is patchy replacement fibrosis, and small thick-walled arterioles can be appreciated grossly, especially in the interventricular septum. IVS, interventricular septum; LV, left ventricle; RV, right ventricle. (Image courtesy of Robert Padera, MD, PhD, Department of Pathology, Brigham and Women’s Hospital, Boston.)

1	FIGURE 287-16 Hypertrophic cardiomyopathy. This echocardiogram of hypertrophic cardiomyopathy shows asymmetric hypertrophy of the septum compared to the lateral wall of the left ventricle (LV). The mitral valve (MV) is moving anteriorly toward the hypertrophied septum in systole. The left atrium (LA) is enlarged. Note that the echocardiographic and pathologic images are vertically opposite, such that the LV is by convention on the top right in the echocardiographic image and bottom right in the pathologic images. (Image courtesy of Justina Wu, MD, Brigham and Women’s Hospital, Boston.) cardiomyopathy with or without obstruction. Prevalence in North America, Japan, and China is about 1:500. It is the leading cause of sudden death in the young and is an important cause of heart failure. Although pediatric presentation is associated with increased early morbidity and mortality, the prognosis for patients diagnosed as adults is generally favorable.

1	The clustering of hypertrophic cardiomyopathy within families has been appreciated since recognition of the disease approximately 55 years ago. Echocardiographic screening of families revealed an autosomal dominant pattern of inheritance. Initial genetic studies using linkage analysis in large families identified disease-causing mutations in sarcomeric genes. A sarcomere mutation is present in ~60% of patients with hypertrophic cardiomyopathy and is more common in those with familial disease and characteristic asymmetric septal hypertrophy. More than nine different sarcomere genes with over 1400 mutations have been implicated, although ~80% of patients have a mutation in either MYH7 or MYBPC3 (Table 287-3), most of which are unique to individual families (“private” mutations).

1	Hypertrophic cardiomyopathy is characterized by age-dependent and incomplete penetrance. The defining phenotype of left ventricular hypertrophy is rarely present at birth and usually develops later in life. Accordingly, screening of family members should begin in adolescence and extend through adulthood. In MYBPC3 mutation carriers, the average age of disease development is 40 years, while 30% remain free from hypertrophy after 70 years. Related individuals who carry the same mutation may have a different extent and pattern of hypertrophy (e.g., asymmetric versus concentric), occurrence of outflow tract obstruction, and associated clinical outcomes (e.g., sudden death, atrial fibrillation).

1	FIGURE 287-17 Hypertrophic cardiomyopathy. Microscopic image of hypertrophic cardiomyopathy showing the characteristic disordered myocyte architecture with swirling and branching rather than the usual parallel arrangement of myocyte fibers. Myocyte nuclei vary markedly in size and interstitial fibrosis is present. (Image courtesy of Robert Padera, MD, PhD, Department of Pathology, Brigham and Women’s Hospital, Boston.)

1	At the level of the sarcomere, hypertrophic cardiomyopathy mutations lead to enhanced calcium sensitivity, maximal force generation, and ATPase activity. Calcium handling is affected through modification of regulatory proteins. Sarcomere mutations lead to abnormal energetics and impaired relaxation, both directly and as a result of hypertrophy. Hypertrophic cardiomyopathy is characterized by misalignment and disarray of the enlarged myofibrils and myocytes (Fig. 287-17), which can also occur to a lesser extent in other cardiac diseases. Although hypertrophy is the defining feature of hypertrophic cardiomyopathy, fibrosis and microvascular disease are also present. Interstitial fibrosis is detectable before overt hypertrophy develops and likely results from early activation of profibrotic pathways. In the majority of patients with overt cardiomyopathy, focal areas of replacement fibrosis can be readily detected with MRI. These areas of “scar” may represent substrate for the development

1	In the majority of patients with overt cardiomyopathy, focal areas of replacement fibrosis can be readily detected with MRI. These areas of “scar” may represent substrate for the development of ventricular arrhythmias. Increased thickness and decreased luminal area of the intramural vessels in hypertrophied myocardium contribute to microvascular ischemia and angina. Microinfarction of hypertrophied myocardium is a hypothesized mechanism for replacement scar formation.

1	Macroscopically, hypertrophy is typically manifest as nonuniform ventricular thickening (Fig. 287-15). The interventricular septum is the typical location of maximal hypertrophy, although other patterns of hypertrophic remodeling include concentric and midventricular. Hypertrophy confined to the ventricular apex (apical hypertrophic cardiomyopathy) is less often familial and has a different genetic substrate, with sarcomere mutations present in only ~15%. Left ventricular outflow tract obstruction represents the most common focus of diagnosis and intervention, although diastolic dysfunction, myocardial fibrosis, and microvascular ischemia also contribute to contractile dysfunction and elevated intracardiac pressures. Obstruction is present in ~30% of patients at rest and can be provoked by exercise in another ~30%. Systolic obstruction is initiated by drag forces, which push an anteriorly displaced and enlarged anterior mitral leaflet into contact with the hypertrophied ventricular

1	by exercise in another ~30%. Systolic obstruction is initiated by drag forces, which push an anteriorly displaced and enlarged anterior mitral leaflet into contact with the hypertrophied ventricular septum. Mitral leaflet coaptation may ensue, leading to posteriorly directed mitral regurgitation. In order to maintain stroke volume across outflow tract obstruction, the ventricle generates higher pressures, leading to higher wall stress and myocardial oxygen demand. Smaller chamber size and increased contractility exacerbate the severity of obstruction. Conditions of low preload, such as dehydration, and low afterload, such as arterial vasodilation, may lead to transient hypotension and near-syncope. The systolic ejection murmur of left ventricular outflow tract obstruction is harsh and late peaking and can be enhanced by bedside maneuvers that diminish ventricular volume and transiently worsen obstruction, such as stand-1569 ing from a squatting position or the Valsalva maneuver.

1	The substantial variability of hypertrophic cardiomyopathy pathology is reflected in the diversity of clinical presentations. Patients may be diagnosed after undergoing evaluations triggered by the abnormal physical findings (murmur) or symptoms of exertional dyspnea, angina, or syncope. Alternatively, diagnosis may follow evaluations prompted by the detection of disease in family members. Cardiac imaging (Fig. 287-16) is central to diagnosis due to the insensitivity of examination and ECG and the need to exclude other causes for hypertrophy. The identification of a disease-causing mutation in a proband can focus family evaluations on mutation carriers, but this strategy requires a high degree of certainty that the mutation is truly pathogenic and not a benign DNA variant. Biopsy is not needed to diagnose hypertrophic cardiomyopathy but can be used to exclude infiltrative and metabolic diseases. Rigorous athletic training (athlete’s heart) may cause intermediate degrees of physiologic

1	to diagnose hypertrophic cardiomyopathy but can be used to exclude infiltrative and metabolic diseases. Rigorous athletic training (athlete’s heart) may cause intermediate degrees of physiologic hypertrophy difficult to differentiate from mild hypertrophic cardiomyopathy. Unlike hyper-trophic cardiomyopathy, hypertrophy in the athlete’s heart regresses with cessation of training, and is accompanied by supernormal exercise capacity (VO2max >50 mL/kg/min), mild ventricular dilation, and normal diastolic function.

1	Management focuses on treatment of symptoms and prevention of sudden death and stroke (Fig. 287-18). Left ventricular outflow tract obstruction can be controlled medically in the majority of patients. β-Adrenergic blocking agents and L-type calcium channel blockers (e.g., verapamil) are first-line agents that reduce the severity of obstruction by slowing heart rate, enhancing diastolic filling, and decreasing contractility. Persistent symptoms of exertional dyspnea or chest pain can sometimes be controlled with the addition of disopyramide, an antiarrhythmic agent with potent negative inotropic properties. Patients with or without obstruction may develop heart failure symptoms due to fluid retention and require diuretic therapies for venous congestion. Severe medically refractory symptoms develop in ~5% of patients, for whom surgical myectomy or alcohol septal ablation may be effective. Developed over 50 years ago, surgical myectomy effectively relieves outflow tract obstruction by

1	develop in ~5% of patients, for whom surgical myectomy or alcohol septal ablation may be effective. Developed over 50 years ago, surgical myectomy effectively relieves outflow tract obstruction by excising part of the septal myocardium involved in the dynamic obstruction. In selected patients, perioperative mortality is extremely low with excellent long-term survival free from recurrent obstruction and symptoms. Mitral valve repair or replacement is usually unnecessary as associated eccentric mitral regurgitation resolves with myectomy alone. Alcohol septal ablation in patients with suitable coronary anatomy can relieve outflow tract obstruction via a controlled infarction of the proximal septum, which produces similar periprocedural outcomes and gradient reduction as surgical myomectomy. Until long-term outcomes are demonstrated for this procedure, it is relegated primarily to patients who wish to avoid surgery or who have limiting comorbidities. Neither procedure has been shown to

1	Until long-term outcomes are demonstrated for this procedure, it is relegated primarily to patients who wish to avoid surgery or who have limiting comorbidities. Neither procedure has been shown to improve outcomes other than symptoms. With both procedures, the most common complication is the development of complete heart block necessitating permanent pacing. However, ventricular pacing as a primary therapy for outflow tract obstruction is ineffective and not generally advised.

1	Patients with hypertrophic cardiomyopathy have an increased risk of sudden cardiac death from ventricular tachyarrhythmias. Vigorous physical activity and competitive sport are prohibited. Factors that increase the risk of sudden death from a baseline of 0.5% per year are presented in Table 287-6. As sudden death has not been reduced by medical or procedural interventions, an implantable cardioverter-defibrillator is advised for patients with two or more risk factors and is advised on a selected basis for patient with one risk factor. Nevertheless, the positive predictive value of most risk factors is low, and many patients receiving a defibrillator

1	Use diuretics with caution to avoid hypovolemia, particularly in presence of outflow gradient Evidence of fluid retention? In all pts, evaluate risk for sudden death Symptomatic? If low follow with serial evaluation Try disopyramide or amiodarone Evidence of severe progressive LV dysfunction? Titrate beta blocker or calcium channel blockerIf high, consider ICD No Yes Yes No Yes Persistent symptoms Outflow gradient? Rarely, consider cardiac transplantation Refractory severe symptoms FIGURE 287-18 Treatment algorithm for hypertrophic cardiomyopathy depending on the presence and severity of symptoms and the presence of an intraventricular gradient with obstruction to outflow. Note that all patients with hypertrophic cardiomyopathy should be evaluated for atrial fibrillation and risk of sudden death, whether or not they require treatment for symptoms. ICD, implantable cardioverter-defibrillator; LV, left ventricular.

1	LV, left ventricular. never receive an appropriate therapy. Long-term use of a defibrillator may be associated with serious device-related complications, particularly in young active patients. Refinement of sudden death risk through the application of contemporary technologies such as cardiac MRI is ongoing. History of cardiac arrest or spontaneous sustained ventricular tachycardiaa Family history of sudden cardiac death Abnormal blood pressure response to exerciseb Nonvagal, often with or after exertion Present in <10% of patients Systolic blood pressure fall or failure to increase at peak exercise History

1	Present in <10% of patients Systolic blood pressure fall or failure to increase at peak exercise History Atrial fibrillation is common in patients with hypertrophic cardiomyopathy and may lead to hemodynamic deterioration and embolic stroke. Rapid ventricular response is poorly tolerated and may worsen outflow tract obstruction. β-Adrenergic blocking agents and L-type calcium channel blockers slow AV nodal conduction and improve symptoms; cardiac glycosides should be avoided, as they may increase contractility and worsen obstruction. Symptoms exacerbated by atrial fibrillation may persist despite adequate rate control due to loss of AV synchrony and may require restoration of sinus rhythm. Disopyramide and amiodarone are the preferred antiarrhythmic agents, with radiofrequency ablation considered for medically refractory cases. Anticoagulation to prevent embolic stroke in atrial fibrillation is recommended.

1	PROGNOSIS The general prognosis for hypertrophic cardiomyopathy is good, better than in early studies of referral populations. For patients diagnosed as adults, survival is comparable to an age-matched population without cardiomyopathy. The sudden death risk is less than 1% per year; however, up to 1 in 20 patients will progress to overt systolic dysfunction with a reduced ejection fraction with or without dilated remodeling (“burned out” or end-stage hypertrophic cardiomyopathy). These patients suffer from low cardiac output and have a high risk of death from progressive heart failure and sudden death unless they undergo cardiac transplantation. aImplantable cardioverter-defibrillator advised for patients with prior arrest or sustained ventricular tachycardia regardless of other risk factors. b Prognostic value most applicable to patients less than 40 years old. Abbreviation: LV, left ventricle.

1	Pericardial Disease Eugene Braunwald NORMAL FUNCTIONS OF THE PERICARDIUM The normal pericardium is a double-layered sac; the visceral peri-cardium is a serous membrane that is separated by a small quantity (15–50 mL) of fluid, an ultrafiltrate of plasma, from the fibrous parietal 288 pericardium. The normal pericardium, by exerting a restraining force, prevents sudden dilation of the cardiac chambers, especially the right atrium and ventricle, during exercise and with hypervolemia. It also restricts the anatomic position of the heart, and probably retards the spread of infections from the lungs and pleural cavities to the heart. Nevertheless, total absence of the pericardium, either congenital or after surgery, does not produce obvious clinical disease. In partial left pericardial defects, the main pulmonary artery and left atrium may bulge through the defect; very rarely, herniation and subsequent strangulation of the left atrium may cause sudden death.

1	Acute pericarditis, by far the most common pathologic process involving the pericardium (Table 288-1), has four principal diagnostic features: 1.

1	Chest pain is usually present in acute infectious pericarditis and in many of the forms presumed to be related to hypersensitivity or autoimmunity. The pain of acute pericarditis is often severe, retrosternal, and left precordial, and referred to the neck, arms, or left shoulder. Frequently the pain is pleuritic, consequent to accompanying pleural inflammation (i.e., sharp and aggravated by inspiration and coughing), but sometimes it is steady, constricting, radiates into either arm or both arms, and resembles that of myocardial ischemia; therefore, confusion with acute myocardial infarction (AMI) is common. Characteristically, however, pericardial pain may be relieved by sitting up and leaning forward and is intensified by lying supine (Chap. 19). Pain is often absent in slowly developing tuberculous, postirradiation, and neoplastic, uremic, and constrictive pericarditis.

1	The differentiation of AMI from acute pericarditis may become perplexing when, with acute pericarditis, serum biomarkers of myocardial damage such as troponin and creatine kinase-MB rise, presumably because of concomitant involvement of the epicardium in the inflammatory process (an epi-myocarditis) with resulting myocyte necrosis. However, these elevations, if they occur, are quite modest given the extensive electrocardiographic ST-segment elevation in pericarditis. This dissociation is useful in differentiating between these conditions. 2. A pericardial friction rub is audible at some point in about 85% of patients with acute pericarditis, may have up to three components per cardiac cycle, is high-pitched, and is described as rasping, scratching, or grating (Chap. 267). It is heard most frequently at end expiration with the patient upright and leaning forward. 3.

1	3. The electrocardiogram (ECG) in acute pericarditis without massive effusion usually displays changes secondary to acute subepicardial inflammation (Fig. 288-1). It typically evolves through four stages. In stage 1, there is widespread elevation of the ST segments, often with upward concavity, involving two or three standard limb leads and V2 to V6, with reciprocal depressions only in aVR and sometimes V1. Also, there is depression of the PR segment below the TP segment, reflecting atrial involvement. Usually there are no significant changes in QRS complexes. After several days, the ST segments return to normal (stage 2), and only then, or even later, do the T waves become inverted (stage 3). Weeks or months after the onset of acute pericarditis, the ECG returns to normal (stage 4). In contrast, in AMI, ST elevations are convex, and reciprocal depression is usually more prominent; these changes may return to normal within a I. Acute pericarditis (<6 weeks) A. Fibrinous

1	I. Acute pericarditis (<6 weeks) A. Fibrinous B. Effusive (serous or sanguineous) II. Subacute pericarditis (6 weeks to 6 months) A. B. III. A. B. C. I. Infectious pericarditis A. Viral (coxsackievirus A and B, echovirus, mumps, adenovirus, hepatitis, HIV) B. Pyogenic (pneumococcus, Streptococcus, Staphylococcus, Neisseria, Legionella) C. Tuberculous D. Fungal (histoplasmosis, coccidioidomycosis, Candida, blastomycosis) E. Other infections (syphilitic, protozoal, parasitic) II. A. B. C. 1. Primary tumors (benign or malignant, mesothelioma) 2. Tumors metastatic to pericardium (lung and breast cancer, lymphoma, leukemia) D. Myxedema E. Cholesterol F. Chylopericardium G. Trauma 1. 2. H. Aortic dissection (with leakage into pericardial sac) I. Postirradiation J. Familial Mediterranean fever K. Familial pericarditis 1. Mulibrey nanisma L. Acute idiopathic M. Whipple’s disease N. Sarcoidosis III.

1	I. Postirradiation J. Familial Mediterranean fever K. Familial pericarditis 1. Mulibrey nanisma L. Acute idiopathic M. Whipple’s disease N. Sarcoidosis III. Pericarditis presumably related to hypersensitivity or autoimmunity A. B. Collagen vascular disease (systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, scleroderma, acute rheumatic fever, granulomatosis with polyangiitis [Wegener’s]) C. Drug-induced (e.g., procainamide, hydralazine, phenytoin, isoniazid, minoxidil, anticoagulants, methysergide) D. 1. 2. 3. aAn autosomal recessive syndrome characterized by growth failure, muscle hypotonia, hepatomegaly, ocular changes, enlarged cerebral ventricles, mental retardation, ventricular hypertrophy, and chronic constrictive pericarditis day or two. Q waves may develop, with loss of R-wave amplitude, and T-wave inversions are usually seen within hours before the ST segments have become isoelectric (Chaps. 294 and 295).

1	4. Pericardial effusion is usually associated with pain and/or the ECG changes mentioned above, as well as electrical alternans. FIGURE 288-1 Acute pericarditis. There are diffuse ST-segment elevations (in this case in leads I, II, aVF, and V2 to V6) due to a ventricular current of injury. There is PR-segment deviation (opposite in polarity to the ST segment) due to a concomitant atrial injury current.

1	Pericardial effusion is especially important clinically when it develops within a relatively short time because it may lead to cardiac tamponade (see below). Differentiation from cardiac enlargement may be difficult on physical examination, but heart sounds may be fainter with pericardial effusion. The friction rub and the apex impulse may disappear. The base of the left lung may be compressed by pericardial fluid, producing Ewart’s sign, a patch of dullness and increased fremitus (and egophony) beneath the angle of the left scapula. The chest roentgenogram may show enlargement of the cardiac silhouette, with a “water bottle” configuration, but may be normal.

1	Diagnosis Echocardiography (Chap. 270e) is the most widely used imaging technique. It is sensitive, specific, simple, noninvasive, may be performed at the bedside, and can identify accompanying cardiac tamponade (see below) (Fig. 288-2). The presence of pericardial fluid is recorded by two-dimensional transthoracic echocardiography as a relatively echo-free space between the posterior pericardium and left ventricular epicardium in patients with small effusions and as a space between the anterior right ventricle and the parietal pericardium just beneath the anterior chest wall. In patients with large effusions, the a patient with a large pericardial effusion. Ao, aorta; LV, left ventricle; heart may swing freely within the pericardial sac. When severe, the pe, pericardial effusion; RV, right ventricle. (From M Imazio: Curr Opin extent of this motion alternates and may be associated with electrical

1	Cardiol 27:308, 2012.) alternans (Fig. 288-3). Echocardiography allows localization and identification of the quantity of pericardial fluid. FIGURE 288-3 Electrical alternans. This tracing was obtained from a patient with a large pericardial effusion with tamponade. (Reproduced from DM Mirvis, AL Goldberger: Electrocardiography, in RO Bonowet al [eds]: Braunwald’s Heart Disease, 9th ed. Philadelphia: Elsevier, 2012.) The diagnosis of pericardial fluid or thickening may be confirmed by computed tomography (CT) or magnetic resonance imaging (MRI). These techniques may be superior to echocardiography in detecting loculated pericardial effusions, pericardial thickening, and the identification of pericardial masses.

1	There is no specific therapy for acute idiopathic pericarditis, but bed rest and anti-inflammatory treatment with aspirin (2–4 g/d), with gastric protection (e.g., omeprazole 20 mg/d), may be given. If this is ineffective, one of the nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen (400–600 mg tid) or indomethacin (25–50 mg tid), should be tried. In responsive patients, these doses should be continued for 1–2 weeks and then tapered over several weeks. In patients who are unresponsive, colchicine (0.5 mg bid, given for 4–8 weeks) has been found to be effective, not only in acute pericarditis, but also in reducing the risk of recurrent pericarditis. Colchicine is concentrated in and interferes with the migration of neutrophils, is contraindicated in patients with hepatic or renal dysfunction, and may cause diarrhea and other gastrointestinal side effects. Glucocorticoids (e.g., prednisone 1 mg/kg per day) usually suppress the clinical manifestations of acute pericarditis

1	dysfunction, and may cause diarrhea and other gastrointestinal side effects. Glucocorticoids (e.g., prednisone 1 mg/kg per day) usually suppress the clinical manifestations of acute pericarditis in patients who have failed therapy with the anti-inflammatory therapies described above, but appear to increase the risk of subsequent recurrence. Therefore, full-dose corticosteroids should be given for only 2–4 days and then tapered. Anticoagulants should be avoided because their use could cause bleeding into the pericardial cavity and tamponade.

1	In patients with recurrences that are multiple, frequent, disabling, continue for more than 2 years, and are not prevented by colchicine and other NSAIDs and are not controlled by glucocorticoids, pericardial stripping may be necessary to terminate the illness, and 1573 usually does so. The accumulation of fluid in the pericardial space in a quantity sufficient to cause serious obstruction of the inflow of blood into the ventricles results in cardiac tamponade. This complication may be fatal if it is not recognized and treated promptly. The most common causes of tamponade are idiopathic pericarditis and pericarditis secondary to neoplastic disease. Tamponade may also result from bleeding into the pericardial space after leakage from an aortic dissection, cardiac operations, trauma, and treatment of patients with acute pericarditis with anticoagulants.

1	The three principal features of tamponade (Beck’s triad) are hypo-tension, soft or absent heart sounds, and jugular venous distention with a prominent x descent but an absent y descent. The limitations of ventricular filling are responsible for a reduction of cardiac output. The quantity of fluid necessary to produce cardiac tamponade may be as small as 200 mL when the fluid develops rapidly to as much as >2000 mL in slowly developing effusions when the pericardium has had the opportunity to stretch and adapt to an increasing volume. Tamponade may also develop more slowly, and in these circumstances, the clinical manifestations can resemble those of heart failure, including dyspnea, orthopnea, and hepatic engorgement.

1	A high index of suspicion for cardiac tamponade is required because in many instances no obvious cause for pericardial disease is apparent, and this diagnosis should be considered in any patient with otherwise unexplained enlargement of the cardiac silhouette, hypotension, and elevation of jugular venous pressure. There may be reduction in amplitude of the QRS complexes, and electrical alternans of the P, QRS, or T waves should raise the suspicion of cardiac tamponade (Fig. 288-3). Table 288-2 lists the features that distinguish acute cardiac tamponade from constrictive pericarditis. Thickened pericardium – +++ – ++ Cardiac catheterization Equalization of diastolic +++ +++ – ++ pressures Abbreviations: +++, always present; ++, usually present; +, rare;–, absent; DC, diastolic collapse; ECG, electrocardiograph; RA, right atrium; RV, right ventricle; RVMI, right ventricular myocardial infarction. Source: Adapted from GM Brockington et al: Cardiol Clin 8:645, 1990, with permission.

1	Source: Adapted from GM Brockington et al: Cardiol Clin 8:645, 1990, with permission. 1574 Paradoxical Pulse This important clue to the presence of cardiac tamponade consists of a greater than normal (10 mmHg) inspiratory decline in systolic arterial pressure. When severe, it may be detected by palpating weakness or disappearance of the arterial pulse during inspiration, but usually sphygmomanometric measurement of systolic pressure during slow respiration is required.

1	Because both ventricles share a tight incompressible covering, i.e., the pericardial sac, the inspiratory enlargement of the right ventricle in cardiac tamponade compresses and reduces left ventricular volume; leftward bulging of the interventricular septum reduces further the left ventricular cavity as the right ventricle enlarges during inspiration. Thus, in cardiac tamponade, the normal inspiratory augmentation of right ventricular volume causes an exaggerated reduction of left ventricular volume, stroke volume, and systolic pressure. Paradoxical pulse also occurs in approximately one-third of patients with constrictive pericarditis (see below), and in some cases of hypovolemic shock, acute and chronic obstructive airway disease, and pulmonary embolus. Right ventricular infarction (Chap. 295) may resemble cardiac tamponade with hypotension, elevated jugular venous pressure, an absent y descent in the jugular venous pulse, and, occasionally, a paradoxical pulse (Table 288-2).

1	Low-pressure tamponade refers to mild tamponade in which the intrapericardial pressure is increased from its slightly subatmospheric levels to +5 to +10 mmHg; in some instances, hypovolemia coexists. As a consequence, the central venous pressure is normal or only slightly elevated, whereas arterial pressure is unaffected and there is no paradoxical pulse. These patients are asymptomatic or complain of mild weakness and dyspnea. The diagnosis is aided by echocardiography, and both hemodynamic and clinical manifestations improve after pericardiocentesis.

1	Diagnosis Because immediate treatment of cardiac tamponade may be lifesaving, prompt measures to establish the diagnosis by echocardiography should be undertaken. When pericardial effusion causes tamponade, Doppler ultrasound shows that tricuspid and pulmonic valve flow velocities increase markedly during inspiration, whereas pulmonic vein, mitral, and aortic flow velocities diminish (as in constrictive pericarditis, see below) (Fig. 288-4). In tamponade, there is late diastolic inward motion (collapse) of the right ventricular free wall and the right atrium. Transesophageal echocardiography, CT, or cardiac MRI may be necessary to diagnose a loculated effusion responsible for cardiac tamponade.

1	FIGURE 288-4 Constrictive pericarditis. Doppler schema of respirophasic changes in mitral and tricuspid inflow. Reciprocal patterns of ventricular filling are assessed on pulsed Doppler examination of mitral valve (MV) and tricuspid valve (TV) inflow. IVC, inferior vena cava; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. (Courtesy of Bernard E. Bulwer, MD; with permission.) Patients with acute pericarditis should be observed frequently for the development of an effusion; if a large effusion is present, pericardiocentesis should be carried out or the patient watched closely for signs of tamponade. Arterial and venous pressures should be monitored and serial echocardiograms obtained.

1	If manifestations of tamponade appear, echocardiographically guided pericardiocentesis using an apical, parasternal, or, most commonly, subxiphoid approach must be carried out at once because reduction of the elevated intrapericardial pressure may be lifesaving. Intravenous saline may be administered as the patient is being readied for the procedure, but the pericardiocentesis must not be delayed. If possible, intrapericardial pressure should be measured before fluid is withdrawn, and the pericardial cavity should be drained as completely as possible. A small, multiholed catheter advanced over the needle inserted into the pericardial cavity may be left in place to allow draining of the pericardial space if fluid reaccumulates. Surgical drainage through a limited (subxiphoid) thoracotomy may be required in recurrent tamponade, when it is necessary to remove loculated effusions, and/or when it is necessary to obtain tissue for diagnosis.

1	Pericardial fluid obtained from an effusion often has the physical characteristics of an exudate. Bloody fluid is most commonly due to neoplasm, renal failure, or dialysis in the United States and tuberculosis in developing nations but may also be found in the effusion of acute rheumatic fever, after cardiac injury, and after myocardial infarction. Transudative pericardial effusions may occur in heart failure. The pericardial fluid should be analyzed for red and white blood cells and cytologic studies, and cultures should be obtained. The presence of DNA of Mycobacterium tuberculosis determined by the polymerase chain reaction strongly supports the diagnosis of tuberculous pericarditis (Chap. 202).

1	In many instances, acute pericarditis occurs in association with illnesses of known or presumed viral origin and probably is caused by the same agent. Commonly, there is an antecedent infection of the respiratory tract, and viral isolation and serologic studies are negative. In some cases, coxsackievirus A or B or the virus of influenza, echovirus, mumps, herpes simplex, chickenpox, adenovirus, or cytomegalovirus has been isolated from pericardial fluid and/or appropriate elevations in viral antibody titers have been noted. Pericardial effusion is a common cardiac manifestation of HIV; it is usually secondary to infection (often mycobacterial) or neoplasm, most often lymphoma. Frequently, a viral cause cannot be established, and the term idiopathic acute pericarditis is then appropriate.

1	Viral or idiopathic acute pericarditis occurs at all ages but is more common in young adults and is often associated with pleural effusions and pneumonitis. The almost simultaneous development of fever and precordial pain, often 10–12 days after a presumed viral illness, constitutes an important feature in the differentiation of acute pericarditis from AMI, in which chest pain precedes fever. The constitutional symptoms are usually mild to moderate, and a pericardial friction rub is often audible. The disease ordinarily runs its course in a few days to 4 weeks. The ST-segment alterations in the ECG usually disappear after 1 or more weeks, but the abnormal T waves may persist for several years and be a source of confusion in persons without a clear history of pericarditis. Pleuritis and pneumonitis frequently accompany viral or idiopathic acute pericarditis. Accumulation of some pericardial fluid is common, and both tamponade and constrictive pericarditis are possible, but infrequent,

1	frequently accompany viral or idiopathic acute pericarditis. Accumulation of some pericardial fluid is common, and both tamponade and constrictive pericarditis are possible, but infrequent, complications.

1	The most frequent complication is recurrent (relapsing) pericarditis, which occurs in about one-fourth of patients with acute idiopathic pericarditis. In a smaller number, there are multiple recurrences. For treatment, see earlier section on treatment of acute pericarditis. Postcardiac Injury Syndrome Acute pericarditis may appear in a variety of circumstances that have one common feature—previous injury to the myocardium with blood in the pericardial cavity. The syndrome may develop after a cardiac operation (postpericardiotomy syndrome), after blunt or penetrating cardiac trauma (Chap. 289e), or after perforation of the heart with a catheter. Rarely, it follows AMI.

1	The clinical picture mimics acute viral or idiopathic pericarditis. The principal symptom is the pain of acute pericarditis, which usually develops 1–4 weeks after the cardiac injury but earlier (1–3 days) after AMI. Recurrences are common and may occur up to 2 years or more following the injury. Fever, pleuritis, and pneumonitis are the outstanding features, and the bout of illness usually subsides in 1 or 2 weeks. The pericarditis may be of the fibrinous variety, or it may be a pericardial effusion, which is often serosanguineous but rarely causes tamponade. ECG changes typical of acute pericarditis may also occur. This syndrome is probably the result of a hypersensitivity reaction to antigen(s) that originate from injured myocardial tissue and/or pericardium.

1	Often no treatment is necessary aside from aspirin and analgesics. When the illness is severe or followed by a series of disabling recurrences, therapy with an NSAID, colchicine, or a glucocorticoid, such as described for treatment of acute pericarditis, is usually effective.

1	Because there is no specific test for acute idiopathic pericarditis, the diagnosis is one of exclusion. Consequently, all other disorders that may be associated with acute fibrinous pericarditis must be considered. A common diagnostic error is mistaking acute viral or idiopathic pericarditis for AMI and vice versa. When acute fibrinous pericarditis is associated with AMI (Chap. 295), it is characterized by fever, pain, and a friction rub in the first 4 days after the development of the infarct. ECG abnormalities (such as the appearance of Q waves, brief ST-segment elevations with reciprocal changes, and earlier T-wave changes in AMI) and the extent of the elevations of markers of myocardial necrosis (higher in AMI) are helpful in differentiating pericarditis from AMI.

1	Pericarditis secondary to postcardiac injury is differentiated from acute idiopathic pericarditis chiefly by timing. If it occurs within a few days or weeks of an AMI, a chest blow, a cardiac perforation, or a cardiac operation, it may be justified to conclude that the two are probably related. It is important to distinguish pericarditis due to collagen vascular disease from acute idiopathic pericarditis. Most important in the differential diagnosis is the pericarditis due to systemic lupus erythematosus (SLE; Chap. 378) or drug-induced (procainamide or hydralazine) lupus. When pericarditis occurs in the absence of any obvious underlying disorder, the diagnosis of SLE may be suggested by a rise in the titer of antinuclear antibodies. Acute pericarditis is an occasional complication of rheumatoid arthritis, scleroderma, and polyarteritis nodosa, and other evidence of these diseases is usually obvious.

1	Pyogenic (purulent) pericarditis is usually secondary to cardiothoracic operations, by extension of infection from the lungs or pleural cavities, from rupture of the esophagus into the pericardial sac, or from rupture of a ring abscess in a patient with infective endocarditis. It may also complicate the viral, pyogenic, mycobacterial, and fungal infections that occur with HIV infection. It is generally accompanied by fever, chills, septicemia, and evidence of infection elsewhere and generally has a poor prognosis. The diagnosis is made by examination of the pericardial fluid. It requires drainage as well as vigorous antibiotic treatment.

1	Pericarditis of renal failure occurs in up to one-third of patients with chronic uremia (uremic pericarditis), and is also seen in patients undergoing chronic dialysis who have normal levels of blood urea and creatinine (dialysis-associated pericarditis). These two forms of pericarditis may be fibrinous and are generally associated with serosanguineous effusions. A pericardial friction rub is common, but pain is usually absent 1575 or mild. Treatment with an NSAID and intensification of dialysis are usually adequate. Occasionally, tamponade occurs and pericardiocentesis is required. When the pericarditis of renal failure is recurrent or persistent, a pericardial window should be created or pericardiectomy may be necessary.

1	Pericarditis due to neoplastic diseases results from extension or invasion of metastatic tumors (most commonly carcinoma of the lung and breast, malignant melanoma, lymphoma, and leukemia) to the pericardium; pain, atrial arrhythmias, and tamponade are complica tions that occur occasionally. Diagnosis is made by pericardial fluid cytology or pericardial biopsy. Mediastinal irradiation for neoplasm may cause acute pericarditis and/or chronic constrictive pericarditis. Unusual causes of acute pericarditis include syphilis, fungal infection (histoplasmosis, blastomycosis, aspergillosis, and candidiasis), and parasitic infestation (amebiasis, toxoplasmosis, echinococcosis, and trichinosis).

1	Chronic pericardial effusions are sometimes encountered in patients without an antecedent history of acute pericarditis. They may cause few symptoms per se, and their presence may be detected by finding an enlarged cardiac silhouette on a chest roentgenogram. Tuberculosis is a common cause. Myxedema may be responsible for chronic pericardial effusion that is sometimes massive but rarely, if ever, causes cardiac tamponade. The cardiac silhouette may be markedly enlarged, and an echocardiogram distinguishes cardiomegaly from pericardial effusion. The diagnosis of myxedema can be confirmed by tests of thyroid function (Chap. 405). Myxedematous pericardial effusion responds to thyroid hormone replacement. Neoplasms, SLE, rheumatoid arthritis, mycotic infections, radiation therapy to the chest, pyogenic infections, and chylopericardium may also cause chronic pericardial effusion and should be considered and specifically sought in such patients.

1	Aspiration and analysis of the pericardial fluid are often helpful in diagnosis. Pericardial fluid should be analyzed as described in pericardiocentesis. Grossly sanguineous pericardial fluid results most commonly from a neoplasm, tuberculosis, renal failure, or slow leakage from an aortic dissection. Pericardiocentesis may resolve large effusions, but pericardiectomy may be required in patients with recurrence. Intrapericardial instillation of sclerosing agents may be used to prevent reaccumulation of fluid.

1	This disorder results when the healing of an acute fibrinous or serofibrinous pericarditis or the resorption of a chronic pericardial effusion is followed by obliteration of the pericardial cavity with the formation of granulation tissue. The latter gradually contracts and forms a firm scar encasing the heart, which may be calcified. In developing nations where the condition is prevalent, a high percentage of cases are of tuberculous origin, but this is now an uncommon cause in North America. Chronic constrictive pericarditis may follow acute or relapsing viral or idiopathic pericarditis, trauma with organized blood clot, or cardiac surgery of any type or result from mediastinal irradiation, purulent infection, histoplasmosis, neoplastic disease (especially breast cancer, lung cancer, and lymphoma), rheumatoid arthritis, SLE, or chronic renal failure treated by chronic dialysis. In many patients, the cause of the pericardial disease is undetermined, and in these patients, an

1	and lymphoma), rheumatoid arthritis, SLE, or chronic renal failure treated by chronic dialysis. In many patients, the cause of the pericardial disease is undetermined, and in these patients, an asymptomatic or forgotten bout of viral pericarditis, acute or idiopathic, may have been the inciting event.

1	The basic physiologic abnormality in patients with chronic constrictive pericarditis is the inability of the ventricles to fill because of the limitations imposed by the rigid, thickened pericardium. Ventricular filling is unimpeded during early diastole but is reduced abruptly when the elastic limit of the pericardium is reached, whereas in cardiac tamponade, ventricular filling is impeded throughout diastole. In both conditions, ventricular end-diastolic and stroke volumes are reduced and the end-diastolic pressures in both ventricles and 1576 the mean pressures in the atria, pulmonary veins, and systemic veins are all elevated to similar levels (i.e., within 5 mmHg of one another). Despite these hemodynamic changes, systolic function may be normal or only slightly impaired. However, in advanced cases, the fibrotic process may extend into the myocardium and cause myocardial scarring and atrophy, and venous congestion may then be due to the combined effects of the pericardial and

1	in advanced cases, the fibrotic process may extend into the myocardium and cause myocardial scarring and atrophy, and venous congestion may then be due to the combined effects of the pericardial and myocardial lesions. In constrictive pericarditis, the right and left atrial pressure pulses display an M-shaped contour, with prominent x and y descents. The y descent, which is absent or diminished in cardiac tamponade, is the most prominent deflection in constrictive pericarditis; it reflects rapid early filling of the ventricles. The y descent is interrupted by a rapid rise in atrial pressure during early diastole, when ventricular filling is impeded by the constricting pericardium. These characteristic changes are transmitted to the jugular veins, where they may be recognized by inspection. In constrictive pericarditis, the ventricular pressure pulses in both ventricles exhibit characteristic “square root” signs during diastole. These hemodynamic changes, although characteristic, are

1	In constrictive pericarditis, the ventricular pressure pulses in both ventricles exhibit characteristic “square root” signs during diastole. These hemodynamic changes, although characteristic, are not pathognomonic of constrictive pericarditis and may also be observed in restrictive cardiomyopathies (Chap. 287, Table 287-2).

1	Weakness, fatigue, weight gain, increased abdominal girth, abdominal discomfort, and edema are common. The patient often appears chronically ill, and in advanced cases, anasarca, skeletal muscle wasting, and cachexia may be present. Exertional dyspnea is common, and orthopnea may occur, although it is usually not severe. Acute left ventricular failure (acute pulmonary edema) is very uncommon. The cervical veins are distended and may remain so even after intensive diuretic treatment, and venous pressure may fail to decline during inspiration (Kussmaul’s sign). The latter is common in chronic pericarditis but may also occur in tricuspid stenosis, right ventricular infarction, and restrictive cardiomyopathy.

1	The pulse pressure is normal or reduced. A paradoxical pulse can be detected in about one-third of cases. Congestive hepatomegaly is pronounced and may impair hepatic function and cause jaundice; ascites is common and is usually more prominent than dependent edema. The apical pulse is reduced and may retract in systole (Broadbent’s sign). The heart sounds may be distant; an early third heart sound (i.e., a pericardial knock, occurring at the cardiac apex 0.09–0.12 s after aortic valve closure) with the abrupt cessation of ventricular filling is often conspicuous.

1	The ECG frequently displays low voltage of the QRS complexes and diffuse flattening or inversion of the T waves. Atrial fibrillation is present in about one-third of patients. The chest roentgenogram shows a normal or slightly enlarged heart. Pericardial calcification is most common in tuberculous pericarditis. Pericardial calcification may, however, occur in the absence of constriction, and constriction may occur without calcification. Inasmuch as the usual physical signs of cardiac disease (murmurs, cardiac enlargement) may be inconspicuous or absent in chronic constrictive pericarditis, hepatic enlargement and dysfunction associated with jaundice and intractable ascites may lead to a mistaken diagnosis of hepatic cirrhosis. This error can be avoided if the neck veins are inspected and found to be distended.

1	The transthoracic echocardiogram typically shows pericardial thickening, dilation of the inferior vena cava and hepatic veins, and a sharp halt in ventricular filling in early diastole, with normal ventricular systolic function and flattening of the left ventricular posterior wall. There is a distinctive pattern of transvalvular flow velocity on Doppler echocardiography. During inspiration, there is an exaggerated reduction in blood flow velocity in the pulmonary veins and across the mitral valve and a leftward shift of the ventricular septum; the opposite occurs during expiration. Diastolic flow velocity in the inferior vena cava into the right atrium and across the tricuspid valve increases in an exaggerated manner during inspiration and declines during expiration (Fig. 288-4). However, echocardiography cannot definitively exclude the diagnosis of constrictive pericarditis. CT and MRI scanning (Fig. 288-5) are more accurate than echocardiography in establishing or excluding the

1	echocardiography cannot definitively exclude the diagnosis of constrictive pericarditis. CT and MRI scanning (Fig. 288-5) are more accurate than echocardiography in establishing or excluding the presence of a thickened pericardium.

1	FIGURE 288-5 Magnetic resonance imaging in chronic constric-tive pericarditis. The arrows point to a thickened pericardium, which shows late enhancement after gadolinium, characteristic of intense inflammation. LV, left ventricle; RV, right ventricle. (From RY Kwong: Cardiovascular magnetic resonance imaging, in RO Bonow et al [eds]: Braunwald’s Heart Disease, 9th ed. Philadelphia: Elsevier, 2012.)

1	Like chronic constrictive pericarditis, cor pulmonale (Chap. 279) may be associated with severe systemic venous hypertension but little pulmonary congestion; the heart is usually not enlarged, and a paradoxical pulse may be present. However, in cor pulmonale, advanced parenchymal pulmonary disease is usually apparent and venous pressure falls during inspiration (i.e., Kussmaul’s sign is negative). Tricuspid stenosis (Chap. 283) may also simulate chronic constrictive pericarditis; congestive hepatomegaly, splenomegaly, ascites, and venous distention may be equally prominent. However, in tricuspid stenosis, a characteristic murmur and the murmur of accompanying mitral stenosis are usually present.

1	Because constrictive pericarditis can be corrected surgically, it is important to distinguish chronic constrictive pericarditis from restrictive cardiomyopathy (Chap. 287), which has a similar physiologic abnormality (i.e., restriction of ventricular filling). The differentiating features are summarized in Table 288-2. When a patient has progressive, disabling, and unresponsive congestive heart failure and displays any of the features of constrictive heart disease, Doppler echocardiography to record respiratory effects on transvalvular flow and an MRI or CT scan should be obtained to detect or exclude constrictive pericarditis, because the latter is usually correctable.

1	Pericardial resection is the only definitive treatment of constrictive pericarditis and should be as complete as possible. Dietary sodium restriction and diuretics are useful during preoperative preparation. Coronary arteriography should be carried out preoperatively in patients older than 50 years to exclude unsuspected accompanying coronary artery disease. The benefits derived from cardiac decortication are usually progressive over a period of months. The risk of this operation depends on the extent of penetration of the myocardium by the fibrotic and calcific process, the severity of myocardial atrophy, the extent of secondary impairment of hepatic and/or renal function, and the patient’s general condition. Operative mortality is in the range of 5 to 10% even in experienced centers; the patients with the most severe disease are at highest risk. Therefore, surgical 1577 treatment should, if possible, be carried out as early as possible in the course.

1	Subacute Effusive-Constrictive Pericarditis This form of pericardial disease is characterized by the combination of a tense effusion in the pericardial space and constriction of the heart by thickened pericardium. It shares a number of features with both chronic pericardial effusion producing cardiac compression and with pericardial constriction. It may be caused by tuberculosis (see below), multiple attacks of acute idiopathic pericarditis, radiation, traumatic pericarditis, renal failure, scleroderma, and neoplasms. The heart is generally enlarged, and a paradoxical pulse and a prominent x descent (without a prominent y descent) are present in the atrial and jugular venous pressure pulses. After pericardiocentesis, the physiologic findings may change from those of cardiac tamponade to those of pericardial constriction. Furthermore, the intrapericardial pressure and the central venous pressure may decline, but not to normal. The diagnosis can be established by pericardiocentesis

1	to those of pericardial constriction. Furthermore, the intrapericardial pressure and the central venous pressure may decline, but not to normal. The diagnosis can be established by pericardiocentesis followed by pericardial biopsy. Wide excision of both the visceral and parietal pericardium is usually effective therapy.

1	Tuberculous Pericardial disease This chronic infection is a common cause of chronic pericardial effusion, although less so in North America than in the developing world where active tuberculosis is endemic. The clinical picture is that of a chronic, systemic illness in a patient with pericardial effusion. It is important to consider this diagnosis in a patient with known tuberculosis, with HIV, and with fever, chest pain, weight loss, and enlargement of the cardiac silhouette of undetermined origin. If the etiology of chronic pericardial effusion remains obscure despite detailed analysis of the pericardial fluid (see above), a pericardial biopsy, preferably by a limited thoracotomy, should be performed. If definitive evidence is still lacking but the specimen shows granulomas with caseation, antituberculous chemotherapy (Chap. 202) is indicated.

1	If the biopsy specimen shows a thickened pericardium after 2–4 weeks of antituberculin therapy, pericardiectomy should be carried out to prevent the development of constriction. Tubercular cardiac constriction should be treated surgically while the patient is receiving antituberculous chemotherapy.

1	Pericardial cysts appear as rounded or lobulated deformities of the cardiac silhouette, most commonly at the right cardiophrenic angle. They usually do not cause symptoms, and their major clinical significance lies in the possibility of confusion with a tumor, ventricular aneurysm, or massive cardiomegaly. Tumors involving the pericardium are most commonly secondary to malignant neoplasms originating in or invading the mediastinum, including carcinoma of the bronchus and breast, lymphoma, and melanoma. Mesothelioma is the most common primary malignant tumor. The usual clinical picture of malignant pericardial tumor is an insidiously developing, often bloody pericardial effusion. Surgical exploration is required to establish a definitive diagnosis and to carry out definitive or, more commonly, palliative treatment. Heart Eric H. Awtry, Wilson S. Colucci TUMORS OF THE HEART PRIMARY TUMORS 289e Tumors and Trauma of the

1	Heart Eric H. Awtry, Wilson S. Colucci TUMORS OF THE HEART PRIMARY TUMORS 289e Tumors and Trauma of the Primary tumors of the heart are rare. Approximately three-quarters are histologically benign, and the majority of these tumors are myxomas. Malignant tumors, almost all of which are sarcomas, account for 25% of primary cardiac tumors. All cardiac tumors, regardless of pathologic type, have the potential to cause life-threatening complications. Many tumors are now surgically curable; thus, early diagnosis is imperative.

1	Clinical Presentation Cardiac tumors may present with a wide array of cardiac and noncardiac manifestations. These manifestations depend in large part on the location and size of the tumor and are often nonspecific features of more common forms of heart disease, such as chest pain, syncope, heart failure, murmurs, arrhythmias, conduction disturbances, and pericardial effusion with or without tamponade. Additionally, embolic phenomena and constitutional symptoms may occur.

1	Myxoma Myxomas are the most common type of primary cardiac tumor in adults, accounting for one-third to one-half of all cases at postmortem examination, and about three-quarters of the tumors treated surgically. They occur at all ages, most commonly in the third through sixth decades, with a female predilection. Approximately 90% of myxomas are sporadic; the remainder are familial with autosomal dominant transmission. The familial variety often occurs as part of a syndrome complex (Carney complex) that includes (1) myxomas (cardiac, skin, and/or breast), (2) lentigines and/or pigmented nevi, and (3) endocrine overactivity (primary nodular adrenal cortical disease with or without Cushing’s syndrome, testicular tumors, and/ or pituitary adenomas with gigantism or acromegaly). Certain constellations of findings have been referred to as the NAME syndrome (nevi, atrial myxoma, myxoid neurofibroma, and ephelides) or the LAMB syndrome (lentigines, atrial myxoma, and blue nevi), although

1	of findings have been referred to as the NAME syndrome (nevi, atrial myxoma, myxoid neurofibroma, and ephelides) or the LAMB syndrome (lentigines, atrial myxoma, and blue nevi), although these syndromes probably represent subsets of the Carney complex. The genetic basis of this complex has not been elucidated completely; however, patients frequently have inactivating mutations in the tumor-suppressor gene PRKAR1A, which encodes the protein kinase A type I-α regulatory subunit.

1	Pathologically, myxomas are gelatinous structures that consist of 289e-1 myxoma cells embedded in a stroma rich in glycosaminoglycans. Most are solitary, arise from the interatrial septum in the vicinity of the fossa ovalis (particularly the left atrium), and are often pedunculated on a fibrovascular stalk. In contrast to sporadic tumors, familial or syndromic tumors tend to occur in younger individuals, are often multiple, may be ventricular in location, and are more likely to recur after initial resection.

1	Myxomas commonly present with obstructive signs and symptoms. The most common clinical presentation mimics that of mitral valve disease: either stenosis owing to tumor prolapse into the mitral orifice or regurgitation resulting from tumor-induced valvular trauma. Ventricular myxomas may cause outflow obstruction similar to that caused by subaortic or subpulmonic stenosis. The symptoms and signs of myxoma may be sudden in onset or positional in nature, owing to the effects of gravity on tumor position. A characteristic low-pitched sound, a “tumor plop,” may be appreciated on auscultation during early or mid-diastole and is thought to result from the impact of the tumor against the mitral valve or ventricular wall. Myxomas also may present with peripheral or pulmonary emboli or with constitutional signs and symptoms, including fever, weight loss, cachexia, malaise, arthralgias, rash, digital clubbing, Raynaud’s phenomenon, hypergammaglobulinemia, anemia, polycythemia, leukocytosis,

1	signs and symptoms, including fever, weight loss, cachexia, malaise, arthralgias, rash, digital clubbing, Raynaud’s phenomenon, hypergammaglobulinemia, anemia, polycythemia, leukocytosis, elevated erythrocyte sedimentation rate, thrombocytopenia, and thrombocytosis. These features account for the frequent misdiagnosis of patients with myxomas as having endocarditis, collagen vascular disease, or a paraneoplastic syndrome.

1	Two-dimensional transthoracic or omniplane transesophageal echocardiography is useful in the diagnosis of cardiac myxoma and allows assessment of tumor size and determination of the site of tumor attachment, both of which are important considerations in the planning of surgical excision (Fig. 289e-1). Computed tomography (CT) and magnetic resonance imaging (MRI) may provide important information regarding size, shape, composition, and surface characteristics of the tumor (Fig. 289e-2).

1	Although cardiac catheterization and angiography were previously performed routinely before tumor resection, they no longer are considered mandatory when adequate noninvasive information is available and other cardiac disorders (e.g., coronary artery disease) are not considered likely. Additionally, catheterization of the chamber from which the tumor arises carries the risk of tumor embolization. Because myxomas may be familial, echocardiographic screening of first-degree relatives is appropriate, particularly if the patient is young and has multiple tumors or evidence of myxoma syndrome. CHAPTER 289e Tumors and Trauma of the Heart FIgURE 289e-1 Transthoracic echocardiogram demonstrating a large atrial myxoma. The myxoma (Myx) fills the entire left atrium in systole (A) and prolapses across the mitral valve and into the left ventricle (LV) during diastole (B). RA, right atrium; RV, right ventricle. (Courtesy of Dr. Michael Tsang; with permission.)

1	PART 10 Disorders of the Cardiovascular System FIgURE 289e-2 Cardiac magnetic resonance imaging demonstrat-ing a rounded mass (M) within the left atrium (LA). Pathologic evaluation at the time of surgery revealed it to be an atrial myxoma. LV, left ventricle; RA, right atrium; RV, right ventricle. Surgical excision using cardiopulmonary bypass is indicated regardless of tumor size and is generally curative. Myxomas recur in 12–22% of familial cases but in only 1–2% of sporadic cases. Tumor recurrence most likely is due to multifocal lesions in the former and inadequate resection in the latter.

1	Other Benign Tumors Cardiac lipomas, although relatively common, are usually incidental findings at postmortem examination; however, they may grow as large as 15 cm and may present with symptoms owing to mechanical interference with cardiac function, arrhythmias, or conduction disturbances or as an abnormality of the cardiac silhouette on chest x-ray. Papillary fibroelastomas are the most common tumors of the cardiac valves. Although usually clinically silent, they can cause valve dysfunction and may embolize distally, resulting in transient ischemic attacks, stroke, or myocardial infarction. In general, these tumors should be resected even when asymptomatic, although a more conservative approach may be considered for small, right-sided lesions. Rhabdomyomas and fibromas are the most common cardiac tumors in infants and children and usually occur in the ventricles, where they may produce mechanical obstruction to blood flow, thereby mimicking valvular stenosis, congestive heart

1	common cardiac tumors in infants and children and usually occur in the ventricles, where they may produce mechanical obstruction to blood flow, thereby mimicking valvular stenosis, congestive heart failure (CHF), restrictive or hypertrophic cardiomyopathy, or pericardial constriction. Rhabdomyomas are probably hamartomatous growths, are multiple in 90% of cases, and are strongly associated with tuberous sclerosis. These tumors have a tendency to regress completely or partially; only tumors that cause obstruction require surgical resection. Fibromas are usually single, are often calcified, tend to grow and cause obstructive symptoms, and should be resected. Hemangiomas and mesotheliomas are generally small tumors, most often intramyocardial in location, and may cause atrioventricular (AV) conduction disturbances and even sudden death as a result of their propensity to develop in the region of the AV node. Other benign tumors arising from the heart include teratoma, chemodectoma,

1	(AV) conduction disturbances and even sudden death as a result of their propensity to develop in the region of the AV node. Other benign tumors arising from the heart include teratoma, chemodectoma, neurilemoma, granular cell myoblastoma, and bronchogenic cysts.

1	Sarcoma Almost all malignant primary cardiac tumors are sarcomas, which may be of several histologic types. In general, these tumors are characterized by rapid progression that culminates in the patient’s death within weeks to months from the time of presentation as a result of hemodynamic compromise, local invasion, or distant metastases. Sarcomas commonly involve the right side of the heart, are characterized by rapid growth, frequently invade the pericardial space, and may obstruct the cardiac chambers or venae cavae. Sarcomas also may occur on the left side of the heart and may be mistaken for myxomas.

1	At the time of presentation, these tumors have often spread too extensively to allow for surgical excision. Although there are scattered reports of palliation with surgery, radiotherapy, and/or chemotherapy, the response of cardiac sarcomas to these therapies is generally poor. The one exception appears to be cardiac lymphosarcomas, which may respond to a combination of chemoand radiotherapy.

1	Tumors metastatic to the heart are much more common than primary tumors, and their incidence is likely to increase as the life expectancy of patients with various forms of malignant neoplasms is extended by more effective therapy. Although cardiac metastases may occur with any tumor type, the relative incidence is especially high in malignant melanoma and, to a somewhat lesser extent, leukemia and lymphoma. In absolute terms, the most common primary originating sites of cardiac metastases are carcinoma of the breast and lung, reflecting the high incidence of those cancers. Cardiac metastases almost always occur in the setting of widespread primary disease, and most often there is either primary or metastatic disease elsewhere in the thoracic cavity. Nevertheless, cardiac metastasis occasionally may be the initial presentation of an extrathoracic tumor.

1	Cardiac metastases may occur via hematogenous or lymphangitic spread or by direct tumor invasion. They generally manifest as small, firm nodules; diffuse infiltration also may occur, especially with sarcomas or hematologic neoplasms. The pericardium is most often involved, followed by myocardial involvement of any chamber and, rarely, by involvement of the endocardium or cardiac valves.

1	Cardiac metastases are clinically apparent only ~10% of the time, are usually not the cause of the patient’s presentation, and rarely are the cause of death. The vast majority occur in the setting of a previously recognized malignant neoplasm. As with primary cardiac tumors, the clinical presentation reflects more the location and size of the tumor than its histologic type. When symptomatic, cardiac metastases may result in a variety of clinical features, including dyspnea, acute pericarditis, cardiac tamponade, ectopic tachyarrhythmias, heart block, and CHF. Importantly, many of these signs and symptoms may also result from myocarditis, pericarditis, or cardiomyopathy induced by radiotherapy or chemotherapy.

1	Electrocardiographic (ECG) findings are nonspecific. On chest x-ray, the cardiac silhouette is most often normal but may be enlarged or exhibit a bizarre contour. Echocardiography is useful for identifying pericardial effusions and visualizing larger metastases, although CT and radionuclide imaging with gallium or thallium may define the tumor burden more clearly. Cardiac MRI offers superb image quality and plays a central role in the diagnostic evaluation of cardiac metastases and cardiac tumors in general. Pericardiocentesis may allow for a specific cytologic diagnosis in patients with malignant pericardial effusions. Angiography is rarely necessary but may delineate discrete lesions.

1	Most patients with cardiac metastases have advanced malignant disease; thus, therapy is generally palliative and consists of treatment of the primary tumor. Symptomatic malignant pericardial effusions should be drained by pericardiocentesis. Concomitant instillation of a sclerosing agent (e.g., tetracycline or bleomycin) may delay or prevent reaccumulation of the effusion, and creation of a pericardial window allows drainage of the effusion to the pleural or peritoneal space. Traumatic cardiac injury may be caused by either penetrating or nonpenetrating trauma. Penetrating injuries most often result from gunshot or knife wounds, and the site of entry is usually obvious. Nonpenetrating injuries most often occur during motor vehicle accidents, either from rapid deceleration or from impact of the chest against the steering wheel, and may be associated with significant cardiac injury even in the absence of external signs of thoracic trauma.

1	Myocardial contusions are the most common form of nonpenetrating cardiac injury and may initially be overlooked in trauma patients as the clinical focus is directed toward other, more obvious injuries. Myocardial necrosis may occur as a direct result of the blunt injury or as a result of traumatic coronary laceration or thrombosis. The contused myocardium is pathologically similar to infarcted myocardium and may be associated with atrial or ventricular arrhythmias; conduction disturbances, including bundle branch block; or ECG abnormalities resembling those of infarction or pericarditis. Thus, it is important to consider contusion as a cause of otherwise unexplained ECG changes in a trauma patient. Serum creatine kinase, myocardial band (CK-MB) isoenzyme levels are increased in ~20% of patients who experience blunt chest trauma but may be falsely elevated in the presence of massive skeletal muscle injury. Cardiac troponin levels are more specific for identifying cardiac injury in this

1	who experience blunt chest trauma but may be falsely elevated in the presence of massive skeletal muscle injury. Cardiac troponin levels are more specific for identifying cardiac injury in this setting. Echocardiography is useful in detecting structural and functional sequelae of contusion, including wall motion abnormalities (most commonly involving the right ventricle, interventricular septum, or left ventricular apex), pericardial effusion, valvular dysfunction, and ventricular rupture.

1	Rupture of the cardiac valves or their supporting structures, most commonly of the tricuspid or mitral valve, leads to acute valvular incompetence. This complication is usually heralded by the development of a loud murmur, may be associated with rapidly progressive heart failure, and can be diagnosed by either transthoracic or trans-esophageal echocardiography.

1	The most serious consequence of nonpenetrating cardiac injury is myocardial rupture, which may result in hemopericardium and tamponade (free wall rupture) or intracardiac shunting (ventricular septal rupture). Although it generally is fatal, up to 40% of patients with cardiac rupture have been reported to survive long enough to reach a specialized trauma center. Hemopericardium also may result from traumatic rupture of a pericardial vessel or a coronary artery. Additionally, a pericardial effusion may develop weeks or even months after blunt chest trauma as a manifestation of the post–cardiac injury syndrome, which resembles the postpericardiotomy syndrome (Chap. 288).

1	Blunt, nonpenetrating, often innocent-appearing injuries to the chest may trigger ventricular fibrillation even in absence of overt signs of injury. This syndrome, referred to as commotio cordis, occurs most often in adolescents during sporting events (e.g., baseball, hockey, football, and lacrosse) and probably results from an impact to the chest wall overlying the heart during the susceptible phase of repolarization just before the peak of the T wave. Survival depends on prompt defibrillation. Sudden emotional or physical trauma, even in the absence of direct cardiac trauma, may precipitate a transient catecholamine-mediated cardiomyopathy referred to as tako-tsubo syndrome or the apical bal-289e-3 looning syndrome (Chap. 287).

1	Rupture or transection of the aorta, usually just above the aortic valve or at the site of the ligamentum arteriosum, is a common consequence of nonpenetrating chest trauma and is the most common vascular deceleration injury. The clinical presentation is similar to that of aortic dissection (Chap. 301); the arterial pressure and pulse amplitude may be increased in the upper extremities and decreased in the lower extremities, and chest x-ray may reveal mediastinal widening. Occasionally, aortic rupture is contained by the aortic adventitia, resulting in a false, or pseudo-, aneurysm that may be discovered months or years after the initial injury.

1	Penetrating injuries of the heart produced by knife or bullet wounds usually result in rapid clinical deterioration and frequently in death as a result of hemopericardium/pericardial tamponade or massive hemorrhage. Nonetheless, up to half of such patients may survive long enough to reach a specialized trauma center if immediate resuscitation is performed. Prognosis in these patients relates to the mechanism of injury, their clinical condition at presentation, and the specific cardiac chamber(s) involved. Iatrogenic cardiac or coronary arterial perforation may complicate placement of central venous or intracardiac catheters, pacemaker leads, or intracoronary stents and is associated with a better prognosis than are other forms of penetrating cardiac trauma.

1	Traumatic rupture of a great vessel from penetrating injury is usually associated with hemothorax and, less often, hemopericardium. Local hematoma formation may compress major vessels and produce ischemic symptoms, and AV fistulas may develop, occasionally resulting in high-output CHF. Occasionally, patients who survive penetrating cardiac injuries may subsequently present with a new cardiac murmur or CHF as a result of mitral regurgitation or an intracardiac shunt (i.e., ventricular or atrial septal defect, aortopulmonary fistula, or coronary AV fistula) that was undetected at the time of the initial injury or developed subsequently. Therefore, trauma patients should be examined carefully several weeks after the injury. If a mechanical complication is suspected, it can be confirmed by echocardiography or cardiac catheterization.

1	The treatment of an uncomplicated myocardial contusion is similar to the medical therapy for a myocardial infarction, except that anticoagulation is contraindicated, and should include monitoring for the development of arrhythmias and mechanical complications such as cardiac rupture (Chap. 295). Acute myocardial failure resulting from traumatic valve rupture usually requires urgent operative correction. Immediate thoracotomy should be carried out for most cases of penetrating injury, or if there is evidence of cardiac tamponade and/or shock regardless of the type of trauma. Pericardiocentesis may be lifesaving in patients with tamponade but is usually only a temporizing measure while awaiting definitive surgical therapy. Pericardial hemorrhage often leads to constriction (Chap. 288), which must be treated by surgical decortication. CHAPTER 289e Tumors and Trauma of the Heart

1	CHAPTER 289e Tumors and Trauma of the Heart Cardiac Manifestations of Systemic Disease Eric H. Awtry, Wilson S. Colucci The common systemic disorders that have associated cardiac manifes-tations are summarized in Table 290e-1. 290e (See also Chap. 417) Diabetes mellitus, both insulinand non-insulindependent, is an independent risk factor for coronary artery disease (CAD; Chap. 291e) and accounts for 14–50% of new cases of cardiovascular disease. Furthermore, CAD is the most common cause of death in adults with diabetes mellitus. In the diabetic population, the incidence of CAD relates to the duration of diabetes and the level of glycemic control, and its pathogenesis involves endothelial dysfunction, increased lipoprotein peroxidation, increased inflammation, a prothrombotic state, and associated metabolic abnormalities.

1	Compared to their nondiabetic counterparts, diabetic patients are more likely to have a myocardial infarction, have a greater burden of CAD, have larger infarct size, and have more postinfarct complications, including heart failure, shock, and death. Importantly, diabetic patients are more likely to have atypical ischemic symptoms; nausea, dyspnea, pulmonary edema, arrhythmias, heart block, or syncope may be their anginal equivalent. Additionally, “silent ischemia,” resulting from autonomic nervous system dysfunction, is more common in diabetic patients, accounting for up to 90% of their ischemic episodes. Thus, one must have a low threshold for suspecting CAD in diabetic patients. The treatment of diabetic patients with CAD must include aggressive risk factor management (Chap. 418). Considerations regarding pharmacologic therapy and revascularization strategies are similar in diabetic and nondiabetic patients except that diabetic patients have higher morbidity and mortality rates

1	regarding pharmacologic therapy and revascularization strategies are similar in diabetic and nondiabetic patients except that diabetic patients have higher morbidity and mortality rates associated with revascularization, have an increased risk of restenosis after percutaneous coronary intervention (PCI), and have improved survival when treated with surgical bypass compared with PCI for multivessel CAD.

1	Patients with diabetes mellitus also may have abnormal left ven-290e-1 tricular systolic and diastolic function, reflecting concomitant epicardial CAD and/or hypertension, coronary microvascular disease, endothelial dysfunction, ventricular hypertrophy, and autonomic dysfunction. Furthermore, the increase in intramyocardial lipid deposition (predominantly nonesterified fatty acids) that is characteristic of diabetic states may contribute to both systolic and diastolic dysfunction by impairing insulin signaling, reducing trans-sarcolemma calcium flux, and inducing myocyte apoptosis. A restrictive cardiomyopathy may be present with abnormal myocardial relaxation and elevated ventricular filling pressures. Histologically, interstitial fibrosis is seen, and intramural arteries may demonstrate intimal thickening, hyaline deposition, and inflammatory changes. Diabetic patients have an increased risk of developing clinical heart failure, which probably contributes to their excessive

1	intimal thickening, hyaline deposition, and inflammatory changes. Diabetic patients have an increased risk of developing clinical heart failure, which probably contributes to their excessive cardiovascular morbidity and mortality rates. There is some evidence that insulin therapy may ameliorate diabetes-related myocardial dysfunction.

1	MALNUTRITION AND VITAMIN DEFICIENCY Malnutrition (See also Chap. 97) In patients whose intake of protein, calories, or both is severely deficient, the heart may become thin, pale, and hypokinetic with myofibrillar atrophy and interstitial edema. The systolic pressure and cardiac output fall, and the pulse pressure narrows. Generalized edema is common and relates to a variety of factors, including reduced serum oncotic pressure and myocardial dysfunction. Such profound states of protein and calorie malnutrition, termed kwashiorkor and marasmus, respectively, are most common in underdeveloped countries. However, significant nutritional heart disease also may occur in developed nations, particularly in patients with chronic diseases such as AIDS, patients with anorexia nervosa, and patients with severe cardiac failure in whom gastrointestinal hypoperfusion and venous congestion may lead to anorexia and malabsorption. Open-heart surgery poses increased risk in malnourished patients; such

1	with severe cardiac failure in whom gastrointestinal hypoperfusion and venous congestion may lead to anorexia and malabsorption. Open-heart surgery poses increased risk in malnourished patients; such patients may benefit from preoperative hyperalimentation.

1	Thiamine Deficiency (Beriberi) (See also Chap. 96e) Generalized malnutrition often is accompanied by thiamine deficiency; however, this hypovitaminosis also may occur in the presence of an adequate protein and caloric intake, particularly in East Asia, where polished rice CHAPTER 290e Cardiac Manifestations of Systemic Disease Abbreviations: CAD, coronary artery disease; CHF, congestive heart failure; CMP, cardiomyopathy; SVT, supraventricular tachycardia.

1	PART 10 Disorders of the Cardiovascular System 290e-2 deficient in thiamine may be a major dietary component. In Western nations where the use of thiamine-enriched flour is widespread, clinical thiamine deficiency is limited primarily to alcoholics, food faddists, and patients receiving chemotherapy. Nonetheless, when thiamine stores are measured using the thiamine-pyrophosphate effect (TPPE), thiamine deficiency has been found in 20–90% of patients with chronic heart failure. This deficiency appears to result from both reduced dietary intake and a diuretic-induced increase in the urinary excretion of thiamine. The acute administration of thiamine to these patients increases the left ventricular ejection fraction and the excretion of salt and water. Clinically, patients with thiamine deficiency usually have evidence of generalized malnutrition, peripheral neuropathy, glossitis, and anemia. The classic associated cardiovascular syndrome is characterized by high-output heart failure,

1	usually have evidence of generalized malnutrition, peripheral neuropathy, glossitis, and anemia. The classic associated cardiovascular syndrome is characterized by high-output heart failure, tachycardia, and often elevated biventricular filling pressures. The major cause of the high-output state is vasomotor depression leading to reduced systemic vascular resistance, the precise mechanism of which is not understood. The cardiac examination may reveal a wide pulse pressure, tachycardia, a third heart sound, and an apical systolic murmur. The electrocardiogram (ECG) may reveal decreased voltage, a prolonged QT interval, and T-wave abnormalities. The chest x-ray generally reveals cardiomegaly and signs of congestive heart failure (CHF). The response to thiamine is often dramatic, with an increase in systemic vascular resistance, a decrease in cardiac output, clearing of pulmonary congestion, and a reduction in heart size often occurring in 12–48 h. Although the response to inotropes and

1	in systemic vascular resistance, a decrease in cardiac output, clearing of pulmonary congestion, and a reduction in heart size often occurring in 12–48 h. Although the response to inotropes and diuretics may be poor before thiamine therapy, these agents may be important after thiamine repletion, since the left ventricle may not be able to handle the increased work load presented by the return of vascular tone.

1	Vitamin B6, B12, and Folate Deficiency (See also Chap. 96e) Vitamin B6, vitamin B12, and folate are cofactors in the metabolism of homocysteine. Their deficiency probably contributes to the majority of cases of hyperhomocysteinemia, a disorder associated with increased atherosclerotic risk. Supplementation of these vitamins has reduced the incidence of hyperhomocysteinemia in the United States; however, the clinical cardiovascular benefit of normalizing elevated homocysteine levels has not been proved.

1	(See also Chap. 415e) Obesity is associated with an increased prevalence of hypertension, glucose intolerance, atherosclerotic CAD, atrial fibrillation, obstructive sleep apnea, and pulmonary hypertension, and is associated with increased cardiovascular morbidity and mortality rates. In addition, obese patients have a distinct hemodynamic profile characterized by increased total and central blood volumes, increased cardiac output, and elevated left ventricular filling pressure. The elevated cardiac output appears to be required to support the metabolic demands of the excess adipose tissue. Left ventricular filling pressure is often at the upper limits of normal at rest and rises excessively with exercise, contributing to exertional dyspnea. In part as a result of chronic volume overload, eccentric cardiac hypertrophy with cardiac dilation and ventricular diastolic and/or systolic dysfunction may develop. In addition, altered levels of adipokines secreted by adipose tissue may

1	eccentric cardiac hypertrophy with cardiac dilation and ventricular diastolic and/or systolic dysfunction may develop. In addition, altered levels of adipokines secreted by adipose tissue may contribute to adverse myocardial remodeling via direct effects on cardiac myocytes and other cells. Pathologically, there is left and, in some cases, right ventricular hypertrophy and generalized cardiac dilation. Pulmonary congestion, peripheral edema, and exercise intolerance may all ensue; however, the recognition of these findings may be difficult in massively obese patients.

1	Treatment with angiotensin-converting enzyme inhibitors, sodium restriction, and diuretics may be useful to control heart failure symptoms. Weight reduction, however, is the most effective therapy and results in reduction in blood volume and the return of cardiac output toward normal. However, rapid weight reduction may be dangerous, as cardiac arrhythmias and sudden death owing to electrolyte imbalance have been described.

1	(See also Chap. 405) Thyroid hormone exerts a major influence on the cardiovascular system by a number of direct and indirect mechanisms, and not surprisingly, cardiovascular effects are prominent in both hypoand hyperthyroidism. Thyroid hormone causes increases in total-body metabolism and oxygen consumption that indirectly increase the cardiac workload. In addition, thyroid hormone exerts direct inotropic, chronotropic, and dromotropic effects that are similar to those seen with adrenergic stimulation (e.g., tachycardia, increased cardiac output); they are mediated at least partly by both transcriptional and nontranscriptional effects of thyroid hormone on myosin, calcium-activated ATPase, Na+-K+-ATPase, and myocardial β-adrenergic receptors.

1	Hyperthyroidism Common cardiovascular manifestations of hyperthyroidism include palpitations, systolic hypertension, and fatigue. Sinus tachycardia is present in ~40% of hyperthyroid patients, and atrial fibrillation is present in ~15%. Physical examination may reveal a hyperdynamic precordium, a widened pulse pressure, increases in the intensity of the first heart sound and the pulmonic component of the second heart sound, and a third heart sound. An increased incidence of mitral valve prolapse has been described in hyperthyroid patients, in which case a midsystolic murmur may be heard at the left sternal border with or without a midsystolic click. A systolic pleuropericardial friction rub (Means-Lerman scratch) may be heard at the left second intercostal space during expiration and is thought to result from the hyperdynamic cardiac motion.

1	Elderly patients with hyperthyroidism may present with only cardiovascular manifestations of thyrotoxicosis such as sinus tachycardia, atrial fibrillation, and hypertension, all of which may be resistant to therapy until the hyperthyroidism is controlled. Angina pectoris and CHF are unusual with hyperthyroidism unless there is coexistent heart disease; in such cases, symptoms often resolve with treatment of the hyperthyroidism.

1	Hypothyroidism Cardiac manifestations of hypothyroidism include a reduction in cardiac output, stroke volume, heart rate, systolic blood pressure, and pulse pressure. Pericardial effusions are present in about one-third of patients, rarely progress to tamponade, and probably result from increased capillary permeability. Other clinical signs include cardiomegaly, bradycardia, weak arterial pulses, distant heart sounds, and pleural effusions. Although the signs and symptoms of myxedema may mimic those of CHF, in the absence of other cardiac disease, myocardial failure is uncommon. The ECG generally reveals sinus bradycardia and low voltage and may show prolongation of the QT interval, decreased P-wave voltage, prolonged AV conduction time, intraventricular conduction disturbances, and nonspecific ST-Twave abnormalities. Chest x-ray may show cardiomegaly, often with a “water bottle” configuration; pleural effusions; and, in some cases, evidence of CHF. Pathologically, the heart is pale

1	ST-Twave abnormalities. Chest x-ray may show cardiomegaly, often with a “water bottle” configuration; pleural effusions; and, in some cases, evidence of CHF. Pathologically, the heart is pale and dilated and often demonstrates myofibrillar swelling, loss of striations, and interstitial fibrosis.

1	Patients with hypothyroidism frequently have elevations of cholesterol and triglycerides, resulting in premature atherosclerotic CAD. Before treatment with thyroid hormone, patients with hypothyroidism frequently do not have angina pectoris, presumably because of the low metabolic demands caused by their condition. However, angina and myocardial infarction may be precipitated during initiation of thyroid hormone replacement, especially in elderly patients with underlying heart disease. Therefore, replacement should be done with care, starting with low doses that are increased gradually.

1	(See also Chap. 113) Carcinoid tumors most often originate in the small bowel and elaborate a variety of vasoactive amines (e.g., serotonin), kinins, indoles, and prostaglandins that are believed to be responsible for the diarrhea, flushing, and labile blood pressure that characterize the carcinoid syndrome. Some 50% of patients with carcinoid syndrome have cardiac involvement, usually manifesting as abnormalities of the tricuspid or pulmonic valves. These patients invariably have hepatic metastases that allow vasoactive substances to circumvent hepatic metabolism. Left-sided cardiac involvement is rare and indicates either pulmonary carcinoid or an intracardiac shunt. Pathologically, carcinoid lesions are fibrous plaques that consist of smooth-muscle cells embedded in a stroma of glycosaminoglycans and collagen. They occur on the cardiac valves, where they cause valvular dysfunction, as well as on the endothelium of the cardiac chambers and great vessels.

1	Carcinoid heart disease most often presents as tricuspid regurgitation, pulmonic stenosis, or both. In some cases, a high cardiac output state may occur, presumably as a result of a decrease in systemic vascular resistance resulting from vasoactive substances released by the tumor. Treatment with somatostatin analogues (e.g., octreotide) or interferon α improves symptoms and survival in patients with carcinoid heart disease but does not appear to improve valvular abnormalities. Treatment with diuretics usually mitigates the symptoms of right heart failure; in some severely symptomatic patients, valve replacement is indicated. Coronary artery spasm, presumably due to a circulating vasoactive substance, may occur in patients with carcinoid syndrome.

1	(See also Chap. 407) In addition to causing labile or sustained hypertension, the high circulating levels of catecholamines resulting from a pheochromocytoma may cause direct myocardial injury. Focal myocardial necrosis and inflammatory cell infiltration are present in ~50% of patients who die with pheochromocytoma and may contribute to clinically significant left ventricular failure and pulmonary edema. In addition, associated hypertension results in left ventricular hypertrophy. Left ventricular dysfunction and CHF may resolve after removal of the tumor.

1	(See also Chap. 401e) Exposure of the heart to excessive growth hormone may cause CHF as a result of high cardiac output, diastolic dysfunction owing to ventricular hypertrophy (with increased left ventricular chamber size or wall thickness), or global systolic dysfunction. Hypertension occurs in up to one-third of patients with acromegaly and is characterized by suppression of the renin-angiotensin-aldosterone axis and increases in total-body sodium and plasma volume. Some form of cardiac disease occurs in about one-third of patients with acromegaly and is associated with a doubling of the risk of cardiac death.

1	RHEUMATOID ARTHRITIS AND THE COLLAGEN VASCULAR DISEASES Rheumatoid Arthritis (See also Chap. 380) Rheumatoid arthritis may be associated with inflammatory changes in any or all cardiac structures, although pericarditis is the most common clinical entity. Pericardial effusions are found on echocardiography in 10–50% of patients with rheumatoid arthritis, particularly those with subcutaneous nodules. Nonetheless, only a small fraction of these patients have symptomatic pericarditis, and when present, it usually follows a benign course, only occasionally progressing to cardiac tamponade or constrictive pericarditis. The pericardial fluid is generally exudative, with decreased concentrations of complement and glucose and ele-290e-3 vated cholesterol. Coronary arteritis with intimal inflammation and edema is present in ~20% of cases but only rarely results in angina pectoris or myocardial infarction. Inflammation and granuloma formation may affect the cardiac valves, most often the mitral

1	and edema is present in ~20% of cases but only rarely results in angina pectoris or myocardial infarction. Inflammation and granuloma formation may affect the cardiac valves, most often the mitral and aortic valves, and may cause clinically significant regurgitation owing to valve deformity. Myocarditis is uncommon and rarely results in cardiac dysfunction.

1	Treatment is directed at the underlying rheumatoid arthritis and may include glucocorticoids. Urgent pericardiocentesis should be performed in patients with tamponade, but pericardiectomy usually is required in cases of pericardial constriction.

1	Seronegative Arthropathies (See also Chap. 384) The seronegative arthropathies, including ankylosing spondylitis, reactive arthritis, psoriatic arthritis, and the arthritides associated with ulcerative colitis and regional enteritis, are all strongly associated with the HLA-B27 histocompatibility antigen and may be accompanied by a pancarditis and proximal aortitis. The aortic inflammation usually is limited to the aortic root but may extend to involve the aortic valve, mitral valve, and ventricular myocardium, resulting in aortic and mitral regurgitation, conduction abnormalities, and ventricular dysfunction. One-tenth of these patients have significant aortic insufficiency, and one-third have conduction disturbances; both are more common in patients with peripheral joint involvement and long-standing disease. Treatment with aortic valve replacement and permanent pacemaker implantation may be required. Occasionally, aortic regurgitation precedes the onset of arthritis, and therefore,

1	long-standing disease. Treatment with aortic valve replacement and permanent pacemaker implantation may be required. Occasionally, aortic regurgitation precedes the onset of arthritis, and therefore, the diagnosis of a seronegative arthritis should be considered in young males with isolated aortic regurgitation.

1	Systemic Lupus Erythematosus (SLE) (See also Chap. 378) A significant percentage of patients with SLE have cardiac involvement. Pericarditis is common, occurring in about two-thirds of patients, and generally follows a benign course, although rarely tamponade or constriction may result. The characteristic endocardial lesions of SLE are verrucous valvular abnormalities known as Libman-Sacks endocarditis. They most often are located on the left-sided cardiac valves, particularly on the ventricular surface of the posterior mitral leaflet, and are made up almost entirely of fibrin. These lesions may embolize or become infected but rarely cause hemodynamically important valvular regurgitation. Myocarditis generally parallels the activity of the disease and, although common histologically, seldom results in clinical heart failure unless associated with hypertension. Although arteritis of epicardial coronary arteries may occur, it rarely results in myocardial ischemia. There is, however, an

1	results in clinical heart failure unless associated with hypertension. Although arteritis of epicardial coronary arteries may occur, it rarely results in myocardial ischemia. There is, however, an increased incidence of coronary atherosclerosis that probably is related more to associated risk factors and glucocorticoid use than to SLE itself. Patients with the antiphospholipid antibody syndrome may have a higher incidence of cardiovascular abnormalities, including valvular regurgitation, venous and arterial thrombosis, premature stroke, myocardial infarction, pulmonary hypertension, and cardiomyopathy.

1	CHAPTER 290e Cardiac Manifestations of Systemic Disease 291e-1 The Pathogenesis, Prevention, and Treatment of Atherosclerosis Peter Libby PATHOGENESIS 291e SEC Tion 5 CoRonARy And PERiPHERAl VASCulAR diSEASE

1	Atherosclerosis remains the major cause of death and premature disability in developed societies. Moreover, current predictions estimate that by the year 2020 cardiovascular diseases, notably atherosclerosis, will become the leading global cause of total disease burden. Although many generalized or systemic risk factors predispose to its development, atherosclerosis affects various regions of the circulation preferentially and has distinct clinical manifestations that depend on the particular circulatory bed affected. Atherosclerosis of the coronary arteries commonly causes myocardial infarction (MI) (Chap. 295) and angina pectoris (Chap. 293). Atherosclerosis of the arteries supplying the central nervous system frequently provokes strokes and transient cerebral ischemia (Chap. 446). In the peripheral circulation, atherosclerosis causes intermittent claudication and gangrene and can jeopardize limb viability. Involvement of the splanchnic circulation can cause mesenteric ischemia.

1	the peripheral circulation, atherosclerosis causes intermittent claudication and gangrene and can jeopardize limb viability. Involvement of the splanchnic circulation can cause mesenteric ischemia. Atherosclerosis can affect the kidneys either directly (e.g., renal artery stenosis) or as a common site of atheroembolic disease (Chap. 301).

1	Even within a particular arterial bed, stenoses due to atherosclerosis tend to occur focally, typically in certain predisposed regions. In the coronary circulation, for example, the proximal left anterior descending coronary artery exhibits a particular predilection for developing atherosclerotic disease. Similarly, atherosclerosis preferentially affects the proximal portions of the renal arteries and, in the extracranial circulation to the brain, the carotid bifurcation. Indeed, atherosclerotic lesions often form at branch points of arteries, regions characterized disturbed hydrodynamics. Not all manifestations of atherosclerosis result from stenotic, occlusive disease. Ectasia and the development of aneurysmal disease, for example, frequently occur in the aorta (Chap. 301). In addition to focal, flow-limiting stenoses, nonocclusive intimal atherosclerosis also occurs diffusely in affected arteries, as shown by intravascular imaging and postmortem studies.

1	Atherogenesis in humans typically occurs over a period of many years, usually many decades. Growth of atherosclerotic plaques probably does not occur in a smooth, linear fashion but discontinuously, with periods of relative quiescence punctuated by periods of rapid evolution. After a generally prolonged “silent” period, atherosclerosis may become clinically manifest. The clinical expressions of atherosclerosis may be chronic, as in the development of stable, effort-induced angina pectoris or predictable and reproducible intermittent claudication. Alternatively, a dramatic acute clinical event such as MI, stroke, or sudden cardiac death may first herald the presence of atherosclerosis. Other individuals may never experience clinical manifestations of arterial disease despite the presence of widespread atherosclerosis demonstrated postmortem.

1	An integrated view of experimental results in animals and studies of human atherosclerosis suggests that the “fatty streak” represents the initial lesion of atherosclerosis. These early lesions most often seem to arise from focal increases in the content of lipoproteins within regions of the intima. In particular, the fraction of lipoproteins related to low-density lipoprotein (LDL) that bear apolipoprotein B appear causally related to atherosclerosis. This accumulation of lipoprotein particles may not result simply from increased permeability, or CHAPTER 291e The Pathogenesis, Prevention, and Treatment of Atherosclerosis Monocyte

1	CHAPTER 291e The Pathogenesis, Prevention, and Treatment of Atherosclerosis Monocyte FIGuRE 291e-1 Cross-sectional view of an artery depicting steps in development of an atheroma, from left to right. The upper panel shows a detail of the boxed area below. The endothelial mono-layer overlying the intima contacts blood. Hypercholesterolemia promotes accumulation of low-density lipoprotein (LDL) particles (yellow spheres) in the intima. The lipoprotein particles often associate with constituents of the extracellular matrix, notably proteoglycans. Sequestration within the intima separates lipoproteins from some plasma antioxidants and favors oxidative modification. Such modified lipoprotein particles (darker spheres) may trigger a local inflammatory response that signals subsequent steps in lesion formation. The augmented expression of various adhesion molecules for leukocytes recruits monocytes to the site of a nascent arterial lesion.

1	Once adherent, some white blood cells migrate into the intima. The directed migration of leukocytes probably depends on chemoattractant factors, including modified lipoprotein particles themselves and chemoattractant cytokines (depicted by the smaller green spheres), such as the chemokine macrophage chemoattractant protein-1 produced by vascular wall cells in response to modified lipoproteins. Leukocytes in the evolving fatty streak can divide and exhibit augmented expression of receptors for modified lipoproteins (scavenger receptors). These mononuclear phagocytes ingest lipids and become foam cells, represented by a cytoplasm filled with lipid droplets. As the fatty streak evolves into a more complicated atherosclerotic lesion, smooth-muscle cells migrate from the media (bottom of lower panel hairline) through the internal elastic membrane (solid wavy line) and accumulate within the expanding intima, where they lay down extracellular matrix that forms the bulk of the advanced lesion

1	panel hairline) through the internal elastic membrane (solid wavy line) and accumulate within the expanding intima, where they lay down extracellular matrix that forms the bulk of the advanced lesion (bottom panel, right side).

1	“leakiness,” of the overlying endothelium (Fig. 291e-1). Rather, the lipoproteins may collect in the intima of arteries because they bind to constituents of the extracellular matrix, increasing the residence time of the lipid-rich particles within the arterial wall. Lipoproteins that accumulate in the extracellular space of the intima of arteries often associate with proteoglycans of the arterial extracellular matrix, an interaction that may slow the egress of these lipid-rich particles from

1	PART 10 Disorders of the Cardiovascular System 291e-2 the intima. Lipoprotein particles in the extracellular space of the intima, particularly those retained by binding to matrix macromolecules, may undergo oxidative modifications. Considerable evidence supports a pathogenic role for products of oxidized lipoproteins in atherogenesis. Lipoproteins sequestered from (plasma) antioxidants in the extracellular space of the intima become particularly susceptible to oxidative modification, giving rise to hydroperoxides, lysophospholipids, oxysterols, and aldehydic breakdown products of fatty acids and phospholipids. Modifications of the apoprotein moieties may include breaks in the peptide backbone as well as derivatization of certain amino acid residues. Local production of hypochlorous acid by myeloperoxidase associated with inflammatory cells within the plaque yields chlorinated species such as chlorotyrosyl moieties. Considerable evidence supports the presence of such oxidation products

1	myeloperoxidase associated with inflammatory cells within the plaque yields chlorinated species such as chlorotyrosyl moieties. Considerable evidence supports the presence of such oxidation products in atherosclerotic lesions.

1	Leukocyte Recruitment Accumulation of leukocytes characterizes the formation of early atherosclerotic lesions (Fig. 291e-1). Thus, from its very inception, atherogenesis involves elements of inflammation, a process that now provides a unifying theme in the pathogenesis of this disease. The inflammatory cell types typically found in the evolving atheroma include monocyte-derived macrophages and dendritic cells, T and B lymphocytes, and mast cells. Hypercholesterolemia augments the portion of particularly proinflammatory monocytes in blood that preferentially enter the nascent atheroma in mice. A number of adhesion molecules or receptors for leukocytes expressed on the surface of the arterial endothelial cell probably participate in the recruitment of leukocytes to the nascent atheroma. Proinflammatory cytokines can augment the expression of leukocyte adhesion molecules.

1	Laminar shear forces such as those encountered in most regions of normal arteries also can suppress the expression of leukocyte adhesion molecules. Sites of predilection for atherosclerotic lesions (e.g., distal to flow dividers) often have low shear stress and/or disturbed flow. Ordered, pulsatile laminar shear of normal blood flow augments the production of nitric oxide by endothelial cells. This molecule, in addition to its vasodilator properties, can act at the low levels constitutively produced by arterial endothelium as a local anti-inflammatory autacoid, e.g., limiting local adhesion molecule expression. Exposure of endothelial cells to laminar shear stress increases the transcription of Krüppel-like factor 2 (KLF2), which augments the activity of numerous salutary endothelial functions including nitric oxide synthase. Laminar shear stress also stimulates endothelial cells to produce super-oxide dismutase, an antioxidant enzyme. These examples indicate how hemodynamic forces

1	including nitric oxide synthase. Laminar shear stress also stimulates endothelial cells to produce super-oxide dismutase, an antioxidant enzyme. These examples indicate how hemodynamic forces may influence the cellular events that underlie atherosclerotic lesion initiation and potentially explain the favored localization of atherosclerotic lesions at sites that experience disturbed flow or low shear stress.

1	Once captured on the surface of the arterial endothelial cell by adhesion receptors, the leukocytes penetrate the endothelial layer and take up residence in the intima. In addition to products of modified lipoproteins, cytokines (protein mediators of inflammation) can regulate the expression of adhesion molecules involved in leukocyte recruitment. For example, interleukin 1 (IL-1) and tumor necrosis factor (TNF) induce or augment the expression of leukocyte adhesion molecules on endothelial cells. Because products of lipoprotein oxidation can induce cytokine release from vascular wall cells, this pathway may provide an additional link between arterial accumulation of lipoproteins and leukocyte recruitment. Chemoattractant cytokines appear to direct the migration of leukocytes into the arterial wall.

1	Foam-Cell Formation Once resident within the intima, the mononuclear phagocytes mature into macrophages and become lipid-laden foam cells, a conversion that requires the uptake of lipoprotein particles by receptor-mediated endocytosis. One might suppose that the “classic” LDL receptor mediates this lipid uptake; however, humans or animals lacking effective LDL receptors due to genetic alterations (e.g., familial hypercholesterolemia) have abundant arterial lesions and extraarterial xanthomata rich in macrophage-derived foam cells. In addition, the exogenous cholesterol suppresses expression of the LDL receptor; thus, the level of this cell-surface receptor for LDL decreases under conditions of cholesterol excess. Candidates for alternative receptors that can mediate lipid loading of foam cells include a number of macrophage “scavenger” receptors, which preferentially endocytose modified lipoproteins, and other receptors for oxidized LDL or very low-density lipoprotein (VLDL). Monocyte

1	include a number of macrophage “scavenger” receptors, which preferentially endocytose modified lipoproteins, and other receptors for oxidized LDL or very low-density lipoprotein (VLDL). Monocyte attachment to the endothelium, migration into the intima, and maturation to form lipid-laden macrophages thus represent key steps in the formation of the fatty streak, the precursor of fully formed atherosclerotic plaques.

1	Although the fatty streak commonly precedes the development of a more advanced atherosclerotic plaque, not all fatty streaks progress to form complex atheromata. By ingesting lipids from the extracellular space, the mononuclear phagocytes bearing such scavenger receptors may remove lipoproteins from the developing lesion. Some lipid-laden macrophages may leave the artery wall, exporting lipid in the process. Lipid accumulation, and hence the propensity to form an atheroma, ensues if the amount of lipid entering the artery wall exceeds that removed by mononuclear phagocytes or other pathways. Macrophages also proliferate in plaques in response to hematopoietic growth factors overexpressed in lesions, another aspect of the dynamic regulation and flux of cells during atherogenesis.

1	Export by phagocytes may constitute one response to local lipid overload in the evolving lesion. Another mechanism, reverse cholesterol transport mediated by high-density lipoproteins (HDLs), probably provides an independent pathway for lipid removal from atheroma. This transfer of cholesterol from the cell to the HDL particle involves specialized cell-surface molecules such as the ATP binding cassette (ABC) transporters. ABCA1, the gene mutated in Tangier disease, a condition characterized by very low HDL levels, transfers cholesterol from cells to nascent HDL particles and ABCG1 to mature HDL particles. “Reverse cholesterol transport” mediated by these ABC transporters allows HDL loaded with cholesterol to deliver it to hepatocytes by binding to scavenger receptor B1 or other receptors. The liver cell can metabolize the sterol to bile acids that can be excreted. Thus, macrophages may play a vital role in the dynamic economy of lipid accumulation in the arterial wall during

1	The liver cell can metabolize the sterol to bile acids that can be excreted. Thus, macrophages may play a vital role in the dynamic economy of lipid accumulation in the arterial wall during atherogenesis.

1	Some lipid-laden foam cells within the expanding intimal lesion perish. Some foam cells may die as a result of programmed cell death, or apoptosis. This death of mononuclear phagocytes results in the formation of the lipid-rich center, often called the necrotic core, in established atherosclerotic plaques. Impaired clearance of dead foam cells (efferocytosis) in plaques may hasten lipid core formation. Macrophages loaded with modified lipoproteins may elaborate microparticles or exosomes (which may contain regulatory microRNAs), cytokines, and growth factors that can further signal some of the cellular events in lesion complication. Whereas accumulation of lipid-laden macrophages characterizes the fatty streak, buildup of fibrous tissue formed by extracellular matrix typifies the more advanced atherosclerotic lesion. The smooth-muscle cell synthesizes the bulk of the extracellular matrix of the complex atherosclerotic lesion. A number of growth factors or cytokines elaborated by

1	advanced atherosclerotic lesion. The smooth-muscle cell synthesizes the bulk of the extracellular matrix of the complex atherosclerotic lesion. A number of growth factors or cytokines elaborated by mononuclear phagocytes can stimulate smooth-muscle cell proliferation and production of extracellular matrix. Cytokines found in the plaque, including IL-1 and TNF, can induce local production of growth factors, including forms of platelet-derived growth factor (PDGF), fibroblast growth factors, and others, which may contribute to plaque evolution and complication. Other cytokines, notably interferon γ (IFN-γ) derived from activated T cells within lesions, can limit the synthesis of interstitial forms of collagen by smooth-muscle cells. These examples illustrate how atherogenesis involves a complex mix of mediators that in the balance determines the characteristics of particular lesions.

1	The accumulation of smooth-muscle cells and their elaboration of extracellular matrix probably provide a critical transition, yielding a fibrofatty lesion in place of a simple accumulation of macrophage-derived foam cells. For example, PDGF elaborated by activated platelets, macrophages, and endothelial cells can stimulate the migration of smooth-muscle cells normally resident in the tunica media into the intima. Such growth factors and cytokines produced locally can stimulate the proliferation of resident smooth-muscle cells or resident stem cells in the intima as well as those that may migrate in from the media. Transforming growth factor β (TGF-β), among other mediators, potently stimulates interstitial collagen production by smooth-muscle cells. These mediators may arise not only from neighboring vascular cells or leukocytes (a “paracrine” pathway), but also, in some instances, from the same cell that responds to the factor (an “autocrine” pathway). Together, these alterations in

1	neighboring vascular cells or leukocytes (a “paracrine” pathway), but also, in some instances, from the same cell that responds to the factor (an “autocrine” pathway). Together, these alterations in smooth-muscle cells, signaled by these mediators acting at short distances, can hasten transformation of the fatty streak into a more fibrous smooth-muscle cell and extracellular matrix—rich lesion.

1	In addition to locally produced mediators, products of blood coagulation and thrombosis likely contribute to atheroma evolution and complication. This involvement justifies the use of the term atherothrombosis to convey the inextricable links between atherosclerosis and thrombosis. Fatty streak formation begins beneath a morphologically intact endothelium. In advanced fatty streaks, however, microscopic breaches in endothelial integrity may occur. Microthrombi rich in platelets can form at such sites of limited endothelial denudation, owing to exposure of the thrombogenic extracellular matrix of the underlying basement membrane. Activated platelets release numerous factors that can promote the fibrotic response, including PDGF and TGF-β. Thrombin not only generates fibrin during coagulation, but also stimulates protease-activated receptors that can signal smooth-muscle migration, proliferation, and extracellular matrix production. Many arterial mural microthrombi resolve without

1	but also stimulates protease-activated receptors that can signal smooth-muscle migration, proliferation, and extracellular matrix production. Many arterial mural microthrombi resolve without clinical manifestation by a process of local fibrinolysis, resorption, and endothelial repair, yet can lead to lesion progression by stimulating these profibrotic functions of smooth-muscle cells (Fig. 291e-2D).

1	Microvessels As atherosclerotic lesions advance, abundant plexi of microvessels develop in connection with the artery’s vasa vasorum. Newly developing microvascular networks may contribute to lesion complications in several ways. These blood vessels provide an abundant surface area for leukocyte trafficking and may serve as the portal for entry and exit of white blood cells from the established atheroma. Microvessels in the plaques may also furnish foci for intraplaque hemorrhage. Like the neovessels in the diabetic retina, microvessels in the atheroma may be friable and prone to rupture and can produce focal hemorrhage. Such a vascular leak can provoke thrombosis in situ, yielding local thrombin generation, which in turn can activate smooth-muscle and endothelial cells through ligation of protease-activated receptors. Atherosclerotic plaques often contain fibrin and hemosiderin, an indication that episodes of intraplaque hemorrhage contribute to plaque complications.

1	CALCIFICATION As they advance, atherosclerotic plaques also accumulate calcium. Microvesicles derived from lesional cells can stimulate calcification, and this process co-localizes with regions of heightened inflammation. Mineralization of the atherosclerotic plaque recapitulates many aspects of bone formation, including the regulatory participation of transcription factors such as Runx2.

1	Plaque Evolution Smooth-muscle cells and macrophages die in the atherosclerotic plaque. Indeed, complex atheromata often have a mostly fibrous character and lack the cellularity of less advanced lesions. This relative paucity of smooth-muscle cells in advanced atheromata may result from the predominance of cytostatic mediators such as TGF-β and IFN-γ (which can inhibit smooth-muscle cell proliferation) and also from smooth-muscle cell apoptosis. Thus, during the evolution of the atherosclerotic plaque, a complex and highly regulated balance between entry and egress of lipoproteins and leukocytes, cell proliferation and cell death, extracellular matrix production, and remodeling, as well as calcification and neovascularization, contribute to lesion formation. Many mediators related to atherogenic risk factors, including those derived from lipoproteins, cigarette smoking, and angiotensin II, provoke the production of proinflammatory cytokines and alter the behavior of the intrinsic

1	atherogenic risk factors, including those derived from lipoproteins, cigarette smoking, and angiotensin II, provoke the production of proinflammatory cytokines and alter the behavior of the intrinsic vascular wall cells and infiltrating leukocytes that underlie the complex pathogenesis of these lesions. Thus, advances in vascular biology have led to increased

1	FIGuRE 291e-2 Plaque rupture, thrombosis, and healing. A. Arterial remodeling during atherogenesis. During the initial part of the life history of an atheroma, growth is often outward, preserving the caliber of the lumen. This phenomenon of “compensatory enlargement” accounts in part for the tendency of coronary arteriography to underestimate the degree of atherosclerosis. B. Rupture of the plaque’s fibrous cap causes thrombosis. Physical disruption of the atherosclerotic plaque commonly causes arterial thrombosis by allowing blood coagulant factors to contact thrombogenic collagen found in the arterial extracellular matrix and tissue factor produced by macrophage-derived foam cells in the lipid core of lesions. In this manner, sites of plaque rupture form the nidus for thrombi. The normal artery wall has several fibrinolytic or antithrombotic mechanisms that tend to resist thrombosis and lyse clots that begin to form in situ. Such antithrombotic or thrombolytic molecules include

1	artery wall has several fibrinolytic or antithrombotic mechanisms that tend to resist thrombosis and lyse clots that begin to form in situ. Such antithrombotic or thrombolytic molecules include thrombomodulin, tissueand urokinase-type plasminogen activators, heparan sulfate proteoglycans, prostacyclin, and nitric oxide.

1	C. When the clot overwhelms the endogenous fibrinolytic mechanisms, it may propagate and lead to arterial occlusion. The consequences of this occlusion depend on the degree of existing collateral vessels. In a patient with chronic multivessel occlusive coronary artery disease (CAD), collateral channels have often formed. In such circumstances, even a total arterial occlusion may not lead to myocardial infarction (MI), or it may produce an unexpectedly modest or a non-ST-segment elevation infarct because of collateral flow. In a patient with less advanced disease and without substantial stenotic lesions to provide a stimulus for collateral vessel formation, sudden plaque rupture and arterial occlusion commonly produces an ST-segment elevation infarction. These are the types of patients who may present with MI or sudden death as a first manifestation of coronary atherosclerosis. In some cases, the thrombus may lyse or organize into a mural thrombus without occluding the vessel. Such

1	who may present with MI or sudden death as a first manifestation of coronary atherosclerosis. In some cases, the thrombus may lyse or organize into a mural thrombus without occluding the vessel. Such instances may be clinically silent. D. The subsequent thrombin-induced fibrosis and healing causes a fibroproliferative response that can lead to a more fibrous lesion that can produce an eccentric plaque that causes a hemodynamically significant stenosis. In this way, a nonocclusive mural thrombus, even if clinically silent or causing unstable angina rather than infarction, can provoke a healing response that can promote lesion fibrosis and luminal encroachment. Such a sequence of events may convert a “vulnerable” atheroma with a thin fibrous cap that is prone to rupture into a more “stable” fibrous plaque with a reinforced cap. Angioplasty of unstable coronary lesions may “stabilize” the lesions by a similar mechanism, producing a wound followed by healing.

1	CHAPTER 291e The Pathogenesis, Prevention, and Treatment of Atherosclerosis PART 10 Disorders of the Cardiovascular System 291e-4 understanding of the mechanisms that link risk factors to the pathogenesis of atherosclerosis and its complications.

1	Atherosclerotic lesions occur ubiquitously in Western societies, and the prevalence of this disease is on the rise globally. Most atheromata produce no symptoms, and many never cause clinical manifestations. Numerous patients with diffuse atherosclerosis may succumb to unrelated illnesses without ever having experienced a clinically significant manifestation of atherosclerosis. Arterial remodeling during atheroma formation accounts for some of this variability in the clinical expression of atherosclerotic disease (Fig. 291e-2A). During the initial phases of atheroma development, the plaque usually grows outward, in an abluminal direction. Vessels affected by atherogenesis tend to increase in diameter, a phenomenon known as compensatory enlargement, a type of vascular remodeling. The growing atheroma does not encroach on the arterial lumen until the burden of atherosclerotic plaque exceeds ~40% of the area encompassed by the internal elastic lamina. Thus, during much of its life

1	growing atheroma does not encroach on the arterial lumen until the burden of atherosclerotic plaque exceeds ~40% of the area encompassed by the internal elastic lamina. Thus, during much of its life history, an atheroma will not cause stenosis that can limit tissue perfusion.

1	Flow-limiting stenoses commonly form later in the history of the plaque. Many such plaques cause stable syndromes such as demand-induced angina pectoris or intermittent claudication in the extremities. In the coronary circulation and other circulations, even total vascular occlusion by an atheroma does not invariably lead to infarction. The hypoxic stimulus of repeated bouts of ischemia characteristically induces formation of collateral vessels in the myocardium, mitigating the consequences of an acute occlusion of an epicardial coronary artery. By contrast, many lesions that cause acute or unstable atherosclerotic syndromes, particularly in the coronary circulation, may arise from atherosclerotic plaques that do not produce a flow-limiting stenosis. Such lesions may produce only minimal luminal irregularities on traditional angiograms and often do not meet the traditional criteria for “significance” by arteriography. Thrombi arising from such nonocclusive stenoses may explain the

1	luminal irregularities on traditional angiograms and often do not meet the traditional criteria for “significance” by arteriography. Thrombi arising from such nonocclusive stenoses may explain the frequency of MI as an initial manifestation of coronary artery disease (CAD) (in at least one-third of cases) in patients who report no prior history of angina pectoris, a syndrome usually caused by flow-limiting stenoses.

1	Plaque Instability and Rupture Postmortem studies afford considerable insight into the microanatomic substrate underlying the “instability” of plaques that do not cause critical stenoses. A superficial erosion of the endothelium or a frank plaque rupture or fissure usually produces the thrombus that causes episodes of unstable angina pectoris or the occlusive and relatively persistent thrombus that causes acute MI (Fig. 291e-2B). Rupture of the plaque’s fibrous cap (Fig. 291e-2C) permits contact between coagulation factors in the blood and highly thrombogenic tissue factor expressed by macrophage foam cells in the plaque’s lipid-rich core. If the ensuing thrombus is nonocclusive or transient, the episode of plaque disruption may not cause symptoms or may result in episodic ischemic symptoms such as rest angina. Occlusive thrombi that endure often cause acute MI, particularly in the absence of a well-developed collateral circulation that supplies the affected territory. Repetitive

1	such as rest angina. Occlusive thrombi that endure often cause acute MI, particularly in the absence of a well-developed collateral circulation that supplies the affected territory. Repetitive episodes of plaque disruption and healing provide one likely mechanism of transition of the fatty streak to a more complex fibrous lesion (Fig. 291e-2D). The healing process in arteries, as in skin wounds, involves the laying down of new extracellular matrix and fibrosis.

1	Not all atheromata exhibit the same propensity to rupture. Pathologic studies of culprit lesions that have caused acute MI reveal several characteristic features. Plaques that have caused thromboses tend to have thin fibrous caps, relatively large lipid cores, a high content of macrophages, outward remodeling, and spotty (rather than dense) calcification. Morphometric studies of such culprit lesions show that at sites of plaque rupture, macrophages and T lymphocytes predominate and contain relatively few smooth-muscle cells. The cells that concentrate at sites of plaque rupture bear markers of inflammatory activation. In addition, patients with active atherosclerosis and acute coronary syndromes display signs of disseminated inflammation. Inflammatory mediators regulate processes that govern the integrity of the plaque’s fibrous cap and, hence, its propensity to rupture. For example, the T cell–derived cytokine IFN-γ, which is found in atherosclerotic plaques, can inhibit growth and

1	the integrity of the plaque’s fibrous cap and, hence, its propensity to rupture. For example, the T cell–derived cytokine IFN-γ, which is found in atherosclerotic plaques, can inhibit growth and collagen synthesis of smooth-muscle cells, as noted above. Cytokines derived from activated macrophages and lesional T cells can boost production of proteolytic enzymes that can degrade the extracellular matrix of the plaque’s fibrous cap. Thus, inflammatory mediators can impair the collagen synthesis required for maintenance and repair of the fibrous cap and trigger degradation of extracellular matrix macromolecules, processes that weaken the plaque’s fibrous cap and enhance its susceptibility to rupture (so-called vulnerable plaques, Fig. 291e-3). In contrast to plaques with these features of vulnerability, those with a dense extracellular matrix and relatively thick fibrous cap without substantial tissue factor–rich lipid cores seem generally resistant to rupture and unlikely to provoke

1	vulnerability, those with a dense extracellular matrix and relatively thick fibrous cap without substantial tissue factor–rich lipid cores seem generally resistant to rupture and unlikely to provoke thrombosis.

1	Functional features of the atheromatous plaque, in addition to its degree of luminal encroachment, influence the clinical manifestations of this disease. This enhanced understanding of plaque biology provides insight into the diverse ways in which atherosclerosis can present clinically and the reasons why the disease may remain silent or stable for prolonged periods, punctuated by acute complications at certain times. Increased understanding of atherogenesis provides new insight into the mechanisms linking it to the risk factors discussed below, indicates the ways in which current therapies may improve outcomes, and suggests new targets for future intervention.

1	The systematic study of risk factors for atherosclerosis emerged from a coalescence of experimental results, as well as from cross-sectional and ultimately longitudinal studies in humans. The prospective, community-based Framingham Heart Study provided rigorous support for the concept that hypercholesterolemia, hypertension, and other factors correlate with cardiovascular risk. Similar observational studies performed worldwide bolstered the concept of “risk factors” for cardiovascular disease.

1	From a practical viewpoint, the cardiovascular risk factors that have emerged from such studies fall into two categories: those modifiable by lifestyle and/or pharmacotherapy, and those that are immutable, such as age and sex. The weight of evidence supporting various risk factors differs. For example, hypercholesterolemia and hypertension certainly predict coronary risk, but the magnitude of the contributions of other so-called nontraditional risk factors, such as levels of homocysteine, levels of lipoprotein (a) [Lp(a)], and infection, remains controversial. Moreover, some biomarkers that predict cardiovascular risk may not participate in the causal pathway for the disease or its complications. Genetic studies using genome-wide association (GWAS) approaches and Mendelian randomization approaches have helped to distinguish between risk markers and factors that contribute causally to the disease. For example, recent genetic studies suggest that C-reactive protein (CRP) does not itself

1	have helped to distinguish between risk markers and factors that contribute causally to the disease. For example, recent genetic studies suggest that C-reactive protein (CRP) does not itself mediate atherogenesis, despite its ability to predict risk, whereas Lp(a) and apolipoprotein C3 have emerged as a causal risk factor. Table 291e-1 lists a number of risk factors implicated in atherosclerosis. The sections below will consider some of these factors and approaches to their modification.

1	Abnormalities in plasma lipoproteins and derangements in lipid metabolism rank among the most firmly established and best understood risk factors for atherosclerosis. Chapter 421 describes the lipoprotein classes and provides a detailed discussion of lipoprotein metabolism. The American College of Cardiology and American Heart Association (ACC/AHA) promulgated new guidelines on risk assessment, lifestyle measures, and cholesterol management in 2013. The panels that produced these guidelines followed an evidence-based approach.

1	FIGuRE 291e-3 Inflammatory pathways that predispose atherosclerotic plaques to rupture and provoke thrombosis. A cross-section of an atheromatous plaque at the bottom of the figure shows the central lipid core that contains macrophage foam cells (yellow) and T cells (blue). The intima and media also contain arterial smooth-muscle cells (red ), which are the source of arterial collagen (depicted as triple helical coiled structures). Activated T cells (of the type 1 helper T cell subtype) secrete cytokine interferon γ, which inhibits the production of the new, interstitial collagen that is required to repair and maintain the plaque’s protective fibrous cap (upper left). The T cells can also activate the macrophages in the intimal lesion by expressing the inflammatory mediator CD40 ligand (CD154), which engages its cognate receptor (CD40) on the phagocyte. This inflammatory signalling causes overproduction of interstitial collagenases (matrix metalloproteinases [MMPs] 1, 8, and 13) that

1	which engages its cognate receptor (CD40) on the phagocyte. This inflammatory signalling causes overproduction of interstitial collagenases (matrix metalloproteinases [MMPs] 1, 8, and 13) that catalyze the initial rate-limiting step in collagen breakdown (top right). CD40 ligation also causes macrophages to overproduce tissue-factor procoagulant. Thus, inflammatory signalling puts the collagen in the plaque’s fibrous cap in double jeopardy—decreasing synthesis and increasing breakdown—rendering the cap susceptible to rupture. Inflammatory activation also boosts tissue-factor production, which triggers thrombus formation in the disrupted plaque. These mechanisms link inflammation in the plaque to the thrombotic complications of atherosclerosis, including the acute coronary syndromes. (Adapted from P Libby: N Engl J Med 368:2004, 2013.)

1	CHAPTER 291e The Pathogenesis, Prevention, and Treatment of Atherosclerosis

1	The 2013 cholesterol guideline focused on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) rather than other classes of lipid-modulating drugs, including fibric acid derivatives, cholesterol absorption inhibitors such as ezetimibe, and niacin products. The guideline cites the lack of contemporary randomized clinical trial evidence that supports the efficacy of these nonstatin lipid-modifying agents in cardiovascular event reduction. The cholesterol guideline defined four statin benefit groups (Table 291e-2): (1) all individuals who have clinical atherosclerotic cardiovascular disease (ASCVD), therefore considered “secondary prevention”; (2) those with LDL cholesterol ≥190 mg/dL without a secondary cause such as a high intake of saturated or trans fats, various drugs, or certain diseases; (3) individuals with diabetes without established cardiovascular disease who are 40–75 years old and have LDL cholesterol of 70–189 mg/dL; and (4) those without

1	various drugs, or certain diseases; (3) individuals with diabetes without established cardiovascular disease who are 40–75 years old and have LDL cholesterol of 70–189 mg/dL; and (4) those without established ASCVD without diabetes who are 40–75 years old and who have LDL cholesterol of 70–189 mg/dL and a calculated ASCVD risk ≥7.5%. An online risk calculator based on pooled cohorts was provided to aid clinicians and patients in calculating their risk (http://my.americanheart.org/ professional/StatementsGuidelines/PreventionGuidelines/PreventionGuidelines_UCM_457698_SubHomePage.jsp). Other validated risk calculators that incorporate family history of CAD and a marker of inflammation (high-sensitivity CRP [hsCRP]) that apply to U.S. women and men exist (http://www.reynoldsriskscore.org). Downloadable applications for risk calculation on handheld devices are readily available.

1	The 2013 guideline emphasized a patient-centered approach and recommended that clinicians and patients engage in a risk-benefit conversation before starting statin therapy and not rely solely on calculated risks or arbitrary category assignment. It further emphasizes that medications do not supplant a healthy lifestyle. The guideline also provides some practical suggestions regarding management of muscle symptoms attributed to statins, an issue of considerable concern to many patients and practitioners alike. In a major departure from prior guidelines, the 2013 guideline eliminates LDL targets as goals of therapy. The panel did so because major clinical trials did not titrate therapy to a goal, but rather used fixed doses of statins. Instead, the new guideline suggests different intensities of statin therapy based on risk category (Fig. 291e-4). The 2013 guideline focus on statins reflects an extensive body of rigorous evidence that supports the effectiveness of this class of drugs

1	The 2013 guideline focus on statins reflects an extensive body of rigorous evidence that supports the effectiveness of this class of drugs PART 10 Disorders of the Cardiovascular System Low HDL cholesterola (<1.0 mmol/L [<40 mg/dL]) Family history of premature CHD aHDL cholesterol ≥1.6 mmol/L (≥60 mg/dL) has been viewed as a “negative” risk factor. Abbreviations: BMI, body mass index; BP, blood pressure; CHD, coronary heart disease; HDL, high-density lipoprotein; LDL, low-density lipoprotein. in cardiovascular event reduction and an acceptable risk-benefit relationship (Fig. 291e-5). Moreover, because almost all statins are now available as generic statins medications, cost has become much less of an impediment to their use.

1	The clinical use of effective pharmacologic strategies for lowering LDL has reduced cardiovascular events markedly, but a considerable burden of residual risk remains even in patients treated with high-intensity statins. Hence, current studies are evaluating other avenues to address the residual burden of cardiovascular disease that persists despite statin treatment. Inhibitors of genetic studies identified proprotein convertase subtilisin kexin-like 9 (PCSK9) as a regulator of LDL levels associated with cardiovascular outcomes. Interaction of the LDL receptor with PCSK9 hastens the receptor’s degradation, and hence yields higher circulating LDL concentrations. Genetic variants that lower PCSK9 activity appear to protect against cardiovascular events. Monoclonal antibodies that neutralize PCSK9 lower LDL levels even in statin-treated patients and are currently under investigation as novel therapeutics to lower cardiovascular risk.

1	LDL-lowering therapies do not appear to exert their beneficial effect on cardiovascular events by causing a marked “regression” of stenoses. Studies of lipid lowering monitored by angiography or by intravascular imaging modalities have shown at best a modest reduction in coronary artery stenoses over the duration of study, despite abundant evidence of event reduction. These results suggest that the beneficial mechanism of lipid lowering by statins does not require a substantial reduction in the fixed stenoses. Rather, the benefit may derive from “stabilization” of atherosclerotic lesions without substantially decreased stenosis. ≥190 mg/dL without secondary cause (e.g., saturated/trans fats, drugs, certain diseases) prevention with diabetes mellitus: age 40–75 years, LDL-C 70–189 mg/dL prevention without diabetes mellitus: age 40–75 years, LDL-C 70–189 mg/dL, estimated ASCVD risk ≥7.5% Abbreviations: ACC/AHA, American College of Cardiology and American Heart Association;

1	Abbreviations: ACC/AHA, American College of Cardiology and American Heart Association; ASCVD, atherosclerotic cardiovascular disease; LDL-C, low-density lipoprotein cholesterol. Source: Adapted from NJ Stone et al: 2013 ACC/AHA Guideline on the Treatment of Blood Cholesterol to Reduce Atherosclerotic Cardiovascular Risk in Adults. J Am Coll Cardiol 2013, doi: 10.1016/j.jacc.2013.11.002. Such stabilization of atherosclerotic lesions and the attendant decrease in coronary events may result from the egress of lipids or from favorably influencing aspects of the biology of atherogenesis discussed above. In addition, as sizable lesions may protrude abluminally rather than into the lumen due to complementary enlargement, shrinkage of such plaques may not be apparent on angiograms. The consistent benefit of statins may depend not only on their salutary effects on the lipid profile, but also on direct modulation of plaque biology independent of lipid lowering.

1	As the prevalence of metabolic syndrome and diabetes increases, many patients present with low concentrations of HDL (HDL cholesterol <1.0 mmol/L [<40 mg/dL]). A baseline measurement of HDL cholesterol indubitably correlates with future cardiovascular risk. Yet, the utility of therapies that raise HDL cholesterol levels in blood as effective interventions to reduce cardiovascular vascular events has come into question. Blood HDL levels vary inversely with those of triglycerides, and the independent role of HDL versus triglycerides as a cardiovascular risk factor remains unsettled. The 2013 guideline does not advocate any specific therapy for raising HDL. Indeed, multiple recent trials failed to show that raising HDL cholesterol levels improves cardiovascular outcomes, and recent genetic studies cast doubt on low HDL as a causal risk factor for atherosclerotic events. Weight loss and physical activity can raise HDL, and these lifestyle measures merit universal adoption (Table 291e-3).

1	cast doubt on low HDL as a causal risk factor for atherosclerotic events. Weight loss and physical activity can raise HDL, and these lifestyle measures merit universal adoption (Table 291e-3). Nicotinic acid, particularly in combination with statins, can robustly raise HDL, but clinical trial data do not support the effectiveness of nicotinic acid in cardiovascular risk reduction. Agonists of nuclear receptors provide another potential avenue for raising HDL levels. Yet patients treated with peroxisome proliferator–activated receptors alpha and gamma (PPAR-α and -γ) agonists have not consistently shown improved cardiovascular outcomes, and at least some PPAR agonists have been associated with worsened cardiovascular outcomes. Other agents in clinical development raise HDL levels by inhibiting cholesteryl ester transfer protein (CETP). Two such agents have undergone large-scale clinical evaluation and have not shown efficacy in improving cardiovascular outcomes. Clinical studies

1	inhibiting cholesteryl ester transfer protein (CETP). Two such agents have undergone large-scale clinical evaluation and have not shown efficacy in improving cardiovascular outcomes. Clinical studies currently under way will assess the effectiveness of two other CETP inhibitors that lack some of the adverse off-target actions encountered with the first agent tested.

1	The mechanism by which elevated LDL levels promote atherogenesis may involve oxidative modification. Yet, rigorous and well-controlled clinical trials have failed to demonstrate that antioxidant vitamin therapy improves coronary heart disease (CHD) outcomes. In regard to nontraditional risk factors including homocysteine and infection, large-scale clinical trials using vitamins to lower homocysteine or using antibiotics have not reduced cardiovascular events. Therefore, the current evidence base does not support the use of vitamins or antibiotics to lower cardiovascular risk. Hypertension (See also Chap. 298) A wealth of epidemiologic data support a relationship between hypertension and atherosclerotic risk, and extensive clinical trial evidence has established that pharmacologic treatment of hypertension can reduce the risk of stroke, heart failure, and CHD events.

1	Diabetes Mellitus, Insulin Resistance, and the Metabolic Syndrome (See also Chap. 417) Most patients with diabetes mellitus die of atherosclerosis and its complications. Aging and rampant obesity underlie a current epidemic of type 2 diabetes mellitus. The abnormal lipoprotein profile associated with insulin resistance, known as diabetic dyslipidemia, accounts for part of the elevated cardiovascular risk in patients with type 2 diabetes. Although diabetic individuals often have LDL cholesterol levels near the average, the LDL particles tend to be smaller and denser and, therefore, more atherogenic. Other features of diabetic dyslipidemia include low HDL and elevated triglyceride levels. Hypertension also frequently accompanies obesity, insulin resistance, and dyslipidemia. This commonly encountered clinical cluster of risk factors has become known as the metabolic syndrome (Chap. 422). Despite legitimate concerns about whether clustered components confer more risk than the individual

1	clinical cluster of risk factors has become known as the metabolic syndrome (Chap. 422). Despite legitimate concerns about whether clustered components confer more risk than the individual components, the metabolic syndrome concept may offer clinical utility.

1	Heart-healthy lifestyle habits are the foundation of ASCVD prevention. In individuals not receiving cholesterol-lowering drug therapy, recalculate estimated 10-y ASCVD risk every 4–6 y in individuals aged 40-75 y without clinical ASCVD or diabetes and with LDL–C 70-189 mg/dL. Age >75 y OR if not candidate for high-intensity statin Moderate-intensity statin High-intensity statin (Moderate-intensity statin if not candidate for high-intensity statin) Moderate-intensity statin Estimated 10-y ASCVD risk ˜7.5% High-intensity statin Estimate 10-y ASCVD risk with pooled cohort equations Clinical ASCVD LDL–C ˜190 mg/dL Diabetes Type 1 or 2 age 40–75 y Yes Yes Yes Yes Age °75 y High-intensity statin (Moderate-intensity statin if not candidate for high-intensity statin) No No No Yes CHAPTER 291e The Pathogenesis, Prevention, and Treatment of Atherosclerosis Moderate-to-high intensity statin ˜7.5% estimated 10-y ASCVD risk and age 40–75 y

1	CHAPTER 291e The Pathogenesis, Prevention, and Treatment of Atherosclerosis Moderate-to-high intensity statin ˜7.5% estimated 10-y ASCVD risk and age 40–75 y ASCVD prevention benefit of statin therapy may be less clear in other groups In selected individuals, consider additional factors influencing ASCVD risk and potential ASCVD risk benefits and adverse effects, drug–drug interactions, and patient preferences for statin treatment FIGuRE 291e-4 Major recommendations for statin therapy for atherosclerotic cardiovascular disease (ASCVD) prevention. LDL-C, low-density lipoprotein cholesterol. (From NJ Stone et al: J Am Coll Cardiol, 2013, doi: 10.1016/j.jacc.2013.11.002.)

1	Therapeutic objectives for intervention in these patients include addressing the underlying causes, including obesity and low physical activity, by initiating lifestyle measures (see below). Establishing that strict glycemic control reduces the risk of macrovascular complications of diabetes has proved much more elusive than the beneficial effects on microvascular complications such as retinopathy and renal disease. Indeed, “tight” glycemic control may increase adverse events in patients with type 2 diabetes, lending even greater importance to aggressive control of other aspects of risk in this patient population. In this regard, multiple clinical trials have demonstrated unequivocal benefit of statin therapy in diabetic patients over all ranges of LDL cholesterol levels (but not those with end-stage renal disease or advanced heart failure). Among the oral hypoglycemic agents, metformin possesses the best evidence base for cardiovascular event reduction. The novel oral hypoglycemic

1	with end-stage renal disease or advanced heart failure). Among the oral hypoglycemic agents, metformin possesses the best evidence base for cardiovascular event reduction. The novel oral hypoglycemic agents tested in sufficiently powered trials, the dipeptidyl peptidase-4 (DPP-4) inhibitors saxagliptin and alogliptin, did not show cardiovascular benefit. Indeed, saxagliptin was associated with a slight increase in heart failure. Diabetic populations appear to derive particular benefit from antihypertensive strategies that block the action of angiotensin II. Thus, the antihypertensive regimen for patients with the metabolic syndrome should include angiotensinconverting enzyme inhibitors or angiotensin receptor blockers when possible. Many of these individuals will require more than one anti-hypertensive agent to reach the 2013 goals for individuals 18 years of age or older with diabetes to achieve a systolic blood pressure of less than 140 mmHg and a diastolic blood pressure of less

1	agent to reach the 2013 goals for individuals 18 years of age or older with diabetes to achieve a systolic blood pressure of less than 140 mmHg and a diastolic blood pressure of less than 90 mmHg.

1	Male Sex/Postmenopausal State Decades of observational studies have verified excess coronary risk in men compared with premenopausal women. After menopause, however, coronary risk accelerates in women. Although observational and experimental studies have suggested that estrogen therapy reduces coronary risk, large-scale randomized clinical trials have not demonstrated a net benefit of estrogen with or without progestins on CHD outcomes. In the Heart LDL cholesterol reduction (mmol/L) with statin treatment 1 0 5 10 15 20 1.5 2 2.5 <5% ˜5% to <10% ˜10% to <20% ˜20% to <30% ˜30% 2.7 2.2 1.2 1.7 46 38 30 20 21 16 11 14 11 8 754 25 17 8 risk of PART 10 Disorders of the Cardiovascular System FIGuRE 291e-5 The Cholesterol Treatment Trialists Collaboration meta-analyzed 27 randomized clinical trials evaluating statin therapy.

1	PART 10 Disorders of the Cardiovascular System FIGuRE 291e-5 The Cholesterol Treatment Trialists Collaboration meta-analyzed 27 randomized clinical trials evaluating statin therapy. They found profound decreases in both major vascular events and vascular death (not shown) proportional to the magnitude of low-density lipoprotein (LDL) cholesterol reduction achieved with statin treatment. This diagram shows the results of this meta-analysis for vascular death. (From Lancet 380:581, 2012.) and Estrogen/Progestin Replacement Study (HERS), postmenopausal female survivors of acute MI were randomized to an estrogen/progestin combination or to placebo. This study showed no overall reduction in recurrent coronary events in the active treatment arm. Indeed, early in the 5-year course of this trial, a trend occurred toward an increase

1	The adult population should be encouraged to practice heart healthy lifestyle behaviors, including: • Consume a dietary pattern that emphasizes intake of vegetables, fruits, and whole grains; include low-fat dairy products, poultry, fish, legumes, nontropical vegetable oils, and nuts; and limit intake of sodium, sweets, sugar-sweetened beverages, and red meats. • Adapt this dietary pattern to appropriate calorie requirements, personal and cultural food preferences, and nutrition therapy for other medical conditions (including diabetes mellitus). • Achieve this pattern by following plans such as the DASH dietary pattern, the USDA Food Pattern, or the AHA Diet.

1	• Achieve this pattern by following plans such as the DASH dietary pattern, the USDA Food Pattern, or the AHA Diet. • Engage in 2 h and 30 min a week of moderate-intensity or 1 h and 15 min (75 min) a week of vigorous-intensity aerobic physical activity, or an equivalent combination of moderateand vigorous-intensity aerobic physical activity. Aerobic activity should be performed in episodes of at least 10 min, preferably spread throughout the week. • Achieve and maintain a healthy weight. Refer to the 2013 Obesity Expert Panel Report for recommendations on weight loss and maintenance. Abbreviations: ACC/AHA, American College of Cardiology and American Heart Association; DASH, Dietary Approaches to Stop Hypertension; USDA, U.S. Department of Agriculture. Source: Adapted from RH Eckel et al: 2013 AHA/ACC Guideline on Lifestyle Management to Reduce Cardiovascular Risk. J Am Coll Cardiol 2013, doi: 10.1016/j.jacc.2013.11.003.

1	in vascular events in the treated women. Extended follow-up of this cohort did not disclose an accrual of benefit in the treatment group. The Women’s Health Initiative (WHI) study arm, using a similar estrogen plus progesterone regimen, was halted due to a small but significant hazard of cardiovascular events, stroke, and breast cancer. The estrogen without progestin arm of WHI (conducted in women without a uterus) was stopped early due to an increase in strokes, and failed to afford protection from MI or CHD death during observation over 7 years. The excess cardiovascular events in these trials may result from an increase in thromboembolism (Chap. 413). Physicians should work with women to provide information and help weigh the small but evident CHD risk of estrogen ± progestin versus the benefits for postmenopausal symptoms and osteoporosis, taking personal preferences into account. Post hoc analyses of observational studies suggest that estrogen therapy in women younger than or

1	the benefits for postmenopausal symptoms and osteoporosis, taking personal preferences into account. Post hoc analyses of observational studies suggest that estrogen therapy in women younger than or closer to menopause than the women enrolled in WHI might confer cardiovascular benefit. Thus, the timing in relation to menopause or the age at which estrogen therapy begins may influence its risk/benefit balance.

1	The lack of efficacy of estrogen therapy in cardiovascular risk reduction highlights the need for redoubled attention to known modifiable risk factors in women. Meta-analysis supports the efficacy of statins to reduce cardiovascular events in women in primary prevention, as well as in those who have already experienced a cardiovascular event. Dysregulated Coagulation or Fibrinolysis Thrombosis ultimately causes the gravest complications of atherosclerosis. The propensity to form thrombi and/or lyse clots once they form influences the manifestations of atherosclerosis. Thrombosis provoked by atheroma rupture and subsequent healing may promote plaque growth. Certain individual characteristics can influence thrombosis or fibrinolysis and have received attention as potential coronary risk factors. For example, 30.0 25.0 20.0 15.0 10.0 5.0 0.0 10–205–100.5–1.01.0–3.03.0–10.0>10.0hsCRP mg/L

1	FIGuRE 291e-6 C-reactive protein (CRP) level adds to the predictive value of the Framingham score. hsCRP, high-sensitivity measurement of CRP. (Adapted from PM Ridker et al: Circulation 109:2818, 2004.) fibrinogen levels correlate with coronary risk and provide information about coronary risk independent of the lipoprotein profile. The stability of an arterial thrombus depends on the balance between fibrinolytic factors, such as plasmin, and inhibitors of the fibrinolytic system, such as plasminogen activator inhibitor 1 (PAI-1). Individuals with diabetes mellitus or the metabolic syndrome have elevated levels of PAI-1 in plasma, and this probably contributes to the increased risk of thrombotic events. Lp(a) (Chap. 421) may modulate fibrinolysis, and individuals with elevated Lp(a) levels have increased CHD risk.

1	Aspirin reduces CHD events in several contexts. Chapter 293 discusses aspirin therapy in stable ischemic heart disease, Chap. 294 reviews recommendations for aspirin treatment in acute coronary syndromes, and Chap. 446 describes aspirin’s role in preventing recurrent ischemic stroke. In primary prevention, pooled trial data show that low-dose aspirin treatment (81 mg/d to 325 mg on alternate days) can reduce the risk of a first MI in men. Although the Women’s Health Study (WHS) showed that aspirin (100 mg on alternate days) reduced strokes by 17%, it did not prevent MI in women. Current AHA guidelines recommend the use of low-dose aspirin (75–160 mg/d) for women with high cardiovascular risk (≥20% 10-year risk), for men with a ≥10% 10-year risk of CHD, and for all aspirin-tolerant patients with established cardiovascular disease who lack contraindications.

1	Inflammation An accumulation of clinical evidence shows that markers of inflammation correlate with coronary risk. For example, plasma levels of CRP, as measured by a high-sensitivity assay (hsCRP), prospectively predict the risk of MI. CRP levels also correlate with the outcome in patients with acute coronary syndromes. In contrast to several other novel risk factors, CRP adds predictive information to that derived from established risk factors, such as those included in the Framingham score (Fig. 291e-6). Mendelian randomization studies do not support a causal role for CRP in cardiovascular disease. Thus, CRP serves as a validated biomarker of risk, but probably not as a direct contributor to pathogenesis. Elevations in acute-phase reactants such as fibrinogen and CRP reflect the overall inflammatory burden, not just vascular foci of inflammation. Visceral adipose tissue releases proinflammatory cytokines that drive CRP production and may represent a major extravascular

1	Placebo 7832 1.11 LDL ˜ 70 mg/dL, hsCRP ˜ 2 mg/L 1384 1.11 LDL < 70 mg/dL, hsCRP ˜ 2 mg/L 2921 0.62 LDL ˜ 70 mg/dL, hsCRP < 2 mg/L 726 0.54 LDL < 70 mg/dL, hsCRP < 2 mg/L 2685 0.38 stimulus to the elevation of inflammatory markers in obese and 291e-9 overweight individuals. Indeed, CRP levels rise with body mass index (BMI) or visceral adipose depot as assessed by imaging, and weight reduction lowers CRP levels. Infectious agents might also furnish inflammatory stimuli related to cardiovascular risk.

1	Statin therapy likely reduces cardiovascular events in part by muting the inflammatory aspects of the pathogenesis of atherosclerosis. For example, in statin trials conducted in both primary (JUPITER) and secondary (PROVE-IT/TIMI-22) prevention populations, prespecified analyses showed that those who achieved lower levels of both LDL and CRP had better clinical outcomes than did those who only reached the lower level of either the inflammatory marker or the atherogenic lipoprotein (Fig. 291e-7). The anti-inflammatory effect of statins appears independent of LDL lowering, because these two variables correlated very poorly in individual subjects in multiple clinical trials.

1	Lifestyle Modification The prevention of atherosclerosis presents a long-term challenge to all health care professionals and for public health policy. Both individual practitioners and organizations providing health care should strive to help patients optimize their risk factor profiles long before atherosclerotic disease becomes manifest. The current accumulation of cardiovascular risk in youth and in certain minority populations presents a particularly vexing concern from a public health perspective.

1	The ACC/AHA 2013 Guideline on Lifestyle Management to Reduce Cardiovascular Risk relied on rigorous evidentiary reviews. Few lifestyle interventions have undergone rigorous evaluation in randomized clinical trials. Therefore, these guidelines reflected judicious analysis of carefully selected observational studies and of intervention studies that relied primarily on biomarkers or surrogate endpoints rather than “hard” cardiovascular outcomes. Table 291e-3 summarizes the ACC/ AHA lifestyle recommendations.

1	The care plan for all patients seen by internists should include measures to assess and minimize cardiovascular risk. Physicians must counsel patients about the health risks of tobacco use and provide guidance and resources regarding smoking cessation. Similarly, physicians should advise all patients about prudent dietary and physical activity habits for maintaining ideal body weight. Both National Institutes of Health (NIH) and AHA statements recommend at least 30 min of moderate-intensity physical activity per day. Obesity, particularly the male pattern of centripetal or visceral fat accumulation, can contribute to the elements of the “metabolic syndrome” cluster. Physicians should encourage their patients to take personal responsibility for behavior related to modifiable risk factors for the development of premature atherosclerotic disease. Conscientious counseling and patient education may forestall the need for pharmacologic measures intended to reduce coronary risk.

1	Issues in Risk Assessment A growing panel of markers of coronary risk presents a perplexing array to the practitioner. Markers measured in peripheral blood include size fractions of LDL particles and concentrations of homocysteine, Lp(a), fibrinogen, CRP, PAI-1, myeloperoxidase, lipoprotein-associated phospholipase A2, and imaging assessment of subclinical atherosclerosis, among many others. In general, such specialized tests add little to the information available from a careful history and physical examination combined with measurement of a plasma lipoprotein profile and fasting blood glucose. The hsCRP measurement may well prove an exception in view of its robustness in risk CHAPTER 291e The Pathogenesis, Prevention, and Treatment of Atherosclerosis P < 0.001

1	FIGuRE 291e-7 Evidence from the JUPITER study that both low-density lipoprotein (LDL)-lowering and anti-inflammatory actions contribute to the benefit of statin therapy in primary prevention. See text for explanation. hsCRP, high-sensitivity measurement of C-reactive protein (CRP). (Adapted from PM Ridker et al: Lancet 373:1175, 2009.) 291e-10 prediction, ease of reproducible and standardized measurement, relative stability in individuals over time, ability to add to the risk information disclosed by standard measurements such as the components of the Framingham risk score, and most importantly, the demonstration in a large-scale trial (JUPITER) that allocating therapy can reduce cardiovascular events in those deemed ineligible by traditional risk assessment criteria. The addition of information regarding a family history of premature atherosclerosis (a simply obtained indicator of genetic susceptibility), together with the inflammation marker hsCRP, permits correct reclassification

1	regarding a family history of premature atherosclerosis (a simply obtained indicator of genetic susceptibility), together with the inflammation marker hsCRP, permits correct reclassification of risk in individuals, especially those whose Framingham scores place them at intermediate risk. Available data do not support the routine use of imaging studies to screen for subclinical disease (e.g., measurement of carotid intimamedia thickness, coronary artery calcification, and use of computed tomographic coronary angiograms [CTA]). Inappropriate use of such imaging modalities may promote excessive alarm in asymptomatic individuals and prompt invasive diagnostic and therapeutic procedures of unproven value for both asymptomatic atherosclerosis and incidental findings. Widespread application of such modalities for screening should await proof that targeting therapies based on their application provides clinical benefit. The 2013 ACC/AHA Guideline on the Assessment of Cardiovascular Risk

1	of such modalities for screening should await proof that targeting therapies based on their application provides clinical benefit. The 2013 ACC/AHA Guideline on the Assessment of Cardiovascular Risk recommends the use of newer risk markers if uncertainty persists after assessing quantitative risk using the pooled cohort calculator. The guideline states that family history, hsCRP, coronary artery calcium (CAC) score, or ankle-brachial index (ABI) may then be considered to inform treatment decision making. It discourages carotid intimamedia thickness (CIMT) for routine measurement in clinical practice for risk assessment for a first ASCVD event. The guideline panel deemed the contribution to risk assessment for a first ASCVD event using apolipoprotein B (ApoB), chronic kidney disease, albuminuria, or cardiorespiratory fitness as uncertain at present.

1	PART 10 Disorders of the Cardiovascular System Progress in human genetics holds considerable promise for risk prediction and for individualization of cardiovascular therapy. Many early reports identified single-nucleotide polymorphisms (SNPs) in candidate genes as predictors of cardiovascular risk. The validation of such genetic markers of risk and drug responsiveness in multiple populations often proved disappointing. The era of GWAS has led to discovery of sites of genetic variation that reproducibly indicate heightened cardiovascular risk (e.g., chromosome 9p21). The advent of technology that permits relatively rapid and inexpensive exome or whole-genome sequencing promises to identify new therapeutic targets, sharpen risk prediction, and deploy preventive or therapeutic measures in a more personalized manner. Despite this considerable promise, genetic scores for risk prediction have not yet demonstrated consistent improvement over algorithms that use traditional tools.

1	THE CHALLENGE OF IMPLEMENTATION: CHANGING PHYSICIAN AND PATIENT BEHAVIOR

1	Despite declining age-adjusted rates of coronary death, cardiovascular mortality worldwide is rising due to aging of the population and due to subsiding of communicable diseases and increased prevalence of risk factors in developing countries. Enormous challenges remain regarding translation of the current evidence base into practice. Physicians must learn how to help individuals adopt a healthy lifestyle in a culturally appropriate manner and to deploy their increasingly powerful pharmacologic tools most economically and effectively. The obstacles to implementation of current evidence-based prevention and treatment of atherosclerosis involve economics, education, physician awareness, and patient adherence to recommended regimens. Future goals in the treatment of atherosclerosis should include more widespread implementation of the current evidence-based guidelines regarding risk factor management and, when appropriate, drug therapy.

1	activation as they apply to the precipitation of thrombotic complica-292e-1 tions of atherosclerosis. Atlas of Atherosclerosis Knowledge about the biology of human atherosclerosis and the risk factors for the disease has expanded considerably. The application of vascular biology to human atherosclerosis has revealed many new insights into the mechanisms that promote clinical events. The series of animated video presentations presented here illustrates some of the evolving information about risk factors for atherosclerosis and the pathophysiology of clinical events.

1	The importance of blood pressure as a risk factor for atherosclerosis and cardiovascular events has long been recognized. More recent clinical information has highlighted the importance of pulse pressure—the difference between the systolic pressure and minimum diastolic arterial pressure—as a prognostic indicator of cardiovascular risk. The video clip on pulse pressure explains the pathophysiology of this readily measured clinical variable.

1	Physicians possess a great deal of knowledge about the role of cholesterol in the prediction of atherosclerosis and its complications, but knowledge about the mechanism that links hypercholesterolemia to cardiovascular events has lagged the epidemiologic and observational findings. Low-density lipoprotein (LDL) provides an example of a well-understood cardiovascular risk factor. Several of the animations included in this series highlight the role of modified LDL as a trigger for inflammation and other aspects of the pathobiology of arterial plaques that lead to their aggravation and clinical events. Physicians have useful tools for modulating LDL, but other aspects of dyslipidemia are on the rise and provide a growing challenge to the practitioner. In particular, low levels of high-density lipoprotein (HDL) and elevated levels of triglycerides characterize the constellation of findings denoted by some as the “metabolic syndrome.” In the wake of increasing obesity worldwide, these

1	lipoprotein (HDL) and elevated levels of triglycerides characterize the constellation of findings denoted by some as the “metabolic syndrome.” In the wake of increasing obesity worldwide, these features of the lipoprotein profile require renewed focus. Several of the animations in this collection discuss the concept of the metabolic syndrome and the role of lipid profile components other than LDL in atherogenesis.

1	The traditional approach to atherosclerosis focused on arterial stenoses as a cause of ischemia and cardiovascular events. Physicians now have effective revascularization modalities for addressing flow-limiting stenoses, but atherosclerotic plaques that do not cause stenoses nonetheless may precipitate clinical events, such as unstable angina and acute myocardial infarction. Thus, it is necessary to add to the traditional focus on stenosis an enlarged appreciation of the pathobiology of atherosclerosis that underlies many acute coronary syndromes. The animation on the development and complication of atherosclerotic plaque explains some of these emerging concepts in plaque From Peter Libby, MD: Changes and Challenges in Cardiovascular Protection: A Special CME Activity for Physicians. Created under an unrestricted educational grant from Merck & Co., Inc. Copyright © 2002, Cardinal Health; used with permission.

1	Video 292e-1 Pulse pressure. Considerable evidence suggests that pulse pressure serves as an important risk factor for future cardiovascular events. This video clip explains the derivation of pulse pressure and some of the pathophysiology that determines this parameter. (With permission from the Academy for Health Care Education.) Video 292e-2 Plaque instability. Most coronary thromboses result from a physical disruption of the atherosclerotic plaque. This animation explains some of the current concepts of the pathophysiology of atherosclerotic plaque disruption and how it triggers arterial thrombosis. Video 292e-3 Lipoprotein menagerie. The lipid profile confers important information regarding cardiovascular risk and the effects of therapies; understanding lipoprotein metabolism provides insight into the pathophysiology of arterial disease. This animation presents the rudiments of lipoprotein metabolism that are important in clinical medicine.

1	Video 292e-4 Formation and complication of atherosclerotic plaques. Physicians now understand the generation of atherosclerotic plaques as a dynamic process involving an interchange between cells of the artery wall, inflammatory cells recruited from blood, and risk factors such as lipoproteins. This animation reviews current thinking about how risk factors alter the biology of the artery wall and can incite initiation and progression of atherosclerosis. It also discusses the importance of inflammation in these processes and portrays the role of inflammation in plaque disruption and thrombosis. Finally, this animation depicts the concept of stabilization of atherosclerotic plaques by interventions such as lipid lowering. Video 292e-5 Atherogenesis. This video clip highlights some of the current thinking about mechanisms of atherogenesis. Video 292e-6 Metabolic syndrome. A number of important cardiovascular risk factors tend to cluster in a pattern that has been described by some as the

1	thinking about mechanisms of atherogenesis. Video 292e-6 Metabolic syndrome. A number of important cardiovascular risk factors tend to cluster in a pattern that has been described by some as the metabolic syndrome. Although controversy persists regarding whether cardiovascular risk due to these factors is additive or synergistic, their clinical importance is growing. This animation discusses some of the metabolic derangements that underlie the metabolic syndrome.

1	CHAPTER 292e Atlas of Atherosclerosis Elliott M. Antman, Joseph Loscalzo Ischemic heart disease (IHD) is a condition in which there is an inadequate supply of blood and oxygen to a portion of the myocardium; it typically occurs when there is an imbalance between myocardial oxygen supply and demand. The most common cause of myocardial ischemia is atherosclerotic disease of an epicardial coronary artery (or arteries) sufficient to cause a regional reduction in myocardial blood flow and inadequate perfusion of the myocardium supplied by the involved coronary artery. Chapter 291e deals with the development and treatment of atherosclerosis. This chapter focuses on the chronic manifestations and treatment of IHD. The subsequent chapters address the acute phases of IHD. economic costs than any other illness in the developed world.

1	economic costs than any other illness in the developed world. IHD is the most common, serious, chronic, life-threatening illness in the United States, where 13 million persons have IHD, >6 million have angina pectoris, and >7 million have sustained a myocardial infarction. Genetic factors, a high-fat and energy-rich diet, smoking, and a sedentary lifestyle are associated with the emergence of IHD (Chap. 291e). In the United States and Western Europe, IHD is growing among low-income groups, but primary prevention has delayed the disease to later in life in all socioeconomic groups. Despite these sobering statistics, it is worth noting that epidemiologic data show a decline in the rate of deaths due to IHD, about half of which is attributable to treatments and half to prevention by risk factor modification.

1	Obesity, insulin resistance, and type 2 diabetes mellitus are increasing and are powerful risk factors for IHD. These trends are occurring in the general context of population growth and as a result of the increase in the average age of the world’s population. With urbanization in countries with emerging economies and a growing middle class, elements of the energy-rich Western diet are being adopted. As a result, the prevalence of risk factors for IHD and the prevalence of IHD itself are both increasing rapidly, so that in analyses of the global burden of disease, there is a shift from communicable to noncommunicable diseases. Population subgroups that appear to be particularly affected are men in South Asian countries, especially India and the Middle East. In light of the projection of large increases in IHD throughout the world, IHD is likely to become the most common cause of death worldwide by 2020.

1	Central to an understanding of the pathophysiology of myocardial ischemia is the concept of myocardial supply and demand. In normal conditions, for any given level of a demand for oxygen, the myocardium will control the supply of oxygen-rich blood to prevent under-perfusion of myocytes and the subsequent development of ischemia and infarction. The major determinants of myocardial oxygen demand (MVO2) are heart rate, myocardial contractility, and myocardial wall tension (stress). An adequate supply of oxygen to the myocardium requires a satisfactory level of oxygen-carrying capacity of the blood (determined by the inspired level of oxygen, pulmonary function, and hemoglobin concentration and function) and an adequate level of coronary blood flow. Blood flows through the coronary arteries in a phasic fashion, with the majority occurring during diastole. About 75% of the total coronary resistance to flow occurs across three sets of arteries: (1) large epicardial arteries (Resistance 1 =

1	a phasic fashion, with the majority occurring during diastole. About 75% of the total coronary resistance to flow occurs across three sets of arteries: (1) large epicardial arteries (Resistance 1 = R1), (2) prearteriolar vessels (R2), and (3) arteriolar and intramyocardial capillary vessels (R3). In the absence of significant flow-limiting atherosclerotic obstructions, R1 is trivial; the major determinant of coronary resistance is found in R2 and R3.

1	The normal coronary circulation is dominated and controlled by the heart’s requirements for oxygen. This need is met by the ability of the coronary vascular bed to vary its resistance (and, therefore, blood flow) considerably while the myocardium extracts a high and relatively fixed percentage of oxygen. Normally, intramyocardial resistance vessels demonstrate a great capacity for dilation (R2 and R3 decrease). For example, the changing oxygen needs of the heart with exercise and emotional stress affect coronary vascular resistance and in this manner regulate the supply of oxygen and substrate to the myocardium (metabolic regulation). The coronary resistance vessels also adapt to physiologic alterations in blood pressure to maintain coronary blood flow at levels appropriate to myocardial needs (autoregulation).

1	By reducing the lumen of the coronary arteries, atherosclerosis limits appropriate increases in perfusion when the demand for flow is augmented, as occurs during exertion or excitement. When the luminal reduction is severe, myocardial perfusion in the basal state is reduced. Coronary blood flow also can be limited by spasm (see Prinzmetal’s angina in Chap. 294), arterial thrombi, and, rarely, coronary emboli as well as by ostial narrowing due to aortitis. Congenital abnormalities such as the origin of the left anterior descending coronary artery from the pulmonary artery may cause myocardial ischemia and infarction in infancy, but this cause is very rare in adults.

1	Myocardial ischemia also can occur if myocardial oxygen demands are markedly increased and particularly when coronary blood flow may be limited, as occurs in severe left ventricular hypertrophy due to aortic stenosis. The latter can present with angina that is indistinguishable from that caused by coronary atherosclerosis largely owing to subendocardial ischemia (Chap. 283). A reduction in the oxygen-carrying capacity of the blood, as in extremely severe anemia or in the presence of carboxyhemoglobin, rarely causes myocardial ischemia by itself but may lower the threshold for ischemia in patients with moderate coronary obstruction.

1	Not infrequently, two or more causes of ischemia coexist in a patient, such as an increase in oxygen demand due to left ventricular hypertrophy secondary to hypertension and a reduction in oxygen supply secondary to coronary atherosclerosis and anemia. Abnormal constriction or failure of normal dilation of the coronary resistance vessels also can cause ischemia. When it causes angina, this condition is referred to as microvascular angina.

1	Epicardial coronary arteries are the major site of atherosclerotic disease. The major risk factors for atherosclerosis (high levels of plasma low-density lipoprotein [LDL], low plasma high-density lipoprotein [HDL], cigarette smoking, hypertension, and diabetes mellitus [Chap. 291e]) disturb the normal functions of the vascular endothelium. These functions include local control of vascular tone, maintenance of an antithrombotic surface, and control of inflammatory cell adhesion and diapedesis. The loss of these defenses leads to inappropriate constriction, luminal thrombus formation, and abnormal interactions between blood cells, especially monocytes and platelets, and the activated vascular endothelium. Functional changes in the vascular milieu ultimately result in the subintimal collections of fat, smooth muscle cells, fibroblasts, and intercellular matrix that define the atherosclerotic plaque. Rather than viewing atherosclerosis strictly as a vascular problem, it is useful to

1	of fat, smooth muscle cells, fibroblasts, and intercellular matrix that define the atherosclerotic plaque. Rather than viewing atherosclerosis strictly as a vascular problem, it is useful to consider it in the context of alterations in the nature of the circulating blood (hyperglycemia; increased concentrations of LDL cholesterol, tissue factor, fibrinogen, von Willebrand factor, coagulation factor VII, and platelet microparticles). The combination of a “vulnerable vessel” in a patient with “vulnerable blood” promotes a state of hypercoagulability and hypofibrinolysis. This is especially true in patients with diabetes mellitus.

1	Atherosclerosis develops at irregular rates in different segments of the epicardial coronary tree and leads eventually to segmental reductions in cross-sectional area, i.e., plaque formation. There is also a predilection for atherosclerotic plaques to develop at sites of increased 1579 turbulence in coronary flow, such as at branch points in the epicardial arteries. When a stenosis reduces the diameter of an epicardial artery by 50%, there is a limitation of the ability to increase flow to meet increased myocardial demand. When the diameter is reduced by ~80%, blood flow at rest may be reduced, and further minor decreases in the stenotic orifice area can reduce coronary flow dramatically to cause myocardial ischemia at rest or with minimal stress.

1	Segmental atherosclerotic narrowing of epicardial coronary arteries is caused most commonly by the formation of a plaque, which is subject to rupture or erosion of the cap separating the plaque from the bloodstream. Upon exposure of the plaque contents to blood, two important and interrelated processes are set in motion: (1) platelets are activated and aggregate, and (2) the coagulation cascade is activated, leading to deposition of fibrin strands. A thrombus composed of platelet aggregates and fibrin strands traps red blood cells and can reduce coronary blood flow, leading to the clinical manifestations of myocardial ischemia.

1	The location of the obstruction influences the quantity of myo cardium rendered ischemic and determines the severity of the clinical manifestations. Thus, critical obstructions in vessels, such as the left main coronary artery and the proximal left anterior descending coronary artery, are particularly hazardous. Chronic severe coronary narrowing and myocardial ischemia frequently are accompanied by the development of collateral vessels, especially when the narrowing develops gradually. When well developed, such vessels can by themselves provide sufficient blood flow to sustain the viability of the myocardium at rest but not during conditions of increased demand.

1	With progressive worsening of a stenosis in a proximal epicardial artery, the distal resistance vessels (when they function normally) dilate to reduce vascular resistance and maintain coronary blood flow. A pressure gradient develops across the proximal stenosis, and poststenotic pressure falls. When the resistance vessels are maximally dilated, myocardial blood flow becomes dependent on the pressure in the coronary artery distal to the obstruction. In these circumstances, ischemia, manifest clinically by angina or electrocardiographically by ST-segment deviation, can be precipitated by increases in myocardial oxygen demand caused by physical activity, emotional stress, and/or tachycardia. Changes in the caliber of the stenosed coronary artery due to physiologic vasomotion, loss of endothelial control of dilation (as occurs in atherosclerosis), pathologic spasm (Prinzmetal’s angina), or small platelet-rich plugs also can upset the critical balance between oxygen supply and demand and

1	control of dilation (as occurs in atherosclerosis), pathologic spasm (Prinzmetal’s angina), or small platelet-rich plugs also can upset the critical balance between oxygen supply and demand and thereby precipitate myocardial ischemia.

1	During episodes of inadequate perfusion caused by coronary atherosclerosis, myocardial tissue oxygen tension falls and may cause transient disturbances of the mechanical, biochemical, and electrical functions of the myocardium (Fig. 293-1). Coronary atherosclerosis is a focal process that usually causes nonuniform ischemia. During ischemia, regional disturbances of ventricular contractility cause segmental hypokinesia, akinesia, or, in severe cases, bulging (dyskinesia), which can reduce myocardial pump function.

1	The abrupt development of severe ischemia, as occurs with total or subtotal coronary occlusion, is associated with almost instantaneous failure of normal muscle relaxation and then contraction. The relatively poor perfusion of the subendocardium causes more intense ischemia of this portion of the wall (compared with the subepicardial region). Ischemia of large portions of the ventricle causes transient left ventricular failure, and if the papillary muscle apparatus is involved, mitral regurgitation can occur. When ischemia is transient, it may be associated with angina pectoris; when it is prolonged, it can lead to myocardial necrosis and scarring with or without the clinical picture of acute myocardial infarction (Chap. 295). A wide range of abnormalities in cell metabolism, function, and structure underlie these mechanical disturbances during ischemia. The normal myocardium metabolizes fatty acids and glucose to carbon

1	FIGURE 293-1 Cascade of mechanisms and manifestations of ischemia. (Modified from LJ Shaw et al: J Am Coll Cardiol 54:1561, 2009. Original figure illustration by Rob Flewell.) dioxide and water. With severe oxygen deprivation, fatty acids cannot be oxidized, and glucose is converted to lactate; intracellular pH is reduced, as are the myocardial stores of high-energy phosphates, i.e., ATP and creatine phosphate. Impaired cell membrane function leads to the leakage of potassium and the uptake of sodium by myocytes as well as an increase in cytosolic calcium. The severity and duration of the imbalance between myocardial oxygen supply and demand determine whether the damage is reversible (≤20 min for total occlusion in the absence of collaterals) or permanent, with subsequent myocardial necrosis (>20 min).

1	Ischemia also causes characteristic changes in the electrocardiogram (ECG) such as repolarization abnormalities, as evidenced by inversion of T waves and, when more severe, displacement of ST segments (Chap. 268). Transient T-wave inversion probably reflects nontransmural, intramyocardial ischemia; transient ST-segment depression often reflects patchy subendocardial ischemia; and ST-segment elevation is thought to be caused by more severe transmural ischemia. Another important consequence of myocardial ischemia is electrical instability, which may lead to isolated ventricular premature beats or even ventricular tachycardia or ventricular fibrillation (Chap. 277). Most patients who die suddenly from IHD do so as a result of ischemia-induced ventricular tachyarrhythmias (Chap. 327).

1	Although the prevalence is decreasing, postmortem studies of accident victims and military casualties in Western countries still show that coronary atherosclerosis can begin before age 20 and is present even among adults who were asymptomatic during life. Exercise stress tests in asymptomatic persons may show evidence of silent myocardial ischemia, i.e., exercise-induced ECG changes not accompanied by angina pectoris; coronary angiographic studies of such persons may reveal coronary artery plaques and previously unrecognized obstructions (Chap. 272). Postmortem examination of patients with such obstructions without a history of clinical manifestations of myocardial ischemia often shows macroscopic scars secondary to myocardial infarction in regions supplied by diseased coronary arteries, with or without collateral circulation. According to population studies, ~25% of patients who survive acute myocardial infarction may not come to medical attention, and these patients have the same

1	with or without collateral circulation. According to population studies, ~25% of patients who survive acute myocardial infarction may not come to medical attention, and these patients have the same adverse prognosis as do those who present with the classic clinical picture of acute myocardial infarction (Chap. 295). Sudden death may be unheralded and is a common presenting manifestation of IHD (Chap. 327).

1	Patients with IHD also can present with cardiomegaly and heart failure secondary to ischemic damage of the left ventricular myocardium that may have caused no symptoms before the development of heart failure; this condition is referred to as ischemic cardiomyopathy. In contrast to the asymptomatic phase of IHD, the symptomatic phase is characterized by chest discomfort due to either angina pectoris or acute myocardial infarction (Chap. 295). Having entered the symptomatic phase, the patient may exhibit a stable or progressive course, revert to the asymptomatic stage, or die suddenly.

1	This episodic clinical syndrome is due to transient myocardial ischemia. Various diseases that cause myocardial ischemia and the numerous forms of discomfort with which it may be confused are discussed in Chap. 19. Males constitute ~70% of all patients with angina pectoris and an even greater proportion of those less than 50 years of age. It is, however, important to note that angina pectoris in women is often atypical in presentation (see below).

1	The typical patient with angina is a man >50 years or a woman >60 years of age who complains of episodes of chest discomfort, usually described as heaviness, pressure, squeezing, smothering, or choking and only rarely as frank pain. When the patient is asked to localize the sensation, he or she typically places a hand over the sternum, sometimes with a clenched fist, to indicate a squeezing, central, substernal discomfort (Levine’s sign). Angina is usually crescendo-decrescendo in nature, typically lasts 2 to 5 min, and can radiate to either shoulder and to both arms (especially the ulnar surfaces of the forearm and hand). It also can arise in or radiate to the back, interscapular region, root of the neck, jaw, teeth, and epigastrium. Angina is rarely localized below the umbilicus or above the mandible. A useful finding in assessing a patient with chest discomfort is the fact that myocardial ischemic discomfort does not radiate to the trapezius muscles; that radiation pattern is more

1	the mandible. A useful finding in assessing a patient with chest discomfort is the fact that myocardial ischemic discomfort does not radiate to the trapezius muscles; that radiation pattern is more typical of pericarditis.

1	Although episodes of angina typically are caused by exertion (e.g., exercise, hurrying, or sexual activity) or emotion (e.g., stress, anger, fright, or frustration) and are relieved by rest, they also may occur at rest (Chap. 294) and while the patient is recumbent (angina decubitus). The patient may be awakened at night by typical chest discomfort and dyspnea. Nocturnal angina may be due to episodic tachycardia, diminished oxygenation as the respiratory pattern changes during sleep, or expansion of the intrathoracic blood volume that occurs with recumbency; the latter causes an increase in cardiac size (end-diastolic volume), wall tension, and myocardial oxygen demand that can lead to ischemia and transient left ventricular failure.

1	The threshold for the development of angina pectoris may vary by time of day and emotional state. Many patients report a fixed threshold for angina, which occurs predictably at a certain level of activity, such as climbing two flights of stairs at a normal pace. In these patients, coronary stenosis and myocardial oxygen supply are fixed, and ischemia is precipitated by an increase in myocardial oxygen demand; they are said to have stable exertional angina. In other patients, the threshold for angina may vary considerably within any particular day and from day to day. In such patients, variations in myocardial oxygen supply, most likely due to changes in coronary vasomotor tone, may play an important role in defining the pattern of angina. A patient may report symptoms upon minor exertion in the morning (a short walk or shaving) yet by midday be capable of much greater effort without symptoms. Angina may also be precipitated by unfamiliar tasks, a heavy meal, exposure to cold, or a

1	in the morning (a short walk or shaving) yet by midday be capable of much greater effort without symptoms. Angina may also be precipitated by unfamiliar tasks, a heavy meal, exposure to cold, or a combination of these factors.

1	Exertional angina typically is relieved in 1–5 min by slowing or ceasing activities and even more rapidly by rest and sublingual nitroglycerin (see below). Indeed, the diagnosis of angina should be suspect if it does not respond to the combination of these measures. The severity of angina can be conveniently summarized by the Canadian Cardiac Society functional classification (Table 293-1). Its impact on the patient’s functional capacity can be described by using the New York Heart Association functional classification (Table 293-1). I Patients have cardiac disease but without the resulting limitations of physical activity. Ordinary physical activity does not cause undue fatigue, palpitation, dyspnea, or anginal pain. II Patients have cardiac disease resulting in slight limitation of physical activity. They are comfortable at rest. Ordinary physical activity results in fatigue, palpitation, dyspnea, or anginal pain.

1	III Patients have cardiac disease resulting in marked limitation of physical activity. They are comfortable at rest. Less than ordinary physical activity causes fatigue, palpitation, dyspnea, or anginal pain. IV Patients have cardiac disease resulting in inability to carry on any physical activity without discomfort. Symptoms of cardiac insufficiency or of the anginal syndrome may be present even at rest. If any physical activity is undertaken, discomfort is increased. Ordinary physical activity, such as walking and climbing stairs, does not cause angina. Angina present with strenuous or rapid or prolonged exertion at work or recreation. Slight limitation of ordinary activity. Walking or climbing stairs rapidly, walking uphill, walking or stair climbing after meals, in cold, or when under emotional stress or only during the few hours after awakening. Walking more than two blocks on the level and climbing more than one flight of stairs at a normal pace and in normal conditions.

1	Marked limitation of ordinary physical activity. Walking one to two blocks on the level and climbing more than one flight of stairs in normal conditions. Inability to carry on any physical activity without discomfort— anginal syndrome may be present at rest. Source: Modified from L Goldman et al: Circulation 64:1227, 1981. Sharp, fleeting chest pain or a prolonged, dull ache localized to the 1581 left submammary area is rarely due to myocardial ischemia. However, especially in women and diabetic patients, angina pectoris may be atypical in location and not strictly related to provoking factors. In addition, this symptom may exacerbate and remit over days, weeks, or months. Its occurrence can be seasonal, occurring more frequently in the winter in temperate climates. Anginal “equivalents” are symptoms of myocardial ischemia other than angina. They include dyspnea, nausea, fatigue, and faintness and are more common in the elderly and in diabetic patients.

1	Systematic questioning of a patient with suspected IHD is important to uncover the features of an unstable syndrome associated with increased risk, such as angina occurring with less exertion than in the past, occurring at rest, or awakening the patient from sleep. Since coronary atherosclerosis often is accompanied by similar lesions in other arteries, a patient with angina should be questioned and examined for peripheral arterial disease (intermittent claudication [Chap. 302]), stroke, or transient ischemic attacks (Chap. 446). It is also important to uncover a family history of premature IHD (<55 years in first-degree male relatives and <65 in female relatives) and the presence of diabetes mellitus, hyperlipidemia, hypertension, cigarette smoking, and other risk factors for coronary atherosclerosis (Chap. 291e).

1	The history of typical angina pectoris establishes the diagnosis of IHD until proven otherwise. The coexistence of advanced age, male sex, the postmenopausal state, and risk factors for atherosclerosis increase the likelihood of hemodynamically significant coronary disease. A particularly challenging problem is the evaluation and management of patients with persistent ischemic-type chest discomfort but no flow-limiting obstructions in their epicardial coronary arteries. This situation arises more often in women than in men. Potential etiologies include microvascular coronary disease (detectable on coronary reactivity testing in response to vasoactive agents such as intracoronary adenosine, acetylcholine, and nitroglycerin) and abnormal cardiac nociception. Treatment of microvascular coronary disease should focus on efforts to improve endothelial function, including nitrates, beta blockers, calcium antagonists, statins, and angiotensin-converting enzyme (ACE) inhibitors. Abnormal

1	coronary disease should focus on efforts to improve endothelial function, including nitrates, beta blockers, calcium antagonists, statins, and angiotensin-converting enzyme (ACE) inhibitors. Abnormal cardiac nociception is more difficult to manage and may be ameliorated in some cases by imipramine.

1	The physical examination is often normal in patients with stable angina when they are asymptomatic. However, because of the increased likelihood of IHD in patients with diabetes and/or peripheral arterial disease, clinicians should search for evidence of atherosclerotic disease at other sites, such as an abdominal aortic aneurysm, carotid arterial bruits, and diminished arterial pulses in the lower extremities. The physical examination also should include a search for evidence of risk factors for atherosclerosis such as xanthelasmas and xanthomas (Chap. 291e). Evidence for peripheral arterial disease should be sought by evaluating the pulse contour at multiple locations and comparing the blood pressure between the arms and between the arms and the legs (ankle-brachial index). Examination of the fundi may reveal an increased light reflex and arteriovenous nicking as evidence of hypertension. There also may be signs of anemia, thyroid disease, and nicotine stains on the fingertips from

1	of the fundi may reveal an increased light reflex and arteriovenous nicking as evidence of hypertension. There also may be signs of anemia, thyroid disease, and nicotine stains on the fingertips from cigarette smoking.

1	Palpation may reveal cardiac enlargement and abnormal contraction of the cardiac impulse (left ventricular dyskinesia). Auscultation can uncover arterial bruits, a third and/or fourth heart sound, and, if acute ischemia or previous infarction has impaired papillary muscle function, an apical systolic murmur due to mitral regurgitation. These auscultatory signs are best appreciated with the patient in the left lateral decubitus position. Aortic stenosis, aortic regurgitation (Chap. 283), pulmonary hypertension (Chap. 304), and hypertrophic cardiomyopathy (Chap. 287) must be excluded, since these disorders may cause angina in the absence of coronary atherosclerosis. Examination during an anginal attack is useful, since ischemia can cause transient left ventricular failure with the appearance of a third 1582 and/or fourth heart sound, a dyskinetic cardiac apex, mitral regurgitation, and even pulmonary edema. Tenderness of the chest wall, localization of the discomfort with a single

1	of a third 1582 and/or fourth heart sound, a dyskinetic cardiac apex, mitral regurgitation, and even pulmonary edema. Tenderness of the chest wall, localization of the discomfort with a single fingertip on the chest, or reproduction of the pain with palpation of the chest makes it unlikely that the pain is caused by myocardial ischemia. A protuberant abdomen may indicate that the patient has the metabolic syndrome and is at increased risk for atherosclerosis.

1	Although the diagnosis of IHD can be made with a high degree of confidence from the history and physical examination, a number of simple laboratory tests can be helpful. The urine should be examined for evidence of diabetes mellitus and renal disease (including microalbuminuria) since these conditions accelerate atherosclerosis. Similarly, examination of the blood should include measurements of lipids (cholesterol—total, LDL, HDL—and triglycerides), glucose (hemoglobin ), creatinine, hematocrit, and, if indicated based on the physical examination, thyroid function. A chest x-ray is important as it may show the consequences of IHD, i.e., cardiac enlargement, ventricular aneurysm, or signs of heart failure. These signs can support the diagnosis of IHD and are important in assessing the degree of cardiac damage. Evidence exists that an elevated level of high-sensitivity C-reactive protein (CRP) (specifically, between 0 and 3 mg/dL) is an independent risk factor for IHD and may be useful

1	of cardiac damage. Evidence exists that an elevated level of high-sensitivity C-reactive protein (CRP) (specifically, between 0 and 3 mg/dL) is an independent risk factor for IHD and may be useful in therapeutic decision making about the initiation of hypolipidemic treatment. The major benefit of high-sensitivity CRP is in reclassifying the risk of IHD in patients in the “intermediate” risk category on the basis of traditional risk factors.

1	A 12-lead ECG recorded at rest may be normal in patients with typical angina pectoris, but there may also be signs of an old myocardial infarction (Chap. 268). Although repolarization abnormalities, i.e., ST-segment and T-wave changes, as well as left ventricular hypertrophy and disturbances of cardiac rhythm or intraventricular conduction are suggestive of IHD, they are nonspecific, since they also can occur in pericardial, myocardial, and valvular heart disease or, in the case of the former, transiently with anxiety, changes in posture, drugs, or esophageal disease. The presence of left ventricular hypertrophy (LVH) is a significant indication of increased risk of adverse outcomes from IHD. Of note, even though LVH and cardiac rhythm disturbances are nonspecific indicators of the development of IHD, they may be contributing factors to episodes of angina in patients in whom IHD has developed as a consequence of conventional risk factors. Dynamic ST-segment and T-wave changes that

1	of IHD, they may be contributing factors to episodes of angina in patients in whom IHD has developed as a consequence of conventional risk factors. Dynamic ST-segment and T-wave changes that accompany episodes of angina pectoris and disappear thereafter are more specific.

1	Electrocardiographic The most widely used test for both the diagnosis of IHD and the estimation of risk and prognosis involves recording the 12-lead ECG before, during, and after exercise, usually on a treadmill (Fig. 293-2). The test consists of a standardized incremental increase in external workload (Table 293-2) while symptoms, the ECG, and arm blood pressure are monitored. Exercise duration is usually symptom-limited, and the test is discontinued upon evidence of chest discomfort, severe shortness of breath, dizziness, severe fatigue, ST-segment depression >0.2 mV (2 mm), a fall in systolic blood pressure >10 mmHg, or the development of a ventricular tachyarrhythmia. This test is used to discover any limitation in exercise performance, detect typical ECG signs of myocardial ischemia, and establish their relationship to chest discomfort. The ischemic ST-segment response generally is defined as flat or downsloping depression of the ST segment >0.1 mV below baseline (i.e., the PR

1	and establish their relationship to chest discomfort. The ischemic ST-segment response generally is defined as flat or downsloping depression of the ST segment >0.1 mV below baseline (i.e., the PR segment) and lasting longer than 0.08 s (Fig. 293-1). Upsloping or junctional ST-segment changes are not considered characteristic of ischemia and do not constitute a positive test. Although T-wave abnormalities, conduction disturbances, and ventricular arrhythmias that develop during exercise should be noted, they are also not diagnostic. Negative exercise tests in which the target heart rate (85% of maximal predicted heart rate for age and sex) is not achieved are considered nondiagnostic.

1	In interpreting ECG stress tests, the probability that coronary artery disease (CAD) exists in the patient or population under study (i.e., pretest probability) should be considered. Overall, false-positive or false-negative results occur in one-third of cases. However, a positive result on exercise indicates that the likelihood of CAD is 98% in males who are >50 years with a history of typical angina pectoris and who develop chest discomfort during the test. The likelihood decreases if the patient has atypical or no chest pain by history and/or during the test.

1	The incidence of false-positive tests is significantly increased in patients with low probabilities of IHD, such as asymptomatic men age <40 or premenopausal women with no risk factors for premature atherosclerosis. It is also increased in patients taking cardioactive drugs, such as digitalis and antiarrhythmic agents, and in those with intraventricular conduction disturbances, resting ST-segment and T-wave abnormalities, ventricular hypertrophy, or abnormal serum potassium levels. Obstructive disease limited to the circumflex coronary artery may result in a false-negative stress test since the lateral portion of the heart that this vessel supplies is not well represented on the surface 12-lead ECG. Since the overall sensitivity of exercise stress electrocardiography is only ~75%, a negative result does not exclude CAD, although it makes the likelihood of three-vessel or left main CAD extremely unlikely.

1	A medical professional should be present throughout the exercise test. It is important to measure total duration of exercise, the times to the onset of ischemic ST-segment change and chest discomfort, the external work performed (generally expressed as the stage of exercise), and the internal cardiac work performed, i.e., by the heart rate–blood pressure product. The depth of the ST-segment depression and the time needed for recovery of these ECG changes are also important. Because the risks of exercise testing are small but real—estimated at one fatality and two nonfatal complications per 10,000 tests—equipment for resuscitation should be available. Modified (heart rate–limited rather than symptom-limited) exercise tests can be performed safely in patients as early as 6 days after uncomplicated myocardial infarction (Table 293-2). Contraindications to exercise stress testing include rest angina within 48 h, unstable rhythm, severe aortic stenosis, acute myocarditis, uncontrolled

1	myocardial infarction (Table 293-2). Contraindications to exercise stress testing include rest angina within 48 h, unstable rhythm, severe aortic stenosis, acute myocarditis, uncontrolled heart failure, severe pulmonary hypertension, and active infective endocarditis.

1	The normal response to graded exercise includes progressive increases in heart rate and blood pressure. Failure of the blood pressure to increase or an actual decrease with signs of ischemia during the test is an important adverse prognostic sign, since it may reflect ischemia-induced global left ventricular dysfunction. The development of angina and/or severe (>0.2 mV) ST-segment depression at a low workload, i.e., before completion of stage II of the Bruce protocol, and/or ST-segment depression that persists >5 min after the termination of exercise increases the specificity of the test and suggests severe IHD and a high risk of future adverse events.

1	Cardiac Imaging (See also Chap. 270e) When the resting ECG is abnormal (e.g., preexcitation syndrome, >1 mm of resting ST-segment depression, left bundle branch block, paced ventricular rhythm), information gained from an exercise test can be enhanced by stress myocardial radionuclide perfusion imaging after the intravenous administration of thallium-201 or 99m-technetium sestamibi during exercise (or with pharmacologic) stress. Contemporary data also suggest positron emission tomography (PET) imaging (with exercise or pharmacologic stress) using N-13 ammonia or rubidium-82 nuclide as another technique for assessing perfusion. Images obtained immediately after cessation of exercise to detect regional ischemia are compared with those obtained at rest to confirm reversible ischemia and regions of persistently absent uptake that signify infarction.

1	A sizable fraction of patients who need noninvasive stress testing to identify myocardial ischemia and increased risk of coronary events cannot exercise because of peripheral vascular or musculoskeletal disease, exertional dyspnea, or deconditioning. In these circumstances, an intravenous pharmacologic challenge is used in place of exercise. For example, dipyridamole or adenosine can be given to create a coronary “steal” by temporarily increasing flow in nondiseased

1	CHAPTER 293 Ischemic Heart DiseaseAEvaluation of the patient with known or suspected IHD Can patient exercise adequately? Are confounding features present on resting ECG? Perform treadmill exercise test An imaging study should be performed Yes No Possible indications for stress testing of patient: 1. Dx of IHD uncertain 2. Assess functional capacity of patient 3. Assess adequacy of treatment program for IHD 4. Markedly abnormal calcium score on EBCT 2-D Echo Nuclear perfusion scan Cardiac MR scan Cardiac PET scan ECHOECG MIBI CMR PET No Yes

1	FIGURE 293-2 Evaluation of the patient with known or suspected ischemic heart disease. On the left of the figure is an algorithm for identifying patients who should be referred for stress testing and the decision pathway for determining whether a standard treadmill exercise with electrocardiogram (ECG) monitoring alone is adequate. A specialized imaging study is necessary if the patient cannot exercise adequately (pharmacologic challenge is given) or if there are confounding features on the resting ECG (symptom-limited treadmill exercise may be used to stress the coronary circulation). Panels B–E, continued on the next page, are examples of the data obtained with ECG monitoring and specialized imaging procedures. CMR, cardiac magnetic resonance; EBCT, electron beam computed tomography; ECHO, echocardiography; IHD, ischemic heart disease; MIBI, methoxyisobutyl isonitrite; MR, magnetic resonance; PET, positron emission tomography. A. Lead V4 at rest (top panel) and after 4.5 min of

1	ECHO, echocardiography; IHD, ischemic heart disease; MIBI, methoxyisobutyl isonitrite; MR, magnetic resonance; PET, positron emission tomography. A. Lead V4 at rest (top panel) and after 4.5 min of exercise (bottom panel). There is 3 mm (0.3 mV) of horizontal ST-segment depression, indicating a positive test for ischemia. (Modified from BR Chaitman, in E Braunwald et al [eds]: Heart Disease, 8th ed, Philadelphia, Saunders, 2008.) B. A 45-year-old avid jogger who began experiencing classic substernal chest pressure underwent an exercise echo study. With exercise the patient’s heart rate increased from 52 to 153 beats/min. The left ventricular chamber dilated with exercise, and the septal and apical portions became akinetic to dyskinetic (red arrow). These findings are strongly suggestive of a significant flow-limiting stenosis in the proximal left anterior descending artery, which was confirmed at coronary angiography. (Modified from SD Solomon, in E. Braunwald et al [eds]: Primary

1	of a significant flow-limiting stenosis in the proximal left anterior descending artery, which was confirmed at coronary angiography. (Modified from SD Solomon, in E. Braunwald et al [eds]: Primary Cardiology, 2nd ed, Philadelphia, Saunders, 2003.) C. Stress and rest myocardial perfusion single-photon emission computed tomography images obtained with 99m-technetium sestamibi in a patient with chest pain and dyspnea on exertion. The images demonstrate a medium-size and severe stress perfusion defect involving the inferolateral and basal inferior walls, showing nearly complete reversibility, consistent with moderate ischemia in the right coronary artery territory (red arrows). (Images provided by Dr. Marcello Di Carli, Nuclear Medicine Division, Brigham and Women’s Hospital, Boston, MA.) D. A patient with a prior myocardial infarction presented with recurrent chest discomfort. On cardiac magnetic resonance (CMR) cine imaging, a large area of anterior akinesia was noted (marked by the

1	D. A patient with a prior myocardial infarction presented with recurrent chest discomfort. On cardiac magnetic resonance (CMR) cine imaging, a large area of anterior akinesia was noted (marked by the arrows in the top left and right images, systolic frame only). This area of akinesia was matched by a larger extent of late gadolinium-DTPA enhancements consistent with a large transmural myocardial infarction (marked by arrows in the middle left and right images). Resting (bottom left) and adenosine vasodilating stress (bottom right) first-pass perfusion images revealed reversible perfusion abnormality that extended to the inferior septum. This patient was found to have an occluded proximal left anterior descending coronary artery with extensive collateral formation. This case illustrates the utility of different modalities in a CMR examination in characterizing ischemic and infarcted myocardium. DTPA, diethylenetriamine penta-acetic acid. (Images provided by Dr. Raymond Kwong,

1	the utility of different modalities in a CMR examination in characterizing ischemic and infarcted myocardium. DTPA, diethylenetriamine penta-acetic acid. (Images provided by Dr. Raymond Kwong, Cardiovascular Division, Brigham and Women’s Hospital, Boston, MA.) E. Stress and rest myocardial perfusion PET images obtained with rubidium-82 in a patient with chest pain on exertion. The images demonstrate a large and severe stress perfusion defect involving the mid and apical anterior, anterolateral, and anteroseptal walls and the left ventricular apex, showing complete reversibility, consistent with extensive and severe ischemia in the mid-left anterior descending coronary artery territory (red arrows). (Images provided by Dr. Marcello Di Carli, Nuclear Medicine Division, Brigham and Women’s Hospital, Boston, MA.)

1	NORMAL AND I BRUCE Modified 3 min Stages BRUCE 3 min Stages MPH %GR MPH %GR 6.0 22 6.0 22 HEALTHY, DEPENDENT ON AGE, ACTIVITY 5.5 20 5.2 20 5.0 18 5.0 18 56.0 16 52.5 15 49.0 14 45.5 13 4.2 16 4.2 16 42.0 12 38.5 11 3.4 14 3.4 14 SEDENTARY HEALTHY35.0 10 31.5 9 28.0 8 LIMITED 24.5 7 2.5 12 2.5 12 II SYMPTOMATIC 21.0 6 17.5 5 1.7 10 1.7 10 III 14.0 4 10.5 3 1.7 5 7.0 2 1.7 0 IV 3.5 1 Abbreviations: GR, grade; MPH, miles per hour. Source: Modified from GF Fletcher et al: Circulation 104:1694, 2001. segments of the coronary vasculature at the expense of diseased segments. Alternatively, a graded incremental infusion of dobutamine may be administered to increase MVO2. A variety of imaging options are available to accompany these pharmacologic stressors (Fig. 293-2). The development of a transient perfusion defect with a tracer such as thallium-201 or 99m-technetium sestamibi is used to detect myocardial ischemia.

1	Echocardiography is used to assess left ventricular function in patients with chronic stable angina and patients with a history of a prior myocardial infarction, pathologic Q waves, or clinical evidence of heart failure. Two-dimensional echocardiography can assess both global and regional wall motion abnormalities of the left ventricle that are transient when due to ischemia. Stress (exercise or dobutamine) echocardiography may cause the emergence of regions of akinesis or dyskinesis that are not present at rest. Stress echocardiography, like stress myocardial perfusion imaging, is more sensitive than exercise electrocardiography in the diagnosis of IHD. Cardiac magnetic resonance (CMR) stress testing is also evolving as an alternative to radionuclide, PET, or echocardiographic stress imaging. CMR stress testing performed with dobutamine infusion can be used to assess wall motion abnormalities accompanying ischemia, as well as myocardial perfusion. CMR can be used to provide more

1	imaging. CMR stress testing performed with dobutamine infusion can be used to assess wall motion abnormalities accompanying ischemia, as well as myocardial perfusion. CMR can be used to provide more complete ventricular evaluation using multislice magnetic resonance imaging (MRI) studies.

1	Atherosclerotic plaques become progressively calcified over time, and coronary calcification in general increases with age. For this reason, methods for detecting coronary calcium have been developed as a measure of the presence of coronary atherosclerosis. These methods involve computed tomography (CT) applications that achieve rapid acquisition of images (electron beam [EBCT] and multidetector [MDCT] detection). Coronary calcium detected by these imaging techniques most commonly is quantified by using the Agatston score, which is based on the area and density of calcification. Although the diagnostic accuracy of this imaging method is high (sensitivity, 90–94%; specificity, 95–97%; negative predictive value, 93–99%), its prognostic utility has not been defined. Thus, its role in CT, EBCT, and MDCT scans for the detection and management of patients with IHD has not been clarified.

1	(See also Chap. 272) This diagnostic method outlines the lumina of the coronary arteries and can be used to detect or exclude serious coronary obstruction. However, coronary arteriography provides no information about the arterial wall, and severe atherosclerosis that does not encroach on the lumen may go undetected. Of note, atherosclerotic plaques characteristically are scattered throughout the coronary tree, tend to occur more frequently at branch points, and grow progressively in the intima and media of an epicardial coronary artery at first without encroaching on the lumen, causing an outward bulging of the artery—a process referred to as remodeling (Chap. 291e). Later in the course of the disease, further growth causes luminal narrowing.

1	Indications Coronary arteriography is indicated in (1) patients with chronic stable angina pectoris who are severely symptomatic despite medical therapy and are being considered for revascularization, i.e., a percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG); (2) patients with troublesome symptoms that present diagnostic difficulties in whom there is a need to confirm or rule out the diagnosis of IHD; (3) patients with known or possible angina pectoris who have survived cardiac arrest; (4) patients with angina or evidence of ischemia on noninvasive testing with clinical or laboratory evidence of ventricular dysfunction; and (5) patients judged to be at high risk of sustaining coronary events based on signs of severe ischemia on noninvasive testing, regardless of the presence or severity of symptoms (see below). Examples of other indications for coronary arteriography include the following: 1.

1	Examples of other indications for coronary arteriography include the following: 1. Patients with chest discomfort suggestive of angina pectoris but a negative or nondiagnostic stress test who require a definitive diagnosis for guiding medical management, alleviating psychological stress, career or family planning, or insurance purposes. 2. Patients who have been admitted repeatedly to the hospital for a suspected acute coronary syndrome (Chaps. 294 and 295), but in whom this diagnosis has not been established and in whom the presence or absence of CAD should be determined. 1586 3. Patients with careers that involve the safety of others (e.g., pilots, firefighters, police) who have questionable symptoms or suspicious or positive noninvasive tests and in whom there are reasonable doubts about the state of the coronary arteries. 4. Patients with aortic stenosis or hypertrophic cardiomyopathy and angina in whom the chest pain could be due to IHD. 5.

1	4. Patients with aortic stenosis or hypertrophic cardiomyopathy and angina in whom the chest pain could be due to IHD. 5. Male patients >45 years and females >55 years who are to undergo a cardiac operation such as valve replacement or repair and who may or may not have clinical evidence of myocardial ischemia. 6. Patients after myocardial infarction, especially those who are at high risk after myocardial infarction because of the recurrence of angina or the presence of heart failure, frequent ventricular premature contractions, or signs of ischemia on the stress test. 7. Patients with angina pectoris, regardless of severity, in whom noninvasive testing indicates a high risk of coronary events (poor exercise performance or severe ischemia). 8. Patients in whom coronary spasm or another nonatherosclerotic cause of myocardial ischemia (e.g., coronary artery anomaly, Kawasaki disease) is suspected.

1	8. Patients in whom coronary spasm or another nonatherosclerotic cause of myocardial ischemia (e.g., coronary artery anomaly, Kawasaki disease) is suspected. Noninvasive alternatives to diagnostic coronary arteriography include CT angiography and CMR angiography (Chap. 270e). Although these new imaging techniques can provide information about obstructive lesions in the epicardial coronary arteries, their exact role in clinical practice has not been rigorously defined. Important aspects of their use that should be noted include the substantially higher radiation exposure with CT angiography compared to conventional diagnostic arteriography and the limitations on CMR imposed by cardiac movement during the cardiac cycle, especially at high heart rates.

1	The principal prognostic indicators in patients known to have IHD are age, the functional state of the left ventricle, the location(s) and severity of coronary artery narrowing, and the severity or activity of myocardial ischemia. Angina pectoris of recent onset, unstable angina (Chap. 294), early postmyocardial infarction angina, angina that is unresponsive or poorly responsive to medical therapy, and angina accompanied by symptoms of congestive heart failure all indicate an increased risk for adverse coronary events. The same is true for the physical signs of heart failure, episodes of pulmonary edema, transient third heart sounds, and mitral regurgitation and for echocardiographic or radioisotopic (or roentgenographic) evidence of cardiac enlargement and reduced (<0.40) ejection fraction.

1	Most important, any of the following signs during noninvasive testing indicates a high risk for coronary events: inability to exercise for 6 min, i.e., stage II (Bruce protocol) of the exercise test; a strongly positive exercise test showing onset of myocardial ischemia at low workloads (≥0.1 mV ST-segment depression before completion of stage II, ≥0.2 mV ST-segment depression at any stage, ST-segment depression for >5 min after the cessation of exercise, a decline in systolic pressure >10 mmHg during exercise, or the development of ventricular tachyarrhythmias during exercise); the development of large or multiple perfusion defects or increased lung uptake during stress radioisotope perfusion imaging; and a decrease in left ventricular ejection fraction during exercise on radionuclide ventriculography or during stress echocardiography. Conversely, patients who can complete stage III of the Bruce exercise protocol and have a normal stress perfusion scan or negative stress

1	ventriculography or during stress echocardiography. Conversely, patients who can complete stage III of the Bruce exercise protocol and have a normal stress perfusion scan or negative stress echocardiographic evaluation are at very low risk for future coronary events. The finding of frequent episodes of ST-segment deviation on ambulatory ECG monitoring (even in the absence of symptoms) is also an adverse prognostic finding.

1	On cardiac catheterization, elevations of left ventricular end-diastolic pressure and ventricular volume and reduced ejection fraction are the most important signs of left ventricular dysfunction and are associated with a poor prognosis. Patients with chest discomfort but normal left ventricular function and normal coronary arteries have an excellent prognosis. Obstructive lesions of the left main (>50% luminal diameter) or left anterior descending coronary artery proximal to the origin of the first septal artery are associated with a greater risk than are lesions of the right or left circumflex coronary artery because of the greater quantity of myocardium at risk. Atherosclerotic plaques in epicardial arteries with fissuring or filling defects indicate increased risk. These lesions go through phases of inflammatory cellular activity, degeneration, endothelial dysfunction, abnormal vasomotion, platelet aggregation, and fissuring or hemorrhage. These factors can temporarily worsen the

1	phases of inflammatory cellular activity, degeneration, endothelial dysfunction, abnormal vasomotion, platelet aggregation, and fissuring or hemorrhage. These factors can temporarily worsen the stenosis and cause thrombosis and/or abnormal reactivity of the vessel wall, thus exacerbating the manifestations of ischemia. The recent onset of symptoms, the development of severe ischemia during stress testing (see above), and unstable angina pectoris (Chap. 294) all reflect episodes of rapid progression in coronary lesions.

1	With any degree of obstructive CAD, mortality is greatly increased when left ventricular function is impaired; conversely, at any level of left ventricular function, the prognosis is influenced importantly by the quantity of myocardium perfused by critically obstructed vessels. Therefore, it is essential to collect all the evidence substantiating past myocardial damage (evidence of myocardial infarction on ECG, echocardiography, radioisotope imaging, or left ventriculography), residual left ventricular function (ejection fraction and wall motion), and risk of future damage from coronary events (extent of coronary disease and severity of ischemia defined by noninvasive stress testing). The larger the quantity of established myocardial necrosis is, the less the heart is able to withstand additional damage and the poorer the prognosis is. Risk estimation must include age, presenting symptoms, all risk factors, signs of arterial disease, existing cardiac damage, and signs of impending

1	additional damage and the poorer the prognosis is. Risk estimation must include age, presenting symptoms, all risk factors, signs of arterial disease, existing cardiac damage, and signs of impending damage (i.e., ischemia).

1	The greater the number and severity of risk factors for coronary atherosclerosis (advanced age [>75 years], hypertension, dyslipidemia, diabetes, morbid obesity, accompanying peripheral and/or cerebrovascular disease, previous myocardial infarction), the worse the prognosis of an angina patient. Evidence exists that elevated levels of CRP in the plasma, extensive coronary calcification on electron beam CT (see above), and increased carotid intimal thickening on ultrasound examination also indicate an increased risk of coronary events.

1	Once the diagnosis of IHD has been made, each patient must be evaluated individually with respect to his or her level of understanding, expectations and goals, control of symptoms, and prevention of adverse clinical outcomes such as myocardial infarction and premature death. The degree of disability and the physical and emotional stress that precipitates angina must be recorded carefully to set treatment goals. The management plan should include the following components: (1) explanation of the problem and reassurance about the ability to formulate a treatment plan, (2) identification and treatment of aggravating conditions, (3) recommendations for adaptation of activity as needed, (4) treatment of risk factors that will decrease the occurrence of adverse coronary outcomes, (5) drug therapy for angina, and (6) consideration of revascularization.

1	Patients with IHD need to understand their condition and realize that a long and productive life is possible even though they have angina pectoris or have experienced and recovered from an acute myocardial infarction. Offering results of clinical trials showing improved outcomes can be of great value in encouraging patients to resume or maintain activity and return to work. A planned program of rehabilitation can encourage patients to lose weight, improve exercise tolerance, and control risk factors with more confidence.

1	A number of conditions may increase oxygen demand or decrease oxygen supply to the myocardium and may precipitate or exacerbate angina in patients with IHD. Left ventricular hypertrophy, aortic valve disease, and hypertrophic cardiomyopathy may cause or contribute to angina and should be excluded or treated. Obesity, hypertension, and hyperthyroidism should be treated aggressively to reduce the frequency and severity of anginal episodes. Decreased myocardial oxygen supply may be due to reduced oxygenation of the arterial blood (e.g., in pulmonary disease or, when carboxyhemoglobin is present, due to cigarette or cigar smoking) or decreased oxygen-carrying capacity (e.g., in anemia). Correction of these abnormalities, if present, may reduce or even eliminate angina pectoris.

1	Myocardial ischemia is caused by a discrepancy between the demand of the heart muscle for oxygen and the ability of the coronary circulation to meet that demand. Most patients can be helped to understand this concept and utilize it in the rational programming of activity. Many tasks that ordinarily evoke angina may be accomplished without symptoms simply by reducing the speed at which they are performed. Patients must appreciate the diurnal variation in their tolerance of certain activities and should reduce their energy requirements in the morning, immediately after meals, and in cold or inclement weather. On occasion, it may be necessary to recommend a change in employment or residence to avoid physical stress.

1	Physical conditioning usually improves the exercise tolerance of patients with angina and has substantial psychological benefits. A regular program of isotonic exercise that is within the limits of the individual patient’s threshold for the development of angina pectoris and that does not exceed 80% of the heart rate associated with ischemia on exercise testing should be strongly encouraged. Based on the results of an exercise test, the number of metabolic equivalent tasks (METs) performed at the onset of ischemia can be estimated (Table 293-2) and a practical exercise prescription can be formulated to permit daily activities that will fall below the ischemic threshold (Table 293-3).

1	A family history of premature IHD is an important indicator of increased risk and should trigger a search for treatable risk factors such as hyperlipidemia, hypertension, and diabetes mellitus. Obesity impairs the treatment of other risk factors and increases the risk of adverse coronary events. In addition, obesity often is accompanied by three other risk factors: diabetes mellitus, hypertension, and hyperlipidemia. The treatment of obesity and these accompanying risk factors is an important component of any management plan. A diet low in saturated and trans-unsaturated fatty acids and a reduced caloric intake to achieve optimal body weight are a cornerstone in the management of chronic IHD. It is especially important to emphasize weight loss and regular exercise in patients with the metabolic syndrome or overt diabetes mellitus.

1	Cigarette smoking accelerates coronary atherosclerosis in both sexes and at all ages and increases the risk of thrombosis, plaque instability, myocardial infarction, and death (Chap. 291e). In addition, by increasing myocardial oxygen needs and reducing oxygen supply, it aggravates angina. Smoking cessation studies have demonstrated important benefits with a significant decline in the occurrence of these adverse outcomes. The physician’s message must be clear and strong and supported by programs that achieve and monitor abstinence (Chap. 470). Hypertension (Chap. 298) is associated with an increased risk of adverse clinical events from coronary atherosclerosis as well as stroke. In addition, the left ventricular hypertrophy that results from sustained hypertension aggravates ischemia. There is evidence that long-term effective treatment of hypertension can decrease the occurrence of adverse coronary events.

1	Diabetes mellitus (Chap. 417) accelerates coronary and peripheral atherosclerosis and is frequently associated with dyslipidemias and increases in the risk of angina, myocardial infarction, and sudden coronary death. Aggressive control of the dyslipidemia (target LDL cholesterol <70 mg/dL) and hypertension (target blood pressure 120/80 mmHg) that are frequently found in diabetic patients is highly effective and therefore essential, as described below. Abbreviation: METs, metabolic equivalent tasks. Source: Modified from WL Haskell: Rehabilitation of the coronary patient, in NK Wenger, HK Hellerstein (eds): Design and Implementation of Cardiac Conditioning Program. New York, Churchill Livingstone, 1978.

1	1588 DYSLIPIDEMIA The treatment of dyslipidemia is central in aiming for long-term relief from angina, reduced need for revascularization, and reduction in myocardial infarction and death. The control of lipids can be achieved by the combination of a diet low in saturated and trans-unsaturated fatty acids, exercise, and weight loss. Nearly always, HMG-CoA reductase inhibitors (statins) are required and can lower LDL cholesterol (25–50%), raise HDL cholesterol (5–9%), and lower triglycerides (5–30%). A powerful treatment effect of statins on atherosclerosis, IHD, and outcomes is seen regardless of the pretreatment LDL cholesterol level. Fibrates or niacin can be used to raise HDL cholesterol and lower triglycerides (Chaps. 291e and 421). Controlled trials with lipid-regulating regimens have shown equal proportional benefit for men, women, the elderly, diabetic patients, and smokers. Compliance with the health-promoting behaviors listed above is generally very poor, and a conscientious

1	shown equal proportional benefit for men, women, the elderly, diabetic patients, and smokers. Compliance with the health-promoting behaviors listed above is generally very poor, and a conscientious physician must not underestimate the major effort required to meet this challenge. Many patients who are discharged from the hospital with proven coronary disease do not receive adequate treatment for dyslipidemia. In light of the proof that treating dyslipidemia brings major benefits, physicians need to establish treatment pathways, monitor compliance, and follow up regularly.

1	The incidence of clinical IHD in premenopausal women is very low; however, after menopause, the atherogenic risk factors increase (e.g., increased LDL, reduced HDL) and the rate of clinical coronary events accelerates to the levels observed in men. Women have not given up cigarette smoking as effectively as have men. Diabetes mellitus, which is more common in women, greatly increases the occurrence of clinical IHD and amplifies the deleterious effects of hypertension, hyperlipidemia, and smoking. Cardiac catheterization and coronary revascularization are underused in women and are performed at a later and more severe stage of the disease than in men. When cholesterol lowering, beta blockers after myocardial infarction, and coronary artery bypass grafting are applied in the appropriate patient groups, women benefit to the same degree as men.

1	The commonly used drugs for the treatment of angina pectoris are summarized in Tables 293-4 through 293-6. Pharmacotherapy for IHD is designed to reduce the frequency of anginal episodes, myocardial infarction, and coronary death. There is a wealth of trial Preparation of Agent Dose Schedule aA 10to 12-h nitrate-free interval is recommended. Source: Modified from DA Morrow, WE Boden: Stable ischemic heart disease. In RO Bonow et al (eds): Braunwald’s Heart Disease: A Textbook of Cardiovascular Medicine. 9th edition. Philadelphia, Saunders, 2012, p. 1224. data to emphasize how important this medical management is when added to the health-promoting behaviors discussed above. To achieve maximum benefit from medical therapy for IHD, it is frequently necessary to combine agents from different classes and titrate the doses as guided by the individual profile of risk factors, symptoms, hemodynamic responses, and side effects.

1	The organic nitrates are a valuable class of drugs in the management of angina pectoris (Table 293-4). Their major mechanisms of action include systemic venodilation with concomitant reduction in left ventricular end-diastolic volume and pressure, thereby reducing myocardial wall tension and oxygen requirements; dilation of epicardial coronary vessels; and increased blood flow in collateral vessels. When metabolized, organic nitrates release nitric oxide (NO) that binds to guanylyl cyclase in vascular smooth muscle cells, leading to an increase in cyclic guanosine monophosphate, which causes relaxation of vascular smooth muscle. Nitrates also exert antithrombotic activity by NO-dependent activation of platelet guanylyl cyclase, impairment of intraplatelet calcium flux, and platelet activation.

1	The absorption of these agents is most rapid and complete through the mucous membranes. For this reason, nitroglycerin is most commonly administered sublingually in tablets of 0.4 or 0.6 mg. Patients with angina should be instructed to take the medication both to relieve angina and also approximately 5 min before stress that is likely to induce an episode. The value of this prophylactic use of the drug cannot be overemphasized. Nitrates improve exercise tolerance in patients with chronic angina and relieve ischemia in patients with unstable angina as well as patients with Prinzmetal’s variant angina (Chap. 294). A diary of angina and nitroglycerin use may be valuable for detecting changes in the frequency, severity, or threshold for discomfort that may signify the development of unstable angina pectoris and/or herald an impending myocardial infarction.

1	Long-Acting Nitrates None of the long-acting nitrates are as effective as sublingual nitroglycerin for the acute relief of angina. These organic nitrate preparations can be swallowed, chewed, or administered as a patch or paste by the transdermal route (Table 293-4). They can provide effective plasma levels for up to 24 h, but the therapeutic response is highly variable. Different preparations and/or administration during the daytime should be tried only to prevent discomfort while avoiding side effects such as headache and dizziness. Individual dose titration is important to prevent side effects. To minimize the effects of tolerance, the minimum effective dose should be used and a minimum of 8 h each day kept free of the drug to restore any useful response(s).

1	β-Adrenergic Blockers These drugs represent an important component of the pharmacologic treatment of angina pectoris (Table 293-5). They reduce myocardial oxygen demand by inhibiting the increases in heart rate, arterial pressure, and myocardial contractility caused by adrenergic activation. Beta blockade reduces these variables most strikingly during exercise but causes only small reductions at rest. Long-acting beta-blocking drugs or sustained-release formulations offer the advantage of once-daily dosing (Table 293-5). The therapeutic aims include relief of angina and ischemia. These drugs also can reduce mortality and reinfarction rates in patients after myocardial infarction and are moderately effective antihypertensive agents.

1	Relative contraindications include asthma and reversible airway obstruction in patients with chronic lung disease, atrioventricular conduction disturbances, severe bradycardia, Raynaud’s phenomenon, and a history of mental depression. Side effects include fatigue, reduced exercise tolerance, nightmares, impotence, cold extremities, intermittent claudication, bradycardia (sometimes severe), impaired atrioventricular conduction, left ventricular failure, bronchial asthma, worsening claudication, and intensification of the hypoglycemia produced by oral hypoglycemic agents and insulin.

1	Acebutolol β1 Yes 200–600 mg twice daily Atenolol β1 No 50–200 mg/d Betaxolol β1 No 10–20 mg/d Bisoprolol β1 No 10 mg/d Esmolol (intravenous)a β1 No 50–300 μg/kg/min Labetalolb None Yes 200–600 mg twice daily Metoprolol β1 No 50–200 mg twice daily Nadolol None No 40–80 mg/d Nebivolol β1 (at low doses) No 5–40 mg/d Pindolol None Yes 2.5–7.5 mg 3 times daily Propranolol None No 80–120 mg twice daily Timolol None No 10 mg twice daily aEsmolol is an ultra-short-acting beta blocker that is administered as a continuous intravenous infusion. Its rapid offset of action makes esmolol an attractive agent to use in patients with relative contraindications to beta blockade. bLabetolol is a combined alpha and beta blocker.

1	Note: This list of beta blockers that may be used to treat patients with angina pectoris is arranged alphabetically. The agents for which there is the greatest clinical experience include atenolol, metoprolol, and propranolol. It is preferable to use a sustained-release formulation that may be taken once daily to improve the patient’s compliance with the regimen. Source: Modified from RJ Gibbons et al: J Am Coll Cardiol 41:159, 2003. Reducing the dose or even discontinuation may be necessary if these side effects develop and persist. Since sudden discontinuation can intensify ischemia, the doses should be tapered over 2 weeks. Beta blockers with relative β1-receptor specificity such as metoprolol and atenolol may be preferable in patients with mild bronchial obstruction and insulin-requiring diabetes mellitus.

1	Calcium Channel Blockers Calcium channel blockers (Table 293-6) are coronary vasodilators that produce variable and dose-dependent reductions in myocardial oxygen demand, contractility, and arterial pressure. These combined pharmacologic effects are advantageous and make these agents as effective as beta blockers in the treatment of angina pectoris. They are indicated when beta blockers are contraindicated, poorly tolerated, or ineffective. Because of differences in the dose-response relationship on cardiac electrical activity between the dihydropyridine and nondihydropyridine calcium channel blockers, verapamil and diltiazem may produce symptomatic disturbances in cardiac conduction and bradyarrhythmias. They also exert negative inotropic actions and are more likely to aggravate left ventricular failure, particularly when used in patients with left ventricular dysfunction, especially if the patients are also receiving beta blockers. Although useful effects usually are achieved when

1	failure, particularly when used in patients with left ventricular dysfunction, especially if the patients are also receiving beta blockers. Although useful effects usually are achieved when calcium channel blockers are combined with beta blockers and nitrates, individual titration of the doses is essential with these combinations. Variant (Prinzmetal’s) angina responds particularly well to calcium channel blockers (especially members of the dihydropyridine class), supplemented when necessary by nitrates (Chap. 294).

1	Verapamil ordinarily should not be combined with beta blockers because of the combined adverse effects on heart rate and contractility. Diltiazem can be combined with beta blockers in patients with normal ventricular function and no conduction disturbances. Amlodipine and beta blockers have complementary actions on coronary blood supply and myocardial oxygen demands. Whereas the former decreases blood pressure and dilates coronary arteries, the latter slows heart rate and decreases contractility. Amlodipine and the other second-generation dihydropyridine calcium antagonists (nicardipine, isradipine, long-acting nifedipine, and felodipine) are aMay be associated with increased risk of mortality if administered during acute myocardial infarction.

1	Note: This list of calcium channel blockers that may be used to treat patients with angina pectoris is divided into two broad classes, dihydropyridines and nondihydropyridines, and arranged alphabetically within each class. Among the dihydropyridines, the greatest clinical experience has been obtained with amlodipine and nifedipine. After the initial period of dose titration with a short-acting formulation, it is preferable to switch to a sustained-release formulation that may be taken once daily to improve patient compliance with the regimen. Source: Modified from RJ Gibbons et al: J Am Coll Cardiol 41:159, 2003. 1590 potent vasodilators and are useful in the simultaneous treatment of angina and hypertension. Short-acting dihydropyridines should be avoided because of the risk of precipitating infarction, particularly in the absence of concomitant beta blocker therapy.

1	Choice Between Beta Blockers and Calcium Channel Blockers for Initial Therapy Since beta blockers have been shown to improve life expectancy after acute myocardial infarction (Chaps. 294 and 295) and calcium channel blockers have not, the former may also be preferable in patients with angina and a damaged left ventricle. However, calcium channel blockers are indicated in patients with the following: (1) inadequate responsiveness to the combination of beta blockers and nitrates; many of these patients do well with a combination of a beta blocker and a dihydropyridine calcium channel blocker; (2) adverse reactions to beta blockers such as depression, sexual disturbances, and fatigue; (3) angina and a history of asthma or chronic obstructive pulmonary disease; (4) sick-sinus syndrome or significant atrioventricular conduction disturbances; (5) Prinzmetal’s angina; or (6) symptomatic peripheral arterial disease.

1	Antiplatelet Drugs Aspirin is an irreversible inhibitor of platelet cyclooxygenase and thereby interferes with platelet activation. Chronic administration of 75–325 mg orally per day has been shown to reduce coronary events in asymptomatic adult men over age 50, patients with chronic stable angina, and patients who have or have survived unstable angina and myocardial infarction. There is a dose-dependent increase in bleeding when aspirin is used chronically. It is preferable to use an enteric-coated formulation in the range of 81–162 mg/d. Administration of this drug should be considered in all patients with IHD in the absence of gastrointestinal bleeding, allergy, or dyspepsia. Clopidogrel (300–600 mg loading and 75 mg/d) is an oral agent that blocks P2Y12 ADP receptor–mediated platelet aggregation. It provides benefits similar to those of aspirin in patients with stable chronic IHD and may be substituted for aspirin if aspirin causes the side effects listed above. Clopidogrel

1	aggregation. It provides benefits similar to those of aspirin in patients with stable chronic IHD and may be substituted for aspirin if aspirin causes the side effects listed above. Clopidogrel combined with aspirin reduces death and coronary ischemic events in patients with an acute coronary syndrome (Chap. 294) and also reduces the risk of thrombus formation in patients undergoing implantation of a stent in a coronary artery (Chap. 296e). Alternative antiplatelet agents that block the P2Y12 platelet receptor such as prasugrel and ticagrelor have been shown to be more effective than clopidogrel for prevention of ischemic events after placement of a stent for an acute coronary syndrome but are associated with an increased risk of bleeding. Although combined treatment with clopidogrel and aspirin for at least a year is recommended in patients with an acute coronary syndrome treated with implantation of a drug-eluting stent, studies have not shown any benefit from the routine addition

1	aspirin for at least a year is recommended in patients with an acute coronary syndrome treated with implantation of a drug-eluting stent, studies have not shown any benefit from the routine addition of clopidogrel to aspirin in patients with chronic stable IHD.

1	The angiotensin-converting enzyme (ACE) inhibitors are widely used in the treatment of survivors of myocardial infarction, patients with hypertension or chronic IHD including angina pectoris, and those at high risk of vascular diseases such as diabetes. The benefits of ACE inhibitors are most evident in IHD patients at increased risk, especially if diabetes mellitus or left ventricle dysfunction is present, and those who have not achieved adequate control of blood pressure and LDL cholesterol on beta blockers and statins. However, the routine administration of ACE inhibitors to IHD patients who have normal left ventricular function and have achieved blood pressure and LDL goals on other therapies does not reduce the incidence of events and therefore is not cost-effective.

1	Despite treatment with nitrates, beta blockers, or calcium channel blockers, some patients with IHD continue to experience angina, and additional medical therapy is now available to alleviate their symptoms. Ranolazine, a piperazine derivative, may be useful for patients with chronic angina despite standard medical therapy. Its antianginal action is believed to occur via inhibition of the late inward sodium current (INa). The benefits of INa inhibition include limitation of the Na overload of ischemic myocytes and prevention of Ca2+ overload via the Na+–Ca2+ exchanger. A dose of 500–1000 mg orally twice daily is usually well tolerated. Ranolazine is contraindicated in patients with hepatic impairment or with conditions or drugs associated with QTc prolongation and when drugs that inhibit the CYP3A metabolic system (e.g., ketoconazole, diltiazem, verapamil, macrolide antibiotics, HIV protease inhibitors, and large quantities of grapefruit juice) are being used.

1	Nonsteroidal anti-inflammatory drug (NSAID) use in patients with IHD may be associated with a small but finite increased risk of myocardial infarction and mortality. For this reason, they generally should be avoided in IHD patients. If they are required for symptom relief, it is advisable to coadminister aspirin and strive to use an NSAID associated with the lowest risk of cardiovascular events, in the lowest dose required, and for the shortest period of time. Another class of agents opens ATP-sensitive potassium channels in myocytes, leading to a reduction of free intracellular calcium ions. The major drug in this class is nicorandil, which typically is administered orally in a dose of 20 mg twice daily for prevention of angina. (Nicorandil is not available for use in the United States but is used in several other countries.)

1	Angina and Heart Failure Transient left ventricular failure with angina can be controlled by the use of nitrates. For patients with established congestive heart failure, the increased left ventricular wall tension raises myocardial oxygen demand. Treatment of congestive heart failure with an ACE inhibitor, a diuretic, and digoxin (Chap. 279) reduces heart size, wall tension, and myocardial oxygen demand, which helps control angina and ischemia. If the symptoms and signs of heart failure are controlled, an effort should be made to use beta blockers not only for angina but because trials in heart failure have shown significant improvement in survival. A trial of the intravenous ultra-short-acting beta blocker esmolol may be useful to establish the safety of beta blockade in selected patients. Nocturnal angina often can be relieved by the treatment of heart failure.

1	The combination of congestive heart failure and angina in patients with IHD usually indicates a poor prognosis and warrants serious consideration of cardiac catheterization and coronary revascularization. Clinical trials have confirmed that with the initial diagnosis of stable IHD, it is first appropriate to initiate a thorough medical regimen as described above. Revascularization should be considered in the presence of unstable phases of the disease, intractable symptoms, severe ischemia or high-risk coronary anatomy, diabetes, and impaired left ventricular (LV) function. Revascularization should be employed in conjunction with but not replace the continuing need to modify risk factors and assess medical therapy. An algorithm for integrating medical therapy and revascularization options in patients with IHD is shown in Fig. 293-3.

1	(See also Chap. 296e) Percutaneous coronary intervention (PCI) involving balloon dilatation usually accompanied by coronary stenting is widely used to achieve revascularization of the myocardium in patients with symptomatic IHD and suitable stenoses of epicardial coronary arteries. Whereas patients with stenosis of the left main coronary artery and those with three-vessel IHD (especially with diabetes and/or impaired LV function) who require revascularization are best treated with CABG, PCI is widely employed in patients with symptoms and evidence of ischemia due to stenoses of one or two vessels and even in selected patients with three-vessel disease (and, perhaps, in some patients with left main disease) and may offer many advantages over surgery.

1	Indications and Patient Selection The most common clinical indication for PCI is symptom-limiting angina pectoris, despite medical therapy, accompanied by evidence of ischemia during a stress test. PCI is more effective than medical therapy for the relief of angina. PCI improves Initiate medical therapy: 1. Decrease demand ischemia 2. Minimize IHD risk factors 3. ASA (clopidogrel if ASA intolerant) Any high-risk features? Low exercise capacity or ischemia at low workload, large area of ischemic myocardium, EF <40%, ACS presentation No Yes Are exertional symptoms controlled? Refer for coronary arteriography Yes No Yes No Single vessel disease LM +/or multi vessel disease PCI Assess: PCI vs CABG Consider unconventional treatments Continue medical therapy periodic stress assessment (see Fig. 293-2) Anatomy suitable for revascularization?

1	FIGURE 293-3 Algorithm for management of a patient with ischemic heart disease. All patients should receive the core elements of medical therapy as shown at the top of the algorithm. If high-risk features are present, as established by the clinical history, exercise test data, and imaging studies, the patient should be referred for coronary arteriography. Based on the number and location of the diseased vessels and their suitability for revascularization, the patient is treated with a percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) surgery or should be considered for unconventional treatments. See text for further discussion. ACS, acute coronary syndrome; ASA, aspirin; EF, ejection fraction; IHD, ischemic heart disease; LM, left main.

1	outcomes in patients with unstable angina or when used early in the course of myocardial infarction with and without cardiogenic shock. However, in patients with stable exertional angina, clinical trials have confirmed that PCI does not reduce the occurrence of death or myocardial infarction compared to optimum medical therapy. PCI can be used to treat stenoses in native coronary arteries as well as in bypass grafts in patients who have recurrent angina after CABG.

1	Risks When coronary stenoses are discrete and symmetric, two and even three vessels can be treated in sequence. However, case selection is essential to avoid a prohibitive risk of complications, which are usually due to dissection or thrombosis with vessel occlusion, uncontrolled ischemia, and ventricular failure (Chap. 296e). Oral aspirin, a P2Y12 antagonist, and an antithrombin agent are given to reduce coronary thrombus formation. Left main coronary artery stenosis generally is regarded as a contraindication to PCI; such patients should be treated with CABG. In selected cases such as patients with prohibitive surgical risks, PCI of an unprotected left main can be considered, but such a procedure should be performed only by a highly skilled operator; importantly, there are regional differences in the use of this 1591 approach internationally.

1	Efficacy Primary success, i.e., adequate dilation (an increase in luminal diameter >20% to a residual diameter obstruction <50%) with relief of angina, is achieved in >95% of cases. Recurrent stenosis of the dilated vessels occurs in ~20% of cases within 6 months of PCI with bare metal stents, and angina will recur within 6 months in 10% of cases. Restenosis is more common in patients with diabetes mellitus, arteries with small caliber, incomplete dilation of the stenosis, long stents, occluded ves sels, obstructed vein grafts, dilation of the left anterior descending coronary artery, and stenoses containing thrombi. In diseased vein grafts, procedural success has been improved by the use of capture devices or filters that prevent embolization, ischemia, and infarction.

1	It is usual clinical practice to administer aspirin indefinitely and a P2Y12 antagonist for 1–3 months after the implantation of a bare metal stent. Although aspirin in combination with a thienopyridine may help prevent coronary thrombosis during and shortly after PCI with stenting, there is no evidence that these medications reduce the incidence of restenosis.

1	The use of drug-eluting stents that locally deliver antiproliferative drugs can reduce restenosis to less than 10%. Advances in PCI, especially the availability of drug-eluting stents, have vastly extended the use of this revascularization option in patients with IHD. Of note, however, the delayed endothelial healing in the region of a drug-eluting stent also extends the period during which the patient is at risk for subacute stent thrombosis. Current recommendations are to administer aspirin indefinitely and a P2Y12 antagonist daily for at least 1 year after implantation of a drug-eluting stent. When a situation arises in which temporary discontinuation of antiplatelet therapy is necessary, the clinical circumstances should be reviewed with the operator who performed the PCI and a coordinated plan should be established for minimizing the risk of late stent thrombus; central to this plan is the discontinuation of antiplatelet therapy for the shortest acceptable period. The risk of

1	plan should be established for minimizing the risk of late stent thrombus; central to this plan is the discontinuation of antiplatelet therapy for the shortest acceptable period. The risk of stent thrombosis is dependent on stent size and length, complexity of the lesions, age, diabetes, and technique. However, compliance with dual antiplatelet therapy and individual responsiveness to platelet inhibition are very important factors as well.

1	Successful PCI produces effective relief of angina in >95% of cases. The majority of patients with symptomatic IHD who require revascularization can be treated initially by PCI. Successful PCI is less invasive and expensive than CABG and permits savings in the initial cost of care. Successful PCI avoids the risk of stroke associated with CABG surgery, allows earlier return to work, and allows the resumption of an active life. However, the early health-related and economic benefit of PCI is reduced over time because of the greater need for follow-up and the increased need for repeat procedures. When directly compared in patients with diabetes or three-vessel or left main CAD, CABG was superior to PCI in preventing major adverse cardiac or cerebrovascular events over a 12-month follow-up.

1	Anastomosis of one or both of the internal mammary arteries or a radial artery to the coronary artery distal to the obstructive lesion is the preferred procedure. For additional obstructions that cannot be bypassed by an artery, a section of a vein (usually the saphenous) is used to form a connection between the aorta and the coronary artery distal to the obstructive lesion. Although some indications for CABG are controversial, certain areas of agreement exist: 1. The operation is relatively safe, with mortality rates <1% in patients without serious comorbid disease and normal LV function and when the procedure is performed by an experienced surgical team. 2. Intraoperative and postoperative mortality rates increase with the severity of ventricular dysfunction, comorbidities, age >80 years, and lack of surgical experience. The effectiveness and risk of CABG vary widely depending on case selection and the skill and experience of the surgical team.

1	1592 3. Occlusion of venous grafts is observed in 10–20% of patients during the first postoperative year and in approximately 2% per year during 5to 7-year follow-up and 4% per year thereafter. Long-term patency rates are considerably higher for internal mammary and radial artery implantations than for saphenous vein grafts. In patients with left anterior descending coronary artery obstruction, survival is better when coronary bypass involves the internal mammary artery rather than a saphenous vein. Graft patency and outcomes are improved by meticulous treatment of risk factors, particularly dyslipidemia. 4. Angina is abolished or greatly reduced in ~90% of patients after complete revascularization. Although this usually is associated with graft patency and restoration of blood flow, the pain may also have been alleviated as a result of infarction of the ischemic segment or a placebo effect. Within 3 years, angina recurs in about one-fourth of patients but is rarely severe. 5.

1	5. Survival may be improved by operation in patients with stenosis of the left main coronary artery as well as in patients with three-or two-vessel disease with significant obstruction of the proximal left anterior descending coronary artery. The survival benefit is greater in patients with abnormal LV function (ejection fraction <50%). Survival may also be improved in the following patients: (a) patients with obstructive CAD who have survived sudden cardiac death or sustained ventricular tachycardia; (b) patients who have undergone previous CABG and have multiple saphenous vein graft stenoses, especially of a graft supplying the left anterior descending coronary artery; and (c) patients with recurrent stenosis after PCI and high-risk criteria on noninvasive testing. 6.

1	6. Minimally invasive CABG through a small thoracotomy and/or off-pump surgery can reduce morbidity and shorten convalescence in suitable patients but does not appear to reduce significantly the risk of neurocognitive dysfunction postoperatively. 7. Among patients with type 2 diabetes mellitus and multivessel coronary disease, CABG surgery plus optimal medical therapy is superior to optimal medical therapy alone in preventing major cardiovascular events, a benefit mediated largely by a significant reduction in nonfatal myocardial infarction. The benefits of CABG are especially evident in diabetic patients treated with an insulin-sensitizing strategy as opposed to an insulin-providing strategy. CABG has also been shown to be superior to PCI (including the use of drug-eluting stents) in preventing death, myocardial infarction, and repeat revascularization in patients with diabetes mellitus and multivessel IHD.

1	Indications for CABG usually are based on the severity of symptoms, coronary anatomy, and ventricular function. The ideal candidate is male, <80 years of age, has no other complicating disease, and has troublesome or disabling angina that is not adequately controlled by medical therapy or does not tolerate medical therapy. The patient wishes to lead a more active life and has severe stenoses of two or three epicardial coronary arteries with objective evidence of myocardial ischemia as a cause of the chest discomfort. Great symptomatic benefit can be anticipated in such patients. Congestive heart failure and/or LV dysfunction, advanced age (>80 years), reoperation, urgent need for surgery, and the presence of diabetes mellitus are all associated with a higher perioperative mortality rate.

1	LV dysfunction can be due to noncontractile or hypocontractile segments that are viable but are chronically ischemic (hibernating myocardium). As a consequence of chronic reduction in myocardial blood flow, these segments downregulate their contractile function. They can be detected by using radionuclide scans of myocardial perfusion and metabolism, PET, cardiac MRI, or delayed scanning with thallium-201 or by improvement of regional functional impairment provoked by low-dose dobutamine. In such patients, revascularization improves myocardial blood flow, can return function, and can improve survival.

1	The Choice Between PCI and CABG All the clinical characteristics of each individual patient must be used to decide on the method of revascularization (e.g., LV function, diabetes, lesion complexity). A number of randomized clinical trials have compared PCI and CABG in patients with multivessel CAD who were suitable technically for both procedures. The redevelopment of angina requiring repeat coronary angiography and repeat revascularization is higher with PCI. This is a result of restenosis in the stented segment (a problem largely solved with drug-eluting stents) and the development of new stenoses in unstented portions of the coronary vasculature. It has been argued that PCI with stenting focuses on culprit lesions, whereas a bypass graft to the target vessel also provides a conduit around future culprit lesions proximal to the anastomosis of the graft to the native vessel (Fig. 293-4). By contrast, stroke rates are lower with PCI.

1	Based on available evidence, it is now recommended that patients with an unacceptable level of angina despite optimal medical management be considered for coronary revascularization. Patients with singleor two-vessel disease with normal LV function and anatomically suitable lesions ordinarily are advised to undergo PCI (Chap. 296e). Patients with three-vessel disease (or two-vessel disease that includes the proximal left descending coronary artery) and impaired global LV function (LV ejection fraction <50%) or diabetes mellitus and those with left main CAD or other lesions unsuitable for catheter-based procedures should be considered for CABG as the initial method of revascularization. In light of the complexity of the decision making, it is desirable to have a multidisciplinary team, including a cardiologist and a cardiac surgeon in conjunction with the patient’s primary care physician, provide input along with ascertaining the patient’s preferences before committing to a particular

1	a cardiologist and a cardiac surgeon in conjunction with the patient’s primary care physician, provide input along with ascertaining the patient’s preferences before committing to a particular revascularization option.

1	On occasion clinicians will encounter a patient who has persistent disabling angina despite maximally tolerated medical therapy and for whom revascularization is not an option (e.g., small diffusely diseased vessels not amenable to stent implantation or acceptable targets for bypass grafting). In such situations, unconventional treatments should be considered. Enhanced external counterpulsation utilizes pneumatic cuffs on the lower extremities to provide diastolic augmentation and systolic unloading of blood pressure to decrease cardiac work and oxygen consumption while enhancing coronary blood flow. Clinical trials have shown that regular application improves angina, exercise capacity, and regional myocardial perfusion. Experimental approaches such as gene and stem cell therapies are also under active study.

1	Obstructive CAD, acute myocardial infarction, and transient myocardial ischemia are frequently asymptomatic. During continuous ambulatory ECG monitoring, the majority of ambulatory patients with typical chronic stable angina are found to have objective evidence of myocardial ischemia (ST-segment depression) during episodes of chest discomfort while they are active outside the hospital. In addition, many of these patients also have more frequent episodes of asymptomatic ischemia. Frequent episodes of ischemia (symptomatic and asymptomatic) during daily life appear to be associated with an increased likelihood of adverse coronary events (death and myocardial infarction). In addition, patients with asymptomatic ischemia after a myocardial infarction are at greater risk for a second coronary event. The widespread use of exercise ECG during routine examinations has also identified some of these previously unrecognized patients with asymptomatic CAD. Longitudinal studies have demonstrated

1	event. The widespread use of exercise ECG during routine examinations has also identified some of these previously unrecognized patients with asymptomatic CAD. Longitudinal studies have demonstrated an increased incidence of coronary events in asymptomatic patients with positive exercise tests.

1	The management of patients with asymptomatic ischemia must be individualized. When coronary disease has been confirmed, the aggressive treatment of hypertension and dyslipidemia is essential and will decrease the risk of infarction and death. In addition, the

1	FIGURE 293-4 Difference in the approach to the lesion with percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG). PCI is targeted at the “culprit” lesion or lesions, whereas CABG is directed at the epicardial vessel, including the culprit lesion or lesions and future culprits, proximal to the insertion of the vein graft, a difference that may account for the superiority of CABG, at least in the intermediate term, in patients with multivessel disease. (Reproduced from BJ Gersh, RL Frye: N Engl J Med 352:2235, 2005.) physician should consider the following: (1) the degree of positivity of the stress test, particularly the stage of exercise at which ECG signs of ischemia appear; the magnitude and number of the ischemic zones of myocardium on imaging; and the change in LV ejection fraction that occurs on radionuclide ventriculography or echocardiography during ischemia and/or during exercise; (2) the ECG leads showing a positive response, with changes in the

1	in LV ejection fraction that occurs on radionuclide ventriculography or echocardiography during ischemia and/or during exercise; (2) the ECG leads showing a positive response, with changes in the anterior precordial leads indicating a less favorable prognosis than changes in the inferior leads; and (3) the patient’s age, occupation, and general medical condition.

1	Most would agree that an asymptomatic 45-year-old commercial airline pilot with significant (0.4-mV) ST-segment depression in leads V1 to V4 during mild exercise should undergo coronary arteriography, whereas an asymptomatic, sedentary 85-year-old retiree with 0.1-mV ST-segment depression in leads II and III during maximal activity need not. However, there is no consensus about the most appropriate approach in the large majority of patients for whom the situation is less extreme. Asymptomatic patients with silent ischemia, three-vessel CAD, and impaired LV function may be considered appropriate candidates for CABG.

1	The treatment of risk factors, particularly lipid lowering and blood pressure control as described above, and the use of aspirin, statins, and beta blockers after infarction have been shown to reduce events and improve outcomes in asymptomatic as well as symptomatic patients with ischemia and proven CAD. Although the incidence of asymptomatic ischemia can be reduced by treatment with beta blockers, calcium channel blockers, and long-acting nitrates, it is not clear whether this is necessary or desirable in patients who have not had a myocardial infarction. 294 Acute Coronary Syndrome (non-ST-Segment Elevation Myocardial infarction and unstable Angina) Christopher P. Cannon, Eugene Braunwald

1	Patients with ischemic heart disease fall into two large groups: patients with chronic coronary artery disease (CAD) who most commonly present with stable angina (Chap. 293) and patients with acute coronary syndromes (ACSs). These include patients with acute myocardial infarction with ST-segment elevation (STEMI) on their presenting electrocardiogram (Chap. 295) and those with non-ST-segment elevation acute coronary syndrome (NSTE-ACS). The latter include patients with non-ST-segment elevation myocardial infarction (NSTEMI), who, by definition, have evidence of myocyte necrosis, and those with unstable angina (UA), who do not. The relative incidence of NSTEMI compared to STEMI appears to be increasing (Fig. 294-1). Every year in the United States, approximately 1.1 million patients are admitted to hospitals with NSTE-ACS as compared with ~300,000 patients with acute STEMI. Women comprise more than one-third of patients with NSTE-ACS, but less than one-fourth of patients with STEMI.

1	NSTE-ACS is most commonly caused by an imbalance between oxygen supply and oxygen demand resulting from a partially occluding thrombus forming on a disrupted atherothrombotic coronary plaque 1594 Trends of STEMI and NSTEMI in NRMI Registry (1990–2006) myocardial infarction (STEMI) and non-ST-segment elevation myocardial infarction (NSTEMI) and of frequency of use of troponin assay to diagnose acute myocardial infarction. NRMI, National Registry of Myocardial Infarction. (From N Arora, RG Brindis, CP Cannon: Acute coronary syndrome in North America, in Theroux P [ed]: Acute Coronary Syndromes, 2nd ed. Philadelphia: Elsevier, 2011.) or on eroded coronary artery endothelium. Severe ischemia or myocardial necrosis may occur consequent to the reduction of coronary blood flow caused by the thrombus and by downstream embolization of platelet aggregates and/or atherosclerotic debris. Other causes of NSTE-ACS include: (1) dynamic obstruction (e.g., coronary spasm, as in Prinzmetal’s variant

1	and by downstream embolization of platelet aggregates and/or atherosclerotic debris. Other causes of NSTE-ACS include: (1) dynamic obstruction (e.g., coronary spasm, as in Prinzmetal’s variant angina [see “Prinzmetal’s Variant Angina” later]); (2) severe mechanical obstruction due to progressive coronary atherosclerosis; and (3) increased myocardial oxygen demand produced by conditions such as fever, tachycardia, and thyrotoxicosis in the presence of fixed epicardial coronary obstruction. More than one of these processes may be involved.

1	Among patients with NSTE-ACS studied at angiography, approximately 10% have stenosis of the left main coronary artery, 35% have three-vessel CAD, 20% have two-vessel disease, 20% have single-vessel disease, and 15% have no apparent critical epicardial coronary artery stenosis; some of the latter may have obstruction of the coronary microcirculation and/or spasm. The “culprit lesion” responsible for ischemia may show an eccentric stenosis with scalloped or overhanging edges and a narrow neck on coronary angiography. Optical coherence tomography (an invasive technique) and contrast-enhanced coronary computed tomographic angiography (CCTA), a noninvasive technique (Fig. 294-2), have shown that culprit lesions are composed of a lipid-rich core with a thin fibrous cap. Patients with NSTE-ACS frequently have multiple such plaques that are at risk of disruption (vulnerable plaques).

1	CLINICAL PRESENTATION Diagnosis The diagnosis of NSTE-ACS is based largely on the clinical presentation. Typically, chest discomfort is severe and has at least one of three features: (1) it occurs at rest (or with minimal exertion), lasting >10 minutes; (2) it is of relatively recent onset (i.e., within the prior 2 weeks); and/or (3) it occurs with a crescendo pattern (i.e., distinctly more severe, prolonged, or frequent than previous episodes). The diagnosis of NSTEMI is established if a patient with these clinical features develops evidence of myocardial necrosis, as reflected in abnormally elevated levels of biomarkers of cardiac necrosis (see below).

1	History and Physical Examination The chest discomfort, often severe enough to be described as frank pain, is typically located in the sub-sternal region or sometimes in the epigastrium, and radiates to the left arm, left shoulder, and/or neck. Anginal “equivalents” such as dyspnea, epigastric discomfort, nausea, or weakness may occur instead of chest pain and appear to be more frequent in women, the elderly, and patients with diabetes mellitus. The physical examination resembles that in patients with stable angina (Chap. 293) and may be unremarkable. If the patient has a large area of myocardial ischemia or a large NSTEMI, the physical findings can include diaphoresis; pale, cool skin; sinus tachycardia; a third and/or fourth heart sound; basilar rales; and, sometimes, hypotension.

1	FIGURE 294-2 Coronary computed tomographic angiogram showing an obstructive plaque in the right coronary artery. (From PJ de Feyter, K Nieman. Multislice computed tomography in acute coro-nary syndromes, in Theroux P [ed]: Acute Coronary Syndromes, 2nd ed. Philadelphia: Elsevier, 2011.) Electrocardiogram ST-segment depression occurs in 20 to 25% of patients; it may be transient in patients without biomarker evidence of myocardial necrosis, but may be persistent for several days in NSTEMI. T-wave changes are common but are less specific signs of ischemia, unless they are new and deep T-wave inversions (≥0.3 mV).

1	Cardiac Biomarkers Patients with NSTEMI have elevated biomarkers of necrosis, such as cardiac troponin I or T, which are specific, sensitive, and the preferred markers of myocardial necrosis. The MB isoform of creatine kinase (CK-MB) is a less sensitive alternative. Elevated levels of these markers distinguish patients with NSTEMI from those with UA. There is a characteristic temporal rise and fall of the plasma concentration of these markers and a direct relationship between the degree of elevation and mortality (see Fig. 294-4B). However, in patients without a clear clinical history of myocardial ischemia, minor cardiac troponin (cTn) elevations have been reported and can be caused by congestive heart failure, myocarditis, or pulmonary embolism, or using high-sensitivity assays, they may occur in ostensibly normal subjects. Thus, in patients with an unclear history, small elevations of cTn, especially if they are persistent, may not be diagnostic of an ACS.

1	With more widespread measurement of troponin, especially using high-sensitivity assays, an increasing fraction of patients with NSTEACS are found to have NSTEMI, whereas the fraction of patients with UA is dwindling. In addition to the clinical examination, three major noninvasive tools are used in the evaluation of NSTEMI-ACS: the electrocardiogram (ECG), cardiac biomarkers, and stress testing. CCTA is an additional emerging option (Fig. 294-2). The goals are to: (1) recognize or exclude myocardial infarction (MI) using cardiac biomarkers, preferably cTn;

1	Definite ACS Possible ACS See Chap. 295 Outpatient follow-up Chronic stable angina See Chap. 293 Noncardiac diagnosis Treatment as indicated by alternative diagnosis Observe 12 h or more from symptom onset Nondiagnostic ECG Normal initial cTn No recurrent pain; negative follow-up studies Recurrent ischemic pain or positive follow-up studies Diagnosis of ACS confirmed Stress study to provoke ischemia consider evaluation of LV function if ischemia is present Negative Potential diagnoses: nonischemic discomfort; low-risk ACS Positive Diagnosis of ACS confirmed or highly likely Admit to hospital Manage via acute ischemia pathway STand/or T-wave changes Ongoing pain or elevated cTn Hemodynamic abnormalities No ST-segment elevation ST-segment elevation Symptoms suggestive of ACS FIGURE 294-3 Algorithm for evaluation and management of patients with suspected acute coronary syndrome (ACS). Follow-up stud-ies refer to ST deviations and elevation of troponin levels. cTn, cardiac troponin; ECG,

1	for evaluation and management of patients with suspected acute coronary syndrome (ACS). Follow-up stud-ies refer to ST deviations and elevation of troponin levels. cTn, cardiac troponin; ECG, electrocardiogram; LV, left ventricular. (Modified from JL Anderson et al: J Am Coll Cardiol 61:e179, 2013.) (2) detect rest ischemia (using serial or continuous ECGs); and (3) detect significant coronary obstruction at rest with CCTA and myocardial ischemia using stress testing (Chap 270e).

1	Patients with a low likelihood of ischemia are usually managed with an emergency department–based critical pathway (which, in some institutions, is carried out in a “chest pain unit”) (Fig. 294-3). Evaluation of such patients includes clinical monitoring for recurrent ischemic discomfort and continuous monitoring of ECGs and cardiac markers, typically obtained at baseline and at 4–6 h and 12 h after presentation. If new elevations in cardiac markers or ST-T-wave changes on the ECG are noted, the patient should be admitted to the hospital. Patients who remain pain free with negative markers may proceed to stress testing to determine the presence of ischemia or CCTA to detect coronary luminal obstruction (Fig. 294-2).

1	Patients with documented NSTE-ACS exhibit a wide spectrum of early (30 days) risk of death, ranging from 1 to 10%, and a recurrent ACS rate of 5 to 15% during the first year. Assessment of risk can be accomplished by clinical risk scoring systems such as that developed from the Thrombolysis in Myocardial Infarction (TIMI) Trials, which includes seven independent risk factors (Fig. 294-4A). The presence of an abnormally elevated cTn is especially important, as is its peak level, which correlates with the extent of myocardial damage (Fig. 294-4B). Other risk factors include diabetes mellitus, left ventricular dysfunction, renal dysfunction, and elevated levels of B-type natriuretic peptides and C-reactive protein. Multimarker strategies are now gaining favor, both to define more fully the pathophysiologic mechanisms underlying a given patient’s presentation and to stratify the patient’s risk further. Patients with ACS without elevated levels of cTn (infrequently encountered with the new

1	mechanisms underlying a given patient’s presentation and to stratify the patient’s risk further. Patients with ACS without elevated levels of cTn (infrequently encountered with the new sensitive troponin assays) are considered to have UA and have a more favorable prognosis than those with cTn elevations (NSTEMI).

1	Early risk assessment is useful both in predicting the risk of recurrent cardiac events and in identifying patients who would derive the greatest benefit from an early invasive strategy. For example, in the TACTICSTIMI 18 Trial, an early invasive strategy conferred a 40% reduction in recurrent cardiac events in patients with an elevated cTn level, whereas no benefit was observed in those without detectable troponin. Patients should be placed at bed rest with continuous ECG monitoring for ST-segment deviation and cardiac arrhythmias. Ambulation is permitted if the patient shows no recurrence of ischemia (symptoms or ECG changes) and does not develop an elevation of a biomarker of necrosis for 12–24 h. Medical therapy involves simultaneous anti-ischemic and antithrombotic treatments and consideration of coronary revascularization.

1	To provide relief and prevention of recurrence of chest pain, initial treatment should include bed rest, nitrates, beta adrenergic blockers, and inhaled oxygen in the presence of hypoxemia. Nitrates These should first be given sublingually or by buccal spray (0.3–0.6 mg) if the patient is experiencing ischemic pain. If pain persists after three doses given 5 min apart, intravenous nitroglycerin (5–10 μg/min using nonabsorbing tubing) is recommended. The rate of the infusion may be increased by 10 μg/min every 3–5 min until symptoms are relieved, systolic arterial pressure falls to This is the second major cornerstone of treatment. There are two components of antithrombotic therapy: antiplatelet drugs and anticoagulants.

1	This is the second major cornerstone of treatment. There are two components of antithrombotic therapy: antiplatelet drugs and anticoagulants. Antiplatelet Drugs (See Chap. 143) Initial treatment should begin with the platelet cyclooxygenase inhibitor aspirin. The typical initial 4.7 8.3 13.2 19.9 26.2 40.9 dose is 325 mg/d, with lower doses (75–100 mg/d) recommended thereafter. Contraindications are active bleeding or aspirin intolerance. “Aspirin resistance” has been noted in 2–8% of patients but frequently has been related to noncompliance.

1	In the absence of a high risk for bleeding, patients with NSTE-ACS, 0/12 3 4 56/7 TIMI risk score UA/NSTEMI % Population 4.3 17.3 32.0 29.3 13.0 3.4 irrespective of whether an invasive or conservative strategy (see below) is selected, should receive a platelet P2Y12 receptor blocker to inhibit platelet activation. The thienopyridine clopidogrel is an inactive prodrug that is converted into an active metabolite that causes irreversible blockade of the platelet P2Y12 receptor. When added to aspirin, so-called dual antiplatelet therapy, it has been shown to confer a 20% relative reduction in cardiovascular death, MI, or stroke, compared to aspirin alone, but to be associated with a moderate (absolute 1%) increase in major bleeding.

1	Continued benefit of treatment with the combination of aspirin and clopidogrel has been observed both in patients treated con servatively and in those who underwent PCI. This regimen should continue for at least 1 year in patients with NSTE-ACS, especially those with a drug-eluting stent, to prevent stent thrombosis. Up to one-third of patients have an inadequate response to clopidogrel, and a substantial proportion of these cases are related to a genetic variant of the cytochrome P450 system. A variant of the 2C19 gene leads to reduced conversion of clopidogrel into its active metabo lite, which, in turn, reduces platelet inhibition and is associated 1.0 Mortality at 6 weeks(% of patients)1.7 3.4 3.7 6.0 7.5 0–<0.4 0.4–<1.0 1.0–<2.0 2.0–<5.0 5.0–<9.0 ˜9.0 831 174 148 134 50 67 0 1 2 3 4 5 6 7 8 Risk ratio 1.0 1.8 3.5 3.9 6.2 7.8 95% confidence – 0.5–6.7 1.2–10.6 1.3–11.7 1.7–22.3 2.6–23.0

1	Risk ratio 1.0 1.8 3.5 3.9 6.2 7.8 95% confidence – 0.5–6.7 1.2–10.6 1.3–11.7 1.7–22.3 2.6–23.0 FIGURE 294-4 A. Death (D), myocardial infarction (MI), or need for urgent revascularization (UR) through 6 weeks by Thrombolysis in Myocardial Infarction (TIMI) Risk Score in the unfractionated heparin arm of the TIMI 11B trial. (From EM Antman et al: JAMA 284:835, 2000.) B. Mortality rate at 42 days by baseline cardiac troponin I levels in the TIMI 3B trial. (From EM Antman et al: N Engl J Med 335:1342, 1996.) <100 mmHg, or the dose reaches 200 μg/min. Topical or oral nitrates (Chap. 293) can be used when the pain has resolved, or they may replace intravenous nitroglycerin when the patient has been pain-free for 12–24 h. The only absolute contraindications to the use of nitrates are hypotension or the use of sildenafil or other phosphodiesterase-5 inhibitors within the previous 24–48 h.

1	Beta Adrenergic Blockers and Other Agents Beta blockers are the other mainstay of anti-ischemic treatment. They may be started by the intravenous route in patients with severe ischemia, but this is contraindicated in the presence of heart failure. Ordinarily, oral beta blockade targeted to a heart rate of 50–60 beats/min is recommended. Heart rate–slowing calcium channel blockers, e.g., verapamil or diltiazem, are recommended for patients who have persistent symptoms or ECG signs of ischemia after treatment with full-dose nitrates and beta blockers and in patients with contraindications to either class of these agents. Additional medical therapy includes angiotensin-converting enzyme (ACE) inhibitors or, if these are not tolerated, angiotensin receptor blockers. Early administration of intensive HMG-CoA reductase inhibitors (statins), such as atorvastatin 80 mg/d, prior to percutaneous coronary intervention (PCI), and continued thereafter, has been shown to reduce complications of the

1	HMG-CoA reductase inhibitors (statins), such as atorvastatin 80 mg/d, prior to percutaneous coronary intervention (PCI), and continued thereafter, has been shown to reduce complications of the procedure and recurrences of ACS.

1	with increases in the incidence of adverse cardiovascular events. Alternate P2Y12 blockers, such as prasugrel or ticagrelor (see below) used with aspirin, should be considered in patients with NSTE-ACS who develop a coronary event while receiving clopidogrel and aspirin or who are hyporesponsive to clopidogrel as identified by platelet and/or genetic testing, although such testing is not yet widespread.

1	A second P2Y12 blocker, prasugrel, also a thienopyridine, achieves a more rapid onset and higher level of platelet inhibition than clopidogrel. It has been approved for ACS patients following angiography in whom PCI is planned. It should be administered at a loading dose of 60 mg followed by 10 mg/d for up to 15 months. The TRITON-TIMI 38 trial showed that relative to clopidogrel, prasugrel reduced the risk of cardiovascular death, MI, or stroke significantly, albeit with an increase in major bleeding. Stent thrombosis was reduced by half. This agent is contraindicated in patients with prior stroke or transient ischemic attack or at high risk for bleeding. It has not been found to be effective in patients treated by a conservative strategy (see below).

1	Ticagrelor is a novel, potent, reversible platelet P2Y12 inhibitor. It has been shown in the PLATO trial to reduce the risk of cardiovascular death, MI, or stroke compared with clopidogrel in ACS patients who are treated by either an invasive or a conservative strategy. This agent reduced mortality but increased the risk of bleeding not associated with coronary artery bypass grafting. After a loading dose of 180 mg, 90 mg bid is administered as maintenance.

1	Prior to the development of the oral P2Y12 receptor blockers, many trials had shown the benefit of intravenous glycoprotein IIb/ IIIa inhibitors. Their benefit, however, has been small (i.e., only a 10% reduction in death or MI, with a significant increase in major bleeding). Two recent studies failed to show a benefit of routine early initiation of a drug in this class compared with their use only in patients who undergo PCI. The addition of these agents to aspirin and a P2Y12 inhibitor (i.e., triple antiplatelet therapy) should be reserved for unstable patients with recurrent rest pain, elevated cTn, and ECG changes, as well as those who have a coronary thrombus evident on angiography when they undergo PCI.

1	aAllergy or prior intolerance is a contraindication for all categories of drugs listed in this chart. bChoice of the specific agent is not as important as ensuring that appropriate candidates receive this therapy. If there are concerns about patient intolerance due to existing pulmonary disease, especially asthma, left ventricular dysfunction, risk of hypotension, or severe bradycardia, initial selection should favor a short-acting agent, such as propranolol or metoprolol or the ultra-short-acting agent esmolol. Mild wheezing or a history of chronic obstructive pulmonary disease should prompt a trial of a short-acting agent at a reduced dose (e.g., 2.5 mg IV metoprolol, 12.5 mg oral metoprolol, or 25 μg/kg per min esmolol as initial doses) rather than complete avoidance of beta blocker therapy.

1	Note: Some of the recommendations in this guide suggest the use of agents for purposes or in doses other than those specified by the U.S. Food and Drug Administration. Such recommendations are made after consideration of concerns regarding nonapproved indications. Where made, such recommendations are based on more recent clinical trials or expert consensus. 2°, second-degree; 3°, third-degree; ECG, electrocardiogram; IV, intravenous. Source: Modified from J Anderson et al: J Am Coll Cardiol 61:e179, 2013.

1	Anticoagulants (See Chap. 143) Four options are available for anti-clinical risk factors, ST-segment deviation, and/or positive biomarkcoagulant therapy to be added to antiplatelet agents: (1) unfrac-ers) (Table 294-3). In this strategy, following treatment with antitionated heparin (UFH), long the mainstay of therapy; (2) the ischemic and antithrombotic agents, coronary arteriography is low-molecular-weight heparin (LMWH), enoxaparin, which has been carried out within ~48 h of presentation, followed by coronary shown to be superior to UFH in reducing recurrent cardiac events, revascularization (PCI or coronary artery bypass grafting), depend-especially in patients managed by a conservative strategy but with ing on the coronary anatomy. In low-risk patients, the outcomes some increase in bleeding; (3) bivalirudin, a direct thrombin inhibi-from an invasive strategy are similar to those obtained from a tor that is similar in efficacy to either UFH or LMWH but causes less conservative

1	bleeding; (3) bivalirudin, a direct thrombin inhibi-from an invasive strategy are similar to those obtained from a tor that is similar in efficacy to either UFH or LMWH but causes less conservative strategy. The latter consists of anti-ischemic and anti-bleeding and is used just prior to and/or during PCI; and (4) the indi-thrombotic therapy followed by “watchful waiting,” in which the rect factor Xa inhibitor, fondaparinux, which is equivalent in efficacy patient is closely observed and coronary arteriography is carried to enoxaparin but appears to have a lower risk of major bleeding. out only if rest pain or ST-segment changes recur, a biomarker of

1	Excessive bleeding is the most important adverse effect of necrosis becomes positive, or there is evidence of severe ischemia all antithrombotic agents, including both antiplatelet agents on a stress test. and anticoagulants. Therefore, attention must be directed to the doses of antithrombotic agents, accounting for body weight, creatinine clearance, and a previous history of excessive bleeding, as The time of hospital discharge is a “teachable moment” for the patient a means of reducing the risk of bleeding. Patients who have experi with NSTE-ACS, when the physician can review and optimize the enced a stroke are at higher risk of intracranial bleeding with potent medical regimen. Risk-factor modification is key, and the caregiver antiplatelet agents and combinations of antithrombotic drugs.

1	should discuss with the patient the importance of smoking cessa tion, achieving optimal weight, daily exercise, blood-pressure control, INVASIVE VERSUS CONSERVATIVE STRATEGY following an appropriate diet, control of hyperglycemia (in diabetic Multiple clinical trials have demonstrated the benefit of an early patients), and lipid management as recommended for patients with invasive strategy in high-risk patients (i.e., patients with multiple chronic stable angina (Chap. 293). Aspirin Initial dose of 325 mg nonenteric formulation followed by 75–100 mg/d of an enteric or a nonenteric formulation Clopidogrel Loading dose of 300–600 mg followed by 75 mg/d Prasugrel Pre-PCI: Loading dose 60 mg followed by 10 mg/d Ticagrelor Loading dose of 180 mg followed by 90 mg twice daily Abciximab 0.25 mg/kg bolus followed by infusion of 0.125 μg/ kg per min (maximum 10 μg/min) for 12–24 h

1	Ticagrelor Loading dose of 180 mg followed by 90 mg twice daily Abciximab 0.25 mg/kg bolus followed by infusion of 0.125 μg/ kg per min (maximum 10 μg/min) for 12–24 h Eptifibatide 180 μg/kg bolus followed 10 min later by second bolus of 180 μg with infusion of 2.0 μg/kg per min for 72–96 h following first bolus Tirofiban 25 μg/kg per min followed by infusion of 0.15 μg/kg per min for 48–96 h Unfractionated hepa-bBolus 70–100 U/kg (maximum 5000 U) IV followed rin (UFH) by infusion of 12–15 U/kg per h (initial maximum 1000 U/h) titrated to ACT 250–300 s Enoxaparin 1 mg/kg SC every 12 h; the first dose may be preceded by a 30-mg IV bolus; renal adjustment to 1 mg/kg once daily if creatine clearance <30 cc/min Fondaparinux 2.5 mg SC qd Bivalirudin Initial IV bolus of 0.75 mg/kg and an infusion of 1.75 mg/kg per h. aOther low-molecular-weight heparins exist beyond those listed. bIf no glycoprotein IIb/IIIa inhibitor planned.

1	Bivalirudin Initial IV bolus of 0.75 mg/kg and an infusion of 1.75 mg/kg per h. aOther low-molecular-weight heparins exist beyond those listed. bIf no glycoprotein IIb/IIIa inhibitor planned. Abbreviations: ACT, activated clotting time for HemoTec; IV, intravenous; SC, subcutaneously. Source: Modified from J Anderson et al: J Am Coll Cardiol 61:e179, 2013. There is evidence of benefit with long-term therapy with five classes of drugs that are directed at different components of the atherothrombotic process. Beta blockers, statins (at a high dose, e.g., atorvastatin 80 mg/d), and ACE inhibitors or angiotensin receptor blockers are recommended for long-term plaque stabilization. Antiplatelet therapy, Class I (Level of Evidence: A) Indications

1	Class I (Level of Evidence: A) Indications Recurrent angina at rest/low-level activity despite treatment Elevated TnT or TnI New ST-segment depression CHF symptoms, rales, MR EF <0.40 Sustained VT PCI <6 months, prior CABG High-risk findings from noninvasive testing Hemodynamic instability Mild-to-moderate renal dysfunction Diabetes mellitus High TIMI Risk Score (>3)b aAny one of the high-risk indicators. bSee Antman (JAMA 284:835, 2000). Abbreviations: CABG, coronary artery bypass grafting; CHF, congestive heart failure; EF, ejection fraction; MR, mitral regurgitation; PCI, percutaneous coronary intervention; TIMI, Thrombolysis in Myocardial Infarction; TnI, troponin I; TnT, troponin T; VT, ventricular tachycardia. Source: Modified from J Anderson et al: J Am Coll Cardiol 61:e179, 2013.

1	Source: Modified from J Anderson et al: J Am Coll Cardiol 61:e179, 2013. now recommended to be the combination of low-dose (75–100 mg/d) aspirin and a P2Y12 inhibitor (clopidogrel, prasugrel, or ticagrelor) for 1 year, with aspirin continued thereafter, prevents or reduces the severity of any thrombosis that would occur if a plaque were to rupture. Registries have shown that women and racial minorities, as well as patients with NSTE-ACS at high risk, including the elderly and patients with diabetes or chronic kidney disease, are less likely to receive evidence-based pharmacologic and interventional therapies with resultant poorer clinical outcomes and quality of life. Special attention should be directed to these groups.

1	In 1959 Prinzmetal et al. described a syndrome of severe ischemic pain that usually occurs at rest and is associated with transient ST-segment elevation. Prinzmetal’s variant angina (PVA) is caused by focal spasm of an epicardial coronary artery, leading to severe transient myocardial ischemia and occasionally infarction. The cause of the spasm is not well defined, but it may be related to hypercontractility of vascular smooth muscle due to adrenergic vasoconstrictors, leukotrienes, or serotonin. For reasons that are not clear, the prevalence of PVA has decreased substantially during the past few decades.

1	Clinical and Angiographic Manifestations Patients with PVA are generally younger and have fewer coronary risk factors (with the exception of cigarette smoking) than do patients with NSTE-ACS. Cardiac examination is usually unremarkable in the absence of ischemia. The clinical diagnosis of PVA is made by the detection of transient ST-segment elevation with rest pain. Many patients also exhibit multiple episodes of asymptomatic ST-segment elevation (silent ischemia). Small elevations of troponin may occur in patients with prolonged attacks.

1	Coronary angiography demonstrates transient coronary spasm as the diagnostic hallmark of PVA. Atherosclerotic plaques in at least one proximal coronary artery occur in about half of patients, and in these patients, spasm usually occurs within 1 cm of the plaque. Focal spasm is most common in the right coronary artery, and it may occur at one or more sites in one artery or in multiple arteries simultaneously. Hyperventilation or intracoronary acetylcholine has been used to provoke focal coronary stenosis on angiography or to provoke rest angina with ST-segment elevation to establish the diagnosis.

1	Nitrates and calcium channel blockers are the main therapeutic agents. Aspirin may actually increase the severity of ischemic episodes, possibly as a result of the sensitivity of coronary tone to modest changes in the synthesis of prostacyclin. The response to beta blockers is variable. Coronary revascularization may be helpful in patients who also have discrete, flow-limiting, proximal fixed obstructive lesions.

1	Prognosis Many patients with PVA pass through an acute, active phase, with frequent episodes of angina and cardiac events during the first 6 months after presentation. Survival at 5 years is excellent (~90–95%). Patients with no or mild fixed coronary obstruction tend to experience a more benign course than do patients with associated severe obstructive lesions. Nonfatal MI occurs in up to 20% of patients by 5 years. Patients with PVA who develop serious arrhythmias during spontaneous episodes of pain are at a higher risk for sudden cardiac death. In most patients who survive an infarction or the initial 3to 6-month period of frequent episodes, there is a tendency for symptoms and cardiac events to diminish over time. ST-Segment Elevation Myocardial infarction Elliott M. Antman, Joseph Loscalzo Acute myocardial infarction (AMI) is one of the most common diag-noses in hospitalized patients in industrialized countries. In the United 295

1	States, approximately 525,000 patients experience a new AMI, and 190,000 experience a recurrent AMI each year. More than half of AMI-related deaths occur before the stricken individual reaches the hospital. The in-hospital mortality rate after admission for AMI has declined from 10% to about 6% over the past decade. The 1-year mortality rate after AMI is about 15%. Mortality is approximately fourfold higher in elderly patients (over age 75) as compared with younger patients.

1	When patients with prolonged ischemic discomfort at rest are first seen, the working clinical diagnosis is that they are suffering from an acute coronary syndrome (Fig. 295-1). The 12-lead electrocardiogram (ECG) is a pivotal diagnostic and triage tool because it is at the center of the decision pathway for management; it permits distinction of those patients presenting with ST-segment elevation from those presenting without ST-segment elevation. Serum cardiac biomarkers are obtained to distinguish unstable angina (UA) from non-ST-segment elevation myocardial infarction (NSTEMI) and to assess the magnitude of an ST-segment elevation myocardial infarction (STEMI). This chapter focuses on the evaluation and management of patients with STEMI, while Chap. 294 discusses UA/NSTEMI. Acute coronary syndrome Presentation Working Dx ECG Biochem. marker Final Dx Myocardial infarction Unstable angina NQMI Qw MI No ST elevation NSTEMI ST elevation Ischemic Discomfort

1	FIGURE 295-1 Acute coronary syndromes. Following disruption of a vulnerable plaque, patients experience ischemic discomfort resulting from a reduction of flow through the affected epicardial coronary artery. The flow reduction may be caused by a completely occlusive thrombus (right) or subtotally occlusive thrombus (left). Patients with ischemic discomfort may present with or without ST-segment elevation. Of patients with ST-segment elevation, the majority (wide red arrow) ultimately develop a Q wave on the ECG (Qw MI), while a minority (thin red arrow) do not develop Q wave and, in older literature, were said to have sustained a non-Q-wave MI (NQMI). Patients who present without ST-segment elevation are suffering from either unstable angina or a non-ST-segment elevation MI (NSTEMI) (wide green arrows), a distinction that is ultimately made based on the presence or absence of a serum cardiac marker such as CK-MB or a cardiac troponin detected in the blood. The majority of patients

1	green arrows), a distinction that is ultimately made based on the presence or absence of a serum cardiac marker such as CK-MB or a cardiac troponin detected in the blood. The majority of patients presenting with NSTEMI do not develop a Q wave on the ECG; a minority develop a Qw MI (thin green arrow). Dx, diagnosis; ECG, electrocardiogram; MI, myocardial infarction. (Adapted from CW Hamm et al: Lancet 358:1533, 2001, and MJ Davies: Heart 83:361, 2000; with permission from the BMJ Publishing Group.)

1	PATHOPHYSIOLOGY: ROLE OF ACUTE PLAQUE RUPTURE 1599

1	STEMI usually occurs when coronary blood flow decreases abruptly after a thrombotic occlusion of a coronary artery previously affected by atherosclerosis. Slowly developing, high-grade coronary artery stenoses do not typically precipitate STEMI because of the development of a rich collateral network over time. Instead, STEMI occurs when a coronary artery thrombus develops rapidly at a site of vascular injury. This injury is produced or facilitated by factors such as cigarette smoking, hypertension, and lipid accumulation. In most cases, STEMI occurs when the surface of an atherosclerotic plaque becomes disrupted (exposing its contents to the blood) and conditions (local or systemic) favor thrombogenesis. A mural thrombus forms at the site of plaque disruption, and the involved coronary artery becomes occluded. Histologic studies indicate that the coronary plaques prone to disruption are those with a rich lipid core and a thin fibrous cap (Chap. 291e). After an initial platelet

1	artery becomes occluded. Histologic studies indicate that the coronary plaques prone to disruption are those with a rich lipid core and a thin fibrous cap (Chap. 291e). After an initial platelet monolayer forms at the site of the disrupted plaque, various agonists (collagen, ADP, epinephrine, serotonin) promote platelet activation. After agonist stimulation of platelets, thromboxane A2 (a potent local vasoconstrictor) is released, further platelet activation occurs, and potential resistance to fibrinolysis develops.

1	In addition to the generation of thromboxane A2, activation of platelets by agonists promotes a conformational change in the glycoprotein IIb/IIIa receptor (Chap. 140). Once converted to its functional state, this receptor develops a high affinity for soluble adhesive proteins (i.e., integrins) such as fibrinogen. Since fibrinogen is a multivalent molecule, it can bind to two different platelets simultaneously, resulting in platelet cross-linking and aggregation.

1	The coagulation cascade is activated on exposure of tissue factor in damaged endothelial cells at the site of the disrupted plaque. Factors VII and X are activated, ultimately leading to the conversion of prothrombin to thrombin, which then converts fibrinogen to fibrin (Chap. 141). Fluid-phase and clot-bound thrombin participates in an autoamplification reaction leading to further activation of the coagulation cascade. The culprit coronary artery eventually becomes occluded by a thrombus containing platelet aggregates and fibrin strands.

1	In rare cases, STEMI may be due to coronary artery occlusion caused by coronary emboli, congenital abnormalities, coronary spasm, and a wide variety of systemic—particularly inflammatory—diseases. The amount of myocardial damage caused by coronary occlusion depends on (1) the territory supplied by the affected vessel, (2) whether or not the vessel becomes totally occluded, (3) the duration of coronary occlusion, (4) the quantity of blood supplied by collateral vessels to the affected tissue, (5) the demand for oxygen of the myocardium whose blood supply has been suddenly limited, (6) endogenous factors that can produce early spontaneous lysis of the occlusive thrombus, and (7) the adequacy of myocardial perfusion in the infarct zone when flow is restored in the occluded epicardial coronary artery.

1	Patients at increased risk for developing STEMI include those with multiple coronary risk factors (Chap. 291e) and those with UA (Chap. 294). Less common underlying medical conditions predisposing patients to STEMI include hypercoagulability, collagen vascular disease, cocaine abuse, and intracardiac thrombi or masses that can produce coronary emboli. There have been major advances in the management of STEMI with recognition that the “chain of survival” involves a highly integrated system starting with prehospital care and extending to early hospital management so as to provide expeditious implementation of a reperfusion strategy. In up to one-half of cases, a precipitating factor appears to be present before STEMI, such as vigorous physical exercise, emotional stress, or a medical or surgical illness. Although STEMI may commence at any time of the day or night, circadian variations have been reported such that clusters are seen in the morning within a few hours of awakening.

1	Pain is the most common presenting complaint in patients with STEMI. The pain is deep and visceral; adjectives commonly used to 1600 describe it are heavy, squeezing, and crushing, although, occasionally, it is described as stabbing or burning (Chap. 19). It is similar in character to the discomfort of angina pectoris (Chap. 293) but commonly occurs at rest, is usually more severe, and lasts longer. Typically, the pain involves the central portion of the chest and/or the epigastrium, and, on occasion, it radiates to the arms. Less common sites of radiation include the abdomen, back, lower jaw, and neck. The frequent location of the pain beneath the xiphoid and epigastrium and the patients’ denial that they may be suffering a heart attack are chiefly responsible for the common mistaken impression of indigestion. The pain of STEMI may radiate as high as the occipital area but not below the umbilicus. It is often accompanied by weakness, sweating, nausea, vomiting, anxiety, and a sense

1	of indigestion. The pain of STEMI may radiate as high as the occipital area but not below the umbilicus. It is often accompanied by weakness, sweating, nausea, vomiting, anxiety, and a sense of impending doom. The pain may commence when the patient is at rest, but when it begins during a period of exertion, it does not usually subside with cessation of activity, in contrast to angina pectoris. The pain of STEMI can simulate pain from acute pericarditis (Chap. 288), pulmonary embolism (Chap. 300), acute aortic dissection (Chap. 301), costochondritis, and gastrointestinal disorders. These conditions should therefore be considered in the differential diagnosis. Radiation of discomfort to the trapezius is not seen in patients with STEMI and may be a useful distinguishing feature that suggests pericarditis is the correct diagnosis. However, pain is not uniformly present in patients with STEMI. The proportion of painless STEMIs is greater in patients with diabetes mellitus, and it increases

1	pericarditis is the correct diagnosis. However, pain is not uniformly present in patients with STEMI. The proportion of painless STEMIs is greater in patients with diabetes mellitus, and it increases with age. In the elderly, STEMI may present as sudden-onset breathlessness, which may progress to pulmonary edema. Other less common presentations, with or without pain, include sudden loss of consciousness, a confusional state, a sensation of profound weakness, the appearance of an arrhythmia, evidence of peripheral embolism, or merely an unexplained drop in arterial pressure.

1	Most patients are anxious and restless, attempting unsuccessfully to relieve the pain by moving about in bed, altering their position, and stretching. Pallor associated with perspiration and coolness of the extremities occurs commonly. The combination of substernal chest pain persisting for >30 min and diaphoresis strongly suggests STEMI. Although many patients have a normal pulse rate and blood pressure within the first hour of STEMI, about one-fourth of patients with anterior infarction have manifestations of sympathetic nervous system hyperactivity (tachycardia and/or hypertension), and up to one-half with inferior infarction show evidence of parasympathetic hyperactivity (bradycardia and/or hypotension).

1	The precordium is usually quiet, and the apical impulse may be difficult to palpate. In patients with anterior wall infarction, an abnormal systolic pulsation caused by dyskinetic bulging of infarcted myocardium may develop in the periapical area within the first days of the illness and then may resolve. Other physical signs of ventricular dysfunction include fourth and third heart sounds, decreased intensity of the first heart sound, and paradoxical splitting of the second heart sound (Chap. 267). A transient midsystolic or late systolic apical systolic murmur due to dysfunction of the mitral valve apparatus may be present. A pericardial friction rub may be heard in patients with transmural STEMI at some time in the course of the disease, if they are examined frequently. The carotid pulse is often decreased in volume, reflecting reduced stroke volume. Temperature elevations up to 38°C may be observed during the first week after STEMI. The arterial pressure is variable; in most

1	pulse is often decreased in volume, reflecting reduced stroke volume. Temperature elevations up to 38°C may be observed during the first week after STEMI. The arterial pressure is variable; in most patients with transmural infarction, systolic pressure declines by approximately 10–15 mmHg from the preinfarction state.

1	STEMI progresses through the following temporal stages: (1) acute (first few hours–7 days), (2) healing (7–28 days), and (3) healed (≥29 days). When evaluating the results of diagnostic tests for STEMI, the temporal phase of the infarction must be considered. The laboratory tests of value in confirming the diagnosis may be divided into four groups: (1) ECG, (2) serum cardiac biomarkers, (3) cardiac imaging, and (4) nonspecific indices of tissue necrosis and inflammation.

1	The electrocardiographic manifestations of STEMI are described in Chap. 268. During the initial stage, total occlusion of an epicardial coronary artery produces ST-segment elevation. Most patients initially presenting with ST-segment elevation ultimately evolve Q waves on the ECG. However, Q waves in the leads overlying the infarct zone may vary in magnitude and even appear only transiently, depending on the reperfusion status of the ischemic myocardium and restoration of transmembrane potentials over time. A small proportion of patients initially presenting with ST-segment elevation will not develop Q waves when the obstructing thrombus is not totally occlusive, obstruction is transient, or if a rich collateral network is present. Among patients presenting with ischemic discomfort but without ST-segment elevation, if a serum cardiac biomarker of necrosis (see below) is detected, the diagnosis of NSTEMI is ultimately made (Fig. 295-1). A minority of patients who present initially

1	without ST-segment elevation, if a serum cardiac biomarker of necrosis (see below) is detected, the diagnosis of NSTEMI is ultimately made (Fig. 295-1). A minority of patients who present initially without ST-segment elevation may develop a Q-wave MI. Previously, it was believed that transmural myocardial infarction (MI) is present if the ECG demonstrates Q waves or loss of R waves, and nontransmural MI may be present if the ECG shows only transient ST-segment and T-wave changes. However, electrocardiographic-pathologic correlations are far from perfect and terms such as Q-wave MI, non-Q-wave MI, transmural MI, and nontransmural MI, have been replaced by STEMI and NSTEMI (Fig. 295-1). Contemporary studies using magnetic resonance imaging (MRI) suggest that the development of a Q wave on the ECG is more dependent on the volume of infarcted tissue rather than the transmurality of infarction.

1	Certain proteins, called serum cardiac biomarkers, are released from necrotic heart muscle after STEMI. The rate of liberation of specific proteins differs depending on their intracellular location, their molecular weight, and the local blood and lymphatic flow. Cardiac biomarkers become detectable in the peripheral blood once the capacity of the cardiac lymphatics to clear the interstitium of the infarct zone is exceeded and spillover into the venous circulation occurs. The temporal pattern of protein release is of diagnostic importance. The criteria for AMI require a rise and/or fall in cardiac biomarker values with at least one value above the 99th percentile of the upper reference limit for normal individuals

1	Cardiac-specific troponin T (cTnT) and cardiac-specific troponin I (cTnI) have amino-acid sequences different from those of the skeletal muscle forms of these proteins. These differences permitted the development of quantitative assays for cTnT and cTnI with highly specific monoclonal antibodies. Since cTnT and cTnI are not normally detectable in the blood of healthy individuals but may increase after STEMI to levels many times higher than the upper reference limit (the highest value seen in 99% of a reference population not suffering from MI), the measurement of cTnT or cTnI is of considerable diagnostic usefulness, and they are now the preferred biochemical markers for MI (Fig. 295-2). With improvements in the assays for the cardiac-specific troponins, it is now possible to detect concentrations <1 ng/L in patients without ischemic-type chest discomfort. The cardiac troponins are particularly valuable when there is clinical suspicion of either skeletal muscle injury or a small MI

1	<1 ng/L in patients without ischemic-type chest discomfort. The cardiac troponins are particularly valuable when there is clinical suspicion of either skeletal muscle injury or a small MI that may be below the detection limit for creatine phosphokinase (CK) and its MB isoenzyme (CK-MB) measurements, and they are, therefore, of particular value in distinguishing UA from NSTEMI. In practical terms, the high-sensitivity troponin assays are of less immediate value in patients with STEMI. Contemporary urgent reperfusion strategies necessitate making a decision (based largely on a combination of clinical and ECG findings) before the results of blood tests have returned from the laboratory. Levels of cTnI and cTnT may remain elevated for 7–10 days after STEMI.

1	CK rises within 4–8 h and generally returns to normal by 48–72 h (Fig. 295-2). An important drawback of total CK measurement is its lack of specificity for STEMI, as CK may be elevated with skeletal muscle disease or trauma, including intramuscular injection. The MB isoenzyme of CK has the advantage over total CK that it is not present in significant concentrations in extracardiac tissue and, therefore, Multiples of the AMI cutoff limit Zone of necrosing myocardium Actin Troponin complex bound to actin filament Lymphatic system Venous system Days after onset of AMI

1	FIGURE 295-2 The zone of necrosing myocardium is shown at the top of the figure, followed in the middle portion of the figure by a diagram of a cardiomyocyte that is in the process of releasing biomarkers. The biomarkers that are released into the interstitium are first cleared by lymphatics followed subsequently by spillover into the venous system. After disruption of the sarcolemmal membrane of the cardiomyocyte, the cytoplasmic pool of biomarkers is released first (left-most arrow in bottom portion of figure). Markers such as myoglobin and CK isoforms are rapidly released, and blood levels rise quickly above the cutoff limit; this is then followed by a more protracted release of biomarkers from the disintegrating myofilaments that may continue for several days. Cardiac troponin levels rise to about 20 to 50 times the upper reference limit (the 99th percentile of values in a reference control group) in patients who have a “classic” acute myocardial infarction (MI) and sustain

1	rise to about 20 to 50 times the upper reference limit (the 99th percentile of values in a reference control group) in patients who have a “classic” acute myocardial infarction (MI) and sustain sufficient myocardial necrosis to result in abnormally elevated levels of the MB fraction of creatine kinase (CK-MB). Clinicians can now diagnose episodes of microinfarction by sensitive assays that detect cardiac troponin elevations above the upper reference limit, even though CK-MB levels may still be in the normal reference range (not shown). CV, coefficient of variation.

1	(Modified from EM Antman: Decision making with cardiac troponin tests. N Engl J Med 346:2079, 2002 and AS Jaffe, L Babiun, FS Apple: Biomarkers in acute cardiac disease: The present and the future. J Am Coll Cardiol 48:1, 2006.) is considerably more specific. However, cardiac surgery, myocarditis, 1601 and electrical cardioversion often result in elevated serum levels of the MB isoenzyme. A ratio (relative index) of CK-MB mass to CK activity ≥2.5 suggests but is not diagnostic of a myocardial rather than a skeletal muscle source for the CK-MB elevation. Many hospitals are using cTnT or cTnI rather than CK-MB as the routine serum cardiac marker for diagnosis of STEMI, although any of these analytes remain clinically acceptable. It is not cost-effective to measure both a cardiac-specific troponin and CK-MB at all time points in every patient.

1	While it has long been recognized that the total quantity of protein released correlates with the size of the infarct, the peak protein concentration correlates only weakly with infarct size. Recanalization of a coronary artery occlusion (either spontaneously or by mechanical or pharmacologic means) in the early hours of STEMI causes earlier peaking of biomarker measurements (Fig. 295-2) because of a rapid washout from the interstitium of the infarct zone, quickly overwhelming lymphatic clearance of the proteins. The nonspecific reaction to myocardial injury is associated with polymorphonuclear leukocytosis, which appears within a few hours after the onset of pain and persists for 3–7 days; the white blood cell count often reaches levels of 12,000–15,000/μL. The erythrocyte sedimentation rate rises more slowly than the white blood cell count, peaking during the first week and sometimes remaining elevated for 1 or 2 weeks.

1	Abnormalities of wall motion on two-dimensional echocardiography (Chap. 270e) are almost universally present. Although acute STEMI cannot be distinguished from an old myocardial scar or from acute severe ischemia by echocardiography, the ease and safety of the procedure make its use appealing as a screening tool in the Emergency Department setting. When the ECG is not diagnostic of STEMI, early detection of the presence or absence of wall motion abnormalities by echocardiography can aid in management decisions, such as whether the patient should receive reperfusion therapy (e.g., fibrinolysis or a percutaneous coronary intervention [PCI]). Echocardiographic estimation of left ventricular (LV) function is useful prognostically; detection of reduced function serves as an indication for therapy with an inhibitor of the renin-angiotensin-aldosterone system. Echocardiography may also identify the presence of right ventricular (RV) infarction, ventricular aneurysm, pericardial effusion, and

1	with an inhibitor of the renin-angiotensin-aldosterone system. Echocardiography may also identify the presence of right ventricular (RV) infarction, ventricular aneurysm, pericardial effusion, and LV thrombus. In addition, Doppler echocardiography is useful in the detection and quantitation of a ventricular septal defect and mitral regurgitation, two serious complications of STEMI.

1	Several radionuclide imaging techniques (Chap. 270e) are available for evaluating patients with suspected STEMI. However, these imaging modalities are used less often than echocardiography because they are more cumbersome and lack sensitivity and specificity in many clinical circumstances. Myocardial perfusion imaging with [201Tl] or [99mTc]-sestamibi, which are distributed in proportion to myocardial blood flow and concentrated by viable myocardium (Chap. 293), reveals a defect (“cold spot”) in most patients during the first few hours after development of a transmural infarct. Although perfusion scanning is extremely sensitive, it cannot distinguish acute infarcts from chronic scars and, thus, is not specific for the diagnosis of acute MI. Radionuclide ventriculography, carried out with [99mTc]-labeled red blood cells, frequently demonstrates wall motion disorders and reduction in the ventricular ejection fraction in patients with STEMI. While of value in assessing the hemodynamic

1	[99mTc]-labeled red blood cells, frequently demonstrates wall motion disorders and reduction in the ventricular ejection fraction in patients with STEMI. While of value in assessing the hemodynamic consequences of infarction and in aiding in the diagnosis of RV infarction when the RV ejection fraction is depressed, this technique is nonspecific, as many cardiac abnormalities other than MI alter the radionuclide ventriculogram.

1	MI can be detected accurately with high-resolution cardiac MRI (Chap. 270e) using a technique referred to as late enhancement. A standard imaging agent (gadolinium) is administered and images are obtained after a 10-min delay. Since little gadolinium enters normal myocardium, where there are tightly packed myocytes, but does percolate into the expanded intercellular region of the infarct zone, there

1	The term acute myocardial infarction (MI) should be used when there is evidence of myocardial necrosis in a clinical setting consistent with acute myocardial ischemia. Under these conditions, any one of the following criteria meets the diagnosis for MI: • Detection of a rise and/or fall of cardiac biomarker values (preferably cardiac troponin [cTn]) with at least one value above the 99th percentile upper reference limit (URL) and with at least one of the following: of pathologic Q waves in the electrocardiogram (ECG) evidence of new loss of viable myocardium or new regional wall death with symptoms suggestive of myocardial ischemia and presumed new ischemic ECG changes of new LBBB, but death occurred before cardiac biomarkers were obtained or before cardiac biomarker values would be increased.

1	• Percutaneous coronary intervention (PCI)–related MI is arbitrarily defined by elevation of cTn values (>5 × 99th percentile URL) in patients with normal baseline values (≤99th percentile URL) or a rise of cTn values >20% if the baseline values are elevated and are stable or falling. In addition, either (i) symptoms suggestive of myocardial ischemia, or (ii) new ischemic ECG changes, or (iii) angiographic findings consistent with a procedural complication, or (iv) imaging demonstration of new loss of viable myocardium or new regional wall motion abnormality are required. thrombosis associated with MI when detected by coronary angiography or autopsy in the setting of myocardial ischemia and with a rise and/ or fall of cardiac biomarker values with at least one value above the 99th percentile URL.

1	artery bypass grafting (CABG)–related MI is arbitrarily defined by elevation of cardiac biomarker values (>10 × 99th percentile URL) in patients with normal baseline cTn values (≤99th percentile URL). In addition, either (i) new pathologic Q waves or new LBBB, or (ii) angiographic documented new graft or new native coronary artery occlusion, or (iii) imaging evidence of new loss of viable myocardium or new regional wall motion abnormality. Any one of the following criteria meets the diagnosis for prior MI: Q waves with or without symptoms in the absence of nonischemic causes. evidence of a region of loss of viable myocardium that is thinned and fails to contract, in the absence of a nonischemic cause. • Pathologic findings of a prior MI. Source: K Thygesen: Eur Heart J 33:2551, 2012. is a bright signal in areas of infarction that appears in stark contrast to the dark areas of normal myocardium.

1	• Pathologic findings of a prior MI. Source: K Thygesen: Eur Heart J 33:2551, 2012. is a bright signal in areas of infarction that appears in stark contrast to the dark areas of normal myocardium. An Expert Consensus Task Force for the Universal Definition of Myocardial Infarction has provided a comprehensive set of criteria for the definition of MI that integrates the clinical and laboratory findings discussed earlier (Table 295-1) as well as a classification of MI into five types that reflect the clinical circumstances in which it may occur (Table 295-2).

1	The prognosis in STEMI is largely related to the occurrence of two general classes of complications: (1) electrical complications (arrhythmias) and (2) mechanical complications (“pump failure”). Most outof-hospital deaths from STEMI are due to the sudden development of ventricular fibrillation. The vast majority of deaths due to ventricular fibrillation occur within the first 24 h of the onset of symptoms, and of these, over half occur in the first hour. Therefore, the major elements of prehospital care of patients with suspected STEMI include (1) recognition of symptoms by the patient and prompt seeking of medical attention; (2) rapid deployment of an emergency medical team capable Type I: Spontaneous Myocardial Infarction

1	Type I: Spontaneous Myocardial Infarction Spontaneous myocardial infarction related to atherosclerotic plaque rupture, ulceration, fissuring, erosion, or dissection with resulting intraluminal thrombus in one or more of the coronary arteries leading to decreased myocardial blood flow or distal platelet emboli with ensuing myocyte necrosis. The patient may have underlying severe coronary artery disease (CAD) but on occasion nonobstructive or no CAD. Type 2: Myocardial Infarction Secondary to an Ischemic Imbalance In instances of myocardial injury with necrosis where a condition other than CAD contributes to an imbalance between myocardial oxygen supply and/ or demand, e.g., coronary endothelial dysfunction, coronary artery spasm, coronary embolism, tachy-brady-arrhythmias, anemia, respiratory failure, hypotension, and hypertension with or without left ventricular hypertrophy. Type 3: Myocardial Infarction Resulting in Death When Biomarker Values Are Unavailable

1	Type 3: Myocardial Infarction Resulting in Death When Biomarker Values Are Unavailable Cardiac death with symptoms suggestive of myocardial ischemia and presumed new ischemic electrocardiogram (ECG) changes or new left bundle branch block (LBBB), but death occurring before blood samples could be obtained or before cardiac biomarker could rise, or in rare cases, cardiac biomarkers were not collected. Type 4a: Myocardial Infarction Related to Percutaneous Coronary Intervention (PCI)

1	Type 4a: Myocardial Infarction Related to Percutaneous Coronary Intervention (PCI) Myocardial infarction associated with PCI is arbitrarily defined by elevation of cardiac troponin (cTn) values >5 × 99th percentile upper reference limit (URL) in patients with normal baseline values (≤99th percentile URL) or a rise of cTn values >20% if the baseline values are elevated and are stable or falling. In addition, either (i) symptoms suggestive of myocardial ischemia, or (ii) new ischemic ECG changes or new LBBB, or (iii) angiographic loss of patency of a major coronary artery or a side branch or persistent slow or no flow or embolization, or (iv) imaging demonstration of new loss of viable myocardium or new regional wall motion abnormality is required. Type 4b: Myocardial Infarction Related to Stent Thrombosis

1	Type 4b: Myocardial Infarction Related to Stent Thrombosis Myocardial infarction associated with stent thrombosis is detected by coronary angiography or autopsy in the setting of myocardial ischemia and with a rise and/or fall of cardiac biomarker values with at least one value above the 99th percentile URL. Type 5: Myocardial Infarction Related to Coronary Artery Bypass Grafting (CABG) Myocardial infarction associated with CABG is arbitrarily defined by elevation of cardiac biomarker values >10 × 99th percentile URL in patients with normal baseline cTn values (≤99th percentile URL). In addition, either (i) new pathologic Q waves or new LBBB, or (ii) angiographic documented new graft or new native coronary artery occlusion, or (iii) imaging evidence of new loss of viable myocardium or new regional wall motion abnormality. Source: K Thygesen: Eur Heart J 33:2551, 2012.

1	Source: K Thygesen: Eur Heart J 33:2551, 2012. of performing resuscitative maneuvers, including defibrillation; (3) expeditious transportation of the patient to a hospital facility that is continuously staffed by physicians and nurses skilled in managing arrhythmias and providing advanced cardiac life support; and (4) expeditious implementation of reperfusion therapy (Fig. 295-3). The greatest delay usually occurs not during transportation to the hospital but, rather, between the onset of pain and the patient’s decision to call for help. This delay can best be reduced by health care professionals educating the public concerning the significance of chest discomfort and the importance of seeking early medical attention. Regular office visits with patients having a history of or who are at risk for ischemic heart disease are important “teachable moments” for clinicians to review the symptoms of STEMI and the appropriate action plan.

1	Increasingly, monitoring and treatment are carried out by trained personnel in the ambulance, further shortening the time between the onset of the infarction and appropriate treatment. General guidelines for initiation of fibrinolysis in the prehospital setting include the ability to transmit 12-lead ECGs to confirm the diagnosis, the presence of paramedics in the ambulance, training of paramedics in the interpretation of ECGs and management of STEMI, and online medical command and control that can authorize the initiation of treatment in the field. Methods of Speeding Time to Reperfusion

1	FIGURE 295-3 Major components of time delay between onset of symptoms from ST-segment elevation myocardial infarction and restoration of flow in the infarct-related artery. Plotted sequentially from left to right are the times for patients to recognize symptoms and seek medical attention, transportation to the hospital, in-hospital decision making, implementation of reperfusion strategy, and restoration of flow once the reperfusion strategy has been initiated. The time to initiate fibrinolytic therapy is the “door-to-needle” (D-N) time; this is followed by the period of time required for pharmacologic restoration of flow. More time is required to move the patient to the catheterization laboratory for a percutaneous coronary interventional (PCI) procedure, referred to as the “door-to-balloon” (D-B) time, but restoration of flow in the epicardial infarct–related artery occurs promptly after PCI. At the bottom is a variety of methods for speeding the time to reperfusion along with the

1	(D-B) time, but restoration of flow in the epicardial infarct–related artery occurs promptly after PCI. At the bottom is a variety of methods for speeding the time to reperfusion along with the goals for the time intervals for the various components of the time delay. (Adapted from CP Cannon et al: J Thromb Thrombol 1:27, 1994.)

1	In the Emergency Department, the goals for the management of patients with suspected STEMI include control of cardiac discomfort, rapid identification of patients who are candidates for urgent reperfusion therapy, triage of lower-risk patients to the appropriate location in the hospital, and avoidance of inappropriate discharge of patients with STEMI. Many aspects of the treatment of STEMI are initiated in the Emergency Department and then continued during the in-hospital phase of management (Fig. 295-4). The overarching goal is to minimize the time from first medical contact to initiation of reperfusion therapy. This may involve transfer from a non-PCI hospital to one that is PCI capable, with a goal of initiating PCI within 120 min of first medical contact (Fig. 295-4).

1	Aspirin is essential in the management of patients with suspected STEMI and is effective across the entire spectrum of acute coronary syndromes (Fig. 295-1). Rapid inhibition of cyclooxygenase-1 in platelets followed by a reduction of thromboxane A2 levels is achieved by buccal absorption of a chewed 160–325-mg tablet in the Emergency Department. This measure should be followed by daily oral administration of aspirin in a dose of 75–162 mg. In patients whose arterial O2 saturation is normal, supplemental O2 is of limited if any clinical benefit and therefore is not cost-effective. However, when hypoxemia is present, O2 should be administered by nasal prongs or face mask (2–4 L/min) for the first 6–12 h after infarction; the patient should then be reassessed to determine if there is a continued need for such treatment.

1	Sublingual nitroglycerin can be given safely to most patients with STEMI. Up to three doses of 0.4 mg should be administered at about 5-min intervals. In addition to diminishing or abolishing chest discomfort, nitroglycerin may be capable of both decreasing myocardial oxygen demand (by lowering preload) and increasing myocardial oxygen supply (by dilating infarct-related coronary vessels or collateral vessels). In patients whose initially favorable response to sublingual nitroglycerin is followed by the return of chest discomfort, particularly if accompanied by other evidence of ongoing ischemia such as further ST-segment or T-wave shifts, the use of intravenous nitroglycerin should be considered. Therapy with nitrates should be avoided in patients who present with low systolic arterial pressure (<90 mmHg) or in whom there is clinical suspicion of RV infarction (inferior infarction on ECG, elevated jugular venous pressure, clear lungs, and hypo-tension). Nitrates should not be

1	pressure (<90 mmHg) or in whom there is clinical suspicion of RV infarction (inferior infarction on ECG, elevated jugular venous pressure, clear lungs, and hypo-tension). Nitrates should not be administered to patients who have taken a phosphodiesterase-5 inhibitor for erectile dysfunction within the preceding 24 h, because it may potentiate the hypotensive effects of nitrates. An idiosyncratic reaction to nitrates, consisting of sudden marked hypotension, sometimes occurs but can usually be reversed promptly by the rapid administration of intravenous atropine.

1	Morphine is a very effective analgesic for the pain associated with STEMI. However, it may reduce sympathetically mediated arteriolar and venous constriction, and the resulting venous pooling may reduce cardiac output and arterial pressure. These hemodynamic disturbances usually respond promptly to elevation of the legs, but in some patients, volume expansion with intravenous saline is required. The patient may experience diaphoresis and nausea, but these events usually pass and are replaced by a feeling of well-being associated with the relief of pain. Morphine also has a vagotonic effect and may cause bradycardia or advanced degrees of heart block, particularly in patients with inferior infarction. These side effects usually respond to atropine (0.5 mg intravenously). Morphine is routinely administered by repetitive (every 5 min) intravenous injection of small doses (2–4 mg), rather than by the subcutaneous administration of a larger quantity, because absorption may be unpredictable

1	administered by repetitive (every 5 min) intravenous injection of small doses (2–4 mg), rather than by the subcutaneous administration of a larger quantity, because absorption may be unpredictable by the latter route.

1	Intravenous beta blockers are also useful in the control of the pain of STEMI. These drugs control pain effectively in some patients, presumably by diminishing myocardial O2 demand and hence ischemia. More important, there is evidence that intravenous beta blockers reduce the risks of reinfarction and ventricular fibrillation (see “Beta-Adrenoceptor Blockers” below). However, patient selection is important when considering beta blockers for STEMI. Oral beta blocker therapy should be initiated in the first 24 h for patients who do not have any of the following: (1) signs of heart failure, (2) evidence of a low-output state, (3) increased risk for cardiogenic shock, or (4) other

1	STEMI patient who is a candidate for reperfusion Diagnostic angiogram Medical therapy only PCI CABG Initially seen at a PCI-capable hospital Initially seen at a non-PCI-capable hospital* Send to cath lab for primary PCI FMC-device time ˜90 min (Class I, LOE: A) Transfer for angiography and revascularization within 3–24 h for other patients as part of an invasive strategy† (Class IIa, LOE: B) Transfer for primary PCI FMC-device time as soon as possible and ˜120 min (Class I, LOE: B) Administer fibrinolytic agent within 30 min of arrival when anticipated FMC-device >120 min (Class I, LOE: B) Urgent transfer for PCI for patients with evidence of failed reperfusion or reocclusion (Class IIa, LOE: B) DIDO time ˜30 min

1	FIGURE 295-4 Reperfusion therapy for patients with ST-segment elevation myocardial infarction (STEMI). The bold arrows and boxes are the preferred strategies. Performance of percutaneous coronary intervention (PCI) is dictated by an anatomically appropriate culprit stenosis. *Patients with cardiogenic shock or severe heart failure initially seen at a non–PCI-capable hospital should be transferred for cardiac catheterization and revascularization as soon as possible, irrespective of time delay from myocardial infarction (MI) onset (Class I, LOE: B). †Angiography and revascularization should not be performed within the first 2 to 3 hours after administration of fibrinolytic therapy. CABG, coronary artery bypass graft; DIDO, door-in–door-out; FMC, first medical contact; LOE, level of evidence; STEMI, ST-elevation myocardial infarction. (Adapted with permission from P O’Gara et al: Circulation 127:e362, 2013.) relative contraindications to beta blockade (PR interval greater than 0.24

1	STEMI, ST-elevation myocardial infarction. (Adapted with permission from P O’Gara et al: Circulation 127:e362, 2013.) relative contraindications to beta blockade (PR interval greater than 0.24 seconds, secondor third-degree heart block, active asthma, or reactive airway disease). A commonly employed regimen is metoprolol, 5 mg every 2–5 min for a total of three doses, provided the patient has a heart rate >60 beats/min, systolic pressure >100 mmHg, a PR interval <0.24 s, and rales that are no higher than 10 cm up from the diaphragm. Fifteen minutes after the last intravenous dose, an oral regimen is initiated of 50 mg every 6 h for 48 h, followed by 100 mg every 12 h.

1	Unlike beta blockers, calcium antagonists are of little value in the acute setting, and there is evidence that short-acting dihydropyridines may be associated with an increased mortality risk. The primary tool for screening patients and making triage decisions is the initial 12-lead ECG. When ST-segment elevation of at least 2 mm in two contiguous precordial leads and 1 mm in two adjacent limb leads is present, a patient should be considered a candidate for reperfusion therapy (Fig. 295-4). The process of selecting patients for fibrinolysis versus primary PCI (angioplasty or stenting; Chap. 296e) is discussed below. In the absence of ST-segment elevation, fibrinolysis is not helpful, and evidence exists suggesting that it may be harmful.

1	The quantity of myocardium that becomes necrotic as a consequence of a coronary artery occlusion is determined by factors other than just the site of occlusion. While the central zone of the infarct contains necrotic tissue that is irretrievably lost, the fate of the surrounding ischemic myocardium (ischemic penumbra) may be improved by timely restoration of coronary perfusion, reduction of myocardial O2 demands, prevention of the accumulation of noxious metabolites, and blunting of the impact of mediators of reperfusion injury (e.g., calcium overload and oxygen-derived free radicals). Up to one-third of patients with STEMI may achieve spontaneous reperfusion of the infarct-related coronary artery within 24 h and experience improved healing of infarcted tissue. Reperfusion, either pharmacologically (by fibrinolysis) or by PCI, accelerates the opening of infarct-related arteries in those patients in whom spontaneous fibrinolysis ultimately would have occurred and also greatly increases

1	(by fibrinolysis) or by PCI, accelerates the opening of infarct-related arteries in those patients in whom spontaneous fibrinolysis ultimately would have occurred and also greatly increases the number of patients in whom restoration of flow in the infarct-related artery is accomplished. Timely restoration of flow in the epicardial infarct–related artery combined with improved perfusion of the downstream zone of infarcted myocardium results in a limitation of infarct size. Protection of the ischemic myocardium by the maintenance of an optimal balance between myocardial O2 supply and demand through pain control, treatment of congestive heart failure (CHF), and minimization of tachycardia and hypertension extends the “window” of time for the salvage of myocardium by reperfusion strategies.

1	Glucocorticoids and nonsteroidal anti-inflammatory agents, with the exception of aspirin, should be avoided in patients with STEMI. They can impair infarct healing and increase the risk of myocardial rupture, and their use may result in a larger infarct scar. In addition, they can increase coronary vascular resistance, thereby potentially reducing flow to ischemic myocardium.

1	(See also Chap. 296e) PCI, usually angioplasty and/or stenting without preceding fibrinolysis, referred to as primary PCI, is effective in restoring perfusion in STEMI when carried out on an emergency basis in the first few hours of MI. It has the advantage of being applicable to patients who have contraindications to fibrinolytic therapy (see below) but otherwise are considered appropriate candidates for reperfusion. It appears to be more effective than fibrinolysis in opening occluded coronary arteries and, when performed by experienced operators in dedicated medical centers, is associated with better short-term and longterm clinical outcomes. Compared with fibrinolysis, primary PCI is generally preferred when the diagnosis is in doubt, cardiogenic shock is present, bleeding risk is increased, or symptoms have been present for at least 2–3 h when the clot is more mature and less easily lysed by fibrinolytic drugs. However, PCI is expensive in terms of personnel and facilities, and

1	or symptoms have been present for at least 2–3 h when the clot is more mature and less easily lysed by fibrinolytic drugs. However, PCI is expensive in terms of personnel and facilities, and its applicability is limited by its availability, around the clock, in only a minority of hospitals (Fig. 295-4).

1	If no contraindications are present (see below), fibrinolytic therapy should ideally be initiated within 30 min of presentation (i.e., doorto-needle time ≤30 min). The principal goal of fibrinolysis is prompt restoration of full coronary arterial patency. The fibrinolytic agents tissue plasminogen activator (tPA), streptokinase, tenecteplase (TNK), and reteplase (rPA) have been approved by the U.S. Food and Drug Administration for intravenous use in patients with STEMI. These drugs all act by promoting the conversion of plasminogen to plasmin, which subsequently lyses fibrin thrombi. Although considerable emphasis was first placed on a distinction between more fibrin-specific agents, such as tPA, and non-fibrin-specific agents, such as streptokinase, it is now recognized that these differences are only relative, as some degree of systemic fibrinolysis occurs with the former agents. TNK and rPA are referred to as bolus fibrinolytics since their administration does not require a

1	differences are only relative, as some degree of systemic fibrinolysis occurs with the former agents. TNK and rPA are referred to as bolus fibrinolytics since their administration does not require a prolonged intravenous infusion.

1	When assessed angiographically, flow in the culprit coronary artery is described by a simple qualitative scale called the Thrombolysis in Myocardial Infarction (TIMI) grading system: grade 0 indicates complete occlusion of the infarct-related artery; grade 1 indicates some penetration of the contrast material beyond the point of obstruction but without perfusion of the distal coronary bed; grade 2 indicates perfusion of the entire infarct vessel into the distal bed, but with flow that is delayed compared with that of a normal artery; and grade 3 indicates full perfusion of the infarct vessel with normal flow. The latter is the goal of reperfusion therapy, because full perfusion of the infarct-related coronary artery yields far better results in terms of limiting infarct size, maintenance of LV function, and reduction of both shortand long-term mortality rates. Additional methods of angiographic assessment of the efficacy of fibrinolysis include counting the number of frames on the

1	of LV function, and reduction of both shortand long-term mortality rates. Additional methods of angiographic assessment of the efficacy of fibrinolysis include counting the number of frames on the cine film required for dye to flow from the origin of the infarct-related artery to a landmark in the distal vascular bed (TIMI frame count) and determining the rate of entry and exit of contrast dye from the microvasculature in the myocardial infarct zone (TIMI myocardial perfusion grade). These methods have an even tighter correlation with outcomes after STEMI than the more commonly employed TIMI flow grade.

1	tPA and the other relatively fibrin-specific plasminogen activators, rPA and TNK, are more effective than streptokinase at restoring full perfusion—i.e., TIMI grade 3 coronary flow—and have a small edge in improving survival as well. The current recommended regimen of tPA consists of a 15-mg bolus followed by 50 mg intravenously over the first 30 min, followed by 35 mg over the next 60 min. Streptokinase is administered as 1.5 million units (MU) intravenously over 1 h. rPA is administered in a double-bolus regimen consisting of a 10-MU bolus given over 2–3 min, followed by a second 10-MU bolus 30 min later. TNK is given as a single weight-based intravenous bolus of 0.53 mg/ kg over 10 s. In addition to the fibrinolytic agents discussed earlier, pharmacologic reperfusion typically involves adjunctive antiplatelet and antithrombotic drugs, as discussed subsequently.

1	Clear contraindications to the use of fibrinolytic agents include a history of cerebrovascular hemorrhage at any time, a nonhemorrhagic stroke or other cerebrovascular event within the past year, marked hypertension (a reliably determined systolic arterial pressure >180 mmHg and/or a diastolic pressure >110 mmHg) at any time during the acute presentation, suspicion of aortic dissection, and active internal bleeding (excluding menses). While advanced age is associated with an increase in hemorrhagic complications, the benefit of fibrinolytic 1605 therapy in the elderly appears to justify its use if no other contraindications are present and the amount of myocardium in jeopardy appears to be substantial.

1	Relative contraindications to fibrinolytic therapy, which require assessment of the risk-to-benefit ratio, include current use of anticoagulants (international normalized ratio ≥2), a recent (<2 weeks) invasive or surgical procedure or prolonged (>10 min) cardiopulmonary resuscitation, known bleeding diathesis, pregnancy, a hemorrhagic ophthalmic condition (e.g., hemorrhagic diabetic retinopathy), active peptic ulcer disease, and a history of severe hypertension that is currently adequately controlled. Because of the risk of an allergic reaction, patients should not receive streptokinase if that agent had been received within the preceding 5 days to 2 years. Allergic reactions to streptokinase occur in ~2% of patients who receive it. While a minor degree of hypotension occurs in 4–10% of patients given this agent, marked hypotension occurs, although rarely, in association with severe allergic reactions.

1	Hemorrhage is the most frequent and potentially the most serious complication. Because bleeding episodes that require transfusion are more common when patients require invasive procedures, unnecessary venous or arterial interventions should be avoided in patients receiving fibrinolytic agents. Hemorrhagic stroke is the most serious complication and occurs in ~0.5–0.9% of patients being treated with these agents. This rate increases with advancing age, with patients >70 years experiencing roughly twice the rate of intracranial hemorrhage as those <65 years. Large-scale trials have suggested that the rate of intracranial hemorrhage with tPA or rPA is slightly higher than with streptokinase.

1	Evidence has emerged that suggests PCI plays an increasingly important role in the management of STEMI. Prior approaches that segregated the pharmacologic and catheter-based approaches to reperfusion have now been replaced with an integrated approach to triage and transfer of STEMI patients to receive PCI (Fig. 295-4). To achieve the degree of integration required to care for a patient with STEMI, all communities should create and maintain a regional system of STEMI care that includes assessment and continuous quality improvement of emergency medical services and hospital-based activities.

1	Cardiac catheterization and coronary angiography should be carried out after fibrinolytic therapy if there is evidence of either (1) failure of reperfusion (persistent chest pain and ST-segment elevation >90 min), in which case a rescue PCI should be considered; or (2) coronary artery reocclusion (re-elevation of ST segments and/or recurrent chest pain) or the development of recurrent ischemia (such as recurrent angina in the early hospital course or a positive exercise stress test before discharge), in which case an urgent PCI should be considered. Routine angiography and elective PCI even in asymptomatic patients following administration of fibrinolytic therapy are used with less frequency, given the numerous technologic advances that have occurred in the catheterization laboratory and the increasing number of skilled interventionalists. Coronary artery bypass surgery should be reserved for patients whose coronary anatomy is unsuited to PCI but in whom revascularization appears to

1	the increasing number of skilled interventionalists. Coronary artery bypass surgery should be reserved for patients whose coronary anatomy is unsuited to PCI but in whom revascularization appears to be advisable because of extensive jeopardized myocardium or recurrent ischemia.

1	These units are routinely equipped with a system that permits continuous monitoring of the cardiac rhythm of each patient and hemodynamic monitoring in selected patients. Defibrillators, respirators, noninvasive transthoracic pacemakers, and facilities for introducing pacing catheters and flow-directed balloon-tipped catheters are also usually available. Equally important is the organization of a highly trained team of nurses who can recognize arrhythmias; adjust the dosage of antiarrhythmic, vasoactive, and anticoagulant drugs; and perform cardiac resuscitation, including electroshock, when necessary.

1	1606 Patients should be admitted to a coronary care unit early in their illness when it is expected that they will derive benefit from the sophisticated and expensive care provided. The availability of electrocardiographic monitoring and trained personnel outside the coronary care unit has made it possible to admit lower-risk patients (e.g., those not hemodynamically compromised and without active arrhythmias) to “intermediate care units.” The duration of stay in the coronary care unit is dictated by the ongoing need for intensive care. If symptoms are controlled with oral therapy, patients may be transferred out of the coronary care unit. Also, patients who have a confirmed STEMI but who are considered to be at low risk (no prior infarction and no persistent chest discomfort, CHF, hypotension, or cardiac arrhythmias) may be safely transferred out of the coronary care unit within 24 h.

1	Activity Factors that increase the work of the heart during the initial hours of infarction may increase the size of the infarct. Therefore, patients with STEMI should be kept at bed rest for the first 6–12 h. However, in the absence of complications, patients should be encouraged, under supervision, to resume an upright posture by dangling their feet over the side of the bed and sitting in a chair within the first 24 h. This practice is psychologically beneficial and usually results in a reduction in the pulmonary capillary wedge pressure. In the absence of hypotension and other complications, by the second or third day, patients typically are ambulating in their room with increasing duration and frequency, and they may shower or stand at the sink to bathe. By day 3 after infarction, patients should be increasing their ambulation progressively to a goal of 185 m (600 ft) at least three times a day.

1	Diet Because of the risk of emesis and aspiration soon after STEMI, patients should receive either nothing or only clear liquids by mouth for the first 4–12 h. The typical coronary care unit diet should provide ≤30% of total calories as fat and have a cholesterol content of ≤300 mg/d. Complex carbohydrates should make up 50–55% of total calories. Portions should not be unusually large, and the menu should be enriched with foods that are high in potassium, magnesium, and fiber, but low in sodium. Diabetes mellitus and hypertriglyceridemia are managed by restriction of concentrated sweets in the diet.

1	Bowel Management Bed rest and the effect of the narcotics used for the relief of pain often lead to constipation. A bedside commode rather than a bedpan, a diet rich in bulk, and the routine use of a stool softener such as dioctyl sodium sulfosuccinate (200 mg/d) are recommended. If the patient remains constipated despite these measures, a laxative can be prescribed. Contrary to prior belief, it is safe to perform a gentle rectal examination on patients with STEMI.

1	Sedation Many patients require sedation during hospitalization to withstand the period of enforced inactivity with tranquility. Diazepam (5 mg), oxazepam (15–30 mg), or lorazepam (0.5–2 mg), given three to four times daily, is usually effective. An additional dose of any of the above medications may be given at night to ensure adequate sleep. Attention to this problem is especially important during the first few days in the coronary care unit, where the atmosphere of 24-h vigilance may interfere with the patient’s sleep. However, sedation is no substitute for reassuring, quiet surroundings. Many drugs used in the coronary care unit, such as atropine, H2 blockers, and narcotics, can produce delirium, particularly in the elderly. This effect should not be confused with agitation, and it is wise to conduct a thorough review of the patient’s medications before arbitrarily prescribing additional doses of anxiolytics.

1	The use of antiplatelet and anticoagulant therapy during the initial phase of STEMI is based on extensive laboratory and clinical evidence that thrombosis plays an important role in the pathogenesis of this condition. The primary goal of treatment with antiplatelet and anticoagulant agents is to maintain patency of the infarct-related artery, in conjunction with reperfusion strategies. A secondary goal is to reduce the patient’s tendency to thrombosis and, thus, the likelihood of mural thrombus formation or deep venous thrombosis, either of which could result in pulmonary embolization. The degree to which antiplatelet and anticoagulant therapy achieves these goals partly determines how effectively it reduces the risk of mortality from STEMI.

1	As noted previously (see “Management in the Emergency Department” earlier), aspirin is the standard antiplatelet agent for patients with STEMI. The most compelling evidence for the benefits of antiplatelet therapy (mainly with aspirin) in STEMI is found in the comprehensive overview by the Antiplatelet Trialists’ Collaboration. Data from nearly 20,000 patients with MI enrolled in 15 randomized trials were pooled and revealed a relative reduction of 27% in the mortality rate, from 14.2% in control patients to 10.4% in patients receiving antiplatelet agents.

1	Inhibitors of the P2Y12 ADP receptor prevent activation and aggregation of platelets. The addition of the P2Y12 inhibitor clopidogrel to background treatment with aspirin to STEMI patients reduces the risk of clinical events (death, reinfarction, stroke) and, in patients receiving fibrinolytic therapy, has been shown to prevent reocclusion of a successfully reperfused infarct artery. New P2Y12 ADP receptor antagonists, such as prasugrel and ticagrelor, are more effective than clopidogrel in preventing ischemic complications in STEMI patients undergoing PCI, but are associated with an increased risk of bleeding. Glycoprotein IIb/IIIa receptor inhibitors appear useful for preventing thrombotic complications in patients with STEMI undergoing PCI.

1	The standard anticoagulant agent used in clinical practice is unfractionated heparin (UFH). The available data suggest that when UFH is added to a regimen of aspirin and a non-fibrin-specific thrombolytic agent such as streptokinase, additional mortality benefit occurs (about 5 lives saved per 1000 patients treated). It appears that the immediate administration of intravenous UFH, in addition to a regimen of aspirin and relatively fibrin-specific fibrinolytic agents (tPA, rPA, or TNK), helps to maintain patency of the infarct-related artery. This effect is achieved at the cost of a small increased risk of bleeding. The recommended dose of UFH is an initial bolus of 60 U/kg (maximum 4000 U) followed by an initial infusion of 12 U/kg per hour (maximum 1000 U/h). The activated partial thromboplastin time during maintenance therapy should be 1.5–2 times the control value.

1	Alternatives to UFH for anticoagulation of patients with STEMI are the low-molecular-weight heparin (LMWH) preparations, a synthetic version of the critical pentasaccharide sequence (fondaparinux), and the direct antithrombin bivalirudin. Advantages of LMWHs include high bioavailability permitting administration subcutaneously, reliable anticoagulation without monitoring, and greater antiXa:IIa activity. Enoxaparin has been shown to reduce significantly the composite endpoints of death/nonfatal reinfarction and death/nonfatal reinfarction/ urgent revascularization compared with UFH in STEMI patients who receive fibrinolysis. Treatment with enoxaparin is associated with higher rates of serious bleeding, but net clinical benefit—a composite endpoint that combines efficacy and safety—still favors enoxaparin over UFH. Interpretation of the data on fondaparinux is difficult because of the complex nature of the pivotal clinical trial evaluating it in STEMI (OASIS-6). Fondaparinux appears

1	favors enoxaparin over UFH. Interpretation of the data on fondaparinux is difficult because of the complex nature of the pivotal clinical trial evaluating it in STEMI (OASIS-6). Fondaparinux appears superior to placebo in STEMI patients not receiving reperfusion therapy, but its relative efficacy and safety compared with UFH is less certain. Due to the risk of catheter thrombosis, fondaparinux should not be used alone at the time of coronary angiography and PCI but should be combined with another anticoagulant with antithrombin activity such as UFH or bivalirudin. Contemporary trials of bivalirudin used an open-label design to evaluate its efficacy and safety compared with UFH plus a glycoprotein IIb/IIIa inhibitor. Bivalirudin was associated with a lower rate of bleeding, largely driven by reductions in vascular access site hematomas ≥5 cm or the administration of blood transfusions.

1	Patients with an anterior location of the infarction, severe LV dysfunction, heart failure, a history of embolism, two-dimensional echocardiographic evidence of mural thrombus, or atrial fibrillation are at increased risk of systemic or pulmonary thromboembolism. Such individuals should receive full therapeutic levels of anticoagulant therapy (LMWH or UFH) while hospitalized, followed by at least 3 months of warfarin therapy.

1	The benefits of beta blockers in patients with STEMI can be divided into those that occur immediately when the drug is given acutely and those that accrue over the long term when the drug is given for secondary prevention after an infarction. Acute intravenous beta blockade improves the myocardial O2 supply-demand relationship, decreases pain, reduces infarct size, and decreases the incidence of serious ventricular arrhythmias. In patients who undergo fibrinolysis soon after the onset of chest pain, no incremental reduction in mortality rate is seen with beta blockers, but recurrent ischemia and reinfarction are reduced.

1	Thus, beta-blocker therapy after STEMI is useful for most patients (including those treated with an angiotensin-converting enzyme [ACE] inhibitor) except those in whom it is specifically contraindicated (patients with heart failure or severely compromised LV function, heart block, orthostatic hypotension, or a history of asthma) and perhaps those whose excellent long-term prognosis (defined as an expected mortality rate of <1% per year, patients <55 years, no previous MI, with normal ventricular function, no complex ventricular ectopy, and no angina) markedly diminishes any potential benefit.

1	ACE inhibitors reduce the mortality rate after STEMI, and the mortality benefits are additive to those achieved with aspirin and beta blockers. The maximum benefit is seen in high-risk patients (those who are elderly or who have an anterior infarction, a prior infarction, and/or globally depressed LV function), but evidence suggests that a short-term benefit occurs when ACE inhibitors are prescribed unselectively to all hemodynamically stable patients with STEMI (i.e., those with a systolic pressure >100 mmHg). The mechanism involves a reduction in ventricular remodeling after infarction (see “Ventricular Dysfunction” later) with a subsequent reduction in the risk of CHF. The rate of recurrent infarction may also be lower in patients treated chronically with ACE inhibitors after infarction.

1	Before hospital discharge, LV function should be assessed with an imaging study. ACE inhibitors should be continued indefinitely in patients who have clinically evident CHF, in patients in whom an imaging study shows a reduction in global LV function or a large regional wall motion abnormality, or in those who are hypertensive.

1	Angiotensin receptor blockers (ARBs) should be administered to STEMI patients who are intolerant of ACE inhibitors and who have either clinical or radiologic signs of heart failure. Long-term aldosterone blockade should be prescribed for STEMI patients without significant renal dysfunction (creatinine ≥2.5 mg/dL in men and ≥2.0 mg/dL in women) or hyperkalemia (potassium ≥5.0 mEq/L) who are already receiving therapeutic doses of an ACE inhibitor, have an LV ejection fraction ≤40%, and have either symptomatic heart failure or diabetes mellitus. A multidrug regimen for inhibiting the reninangiotensin-aldosterone system has been shown to reduce both heart failure–related and sudden cardiac death–related cardiovascular mortality after STEMI, but has not been as thoroughly explored as ACE inhibitors in STEMI patients.

1	Favorable effects on the ischemic process and ventricular remodeling (see below) previously led many physicians to routinely use intravenous nitroglycerin (5–10 μg/min initial dose and up to 200 μg/min as long as hemodynamic stability is maintained) for the first 24–48 h after the onset of infarction. However, the benefits of routine use of intravenous nitroglycerin are less in the contemporary era where betaadrenoceptor blockers and ACE inhibitors are routinely prescribed for patients with STEMI. Results of multiple trials of different calcium antagonists have failed to establish a role for these agents in the treatment of most patients with STEMI. Therefore, the routine use of calcium antagonists cannot be recommended. Strict control of blood glucose in diabetic patients with STEMI has been shown to reduce the mortality rate. Serum magnesium should be measured in all patients on admission, and any demonstrated deficits should be corrected to minimize the risk of arrhythmias.

1	After STEMI, the left ventricle undergoes a series of changes in shape, size, and thickness in both the infarcted and noninfarcted segments. This process is referred to as ventricular remodeling and generally precedes the development of clinically evident CHF in the months to years after infarction. Soon after STEMI, the left ventricle begins to dilate. Acutely, this results from expansion of the infarct, i.e., slippage of muscle bundles, disruption of normal myocardial cells, and tissue loss within the necrotic zone, resulting in disproportionate thinning and elongation of the infarct zone. Later, lengthening of the noninfarcted segments occurs as well. The overall chamber enlargement that occurs is related to the size and location of the infarct, with greater dilation following infarction of the anterior wall and apex of the left ventricle and causing more marked hemodynamic impairment, more frequent heart failure, and a poorer prognosis. Progressive dilation and its clinical

1	of the anterior wall and apex of the left ventricle and causing more marked hemodynamic impairment, more frequent heart failure, and a poorer prognosis. Progressive dilation and its clinical consequences may be ameliorated by therapy with ACE inhibitors and other vasodilators (e.g., nitrates). In patients with an ejection fraction <40%, regardless of whether or not heart failure is present, ACE inhibitors or ARBs should be prescribed (see “Inhibition of the Renin-Angiotensin-Aldosterone System” earlier).

1	Pump failure is now the primary cause of in-hospital death from STEMI. The extent of infarction correlates well with the degree of pump failure and with mortality, both early (within 10 days of infarction) and later. The most common clinical signs are pulmonary rales and S3 and S4 gallop sounds. Pulmonary congestion is also frequently seen on the chest roentgenogram. Elevated LV filling pressure and elevated pulmonary artery pressure are the characteristic hemodynamic findings, but these findings may result from a reduction of ventricular compliance (diastolic failure) and/or a reduction of stroke volume with secondary cardiac dilation (systolic failure) (Chap. 279).

1	A classification originally proposed by Killip divides patients into four groups: class I, no signs of pulmonary or venous congestion; class II, moderate heart failure as evidenced by rales at the lung bases, S3 gallop, tachypnea, or signs of failure of the right side of the heart, including venous and hepatic congestion; class III, severe heart failure, pulmonary edema; and class IV, shock with systolic pressure <90 mmHg and evidence of peripheral vasoconstriction, peripheral cyanosis, mental confusion, and oliguria. When this classification was established in 1967, the expected hospital mortality rate of patients in these classes was as follows: class I, 0–5%; class II, 10–20%; class III, 35–45%; and class IV, 85–95%. With advances in management, the mortality rate in each class has fallen, perhaps by as much as one-third to one-half.

1	Hemodynamic evidence of abnormal global LV function appears when contraction is seriously impaired in 20–25% of the left ventricle. Infarction of ≥40% of the left ventricle usually results in cardiogenic shock (Chap. 326). Positioning of a balloon flotation (Swan-Ganz) catheter in the pulmonary artery permits monitoring of LV filling pressure; this technique is useful in patients who exhibit hypotension and/or clinical evidence of CHF. Cardiac output can also be determined with a pulmonary artery catheter. With the addition of intra-arterial pressure monitoring, systemic vascular resistance can be calculated as a guide to adjusting vasopressor and vasodilator therapy. Some patients with STEMI have markedly elevated LV filling pressures (>22 mmHg) and normal cardiac indices (2.6–3.6 L/[min/m2]), while others have relatively low LV filling pressures (<15 mmHg) and reduced cardiac indices. The former patients usually benefit from diuresis, while the latter may respond to volume

1	L/[min/m2]), while others have relatively low LV filling pressures (<15 mmHg) and reduced cardiac indices. The former patients usually benefit from diuresis, while the latter may respond to volume expansion.

1	This is an easily corrected condition that may contribute to the hypo-tension and vascular collapse associated with STEMI in some patients. It may be secondary to previous diuretic use, to reduced fluid intake during the early stages of the illness, and/or to vomiting associated with pain or medications. Consequently, hypovolemia should be identified and corrected in patients with STEMI and hypotension before 1608 more vigorous forms of therapy are begun. Central venous pressure reflects RV rather than LV filling pressure and is an inadequate guide for adjustment of blood volume, because LV function is almost always affected much more adversely than RV function in patients with STEMI. The optimal LV filling or pulmonary artery wedge pressure may vary considerably among patients. Each patient’s ideal level (generally ~20 mmHg) is reached by cautious fluid administration during careful monitoring of oxygenation and cardiac output. Eventually, the cardiac output level plateaus, and

1	patient’s ideal level (generally ~20 mmHg) is reached by cautious fluid administration during careful monitoring of oxygenation and cardiac output. Eventually, the cardiac output level plateaus, and further increases in LV filling pressure only increase congestive symptoms and decrease systemic oxygenation without raising arterial pressure.

1	The management of CHF in association with STEMI is similar to that of acute heart failure secondary to other forms of heart disease (avoidance of hypoxemia, diuresis, afterload reduction, inotropic support) (Chap. 279), except that the benefits of digitalis administration to patients with STEMI are unimpressive. By contrast, diuretic agents are extremely effective, as they diminish pulmonary congestion in the presence of systolic and/or diastolic heart failure. LV filling pressure falls and orthopnea and dyspnea improve after the intravenous administration of furosemide or other loop diuretics. These drugs should be used with caution, however, as they can result in a massive diuresis with associated decreases in plasma volume, cardiac output, systemic blood pressure, and, hence, coronary perfusion. Nitrates in various forms may be used to decrease preload and congestive symptoms. Oral isosorbide dinitrate, topical nitroglycerin ointment, and intravenous nitroglycerin all have the

1	perfusion. Nitrates in various forms may be used to decrease preload and congestive symptoms. Oral isosorbide dinitrate, topical nitroglycerin ointment, and intravenous nitroglycerin all have the advantage over a diuretic of lowering preload through venodilation without decreasing the total plasma volume. In addition, nitrates may improve ventricular compliance if ischemia is present, as ischemia causes an elevation of LV filling pressure. Vasodilators must be used with caution to prevent serious hypotension. As noted earlier, ACE inhibitors are an ideal class of drugs for management of ventricular dysfunction after STEMI, especially for the long term. (See “Inhibition of the Renin-Angiotensin-Aldosterone System” earlier.)

1	Prompt reperfusion, efforts to reduce infarct size and treatment of ongoing ischemia and other complications of MI appear to have reduced the incidence of cardiogenic shock from 20% to about 7%. Only 10% of patients with this condition present with it on admission, while 90% develop it during hospitalization. Typically, patients who develop cardiogenic shock have severe multivessel coronary artery disease with evidence of “piecemeal” necrosis extending outward from the original infarct zone. The evaluation and management of cardiogenic shock and severe power failure after STEMI are discussed in detail in Chap. 326.

1	Approximately one-third of patients with inferior infarction demonstrate at least a minor degree of RV necrosis. An occasional patient with inferoposterior LV infarction also has extensive RV infarction, and rare patients present with infarction limited primarily to the RV. Clinically significant RV infarction causes signs of severe RV failure (jugular venous distention, Kussmaul’s sign, hepatomegaly [Chap. 267]) with or without hypotension. ST-segment elevations of right-sided precordial ECG leads, particularly lead V4R, are frequently present in the first 24 h in patients with RV infarction. Two-dimensional echocardiography is helpful in determining the degree of RV dysfunction. Catheterization of the right side of the heart often reveals a distinctive hemodynamic pattern resembling constrictive pericarditis (steep right atrial “y” descent and an early diastolic dip and plateau in RV waveforms) (Chap. 288). Therapy consists of volume expansion to maintain adequate RV preload and

1	constrictive pericarditis (steep right atrial “y” descent and an early diastolic dip and plateau in RV waveforms) (Chap. 288). Therapy consists of volume expansion to maintain adequate RV preload and efforts to improve LV performance with attendant reduction in pulmonary capillary wedge and pulmonary arterial pressures.

1	(See also Chaps. 274 and 276) The incidence of arrhythmias after STEMI is higher in patients seen early after the onset of symptoms. The mechanisms responsible for infarction-related arrhythmias include autonomic nervous system imbalance, electrolyte disturbances, ischemia, and slowed conduction in zones of ischemic myocardium. An arrhythmia can usually be managed successfully if trained personnel and appropriate equipment are available when it develops. Since most deaths from arrhythmia occur during the first few hours after infarction, the effectiveness of treatment relates directly to the speed with which patients come under medical observation. The prompt management of arrhythmias constitutes a significant advance in the treatment of STEMI.

1	Ventricular Premature Beats Infrequent, sporadic ventricular premature depolarizations occur in almost all patients with STEMI and do not require therapy. Whereas in the past, frequent, multifocal, or early diastolic ventricular extrasystoles (so-called warning arrhythmias) were routinely treated with antiarrhythmic drugs to reduce the risk of development of ventricular tachycardia and ventricular fibrillation, pharmacologic therapy is now reserved for patients with sustained ventricular arrhythmias. Prophylactic antiarrhythmic therapy (either intravenous lidocaine early or oral agents later) is contraindicated for ventricular premature beats in the absence of clinically important ventricular tachyarrhythmias, because such therapy may actually increase the mortality rate. Beta-adrenoceptor blocking agents are effective in abolishing ventricular ectopic activity in patients with STEMI and in the prevention of ventricular fibrillation. As described earlier (see “Beta-Adrenoceptor

1	blocking agents are effective in abolishing ventricular ectopic activity in patients with STEMI and in the prevention of ventricular fibrillation. As described earlier (see “Beta-Adrenoceptor Blockers”), they should be used routinely in patients without contraindications. In addition, hypokalemia and hypomagnesemia are risk factors for ventricular fibrillation in patients with STEMI; to reduce the risk, the serum potassium concentration should be adjusted to approximately 4.5 mmol/L and magnesium to about 2.0 mmol/L.

1	Ventricular Tachycardia and Fibrillation Within the first 24 h of STEMI, ventricular tachycardia and fibrillation can occur without prior warning arrhythmias. The occurrence of ventricular fibrillation can be reduced by prophylactic administration of intravenous lidocaine. However, prophylactic use of lidocaine has not been shown to reduce overall mortality from STEMI. In fact, in addition to causing possible noncardiac complications, lidocaine may predispose to an excess risk of bradycardia and asystole. For these reasons, and with earlier treatment of active ischemia, more frequent use of beta-blocking agents, and the nearly universal success of electrical cardioversion or defibrillation, routine prophylactic antiarrhythmic drug therapy is no longer recommended.

1	Sustained ventricular tachycardia that is well tolerated hemodynamically should be treated with an intravenous regimen of amiodarone (bolus of 150 mg over 10 min, followed by infusion of 1.0 mg/min for 6 h and then 0.5 mg/min) or procainamide (bolus of 15 mg/kg over 20–30 min; infusion of 1–4 mg/min); if it does not stop promptly, electroversion should be used (Chap. 276). An unsynchronized discharge of 200–300 J (monophasic waveform; approximately 50% of these energies with biphasic waveforms) is used immediately in patients with ventricular fibrillation or when ventricular tachycardia causes hemodynamic deterioration. Ventricular tachycardia or fibrillation that is refractory to electroshock may be more responsive after the patient is treated with epinephrine (1 mg intravenously or 10 mL of a 1:10,000 solution via the intracardiac route) or amiodarone (a 75–150-mg bolus).

1	Ventricular arrhythmias, including the unusual form of ventricular tachycardia known as torsades des pointes (Chaps. 276 and 277), may occur in patients with STEMI as a consequence of other concurrent problems (such as hypoxia, hypokalemia, or other electrolyte disturbances) or of the toxic effects of an agent being administered to the patient (such as digoxin or quinidine). A search for such secondary causes should always be undertaken.

1	Although the in-hospital mortality rate is increased, the long-term survival is excellent in patients who survive to hospital discharge after primary ventricular fibrillation; i.e., ventricular fibrillation that is a primary response to acute ischemia that occurs during the first 48 h and is not associated with predisposing factors such as CHF, shock, bundle branch block, or ventricular aneurysm. This result is in sharp contrast to the poor prognosis for patients who develop ventricular fibrillation secondary to severe pump failure. For patients who develop ventricular tachycardia or ventricular fibrillation late in their hospital course (i.e., after the first 48 h), the mortality rate is increased both in-hospital and during long-term follow-up. Such patients should be considered for electrophysiologic study and implantation of a cardioverter-defibrillator (ICD) (Chap. 276). A more challenging issue is the prevention of sudden cardiac death from ventricular fibrillation late after

1	study and implantation of a cardioverter-defibrillator (ICD) (Chap. 276). A more challenging issue is the prevention of sudden cardiac death from ventricular fibrillation late after STEMI in patients who have not exhibited sustained ventricular tachyarrhythmias during their index hospitalization. An algorithm for selection of patients who warrant prophylactic implantation of an ICD is shown in Fig. 295-5.

1	Accelerated Idioventricular Rhythm Accelerated idioventricular rhythm (AIVR, “slow ventricular tachycardia”), a ventricular rhythm with a rate of 60–100 beats/min, often occurs transiently during fibrinolytic therapy at the time of reperfusion. For the most part, AIVR, whether it occurs in association with fibrinolytic therapy or spontaneously, is benign and does not presage the development of classic ventricular tachycardia. Most episodes of AIVR do not require treatment if the patient is monitored carefully, as degeneration into a more serious arrhythmia is rare. Supraventricular Arrhythmias Sinus tachycardia is the most common supraventricular arrhythmia. If it occurs secondary to another cause

1	FIGURE 295-5 Algorithm for assessment of need for implantation of a cardioverter-defibrillator. The appropriate management is selected based on measurement of left ventricular ejection fraction and assessment of the New York Heart Association (NYHA) functional class. Patients with depressed left ventricular function at least 40 days after ST-segment elevation myocardial infarction (STEMI) are referred for insertion of an implantable cardioverter-defibrillator (ICD) if the left ventricular ejection fraction (LVEF) is <30–40% and they are in NYHA class II–III or if the LVEF is <30–35% and they are in NYHA class I functional status. Patients with preserved left ventricular function (LVEF >40%) do not receive an ICD regardless of NYHA functional class. All patients are treated with medical therapy after STEMI. VF, ventricular fibrillation; VT, ventricular tachycardia. (Adapted from data contained in DP Zipes et al: ACC/AHA/ESC 2006 guidelines for management of patients with ventricular

1	therapy after STEMI. VF, ventricular fibrillation; VT, ventricular tachycardia. (Adapted from data contained in DP Zipes et al: ACC/AHA/ESC 2006 guidelines for management of patients with ventricular arrhythmias and the prevention of sudden cardiac death; a report of the American College of Cardiology/American Heart Association Task Force and the European Society of Cardiology Committee for Practice Guidelines [Writing Committee to Develop Guidelines for Management of Patients with Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death]. J Am Coll Cardiol 48:1064, 2006.) (such as anemia, fever, heart failure, or a metabolic derangement), the 1609 primary problem should be treated first. However, if it appears to be due to sympathetic overstimulation (e.g., as part of a hyperdynamic state), then treatment with a beta blocker is indicated. Other common arrhythmias in this group are atrial flutter and atrial fibrillation, which are often secondary to LV failure. Digoxin is

1	state), then treatment with a beta blocker is indicated. Other common arrhythmias in this group are atrial flutter and atrial fibrillation, which are often secondary to LV failure. Digoxin is usually the treatment of choice for supraventricular arrhythmias if heart failure is present. If heart failure is absent, beta blockers, verapamil, or diltiazem are suitable alternatives for controlling the ventricular rate, as they may also help to control ischemia. If the abnormal rhythm persists for >2 h with a ventricular rate >120 beats/min, or if tachycardia induces heart failure, shock, or ischemia (as manifested by recurrent pain or ECG changes), a synchronized electroshock (100–200 J monophasic waveform) should be used.

1	Accelerated junctional rhythms have diverse causes but may occur in patients with inferoposterior infarction. Digitalis excess must be ruled out. In some patients with severely compromised LV function, the loss of appropriately timed atrial systole results in a marked reduction of cardiac output. Right atrial or coronary sinus pacing is indicated in such instances. Sinus Bradycardia Treatment of sinus bradycardia is indicated if hemodynamic compromise results from the slow heart rate. Atropine is the most useful drug for increasing heart rate and should be given intravenously in doses of 0.5 mg initially. If the rate remains <50–60 beats/min, additional doses of 0.2 mg, up to a total of 2.0 mg, may be given. Persistent bradycardia (<40 beats/min) despite atropine may be treated with electrical pacing. Isoproterenol should be avoided.

1	Atrioventricular and Intraventricular Conduction Disturbances (See also Chap. 274) Both the in-hospital mortality rate and the postdischarge mortality rate of patients who have complete atrioventricular (AV) block in association with anterior infarction are markedly higher than those of patients who develop AV block with inferior infarction. This difference is related to the fact that heart block in inferior infarction is commonly a result of increased vagal tone and/or the release of adenosine and therefore is transient. In anterior wall infarction, however, heart block is usually related to ischemic malfunction of the conduction system, which is commonly associated with extensive myocardial necrosis.

1	Temporary electrical pacing provides an effective means of increasing the heart rate of patients with bradycardia due to AV block. However, acceleration of the heart rate may have only a limited impact on prognosis in patients with anterior wall infarction and complete heart block in whom the large size of the infarct is the major factor determining outcome. It should be carried out if it improves hemodynamics. Pacing does appear to be beneficial in patients with inferoposterior infarction who have complete heart block associated with heart failure, hypotension, marked bradycardia, or significant ventricular ectopic activity. A subgroup of these patients, those with RV infarction, often respond poorly to ventricular pacing because of the loss of the atrial contribution to ventricular filling. In such patients, dual-chamber AV sequential pacing may be required.

1	External noninvasive pacing electrodes should be positioned in a “demand” mode for patients with sinus bradycardia (rate <50 beats/ min) that is unresponsive to drug therapy, Mobitz II second-degree AV block, third-degree heart block, or bilateral bundle branch block (e.g., right bundle branch block plus left anterior fascicular block). Retrospective studies suggest that permanent pacing may reduce the long-term risk of sudden death due to bradyarrhythmias in the rare patient who develops combined persistent bifascicular and transient third-degree heart block during the acute phase of MI. OTHER COMPLICATIONS Recurrent Chest Discomfort Because recurrent or persistent ischemia often heralds extension of the original infarct or reinfarction in a new myocardial zone and is associated with a near tripling of mortality after STEMI, patients with these symptoms should be referred for prompt coronary arteriography and mechanical revascularization.

1	1610 Administration of a fibrinolytic agent is an alternative to early mechanical revascularization.

1	Pericarditis (See also Chap. 288) Pericardial friction rubs and/or pericardial pain are frequently encountered in patients with STEMI involving the epicardium. This complication can usually be managed with aspirin (650 mg four times daily). It is important to diagnose the chest pain of pericarditis accurately, because failure to recognize it may lead to the erroneous diagnosis of recurrent ischemic pain and/or infarct extension, with resulting inappropriate use of anticoagulants, nitrates, beta blockers, or coronary arteriography. When it occurs, complaints of pain radiating to either trapezius muscle is helpful, because such a pattern of discomfort is typical of pericarditis but rarely occurs with ischemic discomfort. Anticoagulants potentially could cause tamponade in the presence of acute pericarditis (as manifested by either pain or persistent rub) and therefore should not be used unless there is a compelling indication.

1	Thromboembolism Clinically apparent thromboembolism complicates STEMI in ~10% of cases, but embolic lesions are found in 20% of patients in necropsy series, suggesting that thromboembolism is often clinically silent. Thromboembolism is considered to be an important contributing cause of death in 25% of patients with STEMI who die after admission to the hospital. Arterial emboli originate from LV mural thrombi, while most pulmonary emboli arise in the leg veins.

1	Thromboembolism typically occurs in association with large infarcts (especially anterior), CHF, and an LV thrombus detected by echocardiography. The incidence of arterial embolism from a clot originating in the ventricle at the site of an infarction is small but real. Two-dimensional echocardiography reveals LV thrombi in about one-third of patients with anterior wall infarction but in few patients with inferior or posterior infarction. Arterial embolism often presents as a major complication, such as hemiparesis when the cerebral circulation is involved or hypertension if the renal circulation is compromised. When a thrombus has been clearly demonstrated by echocardiographic or other techniques or when a large area of regional wall motion abnormality is seen even in the absence of a detectable mural thrombus, systemic anticoagulation should be undertaken (in the absence of contraindications), as the incidence of embolic complications appears to be markedly lowered by such therapy.

1	mural thrombus, systemic anticoagulation should be undertaken (in the absence of contraindications), as the incidence of embolic complications appears to be markedly lowered by such therapy. The appropriate duration of therapy is unknown, but 3–6 months is probably prudent.

1	Left Ventricular Aneurysm The term ventricular aneurysm is usually used to describe dyskinesis or local expansile paradoxical wall motion. Normally functioning myocardial fibers must shorten more if stroke volume and cardiac output are to be maintained in patients with ventricular aneurysm; if they cannot, overall ventricular function is impaired. True aneurysms are composed of scar tissue and neither predispose to nor are associated with cardiac rupture. The complications of LV aneurysm do not usually occur for weeks to months after STEMI; they include CHF, arterial embolism, and ventricular arrhythmias. Apical aneurysms are the most common and the most easily detected by clinical examination. The physical finding of greatest value is a double, diffuse, or displaced apical impulse. Ventricular aneurysms are readily detected by two-dimensional echocardiography, which may also reveal a mural thrombus in an aneurysm.

1	Rarely, myocardial rupture may be contained by a local area of pericardium, along with organizing thrombus and hematoma. Over time, this pseudoaneurysm enlarges, maintaining communication with the LV cavity through a narrow neck. Because a pseudoaneurysm often ruptures spontaneously, it should be surgically repaired if recognized.

1	Many clinical and laboratory factors have been identified that are associated with an increase in cardiovascular risk after initial recovery from STEMI. Some of the most important factors include persistent ischemia (spontaneous or provoked), depressed LV ejection fraction (<40%), rales above the lung bases on physical examination or congestion on chest radiograph, and symptomatic ventricular arrhythmias. Other features associated with increased risk include a history of previous MI, age >75, diabetes mellitus, prolonged sinus tachycardia, hypotension, ST-segment changes at rest without angina (“silent ischemia”), an abnormal signal-averaged ECG, nonpatency of the infarct-related coronary artery (if angiography is undertaken), and persistent advanced heart block or a new intraventricular conduction abnormality on the ECG. Therapy must be individualized on the basis of the relative importance of the risk(s) present.

1	The goal of preventing reinfarction and death after recovery from STEMI has led to strategies to evaluate risk after infarction. In stable patients, submaximal exercise stress testing may be carried out before hospital discharge to detect residual ischemia and ventricular ectopy and to provide the patient with a guideline for exercise in the early recovery period. Alternatively, or in addition, a maximal (symptomlimited) exercise stress test may be carried out 4–6 weeks after infarction. Evaluation of LV function is usually warranted as well. Recognition of a depressed LV ejection fraction by echocardiography or radionuclide ventriculography identifies patients who should receive medications to inhibit the renin-angiotensin-aldosterone system. Patients in whom angina is induced at relatively low workloads, those who have a large reversible defect on perfusion imaging or a depressed ejection fraction, those with demonstrable ischemia, and those in whom exercise provokes symptomatic

1	low workloads, those who have a large reversible defect on perfusion imaging or a depressed ejection fraction, those with demonstrable ischemia, and those in whom exercise provokes symptomatic ventricular arrhythmias should be considered at high risk for recurrent MI or death from arrhythmia (Fig. 295-5). Cardiac catheterization with coronary angiography and/or invasive electrophysiologic evaluation is advised.

1	Exercise tests also aid in formulating an individualized exercise prescription, which can be much more vigorous in patients who tolerate exercise without any of the previously mentioned adverse signs. In addition, predischarge stress testing may provide an important psychological benefit, building the patient’s confidence by demonstrating a reasonable exercise tolerance. In many hospitals, a cardiac rehabilitation program with progressive exercise is initiated in the hospital and continued after discharge. Ideally, such programs should include an educational component that informs patients about their disease and its risk factors.

1	The usual duration of hospitalization for an uncomplicated STEMI is about 5 days. The remainder of the convalescent phase may be accomplished at home. During the first 1–2 weeks, the patient should be encouraged to increase activity by walking about the house and outdoors in good weather. Normal sexual activity may be resumed during this period. After 2 weeks, the physician must regulate the patient’s activity on the basis of exercise tolerance. Most patients will be able to return to work within 2–4 weeks.

1	Various secondary preventive measures are at least partly responsible for the improvement in the long-term mortality and morbidity rates after STEMI. Long-term treatment with an antiplatelet agent (usually aspirin) after STEMI is associated with a 25% reduction in the risk of recurrent infarction, stroke, or cardiovascular mortality (36 fewer events for every 1000 patients treated). An alternative antiplatelet agent that may be used for secondary prevention in patients intolerant of aspirin is clopidogrel (75 mg orally daily). ACE inhibitors or ARBs and, in appropriate patients, aldosterone antagonists should be used indefinitely by patients with clinically evident heart failure, a moderate decrease in global ejection fraction, or a large regional wall motion abnormality to prevent late ventricular remodeling and recurrent ischemic events.

1	The chronic routine use of oral beta-adrenoceptor blockers for at least 2 years after STEMI is supported by well-conducted, placebo-controlled trials.

1	Evidence suggests that warfarin lowers the risk of late mortality and the incidence of reinfarction after STEMI. Most physicians prescribe aspirin routinely for all patients without contraindications and add warfarin for patients at increased risk of embolism (see “Thromboembolism” earlier). Several studies suggest that in patients <75 years old a low dose of aspirin (75–81 mg/d) in combination with 1611 warfarin administered to achieve an international normalized ratio >2.0 is more effective than aspirin alone for preventing recurrent MI and embolic cerebrovascular accident. However, there is an increased risk of bleeding and a high rate of discontinuation of warfarin that has limited clinical acceptance of combination antithrombotic therapy. There is increased risk of bleeding when warfarin is added to dual antiplatelet therapy (aspirin and clopidogrel). However, patients who have had a stent implanted and have an indication for anticoagulation should receive dual antiplatelet

1	warfarin is added to dual antiplatelet therapy (aspirin and clopidogrel). However, patients who have had a stent implanted and have an indication for anticoagulation should receive dual antiplatelet therapies in combination with warfarin. Such patients should also receive a proton pump inhibitor to minimize the risk of gastrointestinal bleeding and should have regular monitoring of their hemoglobin levels and stool hematest while on combination antithrombotic therapy.

1	Finally, risk factors for atherosclerosis (Chap. 265e) should be discussed with the patient and, when possible, favorably modified.

1	Percutaneous Coronary Interventions and Other Interventional Procedures David P. Faxon, Deepak L. Bhatt Percutaneous transluminal coronary angioplasty (PTCA) was first 296e introduced by Andreas Gruentzig in 1977 as an alternative to coronary bypass surgery. The concept was initially demonstrated by Charles Dotter in 1964 in peripheral vessels. The development of a small inelastic balloon catheter by Gruentzig allowed expansion of the technique into smaller peripheral and coronary vessels. Initial coronary experience was limited to single-vessel coronary disease and discrete proximal lesions due to the technical limitations of the equipment. Advances in technology and greater operator experience allowed the procedure to grow rapidly with expanded use in patients with more complex lesions and multivessel disease. The introduction of coronary stents in 1994 was one of the major advances in the field. These devices reduced acute complications and reduced by half the significant problem

1	and multivessel disease. The introduction of coronary stents in 1994 was one of the major advances in the field. These devices reduced acute complications and reduced by half the significant problem of restenosis (or recurrence of the stenosis). Further reductions in restenosis were achieved by the introduction of drug-eluting stents in 2003. These stents slowly release antiproliferative drugs directly into the plaque over a few months. Percutaneous coronary intervention (PCI) is the most common revascularization procedure in the United States and is performed more than twice as often as coronary artery bypass surgery: nearly 600,000 patients a year.

1	Interventional cardiology is a separate discipline in cardiology that requires a dedicated 1-year interventional cardiology fellowship following a 3-year general cardiology fellowship in order to obtain a separate board certification. The discipline has also expanded to include interventions for structural heart disease including treatment of congenital heart disease and valvular heart disease; it also includes interventions to treat peripheral vascular disease, including atherosclerotic and nonatherosclerotic lesions in the carotid, renal, aortic, and peripheral circulations.

1	The initial procedure is performed in a similar manner as a diagnostic cardiac catheterization (Chap. 272). Arterial access is obtained via the femoral or radial artery. To prevent thrombotic complications during the procedure, patients who are anticipated to need an angioplasty are given aspirin (325 mg) and may be given clopidogrel (loading 296e-1 dose of 300–600 mg), prasugrel (loading dose of 60 mg), or ticagrelor (loading dose of 180 mg) before the procedure. During the procedure, anticoagulation is achieved by administration of unfractionated heparin, enoxaparin (a low-molecular-weight heparin), or bivalirudin (a direct thrombin inhibitor). The latter has gained popularity due to the significant reduction in bleeding complications. In patients with ST-elevation myocardial infarction, high-risk acute coronary syndrome, or a large thrombus in the coronary artery, an intravenous glycoprotein IIb/IIIa inhibitor (abciximab, tirofiban, or eptifibatide) may also be given.

1	Following placement of an introducing sheath, preformed guiding catheters are used to cannulate selectively the origins of the coronary arteries. Through the guiding catheter, a flexible, steerable guidewire is negotiated down the coronary artery lumen using fluoroscopic guidance; it is then advanced through the stenosis and into the vessel beyond. This guidewire then serves as a “rail” over which angioplasty balloons, stents, or other therapeutic devices can be advanced to enlarge the narrowed segment of coronary artery. The artery is usually dilated with a balloon catheter followed by placement of a stent. The catheters and introducing sheath are removed and the artery manually held or closed using one of several femoral arterial closure devices to achieve hemostasis. Because PCI is performed under local anesthesia and mild sedation, it requires only a short (1-day) hospitalization or less.

1	Angioplasty works by stretching the artery and displacing the plaque away from the lumen, enlarging the entire vessel (Figs. 296e-1 and 296e-2). The procedure rarely results in embolization of atherosclerotic material. Owing to inelastic elements in the plaque, the stretching of the vessel by the balloon results in small localized dissections that can protrude into the lumen and be a nidus for acute thrombus formation. If the dissections are severe, then they can obstruct the lumen or induce a thrombotic occlusion of the artery (acute closure). Stents have largely prevented this complication by holding the dissection flaps up against the vessel wall (Fig. 296e-1).

1	Stents are currently used in more than 90% of coronary angioplasty procedures. Stents are wire meshes (usually made of stainless steel) that are compressed over a deflated angioplasty balloon. When the balloon is inflated, the stent is enlarged to approximate the “normal” vessel lumen. The balloon is then deflated and removed, leaving the stent behind to provide a permanent scaffold in the artery. Owing to the design of the struts, these devices are flexible, allowing their passage through diseased and tortuous coronary vessels. Stents are rigid enough to prevent elastic recoil of the vessel and have dramatically improved the success and safety of the procedure as a result.

1	ABCDFIgUrE 296e-1 Schematic diagram of the primary mechanisms of balloon angioplasty and stenting. A. A balloon angioplasty catheter is positioned into the stenosis over a guidewire under fluoroscopic guidance. B. The balloon is inflated temporarily occluding the vessel. C. The lumen is enlarged primarily by stretching the vessel, often resulting in small dissections in the neointima. D. A stent mounted on a deflated balloon is placed into the lesion and pressed against the vessel wall with balloon inflation (not shown). The balloon is deflated and removed, leaving the stent permanently against the wall acting as a scaffold to hold the dissections against the wall and prevent vessel recoil. (Adapted from EJ Topol: Textbook of Cardiovascular Medicine, 2nd ed. Philadelphia, Lippincott Williams & Wilkins, 2002.) PART 10 Disorders of the Cardiovascular System

1	PART 10 Disorders of the Cardiovascular System FIgUrE 296e-2 Pathology of acute effects of balloon angioplasty with intimal dissection and vessel stretching (A) and an example of neointimal hyperplasia and restenosis showing renarrowing of the vessel (B). (Panel A from M Ueda et al: Eur Heart J 12:937, 1991; with permission. Panel B from CE Essed et al: Br Heart J 49:393, 1983; with permission.)

1	Drug-eluting stents further enhanced the efficacy of PCI. An antiproliferative agent is attached to the metal stent by use of a thin polymer coating. The antiproliferative drug elutes from the stent over a 1to 3-month period after implantation. Drug-eluting stents have been shown to reduce clinical restenosis by 50%, so that in uncomplicated lesions symptomatic restenosis occurs in 5–10% of patients. Not surprisingly, this led to the rapid acceptance of these devices; currently 80–90% of all stents implanted are drug-eluting. The first-generation devices were coated with either sirolimus or paclitaxel. Second-generation drug-eluting stents use newer agents such as everolimus, biolimus, and zotarolimus. These second-generation drug-eluting stents appear to be more effective with fewer complications, such as early or late stent thrombosis, than the first-generation devices and, therefore, have replaced the first-generation stents. Biodegradable polymers that are used to attach the drugs

1	such as early or late stent thrombosis, than the first-generation devices and, therefore, have replaced the first-generation stents. Biodegradable polymers that are used to attach the drugs to the stents may be superior to permanent polymers in preventing late stent thrombosis and are under investigation. In addition, the everolimus-eluting biodegradable vascular scaffold (BVS) stent has been shown to be safe and effective with gradual degradation over several years with return of normal vessel function. It is currently approved in Europe. Additional stents are under investigation. Other interventional devices include atherectomy devices and thrombectomy catheters. These devices are designed to remove atherosclerotic plaque or thrombus and are used in conjunction with balloon dilatation and stent placement. Rotational atherectomy is the most commonly used adjunctive device and is modeled after a dentist’s drill, with small round burrs of 1.25–2.5 mm at the tip of a flexible wire

1	and stent placement. Rotational atherectomy is the most commonly used adjunctive device and is modeled after a dentist’s drill, with small round burrs of 1.25–2.5 mm at the tip of a flexible wire shaft. They are passed over the guidewire up to the stenosis and drill away atherosclerotic material. Because the atherosclerotic particles are ≤25 μm, they pass through the coronary microcirculation and rarely cause problems. The device is particularly useful in heavily calcified plaques that are resistant to balloon dilatation. Given the current advances in stents, rotational atherectomy is infrequently used. Directional atherectomy catheters are not used in the coronaries any longer but are used in peripheral arterial disease.

1	In acute ST-elevation myocardial infarction, specialized catheters without a balloon are used to aspirate thrombus in order to prevent embolization down the coronary vessel and to improve blood flow before angioplasty and stent placement. Some data suggest that manual catheter thrombus aspiration may reduce mortality in addition to improving blood flow in primary PCI.

1	PCI of degenerated saphenous vein graft lesions has been associated with a significant incidence of distal embolization of atherosclerotic material, unlike PCI of native vessel disease. A number of distal protection devices have been shown to significantly reduce embolization and myocardial infarction in this setting. Most devices work by using a collapsible wire filter at the end of a guidewire that is expanded in the distal vessel before PCI. If atherosclerotic debris is dislodged, the basket captures the material, and at the end of the PCI, the basket is pulled into a delivery catheter and the debris safely removed from the patient.

1	A successful procedure (angiographic success), defined as a reduction of the stenosis to less than a 20% diameter narrowing, occurs in 95–99% of patients. Lower success rates are seen in patients with tortuous, small, or calcified vessels or chronic total occlusions. Chronic total occlusions have the lowest success rates (60–70%), and their recanalization is usually not attempted unless the occlusion is recent (within 3 months) or there are favorable anatomic features. Improvements in equipment and technique have increased the success rates of recanalization of chronic total occlusions.

1	Serious complications are rare but include a mortality rate of 0.1–0.3% for elective cases, a large myocardial infarction in less than 3%, and stroke in less than 0.1%. Patients who are elderly (>65 years), undergoing an emergent or urgent procedure, have chronic kidney disease, present with an ST-segment elevation myocardial infarction (STEMI), or are in shock have significantly higher risk. Scoring systems can help to estimate the risk of the procedure. Myocardial infarction during PCI can occur for multiple reasons including an acute occluding thrombus, severe coronary dissection, embolization of thrombus or atherosclerotic material, or closure of a side branch vessel at the site of angioplasty. Most myocardial infarctions are small and only detected by a rise in the creatine phosphokinase (CPK) or troponin level after the procedure. Only those with significant enzyme elevations (more than three to five times the upper limit of normal) are associated with a less favorable long-term

1	(CPK) or troponin level after the procedure. Only those with significant enzyme elevations (more than three to five times the upper limit of normal) are associated with a less favorable long-term outcome. Coronary stents have largely prevented coronary dissections due to the scaffolding effect of the stent.

1	Metallic stents are also prone to stent thrombosis (1–3%), either acute (<24 h) or subacute (1–30 days), which can be ameliorated by greater attention to full initial stent deployment and the use of dual antiplatelet therapy (DAPT) (aspirin, plus a platelet P2Y12 receptor blocker [clopidogrel, prasugrel, or ticagrelor]). Late (30 days–1 year) and very late stent thromboses (>1 year) occur very infrequently with stents but are slightly more common with first-generation drug-eluting stents, necessitating DAPT for up to 1 year or longer. Use of the second-generation stents is associated with lower rates of late and very late stent thromboses, and shorter durations of DAPT may be possible. Premature discontinuation of DAPT, particularly in the first month after implantation, is associated with a significantly increased risk for stent thrombosis (threeto ninefold greater). Stent thrombosis results in death in 10–20% and myocardial infarction in 30–70% of patients. Elective surgery that

1	with a significantly increased risk for stent thrombosis (threeto ninefold greater). Stent thrombosis results in death in 10–20% and myocardial infarction in 30–70% of patients. Elective surgery that requires discontinuation of antiplatelet therapy after drug-eluting stent implantation should be postponed until after 6 months and preferably after 1 year, if at all possible.

1	Restenosis, or renarrowing of the dilated coronary stenosis, is the most common complication of angioplasty and occurs in 20–50% of patients with balloon angioplasty alone, 10–30% of patients with bare metal stents, and 5–15% of patients with drug-eluting stents within the first year. The fact that stent placement provides a larger acute luminal area than balloon angioplasty alone reduces the incidence of subsequent restenosis. Drug-eluting stents further reduce restenosis through a reduction in excessive neointimal growth over the stent. If restenosis does not occur, the long-term outcome is excellent (Fig. 296e-3). Clinical restenosis is recognized by recurrence of angina or symptoms within 9 months of the procedure. Less frequently, patients with restenosis can present with non-ST-segment elevation myocardial infarction (NSTEMI) (10%) or STEMI (2%) as well. Clinical restenosis requires confirmation of a significant stenosis at the site of the prior PCI. Target lesion

1	non-ST-segment elevation myocardial infarction (NSTEMI) (10%) or STEMI (2%) as well. Clinical restenosis requires confirmation of a significant stenosis at the site of the prior PCI. Target lesion revascularization (TLR) or target vessel revascularization (TVR) is defined as angiographic restenosis with repeat PCI or coronary artery bypass grafting (CABG). By angiography, the incidence of restenosis is significantly higher than clinical restenosis (TLR or TVR) because many patients have mild restenosis that does not result in a recurrence of symptoms. The management of clinical restenosis is usually to repeat the PCI with balloon dilatation and placement of a drug-eluting stent. Once a patient has had restenosis, the risk of a second restenosis is further increased. The risk factors for restenosis are diabetes, myocardial infarction, long lesions, small-diameter vessels, and suboptimal initial PCI result.

1	FIgUrE 296e-3 Long-term results from one of the first patients to receive a sirolimus-eluting stent from early Sao Paulo experience. (From: GW Stone, in D Baim [ed]: Cardiac Catheterization, Angiography and Intervention, 7th ed. Philadelphia, Lippincott Williams & Wilkins, 2006; with permission.)

1	The American College of Cardiology (ACC)/American Heart Association (AHA) guidelines extensively review the indications for PCI in patients with stable angina, unstable angina, NSTEMI, and STEMI and should be referred to for a comprehensive discussion of the indications. Briefly, the two principal indications for coronary revascularization in patients with chronic stable angina (Chap. 293) are (1) to improve anginal symptoms in patients who remain symptomatic despite adequate medical therapy and (2) to reduce mortality rates in patients with severe coronary disease. In patients with stable angina who are well controlled on medical therapy, studies such as the Clinical Outcomes Utilizing Revascularization and Aggressive Drug Evaluation (COURAGE) and Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) trials have shown that initial revascularization does not lead to better outcomes and can be safely delayed until symptoms worsen or evidence of severe ischemia on

1	Investigation 2 Diabetes (BARI 2D) trials have shown that initial revascularization does not lead to better outcomes and can be safely delayed until symptoms worsen or evidence of severe ischemia on noninvasive testing occurs. When revascularization is indicated, the choice of PCI or CABG depends on a number of clinical and anatomic factors (Fig. 296e-4). The Synergy between Percutaneous Coronary Intervention with Taxus and Cardiac Surgery (SYNTAX) trial compared PCI with the paclitaxel drug-eluting stent to CABG in 1800 patients with three-vessel coronary disease or left main disease. The study found no difference in death or myocardial infarction at 1 year, but repeat revascularization was signifi-296e-3 cantly higher in the stent-treated group (13.5 vs. 5.9%), while stroke was significantly higher in the surgical group (2.2 vs. 0.6%). The primary endpoint of death, myocardial infarction, stroke, or revascularization was significantly better with CABG, particularly in those with the

1	higher in the surgical group (2.2 vs. 0.6%). The primary endpoint of death, myocardial infarction, stroke, or revascularization was significantly better with CABG, particularly in those with the most extensive coronary artery disease. The 3-year results confirm these findings. The Future Revascularization Evaluation in Patients With Diabetes Mellitus: Optimal Management of Multivessel Disease (FREEDOM) trial randomized 1900 patients with diabetes and multivessel disease and showed a significantly lower primary endpoint of death, myocardial infarction, or stroke with CABG than PCI. These studies support CABG for those with the most severe left main and three-vessel disease or those with diabetes. Lesser degrees of multivessel disease in patients with or without diabetes have an equal outcome with PCI.

1	The choice of PCI versus CABG is also related to the anticipated procedural success and complications of PCI and the risks of CABG. For PCI, the characteristics of the coronary anatomy are critically important. The location of the lesion in the vessel (proximal or distal), the degree of tortuosity, and the size of the vessel are considered. In addition, the Is there an increased risk for less favorable long-term outcome due to both stent thrombosis and restenosis concerns? Example: Diabetics with multivessel Yes disease, diffuse three vessel disease Is there a low risk for restenosis? Example: Large vessels, focal lesions Are there relative contraindications to DES? Example: • Medical noncompliance • Chronic oral anti-coagulation • Anticipated surgery • High risk for bleeding Consider BMSYesNoNo

1	FIgUrE 296e-4 In patients requiring revascularization, several factors need to be considered in choosing between bare metal stents, drug-eluting stents, or coronary artery bypass surgery. ACS, acute coronary syndrome; BMS, bare metal stent; CABG, coronary artery bypass grafting; DES, drug-eluting stent; IVUS, intravascular ultrasound; STEMI, ST-segment elevation myocardial infarction. (From AA Bavry, DL Bhatt: Circulation 116:696, 2007; with permission.)

1	PART 10 Disorders of the Cardiovascular System 296e-4 lesion characteristics, including the degree of the stenosis, the presence of calcium, lesion length, and presence of thrombus, are assessed. The most common reason to decide not to do PCI is that the lesion felt to be responsible for the patient’s symptoms is not treatable. This is most commonly due to the presence of a chronic total occlusion (>3 months in duration). In this setting, the historical success rate has been low (30–70%) and complications are more common. A lesion classification to characterize the likelihood of success or failure of PCI has been developed by the ACC/AHA. Lesions with the highest success are called type A lesions (such as proximal noncalcified subtotal lesions), and those with the lowest success or highest complication rate are type C lesions (such as chronic total occlusions). Intermediate lesions are classified as type B1 or B2 depending on the number of unfavorable characteristics. Approximately

1	complication rate are type C lesions (such as chronic total occlusions). Intermediate lesions are classified as type B1 or B2 depending on the number of unfavorable characteristics. Approximately 25–30% of patients will not be candidates for PCI due to unfavorable anatomy, whereas only 5% of CABG patients will not be candidates for surgery due to coronary anatomy. The primary reason for being considered inoperable with CABG is the presence of severe comorbidities such as advanced age, frailty, severe chronic obstructive pulmonary disease (COPD), or poor left ventricular function. Another consideration in choosing a revascularization strategy is the degree of revascularization. In patients with multivessel disease, bypass grafts can usually be placed to all vessels with significant steno-sis, whereas PCI may be able to treat only some of the lesions due to the presence of unfavorable anatomy. Assessment of the significance of intermediate lesions using fractional flow reserve (FFR)

1	whereas PCI may be able to treat only some of the lesions due to the presence of unfavorable anatomy. Assessment of the significance of intermediate lesions using fractional flow reserve (FFR) (Chap.

1	272) can assist in determining which lesions should be revascularized. The Fractional Flow Reserve versus Angiography for Multivessel Evaluation (FAME) trial showed a 30% reduction in adverse events when revascularization by PCI was restricted to those lesions that were hemodynamically significant (FFR ≤0.80) rather than when guided by angiography alone. Thus, complete revascularization of all functionally significant lesions should be favored and considered when choosing the optimal revascularization strategy. Given the multiple factors that need to be considered in choosing the best revascularization for an individual patient with multivessel disease, it is optimal to have a discussion among the cardiac surgeon, interventional cardiologist, and the physicians caring for the patient (so-called Heart Team) to properly weigh the choices.

1	Patients with acute coronary syndrome are at excess risk of short-and long-term mortality. Randomized clinical trials have shown that PCI is superior to intensive medical therapy in reducing mortality and myocardial infarction, with the benefit largely confined to those patients who are high risk. High-risk patients are defined as those with any one of the following: refractory ischemia, recurrent angina, positive cardiac-specific enzymes, new ST-segment depression, low ejection fraction, severe arrhythmias, or a recent PCI or CABG. PCI is preferred over surgical therapy in most high-risk patients with acute coronary syndromes unless they have severe multivessel disease or the culprit lesion responsible for the unstable presentation cannot be adequately treated. In STEMI, thrombolysis or PCI (primary PCI) are effective methods to restore coronary blood flow and salvage myocardium within the first 12 h after onset of chest pain. Because PCI is more effective in restoring flow than

1	or PCI (primary PCI) are effective methods to restore coronary blood flow and salvage myocardium within the first 12 h after onset of chest pain. Because PCI is more effective in restoring flow than thrombolysis, it is preferred if readily available. PCI is also performed following thrombolysis to facilitate adequate reperfusion or as a rescue procedure in those who do not achieve reperfusion from thrombolysis or in those who develop cardiogenic shock.

1	Interventional treatment for structural heart disease (adult congenital heart disease and valvular heart disease) is a significant and growing component of the field of interventional cardiology.

1	The most common adult congenital lesion to be treated with percutaneous techniques is closure of atrial septal defects (Chap. 282). The procedure is done as in a diagnostic right heart catheterization with the passage of a catheter up the femoral vein into the right atrium. With echo and fluoroscopic guidance, the size and location of the defect can be accurately defined, and closure is accomplished using one of several approved devices. All devices use a left atrial and right atrial wire mesh or covered disk that are pulled together to capture the atrial septum around the defect and seal it off. The Amplatzer Septal Occluder device (AGA Medical, Minneapolis, Minnesota) is the most commonly used in the United States. The success rate in selected patients is 85–95%, and the device complications are rare and include device embolization, infection, or erosion. Closure of patent foramen ovale (PFO) is done in a similar way. PFO closure may be considered in patients who have had recurrent

1	are rare and include device embolization, infection, or erosion. Closure of patent foramen ovale (PFO) is done in a similar way. PFO closure may be considered in patients who have had recurrent paradoxical stroke or transient ischemic attack (TIA) despite adequate medical therapy including anticoagulation or antiplatelet therapy. The benefit, however, has not been proven. The CLOSURE I trial randomized 909 patients with cryptogenic stroke or TIA who had a PFO. Closure did not reduce the primary endpoint of death within 30 days or death following a neurologic cause during 2 years of follow-up or stroke/TIA within 2 years. Other trials have confirmed these findings. The use in the treatment of migraine is under clinical investigation and is not an approved indication.

1	Similar devices can also be used to close patent ductus arteriosus and ventricular septal defects. Other congenital diseases that can be treated percutaneously include coarctation of the aorta, pulmonic stenosis, peripheral pulmonary stenosis, and other abnormal communications between the cardiac chambers or vessels.

1	The treatment of valvular heart disease is the most rapidly growing area in interventional cardiology. Until recently, the only available techniques were balloon valvuloplasty for the treatment of aortic, mitral, or pulmonic stenosis (Chap. 283). Mitral valvuloplasty is the preferred treatment for symptomatic patients with rheumatic mitral stenosis who have favorable anatomy. The outcome in these patients is equal to that of surgical commissurotomy. The success is highly related to the echocardiographic appearance of the valve. The most favorable setting is commissural fusion without calcification or subchordal fusion and the absence of significant mitral regurgitation. Access is obtained from the femoral vein using a transseptal technique in which a long metal catheter with a needle tip is advanced from the femoral vein through the right atrium and atrial septum at the level of the foramen ovale into the left atrium. A guidewire is advanced into the left ventricle, and a

1	needle tip is advanced from the femoral vein through the right atrium and atrial septum at the level of the foramen ovale into the left atrium. A guidewire is advanced into the left ventricle, and a balloon-dilatation catheter is negotiated across the mitral valve and inflated to a predetermined size to enlarge the valve. The most commonly used dilatation catheter is the Inoue balloon. The technique splits the commissural fusion and commonly results in a doubling of the mitral valve area. The success of the procedure in favorable anatomy is 95% and severe complications are rare (1–2%). The most common complications are tamponade due to puncture into the pericardium or the creation of severe mitral regurgitation.

1	For regurgitant valvular lesions, only severe mitral regurgitation can be effectively treated percutaneously using the MitraClip (Abbott, Abbott Park, Ill) device. The procedure involves the passage of a catheter into the left atrium using the transseptal technique. A special catheter with a metallic clip on the end is passed through the mitral valve and retracted to catch and clip together the mid portion of the anterior and posterior mitral valve leaflet. The clip creates a double opening in the mitral valve and thereby reduces mitral regurgitation similar to the surgical Alfieri repair. In the Endovascular Valve Edge-to-Edge Repair Study (EVEREST II) trial, the device was less effective than surgical repair or replacement but was shown to be safe. It is currently used for patients who are not good candidates for surgical repair, particularly when the regurgitation is due to functional causes.

1	Severe aortic stenosis can be treated with balloon valvuloplasty as well. In this setting, the valvuloplasty balloon catheter is placed retrograde across the aortic valve from the femoral artery and briefly inflated to stretch open the valve. The success is much less favorable, with only 50% achieving an aortic valve area of >1 cm2 and a restenosis rate of 25–50% after 6–12 months. This poor success rate has limited its use to patients who are not surgical candidates or as a bridge to surgery or transcatheter aortic valve replacement (TAVR). In this setting, the intermediate-term mortality rate of the procedure is high (10%). Repeat aortic valvuloplasty as a treatment for aortic valve restenosis has been reported.

1	Percutaneous aortic valve replacement (TAVR) has been shown to be an effective treatment for high-risk and inoperable patients with aortic stenosis. Currently, two valve models, the Edwards SAPIEN valve (Edwards Lifescience, Irvine, California) and the CoreValve ReValving system (Medtronic, Minneapolis, Minnesota) are available. In more than 10,000 cases worldwide, follow-up shows no evidence of restenosis or severe prosthetic valve dysfunction in the midterm. The CoreValve is self-expanding, while the Edwards valve is balloon expanded. The cannulas are large (14–22 French), and retrograde access via the femoral artery is most commonly chosen, if possible. In patients with peripheral artery disease, access via the subclavian artery or transapically through a surgical incision can be used. Following balloon valvuloplasty, the valve is positioned across the valve and deployed with postdeployment balloon inflation to ensure full contact with the aortic annulus. The success rate is

1	used. Following balloon valvuloplasty, the valve is positioned across the valve and deployed with postdeployment balloon inflation to ensure full contact with the aortic annulus. The success rate is 80–90%, and the 30-day mortality rate is 10–15%, not unexpectedly as only high-risk patients are undergoing the procedure currently. The Placement of Aortic Transcatheter Valve (PARTNER) randomized trial of the Edwards valve showed a 55% reduction in 1-year mortality and major adverse events in the extreme-risk group randomized to TAVR compared to medical therapy. In a separate randomized trial, high-risk patients had a similar outcome to surgical valve replacement at 1 year. As a result, this valve is approved for both high-risk and extreme-risk patients with severe aortic stenosis.

1	Pulmonic stenosis can also be effectively treated with balloon valvuloplasty and percutaneously replaced with the Melody stent (Medtronic). Tricuspid valve interventions remain experimental.

1	The use of percutaneous interventions to treat symptomatic patients with arterial obstruction in the carotid, renal, aortic, and peripheral vessels is also part of the field of interventional cardiology. Randomized clinical trial data already support the use of carotid stenting in patients at high risk of complications from carotid endarterectomy (Fig. 296e-5). Recent trials suggest similar outcomes with carotid stenting and carotid endarterectomy in patients at average risk, although depending on the patient’s risk for periprocedural stroke or myocardial infarction, one procedure may be preferred over the other. The success rate of peripheral artery interventional procedures has been improving, including for long segments of occlusive disease historically treated by peripheral bypass surgery (Fig. 296e-6). Peripheral intervention is increasingly part of the training of an interventional cardiologist, and most programs now require an additional year of training after the

1	bypass surgery (Fig. 296e-6). Peripheral intervention is increasingly part of the training of an interventional cardiologist, and most programs now require an additional year of training after the interventional cardiology training year. The techniques and outcomes are described in detail in the chapter on peripheral vascular disease (Chap. 302).

1	The use of circulatory support techniques is occasionally needed in order to safely perform PCI on hemodynamically unstable patients. It also can be useful in helping to stabilize patients before surgical interventions. The most commonly used device is the percutaneous intraaortic balloon pump developed in the early 1960s. A 7to 10-French, 25to 50-mL balloon catheter is placed retrograde from the femoral artery into the descending aorta between the aortic arch and the abdominal aortic bifurcation. It is connected to a helium gas inflation system that synchronizes the inflation to coincide with early diastole with deflation by mid-diastole. As a result, it increases early diastolic pressure, lowers systolic pressure, and lowers late diastolic pressure through displacement of blood from the descending aorta (counterpulsation). This results in an increase in coronary blood flow and a decrease in afterload. It is contraindicated in patients with aortic regurgitation, aortic dissection, or

1	descending aorta (counterpulsation). This results in an increase in coronary blood flow and a decrease in afterload. It is contraindicated in patients with aortic regurgitation, aortic dissection, or severe peripheral artery disease. The major complications are vascular and thrombotic. Intravenous heparin is given in order to reduce thrombotic complications.

1	Another potentially useful tool is the Impella device (Abiomed, Danvers, Massachusetts). The catheter is placed percutaneously from the femoral artery into the left ventricle. The catheter has a small microaxial pump at its tip that can pump up to 2.5–5 L/min from the left ventricle to the aorta. Other support devices include TandemHeart (CardiacAssist, Pittsburgh, Pennsylvania), which involves placement of a large 21-French catheter from the femoral vein through the right atrium into the left atrium using the transseptal technique and a catheter in the femoral artery. A centrifugal pump can deliver 5 L of blood per minute. It may be useful in patients in shock or with STEMI or very-high-risk PCI. Patients can also be placed on peripheral extracorporeal membrane oxygenation using large cannulas placed in the femoral artery and vein.

1	The treatment of deep vein thrombosis is intravenous anticoagulation, with placement of an inferior vena cava filter if recurrent pulmonary emboli occur. Postphlebitic syndrome is a serious condition due to chronic venous obstruction that can lead to chronic leg edema and venous ulcers. Preliminary studies suggest that mechanical treatments may have a role in treatment, and a large trial is ongoing.

1	Pulmonary emboli (PE) should be treated with fibrinolytic agents if massive and in some cases if submassive. Surgical pulmonary embolectomy is an option for the treatment of massive PE with hemodynamic instability in patients who have contraindications for systemic fibrinolysis or those in whom it has failed. Catheter-based therapies for submassive and massive PEs are still evolving, but studies have shown promise. The techniques employed include the use of aspiration of the clot with a large catheter (10 French), intraclot infusion of a thrombolytic agent followed by aspiration, ultrasound-assisted catheter-directed thrombolysis, and use of rheolytic thrombectomy. Success for these techniques has been reported to be 80–90%, with major complications occurring in 2–4% of patients.

1	The recent recognition of the importance of the renal sympathetic nerves in modulating blood pressure has led to a technique to selectively denervate renal sympathetic nerves in patients with refractory hypertension. The procedure involves applying low-power radiofrequency treatment via a catheter along the length of both renal arteries. In the randomized Symplicity HTN-2 trial,AB renal denervation significantly reduced blood FIgUrE 296e-5 An example of a high-risk patient who requires carotid revasculariza-pressure compared with medical therapy. The tion, but who is not a candidate for carotid endarterectomy. Carotid artery stenting Symplicity device (Medtronic) is approved in resulted in an excellent angiographic result. (From M Belkin, DL Bhatt: Circulation 119:2302, Europe, though the randomized and blinded U.S. 2009; with permission.) Symplicity HTN-3 trial showed no effect.

1	Interventional cardiology continues to expand its borders. Treatment for coronary artery disease, including complex anatomic subsets, continues to advance, encroaching on what has traditionally been treated by CABG. Technological advances such as drug-eluting stents, now already in their second generation, and manual thrombus aspiration devices are improving the A BC results of PCI. In particular, PCI has been shown to prevent future ischemic events in acute coronary syndromes. For patients with stable coronary disease, PCI has an important role in symptom alleviation. Treatment of peripheral and cerebrovascular disease has also benefited from the application of percutaneous techniques. Structural heart disease is increasingly being treated with percutaneous options, with a high likelihood that interventional approaches will compete with open-heart surgery in a significant proportion of cases in years to come.

1	FIgUrE 296e-6 Peripheral interventional procedures have become highly effective at treating anatomic lesions previously amenable only to bypass surgery. A. Complete occlusion of the left superficial femoral artery. B. Wire and catheter advanced into subintimal space. C. Intravascular ultrasound positioned in the subintimal space to guide retrograde wire placement through the occluded vessel. D. Balloon dilation of the occlusion. E. Stent placement with excellent angiographic result. (From A Al Mahameed, DL Bhatt: Cleve Clin J Med 73:S45, 2006; with permission.)

1	E. Stent placement with excellent angiographic result. (From A Al Mahameed, DL Bhatt: Cleve Clin J Med 73:S45, 2006; with permission.) Atlas of Percutaneous Revascularization Jane A. Leopold, Deepak L. Bhatt, David P. Faxon Percutaneous coronary intervention (PCI) is the most widely employed coronary revascularization procedure worldwide (Chap. 296e). It 297e is now applied to patients with stable angina; patients with acute coronary syndromes, including unstable angina and non-ST-segment elevation myocardial infarction (NSTEMI); and as a primary treatment strategy in patients with ST-segment elevation myocardial infarction (STEMI). PCI is also applicable to patients with either single-vessel or multivessel disease.

1	In this chapter, the use of PCI will be illustrated in a variety of commonly encountered clinical and anatomic situations, such as chronic total occlusion of a coronary artery, bifurcation disease, acute STEMI, saphenous vein graft disease, left main coronary artery disease, multivessel disease, and stent thrombosis. In addition, the use of interventional techniques to treat structural heart disease will be shown, including closure of an atrial septal defect (ASD) and transcatheter aortic valve replacement (TAVR). CASE 1: CHRONIC TOTAL OCCLUSION (Videos 297e-1 to 297e-7) An 81-year-old male with angina, New York Heart Association (NYHA) class IV congestive heart failure and inferoapicoposterior ischemia on an exercise technetium-99m scan.

1	An 81-year-old male with angina, New York Heart Association (NYHA) class IV congestive heart failure and inferoapicoposterior ischemia on an exercise technetium-99m scan. Diagnostic cardiac catheterization revealed a left dominant system with a totally occluded left circumflex (LCx) artery. The distal LCx filled via collaterals from the left anterior descending (LAD) artery, indicating chronicity of the total occlusion. VIDEO 297e-1 Baseline left coronary angiogram shows an occluded LCx with left-to-left collaterals originating from LAD septal vessels. VIDEO 297e-2 Attempts to cross the total occlusion in the LCx using a hydrophilic wire and an antegrade approach were not successful with the wire tracking to the right of the trajectory.

1	VIDEO 297e-3 The LAD septal collateral is accessed with a guidewire 297e-1 that is directed toward the distal LCx to cross the total occlusion retrograde. VIDEO 297e-4 The total occlusion is crossed retrograde. The wire is snared in the guide, exteriorized, and used to provide antegrade access to the LCx. VIDEO 297e-5 Antegrade flow in the LCx is restored after balloon inflation. VIDEO 297e-6 Following stenting of the total occlusion, blood flow in the distal vessel is improved and a second significant stenosis is seen. VIDEO 297e-7 Final result after LCx stenting. Approximately 15–30% of all patients referred for cardiac catheterization will have a chronic total occlusion (CTO) of a coronary artery. CTO often leads to a surgical referral for complete revascularization. Incomplete revascularization due to an untreated CTO is associated with an increased mortality rate (hazard ratio = 1.36; 95% confidence interval [CI], 1.12–1.66, p <.05).

1	Incomplete revascularization due to an untreated CTO is associated with an increased mortality rate (hazard ratio = 1.36; 95% confidence interval [CI], 1.12–1.66, p <.05). Successful PCI of a CTO leads to a 3.8–8.4% absolute reduction in mortality, symptom relief, and improved left ventricular function. Newer techniques, such as the retrograde approach to crossing total occlusions, are useful when the antegrade approach fails or is not feasible and there are well-developed collateral vessels. (Case contributed with permission by Dr. Frederick G.P. Welt.) CASE 2: BIFURCATION STENTING (Fig. 297e-1; Videos 297e-8 to 297e-16) A 52-year-old male with an acute coronary syndrome and a troponin I = 0.18 (upper limit of normal, <0.04). Diagnostic cardiac catheterization showed single-vessel coronary artery disease with a significant stenosis in the mid-LAD and a bifurcation lesion involving a large diagonal branch.

1	Diagnostic cardiac catheterization showed single-vessel coronary artery disease with a significant stenosis in the mid-LAD and a bifurcation lesion involving a large diagonal branch. VIDEO 297e-8 Baseline angiogram of the left coronary circulation shows the significant stenosis in the mid-LAD and the bifurcation lesion involving a large diagonal branch. CHAPTER 297e Atlas of Percutaneous Revascularization

1	CHAPTER 297e Atlas of Percutaneous Revascularization FIGURE 297e-1 Schematic representation of one-stent and two-stent techniques to treat bifurcation lesions. PTCA, percutaneous transluminal coronary angioplasty 297e-2 VIDEO 297e-9 Both vessels are accessed with guidewires and pretreated with balloon angioplasty. VIDEO 297e-10 Result after balloon angioplasty. VIDEO 297e-11 Stent being positioned in the LAD. VIDEO 297e-12 LAD poststent result. VIDEO 297e-13 Stent deployed in the diagonal branch through the stent struts in the LAD using the “culotte” technique. VIDEO 297e-14 Diagonal branch poststent result. VIDEO 297e-15 Simultaneous inflation of two 2.5-mm “kissing” balloons. VIDEO 297e-16 Final postbifurcation stenting result. Approximately 15–20% of PCIs will involve the treatment of bifurcation lesions. Bifurcation lesions require consideration of PCI strategies that protect side-branch patency.

1	Approximately 15–20% of PCIs will involve the treatment of bifurcation lesions. Bifurcation lesions require consideration of PCI strategies that protect side-branch patency. There are both one-stent and two-stent techniques to treat bifurcation lesions; the selection of technique depends on anatomic considerations, including plaque burden, angle of side-branch take-off, plaque shift during angioplasty, and side-branch distribution. Rates of target lesion revascularization and stent thrombosis are similar between one-stent and two-stent procedures. CASE 3: INFERIOR MYOCARDIAL INFARCTION—THROMBUS AND MANUAL THROMBECTOMY PART 10 Disorders of the Cardiovascular System (Figs. 297e-2 to 297e-4; Videos 297e-17 to 297e-22) A 59-year-old male presented to the emergency room with 2 h of severe midsternal chest pressure. His systolic blood pressure was 100 mmHg, and he was tachycardic in sinus rhythm with a heart rate of 90–100 beats/min.

1	His systolic blood pressure was 100 mmHg, and he was tachycardic in sinus rhythm with a heart rate of 90–100 beats/min. His initial electrocardiogram (ECG) showed inferior ST-segment elevations with lateral ST-segment depressions. He was referred emergently to the cardiac catheterization laboratory for primary PCI. VIDEO 297e-17 The right coronary artery (RCA) is totally occluded with filling defects in the vessel after contrast injection, indicating thrombus is present in the vessel. VIDEO 297e-18 An angioplasty wire is threaded through the thrombotic lesion, but this does not restore blood flow to the distal vessel. VIDEO 297e-19 Result after manual thrombectomy and thrombus extraction. The “culprit” ruptured plaque and residual thrombus are now apparent in the vessel. FIGURE 297e-3 Example of an organized red thrombus retrieved by manual thrombectomy.

1	FIGURE 297e-3 Example of an organized red thrombus retrieved by manual thrombectomy. VIDEO 297e-20 After balloon angioplasty and stenting, thrombus is still present. VIDEO 297e-21 After repeat manual thrombectomy and expansion of the stent, the thrombus is no longer present. VIDEO 297e-22 Final result. An acute STEMI occurs following plaque rupture that promotes thrombotic occlusion of a coronary artery. Despite successful revascularization of the epicardial coronary artery, microemboli liberated during balloon angioplasty and stenting may lead to persistent microvascular dysfunction. When present, microvascular dysfunction is associated with a larger infarct size, heart failure, malignant ventricular arrhythmias, and death. Manual thrombectomy is used to aspirate or remove thrombus in the vessel and limit distal embolization during angioplasty and stenting. Manual thrombectomy in primary PCI is associated with improved myocardial perfusion and a reduction in mortality.

1	Manual thrombectomy in primary PCI is associated with improved myocardial perfusion and a reduction in mortality. Adjunctive antiplatelet and antithrombin agents are important to aid in the resolution of intracoronary thrombus. FIGURE 297e-2 Preprocedure ECG showing inferior ST-segment elevations and lateral ST-segment depressions. FIGURE 297e-4 Postprocedure ECG showing resolution of ST-segment elevations. FIGURE 297e-5 Distal protection device showing captured atherosclerotic debris liberated by initial balloon dilation. CASE 4: SAPHENOUS VEIN GRAFT INTERVENTION WITH DISTAL PROTECTION (Fig. 297e-5; Videos 297e-23 to 297e-26) A 62-year-old male with a history of chronic stable angina.

1	CASE 4: SAPHENOUS VEIN GRAFT INTERVENTION WITH DISTAL PROTECTION (Fig. 297e-5; Videos 297e-23 to 297e-26) A 62-year-old male with a history of chronic stable angina. A four-vessel coronary artery bypass grafting (CABG) surgery was performed 17 years earlier with a left internal mammary artery graft to the LAD, a right internal mammary artery graft to the right coronary artery (RCA), a saphenous vein graft to the first obtuse marginal branch, and a saphenous vein graft to the first diagonal branch. The patient had a recent increase in angina with exertion and was found to have lateral ischemia on an exercise technetium-99m scan. Diagnostic cardiac catheterization revealed a significant stenosis in the body of the saphenous vein graft to the first obtuse marginal branch.

1	Diagnostic cardiac catheterization revealed a significant stenosis in the body of the saphenous vein graft to the first obtuse marginal branch. VIDEO 297e-23 Saphenous vein graft to a first obtuse marginal branch with an 80% eccentric stenosis in the midgraft. VIDEO 297e-24 A distal protection device is deployed past the lesion. VIDEO 297e-25 Angioplasty balloon inflation with the distal protection device in place. VIDEO 297e-26 Final result after stent placement. Saphenous vein grafts have a failure rate of up to 20% after 1 year and as high as 50% by 5 years. Graft failure (after >1 month) results from intimal hyperplasia and atherosclerosis. Saphenous vein graft PCI is associated with distal embolization of 297e-3 atherosclerotic debris and microthrombi leading to microvascular occlusion, reduced antegrade blood flow (the “no-reflow” phenomenon), and myocardial infarction.

1	Embolic distal protection devices decrease the risk of distal embolization, as well as the incidence of no-reflow and myocardial infarction associated with saphenous vein graft interventions. CASE 5: UNPROTECTED LEFT MAIN PCI IN A HIGH-RISK PATIENT (Figs. 297e-6 and 297e-7; Videos 297e-27 to 297e-34) An 89-year-old woman presented with a NSTEMI associated with 5-mm ST-segment depression in the apical leads occurring 2 weeks after hospitalization for a NSTEMI that was treated conservatively. Chronic obstructive lung disease, elderly age, and the patient’s refusal to consider cardiac surgery restricted the choice of therapeutic options to medical and/or percutaneous interventions. Diagnostic catheterization revealed a left dominant circulation with a heavily calcified 80% distal left main coronary artery stenosis extending into the LAD and into the proximal LCx coronary arteries. A 70% proximal LAD lesion was also present.

1	After consultation with the patient, family, and a cardiac surgeon, PCI was performed with intraaortic balloon pump support and a temporary pacemaker in the right ventricle.

1	VIDEO 297e-27 Baseline left coronary artery injection in right anterior oblique (RAO) cranial projection shows a high-grade calcified stenosis in the left main coronary artery and a significant stenosis in the proximal LAD. VIDEO 297e-28 In the left anterior oblique (LAO) caudal view, the left main coronary artery lesion can be seen to extend into the ostia of both the LCx and the LAD. VIDEO 297e-29 Guidewires were placed into both the LCx and LAD. After the left main coronary artery and LCx are dilated with balloon angioplasty, the proximal LAD is dilated, and a long drug-eluting stent is placed to cover a lesion dissection that occurred with wiring of the vessel. VIDEO 297e-30 The bifurcation lesion in the left main coronary artery extending into the LCx and LAD ostia is treated using a “culotte” technique. First, a drug-eluting stent is placed in the left main coronary artery and into the proximal LCx. CHAPTER 297e Atlas of Percutaneous Revascularization

1	CHAPTER 297e Atlas of Percutaneous Revascularization FIGURE 297e-6 During chest pain, the ECG showed diffuse ST-segment depression of up to 5 mm in the inferior and lateral leads. PART 10 Disorders of the Cardiovascular System FIGURE 297e-7 Following resolution of the chest pain, the ST-segment depression is less marked. VIDEO 297e-31 Next, the LAD wire is removed and passed through the stent into the distal LAD. A second drug-eluting stent is deployed through the struts of the left main coronary artery/LCx stent. VIDEO 297e-32 Following rewiring of the LCx, both stents are re-dilated simultaneously (“kissing” balloons). VIDEO 297e-33 The final result in the LAO caudal view. VIDEO 297e-34 The final result in the RAO cranial view showing patent left main, LCx, and LAD coronary arteries. Left main coronary artery disease occurs in 5–10% of patients with symptomatic coronary artery disease.

1	Left main coronary artery disease occurs in 5–10% of patients with symptomatic coronary artery disease. In patients with left main coronary artery disease, revascularization with CABG has been shown to decrease mortality significantly over 5–10 years of follow-up. PCI with drug-eluting stents in selected cases has been shown to have equal in-hospital and 1-year death and myocardial infarction rates compared with CABG in the Synergy between PCI with Taxus and Cardiac Surgery (SYNTAX) trial. Long-term outcome differences between the two treatment strategies are not known.

1	Indications for PCI of left main coronary artery lesions are high-risk surgical patients and patients with protected left main coronary artery disease (i.e., prior CABG with patent bypass grafts). Patients who are good candidates for bypass surgery may also undergo a stenting procedure, but discussion with the patient, the interventional cardiologist, and the cardiac surgeon should be undertaken to determine the best treatment option in an individual case. Outcomes are better for patients with an isolated lesion in the ostium and body of the left main coronary artery where a single stent can be placed compared to bifurcation lesions that involve the ostium of the LAD and LCx. CASE 6: MULTIVESSEL PCI IN A DIABETIC PATIENT (Videos 297e-35 to 297e-42) A 58-year-old man presented with a NSTEMI. The patient has hyperlipidemia and type 2 diabetes mellitus treated with oral hypoglycemic agents.

1	A 58-year-old man presented with a NSTEMI. The patient has hyperlipidemia and type 2 diabetes mellitus treated with oral hypoglycemic agents. Diagnostic catheterization revealed two-vessel disease with a total occlusion of the second obtuse marginal branch that was felt to be responsible for the patient’s symptoms (culprit lesion). In addition, there was a high-grade stenosis in a large ramus intermedius branch, and the RCA had a significant stenosis in the midsegment of the vessel.

1	VIDEO 297e-35 Baseline angiogram of the left coronary circulation in the RAO view shows the total occlusion of the second obtuse marginal branch with delayed retrograde filling via collateral vessels and a high-grade stenosis in the ramus intermedius. VIDEO 297e-36 A guidewire is passed through the total occlusion, and the lesion is pretreated with balloon angioplasty. VIDEO 297e-37 Following placement of a drug-eluting stent in the lesion, the vessel is widely patent. A third obtuse marginal vessel, not previously seen, now fills faintly (Thrombolysis in Myocardial Infarction [TIMI] grade 1 flow) with contrast but was not treated. VIDEO 297e-38 The ramus intermedius lesion was crossed with a guidewire and pretreated with balloon angioplasty. VIDEO 297e-39 A drug-eluting stent is placed across the ramus lesion and deployed. The final result shows no residual stenosis in either the ramus or second obtuse marginal vessels. VIDEO 297e-40 Baseline angiogram of the RCA shows a high-grade

1	across the ramus lesion and deployed. The final result shows no residual stenosis in either the ramus or second obtuse marginal vessels. VIDEO 297e-40 Baseline angiogram of the RCA shows a high-grade lesion in the midsegment of the vessel. VIDEO 297e-41 The lesion was pretreated with balloon dilation followed by stent deployment. VIDEO 297e-42 The final result shows no residual stenosis in the mid-RCA.

1	Multivessel PCI is performed commonly and may be done in one setting or staged with two or more procedures. Acute and long-term studies of multivessel PCI have shown comparable rates of death and myocardial infarction when compared to CABG, but a higher incidence of repeat revascularization as a result of restenosis is associated with PCI. In the randomized Bypass Angioplasty Revascularization Investigation (BARI) trial, diabetic patients treated with PCI had a worse long-term mortality than diabetic patients treated with CABG. However, the BARI registry found that in selected diabetic patients with favorable anatomy, PCI can result in outcomes equal to those observed with CABG. FIGURE 297e-8 Optical coherence tomography image following initial balloon dilation. Residual thrombus that is adherent to the stent struts is seen. CASE 7: VERY LATE STENT THROMBOSIS OF A PROXIMAL LAD DRUG-ELUTING STENT (Figs. 297e-8 and 297e-9; Videos 297e-43 to 297e-46)

1	CASE 7: VERY LATE STENT THROMBOSIS OF A PROXIMAL LAD DRUG-ELUTING STENT (Figs. 297e-8 and 297e-9; Videos 297e-43 to 297e-46) A 62-year-old male had a drug-eluting stent placed in a proximal LAD lesion to treat severe angina. He received dual antiplatelet therapy with aspirin and clopidogrel for 1 year and then discontinued clopidogrel per protocol. He remained asymptomatic until 15 months after the initial stent placement when he presented with severe chest pain due to an acute anterior STEMI. He was taken to the catheterization laboratory within 70 min of presentation, and his initial angiogram showed a total occlusion of the proximal LAD stent. VIDEO 297e-43 Baseline angiogram showing a total occlusion of the proximal LAD within the drug-eluting stent and a significant stenosis at the origin of the LCx.

1	VIDEO 297e-43 Baseline angiogram showing a total occlusion of the proximal LAD within the drug-eluting stent and a significant stenosis at the origin of the LCx. VIDEO 297e-44 The LAO view shows the LCx stenosis with a filling 297e-5 defect indicating that thrombus is present in the vessel lumen. VIDEO 297e-45 The LAD lesion was crossed with a guidewire, which resulted in slow filling of the mid-LAD (TIMI 2 flow) and revealed thrombus filling the stent. VIDEO 297e-46 The final result after LAD and LCx stenting. The LAD lesion was pretreated with balloon angioplasty, and a bare metal stent was deployed to cover the proximal lesion. The LCx ostial lesion was dilated with balloon angioplasty, and a bare metal stent was placed using a “V stenting” technique.

1	Stent thrombosis is an infrequent (1–2%) but serious complication of stent placement. It occurs most commonly within the first month, but rarely can occur as late as 1 year (0.2–0.6%) with bare metal stents. Very late stent thrombosis (VLST), which occurs after 1 year, is very rare with bare metal stents but can occur with drug-eluting stents. Premature discontinuation of dual antiplatelet therapy is the most common cause of early and late stent thrombosis; however, the etiology of VLST is not clear. The majority of patients with stent thrombosis present with acute coronary syndromes or STEMI; this presentation is associated with a high mortality rate (10%). Treatment is immediate PCI with balloon angioplasty or re-stenting. CASE 8: TRANSCATHETER AORTIC VALVE REPLACEMENT CHAPTER 297e Atlas of Percutaneous Revascularization (Figs. 297e-10 to 297e-14; Videos 297e-47 to 297e-50)

1	CASE 8: TRANSCATHETER AORTIC VALVE REPLACEMENT CHAPTER 297e Atlas of Percutaneous Revascularization (Figs. 297e-10 to 297e-14; Videos 297e-47 to 297e-50) A 75-year-old female with symptomatic aortic stenosis and a valve area of 0.58 cm2 by transthoracic echocardiogram. Chronic obstructive pulmonary disease (forced expiratory volume in 1 s [FEV1] = 0.54) and other comorbidities contributed to an unacceptably high cardiac surgical risk (calculated logistic Euroscore = 29.57%) for aortic valve replacement. She was referred for transcatheter aortic valve replacement (TAVR) as part of a clinical trial.

1	She was referred for transcatheter aortic valve replacement (TAVR) as part of a clinical trial. VIDEO 297e-47 Aortogram shows patent coronary arteries and minimal aortic insufficiency. VIDEO 297e-48 Balloon valvuloplasty is performed with rapid ventricular pacing at 180 beats/min. VIDEO 297e-49 A 26-mm Edwards SAPIEN valve is positioned using fluoroscopic and transesophageal echo guidance and deployed. VIDEO 297e-50 Aortogram after valve deployment shows a functional valve with mild aortic insufficiency and without impingement of the coronary ostia. FIGURE 297e-9 Pathologic specimen of late stent thrombosis obtained at autopsy. Thrombus is seen filling the LAD vessel lumen and extending into a diagonal branch (LD). Stent struts occupied the space denoted by the asterisk (*) (left). A magnified view of the vessel reveals thrombus around the stent strut and neointima formation (arrow) (right). Disorders of the Cardiovascular System

1	Disorders of the Cardiovascular System FIGURE 297e-10 Transesophageal echocardiogram shows a calcified trileaflet aortic valve (left) with reduced leaflet excursion and a narrowed orifice in peak systole (right). FIGURE 297e-11 Hemodynamically significant aortic (AO) steno-sis. Simultaneous recording of AO and left ventricle (LV) pressures shows an 82 mmHg peak-to-peak gradient and a 63.3 mmHg mean gradient between the LV (154/9 mmHg) and AO (72/29 mmHg) pres-sures. This is consistent with an aortic valve area of 0.58 cm2. FIGURE 297e-13 The Edwards SAPIEN transcatheter heart valve. (Reprinted with permission from A Zajarias, AG Cribier: J Am Coll Cardiol 53:1829, 2009.) FIGURE 297e-12 After balloon valvuloplasty, the LV-AO mean pres-sure gradient decreased to 37.3 mmHg, indicating that the aortic valve area increased to 0.95 cm2.

1	FIGURE 297e-12 After balloon valvuloplasty, the LV-AO mean pres-sure gradient decreased to 37.3 mmHg, indicating that the aortic valve area increased to 0.95 cm2. FIGURE 297e-14 Once the valve was deployed, the pressure gradi-ent between the LV and AO decreased to 11.6 mmHg, and the func-tional valve area is 1.34 cm2. The prevalence of calcific aortic stenosis is 2–3% in individuals age ≥75 years. Symptomatic aortic stenosis is associated with an average survival of 2–3 years and an increased risk of sudden death; aortic valve replacement improves both symptoms and survival. In high-risk patients with severe aortic stenosis who are not surgical candidates, 1and 5-year survival rates are ~62% and 38%, respectively. TAVR is approved in Europe and was recently approved in the United States as an alternative to surgical aortic valve replacement in high-risk patients. (Case contributed with permission by Dr. Andrew C. Eisenhauer.)

1	(Case contributed with permission by Dr. Andrew C. Eisenhauer.) CASE 9: ATRIAL SEPTAL DEFECT CLOSURE (Figs. 297e-15 to 297e-21; Videos 297e-51 to 297e-53) A 48-year-old female with increased shortness of breath, exercise intolerance, and an 18-mm secundum ASD. Echocardiogram showed a dilated right atrium (RA) and right ventricle (RV) with evidence of right ventricular volume overload.

1	Echocardiogram showed a dilated right atrium (RA) and right ventricle (RV) with evidence of right ventricular volume overload. FIGURE 297e-15 Anatomic location of common atrial septal defects (ASDs). The most common ASDs are the sinus venosus, ostium secundum, and ostium primum ASDs. The sinus venosus ASD is located at the junction of the superior vena cava (SVC) and the right atrium (RA). This ASD is often associated with anomalous drainage of the right-side pulmonary veins into the RA instead of the left atrium. The secundum ASD is located at the foramen ovale and allows blood to flow between the RA and the left atrium. The primum ASD, also known as an atrioventricular septal defect, connects the RA and right ventricle with the left atrium and ventricle. (Illustration by Justin E. Tribuna.) FIGURE 297e-17 Three-dimensional echocardiographic recon-struction of the secundum ASD. The ASD is round and has an acceptable margin of tissue to seat a septal occluder device.

1	FIGURE 297e-17 Three-dimensional echocardiographic recon-struction of the secundum ASD. The ASD is round and has an acceptable margin of tissue to seat a septal occluder device. CHAPTER 297e Atlas of Percutaneous Revascularization FIGURE 297e-18 ASD percutaneous closure devices. The Amplatzer® septal occluder (left) and the HELEX® septal occluder (right) are among the devices used for percutaneous closure of ASDs. The Amplatzer® septal occluder is a plug between two disks that are positioned on each side of the ASD to obstruct blood flow. The HELEX® Septal Occluder is positioned on each side of the ASD to allow circular rings covered with rapidly stretching polytetrafluoroethylene device to limit blood flow. The delivery catheters are detached and the devices are left in place. (Illustration by Justin E. Tribuna.)

1	FIGURE 297e-16 Transesophageal echocardiogram of a secundum ASD. The ASD is seen as “dropout” in the interatrial septum between the left atrium (LA) and RA (left). Doppler color flow imaging shows blue in the RA consistent with left-to-right flow (right). Disorders of the Cardiovascular System FIGURE 297e-19 Transesophageal echocardiogram showing sizing balloon (left) and no flow (right) across the atrial septal defect. FIGURE 297e-20 Amplatzer® septal occluder in place (left). There is no blood flow across the device (right). FIGURE 297e-21 Postprocedure lateral chest x-ray showing the Amplatzer® septal occluder in place. A shunt ratio (Qp/Qs) of 2.3:1 was determined at cardiac catheterization. Based on her symptoms, evidence of right-side chamber dilation, and a moderately sized ASD, the patient was referred for percutaneous closure of the ASD.

1	Based on her symptoms, evidence of right-side chamber dilation, and a moderately sized ASD, the patient was referred for percutaneous closure of the ASD. VIDEO 297e-51 A sizing balloon is placed across the ASD. VIDEO 297e-52 An Amplatzer® septal occluder is being positioned across the ASD. VIDEO 297e-53 The two disks of the device in place across the ASD. • Unrepaired ASDs lead to signs and symptoms of increased pulmonary blood flow and right heart failure, dyspnea, exercise intolerance, fatigue, palpitations and atrial arrhythmias, and pulmonary infections. Percutaneous closure of an ASD may be recommended for indi-297e-9 viduals with a secundum ASD and evidence of RA and RV enlargement with or without symptoms. Percutaneous closure is contraindicated in patients with irreversible pulmonary arterial hypertension and no left-to-right shunt. It is not recommended for closure of sinus venosus, coronary sinus, or primum ASDs.

1	After the atrial septal occluder device is placed, patients are treated with antiplatelet agents and use antibiotic prophylaxis for certain procedures for 6 months. Follow-up echocardiograms to assess for device migration or erosion, residual shunting, thrombus, or pericardial effusion are recommended at 1 day, 1 month, 6 months, 1 year, and periodically thereafter. (Case contributed with permission by Dr. Andrew C. Eisenhauer.) CHAPTER 297e Atlas of Percutaneous Revascularization

1	(Case contributed with permission by Dr. Andrew C. Eisenhauer.) CHAPTER 297e Atlas of Percutaneous Revascularization Hypertensive vascular Disease Theodore A. Kotchen Hypertension is one of the leading causes of the global burden of disease. Approximately 7.6 million deaths (13–15% of the total) and 92 million disability-adjusted life years worldwide were attributable to high blood pressure in 2001. Hypertension doubles the risk of cardio-298 vascular diseases, including coronary heart disease (CHD), congestive heart failure (CHF), ischemic and hemorrhagic stroke, renal failure, and peripheral arterial disease. It often is associated with additional cardiovascular disease risk factors, and the risk of cardiovascular disease increases with the total burden of risk factors. Although anti-hypertensive therapy reduces the risks of cardiovascular and renal disease, large segments of the hypertensive population are either untreated or inadequately treated.

1	Blood pressure levels, the rate of age-related increases in blood pressure, and the prevalence of hypertension vary among countries and among subpopulations within a country. Hypertension is present in all populations except for a small number of individuals living in developing countries. In industrialized societies, blood pressure increases steadily during the first two decades of life. In children and adolescents, blood pressure is associated with growth and maturation. Blood pressure “tracks” over time in children and between adolescence and young adulthood. In the United States, average systolic blood pressure is higher for men than for women during early adulthood, although among older individuals the age-related rate of rise is steeper for women. Consequently, among individuals age 60 and older, systolic blood pressures of women are higher than those of men. Among adults, diastolic blood pressure also increases progressively with age until ~55 years, after which it tends to

1	age 60 and older, systolic blood pressures of women are higher than those of men. Among adults, diastolic blood pressure also increases progressively with age until ~55 years, after which it tends to decrease. The consequence is a widening of pulse pressure (the difference between systolic and diastolic blood pressure) beyond age 60.

1	1612 In the United States, based on results of the National Health and Nutrition Examination Survey (NHANES), approximately 30% (ageadjusted prevalence) of adults, or at least 65 million individuals, have hypertension (defined as any one of the following: systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg, taking antihypertensive medications). Hypertension prevalence is 33.5% in non-Hispanic blacks, 28.9% in non-Hispanic whites, and 20.7% in Mexican Americans. The likelihood of hypertension increases with age, and among individuals age ≥60, the prevalence is 65.4%. Recent evidence suggests that the prevalence of hypertension in the United States may be increasing, possibly as a consequence of increasing obesity. The prevalence of hypertension and stroke mortality rates are higher in the southeastern United States than in other regions. In African Americans, hypertension appears earlier, is generally more severe, and results in higher rates of morbidity and mortality

1	higher in the southeastern United States than in other regions. In African Americans, hypertension appears earlier, is generally more severe, and results in higher rates of morbidity and mortality from stroke, left ventricular hypertrophy, CHF, and end-stage renal disease (ESRD) than in white Americans. Both environmental and genetic factors may contribute to regional and racial variations in hypertension prevalence. Studies of societies undergoing “acculturation” and studies of migrants from a less to a more urbanized setting indicate a profound environmental contribution to blood pressure. Obesity and weight gain are strong, independent risk factors for hypertension. It has been estimated that 60% of hypertensives are >20% overweight. Among populations, hypertension prevalence is related to dietary NaCl intake, and the age-related increase in blood pressure may be augmented by a high NaCl intake. Low dietary intakes of calcium and potassium also may contribute to the risk of

1	related to dietary NaCl intake, and the age-related increase in blood pressure may be augmented by a high NaCl intake. Low dietary intakes of calcium and potassium also may contribute to the risk of hypertension. The urine sodium-to-potassium ratio (an index of both sodium and potassium intakes) is a stronger correlate of blood pressure than is either sodium or potassium alone. Alcohol consumption, psychosocial stress, and low levels of physical activity also may contribute to hypertension. Adoption, twin, and family studies document a significant heritable component to blood pressure levels and hypertension. Family studies controlling for a common environment indicate that blood pressure heritabilities are in the range 15–35%. In twin studies, heritability estimates of blood pressure are ~60% for males and 30–40% for females. High blood pressure before age 55 occurs 3.8 times more frequently among persons with a positive family history of hypertension. However, to date, only a

1	are ~60% for males and 30–40% for females. High blood pressure before age 55 occurs 3.8 times more frequently among persons with a positive family history of hypertension. However, to date, only a fraction of high heritability estimates are accounted for by specific genetic determinants.

1	Although specific genetic variants have been identified in rare Mendelian forms of hypertension (Table 298–5), these variants are not applicable to the vast majority (>98%) of patients with hypertension. For most individuals, it is likely that hypertension represents a polygenic disorder in which a combination of genes acts in concert with environmental exposures to make only a modest contribution to blood pressure. Further, different subsets of genes may lead to different phenotypes associated with hypertension, e.g., obesity, dyslipidemia, insulin resistance.

1	Several strategies are being used in the search for specific hypertension-related genes. Animal models (including selectively bred rats and congenic rat strains) provide a powerful approach for evaluating genetic loci and genes associated with hypertension. Comparative mapping strategies allow for the identification of syntenic genomic regions between the rat and human genomes that may be involved in blood pressure regulation. In association studies, different alleles (or combinations of alleles at different loci) of specific candidate genes or chromosomal regions are compared in hypertensive patients and normotensive control subjects. Current evidence suggests that genes that encode components of the renin-angiotensin-aldosterone system, along with angiotensinogen and angiotensin-converting enzyme (ACE) polymorphisms, may be related to hypertension and to blood pressure sensitivity to dietary NaCl. The alpha-adducin gene is thought to be associated with increased renal tubular

1	enzyme (ACE) polymorphisms, may be related to hypertension and to blood pressure sensitivity to dietary NaCl. The alpha-adducin gene is thought to be associated with increased renal tubular absorption of sodium, and variants of this gene may be associated with hypertension and salt sensitivity of blood pressure. Other genes possibly related to hypertension include genes encoding the AT1 receptor, aldosterone synthase, atrial natriuretic peptide, and the β2 adrenoreceptor. Genomewide association studies involve rapidly scanning markers across the entire genome to identify loci (not specific genes) associated with an observable trait (e.g., blood pressure) or a particular disease. This strategy has been facilitated by the availability of dense genotyping chips and the International HapMap. Results of candidate gene studies often have not been replicated, and in contrast to several other polygenic disorders, genomewide association studies have had limited success in identifying genetic

1	Results of candidate gene studies often have not been replicated, and in contrast to several other polygenic disorders, genomewide association studies have had limited success in identifying genetic determinants of hypertension.

1	Preliminary evidence suggests that there may also be genetic determinants of target organ damage attributed to hypertension. Family studies indicate significant heritability of left ventricular mass, and there is considerable individual variation in the responses of the heart to hypertension. Family studies and variations in candidate genes associated with renal damage suggest that genetic factors also may contribute to hypertensive nephropathy. Specific genetic variants have been linked to CHD and stroke. In the future, it is possible that DNA analysis will predict individual risk for hypertension and target organ damage and will identify responders to specific classes of antihypertensive agents. However, with the exception of the rare, monogenic hypertensive diseases, the genetic variants associated with hypertension remain to be confirmed, and the intermediate steps by which these variants affect blood pressure remain to be determined.

1	To provide a framework for understanding the pathogenesis of and treatment options for hypertensive disorders, it is useful to understand factors involved in the regulation of both normal and elevated arterial pressure. Cardiac output and peripheral resistance are the two determinants of arterial pressure (Fig. 298-1). Cardiac output is determined by stroke volume and heart rate; stroke volume is related to myocardial contractility and to the size of the vascular compartment. Peripheral resistance is determined by functional and anatomic changes in small arteries (lumen diameter 100–400 μm) and arterioles.

1	Sodium is predominantly an extracellular ion and is a primary determinant of the extracellular fluid volume. When NaCl intake exceeds the capacity of the kidney to excrete sodium, vascular volume may initially expand and cardiac output may increase. However, many vascular beds have the capacity to autoregulate blood flow, and if constant blood flow is to be maintained in the face of increased arterial pressure, resistance within that bed must increase, since

1	The initial elevation of blood pressure in response to vascular volume expansion may be related to an increase of cardiac output; however, over time, peripheral resistance increases and cardiac output reverts toward normal. Whether this hypothesized sequence of events occurs in the pathogenesis of hypertension is not clear. What is clear is that salt can activate a number of neural, endocrine/paracrine, and vascular mechanisms, all of which have the potential to increase arterial pressure. The effect of sodium on blood pressure is related to the provision of sodium with chloride; nonchloride salts of sodium have FIGURE 298-1 Determinants of arterial pressure.

1	FIGURE 298-1 Determinants of arterial pressure. little or no effect on blood pressure. As arterial pressure increases in response to a high NaCl intake, urinary sodium excretion increases and sodium balance is maintained at the expense of an increase in arterial pressure. The mechanism for this “pressure-natriuresis” phenomenon may involve a subtle increase in the glomerular filtration rate, decreased absorbing capacity of the renal tubules, and possibly hormonal factors such as atrial natriuretic factor. In individuals with an impaired capacity to excrete sodium, greater increases in arterial pressure are required to achieve natriuresis and sodium balance.

1	NaCl-dependent hypertension may be a consequence of a decreased capacity of the kidney to excrete sodium, due either to intrinsic renal disease or to increased production of a salt-retaining hormone (mineralocorticoid) resulting in increased renal tubular reabsorption of sodium. Renal tubular sodium reabsorption also may be augmented by increased neural activity to the kidney. In each of these situations, a higher arterial pressure may be required to achieve sodium balance. Conversely, salt-wasting disorders are associated with low blood pressure levels. ESRD is an extreme example of volume-dependent hypertension. In ~80% of these patients, vascular volume and hypertension can be controlled with adequate dialysis; in the other 20%, the mechanism of hypertension is related to increased activity of the reninangiotensin system and is likely to be responsive to pharmacologic blockade of renin-angiotensin.

1	Adrenergic reflexes modulate blood pressure over the short term, and adrenergic function, in concert with hormonal and volume-related factors, contributes to the long-term regulation of arterial pressure. Norepinephrine, epinephrine, and dopamine all play important roles in tonic and phasic cardiovascular regulation.

1	The activities of the adrenergic receptors are mediated by guanosine nucleotide-binding regulatory proteins (G proteins) and by intracellular concentrations of downstream second messengers. In addition to receptor affinity and density, physiologic responsiveness to catecholamines may be altered by the efficiency of receptor-effector coupling at a site “distal” to receptor binding. The receptor sites are relatively specific both for the transmitter substance and for the response that occupancy of the receptor site elicits. Based on their physiology and pharmacology, adrenergic receptors have been divided into two principal types: α and β. These types have been differentiated further into α1, α2, β1, and β2 receptors. Recent molecular cloning studies have identified several additional subtypes. α Receptors are occupied and activated more avidly by norepinephrine than by epinephrine, and the reverse is true for β receptors. α1 Receptors are located on postsynaptic cells in smooth muscle

1	α Receptors are occupied and activated more avidly by norepinephrine than by epinephrine, and the reverse is true for β receptors. α1 Receptors are located on postsynaptic cells in smooth muscle and elicit vasoconstriction. α2 Receptors are localized on presynaptic membranes of postganglionic nerve terminals that synthesize norepinephrine. When activated by catecholamines, α2 receptors act as negative feedback controllers, inhibiting further norepinephrine release. In the kidney, activation of α1-adrenergic receptors increases renal tubular reabsorption of sodium. Different classes of antihypertensive agents either inhibit α1 receptors or act as agonists of α2 receptors and reduce systemic sympathetic outflow. Activation of myocardial β1 receptors stimulates the rate and strength of cardiac contraction and consequently increases cardiac output. β1 Receptor activation also stimulates renin release from the kidney. Another class of antihypertensive agents acts by inhibiting β1

1	of cardiac contraction and consequently increases cardiac output. β1 Receptor activation also stimulates renin release from the kidney. Another class of antihypertensive agents acts by inhibiting β1 receptors. Activation of β2 receptors by epinephrine relaxes vascular smooth muscle and results in vasodilation.

1	Circulating catecholamine concentrations may affect the number of adrenoreceptors in various tissues. Downregulation of receptors may be a consequence of sustained high levels of catecholamines and provides an explanation for decreasing responsiveness, or tachyphylaxis, to catecholamines. For example, orthostatic hypotension frequently is observed in patients with pheochromocytoma, possibly due to the lack of norepinephrine-induced vasoconstriction with assumption of the upright posture. Conversely, with chronic reduction of neurotransmitter substances, adrenoreceptors may increase in number or be upregulated, resulting in increased responsiveness to the neurotransmitter. Chronic administration of agents that block adrenergic receptors may result in upregulation, and abrupt withdrawal of those agents may pro-1613 duce a condition of temporary hypersensitivity to sympathetic stimuli. For example, clonidine is an antihypertensive agent that is a centrally acting α2 agonist that inhibits

1	agents may pro-1613 duce a condition of temporary hypersensitivity to sympathetic stimuli. For example, clonidine is an antihypertensive agent that is a centrally acting α2 agonist that inhibits sympathetic outflow. Rebound hypertension may occur with the abrupt cessation of clonidine therapy, probably as a consequence of upregulation of α1 receptors.

1	Several reflexes modulate blood pressure on a minute-to-minute basis. One arterial baroreflex is mediated by stretch-sensitive sensory nerve endings in the carotid sinuses and the aortic arch. The rate of firing of these baroreceptors increases with arterial pressure, and the net effect is a decrease in sympathetic outflow, resulting in decreases in arterial pressure and heart rate. This is a primary mechanism for rapid buffering of acute fluctuations of arterial pressure that may occur during postural changes, behavioral or physiologic stress, and changes in blood volume. However, the activity of the baroreflex declines or adapts to sustained increases in arterial pressure such that the baroreceptors are reset to higher pressures. Patients with autonomic neuropathy and impaired baroreflex function may have extremely labile blood pressures with difficult-to-control episodic blood pressure spikes associated with tachycardia.

1	In both normal-weight and obese individuals, hypertension often is associated with increased sympathetic outflow. Based on recordings of postganglionic muscle nerve activity (detected by a microelectrode inserted in a peroneal nerve in the leg), sympathetic outflow tends to be higher in hypertensive than in normotensive individuals. Sympathetic outflow is increased in obesity-related hypertension and in hypertension associated with obstructive sleep apnea. Baroreceptor activation via electrical stimulation of carotid sinus afferent nerves lowers blood pressure in patients with “resistant” hypertension. Drugs that block the sympathetic nervous system are potent antihypertensive agents, indicating that the sympathetic nervous system plays a permissive, although not necessarily a causative, role in the maintenance of increased arterial pressure.

1	Pheochromocytoma is the most blatant example of hypertension related to increased catecholamine production, in this instance by a tumor. Blood pressure can be reduced by surgical excision of the tumor or by pharmacologic treatment with an α1 receptor antagonist or with an inhibitor of tyrosine hydroxylase, the rate-limiting step in catecholamine biosynthesis.

1	The renin-angiotensin-aldosterone system contributes to the regulation of arterial pressure primarily via the vasoconstrictor properties of angiotensin II and the sodium-retaining properties of aldosterone. Renin is an aspartyl protease that is synthesized as an enzymatically inactive precursor, prorenin. Most renin in the circulation is synthesized in the renal afferent renal arteriole. Prorenin may be secreted directly into the circulation or may be activated within secretory cells and released as active renin. Although human plasma contains two to five times more prorenin than renin, there is no evidence that prorenin contributes to the physiologic activity of this system. There are three primary stimuli for renin secretion: (1) decreased NaCl transport in the distal portion of the thick ascending limb of the loop of Henle that abuts the corresponding afferent arteriole (macula densa), (2) decreased pressure or stretch within the renal afferent arteriole (baroreceptor mechanism),

1	ascending limb of the loop of Henle that abuts the corresponding afferent arteriole (macula densa), (2) decreased pressure or stretch within the renal afferent arteriole (baroreceptor mechanism), and (3) sympathetic nervous system stimulation of renin-secreting cells via β1 adrenoreceptors. Conversely, renin secretion is inhibited by increased NaCl transport in the thick ascending limb of the loop of Henle, by increased stretch within the renal afferent arteriole, and by β1 receptor blockade. In addition, angiotensin II directly inhibits renin secretion due to angiotensin II type 1 receptors on juxtaglomerular cells, and renin secretion increases in response to pharmacologic blockade of either ACE or angiotensin II receptors.

1	Once released into the circulation, active renin cleaves a substrate, angiotensinogen, to form an inactive decapeptide, angiotensin I (Fig. 298-2). A converting enzyme, located primarily but not exclusively in the pulmonary circulation, converts angiotensin I to the active octapeptide, angiotensin II, by releasing the C-terminal histidylleucine dipeptide. The same converting enzyme cleaves a number FIGURE 298-2 Renin-angiotensin-aldosterone axis. ACE, angiotensinconverting enzyme.

1	of other peptides, including and thereby inactivating the vasodilator bradykinin. Acting primarily through angiotensin II type 1 (AT1) receptors on cell membranes, angiotensin II is a potent pressor substance, the primary tropic factor for the secretion of aldosterone by the adrenal zona glomerulosa, and a potent mitogen that stimulates vascular smooth muscle cell and myocyte growth. Independent of its hemodynamic effects, angiotensin II may play a role in the pathogenesis of atherosclerosis through a direct cellular action on the vessel wall. The angiotensin II type 2 (AT2) receptor has the opposite functional effects of the AT1 receptor. The AT2 receptor induces vasodilation, sodium excretion, and inhibition of cell growth and matrix formation. Experimental evidence suggests that the AT2 receptor improves vascular remodeling by stimulating smooth muscle cell apoptosis and contributes to the regulation of glomerular filtration rate. AT1 receptor blockade induces an increase in AT2

1	AT2 receptor improves vascular remodeling by stimulating smooth muscle cell apoptosis and contributes to the regulation of glomerular filtration rate. AT1 receptor blockade induces an increase in AT2 receptor activity.

1	Renin-secreting tumors are clear examples of renin-dependent hypertension. In the kidney, these tumors include benign hemangiopericytomas of the juxtaglomerular apparatus and, infrequently, renal carcinomas, including Wilms’ tumors. Renin-producing carcinomas also have been described in lung, liver, pancreas, colon, and adrenals. In these instances, in addition to excision and/or ablation of the tumor, treatment of hypertension includes pharmacologic therapies targeted to inhibit angiotensin II production or action. Renovascular hypertension is another renin-mediated form of hypertension. Obstruction of the renal artery leads to decreased renal perfusion pressure, thereby stimulating renin secretion. Over time, possibly as a consequence of secondary renal damage, this form of hypertension may become less renin dependent.

1	Angiotensinogen, renin, and angiotensin II are also synthesized locally in many tissues, including the brain, pituitary, aorta, arteries, heart, adrenal glands, kidneys, adipocytes, leukocytes, ovaries, testes, uterus, spleen, and skin. Angiotensin II in tissues may be formed by the enzymatic activity of renin or by other proteases, e.g., tonin, chymase, and cathepsins. In addition to regulating local blood flow, tissue angiotensin II is a mitogen that stimulates growth and contributes to modeling and repair. Excess tissue angiotensin II may contribute to atherosclerosis, cardiac hypertrophy, and renal failure and consequently may be a target for pharmacologic therapy to prevent target organ damage.

1	Angiotensin II is the primary tropic factor regulating the synthesis and secretion of aldosterone by the zona glomerulosa of the adrenal cortex. Aldosterone synthesis is also dependent on potassium, and aldosterone secretion may be decreased in potassium-depleted individuals. Although acute elevations of adrenocorticotropic hormone (ACTH) levels also increase aldosterone secretion, ACTH is not an important tropic factor for the chronic regulation of aldosterone. Aldosterone is a potent mineralocorticoid that increases sodium reabsorption by amiloride-sensitive epithelial sodium channels (ENaC) on the apical surface of the principal cells of the renal cortical collecting duct (Chap. 332e). Electric neutrality is maintained by exchanging sodium for potassium and hydrogen ions. Consequently, increased aldosterone secretion may result in hypokalemia and alkalosis.

1	Cortisol also binds to the mineralocorticoid receptor but normally functions as a less potent mineralocorticoid than aldosterone because cortisol is converted to cortisone by the enzyme 11 β-hydroxysteroid dehydrogenase type 2. Cortisone has no affinity for the mineralocorticoid receptor. Primary aldosteronism is a compelling example of mineralocorticoid-mediated hypertension. In this disorder, adrenal aldosterone synthesis and release are independent of renin-angiotensin, and renin release is suppressed by the resulting volume expansion.

1	Mineralocorticoid receptors are expressed in a number of tissues in addition to the kidney, and mineralocorticoid receptor activation induces structural and functional alterations in the heart, kidney, and blood vessels, leading to myocardial fibrosis, nephrosclerosis, and vascular inflammation and remodeling, perhaps as a consequence of oxidative stress. These effects are amplified by a high salt intake. In animal models, high circulating aldosterone levels stimulate cardiac fibrosis and left ventricular hypertrophy, and spironolactone (an aldosterone antagonist) prevents aldosterone-induced myocardial fibrosis. Pathologic patterns of left ventricular geometry also have been associated with elevations of plasma aldosterone concentration in hypertensive patients. In patients with CHF, low-dose spironolactone reduces the risk of progressive heart failure and sudden death from cardiac causes by 30%. Due to a renal hemodynamic effect, in patients with primary aldosteronism, high

1	CHF, low-dose spironolactone reduces the risk of progressive heart failure and sudden death from cardiac causes by 30%. Due to a renal hemodynamic effect, in patients with primary aldosteronism, high circulating levels of aldosterone also may cause glomerular hyperfiltration and albuminuria. These renal effects are reversible after removal of the effects of excess aldosterone by adrenalectomy or spironolactone.

1	Increased activity of the renin-angiotensin-aldosterone axis is not invariably associated with hypertension. In response to a low-NaCl diet or to volume contraction, arterial pressure and volume homeostasis may be maintained by increased activity of the renin-angiotensinaldosterone axis. Secondary aldosteronism (i.e., increased aldosterone secondary to increased renin-angiotensin), but not hypertension, also is observed in edematous states such as CHF and liver disease.

1	Vascular radius and compliance of resistance arteries are important determinants of arterial pressure. Resistance to flow varies inversely with the fourth power of the radius, and consequently, small decreases in lumen size significantly increase resistance. In hypertensive patients, structural, mechanical, or functional changes may reduce the lumen diameter of small arteries and arterioles. Remodeling refers to geometric alterations in the vessel wall without a change in vessel volume. Hypertrophic (increased cell size, and increased deposition of intercellular matrix) or eutrophic vascular remodeling results in decreased lumen size and, hence, increased peripheral resistance. Apoptosis, low-grade inflammation, and vascular fibrosis also contribute to remodeling. Lumen diameter also is related to elasticity of the vessel. Vessels with a high degree of elasticity can accommodate an increase of volume with relatively little change in pressure, whereas in a semirigid vascular system, a

1	to elasticity of the vessel. Vessels with a high degree of elasticity can accommodate an increase of volume with relatively little change in pressure, whereas in a semirigid vascular system, a small increment in volume induces a relatively large increment of pressure.

1	Hypertensive patients may have stiffer arteries due to arteriosclerosis, and high systolic blood pressures and wide pulse pressures are a consequence of decreased vascular compliance. Due to arterial stiffness, central blood pressures (aortic, carotid) may not correspond to brachial artery pressures. Ejection of blood into the aorta elicits a pressure wave that is propagated at a given velocity. The forward travelling wave generates a reflected wave that travels backward toward the ascending aorta. Although mean arterial pressure is determined by cardiac output and peripheral resistance, pulse pressure is related to the functional properties of large arteries and the amplitude and timing of the incident and reflected waves. Increased arterial stiffness results in increased pulse wave velocity of both incident and reflected waves. Due to the timing of these waves, the consequence is augmentation of aortic systolic pressure and a reduction of aortic diastolic pressure, i.e., an increase

1	of both incident and reflected waves. Due to the timing of these waves, the consequence is augmentation of aortic systolic pressure and a reduction of aortic diastolic pressure, i.e., an increase in pulse pressure. The aortic augmentation index, an index of arterial stiffening, is calculated as the ratio of central arterial pressure-to-pulse pressure. Central blood pressure may be measured directly by placing a sensor in the aorta or noninvasively by radial tonometry using commercially available devices. Central blood pressure and the aortic augmentation index are strong, independent predictors of cardiovascular disease and all-cause mortality.

1	Ion transport by vascular smooth muscle cells may contribute to hypertension-associated abnormalities of vascular tone and vascular growth, both of which are modulated by intracellular pH (pHi). Three ion transport mechanisms participate in the regulation of pHi:

1	Na+-H+ exchange, (2) Na+-dependent HCO3−-Cl− exchange, and cation-independent HCO3−-Cl− exchange. Based on measurements in cell types that are more accessible than vascular smooth muscle (e.g., leukocytes, erythrocytes, platelets, skeletal muscle), activity of the Na+-H+ exchanger is increased in hypertension, and this may result in increased vascular tone by two mechanisms. First, increased sodium entry may lead to increased vascular tone by activating Na+-Ca2+ exchange and thereby increasing intracellular calcium. Second, increased pHi enhances calcium sensitivity of the contractile apparatus, leading to an increase in contractility for a given intracellular calcium concentration. Additionally, increased Na+-H+ exchange may stimulate growth of vascular smooth muscle cells by enhancing sensitivity to mitogens.

1	Vascular endothelial function also modulates vascular tone. The vascular endothelium synthesizes and releases several vasoactive substances, including nitric oxide, a potent vasodilator. Endotheliumdependent vasodilation is impaired in hypertensive patients. This impairment often is assessed with high-resolution ultrasonography before and after the hyperemic phase of reperfusion that follows 5 minutes of forearm ischemia. Alternatively, endothelium-dependent vasodilation may be assessed in response to an intra-arterially infused endothelium-dependent vasodilator, e.g., acetylcholine. Endothelin is a vasoconstrictor peptide produced by the endothelium, and orally active endothelin antagonists may lower blood pressure in patients with resistant hypertension.

1	Currently, it is not known if the hypertension-related vascular abnormalities of ion transport and endothelial function are primary alterations or secondary consequences of elevated arterial pressure. Limited evidence suggests that vascular compliance and endotheliumdependent vasodilation may be improved by aerobic exercise, weight loss, and antihypertensive agents. It remains to be determined whether these interventions affect arterial structure and stiffness via a blood pressure–independent mechanism and whether different classes of antihypertensive agents preferentially affect vascular structure and function. Hypertension is an independent predisposing factor for heart failure, coronary artery disease, stroke, renal disease, and peripheral arterial disease (PAD).

1	Hypertension is an independent predisposing factor for heart failure, coronary artery disease, stroke, renal disease, and peripheral arterial disease (PAD). Heart disease is the most common cause of death in hypertensive patients. Hypertensive heart disease is the result of structural and functional adaptations leading to left ventricular hypertrophy, CHF, abnormalities of blood flow due to atherosclerotic coronary artery disease and microvascular disease, and cardiac arrhythmias. Individuals with left ventricular hypertrophy are at increased risk for CHD, stroke, CHF, and sudden death. Aggressive control of hypertension can regress or reverse left ventricular hypertrophy and reduce the risk of cardiovascular disease. It is not clear whether different 1615 classes of antihypertensive agents have an added impact on reducing left ventricular mass, independent of their blood pressure–lowering effect.

1	CHF may be related to systolic dysfunction, diastolic dysfunction, or a combination of the two. Abnormalities of diastolic function that range from asymptomatic heart disease to overt heart failure are common in hypertensive patients. Approximately one-third of patients with CHF have normal systolic function but abnormal diastolic function. Diastolic dysfunction is an early consequence of hypertension- related heart disease and is exacerbated by left ventricular hypertrophy and ischemia. Cardiac catheterization provides the most accurate assessment of diastolic function. Alternatively, diastolic function can be evaluated by several noninvasive methods, including echocardiography and radionuclide angiography.

1	Stroke is the second most frequent cause of death in the world; it accounts for 5 million deaths each year, with an additional 15 million persons having nonfatal strokes. Elevated blood pressure is the strongest risk factor for stroke. Approximately 85% of strokes are due to infarction, and the remainder are due to either intracerebral or subarachnoid hemorrhage. The incidence of stroke rises progressively with increasing blood pressure levels, particularly systolic blood pressure in individuals >65 years. Treatment of hypertension decreases the incidence of both ischemic and hemorrhagic strokes.

1	Hypertension also is associated with impaired cognition in an aging population, and longitudinal studies support an association between midlife hypertension and late-life cognitive decline. Hypertension-related cognitive impairment and dementia may be a consequence of a single infarct due to occlusion of a “strategic” larger vessel or multiple lacunar infarcts due to occlusive small vessel disease resulting in subcortical white matter ischemia. Several clinical trials suggest that antihypertensive therapy has a beneficial effect on cognitive function, although this remains an active area of investigation.

1	Cerebral blood flow remains unchanged over a wide range of arterial pressures (mean arterial pressure of 50–150 mmHg) through a process termed autoregulation of blood flow. In patients with the clinical syndrome of malignant hypertension, encephalopathy is related to failure of autoregulation of cerebral blood flow at the upper pressure limit, resulting in vasodilation and hyperperfusion. Signs and symptoms of hypertensive encephalopathy may include severe headache, nausea and vomiting (often of a projectile nature), focal neurologic signs, and alterations in mental status. Untreated, hypertensive encephalopathy may progress to stupor, coma, seizures, and death within hours. It is important to distinguish hypertensive encephalopathy from other neurologic syndromes that may be associated with hypertension, e.g., cerebral ischemia, hemorrhagic or thrombotic stroke, seizure disorder, mass lesions, pseudotumor cerebri, delirium tremens, meningitis, acute intermittent porphyria, traumatic

1	hypertension, e.g., cerebral ischemia, hemorrhagic or thrombotic stroke, seizure disorder, mass lesions, pseudotumor cerebri, delirium tremens, meningitis, acute intermittent porphyria, traumatic or chemical injury to the brain, and uremic encephalopathy.

1	The kidney is both a target and a cause of hypertension. Primary renal disease is the most common etiology of secondary hypertension. Mechanisms of kidney-related hypertension include a diminished capacity to excrete sodium, excessive renin secretion in relation to volume status, and sympathetic nervous system overactivity. Conversely, hypertension is a risk factor for renal injury and ESRD. The increased risk associated with high blood pressure is graded, continuous, and present throughout the distribution of blood pressure above optimal pressure. Renal risk appears to be more closely related to systolic than to diastolic blood pressure, and black men are at greater risk than white men for developing ESRD at every level of blood pressure.

1	Atherosclerotic, hypertension-related vascular lesions in the kidney primarily affect preglomerular arterioles, resulting in ischemic changes in the glomeruli and postglomerular structures. Glomerular injury also may be a consequence of direct damage to the glomerular capillaries due to glomerular hyperperfusion. Studies of hypertension-related 1616 renal damage, primarily in experimental animals, suggest that loss of autoregulation of renal blood flow at the afferent arteriole results in transmission of elevated pressures to an unprotected glomerulus with ensuing hyperfiltration, hypertrophy, and eventual focal segmental glomerular sclerosis. With progressive renal injury there is a loss of autoregulation of renal blood flow and glomerular filtration rate, resulting in a lower blood pressure threshold for renal damage and a steeper slope between blood pressure and renal damage. The result may be a vicious cycle of renal damage and nephron loss leading to more severe hypertension,

1	threshold for renal damage and a steeper slope between blood pressure and renal damage. The result may be a vicious cycle of renal damage and nephron loss leading to more severe hypertension, glomerular hyperfiltration, and further renal damage. Glomerular pathology progresses to glomerulosclerosis, and eventually the renal tubules may also become ischemic and gradually atrophic. The renal lesion associated with malignant hypertension consists of fibrinoid necrosis of the afferent arterioles, sometimes extending into the glomerulus, and may result in focal necrosis of the glomerular tuft. Clinically, macroalbuminuria (a random urine albumin/creatinine ratio >300 mg/g) or microalbuminuria (a random urine albumin/ creatinine ratio 30–300 mg/g) are early markers of renal injury. These are also risk factors for renal disease progression and cardiovascular disease.

1	In addition to contributing to the pathogenesis of hypertension, blood vessels are a target organ for atherosclerotic disease secondary to long-standing elevated blood pressure. In hypertensive patients, vascular disease is a major contributor to stroke, heart disease, and renal failure. Further, hypertensive patients with arterial disease of the lower extremities are at increased risk for future cardiovascular disease. Although patients with stenotic lesions of the lower extremities may be asymptomatic, intermittent claudication is the classic symptom of PAD. The ankle-brachial index is a useful approach for evaluating PAD and is defined as the ratio of noninvasively assessed ankle to brachial (arm) systolic blood pressure. An ankle-brachial index <0.90 is considered diagnostic of PAD and is associated with >50% stenosis in at least one major lower limb vessel. An ankle-brachial index <0.80 is associated with elevated blood pressure, particularly systolic blood pressure.

1	From an epidemiologic perspective, there is no obvious level of blood pressure that defines hypertension. In adults, there is a continuous, incremental risk of cardiovascular disease, stroke, and renal disease across levels of both systolic and diastolic blood pressure. The Multiple Risk Factor Intervention Trial (MRFIT), which included >350,000 male participants, demonstrated a continuous and graded influence of both systolic and diastolic blood pressure on CHD mortality, extending down to systolic blood pressures of 120 mmHg. Similarly, results of a meta-analysis involving almost 1 million participants indicate that ischemic heart disease mortality, stroke mortality, and mortality from other vascular causes are directly related to the height of the blood pressure, beginning at 115/75 mmHg, without evidence of a threshold. Cardiovascular disease risk doubles for every 20-mmHg increase in systolic and 10-mmHg increase in diastolic pressure. Among older individuals, systolic blood

1	mmHg, without evidence of a threshold. Cardiovascular disease risk doubles for every 20-mmHg increase in systolic and 10-mmHg increase in diastolic pressure. Among older individuals, systolic blood pressure and pulse pressure are more powerful predictors of cardiovascular disease than is diastolic blood pressure.

1	Clinically, hypertension may be defined as that level of blood pressure at which the institution of therapy reduces blood pressure–related morbidity and mortality. Current clinical criteria for defining hypertension generally are based on the average of two or more seated blood pressure readings during each of two or more outpatient visits. A recent classification recommends blood pressure criteria for defining normal blood pressure, prehypertension, hypertension (stages I and II), and isolated systolic hypertension, which is frequent among the elderly (Table 298-1). In children and adolescents, hypertension generally is defined as systolic and/or diastolic blood pressure consistently >95th percentile for age, sex, and height. Blood pressures between the 90th Blood Pressure Classification Systolic, mmHg Diastolic, mmHg Source: Adapted from AV Chobanian et al: JAMA 289:2560, 2003. and 95th percentiles are considered prehypertensive and are an indication for lifestyle interventions.

1	Home blood pressure and average 24-h ambulatory blood pressure measurements are generally lower than clinic blood pressures. Because ambulatory blood pressure recordings yield multiple readings throughout the day and night, they provide a more comprehensive assessment of the vascular burden of hypertension than do a limited number of office readings. Increasing evidence suggests that home blood pressures, including 24-h blood pressure recordings, more reliably predict target organ damage than do office blood pressures. Blood pressure tends to be higher in the early morning hours, soon after waking, than at other times of day. Myocardial infarction and stroke are more common in the early morning hours. Nighttime blood pressures are generally 10–20% lower than daytime blood pressures, and an attenuated nighttime blood pressure “dip” may be associated with increased cardiovascular disease risk. Recommended criteria for a diagnosis of hypertension, based on 24-h blood pressure monitoring,

1	attenuated nighttime blood pressure “dip” may be associated with increased cardiovascular disease risk. Recommended criteria for a diagnosis of hypertension, based on 24-h blood pressure monitoring, are average awake blood pressure ≥135/85 mmHg and asleep blood pressure ≥120/75 mmHg. These levels approximate a clinic blood pressure of 140/90 mmHg.

1	Approximately 15–20% of patients with stage 1 hypertension (as defined in Table 298-1) based on office blood pressures have average ambulatory readings <135/85 mmHg. This phenomenon, so-called white coat hypertension, also may be associated with an increased risk of target organ damage, although to a lesser extent than in individuals with elevated office and ambulatory readings. Individuals with white coat hypertension are also at increased risk for developing sustained hypertension. Depending on methods of patient ascertainment, ~80–95% of hypertensive patients are diagnosed as having primary, or “essential,” hypertension. In the remaining 5–20% of hypertensive patients, a specific underlying disorder causing the elevation of blood pressure can be identified (Tables 298-2 and 298-3). In individuals with “secondary” hypertension, a specific mechanism for the blood pressure elevation is often more apparent.

1	Primary hypertension tends to be familial and is likely to be the consequence of an interaction between environmental and genetic factors. The prevalence of primary hypertension increases with age, and individuals with relatively high blood pressures at younger ages are at increased risk for the subsequent development of hypertension. It is 1. 2. a. b. c. d. e. f. Renal Parenchymal diseases, renal cysts (includ ing polycystic kidney disease), renal tumors (including renin-secreting tumors), obstructive Renovascular Arteriosclerotic, fibromuscular dysplasia Adrenal Primary aldosteronism, Cushing’s syndrome, 17α-hydroxylase deficiency, 11β-hydroxylase deficiency, 11-hydroxysteroid dehydrogenase deficiency (licorice), pheochromocytoma Neurogenic Psychogenic, diencephalic syndrome, familial dysautonomia, polyneuritis (acute porphyria, lead poisoning), acute increased intracranial pressure, acute spinal cord section

1	Neurogenic Psychogenic, diencephalic syndrome, familial dysautonomia, polyneuritis (acute porphyria, lead poisoning), acute increased intracranial pressure, acute spinal cord section Miscellaneous endocrine Hypothyroidism, hyperthyroidism, hypercalcemia, acromegaly Medications High-dose estrogens, adrenal steroids, decon gestants, appetite suppressants, cyclosporine, tricyclic antidepressants, monoamine oxidase inhibitors, erythropoietin, nonsteroidal anti- inflammatory agents, cocaine Mendelian forms of hyper-See Table 298-4 tension likely that primary hypertension represents a spectrum of disorders with different underlying pathophysiologies. In the majority of patients with established hypertension, peripheral resistance is increased and cardiac output is normal or decreased; however, in younger patients with mild or labile hypertension, cardiac output may be increased and peripheral resistance may be normal.

1	When plasma renin activity (PRA) is plotted against 24-h sodium excretion, ~10–15% of hypertensive patients have high PRA and 25% have low PRA. High-renin patients may have a vasoconstrictor form of hypertension, whereas low-renin patients may have volume-dependent hypertension. Inconsistent associations between plasma aldosterone and blood pressure have been described in patients with primary hypertension. The association between aldosterone and blood pressure is more striking in African Americans, and PRA tends to be low in hypertensive African Americans. This raises the possibility that subtle increases in aldosterone may contribute to hypertension in at least some groups of patients who do not have overt primary aldosteronism. Furthermore, spironolactone, an aldosterone antagonist, may be a particularly effective antihypertensive agent for some patients with primary hypertension, including some patients with “drug-resistant” hypertension.

1	(See also Chap. 422) There is a well-documented association between obesity (body mass index >30 kg/m2) and hypertension. Further, cross-sectional studies indicate a direct linear correlation between body weight (or body mass index) and blood pressure. Centrally located body fat is a more important determinant of blood pressure elevation than is peripheral body fat. In longitudinal studies, a direct correlation exists between change in weight and change in blood pressure over time. Sixty percent of hypertensive adults are more than 20% overweight. It has been established that 60–70% of hypertension in adults may be directly attributable to adiposity.

1	Hypertension and dyslipidemia frequently occur together and in association with resistance to insulin-stimulated glucose uptake. This clustering of risk factors is often, but not invariably, associated with obesity, particularly abdominal obesity. Insulin resistance also is associated with an unfavorable imbalance in the endothelial production of mediators that regulate platelet aggregation, coagulation, fibrinolysis, and vessel tone. When these risk factors cluster, the risks for CHD, stroke, diabetes, and cardiovascular disease mortality are increased further.

1	Depending on the populations studied and the methodologies for 1617 defining insulin resistance, ~25–50% of nonobese, nondiabetic hypertensive persons are insulin resistant. The constellation of insulin resistance, abdominal obesity, hypertension, and dyslipidemia has been designated as the metabolic syndrome. As a group, first-degree relatives of patients with primary hypertension are also insulin resistant, and hyperinsulinemia (a surrogate marker of insulin resistance) may predict the eventual development of hypertension and cardiovascular disease. Although the metabolic syndrome may in part be heritable as a polygenic condition, the expression of the syndrome is modified by environmental factors, such as degree of physical activity and diet. Insulin sensitivity increases and blood pressure decreases in response to weight loss. The recognition that cardiovascular disease risk factors tend to cluster within individuals has important implications for the evaluation and treatment of

1	decreases in response to weight loss. The recognition that cardiovascular disease risk factors tend to cluster within individuals has important implications for the evaluation and treatment of hypertension. Evaluation of both hypertensive patients and individuals at risk for developing hypertension should include assessment of overall cardiovascular disease risk. Similarly, introduction of lifestyle modification strategies and drug therapies should address overall risk and not focus exclusively on hypertension.

1	Virtually all disorders of the kidney may cause hypertension (Table 298-3), and renal disease is the most common cause of secondary hypertension. Hypertension is present in >80% of patients with chronic renal failure. In general, hypertension is more severe in glomerular diseases than in interstitial diseases such as chronic pyelonephritis. Conversely, hypertension may cause nephrosclerosis, and in some instances it may be difficult to determine whether hypertension or renal disease was the initial disorder. Proteinuria >1000 mg/d and an active urine sediment are indicative of primary renal disease. In either instance, the goals are to control blood pressure and retard the rate of progression of renal dysfunction.

1	Hypertension due to an occlusive lesion of a renal artery, renovascular hypertension, is a potentially curable form of hypertension. In the initial stages, the mechanism of hypertension generally is related to activation of the renin-angiotensin system. However, renin activity and other components of the renin-angiotensin system may be elevated only transiently; over time, recruitment of other pressure mechanisms may contribute to elevated arterial pressure. Two groups of patients are at risk for this disorder: older arteriosclerotic patients who have a plaque obstructing the renal artery, frequently at its origin, and patients with fibromuscular dysplasia. Atherosclerosis accounts for the large majority of patients with renovascular hypertension. Although fibromuscular dysplasia may occur at any age, it has a strong predilection for young white women. The prevalence in females is eightfold that in males. There are several histologic variants of fibromuscular dysplasia, including

1	occur at any age, it has a strong predilection for young white women. The prevalence in females is eightfold that in males. There are several histologic variants of fibromuscular dysplasia, including medial fibroplasia, perimedial fibroplasia, medial hyperplasia, and intimal fibroplasia. Medial fibroplasia is the most common variant and accounts for approximately two-thirds of patients. The lesions of fibromuscular dysplasia are frequently bilateral and, in contrast to atherosclerotic renovascular disease, tend to affect more distal portions of the renal artery.

1	Several clues from the history and physical examination may suggest renovascular hypertension. The diagnosis should be considered in patients with other evidence of atherosclerotic vascular disease. Although response to antihypertensive therapy does not exclude the diagnosis, severe or refractory hypertension, recent loss of hypertension control or recent onset of moderately severe hypertension, and unexplained deterioration of renal function or deterioration of renal function associated with an ACE inhibitor should raise the possibility of renovascular hypertension. Approximately 50% of patients with renovascular hypertension have an abdominal or flank bruit, and the bruit is more likely to be hemodynamically significant if it lateralizes or extends throughout systole into diastole.

1	If blood pressure is adequately controlled with a simple antihypertensive regimen and renal function remains stable, there may be little 1618 impetus to pursue an evaluation for renal artery stenosis, particularly in an older patient with atherosclerotic disease and comorbid conditions. Patients with long-standing hypertension, advanced renal insufficiency, or diabetes mellitus are less likely to benefit from renal vascular repair. The most effective medical therapies include an ACE inhibitor or an angiotensin II receptor blocker; however, these agents decrease glomerular filtration rate in a stenotic kidney owing to efferent renal arteriolar dilation. In the presence of bilateral renal artery stenosis or renal artery stenosis to a solitary kidney, progressive renal insufficiency may result from the use of these agents. Importantly, the renal insufficiency is generally reversible after discontinuation of the offending drug. If renal artery stenosis is suspected and if the clinical

1	from the use of these agents. Importantly, the renal insufficiency is generally reversible after discontinuation of the offending drug. If renal artery stenosis is suspected and if the clinical condition warrants an intervention such as percutaneous transluminal renal angioplasty (PTRA), placement of a vascular endoprosthesis (stent), or surgical renal revascularization, imaging studies should be the next step in the evaluation. As a screening test, renal blood flow may be evaluated with a radionuclide [131I]-orthoiodohippurate (OIH) scan, or glomerular filtration rate may be evaluated with a [99mTc]diethylenetriamine pentaacetic acid (DTPA) scan before and after a single dose of captopril (or another ACE inhibitor). The following are consistent with a positive study: (1) decreased relative uptake by the involved kidney, which contributes <40% of total renal function, (2) delayed uptake on the affected side, and (3) delayed washout on the affected side. In patients with normal, or

1	uptake by the involved kidney, which contributes <40% of total renal function, (2) delayed uptake on the affected side, and (3) delayed washout on the affected side. In patients with normal, or nearly normal, renal function, a normal captopril renogram essentially excludes functionally significant renal artery stenosis; however, its usefulness is limited in patients with renal insufficiency (creatinine clearance <20 mL/min) or bilateral renal artery stenosis. Additional imaging studies are indicated if the scan is positive. Doppler ultrasound of the renal arteries produces reliable estimates of renal blood flow velocity and offers the opportunity to track a lesion over time. Positive studies usually are confirmed at angiography, whereas false-negative results occur frequently, particularly in obese patients. Gadolinium-contrast magnetic resonance angiography offers clear images of the proximal renal artery but may miss distal lesions. An advantage is the opportunity to image the renal

1	in obese patients. Gadolinium-contrast magnetic resonance angiography offers clear images of the proximal renal artery but may miss distal lesions. An advantage is the opportunity to image the renal arteries with an agent that is not nephrotoxic. Contrast arteriography remains the “gold standard” for evaluation and identification of renal artery lesions. Potential risks include nephrotoxicity, particularly in patients with diabetes mellitus or preexisting renal insufficiency.

1	Some degree of renal artery obstruction may be observed in almost 50% of patients with atherosclerotic disease, and there are several approaches for evaluating the functional significance of such a lesion to predict the effect of vascular repair on blood pressure control and renal function. Each approach has varying degrees of sensitivity and specificity, and no single test is sufficiently reliable to determine a causal relationship between a renal artery lesion and hypertension. Functionally significant lesions generally occlude more than 70% of the lumen of the affected renal artery. On angiography, the presence of collateral vessels to the ischemic kidney suggests a functionally significant lesion. A lateralizing renal vein renin ratio (ratio >1.5 of affected side/contralateral side) has a 90% predictive value for a lesion that would respond to vascular repair; however, the false-negative rate for blood pressure control is 50–60%. Measurement of the pressure gradient across a renal

1	a 90% predictive value for a lesion that would respond to vascular repair; however, the false-negative rate for blood pressure control is 50–60%. Measurement of the pressure gradient across a renal artery lesion does not reliably predict the response to vascular repair.

1	In the final analysis, a decision concerning vascular repair vs. medical therapy and the type of repair procedure should be individualized. Patients with fibromuscular disease have more favorable outcomes than do patients with atherosclerotic lesions, presumably owing to their younger age, shorter duration of hypertension, and less systemic disease. Because of its low risk-versus-benefit ratio and high success rate (improvement or cure of hypertension in 90% of patients and restenosis rate of 10%), PTRA is the initial treatment of choice for these patients. Surgical revascularization may be undertaken if PTRA is unsuccessful or if a branch lesion is present. In atherosclerotic patients, vascular repair should be considered if blood pressure cannot be controlled adequately despite optimal medical therapy or if renal function deteriorates. Surgery may be the preferred initial approach for younger atherosclerotic patients without comorbid conditions; however, for most atherosclerotic

1	medical therapy or if renal function deteriorates. Surgery may be the preferred initial approach for younger atherosclerotic patients without comorbid conditions; however, for most atherosclerotic patients, depending on the location of the lesion, the initial approach may be PTRA and/or stenting. Surgical revascularization may be indicated if these approaches are unsuccessful, the vascular lesion is not amenable to PTRA or stenting, or concomitant aortic surgery is required, e.g., to repair an aneurysm. A National Institutes of Health–sponsored prospective, randomized clinical trial is in progress comparing medical therapy alone with medical therapy plus renal artery stenting regarding Cardiovascular Outcomes for Renal Atherosclerotic Lesions (CORAL).

1	Excess aldosterone production due to primary aldosteronism is a potentially curable form of hypertension. In patients with primary aldosteronism, increased aldosterone production is independent of the renin-angiotensin system, and the consequences are sodium retention, hypertension, hypokalemia, and low PRA. The reported prevalence of this disorder varies from <2% to ~15% of hypertensive individuals. In part, this variation is related to the intensity of screening and the criteria for establishing the diagnosis.

1	History and physical examination provide little information about the diagnosis. The age at the time of diagnosis is generally the third through fifth decade. Hypertension is usually mild to moderate but occasionally may be severe; primary aldosteronism should be considered in all patients with refractory hypertension. Hypertension in these patients may be associated with glucose intolerance. Most patients are asymptomatic; however, infrequently, polyuria, polydipsia, paresthesias, or muscle weakness may be present as a consequence of hypokalemic alkalosis. Although aldosterone is a salt-retaining hormone, patients with primary aldosteronism rarely have edema. Renal dysfunction and cardiovascular disease are strikingly increased in patients with primary aldosteronism compared to those with primary hypertension.

1	In a hypertensive patient with unprovoked hypokalemia (i.e., unrelated to diuretics, vomiting, or diarrhea), the prevalence of primary aldosteronism approaches 40–50%. In patients on diuretics, serum potassium <3.1 mmol/L (<3.1 meq/L) also raises the possibility of primary aldosteronism; however, serum potassium is an insensitive and nonspecific screening test. Serum potassium is normal in ~25% of patients subsequently found to have an aldosterone-producing adenoma, and higher percentages of patients with other etiologies of primary aldosteronism are not hypokalemic. Additionally, hypokalemic hypertension may be a consequence of secondary aldosteronism, other mineralocorticoidand glucocorticoid-induced hypertensive disorders, and pheochromocytoma.

1	The ratio of plasma aldosterone to plasma renin activity (PA/ PRA) is a useful screening test. These measurements preferably are obtained in ambulatory patients in the morning. A ratio >30:1 in conjunction with a plasma aldosterone concentration >555 pmol/L (>20 ng/dL) reportedly has a sensitivity of 90% and a specificity of 91% for an aldosterone-producing adenoma. In a Mayo Clinic series, an aldosterone-producing adenoma subsequently was confirmed surgically in >90% of hypertensive patients with a PA/PRA ratio ≥20 and a plasma aldosterone concentration ≥415 pmol/L (≥15 ng/dL). There are, however, several caveats to interpreting the ratio. The cutoff for a “high” ratio is laboratoryand assay-dependent. Some anti-hypertensive agents may affect the ratio (e.g., aldosterone antagonists, angiotensin receptor antagonists, and ACE inhibitors may increase renin; aldosterone antagonists may increase aldosterone). Current recommendations are to withdraw aldosterone antagonists for at least

1	receptor antagonists, and ACE inhibitors may increase renin; aldosterone antagonists may increase aldosterone). Current recommendations are to withdraw aldosterone antagonists for at least 4–6 weeks before obtaining these measurements. Because aldosterone biosynthesis is potassium-dependent, hypokalemia should be corrected with oral potassium supplements prior to screening. With these caveats, the ratio has been reported to be useful as a screening test in measurements obtained with patients taking their usual antihypertensive medications. A high ratio in the absence of an elevated plasma aldosterone level is considerably less specific for primary aldosteronism since many patients with primary hypertension have low renin levels in this setting, particularly African Americans and elderly patients.

1	In patients with renal insufficiency, the ratio may also be elevated because of decreased aldosterone clearance. In patients with an elevated PA/PRA ratio, the diagnosis of primary aldosteronism can be confirmed by demonstrating failure to suppress plasma aldosterone to <277 pmol/L (<10 ng/dL) after IV infusion of 2 L of isotonic saline over 4 h; post-saline infusion plasma aldosterone values between 138 and 277 pmol/L (5–10 ng/dL) are not determinant. Alternative confirmatory tests include failure to suppress aldosterone (based on test specific criteria) in response to an oral NaCl load, fludrocortisone, or captopril.

1	Several sporadic and familial adrenal abnormalities may culminate in the syndrome of primary aldosteronism, and appropriate therapy depends on the specific etiology. The two most common causes of sporadic primary aldosteronism are an aldosterone-producing adenoma and bilateral adrenal hyperplasia. Together, they account for >90% of all patients with primary aldosteronism. The tumor is almost always unilateral, and most often measures <3 cm in diameter. Most of the remainder of these patients have bilateral adrenocortical hyperplasia (idiopathic hyperaldosteronism). Rarely, primary aldosteronism may be caused by an adrenal carcinoma or an ectopic malignancy, e.g., ovarian arrhenoblastoma. Most aldosterone-producing carcinomas, in contrast to adrenal adenomas and hyperplasia, produce excessive amounts of other adrenal steroids in addition to aldosterone. Functional differences in hormone secretion may assist in the diagnosis of adenoma vs. hyperplasia. Aldosterone biosynthesis is more

1	amounts of other adrenal steroids in addition to aldosterone. Functional differences in hormone secretion may assist in the diagnosis of adenoma vs. hyperplasia. Aldosterone biosynthesis is more responsive to ACTH in patients with adenoma and more responsive to angiotensin in patients with hyperplasia. Consequently, patients with adenoma tend to have higher plasma aldosterone in the early morning that decreases during the day, reflecting the diurnal rhythm of ACTH, whereas plasma aldosterone tends to increase with upright posture in patients with hyperplasia, reflecting the normal postural response of the renin-angiotensin-aldosterone axis. However, there is overlap in the ability of these measurements to discriminate between adenoma and hyperplasia. Rare familial forms of primary aldosteronism include glucocorticoid-remediable primary aldosteronism and familial aldosteronism types II and III. Genetic testing may assist in the diagnosis of these familial disorders.

1	Adrenal computed tomography (CT) should be carried out in all patients diagnosed with primary aldosteronism. High-resolution CT may identify tumors as small as 0.3 cm and is positive for an adrenal tumor 90% of the time. If the CT is not diagnostic, an adenoma may be detected by adrenal scintigraphy with 6 β-[I131] iodomethyl-19norcholesterol after dexamethasone suppression (0.5 mg every 6 h for 7 days); however, this technique has decreased sensitivity for adenomas <1.5 cm.

1	When carried out by an experienced radiologist, bilateral adrenal venous sampling for measurement of plasma aldosterone is the most accurate means of differentiating unilateral from bilateral forms of primary aldosteronism. The sensitivity and specificity of adrenal venous sampling (95% and 100%, respectively) for detecting unilateral aldosterone hypersecretion are superior to those of adrenal CT; success rates are 90–96%, and complication rates are <2.5%. One frequently used protocol involves sampling for aldosterone and cortisol levels in response to ACTH stimulation. An ipsilateral/contralateral aldosterone ratio >4, with symmetric ACTH-stimulated cortisol levels, is indicative of unilateral aldosterone production.

1	Hypertension generally is responsive to surgery in patients with adenoma but not in patients with bilateral adrenal hyperplasia. Unilateral adrenalectomy, often done via a laparoscopic approach, is curative in 40–70% of patients with an adenoma. Transient hypoaldosteronism may occur up to 3 months postoperatively, resulting in hyperkalemia. Potassium should be monitored during this time, and hyperkalemia should be treated with potassium-wasting diuretics and with fludrocortisone, if needed. Patients with bilateral hyperplasia should be treated medically. The drug regimen for these patients, as well as for patients with an adenoma who are poor surgical candidates, should include an aldosterone antagonist and, if necessary, other potassium-sparing diuretics.

1	Glucocorticoid-remediable hyperaldosteronism is a rare, monogenic autosomal dominant disorder characterized by moderate to severe hypertension, often occurring at an early age. These patients 1619 may have a family history of hemorrhagic stroke at a young age. Hypokalemia is usually mild or absent. Normally, angiotensin II stimulates aldosterone production by the adrenal zona glomerulosa, whereas ACTH stimulates cortisol production in the zona fasciculata. Owing to a chimeric gene on chromosome 8, ACTH also regulates aldosterone secretion by the zona fasciculata in patients with glucocorticoid-remediable hyperaldosteronism. The consequence is overproduction in the zona fasciculata of both aldosterone and hybrid steroids (18-hydroxycortisol and 18-oxocortisol) due to oxidation of cortisol. The diagnosis may be established by urine excretion rates of these hybrid steroids that are 20 to 30 times normal or by direct genetic testing. Therapeutically, suppression of ACTH with low-dose

1	The diagnosis may be established by urine excretion rates of these hybrid steroids that are 20 to 30 times normal or by direct genetic testing. Therapeutically, suppression of ACTH with low-dose glucocorticoids corrects the hyperaldosteronism, hypertension, and hypokalemia. Aldosterone antagonists are also therapeutic options. Patients with familial aldosteronism types II and III are treated with aldosterone antagonists or adrenalectomy.

1	(See also Chap. 406) Cushing’s syndrome is related to excess cortisol production due either to excess ACTH secretion (from a pituitary tumor or an ectopic tumor) or to ACTH-independent adrenal production of cortisol. Hypertension occurs in 75–80% of patients with Cushing’s syndrome. The mechanism of hypertension may be related to stimulation of mineralocorticoid receptors by cortisol and increased secretion of other adrenal steroids. If clinically suspected based on phenotypic characteristics, in patients not taking exogenous glucocorticoids, laboratory screening may be carried out with measurement of 24-h excretion rates of urine free cortisol or an overnight dexamethasone-suppression test. Late night salivary cortisol is also a sensitive and convenient screening test. Further evaluation is required to confirm the diagnosis and identify the specific etiology of Cushing’s syndrome. Appropriate therapy depends on the etiology.

1	(See also Chap. 407) Catecholamine-secreting tumors are located in the adrenal medulla (pheochromocytoma) or in extra-adrenal paraganglion tissue (paraganglioma) and account for hypertension in ~0.05% of patients. If unrecognized, pheochromocytoma may result in lethal cardiovascular consequences. Clinical manifestations, including hypertension, are primarily related to increased circulating catecholamines, although some of these tumors may secrete a number of other vasoactive substances. In a small percentage of patients, epinephrine is the predominant catecholamine secreted by the tumor, and these patients may present with hypotension rather than hypertension. The initial suspicion of the diagnosis is based on symptoms and/or the association of pheochromocytoma with other disorders (Table 298-4). Approximately 20% of pheochromocytomas are familial with autosomal dominant inheritance. Inherited pheochromocytomas may be associated with multiple endocrine neoplasia (MEN) type 2A and

1	298-4). Approximately 20% of pheochromocytomas are familial with autosomal dominant inheritance. Inherited pheochromocytomas may be associated with multiple endocrine neoplasia (MEN) type 2A and type 2B, von Hippel-Lindau disease, and neurofibromatosis (Table 298-4). Each of these syndromes is related to specific, identifiable germ-line mutations. Additionally, mutations of succinate dehydrogenase genes are associated with paraganglioma syndromes, generally characterized by head and neck paragangliomas. Laboratory testing consists of measuring catecholamines in either urine or plasma, e.g., 24-h urine metanephrine excretion or fractionated plasma free metanephrines. The urine measurement is less sensitive but more specific. Genetic screening is available for evaluating patients and relatives suspected of harboring a pheochromocytoma associated with a familial syndrome. Surgical excision is the definitive treatment of pheochromocytoma and results in cure in ~90% of patients.

1	Independent of obesity, hypertension occurs in >50% of individuals with obstructive sleep apnea. The severity of hypertension correlates with the severity of sleep apnea. Approximately 70% of patients with obstructive sleep apnea are obese. Hypertension related to obstructive sleep apnea also should be considered in patients with drug-resistant hypertension and patients with a history of snoring. The diagnosis can be confirmed by polysomnography. In obese patients, weight loss may alleviate or cure sleep apnea and related hypertension. Continuous positive airway pressure (CPAP) or bilevel positive airway pressure (BiPAP) administered during sleep is an effective therapy for obstructive sleep apnea. With CPAP or BiPAP, patients with apparently drug-resistant hypertension may be more responsive to antihypertensive agents.

1	Coarctation of the aorta is the most common congenital cardiovascular cause of hypertension (Chap. 282). The incidence is 1–8 per 1000 live births. It is usually sporadic but occurs in 35% of children with Turner’s syndrome. Even when the anatomic lesion is surgically corrected in infancy, up to 30% of patients develop subsequent hypertension and are at risk of accelerated coronary artery disease and cerebrovascular events. Patients with less severe lesions may not be diagnosed until young adulthood. Physical findings include diminished and delayed femoral pulses and a systolic pressure gradient between the right arm and the legs and, depending on the location of the coarctation, between the right and left arms. A blowing systolic murmur may be heard in the posterior left interscapular areas. The diagnosis may be confirmed by chest x-ray and transesophageal echocardiography. Therapeutic options include surgical repair and balloon angioplasty, with or without placement of an

1	areas. The diagnosis may be confirmed by chest x-ray and transesophageal echocardiography. Therapeutic options include surgical repair and balloon angioplasty, with or without placement of an intravascular stent. Subsequently, many patients do not have a normal life expectancy but may have persistent hypertension, with death due to ischemic heart disease, cerebral hemorrhage, or aortic aneurysm.

1	Several additional endocrine disorders, including thyroid diseases and acromegaly, cause hypertension. Mild diastolic hypertension may be a consequence of hypothyroidism, whereas hyperthyroidism may result in systolic hypertension. Hypercalcemia of any etiology, the most common being primary hyperparathyroidism, may result in hypertension. Hypertension also may be related to a number of prescribed or over-the-counter medications.

1	In addition to glucocorticoid-remediable primary aldosteronism, a number of rare forms of monogenic hypertension have been identified (Table 298–4). These disorders may be recognized by their characteristic phenotypes, and in many instances the diagnosis may be confirmed by genetic analysis. Several inherited defects in adrenal steroid biosynthesis and metabolism result in mineralocorticoid-induced hypertension and hypokalemia. In patients with a 17α-hydroxylase deficiency, synthesis of sex hormones and cortisol is decreased (Fig. 298-3). Consequently, these individuals do not mature sexually; males may present with pseudohermaphroditism and females with primary amenorrhea and absent secondary sexual characteristics. Because cortisol-induced negative feedback on pituitary ACTH production is diminished, ACTH-stimulated adrenal steroid synthesis proximal to the enzymatic block is increased. Hypertension and hypokalemia are consequences of increased synthesis of mineralocorticoids

1	is diminished, ACTH-stimulated adrenal steroid synthesis proximal to the enzymatic block is increased. Hypertension and hypokalemia are consequences of increased synthesis of mineralocorticoids proximal to the enzymatic block, particularly desoxycorticosterone. Increased steroid production and, hence, hypertension may be treated with low-dose glucocorticoids. An 11β-hydroxylase deficiency results in a salt-retaining adrenogenital syndrome that occurs in 1 in 100,000 live births. This enzymatic defect results in decreased cortisol synthesis, increased synthesis of mineralocorticoids (e.g., desoxycorticosterone), and shunting of steroid biosynthesis into the androgen pathway. In the severe form, the syndrome may present early in life, including the

1	FIGURE 298-3 Adrenal enzymatic defects. DHEA, dehydroepiandrosterone.

1	newborn period, with virilization and ambiguous genitalia in females and penile enlargement in males, or in older children as precocious puberty and short stature. Acne, hirsutism, and menstrual irregularities may be the presenting features when the disorder is first recognized in adolescence or early adulthood. Hypertension is less common in the late-onset forms. Patients with an 11β-hydroxysteroid dehydrogenase deficiency have an impaired capacity to metabolize cortisol to its inactive metabolite, cortisone, and hypertension is related to activation of mineralocorticoid receptors by cortisol. This defect may be inherited or acquired, due to licorice-containing glycyrrhizin acid. The same substance is present in the paste of several brands of chewing tobacco. The defect in Liddle’s syndrome (Chaps. 63 and 406) results from constitutive activation of amiloride-sensitive epithelial sodium channels on the distal renal tubule, resulting in excess sodium reabsorption; the syndrome is

1	(Chaps. 63 and 406) results from constitutive activation of amiloride-sensitive epithelial sodium channels on the distal renal tubule, resulting in excess sodium reabsorption; the syndrome is ameliorated by amiloride. Hypertension exacerbated in pregnancy (Chap. 8) may be due to activation of the mineralocorticoid receptor by progesterone.

1	APPROACH TO THE PATIENT: The initial assessment of a hypertensive patient should include a complete history and physical examination to confirm a diagnosis of hypertension, screen for other cardiovascular disease risk factors, screen for secondary causes of hypertension, identify cardiovascular consequences of hypertension and other comorbidities, assess blood pressure–related lifestyles, and determine the potential for intervention.

1	Most patients with hypertension have no specific symptoms referable to their blood pressure elevation. Although popularly considered a symptom of elevated arterial pressure, headache generally occurs only in patients with severe hypertension. Characteristically, a “hypertensive headache” occurs in the morning and is localized to the occipital region. Other nonspecific symptoms that may be related to elevated blood pressure include dizziness, palpitations, easy fatigability, and impotence. When symptoms are present, they are generally related to hypertensive cardiovascular disease or to manifestations of secondary hypertension. Table 298-5 lists salient features that should be addressed in obtaining a history from a hypertensive patient.

1	Reliable measurements of blood pressure depend on attention to the details of the technique and conditions of the measurement. Proper training of observers, positioning of the patient, and selection of cuff size are essential. Owing to recent regulations preventing the use of mercury because of concerns about its potential toxicity, most office measurements are made with aneroid sphygmomanometers or with oscillometric devices. These instruments should be calibrated periodically, and their accuracy confirmed. Before the blood pressure measurement is taken, the individual should be seated quietly in a chair (not the exam table) with feet on the floor for 5 min in a private, quiet setting with a comfortable room temperature. At least two measurements should be made. The center of the cuff should be at heart level, and the width of the bladder cuff Duration of hypertension Previous therapies: responses and side effects Family history of hypertension and cardiovascular disease

1	Duration of hypertension Previous therapies: responses and side effects Family history of hypertension and cardiovascular disease Other risk factors: weight change, dyslipidemia, smoking, diabetes, physical inactivity Evidence of secondary hypertension: history of renal disease; change in appearance; muscle weakness; spells of sweating, palpitations, tremor; erratic sleep, snoring, daytime somnolence; symptoms of hypo-or hyperthyroidism; use of agents that may increase blood pressure Evidence of target organ damage: history of TIA, stroke, transient blindness; angina, myocardial infarction, congestive heart failure; sexual function Abbreviation: TIA, transient ischemic attack.

1	Evidence of target organ damage: history of TIA, stroke, transient blindness; angina, myocardial infarction, congestive heart failure; sexual function Abbreviation: TIA, transient ischemic attack. should equal at least 40% of the arm circumference; the length of the cuff bladder should encircle at least 80% of the arm circumference. It is important to pay attention to cuff placement, stethoscope placement, and the rate of deflation of the cuff (2 mmHg/s). Systolic blood pressure is the first of at least two regular “tapping” Korotkoff sounds, and diastolic blood pressure is the point at which the last regular Korotkoff sound is heard. In current practice, a diagnosis of hypertension generally is based on seated, office measurements.

1	Currently available ambulatory monitors are fully automated, use the oscillometric technique, and typically are programmed to take readings every 15–30 min. Twenty-four-hour ambulatory blood pressure monitoring more reliably predicts cardiovascular disease risk than do office measurements. However, ambulatory monitoring is not used routinely in clinical practice and generally is reserved for patients in whom white coat hypertension is suspected. The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC 7) has also recommended ambulatory monitoring for treatment resistance, symptomatic hypotension, autonomic failure, and episodic hypertension.

1	Body habitus, including weight and height, should be noted. At the initial examination, blood pressure should be measured in both arms and preferably in the supine, sitting, and standing positions to evaluate for postural hypotension. Even if the femoral pulse is normal to palpation, arterial pressure should be measured at least once in the lower extremity in patients in whom hypertension is discovered before age 30. Heart rate also should be recorded. Hypertensive individuals have an increased prevalence of atrial fibrillation. The neck should be palpated for an enlarged thyroid gland, and patients should be assessed for signs of hypoand hyperthyroidism. Examination of blood vessels may provide clues about underlying vascular disease and should include funduscopic examination, auscultation for bruits over the carotid and femoral arteries, and palpation of femoral and pedal pulses. The retina is the only tissue in which arteries and arterioles can be examined directly. With increasing

1	for bruits over the carotid and femoral arteries, and palpation of femoral and pedal pulses. The retina is the only tissue in which arteries and arterioles can be examined directly. With increasing severity of hypertension and atherosclerotic disease, progressive funduscopic changes include increased arteriolar light reflex, arteriovenous crossing defects, hemorrhages and exudates, and, in patients with malignant hypertension, papilledema. Examination of the heart may reveal a loud second heart sound due to closure of the aortic valve and an S4 gallop attributed to atrial contraction against a noncompliant left ventricle. Left ventricular hypertrophy may be detected by an enlarged, sustained, and laterally displaced apical impulse. An abdominal bruit, particularly a bruit that lateralizes and extends throughout systole into diastole, raises the possibility of renovascular hypertension. Kidneys of patients with polycystic kidney disease may be palpable in the abdomen. The physical

1	and extends throughout systole into diastole, raises the possibility of renovascular hypertension. Kidneys of patients with polycystic kidney disease may be palpable in the abdomen. The physical examination also should include evaluation for signs of CHF and a neurologic examination.

1	Table 298-6 lists recommended laboratory tests in the initial evaluation of hypertensive patients. Repeat measurements of renal function, serum electrolytes, fasting glucose, and lipids may be obtained Abbreviations: BUN, blood urea nitrogen; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TSH, thyroid-stimulating hormone. after the introduction of a new antihypertensive agent and then annually or more frequently if clinically indicated. More extensive laboratory testing is appropriate for patients with apparent drug-resistant hypertension or when the clinical evaluation suggests a secondary form of hypertension.

1	Implementation of lifestyles that favorably affect blood pressure has implications for both the prevention and the treatment of hypertension. Health-promoting lifestyle modifications are recommended for individuals with prehypertension and as an adjunct to drug therapy in hypertensive individuals. These interventions should address overall cardiovascular disease risk. Although the impact of lifestyle interventions on blood pressure is more pronounced in persons with hypertension, in short-term trials, weight loss and reduction of dietary NaCl have been shown to prevent the development of hypertension. In hypertensive individuals, even if these interventions do not produce a sufficient reduction in blood pressure to avoid drug therapy, the number of medications or doses required for blood pressure control may be reduced. Dietary modifications that effectively lower blood pressure are weight loss, reduced NaCl intake, increased potassium intake, moderation of alcohol consumption, and an

1	control may be reduced. Dietary modifications that effectively lower blood pressure are weight loss, reduced NaCl intake, increased potassium intake, moderation of alcohol consumption, and an overall healthy dietary pattern (Table 298-7).

1	Prevention and treatment of obesity are important for reducing blood pressure and cardiovascular disease risk. In short-term trials, even modest weight loss can lead to a reduction of blood pressure and an increase in insulin sensitivity. Average blood pressure reductions of 6.3/3.1 mmHg have been observed with a reduction in mean body weight of 9.2 kg. Regular physical activity facilitates weight loss, decreases blood pressure, and reduces the overall risk of cardiovascular disease. Blood pressure may be lowered by 30 min of moderately intense physical activity, such as brisk walking, 6–7 days a week, or by more intense, less frequent workouts.

1	There is individual variability in the sensitivity of blood pressure to NaCl, and this variability may have a genetic basis. Based on results of meta-analyses, lowering of blood pressure by limiting daily NaCl intake to 4.4–7.4 g (75–125 meq) results in blood pressure reductions of 3.7–4.9/0.9–2.9 mmHg in hypertensive individuals and lesser reductions in normotensive individuals. Several long-term, prospective, randomized clinical trials have reported that a reduced salt intake results in a decreased incidence of cardiovascular events. Although reduced salt intakes are generally recommended for both the prevention and treatment of hypertension, overly rigorous salt restriction may have adverse cardiovascular outcomes in diabetic patients and in patients with CHF aggressively treated with diuretics. Potassium and calcium supplementation have inconsistent, modest antihypertensive effects, and, independent of blood pressure, potassium supplementation may be associated with reduced stroke

1	Potassium and calcium supplementation have inconsistent, modest antihypertensive effects, and, independent of blood pressure, potassium supplementation may be associated with reduced stroke mortality. Consuming three or more alcoholic drinks per day (a standard drink contains ~14 g ethanol) is associated with higher blood pressures, and a reduction of alcohol consumption is associated with a

1	Abbreviations: BMI, body mass index; DASH, Dietary Approaches to Stop Hypertension (trial). reduction of blood pressure. In patients with advanced renal disease, dietary protein restriction may have a modest effect in mitigating renal damage by reducing the intrarenal transmission of systemic arterial pressure. The DASH (Dietary Approaches to Stop Hypertension) trial convincingly demonstrated that over an 8-week period a diet high in fruits, vegetables, and low-fat dairy products lowers blood pressure in individuals with high-normal blood pressures or mild hypertension. Reduction of daily NaCl intake to <6 g (100 meq) augmented the effect of this diet on blood pressure. Fruits and vegetables are enriched sources of potassium, magnesium, and fiber, and dairy products are an important source of calcium.

1	Drug therapy is recommended for individuals with blood pressures ≥140/90 mmHg. The degree of benefit derived from antihypertensive agents is related to the magnitude of the blood pressure reduction. Lowering systolic blood pressure by 10–12 mmHg and diastolic blood pressure by 5–6 mmHg confers relative risk reductions of 35–40% for stroke and 12–16% for CHD within 5 years of the initiation of treatment. Risk of heart failure is reduced by >50%. Hypertension control is the single most effective intervention for slowing the rate of progression of hypertension-related kidney disease.

1	There is considerable variation in individual responses to different classes of antihypertensive agents, and the magnitude of response to any single agent may be limited by activation of counter-regulatory mechanisms. Most available agents reduce systolic blood pressure by 7–13 mmHg and diastolic blood pressure by 4–8 mmHg when corrected for placebo effect. More often than not, combinations of agents, with complementary antihypertensive mechanisms, are required to achieve goal blood pressure reductions. Selection of antihypertensive agents and combinations of agents should be individualized, taking into account age, severity of hypertension, other cardiovascular disease risk factors, comorbid conditions, and practical considerations related to cost, side effects, and frequency of dosing (Table 298-8).

1	Diuretics Low-dose thiazide diuretics may be used alone or in combination with other antihypertensive drugs. Thiazides inhibit the Na+/Cl− pump in the distal convoluted tubule and hence increase sodium excretion. In the long term, they also may act as vasodilators. Thiazides are safe, efficacious, inexpensive, and reduce clinical events. They provide additive blood pressure–lowering effects when combined with beta blockers, angiotensin-converting enzyme inhibitors (ACEIs), or angiotensin receptor blockers (ARBs). In contrast, addition of a diuretic to a calcium channel blocker is less effective. Usual doses of hydrochlorothiazide range from 6.25–50 mg/d. Owing to an increased incidence of metabolic side effects (hypokalemia, insulin resistance, increased cholesterol), higher doses generally are not recommended. Chlorthalidone is a diuretic structurally similar to hydrochlorothiazide, and like hydrochlorothiazide, it blocks sodium-chloride cotransport in the early distal tubule.

1	are not recommended. Chlorthalidone is a diuretic structurally similar to hydrochlorothiazide, and like hydrochlorothiazide, it blocks sodium-chloride cotransport in the early distal tubule. However, chlorthalidone has a longer half-life (40–60 h vs. 9–15 h) and an antihypertensive potency ~1.5–2.0 times that of hydrochlorothiazide. Potassium loss is also greater with chlorthalidone. Two potassium-sparing diuretics, amiloride and triamterene, act by inhibiting epithelial sodium channels in the distal nephron. These agents are weak antihypertensive agents but may be used in combination with a thiazide to protect against hypokalemia. The main pharmacologic target for loop diuretics is the Na+-K+-2Cl− cotransporter in the thick ascending limb of the loop of Henle. Loop diuretics generally are reserved for hypertensive patients with reduced glomerular filtration rates (reflected in serum creatinine >220 μmol/L [>2.5 mg/ dL]), CHF, or sodium retention and edema for some other reason, such

1	reserved for hypertensive patients with reduced glomerular filtration rates (reflected in serum creatinine >220 μmol/L [>2.5 mg/ dL]), CHF, or sodium retention and edema for some other reason, such as treatment with a potent vasodilator, e.g., minoxidil.

1	Blockers of the Renin–Angiotensin System ACEIs decrease the production of angiotensin II, increase bradykinin levels, and reduce sympathetic nervous system activity. ARBs provide selective blockade of AT1 receptors, and the effect of angiotensin II on unblocked AT2 receptors 1623 may augment their hypotensive effect. Both classes of agents are effective antihypertensive agents that may be used as monotherapy or in combination with diuretics, calcium antagonists, and alpha blocking agents. ACEIs and ARBs improve insulin action and ameliorate the adverse effects of diuretics on glucose metabolism. Although the overall impact on the incidence of diabetes is modest, compared with amlodipine (a calcium antagonist), valsartan (an ARB) has been shown to reduce the risk of developing diabetes in high-risk hypertensive patients. ACEI/ARB combinations are less effective in lower ing blood pressure than is the case when either class of these agents is used in combination with other classes of

1	high-risk hypertensive patients. ACEI/ARB combinations are less effective in lower ing blood pressure than is the case when either class of these agents is used in combination with other classes of agents. In patients with vascular disease or a high risk of diabetes, combination ACEI/ARB therapy has been associated with more adverse events (e.g., cardiovascular death, myocardial infarction, stroke, and hospitalization for heart failure) without increases in benefit.

1	Side effects of ACEIs and ARBs include functional renal insufficiency due to efferent renal arteriolar dilation in a kidney with a stenotic lesion of the renal artery. Additional predisposing conditions to renal insufficiency induced by these agents include dehydration, CHF, and use of nonsteroidal anti-inflammatory drugs. Dry cough occurs in ~15% of patients, and angioedema occurs in <1% of patients taking ACEIs. Angioedema occurs most commonly in individuals of Asian origin and more commonly in African Americans than in whites. Hyperkalemia due to hypoaldosteronism is an occasional side effect of both ACEIs and ARBs.

1	An alternative approach to blocking the renin-angiotensin system has recently been introduced into clinical practice for the treatment of hypertension: direct renin inhibitors. Blockade of the renin-angiotensin system is more complete with renin inhibitors than with ACEIs or ARBs. Aliskiren is the first of a class of oral, nonpeptide competitive inhibitors of the enzymatic activity of renin. Monotherapy with aliskiren seems to be as effective as an ACEI or ARB for lowering blood pressure, but not more effective. Further blood reductions may be achieved when aliskiren is used in combination with a thiazide diuretic or a calcium antagonist. Currently, aliskiren is not considered a first-line antihypertensive agent.

1	Aldosterone Antagonists Spironolactone is a nonselective aldosterone antagonist that may be used alone or in combination with a thiazide diuretic. It may be a particularly effective agent in patients with low-renin primary hypertension, resistant hypertension, and primary aldosteronism. In patients with CHF, low-dose spironolactone reduces mortality and hospitalizations for heart failure when given in addition to conventional therapy with ACEIs, digoxin, and loop diuretics. Because spironolactone binds to progesterone and androgen receptors, side effects may include gynecomastia, impotence, and menstrual abnormalities. These side effects are circumvented by a newer agent, eplerenone, which is a selective aldosterone antagonist.

1	Beta Blockers β-Adrenergic receptor blockers lower blood pressure by decreasing cardiac output, due to a reduction of heart rate and contractility. Other proposed mechanisms by which beta blockers lower blood pressure include a central nervous system effect and inhibition of renin release. Beta blockers are particularly effective in hypertensive patients with tachycardia, and their hypotensive potency is enhanced by coadministration with a diuretic. In lower doses, some beta blockers selectively inhibit cardiac β1 receptors and have less influence on β2 receptors on bronchial and vascular smooth muscle cells; however, there seems to be no difference in the antihypertensive potencies of cardioselective and nonselective beta blockers. Some beta blockers have intrinsic sympathomimetic activity, although it is uncertain whether this constitutes an overall advantage or disadvantage in cardiac therapy. Beta blockers without intrinsic sympathomimetic activity decrease the rate of sudden

1	although it is uncertain whether this constitutes an overall advantage or disadvantage in cardiac therapy. Beta blockers without intrinsic sympathomimetic activity decrease the rate of sudden death, overall mortality, and recurrent myocardial infarction. In patients with CHF, beta blockers have been shown to reduce the risks of hospitalization and mortality. Overall, beta blockers

1	Diuretics Thiazides Hydrochlorothiazide 6.25–50 mg (1–2) Diabetes, dyslipidemia, hyperuricemia, gout, hypokalemia Chlorthalidone 25–50 mg (1) Loop diuretics Furosemide 40–80 mg (2–3) CHF due to systolic dysfunc-Diabetes, dyslipidemia, hypertion, renal failure uricemia, gout, hypokalemia Aldosterone antagonists Spironolactone 25–100 mg (1–2) CHF due to systolic dysfunc-Renal failure, hyperkalemia tion, primary aldosteronism Renal failure, hyperkalemia Triamterene 50–100 mg (1–2) Beta blockers Asthma, COPD, 2ndor 3rd-degree heart block, sick- Cardioselective Atenolol 25–100 mg (1) Angina, CHF due to systolic sinus syndrome dysfunction, post-MI, sinus tachycardia, ventricular tachyarrhythmias Metoprolol 25–100 mg (1–2) Nonselective Propranolol 40–160 mg (2) Propranolol LA 60–180 (1) Combined alpha/beta Labetalol 200–800 mg (2) ?Post-MI, CHF Carvedilol 12.5–50 mg (2) Alpha antagonists

1	Metoprolol 25–100 mg (1–2) Nonselective Propranolol 40–160 mg (2) Propranolol LA 60–180 (1) Combined alpha/beta Labetalol 200–800 mg (2) ?Post-MI, CHF Carvedilol 12.5–50 mg (2) Alpha antagonists Central Clonidine 0.1–0.6 mg (2) Clonidine patch 0.1–0.3 mg (1/week) Methyldopa 250–1000 mg (2) Reserpine 0.05–0.25 mg (1) Guanfacine 0.5–2 mg (1) ACE inhibitors Captopril 25–200 mg (2) Post-MI, coronary syndromes, Acute renal failure, bilateral CHF with low ejection frac-renal artery stenosis, preg- Lisinopril 10–40 mg (1) tion, nephropathy nancy, hyperkalemia Ramipril 2.5–20 mg (1–2) Angiotensin II antagonists Losartan 25–100 mg (1–2) CHF with low ejection frac-Renal failure, bilateral renal tion, nephropathy, ACE inhibi-artery stenosis, pregnancy, Dihydropyridines Nifedipine (long-acting) 30–60 mg (1) Nondihydropyridines Verapamil (long-acting) 120–360 mg (1–2) Post-MI, supraventricular 2nd-or 3rd-degree heart block tachycardias, angina

1	Dihydropyridines Nifedipine (long-acting) 30–60 mg (1) Nondihydropyridines Verapamil (long-acting) 120–360 mg (1–2) Post-MI, supraventricular 2nd-or 3rd-degree heart block tachycardias, angina Severe coronary artery disease Minoxidil 2.5–80 mg (1–2) aAt the initiation of therapy, lower doses may be preferable for elderly patients and for select combinations of antihypertensive agents. Abbreviations: ACE, angiotensin-converting enzyme; CHF, congestive heart failure; COPD, chronic obstructive pulmonary disease; MI, myocardial infarction.

1	may be less protective against cardiovascular and cerebrovascular α-Adrenergic Blockers Postsynaptic, selective α-adrenoreceptor endpoints, and some beta blockers may have less effect on cen-antagonists lower blood pressure by decreasing peripheral vascular tral aortic pressure than other classes of antihypertensive agents. resistance. They are effective antihypertensive agents used either However, beta blockers remain appropriate therapy for hypertensive as monotherapy or in combination with other agents. However, patients with concomitant heart disease and related comorbidi-in clinical trials of hypertensive patients, alpha blockade has not ties. Carvedilol and labetalol block both β receptors and peripheral been shown to reduce cardiovascular morbidity and mortality α-adrenergic receptors. The potential advantages of combined or to provide as much protection against CHF as other classes of βand α-adrenergic blockade in treating hypertension remain to antihypertensive agents. These

1	The potential advantages of combined or to provide as much protection against CHF as other classes of βand α-adrenergic blockade in treating hypertension remain to antihypertensive agents. These agents are also effective in treating be determined. Nebivolol represents another class of cardioselec-lower urinary tract symptoms in men with prostatic hypertrophy. tive beta blockers that has additional vasodilator actions related to Nonselective α-adrenoreceptor antagonists bind to postsynaptic enhancement of nitric oxide activity. Whether this confers greater and presynaptic receptors and are used primarily for the manage-clinical effectiveness remains to be determined. ment of patients with pheochromocytoma.

1	Sympatholytic Agents Centrally acting α2 sympathetic agonists decrease peripheral resistance by inhibiting sympathetic outflow. They may be particularly useful in patients with autonomic neuropathy who have wide variations in blood pressure due to baroreceptor denervation. Drawbacks include somnolence, dry mouth, and rebound hypertension on withdrawal. Peripheral sympatholytics decrease peripheral resistance and venous constriction by depleting nerve terminal norepinephrine. Although they are potentially effective antihypertensive agents, their usefulness is limited by orthostatic hypotension, sexual dysfunction, and numerous drug-drug interactions. Rebound hypertension is another concern with abrupt cessation of drugs with a short half-life.

1	Calcium Channel Blockers Calcium antagonists reduce vascular resistance through L-channel blockade, which reduces intracellular calcium and blunts vasoconstriction. This is a heterogeneous group of agents that includes drugs in the following three classes: phenylalkylamines (verapamil), benzothiazepines (diltiazem), and 1,4-dihydropyridines (nifedipine-like). Used alone and in combination with other agents (ACEIs, beta blockers, α1-adrenergic blockers), calcium antagonists effectively lower blood pressure; however, it is unclear if adding a diuretic to a calcium blocker results in a further lowering of blood pressure. Side effects of flushing, headache, and edema with dihydropyridine use are related to their potencies as arteriolar dilators; edema is due to an increase in transcapillary pressure gradients, not to net salt and water retention.

1	Direct Vasodilators Direct vasodilators decrease peripheral resistance and concomitantly activate mechanisms that defend arterial pressure, notably the sympathetic nervous system, the renin-angiotensin-aldosterone system, and sodium retention. Usually, they are not considered first-line agents but are most effective when added to a combination that includes a diuretic and a beta blocker. Hydralazine is a potent direct vasodilator that has antioxidant and nitric oxide– enhancing actions, and minoxidil is a particularly potent agent and is used most frequently in patients with renal insufficiency who are refractory to all other drugs. Hydralazine may induce a lupus-like syndrome, and side effects of minoxidil include hypertrichosis and pericardial effusion. Intravenous nitroprusside can be used to treat malignant hypertension and life-threatening left ventricular heart failure associated with elevated arterial pressure.

1	Based on pooling results from clinical trials, meta-analyses of the efficacy of different classes of antihypertensive agents suggest essentially equivalent blood pressure–lowering effects of the following six major classes of antihypertensive agents when used as monotherapy: thiazide diuretics, beta blockers, ACEIs, ARBs, calcium antagonists, and α1 blockers. On average, standard doses of most antihypertensive agents reduce blood pressure by 8–10/4–7 mmHg; however, there may be subgroup differences in responsiveness. Younger patients may be more responsive to beta blockers and ACEIs, whereas patients over age 50 may be more responsive to diuretics and calcium antagonists. There is a limited relationship between plasma renin and blood pressure response. Patients with high-renin hypertension may be more responsive to ACEIs and ARBs than to other classes of agents, whereas patients with low-renin hypertension are more responsive to diuretics and calcium antagonists. Hypertensive African

1	may be more responsive to ACEIs and ARBs than to other classes of agents, whereas patients with low-renin hypertension are more responsive to diuretics and calcium antagonists. Hypertensive African Americans tend to have low renin and may require higher doses of ACEIs and ARBs than whites for optimal blood pressure control, although this difference is abolished when these agents are combined with a diuretic. Beta blockers also appear to be less effective than thiazide diuretics in African Americans than in non-African Americans. Early pharmacogenetic studies, utilizing either a candidate gene approach or genome-wide scans, have shown associations of gene polymorphisms with blood pressure responsiveness to specific antihypertensive drugs. However, the reported effects have generally been too small to affect clinical decisions, and associated polymorphisms remain to be confirmed.

1	Currently, in practical terms, the presence of comorbidities often 1625 influences the selection of antihypertensive agents. A meta-analysis of more than 30 randomized trials of blood pressure–lowering therapy indicates that for a given reduction in blood pressure, the major drug classes seem to produce similar overall net effects on total cardiovascular events. In both non-diabetic and diabetic hypertensive patients, most trials have failed to show significant differences in cardiovascular outcomes with different drug regimens as long as equivalent decreases in blood pressure were achieved. For example, the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) demonstrated that the occurrence of CHD and nonfatal myocardial infarction, as well as overall mortality, was virtually identical in hypertensive patients treated with either an ACEI (lisinopril), a diuretic (chlorthalidone), or a calcium antagonist (amlodipine).

1	However, in specific patient groups, ACEIs may have particular advantages, beyond that of blood pressure control, in reducing cardiovascular and renal outcomes. ACEIs and ARBs decrease intraglomerular pressure and proteinuria and may retard the rate of progression of renal insufficiency, not totally accounted for by their hypotensive effects, in both diabetic and nondiabetic renal diseases. In patients with type 2 diabetes, treatment with an ACEI, an ARB, or aliskiren decreases proteinuria and delays the progression of renal disease. In experimental models of hypertension and diabetes, renal protection with aliskiren is comparable to that with ACEIs and ARBs. However, in patients with type 2 diabetes, addition of aliskiren to an ACEI provides no additional protection against cardiovascular or renal disease and may be associated with more adverse outcomes. Among African Americans with hypertension-related renal disease, ACEIs appear to be more effective than beta blockers or

1	or renal disease and may be associated with more adverse outcomes. Among African Americans with hypertension-related renal disease, ACEIs appear to be more effective than beta blockers or dihydropyridine calcium channel blockers in slowing, although not preventing, the decline of glomerular filtration rate. The renoprotective effect of these renin-angiotensin blockers, compared with other antihypertensive drugs, is less obvious at lower blood pressures. In most patients with hypertension and heart failure due to systolic and/or diastolic dysfunction, the use of diuretics, ACEIs or ARBs, and beta blockers is recommended to improve survival. Independent of blood pressure, in both hypertensive and normotensive individuals, ACEIs attenuate the development of left ventricular hypertrophy, improve symptomatology and risk of death from CHF, and reduce morbidity and mortality rates in post-myocardial infarction patients. Similar benefits in cardiovascular morbidity and mortality rates in

1	improve symptomatology and risk of death from CHF, and reduce morbidity and mortality rates in post-myocardial infarction patients. Similar benefits in cardiovascular morbidity and mortality rates in patients with CHF have been observed with the use of ARBs. ACEIs provide better coronary protection than do calcium channel blockers, whereas calcium channel blockers provide more stroke protection than do either ACEIs or beta blockers. Results of a large, double-blind, prospective clinical trial (Avoiding Cardiovascular Events through Combination Therapy in Patients Living with Systolic Hypertension [ACCOMPLISH Trial]) indicated that combination treatment with an ACEI (benazepril) plus a calcium antagonist (amlodipine) was superior to treatment with the ACEI plus a diuretic (hydrochlorothiazide) in reducing the risk of cardiovascular events and death among high-risk patients with hypertension. However, the combination of an ACEI and a diuretic has recently been shown to produce major

1	in reducing the risk of cardiovascular events and death among high-risk patients with hypertension. However, the combination of an ACEI and a diuretic has recently been shown to produce major reductions in morbidity and mortality in the very elderly.

1	After a stroke, combination therapy with an ACEI and a diuretic, but not with an ARB, has been reported to reduce the rate of recurrent stroke. Some of these apparent differences may reflect differences in trial design and/or patient groups.

1	There is a recent resurgence of interest in two nonpharmacologic, antihypertensive therapies that interrupt sympathetic outflow: (1) device-based carotid baroreflex activation by electrical stimulation of the carotid sinus; and (2) endovascular radiofrequency ablation of the renal sympathetic nerves. Whereas renal denervation is a minimally invasive procedure, carotid baroreceptor stimulation is a surgical procedure, usually performed under general anesthesia, that currently involves implanting electrodes on both the right and left carotid arteries. Both interventions inhibit sympathetic drive and 1626 decrease blood pressure by increasing the capacity of the kidney to excrete sodium and by decreasing renin release. Sustained activation of the baroreflex most likely lowers blood pressure by other mechanisms as well. Clinical experience with these interventions is limited. In the short term, blood pressure is lowered in 75–80% of patients, and the magnitude of the blood pressure

1	by other mechanisms as well. Clinical experience with these interventions is limited. In the short term, blood pressure is lowered in 75–80% of patients, and the magnitude of the blood pressure reduction is similar for both procedures. To date, the most impressive results have been observed in patients with “resistant” hypertension and patients with obesity-related hypertension. Awaiting the results of long-term, multicenter clinical trials to evaluate their efficacy and safety, it remains to be seen whether these interventions will be adopted into clinical practice.

1	Based on clinical trial data, the maximum protection against combined cardiovascular endpoints is achieved with pressures <135– 140 mmHg for systolic blood pressure and <80–85 mmHg for diastolic blood pressure; however, treatment has not reduced cardiovascular disease risk to the level in nonhypertensive individuals. In diabetic patients, effective blood pressure control reduces the risk of cardiovascular events and death as well as the risk for microvascular disease (nephropathy, retinopathy). Although guidelines for hypertension control have recommended more aggressive blood pressure targets (e.g., office or clinic blood pressure <130/80 mmHg) for patients with diabetes, CHD, chronic kidney disease, or additional cardiovascular disease risk factors, recent evidence suggests that overly aggressive targets for blood pressure control may not be advantageous, particularly in high-risk patients. For example, among hypertensive patients with diabetes and coronary heart disease, “tight

1	aggressive targets for blood pressure control may not be advantageous, particularly in high-risk patients. For example, among hypertensive patients with diabetes and coronary heart disease, “tight control” of systolic blood pressure (<130 mmHg) is not associated with improved cardiovascular outcomes. The concept of a “J-curve” suggests that the risk of cardiovascular events increases at blood pressures that are either too high or too low. Theoretically blood pressures that are too low may exceed the autoregulatory capacity of cerebral, coronary, and renal blood flows. There is some suggestive evidence from recent randomized clinical trials for a J-shaped relationship between blood pressure and cardiovascular outcomes (including all-cause mortality) in high-risk patients. Consequently, caution should be exercised in lowering blood pressure <130/80 mmHg in patients with diabetes, CHD, and other high-risk patients. In patients with chronic renal insufficiency, a small, nonprogressive

1	caution should be exercised in lowering blood pressure <130/80 mmHg in patients with diabetes, CHD, and other high-risk patients. In patients with chronic renal insufficiency, a small, nonprogressive increase in the serum creatinine concentration may occur. This generally reflects a hemodynamic response, not structural renal injury, indicating that intraglomerular pressure has been reduced. Blood pressure control should not be allowed to deteriorate in order to prevent the modest creatinine rise. Among older patients with isolated systolic hypertension, further lowering of diastolic blood pressure does not result in harm. However, relatively little information is available concerning the risk-versusbenefit ratio of antihypertensive therapy in individuals >80 years of age, and in this population, gradual blood pressure reduction to a less aggressive target level of control may be appropriate.

1	To achieve recommended blood pressure goals, the majority of individuals with hypertension will require treatment with more than one drug. Three or more drugs frequently are needed in patients with diabetes and renal insufficiency. For most agents, reduction of blood pressure at half-standard doses is only ~20% less than at standard doses. Appropriate combinations of agents at these lower doses may have additive or almost additive effects on blood pressure with a lower incidence of side effects.

1	The term resistant hypertension refers to patients with blood pressures persistently >140/90 mmHg despite taking three or more antihypertensive agents, including a diuretic. Resistant or difficultto-control hypertension is more common in patients >60 years than in younger patients. Resistant hypertension may be related to “pseudoresistance” (high office blood pressures and lower home blood pressures), nonadherence to therapy, identifiable causes of hypertension (including obesity and excessive alcohol intake), and the use of any of a number of nonprescription and prescription drugs (Table 298-3). Rarely, in older patients, pseudohypertension may be related to the inability to measure blood pressure accurately in severely sclerotic arteries. This condition is suggested if the radial pulse remains palpable despite occlusion of the brachial artery by the cuff (Osler maneuver). The actual blood pressure can be determined by direct intra-arterial measurement. Evaluation of patients with

1	remains palpable despite occlusion of the brachial artery by the cuff (Osler maneuver). The actual blood pressure can be determined by direct intra-arterial measurement. Evaluation of patients with resistant hypertension might include home blood pressure monitoring to determine if office blood pressures are representative of the usual blood pressure. A more extensive evaluation for a secondary form of hypertension should be undertaken if no other explanation for hypertension resistance becomes apparent.

1	Probably due to the widespread availability of antihypertensive therapy, in the United States there has been a decline in the numbers of patients presenting with “crisis levels” of blood pressure. Most patients who present with severe hypertension are chronically hypertensive, and in the absence of acute end organ damage, precipitous lowering of blood pressure may result in significant morbidity and should be avoided. The key to successful management of severe hypertension is to differentiate hypertensive crises from hypertensive urgencies. The degree of target organ damage, rather than the level of blood pressure alone, determines the rapidity with which blood pressure should be lowered. Tables 298-9 and 298-10 list a number of hypertension-related emergencies and recommended therapies.

1	Malignant hypertension is a syndrome associated with an abrupt increase of blood pressure in a patient with underlying hypertension or related to the sudden onset of hypertension in a previously normotensive individual. The absolute level of blood pressure is not as important as its rate of rise. Pathologically, the syndrome is associated with diffuse necrotizing vasculitis, arteriolar thrombi, and fibrin deposition in arteriolar walls. Fibrinoid necrosis has been observed in arterioles of kidney, brain, retina, and other organs. Clinically, the syndrome is recognized by progressive retinopathy (arteriolar spasm, hemorrhages, exudates, and papilledema), deteriorating renal function with proteinuria, microangiopathic hemolytic anemia, and encephalopathy. Historic inquiry should include questions about the use of monoamine oxidase inhibitors and recreational drugs (e.g., cocaine, amphetamines).

1	Although blood pressure should be lowered rapidly in patients with hypertensive encephalopathy, there are inherent risks of overly aggressive therapy. In hypertensive individuals, the upper and lower limits of autoregulation of cerebral blood flow are shifted to higher levels of arterial pressure, and rapid lowering of blood pressure to below the lower limit of autoregulation may precipitate cerebral ischemia or infarction as a consequence of decreased cerebral blood flow. Renal and coronary blood flows also may decrease with overly aggressive acute therapy. The initial goal of therapy is to reduce Hypertensive encephalopathy Nitroprusside, nicardipine, labetalol

1	Hypertensive encephalopathy Nitroprusside, nicardipine, labetalol Malignant hypertension (when IV Labetalol, nicardipine, nitroprusside, therapy is indicated) enalaprilat Stroke Nicardipine, labetalol, nitroprusside Myocardial infarction/unstable Nitroglycerin, nicardipine, labetalol, angina esmolol Acute left ventricular failure Nitroglycerin, enalaprilat, loop diuretics Aortic dissection Nitroprusside, esmolol, labetalol Adrenergic crisis Phentolamine, nitroprusside Postoperative hypertension Nitroglycerin, nitroprusside, labetalol, nicardipine Preeclampsia/eclampsia of Hydralazine, labetalol, nicardipine pregnancy Source: Adapted from DG Vidt, in S Oparil, MA Weber (eds): Hypertension, 2nd ed. Philadelphia, Elsevier Saunders, 2005. Nitroprusside Initial 0.3 (μg/kg)/min; usual 2–4 (μg/kg)/min; maximum 10 (μg/kg)/min for 10 min Nicardipine Initial 5 mg/h; titrate by 2.5 mg/h at 5–15 min intervals; max 15 mg/h

1	Nitroprusside Initial 0.3 (μg/kg)/min; usual 2–4 (μg/kg)/min; maximum 10 (μg/kg)/min for 10 min Nicardipine Initial 5 mg/h; titrate by 2.5 mg/h at 5–15 min intervals; max 15 mg/h Labetalol 2 mg/min up to 300 mg or 20 mg over 2 min, then 40–80 mg at 10-min intervals up to 300 Enalaprilat Usual 0.625–1.25 mg over 5 min every 6–8 h; maximum 5 mg/dose Esmolol Initial 80–500 μg/kg over 1 min, then 50–300 (μg/kg)/min Nitroglycerin Initial 5 μg/min, then titrate by 5 μg/min at 3–5-min intervals; if no response is seen at 20 μg/min, incremental increases of aConstant blood pressure monitoring is required. Start with the lowest dose. Subsequent doses and intervals of administration should be adjusted according to the blood pressure response and duration of action of the specific agent.

1	mean arterial blood pressure by no more than 25% within minutes to 2 h or to a blood pressure in the range of 160/100–110 mmHg. This may be accomplished with IV nitroprusside, a short-acting vasodilator with a rapid onset of action that allows for minute-to-minute control of blood pressure. Parenteral labetalol and nicardipine are also effective agents for the treatment of hypertensive encephalopathy. In patients with malignant hypertension without encephalopathy or another catastrophic event, it is preferable to reduce blood pressure over hours or longer rather than minutes. This goal may effectively be achieved initially with frequent dosing of short-acting oral agents such as captopril, clonidine, and labetalol.

1	Acute, transient blood pressure elevations that last days to weeks frequently occur after thrombotic and hemorrhagic strokes. Autoregulation of cerebral blood flow is impaired in ischemic cerebral tissue, and higher arterial pressures may be required to maintain cerebral blood flow. Although specific blood pressure targets have not been defined for patients with acute cerebrovascular events, aggressive reductions of blood pressure are to be avoided. With the increasing availability of improved methods for measuring cerebral blood flow (using CT technology), studies are in progress to evaluate the effects of different classes of antihypertensive agents on both blood pressure and cerebral blood flow after an acute stroke. Currently, in the absence of other indications for acute therapy, for patients with cerebral infarction who are not candidates for thrombolytic therapy, one recommended guideline is to institute antihypertensive therapy only for patients with a systolic blood pressure

1	patients with cerebral infarction who are not candidates for thrombolytic therapy, one recommended guideline is to institute antihypertensive therapy only for patients with a systolic blood pressure >220 mmHg or a diastolic blood pressure >130 mmHg. If thrombolytic therapy is to be used, the recommended goal blood pressure is <185 mmHg systolic pressure and <110 mmHg diastolic pressure. In patients with hemorrhagic stroke, suggested guidelines for initiating antihypertensive therapy are systolic >180 mmHg or diastolic pressure >130 mmHg. The management of hypertension after subarachnoid hemorrhage is controversial. Cautious reduction of blood pressure is indicated if mean arterial pressure is >130 mmHg.

1	In addition to pheochromocytoma, an adrenergic crisis due to catecholamine excess may be related to cocaine or amphetamine overdose, clonidine withdrawal, acute spinal cord injuries, and an interaction of tyramine-containing compounds with monoamine oxidase inhibitors. These patients may be treated with phentolamine or nitroprusside. Treatment of hypertension in patients with acute aortic dissection is discussed in Chap. 301, and treatment of hypertension in pregnancy is discussed in Chap. 8. Stephen C. Textor

1	Treatment of hypertension in patients with acute aortic dissection is discussed in Chap. 301, and treatment of hypertension in pregnancy is discussed in Chap. 8. Stephen C. Textor The renal vasculature is unusually complex with rich arteriolar flow to the cortex in excess of metabolic requirements, consistent with its primary function as a filtering organ. After delivering blood to cortical glomeruli, the postglomerular circulation supplies deeper medullary segments that support energy-dependent solute transport at multiple levels of the renal tubule. These postglomerular vessels carry less blood, and high oxygen consumption leaves the deeper medullary regions at the margin of hypoxemia. Vascular disorders that commonly threaten the blood supply of the kidney include large-vessel atherosclerosis, fibromuscular diseases, and embolic disorders. Microvascular injury, including inflammatory and primary hematologic disorders, is described in Chap. 341.

1	The glomerular capillary endothelium shares susceptibility to oxidative stress, pressure injury, and inflammation with other vascular territories. Rates of urinary albumin excretion (UAE) are predictive of systemic atherosclerotic disease events. Increased UAE may develop years before cardiovascular events. UAE and the risk of cardiovascular events are both reduced with pharmacologic therapy such as statins. Experimental studies demonstrate functional changes and rarefaction of renal microvessels under conditions of accelerated atherosclerosis and/or compromise of proximal perfusion pressures with large-vessel disease (Fig. 299-1).

1	Large-vessel renal artery occlusive disease can result from extrinsic compression of the vessel, fibromuscular dysplasia, or, most commonly, atherosclerotic disease. Any disorder that reduces perfusion pressure to the kidney can activate mechanisms that tend to restore renal pressures at the expense of developing systemic hypertension. Because restoration of perfusion pressures can reverse these pathways, renal artery stenosis is considered a specifically treatable “secondary” cause of hypertension.

1	Renal artery stenosis is common and often has only minor hemodynamic effects. Fibromuscular dysplasia (FMD) is reported in 3–5% of normal subjects presenting as potential kidney donors without hypertension. It may present clinically with hypertension in younger individuals (between age 15 and 50), most often women. FMD does not often threaten kidney function, but sometimes produces total occlusion and can be associated with renal artery aneurysms. Atherosclerotic renal artery stenosis (ARAS) is common in the general population (6.8% of a community-based sample above age 65), and the prevalence increases with age and for patients with other vascular conditions such as coronary artery disease (18–23%) and/or peripheral aortic or lower extremity disease (>30%). If untreated, ARAS progresses in nearly 50% of cases over a 5-year period, sometimes to total occlusion. Intensive treatment of arterial blood pressure and statin therapy appear to slow these rates and improve clinical outcomes.

1	Critical levels of stenosis lead to a reduction in perfusion pressure that activates the renin-angiotensin system, reduces sodium excretion, and activates sympathetic adrenergic pathways. These events lead to systemic hypertension characterized by angiotensin dependence in the early stages, widely varying pressures, loss of circadian blood pressure (BP) rhythms, and accelerated target organ injury, including left ventricular hypertrophy and renal fibrosis. Renovascular hypertension can be treated with agents that block the renin-angiotensin system and other drugs that modify these pressor pathways. It can also be treated with restoration of renal blood flow by either endovascular or surgical revascularization. Most patients require continued antihypertensive drug therapy because revascularization alone rarely lowers BP to normal.

1	ARAS and systemic hypertension tend to affect both the poststenotic and contralateral kidneys, reducing overall glomerular filtration rate (GFR) in ARAS. When kidney function is threatened by

1	FIGURE 299-1 Examples of micro-CT images from vessels defined by radiopaque casts injected into the renal vasculature. These illustrate the complex, dense cortical capillary network supplying the kidney cortex that can either proliferate or succumb to rarefaction under the influence of atherosclerosis and/or occlusive disease. Changes in blood supply are followed by tubulointerstitial fibrosis and loss of kidney function. MV, microvascular. (From LO Lerman, AR Chade: Curr Opin Nephrol Hyper 18:160, 2009, with permission.) large-vessel disease primarily, it has been labeled ischemic nephropathy. Moderately reduced blood flow that develops gradually is associated with reduced GFR and limited oxygen consumption with preserved tissue oxygenation. Hence, kidney function can remain stable during medical therapy, sometimes for years. With more advanced disease, reductions in cortical perfusion and frank tissue hypoxia develop. Unlike FMD, ARAS develops in patients with other risk factors for

1	medical therapy, sometimes for years. With more advanced disease, reductions in cortical perfusion and frank tissue hypoxia develop. Unlike FMD, ARAS develops in patients with other risk factors for atherosclerosis and is commonly superimposed upon preexisting small-vessel disease in the kidney resulting from hypertension, aging, and diabetes. Nearly 85% of patients considered for renal revascularization have stage 3–5 chronic kidney disease (CKD) with GFR below 60 mL/min per 1.73 m2. The presence of ARAS is a strong predictor of morbidityand mortality-related cardiovascular events, independent of whether renal revascularization is undertaken.

1	Diagnostic approaches to renal artery stenosis depend partly on the specific issues to be addressed. Noninvasive characterization of the renal vasculature may be achieved by several techniques, summarized in Table 299-1. Although activation of the renin-angiotensin system is a key step in developing renovascular hypertension, it is transient. Levels of renin activity are therefore subject to timing, the effects of drugs, and sodium intake, and do not reliably predict the response to vascular therapy. Renal artery velocities by Doppler ultrasound above 200 cm/s generally predict hemodynamically important lesions (above 60% vessel lumen occlusion), although treatment trials require velocity above 300 cm/s to avoid false positives. The renal resistive index has predictive value regarding the viability of the kidney. It remains operatorand institution-dependent, however. Captopril-enhanced renography has a strong negative predictive value when entirely normal. Magnetic resonance

1	the viability of the kidney. It remains operatorand institution-dependent, however. Captopril-enhanced renography has a strong negative predictive value when entirely normal. Magnetic resonance angiography (MRA) is now less often used, as gadolinium contrast has been associated with nephrogenic systemic fibrosis. Contrast-enhanced computed tomography (CT) with vascular reconstruction provides excellent vascular images and functional assessment, but carries a small risk of contrast toxicity.

1	While restoring renal blood flow and perfusion seems intuitively beneficial for high-grade occlusive lesions, revascularization procedures also pose hazards and expense. Patients with FMD are commonly younger females with otherwise normal vessels and a Perfusion Studies to Assess Differential Renal Blood Flow Shows the renal arteries and measures flow velocity as a means of assessing the severity of stenosis Shows the renal arteries and perirenal aorta Shows the renal arteries and perirenal aorta Shows location and severity of vascular lesion Inexpensive; widely available Not nephrotoxic, but concerns for gadolinium toxicity exclude use in GFR <30 mL/min/1.73 m2; provides excellent images

1	Shows location and severity of vascular lesion Inexpensive; widely available Not nephrotoxic, but concerns for gadolinium toxicity exclude use in GFR <30 mL/min/1.73 m2; provides excellent images Considered “gold standard” for diagnosis of large-vessel disease, usually performed simultaneous with planned intervention Heavily dependent on operator’s experience; less useful than invasive angiography for the diagnosis of fibromuscular dysplasia and abnormalities in accessory renal arteries Expensive; gadolinium excluded in renal failure, unable to visualize stented vessels Expensive, moderate volume of contrast required, potentially nephrotoxic Expensive, associated hazard of atheroemboli, contrast toxicity, procedure-related complications, e.g., dissection Abbreviation: GFR, glomerular filtration rate.

1	Abbreviation: GFR, glomerular filtration rate. long life expectancy. These patients often respond well to percutaneous renal artery angioplasty. If BP can be controlled to goal levels and kidney function remains stable in patients with ARAS, it may be argued that medical therapy with follow-up for disease progression is equally effective. Prospective trials up to now have failed to identify compelling benefits for interventional procedures regarding short-term results of BP and renal function, and long-term studies regarding cardiovascular outcomes, such as stroke, congestive heart failure, myocardial infarction, and end-stage renal failure, are not yet complete. Medical therapy should include blockade of the renin-angiotensin system, attainment of goal BPs, cessation of tobacco, statins, and aspirin. Renal revascularization is now often reserved for patients failing medical therapy or developing additional complications.

1	Techniques of renal revascularization are improving. With experienced operators, major complications occur in about 9% of cases, including renal artery dissection, capsular perforation, hemorrhage, and occasional atheroembolic disease. Although not common, atheroembolic disease can be catastrophic and accelerate both hypertension and kidney failure, precisely the events that revascularization is intended to prevent. Although renal blood flow usually can be restored by endovascular stenting, recovery of renal function is limited to about 25% of cases, with no change in 50% and some deterioration evident in others. Patients with rapid loss of kidney function, sometimes associated with antihypertensive drug therapy, or with vascular disease affecting the entire functioning kidney mass are more likely to recover function after restoring blood flow. When hypertension is refractory to effective therapy, revascularization offers real benefits. Table 299-2 summarizes currently accepted

1	are more likely to recover function after restoring blood flow. When hypertension is refractory to effective therapy, revascularization offers real benefits. Table 299-2 summarizes currently accepted guidelines for considering renal revascularization.

1	Emboli to the kidneys arise most frequently as a result of cholesterol crystals breaking free of atherosclerotic vascular plaque and lodging in downstream microvessels. Most clinical atheroembolic events follow angiographic procedures, often of the coronary vessels. It has been argued that nearly all vascular interventional procedures lead to plaque fracture and release of microemboli, but clinical manifestations develop only in a fraction of these. The incidence of clinical atheroemboli has been increasing with more vascular procedures and decline in GFR during treatment of systemic hypertension to achieve adequate blood pressure control with optimal medical • Rapid or recurrent decline in the GFR in association with a reduction in in the GFR during therapy with ACE inhibitors or ARBs congestive heart failure in a patient in whom the adequacy of

1	Factors Favoring Medical Therapy and Surveillance of Renal Artery Disease • Controlled blood pressure with stable renal function (e.g., stable renal (e.g., serial duplex ultrasound) risk for or previous experience with atheroembolic disease renal dysfunction (e.g., interstitial nephritis, diabetic nephropathy) Abbreviations: ACE, angiotensin-converting enzyme; ARBs, angiotensin receptor blockers; GFR, glomerular filtration rate.

1	Abbreviations: ACE, angiotensin-converting enzyme; ARBs, angiotensin receptor blockers; GFR, glomerular filtration rate. longer life spans. Atheroembolic renal disease is suspected in more 1629 than 3% of elderly subjects with end-stage renal disease (ESRD) and is likely underdiagnosed. It is more frequent in males with a history of diabetes, hypertension, and ischemic cardiac disease. Atheroemboli in the kidney are strongly associated with aortic aneurysmal disease and renal artery stenosis. Most clinical cases can be linked to precipitating events, such as angiography, vascular surgery, anticoagulation with heparin, thrombolytic therapy, or trauma. Clinical manifestations of this syndrome commonly develop between 1 and 14 days after an inciting event and may continue to develop for weeks thereafter.

1	Systemic embolic disease manifestations, such as fever, abdominal pain, and weight loss, are present in less than half of patients, although cutaneous manifestations including livedo reticularis and localized toe gangrene may be more common. Worsening hypertension and deteriorating kidney function are common, sometimes reaching a malignant phase. Progressive renal failure can occur and require dialytic support. These cases often develop after a stuttering onset over many weeks and have an ominous prognosis. Mortality rate after 1 year reaches 38%, and although some may eventually recover sufficiently to no longer require dialysis, many do not.

1	Beyond the clinical manifestations above, laboratory findings include rising creatinine, transient eosinophilia (60–80%), elevated sedimentation rate, and hypocomplementemia (15%). Establishing this diagnosis can be difficult and is often by exclusion. Definitive diagnosis depends on kidney biopsy demonstrating microvessel occlusion with cholesterol crystals that leave a “cleft” in the vessel. Biopsies obtained from patients undergoing surgical revascularization of the kidney indicate that silent cholesterol emboli are frequently present before any further manipulation is performed.

1	No effective therapy is available for atheroembolic disease once it has developed. Withdrawal of anticoagulation is recommended. Late recovery of kidney function after supportive measures sometimes occurs, and statin therapy may improve outcome. The role of embolic protection devices in the renal circulation is unclear, but a few prospective trials have failed to demonstrate major benefits. These devices are limited to distal protection during the endovascular procedure and offer no protection from embolic debris after removal.

1	Thrombotic occlusion of renal vessels or branch arteries can lead to declining renal function and hypertension. It is difficult to diagnose and is often overlooked, especially in elderly patients. Thrombosis can develop as a result of local vessel abnormalities, such as local dissection, trauma, or inflammatory vasculitis. Local microdissections sometimes lead to patchy, transient areas of infarctions labeled “segmental arteriolar mediolysis.” Although hypercoagulability conditions sometimes present as renal artery thrombosis, this is rare. It can also derive from distant embolic events, e.g., the left atrium in patients with atrial fibrillation or from fat emboli originating from traumatized tissue, most commonly large bone fractures. Cardiac sources include vegetations from subacute bacterial endocarditis. Systemic emboli to the kidneys may also arise from the venous circulation if right-to-left shunting occurs, e.g., through a patent foramen ovale.

1	Clinical manifestations vary depending on the rapidity of onset and extent of occlusion. Acute arterial thrombosis may produce flank pain, fever, leukocytosis, nausea, and vomiting. If kidney infarction results, enzymes such as lactate dehydrogenase (LDH) rise to extreme levels. If both kidneys are affected, renal function will decline precipitously with a drop in urine output. If a single kidney is involved, renal functional changes may be minor. Hypertension related to sudden release of renin from ischemic tissue can develop rapidly, as long as some viable tissue in the “peri-infarct” border zone remains. If the infarct zone demarcates precisely, the rise in BP and renin activity may resolve. Diagnosis of renal infarction may be established by vascular imaging with MRI, CT angiography, or arteriography (Fig. 299-2). Options for interventions of newly detected arterial occlusion include surgical reconstruction, anticoagulation, thrombolytic therapy,

1	Options for interventions of newly detected arterial occlusion include surgical reconstruction, anticoagulation, thrombolytic therapy, FIGURE 299-2 A. CT angiogram illustrating loss of circulation to the upper pole of the right kidney in a patient with fibromuscular disease and a renal artery aneurysm. Activation of the renin-angiotensin system produced rapidly developing hypertension. B. Angiogram illustrating high-grade renal artery stenosis affecting the left kidney. This lesion is often part of widespread atherosclerosis and sometimes is an extension of aortic plaque. This lesion develops in older individuals with preexisting atherosclerotic risk factors.

1	endovascular procedures, and supportive care, particularly antihyper-injury including microangiopathic hemolysis and renal dysfunction tensive drug therapy. Application of these methods depends on the can improve over time. Whereas series reported before the era of drug patient’s overall condition, the precipitating factors (e.g., local trauma therapy suggested that 1-year mortality rates exceeded 90%, current or systemic illness), the magnitude of renal tissue and function at risk, survival over 5 years exceeds 50%. and the likelihood of recurrent events in the future. For unilateral Malignant hypertension is less common in Western countries, disease, e.g., arterial dissection with thrombosis, supportive care with although it persists in parts of the world where medical care and antihyanticoagulation may suffice. Acute, bilateral occlusion is potentially pertensive drug therapy are less available. It most commonly develops catastrophic, producing anuric renal failure. Depending on

1	may suffice. Acute, bilateral occlusion is potentially pertensive drug therapy are less available. It most commonly develops catastrophic, producing anuric renal failure. Depending on the pre-in patients with treated hypertension who neglect to take medications cipitating event, surgical or thrombolytic therapies can sometimes or who may use vasospastic drugs, such as cocaine. Renal abnormalities restore kidney viability. typically include rising serum creatinine and occasionally hematuria and proteinuria. Biochemical findings may include evidence of hemo-MICROVASCULAR INJURY ASSOCIATED WITH HYPERTENSION lysis (anemia, schistocytes, and reticulocytosis) and changes associated with kidney failure. African-American males are more likely to develop rapidly progressive hypertension and kidney failure than are whites in“Malignant” Hypertension Although BP rises with age, it has long been the United States. Genetic polymorphisms (first identified as MYH9,recognized that some individuals

1	failure than are whites in“Malignant” Hypertension Although BP rises with age, it has long been the United States. Genetic polymorphisms (first identified as MYH9,recognized that some individuals develop rapidly progressive BP elevabut now thought to be APOL1) that are common in the Africantions with target organ injury including retinal hemorrhages, encepha-American population predispose to subtle focal sclerosing glomerularlopathy, and declining kidney function. Placebo arms during the disease, with severe hypertension developing at younger ages second-controlled trials of hypertension therapy identified progression to ary to renal disease in this instance.

1	severe levels in 20% of subjects over 5 years. If untreated, patients with target organ injury including papilledema and declining kidney “Hypertensive Nephrosclerosis” Based on experience with malignant function suffered mortality rates in excess of 50% over 6–12 months, hypertension and epidemiologic evidence linking BP with long-term hence the designation “malignant.” Postmortem studies of such risks of kidney failure, it has long been assumed that lesser degrees of patients identified vascular lesions, designated “fibrinoid necrosis,” hypertension induce less severe, but prevalent, changes in kidney veswith breakdown of the vessel wall, deposition of eosinophilic mate-sels and loss of kidney function. As a result, a large portion of patients rial including fibrin, and a perivascular cellular infiltrate. A separate reaching ESRD without a specific etiologic diagnosis are assigned the lesion was identified in the larger interlobular arteries in many patients designation

1	cellular infiltrate. A separate reaching ESRD without a specific etiologic diagnosis are assigned the lesion was identified in the larger interlobular arteries in many patients designation “hypertensive nephrosclerosis.” Pathologic examination with hyperplastic proliferation of the vascular wall cellular elements, commonly identifies afferent arteriolar thickening with deposition of deposition of collagen, and separation of layers, designated the homogeneous eosinophilic material (hyaline arteriolosclerosis) associ“onionskin” lesion. For many of these patients, fibrinoid necrosis led ated with narrowing of vascular lumina. Clinical manifestations include to obliteration of glomeruli and loss of tubular structures. Progressive retinal vessel changes associated with hypertension (arteriolar narkidney failure ensued and, without dialysis support, led to early rowing, crossing changes), left ventricular hypertrophy, and elevated mortality in untreated malignant-phase hypertension. These

1	narkidney failure ensued and, without dialysis support, led to early rowing, crossing changes), left ventricular hypertrophy, and elevated mortality in untreated malignant-phase hypertension. These vascular BP. The role of these vascular changes in kidney function is unclear. changes could develop with pressure-related injury from a variety Postmortem and biopsy samples from normotensive kidney donors of hypertensive pathways, including but not limited to activation of demonstrate similar vessel changes associated with aging, dyslipidemia, the renin-angiotensin system and severe vasospasm associated with and glucose intolerance. Although BP reduction does slow progression catecholamine release. Occasionally, endothelial injury is sufficient to of proteinuric kidney diseases and is warranted to reduce the excessive induce microangiopathic hemolysis, as discussed below. cardiovascular risks associated with CKD, antihypertensive therapy

1	Antihypertensive therapy is the mainstay of therapy for malignant does not alter the course of kidney dysfunction identified specifically as hypertension. With effective BP reduction, manifestations of vascular hypertensive nephrosclerosis. Deep venous Thrombosis and Pulmonary Thromboembolism Samuel Z. Goldhaber EPIDEMIOLOGY Venous thromboembolism (VTE) encompasses deep venous throm-300 FIGURE 300-1 Skin ulceration in the lateral malleolus from post-thrombotic syndrome of the leg.

1	FIGURE 300-1 Skin ulceration in the lateral malleolus from post-thrombotic syndrome of the leg. bosis (DVT) and pulmonary embolism (PE) and causes cardiovascular death and disability. In the United States, the Surgeon General estimates there are 100,000 to 180,000 deaths annually from PE and has declared that PE is the most common preventable cause of death among hospitalized patients. Survivors may succumb to the disabilities of chronic thromboembolic pulmonary hypertension or postthrombotic syndrome. Chronic thromboembolic pulmonary hypertension causes breathlessness, especially with exertion. Postthrombotic syndrome (also known as chronic venous insufficiency) damages the venous valves of the leg and causes ankle or calf swelling and leg aching, especially after prolonged standing. In its most severe form, postthrombotic syndrome causes skin ulceration (Fig. 300-1).

1	PATHOPHYSIOLOGY Inflammation and Platelet Activation Virchow’s triad of inflammation, hypercoagulability, and endothelial injury leads to recruitment of activated platelets, which release microparticles. These microparticles contain proinflammatory mediators that bind neutrophils, stimulating them to release their nuclear material and form web-like extracellular networks called neutrophil extracellular traps. These prothrombotic networks contain histones that stimulate platelet aggregation and promote platelet-dependent thrombin generation. Venous thrombi form and flourish in an environment of stasis, low oxygen tension, and upregulation of proinflammatory genes. Prothrombotic States The two most common autosomal dominant genetic mutations are factor V Leiden, which causes resistance to the FIGURE 300-2 Deep venous thrombosis at autopsy.

1	FIGURE 300-2 Deep venous thrombosis at autopsy. endogenous anticoagulant, activated protein C (which inactivates clotting factors V and VIII), and the prothrombin gene mutation, which increases the plasma prothrombin concentration (Chaps. 78 and 142). Antithrombin, protein C, and protein S are naturally occurring coagulation inhibitors. Deficiencies of these inhibitors are associated with VTE but are rare. Antiphospholipid antibody syndrome is the most common acquired cause of thrombophilia and is associated with venous or arterial thrombosis. Other common predisposing factors include cancer, obesity, cigarette smoking, systemic arterial hypertension, chronic obstructive pulmonary disease, chronic kidney disease, blood transfusion, long-haul air travel, air pollution, oral contraceptives, pregnancy, postmenopausal hormone replacement, surgery, and trauma.

1	Embolization When deep venous thrombi (Fig. 300-2) detach from their site of formation, they embolize to the vena cava, right atrium, and right ventricle, and lodge in the pulmonary arterial circulation, thereby causing acute PE. Paradoxically, these thrombi occasionally embolize to the arterial circulation through a patent foramen ovale or atrial septal defect. Many patients with PE have no evidence of DVT because the clot has already embolized to the lungs. Physiology The most common gas exchange abnormalities are arterial hypoxemia and an increased alveolar-arterial O2 tension gradient, which represents the inefficiency of O2 transfer across the lungs. Anatomic dead space increases because breathed gas does not enter gas exchange units of the lung. Physiologic dead space increases because ventilation to gas exchange units exceeds venous blood flow through the pulmonary capillaries. Other pathophysiologic abnormalities include: 1.

1	Other pathophysiologic abnormalities include: 1. Increased pulmonary vascular resistance due to vascular obstruction or platelet secretion of vasoconstricting neurohumoral agents such as serotonin. Release of vasoactive mediators can produce ventilation-perfusion mismatching at sites remote from the embolus, thereby accounting for discordance between a small PE and a large alveolar-arterial O2 gradient. 2. Impaired gas exchange due to increased alveolar dead space from vascular obstruction, hypoxemia from alveolar hypoventilation relative to perfusion in the nonobstructed lung, right-to-left shunting, or impaired carbon monoxide transfer due to loss of gas exchange surface. 3. Alveolar hyperventilation due to reflex stimulation of irritant receptors. 4. Increased airway resistance due to constriction of airways distal to the bronchi. 5. Decreased pulmonary compliance due to lung edema, lung hemorrhage, or loss of surfactant.

1	5. Decreased pulmonary compliance due to lung edema, lung hemorrhage, or loss of surfactant. Pulmonary Hypertension, Right Ventricular (RV) Dysfunction, and RV Microinfarction Pulmonary artery obstruction causes a rise in pulmonary 1632 artery pressure and in pulmonary vascular resistance. When RV wall tension rises, RV dilation and dysfunction ensue, with release of the cardiac biomarker, brain natriuretic peptide. The interventricular septum bulges into and compresses an intrinsically normal left ventricle (LV). Diastolic LV dysfunction reduces LV distensibility and impairs LV filling. Increased RV wall tension also compresses the right coronary artery, limits myocardial oxygen supply, and precipitates right coronary artery ischemia and RV microinfarction, with release of cardiac biomarkers such as troponin. Underfilling of the LV may lead to a fall in LV cardiac output and systemic arterial pressure, with consequent circulatory collapse and death.

1	CLASSIFICATION OF PULMONARY EMBOLISM AND DEEP VENOUS THROMBOSIS Pulmonary Embolism Massive PE accounts for 5–10% of cases, and is characterized by extensive thrombosis affecting at least half of the pulmonary vasculature. Dyspnea, syncope, hypotension, and cyanosis are hallmarks of massive PE. Patients with massive PE may present in cardiogenic shock and can die from multisystem organ failure. Submassive PE accounts for 20–25% of patients, and is characterized by RV dysfunction despite normal systemic arterial pressure. The combination of right heart failure and release of cardiac biomarkers indicates an increased likelihood of clinical deterioration. Low-risk PE constitutes about 70–75% of cases. These patients have an excellent prognosis.

1	Deep Venous Thrombosis Lower extremity DVT usually begins in the calf and propagates proximally to the popliteal vein, femoral vein, and iliac veins. Leg DVT is about 10 times more common than upper extremity DVT, which is often precipitated by placement of pacemakers, internal cardiac defibrillators, or indwelling central venous catheters. The likelihood of upper extremity DVT increases as the catheter diameter and number of lumens increase. Superficial venous thrombosis usually presents with erythema, tenderness, and a “palpable cord.” Patients are at risk for extension of the thrombosis to the deep venous system.

1	Clinical Evaluation PE is known as “the Great Masquerader.” Diagnosis is difficult because symptoms and signs are nonspecific. The most common symptom is unexplained breathlessness. When occult PE occurs concomitantly with overt congestive heart failure or pneumo nia, clinical improvement often fails to occur despite standard medical treatment of the concomitant illness. This scenario presents a clinical clue to the possible coexistence of PE. With DVT, the most common symptom is a cramp or “charley horse” in the lower calf that persists and intensifies over several days. Point score criteria help estimate the clinical likelihood of DVT and PE (Table 300-1). Patients with a low-to-moderate likelihood of DVT or PE should undergo initial diagnostic evaluation with d-dimer testing alone (see “Blood Tests”) without obligatory imaging tests (Fig. 300-3).

1	PE should undergo initial diagnostic evaluation with d-dimer testing alone (see “Blood Tests”) without obligatory imaging tests (Fig. 300-3). However, patients with a high clinical likelihood of VTE should skip d-dimer testing and undergo imaging as the next step in the diagnostic algorithm. Clinical Pearls Not all leg pain is due to DVT, and not all dyspnea is due to PE (Table 300-2). Sudden, severe calf discomfort suggests a ruptured Baker’s cyst. Fever and chills usually herald cellulitis rather than DVT. Physical findings, if present, may consist only of mild palpation discomfort in the lower calf. However, massive DVT often presents with marked thigh swelling, tenderness, and erythema. If the leg is diffusely edematous, DVT is unlikely. More probable is an acute exacerbation of venous insufficiency due to postthrombotic syndrome. Upper extremity venous thrombosis may present with asymmetry in the supraclavicular fossa or in the circumference of the upper arms.

1	Pulmonary infarction usually indicates a small PE. This condition is exquisitely painful because the thrombus lodges peripherally, near the innervation of pleural nerves. Nonthrombotic PE etiologies include fat embolism after pelvic or long bone fracture, tumor embolism, bone marrow, and air embolism. Cement embolism and bony fragment embolism Low Clinical Likelihood of DVT if Point Score Is Zero or Less; Moderate Likelihood if Score Is 1 to 2; High Likelihood if Score Is 3 or Greater can occur after total hip or knee replacement. Intravenous drug users may inject themselves with a wide array of substances that can embolize such as hair, talc, and cotton. Amniotic fluid embolism occurs when fetal membranes leak or tear at the placental margin.

1	Nonimaging Diagnostic Modalities • Blood tEsts The quantitative plasma d-dimer enzyme-linked immunosorbent assay (ELISA) rises in the presence of DVT or PE because of the breakdown of fibrin by plasmin. Elevation of d-dimer indicates endogenous although often clinically ineffective thrombolysis. The sensitivity of the d-dimer is >80% for DVT (including isolated calf DVT) and >95% for PE. The d-dimer is less sensitive for DVT than for PE because the DVT thrombus size is smaller. A normal d-dimer is a useful “rule out” test. However, the d-dimer assay is not specific. Levels increase in patients with myocardial infarction, pneumonia, sepsis, cancer, and the postoperative state and those in the second or third trimester of pregnancy. Therefore, d-dimer rarely has a useful role among hospitalized patients, because levels are frequently elevated due to systemic illness.

1	FIGURE 300-3 How to decide whether diagnostic imaging is needed. For assessment of clinical likelihood, see Table 300-1. Pneumonia, asthma, chronic obstructive pulmonary disease Pleurisy: “viral syndrome,” costochondritis, musculoskeletal discomfort Rib fracture, pneumothorax ElEvatEd cardiac BiomarkErs Serum troponin and plasma heart-type fatty acid–binding protein levels increase because of RV microinfarction. Myocardial stretch causes release of brain natriuretic peptide or NT-pro-brain natriuretic peptide. ElEctrocardiogram The most frequently cited abnormality, in addition to sinus tachycardia, is the S1Q3T3 sign: an S wave in lead I, a Q wave in lead III, and an inverted T wave in lead III (Chap. 268). This finding is relatively specific but insensitive. RV strain and ischemia cause the most common abnormality, T-wave inversion in leads V1 to V4.

1	Noninvasive Imaging Modalities • vEnous ultrasonography Ultrasonography of the deep venous system relies on loss of vein compressibility as the primary criterion for DVT. When a normal vein is imaged in cross-section, it readily collapses with gentle manual pressure from the ultrasound transducer. This creates the illusion of a “wink.” With acute DVT, the vein loses its compressibility because of passive distention by acute thrombus. The diagnosis of acute DVT is even more secure when thrombus is directly visualized. It appears homogeneous and has low echogenicity (Fig. 300-4). The vein itself often appears mildly dilated, and collateral channels may be absent.

1	Venous flow dynamics can be examined with Doppler imaging. Normally, manual calf compression causes augmentation of the Doppler flow pattern. Loss of normal respiratory variation is caused by an obstructing DVT or by any obstructive process within the pelvis. For patients with a technically poor or nondiagnostic venous ultrasound, one should consider alternative imaging modalities for DVT, such as computed tomography (CT) and magnetic resonance imaging. FIGURE 300-4 Venous ultrasound, with and without compression of the leg veins. CFA, common femoral artery; CFV, common femoral vein; GSV, great saphenous vein; LT, left.

1	FIGURE 300-4 Venous ultrasound, with and without compression of the leg veins. CFA, common femoral artery; CFV, common femoral vein; GSV, great saphenous vein; LT, left. FIGURE 300-5 Large bilateral proximal PE on a coronal chest CT image in a 54-year-old man with lung cancer and brain metastases. He had developed sudden onset of chest heaviness and shortness of breath while at home. There are filling defects in the main and segmental pulmonary arteries bilaterally (white arrows). Only the left upper lobe segmental artery is free of thrombus. chEst roEntgEnography A normal or nearly normal chest x-ray often occurs in PE. Well-established abnormalities include focal oligemia (Westermark’s sign), a peripheral wedged-shaped density above the diaphragm (Hampton’s hump), and an enlarged right descending pulmonary artery (Palla’s sign).

1	chEst ct CT of the chest with intravenous contrast is the principal imaging test for the diagnosis of PE (Fig. 300-5). Multidetector-row spiral CT acquires all chest images with ≤1 mm of resolution during a short breath hold. Sixth-order branches can be visualized with resolution superior to that of conventional invasive contrast pulmonary angiography. The CT scan also provides an excellent four-chamber view of the heart. RV enlargement on chest CT indicates an increased likelihood of death within the next 30 days compared with PE patients who have normal RV size. When imaging is continued below the chest to the knee, pelvic and proximal leg DVT also can be diagnosed by CT scanning. In patients without PE, the lung parenchymal images may establish alternative diagnoses not apparent on chest x-ray that explain the presenting symptoms and signs such as pneumonia, emphysema, pulmonary fibrosis, pulmonary mass, and aortic pathology. Sometimes asymptomatic early-stage lung cancer is

1	on chest x-ray that explain the presenting symptoms and signs such as pneumonia, emphysema, pulmonary fibrosis, pulmonary mass, and aortic pathology. Sometimes asymptomatic early-stage lung cancer is diagnosed incidentally.

1	lung scanning Lung scanning has become a second-line diagnostic test for PE, used mostly for patients who cannot tolerate intravenous contrast. Small particulate aggregates of albumin labeled with a gamma-emitting radionuclide are injected intravenously and are trapped in the pulmonary capillary bed. The perfusion scan defect indicates absent or decreased blood flow, possibly due to PE. Ventilation scans, obtained with a radiolabeled inhaled gas such as xenon or krypton, improve the specificity of the perfusion scan. Abnormal ventilation scans indicate abnormal nonventilated lung, thereby providing possible explanations for perfusion defects other than acute PE, such as asthma and chronic obstructive pulmonary disease. A high-probability scan for PE is defined as two or more segmental perfusion defects in the presence of normal ventilation.

1	The diagnosis of PE is very unlikely in patients with normal and nearly normal scans and is about 90% certain in patients with high-probability scans. Unfortunately, most 1634 patients have nondiagnostic scans, and fewer than one-half of patients with angiographically confirmed PE have a high probability scan. As many as 40% of patients with high clinical suspicion for PE but “lowprobability” scans do, in fact, have PE at angiography. magnEtic rEsonancE (mr) (contrast-EnhancEd) imaging When ultrasound is equivocal, MR venography with gadolinium contrast is an excellent imaging modality to diagnose DVT. MR pulmonary angiography may detect large proximal PE but is not reliable for smaller segmental and subsegmental PE.

1	Echocardiography Echocardiography is not a reliable diagnostic imaging tool for acute PE because most patients with PE have normal echocardiograms. However, echocardiography is a very useful diagnostic tool for detecting conditions that may mimic PE, such as acute myocardial infarction, pericardial tamponade, and aortic dissection. Transthoracic echocardiography rarely images thrombus directly. The best-known indirect sign of PE on transthoracic echocardiography is McConnell’s sign: hypokinesis of the RV free wall with normal or hyperkinetic motion of the RV apex. One should consider trans-esophageal echocardiography when CT scanning facilities are not available or when a patient has renal failure or severe contrast allergy that precludes administration of contrast despite premedication with high-dose steroids. This imaging modality can identify saddle, right main, or left main PE.

1	Invasive Diagnostic Modalities • pulmonary angiography Chest CT with contrast (see above) has virtually replaced invasive pulmonary angiography as a diagnostic test. Invasive catheter-based diagnostic testing is reserved for patients with technically unsatisfactory chest CTs and for those in whom an interventional procedure such as catheter-directed thrombolysis is planned. A definitive diagnosis of PE depends on visualization of an intraluminal filling defect in more than one projection. Secondary signs of PE include abrupt occlusion (“cutoff”) of vessels, segmental oligemia or avascularity, a prolonged arterial phase with slow filling, and tortuous, tapering peripheral vessels. contrast phlEBography Venous ultrasonography has virtually replaced contrast phlebography as the diagnostic test for suspected DVT. Integrated Diagnostic Approach An integrated diagnostic approach (Fig. 300-3) streamlines the workup of suspected DVT and PE (Fig. 300-6).

1	Integrated Diagnostic Approach An integrated diagnostic approach (Fig. 300-3) streamlines the workup of suspected DVT and PE (Fig. 300-6). Primary therapy consists of clot dissolution with pharmacomechanical therapy that usually includes low-dose catheter-directed thrombolysis. This approach is reserved for patients with extensive femoral, iliofemoral, or upper extremity DVT. The open vein hypothesis postulates that patients who receive primary therapy will sustain less long-term damage to venous valves, with consequent lower rates of postthrombotic syndrome. A National Heart, Lung, and Blood Institute–sponsored randomized controlled trial called ATTRACT (NCT00790335) is testing this hypothesis.

1	Anticoagulation or placement of an inferior vena caval filter constitutes secondary prevention of VTE. To lessen the severity of post-thrombotic syndrome of the legs, below-knee graduated compression stockings may be prescribed, 30–40 mmHg, for 2 years after the DVT episode. They should be replaced every 3 months because they lose their elasticity. Hemodynamic instability, RV dysfunction on echocardiography, RV enlargement on chest CT, or elevation of the troponin level Diagnostic Nondiagnostic, unavailable, or unsafe FIGURE 300-6 Imaging tests to diagnose DVT and PE. ECHO, echocardiography. due to RV microinfarction portend a high risk of an adverse clinical outcome. When RV function remains normal in a hemodynamically stable patient, a good clinical outcome is highly likely with anticoagulation alone (Fig. 300-7). Effective anticoagulation is the foundation for successful treatment of DVT and PE. There are three options: (1) the conventional strategy

1	Effective anticoagulation is the foundation for successful treatment of DVT and PE. There are three options: (1) the conventional strategy Risk stratify Normotension plus normal RV Normotension plus RV hypokinesis Hypotension Anticoagulation plus thrombolysis IVC filter Embolectomy: catheter/surgical Anticoagulation alone Secondary prevention Individualize therapy Primary therapy FIGURE 300-7 Acute management of pulmonary thromboembo-lism. RV, right ventricular; IVC, inferior vena cava. Unfractionated heparin, bolus and continuous infusion, to achieve aPTT 2–3 times the upper limit of the laboratory normal, or Enoxaparin 1 mg/kg twice daily with normal renal function, or Dalteparin 200 U/kg once daily or 100 U/kg twice daily, with normal renal function, or

1	Enoxaparin 1 mg/kg twice daily with normal renal function, or Dalteparin 200 U/kg once daily or 100 U/kg twice daily, with normal renal function, or Tinzaparin 175 U/kg once daily with normal renal function, or Fondaparinux weight-based once daily; adjust for impaired renal function Direct thrombin inhibitors: argatroban or bivalirudin Rivaroxaban 15 mg twice daily for 3 weeks, followed by 20 mg once daily with the dinner meal thereafter Apixaban (not yet licensed)

1	Requires 5–10 days of administration to achieve effectiveness as monotherapy (Unfractionated heparin, low-molecular-weight heparin, and fondaparinux are the usual immediately effective “bridging agents” used when initiating warfarin) Usual start dose is 5 mg Titrate to INR, target 2.0–3.0 Continue parenteral anticoagulation for a minimum of 5 days and until two sequential INR values, at least 1 day apart, achieve the target INR range of parenteral therapy “bridged” to warfarin, (2) parenteral therapy “bridged” to a novel oral anticoagulant such as dabigatran (a direct thrombin inhibitor) or edoxaban (an anti-Xa agent), or (3) oral anticoagulation with rivaroxaban or apixaban (both are anti-Xa agents) with a loading dose followed by a maintenance dose as monotherapy without parenteral anticoagulation.

1	The three heparin-based parenteral anticoagulants are (1) unfractionated heparin (UFH), (2) low-molecular-weight heparin (LMWH), and (3) fondaparinux. For patients with suspected or proven heparin-induced thrombocytopenia, there are two parenteral direct thrombin inhibitors: argatroban and bivalirudin (Table 300-3). Unfractionated Heparin UFH anticoagulates by binding to and accelerating the activity of antithrombin, thus preventing additional thrombus formation. UFH is dosed to achieve a target activated partial thromboplastin time (aPTT) of 60–80 s. The most popular nomogram uses an initial bolus of 80 U/kg, followed by an initial infusion rate of 18 U/kg per h. The major advantage of UFH is its short half-life, which is especially useful in patients in whom hour-to-hour control of the intensity of anticoagulation is desired.

1	The major advantage of UFH is its short half-life, which is especially useful in patients in whom hour-to-hour control of the intensity of anticoagulation is desired. Low-Molecular-Weight Heparins These fragments of UFH exhibit less binding to plasma proteins and endothelial cells and consequently have greater bioavailability, a more predictable dose response, and a longer half-life than does UFH. No monitoring or dose adjustment is needed unless the patient is markedly obese or has chronic kidney disease. Fondaparinux Fondaparinux, an anti-Xa pentasaccharide, is administered as a weight-based once-daily subcutaneous injection in a prefilled syringe. No laboratory monitoring is required. Fondaparinux is synthesized in a laboratory and, unlike LMWH or UFH, is not derived from animal products. It does not cause heparin-induced thrombocytopenia. The dose must be adjusted downward for patients with renal dysfunction.

1	Warfarin This vitamin K antagonist prevents carboxylation activation of coagulation factors II, VII, IX, and X. The full effect of warfarin requires at least 5 days, even if the prothrombin time, used for monitoring, becomes elevated more rapidly. If warfarin 1635 is initiated as monotherapy during an acute thrombotic illness, a paradoxical exacerbation of hypercoagulability increases the likelihood of thrombosis. Overlapping UFH, LMWH, fondaparinux, or parenteral direct thrombin inhibitors with warfarin for at least 5 days will nullify the early procoagulant effect of warfarin. warfarin dosing In an average-size adult, warfarin is often initiated in a dose of 5 mg. The prothrombin time is standardized by calculating the international normalized ratio (INR), which assesses the anticoagulant effect of warfarin (Chap. 78). The target INR is usually 2.5, with a range of 2.0–3.0.

1	The warfarin dose is usually titrated empirically to achieve the target INR. Proper dosing is difficult because hundreds of drug-drug and drug-food interactions affect warfarin metabolism. Increasing age and systemic illness reduce the required warfarin dose. Pharmacogenomics may provide more precise initial dosing of warfarin. CYP2C9 variant alleles impair the hydroxylation of S-warfarin, thereby lowering the dose requirement. Variants in the gene encoding the vitamin K epoxide reductase complex 1 (VKORC1) can predict whether patients require low, moderate, or high warfarin doses. Centralized anticoagulation clinics have improved the efficacy and safety of warfarin dosing. Patients can self-monitor their INR with a home point-of-care fingerstick machine and can occasionally be taught to self-dose their warfarin.

1	Novel Oral Anticoagulants Novel oral anticoagulants are administered in a fixed dose, establish effective anticoagulation within hours of ingestion, require no laboratory coagulation monitoring, and have few of the drug-drug or drug-food interactions that make warfarin so difficult to dose. Rivaroxaban, a factor Xa inhibitor, is approved for treatment of acute DVT and acute PE as monotherapy, without a parenteral “bridging” anticoagulant. Apixaban is likely to receive similar approval for oral monotherapy. Dabigatran, a direct thrombin inhibitor, and edoxaban, a factor Xa inhibitor, are likely to be approved for treatment of VTE after an initial course of parenteral anticoagulation.

1	Complications of Anticoagulants The most serious adverse effect of anticoagulation is hemorrhage. For life-threatening or intracranial hemorrhage due to heparin or LMWH, protamine sulfate can be administered. Heparin-induced thrombocytopenia is less common with LMWH than with UFH. There is no specific reversal agent for bleeding caused by fondaparinux, direct thrombin inhibitors, or factor Xa inhibitors. Major bleeding from warfarin is best managed with prothrombin complex concentrate. With serious but non–life-threatening bleeding, fresh-frozen plasma or intravenous vitamin K can be used. Recombinant human coagulation factor VIIa (rFVIIa) is an off-label option to manage catastrophic bleeding from warfarin, but prothrombin complex concentrate is a better choice. Oral vitamin K is effective for managing minor bleeding or an excessively high INR in the absence of bleeding.

1	Duration of Anticoagulation For DVT isolated to an upper extremity or calf that has been provoked by surgery, trauma, estrogen, or an indwelling central venous catheter or pacemaker, 3 months of anticoagulation usually suffice. For an initial episode of provoked proximal leg DVT or PE, 3 to 6 months of anticoagulation are considered sufficient. For patients with cancer and VTE, prescribe LMWH as monotherapy without warfarin and continue anticoagulation indefinitely unless the patient is rendered cancer-free.

1	Among patients with idiopathic, unprovoked VTE, the recurrence rate is high after cessation of anticoagulation. VTE that occurs during long-haul air travel is considered unprovoked. Unprovoked VTE may be caused by an exacerbation of an underlying inflammatory state and can be conceptualized as a chronic illness, with latent periods between flares of recurrent episodes. American College of Chest Physicians (ACCP) guidelines recommend considering 1636 anticoagulation for an indefinite duration with a target INR between 2 and 3 for patients with idiopathic VTE. An alternative approach after the first 6 months of anticoagulation is to reduce the intensity of anticoagulation and to lower the target INR range to between 1.5 and 2. Counterintuitively, the presence of genetic mutations such as heterozygous factor V Leiden and prothrombin gene mutation does not appear to increase the risk of recurrent VTE. However, patients with antiphospholipid antibody syndrome may warrant

1	such as heterozygous factor V Leiden and prothrombin gene mutation does not appear to increase the risk of recurrent VTE. However, patients with antiphospholipid antibody syndrome may warrant indefinite-duration anticoagulation, even if the initial VTE was provoked by trauma or surgery.

1	The two principal indications for insertion of an IVC filter are (1) active bleeding that precludes anticoagulation and (2) recurrent venous thrombosis despite intensive anticoagulation. Prevention of recurrent PE in patients with right heart failure who are not candidates for fibrinolysis and prophylaxis of extremely high-risk patients are “softer” indications for filter placement. The filter itself may fail by permitting the passage of small-to medium-size clots. Large thrombi may embolize to the pulmonary arteries via collateral veins that develop. A more common complication is caval thrombosis with marked bilateral leg swelling.

1	Paradoxically, by providing a nidus for clot formation, filters increase the DVT rate, even though they usually prevent PE (over the short term). Retrievable filters can now be placed for patients with an anticipated temporary bleeding disorder or for patients at temporary high risk of PE, such as individuals undergoing bariatric surgery who have a prior history of perioperative PE. The filters can be retrieved up to several months after insertion unless thrombus forms and is trapped within the filter. The retrievable filter becomes permanent if it remains in place or if, for technical reasons such as rapid endothelialization, it cannot be removed.

1	For patients with massive PE and hypotension, replete volume with 500 mL of normal saline. Additional fluid should be infused with extreme caution because excessive fluid administration exacerbates RV wall stress, causes more profound RV ischemia, and worsens LV compliance and filling by causing further interventricular septal shift toward the LV. Dopamine and dobutamine are first-line inotropic agents for treatment of PE-related shock. Maintain a low threshold for initiating these pressors. Often, a “trial-and-error” approach works best; other agents that may be effective include norepinephrine, vasopressin, or phenylephrine.

1	Successful fibrinolytic therapy rapidly reverses right heart failure and may result in a lower rate of death and recurrent PE by (1) dissolving much of the anatomically obstructing pulmonary arterial thrombus, (2) preventing the continued release of serotonin and other neurohumoral factors that exacerbate pulmonary hypertension, and (3) lysing much of the source of the thrombus in the pelvic or deep leg veins, thereby decreasing the likelihood of recurrent PE. The preferred fibrinolytic regimen is 100 mg of recombinant tissue plasminogen activator (tPA) administered as a continuous peripheral intravenous infusion over 2 h. The sooner thrombolysis is administered, the more effective it is. However, this approach can be used for at least 14 days after the PE has occurred.

1	Contraindications to fibrinolysis include intracranial disease, recent surgery, and trauma. The overall major bleeding rate is about 10%, including a 1–3% risk of intracranial hemorrhage. Careful screening of patients for contraindications to fibrinolytic therapy (Chap. 295) is the best way to minimize bleeding risk. The only Food and Drug Administration–approved indication for PE fibrinolysis is massive PE. For patients with submassive PE, who have preserved systolic blood pressure but moderate or severe RV dysfunction, use of fibrinolysis remains controversial. Results of a 1006-patient European multicentered randomized trial of submassive PE, using the thrombolytic agent tenecteplase, were published in 2014. Death or hemodynamic collapse within 7 days of randomization was reduced by 56% in the tenecteplase group. However, hemorrhagic stroke occurred in 2% of tenecteplase patients versus 0.2% in patients who only received heparin.

1	Many patients have relative contraindications to full-dose thrombolysis. Pharmacomechanical catheter-directed therapy usually combines physical fragmentation or pulverization of thrombus with catheter-directed low-dose thrombolysis. Mechanical techniques include catheter maceration and intentional embolization of clot more distally, suction thrombectomy, rheolytic hydrolysis, and low-energy ultrasound-facilitated thrombolysis. The dose of alteplase can be markedly reduced, usually to a range of 20 to 25 mg instead of the peripheral intravenous systemic dose of 100 mg. The risk of major hemorrhage with systemically administered fibrinolysis has prompted a renaissance of interest in surgical embolectomy, an operation that had almost become extinct. More rapid referral before the onset of irreversible multisystem organ failure and improved surgical technique have resulted in a high survival rate.

1	Chronic thromboembolic pulmonary hypertension develops in 2–4% of acute PE patients. Therefore, PE patients who have initial pulmonary hypertension (usually diagnosed with Doppler echocardiography) should be followed up at about 6 weeks with a repeat echocardiogram to determine whether pulmonary arterial pressure has normalized. Patients impaired by dyspnea due to chronic thromboembolic pulmonary hypertension should be considered for pulmonary thromboendarterectomy, which, if successful, can markedly reduce, and sometimes even cure, pulmonary hypertension (Chap. 304). The operation requires median sternotomy, cardiopulmonary bypass, deep hypothermia, and periods of hypothermic circulatory arrest. The mortality rate at experienced centers is approximately 5%. Inoperable patients should be managed with pulmonary vasodilator therapy.

1	Patients with VTE may feel overwhelmed when they learn that they are suffering from PE or DVT. Some have never previously encountered serious cardiovascular illness. They wonder whether they will be able to adapt to the new limitations imposed by anticoagulation. They worry about the health of their families and the genetic implications of their illness. Those who are advised to discontinue anticoagulation may feel especially vulnerable about the potential for suffering recurrent VTE. At Brigham and Woman’s Hospital, a physician-nurse–facilitated PE support group was initiated to address these concerns and has met monthly for more than 20 years.

1	Prevention of DVT and PE (Table 300-4) is of paramount importance because VTE is difficult to detect and poses a profound medical and economic burden. Low-dose UFH or LMWH is the most common form of in-hospital prophylaxis. Computerized reminder systems can increase the use of preventive measures and, at Brigham and Women’s Hospital, have reduced the symptomatic VTE rate by more than 40%. Audits of hospitals to ensure that prophylaxis protocols are being used will also increase utilization of preventive measures. Duration of prophylaxis is an important consideration. Extended-duration prophylaxis has not been shown to be both effective and safe in medically ill patients after hospital discharge in separate large trials that have tested enoxaparin, apixaban, and rivaroxaban. There is an ongoing trial of a novel oral anticoagulant, betrixaban, for extended-duration VTE prophylaxis in medically ill patients.

1	Dalteparin 2500 or 5000 units daily Cancer surgery, including gynecologic cancer surgery Enoxaparin 40 mg daily, consider 1 month of prophylaxis Major orthopedic surgery Warfarin (target INR 2.0–3.0) Fondaparinux 2.5 mg daily Dabigatran 220 mg daily (not in the United States) Apixaban 2.5 mg bid (not in the United States) Intermittent pneumatic compression (with or without pharmacologic prophylaxis) Medically ill patients, especially if immobilized, with a history of Unfractionated heparin 5000 units bid or tid prior VTE, with an indwelling central venous catheter, or with cancer Enoxaparin 40 mg daily (but without active gastroduodenal ulcer, major bleeding within Dalteparin 2500 or 5000 units daily 3 months, or platelet count <50,000) Fondaparinux 2.5 mg daily Anticoagulation contraindicated Intermittent pneumatic compression devices (but whether graduated compression stockings are effective in medical patients is controversial) Diseases of the Aorta

1	Anticoagulation contraindicated Intermittent pneumatic compression devices (but whether graduated compression stockings are effective in medical patients is controversial) Diseases of the Aorta Patients who have undergone total hip or knee replacement or cancer surgery will benefit from extended pharmacologic VTE prophylaxis after hospital discharge. For hip replacement or extensive cancer surgery, the duration of prophylaxis is usually at least 1 month. Diseases of the Aorta Mark A. Creager, Joseph Loscalzo The aorta is the conduit through which blood ejected from the left ven-tricle is delivered to the systemic arterial bed. In adults, its diameter is approximately 3 cm at the origin and in the ascending portion, 2.5 cm in the descending portion in the thorax, and 1.8–2 cm in the abdomen. 301

1	The aortic wall consists of a thin intima composed of endothelium, subendothelial connective tissue, and an internal elastic lamina; a thick tunica media composed of smooth muscle cells and extracellular matrix; and an adventitia composed primarily of connective tissue enclosing the vasa vasorum and nervi vascularis. In addition to the conduit function of the aorta, its viscoelastic and compliant properties serve a buffering function. The aorta is distended during systole to allow a portion of the stroke volume and elastic energy to be stored, and it recoils during diastole so that blood continues to flow to the periphery. Owing to its continuous exposure to high pulsatile pressure and shear stress, the aorta is particularly prone to injury and disease resulting from mechanical trauma. The aorta is also more prone to rupture than is any other vessel, especially with the development of aneurysmal dilation, since its wall tension, as governed by Laplace’s law (i.e., proportional to the

1	aorta is also more prone to rupture than is any other vessel, especially with the development of aneurysmal dilation, since its wall tension, as governed by Laplace’s law (i.e., proportional to the product of pressure and radius), will be increased.

1	Congenital anomalies of the aorta usually involve the aortic arch and its branches. Symptoms such as dysphagia, stridor, and cough may occur if an anomaly causes a ring around or otherwise compresses the esophagus or trachea. Anomalies associated with symptoms include double aortic arch, origin of the right subclavian artery distal to the left subclavian artery, and right-sided aortic arch with an aberrant left subclavian artery. A Kommerell’s diverticulum is an anatomic remnant of a right aortic arch. Most congenital anomalies of the aorta do not cause symptoms and are detected during catheter-based procedures. The diagnosis of suspected congenital anomalies of the aorta typically is confirmed by computed tomographic (CT) or magnetic resonance (MR) angiography. Surgery is used to treat symptomatic anomalies.

1	An aneurysm is defined as a pathologic dilation of a segment of a blood vessel. A true aneurysm involves all three layers of the vessel wall and is distinguished from a pseudoaneurysm, in which the intimal and medial layers are disrupted and the dilated segment of the aorta is lined by adventitia only and, at times, by perivascular clot. Aneurysms also may be classified according to their gross appearance. A fusiform aneurysm affects the entire circumference of a segment of the vessel, resulting in a diffusely dilated artery. In contrast, a saccular aneurysm involves only a portion of the circumference, resulting in an outpouching of the vessel wall. Aortic aneurysms also are classified according to location, i.e., abdominal versus thoracic. Aneurysms of the descending thoracic aorta are usually contiguous with infradiaphragmatic aneurysms and are referred to as thoracoabdominal aortic aneurysms.

1	Aortic aneurysms result from conditions that cause degradation or abnormal production of the structural components of the aortic wall: elastin and collagen. The causes of aortic aneurysms may be broadly categorized as degenerative disorders, genetic or developmental diseases, vasculitis, infections, and trauma (Table 301-1). Inflammation, oxidative stress, proteolysis, and biomechanical wall stress contribute to the degenerative processes that characterize most aneurysms of the abdominal and descending thoracic aorta. These are mediated by B cell and T cell lymphocytes, macrophages, inflammatory cytokines, and matrix metalloproteinases that degrade elastin and collagen and alter the tensile strength and ability of the aorta to accommodate pulsatile stretch. The associated histopathology demonstrates destruction of elastin and collagen, decreased vascular smooth muscle, in-growth of new blood vessels, and inflammation. Factors associated with degenerative aortic aneurysms include

1	demonstrates destruction of elastin and collagen, decreased vascular smooth muscle, in-growth of new blood vessels, and inflammation. Factors associated with degenerative aortic aneurysms include aging, cigarette smoking, hypercholesterolemia, hypertension, and male sex.

1	DiSEASES oF THE AoRTA: ETiologY AnD ASSoCiATED FACToRS Acute aortic syndromes (aortic dissection, acute intramural hematoma, Mycotic (Salmonella, staphylococcal, streptococcal, fungal) The most common pathologic condition associated with degenerative aortic aneurysms is atherosclerosis. Many patients with aortic aneurysms have coexisting risk factors for atherosclerosis (Chap. 291e), as well as atherosclerosis in other blood vessels.

1	Medial degeneration, previously designated cystic medial necrosis, is the histopathologic term used to describe the degeneration of collagen and elastic fibers in the tunica media of the aorta as well as the loss of medial cells that are replaced by multiple clefts of mucoid material, such as proteoglycans. Medial degeneration characteristically affects the proximal aorta, results in circumferential weakness and dilation, and leads to the development of fusiform aneurysms involving the ascending aorta and the sinuses of Valsalva. This condition is particularly prevalent in patients with Marfan’s syndrome, Loeys-Dietz syndrome, Ehlers-Danlos syndrome type IV (Chap. 427), hypertension, congenital bicuspid aortic valves, and familial thoracic aortic aneurysm syndromes; sometimes it appears as an isolated condition in patients without any other apparent disease.

1	Familial clusterings of aortic aneurysms occur in 20% of patients, suggesting a hereditary basis for the disease. Mutations of the gene that encodes fibrillin-1 are present in patients with Marfan’s syndrome. Fibrillin-1 is an important component of extracellular microfibrils, which support the architecture of elastic fibers and other connective tissue. Deficiency of fibrillin-1 in the extracellular matrix leads to excessive signaling by transforming growth factor β (TGF-β). Loeys-Dietz syndrome is caused by mutations in the genes that encode TGF-β receptors 1 (TGFBR1) and 2 (TGFBR2). Increased signaling by TGF-β and mutations of TGFBR1 and TGFBR2 may cause thoracic aortic aneurysms. Mutations of type III procollagen have been implicated in Ehlers-Danlos type IV syndrome. Mutations of SMAD3, which encodes a downstream signaling protein involved with TGF binding to its receptors, have been described in a syndrome of thoracic aortic aneurysm; craniofacial, skeletal, and cutaneous

1	of SMAD3, which encodes a downstream signaling protein involved with TGF binding to its receptors, have been described in a syndrome of thoracic aortic aneurysm; craniofacial, skeletal, and cutaneous anomalies; and osteoarthritis. Mutations of the genes encoding the smooth muscle–specific alpha-actin (ACTA2), smooth muscle cell–specific myosin heavy chain 11 (MHC11), and myosin light chain kinase (MYLK) and mutations of TGFBR2 and SMAD3 have been reported in some patients with nonsyndromic familial thoracic aortic aneurysms.

1	The infectious causes of aortic aneurysms include syphilis, tuberculosis, and other bacterial infections. Syphilis (Chap. 206) is a relatively uncommon cause of aortic aneurysm. Syphilitic periaortitis and mesoaortitis damage elastic fibers, resulting in thickening and weakening of the aortic wall. Approximately 90% of syphilitic aneurysms are located in the ascending aorta or aortic arch. Tuberculous aneurysms (Chap.

1	202) typically affect the thoracic aorta and result from direct extension of infection from hilar lymph nodes or contiguous abscesses as well as from bacterial seeding. Loss of aortic wall elasticity results from granulomatous destruction of the medial layer. A mycotic aneurysm is a rare condition that develops as a result of staphylococcal, streptococcal, Salmonella, or other bacterial or fungal infections of the aorta, usually at an atherosclerotic plaque. These aneurysms are usually saccular. Blood cultures are often positive and reveal the nature of the infective agent.

1	Vasculitides associated with aortic aneurysm include Takayasu’s arteritis and giant cell arteritis, which may cause aneurysms of the aortic arch and descending thoracic aorta. Spondyloarthropathies such as ankylosing spondylitis, rheumatoid arthritis, psoriatic arthritis, relapsing polychondritis, and reactive arthritis (formerly known as Reiter’s syndrome) are associated with dilation of the ascending aorta. Aortic aneurysms occur in patients with Behçet’s syndrome (Chap. 387), Cogan’s syndrome, and IgG4-related systemic disease. Aortic aneurysms also result from idiopathic aortitis. Traumatic aneurysms may occur after penetrating or nonpenetrating chest trauma and most commonly affect the descending thoracic aorta just beyond the site of insertion of the ligamentum arteriosum. Chronic aortic dissections are associated with weakening of the aortic wall that may lead to the development of aneurysmal dilatation.

1	The clinical manifestations and natural history of thoracic aortic aneurysms depend on their location. Medial degeneration is the most common pathology associated with ascending aortic aneurysms, whereas atherosclerosis is the condition most frequently associated with aneurysms of the descending thoracic aorta. The average growth rate of thoracic aneurysms is 0.1–0.2 cm per year. Thoracic aortic aneurysms associated with Marfan’s syndrome or aortic dissection may expand at a greater rate. The risk of rupture is related to the size of the aneurysm and the presence of symptoms, ranging approximately from 2–3% per year for thoracic aortic aneurysms <4.0 cm in diameter to 7% per year for those >6 cm in diameter. Most thoracic aortic aneurysms are asymptomatic; however, compression or erosion of adjacent tissue by aneurysms may cause symptoms such as chest pain, shortness of breath, cough, hoarseness, and dysphagia. Aneurysmal dilation of the ascending aorta may cause congestive heart

1	of adjacent tissue by aneurysms may cause symptoms such as chest pain, shortness of breath, cough, hoarseness, and dysphagia. Aneurysmal dilation of the ascending aorta may cause congestive heart failure as a consequence of aortic regurgitation, and compression of the superior vena cava may produce congestion of the head, neck, and upper extremities.

1	A chest x-ray may be the first test that suggests the diagnosis of a thoracic aortic aneurysm (Fig. 301-1). Findings include widening of the mediastinal shadow and displacement or compression of the trachea or left main stem bronchus. Echocardiography, particularly transesophageal echocardiography, can be used to assess the proximal ascending aorta and descending thoracic aorta. Contrast-enhanced CT, magnetic resonance imaging (MRI), and conventional invasive aortography are sensitive and specific tests for assessment of aneurysms of the thoracic aorta and involvement of branch vessels (Fig. 301-2). In asymptomatic patients whose aneurysms are too small to justify surgery, noninvasive testing with either contrast-enhanced CT or MRI should be performed at least every 6–12 months to monitor expansion. FIGURE 301-1 A chest x-ray of a patient with a thoracic aortic aneurysm.

1	FIGURE 301-2 A magnetic resonance angiogram demonstrating a fusiform aneurysm of the ascending thoracic aorta. (Courtesy of Dr. Michael Steigner, Brigham and Women’s Hospital, Boston, MA, with permission.) β-Adrenergic blockers currently are recommended for patients with thoracic aortic aneurysms, particularly those with Marfan’s syndrome, who have evidence of aortic root dilatation to reduce the rate of further expansion. Additional medical therapy should be given as necessary to control hypertension. Recent studies indicate that angiotensin receptor antagonists and angiotensin-converting enzyme inhibitors reduce the rate of aortic dilation in patients with Marfan’s syndrome by blocking TGF-β signaling; clinical outcome trials of this treatment approach are in progress. Operative repair with placement of a prosthetic graft is indicated in patients with symptomatic ascending thoracic aortic aneurysms and for most asymptomatic aneurysms when the ascending aortic diameter is >5.5 cm. In

1	placement of a prosthetic graft is indicated in patients with symptomatic ascending thoracic aortic aneurysms and for most asymptomatic aneurysms when the ascending aortic diameter is >5.5 cm. In patients with Marfan’s syndrome or bicuspid aortic valve, ascending thoracic aortic aneurysms of 4–5 cm should be considered for surgery. Operative repair is indicated for patients with descending thoracic aortic aneurysms when the diameter is >6 cm, and endovascular repair should be considered if feasible when the diameter is >5.5 cm. Repair is also recommended when the diameter of an aneurysm has increased >1 cm per year.

1	Abdominal aortic aneurysms occur more frequently in males than in females, and the incidence increases with age. Abdominal aortic aneurysms ≥4.0 cm may affect 1–2% of men older than 50 years. At least 90% of all abdominal aortic aneurysms >4.0 cm are related to atherosclerotic disease, and most of these aneurysms are below the level of the renal arteries. Prognosis is related to both the size of the aneurysm and the severity of coexisting coronary artery and cerebrovascular disease. The risk of rupture increases with the size of the aneurysm: the 5-year risk for aneurysms <5 cm is 1–2%, whereas it is 20–40% for aneurysms >5 cm in diameter. The formation of mural thrombi within aneurysms may predispose to peripheral embolization.

1	An abdominal aortic aneurysm commonly produces no symptoms. It usually is detected on routine examination as a palpable, pulsatile, expansile, and nontender mass, or it is an incidental finding observed on an abdominal imaging study performed for other reasons. As abdominal aortic aneurysms expand, however, they may become painful. Some patients complain of strong pulsations in the abdomen; others experience pain in the chest, lower back, or scrotum. Aneurysmal pain is usually a harbinger of rupture and represents a medical emergency. More often, acute rupture occurs without any prior warning, and this complication is always life-threatening. Rarely, there is leakage of the aneurysm with severe pain and tenderness. Acute pain and hypotension occur with rupture of the aneurysm, which requires an emergency operation.

1	Abdominal radiography may demonstrate the calcified outline of the aneurysm; however, about 25% of aneurysms are not calcified and cannot be visualized by x-ray imaging. An abdominal ultrasound can delineate the transverse and longitudinal dimensions of an abdominal aortic aneurysm and may detect mural thrombus. Abdominal ultrasound is useful for serial documentation of aneurysm size and can be used to screen patients at risk for developing an aortic aneurysm. In one large study, ultrasound screening of men age 65–74 years was associated with a risk reduction in aneurysm-related death of 42%. For this reason, screening by ultrasonography is recommended for men age 65–75 years who have ever smoked. In addition, siblings or offspring of persons with abdominal aortic aneurysms, as well as individuals with thoracic aortic or peripheral arterial aneurysms, should be considered for screening for abdominal aortic aneurysms. CT with contrast and MRI are accurate noninvasive tests to determine

1	with thoracic aortic or peripheral arterial aneurysms, should be considered for screening for abdominal aortic aneurysms. CT with contrast and MRI are accurate noninvasive tests to determine the location and size of abdominal aortic aneurysms and to plan endovascular or open surgical repair (Fig. 301-3). Contrast aortography may be used for the evaluation of patients with aneurysms, but the procedure carries a small risk of complications such as bleeding, allergic reactions, and atheroembolism. Since the presence of mural thrombi may reduce the luminal size, aortography may underestimate the diameter of an aneurysm.

1	Diseases of the Aorta FIGURE 301-3 A computed tomographic angiogram depicting a fusiform abdominal aortic aneurysm before (left) and after (right) treatment with a bifurcated stent graft. (Courtesy of Drs. Elizabeth George and Frank Rybicki, Brigham and Women’s Hospital, Boston, MA, with permission.)

1	Operative repair of the aneurysm with insertion of a prosthetic graft or endovascular placement of an aortic stent graft (Fig. 301-3) is indicated for abdominal aortic aneurysms of any size that are expanding rapidly or are associated with symptoms. For asymptomatic aneurysms, abdominal aortic aneurysm repair is indicated if the diameter is >5.5 cm. In randomized trials of patients with abdominal aortic aneurysms <5.5 cm, there was no difference in the long-term (5to 8-year) mortality rate between those followed with ultrasound surveillance and those undergoing elective surgical repair. Thus, serial noninvasive follow-up of smaller aneurysms (<5 cm) is an alternative to immediate repair. The decision to perform an open surgical operation or endovascular repair is based in part on the vascular anatomy and comorbid conditions. Endovascular repair of abdominal aortic aneurysms has a lower short-term morbidity rate but a comparable long-term mortality rate with open surgical

1	on the vascular anatomy and comorbid conditions. Endovascular repair of abdominal aortic aneurysms has a lower short-term morbidity rate but a comparable long-term mortality rate with open surgical reconstruction. Long-term surveillance with CT or MR aortography is indicated after endovascular repair to detect leaks and possible aneurysm expansion.

1	In surgical candidates, careful preoperative cardiac and general medical evaluations (followed by appropriate therapy for complicating conditions) are essential. Preexisting coronary artery disease, congestive heart failure, pulmonary disease, diabetes mellitus, and advanced age add to the risk of surgery. β-Adrenergic blockers decrease perioperative cardiovascular morbidity and mortality. With careful preoperative cardiac evaluation and postoperative care, the operative mortality rate approximates 1–2%. After acute rupture, the mortality rate of emergent operation is 45–50%. Endovascular repair with stent placement is an alternative approach to treat ruptured aneurysms and may be associated with a lower mortality rate.

1	The four major acute aortic syndromes are aortic rupture (discussed earlier), aortic dissection, intramural hematoma, and penetrating atherosclerotic ulcer. Aortic dissection is caused by a circumferential or, less frequently, transverse tear of the intima. It often occurs along the right lateral wall of the ascending aorta where the hydraulic shear stress is high. Another common site is the descending thoracic aorta just below the ligamentum arteriosum. The initiating event is either a primary intimal tear with secondary dissection into the media or a medial hemorrhage that dissects into and disrupts the intima. The pulsatile aortic flow then dissects along the elastic lamellar plates of the aorta and creates a false lumen. The dissection usually propagates distally down the descending aorta and into its major branches, but it may propagate proximally. Distal propagation may be limited by atherosclerotic plaque. In some cases, a secondary distal intimal disruption occurs, resulting

1	aorta and into its major branches, but it may propagate proximally. Distal propagation may be limited by atherosclerotic plaque. In some cases, a secondary distal intimal disruption occurs, resulting in the reentry of blood from the false to the true lumen.

1	There are at least two important pathologic and radiologic variants of aortic dissection: intramural hematoma without an intimal flap and penetrating atherosclerotic ulcer. Acute intramural hematoma is thought to result from rupture of the vasa vasorum with hemorrhage into the wall of the aorta. Most of these hematomas occur in the descending thoracic aorta. Acute intramural hematomas may progress to dissection and rupture. Penetrating atherosclerotic ulcers are caused by erosion of a plaque into the aortic media, are usually localized, and are not associated with extensive propagation. They are found primarily in the middle and distal portions of the descending thoracic aorta and are associated with extensive atherosclerotic disease. The ulcer can erode beyond the internal elastic lamina, leading to medial hematoma, and may progress to false aneurysm formation or rupture.

1	Several classification schemes have been developed for thoracic aortic dissections. DeBakey and colleagues initially classified aortic dissections as type I, in which an intimal tear occurs in the ascending aorta but involves the descending aorta as well; type II, in which the dissection is limited to the ascending aorta; and type III, in which the intimal tear is located in the descending aorta with distal propagation of the dissection (Fig. 301-4). Another classification (Stanford) is that of type A, in which the dissection involves the ascending aorta (proximal dissection), and type B, in which it is limited to the arch and/or descending aorta (distal dissection). From a management standpoint, classification of aortic dissections and intramural hematomas into type A or B is more practical and useful, since DeBakey types I and II are managed in a similar manner.

1	The factors that predispose to aortic dissection include those associated with medial degeneration and others that increase aortic wall stress (Table 301-1). Systemic hypertension is a coexisting condition in

1	FIGURE 301-4 Classification of aortic dissections. Stanford classification: Type A dissections (top) involve the ascending aorta independent of site of tear and distal extension; type B dissections (bottom) involve transverse and/or descending aorta without involvement of the ascending aorta. DeBakey classification: Type I dissection involves ascending to descending aorta (top left); type II dissection is limited to ascending or transverse aorta, without descending aorta (top center + top right); type III dissection involves descending aorta only (bottom left). (From DC Miller, in RM Doroghazi, EE Slater [eds]: Aortic Dissection. New York, McGraw-Hill, 1983, with permission.) 70% of patients. Aortic dissection is the major cause of morbidity and mortality in patients with Marfan’s syndrome (Chap. 427) or Loeys-Dietz syndrome, and similarly may affect patients with Ehlers-Danlos syndrome. The incidence also is increased in patients with inflammatory aortitis (i.e., Takayasu’s

1	syndrome (Chap. 427) or Loeys-Dietz syndrome, and similarly may affect patients with Ehlers-Danlos syndrome. The incidence also is increased in patients with inflammatory aortitis (i.e., Takayasu’s arteritis, giant cell arteritis), congenital aortic valve anomalies (e.g., bicuspid valve), coarctation of the aorta, and a history of aortic trauma. In addition, the risk of dissection is increased in otherwise normal women during the third trimester of pregnancy. Aortic dissection also may occur as a consequence of weight lifting, cocaine use, or deceleration injury.

1	The peak incidence of aortic dissection is in the sixth and seventh decades. Men are more affected than women by a ratio of 2:1. The presentations of aortic dissection and its variants are the consequences of intimal tear, dissecting hematoma, occlusion of involved arteries, and compression of adjacent tissues. Acute aortic dissection presents with the sudden onset of pain (Chap. 19), which often is described as very severe and tearing and is associated with diaphoresis. The pain may be localized to the front or back of the chest, often the interscapular region, and typically migrates with propagation of the dissection. Other symptoms include syncope, dyspnea, and weakness. Physical findings may include hypertension or hypotension, loss of pulses, aortic regurgitation, pulmonary edema, and neurologic findings due to carotid artery obstruction (hemiplegia, hemianesthesia) or spinal cord ischemia (paraplegia). Bowel ischemia, hematuria, and myocardial ischemia have all been observed.

1	and neurologic findings due to carotid artery obstruction (hemiplegia, hemianesthesia) or spinal cord ischemia (paraplegia). Bowel ischemia, hematuria, and myocardial ischemia have all been observed. These clinical manifestations reflect complications resulting from the dissection occluding the major arteries. Furthermore, clinical manifestations may result from the compression of adjacent structures (e.g., superior cervical ganglia, superior vena cava, bronchus, esophagus) by the expanding dissection, causing aneurysmal dilation, and include Horner’s syndrome, superior vena cava syndrome, hoarseness, dysphagia, and airway compromise. 1641 Hemopericardium and cardiac tamponade may complicate a type A lesion with retrograde dissection. Acute aortic regurgitation is an important and common (>50%) complication of proximal dissection. It is the outcome of either a circumferential tear that widens the aortic root or a disruption of the annulus by a dissecting hematoma that tears a

1	common (>50%) complication of proximal dissection. It is the outcome of either a circumferential tear that widens the aortic root or a disruption of the annulus by a dissecting hematoma that tears a leaflet(s) or displaces it, inferior to the line of closure. Signs of aortic regurgitation include bounding pulses, a wide pulse pressure, a diastolic murmur often radiating along the right sternal border, and evidence of congestive heart failure. The clinical manifestations depend on the severity of the regurgitation.

1	In dissections involving the ascending aorta, the chest x-ray often reveals a widened superior mediastinum. A pleural effusion (usually left-sided) also may be present. This effusion is typically serosanguineous and not indicative of rupture unless accompanied by hypotension and falling hematocrit. In dissections of the descending thoracic aorta, a widened mediastinum may be observed on chest x-ray. In addition, the descending aorta may appear to be wider than the ascending portion. An electrocardiogram that shows no evidence of myocardial ischemia is helpful in distinguishing aortic dissection from myocardial infarction. Rarely, the dissection involves the right or, less commonly, left coronary ostium and causes acute myocardial infarction.

1	The diagnosis of aortic dissection can be established by noninvasive techniques such as echocardiography, CT, and MRI. Aortography is used less commonly because of the accuracy of these noninvasive techniques. Transthoracic echocardiography can be performed simply and rapidly and has an overall sensitivity of 60–85% for aortic dissection. For diagnosing proximal ascending aortic dissections, its sensitivity exceeds 80%; it is less useful for detecting dissection of the arch and descending thoracic aorta. Transesophageal echocardiography requires greater skill and patient cooperation but is very accurate in identifying dissections of the ascending and descending thoracic aorta but not the arch, achieving 98% sensitivity and approximately 90% specificity. Echocardiography also provides important information regarding the presence and severity of aortic regurgitation and pericardial effusion. CT and MRI are both highly accurate in identifying the intimal flap and the extent of the

1	important information regarding the presence and severity of aortic regurgitation and pericardial effusion. CT and MRI are both highly accurate in identifying the intimal flap and the extent of the dissection and involvement of major arteries; each has a sensitivity and specificity >90%. They are useful in recognizing intramural hemorrhage and penetrating ulcers. The relative utility of transesophageal echocardiography, CT, and MRI depends on the availability and expertise in individual institutions as well as on the hemodynamic stability of the patient, with CT and MRI obviously less suitable for unstable patients.

1	Medical therapy should be initiated as soon as the diagnosis is considered. The patient should be admitted to an intensive care unit for hemodynamic monitoring. Unless hypotension is present, therapy should be aimed at reducing cardiac contractility and systemic arterial pressure, and thus shear stress. For acute dissection, unless contraindicated, β-adrenergic blockers should be administered parenterally, using intravenous propranolol, metoprolol, or the short-acting esmolol to achieve a heart rate of approximately 60 beats/ min. This should be accompanied by sodium nitroprusside infusion to lower systolic blood pressure to ≤120 mmHg. Labetalol (Chap. 298), a drug with both βand α-adrenergic blocking properties, also may be used as a parenteral agent in acute therapy for dissection.

1	The calcium channel antagonists verapamil and diltiazem may be used intravenously if nitroprusside or β-adrenergic blockers cannot be employed. The addition of a parenteral angiotensin-converting enzyme (ACE) inhibitor such as enalaprilat to a β-adrenergic blocker also may be considered. Isolated use of a direct vasodilator such as hydralazine is contraindicated because these agents can increase hydraulic shear and may propagate the dissection. Emergent or urgent surgical correction is the preferred treatment for acute ascending aortic dissections and intramural hematomas (type A) and for complicated type B dissections, including

1	CHAPTER 301 Diseases of the Aorta 1642 those characterized by propagation, compromise of major aortic branches, impending rupture, or continued pain. Surgery involves excision of the intimal flap, obliteration of the false lumen, and placement of an interposition graft. A composite valve-graft conduit is used if the aortic valve is disrupted. The overall in-hospital mortality rate after surgical treatment of patients with aortic dissection is reported to be 15–25%. The major causes of perioperative mortality and morbidity include myocardial infarction, paraplegia, renal failure, tamponade, hemorrhage, and sepsis. Endoluminal stent grafts may be considered in selected patients. Other transcatheter techniques, such as fenestration of the intimal flaps and stenting of narrowed branch vessels to increase flow to compromised organs, are used in selected patients. For uncomplicated and stable distal dissections and intramural hematomas (type B), medical therapy is the preferred treatment.

1	to increase flow to compromised organs, are used in selected patients. For uncomplicated and stable distal dissections and intramural hematomas (type B), medical therapy is the preferred treatment. The in-hospital mortality rate of medically treated patients with type B dissection is 10–20%. Long-term therapy for patients with aortic dissection and intramural hematomas (with or without surgery) consists of control of hypertension and reduction of cardiac contractility with the use of beta blockers plus other antihypertensive agents, such as ACE inhibitors or calcium antagonists. Patients with chronic type B dissection and intramural hematomas should be followed on an outpatient basis every 6–12 months with contrast-enhanced CT or MRI to detect propagation or expansion. Patients with Marfan’s syndrome are at high risk for postdissection complications. The long-term prognosis for patients with treated dissections is generally good with careful follow-up; the 10-year survival rate is

1	syndrome are at high risk for postdissection complications. The long-term prognosis for patients with treated dissections is generally good with careful follow-up; the 10-year survival rate is approximately 60%.

1	Atherosclerosis may affect the thoracic and abdominal aorta. Occlusive aortic disease caused by atherosclerosis usually is confined to the distal abdominal aorta below the renal arteries. Frequently the disease extends to the iliac arteries (Chap. 302). Claudication characteristically involves the buttocks, thighs, and calves and may be associated with impotence in males (Leriche’s syndrome). The severity of the symptoms depends on the adequacy of collaterals. With sufficient collateral blood flow, a complete occlusion of the abdominal aorta may occur without the development of ischemic symptoms. The physical findings include the absence of femoral and other distal pulses bilaterally and the detection of an audible bruit over the abdomen (usually at or below the umbilicus) and the common femoral arteries. Atrophic skin, loss of hair, and coolness of the lower extremities usually are observed. In advanced ischemia, rubor on dependency and pallor on elevation can be seen.

1	The diagnosis usually is established by physical examination and noninvasive testing, including leg pressure measurements, Doppler velocity analysis, pulse volume recordings, and duplex ultrasonography. The anatomy may be defined by MRI, CT, or conventional aortography, typically performed when one is considering revascularization. Catheter-based endovascular or operative treatment is indicated in patients with lifestyle-limiting or debilitating symptoms of claudication and patients with critical limb ischemia. Acute occlusion in the distal abdominal aorta constitutes a medical emergency because it threatens the viability of the lower extremities; it usually results from an occlusive (saddle) embolus that almost always originates from the heart. Rarely, acute occlusion may occur as the result of in situ thrombosis in a preexisting severely narrowed segment of the aorta.

1	The clinical picture is one of acute ischemia of the lower extremities. Severe rest pain, coolness, and pallor of the lower extremities and the absence of distal pulses bilaterally are the usual manifestations. Diagnosis should be established rapidly by MRI, CT, or aortography. Emergency thrombectomy or revascularization is indicated. Aortitis, a term referring to inflammatory disease of the aorta, may be caused by large vessel vasculitides such as Takayasu’s arteritis and giant cell arteritis, rheumatic and HLA-B27–associated spondyloarthropathies, Behçet’s syndrome, antineutrophil cytoplasmic antibody (ANCA)–associated vasculitides, Cogan’s syndrome, IgG4-related systemic disease, and infections such as syphilis, tuberculosis, and Salmonella, or may be associated with retroperitoneal fibrosis. Aortitis may result in aneurysmal dilation and aortic regurgitation, occlusion of the aorta and its branch vessels, or acute aortic syndromes.

1	This inflammatory disease often affects the ascending aorta and aortic arch, causing obstruction of the aorta and its major arteries. Takayasu’s arteritis is also termed pulseless disease because of the frequent occlusion of the large arteries originating from the aorta. It also may involve the descending thoracic and abdominal aorta and occlude large branches such as the renal arteries. Aortic aneurysms also may occur. The pathology is a panarteritis characterized by mononuclear cells and occasionally giant cells, with marked intimal hyperplasia, medial and adventitial thickening, and, in the chronic form, fibrotic occlusion. The disease is most prevalent in young females of Asian descent but does occur in women of other geographic and ethnic origins and also in young men. During the acute stage, fever, malaise, weight loss, and other systemic symptoms may be evident. Elevations of the erythrocyte sedimentation rate and C-reactive protein are common. The chronic stages of the

1	the acute stage, fever, malaise, weight loss, and other systemic symptoms may be evident. Elevations of the erythrocyte sedimentation rate and C-reactive protein are common. The chronic stages of the disease, which is intermittently active, present with symptoms related to large artery occlusion, such as upper extremity claudication, cerebral ischemia, and syncope. The process is progressive, and there is no definitive therapy. Glucocorticoids and immunosuppressive agents are effective in some patients during the acute phase. Surgical bypass or endovascular intervention of a critically stenotic artery may be necessary.

1	(See also Chap. 385) This vasculitis occurs in older individuals and affects women more often than men. Primarily large and medium-size arteries are affected. The pathology is that of focal granulomatous lesions involving the entire arterial wall; it may be associated with polymyalgia rheumatica. Obstruction of medium-size arteries (e.g., temporal and ophthalmic arteries) and major branches of the aorta and the development of aortitis and aortic regurgitation are important complications of the disease. High-dose glucocorticoid therapy may be effective when given early.

1	Rheumatoid arthritis (Chap. 380), ankylosing spondylitis (Chap. 384), psoriatic arthritis (Chap. 384), reactive arthritis (formerly known as Reiter’s syndrome) (Chap. 384), relapsing polychondritis, and inflammatory bowel disorders may all be associated with aortitis involving the ascending aorta. The inflammatory lesions usually involve the ascending aorta and may extend to the sinuses of Valsalva, the mitral valve leaflets, and adjacent myocardium. The clinical manifestations are aneurysm, aortic regurgitation, and involvement of the cardiac conduction system.

1	Idiopathic abdominal aortitis is characterized by adventitial and periaortic inflammation with thickening of the aortic wall. It is associated with abdominal aortic aneurysms and idiopathic retroperitoneal fibrosis. Affected individuals may present with vague constitutional symptoms, fever, and abdominal pain. Retroperitoneal fibrosis can cause ureteral obstruction and hydronephrosis. Glucocorticoids and immunosuppressive agents may reduce the inflammation. Infective aortitis may result from direct invasion of the aortic wall by bacterial pathogens such as Staphylococcus, Streptococcus, and Salmonella or by fungi. These bacteria cause aortitis by infecting the aorta at sites of atherosclerotic plaque. Bacterial proteases lead to degradation of collagen, and the ensuing destruction of the aortic wall leads to the formation of a saccular aneurysm referred to as a mycotic

1	Arterial Diseases of the Extremities Mark A. Creager, Joseph Loscalzo PERIPHERAL ARTERY DISEASE Peripheral artery disease (PAD) is defined as a clinical disorder in 302 Arterial Diseases of the Extremities aneurysm. Mycotic aneurysms have a predilection for the suprarenal abdominal aorta. The pathologic characteristics of the aortic wall include acute and chronic inflammation, abscesses, hemorrhage, and necrosis. Mycotic aneurysms typically affect the elderly and occur in men three times more frequently than in women. Patients may present with fever, sepsis, and chest, back, or abdominal pain; there may have been a preceding diarrheal illness. Blood cultures are positive in the majority of patients. Both CT and MRI are useful to diagnose mycotic aneurysms. Treatment includes antibiotic therapy and surgical removal of the affected part of the aorta and revascularization of the lower extremities with grafts placed in uninfected tissue.

1	Syphilitic aortitis is a late manifestation of luetic infection (Chap. 206) that usually affects the proximal ascending aorta, particularly the aortic root, resulting in aortic dilation and aneurysm formation. Syphilitic aortitis occasionally may involve the aortic arch or the descending aorta. The aneurysms may be saccular or fusiform and are usually asymptomatic, but compression of and erosion into adjacent structures may result in symptoms; rupture also may occur.

1	The initial lesion is an obliterative endarteritis of the vasa vasorum, especially in the adventitia. This is an inflammatory response to the invasion of the adventitia by the spirochetes. Destruction of the aortic media occurs as the spirochetes spread into this layer, usually via the lymphatics accompanying the vasa vasorum. Destruction of collagen and elastic tissues leads to dilation of the aorta, scar formation, and calcification. These changes account for the characteristic radiographic appearance of linear calcification of the ascending aorta.

1	The disease typically presents as an incidental chest radiographic finding 15–30 years after initial infection. Symptoms may result from aortic regurgitation, narrowing of coronary ostia due to syphilitic aortitis, compression of adjacent structures (e.g., esophagus), or rupture. Diagnosis is established by a positive serologic test, i.e., rapid plasmin reagin (RPR) or fluorescent treponemal antibody. Treatment includes penicillin and surgical excision and repair.

1	which there is a stenosis or occlusion in the aorta or the arteries of the limbs. Atherosclerosis is the leading cause of PAD in patients >40 years old. Other causes include thrombosis, embolism, vasculitis, fibromuscular dysplasia, entrapment, cystic adventitial disease, and trauma. The highest prevalence of atherosclerotic PAD occurs in the sixth and seventh decades of life. As in patients with atherosclerosis of the coronary and cerebral vasculature, there is an increased risk of developing PAD in cigarette smokers and in persons with diabetes mellitus, hypercholesterolemia, hypertension, or renal insufficiency.

1	Pathology (See also Chap. 291e) Segmental lesions that cause steno-sis or occlusion are usually localized to large and medium-size vessels. The pathology of the lesions includes atherosclerotic plaques with calcium deposition, thinning of the media, patchy destruction of muscle and elastic fibers, fragmentation of the internal elastic lamina, and thrombi composed of platelets and fibrin. The primary sites of involvement are the abdominal aorta and iliac arteries (30% of symptomatic patients), the femoral and popliteal arteries (80–90% of patients), and the more distal vessels, including the tibial and peroneal arteries (40– 50% of patients). Atherosclerotic lesions occur preferentially at arterial branch points, which are sites of increased turbulence, altered shear stress, and intimal injury. Involvement of the distal vasculature is most common in elderly individuals and patients with diabetes mellitus.

1	Clinical Evaluation Fewer than 50% of patients with PAD are symp-1643 tomatic, although many have a slow or impaired gait. The most common symptom is intermittent claudication, which is defined as a pain, ache, cramp, numbness, or a sense of fatigue in the muscles; it occurs during exercise and is relieved by rest. The site of claudication is distal to the location of the occlusive lesion. For example, buttock, hip, thigh, and calf discomfort occurs in patients with aortoiliac disease, whereas calf claudication develops in patients with femoral-popliteal disease. Symptoms are far more common in the lower than in the upper extremities because of the higher incidence of obstructive lesions in the former region. In patients with severe arterial occlusive disease in whom resting blood flow cannot accommodate basal nutritional needs of the tissues, critical limb ischemia may develop. Patients complain of rest pain or a feeling of cold or numbness in the foot and toes. Frequently, these

1	cannot accommodate basal nutritional needs of the tissues, critical limb ischemia may develop. Patients complain of rest pain or a feeling of cold or numbness in the foot and toes. Frequently, these symptoms occur at night when the legs are horizontal and improve when the legs are in a dependent position. With severe ischemia, rest pain may be persistent.

1	Important physical findings of PAD include decreased or absent pulses distal to the obstruction, the presence of bruits over the nar rowed artery, and muscle atrophy. With more severe disease, hair loss, thickened nails, smooth and shiny skin, reduced skin temperature, and pallor or cyanosis are common physical signs. In patients with critical limb ischemia, ulcers or gangrene may occur. Elevation of the legs and repeated flexing of the calf muscles produce pallor of the soles of the feet, whereas rubor, secondary to reactive hyperemia, may develop when the legs are dependent. The time required for rubor to develop or for the veins in the foot to fill when the patient’s legs are transferred from an elevated to a dependent position is related to the severity of the ischemia and the presence of collateral vessels. Patients with severe ischemia may develop peripheral edema because they keep their legs in a dependent position much of the time. Ischemic neuropathy can result in numbness

1	of collateral vessels. Patients with severe ischemia may develop peripheral edema because they keep their legs in a dependent position much of the time. Ischemic neuropathy can result in numbness and hyporeflexia.

1	Noninvasive Testing The history and physical examination are often sufficient to establish the diagnosis of PAD. An objective assessment of the presence and severity of disease is obtained by noninvasive techniques. Arterial pressure can be recorded noninvasively in the legs by placement of sphygmomanometric cuffs at the ankles and the use of a Doppler device to auscultate or record blood flow from the dorsalis pedis and posterior tibial arteries. Normally, systolic blood pressure in the legs and arms is similar. Indeed, ankle pressure may be slightly higher than arm pressure due to pulse-wave amplification. In the presence of hemodynamically significant stenoses, the systolic blood pressure in the leg is decreased. Thus, the ratio of the ankle and brachial artery pressures (termed the ankle:brachial index, or ABI) is 1.00–1.40 in normal individuals. ABI values of 0.91–0.99 are considered “borderline,” and those <0.90 are abnormal and diagnostic of PAD. ABIs >1.40 indicate

1	the ankle:brachial index, or ABI) is 1.00–1.40 in normal individuals. ABI values of 0.91–0.99 are considered “borderline,” and those <0.90 are abnormal and diagnostic of PAD. ABIs >1.40 indicate noncompressible arteries secondary to vascular calcification.

1	Other noninvasive tests include segmental pressure measurements, segmental pulse volume recordings, duplex ultrasonography (which combines B-mode imaging and Doppler flow velocity waveform analysis examination), transcutaneous oximetry, and stress testing (usually using a treadmill). Placement of pneumatic cuffs enables assessment of systolic pressure along the legs. The presence of pressure gradients between sequential cuffs provides evidence of the presence and location of hemodynamically significant stenoses. In addition, the amplitude of the pulse volume contour becomes blunted in the presence of significant PAD. Duplex ultrasonography is used to image and detect stenotic lesions in native arteries and bypass grafts. Treadmill testing allows the physician to assess functional limitations objectively. Decline of the ABI immediately after exercise provides further support for the diagnosis of PAD in patients with equivocal symptoms and findings on examination.

1	Magnetic resonance angiography (MRA), computed tomographic angiography (CTA), and conventional catheter-based angiography should not be used for routine diagnostic testing but are performed before potential revascularization (Fig. 302-1). Each test is useful in defining the anatomy to assist planning for endovascular and surgical revascularization procedures. FIGURE 302-1 Magnetic resonance angiography of a patient with intermittent claudication, showing stenoses of the distal abdominal aorta and right iliac common iliac artery (A) and stenoses of the right and left superficial femoral arteries (B). (Courtesy of Dr. Edwin Gravereaux, with permission.)

1	Prognosis The natural history of patients with PAD is influenced primarily by the extent of coexisting coronary artery and cerebrovascular disease. Approximately one-third to one-half of patients with symptomatic PAD have evidence of coronary artery disease (CAD) based on clinical presentation and electrocardiogram, and over one-half have significant CAD by coronary angiography. Patients with PAD have a 15–30% 5-year mortality rate and a twoto sixfold increased risk of death from coronary heart disease. Mortality rates are highest in those with the most severe PAD. Measurement of ABI is useful for detecting PAD and identifying persons at risk for future atherothrombotic events. The likelihood of symptomatic progression of PAD is lower than the chance of succumbing to CAD. Approximately 75–80% of nondiabetic patients who present with mild to moderate claudication remain symptomatically stable. Deterioration is likely to occur in the remainder, with approximately 1–2% of the group

1	75–80% of nondiabetic patients who present with mild to moderate claudication remain symptomatically stable. Deterioration is likely to occur in the remainder, with approximately 1–2% of the group ultimately developing critical limb ischemia each year. Approximately 25–30% of patients with critical limb ischemia undergo amputation within 1 year. The prognosis is worse in patients who continue to smoke cigarettes or have diabetes mellitus.

1	Patients with PAD should receive therapies to reduce the risk of associated cardiovascular events, such as myocardial infarction and death, and to improve limb symptoms, prevent progression to critical limb ischemia, and preserve limb viability. Risk factor modification and antiplatelet therapy should be initiated to improve cardiovascular outcomes. The importance of discontinuing cigarette smoking cannot be overemphasized. The physician must assume a major role in this lifestyle modification. Counseling and adjunctive drug therapy with the nicotine patch, bupropion, or varenicline increase smoking cessation rates and reduce recidivism. It is important to control blood pressure in hypertensive patients. Angiotensin-converting enzyme inhibitors may reduce the risk of cardiovascular events in patients with symptomatic PAD. β-Adrenergic blockers do not worsen claudication and may be used to treat hypertension, especially in patients with coexistent CAD. Treatment of hypercholesterolemia

1	in patients with symptomatic PAD. β-Adrenergic blockers do not worsen claudication and may be used to treat hypertension, especially in patients with coexistent CAD. Treatment of hypercholesterolemia with statins is advocated to reduce the risk of myocardial infarction, stroke, and death. The 2013 ACC/AHA Guideline on the Treatment of Blood Cholesterol to Reduce Atherosclerotic Cardiovascular Risk in Adults recommends high intensity statin treatment in patients with atherosclerotic disorders, including peripheral artery disease. Platelet inhibitors, including aspirin and clopidogrel, reduce the risk of adverse cardiovascular events in patients with atherosclerosis and are recommended for patients with symptomatic PAD, including those with intermittent claudication or critical limb ischemia or prior lower extremity revascularization. Dual antiplatelet therapy with both aspirin and clopidogrel is not more effective than aspirin alone in reducing cardiovascular morbidity and mortality

1	or prior lower extremity revascularization. Dual antiplatelet therapy with both aspirin and clopidogrel is not more effective than aspirin alone in reducing cardiovascular morbidity and mortality rates in patients with PAD. The anticoagulant warfarin is as effective as antiplatelet therapy in preventing adverse cardiovascular events but causes more major bleeding; therefore, it is not indicated to improve outcomes in patients with chronic PAD.

1	Therapies for intermittent claudication and critical limb ischemia include supportive measures, medications, nonoperative interventions, and surgery. Supportive measures include meticulous care of the feet, which should be kept clean and protected against excessive drying with moisturizing creams. Well-fitting and protective shoes are advised to reduce trauma. Elastic support hose should be avoided, as it reduces blood flow to the skin. In patients with critical limb ischemia, shock blocks under the head of the bed together with a canopy over the feet may improve perfusion pressure and ameliorate some of the rest pain.

1	Patients with claudication should be encouraged to exercise regularly and at progressively more strenuous levels. Supervised exercise training programs for 30to 45-min sessions, three to five times per week for at least 12 weeks, prolong walking distance. Patients also should be advised to walk until nearly maximum claudication discomfort occurs and then rest until the symptoms resolve before resuming ambulation. The beneficial effect of supervised exercise training on walking performance in patients with claudication often is similar to or greater than that realized after a revascularization procedure. Pharmacologic treatment of PAD has not been as successful as the medical treatment of CAD (Chap. 293). In particular, vasodilators as a class have not proved to be beneficial. During exercise, peripheral vasodilation occurs distal to sites of significant arterial stenoses. As a result, perfusion pressure falls, often to levels lower than that generated in the interstitial tissue by the

1	peripheral vasodilation occurs distal to sites of significant arterial stenoses. As a result, perfusion pressure falls, often to levels lower than that generated in the interstitial tissue by the exercising muscle. Drugs such as α-adrenergic blocking agents, calcium channel antagonists, and other vasodilators have not been shown to be effective in patients with PAD.

1	Cilostazol, a phosphodiesterase inhibitor with vasodilator and antiplatelet properties, increases claudication distance by 40–60% and improves measures of quality of life. The mechanism of action accounting for its beneficial effects is not known. Pentoxifylline, a substituted xanthine derivative, increases blood flow to the micro-circulation and enhances tissue oxygenation. Although several placebo-controlled studies have found that pentoxifylline increases the duration of exercise in patients with claudication, its efficacy has not been confirmed in all clinical trials. Statins and angiotensinconverting enzyme inhibitors appear promising for treatment of intermittent claudication in initial clinical trials, but more studies are needed to confirm the efficacy of each class of drugs. There is no definitive medical therapy for critical limb ischemia, although several studies have suggested that long-term parenteral administration of vasodilator prostaglandins decreases pain and

1	There is no definitive medical therapy for critical limb ischemia, although several studies have suggested that long-term parenteral administration of vasodilator prostaglandins decreases pain and facilitates healing of ulcers. Enthusiasm for therapy with angiogenic growth factors abated when clinical trials of intramuscular gene transfer of DNA encoding vascular endothelial growth factor, fibroblast growth factor, hepatocyte growth factor, or hypoxia-inducible factor 1α failed to demonstrate improvement in symptoms or outcomes in patients with intermittent claudication or critical limb ischemia. Clinical trials assessing the ability of bone marrow–derived vascular progenitor cells to promote angiogenesis and preserve limb viability in patients with critical limb ischemia are ongoing.

1	Revascularization procedures, including catheter-based and surgical interventions, are usually indicated for patients with disabling, progressive, or severe symptoms of intermittent claudication despite medical therapy and for those with critical limb ischemia. MRA, CTA, or conventional angiography should be performed to assess vascular anatomy in patients who are being considered for revascularization. Nonoperative interventions include percutaneous transluminal angiography (PTA) and stent placement (Chap. 296e). PTA and stenting of the iliac artery are associated with higher success rates than are PTA and stenting of the femoral and popliteal arteries. Approximately 90–95% of iliac PTAs are initially successful, and the 3-year patency rate is >75%. Patency rates may be higher if a stent is placed in the iliac artery. The initial success rates for femoralpopliteal PTA and stenting are approximately 80%, with 60% 3-year patency rates. Patency rates are influenced by the severity of

1	is placed in the iliac artery. The initial success rates for femoralpopliteal PTA and stenting are approximately 80%, with 60% 3-year patency rates. Patency rates are influenced by the severity of pretreatment stenoses; the prognosis of occlusive lesions is worse than that of nonocclusive stenotic lesions. The role of drug-eluting stents and drug-coated balloons in PAD is under investigation.

1	Several operative procedures are available for treating patients with aortoiliac and femoral-popliteal artery disease. The preferred operative procedure depends on the location and extent of the obstruction(s) and the general medical condition of the patient. Operative procedures for aortoiliac disease include aortobifemoral bypass, axillofemoral bypass, femoro-femoral bypass, and aortoiliac endarterectomy. The most frequently used procedure is the aortobifemoral bypass using knitted Dacron grafts. Immediate graft patency approaches 99%, and 5and 10-year graft patency rates in survivors are >90% and 80%, respectively. Operative complications include myocardial infarction and stroke, infection of the graft, peripheral embolization, and sexual dysfunction from interruption of autonomic nerves in the pelvis. The operative mortality rate ranges from 1–3%, mostly due to ischemic heart disease.

1	Operative therapy for femoral-popliteal artery disease includes in situ and reverse autogenous saphenous vein bypass grafts, placement of polytetrafluoroethylene (PTFE) or other synthetic grafts, and thromboendarterectomy. The operative mortality rate ranges from 1–3%. The long-term patency rate depends on the type of graft used, the location of the distal anastomosis, and the patency of runoff vessels beyond the anastomosis. Patency rates of femoral-popliteal saphenous vein bypass grafts approach 90% at 1 year and 70–80% at 5 years. Five-year patency rates of infrapopliteal saphenous vein bypass grafts are 60–70%. In contrast, 5-year patency rates of infrap-1645 opliteal PTFE grafts are <30%.

1	Preoperative cardiac risk assessment may identify individuals who are especially likely to experience an adverse cardiac event during the perioperative period. Patients with angina, prior myocardial infarction, ventricular ectopy, heart failure, or diabetes are among those at increased risk. Stress testing with treadmill exercise (if feasible), radionuclide myocardial perfusion imaging, or echocardiography permits further stratification of risk in these patients (Chap. 296e). Patients with abnormal test results require close supervision and adjunctive management with anti-ischemic medications. β-Adrenergic blockers and statins reduce the risk of postoperative cardiovascular complications. Coronary angiography and coronary artery revascularization compared with optimal medical therapy do not improve outcomes in most patients undergoing peripheral vascular surgery, but cardiac catheterization should be considered in patients with unstable angina and angina refractory to medical therapy

1	improve outcomes in most patients undergoing peripheral vascular surgery, but cardiac catheterization should be considered in patients with unstable angina and angina refractory to medical therapy as well as those suspected of having left main or three-vessel CAD.

1	Fibromuscular dysplasia is a hyperplastic disorder that affects medium-size and small arteries. It occurs predominantly in females and usually involves the renal and carotid arteries but can affect extremity vessels such as the iliac and subclavian arteries. The histologic classification includes intimal fibroplasia (also classified as focal), medial dysplasia (multifocal), and adventitial hyperplasia. Medial dysplasia is subdivided into medial fibroplasia, perimedial fibroplasia, and medial hyperplasia. Medial fibroplasia is the most common type and is characterized by alternating areas of thinned media and fibromuscular ridges. The internal elastic lamina usually is preserved. The iliac arteries are the limb arteries most likely to be affected by fibromuscular dysplasia. It is identified angiographically by a “string of beads” appearance caused by thickened fibromuscular ridges contiguous with thin, less-involved portions of the arterial wall, which is typical of medial fibroplasia.

1	angiographically by a “string of beads” appearance caused by thickened fibromuscular ridges contiguous with thin, less-involved portions of the arterial wall, which is typical of medial fibroplasia. When limb vessels are involved, clinical manifestations are similar to those for atherosclerosis, including claudication and rest pain. PTA and surgical reconstruction have been beneficial in patients with debilitating symptoms or threatened limbs.

1	Thromboangiitis obliterans (Buerger’s disease) is an inflammatory occlusive vascular disorder involving small and medium-size arteries and veins in the distal upper and lower extremities. Cerebral, visceral, and coronary vessels may be affected rarely. This disorder develops most frequently in men <40 years of age. The prevalence is higher in Asians and individuals of Eastern European descent. Although the cause of thromboangiitis obliterans is not known, there is a definite relationship to cigarette smoking in patients with this disorder.

1	In the initial stages of thromboangiitis obliterans, polymorphonuclear leukocytes infiltrate the walls of the small and medium-size arteries and veins. The internal elastic lamina is preserved, and a cellular, inflammatory thrombus develops in the vascular lumen. As the disease progresses, mononuclear cells, fibroblasts, and giant cells replace the neutrophils. Later stages are characterized by perivascular fibrosis, organized thrombus, and recanalization.

1	The clinical features of thromboangiitis obliterans often include a triad of claudication of the affected extremity, Raynaud’s phenomenon, and migratory superficial vein thrombophlebitis. Claudication usually is confined to the calves and feet or the forearms and hands because this disorder primarily affects distal vessels. In the presence of severe digital ischemia, trophic nail changes, painful ulcerations, and gangrene may develop at the tips of the fingers or toes. The physical examination shows normal brachial and popliteal pulses but reduced or absent radial, ulnar, and/or tibial pulses. MRA, CTA, and conventional arteriography are helpful in making the diagnosis. Smooth, tapering segmental lesions in the distal vessels are characteristic, as are

1	Arterial Diseases of the Extremities 1646 collateral vessels at sites of vascular occlusion. Proximal atherosclerotic disease is usually absent. The diagnosis can be confirmed by excisional biopsy and pathologic examination of an involved vessel. There is no specific treatment except abstention from tobacco. The prognosis is worse in individuals who continue to smoke, but results are discouraging even in those who stop smoking. Arterial bypass of the larger vessels may be used in selected instances, as well as local debridement, depending on the symptoms and severity of ischemia. Antibiotics may be useful; anticoagulants and glucocorticoids are not helpful. If these measures fail, amputation may be required. Other vasculitides may affect the arteries that supply the upper and lower extremities. Takayasu’s arteritis and giant cell (temporal) arteritis are discussed in Chap. 385.

1	Other vasculitides may affect the arteries that supply the upper and lower extremities. Takayasu’s arteritis and giant cell (temporal) arteritis are discussed in Chap. 385. Acute limb ischemia occurs when arterial occlusion results in the sudden cessation of blood flow to an extremity. The severity of ischemia and the viability of the extremity depend on the location and extent of the occlusion and the presence and subsequent development of collateral blood vessels. Principal causes of acute arterial occlusion include embolism, thrombus in situ, arterial dissection, and trauma.

1	The most common sources of arterial emboli are the heart, aorta, and large arteries. Cardiac disorders that cause thromboembolism include atrial fibrillation, both chronic and paroxysmal; acute myocardial infarction; ventricular aneurysm; cardiomyopathy; infectious and marantic endocarditis; thrombi associated with prosthetic heart valves; and atrial myxoma. Emboli to the distal vessels may also originate from proximal sites of atherosclerosis and aneurysms of the aorta and large vessels. Less frequently, an arterial occlusion results paradoxically from a venous thrombus that has entered the systemic circulation via a patent foramen ovale or another septal defect. Arterial emboli tend to lodge at vessel bifurcations because the vessel caliber decreases at those sites; in the lower extremities, emboli lodge most frequently in the femoral artery, followed by the iliac artery, aorta, and popliteal and tibioperoneal arteries.

1	Acute arterial thrombosis in situ occurs most frequently in atherosclerotic vessels at the site of an atherosclerotic plaque or aneurysm and in arterial bypass grafts. Trauma to an artery may disrupt continuity of blood flow and cause acute limb ischemia via formation of an acute arterial thrombus or by disruption of an artery’s integrity and extravasation of blood. Arterial occlusion may complicate arterial punctures and placement of catheters; it also may result from arterial dissection if the intimal flap obstructs the artery. Less common causes include thoracic outlet compression syndrome, which causes subclavian artery occlusion, and entrapment of the popliteal artery by abnormal placement of the medial head of the gastrocnemius muscle. Polycythemia and hypercoagulable disorders (Chaps. 131 and 141) are also associated with acute arterial thrombosis.

1	The symptoms of an acute arterial occlusion depend on the location, duration, and severity of the obstruction. Often, severe pain, paresthesia, numbness, and coldness develop in the involved extremity within 1 hour. Paralysis may occur with severe and persistent ischemia. Physical findings include loss of pulses distal to the occlusion, cyanosis or pallor, mottling, decreased skin temperature, muscle stiffening, loss of sensation, weakness, and/or absent deep tendon reflexes. If acute arterial occlusion occurs in the presence of an adequate collateral circulation, as is often the case in acute graft occlusion, the symptoms and findings may be less impressive. In this situation, the patient complains about an abrupt decrease in the distance walked before claudication occurs or of modest pain and paresthesia. Pallor and coolness are evident, but sensory and motor functions generally are preserved. The diagnosis of acute limb ischemia is usually apparent from the clinical presentation.

1	and paresthesia. Pallor and coolness are evident, but sensory and motor functions generally are preserved. The diagnosis of acute limb ischemia is usually apparent from the clinical presentation. In most circumstances, MRA, CTA, or catheter-based arteriography is used to confirm the diagnosis and demonstrate the location and extent of arterial occlusion.

1	Once the diagnosis is made, the patient should be anticoagulated with intravenous heparin to prevent propagation of the clot. In cases of severe ischemia of recent onset, particularly when limb viability is jeopardized, immediate intervention to ensure reperfusion is indicated. Catheter-directed thrombolysis/thrombectomy, surgical thromboembolectomy, and arterial bypass procedures are used to restore blood flow to the ischemic extremity promptly, particularly when a large proximal vessel is occluded.

1	Intraarterial thrombolytic therapy with recombinant tissue plasminogen activator, reteplase, or tenecteplase is most effective when acute arterial occlusion is recent (<2 weeks) and caused by a thrombus in an atherosclerotic vessel, arterial bypass graft, or occluded stent. Thrombolytic therapy is also indicated when the patient’s overall condition contraindicates surgical intervention or when smaller distal vessels are occluded, thus preventing surgical access. Meticulous observation for hemorrhagic complications is required during intraarterial thrombolytic therapy. Another endovascular approach to thrombus removal is percutaneous mechanical thrombectomy using devices that employ hydrodynamic forces or rotating baskets to fragment and remove the clot. These treatments may be used alone but usually are used in conjunction with pharmacologic thrombolysis. Surgical revascularization is preferred when restoration of blood flow must occur within 24 h to prevent limb loss or when symptoms

1	usually are used in conjunction with pharmacologic thrombolysis. Surgical revascularization is preferred when restoration of blood flow must occur within 24 h to prevent limb loss or when symptoms of occlusion have been present for more than 2 weeks. Amputation is performed when the limb is not viable, as characterized by loss of sensation, paralysis, and the absence of Doppler-detected blood flow in both arteries and veins.

1	If the limb is not in jeopardy, a more conservative approach that includes observation and administration of anticoagulants may be taken. Anticoagulation prevents recurrent embolism and reduces the likelihood of thrombus propagation; it can be initiated with intravenous heparin and followed by oral warfarin. Recommended doses are the same as those used for deep vein thrombosis (Chap. 300). Emboli resulting from infective endocarditis, the presence of prosthetic heart valves, or atrial myxoma often require surgical intervention to remove the cause.

1	Atheroembolism is another cause of limb ischemia. In this condition, multiple small deposits of fibrin, platelets, and cholesterol debris embolize from proximal atherosclerotic lesions or aneurysmal sites. Large protruding aortic atheromas are a source of emboli that may lead to limb ischemia, as well as stroke and renal insufficiency. Atheroembolism may occur after intraarterial procedures. Since atheroemboli to limbs tend to lodge in the small vessels of the muscle and skin and may not occlude the large vessels, distal pulses usually remain palpable. Patients complain of acute pain and tenderness at the site of embolization. Digital vascular occlusion may result in ischemia and the “blue toe” syndrome; digital necrosis and gangrene may develop (Fig. 302-2). Localized areas of tenderness, pallor, and livedo reticularis (see below) occur at sites of emboli. Skin or muscle biopsy may demonstrate cholesterol crystals.

1	Ischemia resulting from atheroemboli is notoriously difficult to treat. Usually neither surgical revascularization procedures nor thrombolytic therapy is helpful because of the multiplicity, composition, and distal location of the emboli. There is limited evidence that anti-thrombotic therapy with platelet inhibitors or anticoagulants prevents atheroembolism. Statins may stabilize plaque and potentially reduce the risk of atheroembolism. Surgical intervention to remove or bypass the atherosclerotic vessel or aneurysm that causes the recurrent atheroemboli may be necessary.

1	This is a symptom complex resulting from compression of the neurovascular bundle (artery, vein, or nerves) at the thoracic outlet as it courses through the neck and shoulder. Cervical ribs, abnormalities of the scalenus anticus muscle, proximity of the clavicle to the first rib, or abnormal insertion of the pectoralis minor muscle may compress the subclavian artery, subclavian vein, and brachial plexus as these structures pass from the thorax to the arm. Depending on the structures affected, thoracic outlet compression syndrome is divided into arterial, venous, and neurogenic forms. Patients with neurogenic thoracic outlet compression may develop shoulder and arm pain, weakness, and paresthesias. Patients with arterial compression may experience claudication, Raynaud’s phenomenon, and even ischemic tissue loss and gangrene. Venous compression may cause thrombosis of the subclavian and axillary veins; this is often associated with effort and is referred to as Paget-Schroetter syndrome.

1	FIGURE 302-2 Atheroembolism causing cyanotic discoloration and impending necrosis of the toes (“blue toe” syndrome). Examination of a patient with arterial thoracic outlet compression syndrome is often normal unless provocative maneuvers are performed. Occasionally, distal pulses are decreased or absent and digital cyanosis and ischemia may be evident.

1	Several maneuvers that support the diagnosis of arterial thoracic outlet compression syndrome may be used to precipitate symptoms, cause a subclavian artery bruit, and diminish arm pulses. These maneuvers include the abduction and external rotation test, in which the affected arm is abducted by 90° and the shoulder is externally rotated; the scalene maneuver (extension of the neck and rotation of the head to the side of the symptoms); the costoclavicular maneuver (posterior rotation of shoulders); and the hyperabduction maneuver (raising the arm 180°). A chest x-ray will indicate the presence of cervical ribs. Duplex ultrasonography, MRA, and contrast angiography can be performed during provocative maneuvers to demonstrate thoracic outlet compression of the subclavian artery. Neurophysiologic tests such as the electromyogram, nerve conduction studies, and somatosensory evoked potentials may be abnormal if the brachial plexus is involved, but the diagnosis of neurogenic thoracic outlet

1	tests such as the electromyogram, nerve conduction studies, and somatosensory evoked potentials may be abnormal if the brachial plexus is involved, but the diagnosis of neurogenic thoracic outlet syndrome is not necessarily excluded if these tests are normal owing to their low sensitivity.

1	Most patients can be managed conservatively. They should be advised to avoid the positions that cause symptoms. Many patients benefit from shoulder girdle exercises. Surgical procedures such as removal of the first rib and resection of the scalenus anticus muscle are necessary occasionally for relief of symptoms or treatment of ischemia. Popliteal artery entrapment typically affects young athletic men and women when the gastrocnemius or popliteus muscle compresses the popliteal artery and causes intermittent claudication. Thrombosis, 1647 embolism, or popliteal artery aneurysm may occur. The pulse examination may be normal unless provocative maneuvers such as ankle dorsiflexion and plantar flexion are performed. The diagnosis is confirmed by duplex ultrasound, CTA, MRA, or conventional angiography. Treatment involves surgical release of the popliteal artery or vascular reconstruction.

1	Popliteal artery aneurysms are the most common peripheral artery aneurysms. Approximately 50% are bilateral. Patients with popliteal artery aneurysms often have aneurysms of other arteries, especially the aorta. The most common clinical presentation is limb ischemia secondary to thrombosis or embolism. Rupture occurs less frequently. Other complications include compression of the adjacent popliteal vein or peroneal nerve. Popliteal artery aneurysm can be detected by palpation and confirmed by duplex ultrasonography. Repair is indicated for symptomatic aneurysms or when the diameter exceeds 2–3 cm, owing to the risk of thrombosis, embolism, or rupture.

1	Abnormal communications between an artery and a vein, bypassing the capillary bed, may be congenital or acquired. Congenital arteriovenous fistulas are a result of persistent embryonic vessels that fail to differentiate into arteries and veins; they may be associated with birthmarks, can be located in almost any organ of the body, and frequently occur in the extremities. Acquired arteriovenous fistulas either are created to provide vascular access for hemodialysis or occur as a result of a penetrating injury such as a gunshot or knife wound or as complications of arterial catheterization or surgical dissection. An uncommon cause of arteriovenous fistula is rupture of an arterial aneurysm into a vein.

1	The clinical features depend on the location and size of the fistula. Frequently, a pulsatile mass is palpable, and a thrill and a bruit lasting throughout systole and diastole are present over the fistula. With long-standing fistulas, clinical manifestations of chronic venous insufficiency, including peripheral edema; large, tortuous varicose veins; and stasis pigmentation become apparent because of the high venous pressure. Evidence of ischemia may occur in the distal portion of the extremity. Skin temperature is higher over the arteriovenous fistula. Large arteriovenous fistulas may result in an increased cardiac output with consequent cardiomegaly and high-output heart failure (Chap. 279).

1	The diagnosis is often evident from the physical examination. Compression of a large arteriovenous fistula may cause reflex slowing of the heart rate (Nicoladoni-Branham sign). Duplex ultrasonography may detect an arteriovenous fistula, especially one that affects the femoral artery and vein at the site of catheter access. CTA and conventional angiography can confirm the diagnosis and are useful in demonstrating the site and size of the arteriovenous fistula.

1	Management of arteriovenous fistulas may involve surgery, radiotherapy, or embolization. Congenital arteriovenous fistulas are often difficult to treat because the communications may be numerous and extensive, and new communications frequently develop after ligation of the most obvious ones. Many of these lesions are best treated conservatively using elastic support hose to reduce the consequences of venous hypertension. Occasionally, embolization with autologous material, such as fat or muscle, or with hemostatic agents, such as gelatin sponges or silicon spheres, is used to obliterate the fistula. Acquired arteriovenous fistulas are usually amenable to surgical treatment that involves division or excision of the fistula. Occasionally, autogenous or synthetic grafting is necessary to reestablish continuity of the artery and vein.

1	Raynaud’s phenomenon is characterized by episodic digital ischemia, manifested clinically by the sequential development of digital blanching, cyanosis, and rubor of the fingers or toes after cold exposure Arterial Diseases of the Extremities FIGURE 302-3 Vascular diseases associated with temperature: (A) Raynaud’s phenomenon; (B) acrocyanosis; (C) livedo reticularis; (D) pernio; (E) erythromelalgia; and (F) frostbite.

1	FIGURE 302-3 Vascular diseases associated with temperature: (A) Raynaud’s phenomenon; (B) acrocyanosis; (C) livedo reticularis; (D) pernio; (E) erythromelalgia; and (F) frostbite. and subsequent rewarming. Emotional stress may also precipitate Raynaud’s phenomenon. The color changes are usually well demarcated and are confined to the fingers or toes. Typically, one or more digits will appear white when the patient is exposed to a cold environment or touches a cold object (Fig. 302-3A). The blanching, or pallor, represents the ischemic phase of the phenomenon and results from vasospasm of digital arteries. During the ischemic phase, capillaries and venules dilate, and cyanosis results from the deoxygenated blood that is present in these vessels. A sensation of cold or numbness or paresthesia of the digits often accompanies the phases of pallor and cyanosis.

1	With rewarming, the digital vasospasm resolves, and blood flow into the dilated arterioles and capillaries increases dramatically. This “reactive hyperemia” imparts a bright red color to the digits. In addition to rubor and warmth, patients often experience a throbbing, painful sensation during the hyperemic phase. Although the triphasic color response is typical of Raynaud’s phenomenon, some patients may develop only pallor and cyanosis; others may experience only cyanosis. Raynaud’s phenomenon is broadly separated into two categories: idiopathic, termed primary Raynaud’s phenomenon, and secondary Raynaud’s phenomenon, which is associated with other disease states or known causes of vasospasm (Table 302-1).

1	Primary Raynaud’s Phenomenon This appellation is applied when the secondary causes of Raynaud’s phenomenon have been excluded. Over 50% of patients with Raynaud’s phenomenon have the primary form. Women are affected about five times more often than men, and the age of presentation is usually between 20 and 40 years. The fingers are involved more frequently than the toes. Initial episodes may involve only one or two fingertips, but subsequent attacks may involve the entire finger and may include all the fingers. The toes are affected in 40% of patients. Although vasospasm of the toes usually occurs in patients with symptoms in the fingers, it may happen alone. Rarely, the earlobes, the tip of the nose, and the penis are involved. Raynaud’s phenomenon occurs frequently in patients who also have migraine headaches or variant angina. These associations suggest that there may be a common predisposing cause for the vasospasm.

1	Results of physical examination are often entirely normal; the radial, ulnar, and pedal pulses are normal. The fingers and toes may Collagen vascular diseases: scleroderma, systemic lupus erythematosus, rheumatoid arthritis, dermatomyositis, polymyositis, mixed connective tissue disease, Sjögren’s syndrome Arterial occlusive diseases: atherosclerosis of the extremities, thromboangiitis obliterans, acute arterial occlusion, thoracic outlet syndrome Neurologic disorders: intervertebral disk disease, syringomyelia, spinal cord tumors, stroke, poliomyelitis, carpal tunnel syndrome, complex regional pain syndrome Blood dyscrasias: cold agglutinins, cryoglobulinemia, cryofibrinogenemia, myeloproliferative disorders, lymphoplasmacytic lymphoma Trauma: vibration injury, hammer hand syndrome, electric shock, cold injury, typing, piano playing

1	Trauma: vibration injury, hammer hand syndrome, electric shock, cold injury, typing, piano playing Drugs and toxins: ergot derivatives, methysergide, β-adrenergic receptor blockers, bleomycin, vinblastine, cisplatin, gemcitabine, vinyl chloride be cool between attacks and may perspire excessively. Thickening and tightening of the digital subcutaneous tissue (sclerodactyly) develop in 10% of patients. Angiography of the digits for diagnostic purposes is not indicated. In general, patients with primary Raynaud’s disease have milder clinical manifestations. Fewer than 1% of these patients lose a part of a digit. After the diagnosis is made, the disease improves spontaneously in approximately 15% of patients and progresses in about 30%.

1	Secondary Causes of Raynaud’s Phenomenon Raynaud’s phenomenon occurs in 80–90% of patients with systemic sclerosis (scleroderma) and is the presenting symptom in 30% (Chap. 382). It may be the only symptom of scleroderma for many years. Abnormalities of the digital vessels may contribute to the development of Raynaud’s phenomenon in this disorder. Ischemic fingertip ulcers may develop and progress to gangrene and autoamputation. About 20% of patients with systemic lupus erythematosus (SLE) have Raynaud’s phenomenon (Chap. 378). Occasionally, persistent digital ischemia develops and may result in ulcers or gangrene. In most severe cases, the small vessels are occluded by a proliferative endarteritis. Raynaud’s phenomenon occurs in about 30% of patients with dermatomyositis or polymyositis (Chap. 388). It frequently develops in patients with rheumatoid arthritis and may be related to the intimal proliferation that occurs in the digital arteries.

1	Atherosclerosis of the extremities is a common cause of Raynaud’s phenomenon in men >50 years. Thromboangiitis obliterans is an uncommon cause of Raynaud’s phenomenon but should be considered in young men, particularly those who are cigarette smokers. The development of cold-induced pallor in these disorders may be confined to one or two digits of the involved extremity. Occasionally, Raynaud’s phenomenon may follow acute occlusion of large and medium-size arteries by a thrombus or embolus. Embolization of atheroembolic debris may cause digital ischemia. The latter situation often involves one or two digits and should not be confused with Raynaud’s phenomenon. In patients with thoracic outlet compression syndrome, Raynaud’s phenomenon may result from diminished intravascular pressure, stimulation of sympathetic fibers in the brachial plexus, or a combination of both. Raynaud’s phenomenon occurs in patients with primary pulmonary hypertension (Chap. 304); this is more than coincidental

1	of sympathetic fibers in the brachial plexus, or a combination of both. Raynaud’s phenomenon occurs in patients with primary pulmonary hypertension (Chap. 304); this is more than coincidental and may reflect a neurohumoral abnormality that affects both the pulmonary and digital circulations.

1	A variety of blood dyscrasias may be associated with Raynaud’s phenomenon. Cold-induced precipitation of plasma proteins, hyper-viscosity, and aggregation of red cells and platelets may occur in patients with cold agglutinins, cryoglobulinemia, or cryofibrinogenemia. Hyperviscosity syndromes that accompany myeloproliferative disorders and lymphoplasmacytic lymphoma (Waldenström’s macroglobulinemia) should also be considered in the initial evaluation of patients with Raynaud’s phenomenon. Raynaud’s phenomenon occurs often in patients whose vocations require the use of vibrating hand tools, such as chain saws or jackhammers. The frequency of Raynaud’s phenomenon also seems to be increased in pianists and keyboard operators. Electric shock injury to the hands or frostbite may lead to the later development of Raynaud’s phenomenon.

1	Several drugs have been causally implicated in Raynaud’s phenomenon. They include ergot preparations, methysergide, β-adrenergic receptor antagonists, and the chemotherapeutic agents bleomycin, vinblastine, cisplatin, and gemcitabine. Most patients with Raynaud’s phenomenon experience only mild and infrequent episodes. These patients need reassurance and should be instructed to dress warmly and avoid unnecessary cold exposure. In addition to gloves and mittens, patients should protect the trunk, head, and feet with warm clothing to prevent cold-induced reflex vasoconstriction. Tobacco use is contraindicated.

1	Drug treatment should be reserved for severe cases. 1649 Dihydropyridine calcium channel antagonists such as nifedipine, isradipine, felodipine, and amlodipine decrease the frequency and severity of Raynaud’s phenomenon. Diltiazem may be considered but is less effective. The postsynaptic α1-adrenergic antagonist prazosin has been used with favorable responses; doxazosin and terazosin may also be effective. Phosphodiesterase type 5 inhibitors such as sildenafil and tadalafil may improve symptoms in patients with secondary Raynaud’s phenomenon, as occurs with systemic sclerosis. Digital sympathectomy is helpful in some patients who are unresponsive to medical therapy.

1	In this condition, there is arterial vasoconstriction and secondary dilation of the capillaries and venules with resulting persistent cyanosis of the hands and, less frequently, the feet. Cyanosis may be intensified by exposure to a cold environment. Acrocyanosis may be categorized as primary or secondary to an underlying condition. In primary acrocyanosis, women are affected much more frequently than men, and the age of onset is usually <30 years. Generally, patients are asymptomatic but seek medical attention because of the discoloration. The prognosis is favorable, and pain, ulcers, and gangrene do not occur. Examination reveals normal pulses, peripheral cyanosis, and moist palms (Fig. 302-3B). Trophic skin changes and ulcerations do not occur. The disorder can be distinguished from Raynaud’s phenomenon because it is persistent and not episodic, the discoloration extends proximally from the digits, and blanching does not occur. Ischemia secondary to arterial occlusive disease can

1	phenomenon because it is persistent and not episodic, the discoloration extends proximally from the digits, and blanching does not occur. Ischemia secondary to arterial occlusive disease can usually be excluded by the presence of normal pulses. Central cyanosis and decreased arterial oxygen saturation are not present. Patients should be reassured and advised to dress warmly and avoid cold exposure. Pharmacologic intervention is not indicated.

1	Secondary acrocyanosis may result from hypoxemia, vasopressor medications, connective tissue diseases, atheroembolism, antiphospholipid antibodies, cold agglutinins, or cryoglobulins and is associated with anorexia nervosa and postural orthostatic tachycardia syndrome. Treatment should be directed at the underlying disorder.

1	In this condition, localized areas of the extremities develop a mottled or rete (netlike) appearance of reddish to blue discoloration (Fig. 302-3C). The mottled appearance may be more prominent after cold exposure. There are primary and secondary forms of livedo reticularis. The primary, or idiopathic, form of this disorder may be benign or associated with ulcerations. The benign form occurs more frequently in women than in men, and the most common age of onset is the third decade. Patients with the benign form are usually asymptomatic and seek attention for cosmetic reasons. These patients should be reassured and advised to avoid cold environments. No drug treatment is indicated. Primary livedo reticularis with ulceration is also called atrophie blanche en plaque. The ulcers are painful and may take months to heal. Secondary livedo reticularis can occur with atheroembolism (see above), SLE and other vasculitides, anticardiolipin antibodies, hyper-viscosity, cryoglobulinemia, and

1	and may take months to heal. Secondary livedo reticularis can occur with atheroembolism (see above), SLE and other vasculitides, anticardiolipin antibodies, hyper-viscosity, cryoglobulinemia, and Sneddon’s syndrome (ischemic stroke and livedo reticularis). Rarely, skin ulcerations develop.

1	Pernio is a vasculitic disorder associated with exposure to cold; acute forms have been described. Raised erythematous lesions develop on the lower part of the legs and feet in cold weather (Fig. 302-3D). They are associated with pruritus and a burning sensation, and they may blister and ulcerate. Pathologic examination demonstrates angiitis characterized by intimal proliferation and perivascular infiltration of mononuclear and polymorphonuclear leukocytes. Giant cells may be present in the subcutaneous tissue. Patients should avoid exposure to cold, and ulcers should be kept clean and protected with sterile dressings. Sympatholytic drugs and dihydropyridine calcium channel antagonists may be effective in some patients.

1	Arterial Diseases of the Extremities 1650 ERYTHROMELALGIA This disorder is characterized by burning pain and erythema of the extremities (Fig. 302-3E). The feet are involved more frequently than the hands, and males are affected more frequently than females. Erythromelalgia may occur at any age but is most common in middle age. It may be primary (also termed erythermalgia) or secondary. Mutations in the SCN9A gene, which encodes the Nav1.7 voltage-gated sodium channel expressed in sensory and sympathetic nerves, has been described in inherited forms of erythromelalgia. The most common causes of secondary erythromelalgia are myeloproliferative disorders such as polycythemia vera and essential thrombocytosis. Less common causes include drugs, such as calcium channel blockers, bromocriptine, and pergolide; neuropathies; connective tissue diseases such as SLE; and paraneoplastic syndromes. Patients complain of burning in the extremities that is precipitated by exposure to a warm

1	and pergolide; neuropathies; connective tissue diseases such as SLE; and paraneoplastic syndromes. Patients complain of burning in the extremities that is precipitated by exposure to a warm environment and aggravated by a dependent position. The symptoms are relieved by exposing the affected area to cool air or water or by elevation. Erythromelalgia can be distinguished from ischemia secondary to peripheral arterial disorders because the peripheral pulses are present. There is no specific treatment; aspirin may produce relief in patients with erythromelalgia secondary to myeloproliferative disease. Treatment of associated disorders in secondary erythromelalgia may be helpful.

1	In this condition, tissue damage results from severe environmental cold exposure or from direct contact with a very cold object. Tissue injury results from both freezing and vasoconstriction. Frostbite usually affects the distal aspects of the extremities or exposed parts of the face, such as the ears, nose, chin, and cheeks. Superficial frostbite involves the skin and subcutaneous tissue. Patients experience pain or paresthesia, and the skin appears white and waxy. After rewarming, there is cyanosis and erythema, wheal-and-flare formation, edema, and superficial blisters. Deep frostbite involves muscle, nerves, and deeper blood vessels. It may result in edema of the hand or foot, vesicles and bullae, tissue necrosis, and gangrene (Fig. 302-3F).

1	Initial treatment is rewarming, performed in an environment where reexposure to freezing conditions will not occur. Rewarming is accomplished by immersion of the affected part in a water bath at temperatures of 40°–44°C (104°–111°F). Massage, application of ice water, and extreme heat are contraindicated. The injured area should be cleansed with soap or antiseptic, and sterile dressings should be applied. Analgesics are often required during rewarming. Antibiotics are used if there is evidence of infection. The efficacy of sympathetic may exhibit increased sensitivity to cold. lymphedema CHRONIC VENOUS DISEASE 303 blocking drugs is not established. After recovery, the affected extremity Mark A. Creager, Joseph Loscalzo

1	Mark A. Creager, Joseph Loscalzo Chronic venous diseases range from telangiectasias and reticular veins, to varicose veins, to chronic venous insufficiency with edema, skin changes, and ulceration. This section of the chapter will focus on identification and treatment of varicose veins and chronic venous insufficiency, since these problems are encountered frequently by the internist. The estimated prevalence of varicose veins in the United States is approximately 15% in men and 30% in women. Chronic venous insufficiency with edema affects approximately 7.5% of men and 5% of women, and the prevalence increases with age ranging from 2% among those less than 50 years of age to 10% of those 70 years of age. Approximately 20% of patients with chronic venous insufficiency develop venous ulcers.

1	Veins in the extremities can be broadly classified as either superficial or deep. The superficial veins are located between the skin and deep fascia. In the legs, these include the great and small saphenous veins and their tributaries. The great saphenous vein is the longest vein in the body. It originates on the medial side of the foot and ascends anterior to the medial malleolus and then along the medial side of the calf and thigh, and drains into the common femoral vein. The small saphenous vein originates on the dorsolateral aspect of the foot, ascends posterior to the lateral malleolus and along the posterolateral aspect of the calf, and drains into the popliteal vein. The deep veins of the leg accompany the major arteries. There are usually paired peroneal, anterior tibial, and posterior tibial veins in the calf, which converge to form the popliteal vein. Soleal tributary veins drain into the posterior tibial or peroneal veins, and gastrocnemius tributary veins drain into the

1	tibial veins in the calf, which converge to form the popliteal vein. Soleal tributary veins drain into the posterior tibial or peroneal veins, and gastrocnemius tributary veins drain into the popliteal vein. The popliteal vein ascends in the thigh as the femoral vein. The confluence of the femoral vein and deep femoral vein form the common femoral vein, which ascends in the pelvis as the external iliac and then common iliac vein, which converges with the contralateral common iliac vein at the inferior vena cava. Perforating veins connect the superficial and deep systems in the legs at multiple locations, normally allowing blood to flow from the superficial to deep veins. In the arms, the superficial veins include the basilic, cephalic, and median cubital veins and their tributaries. The basilic and cephalic veins course along the medial and lateral aspects of the arm, respectively, and these are connected via the median cubital vein in the antecubital fossa. The deep veins of the arms

1	and cephalic veins course along the medial and lateral aspects of the arm, respectively, and these are connected via the median cubital vein in the antecubital fossa. The deep veins of the arms accompany the major arteries and include the radial, ulnar, brachial, axillary, and subclavian veins. The subclavian vein converges with the internal jugular vein to form the brachiocephalic vein, which joins the contralateral brachiocephalic vein to form the superior vena cava. Bicuspid valves are present throughout the venous system to direct the flow of venous blood centrally.

1	Pathophysiology of Chronic Venous Disease Varicose veins are dilated, bulging, tortuous superficial veins, measuring at least 3 mm in diameter. The smaller and less tortuous reticular veins are dilated intradermal veins, which appear blue-green, measure 1 to 3 mm in diameter, and do not protrude from the skin surface. Telangiectasias, or spider veins, are small, dilated veins, less than 1 mm in diameter, located near the skin surface, and form blue, purple, or red linear, branching, or spider-web patterns.

1	Varicose veins can be categorized as primary or secondary. Primary varicose veins originate in the superficial system and result from defective structure and function of the valves of the saphenous veins, intrinsic weakness of the vein wall, and high intraluminal pressure. Approximately one-half of these patients have a family history of varicose veins. Other factors associated with primary varicose veins include aging, pregnancy, hormonal therapy, obesity, and prolonged standing. Secondary varicose veins result from venous hypertension, associated with deep venous insufficiency or deep venous obstruction, and incompetent perforating veins that cause enlargement of superficial veins. Arteriovenous fistulas also cause varicose veins in the affected limb.

1	Chronic venous insufficiency is a consequence of incompetent veins in which there is venous hypertension and extravasation of fluid and blood elements into the tissue of the limb. It may occur in patients with varicose veins but usually is caused by disease in the deep veins. It also is categorized as primary or secondary. Primary deep venous insufficiency is a consequence of an intrinsic structural or functional abnormality in the vein wall or venous valves leading to valvular reflux. Secondary deep venous insufficiency is caused by obstruction and/or valvular incompetence from previous deep vein thrombosis (Chap. 300). Deep venous insufficiency occurs following deep vein thrombosis, as the delicate valve leaflets become thickened and contracted and can no longer prevent retrograde flow of blood and the vein itself becomes rigid and thick walled. Although most veins recanalize after an episode of thrombosis, the large proximal veins may remain occluded. Secondary incompetence

1	flow of blood and the vein itself becomes rigid and thick walled. Although most veins recanalize after an episode of thrombosis, the large proximal veins may remain occluded. Secondary incompetence develops in distal valves because high pressures distend the vein and separate the leaflets. Other causes of secondary deep venous insufficiency include May-Thurner syndrome, where the left iliac vein is occluded or stenosed by extrinsic compression from the overlapping right common iliac artery; arteriovenous fistulas resulting in increased venous pressure; congenital deep vein agenesis or hypoplasia; and venous malformations as may occur in Klippel-Trénaunay-Weber and Parkes-Weber syndromes.

1	Clinical Presentation Patients with venous varicosities are often asymptomatic but still concerned about the cosmetic appearance of their legs. Superficial venous thrombosis may be a recurring problem, and, rarely, a varicosity ruptures and bleeds. Symptoms in patients with varicose veins or venous insufficiency, when they occur, include a dull ache, throbbing or heaviness, or pressure sensation in the legs typically after prolonged standing; these symptoms usually are relieved with leg elevation. Additional symptoms may include cramping, burning, pruritus, leg swelling, and skin ulceration.

1	The legs are examined in both the supine and standing positions. Visual inspection and palpation of the legs in the standing position confirm the presence of varicose veins. The location and extent of the varicose veins should be noted. Edema, stasis dermatitis, and skin ulceration near the ankle may be present if there is superficial venous insufficiency and venous hypertension. Findings of deep venous insufficiency include increased leg circumference, venous varicosities, edema, and skin changes. The edema, which is usually pitting, may be confined to the ankles, extend above the ankles to the knees, or involve the thighs in severe cases. Over time, the edema may become less pitting and more indurated. Dermatologic findings associated with venous stasis include hyperpigmentation, erythema, eczema, lipodermatosclerosis, atrophie blanche, and a phlebectasia corona. Lipodermatosclerosis is the combination of induration, hemosiderin deposition, and inflammation, and typically occurs in

1	eczema, lipodermatosclerosis, atrophie blanche, and a phlebectasia corona. Lipodermatosclerosis is the combination of induration, hemosiderin deposition, and inflammation, and typically occurs in the lower part of the leg just above the ankle. Atrophie blanche is a white patch of scar tissue, often with focal telangiectasias and a hyperpigmented border; it usually develops near the medial malleolus. A phlebectasia corona is a fan-shaped pattern of intradermal veins near the ankle or on the foot. Skin ulceration may occur near the medial and lateral malleoli. A venous ulcer is often shallow and characterized by an irregular border, a base of granulation tissue, and the presence of exudate (Fig. 303-1).

1	Bedside maneuvers can be used to distinguish primary varicose veins from secondary varicose veins caused by deep venous insufficiency. With the contemporary use of venous ultrasound (see below), however, these maneuvers are employed infrequently. The Brodie-Trendelenburg

1	FIGURE 303-1 Venous insufficiency with active venous ulcer near the medial malleolus. (Courtesy of Dr. Steven Dean, with permission.) test is used to determine whether varicose veins are secondary to deep 1651 venous insufficiency. As the patient is lying supine, the leg is elevated and the veins allowed to empty. Then, a tourniquet is placed on the proximal part of the thigh and the patient is asked to stand. Filling of the varicose veins within 30 s indicates that the varicose veins are caused by deep venous insufficiency and incompetent perforating veins. Primary varicose veins with superficial venous insufficiency are the likely diagnosis if venous refilling occurs promptly after tourniquet removal. The Perthes test assesses the possibility of deep venous obstruction. A tourniquet is placed on the midthigh after the patient has stood, and the varicose veins are filled. The patient is then instructed to walk for 5 min. A patent deep venous system and competent perforating veins

1	is placed on the midthigh after the patient has stood, and the varicose veins are filled. The patient is then instructed to walk for 5 min. A patent deep venous system and competent perforating veins enable the superficial veins below the tourniquet to collapse. Deep venous obstruction is likely to be present if the superficial veins distend further with walking.

1	Differential Diagnosis The duration of leg edema helps to distinguish chronic venous insufficiency from acute deep vein thrombosis. Lymphedema, as discussed later in this chapter, is often confused with chronic venous insufficiency, and both may occur together. Other disorders that cause leg swelling should be considered and excluded when evaluating a patient with presumed venous insufficiency. Bilateral leg swelling occurs in patients with congestive heart failure, hypoalbuminemia secondary to nephrotic syndrome or severe hepatic disease, myxedema caused by hypothyroidism or pretibial myxedema associated with Graves’ disease, and with drugs such as dihydropyridine calcium channel blockers and thiazolidinediones. Unilateral causes of leg swelling also include ruptured leg muscles, hematomas secondary to trauma, and popliteal cysts. Cellulitis may cause erythema and swelling of the affected limb. Leg ulcers may be caused by severe peripheral artery disease and critical limb ischemia;

1	secondary to trauma, and popliteal cysts. Cellulitis may cause erythema and swelling of the affected limb. Leg ulcers may be caused by severe peripheral artery disease and critical limb ischemia; neuropathies, particularly those associated with diabetes; and less commonly, skin cancer, vasculitis, or rarely as a complication of hydroxyurea. The location and characteristics of venous ulcers help to differentiate these from other causes.

1	Classification of Chronic Venous Disease The CEAP (clinical, etiologic, anatomic, pathophysiologic) classification schema incorporates the range of symptoms and signs of chronic venous disease to characterize its severity. It also broadly categorizes the etiology as congenital, primary, or secondary; identifies the affected veins as superficial, deep, or perforating; and characterizes the pathophysiology as reflux, obstruction, both, or neither (Table 303-1).

1	Diagnostic Testing The principal diagnostic test to evaluate patients with chronic venous disease is venous duplex ultrasonography. A venous duplex ultrasound examination uses a combination of B-mode imaging and spectral Doppler to detect the presence of venous obstruction and venous reflux in superficial and deep veins. Color-assisted Doppler ultrasound is useful to visualize venous flow patterns. Obstruction may be diagnosed by absence of flow, the presence of an echogenic thrombus within the vein, or failure of the vein to collapse when a compression maneuver is applied by the sonographer, the last implicating the presence of an intraluminal thrombus. Venous reflux is detected by prolonged reversal of venous flow direction during a Valsalva maneuver, particularly for the common femoral vein or saphenofemoral junction, or after compression and release of a cuff placed on the limb distal to the area being interrogated.

1	Some vascular laboratories use air or strange gauge plethysmography to assess the severity of venous reflux and complement findings from the venous ultrasound examination. Venous volume and venous refilling time are measured when the legs are placed in a dependent position and after calf exercise to quantify the severity of venous reflux and the efficiency of the calf muscle pump to affect venous return. Magnetic resonance, computed tomographic, and conventional venography are rarely required to determine the cause and plan treatment for chronic venous insufficiency unless there is suspicion for pathology that might warrant intervention. These modalities are used to identify obstruction or stenosis of the inferior vena cava and iliofemoral veins, as may occur in patients with previous proximal TABlE 303-1 CEAP (CliniCAl, ETiologiC, AnAToMiC, PATHoPHYSiologiC) ClASSiFiCATion

1	TABlE 303-1 CEAP (CliniCAl, ETiologiC, AnAToMiC, PATHoPHYSiologiC) ClASSiFiCATion C0 No visible or palpable signs of venous disease C1 Telangiectasias, reticular veins C2 Varicose veins C3 Edema without skin changes C4 Skin changes, including pigmentation, eczema, lipodermatosclerosis, and Pr Reflux Po Obstruction Pr,o Reflux and obstruction Pn No venous pathophysiology identifiable Source: B Eklöf et al: J Vasc Surg 40:1248, 2004. deep vein thrombosis; occlusion of inferior vena cava filters; extrinsic compression from tumors; and May-Thurner syndrome.

1	Source: B Eklöf et al: J Vasc Surg 40:1248, 2004. deep vein thrombosis; occlusion of inferior vena cava filters; extrinsic compression from tumors; and May-Thurner syndrome. Varicose veins usually are treated with conservative measures. Symptoms often decrease when the legs are elevated periodically, prolonged standing is avoided, and elastic support hose are worn. External compression with elastic stockings or stretch bandages provides a counterbalance to the hydrostatic pressure in the veins. Although compression garments may improve symptoms, they do not prevent progression of varicose veins. Graduated compression stockings with pressures of 20–30 mmHg are suitable for most patients with simple varicose veins, although pressures of 30–40 mmHg may be required for patients with manifestations of venous insufficiency such as edema and ulcers.

1	Patients with chronic venous insufficiency also should be advised to avoid prolonged standing or sitting; frequent leg elevation is helpful. Graded compression therapy consisting of stockings or multilayered compression bandages is the standard of care for advanced chronic venous insufficiency characterized by edema, skin changes, or venous ulcers defined as CEAP clinical class C3– C6. Graduated compression stockings of 30–40 mmHg are more effective than lesser grades for healing venous ulcers. The length of stocking depends on the distribution of edema. Calf-length stockings are tolerated better by most patients, particularly elderly patients; for patients with varicose veins or edema extending to the thigh, thigh-length stockings or panty hose should be considered. Overweight and obese patients should be advised to lose weight via caloric restriction and exercise.

1	In addition to a compression bandage or stocking, patients with venous ulcers also may be treated with low adherent absorbent dressings that take up exudates while maintaining a moist environment. Other types of dressings include hydrocolloid (an adhesive dressing comprised of polymers such as carboxymethylcellulose that absorbs exudates by forming a gel), hydrogel (a nonabsorbent dressing comprising over 80% water or glycerin that moisturizes wounds), foam (an absorbent dressing made with polymers such as polyurethane), and alginate (an absorbent, biodegradable dressing that is derived from seaweed), but there is little evidence that these are more effective than low adherent absorbent dressings. The choice of specific dressing depends on the amount of drainage, presence of infection, and integrity of the skin surrounding the ulcer. Antibiotics are not indicated unless the ulcer is infected. The multilayered compression bandage or graduated compression garment is then put over the

1	and integrity of the skin surrounding the ulcer. Antibiotics are not indicated unless the ulcer is infected. The multilayered compression bandage or graduated compression garment is then put over the dressing.

1	There are no drugs approved by the U.S. Food and Drug Administration for the treatment of chronic venous insufficiency. Diuretics may reduce edema, but at the risk of volume depletion and compromise in renal function. Topical steroids may be used for a short period of time to treat inflammation associated with stasis dermatitis. Several herbal supplements, such as horse chestnut seed extract (aescin); flavonoids including diosmin, hesperidin, or the two combined as micronized purified flavonoid fraction; and French maritime pine bark extract, are touted to have venoconstrictive and anti-inflammatory properties. Although meta-analyses have suggested that aescin reduces edema, pruritus, and pain and that micronized purified flavonoid fraction in conjunction with compression therapy facilitates venous ulcer healing, there is insufficient evidence to recommend the general use of these substances in patients with chronic venous insufficiency.

1	Ablative procedures, including endovenous thermal ablation, sclerotherapy, and surgery, are used to treat varicose veins in selected patients who have persistent symptoms, great saphenous vein incompetency, and complications of venous insufficiency including dermatitis, edema, and ulcers. Ablative therapy may also be indicated for cosmetic reasons.

1	Endovenous thermal ablation procedures of the saphenous veins include endovenous laser therapy and radiofrequency ablation. To ablate the great saphenous vein, a catheter is placed percutaneously and advanced from the level of the knee to just below the saphenofemoral junction via ultrasound guidance. Thermal energy is then delivered as the catheter is pulled back. The heat injures the endothelium and media and promotes thrombosis and fibrosis, resulting in venous occlusion. Average 1and 5-year occlusion rates exceed 90% following endovenous laser therapy and are slightly less after radiofrequency ablation. Deep vein thrombosis of the common femoral vein adjacent to the saphenofemoral junction is an uncommon but potential complication of endovenous thermal ablation. Other adverse effects of thermal ablation procedures include pain, paresthesias, bruising, hematoma, and hyperpigmentation.

1	Sclerotherapy involves the injection of a chemical into a vein to cause fibrosis and obstruction. Sclerosing agents approved by the U.S. Food and Drug Administration include sodium tetradecyl sulfate, polidocanol, sodium morrhuate, and glycerin. The sclerosing agent is administered as a liquid or mixed with air or CO2/O2 to create a foam. It first is injected into the great saphenous vein or its affected tributaries, often with ultrasound guidance. Thereafter, smaller more distal veins and incompetent perforating veins are injected. Following completion of the procedure, elastic bandages are applied, or 30–40 mmHg compression stockings are worn for 1–2 weeks. Average 1and 5-year occlusion rates are 81% and 74%, respectively, following sclerotherapy. Complications are uncommon and include deep vein thrombosis, hematomas, damage to adjacent saphenous or sural nerves, and infection. Anaphylaxis is a very rare but severe complication.

1	Surgical therapy usually involves ligation and stripping of the great and small saphenous veins. The procedure is performed under general anesthesia. Incisions are made at the groin and the upper calf. The great saphenous vein is ligated below the saphenofemoral junction, and a wire is inserted into the great saphenous vein and advanced distally. The proximal part of the great saphenous vein is secured to the wire and retrieved, i.e., stripped, via the calf incision. Stripping of the great saphenous vein below the knee and stripping of the small saphenous vein usually are not performed because of the respective risks of saphenous and sural nerve injury. Complications of great saphenous vein ligation and stripping include deep vein thrombosis, bleeding, hematoma, infection, and nerve injury. Recurrent varicose veins occur in up to 50% patients by 5 years, due to technical failures, deep venous insufficiency, and incompetent perforating veins.

1	Stab phlebectomy is another surgical treatment for of varicose veins. A small incision is made alongside the varicose vein, and it is avulsed by means of a forceps or hook. This procedure may be performed in conjunction with saphenous vein ligation and stripping or thermal ablation. Subfascial endoscopic perforator surgery (SEPS) uses endoscopy to identify and occlude incompetent perforating veins. It also may be performed along with other ablative procedures.

1	Endovascular interventions, surgical bypass, and reconstruction of the valves of the deep veins are performed when feasible to treat patients with advanced chronic venous insufficiency who have not responded to other therapies. Catheter-based interventions, usually involving placement of endovenous stents, may be considered to treat some patients with chronic occlusions of the iliac veins. Technical success rates exceed 85% in most series, and long-term patency is achieved in approximately 75% of these patients. Iliocaval bypass, femoroiliac venous bypass, and femorofemoral crossover venous bypass are procedures used occasionally to treat iliofemoral vein occlusion; saphenopopliteal vein bypass can be used to treat chronic femoropopliteal vein obstruction. Long-term patency rates for venous bypass procedures generally exceed 60% and are associated with improvement in symptoms. Surgical reconstruction of the valves of the deep veins and valve transfer procedures are used to treat

1	venous bypass procedures generally exceed 60% and are associated with improvement in symptoms. Surgical reconstruction of the valves of the deep veins and valve transfer procedures are used to treat valvular incompetence. Valvuloplasty involves tightening the valve by commissural apposition. With valve transfer procedures, a segment of vein with a competent valve, such as a brachial or axillary vein, or adjacent saphenous or deep femoral vein, is inserted as an interposition graft in the incompetent vein. Both valvuloplasty and vein transfer operations result in ulcer healing in the majority of patients, although success rates are somewhat better with valvuloplasty.

1	Lymphedema Lymphedema is a chronic condition caused by impaired transport of lymph and characterized by swelling of one or more limbs and occasionally the trunk and genitalia. Fluid accumulates in interstitial tissues when there is an imbalance between lymph production and lymph absorption, a process governed in large part by Starling forces. Deficiency, reflux, or obstruction of lymph vessels perturbs the ability of the lymphatic system to reabsorb proteins that had been filtered by blood vessels, and the tissue osmotic load promotes interstitial accumulation of water. Persistent lymphedema leads to inflammatory and immune responses characterized by infiltration of mononuclear cells, fibroblasts, and adipocytes, leading to adipose and collagen deposition in the skin and subcutaneous tissues.

1	Lymphatic Anatomy Lymphatic capillaries are blind-ended tubes formed by a single layer of endothelial cells. The absent or widely fenestrated basement membrane of lymphatic capillaries allows access to interstitial proteins and particles. Lymphatic capillaries merge to form microlymphatic precollector vessels, which contain few smooth muscle cells. The precollector vessels drain into collecting lymphatic vessels, which comprise endothelial cells, a basement membrane, smooth muscle, and bileaflet valves. The collecting lymphatic vessels in term merge to form larger lymphatic conduits. Analogous to venous anatomy, there are superficial and deep lymphatic vessels in the legs, which communicate at the popliteal and inguinal lymph nodes. Pelvic lymphatic vessels drain into the thoracic duct, which ascends from the abdomen to the thorax and connects with the left brachiocephalic vein. Lymph is propelled centrally by the phasic contractile activity 1653 of lymphatic smooth muscle and

1	duct, which ascends from the abdomen to the thorax and connects with the left brachiocephalic vein. Lymph is propelled centrally by the phasic contractile activity 1653 of lymphatic smooth muscle and facilitated by the contractions of contiguous skeletal muscle. The presence of lymphatic valves ensures unidirectional flow.

1	Etiology Lymphedema may be categorized as primary or secondary (Table 303-2). The prevalence of primary lymphedema is approximately 1.15 per 100,000 persons less than 20 years of age. Females are affected more frequently than males. Primary lymphedema may be caused by agenesis, hypoplasia, hyperplasia, or obstruction of the lymphatic vessels. There are three clinical subtypes: congenital lymph-edema, which appears shortly after birth; lymphedema praecox, which has its onset at the time of puberty; and lymphedema tarda, which usually begins after age 35. Familial forms of congenital lymphedema (Milroy’s disease) and lymphedema praecox (Meige’s disease) may be inherited in an autosomal dominant manner with variable penetrance; autosomal or sex-linked recessive forms are less common. Mutations in genes expressing vascular endothelial growth factor receptor 3 (VEGFR3), which is a determinant of lymphangiogenesis, have been described in patients with Milroy’s disease. A mutation on

1	Mutations in genes expressing vascular endothelial growth factor receptor 3 (VEGFR3), which is a determinant of lymphangiogenesis, have been described in patients with Milroy’s disease. A mutation on chromosome 15q is associated with the cholestasis-lymphedema syndrome. A mutation in the FOXC2 gene, which encodes a transcription factor

1	Klinefelter’s syndrome Trisomy 13, 18, or 21 Noonan’s syndrome Infection Bacterial lymphangitis (Streptococcus pyogenes, Staphylococcus aureus) Lymphogranuloma venereum (Chlamydia trachomatis) Filariasis (Wucheria bancrofti, Brugia malayi, B. timori) Tuberculosis Neoplastic infiltration of lymph nodes Lymphoma Prostate Others Surgery or irradiation of axillary or inguinal lymph nodes for treatment of cancer

1	Iatrogenic Lymphatic division (during peripheral bypass surgery, varicose vein surgery, or harvesting of saphenous veins) 1654 that interacts with a signaling pathway involved in the development of lymphatic vessels, has been reported in patients with the lymphedema-distichiasis syndrome, in which lymphedema praecox occurs in patients who also have a double row of eyelashes. A mutation of SOX18, a transcription factor upstream of lymphatic endothelial cell differentiation, has been described in patients with lymphedema, alopecia, and telangiectasias (hypotrichosis, lymphedema, telangiectasia syndrome). Patients with a chromosomal aneuploidy, such as Turner’s syndrome, Klinefelter’s syndrome, or trisomy 18, 13, or 21, may develop lymphedema. Syndromic vascular anomalies associated with lymphedema include Klippel-Trénaunay syndrome, Parkes-Weber syndrome, and Hennekam’s syndrome. Other disorders associated with lymphedema include Noonan’s syndrome, yellow nail syndrome, intestinal

1	lymphedema include Klippel-Trénaunay syndrome, Parkes-Weber syndrome, and Hennekam’s syndrome. Other disorders associated with lymphedema include Noonan’s syndrome, yellow nail syndrome, intestinal lymphangiectasia syndrome, lymphangiomyomatosis, and neurofibromatosis type 1. Secondary lymphedema is an acquired condition that results from damage to or obstruction of previously normal lymphatic channels. Recurrent episodes of bacterial lymphangitis, usually caused by streptococci, are a very common cause of lymphedema. The most common cause of secondary lymphedema worldwide is lymphatic filariasis, affecting approximately 129 million children and adults worldwide and causing lymphedema and elephantiasis in 14 million of these affected individuals (Chap. 258). Recurrent bacterial lymphangitis by Streptococcus may result in chronic lymphedema. Other infectious causes include lymphogranuloma venereum and tuberculosis. In developed countries, the most common secondary cause of lymphedema

1	by Streptococcus may result in chronic lymphedema. Other infectious causes include lymphogranuloma venereum and tuberculosis. In developed countries, the most common secondary cause of lymphedema is surgical excision or irradiation of axillary and inguinal lymph nodes for treatment of cancers, such as breast, cervical, endometrial, and prostate cancer, sarcomas, and malignant melanoma. Lymphedema of the arm occurs in 13% of breast cancer patients after axillary node dissection and in 22% after both surgery and radiotherapy. Lymphedema of the leg affects approximately 15% of patients with cancer after inguinal lymph node dissection. Tumors, such as prostate cancer and lymphoma, also can infiltrate and obstruct lymphatic vessels. Less common causes include contact dermatitis, rheumatoid arthritis, pregnancy, and self-induced or factitious lymph-edema after application of tourniquets.

1	Clinical Presentation Lymphedema is generally a painless condition, but patients may experience a chronic dull, heavy sensation in the leg, and most often they are concerned about the appearance of the leg. Lymphedema of the lower extremity initially involves the foot and gradually progresses up the leg so that the entire limb becomes edematous (Fig. 303-2). In the early stages, the edema is soft and pits easily with pressure. Over time, subcutaneous adipose tissue accumulates, the limb enlarges further and loses its normal contour, and the toes appear square. Thickening of the skin is detected by Stemmer’s sign, which is the inability to tent the skin at the base of the toes. Peau d’orange is a term used to describe dimpling of the skin, resembling that of an orange peel, caused by lymphedema. In the chronic stages, the edema no longer pits and the limb acquires a woody texture as the tissues become indurated and fibrotic. The International Society of Lymphology describes four

1	lymphedema. In the chronic stages, the edema no longer pits and the limb acquires a woody texture as the tissues become indurated and fibrotic. The International Society of Lymphology describes four clinical stages of lymphedema (Table 303-3).

1	Differential Diagnosis Lymphedema should be distinguished from other disorders that cause unilateral leg swelling, such as deep vein thrombosis and chronic venous insufficiency. In the latter condition, the edema is softer, and there is often evidence of a stasis dermatitis, hyperpigmentation, and superficial venous varicosities, as described earlier. Other causes of leg swelling that resemble lymphedema are myxedema and lipedema. Lipedema usually occurs in women and is caused by accumulation of adipose tissue in the leg from the thigh to the ankle with sparing of the feet. Diagnostic Testing The evaluation of patients with lymphedema should include diagnostic studies to clarify the cause. Abdominal and pelvic ultrasound and computed tomography (CT) can be used to detect obstructing lesions such as neoplasms. Magnetic resonance imaging (MRI) of the affected limb may reveal a honeycomb pattern

1	FIGURE 303-2 A. Lymphedema characterized by swelling of the leg, nonpitting edema, and squaring of the toes. (Courtesy of Dr. Marie Gerhard-Herman, with permission.) B. Advanced chronic stage of lymphedema illustrating the woody appearance of the leg with acanthosis and verrucous overgrowths. (Courtesy of Dr. Jeffrey Olin, with permission.) characteristic of lymphedema in the epifascial compartment and identify enlarged lymphatic channels and lymph nodes. MRI also is useful to distinguish lymphedema from lipedema. Lymphoscintigraphy and lymphangiography are rarely indicated, but either can be used to confirm the diagnosis or differentiate primary from secondary lymph-edema. Lymphoscintigraphy involves the injection of radioactively labeled technetium-containing colloid into the distal subcutaneous tissue of the affected extremity, which is imaged with a scintigraphic camera to visualize lymphatic vessels and lymph nodes. Findings indicative of primary lymphedema include absent or

1	subcutaneous tissue of the affected extremity, which is imaged with a scintigraphic camera to visualize lymphatic vessels and lymph nodes. Findings indicative of primary lymphedema include absent or delayed filling of the lymphatic vessels or dermal back flow caused by lymphatic reflux. Findings of secondary lymphedema include dilated lymphatic vessels distal to an area of obstruction. In lymphangiography, iodinated radio-contrast material is injected into a distal lymphatic vessel that has been isolated and cannulated. In primary lymphedema, lymphatic channels are absent, hypoplastic, or ectatic. In secondary lymphedema, lymphatic channels often appear dilated beneath the level of obstruction.

1	A latent or subclinical condition where swelling is not evident despite impaired lymph transport. It may exist for months or years before overt edema occurs. Early accumulation of fluid relatively high in protein content that subsides with limb elevation. Pitting may occur. An increase in proliferating cells may also be seen. Limb elevation alone rarely reduces tissue swelling, and pitting is manifest. Late in stage II, the limb may or may not pit as excess fat and fibrosis supervene. Lymphostatic elephantiasis where pitting can be absent and trophic skin changes such as acanthosis, further deposition of fat and fibrosis, and warty overgrowths have developed. Source: Adapted from The 2013 Consensus Document of the International Society of Lymphology: Lymphology 46:1, 2013.

1	Source: Adapted from The 2013 Consensus Document of the International Society of Lymphology: Lymphology 46:1, 2013. The complexities of lymphatic cannulation and the risk of lymphangitis associated with the contrast agent limit the utility of lymphangiography. A novel technique of optical imaging with a near-infrared fluorescence dye may enable quantitative imaging of lymph flow. Patients with lymphedema of the lower extremities must be instructed to take meticulous care of their feet to prevent recurrent lymphangitis. Skin hygiene is important, and emollients can be used to prevent drying. Prophylactic antibiotics are often helpful, and fungal infection should be treated aggressively. Patients should be encouraged to participate in physical activity; frequent leg elevation can reduce the amount of edema. Psychosocial support is indicated to assist patients cope with anxiety or depression related to body image, self-esteem, functional disability, and fear of limb loss.

1	Physical therapy, including massage to facilitate lymphatic drainage, may be helpful. The type of massage used in decongestive physiotherapy for lymphedema involves mild compression of the skin of the affected extremity to dilate the lymphatic channels and enhance lymphatic motility. Multilayered, compressive bandages are applied after each massage session to reduce recurrent edema. After optimal reduction in limb volume by decongestive physiotherapy, patients can be fitted with graduated compression hose. Occasionally, intermittent pneumatic compression devices can be applied at home to facilitate reduction of the edema. Diuretics are contraindicated and may cause depletion of intravascular volume and metabolic abnormalities.

1	Liposuction in conjunction with decongestive physiotherapy may be considered to treat lymphedema, particularly postmastectomy lymphedema. Other surgical interventions are rarely used and often not successful in ameliorating lymphedema. Microsurgical lymphaticovenous anastomotic procedures have been performed to rechannel lymph flow from obstructed lymphatic vessels into the venous system. Limb reduction procedures to resect subcutaneous tissue and excessive skin are performed occasionally in severe cases of lymphedema to improve mobility.

1	Therapeutic lymphangiogenesis has been studied in rodent models of lymphedema, but not as yet in humans. Overexpression of vascular endothelial growth factor (VEGF) C generates new lymphatic vessels and improves lymphedema in a murine model of primary lymphedema, and administration of recombinant VEGF-C or VEGF-D stimulated lymphatic growth in preclinical models of post-surgical lymphedema. Clinical trials in patients with lymphedema are required to determine efficacy of gene transfer (cell-based) therapies for lymphedema. Pulmonary Hypertension Aaron B. Waxman, Joseph Loscalzo Pulmonary hypertension (PH) is a spectrum of diseases involving the pulmonary vasculature, and is defined as an elevation in pulmonary arterial pressures (mean pulmonary artery pressure >22 mmHg). Pulmonary arterial hypertension (PAH) is a relatively rare form of 304

1	PH and is characterized by symptoms of dyspnea, chest pain, and syncope. If left untreated, the disease carries a high mortality rate, with the most common cause of death being decompensated right heart failure. There have been significant advances in this field in regard to understanding the pathogenesis, diagnosis, and classification of PAH. Despite these significant advances, there is still a substantial delay in diagnosis of up to 2 years. In many cases, patients whose primary complaint is dyspnea on exertion are frequently misdiagnosed with more common diseases such as asthma or chronic obstructive pulmonary 1655 disease. The availability of newer drugs has resulted in a radical change in the management of this disease with significant improvement in both quality of life and mortality. A delay in diagnosis results in an obvious delay in the initiation of appropriate treatment. Clinicians should be able to recognize the signs and symptoms of PH and to complete a systematic workup

1	A delay in diagnosis results in an obvious delay in the initiation of appropriate treatment. Clinicians should be able to recognize the signs and symptoms of PH and to complete a systematic workup in patients suspected of having it. In this way, early diagnosis, prompt treatment, and improved outcomes for patients become achievable.

1	Vasoconstriction, vascular proliferation, thrombosis, and inflammation appear to underlie the development of PAH (Fig. 304-1). In long-standing PH, intimal proliferation and fibrosis, medial hypertrophy, and in situ thrombosis characterize the pathologic findings in the pulmonary vasculature. Vascular remodeling at earlier stages may be confined to the small pulmonary arteries. As the disease advances, intimal proliferation and pathologic remodeling progress, resulting in decreased compliance and increased elastance of the pulmonary vas culature. The outcome is a progressive increase in the right ventricular afterload or total pulmonary vascular resistance (PVR) and, thus, right ventricular work. In subjects with moderate to severe pulmonary vascular disease with significantly increased PVR, as the resting PVR increases, there will be a corresponding increase in mean pulmonary artery pressure (PAP) until the cardiac output (CO) is compromised and starts to fall. With a decline in CO,

1	as the resting PVR increases, there will be a corresponding increase in mean pulmonary artery pressure (PAP) until the cardiac output (CO) is compromised and starts to fall. With a decline in CO, the PAP will fall. As CO declines as a result of increased afterload and decreased contractility, tachycardia is a compensatory response. Tachycardia decreases filling time and, thus, preload, and results in a reduced fraction of stroke volume available to distend the pulmonary vascular tree.

1	Abnormalities in multiple molecular pathways and genes that regulate the pulmonary vascular endothelial and smooth muscle cells have been identified (Table 304-1). These abnormalities include decreased expression of the voltage-regulated potassium channel, mutations in the bone morphogenetic protein receptor-2, increased tissue factor expression, overactivation of the serotonin transporter, hypoxia-induced activation of hypoxia-inducible factor-1α, and activation of nuclear factor of activated T cells. As a result, there is a decrease in apoptosis of the smooth muscle cells and the emergence of apoptosis-resistant endothelial cells that promote their accumulation and can obliterate the vascular lumen. In addition, thrombin deposition in the pulmonary vasculature from the prothrombotic state that develops as an independent abnormality or as a result of endothelial dysfunction may amplify vascular cell proliferation and the obliterative arteriopathy.

1	The diagnosis of PH can be missed without a reasonable index of suspicion. Dyspnea is the most common presenting symptom, but this complaint is far from specific for the diagnosis of PH. PH symptoms are insidious and overlap considerably with many common conditions, including asthma and other lung disease and cardiac disease. The symptoms of PH are often nonspecific and variable. Most patients will present with dyspnea and/or fatigue, whereas edema, chest pain, presyncope, and frank syncope are less common and associated with more advanced disease. On examination, there may be evidence of right ventricular failure with elevated jugular venous pressure, lower extremity edema, and ascites. Additionally, the cardiovascular examination may reveal an accentuated P2 component of the second heart sound, a right-sided S3 or S4, and a holosystolic tricuspid regurgitant murmur. It is also important to seek signs of the diseases that are often concurrent with PH: clubbing may be seen in some

1	sound, a right-sided S3 or S4, and a holosystolic tricuspid regurgitant murmur. It is also important to seek signs of the diseases that are often concurrent with PH: clubbing may be seen in some chronic lung diseases, sclerodactyly and telangiectasia may signify scleroderma, and crackles and systemic hypertension may be clues to left-sided systolic or diastolic heart failure.

1	Once clinical suspicion is raised, a systematic approach to diagnosis and assessment is essential. An echocardiogram with (if indicated) a bubble study is the most important screening test. Echocardiography is important for the diagnosis of PH and often essential for determining the cause. All forms of PH may demonstrate a hypertrophied and FIGURE 304-1 The left panels show examples of plexogenic pulmonary arteriopathy. These are obstructive and proliferative lesions of the small muscular pulmonary arteries, composed primarily of endothelial cells with intermixed inflammatory cells, myofibroblasts, and connective tissue components. The lower left panel demonstrates proliferating cells (red PCNA stained cells). Panels on the right demonstrate medial hypertrophy of muscular pulmonary arteries. (Photographs on the left are courtesy of Dr. Stephen Archer, Queen’s University School of Medicine, Kingston,

1	Ontario, Canada.) dilated right ventricle (Fig. 304-2) with elevated estimated pulmonary artery systolic pressure. Important additional information can be gleaned about specific etiologies of PH such as valvular disease, left ventricular systolic and diastolic function, intracardiac shunts, and other cardiac diseases.

1	Although the accuracy of Doppler echocardiography is often debated, a high-quality echocardiogram that is absolutely normal may obviate the need for further evaluation for PH. An echocardiogram is a screening test, whereas invasive hemodynamic monitoring is the gold standard for diagnosis and assessment of disease severity. With a normal echo-cardiogram, there may still be some concern for PH; this is particularly true if there is unexplained dyspnea or hypoxemia. In this setting, it is reasonable to proceed to right heart catheterization for definitive diagnosis. Alternatively, if the patient has a reasonable functional capacity, a cardiopulmonary exercise test may help to identify a true physiologic limitation as well as differentiate between cardiac and pulmonary causes of dyspnea. If this test is normal, there is no indication for a right heart catheterization. If a cardiovascular limitation to exercise is found, a right heart catheterization should be pursued.

1	If the echocardiogram or cardiopulmonary exercise test (CPET) suggests PH and the diagnosis is confirmed by catheterization, a reasonable effort must be made to establish the etiology because this will largely determine the therapeutic approach. A stepwise approach to evaluation is outlined below.

1	Chest imaging and lung function tests are essential because lung disease is an important cause of PH. A sign of PH that may be evident on chest x-ray include enlargement of the central pulmonary arteries associated with “vascular pruning,” a relative paucity of peripheral vessels (Fig. 304-3). Cardiomegaly, with specific evidence of right atrial and ventricular enlargement, can often be observed. The chest x-ray may also demonstrate significant interstitial lung disease or suggest hyperinflation from obstructive lung disease, which may be the underlying cause or contributor to the development of PH. High-resolution computed tomography (CT) may provide additional useful information. Classic findings of PH on CT include those found on chest x-ray: enlarged pulmonary arteries (Fig. 304-4), peripheral pruning of the small vessels, and enlarged right ventricle and atrium. However, high-resolution CT may also reveal signs of venous congestion including centrilobular ground-glass infiltrate

1	pruning of the small vessels, and enlarged right ventricle and atrium. However, high-resolution CT may also reveal signs of venous congestion including centrilobular ground-glass infiltrate and thickened septal lines. In the absence of left heart disease, these findings suggest pulmonary veno-occlusive disease, a rare cause of PAH that can be quite challenging to diagnose.

1	CT angiograms are commonly used to evaluate acute thromboembolic disease and have demonstrated excellent sensitivity and specificity for that purpose. Ventilation-perfusion (V/Q) scanning has traditionally been used for screening because of its high sensitivity and its role in qualifying patients for surgical intervention. The role of CT angiograms in the diagnosis of chronic thromboembolic pulmonary hypertension (CTEPH) remains controversial, even with the advent of spiral CT. Although a negative V/Q virtually rules out CTEPH, some cases may be missed through the use of CT angiograms. Pulmonary function tests are an important component of the evaluation. Although an isolated reduction in DLCO is the classic finding in PAH, results of pulmonary function tests may also suggest restrictive or obstructive lung diseases as the cause of dyspnea or PH. The 6-minute walk test Alterations in regulators of proliferation

1	Alterations in regulators of proliferation Abbreviations: PDGF, platelet-derived growth factor; EGF, epidermal-derived growth factor; FGF, fetal-derived growth factor; VEGF, vascular endothelial-derived growth factor; MCP-1, monocyte chemoattractant protein-1; IL, interleukin. FIGURE 304-2 A. Representative echocardiogram showing the apical four-chamber view from a patient with pulmonary hypertension demonstrating an enlarged right atrium and ventricle with some compression of the left side of the heart. B. Same echocardiographic view showing a normal echocardiogram. is also important to evaluate the degree of exertional hypoxemia and 1657 limitation and to monitor progression and response to therapy.

1	Sleep-disordered breathing is another important cause of PH, but a sleep study is generally necessary only when indicated by the patient’s history. Nocturnal desaturation is a common finding in PH, even in the absence of sleep-disordered breathing. Thus, all patients should undergo nocturnal oximetry screening, regardless of whether classic symptoms of obstructive sleep apnea or obesity-hypoventilation syndrome are observed. Laboratory tests that are important for screening include an HIV test when clinically indicated. In addition, all patients should have antinuclear antibodies, rheumatoid factor, and scl-70 antibodies assessed to screen for the most common rheumatologic diseases associated with PH if clinically indicated. Liver function and hepatitis serology tests are important to screen for underlying liver disease. Finally, there is an increasing role for brain natriuretic peptide (BNP) testing in the diagnosis and management of PH. BNP and the N-terminus of its propeptide

1	screen for underlying liver disease. Finally, there is an increasing role for brain natriuretic peptide (BNP) testing in the diagnosis and management of PH. BNP and the N-terminus of its propeptide (NT-proBNP) correlate with right ventricular function, hemodynamic severity, and functional status in PAH.

1	Right heart catheterization with pulmonary vasodilator testing remains the gold standard both to establish the diagnosis of PH and to enable selection of appropriate medical therapy. The definition of precapillary PH or PAH requires (1) an increased mean pulmonary artery pressure (mPAP ≥25 mmHg); (2) a pulmonary capillary wedge pressure (PCWP), left atrial pressure, or left ventricular end-diastolic pressure ≤15 mmHg; and (3) PVR >3 Wood units. Postcapillary PH is differentiated from precapillary PH by a PCWP of ≥15 mmHg; this is further differentiated into passive, based on a transpulmonary gradient <12 mmHg, or reactive, based on a transpulmonary gradient >12 mmHg and an increased PVR. In either case, the CO may be normal or reduced.

1	Vasodilators with a short duration of action, such as inhaled nitric oxide, inhaled epoprostenol, or intravenous adenosine, are preferred for vasodilator testing. A decrease in mPAP by ≥10 mmHg to an absolute level of ≤40 mmHg without a decrease in CO is defined as a positive pulmonary vasodilator response, and responders are considered for long-term treatment with calcium channel blockers (CCBs). Less than 12% of patients are deemed vasoreactive during testing, and even fewer exhibit long-term responsiveness to CCBs. Acute vasodilator-induced reductions in PVR and mPAP predict better long-term survival even among patients not treated with CCBs. The need for invasive hemodynamic measurements to diagnose PH accurately poses an additional problem when evaluating older patients. Physicians are often reluctant to refer older patients for invasive procedures. However, the diagnosis of PH is increasing in the older population, at least in part because of increased awareness of this disease

1	often reluctant to refer older patients for invasive procedures. However, the diagnosis of PH is increasing in the older population, at least in part because of increased awareness of this disease in the elderly and increased use of screening echocardiograms. Furthermore, the increased availability of oral and less complicated therapeutic options has encouraged the referral of older patients for evaluation and treatment.

1	FIGURE 304-3 Posteroanterior (left) and lateral (right) chest radiograph showing enlarged pulmonary arteries (black arrows) and pruning of the distal pulmonary vasculature (white arrow) commonly seen with advanced pulmonary arterial hypertension.

1	PAH is just one of a number of disease classifications that affect the pulmonary vascular bed. PH was previously classified as primary or secondary, but as understanding of the various contributing diseases has increased, classification systems have attempted to group these diseases by clinical features to aid in diagnosis. The World Health Organization (WHO) formulated a clinical classification of the various manifestations of PH, of which PAH is a subgroup, according to similarities in pathophysiologic mechanisms and clinical presentation. PH is a diverse mix of pathologies in which the only unifying theme is elevated PAP relative to left atrial pressure. The categorization of PH was designed by convenience for the purpose of facilitating novel treatments to be tested across different presentations and is not based on a molecular understanding of the pathology and is not a guide for management decisions.

1	FIGURE 304-4 Representative computed tomography scan of the chest demonstrating enlarged main pulmonary arteries. There is also a mosaic pattern evident in both lungs. The current classification system, last revised in 2013 during the Fifth World Symposium on Pulmonary Hypertension, recognizes five categories of PH, including PAH, PH due to left heart disease, PH due to chronic lung disease, PH associated with chronic thromboemboli, and a group of miscellaneous diseases that only rarely cause PH.

1	Pulmonary Arterial Hypertension WHO Group I PH, pulmonary arterial hypertension (PAH), is a relatively rare cause of PH. PAH includes a group of diseases that result in pulmonary arterial precapillary remodeling marked by intimal fibrosis, increased medial thickness, pulmonary arteriolar occlusion, and classic plexiform lesions. PAH is defined as a sustained elevation in resting mPAP ≥25�mmHg, PVR>240�dyne·s/cm5, and PCWP or left ventricle end-diastolic pressure of ≤15�mmHg based on a right heart catheterization. With a normal PCWP and an elevated mPAP, these diseases demonstrate an increased transpulmonary gradient (mPAP – PCWP); in addition, the PVR is elevated.

1	Idiopathic pulmonary arterial hypertension (IPAH) is a progressive disease that leads to right heart failure and death. It is typically seen in young women. The National Institutes of Health registry, the first large registry of patients with PAH, reported that the average age at diagnosis was 36 years, with only 9% of patients with IPAH over the age of 60 at diagnosis. However, the more current clinical data suggest that the patient demographics are changing. The Pulmonary Hypertension Connection registry found that the average age of diagnosis for IPAH was 45 years, with 8.5% of patients older than 70 years at diagnosis. This finding is supported by data from the Registry to Evaluate Early and Long-Term PAH Disease Management (REVEAL), the largest cohort of PAH to date, which reported that the average age at diagnosis of IPAH was 44.9±0.6 years.

1	Other forms of PAH that deserve specific consideration in patients are those associated with HIV, connective tissue disease, and portal hypertension. Although HIV is a rare cause of PAH, this form of PAH is indistinguishable from IPAH and is an important cause of mortality in the HIV-infected population. Importantly, there is no correlation between the stage of HIV infection and the development of PAH. Among connective tissue diseases, the prevalence of PAH has been established only for systemic sclerosis, especially in those with limited cutaneous scleroderma. Although the average age of scleroderma onset is 30 to 50 years old, patients who eventually develop sclerodermaassociated PAH tend to be older at the time of scleroderma diagnosis. Outcomes of scleroderma are closely linked to the development of PAH and are associated with a poor prognosis, although modern therapies have improved outcomes.

1	Portopulmonary hypertension occurs in 2–10% of patients with established portal hypertension. Its occurrence appears to be independent of the cause of liver disease and is observed in patients with nonhepatic causes of portal hypertension. A hyperdynamic circulatory state is common, as in most patients with advanced liver disease; however, the same pulmonary vascular remodeling observed in other forms of PAH is seen in the pulmonary vascular bed in portopulmonary hypertension. It is important to distinguish this process from hepatopulmonary syndrome, which can also manifest with dyspnea and hypoxemia but is pathophysiologically distinct from portopulmonary hypertension in that abnormal vasodilation of the pulmonary vasculature leads to intrapulmonary shunting.

1	Pulmonary Hypertension Associated with Left Heart Disease WHO Group II PH includes patients with left heart systolic failure, aortic and mitral valve disease, and heart failure with preserved ejection fraction (HFpEF). PH can develop as a result of all of these conditions. The hallmark of Group II PH (i.e., PH due to left heart disease) is elevated left atrial pressure with resulting pulmonary venous hypertension. In general, the transpulmonary gradient and PVR remain normal. Although this phenomenon is well described in both left-sided valvular disease and left-sided systolic heart failure, studies suggest that HFpEF may carry a higher overall risk of PH.

1	Whatever the cause of elevated left atrial pressure (i.e., systolic or diastolic heart failure or valvular disease), the increased pulmonary venous pressure indirectly leads to a rise in pulmonary arterial pressure. The presence of PH portends a poor prognosis in all forms of heart failure. In particular, chronic pulmonary venous hypertension may lead to a reactive pulmonary arterial vasculopathy, seen as an elevated transpulmonary gradient (>12 mmHg) and elevated PVR (>3 Wood units). Pathologically, this process is marked by pulmonary arteriolar remodeling with intimal fibrosis and medial hyperplasia akin to that seen in PAH.

1	Pulmonary Hypertension Associated with Lung Disease Intrinsic lung disease is the second most common cause of PH, although its actual prevalence is difficult to ascertain. PH has been observed in both chronic obstructive lung disease and interstitial lung disease. It can also be seen in diseases with mixed obstructive/restrictive physiology: bronchiectasis, cystic fibrosis, mixed obstructive restrictive disease marked by fibrosis in the lower lung zones, and emphysema predominantly in the upper lung zones. As in patients with left heart disease, PH associated with chronic lung disease is usually modest; however, some of these patients appear to have PH “out of proportion” to their parenchymal lung disease, suggesting intrinsic pulmonary arterial disease. These patients typically have more severe PH, with results of pulmonary function tests demonstrating a very low DLCO.

1	Although PH is described in most forms of interstitial lung disease, it has been most extensively studied in idiopathic pulmonary fibrosis; however, the individual studies have been small. Early echocardiographic data suggested that the prevalence of PH in interstitial lung diseases was high, but invasive hemodynamic monitoring suggests that the incidence is considerably lower than originally believed. The diagnosis of PH portends poor outcome in pulmonary fibrosis. Also included in Group III PH is sleep-disordered breathing. Sleep apnea has long been associated with PH. However, PH associated with sleep-disordered breathing is generally mild.

1	Also included in Group III PH is sleep-disordered breathing. Sleep apnea has long been associated with PH. However, PH associated with sleep-disordered breathing is generally mild. Pulmonary Hypertension Associated with Chronic Thromboembolic Disease The development of PH after chronic thromboembolic obstruction of the pulmonary arteries is well described, but its incidence is not known. The incidence of PH after a single pulmonary embolic event is thought to be quite low and likely increases following recurrent embolism. The risk factors for developing CTEPH are unclear. Many patients have no history of clinical venous thromboembolism. The pathogenesis of CTEPH is poorly understood. Obstruction of the proximal pulmonary vasculature is important and often the dominant factor; however, additional pulmonary vascular remodeling occurs. Approximately 10–15% of patients will develop a disease very similar clinically and pathologically to PAH after resection of the proximal thrombus.

1	OTHER DISORDERS AFFECTING THE PULMONARY VASCULATURE Sarcoidosis Patients with sarcoidosis can develop PH as a result of lung involvement. Consequently, patients with sarcoidosis who present with progressive dyspnea and PH require a thorough evaluation. Although the majority of sarcoidosis patients with PH generally do not respond to therapy for PAH, a subset of patients with sarcoidosis and severe PH do have a beneficial response to therapy. Sickle Cell Disease Cardiovascular system abnormalities are prominent in the clinical spectrum of sickle cell disease, including PH. The etiology is multifactorial, including hemolysis, hypoxemia, thromboembolism, chronic high CO, and chronic liver disease. The presence of PH in patients with sickle cell disease is rare.

1	Schistosomiasis Globally, schistosomiasis is one of the most com-1659 mon causes of PH. The development of PH occurs in the setting of hepatosplenic disease and portal hypertension. Studies suggest that inflammation from the infection triggers the pulmonary vascular changes that occur. The diagnosis is confirmed by finding the parasite ova in the urine or stool of patients with symptoms, which can be difficult. The efficacy of therapies directed toward PH in these patients is unknown.

1	PH was a consistently fatal condition with no effective medical treatment options before 1996; however, since that time, there has been an upsurge in the development of novel therapeutic agents for PAH. There are several approved agents for PAH, including prostacyclin and prostacyclin analogues, phosphodiesterase-5 inhibitors, a soluble guanylyl cyclase stimulator, and endothelin receptor antagonists, that have improved the outlook dramatically. Although there is no cure for PAH, current pharmacologic therapies improve morbidity and, in some cases, mortality.

1	In PAH, endothelial dysfunction and platelet activation cause an imbalance of arachidonic acid metabolites with reduced prostacyclin levels and increased thromboxane A2 production. Prostacyclin (PGI2) activates cyclic adenosine monophosphate (cAMP)-dependent pathways that mediate vasodilation. PGI2 also has antiproliferative effects on vascular smooth muscle and inhibits platelet aggregation. Protein levels of prostacyclin synthase are decreased in pulmonary arteries of patients with PAH. This imbalance of mediators is addressed by the exogenous administration of prostanoids as therapy in advanced PAH.

1	Epoprostenol was the first prostanoid available for the management of PAH. Epoprostenol delivered as a continuous intravenous infusion improves functional capacity and survival in PAH. The efficacy of epoprostenol in WHO functional class 3 and 4 PAH patients was demonstrated in a clinical trial that showed improved quality of life, mPAP, PVR, 6-minute walk distance (6MWD), and mortality. Treprostinil has a longer half-life than epoprostenol (~4 h vs ~6 min), which allows for continuous subcutaneous and intravenous administration. Treprostinil has been shown to improve pulmonary hemodynamics, symptoms, exercise capacity, and survival in PAH.

1	Inhaled prostacyclins provide the beneficial effects of infused prostacyclin therapy without the inconvenience and side effects (risk of infection and infusion site reactions) of infusion catheters. Both inhaled iloprost and treprostinil have been approved for patients with WHO class 3 and 4 PAH. The main advantage of treprostinil is less frequent administration. Inhaled formulations can be efficacious in moderately symptomatic patients with PAH and may be appropriate when used in combination with an oral medication. Phosphodiesterase-5 (PDE5) inhibitors (e.g., sildenafil) increase cyclic guanosine mono-phosphate (cGMP) levels and activate cGMP-dependent signaling pathways that also mediate vasodilation and platelet inhibition. Thus, the addition of a PDE5 inhibitor augments the pulmonary hemodynamic and functional capacity benefits of prostanoids in PAH.

1	Endothelin Receptor Antagonists Endothelin receptor antagonists (ERAs) target endothelin-1 (ET-1), a potent endogenous vasoconstrictor and vascular smooth muscle mitogen that is elevated in PAH patients. Endothelin levels are increased coincident with increased PVR and mPAP and decreased CO and 6MWD. ERAs block the binding of ET-1 to either endothelin receptor A (ET-A) and/or B (ET-B). ET-A receptors found on pulmonary artery smooth muscle cells mediate vasoconstriction. In the normal pulmonary vasculature, ET-B receptors are found on endothelial cells and mediate vasodilation via production of prostacyclin and nitric oxide as well as ET-1 clearance. Three ERAs approved for use in the United States are bosentan and macitentan both, nonselective receptor antagonists, and ambrisentan, a selective ET-A receptor antagonist. Studies have shown that both bosentan and macitentan improve hemodynamics and exercise capacity and delay clinical worsening.

1	Studies have shown that both bosentan and macitentan improve hemodynamics and exercise capacity and delay clinical worsening. Route of Generic Name Administration Drug Class Indication stimulator Abbreviations: FDA, U.S. Food and Drug Administration; NYHA, New York Heart Association; PAH, pulmonary arterial hypertension; PDES, phosphodiesterase-5. The randomized, placebo-controlled, phase III Bosentan Randomized Trial of Endothelin Antagonist Therapy (BREATHE)-1 comparing bosentan with placebo demonstrated�improved symptoms, 6MWD, and WHO functional class. The Endothelin Antagonist Trial in Mildly Symptomatic Pulmonary Arterial Hypertension Patients (EARLY) comparing bosentan with placebo demonstrated improved PVR and 6MWD.

1	Several studies, including the phase III, placebo-controlled Ambrisentan in Pulmonary Arterial Hypertension-1 (ARIES-1) trial, suggest that ambrisentan improves exercise tolerance, WHO functional class, hemodynamics, and quality of life in patients with PAH. There are no trial data to evaluate whether the selective ET-A receptor antagonism of ambrisentan has any advantage over the nonselective ET receptor antagonism of bosentan.

1	Phosphodiesterase Type-5 Inhibitors Nitric oxide derived from endothelial cells activates guanylyl cyclase, which, in turn, generates cGMP in vascular smooth muscle cells and platelets. cGMP is a second messenger that induces vasodilation through relaxation of the arterial smooth muscle cells and inhibits platelet activation. PDE5 enzymes metabolize cGMP. Therefore, cGMP PDE5 inhibitors prolong the vasodilatory effect of nitric oxide, especially within the pulmonary arterial bed where high concentrations of cGMP are found. There are currently two PDE5 inhibitors used for the treatment of PAH, sildenafil and tadalafil. Both agents have been shown to improve hemodynamics and 6MWD. Recently, the oral soluble guanylyl cyclase stimulator, riociguat, was approved for the treatment of both PAH and CTEPH.

1	Unmet and Future Research Needs in Pulmonary Hypertension Presently there are only three classes of therapy for patients with PAH, and even with therapy, the median survival for a person with PAH is only 5–6 years (Table 304-2). Although there are five subtypes of PH, current approved therapies only address one subtype. Not only do we need to expand the treatment options for patients with PAH, we also need to develop effective therapies for all patients with PH. Limited survival is, in part, a result of delay in diagnosis. Improved awareness among clinicians and patients could lead to more timely diagnosis that will affect the response to therapy and survival. PH needs to be diagnosed in a timely manner so that therapy can be initiated as soon as possible. Patients should also have the option of referral to a specialty center that focuses on treatment of patients with pulmonary vascular disease, which will ensure their access to state-of-the-art care and a multidisciplinary approach

1	of referral to a specialty center that focuses on treatment of patients with pulmonary vascular disease, which will ensure their access to state-of-the-art care and a multidisciplinary approach to care. Finally, there need to be continued efforts at developing new therapies that target the increasingly complex and overlapping pathways involved in the various forms of PH.

1	approach to the patient with 305 Disease of the respiratory System Patricia A. Kritek, Augustine M. K. Choi

1	Patricia A. Kritek, Augustine M. K. Choi The majority of diseases of the respiratory system fall into one of three major categories: (1) obstructive lung diseases; (2) restrictive disorders; and (3) abnormalities of the vasculature. Obstructive lung diseases are most common and primarily include disorders of the airways, such as asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, and bronchiolitis. Diseases resulting in restrictive pathophysiology include parenchymal lung diseases, abnormalities of the chest wall and pleura, and neuromuscular disease. Disorders of the pulmonary vasculature include pulmonary embolism, pulmonary hypertension, and pulmonary veno-occlusive disease. Although many specific diseases fall into these major categories, both infective and neoplastic processes can affect the respiratory system and result in myriad pathologic findings, including those listed in the three categories above (Table 305-1).

1	Disorders can also be grouped according to gas exchange abnormalities, including hypoxemic, hypercarbic, or combined impairment. However, many diseases of the lung do not manifest as gas exchange abnormalities. As with the evaluation of most patients, the approach to a patient with disease of the respiratory system begins with a thorough history and a focused physical examination. Many patients will subsequently undergo pulmonary function testing, chest imaging, blood and sputum analysis, a variety of serologic or microbiologic studies, and diagnostic procedures, such as bronchoscopy. This stepwise approach is discussed in detail below.

1	HISTORY Dyspnea and Cough The cardinal symptoms of respiratory disease are dyspnea and cough (Chaps. 47e and 48). Dyspnea has many causes, some of which are not predominantly due to lung pathology. The words a patient uses to describe shortness of breath can suggest certain etiologies for dyspnea. Patients with obstructive lung disease often complain of “chest tightness” or “inability to get a deep breath,” whereas patients with congestive heart failure more commonly report “air hunger” or a sense of suffocation.

1	The tempo of onset and the duration of a patient’s dyspnea are likewise helpful in determining the etiology. Acute shortness of breath is usually associated with sudden physiologic changes, such as laryngeal edema, bronchospasm, myocardial infarction, pulmonary embolism, or pneumothorax. Patients with COPD and idiopathic pulmonary fibrosis (IPF) experience a gradual progression of dyspnea on exertion, punctuated by acute exacerbations of shortness of breath. In contrast, most asthmatics have normal breathing the majority of the time with recurrent episodes of dyspnea that are usually associated with specific triggers, such as an upper respiratory tract infection or exposure to allergens.

1	Specific questioning should focus on factors that incite dyspnea as well as on any intervention that helps resolve the patient’s shortness of breath. Asthma is commonly exacerbated by specific triggers, although this can also be true of COPD. Many patients with lung disease report dyspnea on exertion. Determining the degree of activity that results in shortness of breath gives the clinician a gauge of the patient’s degree of disability. Many patients adapt their level of activity to accommodate progressive limitation. For this reason, it is important, particularly in older patients, to delineate the activities in which they engage and how these activities have changed over time. Dyspnea on exertion is often an early symptom of underlying lung or heart disease and warrants a thorough evaluation.

1	Cough generally indicates disease of the respiratory system. The clinician should inquire about the duration of the cough, whether or not it is associated with sputum production, and any specific triggers that induce it. Acute cough productive of phlegm is often a symptom of infection of the respiratory system, including processes affecting the upper airway (e.g., sinusitis, tracheitis), the lower airways (e.g., bronchitis, bronchiectasis), and the lung parenchyma (e.g., pneumonia). Both the quantity and quality of the sputum, including whether it is blood-streaked or frankly bloody, should be determined. Hemoptysis warrants an evaluation as delineated in Chap. 48.

1	Chronic cough (defined as that persisting for >8 weeks) is commonly associated with obstructive lung diseases, particularly asthma and chronic bronchitis, as well as “nonrespiratory” diseases, such as gastroesophageal reflux and postnasal drip. Diffuse parenchymal lung diseases, including IPF, frequently present as a persistent, nonproductive cough. As with dyspnea, all causes of cough are not respiratory in origin, and assessment should encompass a broad differential, including cardiac and gastrointestinal diseases as well as psychogenic causes.

1	Additional Symptoms Patients with respiratory disease may report wheezing, which is suggestive of airways disease, particularly asthma. Hemoptysis can be a symptom of a variety of lung diseases, including infections of the respiratory tract, bronchogenic carcinoma, and pulmonary embolism. In addition, chest pain or discomfort is often thought to be respiratory in origin. As the lung parenchyma is not innervated with pain fibers, pain in the chest from respiratory disorders usually results from either diseases of the parietal pleura (e.g., pneumothorax) or pulmonary vascular diseases (e.g., pulmonary hypertension). As many diseases of the lung can result in strain on Approach to the Patient with Disease of the Respiratory System 1662 the right side of the heart, patients may also present with symptoms of cor pulmonale, including abdominal bloating or distention and pedal edema (Chap. 279).

1	Additional History A thorough social history is an essential component of the evaluation of patients with respiratory disease. All patients should be asked about current or previous cigarette smoking, as this exposure is associated with many diseases of the respiratory system, most notably COPD and bronchogenic lung cancer but also a variety of diffuse parenchymal lung diseases (e.g., desquamative interstitial pneumonitis and pulmonary Langerhans cell histiocytosis). For most disorders, longer duration and greater intensity of exposure to cigarette smoke increases the risk of disease. There is growing evidence that “second-hand smoke” is also a risk factor for respiratory tract pathology; for this reason, patients should be asked about parents, spouses, or housemates who smoke. Possible inhalational exposures should be explored, including those at the work place (e.g., asbestos, wood smoke) and those associated with leisure (e.g., excrement from pet birds) (Chap. 311). Travel

1	inhalational exposures should be explored, including those at the work place (e.g., asbestos, wood smoke) and those associated with leisure (e.g., excrement from pet birds) (Chap. 311). Travel predisposes to certain infections of the respiratory tract, most notably the risk of tuberculosis. Potential exposure to fungi found in specific geographic regions or climates (e.g., Histoplasma capsulatum) should be explored.

1	Associated symptoms of fever and chills should raise the suspicion of infective etiologies, both pulmonary and systemic. A comprehensive review of systems may suggest rheumatologic or autoimmune disease presenting with respiratory tract manifestations. Questions should focus on joint pain or swelling, rashes, dry eyes, dry mouth, or constitutional symptoms. In addition, carcinomas from a variety of primary sources commonly metastasize to the lung and cause respiratory symptoms. Finally, therapy for other conditions, including both irradiation and medications, can result in diseases of the chest.

1	Physical Examination The clinician’s suspicion of respiratory disease often begins with a patient’s vital signs. The respiratory rate is often informative, whether elevated (tachypnea) or depressed (hypopnea). In addition, pulse oximetry should be measured, as many patients with respiratory disease have hypoxemia, either at rest or with exertion. The classic structure of the respiratory examination proceeds through inspection, percussion, palpation, and auscultation as described below. Often, however, auscultatory findings will lead the clinician to perform further percussion or palpation in order to clarify these findings.

1	The first step of the physical examination is inspection. Patients with respiratory disease may be in distress, often using accessory muscles of respiration to breathe. Severe kyphoscoliosis can result in restrictive pathophysiology. Inability to complete a sentence in conversation is generally a sign of severe impairment and should result in an expedited evaluation of the patient. Percussion of the chest is used to establish diaphragm excursion and lung size. In the setting of decreased breath sounds, percussion is used to distinguish between pleural effusions (dull to percussion) and pneumothorax (hyper-resonant note). The role of palpation is limited in the respiratory examination. Palpation can demonstrate subcutaneous air in the setting of barotrauma. It can also be used as an adjunctive assessment to determine whether an area of decreased breath sounds is due to consolidation (increased tactile fremitus) or a pleural effusion (decreased tactile fremitus).

1	The majority of the manifestations of respiratory disease present as abnormalities of auscultation. Wheezes are a manifestation of airway obstruction. While most commonly a sign of asthma, peribronchial edema in the setting of congestive heart failure can also result in diffuse wheezes, as can any other process that causes narrowing of small airways. For this reason, clinicians must take care not to attribute all wheezing to asthma. Rhonchi are a manifestation of obstruction of medium-sized airways, most often with secretions. In the acute setting, this manifestation may be a sign of viral or bacterial bronchitis. Chronic rhonchi suggest bronchiectasis or COPD. Stridor, a high-pitched, focal inspiratory wheeze, usually heard over the neck, is a manifestation of upper airway obstruction and should prompt expedited evaluation of the patient, as it can precede complete upper airway obstruction and respiratory failure.

1	Crackles, or rales, are commonly a sign of alveolar disease. A variety of processes that fill the alveoli with fluid may result in crackles. Pneumonia can cause focal crackles. Pulmonary edema is associated with crackles, generally more prominent at the bases. Interestingly, diseases that result in fibrosis of the interstitium (e.g., IPF) also result in crackles often sounding like Velcro being ripped apart. Although some clinicians make a distinction between “wet” and “dry” crackles, this distinction has not been shown to be a reliable way to differentiate among etiologies of respiratory disease.

1	One way to help distinguish between crackles associated with alveolar fluid and those associated with interstitial fibrosis is to assess for egophony. Egophony is the auscultation of the sound “AH” instead of “EEE” when a patient phonates “EEE.” This change in note is due to abnormal sound transmission through consolidated parenchyma and is present in pneumonia but not in IPF. Similarly, areas of alveolar filling have increased whispered pectoriloquy as well as transmission of larger-airway sounds (i.e., bronchial breath sounds in a lung zone where vesicular breath sounds are expected). The lack or diminution of breath sounds can also help determine the etiology of respiratory disease. Patients with emphysema often have a quiet chest with diffusely decreased breath sounds. A pneumothorax or pleural effusion may present with an area of absent breath sounds.

1	Other Systems Pedal edema, if symmetric, may suggest cor pulmonale; if asymmetric, it may be due to deep venous thrombosis and associated pulmonary embolism. Jugular venous distention may also be a sign of volume overload associated with right heart failure. Pulsus paradoxus is an ominous sign in a patient with obstructive lung disease, as it is associated with significant negative intrathoracic (pleural) pressures required for ventilation and impending respiratory failure. As stated earlier, rheumatologic disease may manifest primarily as lung disease. Owing to this association, particular attention should be paid to joint and skin examination. Clubbing can be found in many lung diseases, including cystic fibrosis, IPF, and lung cancer. Cyanosis is seen in hypoxemic respiratory disorders that result in >5 g of deoxygenated hemoglobin/dL.

1	The sequence of studies is dictated by the clinician’s differential diagnosis, as determined by the history and physical examination. Acute respiratory symptoms are often evaluated with multiple tests performed at the same time in order to diagnose any life-threatening diseases rapidly (e.g., pulmonary embolism or multilobar pneumonia). In contrast, chronic dyspnea and cough can be evaluated in a more protracted, stepwise fashion.

1	Pulmonary Function Testing (See also Chap. 307) The initial pulmonary function test obtained is spirometry. This study is an effort-dependent test used to assess for obstructive pathophysiology as seen in asthma, COPD, and bronchiectasis. A diminished-forced expiratory volume in 1 sec (FEV1)/forced vital capacity (FVC) (often defined as <70% of the predicted value) is diagnostic of obstruction. In addition to measuring FEV1 and FVC, the clinician should examine the flow-volume loop (which is effort-independent). A plateau of the inspiratory and expiratory curves suggests large-airway obstruction in extrathoracic and intrathoracic locations, respectively.

1	Spirometry with symmetric decreases in FEV1 and FVC warrants further testing, including measurement of lung volumes and the diffusion capacity of the lung for carbon monoxide (DLCO). A total lung capacity <80% of the predicted value for a patient’s age, race, sex, and height defines restrictive pathophysiology. Restriction can result from parenchymal disease, neuromuscular weakness, or chest wall or pleural diseases. Restriction with impaired gas exchange, as indicated by a decreased DLCO, suggests parenchymal lung disease. Additional testing, such as measurements of maximal expiratory pressure and maximal inspiratory pressure, can help diagnose neuromuscular weakness. Normal spirometry, normal lung volumes, and a low DLCO should prompt further evaluation for pulmonary vascular disease.

1	Arterial blood gas testing is often helpful in assessing respiratory 1663 disease. Hypoxemia, while usually apparent with pulse oximetry, can be further evaluated with the measurement of arterial PO2 and the calculation of an alveolar gas and arterial blood oxygen tension difference ([A–a]DO2). Patients with diseases that cause ventilation-perfusion mismatch or shunt physiology have an increased (A–a) DO2 at rest. Arterial blood gas testing also allows the measurement of arterial PCO2. Hypercarbia can accompany severe airway obstruction (e.g., COPD) or progressive restrictive physiology, as in patients with neuromuscular weakness.

1	Chest Imaging (See Chap. 308e) Most patients with disease of the respiratory system undergo imaging of the chest as part of the initial evaluation. Clinicians should generally begin with a plain chest radio-graph, preferably posterior-anterior and lateral films. Several findings, including opacities of the parenchyma, blunting of the costophrenic angles, mass lesions, and volume loss, can be very helpful in determining an etiology. However, many diseases of the respiratory system, particularly those of the airways and pulmonary vasculature, are asso ciated with a normal chest radiograph. CT of the chest is often performed subsequently and allows better delineation of parenchymal processes, pleural disease, masses or nodules, and large airways. If the test includes administration of contrast, the pulmonary vasculature can be assessed with particular utility for determination of pulmonary emboli. Intravenous contrast also allows lymph nodes to be delineated in greater detail.

1	Depending on the clinician’s suspicion, a variety of other studies may be done. Concern about large-airway lesions may warrant bronchoscopy. This procedure may also be used to sample the alveolar space with bronchoalveolar lavage or to obtain nonsurgical lung biopsies. Blood testing may include assessment for hypercoagulable states in the setting of pulmonary vascular disease, serologic testing for infectious or rheumatologic disease, or assessment of inflammatory markers or leukocyte counts (e.g., eosinophils). Sputum evaluation for malignant cells or microorganisms may be appropriate. An echocardiogram to assess rightand left-sided heart function is often obtained. Finally, at times, a surgical lung biopsy is needed to diagnose certain diseases of the respiratory system. All of these studies will be guided by the preceding history, physical examination, pulmonary function testing, and chest imaging. Approach to the Patient with Disease of the Respiratory System

1	Disturbances of Respiratory Function Edward T. Naureckas, Julian Solway The primary functions of the respiratory system—to oxygenate blood and eliminate carbon dioxide—require virtual contact between blood 306e and fresh air, which facilitates diffusion of respiratory gases between blood and gas. This process occurs in the lung alveoli, where blood flowing through alveolar wall capillaries is separated from alveolar gas by an extremely thin membrane of flattened endothelial and epithelial cells, across which respiratory gases diffuse and equilibrate. Blood flow through the lung is unidirectional via a continuous vascular path, along which venous blood absorbs oxygen from and loses CO2 to inspired gas. The path for airflow, in contrast, reaches a dead end at the alveolar walls; thus the alveolar space must be ventilated tidally, with inflow of fresh gas and outflow of alveolar gas alternating periodically at the respiratory rate (RR). To provide an enormous alveolar surface area

1	the alveolar space must be ventilated tidally, with inflow of fresh gas and outflow of alveolar gas alternating periodically at the respiratory rate (RR). To provide an enormous alveolar surface area (typically 70 m2) for blood-gas diffusion within the modest volume of a thoracic cavity (typically 7 L), nature has distributed both blood flow and ventilation among millions of tiny alveoli through multigenerational branching of both pulmonary arteries and bronchial airways. As a consequence of variations in tube lengths and calibers along these pathways as well as the effects of gravity, tidal pressure fluctuations, and anatomic constraints from the chest wall, the alveoli vary in their relative ventilations and perfusions. Not surprisingly, for the lung to be most efficient in exchanging gas, the fresh gas ventilation of a given alveolus must be matched to its perfusion.

1	For the respiratory system to succeed in oxygenating blood and eliminating CO2, it must be able to ventilate the lung tidally and thus to freshen alveolar gas; it must provide for perfusion of the individual alveolus in a manner proportional to its ventilation; and it must allow adequate diffusion of respiratory gases between alveolar gas and capillary blood. Furthermore, it must accommodate severalfold increases in the demand for oxygen uptake or CO2 elimination imposed by metabolic needs or acid-base derangement. Given these multiple requirements for normal operation, it is not surprising that many diseases disturb respiratory function. This chapter considers in some detail the physiologic determinants of lung ventilation and perfusion, elucidates how the matching distributions of these processes and rapid gas diffusion allow normal gas exchange, and discusses how common diseases derange these normal functions, thereby impairing gas exchange—or at least increasing the work required

1	processes and rapid gas diffusion allow normal gas exchange, and discusses how common diseases derange these normal functions, thereby impairing gas exchange—or at least increasing the work required by the respiratory muscles or heart to maintain adequate respiratory function.

1	It is useful to think about the respiratory system as three independently functioning components: the lung, including its airways; the neuromuscular system; and the chest wall, which includes everything that is not lung or active neuromuscular system. Accordingly, the mass of the respiratory muscles is part of the chest wall, while the force these muscles generate is part of the neuromuscular system; the abdomen (especially an obese abdomen) and the heart (especially an enlarged heart) are, for these purposes, part of the chest wall. Each of these three components has mechanical properties that FIguRE 306e-1 Pressure-volume curves of the isolated lung, isolated chest wall, combined respiratory system, inspiratory muscles, and expiratory muscles. FRC, functional residual capacity; RV, residual volume; TLC, total lung capacity.

1	relate to its enclosed volume (or—in the case of the neuromuscular 306e-1 system—the respiratory system volume at which it is operating) and to the rate of change of its volume (i.e., flow).

1	Volume-Related Mechanical Properties—Statics Figure 306e-1 shows the volume-related properties of each component of the respiratory system. Due both to surface tension at the air-liquid interface between alveolar wall lining fluid and alveolar gas and to elastic recoil of the lung tissue itself, the lung requires a positive transmural pressure difference between alveolar gas and its pleural surface to stay inflated; this difference is called the elastic recoil pressure of the lung, and it increases with lung volume. The lung becomes rather stiff at high volumes, so that relatively small volume changes are accompanied by large changes in transpulmonary pressure; in contrast, the lung is compliant at lower volumes, including those at which tidal breathing normally occurs. At zero inflation pressure, even normal lungs retain some air in the alveoli because the small peripheral airways are tethered open by radially outward pull from inflated lung parenchyma attached to adventitia; as the

1	pressure, even normal lungs retain some air in the alveoli because the small peripheral airways are tethered open by radially outward pull from inflated lung parenchyma attached to adventitia; as the lung deflates during exhalation, those small airways are pulled open progressively less, and eventually they close, trapping some gas in the alveoli. This effect can be exaggerated with age and especially with obstructive airway diseases, resulting in gas trapping at quite large lung volumes.

1	The elastic behavior of the passive chest wall (i.e., in the absence of neuromuscular activation) differs markedly from that of the lung. Whereas the lung tends toward full deflation with no distending (transmural) pressure, the chest wall encloses a large volume when pleural pressure equals body surface (atmospheric) pressure. Furthermore, the chest wall is compliant at high enclosed volumes, readily expanding even further in response to increases in transmural pressure. The chest wall also remains compliant at small negative transmural pressures (i.e., when pleural pressure falls slightly below atmospheric pressure), but as the volume enclosed by the chest wall becomes quite small in response to large negative transmural pressures, the passive chest wall becomes stiff due to squeezing together of ribs and intercostal muscles, diaphragm stretch, displacement of abdominal contents, and straining of ligaments and bony articulations. Under normal circumstances, the lung and the passive

1	of ribs and intercostal muscles, diaphragm stretch, displacement of abdominal contents, and straining of ligaments and bony articulations. Under normal circumstances, the lung and the passive chest wall enclose essentially the same volume, the only difference being the volumes of the pleural fluid and of the lung parenchyma (both quite small). For this reason and because the lung and chest wall function in mechanical series, the pressure required to displace the passive respiratory system (lungs plus chest wall) at any volume is simply the sum of the elastic recoil pressure of the lungs and the transmural pressure across the chest wall. When plotted against respiratory system volume, this relationship assumes a sigmoid shape, exhibiting stiffness at high lung volumes (imparted by the lung), stiffness at low lung volumes (imparted by the chest wall or sometimes by airway closure), and compliance in the middle range of lung volumes.

1	CHAPTER 306e Disturbances of Respiratory Function FIguRE 306e-2 Spirogram demonstrating a slow vital capacity maneuver and various lung volumes. In addition, a passive resting point of the respiratory system is attained when alveolar gas pressure equals body surface pressure (i.e., when the transrespiratory system pressure is zero). At this volume (called the functional residual capacity [FRC]), the outward recoil of the chest wall is balanced exactly by the inward recoil of the lung. As these recoils are transmitted through the pleural fluid, the lung is pulled both outward and inward simultaneously at FRC, and thus its pressure falls below atmospheric pressure (typically, −5 cmH2O).

1	The normal passive respiratory system would equilibrate at the FRC and remain there were it not for the actions of respiratory muscles. The inspiratory muscles act on the chest wall to generate the equivalent of positive pressure across the lungs and passive chest wall, while the expiratory muscles generate the equivalent of negative transrespiratory pressure. The maximal pressures these sets of muscles can generate vary with the lung volume at which they operate. This variation is due to length-tension relationships in striated muscle sarcomeres and to changes in mechanical advantage as the angles of insertion change with lung volume (Fig. 306e-1). Nonetheless, under normal conditions, the respiratory muscles are substantially “overpowered” for their roles and generate more than adequate force to drive the respiratory system to its stiffness extremes, as determined by the lung (total lung capacity [TLC]) or by chest wall or airway closure (residual volume [RV]); the airway closure

1	force to drive the respiratory system to its stiffness extremes, as determined by the lung (total lung capacity [TLC]) or by chest wall or airway closure (residual volume [RV]); the airway closure always prevents the adult lung from emptying completely under normal circumstances. The excursion between full and minimal lung inflation is called vital capacity (VC; Fig. 306e-2) and is readily seen to be the difference between volumes at two unrelated stiffness extremes—one determined by the lung (TLC) and the other by the chest wall or airways (RV). Thus, although VC is easy to measure (see below), it provides little information about the intrinsic properties of the respiratory system. As will become clear, it is much more useful for the clinician to consider TLC and RV individually.

1	Flow-Related Mechanical Properties—Dynamics The passive chest wall and active neuromuscular system do exhibit mechanical behaviors related to the rate of change of volume, but these behaviors become quantitatively important only at markedly supraphysiologic breathing frequencies (e.g., during high-frequency mechanical ventilation) and thus will not be addressed here. In contrast, the dynamic airflow properties of the lung substantially affect its ability to ventilate and contribute importantly to the work of breathing, and these properties are often deranged by disease. Understanding dynamic airflow properties is therefore worthwhile.

1	As with the flow of any fluid (gas or liquid) in any tube, maintenance of airflow within the pulmonary airways requires a pressure gradient that falls along the direction of flow, the magnitude of which is determined by the flow rate and the frictional resistance to flow. During quiet tidal breathing, the pressure gradients driving inspiratory or expiratory flow are small owing to the very low frictional resistance of normal pulmonary airways (Raw, normally <2 cmH2O/L per second). However, during rapid exhalation, another phenomenon reduces flow below that which would have been expected if frictional resistance were the only impediment to flow. This phenomenon is called dynamic FIguRE 306e-3 Luminal area versus transmural pressure relationship. Transmural pressure represents the pressure difference across the airway wall from inside to outside.

1	airflow limitation, and it occurs because the bronchial airways through which air is exhaled are collapsible rather than rigid (Fig. 306e-3). An important anatomic feature of the pulmonary airways is its treelike branching structure. While the individual airways in each successive generation, from most proximal (trachea) to most distal (respiratory bronchioles), are smaller than those of the parent generation, their number increases exponentially such that the summed cross-sectional area of the airways becomes very large toward the lung periphery. Because flow (volume/time) is constant along the airway tree, the velocity of airflow (flow/summed cross-sectional area) is much greater in the central airways than in the peripheral airways. During exhalation, gas leaving the alveoli must therefore gain velocity as it proceeds toward the mouth. The energy required for this “convective” acceleration is drawn from the component of gas energy manifested as its local pressure, which reduces

1	gain velocity as it proceeds toward the mouth. The energy required for this “convective” acceleration is drawn from the component of gas energy manifested as its local pressure, which reduces intraluminal gas pressure, airway transmural pressure, airway size (Fig. 306e-3), and flow. This is the Bernoulli effect, the same effect that keeps an airplane airborne, generating a lifting force by decreasing pressure above the curved upper surface of the wing due to acceleration of air flowing over the wing. If an individual tries to exhale more forcefully, the local velocity increases further and reduces airway size further, resulting in no net increase in flow. Under these circumstances, flow has reached its maximum possible value, or its flow limit. Lungs normally exhibit such dynamic airflow limitation. This limitation can be assessed by spirometry, in which an individual inhales fully to TLC and then forcibly exhales to RV. One useful spirometric measure is the volume of air exhaled

1	limitation. This limitation can be assessed by spirometry, in which an individual inhales fully to TLC and then forcibly exhales to RV. One useful spirometric measure is the volume of air exhaled during the first second of expiration (FEV1), as discussed later. Maximal expiratory flow at any lung volume is determined by gas density, airway cross-section and distensibility, elastic recoil pressure of the lung, and frictional pressure loss to the flow-limiting airway site. Under normal conditions, maximal expiratory flow falls with lung volume (Fig. 306e-4), primarily because of the dependence of lung recoil pressure on lung volume (Fig. 306e-1). In pulmonary fibrosis, lung recoil pressure is increased at any lung volume, and thus the maximal expiratory flow is elevated when considered in relation to lung volume. Conversely, in emphysema, lung recoil pressure is reduced; this reduction is a principal mechanism by which maximal expiratory flows fall. Diseases that narrow the airway lumen

1	to lung volume. Conversely, in emphysema, lung recoil pressure is reduced; this reduction is a principal mechanism by which maximal expiratory flows fall. Diseases that narrow the airway lumen at any transmural pressure (e.g., asthma or chronic bronchitis) or that cause excessive airway collapsibility (e.g., tracheomalacia) also reduce maximal expiratory flow.

1	The Bernoulli effect also applies during inspiration, but the more negative pleural pressures during inspiration lower the pressure outside of airways, thereby increasing transmural pressure and promoting airway expansion. Thus inspiratory airflow limitation seldom occurs due to diffuse pulmonary airway disease. Conversely, extrathoracic airway narrowing (e.g., due to a tracheal adenoma or post-tracheostomy stricture) can lead to inspiratory airflow limitation (Fig. 306e-4). The Work of Breathing In health, the elastic (volume change–related) and dynamic (flow-related) loads that must be overcome to ventilate the lungs at rest are small, and the work required of the respiratory muscles is minimal. However, the work of breathing can

1	A BC This volume is called the anatomic 306e-3 dead space (VD). Quiet breathing with tidal volumes smaller than the anatomic dead space introduces no fresh gas into the alveoli at all; only that part of the inspired tidal volume (VT) that is greater than the VD introduces fresh gas into the alveoli. The dead space can be further increased functionally if some of the inspired tidal volume is delivered to a part of the lung that receives no pulmonary blood flow and ˜ Increasing volume thus cannot contribute to gas exchange (e.g., the portion of the lung distal to a FIguRE 306e-4 Flow-volume loops. A. Normal. B. Airflow obstruction. C. Fixed central airway large pulmonary embolus). In this situ- obstruction. RV, residual volume; TLC, total lung capacity. E˙V(exhaled minute ventilationation,

1	VT × RR) includes a component of dead increase considerably due to a metabolic requirement for substantially increased ventilation, an abnormally increased mechanical load, or both. As discussed below, the rate of ventilation is primarily set by the need to eliminate carbon dioxide, and thus ventilation increases during exercise (sometimes by more than twentyfold) and during metabolic acidosis as a compensatory response. Naturally, the work rate required to overcome the elasticity of the respiratory system increases with both the depth and the frequency of tidal breaths, while the work required to overcome the dynamic load increases with total ventilation. A modest increase of ventilation is most efficiently achieved by increasing tidal volume but not respiratory rate, which is the normal ventilatory response to lower-level exercise. At high levels of exercise, deep breathing persists, but respiratory rate also increases. The pattern chosen by the respiratory controller minimizes the

1	ventilatory response to lower-level exercise. At high levels of exercise, deep breathing persists, but respiratory rate also increases. The pattern chosen by the respiratory controller minimizes the work of breathing.

1	The work of breathing also increases when disease reduces the compliance of the respiratory system or increases the resistance to airflow. The former occurs commonly in diseases of the lung parenchyma (interstitial processes or fibrosis, alveolar filling diseases such ˙VA ( a component of fresh gas alveolar ventilation

1	CO2 elimination from the alveoli is equal to ˙VA times the difference in CO2 fraction between inspired air (essentially zero) and alveolar gas (typically ~5.6% after correction for humidification of inspired air, corresponding to 40 mmHg). In the steady state, the alveolar fraction of CO2 is equal to metabolic CO2 production divided by alveolar ventilation. Because, as discussed below, alveolar and arterial CO2 tensions are equal, and because the respiratory controller normally strives to maintain arterial PCO (PaCO2) at ~40 mmHg, the adequacy of alveolar ventilation is reflected in PaCO2. If the PaCO2 falls much below 40 mmHg, alveolar hyperventilation is present; if the PaCO2 exceeds 40 mmHg, then alveolar hypoventilation is present. Ventilatory failure is characterized by extreme alveolar hypoventilation.

1	As a consequence of oxygen uptake of alveolar gas into capillary blood, alveolar oxygen tension falls below that of inspired gas. The rate of oxygen uptake (determined by the body’s metabolic oxygen consumption) is related to the average rate of metabolic CO2 production, = [VT – VD] × RR). ˙VO2 / and their ratio—the “respiratory quotient” (R = ˙ largely on the fuel being metabolized. For a typical American diet, )—depends

1	CHAPTER 306e Disturbances of Respiratory Function as pulmonary edema or pneumonia, or substantial lung resection), and the latter occurs in obstructive airway diseases such as asthma, chronic bronchitis, emphysema, and cystic fibrosis. Furthermore, severe airflow obstruction can functionally reduce the compliance of the respiratory system by leading to dynamic hyperinflation. In this scenario, expiratory flows slowed by the obstructive airways disease may be insufficient to allow complete exhalation during the expiratory phase of tidal breathing; as a result, the “functional residual capacity” from which the next breath is inhaled is greater than the static FRC. With repetition of incomplete exhalations of each tidal breath, the operating FRC becomes dynamically elevated, sometimes to a level that approaches TLC. At these high lung volumes, the respiratory system is much less compliant than at normal breathing volumes, and thus the elastic work of each tidal breath is also increased.

1	that approaches TLC. At these high lung volumes, the respiratory system is much less compliant than at normal breathing volumes, and thus the elastic work of each tidal breath is also increased. The dynamic pulmonary hyperinflation that accompanies severe airflow obstruction causes patients to sense difficulty in inhaling—even though the root cause of this pathophysiologic abnormality is expiratory airflow obstruction.

1	Adequacy of Ventilation As noted above, the respiratory control system that sets the rate of ventilation responds to chemical signals, including arterial CO2 and oxygen tensions and blood pH, and to volitional needs, such as the need to inhale deeply before playing a long phrase on the trumpet. Disturbances in ventilation are discussed in Chap. 318. The focus of this chapter is on the relationship between ventilation of the lung and CO2 elimination. At the end of each tidal exhalation, the conducting airways are filled with alveolar gas that had not reached the mouth when expiratory flow stopped. During the ensuing inhalation, fresh gas immediately enters the airway tree at the mouth, but the gas first entering the alveoli at the start of inhalation is that same alveolar gas in the conducting airways that had just left the alveoli. Accordingly, fresh gas does not enter the alveoli until the volume of the conducting airways has been inspired.

1	R is usually around 0.85, and more oxygen is absorbed than CO2 is excreted. Together, these phenomena allow the estimation of alveolar oxygen tension, according to the following relationship, known as the alveolar gas equation: The alveolar gas equation also highlights the influences of inspired oxygen fraction (Fi ), barometric pressure (P ), and vapor pressure of water (PH2O = 47 mmHg at 37°C) in addition to alveolar ventilation (which sets Pa ) in determining Pa . An implication of the alveolar gas equation is that severe arterial hypoxemia rarely occurs as a pure consequence of alveolar hypoventilation at sea level while an individual is breathing air. The potential for alveolar hypoventilation to induce severe hypoxemia with otherwise normal lungs increases as falls with increasing altitude.

1	gAS EXCHANgE Diffusion For oxygen to be delivered to the peripheral tissues, it must pass from alveolar gas into alveolar capillary blood by diffusing through alveolar membrane. The aggregate alveolar membrane is highly optimized for this process, with a very large surface area and minimal thickness. Diffusion through the alveolar membrane is so efficient in the human lung that in most circumstances a red blood cell’s hemoglobin becomes fully oxygen saturated by the time the cell has traveled just one-third the length of the alveolar capillary. Thus the uptake of alveolar oxygen is ordinarily limited by the amount of blood transiting the alveolar capillaries rather than by the rapidity with which oxygen can diffuse across the membrane; consequently, oxygen uptake from the lung is said to be “perfusion limited.” CO2 also equilibrates rapidly across the alveolar membrane. Therefore, the oxygen and CO2 tensions in capillary blood leaving a normal alveolus are essentially equal to those

1	be “perfusion limited.” CO2 also equilibrates rapidly across the alveolar membrane. Therefore, the oxygen and CO2 tensions in capillary blood leaving a normal alveolus are essentially equal to those in alveolar gas. Only in rare circumstances (e.g., at high altitude or in high-performance athletes exerting maximal effort) is exiting ventilated units increases only slightly, as hemoglobin will oxygen uptake from normal lungs diffusion limited. Diffusion limita-already have been nearly fully saturated and the solubility of oxygen in tion can also occur in interstitial lung disease if substantially thickened plasma is quite small. alveolar walls remain perfused. A more common occurrence than the two extreme examples given above is a widening of the distribution of ventilation/perfusion ratios;

1	Ventilation/Perfusion Heterogeneity As noted above, for gas exchange to be most efficient, ventilation to each individual alveolus (among the millions of alveoli) should match perfusion to its accompanying capil lung disease.ofconsequencea commonisQ˙heterogeneity /˙Vsuch In this circumstance, perfusion of relatively underventilated alveoli laries. Because of the differential effects of gravity on lung mechanics and blood flow throughout the lung and because of differences in airway and vascular architecture among various respiratory paths, results in the incomplete oxygenation of exiting blood. When mixed Q˙regions, this partially /with well-oxygenated blood leaving higher ˙V reoxygenated blood disproportionately lowers arterial PaO2, although to a lesser extent than does a similar perfusion fraction of bloodthere is minor ventilation/perfusion heterogeneity even in the nor-

1	Q˙heterogeneity can be particularly marked in /mal lung; however, ˙V disease. Two extreme examples are (1) ventilation of unperfused lung leaving regions of pure shunt. In addition, in contrast to shunt regions, inhalation of supplemental oxygen does raise the PAO2, even in rela Q˙regions, and so the arterial hypoxemia /tively underventilated low ˙V distal to a pulmonary embolus, in which ventilation of the physiologic dead space is “wasted” in the sense that it does not contribute to gas exchange; and (2) perfusion of nonventilated lung (a “shunt”), which allows venous blood to pass through the lung unaltered. When mixed with fully oxygenated blood leaving other well-ventilated lung units, shunted venous blood disproportionately lowers the mixed arterial PaO2 as a result of the nonlinear oxygen content versus PO2 relationship of hemoglobin (Fig. 306e-5). Furthermore, the resulting arterial Q˙heterogeneity is typically responsive to oxygen therapy /induced by ˙V (Fig. 306e-5).

1	Q˙heterogeneity is typically responsive to oxygen therapy /induced by ˙V (Fig. 306e-5). In sum, arterial hypoxemia can be caused by substantial reduc tion of inspired oxygen tension; by severe alveolar hypoventilation; by perfusion of relatively underventilated (low ˙ unventilated (shunt) lung regions; and, in unusual circumstances, by limitation of gas diffusion. hypoxemia is refractory to supplemental inspired oxygen. The reason is that (1) raising the inspired FiO2 has no effect on alveolar gas ten-PATHOPHYSIOLOgY sions in nonventilated alveoli and (2) while raising inspired FiO2 does Although many diseases injure the respiratory system, this system increase PaCO2 in ventilated alveoli, the oxygen content of blood responds to injury in relatively few ways. For this reason, the pattern of

1	FIO2 = 0.21 FIO2 = 1 99 mmHg 40 mmHg40 mmHg (75%) 40 mmHg (75%) 55 mmHg (87.5%) 40 mmHg (75%) 99 mmHg (100%) 650 mmHg 40 mmHg 40 mmHg (75%) 40 mmHg (75%) 56 mmHg (88%) 40 mmHg (75%) 650 mmHg (100%) 99 mmHg 40 mmHg40 mmHg (75%) 45 mmHg (79%) 40 mmHg (75%) 99 mmHg (100%) FIO2 = 0.21 650 mmHg 200 mmHg40 mmHg (75%) 200 mmHg (100%) 40 mmHg (75%) 650 mmHg (100%) FIO2 = 1V/QHeterogeneity.. 58 mmHg 350 mmHg (89.5%) (100%) FIguRE 306e-5 Influence of air versus oxygen breathing on mixed arterial oxygenation in shunt and ventilation/perfusion heterogeneity. Partial pressure of oxygen (mmHg) and oxygen saturations are shown for mixed venous blood, for end capillary blood (normal versus affected alveoli), and for mixed arterial blood. FIO2, fraction of inspired oxygen; ˙V/Q˙ , ventilation/perfusion.

1	CHAPTER 306e Flow Volume Flow Volume Flow Volume Flow Volume Flow Volume FIguRE 306e-6 Common abnormalities of pulmonary function (see text). Pulmonary function values are expressed as a percentage of normal predicted values, except for Raw, which is expressed as cmH2O/L per sec (normal, <2 cmH2O/L per second). The figures at the bottom of each column show the typical configuration of flow-volume loops in each condition, including the flow-volume relationship during tidal breathing. b.d., bronchodilator; DL co, diffusion capacity of lung for carbon monoxide; FEV1, forced expiratory volume in 1 sec; FRC, functional residual capacity; FVC, forced vital capacity; Raw, airways resistance; RV, residual volume; TLC, total lung capacity. physiologic abnormalities may or may not provide sufficient information by which to discriminate among conditions.

1	physiologic abnormalities may or may not provide sufficient information by which to discriminate among conditions. Figure 306e-6 lists abnormalities in pulmonary function testing that are typically found in a number of common respiratory disorders and highlights the simultaneous occurrence of multiple physiologic abnormalities. The coexistence of some of these respiratory disorders results in more complex superposition of these abnormalities. Methods to measure respiratory system function clinically are described later in this chapter.

1	Ventilatory Restriction Due to Increased Elastic Recoil—Example: Idiopathic Pulmonary Fibrosis Idiopathic pulmonary fibrosis raises lung recoil at all lung volumes, thereby lowering TLC, FRC, and RV as well as forced vital capacity (FVC). Maximal expiratory flows are also reduced from normal values but are elevated when considered in relation to lung volumes. Increased flow occurs both because the increased lung recoil drives greater maximal flow at any lung volume and because airway diameters are relatively increased due to greater radially outward traction exerted on bronchi by the stiff lung parenchyma. For the same reason, airway resistance is also normal. Destruction of the pulmonary capillaries by the fibrotic process results in a marked reduction in diffusing capacity (see below). Oxygenation is often severely reduced by persistent perfusion of alveolar units that are relatively underventilated due to fibrosis of nearby (and mechanically linked) lung. The flow-volume loop (see

1	Oxygenation is often severely reduced by persistent perfusion of alveolar units that are relatively underventilated due to fibrosis of nearby (and mechanically linked) lung. The flow-volume loop (see below) looks like a miniature version of a normal loop but is shifted toward lower absolute lung volumes and displays maximal expiratory flows that are increased for any given volume over the normal tracing.

1	Ventilatory Restriction Due to Chest Wall Abnormality—Example: Moderate Obesity As the size of the average American continues to increase, this pattern may become the most common of pulmonary function abnormalities. In moderate obesity, the outward recoil of the chest wall is blunted by the weight of chest wall fat and the space occupied by intraabdominal fat. In this situation, preserved inward recoil of the lung overbalances the reduced outward recoil of the chest wall, and FRC falls. Because respiratory muscle strength and lung recoil remain normal, TLC is typically unchanged (although it may fall in massive obesity) and RV is normal (but may be reduced in massive obesity).

1	Mild hypoxemia may be present due to perfusion of alveolar units that are poorly ventilated because of airway closure in dependent portions of the lung during breathing near the reduced FRC. Flows remain normal, as does the diffusion capacity of the lung for carbon monoxide (DlCO), unless obstructive sleep apnea (which often accompanies obesity) and associated chronic intermittent hypoxemia have induced pulmonary arterial hypertension, in which case DlCO may be low.

1	Ventilatory Restriction Due to Reduced Muscle Strength—Example: Myasthenia gravis In this circumstance, FRC remains normal, as both lung recoil and passive chest wall recoil are normal. However, TLC is low and RV is elevated because respiratory muscle strength is insufficient to push the passive respiratory system fully toward either volume extreme. Caught between the low TLC and the elevated RV, FVC and FEV1 are reduced as “innocent bystanders.” As airway size and lung vasculature are unaffected, both Raw and DlCO are normal. Oxygenation is normal unless weakness becomes so severe that the patient has insufficient strength to reopen collapsed alveoli during sighs, with resulting atelectasis.

1	Airflow Obstruction Due to Decreased Airway Diameter—Example: Acute Asthma During an episode of acute asthma, luminal narrowing due to smooth muscle constriction as well as inflammation and thickening within the smalland medium-sized bronchi raise frictional resistance and reduce airflow. “Scooping” of the flow-volume loop is caused by reduction of airflow, especially at lower lung volumes. Often, airflow obstruction can be reversed by inhalation of β2-adrenergic agonists acutely or by treatment with inhaled steroids chronically. TLC usually remains normal (although elevated TLC is sometimes seen in long-standing asthma), but FRC may be dynamically elevated. RV is often increased due to exaggerated airway closure at low lung volumes, and this elevation of RV reduces FVC. Because central airways are narrowed, Raw is usually elevated. Mild arterial hypoxemia is often present due to perfusion of relatively underventilated alveoli distal to obstructed airways (and is responsive to oxygen

1	are narrowed, Raw is usually elevated. Mild arterial hypoxemia is often present due to perfusion of relatively underventilated alveoli distal to obstructed airways (and is responsive to oxygen supplementation), but DlCO is normal or mildly elevated.

1	Airflow Obstruction Due to Decreased Elastic Recoil—Example: Severe Emphysema Loss of lung elastic recoil in severe emphysema results in Disturbances of Respiratory Function

1	Q˙units has /these circumstances, any venous admixture through low ˙V pulmonary hyperinflation, of which elevated TLC is the hallmark. FRC is more severely elevated due both to loss of lung elastic recoil and to dynamic hyperinflation—the same phenomenon as autoPEEP, which is the positive end-expiratory alveolar pressure that occurs when a new breath is initiated before the lung volume is allowed to return to FRC. Residual volume is very severely elevated because of airway closure and because exhalation toward RV may take so long that RV cannot be reached before the patient must inhale again. Both FVC and FEV1 are markedly decreased, the former because of the severe elevation of RV and the latter because loss of lung elastic recoil reduces the pressure driving maximal expiratory flow and also reduces tethering open of small intrapulmonary airways. The flow-volume loop demonstrates marked scooping, with an initial transient spike of flow attributable largely to expulsion of air from

1	also reduces tethering open of small intrapulmonary airways. The flow-volume loop demonstrates marked scooping, with an initial transient spike of flow attributable largely to expulsion of air from collapsing central airways at the onset of forced exhalation. Otherwise, the central airways remain relatively unaffected, so Raw is normal in “pure” emphysema. Loss of alveolar surface and capillaries in the alveolar walls reduces DlCO; however, because poorly ventilated emphysematous acini are also poorly perfused (due to loss of their capillaries), arterial hypoxemia usually is not seen at rest until emphysema becomes very severe. However, during exercise, PaO2 may fall precipitously if extensive destruction of the pulmonary vasculature prevents a sufficient increase in cardiac output and mixed venous oxygen content falls substantially. Under a particularly marked effect in lowering mixed arterial oxygen tension.

1	FuNCTIONAL MEASuREMENTS Measurement of Ventilatory Function • Lung voLumes Figure 306e-2 demonstrates a spirometry tracing in which the volume of air entering or exiting the lung is plotted over time. In a slow vital capacity maneuver, the subject inhales from FRC, fully inflating the lungs to TLC, and then exhales slowly to RV; VC, the difference between TLC and RV, represents the maximal excursion of the respiratory system. Spirometry discloses relative volume changes during these maneuvers but cannot reveal the absolute volumes at which they occur. To determine absolute lung volumes, two approaches are commonly used: inert gas dilution and body plethysmography. In the former, a known amount of a nonabsorbable inert gas (usually helium or neon) is inhaled in a single large breath or is rebreathed from a closed circuit; the inert gas is diluted by the gas resident in the lung at the time of inhalation, and its final concentration reveals the volume of resident gas contributing to the

1	from a closed circuit; the inert gas is diluted by the gas resident in the lung at the time of inhalation, and its final concentration reveals the volume of resident gas contributing to the dilution. A drawback of this method is that regions of the lung that ventilate poorly (e.g., due to airflow obstruction) may not receive much inspired inert gas and so do not contribute to its dilution. Therefore, inert gas dilution (especially in the single-breath method) often underestimates true lung volumes.

1	In the second approach, FRC is determined by measuring the compressibility of gas within the chest, which is proportional to the volume of gas being compressed. The patient sits in a body plethysmograph (a chamber usually made of transparent plastic to minimize claustrophobia) and, at the end of a normal tidal breath (i.e., when lung volume is at FRC), is instructed to pant against a closed shutter, thus periodically compressing air within the lung slightly. Pressure fluctuations at the mouth and volume fluctuations within the body box (equal but opposite to those in the chest) are determined, and from these measurements the thoracic gas volume is calculated by means of Boyle’s law. Once FRC is obtained, TLC and RV are calculated by adding the value for inspiratory capacity and subtracting the value for expiratory reserve volume, respectively (both values having been obtained during spirometry) (Fig. 306e-2). The most important determinants of healthy individuals’ lung volumes are

1	the value for expiratory reserve volume, respectively (both values having been obtained during spirometry) (Fig. 306e-2). The most important determinants of healthy individuals’ lung volumes are height, age, and sex, but there is considerable additional normal variation beyond that accounted for by these parameters. In addition, race influences lung volumes; on average, TLC values are ~12% lower in African Americans and 6% lower in Asian Americans than in Caucasian Americans. In practice, a mean “normal” value is predicted by multivariate regression equations using height, age, and sex, and the patient’s value is divided by the predicted value (often with “race correction” applied) to determine “percent predicted.” For most measures of lung function, 85–115% of the predicted value can be normal; however, in health, the various lung volumes tend to scale together. For example, if one is “normal big” with a TLC 110% of the predicted value, then all other lung volumes and spirometry

1	be normal; however, in health, the various lung volumes tend to scale together. For example, if one is “normal big” with a TLC 110% of the predicted value, then all other lung volumes and spirometry values will also approximate 110% of their respective predicted values. This pattern is particularly helpful in evaluating airflow, as discussed below.

1	Air FLow As noted above, spirometry plays a key role in lung volume determination. Even more often, spirometry is used to measure airflow, which reflects the dynamic properties of the lung. During an FVC maneuver, the patient inhales to TLC and then exhales rapidly and forcefully to RV; this method ensures that flow limitation has been achieved, so that the precise effort made has little influence on actual flow. The total amount of air exhaled is the FVC, and the amount of air exhaled in the first second is the FEV1; the FEV1 is a flow rate, revealing volume change per time. Like lung volumes, an individual’s maximal expiratory flows should be compared with predicted values based on height, age, and sex. While the FEV1/FVC ratio is typically reduced in airflow obstruction, this condition can also reduce FVC by raising RV, sometimes rendering the FEV1/FVC ratio “artifactually normal” with the erroneous implication that airflow obstruction is absent. To circumvent this problem, it is

1	can also reduce FVC by raising RV, sometimes rendering the FEV1/FVC ratio “artifactually normal” with the erroneous implication that airflow obstruction is absent. To circumvent this problem, it is useful to compare FEV1 as a fraction of its predicted value with TLC as a fraction of its predicted value. In health, the results are usually similar. In contrast, even an FEV1 value that is 95% of its predicted value may actually be relatively low if TLC is 110% of its respective predictied value. In this case, airflow obstruction may be present, despite the “normal” value for FEV1.

1	The relationships among volume, flow, and time during spirometry are best displayed in two plots—the spirogram (volume vs. time) and the flow-volume loop (flow vs. volume) (Fig. 306e-4). In conditions that cause airflow obstruction, the site of obstruction is sometimes correlated with the shape of the flow-volume loop. In diseases that cause lower airway obstruction, such as asthma and emphysema, flows decrease more rapidly with declining lung volumes, leading to a characteristic scooping of the flow-volume loop. In contrast, fixed upper-airway obstruction typically leads to inspiratory and/or expiratory flow plateaus (Fig. 306e-4).

1	AirwAys resistAnce The total resistance of the pulmonary and upper airways is measured in the same body plethysmograph used to measure FRC. The patient is asked once again to pant, but this time against a closed and then opened shutter. Panting against the closed shutter reveals the thoracic gas volume as described above. When the shutter is opened, flow is directed to and from the body box, so that volume fluctuations in the box reveal the extent of thoracic gas compression, which in turn reveals the pressure fluctuations driving flow. Simultaneous measurement of flow allows the calculation of lung resistance (as flow divided by pressure). In health, Raw is very low (<2 cmH2O/L per second), and half of the detected resistance resides within the upper airway. In the lung, most resistance originates in the central airways. For this reason, airways resistance measurement tends to be insensitive to peripheral airflow obstruction.

1	respirAtory muscLe strength To measure respiratory muscle strength, the patient is instructed to exhale or inhale with maximal effort against a closed shutter while pressure is monitored at the mouth. Pressures greater than ±60 cmH2O at FRC are considered adequate and make it unlikely that respiratory muscle weakness accounts for any other resting ventilatory dysfunction that is identified.

1	Measurement of gas Exchange • DiFFusing cApAcity (DL co) This test uses a small (and safe) amount of carbon monoxide (CO) to measure gas exchange across the alveolar membrane during a 10-sec breath hold. CO in exhaled breath is analyzed to determine the quantity of CO crossing the alveolar membrane and combining with hemoglobin in red blood cells. This “single-breath diffusing capacity” (Dlco) value increases with the surface area available for diffusion and the amount of hemoglobin within the capillaries, and it varies inversely with alveolar membrane thickness. Thus, Dlco decreases in diseases that thicken or destroy alveolar membranes (e.g., pulmonary fibrosis, emphysema), curtail the pulmonary vasculature (e.g., pulmonary hypertension), or reduce alveolar capillary hemoglobin (e.g., anemia). Single-breath diffusing capacity may be elevated in acute congestive heart failure, asthma, polycythemia, and pulmonary hemorrhage.

1	ArteriAL BLooD gAses The effectiveness of gas exchange can be assessed by measuring the partial pressures of oxygen and CO2 in a sample of blood obtained by arterial puncture. The oxygen content of blood (Ca ) depends upon arterial saturation (%O Sat), which is set by Pa , pH, and PaCO2 according to the oxyhemoglobin dissociation curve. CaO2can also be measured by oximetry (see below): CaO2 (mL/dL) = 1.39 (mL/dL) × [hemoglobin](g) × % O2 Sat + 0.003 (mL/dL/mmHg) × PaO2 (mmHg) If hemoglobin saturation alone needs to be determined, this task can 306e-7 be accomplished noninvasively with pulse oxymetry. The authors wish to acknowledge the contributions of Drs. Steven E. Weinberger and Irene M. Rosen to this chapter in previous editions as well as the helpful contributions of Drs. Mary Strek and Jeffrey Jacobson. CHAPTER 306e Disturbances of Respiratory Function

1	CHAPTER 306e Disturbances of Respiratory Function Diagnostic procedures in respiratory Disease Anne L. Fuhlbrigge, Augustine M. K. Choi The diagnostic modalities available for assessing the patient with suspected or known respiratory system disease include imaging stud-307 ies and techniques for acquiring biologic specimens, some of which involve direct visualization of part of the respiratory system. Methods to characterize the functional changes developing as a result of disease, including pulmonary function tests and measurements of gas exchange, are discussed in Chap. 306e.

1	Routine chest radiography, including both posteroanterior (PA) and lateral views, is an integral part of the diagnostic evaluation of diseases involving the pulmonary parenchyma, the pleura, and, to a lesser extent, the airways and the mediastinum (see Chaps. 305 and 308e). Lateral decubitus views are useful for determining whether pleural abnormalities represent freely flowing fluid, whereas apical lordotic views can visualize disease at the lung apices better than the standard PA view. Portable equipment is often used for acutely ill patients who cannot be transported to a radiology suite but are more difficult to interpret owing to several limitations: (1) the single anteroposterior (AP) projection obtained; (2) variability in overand underexposure of film; (3) a shorter focal spot-film distance leading to lack of edge sharpness and loss of fine detail; and (4) magnification of the cardiac silhouette and other anterior structures by the AP projection. Common radiographic patterns

1	distance leading to lack of edge sharpness and loss of fine detail; and (4) magnification of the cardiac silhouette and other anterior structures by the AP projection. Common radiographic patterns and their clinical correlates are reviewed in Chap. 308e.

1	Advances in computer technology have allowed the development of digital or computed radiography, which has several benefits: (1) immediate availability of the images; (2) significant postprocessing analysis of images to improve diagnostic information; and (3) ability to store images electronically and to transfer them within or between health care systems.

1	Diagnostic ultrasound (US) produces images using echoes or reflection of the US beam from interfaces between tissues with differing acoustic properties. US is nonionizing and safe to perform on pregnant patients and children. It can detect and localize pleural abnormalities and is a quick and effective way of guiding percutaneous needle biopsy of peripheral lung, pleural, or chest wall lesions. US is also helpful in identifying septations within loculated collections and can facilitate placement of a needle for sampling of pleural liquid (i.e., for thoracentesis), improving the yield and safety of the procedure. Bedside availability makes it valuable in the intensive care setting. Real-time imaging can be used to assess the movement of the diaphragm. Because US energy is rapidly dissipated in air, it is not useful for evaluation of 1664 the pulmonary parenchyma and cannot be used if there is any aerated lung between the US probe and the abnormality of interest. Endobronchial US, in

1	in air, it is not useful for evaluation of 1664 the pulmonary parenchyma and cannot be used if there is any aerated lung between the US probe and the abnormality of interest. Endobronchial US, in which the US probe is passed through a bronchoscope, is a valuable adjunct to bronchoscopy, allowing identification and localization of pathology adjacent to airway walls or within the mediastinum.

1	Nuclear imaging depends on the selective uptake of various compounds by organs of the body. In thoracic imaging, these compounds are concentrated by one of three mechanisms: blood pool or compartmentalization (e.g., within the heart), physiologic incorporation (e.g., bone or thyroid) and capillary blockage (e.g., lung scan). Radioactive isotopes can be administered by either the IV or inhaled routes or both. When injected intravenously, albumin macroaggregates labeled with technetium-99m (99mTc) become lodged in pulmonary capillaries; the distribution of the trapped radioisotope follows the distribution of blood flow. When inhaled, radiolabeled xenon gas can be used to demonstrate the distribution of ventilation. Using these techniques, ventilation-perfusion lung scanning was a commonly used technique for the evaluation of pulmonary embolism. Pulmonary thromboembolism produces one or more regions of ventilation-perfusion mismatch (i.e., regions in which there is a defect in perfusion

1	technique for the evaluation of pulmonary embolism. Pulmonary thromboembolism produces one or more regions of ventilation-perfusion mismatch (i.e., regions in which there is a defect in perfusion that follows the distribution of a vessel and that is not accompanied by a corresponding defect in ventilation [Chap. 300]). However, with advances in computed tomography (CT) scanning, scintigraphic imaging has been largely replaced by CT angiography in patients with suspected pulmonary embolism.

1	Another common use of ventilation-perfusion scans is in patients with impaired lung function, who are being considered for lung resection. Many patients with bronchogenic carcinoma have coexisting chronic obstructive pulmonary disease (COPD), and the question arises as to whether or not a patient can tolerate lung resection. The distribution of the isotope(s) can be used to assess the regional distribution of blood flow and ventilation, allowing the physician to estimate the level of postoperative lung function. CT offers several advantages over routine chest radiography (Figs. 307-1A, B and 307-2A, B; see also Figs. 315-3, 315-4, and 322-4). First, the use of cross-sectional images allows distinction between densities that would be superimposed on plain radiographs. Second, CT is far better than routine radiographic studies at characterizing tissue density and providing accurate size assessment of lesions.

1	CT is particularly valuable in assessing hilar and mediastinal disease (often poorly characterized by plain radiography), in identifying and characterizing disease adjacent to the chest wall or spine (including pleural disease), and in identifying areas of fat density or calcification in pulmonary nodules (Fig. 307-2). Its utility in the assessment of mediastinal disease has made CT an important tool in the staging of lung cancer (Chap. 107). With the additional use of contrast material, CT also makes it possible to distinguish vascular from nonvascular structures, which is particularly important in distinguishing lymph nodes and masses from vascular structures primarily in the mediastinum, and vascular disorders such as pulmonary embolism.

1	In high-resolution CT (HRCT), the thickness of individual cross-sectional images is ~1–2 mm, rather than the usual 7–10 mm in conventional CT. The visible detail on HRCT scans allows better recognition of subtle parenchymal and airway disease, thickened interlobular septa, ground-glass opacification, small nodules, and the abnormally thickened or dilated airways seen in bronchiectasis. Using HRCT, characteristic patterns are recognized for many interstitial lung diseases such as lymphangitic carcinoma, idiopathic pulmonary fibrosis, sarcoidosis, and eosinophilic granuloma. However, there is debate about the settings in which the presence of a characteristic pattern on HRCT eliminates the need for obtaining lung tissue to make a diagnosis. Helical CT and Multidetector CT Helical scanning is currently the standard method for thoracic CT. Helical CT technology results in faster scans with improved contrast enhancement and thinner collimation.

1	FIGURE 307-1 Chest x-ray (A) and computed tomography (CT) scan (B) from a patient with emphysema. The extent and distribution of emphysema are not well appreciated on plain film but clearly evident on the CT scan obtained. Images are obtained during a single breath-holding maneuver that allows less motion artifact and collection of continuous data over a larger volume of lung than is possible with conventional CT. Data from the imaging procedure can be reconstructed in coronal or sagittal planes (Fig. 307-3A), as well as the traditional cross-sectional (axial) view. Further refinements in detector technology have allowed production of scanners with additional detectors along the scanning axis (z-axis). These multidetector CT (MDCT) scanners can obtain multiple slices in a single rotation that are thinner and can be acquired in a shorter period of time. This results in enhanced resolution and

1	FIGURE 307-2 Chest x-ray (A) and computed tomography (CT) scan (B) demonstrating a right lower-lobe mass. The mass is not well appreciated on the plain film because of the hilar structures and known calcified adenopathy. CT is superior to plain radiography for the detection of abnormal mediastinal densities and the distinction of masses from adjacent vascular structures. increased image reconstruction ability. As the technology has progressed, higher numbers (currently up to 64) of detectors are used to produce clearer final images. MDCT allows for even shorter breath holds, which are beneficial for all patients but especially children, the elderly, and the critically ill. However, it should be noted that despite the advantages of MDCT, there is an increase in radiation dose compared to single-detector CT to consider.

1	FIGURE 307-3 Spiral computed tomography (CT) with reconstruction of images in planes other than axial view. Spiral CT in a lung transplant patient with a dehiscence and subsequent aneurysm of the anastomosis. CT images were reconstructed in the sagittal view (A) and using digital subtraction to view images of the airways only (B), which demonstrate the exact location and extent of the abnormality. In MDCT, the additional detectors along the z-axis result in improved use of the contrast bolus. This and the faster scanning times and increased resolution have all led to improved imaging of the pulmonary vasculature and the ability to detect segmental and subsegmental emboli. CT pulmonary angiography (CTPA) also allows simultaneous detection of parenchymal abnormalities that may be contributing to a patient’s clinical presentation. Secondary to these

1	FIGURE 307-4 Virtual bronchoscopic image of the trachea. The view projected is one that would be obtained from the trachea looking down to the carina. The left and right main stem airways are seen bifurcating from the carina. advantages and increasing availability, CTPA has rapidly become the test of choice for many clinicians in the evaluation of pulmonary embolism; compared with pulmonary angiography, it is considered equal in terms of accuracy and with less associated risks.

1	The three-dimensional (3D) image of the thorax obtained by MDCT can be digitally stored, reanalyzed, and displayed as 3D reconstructions of the airways down to the sixth to seventh generation. Using these reconstructions, a “virtual” bronchoscopy can be performed (Fig. 307-4). Virtual bronchoscopy has been proposed as an adjunct to conventional bronchoscopy in several clinical situations: It can allow accurate assessment of the extent and length of an airway stenosis, including the airway distal to the narrowing; it can provide useful information about the relationship of the airway abnormality to adjacent mediastinal structures; and it allows preprocedure planning for therapeutic bronchoscopy to help ensure the appropriate equipment is available for the procedure.

1	Virtual bronchoscopy can be used to help target the area of peripheral lung for endobronchial lung volume reduction surgery that is being used in the management of pulmonary emphysema. The extent of emphysema in each segmental region together with other anatomic details may help in choosing the most appropriate subsegments. However, software packages for the generation of virtual bronchoscopic images are relatively early in development, and their utilization and potential impact on patient care are still unknown. Electromagnetic navigational bronchoscopy systems (EMN or ENB) using virtual bronchoscopy have been developed to allow accurate navigation to peripheral pulmonary target lesions, using technology similar to a car global positioning system (GPS) unit.

1	Positron emission tomographic (PET) scanning is commonly used to identify malignant lesions in the lung, based on their increased uptake and metabolism of glucose. The technique involves injection of a radiolabeled glucose analogue, [18F]-fluoro-2-deoxyglucose (FDG), which is taken up by metabolically active malignant cells. However, FDG is trapped within the cells following phosphorylation, and the unstable [18F] decays by emission of positrons, which can be detected by a specialized PET camera or by a gamma camera that has been adapted for imaging of positron-emitting nuclides. This technique has been used in the evaluation of solitary pulmonary nodules and in staging lung cancer. Detection or exclusion of mediastinal lymph node involvement and identification of extrathoracic disease can be achieved. The limited anatomical definition of radionuclide imaging has been improved by the development of hybrid imaging that allows the superimposition of PET and CT images, a technique known

1	be achieved. The limited anatomical definition of radionuclide imaging has been improved by the development of hybrid imaging that allows the superimposition of PET and CT images, a technique known as functional–anatomical mapping. Hybrid PET/CT scans provide images that help pinpoint the abnormal metabolic activity to anatomical structures seen on CT and provide more accurate diagnoses than the two scans performed separately. FDG-PET can differentiate benign from malignant lesions as small as 1 cm. However, false-negative findings can occur in lesions with low metabolic activity such as carcinoid tumors and bronchioloalveolar cell carcinomas, or in lesions <1 cm in which the required threshold of metabolically active malignant cells is not present for PET diagnosis. False-positive results can be seen due to FDG uptake in inflammatory conditions such as pneumonia and granulomatous diseases.

1	The role of magnetic resonance imaging (MRI) in the evaluation of respiratory system disease is less well-defined than that of CT. Magnetic resonance (MR) provides poorer spatial resolution and less detail of the pulmonary parenchyma and, for these reasons, is currently not considered a substitute for CT in imaging the thorax. However, the use of hyperpolarized gas in conjunction with MR has led to the investigational use of MR for imaging the lungs, particularly in obstructive lung disease. In addition, imaging performed during an inhalation and exhalation can provide dynamic information on lung function. Of note, MR examinations are difficult to obtain among several subgroups of patients. Patients who cannot lie still or who cannot lie on their backs may have MRIs that are of poor quality; some tests require patients to hold their breaths for 15–25 seconds at a time in order to get good MRIs. MRI is generally avoided in unstable and/ or ventilated patients and those with severe

1	some tests require patients to hold their breaths for 15–25 seconds at a time in order to get good MRIs. MRI is generally avoided in unstable and/ or ventilated patients and those with severe trauma because of the hazards of the MR environment and the difficulties in monitoring patients within the MR room. The presence of metallic foreign bodies, pacemakers, and intracranial aneurysm clips also preclude use of MRI.

1	An advantage of MR is the use of nonionizing electromagnetic radiation. Additionally, MR is well suited to distinguish vascular from nonvascular structures without the need for contrast. Blood vessels appear as hollow tubular structures because flowing blood does not produce a signal on MRI. Therefore, MR can be useful in demonstrating pulmonary emboli, defining aortic lesions such as aneurysms or dissection, or other vascular abnormalities (Fig. 307-5) if radiation and IV contrast medium cannot be used. Gadolinium can be used as an intravascular contrast agent for MR angiography (MRA); however, synchronization of data acquisition with the peak arterial bolus is one of the major challenges of MRA. The flow of contrast medium from the peripheral injection site to the vessel of interest is affected by a number of factors including heart rate, stroke volume, and the presence of proximal stenotic lesions.

1	The pulmonary arterial system can be visualized by pulmonary angiography, in which radiopaque contrast medium is injected through a catheter placed in the pulmonary artery. When performed in cases of pulmonary embolism, pulmonary angiography demonstrates the consequences of an intravascular thrombus—either a defect in the lumen of a vessel (a filling defect) or an abrupt termination (cutoff) of the vessel. Other, less common indications for pulmonary angiography include visualization of a suspected pulmonary arteriovenous malformation and assessment of pulmonary arterial invasion by a neoplasm. The risks associated with modern arteriography are small, generally of greatest concern in patients with severe pulmonary hypertension or chronic kidney disease. With advances in CT scanning, MDCT

1	FIGURE 307-5 Magnetic resonance angiography image of the vasculature of a patient after lung transplant. The image demonstrates the detailed view of the vasculature that can be obtained using digital subtraction techniques. Images from a patient after lung transplant show the venous and arterial anastomosis on the right; a slight narrowing is seen at the site of the anastomosis, which is considered within normal limits and not suggestive of obstruction. angiography (MDCTA) is replacing conventional angiography for the diagnosis of pulmonary embolism.

1	angiography (MDCTA) is replacing conventional angiography for the diagnosis of pulmonary embolism. Sputum can be collected either by spontaneous expectoration or induced (after inhalation of an irritating aerosol such as hypertonic saline). Sputum induction is used either because sputum is not spontaneously being produced or because of an expected higher yield of certain types of findings. Because sputum consists mainly of secretions from the tracheobronchial tree rather than the upper airway, the finding of alveolar macrophages and other inflammatory cells is consistent with a lower respiratory tract origin of the sample, whereas the presence of squamous epithelial cells in a “sputum” sample indicates contamination by secretions from the upper airways.

1	In addition to processing for routine bacterial pathogens by Gram’s method and culture, sputum can be processed for a variety of other pathogens, including staining and culture for mycobacteria or fungi, culture for viruses, and staining for Pneumocystis jiroveci. In the specific case of sputum obtained for evaluation of P. jiroveci pneumonia, for example, sputum should be collected by induction rather than spontaneous expectoration, and an immunofluorescent stain should be used to detect the organisms. Traditional stains and cultures are now also being supplemented in some cases by immunologic techniques and by molecular biologic methods, including the use of polymerase chain reaction amplification and DNA probes. Cytologic staining of sputum for malignant cells, using the traditional Papanicolaou method, allows noninvasive evaluation for suspected lung cancer.

1	A needle can be inserted through the chest wall into a pulmonary lesion to obtain an aspirate or tissue core for cytologic/histologic or micro-biologic analysis. Aspiration can be performed to obtain a diagnosis or to decompress and/or drain a fluid collection. The procedure is usually carried out under CT or ultrasound guidance to assist positioning of the needle and assure localization in the lesion. The low potential risk 1667 of this procedure (intrapulmonary bleeding or creation of a pneumothorax with collapse of the underlying lung) in experienced hands is usually acceptable compared with the information obtained. However, a limitation of the technique is sampling error due to the small size of the tissue sample. Thus, findings other than a specific cytologic or microbiologic diagnosis are of limited clinical value.

1	Sampling of pleural liquid by thoracentesis is commonly performed for diagnostic purposes or, in the case of a large effusion, for palliation of dyspnea. Diagnostic sampling, either by blind needle aspiration or after localization by US, allows the collection of liquid for micro-biologic and cytologic studies. Analysis of the fluid obtained for its cellular composition and chemical constituents allows classification of the effusion and can help with diagnosis and treatment (Chap. 316).

1	Bronchoscopy is the process of direct visualization of the tracheobronchial tree. Although bronchoscopy is now performed almost exclusively with flexible fiberoptic instruments, rigid bronchoscopy, generally performed in an operating room on a patient under general anesthesia, still has a role in selected circumstances, primarily because of a larger suction channel and the fact that the patient can be ventilated through the bronchoscope channel. These situations include the retrieval of a foreign body and the suctioning of a massive hemorrhage, for which the small suction channel of the bronchoscope may be insufficient.

1	This outpatient procedure is usually performed in an awake but sedated patient (conscious sedation). The bronchoscope is passed through either the mouth or the nose, between the vocal cords, and into the trachea. The ability to flex the scope makes it possible to visualize virtually all airways to the level of subsegmental bronchi. The bronchoscopist is able to identify endobronchial pathology, including tumors, granulomas, bronchitis, foreign bodies, and sites of bleeding. Samples from airway lesions can be taken by several methods, including washing, brushing, and biopsy. Washing involves instillation of sterile saline through a channel of the bronchoscope and onto the surface of a lesion. A portion of the liquid is collected by suctioning through the bronchoscope, and the recovered material can be analyzed for cells (cytology) or organisms (by standard stains and cultures). Brushing or biopsy of the surface of the lesion, using a small brush or biopsy forceps at the end of a long

1	can be analyzed for cells (cytology) or organisms (by standard stains and cultures). Brushing or biopsy of the surface of the lesion, using a small brush or biopsy forceps at the end of a long cable inserted through a channel of the bronchoscope, allows recovery of cellular material or tissue for analysis by standard cytologic and histopathologic methods.

1	The bronchoscope can be used to sample material not only from the regions that can be directly visualized (i.e., the airways) but also from the more distal pulmonary parenchyma. With the bronchoscope wedged into a subsegmental airway, aliquots of sterile saline can be instilled through the scope, allowing sampling of cells and organisms from alveolar spaces. This procedure, called bronchoalveolar lavage, has been particularly useful for the recovery of organisms such as P. jiroveci.

1	Brushing and biopsy of the distal lung parenchyma can also be performed with the same instruments that are used for endobronchial sampling. These instruments can be passed through the scope into small airways. When biopsies are performed, the forceps penetrate the airway wall, allowing biopsy of peribronchial alveolar tissue. This procedure, called transbronchial biopsy, is used when there is either relatively diffuse disease or a localized lesion of adequate size. With the aid of fluoroscopic imaging, the bronchoscopist is able to determine not only whether and when the instrument is in the area of abnormality, but also the proximity of the instrument to the pleural surface. If the forceps are too close to the pleural surface, there is a risk of violating the visceral pleura and creating a pneumothorax; the other potential complication of transbronchial biopsy is pulmonary hemorrhage. The incidence of these complications is less than several percent.

1	1668 TRANSBRONCHIAL NEEDLE ASPIRATION (TBNA) Another procedure involves use of a hollow-bore needle passed through the bronchoscope for sampling of tissue adjacent to the trachea or a large bronchus. The needle is passed through the airway wall (transbronchial), and cellular material can be aspirated from mass lesions or enlarged lymph nodes, generally in a search for malignant cells. Mediastinoscopy has been considered the gold standard for mediastinal staging; however, transbronchial needle aspiration (TBNA) allows sampling from the lungs and surrounding lymph nodes without the need for surgery or general anesthesia.

1	Further advances in needle aspiration techniques have been accomplished with the development of endobronchial ultrasound (EBUS). The technology uses an ultrasonic bronchoscope fitted with a probe that allows for needle aspiration of mediastinal and hilar lymph nodes guided by real-time US images. EBUS allows sampling of mediastinal lymph nodes and masses under direct vision to better identify and localize peribronchial and mediastinal pathology and offers access to more difficult-to-reach areas and smaller lymph nodes in the staging of malignancies. EBUS-TBNA has the potential to access the same paratracheal and subcarinal lymph node stations as mediastinoscopy, but also extends out to the hilar lymph nodes (levels 10 and 11). The usefulness of EBUS for clinical indications other than lung cancer is improving and has been recommended in the evaluation of mediastinal masses of unknown origin early in the diagnostic process.

1	Emerging techniques that can be performed using bronchoscopy include video/autofluorescence bronchoscopy (AFB), narrow band imaging (NBI), optical coherence tomography (OCT), and endomicroscopy using confocal fluorescent laser microscopy (CFM). AFB uses bronchoscopy with an additional light source to screen high-risk individuals and identify premalignant lesions (airway dysplasia) and carcinoma in situ. NBI capitalizes on the increased absorption of blue and green wavelengths of light by hemoglobin to enhance the visibility of vessels of the mucosa and differentiate between inflammatory versus malignant mucosal lesions. CFM uses a blue laser to induce fluorescence, and its high degree of resolution provides a real-time view of living tissue at an almost histologic resolution. OCT uses near-infrared light source and has spatial resolution advantages over CT and MRI. It can penetrate the airway wall up to three times deeper than CFM and is less susceptible to motion artifacts from

1	near-infrared light source and has spatial resolution advantages over CT and MRI. It can penetrate the airway wall up to three times deeper than CFM and is less susceptible to motion artifacts from cardiac pulsation and respiratory movements. However, careful assessment is required before these methods find a place in the evaluation strategy of early lung cancer and other lung diseases.

1	The bronchoscope may provide the opportunity for treatment as well as diagnosis. A central role of the interventional pulmonology (IP) physician is the performance of therapeutic bronchoscopy. For example, an aspirated foreign body may be retrieved with an instrument passed through the bronchoscope (either flexible or rigid), and bleeding may be controlled with a balloon catheter similarly introduced. Newer interventional techniques performed through a bronchoscope include methods for achieving and maintaining patency of airways that are partially or completely occluded, especially by tumors. These techniques include laser therapy, cryotherapy, argon plasma coagulation, electrocautery, balloon bronchoplasty and dilation, and stent placement. Many IP physicians are also trained in performing percutaneous tracheotomy.

1	Medical thoracoscopy (or pleuroscopy) focuses on the diagnosis of pleural-based problems. The procedure is performed with a conventional rigid or a semi-rigid pleuroscope (similar in design to a bronchoscope and enabling the operator to inspect the pleural surface, sample and/or drain pleural fluid, or perform targeted biopsies of the parietal pleura). Medical thoracoscopy can be performed in the endoscopy suite or operating room with the patient under conscious sedation and local anesthesia. In contrast, video-assisted thoracoscopic surgery (VATS) requires general anesthesia and is only performed in the operating room. A common diagnostic indication for medical thoracoscopy is the evaluation of a pleural effusion or biopsy of presumed parietal pleural carcinomatosis. It can also be used to place a chest tube under visual guidance, or perform chemical or talc pleurodesis, a therapeutic intervention to prevent a recurrent pleural effusion (usually malignant) or recurrent pneumothorax.

1	The increasing availability of advanced bronchoscopic and pleuroscopic techniques has motivated the development of IP programs. IP can be defined as “the art and science of medicine as related to the performance of diagnostic and invasive therapeutic procedures, that which require additional training and expertise beyond that which required in a standard pulmonary medicine training program.” IP physicians provide alternatives to surgery for patients with a wide variety of thoracic disorders and problems. Evaluation and diagnosis of disorders of the chest commonly involve collaboration between pulmonologists and thoracic surgeons. Although procedures such as mediastinoscopy, VATS, and thoracotomy are performed by thoracic surgeons, there is overlap in many minimally invasive techniques that can be performed by a pulmonologist, an interventional pulmonologist, or a thoracic surgeon.

1	Proper staging of lung cancer is of paramount concern when determining a treatment regimen. Although CT and PET scanning are useful for determining the size and nature of mediastinal lymph nodes as part of the staging of lung cancer, tissue biopsy and histopathologic examination are often critical for the diagnosis of mediastinal masses or enlarged mediastinal lymph nodes. The two major surgical procedures used to obtain specimens from masses or nodes in the mediastinum are mediastinoscopy (via a suprasternal approach) and mediastinotomy (via a parasternal approach). Both procedures are performed under general anesthesia by a qualified surgeon. In the case of suprasternal mediastinoscopy, a rigid mediastinoscope is inserted at the suprasternal notch and passed into the mediastinum along a pathway just anterior to the trachea. Tissue can be obtained with biopsy forceps passed through the scope, sampling masses or nodes that are in a paratracheal or pretracheal position (levels 2R, 2L,

1	pathway just anterior to the trachea. Tissue can be obtained with biopsy forceps passed through the scope, sampling masses or nodes that are in a paratracheal or pretracheal position (levels 2R, 2L, 3, 4R, 4L). Aortopulmonary lymph nodes (levels 5, 6) are not accessible by this route and thus are commonly sampled by parasternal mediastinotomy (the Chamberlain procedure). This approach involves a parasternal incision and dissection directly down to a mass or node that requires biopsy.

1	As an alternative to surgery, a bronchoscope can be used to perform TBNA to obtain tissue from the mediastinum, and, when combined with EBUS, can allow access to the same lymph node stations associated with mediastinoscopy, but also extend access out to the hilar lymph nodes (levels 10, 11). Finally, endoscopic ultrasound (EUS)– fine-needle aspiration (FNA) is a second procedure that complements EBUS-FNA in the staging of lung cancer. EUS-FNA is performed via the esophagus and is ideally suited for sampling lymph nodes in the posterior mediastinum (levels 7, 8, 9). Because US imaging cannot penetrate air-filled spaces, the area directly anterior to the trachea cannot accurately be assessed and is a “blind spot” for EUS-FNA. However, EBUS-FNA can visualize the anterior lymph nodes and can complement EUS-FNA. The combination of EUS-FNA and EBUSFNA is a technique that is becoming an alternative to surgery for staging the mediastinum in thoracic malignancies.

1	VATS has become a standard technique for the diagnosis and management of pleural as well as parenchymal lung disease. This procedure is performed in the operating room using single-lung ventilation with double-lumen endotracheal intubation and involves the passage of a rigid scope with a distal lens through a trocar inserted into the pleura. A high-quality image is shown on a monitor screen, allowing the operator to manipulate instruments passed into the pleural space through separate small intercostal incisions. With these instruments the operator can biopsy lesions of the pleura under direct visualization. In addition, this procedure is now used commonly to biopsy peripheral lung tissue or to remove peripheral nodules for both diagnostic and therapeutic purposes. This much less invasive procedure has largely supplanted the traditional “open lung biopsy” performed via thoracotomy. The decision to use a VATS technique versus performing an open thoracotomy is made by the thoracic

1	procedure has largely supplanted the traditional “open lung biopsy” performed via thoracotomy. The decision to use a VATS technique versus performing an open thoracotomy is made by the thoracic surgeon and is based on whether a patient can tolerate the single-lung ventilation that is required to allow adequate visualization of the lung. With further advances in instrumentation and experience, VATS can be used to perform procedures previously requiring thoracotomy, including stapled lung biopsy, resection of pulmonary nodules, lobectomy, pneumonectomy, pericardial window, or other standard thoracic surgical procedures, but allows them to be performed in a minimally invasive manner.

1	Although frequently replaced by VATS, thoracotomy remains an option for the diagnostic sampling of lung tissue. It provides the largest amount of material, and it can be used to biopsy and/or excise lesions 166that are too deep or too close to vital structures for removal by VATS. The choice between VATS and thoracotomy needs to be made on a case-by-case basis. Atlas of Chest Imaging Patricia A. Kritek, John J. Reilly, Jr. This atlas of chest imaging is a collection of interesting chest radiographs and computed tomograms (CTs) of the chest. The readings of the films are meant to be illustrative of specific, major findings. The associated text is not intended as a comprehensive assessment of the images. 308e

1	FIGURE 308e-1 Normal chest radiograph—review of anatomy. 1. Trachea. 2. Carina. 3. Right atrium. 4. Right hemidiaphragm. 5. Aortic knob. 6. Left hilum. 7. Left ventricle. 8. Left hemidiaphragm (with stomach bubble). 9. Retrosternal clear space. 10. Right ventricle. 11. Left hemidiaphragm (with stomach bubble). 12. Left upper lobe bronchus. FIGURE 308e-2 Normal chest tomogram—note anatomy. 1. Superior vena cava. 2. Trachea. 3. Aortic arch. 4. Ascending aorta. 5. Right mainstem bronchus. 6. Descending aorta. 7. Left mainstem bronchus. 8. Main pulmonary artery. 9. Heart. 10. Esophagus. 11. Pericardium. 12. Descending aorta. CT scan demonstrating left upper lobe collapse. FIGURE 308e-5 Left upper lobe scarring with hilar retraction with less prominent scarring in right upper lobe as well. Findings consistent with previous tuberculosis infection in an immigrant from Ecuador. CHAPTER 308e Atlas of Chest Imaging

1	CHAPTER 308e Atlas of Chest Imaging The patient was found to have an endobronchial lesion (not visible on the CT scan) resulting in this finding. The superior vena cava (black arrow) is partially opacified by intravenous contrast. FIGURE 308e-4 CT scan revealing chronic left lower lobe collapse. Note dramatic volume loss with minimal aeration. There is subtle mediastinal shift to the left. FIGURE 308e-6 Apical scarring, traction bronchiectasis (red arrow), and decreased lung volume consistent with previous tuberculosis infection. Findings most significant in left lung. FIGURE 308e-7 Chest radiograph demonstrating right upper lobe collapse (yellow arrow). Note the volume loss as demonstrated by the elevated right hemidiaphragm as well as mediastinal shift to the right. Also apparent on the film are an endotracheal tube (red arrow) and a central venous catheter (black arrow).

1	FIGURE 308e-8 Opacity in the right upper lobe. Note the volume loss as indicated by the elevation of the right hemidiaphragm, eleva-tion of minor fissure (yellow arrow), and deviation of the trachea to the right (blue arrow). FIGURE 308e-9 CT scan of the same right upper lobe opacity. Note the air bronchograms and areas of consolidation. FIGURE 308e-10 Emphysema with increased lucency, flattened diaphragms (black arrows), increased anteroposterior (AP) diameter, and increased retrosternal clear space (red arrow). FIGURE 308e-11 CT scan of diffuse, bilateral emphysema. FIGURE 308e-12 CT scan of bullous emphysema. Atlas of Chest Imaging FIGURE 308e-13 Lymphangioleiomyomatosis—note multiple thin-walled parenchymal cysts.

1	FIGURE 308e-12 CT scan of bullous emphysema. Atlas of Chest Imaging FIGURE 308e-13 Lymphangioleiomyomatosis—note multiple thin-walled parenchymal cysts. FIGURE 308e-14 Two cavities on posteroanterior (PA) and lateral chest radiograph. Cavities and air-fluid levels identified by red arrows. The smaller cavity is in the right lower lobe (located below the major fissure, identified with the yellow arrow), and the larger cavity is located in the right middle lobe, which is located between the minor (red arrow) and major fissures. There is an associated opacity surrounding the cavity in the right lower lobe. FIGURE 308e-15 CT scan of parenchymal cavity. FIGURE 308e-16 Thick-walled cavitary lung lesions. The mass in the right lung has thick walls and advanced cavitation, whereas the smaller nodule on the left has early cavitary changes (arrow). This patient was diagnosed with Nocardia infection.

1	FIGURE 308e-17 Mild congestive heart failure. Note the Kerley B lines (black arrow) and perivascular cuffing (yellow arrow) as well as the pulmonary vascular congestion (red arrow). FIGURE 308e-18 Pulmonary edema. Note indistinct vasculature, perihilar opacities, and peripheral interstitial reticular opacities. Although this is an anteroposterior film making cardiac size more difficult to assess, the cardiac silhouette still appears enlarged. CHAPTER 308e Atlas of Chest Imaging FIGURE 308e-19 Chest radiograph demonstrates reticular nodular opacities bilaterally with small lung volumes consistent with usual interstitial pneumonitis (UIP) on pathology. Clinically, UIP is used interchangeably with idiopathic pulmonary fibrosis (IPF).

1	FIGURE 308e-20 CT scan of usual interstitial pneumonitis (UIP), also known as idiopathic pulmonary fibrosis (IPF). Classic findings include traction bronchiectasis (black arrow) and honeycombing (red arrows). Note subpleural, basilar predominance of the honeycombing. FIGURE 308e-21 A. PA chest film—note presence of paratracheal (blue arrow), aortopulmonary window (yellow arrow), and hilar lymphadenopathy (purple arrows). B. Lateral film—note hilar lymphadenopathy (purple arrow). FIGURE 308e-22 Sarcoid—CT scan of stage I demonstrating bulky hilar and mediastinal lymphadenopathy (red arrows). 4.86 mm‡ A FIGURE 308e-23 Sarcoid—chest radiograph of stage II. A. PA film with hilar lymphadenopathy (black arrows) and parenchymal changes. B. Lateral film with hilar adenopathy (black arrow) and parenchymal changes. FIGURE 308e-24 Sarcoid—CT scan of stage II (calcified lymphade tracking along bronchovascular bundles). nopathy, parenchymal infiltrates).

1	FIGURE 308e-24 Sarcoid—CT scan of stage II (calcified lymphade tracking along bronchovascular bundles). nopathy, parenchymal infiltrates). CHAPTER 308e Atlas of Chest Imaging FIGURE 308e-26 Sarcoid—stage IV with fibrotic lung disease and cavitary areas (yellow arrow). FIGURE 308e-27 Right middle lobe opacity illustrates major (black arrow) and minor fissures (red arrows) as well as the “silhouette sign” on the right heart border. The silhouette sign is the loss of clear demarcation between normal lung and soft tissue (e.g., heart, diaphragm). This occurs when the lung parenchyma is no longer filled with air and the contrast between air and soft tissue is lost. FIGURE 308e-28 Right lower lobe pneumonia—subtle opacity on PA film (red arrow), while the lateral film illustrates the “spine sign” (black arrow) where the lower spine does not become more lucent.

1	FIGURE 308e-28 Right lower lobe pneumonia—subtle opacity on PA film (red arrow), while the lateral film illustrates the “spine sign” (black arrow) where the lower spine does not become more lucent. FIGURE 308e-30 Chest radiograph reveals diffuse, bilateral alveolar opacities without pleural effusions, consistent with acute respiratory distress syndrome (ARDS). Note that the patient has an endotracheal tube (red arrow) and a central venous catheter (black arrow). FIGURE 308e-29 CT scan of diffuse, bilateral “ground-glass” opacities. This finding is consistent with fluid density in the alveolar space. FIGURE 308e-31 CT scan of ARDS demonstrates “ground-glass” opacities with more consolidated areas in the dependent lung zones. Atlas of Chest Imaging FIGURE 308e-32 Three examples of air bronchograms (red arrows) on chest CT. BRONCHIECTASIS AND AIRWAY ABNORMALITIES FIGURE 308e-33 Cystic fibrosis with bronchiectasis, apical disease.

1	FIGURE 308e-32 Three examples of air bronchograms (red arrows) on chest CT. BRONCHIECTASIS AND AIRWAY ABNORMALITIES FIGURE 308e-33 Cystic fibrosis with bronchiectasis, apical disease. FIGURE 308e-34 CT scan of diffuse, cystic bronchiectasis (red arrows) in a patient with cystic fibrosis. FIGURE 308e-35 CT scan of focal right middle lobe and lingular bronchiectasis (yellow arrows). Note that there is near total collapse of the right middle lobe (red arrow). FIGURE 308e-36 “Tree in bud” opacities (red arrows) and bronchiectasis (yellow arrow) consistent with atypical mycobacterial infection. “Tree in bud” refers to small nodules clustered around the centrilobular arteries as well as increased prominence of the centrilobular branching. These findings are consistent with bronchiolitis. FIGURE 308e-37 CT scan demonstrating tracheomalacia (yellow arrow). Tracheomalacia is dynamic collapse of the trachea, most prominent during exhalation, due to loss of cartilaginous support.

1	FIGURE 308e-37 CT scan demonstrating tracheomalacia (yellow arrow). Tracheomalacia is dynamic collapse of the trachea, most prominent during exhalation, due to loss of cartilaginous support. FIGURE 308e-38 Large right pneumothorax with near complete collapse of right lung. Pleural reflection highlighted with red arrows. CHAPTER 308e Atlas of Chest Imaging FIGURE 308e-39 Basilar pneumothorax with visible pleural reflection (red arrows). Also note, patient has subcutaneous emphysema (yellow arrow). FIGURE 308e-40 CT scan of large right-sided pneumothorax. Note significant collapse of right lung with adhesion to anterior chest wall. Pleural reflection highlighted with red arrows. The patient has severe underlying emphysema.

1	FIGURE 308e-41 Small right pleural effusion (red arrows highlight blunted right costophrenic angles) with associated pleural thickening. Note fluid in the major fissure (black arrow) visible on the lateral film as well as the meniscus of the right pleural effusion. FIGURE 308e-42 Left pleural effusion with clear meniscus seen on both PA and lateral chest radiographs. FIGURE 308e-43 Asbestosis. Note calcified pleural plaques (red arrows), pleural thickening (black arrow), and subpleural atelectasis (green arrows). CHAPTER 308e Atlas of Chest Imaging FIGURE 308e-44 Left upper lobe mass, which biopsy revealed to be squamous cell carcinoma. FIGURE 308e-45 Solitary pulmonary nodule on the right (red arrow) with a spiculated pattern concerning for lung cancer. Note also that the patient is status post left upper lobectomy with resultant volume loss and associated effusion (black arrow).

1	FIGURE 308e-47 Left lower lobe lung mass (red arrow) abutting pleura. Biopsy demonstrated small-cell lung cancer. FIGURE 308e-46 Metastatic sarcoma. Note the multiple, well-circumscribed nodules of different size. FIGURE 308e-48 CT scan of soft tissue mass encircling the trachea (red arrow) and invading tracheal lumen. Biopsy demonstrated ade-noid cystic carcinoma (cylindroma). FIGURE 308e-49 Mycetoma. Fungal ball (red arrow) growing in preexisting cavity on the left. Right upper lobe has a large bulla (black arrow). FIGURE 308e-50 Pulmonary arteriovenous malformation (AVM) demonstrated on reformatted CT angiogram (red arrow). CHAPTER 308e Atlas of Chest Imaging FIGURE 308e-51 Large bilateral pulmonary emboli (intravascular filling defects in contrast scan identified by red arrows). FIGURE 308e-52 Chest radiograph of a patient with severe pulmonary hypertension. Note the enlarged pulmonary arteries (red arrows) visible on both PA and lateral films.

1	FIGURE 308e-52 Chest radiograph of a patient with severe pulmonary hypertension. Note the enlarged pulmonary arteries (red arrows) visible on both PA and lateral films. FIGURE 308e-53 CT scan of the same patient as in Fig. 308e-52. Note the markedly enlarged pulmonary arteries (red arrow).

1	FIGURE 308e-53 CT scan of the same patient as in Fig. 308e-52. Note the markedly enlarged pulmonary arteries (red arrow). asthma Peter J. Barnes Asthma is a syndrome characterized by airflow obstruction that varies markedly, both spontaneously and with treatment. Asthmatics harbor a special type of inflammation in the airways that makes them more responsive than nonasthmatics to a wide range of triggers, leading to 309 SeCtion 2 excessive narrowing with consequent reduced airflow and symptomatic wheezing and dyspnea. Narrowing of the airways is usually reversible, but in some patients with chronic asthma there may be an element of irreversible airflow obstruction. The increasing global prevalence of asthma, the large burden it now imposes on patients, and the high health care costs have led to extensive research into its mechanisms and treatment.

1	Asthma is one of the most common chronic diseases globally and currently affects approximately 300 million people worldwide. The prevalence of asthma has risen in affluent countries over the last 30 years but now appears to have stabilized, with approximately 10–12% of adults and 15% of children affected by the disease. In developing countries where the prevalence of asthma had been much lower, there is a rising prevalence, which is associated with increased urbanization. The prevalence of atopy and other allergic diseases has also increased over the same time, suggesting that the reasons for the increase are likely to be systemic rather than confined to the lungs. Most patients with asthma in affluent countries are atopic, with allergic sensitization to the house dust mite Dermatophagoides pteronyssinus and other environmental allergens, such as animal fur and pollens.

1	Asthma can present at any age, with a peak age of 3 years. In childhood, twice as many males as females are asthmatic, but by adulthood the sex ratio has equalized. Long-term studies that have followed children until they reach the age of 40 years suggest that many with asthma become asymptomatic during adolescence but that asthma returns in some during adult life, particularly in those with persistent symptoms and severe asthma. Adults with asthma, including those with onset during adulthood, rarely become permanently asymptomatic. The severity of asthma does not vary significantly within a given patient; those with mild asthma rarely progress to more severe disease, whereas those with severe asthma usually have severe disease at the onset.

1	Deaths from asthma are uncommon, and in many affluent countries have been steadily declining over the last decade. A rise in asthma mortality seen in several countries during the 1960s was associated with increased use of short-acting inhaled β2-adrenergic agonists (as rescue therapy), but there is now compelling evidence that the more widespread use of inhaled corticosteroids (ICS) in patients with persistent asthma is responsible for the decrease in mortality in recent years. Major risk factors for asthma deaths are poorly controlled disease with frequent use of bronchodilator inhalers, lack of or poor compliance with ICS therapy, and previous admissions to hospital with near-fatal asthma. It has proved difficult to agree on a definition of asthma, but there is good agreement on the description of the clinical syndrome and disease pathology. Until the etiologic mechanisms of the disease are better understood, it will be difficult to provide an accurate definition.

1	Asthma is a heterogeneous disease with interplay between genetic and environmental factors. Several risk factors that predispose to asthma have been identified (Table 309-1). These should be distinguished from triggers, which are environmental factors that worsen asthma in a patient with established disease. Atopy Atopy is the major risk factor for asthma, and nonatopic individuals have a very low risk of developing asthma. Patients with asthma commonly suffer from other atopic diseases, particularly allergic rhinitis, which may be found in over 80% of asthmatic patients, and atopic dermatitis (eczema). Atopy may be found in 40–50% of the population in affluent countries, with only a proportion of atopic Drugs (β blockers, aspirin)

1	Drugs (β blockers, aspirin) Irritants (household sprays, paint fumes) individuals becoming asthmatic. This observation suggests that some other environmental or genetic factor(s) predispose to the development of asthma in atopic individuals. The allergens that lead to sensitization are usually proteins that have protease activity, and the most common allergens are derived from house dust mites, cat and dog fur, cockroaches (in inner cities), grass and tree pollens, and rodents (in laboratory workers). Atopy is due to the genetically determined production of specific IgE antibody, with many patients showing a family history of allergic diseases.

1	Genetic Predisposition The familial association of asthma and a high degree of concordance for asthma in identical twins indi cate a genetic predisposition to the disease; however, whether or not the genes predisposing to asthma are similar or in addition to those predisposing to atopy is not yet clear. It now seems likely that different genes may also contribute to asthma specifically, and there is increasing evidence that the severity of asthma is also genetically determined. Genetic screens with classical linkage analysis and single-nucleotide polymorphisms of various candidate genes indicate that asthma is polygenic, with each gene identified having a small effect that is often not replicated in different populations. This observation suggests that the interaction of many genes is important, and these may differ in different populations. The most consistent findings have been associations with polymorphisms of genes on chromosome 5q, including the T helper 2 (TH2) cells

1	is important, and these may differ in different populations. The most consistent findings have been associations with polymorphisms of genes on chromosome 5q, including the T helper 2 (TH2) cells interleukin (IL)-4, IL-5, IL-9, and IL-13, which are associated with atopy. There is increasing evidence for a complex interaction between genetic polymorphisms and environmental factors that will require very large population studies to unravel. Novel genes that have been associated with asthma, including ADAM-33, and DPP-10, have also been identified by positional cloning, but their function in disease pathogenesis is not yet clear. Recent genome-wide association studies have identified further novel genes, such as ORMDL3, although their functional role is not yet clear. Genetic polymorphisms may also be important in determining the response to asthma therapy. For example, the Arg-Gly-16 variant in the β2-receptor has been associated with reduced response to β2-agonists, and repeats of an

1	may also be important in determining the response to asthma therapy. For example, the Arg-Gly-16 variant in the β2-receptor has been associated with reduced response to β2-agonists, and repeats of an Sp1 recognition sequence in the promoter region of 5-lipoxygenase may affect the response to antileukotrienes. However, these effects are small and inconsistent and do not yet have any implications for asthma therapy.

1	It is likely that environmental factors in early life determine which atopic individuals become asthmatic. The increasing prevalence of asthma, particularly in developing countries, over the last few decades also indicates the importance of environmental mechanisms interacting with a genetic predisposition. Infections Although viral infections (especially rhinovirus) are common triggers of asthma exacerbations, it is uncertain whether they play a role in etiology. There is some association between respiratory syncytial virus infection in infancy and the development of asthma, but the specific pathogenesis is difficult to elucidate because this infection is very common in children. Atypical bacteria, such as Mycoplasma and Chlamydophila, have been implicated in the mechanism of severe asthma, but thus far, the evidence is not very convincing of a true association.

1	The observation that allergic sensitization and asthma were less common in children with older siblings first suggested that lower levels of infection may be a factor in affluent societies that increase the risks of asthma. This “hygiene hypothesis” proposes that lack of infections in early childhood preserves the TH2 cell bias at birth, whereas exposure to infections and endotoxin results in a shift toward a predominant protective TH1 immune response. Children brought up on farms who are exposed to a high level of endotoxin are less likely to develop allergic sensitization than children raised on dairy farms. Intestinal parasite infection, such as hookworm, may also be associated with a reduced risk of asthma. Although there is considerable epidemiologic support for the hygiene hypothesis, it cannot account for the parallel increase in TH1-driven diseases such as diabetes mellitus over the same period.

1	Diet The role of dietary factors is controversial. Observational studies have shown that diets low in antioxidants such as vitamin C and vitamin A, magnesium, selenium, and omega-3 polyunsaturated fats (fish oil) or high in sodium and omega-6 polyunsaturated fats are associated with an increased risk of asthma. Vitamin D deficiency may also predispose to the development of asthma. However, interventional studies with supplementary diets have not supported an important role for these dietary factors. Obesity is also an independent risk factor for asthma, particularly in women, but the mechanisms are thus far unknown.

1	Air Pollution Air pollutants, such as sulfur dioxide, ozone, and diesel particulates, may trigger asthma symptoms, but the role of different air pollutants in the etiology of the disease is much less certain. Most evidence argues against an important role for air pollution because asthma is no more prevalent in cities with a high ambient level of traffic pollution than in rural areas with low levels of pollution. Asthma had a much lower prevalence in East Germany compared to West Germany despite a much higher level of air pollution, but since reunification, these differences have decreased as eastern Germany has become more affluent. Indoor air pollution may be more important with exposure to nitrogen oxides from cooking stoves and exposure to passive cigarette smoke. There is some evidence that maternal smoking is a risk factor for asthma, but it is difficult to dissociate this association from an increased risk of respiratory infections.

1	Allergens Inhaled allergens are common triggers of asthma symptoms and have also been implicated in allergic sensitization. Exposure to house dust mites in early childhood is a risk factor for allergic sensitization and asthma, but rigorous allergen avoidance has not shown any evidence for a reduced risk of developing asthma. The increase in house dust mites in centrally heated poorly ventilated homes with fitted carpets has been implicated in the increasing prevalence of asthma in affluent countries. Domestic pets, particularly cats, have also been associated with allergic sensitization, but early exposure to cats in the home may be protective through the induction of tolerance.

1	Occupational Exposure Occupational asthma is relatively common and may affect up to 10% of young adults. Over 300 sensitizing agents have been identified. Chemicals such as toluene diisocyanate and trimellitic anhydride, may lead to sensitization independent of atopy. Individuals may also be exposed to allergens in the workplace such as small animal allergens in laboratory workers and fungal amylase in wheat flour in bakers. Occupational asthma may be suspected when symptoms improve during weekends and holidays. Obesity Asthma occurs more frequently in obese people (body mass index >30 kg/m2) and is often more difficult to control. Although mechanical factors may contribute, it may also be linked to the pro-inflammatory adipokines and reduced anti-inflammatory adipokines that are released from fat stores.

1	Other Factors Several other factors have been implicated in the etiology of asthma, including lower maternal age, duration of breast-feeding, prematurity and low birthweight, and inactivity, but are unlikely to contribute to the recent global increase in asthma prevalence. There is also an association with acetaminophen (paracetamol) consumption in childhood, which may be linked to increased oxidative stress.

1	Intrinsic Asthma A minority of asthmatic patients (approximately 10%) have negative skin tests to common inhalant allergens and normal serum concentrations of IgE. These patients, with nonatopic or intrinsic asthma, usually show later onset of disease (adult-onset asthma), commonly have concomitant nasal polyps, and may be aspirin-sensitive. They usually have more severe, persistent asthma. Little is understood about mechanism, but the immunopathology in bronchial biopsies and sputum appears to be identical to that found in atopic asthma. There is recent evidence for increased local production of IgE in the airways, suggesting that there may be common IgEmediated mechanisms; staphylococcal enterotoxins, which serve as “superantigens,” have been implicated.

1	Asthma Triggers Several stimuli trigger airway narrowing, wheezing, and dyspnea in asthmatic patients. Although a previous view held that these stimuli should be avoided, the triggering of asthma by these stimuli is now seen as evidence for poor control and an indicator of the need to increase controller (preventive) therapy.

1	ALLERGENS Inhaled allergens activate mast cells with bound IgE directly leading to the immediate release of bronchoconstrictor mediators, resulting in the early response that is reversed by bronchodilators. Often, experimental allergen challenge is followed by a late response when there is airway edema and an acute inflammatory response with increased eosinophils and neutrophils that are not very reversible with bronchodilators. The most common allergens to trigger asthma are Dermatophagoides species, and environmental exposure leads to low-grade chronic symptoms that are perennial. Other perennial allergens are derived from cats and other domestic pets, as well as cockroaches. Other allergens, including grass pollen, ragweed, tree pollen, and fungal spores, are seasonal. Pollens usually cause allergic rhinitis rather than asthma, but in thunderstorms, the pollen grains are disrupted and the particles that may be released can trigger severe asthma exacerbations (thunderstorm asthma).

1	VIRUS INFECTIONS Upper respiratory tract virus infections such as rhinovirus, respiratory syncytial virus, and coronavirus are the most common triggers of acute severe exacerbations and may invade epithelial cells of the lower as well as the upper airways. The mechanism whereby these viruses cause exacerbations is poorly understood, but there is an increase in airway inflammation with increased numbers of eosinophils and neutrophils. There is evidence for reduced production of type I interferons by epithelial cells from asthmatic patients, resulting in increased susceptibility to these viral infections and a greater inflammatory response.

1	PHARMACOLOGIC AGENTS Several drugs may trigger asthma. Betaadrenergic blockers commonly acutely worsen asthma, and their use may be fatal. The mechanisms are not clear, but are likely mediated through increased cholinergic bronchoconstriction. All β blockers need to be avoided, and even selective β2 blockers or topical application (e.g., timolol eye drops) may be dangerous. Angiotensin-converting enzyme inhibitors are theoretically detrimental as they inhibit breakdown of kinins, which are bronchoconstrictors; however, they rarely worsen asthma, and the characteristic cough is no more frequent in asthmatics than in nonasthmatics. Aspirin may worsen asthma in some patients (aspirin-sensitive asthma is discussed below under “Special Considerations”).

1	EXERCISE Exercise is a common trigger of asthma, particularly in children. The mechanism is linked to hyperventilation, which results in increased osmolality in airway lining fluid and triggers mast cell 1671 mediator release, resulting in bronchoconstriction. Exercise-induced asthma (EIA) typically begins after exercise has ended and resolves spontaneously within about 30 min. EIA is worse in cold, dry climates than in hot, humid conditions. It is, therefore, more common in sports activities such as cross-country running in cold weather, overland skiing, and ice hockey than in swimming. It may be prevented by prior administration of β2-agonists and antileukotrienes, but is best prevented by regular treatment with ICSs, which reduce the population of surface mast cells required for this response.

1	PHYSICAL FACTORS Cold air and hyperventilation may trigger asthma through the same mechanisms as exercise. Laughter may also be a trigger. Many patients report worsening of asthma in hot weather and when the weather changes. Some asthmatics become worse when exposed to strong smells or perfumes, but the mechanism of this response is uncertain.

1	FOOD AND DIET There is little evidence that allergic reactions to food lead to increased asthma symptoms, despite the belief of many patients that their symptoms are triggered by particular food constituents. Exclusion diets are usually unsuccessful at reducing the frequency of episodes. Some foods such as shellfish and nuts may induce anaphylactic reactions that may include wheezing. Patients with aspirin-induced asthma may benefit from a salicylate-free diet, but these are difficult to maintain. Certain food additives may trigger asthma. Metabisulfite, which is used as a food preservative, may trigger asthma through the release of sulfur dioxide gas in the stomach. Tartrazine, a yellow food- coloring agent, was believed to be a trigger for asthma, but there is little convincing evidence for this. AIR POLLUTION Increased ambient levels of sulfur dioxide, ozone, and nitrogen oxides are associated with increased asthma symptoms.

1	AIR POLLUTION Increased ambient levels of sulfur dioxide, ozone, and nitrogen oxides are associated with increased asthma symptoms. OCCUPATIONAL FACTORS Several substances found in the workplace may act as sensitizing agents, as discussed above, but may also act as triggers of asthma symptoms. Occupational asthma is characteristically associated with symptoms at work with relief on weekends and holidays. If removed from exposure within the first 6 months of symptoms, there is usually complete recovery. More persistent symptoms lead to irreversible airway changes, and thus, early detection and avoidance are important.

1	HORMONES Some women show premenstrual worsening of asthma, which can occasionally be very severe. The mechanisms are not completely understood, but are related to a fall in progesterone and in severe cases may be improved by treatment with high doses of progesterone or gonadotropin-releasing factors. Thyrotoxicosis and hypothyroidism can both worsen asthma, although the mechanisms are uncertain. GASTROESOPHAGEAL REFLUX Gastroesophageal reflux is common in asthmatic patients because it is increased by bronchodilators. Although acid reflux might trigger reflex bronchoconstriction, it rarely causes asthma symptoms, and antireflux therapy usually fails to reduce asthma symptoms in most patients. STRESS Many asthmatics report worsening of symptoms with stress. Psychological factors can induce bronchoconstriction through cholinergic reflex pathways. Paradoxically, very severe stress such as bereavement usually does not worsen, and may even improve, asthma symptoms.

1	Asthma is associated with a specific chronic inflammation of the mucosa of the lower airways. One of the main aims of treatment is to reduce this inflammation.

1	Pathology The pathology of asthma has been revealed through examining the lungs of patients who have died of asthma and from bronchial biopsies. The airway mucosa is infiltrated with activated eosinophils and T lymphocytes, and there is activation of mucosal mast cells. The degree of inflammation is poorly related to disease 1672 severity and may even be found in atopic patients without asthma symptoms. This inflammation is usually reduced by treatment with ICS. There are also structural changes in the airways (described as remodeling). A characteristic finding is thickening of the basement membrane due to subepithelial collagen deposition. This feature is also found in patients with eosinophilic bronchitis presenting as cough who do not have asthma and is, therefore, likely to be a marker of eosinophilic inflammation in the airway as eosinophils release fibrogenic mediators. The epithelium is often shed or friable, with reduced attachments to the airway wall and increased numbers of

1	of eosinophilic inflammation in the airway as eosinophils release fibrogenic mediators. The epithelium is often shed or friable, with reduced attachments to the airway wall and increased numbers of epithelial cells in the lumen. The airway wall itself may be thickened and edematous, particularly in fatal asthma. Another common finding in fatal asthma is occlusion of the airway lumen by a mucous plug, which is comprised of mucous glycoproteins secreted from goblet cells and plasma proteins from leaky bronchial vessels (Fig. 309-1). There is also vasodilation and increased numbers of blood vessels (angiogenesis). Direct observation by bronchoscopy indicates that the airways may be narrowed, erythematous, and edematous. The pathology of asthma is remarkably uniform in different phenotypes of asthma, including atopic (extrinsic), nonatopic (intrinsic), occupational, aspirin-sensitive, and pediatric asthma. These pathologic changes are found in all airways, but do not extend to the lung

1	of asthma, including atopic (extrinsic), nonatopic (intrinsic), occupational, aspirin-sensitive, and pediatric asthma. These pathologic changes are found in all airways, but do not extend to the lung parenchyma; peripheral airway inflammation is found particularly in patients with severe asthma. The involvement of airways may be patchy, and this is consistent with bronchographic findings of uneven narrowing of the airways.

1	Airway Inflammation There is inflammation in the respiratory mucosa from the trachea to terminal bronchioles, but with a predominance in the bronchi (cartilaginous airways); however, it is still uncertain as to how inflammatory cells interact and how inflammation translates into the symptoms of asthma (Fig. 309-2). There is good evidence that the specific pattern of airway inflammation in asthma is associated with airway hyperresponsiveness (AHR), the physiologic abnormality of asthma, which is correlated with variable airflow obstruction. The pattern of inflammation in asthma is characteristic of allergic diseases, with similar inflammatory cells seen in the nasal mucosa in rhinitis. However, an indistinguishable pattern of inflammation is found in intrinsic asthma, and this may reflect local rather than systemic IgE production. Although most attention has focused on the acute inflammatory changes seen in asthma, this is a chronic condition, with inflammation persisting over many

1	local rather than systemic IgE production. Although most attention has focused on the acute inflammatory changes seen in asthma, this is a chronic condition, with inflammation persisting over many years in most patients. The mechanisms involved in persistence of inflammation in asthma are still poorly understood. Superimposed on this chronic inflammatory state are acute inflammatory episodes, which correspond to exacerbations of asthma. Although the common pattern of inflammation in asthma is characterized by eosinophil infiltration, some patients with severe asthma show a neutrophilic pattern of inflammation that is less sensitive to corticosteroids. However, many inflammatory cells are involved in asthma with no key cell that is predominant (Fig. 309-3).

1	MAST CELLS Mast cells are important in initiating the acute bronchoconstrictor responses to allergens and several other indirectly acting stimuli, such as exercise and hyperventilation (via osmolality changes), as well as fog. Activated mucosal mast cells are found at the airway surface in asthma patients and also in the airway smooth-muscle layer, whereas this is not seen in normal subjects or patients with eosinophilic bronchitis. Mast cells are activated by allergens through an IgE-dependent mechanism, and binding of specific IgE to mast cells renders them more sensitive to activation by physical stimuli such as osmolality. The importance of IgE in the pathophysiology of asthma has been highlighted by clinical studies with humanized anti-IgE antibodies, which inhibit IgE-mediated effects, reduce asthma symptoms, and reduce exacerbations. There are, however, uncertainties about the role of mast cells in more chronic allergic inflammatory events. Mast cells release several

1	effects, reduce asthma symptoms, and reduce exacerbations. There are, however, uncertainties about the role of mast cells in more chronic allergic inflammatory events. Mast cells release several bronchoconstrictor mediators, including histamine, prostaglandin D2, and cysteinyl-leukotrienes, but also several cytokines, chemokines, growth factors, and neurotrophins.

1	FIGURE 309-1 Histopathology of a small airway in fatal asthma. The lumen is occluded with a mucous plug, there is goblet cell metaplasia, and the airway wall is thickened, with an increase in basement membrane thickness and airway smooth muscle. (Courtesy of Dr. J. Hogg, University of British Colombia.)

1	MACROPHAGES AND DENDRITIC CELLS Macrophages, which are derived from blood monocytes, may traffic into the airways in asthma and may be activated by allergens via low-affinity IgE receptors (FcεRII). Macrophages have the capacity to initiate a type of inflammatory response via the release of a certain pattern of cytokines, but these cells also release anti-inflammatory mediators (e.g., IL-10), and thus, their roles in asthma are uncertain. Dendritic cells are specialized macrophage-like cells in the airway epithelium, which are the major antigen-presenting cells. Dendritic cells take up allergens, process them to peptides, and migrate to local lymph nodes where they present the allergenic peptides to uncommitted T lymphocytes to program the production of allergen-specific T cells. Immature dendritic cells in the respiratory tract promote TH2 cell differentiation and require cytokines, such as IL-12 and tumor necrosis factor α (TNF-α), to promote the normally preponderant TH1 response.

1	dendritic cells in the respiratory tract promote TH2 cell differentiation and require cytokines, such as IL-12 and tumor necrosis factor α (TNF-α), to promote the normally preponderant TH1 response. The cytokine thymic stromal lymphopoietin (TSLP) released from epithelial cells in asthmatic patients instructs dendritic cells to release chemokines that attract TH2 cells into the airways.

1	Allergens Sensitizers Viruses Air pollutants? InflammationChronic eosinophilic bronchitis SymptomsCough Wheeze Chest tightness Dyspnea TriggersAllergens Exercise Cold air SO2 Particulates Airwayhyperresponsiveness FIGURE 309-2 Inflammation in the airways of asthmatic patients leads to airway hyperresponsiveness and symptoms. SO2, sulfur dioxide. Allergen bronchial biopsies have demonstrated a 1673 preponderance of natural killer CD4+ T lymphocytes that express high levels of IL-4. Regulatory T cells play an important role in determining the expression of other T cells, and there is evidence for a reduction in a certain subset of regulatory T cells (CD4+CD25+) in asthma that is associated with increased TH2 cells. Recently, innate T cells (ILC2) without T cell receptors have been identified that release TH2 cytokines and are Eosinophil Neutrophil regulated by epithelial cytokines, such as IL-25 and IL-33. STRUCTURAL CELLS Structural cells of the

1	Eosinophil Neutrophil regulated by epithelial cytokines, such as IL-25 and IL-33. STRUCTURAL CELLS Structural cells of the Epithelial airways, including epithelial cells, fibroshedding blasts, and airway smooth-muscle cells, are also important sources of inflam- Nerve activation matory mediators, such as cytokines and lipid mediators, in asthma. Indeed, because structural cells far outnumber Subepithelial fibrosis inflammatory cells, they may become the major sources of mediators driv-Plasma leak Myofibroblast airways. In addition, epithelial cells may inflammatory response and are probably major target cells for ICS. Brochoconstriction asthma, and they may have a variety of effects on the airways that account for the pathologic features of asthma (Fig. FIGURE 309-3 The pathophysiology of asthma is complex with participation of several interact 309-4). Mediators such as histamine, pros ing inflammatory cells, which result in acute and chronic inflammatory effects on the airway.

1	EOSINOPHILS Eosinophil infiltration is a characteristic feature of asthmatic airways. Allergen inhalation results in a marked increase in activated eosinophils in the airways at the time of the late reaction. Eosinophils are linked to the development of AHR through the release of basic proteins and oxygen-derived free radicals. Eosinophil recruitment involves adhesion of eosinophils to vascular endothelial cells in the airway circulation due to interaction between adhesion molecules, migration into the submucosa under the direction of chemokines, and their subsequent activation and prolonged survival. Blocking antibodies to IL-5 causes a profound and prolonged reduction in circulating and sputum eosinophils, but is not associated with reduced AHR or asthma symptoms, although in selected patients with steroid-resistant airway eosinophils, there is a reduction in exacerbations. Eosinophils may be important in release of growth factors involved in airway remodeling and in exacerbations

1	patients with steroid-resistant airway eosinophils, there is a reduction in exacerbations. Eosinophils may be important in release of growth factors involved in airway remodeling and in exacerbations but probably not in AHR.

1	NEUTROPHILS Increased numbers of activated neutrophils are found in sputum and airways of some patients with severe asthma and during exacerbations, although there is a proportion of patients even with mild or moderate asthma who have a predominance of neutrophils. The roles of neutrophils in asthma that are resistant to the anti-inflammatory effects of corticosteroids are currently unknown.

1	T LYMPHOCYTES T lymphocytes play a very important role in coordinating the inflammatory response in asthma through the release of specific patterns of cytokines, resulting in the recruitment and survival of eosinophils and in the maintenance of a mast cell population in the airways. The naïve immune system and the immune system of asthmatics are skewed to express the TH2 phenotype, whereas in normal airways, TH1 cells predominate. TH2 cells, through the release of IL-5, are associated with eosinophilic inflammation and, through the release of IL-4 and IL-13, are associated with increased IgE formation. Recently, taglandin D2, and cysteinyl-leukotrienes contract airway smooth muscle, increase microvascular leakage, increase airway mucus secretion, and attract other inflammatory cells. Because each mediator has many effects, the role of individual mediators in the pathophysiology of asthma is not yet clear. Although the multiplicity of mediators makes it unlikely that preventing the

1	each mediator has many effects, the role of individual mediators in the pathophysiology of asthma is not yet clear. Although the multiplicity of mediators makes it unlikely that preventing the synthesis or action of a single mediator will have a major impact in clinical asthma, recent clinical studies with antileukotrienes suggest that cysteinyl-leukotrienes have clinically important effects.

1	CYTOKINES Multiple cytokines regulate the chronic inflammation of asthma. The TH2 cytokines IL-4, IL-5, and IL-13 mediate allergic inflammation, whereas proinflammatory cytokines, such as TNF-α and IL-1β, amplify the inflammatory response and play a role in more FIGURE 309-4 Many cells and mediators are involved in asthma and lead to several effects on the airways. AHR, airway hyperresponsiveness; PAF, platelet-activating factor. Dendritic cell CCL17, CCL22 CCL11 TSLP Allergens Viruses IL-25, IL-33 ILC2TH2 IL-5 IL-5 Eosinophils

1	Dendritic cell CCL17, CCL22 CCL11 TSLP Allergens Viruses IL-25, IL-33 ILC2TH2 IL-5 IL-5 Eosinophils FIGURE 309-5 T lymphocytes in asthma. Allergen interacts with dendritic cells and releases thymus stimulated lymphopoeitin (TSLP), which stimulates activated dendritic cells to release the chemokines CCL17 and CCL22, which attract T helper 2 (TH2) lymphocytes. Allergens and viral infection may release interleukin (IL)-25 and -33, which recruit and activate type 2 innate lymphoid cells (ILC2). Both TH2 and ILC2 cells release IL-5 and epithelial cells release CCL11 (eotaxin), which together lead to recruitment of eosinophils into the airways. severe disease. TSLP is an upstream cytokine released from epithelial cells of asthmatics that orchestrates the release of chemokines that selectively attract TH2 cells. Some cytokines such as IL-10 and IL-12 are anti-inflammatory and may be deficient in asthma.

1	CHEMOKINES Chemokines are involved in attracting inflammatory cells from the bronchial circulation into the airways. Eotaxin (CCL11) is selectively attractant to eosinophils via CCR3 and is expressed by epithelial cells of asthmatics, whereas CCL17 (TARC) and CCL22 (MDC) from epithelial cells attract TH2 cells via CCR4 (Fig. 309-5). OXIDATIVE STRESS Activated inflammatory cells such as macrophages and eosinophils produce reactive oxygen species. Evidence for increased oxidative stress in asthma is provided by the increased concentrations of 8-isoprostane (a product of oxidized arachidonic acid) in exhaled breath condensates and increased ethane (a product of lipid peroxidation) in the expired air of asthmatic patients. Increased oxidative stress is related to disease severity, may amplify the inflammatory response, and may reduce responsiveness to corticosteroids.

1	NITRIC OXIDE Nitric oxide (NO) is produced by NO synthases in several cells in the airway, particularly airway epithelial cells and macrophages. The level of NO in the expired air of patients with asthma is higher than normal and is related to the eosinophilic inflammation. Increased NO may contribute to the bronchial vasodilation observed in asthma. Fractional exhaled NO (FENO) is increasingly used in the diagnosis and monitoring of asthmatic inflammation, although it is not yet used routinely in clinical practice. TRANSCRIPTION FACTORS Proinflammatory transcription factors, such as nuclear factor-κB (NF-κB) and activator protein-1, are activated in asthmatic airways and orchestrate the expression of multiple inflammatory genes. More specific transcription factors that are involved include nuclear factor of activated T cells and GATA-3, which regulate the expression of TH2 cytokines in T cells.

1	Effects of Inflammation The chronic inflammatory response has several effects on the target cells of the airways, resulting in the characteristic pathophysiologic and remodeling changes associated with asthma. Asthma may be regarded as a disease with continuous inflammation and repair proceeding simultaneously, although the relationship between chronic inflammatory processes and asthma symptoms is often obscure.

1	AIRWAY EPITHELIUM Airway epithelial shedding may be important in contributing to AHR and may explain how several mechanisms, such as ozone exposure, virus infections, chemical sensitizers, and allergens (usually proteases), can lead to its development, as all of these stimuli may lead to epithelial disruption. Epithelial damage may contribute to AHR in a number of ways, including loss of its barrier function to allow penetration of allergens; loss of enzymes (such as neutral endopeptidase) that degrade certain peptide inflammatory mediators; loss of a relaxant factor (so called epithelial-derived relaxant factor); and exposure of sensory nerves, which may lead to reflex neural effects on the airway.

1	FIBROSIS In all asthmatic patients, the basement membrane is apparently thickened due to subepithelial fibrosis with deposition of types III and V collagen below the true basement membrane and is associated with eosinophil infiltration, presumably through the release of profibrotic mediators such as transforming growth factor-β. Mechanical manipulations can alter the phenotype of airway epithelial cells in a profibrotic fashion. In more severe patients, there is also fibrosis within the airway wall, which may contribute to irreversible narrowing of the airways.

1	AIRWAY SMOOTH MUSCLE In vitro airway smooth muscle from asthmatic patients usually shows no increased responsiveness to constrictors. Reduced responsiveness to β-agonists has also been reported in postmortem or surgically removed bronchi from asthmatics, although the number of β-receptors is not reduced, suggesting that β-receptors have been uncoupled. These abnormalities of airway smooth muscle may be secondary to the chronic inflammatory process. Inflammatory mediators may modulate the ion channels that serve to regulate the resting membrane potential of airway smooth-muscle cells, thus altering the level of excitability of these cells. In asthmatic airways there is also a characteristic hypertrophy and hyperplasia of airway smooth muscle, which is presumably the result of stimulation of airway smooth-muscle cells by various growth factors such as platelet-derived growth factor (PDGF) or endothelin-1 released from inflammatory or epithelial cells.

1	VASCULAR RESPONSES There is increased airway mucosal blood flow in asthma, which may contribute to airway narrowing. There is an increase in the number of blood vessels in asthmatic airways as a result of angiogenesis in response to growth factors, particularly vascular endothelial growth factor. Microvascular leakage from postcapillary venules in response to inflammatory mediators is observed in asthma, resulting in airway edema and plasma exudation into the airway lumen. MUCUS HYPERSECRETION Increased mucus secretion contributes to the viscid mucous plugs that occlude asthmatic airways, particularly in fatal asthma. There is hyperplasia of submucosal glands that are confined to large airways and of increased numbers of epithelial goblet cells. IL-13 induces mucus hypersecretion in experimental models of asthma.

1	NEURAL REGULATION Various defects in autonomic neural control may contribute to AHR in asthma, but these are likely to be secondary to the disease, rather than primary defects. Cholinergic pathways, through the release of acetylcholine acting on muscarinic receptors, cause bronchoconstriction and may be activated reflexly in asthma. predominant nonproductive cough (cough-variant asthma). There 1675 may be no abnormal physical findings when asthma is under control. The diagnosis of asthma is usually apparent from the symptoms of variable and intermittent airways obstruction, but must be confirmed by objective measurements of lung function.

1	The diagnosis of asthma is usually apparent from the symptoms of variable and intermittent airways obstruction, but must be confirmed by objective measurements of lung function. Lung Function Tests Simple spirometry confirms airflow limitation with a reduced FEV1, FEV1/FVC ratio, and PEF (Fig. 309-6). Reversibility is demonstrated by a >12% and 200-mL increase in FEV1 15 min after an inhaled short-acting β2-agonist or in some patients by a 2to 4-week trial of oral corticosteroids (OCS) (prednisone or prednisolone 30–40 mg daily). Measurements of PEF twice daily may confirm the diurnal variations in airflow obstruction. Flow-volume loops show reduced peak flow and reduced maximum expiratory flow. Further lung function tests are rarely necessary, but whole-body plethysmography shows increased airway resistance and may show increased total lung capacity and residual volume. Gas diffusion is usually normal, but there may be a small increase in gas transfer in some patients.

1	Inflammatory mediators may activate sensory nerves, resulting in reflex cholinergic bronchoconstriction or release of inflammatory neuropeptides. Inflammatory products may also sensitize sensory nerve endings in the airway epithelium such that the nerves become hyperalgesic. Neurotrophins, which may be released from various cell types in airways, including epithelial cells and mast cells, may cause proliferation and sensitization of airway sensory nerves. Airway nerves may also release neurotransmitters, such as substance P, which have inflammatory effects. Airway Remodeling Several changes in the structure of the airway are characteristically found in asthma, and these may lead to irreversible narrowing of the airways. Population studies have shown a greater decline in lung function over time than in normal subjects; however, most patients with asthma preserve normal or near-normal lung function throughout life if appropriately treated.

1	The accelerated decline in lung function occurs in a smaller proportion of asthmatics, and these are usually patients with more severe disease. There is some evidence that the early use of ICS may reduce the decline in lung function. The characteristic structural changes are increased airway smooth muscle, fibrosis, angiogenesis, and mucus hyperplasia.

1	Physiology Limitation of airflow is due mainly to bronchoconstriction, but airway edema, vascular congestion, and luminal occlusion with exudate may contribute. This results in a reduction in forced expiratory volume in 1 second (FEV1), FEV1/forced vital capacity (FVC) ratio, and peak expiratory flow (PEF), as well as an increase in airway resistance. Early closure of peripheral airway results in lung hyperinflation (air trapping) and increased residual volume, particularly during acute exacerbations and in severe persistent asthma. In more severe asthma, reduced ventilation and increased pulmonary blood flow result in mismatching of ventilation and perfusion and in bronchial hyperemia. Ventilatory failure is very uncommon, even in patients with severe asthma, and arterial PCO2 tends to be low due to increased ventilation.

1	Airway Hyperresponsiveness AHR is the characteristic physiologic abnormality of asthma and describes the excessive bronchoconstrictor response to multiple inhaled triggers that would have no effect on normal airways. The increase in AHR is linked to the frequency of asthma symptoms, and, thus, an important aim of therapy is to reduce AHR. Increased bronchoconstrictor responsiveness is seen with direct bronchoconstrictors such as histamine and methacholine, which contract airway smooth muscle, but is characteristically also seen with many indirect stimuli, which release bronchoconstrictors from mast cells or activate sensory nerves. Most of the triggers for asthma symptoms appear to act indirectly, including allergens, exercise, hyperventilation, fog (via mast cell activation), irritant dusts, and sulfur dioxide (via a cholinergic reflex).

1	The characteristic symptoms of asthma are wheez ing, dyspnea, and coughing, which are variable, both spontaneously and with therapy. Symptoms may be worse at night, and patients typically awake in the early morning hours. Patients may report difficulty in filling their lungs with air. There is increased mucus production in some patients, with typically tenacious mucus that is difficult to expectorate. There may be increased ventilation and use of accessory muscles of ventilation. Prodromal symptoms may precede

1	Airway Responsiveness The increased AHR is normally measured by methacholine or histamine challenge with calculation of the provocative concentration that reduces FEV1 by 20% (PC20). This is rarely useful in clinical practice, but can be used in the differential diagnosis of chronic cough and when the diagnosis is in doubt in the setting of normal pulmonary function tests. Occasionally exercise testing is done to demonstrate the postexercise bronchoconstriction if there is a predominant history of EIA. Allergen challenge is rarely necessary and should only be undertaken by a specialist if specific occupational agents are to be identified. Hematologic Tests Blood tests are not usually helpful. Total serum IgE and specific IgE to inhaled allergens (radioallergosorbent test [RAST]) may be measured in some patients.

1	Hematologic Tests Blood tests are not usually helpful. Total serum IgE and specific IgE to inhaled allergens (radioallergosorbent test [RAST]) may be measured in some patients. Imaging Chest roentgenography is usually normal but in more severe patients may show hyperinflated lungs. In exacerbations, there may be evidence of a pneumothorax. Lung shadowing usually indicates pneumonia or eosinophilic infiltrates in patients with bronchopulmonary aspergillosis. High-resolution computed tomography (CT) may show areas of bronchiectasis in patients with severe asthma, and there may be thickening of the bronchial walls, but these changes are not diagnostic of asthma. Skin Tests Skin prick tests to common inhalant allergens (house dust mite, cat fur, grass pollen) are positive in allergic asthma and negative

1	Skin Tests Skin prick tests to common inhalant allergens (house dust mite, cat fur, grass pollen) are positive in allergic asthma and negative Flow (liters/min) FEV1 = 3.5 L FVC = 4.0 L FEV1 = 2.2 L FVC = 3.6 L Normal Asthma Spirometry 10 5 0 Normal Asthma Flow-volume loop FEV1/FVC = 61% PEF an attack, with itching under the chin, discomfort between the scapulae, or inexplicable fear (impend ing doom). FIGURE 309-6 Spirometry and flow-volume loop in asthmatic compared to nor- Typical physical signs are inspiratory, and to mal subject. There is a reduction in forced expiratory volume in 1 second (FEV1) but a greater extent expiratory, rhonchi throughout less reduction in forced vital capacity (FVC), giving a reduced FEV1/FVC ratio (<70%). the chest, and there may be hyperinflation. Some The flow-volume loop shows reduced peak expiratory flow and a typical scalloped patients, particularly children, may present with a appearance indicating widespread airflow obstruction.

1	1676 in intrinsic asthma, but are not helpful in diagnosis. Positive skin responses may be useful in persuading patients to undertake allergen avoidance measures. Exhaled Nitric Oxide FENO is now being used as a noninvasive test to measure airway inflammation. The typically elevated levels in asthma are reduced by ICS, so this may be a test of compliance with therapy. It may also be useful in demonstrating insufficient anti-inflammatory therapy and may be useful in down-titrating ICS. However, studies in unselected patients have not convincingly demonstrated improved clinical outcomes, and it may be necessary to select patients who are poorly controlled.

1	Differential Diagnosis It is usually not difficult to differentiate asthma from other conditions that cause wheezing and dyspnea. Upper airway obstruction by a tumor or laryngeal edema can mimic severe asthma, but patients typically present with stridor localized to large airways. The diagnosis is confirmed by a flow-volume loop that shows a reduction in inspiratory as well as expiratory flow, and bronchoscopy to demonstrate the site of upper airway narrowing. Persistent wheezing in a specific area of the chest may indicate endobronchial obstruction with a foreign body. Left ventricular failure may mimic the wheezing of asthma, but basilar crackles are present in contrast to asthma. Vocal chord dysfunction may mimic asthma and is thought to be an hysterical conversion syndrome.

1	Eosinophilic pneumonias and systemic vasculitis, including Churg-Strauss syndrome and polyarteritis nodosa, may be associated with wheezing. Chronic obstructive pulmonary disease (COPD) is usually easy to differentiate from asthma as symptoms show less variability, never completely remit, and show much less (or no) reversibility to bronchodilators. Approximately 10% of COPD patients have features of asthma, with increased sputum eosinophils and a response to OCSs; these patients probably have both diseases concomitantly.

1	The treatment of asthma is straightforward, and the majority of patients are now managed by internists and family doctors with effective and safe therapies. There are several aims of therapy (Table 309-2). Most emphasis has been placed on drug therapy, but several nonpharmacologic approaches have also been used. The main drugs for asthma can be divided into bronchodilators, which give rapid relief of symptoms mainly through relaxation of airway smooth muscle, and controllers, which inhibit the underlying inflammatory process.

1	Bronchodilators act primarily on airway smooth muscle to reverse the bronchoconstriction of asthma. This gives rapid relief of symptoms but has little or no effect on the underlying inflammatory process. Thus, bronchodilators are not sufficient to control asthma in patients with persistent symptoms. There are three classes of bronchodilators in current use: β2-adrenergic agonists, anticholinergics, and theophylline; of these, β2-agonists are by far the most effective. β2-Agonists β2-Agonists activate β2-adrenergic receptors, which are widely expressed in the airways. β2-Receptors are coupled through a stimulatory G protein to adenylyl cyclase, resulting in increased aiMS of aSthMa therapy (ideally no) chronic symptoms, including nocturnal (ideally no) use of a required β2-agonist limitations on activities, including exercise intracellular cyclic adenosine monophosphate (AMP), which relaxes smooth-muscle cells and inhibits certain inflammatory cells, particularly mast cells.

1	MODE OF ACTION The primary action of β2-agonists is to relax airway smooth-muscle cells of all airways, where they act as functional antagonists, reversing and preventing contraction of airway smooth-muscle cells by all known bronchoconstrictors. This generalized action is likely to account for their great efficacy as bronchodilators in asthma. There are also additional nonbronchodilator effects that may be clinically useful, including inhibition of mast cell mediator release, reduction in plasma exudation, and inhibition of sensory nerve activation. Inflammatory cells express small numbers of β2receptors, but these are rapidly downregulated with β2-agonist activation so that, in contrast to corticosteroids, there are no effects on inflammatory cells in the airways and there is no reduction in AHR.

1	CLINICAL USE β2-Agonists are usually given by inhalation to reduce side effects. Short-acting β2-agonists (SABAs) such as albuterol and terbutaline have a duration of action of 3–6 h. They have a rapid onset of bronchodilatation and are, therefore, used as needed for symptom relief. Increased use of SABA indicates that asthma is not controlled. They are also useful in preventing EIA if taken prior to exercise. SABAs are used in high doses by nebulizer or via a metered-dose inhaler with a spacer. Long-acting β2-agonists (LABAs) include salmeterol and formoterol, both of which have a duration of action over 12 h and are given twice daily by inhalation; indacaterol is given once daily. LABAs have replaced the regular use of SABAs, but LABAs should not be given in the absence of ICS therapy because they do not control the underlying inflammation. They do, however, improve asthma control and reduce exacerbations when added to ICS, which allows asthma to be controlled at lower doses of

1	because they do not control the underlying inflammation. They do, however, improve asthma control and reduce exacerbations when added to ICS, which allows asthma to be controlled at lower doses of corticosteroids. This observation has led to the widespread use of fixed-combination inhalers that contain a corticosteroid and a LABA, which have proved to be highly effective in the control of asthma.

1	SIDE EFFECTS Adverse effects are not usually a problem with β2agonists when given by inhalation. The most common side effects are muscle tremor and palpitations, which are seen more commonly in elderly patients. There is a small fall in plasma potassium due to increased uptake by skeletal muscle cells, but this effect does not usually cause any clinical problem. TOLERANCE Tolerance is a potential problem with any agonist given chronically, but although there is downregulation of β2-receptors, this does not reduce the bronchodilator response because there is a large receptor reserve in airway smooth-muscle cells. By contrast, mast cells become rapidly tolerant, but their tolerance may be prevented by concomitant administration of ICS.

1	SAFETY The safety of β2-agonists has been an important issue. There is an association between asthma mortality and the amount of SABA used, but careful analysis demonstrates that the increased use of rescue SABA reflects poor asthma control, which is a risk factor for asthma death. The slight excess in mortality that has been associated with the use of LABA is related to the lack of use of concomitant ICS, as the LABA therapy fails to suppress the underlying inflammation. This highlights the importance of always using an ICS when LABAs are given, which is most conveniently achieved by using a combination inhaler.

1	Anticholinergics Muscarinic receptor antagonists such as ipratropium bromide prevent cholinergic nerve-induced bronchoconstriction and mucus secretion. They are less effective than β2-agonists in asthma therapy because they inhibit only the cholinergic reflex component of bronchoconstriction, whereas β2-agonists prevent all bronchoconstrictor mechanisms. Anticholinergics, including once-daily tiotropium bromide, may be used as an additional bronchodilator in patients with asthma that is not controlled by ICS and LABA combinations. High doses may be given by nebulizer in treating acute severe asthma but should only be given following β2-agonists, because they have a slower onset of bronchodilatation. Side effects are not usually a problem because there is little or no systemic absorption. The most common side effect is dry mouth; in elderly patients, urinary retention and glaucoma may also be observed.

1	Theophylline Theophylline was widely prescribed as an oral bronchodilator several years ago, especially because it was inexpensive. It has now fallen out of favor because side effects are common and inhaled β2-agonists are much more effective as bronchodilators. The bronchodilator effect is due to inhibition of phosphodiesterases in airway smooth-muscle cells, which increases cyclic AMP, but doses required for bronchodilatation commonly cause side effects that are mediated mainly by phosphodiesterase inhibition. There is increasing evidence that theophylline at lower doses has anti-inflammatory effects, and these are likely to be mediated through different molecular mechanisms. Theophylline activates the key nuclear enzyme histone deacetylase-2 (HDAC2), which is a critical mechanism for switching off activated inflammatory genes and may, therefore, reduce corticosteroid insensitivity in severe asthma.

1	CLINICAL USE Oral theophylline is usually given as a slow-release preparation once or twice daily because this gives more stable plasma concentrations than normal theophylline tablets. It may be used as an additional bronchodilator in patients with severe asthma when plasma concentrations of 10–20 mg/L are required, although these concentrations are often associated with side effects. Low doses of theophylline, giving plasma concentrations of 5–10 mg/L, have additive effects to ICS and are particularly useful in patients with severe asthma. Indeed, withdrawal of theophylline from these patients may result in marked deterioration in asthma control. At low doses, the drug is well tolerated. IV aminophylline (a soluble salt of theophylline) was used for the treatment of severe asthma but has now been largely replaced by high doses of inhaled SABA, which are more effective and have fewer side effects. Aminophylline is occasionally used (via slow IV infusion) in patients with severe

1	has now been largely replaced by high doses of inhaled SABA, which are more effective and have fewer side effects. Aminophylline is occasionally used (via slow IV infusion) in patients with severe exacerbations that are refractory to SABA.

1	SIDE EFFECTS Oral theophylline is well absorbed and is largely inactivated in the liver. Side effects are related to plasma concentrations; measurement of plasma theophylline may be useful in determining the correct dose. The most common side effects are nausea, vomiting, and headaches and are due to phosphodiesterase inhibition. Diuresis and palpitations may also occur, and at high concentrations, cardiac arrhythmias, epileptic seizures, and death may occur due to adenosine A1-receptor antagonism. Theophylline side effects are related to plasma concentration and are rarely observed at plasma concentrations below 10 mg/L. Theophylline is metabolized by CYP450 in the liver, and thus, plasma concentrations may be elevated by drugs that block CYP450 such as erythromycin and allopurinol. Other drugs may also reduce clearance by other mechanisms leading to increased plasma concentrations (Table 309-3).

1	induction (rifampicin, phenobarbitone, ethanol) (tobacco, marijuana) • High-protein, • Enzyme inhibition (cimetidine, erythromycin, ciprofloxacin, allopurinol, zileuton, zafirlukast) CONTROLLER THERAPIES 1677 Inhaled Corticosteroids ICSs are by far the most effective controllers for asthma, and their early use has revolutionized asthma therapy. MODE OF ACTION ICSs are the most effective anti-inflammatory agents used in asthma therapy, reducing inflammatory cell numbers and their activation in the airways. ICSs reduce eosinophils in the airways and sputum and the numbers of activated T lymphocytes and surface mast cells in the airway mucosa. These effects may account for the reduction in AHR that is seen with chronic ICS therapy.

1	The molecular mechanism of action of corticosteroids involves several effects on the inflammatory process. The major effect of corticosteroids is to switch off the transcription of multiple activated genes that encode inflammatory proteins such as cytokines, chemokines, adhesion molecules, and inflammatory enzymes. This effect involves several mechanisms, including inhibition of the transcription factor NF-κB, but an important mechanism is recruitment of HDAC2 to the inflammatory gene complex, which reverses the histone acetylation associated with increased gene transcrip tion. Corticosteroids also activate anti-inflammatory genes, such as mitogen-activated protein (MAP) kinase phosphatase-1, and increase the expression of β2-receptors. Most of the metabolic and endocrine side effects of corticosteroids are also mediated through transcriptional activation.

1	CLINICAL USE ICSs are by far the most effective controllers in the management of asthma and are beneficial in treating asthma of any severity and age. ICSs are usually given twice daily, but some may be effective once daily in mildly symptomatic patients. ICSs rapidly improve the symptoms of asthma, and lung function improves over several days. They are effective in preventing asthma symptoms, such as EIA and nocturnal exacerbations, but also prevent severe exacerbations. ICSs reduce AHR, but maximal improvement may take several months of therapy. Early treatment with ICS appears to prevent irreversible changes in airway function that occur with chronic asthma. Withdrawal of ICS results in slow deterioration of asthma control, indicating that they suppress inflammation and symptoms, but do not cure the underlying condition. ICSs are now given as first-line therapy for patients with persistent asthma, but if they do not control symptoms at low doses, it is usual to add a LABA as the

1	do not cure the underlying condition. ICSs are now given as first-line therapy for patients with persistent asthma, but if they do not control symptoms at low doses, it is usual to add a LABA as the next step.

1	SIDE EFFECTS Local side effects include hoarseness (dysphonia) and oral candidiasis, which may be reduced with the use of a large-volume spacer device. There has been concern about systemic side effects from lung absorption, but many studies have demonstrated that ICS have minimal systemic effects (Fig. 309-7). At the highest recommended doses, there may be some suppression of plasma and urinary cortisol concentrations, but there is no convincing evidence that long-term treatment leads to impaired growth in children or to osteoporosis in adults. Indeed effective control of asthma with ICS reduces the number of courses of OCS that are needed and, thus, reduces systemic exposure to ICS.

1	Systemic Corticosteroids Corticosteroids are used intravenously (hydrocortisone or methylprednisolone) for the treatment of acute severe asthma, although several studies now show that OCSs are as effective and easier to administer. A course of OCS (usually prednisone or prednisolone 30–45 mg once daily for 5–10 days) is used to treat acute exacerbations of asthma; no tapering of the dose is needed. Approximately 1% of asthma patients may require maintenance treatment with OCS; the lowest dose necessary to maintain control needs to be determined. Systemic side effects, including truncal obesity, bruising, osteoporosis, diabetes, hypertension, gastric ulceration, proximal myopathy, depression, and cataracts, may be a major problem, and steroid-sparing therapies may be considered if side effects are a significant problem. If patients require maintenance treatment with OCS, it is important to monitor bone density so that preventive treatment with bisphosphonates or estrogen in

1	if side effects are a significant problem. If patients require maintenance treatment with OCS, it is important to monitor bone density so that preventive treatment with bisphosphonates or estrogen in postmenopausal women may be initiated if bone density is low.

1	FIGURE 309-7 Pharmacokinetics of inhaled corticosteroids. GI, gastrointestinal; MDI, metered-dose inhaler. Intramuscular triamcinolone acetonide is a depot preparation that is occasionally used in noncompliant patients, but proximal myopathy is a major problem with this therapy.

1	Antileukotrienes Cysteinyl-leukotrienes are potent bronchoconstrictors, cause microvascular leakage, and increase eosinophilic inflammation through the activation of cys-LT1-receptors. These inflammatory mediators are produced predominantly by mast cells and, to a lesser extent, eosinophils in asthma. Antileukotrienes, such as montelukast, block cys-LT1-receptors and provide modest clinical benefit in asthma. They are less effective than ICS in controlling asthma and have less effect on airway inflammation, but are useful as an add-on therapy in some patients not controlled with low doses of ICS, although less effective than LABA. They are given orally once or twice daily and are well tolerated. Some patients show a better response than others to antileukotrienes, but this has not been convincingly linked to any genomic differences in the leukotriene pathway.

1	Cromones Cromolyn sodium and nedocromil sodium are asthma controller drugs that appear to inhibit mast cell and sensory nerve activation and are, therefore, effective in blocking trigger-induced asthma such as EIA and allergenand sulfur dioxide–induced symptoms. Cromones have relatively little benefit in the long-term control of asthma due to their short duration of action (at least four times daily by inhalation). They are very safe and were popular in the treatment of childhood asthma, although now low doses of ICS are preferred because they are more effective and have a proven safety profile.

1	Steroid-Sparing Therapies Various immunomodulatory treatments have been used to reduce the requirement for OCS in patients with severe asthma who have serious side effects with this therapy. Methotrexate, cyclosporin A, azathioprine, gold, and IV gamma globulin have all been used as steroid-sparing therapies, but none of these treatments has any long-term benefit, and each is associated with a relatively high risk of side effects.

1	Anti-IgE Omalizumab is a blocking antibody that neutralizes circulating IgE without binding to cell-bound IgE and, thus, inhibits IgE-mediated reactions. This treatment has been shown to reduce the number of exacerbations in patients with severe asthma and may improve asthma control. However, the treatment is very expensive and is only suitable for highly selected patients who are not controlled on maximal doses of inhaler therapy and have a circulating IgE within a specified range. Patients should be given a 3to 4-month trial of therapy to show objective benefit. Omalizumab is usually given as a subcutaneous injection every 2–4 weeks and appears not to have significant side effects, although anaphylaxis is very occasionally seen.

1	Immunotherapy Specific immunotherapy using injected extracts of pollens or house dust mites has not been very effective in controlling asthma and may cause anaphylaxis. Side effects may be reduced by sublingual dosing. It is not recommended in most asthma treatment guidelines because of lack of evidence of clinical efficacy. Alternative Therapies Nonpharmacologic treatments, including hypnosis, acupuncture, chiropraxis, breathing control, yoga, and speleotherapy, may be popular with some patients. However, placebo-controlled studies have shown that each of these treatments lacks efficacy and cannot be recommended. However, they are not detrimental and may be used as long as conventional pharmacologic therapy is continued.

1	Future Therapies It has proved very difficult to discover novel pharmaceutical therapies, particularly because current therapy with corticosteroids and β2-agonists is so effective in the majority of patients. There is, however, a need for the development of new therapies for patients with refractory asthma who have side effects with systemic corticosteroids. Antagonists of specific mediators have little or no benefit in asthma, apart from antileukotrienes, which have rather weak effects, presumably reflecting the fact that multiple mediators are involved. Blocking antibodies against IL-5 may reduce exacerbations in highly selected patients who have sputum eosinophils despite high doses of corticosteroids, whereas anti-TNF-α antibodies are not effective in severe asthma. Novel anti-inflammatory treatments that are in clinical development include inhibitors of phosphodiesterase-4, NF-κB, and p38 MAP kinase. However, these drugs, which act on signal transduction pathways common to many

1	treatments that are in clinical development include inhibitors of phosphodiesterase-4, NF-κB, and p38 MAP kinase. However, these drugs, which act on signal transduction pathways common to many cells, are likely to have troublesome side effects, necessitating their delivery by inhalation. Safer and more effective immunotherapy using T cell peptide fragments of allergens or DNA vaccination is also being investigated. Bacterial products, such as CpG oligonucleotides that stimulate TH1 immunity or regulatory T cells, are also currently under evaluation.

1	There are several aims of chronic therapy in asthma (Table 309-2). It is important to establish the diagnosis objectively using spirometry or PEF measurements at home. Triggers that worsen asthma control, such as allergens or occupational agents, should be avoided, whereas triggers, such as exercise and fog, which result in transient symptoms, provide an indication that more controller therapy is needed. It is important to assess asthma control, determined by Controlled (all of Partly Characteristic the following) Controlled Uncontrolled features of partly controlled Limitation of None Any activities Abbreviations: FEV1, forced expiratory volume in 1 s; PEF, peak expiratory flow.

1	Limitation of None Any activities Abbreviations: FEV1, forced expiratory volume in 1 s; PEF, peak expiratory flow. symptoms, night awakening, need for reliever inhalers, limitation of activity, and lung function (Table 309-4). Avoidance of side effects and expense of medications are also important. There are several validated questionnaires for quantifying asthma control, such as the Asthma Quality of Life Questionnaire (AQLQ) and Asthma Control Test (ACT).

1	Stepwise Therapy For patients with mild, intermittent asthma, a short-acting β2-agonist is all that is required (Fig. 309-8). However, use of a reliever medication more than twice a week indicates the need for regular controller therapy. The treatment of choice for all patients is an ICS given twice daily. It is usual to start with an intermediate dose (e.g., 200 μg bid of beclomethasone dipropionate [BDP]) or equivalent and to decrease the dose if symptoms are controlled after 3 months. If symptoms are not controlled, a LABA should be added, which is most conveniently given by switching to a combination inhaler. The dose of controller should be adjusted accordingly, as judged by the need for a rescue inhaler. Low doses of theophylline or an antileukotriene may also be considered as an add-on therapy, but these are less effective than LABA. In patients with severe asthma, low-dose oral theophylline is also helpful, and when there is irreversible airway narrowing, the long-acting

1	an add-on therapy, but these are less effective than LABA. In patients with severe asthma, low-dose oral theophylline is also helpful, and when there is irreversible airway narrowing, the long-acting anticholinergic tiotropium bromide may be tried. If asthma is not controlled despite the maximal recommended dose of inhaled therapy, it is important to check compliance and inhaler technique. In these patients, maintenance treatment with an OCS may be needed, and the lowest dose that maintains control should be used. Occasionally omalizumab may be tried in steroid-dependent asthmatics who are not well controlled. Once asthma is controlled, it is important to slowly decrease therapy in order to find the optimal dose to control symptoms.

1	Education Patients with asthma need to understand how to use their medications and the difference between reliever and controller FIGURE 309-8 Stepwise approach to asthma therapy according to the severity of asthma and ability to control symptoms. ICS, inhaled corticosteroids; LABA, long-acting β2-agonist; OCS, oral corticosteroid. therapies. Education may improve compliance, particularly with ICS. 1679 All patients should be taught how to use their inhalers correctly. In particular, they need to understand how to recognize worsening of asthma and how to step up therapy. Written action plans have been shown to reduce hospital admissions and morbidity rates in adults and children, and are recommended particularly in patients with unstable disease who have frequent exacerbations.

1	Exacerbations of asthma are feared by patients and may be life threatening. One of the main aims of controller therapy is to prevent exacerbations; in this respect, ICS and combination inhalers are very effective.

1	Clinical Features Patients are aware of increasing chest tightness, wheezing, and dyspnea that are often not or poorly relieved by their usual reliever inhaler. In severe exacerbations, patients may be so breathless that they are unable to complete sentences and may become cyanotic. Examination usually shows increased ventilation, hyperinflation, and tachycardia. Pulsus paradoxus may be present, but this is rarely a useful clinical sign. There is a marked fall in spirometric values and PEF. Arterial blood gases on air show hypoxemia, and PCO2 is usually low due to hyperventilation. A normal or rising PCO2 is an indication of impending respiratory failure and requires immediate monitoring and therapy. A chest roentgenogram is not usually informative but may show pneumonia or pneumothorax.

1	A high concentration of oxygen should be given by face mask to achieve oxygen saturation of >90%. The mainstay of treatment are high doses of SABA given either by nebulizer or via a metered-dose inhaler with a spacer. In severely ill patients with impending respiratory failure, IV β2-agonists may be given. A nebulized anticholinergic may be added if there is not a satisfactory response to β2-agonists alone, as there are additive effects. In patients who are refractory to inhaled therapies, a slow infusion of aminophylline may be effective, but it is important to monitor blood levels, especially if patients have already been treated with oral theophylline. Magnesium sulfate given intravenously or by nebulizer is effective when added to inhaled β2-agonists, and is relatively well tolerated but is not routinely recommended. Prophylactic intubation may be indicated for impending respiratory failure, when the PCO2 is normal or rises. For patients with respiratory failure, it is necessary

1	but is not routinely recommended. Prophylactic intubation may be indicated for impending respiratory failure, when the PCO2 is normal or rises. For patients with respiratory failure, it is necessary to intubate and institute ventilation. These patients may benefit from an anesthetic such as halothane if they have not responded to conventional bronchodilators. Sedatives should never be given because they may depress ventilation. Antibiotics should not be used routinely unless there are signs of pneumonia.

1	SPECIAL CONSIDERATIONS Refractory Asthma Although most patients with asthma are easily controlled with appropriate medication, a small proportion of patients (approximately 5–10% of asthmatics) are difficult to control despite maximal inhaled therapy. Some of these patients will require maintenance treatment with OCS. In managing these patients, it is important to investigate and correct any mechanisms that may be aggravating asthma. There are two major patterns of difficult asthma: some patients have persistent symptoms and poor lung function, despite appropriate therapy, whereas others may have normal or near-normal lung function but intermittent, severe (sometimes life-threatening) exacerbations.

1	MECHANISMS The most common reason for poor control of asthma is noncompliance with medication, particularly ICS. Compliance with ICS may be low because patients do not feel any immediate clinical benefit or may be concerned about side effects. Compliance with ICS is difficult to monitor because there are no useful plasma measurements that can be made, but measuring the fractional excretion of 1680 induced NO (FENO) may identify the problem. Compliance may be improved by giving the ICS as a combination with a LABA that gives symptom relief. Compliance with OCS may be measured by suppression of plasma cortisol and the expected concentration of prednisone/ prednisolone in the plasma. There are several factors that may make asthma more difficult to control, including exposure to high, ambient levels of allergens or unidentified occupational agents. Severe rhino-sinusitis may make asthma more difficult to control; upper airway disease should be vigorously treated. Drugs such as

1	ambient levels of allergens or unidentified occupational agents. Severe rhino-sinusitis may make asthma more difficult to control; upper airway disease should be vigorously treated. Drugs such as beta-adrenergic blockers, aspirin, and other cyclooxygenase (COX) inhibitors may worsen asthma. Some women develop severe premenstrual worsening of asthma, which is unresponsive to corticosteroids and requires treatment with progesterone or gonadotropin-releasing factors. Few systemic diseases make asthma more difficult to control, but hyper-and hypothyroidism may increase asthma symptoms and should be investigated if suspected. Bronchial biopsy studies in refractory asthma may show the typical eosinophilic pattern of inflammation, whereas others have a predominantly neutrophilic pattern. There may be an increase in TH1 cells, TH17 cells, and CD8 lymphocytes compared to mild asthma and increased expression of TNF-α. Structural changes in the airway, including fibrosis, angiogenesis, and

1	may be an increase in TH1 cells, TH17 cells, and CD8 lymphocytes compared to mild asthma and increased expression of TNF-α. Structural changes in the airway, including fibrosis, angiogenesis, and airway smooth-muscle thickening, are more commonly seen in these patients.

1	Corticosteroid-Resistant Asthma A few patients with asthma show a poor response to corticosteroid therapy and may have various molecular abnormalities that impair the anti-inflammatory action of corticosteroids. Complete resistance to corticosteroids is extremely uncommon and affects less than 1 in 1000 patients. It is defined by a failure to respond to a high dose of oral prednisone/prednisolone (40 mg once daily over 2 weeks), ideally with a 2-week run-in with matched placebo. More common is reduced responsiveness to corticosteroids where control of asthma requires OCS (corticosteroiddependent asthma). In patients with poor responsiveness to corticosteroids, there is a reduction in the response of circulating monocytes and lymphocytes to the anti-inflammatory effects of corticosteroids in vitro and reduced skin blanching in response to topical corticosteroids. There are several mechanisms that have been described, including an increase in the alternatively spliced form of the

1	in vitro and reduced skin blanching in response to topical corticosteroids. There are several mechanisms that have been described, including an increase in the alternatively spliced form of the glucocorticoid receptor (GR)-β, an abnormal pattern of histone acetylation in response to corticosteroids, a defect in IL-10 production, and a reduction in HDAC2 activity (as in COPD). These observations suggest that there are likely to be heterogeneous mechanisms for corticosteroid resistance; whether these mechanisms are genetically determined has yet to be decided.

1	Brittle Asthma Some patients show chaotic variations in lung function despite taking appropriate therapy. Some show a persistent pattern of variability and may require oral corticosteroids or, at times, continuous infusion of β2-agonists (type 1 brittle asthma), whereas others have generally normal or near-normal lung function but precipitous, unpredictable falls in lung function that may result in death (type 2 brittle asthma). These latter patients are difficult to manage because they do not respond well to corticosteroids, and the worsening of asthma does not reverse well with inhaled bronchodilators. The most effective therapy is subcutaneous epinephrine, which suggests that the worsening is likely to be a localized airway anaphylactic reaction with edema. In some of these patients, there may be allergy to specific foods. These patients should be taught to self-administer epinephrine and should carry a medical warning accordingly.

1	Refractory asthma is difficult to control, by definition. It is important to check compliance and the correct use of inhalers and to identify and eliminate any underlying triggers. Low doses of theophylline may be helpful in some patients, and theophylline withdrawal has been found to worsen in many patients. Most of these patients will require maintenance treatment with oral corticosteroids, and the minimal dose that achieves satisfactory control should be determined by careful dose titration. Steroid-sparing therapies are rarely effective. In some patients with allergic asthma, omalizumab is effective, particularly when there are frequent exacerbations. Anti-TNF therapy is not effective in severe asthma and should not be used. A few patients may benefit from infusions of β2-agonists. New therapies are needed for these patients, who currently consume a disproportionate amount of health care spending.

1	Aspirin-Sensitive Asthma A small proportion (1–5%) of asthmatics become worse with aspirin and other COX inhibitors, although this is much more commonly seen in severe cases and in patients with frequent hospital admission. Aspirin-sensitive asthma is a well-defined phenotype of asthma that is usually preceded by perennial rhinitis and nasal polyps in nonatopic patients with a late onset of the disease. Aspirin, even in small doses, characteristically provokes rhinorrhea, conjunctival injection, facial flushing, and wheezing. There is a genetic predisposition to increased production of cysteinyl-leukotrienes with functional polymorphism of cys-leukotriene C4 synthase. Asthma is triggered by COX inhibitors but is persistent even in their absence. All nonselective COX inhibitors should be avoided, but selective COX2 inhibitors are safe to use when an anti-inflammatory analgesic is needed. Aspirin-sensitive asthma responds to usual therapy with ICS. Although antileukotrienes should be

1	avoided, but selective COX2 inhibitors are safe to use when an anti-inflammatory analgesic is needed. Aspirin-sensitive asthma responds to usual therapy with ICS. Although antileukotrienes should be effective in these patients, they are no more effective than in allergic asthma. Occasionally, aspirin desensitization is necessary, but this should only be undertaken in specialized centers.

1	Asthma in the Elderly Asthma may start at any age, including in elderly patients. The principles of management are the same as in other asthmatics, but side effects of therapy may be a problem, including muscle tremor with β2-agonists and more systemic side effects with ICS. Comorbidities are more frequent in this age group, and interactions with drugs such as β2-blockers, COX inhibitors, and agents that may affect theophylline metabolism need to be considered. COPD is more likely in elderly patients and may coexist with asthma. A trial of OCS may be very useful in documenting the steroid responsiveness of asthma.

1	Pregnancy Approximately one-third of asthmatic patients who are pregnant improve during the course of a pregnancy, one-third deteriorate, and one-third are unchanged. It is important to maintain good control of asthma because poor control may have adverse effects on fetal development. Compliance may be a problem because there is often concern about the effects of antiasthma medications on fetal development. The drugs that have been used for many years in asthma therapy have now been shown to be safe and without teratogenic potential. These drugs include SABA, ICS, and theophylline; there is less safety information about newer classes of drugs such as LABA, antileukotrienes, and anti-IgE. If an OCS is needed, it is better to use prednisone rather than prednisolone because it cannot be converted to the active prednisolone by the fetal liver, thus protecting the fetus from systemic effects of the corticosteroid. There is no contraindication to breast-feeding when patients are using these

1	to the active prednisolone by the fetal liver, thus protecting the fetus from systemic effects of the corticosteroid. There is no contraindication to breast-feeding when patients are using these drugs.

1	Cigarette Smoking Approximately 20% of asthmatics smoke, which may adversely affect asthma in several ways. Smoking asthmatics have more severe disease, more frequent hospital admissions, a faster decline in lung function, and a higher risk of death from asthma than nonsmoking asthmatics. There is evidence that smoking interferes with the anti-inflammatory actions of corticosteroids by reducing HDAC2, necessitating higher doses for asthma control. Smoking cessation improves lung function and reduces the steroid resistance, and thus, vigorous smoking cessation strategies should be used. Some patients report a temporary worsening of asthma when they first stop smoking, possibly due to the loss of the bronchodilating effect of NO in cigarette smoke.

1	Surgery If asthma is well controlled, there is no contraindication to general anesthesia and intubation. Patients who are treated with OCS will have adrenal suppression and should be treated with an increased dose of OCS immediately prior to surgery. Patients with FEV1 <80% of their normal levels should also be given a boost of OCS prior to surgery. High-maintenance doses of corticosteroids may be a contraindication to surgery because of increased risks of infection and delayed wound healing. hypersensitivity pneumonitis and pulmonary infiltrates with eosinophilia Praveen Akuthota, Michael E. Wechsler HYPERSENSITIVITY PNEUMONITIS 310

1	Bronchopulmonary Aspergillosis Bronchopulmonary aspergillosis (BPA) is uncommon and results from an allergic pulmonary reaction to inhaled spores of Aspergillus fumigatus and, occasionally, other Aspergillus species. A skin prick test to A. fumigatus is always positive, whereas serum Aspergillus precipitins are low or undetectable. Characteristically, there are fleeting eosinophilic infiltrates in the lungs, particularly in the upper lobes. Airways become blocked with mucoid plugs rich in eosinophils, and patients may cough up brown plugs and have hemoptysis. BPA may result in bronchiectasis, particularly affecting central airways, if not suppressed by corticosteroids. Asthma is controlled in the usual way by ICS, but it is necessary to give a course of OCS if any sign of worsening or pulmonary shadowing is found. Treatment with the oral antifungal itraconazole is beneficial in preventing exacerbations.

1	Hypersensitivity pneumonitis (HP), also referred to as extrinsic allergic alveolitis, is a pulmonary disease that occurs due to inhalational exposure to a variety of antigens leading to an inflammatory response of the alveoli and small airways. Systemic manifestations such as fever and fatigue can accompany respiratory symptoms. Although sensitization to an inhaled antigen as manifested by specific circulating IgG antibodies is necessary for the development of HP, sensitization alone is not sufficient as a defining characteristic, because many sensitized individuals do not develop HP. The incidence and prevalence of HP are variable, depending on geography, occupation, avocation, and environment of the cohort being studied. As yet unexplained is the decreased risk of developing HP in smokers.

1	HP can be caused by any of a large list of potential offending inhaled antigens (Table 310-1). The various antigens and environmental conditions described to be associated with HP give rise to an expansive list of monikers given to specific forms of HP. Antigens derived from fungal, bacterial, mycobacterial, bird-derived, and chemical sources have all been implicated in causing HP.

1	Categories of individuals at particular risk in the United States include farmers, bird owners, industrial workers, and hot tub users. Farmer’s lung occurs as a result of exposure to one of several possible sources of bacterial or fungal antigens such as grain, moldy hay, or silage. Potential offending antigens include thermophilic actinomycetes or Aspergillus species. Bird fancier’s lung (also referred to by names corresponding to specific birds) must be considered in patients who give a history of keeping birds in their home and is precipitated by exposure to antigens derived from feathers, droppings, and serum proteins. Occupational exposure to birds may also cause HP, as is seen in poultry worker’s lung. Chemical worker’s lung is provoked by exposure to occupational chemical antigens such as diphenylmethane diisocyanate and toluene diisocyanate. Mycobacteria may cause HP rather than frank infection, a phenomenon observed in hot tub lung and in HP due to metalworking fluid.

1	Grain, moldy hay, silage Thermophilic actinomycetes (e.g., Bird fancier’s lung (also Proteins derived by named by specific bird parakeets, pigeons, exposures) budgerigars Duck fever Duck feathers, serum proteins Fish meal worker’s lung Fish meal dust Furrier’s lung Dust from animal furs Laboratory worker’s lung Rat urine, serum, fur Pituitary snuff taker’s lung Animal proteins Bird feathers, droppings, serum proteins Hypersensitivity Pneumonitis and Pulmonary Infiltrates with Eosinophilia Humidifier fever (and air Several microorconditioner lung) ganisms including: Woodworker’s lung Alternaria species; Bacillus subtilis Polyurethane foam, varnish, lacquer Contaminated water, mold on ceiling House dust mites, bird droppings

1	Oak, cedar, pine, mahogany dusts 1682 PATHOPHYSIOLOGY The pathophysiology of HP has not been characterized in depth on an immunologic level, although it has been established that HP is an immune-mediated condition that occurs in response to inhaled antigens that are small enough to deposit in distal airways and alveoli. From a lymphocyte perspective, HP has been categorized as a condition with a TH1 inflammatory pattern. However, emerging evidence suggests that TH17 lymphocyte subsets may be involved in the pathogenesis of the disease as well. Although the presence of precipitating IgG antibodies against specific antigens in HP suggests a prominent role for adaptive immunity in the pathophysiology of HP, innate immune mechanisms may also make an important contribution. This is highlighted by the observation that Toll-like receptors and downstream signaling proteins such as MyD88 are activated in HP. Although no clear genetic basis for HP has been established, in specific cohorts,

1	by the observation that Toll-like receptors and downstream signaling proteins such as MyD88 are activated in HP. Although no clear genetic basis for HP has been established, in specific cohorts, polymorphisms in genes involved in antigen processing and presentation, including TAP1 and major histocompatibility complex type II, have been observed.

1	Given the heterogeneity among patients, variability in offending antigens, and differences in the intensity and duration of exposure to antigen, the presentation of HP is accordingly variable. Although these categories are not fully satisfactory in capturing this variability, HP has been traditionally categorized as having acute, subacute, and chronic forms. Acute HP usually manifests itself 4–8 h following exposure to the inciting antigen, often intense in nature. Systemic symptoms, including fevers, chills, and malaise, are prominent and are accompanied by dyspnea. Symptoms resolve within hours to days if no further exposure to the offending antigen occurs. In subacute HP resulting from ongoing antigen exposure, the onset of respiratory and systemic symptoms is typically more gradual over the course of weeks. A similar presentation may occur as a culmination of intermittent episodes of acute HP. Although respiratory impairment may be quite severe, antigen avoidance generally results

1	the course of weeks. A similar presentation may occur as a culmination of intermittent episodes of acute HP. Although respiratory impairment may be quite severe, antigen avoidance generally results in resolution of the symptoms, although with a slower time course, on the order of weeks to months, than that seen with acute HP. Chronic HP can present with an even more gradual onset of symptoms than subacute HP, with progressive dyspnea, cough, fatigue, weight loss, and clubbing of the digits. The insidious onset of symptoms and frequent lack of an anteceding episode of acute HP make diagnosing chronic HP a challenge. Unlike with the other forms of HP, there can be an irreversible component to the respiratory impairment that is not responsive to removal of the responsible antigen from the patient’s environment. The disease progression to hypoxemic respiratory failure can mirror that seen in idiopathic pulmonary fibrosis (IPF). Fibrotic lung disease is a potential feature of chronic HP

1	patient’s environment. The disease progression to hypoxemic respiratory failure can mirror that seen in idiopathic pulmonary fibrosis (IPF). Fibrotic lung disease is a potential feature of chronic HP due to exposure to bird antigens, whereas an emphysematous phenotype may be seen in farmer’s lung.

1	The categories of acute, subacute, and chronic HP are not completely sufficient in classifying HP. The HP Study Group found on cluster analysis that a cohort of HP patients is best described in bipartite fashion, with one group featuring recurrent systemic signs and symptoms and the other featuring more severe respiratory findings. Concordant with the variability in the presentation of HP is the observed variability in outcome. HP that has not progressed to chronic lung disease has a more favorable outcome with likely resolution if antigen avoidance can be achieved. However, chronic HP resulting in lung fibrosis has a poorer prognosis, with patients with chronic pigeon breeder’s lung having demonstrated a similar mortality as seen in IPF.

1	Although there is no set of universally accepted criteria for arriving at a diagnosis of HP, diagnosis depends foremost on establishing a history of exposure to an offending antigen that correlates with respiratory and systemic symptoms. A careful occupational and home exposure history should be taken and may be supplemented if necessary by a clinician visit to the work or home environment. Specific inquiries will be influenced by geography and the occupation of the patient. When HP is suspected by history, the additional workup is aimed at establishing an immunologic and physiologic response to inhalational antigen exposure with chest imaging, pulmonary function testing, serologic studies, bronchoscopy, and, on occasion, lung biopsy.

1	Chest Imaging Chest x-ray findings in HP are nonspecific and can even lack any discernible abnormalities. In cases of acute and subacute HP, findings may be transient and can include ill-defined micronodular opacities or hazy ground-glass airspace opacities. Findings on chest x-ray will often resolve with removal from the offending antigen, although the time course of resolution may vary. With chronic HP, the abnormalities seen on the chest radiograph are frequently more fibrotic in nature and may be difficult to distinguish from IPF.

1	With the wide availability of high-resolution computed tomography (HRCT), this modality has become a common component in the diagnostic workup for HP. Although the HRCT may be normal in acute forms of HP, this may be due to lack of temporal correlation between exposure to the offending antigen and obtaining the imaging. Additionally, because of the transient nature of acute HP, HRCT is not always performed. In subacute forms of the disease, ground-glass airspace opacities are characteristic, as is the presence of centrilobular nodules. Expiratory images may show areas of air trapping that are likely caused by involvement of the small airways (Fig. 310-1). Reticular changes and traction bronchiectasis can be observed in chronic HP. Subpleural honeycombing similar to that seen in IPF may be present in advanced cases, although unlike in IPF, the lung bases are frequently spared.

1	Pulmonary Function Testing (PFT) Either restrictive or obstructive PFTs can be present in HP, so the pattern of PFT change is not useful in establishing the diagnosis of HP. However, obtaining PFTs is of use in characterizing the physiologic impairment of an individual patient and in gauging the response to antigen avoidance and/or corticosteroid therapy. Diffusion capacity for carbon monoxide may be significantly impaired, particularly in cases of chronic HP with fibrotic pulmonary parenchymal changes.

1	Serum Precipitins Assaying for precipitating IgG antibodies against specific antigens can be a useful adjunct in the diagnosis of HP. However, the presence of an immunologic response alone is not sufficient for establishing the diagnosis, because many asymptomatic individuals with high levels of exposure to antigen may display serum precipitins, as has been observed in farmers and in pigeon breeders. It should also be noted that panels that test for several specific serum precipitins often provide false-negative results, because they represent an extremely limited proportion of the universe of potential offending environmental antigens. FIGURE 310-1 Chest computed tomography scan of a patient with subacute hypersensitivity pneumonitis in which scattered regions of ground-glass infiltrates in a mosaic pattern consistent with air trapping are seen bilaterally. This patient had bird fancier’s lung. (Courtesy of TJ Gross; with permission.)

1	FIGURE 310-2 Open-lung biopsy from a patient with subacute hypersensitivity pneumonitis demonstrating a loose, nonnecrotiz-ing granuloma made up of histiocytes and multinucleated giant cells. Peribronchial inflammatory infiltrate made up of lymphocytes and plasma cells is also seen. (Courtesy of TJ Gross; with permission.) Bronchoscopy Bronchoscopy with bronchoalveolar lavage (BAL) may be used in the evaluation of HP. Although not a specific finding, BAL lymphocytosis is characteristic of HP. However, in active smokers, a lower threshold should be used to establish BAL lymphocytosis, because smoking will result in lower lymphocyte percentages. Most cases of HP have a CD4+/CD8+ lymphocyte ratio of less than 1, but again, this is not a specific finding and has limited utility in the diagnosis of HP.

1	Lung Biopsy Tissue samples may be obtained by a bronchoscopic approach using transbronchial biopsy, or more architecturally preserved specimens may be obtained by a surgical approach (videoassisted thoracoscopy or open approach). As is the case with BAL, histologic specimens are not absolutely necessary to establish the diagnosis of HP, but they can be useful in the correct clinical context. A common histologic feature in HP is the presence of noncaseating granulomas in the vicinity of small airways (Fig. 310-2). As opposed to pulmonary sarcoidosis, in which noncaseating granulomas are well defined, the granulomas seen in HP are loose and poorly defined in nature. Within the alveolar spaces and in the interstitium, a mixed cellular infiltrate with a lymphocytic predominance is observed that is frequently patchy in distribution. Bronchiolitis with the presence of organizing exudate is also often observed. Fibrosis may be present as well, particularly as the disease progresses to its

1	that is frequently patchy in distribution. Bronchiolitis with the presence of organizing exudate is also often observed. Fibrosis may be present as well, particularly as the disease progresses to its chronic form. Fibrotic changes may be focal but can be diffuse and severe with honeycombing in advanced cases, similar to findings in IPF.

1	Clinical Prediction Rule Although not meant as a set of validated diagnostic criteria, a clinical prediction rule for predicting the presence of HP has been published by the HP Study Group. They identified six statistically significant predictors for HP, the strongest of which was exposure to an antigen known to cause HP. Other predictive criteria were the presence of serum precipitins, recurrent symptoms, symptoms occurring 4–8 h after antigen exposure, crackles on inspiration, and weight loss.

1	Differentiating HP from other conditions that cause a similar constellation of respiratory and systemic symptoms requires an increased index of suspicion based on obtaining a history of possible exposure to an offending antigen. Presentations of acute or subacute HP can be mistaken for respiratory infection. In cases of chronic disease, HP must be differentiated from interstitial lung disease, such as IPF or 1683 nonspecific interstitial pneumonitis (NSIP); this can be a difficult task even with lung biopsy. Given the presence of pulmonary infiltrates and noncaseating granulomas on biopsy, sarcoidosis is also a consideration in the differential diagnosis of HP. Unlike in HP, however, hilar adenopathy may be prominent on chest x-ray, organs other than the lung may be involved, and noncaseating granulomas in pathologic specimens tend to be well formed. Other inhalational syndromes, such as organic toxic dust syndrome (OTDS), can be misdiagnosed as HP. OTDS occurs with exposure to

1	granulomas in pathologic specimens tend to be well formed. Other inhalational syndromes, such as organic toxic dust syndrome (OTDS), can be misdiagnosed as HP. OTDS occurs with exposure to organic dusts, including those produced by grains or mold silage, but neither requires prior antigen sensitization nor is characterized by positive serum precipitins.

1	The mainstay of treatment for HP is antigen avoidance. A careful exposure history must be obtained to attempt to identify the potential offending antigen and to identify the location where a patient is exposed. Once a potential antigen and location are identified, efforts should be made to modify the environment to minimize patient exposure. This may be accomplished with measures such as removal of birds, removal of molds, and improved ventilation. Personal protective equipment including respirators and ventilated helmets can be used but may not provide adequate protection for sensitized individuals. In some cases, fully avoiding specific environments may be necessary, although such a recommendation must be balanced against the effects to an individual’s lifestyle or occupation. It is not uncommon for patients with HP due to exposure to household birds to be unwilling to remove them from the home.

1	Because acute HP is generally a self-limited disease after a discrete exposure to an offending antigen, pharmacologic therapy is generally not necessary. However, in so-called subacute and chronic forms of the disease, there is a role for glucocorticoid therapy. In patients with particularly severe symptoms as a result of subacute HP, antigen avoidance may be insufficient after establishing the diagnosis. Although glucocorticoids do not change the long-term outcome in these patients, they can accelerate the resolution of symptoms. While there is significant variability in the approach to glucocorticoid therapy by individual clinicians, prednisone therapy can be initiated at 0.5–1 mg/kg of ideal body weight per day (not to exceed 60 mg/d or alternative glucocorticoid equivalent) over a duration of 1–2 weeks, followed by a taper over the next 2–6 weeks. In chronic HP, a similar trial of corticosteroids may be used, although a variable component of fibrotic disease may be irreversible.

1	As the ever-expanding list of antigens and exposures associated with the development of HP suggests, populations at risk for HP will vary globally based on specifics of local occupational, avocational, and environmental factors. Specific examples of geographically limited HP include summer-type pneumonitis seen in Japan and suberosis seen in cork workers in Portugal and Spain. Although eosinophils are normal constituents of the lungs, there are several pulmonary eosinophilic syndromes that are characterized by pulmonary infiltrates on imaging along with an increased number of eosinophils in lung tissue, in sputum, and/or in BAL fluid, with resultant increased respiratory symptoms and the potential for systemic manifestations. Because the eosinophil plays such an important role in each of these syndromes, it is often difficult to distinguish between them, but there are important clinical and pathologic differences as well as differences in prognosis and treatment paradigms.

1	Because there are so many different diagnoses associated with pulmonary infiltrates with eosinophilia, the first step in classifying pulmonary Hypersensitivity Pneumonitis and Pulmonary Infiltrates with Eosinophilia DiagnoStiC Criteria of aCute eoSinophiLiC pneuMonia Acute eosinophilic pneumonia Chronic eosinophilic pneumonia Eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome) Hypereosinophilic syndrome Pulmonary Disorders of Known Cause Associated with Eosinophilia Lung Diseases Associated with Eosinophilia Malignant Neoplasms Associated with Eosinophilia Leukemia Lymphoma Lung cancer Adenocarcinoma of various organs Squamous cell carcinoma of various organs

1	Lung Diseases Associated with Eosinophilia Malignant Neoplasms Associated with Eosinophilia Leukemia Lymphoma Lung cancer Adenocarcinoma of various organs Squamous cell carcinoma of various organs Systemic Disease Associated with Eosinophilia eosinophilic syndromes is distinguishing between primary pulmonary eosinophilic lung disorders and those with eosinophilia that are secondary to a specific cause such as a drug reaction, an infection, a malignancy, or another pulmonary condition such as asthma. Table 310-2 lists primary and secondary pulmonary eosinophilic disorders.

1	For each patient, a detailed history is of utmost importance and can help elucidate what the underlying disease is. Details regarding onset, timing, and precipitants of specific symptoms can help discern one diagnosis from another. History regarding pharmacologic, occupational, and environmental exposures is instructive, and family and travel history are crucial. In addition to details about the sinuses and lungs, it is important to inquire about systemic manifestations and assess for physical findings of cardiac, gastrointestinal (GI), neurologic, dermatologic, and genitourinary involvement, all of which may give clues to specific diagnoses. Once the details from history and physical are teased out, laboratory testing (including measurements of blood eosinophils, cultures, and markers of inflammation), spirometry and radiographic imaging can help distinguish between different diseases. Often, however, BAL, transbronchial, or open lung biopsies are required. In many cases, biopsies or

1	inflammation), spirometry and radiographic imaging can help distinguish between different diseases. Often, however, BAL, transbronchial, or open lung biopsies are required. In many cases, biopsies or noninvasive diagnostic studies of other organs (e.g., echocardiogram, electromyogram, or bone marrow biopsy) can be helpful.

1	Pathologically, the pulmonary eosinophilic syndromes are characterized by tissue infiltration by eosinophils (Fig. 310-2). In eosinophilic granulomatosis with polyangiitis (EGPA), extravascular granulomas and necrotizing vasculitis may occur in the lungs, as well as in the heart, skin, muscle, liver, spleen, and kidneys, and may be associated with fibrinoid necrosis and thrombosis. The exact etiology of the various pulmonary eosinophilic syndromes is unknown; however, it is felt that these syndromes result from dysregulated eosinophilopoiesis or an autoimmune process because of Acute febrile illness with respiratory manifestations of <1 month in duration Absence of parasitic, fungal, or other infection Absence of drugs known to cause pulmonary eosinophilia Quick clinical response to corticosteroids

1	Absence of parasitic, fungal, or other infection Absence of drugs known to cause pulmonary eosinophilia Quick clinical response to corticosteroids Failure to relapse after discontinuation of corticosteroids the prominence of allergic features and the presence of immune complexes, heightened T cell immunity, and altered humoral immunity as evidenced by elevated IgE and rheumatoid factor. Because of its integral involvement in eosinophilopoiesis, interleukin 5 (IL-5) has been hypothesized to play an etiologic role, and efforts to block this cytokine are being investigated. Antineutrophil cytoplasmic antibodies (ANCAs) are present in about half of patents with EGPA; binding of ANCAs to vascular walls likely contributes to vascular inflammation and injury as well as chemotaxis of inflammatory cells.

1	Acute eosinophilic pneumonia is a syndrome characterized by fevers, acute respiratory failure that often requires mechanical ventilation, diffuse pulmonary infiltrates, and pulmonary eosinophilia in a previously healthy individual (Table 310-3).

1	Clinical Features and Etiology At presentation, acute eosinophilic pneumonia is often mistaken for acute lung injury or acute respiratory distress syndrome (ARDS), until a BAL is performed and reveals >25% eosinophils. Although the predominant symptoms of acute eosinophilic pneumonia are cough, dyspnea, malaise, myalgias, night sweats, and pleuritic chest pain, physical exam findings include high fevers, basilar rales, and rhonchi on forced expiration. Acute eosinophilic pneumonia most often affects males between age 20 and 40 with no history of asthma. Although no clear etiology has been identified, several case reports have linked acute eosinophilic pneumonia to recent initiation of tobacco smoking or exposure to other environmental stimuli including dust from indoor renovations.

1	In addition to a suggestive history, the key to establishing a diagnosis of acute eosinophilic pneumonia is the presence of >25% eosinophilia on BAL fluid. While lung biopsies show eosinophilic infiltration with acute and organizing diffuse alveolar damage, it is generally not necessary to proceed to biopsy to establish a diagnosis. Although patients present with an elevated white blood cell count, in contrast to other pulmonary eosinophilic syndromes, acute eosinophilic pneumonia is often not associated with peripheral eosinophilia upon presentation. However, between 7 and 30 days of disease onset, peripheral eosinophilia often occurs with mean eosinophil counts of 1700. Erythrocyte sedimentation rate (ESR), C-reactive protein, and IgE levels are high but nonspecific, whereas HRCT is always abnormal with bilateral random patchy ground-glass or reticular opacities, and small pleural effusions in as many as two-thirds of patients. Pleural fluid is characterized by a high pH with marked

1	abnormal with bilateral random patchy ground-glass or reticular opacities, and small pleural effusions in as many as two-thirds of patients. Pleural fluid is characterized by a high pH with marked eosinophilia.

1	Clinical Course and Response to Therapy Although some patients improve spontaneously, most patients require admission to an intensive care unit and respiratory support with either invasive (intubation) or noninvasive mechanical ventilation. However, what distinguishes acute eosinophilic pneumonia from both other cases of acute lung injury as well as some of the other pulmonary eosinophilic syndromes is the absence of organ dysfunction or multisystem organ failure other than respiratory failure. One of the characteristic features of acute eosinophilic pneumonia is the high degree of corticosteroid responsiveness and the excellent prognosis. Another distinguishing feature of acute eosinophilic pneumonia is that complete clinical and radiographic recovery without recurrence or residual sequelae occurs in almost all patients within several weeks of initiation of therapy.

1	In contrast to acute eosinophilic pneumonia, chronic eosinophilic pneumonia is a more indolent syndrome that is characterized by pulmonary infiltrates and eosinophilia in both the tissue and blood. Most patients are female nonsmokers with a mean age of 45, and patients do not usually develop the acute respiratory failure and significant hypoxemia appreciated in acute eosinophilic pneumonia. Similar to EGPA, a majority have asthma, with many having a history of allergies.

1	Patients present with a subacute illness over weeks to months, with cough, low-grade fevers, progressive dyspnea, weight loss, wheezing, malaise, and night sweats, and a chest x-ray with migratory bilateral peripheral or pleural-based opacities. Although this “photographic negative pulmonary edema” appearance on chest x-ray and chest CT is pathognomonic of chronic eosinophilic pneumonia, less than 25% of patients present with this finding. Other radiographic findings include atelectasis, pleural effusions, lymphadenopathy, and septal line thickening.

1	Almost 90% of patients have peripheral eosinophilia, with mean eosinophil counts of over 30% of total white blood cell count. BAL eosinophilia is also an important distinguishing feature with mean BAL eosinophil counts of close to 60%. Both peripheral and BAL eosinophilia are very responsive to treatment with corticosteroids. Other laboratory features of chronic eosinophilic pneumonia include increased ESR, C-reactive protein, platelets, and IgE. Lung biopsy is also often not required to establish a diagnosis, but may show accumulation of eosinophils and histiocytes in the lung parenchyma and interstitium, as well as cryptogenic organizing pneumonia, but with minimal fibrosis. Nonrespiratory manifestations are uncommon, but arthralgias, neuropathy, and skin and GI symptoms have all been reported; their presence may suggest EGPA or hypereosinophilic syndrome. Another similarity is the rapid response to corticosteroids with quick resolution of peripheral and BAL eosinophilia and

1	been reported; their presence may suggest EGPA or hypereosinophilic syndrome. Another similarity is the rapid response to corticosteroids with quick resolution of peripheral and BAL eosinophilia and improvement in symptoms. In contrast to acute eosinophilic pneumonia, though, over 50% of patients relapse, and many require prolonged courses of corticosteroids for months to years.

1	Previously known as allergic angiitis granulomatosis or Churg-Strauss syndrome, this complex syndrome is characterized by eosinophilic vasculitis that may involve multiple organ systems including the lungs, heart, skin, GI tract, and nervous system. Although EGPA is characterized by peripheral and pulmonary eosinophilia with infiltrates on chest x-ray, the primary features that distinguish EGPA from other pulmonary eosinophilic syndromes are the presence of eosinophilic vasculitis in the setting of asthma and involvement of multiple end organs (a feature it shares with hypereosinophilic syndrome). Although perceived to be quite rare, in the last few years, there has appeared to be an increased incidence of this disease, particularly in association with various asthma therapies.

1	The primary features of EGPA include asthma, peripheral eosinophilia, neuropathy, pulmonary infiltrates, paranasal sinus abnormality, and presence of eosinophilic vasculitis. It typically occurs in several phases. The prodromal phase is characterized by asthma and allergic rhinitis, and usually begins when the individual is in his or her twenties or thirties, typically persisting for many years. The eosinophilic infiltrative phase is characterized by peripheral eosinophilia and eosinophilic tissue infiltration of various organs including the lungs and GI tract. The third phase is the vasculitic phase and may be associated with constitutional signs and symptoms including fever, weight loss, malaise, and fatigue. The mean age at diagnosis is 48 years, with a range of 14 to 74 years; the average length of time between diagnosis of asthma and vasculitis is 9 years.

1	Similar to other pulmonary eosinophilic syndromes, constitutional symptoms are very common in EGPA and include weight loss of 10–20 lb, fevers, and diffuse myalgias and migratory polyarthralgias. Myositis may be present with evidence of vasculitis on muscle biopsies. In contrast to the eosinophilic pneumonias, EGPA involves many organ systems including the lungs, skin, nerves, heart, GI tract, and kidneys. Symptoms and Clinical Manifestations • RESPIRATORY Most EGPA 1685 patients have asthma that arises later in life and in individuals who have no family history of atopy. The asthma can often be severe, and oral corticosteroids are often required to control symptoms but may lead to suppression of vasculitic symptoms. In addition to the more common symptoms of cough, dyspnea, sinusitis, and allergic rhinitis, alveolar hemorrhage and hemoptysis may also occur.

1	NEUROLOGIC Over three-fourths of EGPA patients have neurologic manifestations. Mononeuritis multiplex most commonly involves the peroneal nerve, but also involves the ulnar, radial, internal popliteal, and occasionally, cranial nerves. Cerebral hemorrhage and infarction may also occur and are important causes of death. Despite treatment, neurologic sequelae often do not completely resolve. DERMATOLOGIC Approximately half of EGPA patients develop dermatologic manifestations. These include palpable purpura, skin nodules, urticarial rashes, and livedo.

1	DERMATOLOGIC Approximately half of EGPA patients develop dermatologic manifestations. These include palpable purpura, skin nodules, urticarial rashes, and livedo. CARDIOVASCULAR Granulomas, vasculitis, and widespread myocardial damage may be found on biopsy or at autopsy, and cardiomyopathy and heart failure may be seen in up to half of all patients but are often at least partially reversible. Acute pericarditis, constrictive pericarditis, myocardial infarction, and other electrocardiographic changes all may occur. The heart is a primary target organ in EGPA, and cardiac involvement often portends a worse prognosis. GI GI symptoms are common in EGPA and likely represent an eosinophilic gastroenteritis characterized by abdominal pain, diarrhea, GI bleeding, and colitis. Ischemic bowel, pancreatitis, and cholecystitis have also been reported in association with EGPA and usually portend a worse prognosis.

1	GI bleeding, and colitis. Ischemic bowel, pancreatitis, and cholecystitis have also been reported in association with EGPA and usually portend a worse prognosis. RENAL Renal involvement is more common than once thought, and approximately 25% of patients have some degree of renal involvement. This may include proteinuria, glomerulonephritis, renal insufficiency, and rarely, renal infarct. Lab Abnormalities Systemic eosinophilia is the hallmark laboratory finding in patients with EGPA and reflects the likely pathogenic role that the eosinophil plays in this disease. Eosinophilia greater than 10% is one of the defining features of this illness and may be as high as 75% of the peripheral white blood cell count. It is present at the time of diagnosis in over 80% of patients but may respond quickly (often within 24 h) to initiation of systemic corticosteroid therapy. Even in the absence of systemic eosinophilia, tissue eosinophilia may be present.

1	Although not specific to EGPA, ANCAs are present in up to two-thirds of patients, mostly with a perinuclear staining pattern. Nonspecific lab abnormalities that may be present in patients with EGPA include a marked elevation in ESR, a normochromic normocytic anemia, an elevated IgE, hypergammaglobulinemia, and positive rheumatoid factor and antinuclear antibodies (ANA). Although BAL often reveals significant eosinophilia, this may be seen in other eosinophilic lung diseases. Similarly, PFT often reveals an obstructive defect similar to asthma.

1	Radiographic Features Chest x-ray abnormalities are extremely common in EGPA and consist of bilateral, nonsegmental, patchy infiltrates that often migrate and may be interstitial or alveolar in appearance. Reticulonodular and nodular disease without cavitation can be seen, as can pleural effusions and hilar adenopathy. The most common CT findings include bilateral ground-glass opacity and airspace consolidation that is predominantly subpleural. Other CT findings include bronchial wall thickening, hyperinflation, interlobular septal thickening, lymph node enlargement, and pericardial and pleural effusions. Angiography may be used diagnostically and may show signs of vasculitis in the coronary, central nervous system, and peripheral vasculature. Treatment and Prognosis of EGPA Most patients diagnosed with EGPA have previously been diagnosed with asthma, rhinitis, and sinusitis, and have received treatment with inhaled or systemic corticosteroids.

1	Hypersensitivity Pneumonitis and Pulmonary Infiltrates with Eosinophilia 1686 Because these agents are also the initial treatment of choice for EGPA patients, institution of these therapies in patients with EGPA who are perceived to have severe asthma may delay the diagnosis of EGPA because signs of vasculitis may be masked. Corticosteroids dramatically alter the course of EGPA: up to 50% of those who are untreated die within 3 months of diagnosis, whereas treated patients have a 6-year survival of over 70%. Common causes of death include heart failure, cerebral hemorrhage, renal failure, and GI bleeding. Recent data suggest that clinical remission may be obtained in over 90% of patients treated; approximately 25% of those patients may relapse, often due to corticosteroid tapering, with a rising eosinophil count heralding the relapse. Myocardial, GI, and renal involvement most often portend a poor prognosis. In such cases, treatment with higher doses of corticosteroids or the addition

1	rising eosinophil count heralding the relapse. Myocardial, GI, and renal involvement most often portend a poor prognosis. In such cases, treatment with higher doses of corticosteroids or the addition of cytotoxic agents such as cyclophosphamide is often warranted. Although survival does not differ between those treated or untreated with cyclophosphamide, cyclophosphamide is associated with a reduced incidence of relapse and an improved clinical response to treatment. Other therapies that have been used successfully in the management of EGPA include azathioprine, methotrexate, intravenous gamma globulin, and interferon α. Plasma exchange has not been shown to provide any additional benefit. Recent studies examining the efficacy of anti-IL-5 therapy have shown promise.

1	Hypereosinophilic syndromes (HES) constitute a heterogeneous group of disease entities manifest by persistent eosinophilia >1500 eosinophils/μL in association with end organ damage or dysfunction, in the absence of secondary causes of eosinophilia. In addition to familial, undefined, and overlap syndromes with incomplete criteria, the predominant HES subtypes are the myeloproliferative and lymphocytic variants. The myeloproliferative variant may be divided into three subgroups: (1) chronic eosinophilic leukemia with demonstrable cytogenetic abnormalities and/or blasts on peripheral smear; (2) the platelet-derived growth factor receptor α (PDGFRα)–associated HES, attributed to a constitutively activated tyrosine kinase fusion protein (Fip1L1-PDGFRα) due to a chromosomal deletion on 4q12; this variant is often responsive to imatinib; and (3) the FIP1-negative variant associated with clonal eosinophilia and at least four of the following: dysplastic peripheral eosinophils, increased

1	this variant is often responsive to imatinib; and (3) the FIP1-negative variant associated with clonal eosinophilia and at least four of the following: dysplastic peripheral eosinophils, increased serum vitamin B12, increased tryptase, anemia, thrombocytopenia, splenomegaly, bone marrow cellularity >80%, spindle-shaped mast cells, and myelofibrosis.

1	Extrapulmonary Manifestations of HES More common in men than in women, HES occurs between the ages of 20 and 50 and is characterized by significant extrapulmonary involvement, including infiltration of the heart, GI tract, kidney, liver, joints, and skin. Cardiac involvement includes myocarditis and/or endomyocardial fibrosis, as well as a restrictive cardiomyopathy. Pulmonary Manifestations of HES Similar to the other pulmonary eosinophilic syndromes, these HES are manifest by high levels of blood, BAL, and tissue eosinophilia. Lung involvement occurs in 40% of these patients and is characterized by cough and dyspnea, as well as pulmonary infiltrates. Although it is often difficult to discern the pulmonary infiltrates and effusions seen on chest x-ray from pulmonary edema resulting from cardiac involvement, CT scan findings include interstitial infiltrates, ground-glass opacities, and small nodules. HES are typically not associated with ANCA or elevated IgE.

1	Course and Response to Therapy Unlike the other pulmonary eosinophilic syndromes, less than half of patients with these HES respond to corticosteroids as first-line therapy. Although other treatment options include hydroxyurea, cyclosporine, and interferon, the tyrosine kinase inhibitor imatinib has emerged as an important therapeutic option for patients with the myeloproliferative variant. Anti-IL-5 therapy with mepolizumab also holds promise for these patients and is currently being investigated.

1	Allergic bronchopulmonary aspergillosis (ABPA) is an eosinophilic pulmonary disorder that occurs in response to allergic sensitization to antigens from Aspergillus species fungi. The predominant clinical presentation of ABPA is an asthmatic phenotype, often accompanied by cough with production of brownish plugs of mucus. ABPA has also been well described as a complication of cystic fibrosis. A workup for ABPA may be beneficial in patients who carry a diagnosis of asthma but have proven refractory to usual therapy. ABPA is a distinct diagnosis from simple asthma, characterized by prominent peripheral eosinophilia and elevated circulating levels of IgE (>417 IU/mL). Establishing a diagnosis of ABPA also requires establishing sensitivity to Aspergillus antigens by skin test reactivity, positive serum precipitins for Aspergillus, and/or direct measurement of circulating specific IgG and IgE to Aspergillus. Central bronchiectasis is described as a classic finding on chest imaging in ABPA

1	serum precipitins for Aspergillus, and/or direct measurement of circulating specific IgG and IgE to Aspergillus. Central bronchiectasis is described as a classic finding on chest imaging in ABPA but is not necessary for making a diagnosis. Other possible findings on chest imaging include patchy infiltrates and evidence of mucus impaction.

1	Systemic glucocorticoids may be used in the treatment of ABPA that is persistently symptomatic despite the use of inhaled therapies for asthma. Courses of glucocorticoids should be tapered over 3–6 months, and their use must be balanced against the risks of prolonged steroid therapy. Antifungal agents such as fluconazole and voriconazole given over a 4-month course reduce the antigenic stimulus in ABPA and may therefore modulate disease activity in selected patients. The use of monoclonal antibody against IgE (omalizumab) has been described in treating severe ABPA, particularly in individuals with ABPA as a complication of cystic fibrosis. ABPA-like syndromes have been reported as a result to sensitization to several non-Aspergillus species fungi. However, these conditions are substantially rarer than ABPA, which may be present in a significant proportion of patients with refractory asthma.

1	Infectious etiologies of pulmonary eosinophilia are largely due to helminths and are of particular importance in the evaluation of pulmonary eosinophilia in tropical environments and in the developing world (Table 310-4). These infectious conditions may also be considered in recent travelers to endemic regions. Loffler syndrome refers to transient pulmonary infiltrates with eosinophilia that occurs in response to passage of helminthic larvae through the lungs, most Immunologic Response to Organisms in Lungs Tuberculosis Source: Adapted from P Akuthota, PF Weller: Clin Microbiol Rev 25:649, 2012.

1	Tuberculosis Source: Adapted from P Akuthota, PF Weller: Clin Microbiol Rev 25:649, 2012. occupational and environmental Lung Disease John R. Balmes, Frank E. Speizer Occupational and environmental lung diseases are difficult to dis-tinguish from those of nonenvironmental origin. Virtually all major 311 commonly larvae of Ascaris species (roundworm). Symptoms are generally self-limited and may include dyspnea, cough, wheeze, and hemoptysis. Loffler syndrome may also occur in response to hookworm infection with Ancylostoma duodenale or Necator americanus. Chronic Strongyloides stercoralis infection can lead to recurrent respiratory symptoms with peripheral eosinophilia between flares. In immunocompromised hosts, including patients on glucocorticoids, a severe, potentially fatal, hyperinfection syndrome can result from Strongyloides infection. Paragonimiasis, filariasis, and visceral larval migrans can all cause pulmonary eosinophilia as well.

1	A host of medications are associated with the development of pulmonary infiltrates with peripheral eosinophilia. Therefore, drug reaction must always be included in the differential diagnosis of pulmonary eosinophilia. Although the list of medications associated with pulmonary eosinophilia is ever expanding, common culprits include nonsteroidal anti-inflammatory medications and systemic antibiotics, most specifically nitrofurantoin. Additionally, various and diverse environmental exposures such as particulate metals, scorpion stings, and inhalational drugs of abuse may also cause pulmonary eosinophilia. Radiation therapy for breast cancer has been linked with eosinophilic pulmonary infiltration as well. The mainstay of treatment is removal of the offending exposure, although glucocorticoids may be necessary if respiratory symptoms are severe.

1	In the United States, drug-induced eosinophilic pneumonias are the most common cause of eosinophilic pulmonary infil trates. A travel history or evidence of recent immigration should prompt the consideration of parasite-associated disorders. Tropical eosinophilia is usually caused by filarial infection; however, eosinophilic pneumonias also occur with other parasites such as Ascaris spp., Ancylostoma spp., Toxocara spp., and Strongyloides stercoralis. Tropical eosinophilia due to Wuchereria bancrofti or Wuchereria malayi occurs most commonly in southern Asia, Africa, and South America and is treated successfully with diethylcarbamazine. In the United States, Strongyloides is endemic to the southeastern and Appalachian regions. We acknowledge the contributions of Dr. Alicia K. Gerke and Dr. Gary W. Hunninghake to the previous edition of this chapter.

1	We acknowledge the contributions of Dr. Alicia K. Gerke and Dr. Gary W. Hunninghake to the previous edition of this chapter. categories of pulmonary disease can be caused by environmental agents, and environmentally related disease usually presents clinically in a manner indistinguishable from that of disease not caused by such agents. In addition, the etiology of many diseases may be multifactorial; occupational and environmental factors may interact with other factors (such as smoking and genetic risk). It is often only after a careful exposure history is taken that the underlying workplace or general environmental exposure is uncovered.

1	Why is knowledge of occupational or environmental etiology so important? Patient management and prognosis are affected significantly by such knowledge. For example, patients with occupational asthma or hypersensitivity pneumonitis often cannot be managed 1687 adequately without cessation of exposure to the offending agent. Establishment of cause may have significant legal and financial implications for a patient who no longer can work in his or her usual job. Other exposed people may be identified as having the disease or prevented from getting it. In addition, new associations between exposure and disease may be identified (e.g., nylon flock worker’s lung disease and diacetyl-induced bronchiolitis obliterans).

1	Although the exact proportion of lung disease due to occupational and environmental factors is unknown, a large number of individuals are at risk. For example, 15–20% of the burden of adult asthma and chronic obstructive pulmonary disease (COPD) has been estimated to be due to occupational factors.

1	The patient’s history is of paramount importance in assessing any potential occupational or environmental exposure. Inquiry into specific work practices should include questions about the specific contami nants involved, the presence of visible dusts, chemical odors, the size and ventilation of workspaces, the use of respiratory protective equipment, and whether co-workers have similar complaints. The temporal association of exposure at work and symptoms may provide clues to occupation-related disease. In addition, the patient must be questioned about alternative sources of exposure to potentially toxic agents, including hobbies, home characteristics, exposure to secondhand smoke, and proximity to traffic or industrial facilities. Short-term and long-term exposures to potential toxic agents in the distant past also must be considered.

1	Workers in the United States have the right to know about potential hazards in their workplaces under federal Occupational Safety and Health Administration (OSHA) regulations. Employers must provide specific information about potential hazardous agents in products being used through Material Safety Data Sheets as well as training in personal protective equipment and environmental control procedures. However, the introduction of new processes and/or new chemical compounds may change exposure significantly, and often only the employee on the production line is aware of the change. For the physician caring for a patient with a suspected work-related illness, a visit to the work site can be very instructive. Alternatively, an affected worker can request an inspection by OSHA. If reliable environmental sampling data are available, that information should be used in assessing a patient’s exposure. Because many of the chronic diseases result from exposure over many years, current

1	environmental sampling data are available, that information should be used in assessing a patient’s exposure. Because many of the chronic diseases result from exposure over many years, current environmental measurements should be combined with work histories to arrive at estimates of past exposure.

1	Exposures to inorganic and organic dusts can cause interstitial lung disease that presents with a restrictive pattern and a decreased diffusing capacity (Chap. 306e). Similarly, exposures to a number of organic dusts or chemical agents may result in occupational asthma or COPD that is characterized by airway obstruction. Measurement of change in forced expiratory volume (FEV1) before and after a working shift can be used to detect an acute bronchoconstrictive response.

1	The chest radiograph is useful in detecting and monitoring the pulmonary response to mineral dusts, certain metals, and organic dusts capable of inducing hypersensitivity pneumonitis. The International Labour Organisation (ILO) International Classification of Radiographs of Pneumoconioses classifies chest radiographs by the nature and size of opacities seen and the extent of involvement of the parenchyma. In general, small rounded opacities are seen in silicosis or coal worker’s pneumoconiosis, and small linear opacities are seen in asbestosis. Although useful for epidemiologic studies and screening large numbers of workers, the ILO system can be problematic when applied to an individual worker’s chest radiograph. With dusts causing rounded opacities, the degree of involvement on the chest radiograph may be extensive, whereas pulmonary function may be only minimally impaired. In contrast, in pneumoconiosis causing linear, irregular opacities like those seen in asbestosis, the

1	the chest radiograph may be extensive, whereas pulmonary function may be only minimally impaired. In contrast, in pneumoconiosis causing linear, irregular opacities like those seen in asbestosis, the radiograph may lead to 1688 underestimation of the severity of the impairment until relatively late in the disease. For patients with a history of asbestos exposure, conventional computed tomography (CT) is more sensitive for the detection of pleural thickening, and high-resolution CT (HRCT) improves the detection of asbestosis. Other procedures that may be of use in identifying the role of environmental exposures in causing lung disease include skin prick testing or specific IgE antibody titers for evidence of immediate hypersensitivity to agents capable of inducing occupational asthma (flour antigens in bakers), specific IgG precipitating antibody titers for agents capable of causing hypersensitivity pneumonitis (pigeon antigen in bird handlers), and assays for specific cell-mediated

1	antigens in bakers), specific IgG precipitating antibody titers for agents capable of causing hypersensitivity pneumonitis (pigeon antigen in bird handlers), and assays for specific cell-mediated immune responses (beryllium lymphocyte proliferation testing in nuclear workers or tuberculin skin testing in health care workers). Sometimes a bronchoscopy to obtain transbronchial biopsies of lung tissue may be required for histologic diagnosis (chronic beryllium disease). Rarely, video-assisted thoracoscopic surgery to obtain a larger sample of lung tissue may be required to determine the specific diagnosis of environmentally induced lung disease (hypersensitivity pneumonitis or giant cell interstitial pneumonitis due to cobalt exposure).

1	The chemical and physical characteristics of inhaled agents affect both the dose and the site of deposition in the respiratory tract. Water-soluble gases such as ammonia and sulfur dioxide are absorbed in the lining fluid of the upper and proximal airways and thus tend to produce irritative and bronchoconstrictive responses. In contrast, nitrogen dioxide and phosgene, which are less soluble, may penetrate to the bronchioles and alveoli in sufficient quantities to produce acute chemical pneumonitis.

1	Particle size of air contaminants must also be considered. Because of their settling velocities in air, particles >10–15 μm in diameter do not penetrate beyond the nose and throat. Particles <10 μm in size are deposited below the larynx. These particles are divided into three size fractions on the basis of their size characteristics and sources. Particles ~2.5–10 μm (coarse-mode fraction) contain crustal elements such as silica, aluminum, and iron. These particles mostly deposit relatively high in the tracheobronchial tree. Although the total mass of an ambient sample is dominated by these larger respirable particles, the number of particles, and therefore the surface area on which potential toxic agents can deposit and be carried to the lower airways, is dominated by particles <2.5 μm (fine-mode fraction). These fine particles are created primarily by the burning of fossil fuels or high-temperature industrial processes resulting in condensation products from gases, fumes, or vapors.

1	fraction). These fine particles are created primarily by the burning of fossil fuels or high-temperature industrial processes resulting in condensation products from gases, fumes, or vapors. The smallest particles, those <0.1 μm in size, represent the ultrafine fraction and make up the largest number of particles; they tend to remain in the airstream and deposit in the lung only on a random basis as they come into contact with the alveolar walls. If they do deposit, however, particles of this size range may penetrate into the circulation and be carried to extrapulmonary sites. New technologies create particles of this size (“nanoparticles”) for use in many commercial applications. Besides the size characteristics of particles and the solubility of gases, the actual chemical composition, mechanical properties, and immunogenicity or infectivity of inhaled material determine in large part the nature of the diseases found among exposed persons.

1	Table 311-1 provides broad categories of exposure in the workplace and diseases associated with chronic exposure in those industries. Asbestos is a generic term for several different mineral silicates, including chrysolite, amosite, anthophyllite, and crocidolite. In addition to workers involved in the production of asbestos products (mining, milling, and manufacturing), many workers in the shipbuilding and construction trades, including pipe fitters and boilermakers, were occupationally exposed because asbestos was widely used during the twentieth century for its thermal and electrical insulation properties. Asbestos also was used in the manufacture of fire-resistant textiles, in cement and floor tiles, and in friction materials such as brake and clutch linings. Occupational Exposures Nature of Respiratory Responses Comment Asbestos: mining, processing, construction, ship repair Silica: mining, stone cutting, sandblasting, quarrying Coal dust: mining

1	Occupational Exposures Nature of Respiratory Responses Comment Asbestos: mining, processing, construction, ship repair Silica: mining, stone cutting, sandblasting, quarrying Coal dust: mining Beryllium: processing alloys for high-tech industries Other metals: aluminum, chromium, cobalt, nickel, titanium, tungsten carbide, or “hard metal” (contains cobalt) Fibrosis (asbestosis), pleural disease, cancer, mesothelioma Fibrosis (silicosis), progressive massive fibrosis (PMF), cancer, tuberculosis, chronic obstructive pulmonary disease (COPD) Fibrosis (coal worker’s pneumoconiosis), PMF, COPD Acute pneumonitis (rare), chronic granulomatous disease, lung cancer (highly suspect) Wide variety of conditions from acute pneumonitis to lung cancer and asthma Virtually all new mining and construction with asbestos done in developing countries Risk persists in certain areas of United States, increasing in countries where new mines open New diseases appear with new process

1	Risk persists in certain areas of United States, increasing in countries where new mines open New diseases appear with new process Cotton dust: milling, processing Grain dust: elevator agents, dock workers, milling, bakers Other agricultural dusts: fungal spores, vegetable products, insect fragments, animal dander, bird and rodent feces, endotoxins, microorganisms, pollens Toxic chemicals: wide variety of industries; see Table 311-2 Other respiratory environmental agents: uranium and radon daughters, secondhand tobacco smoke, polycyclic aromatic hydrocarbons (PAHs), biomass smoke, diesel exhaust, welding fumes, wood finishing Byssinosis (an asthma-like syndrome), chronic bronchitis, COPD Asthma, chronic bronchitis, COPD Hypersensitivity pneumonitis (farmer’s lung), asthma, chronic bronchitis Asthma, chronic bronchitis, COPD, hypersensitivity pneumonitis, pneumoconiosis, and cancer

1	Asthma, chronic bronchitis, COPD Hypersensitivity pneumonitis (farmer’s lung), asthma, chronic bronchitis Asthma, chronic bronchitis, COPD, hypersensitivity pneumonitis, pneumoconiosis, and cancer Occupational exposures estimated to contribute to up to 10% of all lung cancers; chronic bronchitis, COPD, and fibrosis Increasing risk in developing countries with drop in United States as jobs shift overseas Risk shifting more to migrant labor pool Reduced risk with recognized hazards; increasing risk for developing countries where controlled labor practices are less stringent In-home exposures important; in developing countries, biomass smoke is a major risk factor for COPD among women

1	In-home exposures important; in developing countries, biomass smoke is a major risk factor for COPD among women Exposure to asbestos is not limited to persons who directly handle the material. Cases of asbestos-related diseases have been encountered in individuals with only bystander exposure, such as painters and electricians who worked alongside insulation workers in a shipyard. Community exposure resulted from the use of asbestos-containing mine and mill tailings as landfill, road surface, and playground material (e.g., Libby, MT, the site of a vermiculite mine in which the ore was contaminated with asbestos). Finally, exposure can occur from the disturbance of naturally occurring asbestos (e.g., from increasing residential development in the foothills of the Sierra Mountains in California).

1	Asbestos has largely been replaced in the developed world with synthetic mineral fibers such as fiberglass and refractory ceramic fibers, but it continues to be used in the developing world. The major health effects from exposure to asbestos are pleural and pulmonary fibrosis, cancers of the respiratory tract, and pleural and peritoneal mesothelioma. Asbestosis is a diffuse interstitial fibrosing disease of the lung that is directly related to the intensity and duration of exposure. The disease resembles other forms of diffuse interstitial fibrosis (Chap. 315). Usually, exposure has taken place for at least 10 years before the disease becomes manifest. The mechanisms by which asbestos fibers induce lung fibrosis are not completely understood but are known to involve oxidative injury due to the generation of reactive oxygen species by the transition metals on the surface of the fibers as well as from cells engaged in phagocytosis.

1	Past exposure to asbestos is specifically indicated by pleural plaques on chest radiographs, which are characterized by either thickening or calcification along the parietal pleura, particularly along the lower lung fields, the diaphragm, and the cardiac border. Without additional manifestations, pleural plaques imply only exposure, not pulmonary impairment. Benign pleural effusions also may occur. The fluid is typically a serous or bloody exudate. The effusion may be slowly progressive or may resolve spontaneously. Irregular or linear opacities that usually are first noted in the lower lung fields are the chest radiographic hallmark of asbestosis. An indistinct heart border or a “ground-glass” appearance in the lung fields may be seen. HRCT may show distinct changes of subpleural curvilinear lines 5–10 mm in length that appear to be parallel to the pleural surface (Fig. 311-1).

1	Pulmonary function testing in asbestosis reveals a restrictive pattern with a decrease in both lung volumes and diffusing capacity. There may also be evidence of mild airflow obstruction (due to peribronchiolar fibrosis). Because no specific therapy is available for asbestosis, supportive care is the same as that given to any patient with diffuse interstitial fibrosis of any cause. In general, newly diagnosed cases will have resulted from exposures that occurred many years before.

1	Lung cancer (Chap. 107) is the most common cancer associated with asbestos exposure. The excess frequency of lung cancer (all histologic types) in asbestos workers is associated with a minimum latency of 15–19 years between first exposure and development of the disease. Persons with more exposure are at greater risk of disease. In addition, there is a significant interactive effect of smoking and asbestos exposure that results in greater risk than what would be expected from the additive effect of each factor.

1	Mesotheliomas (Chap. 316), both pleural and peritoneal, are also associated with asbestos exposure. In contrast to lung cancers, these tumors do not appear to be associated with smoking. Relatively short-term asbestos exposures of ≤1–2 years, occurring up to 40 years in the past, have been associated with the development of mesotheliomas (an observation that emphasizes the importance of obtaining a complete environmental exposure history). Although the risk of mesothelioma is much less than that of lung cancer among asbestos-exposed workers, over 2000 cases were reported in the United States per year at the start of the twenty-first century. Because epidemiologic studies have shown that >80% of mesotheliomas may be associated with asbestos exposure, documented mesothelioma in a patient with occupational or environmental exposure to asbestos may be compensable.

1	FIGURE 311-1 Asbestosis. A. Frontal chest radiograph shows bilateral calcified pleural plaques consistent with asbestos-related pleural disease. Poorly defined linear and reticular abnormalities are seen in the lower lobes bilaterally. B. Axial high-resolution computed tomography of the thorax obtained through the lung bases shows bilateral, subpleural reticulation (black arrows), representing fibrotic lung disease due to asbestosis. Subpleural lines are also present (arrowheads), characteristic of, though not specific for, asbestosis. Calcified pleural plaques representing asbestos-related pleural disease (white arrows) are also evident.

1	Despite being one of the oldest known occupational pulmonary hazards, free silica (SiO2), or crystalline quartz, is still a major cause of disease. The major occupational exposures include mining; stonecutting; sand blasting; glass and cement manufacturing; foundry work; packing of silica flour; and quarrying, particularly of granite. Most often, pulmonary fibrosis due to silica exposure (silicosis) occurs in a dose-response fashion after many years of exposure. Workers heavily exposed through sandblasting in confined spaces, tunneling through rock with a high quartz content (15–25%), or the manufacture of abrasive soaps may develop acute silicosis with as little as 10 months of exposure. The clinical and pathologic features of acute silicosis are similar to those of pulmonary alveolar proteinosis

1	FIGURE 311-2 Acute silicosis. This high-resolution computed tomog-raphy scan shows multiple small nodules consistent with silicosis but also diffuse ground-glass densities with thickened intralobular and interlobular septa producing polygonal shapes. This has been referred to as “crazy paving.” (Chap. 315). The chest radiograph may show profuse miliary infiltration or consolidation, and there is a characteristic HRCT pattern known as “crazy paving” (Fig. 311-2). The disease may be quite severe and progressive despite the discontinuation of exposure. Whole-lung lavage may provide symptomatic relief and slow the progression.

1	With long-term, less intense exposure, small rounded opacities in the upper lobes may appear on the chest radiograph after 15–20 years of exposure, usually without associated impairment of lung function (simple silicosis). Calcification of hilar nodes may occur in as many as 20% of cases and produces a characteristic “eggshell” pattern. Silicotic nodules may be identified more readily by HRCT (Fig. 311-3). The nodular fibrosis may be progressive in the absence of further exposure, with coalescence and formation of nonsegmental conglomerates of irregular masses >1 cm in diameter (complicated silicosis). These masses can become quite large, and when this occurs, the term progressive massive fibrosis (PMF) is applied. Significant functional impairment with both restrictive and obstructive components may be associated with PMF.

1	Because silica is cytotoxic to alveolar macrophages, patients with silicosis are at greater risk of acquiring lung infections that involve these cells as a primary defense (Mycobacterium tuberculosis, atypical mycobacteria and fungi). Because of the increased risk of active tuberculosis, the recommended treatment of latent tuberculosis in these patients is longer. Another potential clinical complication of silicosis is autoimmune connective tissue disorders such as rheumatoid arthritis and scleroderma. In addition, there are sufficient epidemiologic data that the International Agency for Research on Cancer lists silica as a probable lung carcinogen.

1	Other, less hazardous silicates include fuller’s earth, kaolin, mica, diatomaceous earths, silica gel, soapstone, carbonate dusts, and cement dusts. The production of fibrosis in workers exposed to these agents is believed to be related either to the free silica content of these dusts or, for substances that contain no free silica, to the potentially large dust loads to which these workers may be exposed. Some silicates, including talc and vermiculite, may be contaminated with asbestos. Fibrosis of lung or pleura, lung cancer, and mesothelioma have been associated with chronic exposure to talc and vermiculite dusts.

1	FIGURE 311-3 Chronic silicosis. A. Frontal chest radiograph in a patient with silicosis shows variably sized, poorly defined nodules (arrows) predominating in the upper lobes. B. Axial thoracic com-puted tomography image through the lung apices shows numerous small nodules, more pronounced in the right upper lobe. A number of the nodules are subpleural in location (arrows). Occupational exposure to coal dust can lead to CWP, which has enormous social, economic, and medical significance in every nation in which coal mining is an important industry. Simple radiographically identified CWP is seen in ~10% of all coal miners and in as many as 50% of anthracite miners with more than 20 years of work on the coal face. The prevalence of disease is lower in workers in bituminous coal mines.

1	With prolonged exposure to coal dust (i.e., 15–20 years), small, rounded opacities similar to those of silicosis may develop. As in silicosis, the presence of these nodules (simple CWP) usually is not associated with pulmonary impairment. In addition to CWP, coal dust can cause chronic bronchitis and COPD (Chap. 314). The effects of coal dust are additive to those of cigarette smoking. Complicated CWP is manifested by the appearance on the chest radiograph of nodules ≥1 cm in diameter generally confined to the upper half of the lungs. As in silicosis, this condition can progress to PMF that is accompanied by severe lung function deficits and associated with premature mortality. Despite improvements in technology to protect coal miners, cases of PMF still occur in the United States at a disturbing rate.

1	Caplan syndrome (Chap. 380), first described in coal miners but subsequently in patients with silicosis, is the combination of pneumoconiotic nodules and seropositive rheumatoid arthritis. Silica has immunoadjuvant properties and is often present in anthracitic coal dust.

1	Beryllium is a lightweight metal with tensile strength, good electrical conductivity, and value in the control of nuclear reactions through its ability to quench neutrons. Although beryllium may produce an acute pneumonitis, it is far more commonly associated with a chronic granulomatous inflammatory disease that is similar to sarcoidosis (Chap. 390). Unless one inquires specifically about occupational exposures to beryllium in the manufacture of alloys, ceramics, or high-technology electronics in a patient with sarcoidosis, one may miss entirely the etiologic relationship to the occupational exposure. What distinguishes chronic beryllium disease (CBD) from sarcoidosis is evidence of a specific cell-mediated immune response (i.e., delayed hypersensitivity) to beryllium.

1	The test that usually provides this evidence is the beryllium lymphocyte proliferation test (BeLPT). The BeLPT compares the in vitro proliferation of lymphocytes from blood or bronchoalveolar lavage in the presence of beryllium salts with that of unstimulated cells. Proliferation is usually measured by lymphocyte uptake of radiolabeled thymidine.

1	Chest imaging findings are similar to those of sarcoidosis (nodules along septal lines) except that hilar adenopathy is somewhat less common. As with sarcoidosis, pulmonary function test results may show restrictive and/or obstructive ventilatory deficits and decreased diffusing capacity. With early disease, both chest imaging studies and pulmonary function tests may be normal. Fiberoptic bronchoscopy with transbronchial lung biopsy usually is required to make the diagnosis of CBD. In a beryllium-sensitized individual, the presence of noncaseating granulomas or monocytic infiltration in lung tissue establishes the diagnosis. Accumulation of beryllium-specific CD4+ T cells occurs in the granulomatous inflammation seen on lung biopsy. Susceptibility to CBD is highly associated with human leukocyte antigen DP (HLA-DP) alleles that have a glutamic acid in position 69 of the β chain.

1	Aluminum and titanium dioxide have been rarely associated with a sarcoid-like reaction in lung tissue. Exposure to dust containing tungsten carbide, also known as “hard metal,” may produce giant cell interstitial pneumonitis. Cobalt is a constituent of tungsten carbide and is the likely etiologic agent of both the interstitial pneumonitis and the occupational asthma that may occur. The most common exposures to tungsten carbide occur in tool and dye, saw blade, and drill bit manufacture. Diamond polishing may also involve exposure to cobalt dust. In patients with interstitial lung disease, one should always inquire about exposure to metal fumes and/or dusts. Especially when sarcoidosis appears to be the diagnosis, one should always consider possible CBD.

1	Most of the inorganic dusts discussed thus far are associated with the production of either dust macules or interstitial fibrotic changes in the lung. Other inorganic and organic dusts (see categories in Table 311-1), along with some of the dusts previously discussed, are associated with chronic mucus hypersecretion (chronic bronchitis), with or without reduction of expiratory flow rates. Cigarette smoking is the major cause of these conditions, and any effort to attribute some component of the disease to occupational and environmental exposures must take cigarette smoking into account. Most studies suggest an additive effect of dust exposure and smoking. The pattern of the irritant dust effect is similar to that of cigarette smoking, suggesting that small airway inflammation may be the initial site of pathologic response in those cases and continued exposure may lead to chronic bronchitis and COPD.

1	Some of the specific diseases associated with organic dusts are discussed in detail in the chapters on asthma (Chap. 309) and hypersensitivity pneumonitis (Chap. 310). Many of these diseases are named for the specific setting in which they are found, e.g., farmer’s lung, malt worker’s disease, and mushroom worker’s disease. Often the temporal relation of symptoms to exposure furnishes the best evidence for the diagnosis. Three occupational exposures are singled out for discussion 1691 here because they affect the largest proportions of workers.

1	Cotton Dust (Byssinosis) Workers occupationally exposed to cotton dust (but also to flax, hemp, or jute dust) in the production of yarns for textiles and rope making are at risk for an asthma-like syndrome known as byssinosis. Exposure occurs throughout the manufacturing process but is most pronounced in the portions of the factory involved with the treatment of the cotton before spinning, i.e., blowing, mixing, and carding (straightening of fibers). The risk of byssinosis is associated with both cotton dust and endotoxin levels in the workplace environment.

1	Byssinosis is characterized clinically as occasional (early-stage) and then regular (late-stage) chest tightness toward the end of the first day of the workweek (“Monday chest tightness”). Exposed workers may show a significant drop in FEV1 over the course of a Monday work-shift. Initially the symptoms do not recur on subsequent days of the week. However, in 10–25% of workers, the disease may be progressive, with chest tightness recurring or persisting throughout the workweek. After >10 years of exposure, workers with recurrent symptoms are more likely to have an obstructive pattern on pulmonary function testing. The highest grades of impairment generally are seen in smokers.

1	Dust exposure can be reduced by the use of exhaust hoods, general increases in ventilation, and wetting procedures, but respiratory protective equipment may be required during certain operations. Regular surveillance of pulmonary function in cotton dust–exposed workers using spirometry before and after the workshift is required by OSHA. All workers with persistent symptoms or significantly reduced levels of pulmonary function should be moved to areas of lower risk of exposure. Grain Dust Worldwide, many farmers and workers in grain storage facilities are exposed to grain dust. The presentation of obstructive airway disease in grain dust–exposed workers is virtually identical to the characteristic findings in cigarette smokers, i.e., persistent cough, mucus hypersecretion, wheeze and dyspnea on exertion, and reduced FEV1 and FEV1/FVC (forced vital capacity) ratio (Chap. 306e).

1	Dust concentrations in grain elevators vary greatly but can be >10,000 μg/m3 with many particles in the respirable size range. The effect of grain dust exposure is additive to that of cigarette smoking, with ~50% of workers who smoke having symptoms. Smoking grain dust–exposed workers are more likely to have obstructive ventilatory deficits on pulmonary function testing. As in byssinosis, endotoxin may play a role in grain dust–induced chronic bronchitis and COPD.

1	Farmer’s Lung This condition results from exposure to moldy hay containing spores of thermophilic actinomycetes that produce a hypersensitivity pneumonitis (Chap. 310). A patient with acute farmer’s lung presents 4–8 h after exposure with fever, chills, malaise, cough, and dyspnea without wheezing. The history of exposure is obviously essential to distinguish this disease from influenza or pneumonia with similar symptoms. In the chronic form of the disease, the history of repeated attacks after similar exposure is important in differentiating this syndrome from other causes of patchy fibrosis (e.g., sarcoidosis). A wide variety of other organic dusts are associated with the occurrence of hypersensitivity pneumonitis (Chap. 310). For patients who present with hypersensitivity pneumonitis, specific and careful inquiry about occupations, hobbies, and other home environmental exposures is necessary to uncover the source of the etiologic agent.

1	Exposure to toxic chemicals affecting the lung generally involves gases and vapors. A common accident is one in which the victim is trapped in a confined space where the chemicals have accumulated to harmful levels. In addition to the specific toxic effects of the chemical, the victim often sustains considerable anoxia, which can play a dominant role in determining whether the individual survives. Table 311-2 lists a variety of toxic agents that can produce acute and sometimes life-threatening reactions in the lung. All these agents in sufficient concentrations have been demonstrated, at least in animal studies, to affect the lower airways and disrupt alveolar architecture, either acutely or as a result of chronic exposure. Some of these agents may be generated acutely in the environment (see below).

1	Acid fumes: H2SO4, Manufacture of fertilizers, chlorinated organic Mucous membrane irritation, followed by Bronchitis and suggestion of mildly HNO3 compounds, dyes, explosives, rubber products, chemical pneumonitis 2–3 days later reduced pulmonary function in children metal etching, plastics with lifelong residential exposure to high levels Acrolein and other By-product of burning plastics, woods, tobacco Mucous membrane irritant, decrease in Upper respiratory tract irritation aldehydes smoke lung function Ammonia Refrigeration; petroleum refining; manufacture Same as for acid fumes, but bronchiectasis Upper respiratory tract irritation, chronic of fertilizers, explosives, plastics, and other also has been reported bronchitis chemicals Cadmium fumes Smelting, soldering, battery production Mucous membrane irritant, acute respira-Chronic obstructive pulmonary disease tory distress syndrome (ARDS) (COPD)

1	Cadmium fumes Smelting, soldering, battery production Mucous membrane irritant, acute respira-Chronic obstructive pulmonary disease tory distress syndrome (ARDS) (COPD) Formaldehyde Manufacture of resins, leathers, rubber, metals, Same as for acid fumes Nasopharyngeal cancer and woods; laboratory workers, embalmers; emission from urethane foam insulation Halides and acid Bleaching in pulp, paper, textile industry; manu-Mucous membrane irritation, pulmonary Upper respiratory tract irritation, epistaxis, salts (Cl, Br, F) facture of chemical compounds; synthetic rub-edema; possible reduced forced vital tracheobronchitis ber, plastics, disinfectant, rocket fuel, gasoline capacity (FVC) 1–2 years after exposure Hydrogen sulfide By-product of many industrial processes, oil, Increase in respiratory rate followed by Conjunctival irritation, chronic bronchitis, other petroleum processes and storage respiratory arrest, lactic acidosis, pulmo-recurrent pneumonitis nary edema, death

1	Isocyanates (TDI, Production of polyurethane foams, plastics, Mucous membrane irritation, dyspnea, Upper respiratory tract irritation, cough, HDI, MDI) adhesives, surface coatings cough, wheeze, pulmonary edema asthma, hypersensitivity pneumonitis, reduced lung function Nitrogen dioxide Silage, metal etching, explosives, rocket fuels, Cough, dyspnea, pulmonary edema may Emphysema in animals, ? chronic bronchi- welding, by-product of burning fossil fuels be delayed 4–12 h; possible result from tis, associated with reduced lung function acute exposure: bronchiolitis obliterans in in children with lifelong residential 2–6 weeks exposure Ozone Arc welding, flour bleaching, deodorizing, emis-Mucous membrane irritant, pulmonary Excess cardiopulmonary mortality rates sions from copying equipment, photochemical hemorrhage and edema, reduced pulmoair pollutant nary function transiently in children and adults, and increased hospitalization with exposure to summer haze

1	Phosgene Organic compound, metallurgy, volatilization of Delayed onset of bronchiolitis and pulmo-Chronic bronchitis chlorine-containing compounds nary edema Sulfur dioxide Manufacture of sulfuric acid, bleaches, coating Mucous membrane irritant, epistaxis, Chronic bronchitis of nonferrous metals, food processing, refriger-bronchospasm (especially in people with ant, burning of fossil fuels, wood pulp industry asthma) Abbreviations: HDI, hexamethylene diisocyanate; MDI, methylene diphenyl diisocyanate; TDI, toluene diisocyanate.

1	Firefighters and fire victims are at risk of smoke inhalation, an Two other agents have been associated with potentially severe lung important cause of acute cardiorespiratory failure. Smoke inhalation disease. Occupational exposure to nylon flock has been shown to kills more fire victims than does thermal injury. Carbon monoxide induce a lymphocytic bronchiolitis, and workers exposed to diacetyl, poisoning with resulting significant hypoxemia can be life-threatening which is used to provide “butter” flavor in the manufacture of micro(Chap. 473e). Synthetic materials (plastic, polyurethanes), when wave popcorn and other foods, have developed bronchiolitis obliterans burned, may release a variety of other toxic agents (such as cyanide and (Chap. 315). hydrochloric acid), and this must be considered in evaluating smoke inhalation victims. Exposed victims may have some degree of lower World Trade Center Disaster A consequence of the attack on the World respiratory tract inflammation

1	be considered in evaluating smoke inhalation victims. Exposed victims may have some degree of lower World Trade Center Disaster A consequence of the attack on the World respiratory tract inflammation and/or pulmonary edema. Trade Center (WTC) on September 11, 2001, was relatively heavy Exposure to certain highly reactive, low-molecular-weight agents exposure of a large number of firefighters and other rescue workers used in the manufacture of synthetic polymers, paints, and coatings to the dust generated by the collapse of the buildings. Environmental (diisocyanates in polyurethanes, aromatic amines and acid anhydrides monitoring and chemical characterization of WTC dust has revealed in epoxies) is associated with a high risk of occupational asthma. a wide variety of potentially toxic constituents, although much of the Although this occupational asthma manifests clinically as if sensitiza-dust was pulverized cement. Possibly because of the high alkalinity tion has occurred, an IgE

1	constituents, although much of the Although this occupational asthma manifests clinically as if sensitiza-dust was pulverized cement. Possibly because of the high alkalinity tion has occurred, an IgE antibody–mediated mechanism is not neces-of WTC dust, significant cough, wheeze, and phlegm production sarily involved. Hypersensitivity pneumonitis–like reactions also have occurred among firefighters and cleanup crews. New cough and been described in diisocyanate and acid anhydride–exposed workers. wheeze syndromes also occurred among local residents. Heavier expo-

1	Fluoropolymers such as Teflon, which at normal temperatures sure to WTC dust among New York City firefighters was associated produce no reaction, become volatilized upon heating. The inhaled with accelerated decline of lung function over the first year after the agents cause a characteristic syndrome of fever, chills, malaise, and disaster. More recently, concerns have been raised about risk of inter- occasionally mild wheezing, leading to the diagnosis of polymer fume stitial lung disease, especially of a granulomatous nature. fever. A similar self-limited, influenza-like syndrome—metal fume fever—results from acute exposure to fumes containing zinc oxide, OCCUPATIONAL RESPIRATORY CARCINOGENS typically from welding of galvanized steel. These inhalational fever Exposures at work have been estimated to contribute to 10% of all syndromes may begin several hours after work and resolve within lung cancer cases. In addition to asbestos, other agents either proven 24 h, only to return on

1	estimated to contribute to 10% of all syndromes may begin several hours after work and resolve within lung cancer cases. In addition to asbestos, other agents either proven 24 h, only to return on repeated exposure. or suspected to be respiratory carcinogens include acrylonitrile, arsenic compounds, beryllium, bis(chloromethyl) ether, chromium (hexavalent), formaldehyde (nasal), isopropanol (nasal sinuses), mustard gas, nickel carbonyl (nickel smelting), polycyclic aromatic hydrocarbons (coke oven emissions and diesel exhaust), secondhand tobacco smoke, silica (both mining and processing), talc (possible asbestos contamination in both mining and milling), vinyl chloride (sarcomas), wood (nasal cancer only), and uranium. Workers at risk of radiation-related lung cancer include not only those involved in mining or processing uranium but also those exposed in underground mining operations of other ores where radon daughters may be emitted from rock formations.

1	Disability is the term used to describe the decreased ability to work due to the effects of a medical condition. Physicians are generally able to assess physiologic dysfunction, or impairment, but the rating of disability for compensation of loss of income also involves nonmedical factors such as the education and employability of the individual. The disability rating scheme differs with the compensation-granting agency. For example, the U.S. Social Security Administration requires that an individual be unable to do any work (i.e., total disability) before he or she will receive income replacement payments. Many state workers’ compensation systems allow for payments for partial disability. In the Social Security scheme, no determination of cause is done, whereas work-relatedness must be established in workers’ compensation systems.

1	For respiratory impairment rating, resting pulmonary function tests (spirometry and diffusing capacity) are used as the initial assessment tool, with cardiopulmonary exercise testing (to assess maximal oxygen consumption) used if the results of the resting tests do not correlate with the patient’s symptoms. Methacholine challenge (to assess airway reactivity) can also be useful in patients with asthma who have normal spirometry when evaluated. Some compensation agencies (e.g., Social Security) have proscribed disability classification schemes based on pulmonary function test results. When no specific scheme is proscribed, the Guidelines of the American Medical Association should be used.

1	In 1971, the U.S. government established national air quality standards for several pollutants believed to be responsible for excess cardiorespiratory diseases. Primary standards regulated by the U.S. Environmental Protection Agency (EPA) designed to protect the public health with an adequate margin of safety exist for sulfur dioxide, particulates matter, nitrogen dioxide, ozone, lead, and carbon monoxide. Standards for each of these pollutants are updated regularly through an extensive review process conducted by the EPA. (For details on current standards, go to http://www.epa.gov/air/criteria.html.)

1	Pollutants are generated from both stationary sources (power plants and industrial complexes) and mobile sources (motor vehicles), and none of the regulated pollutants occurs in isolation. Furthermore, pollutants may be changed by chemical reactions after being emitted. For example, sulfur dioxide and particulate matter emissions from a coal-fired power plant may react in air to produce acid sulfates and aerosols, which can be transported long distances in the atmosphere. Oxides of nitrogen and volatile organic compounds from automobile exhaust react with sunlight to produce ozone. Although originally thought to be confined to Los Angeles, photochemically derived pollution (“smog”) is now known to be a problem throughout the United States and in many other countries. Both acute and chronic effects of these exposures have been documented in large population studies.

1	The symptoms and diseases associated with air pollution are the same as conditions commonly associated with cigarette smoking. In addition, decreased growth of lung function and asthma have been associated with chronic exposure to only modestly elevated levels of traffic-related gases and respirable particles. Multiple population-based time-series studies within cities have demonstrated excess health care utilization for asthma and other cardiopulmonary conditions as well as increased mortality rates. Cohort studies comparing cities that have relatively high levels of particulate exposures with less polluted 1693 communities suggest excess morbidity and mortality rates from cardiopulmonary conditions in long-term residents of the former. The strong epidemiologic evidence that fine particulate matter is a risk factor for cardiovascular morbidity and mortality has prompted toxicologic investigations into the underlying mechanisms. The inhalation of fine particles from combustion sources

1	matter is a risk factor for cardiovascular morbidity and mortality has prompted toxicologic investigations into the underlying mechanisms. The inhalation of fine particles from combustion sources probably generates oxidative stress followed by local injury and inflammation in the lungs that in turn lead to autonomic and systemic inflammatory responses that can induce endothelial dysfunction and/or injury. Recent research findings on the health effects of air pollutants has led to stricter U.S. ambient air quality standards for ozone, oxides of nitrogen, and particulate matter as well as greater emphasis on publicizing pollution alerts to encourage individuals with significant cardiopulmonary impairment to stay indoors during high-pollution episodes.

1	Secondhand tobacco smoke (Chap. 470), radon gas, wood smoke, and other biologic agents generated indoors must be considered. Several studies have shown that the respirable particulate load in any household is directly proportional to the number of cigarette smokers living in that home. Increases in prevalence of respiratory illnesses, especially asthma, and reduced levels of pulmonary function measured with simple spirometry have been found in the children of smoking parents in a number of studies. Recent meta-analyses for lung cancer and cardiopulmonary diseases, combining data from multiple secondhand tobacco smoke epidemiologic studies, suggest an ~25% increase in relative risk for each condition, even after adjustment for major potential confounders.

1	Exposure to radon gas in homes is a risk factor for lung cancer. The main radon product (radon-222) is a gas that results from the decay series of uranium-238, with the immediate precursor being radium-226. The amount of radium in earth materials determines how much radon gas will be emitted. Levels associated with excess lung cancer risk may be present in as many as 10% of the houses in the United States. When smokers reside in the home, the problem is potentially greater, because the molecular size of radon particles allows them to attach readily to smoke particles that are inhaled. Fortunately, technology is available for assessing and reducing the level of exposure.

1	Other indoor exposures of concern are bioaerosols that contain antigenic material (fungi, cockroaches, dust mites, and pet danders) associated with an increased risk of atopy and asthma. Indoor chemical agents include strong cleaning agents (bleach, ammonia), formaldehyde, perfumes, pesticides, and oxides of nitrogen from gas appliances. Nonspecific responses associated with “tight-building syndrome,” perhaps better termed “building-associated illness,” in which no particular agent has been implicated, have included a wide variety of complaints, among them respiratory symptoms that are relieved only by avoiding exposure in the building in question. The degree to which “smells” and other sensory stimuli are involved in the triggering of potentially incapacitating psychological or physical responses has yet to be determined, and the long-term consequences of such environmental exposures are unknown.

1	Indoor exposure to biomass smoke (wood, dung, crop resi dues, charcoal) is estimated to be responsible for >4% of worldwide disability-adjusted life-years (DALYs) lost, due to acute lower respiratory infections in children, COPD and lung cancer in women, and cardiovascular disease among men. This burden of disease places indoor exposure to biomass smoke as the leading environmental hazard for poor health and the third most important risk factor overall. Almost one-half of the world’s population uses biomass fuel for cooking, heating, or baking. This occurs predominantly in the rural areas of developing countries. Because many families burn biomass fuels in open stoves, which are highly inefficient, and inside homes with poor ventilation, women and young children are exposed on a daily basis to high levels of smoke. In these homes, 24-h mean levels

1	FIGURE 311-4 Histopathologic features of biomass smoke–induced interstitial lung disease. A. Anthracitic pigment is seen accumulating along alveolar septae (arrowheads) and within a pigmented dust macule (single arrow). B. A high-power photomicrograph contains a mixture of fibroblasts and carbon-laden macrophages. of fine particulate matter, a component of biomass smoke, have been reported to be 2–30 times higher than the National Ambient Air Quality Standards set by the U.S. EPA.

1	of fine particulate matter, a component of biomass smoke, have been reported to be 2–30 times higher than the National Ambient Air Quality Standards set by the U.S. EPA. Epidemiologic studies have consistently shown associations between exposure to biomass smoke and both chronic bronchitis and COPD, with odds ratios ranging between 3 and 10 and increasing with longer exposures. In addition to the common occupational exposure to biomass smoke of women in developing countries, men from such countries may be occupationally exposed. Because of increased migration to the United States from developing countries, clinicians need to be aware of the chronic respiratory effects of exposure to biomass smoke, which can include interstitial lung disease (Fig. 311-4). Evidence is beginning to emerge that improved stoves with chimneys can reduce

1	Disorders of the Respiratory System biomass smoke–induced respiratory illness in both children and women. Bronchiectasis Rebecca M. Baron, Miriam Baron Barshak Bronchiectasis refers to an irreversible airway dilation that involves the lung in either a focal or a diffuse manner and that classically has been categorized as cylindrical or tubular (the most common form), varicose, or cystic. 312

1	Bronchiectasis can arise from infectious or noninfectious causes (Table 312-1). Clues to the underlying etiology are often provided by the pattern of lung involvement. Focal bronchiectasis refers to bronchiectatic changes in a localized area of the lung and can be a consequence of obstruction of the airway—either extrinsic (e.g., due to compression by adjacent lymphadenopathy or parenchymal tumor mass) or intrinsic (e.g., due to an airway tumor or aspirated foreign body, a scarred/stenotic airway, or bronchial atresia from congenital underdevelopment of the airway). Diffuse bronchiectasis is characterized by widespread bronchiectatic changes throughout the lung and often arises from an underlying systemic or infectious disease process.

1	More pronounced involvement of the upper lung fields is most common in cystic fibrosis (CF) and is also observed in postradiation fibrosis, corresponding to the lung region encompassed by the radiation port. Bronchiectasis with predominant involvement of the lower lung fields usually has its source in chronic recurrent aspiration (e.g., due to esophageal motility disorders like those in scleroderma), end-stage fibrotic lung disease (e.g., traction bronchiectasis from idiopathic Pattern of Lung Etiology by Category Involvement (Examples) Workup Focal Obstruction (aspirated foreign body, tumor mass) Diffuse Infection (bacterial, nontuberculous mycobacterial) Immunodeficiency (hypogammaglobulinemia, HIV infection, bronchiolitis obliterans after lung transplantation) Genetic causes (cystic fibrosis, Kartagener’s syndrome, α1 antitrypsin deficiency)

1	Immunodeficiency (hypogammaglobulinemia, HIV infection, bronchiolitis obliterans after lung transplantation) Genetic causes (cystic fibrosis, Kartagener’s syndrome, α1 antitrypsin deficiency) Autoimmune or rheumatologic causes (rheumatoid arthritis, Sjögren’s syndrome, inflammatory bowel disease); immune-mediated disease (allergic bronchopulmonary aspergillosis) Miscellaneous (yellow nail syndrome, traction bronchiectasis from postradiation fibrosis or idiopathic pulmonary fibrosis) Sputum Gram’s stain/culture; stains/cultures for acid-fast bacilli and fungi. If no pathogen is identified, consider bronchoscopy with bronchoalveolar lavage. Complete blood count with differential; immunoglobulin measurement; HIV testing Measurement of chloride levels in sweat (for cystic fibrosis), α1 antitrypsin levels; nasal or respiratory tract brush/biopsy (for dyskinetic/ immotile cilia syndrome); genetic testing

1	Measurement of chloride levels in sweat (for cystic fibrosis), α1 antitrypsin levels; nasal or respiratory tract brush/biopsy (for dyskinetic/ immotile cilia syndrome); genetic testing Clinical examination with careful joint exam, serologic testing (e.g., for rheumatoid factor). Consider workup for allergic bronchopulmonary aspergillosis, especially in patients with refractory asthma.a Test of swallowing function and general neuromuscular strength Exclusion of other causes aSkin testing for Aspergillus reactivity; measurement of serum precipitins for Aspergillus, serum IgE levels, serum eosinophils, etc.

1	Exclusion of other causes aSkin testing for Aspergillus reactivity; measurement of serum precipitins for Aspergillus, serum IgE levels, serum eosinophils, etc. pulmonary fibrosis), or recurrent immunodeficiency-associated infections (e.g., hypogammaglobulinemia). Bronchiectasis resulting from infection by nontuberculous mycobacteria (NTM), most commonly the Mycobacterium avium-intracellulare complex (MAC), often preferentially affects the midlung fields. Congenital causes of bronchiectasis with predominant midlung field involvement include the dyskinetic/ immotile cilia syndrome. Finally, predominant involvement of the central airways is reported in association with allergic bronchopulmonary aspergillosis (ABPA), in which an immune-mediated reaction to Aspergillus damages the bronchial wall. Congenital causes of central airway–predominant bronchiectasis resulting from cartilage deficiency include tracheobronchomegaly (Mounier-Kuhn syndrome) and Williams-Campbell syndrome.

1	In many cases, the etiology of bronchiectasis is not determined. In case series, as many as 25–50% of patients referred for bronchiectasis have idiopathic disease. The overall reported prevalence of bronchiectasis in the United States has recently increased, but the epidemiology of bronchiectasis varies greatly with the underlying etiology. For example, patients born with CF often develop significant clinical bronchiectasis in late adolescence or early adulthood, although atypical presentations of CF in adults in their thirties and forties are also possible. In contrast, bronchiectasis resulting from MAC infection classically affects nonsmoking women >50 years of age. In general, the incidence of bronchiectasis increases with age. Bronchiectasis is more common among women than among men. In areas where tuberculosis is prevalent, bronchiectasis more frequently occurs as a sequela of granulomatous infection.

1	In areas where tuberculosis is prevalent, bronchiectasis more frequently occurs as a sequela of granulomatous infection. Focal bronchiectasis can arise from extrinsic compression of the airway by enlarged granulomatous lymph nodes and/or from development of intrinsic obstruction as a result of erosion of a calcified lymph node through the airway wall (e.g., broncholithiasis). Especially in reactivated tuberculosis, parenchymal destruction from infection can result in areas of more diffuse bronchiectasis. Apart from cases associated with tuberculosis, an increased incidence of non-CF bronchiectasis with an unclear underlying mechanism has been reported as a significant problem in developing nations. It has been suggested that the high incidence of malnutrition in certain areas may predispose to immune dysfunction and development of bronchiectasis.

1	The most widely cited mechanism of infectious bronchiectasis is the “vicious cycle hypothesis,” in which susceptibility to infection and poor mucociliary clearance result in microbial colonization of the bronchial tree. Some organisms, such as Pseudomonas aeruginosa, exhibit a particular propensity for colonizing damaged airways and evading host defense mechanisms. Impaired mucociliary clearance can result from inherited conditions such as CF or dyskinetic cilia syndrome, and it has been proposed that a single severe infection (e.g., pneumonia caused by Bordetella pertussis or Mycoplasma pneumoniae) can result in significant airway damage and poor secretion clearance. The presence of the microbes incites continued chronic inflammation, with consequent damage to the airway wall, continued impairment of secretion and microbial clearance, and ongoing propagation of the infectious/inflammatory cycle. Moreover, it has been proposed that mediators released directly from bacteria can

1	impairment of secretion and microbial clearance, and ongoing propagation of the infectious/inflammatory cycle. Moreover, it has been proposed that mediators released directly from bacteria can interfere with mucociliary clearance.

1	Classic studies of the pathology of bronchiectasis from the 1950s demonstrated significant small-airway wall inflammation and larger-airway wall destruction as well as dilation, with loss of elastin, smooth muscle, and cartilage. It has been proposed that inflammatory cells in the small airways release proteases and other mediators, such as reactive oxygen species and proinflammatory cytokines, that damage the larger-airway walls. Furthermore, the ongoing inflammatory process in the smaller airways results in airflow obstruction. It is thought that antiproteases, such as α1 antitrypsin, play an important role in neutralizing the damaging effects of neutrophil elastase and in enhancing bacterial killing. Bronchiectasis and emphysema have been observed in patients with α1 antitrypsin deficiency.

1	Proposed mechanisms for noninfectious bronchiectasis include immune-mediated reactions that damage the bronchial wall (e.g., those associated with systemic autoimmune conditions such as Sjögren’s syndrome and rheumatoid arthritis). Traction bronchiectasis refers to 1695 dilated airways arising from parenchymal distortion as a result of lung fibrosis (e.g., postradiation fibrosis or idiopathic pulmonary fibrosis).

1	The most common clinical presentation is a persistent productive cough with ongoing production of thick, tenacious sputum. Physical findings often include crackles and wheezing on lung auscultation, and some patients with bronchiectasis exhibit clubbing of the digits. Mild to moderate airflow obstruction is often detected on pulmonary function tests, overlapping with that seen at presentation with other conditions, such as chronic obstructive pulmonary disease (COPD). Acute exacerbations of bronchiectasis are usually characterized by changes in the nature of sputum production, with increased volume and purulence. However, typical signs and symptoms of lung infection, such as fever and new infiltrates, may not be present.

1	The diagnosis is usually based on presentation with a persistent chronic cough and sputum production accompanied by consistent radiographic features. Although chest radiographs lack sensitivity, the presence of “tram tracks” indicating dilated airways is consistent with bronchiectasis. Chest computed tomography (CT) is more specific for bronchiectasis and is the imaging modality of choice for confirming the diagnosis. CT findings include airway dilation (detected as parallel “tram tracks” or as the “signet-ring sign”—a cross-sectional area of the airway with a diameter at least 1.5 times that of the adjacent vessel), lack of bronchial tapering (including the presence of tubular structures within 1 cm from the pleural surface), bronchial wall thickening in dilated airways, inspissated secretions (e.g., the “tree-in-bud” pattern), or cysts emanating from the bronchial wall (especially pronounced in cystic bronchiectasis; Fig. 312-1). APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: The evaluation of a patient with bronchiectasis entails elicitation of a clinical history, chest imaging, and a workup to determine the underlying etiology. Evaluation of focal bronchiectasis almost always requires bronchoscopy to exclude airway obstruction by an underlying mass or foreign body. A workup for diffuse bronchiectasis includes analysis for the major etiologies (Table 312-1), with an initial focus on excluding CF. Pulmonary function testing is an important component of a functional assessment of the patient. FIGURE 312-1 Representative chest computed tomography (CT) image of severe bronchiectasis. This patient’s CT demonstrates many severely dilated airways, seen both longitudinally (arrowhead) and in cross-section (arrow).

1	Treatment of infectious bronchiectasis is directed at the control of active infection and improvements in secretion clearance and bronchial hygiene so as to decrease the microbial load within the airways and minimize the risk of repeated infections.

1	Antibiotics targeting the causative or presumptive pathogen (with Haemophilus influenzae and P. aeruginosa isolated commonly) should be administered in acute exacerbations, usually for a minimum of 7–10 days and perhaps for as long as 14 days. Decisions about treatment of NTM infection can be difficult, given that these organisms can be colonizers as well as pathogens and the prolonged treatment course often is not well tolerated. Consensus guidelines have advised that diagnostic criteria for true clinical infection with NTM should be considered in patients with symptoms and radiographic findings of lung disease who have at least two sputum samples positive on culture; at least one bronchoalveolar lavage (BAL) fluid sample positive on culture; a biopsy sample displaying histopathologic features of NTM infection (e.g., granuloma or a positive stain for acid-fast bacilli) along with one positive sputum culture; or a pleural fluid sample (or a sample from another sterile extrapulmonary

1	of NTM infection (e.g., granuloma or a positive stain for acid-fast bacilli) along with one positive sputum culture; or a pleural fluid sample (or a sample from another sterile extrapulmonary site) positive on culture. MAC strains are the most common NTM pathogens, and the recommended regimen for HIV-negative patients includes a macrolide combined with rifampin and ethambutol. Consensus guidelines also recommend macrolide susceptibility testing for clinically significant MAC isolates.

1	The numerous approaches used to enhance secretion clearance in bronchiectasis include hydration and mucolytic administration, aerosolization of bronchodilators and hyperosmolar agents (e.g., hypertonic saline), and chest physiotherapy (e.g., postural drainage, traditional mechanical chest percussion via hand clapping to the chest, or use of devices such as an oscillatory positive expiratory pressure flutter valve or a high-frequency chest wall oscillation vest). Pulmonary rehabilitation and a regular exercise program may assist with secretion clearance as well as with other aspects of bronchiectasis, including improved exercise capacity and quality of life. The mucolytic dornase (DNase) is recommended routinely in CF-related bronchiectasis but not in non-CF bronchiectasis, given concerns about lack of efficacy and potential harm in the non-CF population.

1	It has been proposed that control of the inflammatory response may be of benefit in bronchiectasis, and relatively small-scale trials have yielded evidence of alleviated dyspnea, decreased need for inhaled β-agonists, and reduced sputum production with inhaled glucocorticoids. However, no significant differences in lung function or bronchiectasis exacerbation rates have been observed. Risks of immunosuppression and adrenal suppression must be carefully considered with use of anti-inflammatory therapy in infectious bronchiectasis. Nevertheless, administration of oral/systemic glucocorticoids may be important in treatment of bronchiectasis due to certain etiologies, such as ABPA, or of noninfectious bronchiectasis due to underlying conditions, especially that in which an autoimmune condition is believed to be active (e.g., rheumatoid arthritis or Sjögren’s syndrome). Patients with ABPA may also benefit from a prolonged course of treatment with the oral antifungal agent itraconazole.

1	In select cases, surgery can be considered, with resection of a focal area of suppuration. In advanced cases, lung transplantation can be considered. In more severe cases of infectious bronchiectasis, recurrent infections and repeated courses of antibiotics can lead to microbial resistance to antibiotics. In certain cases, combinations of antibiotics that have their own independent toxicity profiles may be necessary to treat resistant organisms. Recurrent infections can result in injury to superficial mucosal vessels, with bleeding and, in severe cases, life-threatening hemoptysis. Management of massive hemoptysis usually requires intubation to stabilize the patient, identification of the source of bleeding, and protection of the nonbleeding lung. Control of bleeding often necessitates bronchial artery embolization and, in severe cases, surgery.

1	Outcomes of bronchiectasis can vary widely with the underlying etiology and may also be influenced by the frequency of exacerbations and (in infectious cases) the specific pathogens involved. In one study, the decline of lung function in patients with non-CF bronchiectasis was similar to that in patients with COPD, with the forced expiratory volume in 1 s (FEV1) declining by 50–55 mL per year as opposed to 20–30 mL per year for healthy controls. Reversal of an underlying immunodeficient state (e.g., by administration of gamma globulin for immunoglobulin-deficient patients) and vaccination of patients with chronic respiratory conditions (e.g., influenza and pneumococcal vaccines) can decrease the risk of recurrent infections. Patients who smoke should be counseled about smoking cessation.

1	After resolution of an acute infection in patients with recurrences (e.g., ≥3 episodes per year), the use of suppressive antibiotics to minimize the microbial load and reduce the frequency of exacerbations has been proposed, although there is less consensus with regard to this approach in non-CF-associated bronchiectasis than in patients with CF-related bronchiectasis. Possible suppressive treatments include (1) administration of an oral antibiotic (e.g., ciprofloxacin) daily for 1–2 weeks per month; (2) use of a rotating schedule of oral antibiotics (to minimize the risk of development of drug resistance); (3) administration of a macrolide antibiotic (see below) daily or three times per week (with mechanisms of possible benefit related to non-antimicrobial properties, such as anti-inflammatory effects and reduction of gram-negative bacillary biofilms); (4) inhalation of aerosolized antibiotics (e.g., tobramycin inhalation solution) by select patients on a rotating schedule (e.g., 30

1	effects and reduction of gram-negative bacillary biofilms); (4) inhalation of aerosolized antibiotics (e.g., tobramycin inhalation solution) by select patients on a rotating schedule (e.g., 30 days on, 30 days off ), with the goal of decreasing the microbial load without eliciting the side effects of systemic drug administration; and (5) intermittent administration of IV antibiotics (e.g., “clean-outs”) for patients with more severe bronchiectasis and/or resistant pathogens. In relation to macrolide therapy (point 3 above), a number of double-blind, placebo-controlled, randomized trials have recently been published in non-CF bronchiectasis and support a benefit of long-term macrolides (6–12 months of azithromycin or erythromycin) in decreasing rates of bronchiectasis exacerbation, mucus production, and decline in lung function. However, two of these studies also reported increased macrolide resistance in commensal pathogens, dampening enthusiasm for universal use of macrolides in this

1	and decline in lung function. However, two of these studies also reported increased macrolide resistance in commensal pathogens, dampening enthusiasm for universal use of macrolides in this setting and raising the question of whether there might be select non-CF bronchiectasis patients with higher morbidity for whom benefits of long-term macrolides might outweigh the risks of emergence of antibiotic resistance. In particular, development of macrolide-resistant NTM is a significant concern, making treatment of that pathogen much more difficult. Therefore, it is advised to rule out NTM infection before chronic macrolide therapy is considered.

1	In addition, ongoing consistent attention to bronchial hygiene can promote secretion clearance and decrease the microbial load in the airways. Cystic fibrosis Eric J. Sorscher CLINICAL FEATURES Cystic fibrosis (CF) is an autosomal recessive exocrinopathy affecting multiple epithelial tissues. The gene product responsible for CF (the cystic fibrosis transmembrane conductance regulator [CFTR]) 313 serves as an anion channel in the apical (luminal) plasma membranes of epithelial cells and regulates volume and composition of exocrine secretion. An increasingly sophisticated understanding of CFTR molecular genetics and membrane protein biochemistry has facilitated CF drug discovery, with a number of new agents advancing through the clinical testing phase.

1	Respiratory Manifestations The major morbidity and mortality associated with CF is attributable to respiratory compromise, characterized by copious hyperviscous and adherent pulmonary secretions that obstruct small and medium-sized airways. CF airway secretions are exceedingly difficult to clear, and a complex bacterial flora that includes Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa (among other pathogens) is routinely cultured from CF sputum. Robust pulmonary inflammation in the setting of inspissated mucus and chronic bacterial infection leads to collateral tissue injury and further aggravates respiratory decline. Organisms such as P. aeruginosa exhibit a stereotypic mode of pathogenesis; a sentinel and early colonization event often engenders lifelong pulmonary infection by the same genetic strain. Over the course of many years,

1	P. aeruginosa evolves in CF lungs to adopt a mucoid phenotype (attributable to release of alginate exoproduct) that confers selective advantage for the pathogen and poor prognosis for the host. Infection with other bacterial organisms such as Burkholderia cepacia also indicates a less favorable pulmonary outlook. Strategies to eradicate organisms such as P. aeruginosa early in the pathogenesis cascade have been successful and are thought to improve prognosis significantly if sustained.

1	Pancreatic Findings The complete name of the disease, cystic fibrosis of the pancreas, refers to profound tissue destruction of the exocrine pancreas, with fibrotic scarring and/or fatty replacement, cyst proliferation, loss of acinar tissue, and ablation of normal pancreatic architecture. As in the lung, tenacious exocrine secretions (sometimes termed concretions) obstruct pancreatic ducts and impair production and flow of digestive enzymes to the duodenum. The sequelae of exocrine pancreatic insufficiency include chronic malabsorption, poor growth, fat-soluble vitamin insufficiency, high levels of serum immunoreactive trypsinogen (a diagnostic test used in newborn screening), and loss of pancreatic islet cell mass. CF-related diabetes mellitus is a manifestation in over 30% of adults with the disease and is likely multifactorial in nature (attributable to progressive destruction of the endocrine pancreas, insulin resistance due to stress hormones, and other factors).

1	Other Organ System Damage As in CF lung and pancreas, thick and tenacious secretions compromise numerous other exocrine tissues. Obstruction of intrahepatic bile ducts and parenchymal fibrosis are commonly observed in pathologic specimens, with multilobular cirrhosis in 4–15% of patients with CF and significant hepatic insufficiency as a resulting manifestation among adults. Contents of the intestinal lumen are often difficult to excrete, leading to meconium ileus (a presentation in approximately 10–20% of newborns with CF) or distal intestinal obstructive syndrome in older individuals. Men typically exhibit complete involution of the vas deferens and infertility (despite functioning spermatogenesis), and approximately 99% of males with CF are infertile. The etiology of this dramatic anatomic defect in the male genitourinary system is not understood but may represent a developmental abnormality secondary to secretory obstruction of the vas. Abnormalities of female reproductive tract

1	defect in the male genitourinary system is not understood but may represent a developmental abnormality secondary to secretory obstruction of the vas. Abnormalities of female reproductive tract secretions are likely contributors to an increased incidence of infertility among women with CF. Radiographic evidence of sinusitis occurs in most CF patients and is associated with pathogens similar to those recovered from lower airways, suggesting that the sinus may serve as a reservoir 1697 for bacterial seeding.

1	PATHOGENESIS Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) CFTR is an integral membrane protein that functions as an epithelial anion channel. The ~1480-amino-acid molecule encodes a passive conduit for chloride and bicarbonate transport across plasma membranes of epithelial tissues, with direction of ion flow dependent on the electrochemical driving force. Gating of CFTR involves conformational cycling between an open and closed configuration and is augmented by hydrolysis of adenosine triphosphate (ATP). Anion flux mediated by CFTR does not involve active transport against a concentration gradient but utilizes the energy provided from ATP hydrolysis as a central feature of ion channel mechanochemistry and gating.

1	CFTR is situated in the apical plasma membranes of acinar and other epithelial cells where it regulates the amount and composition of secretion by exocrine glands. In numerous epithelia, chloride and bicarbonate release is followed passively by the flow of water, allowing for mobilization and clearance of exocrine products. Along respiratory mucosa, CFTR is necessary to provide sufficient depth of the periciliary fluid layer (PCL), allowing normal ciliary extension and mucociliary transport. CFTR-deficient airway cells exhibit depleted PCL, causing ciliary collapse and failure to clear overlying mucus (Video 313-1). In airway submucosal glands, CFTR is highly expressed in acini and may participate both in the formation of mucus and extrusion of glandular secretion onto the airway surface (Fig. 313-1). In other exocrine glands characterized by abrogated mucus transport (e.g., pancreatic acini and ducts, bile canaliculi, intestinal lumen), similar pathogenic mechanisms have been

1	(Fig. 313-1). In other exocrine glands characterized by abrogated mucus transport (e.g., pancreatic acini and ducts, bile canaliculi, intestinal lumen), similar pathogenic mechanisms have been implicated. In these tissues, a driving force for apical chloride and/or bicarbonate secretion is believed to promote CFTR-mediated fluid and electrolyte release into the lumen, which confers proper rheology of mucins and other exocrine products. Failure of this mechanism disrupts normal hydration and transport of glandular secretion and is widely viewed as a proximate cause of ductular obstruction, with concomitant tissue injury.

1	Pulmonary Inflammation and Remodeling The CF airway is characterized by an aggressive, unrelenting, neutrophilic inflammatory response with release of proteases and oxidants leading to airway remodeling and bronchiectasis. Intense pulmonary inflammation is largely driven by chronic respiratory infection. Macrophages resident in CF lungs augment elaboration of proinflammatory cytokines, which contribute to innate and adaptive immune reactivity. CFTR-dependent abnormalities of airway surface fluid composition (e.g., pH) have been reported as contributors to impaired bacterial killing in CF lungs. The role of CFTR as a direct mediator of inflammatory responsiveness and/or pulmonary remodeling represents an important and topical area of investigation.

1	DNA sequencing of CFTR from patients (and others) worldwide has revealed almost 2000 allelic variants; however, only about 10% of these have been well-characterized as disease-causing mutations. Distinguishing the single nucleotide transversions or other polymorphisms with causal relevance often presents a significant challenge. The CFTR2 resource (www.cftr2.org/) delineates gene variants with a clear etiologic role.

1	CFTR defects known to elicit disease are often categorized based on molecular mechanism. For example, the common F508del mutation (nomenclature denotes omission of a single phenylalanine residue [F] at CFTR position 508) leads to a folding abnormality recognized by cellular quality control pathways. CFTR encoding F508del retains partial ion channel function, but protein maturation is arrested in the endoplasmic reticulum, and CFTR fails to arrive at the plasma membrane. Instead, F508del CFTR is misrouted and undergoes endoplasmic reticulum–associated degradation via the proteasome. CFTR mutations that disrupt protein maturation are termed class II defects and are by far the most common genetic abnormalities. F508del alone accounts for ~70% of defective CFTR alleles in the United States, where 1698 approximately 90% of individuals with CF carry at least one F508del mutation.

1	Other gene defects include CFTR ion channels properly trafficked to the apical cell surface but unable to open and/or gate. Such channel proteins include G551D (a glycine to aspartic acid replacement at CFTR position 551), which leads to an inability to transport Cl– or

1	HCO3 in the presence of ATP (a class III abnormality). Individuals with at least one G551D allele represent 4–5% of CF patients in North America. CFTR nonsense alleles such as G542X, R553X, and W1282X (premature termination codon replaces glycine, arginine, or tryptophan at positions 542, 553, or 1282, respectively) are among the common class I defects, in addition to large deletions or other major disruptions of the gene. The W1282X mutation, for example, is prevalent among individuals of Ashkenazi descent and is a predominant CF genotype in Israel. Additional categories of CFTR mutation include defects in the ion channel pore (class IV), RNA splicing (class V), and increased plasma membrane turnover (class VI) (Fig. 313-2).

1	The diagnosis of CF is based in part on clinical symptoms, family history, or positive newborn screening. CFTR mutation analysis together with sweat electrolyte measurements represent cardinal diagnostic tests. DNA-based evaluation typically surveys numerous disease-associated mutations; panels that identify 20–80 gene defects are FIGURE 313-1 Extrusion of mucus secretion onto the epithelial surface of airways in cystic fibrosis. A. Schematic of the surface epithelium and supporting glandular structure of the human airway.

1	B. The submucosal glands of a patient with cystic fibrosis are filled with mucus, and mucopurulent debris overlies the airway surfaces, essentially burying the epithelium. C. A higher magnification view of a mucus plug tightly adhering to the airway surface, with arrows indicating the interface between infected and inflamed secretions and the underlying epithelium to which the secretions adhere. (Both B and C were stained with hematoxylin and eosin, with the colors modified to highlight structures.) Infected secretions obstruct airways and, over time, dramatically disrupt the normal architecture of the lung. D. CFTR is expressed in surface epithelium and serous cells at the base of submucosal glands in a porcine lung sample, as shown by the dark staining, signifying binding by CFTR antibodies to epithelial structures (aminoethylcarbazole detection of horseradish peroxidase with hematoxylin counterstain). (From SM Rowe, S Miller, EJ Sorscher: N Engl J Med 352:1992, 2005.)

1	FIGURE 313-2 Categories of CFTR mutations. Classes of defects in the CFTR gene include the absence of synthesis (class I); defective protein maturation and premature degradation (class II); disordered gating/regulation, such as diminished adenosine triphosphate (ATP) binding and hydrolysis (class III); defective conductance through the ion channel pore (class IV); a reduced number of CFTR transcripts due to a promoter or splicing abnormality (class V); and accelerated turnover from the cell surface (class VI). (From SM Rowe, S Miller, EJ Sorscher: N Engl J Med 352:1992, 2005.) available through commercial sources. For difficult cases, complete CFTR exonic sequencing together with analysis of splice junctions and key regulatory elements can be obtained. Sweat electrolytes following pilocarpine iontophoresis comprise an invaluable diagnostic measurement, with levels of chloride markedly elevated in CF compared to non-CF individuals. The sweat test result is highly specific and served as

1	iontophoresis comprise an invaluable diagnostic measurement, with levels of chloride markedly elevated in CF compared to non-CF individuals. The sweat test result is highly specific and served as the mainstay of diagnosis for many decades prior to availability of CFTR genotyping. Notably, hyperviscosity of eccrine sweat is not a clinical feature of the disease. Sweat ducts function to reabsorb chloride from a primary sweat secretion produced by the glandular coil. Malfunction of CFTR leads to diminished chloride uptake from the ductular lumen, and sweat emerges on the skin with markedly elevated levels of chloride. For the unusual situation in which both CFTR genotype and sweat electrolytes are inconclusive, in vivo measurement of ion transport across the nasal airways can serve as a specific test for CF and is used by a number of referral centers. For example, elevated (sodium-dependent) transepithelial charge separation across airway epithelial tissue and failure of

1	serve as a specific test for CF and is used by a number of referral centers. For example, elevated (sodium-dependent) transepithelial charge separation across airway epithelial tissue and failure of isoproterenol-dependent chloride secretion (via CFTR) represent bioelectric findings highly specific for the disease. Measurements of CFTR activity in excised rectal mucosal biopsies can also be obtained.

1	CF classically presents in childhood with chronic productive cough, malabsorption including steatorrhea, and failure to thrive. The disease is most common among whites (~1 in 3300 live births) and much less frequent among African-American (~1 in 15,000) or Asian populations (~1 in 33,000). Several “severe” defects that impair CFTR activity (including F508del, G551D, and truncation alleles) are predictive of pancreatic insufficiency, which is clinically evident in 80–90% of individuals with CF. These few specific genotype-phenotype correlations notwithstanding, genotype is, in general, a poor predictor of overall respiratory prognosis.

1	A spectrum of CFTR-related diseases with features resembling classic CF has been well described. In addition to multiorgan involvement, forme frustes, such as isolated congenital bilateral absence of the vas deferens or pancreatitis (without other organ system findings), are strongly associated with CFTR mutations in at least one allele. Although CF is a classic monogenic disease, the importance of non-CFTR gene modifiers and proteins that regulate ion flux, inflammatory pathways, and airway remodeling has been increasingly appreciated as influencing clinical course. For example, the magnitude of transepithelial sodium reabsorption in CF airways, which helps control periciliary fluid depth and composition, is strongly influenced by CFTR and represents a molecular target for disease intervention.

1	Standard care for outpatients with CF is intensive, with regimens that include exogenous pancreatic enzymes taken with meals, nutritional supplementation, anti-inflammatory medication, bronchodilators, and chronic or periodic administration of oral or aerosolized antibiotics (e.g., as maintenance therapy for patients with P. aeruginosa). Recombinant DNAse aerosols (degraded DNA strands that contribute to mucus viscosity) and nebulized hypertonic saline (serves to augment PCL depth, activate mucociliary clearance, and mobilize inspissated airway secretions) are administered routinely. Chest physiotherapy several times each day is a standard means to promote clearance of airway mucus. Among older individuals with CF, malabsorption, chronic inflammation, and endocrine abnormalities can lead to poor bone mineralization, requiring treatment with vitamin D, calcium, and other measures. The time, complexity, and expense of home care are considerable and take a significant toll on patients

1	to poor bone mineralization, requiring treatment with vitamin D, calcium, and other measures. The time, complexity, and expense of home care are considerable and take a significant toll on patients and their families.

1	Severe respiratory exacerbation is commonly managed by hospital admission for frequent chest physiotherapy and parenteral antibiotics directed against serious (and often multiply resistant) bacterial pathogens. Aggressive intervention in this setting can restore a large component of lung function, but ongoing and cumulative loss of pulmonary reserve reflects the natural history of the disease. Poor prognostic indicators such as sputum culture containing B. cepacia, mucoid P. aeruginosa, or atypical mycobacteria are rigorously monitored in the 1699 CF patient population. An increasing incidence of methicillin-resistant

1	S. aureus has also been observed, although the clinical significance of this finding has not been fully elucidated. Typical inpatient antibiotic coverage includes combination drug therapy with an aminoglycoside and β-lactam for up to 14 days. Maximal improvement in lung function is often achieved by 8–10 days in this setting. Many families elect parenteral antibiotic treatment at home, and additional studies are needed to evaluate specific drug combinations, duration of therapy, and home versus inpatient management. Other CF respiratory sequelae that may require hospitalization include hemoptysis and pneumothorax. Hypersensitivity to Aspergillus (allergic bronchopulmonary aspergillosis) occurs in approximately 5% of individuals with the disease and should be suspected in the absence of a response to conventional treatment.

1	Lung transplantation remains a viable therapeutic option in the setting of end-stage CF pulmonary failure, with 5-year postoperative survival rates on the order of 50–60%. Determining the optimal tim ing for surgery presents a substantial challenge, particularly because overall prognosis for individuals with severe lung disease is sometimes difficult to predict, and mortality associated with transplantation is significant (1-year survival rates of approximately 80%). Forced expiratory volume in 1 s (FEV1) measurements less than 30% predicted, together with an assortment of other clinical features, are often used as thresholds for entry onto transplantation lists, although waiting periods for healthy donor lungs can be quite protracted. Based on clinical outcome and limited access to healthy donor lungs, many CF patients and their families do not pursue this option.

1	CFTR MODULATION Potentiation of Mutant CFTR Gating A massive effort directed toward high-throughput drug analysis of large compound libraries (containing millions of individual agents) has identified novel and promising approaches to CF therapy. The approved compound ivacaftor, for example, robustly potentiates CFTR channel opening and stimulates ion transport. Ivacaftor overcomes the G551D CFTR gating defect, and individuals carrying this mutation exhibit dramatic improvement in lung function, weight gain, and other clinical parameters after only a few weeks of oral therapy. Remarkably, sweat chloride values are significantly improved with this treatment in patients with G551D CFTR. No clinical intervention of any sort has previously been shown to normalize the CF sweat chloride abnormality. Long-term studies of the drug in patients with G551D CFTR are ongoing. Ivacaftor has been viewed as the harbinger of a new era for CF therapeutics directed at treating the most fundamental causes

1	Long-term studies of the drug in patients with G551D CFTR are ongoing. Ivacaftor has been viewed as the harbinger of a new era for CF therapeutics directed at treating the most fundamental causes of the disease.

1	Correction of the F508del Processing Abnormality Advancement of new drugs that address specific CFTR defects in protein folding and maturation has been bolstered by clinical studies of F508del rescue in combination with ivacaftor. So-called “corrector” molecules (as distinct from CFTR gating “potentiators” such as ivacaftor) discovered through compound library screening are suitable for promoting cell surface localization of the F508del protein. Significant improvement in pulmonary function of F508del homozygous individuals has been achieved with potentiator/corrector combination therapy in early clinical trials, and several candidate molecules are under evaluation.

1	Personalized Molecular Therapies The advent of modulators with robust clinical impact has engendered new optimism regarding care of patients with CF. It is clear that future interventions will be tailored to specific genotypic abnormalities. Drug screening campaigns and other research programs have identified agents capable of suppressing CFTR nonsense alleles, augmenting potentiator activity, and promoting F508del correction. Efforts to apply these compounds in a fashion that will benefit CF subjects carrying a single copy of F508del (i.e., with a distinct or unusual CFTR mutation on the second allele) comprise an essential priority for the future. Progress in CF drug discovery is emblematic of what might be accomplished in other refractory genetic diseases using an approach grounded in molecular mechanism and unbiased compound library screening.

1	Chronic obstructive pulmonary Disease John J. Reilly, Jr., Edwin K. Silverman, Steven D. Shapiro Chronic obstructive pulmonary disease (COPD) is defined as a disease state characterized by airflow limitation that is not fully reversible 314 1700 CF QUALITY IMPROVEMENT, INCLUDING ASPECTS OF GLOBAL HEALTH As a direct result of advances in basic research, new therapies have transformed CF from a disease typically leading to death in early childhood to a condition with frequent survival well into the fourth decade of life. It has also become increasingly clear that specified approaches to patient management can have an impact on overall prognosis. For example, standardization of clinical intervention throughout the United States has led to remarkable benefit among the CF population. Well-defined measures for outpatient care are now established, including thresholds for hospital admission, antibiotic regimens, nutritional guidelines, periodicity of diagnostic tests, and other clinical

1	measures for outpatient care are now established, including thresholds for hospital admission, antibiotic regimens, nutritional guidelines, periodicity of diagnostic tests, and other clinical parameters. These therapeutic recommendations have become standardized throughout approximately 110 specialized CF care centers and 55 affiliated programs. The initiative has improved endpoints such as weight gain, body mass index, and pulmonary function. Information regarding standardized protocols for CF therapy can be accessed at (www.cff.org/treatments/cfcareguidelines/) or through a number of excellent reviews. Newborn screening for CF is now universal throughout the United States, most of the Canadian provinces, Australia, New Zealand, and much of Europe, and will facilitate early CF intervention. Based on data indicating that early nutritional and other therapies can be beneficial, newborn diagnosis is expected to significantly promote health in the CF population. Export of quality control

1	Based on data indicating that early nutritional and other therapies can be beneficial, newborn diagnosis is expected to significantly promote health in the CF population. Export of quality control measures and novel therapeutics worldwide has become an increasing imperative. For example, median survival of individuals with CF is less than 20 years in much of Central and South America (compared to ~40 years in the United States and Canada), and efforts to apply state-of-the-art management to underdiagnosed and underserved CF patient populations are expected to improve outcome and mitigate CF health disparities in the future.

1	VIDEO 313-1 Initial video sequences describe establishment of the normal periciliary fluid layer bathing the surface airway epithelium, with spheres representing chloride and bicarbonate ions secreted through CFTR and across the apical (mucosal) respiratory surface. Later video sequences depict failure of CFTR anion transport and resulting depletion of the periciliary layer, “plastering” of cilia against the mucosal surface, and accumulation of mucus in the airway with resulting bacterial infection. (Video courtesy of the Cystic Fibrosis Foundation.) (http://www.goldcopd.com/). COPD includes emphysema, an anatomically defined condition characterized by destruction and enlargement of the lung alveoli; chronic bronchitis, a clinically defined condition with chronic cough and phlegm; and small airways disease, a condition in which small bronchioles are narrowed. COPD is present only if chronic airflow obstruction occurs; chronic bronchitis without chronic airflow obstruction is not

1	small airways disease, a condition in which small bronchioles are narrowed. COPD is present only if chronic airflow obstruction occurs; chronic bronchitis without chronic airflow obstruction is not included within COPD.

1	COPD is the third leading cause of death and affects >10 million persons in the United States. COPD is also a disease of increasing public health importance around the world. Estimates suggest that COPD will rise from the sixth to the third most common cause of death worldwide by 2020. Airflow limitation, the major physiologic change in COPD, can result from both small airway obstruction and emphysema. As described below, small airways may become narrowed by cells (hyperplasia and accumulation), mucus, and fibrosis. Of note, activation of transforming growth factor β (TGF-β) contributes to airway fibrosis, while lack of TGF-β may contribute to parenchymal inflammation and emphysema. Largely due to greater similarity of animal air spaces than airways to humans, we know more about mechanisms involved in emphysema than small airway obstruction.

1	The dominant paradigm of the pathogenesis of emphysema comprises four interrelated events (Fig. 314-1): (1) Chronic exposure to cigarette smoke leads to inflammatory and immune cell recruitment within the terminal air spaces of the lung. (2) These inflammatory cells release elastolytic and other proteinases that damage the extracellular matrix of the lung. (3) Structural cell death (endothelial and epithelial cells) occurs directly through oxidant-induced cigarette smoke damage and senescence as well as indirectly via proteolytic loss of matrix attachment. (4) Ineffective repair of elastin and other extracellular matrix components result in air space enlargement that defines pulmonary emphysema. THE ELASTASE:ANTIELASTASE HYPOTHESIS

1	THE ELASTASE:ANTIELASTASE HYPOTHESIS Elastin, the principal component of elastic fibers, is a highly stable component of the extracellular matrix that is critical to the integrity of the lung. The elastase:antielastase hypothesis proposed in the mid1960s states that the balance of elastin-degrading enzymes and their inhibitors determines the susceptibility of the lung to destruction resulting in air space enlargement. This hypothesis was based on the clinical observation that patients with genetic deficiency in α1 antitrypsin (α1AT), the inhibitor of the serine proteinase neutrophil elastase, were at increased risk of emphysema, and that instillation of elastases, including neutrophil elastase, into experimental animals results in emphysema. The elastase:antielastase hypothesis remains a prevailing mechanism for the development of emphysema. However, a complex

1	FIGURE 314-1 Pathogenesis of emphysema. Upon long-term exposure to cigarette smoke, inflammatory cells are recruited to the lung; they release proteinases in excess of inhibitors, and if repair is abnormal, this leads to air space destruction and enlargement or emphysema. ECM, extracellular matrix; MMP, matrix metalloproteinase. network of immune and inflammatory cells and additional proteinases that contribute to emphysema have subsequently been identified.

1	Upon exposure to oxidants from cigarette smoke, macrophages and epithelial cells become activated, producing proteinases and chemokines that attract other inflammatory and immune cells. One mechanism of macrophage activation occurs via oxidant-induced inactivation of histone deacetylase-2, shifting the balance toward acetylated or loose chromatin, exposing nuclear factor-κB sites, and resulting in transcription of matrix metalloproteinases, proinflammatory cytokines such as interleukin 8 (IL-8), and tumor necrosis factor α (TNF-α); this leads to neutrophil recruitment. CD8+ T cells are also recruited in response to cigarette smoke and release interferon-inducible protein-10 (IP-10, CXCL-7), which in turn leads to macrophage production of macrophage elastase (matrix metalloproteinase-12 [MMP-12]). Matrix metalloproteinases and serine proteinases, most notably neutrophil elastase, work together by degrading the inhibitor of the other, leading to lung destruction. Proteolytic cleavage

1	[MMP-12]). Matrix metalloproteinases and serine proteinases, most notably neutrophil elastase, work together by degrading the inhibitor of the other, leading to lung destruction. Proteolytic cleavage products of elastin also serve as a macrophage chemokine, fueling this destructive positive feedback loop.

1	Autoimmune mechanisms may promote the progression of disease. Increased B cells and lymphoid follicles are present in patients, particularly those with advanced disease. Antibodies have been found against elastin fragments as well; IgG autoantibodies with avidity for pulmonary epithelium and the potential to mediate cytotoxicity have been detected. Concomitant cigarette smoke–induced loss of cilia in the airway epithelium and impaired macrophage phagocytosis predispose to bacterial infection with neutrophilia. In end-stage lung disease, long after smoking cessation, there remains an exuberant inflammatory response, suggesting that mechanisms of cigarette smoke–induced inflammation that initiate the disease differ from mechanisms that sustain inflammation after smoking cessation.

1	Cell Death Cigarette smoke oxidant-mediated structural cell death occurs via a variety of mechanisms including rt801 inhibition of mammalian target of rapamycin (mTOR), leading to cell death as well as inflammation and proteolysis. Involvement of mTOR and other senescence markers has led to the recent concept that emphysema resembles premature aging of the lung. Uptake of apoptotic cells by macrophages results in production of growth factors and dampens inflammation, promoting lung repair. Cigarette smoke impairs macrophage uptake of apoptotic cells, limiting repair.

1	Ineffective Repair The ability of the adult lung to repair damaged alveoli appears limited. It is unlikely that the process of septation that is responsible for alveologenesis during lung development can be reinitiated. The capacity of stem cells to repopulate the lung is under active investigation. It appears difficult for an adult human to completely restore an appropriate extracellular matrix, particularly functional elastic fibers. Cigarette smoke exposure may affect the large airways, small airways (≤2 mm diameter), and alveoli. Changes in large airways cause cough and sputum, while changes in small airways and alveoli are responsible for physiologic alterations. Emphysema and small airway pathology are both present in most persons with COPD; however, they do not appear to be mechanistically related to each other, and their relative contributions to obstruction vary from one person to another.

1	Cigarette smoking often results in mucus gland enlargement and goblet cell hyperplasia, leading to cough and mucus production that define chronic bronchitis, but these abnormalities are not related to airflow limitation. Goblet cells not only increase in number but in extent through the bronchial tree. Bronchi also undergo squamous metaplasia, predisposing to carcinogenesis and disrupting mucociliary clearance. Although not as prominent as in asthma, patients may have smooth-muscle hypertrophy and bronchial hyperreactivity leading to airflow limitation. Neutrophil influx has been associ-1701 ated with purulent sputum of upper respiratory tract infections. Independent of its proteolytic activity, neutrophil elastase is among the most potent secretagogues identified.

1	The major site of increased resistance in most individuals with COPD is in airways ≤2 mm diameter. Characteristic cellular changes include goblet cell metaplasia, with these mucus-secreting cells replacing surfactant-secreting Clara cells. Smooth-muscle hypertrophy may also be present. These abnormalities may cause luminal narrowing by fibrosis, excess mucus, edema, and cellular infiltration. Reduced surfactant may increase surface tension at the air-tissue interface, predisposing to airway narrowing or collapse. Respiratory bronchiolitis with mononuclear inflammatory cells collecting in distal airway tissues may cause proteolytic destruction of elastic fibers in the respiratory bronchioles and alveolar ducts where the fibers are concentrated as rings around alveolar entrances. Narrowing and drop-out of small airways precede the onset of emphysematous destruction.

1	Emphysema is characterized by destruction of gas-exchanging air spaces, i.e., the respiratory bronchioles, alveolar ducts, and alveoli. Their walls become perforated and later obliterated with coalescence of small distinct air spaces into abnormal and much larger air spaces. Macrophages accumulate in respiratory bronchioles of essentially all young smokers. Bronchoalveolar lavage fluid from such individuals contains roughly five times as many macrophages as lavage from non smokers. In smokers’ lavage fluid, macrophages comprise >95% of the total cell count, and neutrophils, nearly absent in nonsmokers’ lavage, account for 1–2% of the cells. T lymphocytes, particularly CD8+ cells, are also increased in the alveolar space of smokers.

1	Emphysema is classified into distinct pathologic types, the most important being centriacinar and panacinar. Centriacinar emphysema, the type most frequently associated with cigarette smoking, is characterized by enlarged air spaces found (initially) in association with respiratory bronchioles. Centriacinar emphysema is usually most prominent in the upper lobes and superior segments of lower lobes and is often quite focal. Panacinar emphysema refers to abnormally large air spaces evenly distributed within and across acinar units. Panacinar emphysema is usually observed in patients with α1AT deficiency, which has a predilection for the lower lobes. Persistent reduction in forced expiratory flow rates is the most typical finding in COPD. Increases in the residual volume and the residual volume/total lung capacity ratio, nonuniform distribution of ventilation, and ventilation-perfusion mismatching also occur.

1	Airflow limitation, also known as airflow obstruction, is typically determined by spirometry, which involves forced expiratory maneuvers after the subject has inhaled to total lung capacity. Key parameters obtained from spirometry include the volume of air exhaled within the first second of the forced expiratory maneuver (FEV1) and the total volume of air exhaled during the entire spirometric maneuver (forced vital capacity [FVC]). Patients with airflow obstruction related to COPD have a chronically reduced ratio of FEV1/FVC. In contrast to asthma, the reduced FEV1 in COPD seldom shows large responses to inhaled bronchodilators, although improvements up to 15% are common. Asthma patients can also develop chronic (not fully reversible) airflow obstruction.

1	Airflow during forced exhalation is the result of the balance between the elastic recoil of the lungs promoting flow and the resistance of the airways limiting flow. In normal lungs, as well as in lungs affected by COPD, maximal expiratory flow diminishes as the lungs empty because the lung parenchyma provides progressively less elastic recoil and because the cross-sectional area of the airways falls, raising the resistance to airflow. The decrease in flow coincident with decreased lung 1702 volume is readily apparent on the expiratory limb of a flow-volume curve. In the early stages of COPD, the abnormality in airflow is only evident at lung volumes at or below the functional residual capacity (closer to residual volume), appearing as a scooped-out lower part of the descending limb of the flow-volume curve. In more advanced disease, the entire curve has decreased expiratory flow compared to normal.

1	Lung volumes are also routinely assessed in pulmonary function testing. In COPD there is often “air trapping” (increased residual volume and increased ratio of residual volume to total lung capacity) and progressive hyperinflation (increased total lung capacity) late in the disease. Hyperinflation of the thorax during tidal breathing preserves maximum expiratory airflow, because as lung volume increases, elastic recoil pressure increases, and airways enlarge so that airway resistance decreases.

1	Despite compensating for airway obstruction, hyperinflation can push the diaphragm into a flattened position with a number of adverse effects. First, by decreasing the zone of apposition between the diaphragm and the abdominal wall, positive abdominal pressure during inspiration is not applied as effectively to the chest wall, hindering rib cage movement and impairing inspiration. Second, because the muscle fibers of the flattened diaphragm are shorter than those of a more normally curved diaphragm, they are less capable of generating inspiratory pressures than normal. Third, the flattened diaphragm (with increased radius of curvature, r) must generate greater tension (t) to develop the transpulmonary pressure (p) required to produce tidal breathing. This follows from Laplace’s law, p = 2t/r. Also, because the thoracic cage is distended beyond its normal resting volume, during tidal breathing the inspiratory muscles must do work to overcome the resistance of the thoracic cage to

1	p = 2t/r. Also, because the thoracic cage is distended beyond its normal resting volume, during tidal breathing the inspiratory muscles must do work to overcome the resistance of the thoracic cage to further inflation instead of gaining the normal assistance from the chest wall recoiling outward toward its resting volume.

1	Although there is considerable variability in the relationships between the FEV1 and other physiologic abnormalities in COPD, certain generalizations may be made. The partial pressure of oxygen in arterial blood Pao2 usually remains near normal until the FEV1 is decreased to ~50% of predicted, and even much lower FEV1 values can be associated with a normal Pao2, at least at rest. An elevation of arterial level of carbon dioxide (Paco2) is not expected until the FEV1 is <25% of predicted and even then may not occur. Pulmonary hypertension severe enough to cause cor pulmonale and right ventricular failure due to COPD typically occurs in individuals who have marked decreases in FEV1 (<25% of predicted) and chronic hypoxemia (Pao2 <55 mmHg); however, recent evidence suggests that some patients will develop significant pulmonary hypertension independent of COPD severity (Chap. 304).

1	Nonuniform ventilation and ventilation-perfusion mismatching are characteristic of COPD, reflecting the heterogeneous nature of the disease process within the airways and lung parenchyma. Physiologic studies are consistent with multiple parenchymal compartments having different rates of ventilation due to regional differences in compliance and airway resistance. Ventilation-perfusion mismatching accounts for essentially all of the reduction in Pao2 that occurs in COPD; shunting is minimal. This finding explains the effectiveness of modest elevations of inspired oxygen in treating hypoxemia due to COPD and therefore the need to consider problems other than COPD when hypoxemia is difficult to correct with modest levels of supplemental oxygen.

1	By 1964, the Advisory Committee to the Surgeon General of the United States had concluded that cigarette smoking was a major risk factor for mortality from chronic bronchitis and emphysema. Subsequent longitudinal studies have shown accelerated decline in FEV1 in a dose-response relationship to the intensity of cigarette smoking, which is typically expressed as pack-years (average number of packs of cigarettes smoked per day multiplied by the total number –1 S.D. Mean +1 S.D.

1	% of Population (FEV1) values in a general population sample, stratified by pack-years of smoking. Means, medians, and ±1 standard deviation of percent predicted FEV1 are shown for each smoking group. Although a dose-response relationship between smoking intensity and FEV1 was found, marked variability in pulmonary function was observed among subjects with similar smoking histories. (From B Burrows et al: Am Rev Respir Dis 115:95, 1977; with permission.) of years of smoking). This dose-response relationship between reduced pulmonary function and cigarette smoking intensity accounts for the higher prevalence rates of COPD with increasing age. The historically higher rate of smoking among males is the likely explanation for the higher prevalence of COPD among males; however, the prevalence of COPD among females is increasing as the gender gap in smoking rates has diminished in the past 50 years.

1	Although the causal relationship between cigarette smoking and the development of COPD has been absolutely proved, there is considerable variability in the response to smoking. Although pack-years of cigarette smoking is the most highly significant predictor of FEV1 (Fig. 314-2), only 15% of the variability in FEV1 is explained by pack-years. This finding suggests that additional environmental and/or genetic factors contribute to the impact of smoking on the development of airflow obstruction. Although cigar and pipe smoking may also be associated with the development of COPD, the evidence supporting such associations is less compelling, likely related to the lower dose of inhaled tobacco byproducts during cigar and pipe smoking.

1	A tendency for increased bronchoconstriction in response to a variety of exogenous stimuli, including methacholine and histamine, is one of the defining features of asthma (Chap. 309). However, many patients with COPD also share this feature of airway hyperresponsiveness. The considerable overlap between persons with asthma and those with COPD in airway responsiveness, airflow obstruction, and pulmonary symptoms led to the formulation of the Dutch hypothesis. This suggests that asthma, chronic bronchitis, and emphysema are variations of the same basic disease, which is modulated by environmental and genetic factors to produce these pathologically distinct entities. The alternative British hypothesis contends that asthma and COPD are fundamentally different diseases: Asthma is viewed as largely an allergic phenomenon, whereas COPD results from smoking-related inflammation and damage. Determination of the validity of the Dutch hypothesis versus the British hypothesis awaits

1	viewed as largely an allergic phenomenon, whereas COPD results from smoking-related inflammation and damage. Determination of the validity of the Dutch hypothesis versus the British hypothesis awaits identification of all of the genetic predisposing factors for asthma and/or COPD, as well as the interactions between these postulated genetic factors and environmental risk factors.

1	Longitudinal studies that compared airway responsiveness at the beginning of the study to subsequent decline in pulmonary function have demonstrated that increased airway responsiveness is clearly a significant predictor of subsequent decline in pulmonary function. Thus, airway hyperresponsiveness is a risk factor for COPD. The impact of adult respiratory infections on decline in pulmonary function is controversial, but significant long-term reductions in pulmonary function are not typically seen following an episode of bronchitis or pneumonia. The impact of the effects of childhood respiratory illnesses on the subsequent development of COPD has been difficult to assess due to a lack of adequate longitudinal data. Thus, although respiratory infections are important causes of exacerbations of COPD, the association of both adult and childhood respiratory infections with the development and progression of COPD remains to be proven.

1	Increased respiratory symptoms and airflow obstruction have been suggested to result from exposure to dust and fumes at work. Several specific occupational exposures, including coal mining, gold mining, and cotton textile dust, have been suggested as risk factors for chronic airflow obstruction. Although nonsmokers in these occupations can develop some reductions in FEV1, the importance of dust exposure as a risk factor for COPD, independent of cigarette smoking, is not certain for most of these exposures. However, among coal miners, coal mine dust exposure was a significant risk factor for emphysema in both smokers and nonsmokers. In most cases, the magnitude of these occupational exposures on COPD risk is likely substantially less important than the effect of cigarette smoking.

1	Some investigators have reported increased respiratory symptoms in those living in urban compared to rural areas, which may relate to increased pollution in the urban settings. However, the relationship of air pollution to chronic airflow obstruction remains unproved. Prolonged exposure to smoke produced by biomass combustion—a common mode of cooking in some countries—also appears to be a significant risk factor for COPD among women in those countries. However, in most populations, ambient air pollution is a much less important risk factor for COPD than cigarette smoking. PASSIVE, OR SECOND-HAND, SMOKING EXPOSURE

1	PASSIVE, OR SECOND-HAND, SMOKING EXPOSURE Exposure of children to maternal smoking results in significantly reduced lung growth. In utero, tobacco smoke exposure also contributes to significant reductions in postnatal pulmonary function. Although passive smoke exposure has been associated with reductions in pulmonary function, the importance of this risk factor in the development of the severe pulmonary function reductions in COPD remains uncertain. Although cigarette smoking is the major environmental risk fac tor for the development of COPD, the development of airflow obstruction in smokers is highly variable. Severe α1AT deficiency is a proven genetic risk factor for COPD; there is increasing evidence that other genetic determinants also exist.

1	α1 Antitrypsin Deficiency Many variants of the protease inhibitor (PI or SERPINA1) locus that encodes α1AT have been described. The common M allele is associated with normal α1AT levels. The S allele, associated with slightly reduced α1AT levels, and the Z allele, associated with markedly reduced α1AT levels, also occur with frequencies of >1% in 1703 most white populations. Rare individuals inherit null alleles, which lead to the absence of any α1AT production through a heterogeneous collection of mutations. Individuals with two Z alleles or one Z and one null allele are referred to as PiZ, which is the most common form of severe α1AT deficiency.

1	Although only approximately 1% of COPD patients are found to have severe α1AT deficiency as a contributing cause of COPD, these patients demonstrate that genetic factors can have a profound influence on the susceptibility for developing COPD. PiZ individuals often develop early-onset COPD, but the ascertainment bias in the published series of PiZ individuals—which have usually included many PiZ subjects who were tested for α1AT deficiency because they had COPD— means that the fraction of PiZ individuals who will develop COPD and the age-of-onset distribution for the development of COPD in PiZ subjects remain unknown. Approximately 1 in 3000 individuals in the United States inherits severe α1AT deficiency, but only a small minority of these individuals has been identified. The clinical laboratory test used most frequently to screen for α1AT deficiency is measurement of the immunologic level of α1AT in serum (see “Laboratory Findings”).

1	A significant percentage of the variability in pulmonary function among PiZ individuals is explained by cigarette smoking; cigarette smokers with severe α1AT deficiency are more likely to develop COPD at early ages. However, the development of COPD in PiZ subjects, even among current or ex-smokers, is not absolute. Among PiZ nonsmokers, impressive variability has been noted in the development of airflow obstruction. Asthma and male gender also appear to increase the risk of COPD in PiZ subjects. Other genetic and/or environmental factors likely contribute to this variability. Specific treatment in the form of α1AT augmentation therapy is available for severe α1AT deficiency as a weekly IV infusion (see “Treatment,” below).

1	Specific treatment in the form of α1AT augmentation therapy is available for severe α1AT deficiency as a weekly IV infusion (see “Treatment,” below). The risk of lung disease in heterozygous PiMZ individuals, who have intermediate serum levels of α1AT (~60% of PiMM levels), is controversial. Several recent large studies have suggested that PiMZ subjects are at slightly increased risk for the development of airflow obstruction, but it remains unclear if all PiMZ subjects are at slightly increased risk for COPD or if a subset of PiMZ subjects are at substantially increased risk for COPD due to other genetic or environmental factors. Other Genetic Risk Factors Studies of pulmonary function measurements performed in general population samples have suggested that genetic factors other than PI type influence variation in pulmonary function. Familial aggregation of airflow obstruction within families of COPD patients has also been demonstrated.

1	Association studies have compared the distribution of variants in candidate genes hypothesized to be involved in the development of COPD in COPD patients and control subjects. However, the results have been quite inconsistent, often due to underpowered studies. However, a well-powered association study comprising 8300 patients and 7 separate cohorts found that a minor allele single nucleotide polymorphism (SNP) of MMP12 (rs2276109) associated with decreased MMP12 expression has a positive effect on lung function in children with asthma and in adult smokers. Recent genome-wide association studies have identified several COPD susceptibility loci, including a region near the hedgehog interacting protein (HHIP) gene on chromosome 4, a cluster of genes on chromosome 15 (including components of the nicotinic acetylcholine receptor), and a region within a gene of unknown function (FAM13A). A regulatory SNP upstream from the HHIP gene has been identified as one potential functional variant;

1	the nicotinic acetylcholine receptor), and a region within a gene of unknown function (FAM13A). A regulatory SNP upstream from the HHIP gene has been identified as one potential functional variant; the specific genetic determinants in the other genomic regions have yet to be definitively identified.

1	The effects of cigarette smoking on pulmonary function appear to depend on the intensity of smoking exposure, the timing of smoking exposure during growth, and the baseline lung function of the individual; other environmental factors may have similar effects. Most individuals follow a steady trajectory of increasing pulmonary function with FEV1, % normal level at age 20 Age, year FIGURE 314-3 Hypothetical tracking curves of forced expiratory volume in 1 s (FEV1) for individuals throughout their life spans.

1	The normal pattern of growth and decline with age is shown by curve A. Significantly reduced FEV1 (<65% of predicted value at age 20) can develop from a normal rate of decline after a reduced pulmonary function growth phase (curve C), early initiation of pulmonary function decline after normal growth (curve B), or accelerated decline after normal growth (curve D). (From B Rijcken: Doctoral dissertation, p 133, University of Groningen, 1991; with permission.) growth during childhood and adolescence, followed by a gradual decline with aging. Individuals appear to track in their quantile of pulmonary function based on environmental and genetic factors that put them on different tracks. The risk of eventual mortality from COPD is closely associated with reduced levels of FEV1. A graphic depiction of the natural history of COPD is shown as a function of the influences on tracking curves of FEV1 in Fig. 314-3. Death or disability from COPD can result from a normal rate of decline after a

1	of the natural history of COPD is shown as a function of the influences on tracking curves of FEV1 in Fig. 314-3. Death or disability from COPD can result from a normal rate of decline after a reduced growth phase (curve C), an early initiation of pulmonary function decline after normal growth (curve B), or an accelerated decline after normal growth (curve D). The rate of decline in pulmonary function can be modified by changing environmental exposures (i.e., quitting smoking), with smoking cessation at an earlier age providing a more beneficial effect than smoking cessation after marked reductions in pulmonary function have already developed. Genetic factors likely contribute to the level of pulmonary function achieved during growth and to the rate of decline in response to smoking and potentially to other environmental factors as well.

1	The three most common symptoms in COPD are cough, sputum production, and exertional dyspnea. Many patients have such symptoms for months or years before seeking medical attention. Although the development of airflow obstruction is a gradual process, many patients date the onset of their disease to an acute illness or exacerbation. A careful history, however, usually reveals the presence of symptoms prior to the acute exacerbation. The development of exertional dyspnea, often described as increased effort to breathe, heaviness, air hunger, or gasping, can be insidious. It is best elicited by a careful history focused on typical physical activities and how the patient’s ability to perform them has changed. Activities involving significant arm work, particularly at or above shoulder level, are particularly difficult for patients with COPD. Conversely, activities that allow the patient to brace the arms and use accessory muscles of respiration are better tolerated. Examples of such

1	are particularly difficult for patients with COPD. Conversely, activities that allow the patient to brace the arms and use accessory muscles of respiration are better tolerated. Examples of such activities include pushing a shopping cart or walking on a treadmill. As COPD advances, the principal feature is worsening dyspnea on exertion with increasing intrusion on the ability to perform vocational or avocational activities. In the most advanced stages, patients are breathless doing simple activities of daily living.

1	Accompanying worsening airflow obstruction is an increased frequency of exacerbations (described below). Patients may also develop resting hypoxemia and require institution of supplemental oxygen. In the early stages of COPD, patients usually have an entirely normal physical examination. Current smokers may have signs of active smoking, including an odor of smoke or nicotine staining of fingernails. In patients with more severe disease, the physical examination is notable for a prolonged expiratory phase and may include expiratory wheezing. In addition, signs of hyperinflation include a barrel chest and enlarged lung volumes with poor diaphragmatic excursion as assessed by percussion. Patients with severe airflow obstruction may also exhibit use of accessory muscles of respiration, sitting in the characteristic “tripod” position to facilitate the actions of the sternocleidomastoid, scalene, and intercostal muscles. Patients may develop cyanosis, visible in the lips and nail beds.

1	Although traditional teaching is that patients with predominant emphysema, termed “pink puffers,” are thin and noncyanotic at rest and have prominent use of accessory muscles, and patients with chronic bronchitis are more likely to be heavy and cyanotic (“blue bloaters”), current evidence demonstrates that most patients have elements of both bronchitis and emphysema and that the physical examination does not reliably differentiate the two entities.

1	Advanced disease may be accompanied by cachexia, with significant weight loss, bitemporal wasting, and diffuse loss of subcutaneous adipose tissue. This syndrome has been associated with both inadequate oral intake and elevated levels of inflammatory cytokines (TNF-α). Such wasting is an independent poor prognostic factor in COPD. Some patients with advanced disease have paradoxical inward movement of the rib cage with inspiration (Hoover’s sign), the result of alteration of the vector of diaphragmatic contraction on the rib cage as a result of chronic hyperinflation. Signs of overt right heart failure, termed cor pulmonale, are relatively infrequent since the advent of supplemental oxygen therapy. Clubbing of the digits is not a sign of COPD, and its presence should alert the clinician to initiate an investigation for causes of clubbing. In this population, the development of lung cancer is the most likely explanation for newly developed clubbing.

1	The hallmark of COPD is airflow obstruction (discussed above). Pulmonary function testing shows airflow obstruction with a reduction in FEV1 and FEV1/FVC (Chap. 306e). With worsening disease severity, lung volumes may increase, resulting in an increase in total lung capacity, functional residual capacity, and residual volume. In patients with emphysema, the diffusing capacity may be reduced, reflecting the lung parenchymal destruction characteristic of the disease. The degree of airflow obstruction is an important prognostic factor in COPD and is the basis for the Global Initiative for Lung Disease (GOLD) severity classification (Table 314-1). More recently it has been shown that a multifactorial index incorporating airflow obstruction, exercise performance, dyspnea, and body mass index is a better predictor of mortality rate than pulmonary function alone. In 2011, the GOLD added an additional classification system incorporating symptoms and exacerbation history; the utility of this

1	is a better predictor of mortality rate than pulmonary function alone. In 2011, the GOLD added an additional classification system incorporating symptoms and exacerbation history; the utility of this system remains to be defined.

1	Arterial blood gases and oximetry may demonstrate resting or exertional hypoxemia. Arterial blood gases provide additional information I Mild FEV1/FVC <0.7 and FEV1 ≥80% predicted II Moderate FEV1/FVC <0.7 and FEV1 ≥50% but <80% predicted III Severe FEV1/FVC <0.7 and FEV1 ≥30% but <50% predicted IV Very severe FEV1/FVC <0.7 and FEV1 <30% predicted Abbreviations: COPD, chronic obstructive pulmonary disease; GOLD, Global Initiative for Lung Disease. Source: From the Global Strategy for Diagnosis, Management and Prevention of COPD 2014, © Global Initiative for Chronic Obstructive Lung Disease (GOLD), all rights reserved. Available from http://www.goldcopd.org.

1	FIGURE 314-4 Chest computed tomography scan of a patient with chronic obstructive pulmonary disease who underwent a left single-lung transplant. Notethereducedparenchymalmark-ings in the right lung (left side of figure) as compared to the left lung, representing emphysematous destruction of the lung, and mediasti-nal shift to the left, indicative of hyperinflation. about alveolar ventilation and acid-base status by measuring arterial Pco2 and pH. The change in pH with Pco2 is 0.08 units/10 mmHg acutely and 0.03 units/10 mmHg in the chronic state. Knowledge of the arterial pH therefore allows the classification of ventilatory failure, defined as Pco2 >45 mmHg, into acute or chronic conditions. The arterial blood gas is an important component of the evaluation of patients presenting with symptoms of an exacerbation. An elevated hematocrit suggests the presence of chronic hypoxemia, as does the presence of signs of right ventricular hypertrophy.

1	Radiographic studies may assist in the classification of the type of COPD. Obvious bullae, paucity of parenchymal markings, or hyperlucency suggests the presence of emphysema. Increased lung volumes and flattening of the diaphragm suggest hyperinflation but do not provide information about chronicity of the changes. Computed tomography (CT) scan is the current definitive test for establishing the presence or absence of emphysema in living subjects (Fig. 314-4). From a practical perspective, the CT scan currently does little to influence therapy of COPD except in individuals considering surgical therapy for their disease (described below) and as screening for lung cancer.

1	Recent guidelines have suggested testing for α1AT deficiency in all subjects with COPD or asthma with chronic airflow obstruction. Measurement of the serum α1AT level is a reasonable initial test. For subjects with low α1AT levels, the definitive diagnosis of α1AT deficiency requires protease inhibitor (PI) type determination. This is typically performed by isoelectric focusing of serum, which reflects the genotype at the PI locus for the common alleles and many of the rare PI alleles as well. Molecular genotyping of DNA can be performed for the common PI alleles (M, S, and Z).

1	Only three interventions—smoking cessation, oxygen therapy in chronically hypoxemic patients, and lung volume reduction surgery in selected patients with emphysema—have been demonstrated to influence the natural history of patients with COPD. There is 1705 currently suggestive, but not definitive, evidence that the use of inhaled glucocorticoids may alter mortality rate (but not lung function). All other current therapies are directed at improving symptoms and decreasing the frequency and severity of exacerbations. The institution of these therapies should involve an assessment of symptoms, potential risks, costs, and benefits of therapy. This should be followed by an assessment of response to therapy, and a decision should be made whether or not to continue treatment.

1	PHARMACOTHERAPY Smoking Cessation (See also Chap. 470) It has been shown that middle-aged smokers who were able to successfully stop smoking experienced a significant improvement in the rate of decline in pulmonary function, returning to annual changes similar to that of nonsmoking patients. Thus, all patients with COPD should be strongly urged to quit smoking and educated about the benefits of quitting. An emerging body of evidence demonstrates that combining phar macotherapy with traditional supportive approaches considerably enhances the chances of successful smoking cessation. There are three principal pharmacologic approaches to the problem: bupropion; nicotine replacement therapy available as gum, transdermal patch, lozenge, inhaler, and nasal spray; and varenicline, a nicotinic acid receptor agonist/antagonist. Current recommendations from the U.S. Surgeon General are that all adult, nonpregnant smokers considering quitting be offered pharmacotherapy, in the absence of any

1	acid receptor agonist/antagonist. Current recommendations from the U.S. Surgeon General are that all adult, nonpregnant smokers considering quitting be offered pharmacotherapy, in the absence of any contraindication to treatment.

1	Bronchodilators In general, bronchodilators are used for symptomatic benefit in patients with COPD. The inhaled route is preferred for medication delivery because the incidence of side effects is lower than that seen with the use of parenteral medication delivery.

1	Anticholinergic Agents Ipratropium bromide improves symptoms and produces acute improvement in FEV1. Tiotropium, a long-acting anticholinergic, has been shown to improve symptoms and reduce exacerbations. Studies of both ipratropium and tiotropium have failed to demonstrate that either influences the rate of decline in FEV1. In a large randomized clinical trial, there was a trend toward reduced mortality rate in the tiotropium-treated patients that approached, but did not reach, statistical significance. Side effects are minor, and a trial of inhaled anticholinergics is recommended in symptomatic patients with COPD. Recent retrospective analyses raised the possibility that anticholinergic use is associated with increased cardiovascular events in the COPD population. This was not demonstrated in a large, prospective randomized trial of tiotropium.

1	Beta Agonists These provide symptomatic benefit. The main side effects are tremor and tachycardia. Long-acting inhaled β agonists, such as salmeterol or formoterol, have benefits comparable to ipratropium bromide. Their use is more convenient than short-acting agents. The addition of a β agonist to inhaled anticholinergic therapy has been demonstrated to provide incremental benefit. A recent report in asthma suggests that those patients, particularly African Americans, using a long-acting β agonist without concomitant inhaled corticosteroids have an increased risk of deaths from respiratory causes. The applicability of these data to patients with COPD is unclear.

1	Inhaled Glucocorticoids Although a recent trial demonstrated an apparent benefit from the regular use of inhaled glucocorticoids on the rate of decline of lung function, a number of other well-designed randomized trials have not. Patients studied included those with mild to severe airflow obstruction and current and ex-smokers. Patients with significant acute response to inhaled β agonists were excluded from many of these trials, which may impact the generalizability of the findings. Their use has been associated with increased rates of oropharyngeal candidiasis and an increased rate of loss of bone density. Available data suggest that inhaled glucocorticoids reduce exacerbation frequency by ~25%. The impact of 1706 inhaled corticosteroids on mortality rates in COPD is controversial. A meta-analysis and several retrospective studies suggest a mortality benefit, but in a recently published randomized trial, differences in mortality rate approached, but did not reach, conventional

1	A meta-analysis and several retrospective studies suggest a mortality benefit, but in a recently published randomized trial, differences in mortality rate approached, but did not reach, conventional criteria for statistical significance. A trial of inhaled glucocorticoids should be considered in patients with frequent exacerbations, defined as two or more per year, and in patients who demonstrate a significant amount of acute reversibility in response to inhaled bronchodilators.

1	Oral Glucocorticoids The chronic use of oral glucocorticoids for treatment of COPD is not recommended because of an unfavorable benefit/risk ratio. The chronic use of oral glucocorticoids is associated with significant side effects, including osteoporosis, weight gain, cataracts, glucose intolerance, and increased risk of infection. A recent study demonstrated that patients tapered off chronic low-dose prednisone (~10 mg/d) did not experience any adverse effect on the frequency of exacerbations, health-related quality of life, or lung function. On average, patients lost ~4.5 kg (~10 lb) when steroids were withdrawn.

1	Theophylline Theophylline produces modest improvements in expiratory flow rates and vital capacity and a slight improvement in arterial oxygen and carbon dioxide levels in patients with moderate to severe COPD. Nausea is a common side effect; tachycardia and tremor have also been reported. Monitoring of blood theophylline levels is typically required to minimize toxicity. The selective phosphodiesterase 4 (PDE4) inhibitor roflumilast has been demonstrated to reduce exacerbation frequency in COPD patients with chronic bronchitis and a prior history of exacerbations; its effects on airflow obstruction and symptoms are modest.

1	Antibiotics As outlined below, there are strong data implicating bacterial infection as a precipitant of a substantial portion of exacerbations. Early trials of prophylactic or suppressive antibiotics, given either seasonally or year round, failed to show a positive impact on exacerbation occurrence. More recently, a randomized clinical trial of azithromycin, chosen for both its anti-inflammatory and antimicrobial properties, administered daily to subjects with a history of exacerbation in the past 6 months demonstrated a reduced exacerbation frequency and longer time to first exacerbation in the macrolide-treated cohort (hazard ratio, 0.73).

1	Oxygen Supplemental O2 is the only pharmacologic therapy demonstrated to unequivocally decrease mortality rates in patients with COPD. For patients with resting hypoxemia (resting O2 saturation ≤88% or <90% with signs of pulmonary hypertension or right heart failure), the use of O2 has been demonstrated to have a significant impact on mortality rate. Patients meeting these criteria should be on continual oxygen supplementation because the mortality benefit is proportional to the number of hours per day oxygen is used. Various delivery systems are available, including portable systems that patients may carry to allow mobility outside the home. Supplemental O2 is commonly prescribed for patients with exertional hypoxemia or nocturnal hypoxemia. Although the rationale for supplemental O2 in these settings is physiologically sound, the benefits of such therapy are not well substantiated.

1	Other Agents N-acetyl cysteine has been used in patients with COPD for both its mucolytic and antioxidant properties. A prospective trial failed to find any benefit with respect to decline in lung function or prevention of exacerbations. Specific treatment in the form of IV α1AT augmentation therapy is available for individuals with severe α1AT deficiency. Despite sterilization procedures for these blood-derived products and the absence of reported cases of viral infection from therapy, some physicians recommend hepatitis B vaccination prior to starting augmentation therapy. Although biochemical efficacy of α1AT augmentation therapy has been shown, a randomized controlled trial of α1AT augmentation therapy has not definitively established the efficacy of augmentation therapy in reducing decline of pulmonary function. Eligibility for α1AT augmentation therapy requires a serum α1AT level <11 μM (approximately 50 mg/dL). Typically, PiZ individuals will qualify, although other rare types

1	of pulmonary function. Eligibility for α1AT augmentation therapy requires a serum α1AT level <11 μM (approximately 50 mg/dL). Typically, PiZ individuals will qualify, although other rare types associated with severe deficiency (e.g., null-null) are also eligible. Because only a fraction of individuals with severe α1AT deficiency will develop COPD, α1AT augmentation therapy is not recommended for severely α1AT-deficient persons with normal pulmonary function and a normal chest CT scan.

1	NONPHARMACOLOGIC THERAPIES General Medical Care Patients with COPD should receive the influenza vaccine annually. Polyvalent pneumococcal vaccine is also recommended, although proof of efficacy in this patient population is not definitive. Similar recommendations and limitations of evidence also exist for vaccination for Bordetella pertussis. Pulmonary Rehabilitation This refers to a treatment program that incorporates education and cardiovascular conditioning. In COPD, pulmonary rehabilitation has been demonstrated to improve health-related quality of life, dyspnea, and exercise capacity. It has also been shown to reduce rates of hospitalization over a 6to 12-month period.

1	Lung Volume Reduction Surgery (LVRS) Surgery to reduce the volume of lung in patients with emphysema was first introduced with minimal success in the 1950s and was reintroduced in the 1990s. Patients are excluded if they have significant pleural disease, a pulmonary artery systolic pressure >45 mmHg, extreme deconditioning, congestive heart failure, or other severe comorbid conditions. Patients with an FEV1 <20% of predicted and either diffusely distributed emphysema on CT scan or diffusing capacity of lung for carbon monoxide (DLCO) <20% of predicted have an increased mortality rate after the procedure and thus are not candidates for LVRS.

1	The National Emphysema Treatment trial demonstrated that LVRS offers both a mortality benefit and a symptomatic benefit in certain patients with emphysema. The anatomic distribution of emphysema and post-rehabilitation exercise capacity are important prognostic characteristics. Patients with upper lobe–predominant emphysema and a low post-rehabilitation exercise capacity are most likely to benefit from LVRS. Lung Transplantation (See also Chap. 320e) COPD is currently the second leading indication for lung transplantation (Fig. 314-4). Current recommendations are that candidates for lung transplantation should have severe disability despite maximal medical therapy and be free of comorbid conditions such as liver, renal, or cardiac disease. In contrast to LVRS, the anatomic distribution of emphysema and the presence of pulmonary hypertension are not contraindications to lung transplantation.

1	Exacerbations are a prominent feature of the natural history of COPD. Exacerbations are episodes of increased dyspnea and cough and change in the amount and character of sputum. They may or may not be accompanied by other signs of illness, including fever, myalgias, and sore throat. Self-reported health-related quality of life correlates with frequency of exacerbations more closely than it does with the degree of airflow obstruction. Economic analyses have shown that >70% of COPD-related health care expenditures go to emergency department visits and hospital care; this translates to >$10 billion annually in the United States. The frequency of exacerbations increases as airflow obstruction increases; patients with moderate to severe airflow obstruction (GOLD stage III or IV; Table 314-1) on average have one to three episodes per year. However, some individuals with very severe airflow obstruction do not have frequent exacerbations; the history of prior exacerbations is a strong

1	on average have one to three episodes per year. However, some individuals with very severe airflow obstruction do not have frequent exacerbations; the history of prior exacerbations is a strong predictor of future exacerbations. Recently, an elevated ratio of the diameter of the pulmonary artery to aorta on chest CT has been associated with increased risk of COPD exacerbations.

1	The approach to the patient experiencing an exacerbation includes an assessment of the severity of the patient’s illness, both acute and chronic components; an attempt to identify the precipitant of the exacerbation; and the institution of therapy. Precipitating Causes and Strategies to Reduce Frequency of Exacerbations A variety of stimuli may result in the final common pathway of airway inflammation and increased symptoms that are characteristic of COPD exacerbations. Studies suggest that acquiring a new strain of bacteria is associated with increased near-term risk of exacerbation and that bacterial infection/superinfection is involved in over 50% of exacerbations. Viral respiratory infections are present in approximately one-third of COPD exacerbations. In a significant minority of instances (20–35%), no specific precipitant can be identified.

1	The role of pharmacotherapy in reducing exacerbation frequency is less well studied. Chronic oral glucocorticoids are not recommended for this purpose. Inhaled glucocorticoids reduce the frequency of exacerbations by 25–30% in most analyses. The use of inhaled glucocorticoids should be considered in patients with frequent exacerbations or those who have an asthmatic component, i.e., significant reversibility on pulmonary function testing or marked symptomatic improvement after inhaled bronchodilators. Similar magnitudes of reduction have been reported for anticholinergic and long-acting β-agonist therapy. The influenza vaccine has been shown to reduce exacerbation rates in patients with COPD. As outlined above, daily azithromycin administered to subjects with COPD and an exacerbation history reduces exacerbation frequency.

1	Patient Assessment An attempt should be made to establish the severity of the exacerbation as well as the severity of preexisting COPD. The more severe either of these two components, the more likely that the patient will require hospital admission. The history should include quantification of the degree of dyspnea by asking about breathlessness during activities of daily living and typical activities for the patient. The patient should be asked about fever; change in character of sputum; any ill contacts; and associated symptoms such as nausea, vomiting, diarrhea, myalgias, and chills. Inquiring about the frequency and severity of prior exacerbations can provide important information.

1	The physical examination should incorporate an assessment of the degree of distress of the patient. Specific attention should be focused on tachycardia, tachypnea, use of accessory muscles, signs of perioral or peripheral cyanosis, the ability to speak in complete sentences, and the patient’s mental status. The chest examination should establish the presence or absence of focal findings, degree of air movement, presence or absence of wheezing, asymmetry in the chest examination (suggesting large airway obstruction or pneumothorax mimicking an exacerbation), and the presence or absence of paradoxical motion of the abdominal wall.

1	Patients with severe underlying COPD, who are in moderate or severe distress, or those with focal findings should have a chest x-ray. Approximately 25% of x-rays in this clinical situation will be abnormal, with the most frequent findings being pneumonia and congestive heart failure. Patients with advanced COPD, those with a history of hypercarbia, those with mental status changes (confusion, sleepiness), or those in significant distress should have an arterial blood-gas measurement. The presence of hypercarbia, defined as a PCO2 >45 mmHg, has important implications for treatment (discussed below). In contrast to its utility in the management of exacerbations of asthma, measurement of pulmonary function has not been demonstrated to be helpful in the diagnosis or management of exacerbations of COPD.

1	There are no definitive guidelines concerning the need for inpatient treatment of exacerbations. Patients with respiratory acidosis and hypercarbia, significant hypoxemia, or severe underlying disease or those whose living situation is not conducive to careful observation and the delivery of prescribed treatment should be admitted to the hospital. ACUTE EXACERBATIONS Bronchodilators Typically, patients are treated with an inhaled β agonist, often with the addition of an anticholinergic agent. These may be administered separately or together, and the frequency of administration depends on the severity of the exacerbation.

1	Patients are often treated initially with nebulized therapy, as such 1707 treatment is often easier to administer in older patients or to those in respiratory distress. It has been shown, however, that conversion to metered-dose inhalers is effective when accompanied by education and training of patients and staff. This approach has significant economic benefits and also allows an easier transition to outpatient care. The addition of methylxanthines (such as theophylline) to this regimen can be considered, although convincing proof of its efficacy is lacking. If added, serum levels should be monitored in an attempt to minimize toxicity.

1	Antibiotics Patients with COPD are frequently colonized with potential respiratory pathogens, and it is often difficult to identify conclusively a specific species of bacteria responsible for a particular clinical event. Bacteria frequently implicated in COPD exacerbations include Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. In addition, Mycoplasma pneumoniae or Chlamydia pneumoniae are found in 5–10% of exacerbations. The choice of antibiotic should be based on local patterns of antibiotic susceptibility of the above pathogens as well as the patient’s clinical condition. Most practitioners treat patients with moderate or severe exacerbations with antibiotics, even in the absence of data implicating a specific pathogen.

1	Glucocorticoids Among patients admitted to the hospital, the use of glucocorticoids has been demonstrated to reduce the length of stay, hasten recovery, and reduce the chance of subsequent exacerbation or relapse for a period of up to 6 months. One study demonstrated that 2 weeks of glucocorticoid therapy produced benefit indistinguishable from 8 weeks of therapy. The GOLD guidelines recommend 30–40 mg of oral prednisolone or its equivalent for a period of 10–14 days. Hyperglycemia, particularly in patients with preexisting diagnosis of diabetes, is the most frequently reported acute complication of glucocorticoid treatment.

1	Oxygen Supplemental O2 should be supplied to keep arterial saturations ≥90%. Hypoxemic respiratory drive plays a small role in patients with COPD. Studies have demonstrated that in patients with both acute and chronic hypercarbia, the administration of supplemental O2 does not reduce minute ventilation. It does, in some patients, result in modest increases in arterial PCO2, chiefly by altering ventilation-perfusion relationships within the lung. This should not deter practitioners from providing the oxygen needed to correct hypoxemia.

1	Mechanical Ventilatory Support The initiation of noninvasive positive-pressure ventilation (NIPPV) in patients with respiratory failure, defined as PaCO2 >45 mmHg, results in a significant reduction in mortality rate, need for intubation, complications of therapy, and hospital length of stay. Contraindications to NIPPV include cardiovascular instability, impaired mental status or inability to cooperate, copious secretions or the inability to clear secretions, craniofacial abnormalities or trauma precluding effective fitting of mask, extreme obesity, or significant burns.

1	Invasive (conventional) mechanical ventilation via an endotracheal tube is indicated for patients with severe respiratory distress despite initial therapy, life-threatening hypoxemia, severe hypercarbia and/or acidosis, markedly impaired mental status, respiratory arrest, hemodynamic instability, or other complications. The goal of mechanical ventilation is to correct the aforementioned conditions. Factors to consider during mechanical ventilatory support include the need to provide sufficient expiratory time in patients with severe airflow obstruction and the presence of auto-PEEP (positive end-expiratory pressure), which can result in patients having to generate significant respiratory effort to trigger a breath during a demand mode of ventilation. The mortality rate of patients requiring mechanical ventilatory support is 17–30% for that particular hospitalization. For patients age >65 admitted to the intensive care unit for treatment, the mortality rate doubles over the next year

1	mechanical ventilatory support is 17–30% for that particular hospitalization. For patients age >65 admitted to the intensive care unit for treatment, the mortality rate doubles over the next year to 60%, regardless of whether mechanical ventilation was required.

1	Talmadge E. King, Jr. taBLe 315-1 Major CategorieS of aLVeoLar anD interStitiaL infLaMMatory Lung DiSeaSe Lung Response: Alveolitis, Interstitial Inflammation, and Fibrosis Known Cause Patients with interstitial lung diseases (ILDs) come to medical attention mainly because of the onset of progressive exertional dyspnea or a persistent nonproductive cough. Hemoptysis, wheezing, and chest pain may be present. Often, the identification of interstitial opacities on chest x-ray focuses the diagnostic approach on one of the ILDs.

1	ILDs represent a large number of conditions that involve the parenchyma of the lung—the alveoli, the alveolar epithelium, the capillary endothelium, and the spaces between those structures—as well as the perivascular and lymphatic tissues. The disorders in this heterogeneous group are classified together because of similar clinical, roentgenographic, physiologic, or pathologic manifestations. These disorders often are associated with considerable rates of morbidity and mortality, and there is little consensus regarding the best management of most of them.

1	ILDs have been difficult to classify because >200 known individual diseases are characterized by diffuse parenchymal lung involvement, either as the primary condition or as a significant part of a multiorgan process, as may occur in the connective tissue diseases (CTDs). One useful approach to classification is to separate the ILDs into two groups based on the major underlying histopathology: (1) those associated with predominant inflammation and fibrosis and (2) those with a predominantly granulomatous reaction in interstitial or vascular areas (Table 315-1). Each of these groups can be subdivided further according to whether the cause is known or unknown. For each ILD there may be an acute phase, and there is usually a chronic one as well. Rarely, some are recurrent, with intervals of subclinical disease.

1	Sarcoidosis (Chap. 390), idiopathic pulmonary fibrosis (IPF), and pulmonary fibrosis associated with CTDs (Chaps. 378, 382, 388, and 427) are the most common ILDs of unknown etiology. Among the ILDs of known cause, the largest group includes occupational and environmental exposures, especially the inhalation of inorganic dusts, organic dusts, and various fumes or gases (Chap. 311). A multidisciplinary approach—requiring close communication between clinician, radiologist, and when appropriate, pathologist—is often required to make the diagnosis. High-resolution computed tomography (HRCT) scanning improves the diagnostic accuracy and may eliminate the need for tissue examination in many cases, especially in IPF. For other forms, tissue examination, usually obtained by thoracoscopic lung biopsy, is critical to confirmation of the diagnosis.

1	The ILDs are nonmalignant disorders and are not caused by identified infectious agents. The precise pathway(s) leading from injury to fibrosis is not known. Although there are multiple initiating agent(s) of injury, the immunopathogenic responses of lung tissue are limited, and the mechanisms of repair have common features (Fig. 315-1). As mentioned above, the two major histopathologic patterns are a granulomatous pattern and a pattern in which inflammation and fibrosis predominate. Granulomatous Lung Disease This process is characterized by an accumulation of T lymphocytes, macrophages, and epithelioid cells organized into discrete structures (granulomas) in the lung parenchyma. The granulomatous lesions can progress to fibrosis. Many patients with granulomatous lung disease remain free of severe impairment of lung function or, when symptomatic, improve after treatment. The main differential diagnosis is between sarcoidosis (Chap. 390) and hypersensitivity pneumonitis (Chap. 310).

1	Inflammation and Fibrosis The initial insult is an injury to the epithelial surface that causes inflammation in the air spaces and alveolar walls. If the disease becomes chronic, inflammation spreads to adjacent portions of the interstitium and vasculature and eventually causes interstitial fibrosis. Important histopathologic patterns found in the ILDs include Asbestos Residual of acute respiratory distress syndrome Fumes, gases Smoking-related Drugs (antibiotics, amiodarone, Desquamative interstitial pneumonia gold) and chemotherapy drugs Idiopathic pulmonary fibrosis Lymphocytic infiltrative disorders (lympho(usual interstitial pneumonia) cytic interstitial pneumonitis associated with connective tissue disease) Tuberous sclerosis, neurofibromatosis, Idiopathic lymphocytic Niemann-Pick disease, Gaucher disease, interstitial pneumonia Hermansky-Pudlak syndrome Bronchiolocentric patterns of interstitial pneumonia

1	Bronchiolocentric patterns of interstitial pneumonia Connective tissue diseases Gastrointestinal or liver diseases (Crohn disease, primary biliary cirrhosis, chronic Systemic lupus erythemaactive hepatitis, ulcerative colitis) tosus, rheumatoid arthritis, ankylosing spondylitis, systemic sclerosis, Sjögren syndrome, polymyositisdermatomyositis Goodpasture syndrome, idiopathic pulmonary hemosiderosis, isolated pulmonary capillaritis Lung Response: Granulomatous Hypersensitivity pneumonitis Inorganic dusts: beryllium, silica (organic dusts) Granulomatosis with polyangiitis (Wegener)

1	Lung Response: Granulomatous Hypersensitivity pneumonitis Inorganic dusts: beryllium, silica (organic dusts) Granulomatosis with polyangiitis (Wegener) Eosinophilic granulomatosis with polyangiitis (Churg-Strauss) usual interstitial pneumonia (UIP), nonspecific interstitial pneumonia, respiratory bronchiolitis/desquamative interstitial pneumonia, organizing pneumonia, diffuse alveolar damage (acute or organizing), and lymphocytic interstitial pneumonia. The development of irreversible scarring (fibrosis) of alveolar walls, airways, or vasculature is the most feared outcome in all of these conditions because it is often progressive and leads to significant derangement of ventilatory function and gas exchange. Dust, fumes, cigarette smoke, autoimmune conditions Drugs, infections, radiation, other diseases

1	Dust, fumes, cigarette smoke, autoimmune conditions Drugs, infections, radiation, other diseases FIGURE 315-1 Proposed mechanism for the pathogenesis of pulmonary fibrosis. The lung is naturally exposed to repetitive injury from a variety of exogenous and endogenous stimuli. Several local and systemic factors (e.g., fibroblasts, circulating fibrocytes, chemokines, growth factors, and clotting factors) contribute to tissue healing and functional recovery. Dysregulation of this intricate network through genetic predisposition, autoimmune conditions, or superimposed diseases can lead to aberrant wound healing, with the result of pulmonary fibrosis. Alternatively, excessive injury to the lung may overwhelm even intact reparative mechanisms and lead to pulmonary fibrosis. (From S Garantziotis et al: J Clin Invest 114:319, 2004.)

1	HISTORY Duration of Illness Acute presentation (days to weeks), although unusual, occurs with allergy (drugs, fungi, helminths), acute interstitial pneumonia (AIP), eosinophilic pneumonia, and hypersensitivity pneumonitis. These conditions may be confused with atypical pneumonias because of diffuse alveolar opacities on chest x-ray. Subacute presentation (weeks to months) may occur in all ILDs but is seen especially in sarcoidosis, drug-induced ILDs, the alveolar hemorrhage syndromes, cryptogenic organizing pneumonia (COP), and the acute immunologic pneumonia that complicates systemic lupus erythematosus (SLE) or polymyositis. In most ILDs, the symptoms and signs form a chronic presentation (months to years). Examples include IPF, sarcoidosis, pulmonary Langerhans cell histiocytosis (PLCH), pneumoconioses, and CTDs. Episodic presentations are unusual and include eosinophilic pneumonia, hypersensitivity pneumonitis, COP, vasculitides, pulmonary hemorrhage, and Churg-Strauss syndrome.

1	Age Most patients with sarcoidosis, ILD associated with CTD, lymphangioleiomyomatosis (LAM), PLCH, and inherited forms of ILD (familial IPF, Gaucher disease, Hermansky-Pudlak syndrome) present between the ages of 20 and 40 years. Most patients with IPF are older than 60 years. Gender LAM and pulmonary involvement in tuberous sclerosis occur exclusively in premenopausal women. In addition, ILD in Hermansky-Pudlak syndrome and in the CTDs is more common in women; an exception is ILD in rheumatoid arthritis (RA), which is more common in men. IPF is more common in men. Because of occupational exposures, pneumoconioses also occur more frequently in men.

1	Family History Familial lung fibrosis has been associated with mutations in the surfactant protein C gene, the surfactant protein A2 gene, telomerase reverse transcriptase (TERT), telomerase RNA component (TERC), and the promoter of a mucin gene (MUC5B). Familial lung fibrosis is characterized by several patterns of interstitial pneumonia, including nonspecific interstitial pneumonia, desquamative interstitial pneumonia, and UIP. Older age, male sex, and a history of cigarette smoking have been identified as risk factors for familial lung fibro-1709 sis. Family associations (with an autosomal dominant pattern) have been identified in tuberous sclerosis and neurofibromatosis. Familial clustering has been identified increasingly in sarcoidosis. The genes responsible for several rare ILDs have been identified, i.e., alveolar microlithiasis, Gaucher disease, Hermansky-Pudlak syndrome, and Niemann-Pick disease, along with the genes for surfactant homeostasis in pulmonary alveolar

1	ILDs have been identified, i.e., alveolar microlithiasis, Gaucher disease, Hermansky-Pudlak syndrome, and Niemann-Pick disease, along with the genes for surfactant homeostasis in pulmonary alveolar proteinosis and for control of cell growth and differentiation in LAM.

1	Smoking History Two-thirds to 75% of patients with IPF and familial lung fibrosis have a history of smoking. Patients with PLCH, respiratory bronchiolitis/desquamative interstitial pneumonia (DIP), Goodpasture syndrome, respiratory bronchiolitis, and pulmonary alveolar proteinosis are usually current or former smokers. Occupational and Environmental History A strict chronologic listing of the patient’s lifelong employment must be sought, including specific duties and known exposures. In hypersensitivity pneumonitis (see Fig. 310-1), respiratory symptoms, fever, chills, and an abnormal chest roentgenogram are often temporally related to a hobby (pigeon breeder’s disease) or to the workplace (farmer’s lung) (Chap. 310). Symptoms may diminish or disappear after the patient leaves the site of exposure for several days; similarly, symptoms may reappear when the patient returns to the exposure site.

1	Other Important Past History Parasitic infections may cause pulmonary eosinophilia, and therefore a travel history should be taken in patients with known or suspected ILD. History of risk factors for HIV infection should be elicited because several processes may occur at the time of initial presentation or during the clinical course, e.g., HIV infection, organizing pneumonia, AIP, lymphocytic interstitial pneumonitis, and diffuse alveolar hemorrhage.

1	Respiratory Symptoms and Signs Dyspnea is a common and prominent complaint in patients with ILD, especially the idiopathic interstitial pneumonias, hypersensitivity pneumonitis, COP, sarcoidosis, eosinophilic pneumonias, and PLCH. Some patients, especially those with sarcoidosis, silicosis, PLCH, hypersensitivity pneumonitis, lipoid pneumonia, or lymphangitis carcinomatosis, may have extensive parenchymal lung disease on chest imaging studies without significant dyspnea, especially early in the course of the illness. Wheezing is an uncommon manifestation of ILD but has been described in patients with chronic eosinophilic pneumonia, Churg-Strauss syndrome, respiratory bronchiolitis, and sarcoidosis. Clinically significant chest pain is uncommon in most ILDs. However, substernal discomfort is common in sarcoidosis. Sudden worsening of dyspnea, especially if associated with acute chest pain, may indicate a spontaneous pneumothorax, which occurs in PLCH, tuberous sclerosis, LAM, and

1	is common in sarcoidosis. Sudden worsening of dyspnea, especially if associated with acute chest pain, may indicate a spontaneous pneumothorax, which occurs in PLCH, tuberous sclerosis, LAM, and neurofibromatosis. Frank hemoptysis and blood-streaked sputum are rarely presenting manifestations of ILD but can be seen in the diffuse alveolar hemorrhage (DAH) syndromes, LAM, tuberous sclerosis, and the granulomatous vasculitides. Fatigue and weight loss are common in all ILDs.

1	The findings are usually not specific. Most commonly, physical examination reveals tachypnea and bibasilar end-inspiratory dry crackles, which are common in most forms of ILD associated with inflammation but are less likely to be heard in the granulomatous lung diseases. Crackles may be present in the absence of radiographic abnormalities on the chest radiograph. Scattered late inspiratory high-pitched rhonchi—so-called inspiratory squeaks—are heard in patients with bronchiolitis. The cardiac examination is usually normal except in the middle or late stages of the disease, when findings of pulmonary hypertension and cor pulmonale may become evident (Chap. 304). Cyanosis and clubbing of the digits occur in some patients with advanced disease.

1	Antinuclear antibodies and anti-immunoglobulin antibodies (rheumatoid factors) are identified in some patients, even in the absence 1710 of a defined CTD. A raised lactate dehydrogenase (LDH) level is a nonspecific finding common to ILDs. Elevation of the serum level of angiotensin-converting enzyme is common in ILDs, especially sarcoidosis. Serum precipitins confirm exposure when hypersensitivity pneumonitis is suspected, although they are not diagnostic of the process. Antineutrophil cytoplasmic or anti-basement membrane antibodies are useful if vasculitis is suspected. The electrocardiogram is usually normal unless pulmonary hypertension is present; then it demonstrates right-axis deviation, right ventricular hypertrophy, or right atrial enlargement or hypertrophy. Echocardiography also reveals right ventricular dilation and/or hypertrophy in the presence of pulmonary hypertension.

1	CHEST IMAGING STUDIES Chest X-Ray ILD may be first suspected on the basis of an abnormal chest radiograph, which most commonly reveals a bibasilar reticular pattern. A nodular or mixed pattern of alveolar filling and increased reticular markings also may be present. Subgroups of ILDs exhibit nodular opacities with a predilection for the upper lung zones (sarcoidosis, PLCH, chronic hypersensitivity pneumonitis, silicosis, berylliosis, RA [necrobiotic nodular form], ankylosing spondylitis). The chest x-ray correlates poorly with the clinical or histopathologic stage of the disease. The radiographic finding of honeycombing correlates with pathologic findings of small cystic spaces and progressive fibrosis; when present, it portends a poor prognosis. In most cases, the chest radiograph is nonspecific and usually does not allow a specific diagnosis.

1	Computed Tomography HRCT is superior to the plain chest x-ray for early detection and confirmation of suspected ILD (Fig. 315-2). In addition, HRCT allows better assessment of the extent and distribution of disease, and it is especially useful in the investigation of patients with a normal chest radiograph. Coexisting disease is often best recognized on HRCT scanning, e.g., mediastinal adenopathy, carcinoma, or emphysema. In the appropriate clinical setting, HRCT may be sufficiently characteristic to preclude the need for lung biopsy in IPF, sarcoidosis, hypersensitivity pneumonitis, asbestosis, lymphangitic carcinoma, and PLCH. When a lung biopsy is required, HRCT scanning is useful for determining the most appropriate area from which biopsy samples should be taken.

1	PULMONARY FUNCTION TESTING Spirometry and Lung Volumes Measurement of lung function is important in assessing the extent of pulmonary involvement in patients with ILD. Most forms of ILD produce a restrictive defect with reduced total lung capacity (TLC), functional residual capacity, and residual volume FIGURE 315-2 Idiopathic pulmonary fibrosis. High-resolution computed tomography image shows bibasal, peripheral predominant reticular abnormality with traction bronchiectasis and honeycombing. The lung biopsy showed the typical features of usual interstitial pneumonia.

1	(Chap. 306e). Forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) are reduced, but these changes are related to the decreased TLC. The FEV1/FVC ratio is usually normal or increased. Lung volumes decrease as lung stiffness worsens with disease progression. A few disorders produce interstitial opacities on chest x-ray and obstructive airflow limitation on lung function testing (uncommon in sarcoidosis and hypersensitivity pneumonitis but common in tuberous sclerosis and LAM). Pulmonary function studies have been proved to have prognostic value in patients with idiopathic interstitial pneumonias, particularly IPF and nonspecific interstitial pneumonia (NSIP).

1	Diffusing Capacity A reduction in the diffusing capacity of the lung for carbon monoxide (DlCO) is a common but nonspecific finding in most ILDs. This decrease is due in part to effacement of the alveolar capillary units but, more important, to mismatching of ventilation and perfusion (V. /Q. ). Lung regions with reduced compliance due to either fibrosis or cellular infiltration may be poorly ventilated but may still maintain adequate blood flow, and the ventilation-perfusion mismatch in these regions acts like true venous admixture. The severity of the reduction in DlCO does not correlate with disease stage.

1	Arterial Blood Gas The resting arterial blood gas may be normal or reveal hypoxemia (secondary to a mismatching of ventilation to perfusion) and respiratory alkalosis. A normal arterial O2 tension (or saturation by oximetry) at rest does not rule out significant hypoxemia during exercise or sleep. Carbon dioxide (CO2) retention is rare and is usually a manifestation of end-stage disease.

1	Because hypoxemia at rest is not always present and because severe exercise-induced hypoxemia may go undetected, it is useful to perform exercise testing with measurement of arterial blood gases to detect abnormalities of gas exchange. Arterial oxygen desaturation, a failure to decrease dead space appropriately with exercise (i.e., a high Vd/Vt [dead space/tidal volume] ratio [Chap. 306e]), and an excessive increase in respiratory rate with a lower than expected recruitment of tidal volume provide useful information about physiologic abnormalities and extent of disease. Serial assessment of resting and exercise gas exchange is an excellent method for following disease activity and responsiveness to treatment, especially in patients with IPF. Increasingly, the 6-min walk test is used to obtain a global evaluation of submaximal exercise capacity in patients with ILD. The walk distance and level of oxygen desaturation tend to correlate with the patient’s baseline lung function and mirror

1	a global evaluation of submaximal exercise capacity in patients with ILD. The walk distance and level of oxygen desaturation tend to correlate with the patient’s baseline lung function and mirror the patient’s clinical course.

1	In selected diseases (e.g., sarcoidosis, hypersensitivity pneumonitis, DAH syndrome, cancer, pulmonary alveolar proteinosis), cellular analysis of BAL fluid may be useful in narrowing the differential diagnostic possibilities among various types of ILD (Table 315-2). The role of BAL in defining the stage of disease and assessment of disease progression or response to therapy remains poorly understood, and the usefulness of BAL in the clinical assessment and management remains to be established.

1	Lung biopsy is the most effective method for confirming the diagnosis and assessing disease activity. The findings may identify a more treatable process than originally suspected, particularly chronic hypersensitivity pneumonitis, COP, respiratory bronchiolitis–associated ILD, or sarcoidosis. Biopsy should be obtained before the initiation of treatment. A definitive diagnosis avoids confusion and anxiety later in the clinical course if the patient does not respond to therapy or experiences serious side effects from it. Fiberoptic bronchoscopy with multiple transbronchial lung biopsies (four to eight biopsy samples) is often the initial procedure of choice, especially when sarcoidosis, lymphangitic carcinomatosis, eosinophilic pneumonia, Goodpasture syndrome, or infection is suspected. If a specific diagnosis is not made by transbronchial biopsy, surgical lung biopsy by video-assisted thoracic surgery or open thoracotomy is

1	Sarcoidosis Lymphocytosis; CD4:CD8 ratio >3.5 most specific of diagnosis Organizing pneumonia Foamy macrophages; mixed pattern of increased cells characteristic; decreased CD4:CD8 ratio Diffuse alveolar bleeding Hemosiderin-laden macrophages, red blood cells Diffuse alveolar damage, Atypical hyperplastic type II pneumocytes drug toxicity Opportunistic infections Pneumocystis carinii, fungi, cytomegalovirustransformed cells Lymphangitic carcinomatosis, Malignant cells alveolar cell carcinoma, pulmonary lymphoma Alveolar proteinosis Milky effluent, foamy macrophages and lipoproteinaceous intraalveolar material (periodic acid–Schiff stain–positive) Pulmonary Langerhans cell Increased CD1+ Langerhans cells, electron histiocytosis microscopy demonstrating Birbeck granule in lavaged macrophage (expensive and difficult to perform) Asbestos-related pulmonary Dust particles, ferruginous bodies disease Berylliosis Positive lymphocyte transformation test to beryllium

1	Asbestos-related pulmonary Dust particles, ferruginous bodies disease Berylliosis Positive lymphocyte transformation test to beryllium Lipoidosis Accumulation of specific lipopigment in alveolar macrophages indicated. Adequate-sized biopsies from multiple sites, usually from two lobes, should be obtained. Relative contraindications to lung biopsy include serious cardiovascular disease, honeycombing and other roentgenographic evidence of diffuse end-stage disease, severe pulmonary dysfunction, and other major operative risks, especially in the elderly.

1	Although the course of ILD is variable, progression is common and often insidious. All treatable possibilities should be carefully considered. Because therapy does not reverse fibrosis, the major goals of treatment are permanent removal of the offending agent, when known, and early identification and aggressive suppression of the acute and chronic inflammatory process, thereby reducing further lung damage. Hypoxemia (PaO2 <55 mmHg) at rest and/or with exercise should be managed with supplemental oxygen. Management of cor pulmonale may be required as the disease progresses (Chaps. 280 and 304). Pulmonary rehabilitation has been shown to improve the quality of life in patients with ILD.

1	Glucocorticoids are the mainstay of therapy for suppression of the inflammation present in ILD, but the success rate is low. There have been no placebo-controlled trials of glucocorticoids in ILD, and so there is no direct evidence that steroids improve survival in many of the diseases for which they are commonly used. Glucocorticoid therapy is recommended for symptomatic ILD patients with eosinophilic pneumonias, COP, CTD, sarcoidosis, hypersensitivity pneumonitis, acute inorganic dust exposures, acute radiation pneumonitis, DAH, and drug-induced ILD. In organic dust disease, glucocorticoids are recommended for both the acute and chronic stages.

1	The optimal dose and proper length of therapy with glucocorticoids in the treatment of most ILDs are not known. A common starting dose is prednisone, 0.5–1 mg/kg in a once-daily oral dose 1711 (based on the patient’s lean body weight). This dose is continued for 4–12 weeks, at which time the patient is reevaluated. If the patient is stable or improved, the dose is tapered to 0.25–0.5 mg/kg and is maintained at this level for an additional 4–12 weeks, depending on the course. Rapid tapering or a shortened course of glucocorticoid treatment can result in recurrence. If the patient’s condition continues to decline on glucocorticoids, a second agent (see below) often is added and the prednisone dose is lowered to or maintained at 0.25 mg/kg per day.

1	Cyclophosphamide, azathioprine (1–2 mg/kg lean body weight per day), and mycophenolate mofetil, with or without glucocorticoids, have been tried with variable success in IPF, vasculitis, progressive systemic sclerosis, and other ILDs. An objective response usually requires at least 8–12 weeks to occur. In situations in which these drugs have failed or could not be tolerated, other agents, including methotrexate and cyclosporine, have been tried. However, their role in the treatment of ILDs remains to be determined. Many cases of ILD are chronic and irreversible despite the therapy discussed above, and lung transplantation may then be considered (Chap. 320e). IPF is the most common form of idiopathic interstitial pneumonia. Separating IPF from other forms of lung fibrosis is an important step in the evaluation of all patients presenting with ILD. IPF has a distinctly poor response to therapy and a bad prognosis.

1	Clinical Manifestations Exertional dyspnea, a nonproductive cough, and inspiratory crackles with or without digital clubbing may be present on physical examination. HRCT lung scans typically show patchy, predominantly basilar, subpleural reticular opacities, often associated with traction bronchiectasis and honeycombing (Fig. 315-2). A definite UIP pattern on HRCT is highly accurate for the presence of a UIP pattern on surgical lung biopsy. Atypical findings that should suggest an alternative diagnosis include extensive ground-glass abnormality, nodular opacities, upper or midzone predominance, and prominent hilar or mediastinal lymphadenopathy. Pulmonary function tests often reveal a restrictive pattern, a reduced DlCO, and arterial hypoxemia that is exaggerated or elicited by exercise.

1	Histologic Findings Confirmation of the presence of the UIP pattern on histologic examination is essential to confirm this diagnosis. Transbronchial biopsies are not helpful in making the diagnosis of UIP, and surgical biopsy usually is required. The histologic hallmark and chief diagnostic criterion of UIP is a heterogeneous appearance at low magnification with alternating areas of normal lung, interstitial inflammation, foci of proliferating fibroblasts, dense collagen fibrosis, and honeycomb changes. These histologic changes affect the peripheral, subpleural parenchyma most severely. The interstitial inflammation is usually patchy and consists of a lymphoplasmacytic infiltrate in the alveolar septa, associated with hyperplasia of type 2 pneumocytes. The fibrotic zones are composed mainly of dense collagen, although scattered foci of proliferating fibroblasts are a consistent finding. The extent of fibroblastic proliferation is predictive of disease progression. Areas of honeycomb

1	of dense collagen, although scattered foci of proliferating fibroblasts are a consistent finding. The extent of fibroblastic proliferation is predictive of disease progression. Areas of honeycomb change are composed of cystic fibrotic air spaces that frequently are lined by bronchiolar epithelium and filled with mucin. Smooth-muscle hyperplasia is commonly seen in areas of fibrosis and honeycomb change. A fibrotic pattern with some features similar to UIP may be found in the chronic stage of several specific disorders, such as pneumoconioses (e.g., asbestosis), radiation injury, certain drug-induced lung diseases (e.g., nitrofurantoin), chronic aspiration, sarcoidosis, chronic hypersensitivity pneumonitis, organized chronic eosinophilic pneumonia, and PLCH. Commonly, other histopathologic features are present in these situations, thus allowing separation of these lesions from the UIP-like pattern. Consequently, the term usual interstitial pneumonia is used for patients in whom the

1	features are present in these situations, thus allowing separation of these lesions from the UIP-like pattern. Consequently, the term usual interstitial pneumonia is used for patients in whom the lesion is idiopathic and not associated with another condition.

1	Untreated patients with IPF show continued progression of their disease and have a high mortality rate. There is no effective therapy for IPF. Thalidomide appears to improve cough in patients with IPF. Chronic microaspiration secondary to gastroesophageal reflux may play a role in the pathogenesis and natural history of IPF. Gastroesophageal reflux (GER) therapy may be of benefit in IPF. In patients with IPF, treatment with the three-drug regimen of prednisone, azathioprine, and N-acetylcysteine (NAC) or warfarin (in IPF patients who lacked other indications for anticoagulation) has been shown to increase the risks of hospitalization and death. Patients with IPF and coexisting emphysema (combined pulmonary fibrosis and emphysema [CPFE]) are more likely to require longterm oxygen therapy and develop pulmonary hypertension and may have a more dismal outcome than those without emphysema.

1	Patients with IPF may have acute deterioration secondary to infections, pulmonary embolism, or pneumothorax. Heart failure and ischemic heart disease are common problems in patients with IPF, accounting for nearly one-third of deaths. These patients also commonly experience an accelerated phase of rapid clinical decline that is associated with a poor prognosis (so-called acute exacerbations of IPF). These acute exacerbations are defined by worsening of dyspnea within a few days to 4 weeks; newly developing diffuse ground-glass abnormality and/or consolidation superimposed on a background reticular or honeycomb pattern consistent with the UIP pattern; worsening hypoxemia; and absence of infectious pneumonia, heart failure, and sepsis. The rate of these acute exacerbations ranges from 10–57%, apparently depending on the length of follow-up. During these episodes, the histopathologic pattern of diffuse alveolar damage is often found on the background of UIP. No therapy has been found to

1	apparently depending on the length of follow-up. During these episodes, the histopathologic pattern of diffuse alveolar damage is often found on the background of UIP. No therapy has been found to be effective in the management of acute exacerbations of IPF. Often mechanical ventilation is required, but it is usually not successful, with a hospital mortality rate of up to three-fourths of patients. In those who survive, a recurrence of acute exacerbation is common and usually results in death at those times.

1	Patients should be referred early for lung transplant because of the unpredictability of disease progression (e.g., acute exacerbations) (Chap. 320e). This condition defines a subgroup of the idiopathic interstitial pneumonias that can be distinguished clinically and pathologically from UIP, DIP, AIP, and COP. Importantly, many cases with this histopathologic pattern occur in the context of an underlying disorder, such as a CTD, drug-induced ILD, or chronic hypersensitivity pneumonitis.

1	Clinical Manifestations Patients with idiopathic NSIP have clinical, serologic, radiographic, and pathologic characteristics highly suggestive of autoimmune disease and meet the criteria for undifferentiated CTD. Idiopathic NSIP is a subacute restrictive process with a presentation similar to that of IPF but usually at a younger age, most commonly in women who have never smoked. It is often associated with a febrile illness. HRCT shows bilateral, subpleural ground-glass opacities, often associated with lower lobe volume loss (Fig. 315-3). Patchy areas of airspace consolidation and reticular abnormalities may be present, but honeycombing is unusual. Histologic Findings The key histopathologic feature of NSIP is the uniformity of interstitial involvement across the biopsy section, and this may be predominantly cellular or fibrosing. There is less temporal and spatial heterogeneity than in UIP, and little or no honeycombing is found. The cellular variant is rare.

1	Treatment The majority of patients with NSIP have a good prognosis (5-year mortality rate estimated at <15%), with most showing improvement after treatment with glucocorticoids, often used in combination with azathioprine or mycophenolate mofetil. FIGURE 315-3 Nonspecific interstitial pneumonia. High-resolution computed tomography through the lower lung shows volume loss with extensive ground-glass abnormality, reticular abnormality, and traction bronchiectasis. There is sparing on the lung immediately adja-cent to the pleura. Histology showed a combination of inflammation and mild fibrosis.

1	ACUTE INTERSTITIAL PNEUMONIA (HAMMAN-RICH SYNDROME) Clinical Manifestations AIP is a rare, fulminant form of lung injury characterized histologically by diffuse alveolar damage on lung biopsy. Most patients are older than 40 years. AIP is similar in presentation to the acute respiratory distress syndrome (ARDS) (Chap. 322) and probably corresponds to the subset of cases of idiopathic ARDS. The onset is usually abrupt in a previously healthy individual. A prodromal illness, usually lasting 7–14 days before presentation, is common. Fever, cough, and dyspnea are common manifestations at presentation. Diffuse, bilateral, air-space opacification is present on the chest radiograph. HRCT scans show bilateral, patchy, symmetric areas of ground-glass attenuation. Bilateral areas of air-space consolidation also may be present. A predominantly subpleural distribution may be seen.

1	Histologic Findings The diagnosis of AIP requires the presence of a clinical syndrome of idiopathic ARDS and pathologic confirmation of organizing diffuse alveolar damage. Therefore, lung biopsy is required to confirm the diagnosis. Treatment Most patients have moderate to severe hypoxemia and develop respiratory failure. Mechanical ventilation is often required. The mortality rate is high (>60%), with most patients dying within 6 months of presentation. Recurrences have been reported. However, those who recover often have substantial improvement in lung function. The main treatment is supportive. It is not clear that glucocorticoid therapy is effective.

1	CRYPTOGENIC ORGANIZING PNEUMONIA Clinical Manifestations COP is a clinicopathologic syndrome of unknown etiology. The onset is usually in the fifth and sixth decades. The presentation may be of a flulike illness with cough, fever, malaise, fatigue, and weight loss. Inspiratory crackles are frequently present on examination. Pulmonary function is usually impaired, with a restrictive defect and arterial hypoxemia being most common. The roentgenographic manifestations are distinctive, revealing bilateral, patchy, or diffuse alveolar opacities in the presence of normal lung volume. Recurrent and migratory pulmonary opacities are common. HRCT shows areas of air-space consolidation, ground-glass opacities, small nodular opacities, and bronchial wall thickening and dilation. These changes occur more frequently in the periphery of the lung and in the lower lung zone.

1	Histologic Findings Lung biopsy shows granulation tissue within small airways, alveolar ducts, and airspaces, with chronic inflammation in the surrounding alveoli. Foci of organizing pneumonia are a nonspecific reaction to lung injury found adjacent to other pathologic processes or as a component of other primary pulmonary disorders (e.g., cryptococcosis, granulomatosis with polyangiitis [Wegener], lymphoma, hypersensitivity pneumonitis, and eosinophilic pneumonia). Consequently, the clinician must carefully reevaluate any patient found to have this histopathologic lesion to rule out these possibilities. Treatment Glucocorticoid therapy induces clinical recovery in two-thirds of patients. A few patients have rapidly progressive courses with fatal outcomes despite glucocorticoids.

1	Treatment Glucocorticoid therapy induces clinical recovery in two-thirds of patients. A few patients have rapidly progressive courses with fatal outcomes despite glucocorticoids. ILD ASSOCIATED WITH CIGARETTE SMOKING Desquamative Interstitial Pneumonia • CLINICAL MANIFESTATIONS DIP is a rare but distinct clinical and pathologic entity found almost exclusively in cigarette smokers. The histologic hallmark is the extensive accumulation of macrophages in intraalveolar spaces with minimal interstitial fibrosis. The peak incidence is in the fourth and fifth decades. Most patients present with dyspnea and cough. Lung function testing shows a restrictive pattern with reduced DlCO and arterial hypoxemia. The chest x-ray and HRCT scans usually show diffuse hazy opacities.

1	HISTOLOGIC FINDINGS A diffuse and uniform accumulation of macrophages in the alveolar spaces is the hallmark of DIP. The macrophages contain golden, brown, or black pigment of tobacco smoke. There may be mild thickening of the alveolar walls by fibrosis and scanty inflammatory cell infiltration. TREATMENT Clinical recognition of DIP is important because the process is associated with a better prognosis (10-year survival rate is ~70%) in response to smoking cessation. There are no clear data showing that systemic glucocorticoids are effective in DIP.

1	Respiratory bronchiolitis–associated ILD (RB-ILD) is considered to be a subset of DIP and is characterized by the accumulation of macrophages in peribronchial alveoli. The clinical presentation is similar to that of DIP. Crackles are often heard on chest examination and occur throughout inspiration; sometimes they continue into expiration. The process is best seen on HRCT lung scanning, which shows bronchial wall thickening, centrilobular nodules, ground-glass opacity, and emphysema with air trapping. There is a spectrum of CT features in asymptomatic smokers (and elderly asymptomatic individuals) that may not necessarily represent clinically relevant disease.

1	HISTOLOGIC FINDINGS The histologic findings in RB-ILD include alveolar macrophage accumulation in respiratory bronchioles, with a variable chronic inflammatory cell infiltrate in bronchiolar and surrounding alveolar walls and occasional peribronchial alveolar septal fibrosis. The pulmonary parenchyma may show presence of smoking-related emphysema. TREATMENT RB-ILD appears to resolve in most patients after smoking cessation alone.

1	Pulmonary Langerhans Cell Histiocytosis • CLINICAL MANIFESTATIONS This is a rare, smoking-related, diffuse lung disease that primarily affects men between the ages of 20 and 40 years. The clinical presentation varies from an asymptomatic state to a rapidly progressive condition. The most common clinical manifestations at presentation are cough, dyspnea, chest pain, weight loss, and fever. Pneumothorax occurs in ~25% of patients. Hemoptysis and diabetes insipidus are rare manifestations. The radiographic features vary with the stage of the disease. The combination of ill-defined or stellate nodules (2–10 mm in diameter), reticular or nodular opacities, bizarre-shaped upper zone cysts, preservation of lung volume, and sparing of the costophrenic angles are characteristics of PLCH. HRCT that reveals a combination of nodules and thin-walled cysts is virtually diagnostic of PLCH. The most common pulmonary function abnormality is a markedly reduced DlCO, although varying degrees of

1	that reveals a combination of nodules and thin-walled cysts is virtually diagnostic of PLCH. The most common pulmonary function abnormality is a markedly reduced DlCO, although varying degrees of restrictive disease, airflow limita-1713 tion, and diminished exercise capacity may occur.

1	HISTOLOGIC FINDINGS The characteristic histopathologic finding in PLCH is the presence of nodular sclerosing lesions that contain Langerhans cells accompanied by mixed cellular infiltrates. The nodular lesions are poorly defined and are distributed in a bronchiolocentric fashion with intervening normal lung parenchyma. As the disease advances, fibrosis progresses to involve adjacent lung tissue, leading to pericicatricial air space enlargement, which accounts for the concomitant cystic changes. TREATMENT Discontinuance of smoking is the key treatment, resulting in clinical improvement in one-third of patients. Most patients with PLCH experience persistent or progressive disease. Death due to respiratory failure occurs in ~10% of patients.

1	Clinical findings suggestive of a CTD (musculoskeletal pain, weak ness, fatigue, fever, joint pain or swelling, photosensitivity, Raynaud’s phenomenon, pleuritis, dry eyes, dry mouth) should be sought in any patient with ILD. The CTDs may be difficult to rule out since the pulmonary manifestations occasionally precede the more typical systemic manifestations by months or years. The most common form of pulmonary involvement is the nonspecific interstitial pneumonia histopathologic pattern. However, determining the precise nature of lung involvement in most of the CTDs is difficult due to the high incidence of lung involvement caused by disease-associated complications of esophageal dysfunction (predisposing to aspiration and secondary infections), respiratory muscle weakness (atelectasis and secondary infections), complications of therapy (opportunistic infections), and associated malignancies. For the majority of CTDs, with the exception of progressive system sclerosis, recommended

1	and secondary infections), complications of therapy (opportunistic infections), and associated malignancies. For the majority of CTDs, with the exception of progressive system sclerosis, recommended initial treatment for ILD includes oral glucocorticoids often in association with an immunosuppressive agent (usually oral or intravenous cyclophosphamide or oral azathioprine) or mycophenolate mofetil.

1	Progressive Systemic Sclerosis (PSS) • CLINICAL MANIFESTATIONS (See also Chap. 382) Clinical evidence of ILD is present in about one-half of patients with PSS, and pathologic evidence in three-quarters. Pulmonary function tests show a restrictive pattern and impaired diffusing capacity, often before any clinical or radiographic evidence of lung disease appears. The HRCT features of lung disease in PSS range from predominant ground-glass attenuation to a predominant reticular pattern and are mostly similar to idiopathic NSIP. HISTOLOGIC FINDINGS NSIP is the histopathologic pattern in most patients (~75%); the UIP pattern is rare (<10%).

1	HISTOLOGIC FINDINGS NSIP is the histopathologic pattern in most patients (~75%); the UIP pattern is rare (<10%). TREATMENT Therapy is similar to that in idiopathic NSIP. UIP in PSS has a better outcome than IPF. The most widely used initial treatment regimen is low-dose glucocorticoid therapy and an immunosuppressive agent, usually oral or pulse cyclophosphamide. There are no convincing data showing this regime to be efficacious, and there is concern that the risk of renal crisis rises substantially with corticosteroids. Pulmonary vascular disease alone or in association with pulmonary fibrosis, pleuritis, or recurrent aspiration pneumonitis is strikingly resistant to current modes of therapy. Rheumatoid Arthritis • CLINICAL MANIFESTATIONS (See also Chap. 380)

1	Rheumatoid Arthritis • CLINICAL MANIFESTATIONS (See also Chap. 380) ILD associated with RA is more common in men. Pulmonary manifestations of RA include pleurisy with or without effusion, ILD in up to 20% of cases, necrobiotic nodules (nonpneumoconiotic intrapulmonary rheumatoid nodules) with or without cavities, Caplan syndrome (rheumatoid pneumoconiosis), pulmonary hypertension secondary to rheumatoid pulmonary vasculitis, organized pneumonia, and upper airway obstruction due to cricoarytenoid arthritis. HISTOLOGIC FINDINGS There are two primary histopathologic patterns of ILD that are observed in patients with ILD associated with RA: NSIP pattern and UIP pattern.

1	HISTOLOGIC FINDINGS There are two primary histopathologic patterns of ILD that are observed in patients with ILD associated with RA: NSIP pattern and UIP pattern. 1714 TREATMENT Little data exist to support the management of ILD in RA. Initial treatment of rheumatoid ILD, if required, is typically with oral glucocorticoids, which should be tried for 1–3 months. The potential benefit of anti-tumor necrosis factor α (TNF-α) therapy has been clouded by concerns about the development of a rapid and occasionally fatal lung disease in patients with RA-associated ILD treated with anti-TNF-α therapy.

1	Systemic Lupus Erythematosus • CLINICAL MANIFESTATIONS (See also Chap. 378) Lung disease is a common complication in SLE. Pleuritis with or without effusion is the most common pulmonary manifestation. Other lung manifestations include the following: atelectasis, diaphragmatic dysfunction with loss of lung volumes, pulmonary vascular disease, pulmonary hemorrhage, uremic pulmonary edema, infectious pneumonia, and organized pneumonia. Acute lupus pneumonitis characterized by pulmonary capillaritis leading to alveolar hemorrhage is uncommon. Chronic, progressive ILD is uncommon (<10%). It is important to exclude pulmonary infection. Although pleuropulmonary involvement may not be evident clinically, pulmonary function testing, particularly DlCO, reveals abnormalities in many patients with SLE. HISTOLOGIC FINDINGS The most common pathologic patterns seen include NSIP, UIP, LIP, and, on occasion, organizing pneumonia and amyloidosis.

1	HISTOLOGIC FINDINGS The most common pathologic patterns seen include NSIP, UIP, LIP, and, on occasion, organizing pneumonia and amyloidosis. TREATMENT There have been no controlled trials of treatment for ILD in SLE. Treatment involves the use of a glucocorticoid, either alone or, more often, in combination with an additional immunomodulating agent. (See also Chap. 388) ILD occurs in ~10% of patients with PM/DM. Diffuse reticular or nodular opacities with or without an alveolar component occur radiographically, with a predilection for the lung bases (NSIP pattern). ILD occurs more commonly in the subgroup of patients with an anti-Jo-1 antibody that is directed to histidyl tRNA synthetase. Weakness of respiratory muscles contributing to aspiration pneumonia may be present. A rapidly progressive illness characterized by diffuse alveolar damage may cause respiratory failure.

1	HISTOLOGIC FINDINGS NSIP predominates over UIP, organizing pneumonia, or other patterns of interstitial pneumonia. TREATMENT The optimal treatment is unknown. The most widely used initial treatment is oral glucocorticoids. Fulminant disease may require high-dose intravenous methylprednisolone (1.0 g/d) for 3–5 days. Sjögren Syndrome • CliniCal manifestations (See also Chap. 383) General dryness and lack of airway secretion cause the major problems of hoarseness, cough, and bronchitis. HISTOLOGIC FINDINGS Lung biopsy is frequently required to establish a precise pulmonary diagnosis. Fibrotic NSIP is most common. Lymphocytic interstitial pneumonitis, lymphoma, pseudolymphoma, bronchiolitis, and bronchiolitis obliterans are associated with this condition. TREATMENT Glucocorticoids have been used in the management of ILD associated with Sjögren syndrome with some degree of clinical success.

1	DRUG-INDUCED ILD Clinical Manifestations Many classes of drugs have the potential to induce diffuse ILD, which is manifest most commonly as exertional dyspnea and nonproductive cough. A detailed history of the medications taken by the patient is needed to identify drug-induced disease, including over-the-counter medications, oily nose drops, and petroleum products (mineral oil). In most cases, the pathogenesis is unknown, although a combination of direct toxic effects of the drug (or its metabolite) and indirect inflammatory and immunologic events are likely. The onset of the illness may be abrupt and fulminant, or it may be insidious, extending over weeks to months. The drug may have been taken for several years before a reaction develops (e.g., amiodarone), or the lung disease may occur weeks to years after the drug has been discontinued (e.g., carmustine). The extent and severity of disease are usually dose-related.

1	Histologic Findings The patterns of lung injury vary widely and depend on the agent. Treatment Treatment consists of discontinuation of any possible offending drug and supportive care. (See Chap. 310)

1	PULMONARY ALVEOLAR PROTEINOSIS (PAP) Clinical Manifestations Although not strictly an ILD, PAP resembles and is therefore considered with these conditions. It has been proposed that a defect in macrophage function, more specifically an impaired ability to process surfactant, may play a role in the pathogenesis of PAP. PAP is an autoimmune disease with a neutralizing antibody of immunoglobulin G isotype against granulocyte-macrophage colony-stimulating factor (GM-CSF). These findings suggest that neutralization of GM-CSF bioactivity by the antibody causes dysfunction of alveolar macrophages, which results in reduced surfactant clearance. There are three distinct classes of PAP: acquired (>90% of all cases), congenital, and secondary. Congenital PAP is transmitted in an autosomal recessive manner and is caused by homozygosity for a frame-shift mutation (121ins2) in the SP-B gene, which leads to an unstable SP-B mRNA, reduced protein levels, and secondary disturbances of SP-C processing.

1	and is caused by homozygosity for a frame-shift mutation (121ins2) in the SP-B gene, which leads to an unstable SP-B mRNA, reduced protein levels, and secondary disturbances of SP-C processing. Secondary PAP is rare among adults and is caused by lysinuric protein intolerance, acute silicosis and other inhalational syndromes, immunodeficiency disorders, and malignancies (almost exclusively of hematopoietic origin) and hematopoietic disorders.

1	The typical age of presentation is 30–50 years, and males predominate. The clinical presentation is usually insidious and is manifested by progressive exertional dyspnea, fatigue, weight loss, and low-grade fever. A nonproductive cough is common, but occasionally expectoration of “chunky” gelatinous material may occur. Polycythemia, hypergammaglobulinemia, and increased LDH levels are common. Markedly elevated serum levels of lung surfactant proteins A and D have been found in PAP. In the absence of any known secondary cause of PAP, an elevated serum anti-GM-CSF titer is highly sensitive and specific for the diagnosis of acquired PAP. BAL fluid levels of anti-GM-CSF antibodies correlate better with the severity of PAP than do serum titers. Radiographically, bilateral symmetric alveolar opacities located centrally in middle and lower lung zones result in a “bat-wing” distribution. HRCT shows a ground-glass opacification and thickened intralobular structures and interlobular septa.

1	Histologic Findings This diffuse disease is characterized by the accumulation of an amorphous, periodic acid–Schiff–positive lipoproteinaceous material in the distal air spaces. There is little or no lung inflammation, and the underlying lung architecture is preserved. Treatment Whole-lung lavage(s) through a double-lumen endotracheal tube provides relief to many patients with dyspnea or progressive hypoxemia and also may provide long-term benefit.

1	PULMONARY LYMPHANGIOLEIOMYOMATOSIS Clinical Manifestations Pulmonary LAM is a rare condition that afflicts premenopausal women and should be suspected in young women with “emphysema,” recurrent pneumothorax, or chylous pleural effusion. It is often misdiagnosed as asthma or chronic obstructive pulmonary disease. Whites are affected much more commonly than are members of other racial groups. The disease accelerates during pregnancy and abates after oophorectomy. Common complaints at presentation are dyspnea, cough, and chest pain. Hemoptysis may be life threatening. Spontaneous pneumothorax occurs in 50% of patients; it may be bilateral and necessitate pleurodesis. Meningioma and renal angiomyolipomas (hamartomas), characteristic findings in the genetic disorder tuberous sclerosis, are also common in patients with LAM. Chylothorax, chyloperitoneum (chylous ascites), chyluria, and chylopericardium are other complications. Pulmonary function testing usually reveals an obstructive or

1	also common in patients with LAM. Chylothorax, chyloperitoneum (chylous ascites), chyluria, and chylopericardium are other complications. Pulmonary function testing usually reveals an obstructive or mixed obstructive-restrictive pattern, and gas exchange is often abnormal. HRCT shows thin-walled cysts surrounded by normal lung without zonal predominance.

1	Histologic Findings Pathologically, LAM is characterized by the proliferation of atypical pulmonary interstitial smooth muscle and cyst formation. The immature-appearing smooth-muscle cells react with monoclonal antibody HMB45, which recognizes a 100-kDa glycoprotein (gp100) originally found in human melanoma cells.

1	Treatment Progression is common, with a median survival of 8–10 years from diagnosis. No therapy is of proven benefit in LAM. Sirolimus, an inhibitor of the mammalian target of rapamycin (mTOR), appears to be an active agent for LAM. After 12 months, it stabilized lung function (FVC, FEV1, and functional residual capacity) and was associated with a reduction in symptoms and improvement in quality of life. Adverse effects (e.g., mucositis, diarrhea, nausea, hypercholesterolemia, acneiform rash, peripheral edema) were more common in the sirolimus group, but serious adverse effects were not increased. Subjects were followed off sirolimus for an additional 12 months, during which time pulmonary function declined at the same rate as in the placebo group. Progesterone and luteinizing hormone–releasing hormone analogues have been used. Oophorectomy is no longer recommended, and estrogen-containing drugs should be discontinued. Lung transplantation offers the only hope for cure despite

1	hormone analogues have been used. Oophorectomy is no longer recommended, and estrogen-containing drugs should be discontinued. Lung transplantation offers the only hope for cure despite reports of recurrent disease in the transplanted lung.

1	SYNDROMES OF ILD WITH DIFFUSE ALVEOLAR HEMORRHAGE Clinical Manifestations The clinical onset is often abrupt, with cough, fever, and dyspnea. Severe respiratory distress requiring ventilatory support may be evident at initial presentation. Although hemoptysis is expected, it can be absent at the time of presentation in one-third of the cases. For patients without hemoptysis, new alveolar opacities, a falling hemoglobin level, and hemorrhagic BAL fluid point to the diagnosis. The chest radiograph is nonspecific and most commonly shows new patchy or diffuse alveolar opacities. Recurrent episodes of DAH may lead to pulmonary fibrosis, resulting in interstitial opacities on the chest radiograph. An elevated white blood cell count and falling hematocrit are common. Evidence for impaired renal function caused by focal segmental necrotizing glomerulonephritis, usually with crescent formation, also may be present. Varying degrees of hypoxemia may occur and are often severe enough to require

1	function caused by focal segmental necrotizing glomerulonephritis, usually with crescent formation, also may be present. Varying degrees of hypoxemia may occur and are often severe enough to require ventilatory support. DlCO may be increased, resulting from the increased hemoglobin within the alveoli compartment.

1	Histologic Findings Injury to arterioles, venules, and the alveolar septal (alveolar wall or interstitial) capillaries can result in hemoptysis secondary to disruption of the alveolar-capillary basement membrane. This results in bleeding into the alveolar spaces, which characterizes DAH. Pulmonary capillaritis, characterized by a neutrophilic infiltration of the alveolar septae, may lead to necrosis of these structures, loss of capillary structural integrity, and the pouring of red blood cells into the alveolar space. Fibrinoid necrosis of the interstitium and red blood cells within the interstitial space are sometimes seen. Bland pulmonary hemorrhage (i.e., DAH without inflammation of the alveolar structures) also may occur.

1	Evaluation of either lung or renal tissue by immunofluorescent techniques indicates an absence of immune complexes (pauci-immune) in granulomatosis with polyangiitis (Wegener), microscopic polyangiitis, pauci-immune glomerulonephritis, and isolated pulmonary capillaritis. A granular pattern is found in the CTDs, particularly SLE, and a characteristic linear deposition is found in Goodpasture syndrome. Granular deposition of IgA-containing immune complexes is present in Henoch-Schönlein purpura.

1	Treatment The mainstay of therapy for the DAH associated with sys-1715 temic vasculitis, CTD, Goodpasture syndrome, and isolated pulmonary capillaritis is IV methylprednisolone, 0.5–2 g daily in divided doses for up to 5 days, followed by a gradual tapering, and then maintenance on an oral preparation. Prompt initiation of therapy is important, particularly in the face of renal insufficiency, because early initiation of therapy has the best chance of preserving renal function. The decision to start other immunosuppressive therapy (cyclophosphamide or azathioprine) acutely depends on the severity of illness.

1	Goodpasture Syndrome • CLINICAL MANIFESTATIONS Pulmonary hemorrhage and glomerulonephritis are features in most patients with this disease. Autoantibodies to renal glomerular and lung alveolar basement membranes are present. This syndrome can present and recur as DAH without an associated glomerulonephritis. In such cases, circulating anti-basement membrane antibody is often absent, and the only way to establish the diagnosis is by demonstrating linear immunofluorescence in lung tissue. HISTOLOGIC FINDINGS The underlying histology may be bland hemorrhage or DAH associated with capillaritis. TREATMENT Plasmapheresis has been recommended as adjunctive treatment.

1	HISTOLOGIC FINDINGS The underlying histology may be bland hemorrhage or DAH associated with capillaritis. TREATMENT Plasmapheresis has been recommended as adjunctive treatment. Pulmonary opacities and respiratory symptoms typical of ILD can develop in related family members and in several inherited diseases. These diseases include the phakomatoses, tuberous sclerosis and neurofibromatosis (Chap. 118), and the lysosomal storage diseases, Niemann-Pick disease and Gaucher disease (Chap. 432e). The Hermansky-Pudlak syndrome is an autosomal recessive disorder in which granulomatous colitis and ILD may occur. It is characterized by oculocutaneous albinism, bleeding diathesis secondary to platelet dysfunction, and the accumulation of a chromolipid, lipofuscin material in cells of the reticuloendothelial system. A fibrotic pattern is found on lung biopsy, but the alveolar macrophages may contain cytoplasmic ceroid-like inclusions.

1	Inhalation of organic dusts, which cause hypersensitivity pneumonitis, or of inorganic dust, such as silica, which elicits a granulomatous inflammatory reaction leading to ILD, produces diseases of known etiology (Table 315-1) that are discussed in Chaps. 310 and 311. Sarcoidosis (Chap. 390) is prominent among granulomatous diseases of unknown cause in which ILD is an important feature.

1	Granulomatous Vasculitides (See also Chap. 385) The granulomatous vasculitides are characterized by pulmonary angiitis (i.e., inflammation and necrosis of blood vessels) with associated granuloma formation (i.e., infiltrates of lymphocytes, plasma cells, epithelioid cells, or histiocytes, with or without the presence of multinucleated giant cells, sometimes with tissue necrosis). The lungs are almost always involved, although any organ system may be affected. Granulomatosis with polyangiitis (Wegener) and Eosinophilic granulomatosis with polyangiitis (Churg-Strauss) primarily affect the lung but are associated with a systemic vasculitis as well. The granulomatous vasculitides generally limited to the lung include necrotizing sarcoid granulomatosis and benign lymphocytic angiitis and granulomatosis. Granulomatous infection and pulmonary angiitis due to irritating embolic material (e.g., talc) are important known causes of pulmonary vasculitis.

1	This group of disorders features lymphocyte and plasma cell infiltration of the lung parenchyma. The disorders either are benign or can behave as low-grade lymphomas. Included is angioimmunoblastic lymphadenopathy with dysproteinemia, a rare lymphoproliferative disorder characterized by diffuse lymphadenopathy, fever, hepatosplenomegaly, and hemolytic anemia, with ILD in some cases. 1716 Lymphocytic Interstitial Pneumonitis This rare form of ILD occurs in adults, some of whom have an autoimmune disease or dysproteinemia. It has been reported in patients with Sjögren syndrome and HIV infection.

1	Lymphomatoid Granulomatosis • CLINICAL MANIFESTATIONS Pulmonary lymphomatoid granulomatosis generally presents predominantly in men between the ages of 30 and 50, although patients can be affected at any age. The effects of race and geography on disease incidence are not known, although a higher diagnosis rate is reported in Western countries. Although it may affect virtually any organ, it is most frequently characterized by pulmonary (>90%), skin, and central nervous system involvement. The most common presenting symptoms and signs include cough, fever, rash/nodules, malaise, weight loss, neurologic abnormalities, dyspnea, and chest pain. HISTOLOGIC FINDINGS This multisystem disorder of unknown etiology is an angiocentric malignant (T cell) lymphoma characterized by a polymorphic lymphoid infiltrate, an angiitis, and granulomatosis.

1	HISTOLOGIC FINDINGS This multisystem disorder of unknown etiology is an angiocentric malignant (T cell) lymphoma characterized by a polymorphic lymphoid infiltrate, an angiitis, and granulomatosis. TREATMENT The clinical course of lymphomatoid granulomatosis ranges from remission without treatment to death from malignant lymphoma within 2 years. The choice of a treatment strategy should be based upon the presence of symptoms, history of using an inciting medication, extent of extrapulmonary involvement, and careful assessment of the histopathologic grade of the lesion. Referral to a hematology oncology specialist for consultation is recommended.

1	BRONCHOCENTRIC GRANULOMATOSIS Clinical Manifestations Rather than a specific clinical entity, bronchocentric granulomatosis (BG) is a descriptive histologic term that is applied to an uncommon and nonspecific pathologic response to a variety of airway injuries. There is evidence that BG is caused by a hypersensitivity reaction to Aspergillus or other fungi in patients with asthma. About one-half of the patients described have had chronic asthma with severe wheezing and peripheral blood eosinophilia. In patients with asthma, BG probably represents one pathologic manifestation of allergic bronchopulmonary aspergillosis or another allergic mycosis. In patients without asthma, BG has been associated with RA and a variety of infections, including tuberculosis, echinococcosis, histoplasmosis, coccidioidomycosis, and nocardiosis. The chest roentgenogram reveals irregularly shaped nodular or mass lesions with ill-defined margins, which are usually unilateral and solitary, with upper lobe

1	coccidioidomycosis, and nocardiosis. The chest roentgenogram reveals irregularly shaped nodular or mass lesions with ill-defined margins, which are usually unilateral and solitary, with upper lobe predominance.

1	Histologic Findings Bronchocentric granulomatosis is characterized by peribronchial and peribronchiolar necrotizing granulomatous inflammation. Destruction of airway walls and adjacent parenchyma leads to granulomatous replacement of mucosa and submucosa by palisading, epithelioid, and multinucleated histiocytes. Bronchocentric granulomatosis does not typically involve the pulmonary arteries. Treatment Glucocorticoids are the treatment of choice, often with an excellent outcome, although recurrences may occur as therapy is tapered or stopped. Limited epidemiologic data exist describing the prevalence or incidence of ILD in the general population. With a few exceptions, e.g., sarcoidosis and certain occupational and environmental exposures, there appear to be no significant differences in the prevalence or incidence of ILD among various populations. For sarcoidosis, there are important environmental, racial, and genetic differences (Chap. 390). Disorders of the pleura

1	Disorders of the pleura Richard W. Light The pleural space lies between the lung and the chest wall and normally contains a very thin layer of fluid, which serves as a coupling system. A pleural effusion is present when there is an excess quantity of fluid in the pleural space.

1	Etiology Pleural fluid accumulates when pleural fluid formation exceeds pleural fluid absorption. Normally, fluid enters the pleural space from the capillaries in the parietal pleura and is removed via the lymphatics in the parietal pleura. Fluid also can enter the pleural space from the interstitial spaces of the lung via the visceral pleura or from the peritoneal cavity via small holes in the diaphragm. The lymphatics have the capacity to absorb 20 times more fluid than is formed normally. Accordingly, a pleural effusion may develop when there is excess pleural fluid formation (from the interstitial spaces of the lung, the parietal pleura, or the peritoneal cavity) or when there is decreased fluid removal by the lymphatics.

1	Diagnostic Approach Patients suspected of having a pleural effusion should undergo chest imaging to diagnose its extent. Chest ultrasound has replaced the lateral decubitus x-ray in the evaluation of suspected pleural effusions and as a guide to thoracentesis. When a patient is found to have a pleural effusion, an effort should be made to determine the cause (Fig. 316-1). The first step is to determine whether the effusion is a transudate or an exudate. A transudative pleural effusion occurs when systemic factors that influence the formation and absorption of pleural fluid are altered. The leading causes of transudative pleural effusions in the United States are left ventricular failure and cirrhosis. An exudative pleural effusion occurs when local factors that influence the formation and absorption of pleural fluid are altered. The leading causes of exudative pleural effusions are bacterial pneumonia, malignancy, viral infection, and pulmonary embolism. The primary reason for making

1	absorption of pleural fluid are altered. The leading causes of exudative pleural effusions are bacterial pneumonia, malignancy, viral infection, and pulmonary embolism. The primary reason for making this differentiation is that additional diagnostic procedures are indicated with exudative effusions to define the cause of the local disease.

1	Transudative and exudative pleural effusions are distinguished by measuring the lactate dehydrogenase (LDH) and protein levels in the pleural fluid. Exudative pleural effusions meet at least one of the following criteria, whereas transudative pleural effusions meet none: 1. Pleural fluid protein/serum protein >0.5 2. Pleural fluid LDH/serum LDH >0.6 3. Pleural fluid LDH more than two-thirds the normal upper limit for serum These criteria misidentify ~25% of transudates as exudates. If one or more of the exudative criteria are met and the patient is clinically thought to have a condition producing a transudative effusion, the difference between the protein levels in the serum and the pleural fluid should be measured. If this gradient is >31 g/L (3.1 g/dL), the exudative categorization by these criteria can be ignored because almost all such patients have a transudative pleural effusion.

1	If a patient has an exudative pleural effusion, the following tests on the pleural fluid should be obtained: description of the appearance of the fluid, glucose level, differential cell count, microbiologic studies, and cytology. Effusion Due to Heart Failure The most common cause of pleural effusion is left ventricular failure. The effusion occurs because the increased amounts of fluid in the lung interstitial spaces exit in part across the visceral pleura; this overwhelms the capacity of the lymphatics in the parietal pleura to remove fluid. In patients with heart failure,

1	Perform diagnostic thoracentesis Measure pleural fluid protein and LDH Exudate Further diagnostic procedures Transudate Treat CHF, cirrhosis, nephrosis Measure PF glucose Obtain PF cytology Obtain differential cell count Culture, stain PF PF marker for TB Consider:Malignancy Bacterial infections Rheumatoid pleuritis Glucose < 60 mg/dL No diagnosis Consider pulmonary embolus (spiral CT or lung scan) Treat for PE Treat for TB PF marker for TB Observe Consider thoracoscopy or image-guided pleural biopsy Any of following met? PF/serum protein > 0.5 PF/serum LDH > 0.6 PF LDH > 2/3 upper normal serum limit Pleural effusion Yes No Yes Yes Yes No No No SYMPTOMS IMPROVING FIGURE 316-1 Approach to the diagnosis of pleural effusions. CHF, congestive heart failure; CT, computed tomography; LDH, lactate dehydrogenase; PE, pulmonary embolism; PF, pleural fluid; TB, tuberculosis.

1	CHF, congestive heart failure; CT, computed tomography; LDH, lactate dehydrogenase; PE, pulmonary embolism; PF, pleural fluid; TB, tuberculosis. a diagnostic thoracentesis should be performed if the effusions are not bilateral and comparable in size, if the patient is febrile, or if the patient has pleuritic chest pain to verify that the patient has a transudative effusion. Otherwise the patient’s heart failure is treated. If the effusion persists despite therapy, a diagnostic thoracentesis should be performed. A pleural fluid N-terminal pro-brain natriuretic peptide (NT-proBNP) >1500 pg/mL is virtually diagnostic that the effusion is secondary to congestive heart failure.

1	Hepatic Hydrothorax Pleural effusions occur in ~5% of patients with cirrhosis and ascites. The predominant mechanism is the direct movement of peritoneal fluid through small openings in the diaphragm into the pleural space. The effusion is usually right-sided and frequently is large enough to produce severe dyspnea. Parapneumonic Effusion Parapneumonic effusions are associated with bacterial pneumonia, lung abscess, or bronchiectasis and are probably the most common cause of exudative pleural effusion in the United States. Empyema refers to a grossly purulent effusion. Patients with aerobic bacterial pneumonia and pleural effusion 1717 present with an acute febrile illness consisting of chest pain, sputum production, and leukocytosis. Patients with anaerobic infections present with a subacute illness with weight loss, a brisk leukocytosis, mild anemia, and a history of some factor that predisposes them to aspiration.

1	The possibility of a parapneumonic effusion should be considered whenever a patient with bacterial pneumonia is initially evaluated. The presence of free pleural fluid can be demonstrated with a lateral decubitus radiograph, computed tomography (CT) of the chest, or ultrasound. If the free fluid separates the lung from the chest wall by >10 mm, a therapeutic thoracentesis should be performed. Factors indicating the likely need for a procedure more invasive than a thoracentesis (in increasing order of importance) include the following: 1. 2. Pleural fluid pH <7.20 3. Pleural fluid glucose <3.3 mmol/L (<60 mg/dL) 4. Positive Gram stain or culture of the pleural fluid 5. Presence of gross pus in the pleural space

1	2. Pleural fluid pH <7.20 3. Pleural fluid glucose <3.3 mmol/L (<60 mg/dL) 4. Positive Gram stain or culture of the pleural fluid 5. Presence of gross pus in the pleural space If the fluid recurs after the initial therapeutic thoracentesis and if any of these characteristics are present, a repeat thoracentesis should be performed. If the fluid cannot be completely removed with the therapeutic thoracentesis, consideration should be given to inserting a chest tube and instilling the combination of a fibrinolytic agent (e.g., tissue plasminogen activator, 10 mg) and deoxyribonuclease (5 mg) or performing a thoracoscopy with the breakdown of adhesions. Decortication should be considered when these measures are ineffective.

1	Effusion Secondary to Malignancy Malignant pleural effusions secondary to metastatic disease are the second most common type of exudative pleural effusion. The three tumors that cause ~75% of all malignant pleural effusions are lung carcinoma, breast carcinoma, and lymphoma. Most patients complain of dyspnea, which is frequently out of proportion to the size of the effusion. The pleural fluid is an exudate, and its glucose level may be reduced if the tumor burden in the pleural space is high.

1	The diagnosis usually is made via cytology of the pleural fluid. If the initial cytologic examination is negative, thoracoscopy is the best next procedure if malignancy is strongly suspected. At the time of thoracoscopy, a procedure such as pleural abrasion should be performed to effect a pleurodesis. An alternative to thoracoscopy is CTor ultrasound-guided needle biopsy of pleural thickening or nodules. Patients with a malignant pleural effusion are treated symptomatically for the most part, since the presence of the effusion indicates disseminated disease and most malignancies associated with pleural effusion are not curable with chemotherapy. The only symptom that can be attributed to the effusion itself is dyspnea. If the patient’s lifestyle is compromised by dyspnea and if the dyspnea is relieved with a therapeutic thoracentesis, one of the following procedures should be considered: (1) insertion of a small indwelling catheter or (2) tube thoracostomy with the instillation of a

1	is relieved with a therapeutic thoracentesis, one of the following procedures should be considered: (1) insertion of a small indwelling catheter or (2) tube thoracostomy with the instillation of a sclerosing agent such as doxycycline (500 mg).

1	Mesothelioma Malignant mesotheliomas are primary tumors that arise from the mesothelial cells that line the pleural cavities; most are related to asbestos exposure. Patients with mesothelioma present with chest pain and shortness of breath. The chest radiograph reveals a pleural effusion, generalized pleural thickening, and a shrunken hemithorax. The diagnosis is usually established with image-guided needle biopsy or thoracoscopy.

1	Effusion Secondary to Pulmonary Embolization The diagnosis most commonly overlooked in the differential diagnosis of a patient with an undiagnosed pleural effusion is pulmonary embolism. Dyspnea is the most common symptom. The pleural fluid is almost always an exudate. The diagnosis is established by spiral CT scan or pulmonary arteriography (Chap. 300). Treatment of a patient with a pleural effusion secondary to pulmonary embolism is the same as it is for any patient with pulmonary emboli. If the pleural effusion increases in size after anticoagulation, the patient probably has recurrent emboli or another complication, such as a hemothorax or a pleural infection.

1	Disorders of the Pleura 1718 Tuberculous Pleuritis (See also Chap. 202) In many parts of the world, the most common cause of an exudative pleural effusion is tuberculosis (TB), but tuberculous effusions are relatively uncommon in the United States. Tuberculous pleural effusions usually are associated with primary TB and are thought to be due primarily to a hypersensitivity reaction to tuberculous protein in the pleural space. Patients with tuberculous pleuritis present with fever, weight loss, dyspnea, and/or pleuritic chest pain. The pleural fluid is an exudate with predominantly small lymphocytes. The diagnosis is established by demonstrating high levels of TB markers in the pleural fluid (adenosine deaminase >40 IU/L or interferon γ >140 pg/mL). Alternatively, the diagnosis can be established by culture of the pleural fluid, needle biopsy of the pleura, or thoracoscopy. The recommended treatments of pleural and pulmonary TB are identical (Chap. 202).

1	Effusion Secondary to Viral Infection Viral infections are probably responsible for a sizable percentage of undiagnosed exudative pleural effusions. In many series, no diagnosis is established for ~20% of exudative effusions, and these effusions resolve spontaneously with no long-term residua. The importance of these effusions is that one should not be too aggressive in trying to establish a diagnosis for the undiagnosed effusion, particularly if the patient is improving clinically.

1	Chylothorax A chylothorax occurs when the thoracic duct is disrupted and chyle accumulates in the pleural space. The most common cause of chylothorax is trauma (most frequently thoracic surgery), but it also may result from tumors in the mediastinum. Patients with chylothorax present with dyspnea, and a large pleural effusion is present on the chest radiograph. Thoracentesis reveals milky fluid, and biochemical analysis reveals a triglyceride level that exceeds 1.2 mmol/L (110 mg/ dL). Patients with chylothorax and no obvious trauma should have a lymphangiogram and a mediastinal CT scan to assess the mediastinum for lymph nodes. The treatment of choice for most chylothoraxes is insertion of a chest tube plus the administration of octreotide. If these modalities fail, a pleuroperitoneal shunt should be placed unless the patient has chylous ascites. Alternative treatments are ligation of the thoracic duct and percutaneous transabdominal thoracic duct blockage. Patients with

1	shunt should be placed unless the patient has chylous ascites. Alternative treatments are ligation of the thoracic duct and percutaneous transabdominal thoracic duct blockage. Patients with chylothoraxes should not undergo prolonged tube thoracostomy with chest tube drainage because this will lead to malnutrition and immunologic incompetence.

1	Hemothorax When a diagnostic thoracentesis reveals bloody pleural fluid, a hematocrit should be obtained on the pleural fluid. If the hematocrit is more than one-half of that in the peripheral blood, the patient is considered to have a hemothorax. Most hemothoraxes are the result of trauma; other causes include rupture of a blood vessel or tumor. Most patients with hemothorax should be treated with tube thoracostomy, which allows continuous quantification of bleeding. If the bleeding emanates from a laceration of the pleura, apposition of the two pleural surfaces is likely to stop the bleeding. If the pleural hemorrhage exceeds 200 mL/h, consideration should be given to thoracoscopy or thoracotomy.

1	Miscellaneous Causes of Pleural Effusion There are many other causes of pleural effusion (Table 316-1). Key features of some of these conditions are as follows: If the pleural fluid amylase level is elevated, the diagnosis of esophageal rupture or pancreatic disease is likely. If the patient is febrile, has predominantly polymorphonuclear cells in the pleural fluid, and has no pulmonary parenchymal abnormalities, an intraabdominal abscess should be considered.

1	The diagnosis of an asbestos pleural effusion is one of exclusion. Benign ovarian tumors can produce ascites and a pleural effusion (Meigs’ syndrome), as can the ovarian hyperstimulation syndrome. Several drugs can cause pleural effusion; the associated fluid is usually eosinophilic. Pleural effusions commonly occur after coronary artery bypass surgery. Effusions occurring within the first weeks are typically left-sided and bloody, with large numbers of eosinophils, and respond to one or two therapeutic thoracenteses. Effusions occurring after the first few weeks are typically left-sided and clear yellow, with predominantly small lymphocytes, and tend to recur. Other medical manipulations that induce pleural effusions include abdominal 1. 2. 3. Nephrotic syndrome 4. 5. 6. 7. 1. Neoplastic diseases a. b. 2. Infectious diseases a. b. c. d. e. 3. 4. a. b. c. d. e. f. g. 5. Collagen vascular diseases a. b. c. d. e. f.

1	5. 6. 7. 1. Neoplastic diseases a. b. 2. Infectious diseases a. b. c. d. e. 3. 4. a. b. c. d. e. f. g. 5. Collagen vascular diseases a. b. c. d. e. f. Granulomatosis with polyangiitis (Wegener) g. 6. 7. 8. 9. 10. 11. 12. a. b. c. d. e. f. g. 13. 14. 15. 16. 17. 18. 19. 20. surgery; radiation therapy; liver, lung, or heart transplantation; and the 1719 intravascular insertion of central lines. Disorders of the Mediastinum PNEUMOTHORAX Richard W. Light

1	18. 19. 20. surgery; radiation therapy; liver, lung, or heart transplantation; and the 1719 intravascular insertion of central lines. Disorders of the Mediastinum PNEUMOTHORAX Richard W. Light Pneumothorax is the presence of gas in the pleural space. A spontaneous pneumothorax is one that occurs without antecedent trauma to the thorax. A primary spontaneous pneumothorax occurs in the absence of underlying lung disease, whereas a secondary pneumothorax occurs in its presence. A traumatic pneumothorax results from penetrating or nonpenetrating chest injuries. A tension pneumothorax is a pneumothorax in which the pressure in the pleural space is positive throughout the respiratory cycle.

1	Primary Spontaneous Pneumothorax Primary spontaneous pneumothoraxes are usually due to rupture of apical pleural blebs, small cystic spaces that lie within or immediately under the visceral pleura. Primary spontaneous pneumothoraxes occur almost exclusively in smokers; this suggests that these patients have subclinical lung disease. Approximately one-half of patients with an initial primary spontaneous pneumothorax will have a recurrence. The initial recommended treatment for primary spontaneous pneumothorax is simple aspiration. If the lung does not expand with aspiration or if the patient has a recurrent pneumothorax, thoracoscopy with stapling of blebs and pleural abrasion is indicated. Thoracoscopy or thoracotomy with pleural abrasion is almost 100% successful in preventing recurrences.

1	Secondary Pneumothorax Most secondary pneumothoraxes are due to chronic obstructive pulmonary disease, but pneumothoraxes have been reported with virtually every lung disease. Pneumothorax in patients with lung disease is more life-threatening than it is in normal individuals because of the lack of pulmonary reserve in these patients. Nearly all patients with secondary pneumothorax should be treated with tube thoracostomy. Most should also be treated with thoracoscopy or thoracotomy with the stapling of blebs and pleural abrasion. If the patient is not a good operative candidate or refuses surgery, pleurodesis should be attempted by the intrapleural injection of a sclerosing agent such as doxycycline.

1	Traumatic Pneumothorax Traumatic pneumothoraxes can result from both penetrating and nonpenetrating chest trauma. Traumatic pneumothoraxes should be treated with tube thoracostomy unless they are very small. If a hemopneumothorax is present, one chest tube should be placed in the superior part of the hemithorax to evacuate the air and another should be placed in the inferior part of the hemithorax to remove the blood. Iatrogenic pneumothorax is a type of traumatic pneumothorax that is becoming more common. The leading causes are transthoracic needle aspiration, thoracentesis, and the insertion of central intravenous catheters. Most can be managed with supplemental oxygen or aspiration, but if these measures are unsuccessful, a tube thoracostomy should be performed.

1	Tension Pneumothorax This condition usually occurs during mechanical ventilation or resuscitative efforts. The positive pleural pressure is life-threatening both because ventilation is severely compromised and because the positive pressure is transmitted to the mediastinum, resulting in decreased venous return to the heart and reduced cardiac output.

1	Difficulty in ventilation during resuscitation or high peak inspiratory pressures during mechanical ventilation strongly suggest the diagnosis. The diagnosis is made by physical examination showing an enlarged hemithorax with no breath sounds, hyperresonance to percussion, and shift of the mediastinum to the contralateral side. Tension pneumothorax must be treated as a medical emergency. If the tension in the pleural space is not relieved, the patient is likely to die from inadequate cardiac output or marked hypoxemia. A large-bore needle should be inserted into the pleural space through the second anterior intercostal space. If large amounts of gas escape from the needle after insertion, the diagnosis is confirmed. The needle should be left in place until a thoracostomy tube can be inserted.

1	The mediastinum is the region between the pleural sacs. It is separated into three compartments (Table 317-1). The anterior mediastinum extends from the sternum anteriorly to the pericardium and brachiocephalic vessels posteriorly. It contains the thymus gland, the anterior mediastinal lymph nodes, and the internal mammary arteries and veins. The middle mediastinum lies between the anterior and posterior mediastina and contains the heart; the ascending and transverse arches of the aorta; the venae cavae; the brachiocephalic arteries and veins; the phrenic nerves; the trachea, the main bronchi, and their contiguous lymph nodes; and the pulmonary arteries and veins. The posterior mediastinum is bounded by the pericardium and trachea anteriorly and the vertebral column posteriorly. It contains the descending thoracic aorta, the esophagus, the thoracic duct, the azygos and hemiazygos veins, and the posterior group of mediastinal lymph nodes.

1	The first step in evaluating a mediastinal mass is to place it in one of the three mediastinal compartments, since each has different characteristic lesions (Table 317-1). The most common lesions in the anterior mediastinum are thymomas, lymphomas, teratomatous neoplasms, and thyroid masses. The most common masses in the middle mediastinum are vascular masses, lymph node enlargement from metastases or granulomatous disease, and pleuropericardial and bronchogenic cysts. In the posterior mediastinum, neurogenic tumors, meningoceles, meningomyeloceles, gastroenteric cysts, and esophageal diverticula are commonly found.

1	Computed tomography (CT) scanning is the most valuable imaging technique for evaluating mediastinal masses and is the only imaging technique that should be done in most instances. Barium studies of the gastrointestinal tract are indicated in many patients with posterior mediastinal lesions, because hernias, diverticula, and achalasia are readily diagnosed in this manner. An iodine-131 scan can efficiently establish the diagnosis of intrathoracic goiter.

1	A definite diagnosis can be obtained with mediastinoscopy or anterior mediastinotomy in many patients with masses in the anterior or middle mediastinal compartments. A diagnosis can be established without thoracotomy via percutaneous fine-needle aspiration biopsy or endoscopic transesophageal or endobronchial ultrasound-guided biopsy of mediastinal masses in most cases. An alternative way to establish the diagnosis is video-assisted thoracoscopy. In many cases, the diagnosis can be established and the mediastinal mass removed with video-assisted thoracoscopy.

1	Most cases of acute mediastinitis either are due to esophageal perforation or occur after median sternotomy for cardiac surgery. Patients with esophageal rupture are acutely ill with chest pain and dyspnea due to the mediastinal infection. The esophageal rupture can occur spontaneously or as a complication of esophagoscopy or the insertion of a Blakemore tube. Appropriate treatment consists of exploration of the mediastinum with primary repair of the esophageal tear and drainage of the pleural space and the mediastinum. The incidence of mediastinitis after median sternotomy is 0.4–5.0%. Patients most commonly present with wound drainage. Other presentations include sepsis and a widened mediastinum. The diagnosis usually is established with mediastinal needle aspiration. Treatment includes immediate drainage, debridement, and parenteral antibiotic therapy, but the mortality rate still exceeds 20%.

1	The spectrum of chronic mediastinitis ranges from granulomatous inflammation of the lymph nodes in the mediastinum to fibrosing Disorders of the Mediastinum Common abnormalities Manubrium and sternum anteriorly, Anterior mediastinum anteriorly, posterior Pericardium and trachea anteriorly; pericardium, aorta, and brachiocephalic mediastinum posteriorly vertebral column posteriorly vessels posteriorly Thymus gland, anterior mediastinal lymph Pericardium, heart, ascending and transverse arch Descending thoracic aorta, esophagus, nodes, internal mammary arteries and of aorta, superior and inferior vena cavae, brachio-thoracic duct, azygos and hemiazygos veins cephalic arteries and veins, phrenic nerves, trachea, veins, sympathetic chains, and the posterior and main bronchi and their contiguous lymph group of mediastinal lymph nodes nodes, pulmonary arteries, and veins

1	Thymoma, lymphomas, teratomatous Metastatic lymph node enlargement, granuloma-Neurogenic tumors, meningocele, meninneoplasms, thyroid masses, parathyroid tous lymph node enlargement, pleuropericardial gomyelocele, gastroenteric cysts, esophamasses, mesenchymal tumors, giant cysts, bronchogenic cysts, masses of vascular origin geal diverticula, hernia through foramen of lymph node hyperplasia, hernia through foramen of Morgagni mediastinitis. Most cases are due to histoplasmosis or tuberculosis, but sarcoidosis, silicosis, and other fungal diseases are at times causative. Patients with granulomatous mediastinitis are usually asymptomatic. Those with fibrosing mediastinitis usually have signs of compression of a mediastinal structure such as the superior vena cava or large airways, phrenic or recurrent laryngeal nerve paralysis, or obstruction of the pulmonary artery or proximal pulmonary veins. Other than antituberculous therapy for tuberculous mediastinitis, no medical or surgical

1	or recurrent laryngeal nerve paralysis, or obstruction of the pulmonary artery or proximal pulmonary veins. Other than antituberculous therapy for tuberculous mediastinitis, no medical or surgical therapy has been demonstrated to be effective for mediastinal fibrosis.

1	In this condition, there is gas in the interstices of the mediastinum. The three main causes are (1) alveolar rupture with dissection of air into the mediastinum; (2) perforation or rupture of the esophagus, trachea, or main bronchi; and (3) dissection of air from the neck or the abdomen into the mediastinum. Typically, there is severe substernal chest pain with or without radiation into the neck and arms. The physical examination usually reveals subcutaneous emphysema in the suprasternal notch and Hamman’s sign, which is a crunching or clicking noise synchronous with the heartbeat and is best heard in the left lateral decubitus position. The diagnosis is confirmed with the chest radiograph. Usually no treatment is required, but the mediastinal air will be absorbed faster if the patient inspires high concentrations of oxygen. If mediastinal structures are compressed, the compression can be relieved with needle aspiration.

1	Disorders of Ventilation John F. McConville, Babak Mokhlesi, Julian Solway DEFINITION AND PHYSIOLOGY In health the arterial level of carbon dioxide (PaCO2) is maintained between 37 and 43 mmHg at sea level. All disorders of ventilation result in abnormal measurements of PaCO2. This chapter reviews 318 chronic ventilatory disorders. The continuous production of CO2 by cellular metabolism necessitates its efficient elimination by the respiratory system. The relationship between CO2 production and PaCO2 is described by the equation:

1	PaCO2 = (k) (V CO2)/V A, where V CO2 represents the carbon dioxide production, k is a constant, and VA is fresh gas alveolar ventilation (Chap. 306e). VA can be calculated as minute ventilation × (1 – Vd/ Vt), where the dead space fraction Vd/Vt represents the portion of a tidal breath that remains within the conducting airways at the conclusion of inspiration and so does not contribute to alveolar ventilation. As such, all disturbances of PaCO2 must reflect altered CO2 production, minute ventilation, or dead space fraction. Bochdalek, extramedullary hematopoiesis Diseases that alter VCO2 are often acute (e.g., sepsis, burns, or pyrexia), and their contribution to ventilatory abnormalities and/or respiratory failure is reviewed elsewhere. Chronic ventilatory disorders typically involve inappropriate levels of minute ventilation or increased dead space fraction. Characterization of these disorders requires a review of the normal respiratory cycle.

1	The spontaneous cycle of inspiration and expiration is automatically generated in the brainstem. Two groups of neurons located within the medulla are particularly important: the dorsal respiratory group (DRG) and the ventral respiratory column (VRC). These neurons have widespread projections including the descending projections into the contralateral spinal cord where they perform many functions. They initiate activity in the phrenic nerve/diaphragm, project to the upper airway muscle groups and spinal respiratory neurons, and innervate the intercostal and abdominal muscles that participate in normal respiration. The DRG acts as the initial integration site for many of the afferent nerves relaying information about PaO2, PaCO2, pH, and blood pressure from the carotid and aortic chemoreceptors and baroreceptors to the central nervous system (CNS). In addition, the vagus nerve relays information from stretch receptors and juxtapulmonary-capillary receptors in the lung parenchyma and

1	and baroreceptors to the central nervous system (CNS). In addition, the vagus nerve relays information from stretch receptors and juxtapulmonary-capillary receptors in the lung parenchyma and chest wall to the DRG. The respiratory rhythm is generated within the VRC as well as the more rostrally located parafacial respiratory group (pFRG), which is particularly important for the generation of active expiration. One particularly important area within the VRC is the so-called pre-Bötzinger complex. This area is responsible for the generation of various forms of inspiratory activity, and lesioning of the pre-Bötzinger complex leads to the complete cessation of breathing. The neural output of these medullary respiratory networks can be voluntarily suppressed or augmented by input from higher brain centers and the autonomic nervous system. During normal sleep, there is an attenuated response to hypercapnia and hypoxemia, resulting in mild nocturnal hypoventilation that corrects upon

1	brain centers and the autonomic nervous system. During normal sleep, there is an attenuated response to hypercapnia and hypoxemia, resulting in mild nocturnal hypoventilation that corrects upon awakening.

1	Once neural input has been delivered to the respiratory pump muscles, normal gas exchange requires an adequate amount of respiratory muscle strength to overcome the elastic and resistive loads of the respiratory system (Fig. 318-1A) (Chap. 306e). In health, the strength of the respiratory muscles readily accomplishes this, and normal respiration continues indefinitely. Reduction in respiratory drive or neuromuscular competence or substantial increase in respiratory load can diminish minute ventilation, resulting in hypercapnia (Fig. 318-1B). Alternatively, if normal respiratory muscle strength is coupled with excessive respiratory drive, then alveolar hyperventilation ensues and leads to hypocapnia (Fig. 318-1C).

1	Diseases that reduce minute ventilation or increase dead space fall into four major categories: parenchymal lung and chest wall disease, sleep-disordered breathing, neuromuscular disease, and respiratory drive disorders (Fig. 318-1B). The clinical manifestations of hypoventilation Increased drive with acceptable strength strength and load. A. Excess respiratory muscle strength in health. B. Load greater than strength. C. Increased drive with acceptable strength.

1	Increased drive with acceptable strength strength and load. A. Excess respiratory muscle strength in health. B. Load greater than strength. C. Increased drive with acceptable strength. syndromes are nonspecific (Table 318-1) and vary depending on the severity of hypoventilation, the rate at which hypercapnia develops, the degree of compensation for respiratory acidosis, and the underlying disorder. Patients with parenchymal lung or chest wall disease typically present with shortness of breath and diminished exercise tolerance. Episodes of increased dyspnea and sputum production are hallmarks of obstructive lung diseases such as chronic obstructive pulmonary disease, whereas progressive dyspnea and cough are common in interstitial lung diseases. Excessive daytime somnolence, poor-quality sleep, and snoring are common among patients with sleep-disordered breathing. Sleep disturbance and orthopnea are also described in SignS anD SyMptoMS of hypoVentiLation

1	SignS anD SyMptoMS of hypoVentiLation Dyspnea during activities of daily living neuromuscular disorders. As neuromuscular weakness progresses, the respiratory muscles, including the diaphragm, are placed at a mechanical disadvantage in the supine position due to the upward movement of the abdominal contents. New-onset orthopnea is frequently a sign of reduced respiratory muscle force generation. More commonly, however, extremity weakness or bulbar symptoms develop prior to sleep disturbance in neuromuscular diseases such as amyotrophic lateral sclerosis (ALS) or muscular dystrophy. Patients with respiratory drive disorders do not have symptoms distinguishable from other causes of chronic hypoventilation.

1	The clinical course of patients with chronic hypoventilation from neuromuscular or chest wall disease follows a characteristic sequence: an asymptomatic stage where daytime PaO2 and PaCO2 are normal followed by nocturnal hypoventilation, initially during rapid eye movement (REM) sleep and later in non-REM sleep. Finally, if vital capacity drops further, daytime hypercapnia develops. Symptoms can develop at any point along this time course and often depend on the pace of respiratory muscle functional decline. Regardless of cause, the hallmark of all alveolar hypoventilation syndromes is an increase in alveolar PCO2 (PACO2) and therefore in PaCO2. The resulting respiratory acidosis eventually leads to a compensatory increase in plasma bicarbonate concentration. The increase in PACO2 results in an obligatory decrease in PAO2, often resulting in hypoxemia. If severe, the hypoxemia manifests clinically as cyanosis and can stimulate erythropoiesis and thus induce secondary erythrocytosis.

1	in an obligatory decrease in PAO2, often resulting in hypoxemia. If severe, the hypoxemia manifests clinically as cyanosis and can stimulate erythropoiesis and thus induce secondary erythrocytosis. The combination of chronic hypoxemia and hypercapnia may also induce pulmonary vasoconstriction, leading eventually to pulmonary hypertension, right ventricular hypertrophy, and right heart failure.

1	Elevated plasma bicarbonate in the absence of volume depletion is suggestive of hypoventilation. An arterial blood gas demonstrating elevated PaCO2 with a normal pH confirms chronic alveolar hypoventilation. The subsequent evaluation to identify an etiology should initially focus on whether the patient has lung disease or chest wall abnormalities. Physical examination, imaging studies (chest x-ray and/or computed tomography [CT] scan), and pulmonary function tests are sufficient to identify most lung/chest wall disorders leading to hypercapnia. If these evaluations are unrevealing, then the clinician should screen for obesity hypoventilation syndrome (OHS), the most frequent sleep disorder leading to chronic hypoventilation, which is typically accompanied by obstructive sleep apnea (OSA). Several screening tools have been developed to identify patients at risk for OSA. The Berlin Questionnaire has been validated in a primary care setting and identifies patients likely to have OSA. The

1	Several screening tools have been developed to identify patients at risk for OSA. The Berlin Questionnaire has been validated in a primary care setting and identifies patients likely to have OSA. The Epworth Sleepiness Scale (ESS) and the STOP-Bang questionnaires have not been validated in outpatient primary care settings but are quick and easy to use. The ESS measures daytime sleepiness, with a score of ≥10 indentifying individuals who warrant additional investigation. The STOP-Bang survey has been used in preoperative clinics to identify patients at risk of having OSA. In this population, it has 93% sensitivity and 90% negative predictive value.

1	If the ventilatory apparatus (lungs, airways, chest wall) is not responsible for chronic hypercapnia, then the focus should shift to respiratory drive and neuromuscular disorders. There is an attenuated increase in minute ventilation in response to elevated CO2 and/ or low O2 in respiratory drive disorders. These diseases are difficult to diagnose and should be suspected when patients with hypercapnia are

1	Disorders of Ventilation 1722 found to have normal respiratory muscle strength, normal pulmonary function, and normal alveolar-arterial PO2 difference. Hypoventilation is more marked during sleep in patients with respiratory drive defects, and polysomnography often reveals central apneas, hypopneas, or hypoventilation. Brain imaging (CT scan or magnetic resonance imaging [MRI]) can sometimes identify structural abnormalities in the pons or medulla that result in hypoventilation. Chronic narcotic use or significant hypothyroidism can depress the central respiratory drive and lead to chronic hypercapnia as well. Respiratory muscle weakness has to be profound before lung volumes are compromised and hypercapnia develops. Typically physical examination reveals decreased strength in major muscle groups prior to the development of hypercapnia. Measurement of maximum inspiratory and expiratory pressures or forced vital capacity (FVC) can be used to monitor for respiratory muscle involvement

1	groups prior to the development of hypercapnia. Measurement of maximum inspiratory and expiratory pressures or forced vital capacity (FVC) can be used to monitor for respiratory muscle involvement in diseases with progressive muscle weakness. These patients also have increased risk for sleep-disordered breathing, including hypopneas, central and obstructive apneas, and hypoxemia. Nighttime oximetry and capnometry during polysomnography are helpful in better characterizing sleep disturbances in this patient population.

1	Nocturnal noninvasive positive-pressure ventilation (NIPPV) has been used successfully in the treatment of hypoventilation and apneas, both central and obstructive, in patients with neuromuscular and chest wall disorders. Nocturnal NIPPV has been shown to improve daytime hypercapnia, prolong survival, and improve health-related quality of life when daytime hypercapnia is documented. ALS guidelines recommend consideration of nocturnal NIPPV if symptoms of hypoventilation exist and one of the following criteria is present: PaCO2 ≥45 mmHg; nocturnal oximetry demonstrates oxygen saturation ≤88% for 5 consecutive min; maximal inspiratory pressure <60 cmH2O; FVC <50% predicted; or sniff nasal pressure <40 cmH2O. However, at present, there is inconclusive evidence to support preemptive nocturnal NIPPV use in all patients with neuromuscular and chest wall disorders who demonstrate nocturnal but not daytime hypercapnia. Nevertheless, at some point, the institution of full-time ventilatory

1	NIPPV use in all patients with neuromuscular and chest wall disorders who demonstrate nocturnal but not daytime hypercapnia. Nevertheless, at some point, the institution of full-time ventilatory support with either pressure or volume-preset modes is required in progressive neuromuscular disorders. There is less evidence to direct the timing of this decision, but ventilatory failure requiring mechanical ventilation and chest infections related to ineffective cough are frequent triggers for the institution of full-time ventilatory support.

1	Treatment of chronic hypoventilation from lung or neuromuscular diseases should be directed at the underlying disorder. Pharmacologic agents that stimulate respiration, such as medroxyprogesterone and acetazolamide, have been poorly studied in chronic hypoventilation and should not replace treatment of the underlying disease process. Regardless of the cause, excessive metabolic alkalosis should be corrected, because plasma bicarbonate levels elevated out of proportion for the degree of chronic respiratory acidosis can result in additional hypoventilation. When indicated, administration of supplemental oxygen is effective in attenuating hypoxemia, polycythemia, and pulmonary hypertension. However, in some patients, supplemental oxygen can worsen hypercapnia.

1	Phrenic nerve or diaphragm pacing is a potential therapy for patients with hypoventilation from high cervical spinal cord lesions or respiratory drive disorders. Prior to surgical implantation, patients should have nerve conduction studies to ensure normal bilateral phrenic nerve function. Small case series suggest that effective diaphragmatic pacing can improve quality of life in these patients.

1	The diagnosis of OHS requires body mass index (BMI) ≥30 kg/m2 and chronic daytime alveolar hypoventilation, defined as PaCO2 ≥45 mmHg at sea level in the absence of other known causes of hypercapnia. In almost 90% of cases, the sleep-disordered breathing is in the form of OSA. Several international studies in different populations confirm that the overall prevalence of OSA syndrome, defined by an apnea-hypopnea index (AHI) ≥5 and daytime sleepiness, is approximately 3–4% in middle-aged men and 2% in middle-aged women. Thus, the population at risk for the development of OHS continues to rise as the worldwide obesity epidemic persists. Although no population-based prevalence studies of OHS have been performed, some estimates suggest there may be as many as 500,000 individuals with OHS in the United States.

1	Some, but not all, studies suggest that severe obesity (BMI >40 kg/ m2) and severe OSA (AHI >30 events per hour) are risk factors for the development of OHS. The pathogenesis of hypoventilation in these patients is the result of multiple physiologic variables and conditions including OSA, increased work of breathing, respiratory muscle impairment, ventilation-perfusion mismatching, and depressed central ventilatory responsiveness to hypoxemia and hypercapnia. These defects in central respiratory drive often improve with treatment, which suggests that decreased ventilatory responsiveness is a consequence rather than a primary cause of OHS. The treatment of OHS is similar to that for OSA: weight reduction and nocturnal NIPPV. There is evidence that weight loss alone lowers PaCO2 in patients with OHS. However, treatment with NIPPV should never be delayed while the patient attempts to lose weight. Continuous positive airway pressure (CPAP) improves daytime hypercapnia and hypoxemia in

1	with OHS. However, treatment with NIPPV should never be delayed while the patient attempts to lose weight. Continuous positive airway pressure (CPAP) improves daytime hypercapnia and hypoxemia in more than half of patients with OHS and concomitant OSA. Bilevel positive airway pressure should be reserved for patients not able to tolerate high levels of CPAP support or patients who remain hypoxemic despite resolution of obstructive respiratory events. NIPPV with bilevel positive airway pressure should be strongly considered if hypercapnia persists after several weeks of CPAP therapy with objectively proven adherence. Patients with OHS and no evidence of OSA are typically started on bilevel positive airway pressure, as are patients presenting with acute decompensated OHS. Finally, comorbid conditions that impair ventilation, such as chronic obstructive pulmonary disease, should be aggressively treated in conjunction with coexisting OHS.

1	This syndrome can present later in life or in the neonatal period where it is often called Ondine’s curse or congenital central hypoventilation syndrome. Abnormalities in the gene encoding PHOX2b, a transcription factor with a role in neuronal development, have been implicated in the pathogenesis of congenital central hypoventilation syndrome. Regardless of the age of onset, these patients have absent respiratory response to hypoxia or hypercapnia, mildly elevated PaCO2 while awake, and markedly elevated PaCO2 during non-REM sleep. Interestingly these patients are able to augment their ventilation and “normalize” PaCO2 during exercise and during REM sleep. These patients typically require NIPPV or mechanical ventilation as therapy and should be considered for phrenic nerve or diaphragmatic pacing at centers with experience performing these procedures.

1	Hyperventilation is defined as ventilation in excess of metabolic requirements (CO2 production) leading to a reduction in PaCO2. The physiology of patients with chronic hyperventilation is poorly understood, and there is no typical clinical presentation. Symptoms can include dyspnea, paresthesias, tetany, headache, dizziness, visual disturbances, and atypical chest pain. Because symptoms can be so diverse, patients with chronic hyperventilation present to a variety of health care providers, including internists, neurologists, psychologists, psychiatrists, and pulmonologists.

1	It is helpful to think of hyperventilation as having initiating and sustaining factors. Some investigators believe that an initial event leads to increased alveolar ventilation and a drop in PaCO2 to ~20 mmHg. The ensuing onset of chest pain, breathlessness, paresthesia, or altered consciousness can be alarming. The resultant increase in minute volume to relieve these acute symptoms only serves to exacerbate symptoms that are often misattributed by the patient and health care workers to cardiopulmonary disorders. An unrevealing evaluation for causes of these symptoms often results in patients being anxious and fearful of additional attacks. It is important to note that anxiety disorders and panic attacks are not synonymous with hyperventilation. Anxiety disorders can be both an initiating and sustaining factor in the pathogenesis of chronic hyperventilation, but these are not necessary for the development of chronic hypocapnia.

1	Respiratory symptoms associated with acute hyperventilation can be the initial manifestation of systemic illnesses such as diabetic ketoacidosis. Causes of acute hyperventilation need to be excluded before a diagnosis of chronic hyperventilation is considered. Arterial blood gas sampling that demonstrates a compensated respiratory alkalosis with a near normal pH, low PaCO2, and low calculated bicarbonate is necessary to confirm chronic hyperventilation. Other causes of respiratory alkalosis, such as mild asthma, need to be diagnosed and treated before chronic hyperventilation can be considered. A high index of suspicion is required because increased minute ventilation can be difficult to detect on physical examination. Once chronic hyperventilation is established, a sustained 10% increase in alveolar ventilation is enough to perpetuate hypocapnia. This increase can be accomplished with subtle changes in the respiratory pattern, such as occasional sigh breaths or yawning two to three

1	in alveolar ventilation is enough to perpetuate hypocapnia. This increase can be accomplished with subtle changes in the respiratory pattern, such as occasional sigh breaths or yawning two to three times per minute.

1	There are few well-controlled treatment studies of chronic hyperventilation due to its diverse features and the lack of a universally accepted diagnostic process. Clinicians often spend considerable time identifying initiating factors, excluding alternative diagnoses, and discussing the patient’s concerns and fears. In some patients, reassurance and frank discussion about hyperventilation can be liberating. Identifying and eliminating habits that perpetuate hypocapnia, such as frequent yawning or sigh breathing, can be helpful. Some evidence suggests that breathing exercises and diaphragmatic retraining may be beneficial for some patients. The evidence for using medications to treat hyperventilation is scant. Beta blockers may be helpful in patients with sympathetically mediated symptoms such as palpitations and tremors. We would like to acknowledge Eliot A. Phillipson for earlier versions of this chapter and Jan-Marino Ramirez for his careful critique and helpful suggestions.

1	We would like to acknowledge Eliot A. Phillipson for earlier versions of this chapter and Jan-Marino Ramirez for his careful critique and helpful suggestions. Andrew Wellman, Susan Redline

1	Andrew Wellman, Susan Redline Obstructive sleep apnea/hypopnea syndrome (OSAHS) and central sleep apnea (CSA) are both classified as sleep-related breathing disorders. OSAHS and CSA share some risk factors and physiological bases but also have unique features. Each disorder is associated with impaired ventilation during sleep and disruption of sleep, and each diagnosis requires careful elicitation of the patient’s history, physical examination, and physiological testing. OSAHS, the more common disorder, causes daytime sleepiness, impairs daily function, and is a major contributor to cardiovascular disease in adults and to behavioral 1723 problems in children. CSA is less common and may occur in combination with obstructive sleep apnea, as a primary condition, or secondary to a medical condition or medication. CSA impairs overnight gas exchange and may result in symptoms of either insomnia or excessive sleepiness.

1	OBSTRUCTIVE SLEEP APNEA/HYPOPNEA SYNDROME (OSAHS) Definition OSAHS is defined on the basis of nocturnal and daytime symptoms as well as sleep study findings. Diagnosis requires the patient to have (1) either symptoms of nocturnal breathing disturbances (snoring, snorting, gasping, or breathing pauses during sleep) or daytime sleepiness or fatigue that occurs despite sufficient opportunities to sleep and is unexplained by other medical problems; and (2) five or more episodes of obstructive apnea or hypopnea per hour of sleep (the apnea-hypopnea index [AHI], calculated as the number of episodes divided by the number of hours of sleep) documented during a sleep study. OSAHS also may be diagnosed in the absence of symptoms if the AHI is above 15. Each episode of apnea or hypopnea represents a reduction in breathing for at least 10 sec. OSAHS is often identified when associated with a ≥3% drop in oxygen saturation and/ or a brain cortical arousal. OSAHS severity is based on the frequency

1	a reduction in breathing for at least 10 sec. OSAHS is often identified when associated with a ≥3% drop in oxygen saturation and/ or a brain cortical arousal. OSAHS severity is based on the frequency of breathing disturbances (AHI), the amount of oxygen desaturation with respiratory events, the duration of apneas and hypopneas, the degree of sleep fragmentation, and the level of daytime sleepiness.

1	Pathophysiology During inspiration, intraluminal pharyngeal pressure becomes increasingly negative, creating a “suctioning” force. Because the pharyngeal airway has no bone or cartilage, airway patency is dependent on the stabilizing influence of the pharyngeal dilator muscles. Although these muscles are continuously activated during wakefulness, neuromuscular output declines with sleep onset. In patients with a collapsible airway, the reduction in neuromuscular output results in transient episodes of pharyngeal collapse (manifesting as an “apnea”) or near collapse (manifesting as a “hypopnea”). The episodes of collapse are terminated when ventilatory reflexes are activated and cause arousal, thus stimulating an increase in neuromuscular activity and opening of the airway. The airway may collapse at various levels: the soft palate (most common), tongue base, lateral pharyngeal walls, and/or epiglottis (Fig. 319-1). OSAHS may be most

1	TongueFIGURE 319-1 Common sites of airway collapse. For example, the palate, tongue, and/or epiglottis (Ep) can be posteriorly displaced, and the lateral pharyngeal walls (LW) can collapse.

1	1724 severe during REM (rapid eye movement) sleep, when neuromuscular output to the skeletal muscles is particularly low, and in the supine position due to gravitational forces. Individuals with a small pharyngeal lumen require relatively high levels of neuromuscular innervation to maintain patency during wakefulness and thus are predisposed to excessive airway collapsibility during sleep. The airway lumen may be narrowed with enlargement of soft tissue structures (tongue, palate, and uvula) due to fat deposition, increased lymphoid tissue, or genetic variation. Craniofacial factors such as mandibular retroposition or micrognathia, reflecting genetic variation or developmental influences, also can reduce lumen dimensions. In addition, lung volumes influence the caudal traction on the pharynx and consequently the stiffness of the pharyngeal wall. Accordingly, low lung volume in the recumbent position, which is particularly pronounced in the obese, contributes to collapse. A high degree

1	and consequently the stiffness of the pharyngeal wall. Accordingly, low lung volume in the recumbent position, which is particularly pronounced in the obese, contributes to collapse. A high degree of nasal resistance (e.g., due to nasal septal deviation or polyps) can contribute to airway collapse by increasing the negative intraluminal suction pressure. High-level nasal resistance also may trigger mouth opening during sleep, which breaks the seal between the tongue and the teeth and allows the tongue to fall posteriorly and occlude the airway. Pharyngeal muscle activation is integrally linked to ventilatory drive. Thus, factors related to ventilatory control, particularly ventilatory sensitivity, arousal threshold, and neuromuscular responses to CO2, contribute to the pathogenesis of OSAHS. A buildup in CO2 during sleep activates both the diaphragm and the pharyngeal muscles, which stiffen the upper airway and can counteract inspiratory suction pressures and maintain airway patency

1	A buildup in CO2 during sleep activates both the diaphragm and the pharyngeal muscles, which stiffen the upper airway and can counteract inspiratory suction pressures and maintain airway patency to an extent that depends on the anatomic predisposition to collapse. However, pharyngeal collapse can occur when the ventilatory control system is overly sensitive to CO2, with resultant wide fluctuations in ventilation and ventilatory drive and in upper airway instability. Moreover, increasing levels of CO2 during sleep result in central nervous system arousal, causing the individual to move from a deeper to a lighter level of sleep or to awaken. A low arousal threshold (i.e., awaken to a low level of CO2 or ventilatory drive) can preempt the CO2-mediated process of pharyngeal muscle compensation and prevent airway stabilization. A high arousal threshold, conversely, may prevent appropriate termination of apneas, prolonging apnea duration and oxyhemoglobin desaturation severity. Finally, any

1	and prevent airway stabilization. A high arousal threshold, conversely, may prevent appropriate termination of apneas, prolonging apnea duration and oxyhemoglobin desaturation severity. Finally, any impairment in the ability of the muscles to compensate during sleep can contribute to collapse of the pharynx. The relative contributions of risk factors vary among individuals. Approaches to the measurement of these factors in clinical settings, with consequent enhancement of “personalized” therapeutic interventions, are being actively investigated.

1	Risk Factors and Prevalence The major risk factors for OSAHS are obesity and male sex. Additional risk factors include mandibular retrognathia and micrognathia, a positive family history of OSAHS, genetic syndromes that reduce upper airway patency (e.g., Down syndrome, Treacher-Collins syndrome), adenotonsillar hypertrophy (especially in children), menopause (in women), and various endocrine syndromes (e.g., acromegaly, hypothyroidism).

1	Approximately 40–60% of cases of OSAHS are attributable to excess weight. Obesity predisposes to OSAHS through the narrowing effects of upper airway fat on the pharyngeal lumen. Obesity also reduces chest wall compliance and decreases lung volumes, resulting in a loss of caudal traction on upper airway structures. Obese individuals are at a fourfold or greater risk for OSAHS than their normal-weight counterparts. A 10% weight gain is associated with a >30% increase in AHI. Even modest weight loss or weight gain can influence the risk and severity of OSAHS. However, the absence of obesity does not exclude this diagnosis.

1	The prevalence of OSAHS is twoto fourfold higher among men than among women. Factors that predispose men to OSAHS include android patterns of obesity (resulting in upper-airway fat deposition) and relatively great pharyngeal length, which exacerbates collapsibility. Premenopausal women are relatively protected from OSAHS by the influence of sex hormones on ventilatory drive. The decline in sex differences in older age is associated with an increased OSAHS prevalence in women after menopause. Variations in craniofacial morphology that reduce the size of the posterior airway space increase OSAHS risk. The contribution of hard-tissue structural features to OSAHS is most evident in nonobese patients. Identification of features such as retrognathia can influence therapeutic decision-making.

1	OSAHS has a strong genetic basis, as evidenced by its significant familial aggregation and heritability. For a first-degree relative of a patient with OSAHS, the odds ratio of having OSAHS is approximately twofold higher than that for someone without an affected relative. OSAHS prevalence varies with age, from 2–15% among middle-aged adults to >20% among elderly individuals. There is a peak due to lymphoid hypertrophy among children between the ages of 3 and 8 years; with airway growth and lymphoid tissue regression during later childhood, prevalence declines. Then, as obesity prevalence increases in middle life and women enter menopause, OSAHS again increases.

1	The prevalence of OSAHS may be especially high among patients with diabetes or hypertension. Individuals of Asian ancestry appear to be at increased risk of OSAHS at relatively low levels of body mass index, possibly because of the influence of craniofacial risk factors that narrow the nasopharynx. In the United States, African Americans, especially children and young adults, are at higher risk for OSAHS than their Caucasian counterparts. In a majority of adults with OSAHS, the disorder is undiagnosed. Course of the Disorder The precise onset of OSAHS is usually hard to identify. A person may snore for many years, often beginning in childhood, before OSAHS is identified. Weight gain may precipitate an increase in symptoms, which in turn may lead the patient to pursue an evaluation. OSAHS may become less severe with weight loss, particularly after bariatric surgery. Marked increases and decreases in the AHI are uncommon unless accompanied by weight change. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: An evaluation for OSAHS should be considered in patients with symptoms of OSAHS and one or more risk factors. Screening also should be considered in patients who report symptoms consistent with OSAHS and who are at high risk for OSAHS-related morbidities, such as hypertension, diabetes mellitus, and cardiac and cerebrovascular diseases.

1	When possible, a sleep history should be obtained in the presence of a bed partner. Snoring is the most common complaint; however, its absence does not exclude the diagnosis, as pharyngeal collapse may occur without tissue vibration. Gasping or snorting during sleep may also be reported, reflecting termination of individual apneas with abrupt airway opening. Dyspnea is unusual, and its absence generally distinguishes OSAHS from paroxysmal nocturnal dyspnea, nocturnal asthma, and acid reflux with laryngospasm. Patients also may describe frequent awakening or sleep disruption, which is more common among women and older adults. The most common daytime symptom is sleepiness. This symptom can be difficult to elicit and may be hard to distinguish from exercise-related fatigue, deconditioning, and malaise. In contrast to true sleepiness, the latter symptoms generally improve with rest. Other symptoms include a dry mouth, nocturnal heartburn, diaphoresis of the chest and neck, nocturia,

1	and malaise. In contrast to true sleepiness, the latter symptoms generally improve with rest. Other symptoms include a dry mouth, nocturnal heartburn, diaphoresis of the chest and neck, nocturia, morning headaches, trouble concentrating, irritability, and mood disturbances. Several questionnaires that evaluate snoring frequency, self-reported apneas, and daytime sleepiness can facilitate OSAHS screening. The predictive ability of a questionnaire can be enhanced by a consideration of whether the patient is male or has risk factors such as obesity or hypertension.

1	Physical findings often reflect the etiologic factors for the disorder as well as comorbid conditions, particularly vascular disease. On examination, patients may exhibit hypertension and regional (central) obesity, as indicated by a large waist and neck circumference. The oropharynx may reveal a small orifice with crowding due to an enlarged tongue, a low-lying soft palate with a bulky uvula, large tonsils, a high arched palate, and/or micro/retrognathia. Since high-level nasal resistance can increase pharyngeal collapsibility, the nasal cavity should be inspected for polyps, septal deviation, and other signs of obstruction. Because patients with heart failure are at increased risk for both OSAHS and CSA, a careful cardiac examination should be conducted to detect possible leftor right-sided cardiac dysfunction. Evidence of cor pulmonale suggests severe OSAHS or a comorbid cardiopulmonary condition. A neurologic evaluation is needed to evaluate for conditions such as neuromuscular

1	cardiac dysfunction. Evidence of cor pulmonale suggests severe OSAHS or a comorbid cardiopulmonary condition. A neurologic evaluation is needed to evaluate for conditions such as neuromuscular and cerebrovascular diseases, which increase OSAHS risk.

1	predict the severity of sleep-related breathing disturbances, specific diagnosis and categorization of OSAHS severity require objective measurement of breathing during the period of sleep. The gold standard for diagnosis of OSAHS is an overnight polysomnogram (PSG). A negative in-laboratory PSG rules out OSAHS except in unusual circumstances—e.g., with insufficient REM sleep or supine sleep. Home sleep tests that record only a few respiratory and cardiac channels commonly are used as a cost-effective means a high pretest probability of OSAHS. However, a home study may yield a false-negative result if sleep time is not accurately estimated, and further evaluation may therefore be required.

1	The key physiological information collected during a sleep study for OSAHS assessment includes measurement of breathing (changes in airflow, respiratory excursion), oxygenation (hemoglobin oxygen saturation), body position, and cardiac rhythm. In addition, PSGs stages (by electroencephalography, chin electromyography, and electro-oculography), limb movements (by leg sensors), and snor ing intensity. This information is used to quantify the frequency and subtypes of abnormal respiratory events during sleep as well as associated changes in oxygen saturation, arousals, and sleep stage distributions. Tables 319-1 and 319-2 define the respiratory events scored and the severity guidelines employed during a sleep study. Figure 319-2 shows examples of sleep-related respiratory events. A typical sleep study report provides quantitative data such as the

1	Figure 319-2 shows examples of sleep-related respiratory events. A typical sleep study report provides quantitative data such as the AHI and the profile of oxygen saturation over the night (mean, nadir, time at low levels). Reports may also include the respira tory disturbance index, which includes the number of respiratory effort–related arousals in addition to the number of apneas plus hypopneas. In-laboratory PSG also quantifies sleep latency (time from “lights off ” to first sleep onset), sleep efficiency (percentage of • Apnea: Cessation of airflow for ≥10 sec during sleep, accompanied by: respiratory effort (obstructive apneas, Fig. 319-2A), or of respiratory effort (central apneas, Fig. 319-2B) • Hypopnea:

1	Cessation of airflow for ≥10 sec during sleep, accompanied by: respiratory effort (obstructive apneas, Fig. 319-2A), or of respiratory effort (central apneas, Fig. 319-2B) • Hypopnea: A ≥30% reduction in airflow for at least 10 sec during sleep that is accompanied by either a ≥3% desaturation or an arousal (Fig. 319-2C) effort–related arousal (RERA): A partially obstructed breath that does not meet the criteria for hypopnea but provides evidence of increasing inspiratory effort (usually through pleural pressure monitoring) punctuated by an arousal (Fig. 319-2D) breath: A partially obstructed breath, typically within a hypopnea or RERA, identified by a flattened or “scooped-out” inspiratory flow shape (Fig. 319-3) oBStruCtiVe SLeep apnea/hypopnea SynDroMe (oSahS): QuantifiCation anD SeVerity SCaLe • Apnea-hypopnea index (AHI):a Number of apneas plus hypopneas per hour • Respiratory disturbance index (RDI): Number of apneas plus hypopneas plus RERAs per hour of sleep

1	RERAs per hour of sleep OSAHS: AHI of 5–14 events/h OSAHS: AHI of 15–29 events/h OSAHS: AHI of ≥30 events/h aEach level of AHI can be further quantified by level of sleepiness and associated hypoxemia. time asleep relative to time in bed), arousal index (number of cortical arousals per hour of sleep), time in each sleep stage, and periodic limb movement index. OSAHS severity can be further characterized according to the degree of sleep fragmentation associated with respiratory disturbances. Relevant metrics include the frequency of cortical micro-arousals or awakenings per sleep hour, reduction in sleep continuity (low sleep efficiency), reduction of time in deeper stages of sleep (stage N3 and REM sleep) and increases in light sleep (stage N1). The detection of autonomic arousals, such as surges in blood pressure, changes in heart rate, and abnormalities in cardiac rhythm, also provides relevant information on OSAHS severity.

1	Other Laboratory Findings Various imaging studies, including cephalometric radiography, MRI, CT, and fiberoptic endoscopy, can be used to identify anatomic risk factors for OSAHS. Cardiac testing may yield evidence of impaired systolic or diastolic ventricular function or abnormal cardiac structure. Overnight blood pressure monitoring often displays a “non-dipping” pattern (absence of the typical 10-mmHg fall during sleep from blood pressure while awake). Arterial blood gas measurements made during wakefulness are usually normal. Waking hypoxemia or hypercarbia suggests coexisting lung disease or hypoventilation syndrome. Patients with severe nocturnal hypoxemia may have elevated hemoglobin values. A multiple sleep latency test or a maintenance of wakefulness test can be useful in quantifying sleepiness and helping to distinguish OSAHS from narcolepsy.

1	Health Consequences and Comorbidities OSAHS is a major contributor to cardiac, cerebrovascular, and metabolic disorders as well as to premature death. It is the most common medical cause of daytime sleepiness and negatively influences quality of life. This broad range of health effects is attributable to the impact of sleep fragmentation, cortical arousal, and intermittent hypoxemia on vascular, cardiac, metabolic, and neurologic functions. OSAHS-related respiratory events stimulate sympathetic overactivity, leading to acute blood pressure surges during sleep, endothelial damage, and nocturnal as well as daytime hypertension. OSAHS-related hypoxemia also stimulates release of acute-phase proteins and reactive oxygen species that exacerbate insulin resistance and lipolysis and cause an augmented prothrombotic and proinflammatory state. Inspiratory effort against an occluded airway causes large intrathoracic negative pressure swings, altering cardiac preload and afterload and resulting

1	prothrombotic and proinflammatory state. Inspiratory effort against an occluded airway causes large intrathoracic negative pressure swings, altering cardiac preload and afterload and resulting in cardiac remodeling and reduced cardiac function. Hypoxemia and sympathetic-parasympathetic imbalance also may cause electrical remodeling of the heart and myocyte injury.

1	HYPERTENSION OSAHS can raise blood pressure to prehypertensive and hypertensive ranges, increase the prevalence of a non-dipping overnight blood pressure pattern, and increase the risk of resistant hypertension. Elevations in blood pressure are due to augmented sympathetic nervous system activation as well as alterations in the rennin–angiotensin–aldosterone system and fluid balance. Treatment of OSAHS with nocturnal continuous positive airway pressure (CPAP) has been shown to reduce 24-h ambulatory blood pressure. Although the overall impact of CPAP on blood pressure levels is relatively modest (averaging 2–4 mmHg), larger improvements are observed among patients with high AHIs and sleepiness. t. flow snore flown. p. flow t. flowchest n. p. flow abdomen

1	t. flow snore flown. p. flow t. flowchest n. p. flow abdomen FIGURE 319-2 A. Obstructive apnea. There are 30 sec of no airflow, as shown in the nasal pressure (n. p. flow) and thermistor-measured flow (t. flow). Note the presence of chest-abdomen motion, indicating respiratory effort against an occluded airway. B. Central apnea in a patient with Cheyne-Stokes respiration due to congestive heart failure. The flat chest-abdomen tracings indicate the absence of inspiratory effort during the central apneas. C. Hypopnea. Partial obstruction of the pharyngeal airway can limit ventilation, leading to desaturation (a mild decrease in this patient, from 93% to 90%) and arousal. D. Respiratory effort–related arousal (RERA). Minimal flow reduction terminated by an arousal (Ar) without desaturation constitutes a RERA. EEG, electroencephalogram; EOG, electro-oculogram; EKG, electrocardiogram.

1	CARDIOVASCULAR, CEREBROVASCULAR, AND METABOLIC DISEASES Among the SLEEPINESS More than 50% of patients with moderate to severe most serious health consequences of OSAHS is its impact on cardiac OSAHS report daytime sleepiness. Patients with OSAHS symptoms and metabolic functions. Strong epidemiologic evidence indicates that have a twofold increased risk of occupational accidents. Individuals OSAHS significantly increases the risk of coronary artery disease, heart with elevated AHIs are involved in motor vehicle crashes as failure with and without reduced ejection fraction, atrial and ventricu-much as seven times more often than persons with normal AHIs.

1	lar arrhythmias, atherosclerosis and coronary artery disease, stroke, Randomized controlled trials have shown that treatment of OSAHS and diabetes. Treatment of OSAHS has been shown to reduce several with nasal CPAP therapy alleviates sleepiness as measured by either markers of cardiovascular risk, improve insulin resistance, decrease the questionnaire or objective testing. However, the degree of improve- recurrence rate of atrial fibrillation, and improve various outcomes in ment varies widely. Residual sleepiness may be due to several factors, patients with active cardiovascular disease. Large-scale trials are under including suboptimal treatment adherence, insufficient sleep time, way to evaluate the role of OSAHS treatment in reducing cardiac event other sleep disorders, or prior hypoxic-mediated damage in brain rates and in prolonging the survival of patients with cardiac disease. areas involved in alertness. Visceral adipose tissue, whose amounts are increased in patients with

1	damage in brain rates and in prolonging the survival of patients with cardiac disease. areas involved in alertness. Visceral adipose tissue, whose amounts are increased in patients with OSAHS, releases somnogenic cyto kines that may contribute to sleepiness. Thus, even after treatment, it is important to assess and monitor patients for residual sleepiness and to evaluate the necessity of optimizing treatment adherence, improving sleep patterns, and identifying other disorders contributing to sleepiness.

1	QUALITY OF LIFE AND MOOD Reductions in health-related quality of life are common in patients with OSAHS, with the largest decrements on FIGURE 319-3 Example of flow limitation. The inspiratory flow pat-the physical and vitality subscales. Treatment with CPAP often results tern in a patent airway is rounded and peaks in the middle. In con-in improvement in these patient-reported outcomes. Depression, in trast, a partially obstructed airway exhibits an early peak followed by particular symptoms of somatic depression (irritability, fatigue, lack of mid-inspiratory flattening, yielding a scooped-out appearance. energy) is commonly reported in OSAHS.

1	A comprehensive approach to the management of OSAHS is needed to reduce risk factors and comorbidities. The clinician should seek to identify and address lifestyle and behavioral factors as well as comorbidities that may be exacerbating OSAHS. As appropriate, treatment should aim to reduce weight; optimize sleep duration (7–9 hours); regulate sleep schedules (with similar bedtimes and wake times across the week); encourage the patient to avoid sleeping in the supine position; treat nasal allergies; increase physical activity; eliminate alcohol ingestion within 3 h of bedtime; and minimize use of sedating medications. Patients should be counseled to avoid drowsy driving.

1	CPAP is the standard medical therapy with the highest level of evidence for efficacy. Delivered through a nasal or nasal-oral mask, CPAP works as a mechanical splint to hold the airway open, thus maintaining airway patency during sleep. An overnight CPAP titration study, performed either in a laboratory or with a home “autotitrating” device, is required to determine the optimal pressure setting that reduces the number of apneas/hypopneas during sleep, improves gas exchange, and reduces arousals. Rates of adherence to CPAP treatment are highly variable (average, 50–80%) and may be improved with support by a skilled health care team who can address side effects (Table 319-3). Despite the limitations of CPAP, controlled studies have demonstrated its beneficial effect on blood pressure, alertness, mood, and insulin sensitivity. Uncontrolled studies also indicate a favorable effect on cardiovascular outcomes, cardiac ejection fraction, atrial fibrillation recurrence, and mortality risk.

1	Oral appliances for OSAHS work by advancing the mandible, thus opening the airway by repositioning the lower jaw and pulling the tongue forward. These devices generally work better when customized for patient use; maximal adaptation can take several weeks. Efficacy studies show that these devices can reduce the AHI by γ50% in two-thirds of individuals, although these data are based largely on patients with mild OSAHS. Side effects of oral appliances include temporomandibular joint pain and tooth movement. Oral appliances are most often used for treating patients with mild OSAHS or patients who do not tolerate CPAP. However, since adherence to the use of oral appliances sometimes exceeds CPAP adherence, these devices are under investigation for treatment of more severe disease.

1	Upper airway surgery for OSAHS is less effective than CPAP and is mostly reserved for the treatment of patients who snore, have mild OSAHS, and cannot tolerate CPAP. Uvulopalatopharyngoplasty (removal of the uvula and the margin of the soft palate) is the most common surgery and, although results vary greatly, has a success Nasal congestion Provide heated humidification, administer saline/ steroid nasal sprays Claustrophobia Change mask interface (e.g., to nasal prongs), promote habituation (i.e., practice breathing on CPAP while awake) Difficulty exhaling Temporarily reduce pressure, provide bilevel positive airway pressure

1	Difficulty exhaling Temporarily reduce pressure, provide bilevel positive airway pressure Bruised nasal ridge Change mask interface, provide protective padding rate similar to or slightly lower than treatment with oral appliances. 1Upper airway surgery is less effective in severe OSAHS and in obese patients. Success rates may be higher for multilevel surgery (involving more than one site/structure) performed by an experienced surgeon, but the selection of patients is an important factor and relies on careful targeting of culprit areas for surgical resection. Bariatric surgery is an option for obese patients with OSAHS and can improve not only OSAHS but also other obesity-associated health conditions. Other procedures that can decrease snoring but have minimal effects on OSAHS include injection of the soft palate (resulting in stiffening), radiofrequency ablation, laser-assisted uvulopalatoplasty, and palatal implants.

1	Supplemental oxygen can improve oxygen saturation, but there is little evidence that it improves OSAHS symptoms or the AHI.

1	CSA, which is less common than OSAHS, may occur in isolation or, more often, in combination with obstructive events in the form of “mixed” apneas. CSA is often caused by an increased sensitivity to pCO2, which leads to an unstable breathing pattern that manifests as hyperventilation alternating with apnea. A prolonged circulation delay between the pulmonary capillaries and carotid chemoreceptors is also a contributing cause; thus individuals with congestive heart failure are at risk for CSA. With prolonged circulation delay, there is a crescendo-decrescendo breathing pattern known as Cheyne-Stokes respiration (Fig. 319–2B). Other risk factors for CSA include opioid medications (which appear to have a dose-dependent effect on CSA) and hypoxia (e.g., breathing at high altitude). In some individuals, CPAP— particularly at high pressures—seems to induce central apnea; this condition is referred to as complex sleep apnea. Rarely, CSA may be caused by blunted chemosensitivity due to

1	individuals, CPAP— particularly at high pressures—seems to induce central apnea; this condition is referred to as complex sleep apnea. Rarely, CSA may be caused by blunted chemosensitivity due to congenital disorders (congenital central hypoventilation syndrome) or acquired factors. Treatment of CSA is difficult and depends on the underlying cause. Limited data suggest that supplemental oxygen can reduce the frequency of central apneas, particularly in patients with hypoxemia. Cheyne-Stokes respiration is treated by optimizing therapy for heart failure and, in some cases, using CPAP with or without supplemental oxygen. Adaptive servoventilation, a form of ventilatory support that dynamically changes inspiratory support levels across periods of apnea and hypopnea, can minimize large fluctuations in PCO2 that produce central apnea and can be effective for the treatment of CSA.

1	Lung transplantation Elbert P. Trulock Lung transplantation is a therapeutic consideration for many patients with nonmalignant end-stage lung disease, and it prolongs survival and improves quality of life in appropriately selected recipients. Since 1985 almost 40,000 procedures have been recorded worldwide, and 320e since 2009 more than 3000 transplants have been reported annually. The indications span the gamut of lung diseases, but in some respects the distribution of indications differs among countries. According to aggregate international data, the most common indications in the last few years have been chronic obstructive pulmonary disease (COPD), ~29%; idiopathic pulmonary fibrosis (IPF), ~28%; cystic fibrosis (CF), ~16%; α1-antitrypsin deficiency emphysema, ~3.5%; and idiopathic pulmonary arterial hypertension (IPAH), ~3%. Other diseases have made up the balance of primary indications, and retransplantation has accounted for ~3% of procedures.

1	Transplantation should be considered when other therapeutic options have been exhausted and when the patient’s prognosis is expected to improve as a result of the procedure. Survival rates after transplantation can be compared with predictive indices for the patient’s disease, but each patient’s clinical course must be integrated into the assessment as well. Moreover, quality of life is a primary motive for transplantation for many patients, and the prospect of improved quality-adjusted survival is often attractive even if the survival advantage itself may be marginal.

1	Disease-specific consensus guidelines for referring patients for evaluation and for proceeding with transplantation are summarized in Table 320e-1 and are linked to clinical, physiologic, radiographic, and pathologic features that influence the prognosis of the respective diseases. Candidates for lung transplantation are also thoroughly screened for comorbidities that might affect the outcome adversely. Conditions such as systemic hypertension, diabetes mellitus, gastroesophageal reflux, and osteoporosis are not unusual; however, if uncomplicated and adequately managed, they do not disqualify patients from transplantation. The upper age limit is ~70 years at most centers, but the median age of recipients has been increasing steadily over the last decade. In the United States in 2009, 22% of recipients were ≥65 years old.

1	Standard exclusions include HIV infection, chronic active hepatitis B or C infection, uncontrolled or untreatable pulmonary or extrapulmonary infection, uncured malignancy, active cigarette smoking, drug or alcohol dependency, irreversible physical deconditioning, chronic nonadherence with medical care, significant disease of another vital organ (e.g., heart, liver, or kidney), and psychiatric or psychosocial situations that could substantially interfere with post-transplantation management. Other problems that may compromise outcome constitute relative contraindications. Some typical issues are ventilator-dependent respiratory failure, previous thoracic surgical procedures, obesity, and coronary artery disease. Chronic infection with antibiotic-resistant Pseudomonas species, Burkholderia species, Aspergillus species, or nontuberculous mycobacteria is a unique concern in some patients with CF. The potential impact of these and other factors must be judged in the clinical context to

1	species, Aspergillus species, or nontuberculous mycobacteria is a unique concern in some patients with CF. The potential impact of these and other factors must be judged in the clinical context to determine an individual candidate’s suitability for transplantation.

1	Organ allocation policies are influenced by medical, ethical, geographic, and political factors, with systems varying from country to country. Regardless of the system, potential recipients are placed on a waiting list and must be matched for blood group compatibility and, with some latitude, for lung size with an acceptable donor. Most lungs are procured from deceased donors after total brain failure (“brain Any of the following criteria: Hospitalization for exacerbation, with PaCO2 >50 mmHg Pulmonary hypertension or cor pulmonale, despite oxygen therapy FEV1 <20% with either DLCO <20% or diffuse emphysema Referral FEV1 <30% or rapidly declining FEV1 Hospitalization in ICU for exacerbation Increasing frequency of exacerbations Refractory or recurrent pneumothorax Recurrent hemoptysis not controlled by bronchial artery embolization Referral Pathologic or radiographic evidence of UIP, regardless of vital capacity Transplantation Pathologic or radiographic evidence of UIP

1	Referral Pathologic or radiographic evidence of UIP, regardless of vital capacity Transplantation Pathologic or radiographic evidence of UIP Any of the following criteria: DLCO <39% Decrement in FVC ≥10% during 6 months of follow-up Decrease in SpO2 to <88% during a 6-min walk test Honeycombing on HRCT (fibrosis score >2) NYHA functional class III or IV, regardless of therapy Failure of therapy with IV epoprostenol (or equivalent drug) Abbreviations: BODE, body mass index (B), airflow obstruction (O), dyspnea (D), exercise capacity (E); DLCO, diffusing capacity for carbon monoxide; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; HRCT, high-resolution computed tomography; ICU, intensive care unit; NYHA, New York Heart Association; PaCO2, partial pressure of carbon dioxide in arterial blood; SpO2, arterial oxygen saturation by pulse oximetry; UIP, usual interstitial pneumonitis.

1	Source: Summarized from JB Orens et al: J Heart Lung Transplant 25:745, 2006. For BODE index, BR Celli et al: N Engl J Med 350:1005, 2004. death”), but only ~15–20% of brain-death organ donors yield either one or two lungs suitable for transplantation. Lungs from donors after cardiac death have been utilized to a limited extent (~2% of lung donors in the United States in 2009). Recently, ex vivo lung perfusion has been used by some centers to assess donor lungs that are marginal or high-risk for implantation by standard criteria; if the results of ex vivo testing are satisfactory, these lungs have been transplanted successfully.

1	In the United States, a lung allocation scoring system is used to prioritize patients on the waiting list. The lung allocation score (LAS) for a patient is based on the patient’s risk of death during 1 year on the waiting list and the patient’s likelihood of survival for 1 year after transplantation. The LAS can range from 0 to 100, and precedence for transplantation is ranked from highest to lowest scores. Both the lung disease and its severity affect a patient’s LAS; parameters in the LAS must be updated biannually but can be submitted for recalculation whenever the patient’s condition changes. The median LAS for all candidates on the waiting list is usually ~35, but the LAS tends to be higher among patients with IPF and CF than among patients with COPD and IPAH.

1	Under this system in the United States, the median waiting time for transplantation has been ~135 days. The overall death rate on the waiting list has been ~6.5%, but death rates vary substantially with the diagnosis (e.g., COPD, ~3%; IPF, ~7%) and with the LAS (e.g., 40–49, ~7%; 50–59, ~15%; ≥60, ~25%). The indications for transplantation depend not only on the prevalence and natural history of the various lung diseases but also on the LAS typically associated with these diseases. While patients with IPF constitute ~20% of the waiting list, they make up ~34% of recipients because their allocation scores are typically higher than those of patients with other diseases.

1	Bilateral transplantation is mandatory for CF and other forms of bronchiectasis because the risk of spillover infection from a remaining native lung precludes single-lung transplantation. Heart-lung transplantation is obligatory for Eisenmenger syndrome with complex anomalies that cannot readily be repaired in conjunction with lung transplantation and for concomitant end-stage lung and heart disease. However, cardiac replacement is not necessary for cor pulmonale because right ventricular function will recover when pulmonary vascular afterload is normalized by lung transplantation.

1	Either bilateral or single-lung transplantation is an option for other diseases unless there is a special consideration, but bilateral transplantation has been utilized increasingly for most indications. Recently, ~65% of procedures in the United States have been bilateral, and ~70% of transplants for COPD, ~55% of those for IPF, and ~95% of those for IPAH in the international registry have been bilateral. Living-donor lobar transplantation has had a limited role in adult lung transplantation but is now rarely performed. It has been used predominantly for teenagers or young adults with CF and has usually been reserved for patients who were unlikely to survive the wait for a deceased-donor organ.

1	Induction therapy with an antilymphocyte globulin or an interleukin 2 receptor antagonist is utilized by ~55% of centers, and a three-drug maintenance immunosuppressive regimen that includes a calcineurin inhibitor (cyclosporine or tacrolimus), a purine synthesis antagonist (azathioprine or a mycophenolic acid precursor), and prednisone is traditional. Subsequently, other drugs (e.g., sirolimus) may be substituted into the regimen for various reasons. Prophylaxis against Pneumocystis jirovecii pneumonia is standard, and prophylaxis against cytomegalovirus (CMV) infection and fungal infection is part of many protocols. The dose of cyclosporine, tacrolimus, or sirolimus is adjusted by blood-level monitoring. All of these agents are metabolized by the hepatic cytochrome P450 system, and interactions with medications that affect this pathway can significantly alter their clearance and blood level.

1	Routine management focuses on monitoring of the allograft, regulation of immunosuppressive therapy, and expeditious detection of problems or complications. Regular contact with a nurse coordinator, physician follow-up, chest radiography, blood tests, and spirometry are customary, and periodic surveillance bronchoscopies are employed in some programs. If recovery is uncomplicated, lung function rapidly improves and then stabilizes by 3–6 months after transplantation. Subsequently, the variation in spirometric measurements is small, and a sustained decline of ≥10–15% signals a potentially significant problem.

1	OUTCOMES Survival Major registries publish survival rates (Table 320e-2) and other outcomes annually (www.ishlt.org; www.srtr.org). In the international registry, the survival half-life for recipients with IPF is 4.4 years; IPAH, 5 years; COPD, 5.3 years; and CF, 7.5 years. However, age and transplantation procedure have a significant impact on outcome. For recipients 18–59 years of age, the survival half-life is 5–6 years, but this figure decreases to 4.4 years among patients 60–65 years old and to 3.6 years for those >65 years old. Survival rates at >15 years have been significantly higher after bilateral transplantation than after unilateral transplantation for COPD, α1-antitrypsin deficiency emphysema, IPF, and IPAH.

1	The main sources of perioperative mortality include technical complications of the operation, primary graft dysfunction, and infections. Acute rejection and CMV infection are common problems in the first year, but neither is usually fatal. Beyond the first year, chronic rejection and non-CMV infections cause the majority of deaths. Risk factors for mortality have been analyzed in the international and U. S. registries. In these analyses, factors associated with an increased risk of death, especially in the first year after transplantation, have included the following: recipients hospitalized at the time of transplantation; recipients supported by mechanical ventilation, extra-corporeal membrane oxygenation, inotropic drugs, or dialysis at the time of transplantation; and recipients undergoing retransplantation. taBLe 320e-2 reCipient survivaL, By pretranspLantation DiaGnosis (1990–2010) Survival Rate, %

1	taBLe 320e-2 reCipient survivaL, By pretranspLantation DiaGnosis (1990–2010) Survival Rate, % However, other factors have contributed as well. The mortality risk has been higher at centers with <20–30 transplantations per year. Function Regardless of the disease, successful transplantation impressively restores cardiopulmonary function. After bilateral transplantation, pulmonary function tests are typically normal; after unilateral transplantation, a mild abnormality characteristic of the remaining diseased lung is still apparent. Formal exercise testing usually demonstrates some impairment in maximal work rate and maximal oxygen uptake, but few recipients report any limitation to activities of daily living.

1	Quality of Life Both overall and health-related quality-of-life scores are enhanced. With multidimensional profiles, improvements extend across most domains and are sustained longitudinally unless chronic rejection or some other complication develops. Other problems that detract from quality of life include renal dysfunction and drug side effects.

1	Cost The cost of transplantation depends on the health care system, other health care policies, and economic factors that vary from country to country. In the United States in 2011, the average billed charge for the period from 30 days before bilateral lung transplantation until 180 days after discharge from the transplantation admission was $797,300. The total cost included the following charges: all care during 30 days before transplantation, $21,400; organ procurement, $90,300; hospital transplantation admission, $458,500; physician fees during transplantation admission, $56,300; all inpatient and outpatient care for 180 days after discharge, $142,600; and all outpatient drugs (including immunosuppressants) for 180 days after discharge, $28,200. Lung transplantation can be complicated by a variety of problems (Table 320e-3). Aside from predicaments that are unique to transplantation,

1	Lung transplantation can be complicated by a variety of problems (Table 320e-3). Aside from predicaments that are unique to transplantation, Allograft Primary graft dysfunction; anastomotic dehiscence or stenosis; ischemic airway injury with bronchostenosis or bronchomalacia; rejection; infection; recurrence of primary disease (sarcoidosis, lymphangioleiomyomatosis, giant cell interstitial pneumonitis, diffuse panbronchiolitis, pulmonary alveolar proteinosis, Langerhans cell histiocytosis) Gastrointestinal Esophagitis (especially Candida, herpesvirus, or cytomegalovirus [CMV]); gastroparesis; gastroesophageal reflux; diarrhea (Clostridium difficile; medications, especially mycophenolate mofetil and sirolimus); colitis (C. difficile; CMV) side effects and toxicities of immunosuppressive medications can cause 320e-3 new medical problems or aggravate preexisting conditions.

1	Graft Dysfunction Primary graft dysfunction (PGD), an acute lung injury, is a manifestation of multiple potential insults to the donor organ that are inherent in the transplantation process. The principal clinical features are diffuse pulmonary infiltrates and hypoxemia within 72 h of transplantation; however, the presentation can be mimicked by pulmonary venous obstruction, hyperacute rejection, pulmonary edema, and pneumonia.

1	The severity is variable, and a standardized grading system has been established. Up to 50% of lung transplant recipients may have some degree of PGD, and ~10–20% have severe PGD. The treatment follows the conventional supportive paradigm for acute lung injury. Inhalation of nitric oxide and extracorporeal membrane oxygenation have been used in severe cases; retransplantation has also been performed, but when undertaken in the first 30 days this procedure is associated with a poor survival rate (~30% at 1 year). Most recipients with mild PGD recover, but the mortality rate for severe PGD has been ~40–60%. PGD is also associated with longer postoperative ventilator support, longer intensive care unit and hospital stays, higher costs, and excess morbidity, and severe PGD is a risk factor for the later development of chronic rejection (see below).

1	Airway Complications The bronchial blood supply to the donor lung is disrupted during procurement. Bronchial revascularization during transplantation is technically feasible in some cases, but it is not widely practiced. Consequently, after implantation, the donor bronchus is dependent on retrograde bronchial blood flow from the pulmonary circulation and is vulnerable to ischemia. The spectrum of airway problems includes anastomotic necrosis and dehiscence, occlusive granulation tissue, anastomotic or bronchial stenosis, and bronchomalacia. The incidence has been in the range of 7–18%, but the associated mortality rate has been low. These problems usually can be managed bronchoscopically with techniques such as simple endoscopic debridement, laser photoresection, balloon dilation, and bronchial stenting.

1	Rejection Rejection is the main deterrent to higher mediumand long-term survival rates. In this immunologic response to alloantigen recognition, both cell-mediated and antibody-mediated (humoral) cascades can play a role. Cellular rejection is effected by T lymphocyte interactions with donor alloantigens, mainly in the major histocompatibility complex (MHC), whereas humoral rejection is driven by antibodies to donor MHC alloantigens or possibly to non-MHC antigens on epithelial or endothelial cells. Rejection is often categorized as acute or chronic without reference to the underlying mechanism. Acute rejection is cell-mediated, and its incidence is highest in the first 6–12 months after transplantation. In contrast, chronic rejection generally emerges later, and both alloimmune and non-alloimmune fibroproliferative reactions may contribute to its pathogenesis.

1	Acute Cellular Rejection With current immunosuppressive regimens, ~30–40% of recipients experience acute rejection in the first year. Acute cellular rejection (ACR) can be clinically silent or can be manifested by nonspecific symptoms or signs that may include cough, low-grade fever, dyspnea, hypoxemia, inspiratory crackles, interstitial infiltrates, and declining lung function; however, clinical impressions are not reliable. The diagnosis is confirmed by transbronchial biopsies showing the characteristic lymphocytic infiltrates around arterioles or bronchioles, and a standardized pathologic scheme is used to grade the biopsies.

1	Minimal ACR on a surveillance biopsy in a clinically stable recipient is often left untreated, but higher grades generally are treated regardless of the clinical situation. Treatment usually includes a short course of high-dose glucocorticoids and adjustment of the maintenance immunosuppressive regimen. Most episodes respond to this approach; however, more intensive therapy is sometimes necessary for persistent or recurrent episodes.

1	Chronic Rejection This complication is the main impediment to longterm survival and is the source of substantial morbidity because of its impact on lung function and quality of life. Clinically, chronic rejection is characterized physiologically by airflow limitation and pathologically by bronchiolitis obliterans; the process is designated bronchiolitis obliterans syndrome (BOS). Transbronchial biopsies are relatively insensitive for detecting bronchiolitis obliterans, and pathologic confirmation is not required for diagnosis. Thus, after other causes of graft dysfunction have been excluded, the diagnosis of BOS is based primarily on a sustained decrement (≥20%) in forced expiratory volume in 1 s (FEV1), although smaller declines in FEV1 (≥10%) or in midexpiratory flow rate (FEF ) may presage BOS. Spirometric cri teria for diagnosis and staging of BOS have been standardized.

1	The prevalence of BOS approaches 50% by 5 years after transplantation. Antecedent ACR is the main risk factor, but PGD, CMV pneumonitis, other community-acquired respiratory viral infections, and gastroesophageal reflux have been implicated as well. BOS can present acutely and imitate infectious bronchitis, or it can manifest as an insidious decline in lung function. The chest radiograph is typically unchanged; CT may reveal mosaic perfusion, air trapping, ground-glass opacities, or bronchiolectasis. Bronchoscopy is indicated to rule out other processes, but transbronchial biopsies identify bronchiolitis obliterans in a minority of cases.

1	BOS usually is treated with augmented immunosuppression, but there is no consensus about therapy. Strategies include changes in the maintenance drug regimen, including the addition of azithromycin, antilymphocyte globulin, photopheresis, and total lymphoid irradiation. Although therapy may stabilize lung function, the overall results of treatment have been disappointing; the median survival period after onset has been ~3–4 years. Retransplantation is a consideration if clinical circumstances and other comorbidities are not prohibitive, but survival rates have been inferior to those with primary transplantation.

1	Humoral Rejection Consensus on the role of antibody-mediated rejection is still evolving. Hyperacute rejection is caused by preformed HLA antibodies in the recipient, but it is minimized by pretransplantation antibody screening coupled with virtual or direct cross-matching with any potential donor. Donor-specific HLA antibodies develop after transplantation in up to 50% of recipients, and their presence has been associated with an increased risk of both ACR and BOS and with poorer overall survival. However, the mechanisms by which these antibodies could contribute to ACR or BOS or could otherwise be detrimental have not been unraveled. Formal criteria for antibody-mediated rejection have been defined for renal transplantation, but few cases in lung transplantation fulfill these criteria. Nonetheless, episodes of acute lung allograft dysfunction occasionally have been attributed directly to antibody-mediated injury. If treatment is indicated, potential therapies include plasmapheresis

1	Nonetheless, episodes of acute lung allograft dysfunction occasionally have been attributed directly to antibody-mediated injury. If treatment is indicated, potential therapies include plasmapheresis and administration of IV immune globulin, rituximab, bortezomib, or eculizumab.

1	Infection The lung allograft is especially susceptible to infection, which has been one of the leading causes of death in recipients. In addition to a blunted immune response from immunosuppressive drugs, other normal defenses are compromised: the cough reflex is diminished, and mucociliary clearance is impaired in the transplanted lung. The spectrum of infections includes both opportunistic and non-opportunistic pathogens. Bacterial bronchitis or pneumonia can occur at any time but is very common in the perioperative period. Later, bronchitis occurs frequently in recipients with BOS, and Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus is often the culprit.

1	CMV is the most common cause of viral infection. Although gastroenteritis, colitis, and hepatitis can occur, CMV viremia and CMV pneumonia are the main illnesses. Most episodes occur in the first 6 months, and treatment with ganciclovir is effective unless resistance develops. Other community-acquired viruses, such as influenza, parainfluenza, and respiratory syncytial viruses, also contribute to respiratory complications. The most problematic fungal infections are caused by Aspergillus species. The spectrum encompasses simple pulmonary colonization, tracheobronchitis, invasive pulmonary aspergillosis, and disseminated aspergillosis, and the clinical scenario dictates treatment.

1	Other Complications Other potential complications are listed in Table 320e-3. Many of them are related to side effects or toxicities of immunosuppressive drugs. Management of these general medical problems is guided by standard practices, but the complex milieu of transplantation requires close collaboration and good communication among health care providers.

1	Approach to the Patient with Critical Illness John P. Kress, Jesse B. Hall The care of critically ill patients requires a thorough understanding of pathophysiology and centers initially on the resuscitation of patients at the extremes of physiologic deterioration. This resuscitation is often fast-paced and occurs early, without a detailed awareness of the patient’s chronic medical problems. While physiologic stabilization is taking place, intensivists attempt to gather important background med-ical information to supplement the real-time assessment of the patient’s current physiologic conditions. Numerous tools are available to assist intensivists in the accurate assessment of pathophysiology and manage-ment of incipient organ failure, offering a window of opportunity for diagnosing and treating underlying disease(s) in a stabilized patient. Indeed, the use of invasive interventions such as mechanical ventilation and renal replacement therapy is commonplace in the intensive care unit

1	underlying disease(s) in a stabilized patient. Indeed, the use of invasive interventions such as mechanical ventilation and renal replacement therapy is commonplace in the intensive care unit (ICU). An appreciation of the risks and benefits of such aggressive and often invasive interventions is vital to ensure an optimal outcome. Nonetheless, intensivists must recognize when a patient’s chances for recovery are remote or nonexistent and must counsel and comfort dying patients and their significant others. Critical care physicians often must redirect the goals of care from resuscitation and cure to comfort when the resolution of an underlying illness is not possible. ASSESSMENT OF ILLNESS SEVERITY 321 SEC TIon 1 RESPIRAToRy CRITICAl CARE

1	Approach to the Patient with Critical Illness

1	In the ICU, illnesses are frequently categorized by degree of severity. Numerous severity-of-illness (SOI) scoring systems have been developed and validated over the past three decades. Although these scoring systems have been validated as tools to assess populations of critically ill patients, their utility in predicting individual patient outcomes is not clear. SOI scoring systems are important for defining populations of critically ill patients. Such systematic scoring allows effective comparison of groups of patients enrolled in clinical trials. In verifying a purported benefit of therapy, investigators must be confident that different groups involved in a clinical trial have similar illness severities. SOI scores are also useful in guiding hospital administrative policies, directing the allocation of resources such as nursing and ancillary care and assisting in assessments of quality of ICU care over time. Scoring system validations are based on the premise that age, chronic

1	the allocation of resources such as nursing and ancillary care and assisting in assessments of quality of ICU care over time. Scoring system validations are based on the premise that age, chronic medical illnesses, and derangements from normal physiology are associated with increased mortality rates. All existing SOI scoring systems are derived from patients who have already been admitted to the ICU.

1	SOI scoring systems cannot be used to predict survival in individual patients. No established scoring systems that purport to direct clinicians’ decision-making regarding criteria for admission to an ICU are available, although such models are being developed. Thus the use of SOI scoring systems to direct therapy and clinical decision-making cannot be recommended at present. Instead, these tools should be used as a source of important data to complement clinical bedside decision-making. The most commonly utilized scoring systems are the APACHE (Acute Physiology and Chronic Health Evaluation) and the SAPS (Simplified Acute Physiology Score) systems.

1	The most commonly utilized scoring systems are the APACHE (Acute Physiology and Chronic Health Evaluation) and the SAPS (Simplified Acute Physiology Score) systems. The APACHE II system is the most commonly used SOI scoring system in North America. Age, type of ICU admission (after elective surgery vs. nonsurgical or after emergency surgery), chronic health problems, and 12 physiologic variables (the worst values for each in the first 24 h after ICU admission) are used to derive a score. The predicted hospital mortality rate is derived from a formula that takes into account the APACHE II score, the need for emergency surgery, and a weighted, disease-specific diagnostic category (Table 321–1). The relationship between APACHE II score and mortality risk is illustrated in Fig. 321-1. Updated versions of the APACHE scoring system (APACHE III and APACHE IV) have been published.

1	The SAPS II score, used more frequently in Europe than in the United States, was derived in a manner similar to the APACHE score. This score is not disease specific but rather incorporates three underlying disease variables: AIDS, metastatic cancer, and hematologic malignancy. SAPS 3, which utilizes a 1-h rather than a 24-h window for measuring physiologic derangement scores, was developed in 2005. See also Chap. 324.

1	Shock, a common condition necessitating ICU admission or occurring in the course of critical care, is defined by the presence of multisystem end-organ hypoperfusion. Clinical indicators include reduced mean arterial pressure (MAP), tachycardia, tachypnea, cool skin and extremities, acute altered mental status, and oliguria. Hypotension is usually, though not always, present. The end result of multiorgan hypoperfusion is tissue hypoxia, often with accompanying lactic acidosis. Since the MAP is the product of cardiac output and systemic vascular resistance (SVR), reductions in blood pressure can be caused by decreases in cardiac output and/or SVR. Accordingly, once shock is contemplated, the initial evaluation of a hypotensive patient should include an early bedside assessment of the adequacy of cardiac output (Fig. 321-2). Clinical evidence of diminished cardiac output includes a narrow pulse pressure—a marker that correlates with stroke volume— and cool extremities with delayed

1	adequacy of cardiac output (Fig. 321-2). Clinical evidence of diminished cardiac output includes a narrow pulse pressure—a marker that correlates with stroke volume— and cool extremities with delayed capillary refill. Signs of increased cardiac output include a widened pulse pressure (particularly with a reduced diastolic pressure), warm extremities with bounding pulses, and rapid capillary refill. If a hypotensive patient has clinical signs of increased cardiac output, it can be inferred that the reduced blood pressure is from decreased SVR.

1	In hypotensive patients with signs of reduced cardiac output, an assessment of intravascular volume status is appropriate. A hypotensive patient with decreased intravascular volume status may have a history suggesting hemorrhage or other volume losses (e.g., vomiting, diarrhea, polyuria). Although evidence of a reduced jugular venous pressure (JVP) is often sought, static measures of right atrial pressure do not predict fluid responsiveness reliably; the change in right atrial pressure as a function of spontaneous respiration is a better predictor of fluid responsiveness (Fig. 321-3). Patients with fluid-responsive (i.e., hypovolemic) shock also may manifest large changes in pulse pressure as a function of respiration during mechanical ventilation (Fig. 321-4). A hypotensive patient with increased intravascular volume and cardiac dysfunction may have S3 and/or S4 gallops on examination, increased JVP, extremity edema, and crackles on lung auscultation. The chest x-ray may show

1	with increased intravascular volume and cardiac dysfunction may have S3 and/or S4 gallops on examination, increased JVP, extremity edema, and crackles on lung auscultation. The chest x-ray may show cardiomegaly, widening of the vascular pedicle, Kerley B lines, and pulmonary edema. Chest pain and electrocardiographic changes consistent with ischemia may be noted (Chap. 326).

1	In hypotensive patients with clinical evidence of increased cardiac output, a search for causes of decreased SVR is appropriate. The If patient is admitted after elective surgery

1	If patient is admitted after elective surgery If patient is admitted after emergency surgery or for reason other than after elective surgery aThe APACHE II score is the sum of the acute physiology score (vital signs, oxygenation, laboratory values), the Glasgow coma score, age, and chronic health points. The worst values during the first 24 h in the ICU should be used. bGlasgow coma score (GCS) = eye-opening score + verbal (intubated or nonintubated) score + motor score. cFor GCS component of acute physiology score, subtract GCS from 15 to obtain points assigned. dHepatic: cirrhosis with portal hypertension or encephalopathy; cardiovascular: class IV angina (at rest or with minimal self-care activities); pulmonary: chronic hypoxemia or hypercapnia, polycythemia, ventilator dependence; renal: chronic peritoneal or hemodialysis; immune: immunocompromised host.

1	Abbreviations: (A − a) DO2, alveolar-arterial oxygen difference; FIO2, fraction of inspired oxygen; PaO2, partial pressure of oxygen; WBC, white blood cell count. Mortality rate, % FIGURE 321-1 APACHE II survival curve. Blue, nonoperative; green, (See also Chap. 323) During the initial resuscitation of patients in postoperative. shock, principles of advanced cardiac life support should be followed. most common cause of high-cardiac-output hypotension is sepsis (Chap. 325). Other causes include liver failure, severe pancreatitis, burns and other trauma that elicit the systemic inflammatory response syndrome (SIRS), anaphylaxis, thyrotoxicosis, and peripheral arteriovenous shunts. In summary, the most common categories of shock are hypovolemic, cardiogenic, and high-cardiac-output with decreased SVR (highoutput hypotension). Certainly more than one category can occur simultaneously (e.g., hypovolemic and septic shock).

1	The initial assessment of a patient in shock should take only a few minutes. It is important that aggressive resuscitation is instituted on the basis of the initial assessment, particularly since early resuscitation from septic and cardiogenic shock may improve survival (see below). If the initial bedside assessment yields equivocal or confounding data, more objective assessments such as echocardiography and/or invasive vascular monitoring may be useful. The goal of early resuscitation is to reestablish adequate tissue perfusion and thus to prevent or minimize 5-9 15-19 25-29 35+ end-organ injury. APACHE II Score

1	Inotropes, afterload reduction Heart is “full” (cardiogenic shock) Evaluate for myocardial ischemia Cold, clammy extremities Warm, bounding extremities High cardiac output No improvement What does not fit? Adrenal crisis, right heart syndrome, pericardial disease Consider echocardiogram, invasive vascular monitoring Consider echocardiogram, invasive vascular monitoring Septic shock, liver failure Low cardiac output JVP, crackles JVP, orthostasis Intravenous fluids Antibiotics, EGDT May convert to SHOCK Heart is “empty” (hypovolemic shock) FIGURE 321-2 Approach to the patient in shock. EGDT, early goal-directed therapy; JVP, jugular venous pulse.

1	As such patients may be obtunded and unable to protect the airway, an early assessment of the airway is mandatory. Early intubation and mechanical ventilation often are required. Reasons for the institution of endotracheal intubation and mechanical ventilation include acute hypoxemic respiratory failure and ventilatory failure, which frequently accompany shock. Acute hypoxemic respiratory failure may occur in patients with cardiogenic shock and pulmonary edema (Chap. 326) as well as in those who are in septic shock with pneumonia or acute respiratory distress syndrome (ARDS) (Chaps. 322 and 325). Ventilatory failure often occurs as a consequence of an increased load on the respiratory system in the form of acute metabolic (often lactic) acidosis or decreased lung compliance due to pulmonary edema. Inadequate perfusion to respiratory muscles in the setting of shock may be another reason for early intubation and mechanical ventilation. Normally, the respiratory muscles receive a very

1	edema. Inadequate perfusion to respiratory muscles in the setting of shock may be another reason for early intubation and mechanical ventilation. Normally, the respiratory muscles receive a very small percentage of the cardiac output. However, in patients who are in shock with respiratory distress, the percentage of cardiac output dedicated to respiratory muscles may increase by tenfold or more. Lactic acid production from inefficient respiratory muscle activity presents an additional ventilatory load.

1	Mechanical ventilation may relieve the work of breathing and 1731 allow redistribution of a limited cardiac output to other vital organs. Patients demonstrate respiratory distress by an inability to speak full sentences, accessory use of respiratory muscles, paradoxical abdominal muscle activity, extreme tachypnea (>40 breaths/min), and decreasing respiratory rate despite an increasing drive to breathe. When patients with shock are treated with mechanical ventilation, a major goal is for the ventilator to assume all or the majority of the work of breathing, facilitating a state of minimal respiratory muscle work. With the institution of mechanical ventilation for shock, further declines in MAP are frequently seen. The reasons include impeded venous return from positive-pressure ventilation, reduced endogenous catecholamine secretion once the stress associated with respiratory failure abates, and the actions of drugs used to facilitate endotracheal intubation (e.g., propofol, opiates).

1	reduced endogenous catecholamine secretion once the stress associated with respiratory failure abates, and the actions of drugs used to facilitate endotracheal intubation (e.g., propofol, opiates). Accordingly, hypotension should be anticipated during endotracheal intubation. Because many of these patients may be fluid responsive, IV volume administration should be considered. Figure 321-2 summarizes the diagnosis and treatment of different types of shock For further discussion of individual forms of shock, see Chaps. 324, 325, and 326.

1	Respiratory failure is one of the most common reasons for ICU admission. In some ICUs, ≥75% of patients require mechanical ventilation during their stay. Respiratory failure can be categorized mechanistically on the basis of pathophysiologic derangements in respiratory function. TYPE I: ACUTE HYPOXEMIC RESPIRATORY FAILURE

1	This type of respiratory failure occurs with alveolar flooding and subsequent intrapulmonary shunt physiology. Alveolar flooding may be a consequence of pulmonary edema, pneumonia, or alveolar hemorrhage. Pulmonary edema can be further categorized as occurring due to elevated pulmonary microvascular pressures, as seen in heart failure and intravascular volume overload or ARDS (“low-pressure pulmonary edema,” Chap. 322). This syndrome is defined by acute onset (≤1 week) of bilateral opacities on chest imaging that are not fully explained by cardiac failure or fluid overload and of shunt physiology requiring positive end-expiratory pressure (PEEP). Type I respiratory failure occurs in clinical settings such as sepsis, gastric aspiration, pneumonia, near-drowning, multiple blood transfusions, and pancreatitis. The mortality rate among patients with ARDS was traditionally very high (50–70%), although changes in patient care have led to mortality rates closer to 30% (see below).

1	For many years, physicians have suspected that mechanical ventilation of patients with ARDS may propagate lung injury. Cyclical collapse and reopening of alveoli may be partly responsible for this adverse effect. As seen in Fig. 321-5, the pressure-volume relationship of the lung in ARDS is not linear. Alveoli may collapse at very low lung volumes. Animal studies have suggested that stretching and overdistention of injured alveoli during mechanical ventilation can further injure the lung. Concern over this alveolar overdistention, termed ventilator-induced “volutrauma,” led to a multicenter, randomized, prospective trial comparing traditional ventilator strategies for ARDS (large tidal volume: 12 mL/kg of ideal body weight) with a low tidal volume (6 mL/kg of ideal body weight). This study showed a dramatic reduction in mortality rate in the low-tidal-volume group from that in the high-tidalvolume group (31% versus 39.8%). In addition, a “fluid-conservative” management strategy

1	study showed a dramatic reduction in mortality rate in the low-tidal-volume group from that in the high-tidalvolume group (31% versus 39.8%). In addition, a “fluid-conservative” management strategy (maintaining a low central venous pressure [CVP] or pulmonary capillary wedge pressure

1	Approach to the Patient with Critical Illness This form of respiratory failure results from lung atelectasis. Because atelectasis occurs so commonly in the perioperative period, this form is also called periop erative respiratory failure. After general anesthesia, decreases in functional residual capacity lead to collapse of depen dent lung units. Such atelectasis can be treated by frequent changes in position, FIGURE 321-4 Pulse pressure change during mechanical ventilation in a patient with shock chest physiotherapy, upright positioning, whose cardiac output will increase in response to intravenous fluid administration. The pulse pres- and control of incisional and/or abdomi nal pain. Noninvasive positive-pressure with septic shock.

1	This type of respiratory failure is a consequence of alveolar hypoventilation and results from the inability to eliminate carbon dioxide effectively. Mechanisms are categorized by impaired central nervous system (CNS) drive to breathe, impaired strength with failure of neuromuscular function in the respiratory system, and increased load(s) on the respiratory system. Reasons for diminished CNS drive to breathe include drug overdose, brainstem injury, sleep-disordered breathing, and severe hypothyroidism. Reduced strength can be due to impaired neuromuscular transmission (e.g., myasthenia gravis, Guillain-Barré syndrome, amyotrophic lateral sclerosis) or respiratory muscle weakness (e.g., myopathy, electrolyte derangements, fatigue).

1	The overall load on the respiratory system can be subclassified into resistive loads (e.g., bronchospasm), loads due to reduced lung compliance (e.g., alveolar edema, atelectasis, intrinsic positive end-expiratory pressure [auto-PEEP]—see below), loads due to reduced chest wall compliance (e.g., pneumothorax, pleural effusion, abdominal distention), and loads due to increased minute ventilation requirements (e.g., pulmonary embolus with increased dead-space fraction, sepsis).

1	The mainstays of therapy for type II respiratory failure are directed at reversing the underlying cause(s) of ventilatory failure. Noninvasive positive-pressure ventilation with a tight-fitting facial or nasal mask, with avoidance of endotracheal intubation, often stabilizes these patients. This approach has been shown to be beneficial in treating patients with exacerbations of chronic obstructive pulmonary disease; it has been tested less extensively in other kinds of respiratory failure but may be attempted nonetheless in the absence of contraindications (hemodynamic instability, inability to protect the airway, respiratory arrest). ventilation may also be used to reverse regional atelectasis.

1	ventilation may also be used to reverse regional atelectasis. This form results from hypoperfusion of respiratory muscles in patients in shock. Normally, respiratory muscles consume <5% of total cardiac output and oxygen delivery. Patients in shock often experience respiratory distress due to pulmonary edema (e.g., in cardiogenic shock), lactic acidosis, and anemia. In this setting, up to 40% of cardiac output may be distributed to the respiratory muscles. Intubation and mechanical ventilation can allow redistribution of the cardiac output away from the respiratory muscles and back to vital organs while the shock is treated.

1	(See also Chap. 323) Whereas a thorough understanding of the pathophysiology of respiratory failure is essential for optimal patient care, recognition of a patient’s readiness to be liberated from mechanical ventilation is likewise important. Several studies have shown that daily spontaneous breathing trials can identify patients who are ready for extubation. Accordingly, all intubated, mechanically ventilated patients should undergo daily screening of respiratory function. If oxygenation is stable (i.e., PaO2/FIO2 [partial pressure of oxygen/fraction of inspired oxygen] >200 and PEEP ≤5 cmH2O), cough and airway reflexes are intact, and no vasopressor agents or sedatives are being administered, the patient has passed the screening test and should undergo a spontaneous breathing trial. This trial consists of a period of breathing through the endotracheal tube without ventilator support (both continuous positive airway pressure [CPAP] of 5 cmH2O and an open T-piece breathing system can

1	trial consists of a period of breathing through the endotracheal tube without ventilator support (both continuous positive airway pressure [CPAP] of 5 cmH2O and an open T-piece breathing system can be used) for 30–120 min. The spontaneous breathing trial is declared a failure and stopped if any of the following occur: (1) respiratory rate >35/min for >5 min, (2) O2 saturation <90%, (3) heart rate >140/min or a 20% increase or decrease from baseline, (4) systolic blood pressure <90 mmHg or >180 mmHg, or (5) increased anxiety or diaphoresis. If, at the end of the spontaneous breathing trial, none of the above events has occurred and the ratio of the respiratory rate and tidal volume in liters (f/VT) is <105, the patient can be extubated. Such protocol-driven approaches to patient care can have an important impact on the duration of mechanical ventilation and ICU stay. In spite of such a careful approach to liberation from mechanical ventilation, up to 10% of patients develop respiratory

1	an important impact on the duration of mechanical ventilation and ICU stay. In spite of such a careful approach to liberation from mechanical ventilation, up to 10% of patients develop respiratory distress after extubation and may require resumption of mechanical ventilation. Many of these patients will require reintubation. The use

1	Volume, mL of noninvasive ventilation in patients in whom extubation fails may be associated with worse outcomes than are obtained with immediate reintubation. Mechanically ventilated patients frequently require sedatives and analgesics. Opiates are the mainstay of therapy for pain control in mechanically ventilated patients. After adequate pain control has been Pressure, cmH2O ensured, additional indications for sedation include anxiolysis; treat-

1	FIGURE 321-5 Pressure-volume relationship in the lungs of a ment of subjective dyspnea; psychosis; facilitation of nursing care; patient with acute respiratory distress syndrome (ARDS). At reduction of autonomic hyperactivity, which may precipitate myocarthe lower inflection point, collapsed alveoli begin to open and lung dial ischemia; and reduction of total O2 consumption (VO2). compliance changes. At the upper deflection point, alveoli become Neuromuscular blocking agents are occasionally needed to facilitate overdistended. The shape and size of alveoli are illustrated at the top mechanical ventilation in patients with profound ventilator dyssynof the figure. chrony despite optimal sedation, particularly in the setting of severe ARDS. Use of these agents may result in prolonged weakness—a myopathy known as the postparalytic syndrome. For this reason, neuromuscular blocking agents typically are used as a last resort when aggressive sedation fails to achieve patient-ventilator

1	myopathy known as the postparalytic syndrome. For this reason, neuromuscular blocking agents typically are used as a last resort when aggressive sedation fails to achieve patient-ventilator synchrony. Because neuromuscular blocking agents result in pharmacologic paralysis without altering mental status, sedative-induced amnesia is mandatory when these agents are administered.

1	Amnesia can be achieved reliably with benzodiazepines such as lorazepam and midazolam as well as the the IV anesthetic agent propofol. Outside the setting of pharmacologic paralysis, few data support the idea that amnesia is mandatory in all patients who require intubation and mechanical ventilation. Since many of these critically patients have impaired hepatic and renal function, sedatives and opiates may accumulate when given for prolonged periods. A nursing protocol– driven approach to sedation of mechanically ventilated patients or daily interruption of sedative infusions paired with daily spontaneous breathing trials has been shown to prevent excessive drug accumulation and shorten the duration of both mechanical ventilation and ICU stay.

1	Multiorgan system failure, which is commonly associated with critical illness, is defined by the simultaneous presence of physiologic dysfunction and/or failure of two or more organs. Typically, this syndrome occurs in the setting of severe sepsis, shock of any kind, severe inflammatory conditions such as pancreatitis, and trauma. The fact that multiorgan system failure occurs commonly in the ICU is a testament to our current ability to stabilize and support single-organ failure. The ability to support single-organ failure aggressively (e.g., by mechanical ventilation or by renal replacement therapy) has reduced rates of early mortality in critical illness. As a result, it is uncommon for critically ill patients to die in the initial stages of resuscitation. Instead, many patients succumb to critical illness later in the ICU stay, after the initial presenting problem has been stabilized.

1	Although there is debate regarding specific definitions of organ failure, several general principles governing the syndrome of multiorgan system failure apply. First, organ failure, no matter how it is defined, must persist beyond 24 h. Second, mortality risk increases with the accrual of failing organs. Third, the prognosis worsens with increased duration of organ failure. These observations remain true across various critical care settings (e.g., medical versus surgical). SIRS is a common basis for multiorgan system failure. Although infection is a common cause of SIRS, “sterile” triggers such as pancreatitis, trauma, and burns often are invoked to explain multiorgan system failure.

1	Because respiratory failure and circulatory failure are common in critically ill patients, monitoring of the respiratory and cardiovascular systems is undertaken frequently. Evaluation of respiratory gas exchange is routine in critical illness. The “gold standard” remains arterial blood-gas analysis, in which pH, PaO2, partial pressure of carbon dioxide (PCO2), and O2 saturation are measured directly. With arterial blood-gas analysis, the two main functions of the lung—oxygenation of arterial blood and elimination of CO2—can be assessed directly. In fact, the blood pH, which has a profound effect on the drive to breathe, can be assessed only by such sampling. Although sampling of arterial blood is generally safe, it may be painful and cannot provide continuous information. In light of these limitations, noninvasive monitoring of respiratory function is often employed.

1	The most commonly utilized noninvasive technique for monitoring respiratory function, pulse oximetry takes advantage of differences in the absorptive properties of oxygenated and deoxygenated hemoglobin. At wavelengths of 660 nm, oxyhemoglobin reflects light more effectively than does deoxyhemoglobin, whereas the reverse is true in the infrared spectrum (940 nm). A pulse oximeter passes both wavelengths of light through a perfused digit such as a finger, and the relative intensity of light transmission at these two wavelengths is recorded. From this information, the relative percentage of oxyhe-1733 moglobin is derived. Since arterial pulsations produce phasic changes in the intensity of transmitted light, the pulse oximeter is designed to detect only light of alternating intensity. This feature allows distinction of arterial and venous blood O2 saturations.

1	Respiratory system mechanics can be measured in patients during mechanical ventilation (Chap. 323). When volume-controlled modes of mechanical ventilation are used, accompanying airway pressures can easily be measured as long as the patient is passive. The peak airway pressure is determined by two variables: airway resistance and respiratory system compliance. At the end of inspiration, inspiratory flow can be stopped transiently. This end-inspiratory pause (plateau pressure) is a static measurement, affected only by respiratory system compliance and not by airway resistance. Therefore, during volume-controlled ventilation, the difference between the peak (airway resistance + respiratory system compliance) and plateau (respiratory system compliance only) airway pressures provides a quantitative assessment of airway resistance. Accordingly, during volume-controlled ventilation, patients with increases in airway resistance typically have increased peak airway pressures as well as

1	quantitative assessment of airway resistance. Accordingly, during volume-controlled ventilation, patients with increases in airway resistance typically have increased peak airway pressures as well as abnormally high gradients between peak and plateau airway pressures (typically >15 cmH2O) at an inspiratory flow rate of 1 L/sec. The compliance of the respiratory system is defined by the change in pressure of the respiratory system per unit change in volume.

1	The respiratory system can be divided into two components: the lungs and the chest wall. Normally, respiratory system compliance is ~100 mL/cmH2O. Pathophysiologic processes such as pleural effusions, pneumothorax, and increased abdominal girth all reduce chest wall compliance. Lung compliance may be reduced by pneumonia, pulmonary edema, interstitial lung disease, or auto-PEEP. Accordingly, patients with abnormalities in compliance of the respiratory system (lungs and/or chest wall) typically have elevated peak and plateau airway pressures but a normal gradient between these two pressures. Auto-PEEP occurs when there is insufficient time for emptying of alveoli before the next inspiratory cycle. Since the alveoli have not decompressed completely, alveolar pressure remains positive at the end of exhalation (functional residual capacity). This phenomenon results most commonly from critical narrowing of distal airways in disease processes such as asthma and COPD. Auto-PEEP with

1	at the end of exhalation (functional residual capacity). This phenomenon results most commonly from critical narrowing of distal airways in disease processes such as asthma and COPD. Auto-PEEP with resulting alveolar overdistention may result in diminished lung compliance, reflected by abnormally increased plateau airway pressures. Modern mechanical ventilators allow breath-to-breath display of pressure and flow, permitting detection of problems such as patient-ventilator dyssynchrony, airflow obstruction, and auto-PEEP (Fig. 321–6).

1	Oxygen delivery (QO2) is a function of cardiac output and the content of O2 in the arterial blood (CaO2). The CaO2 is determined by the hemoglobin concentration, the arterial hemoglobin saturation, and dissolved O2 not bound to hemoglobin. For normal adults: QO2 = 50 dL/min × (1.39 × 15 g/dL [hemoglobin concentration] × 1.0 [hemoglobin % saturation] + 0.0031 × 100 [PaO2]) = 50 dL/min (cardiac output) × 21.6 mL O2 per dL blood (CaO2) It is apparent that nearly all of the O2 delivered to tissues is bound to hemoglobin and that the dissolved O2 (PaO2) contributes very little to O2 content in arterial blood or to O2 delivery. Normally, the content of O2 in mixed venous blood (C–vO2) is 15.76 mL/dL since the mixed venous blood is 75% saturated. Therefore, the normal tissue extraction ratio for O2 is CaO2 – C–vO2/CaO2 ([21.16–15.76]/21.16) or ~25%. A pulmonary artery catheter allows measurements of O2 delivery and the O2 extraction ratio.

1	Information on the mixed venous O2 saturation allows assessment of global tissue perfusion. A reduced mixed venous O2 saturation may be caused by inadequate cardiac output, reduced hemoglobin concentration, and/or reduced arterial O2 saturation. An abnormally high VO2may also lead to a reduced mixed venous O2 saturation if O2 delivery is Approach to the Patient with Critical Illness 1.2 -1.2 FIGURE 321-6 Increased airway resistance with auto-PEEP. The top waveform (airway pressure vs. time) shows a large difference between the peak airway pressure (80 cmH2O) and the plateau airway pressure (20 cmH2O). The bottom waveform (flow vs. time) demonstrates airflow throughout expiration (reflected by the flow tracing on the negative portion of the abscissa) that persists up to the next inspiratory effort. not concomitantly increased. Abnormally increased VO2 in peripheral tissues may be caused by problems such as fever, agitation, shivering, and thyrotoxicosis.

1	not concomitantly increased. Abnormally increased VO2 in peripheral tissues may be caused by problems such as fever, agitation, shivering, and thyrotoxicosis. The pulmonary artery catheter originally was designed as a tool to guide therapy for acute myocardial infarction but has been used in the ICU for evaluation and treatment of a variety of other conditions, such as ARDS, septic shock, congestive heart failure, and acute renal failure. This device has never been validated as a tool associated with reduction in morbidity and mortality rates. Indeed, despite numerous prospective studies, mortality or morbidity rate benefits associated with use of the pulmonary artery catheter have never been reported in any setting. Accordingly, it appears that routine pulmonary artery catheterization is not indicated as a means of monitoring and characterizing circulatory status in most critically ill patients.

1	Static measurements of circulatory parameters (e.g., CVP, PCWP) do not provide reliable information on the circulatory status of critically ill patients. In contrast, dynamic assessments measuring the impact of breathing on the circulation are more reliable predictors of responsiveness to IV fluid administration. A decrease in CVP of >1 mmHg during inspiration in a spontaneously breathing patient may predict an increase in cardiac output after IV fluid administration. Similarly, a changing pulse pressure during mechanical ventilation has been shown to predict an increase in cardiac output after IV fluid administration in patients with septic shock.

1	(See also Chap. 325) Sepsis, defined as the presence of SIRS in the setting of known or suspected infection, is a significant problem in the care of critically ill patients, who often progress to severe sepsis with the failure of one or more organs. Sepsis is the leading cause of death in noncoronary ICUs in the United States, with case rates expected to increase as the population ages and a higher percentage of people are vulnerable to infection.

1	Many therapeutic interventions in the ICU are invasive and predispose patients to infectious complications. These interventions include endotracheal intubation, indwelling vascular catheters, transurethral bladder catheters, and other catheters placed into sterile body cavities (e.g., tube thoracostomy, percutaneous intraabdominal drainage catheterization). The longer such devices remain in place, the more prone to these infections patients become. For example, ventilator-associated pneumonia correlates strongly with the duration of intubation and mechanical ventilation. Therefore, an important aspect of preventive care is the timely removal of invasive devices as soon as they are no longer needed. Moreover, multidrug-resistant organisms are commonplace in the ICU.

1	Infection control is critical in the ICU. Care bundles, which include measures such as frequent hand washing, are effective but underutilized strategies. Other components of care bundles, such as protective isolation of patients colonized or infected by drug-resistant organisms, are also commonly used. Silver-coated endotracheal tubes reportedly reduce the incidence of ventilator-associated pneumonia. Studies evaluating multifaceted, evidence-based strategies to decrease catheter-related bloodstream infections have shown improved outcomes with strict adherence to measures such as hand washing, full-barrier precautions during catheter insertion, chlorhexidine skin preparation, avoidance of the femoral site, and timely catheter removal.

1	(See also Chap. 300) All ICU patients are at high risk for this complication because of their predilection for immobility. Therefore, all should receive some form of prophylaxis against DVT. The most commonly employed forms of prophylaxis are subcutaneous low-dose heparin injections and sequential compression devices for the lower extremities. Observational studies report an alarming incidence of DVTs despite the use of these standard prophylactic regimens. Furthermore, heparin prophylaxis may result in heparin-induced thrombocytopenia, another nosocomial complication in critically ill patients. Low-molecular-weight heparins such as enoxaparin are more effective than unfractionated heparin for DVT prophylaxis in high-risk patients (e.g., those undergoing orthopedic surgery) and are associated with a lower incidence of heparin-induced thrombocytopenia. Fondaparinux, a selective factor Xa inhibitor, is even more effective than enoxaparin in high-risk orthopedic patients.

1	Prophylaxis against stress ulcers is frequently administered in most ICUs; typically, histamine-2 antagonists or proton pump inhibitors are given. Available data suggest that high-risk patients, such as those with coagulopathy, shock, or respiratory failure requiring mechanical ventilation, benefit from such prophylactic treatment.

1	These are important issues that may be associated with respiratory failure, impaired wound healing, and dysfunctional immune response in critically ill patients. Early enteral feeding is reasonable, though no data are available to suggest that this treatment improves patient outcome per se. Certainly, enteral feeding, if possible, is preferred over parenteral nutrition, which is associated with numerous complications, including hyperglycemia, fatty liver, cholestasis, and sepsis. When parenteral feeding is necessary to supplement enteral nutrition, delaying this intervention until day 8 in the ICU results in better recovery and fewer ICU-related complications. Tight glucose control is an area of controversy in critical care. Although one study showed a significant mortality benefit when glucose levels were aggressively normalized in a large group of surgical ICU patients, more recent data for a large population of both medical and surgical ICU patients suggested that tight glucose

1	glucose levels were aggressively normalized in a large group of surgical ICU patients, more recent data for a large population of both medical and surgical ICU patients suggested that tight glucose control resulted in increased rates of mortality.

1	ICU-acquired weakness occurs frequently in patients who survive critical illness, particularly those with SIRS and/or sepsis. Both neuropathies and myopathies have been described, most commonly after ~1 week in the ICU. The mechanisms behind ICU-acquired weakness syndromes are poorly understood. Intensive insulin therapy may reduce polyneuropathy in critical illness. Very early physical and occupational therapy in mechanically ventilated patients reportedly results in significant improvements in functional independence at hospital discharge as well as in reduced durations of mechanical ventilation and delirium.

1	Studies have shown that most ICU patients are anemic as a result of chronic inflammation. Phlebotomy also contributes to ICU anemia. A large multicenter study involving patients in many different ICU settings challenged the conventional notion that a hemoglobin level of 100 g/L (10 g/dL) is needed in critically ill patients, with similar outcomes noted in those whose transfusion trigger was 7 g/dL. Red blood cell transfusion is associated with impairment of immune function and increased risk of infections as well as of ARDS and volume overload, all of which may explain the findings in this study. Recently, a conservative transfusion strategy enhanced survival among patients with active upper gastrointestinal hemorrhage.

1	(See also Chap. 334) Acute renal failure occurs in a significant percentage of critically ill patients. The most common underlying etiology is acute tubular necrosis, usually precipitated by hypoperfusion and/or nephrotoxic agents. Currently, no pharmacologic agents are available for prevention of renal injury in critical illness. Studies have shown convincingly that low-dose dopamine is not effective in protecting the kidneys from acute injury.

1	(See also Chaps. 34 and 328) This state is defined by (1) an acute onset of changes or fluctuations in mental status, (2) inattention, (3) disorganized thinking, and (4) an altered level of consciousness (i.e., a state other than alertness). Delirium is reported to occur in a wide range of mechanically ventilated ICU patients and can be detected by the Confusion Assessment Method (CAM)-ICU or the Intensive Care Delirium Screening Checklist. These tools are used to ask patients to answer simple questions and perform simple tasks and can be used readily at the bedside. The differential diagnosis of delirium in ICU patients is broad and includes infectious etiologies (including sepsis), medications (particularly sedatives and analgesics), drug withdrawal, metabolic/electrolyte derangements, intracranial pathology (e.g., stroke, intracranial hemorrhage), seizures, hypoxia, hypertensive crisis, shock, and vitamin deficiencies (particularly thiamine). Patients with ICU delirium have

1	intracranial pathology (e.g., stroke, intracranial hemorrhage), seizures, hypoxia, hypertensive crisis, shock, and vitamin deficiencies (particularly thiamine). Patients with ICU delirium have increases in length of hospital stay, time on mechanical ventilation, cognitive impairment at hospital discharge, and 6-month mortality rate. Interventions to reduce ICU delirium are limited. The sedative dexmedetomidine has been less strongly associated with ICU delirium than midazolam. In addition, as mentioned above, very early physical and occupational therapy in mechanically ventilated patients has been demonstrated to reduce delirium.

1	(See also Chap. 330) This condition is common after cardiac arrest and often results in severe and permanent brain injury in survivors. Active cooling of patients after cardiac arrest has been shown to improve neurologic outcomes. Therefore, patients who present to the ICU after circulatory arrest from ventricular fibrillation or pulseless ventricular tachycardia should be actively cooled to achieve a core body temperature of 32–34°C.

1	(See also Chap. 446) Stroke is a common cause of neurologic critical illness. Hypertension must be managed carefully, since abrupt reductions in blood pressure may be associated with further brain ischemia and injury. Acute ischemic stroke treated with tissue plasminogen activator (tPA) has an improved neurologic outcome when treatment is given within 3 h of onset of symptoms. The mortality rate is not reduced when tPA is compared with placebo, despite the improved neurologic outcome. The risk of cerebral hemorrhage is significantly higher in patients given tPA. No benefit is seen when tPA therapy is given beyond 3 h after symptom onset. Heparin has not been convincingly shown to improve outcomes in patients with acute ischemic stroke. Decompressive cra-1735 niectomy is a surgical procedure that relieves increased intracranial pressure in the setting of space-occupying brain lesions or brain swelling from stroke; available evidence suggests that this procedure may improve survival

1	that relieves increased intracranial pressure in the setting of space-occupying brain lesions or brain swelling from stroke; available evidence suggests that this procedure may improve survival among select patients (≤55 years or age), albeit at a cost of increased disability for some.

1	(See also Chap. 446) Subarachnoid hemorrhage may occur secondary to aneurysm rupture and is often complicated by cerebral vasospasm, re-bleeding, and hydrocephalus. Vasospasm can be detected by either transcranial Doppler assessment or cerebral angiography; it is typically treated with the calcium channel blocker nimodipine, aggressive IV fluid administration, and therapy aimed at increasing blood pressure, typically with vasoactive drugs such as phenylephrine. The IV fluids and vasoactive drugs (hypertensive hypervolemic therapy) are used to overcome the cerebral vasospasm. Early surgical clipping or endovascular coiling of aneurysms is advocated to prevent complications related to re-bleeding. Hydrocephalus, typically heralded by a decreased level of consciousness, may require ventriculostomy drainage.

1	(See also Chap. 445) Recurrent or relentless seizure activity is a medical emergency. Cessation of seizure activity is required to prevent irreversible neurologic injury. Lorazepam is the most effective benzodiazepine for treating status epilepticus and is the treatment of choice for controlling seizures acutely. Phenytoin or fosphenytoin should be given concomitantly since lorazepam has a short half-life. Other drugs, such as gabapentin, carbamazepine, and phenobarbital, should be reserved for patients with contraindications to phenytoin (e.g., allergy or pregnancy) or ongoing seizures despite phenytoin.

1	(See also Chap. 330) Although deaths of critically ill patients usually are attributable to irreversible cessation of circulatory and respiratory function, a diagnosis of death also may be established by irreversible cessation of all functions of the entire brain, including the brainstem, even if circulatory and respiratory functions remain intact on artificial life support. Such a diagnosis requires demonstration of the absence of cerebral function (no response to any external stimulus) and brainstem functions (e.g., unreactive pupils, lack of ocular movement in response to head turning or ice-water irrigation of ear canals, positive apnea test [no drive to breathe]). Absence of brain function must have an established cause and be permanent without possibility of recovery; a sedative effect, hypothermia, hypoxemia, neuromuscular paralysis, and severe hypotension must be ruled out. If there is uncertainty about the cause of coma, studies of cerebral blood flow and

1	a sedative effect, hypothermia, hypoxemia, neuromuscular paralysis, and severe hypotension must be ruled out. If there is uncertainty about the cause of coma, studies of cerebral blood flow and electroencephalography should be performed.

1	(See also Chap. 10) Withholding or withdrawal of care occurs commonly in the ICU setting. The Task Force on Ethics of the Society of Critical Care Medicine reported that it is ethically sound to withhold or withdraw care if a patient or the patient’s surrogate makes such a request or if the physician judges that the goals of therapy are not achievable. Since all medical treatments are justified by their expected benefits, the loss of such an expectation justifies the act of withdrawing or withholding such treatment; these two actions are judged to be fundamentally similar. An underlying stipulation derived from this report is that an informed patient should have his or her wishes respected with regard to life-sustaining therapy. Implicit in this stipulation is the need to ensure that patients are thoroughly and accurately informed regarding the plausibility and expected results of various therapies.

1	The act of informing patients and/or surrogate decision-makers is the responsibility of the physician and other health care providers. If a patient or surrogate desires therapy deemed futile by the treating physician, the physician is not obligated ethically to provide such treatment. Rather, arrangements may be made to transfer the patient’s care to another care provider. Whether the decision to withdraw

1	Approach to the Patient with Critical Illness 1736 life support should be initiated by the physician or left to surrogate decision-makers alone is not clear. One study reported that slightly more than half of surrogate decision-makers preferred to receive such a recommendation, whereas the rest did not. Critical care providers should meet regularly with patients and/or surrogates to discuss prognosis when the withholding or withdrawal of care is being considered. After a consensus among caregivers has been reached, this information should be relayed to the patient and/or surrogate decision-maker. If a decision to withhold or withdraw life-sustaining care for a patient has been made, aggressive attention to analgesia and anxiolysis is needed. Syndrome 322 Bruce D. Levy, Augustine M. K. Choi

1	Bruce D. Levy, Augustine M. K. Choi Acute respiratory distress syndrome (ARDS) is a clinical syndrome of severe dyspnea of rapid onset, hypoxemia, and diffuse pulmonary infiltrates leading to respiratory failure. ARDS is caused by diffuse lung injury from many underlying medical and surgical disorders. The lung injury may be direct, as occurs in toxic inhalation, or indirect, as occurs in sepsis (Table 322-1). The clinical features of ARDS are listed in Table 322-2. By expert consensus, ARDS is defined by three categories based on the degrees of hypoxemia (Table 322-2). These stages of mild, moderate, and severe ARDS are associated with mortality risk and with the duration of mechanical ventilation in survivors. The annual incidence of ARDS is estimated to be as high as 60 cases/100,000 population. Approximately 10% of all intensive care unit (ICU) admissions involve patients with acute respiratory failure; ~20% of these patients meet the criteria for ARDS.

1	While many medical and surgical illnesses have been associated with the development of ARDS, most cases (>80%) are caused by a relatively small number of clinical disorders: severe sepsis syndrome and/or bacterial pneumonia (~40–50%), trauma, multiple transfusions, aspiration of gastric contents, and drug overdose. Among patients with trauma, the most frequently reported surgical conditions in ARDS are pulmonary contusion, multiple bone fractures, and chest wall trauma/flail chest, whereas head trauma, near-drowning, toxic inhalation, and burns are rare causes. The risks of developing ARDS are increased in patients with more than one predisposing medical or surgical condition.

1	Several other clinical variables have been associated with the development of ARDS. These include older age, chronic alcohol abuse, metabolic acidosis, and severity of critical illness. Trauma patients with an Acute Physiology and Chronic Health Evaluation (APACHE) II score ≥16 (Chap. 321) have a 2.5-fold increased risk of developing ARDS, and those with a score >20 have an incidence of ARDS that Direct Lung Injury Indirect Lung Injury Chest Absence of Left Severity: Oxygenation Onset Radiograph Atrial Hypertension Abbreviations: ARDS, acute respiratory distress syndrome; FIO2, inspired O2 percentage; PaO2, arterial partial pressure of O2; PCWP, pulmonary capillary wedge pressure. is more than threefold greater than the incidence among those with APACHE II scores ≤9. The natural history of ARDS is marked by three phases—exudative, proliferative, and fibrotic—that each have characteristic clinical and pathologic features (Fig. 322-1).

1	Exudative Phase In this phase (Fig. 322-2), alveolar capillary endothelial cells and type I pneumocytes (alveolar epithelial cells) are injured, with consequent loss of the normally tight alveolar barrier to fluid and macromolecules. Edema fluid that is rich in protein accumulates in the interstitial and alveolar spaces. Significant concentrations of cytokines (e.g., interleukin 1, interleukin 8, and tumor necrosis factor α) and lipid mediators (e.g., leukotriene B4) are present in the lung in this acute phase. In response to proinflammatory mediators, leukocytes (especially neutrophils) traffic into the pulmonary interstitium and alveoli. In addition, condensed plasma proteins aggregate in the air spaces with cellular debris and dysfunctional pulmonary surfactant to form hyaline membrane whorls. Pulmonary vascular injury also occurs early in ARDS, with vascular obliteration by microthrombi and fibrocellular proliferation (Fig. 322-3).

1	Alveolar edema predominantly involves dependent portions of the lung, with diminished aeration and atelectasis. Collapse of large sections of dependent lung markedly decreases lung compliance. Consequently, intrapulmonary shunting and hypoxemia develop and the work of breathing increases, leading to dyspnea. The pathophysiologic alterations in alveolar spaces are exacerbated by microvascular occlusion that results in reductions in pulmonary arterial blood flow to ventilated portions of the lung (and thus in increased dead space) and in pulmonary hypertension. Thus, in addition to severe hypoxemia, hypercapnia secondary to an increase in pulmonary dead space is prominent in early ARDS.

1	The exudative phase encompasses the first 7 days of illness after exposure to a precipitating ARDS risk factor, with the patient experiencing the onset of respiratory symptoms. Although usually presenting within 12–36 h after the initial insult, symptoms can be delayed by 5–7 days. Dyspnea develops, with a sensation of rapid shallow breathing Day: 02 7 14 21. . . FIGURE 322-1 Diagram illustrating the time course for the development and resolution of ARDS. The exudative phase is notable for early alveolar edema and neutrophil-rich leukocytic infiltration of the lungs, with subsequent formation of hyaline membranes from diffuse alveolar damage. Within 7 days, a proliferative phase ensues with prominent interstitial inflammation and early fibrotic changes. Approximately 3 weeks after the initial pulmonary injury, most patients recover. However, some patients enter the fibrotic phase, with substantial fibrosis and bullae formation.

1	FIGURE 322-2 A representative anteroposterior chest x-ray in the exudative phase of ARDS shows diffuse interstitial and alveolar infiltrates that can be difficult to distinguish from left ventricular failure. and an inability to get enough air. Tachypnea and increased work of 1737 breathing result frequently in respiratory fatigue and ultimately in respiratory failure. Laboratory values are generally nonspecific and are primarily indicative of underlying clinical disorders. The chest radio-graph usually reveals alveolar and interstitial opacities involving at least three-quarters of the lung fields (Fig. 322-2). While characteristic for ARDS, these radiographic findings are not specific and can be indistinguishable from cardiogenic pulmonary edema (Chap. 326). Unlike the latter, however, the chest x-ray in ARDS rarely shows cardiomegaly, pleural effusions, or pulmonary vascular redistribution. Chest CT in ARDS reveals extensive heterogeneity of lung involvement (Fig. 322-4).

1	Because the early features of ARDS are nonspecific, alternative diagnoses must be considered. In the differential diagnosis of ARDS, the most common disorders are cardiogenic pulmonary edema, diffuse pneumonia, and alveolar hemorrhage. Less common diagnoses to consider include acute interstitial lung diseases (e.g., acute interstitial pneumonitis; Chap. 315), acute immunologic injury (e.g., hypersensitivity pneumonitis; Chap. 310), toxin injury (e.g., radiation pneumonitis; Chap. 263), and neurogenic pulmonary edema (Chap. 47e). Proliferative Phase This phase of ARDS usually lasts from day 7 to day 21. Most patients recover rapidly and are liberated from mechanical

1	Proliferative Phase This phase of ARDS usually lasts from day 7 to day 21. Most patients recover rapidly and are liberated from mechanical Alveolar air space Protein-rich edema fluid Inactivated surfactant Activated neutrophil Alveolar macrophages Type I cell Type II cell Endothelial basement membrane FibroblastsNeutrophils Gap formation Procollagen Proteases IL-8 TNF-˜, IL-8 MIF IL-6, IL-8 Leukotrienes Oxidants PAF Proteases IL-8 Migrating neutrophil Fibroblasts Capillary Swollen, injured endothelial cell Platelets Interstitium Widened edematous interstitium Epithelial basement membrane Sloghing of bronchial epithelium Necrotic or apoptotic type I cell Red blood cell Intact type II cell Cellular debris Fibrin Denuded basement membrane Hyaline membrane Surfactant layer Normal alveolus Injured alveolus during the acute phase

1	FIGURE 322-3 The normal alveolus (left) and the injured alveolus in the acute phase of acute lung injury and the acute respiratory distress syndrome (right). In the acute phase of the syndrome (right), there is sloughing of both the bronchial and alveolar epithelial cells, with the formation of protein-rich hyaline membranes on the denuded basement membrane. Neutrophils are shown adhering to the injured capillary endothelium and transmigrating through the interstitium into the air space, which is filled with protein-rich edema fluid. In the air space, an alveolar macrophage is secreting cytokines—i.e., interleukins 1, 6, 8, and 10 (IL-1, -6, -8, and -10) and tumor necrosis factor α (TNF-α)—that act locally to stimulate chemotaxis and activate neutrophils. Macrophages also secrete other cytokines, including IL-1, -6, and -10. IL-1 can also stimulate the production of extracellular matrix by fibroblasts. Neutrophils can release oxidants, proteases, leukotrienes, and other

1	other cytokines, including IL-1, -6, and -10. IL-1 can also stimulate the production of extracellular matrix by fibroblasts. Neutrophils can release oxidants, proteases, leukotrienes, and other proinflammatory molecules, such as platelet-activating factor (PAF). A number of antiinflammatory mediators are also present in the alveolar milieu, including the IL-1-receptor antagonist, soluble TNF-α receptor, autoantibodies to IL-8, and cytokines such as IL-10 and IL-11 (not shown). The influx of protein-rich edema fluid into the alveolus has led to the inactivation of surfactant. MIF, macrophage inhibitory factor. (From LB Ware, MA Matthay: N Engl J Med 342:1334, 2000, with permission.)

1	FIGURE 322-4 A representative CT scan of the chest during the exudative phase of ARDS, in which dependent alveolar edema and atelectasis predominate. ventilation during this phase. Despite this improvement, many patients still experience dyspnea, tachypnea, and hypoxemia. Some patients develop progressive lung injury and early changes of pulmonary fibrosis during the proliferative phase. Histologically, the first signs of resolution are often evident in this phase, with the initiation of lung repair, the organization of alveolar exudates, and a shift from a neutrophilto a lymphocyte-predominant pulmonary infiltrate. As part of the reparative process, type II pneumocytes proliferate along alveolar basement membranes. These specialized epithelial cells synthesize new pulmonary surfactant and differentiate into type I pneumocytes.

1	Fibrotic Phase While many patients with ARDS recover lung function 3–4 weeks after the initial pulmonary injury, some enter a fibrotic phase that may require long-term support on mechanical ventilators and/or supplemental oxygen. Histologically, the alveolar edema and inflammatory exudates of earlier phases are now converted to extensive alveolar-duct and interstitial fibrosis. Marked disruption of acinar architecture leads to emphysema-like changes, with large bullae. Intimal fibroproliferation in the pulmonary microcirculation causes progressive vascular occlusion and pulmonary hypertension. The physiologic consequences include an increased risk of pneumothorax, reductions in lung compliance, and increased pulmonary dead space. Patients in this late phase experience a substantial burden of excess morbidity. Lung biopsy evidence for pulmonary fibrosis in any phase of ARDS is associated with increased mortality risk.

1	Recent reductions in ARDS mortality rates are largely the result of general advances in the care of critically ill patients (Chap. 321). Thus, caring for these patients requires close attention to (1) the recognition and treatment of underlying medical and surgical disorders (e.g., sepsis, aspiration, trauma); (2) the minimization of procedures and their complications; (3) prophylaxis against venous thromboembolism, gastrointestinal bleeding, aspiration, excessive sedation, and central venous catheter infections; (4) prompt recognition of nosocomial infections; and (5) provision of adequate nutrition. (See also Chap. 323) Patients meeting clinical criteria for ARDS frequently become fatigued from increased work of breathing and progressive hypoxemia, requiring mechanical ventilation for support.

1	Ventilator-Induced Lung Injury Despite its life-saving potential, mechanical ventilation can aggravate lung injury. Experimental models have demonstrated that ventilator-induced lung injury appears to require two processes: repeated alveolar overdistention and recurrent alveolar collapse. As is clearly evident from chest CT (Fig. 322-4), ARDS is a heterogeneous disorder, principally involving dependent portions of the lung with relative sparing of other regions. Because compliance differs in affected versus more “normal” areas of the lung, attempts to fully inflate the consolidated lung may lead to overdistention of and injury to the more normal areas. Ventilator-induced injury can be demonstrated in experimental models of acute lung injury, with high-tidal-volume (VT) ventilation resulting in additional, synergistic alveolar damage.

1	A large-scale, randomized controlled trial sponsored by the National Institutes of Health and conducted by the ARDS Network compared low VT ventilation (6 mL/kg of predicted body weight) to conventional VT ventilation (12 mL/kg predicted body weight). The mortality rate was significantly lower in the low VT patients (31%) than in the conventional VT patients (40%). This improvement in survival represents the most substantial ARDS-mortality benefit that has been demonstrated for any therapeutic intervention to date.

1	Prevention of Alveolar Collapse In ARDS, the presence of alveolar and interstitial fluid and the loss of surfactant can lead to a marked reduction of lung compliance. Without an increase in end-expiratory pressure, significant alveolar collapse can occur at end-expiration, with consequent impairment of oxygenation. In most clinical settings, positive end-expiratory pressure (PEEP) is empirically set to minimize FIO2 (inspired O2 percentage) and maximize PaO2 (arterial partial pressure of O2). On most modern mechanical ventilators, it is possible to construct a static pressure–volume curve for the respiratory system. The lower inflection point on the curve represents alveolar opening (or “recruitment”). The pressure at this point, usually 12–15 mmHg in ARDS, is a theoretical “optimal PEEP” for alveolar recruitment. Titration of the PEEP to the lower inflection point on the static pressure–volume curve has been hypothesized to keep the lung open, improving oxygenation and protecting

1	for alveolar recruitment. Titration of the PEEP to the lower inflection point on the static pressure–volume curve has been hypothesized to keep the lung open, improving oxygenation and protecting against lung injury. Three large randomized trials have investigated the utility of PEEP-based strategies to keep the lung open. In all three trials, improvement in lung function was evident but overall mortality rates were not altered significantly. Until more data become available on the clinical utility of high PEEP, it is advisable to set PEEP to minimize FIO2 and optimize PaO2 (Chap. 323). Measurement of esophageal pressures to estimate transpulmonary pressure may help identify an optimal PEEP in some cases.

1	Oxygenation can also be improved by increasing mean airway pressure with inverse-ratio ventilation. In this technique, the inspiratory time (I) is lengthened so that it is longer than the expiratory time (E)— that is, I:E > 1:1. With diminished time to exhale, dynamic hyperinflation leads to increased end-expiratory pressure, similar to ventilator-prescribed PEEP. This mode of ventilation has the advantage of improving oxygenation with lower peak pressures than are required for conventional ventilation. Although inverse-ratio ventilation can improve oxygenation and can help reduce FIO2 to ≤0.60, thus avoiding possible oxygen toxicity, no benefit in ARDS mortality risk has been demonstrated. Recruitment maneuvers that transiently increase PEEP to “recruit” atelectatic lung can also increase oxygenation, but a mortality benefit has not been established.

1	In several randomized trials, mechanical ventilation in the prone position improved arterial oxygenation, but its effect on survival and other important clinical outcomes remains uncertain. Moreover, unless the critical-care team is experienced in “proning,” repositioning critically ill patients can be hazardous, leading to accidental endotracheal extubation, loss of central venous catheters, and orthopedic injury.

1	Several additional mechanical-ventilation strategies that use specialized equipment have been tested in ARDS patients; most of these approaches have had mixed or disappointing results in adults. High-frequency ventilation (HFV) entails ventilating at extremely high respiratory rates (5–20 cycles per second) and low VTs (1–2 mL/ kg). Use of partial liquid ventilation (PLV) with perfluorocarbon—an inert, high-density liquid that easily solubilizes oxygen and carbon dioxide—has yielded promising preliminary results, enhancing pulmonary function in patients with ARDS, but also has provided no survival benefit. Lung-replacement therapy with extracorporeal membrane oxygenation (ECMO), which provides a clear survival benefit in neonatal respiratory distress syndrome, may also have utility in selected adult patients with ARDS.

1	Data supporting the efficacy of “adjunctive” ventilator therapies (e.g., high PEEP, inverse ratio ventilation, recruitment maneuvers, prone positioning, HFV, ECMO, and PLV) remain incomplete. Accordingly, these modalities are reserved for use as rescue rather than primary therapies.

1	(See also Chap. 321) Increased pulmonary vascular permeability leading to interstitial and alveolar edema fluid rich in protein is a central feature of ARDS. In addition, impaired vascular integrity augments the normal increase in extravascular lung water that occurs with increasing left atrial pressure. Maintaining a low left atrial filling pressure minimizes pulmonary edema and prevents further decrements in arterial oxygenation and lung compliance; improves pulmonary mechanics; shortens ICU stay and the duration of mechanical ventilation; and is associated with a lower mortality rate in both medical and surgical ICU patients. Thus, aggressive attempts to reduce left atrial filling pressures with fluid restriction and diuretics should be an important aspect of ARDS management, limited only by hypotension and hypoperfusion of critical organs such as the kidneys.

1	In severe ARDS, sedation alone can be inadequate for the patient-ventilator synchrony required for lung-protective ventilation. This clinical problem was recently addressed in a multicenter, randomized, placebo-controlled trial of early neuromuscular blockade (with cisatracurium besylate) for 48 h. In severe ARDS, early neuromuscular blockade increased the rate of survival and ventilator-free days without increasing ICU-acquired paresis. These promising findings support the early administration of neuromuscular blockade if needed to facilitate mechanical ventilation in severe ARDS; however, these results must be replicated prior to their widespread application in clinical practice. Many attempts have been made to treat both early and late ARDS with glucocorticoids, with the goal of reducing potentially deleterious pulmonary inflammation. Few studies have shown any benefit. Current evidence does not support the use of high-dose glucocorticoids in the care of ARDS patients.

1	Clinical trials of surfactant replacement and multiple other medical therapies have proved disappointing. Inhaled nitric oxide and inhaled epoprostenol sodium can transiently improve oxygenation but do not improve survival or decrease time on mechanical ventilation. Many clinical trials have been undertaken to improve the outcome of patients with ARDS; most have been unsuccessful in modifying the natural history. While results of large clinical trials must be judiciously applied to individual patients, evidence-based recommendations are summarized in Table 322-3, and an algorithm for the initial therapeutic goals and limits in ARDS management is provided in Fig. 322-5.

1	PROGNOSIS Mortality Recent mortality estimates for ARDS range from 26% to 44%. There is substantial variability, but a trend toward improved ARDS outcomes appears evident. Of interest, mortality in ARDS is largely attributable to nonpulmonary causes, with sepsis and nonpulmonary organ failure accounting for >80% of deaths. Thus, improvement in survival is aKey: A, recommended therapy based on strong clinical evidence from randomized clinical trials; B, recommended therapy based on supportive but limited clinical data; C, recommended only as alternative therapy on the basis of indeterminate evidence; D, not recommended on the basis of clinical evidence against efficacy of therapy. Abbreviations: ARDS, acute respiratory distress syndrome; ECMO, extracorporeal membrane oxygenation; NO, nitric oxide; NSAIDs, nonsteroidal anti-inflammatory drugs; PEEP, positive end-expiratory pressure; PGE1, prostaglandin E1.

1	likely secondary to advances in the care of septic/infected patients and those with multiple organ failure (Chap. 321). The major risk factors for ARDS mortality are nonpulmonary. Advanced age is an important risk factor. Patients >75 years of age have a substantially higher mortality risk (~60%) than those <45 (~20%). Moreover, patients >60 years of age with ARDS and sepsis have a threefold higher mortality risk than those <60. Other risk factors include preexisting organ dysfunction from chronic medical illness—in particular, chronic liver disease, cirrhosis, chronic alcohol abuse, chronic immunosuppression, sepsis, chronic renal disease, failure of any nonpulmonary organ, and increased APACHE III scores (Chap. 321). Patients with ARDS arising from direct lung injury (including pneumonia, pulmonary contusion, and aspiration; FIO2 ˜ 0.6 PEEP ˜ 10 cmH2O SpO2 88 – 95% pH ° 7.30 RR ˜ 35 bpm FIGURE 322-5 Algorithm for the initial management of ARDS.

1	FIO2 ˜ 0.6 PEEP ˜ 10 cmH2O SpO2 88 – 95% pH ° 7.30 RR ˜ 35 bpm FIGURE 322-5 Algorithm for the initial management of ARDS. Clinical trials have provided evidence-based therapeutic goals for a stepwise approach to the early mechanical ventilation, oxygenation, and correction of acidosis and diuresis of critically ill patients with ARDS. FIO2, inspired O2 percentage; MAP, mean arterial pressure; PBW, predicted body weight; PEEP, positive end expiratory pressure; RR, respiratory rate; SpO2, arterial oxyhemoglobin saturation measured by pulse oximetry.

1	mechanical ventilatory Support Bartolome R. Celli MECHANICAL VENTILATORY SUPPORT Mechanical ventilation is used to assist or replace spontaneous breath-ing. It is implemented with special devices that can support ventilatory 323 1740 Table 322-1) are nearly twice as likely to die as those with indirect causes of lung injury, while surgical and trauma patients with ARDS— especially those without direct lung injury—have a higher survival rate than other ARDS patients. An early (within 24 h of presentation) elevation in pulmonary dead space (>0.60) and severe arterial hypoxemia (Pao2/Fio2, <100 mmHg) predict increased mortality risk from ARDS; however, there is surprisingly little additional value in predicting ARDS mortality from other measures of the severity of lung injury, including the level of PEEP (≥10 cm H2O), respiratory system compliance (≤40 mL/cm H2O), the extent of alveolar infiltrates on chest radiography, and the corrected expired volume per minute (≥10 L/min).

1	Functional Recovery in ARDS Survivors While it is common for patients with ARDS to experience prolonged respiratory failure and remain dependent on mechanical ventilation for survival, it is a testament to the resolving powers of the lung that the majority of patients recover nearly normal lung function. Patients usually recover maximal lung function within 6 months. One year after endotracheal extubation, more than one-third of ARDS survivors have normal spirometry values and diffusion capacity. Most of the remaining patients have only mild abnormalities in pulmonary function. Unlike mortality risk, recovery of lung function is strongly associated with the extent of lung injury in early ARDS. Low static respiratory compliance, high levels of required PEEP, longer durations of mechanical ventilation, and high lung injury scores are all associated with less recovery of pulmonary function. Of note, when physical function is assessed 5 years after ARDS, exercise limitation and decreased

1	ventilation, and high lung injury scores are all associated with less recovery of pulmonary function. Of note, when physical function is assessed 5 years after ARDS, exercise limitation and decreased physical quality of life are often documented despite normal or nearly normal pulmonary function. When caring for ARDS survivors, it is important to be aware of the potential for a substantial burden of psychological problems in patients and family caregivers, including significant rates of depression and posttraumatic stress disorder.

1	ARDS Support Center for patient-oriented education: www.ards.org NHLBI ARDS Clinical Trials information: www.ardsnet.org ARDS Foundation: www.ardsusa.org The authors acknowledge the contribution to this chapter by the previous author, Dr. Steven D. Shapiro.

1	The authors acknowledge the contribution to this chapter by the previous author, Dr. Steven D. Shapiro. function and improve oxygenation through the application of highoxygen-content gas and positive pressure. The primary indication for initiation of mechanical ventilation is respiratory failure, of which there are two basic types: (1) hypoxemic, which is present when arterial O2 saturation (Sao2) <90% occurs despite an increased inspired O2 fraction and usually results from ventilation-perfusion mismatch or shunt; and (2) hypercarbic, which is characterized by elevated arterial carbon dioxide partial pressure (PCO2) values (usually >50 mmHg) resulting from conditions that decrease minute ventilation or increase physiologic dead space such that alveolar ventilation is inadequate to meet metabolic demands. When respiratory failure is chronic, neither of the two types is obligatorily treated with mechanical ventilation, but when it is acute, mechanical ventilation may be lifesaving.

1	The most common reasons for instituting mechanical ventilation are acute respiratory failure with hypoxemia (acute respiratory distress syndrome, heart failure with pulmonary edema, pneumonia, sepsis, complications of surgery and trauma), which accounts for ~65% of all ventilated cases, and hypercarbic ventilatory failure—e.g., due to coma (15%), exacerbations of chronic obstructive pulmonary disease (COPD; 13%), and neuromuscular diseases (5%). The primary objectives of mechanical ventilation are to decrease the work of breathing, thus avoiding respiratory muscle fatigue, and to reverse life-threatening hypoxemia and progressive respiratory acidosis.

1	In some cases, mechanical ventilation is used as an adjunct to other forms of therapy. For example, it is used to reduce cerebral blood flow in patients with increased intracranial pressure. Mechanical ventilation also is used frequently in conjunction with endotracheal intubation for airway protection to prevent aspiration of gastric contents in otherwise unstable patients during gastric lavage for suspected drug overdose or during gastrointestinal endoscopy. In critically ill patients, intubation and mechanical ventilation may be indicated before the performance of essential diagnostic or therapeutic studies if it appears that respiratory failure may occur during those maneuvers. There are two basic methods of mechanical ventilation: noninvasive ventilation (NIV) and invasive (or conventional mechanical) ventilation (MV).

1	Noninvasive Ventilation NIV has gained acceptance because it is effective in certain conditions, such as acute or chronic respiratory failure, and is associated with fewer complications—namely, pneumonia and tracheolaryngeal trauma. NIV usually is provided with a tight-fitting face mask or nasal mask similar to the masks traditionally used for treatment of sleep apnea. NIV has proved highly effective in patients with respiratory failure arising from acute exacerbations of chronic obstructive pulmonary disease. It is most frequently implemented as bilevel positive airway pressure ventilation or pressure-support ventilation. Both modes, which apply a preset positive pressure during inspiration and a lower pressure during expiration at the mask, are well tolerated by a conscious patient and optimize patient-ventilator synchrony. The major limitation to the widespread application of NIV has been patient intolerance: the tight-fitting mask required for NIV can cause both physical and

1	and optimize patient-ventilator synchrony. The major limitation to the widespread application of NIV has been patient intolerance: the tight-fitting mask required for NIV can cause both physical and psychological discomfort. In addition, NIV has had limited success in patients with acute hypoxemic respiratory failure, for whom endotracheal intubation and conventional MV remain the ventilatory method of choice.

1	The most important group of patients who benefit from a trial of NIV are those with exacerbations of COPD and respiratory acidosis (pH <7.35). Experience from several randomized trials has shown that, in patients with ventilatory failure characterized by blood pH levels between 7.25 and 7.35, NIV is associated with low failure rates (15–20%) and good outcomes (as judged by intubation rate, length of stay in intensive care, and—in some series—mortality rates). In more severely ill patients with a blood pH <7.25, the rate of NIV failure is inversely related to the severity of respiratory acidosis, with higher failure rates as the pH decreases. In patients with milder acidosis (pH >7.35), NIV is not better than conventional treatment that includes controlled oxygen delivery and pharmacotherapy for exacerbations of COPD (systemic glucocorticoids, bronchodilators, and, if needed, antibiotics).

1	Despite its benign outcomes, NIV is not useful in the majority of cases of respiratory failure and is contraindicated in patients with the conditions listed in Table 323-1. NIV can delay lifesaving ventilatory support in those cases and, in fact, can actually result in aspiration or hypoventilation. Once NIV is initiated, patients should be monitored; a reduction in respiratory frequency and a decrease in the use of accessory muscles (scalene, sternomastoid, and intercostals) are good clinical indicators of adequate therapeutic benefit. Arterial blood gases should be determined at least within hours of the initiation of therapy to ensure that NIV is having the desired effect. Lack of benefit within High-risk aspiration and/or inability to protect airways Inability to clear secretions that time frame should alert the physician to the possible need for conventional MV.

1	Conventional Mechanical Ventilation Conventional MV is implemented once a cuffed tube is inserted into the trachea to allow conditioned gas (warmed, oxygenated, and humidified) to be delivered to the airways and lungs at pressures above atmospheric pressure. Care should be taken during intubation to avoid brain-damaging hypoxia. In most cases, the administration of mild sedation may facilitate the procedure. Opiates and benzodiazepines are good choices but can have a deleterious effect on hemodynamics in patients with depressed cardiac function or low systemic vascular resistance. Morphine can promote histamine release from tissue mast cells and may worsen bronchospasm in patients with asthma; fentanyl, sufentanil, and alfentanil are acceptable alternatives. Ketamine may increase systemic arterial pressure and has been associated with hallucinatory responses. The shorter-acting agents etomidate and propofol have been used for both induction and maintenance of anesthesia in ventilated

1	arterial pressure and has been associated with hallucinatory responses. The shorter-acting agents etomidate and propofol have been used for both induction and maintenance of anesthesia in ventilated patients because they have fewer adverse hemodynamic effects, but both are significantly more expensive than older agents. Great care must be taken to avoid the use of neuromuscular paralysis during intubation of patients with renal failure, tumor lysis syndrome, crush injuries, medical conditions associated with elevated serum potassium levels, and muscular dystrophy syndromes; in particular, the use of agents whose mechanism of action includes depolarization at the neuromuscular junction, such as succinylcholine chloride, must be avoided.

1	Once the patient has been intubated, the basic goals of MV are to optimize oxygenation while avoiding ventilator-induced lung injury due to overstretch and collapse/re-recruitment. This concept, known as the “protective ventilatory strategy” (see below and Fig. 323-1) is supported by evidence linking high airway pressures and volumes and overstretching of the lung as well as collapse/re-recruitment to poor clinical outcomes (barotrauma and volume trauma). Although normalization of pH through elimination of CO2 is desirable, the risk of lung damage associated with the large volume and high pressures needed to achieve this goal has led to the acceptance of permissive hypercapnia. This condition is well tolerated when care is taken to avoid excess acidosis by pH buffering.

1	Mode refers to the manner in which ventilator breaths are triggered, cycled, and limited. The trigger, either an inspiratory effort or a time-based signal, defines what the ventilator senses to initiate an assisted breath. Cycle refers to the factors that determine the end of inspiration. For example, in volume-cycled ventilation, inspiration ends when a specific tidal volume is delivered. Other types of cycling include pressure cycling and time cycling. The limiting factors are operator-specified values, such as airway pressure, that are monitored by transducers internal to the ventilator circuit throughout the respiratory cycle; if the specified values are exceeded, inspiratory flow is terminated, and the ventilator circuit is vented to atmospheric pressure or the specified pressure at the end of expiration (positive end-expiratory pressure, or PEEP). Most patients are ventilated with assist-control ventilation, B 0.8 Alveolar 0.6 0.4 0.2 A Pressure (cm of water)

1	B 0.8 Alveolar 0.6 0.4 0.2 A Pressure (cm of water) FIGURE 323-1 Hypothetical pressure-volume curve of the lung in a patient undergoing mechanical ventilation. Alveoli tend to close if the distending pressure falls below the lower inflection point (A), whereas they overstretch if the pressure within them is higher than that of the upper inflection point (B). Collapse and opening of ventilated alveoli are associated with poor outcomes in patients with acute respiratory failure. Protective ventilation (purple shaded area), using a lower tidal volume (6 mL/kg of ideal body weight) and maintaining positive end-expiratory pressure to prevent overstretching and collapse/opening of alveoli, has resulted in improved survival rates among patients receiving mechanical ventilatory support. intermittent mandatory ventilation, or pressure-support ventilation, with the latter two modes often used simultaneously (Table 323-2).

1	intermittent mandatory ventilation, or pressure-support ventilation, with the latter two modes often used simultaneously (Table 323-2). Assist-Control Ventilation (ACMV) ACMV is the most widely used mode of ventilation. In this mode, an inspiratory cycle is initiated either by the patient’s inspiratory effort or, if none is detected within a specified time window, by a timer signal within the ventilator. Every breath delivered, whether patientor timer-triggered, consists of the operator-specified tidal volume. Ventilatory rate is determined either by the patient or by the operator-specified backup rate, whichever is of higher frequency. ACMV is commonly used for initiation of mechanical ventilation because it ensures a backup minute ventilation in the absence of an intact respiratory drive and allows for synchronization of the ventilator cycle with the patient’s inspiratory effort.

1	Problems can arise when ACMV is used in patients with tachypnea due to nonrespiratory or nonmetabolic factors, such as anxiety, pain, and airway irritation. Respiratory alkalemia may develop and trigger myoclonus or seizures. Dynamic hyperinflation leading to increased intrathoracic pressures (so-called auto-PEEP) may occur if the patient’s respiratory mechanics are such that inadequate time is available for complete exhalation between inspiratory cycles. Auto-PEEP can limit venous return, decrease cardiac output, and increase airway pressures, predisposing to barotrauma.

1	Intermittent Mandatory Ventilation (IMV) With this mode, the operator sets the number of mandatory breaths of fixed volume to be delivered by the ventilator; between those breaths, the patient can breathe spontaneously. In the most frequently used synchronized mode (SIMV), mandatory breaths are delivered in synchrony with the patient’s inspiratory efforts at a frequency determined by the operator. If the patient fails to initiate a breath, the ventilator delivers a fixed-tidal-volume breath and resets the internal timer for the next inspiratory cycle. SIMV differs from ACMV in that only a preset number of breaths are ventilator-assisted. SIMV allows patients with an intact respiratory drive to exercise inspiratory muscles between assisted breaths; thus it is useful for both supporting and weaning intubated patients. SIMV may be difficult to Peak, mean, and plateau airway pressures VE ABG I/E ratio Peak, mean, and plateau airway pressures VE ABG I/E ratio

1	Peak, mean, and plateau airway pressures VE ABG I/E ratio Peak, mean, and plateau airway pressures VE ABG I/E ratio Mask interface may cause discomfort and facial bruising Leaks are common Hypoventilation Abbreviations: ABG, arterial blood gases; FIO2, fraction of inspired oxygen; PEEP, positive end-expiratory pressure; I/E, inspiratory to expiratory time ratio; VE, minute ventilation. use in patients with tachypnea because they may attempt to exhale during the ventilator-programmed inspiratory cycle. Consequently, the airway pressure may exceed the inspiratory pressure limit, the ventilator-assisted breath will be aborted, and minute volume may drop below that programmed by the operator. In this setting, if the tachypnea represents a response to respiratory or metabolic acidosis, a change in ACMV will increase minute ventilation and help normalize the pH while the underlying process is further evaluated and treated.

1	Pressure-Support Ventilation (PSV) This form of ventilation is patient-triggered, flow-cycled, and pressure-limited. It provides graded assistance and differs from the other two modes in that the operator sets the pressure level (rather than the volume) to augment every spontaneous respiratory effort. The level of pressure is adjusted by observing the patient’s respiratory frequency. During PSV, the inspiration is terminated when inspiratory airflow falls below a certain level; in most ventilators, this flow rate cannot be adjusted by the operator. With PSV, patients receive ventilator assistance only when the ventilator detects an inspiratory effort. PSV is often used in combination with SIMV to ensure volume-cycled backup for patients whose respiratory drive is depressed. PSV is well tolerated by most patients who are being weaned from MV; PSV parameters can be set to provide full ventilatory support and can be withdrawn to load the respiratory muscles gradually.

1	Other Modes of Ventilation There are other modes of ventilation, each with its own acronym and each with specific modifications of the manner and duration in which pressure is applied to the airway and lungs and of the interaction between the mechanical assistance provided by the ventilator and the patient’s respiratory effort. Although their use in acute respiratory failure is limited, the following modes have been used with varying levels of enthusiasm and adoption.

1	Pressure-control ventilation (Pcv) This form of ventilation is time-triggered, time-cycled, and pressure-limited. A specified pressure is imposed at the airway opening throughout inspiration. Since the inspiratory pressure is specified by the operator, tidal volume and inspiratory flow rate are dependent, rather than independent, variables and are not operator-specified. PCV is the preferred mode of ventilation for patients in whom it is desirable to regulate peak airway pressures, such as those with preexisting barotrauma, and for post– thoracic surgery patients, in whom the shear forces across a fresh suture line should be limited. When PCV is used, minute ventilation is altered through changes in rate or in the pressure-control value, with consequent changes in tidal volume.

1	inverse-ratio ventilation (irv) This mode is a variant of PCV that incorporates the use of a prolonged inspiratory time with the appropriate shortening of the expiratory time. IRV has been used in patients with severe hypoxemic respiratory failure. This approach increases mean distending pressures without increasing peak airway pressures. It is thought to work in conjunction with PEEP to open collapsed alveoli and improve oxygenation. However, no clinical-trial data have shown that IRV improves outcomes.

1	continuous Positive airway Pressure (cPaP) CPAP is not a true support mode of ventilation because all ventilation occurs through the patient’s spontaneous efforts. The ventilator provides fresh gas to the breathing circuit with each inspiration and sets the circuit to a constant, operator-specified pressure. CPAP is used to assess extubation potential in patients who have been effectively weaned and who require little ventilatory support and in patients with intact respiratory system function who require an endotracheal tube for airway protection.

1	Nonconventional Ventilatory Strategies Several nonconventional strategies have been evaluated for their ability to improve oxygenation and reduce mortality rates in patients with advanced hypoxemic respiratory failure. These strategies include high-frequency oscillatory ventilation (HFOV), airway pressure release ventilation (APRV), extracorporeal membrane oxygenation (ECMO), and partial liquid ventilation (PLV) using perfluorocarbons. Although case reports and small uncontrolled cohort studies have shown benefit, randomized controlled trials have failed to demonstrate consistent improvements in outcome with most of these strategies. A recent randomized trial of ECMO documented positive outcomes, but the technique remains controversial because older studies failed to document positive results. Currently, these approaches should be thought of as “salvage” techniques and considered for patients with hypoxemia refractory to conventional therapy. Prone positioning of patients with

1	results. Currently, these approaches should be thought of as “salvage” techniques and considered for patients with hypoxemia refractory to conventional therapy. Prone positioning of patients with refractory hypoxemia has also been explored because, in theory, lying prone should improve ventilation-perfusion matching. Several randomized trials in patients with acute lung injury did not demonstrate a survival advantage with prone positioning despite demonstration of a transient physiologic benefit. The administration of nitric oxide gas, which has bronchodilator and pulmonary vasodilator effects when delivered through the airways and improves arterial oxygenation in many patients with advanced hypoxemic respiratory failure, also failed to improve outcomes in these patients with acute lung injury.

1	The design of new ventilator modes reflect attempts to improve patient-ventilator synchrony—a major practical issue during MV—by allowing patients to trigger the ventilator with their own effort while also incorporating flow algorithms that terminate the cycles once certain preset criteria are reached; this approach has greatly improved patient comfort. New modes of ventilation that synchronize not only the timing but also the levels of assistance to match the patient’s effort have been developed. Proportional assist ventilation (PAV) and neurally adjusted ventilatory-assist ventilation (NAV) are two modes that are designed to deliver assisted breaths through algorithms incorporating not only pressure, volume, and time but also overall respiratory resistance as well as compliance (in the case of PAV) and neural activation of the diaphragm (in the case of NAV). Although these modes enhance patient-ventilator synchrony, their practical use in the everyday management of patients

1	(in the case of PAV) and neural activation of the diaphragm (in the case of NAV). Although these modes enhance patient-ventilator synchrony, their practical use in the everyday management of patients undergoing MV needs further study.

1	Whichever mode of MV is used in acute respiratory failure, the evidence from several important controlled trials indicates that a protective ventilation approach guided by the following principles (and summarized in Fig. 323-1) is safe and offers the best chance of a good outcome: (1) Set a target tidal volume close to 6 mL/kg of ideal body weight. (2) Prevent plateau pressure (static pressure in the airway at the end of inspiration) exceeding 30 cm H2O. (3) Use the lowest possible fraction of inspired oxygen (Fio2) to keep the Sao2 at ≥90%. (4) Adjust the PEEP to maintain alveolar patency while preventing overdistention and closure/reopening. With the application of these techniques, the mortality rate among patients with acute hypoxemic respiratory failure has decreased to ~30% from close to 50% a decade ago.

1	Once the patient has been stabilized with respect to gas exchange, definitive therapy for the underlying process responsible for respiratory failure is initiated. Subsequent modifications in ventilator therapy must be provided in parallel with changes in the patient’s clinical status. As improvement in respiratory function is noted, the first priority is to reduce the level of mechanical ventilatory support. Patients on full ventilatory support should be monitored frequently, with the goal of switching to a mode that allows for weaning as soon as possible. Protocols and guidelines that can be applied by paramedical personnel when physicians are not readily available have proved to be of value in shortening ventilator and intensive care unit (ICU) time, with very good outcomes. Patients whose condition continues to deteriorate after ventilatory support is initiated may require increased O2, PEEP, or one of the alternative modes of ventilation.

1	Patients for whom mechanical ventilation has been initiated usually require sedation and analgesia to maintain an acceptable level of comfort. Often, this treatment consists of a combination of a benzodiazepine and an opiate administered intravenously. Medications commonly used for this purpose include lorazepam, midazolam, diazepam, morphine, and fentanyl. Oversedation must be avoided in the ICU because most (but not all) studies show that daily interruption of sedation in patients with improved ventilatory status results in a shorter time on 1743 the ventilator and a shorter ICU stay.

1	Immobilized patients receiving mechanical ventilatory support are at risk for deep venous thrombosis and decubitus ulcers. Venous thrombosis should be prevented with the use of subcutaneous heparin and/or pneumatic compression boots. Fractionated low-molecularweight heparin appears to be equally effective for this purpose. To help prevent decubitus ulcers, frequent changes in body position and the use of soft mattress overlays and air mattresses are employed. Prophylaxis against diffuse gastrointestinal mucosal injury is indicated for patients undergoing MV. Histamine-receptor (H2-receptor) antagonists, antacids, and cytoprotective agents such as sucralfate have all been used for this purpose and appear to be effective. Nutritional support by enteral feeding through either a nasogastric or an orogastric tube should be initiated and maintained whenever possible. Delayed gastric emptying is common in critically ill patients taking sedative medications but often responds to promotility

1	or an orogastric tube should be initiated and maintained whenever possible. Delayed gastric emptying is common in critically ill patients taking sedative medications but often responds to promotility agents such as metoclopramide. Parenteral nutrition is an alternative to enteral nutrition in patients with severe gastrointestinal pathology who need prolonged MV.

1	Endotracheal intubation and mechanical ventilation have direct and indirect effects on the lung and upper airways, the cardiovascular system, and the gastrointestinal system. Pulmonary complications include barotrauma, nosocomial pneumonia, oxygen toxicity, tracheal stenosis, and deconditioning of respiratory muscles. Barotrauma and volutrauma overdistend and disrupt lung tissue; may be clinically manifest by interstitial emphysema, pneumomediastinum, subcutaneous emphysema, or pneumothorax; and can result in the liberation of cytokines from overdistended tissues, further promoting tissue injury. Clinically significant pneumothorax requires tube thoracostomy. Intubated patients are at high risk for ventilator-associated pneumonia as a result of aspiration from the upper airways through small leaks around the endotracheal tube cuff; the most common organisms responsible for this condition are Pseudomonas aeruginosa, enteric gram-negative rods, and Staphylococcus aureus. Given the high

1	leaks around the endotracheal tube cuff; the most common organisms responsible for this condition are Pseudomonas aeruginosa, enteric gram-negative rods, and Staphylococcus aureus. Given the high associated mortality rates, early initiation of empirical antibiotics directed against likely pathogens is recommended. Hypotension resulting from elevated intrathoracic pressures with decreased venous return is almost always responsive to intravascular volume repletion. In patients who are judged to have respiratory failure on the basis of alveolar edema but in whom the cardiac or pulmonary origin of the edema is unclear, hemodynamic monitoring with a pulmonary arterial catheter may be of value in helping to clarify the cause of the edema. Gastrointestinal effects of positive-pressure ventilation include stress ulceration and mild to moderate cholestasis.

1	WEANING FROM MECHANICAL VENTILATION The Decision to Wean It is important to consider discontinuation of mechanical ventilation once the underlying respiratory disease begins to reverse. Although the predictive capacities of multiple clinical and physiologic variables have been explored, the consensus from a ventilatory weaning task force cites the following conditions as indicating amenability to weaning: (1) Lung injury is stable or resolving. (2) Gas exchange is adequate, with low PEEP/Fio2 (<8 cmH2O) and Fio2(<0.5).

1	(3) Hemodynamic variables are stable, and the patient is no longer receiving vasopressors). (4) The patient is capable of initiating spontaneous breaths. A “wean screen” based on these variables should be done at least daily. If the patient is deemed capable of beginning to wean, the recommendation is to perform a spontaneous breathing trial (SBT), whose value is supported by several randomized trials (Fig. 323-2). The SBT involves an integrated patient assessment during spontaneous breathing with little or no ventilatory support. The SBT is usually implemented with a T-piece using 1–5 cmH2O CPAP with 5–7 cmH2O or PSV from the ventilator to offset resistance from the endotracheal tube. Once it is determined that the patient can breathe spontaneously, a decision must be made about the removal of the artificial airway, which should be undertaken only when it is concluded that the patient has the ability to protect the airway, is able to cough and clear

1	FIGURE 323-2 Flow chart to guide the daily approach to management of patients being considered for weaning off mechanical ventilation (MV). If attempts at extubation fail, a tracheostomy should be considered. SBT, spontaneous breathing trial.

1	Critical Care Medicine Approach to the Patient with Shock Ronald V. Maier Shock is the clinical syndrome that results from inadequate tissue perfusion. Irrespective of cause, the hypoperfusion-induced imbal-ance between the delivery of and requirements for oxygen and substrate leads to cellular dysfunction. The cellular injury created by the inadequate delivery of oxygen and substrates also induces the production and release of damage-associated molecular patterns (DAMPs or “danger signals”) and inflammatory mediators that further compromise perfusion through functional and structural changes within the microvasculature. This leads to a vicious cycle in which impaired perfusion is responsible for cellular injury that causes maldistribution of blood flow, further compromising cellular perfusion; the latter ultimately causes multiple organ failure (MOF) and, if the process is not interrupted, leads to death. The clinical manifestations of shock are also the result, in part, of autonomic

1	the latter ultimately causes multiple organ failure (MOF) and, if the process is not interrupted, leads to death. The clinical manifestations of shock are also the result, in part, of autonomic neuroendocrine responses to hypoperfusion as well as the break-down in organ function induced by severe cellular dysfunction (Fig. 324-1). When very severe and/or persistent, inadequate oxygen delivery leads to irreversible cell injury, only rapid restoration of oxygen 324 SEC TIon 2 SHoCK And CARdIAC ARREST delivery can reverse the progression of the shock state. The funda- 1744 Daily wean screen (resolving disease, adequate gas exchange, stable hemodynamics, spontaneous breathing ability) Pass Assess for extubation Fail NoYes SBT Pass Fail Continue MV Treat reversible elements Repeat daily screen Extubate Consider tracheostomy secretions, and is alert enough to follow commands. In addition, other factors must be taken into account, such as the possible difficulty of replacing the tube if that

1	Extubate Consider tracheostomy secretions, and is alert enough to follow commands. In addition, other factors must be taken into account, such as the possible difficulty of replacing the tube if that maneuver is required. If upper airway difficulty is suspected, an evaluation using a “cuff-leak” test (assessing the presence of air movement around a deflated endotracheal tube cuff) is supported by some internists. Despite all precautions, ~10–15% of extubated patients require reintubation. Several studies suggest that NIV can be used to obviate reintubation, particularly in patients with ventilatory failure secondary to COPD exacerbation; in this setting, earlier extubation with the use of prophylactic NIV has yielded good results. The use of NIV to facilitate weaning in respiratory failure of other etiologies is not currently indicated.

1	Prolonged Mechanical Ventilation and Tracheostomy From 5% to 13% of patients undergoing MV will go on to require prolonged MV (>21 days). In these instances, critical care personnel must decide whether and when to perform a tracheostomy. This decision is individualized and is based on the risk and benefits of tracheostomy and prolonged intubation as well as the patient’s preferences and expected outcomes. A tracheostomy is thought to be more comfortable, to require less sedation, and to provide a more secure airway and may also reduce weaning time. However, tracheostomy carries the risk of complications, which occur in 5–40% of these procedures and include bleeding, cardiopulmonary arrest, hypoxia, structural damage, pneumothorax, pneumomediastinum, and wound infection. In patients with long-term tracheostomy, complex complications include tracheal stenosis, granulation, and erosion of the innominate artery. In general, if a patient needs MV for more than 10–14 days, a tracheostomy,

1	long-term tracheostomy, complex complications include tracheal stenosis, granulation, and erosion of the innominate artery. In general, if a patient needs MV for more than 10–14 days, a tracheostomy, planned under optimal conditions, is indicated. Whether it is completed at the bedside or as an operative procedure depends on local resources and experience. Some 5–10% of patients are deemed unable to wean in the ICU. These patients may benefit from transfer to special units where a multidisciplinary approach, including nutrition optimization, physical therapy with rehabilitation, and slower weaning methods (including SIMV with PSV), results in successful weaning rates of up to 30%. Unfortunately, close to 2% of ventilated patients may ultimately become dependent on ventilatory support to maintain life. Most of these patients remain in chronic care institutions, although some with strong social, economic, and family support may live a relatively fulfilling life with at-home ventilation.

1	mental approach to management, therefore, is to recognize overt and impending shock in a timely fashion and to intervene emergently to restore perfusion. Doing so often requires the expansion or reexpansion of intravascular blood volume. Control of any inciting pathologic process (e.g., continued hemorrhage, impairment of cardiac function, or infection) must occur simultaneously. Clinical shock is usually accompanied by hypotension (i.e., a mean arterial pressure [MAP] <60 mmHg in previously normotensive persons). Multiple classification schemes have been developed in an attempt to synthesize the seemingly dissimilar processes leading to shock. Strict adherence to a classification scheme may be difficult from a clinical standpoint because of the frequent combination of two or more causes of shock in any individual patient, but the classification shown in Table 324-1 provides a useful reference point from which to discuss and further delineate the underlying processes.

1	Normally when cardiac output falls, systemic vascular resistance rises to maintain a level of systemic pressure that is adequate for perfusion of the heart and brain at the expense of other tissues such as muscle, skin, and especially the gastrointestinal (GI) tract. Systemic vascular resistance is determined primarily by the luminal diameter of arterioles. The metabolic rates of the heart and brain are high, and their stores of energy substrate are low. These organs are critically dependent on a FIGURE 324-1 Shock-induced vicious cycle. continuous supply of oxygen and nutrients, and neither tolerates severe ischemia for more than brief periods (minutes). Autoregulation (i.e., the maintenance of blood flow over a wide range of perfusion pressures) is critical in sustaining cerebral and coronary perfusion despite significant hypotension. However, when MAP drops to ≤60 mmHg, blood flow to these organs falls, and their function deteriorates.

1	Arteriolar vascular smooth muscle has both αand β-adrenergic receptors. The α1 receptors mediate vasoconstriction, while the β2 receptors mediate vasodilation. Efferent sympathetic fibers release norepinephrine, which acts primarily on α1 receptors as one of the most fundamental compensatory responses to reduced perfusion pressure. Other constrictor substances that are increased in most forms of shock include angiotensin II, vasopressin, endothelin 1, and thromboxane A2. Both norepinephrine and epinephrine are released by the adrenal medulla, and the concentrations of these catecholamines in the bloodstream rise. Circulating vasodilators in shock include prostacyclin (prostaglandin [PG] I2), nitric oxide (NO), and, importantly, products of local metabolism such as adenosine that match flow to the tissue’s metabolic needs. The balance between these various vasoconstrictors and vasodilators influences the microcirculation and determines local perfusion.

1	Transport to cells depends on microcirculatory flow; capillary permeability; the diffusion of oxygen, carbon dioxide, nutrients, and products of metabolism through the interstitium; and the exchange of these products across cell membranes. Impairment of the micro-circulation that is central to the pathophysiologic responses in the late stages of all forms of shock results in the derangement of cellular metabolism that is ultimately responsible for organ failure. The endogenous response to mild or moderate hypovolemia is an attempt at restitution of intravascular volume through alterations in hydrostatic pressure and osmolarity. Constriction of arterioles leads to reductions in both the capillary hydrostatic pressure and the number of capillary beds perfused, thereby limiting the capillary surface area across which filtration occurs. When filtration is reduced while intravascular oncotic pressure remains constant or rises, there is net

1	ClASSIfICATIon of SHoCK reabsorption of fluid into the vascular bed, in 1745 accord with Starling’s law of capillary interstitial liquid exchange. Metabolic changes (including hyperglycemia and elevations in the products of glycolysis, lipolysis, and proteolysis) raise extracellular osmolarity, leading to an osmotic gradient that increases interstitial and intravascular volume at the expense of intracellular volume.

1	Interstitial transport of nutrients is impaired in shock, leading to a decline in intracellular high-energy phosphate stores. Mitochondrial dysfunction and uncoupling of oxidative phosphorylation are the most likely causes for decreased amounts of adenosine triphosphate (ATP). As a consequence, there is an accumulation of hydrogen ions, lactate, reactive oxygen species, and other products of anaerobic metabolism. As shock progresses, these vasodilator metabolites override vasomotor tone, causing fur ther hypotension and hypoperfusion. Dysfunction of cell membranes is thought to represent a common end-stage pathophysiologic pathway in the various forms of shock. Normal cellular transmembrane potential falls, and there is an associated increase in intracellular sodium and water, leading to cell swelling that interferes further with microvascular perfusion. In a preterminal event, homeostasis of calcium via membrane channels is lost with flooding of calcium into the cytosol and

1	to cell swelling that interferes further with microvascular perfusion. In a preterminal event, homeostasis of calcium via membrane channels is lost with flooding of calcium into the cytosol and concomitant extracellular hypocalcemia. There is also evidence for a widespread but selective apoptotic (programmed cell death) loss of cells, contributing to organ and immune failure.

1	Hypovolemia, hypotension, and hypoxia are sensed by baroreceptors and chemoreceptors that contribute to an autonomic response that attempts to restore blood volume, maintain central perfusion, and mobilize metabolic substrates. Hypotension disinhibits the vasomotor center, resulting in increased adrenergic output and reduced vagal activity. Release of norepinephrine from adrenergic neurons induces significant peripheral and splanchnic vasoconstriction, a major contributor to the maintenance of central organ perfusion, while reduced vagal activity increases the heart rate and cardiac output. Loss of vagal activity is also recognized to upregulate the innate immune inflammatory response. The effects of circulating epinephrine released by the adrenal medulla in shock are largely metabolic, causing increased glycogenolysis and gluconeogenesis and reduced pancreatic insulin release. However, epinephrine also inhibits production and release of inflammatory mediators through stimulation of

1	causing increased glycogenolysis and gluconeogenesis and reduced pancreatic insulin release. However, epinephrine also inhibits production and release of inflammatory mediators through stimulation of β-adrenergic receptors on innate immune cells.

1	Severe pain or other stresses cause the hypothalamic release of adrenocorticotropic hormone (ACTH). This stimulates cortisol secretion that contributes to decreased peripheral uptake of glucose and amino acids, enhances lipolysis, and increases gluconeogenesis. Increased pancreatic secretion of glucagon during stress accelerates hepatic gluconeogenesis and further elevates blood glucose concentration. These hormonal actions act synergistically to increase blood glucose for both selective tissue metabolism and the maintenance of blood volume. Many critically ill patients have recently been shown to exhibit low plasma cortisol levels and an impaired response to ACTH stimulation, which is linked to a decrease in survival. The importance of the cortisol response to stress is illustrated by the profound circulatory collapse that occurs in patients with adrenocortical insufficiency (Chap. 406).

1	Renin release is increased in response to adrenergic discharge and reduced perfusion of the juxtaglomerular apparatus in the kidney. Renin induces the formation of angiotensin I that is then converted to angiotensin II by the angiotensin converting enzyme; angiotensin II is an extremely potent vasoconstrictor and stimulator of aldosterone release by the adrenal cortex and of vasopressin by the posterior pituitary. Aldosterone contributes to the maintenance of intravascular Approach to the Patient with Shock 1746 volume by enhancing renal tubular reabsorption of sodium, resulting in the excretion of a low-volume, concentrated, sodium-free urine. Vasopressin has a direct action on vascular smooth muscle, contributing to vasoconstriction, and acts on the distal renal tubules to enhance water reabsorption.

1	Three variables—ventricular filling (preload), the resistance to ventricular ejection (afterload), and myocardial contractility—are paramount in controlling stroke volume (Chap. 265e). Cardiac output, the major determinant of tissue perfusion, is the product of stroke volume and heart rate. Hypovolemia leads to decreased ventricular preload that, in turn, reduces the stroke volume. An increase in heart rate is a useful but limited compensatory mechanism to maintain cardiac output. A shock-induced reduction in myocardial compliance is frequent, reducing ventricular end-diastolic volume and, hence, stroke volume at any given ventricular filling pressure. Restoration of intravascular volume may return stroke volume to normal but only at elevated filling pressures. Increased filling pressures stimulate release of brain natriuretic peptide (BNP) to secrete sodium and volume to relieve the pressure on the heart. Levels of BNP correlate with outcome following severe stress. In addition,

1	stimulate release of brain natriuretic peptide (BNP) to secrete sodium and volume to relieve the pressure on the heart. Levels of BNP correlate with outcome following severe stress. In addition, sepsis, ischemia, myocardial infarction (MI), severe tissue trauma, hypothermia, general anesthesia, prolonged hypotension, and acidemia may all also impair myocardial contractility and reduce the stroke volume at any given ventricular end-diastolic volume. The resistance to ventricular ejection is significantly influenced by the systemic vascular resistance, which is elevated in most forms of shock. However, resistance is decreased in the early hyperdynamic stage of septic shock or neurogenic shock (Chap. 325), thereby initially allowing the cardiac output to be maintained or elevated.

1	The venous system contains nearly two-thirds of the total circulating blood volume, most in the small veins, and serves as a dynamic reservoir for autoinfusion of blood. Active venoconstriction as a consequence of α-adrenergic activity is an important compensatory mechanism for the maintenance of venous return and, therefore, of ventricular filling during shock. By contrast, venous dilation, as occurs in neurogenic shock, reduces ventricular filling and hence stroke volume and potentially cardiac output.

1	The response of the pulmonary vascular bed to shock parallels that of the systemic vascular bed, and the relative increase in pulmonary vascular resistance, particularly in septic shock, may exceed that of the systemic vascular resistance, leading to right heart failure. Shock-induced tachypnea reduces tidal volume and increases both dead space and minute ventilation. Relative hypoxia and the subsequent tachypnea induce a respiratory alkalosis. Recumbency and involuntary restriction of ventilation secondary to pain reduce functional residual capacity and may lead to atelectasis. Shock and, in particular, resuscitation-induced reactive oxygen species (oxidant radical) generation are recognized as major causes of acute lung injury and subsequent acute respiratory distress syndrome (ARDS; Chap. 322). These disorders are characterized by noncardiogenic pulmonary edema secondary to diffuse pulmonary capillary endothelial and alveolar epithelial injury, hypoxemia, and bilateral diffuse

1	Chap. 322). These disorders are characterized by noncardiogenic pulmonary edema secondary to diffuse pulmonary capillary endothelial and alveolar epithelial injury, hypoxemia, and bilateral diffuse pulmonary infiltrates. Hypoxemia results from perfusion of underventilated and nonventilated alveoli. Loss of surfactant and lung volume in combination with increased interstitial and alveolar edema reduces lung compliance. The work of breathing and the oxygen requirements of respiratory muscles increase.

1	Acute kidney injury (Chap. 334), a serious complication of shock and hypoperfusion, occurs less frequently than heretofore because of early aggressive volume repletion. Acute tubular necrosis is now more frequently seen as a result of the interactions of shock, sepsis, the administration of nephrotoxic agents (such as aminoglycosides and angiographic contrast media), and rhabdomyolysis; the latter may be particularly severe in skeletal muscle trauma. The physiologic response of the kidney to hypoperfusion is to conserve salt and water. In addition to decreased renal blood flow, increased afferent arteriolar resistance accounts for diminished glomerular filtration rate (GFR) that, together with increased aldosterone and vasopressin, is responsible for reduced urine formation. Toxic injury causes necrosis of tubular epithelium and tubular obstruction by cellular debris with back leak of filtrate. The depletion of renal ATP stores that occurs with prolonged renal hypoperfusion

1	injury causes necrosis of tubular epithelium and tubular obstruction by cellular debris with back leak of filtrate. The depletion of renal ATP stores that occurs with prolonged renal hypoperfusion contributes to subsequent impairment of renal function.

1	During shock, there is disruption of the normal cycles of carbohydrate, lipid, and protein metabolism. Through the citric acid cycle, alanine in conjunction with lactate, which is converted from pyruvate in the periphery in the presence of oxygen deprivation, enhances the hepatic production of glucose. With reduced availability of oxygen, the breakdown of glucose to pyruvate, and ultimately lactate, represents an inefficient cycling of substrate with minimal net energy production. An elevated plasma lactate/pyruvate ratio is preferable to lactate alone as a measure of anaerobic metabolism and reflects inadequate tissue perfusion. Decreased clearance of exogenous triglycerides coupled with increased hepatic lipogenesis causes a significant rise in serum triglyceride concentrations. There is increased protein catabolism as energy substrate, a negative nitrogen balance, and, if the process is prolonged, severe muscle wasting.

1	Activation of an extensive network of proinflammatory mediator pathways by the innate immune system plays a significant role in the progression of shock and contributes importantly to the development of multiple organ injury, multiple organ dysfunction (MOD), and MOF (Fig. 324-2). In those surviving the acute insult, there is a prolonged endogenous counterregulatory response to “turn off” or balance the excessive proinflammatory response. If balance is restored, the patient does well. If the response is excessive, adaptive immunity is suppressed and the patient is highly susceptible to secondary nosocomial infections, which may then drive the inflammatory response and lead to delayed MOF.

1	Multiple humoral mediators are activated during shock and tissue injury. The complement cascade, activated through both the classic and alternate pathways, generates the anaphylatoxins C3a and C5a (Chap. 372e). Direct complement fixation to injured tissues can progress to the C5-C9 attack complex, causing further cell damage. Activation of the coagulation cascade (Chap. 141) causes microvascular thrombosis, with subsequent fibrinolysis leading to repeated episodes of ischemia and reperfusion. Components of the coagulation system (e.g., thrombin) are potent proinflammatory mediators that cause expression of adhesion molecules on endothelial cells and activation of neutrophils, leading to microvascular injury. Coagulation also activates the kallikrein-kininogen cascade, contributing to hypo-tension.

1	Eicosanoids are vasoactive and immunomodulatory products of arachidonic acid metabolism that include cyclooxygenase-derived prostaglandins (PGs) and thromboxane A2, as well as lipoxygenasederived leukotrienes and lipoxins. Thromboxane A2 is a potent vasoconstrictor that contributes to the pulmonary hypertension and acute tubular necrosis of shock. PGI2 and PGE2 are potent vasodilators that enhance capillary permeability and edema formation. The cysteinyl leukotrienes LTC4 and LTD4 are pivotal mediators of the vascular sequelae of anaphylaxis, as well as of shock states resulting from sepsis or tissue injury. LTB4 is a potent neutrophil chemoattractant and secretagogue that stimulates the formation of reactive oxygen species. Platelet-activating factor, an ether-linked, arachidonyl-containing phospholipid mediator, causes pulmonary vasoconstriction, bronchoconstriction, systemic vasodilation, increased capillary permeability, and the priming of macrophages and neutrophils to produce

1	phospholipid mediator, causes pulmonary vasoconstriction, bronchoconstriction, systemic vasodilation, increased capillary permeability, and the priming of macrophages and neutrophils to produce enhanced levels of inflammatory mediators.

1	Tumor necrosis factor α (TNF-α), produced by activated macrophages, reproduces many components of the shock state, including • TNF-˜, IL-1°, IL-6, IL-12, IL-18, PGE2, TGF-° Lymphocytes • TH1˙TH2 • ˛IL-2, IL-2R, IFN-ˆ, TNF-°• ˝IL-4, IL-10, IL-5, IL-13 Coagulation/ Complement ˝C3a ˝C5a ˝Thrombin ˝D-Dimers ˛Antithrombin III (AT III) ˛Activated protein C (APC) FIGURE 324-2 A schematic of the host immunoinflammatory response to shock. IFN, interferon; IL, interleukin; PG, prostaglandin; TGF, tumor growth factor; TNF, tumor necrosis factor.

1	hypotension, lactic acidosis, and respiratory failure. Interleukin 1β (IL-1β), originally defined as “endogenous pyrogen” and produced by tissue-fixed macrophages, is critical to the inflammatory response. Both are significantly elevated immediately following trauma and shock. IL-6, also produced predominantly by the macrophage, has a slightly delayed peak response but is the best single predictor of prolonged recovery and development of MOF following shock. Chemokines such as IL-8 are potent neutrophil chemoattractants and activators that upregulate adhesion molecules on the neutrophil to enhance aggregation, adherence, and damage to the vascular endothelium. While the endothelium normally produces low levels of NO, the inflammatory response stimulates the inducible isoform of NO synthase (iNOS), which is overexpressed and produces toxic nitroxyland oxygen-derived free radicals that contribute to the hyperdynamic cardiovascular response and tissue injury in sepsis.

1	Multiple inflammatory cells, including neutrophils, macrophages, and platelets, are major contributors to inflammation-induced injury. Margination of activated neutrophils in the microcirculation is a common pathologic finding in shock, causing secondary injury due to the release of toxic oxygen radicals, lipases (primarily PLA2), and proteases. Release of high levels of reactive oxygen intermediates/species (ROI/ROS) rapidly consumes endogenous essential antioxidants and generates diffuse oxygen radical damage. Newer efforts to control ischemia/reperfusion injury include treatment with carbon monoxide, hydrogen sulfide, or other agents to reduce oxidant stress. Tissue-fixed macrophages produce virtually all major mediators of the inflammatory response and orchestrate the progression and duration of the inflammatory response. A major source of activation of the monocyte/macrophage is through the highly conserved membrane toll-like receptors (TLRs) that recognize DAMPs, such as HMGB-1,

1	of the inflammatory response. A major source of activation of the monocyte/macrophage is through the highly conserved membrane toll-like receptors (TLRs) that recognize DAMPs, such as HMGB-1, and pathogen-associated molecular patterns (PAMPs), such as endotoxins released following tissue injury, and by pathogenic microbial organisms, respectively. TLRs also appear important in the chronic inflammation seen in Crohn’s disease, ulcerative colitis, and transplant rejection. The variability in individual responses is a genetic predisposition that, in part, is due to variants in genetic sequences affecting the function and production of various inflammatory mediators.

1	Patients in shock require care in an intensive care unit (ICU). Careful and continuous assessment of the physiologic status is necessary. Arterial pressure through an indwelling line, pulse, and respiratory rate should be monitored continuously; a Foley catheter should be inserted to follow urine flow; and mental status should be assessed frequently. Sedated patients should be allowed to awaken (“drug holiday”) daily to assess their neurologic status and to shorten duration of ventilator support.

1	There is ongoing debate as to the indications for using the flow-directed pulmonary artery catheter (PAC; Swan-Ganz catheter) in the ICU. A recent Cochrane analysis showed that the use of a PAC did not alter mortality, length of stay, or cost for adult ICU patients. Most patients in the ICU can be safely managed without the use of a PAC. However, in shock with significant ongoing blood loss, fluid shifts, and underlying cardiac dysfunction, a PAC may be useful. The PAC is placed percutaneously via the subclavian or jugular vein through the central venous circulation and right heart into the pulmonary artery. There are ports both proximal in the right atrium and distal in the pulmonary artery to provide access for infusions and for cardiac output measurements. Right atrial and pulmonary artery pressures (PAPs) are measured, and the pulmonary capillary wedge pressure (PCWP) serves as an approximation of the left atrial pressure. Normal hemodynamic parameters and their derivation are

1	artery pressures (PAPs) are measured, and the pulmonary capillary wedge pressure (PCWP) serves as an approximation of the left atrial pressure. Normal hemodynamic parameters and their derivation are summarized in Table 272-2 and Table 324-2.

1	Cardiac output is determined by the thermodilution technique, and high-resolution thermistors can also be used to determine right ventricular end-diastolic volume to monitor further the response of the right heart to fluid resuscitation. A PAC with an oximeter port offers the additional advantage of online monitoring of the mixed venous oxygen saturation, an important index of overall tissue perfusion. Systemic and pulmonary vascular resistances are calculated as the ratio of the pressure drop across these vascular beds to the cardiac output (Chap. 272). Determinations of oxygen content in arterial and venous blood, together with cardiac output and hemoglobin concentration, allow calculation of oxygen delivery, Approach to the Patient with Shock Cardiac index (CI) CO/BSA 2.6–4.2 (L/min)/m2 Left ventricular stroke SV(MAP – PCWP) × 60–80 g-m/beat work (LVSW) 0.0136

1	Approach to the Patient with Shock Cardiac index (CI) CO/BSA 2.6–4.2 (L/min)/m2 Left ventricular stroke SV(MAP – PCWP) × 60–80 g-m/beat work (LVSW) 0.0136 Abbreviations: BSA, body surface area; HR, heart rate; MAP, mean arterial pressure; PAPm, pulmonary artery pressure—mean; PCWP, pulmonary capillary wedge pressure; RAP, right atrial pressure. oxygen consumption, and oxygen-extraction ratio (Table 324-3). The hemodynamic patterns associated with the various forms of shock are shown in Table 324-4.

1	In resuscitation from shock, it is critical to restore tissue perfusion and optimize oxygen delivery, hemodynamics, and cardiac function rapidly. A reasonable goal of therapy is to achieve a normal mixed venous oxygen-saturation and arteriovenous oxygen-extraction ratio. To enhance oxygen delivery, red cell mass, arterial oxygen saturation, and cardiac output may be augmented singly or simultaneously. An increase in oxygen delivery not accompanied by an increase in oxygen consumption implies that oxygen availability is adequate and that oxygen consumption is not flow dependent. Conversely, an elevation of oxygen consumption with increased delivery implies that the oxygen supply was inadequate. However, cautious interpretation is required due to the link among increased oxygen delivery, cardiac work, and oxygen consumption. A reduction in systemic vascular resistance accompanying an increase in cardiac output indicates that compensatory vasoconstriction is reversing due to improved

1	cardiac work, and oxygen consumption. A reduction in systemic vascular resistance accompanying an increase in cardiac output indicates that compensatory vasoconstriction is reversing due to improved tissue perfusion. The determination of stepwise expansion of blood volume on cardiac performance allows identification of the optimum preload (Starling’s law). An algorithm for the resuscitation of the patient in shock is shown in Fig. 324-3.

1	1.39 mL/g of hemoglobin Plasma O2 concentration PO2 × 0.0031 Arterial O2 concentration 1.39 SaO2 + 0.0031 PaO2 20 vol% (CaO2) Venous O2 concentration 1.39 SvO2 + 0.0031 PvO2 15.5 vol% (CvO2) Arteriovenous O2 1.39 (SaO2 – SvO2) + 3.5 vol% difference (CaO2 – CvO2) 0.0031 (PaO2 – PvO2) 1.39 SaO2 × CO × 10 1.39 (SaO2 – SvO2) × CO × 10 Abbreviations: BSA, body surface area; CO, cardiac output; PO2, partial pressure of oxygen; PaO2, partial pressure of oxygen in arterial blood; PvO2, partial pressure of oxygen in venous blood; SaO2, saturation of hemoglobin with oxygen in arterial blood; SvO2, saturation of hemoglobin with oxygen in venous blood. CVP and Cardiac Systemic Vascular Venous O2 Type of Shock PCWP Output Resistance Saturation Hyperdynamic ↓↑↑↓ ↑ Hypodynamic ↓↑↓↑ ↓↑ Traumatic ↓↓↑↑↓ ↓ Neurogenic ↓↓↓ ↓ Hypoadrenal ↓↓ =↓↓ Abbreviations: CVP, central venous pressure; PCWP, pulmonary capillary wedge pressure.

1	This most common form of shock results either from the loss of red blood cell mass and plasma from hemorrhage or from the loss of plasma volume alone due to extravascular fluid sequestration or GI, urinary, and insensible losses. The signs and symptoms of nonhemorrhagic hypovolemic shock are the same as those of hemorrhagic shock, although they may have a more insidious onset. The normal physiologic response to hypovolemia is to maintain perfusion of the brain and heart while attempting to restore an effective circulating blood volume. There is an increase in sympathetic activity, hyperventilation, collapse of venous capacitance vessels, release of stress hormones, and an attempt to replace the loss of intravascular volume through the recruitment of interstitial and intracellular fluid and by reduction of urine output.

1	Mild hypovolemia (≤20% of the blood volume) generates mild tachycardia but relatively few external signs, especially in a supine young patient (Table 324-5). With moderate hypovolemia (~20–40% of the blood volume), the patient becomes increasingly anxious and tachycardic; although normal blood pressure may be maintained in the supine position, there may be significant postural hypotension and tachycardia. If hypovolemia is severe (≥40% of the blood volume), the classic signs of shock appear; the blood pressure declines and becomes unstable even in the supine position, and the patient develops marked tachycardia, oliguria, and agitation or confusion. Perfusion of the central nervous system is well maintained until shock becomes severe. Hence, mental obtundation is an ominous clinical sign. The transition from mild to severe hypovolemic shock can be insidious or extremely rapid. If severe shock is not reversed rapidly, especially in elderly patients and those with comorbid illnesses,

1	The transition from mild to severe hypovolemic shock can be insidious or extremely rapid. If severe shock is not reversed rapidly, especially in elderly patients and those with comorbid illnesses, death is imminent. A very narrow time frame separates the derangements found in severe shock that can be reversed with aggressive resuscitation from those of progressive decompensation and irreversible cell injury.

1	Diagnosis Hypovolemic shock is readily diagnosed when there are signs of hemodynamic instability and the source of volume loss is obvious. The diagnosis is more difficult when the source of blood loss is occult, as into the GI tract, or when plasma volume alone is depleted. Even after acute hemorrhage, hemoglobin and hematocrit values do not change until compensatory fluid shifts have occurred or exogenous fluid is administered. Thus, an initial normal hematocrit does not disprove the presence of significant blood loss. Plasma losses cause hemoconcentration, and free water loss leads to hypernatremia. These findings should suggest the presence of hypovolemia.

1	It is essential to distinguish between hypovolemic and cardiogenic shock (Chap. 326) because, although both may respond to volume initially, definitive therapy differs significantly. Both forms are associated with a reduced cardiac output and a compensatory sympathetic mediated response characterized by tachycardia and elevated systemic vascular resistance. However, the findings in cardiogenic shock of jugular venous distention, rales, and an S3 gallop distinguish it from hypovolemic shock and signify that ongoing volume expansion is undesirable and may cause further organ dysfunction. CI <3.5; PCWP <15Administer crystalloid+/– blood PCWP >15, Hct >30 CI <3.5; 15< PCWP <20CI <3.5; PCWP >20 Administer 500 mL crystalloidboluses until preload maximal CI • Inotrope as indicated(Starling’s curve)• Consider ECHO

1	CI <3.5; 15< PCWP <20CI <3.5; PCWP >20 Administer 500 mL crystalloidboluses until preload maximal CI • Inotrope as indicated(Starling’s curve)• Consider ECHO FIGURE 324-3 An algorithm for the resuscitation of the patient in shock. *Monitor SvO2, SVRI, and RVEDVI as additional markers of correction for perfusion and hypovolemia. Consider age-adjusted CI. CI, cardiac index in (L/min) per m2; CVP, central venous pressure; ECHO, echocardiogram; Hct, hematocrit; HR, heart rate; PAC, pulmonary artery catheter; PCWP, pulmonary capillary wedge pressure in mmHg; RVEDVI, right ventricular end-diastolic volume index; SBP, systolic blood pressure; SvO2, saturation of hemoglobin with O2 in venous blood; SVRI, systemic vascular resistance index; VS, vital signs; W/U, workup.

1	Initial resuscitation requires rapid reexpansion of the circulating intravascular blood volume along with interventions to control ongoing losses. In accordance with Starling’s law (Chap. 265e), stroke volume and cardiac output rise with the increase in preload. After resuscitation, the compliance of the ventricles may remain reduced due to increased interstitial fluid in the myocardium. Therefore, elevated filling pressures are frequently required to maintain adequate ventricular performance.

1	Volume resuscitation is initiated with the rapid infusion of either isotonic saline (although care must be taken to avoid hyperchloremic acidosis from loss of bicarbonate buffering capacity and replacement with excess chloride) or a balanced salt solution such as Ringer’s lactate (being cognizant of the presence of potassium and potential renal dysfunction) through large-bore intravenous lines. Data, particularly on severe traumatic brain injury (TBI), regarding benefits of small volumes of hypertonic saline that more rapidly restore blood pressure are variable but tend to show improved survival thought to be linked to immunomodulation. No distinct benefit from the use of colloid has been demonstrated, and in trauma patients, it is associated with a higher mortality particularly in patients with TBI. The infusion of 2–3 L of salt solution over 20–30 min should restore normal hemodynamic parameters. Continued hemodynamic instability implies that shock has not been reversed and/or there

1	with TBI. The infusion of 2–3 L of salt solution over 20–30 min should restore normal hemodynamic parameters. Continued hemodynamic instability implies that shock has not been reversed and/or there are significant ongoing blood or other volume losses. Continuing acute blood loss with hemoglobin concentrations declining to ≤100 g/L (10 g/dL) should initiate blood transfusion preferably as fully cross-matched, recently banked (<14 days old) blood. Resuscitated patients are often coagulopathic due

1	Approach to the Patient with Shock Same, plus: Tachycardia Tachypnea Oliguria Postural

1	Same, plus: 1750 to deficient clotting factors in crystalloids and banked packed red blood cells (PRBCs). Early administration of component therapy during massive transfusion (fresh-frozen plasma [FFP] and platelets) approaching a 1:1 ratio of PRBC/FFP appears to improve survival. In extreme emergencies, type-specific or O-negative packed red cells may be transfused. Following severe and/or prolonged hypovolemia, inotropic support with norepinephrine, vasopressin, or dopamine may be required to maintain adequate ventricular performance but only after blood volume has been restored. Increases in peripheral vasoconstriction with inadequate resuscitation lead to tissue loss and organ failure. Once hemorrhage is controlled and the patient has stabilized, blood transfusions should not be continued unless the hemoglobin is <~7 g/dL. Studies have demonstrated an increased survival in patients treated with this restrictive blood transfusion protocol. Successful resuscitation also requires

1	unless the hemoglobin is <~7 g/dL. Studies have demonstrated an increased survival in patients treated with this restrictive blood transfusion protocol. Successful resuscitation also requires support of respiratory function. Supplemental oxygen should always be provided, and endotracheal intubation may be necessary to maintain arterial oxygenation. Following resuscitation from isolated hemorrhagic shock, end-organ damage is frequently less than following septic or traumatic shock. This may be due to the absence of massive activation of the inflammatory innate immune response and consequent nonspecific organ injury and failure.

1	Shock following trauma is, in large measure, due to hemorrhage. However, even when hemorrhage has been controlled, patients can continue to suffer loss of plasma volume into the interstitium of injured tissues. These fluid losses are compounded by injury-induced inflammatory responses, which contribute to the secondary microcirculatory injury. Proinflammatory mediators are induced by DAMPs released from injured tissue and are recognized by the highly conserved membrane receptors of the TLR family (see “Inflammatory Responses” above). These receptors on cells of the innate immune system, particularly the circulating monocyte, tissue-fixed macrophage, and dendritic cell, are potent activators of an excessive proinflammatory phenotype in response to cellular injury. This causes secondary tissue injury and maldistribution of blood flow, intensifying tissue ischemia and leading to multiple organ system failure. In addition, direct structural injury to the heart, chest, or head can also

1	tissue injury and maldistribution of blood flow, intensifying tissue ischemia and leading to multiple organ system failure. In addition, direct structural injury to the heart, chest, or head can also contribute to shock. For example, pericardial tamponade or tension pneumothorax impairs ventricular filling, whereas myocardial contusion depresses myocardial contractility.

1	Inability of the patient to maintain a systolic blood pressure ≥90 mmHg after trauma-induced hypovolemia is associated with a mortality rate up to ~50%. To prevent this decompensation of homeostatic mechanisms, therapy must be promptly administered. The initial management of the seriously injured patient requires attention to the “ABCs” of resuscitation: assurance of an airway (A), adequate ventilation (breathing, B), and establishment of an adequate blood volume to support the circulation (C). Control of ongoing hemorrhage requires immediate attention. Early stabilization of fractures, debridement of devitalized or contaminated tissues, and evacuation of hematomata all reduce the subsequent inflammatory response to the initial insult and minimize damaged tissue release of DAMPs and subsequent diffuse organ injury. Supplementation of depleted endogenous antioxidants also reduces subsequent organ failure and mortality. See Chap. 326.

1	See Chap. 326. With extrinsic compression, the heart and surrounding structures are less compliant, and therefore, normal filling pressures generate inadequate diastolic filling and stroke volume. Blood or fluid within the poorly distensible pericardial sac may cause tamponade (Chap. 288). Any cause of increased intrathoracic pressure, such as tension pneumothorax, herniation of abdominal viscera through a diaphragmatic hernia, or excessive positive-pressure ventilation to support pulmonary function, can also initiate compressive cardiogenic shock while simultaneously impeding venous return and preload. Although initially responsive to increased filling pressures produced by volume expansion, as compression increases, cardiogenic shock recurs. The window of opportunity gained by volume loading may be very brief until irreversible shock recurs. Diagnosis and intervention must occur urgently.

1	The diagnosis of compressive cardiogenic shock is most frequently based on clinical findings, the chest radiograph, and an echocardiogram. The diagnosis of compressive cardiac shock may be more difficult to establish in the setting of trauma when hypovolemia and cardiac compression are present simultaneously. The classic findings of pericardial tamponade include the triad of hypotension, neck vein distention, and muffled heart sounds (Chap. 288). Pulsus paradoxus (i.e., an inspiratory reduction in systolic pressure >10 mmHg) may also be noted. The diagnosis is confirmed by echocardiography, and treatment consists of immediate pericardiocentesis or the creation of an open subxiphoid pericardial window. A tension pneumothorax produces ipsilateral decreased breath sounds, tracheal deviation away from the affected thorax, and jugular venous distention. Radiographic findings include increased intrathoracic volume, depression of the diaphragm of the affected hemithorax, and shifting of the

1	away from the affected thorax, and jugular venous distention. Radiographic findings include increased intrathoracic volume, depression of the diaphragm of the affected hemithorax, and shifting of the mediastinum to the contralateral side. Chest decompression must be carried out immediately and, ideally, should occur based on clinical findings rather than awaiting a chest radiograph. Release of air and restoration of normal cardiovascular dynamics are both diagnostic and therapeutic.

1	See Chap. 325. Interruption of sympathetic vasomotor input after a high cervical spinal cord injury, inadvertent cephalad migration of spinal anesthesia, or devastating head injury may result in neurogenic shock. In addition to arteriolar dilation, venodilation causes pooling in the venous system, which decreases venous return and cardiac output. The extremities are often warm, in contrast to the usual sympathetic vasoconstrictioninduced coolness in hypovolemic or cardiogenic shock. Treatment involves a simultaneous approach to the relative hypovolemia and to the loss of vasomotor tone. Excessive volumes of fluid may be required to restore normal hemodynamics if given alone. Once hemorrhage has been ruled out, norepinephrine or a pure α-adrenergic agent (phenylephrine) may be necessary to augment vascular resistance and maintain an adequate MAP.

1	(See also Chap. 406) The normal host response to the stress of illness, operation, or trauma requires that the adrenal glands hypersecrete cortisol in excess of that normally required. Hypoadrenal shock occurs in settings in which unrecognized adrenal insufficiency complicates the host response to the stress induced by acute illness or major surgery. Adrenocortical insufficiency may occur as a consequence of the chronic administration of high doses of exogenous glucocorticoids. In addition, recent studies have shown that critical illness, including trauma and sepsis, may also induce a relative hypoadrenal state. Other, less common causes include adrenal insufficiency secondary to idiopathic atrophy, use of etomidate for intubation, tuberculosis, metastatic disease, bilateral hemorrhage, and amyloidosis. The shock produced by adrenal insufficiency is characterized by loss of homeostasis with reductions in systemic vascular resistance, hypovolemia, and reduced cardiac output. The

1	and amyloidosis. The shock produced by adrenal insufficiency is characterized by loss of homeostasis with reductions in systemic vascular resistance, hypovolemia, and reduced cardiac output. The diagnosis of adrenal insufficiency may be established by means of an ACTH stimulation test.

1	In the persistently hemodynamically unstable patient, dexamethasone sodium phosphate, 4 mg, should be given intravenously. This agent is preferred if empiric therapy is required because, unlike hydrocortisone, it does not interfere with the ACTH stimulation test. If the diagnosis of absolute or relative adrenal insufficiency is established as shown by nonresponse to corticotropin stimulation (cortisol ≤9 μg/dL change after stimulation), the patient has a reduced risk of death if treated with hydrocortisone, 100 mg every 6–8 h, and tapered as the patient achieves hemodynamic stability. Simultaneous volume resuscitation and pressor support are required. The need for simultaneous mineralocoid is unclear.

1	The sympathomimetic amines dobutamine, dopamine, and nor-epinephrine are widely used in the treatment of all forms of shock. Dobutamine is inotropic with simultaneous afterload reduction, thus minimizing cardiac-oxygen consumption increases as cardiac output increases. Dopamine is an inotropic and chronotropic agent that also supports vascular resistance in those whose blood pressure will not tolerate peripheral vascular dilation. Norepinephrine primarily supports blood pressure through vasoconstriction and increases myocardial oxygen consumption while placing marginally perfused tissues, such as extremities and splanchnic organs, at risk for ischemia or necrosis, but it is also inotropic without significant chronotropy. Argininevasopressin (antidiuretic hormone) is being used increasingly to increase afterload and may better protect vital organ blood flow and prevent pathologic vasodilation.

1	Hypothermia is a frequent adverse consequence of massive volume resuscitation (Chap. 478e). The infusion of large volumes of refrigerated blood products and room temperature crystalloid solutions can rapidly drop core temperatures if fluid is not run through warming devices. Hypothermia may depress cardiac contractility and thereby further impair cardiac output and oxygen delivery/utilization. Hypothermia, particularly temperatures <35°C (<95°F), directly impairs the coagulation pathway, sometimes causing a significant coagulopathy. Rapid rewarming to >35°C (>95°F) significantly decreases the requirement for blood products and produces an improvement in cardiac function. The most effective method for rewarming is endovascular countercurrent warmers through femoral vein cannulation. This process does not require a pump and can rewarm a patient from 30° to 35°C (86° to and tachycardia are cardinal signs of the systemic response. To date, attempts to devise precise definitions for the

1	process does not require a pump and can rewarm a patient from 30° to 35°C (86° to and tachycardia are cardinal signs of the systemic response. To date, attempts to devise precise definitions for the harmful systemic reaction to infection (“sepsis”) have not resulted in a clinically useful level of specificity, in part because the systemic responses to infection, trauma, and other major stresses can be so similar. In general, when an infectious etiology is proven or strongly suspected and the response results in hypofunction of uninfected organs, the term sepsis (or severe sepsis) should be used. Septic shock refers to sepsis accompanied by hypotension that cannot be corrected by the infusion of fluids.

1	Bacteremia Presence of bacteria in blood, as evidenced by positive blood cultures Signs of possibly harmful systemic Two or more of the following condi response tions: (1) fever (oral temperature >38°C [>100.4°F]) or hypothermia (<36°C [<96.8°F]); (2) tachypnea (>24 breaths/min); (3) tachycardia (heart rate >90 beats/min); (4) leukocytosis (>12,000/μL), leukopenia (<4000/μL), or >10% bands Sepsis (or severe sepsis) The harmful host response to infection; systemic response to proven or suspected infection plus some degree of organ hypofunction, i.e.: 1. Cardiovascular: Arterial systolic blood pressure ≤90 mmHg or mean arterial pressure ≤70 mmHg that responds to administration of IV fluid 2. Renal: Urine output <0.5 mL/kg per hour for 1 h despite adequate fluid resuscitation 3. Respiratory: PaO2/FIO2 ≤250 or, if the lung is the only dysfunctional organ, ≤200 4.

1	Renal: Urine output <0.5 mL/kg per hour for 1 h despite adequate fluid resuscitation 3. Respiratory: PaO2/FIO2 ≤250 or, if the lung is the only dysfunctional organ, ≤200 4. Hematologic: Platelet count <80,000/μL or 50% decrease in platelet count from highest value recorded over previous 3 days 5. Unexplained metabolic acidosis: A pH ≤7.30 or a base deficit ≥5.0 mEq/L and a plasma lactate level >1.5 times upper limit of normal for reporting lab Septic shock Sepsis with hypotension (arterial blood pressure <90 mmHg systolic, or 40 mmHg less than patient’s normal blood pressure) for at least 1 h despite adequate fluid resuscitationa Need for vasopressors to maintain systolic blood pressure ≥90 mmHg or mean arterial pressure ≥70 mmHg Refractory septic shock Septic shock that lasts for >1 h and does not respond to fluid or pressor administration aFluid resuscitation is considered adequate when the pulmonary artery wedge pressure is ≥12 mmHg or the central venous pressure is ≥8 mmHg.

1	The systemic response to any class of microorganism can be harmful. Microbial invasion of the bloodstream is not essential because local inflammation can also elicit distant organ dysfunction and hypotension. In fact, blood cultures yield bacteria or fungi in only ~20–40% of cases of severe sepsis and 40–70% of cases of septic shock. In a prevalence study of 14,414 patients in intensive care units (ICUs) from 75 countries in 2007, 51% of patients were considered infected. Respiratory infection was most common (64%). Microbiologic results were positive in 70% of individuals considered infected; of the isolates, 62% were gram-negative bacteria (Pseudomonas species and Escherichia coli were most common), 47% were gram-positive bacteria (Staphylococcus aureus was most common), and 19% were fungi (Candida species). This distribution is similar to that reported a decade earlier from eight academic centers in the United States (Table 325-2). In patients whose blood cultures are negative, the

1	(Candida species). This distribution is similar to that reported a decade earlier from eight academic centers in the United States (Table 325-2). In patients whose blood cultures are negative, the etiologic agent is often established by culture or microscopic examination of infected material from a local site; specific identification of microbial DNA or RNA in blood or tissue samples is also used. In some case series, a majority of patients with a clinical picture of severe sepsis or septic shock have had negative microbiologic data.

1	95°F) in 30–60 min. Severe Sepsis and Septic Shock Robert S. Munford DEFINITIONS (Table 325-1) Animals mount both local and systemic responses to microbes that traverse their epithelial barriers and enter underlying tissues. Fever or hypothermia, leukocytosis or leukopenia, tachypnea, 325 a Enterobacteriaceae, pseudomonads, Haemophilus spp., other gram-negative bacteria. b Staphylococcus aureus, coagulase-negative staphylococci, enterococci, Streptococcus pneumoniae, other streptococci, other gram-positive bacteria. cSuch as Neisseria meningitidis, S. pneumoniae, Haemophilus influenzae, and Streptococcus pyogenes. Source: Adapted from KE Sands et al: JAMA 278:234, 1997.

1	Severe sepsis is a contributing factor in >200,000 deaths per year in the United States. The incidence of severe sepsis and septic shock has increased over the past 30 years, and the annual number of cases is now >750,000 (~3 per 1000 population). Approximately two-thirds of the cases occur in patients with significant underlying illness. Sepsis-related incidence and mortality rates increase with age and preexisting comorbidity. The rising incidence of severe sepsis in the United States has been attributable to the aging of the population, the increasing longevity of patients with chronic diseases, and the relatively high frequency with which sepsis has occurred in patients with AIDS. The widespread use of immunosuppressive drugs, indwelling catheters, and mechanical devices has also played a role. In the aforementioned international ICU prevalence study, the case–fatality rate among infected patients (33%) greatly exceeded that among uninfected patients (15%).

1	Invasive bacterial infections are prominent causes of death around the world, particularly among young children. In sub-Saharan Africa, for example, careful screening for positive blood cultures found that community-acquired bacteremia accounted for at least one-fourth of deaths of children >1 year of age. Nontyphoidal Salmonella species, Streptococcus pneumoniae, Haemophilus influenzae, and E. coli were the most commonly isolated bacteria. Bacteremic children often had HIV infection or were severely malnourished.

1	Sepsis is triggered most often by bacteria or fungi that do not ordinarily cause systemic disease in immunocompetent hosts (Table 325-2). To survive within the human body, these microbes often exploit acquired deficiencies in host defenses, indwelling catheters or other foreign matter, or obstructed fluid drainage conduits. Microbial pathogens, in contrast, can circumvent innate defenses because they (1) lack molecules that can be recognized by host receptors (see below) or (2) elaborate toxins or other virulence factors. In both cases, the body can mount a vigorous inflammatory reaction that results in sepsis or septic shock yet fails to kill the invaders. The septic response may also be induced by microbial exotoxins that act as superantigens (e.g., toxic shock syndrome toxin 1; Chap. 172) as well as by many pathogenic viruses.

1	Host Mechanisms for Sensing Microbes Animals have exquisitely sensitive mechanisms for recognizing and responding to certain highly conserved microbial molecules. Recognition of the lipid A moiety of lipopolysaccharide (LPS, also called endotoxin; Chap. 145e) is the best-studied example. A host protein (LPS-binding protein) binds lipid A and transfers the LPS to CD14 on the surfaces of monocytes, macrophages, and neutrophils. LPS then is passed to MD-2, a small receptor protein that is bound to Toll-like receptor (TLR) 4 to form a molecular complex that transduces the LPS recognition signal to the interior of the cell. This signal rapidly triggers the production and release of mediators, such as tumor necrosis factor (TNF; see below), that amplify the LPS signal and transmit it to other cells and tissues. Bacterial peptidoglycan and lipopeptides elicit responses in animals that are generally similar to those induced by LPS, although they interact with different TLRs. Having numerous

1	cells and tissues. Bacterial peptidoglycan and lipopeptides elicit responses in animals that are generally similar to those induced by LPS, although they interact with different TLRs. Having numerous TLR-based receptor complexes (10 different TLRs have been identified in humans) allows animals to recognize many conserved microbial molecules; others include lipopeptides (TLR2/1, TLR2/6), flagellin (TLR5), undermethylated DNA CpG sequences (TLR9), single-stranded RNA (TLR7, 8), and double-stranded RNA (TLR3). The ability of some TLRs to serve as receptors for host ligands (e.g., hyaluronans, heparan sulfate, saturated fatty acids, high-mobility group box 1) raises the possibility that they also play a role in producing noninfectious sepsis-like states. Other host pattern-recognition proteins that are important for sensing microbes include the intracellular NOD1 and NOD2 proteins, which recognize discrete fragments of bacterial peptidoglycan; the inflammasome, which senses some pathogens

1	that are important for sensing microbes include the intracellular NOD1 and NOD2 proteins, which recognize discrete fragments of bacterial peptidoglycan; the inflammasome, which senses some pathogens and produces interleukin (IL) 1β and IL-18; early complement components (principally in the alternative pathway); mannose-binding lectin and C-reactive protein, which activate the classic complement pathway; and Dectin-1 and complement receptor 3, which sense fungal β-glucan.

1	A host’s ability to recognize certain microbial molecules may influence both the potency of its own defenses and the pathogenesis of severe sepsis. For example, MD-2–TLR4 best senses LPS that has a bisphosphorylated, hexaacyl lipid A moiety (i.e., one with two phosphates and six fatty acyl chains). Most of the commensal aerobic and facultatively anaerobic gram-negative bacteria that trigger severe sepsis and shock (including E. coli, Klebsiella, and Enterobacter) make this lipid A structure. When they invade human hosts, often through breaks in an epithelial barrier, they are typically confined to the sub-epithelial tissue by a localized inflammatory response. Bacteremia, if it occurs, is intermittent and low grade because these bacteria are efficiently cleared from the bloodstream by TLR4-expressing Kupffer cells and splenic macrophages. These mucosal commensals seem to induce severe sepsis most often by triggering severe local tissue inflammation rather than by circulating within

1	Kupffer cells and splenic macrophages. These mucosal commensals seem to induce severe sepsis most often by triggering severe local tissue inflammation rather than by circulating within the bloodstream. One exception is Neisseria meningitidis. Its hexaacyl LPS seems to be shielded from host recognition by its polysaccharide capsule. This protection may allow meningococci to transit undetected from the nasopharyngeal mucosa into the bloodstream, where they can infect vascular endothelial cells and release large amounts of endotoxin and DNA. Host recognition of lipid A may nonetheless influence pathogenesis, as meningococci that produce pentaacyl LPS were isolated from the blood of patients with less severe coagulopathy than was found in patients whose isolates produced hexaacyl lipid A; underacylated N. meningitidis LPS has also been found in many isolates from patients with chronic meningococcemia. In contrast, gram-negative bacteria that make lipid A with fewer than six acyl chains

1	N. meningitidis LPS has also been found in many isolates from patients with chronic meningococcemia. In contrast, gram-negative bacteria that make lipid A with fewer than six acyl chains (Yersinia pestis, Francisella tularensis, Vibrio vulnificus, Pseudomonas aeruginosa, and Burkholderia pseudomallei, among others) are poorly recognized by MD-2–TLR4. When these bacteria enter the body, they may initially induce relatively little inflammation. When they do trigger severe sepsis, it is often after they have multiplied to high density in tissues and blood. The importance of LPS recognition in disease pathogenesis was demonstrated by engineering of a virulent strain of Y. pestis that makes tetraacyl LPS at 37°C to produce hexaacyl LPS; unlike its virulent parent, the mutant strain stimulated local inflammation and was rapidly cleared from tissues. These findings were subsequently replicated in F. tularensis. For at least one large class of microbes—gram-negative aerobic bacteria—the

1	local inflammation and was rapidly cleared from tissues. These findings were subsequently replicated in F. tularensis. For at least one large class of microbes—gram-negative aerobic bacteria—the pathogenesis of sepsis thus depends, at least in part, on whether the bacterium’s major signal molecule, LPS, can be sensed by the host.

1	Local and Systemic Host Responses to Invading Microbes Recognition of microbial molecules by tissue phagocytes triggers the production and/or release of numerous host molecules (cytokines, chemokines, prostanoids, leukotrienes, and others) that increase blood flow to the infected tissue (rubor), enhance the permeability of local blood vessels (tumor), recruit neutrophils and other cells to the site of infection (calor), and elicit pain (dolor). These reactions are familiar elements of local inflammation, the body’s frontline innate immune mechanism for eliminating microbial invaders. Systemic responses are activated by neural and/or humoral communication with the hypothalamus and brainstem; these responses enhance local defenses by increasing blood flow to the infected area, augmenting the number of circulating neutrophils, and elevating blood levels of numerous molecules (such as the microbial recognition proteins discussed above) that have anti-infective functions.

1	cytokines and other mediators Cytokines can exert endocrine, paracrine, and autocrine effects (Chap. 372e). TNF-α stimulates leukocytes and vascular endothelial cells to release other cytokines (as well as additional TNF-α), to express cell-surface molecules that enhance neutrophil endothelial adhesion at sites of infection, and to increase prostaglandin and leukotriene production. Whereas blood levels of TNF-α are not elevated in individuals with localized infections, they increase in most patients with severe sepsis or septic shock. Moreover, IV infusion of TNF-α can elicit fever, tachycardia, hypotension, and other responses. In animals, larger doses of TNF-α induce shock and death.

1	Although TNF-α is a central mediator, it is only one of many proinflammatory molecules that contribute to innate host defense. Chemokines, most prominently IL-8 and IL-17, attract circulating neutrophils to the infection site. IL-1β exhibits many of the same activities as TNF-α. TNF-α, IL-1β, interferon γ, IL-12, IL-17, and other proinflammatory cytokines probably interact synergistically with one another and with additional mediators. The nonlinearity and multiplicity of these interactions have made it difficult to interpret the roles played by individual mediators in both tissues and blood.

1	coagulation factors Intravascular thrombosis, a hallmark of the local inflammatory response, may help wall off invading microbes and prevent infection and inflammation from spreading to other tissues. IL-6 and other mediators promote intravascular coagulation initially by inducing blood monocytes and vascular endothelial cells to express tissue factor (Chap. 78). When tissue factor is expressed on cell surfaces, it binds to factor VIIa to form an active complex that can convert factors X and IX to their enzymatically active forms. The result is activation of both extrinsic and intrinsic clotting pathways, culminating in the generation of fibrin. Clotting is also favored by impaired function of the protein C–protein S inhibitory pathway and depletion of antithrombin and proteins C and S, whereas fibrinolysis is reduced by increases in plasma levels of plasminogen activator inhibitor 1. Thus, there may be a striking propensity toward intravascular fibrin deposition, thrombosis, and

1	S, whereas fibrinolysis is reduced by increases in plasma levels of plasminogen activator inhibitor 1. Thus, there may be a striking propensity toward intravascular fibrin deposition, thrombosis, and bleeding; this propensity has been most apparent in patients with intravascular endothelial infections such as meningococcemia (Chap. 180). Evidence points to tissue factor–expressing microparticles derived from leukocytes as a potential trigger for intra-vascular coagulation. The contact system is activated during sepsis but contributes more to the development of hypotension than to that of disseminated intravascular coagulation (DIC).

1	Neutrophil extracellular traps (NETs) are produced when neutrophils, stimulated by microbial agonists or IL-8, release granule proteins and chromatin to form an extracellular fibrillar matrix. NETs kill bacteria and fungi with antimicrobial granule proteins (e.g., elastase) and histones. It has been reported that NETs can form within hepatic sinusoids in animals injected with large amounts of LPS, and platelets can induce NET formation without killing neutrophils. A role played by NETs in organ hypofunction during sepsis has been proposed but not established. control mechanisms Elaborate control mechanisms operate within 1753 both local sites of inflammation and the systemic compartment.

1	Local control mechanisms Host recognition of invading microbes within subepithelial tissues typically ignites immune responses that rapidly kill the invaders and then subside to allow tissue recovery. The forces that put out the fire and clean up the battleground include molecules that neutralize or inactivate microbial signals. Among these molecules are intracellular factors (e.g., suppressor of cytokine signaling 3 and IL-1 receptor–associated kinase 3) that diminish the production of proinflammatory mediators by neutrophils and macrophages; anti-inflammatory cytokines (IL-10, IL-4); and molecules derived from essential polyunsaturated fatty acids (lipoxins, resolvins, and protectins) that promote tissue restoration. Enzymatic inactivation of microbial signal molecules (e.g., LPS) may be required to restore homeostasis; a leukocyte enzyme, acyloxyacyl hydrolase, has been shown to prevent prolonged inflammation in mice by inactivating LPS.

1	Systemic control mechanisms The signaling apparatus that links microbial recognition to cellular responses in tissues is less active in the blood. For example, whereas LPS-binding protein plays a role in recognizing LPS, in plasma it also prevents LPS signaling by transferring LPS molecules into plasma lipoprotein particles that sequester the lipid A moiety so that it cannot interact with cells. At the high concentrations found in blood, LPS-binding protein also inhibits monocyte responses to LPS, and the soluble (circulating) form of CD14 strips off LPS that has bound to monocyte surfaces.

1	Systemic responses to infection also diminish cellular responses to microbial molecules. Circulating levels of cortisol and anti-inflammatory cytokines (e.g., IL-6 and IL-10) increase even in patients with minor infections. Glucocorticoids inhibit cytokine synthesis by monocytes in vitro; the increase in blood cortisol levels that occurs early in the systemic response presumably plays a similarly inhibitory role. Epinephrine inhibits the TNF-α response to endotoxin infusion in humans while augmenting and accelerating the release of IL-10; prostaglandin E2 has a similar “reprogramming” effect on the responses of circulating monocytes to LPS and other bacterial agonists. Cortisol, epinephrine, IL-10, and C-reactive protein reduce the ability of neutrophils to attach to vascular endothelium, favoring their demargination and thus contributing to leukocytosis while preventing neutrophilendothelial adhesion in uninflamed organs. Studies in rodents have found that macrophage cytokine

1	favoring their demargination and thus contributing to leukocytosis while preventing neutrophilendothelial adhesion in uninflamed organs. Studies in rodents have found that macrophage cytokine synthesis is inhibited by acetylcholine that is produced by choline acetyltransferase–secreting CD4+ T cells in response to stimulation by norepinephrine, whereas acetylcholineproducing B cells reduce neutrophil infiltration into tissues. Several lines of evidence thus suggest that the body’s neuroendocrine responses to injury and infection normally prevent inflammation within organs distant from a site of infection. There is also evidence that these responses may be immunosuppressive.

1	IL-6 plays important roles in the systemic compartment. Released by many different cell types, IL-6 is an important stimulus to the hypothalamic-pituitary-adrenal axis, is the major procoagulant cytokine, and is a principal inducer of the acute-phase response, which increases the blood concentrations of numerous molecules that have anti-infective, procoagulant, or anti-inflammatory actions. Blood levels of IL-1 receptor antagonist often greatly exceed those of circulating IL-1β, for example, and this excess may inhibit the binding of IL-1β to its receptors. High levels of soluble TNF receptors neutralize TNF-α that enters the circulation. Other acute-phase proteins are protease inhibitors or antioxidants; these may neutralize potentially harmful molecules released from neutrophils and other inflammatory cells. Increased hepatic production of hepcidin (stimulated largely by IL-6) promotes the sequestration of iron in hepatocytes, intestinal epithelial cells, and erythrocytes; this

1	other inflammatory cells. Increased hepatic production of hepcidin (stimulated largely by IL-6) promotes the sequestration of iron in hepatocytes, intestinal epithelial cells, and erythrocytes; this effect reduces iron acquisition by invading microbes while contributing to the normocytic, normochromic anemia associated with inflammation.

1	It may thus be said that both local and systemic responses to infectious agents benefit the host in important ways. Most of these responses and the molecules responsible for them have been highly conserved during animal evolution and therefore may be adaptive. 1754 Elucidating how they become maladaptive and contribute to lethality remains a major challenge for sepsis research.

1	Organ Dysfunction and Shock As the body’s responses to infection intensify, the mixture of circulating cytokines and other molecules becomes very complex: elevated blood levels of more than 60 molecules have been found in patients with septic shock. Although high concentrations of both proand anti-inflammatory molecules are found, the net mediator balance in the plasma of these extremely sick patients seems to be anti-inflammatory. For example, blood leukocytes from patients with severe sepsis are often hyporesponsive to agonists such as LPS. In patients with severe sepsis, persistence of leukocyte hyporesponsiveness has been associated with an increased risk of dying; at this time, the most predictive biomarker is a decrease in the expression of HLA-DR (class II) molecules on the surfaces of circulating monocytes, a response that seems to be induced by cortisol and/or IL-10. Apoptotic death of B cells, follicular dendritic cells, and CD4+ T lymphocytes also may contribute

1	the surfaces of circulating monocytes, a response that seems to be induced by cortisol and/or IL-10. Apoptotic death of B cells, follicular dendritic cells, and CD4+ T lymphocytes also may contribute significantly to the immunosuppressive state.

1	endothelial injury Given the vascular endothelium’s important roles in regulating vascular tone, vascular permeability, and coagulation, many investigators have favored widespread vascular endothelial injury as the major mechanism for multiorgan dysfunction. In keeping with this idea, one study found high numbers of vascular endothelial cells in the peripheral blood of septic patients. Leukocyte-derived mediators and platelet-leukocyte-fibrin thrombi may contribute to vascular injury, but the vascular endothelium also seems to play an active role. Stimuli such as TNF-α induce vascular endothelial cells to produce and release cytokines, procoagulant molecules, platelet-activating factor, nitric oxide, and other mediators. In addition, regulated cell-adhesion molecules promote the adherence of neutrophils to endothelial cells. Although these responses can attract phagocytes to infected sites and activate their antimicrobial arsenals, endothelial cell activation can also promote

1	of neutrophils to endothelial cells. Although these responses can attract phagocytes to infected sites and activate their antimicrobial arsenals, endothelial cell activation can also promote increased vascular permeability, microvascular thrombosis, DIC, and hypotension.

1	Tissue oxygenation may decrease as the number of functional capillaries is reduced by luminal obstruction due to swollen endothelial cells, decreased deformability of circulating erythrocytes, leukocyte-platelet-fibrin thrombi, or compression by edema fluid. On the other hand, studies using orthogonal polarization spectral imaging of the microcirculation in the tongue found that sepsis-associated derangements in capillary flow could be reversed by applying acetylcholine to the surface of the tongue or by giving nitroprusside intravenously; these observations suggest a neuroendocrine basis for the loss of capillary filling. Oxygen utilization by tissues may also be impaired by changes (possibly induced by nitric oxide) that decrease oxidative phosphorylation and ATP production while increasing glycolysis. The local accumulation of lactic acid, a consequence of increased glycolysis, may decrease extracellular pH and contribute to the slowdown in cellular metabolism that occurs within

1	glycolysis. The local accumulation of lactic acid, a consequence of increased glycolysis, may decrease extracellular pH and contribute to the slowdown in cellular metabolism that occurs within affected tissues.

1	Remarkably, poorly functioning “septic” organs usually appear normal at autopsy. There is typically very little necrosis or thrombosis, and apoptosis is largely confined to lymphoid organs and the gastrointestinal tract. Moreover, organ function usually returns to normal if patients recover. These points suggest that organ dysfunction during severe sepsis has a basis that is principally biochemical, not structural.

1	sePtic shock The hallmark of septic shock is a decrease in peripheral vascular resistance that occurs despite increased levels of vasopressor catecholamines. Before this vasodilatory phase, many patients experience a period during which oxygen delivery to tissues is compromised by myocardial depression, hypovolemia, and other factors. During this “hypodynamic” period, the blood lactate concentration is elevated and central venous oxygen saturation is low. Fluid administration is usually followed by the hyperdynamic vasodilatory phase, during which cardiac output is normal (or even high) and oxygen consumption declines despite adequate oxygen delivery. The blood lactate level may be normal or increased, and normalization of central venous oxygen saturation may reflect improved oxygen delivery, decreased oxygen uptake by tissues, or left-to-right shunting.

1	Prominent hypotensive molecules include nitric oxide, β-endorphin, bradykinin, platelet-activating factor, and prostacyclin. Agents that inhibit the synthesis or action of each of these mediators can prevent or reverse endotoxic shock in animals. However, in clinical trials, neither a platelet-activating factor receptor antagonist nor a bradykinin antagonist improved survival rates among patients with septic shock, and a nitric oxide synthase inhibitor, L-NG-methylarginine HCl, actually increased the mortality rate.

1	Severe Sepsis: A Single Pathogenesis? In some cases, circulating bacteria and their products almost certainly elicit multiorgan dysfunction and hypotension by directly stimulating inflammatory responses within the vasculature. In patients with fulminant meningococcemia, for example, mortality rates have correlated directly with blood levels of endotoxin and bacterial DNA and with the occurrence of DIC (Chap. 180). In most patients infected with other gram-negative bacteria, in contrast, circulating bacteria or bacterial molecules may reflect uncontrolled infection at a local tissue site and have little or no direct impact on distant organs; in these patients, inflammatory mediators or neural signals arising from the local site seem to be the key triggers for severe sepsis and septic shock. In a large series of patients with positive blood cultures, the risk of developing severe sepsis was strongly related to the site of primary infection: bacteremia arising from a pulmonary or

1	shock. In a large series of patients with positive blood cultures, the risk of developing severe sepsis was strongly related to the site of primary infection: bacteremia arising from a pulmonary or abdominal source was eightfold more likely to be associated with severe sepsis than was bacteremic urinary tract infection, even after the investigators controlled for age, the kind of bacteria isolated from the blood, and other factors. A third pathogenesis may be represented by severe sepsis due to superantigen-producing S. aureus or Streptococcus pyogenes; the T cell activation induced by these toxins produces a cytokine profile that differs substantially from that elicited by gram-negative bacterial infection. Further evidence for different pathogenetic pathways has come from observations that the pattern of mRNA expression in peripheral-blood leukocytes from children with sepsis is different for gram-positive, gram-negative, and viral pathogens.

1	The pathogenesis of severe sepsis thus may differ according to the infecting microbe, the ability of the host’s innate defense mechanisms to sense and respond to it, the site of the primary infection, the presence or absence of immune defects, and the prior physiologic status of the host. Genetic factors are probably important as well, yet despite much study very few allelic polymorphisms have been associated with sepsis severity in more than one or two analyses. Further studies in this area are needed. The manifestations of the septic response are superimposed on the symptoms and signs of the patient’s underlying illness and primary infection. The rate at which severe sepsis develops may differ from patient to patient, and there are striking individual variations in presentation. For example, some patients with sepsis are normoor hypothermic; the absence of fever is most common in neonates, in elderly patients, and in persons with uremia or alcoholism.

1	Hyperventilation, producing respiratory alkalosis, is often an early sign of the septic response. Disorientation, confusion, and other manifestations of encephalopathy may also develop early on, particularly in the elderly and in individuals with preexisting neurologic impairment. Focal neurologic signs are uncommon, although preexisting focal deficits may become more prominent.

1	Hypotension and DIC predispose to acrocyanosis and ischemic necrosis of peripheral tissues, most commonly the digits. Cellulitis, pustules, bullae, or hemorrhagic lesions may develop when hematogenous bacteria or fungi seed the skin or underlying soft tissue. Bacterial toxins may also be distributed hematogenously and elicit diffuse cutaneous reactions. On occasion, skin lesions may suggest specific pathogens. When sepsis is accompanied by cutaneous petechiae or purpura, infection with N. meningitidis (or, less commonly, H. influenzae) should be suspected (see Fig. 25e-42); in a patient who has been bitten by a tick while in an endemic area, petechial lesions also suggest Rocky Mountain spotted fever (see Fig. 211-1). A cutaneous lesion seen almost exclusively in neutropenic patients is ecthyma gangrenosum, often caused by P. aeruginosa. This bullous lesion surrounded by edema undergoes central hemorrhage and necrosis (see Fig. 189-1).

1	Histopathologic examination shows bacteria in and around the wall of a small vessel, with little or no neutrophilic response. Hemorrhagic or bullous lesions in a septic patient who has recently eaten raw oysters suggest V. vulnificus bacteremia, whereas such lesions in a patient who has recently sustained a dog bite may indicate bloodstream infection due to Capnocytophaga canimorsus or Capnocytophaga cynodegmi. Generalized erythroderma in a septic patient suggests the toxic shock syndrome due to S. aureus or S. pyogenes.

1	Gastrointestinal manifestations such as nausea, vomiting, diarrhea, and ileus may suggest acute gastroenteritis. Stress ulceration can lead to upper gastrointestinal bleeding. Cholestatic jaundice, with elevated levels of serum bilirubin (mostly conjugated) and alkaline phosphatase, may precede other signs of sepsis. Hepatocellular or canalicular dysfunction appears to underlie most cases, and the results of hepatic function tests return to normal with resolution of the infection. Prolonged or severe hypotension may induce acute hepatic injury or ischemic bowel necrosis.

1	Many tissues may be unable to extract oxygen normally from the blood, so that anaerobic metabolism occurs despite near-normal mixed venous oxygen saturation. Blood lactate levels rise early because of increased glycolysis as well as impaired clearance of the resulting lactate and pyruvate by the liver and kidneys. The blood glucose concentration often increases, particularly in patients with diabetes, although impaired gluconeogenesis and excessive insulin release on occasion produce hypoglycemia. The cytokine-driven acute-phase response inhibits the synthesis of transthyretin while enhancing the production of C-reactive protein, fibrinogen, and complement components. Protein catabolism is often markedly accelerated. Serum albumin levels decline as a result of decreased hepatic synthesis and the movement of albumin into interstitial spaces.

1	MAJOR COMPLICATIONS Cardiopulmonary Complications Ventilation-perfusion mismatching produces a fall in arterial Po2 early in the course. Increasing alveolar epithelial injury and capillary permeability result in increased pulmonary water content, which decreases pulmonary compliance and interferes with oxygen exchange. In the absence of pneumonia or heart failure, progressive diffuse pulmonary infiltrates and arterial hypoxemia occurring within 1 week of a known insult indicate the development of mild acute respiratory distress syndrome (ARDS) (200 mmHg < Pao2/Fio2 ≤ 300 mmHg), moderate ARDS (100 mmHg < Pao2/Fio2 ≤ 200 mmHg), or severe ARDS (Pao2/Fio2 ≤100 mmHg). Acute lung injury or ARDS develops in ~50% of patients with severe sepsis or septic shock. Respiratory muscle fatigue can exacerbate hypoxemia and hypercapnia. An elevated pulmonary capillary wedge pressure (>18 mmHg) suggests fluid volume overload or cardiac failure rather than ARDS. Pneumonia caused by viruses or by

1	exacerbate hypoxemia and hypercapnia. An elevated pulmonary capillary wedge pressure (>18 mmHg) suggests fluid volume overload or cardiac failure rather than ARDS. Pneumonia caused by viruses or by Pneumocystis may be clinically indistinguishable from ARDS.

1	Sepsis-induced hypotension (see “Septic Shock,” above) usually results initially from a generalized maldistribution of blood flow and blood volume and from hypovolemia that is due, at least in part, to diffuse capillary leakage of intravascular fluid. Other factors that may decrease effective intravascular volume include dehydration from antecedent disease or insensible fluid losses, vomiting or diarrhea, and polyuria. During early septic shock, systemic vascular resistance is usually elevated and cardiac output may be low. After fluid repletion, in contrast, cardiac output typically increases and systemic vascular resistance falls. Indeed, normal or increased cardiac output and decreased systemic vascular resistance distinguish septic shock from cardiogenic, extracardiac obstructive, and hypovolemic shock; other processes that can produce this combination include anaphylaxis, beriberi, cirrhosis, and overdoses of nitroprusside or narcotics.

1	Depression of myocardial function, manifested as increased end-diastolic and systolic ventricular volumes with a decreased ejection fraction, develops within 24 h in most patients with severe sepsis. Cardiac output is maintained despite the low ejection fraction because ventricular dilation permits a normal stroke volume. In survivors, myocardial function returns to normal over several days. Although myocardial dysfunction may contribute to hypotension, refractory hypotension is usually due to low systemic vascular resistance, and 1755 death most often results from refractory shock or the failure of multiple organs rather than from cardiac dysfunction per se.

1	Adrenal Insufficiency The diagnosis of adrenal insufficiency may be very difficult in critically ill patients. Whereas a plasma cortisol level of ≤15 μg/mL (≤10 μg/mL if the serum albumin concentration is <2.5 mg/dL) indicates adrenal insufficiency (inadequate production of cortisol), many experts now feel that the adrenocorticotropic hormone (CoSyntropin®) stimulation test is not useful for detecting less profound degrees of corticosteroid deficiency in patients who are critically ill.

1	The concept of critical illness–related corticosteroid insufficiency (CIRCI) was proposed to encompass the different mechanisms that may produce corticosteroid activity that is inadequate for the severity of a patient’s illness. Although CIRCI may result from structural damage to the adrenal gland, it is more commonly due to reversible dysfunction of the hypothalamic-pituitary axis or to tissue corticosteroid resistance resulting from abnormalities of the glucocorticoid receptor or increased conversion of cortisol to cortisone. The major clinical manifestation of CIRCI is hypotension that is refractory to fluid replacement and requires pressor therapy. Some classic features of adrenal insufficiency, such as hyponatremia and hyperkalemia, are usually absent; others, such as eosinophilia and modest hypoglycemia, may sometimes be found. Specific etiologies include fulminant N.

1	N. meningitidis bacteremia, disseminated tuberculosis, AIDS (with cytomegalovirus, Mycobacterium avium-intracellulare, or Histoplasma capsulatum disease), or the prior use of drugs that diminish glucocor ticoid production, such as glucocorticoids, megestrol, etomidate, or ketoconazole. Renal Complications Oliguria, azotemia, proteinuria, and nonspecific urinary casts are frequently found. Many patients are inappropriately polyuric; hyperglycemia may exacerbate this tendency. Most renal failure is due to acute tubular necrosis induced by hypovolemia, arterial hypotension, or toxic drugs, although some patients also have glomerulonephritis, renal cortical necrosis, or interstitial nephritis. Drug-induced renal damage may greatly complicate therapy, particu larly when hypotensive patients are given aminoglycoside antibiotics. Nosocomial sepsis following acute renal injury is associated with a high mortality rate.

1	Coagulopathy Although thrombocytopenia occurs in 10–30% of patients, the underlying mechanisms are not understood. Platelet counts are usually very low (<50,000/μL) in patients with DIC; these low counts may reflect diffuse endothelial injury or microvascular thrombosis, yet thrombi have only infrequently been found on biopsy of septic organs. Neurologic Complications Delirium (acute encephalopathy) is often an early manifestation of sepsis. Depending on the diagnostic criteria used, it occurs in 10–70% of septic patients at some point during the hospital course. When the septic illness lasts for weeks or months, “critical illness” polyneuropathy may prevent weaning from ventilatory support and produce distal motor weakness. Electrophysiologic studies are diagnostic. Guillain-Barré syndrome, metabolic disturbances, and toxin activity must be ruled out. Recent studies have documented long-term cognitive loss in survivors of severe sepsis.

1	Immunosuppression Patients with severe sepsis often become profoundly immunosuppressed. Manifestations include loss of delayed-type hypersensitivity reactions to common antigens, failure to control the primary infection, and increased risk for secondary infections (e.g., by opportunists such as Stenotrophomonas maltophilia, Acinetobacter calcoaceticus-baumannii, and Candida albicans). Approximately one-third of patients experience reactivation of herpes simplex virus, varicellazoster virus, or cytomegalovirus infections; the latter are thought to contribute to adverse outcomes in some instances.

1	Abnormalities that occur early in the septic response may include leukocytosis with a left shift, thrombocytopenia, hyperbilirubinemia, and 1756 proteinuria. Leukopenia may develop. The neutrophils may contain toxic granulations, Döhle bodies, or cytoplasmic vacuoles. As the septic response becomes more severe, thrombocytopenia worsens (often with prolonged thrombin time, decreased fibrinogen, and the presence of d-dimers, suggesting DIC), azotemia and hyperbilirubinemia become more prominent, and levels of aminotransferases rise. Active hemolysis suggests clostridial bacteremia, malaria, a drug reaction, or DIC; in the case of DIC, microangiopathic changes may be seen on a blood smear. During early sepsis, hyperventilation induces respiratory alkalosis. With respiratory muscle fatigue and the accumulation of lactate, metabolic acidosis (with increased anion gap) typically supervenes. Evaluation of arterial blood gases reveals hypoxemia that is initially correctable with supplemental

1	the accumulation of lactate, metabolic acidosis (with increased anion gap) typically supervenes. Evaluation of arterial blood gases reveals hypoxemia that is initially correctable with supplemental oxygen but whose later refractoriness to 100% oxygen inhalation indicates right-to-left shunting. The chest radiograph may be normal or may show evidence of underlying pneumonia, volume overload, or the diffuse infiltrates of ARDS. The electrocardiogram may show only sinus tachycardia or nonspecific ST–T wave abnormalities. Most diabetic patients with sepsis develop hyperglycemia. Severe infection may precipitate diabetic ketoacidosis that may exacerbate hypotension (Chap. 417). Hypoglycemia occurs rarely and may indicate adrenal insufficiency. The serum albumin level declines as sepsis continues. Hypocalcemia is rare.

1	There is no specific diagnostic test for sepsis. Diagnostically sensitive findings in a patient with suspected or proven infection include fever or hypothermia, tachypnea, tachycardia, and leukocytosis or leukopenia (Table 325-1); acutely altered mental status, thrombocytopenia, an elevated blood lactate level, respiratory alkalosis, or hypotension also should suggest the diagnosis. The systemic response can be quite variable, however. In one study, 36% of patients with severe sepsis had a normal temperature, 40% had a normal respiratory rate, 10% had a normal pulse rate, and 33% had normal white blood cell counts. Moreover, the systemic responses of uninfected patients with other conditions may be similar to those characteristic of sepsis. Examples include pancreatitis, burns, trauma, adrenal insufficiency, pulmonary embolism, dissecting or ruptured aortic aneurysm, myocardial infarction, occult hemorrhage, cardiac tamponade, postcardiopulmonary bypass syndrome, anaphylaxis,

1	adrenal insufficiency, pulmonary embolism, dissecting or ruptured aortic aneurysm, myocardial infarction, occult hemorrhage, cardiac tamponade, postcardiopulmonary bypass syndrome, anaphylaxis, tumor-associated lactic acidosis, and drug overdose.

1	Definitive etiologic diagnosis requires identification of the causative microorganism from blood or a local site of infection. At least two blood samples should be obtained (from two different venipuncture sites) for culture; in a patient with an indwelling catheter, one sample should be collected from each lumen of the catheter and another via venipuncture. In many cases, blood cultures are negative; this result can reflect prior antibiotic administration, the presence of slow-growing or fastidious organisms, or the absence of microbial invasion of the bloodstream. In these cases, Gram’s staining and culture of material from the primary site of infection or from infected cutaneous lesions may help establish the microbial etiology. Identification of microbial DNA in peripheral blood or tissue samples by polymerase chain reaction may also be definitive. The skin and mucosae should be examined carefully and repeatedly for lesions that might yield diagnostic information. With

1	blood or tissue samples by polymerase chain reaction may also be definitive. The skin and mucosae should be examined carefully and repeatedly for lesions that might yield diagnostic information. With overwhelming bacteremia (e.g., pneumococcal sepsis in splenectomized individuals; fulminant meningococcemia; or infection with V. vulnificus, B. pseudomallei, or Y. pestis), microorganisms are sometimes visible on buffy coat smears of peripheral blood.

1	Patients in whom sepsis is suspected must be managed expeditiously. This task is best accomplished by personnel who are experienced in the care of the critically ill. Successful management requires urgent measures to treat the infection, to provide hemodynamic and respiratory support, and to remove or drain infected tissues. These measures should be initiated within 1 h of the patient’s presentation with severe sepsis or septic shock. Rapid assessment and diagnosis are therefore essential.

1	Antimicrobial chemotherapy should be started as soon as samples of blood and other relevant sites have been obtained for culture. A large retrospective review of patients who developed septic shock found that the interval between the onset of hypotension and the administration of appropriate antimicrobial chemotherapy was the major determinant of outcome; a delay of as little as 1 h was associated with lower survival rates. Use of “inappropriate” antibiotics, defined on the basis of local microbial susceptibilities and published guidelines for empirical therapy (see below), was associated with fivefold lower survival rates, even among patients with negative cultures.

1	It is therefore very important to promptly initiate empirical antimicrobial therapy that is effective against both gram-positive and gram-negative bacteria (Table 325-3). Maximal recommended doses of antimicrobial drugs should be given intravenously, with adjustment for impaired renal function when necessary. Available information about patterns of antimicrobial susceptibility among bacterial isolates from the community, the hospital, and the patient should be taken into account. When culture results become available, the regimen can often be simplified because a single antimicrobial agent is usually adequate for the treatment of a known pathogen. Meta-analyses have concluded that, with one exception, combination antimicrobial therapy is not superior to monotherapy for treating gram-negative bacteremia; the exception is that aminoglycoside monotherapy for P. aeruginosa bacteremia is less effective than the combination of an aminoglycoside with an antipseudomonal β-lactam agent.

1	bacteremia; the exception is that aminoglycoside monotherapy for P. aeruginosa bacteremia is less effective than the combination of an aminoglycoside with an antipseudomonal β-lactam agent. Empirical antifungal therapy should be strongly considered if the septic patient is already receiving broad-spectrum antibiotics or parenteral nutrition, has been neutropenic for ≥5 days, has had a long-term central venous catheter in place, or has been hospitalized in an ICU for a prolonged period. The chosen antimicrobial regimen should be reconsidered daily in order to provide maximal efficacy with minimal resistance, toxicity, and cost.

1	Most patients require antimicrobial therapy for at least 1 week. The duration of treatment is typically influenced by factors such as the site of tissue infection, the adequacy of surgical drainage, the patient’s underlying disease, and the antimicrobial susceptibility of the microbial isolate(s). The absence of an identified microbial pathogen is not necessarily an indication for discontinuing antimicrobial therapy because “appropriate” antimicrobial regimens seem to be beneficial in both culture-negative and culture-positive cases.

1	Removal or drainage of a focal source of infection is essential. In one series, a focus of ongoing infection was found in ~80% of surgical ICU patients who died of severe sepsis or septic shock. Sites of occult infection should be sought carefully, particularly in the lungs, abdomen, and urinary tract. Indwelling IV or arterial catheters should be removed and the tip rolled over a blood agar plate for quantitative culture; after antibiotic therapy has been initiated, a new catheter should be inserted at a different site. Foley and drainage catheters should be replaced. The possibility of paranasal sinusitis (often caused by gram-negative bacteria) should be considered if the patient has undergone nasal intubation or has an indwelling nasogastric or feeding tube. Even in patients without abnormalities on chest radiographs, computed tomography (CT) of the chest may identify unsuspected parenchymal, mediastinal, or pleural disease. In the neutropenic patient, cutaneous sites of

1	without abnormalities on chest radiographs, computed tomography (CT) of the chest may identify unsuspected parenchymal, mediastinal, or pleural disease. In the neutropenic patient, cutaneous sites of tenderness and erythema, particularly in the perianal region, must be carefully sought. In patients with sacral or ischial decubitus ulcers, it is important to exclude pelvic or other soft tissue pus collections with CT or magnetic resonance imaging (MRI). In patients with severe sepsis arising from the urinary tract, sonography or CT should be used to rule out ureteral obstruction, perinephric abscess, and renal abscess. Sonographic or CT imaging of the upper abdomen may disclose

1	Immunocompetent adult The many acceptable regimens include (1) piperacillin-tazobactam (3.375 g q4–6h); (2) imipenem-cilastatin (0.5 g q6h), ertapenem (1 g q24h), or meropenem (1 g q8h); or (3) cefepime (2 g q12h). If the patient is allergic to β-lactam agents, use ciprofloxacin (400 mg q12h) or levofloxacin (500–750 mg q12h) plus clindamycin (600 mg q8h). Vancomycin (15 mg/kg q12h) should be added to each of the above regimens.

1	Neutropenia (<500 neutrophils/μL) Regimens include (1) imipenemcilastatin (0.5 g q6h) or meropenem (1 g q8h) or cefepime (2 g q8h) or (2) piperacillin-tazobactam (3.375 g q4h) plus tobramycin (5–7 mg/kg q24h). Vancomycin (15 mg/kg q12h) should be added if the patient has an indwelling vascular catheter, has received quinolone prophylaxis, or has received intensive chemotherapy that produces mucosal damage; if staphylococci are suspected; if the institution has a high incidence of MRSA infections; or if there is a high prevalence of MRSA isolates in the community. Empirical antifungal therapy with an echinocandin (for caspofungin: a 70-mg loading dose, then 50 mg daily), voriconazole (6 mg/kg q12h for 2 doses, then 3 mg/ kg q12h), or a lipid formulation of amphotericin B should be added if the patient is hypotensive, has been receiving broad-spectrum antibacterial drugs, or remains febrile 5 days after initiation of empirical antibacterial therapy.

1	Splenectomy Cefotaxime (2 g q6–8h) or ceftriaxone (2 g q12h) should be used. If the local prevalence of cephalosporinresistant pneumococci is high, add vancomycin. If the patient is allergic to β-lactam drugs, vancomycin (15 mg/kg q12h) plus either moxifloxacin (400 mg q24h) or levofloxacin (750 mg q24h) should be used. IV drug user Vancomycin (15 mg/kg q12h) is essential. AIDS Cefepime alone (2 g q8h) or piperacillintazobactam (3.375 g q4h) plus tobramycin (5–7 mg/kg q24h) should be used. If the patient is allergic to β-lactam drugs, ciprofloxacin (400 mg q12h) or levofloxacin (750 mg q12h) plus vancomycin (15 mg/kg q12h) plus tobramycin should be used. Abbreviation: MRSA, methicillin-resistant Staphylococcus aureus. Source: Adapted in part from DN Gilbert et al: The Sanford Guide to Antimicrobial Therapy, 43rd ed, 2013. evidence of cholecystitis, bile duct dilation, and pus collections in the liver, subphrenic space, or spleen. HEMODYNAMIC, RESPIRATORY, AND METABOLIC SUPPORT

1	The primary goals are to restore adequate oxygen and substrate delivery to the tissues as quickly as possible and to improve tissue oxygen utilization and cellular metabolism. Adequate organ perfusion is thus essential. Circulatory adequacy is assessed by measurement 1757 of arterial blood pressure and monitoring of parameters such as mentation, urine output, and skin perfusion. Indirect indices of oxygen delivery and consumption, such as central venous oxygen saturation, may also be useful. Initial management of hypotension should include the administration of IV fluids, typically beginning with 1–2 L of normal saline over 1–2 h. To avoid pulmonary edema, the central venous pressure should be maintained at 8–12 cmH2O. The urine output rate should be kept at >0.5 mL/kg per hour by continuing fluid administration; a diuretic such as furosemide may be used if needed. In about one-third of patients, hypotension and organ hypoperfusion respond to fluid resuscitation; a reasonable goal is

1	fluid administration; a diuretic such as furosemide may be used if needed. In about one-third of patients, hypotension and organ hypoperfusion respond to fluid resuscitation; a reasonable goal is to maintain a mean arterial blood pressure of >65 mmHg (systolic pressure >90 mmHg). If these guidelines cannot be met by volume infusion, vasopressor therapy is indicated (Chap. 326). Titrated doses of norepinephrine should be administered through a central catheter. If myocardial dysfunction produces elevated cardiac filling pressures and low cardiac output, inotropic therapy with dobutamine is recommended. Dopamine is rarely used.

1	In patients with septic shock, plasma vasopressin levels increase transiently but then decrease dramatically. Early studies found that vasopressin infusion can reverse septic shock in some patients, reducing or eliminating the need for catecholamine pressors. Although vasopressin may benefit patients who require less nor-epinephrine, its role in the treatment of septic shock seems to be a minor one overall. CIRCI (see “Adrenal Insufficiency,” above) should be strongly con sidered in patients who develop hypotension that does not respond to fluid replacement therapy. Hydrocortisone (50 mg IV every 6 h) should be given; if clinical improvement occurs over 24–48 h, most experts would continue hydrocortisone therapy for 5–7 days before slowly tapering and discontinuing it. Meta-analyses of recent clinical trials have concluded that hydrocortisone therapy hastens recovery from sepsis-induced hypotension without increasing long-term survival.

1	Ventilator therapy is indicated for progressive hypoxemia, hyper capnia, neurologic deterioration, or respiratory muscle failure. Sustained tachypnea (respiratory rate, >30 breaths/min) is frequently a harbinger of impending respiratory collapse; mechanical ventilation is often initiated to ensure adequate oxygenation, to divert blood from the muscles of respiration, to prevent aspiration of oropharyngeal contents, and to reduce the cardiac afterload. The results of recent studies favor the use of low tidal volumes (6 mL/kg of ideal body weight, or as low as 4 mL/kg if the plateau pressure exceeds 30 cmH2O). Patients undergoing mechanical ventilation require careful sedation, with daily interruptions; elevation of the head of the bed helps to prevent nosocomial pneumonia. Stress-ulcer prophylaxis with a histamine H2-receptor antagonist may decrease the risk of gastrointestinal hemorrhage in ventilated patients.

1	Erythrocyte transfusion is generally recommended when the blood hemoglobin level decreases to ≤7 g/dL, with a target level of 9 g/dL in adults. Erythropoietin is not used to treat sepsis-related anemia. Bicarbonate is sometimes administered for severe metabolic acidosis (arterial pH <7.2), but there is little evidence that it improves either hemodynamics or the response to vasopressor hormones. DIC, if complicated by major bleeding, should be treated with transfusion of fresh-frozen plasma and platelets. Successful treatment of the underlying infection is essential to reverse both acidosis and DIC. Patients who are hypercatabolic and have acute renal failure may benefit greatly from intermittent hemodialysis or continuous veno-venous hemofiltration.

1	In patients with prolonged severe sepsis (i.e., that lasting more than 2 or 3 days), nutritional supplementation may reduce the impact of protein hypercatabolism; the available evidence favors the enteral delivery route. Prophylactic heparinization to prevent deep venous thrombosis is indicated for patients who do not have active bleeding or coagulopathy; when heparin is contraindicated, compression 1758 stockings or an intermittent compression device should be used. Recovery is also assisted by prevention of skin breakdown, nosocomial infections, and stress ulcers. The role of tight control of the blood glucose concentration in recovery from critical illness has been addressed in numerous controlled trials. Meta-analyses of these trials have concluded that use of insulin to lower blood glucose levels to 100–120 mg/dL is potentially harmful and does not improve survival rates. Most experts now recommend using insulin only if it is needed to maintain the blood glucose concentration

1	glucose levels to 100–120 mg/dL is potentially harmful and does not improve survival rates. Most experts now recommend using insulin only if it is needed to maintain the blood glucose concentration below ~180 mg/dL. Patients receiving intravenous insulin must be monitored frequently (every 1–2 h) for hypoglycemia.

1	Despite aggressive management, many patients with severe sepsis or septic shock die. Numerous interventions have been tested for their ability to improve survival rates among patients with severe sepsis. The list includes endotoxin-neutralizing proteins, inhibitors of cyclooxygenase or nitric oxide synthase, anticoagulants, polyclonal immunoglobulins, glucocorticoids, a phospholipid emulsion, and antagonists to TNF-α, IL-1, platelet-activating factor, and bradykinin. Unfortunately, none of these agents has improved rates of survival among patients with severe sepsis/septic shock in more than one large-scale, randomized, placebo-controlled clinical trial. Many factors have contributed to this lack of reproducibility, including (1) heterogeneity of the patient populations studied, the primary infection sites, the preexisting illnesses, and the inciting microbes; and (2) the nature of the “standard” therapy also used. A dramatic example of this problem was seen in a trial of tissue

1	primary infection sites, the preexisting illnesses, and the inciting microbes; and (2) the nature of the “standard” therapy also used. A dramatic example of this problem was seen in a trial of tissue factor pathway inhibitor. Whereas the drug appeared to improve survival rates after 722 patients had been studied ( p = .006), it did not do so in the next 1032 patients, and the overall result was negative. This inconsistency argues that the results of a clinical trial may not apply to individual patients, even within a carefully selected patient population. It also suggests that, at a minimum, a sepsis intervention should show a significant survival benefit in more than one placebo-controlled, randomized clinical trial before it is accepted as routine clinical practice. In one attempt to reduce patient heterogeneity in clinical trials, experts have called for changes that would restrict these trials to patients who have similar underlying diseases (e.g., major trauma) and inciting

1	to reduce patient heterogeneity in clinical trials, experts have called for changes that would restrict these trials to patients who have similar underlying diseases (e.g., major trauma) and inciting infections (e.g., pneumonia). Other investigators have proposed using specific biobe harmful to patients whose adaptive defense mechanisms are working well. This analysis suggests that if more aggressive early resuscitation improves survival rates among sicker patients, it will become more difficult to obtain additional benefit from other therapies; that is, if an intervention improves patients’ risk status, moving them into a “less severe illness” category, it will be harder to show that adding another agent to the therapeutic regimen is beneficial.

1	An international consortium has advocated “bundling” of multiple therapeutic maneuvers into a unified algorithmic approach that will become the standard of care for severe sepsis. In theory, such a strategy would improve care by mandating measures that seem to bring maximal benefit, such as the rapid administration of appropriate antimicrobial therapy, fluids, and blood pressure support. Caution may be engendered by the fact that three of the key elements of the initial algorithm were eventually withdrawn for lack of evidence; moreover, the benefit of the current sepsis bundles has not been established in randomized controlled clinical trials.

1	Approximately 20–35% of patients with severe sepsis and 40–60% of patients with septic shock die within 30 days. Others die within the ensuing 6 months. Late deaths often result from poorly controlled infection, immunosuppression, complications of intensive care, failure of multiple organs, or the patient’s underlying disease. Case–fatality rates are similar for culture-positive and culture-negative severe sepsis. Prognostic stratification systems such as APACHE II indicate that factoring in the patient’s age, underlying condition, and various physiologic variables can yield useful estimates of the risk of dying of severe sepsis. Age and prior health status are probably the most important risk factors (Fig. 325-1). In patients with no known preexisting morbidity, the case–fatality rate remains <10% until the fourth decade of life, after which it gradually increases to >35% in the very elderly. Death is significantly more likely in severely septic patients with preexisting illness.

1	remains <10% until the fourth decade of life, after which it gradually increases to >35% in the very elderly. Death is significantly more likely in severely septic patients with preexisting illness. Septic shock is also a strong predictor of markers, such as IL-6 levels in blood or the expression of HLA-DR on peripheral-blood monocytes, to identify the patients most likely to benefit from certain interventions.

1	Recombinant activated protein C (aPC) was the first immunomodulatory drug to be approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with severe sepsis or septic shock. Approval was based on the results of a single randomized controlled trial in which the drug was given within 24 h of the patient’s first sepsis-related organ dysfunction; the 28-day survival rate was significantly higher among aPC recipients

1	Case-fatality rate, % who were very sick (APACHE II score, ≥25) before infusion of the protein than among placebo-treated controls. Subsequent trials failed to show a benefit of aPC treatment in patients who were less sick (APACHE II score, <25) or in children, and, a decade after its licensure by the FDA, the drug was withdrawn from the market when a European trial failed to confirm its efficacy in adults with sepsis. Agents in ongoing or planned clinical trials include intravenous immunoglobulin, a polymyxin B hemofiltration column, and FIGURE 325-1 Age, years Influence of age and prior health status on outcome granulocyte-macrophage colony-stimulating factor, which has been reported to restore monocyte immunocompetence in patients with sepsis-associated immunosuppression.

1	A careful retrospective analysis found that the apparent efficacy of all sepsis therapeutics studied to date has been greatest among the patients at greatest risk of dying before treatment; conversely, use of many of these drugs has been associated with increased mortality rates among patients who are less ill. It is possible that neutralizing one of many different mediators may help patients who are very sick, whereas disrupting the mediator balance may of severe sepsis. With modern therapy, fewer than 10% of previously healthy young individuals (below 35 years of age) die with severe sepsis; the case–fatality rate then increases slowly through middle and old age. The most commonly identified etiologic agents in patients who die are Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, and Neisseria meningitidis. Individuals with preexisting comorbidities are at greater risk of dying of severe sepsis at any age. The etiologic agents in these cases are likely to be

1	pneumoniae, and Neisseria meningitidis. Individuals with preexisting comorbidities are at greater risk of dying of severe sepsis at any age. The etiologic agents in these cases are likely to be S. aureus, Pseudomonas aeruginosa, various Enterobacteriaceae, enterococci, or fungi. (Adapted from DC Angus et al: Crit Care Med 29:1303, 2001.) both short-and long-term mortality. Cognitive impairment may be significant in survivors, particularly those who are elderly.

1	Prevention offers the best opportunity to reduce morbidity and mortality from severe sepsis. In developed countries, most episodes of severe sepsis and septic shock are complications of nosocomial infections. These cases might be prevented by reducing the number of invasive procedures undertaken, by limiting the use (and duration of use) of indwelling vascular and bladder catheters, by reducing the incidence and duration of profound neutropenia (<500 neutrophils/ μL), and by more aggressively treating localized nosocomial infections. Indiscriminate use of antimicrobial agents and glucocorticoids should be avoided, and optimal infection-control measures (Chap. 168) should be used. Studies indicate that 50–70% of patients who develop nosocomial severe sepsis or septic shock have experienced a less severe stage of the septic response on at least one previous day in the hospital. Research is needed to identify patients at increased risk and to develop adjunctive agents that can modulate

1	a less severe stage of the septic response on at least one previous day in the hospital. Research is needed to identify patients at increased risk and to develop adjunctive agents that can modulate the septic response before organ dysfunction or hypotension occurs. Cardiogenic Shock and Pulmonary Edema Judith S. Hochman, David H. Ingbar Cardiogenic shock and pulmonary edema are life-threatening condi-tions that should be treated as medical emergencies. The most com-mon joint etiology is severe left ventricular (LV) dysfunction that leads 326 to pulmonary congestion and/or systemic hypoperfusion (Fig. 326-1). The pathophysiology of pulmonary edema and shock is discussed in Chaps. 47e and 324, respectively.

1	Cardiogenic shock (CS) is characterized by systemic hypoperfusion due to severe depression of the cardiac index (<2.2 [L/min]/m2) and sustained systolic arterial hypotension (<90 mmHg) despite an elevated filling pressure (pulmonary capillary wedge pressure [PCWP] >18 mmHg). It is associated with in-hospital mortality rates >50%. The major causes of CS are listed in Table 326-1. Circulatory failure based on cardiac dysfunction may be caused by primary myocardial failure, most commonly secondary to acute myocardial infarction (MI) (Chap. 295), and less frequently by cardiomyopathy or myocarditis (Chap. 287), cardiac tamponade (Chap. 288), or critical valvular heart disease (Chap. 283).

1	Incidence The rate of CS complicating acute MI was 20% in the 1960s, stayed at ~8% for >20 years, but decreased to 5–7% in the first decade of this millennium largely due to increasing use of early reperfusion therapy for acute MI. Shock is more common with ST elevation MI (STEMI) than with non-ST elevation MI (Chap. 295). LV failure accounts for ~80% of cases of CS complicating acute MI. Acute severe mitral regurgitation (MR), ventricular septal rupture (VSR), predominant right ventricular (RV) failure, and free wall rupture or tamponade account for the remainder.

1	Pathophysiology CS is characterized by a vicious circle in which depression of myocardial contractility, usually due to ischemia, results in reduced cardiac output and arterial blood pressure (BP), which result in hypoperfusion of the myocardium and further ischemia and depression of cardiac output (Fig. 326-1). Systolic myocardial dysfunction reduces stroke volume and, together with diastolic dysfunction, leads to elevated LV end-diastolic pressure and PCWP as well as to pulmonary congestion. Reduced coronary perfusion leads to worsening ischemia and progressive myocardial dysfunction and a rapid

1	FIGURE 326-1 Pathophysiology of cardiogenic shock. Systolic and diastolic myocardial dysfunction results in a reduction in cardiac output and often pulmonary congestion. Systemic and coronary hypo-perfusion occur, resulting in progressive ischemia. Although a number of compensatory mechanisms are activated in an attempt to support the circulation, these compensatory mechanisms may become maladaptive and produce a worsening of hemodynamics. *Release of inflammatory cytokines after myocardial infarction may lead to inducible nitric oxide expression, excess nitric oxide, and inappropriate vasodilation. This causes further reduction in systemic and coronary perfusion. A vicious spiral of progressive myocardial dysfunction occurs that ultimately results in death if it is not interrupted. LVEDP, left ventricular end-diastolic pressure. (From SM Hollenberg et al: Ann Intern Med 131:47, 1999.) downward spiral, which, if uninterrupted, is often fatal. A systemic inflammatory response syndrome

1	left ventricular end-diastolic pressure. (From SM Hollenberg et al: Ann Intern Med 131:47, 1999.) downward spiral, which, if uninterrupted, is often fatal. A systemic inflammatory response syndrome may accompany large infarctions and shock. Inflammatory cytokines, inducible nitric oxide synthase, and excess nitric oxide and peroxynitrite may contribute to the genesis of CS as they do to that of other forms of shock (Chap. 324). Lactic acidosis and hypoxemia from CS contribute to the vicious circle by worsening myocardial ischemia and hypotension. Severe acidosis reduces the efficacy of endogenous and exogenously administered catecholamines. Refractory sustained ventricular or atrial tachyarrhythmias can cause or exacerbate CS.

1	Patient Profile Older age, female sex, prior MI, diabetes, anterior MI location, and extensive coronary artery stenoses are associated with an increased risk of CS complicating MI. Shock associated with a first inferior MI should prompt a search for a mechanical cause. CS may rarely occur in the absence of significant stenosis, as seen in LV apical ballooning/Takotsubo’s cardiomyopathy. Timing Shock is present on admission in only one-quarter of patients who develop CS complicating MI; one-quarter develop it rapidly thereafter, within 6 h of MI onset. Another quarter develop shock later on the first day. Subsequent onset of CS may be due to reinfarction, marked infarct expansion, or a mechanical complication.

1	Diagnosis Due to the unstable condition of these patients, supportive therapy must be initiated simultaneously with diagnostic evaluation (Fig. 326-2). A focused history and physical examination should be performed, blood specimens sent to the laboratory, and an electrocardiogram (ECG) and chest x-ray obtained. Etiologies of Cardiogenic Shock or Pulmonary Edema Acute myocardial infarction/ischemia LV failure Ventricular septal rupture Papillary muscle/chordal rupture–severe MR Ventricular free wall rupture with subacute tamponade Other conditions complicating large MIs Post-cardiac arrest Post-cardiotomy Refractory sustained tachyarrhythmias Acute fulminant myocarditis End-stage cardiomyopathy LV apical ballooning Takotsubo’s cardiomyopathy Hypertrophic cardiomyopathy with severe outflow obstruction Aortic dissection with aortic insufficiency or tamponade Severe valvular heart disease Other Etiologies of Cardiogenic Shockb RV failure due to:

1	Other Etiologies of Cardiogenic Shockb RV failure due to: Severe acidosis, severe hypoxemia aThe etiologies of CS are listed. Most of these can cause pulmonary edema instead of shock or pulmonary edema with CS. bThese cause CS but not pulmonary edema. Abbreviations: LV, left ventricular; MI, myocardial infarction; MR, mitral regurgitation; RV, right ventricular; VSR, ventricular septal rupture. Echocardiography is an invaluable diagnostic tool in patients with suspected CS.

1	Echocardiography is an invaluable diagnostic tool in patients with suspected CS. clinical findings Most patients have dyspnea and appear pale, apprehensive, and diaphoretic, and mental status may be altered. The pulse is typically weak and rapid, often in the range of 90–110 beats/min, or severe bradycardia due to high-grade heart block may be present. Systolic BP is reduced (<90 mmHg or ≥30 mmHg below baseline) with a narrow pulse pressure (<30 mmHg), but occasionally BP may be maintained by very high systemic vascular resistance. Tachypnea, Cheyne-Stokes respirations, and jugular venous distention may be present. There is typically a weak apical pulse and soft S1, and an S3 gallop may be audible. Acute, severe MR and VSR usually are associated with characteristic systolic murmurs (Chap. 295). Rales are audible in most patients with LV failure. Oliguria is common.

1	laboratory findings The white blood cell count is typically elevated with a left shift. Renal function is initially unchanged, but blood urea nitrogen and creatinine rise progressively. Hepatic transaminases may be markedly elevated due to liver hypoperfusion. The lactic acid level is elevated. Arterial blood gases usually demonstrate hypoxemia and anion gap metabolic acidosis, which may be compensated by respiratory alkalosis. Cardiac markers, creatine phosphokinase and its MB fraction, and troponins I and T are typically markedly elevated. electrocardiogram In CS due to acute MI with LV failure, Q waves and/or >2-mm ST elevation in multiple leads or left bundle branch block are usually present. More than one-half of all infarcts associated with shock are anterior. Global ischemia due to severe left main stenosis usually is accompanied by severe (e.g., >3 mm) ST depressions in multiple leads.

1	chest roentgenogram The chest x-ray typically shows pulmonary vascular congestion and often pulmonary edema, but these findings may be absent in up to a third of patients. The heart size is usually normal when CS results from a first MI but is enlarged when it occurs in a patient with a previous MI. echocardiogram A two-dimensional echocardiogram with color-flow Doppler (Chap. 270e) should be obtained promptly in patients with suspected CS to help define its etiology. Doppler mapping demonstrates a left-to-right shunt in patients with VSR and the severity of MR when the latter is present. Proximal aortic dissection with aortic regurgitation or tamponade may be visualized, or evidence for pulmonary embolism may be obtained (Chap. 300).

1	Pulmonary artery catheterization The use of pulmonary artery (Swan-Ganz) catheters in patients with established or suspected CS is controversial (Chaps. 272 and 321). Their use is generally recommended for measurement of filling pressures and cardiac output to confirm the diagnosis and to optimize the use of IV fluids, inotropic agents, and vasopressors in persistent shock (Table 326-2). O2 saturation measurement from right atrial, RV, and pulmonary arterial blood samples can rule out a left-to-right shunt. In CS, low mixed venous O2 saturations and elevated arteriovenous (AV) O2 differences reflect low cardiac index and high fractional O2 extraction. However, when sepsis accompanies CS, AV O2 differences may not be elevated (Chap. 324). The PCWP is elevated. Use of sympathomimetic amines may return these measurements and the systemic BP to normal. Systemic vascular resistance may be low, normal, or elevated in CS. Equalization of rightand left-sided filling pressures (right atrial

1	may return these measurements and the systemic BP to normal. Systemic vascular resistance may be low, normal, or elevated in CS. Equalization of rightand left-sided filling pressures (right atrial and PCWP) suggests cardiac tamponade as the cause of CS (Chap. 288).

1	left heart catheterization and coronary angiograPhy Measurement of LV pressure and definition of the coronary anatomy provide useful information and are indicated in most patients with CS complicating MI. Cardiac catheterization should be performed when there is a plan and capability for immediate coronary intervention (see below) or when a definitive diagnosis has not been made by other tests.

1	(Fig. 326-2) In addition to the usual treatment of acute MI (Chap. 295), initial therapy is aimed at maintaining adequate systemic and coronary perfusion by raising systemic BP with vasopressors and adjusting volume status to a level that ensures optimum LV filling pressure. There is interpatient variability, but the values that generally are associated with adequate perfusion are systolic BP ~90 mmHg or mean BP >60 mmHg and PCWP >20 mmHg. Hypoxemia and acidosis must be corrected; most patients require ventilatory support (see “Pulmonary Edema,” below). Negative inotropic agents should be discontinued and the doses of renally cleared medications adjusted. Hyperglycemia should be controlled with insulin. Bradyarrhythmias may require transvenous pacing. Recurrent ventricular tachycardia or rapid atrial fibrillation may require immediate treatment (Chap. 276).

1	Various IV drugs may be used to augment BP and cardiac output in patients with CS. All have important disadvantages, and none has been shown to change the outcome in patients with established shock. Norepinephrine is a potent vasoconstrictor and inotropic stimulant that is useful for patients with CS. As first line of therapy norepinephrine was associated with fewer adverse events, including arrhythmias, compared to a dopamine randomized trial of patients Third line of action

1	Clinical signs: Shock, hypoperfusion, congestive heart failure, acute pulmonary edema Most likely major underlying disturbance? Acute pulmonary edema Check blood pressure Systolic BP Greater than 100 mmHg and not less than 30 mmHg below baseline ACE Inhibitors Short-acting agent such as captopril (1 to 6.25 mg) Low output-cardiogenic shock Check blood pressure Section 9.5 – 2013 American College of Cardiology Foundation /American Heart Association Guidelines for Management of ST-Elevation Myocardial Infarction andFigures 3 and 4 – 2010 American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Part 8: Adult Advanced Cardiovascular Life Support Hypovolemia BradycardiaTachycardiaAdminister • Fluids• Blood transfusions• Cause-specific interventionsConsider vasopressorsArrhythmia Systolic BP Greater than 100 mmHgDopamine, 5 to 15 ˜g/kg per minute IV Nitroglycerin 10to 20 ˜g/min IVDobutamine Systolic BP 70 to 100 mmHgSystolic BP NO

1	interventionsConsider vasopressorsArrhythmia Systolic BP Greater than 100 mmHgDopamine, 5 to 15 ˜g/kg per minute IV Nitroglycerin 10to 20 ˜g/min IVDobutamine Systolic BP 70 to 100 mmHgSystolic BP NO signs/symptoms of shocksigns/symptoms of shock* 2 to 20 ˜g/kg per minute IVless than 100 mmHg *Norepinephrine 0.5 to 30 ˜g/min IV or Administer • Furosemide IV 0.5 to 1.0 mg/kg• Morphine IV 2 to 4 mg• Oxygen/intubation as needed• Nitroglycerin SL, then 10to 20 ˜g/min IV if SBP greater than 100 mmHg• *Norepinephrine, 0.5 to 30 ˜g/min IV or Dopamine, 5 to 15 ˜g/kg per minute IV if SBP <100 mmHg and signs/symptoms of shock present • Dobutamine 2 to 20 ˜g/kg per minute IV if SBP 70to 100 mmHg and no signs/symptoms of shockFirst line of actionSecond line of actionFurther diagnostic/therapeutic considerations (should be consideredin nonhypovolemic shock)Therapeutic • Intraaortic balloon pump or othercirculatory assist device• Reperfusion/revascularization

1	FIGURE 326-2 The emergency management of patients with cardiogenic shock, acute pulmonary edema, or both is outlined.

1	*Furosemide: <0.5 mg/kg for new-onset acute pulmonary edema without hypervolemia; 1 mg/kg for acute on chronic volume overload, renal insufficiency. †For management of bradycardia and tachycardia, see Chaps. 274 and 276. Additional information can also be found in Section 9.5 of the 2013 American College of Cardiology Foundation/American Heart Association Guidelines for Management of ST-Elevation Myocardial Infarction and Figures 3 and 4 of the 2010 American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Part 8: Adult Advanced Cardiovascular Life Support. *Indicates modification from published guidelines. ACE, angiotensinconverting enzyme; BP, blood pressure; MI, myocardial infarction. (Modified from Guidelines 2000 for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Part 7: The era of reperfusion: Section 1: Acute coronary syndromes [acute myocardial infarction]. The American Heart Association in collaboration with

1	and Emergency Cardiovascular Care. Part 7: The era of reperfusion: Section 1: Acute coronary syndromes [acute myocardial infarction]. The American Heart Association in collaboration with the International Liaison Committee on Resuscitation. Circulation 102:I172, 2000.) with several etiologies of circulatory shock. Although it did not significantly improve survival compared to dopamine, its relative safety suggests that norepinephrine is reasonable as initial vasopressor therapy. Norepinephrine should be started at a dose of 2 to 4 μg/ min and titrated upward as necessary. If systemic perfusion or systolic pressure cannot be maintained at >90 mmHg with a dose of 15 μg/min, it is unlikely that a further increase will be beneficial.

1	Dopamine has varying hemodynamic effects based on the dose; at low doses (≤ 2 μg/kg per min), it dilates the renal vascular bed, although its outcome benefits at this low dose have not been demonstrated conclusively; at moderate doses (2–10 μg/kg per min), it has positive chronotropic and inotropic effects as a consequence of β-adrenergic receptor stimulation. At higher doses, a vasoconstrictor effect results from α-receptor stimulation. It is started at an infusion rate of 2–5 μg/kg per min, and the dose is increased every 2–5 min to a maximum of 20–50 μg/kg per min. Dobutamine is a synthetic sympathomimetic amine with positive inotropic action and minimal positive chronotropic activity at low doses (2.5 μg/kg per min) but moderate chronotropic activity at higher doses. Although the usual dose is up to 10 μg/kg per min, its vasodilating activity precludes its use when a vasoconstrictor effect is required.

1	Circulatory assist devices can be placed percutaneously or surgically and can be used to support the left, right, or both ventricles. Venoarterial extracorporeal membrane oxygenation (VA ECMO, a pump in combination with an oxygenator) may be used when respiratory failure accompanies biventricular failure. Temporary percutaneous devices can be used as a bridge to surgically implanted devices in community hospital settings or when neurologic status is uncertain. The most commonly used device is an intraaortic balloon pump (IABP), which is inserted into the aorta via the femoral artery and provides temporary hemodynamic support. However, routine IABP use in conjunction with early revascularization (predominantly with percutaneous coronary intervention [PCI]) did not reduce 30-day mortality in the IABP-SHOCK II trial. Although other percutaneous devices, including VA ECMO, result in better hemodynamic support compared to IABP, the effects on clinical outcomes are unknown. Surgically

1	in the IABP-SHOCK II trial. Although other percutaneous devices, including VA ECMO, result in better hemodynamic support compared to IABP, the effects on clinical outcomes are unknown. Surgically implanted devices can support the circulation as bridging therapy for cardiac transplant candidates or as destination therapy (Chap. 281). Assist devices should be used selectively in suitable patients in consultation with advanced heart failure specialists.

1	SVR, (dyn · s)/ RA, mmHg RVS, mmHg RVD, mmHg PAS, mmHg PAD, mmHg PCW, mmHg CI, (L/min)/m2 cm5 Normal values <6 <25 0–12 <25 0–12 <6–12 ≥2.5 (800–1600) MI without pul-– – – – – ~13 (5–18) ~2.7 (2.2–4.3) – monary edemab Pulmonary ↔↑↔↑↔↑↑ ↑ ↑ ↔↓↑ Septic shock ↓↔↓↔↓↓ ↓↓↑↓ aThere is significant patient-to-patient variation. Pressure may be normalized if cardiac output is low. bForrester et al classified nonreperfused MI patients into four hemodynamic subsets. (From JS Forrester et al: N Engl J Med 295:1356, 1976.) PCW pressure and CI in clinically stable subset 1 patients are shown. Values in parentheses represent range. c”Isolated” or predominant RV failure. dPCW and pulmonary artery pressures may rise in RV failure after volume loading due to RV dilation and right-to-left shift of the interventricular septum, resulting in impaired LV filling. When biventricular failure is present, the patterns are similar to those shown for LV failure.

1	Abbreviations: CI, cardiac index; MI, myocardial infarction; P/SBF, pulmonary/systemic blood flow; PAS/D, pulmonary artery systolic/diastolic; PCW, pulmonary capillary wedge; RA, right atrium; RVS/D, right ventricular systolic/diastolic; SVR, systemic vascular resistance. Source: Table prepared with the assistance of Krishnan Ramanathan, MD. The rapid establishment of blood flow in the infarct-related artery is essential in the management of CS and forms the centerpiece of management. The randomized SHOCK Trial demonstrated that 132 lives were saved per 1000 patients treated with early revascularization with PCI or coronary artery bypass graft (CABG) compared with initial medical therapy including IABP with fibrinolytics followed by delayed revascularization. The benefit is seen across the risk strata and is sustained up to 11 years after an MI. Early revascularization with PCI or CABG is recommended in candidates suitable for aggressive care.

1	Prognosis Within this high-risk condition, there is a wide range of expected death rates based on age, severity of hemodynamic abnormalities, severity of the clinical manifestations of hypoperfusion, and the performance of early revascularization.

1	Although transient hypotension is common in patients with RV infarction and inferior MI (Chap. 295), persistent CS due to RV failure accounts for only 3% of CS complicating MI. The salient features of RV shock are absence of pulmonary congestion, high right atrial pressure (which may be seen only after volume loading), RV dilation and dysfunction, only mildly or moderately depressed LV function, and predominance of single-vessel proximal right coronary artery occlusion. Management includes IV fluid administration to optimize right atrial pressure (10–15 mmHg); avoidance of excess fluids, which cause a shift of the interventricular septum into the LV; sympathomimetic amines; the early reestablishment of infarct-artery flow; and assist devices.

1	(See also Chap. 295) Acute severe MR due to papillary muscle dysfunction and/or rupture may complicate MI and result in CS and/or pulmonary edema. This complication most often occurs on the first day, with a second peak several days later. The diagnosis is confirmed by echo-Doppler. Rapid stabilization with IABP is recommended, with administration of dobutamine as needed to raise cardiac output. Reducing the load against which the LV pumps (afterload) reduces the volume of regurgitant flow of blood into the left atrium. Mitral valve surgery is the definitive therapy and should be performed early in the course in suitable candidates. (See also Chap. 295) Echo-Doppler demonstrates shunting of blood from the left to the right ventricle and may visualize the opening in the interventricular septum. Timing and management are similar to those for MR with IABP support and surgical correction for suitable candidates.

1	Myocardial rupture is a dramatic complication of STEMI that is most likely to occur during the first week after the onset of symptoms; its frequency increases with the age of the patient. The clinical presentation typically is a sudden loss of pulse, blood pressure, and consciousness but sinus rhythm on ECG (pulseless electrical activity) due to cardiac tamponade (Chap. 288). Free wall rupture may also result in CS due to subacute tamponade when the pericardium temporarily seals the rupture sites. Definitive surgical repair is required.

1	(See also Chap. 287) Myocarditis can mimic acute MI with ST deviation or bundle branch block on the ECG and marked elevation of cardiac markers. Acute myocarditis causes CS in a small proportion of cases. These patients are typically younger than those with CS due to acute MI and often do not have typical ischemic chest pain. Echocardiography usually shows global LV dysfunction. Initial management is the same as for CS complicating acute MI (Fig. 326-2) but does not involve coronary revascularization. Endomyocardial biopsy is recommended to determine the diagnosis and need for immunosuppressives for entities such as giant cell myocarditis. Refractory CS can be managed with assist devices with or without ECMO. The etiologies and pathophysiology of pulmonary edema are discussed in Chap. 47e.

1	The etiologies and pathophysiology of pulmonary edema are discussed in Chap. 47e. Diagnosis Acute pulmonary edema usually presents with the rapid onset of dyspnea at rest, tachypnea, tachycardia, and severe hypoxemia. Crackles and wheezing due to alveolar flooding and airway compression from peribronchial cuffing may be audible. Release of endogenous catecholamines often causes hypertension. It is often difficult to distinguish between cardiogenic and noncardiogenic causes of acute pulmonary edema. Echocardiography may identify systolic and diastolic ventricular dysfunction and valvular lesions. Electrocardiographic ST elevation and evolving Q waves are usually diagnostic of acute MI and should prompt immediate institution of MI protocols and coronary artery reperfusion therapy (Chap. 295). Brain natriuretic peptide levels, when substantially elevated, support heart failure as the etiology of acute dyspnea with pulmonary edema (Chap. 279).

1	The use of a Swan-Ganz catheter permits measurement of PCWP and helps differentiate high-pressure (cardiogenic) from normal-pressure (noncardiogenic) causes of pulmonary edema. Pulmonary artery catheterization is indicated when the etiology of the pulmonary edema is uncertain, when edema is refractory to therapy, or when it is accompanied by hypotension. Data derived from use of a catheter often alter the treatment plan, but no impact on mortality rates has been demonstrated. The treatment of pulmonary edema depends on the specific etiology. As an acute, life-threatening condition, a number of measures must be applied immediately to support the circulation, gas exchange, and lung mechanics. Simultaneously, conditions that frequently complicate pulmonary edema, such as infection, acidemia, anemia, and acute kidney dysfunction, must be corrected.

1	Patients with acute cardiogenic pulmonary edema generally have an identifiable cause of acute LV failure—such as arrhythmia, ischemia/infarction, or myocardial decompensation (Chap. 279)— that may be rapidly treated, with improvement in gas exchange. In contrast, noncardiogenic edema usually resolves much less quickly, and most patients require mechanical ventilation. Oxygen Therapy Support of oxygenation is essential to ensure adequate O2 delivery to peripheral tissues, including the heart.

1	Positive-Pressure Ventilation Pulmonary edema increases the work of breathing and the O2 requirements of this work, imposing a significant physiologic stress on the heart. When oxygenation or ventilation is not adequate in spite of supplemental O2, positive-pressure ventilation by face or nasal mask or by endotracheal intubation should be initiated. Noninvasive ventilation (Chap. 323) can rest the respiratory muscles, improve oxygenation and cardiac function, and reduce the need for intubation. In refractory cases, mechanical ventilation can relieve the work of breathing more completely than can noninvasive ventilation. Mechanical ventilation with positive end-expiratory pressure can have multiple beneficial effects on pulmonary edema: (1) decreases both preload and afterload, thereby improving cardiac function; (2) redistributes lung water from the intraalveolar to the extraalveolar space, where the fluid interferes less with gas exchange; and (3) increases lung volume to avoid

1	improving cardiac function; (2) redistributes lung water from the intraalveolar to the extraalveolar space, where the fluid interferes less with gas exchange; and (3) increases lung volume to avoid atelectasis.

1	In most forms of pulmonary edema, the quantity of extravascular lung water is determined by both the PCWP and the intravascular volume status. Diuretics The “loop diuretics” furosemide, bumetanide, and torsemide are effective in most forms of pulmonary edema, even in the presence of hypoalbuminemia, hyponatremia, or hypochloremia. Furosemide is also a venodilator that rapidly reduces preload before any diuresis, and is the diuretic of choice. The initial dose of furosemide should be ≤0.5 mg/kg, but a higher dose (1 mg/kg) is required in patients with renal insufficiency, chronic diuretic use, or hypervol-1763 emia or after failure of a lower dose.

1	Nitrates Nitroglycerin and isosorbide dinitrate act predominantly as venodilators but have coronary vasodilating properties as well. They are rapid in onset and effective when administered by a variety of routes. Sublingual nitroglycerin (0.4 mg × 3 every 5 min) is first-line therapy for acute cardiogenic pulmonary edema. If pulmonary edema persists in the absence of hypotension, sublingual may be followed by IV nitroglycerin, commencing at 5–10 μg/min. IV nitroprusside (0.1–5 μg/kg per min) is a potent venous and arterial vasodilator. It is useful for patients with pulmonary edema and hypertension but is not recommended in states of reduced coronary artery perfusion. It requires close monitoring and titration using an arterial catheter for continuous BP measurement.

1	Morphine Given in 2to 4-mg IV boluses, morphine is a transient venodilator that reduces preload while relieving dyspnea and anxiety. These effects can diminish stress, catecholamine levels, tachycardia, and ventricular afterload in patients with pulmonary edema and systemic hypertension. Angiotensin-Converting Enzyme (ACE) Inhibitors ACE inhibitors reduce both afterload and preload and are recommended for hypertensive patients. A low dose of a short-acting agent may be initiated and followed by increasing oral doses. In acute MI with heart failure, ACE inhibitors reduce shortand long-term mortality rates. tide (nesiritide) is a potent vasodilator with diuretic properties and is effective in the treatment of cardiogenic pulmonary edema. It should be reserved for refractory patients and is not recommended in the setting of ischemia or MI.

1	Physical Methods In nonhypotensive patients, venous return can be reduced by use of the sitting position with the legs dangling along the side of the bed. Inotropic and Inodilator Drugs The sympathomimetic amines dopa mine and dobutamine (see above) are potent inotropic agents. The bipyridine phosphodiesterase-3 inhibitors (inodilators), such as milrinone (50 μg/kg followed by 0.25–0.75 μg/kg per min), stimulate myocardial contractility while promoting peripheral and pulmonary vasodilation. Such agents are indicated in patients with cardiogenic pulmonary edema and severe LV dysfunction.

1	Digitalis Glycosides Once a mainstay of treatment because of their positive inotropic action (Chap. 279), digitalis glycosides are rarely used at present. However, they may be useful for control of ventricular rate in patients with rapid atrial fibrillation or flutter and LV dysfunction, because they do not have the negative inotropic effects of other drugs that inhibit atrioventricular nodal conduction. Intraaortic Balloon Counterpulsation IABP or other LV-assist devices (Chap. 281) may help relieve cardiogenic pulmonary edema and are indicated when refractory pulmonary edema results from the etiologies discussed in the CS section, especially in preparation for surgical repair.

1	Treatment of Tachyarrhythmias and Atrial-Ventricular Resynchronization (See also Chap. 277) Sinus tachycardia or atrial fibrillation can result from elevated left atrial pressure and sympathetic stimulation. Tachycardia itself can limit LV filling time and raise left atrial pressure further. Although relief of pulmonary congestion will slow the sinus rate or ventricular response in atrial fibrillation, a primary tachyarrhythmia may require cardioversion. In patients with reduced LV function and without atrial contraction or with lack of synchronized atrioventricular contraction, placement of an atrioventricular sequential pacemaker should be considered (Chap. 274). Stimulation of Alveolar Fluid Clearance A variety of drugs can stimulate alveolar epithelial ion transport and upregulate the clearance of alveolar solute and water, but this strategy has not been proven beneficial in clinical trials thus far.

1	Critical Care Medicine Cardiovascular Collapse, Cardiac Arrest, and Sudden Cardiac death Robert J. Myerburg, Agustin Castellanos OVERVIEW AND DEFINITIONS Sudden cardiac death (SCD) is defined as natural death due to cardiac 327 1764 SPECIAL CONSIDERATIONS Risk of Iatrogenic Cardiogenic Shock In the treatment of pulmonary edema, vasodilators lower BP, and their use, particularly in combination, may lead to hypotension, coronary artery hypoperfusion, and shock (Fig. 326-1). In general, patients with a hypertensive response to pulmonary edema tolerate and benefit from these medications. In normotensive patients, low doses of single agents should be instituted sequentially, as needed. Acute Coronary Syndromes (See also Chap. 295) Acute STEMI complicated by pulmonary edema is associated with in-hospital mortality rates of 20–40%. After immediate stabilization, coronary artery blood flow must be reestablished rapidly. When available, primary PCI is preferable; alternatively, a fibrinolytic

1	mortality rates of 20–40%. After immediate stabilization, coronary artery blood flow must be reestablished rapidly. When available, primary PCI is preferable; alternatively, a fibrinolytic agent should be administered. Early coronary angiography and revascularization by PCI or CABG also are indicated for patients with non-ST elevation acute coronary syndrome. Assist devices may be used selectively as noted for refractory pulmonary edema.

1	Extracorporeal Membrane Oxygenation For patients with acute, severe noncardiogenic edema with a potential rapidly reversible cause, ECMO may be considered as a temporizing supportive measure to achieve adequate gas exchange. Usually venovenous ECMO is used in this setting. Unusual Types of Edema Specific etiologies of pulmonary edema may require particular therapy. Reexpansion pulmonary edema can develop after removal of longstanding pleural space air or fluid. These patients may develop hypotension or oliguria resulting from rapid fluid shifts into the lung. Diuretics and preload reduction are contraindicated, and intravascular volume repletion often is needed while supporting oxygenation and gas exchange.

1	High-altitude pulmonary edema often can be prevented by use of dexamethasone, calcium channel–blocking drugs, or long-acting inhaled β2-adrenergic agonists. Treatment includes descent from altitude, bed rest, oxygen, and, if feasible, inhaled nitric oxide; nifedipine may also be effective. For pulmonary edema resulting from upper airway obstruction, recognition of the obstructing cause is key, because treatment then is to relieve or bypass the obstruction. causes in a person who may or may not have previously recognized heart disease but in whom the time and mode of death are unexpected. The term “sudden,” in the context of SCD, is defined for most clinical and epidemiologic purposes as 1 h or less between a change in clinical status heralding the onset of the terminal clinical event and the cardiac arrest itself. One exception is unwitnessed deaths, in which pathologists may expand the temporal definition to 24 h after the victim was last seen to be alive and stable.

1	Another exception is the variable interval between cardiac arrest and biological death that results from community-based interventions, following which victims may remain biologically alive for days or even weeks after a cardiac arrest that has resulted in irreversible central nervous system damage. Confusion in terms can be avoided by adhering strictly to definitions of cardiovascular collapse, cardiac arrest, and death (Table 327-1). Although cardiac arrest is often potentially reversible by appropriate and timely interventions, death is biologically, legally, and literally an absolute and irreversible event. Biological death may be delayed by interventions, but the relevant pathophysiologic event remains the sudden and unexpected cardiac arrest. Accordingly, for statistical purposes, deaths that occur during hospitalization or within 30 days after resuscitated cardiac arrest are counted as sudden deaths.

1	The majority of natural deaths are caused by cardiac disorders. However, it is common for underlying heart diseases—often far advanced—to go unrecognized before the fatal event. As a result, up to two-thirds of all SCDs occur as the first clinical expression of previously undiagnosed disease or in patients with known heart disease, the extent of which suggests low individual risk. The magnitude of sudden cardiac death as a public health problem is highlighted by the estimate that ~50% of all cardiac deaths are sudden and unexpected, accounting for a total SCD burden estimated to range from <200,000 to >450,000 deaths each year in the United States. SCD is a direct consequence of cardiac arrest, which may be reversible if addressed promptly. Because resuscitation techniques and emergency rescue systems are available to respond to victims of out-of-hospital cardiac arrest, which was uniformly fatal in the past, understanding the SCD problem has practical clinical importance.

1	Cardiovascular collapse is a general term connoting loss of sufficient cerebral blood flow to maintain consciousness due to acute dysfunction of the heart and/or peripheral vasculature. It may be caused by vasodepressor syncope (vasovagal syncope, postural hypotension with syncope, neurocardiogenic syncope; Chap. 27), a transient severe bradycardia, or cardiac arrest. The latter is distinguished from the transient forms of cardiovascular collapse in that it usually requires an active intervention to restore spontaneous blood flow. In contrast, vasodepressor syncope and other primary bradyarrhythmic syncopal events are transient and non-life-threatening, with spontaneous return of consciousness.

1	In the past, the most common electrical mechanism for cardiac arrest was ventricular fibrillation (VF) or pulseless sustained ventricular tachycardia (PVT). These were the initial rhythms recorded in 60–80% of cardiac arrests, with VF being the far more common of the two. Severe persistent bradyarrhythmias, asystole, and pulseless electrical activity (PEA; organized electrical activity, unusually slow, without mechanical response, formerly called electromechanical dissociation [EMD]) caused another 20–30%. Currently, asystole has emerged as the most common mechanism recorded at initial contact (45–50% of cases). PEA accounts for 20–25%, and VF is now present on initial contact in 25–35%. Undoubtedly, a significant proportion of the asystole cases began as VF and deteriorated to asystole because of long response times, but there are data suggesting an absolute reduction in VF as well. Acute low cardiac output states, having a precipitous onset, also may present clinically as a cardiac

1	of long response times, but there are data suggesting an absolute reduction in VF as well. Acute low cardiac output states, having a precipitous onset, also may present clinically as a cardiac arrest. These hemodynamic causes include massive acute pulmonary emboli, internal blood loss from a ruptured aortic aneurysm, intense anaphylaxis, and cardiac rupture with tamponade after myocardial infarction (MI).

1	ETIOLOGY, INITIATING EVENTS, AND CLINICAL EPIDEMIOLOGY Clinical, epidemiologic, and pathologic studies have provided information on the underlying structural substrates in victims of SCD and identified subgroups at high risk for SCD. In addition, studies of clinical physiology have begun to identify transient functional factors that may convert a long-standing underlying structural abnormality from a stable to an unstable state, leading to the onset of cardiac arrest (Table 327-2).

1	Cardiac disorders constitute the most common causes of sudden natural death. After an initial peak incidence of sudden death between birth and 6 months of age (sudden infant death syndrome [SIDS]), the incidence of sudden death declines sharply and remains low through childhood and adolescence. Among adolescents and young adults, the incidence of SCD is approximately 1 per 100,000 population per year. The incidence begins to increase in adults over age 30 years, reaching a second peak in the age range of 45–75 years, when it approximates 1–2 per 1000 per year among the unselected adult population. Increasing 1765TABlE 327-1 dISTInCTIon BETwEEn CARdIovASCulAR CollAPSE, CARdIAC ARREST, And dEATH Source: Modified from RJ Myerburg, A Castellanos: Cardiac arrest and sudden cardiac death, in P Libby et al (eds): Braunwald’s Heart Disease, 8th ed. Philadelphia, Saunders, 2008. I. Coronary heart disease A. Coronary artery abnormalities 1. 2.

1	I. Coronary heart disease A. Coronary artery abnormalities 1. 2. Active lesions (plaque fissuring, platelet aggregation, acute thrombosis) 3. B. Myocardial infarction 1. 2. II. A. B. 1. 2. III. Dilated cardiomyopathy—primary muscle disease IV. A. B. C. V. VI. Electrophysiologic abnormalities, structural A. B. VII. Inherited disorders associated with electrophysiologic abnormalities (congenital long QT syndromes, right ventricular dysplasia, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia, etc.) Triggers for Expression of Cardiac Arrest I. Alterations of coronary blood flow A. Transient ischemia B. Reperfusion after ischemia II. A. 1. 2. B. Shock III. A. Electrolyte imbalance (e.g., hypokalemia) B. Hypoxemia, acidosis IV. A. Autonomic fluctuations: central, neural, humoral B. V. A. B. Cardiac toxins (e.g., cocaine, digitalis intoxication) C.

1	B. Hypoxemia, acidosis IV. A. Autonomic fluctuations: central, neural, humoral B. V. A. B. Cardiac toxins (e.g., cocaine, digitalis intoxication) C. age within this range is associated with increasing risk for sudden cardiac death (Fig. 327-1A). From 1 to 13 years of age, only one of five sudden natural deaths is due to cardiac causes. Between 14 and 21 years of age, the proportion increases to 30%, and it rises to 88% in the middle-aged and elderly.

1	Young and middle-aged men and women have different susceptibilities to SCD, but the sex differences decrease and ultimately disappear with advancing age. The difference in risk for SCD parallels the differences in age-related risks for other manifestations of coronary heart disease (CHD) between men and women. As the gender gap for manifestations of CHD closes in the sixth to eighth decades of life, the excess risk of SCD in males progressively narrows. Despite the lower incidence among younger women, coronary risk factors such as cigarette smoking, diabetes, hyperlipidemia, and hypertension are highly influential, and SCD remains an important clinical and epidemiologic problem. The incidence of SCD among the African-American population appears to be higher than it is among the white population; the reasons remain uncertain.

1	Genetic factors contribute to the risk of acquiring CHD, and a genetic basis for its expression as SCD is being explored. A genetic hypothesis for at least part of the SCD risk is supported by data suggesting a familial predisposition to SCD as a specific form of expression of CHD. A parental history of SCD as a first cardiac event increases the probability that an acute coronary event in the offspring will express similarly. In a number of less common syndromes, such as hypertrophic cardiomyopathy, congenital long QT interval syndromes, right ventricular dysplasia, and the syndrome of right bundle branch block and nonischemic ST-segment elevations (Brugada syndrome), and other more rare syndromes, there is a specific inherited risk of ventricular arrhythmias and SCD (Chap. 277).

1	The etiologic structural substrates and functional factors contributing to expression of the SCD syndrome are listed in Table 327-2. Worldwide, and especially in Western cultures, coronary atherosclerotic heart disease is the most common structural abnormality associated with SCD in middle-aged and older adults. Up to 80% of all SCDs in the United States are due to the consequences of coronary atherosclerosis. The nonischemic cardiomyopathies (dilated and hypertrophic, collectively; Chap. 273e) account for another 10–15% of SCDs, and all the remaining diverse etiologies cause only 5–10% of all SCDs. The inherited arrhythmia syndromes (see above and Table 327-2) are proportionally more common causes in adolescents and young adults. For some of these syndromes, such as hypertrophic cardiomyopathy (Chap. 287), the risk of SCD increases significantly after the onset of puberty.

1	Transient ischemia in a previously scarred or hypertrophied heart, hemodynamic and fluid and electrolyte disturbances, fluctuations in autonomic nervous system activity, and transient electrophysiologic changes caused by drugs or other chemicals (e.g., proarrhythmia) have all been implicated as mechanisms responsible for the transition from electrophysiologic stability to instability. In addition, reperfusion of ischemic myocardium may cause transient electrophysiologic instability and arrhythmias. Cardiovascular Collapse, Cardiac Arrest, and Sudden Cardiac Death Data from postmortem examinations of SCD victims parallel the clinical observations on the prevalence of CHD as the major structural etiologic factor. More than 80% of SCD victims have pathologic findings of CHD. The pathologic description often includes a combination of long-standing, extensive atherosclerosis of the epicardial coronary 0.1%/Year prior cardiac arrest.

1	Adolescents/ young adults [1 per 100,000] General population >35 years of age [1 per 500–1000] arteries and unstable coronary artery lesions, which include various permutations of eroded, fissured, or ruptured plaques; platelet aggregates; hemorrhage; and/or thrombosis. As many as 70–75% of males who die suddenly have preexisting healed MIs, whereas only 20–30% have recent acute MIs, despite the prevalence of unstable plaques and thrombi. The latter suggests transient ischemia as the mechanism of 0.001%/Year SCDs: SCDs: [Annual incidence] [Annual number] General population High coronary risk profile Prior coronary event Cardiac arrest survivor Post-MI: low EF, VT EF <35%; CHF 0 5 10 15 20 25 30 0 100,000300,000200,000 onset. Regional or global left ventricular (LV) hypertrophy often coexists with prior MIs.

1	SCD accounts for approximately one-half the total number of cardiovascular deaths. As shown in Fig. 327-1B, the very-high-risk subgroups consist of more focused populations at higher risk of cardiac arrest or SCD, with better individual prediction, but the representation of such subgroups within the overall population burden of SCD is small. This is indicated by the absolute number of events (“events per year”), in contrast to the percentage per year in the subgroup. To achieve a major population impact, effective prevention of underlying diseases and the development of new epidemiologic and clinical probes that will allow better individual risk prediction by identifying specific high-risk subgroups within the large general populations are needed.

1	Strategies for predicting and preventing SCD are classified as primary and secondary. Primary prevention refers to the attempt to identify individual patients at specific risk for SCD and institute preventive strategies. Secondary prevention refers to measures taken to prevent The effectiveness of the prevention strategies currently used depends on the magnitude of risk among the various population subgroups. diac death (SCD). For the general population age 35 years and older, Because the annual incidence of SCD among the unselected adult

1	diac death (SCD). For the general population age 35 years and older, Because the annual incidence of SCD among the unselected adult SCD risk is 0.1–0.2% per year (1 per 500–1000 population). Among the population is limited to approximately 1 per 1000 population per year general population of adolescents and adults younger than age 30 (Fig. 327-1) and ~50% of all SCDs due to coronary artery disease occur years, the overall risk of SCD is 1 per 100,000 population, or 0.001% as the first clinical manifestation of the disease (Fig. 327-2A), the only per year. The risk of SCD increases dramatically beyond age 35 years. currently practical strategies are profiling for risk of developing CHD

1	currently practical strategies are profiling for risk of developing CHD The greatest rate of increase is between 40 and 65 years and risk factor control (Fig. 327-2B). The most powerful long-term (vertical axis is discontinuous). Among patients older than 30 years of risk factors include age, cigarette smoking, elevated serum cholesterol, age, with advanced structural heart disease and markers of high risk for diabetes mellitus, elevated blood pressure, LV hypertrophy, and non- cardiac arrest, the event rate may exceed 25% per year, and age-related specific electrocardiographic abnormalities. Markers of inflammation risk attenuates. (Modified from RJ Myerburg, A Castellanos: Cardiac (e.g., levels of C-reactive protein) that may predict plaque destabiliza arrest and sudden cardiac death, in P Libby et al [eds]: Braunwald’s Heart tion have been added to risk classifications. The presence of multiple

1	Disease, 8th ed. Philadelphia, Saunders, 2008.) Panel B demonstrates risk factors progressively increases incidence, but not sufficiently or the incidence of SCD in population subgroups and the relation of specifically enough to warrant therapies targeted to potentially fatal total number of events per year to incidence figures. Approximations arrhythmias (Fig. 327-1A). However, recent studies suggesting familial of subgroup incidence figures and the related population pool from clustering of SCD associated with a first acute coronary syndrome offer which they are derived are presented. Approximately 50% of all hope that genetic markers for specific risk may be forthcoming. cardiac deaths are sudden and unexpected. The incidence bars on

1	After coronary artery disease has been identified in a patient, addi the left (percent/year) indicate the approximate percentage of sud tional strategies for risk profiling become available (Fig. 327-2B), but den and nonsudden deaths in each of the population subgroups the majority of SCDs occur among the large unselected groups rather indicated, ranging from the lowest percentage in unselected adult than in the specific high-risk subgroups that become evident among populations (0.1–2% per year) to the highest percentage in patients populations with established disease (compare events per year with percentage per year in Fig. 327-1B). After a major cardiovascular mately 25% per year). The bars on the right indicate the total number event, such as acute MI, recent onset of heart failure, or survival after of events per year in each of these groups with the population impact out-of-hospital cardiac arrest, the highest risk of death occurs dur size of each of the subgroups. The highest

1	or survival after of events per year in each of these groups with the population impact out-of-hospital cardiac arrest, the highest risk of death occurs dur size of each of the subgroups. The highest risk categories identify the ing the initial 6–18 months after the event and then plateaus toward smallest number of total annual events, and the lowest incidence the baseline risk associated with the extent of underlying disease.

1	category accounts for the largest number of events per year. CHF, con- However, many of the early deaths are nonsudden, diluting the poten gestive heart failure; EF, ejection fraction; MI, myocardial infarction; VT, tial benefit of strategies targeted specifically to SCD. Thus, although ventricular tachycardia. (After RJ Myerburg et al: Circulation 85:2, 1992.) post-MI beta blocker therapy has an identifiable benefit for both early SCD and nonsudden mortality risk, a total mortality benefit for implantable cardioverter-defibrillator (ICD) therapy early after MI has not been observed. 1767 A100 40 50 30 20 10 0 5–10%Arrhythmic risk markers 7–15%Hemodynamic risk markers ˜20%Acute M.I.; unstable AP °30%Know disease; low power or non-specific markers ~50%First clinical event

1	FIGURE 327-2 Population subsets, risk predictors, and distribution of sudden cardiac deaths (SCDs) according to clinical circumstances. A. The population subset with high-risk arrhythmia markers in conjunction with low ejection fraction is a group at high risk of SCD but accounts for <10% of the total SCD burden attributable to coronary artery disease. In contrast, 50% of all SCD victims present with SCD as the first and only manifestation of underlying disease, and up to 30% have known disease but are considered relatively low risk because of the absence of high-risk markers. B. Profiling for individual prediction and prevention of SCD is difficult. The highest absolute numbers of events occur among the general population who may have risk factors for coronary heart disease or expressions of disease that do not predict high risk. This results in a low sensitivity for predicting and preventing SCD. New approaches that include epidemiologic modeling of transient risk factors and

1	of disease that do not predict high risk. This results in a low sensitivity for predicting and preventing SCD. New approaches that include epidemiologic modeling of transient risk factors and genetic predictors of individual patient risk offer hope for greater sensitivity in the future. AM, ambulatory monitoring; AP, angina pectoris; ASHD, arteriosclerotic heart disease; CAD, coronary artery disease; CT, computed tomography; EF, ejection fraction; EP, electrophysiologic; EPS, electrophysiologic study; MI, myocardial infarction. (Modified from RJ Myerburg: J Cardiovasc Electrophysiol 12:369–381, 2001.)

1	Among patients in the acute, convalescent, and chronic phases of MI (Chap. 295), subgroups at high absolute risk of SCD can be identified. During the acute phase, the potential risk of cardiac arrest from onset through the first 48 h used to be as high as 15%, but is now reported in the range of 2.3–4.4% because of early patient awareness of the significance of symptoms and the availability of emergency revascularization strategies. Those who survive acute-phase VF are not at continuing risk for recurrent cardiac arrest indexed to that event. During the convalescent phase after MI (3 days to ~6 weeks), an episode of sustained ventricular tachycardia (VT) or VF, which is usually associated with a large infarct, predicts a natural history mortality risk of >25% at 12 months. At least one-half of the deaths are sudden. Aggressive intervention techniques may reduce this incidence.

1	During the chronic phase after MI, the longer-term risk for total mortality and SCD mortality is predicted by a number of factors (Fig. 327-2B). The most important for both SCD and nonsudden death is the extent of myocardial damage sustained as a result of the acute MI. This is measured by the magnitude of reduction of the ejection fraction (EF) and/or the occurrence of heart failure. Various studies have demonstrated that ventricular arrhythmias identified by ambulatory monitoring contribute significantly to this risk, especially in patients with an EF <40%. In addition, inducibility of VT or VF during electrophysiologic testing of patients who have ambient ventricular arrhythmias (premature ventricular contractions [PVCs] and nonsustained VT) and an EF <35% is a strong predictor of SCD risk. Patients in this subgroup are now considered candidates for ICDs (see below). Risk falls off sharply with EFs >35% and the absence of ambient arrhythmias after MI, and conversely is high with

1	risk. Patients in this subgroup are now considered candidates for ICDs (see below). Risk falls off sharply with EFs >35% and the absence of ambient arrhythmias after MI, and conversely is high with EFs <30% even without the ambient arrhythmia markers.

1	The cardiomyopathies (dilated and hypertrophic, Chap. 287) are the second most common category of diseases associated with risk of SCD (Table 327-2). Some risk factors have been identified, largely related to extent of disease, presence of heart failure, documented ventricular arrhythmias, and syncope thought to be due to arrhythmias. The less common causes of SCD include valvular heart disease (primarily aortic) and inflammatory and infiltrative disorders of the myocardium. The latter include viral myocarditis, sarcoidosis, and amyloidosis.

1	Among adolescents and young adults, rare inherited disorders such as hypertrophic cardiomyopathy, the long QT interval syndromes, right ventricular dysplasia, and the Brugada syndrome have received attention as important causes of SCD, as has acute myocarditis and other less common acquired diseases. Among the subgroup of young competitive athletes, the incidence of SCD may be higher than it is for the general adolescent and young adult population, perhaps up to 1 in 75,000–100,000. Hypertrophic cardiomyopathy (Chap. 287) is the most common cause in the United States.

1	Secondary prevention strategies should be applied to survivors of cardiac arrest that was not associated with an acute MI or other controllable transient risk factors, such as certain drug exposures and correctable electrolyte imbalances. Multivessel coronary artery disease and dilated cardiomyopathy, especially with markedly reduced left ventricular EF, predict a high risk of recurrence of cardiac arrest or SCD and are indications for specific interventions, such as ICDs (see Cardiovascular Collapse, Cardiac Arrest, and Sudden Cardiac Death 1768 below). The occurrence of otherwise unexplained syncope or documented life-threatening arrhythmias in patients with long QT syndromes or right ventricular dysplasia are also associated with increased risk of SCD. PRODROME, ONSET, ARREST, DEATH

1	PRODROME, ONSET, ARREST, DEATH SCD may be presaged by days to months of increasing angina, dyspnea, palpitations, easy fatigability, and other nonspecific complaints. However, these prodromal symptoms are generally predictive of any major cardiac event; they are not specific for predicting SCD. The onset of the clinical transition, leading to cardiac arrest, is defined as an acute change in cardiovascular status preceding cardiac arrest by up to 1 h. When the onset is instantaneous or abrupt, the probability that the arrest is cardiac in origin is >95%. Continuous electrocardiogram (ECG) recordings fortuitously obtained at the onset of a cardiac arrest commonly demonstrate a tendency for the heart rate to increase and for advanced grades of PVCs to evolve during the minutes or hours before the event.

1	The probability of achieving successful resuscitation from cardiac arrest is related to the interval from onset of loss of circulation to return of spontaneous circulation (ROSC), the setting in which the event occurs, the mechanism (VF, VT, PEA, asystole), and the clinical status of the patient before the cardiac arrest. ROSC and survival rates as a result of defibrillation decrease almost linearly from the first minute to 10 min. After 4-5 min, survival rates are no better than 25–30% in out-of-hospital settings without bystander cardiopulmonary resuscitation (CPR). Those settings in which it is possible to institute prompt CPR followed by prompt defibrillation provide a better chance of a successful outcome. The outcome in intensive care units and other in-hospital environments is heavily influenced by the patient’s preceding clinical status. The immediate outcome is good for cardiac arrest occurring in the intensive care unit in the presence of an acute cardiac event or transient

1	influenced by the patient’s preceding clinical status. The immediate outcome is good for cardiac arrest occurring in the intensive care unit in the presence of an acute cardiac event or transient metabolic disturbance, but survival among patients with far-advanced chronic cardiac disease or advanced noncardiac diseases (e.g., renal failure, pneumonia, sepsis, diabetes, cancer) is low and not much better in the in-hospital setting. Survival rates after unexpected cardiac arrest in unmonitored areas in a hospital do not differ from witnessed out-of-hospital arrests. Since implementation of community response systems, survival from out-of-hospital cardiac arrest has improved, although it still remains low, under most circumstances. Survival probabilities in public sites exceed those in the home environment, where the majority of cardiac arrests occur.

1	The success rate for initial resuscitation and survival to hospital discharge after an out-of-hospital cardiac arrest depends heavily on the mechanism of the event. When the mechanism is pulseless VT, the outcome is best; VF is the next most successful; and asystole and PEA, now the most common mechanisms, generate dismal outcome statistics. Advanced age also adversely influences the chances of successful resuscitation.

1	The probability of progression to biologic death is a function of the mechanism of cardiac arrest and the length of the delay before interventions. VF without CPR within the first 4–6 min has a poor outcome even if defibrillation is successful because of secondary brain damage; the prompt interposition of bystander CPR (basic life support; see below) improves outcome at any point along the time scale, especially when followed by early successful defibrillation. However, there are few survivors among patients who had no life support activities for the first 8 min after onset. Evaluations of deployment of automatic external defibrillators (AEDs) in communities (e.g., police vehicles, large buildings, airports, and stadiums) are beginning to generate encouraging data, but the data for home deployment has been have been less impressive.

1	Death during the hospitalization after a successfully resuscitated cardiac arrest relates closely to the severity of central nervous system injury. Anoxic encephalopathy and infections subsequent to prolonged respirator dependence account for 60% of the deaths. Another 30% occur as a consequence of low cardiac output states that fail to respond to interventions. Recurrent arrhythmias are the least common cause of death, accounting for only 10% of in-hospital deaths.

1	In the setting of acute MI (Chap. 295), it is important to distinguish between primary and secondary cardiac arrests. Primary cardiac arrests are those that occur in the absence of hemodynamic instability, and secondary cardiac arrests are those that occur in patients in whom abnormal hemodynamics dominate the clinical picture before cardiac arrest. The success rate for immediate resuscitation in primary cardiac arrest during acute MI in a monitored setting should exceed 90%. In contrast, as many as 70% of patients with secondary cardiac arrest succumb immediately or during the same hospitalization.

1	An individual who collapses suddenly is managed in five stages: initial evaluation and basic life support if cardiac arrest is confirmed, public access defibrillation (when available), (3) advanced life support, (4) postresuscitation care, and (5) long-term management. The initial response, including confirmation of loss of circulation, followed by basic life support and public access defibrillation, can be carried out by physicians, nurses, paramedical personnel, and trained laypersons.

1	Confirmation that a sudden collapse with loss of consciousness (LOC) is due to a cardiac arrest includes prompt observations of the state of consciousness, respiratory movements, skin color, and the presence or absence of pulses in the carotid or femoral arteries. For lay responders, the pulse check is no longer recommended because it is unreliable. As soon as a cardiac arrest is suspected, confirmed, or even considered to be impending, calling an emergency rescue system (e.g., 911) is the immediate priority. With the development of AEDs that are easily used by nonconventional emergency responders, an additional layer for response has evolved (see below).

1	Careful attention to the respiratory status after abrupt LOC is important. Although normal breathing or tachypnea after LOC makes cardiac arrest less likely, gasping respiratory movements may persist during a true cardiac arrest, and their presence should not deter appropriate responses. In fact, continued gasping is considered a good prognostic sign for successful outcome. It is also important to observe for severe stridor with a persistent pulse as a clue to aspiration of a foreign body or food. If this is suspected, a Heimlich maneuver (see below) may dislodge the obstructing body. A precordial blow, or “thump,” delivered firmly with a clenched fist to the junction of the middle and lower thirds of the sternum may occasionally revert VT or VF, but there is concern about converting VT to VF. Therefore, it is recommended to use precordial thumps as a life support technique only when monitoring and defibrillation are available. This conservative application of the technique remains

1	to VF. Therefore, it is recommended to use precordial thumps as a life support technique only when monitoring and defibrillation are available. This conservative application of the technique remains controversial.

1	The third action during the initial response is to clear the airway. The head is tilted back and the chin lifted so that the oropharynx can be explored to clear the airway. Dentures or foreign bodies are removed, and the Heimlich maneuver is performed if there is reason to suspect that a foreign body is lodged in the oropharynx. If respiratory arrest precipitating cardiac arrest is suspected, a second precordial thump is delivered after the airway is cleared.

1	Basic life support, more popularly known as CPR, is intended to maintain organ perfusion until definitive interventions can be instituted. The initial and primary element of CPR is maintenance of perfusion until spontaneous circulation can be restored. Closed chest cardiac compression maintains a pump function by sequential filling and emptying of the chambers, with competent valves maintaining forward direction of flow. The palm of one hand is placed over the lower sternum, with the heel of the other resting on the dorsum of the lower hand. The sternum is depressed, with the arms remaining straight, at a rate of 100 per minute. Sufficient force is applied to depress the sternum 4–5 cm, and relaxation is abrupt.

1	Until recently, providing ventilation of the lungs by mouth-tomouth respiration was used if no specific rescue equipment was immediately available (e.g., plastic oropharyngeal airways, esophageal obturators, masked Ambu bag). However, ventilatory support during CPR has yielded to evidence that continuous chest compressions (“hands only” CPR) results in better outcomes. Compressions are interrupted only for single shocks from an AED when available, with 2 min of CPR between each single shock.

1	AEDs that are easily used by nonconventional responders, such as nonparamedic firefighters, police officers, ambulance drivers, trained security guards, and minimally trained or untrained laypersons, have been developed. This advance has inserted another level of response into the cardiac arrest paradigm. A number of studies have demonstrated that AED use by nonconventional responders in strategic response systems and public access lay responders can improve cardiac arrest survival rates. The rapidity with which defibrillation/cardioversion is achieved is an important element for successful resuscitation, both for ROSC and for protection of the central nervous system. Chest compressions should be carried out while the defibrillator is being charged. As soon as a diagnosis of VF or VT is established, a biphasic waveform shock of 150–200 J (360 J if a monophasic waveform device is used) should be delivered. If 5 min has elapsed between collapse and first contact with the victim, there

1	established, a biphasic waveform shock of 150–200 J (360 J if a monophasic waveform device is used) should be delivered. If 5 min has elapsed between collapse and first contact with the victim, there is some evidence that 60–90 s of CPR before the first shock may improve probability of survival without neurologic damage. If the initial shock does not successfully revert VT or VF, chest compression at a rate of 100 per minute is resumed for 2 min, and then

1	If return of circulation fails If return of circulation fails Continue chest compressions, intubate, IV access Epinephrine, 1 mg IV or vasopressin, 40 units IV; follow with repeat defibrillation at maximum energy within 30–60 seconds as required; repeat epinephrine If return of circulation fails Epinephrine, °Dose NaHCO3, 1 meq/kg (° K+) (no longer for routine use; may be used for persistent acidosis -see text) Antiarrhythmics If return of circulation fails Defibrillate, CPR: Drug – Shock – Drug – Shock a second shock is delivered. Multiple shocks given in sequence are 1769 no longer recommended, in order to minimize interruptions of chest compressions. This sequence is continued until personnel capable of, and equipped for, advanced life support are available, although not much data support the notion that shocks and chest compressions alone will revert VF after three shocks have failed.

1	ACLS is intended to achieve and maintain organ perfusion and adequate ventilation, control cardiac arrhythmias, and stabilize blood pressure and cardiac output. The activities carried out to achieve these goals include (1) defibrillation/cardioversion and/or pacing, (2) intubation with an endotracheal tube, and (3) insertion of an intravenous line. As in basic life support, the major emphasis during ACLS is minimizing interruptions of chest compressions until ROSC is achieved. After two or three unsuccessful defibrillation attempts, epinephrine, 1 mg IV, is given and attempts to defibrillate are repeated. The dose of epinephrine may be repeated after intervals of 3–5 min (Fig. 327-3A). Vasopressin (a single 40-unit dose given IV) has been suggested as an alternative to epinephrine.

1	If the patient is less than fully conscious upon reversion or if two or three attempts fail, prompt intubation, ventilation, and arterial blood gas analysis should be carried out. Ventilation with O2 (room air if O2 is not immediately available) may promptly reverse hypoxemia and acidosis. Quantitative waveform capnography is now recommended for confirmation and monitoring of endotracheal tube placement. A patient who is persistently acidotic after successful defibrillation and intubation or had acidosis prior to arrest, may be CPR, intubate, IV access [Confirm asystole] [Assess blood flow] Cardiovascular Collapse, Cardiac Arrest, and Sudden Cardiac Death

1	CPR, intubate, IV access [Confirm asystole] [Assess blood flow] Cardiovascular Collapse, Cardiac Arrest, and Sudden Cardiac Death FIGURE 327-3 A. The algorithm of ventricular fibrillation or pulseless ventricular tachycardia begins with and initial defibrillate on attempt. If a single shock fails to restore a pulse, it is followed by 2 min of cardiopulmonary resuscitation (CPR; chest compressions), followed by another single shock. After three such sequences, epinephrine and then antiarrhythmic drugs are added to the protocol. See text for details. B. The algorithms for bradyarrhythmia/asystole (left) or pulseless electrical activity (right) are dominated first by continued life support and a search for reversible causes. Subsequent therapy is nonspecific and is accompanied by a low success rate. See text for details. MI, myocardial infarction; VT, ventricular tachycardia.

1	1770 given 1 meq/kg NaHCO3 initially and an additional 50% of the dose repeated every 10–15 min. However, it should not be used routinely. After initial unsuccessful defibrillation attempts or with persistent/ recurrent electrical instability, antiarrhythmic therapy should be instituted. Intravenous amiodarone has emerged as the initial treatment of choice (150 mg over 10 min, followed by 1 mg/min for up to 6 h and 0.5 mg/min thereafter) (Fig. 327-3A). For cardiac arrest due to VF in the early phase of an acute coronary syndrome, a bolus of 1 mg/kg of lidocaine may be given intravenously as an alternative, and the dose may be repeated in 2 min. It also may be tried in patients in whom amiodarone is unsuccessful. Intravenous procainamide (loading infusion of 100 mg/5 min to a total dose of 500–800 mg, followed by continuous infusion at 2–5 mg/min) is now rarely used in this setting but may be tried for persisting, hemodynamically stable arrhythmias. Intravenous calcium gluconate is no

1	500–800 mg, followed by continuous infusion at 2–5 mg/min) is now rarely used in this setting but may be tried for persisting, hemodynamically stable arrhythmias. Intravenous calcium gluconate is no longer considered safe or necessary for routine administration. It is used only in patients in whom acute hyperkalemia is known to be the triggering event for resistant VF, in the presence of known hypocalcemia, or in patients who have received toxic doses of calcium channel antagonists. Cardiac arrest due to bradyarrhythmias or asystole (B/A cardiac arrest) is managed differently (Fig. 327-3B). The patient is promptly intubated, CPR is continued, and an attempt is made to control hypoxemia and acidosis and identify other reversible causes. Epinephrine may be given intravenously or by an intraosseous route. Atropine is no longer considered effective for asystole or PEA, but can be used for bradyarrhythmias. External pacing devices are used to attempt to establish a regular rhythm when

1	route. Atropine is no longer considered effective for asystole or PEA, but can be used for bradyarrhythmias. External pacing devices are used to attempt to establish a regular rhythm when atropine fails for a bradyarrhythmia, but chronotropic agents given intravenously are now recognized as an equally effective alternative. The success rate may be good when B/A arrest is due to acute inferior wall MI or to correctable airway obstruction or drug-induced respiratory depression or with prompt resuscitation efforts. For acute airway obstruction, prompt removal of foreign bodies by the Heimlich maneuver or, in hospitalized patients, by intubation and suctioning of obstructing secretions in the airway is often successful. The prognosis is generally very poor in other causes of this form of cardiac arrest, such as end-stage cardiac or noncardiac diseases. Treatment of PEA is similar to that for bradyarrhythmias, but its outcome is also dismal.

1	After return of spontaneous or stable assisted circulation, attention shifts to the diagnostic and therapeutic elements of the post-cardiac arrest syndrome. This recently developed clinical classification emerged from the organization of the elements of injury following cardiac arrest into a multidisciplinary continuum. The four components of post-cardiac arrest syndrome include brain injury, myocardial dysfunction, systemic ischemia/reperfusion responses, and control of persistent precipitating factors. The therapeutic goal is to maintain a stable electrical, hemodynamic, and central nervous system status.

1	Postresuscitation care is determined by the specific clinical circumstances. The most pressing is the presence of anoxic encephalopathy, which is a strong predictor of in-hospital death and postarrest disability. Mild therapeutic hypothermia is indicated for resuscitated cardiac arrest victims who are hemodynamically stable, but remain comatose. Core body temperature is decreased to 32–34°C, by several available techniques (external and/or internal [core]), as soon as practical after resuscitation and maintained for a minimum of 12–24 h. By reducing metabolic demands and cerebral edema, this intervention improves probability of survival with better neurologic outcome.

1	Primary VF in acute MI (not accompanied by low-output states) (Chap. 295) is generally very responsive to life support techniques and easily controlled after the initial event. In the in-hospital setting, respirator support is usually not necessary or is needed for only a short time, and hemodynamics stabilize promptly after defibrillation or cardioversion. In secondary VF in acute MI (those events in which hemodynamic abnormalities predispose to the potentially fatal arrhythmia), resuscitative efforts are less often successful, and in patients who are successfully resuscitated, the recurrence rate is high. The clinical picture and outcome are dominated by hemodynamic instability and the ability to control hemodynamic dysfunction. Bradyarrhythmias, asystole, and PEA are commonly secondary events in hemodynamically unstable patients.

1	The outcome after in-hospital cardiac arrest associated with noncardiac diseases is poor, and in the few successfully resuscitated patients, the postresuscitation course is dominated by the nature of the underlying disease. Patients with end-stage cancer, renal failure, acute central nervous system disease, and uncontrolled infections, as a group, have a survival rate of <10% after in-hospital cardiac arrest. Some major exceptions are patients with transient airway obstruction, electrolyte disturbances, proarrhythmic effects of drugs, and severe metabolic abnormalities, most of whom may have a good chance of survival if they can be resuscitated promptly and stabilized while the transient abnormalities are being corrected.

1	Patients who survive cardiac arrest without irreversible damage to the central nervous system and who achieve hemodynamic stability should have diagnostic testing to define appropriate therapeutic interventions for their long-term management. This approach is driven by the fact that survival after out-of-hospital cardiac arrest is followed by a 10–25% mortality rate during the first 2 years after the event, and there are data suggesting that significant survival benefits can be achieved by prescription of an ICD.

1	Among patients in whom an acute ST elevation MI or transient and reversible myocardial ischemia is identified as the specific mechanism triggering an out-of-hospital cardiac arrest, the management is dictated in part by the transient nature of life-threatening arrhythmia risk during the acute coronary syndrome (ACS) and in part by the extent of permanent myocardial damage that results. Cardiac arrest during the acute ischemic phase is not an ICD indication, but survivors of cardiac arrest not associated with an ACS do benefit. In addition, patients who survive MI with an EF less than 30–35% appear to benefit from ICDs. For patients with cardiac arrest determined to be due to a treatable transient ischemic mechanism, particularly with higher EFs, catheter interventional, surgical, and/or pharmacologic antiischemic therapy is generally accepted for long-term management.

1	Survivors of cardiac arrest due to other categories of disease, such as the hypertrophic or dilated cardiomyopathies and the various rare inherited disorders (e.g., right ventricular dysplasia, long QT syndrome, Brugada syndrome, catecholaminergic polymorphic VT, and so-called idiopathic VF), are all considered ICD candidates.

1	Post-MI patients with EFs <35% and other markers of risk such as ambient ventricular arrhythmias, inducible ventricular tachyarrhythmias in the electrophysiology laboratory, and a history of heart failure are considered candidates for ICDs 40 days or more after the MI. Total mortality benefits in the range of a 20–35% reduction over 2–5 years have been observed in a series of clinical trials. One study suggested that an EF <30% was a sufficient marker of risk to indicate ICD benefit, and another demonstrated benefit for patients with Functional Class 2 or 3 heart failure and EFs ≤35%, regardless of etiology (ischemic or nonischemic) or the presence of ambient or induced arrhythmias (Chaps. 277 and 279). For patients with newly diagnosed heart failure and an EF <35%, the required delay between diagnosis and institution of medical therapy, and subsequent implantation of an ICD, is 90 days. In general, there appears to be a gradient of increasing ICD benefit with EFs ranging lower than

1	diagnosis and institution of medical therapy, and subsequent implantation of an ICD, is 90 days. In general, there appears to be a gradient of increasing ICD benefit with EFs ranging lower than the threshold indications. However, patients with very low EFs (e.g., <20%) may receive less benefit.

1	Decision making for primary prevention in disorders other than the rare disorders listed above, indicators of arrhythmic risk such as 1771 coronary artery disease and dilated cardiomyopathy is generally driven syncope, documented ventricular tachyarrhythmias, aborted cardiac by observational data and judgment based on clinical observations. arrest, or a family history of premature SCD in some conditions, and Controlled clinical trials providing evidence-based indicators for ICDs a number of other clinical or ECG markers, may be used as indicators are lacking for these smaller population subgroups. In general, for for ICDs. Allan H. Ropper Coma is among the most common and striking problems in general medicine. It accounts for a substantial portion of admissions to emergency wards and occurs on all hospital services. It demands immediate attention and requires an organized approach.

1	There is a continuum of states of reduced alertness, the most severe form being coma, defined as a deep sleeplike state from which the patient cannot be aroused. Stupor refers to a higher degree of arousability in which the patient can be transiently awakened by vigorous stimuli, accompanied by motor behavior that leads to avoidance of uncomfortable or aggravating stimuli. Drowsiness, which is familiar to all persons, simulates light sleep and is characterized by easy arousal and the persistence of alertness for brief periods. Drowsiness and stupor are usually accompanied by some degree of confusion (Chap. 34). A precise narrative description of the level of arousal and of the type of responses evoked by various stimuli as observed at the bedside is preferable to ambiguous terms such as lethargy, semicoma, or obtundation.

1	Several conditions that render patients unresponsive and simulate coma are considered separately because of their special significance. The vegetative state signifies an awake-appearing but nonresponsive state in a patient who has emerged from coma. In the vegetative state, the eyelids may open, giving the appearance of wakefulness. Respiratory and autonomic functions are retained. Yawning, coughing, swallowing, and limb and head movements persist, and the patient may follow visually presented objects, but there are few, if any, meaningful responses to the external and internal environment—in essence, an “awake coma.” The term vegetative is unfortunate because it is subject to misinterpretation. There are always accompanying signs that indicate extensive damage in both cerebral hemispheres, e.g., decerebrate or decorticate limb posturing and absent responses to visual stimuli (see below). In the closely related but less severe minimally conscious state, the patient displays

1	hemispheres, e.g., decerebrate or decorticate limb posturing and absent responses to visual stimuli (see below). In the closely related but less severe minimally conscious state, the patient displays rudimentary vocal or motor behaviors, often spontaneous, but some in response to touch, visual stimuli, or command. Cardiac arrest with cerebral hypoperfusion and head injuries are the most common causes of the vegetative and minimally conscious states (Chaps. 327 and 330). The prognosis for regaining mental faculties once the vegetative state has supervened for several months is very poor, and after a year, almost nil; hence the term persistent vegetative state. Most reports of dramatic recovery, when investigated carefully, are found to yield to the usual rules for prognosis, but there have been rare instances in which recovery has occurred to a severely disabled condition and, in rare childhood cases, to an even better state. The possibility of incorrectly attributing meaningful

1	have been rare instances in which recovery has occurred to a severely disabled condition and, in rare childhood cases, to an even better state. The possibility of incorrectly attributing meaningful behavior to patients in the vegetative and minimally conscious states creates inordinate problems and anguish. On the other hand, the question of whether these patients lack any capability for cognition has been reopened by functional imaging studies that have demonstrated, in a small proportion of posttraumatic cases, meaningful cerebral activation in response to verbal and other stimuli.

1	Apart from the above conditions, several syndromes that affect alertness are prone to be misinterpreted as stupor or coma. Akinetic mutism refers to a partially or fully awake state in which the patient is able to form impressions and think, as demonstrated by later recounting of events, but remains virtually immobile and mute. The condition results from damage in the regions of the medial thalamic nuclei or the frontal lobes (particularly lesions situated deeply or on the orbitofrontal surfaces) or from extreme hydrocephalus. The term abulia describes a milder form of akinetic mutism characterized by mental and physical slowness and diminished ability to initiate activity. It is also usually the result of damage to the frontal lobes and its connections (Chap. 36).

1	Catatonia is a curious hypomobile and mute syndrome that occurs as part of a major psychosis, usually schizophrenia or major depression. Catatonic patients make few voluntary or responsive movements, although they blink, swallow, and may not appear distressed. There are nonetheless signs that the patient is responsive, although it may take ingenuity on the part of the examiner to demonstrate them. For example, eyelid elevation is actively resisted, blinking occurs in response to a visual threat, and the eyes move concomitantly with head rotation, all of which are inconsistent with the presence of a brain lesion causing unresponsiveness. It is characteristic but not invariable in catatonia for the limbs to retain the postures in which they have been placed by the examiner (“waxy flexibility,” or catalepsy). With recovery, patients often have some memory of events that occurred during their catatonic stupor. Catatonia is superficially similar to akinetic mutism, but clinical evidence of

1	or catalepsy). With recovery, patients often have some memory of events that occurred during their catatonic stupor. Catatonia is superficially similar to akinetic mutism, but clinical evidence of cerebral damage such as Babinski signs and hypertonicity of the limbs is lacking. The special problem of coma in brain death is discussed below.

1	The locked-in state describes yet another type of pseudocoma in which an awake patient has no means of producing speech or volitional movement but retains voluntary vertical eye movements and lid elevation, thus allowing the patient to signal with a clear mind. The pupils are normally reactive. Such individuals have written entire treatises using Morse code. The usual cause is an infarction or hemorrhage of the ventral pons that transects all descending motor (corticospinal and corticobulbar) pathways. A similar awake but de-efferented state occurs as a result of total paralysis of the musculature in severe cases of Guillain-Barré syndrome (Chap. 460), critical illness neuropathy (Chap. 330), and pharmacologic neuromuscular blockade.

1	Almost all instances of diminished alertness can be traced to widespread abnormalities of the cerebral hemispheres or to reduced activity of a special thalamocortical alerting system termed the reticular activating system (RAS). The proper functioning of this system, its ascending projections to the cortex, and the cortex itself are required to maintain alertness and coherence of thought. It follows that the principal causes of coma are (1) lesions that damage the RAS in the upper midbrain or its projections; (2) destruction of large portions of both cerebral hemispheres; or (3) suppression of reticulocerebral function by drugs, toxins, or metabolic derangements such as hypoglycemia, anoxia, uremia, and hepatic failure.

1	The proximity of the RAS to midbrain structures that control pupillary function and eye movements permits clinical localization of the cause of coma in many cases. Pupillary enlargement with loss of light 1772 reaction and loss of vertical and adduction movements of the eyes suggests that the lesion is in the upper brainstem where the nuclei subserving these functions reside. Conversely, preservation of pupillary light reactivity and of eye movements absolves the upper brainstem and indicates that widespread structural lesions or metabolic suppression of the cerebral hemispheres is responsible for coma.

1	Coma Due to Cerebral Mass Lesions and Herniations In addition to the fixed restriction of the skull, the cranial cavity is separated into compartments by infoldings of the dura. The two cerebral hemispheres are separated by the falx, and the anterior and posterior fossae by the tentorium. Herniation refers to displacement of brain tissue by an overlying or adjacent mass into a contiguous compartment that it normally does not occupy. Coma and many of its associated signs can be attributed to these tissue shifts, and certain clinical features are characteristic of specific configurations of herniation (Fig. 328-1). They are in essence “false localizing” signs because they derive from compression of brain structures at a distance from the mass.

1	In the most common form of herniation, brain tissue is displaced from the supratentorial to the infratentorial compartment through the tentorial opening; this is referred to as transtentorial herniation. Uncal transtentorial herniation refers to impaction of the anterior medial temporal gyrus (the uncus) into the tentorial opening just anterior to and adjacent to the midbrain (Fig. 328-1A). The uncus compresses the third nerve as the nerve traverses the subarachnoid space, causing enlargement of the ipsilateral pupil (the fibers sub-serving parasympathetic pupillary function are located peripherally in the nerve). The coma that follows is due to compression of the midbrain against the opposite tentorial edge by the displaced parahippocampal gyrus (Fig. 328-2). Lateral displacement of the midbrain may compress the opposite cerebral peduncle against the tentorial edge, producing a Babinski sign and hemiparesis contralateral to the hemiparesis that resulted from the mass (the

1	of the midbrain may compress the opposite cerebral peduncle against the tentorial edge, producing a Babinski sign and hemiparesis contralateral to the hemiparesis that resulted from the mass (the Kernohan-Woltman sign). Herniation may also compress the anterior and posterior cerebral arteries as they pass over the tentorial reflections, with resultant brain infarction. The distortions may also entrap portions of the ventricular system, resulting in hydrocephalus.

1	Central transtentorial herniation denotes a symmetric downward movement of the thalamic structures through the tentorial opening with compression of the upper midbrain (Fig. 328-1B). Miotic pupils and drowsiness are the heralding signs, in contrast to a unilaterally FIGURE 328-1 Types of cerebral herniation: (A) uncal; (B) central; (C) transfalcial; and (D) foraminal. FIGURE 328-2 Coronal (A) and axial (B) magnetic resonance images from a stuporous patient with a left third nerve palsy as a result of a large left-sided subdural hematoma (seen as a gray-white rim). The upper midbrain and lower thalamic regions are compressed and displaced horizontally away from the mass, and there is transtentorial herniation of the medial temporal lobe structures, including the uncus anteriorly. The lateral ventricle opposite to the hematoma has become enlarged as a result of compression of the third ventricle.

1	enlarged pupil of the uncal syndrome. Both uncal and central transtentorial herniations cause progressive compression of the brainstem, with initial damage to the midbrain, then the pons, and finally the medulla. The result is an approximate sequence of neurologic signs that corresponds to each affected level. Other forms of herniation are transfalcial herniation (displacement of the cingulate gyrus under the falx and across the midline, Fig. 328-1C) and foraminal herniation (downward forcing of the cerebellar tonsils into the foramen magnum, Fig. 328-1D), which causes compression of the medulla, respiratory arrest, and death.

1	A direct relationship between the various configurations of transtentorial herniation and coma is not always found. Drowsiness and stupor can occur with moderate horizontal displacement of the diencephalon (thalamus), before transtentorial herniation is evident. This lateral shift may be quantified on axial images of computed tomography (CT) and magnetic resonance imaging (MRI) scans (Fig. 328-2). In cases of acutely enlarging masses, horizontal displacement of the pineal calcification of 3–5 mm is generally associated with drowsiness, 6–8 mm with stupor, and >9 mm with coma. Intrusion of the medial temporal lobe into the tentorial opening is also apparent on MRI and CT scans as obliteration of the cisterna that surrounds the upper brainstem.

1	Coma due to Metabolic Disorders Many systemic metabolic abnormalities cause coma by interrupting the delivery of energy substrates (e.g., oxygen, glucose) or by altering neuronal excitability (drugs and alcohol, anesthesia, and epilepsy). The metabolic abnormalities that produce coma may, in milder forms, induce an acute confusional state. Thus, in metabolic encephalopathies, clouded consciousness and coma are in a continuum.

1	Cerebral neurons are fully dependent on cerebral blood flow (CBF) and the delivery of oxygen and glucose. CBF is ~75 mL per 100 g/ min in gray matter and 30 mL per 100 g/min in white matter (mean ~55 mL per 100 g/min); oxygen consumption is 3.5 mL per 100 g/ min, and glucose utilization is 5 mg per 100 g/min. Brain stores of glucose are able to provide energy for ~2 min after blood flow is interrupted, and oxygen stores last 8–10 s after the cessation of blood flow. Simultaneous hypoxia and ischemia exhaust glucose more rapidly. The electroencephalogram (EEG) rhythm in these circumstances becomes diffusely slowed, typical of metabolic encephalopathies, and as substrate delivery worsens, eventually brain electrical activity ceases.

1	Unlike hypoxia-ischemia, which causes neuronal destruction, most metabolic disorders such as hypoglycemia, hyponatremia, hyperosmolarity, hypercapnia, hypercalcemia, and hepatic and renal failure cause only minor neuropathologic changes. The reversible effects of these conditions on the brain are not understood but may result from impaired energy supplies, changes in ion fluxes across neuronal membranes, and neurotransmitter abnormalities. For example, the high ammonia concentration of hepatic coma interferes with cerebral energy metabolism and with the Na+, K+-ATPase pump, increases the number and size of astrocytes, and causes increased concentrations of potentially toxic products of ammonia metabolism; it may also affect neurotransmitters, including the production of putative “false” neurotransmitters that are active at receptor sites. Apart from hyperammonemia, which of these mechanisms is of critical importance is not clear. The mechanism of the encephalopathy of renal failure is

1	that are active at receptor sites. Apart from hyperammonemia, which of these mechanisms is of critical importance is not clear. The mechanism of the encephalopathy of renal failure is also not known. Unlike ammonia, urea does not produce central nervous system (CNS) toxicity, and a multifactorial causation has been proposed for the encephalopathy, including increased permeability of the blood-brain barrier to toxic substances such as organic acids and an increase in brain calcium and cerebrospinal fluid (CSF) phosphate content.

1	Coma and seizures are common accompaniments of large shifts in sodium and water balance in the brain. These changes in osmolarity arise from systemic medical disorders, including diabetic ketoacidosis, the nonketotic hyperosmolar state, and hyponatremia from any cause (e.g., water intoxication, excessive secretion of antidiuretic hormone, or atrial natriuretic peptides). Sodium levels <125 mmol/L induce confusion, and levels <115 mmol/L are typically associated with coma and convulsions. In hyperosmolar coma, the serum osmolarity is generally >350 mosmol/L. Hypercapnia depresses the level of consciousness in proportion to the rise in carbon dioxide (CO2) tension in the blood. In all of these metabolic encephalopathies, the degree of neurologic change depends to a large extent on the rapidity with which the serum changes occur. The pathophysiology of other metabolic encephalopathies such as those due to hypercalcemia, hypothyroidism, vitamin B12 deficiency, and hypothermia are

1	rapidity with which the serum changes occur. The pathophysiology of other metabolic encephalopathies such as those due to hypercalcemia, hypothyroidism, vitamin B12 deficiency, and hypothermia are incompletely understood but must reflect derangements of CNS biochemistry, membrane function, or neurotransmitters.

1	Epileptic Coma Generalized electrical seizures are associated with coma, even in the absence of motor convulsions (nonconvulsive status epilepticus). The self-limited coma that follows a seizure, the postictal state, may be due to exhaustion of energy reserves or effects of locally toxic molecules that are the by-product of seizures. The postictal state produces continuous, generalized slowing of the background EEG activity similar to that of metabolic encephalopathies.

1	Toxic (Including Drug-Induced) Coma This common class of encephalopathy is in large measure reversible and leaves no residual damage provided there has not been cardiorespiratory failure. Many drugs and toxins are capable of depressing nervous system function. Some produce coma by affecting both the brainstem nuclei, including the RAS, and the cerebral cortex. The combination of cortical and brainstem signs, which occurs in certain drug overdoses, may lead to an incorrect diagnosis of structural brainstem disease. Overdose of medications that have atropinic actions produces signs such as dilated pupils, tachycardia, and dry skin; opiate overdose produces pinpoint pupils <1 mm in diameter.

1	Coma due to Widespread Damage to the Cerebral Hemispheres This category, comprising a number of unrelated disorders, results from widespread structural cerebral damage that simulates a metabolic disorder of the cortex. Hypoxia-ischemia is perhaps the best characterized and one in which it is not possible initially to distinguish the acute reversible effects of oxygen deprivation of the brain from the subsequent effects of anoxic neuronal damage. Similar widespread cerebral damage may be produced by disorders that occlude small blood vessels throughout the brain; examples include cerebral malaria, thrombotic thrombocytopenic purpura, and hyperviscosity. Diffuse white matter damage from cranial trauma or inflammatory demyelinating diseases can cause a similar coma syndrome. APPROACH TO THE PATIENT: A video examination of the comatose patient is shown in Chap. 329e.

1	APPROACH TO THE PATIENT: A video examination of the comatose patient is shown in Chap. 329e. Acute respiratory and cardiovascular problems should be attended to prior to neurologic assessment. In most instances, a complete medical evaluation, except for vital signs, funduscopy, and examination for nuchal rigidity, may be deferred until the neurologic evaluation has established the severity and nature of coma. The approach to the patient with coma from cranial trauma is discussed in Chap. 457e.

1	The cause of coma may be immediately evident as in cases of trauma, cardiac arrest, or observed drug ingestion. In the remainder, certain points are useful: (1) the circumstances and rapidity with which neurologic symptoms developed; (2) the antecedent symptoms (confusion, weakness, headache, fever, seizures, dizziness, double vision, or vomiting); (3) the use of medications, drugs, or alcohol; and (4) chronic liver, kidney, lung, heart, or other medical disease. Direct interrogation of family, observers, and ambulance technicians on the scene, in person or by telephone, is an important part of the evaluation when possible.

1	Fever suggests a systemic infection, bacterial meningitis, encephalitis, heat stroke, neuroleptic malignant syndrome, malignant hyperthermia due to anesthetics, or anticholinergic drug intoxication. Only rarely is fever attributable to a lesion that has disturbed hypothalamic temperature-regulating centers (“central fever”). A slight elevation in temperature may follow vigorous convulsions. Hypothermia is observed with exposure that attends alcohol, barbiturate, sedative, or phenothiazine intoxication; hypoglycemia; peripheral circulatory failure; or extreme hypothyroidism. Hypothermia itself causes coma when the temperature is <31°C (87.8°F). Tachypnea may indicate systemic acidosis or pneumonia or, rarely, infiltration of the brain with lymphoma. Aberrant respiratory patterns that reflect brainstem disorders are discussed below. Marked hypertension suggests hypertensive encephalopathy or cerebral hemorrhage or head injury. Hypotension is characteristic of coma from alcohol or

1	reflect brainstem disorders are discussed below. Marked hypertension suggests hypertensive encephalopathy or cerebral hemorrhage or head injury. Hypotension is characteristic of coma from alcohol or barbiturate intoxication, internal hemorrhage, myocardial infarction, sepsis, profound hypothyroidism, or Addisonian crisis. The funduscopic examination can detect subarachnoid hemorrhage (subhyaloid hemorrhages), hypertensive encephalopathy (exudates, hemorrhages, vessel-crossing changes, papilledema), and increased intracranial pressure (ICP) (papilledema). Cutaneous petechiae suggest thrombotic thrombocytopenic purpura, meningococcemia, or a bleeding diathesis associated with an intracerebral hemorrhage. Cyanosis and reddish or anemic skin coloration are other indications of an underlying systemic disease or carbon monoxide as responsible for the coma.

1	The patient should be observed without intervention by the examiner. Tossing about in the bed, reaching up toward the face, crossing legs, yawning, swallowing, coughing, or moaning reflect a drowsy state that is close to normal awakeness. Lack of restless movements on one side or an outturned leg suggests a hemiplegia. Intermittent twitching movements of a foot, finger, or facial muscle may be the only sign of seizures. Multifocal myoclonus almost always indicates a metabolic disorder, particularly uremia, anoxia, drug intoxication (especially with lithium or haloperidol), or a prion disease (Chap. 453e). In a drowsy and confused patient, bilateral asterixis is a certain sign of metabolic encephalopathy or drug intoxication.

1	Decorticate rigidity and decerebrate rigidity, or “posturing,” describe stereotyped arm and leg movements occurring spontaneously or elicited by sensory stimulation. Flexion of the elbows and wrists and supination of the arm (decorticate posturing) suggests bilateral damage rostral to the midbrain, whereas extension of the elbows and wrists with pronation (decerebrate posturing) indicates damage to motor tracts in the midbrain or caudal diencephalon. The less frequent combination of arm extension with leg flexion or flaccid legs is associated with lesions in the pons. These concepts have been adapted from animal work and cannot be applied with precision to coma in humans. In fact, acute and widespread disorders of any type, regardless of location, frequently cause limb extension, and almost all extensor posturing becomes predominantly flexor as time passes.

1	A sequence of increasingly intense stimuli is used to determine the threshold for arousal and the motor response of each side of the body. The results of testing may vary from minute to minute, and serial examinations are useful. Tickling the nostrils with a cotton wisp is a moderate stimulus to arousal—all but deeply stuporous and comatose patients will move the head away and arouse to some degree. An even greater degree of responsiveness is present if the patient uses his hand to remove an offending stimulus. Pressure on the knuckles or bony prominences and pinprick stimulation are humane forms of noxious stimuli; pinching the skin causes unsightly ecchymoses and is generally not necessary but may be useful in eliciting abduction withdrawal movements of the limbs. Posturing in response to noxious stimuli indicates severe damage to the corticospinal system, whereas abduction-avoidance movement of a limb is usually purposeful and denotes an intact corticospinal system. Posturing may

1	to noxious stimuli indicates severe damage to the corticospinal system, whereas abduction-avoidance movement of a limb is usually purposeful and denotes an intact corticospinal system. Posturing may also be unilateral and coexist with purposeful limb movements, reflecting incomplete damage to the motor system.

1	Assessment of brainstem function is essential to localization of the lesion in coma (Fig. 328-3). The brainstem reflexes that are examined are pupillary size and reaction to light, spontaneous and elicited eye movements, corneal responses, and the respiratory pattern. As a rule, coma due to bilateral hemispheral disease preserves these brainstem activities, particularly the pupillary reactions and eye movements. However, the presence of abnormal brainstem signs does not always indicate that the primary lesion is in the brainstem because hemispheral masses can cause secondary brainstem damage by the earlier described transtentorial herniations.

1	Pupillary Signs Pupillary reactions are examined with a bright, diffuse light (preferably not an ophthalmoscope, which illuminates only a limited part of the retina). Reactive and round pupils of midsize (2.5–5 mm) essentially exclude midbrain damage, either primary or secondary to compression. A response to light may be difficult to appreciate in pupils <2 mm in diameter, and bright room lighting mutes pupillary reactivity. One enlarged and poorly reactive pupil (>6 mm) signifies compression or stretching of the third nerve from the effects of a cerebral mass above. Enlargement of the pupil contralateral to a hemispheral mass may occur but is infrequent. An oval and slightly eccentric pupil is a transitional sign that accompanies early midbrain–third nerve compression. The most extreme pupillary sign, bilaterally dilated and unreactive pupils, indicates severe midbrain damage, usually from compression by a supratentorial mass. Ingestion of drugs with anticholinergic activity, the use

1	sign, bilaterally dilated and unreactive pupils, indicates severe midbrain damage, usually from compression by a supratentorial mass. Ingestion of drugs with anticholinergic activity, the use of mydriatic eye drops, and direct ocular trauma are among the causes of misleading pupillary enlargement.

1	Unilateral miosis in coma has been attributed to dysfunction of sympathetic efferents originating in the posterior hypothalamus and descending in the tegmentum of the brainstem to the cervical cord. It is therefore of limited localizing value but is an occasional finding in patients with a large cerebral hemorrhage that affects the thalamus. Reactive and bilaterally small (1–2.5 mm) but not pinpoint pupils are seen in metabolic encephalopathies or in deep bilateral hemispheral lesions such as hydrocephalus or thalamic

1	FIGURE 328-3 Examination of brainstem reflexes in coma. Midbrain and third nerve function are tested by pupillary reaction to light, pontine function by spontaneous and reflex eye movements and corneal responses, and medullary function by respiratory and pharyngeal responses. Reflex conjugate, horizontal eye movements are dependent on the medial longitudinal fasciculus (MLF) interconnecting the sixth and contralateral third nerve nuclei. Head rotation (oculocephalic reflex) or caloric stimulation of the labyrinths (oculovestibular reflex) elicits contraversive eye movements (for details see text). hemorrhage. Even smaller reactive pupils (<1 mm) characterize narcotic or barbiturate overdoses but also occur with extensive pontine hemorrhage. The response to naloxone and the presence of reflex eye movements (see below) assist in distinguishing between these.

1	Ocular Movements The eyes are first observed by elevating the lids and observing the resting position and spontaneous movements of the globes. Lid tone, tested by lifting the eyelids and noting their resistance to opening and the speed of closure, is progressively reduced as unresponsiveness progresses. Horizontal divergence of the eyes at rest is normal in drowsiness. As coma deepens, the ocular axes may become parallel again.

1	Spontaneous eye movements in coma often take the form of conjugate horizontal roving. This finding alone exonerates damage in the midbrain and pons and has the same significance as normal reflex eye movements (see below). Conjugate horizontal ocular deviation to one side indicates damage to the pons on the opposite side or, alternatively, to the frontal lobe on the same side. This phenomenon is summarized by the following maxim: The eyes look toward a hemispheral lesion and away from a brainstem lesion. Seizures also drive the eyes to one side but usually with superimposed clonic movements of the globes. The eyes may occasionally turn paradoxically away from the side of a deep hemispheral lesion (“wrong-way eyes”). The eyes turn down and inward with thalamic and upper midbrain lesions, typically thalamic hemorrhage.

1	“Ocular bobbing” describes brisk downward and slow upward movements of the eyes associated with loss of horizontal eye movements and is diagnostic of bilateral pontine damage, usually from thrombosis of the basilar artery. “Ocular dipping” is a slower, arrhythmic downward movement followed by a faster upward movement in patients with normal reflex horizontal gaze; it usually indicates diffuse cortical anoxic damage.

1	The oculocephalic reflexes, elicited by moving the head from side to side or vertically and observing eye movements in the direction opposite to the head movement, depend on the integrity of the ocular motor nuclei and their interconnecting tracts that extend from the midbrain to the pons and medulla (Fig. 328-3). The movements, called somewhat inappropriately “doll’s eyes” (which refers more accurately to the reflex elevation of the eyelids with flexion of the neck), are normally suppressed in the awake patient. The ability to elicit them therefore reflects both reduced cortical influence on the brainstem and intact brainstem pathways, indicating that coma is caused by a lesion or dysfunction in the cerebral hemispheres. The opposite, an absence of reflex eye movements, usually signifies damage within the brainstem but can result from overdoses of certain drugs. In this circumstance, normal pupillary size and light reaction distinguishes most drug-induced comas from structural

1	damage within the brainstem but can result from overdoses of certain drugs. In this circumstance, normal pupillary size and light reaction distinguishes most drug-induced comas from structural brainstem damage.

1	Thermal, or “caloric,” stimulation of the vestibular apparatus (oculovestibular response) provides a more intense stimulus for the oculocephalic reflex but provides essentially the same information. The test is performed by irrigating the external auditory canal with cool water in order to induce convection currents in the labyrinths. After a brief latency, the result is tonic deviation of both eyes to the side of cool-water irrigation and nystagmus in the opposite direction. (The acronym “COWS” has been used to remind generations of medical students of the direction of nystagmus—“cold water opposite, warm water same.”) The loss of induced conjugate ocular movements indicates brainstem damage. The presence of corrective nystagmus indicates that the frontal lobes are functioning and connected to the brainstem; thus catatonia or hysterical coma is likely.

1	By touching the cornea with a wisp of cotton, a response consisting of brief bilateral lid closure is normally observed. The corneal reflex depends on the integrity of pontine pathways between the fifth (afferent) and both seventh (efferent) cranial nerves; in conjunction with reflex eye movements, it is a useful test of pontine function. CNS-depressant drugs diminish or eliminate the corneal responses soon after reflex eye movements are paralyzed but before the pupils become unreactive to light. The corneal (and pharyngeal) response may be lost for a time on the side of an acute hemiplegia.

1	Respiratory Patterns These are of less localizing value in comparison to other brainstem signs. Shallow, slow, but regular breathing suggests metabolic or drug depression. Cheyne-Stokes respiration in its typical cyclic form, ending with a brief apneic period, signifies bihemispheral damage or metabolic suppression and commonly accompanies light coma. Rapid, deep (Kussmaul) breathing usually implies metabolic acidosis but may also occur with pontomesencephalic lesions. Agonal gasps are the result of lower brainstem (medullary) damage and are recognized as the terminal respiratory pattern of severe brain damage. A number of other cyclic breathing variations have been described but are of lesser significance.

1	The studies that are most useful in the diagnosis of coma are chemicaltoxicologic analysis of blood and urine, cranial CT or MRI, EEG, and CSF examination. Arterial blood gas analysis is helpful in patients with lung disease and acid-base disorders. The metabolic aberrations commonly encountered in clinical practice are usually exposed by measurement of electrolytes, glucose, calcium, osmolarity, and renal (blood urea nitrogen) and hepatic (NH3) function. Toxicologic analysis may be necessary in any case of acute coma where the diagnosis is not immediately clear. However, the presence of exogenous drugs or toxins, especially alcohol, does not exclude the possibility that other factors, particularly head trauma, are also contributing to the 1775 clinical state. An ethanol level of 43 mmol/L (0.2 g/dL) in nonhabituated patients generally causes impaired mental activity; a level of >65 mmol/L (0.3 g/dL) is associated with stupor. The development of tolerance may allow the chronic

1	mmol/L (0.2 g/dL) in nonhabituated patients generally causes impaired mental activity; a level of >65 mmol/L (0.3 g/dL) is associated with stupor. The development of tolerance may allow the chronic alcoholic to remain awake at levels >87 mmol/L (0.4 g/dL).

1	The availability of CT and MRI has focused attention on causes of coma that are detectable by imaging (e.g., hemorrhage, tumor, or hydrocephalus). Resorting primarily to this approach, although at times expedient, is imprudent because most cases of coma (and confusion) are metabolic or toxic in origin. Furthermore, the notion that a normal CT scan excludes an anatomic lesion as the cause of coma is erroneous. Bilateral hemisphere infarction, acute brainstem infarction, encephalitis, meningitis, mechanical shearing of axons as a result of closed head trauma, sagittal sinus thrombosis, and subdural hematoma isodense to adjacent brain are some of the disorders that may not be detected. Nevertheless, if the source of coma remains unknown, a scan should be obtained.

1	The EEG (Chap. 442e) is useful in metabolic or drug-induced states but is rarely diagnostic. However, it is the essential test to reveal coma that is due to clinically unrecognized, nonconvulsive seizures, and shows fairly characteristic patterns in herpesvirus encephalitis and prion (Creutzfeldt-Jakob) disease. The EEG may be further helpful in disclosing generalized slowing of the background activity, a reflection of the severity of an encephalopathy. Predominant high-voltage slowing (δ or triphasic waves) in the frontal regions is typical of metabolic coma, as from hepatic failure, and widespread fast (β) activity implicates seda tive drugs (e.g., benzodiazepines). A special pattern of “alpha coma,” defined by widespread, variable 8to 12-Hz activity, superficially resembles the normal α rhythm of waking but, unlike normal α activity, is not altered by environmental stimuli. Alpha coma results from pontine or diffuse cortical damage and is associated with a poor prognosis. Normal α

1	α rhythm of waking but, unlike normal α activity, is not altered by environmental stimuli. Alpha coma results from pontine or diffuse cortical damage and is associated with a poor prognosis. Normal α activity on the EEG, which is suppressed by stimulating the patient, also alerts the clinician to the locked-in syndrome or to hysteria or catatonia. Still, the most important use of EEG recordings in coma is to reveal clinically inapparent epileptic discharges.

1	Lumbar puncture is performed less frequently than in the past for coma diagnosis because neuroimaging effectively excludes intracerebral and extensive subarachnoid hemorrhage. However, examination of the CSF remains indispensable in the diagnosis of meningitis and encephalitis. For patients with an altered level of consciousness, it is generally recommended that an imaging study be performed prior to lumbar puncture to exclude a large intracranial mass lesion. Blood culture and antibiotic administration usually precede the imaging study if meningitis is suspected (Chap. 164).

1	(Table 328-1) The causes of coma can be divided into three broad categories: those cases without focal neurologic signs (e.g., metabolic and toxic encephalopathies); meningitis syndromes, characterized by fever or stiff neck and an excess of cells in the spinal fluid (e.g., bacterial meningitis, subarachnoid hemorrhage, encephalitis); and diseases associated with prominent focal signs (e.g., stroke, cerebral hemorrhage). Conditions that cause sudden coma include drug ingestion, cerebral hemorrhage, trauma, cardiac arrest, epilepsy, and basilar artery occlusion from an embolism. Coma that appears subacutely is usually related to a preexisting medical or neurologic problem or, less often, to secondary brain swelling surrounding a mass such as tumor or cerebral infarction.

1	The diagnosis of coma due to cerebrovascular disease can be difficult (Chap. 446). The most common diseases are (1) basal ganglia and thalamic hemorrhage (acute but not instantaneous onset, vomiting, headache, hemiplegia, and characteristic eye signs); (2) pontine hemorrhage (sudden onset, pinpoint pupils, loss of reflex eye movements and corneal responses, ocular bobbing, posturing, and hyperventilation); (3) cerebellar hemorrhage (occipital headache, vomiting, gaze paresis, and inability to stand and walk); (4) basilar artery thrombosis (neurologic prodrome or warning spells, diplopia, dysarthria, vomiting, eye movement and corneal response abnormalities, and asymmetric limb paresis); and (5) subarachnoid hemorrhage (precipitous dIffEREnTIAl dIAgnoSIS of ComA 1. Diseases that cause no focal or lateralizing neurologic signs, usually with normal brainstem functions; CT scan and cellular content of the CSF are normal a. Intoxications: alcohol, sedative drugs, opiates, etc. b.

1	Intoxications: alcohol, sedative drugs, opiates, etc. b. Metabolic disturbances: anoxia, hyponatremia, hypernatremia, hypercalcemia, diabetic acidosis, nonketotic hyperosmolar hyperglycemia, hypoglycemia, uremia, hepatic coma, hypercarbia, Addisonian crisis, hypoand hyperthyroid states, profound nutritional deficiency c. Severe systemic infections: pneumonia, septicemia, typhoid fever, malaria, Waterhouse-Friderichsen syndrome d. e. Postseizure states, status epilepticus, nonconvulsive status epilepticus f. Hypertensive encephalopathy, eclampsia g. Severe hyperthermia, hypothermia h. i. 2. Diseases that cause meningeal irritation with or without fever, and with an excess of WBCs or RBCs in the CSF, usually without focal or lateralizing cerebral or brainstem signs; CT or MRI shows no mass lesion a. Subarachnoid hemorrhage from ruptured aneurysm, arteriovenous malformation, trauma b. c. d.

1	Subarachnoid hemorrhage from ruptured aneurysm, arteriovenous malformation, trauma b. c. d. Miscellaneous: fat embolism, cholesterol embolism, carcinomatous and lymphomatous meningitis, etc. 3. Diseases that cause focal brainstem or lateralizing cerebral signs, with or without changes in the CSF; CT and MRI are abnormal a. Hemispheral hemorrhage (basal ganglionic, thalamic) or infarction (large middle cerebral artery territory) with secondary brainstem compression b. Brainstem infarction due to basilar artery thrombosis or embolism c. Brain abscess, subdural empyema d. Epidural and subdural hemorrhage, brain contusion e. Brain tumor with surrounding edema f. g. h. Metabolic coma (see above) with preexisting focal damage i.

1	Brain abscess, subdural empyema d. Epidural and subdural hemorrhage, brain contusion e. Brain tumor with surrounding edema f. g. h. Metabolic coma (see above) with preexisting focal damage i. Miscellaneous: Cortical vein thrombosis, herpes simplex encephalitis, multiple cerebral emboli due to bacterial endocarditis, acute hemorrhagic leukoencephalitis, acute disseminated (postinfectious) encephalomyelitis, thrombotic thrombocytopenic purpura, cerebral vasculitis, gliomatosis cerebri, pituitary apoplexy, intravascular lymphoma, etc. Abbreviations: CSF, cerebrospinal fluid; CT, computed tomography; MRI, magnetic resonance imaging; RBCs, red blood cells; WBCs, white blood cells. coma after sudden severe headache and vomiting). The most common stroke, infarction in the territory of the middle cerebral artery, does not cause coma, but edema surrounding large infarctions may expand over several days and cause coma from mass effect.

1	The syndrome of acute hydrocephalus accompanies many intracranial diseases, particularly subarachnoid hemorrhage. It is characterized by headache and sometimes vomiting that may progress quickly to coma with extensor posturing of the limbs, bilateral Babinski signs, small unreactive pupils, and impaired oculocephalic movements in the vertical direction. The majority of medical causes of coma can be established without a neuroimaging study but if the history and examination do not indicate the cause of coma, CT or MRI is needed. Sometimes imaging results can be misleading such as when small subdural hematomas or old strokes are found, but the patient’s coma is due to intoxication.

1	This is a state of irreversible cessation of all cerebral function with preservation of cardiac activity and maintenance of respiratory and somatic function by artificial means. It is the only type of brain damage recognized as equivalent to death. Criteria have been advanced for the diagnosis of brain death, and it is essential to adhere to standards endorsed by the local medical community. Ideal criteria are simple, can be assessed at the bedside, and allow no chance of diagnostic error. They contain three essential elements: (1) widespread cortical destruction that is reflected by deep coma and unresponsiveness to all forms of stimulation; (2) global brainstem damage demonstrated by absent pupillary light reaction and by the loss of oculovestibular and corneal reflexes; and (3) destruction of the medulla, manifested by complete and irreversible apnea. The heart rate is invariant and does not accelerate to atropine. Diabetes insipidus is usually present but may only develop hours or

1	of the medulla, manifested by complete and irreversible apnea. The heart rate is invariant and does not accelerate to atropine. Diabetes insipidus is usually present but may only develop hours or days after the other clinical signs of brain death. The pupils are usually midsized but may be enlarged; they should not, however, be small. Loss of deep tendon reflexes is not required because the spinal cord remains functional. Babinski signs are generally absent and the toe response is instead, often flexor.

1	Demonstration that apnea is due to structural medullary damage requires that the Pco2 be high enough to stimulate respiration during a test of spontaneous breathing. Apnea testing can be done safely by the use of diffusion oxygenation prior to removing the ventilator. This is accomplished by preoxygenation with 100% oxygen, which is then sustained during the test by oxygen administered through a tracheal cannula. CO2 tension increases ~0.3–0.4 kPa/min (2–3 mmHg/min) during apnea. At the end of a period of observation, typically several minutes, arterial Pco2 should be at least >6.6–8.0 kPa (50–60 mmHg) for the test to be valid. Apnea is confirmed if no respiratory effort has been observed in the presence of a sufficiently elevated Pco2. Other techniques, including the administration of CO2 to accelerate the test, are used in special circumstances. The apnea test is usually stopped if there is serious cardiovascular instability.

1	An isoelectric EEG may be used as a confirmatory test for total cerebral damage. Radionuclide brain scanning, cerebral angiography, or transcranial Doppler measurements may also be included to demonstrate the absence of CBF, but they have not been as extensively correlated with pathologic changes. The possibility of profound drug-induced or hypothermic depression of the nervous system must be excluded, and some period of observation, usually 6–24 h, is desirable, during which the clinical signs of brain death are sustained. It is advisable to delay clinical testing for at least 24 h if a cardiac arrest has caused brain death or if the inciting disease is not known.

1	Although it is largely accepted in Western society that the respirator can be disconnected from a brain-dead patient and that organ donation is subsequently possible, problems frequently arise because of poor communication and inadequate preparation of the family by the physician. Reasonable medical practice, ideally with the agreement of the family, also allows the removal of support or transfer out of an intensive care unit of patients who are not brain dead but whose neurologic conditions are nonetheless hopeless.

1	The immediate goal in a comatose patient is prevention of further nervous system damage. Hypotension, hypoglycemia, hypercalcemia, hypoxia, hypercapnia, and hyperthermia should be corrected rapidly. An oropharyngeal airway is adequate to keep the pharynx open in a drowsy patient who is breathing normally. Tracheal intubation is indicated if there is apnea, upper airway obstruction, hypoventilation, or emesis, or if the patient is liable to aspirate because of coma. Mechanical ventilation is required if there is hypoventilation or a need to induce hypocapnia in order to lower ICP. IV access is established, and naloxone and dextrose are administered if narcotic overdose or hypoglycemia is a possibility; thiamine is given along with glucose to avoid provoking Wernicke’s disease in malnourished patients. In cases of suspected basilar thrombosis with brainstem ischemia, IV heparin or a thrombolytic agent is often used, after cerebral hemorrhage has been excluded by a neuroimaging study.

1	patients. In cases of suspected basilar thrombosis with brainstem ischemia, IV heparin or a thrombolytic agent is often used, after cerebral hemorrhage has been excluded by a neuroimaging study. Physostigmine may awaken patients with anticholinergictype drug overdose but should be used only with careful monitoring; many physicians believe that it should only be used to treat anticholinergic overdose–associated cardiac arrhythmias. The use of 1777 benzodiazepine antagonists offers some prospect of improvement after overdose of soporific drugs and has transient benefit in hepatic encephalopathy. Certain other toxic and drug-induced comas have specific treatments such as fomepizole for ethylene glycol ingestion.

1	Administration of hypotonic intravenous solutions should be monitored carefully in any serious acute brain illness because of the potential for exacerbating brain swelling. Cervical spine injuries must not be overlooked, particularly before attempting intubation or evaluation of oculocephalic responses. Fever and meningismus indicate an urgent need for examination of the CSF to diagnose meningitis. If the lumbar puncture in a case of suspected meningitis is delayed, an antibiotic such as a third-generation cephalosporin may be administered, preferably after obtaining blood cultures. The management of raised ICP is discussed in Chap. 330.

1	One hopes to avoid the difficult outcome of a patient who is left severely disabled or vegetative. Children and young adults may have ominous early clinical findings such as abnormal brainstem reflexes and yet recover; temporization in offering a prognosis in this group of patients is wise. Metabolic comas have a far better prognosis than traumatic ones. All systems for estimating prognosis in adults should be taken as approximations, and medical judgments must be tempered by factors such as age, underlying systemic disease, and general medical condition. In an attempt to collect prognostic information from large numbers of patients with head injury, the Glasgow Coma Scale was devised; empirically, it has predictive value in cases of brain trauma (see Table 457e-2). For anoxic and metabolic coma, clinical signs such as the pupillary and motor responses after 1 day, 3 days, and 1 week have been shown to have predictive value. Other studies suggest that the absence of corneal responses

1	coma, clinical signs such as the pupillary and motor responses after 1 day, 3 days, and 1 week have been shown to have predictive value. Other studies suggest that the absence of corneal responses may have the most discriminative value. The absence of the cortical waves of the somatosensory evoked potentials has also proved a strong indicator of poor outcome in coma from any cause.

1	The uniformly poor outcome of the prolonged vegetative state has already been mentioned, but recent reports that a small number of such patients display consistent cortical activation on functional MRI in response to salient stimuli have begun to alter the perception of the possible internal mental milieu of such individuals. These findings do not change the poor prognosis. For example, in one series, about 10% of vegetative patients after traumatic brain injury could activate their frontal or temporal lobes in response to requests by an examiner to imagine certain visuospatial tasks. In one case, a rudimentary form of communication could be established. There are also reports in exceptional patients of improvement in cognitive function with the implantation of thalamic-stimulating electrodes. It is prudent to avoid generalizations from these findings.

1	Examination of the Comatose Patient S. Andrew Josephson This chapter features a video illustrating the examination of a coma-tose patient. Proper techniques are demonstrated and supplemented with a discussion of interpretation of findings and implications for management. Also included is an overview of coma and its anatomic 329e basis. CHAPTER 329e Examination of the Comatose Patient 1777 neurologic Critical Care, Including Hypoxic-Ischemic Encephalopathy, and Subarachnoid Hemorrhage J. Claude Hemphill, III, Wade S. Smith, Daryl R. Gress 330

1	Life-threatening neurologic illness may be caused by a primary disorder affecting any region of the neuraxis or may occur as a consequence of a systemic disorder such as hepatic failure, multisystem organ failure, or cardiac arrest (Table 330-1). Neurologic critical care focuses on preservation of neurologic tissue and prevention of secondary brain injury caused by ischemia, hemorrhage, edema, herniation, and elevated intracranial pressure (ICP). Management of other organ systems proceeds concurrently and may need to be modified in order to maintain the overall focus on neurologic issues.

1	PATHOPHYSIOLOGY Brain Edema Swelling, or edema, of brain tissue occurs with many types of brain injury. The two principal types of edema are vasogenic and cytotoxic. Vasogenic edema refers to the influx of fluid and solutes into the brain through an incompetent blood-brain barrier (BBB). In the normal cerebral vasculature, endothelial tight junctions associated with astrocytes create an impermeable barrier (the BBB), through which access into the brain interstitium is dependent upon specific transport mechanisms. The BBB may be compromised in ischemia, trauma, infection, and metabolic derangements. Vasogenic edema results from abnormal permeability of the BBB, and typically develops rapidly following injury. Cytotoxic edema results from cellular swelling, membrane breakdown, and ultimately cell death. Clinically significant brain edema usually represents a combination of vasogenic and cytotoxic components. Edema can lead to increased ICP as well as tissue shifts and brain displacement

1	cell death. Clinically significant brain edema usually represents a combination of vasogenic and cytotoxic components. Edema can lead to increased ICP as well as tissue shifts and brain displacement or herniation from focal processes (Chap. 328). These tissue shifts can cause injury by mechanical distention and compression in addition to the ischemia of impaired perfusion consequent to the elevated ICP.

1	Ischemic Cascade and Cellular Injury When delivery of substrates, principally oxygen and glucose, is inadequate to sustain cellular function, a series of interrelated biochemical reactions known as the ischemic cascade is initiated (see Fig. 446-2). The release of excitatory amino acids, especially glutamate, leads to influx of calcium and sodium ions, which disrupt cellular homeostasis. An increased intracellular calcium concentration may activate proteases and lipases, which then lead to lipid peroxidation and free radical–mediated cell membrane injury. Cytotoxic edema ensues, and ultimately necrotic cell death and tissue infarction occur. This pathway to irreversible cell death is common to ischemic stroke, global cerebral ischemia, and traumatic brain injury.

1	Penumbra refers to areas of ischemic brain tissue that have not yet undergone irreversible infarction, implying that these regions are potentially salvageable if ischemia can be reversed. Factors that may exacerbate ischemic brain injury include systemic hypotension and hypoxia, which further reduce substrate delivery to vulnerable brain tissue, and fever, seizures, and hyperglycemia, which can increase cellular metabolism, outstripping compensatory processes. Clinically, these events are known as secondary brain insults because they lead to exacerbation of the primary brain injury. Prevention, identification, and treatment of secondary brain insults are fundamental goals of management.

1	An alternative pathway of cellular injury is apoptosis. This process implies programmed cell death, which may occur in the setting of ischemic stroke, global cerebral ischemia, traumatic brain injury, and possibly intracerebral hemorrhage. Apoptotic cell death can be distinguished histologically from the necrotic cell death of ischemia and is mediated through a different set of biochemical pathways; apoptotic cell death occurs without cerebral edema and therefore is often not seen on brain imaging. At present, interventions for prevention and Brain: Cerebral Global encephalopathy treatment of apoptotic cell death remain less well defined than those for ischemia. Excitotoxicity and mechanisms of cell death are dis cussed in more detail in Chap. 444e. hemispheres Delirium Sepsis Organ failure—hepatic, renal Medication related—sedatives, hypnotics, analgesics, H2 blockers, antihypertensives Drug overdose Electrolyte disturbance—hyponatremia,

1	hemispheres Delirium Sepsis Organ failure—hepatic, renal Medication related—sedatives, hypnotics, analgesics, H2 blockers, antihypertensives Drug overdose Electrolyte disturbance—hyponatremia, Focal deficits Ischemic stroke Tumor Abscess, subdural empyema Intraparenchymal hemorrhage Subdural/epidural hematoma

1	Cerebral Perfusion and Autoregulation Brain tissue requires constant perfusion in order to ensure adequate delivery of substrate. The hemodynamic response of the brain has the capacity to preserve perfusion across a wide range of systemic blood pressures. Cerebral perfusion pressure (CPP), defined as the mean systemic arterial pressure (MAP) minus the ICP, provides the driving force for circulation across the capillary beds of the brain. Autoregulation refers to the physiologic response whereby cerebral blood flow (CBF) is regulated via alterations in cerebrovascular resistance in order to maintain perfusion over wide physiologic changes such as neuronal activation or changes in hemodynamic function. If systemic blood pressure drops, cerebral perfusion is preserved through vasodilation of arterioles in the brain; likewise, arteriolar vasoconstriction occurs at high systemic pressures to prevent hyperperfusion, resulting in fairly constant perfusion across a wide range of systemic

1	of arterioles in the brain; likewise, arteriolar vasoconstriction occurs at high systemic pressures to prevent hyperperfusion, resulting in fairly constant perfusion across a wide range of systemic blood pressures (Fig. 330-1). At the extreme limits of MAP or CPP (high or low), flow becomes directly related to perfusion pressure. These autoregulatory changes occur in the micro-circulation and are mediated by vessels below the resolution of those seen on angiography. CBF is also strongly influenced by pH and Paco2. CBF increases with hypercapnia and acidosis and decreases with hypocapnia and alkalosis because of pH related changes in cerebral vascular resistance. This forms the basis for the use of hyperventilation to lower ICP, and this effect on ICP is mediated through a decrease in both CBF and intracranial blood volume. Cerebral autoregulation is a complex process critical to the normal homeostatic functioning of the brain, and this process may be disordered focally and

1	in both CBF and intracranial blood volume. Cerebral autoregulation is a complex process critical to the normal homeostatic functioning of the brain, and this process may be disordered focally and unpredictably in disease states such as traumatic brain injury and severe focal cerebral ischemia.

1	Cerebrospinal Fluid and Intracranial Pressure The cranial contents consist essentially of brain, cerebrospinal fluid (CSF), and blood. CSF is produced principally in the choroid plexus of each lateral ventricle, exits the brain via the foramens of Luschka and Magendie, and flows over the cortex to be absorbed into the venous system along the superior sagittal sinus. In adults, approximately 150 mL of CSF are contained within the ventricles and surrounding the brain and spinal cord; the cerebral blood volume is also ~150 mL. The bony skull offers excellent protection for the brain but allows little tolerance for additional volume. Significant Medication effects—chemotherapeutic, antiretroviral Demyelinating Guillain-Barré syndrome Chronic inflammatory demyelinating polyneuropathy Neuromuscular Prolonged effect of neuromuscular blockade junction Medication effects—aminoglycosides CBF, mL/100g per min

1	CBF, mL/100g per min Myasthenia gravis, Lambert-Eaton syndrome, botulism Muscle Critical illness myopathy Cachectic myopathy Acute necrotizing myopathy Thick-filament myopathy Electrolyte disturbances—hypokalemia/ hyperkalemia, hypophosphatemia BP, mmHg FIGURE 330-1 Autoregulation of cerebral blood flow (solid line). Cerebral perfusion is constant over a wide range of systemic blood pressure. Perfusion is increased in the setting of hypoxia or hypercarbia. BP, blood pressure; CBF, cerebral blood flow. (Reprinted with permission from HM Shapiro: Anesthesiology 43:447, 1975. Copyright 1975, Lippincott Company.).

1	FIGURE 330-2 Ischemia and vasodilatation. Reduced cerebral perfusion pressure (CPP) leads to increased ischemia, vasodilation, increased intracranial pressure (ICP), and further reductions in CPP, a cycle leading to further neurologic injury. CBV, cerebral blood volume; CMR, cerebral metabolic rate; CSF, cerebrospinal fluid; SABP, systolic arterial blood pressure. (Adapted from MJ Rosner et al: J Neurosurg 83:949, 1995; with permission.) increases in volume eventually result in increased ICP. Obstruction of CSF outflow, edema of cerebral tissue, or increases in volume from tumor or hematoma may increase ICP. Elevated ICP diminishes cerebral perfusion and can lead to tissue ischemia. Ischemia in turn may lead to vasodilation via autoregulatory mechanisms designed to restore cerebral perfusion. However, vasodilation also increases cerebral blood volume, which in turn then increases ICP, lowers CPP, and provokes further ischemia (Fig. 330-2). This vicious cycle is commonly seen in

1	perfusion. However, vasodilation also increases cerebral blood volume, which in turn then increases ICP, lowers CPP, and provokes further ischemia (Fig. 330-2). This vicious cycle is commonly seen in traumatic brain injury, massive intracerebral hemorrhage, and large hemispheric infarcts with significant tissue shifts.

1	APPROACH TO THE PATIENT: Critically ill patients with severe central nervous system (CNS) dysfunction require rapid evaluation and intervention in order to limit primary and secondary brain injury. Initial neurologic evaluation should be performed concurrent with stabilization of basic respiratory, cardiac, and hemodynamic parameters. Significant barriers may exist to neurologic assessment in the critical care unit, including endotracheal intubation and the use of sedative or paralytic agents to facilitate procedures.

1	An impaired level of consciousness is common in critically ill patients. The essential first task in assessment is to determine whether the cause of dysfunction is related to a diffuse, usually metabolic, process or whether a focal, usually structural, process is implicated. Examples of diffuse processes include metabolic encephalopathies related to organ failure, drug overdose, or hypoxia-ischemia. Focal processes include ischemic and hemorrhagic stroke and traumatic brain injury, especially with intracranial hematomas. Because these two categories of disorders have fundamentally different causes, treatments, and prognoses, the initial focus is on making this distinction rapidly and accurately. The approach to the comatose patient is discussed in Chap. 328; etiologies are listed in Table 328-1.

1	Minor focal deficits may be present on the neurologic examination in patients with metabolic encephalopathies. However, the finding of prominent focal signs such as pupillary asymmetry, hemiparesis, gaze palsy, or paraplegia should suggest the possibility of a structural lesion. All patients with a decreased level of consciousness associated with focal findings should undergo an urgent neuroimaging procedure, as should all patients with coma of unknown etiology. Computed tomography (CT) scanning is usually the most appropriate initial study because it can be performed quickly in critically ill patients and demonstrates hemorrhage, hydrocephalus, and intracranial tissue shifts well. Magnetic resonance imaging (MRI) may provide more specific information in some situations, such as acute ischemic stroke (diffusion-weighted imaging [DWI]) and cerebral venous sinus thrombosis (magnetic resonance venography [MRV]). Any suggestion of trauma from the history or examination should alert the

1	stroke (diffusion-weighted imaging [DWI]) and cerebral venous sinus thrombosis (magnetic resonance venography [MRV]). Any suggestion of trauma from the history or examination should alert the examiner to the possibility of cervical spine injury and prompt an imaging evaluation using plain x-rays, CT, or MRI.

1	Acute brainstem ischemia due to basilar artery thrombosis may cause brief episodes of spontaneous extensor posturing superficially resembling generalized seizures. Coma of sudden onset, accompanied by these movements and cranial nerve abnormalities, necessitates emergency imaging. A noncontrast CT scan of the brain may reveal a hyperdense basilar artery indicating thrombus in the vessel, and subsequent CT or MR angiography can assess basilar artery patency.

1	Other diagnostic studies are best used in specific circumstances, usually when neuroimaging studies fail to reveal a structural lesion and the etiology of the altered mental state remains uncertain. Electroencephalography (EEG) can be important in the evaluation of critically ill patients with severe brain dysfunction. The EEG of metabolic encephalopathy typically reveals generalized slowing. One of the most important uses of EEG is to help exclude inapparent seizures, especially nonconvulsive status epilepticus. Untreated continuous or frequently recurrent seizures may cause neuronal injury, making the diagnosis and treatment of seizures crucial in this patient group. Lumbar puncture (LP) may be necessary to exclude infectious or inflammatory processes, and an elevated opening pressure may be an important clue to cerebral venous sinus thrombosis. In patients with coma or profound encephalopathy, it is preferable to perform a neuroimaging study prior to LP. If bacterial meningitis is

1	be an important clue to cerebral venous sinus thrombosis. In patients with coma or profound encephalopathy, it is preferable to perform a neuroimaging study prior to LP. If bacterial meningitis is suspected, an LP may be performed first or antibiotics may be empirically administered before the diagnostic studies are completed. Standard laboratory evaluation of critically ill patients should include assessment of serum electrolytes (especially sodium and calcium), glucose, renal and hepatic function, complete blood count, and coagulation. Serum or urine toxicology screens should be performed in patients with encephalopathy of unknown cause. EEG, LP, and other specific laboratory tests are most useful when the mechanism of the altered level of consciousness is uncertain; they are not routinely performed in clear-cut cases of stroke or traumatic brain injury.

1	Monitoring of ICP can be an important tool in selected patients. In general, patients who should be considered for ICP monitoring are those with primary neurologic disorders, such as stroke or traumatic brain injury, who are at significant risk for secondary brain injury due to elevated ICP and decreased CPP. Included are patients with the following: severe traumatic brain injury (Glasgow Coma Scale [GCS] score ≤8 [see Table 457e-2]); large tissue shifts from supratentorial ischemic or hemorrhagic stroke; or hydrocephalus from subarachnoid hemorrhage (SAH), intraventricular hemorrhage, or posterior fossa stroke. An additional disorder in which ICP monitoring can add important information is fulminant hepatic failure, in which elevated ICP may be treated with barbiturates or, eventually, liver transplantation. In general, ventriculostomy is preferable to ICP monitoring devices that are placed in the brain parenchyma, because ventriculostomy allows CSF drainage as a method of treating

1	liver transplantation. In general, ventriculostomy is preferable to ICP monitoring devices that are placed in the brain parenchyma, because ventriculostomy allows CSF drainage as a method of treating elevated ICP. However, parenchymal ICP monitoring is most appropriate for patients with diffuse edema and small ventricles (which may make ventriculostomy placement more difficult) or any degree of coagulopathy (in which ventriculostomy carries a higher risk of hemorrhagic complications) (Fig 330-3).

1	Treatment of Elevated ICP Elevated ICP may occur in a wide range of disorders, including head trauma, intracerebral hemorrhage, SAH with hydrocephalus, and fulminant hepatic failure. Because CSF and blood volume can be redistributed initially, by the time FIGURE 330-3 Intracranial pressure and brain tissue oxygen mon-itoring. A ventriculostomy allows for drainage of cerebrospinal fluid to treat elevated intracranial pressure (ICP). Fiberoptic ICP and brain tissue oxygen monitors are usually secured using a screwlike skull bolt. Cerebral blood flow and microdialysis probes (not shown) may be placed in a manner similar to the brain tissue oxygen probe.

1	elevated ICP occurs, intracranial compliance is severely impaired. At this point, any small increase in the volume of CSF, intravascular blood, edema, or a mass lesion may result in a significant increase in ICP and a decrease in cerebral perfusion. This is a fundamental mechanism of secondary ischemic brain injury and constitutes an emergency that requires immediate attention. In general, ICP should be maintained at <20 mmHg and CPP should be maintained at ≥60 mmHg. Interventions to lower ICP are ideally based on the underlying mechanism responsible for the elevated ICP (Table 330-2). For STEPwISE APPRoACH To TREATmEnT of ElEvATEd InTRACRAnIAl PRESSuRE (ICP)a General goals: maintain ICP <20 mmHg and CPP ≥60 mmHg. For ICP >20–25 mmHg for >5 min: 1. Elevate head of the bed; midline head position 2. 3. Osmotherapy—mannitol 25–100 g q4h as needed (maintain serum osmolality <320 mosmol) or hypertonic saline (30 mL, 23.4% NaCl bolus) 4.

1	Elevate head of the bed; midline head position 2. 3. Osmotherapy—mannitol 25–100 g q4h as needed (maintain serum osmolality <320 mosmol) or hypertonic saline (30 mL, 23.4% NaCl bolus) 4. Glucocorticoids—dexamethasone 4 mg q6h for vasogenic edema from tumor, abscess (avoid glucocorticoids in head trauma, ischemic and hemorrhagic stroke) 5. Sedation (e.g., morphine, propofol, or midazolam); add neuromuscular paralysis if necessary (patient will require endotracheal intubation and mechanical ventilation at this point, if not before) 6. 7. Pressor therapy—phenylephrine, dopamine, or norepinephrine to maintain adequate MAP to ensure CPP ≥60 mmHg (maintain euvolemia to minimize deleterious systemic effects of pressors). May adjust target CPP in individual patients based on autoregulation status. 8. a. b. c.

1	8. a. b. c. Hypothermia to 33°C aThroughout ICP treatment algorithm, consider repeat head computed tomography to identify mass lesions amenable to surgical evacuation. May alter order of steps based on directed treatment to specific cause of elevated ICP. Abbreviations: CPP, cerebral perfusion pressure; CSF, cerebrospinal fluid; MAP, mean arterial pressure; PaCO2, arterial partial pressure of carbon dioxide.

1	Abbreviations: CPP, cerebral perfusion pressure; CSF, cerebrospinal fluid; MAP, mean arterial pressure; PaCO2, arterial partial pressure of carbon dioxide. example, in hydrocephalus from SAH, the principal cause of elevated ICP is impairment of CSF drainage. In this setting, ventricular drainage of CSF is likely to be sufficient and most appropriate. In head trauma and stroke, cytotoxic edema may be most responsible, and the use of osmotic agents such as mannitol or hypertonic saline becomes an appropriate early step. As described above, elevated ICP may cause tissue ischemia, and, if cerebral autoregulation is intact, the resulting vasodilation can lead to a cycle of worsening ischemia. Paradoxically, administration of vasopressor agents to increase mean arterial pressure may actually lower ICP by improving perfusion, thereby allowing autoregulatory vasoconstriction as ischemia is relieved and ultimately decreasing intracranial blood volume.

1	Early signs of elevated ICP include drowsiness and a diminished level of consciousness. Neuroimaging studies may reveal evidence of edema and mass effect. Hypotonic IV fluids should be avoided, and elevation of the head of the bed is recommended. Patients must be carefully observed for risk of aspiration and compromise of the airway as the level of alertness declines. Coma and unilateral pupillary changes are late signs and require immediate intervention. Emergent treatment of elevated ICP is most quickly achieved by intubation and hyperventilation, which causes vasoconstriction and reduces cerebral blood volume. To avoid provoking or worsening cerebral ischemia, hyperventilation, if used at all, is best administered only for short periods of time until a more definitive treatment can be instituted. Furthermore, the effects of hyperventilation on ICP are short-lived, often lasting only for several hours because of the buffering capacity of the cerebral interstitium, and rebound

1	be instituted. Furthermore, the effects of hyperventilation on ICP are short-lived, often lasting only for several hours because of the buffering capacity of the cerebral interstitium, and rebound elevations of ICP may accompany abrupt discontinuation of hyperventilation. As the level of consciousness declines to coma, the ability to follow the neurologic status of the patient by examination lessens and measurement of ICP assumes greater importance. If a ventriculostomy device is in place, direct drainage of CSF to reduce ICP is possible. Finally, high-dose barbiturates, decompressive hemicraniectomy, and hypothermia are sometimes used for refractory elevations of ICP, although these have significant side effects and have not been proven to improve outcome.

1	Secondary Brain Insults Patients with primary brain injuries, whether due to trauma or stroke, are at risk for ongoing secondary ischemic brain injury. Because secondary brain injury can be a major determinant of a poor outcome, strategies for minimizing secondary brain insults are an integral part of the critical care of all patients. Although elevated ICP may lead to secondary ischemia, most secondary brain injury is mediated through other clinical events that exacerbate the ischemic cascade already initiated by the primary brain injury. Episodes of secondary brain insults are usually not associated with apparent neurologic worsening. Rather, they lead to cumulative injury limiting eventual recovery, which manifests as a higher mortality rate or worsened long-term functional outcome. Thus, close monitoring of vital signs is important, as is early intervention to prevent secondary ischemia. Avoiding hypotension and hypoxia is critical, as significant hypotensive events (systolic

1	Thus, close monitoring of vital signs is important, as is early intervention to prevent secondary ischemia. Avoiding hypotension and hypoxia is critical, as significant hypotensive events (systolic blood pressure <90 mmHg) as short as 10 min in duration have been shown to adversely influence outcome after traumatic brain injury. Even in patients with stroke or head trauma who do not require ICP monitoring, close attention to adequate cerebral perfusion is warranted. Hypoxia (pulse oximetry saturation <90%), particularly in combination with hypotension, also leads to secondary brain injury. Likewise, fever and hyperglycemia both worsen experimental ischemia and have been associated with worsened clinical outcome after stroke and head trauma. Aggressive control of fever with a goal of normothermia is warranted but may be difficult to achieve with antipyretic medications and cooling blankets. The value of newer surface or intravascular temperature control devices for the management of

1	is warranted but may be difficult to achieve with antipyretic medications and cooling blankets. The value of newer surface or intravascular temperature control devices for the management of refractory fever is under investigation. The use of IV insulin infusion is encouraged for control of hyperglycemia because this allows better regulation of serum glucose levels than SC insulin. A reasonable goal is to maintain the serum glucose level at <10.0 mmol/L (<180 mg/dL), although episodes of hypoglycemia appear equally detrimental and the optimal targets remain uncertain. New cerebral monitoring tools that allow continuous evaluation of brain tissue oxygen tension, CBF, and metabolism (via microdialysis) may further improve the management of secondary brain injury.

1	This occurs from lack of delivery of oxygen to the brain because of extreme hypotension (hypoxia-ischemia) or hypoxia due to respiratory failure. Causes include myocardial infarction, cardiac arrest, shock, asphyxiation, paralysis of respiration, and carbon monoxide or cyanide poisoning. In some circumstances, hypoxia may predominate. Carbon monoxide and cyanide poisoning are sometimes termed histotoxic hypoxia because they cause a direct impairment of the respiratory chain.

1	Clinical Manifestations Mild degrees of pure hypoxia, such as occur at high altitudes, cause impaired judgment, inattentiveness, motor incoordination, and, at times, euphoria. However, with hypoxia-ischemia, such as occurs with circulatory arrest, consciousness is lost within seconds. If circulation is restored within 3–5 min, full recovery may occur, but if hypoxia-ischemia lasts beyond 3–5 min, some degree of permanent cerebral damage usually results. Except in extreme cases, it may be difficult to judge the precise degree of hypoxia-ischemia, and some patients make a relatively full recovery after even 8–10 min of global cerebral ischemia. The brain is more tolerant to pure hypoxia than it is to hypoxia-ischemia. For example, a Pao2 as low as 20 mmHg (2.7 kPa) can be well tolerated if it develops gradually and normal blood pressure is maintained, whereas short durations of very low or absent cerebral circulation usually result in permanent impairment.

1	Clinical examination at different time points after a hypoxicischemic insult (especially cardiac arrest) is useful in assessing prognosis for long-term neurologic outcome. The prognosis is better for patients with intact brainstem function, as indicated by normal pupillary light responses and intact oculocephalic (doll’s eyes), oculovestibular (caloric), and corneal reflexes. Absence of these reflexes and the presence of persistently dilated pupils that do not react to light are grave prognostic signs. A low likelihood of a favorable outcome from hypoxic-ischemic coma is strongly suggested by an absent pupillary light reflex or extensor or absent motor response to pain on day 3 following the injury, excluding patients with metabolic disturbances and those treated with high-dose barbiturates or hypothermia, which confound interpretation of these signs. Electrophysiologically, the bilateral absence of the N20 component of the somatosensory evoked potential (SSEP) in the first several

1	or hypothermia, which confound interpretation of these signs. Electrophysiologically, the bilateral absence of the N20 component of the somatosensory evoked potential (SSEP) in the first several days also conveys a poor prognosis. A very elevated serum level (>33 μg/L) of the biochemical marker neuron-specific enolase (NSE) is indicative of brain damage after resuscitation from cardiac arrest and predicts a poor outcome. However, at present, SSEPs and NSE levels may be difficult to obtain in a timely fashion, with SSEP testing requiring substantial expertise in interpretation and NSE measurements not yet standardized. Recent studies suggest that the administration of mild hypothermia after cardiac arrest (see “Treatment”) may affect the time points when these clinical and electrophysiologic predictors become reliable in identifying patients with a very low likelihood of clinically meaningful recovery. For example, the false-positive rate for incorrect prediction of poor neurologic

1	predictors become reliable in identifying patients with a very low likelihood of clinically meaningful recovery. For example, the false-positive rate for incorrect prediction of poor neurologic outcome may be as high as 21% (95% confidence interval [CI] 8–43%) for patients treated with mild hypothermia who exhibit 3-day motor function no better than extensor posturing. Long-term consequences of hypoxic-ischemic encephalopathy include persistent coma or a vegetative state (Chap. 328), dementia, visual agnosia (Chap. 36), parkinsonism, choreoathetosis, cerebellar ataxia, myoclonus, seizures, and an amnestic state, which may be a consequence of selective damage to the hippocampus.

1	Pathology Principal histologic findings are extensive multifocal or diffuse laminar cortical necrosis (Fig. 330-4), with frequent involvement of the hippocampus. The hippocampal CA1 neurons are vulnerable to even brief episodes of hypoxia-ischemia, perhaps explaining why selective persistent memory deficits may occur after brief cardiac arrest. Scattered small areas of infarction or neuronal loss may be present in the basal ganglia, hypothalamus, or brainstem. In some cases, extensive bilateral thalamic scarring may affect pathways that mediate arousal, and this pathology may be responsible for the persistent vegetative state. A specific form of hypoxic-ischemic encephalopathy, so-called watershed infarcts, occurs at the distal territories between the major cerebral arteries and can cause cognitive deficits, including visual agnosia, and weakness that is greater in proximal than in distal muscle groups.

1	FIGURE 330-4 Cortical laminar necrosis in hypoxic-ischemic encephalopathy. T1-weighted postcontrast magnetic resonance imaging shows cortical enhancement in a watershed distribution consistent with laminar necrosis. Diagnosis Diagnosis is based on the history of a hypoxic-ischemic event such as cardiac arrest. Blood pressure <70 mmHg systolic or Pao2 <40 mmHg is usually necessary, although both absolute levels and duration of exposure are important determinants of cellular injury. Carbon monoxide intoxication can be confirmed by measurement of carboxyhemoglobin and is suggested by a cherry red color of the venous blood and skin, although the latter is an inconsistent clinical finding.

1	Treatment should be directed at restoration of normal cardiorespiratory function. This includes securing a clear airway, ensuring adequate oxygenation and ventilation, and restoring cerebral perfusion, whether by cardiopulmonary resuscitation, fluid, pressors, or cardiac pacing. Hypothermia may target the neuronal cell injury cascade and has substantial neuroprotective properties in experimental models of brain injury. In two trials, mild hypothermia (33°C) improved functional outcome in patients who remained comatose after resuscitation from a cardiac arrest. Treatment was initiated within minutes of cardiac resuscitation and continued for 12 h in one study and 24 h in the other. Potential complications of hypothermia include coagulopathy and an increased risk of infection. Based on these studies, the International Liaison Committee on Resuscitation issued the following advisory statement: “Unconscious adult patients with spontaneous circulation after out-of-hospital cardiac arrest

1	studies, the International Liaison Committee on Resuscitation issued the following advisory statement: “Unconscious adult patients with spontaneous circulation after out-of-hospital cardiac arrest should be cooled to 32°–34°C for 12–24 h when the initial rhythm was ventricular fibrillation. Such cooling may also be beneficial for other rhythms or in-hospital cardiac arrest.” 1782 Severe carbon monoxide intoxication may be treated with hyperbaric oxygen. Anticonvulsants may be needed to control seizures, although these are not usually given prophylactically. Posthypoxic myoclonus may respond to oral administration of clonazepam at doses of 1.5–10 mg daily or valproate at doses of 300–1200 mg daily in divided doses. Myoclonic status epilepticus within 24 h after a primary circulatory arrest generally portends a very poor prognosis, even if seizures are controlled. Carbon monoxide and cyanide intoxication can also cause a delayed encephalopathy. Little clinical impairment is evident when

1	generally portends a very poor prognosis, even if seizures are controlled. Carbon monoxide and cyanide intoxication can also cause a delayed encephalopathy. Little clinical impairment is evident when the patient first regains consciousness, but a parkinsonian syndrome characterized by akinesia and rigidity without tremor may develop. Symptoms can worsen over months, accompanied by increasing evidence of damage in the basal ganglia as seen on both CT and MRI.

1	Altered mental states, variously described as confusion, delirium, disorientation, and encephalopathy, are present in many patients with severe illness in an intensive care unit (ICU). Older patients are particularly vulnerable to delirium, a confusional state characterized by disordered perception, frequent hallucinations, delusions, and sleep disturbance. This is often attributed to medication effects, sleep deprivation, pain, and anxiety. The presence of delirium is associated with worsened outcome in critically ill patients, even in those without an identifiable CNS pathology such as stroke or brain trauma. In these patients, the cause of delirium is often multifactorial, resulting from organ dysfunction, sepsis, and especially the use of medications given to treat pain, agitation, or anxiety. Critically ill patients are often treated with a variety of sedative and analgesic medications, including opiates, benzodiazepines, neuroleptics, and sedative-anesthetic medications, such as

1	anxiety. Critically ill patients are often treated with a variety of sedative and analgesic medications, including opiates, benzodiazepines, neuroleptics, and sedative-anesthetic medications, such as propofol. In critically ill patients requiring sedation, use of the centrally acting α2 agonist dexmedetomidine may reduce delirium and shorten the duration of mechanical ventilation compared to the use of benzodiazepines such as lorazepam or midazolam. The presence of family members in the ICU may also help to calm and orient agitated patients, and in severe cases, low doses of neuroleptics (e.g., haloperidol 0.5–1 mg) can be useful. Current strategies focus on limiting the use of sedative medications when this can be done safely.

1	In the ICU setting, several metabolic causes of an altered level of consciousness predominate. Hypercarbic encephalopathy can present with headache, confusion, stupor, or coma. Hypoventilation syndrome occurs most frequently in patients with a history of chronic CO2 retention who are receiving oxygen therapy for emphysema or chronic pulmonary disease (Chap. 318). The elevated Paco2 leading to CO2 narcosis may have a direct anesthetic effect, and cerebral vasodilation from increased Paco2 can lead to increased ICP. Hepatic encephalopathy is suggested by asterixis and can occur in chronic liver failure or acute fulminant hepatic failure. Both hyperglycemia and hypoglycemia can cause encephalopathy, as can hypernatremia and hyponatremia. Confusion, impairment of eye movements, and gait ataxia are the hallmarks of acute Wernicke’s disease (see below).

1	SEPSIS-ASSOCIATED ENCEPHALOPATHY Pathogenesis In patients with sepsis, the systemic response to infectious agents leads to the release of circulating inflammatory mediators that appear to contribute to encephalopathy. Critical illness, in association with the systemic inflammatory response syndrome (SIRS), can lead to multisystem organ failure. This syndrome can occur in the setting of apparent sepsis, severe burns, or trauma, even without clear identification of an infectious agent. Many patients with critical illness, sepsis, or SIRS develop encephalopathy without obvious explanation. This condition is broadly termed sepsis-associated encephalopathy. Although the specific mediators leading to neurologic dysfunction remain uncertain, it is clear that the encephalopathy is not simply the result of metabolic derangements of multiorgan failure. The cytokines tumor necrosis factor, interleukin (IL)-1, IL-2, and IL-6 are thought to play a role in this syndrome.

1	Diagnosis Sepsis-associated encephalopathy presents clinically as a diffuse dysfunction of the brain without prominent focal findings. Confusion, disorientation, agitation, and fluctuations in level of alertness are typical. In more profound cases, especially with hemodynamic compromise, the decrease in level of alertness can be more prominent, at times resulting in coma. Hyperreflexia and frontal release signs such as a grasp or snout reflex (Chap. 36) can be seen. Abnormal movements such as myoclonus, tremor, or asterixis can occur. Sepsis-associated encephalopathy is quite common, occurring in the majority of patients with sepsis and multisystem organ failure. Diagnosis is often difficult because of the multiple potential causes of neurologic dysfunction in critically ill patients and requires exclusion of structural, metabolic, toxic, and infectious (e.g., meningitis or encephalitis) causes. The mortality rate of patients with sepsis-associated encephalopathy severe enough to

1	and requires exclusion of structural, metabolic, toxic, and infectious (e.g., meningitis or encephalitis) causes. The mortality rate of patients with sepsis-associated encephalopathy severe enough to produce coma approaches 50%, although this principally reflects the severity of the underlying critical illness and is not a direct result of the encephalopathy. Patients dying from severe sepsis or septic shock may have elevated levels of the serum brain injury biomarker S-100β and neuropathologic findings of neuronal apoptosis and cerebral ischemic injury. Successful treatment of the underlying critical illness almost always results in substantial improvement of the encephalopathy. However, although severe disability to the level of chronic vegetative or minimally conscious states is uncommon, long-term cognitive dysfunction clinically similar to dementia is being increasingly recognized in some survivors.

1	This disorder typically presents in a devastating fashion as quadriplegia and pseudobulbar palsy. Predisposing factors include severe underlying medical illness or nutritional deficiency; most cases are associated with rapid correction of hyponatremia or with hyperosmolar states. The pathology consists of demyelination without inflammation in the base of the pons, with relative sparing of axons and nerve cells. MRI is useful in establishing the diagnosis (Fig. 330-5) and may also identify partial forms that present as confusion, dysarthria, and/or disturbances of conjugate gaze without quadriplegia. Occasional cases present with lesions outside of the brainstem. Therapeutic guidelines for the restoration of severe hyponatremia should aim for gradual correction, i.e., by ≤10 mmol/L (10 meq/L) within 24 h and 20 mmol/L (20 meq/L) within 48 h.

1	FIGURE 330-5 Central pontine myelinolysis. Axial T2-weighted magnetic resonance scan through the pons reveals a symmetric area of abnormal high signal intensity within the basis pontis (arrows).

1	Wernicke’s disease is a common and preventable disorder due to a deficiency of thiamine (Chap. 96e). In the United States, alcoholics account for most cases, but patients with malnutrition due to hyperemesis, starvation, renal dialysis, cancer, AIDS, or rarely gastric surgery are also at risk. The characteristic clinical triad is that of ophthalmoplegia, ataxia, and global confusion. However, only one-third of patients with acute Wernicke’s disease present with the classic clinical triad. Most patients are profoundly disoriented, indifferent, and inattentive, although rarely they have an agitated delirium related to ethanol withdrawal. If the disease is not treated, stupor, coma, and death may ensue. Ocular motor abnormalities include horizontal nystagmus on lateral gaze, lateral rectus palsy (usually bilateral), conjugate gaze palsies, and rarely ptosis. Gait ataxia probably results from a combination of polyneuropathy, cerebellar involvement, and vestibular paresis. The pupils are

1	palsy (usually bilateral), conjugate gaze palsies, and rarely ptosis. Gait ataxia probably results from a combination of polyneuropathy, cerebellar involvement, and vestibular paresis. The pupils are usually spared, but they may become miotic with advanced disease.

1	Wernicke’s disease is usually associated with other manifestations of nutritional disease, such as polyneuropathy. Rarely, amblyopia or myelopathy occurs. Tachycardia and postural hypotension may be related to impaired function of the autonomic nervous system or to the coexistence of cardiovascular beriberi. Patients who recover show improvement in ocular palsies within hours after the administration of thiamine, but horizontal nystagmus may persist. Ataxia improves more slowly than the ocular motor abnormalities. Approximately half recover incompletely and are left with a slow, shuffling, wide-based gait and an inability to tandem walk. Apathy, drowsiness, and confusion improve more gradually. As these symptoms recede, an amnestic state with impairment in recent memory and learning may become more apparent (Korsakoff’s psychosis). Korsakoff’s psychosis is frequently persistent; the residual mental state is characterized by gaps in memory, confabulation, and disordered temporal

1	may become more apparent (Korsakoff’s psychosis). Korsakoff’s psychosis is frequently persistent; the residual mental state is characterized by gaps in memory, confabulation, and disordered temporal sequencing.

1	Pathology Periventricular lesions surround the third ventricle, aqueduct, and fourth ventricle, with petechial hemorrhages in occasional acute cases and atrophy of the mammillary bodies in most chronic cases. There is frequently endothelial proliferation, demyelination, and some neuronal loss. These changes may be detected by MRI scanning (Fig. 330-6). The amnestic defect is related to lesions in the dorsal medial nuclei of the thalamus. Pathogenesis Thiamine is a cofactor of several enzymes, including transketolase, pyruvate dehydrogenase, and α-ketoglutarate FIGURE 330-6 Wernicke’s disease. Coronal T1-weighted postcontrast magnetic resonance imaging reveals abnormal enhancement of the mammillary bodies (arrows), typical of acute Wernicke’s encephalopathy.

1	dehydrogenase. Thiamine deficiency produces a diffuse decrease in 1783 cerebral glucose utilization and results in mitochondrial damage. Glutamate accumulates due to impairment of α-ketoglutarate dehydrogenase activity and, in combination with the energy deficiency, may result in excitotoxic cell damage. Wernicke’s disease is a medical emergency and requires immediate administration of thiamine, in a dose of 100 mg either IV or IM. The dose should be given daily until the patient resumes a normal diet and should be begun prior to treatment with IV glucose solutions. Larger doses, 100 mg four times a day or more, have been advocated by some. Glucose infusions may precipitate Wernicke’s disease in a previously unaffected patient or cause a rapid worsening of an early form of the disease. For this reason, thiamine should be administered to all alcoholic patients requiring parenteral glucose.

1	Critical illness with disorders of the peripheral nervous system (PNS) arises in two contexts: (1) primary neurologic diseases that require critical care interventions such as intubation and mechanical ventilation, and (2) secondary PNS manifestations of systemic critical illness, often involving multisystem organ failure. The former include acute polyneuropathies such as Guillain-Barré syndrome (Chap. 460), neuromuscular junction disorders including myasthenia gravis (Chap. 461) and botulism (Chap. 178), and primary muscle disorders such as polymyositis (Chap. 462e). The latter result either from the systemic disease itself or as a consequence of interventions.

1	General principles of respiratory evaluation in patients with PNS involvement, regardless of cause, include assessment of pulmonary mechanics, such as maximal inspiratory force (MIF) and vital capacity (VC), and evaluation of strength of bulbar muscles. Regardless of the cause of weakness, endotracheal intubation should be considered when the MIF falls to <–25 cmH2O or the VC is <1 L. Also, patients with severe palatal weakness may require endotracheal intubation in order to prevent acute upper airway obstruction or recurrent aspiration. Arterial blood gases and oxygen saturation from pulse oximetry are used to follow patients with potential respiratory compromise from PNS dysfunction. However, intubation and mechanical ventilation should be undertaken based on clinical assessment rather than waiting until oxygen saturation drops or CO2 retention develops from hypoventilation. Noninvasive mechanical ventilation may be considered initially in lieu of endotracheal intubation but is

1	than waiting until oxygen saturation drops or CO2 retention develops from hypoventilation. Noninvasive mechanical ventilation may be considered initially in lieu of endotracheal intubation but is generally insufficient in patients with severe bulbar weakness or ventilatory failure with hypercarbia. Principles of mechanical ventilation are discussed in Chap. 323.

1	Although encephalopathy may be the most obvious neurologic dysfunction in critically ill patients, dysfunction of the PNS is also quite common. It is typically present in patients with prolonged critical illnesses lasting several weeks and involving sepsis; clinical suspicion is aroused when there is failure to wean from mechanical ventilation despite improvement of the underlying sepsis and critical illness. Critical illness polyneuropathy refers to the most common PNS complication related to critical illness; it is seen in the setting of prolonged critical illness, sepsis, and multisystem organ failure. Neurologic findings include diffuse weakness, decreased reflexes, and distal sensory loss. Electrophysiologic studies demonstrate a diffuse, symmetric, distal axonal sensorimotor neuropathy, and pathologic studies have confirmed axonal degeneration. The precise mechanism of critical illness polyneuropathy remains unclear, but circulating factors such as cytokines, which are

1	neuropathy, and pathologic studies have confirmed axonal degeneration. The precise mechanism of critical illness polyneuropathy remains unclear, but circulating factors such as cytokines, which are associated with sepsis and SIRS, are thought to play a role. It has been reported that up to 70% of patients with the sepsis syndrome have some degree of neuropathy, although far fewer 1784 have a clinical syndrome profound enough to cause severe respiratory muscle weakness requiring prolonged mechanical ventilation or resulting in failure to wean. Aggressive glycemic control with insulin infusions appears to decrease the risk of critical illness polyneuropathy. Treatment is otherwise supportive, with specific intervention directed at treating the underlying illness. Although spontaneous recovery is usually seen, the time course may extend over weeks to months and necessitate long-term ventilatory support and care even after the underlying critical illness has resolved.

1	A defect in neuromuscular transmission may be a source of weakness in critically ill patients. Botulism (Chap. 178) may be acquired by ingesting botulinum toxin from improperly stored food or may arise from an anaerobic abscess from Clostridium botulinum (wound botulism). Infants can present with generalized weakness from gut-derived Clostridium infection, especially if they are fed honey. Diplopia and dysphagia are early signs of foodborne botulism. Treatment is mostly supportive, although use of antitoxin early in the course may limit the duration of the neuromuscular blockade. General ICU care is similar to patients with Guillain-Barré syndrome or myasthenia gravis with focused care to avoid ulcer formation at pressure points, deep venous thromboprophylaxis, and infection prevention. Public health officers should be rapidly informed when the diagnosis is made to prevent further exposure to others from the tainted food or source of wound botulism (such as injection drug use).

1	Undiagnosed myasthenia gravis (Chap. 461) may be a consideration in weak ICU patients; however, persistent weakness secondary to impaired neuromuscular junction transmission is almost always due to administration of drugs. A number of medications impair neuromuscular transmission; these include antibiotics, especially aminoglycosides, and beta-blocking agents. In the ICU, the nondepolarizing neuromuscular blocking agents (nd-NMBAs), also known as muscle relaxants, are most commonly responsible. Included in this group of drugs are such agents as pancuronium, vecuronium, rocuronium, and cisatracurium. They are often used to facilitate mechanical ventilation or other critical care procedures, but with prolonged use persistent neuromuscular blockade may result in weakness even after discontinuation of these agents hours or days earlier. Risk factors for this prolonged action of neuromuscular blocking agents include female sex, metabolic acidosis, and renal failure.

1	Prolonged neuromuscular blockade does not appear to produce permanent damage to the PNS. Once the offending medications are discontinued, full strength is restored, although this may take days. In general, the lowest dose of neuromuscular blocking agent should be used to achieve the desired result and, when these agents are used in the ICU, a peripheral nerve stimulator should be used to monitor neuromuscular junction function.

1	Critically ill patients, especially those with sepsis, frequently develop muscle weakness and wasting, often in the face of seemingly adequate nutritional support. Critical illness myopathy is an overall term that describes several different discrete muscle disorders that may occur in critically ill patients. The assumption has been that a catabolic myopathy may develop as a result of multiple factors, including elevated cortisol and catecholamine release and other circulating factors induced by the SIRS. In this syndrome, known as cachectic myopathy, serum creatine kinase levels and electromyography (EMG) are normal. Muscle biopsy shows type II fiber atrophy. Panfascicular muscle fiber necrosis may also occur in the setting of profound sepsis. This less common acute necrotizing intensive care myopathy is characterized clinically by weakness progressing to a profound level over just a few days. There may be associated elevations in serum creatine kinase and urine myoglobin. Both EMG

1	care myopathy is characterized clinically by weakness progressing to a profound level over just a few days. There may be associated elevations in serum creatine kinase and urine myoglobin. Both EMG and muscle biopsy may be normal initially but eventually show abnormal spontaneous activity and panfascicular necrosis with an accompanying inflammatory reaction. Acute rhabdomyolysis can occur from alcohol ingestion or from compartment syndromes.

1	A thick-filament myopathy may occur in the setting of glucocorticoid and nd-NMBA use. The most frequent scenario in which this is encountered is the asthmatic patient who requires high-dose glucocorticoids and nd-NMBA to facilitate mechanical ventilation. This muscle disorder is not due to prolonged action of nd-NMBAs at the neuromuscular junction but, rather, is an actual myopathy with muscle damage; it has occasionally been described with high-dose glucocorticoid use or sepsis alone. Clinically this syndrome is most often recognized when a patient fails to wean from mechanical ventilation despite resolution of the primary pulmonary process. Pathologically, there may be loss of thick (myosin) filaments. Thick-filament critical illness myopathy has a good prognosis. If patients survive their underlying critical illness, the myopathy invariably improves and most patients return to normal. However, because this syndrome is a result of true muscle damage, not just prolonged blockade at

1	underlying critical illness, the myopathy invariably improves and most patients return to normal. However, because this syndrome is a result of true muscle damage, not just prolonged blockade at the neuromuscular junction, this process may take weeks or months, and tracheotomy with prolonged ventilatory support may be necessary. Some patients do have residual long-term weakness, with atrophy and fatigue limiting ambulation. At present, it is unclear how to prevent this myopathic complication, except by avoiding use of nd-NMBAs, a strategy not always possible. Monitoring with a peripheral nerve stimulator can help to avoid the overuse of these agents. However, this is more likely to prevent the complication of prolonged neuromuscular junction blockade than it is to prevent this myopathy.

1	Subarachnoid hemorrhage (SAH) renders the brain critically ill from both primary and secondary brain insults. Excluding head trauma, the most common cause of SAH is rupture of a saccular aneurysm. Other causes include bleeding from a vascular malformation (arteriovenous malformation or dural arteriovenous fistula) and extension into the subarachnoid space from a primary intracerebral hemorrhage. Some idiopathic SAHs are localized to the perimesencephalic cisterns and are benign; they probably have a venous or capillary source, and angiography is unrevealing.

1	Autopsy and angiography studies have found that about 2% of adults harbor intracranial aneurysms, for a prevalence of 4 million persons in the United States; the aneurysm will rupture, producing SAH, in 25,000–30,000 cases per year. For patients who arrive alive at hospital, the mortality rate over the next month is about 45%. Of those who survive, more than half are left with major neurologic deficits as a result of the initial hemorrhage, cerebral vasospasm with infarction, or hydrocephalus. If the patient survives but the aneurysm is not obliterated, the rate of rebleeding is about 20% in the first 2 weeks, 30% in the first month, and about 3% per year afterward. Given these alarming figures, the major therapeutic emphasis is on preventing the predictable early complications of the SAH.

1	Unruptured, asymptomatic aneurysms are much less dangerous than a recently ruptured aneurysm. The annual risk of rupture for aneurysms <10 mm in size is ~0.1%, and for aneurysms ≥10 mm in size is ~0.5–1%; the surgical morbidity rate far exceeds these percentages. Because of the longer length of exposure to risk of rupture, younger patients with aneurysms >10 mm in size may benefit from prophylactic treatment. As with the treatment of asymptomatic carotid stenosis, this risk-benefit ratio strongly depends on the complication rate of treatment.

1	Giant aneurysms, those >2.5 cm in diameter, occur at the same sites (see below) as small aneurysms and account for 5% of cases. The three most common locations are the terminal internal carotid artery, middle cerebral artery (MCA) bifurcation, and top of the basilar artery. Their risk of rupture is ~6% in the first year after identification and may remain high indefinitely. They often cause symptoms by compressing the adjacent brain or cranial nerves. Mycotic aneurysms are usually located distal to the first bifurcation of major arteries of the circle of Willis. Most result from infected emboli due to bacterial endocarditis causing septic degeneration of arteries and subsequent dilation and rupture. Whether these lesions should be sought and repaired prior to rupture or left to heal spontaneously with antibiotic treatment is controversial.

1	Pathophysiology Saccular aneurysms occur at the bifurcations of the largeto medium-sized intracranial arteries; rupture is into the subarachnoid space in the basal cisterns and often into the parenchyma of the adjacent brain. Approximately 85% of aneurysms occur in the anterior circulation, mostly on the circle of Willis. About 20% of patients have multiple aneurysms, many at mirror sites bilaterally. As an aneurysm develops, it typically forms a neck with a dome. The length of the neck and the size of the dome vary greatly and are important factors in planning neurosurgical obliteration or endovascular embolization. The arterial internal elastic lamina disappears at the base of the neck. The media thins, and connective tissue replaces smooth-muscle cells. At the site of rupture (most often the dome), the wall thins, and the tear that allows bleeding is often ≤0.5 mm long. Aneurysm size and site are important in predicting risk of rupture. Those >7 mm in diameter and those at the top

1	the dome), the wall thins, and the tear that allows bleeding is often ≤0.5 mm long. Aneurysm size and site are important in predicting risk of rupture. Those >7 mm in diameter and those at the top of the basilar artery and at the origin of the posterior communicating artery are at greater risk of rupture.

1	Clinical Manifestations Most unruptured intracranial aneurysms are completely asymptomatic. Symptoms are usually due to rupture and resultant SAH, although some unruptured aneurysms present with mass effect on cranial nerves or brain parenchyma. At the moment of aneurysmal rupture with major SAH, the ICP suddenly rises. This may account for the sudden transient loss of consciousness that occurs in nearly half of patients. Sudden loss of consciousness may be preceded by a brief moment of excruciating headache, but most patients first complain of headache upon regaining consciousness. In 10% of cases, aneurysmal bleeding is severe enough to cause loss of consciousness for several days. In ~45% of cases, severe headache associated with exertion is the presenting complaint. The patient often calls the headache “the worst headache of my life”; however, the most important characteristic is sudden onset. Occasionally, these ruptures may present as headache of only moderate intensity or as a

1	calls the headache “the worst headache of my life”; however, the most important characteristic is sudden onset. Occasionally, these ruptures may present as headache of only moderate intensity or as a change in the patient’s usual headache pattern. The headache is usually generalized, often with neck stiffness, and vomiting is common.

1	Although sudden headache in the absence of focal neurologic symptoms is the hallmark of aneurysmal rupture, focal neurologic deficits may occur. Anterior communicating artery or MCA bifurcation aneurysms may rupture into the adjacent brain or subdural space and form a hematoma large enough to produce mass effect. The deficits that result can include hemiparesis, aphasia, and abulia.

1	Occasionally, prodromal symptoms suggest the location of a progressively enlarging unruptured aneurysm. A third cranial nerve palsy, particularly when associated with pupillary dilation, loss of ipsilateral (but retained contralateral) light reflex, and focal pain above or behind the eye, may occur with an expanding aneurysm at the junction of the posterior communicating artery and the internal carotid artery. A sixth nerve palsy may indicate an aneurysm in the cavernous sinus, and visual field defects can occur with an expanding supraclinoid carotid or anterior cerebral artery aneurysm. Occipital and posterior cervical pain may signal a posterior inferior cerebellar artery or anterior inferior cerebellar artery aneurysm (Chap. 446). Pain in or behind the eye and in the low temple can occur with an expanding MCA aneurysm. Thunderclap headache is a variant of migraine that simulates an SAH. Before concluding that a patient with sudden, severe headache has thunderclap migraine, a

1	occur with an expanding MCA aneurysm. Thunderclap headache is a variant of migraine that simulates an SAH. Before concluding that a patient with sudden, severe headache has thunderclap migraine, a definitive workup for aneurysm or other intracranial pathology is required.

1	Aneurysms can undergo small ruptures and leaks of blood into the subarachnoid space, so-called sentinel bleeds. Sudden unexplained headache at any location should raise suspicion of SAH and be investigated, because a major hemorrhage may be imminent. The initial clinical manifestations of SAH can be graded using the Hunt-Hess or World Federation of Neurosurgical Societies classification schemes (Table 330-3). For ruptured aneurysms, prognosis for good outcomes falls as the grade increases. For example, it is unusual for a Hunt-Hess grade 1 patient to die if the aneurysm is treated, but the mortality rate for grade 4 and 5 patients may be as high as 80%.

1	World Federation of Neurosurgical Societies Grade Hunt-Hess Scale (WFNS) Scale 1 Mild headache, normal mental status, no cranial nerve or motor findings 2 Severe headache, normal mental status, may have cranial nerve deficit 3 Somnolent, confused, may have cranial nerve or mild motor deficit 4 Stupor, moderate to severe motor deficit, may have intermittent reflex posturing 5 Coma, reflex posturing or flaccid aGlasgow Coma Scale; see Table 457e-1. GCSa score 15, no motor deficits GCS score 13–14, no motor deficits GCS score 13–14, with motor deficits GCS score 7–12, with or without motor deficits GCS score 3–6, with or without motor deficits Delayed Neurologic Deficits There are four major causes of delayed neurologic deficits: rerupture, hydrocephalus, vasospasm, and hyponatremia. 1.

1	GCS score 3–6, with or without motor deficits Delayed Neurologic Deficits There are four major causes of delayed neurologic deficits: rerupture, hydrocephalus, vasospasm, and hyponatremia. 1. Rerupture. The incidence of rerupture of an untreated aneurysm in the first month following SAH is ~30%, with the peak in the first 7 days. Rerupture is associated with a 60% mortality rate and poor outcome. Early treatment eliminates this risk. 2.

1	2. Hydrocephalus. Acute hydrocephalus can cause stupor and coma and can be mitigated by placement of an external ventricular drain. More often, subacute hydrocephalus may develop over a few days or weeks and causes progressive drowsiness or slowed mentation (abulia) with incontinence. Hydrocephalus is differentiated from cerebral vasospasm with a CT scan, CT angiogram, transcranial Doppler (TCD) ultrasound, or conventional x-ray angiography. Hydrocephalus may clear spontaneously or require temporary ventricular drainage. Chronic hydrocephalus may develop weeks to months after SAH and manifest as gait difficulty, incontinence, or impaired mentation. Subtle signs may be a lack of initiative in conversation or a failure to recover independence. 3.

1	3. Vasospasm. Narrowing of the arteries at the base of the brain following SAH causes symptomatic ischemia and infarction in ~30% of patients and is the major cause of delayed morbidity and death. Signs of ischemia appear 4–14 days after the hemorrhage, most often at 7 days. The severity and distribution of vasospasm determine whether infarction will occur. 4. Delayed vasospasm is believed to result from direct effects of clotted blood and its breakdown products on the arteries within the subarachnoid space. In general, the more blood that surrounds the arteries, the greater the chance of symptomatic vasospasm. Spasm of major arteries produces symptoms referable to the appropriate vascular territory (Chap. 446). All of these focal symptoms may present abruptly, fluctuate, or develop over a few days. In most cases, focal spasm is preceded by a decline in mental status. 5.

1	5. Vasospasm can be detected reliably with conventional x-ray angiography, but this invasive procedure is expensive and carries the risk of stroke and other complications. TCD ultrasound is based on the principle that the velocity of blood flow within an artery will rise as the lumen diameter is narrowed. By directing the probe along the MCA and proximal anterior cerebral artery (ACA), carotid terminus, and vertebral and basilar arteries on a daily or every-other-day basis, vasospasm can be reliably detected and treatments initiated to prevent cerebral ischemia (see below). CT angiography is another method that can detect vasospasm. 6. Severe cerebral edema in patients with infarction from vasospasm may increase the ICP enough to reduce cerebral perfusion pressure. Treatment may include mannitol, hyperventilation, and hemicraniectomy; moderate hypothermia may have a role as well. 7.

1	7. Hyponatremia. Hyponatremia may be profound and can develop quickly in the first 2 weeks following SAH. There is both natriuresis and volume depletion with SAH, so that patients become both hyponatremic and hypovolemic. Both atrial natriuretic peptide and brain natriuretic peptide have a role in producing this “cerebral FIGURE 330-7 Subarachnoid hemorrhage. A. Computed tomography (CT) angiography revealing an aneurysm of the left superior cerebellar artery. B. Noncontrast CT scan at the level of the third ventricle revealing subarachnoid blood (bright) in the left sylvian fissure and within the left lateral ventricle. C. Conventional anteroposterior x-ray angiogram of the right vertebral and basilar artery showing the large aneurysm. D. Conventional angiogram following coil embolization of the aneurysm, whereby the aneurysm body is filled with platinum coils delivered through a microcatheter navigated from the femoral artery into the aneurysm neck.

1	salt-wasting syndrome.” Typically, it clears over the course of 1–2 weeks and, in the setting of SAH, should not be treated with free-water restriction as this may increase the risk of stroke (see below). Laboratory Evaluation and Imaging (Fig. 330-7) The hallmark of aneurysmal rupture is blood in the CSF. More than 95% of cases have enough blood to be visualized on a high-quality noncontrast CT scan obtained within 72 h. If the scan fails to establish the diagnosis of SAH and no mass lesion or obstructive hydrocephalus is found, a lumbar puncture should be performed to establish the presence of subarachnoid blood. Lysis of the red blood cells and subsequent conversion of hemoglobin to bilirubin stains the spinal fluid yellow within 6–12 h. This xanthochromic spinal fluid peaks in intensity at 48 h and lasts for 1–4 weeks, depending on the amount of subarachnoid blood.

1	The extent and location of subarachnoid blood on a noncontrast CT scan help locate the underlying aneurysm, identify the cause of any neurologic deficit, and predict delayed vasospasm. A high incidence of symptomatic vasospasm in the MCA and ACA has been found when early CT scans show subarachnoid clots >5 × 3 mm in the basal cisterns, or layers of blood >1 mm thick in the cerebral fissures. CT scans less reliably predict vasospasm in the vertebral, basilar, or posterior cerebral arteries.

1	Lumbar puncture prior to an imaging procedure is indicated only if a CT scan is not available at the time of the suspected SAH. Once the diagnosis of hemorrhage from a ruptured saccular aneurysm is suspected, four-vessel conventional x-ray angiography (both carotids and both vertebrals) is generally performed to localize and define the anatomic details of the aneurysm and to determine if other unruptured aneurysms exist (Fig. 330-7C). At some centers, the ruptured aneurysm can be treated using endovascular techniques at the time of the initial angiogram as a way to expedite treatment and minimize the number of invasive procedures. CT angiography is an alternative method for locating the aneurysm and may be sufficient to plan definitive therapy. Close monitoring (daily or twice daily) of electrolytes is important because hyponatremia can occur precipitously during the first 2 weeks following SAH (see above).

1	The electrocardiogram (ECG) frequently shows ST-segment and T-wave changes similar to those associated with cardiac ischemia. A prolonged QRS complex, increased QT interval, and prominent “peaked” or deeply inverted symmetric T waves are usually secondary to the intracranial hemorrhage. There is evidence that structural myocardial lesions produced by circulating catecholamines and excessive discharge of sympathetic neurons may occur after SAH, causing these ECG changes and a reversible cardiomyopathy sufficient to cause shock or congestive heart failure. Echocardiography reveals a pattern of regional wall motion abnormalities that follow the distribution of sympathetic nerves rather than the major coronary arteries, with relative sparing of the ventricular wall apex. The sympathetic nerves themselves appear to be injured by direct toxicity from the excessive catecholamine release. An asymptomatic troponin elevation is common. Serious ventricular dysrhythmias occurring in-hospital are

1	themselves appear to be injured by direct toxicity from the excessive catecholamine release. An asymptomatic troponin elevation is common. Serious ventricular dysrhythmias occurring in-hospital are unusual.

1	Early aneurysm repair prevents rerupture and allows the safe application of techniques to improve blood flow (e.g., induced hypertension) should symptomatic vasospasm develop. An aneurysm can be “clipped” by a neurosurgeon or “coiled” by an endovascular surgeon. Surgical repair involves placing a metal clip across the aneurysm neck, thereby immediately eliminating the risk of rebleeding. This approach requires craniotomy and brain retraction, which is associated with neurologic morbidity. Endovascular techniques involve placing platinum coils, or other embolic material, within the aneurysm via a catheter that is passed from the femoral artery. The aneurysm is packed tightly to enhance thrombosis and over time is walled off from the circulation (Fig. 330-7D). There have been two prospective randomized trials of surgery versus endovascular treatment for ruptured aneurysms: the first was the International Subarachnoid Aneurysm Trial (ISAT), which was terminated early when 24% of patients

1	randomized trials of surgery versus endovascular treatment for ruptured aneurysms: the first was the International Subarachnoid Aneurysm Trial (ISAT), which was terminated early when 24% of patients treated with endovascular therapy were dead or dependent at 1 year compared to 31% treated with surgery, a significant 23% relative reduction. After 5 years, risk of death was lower in the coiling group, although the proportion of survivors who were independent was the same in both groups. Risk of rebleeding was low, but more common in the coiling group. These results favoring coiling at 1 year were confirmed in a second trial, although the differences in functional outcome were no longer significant at 3 years. Because some aneurysms have a morphology that is not amenable to endovascular treatment, surgery remains an important treatment option. Centers that combine both endovascular and neurosurgical expertise likely offer the best outcomes for patients, and there are reliable data

1	treatment, surgery remains an important treatment option. Centers that combine both endovascular and neurosurgical expertise likely offer the best outcomes for patients, and there are reliable data showing that specialized aneurysm treatment centers can improve mortality rates.

1	The medical management of SAH focuses on protecting the airway, managing blood pressure before and after aneurysm treatment, preventing rebleeding prior to treatment, managing vasospasm, treating hydrocephalus, treating hyponatremia, limiting secondary brain insults, and preventing pulmonary embolus (PE). Intracranial hypertension following aneurysmal rupture occurs secondary to subarachnoid blood, parenchymal hematoma, acute hydrocephalus, or loss of vascular autoregulation. Patients who are stuporous should undergo emergent ventriculostomy to measure ICP and to treat high ICP in order to prevent cerebral ischemia. Medical therapies designed to combat raised ICP (e.g., osmotic therapy and sedation) can also be used as needed. High ICP refractory to treatment is a poor prognostic sign.

1	Prior to definitive treatment of the ruptured aneurysm, care is required to maintain adequate cerebral perfusion pressure while avoiding excessive elevation of arterial pressure. If the patient is alert, it is reasonable to lower the systolic blood pressure to below 160 mmHg using nicardipine, labetalol, or esmolol. If the patient has a depressed level of consciousness, ICP should be measured and the cerebral perfusion pressure targeted to 60–70 mmHg. If headache or neck pain is severe, mild sedation and analgesia are prescribed. Extreme sedation is avoided if possible because it can obscure the ability to clinically detect changes in neurologic status. Adequate hydration is necessary to avoid a decrease in blood volume predisposing to brain ischemia.

1	Seizures are uncommon at the onset of aneurysmal rupture. The quivering, jerking, and extensor posturing that often accompany loss of consciousness with SAH are probably related to the sharp rise in ICP rather than seizures. However, anticonvulsants are sometimes given as prophylactic therapy because a seizure could theoretically promote rebleeding. Glucocorticoids may help reduce the head and neck ache caused by the irritative effect of the subarachnoid blood. There is no good evidence that they reduce cerebral edema, are neuroprotective, or reduce vascular injury, and their routine use therefore is not recommended.

1	Antifibrinolytic agents are not routinely prescribed but may be considered in patients in whom aneurysm treatment cannot proceed immediately. They are associated with a reduced incidence of aneurysmal rerupture but may also increase the risk of delayed cerebral infarction and deep vein thrombosis (DVT). Several recent studies suggest that a shorter duration of use (until the aneurysm is secured or for the first 3 days) may decrease rerupture and be safer than found in earlier studies of longer duration treatment.

1	Vasospasm remains the leading cause of morbidity and mortality following aneurysmal SAH. Treatment with the calcium channel antagonist nimodipine (60 mg PO every 4 h) improves outcome, perhaps by preventing ischemic injury rather than reducing the risk of vasospasm. Nimodipine can cause significant hypotension in some patients, which may worsen cerebral ischemia in patients with vasospasm. Symptomatic cerebral vasospasm can also be treated by increasing the cerebral perfusion pressure by raising mean arterial pressure through plasma volume expansion and the judicious use of IV vasopressor agents, usually phenylephrine or norepinephrine. Raised perfusion pressure has been associated with clinical improvement in many patients, but high arterial pressure may promote rebleeding in unprotected aneurysms. Treatment with oncologic Emergencies Rasim Gucalp, Janice P. Dutcher Emergencies in patients with cancer may be classified into three groups: pressure or obstruction caused by a

1	aneurysms. Treatment with oncologic Emergencies Rasim Gucalp, Janice P. Dutcher Emergencies in patients with cancer may be classified into three groups: pressure or obstruction caused by a space-occupying lesion, metabolic or hormonal problems (paraneoplastic syndromes, Chap. 121), and treatment-related complications. 331 SEC TIon 4

1	Superior vena cava syndrome (SVCS) is the clinical manifestation of superior vena cava (SVC) obstruction, with severe reduction in venous return from the head, neck, and upper extremities. Malignant induced hypertension and hypervolemia generally requires moni-1787 toring of arterial and central venous pressures; it is best to infuse pressors through a central venous line as well. Volume expansion helps prevent hypotension and augments cardiac output.

1	If symptomatic vasospasm persists despite optimal medical therapy, intraarterial vasodilators and percutaneous transluminal angioplasty are considered. Vasodilatation by direct angioplasty appears to be permanent, allowing hypertensive therapy to be tapered sooner. The pharmacologic vasodilators (verapamil and nicardipine) do not last more than about 24 h, and therefore multiple treatments may be required until the subarachnoid blood is reabsorbed. Although intraarterial papaverine is an effective vasodilator, there is evidence that papaverine may be neurotoxic, so its use should generally be avoided. Acute hydrocephalus can cause stupor or coma. It may clear spontaneously or require temporary ventricular drainage. When chronic hydrocephalus develops, ventricular shunting is the treatment of choice.

1	Free-water restriction is contraindicated in patients with SAH at risk for vasospasm because hypovolemia and hypotension may occur and precipitate cerebral ischemia. Many patients continue to experience a decline in serum sodium despite receiving parenteral fluids containing normal saline. Frequently, supplemental oral salt coupled with normal saline will mitigate hyponatremia, but often patients also require intravenous hypertonic saline. Care must be taken not to correct serum sodium too quickly in patients with marked hyponatremia of several days’ duration, as central pontine myelinolysis may occur.

1	All patients should have pneumatic compression stockings applied to prevent pulmonary embolism. Unfractionated heparin administered subcutaneously for DVT prophylaxis can be initiated immediately following endovascular treatment and within days following craniotomy with surgical clipping and is a useful adjunct to pneumatic compression stockings. Treatment of pulmonary embolus depends on whether the aneurysm has been treated and whether or not the patient has had a craniotomy. Systemic anticoagulation with heparin is contraindicated in patients with ruptured and untreated aneurysms. It is a relative contraindication following craniotomy for several days, and it may delay thrombosis of a coiled aneurysm. If DVT or PE occurs within the first days following craniotomy, use of an inferior vena cava filter may be considered to prevent additional pulmonary emboli, whereas systemic anticoagulation with heparin is preferred following successful endovascular treatment.

1	tumors, such as lung cancer, lymphoma, and metastatic tumors, are responsible for the majority of SVCS cases. With the expanding use of intravascular devices (e.g., permanent central venous access catheters, pacemaker/defibrillator leads), the prevalence of benign causes of SVCS is increasing now, accounting for at least 40% of cases. Lung cancer, particularly of small-cell and squamous cell histologies, accounts for approximately 85% of all cases of malignant origin. In young adults, malignant lymphoma is a leading cause of SVCS. Hodgkin’s lymphoma involves the mediastinum more commonly than other lymphomas but rarely causes SVCS. When SVCS is noted in a young man with a mediastinal mass, the differential diagnosis is lymphoma versus primary mediastinal germ cell tumor. Metastatic cancers to the mediastinal lymph nodes, such as testicular and breast carcinomas, account for a small proportion of cases. Other causes include benign tumors, aortic aneurysm, thyromegaly, thrombosis, and

1	to the mediastinal lymph nodes, such as testicular and breast carcinomas, account for a small proportion of cases. Other causes include benign tumors, aortic aneurysm, thyromegaly, thrombosis, and fibrosing mediastinitis from prior irradiation, histoplasmosis, or Behçet’s syndrome. SVCS as 1788 the initial manifestation of Behçet’s syndrome may be due to inflammation of the SVC associated with thrombosis. Patients with SVCS usually present with neck and facial swelling (especially around the eyes), dyspnea, and cough. Other symptoms include hoarseness, tongue swelling, headaches, nasal congestion, epistaxis, hemoptysis, dysphagia, pain, dizziness, syncope, and lethargy. Bending forward or lying down may aggravate the symptoms. The characteristic physical findings are dilated neck veins; an increased number of collateral veins covering the anterior chest wall; cyanosis; and edema of the face, arms, and chest. Facial swelling and plethora are typically exacerbated when the patient is

1	an increased number of collateral veins covering the anterior chest wall; cyanosis; and edema of the face, arms, and chest. Facial swelling and plethora are typically exacerbated when the patient is supine. More severe cases include proptosis, glossal and laryngeal edema, and obtundation. The clinical picture is milder if the obstruction is located above the azygos vein. Symptoms are usually progressive, but in some cases, they may improve as collateral circulation develops. Signs and symptoms of cerebral and/or laryngeal edema, though rare, are associated with a poorer prognosis and require urgent evaluation. Seizures are more likely related to brain metastases than to cerebral edema from venous occlusion. Patients with small-cell lung cancer and SVCS have a higher incidence of brain metastases than those without SVCS. Cardiorespiratory symptoms at rest, particularly with positional changes, suggest significant airway and vascular obstruction and limited physiologic reserve. Cardiac

1	than those without SVCS. Cardiorespiratory symptoms at rest, particularly with positional changes, suggest significant airway and vascular obstruction and limited physiologic reserve. Cardiac arrest or respiratory failure can occur, particularly in patients receiving sedatives or undergoing general anesthesia. Rarely, esophageal varices may develop. These are “downhill” varices based on the direction of blood flow from cephalad to caudad (in contrast to “uphill” varices associated with caudad to cephalad flow from portal hypertension). If the obstruction to the SVC is proximal to the azygous vein, varices develop in the upper one-third of the esophagus. If the obstruction involves or is distal to the azygous vein, varices occur in the entire length of the esophagus. Variceal bleeding may be a late complication of chronic SVCS. Superior vena cava obstruction may lead to bilateral breast edema with bilateral enlarged breast. Unilateral breast dilatation may be seen as a consequence of

1	be a late complication of chronic SVCS. Superior vena cava obstruction may lead to bilateral breast edema with bilateral enlarged breast. Unilateral breast dilatation may be seen as a consequence of axillary or subclavian vein blockage. The diagnosis of SVCS is a clinical one. The most significant chest radiographic finding is widening of the superior mediastinum, most commonly on the right side. Pleural effusion occurs in only 25% of patients, often on the right side. The majority of these effusions are exudative and occasionally chylous. However, a normal chest radio-graph is still compatible with the diagnosis if other characteristic findings are present. Computed tomography (CT) provides the most reliable view of the mediastinal anatomy. The diagnosis of SVCS requires diminished or absent opacification of central venous structures with prominent collateral venous circulation. Magnetic resonance imaging (MRI) has no advantages over CT. Invasive procedures, including bronchoscopy,

1	opacification of central venous structures with prominent collateral venous circulation. Magnetic resonance imaging (MRI) has no advantages over CT. Invasive procedures, including bronchoscopy, percutaneous needle biopsy, mediastinoscopy, and even thoracotomy, can be performed by a skilled clinician without any major risk of bleeding. Endobronchial or esophageal ultrasound-guided needle aspiration may establish the diagnosis safely. For patients with a known cancer, a detailed workup usually is not necessary, and appropriate treatment may be started after obtaining a CT scan of the thorax. For those with no history of malignancy, a detailed evaluation is essential to rule out benign causes and determine a specific diagnosis to direct the appropriate therapy.

1	The one potentially life-threatening complication of a superior mediastinal mass is tracheal obstruction. Upper airway obstruction demands emergent therapy. Diuretics with a low-salt diet, head elevation, and oxygen may produce temporary symptomatic relief. Glucocorticoids may be useful at shrinking lymphoma masses; they are of no benefit in patients with lung cancer.

1	Radiation therapy is the primary treatment for SVCS caused by non-small-cell lung cancer and other metastatic solid tumors. Chemotherapy is effective when the underlying cancer is small-cell carcinoma of the lung, lymphoma, or germ cell tumor. SVCS recurs in 10–30% of patients; it may be palliated with the use of intravascular self-expanding stents (Fig. 331-1). Early stenting may be necessary in patients with severe symptoms; however, the prompt increase in venous return after stenting may precipitate heart failure and pulmonary edema. Other complications of stent placement include hematoma at the insertion site, SVC perforation, stent migration in the right ventricle, stent fracture, and pulmonary embolism. Surgery may provide immediate relief for patients in whom a benign process is the cause.

1	Clinical improvement occurs in most patients, although this improvement may be due to the development of adequate collateral circulation. The mortality associated with SVCS does not relate to caval obstruction but rather to the underlying cause. The use of long-term central venous catheters has become common practice in patients with cancer. Major vessel thrombosis may occur. In these cases, catheter removal should be combined with anticoagulation to prevent embolization. SVCS in this setting, if detected early, can be treated by fibrinolytic therapy without sacrificing the catheter. The routine use of low-dose warfarin or low-molecularweight heparin to prevent thrombosis related to permanent central venous access catheters in cancer patients is not recommended.

1	Malignant pericardial disease is found at autopsy in 5–10% of patients with cancer, most frequently with lung cancer, breast cancer, leukemias, and lymphomas. Cardiac tamponade as the initial presentation of extrathoracic malignancy is rare. The origin is not malignancy in about 50% of cancer patients with symptomatic pericardial disease, but it can be related to irradiation, drug-induced pericarditis, hypothyroidism, idiopathic pericarditis, infection, or autoimmune diseases. Two types of radiation pericarditis occur: an acute inflammatory, effusive pericarditis occurring within months of irradiation, which usually resolves spontaneously, and a chronic effusive pericarditis that may appear up to 20 years after radiation therapy and is accompanied by a thickened pericardium.

1	Most patients with pericardial metastasis are asymptomatic. However, the common symptoms are dyspnea, cough, chest pain, orthopnea, and weakness. Pleural effusion, sinus tachycardia, jugular venous distention, hepatomegaly, peripheral edema, and cyanosis are the most frequent physical findings. Relatively specific diagnostic findings, such as paradoxical pulse, diminished heart sounds, pulsus alternans (pulse waves alternating between those of greater and lesser amplitude with successive beats), and friction rub are less common than with nonmalignant pericardial disease. Chest radiographs and electrocardiogram (ECG) reveal abnormalities in 90% of patients, but half of these abnormalities are nonspecific. Echocardiography is the most helpful diagnostic test. Pericardial fluid may be serous, serosanguineous, or hemorrhagic, and cytologic examination of pericardial fluid is diagnostic in most patients. Measurements of tumor markers in the pericardial fluid are not helpful in the

1	serous, serosanguineous, or hemorrhagic, and cytologic examination of pericardial fluid is diagnostic in most patients. Measurements of tumor markers in the pericardial fluid are not helpful in the diagnosis of malignant pericardial fluid. Pericardioscopy (not widely available) with targeted pericardial and epicardial biopsy may differentiate neoplastic and benign pericardial disease. A combination of cytology, pericardial and epicardial biopsy, and guided pericardioscopy gives the best diagnostic yield. CT scan findings of irregular pericardial thickening and mediastinal lymphadenopathy suggest this is a malignant pericardial effusion. Cancer patients with pericardial effusion containing malignant cells on cytology have a very poor survival, about 7 weeks.

1	Pericardiocentesis with or without the introduction of sclerosing agents, the creation of a pericardial window, complete pericardial stripping, cardiac irradiation, or systemic chemotherapy are effective treatments. Acute pericardial tamponade with life-threatening hemodynamic instability requires immediate drainage of fluid. This FIGURE 331–1 Superior vena cava syndrome (SVCS). A. Chest radiographs of a 59-year-old man with recurrent SVCS caused by non-small-cell lung cancer showing right paratracheal mass with right pleural effusion. B. Computed tomography of same patient demonstrating obstruction of the superior vena cava with thrombosis (arrow) by the lung cancer (square) and collaterals (arrowheads). C. Balloon angioplasty (arrowhead) with Wallstent (arrow) in same patient.

1	can be quickly achieved by pericardiocentesis. The recurrence rate 1789 after percutaneous catheter drainage is about 20%. Sclerotherapy (pericardial instillation of bleomycin, mitomycin C, or tetracycline) may decrease recurrences. Alternatively, subxiphoid pericardiotomy can be performed in 45 min under local anesthesia. Thoracoscopic pericardial fenestration can be employed for benign causes; however, 60% of malignant pericardial effusions recur after this procedure. In a subset of patients, drainage of the pericardial effusion is paradoxically followed by worsening hemodynamic instability. This so-called “postoperative low cardiac output syndrome” occurs in up to 10% of patients undergoing surgical drainage and carries poor short-term survival.

1	Intestinal obstruction and reobstruction are common problems in patients with advanced cancer, particularly colorectal or ovarian carcinoma. However, other cancers, such as lung or breast cancer and melanoma, can metastasize within the abdomen, leading to intestinal obstruction. Metastatic disease from colorectal, ovarian, pancreatic, gastric, and occasionally breast cancer can lead to peritoneal carcinomatosis, with infiltration of the omentum and peritoneal surface, thus limiting bowel motility. Typically, obstruction occurs at multiple sites in peritoneal carcinomatosis. Melanoma has a predilection to involve the small bowel; this involvement may be isolated, and resection may result in prolonged survival. Intestinal pseudoobstruction is caused by infiltration of the mesentery or bowel muscle by tumor, involvement of the celiac plexus, or paraneoplastic neuropathy in patients with small-cell lung cancer. Paraneoplastic neuropathy is associated with IgG antibodies reactive to

1	bowel muscle by tumor, involvement of the celiac plexus, or paraneoplastic neuropathy in patients with small-cell lung cancer. Paraneoplastic neuropathy is associated with IgG antibodies reactive to neurons of the myenteric and submucosal plexuses of the jejunum and stomach. Ovarian cancer can lead to authentic luminal obstruction or to pseudoobstruction that results when circumferential invasion of a bowel segment arrests the forward progression of peristaltic contractions.

1	The onset of obstruction is usually insidious. Pain is the most common symptom and is usually colicky in nature. Pain can also be due to abdominal distention, tumor masses, or hepatomegaly. Vomiting can be intermittent or continuous. Patients with complete obstruction usually have constipation. Physical examination may reveal abdominal distention with tympany, ascites, visible peristalsis, high-pitched bowel sounds, and tumor masses. Erect plain abdominal films may reveal multiple air-fluid levels and dilation of the small or large bowel. Acute cecal dilation to >12–14 cm is considered a surgical emergency because of the high likelihood of rupture. CT scan is useful in defining the extent of disease and the exact nature of the obstruction and differentiating benign from malignant causes of obstruction in patients who have undergone surgery for malignancy. Malignant obstruction is suggested by a mass at the site of obstruction or prior surgery, adenopathy, or an abrupt transition zone

1	obstruction in patients who have undergone surgery for malignancy. Malignant obstruction is suggested by a mass at the site of obstruction or prior surgery, adenopathy, or an abrupt transition zone and irregular bowel thickening at the obstruction site. Benign obstruction is more likely when CT shows mesenteric vascular changes, a large volume of ascites, or a smooth transition zone and smooth bowel thickening at the obstruction site. In challenging patients with obstructive symptoms, particularly low-grade small-bowel obstruction (SBO), CT enteroclysis often can help establish the diagnosis by providing distention of small-bowel loops. In this technique, water-soluble contrast is infused through a nasoenteric tube into the duodenum or proximal small bowel followed by CT images. The prognosis for the patient with cancer who develops intestinal obstruction is poor; median survival is 3–4 months. About 25–30% of patients are found to have intestinal obstruction due to causes other than

1	for the patient with cancer who develops intestinal obstruction is poor; median survival is 3–4 months. About 25–30% of patients are found to have intestinal obstruction due to causes other than cancer. Adhesions from previous operations are a common benign cause. Ileus induced by vinca alkaloids, narcotics, or other drugs is another reversible cause.

1	The management of intestinal obstruction in patients with advanced malignancy depends on the extent of the underlying malignancy, options for further antineoplastic therapy, estimated life expectancy, 1790 the functional status of the major organs, and the extent of the obstruction. The initial management should include surgical evaluation. Operation is not always successful and may lead to further complications with a substantial mortality rate (10–20%). Laparoscopy can diagnose and treat malignant bowel obstruction in some cases. Self-expanding metal stents placed in the gastric outlet, duodenum, proximal jejunum, colon, or rectum may palliate obstructive symptoms at those sites without major surgery. Patients known to have advanced intraabdominal malignancy should receive a prolonged course of conservative management, including nasogastric decompression. Percutaneous endoscopic or surgical gastrostomy tube placement is an option for palliation of nausea and vomiting, the so-called

1	of conservative management, including nasogastric decompression. Percutaneous endoscopic or surgical gastrostomy tube placement is an option for palliation of nausea and vomiting, the so-called “venting gastrostomy.” Treatment with antiemetics, antispasmodics, and analgesics may allow patients to remain outside the hospital. Octreotide may relieve obstructive symptoms through its inhibitory effect on gastrointestinal secretion. Glucocorticoids have anti-inflammatory effects and may help the resolution of bowel obstruction. They also have antiemetic effects.

1	Urinary obstruction may occur in patients with prostatic or gynecologic malignancies, particularly cervical carcinoma; metastatic disease from other primary sites such as carcinomas of the breast, stomach, lung, colon, and pancreas; or lymphomas. Radiation therapy to pelvic tumors may cause fibrosis and subsequent ureteral obstruction. Bladder outlet obstruction is usually due to prostate and cervical cancers and may lead to bilateral hydronephrosis and renal failure.

1	Flank pain is the most common symptom. Persistent urinary tract infection, persistent proteinuria, or hematuria in patients with cancer should raise suspicion of ureteral obstruction. Total anuria and/or anuria alternating with polyuria may occur. A slow, continuous rise in the serum creatinine level necessitates immediate evaluation. Renal ultrasound is the safest and cheapest way to identify hydronephrosis. The function of an obstructed kidney can be evaluated by a nuclear scan. CT scan can reveal the point of obstruction and identify a retro-peritoneal mass or adenopathy.

1	Obstruction associated with flank pain, sepsis, or fistula formation is an indication for immediate palliative urinary diversion. Internal ureteral stents can be placed under local anesthesia. Percutaneous nephrostomy offers an alternative approach for drainage. The placement of a nephrostomy is associated with a significant rate of pyelonephritis. In the case of bladder outlet obstruction due to malignancy, a suprapubic cystostomy can be used for urinary drainage. An aggressive intervention with invasive approaches to improve the obstruction should be weighed against the likelihood of antitumor response, and the ability to reverse renal insufficiency should be evaluated.

1	This common clinical problem can be caused by a primary carcinoma arising in the pancreas, ampulla of Vater, bile duct, or liver or by metastatic disease to the periductal lymph nodes or liver parenchyma. The most common metastatic tumors causing biliary obstruction are gastric, colon, breast, and lung cancers. Jaundice, light-colored stools, dark urine, pruritus, and weight loss due to malabsorption are usual symptoms. Pain and secondary infection are uncommon in malignant biliary obstruction. Ultrasound, CT scan, or percutaneous transhepatic or endoscopic retrograde cholangiography will identify the site and nature of the biliary obstruction.

1	Palliative intervention is indicated only in patients with disabling pruritus resistant to medical treatment, severe malabsorption, or infection. Stenting under radiographic control, surgical bypass, or radiation therapy with or without chemotherapy may alleviate the obstruction. The choice of therapy should be based on the site of obstruction (proximal vs distal), the type of tumor (sensitive to radiotherapy, chemotherapy, or neither), and the general condition of the patient. In the absence of pruritus, biliary obstruction may be a largely asymptomatic cause of death.

1	Malignant spinal cord compression (MSCC) is defined as compression of the spinal cord and/or cauda equina by an extradural tumor mass. The minimum radiologic evidence for cord compression is indentation of the theca at the level of clinical features. Spinal cord compression occurs in 5–10% of patients with cancer. Epidural tumor is the first manifestation of malignancy in about 10% of patients. The underlying cancer is usually identified during the initial evaluation; lung cancer is the most common cause of MSCC.

1	Metastatic tumor involves the vertebral column more often than any other part of the bony skeleton. Lung, breast, and prostate cancer are the most frequent offenders. Multiple myeloma also has a high incidence of spine involvement. Lymphomas, melanoma, renal cell cancer, and genitourinary cancers also cause cord compression. The thoracic spine is the most common site (70%), followed by the lumbosacral spine (20%) and the cervical spine (10%). Involvement of multiple sites is most frequent in patients with breast and prostate carcinoma. Cord injury develops when metastases to the vertebral body or pedicle enlarge and compress the underlying dura. Another cause of cord compression is direct extension of a paravertebral lesion through the intervertebral foramen. These cases usually involve a lymphoma, myeloma, or pediatric neoplasm. Parenchymal spinal cord metastasis due to hematogenous spread is rare. Intramedullary metastases can be seen in lung cancer, breast cancer, renal cancer,

1	a lymphoma, myeloma, or pediatric neoplasm. Parenchymal spinal cord metastasis due to hematogenous spread is rare. Intramedullary metastases can be seen in lung cancer, breast cancer, renal cancer, melanoma, and lymphoma and are frequently associated with brain metastases and leptomeningeal disease.

1	Expanding extradural tumors induce injury through several mechanisms. Obstruction of the epidural venous plexus leads to edema. Local production of inflammatory cytokines enhances blood flow and edema formation. Compression compromises blood flow, leading to ischemia. Production of vascular endothelial growth factor is associated with spinal cord hypoxia and has been implicated as a potential cause of damage after spinal cord injury.

1	The most common initial symptom in patients with spinal cord compression is localized back pain and tenderness due to involvement of vertebrae by tumor. Pain is usually present for days or months before other neurologic findings appear. It is exacerbated by movement and by coughing or sneezing. It can be differentiated from the pain of disk disease by the fact that it worsens when the patient is supine. Radicular pain is less common than localized back pain and usually develops later. Radicular pain in the cervical or lumbosacral areas may be unilateral or bilateral. Radicular pain from the thoracic roots is often bilateral and is described by patients as a feeling of tight, band-like constriction around the thorax and abdomen. Typical cervical radicular pain radiates down the arm; in the lumbar region, the radiation is down the legs. Lhermitte’s sign, a tingling or electric sensation down the back and upper and lower limbs upon flexing or extending the neck, may be an early sign of

1	lumbar region, the radiation is down the legs. Lhermitte’s sign, a tingling or electric sensation down the back and upper and lower limbs upon flexing or extending the neck, may be an early sign of cord compression. Loss of bowel or bladder control may be the presenting symptom but usually occurs late in the course. Occasionally patients present with ataxia of gait without motor and sensory involvement due to involvement of the spinocerebellar tract.

1	On physical examination, pain induced by straight leg raising, neck flexion, or vertebral percussion may help to determine the level of cord compression. Patients develop numbness and paresthesias in the extremities or trunk. Loss of sensibility to pinprick is as common as loss of sensibility to vibration or position. The upper limit of the zone of sensory loss is often one or two vertebrae below the site of compression. Motor findings include weakness, spasticity, and abnormal muscle stretching. An extensor plantar reflex reflects significant compression. Deep tendon reflexes may be brisk. Motor and sensory loss usually precedes sphincter disturbance. Patients with autonomic dysfunction may present with decreased anal tonus, decreased perineal sensibility, and a distended bladder. The absence of the anal wink reflex or the bulbocavernosus reflex confirms cord involvement. In doubtful cases, evaluation of postvoiding urinary residual volume can be helpful. A residual volume of >150 mL

1	of the anal wink reflex or the bulbocavernosus reflex confirms cord involvement. In doubtful cases, evaluation of postvoiding urinary residual volume can be helpful. A residual volume of >150 mL suggests bladder dysfunction. Autonomic dysfunction is an unfavorable prognostic factor. Patients with progressive neurologic symptoms should have frequent neurologic examinations and rapid therapeutic intervention. Other illnesses that may mimic cord compression include osteoporotic vertebral collapse, disk disease, pyogenic abscess or vertebral tuberculosis, radiation myelopathy, neoplastic leptomeningitis, benign tumors, epidural hematoma, and spinal lipomatosis.

1	Cauda equina syndrome is characterized by low back pain; diminished sensation over the buttocks, posterior-superior thighs, and perineal area in a saddle distribution; rectal and bladder dysfunction; sexual impotence; absent bulbocavernous, patellar, and Achilles’ reflexes; and variable amount of lower-extremity weakness. This reflects compression of nerve roots as they form the cauda equina after leaving the spinal cord. The majority of cauda equine tumors are primary tumors of glial or nerve sheath origin; metastases are very rare.

1	Patients with cancer who develop back pain should be evaluated for spinal cord compression as quickly as possible (Fig. 331-2). Treatment is more often successful in patients who are ambulatory and still have sphincter control at the time treatment is initiated. Patients should have a neurologic examination and plain films of the spine. Those whose physical examination suggests cord compression should receive dexamethasone (6 mg intravenously every 6 h), starting immediately.

1	Erosion of the pedicles (the “winking owl” sign) is the earliest radiologic finding of vertebral tumor. Other radiographic changes include increased intrapedicular distance, vertebral destruction, lytic or sclerotic lesions, scalloped vertebral bodies, and vertebral body collapse. Vertebral collapse is not a reliable indicator of the presence of tumor; about 20% of cases of vertebral collapse, particularly those in older patients and postmenopausal women, are due not to cancer but 1791 to osteoporosis. Also, a normal appearance on plain films of the spine does not exclude the diagnosis of cancer. The role of bone scans in the detection of cord compression is not clear; this method is sensitive but less specific than spinal radiography.

1	The full-length image of the cord provided by MRI is the imaging procedure of choice. Multiple epidural metastases are noted in 25% of patients with cord compression, and their presence influences treatment plans. On T1-weighted images, good contrast is noted between the cord, cerebrospinal fluid, and extradural lesions. Owing to its sensitivity in demonstrating the replacement of bone marrow by tumor, MRI can show which parts of a vertebra are involved by tumor. MRI also visualizes intraspinal extradural masses compressing the cord. T2-weighted images are most useful for the demonstration of intramedullary pathology. Gadolinium-enhanced MRI can help to delineate intramedullary disease. MRI is as good as or better than myelography plus postmyelogram CT scan in detecting metastatic epidural disease with cord compression. Myelography should be reserved for patients who have poor MRIs or who cannot undergo MRI promptly. CT scan in conjunction with myelography enhances the detection of

1	disease with cord compression. Myelography should be reserved for patients who have poor MRIs or who cannot undergo MRI promptly. CT scan in conjunction with myelography enhances the detection of small areas of spinal destruction.

1	In patients with cord compression and an unknown primary tumor, a simple workup including chest radiography, mammography, measurement of prostate-specific antigen, and abdominal CT usually reveals the underlying malignancy. The treatment of patients with spinal cord compression is aimed at relief of pain and restoration/preservation of neurologic function (Fig. 331-2). Management of MSCC requires a multidisciplinary approach. CHAPTER 331 Symptomatic therapy Back pain Neurologic exam Plain spine x-ray High-dose dexamethasone MRI of spine Bone metastases but no epidural metastases Symptomatic therapy ±radiation therapy Epidural metastases No metastases Surgery followed by radiation therapy or radiation therapy alone Symptomatic therapy Pain crescendo pattern Lhermitte’s sign Pain aggravated with cough, Valsalva, and recumbency Abnormal Normal Normal Suspicious for myelopathy FIGURE 331-2 Management of cancer patients with back pain.

1	1792 Radiation therapy plus glucocorticoids is generally the initial treatment of choice for most patients with spinal cord compression. Up to 75% of patients treated when still ambulatory remain ambulatory, but only 10% of patients with paraplegia recover walking capacity. Indications for surgical intervention include unknown etiology, failure of radiation therapy, a radioresistant tumor type (e.g., melanoma or renal cell cancer), pathologic fracture dislocation, and rapidly evolving neurologic symptoms. Laminectomy is done for tissue diagnosis and for the removal of posteriorly localized epidural deposits in the absence of vertebral body disease. Because most cases of epidural spinal cord compression are due to anterior or anterolateral extradural disease, resection of the anterior vertebral body along with the tumor, followed by spinal stabilization, has achieved good results. A randomized trial showed that patients who underwent an operation followed by radiotherapy (within 14

1	body along with the tumor, followed by spinal stabilization, has achieved good results. A randomized trial showed that patients who underwent an operation followed by radiotherapy (within 14 days) retained the ability to walk significantly longer than those treated with radiotherapy alone. Surgically treated patients also maintained continence and neurologic function significantly longer than patients in the radiation group. The length of survival was not significantly different in the two groups, although there was a trend toward longer survival in the surgery group. The study drew some criticism for the poorer than expected results in the patients who did not go to surgery. The benefit of surgery over radiotherapy decreased in patients over age 65 years. However, patients should be evaluated for surgery if they are expected to survive longer than 3 months. Conventional radiotherapy has a role after surgery. Chemotherapy may have a role in patients with chemosensitive tumors who have

1	for surgery if they are expected to survive longer than 3 months. Conventional radiotherapy has a role after surgery. Chemotherapy may have a role in patients with chemosensitive tumors who have had prior radiotherapy to the same region and who are not candidates for surgery. Most patients with prostate cancer who develop cord compression have already had hormonal therapy; however, for those who have not, androgen deprivation is combined with surgery and radiotherapy. Patients who previously received radiotherapy for MSCC with an in-field tumor progression can be treated with reirradiation if they are not surgical candidates. Patients with metastatic vertebral tumors may benefit from percutaneous vertebroplasty or kyphoplasty, the injection of acrylic cement into a collapsed vertebra to stabilize the fracture. Pain palliation is common, and local antitumor effects have been noted. Cement leakage may cause symptoms in about 10% of patients. Bisphosphonates may be helpful in prevention

1	the fracture. Pain palliation is common, and local antitumor effects have been noted. Cement leakage may cause symptoms in about 10% of patients. Bisphosphonates may be helpful in prevention of SCC in patients with bony involvement. The histology of the tumor is an important determinant of both recovery and survival. Rapid onset and progression of signs and symptoms are poor prognostic features.

1	About 25% of patients with cancer die with intracranial metastases. The cancers that most often metastasize to the brain are lung and breast cancers and melanoma. Brain metastases often occur in the presence of systemic disease, and they frequently cause major symptoms, disability, and early death. The initial presentation of brain metastases from a previously unknown primary cancer is common. Lung cancer is most commonly the primary malignancy. Chest/ abdomen CT scans and brain MRI as the initial diagnostic studies can identify a biopsy site in most patients.

1	The signs and symptoms of a metastatic brain tumor are similar to those of other intracranial expanding lesions: headache, nausea, vomiting, behavioral changes, seizures, and focal, progressive neurologic changes. Occasionally the onset is abrupt, resembling a stroke, with the sudden appearance of headache, nausea, vomiting, and neurologic deficits. This picture is usually due to hemorrhage into the metastasis. Melanoma, germ cell tumors, and renal cell cancers have a particularly high incidence of intracranial bleeding. The tumor mass and surrounding edema may cause obstruction of the circulation of cerebrospinal fluid, with resulting hydrocephalus. Patients with increased intracranial pressure may have papilledema with visual disturbances and neck stiffness. As the mass enlarges, brain tissue may be displaced through the fixed cranial openings, producing various herniation syndromes.

1	CT scan and MRI are equally effective in the diagnosis of brain metastases. CT scan with contrast should be used as a screening procedure. The CT scan shows brain metastases as multiple enhancing lesions of various sizes with surrounding areas of low-density edema. If a single lesion or no metastases are visualized by contrast-enhanced CT, MRI of the brain should be performed. Gadolinium-enhanced MRI is more sensitive than CT at revealing meningeal involvement and small lesions, particularly in the brainstem or cerebellum. Intracranial hypertension (“pseudotumor cerebri”) secondary to tretinoin therapy has been reported.

1	Dexamethasone is the best initial treatment for all symptomatic patients with brain metastases. Patients with multiple lesions should usually receive whole-brain radiation. Patients with a single brain metastasis and with controlled extracranial disease may be treated with surgical excision followed by whole-brain radiation therapy, especially if they are younger than 60 years. Radioresistant tumors should be resected if possible. Stereotactic radiosurgery (SRS) is recommended in patients with a limited number of brain metastases (one to four) who have stable, systemic disease or reasonable systemic treatment options and for patients who have a small number of metastatic lesions in whom whole-brain radiation therapy has failed. With a gamma knife or linear accelerator, multiple small, well-collimated beams of ionizing radiation destroy lesions seen on MRI. Some patients with increased intracranial pressure associated with hydrocephalus may benefit from shunt placement. If neurologic

1	beams of ionizing radiation destroy lesions seen on MRI. Some patients with increased intracranial pressure associated with hydrocephalus may benefit from shunt placement. If neurologic deterioration is not reversed with medical therapy, ventriculotomy to remove cerebrospinal fluid (CSF) or craniotomy to remove tumors or hematomas may be necessary.

1	Tumor involving the leptomeninges is a complication of both primary central nervous system (CNS) tumors and tumors that metastasize to the CNS. The incidence is estimated at 3–8% of patients with cancer. Melanoma, breast and lung cancer, lymphoma (including AIDS-associated), and acute leukemia are the most common causes. Synchronous intraparenchymal brain metastases are evident in 11–31% of patients with neoplastic meningitis. Leptomeningeal seeding is frequent in patients undergoing resection of brain metastases or receiving stereotactic radiotherapy for brain metastases. Patients typically present with multifocal neurologic signs and symptoms, including headache, gait abnormality, mental changes, nausea, vomiting, seizures, back or radicular pain, and limb weakness. Signs include cranial nerve palsies, extremity weakness, paresthesia, and decreased deep tendon reflexes.

1	Diagnosis is made by demonstrating malignant cells in the CSF; however, up to 40% of patients may have false-negative CSF cytology. An elevated CSF protein level is nearly always present (except in HTLV-1–associated adult T cell leukemia). Patients with neurologic signs and symptoms consistent with neoplastic meningitis who have a negative CSF cytology but an elevated CSF protein level should have the spinal tap repeated at least three times for cytologic examination before the diagnosis is rejected. MRI findings suggestive of neoplastic meningitis include leptomeningeal, subependymal, dural, or cranial nerve enhancement; superficial cerebral lesions; intradural nodules; and communicating hydrocephalus. Spinal cord imaging by MRI is a necessary component of the evaluation of nonleukemia neoplastic meningitis because ~20% of patients have cord abnormalities, including intradural enhancing nodules that are diagnostic for leptomeningeal involvement. Cauda equina lesions are common, but

1	neoplastic meningitis because ~20% of patients have cord abnormalities, including intradural enhancing nodules that are diagnostic for leptomeningeal involvement. Cauda equina lesions are common, but lesions may be seen anywhere in the spinal canal. The value of MRI for the diagnosis of leptomeningeal disease is limited in patients with hematopoietic malignancy. Radiolabeled CSF flow studies are abnormal in up to 70% of patients with neoplastic meningitis; ventricular outlet obstruction, abnormal flow in the spinal canal, or impaired flow over the cerebral convexities may affect distribution of intrathecal chemotherapy, resulting in decreased efficacy or increased toxicity. Radiation therapy may correct CSF flow abnormalities before use of intrathecal chemotherapy. Neoplastic meningitis can also lead to intracranial hypertension and hydrocephalus. Placement of a ventriculoperitoneal shunt may effectively palliate symptoms in these patients.

1	The development of neoplastic meningitis usually occurs in the setting of uncontrolled cancer outside the CNS; thus, prognosis is poor (median survival 10–12 weeks). However, treatment of the neoplastic meningitis may successfully alleviate symptoms and control the CNS spread. Intrathecal chemotherapy, usually methotrexate, cytarabine, or thiotepa, is delivered by lumbar puncture or by an intraventricular reservoir (Ommaya). An extended-release preparation of cytarabine (Depocyte) has a longer half-life and is more effective than other formulations. Among solid tumors, breast cancer responds best to therapy. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) may be effective in non-small-cell lung cancer patients with EGFR mutations and leptomeningeal involvement. Patients with neoplastic meningitis from either acute leukemia or lymphoma may be cured of their CNS disease if the systemic disease can be eliminated.

1	Seizures occurring in a patient with cancer can be caused by the tumor itself, by metabolic disturbances, by radiation injury, by cerebral infarctions, by chemotherapy-related encephalopathies, or by CNS infections. Metastatic disease to the CNS is the most common cause of seizures in patients with cancer. However, seizures occur more frequently in primary brain tumors than in metastatic brain lesions. Seizures are a presenting symptom of CNS metastasis in 6–29% of cases. Approximately 10% of patients with CNS metastasis eventually develop seizures. Tumors that affect the frontal, temporal, and parietal lobes are more commonly associated with seizures than are occipital lesions. The presence of frontal lesions correlates with early seizures, and the presence of hemispheric symptoms increases the risk for late seizures. Both early and late seizures are uncommon in patients with posterior fossa and sellar lesions. Seizures are common in patients with CNS metastases from melanoma and

1	the risk for late seizures. Both early and late seizures are uncommon in patients with posterior fossa and sellar lesions. Seizures are common in patients with CNS metastases from melanoma and low-grade primary brain tumors. Very rarely, cytotoxic drugs such as etoposide, busulfan, ifosfamide, and chlorambucil cause seizures. Another cause of seizures related to drug therapy is reversible posterior leukoencephalopathy syndrome (RPLS). RPLS is associated rarely with administration of cisplatin, 5-fluorouracil, bleomycin, vinblastine, vincristine, etoposide, paclitaxel, ifosfamide, cyclophosphamide, doxorubicin, cytarabine, methotrexate, oxaliplatin, cyclosporine, tacrolimus, and vascular endothelial growth factor inhibitors including bevacizumab, aflibercept, sunitinib, sorafenib, pazopanib, and axitinib. RPLS occurs in patients undergoing allogeneic bone marrow or solid-organ transplantation. RPLS is characterized by headache, altered consciousness, generalized seizures, visual

1	and axitinib. RPLS occurs in patients undergoing allogeneic bone marrow or solid-organ transplantation. RPLS is characterized by headache, altered consciousness, generalized seizures, visual disturbances, hypertension, and posterior cerebral white matter vasogenic edema on CT/MRI. Seizures may begin focally but are typically generalized.

1	Patients in whom seizures due to CNS metastases have been demonstrated should receive anticonvulsive treatment with phenytoin or levetiracetam. If this is not effective, valproic acid can be added. Prophylactic anticonvulsant therapy is not recommended. In postcraniotomy patients, prophylactic antiepileptic drugs should be withdrawn during the first week after surgery. Most antiseizure medications including phenytoin induce cytochrome P450 (CYP450), which alters the metabolism of many antitumor agents, including irinotecan, taxanes, and etoposide as well as molecular 1793 targeted agents, including imatinib, gefitinib, erlotinib, tipifarnib, sorafenib, sunitinib, temsirolimus, everolimus, and vemurafenib. Levetiracetam and topiramate are anticonvulsant agents not metabolized by the hepatic CYP450 system and do not alter the metabolism of antitumor agents. They have become the preferred drugs. Surgical resection and other antitumor treatments such as radiotherapy and chemotherapy may

1	CYP450 system and do not alter the metabolism of antitumor agents. They have become the preferred drugs. Surgical resection and other antitumor treatments such as radiotherapy and chemotherapy may improve seizure control.

1	Hyperleukocytosis and the leukostasis syndrome associated with it is a potentially fatal complication of acute leukemia (particularly myeloid leukemia) that can occur when the peripheral blast cell count is >100,000/mL. The frequency of hyperleukocytosis is 5–13% in acute myeloid leukemia (AML) and 10–30% in acute lymphoid leukemia; however, leukostasis is rare in lymphoid leukemia. At such high blast cell counts, blood viscosity is increased, blood flow is slowed by aggregates of tumor cells, and the primitive myeloid leukemic cells are capable of invading through the endothelium and causing hemorrhage. Brain and lung are most commonly affected. Patients with brain leukostasis may experience stupor, headache, dizziness, tinnitus, visual disturbances, ataxia, confusion, coma, or sudden death. On examination, papilledema, retinal vein distension, retinal hemorrhages, and focal deficit may be present. Administration of 600 cGy of whole-brain irradiation can protect against this

1	death. On examination, papilledema, retinal vein distension, retinal hemorrhages, and focal deficit may be present. Administration of 600 cGy of whole-brain irradiation can protect against this complication and can be followed by rapid institution of antileukemic therapy. Hydroxyurea, 3–5 g, can rapidly reduce a high blast cell count while the accurate diagnostic workup is in progress. Pulmonary leukostasis may present as respiratory distress and hypoxemia, and progress to respiratory failure. Chest radiographs may be normal but usually show interstitial or alveolar infiltrates. Hyperleukocytosis rarely may cause acute leg ischemia, renal vein thrombosis, myocardial ischemia, bowel infraction, and priapism. Arterial blood gas results should be interpreted cautiously. Rapid consumption of plasma oxygen by the markedly increased number of white blood cells can cause spuriously low arterial oxygen tension. Pulse oximetry is the most accurate way of assessing oxygenation in patients with

1	plasma oxygen by the markedly increased number of white blood cells can cause spuriously low arterial oxygen tension. Pulse oximetry is the most accurate way of assessing oxygenation in patients with hyperleukocytosis. Leukapheresis may be helpful in decreasing circulating blast counts. Treatment of the leukemia can result in pulmonary hemorrhage from lysis of blasts in the lung, called leukemic cell lysis pneumopathy. Intravascular volume depletion and unnecessary blood transfusions may increase blood viscosity and worsen the leukostasis syndrome. Leukostasis is very rarely a feature of the high white cell counts associated with chronic lymphoid or chronic myeloid leukemia.

1	When acute promyelocytic leukemia is treated with differentiating agents like tretinoin and arsenic trioxide, cerebral or pulmonary leukostasis may occur as tumor cells differentiate into mature neutrophils. This complication can be largely avoided by using cytotoxic chemotherapy or arsenic together with the differentiating agents.

1	Hemoptysis may be caused by nonmalignant conditions, but lung cancer accounts for a large proportion of cases. Up to 20% of patients with lung cancer have hemoptysis some time in their course. Endobronchial metastases from carcinoid tumors, breast cancer, colon cancer, kidney cancer, and melanoma may also cause hemoptysis. The volume of bleeding is often difficult to gauge. Massive hemoptysis is defined as >200–600 mL of blood produced in 24 h. However, any hemoptysis should be considered massive if it threatens life. When respiratory difficulty occurs, hemoptysis should be treated emergently. The first priorities are to maintain the airway, optimize oxygenation, and stabilize the hemodynamic status. If the bleeding side is known, the patient should be placed in a lateral decubitus position, with the bleeding side down to prevent aspiration into the unaffected lung, and given supplemental oxygen. If large-volume bleeding continues or the airway is compromised, the patient should be

1	with the bleeding side down to prevent aspiration into the unaffected lung, and given supplemental oxygen. If large-volume bleeding continues or the airway is compromised, the patient should be intubated and undergo emergency bronchoscopy. If the site of bleeding is detected, either the patient undergoes a definitive surgical procedure or the lesion is treated with a neodymium:yttrium-aluminum-garnet (Nd:YAG) laser, argon plasma coagulation, or electrocautery. In stable 1794 patients, multidetector CT angiography delineates bronchial and non-bronchial systemic arteries and identifies the source of bleeding and underlying pathology with high sensitivity. Massive hemoptysis usually originates from the high-pressure bronchial circulation. Bronchial artery embolization is considered a first-line definite procedure for managing hemoptysis. Bronchial artery embolization may control brisk bleeding in 75–90% of patients, permitting the definitive surgical procedure to be done more safely.

1	definite procedure for managing hemoptysis. Bronchial artery embolization may control brisk bleeding in 75–90% of patients, permitting the definitive surgical procedure to be done more safely. Embolization without definitive surgery is associated with rebleeding in 20–50% of patients. Recurrent hemoptysis usually responds to a second embolization procedure. A postembolization syndrome characterized by pleuritic pain, fever, dysphagia, and leukocytosis may occur; it lasts 5–7 days and resolves with symptomatic treatment. Bronchial or esophageal wall necrosis, myocardial infarction, and spinal cord infarction are rare complications. Surgery, as a salvage strategy, is indicated after failure of embolization and is associated with better survival when performed in a nonurgent setting. Pulmonary hemorrhage with or without hemoptysis in hematologic malignancies is often associated with fungal infections, particularly Aspergillus sp. After granulocytopenia resolves, the lung infiltrates in

1	hemorrhage with or without hemoptysis in hematologic malignancies is often associated with fungal infections, particularly Aspergillus sp. After granulocytopenia resolves, the lung infiltrates in aspergillosis may cavitate and cause massive hemoptysis. Thrombocytopenia and coagulation defects should be corrected, if possible. Surgical evaluation is recommended in patients with aspergillosis-related cavitary lesions. Bevacizumab, an antibody to vascular endothelial growth factor (VEGF) that inhibits angiogenesis, has been associated with life-threatening hemoptysis in patients with non-small-cell lung cancer, particularly of squamous cell histology. Non-small-cell lung cancer patients with cavitary lesions or previous hemoptysis (≥2.5 mL) within the past 3 months have higher risk for pulmonary hemorrhage.

1	Airway obstruction refers to a blockage at the level of the mainstem bronchi or above. It may result either from intraluminal tumor growth or from extrinsic compression of the airway. The most common cause of malignant upper airway obstruction is invasion from an adjacent primary tumor, most commonly lung cancer, followed by esophageal, thyroid, and mediastinal malignancies including lymphomas. Extrathoracic primary tumors such as renal, colon, or breast cancer can cause airway obstruction through endobronchial and/or mediastinal lymph node metastases. Patients may present with dyspnea, hemoptysis, stridor, wheezing, intractable cough, postobstructive pneumonia, or hoarseness. Chest radiographs usually demonstrate obstructing lesions. CT scans reveal the extent of tumor. Cool, humidified oxygen, glucocorticoids, and ventilation with a mixture of helium and oxygen (Heliox) may provide temporary relief. If the obstruction is proximal to the larynx, a tracheostomy may be lifesaving. For

1	oxygen, glucocorticoids, and ventilation with a mixture of helium and oxygen (Heliox) may provide temporary relief. If the obstruction is proximal to the larynx, a tracheostomy may be lifesaving. For more distal obstructions, particularly intrinsic lesions incompletely obstructing the airway, bronchoscopy with mechanical debulking and dilatation or ablational treatments including laser treatment, photodynamic therapy, argon plasma coagulation, electrocautery, or stenting can produce immediate relief in most patients (Fig. 331-3). However, radiation therapy (either external-beam irradiation or brachytherapy) given together with glucocorticoids may also open the airway. Symptomatic extrinsic compression may be palliated by stenting. Patients with primary airway tumors such as squamous cell carcinoma, carcinoid tumor, adenocystic carcinoma, or non-small-cell lung cancer, if resectable, should have surgery.

1	Hypercalcemia is the most common paraneoplastic syndrome. Its pathogenesis and management are discussed fully in Chaps. 121 and 424. Hyponatremia is a common electrolyte abnormality in cancer patients, and SIADH is the most common cause among patients with cancer. SIADH is discussed fully in Chaps. 121 and 401e. FIGURE 331-3 Airway obstruction. A. Computed tomography scan of a 62-year-old man with tracheal obstruction caused by renal carci-noma showing paratracheal mass with tracheal invasion/obstruction (arrow). B. Chest x-ray of same patient after stent (arrows) placement.

1	Lactic acidosis is a rare and potentially fatal metabolic complication of cancer. Lactic acidosis associated with sepsis and circulatory failure is a common preterminal event in many malignancies. Lactic acidosis in the absence of hypoxemia may occur in patients with leukemia, lymphoma, or solid tumors. In some cases, hypoglycemia also is present. Extensive involvement of the liver by tumor is often present. In most cases, decreased metabolism and increased production by the tumor both contribute to lactate accumulation. Tumor cell overexpression of certain glycolytic enzymes and mitochondrial dysfunction can contribute to its increased lactate production. HIV-infected patients have an increased risk of aggressive lymphoma; lactic acidosis that occurs in such patients may be related either to the rapid growth of the tumor or from toxicity of nucleoside reverse transcriptase inhibitors. Symptoms of lactic acidosis include tachypnea, tachycardia, change of mental status, and

1	either to the rapid growth of the tumor or from toxicity of nucleoside reverse transcriptase inhibitors. Symptoms of lactic acidosis include tachypnea, tachycardia, change of mental status, and hepatomegaly. The serum level of lactic acid may reach 10–20 mmol/L (90–180 mg/dL). Treatment is aimed at the underlying disease. The danger from lactic acidosis is from the acidosis, not the lactate. Sodium bicarbonate should be added if acidosis is very severe or if hydrogen ion production is very rapid and uncontrolled. Other treatment options include renal replacement therapy, such as hemodialysis, and thiamine replacement. The prognosis is poor regardless of the treatment offered.

1	Persistent hypoglycemia is occasionally associated with tumors other than pancreatic islet cell tumors. Usually these tumors are large; tumors of mesenchymal origin, hepatomas, or adrenocortical tumors may cause hypoglycemia. Mesenchymal tumors are usually located in the retroperitoneum or thorax. Obtundation, confusion, and behavioral aberrations occur in the postabsorptive period and may precede the diagnosis of the tumor. These tumors often secrete incompletely processed insulin-like growth factor II (IGF-II), a hormone capable of activating insulin receptors and causing hypoglycemia. Tumors secreting incompletely processed big IGF-II are characterized by an increased IGF-II to IGF-I ratio, suppressed insulin and C-peptide level, and inappropriately low growth hormone and β-hydroxybutyrate concentrations. Rarely, hypoglycemia is due to insulin secretion by a non-islet cell carcinoma. The development of hepatic dysfunction from liver metastases and increased glucose consumption by

1	concentrations. Rarely, hypoglycemia is due to insulin secretion by a non-islet cell carcinoma. The development of hepatic dysfunction from liver metastases and increased glucose consumption by the tumor can contribute to hypoglycemia. If the tumor cannot be resected, hypoglycemia symptoms may be relieved by the administration of glucose, glucocorticoids, or glucagon.

1	Hypoglycemia can be artifactual; hyperleukocytosis from leukemia, myeloproliferative diseases, leukemoid reactions, or colony-stimulating factor treatment can increase glucose consumption in the test tube after blood is drawn, leading to pseudohypoglycemia.

1	In patients with cancer, adrenal insufficiency may go unrecognized because the symptoms, such as nausea, vomiting, anorexia, and orthostatic hypotension, are nonspecific and may be mistakenly attributed to progressive cancer or to therapy. Primary adrenal insufficiency may develop owing to replacement of both glands by metastases (lung, breast, colon, or kidney cancer; lymphoma), to removal of both glands, or to hemorrhagic necrosis in association with sepsis or anticoagulation. Impaired adrenal steroid synthesis occurs in patients being treated for cancer with mitotane, ketoconazole, or aminoglutethimide or undergoing rapid reduction in glucocorticoid therapy. Rarely, metastatic replacement causes primary adrenal insufficiency as the first manifestation of an occult malignancy. Metastasis to the pituitary or hypothalamus is found at autopsy in up to 5% of patients with cancer, but associated secondary adrenal insufficiency is rare. On the other hand, ipilimumab, an anti-CTLA-4

1	to the pituitary or hypothalamus is found at autopsy in up to 5% of patients with cancer, but associated secondary adrenal insufficiency is rare. On the other hand, ipilimumab, an anti-CTLA-4 antibody used for treatment of malignant melanoma, may cause autoimmunity including autoimmune-like enterocolitis, hypophysitis, and hepatitis. Autoimmune hypophysitis may present with headache, visual field defects, and pituitary hormone deficiencies manifesting as hypopituitarism, adrenal insufficiency (including adrenal crisis), or hypothyroidism. Anti-CTLA-4-associated hypophysitis symptoms occur at an average of 6–12 weeks after initiation of therapy. The treatment of severe autoimmune toxicity is glucocorticoids. Almost all patients with hypophysitis respond to withdrawal of ipilimumab and glucocorticoid therapy in several days. However, pituitary dysfunction may resolve or may be permanent, requiring longterm therapy and thyroid and testosterone replacement. Peripheral Addison’s disease

1	therapy in several days. However, pituitary dysfunction may resolve or may be permanent, requiring longterm therapy and thyroid and testosterone replacement. Peripheral Addison’s disease can also be observed with anti-CTLA-4 antibodies. Megestrol acetate, used to manage cancer and HIV-related cachexia, may suppress plasma levels of cortisol and adrenocorticotropic hormone (ACTH). Patients taking megestrol may develop adrenal insufficiency, and even those whose adrenal dysfunction is not symptomatic may have inadequate adrenal reserve if they become seriously ill. Paradoxically, some patients may develop Cushing’s syndrome and/or hyperglycemia because of the glucocorticoid-like activity of megestrol acetate. Cranial irradiation for childhood brain tumors may affect the hypothalamus-pituitary-adrenal axis, resulting in secondary adrenal insufficiency.

1	Acute adrenal insufficiency is potentially lethal. Treatment of suspected adrenal crisis is initiated after the sampling of serum cortisol and ACTH levels (Chap. 406). Tumor lysis syndrome (TLS) is characterized by hyperuricemia, hyperkalemia, hyperphosphatemia, and hypocalcemia and is caused by the destruction of a large number of rapidly proliferating neoplastic cells. Acidosis may also develop. Acute renal failure occurs frequently.

1	TLS is most often associated with the treatment of Burkitt’s lym-1795 phoma, acute lymphoblastic leukemia, and other rapidly proliferating lymphomas, but it also may be seen with chronic leukemias and, rarely, with solid tumors. This syndrome has been seen in patients with chronic lymphocytic leukemia after treatment with nucleosides like fludarabine. TLS has been observed with administration of glucocorticoids, hormonal agents such as letrozole and tamoxifen, and monoclonal antibodies such as rituximab and gemtuzumab. TLS usually occurs during or shortly (1–5 days) after chemotherapy. Rarely, spontaneous necrosis of malignancies causes TLS.

1	Hyperuricemia may be present at the time of chemotherapy. Effective treatment kills malignant cells and leads to increased serum uric acid levels from the turnover of nucleic acids. Owing to the acidic local environment, uric acid can precipitate in the tubules, medulla, and collecting ducts of the kidney, leading to renal failure. Lactic acidosis and dehydration may contribute to the precipitation of uric acid in the renal tubules. The finding of uric acid crystals in the urine is strong evidence for uric acid nephropathy. The ratio of urinary uric acid to urinary creatinine is >1 in patients with acute hyperuricemic nephropathy and <1 in patients with renal failure due to other causes.

1	Hyperphosphatemia, which can be caused by the release of intracellular phosphate pools by tumor lysis, produces a reciprocal depression in serum calcium, which causes severe neuromuscular irritability and tetany. Deposition of calcium phosphate in the kidney and hyperphosphatemia may cause renal failure. Potassium is the principal intracellular cation, and massive destruction of malignant cells may lead to hyperkalemia. Hyperkalemia in patients with renal failure may rapidly become life-threatening by causing ventricular arrhythmias and sudden death.

1	The likelihood that TLS will occur in patients with Burkitt’s lymphoma is related to the tumor burden and renal function. Hyperuricemia and high serum levels of lactate dehydrogenase (LDH >1500 U/L), both of which correlate with total tumor burden, also correlate with the risk of TLS. In patients at risk for TLS, pretreatment evaluations should include a complete blood count, serum chemistry evaluation, and urine analysis. High leukocyte and platelet counts may artificially elevate potassium levels (“pseudohyperkalemia”) due to lysis of these cells after the blood is drawn. In these cases, plasma potassium instead of serum potassium should be followed. In pseudohyperkalemia, no electrocardiographic abnormalities are present. In patients with abnormal baseline renal function, the kidneys and retroperitoneal area should be evaluated by sonography and/or CT to rule out obstructive uropathy. Urine output should be watched closely.

1	Recognition of risk and prevention are the most important steps in the management of this syndrome (Fig. 331-4). The standard preventive approach consists of allopurinol, urinary alkalinization, and aggressive hydration. Urinary alkalization with sodium bicarbonate is controversial. It increases uric acid solubility, but decreases calcium phosphate solubility. If it is used, it should be discontinued when hyperphosphatemia develops. Intravenous allopurinol may be given in patients who cannot tolerate oral therapy. In some cases, uric acid levels cannot be lowered sufficiently with the standard preventive approach. Rasburicase (recombinant urate oxidase) can be effective in these instances, particularly when renal failure is present. Urate oxidase is missing from primates and catalyzes the conversion of poorly soluble uric acid to readily soluble allantoin. Rasburicase acts rapidly, decreasing uric acid levels within hours; however, it may cause hypersensitivity reactions such as

1	the conversion of poorly soluble uric acid to readily soluble allantoin. Rasburicase acts rapidly, decreasing uric acid levels within hours; however, it may cause hypersensitivity reactions such as bronchospasm, hypoxemia, and hypotension. Rasburicase should also be administered to high-risk patients for TLS prophylaxis. Rasburicase is contraindicated in patients with glucose-6-phosphate dehydrogenase deficiency who are unable to break down hydrogen peroxide, an end product of the urate oxidase reaction. Rasburicase is known to cause ex vivo enzymatic degradation of uric acid in test tube at room temperature. This leads to spuriously low uric acid levels monocytes/macrophages and T and B lymphocytes.

1	Severe reactions from rituximab have occurred with Maintain hydration by administration of normal or 1/2 normal saline at 3000 mL/m2 per day high numbers (>50 × 109 lymphocytes) of circulating –/+ Keep urine pH at 7.0 or greater by administration of sodium bicarbonate* cells bearing the target antigen (CD20) and have been Administer allopurinol at 300 mg/m2 per day associated with a rapid fall in circulating tumor cells, mild electrolyte evidence of TLS, and very rarely, death. In addition, increased liver enzymes, D-dimer, and LDH and prolongation of the prothrombin time may occur. Diphenhydramine, hydrocortisone, and acetaminophen can often prevent or suppress the infusion-related symptoms. If they occur, the infusion is stopped and restarted at half the initial infusion rate after the symptoms have abated. Severe CRS may require intensive support for acute respiratory distress syndrome (ARDS) and resistant hypotension.

1	Serum uric acid >8 mg/dL Serum creatinine >1.6 mg/dL If, after 24–48 h Serum uric acid >8 mg/dL Serum creatinine >1.6 mg/dL Correct treatable renal failure (obstruction) Start rasburicase 0.2 mg/kg daily Serum uric acid ˜8.0 mg/dL Serum creatinine ˜1.6 mg/dL Urine pH °7.0 Delay chemotherapy if feasible or start hemodialysis Start chemotherapy ± chemotherapy Monitor serum chemistry every 6–12 h Discontinue bicarbonate administration* If serum potassium >6 meq/L Serum uric acid >10 mg/dL Serum creatinine >10 mg/dL Serum phosphate >10 mg/dL or increasing Symptomatic hypocalcemia present

1	Hemolytic-uremic syndrome (HUS) and, less commonly, thrombotic thrombocytopenic purpura (TTP) (Chap. 341) may rarely occur after treatment with antineoplastic drugs, including mitomycin, gemcitabine, cisplatin, and bleomycin, and with VEGF inhibitors. It occurs most often in patients with gastric, lung, colorectal, pancreatic, and breast carcinoma. In one series, 35% of patients were without evident cancer at the time this syndrome appeared. Secondary HUS/TTP has also been reported as a rare but sometimes fatal complication of bone marrow transplantation. HUS usually has its onset 4–8 weeks after the last dose of chemotherapy, but it is not rare to detect it several months later. HUS is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. Dyspnea, weakness, fatigue, oliguria, and purpura are also common initial symptoms and findings. Systemic hypertension and pulmonary

1	FIGURE 331-4 Management of patients at high risk for the tumor lysis syndrome. edema frequently occur. Severe hypertension, pul *See text. monary edema, and rapid worsening of hemolysis and renal function may occur after a blood or blood product transfusion. Cardiac findings include during laboratory monitoring of the patient with TLS. Samples atrial arrhythmias, pericardial friction rub, and pericardial effusion. must be cooled immediately to deactivate the urate oxidase. Despite Raynaud’s phenomenon is part of the syndrome in patients treated aggressive prophylaxis, TLS and/or oliguric or anuric renal failure with bleomycin. may occur. Care should be taken to prevent worsening of symptom-Laboratory findings include severe to moderate anemia associatic hypocalcemia by induction of alkalosis during bicarbonate infu-ated with red blood cell fragmentation and numerous schistocytes sion. Administration of sodium bicarbonate may also lead to urinary on peripheral smear. Reticulocytosis,

1	during bicarbonate infu-ated with red blood cell fragmentation and numerous schistocytes sion. Administration of sodium bicarbonate may also lead to urinary on peripheral smear. Reticulocytosis, decreased plasma haptoglobin, precipitation of calcium phosphate, which is less soluble at alkaline and an LDH level document hemolysis. The serum bilirubin level is pH. Dialysis is often necessary and should be considered early in the usually normal or slightly elevated. The Coombs’ test is negative. The course. Hemodialysis is preferred. Hemofiltration offers a gradual, white cell count is usually normal, and thrombocytopenia (<100,000/ continuous method of removing cellular by-products and fluid. The μL) is almost always present. Most patients have a normal coagulation prognosis is excellent, and renal function recovers after the uric acid profile, although some have mild elevations in thrombin time and in level is lowered to ≤10 mg/dL. levels of fibrin degradation products. The serum

1	and renal function recovers after the uric acid profile, although some have mild elevations in thrombin time and in level is lowered to ≤10 mg/dL. levels of fibrin degradation products. The serum creatinine level is elevated at presentation and shows a pattern of subacute worsening within weeks of the initial azotemia. The urinalysis reveals hematuria,

1	HUMAN ANTIBODY INFUSION REACTIONS proteinuria, and granular or hyaline casts; and circulating immune The initial infusion of human or humanized antibodies (e.g., ritux-complexes may be present. imab, gemtuzumab, trastuzumab, alemtuzumab, panitumumab, bren-The basic pathologic lesion appears to be deposition of fibrin in tuximab vedotin) is associated with fever, chills, nausea, asthenia, the walls of capillaries and arterioles, and these deposits are similar to and headache in up to half of treated patients. Bronchospasm and those seen in HUS due to other causes. These microvascular abnorhypotension occur in 1% of patients. Severe manifestations including malities involve mainly the kidneys and rarely occur in other organs. pulmonary infiltrates, acute respiratory distress syndrome, and cardio-The pathogenesis of cancer treatment–related HUS is not completely genic shock occur rarely. Laboratory manifestations include elevated understood, but probably the most important factor is

1	cardio-The pathogenesis of cancer treatment–related HUS is not completely genic shock occur rarely. Laboratory manifestations include elevated understood, but probably the most important factor is endothelial hepatic aminotransferase levels, thrombocytopenia, and prolongation damage. Primary forms of HUS/TTP are related to a decrease in proof prothrombin time. The pathogenesis is thought to be activation cessing of von Willebrand factor by a protease called ADAMTS13. of immune effector processes (cells and complement) and release of The case fatality rate is high; most patients die within a few months. inflammatory cytokines, such as tumor necrosis factor α, interferon There is no consensus on the optimal treatment for chemotherapy-gamma, interleukin 6, and interleukin 10 (cytokine release syndrome induced HUS. Treatment modalities for HUS/TTP including immu[CRS]). Although its origins are not completely understood, CRS is nocomplex removal (plasmapheresis, immunoadsorption, or

1	release syndrome induced HUS. Treatment modalities for HUS/TTP including immu[CRS]). Although its origins are not completely understood, CRS is nocomplex removal (plasmapheresis, immunoadsorption, or exchange believed to be due to activation of a variety of cell types including transfusion), antiplatelet/anticoagulant therapies, immunosuppressive therapies, and plasma exchange have varying degrees of success. The outcome with plasma exchange is generally poor, as in many other cases of secondary TTP. Rituximab is successfully used in patients with chemotherapy-induced HUS as well as in ADAMTS13deficient TTP.

1	These remain the most common serious complications of cancer therapy. They are covered in detail in Chap. 104.

1	Patients with cancer may present with dyspnea associated with diffuse interstitial infiltrates on chest radiographs. Such infiltrates may be due to progression of the underlying malignancy, treatment-related toxicities, infection, and/or unrelated diseases. The cause may be multifactorial; however, most commonly they occur as a consequence of treatment. Infiltration of the lung by malignancy has been described in patients with leukemia, lymphoma, and breast and other solid cancers. Pulmonary lymphatics may be involved diffusely by neoplasm (pulmonary lymphangitic carcinomatosis), resulting in a diffuse increase in interstitial markings on chest radiographs. The patient is often mildly dyspneic at the onset, but pulmonary failure develops over a period of weeks. In some patients, dyspnea precedes changes on the chest radiographs and is accompanied by a nonproductive cough. This syndrome is characteristic of solid tumors. In patients with leukemia, diffuse microscopic neoplastic

1	precedes changes on the chest radiographs and is accompanied by a nonproductive cough. This syndrome is characteristic of solid tumors. In patients with leukemia, diffuse microscopic neoplastic peribronchial and peribronchiolar infiltration is frequent but may be asymptomatic. However, some patients present with diffuse interstitial infiltrates, an alveolar capillary block syndrome, and respiratory distress. In these situations, glucocorticoids can provide symptomatic relief, but specific chemotherapy should always be started promptly.

1	Several cytotoxic agents, such as bleomycin, methotrexate, busulfan, nitrosoureas, gemcitabine, mitomycin, vinorelbine, docetaxel, paclitaxel, fludarabine, pentostatin, and ifosfamide may cause pulmonary damage. The most frequent presentations are interstitial pneumonitis, alveolitis, and pulmonary fibrosis. Some cytotoxic agents, including methotrexate and procarbazine, may cause an acute hypersensitivity reaction. Cytosine arabinoside has been associated with noncardiogenic pulmonary edema. Administration of multiple cytotoxic drugs, as well as radiotherapy and preexisting lung disease, may potentiate the pulmonary toxicity. Supplemental oxygen may potentiate the effects of drugs and radiation injury. Patients should always be managed with the lowest FIO2 that is sufficient to maintain hemoglobin saturation.

1	The onset of symptoms may be insidious, with symptoms including dyspnea, nonproductive cough, and tachycardia. Patients may have bibasilar crepitant rales, end-inspiratory crackles, fever, and cyanosis. The chest radiograph generally shows an interstitial and sometimes an intraalveolar pattern that is strongest at the lung bases and may be symmetric. A small effusion may occur. Hypoxemia with decreased carbon monoxide diffusing capacity is always present. Glucocorticoids may be helpful in patients in whom pulmonary toxicity is related to radiation therapy or to chemotherapy. Treatment is otherwise supportive.

1	Molecular targeted agents, imatinib, erlotinib, and gefitinib are potent inhibitors of tyrosine kinases. These drugs may cause interstitial lung disease (ILD). In the case of gefitinib, preexisting fibrosis, poor performance status, and prior thoracic irradiation are independent risk factors; this complication has a high fatality rate. In Japan, incidence of interstitial lung disease associated with gefitinib was about 4.5% compared to 0.5% in the United States. Temsirolimus and everolimus, both esters a derivative of rapamycin, are agents that block the effects of mammalian target of rapamycin (mTOR), an enzyme that has an important role in regulating the synthesis of proteins that control cell division. It may cause ground-glass opacities in the lung with or without diffuse interstitial disease and lung parenchymal consolidation. Patients may be asymptomatic with only radiologic findings or may be symptomatic. Symptoms include cough, dyspnea, and/or hypoxemia, and sometimes patients

1	disease and lung parenchymal consolidation. Patients may be asymptomatic with only radiologic findings or may be symptomatic. Symptoms include cough, dyspnea, and/or hypoxemia, and sometimes patients present with systemic symptoms such as fever and fatigue. The incidence of everolimus-induced interstitial lung disease also appears to be higher in Japanese patients. 1797 Treatment includes dose reduction or withdrawal and, in some cases, the addition of glucocorticoids.

1	Radiation pneumonitis and/or fibrosis is a relatively frequent side effect of thoracic radiation therapy. It may be acute or chronic. Radiation-induced lung toxicity is a function of the irradiated lung volume, dose per fraction, and radiation dose. The larger the irradiated lung field, the higher is the risk for radiation pneumonitis. The use of concurrent chemoradiation, particularly regimens including paclitaxel, increases pulmonary toxicity. Radiation pneumonitis usually develops 2–6 months after completion of radiotherapy. The clinical syndrome, which varies in severity, consists of dyspnea, cough with scanty sputum, low-grade fever, and an initial hazy infiltrate on chest radiographs. The infiltrate and tissue damage usually are confined to the radiation field. The patients subsequently may develop a patchy alveolar infiltrate and air bronchograms, which may progress to acute respiratory failure that is sometimes fatal. A lung biopsy may be necessary to make the diagnosis.

1	may develop a patchy alveolar infiltrate and air bronchograms, which may progress to acute respiratory failure that is sometimes fatal. A lung biopsy may be necessary to make the diagnosis. Asymptomatic infiltrates found incidentally after radiation therapy need not be treated. However, prednisone should be administered to patients with fever or other symptoms. The dosage should be tapered slowly after the resolution of radiation pneumonitis, because abrupt withdrawal of glucocorticoids may cause an exacerbation of pneumonia. Delayed radiation fibrosis may occur years after radiation therapy and is signaled by dyspnea on exertion. Often it is mild, but it can progress to chronic respiratory failure. Therapy is supportive.

1	Classical radiation pneumonitis that leads to pulmonary fibrosis is due to radiation-induced production of local cytokines such as platelet-derived growth factor β, tumor necrosis factor, interleukins, and transforming growth factor β in the radiation field. An immunologically mediated sporadic radiation pneumonitis occurs in about 10% of patients; bilateral alveolitis mediated by T cells results in infiltrates outside the radiation field. This form of radiation pneumonitis usually resolves without sequelae.

1	Pneumonia is a common problem in patients undergoing treatment for cancer. Bacterial pneumonia typically causes a localized infiltrate on chest radiographs. Therapy is tailored to the causative organism. When diffuse interstitial infiltrates appear in a febrile patient, the differential diagnosis is extensive and includes pneumonia due to infection with Pneumocystis carinii; viral infections including cytomegalovirus, adenovirus, herpes simplex virus, herpes zoster, respiratory syncytial virus, or intracellular pathogens such as Mycoplasma and Legionella; effects of drugs or radiation; tumor progression; nonspecific pneumonitis; and fungal disease. Detection of opportunistic pathogens in pulmonary infections is still a challenge. Diagnostic tools include chest radiographs, CT scans, bronchoscopy with bronchoalveolar lavage, brush cytology, transbronchial biopsy, fine-needle aspiration, and open lung biopsy. In addition to the culture, evaluation of bronchoalveolar lavage fluid for P.

1	with bronchoalveolar lavage, brush cytology, transbronchial biopsy, fine-needle aspiration, and open lung biopsy. In addition to the culture, evaluation of bronchoalveolar lavage fluid for P. carinii by polymerase chain reaction (PCR) and serum galactomannan test improve the diagnostic yield. Patients with cancer who are neutropenic and have fever and local infiltrates on chest radiograph should be treated initially with broad-spectrum antibiotics. A new or persistent focal infiltrate not responding to broad-spectrum antibiotics argues for initiation of empiric antifungal therapy. When diffuse bilateral infiltrates develop in patients with febrile neutropenia, broad-spectrum antibiotics plus trimethoprim-sulfamethoxazole, with or without erythromycin, should be initiated. Addition of an antiviral agent is necessary in some settings, such as patients undergoing allogeneic hematopoietic stem cell transplantation. If the patient does not improve in 4 days, open lung biopsy is the

1	of an antiviral agent is necessary in some settings, such as patients undergoing allogeneic hematopoietic stem cell transplantation. If the patient does not improve in 4 days, open lung biopsy is the procedure of choice. Bronchoscopy with bronchoalveolar lavage may be used in patients who are poor candidates for surgery.

1	In patients with pulmonary infiltrates who are afebrile, heart failure and multiple pulmonary emboli are in the differential diagnosis. Neutropenic enterocolitis (typhlitis) is the inflammation and necrosis of the cecum and surrounding tissues that may complicate the treatment FIGURE 331-5 Abdominal computed tomography (CT) scans of a 72-year-old woman with neutropenic enterocolitis secondary to chemotherapy. A. Air in inferior mesenteric vein (arrow) and bowel wall with pneumatosis intestinalis. B. CT scan of upper abdomen demonstrating air in portal vein (arrows).

1	of acute leukemia. Nevertheless, it may involve any segment of the gastrointestinal tract including small intestine, appendix, and colon. This complication has also been seen in patients with other forms of cancer treated with taxanes, 5-fluorouracil, irinotecan, vinorelbine, cisplatin, carboplatin, and high-dose chemotherapy (Fig. 331-5). It also has been reported in patients with AIDS, aplastic anemia, cyclic neutropenia, idiosyncratic drug reactions involving antibiotics, and immunosuppressive therapies. The patient develops right lower quadrant abdominal pain, often with rebound tenderness and a tense, distended abdomen, in a setting of fever and neutropenia. Watery diarrhea (often containing sloughed mucosa) and bacteremia are common, and bleeding may occur. Plain abdominal films are generally of little value in the diagnosis; CT scan may show marked bowel wall thickening, particularly in the cecum, with bowel wall edema, mesenteric stranding, and ascites, and may help to

1	are generally of little value in the diagnosis; CT scan may show marked bowel wall thickening, particularly in the cecum, with bowel wall edema, mesenteric stranding, and ascites, and may help to differentiate neutropenic colitis from other abdominal disorders such as appendicitis, diverticulitis, and Clostridium difficile–associated colitis in this high-risk population. Patients with bowel wall thickness >10 mm on ultrasonogram have higher mortality rates. However, bowel wall thickening is significantly more prominent in patients with C. difficile colitis. Pneumatosis intestinalis is a more specific finding, seen only in those with neutropenic enterocolitis and ischemia. The combined involvement of the small and large bowel suggests a diagnosis of neutropenic enterocolitis.

1	Rapid institution of broad-spectrum antibiotics, bowel rest, and nasogastric suction may reverse the process. Use of myeloid growth factors improved outcome significantly. Surgical intervention is reserved for severe cases of neutropenic enterocolitis with evidence of perforation, peritonitis, gangrenous bowel, or gastrointestinal hemorrhage despite correction of any coagulopathy. C. difficile colitis is increasing in incidence. Newer strains of C. difficile produce about 20 times more of toxins A and B compared to previously studied strains. C. difficile risk is also increased with chemotherapy. Antibiotic coverage for C. difficile should be added if pseudomembranous colitis cannot be excluded.

1	Hemorrhagic cystitis can develop in patients receiving cyclophosphamide or ifosfamide. Both drugs are metabolized to acrolein, which is a strong chemical irritant that is excreted in the urine. Prolonged contact or high concentrations may lead to bladder irritation and hemorrhage. Symptoms include gross hematuria, frequency, dysuria, burning, urgency, incontinence, and nocturia. The best management is prevention. Maintaining a high rate of urine flow minimizes exposure. In addition, 2-mercaptoethanesulfonate (mesna) detoxifies the metabolites and can be coadministered with the instigating drugs. Mesna usually is given three times on the day of ifosfamide administration in doses that are each 20% of the total ifosfamide dose. If hemorrhagic cystitis develops, the maintenance of a high urine flow may be sufficient supportive care. If conservative management is not effective, irrigation of the bladder with a 0.37–0.74% formalin solution for 10 min stops the bleeding in most cases.

1	urine flow may be sufficient supportive care. If conservative management is not effective, irrigation of the bladder with a 0.37–0.74% formalin solution for 10 min stops the bleeding in most cases. N-Acetylcysteine may also be an effective irrigant. Prostaglandin (carboprost) can inhibit the process. In extreme cases, ligation of the hypogastric arteries, urinary diversion, or cystectomy may be necessary.

1	Hemorrhagic cystitis also occurs in patients who undergo bone marrow transplantation (BMT). In the BMT setting, early-onset hemorrhagic cystitis is related to drugs in the treatment regimen (e.g., cyclophosphamide), and late-onset hemorrhagic cystitis is usually due to the polyoma virus BKV or adenovirus type 11. BKV load in urine alone or in combination with acute graft-versus-host disease correlates with development of hemorrhagic cystitis. Viral causes are usually detected by PCR-based diagnostic tests. Treatment of viral hemorrhagic cystitis is largely supportive, with reduction in doses of immunosuppressive agents, if possible. No antiviral therapy is approved, although cidofovir is reported to be effective in a small series. Hyperbaric oxygen therapy has been used successfully in patients with BKV-associated and cyclophosphamide-induced hemorrhagic cystitis during hematopoietic stem cell transplantation, as well as in hemorrhagic radiation cystitis.

1	Many antineoplastic drugs may cause hypersensitivity reaction. These reactions are unpredictable and potentially life-threatening. Most reactions occur during or within hours of parenteral drug administration. Taxanes, platinum compounds, asparaginase, etoposide, procarbazine, and biologic agents, including rituximab, bevacizumab, trastuzumab, gemtuzumab, cetuximab, and alemtuzumab, are more commonly associated with acute hypersensitivity reactions than are other agents. Acute hypersensitivity reactions to some drugs, such as taxanes, occur during the first or second dose administered. Hypersensitivity to platinum compounds occurs after prolonged exposure. Skin testing may identify patients with high risk for hypersensitivity after carboplatin exposure. Premedication with histamine H1 and H2 receptor antagonists and glucocorticoids reduces the incidence of hypersensitivity reaction to taxanes, particularly paclitaxel. Despite premedication, hypersensitivity reactions may still occur.

1	H2 receptor antagonists and glucocorticoids reduces the incidence of hypersensitivity reaction to taxanes, particularly paclitaxel. Despite premedication, hypersensitivity reactions may still occur. In these cases, rapid desensitization in the intensive care unit setting or re-treatment may be attempted with care, but the use of alternative agents may be required. Candidate patients for desensitization include those who have mild to severe hypersensitivity type I, with mast cell–mediated and IgE-dependent reactions occurring during a chemotherapy infusion or shortly thereafter.

1	332e-1 Cellular and Molecular Biology of the Kidney Alfred L. George, Jr., Eric G. Neilson The kidney is one of the most highly differentiated organs in the body. At the conclusion of embryologic development, nearly 30 different cell types form a multitude of filtering capillaries and segmented nephrons secreted by adjacently developing podocytes. Epithelial podocytes facing the urinary space envelop the exterior basement membrane sup-porting these emerging endothelial capillaries. Podocytes are partially polarized and periodically fall off into the urinary space by epithelial-mesenchymal transition, and to a lesser extent apoptosis, only to be replenished by migrating parietal epithelia from Bowman capsule. Impaired replenishment results in heavy proteinuria. Podocytes attach to the basement membrane by special foot processes and share a slit-pore membrane with their neighbor. The slit-pore membrane forms a filter for plasma water and solute by the synthetic interaction of neph-332e

1	membrane by special foot processes and share a slit-pore membrane with their neighbor. The slit-pore membrane forms a filter for plasma water and solute by the synthetic interaction of neph-332e PaRT 13: Disorders of the Kidney and Urinary Tract enveloped by a dynamic interstitium. This cellular diversity modulates a variety of complex physiologic processes. Endocrine functions, the regulation of blood pressure and intraglomerular hemodynamics, solute and water transport, acid-base balance, and removal of drug metabolites are all accomplished by intricate mechanisms of renal response. This breadth of physiology hinges on the clever ingenuity of nephron architecture that evolved as complex organisms came out of water to live on land.

1	Kidneys develop from intermediate mesoderm under the timed or sequential control of a growing number of genes, described in Fig. 332e-1. The transcription of these genes is guided by morphogenic cues that invite two ureteric buds to each penetrate bilateral metanephric blastema, where they induce primary mesenchymal cells to form early nephrons. The two ureteric buds emerge from posterior nephric ducts and mature into separate collecting systems that eventually form a renal pelvis and ureter. Induced mesenchyme undergoes mesenchymal epithelial transitions to form comma-shaped bodies at the proximal end of each ureteric bud leading to the formation of S-shaped nephrons that cleft and enjoin with penetrating endothelial cells derived from sprouting angioblasts. Under the influence of vascular endothelial growth factor A (VEGF-A), these penetrating cells form capillaries with surrounding mesangial cells that differentiate into a glomerular filter for plasma water and solute. The ureteric

1	endothelial growth factor A (VEGF-A), these penetrating cells form capillaries with surrounding mesangial cells that differentiate into a glomerular filter for plasma water and solute. The ureteric buds branch, and each branch produce a new set of nephrons. The number of branching events ultimately determines the total number of nephrons in each kidney. There are approximately 900,000 glomeruli in each kidney in normal birth weight adults and as few as 225,000 in low-birth-weight adults, with the latter producing numerous comorbid risks.

1	Glomeruli evolve as complex capillary filters with fenestrated endothelia under the guiding influence of VEGF-A and angiopoietin-1 rin, annexin-4, CD2AP, FAT, ZO-1, P-cadherin, podocin, TRPC6, PLCE1, and Neph 1-3 proteins. Mutations in many of these proteins also result in heavy proteinuria. The glomerular capillaries are embedded in a mesangial matrix shrouded by parietal and proximal tubular epithelia forming Bowman capsule. Mesangial cells have an embryonic lineage consistent with arteriolar or juxtaglomerular cells and contain contractile actin-myosin fibers. These mesangial cells make contact with glomerular capillary loops, and their local matrix holds them in condensed arrangement.

1	Between nephrons lies the renal interstitium. This region forms a functional space surrounding glomeruli and their downstream tubules, which are home to resident and trafficking cells such as fibroblasts, dendritic cells, occasional lymphocytes, and lipid-laden macrophages. The cortical and medullary capillaries, which siphon off solute and water following tubular reclamation of glomerular filtrate, are also part of the interstitial fabric as well as a web of connective tissue that supports the kidney’s emblematic architecture of folding tubules. The relational precision of these structures determines the unique physiology of the kidney.

1	Each nephron is partitioned during embryologic development into a proximal tubule, descending and ascending limbs of the loop of Henle, distal tubule, and the collecting duct. These classic tubular segments build from subsegments lined by highly unique epithelia serving regional physiology. All nephrons have the same structural components, but there are two types whose structures depend on their location within the kidney. The majority of nephrons are cortical, with glomeruli located in the mid-to-outer cortex. Fewer nephrons are juxtamedullary, with glomeruli at the boundary of the cortex and outer medulla. Cortical nephrons have short loops of Henle, whereas juxtamedullary nephrons have long loops of Henle. There are critical differences in blood supply as well. The peritubular capillaries surrounding cortical nephrons are shared among adjacent nephrons. By contrast, juxtamedullary nephrons depend on individual capillaries Cellular and Molecular Biology of the Kidney

1	Cellular and Molecular Biology of the Kidney FIGuRE 332e-1 Genes controlling renal nephrogenesis. A growing number of genes have been identified at various stages of glomerulotubular development in the mammalian kidney. The genes listed have been tested in various genetically modified mice, and their location corresponds to the classical stages of kidney development postulated by Saxen in 1987. called vasa recta. Cortical nephrons perform most of the glomerular filtration because there are more of them and because their afferent arterioles are larger than their respective efferent arterioles. The juxtamedullary nephrons, with longer loops of Henle, create an osmotic gradient for concentrating urine. How developmental instructions specify the differentiation of all these unique epithelia among various tubular segments is still unknown.

1	Renal blood flow normally drains approximately 20% of the cardiac output, or 1000 mL/min. Blood reaches each nephron through the afferent arteriole leading into a glomerular capillary where large amounts of fluid and solutes are filtered to form the tubular fluid. The distal ends of the glomerular capillaries coalesce to form an efferent arteriole leading to the first segment of a second capillary network (cortical peritubular capillaries or medullary vasa recta) surrounding the tubules (Fig. 332e-2A). Thus, nephrons have two capillary beds arranged in a series separated by the efferent arteriole that regulates the hydrostatic pressure in both capillary beds. The distal capillaries empty into small venous branches that coalesce into larger veins to eventually form the renal vein.

1	The hydrostatic pressure gradient across the glomerular capillary wall is the primary driving force for glomerular filtration. Oncotic pressure within the capillary lumen, determined by the concentration of unfiltered plasma proteins, partially offsets the hydrostatic pressure gradient and opposes filtration. As the oncotic pressure rises along the length of the glomerular capillary, the driving force for filtration falls to zero on reaching the efferent arteriole. Approximately 20% of the renal plasma flow is filtered into Bowman space, and the ratio of glomerular filtration rate (GFR) to renal plasma flow determines the filtration fraction. Several factors, mostly hemodynamic, contribute to the regulation of filtration under physiologic conditions.

1	Although glomerular filtration is affected by renal artery pressure, this relationship is not linear across the range of physiologic blood pressures due to autoregulation of GFR. Autoregulation of glomerular filtration is the result of three major factors that modulate either afferent or efferent arteriolar tone: these include an autonomous vasoreactive (myogenic) reflex in the afferent arteriole, tubuloglomerular feedback, and angiotensin II-mediated vasoconstriction of the efferent arteriole. The myogenic reflex is a first line of defense against fluctuations in renal blood flow. Acute changes in renal perfusion pressure evoke reflex constriction or dilatation of the afferent arteriole in response to increased or decreased pressure, respectively. This phenomenon helps protect the glomerular capillary from sudden changes in systolic pressure.

1	Tubuloglomerular feedback (TGF) changes the rate of filtration and tubular flow by reflex vasoconstriction or dilatation of the afferent arteriole. TGF is mediated by specialized cells in the thick ascending limb of the loop of Henle called the macula densa that act as sensors of solute concentration and tubular flow rate. With high tubular flow rates, a proxy for an inappropriately high filtration rate, there is increased solute delivery to the macula densa (Fig. 332e-2B) that evokes vasoconstriction of the afferent arteriole causing GFR to return toward normal. One component of the soluble signal from the macula densa is adenosine triphosphate (ATP) released by the cells during increased NaCl reabsorption. ATP is metabolized in the extracellular space to generate adenosine, a potent vasoconstrictor of the afferent arteriole. During conditions associated with a fall in filtration rate, reduced solute delivery to the macula densa attenuates TGF, allowing afferent arteriolar dilatation

1	of the afferent arteriole. During conditions associated with a fall in filtration rate, reduced solute delivery to the macula densa attenuates TGF, allowing afferent arteriolar dilatation and restoring glomerular filtration to normal levels. Angiotensin II and reactive oxygen species enhance, while nitric oxide (NO) blunts, TGF.

1	The third component underlying autoregulation of GFR involves angiotensin II. During states of reduced renal blood flow, renin is released from granular cells within the wall of the afferent arteriole near the macula densa in a region called the juxtaglomerular apparatus (Fig. 332e-2B). Renin, a proteolytic enzyme, catalyzes the conversion of angiotensinogen to angiotensin I, which is subsequently converted to angiotensin II by angiotensin-converting enzyme (ACE) (Fig. 332e-2C). FIGuRE 332e-2 Renal microcirculation and the renin-angiotensin system. A. Diagram illustrating relationships of the nephron with glomerular and peritubular capillaries. B. Expanded view of the glomerulus with its juxtaglomerular apparatus including the macula densa and adjacent afferent arteriole. C. Proteolytic processing steps in the generation of angiotensins.

1	Angiotensin II evokes vasoconstriction of the efferent arteriole, and especially proteins mediating transport processes, provides the machin-332e-3 the resulting increased glomerular hydrostatic pressure elevates filtra-ery for directional movement of fluid and solutes by the nephron. tion to normal levels.

1	The renal tubules are composed of highly differentiated epithelia that vary dramatically in morphology and function along the nephron (Fig. 332e-3). The cells lining the various tubular segments form monolayers connected to one another by a specialized region of the adjacent lateral membranes called the tight junction. Tight junctions form an occlusive barrier that separates the lumen of the tubule from the interstitial spaces surrounding the tubule and also apportions the cell membrane into discrete domains: the apical membrane facing the tubular lumen and the basolateral membrane facing the interstitium. This regionalization allows cells to allocate membrane proteins and lipids asymmetrically. Owing to this feature, renal epithelial cells are said to be polarized. The asymmetric assignment of membrane proteins, There are two types of epithelial transport. Movement of fluid and solutes sequentially across the apical and basolateral cell membranes (or vice versa) mediated by

1	assignment of membrane proteins, There are two types of epithelial transport. Movement of fluid and solutes sequentially across the apical and basolateral cell membranes (or vice versa) mediated by transporters, channels, or pumps is called cellular transport. By contrast, movement of fluid and solutes through the narrow passageway between adjacent cells is called paracellular transport. Paracellular transport occurs through tight junctions, indicating that they are not completely “tight.” Indeed, some epithelial cell layers allow rather robust paracellular transport to occur (leaky epithelia), whereas other epithelia have more effective tight junctions (tight epithelia). In addition, because the ability of ions to flow through the paracellular pathway determines the electrical resistance across the epithelial monolayer, leaky and tight epithelia are also referred to as lowor high-resistance epithelia, respectively. The proximal tubule

1	Chapter 332e Cellular and Molecular Biology of the Kidney H2O, solutes

1	FIGuRE 332e-3 Transport activities of the major nephron segments. Representative cells from five major tubular segments are illustrated with the lumen side (apical membrane) facing left and interstitial side (basolateral membrane) facing right. A. Proximal tubular cells. B. Typical cell in the thick ascending limb of the loop of Henle. C. Distal convoluted tubular cell. D. Overview of entire nephron. E. Cortical collecting duct cells. F. Typical cell in the inner medullary collecting duct. The major membrane transporters, channels, and pumps are drawn with arrows indicating the direction of solute or water movement. For some events, the stoichiometry of transport is indicated by numerals preceding the solute. Targets for major diuretic agents are labeled. The actions of hormones are illustrated by arrows with plus signs for stimulatory effects and lines with perpendicular ends for inhibitory events. Dotted lines indicate free diffusion across cell membranes. The dashed line indicates

1	by arrows with plus signs for stimulatory effects and lines with perpendicular ends for inhibitory events. Dotted lines indicate free diffusion across cell membranes. The dashed line indicates water impermeability of cell membranes in the thick ascending limb and distal convoluted tubule.

1	Lumen Interstitium 3NaThiazidesDISTAL CONVOLUTED TUBULE H2OCa2KClNaClCa3NaC+ Ca, Mg 3Na Loop diuretics THICK ASCENDING LIMB BCa H2O K 2K Cl Na 2Cl K Chapter 332e Cellular and Molecular Biology of the Kidney Loop of Henle: contains leaky epithelia, whereas distal nephron segments, such as the collecting duct, contain tight epithelia. Leaky epithelia are most well suited for bulk fluid reabsorption, whereas tight epithelia allow for more refined control and regulation of transport.

1	Cell membranes are composed of hydrophobic lipids that repel water and aqueous solutes. The movement of solutes and water across cell membranes is made possible by discrete classes of integral membrane proteins, including channels, pumps, and transporters. These different mechanisms mediate specific types of transport activities, including active transport (pumps), passive transport (channels), facilitated diffusion (transporters), and secondary active transport (cotransporters). Active transport requires metabolic energy generated by the hydrolysis of ATP. Active transport pumps are ion-translocating ATPases, including the ubiquitous Na+/K+-ATPase, the H+-ATPases, and Ca2+-ATPases. Active transport creates asymmetric ion concentrations across a cell membrane and can move ions against a chemical gradient. The potential energy stored in a concentration gradient of an ion such as Na+ can be used to drive transport through other mechanisms (secondary active transport). Pumps are often

1	chemical gradient. The potential energy stored in a concentration gradient of an ion such as Na+ can be used to drive transport through other mechanisms (secondary active transport). Pumps are often electrogenic, meaning they can create an asymmetric distribution of electrostatic charges across the membrane and establish a voltage or membrane potential. The movement of solutes through a membrane protein by simple diffusion is called passive transport. This activity is mediated by channels created by selectively permeable membrane proteins, and it allows solute or water to move across a membrane driven by favorable concentration gradients or electrochemical potential. Facilitated diffusion is a specialized type of passive transport mediated by simple transporters called carriers or uniporters. For example, hexose transporters such as GLUT2 mediate glucose transport by tubular cells. These transporters are driven by the concentration gradient for glucose that is highest in extracellular

1	For example, hexose transporters such as GLUT2 mediate glucose transport by tubular cells. These transporters are driven by the concentration gradient for glucose that is highest in extracellular fluids and lowest in the cytoplasm due to rapid metabolism. Many other transporters operate by translocating two or more ions/solutes in concert either in the same direction (symporters or cotransporters) or in opposite directions (antiporters or exchangers) across the cell membrane. The movement of two or more ions/solutes may produce no net change in the balance of electrostatic charges across the membrane (electroneutral), or a transport event may alter the balance of charges (electrogenic). Several inherited disorders of renal tubular solute and water transport occur as a consequence of mutations in genes encoding a variety of channels, transporter proteins, and their regulators (Table 332e-1).

1	Each anatomic segment of the nephron has unique characteristics and specialized functions enabling selective transport of solutes and water (Fig. 332e-3). Through sequential events of reabsorption and secretion along the nephron, tubular fluid is progressively conditioned into urine. Knowledge of the major tubular mechanisms responsible for solute and water transport is critical for understanding hormonal regulation of kidney function and the pharmacologic manipulation of renal excretion. The proximal tubule is responsible for reabsorbing ~60% of filtered NaCl and water, as well as ~90% of filtered bicarbonate and most critical nutrients such as glucose and amino acids. The proximal tubule uses both cellular and paracellular transport mechanisms. The apical membrane of proximal tubular cells has an expanded surface area available for reabsorptive work created by a dense array of microvilli called the brush border, and leaky tight junctions enable high-capacity fluid reabsorption.

1	Solute and water pass through these tight junctions to enter the lateral intercellular space where absorption by the peritubular capillaries occurs. Bulk fluid reabsorption by the proximal tubule is driven by high oncotic pressure and low hydrostatic pressure within the peritubular capillaries. Cellular transport of most solutes by the proximal tubule is coupled to the Na+ concentration gradient established by the activity of a basolateral Na+/K+-ATPase (Fig. 332e-3A). This active transport mechanism maintains a steep Na+ gradient by keeping intracellular Na+ concentrations low. Solute reabsorption is coupled to the Na+ gradient by Na+-dependent transporters such as Na+-glucose and Na+-phosphate cotransporters. In addition to the paracellular route, water reabsorption also occurs through the cellular pathway enabled by constitutively active water channels (aquaporin-1) present on both apical and basolateral membranes.

1	Proximal tubular cells reclaim bicarbonate by a mechanism dependent on carbonic anhydrases. Filtered bicarbonate is first titrated by protons delivered to the lumen by Na+/H+ exchange. The resulting carbonic acid (H2CO3) is metabolized by brush border carbonic anhydrase to water and carbon dioxide. Dissolved carbon dioxide then diffuses into the cell, where it is enzymatically hydrated by cytoplasmic carbonic anhydrase to re-form carbonic acid. Finally, intracellular carbonic acid dissociates into free protons and bicarbonate anions, and bicarbonate exits the cell through a basolateral Na+/HCO3− cotransporter. Chapter 332e Cellular and Molecular Biology of the Kidney aOnline Mendelian Inheritance in Man database (http://www.ncbi.nlm.nih.gov/Omim).

1	Chapter 332e Cellular and Molecular Biology of the Kidney aOnline Mendelian Inheritance in Man database (http://www.ncbi.nlm.nih.gov/Omim). This process is saturable, resulting in urinary bicarbonate excretion Filtered hydrogen phosphate ion (HPO42-) is also titrated in the proxiwhen plasma levels exceed the physiologically normal range (24-26 mal tubule by secreted H+ to form H2PO4 -, and this reaction constitutes meq/L). Carbonic anhydrase inhibitors such as acetazolamide, a class a major component of the urinary buffer referred to as titratable acid. of weak diuretic agents, block proximal tubule reabsorption of bicar-Most filtered phosphate ion is reabsorbed by the proximal tubule bonate and are useful for alkalinizing the urine. through a sodium-coupled cotransport process that is regulated by

1	The proximal tubule contributes to acid secretion by two mecha-parathyroid hormone. nisms involving the titration of the urinary buffers ammonia (NH3) Chloride is poorly reabsorbed throughout the first segment of the and phosphate. Renal NH3 is produced by glutamine metabolism in proximal tubule, and a rise in Cl− concentration counterbalances the the proximal tubule. Subsequent diffusion of NH3 out of the proxi-removal of bicarbonate anion from tubular fluid. In later proximal mal tubular cell enables trapping of H+ secreted by sodium-proton tubular segments, cellular Cl− reabsorption is initiated by apical exchange in the lumen as ammonium ion (NH4+). Cellular K+ levels exchange of cellular formate for higher luminal concentrations of inversely modulate proximal tubular ammoniagenesis, and in the Cl-. Once in the lumen, formate anions are titrated by H+ (provided setting of high serum K+ from hypoaldosteronism, reduced ammo-by Na+/H+ exchange) to generate neutral formic acid, which

1	and in the Cl-. Once in the lumen, formate anions are titrated by H+ (provided setting of high serum K+ from hypoaldosteronism, reduced ammo-by Na+/H+ exchange) to generate neutral formic acid, which can difniagenesis facilitates the appearance of type IV renal tubular acidosis. fuse passively across the apical membrane back into the cell where it dissociates a proton and is recycled. Basolateral Cl− exit is mediated by a K+/Cl− cotransporter.

1	Reabsorption of glucose is nearly complete by the end of the proximal tubule. Cellular transport of glucose is mediated by apical Na+-glucose cotransport coupled with basolateral, facilitated diffusion by a glucose transporter. This process is also saturable, leading to glycosuria when plasma levels exceed 180-200 mg/dL, as seen in untreated diabetes mellitus.

1	The proximal tubule possesses specific transporters capable of secreting a variety of organic acids (carboxylate anions) and bases (mostly primary amine cations). Organic anions transported by these systems include urate, dicarboxylic acid anions (succinate), ketoacid anions, and several protein-bound drugs not filtered at the glomerulus (penicillins, cephalosporins, and salicylates). Probenecid inhibits renal organic anion secretion and can be clinically useful for raising plasma concentrations of certain drugs like penicillin and oseltamivir. Organic cations secreted by the proximal tubule include various biogenic amine neurotransmitters (dopamine, acetylcholine, epinephrine, norepinephrine, and histamine) and creatinine. The ATP-dependent transporter P-glycoprotein is highly expressed in brush border membranes and secretes several medically important drugs, including cyclosporine, digoxin, tacrolimus, and various cancer chemotherapeutic agents. Certain drugs like cimetidine and

1	in brush border membranes and secretes several medically important drugs, including cyclosporine, digoxin, tacrolimus, and various cancer chemotherapeutic agents. Certain drugs like cimetidine and trimethoprim compete with endogenous compounds for transport by the organic cation pathways. Although these drugs elevate serum creatinine levels, there is no change in the actual GFR.

1	The proximal tubule, through distinct classes of Na+-dependent and Na+-independent transport systems, reabsorbs amino acids efficiently. These transporters are specific for different groups of amino acids. For example, cystine, lysine, arginine, and ornithine are transported by a system comprising two proteins encoded by the SLC3A1 and SLC7A9 genes. Mutations in either SLC3A1 or SLC7A9 impair reabsorption of these amino acids and cause the disease cystinuria. Peptide hormones, such as insulin and growth hormone, β2-microglobulin, albumin, and other small proteins, are taken up by the proximal tubule through a process of absorptive endocytosis and are degraded in acidified endocytic lysosomes. Acidification of these vesicles depends on a vacuolar H+-ATPase and Cl− channel. Impaired acidification of endocytic vesicles because of mutations in a Cl− channel gene (CLCN5) causes low-molecular-weight proteinuria in Dent disease.

1	The loop of Henle consists of three major segments: descending thin limb, ascending thin limb, and ascending thick limb. These divisions are based on cellular morphology and anatomic location, but also correlate with specialization of function. Approximately 15–25% of filtered NaCl is reabsorbed in the loop of Henle, mainly by the thick ascending limb. The loop of Henle has an important role in urinary concentration by contributing to the generation of a hypertonic medullary interstitium in a process called countercurrent multiplication. The loop of Henle is the site of action for the most potent class of diuretic agents (loop diuretics) and also contributes to reabsorption of calcium and magnesium ions.

1	The descending thin limb is highly water permeable owing to dense expression of constitutively active aquaporin-1 water channels. By contrast, water permeability is negligible in the ascending limb. In the thick ascending limb, there is a high level of secondary active salt transport enabled by the Na+/K+/2Cl− cotransporter on the apical membrane in series with basolateral Cl− channels and Na+/K+-ATPase (Fig. 332e-3B). The Na+/K+/2Cl− cotransporter is the primary target for loop diuretics. Tubular fluid K+ is the limiting substrate for this cotransporter (tubular concentration of K+ is similar to plasma, about 4 meq/L), but transporter activity is maintained by K+ recycling through an apical potassium channel. The cotransporter also enables reabsorption of NH4+ in lieu of K+, and this leads to accumulation of both NH4+ and NH3 in the medullary interstitium. An inherited disorder of the thick ascending limb, Bartter syndrome, also results in a salt-wasting renal disease associated with

1	to accumulation of both NH4+ and NH3 in the medullary interstitium. An inherited disorder of the thick ascending limb, Bartter syndrome, also results in a salt-wasting renal disease associated with hypokalemia and metabolic alkalosis; loss-of-function mutations in one of five distinct genes encoding components of the Na+/K+/2Cl− cotransporter (NKCC2), apical K+ channel (KCNJ1), basolateral Cl− channel (CLCNKB, BSND), or calcium-sensing receptor (CASR) can cause Bartter syndrome.

1	Potassium recycling also contributes to a positive electrostatic charge in the lumen relative to the interstitium that promotes divalent cation (Mg2+ and Ca2+) reabsorption through a paracellular pathway. A Ca2+-sensing, G-protein-coupled receptor (CaSR) on basolateral membranes regulates NaCl reabsorption in the thick ascending limb through dual signaling mechanisms using either cyclic AMP or eicosanoids. This receptor enables a steep relationship between plasma Ca2+ levels and renal Ca2+ excretion. Loss-of-function mutations in CaSR cause familial hypercalcemic hypocalciuria because of a blunted response of the thick ascending limb to extracellular Ca2+. Mutations in CLDN16 encoding paracellin-1, a transmembrane protein located within the tight junction complex, leads to familial hypomagnesemia with hypercalciuria and nephrocalcinosis, suggesting that the ion conductance of the paracellular pathway in the thick limb is regulated.

1	The loop of Henle contributes to urine-concentrating ability by establishing a hypertonic medullary interstitium that promotes water reabsorption by the downstream inner medullary collecting duct. Countercurrent multiplication produces a hypertonic medullary interstitium using two countercurrent systems: the loop of Henle (opposing descending and ascending limbs) and the vasa recta (medullary peritubular capillaries enveloping the loop). The countercurrent flow in these two systems helps maintain the hypertonic environment of the inner medulla, but NaCl reabsorption by the thick ascending limb is the primary initiating event. Reabsorption of NaCl without water dilutes the tubular fluid and adds new osmoles to medullary interstitial fluid. Because the descending thin limb is highly water permeable, osmotic equilibrium occurs between the descending limb tubular fluid and the interstitial space, leading to progressive solute trapping in the inner medulla. Maximum medullary interstitial

1	permeable, osmotic equilibrium occurs between the descending limb tubular fluid and the interstitial space, leading to progressive solute trapping in the inner medulla. Maximum medullary interstitial osmolality also requires partial recycling of urea from the collecting duct.

1	The distal convoluted tubule reabsorbs ~5% of the filtered NaCl. This segment is composed of a tight epithelium with little water permeability. The major NaCl-transporting pathway uses an apical membrane, electroneutral thiazide-sensitive Na+/Cl− cotransporter in tandem with basolateral Na+/K+-ATPase and Cl− channels (Fig. 332e-3C). Apical Ca2+-selective channels (TRPV5) and basolateral Na+/Ca2+ exchange mediate calcium reabsorption in the distal convoluted tubule. Ca2+ reabsorption is inversely related to Na+ reabsorption and is stimulated by parathyroid hormone. Blocking apical Na+/Cl− cotransport will reduce intracellular Na+, favoring increased basolateral Na+/ Ca2+ exchange and passive apical Ca2+ entry. Loss-of-function mutations of SLC12A3 encoding the apical Na+/Cl− cotransporter cause Gitelman syndrome, a salt-wasting disorder associated with hypokalemic alkalosis and hypocalciuria. Mutations in genes encoding WNK kinases, WNK-1 and WNK-4, cause pseudohypoaldosteronism type

1	cause Gitelman syndrome, a salt-wasting disorder associated with hypokalemic alkalosis and hypocalciuria. Mutations in genes encoding WNK kinases, WNK-1 and WNK-4, cause pseudohypoaldosteronism type II or Gordon syndrome characterized by familial hypertension with hyperkalemia. WNK kinases influence the activity of several tubular ion transporters. Mutations in this disorder lead to overactivity of the apical Na+/Cl− cotransporter in the distal convoluted tubule as the primary stimulus for increased salt reabsorption, extracellular volume expansion, and hypertension. Hyperkalemia may be caused by diminished activity of apical K+ channels in the collecting duct, a primary route for K+ secretion. Mutations in TRPM6 encoding Mg2+ permeable ion channels also cause familial hypomagnesemia with hypocalcemia. A molecular complex of TRPM6 and TRPM7 proteins is critical for Mg2+ reabsorption in the distal convoluted tubule.

1	The collecting duct modulates the final composition of urine. The two major divisions, the cortical collecting duct and inner medullary collecting duct, contribute to reabsorbing ~4-5% of filtered Na+ and are important for hormonal regulation of salt and water balance. The cortical collecting duct contains high-resistance epithelia with two cell types. Principal cells are the main water, Na+-reabsorbing, and K+-secreting cells, and the site of action of aldosterone, K+-sparing diuretics, and mineralocorticoid receptor antagonists such as spironolactone. The other cells are type A and B intercalated cells. Type A intercalated cells mediate acid secretion and bicarbonate reabsorption also under the influence of aldosterone. Type B intercalated cells mediate bicarbonate secretion and acid reabsorption.

1	Virtually all transport is mediated through the cellular pathway for both principal cells and intercalated cells. In principal cells, passive apical Na+ entry occurs through the amiloride-sensitive, epithelial Na+ channel (ENaC) with basolateral exit via the Na+/K+-ATPase (Fig. 332e-3E). This Na+ reabsorptive process is tightly regulated by aldosterone and is physiologically activated by a variety of proteolytic enzymes that cleave extracellular domains of ENaC; plasmin in the tubular fluid of nephrotic patients, for example, activates ENaC, leading to sodium retention. Aldosterone enters the cell across the basolateral membrane, binds to a cytoplasmic mineralocorticoid receptor, and then translocates into the nucleus, where it modulates gene transcription, resulting in increased Na+ reabsorption and K+ secretion. Activating mutations in ENaC increase Na+ reclamation and produce hypokalemia, hypertension, and metabolic alkalosis (Liddle’s syndrome). The potassium-sparing diuretics

1	and K+ secretion. Activating mutations in ENaC increase Na+ reclamation and produce hypokalemia, hypertension, and metabolic alkalosis (Liddle’s syndrome). The potassium-sparing diuretics amiloride and triamterene block ENaC, causing reduced Na+ reabsorption.

1	Principal cells secrete K+ through an apical membrane potassium channel. Several forces govern the secretion of K+. Most importantly, the high intracellular K+ concentration generated by Na+/K+-ATPase creates a favorable concentration gradient for K+ secretion into tubular fluid. With reabsorption of Na+ without an accompanying anion, the tubular lumen becomes negative relative to the cell interior, creating a favorable electrical gradient for secretion of potassium. When Na+ reabsorption is blocked, the electrical component of the driving force for K+ secretion is blunted, and this explains lack of excess urinary K+ loss during treatment with potassium-sparing diuretics or mineralocorticoid receptor antagonists. K+ secretion is also promoted by aldosterone actions that increase regional Na+ transport favoring more electronegativity and by increasing the number and activity of potassium channels. Fast tubular fluid flow rates that occur during volume expansion or diuretics acting

1	Na+ transport favoring more electronegativity and by increasing the number and activity of potassium channels. Fast tubular fluid flow rates that occur during volume expansion or diuretics acting “upstream” of the cortical collecting duct also increase K+ secretion, as does the presence of relatively nonreabsorbable anions (including bicarbonate and semisynthetic penicillins) that contribute to the lumen-negative potential. Off-target effects of certain antibiotics, such as trimethoprim and pentamidine, block ENaCs and predispose to hyperkalemia, especially when renal K+ handling is impaired for other reasons. Principal cells, as described below, also participate in water reabsorption by increased water permeability in response to vasopressin.

1	Intercalated cells do not participate in Na+ reabsorption but, instead, mediate acid-base secretion. These cells perform two types of transport: active H+ transport mediated by H+-ATPase (proton pump), and Cl-/HCO3− exchange. Intercalated cells arrange the two transport mechanisms on opposite membranes to enable either acid or base secretion. Type A intercalated cells have an apical proton pump that mediates acid secretion and a basolateral Cl-/HCO3 anion exchanger for bicarbonate reabsorption (Fig. 332e-3E); aldosterone increases the number of H+-ATPase pumps, sometimes contributing to the development of metabolic alkalosis. Secreted H+ is buffered by NH3 that has diffused into the collecting duct lumen from the surrounding interstitium. By contrast, type B intercalated cells have the anion exchanger on the apical membrane to mediate bicarbonate secretion while the proton pump resides on the basolateral membrane to enable acid reabsorption. Under conditions of acidemia, the kidney

1	anion exchanger on the apical membrane to mediate bicarbonate secretion while the proton pump resides on the basolateral membrane to enable acid reabsorption. Under conditions of acidemia, the kidney preferentially uses type A intercalated cells to secrete the excess H+ and generate more

1	HCO3 . The opposite is true in states of bicarbonate excess with alkalemia where the type B intercalated cells predominate. An extracellular protein called hensin mediates this adaptation.

1	Inner medullary collecting duct cells share many similarities with principal cells of the cortical collecting duct. They have apical Na+ and K+ channels that mediate Na+ reabsorption and K+ secretion, respectively (Fig. 332e-3F). Inner medullary collecting duct cells also 332e-9 have vasopressin-regulated water channels (aquaporin-2 on the apical membrane, aquaporin-3 and -4 on the basolateral membrane). The antidiuretic hormone vasopressin binds to the V2 receptor on the basolateral membrane and triggers an intracellular signaling cascade through G-protein-mediated activation of adenylyl cyclase, resulting in an increase in the cellular levels of cyclic AMP. This signaling cascade stimulates the insertion of water channels into the apical membrane of the inner medullary collecting duct cells to promote increased water permeability. This increase in permeability enables water reabsorption and production of concentrated urine. In the absence of vasopressin, inner medullary collecting

1	cells to promote increased water permeability. This increase in permeability enables water reabsorption and production of concentrated urine. In the absence of vasopressin, inner medullary collecting duct cells are water impermeable, and urine remains dilute.

1	Sodium reabsorption by inner medullary collecting duct cells is also inhibited by the natriuretic peptides called atrial natriuretic peptide or renal natriuretic peptide (urodilatin); the same gene encodes both peptides but uses different posttranslational processing of a common preprohormone to generate different proteins. Atrial natriuretic peptides are secreted by atrial myocytes in response to volume expansion, whereas urodilatin is secreted by renal tubular epithelia. Natriuretic peptides interact with either apical (urodilatin) or basolateral (atrial natriuretic peptides) receptors on inner medullary collecting duct cells to stimulate guanylyl cyclase and increase levels of cytoplasmic cGMP. This effect in turn reduces the activity of the apical Na+ channel in these cells and attenuates net Na+ reabsorption, producing natriuresis.

1	The inner medullary collecting duct transports urea out of the lumen, returning urea to the interstitium, where it contributes to the hypertonicity of the medullary interstitium. Urea is recycled by diffusing from the interstitium into the descending and ascending limbs of the loop of Henle. The balance of solute and water in the body is determined by the amounts ingested, distributed to various fluid compartments, and excreted by skin, bowel, and kidneys. Tonicity, the osmolar state determining the volume behavior of cells in a solution, is regulated by water balance (Fig. 332e-4A), and extracellular blood volume is regulated by Na+ balance (Fig. 332e-4B). The kidney is a critical modulator of both physiologic processes.

1	Tonicity depends on the variable concentration of effective osmoles inside and outside the cell causing water to move in either direction across its membrane. Classic effective osmoles, like Na+, K+, and their anions, are solutes trapped on either side of a cell membrane, where they collectively partition and obligate water to move and find equilibrium in proportion to retained solute; Na+/K+-ATPase keeps most K+ inside cells and most Na+ outside. Normal tonicity (~280 mosmol/L) is rigorously defended by osmoregulatory mechanisms that control water balance to protect tissues from inadvertent dehydration (cell shrinkage) or water intoxication (cell swelling), both of which are deleterious to cell function (Fig. 332e-4A).

1	The mechanisms that control osmoregulation are distinct from those governing extracellular volume, although there is some shared physiology in both processes. While cellular concentrations of K+ have a determinant role in any level of tonicity, the routine surrogate marker for assessing clinical tonicity is the concentration of serum Na+. Any reduction in total body water, which raises the Na+ concentration, triggers a brisk sense of thirst and conservation of water by decreasing renal water excretion mediated by release of vasopressin from the posterior pituitary. Conversely, a decrease in plasma Na+ concentration triggers an increase in renal water excretion by suppressing the secretion of vasopressin. Whereas all cells expressing mechanosensitive TRPV1, 2, or 4 channels, among potentially other sensors, respond to changes in tonicity by altering their volume and Ca2+ concentration, only TRPV+ neuronal cells connected to the organum vasculosum of the lamina terminalis are

1	potentially other sensors, respond to changes in tonicity by altering their volume and Ca2+ concentration, only TRPV+ neuronal cells connected to the organum vasculosum of the lamina terminalis are osmoreceptive. Only these cells, because of their neural

1	Chapter 332e Cellular and Molecular Biology of the Kidney

1	FIGuRE 332e-4 Determinants of sodium and water balance. A. Plasma Na+ concentration is a surrogate marker for plasma tonicity, the volume behavior of cells in a solution. Tonicity is determined by the number of effective osmoles in the body divided by the total body H2O (TB H2O), which translates simply into the total body Na (TB Na+) and anions outside the cell separated from the total body K (TB K+) inside the cell by the cell membrane. Net water balance is determined by the integrated functions of thirst, osmoreception, Na reabsorption, vasopressin release, and the strength of the medullary gradient in the kidney, keeping tonicity within a narrow range of osmolality around 280 mosmol/L. When water metabolism is disturbed and total body water increases, hyponatremia, hypotonicity, and water intoxication occur; when total body water decreases, hypernatremia, hypertonicity, and dehydration occur. B. Extracellular blood volume and pressure are an integrated function of total body Na+

1	water intoxication occur; when total body water decreases, hypernatremia, hypertonicity, and dehydration occur. B. Extracellular blood volume and pressure are an integrated function of total body Na+ (TB Na+), total body H2O (TB H2O), vascular tone, heart rate, and stroke volume that modulates volume and pressure in the vascular tree of the body. This extracellular blood volume is determined by net Na balance under the control of taste, baroreception, habit, Na+ reabsorption, macula densa/tubuloglomerular feedback, and natriuretic peptides. When Na+ metabolism is disturbed and total body Na+ increases, edema occurs; when total body Na+ is decreased, volume depletion occurs. ADH, antidiuretic hormone; AQP2, aquaporin-2.

1	connectivity and adjacency to a minimal blood-brain barrier, modulate the downstream release of vasopressin by the posterior lobe of the pituitary gland. Secretion is stimulated primarily by changing tonicity and secondarily by other nonosmotic signals such as variable blood volume, stress, pain, nausea, and some drugs. The release of vasopressin by the posterior pituitary increases linearly as plasma tonicity rises above normal, although this varies, depending on the perception of extracellular volume (one form of cross-talk between mechanisms that adjudicate blood volume and osmoregulation). Changing the intake or excretion of water provides a means for adjusting plasma tonicity; thus, osmoregulation governs water balance.

1	The kidneys play a vital role in maintaining water balance through the regulation of renal water excretion. The ability to concentrate urine to an osmolality exceeding that of plasma enables water conservation, whereas the ability to produce urine more dilute than plasma promotes excretion of excess water. For water to enter or exit a cell, the cell membrane must express aquaporins. In the kidney, aquaporin-1 is constitutively active in all water-permeable segments of the proximal and distal tubules, whereas vasopressin-regulated aquaporin-2, -3, and -4 in the inner medullary collecting duct promote rapid water permeability. Net water reabsorption is ultimately driven by the osmotic gradient between dilute tubular fluid and a hypertonic medullary interstitium.

1	The perception of extracellular blood volume is determined, in part, by the integration of arterial tone, cardiac stroke volume, heart rate, and the water and solute content of extracellular fluid. Na+ and accompanying anions are the most abundant extracellular effective osmoles and together support a blood volume around which pressure is generated. Under normal conditions, this volume is regulated by sodium balance (Fig. 332e-4B), and the balance between daily Na+ intake and excretion is under the influence of baroreceptors in regional blood vessels and vascular hormone sensors modulated by atrial natriuretic peptides, the renin-angiotensin-aldosterone system, Ca2+ signaling, adenosine, vasopressin, and the neural adrenergic axis. If Na+ intake exceeds Na+ excretion (positive Na+ balance), then an increase in blood volume will trigger a proportional increase in urinary Na+ excretion. Conversely, when Na+ intake is less than urinary excretion (negative Na+ balance), blood volume will

1	then an increase in blood volume will trigger a proportional increase in urinary Na+ excretion. Conversely, when Na+ intake is less than urinary excretion (negative Na+ balance), blood volume will decrease and trigger enhanced renal Na+ reabsorption, leading to decreased urinary Na+ excretion.

1	The renin-angiotensin-aldosterone system is the best-understood hormonal system modulating renal Na+ excretion. Renin is synthesized and secreted by granular cells in the wall of the afferent arteriole. Its secretion is controlled by several factors, including β1-adrenergic stimulation to the afferent arteriole, input from the macula densa, and prostaglandins. Renin and ACE activity eventually produce angiotensin II that directly or indirectly promotes renal Na+ and water reabsorption. Stimulation of proximal tubular Na+/H+ exchange by angiotensin II directly increases Na+ reabsorption. Angiotensin II also promotes Na+ reabsorption along the collecting duct by stimulating aldosterone secretion by the adrenal cortex. Constriction of the efferent glomerular arteriole by angiotensin II indirectly increases the filtration fraction and raises peritubular capillary oncotic pressure to promote tubular Na+ reabsorption. Finally, angiotensin II inhibits renin secretion through a negative

1	indirectly increases the filtration fraction and raises peritubular capillary oncotic pressure to promote tubular Na+ reabsorption. Finally, angiotensin II inhibits renin secretion through a negative feedback loop. Alternative metabolism of angiotensin by ACE2 generates the vasodilatory peptide angiotensin 1-7 that acts through Mas receptors to counterbalance several actions of angiotensin II on blood pressure and renal function (Fig. 332e-2C).

1	Aldosterone is synthesized and secreted by granulosa cells in the adrenal cortex. It binds to cytoplasmic mineralocorticoid receptors in the collecting duct principal cells that increase activity of ENaC, apical membrane K+ channel, and basolateral Na+/K+-ATPase. These effects 332e-11 are mediated in part by aldosterone-stimulated transcription of the gene encoding serum/glucocorticoid-induced kinase 1 (SGK1). The activity of ENaC is increased by SGK1-mediated phosphorylation of Nedd4-2, a protein that promotes recycling of the Na+ channel from the plasma membrane. Phosphorylated Nedd4-2 has impaired interactions with ENaC, leading to increased channel density at the plasma membrane and increased capacity for Na+ reabsorption by the collecting duct.

1	Chronic exposure to aldosterone causes a decrease in urinary Na+ excretion lasting only a few days, after which Na+ excretion returns to previous levels. This phenomenon, called aldosterone escape, is explained by decreased proximal tubular Na+ reabsorption following blood volume expansion. Excess Na+ that is not reabsorbed by the proximal tubule overwhelms the reabsorptive capacity of more distal nephron segments. This escape may be facilitated by atrial natriuretic peptides that lose their effectiveness in the clinical settings of heart failure, nephrotic syndrome, and cirrhosis, leading to severe Na+ retention and volume overload.

1	Chapter 332e Cellular and Molecular Biology of the Kidney adaptation of the Kidney to Injury Joseph V. Bonventre Many years ago Claude Bernard (1878) introduced the concepts of milieu extérieur (the environment where an organism lives) and a 333e milieu intérieur (the environment in which the tissues of that organism live). He argued that the milieu intérieur varied very little and that there were vital mechanisms that functioned to maintain this internal environment constant. Walter B. Cannon later extended these concepts by recognizing that the constancy of the internal state, which he termed the homeostatic state, was evidence of physiologic mechanisms that act to maintain this minimal variability. In higher animals, the plasma is maintained remarkably constant in composition both within an individual and among individuals. The kidney plays a vital role in this constancy. The kidney changes the composition of the urine to maintain electrolyte and acid-base balance and can produce

1	an individual and among individuals. The kidney plays a vital role in this constancy. The kidney changes the composition of the urine to maintain electrolyte and acid-base balance and can produce hormones that can maintain constancy of blood hemoglobin and mineral metabolism. When the kidney is injured, the remaining functional mass responds and attempts to continue to maintain the milieu intérieur. It is remarkable how well the residual nephrons can perform in this task so that in many cases homeostasis is maintained until the glomerular filtration rate (GFR) drops to very low levels. At this point, the functional tissue can no longer compensate. In this chapter, we will discuss a number of these compensatory adaptations that the kidney makes in response to injury in an attempt to protect itself and protect the milieu intérieur. A theme that permeates, however, is that these adaptive processes can often be maladaptive and contribute to enhanced renal dysfunction, facilitating a

1	itself and protect the milieu intérieur. A theme that permeates, however, is that these adaptive processes can often be maladaptive and contribute to enhanced renal dysfunction, facilitating a positive feedback process that is inherently unstable.

1	Renal disease is associated with a reduction in functional nephrons. The rest of the kidney adapts to this reduction by increasing blood flow to and the size of the remaining glomeruli and increasing size and function of the remaining tubules. Robert Platt, in 1936, argued that “…a high glomerular pressure, together with loss of nephrons (destroyed by disease) [is] an explanation of the peculiarities of renal function in this stage of kidney disease.” The raised glomerular pressure will increase the amount of filtrate produced by each nephron and thus compensate for a time for the destruction of part of the kidney. But eventually there are too few nephrons remaining to produce an adequate filtrate, even though they may work under the highest possible pressure, associated with a high systemic blood pressure. The responses to kidney injury can be both adaptive and maladaptive, and in many cases, the early adaptive responses can become maladaptive over time, leading to progressive

1	systemic blood pressure. The responses to kidney injury can be both adaptive and maladaptive, and in many cases, the early adaptive responses can become maladaptive over time, leading to progressive decline in the anatomic and functional integrity of the kidney. As described previously, the early responses are likely in many cases motivated by attempts to maintain the constancy of the milieu intérieur for the survival of the organism (Claude Bernard).

1	Barry Brenner in the 1960s and 1970s carried out micropuncture experiments to define the pressures in glomerular capillaries as well as afferent and efferent resistances and modeled the behavior of the factors that governed glomerular filtration in health and disease. According to the Brenner Hyperfiltration Hypothesis, a reduction in the number of nephrons results in glomerular hypertension, hyperfiltration, and enlargement of glomeruli and this hyperfiltration results in damage to those glomeruli over time and ultimately decreased kidney function. According to this hypothesis, a positive feedback process is set into motion whereby injury to the glomeruli will result in further hyperfiltration to other glomeruli and hence more accelerated injury to those glomeruli. Since nephrons are not generated after 34–36 weeks of gestation or after birth (if earlier than 34–36 weeks) in humans, this hypothesis implies a deterministic effect of low nephron numbers at birth. There is over a

1	not generated after 34–36 weeks of gestation or after birth (if earlier than 34–36 weeks) in humans, this hypothesis implies a deterministic effect of low nephron numbers at birth. There is over a 10-fold variation in the number of nephrons per kidney in the population (200,000 to over 2.5 million). This variation 333e-1 is not explained by kidney size in the adult. Children born with low birth weights would be more prone to kidney disease as adults. There are many reasons why there might be reduced nephron numbers at birth: developmental abnormalities, genetic predisposition, and environmental factors, such as malnutrition. There are thought to be interactions between these various factors. Reduced nephron mass can also occur with chronic kidney disease (CKD) in the adult, and the response of the kidney is similar qualitatively with hyperfiltration of the remaining nephrons.

1	Developmental Abnormalities There are many congenital abnormalities of the kidney and urinary tract (CAKUT). Dysplastic kidneys have varying degrees of abnormalities that interfere with their function. Anatomically abnormal kidneys can be associated with abnormalities of the lower urinary tract. Urinary tract abnormalities resulting in obstruction or vesicoureteric reflux can dramatically alter the normal development of the kidney nephrons. Dysplastic or hypoplastic kidneys can be cystic in patterns that are distinct from polycystic kidney disease. Of course, autosomal recessive kidney disease can result in widespread cyst formation.

1	Hypoplastic kidneys are characterized by a reduced number of functional nephrons. One definition of hypoplastic kidneys is as follows: “Kidney mass below two standard deviations of that of age-matched normal [individuals] or a combined kidney mass of less than half normal for the patient’s age.” Renal agenesis and cystic dysplasia often affects only one kidney. This results in hypertrophy of the other kidney if it is unaffected by any congenital abnormality itself. Although there is hypertrophy in size, it is not clear if this is associated with an increase in the number of nephrons on the contralateral side.

1	The prevalence of CAKUT has been generally found to be between 0.003 and 0.2%, depending on the population studied. This excludes fetuses with transient upper renal tract dilatation likely related to the high rate of fetal urine flow rate. In the adult U.S. Renal Data System (USRDS) of patients with end-stage kidney disease, approximately 0.6% are listed as having dysplastic or hypoplastic kidneys as a primary cause of the disease. This is likely an underestimate, however, because many patients with “small kidneys” may be misdiagnosed with chronic glomerulonephritis or chronic pyelonephritis.

1	Environmental Contributions to Reduced Nephron Mass The most important environmental factor responsible for reduced nephron number is growth restriction within the uterus. This has been associated with disease processes such as diabetes mellitus in the mother, but there also is a strong genetic disposition. Low-birth-weight children are more likely to be born to mothers who, themselves, were born with low birth weight. There are clearly other environmental factors. Caloric restriction during pregnancy in humans has been associated with altered glucose as adults and increased risk for hypertension. In one study, it was found that if women were calorie restricted in midgestation, the time of most rapid nephrogenesis, there was a threefold incidence of albuminuria in their children when they were tested as adults. Factors such as deficiency in vitamin A, sodium, zinc, or iron have been implicated as predisposing to abnormal kidney development. Other environmental factors that can

1	they were tested as adults. Factors such as deficiency in vitamin A, sodium, zinc, or iron have been implicated as predisposing to abnormal kidney development. Other environmental factors that can influence kidney development are medications taken by the mother, such as dexamethasone, angiotensin-converting enzyme inhibitors, and angiotensin receptor antagonists (Table 333e-1). Protein restriction in mice during pregnancy can reduce lifespan of the offspring by 200 days. Obesity may play an important role in determining kidney outcome long term in patients with reduced kidney mass. It has been shown in mice fed a high-fat diet that the rodents that had reduced nephron number had a greater incidence of hypertension and renal fibrosis.

1	Chapter 333e Adaptation of the Kidney to Injury Implications of Low Nephron Number at Birth David Barker was the first to describe the association between low birth weight and later cardiovascular death. This was followed by studies relating low birth weight to risk for diabetes, stroke, hypertension, and CKD. It has been found that there is an inverse relationship between nephron number and blood pressure in adults. This relationship was found in Caucasians but not in African Americans. Approximately one-third of children with a single functioning kidney at the age of 10 years had signs of renal injury as determined by the presence of hypertension, albuminuria, or the use of renoprotective drugs. Another study revealed that 20–40% of patients born with a single functional kidney had renal failure requiring dialysis by 30 years of age.

1	In the early stages of CKD, there are many adaptations structurally and functionally that limit the consequences of the loss of nephrons on total-body homeostasis. In later stages of disease, however, these adaptations are insufficient to counteract the consequences of nephron loss and in fact often become maladaptive.

1	Counterbalance Renal counterbalance was defined by Hinman in 1923 as “an attempt on the part of the less injured or uninjured portion (of the kidney) to take over the work of the more injured portion.” Hinman defined “renal reserve” to be of two types: “native reserve, which is the normal physiological response to stimulation . . . and acquired reserve, which involves growth or compensation due to overstimulation.” It was known that removal of one kidney results in an increase in size of the contralateral kidney. If, instead of nephrectomy, one kidney is rendered ischemic and the other left intact, there is a resultant atrophy of the postischemic kidney. If the contralateral kidney is removed, however, before the atrophy becomes too severe, then the postischemic kidney increases markedly in size. With the contralateral kidney in place, there is vasoconstriction and reduced renal blood flow to the postischemic kidney. This is rapidly reversed, however, when the contralateral normal

1	in size. With the contralateral kidney in place, there is vasoconstriction and reduced renal blood flow to the postischemic kidney. This is rapidly reversed, however, when the contralateral normal kidney is removed. The factors responsible for the persistent initial (prenephrectomy) vasoconstriction and those responsible for the rapid vasodilation and enhanced growth after contralateral nephrectomy are unknown.

1	Hypertrophy Because nephrons of mammals, in contrast to those of fish, cannot regenerate, the loss of functional units of the kidney, either due to disease or surgery, results in anatomic and functional changes in the remaining nephrons. As described above, there is increased blood flow to remaining glomeruli with potentially adverse effects over time of the resultant increased size of the remaining glomeruli and hyperfiltration (Fig. 333e-1). In addition, there is hypertrophy of the tubules. Some of the mediators of this hypertrophy of the remaining functional tubules are listed in Table 333e-2. In the adult, within a few weeks after unilateral nephrectomy for donation of a kidney, the GFR is approximately 70% of the prenephrectomy value. It then remains relatively stable for most patients over 15–20 years. The hyperfiltration is related to an increase in renal blood flow likely secondary to dilatation of the afferent arterioles potentially due to increases in nitric oxide (NO)

1	over 15–20 years. The hyperfiltration is related to an increase in renal blood flow likely secondary to dilatation of the afferent arterioles potentially due to increases in nitric oxide (NO) production. The rate of increase in GFR is slower in the adult than it is in the young after nephrectomy. There are a number of factors that have been implicated at the cellular and nephron level to account for the compensatory hypertrophy that ensues after removal of functional nephrons (Table 333e-2).

1	With increased blood flow to the kidney, there is glomerular hypertension (i.e., an increase in glomerular capillary pressure). There is increased wall tension and force on the capillary wall that is counteracted by contractile properties of the endothelium and elastic properties of the glomerular basement membrane. The force is conveyed to podocytes, which adapt by reinforcing cell cycle arrest and increasing cell adhesion in an adaptive attempt to maintain the delicate architecture of the interdigitating foot processes. Over time, however, these increased forces due to glomerular hypertension lead to podocyte damage and glomerulosclerosis. FIGURE 333e-1 Some of the pathophysiologic mechanisms involved with the maladaptive response to a reduction in the number of functional nephrons due to prenatal factors or postnatal disease processes.

1	Other Systemic and Renal Adaptations to Reduced Nephron Function With reduced functional nephrons, as is seen in CKD, there are many other systemic adaptations that occur to preserve the milieu intérieur because the kidney is involved in so many regulatory networks that are then stressed when there is dysfunction. In the 1960s, Neil Bricker introduced the “intact nephron hypothesis.” According to his concept, with decreases in the number of functioning nephrons, each remaining nephron has to adapt to carry a larger burden of transport, synthetic function, and regulatory function.

1	Potassium Under normal and abnormal conditions, most of the filtered potassium is reabsorbed in the proximal tubule so that excretion is determined by secretion by the distal nephron. Potassium handling is altered in CKD protecting the organism somewhat from lethal hyperkalemia. Hyperkalemia is a common feature of individuals with CKD. Hyperkalemia (if not severe and dangerous) is adaptive in that it promotes potassium secretion by the principal cells of the collecting duct. When patients with CKD are given a potassium load, they can excrete it at the same rate as patients with normal renal function except that they do so at a higher serum potassium, consistent with the view that the hyperkalemia facilitates potassium excretion. The direct effect of hyperkalemia on potassium secretion by the distal nephron is independent of changes in aldosterone levels, but “normal” levels of aldosterone are necessary to see the effect of hyperkalemia on potassium excretion. Elevated potassium

1	by the distal nephron is independent of changes in aldosterone levels, but “normal” levels of aldosterone are necessary to see the effect of hyperkalemia on potassium excretion. Elevated potassium stimulates the production of aldosterone, and this effect is also seen in patients with CKD. Aldosterone increases the density and activity of the basolateral Na+-K+ ATPase and

1	Increased renal blood flow Increased tubular absorption of Na with decreased distal delivery and decreased afferent arterial resistance due to adaptive tubuloglomerular feedback Hepatocyte growth factor Glucose transporters Increased renal nerve activity Insulin-like growth factor Mammalian target of rapamycin (mTOR) signaling pathway activation p21Waf1, p27kip1, and p57kip2 Transforming growth factor β the number of Na+ channels in the apical membrane of the collecting duct. In CKD, the excretion of the dietary load of potassium occurs at the expense of an elevation in serum potassium concentrations.

1	sodium As renal function is reduced with CKD, there is a reduced ability to excrete sodium. Thus, patients with advanced kidney disease are often fluid overloaded. In early disease, however, there are functional adaptations that the kidney assumes to help to maintain the milieu intérieur. With loss of functional nephrons, the remaining nephrons are hyperperfused and are hyperfiltering in a manner that can be influenced by dietary protein intake. Although protein restriction can decrease this compensatory hyperperfusion, there is generally more sodium and water filtered and delivered to the remaining nephrons. There is some preservation of glomerulotubular balance with increased proximal tubule sodium and water reabsorption associated with increased levels of the Na/H exchanger in apical membranes of the tubule. The tubuloglomerular feedback (TGF) of the remaining nephrons is sensitive to sodium intake. With high sodium intake in normal renal function, a negative feedback process

1	membranes of the tubule. The tubuloglomerular feedback (TGF) of the remaining nephrons is sensitive to sodium intake. With high sodium intake in normal renal function, a negative feedback process occurs by which increased distal delivery results in reduced GFR and hence filtration of sodium. In CKD, the TGF becomes a positive feedback process by which increased distal delivery results in increased filtration so that the need to excrete an increased amount of sodium per nephron is achieved. This conversion from a negative feedback process to a positive feedback process may be due to conversion of an adenosine-dominated vasoconstrictive feedback on the afferent arteriole of the glomerulus to a NO-dominated vasodilatory feedback. Like so many of these adaptive responses, this one may turn maladaptive, resulting in higher intraglomerular hydrostatic pressures with increased mechanical strain on the glomerular capillary wall and podocytes and increased glomerulosclerosis as a consequence.

1	acid-base homeostasis The kidneys excrete approximately 1 mEq/kg per day of dietary acid load under normal dietary conditions. With decreased kidney functional mass, there is an adaptive response to increase H+ excretion by the remaining functional nephrons. This takes the form of enhanced nephron ammoniagenesis and increased distal nephron H+ ion secretion, which is mediated by the reninangiotensin system and endothelin-1. NH3 is produced by deamidization of glutamine in the proximal tubule. NH3 is converted to NH4+ in the collecting duct, where it buffers the secreted H+. It has been argued, however, that these mechanistic attempts to enhance H+ secretion can be maladaptive in that they can contribute to kidney inflammation and fibrosis and hence facilitate the progression of CKD.

1	mineral metabolism In CKD, there is a decrease in the ability of the kidney to excrete phosphate and produce 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. There is a resultant increase in serum phosphate and reduction in serum calcium (Fig. 333e-2). In response, the body adapts by increasing production of parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF-23) in an attempt to increase phosphaturia. The elevated levels of PTH act on bone to increase bone resorption and on osteocytes to increase FGF-23 expression. Elevated levels of PTH increase FGF-23 expression by activating protein kinase A and wnt signaling in osteoblast-like cells. There are a number of other factors that increase bone FGF-23 production in CKD including systemic acidosis, altered hydroxyapatite metabolism, changes in bone matrix, and release of low-molecular-weight FGFs. Although the production of PTH and FGF-23 initially are adaptive attempts to maintain body phosphate levels by enhancing excretion by the

1	in bone matrix, and release of low-molecular-weight FGFs. Although the production of PTH and FGF-23 initially are adaptive attempts to maintain body phosphate levels by enhancing excretion by the kidney, they become maladaptive due to systemic effects on the cardiovascular system and bone, as renal function continues to deteriorate. PTH and FGF-23 decrease the kidney’s ability to reabsorb phosphate by decreasing the levels of the sodium-phosphate cotransporters NaPi2a and NaPi2c on the apical and basolateral membranes of the renal tubule. FGF-23 also reduces the ability of the kidney to generate 1,25(OH)2D3. In the parathyroid gland, the FGF-23 receptor, the klotho-fibroblast growth factor 1 complex, is downregulated with a consequent loss of the normal action of FGF-23 to downregulate PTH production. PTH and FGF-23 have been implicated in the cardiovascular disease that is so characteristic of patients with CKD. With CKD, there is less klotho expression in ˜Renal 1,25 (OH)2D3

1	FIGURE 333e-2 Modification of the trade-off hypothesis of Slatopolsky and Bricker as it relates to the adaptation of the body to decreased functional renal mass in an attempt to maintain calcium and phosphate stores and serum levels. 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; FGF-23, fibroblast growth factor-23; GI, gastrointestinal; PTH, parathyroid hormone. the kidney and the parathyroid glands. Klotho deficiency contributes to soft tissue calcifications in CKD. FGF-23 has been associated with increased mortality in CKD and has been reported to be involved causally in the development of left ventricular hypertrophy. PTH also has been reported to directly affect rat myocardial cells, increasing calcium entry into the cells and contributing to death of the cells.

1	Preconditioning represents activation by the organism of intrinsic defense mechanisms to cope with pathologic conditions. Ischemic preconditioning is the phenomenon whereby a prior ischemic insult renders the organ resistant to a subsequent ischemic insult. Renal protection afforded by prior renal injury was described approximately 100 years ago, in 1912, by Suzuki, who noted that the kidney became resistant to uranium nephrotoxicity if the animal had previously been exposed to a sublethal dose of uranium. This resistance of the renal epithelium to recurrent toxic injury was proposed to be a defense mechanism of the kidney. There have been a number of studies over the years demonstrating that preconditioning with a number of renal toxicants leads to protection against injury associated with a second exposure to the same toxicant or to another nephrotoxicant. It is not, however, a universal finding that toxins confer resistance to subsequent insults.

1	Kidney ischemic preconditioning is the conveyance of protection against ischemia due to prior exposure of the kidney to sublethal episodes of ischemia. In some experiments in rodents, these prior exposures were short (e.g., 5 min) and repeated or longer. Subsequent protection was generally found at 1–2 h or up to 48 h, but there has been a report of protection in the mouse for up to 12 weeks after the preconditioning exposures. Unilateral ischemia, with the contralateral kidney left alone, was also protective against a subsequent ischemic insult to the postischemic kidney, revealing that systemic uremia was not necessary for protection.

1	Remote Ischemic Preconditioning Remote ischemic preconditioning is a therapeutic strategy by which protection can be afforded in one vascular bed by ischemia to another vascular bed in the same organ or a different organ. A large number of studies have demonstrated that ischemia to one organ protects against ischemia to another. There are very few mechanistic studies of remote preconditioning in the kidney. In one study, naloxone blocked preconditioning in the kidney, implicating opiates as effectors. Remote preconditioning induced by ischemia to the muscle of the arm induced by a blood pressure cuff can result in protection of the kidney against a subsequent insult, such as one related to contrast agents in humans. Some of the cellular processes and signaling mechanisms proposed to explain preconditioning in the kidney and other organs are listed in Table 333e-3. These protective Chapter 333e Adaptation of the Kidney to Injury

1	Chapter 333e Adaptation of the Kidney to Injury FaCtors anD proCesses ImpLICateD as proteCtIve meDIators oF IsChemIC preConDItIonIng

1	Decrease in genes regulating inflammation (cytokine synthesis, leukocyte chemotaxis, adhesion, exocytosis, innate immune signaling pathways) processes, most of which have been identified in the heart, involve multiple signaling pathways that affect decreased apoptosis, inhibition of mitochondrial permeability transition pores, activation of survival pathways, autophagy, and other pathways involved in reducing energy consumption or reactive oxygen production. In a study from our laboratory, inducible NO synthase was found to be an important contributor to the adaptive response to kidney injury, which results in protection against a subsequent insult. Identification of the responsible protective factor(s) mediating the advantageous adaptive response to remote ischemic preconditioning would provide a therapeutic approach for prevention of acute kidney injury or facilitation of a protective adaptation to kidney injury.

1	ADAPTIVE RESPONSE OF THE KIDNEY TO ACUTE INJURY Adaptive Response to Hypoxic Injury Hypoxia plays a role in ischemic, septic, and toxic acute kidney injury. Many conditions result in a global or regional impairment of oxygen delivery. This is particularly important in the outer medulla where there is baseline reduced oxygen tension and a complex capillary network that, by its nature, is susceptible to interruption. In addition, the S3 segment of the proximal tubule is very dependent on oxidative metabolism, whereas the medullary thick ascending limb of the nephron that also traverses the outer medulla can adapt to hypoxia by converting to glycolysis as a primary energy source.

1	One proposed adaptive response to hypoxia is a reduction in glomerular filtration with consequent reduction in “work” requirement for reabsorption of solutes by the tubule. This was termed acute renal success by Thurau many years ago. The importance of this has been questioned, however, because there is no significant reduction in renal oxygen consumption in post–cardiac surgery patients with acute kidney injury in the setting of reduced GFR and renal blood flow.

1	If hypoxia or other influences, such as toxins, damage the proximal tubule and interfere with reabsorption of sodium and water, it is important that the kidney adapt in such a way so that there is not a large natriuresis that might compromise intravascular volume and blood pressure. This is accomplished, at least in part, by tubuloglomerular feedback (TGF). The increased distal delivery of salt and water results in a homeostatic adaptation to decrease glomerular filtration and hence decrease tubular delivery of salt and water through the glomerulus and reduce the delivery to the distal nephron. This adaptive response to acute injury is different from the role of TGF in CKD, as we have discussed previously in this chapter. In chronic disease with reduced nephron function, there is a steady-state need to increase excretion of sodium, whereas with acute injury, excretion of sodium is reduced.

1	Many genes are activated by hypoxia that are adaptive in serving to protect the cell and organ. With hypoxia, hypoxia-inducible factor (HIF) 1α rapidly accumulates due to the inhibition of the HIF prolylhydroxylases, which normally promote HIF1α proteasomal degradation. HIF1α then dimerizes with HIF1β and the dimer moves to the nucleus, where it upregulates a number of genes whose protein products are involved in energy metabolism, angiogenesis, and apoptosis, enhancing oxygen delivery and metabolic adaptation to hypoxia. This takes the form of a complex interplay among factors that regulate perfusion, cellular redox state, and mitochondrial function. For example, upregulation of NO production by sepsis results in vasodilatation and reduction in mitochondrial respiration and oxygen consumption. In addition, HIF1 activation in endothelial cells may be important for adaptive preservation of the microvasculature during and after hypoxia. Better understanding of the role that the HIFs

1	In addition, HIF1 activation in endothelial cells may be important for adaptive preservation of the microvasculature during and after hypoxia. Better understanding of the role that the HIFs play in protective adaptation has led to an aggressive development of HIF prolyl-hydroxylase inhibitors by biotechnology and pharmaceutical companies for clinical use.

1	Adaptive Response to Toxic Injury Specific to the Proximal Tubule One can model an acute kidney injury by genetically inserting a Simian diphtheria toxin (DT) receptor into the proximal tubule and then adding either a single dose of DT or multiple doses of the toxin. Repair of the kidney after a single dose of DT can be shown to be adaptive with few longer term sequelae. There is a very robust proliferative response of the proximal tubule cells to replace the cells that die as a result of the DT. Ultimately the inflammation resolves, and there is little, if any, residual interstitial inflammation, expansion, or matrix deposition.

1	Maladaptive Response of the Kidney to Acute Injury By contrast to the above adaptive repair that occurs after a single insult, after three doses of DT administered at weekly intervals, there is maladaptive repair with development over time of a chronic interstitial infiltrate, increased myofibroblast proliferation, tubulointerstitial fibrosis, and tubular atrophy, as well as an increase in serum creatinine (0.6 ± 0.1 mg/dL vs 0.18 ± 0.02 mg/dL in control mice) by week 5, 2 weeks after the last dose in the thrice-treated animals. There is a dramatic increase in the number of interstitial cells that expressed the platelet-derived growth factor receptor β (pericytes/perivascular fibroblasts), αSMA (myofibroblasts), FSP-1/S100A4 (fibroblast specific protein-1), and F4/80 (macrophages). In addition, there is loss of endothelial cells, interstitial capillaries, and development of focal global and segmental glomerulosclerosis.

1	It has become increasingly recognized as a result of large epidemiologic studies that even mild forms of acute kidney injury are associated with adverse shortand long-term outcomes including onset or progression of CKD and more rapid progression to end-stage kidney disease. Experimental models in animals, such as the DT model described above, provide pathophysiologic explanations for how the effects of acute injury can lead to chronic inflammation, vascular rarefaction, tubular cell atrophy, interstitial fibrosis, and glomerulosclerosis. Recurrent specific tubular injury leads to a pattern very typical of CKD in humans: tubular atrophy, interstitial chronic inflammation and fibrosis, vascular rarefaction, and glomerulosclerosis. The mechanisms involved in the development of glomerulosclerosis evoked by primary tubular injury may be multifactorial. Damage to nephron segments may lead to sluffing of cells into the lumen and to tubular obstruction. Progressive narrowing of the early

1	evoked by primary tubular injury may be multifactorial. Damage to nephron segments may lead to sluffing of cells into the lumen and to tubular obstruction. Progressive narrowing of the early proximal tubule near the glomerular tuft can lead to a sclerotic atubular glomerulus like those that are seen with ureteral obstruction. There may be paracrine signaling from injured and regenerating/undifferentiated epithelium to directly impact the glomerulus. Alternatively, a progressive tubulointerstitial reaction originating around atrophic and undifferentiated tubules may directly encroach upon the glomerular tuft. The loss of interstitial capillaries may lead to a progressive reduction of glomerular blood flow with ischemia to the glomerulus and to the kidney regions perfused by the postglomerular capillaries. This speaks to the fact that primary tubular injury can trigger a response that adversely affects multiple compartments of the kidney and leads to a positive feedback process,

1	capillaries. This speaks to the fact that primary tubular injury can trigger a response that adversely affects multiple compartments of the kidney and leads to a positive feedback process, involving loss of capillaries, glomerulosclerosis, persistent ischemia, tubular atrophy, increased fibrosis, and ultimately kidney failure.

1	Sushrut S. Waikar, Joseph V. Bonventre

1	Acute kidney injury (AKI), previously known as acute renal failure, is characterized by the sudden impairment of kidney function resulting in the retention of nitrogenous and other waste products normally cleared by the kidneys. AKI is not a single disease but, rather, a designation for a heterogeneous group of conditions that share common diagnostic features: specifically, an increase in the blood urea nitrogen (BUN) concentration and/or an increase in the plasma or serum creatinine (SCr) concentration, often associated with a reduction in urine volume. It is important to recognize that AKI is a clinical diagnosis and not a structural one. A patient may have AKI without injury to the kidney parenchyma. AKI can range in severity from asymptomatic and transient changes in laboratory parameters of glomerular filtration rate (GFR), to overwhelming and rapidly fatal derangements in effective circulating volume regulation and electrolyte and acid-base composition of the plasma.

1	AKI complicates 5–7% of acute care hospital admissions and up to 30% of admissions to the intensive care unit, particularly in the setting of diarrheal illnesses, infectious diseases like malaria and leptospirosis, and natural disasters such as earthquakes. The incidence of AKI has grown by more than fourfold in the United States since 1988 and is estimated to have a yearly incidence of 500 per 100,000 population, higher than the yearly incidence of stroke. AKI is associated with a markedly increased risk of death in hospitalized individuals, particularly in those admitted to the ICU where in-hospital mortality rates may exceed 50%. AKI increases the risk for the development or worsening of chronic kidney disease. Patients who survive and recover from an episode of severe AKI requiring dialysis are at increased risk for the later development of dialysis-requiring end-stage kidney disease. AKI may be community-acquired or hospital-acquired. Common causes of community-acquired AKI

1	dialysis are at increased risk for the later development of dialysis-requiring end-stage kidney disease. AKI may be community-acquired or hospital-acquired. Common causes of community-acquired AKI include volume depletion, adverse effects of medications, and obstruction of the urinary tract. The most common clinical settings for hospital-acquired AKI are sepsis, major surgical procedures, critical illness involving heart or liver failure, intravenous iodinated contrast administration, and nephrotoxic medication administration.

1	AKI is also a major medical complication in the developing world, where the epidemiology differs from that in developed countries due to differences in demographics, economics, geography, and comorbid disease burden. While certain features of AKI are common to both—particularly since urban centers of some developing countries increasingly resemble those in the developed world—many etiologies for AKI are region-specific such as envenomations from snakes, spiders, caterpillars, and bees; infectious causes such as malaria and leptospirosis; and crush injuries and resultant rhabdomyolysis from earthquakes. The causes of AKI have traditionally been divided into three broad categories: prerenal azotemia, intrinsic renal parenchymal disease, and postrenal obstruction (Fig. 334-1).

1	The causes of AKI have traditionally been divided into three broad categories: prerenal azotemia, intrinsic renal parenchymal disease, and postrenal obstruction (Fig. 334-1). Prerenal azotemia (from “azo,” meaning nitrogen, and “-emia”) is the most common form of AKI. It is the designation for a rise in SCr or BUN concentration due to inadequate renal plasma flow and intraglomerular hydrostatic pressure to support normal glomerular Nephrotoxins Exogenous: Iodinated contrast, aminoglycosides, cisplatin, amphotericin B Endogenous: Hemolysis, rhabdomyolysis, myeloma, intratubular crystals FIGuRE 334-1 Classification of the major causes of acute kidney injury. ACE-I, angiotensin-converting enzyme inhibitor-I; ARB, angiotensin receptor blocker; NSAIDs, nonsteroidal anti-inflammatory drugs; TTP-HUS, thrombotic thrombocytopenic purpura–hemolytic-uremic syndrome.

1	filtration. The most common clinical conditions associated with prerenal azotemia are hypovolemia, decreased cardiac output, and medications that interfere with renal autoregulatory responses such as nonsteroidal anti-inflammatory drugs (NSAIDs) and inhibitors of angiotensin II (Fig. 334-2). Prerenal azotemia may coexist with other forms of intrinsic AKI associated with processes acting directly on the renal parenchyma. Prolonged periods of prerenal azotemia may lead to ischemic injury, often termed acute tubular necrosis (ATN). By definition, prerenal azotemia involves no parenchymal damage to the kidney and is rapidly reversible once intraglomerular hemodynamics are restored.

1	Normal GFR is maintained in part by the relative resistances of the afferent and efferent renal arterioles, which determine the glomerular plasma flow and the transcapillary hydraulic pressure gradient that drive glomerular ultrafiltration. Mild degrees of hypovolemia and reductions in cardiac output elicit compensatory renal physiologic changes. Because renal blood flow accounts for 20% of the cardiac output, renal vasoconstriction and salt and water reabsorption occur as homeostatic responses to decreased effective circulating volume or cardiac output in order to maintain blood pressure and increase intravascular volume to sustain perfusion to the cerebral and coronary vessels. Mediators of this response include angiotensin II, norepinephrine, and vasopressin (also termed antidiuretic hormone). Glomerular filtration can be maintained despite reduced renal blood flow by angiotensin II–mediated renal efferent vasoconstriction, which maintains glomerular capillary hydrostatic pressure

1	Glomerular filtration can be maintained despite reduced renal blood flow by angiotensin II–mediated renal efferent vasoconstriction, which maintains glomerular capillary hydrostatic pressure closer to normal and thereby prevents marked reductions in GFR if renal blood flow reduction is not excessive.

1	In addition, a myogenic reflex within the afferent arteriole leads to dilation in the setting of low perfusion pressure, thereby maintaining glomerular perfusion. Intrarenal biosynthesis of vasodilator prostaglandins (prostacyclin, prostaglandin E2), kallikrein and kinins, and possibly nitric oxide (NO) also increase in response to low renal perfusion pressure. Autoregulation is also accomplished by tubuloglomerular feedback, in which decreases in solute delivery to the macula densa (specialized cells within the distal tubule) elicit dilation of the juxtaposed afferent arteriole in order to maintain glomerular perfusion, a mechanism mediated, in part, by NO. There is a limit, however, to the ability of these counterregulatory mechanisms to maintain GFR in the face of systemic hypotension. Even in healthy adults, renal autoregulation usually fails once the systolic blood pressure falls below 80 mmHg.

1	A number of factors determine the robustness of the autoregulatory response and the risk of prerenal azotemia. Atherosclerosis, long-standing hypertension, and older age can lead to hyalinosis and myointimal hyperplasia, causing structural narrowing of the intrarenal arterioles and impaired capacity for renal afferent vasodilation. In chronic kidney disease, renal afferent vasodilation may be operating at maximal capacity in order to maximize GFR in response to reduced functional renal mass. Drugs can affect the compensatory changes evoked to maintain GFR. NSAIDs inhibit renal prostaglandin production, limiting renal afferent vasodilation. Angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) limit renal efferent vasoconstriction; this effect is particularly pronounced in patients with bilateral renal artery stenosis or unilateral renal artery stenosis (in the case of a solitary functioning kidney) because renal efferent vasoconstriction is needed to

1	pronounced in patients with bilateral renal artery stenosis or unilateral renal artery stenosis (in the case of a solitary functioning kidney) because renal efferent vasoconstriction is needed to maintain GFR due to low renal perfusion. The combined use of NSAIDs with ACE inhibitors or ARBs poses a particularly high risk for developing prerenal azotemia.

1	Many individuals with advanced cirrhosis exhibit a unique hemodynamic profile that resembles prerenal azotemia despite total-body volume overload. Systemic vascular resistance is markedly reduced due to primary arterial vasodilation in the splanchnic circulation, resulting ultimately in activation of vasoconstrictor responses similar to those seen in hypovolemia. AKI is a common complication in this setting, and it can be triggered by volume depletion and spontaneous bacterial peritonitis. A particularly poor prognosis is seen in the case of type 1 hepatorenal syndrome, in which AKI without an alternate cause (e.g., shock and nephrotoxic drugs) persists despite volume administration and withholding of diuretics. Type 2 hepatorenal syndrome is a less severe form characterized mainly by refractory ascites.

1	The most common causes of intrinsic AKI are sepsis, ischemia, and nephrotoxins, both endogenous and exogenous (Fig. 334-3). In many cases, prerenal azotemia advances to tubular injury. Although classically termed “acute tubular necrosis,” human biopsy confirmation of tubular necrosis is, in general, often lacking in cases of sepsis and ischemia; indeed, processes such as inflammation, apoptosis, and altered regional perfusion may be important contributors pathophysiologically. Other causes of intrinsic AKI are less common and can be conceptualized anatomically according to the major site of renal parenchymal damage: glomeruli, tubulointerstitium, and vessels.

1	FIGuRE 334-2 Intrarenal mechanisms for autoregulation of the glomerular filtration rate (GFR) under decreased perfusion pressure and reduction of the GFR by drugs. A. Normal conditions and a normal GFR. B. Reduced perfusion pressure within the autoregulatory range. Normal glomerular capillary pressure is maintained by afferent vasodilatation and efferent vasoconstriction. C. Reduced perfusion pressure with a nonsteroidal anti-inflammatory drug (NSAID). Loss of vasodilatory prostaglandins increases afferent resistance; this causes the glomerular capillary pressure to drop below normal values and the GFR to decrease. D. Reduced perfusion pressure with an angiotensin-converting enzyme inhibitor (ACE-I) or an angiotensin receptor blocker (ARB). Loss of angiotensin II action reduces efferent resistance; this causes the glomerular capillary pressure to drop below normal values and the GFR to decrease. (From JG Abuelo: N Engl J Med 357:797-805, 2007; with permission.)

1	In the United States, more than 700,000 cases of sepsis occur each year. AKI complicates more than 50% of cases of severe sepsis and greatly increases the risk of death. Sepsis is also a very important cause of AKI in the developing world. Decreases in GFR with sepsis can occur even in the absence of overt hypotension, although most cases of severe AKI typically occur in the setting of hemodynamic collapse requiring vasopressor support. While there is clearly tubular injury associated with AKI in sepsis as manifest by the presence of tubular debris and casts in the urine, postmortem examinations of kidneys from individuals with severe sepsis suggest that other factors, perhaps related to inflammation, mitochondrial dysfunction, and interstitial edema, must be considered in the pathophysiology of sepsis-induced AKI.

1	The hemodynamic effects of sepsis—arising from generalized arterial vasodilation, mediated in part by cytokines that upregulate the expression of inducible NO synthase in the vasculature—can lead to a reduction in GFR. The operative mechanisms may be excessive efferent arteriole vasodilation, particularly early in the course of sepsis, or renal vasoconstriction from activation of the sympathetic nervous system, the renin-angiotensin-aldosterone system, vasopressin, and endothelin. Sepsis may lead to endothelial damage, which results in microvascular thrombosis, activation of reactive oxygen species, and leukocyte adhesion and migration, all of which may injure renal tubular cells.

1	Healthy kidneys receive 20% of the cardiac output and account for 10% of resting oxygen consumption, despite constituting only 0.5% of the human body mass. The kidneys are also the site of one of the most hypoxic regions in the body, the renal medulla. The outer medulla is particularly vulnerable to ischemic damage because of the Glomerulonephritis • Atheroemboli (rhabdomyolysis, • Uric acid (tumor

1	Glomerulonephritis • Atheroemboli (rhabdomyolysis, • Uric acid (tumor Cortex Medulla Outer Inner Loop of Henle Loop of Henle Collecting duct Thin descending limb Thick ascending limb Thick ascending limb Pars recta tubule Proximal convoluted tubule Distal convoluted tubule Pars recta glomerulus Juxtamedullary glomerulus Distal convoluted tubule • TTP/HUS • DIC• Calcineurin inhibitors • Sepsis• Cellular debris • Exogenous • Acyclovir, methotrexate Large vessels • Renal artery embolus, dissection, vasculitis • Renal vein thrombosis • Abdominal compartment syndrome Interstitium • Allergic (PCN, rifampin, etc.) • Infection (severe pyelonephritis, Legionella, sepsis) • Infiltration (lymphoma. leukemia) • Inflammatory (Sjogren’s, tubulointerstitial nephritis uveitis), sepsis • Exogenous (contrast, cisplatin, gentamicin) • Ischemic ATN • Sepsis

1	FIGuRE 334-3 Major causes of intrinsic acute kidney injury. ATN, acute tubular necrosis; DIC, disseminated intravascular coagulation; HTN, hypertension; PCN, penicillin; TTP/HUS, thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome; TINU, tubulointerstitial nephritis-uveitis.

1	architecture of the blood vessels that supply oxygen and nutrients to the tubules. Enhanced leukocyte-endothelial interactions in the small vessels lead to inflammation and reduced local blood flow to the metabolically very active S3 segment of the proximal tubule, which depends on oxidative metabolism for survival. Ischemia alone in a normal kidney is usually not sufficient to cause severe AKI, as evidenced by the relatively low risk of severe AKI even after total interruption of renal blood flow during suprarenal aortic clamping or cardiac arrest. Clinically, AKI more commonly develops when ischemia occurs in the context of limited renal reserve (e.g., chronic kidney disease or older age) or coexisting insults such as sepsis, vasoactive or nephrotoxic drugs, rhabdomyolysis, or the systemic inflammatory states associated with burns and pancreatitis. Prerenal azotemia and ischemia-associated AKI represent a continuum of the manifestations of renal hypoperfusion. Persistent

1	the systemic inflammatory states associated with burns and pancreatitis. Prerenal azotemia and ischemia-associated AKI represent a continuum of the manifestations of renal hypoperfusion. Persistent preglomerular vasoconstriction may be a common underlying cause of the reduction in GFR seen in AKI; implicated factors for vasoconstriction include activation of tubuloglomerular feedback from enhanced delivery of solute to the macula densa following proximal tubule injury, increased basal vascular tone and reactivity to vasoconstrictive agents, and decreased vasodilator responsiveness. Other contributors to low GFR include backleak of filtrate across damaged and denuded tubular epithelium and mechanical obstruction of tubules from necrotic debris (Fig. 334-4).

1	Postoperative AKI Ischemia-associated AKI is a serious complication in the postoperative period, especially after major operations involving significant blood loss and intraoperative hypotension. The procedures most commonly associated with AKI are cardiac surgery with cardiopulmonary bypass (particularly for combined valve and bypass procedures), vascular procedures with aortic cross clamping, and intraperitoneal procedures. Severe AKI requiring dialysis occurs in approximately 1% of cardiac and vascular surgery procedures. The risk of severe AKI has been less well studied for major intraperitoneal procedures but appears to be of comparable magnitude. Common risk factors for postoperative AKI include underlying chronic kidney disease, older age, diabetes mellitus, congestive heart failure, and emergency procedures. The pathophysiology of AKI following cardiac surgery is multifactorial. Major AKI risk factors are common in the population undergoing cardiac surgery. The use of

1	failure, and emergency procedures. The pathophysiology of AKI following cardiac surgery is multifactorial. Major AKI risk factors are common in the population undergoing cardiac surgery. The use of nephrotoxic agents including iodinated contrast for cardiac imaging prior to surgery may increase the risk of AKI. Cardiopulmonary bypass is a unique hemodynamic state characterized by nonpulsatile flow and exposure of the circulation to extracorporeal circuits. Longer duration of cardiopulmonary bypass is a risk factor for AKI. In addition to ischemic

1	Pathophysiology of Ischemic Acute Renal Failure 1803 Vasoconstriction in response to: endothelin, adenosine, angiotensin II, thromboxane A2, leukotrienes, sympathetic nerve activity Vasodilation in response to: nitric oxide, PGE2, acetylcholine, bradykinin Leukocyte-endothelial adhesion, vascular obstruction, leukocyte activation, and inflammation Cytoskeletal breakdown Loss of polarity Apoptosis and necrosis Desquamation of viable FIGuRE 334-4 Interacting microvascular and tubular events contributing to the pathophysiology of ischemic acute kidney injury.

1	PGE2, prostaglandin E2. (From JV Bonventre, JM Weinberg: J Am Soc Nephrol 14:2199, 2003.) injury from sustained hypoperfusion, cardiopulmonary bypass may cause AKI through a number of mechanisms including extracorporeal circuit activation of leukocytes and inflammatory processes, hemolysis with resultant pigment nephropathy (see below), and aortic injury with resultant atheroemboli. AKI from atheroembolic disease, which can also occur following percutaneous catheterization of the aorta, or spontaneously, is due to cholesterol crystal embolization resulting in partial or total occlusion of multiple small arteries within the kidney. Over time, a foreign body reaction can result in intimal proliferation, giant cell formation, and further narrowing of the vascular lumen, accounting for the generally sub-acute (over a period of weeks rather than days) decline in renal function.

1	Burns and Acute Pancreatitis Extensive fluid losses into the extravascular compartments of the body frequently accompany severe burns and acute pancreatitis. AKI is an ominous complication of burns, affecting 25% of individuals with more than 10% total body surface area involvement. In addition to severe hypovolemia resulting in decreased cardiac output and increased neurohormonal activation, burns and acute pancreatitis both lead to dysregulated inflammation and an increased risk of sepsis and acute lung injury, all of which may facilitate the development and progression of AKI. Individuals undergoing massive fluid resuscitation for trauma, burns, and acute pancreatitis can also develop the abdominal compartment syndrome, where markedly elevated intraabdominal pressures, usually higher than 20 mmHg, lead to renal vein compression and reduced GFR.

1	Diseases of the Microvasculature Leading to Ischemia Microvascular causes of AKI include the thrombotic microangiopathies (antiphospholipid antibody syndrome, radiation nephritis, malignant nephrosclerosis, and thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome [TTP-HUS]), scleroderma, and atheroembolic disease. Large-vessel diseases associated with AKI include renal artery dissection, thromboembolism, thrombosis, and renal vein compression or thrombosis.

1	The kidney has very high susceptibility to nephrotoxicity due to extremely high blood perfusion and concentration of circulating substances along the nephron where water is reabsorbed and in the medullary interstitium; this results in high-concentration exposure of toxins to tubular, interstitial, and endothelial cells. Nephrotoxic injury occurs in response to a number of pharmacologic compounds with diverse structures, endogenous substances, and environmental exposures. All structures of the kidney are vulnerable to toxic injury, including the tubules, interstitium, vasculature, and collecting system. As with other forms of AKI, risk factors for nephrotoxicity include older age, chronic kidney disease (CKD), and prerenal azotemia. Hypoalbuminemia may increase the risk of some forms of nephrotoxin-associated AKI due to increased free circulating drug concentrations.

1	Contrast Agents Iodinated contrast agents used for cardiovascular and computed tomography (CT) imaging are a leading cause of AKI. The risk of AKI, or “contrast nephropathy,” is negligible in those with normal renal function but increases markedly in the setting of CKD, particularly diabetic nephropathy. The most common clinical course of contrast nephropathy is characterized by a rise in SCr beginning 24–48 h following exposure, peaking within 3–5 days, and resolving within 1 week. More severe, dialysis-requiring AKI is uncommon except in the setting of significant preexisting CKD, often in association with congestive heart failure or other coexisting causes for ischemia-associated AKI. Patients with multiple myeloma and renal disease are particularly susceptible. Low fractional excretion of sodium and relatively benign urinary sediment without features of tubular necrosis (see below) are common findings. Contrast nephropathy is thought to occur from a combination of factors,

1	of sodium and relatively benign urinary sediment without features of tubular necrosis (see below) are common findings. Contrast nephropathy is thought to occur from a combination of factors, including (1) hypoxia in the renal outer medulla due to perturbations in renal microcirculation and occlusion of small vessels; (2) cytotoxic damage to the tubules directly or via the generation of oxygen free radicals, especially because the concentration of the agent within the tubule is markedly increased; and (3) transient tubule obstruction with precipitated contrast material. Other diagnostic agents implicated as a cause of AKI are high-dose gadolinium used for magnetic resonance imaging (MRI) and oral sodium phosphate solutions used as bowel purgatives.

1	Antibiotics Several antimicrobial agents are commonly associated with AKI. Aminoglycosides and amphotericin B both cause tubular necrosis. Nonoliguric AKI (i.e., without a significant reduction in urine volume) accompanies 10–30% of courses of aminoglycoside antibiotics, even when plasma levels are in the therapeutic range. Aminoglycosides are freely filtered across the glomerulus and then accumulate within the renal cortex, where concentrations can greatly exceed those of the plasma. AKI typically manifests after 5–7 days of therapy and can present even after the drug has been discontinued. Hypomagnesemia is a common finding.

1	Amphotericin B causes renal vasoconstriction from an increase in tubuloglomerular feedback as well as direct tubular toxicity mediated by reactive oxygen species. Nephrotoxicity from amphotericin B is dose and duration dependent. This drug binds to tubular membrane cholesterol and introduces pores. Clinical features of amphotericin B nephrotoxicity include polyuria, hypomagnesemia, hypocalcemia, and nongap metabolic acidosis.

1	Vancomycin may be associated with AKI, particularly when trough levels are high, but a causal relationship with AKI has not been definitively 1804 established. Acyclovir can precipitate in tubules and cause AKI by tubular obstruction, particularly when given as an intravenous bolus at high doses (500 mg/m2) or in the setting of hypovolemia. Foscarnet, pentamidine, tenofovir, and cidofovir are also frequently associated with AKI due to tubular toxicity. AKI secondary to acute interstitial nephritis can occur as a consequence of exposure to many antibiotics, including penicillins, cephalosporins, quinolones, sulfonamides, and rifampin.

1	Chemotherapeutic Agents Cisplatin and carboplatin are accumulated by proximal tubular cells and cause necrosis and apoptosis. Intensive hydration regimens have reduced the incidence of cisplatin nephrotoxicity, but it remains a dose-limiting toxicity. Ifosfamide may cause hemorrhagic cystitis and tubular toxicity, manifested as type II renal tubular acidosis (Fanconi’s syndrome), polyuria, hypokalemia, and a modest decline in GFR. Antiangiogenesis agents, such as bevacizumab, can cause proteinuria and hypertension via injury to the glomerular microvasculature (thrombotic microangiopathy). Other antineoplastic agents such as mitomycin C and gemcitabine may cause thrombotic microangiopathy with resultant AKI.

1	Toxic Ingestions Ethylene glycol, present in automobile antifreeze, is metabolized to oxalic acid, glycolaldehyde, and glyoxylate, which may cause AKI through direct tubular injury. Diethylene glycol is an industrial agent that has been the cause of outbreaks of severe AKI around the world due to adulteration of pharmaceutical preparations. The metabolite 2-hydroxyethoxyacetic acid (HEAA) is thought to be responsible for tubular injury. Melamine contamination of foodstuffs has led to nephrolithiasis and AKI, either through intratubular obstruction or possibly direct tubular toxicity. Aristolochic acid was found to be the cause of “Chinese herb nephropathy” and “Balkan nephropathy” due to contamination of medicinal herbs or farming. The list of environmental toxins is likely to grow and contribute to a better understanding of previously catalogued “idiopathic” chronic tubular interstitial disease, a common diagnosis in both the developed and developing world.

1	Endogenous Toxins AKI may be caused by a number of endogenous compounds, including myoglobin, hemoglobin, uric acid, and myeloma light chains. Myoglobin can be released by injured muscle cells, and hemoglobin can be released during massive hemolysis leading to pigment nephropathy. Rhabdomyolysis may result from traumatic crush injuries, muscle ischemia during vascular or orthopedic surgery, compression FIGuRE 334-5 Anatomic sites and causes of obstruction leading to postrenal acute kidney injury.

1	during coma or immobilization, prolonged seizure activity, excessive exercise, heat stroke or malignant hyperthermia, infections, metabolic disorders (e.g., hypophosphatemia, severe hypothyroidism), and myopathies (drug-induced, metabolic, or inflammatory). Pathogenic factors for AKI include intrarenal vasoconstriction, direct proximal tubular toxicity, and mechanical obstruction of the distal nephron lumen when myoglobin or hemoglobin precipitates with Tamm-Horsfall protein (uromodulin, the most common protein in urine and produced in the thick ascending limb of the loop of Henle), a process favored by acidic urine. Tumor lysis syndrome may follow initiation of cytotoxic therapy in patients with high-grade lymphomas and acute lymphoblastic leukemia; massive release of uric acid (with serum levels often exceeding 15 mg/dL) leads to precipitation of uric acid in the renal tubules and AKI (Chap. 331). Other features of tumor lysis syndrome include hyperkalemia and hyperphosphatemia. The

1	levels often exceeding 15 mg/dL) leads to precipitation of uric acid in the renal tubules and AKI (Chap. 331). Other features of tumor lysis syndrome include hyperkalemia and hyperphosphatemia. The tumor lysis syndrome can also occasionally occur spontaneously or with treatment for solid tumors or multiple myeloma. Myeloma light chains can also cause AKI by direct tubular toxicity and by binding to Tamm-Horsfall protein to form obstructing intratubular casts. Hypercalcemia, which can also be seen in multiple myeloma, may cause AKI by intense renal vasoconstriction and volume depletion.

1	Allergic Acute Tubulointerstitial Disease and Other Causes of Intrinsic AKI While many of the ischemic and toxic causes of AKI previously described result in tubulointerstitial disease, many drugs are also associated with the development of an allergic response characterized by an inflammatory infiltrate and often peripheral and urinary eosinophilia. AKI may be caused by severe infections and infiltrative diseases. Diseases of the glomeruli or vasculature can lead to AKI by compromising blood flow within the renal circulation. Glomerulonephritis and vasculitis are less common causes of AKI. It is particularly important to recognize these diseases early because they require timely treatment with immunosuppressive agents or therapeutic plasma exchange.

1	(See also Chap. 343) Postrenal AKI occurs when the normally unidirectional flow of urine is acutely blocked either partially or totally, leading to increased retrograde hydrostatic pressure and interference with glomerular filtration. Obstruction to urinary flow may be caused by functional or structural derangements anywhere from the renal pelvis to the tip of the urethra (Fig. 334-5). Normal urinary flow rate Stones, blood clots, external compression, tumor, retroperitoneal fibrosis

1	Prostatic enlargement, blood clots, cancer does not rule out the presence of partial obstruction, because the GFR is normally two orders of magnitude higher than the urinary flow rate. For AKI to occur in healthy individuals, obstruction must affect both kidneys unless only one kidney is functional, in which case unilateral obstruction can cause AKI. Unilateral obstruction may cause AKI in the setting of significant underlying CKD or, in rare cases, from reflex vasospasm of the contralateral kidney. Bladder neck obstruction is a common cause of postrenal AKI and can be due to prostate disease (benign prostatic hypertrophy or prostate cancer), neurogenic bladder, or therapy with anticholinergic drugs. Obstructed Foley catheters can cause postrenal AKI if not recognized and relieved. Other causes of lower tract obstruction are blood clots, calculi, and urethral strictures. Ureteric obstruction can occur from intraluminal obstruction (e.g., calculi, blood clots, sloughed renal papillae),

1	of lower tract obstruction are blood clots, calculi, and urethral strictures. Ureteric obstruction can occur from intraluminal obstruction (e.g., calculi, blood clots, sloughed renal papillae), infiltration of the ureteric wall (e.g., neoplasia), or external compression (e.g., retroperitoneal fibrosis, neoplasia, abscess, or inadvertent surgical damage). The pathophysiology of postrenal AKI involves hemodynamic alterations triggered by an abrupt increase in intratubular pressures. An initial period of hyperemia from afferent arteriolar dilation is followed by intrarenal vasoconstriction from the generation of angiotensin II, thromboxane A2, and vasopressin, and a reduction in NO production. Reduced GFR is due to underperfusion of glomeruli and, possibly, changes in the glomerular ultrafiltration coefficient.

1	The presence of AKI is usually inferred by an elevation in the SCr concentration. AKI is currently defined by a rise from baseline of at least 0.3 mg/dL within 48 h or at least 50% higher than baseline within 1 week, or a reduction in urine output to less than 0.5 mL/kg per hour for longer than 6 h. It is important to recognize that given this definition, some patients with AKI will not have tubular or glomerular damage (e.g., prerenal azotemia). The distinction between AKI and CKD is important for proper diagnosis and treatment. The distinction is straightforward when a recent baseline SCr concentration is available, but more difficult in the many instances in which the baseline is unknown. In such cases, clues suggestive of CKD can come from radiologic studies (e.g., small, shrunken kidneys with cortical thinning on renal ultrasound, or evidence of renal osteodystrophy) or laboratory tests such as normocytic anemia in the absence of blood loss or secondary hyperparathyroidism with

1	with cortical thinning on renal ultrasound, or evidence of renal osteodystrophy) or laboratory tests such as normocytic anemia in the absence of blood loss or secondary hyperparathyroidism with hyperphosphatemia and hypocalcemia, consistent with CKD. No set of tests, however, can rule out AKI superimposed on CKD because AKI is a frequent complication in patients with CKD, further complicating the distinction. Serial blood tests showing continued substantial rise of SCr represents clear evidence of AKI. Once the diagnosis of AKI is established, its cause needs to be determined.

1	The clinical context, careful history taking, and physical examination often narrow the differential diagnosis for the cause of AKI. Prerenal azotemia should be suspected in the setting of vomiting, diarrhea, glycosuria causing polyuria, and several medications including diuretics, NSAIDs, ACE inhibitors, and ARBs. Physical signs of orthostatic hypotension, tachycardia, reduced jugular venous pressure, decreased skin turgor, and dry mucous membranes are often present in prerenal azotemia. A history of prostatic disease, nephrolithiasis, or pelvic or paraaortic malignancy would suggest the possibility of postrenal AKI. Whether or not symptoms are present early during obstruction of the urinary tract depends on the location of obstruction. Colicky flank pain radiating to the groin suggests acute ureteric obstruction. Nocturia and urinary frequency or hesitancy can be seen in prostatic disease. Abdominal fullness and suprapubic pain can accompany massive bladder enlargement. Definitive

1	acute ureteric obstruction. Nocturia and urinary frequency or hesitancy can be seen in prostatic disease. Abdominal fullness and suprapubic pain can accompany massive bladder enlargement. Definitive diagnosis of obstruction requires radiologic investigations.

1	A careful review of all medications is imperative in the evaluation of an individual with AKI. Not only are medications frequently a cause of AKI, but doses of administered medications must be adjusted for estimated GFR. Idiosyncratic reactions to a wide variety of medications can lead to allergic interstitial nephritis, which may be accompanied 1805 by fever, arthralgias, and a pruritic erythematous rash. The absence of systemic features of hypersensitivity, however, does not exclude the diagnosis of interstitial nephritis. AKI accompanied by palpable purpura, pulmonary hemorrhage, or sinusitis raises the possibility of systemic vasculitis with glomerulonephritis. Atheroembolic disease can be associated with livedo reticularis and other signs of emboli to the legs. A tense abdomen should prompt consideration of acute abdominal compartment syndrome, which requires measurement of bladder pressure. Signs of limb ischemia may be clues to the diagnosis of rhabdomyolysis.

1	Complete anuria early in the course of AKI is uncommon except in the following situations: complete urinary tract obstruction, renal artery occlusion, overwhelming septic shock, severe ischemia (often with cortical necrosis), or severe proliferative glomerulonephritis or vasculitis. A reduction in urine output (oliguria, defined as <400 mL/24 h) usually denotes more severe AKI (i.e., lower GFR) than when urine output is preserved. Oliguria is associated with worse clinical outcomes. Preserved urine output can be seen in nephrogenic diabetes insipidus characteristic of longstanding urinary tract obstruction, tubulointerstitial disease, or nephrotoxicity from cisplatin or aminoglycosides, among other causes. Red or brown urine may be seen with or without gross hematuria; if the color persists in the supernatant after centrifugation, then pigment nephropathy from rhabdomyolysis or hemolysis should be suspected.

1	The urinalysis and urine sediment examination are invaluable tools, but they require clinical correlation because of generally limited sensitivity and specificity (Fig. 334-6) (Chap. 62e). In the absence of preexisting proteinuria from CKD, AKI from ischemia or nephrotoxins leads to mild proteinuria (<1 g/d). Greater proteinuria in AKI suggests damage to the glomerular ultrafiltration barrier or excretion of myeloma light chains; the latter are not detected with conventional urine dipsticks (which detect albumin) and require the sulfosalicylic acid test or immunoelectrophoresis. Atheroemboli can cause a variable degree of proteinuria. Extremely heavy proteinuria (“nephrotic range,” >3.5 g/d) can occasionally be seen in glomerulonephritis, vasculitis, or interstitial nephritis (particularly from NSAIDs). AKI can also complicate cases of minimal change disease, a cause of the nephrotic syndrome (Chap. 332e). If the dipstick is positive for hemoglobin but few red blood cells are evident

1	from NSAIDs). AKI can also complicate cases of minimal change disease, a cause of the nephrotic syndrome (Chap. 332e). If the dipstick is positive for hemoglobin but few red blood cells are evident in the urine sediment, then rhabdomyolysis or hemolysis should be suspected.

1	Prerenal azotemia may present with hyaline casts or an unremarkable urine sediment exam. Postrenal AKI may also lead to an unremarkable sediment, but hematuria and pyuria may be seen depending on the cause of obstruction. AKI from ATN due to ischemic injury, sepsis, or certain nephrotoxins has characteristic urine sediment findings: pigmented “muddy brown” granular casts and tubular epithelial cell casts. These findings may be absent in more than 20% of cases, however. Glomerulonephritis may lead to dysmorphic red blood cells or red blood cell casts. Interstitial nephritis may lead to white blood cell casts. The urine sediment findings overlap somewhat in glomerulonephritis and interstitial nephritis, and a diagnosis is not always possible on the basis of the urine sediment alone. Urine eosinophils have a limited role in differential diagnosis; they can be seen in interstitial nephritis, pyelonephritis, cystitis, atheroembolic disease, or glomerulonephritis. Crystalluria may be

1	eosinophils have a limited role in differential diagnosis; they can be seen in interstitial nephritis, pyelonephritis, cystitis, atheroembolic disease, or glomerulonephritis. Crystalluria may be important diagnostically. The finding of oxalate crystals in AKI should prompt an evaluation for ethylene glycol toxicity. Abundant uric acid crystals may be seen in the tumor lysis syndrome.

1	Certain forms of AKI are associated with characteristic patterns in the rise and fall of SCr. Prerenal azotemia typically leads to modest rises in SCr that return to baseline with improvement in hemodynamic status. Contrast nephropathy leads to a rise in SCr within 24–48 h, peak within 3–5 days, and resolution within 5–7 days. In comparison, atheroembolic disease usually manifests with more subacute rises in 1806 taBLe 334-1 MajOr Causes, CLInICaL Features, anD DIagnOstIC stuDIes FOr prerenaL anD IntrInsIC aCute KIDney Injury Nephrotoxin-Associated AKI: Endogenous BUN/creatinine ratio above 20, FeNa <1%, hyaline casts in urine sediment, urine specific gravity >1.018, urine osmolality >500 mOsm/kg Positive culture from normally sterile body fluid; urine sediment often contains granular casts, renal tubular epithelial cell casts Urine sediment often contains granular casts, renal tubular epithelial cell casts. FeNa typically >1%.

1	Urine sediment often contains granular casts, renal tubular epithelial cell casts. FeNa typically >1%. Low FeNa, high specific gravity and osmolality may not be seen in the setting of CKD, diuretic use; BUN elevation out of proportion to creatinine may alternatively indicate upper GI bleed or increased catabolism. Response to restoration of hemodynamics is most diagnostic. FeNa may be low (<1%), particularly early in the course, but is usually >1% with osmolality <500 mOsm/kg toms, bone pain Nephrotoxin-Associated AKI: Exogenous Elevated myoglobin, creatine kinase; urine heme positive with few red blood cells Anemia, elevated LDH, low haptoglobin Hyperphosphatemia, hypocalcemia, hyperuricemia Other Causes of Intrinsic AKI Exposure to iodinated contrast Aminoglycoside antibiotics, cisplatin, tenofovir, zoledronate, ethylene glycol, aristolochic acid, and melamine (to name a few)

1	Other Causes of Intrinsic AKI Exposure to iodinated contrast Aminoglycoside antibiotics, cisplatin, tenofovir, zoledronate, ethylene glycol, aristolochic acid, and melamine (to name a few) Recent medication exposure; can have fever, rash, arthralgias Characteristic course is rise in SCr within 1–2 d, peak within 3–5 d, recovery within 7 d Urine sediment often contains granular casts, renal tubular epithelial cell casts. FeNa typically >1%. Eosinophilia, sterile pyuria; often nonoliguric FeNa may be low (<1%) Urine eosinophils have limited diagnostic accuracy; systemic signs of drug reaction often absent; kidney biopsy may be helpful Postrenal AKI Variable (Chap. 338) features include skin rash, arthralgias, sinusitis (AGBM disease), lung hemorrhage (AGBM, ANCA, lupus), recent skin infection or pharyngitis (poststreptococcal) Nondrug-related causes include tubulointerstitial nephritis-uveitis (TINU) syndrome, Legionella infection

1	Nondrug-related causes include tubulointerstitial nephritis-uveitis (TINU) syndrome, Legionella infection Neurologic abnormalities and/or AKI; recent diarrheal illness; use of calcineurin inhibitors; pregnancy or postpartum; spontaneous Recent manipulation of the aorta or other large vessels; may occur spontaneously or after anticoagulation; retinal plaques, palpable purpura, livedo reticularis, GI bleed History of kidney stones, prostate disease, obstructed bladder catheter, retroperitoneal or pelvic neoplasm ANA, ANCA, AGBM antibody, hepatitis serologies, cryoglobulins, blood culture, decreased complement levels, ASO titer (abnormalities of these tests depending on etiology) Eosinophilia, sterile pyuria; often nonoliguric Schistocytes on peripheral blood smear, elevated LDH, anemia, thrombocytopenia

1	Eosinophilia, sterile pyuria; often nonoliguric Schistocytes on peripheral blood smear, elevated LDH, anemia, thrombocytopenia Hypocomplementemia, eosinophiluria (variable), variable amounts of proteinuria “Typical HUS” refers to AKI with a diarrheal prodrome, often due to Shiga toxin released from Escherichia coli or other bacteria; “atypical HUS” is due to inherited or acquired complement dysregulation. “TTP-HUS” refers to sporadic cases in adults. Diagnosis may involve screening for ADAMTS13 activity, Shiga toxin–producing E. coli, genetic evaluation of complement regulatory proteins, and kidney biopsy. Imaging with computed tomography or ultrasound

1	Imaging with computed tomography or ultrasound Abbreviations: ACE-I, angiotensin-converting enzyme inhibitor-I; AGBM, antiglomerular basement membrane; AKI, acute kidney injury; ANA, antinuclear antibody; ANCA, antineutrophilic cytoplasmic antibody; ARB, angiotensin receptor blocker; ASO, antistreptolysin O; BUN, blood urea nitrogen; CKD, chronic kidney disease; FeNa, fractional excretion of sodium; GI, gastrointestinal; LDH, lactate dehydrogenase; NSAID, nonsteroidal anti-inflammatory drug; TTP/HUS, thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome.

1	Acute Kidney InjuryUrinary sediment in AKI Normal or few RBC or WBC or hyaline casts Prerenal Postrenal Arterial thrombosis or embolism Preglomerular vasculitis HUS or TTP Scleroderma crisis RBCs RBC casts GN Vasculitis Malignant hypertension Thrombotic microangiopathy Interstitial nephritis GN Pyelonephritis Allograft rejection Malignant infiltration of the kidney ATN Tubulointerstitial nephritis Acute cellular allograft rejection Myoglobinuria Hemoglobinuria ATN GN Vasculitis Tubulo interstitial nephritis Allergic interstitial nephritis Atheroembolic disease Pyelonephritis Cystitis Glomerulo nephritis Acute uric acid nephropathy Calcium oxalate (ethylene glycol intoxication) Drugs or toxins (acyclovir, indinavir, sulfadiazine, amoxicillin) Abnormal WBCs WBC casts Renal tubular epithelial (RTE) cells RTE casts Pigmented casts Granular casts Eosinophiluria Crystalluria FIGuRE 334-6 Interpretation of urinary sediment findings in acute kidney injury (AKI). ATN, acute

1	tubular epithelial (RTE) cells RTE casts Pigmented casts Granular casts Eosinophiluria Crystalluria FIGuRE 334-6 Interpretation of urinary sediment findings in acute kidney injury (AKI). ATN, acute tubular necrosis; GN, glomerulonephri-tis; HUS, hemolytic-uremic syndrome; RBCs, red blood cells; RTE, renal tubular epithelial; TTP, thrombotic thrombocytopenic purpura; WBCs, white blood cells. (Adapted from L Yang, JV Bonventre: Diagnosis and clinical evaluation of acute kidney injury. In Comprehensive Nephrology, 4th ed. J Floege et al [eds]. Philadelphia, Elsevier, 2010.)

1	SCr, although severe AKI with rapid increases in SCr can occur in this setting. With many of the epithelial cell toxins such as aminoglycoside antibiotics and cisplatin, the rise in SCr is characteristically delayed for 3–5 days to 2 weeks after initial exposure.

1	A complete blood count may provide diagnostic clues. Anemia is common in AKI and is usually multifactorial in origin. It is not related to an effect of AKI solely on production of red blood cells because this effect in isolation takes longer to manifest. Peripheral eosinophilia can accompany interstitial nephritis, atheroembolic disease, polyarteritis nodosa, and Churg-Strauss vasculitis. Severe anemia in the absence of bleeding may reflect hemolysis, multiple myeloma, or thrombotic microangiopathy (e.g., HUS or TTP). Other laboratory findings of thrombotic microangiopathy include thrombocytopenia, schistocytes on peripheral blood smear, elevated lactate dehydrogenase level, and low haptoglobin content. Evaluation of patients suspected of having TTP-HUS includes measurement of levels of the von Willebrand factor cleaving protease (ADAMTS13) and testing for Shiga toxin–producing Escherichia coli. “Atypical HUS” constitutes the majority of adult cases of HUS; genetic testing is

1	of the von Willebrand factor cleaving protease (ADAMTS13) and testing for Shiga toxin–producing Escherichia coli. “Atypical HUS” constitutes the majority of adult cases of HUS; genetic testing is important because it is estimated that 60–70% of atypical HUS patients have mutations in genes encoding proteins that regulate the alternative complement pathway.

1	AKI often leads to hyperkalemia, hyperphosphatemia, and hypocalcemia. Marked hyperphosphatemia with accompanying hypocalcemia, however, suggests rhabdomyolysis or the tumor lysis syndrome. Creatine phosphokinase levels and serum uric acid are elevated in rhabdomyolysis, while tumor lysis syndrome shows normal or marginally elevated creatine kinase and markedly elevated serum uric acid. The anion gap may be increased with any cause of uremia due to retention of anions such as phosphate, hippurate, sulfate, and urate. The co-occurrence of an increased anion gap and an osmolal gap may suggest ethylene glycol poisoning, which may also cause oxalate crystalluria. Low anion gap may provide a clue to the diagnosis of multiple myeloma due to the presence of unmeasured cationic proteins. Laboratory blood tests helpful for the diagnosis of glomerulonephritis and vasculitis include depressed complement levels and high titers of antinuclear antibodies (ANAs), antineutrophilic cytoplasmic

1	blood tests helpful for the diagnosis of glomerulonephritis and vasculitis include depressed complement levels and high titers of antinuclear antibodies (ANAs), antineutrophilic cytoplasmic antibodies (ANCAs), antiglomerular basement membrane (AGBM) antibodies, and cryoglobulins.

1	Several indices have been used to help differentiate prerenal azotemia from intrinsic AKI when the tubules are malfunctioning. The low tubular flow rate and increased renal medullary recycling of urea seen in prerenal azotemia may cause a disproportionate elevation of the BUN compared to creatinine. Other causes of disproportionate BUN elevation need to be kept in mind, however, including upper gastrointestinal bleeding, hyperalimentation, increased tissue catabolism, and glucocorticoid use.

1	The fractional excretion of sodium (FeNa) is the fraction of the filtered sodium load that is reabsorbed by the tubules, and is a measure of both the kidney’s ability to reabsorb sodium as well as endogenously and exogenously administered factors that affect tubular reabsorption. As such, it depends on sodium intake, effective intravascular volume, GFR, diuretic intake, and intact tubular reabsorptive mechanisms. With prerenal azotemia, the FeNa may be below 1%, suggesting avid tubular sodium reabsorption. In patients with CKD, a FeNa significantly above 1% can be present despite a superimposed prerenal state. The FeNa may also be above 1% despite hypovolemia due to treatment with diuretics. Low FeNa is often seen early in glomerulonephritis and other disorders and, hence, should not be taken as prima facie evidence of prerenal azotemia. Low FeNa is therefore suggestive, but not synonymous, with effective intravascular volume depletion, and should not be used as the sole guide for

1	taken as prima facie evidence of prerenal azotemia. Low FeNa is therefore suggestive, but not synonymous, with effective intravascular volume depletion, and should not be used as the sole guide for volume management. The response of urine output to crystalloid or colloid fluid administration may be both diagnostic and therapeutic in prerenal azotemia. In ischemic AKI, the FeNa is frequently above 1% because of tubular injury and resultant inability to reabsorb sodium. Several causes of ischemia-associated and nephrotoxin-associated AKI can present with FeNa below 1%, however, including sepsis (often early in the course), rhabdomyolysis, and contrast nephropathy.

1	The ability of the kidney to produce a concentrated urine is dependent upon many factors and reliant on good tubular function in multiple regions of the kidney. In the patient not taking diuretics and with good baseline kidney function, urine osmolality may be above 500 mOsm/kg in prerenal azotemia, consistent with an intact medullary gradient and elevated serum vasopressin levels causing water reabsorption resulting in concentrated urine. In elderly patients and those 1808 with CKD, however, baseline concentrating defects may exist, making urinary osmolality unreliable in many instances. Loss of concentrating ability is common in septic or ischemic AKI, resulting in urine osmolality below 350 mOsm/kg, but the finding is not specific.

1	Postrenal AKI should always be considered in the differential diagnosis of AKI because treatment is usually successful if instituted early. Simple bladder catheterization can rule out urethral obstruction. Imaging of the urinary tract with renal ultrasound or CT should be undertaken to investigate obstruction in individuals with AKI unless an alternate diagnosis is apparent. Findings of obstruction include dilation of the collecting system and hydroureteronephrosis. Obstruction can be present without radiologic abnormalities in the setting of volume depletion, retroperitoneal fibrosis, encasement with tumor, and also early in the course of obstruction. If a high clinical index of suspicion for obstruction persists despite normal imaging, antegrade or retrograde pyelography should be performed. Imaging may also provide additional helpful information about kidney size and echogenicity to assist in the distinction between acute versus CKD. In CKD, kidneys are usually smaller unless the

1	Imaging may also provide additional helpful information about kidney size and echogenicity to assist in the distinction between acute versus CKD. In CKD, kidneys are usually smaller unless the patient has diabetic nephropathy, HIV-associated nephropathy, or infiltrative diseases. Normal sized kidneys are expected in AKI. Enlarged kidneys in a patient with AKI suggests the possibility of acute interstitial nephritis. Vascular imaging may be useful if venous or arterial obstruction is suspected, but the risks of contrast administration should be kept in mind. MRI with gadolinium-based contrast agents should be avoided if possible in severe AKI due to the possibility of inducing nephrogenic system fibrosis, a rare but serious complication seen most commonly in patients with end-stage renal disease.

1	If the cause of AKI is not apparent based on the clinical context, physical examination, laboratory studies, and radiologic evaluation, kidney biopsy should be considered. The kidney biopsy can provide definitive diagnostic and prognostic information about acute kidney disease and CKD. The procedure is most often used in AKI when prerenal azotemia, postrenal AKI, and ischemic or nephrotoxic AKI have been deemed unlikely, and other possible diagnoses are being considered such as glomerulonephritis, vasculitis, interstitial nephritis, myeloma kidney, HUS and TTP, and allograft dysfunction. Kidney biopsy is associated with a risk of bleeding, which can be severe and organor life-threatening in patients with thrombocytopenia or coagulopathy.

1	BUN and creatinine are functional biomarkers of glomerular filtration rather than tissue injury biomarkers and, therefore, may be suboptimal for the diagnosis of actual parenchymal kidney damage. BUN and creatinine are also relatively slow to rise after kidney injury. Several novel kidney injury biomarkers have been investigated and show promise for earlier and accurate diagnosis of AKI. Kidney injury molecule-1 (KIM-1) is a type 1 transmembrane protein that is abundantly expressed in proximal tubular cells injured by ischemia or nephrotoxins such as cisplatin. KIM-1 is not expressed in appreciable quantities in the absence of tubular injury or in extrarenal tissues. KIM-1’s functional role may be to confer phagocytic properties to tubular cells, enabling them to clear debris from the tubular lumen after kidney injury. KIM-1 can be detected shortly after ischemic or nephrotoxic injury in the urine and, therefore, may be an easily tested biomarker in the clinical setting. Neutrophil

1	lumen after kidney injury. KIM-1 can be detected shortly after ischemic or nephrotoxic injury in the urine and, therefore, may be an easily tested biomarker in the clinical setting. Neutrophil gelatinase associated lipocalin (NGAL, also known as lipocalin-2 or siderocalin) is another novel biomarker of AKI. NGAL was first discovered as a protein in granules of human neutrophils. NGAL can bind to iron siderophore complexes and may have tissue-protective effects in the proximal tubule. NGAL is highly upregulated after inflammation and kidney injury and can be detected in the plasma and urine within 2 h of cardiopulmonary bypass–associated AKI. Other candidate biomarkers of AKI include interleukin (IL) 18, a proinflammatory cytokine of the IL-1 superfamily that may mediate ischemic proximal tubular injury, and L-type fatty acid binding protein, which is expressed in ischemic proximal tubule cells and may be renoprotective by binding free fatty acids and lipid peroxidation products. A

1	tubular injury, and L-type fatty acid binding protein, which is expressed in ischemic proximal tubule cells and may be renoprotective by binding free fatty acids and lipid peroxidation products. A number of other biomarkers are under investigation for early and accurate identification of AKI and for risk stratification to identify individuals at increased risk. The optimal use of novel AKI biomarkers in clinical settings is an area of ongoing investigation.

1	The kidney plays a central role in homeostatic control of volume status, blood pressure, plasma electrolyte composition, and acid-base balance, and for excretion of nitrogenous and other waste products. Complications associated with AKI are, therefore, protean, and depend on the severity of AKI and other associated conditions. Mild to moderate AKI may be entirely asymptomatic, particularly early in the course.

1	Buildup of nitrogenous waste products, manifested as an elevated BUN concentration, is a hallmark of AKI. BUN itself poses little direct toxicity at levels below 100 mg/dL. At higher concentrations, mental status changes and bleeding complications can arise. Other toxins normally cleared by the kidney may be responsible for the symptom complex known as uremia. Few of the many possible uremic toxins have been definitively identified. The correlation of BUN and SCr concentrations with uremic symptoms is extremely variable, due in part to differences in urea and creatinine generation rates across individuals.

1	Expansion of extracellular fluid volume is a major complication of oliguric and anuric AKI, due to impaired salt and water excretion. The result can be weight gain, dependent edema, increased jugular venous pressure, and pulmonary edema; the latter can be life threatening. Pulmonary edema can also occur from volume overload and hemorrhage in pulmonary renal syndromes. AKI may also induce or exacerbate acute lung injury characterized by increased vascular permeability and inflammatory cell infiltration in lung parenchyma. Recovery from AKI can sometimes be accompanied by polyuria, which, if untreated, can lead to significant volume depletion. The polyuric phase of recovery may be due to an osmotic diuresis from retained urea and other waste products as well as delayed recovery of tubular reabsorptive functions.

1	Administration of excessive hypotonic crystalloid or isotonic dextrose solutions can result in hypoosmolality and hyponatremia, which, if severe, can cause neurologic abnormalities, including seizures. Abnormalities in plasma electrolyte composition can be mild or life threatening. Frequently the most concerning complication of AKI is hyperkalemia. Marked hyperkalemia is particularly common in rhabdomyolysis, hemolysis, and tumor lysis syndrome due to release of intracellular potassium from damaged cells. Potassium affects the cellular membrane potential of cardiac and neuromuscular tissues. Muscle weakness may be a symptom of hyperkalemia. The more serious complication of hyperkalemia is due to effects on cardiac conduction, leading to potentially fatal arrhythmias.

1	Metabolic acidosis, usually accompanied by an elevation in the anion gap, is common in AKI, and can further complicate acid-base and potassium balance in individuals with other causes of acidosis, including sepsis, diabetic ketoacidosis, or respiratory acidosis. AKI can lead to hyperphosphatemia, particularly in highly catabolic patients or those with AKI from rhabdomyolysis, hemolysis, and tumor lysis syndrome. Metastatic deposition of calcium phosphate can lead to hypocalcemia. AKI-associated hypocalcemia may also arise from derangements in the vitamin D–parathyroid hormone–fibroblast growth factor-23 axis. Hypocalcemia is often asymptomatic but can lead to perioral paresthesias, muscle cramps, seizures, carpopedal spasms, and prolongation of the QT interval on electrocardiography. Calcium levels should be corrected for the degree of hypoalbuminemia, if present, or ionized calcium levels should be followed. Mild, asymptomatic hypocalcemia does not require treatment.

1	Hematologic complications of AKI include anemia and bleeding, both of which are exacerbated by coexisting disease processes such as sepsis, liver disease, and disseminated intravascular coagulation. Direct hematologic effects from AKI-related uremia include decreased erythropoiesis and platelet dysfunction. Infections are a common precipitant of AKI and also a dreaded complication of AKI. Impaired host immunity has been described in end-stage renal disease and may be operative in severe AKI. The major cardiac complications of AKI are arrhythmias, pericarditis, and pericardial effusion. AKI is often a severely hypercatabolic state, and therefore, malnutrition is a major complication.

1	AKI is often a severely hypercatabolic state, and therefore, malnutrition is a major complication. The management of individuals with and at risk for AKI varies according to the underlying cause (Table 334-2). Common to all are several principles. Optimization of hemodynamics, correction of fluid and electrolyte imbalances, discontinuation of nephrotoxic medications, and dose adjustment of administered medications are all critical. Common causes of AKI such as sepsis and ischemic ATN do not yet have specific therapies once injury is established, but meticulous clinical attention is needed to support the patient until (if ) AKI resolves. The kidney possesses remarkable capacity to repair itself after even severe, dialysis-requiring AKI. However, many patients with AKI do not recover fully and may remain dialysis dependent. It has become increasingly apparent that AKI predisposes to accelerated progression of CKD, and CKD is an important risk factor for AKI.

1	Prerenal Azotemia Prevention and treatment of prerenal azotemia require optimization of renal perfusion. The composition of replacement fluids should be targeted to the type of fluid lost. Severe acute blood loss should be treated with packed red blood cells. Isotonic crystalloid and/or colloid should be used for less severe acute hemorrhage or plasma loss in the case of burns and pancreatitis. Crystalloid solutions are less expensive and probably equally efficacious as colloid solutions. Hydroxyethyl starch solutions increase the risk of severe AKI and are contraindicated. Crystalloid has been reported to be preferable to albumin in the setting of traumatic brain injury. Isotonic crystalloid (e.g., 0.9% saline) or colloid should be used for volume resuscitation in severe hypovolemia, whereas hypotonic crystalloids (e.g., 0.45% saline) suffice for less severe hypovolemia. Excessive chloride administration from 0.9% saline may lead to hyperchloremic metabolic acidosis and may impair

1	hypotonic crystalloids (e.g., 0.45% saline) suffice for less severe hypovolemia. Excessive chloride administration from 0.9% saline may lead to hyperchloremic metabolic acidosis and may impair GFR. Bicarbonate-containing solutions (e.g., dextrose water with 150 mEq sodium bicarbonate) should be used if metabolic acidosis is a concern.

1	Optimization of cardiac function in AKI may require use of inotropic agents, preloadand afterload-reducing agents, antiarrhythmic drugs, and mechanical aids such as an intraaortic balloon pump. Invasive hemodynamic monitoring to guide therapy may be necessary. Cirrhosis and Hepatorenal Syndrome Fluid management in individuals with cirrhosis, ascites, and AKI is challenging because of the frequent 1. Optimization of systemic and renal hemodynamics through volume resuscitation and judicious use of vasopressors 2. Elimination of nephrotoxic agents (e.g., ACE inhibitors, ARBs, NSAIDs, aminoglycosides) if possible 3. Initiation of renal replacement therapy when indicated 1. Nephrotoxin-specific a. Rhabdomyolysis: aggressive intravenous fluids; consider forced alkaline diuresis b. Tumor lysis syndrome: aggressive intravenous fluids and allopurinol or rasburicase 2. Volume overload a. b. c. 3. Hyponatremia a.

1	Tumor lysis syndrome: aggressive intravenous fluids and allopurinol or rasburicase 2. Volume overload a. b. c. 3. Hyponatremia a. Restriction of enteral free water intake, minimization of hypotonic intravenous solutions including those containing dextrose b. Hypertonic saline is rarely necessary in AKI. Vasopressin antagonists are generally not needed. 4. Hyperkalemia a. Restriction of dietary potassium intake b. Discontinuation of potassium-sparing diuretics, ACE inhibitors, ARBs, NSAIDs c. Loop diuretics to promote urinary potassium loss d. e. Insulin (10 units regular) and glucose (50 mL of 50% dextrose) to promote entry of potassium intracellularly f. Inhaled beta-agonist therapy to promote entry of potassium intracellularly g. Calcium gluconate or calcium chloride (1 g) to stabilize the myocardium 5. Metabolic acidosis a. Sodium bicarbonate (if pH <7.2 to keep serum bicarbonate >15 mmol/L) b. Administration of other bases, e.g., THAM c. 6. Hyperphosphatemia a.

1	Sodium bicarbonate (if pH <7.2 to keep serum bicarbonate >15 mmol/L) b. Administration of other bases, e.g., THAM c. 6. Hyperphosphatemia a. Restriction of dietary phosphate intake b. Phosphate binding agents (calcium acetate, sevelamer hydrochloride, aluminum hydroxide—taken with meals) 7. Hypocalcemia a. Calcium carbonate or calcium gluconate if symptomatic 8. Hypermagnesemia a. Discontinue Mg2+ containing antacids 9. Hyperuricemia a. Acute treatment is usually not required except in the setting of tumor lysis syndrome (see above) 10. Nutrition a. Sufficient protein and calorie intake (20–30 kcal/kg per day) to avoid negative nitrogen balance. Nutrition should be provided via the enteral route if possible. 11. Drug dosing a. Careful attention to dosages and frequency of administration of drugs, adjustment for degree of renal failure b. Note that serum creatinine concentration may overestimate renal function in the non–steady state characteristic of patients with AKI

1	Note that serum creatinine concentration may overestimate renal function in the non–steady state characteristic of patients with AKI Abbreviations: ACE, angiotensin-converting enzyme; ARBs, angiotensin receptor blocker; NSAIDs, nonsteroidal anti-inflammatory drug; THAM, tris (hydroxymethyl) aminomethane.

1	difficulty in ascertaining intravascular volume status. Administration of intravenous fluids as a volume challenge may be required diagnostically as well as therapeutically. Excessive volume administration may, however, result in worsening ascites and pulmonary compromise in 1810 the setting of hepatorenal syndrome or AKI due to superimposed spontaneous bacterial peritonitis. Peritonitis should be ruled out by culture of ascitic fluid. Albumin may prevent AKI in those treated with antibiotics for spontaneous bacterial peritonitis. The definitive treatment of the hepatorenal syndrome is orthotopic liver transplantation. Bridge therapies that have shown promise include terlipressin (a vasopressin analog), combination therapy with octreotide (a somatostatin analog) and midodrine (an α1-adrenergic agonist), and norepinephrine, in combination with intravenous albumin (25–50 g, maximum 100 g/d).

1	Intrinsic AKI Several agents have been tested and have failed to show benefit in the treatment of acute tubular injury. These include atrial natriuretic peptide, low-dose dopamine, endothelin antagonists, loop diuretics, calcium channel blockers, α-adrenergic receptor blockers, prostaglandin analogs, antioxidants, antibodies against leukocyte adhesion molecules, and insulin-like growth factor, among many others. Most studies have enrolled patients with severe and well-established AKI, and treatment may have been initiated too late. Novel kidney injury biomarkers may provide an opportunity to test agents earlier in the course of AKI.

1	AKI due to acute glomerulonephritis or vasculitis may respond to immunosuppressive agents and/or plasmapheresis (Chap. 332e). Allergic interstitial nephritis due to medications requires discontinuation of the offending agent. Glucocorticoids have been used, but not tested in randomized trials, in cases where AKI persists or worsens despite discontinuation of the suspected medication. AKI due to scleroderma (scleroderma renal crisis) should be treated with ACE inhibitors. Idiopathic TTP-HUS is a medical emergency and should be treated promptly with plasma exchange. Pharmacologic blockade of complement activation may be effective in atypical HUS.

1	Early and aggressive volume repletion is mandatory in patients with rhabdomyolysis, who may initially require 10 L of fluid per day. Alkaline fluids (e.g., 75 mmol/L sodium bicarbonate added to 0.45% saline) may be beneficial in preventing tubular injury and cast formation, but carry the risk of worsening hypocalcemia. Diuretics may be used if fluid repletion is adequate but unsuccessful in achieving urinary flow rates of 200–300 mL/h. There is no specific therapy for established AKI in rhabdomyolysis, other than dialysis in severe cases or general supportive care to maintain fluid and electrolyte balance and tissue perfusion. Careful attention must be focused on calcium and phosphate status because of precipitation in damaged tissue and release when the tissue heals.

1	Postrenal AKI Prompt recognition and relief of urinary tract obstruction can forestall the development of permanent structural damage induced by urinary stasis. The site of obstruction defines the treatment approach. Transurethral or suprapubic bladder catheterization may be all that is needed initially for urethral strictures or functional bladder impairment. Ureteric obstruction may be treated by percutaneous nephrostomy tube placement or ureteral stent placement. Relief of obstruction is usually followed by an appropriate diuresis for several days. In rare cases, severe polyuria persists due to tubular dysfunction and may require continued administration of intravenous fluids and electrolytes for a period of time.

1	SuPPORTIVE MEASuRES Volume Management Hypervolemia in oliguric or anuric AKI may be life threatening due to acute pulmonary edema, especially because many patients have coexisting pulmonary disease, and AKI likely increases pulmonary vascular permeability. Fluid and sodium should be restricted, and diuretics may be used to increase the urinary flow rate. There is no evidence that increasing urine output itself improves the natural history of AKI, but diuretics may help to avoid the need for dialysis in some cases. In severe cases of volume overload, furosemide may be given as a bolus (200 mg) followed by an intravenous drip (10–40 mg/h), with or without a thiazide diuretic. In decompensated heart failure, stepped diuretic therapy was found to be superior to ultrafiltration in preserving renal function. Diuretic therapy should be stopped if there is no response. Dopamine in low doses may transiently increase salt and water excretion by the kidney in prerenal states, but clinical trials

1	function. Diuretic therapy should be stopped if there is no response. Dopamine in low doses may transiently increase salt and water excretion by the kidney in prerenal states, but clinical trials have failed to show any benefit in patients with intrinsic AKI. Because of the risk of arrhythmias and potential bowel ischemia, it has been argued that the risks of dopamine outweigh the benefits in the treatment or prevention of AKI.

1	Electrolyte and Acid-Base Abnormalities The treatment of dysnatremias and hyperkalemia is described in Chap. 63. Metabolic acidosis is generally not treated unless severe (pH <7.20 and serum bicarbonate <15 mmol/L). Acidosis can be treated with oral or intravenous sodium bicarbonate (Chap. 66), but overcorrection should be avoided because of the possibility of metabolic alkalosis, hypocalcemia, hypokalemia, and volume overload. Hyperphosphatemia is common in AKI and can usually be treated by limiting intestinal absorption of phosphate using phosphate binders (calcium carbonate, calcium acetate, lanthanum, sevelamer, or aluminum hydroxide). Hypocalcemia does not usually require therapy unless symptoms are present. Ionized calcium should be monitored rather than total calcium when hypoalbuminemia is present.

1	Malnutrition Protein energy wasting is common in AKI, particularly in the setting of multisystem organ failure. Inadequate nutrition may lead to starvation ketoacidosis and protein catabolism. Excessive nutrition may increase the generation of nitrogenous waste and lead to worsening azotemia. Total parenteral nutrition requires large volumes of fluid administration and may complicate efforts at volume control. According to the Kidney Disease Improving Global Outcomes (KDIGO) guidelines, patients with AKI should achieve a total energy intake of 20–30 kcal/kg per day. Protein intake should vary depending on the severity of AKI: 0.8–1.0 g/kg per day in noncatabolic AKI without the need for dialysis; 1.0–1.5 g/kg per day in patients on dialysis; and up to a maximum of 1.7 g/kg per day if hypercatabolic and receiving continuous renal replacement therapy. Trace elements and water-soluble vitamins should also be supplemented in AKI patients treated with dialysis and continuous renal

1	if hypercatabolic and receiving continuous renal replacement therapy. Trace elements and water-soluble vitamins should also be supplemented in AKI patients treated with dialysis and continuous renal replacement therapy.

1	Anemia The anemia seen in AKI is usually multifactorial and is not improved by erythropoiesis-stimulating agents, due to their delayed onset of action and the presence of bone marrow resistance in critically ill patients. Uremic bleeding may respond to desmopressin or estrogens, but may require dialysis for treatment in the case of longstanding or severe uremia. Gastrointestinal prophylaxis with proton pump inhibitors or histamine (H2) receptor blockers is required. Venous thromboembolism prophylaxis is important and should be tailored to the clinical setting; low-molecular-weight heparins and factor Xa inhibitors have unpredictable pharmacokinetics in severe AKI and should be avoided.

1	Dialysis Indications and Modalities (See also Chap. 336) Dialysis is indicated when medical management fails to control volume overload, hyperkalemia, or acidosis; in some toxic ingestions; and when there are severe complications of uremia (asterixis, pericardial rub or effusion, encephalopathy, uremic bleeding). The timing of dialysis is still a matter of debate. Late initiation of dialysis carries the risk of avoidable volume, electrolyte, and metabolic complications of AKI. On the other hand, initiating dialysis too early may unnecessarily expose individuals to intravenous lines and invasive procedures, with the attendant risks of infection, bleeding, procedural complications, and hypotension. The initiation of dialysis should not await the development of a life-threatening complication of renal failure. Many nephrologists initiate dialysis for AKI empirically when the BUN exceeds a certain value (e.g., 100 mg/dL) in patients without clinical signs of recovery of kidney function.

1	of renal failure. Many nephrologists initiate dialysis for AKI empirically when the BUN exceeds a certain value (e.g., 100 mg/dL) in patients without clinical signs of recovery of kidney function. The available modes for renal replacement therapy in AKI require either access to the peritoneal cavity (for peritoneal dialysis) or the large blood vessels (for hemodialysis, hemofiltration, and other hybrid procedures). Small solutes are removed across a semipermeable membrane down their concentration gradient (“diffusive” clearance) and/or along with the movement of plasma water (“convective” clearance). The choice of modality is often dictated by the immediate availability of technology and the expertise of medical staff. Peritoneal dialysis is performed through a temporary intraperitoneal catheter. It is rarely used in the United States for AKI in adults but has enjoyed widespread use internationally, particularly when hemodialysis technology is not available. Dialysate solution is

1	catheter. It is rarely used in the United States for AKI in adults but has enjoyed widespread use internationally, particularly when hemodialysis technology is not available. Dialysate solution is instilled into and removed from the peritoneal cavity at regular intervals in order to achieve diffusive and convective clearance of solutes across the peritoneal membrane; ultrafiltration of water is achieved by the presence of an osmotic gradient across the peritoneal membrane achieved by high concentrations of dextrose in the dialysate solution. Because of its continuous nature, it is often better tolerated than intermittent procedures like hemodialysis in hypotensive patients. Peritoneal dialysis may not be sufficient for hypercatabolic patients due to inherent limitations in dialysis efficacy.

1	Hemodialysis can be used intermittently or continuously and can be done through convective clearance, diffusive clearance, or a combination of the two. Vascular access is through the femoral, internal jugular, or subclavian veins. Hemodialysis is an intermittent procedure that removes solutes through diffusive and convective clearance. Hemodialysis is typically performed 3–4 h per day, three to four times per week, and is the most common form of renal replacement therapy for AKI. One of the major complications of hemodialysis is hypotension, particularly in the critically ill.

1	Continuous intravascular procedures were developed in the early 1980s to treat hemodynamically unstable patients without inducing the rapid shifts of volume, osmolarity, and electrolytes characteristic of intermittent hemodialysis. Continuous renal replacement therapy (CRRT) can be performed by convective clearance (continuous venovenous hemofiltration [CVVH]), in which large volumes of plasma water (and accompanying solutes) are forced across the semipermeable membrane by means of hydrostatic pressure; the plasma water is then replaced by a physiologic crystalloid solution. CRRT can also be performed by diffusive clearance (continuous venovenous hemodialysis [CVVHD]), a technology similar to hemodialysis except at lower blood flow and dialysate flow rates. A hybrid therapy combines both diffusive and convective clearance (continuous venovenous hemodiafiltration [CVVHDF]). To achieve some of the advantages of CRRT without the need for 24-h staffing of the procedure, some physicians

1	diffusive and convective clearance (continuous venovenous hemodiafiltration [CVVHDF]). To achieve some of the advantages of CRRT without the need for 24-h staffing of the procedure, some physicians favor slow low-efficiency dialysis (SLED) or extended daily dialysis (EDD). In this therapy, blood flow and dialysate flow are higher than in CVVHD, but the treatment time is reduced to 12 h or less.

1	The optimal dose of dialysis for AKI is not clear. Daily intermittent hemodialysis and high-dose CRRT do not confer a demonstrable survival or renal recovery advantage, but care should be taken to avoid undertreatment. Studies have failed to show that continuous therapies are superior to intermittent therapies. If available, CRRT is often preferred in patients with severe hemodynamic instability, cerebral edema, or significant volume overload.

1	The development of AKI is associated with a significantly increased risk of in-hospital and long-term mortality, longer length of stay, and increased costs. Prerenal azotemia, with the exception of the cardiorenal and hepatorenal syndromes, and postrenal azotemia carry a better prognosis than most cases of intrinsic AKI. The kidneys may recover even after severe, dialysis-requiring AKI. Survivors of an episode of AKI requiring temporary dialysis, however, are at extremely high risk for progressive CKD, and up to 10% may develop end-stage renal disease. Postdischarge care under the supervision of a nephrologist for aggressive secondary prevention of kidney disease is prudent. Patients with AKI are more likely to die prematurely after they leave the hospital even if their kidney function has recovered. Joanne M. Bargman, Karl Skorecki

1	Joanne M. Bargman, Karl Skorecki Chronic kidney disease (CKD) encompasses a spectrum of different pathophysiologic processes associated with abnormal kidney function and a progressive decline in glomerular filtration rate (GFR). Figure 335-1 provides a recently updated classification, in which stages of CKD are stratified by both estimated GFR and the degree of albuminuria, in order to predict risk of progression of CKD. Previously, CKD had been staged solely by the GFR. However, the risk of worsening of kidney function is closely linked to the amount of albuminuria, and so it has been incorporated into the classification.

1	The pathophysiologic processes, adaptations, clinical presentations, assessment, and therapeutic interventions associated with CKD will be the focus of this chapter. The dispiriting term end-stage renal disease represents a stage of CKD where the accumulation of toxins, fluid, and electrolytes normally excreted by the kidneys results in the uremic syndrome. This syndrome leads to death unless the toxins are removed by renal replacement therapy, using dialysis or kidney transplantation. These interventions are discussed in Chaps. 336 and 337. End-stage renal disease will be supplanted in this chapter by the term stage 5 CKD.

1	The pathophysiology of CKD involves two broad sets of mechanisms of damage: (1) initiating mechanisms specific to the underlying etiology (e.g., genetically determined abnormalities in kidney development or integrity, immune complex deposition and inflammation in certain types of glomerulonephritis, or toxin exposure in certain diseases of the renal tubules and interstitium) and (2) a set of progressive mechanisms, involving hyperfiltration and hypertrophy of the remaining viable nephrons, that are a common consequence following long-term reduction of renal mass, irrespective of underlying etiology (Chap. 333e). The responses to reduction in nephron number are mediated by vasoactive hormones, cytokines, and growth factors. Eventually, these short-term adaptations of hypertrophy and hyperfiltration become maladaptive as the increased pressure and flow within the nephron predisposes to distortion of glomerular architecture, abnormal podocyte function, and disruption of the filtration

1	become maladaptive as the increased pressure and flow within the nephron predisposes to distortion of glomerular architecture, abnormal podocyte function, and disruption of the filtration barrier leading to sclerosis and dropout of the remaining nephrons (Fig. 335-2). Increased intrarenal activity of the renin-angiotensin system (RAS) appears to contribute both to the initial adaptive hyperfiltration and to the subsequent maladaptive hypertrophy and sclerosis. This process explains why a reduction in renal mass from an isolated insult may lead to a progressive decline in renal function over many years (Fig. 335-3).

1	It is important to identify factors that increase the risk for CKD, even in individuals with normal GFR. Risk factors include small for gestation birth weight, childhood obesity, hypertension, diabetes mellitus, autoimmune disease, advanced age, African ancestry, a family history of kidney disease, a previous episode of acute kidney injury, and the presence of proteinuria, abnormal urinary sediment, or structural abnormalities of the urinary tract.

1	Many rare inherited forms of CKD follow a Mendelian inheritance pattern, often as part of a systemic syndrome, with the most common in this category being autosomal dominant polycystic kidney disease. In addition, recent research in the genetics of predisposition to common complex diseases (Chap. 82) has revealed DNA sequence variants at a number of genetic loci that are associated with common forms of CKD. A striking example is the finding of allelic versions of the APOL1 gene, of West African population ancestry, which contributes to the several-fold higher frequency of certain common etiologies of nondiabetic CKD (e.g., focal segmental glomerulosclerosis) observed among African and Hispanic Americans. The prevalence in West African populations seems to have arisen as an evolutionary adaptation conferring protection from tropical pathogens. As in other common diseases

1	FIGuRE 335-2 Left: Schema of the normal glomerular architecture. Right: Secondary glomerular changes associated with a reduction in nephron number, including enlargement of capillary lumens and focal adhesions, which are thought to occur consequent to compensatory hyperfiltration and hypertrophy in the remaining nephrons. (Modified from JR Ingelfinger: N Engl J Med 348:99, 2003.) FIGuRE 335-1 Kidney Disease Improving Global Outcome (KDIGO) classification of chronic kidney disease (CKD). Gradation of color from green to red corresponds to increasing risk and progression of CKD. GFR, glomerular filtration rate. (Reproduced with permission from Kidney Int Suppl 3:5-14, 2013.) with a heritable component, an environmental trigger (such as a viral pathogen) is required to transform genetic risk into disease.

1	Int Suppl 3:5-14, 2013.) with a heritable component, an environmental trigger (such as a viral pathogen) is required to transform genetic risk into disease. To stage CKD, it is necessary to estimate the GFR rather than relying on serum creatinine concentration (Table 335-1). Many laboratories now report an estimated GFR, or eGFR, using one of these equations.

1	The normal annual mean decline in GFR with age from the peak GFR (~120 mL/min per 1.73 m2) attained during the third decade of life is ~1 mL/min per year per 1.73 m2, reaching a mean value of 70 mL/min per 1.73 m2 at age 70. Although reduced GFR occurs with human aging, the lower GFR signifies a true loss of kidney function, with all of the implications that apply to the corresponding stage of CKD. The mean GFR is lower in women than in men. For example, a woman in her 80s with a normal serum creatinine may have a GFR of just 50 mL/min per 1.73 m2. Thus, even a mild elevation in serum creatinine concentration (e.g., 130 μmol/L [1.5 mg/dL]) often signifies a substantial reduction in GFR in most individuals.

1	FIGuRE 335-3 Left: Low-power photomicrograph of a normal kidney showing normal glomeruli and healthy tubulointerstitium without fibrosis. Right: Low-power photomicrograph of chronic kidney disease with sclerosis of many glomeruli and severe tubulointerstitial fibrosis (Masson trichrome, ×40 magnification). (Slides courtesy of the late Dr. Andrew Herzenberg.) The equations for estimating GFR are valid only if the patient is in steady state, that is, the serum creatinine is neither rising nor falling over days.

1	Measurement of albuminuria is also helpful for monitoring nephron injury and the response to therapy in many forms of CKD, especially chronic glomerular diseases. Although an accurate 24-h urine collection is the standard for measurement of albuminuria, the measurement of protein-to-creatinine ratio in a spot first-morning urine sample is often more practical to obtain and correlates well, but not perfectly, with 24-h urine collections. Microalbuminuria (Fig. 335-1, stage A2) refers to the excretion of amounts of albumin too small to detect by urinary dipstick or conventional measures of urine protein. It is a good screening test for early detection of renal disease, and may be a marker for the presence of microvascular disease in general. If a patient has a large amount of excreted albumin, there is no reason to test for microalbuminuria.

1	Stages 1 and 2 CKD are usually not associated with any symptoms arising from the decrement in GFR. If the decline in GFR progresses to stages 3 and 4, clinical and laboratory complications of CKD become reCOMMenDeD equatIOns FOr estIMatIOn OF gLOMeruLar FILtratIOn rate (gFr) usIng seruM CreatInIne COnCentratIOn (sCr), age, sex, raCe, anD BODy WeIght 1. Equation from the Modification of Diet in Renal Disease study Estimated GFR (mL/min per 1.73 m2) = 1.86 × (SCr) −1.154 × (age)−0.203 Multiply by 0.742 for women Multiply by 1.21 for African ancestry 2. CKD-EPI equation GFR = 141 × min(SCr/κ, 1)α × max(SCr/κ, 1)–1.209 × 0.993Age Multiply by 1.018 for women Multiply by 1.159 for African ancestry where SCr is serum creatinine in mg/dL, κ is 0.7 for females and 0.9 for males, α is –0.329 for females and –0.411 for males, min indicates the minimum of SCr/κ or 1, and max indicates the maximum of SCr/κ or 1. Abbreviation: CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration.

1	more prominent. Virtually all organ systems are affected, but the most 1813 evident complications include anemia and associated easy fatigability; decreased appetite with progressive malnutrition; abnormalities in calcium, phosphorus, and mineral-regulating hormones, such as 1,25(OH)2D3 (calcitriol), parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF-23); and abnormalities in sodium, potassium, water, and acid-base homeostasis. Many patients, especially the elderly, will have eGFR values compatible with stage 2 or 3 CKD. However, the majority of these patients will show no further deterioration of renal function. The primary care physician is advised to recheck kidney function, and if it is stable and not associated with proteinuria, the patient can usually be managed in this setting. However, if there is evidence of decline of GFR, uncontrolled hypertension, or proteinuria, referral to a nephrologist is appropriate. If the patient progresses to stage 5 CKD, toxins

1	this setting. However, if there is evidence of decline of GFR, uncontrolled hypertension, or proteinuria, referral to a nephrologist is appropriate. If the patient progresses to stage 5 CKD, toxins accumulate such that patients usually experience a marked disturbance in their activities of daily living, well-being, nutritional status, and water and electrolyte homeostasis, eventuating in the uremic syndrome.

1	It has been estimated from population survey data that at least 6% of the adult population in the United States has CKD at stages 1 and 2. An additional 4.5% of the U.S. population is estimated to have stages 3 and 4 CKD. Table 335-2 lists the five most frequent categories of causes of CKD, cumulatively accounting for greater than 90% of the CKD disease burden worldwide. The relative contribution of each category varies among different geographic regions. The most frequent cause of CKD in North America and Europe is diabetic nephropathy, most often secondary to type 2 diabetes mellitus. Patients with newly diagnosed CKD often also present with hypertension. When no overt evidence for a primary glomerular or tubulointerstitial kidney disease process is present, CKD is often attributed to hypertension. However, it is now appreciated that such individuals can be considered in two categories. The first includes patients with a silent primary glomerulopathy, such as focal segmental

1	to hypertension. However, it is now appreciated that such individuals can be considered in two categories. The first includes patients with a silent primary glomerulopathy, such as focal segmental glomerulosclerosis, without the overt nephrotic or nephritic manifestations of glomerular disease (Chap. 338). The second includes patients in whom progressive nephrosclerosis and hypertension is the renal correlate of a systemic vascular disease, often also involving largeand small-vessel cardiac and cerebral pathology. This latter combination is especially common in the elderly, in whom chronic renal ischemia as a cause of CKD may be underdiagnosed. The increasing incidence of CKD in the elderly has been ascribed, in part, to decreased mortality rate from the cardiac and cerebral complications of atherosclerotic vascular disease, enabling a greater segment of the population to eventually manifest the renal component of generalized vascular disease. Nevertheless, it should be appreciated

1	of atherosclerotic vascular disease, enabling a greater segment of the population to eventually manifest the renal component of generalized vascular disease. Nevertheless, it should be appreciated that the vast majority of such patients with early stages of CKD will succumb to the cardiovascular and cerebrovascular consequences of the vascular disease before they can progress to the most advanced stages of CKD. Indeed, even a minor decrement in GFR or the presence of albuminuria is now recognized as a major risk factor for cardiovascular disease.

1	Although serum urea and creatinine concentrations are used to measure the excretory capacity of the kidneys, accumulation of these two molecules themselves does not account for the many symptoms and signs that characterize the uremic syndrome in advanced renal failure. disease and primary glomerular disease with associated hypertension) aRelative contribution of each category varies with geographic region and race.

1	1814 Hundreds of toxins that accumulate in renal failure have been implicated in the uremic syndrome. These include water-soluble, hydrophobic, protein-bound, charged, and uncharged compounds. Additional categories of nitrogenous excretory products include guanidino compounds, urates and hippurates, products of nucleic acid metabolism, polyamines, myoinositol, phenols, benzoates, and indoles. It is thus evident that the serum concentrations of urea and creatinine should be viewed as being readily measured, but incomplete, surrogate markers for these compounds, and monitoring the levels of urea and creatinine in the patient with impaired kidney function represents a vast oversimplification of the uremic state. The uremic syndrome and the disease state associated with advanced renal impairment involve more than renal excretory failure. A host of metabolic and endocrine functions normally performed by the kidneys is also impaired or suppressed, and this results in anemia, malnutrition,

1	involve more than renal excretory failure. A host of metabolic and endocrine functions normally performed by the kidneys is also impaired or suppressed, and this results in anemia, malnutrition, and abnormal metabolism of carbohydrates, fats, and proteins. Furthermore, plasma levels of many hormones, including PTH, FGF23, insulin, glucagon, steroid hormones including vitamin D and sex hormones, and prolactin, change with CKD as a result of reduced excretion, decreased degradation, or abnormal regulation. Finally, CKD is associated with worsening systemic inflammation. Elevated levels of C-reactive protein are detected along with other acute-phase reactants, whereas levels of so-called negative acute-phase reactants, such as albumin and fetuin, decline with progressive reduction in GFR. Thus, the inflammation associated with CKD is important in the malnutrition-inflammation-atherosclerosis/calcification syndrome, which contributes in turn to the acceleration of vascular disease and

1	Thus, the inflammation associated with CKD is important in the malnutrition-inflammation-atherosclerosis/calcification syndrome, which contributes in turn to the acceleration of vascular disease and comorbidity associated with advanced kidney disease. In summary, the pathophysiology of the uremic syndrome can be divided into manifestations in three spheres of dysfunction: (1) those consequent to the accumulation of toxins that normally undergo renal excretion, including products of protein metabolism; (2) those consequent to the loss of other kidney functions, such as fluid and electrolyte homeostasis and hormone regulation; and (3) progressive systemic inflammation and its vascular and nutritional consequences.

1	Uremia leads to disturbances in the function of virtually every organ system. Chronic dialysis can reduce the incidence and severity of many of these disturbances, so that the overt and florid manifestations of uremia have largely disappeared in the modern health setting. However, even optimal dialysis therapy is not completely effective as renal replacement therapy, because some disturbances resulting from impaired kidney function fail to respond to dialysis.

1	FLuID, ELECTROLYTE, AND ACID-BASE DISORDERS Sodium and Water Homeostasis In most patients with stable CKD, the total-body content of sodium and water is modestly increased, although this may not be apparent on clinical examination. With normal renal function, the tubular reabsorption of filtered sodium and water is adjusted so that urinary excretion matches intake. Many forms of kidney disease (e.g., glomerulonephritis) disrupt this balance such that dietary intake of sodium exceeds its urinary excretion, leading to sodium retention and attendant extracellular fluid volume (ECFV) expansion. This expansion may contribute to hypertension, which itself can accelerate the nephron injury. As long as water intake does not exceed the capacity for water clearance, the ECFV expansion will be isotonic and the patient will have a normal plasma sodium concentration (Chap. 333e). Hyponatremia is not commonly seen in CKD patients but, when present, often responds to water restriction. The patient

1	and the patient will have a normal plasma sodium concentration (Chap. 333e). Hyponatremia is not commonly seen in CKD patients but, when present, often responds to water restriction. The patient with ECFV expansion (peripheral edema, sometimes hypertension poorly responsive to therapy) should be counseled regarding salt restriction. Thiazide diuretics have limited utility in stages 3–5 CKD, such that administration of loop diuretics, including furosemide, bumetanide, or torsemide, may also be needed. Resistance to loop diuretics in CKD often mandates use of higher doses than those used in patients with more normal kidney function. The combination of loop diuretics with metolazone, which inhibits the sodium chloride co-transporter of the distal convoluted tubule, can promote renal salt excretion. Diuretic resistance with intractable edema and hypertension in advanced CKD may serve as an indication to initiate dialysis.

1	In addition to problems with salt and water excretion, some patients with CKD may instead have impaired renal conservation of sodium and water. When an extrarenal cause for fluid loss, such as gastrointestinal (GI) loss, is present, these patients may be prone to ECFV depletion because of the inability of the failing kidney to reclaim filtered sodium adequately. Furthermore, depletion of ECFV, whether due to GI losses or overzealous diuretic therapy, can further compromise kidney function through underperfusion, or a “prerenal” basis, leading to acute-on-chronic kidney failure. In this setting, cautious volume repletion with normal saline may return the ECFV to normal and restore renal function to baseline without having to intervene with dialysis.

1	Potassium Homeostasis In CKD, the decline in GFR is not necessarily accompanied by a parallel decline in urinary potassium excretion, which is predominantly mediated by aldosterone-dependent secretion in the distal nephron. Another defense against potassium retention in these patients is augmented potassium excretion in the GI tract. Notwithstanding these two homeostatic responses, hyperkalemia may be precipitated in certain settings. These include increased dietary potassium intake, protein catabolism, hemolysis, hemorrhage, transfusion of stored red blood cells, and metabolic acidosis. In addition, a host of medications can inhibit renal potassium excretion and lead to hyperkalemia. The most important medications in this respect include the RAS inhibitors and spironolactone and other potassium-sparing diuretics such as amiloride, eplerenone, and triamterene.

1	Certain causes of CKD can be associated with earlier and more severe disruption of potassium-secretory mechanisms in the distal nephron, out of proportion to the decline in GFR. These include conditions associated with hyporeninemic hypoaldosteronism, such as diabetes, and renal diseases that preferentially affect the distal nephron, such as obstructive uropathy and sickle cell nephropathy. Hypokalemia is not common in CKD and usually reflects markedly reduced dietary potassium intake, especially in association with excessive diuretic therapy or concurrent GI losses. The use of potassium supplements and potassium-sparing diuretics may be risky in patients with impaired renal function, and should be constantly reevaluated as GFR declines.

1	Metabolic Acidosis Metabolic acidosis is a common disturbance in advanced CKD. The majority of patients can still acidify the urine, but they produce less ammonia and, therefore, cannot excrete the normal quantity of protons in combination with this urinary buffer. Hyperkalemia, if present, further depresses ammonia production. The combination of hyperkalemia and hyperchloremic metabolic acidosis is often present, even at earlier stages of CKD (stages 1–3), in patients with diabetic nephropathy or in those with predominant tubulointerstitial disease or obstructive uropathy; this is a non-anion-gap metabolic acidosis.

1	With worsening renal function, the total urinary net daily acid excretion is usually limited to 30–40 mmol, and the anions of retained organic acids can then lead to an anion-gap metabolic acidosis. Thus, the non-anion-gap metabolic acidosis that can be seen in earlier stages of CKD may be complicated by the addition of an anion-gap metabolic acidosis as CKD progresses. In most patients, the metabolic acidosis is mild; the pH is rarely <7.35 and can usually be corrected with oral sodium bicarbonate supplementation. Animal and human studies have suggested that even modest degrees of metabolic acidosis may be associated with the development of protein catabolism. Alkali supplementation may attenuate the catabolic state and possibly slow CKD progression and accordingly is recommended when the serum bicarbonate concentration falls below 20–23 mmol/L. The concomitant sodium load mandates careful attention to volume status and the need for diuretic agents.

1	treatMent Fluid, electrolyte, And Acid-BAse disorders

1	Dietary salt restriction and the use of loop diuretics, occasionally in combination with metolazone, may be needed to maintain euvolemia. In contrast, overzealous salt restriction or diuretic use can lead to ECFV depletion and precipitate a further decline in GFR. The rare patient with salt-losing nephropathy may require a sodium-rich diet or salt supplementation. Water restriction is indicated only if there is a problem with hyponatremia. Intractable ECFV expansion, despite dietary salt restriction and diuretic therapy, may be an indication to start renal replacement therapy. Hyperkalemia often responds to dietary restriction of potassium, the use of kaliuretic diuretics, and avoidance of both potassium supplements (including occult sources, such as dietary salt substitutes) and potassium-retaining medications (especially angiotensin-converting enzyme [ACE] inhibitors or angiotensin receptor blockers [ARBs]). Kaliuretic diuretics promote urinary potassium excretion, whereas

1	potassium-retaining medications (especially angiotensin-converting enzyme [ACE] inhibitors or angiotensin receptor blockers [ARBs]). Kaliuretic diuretics promote urinary potassium excretion, whereas potassium-binding resins, such as calcium resonium or sodium polystyrene, can promote potassium loss through the GI tract and may reduce the incidence of hyperkalemia. Intractable hyperkalemia is an indication (although uncommon) to consider institution of dialysis in a CKD patient. The renal tubular acidosis and subsequent anion-gap metabolic acidosis in progressive CKD will respond to alkali supplementation, typically with sodium bicarbonate. Recent studies suggest that this replacement should be considered when the serum bicarbonate concentration falls below 20–23 mmol/L to avoid the protein catabolic state seen with even mild degrees of metabolic acidosis and to slow the progression of CKD.

1	The principal complications of abnormalities of calcium and phosphate metabolism in CKD occur in the skeleton and the vascular bed, with occasional severe involvement of extraosseous soft tissues. It is likely that disorders of bone turnover and disorders of vascular and soft tissue calcification are related to each other (Fig. 335-3). Bone Manifestations of CKD The major disorders of bone disease can be classified into those associated with high bone turnover with increased PTH levels (including osteitis fibrosa cystica, the classic lesion of secondary hyperparathyroidism) and low bone turnover with low or normal PTH levels (adynamic bone disease and osteomalacia).

1	The pathophysiology of secondary hyperparathyroidism and the consequent high-turnover bone disease is related to abnormal mineral metabolism through the following events: (1) declining GFR leads to reduced excretion of phosphate and, thus, phosphate retention; (2) the retained phosphate stimulates increased synthesis of both FGF-23 by osteocytes and PTH and stimulates growth of parathyroid gland mass; and (3) decreased levels of ionized calcium, resulting from suppression of calcitriol production by FGF-23 and by the failing kidney, as well as phosphate retention, also stimulate PTH production. Low calcitriol levels contribute to hyperparathyroidism, both by leading to hypocalcemia and also by a direct effect on PTH gene transcription. These changes start to occur when the GFR falls below 60 mL/min.

1	FGF-23 is part of a family of phosphatonins that promotes renal phosphate excretion. Recent studies have shown that levels of this hormone, secreted by osteocytes, increase early in the course of CKD, even before phosphate retention and hyperphosphatemia. FGF-23 may defend normal serum phosphorus in at least three ways: (1) increased renal phosphate excretion; (2) stimulation of PTH, which also increases renal phosphate excretion; and (3) suppression of the formation of 1,25(OH)2D3, leading to diminished phosphorus absorption from the GI tract. Interestingly, high levels of FGF-23 are also an independent risk factor for left ventricular hypertrophy and mortality in CKD, dialysis, and renal transplant patients. Moreover, elevated levels of FGF-23 may indicate the need for therapeutic intervention (e.g., phosphate restriction), even when serum phosphate levels are within the normal range.

1	FIGuRE 335-4 Tumoral calcinosis. This patient was on hemodialysis for many years and was nonadherent to dietary phosphorus restric-tion or the use of phosphate binders. He was chronically severely hyperphosphatemic. He developed an enlarging painful mass on his arm that was extensively calcified. Hyperparathyroidism stimulates bone turnover and leads to osteitis fibrosa cystica. Bone histology shows abnormal osteoid, bone and bone marrow fibrosis, and in advanced stages, the formation of bone cysts, sometimes with hemorrhagic elements so that they appear brown in color, hence the term brown tumor. Clinical manifestations of severe hyperparathyroidism include bone pain and fragility, brown tumors, compression syndromes, and erythropoietin resistance in part related to the bone marrow fibrosis. Furthermore, PTH itself is considered a uremic toxin, and high levels are associated with muscle weakness, fibrosis of cardiac muscle, and nonspecific constitutional symptoms.

1	Low-turnover bone disease can be grouped into two categories— adynamic bone disease and osteomalacia. Adynamic bone disease is increasing in prevalence, especially among diabetics and the elderly. It is characterized by reduced bone volume and mineralization and may result from excessive suppression of PTH production, chronic inflammation, or both. Suppression of PTH can result from the use of vitamin D preparations or from excessive calcium exposure in the form of calcium-containing phosphate binders or high-calcium dialysis solutions. Complications of adynamic bone disease include an increased incidence of fracture and bone pain and an association with increased vascular and cardiac calcification. Occasionally the calcium will precipitate in the soft tissues into large concretions termed “tumoral calcinosis” (Fig. 335-4).

1	Calcium, Phosphorus, and the Cardiovascular System Recent epidemiologic evidence has shown a strong association between hyperphosphatemia and increased cardiovascular mortality rate in patients with stage 5 CKD and even in patients with earlier stages of CKD. Hyperphosphatemia and hypercalcemia are associated with increased vascular calcification, but it is unclear whether the excessive mortality rate is mediated by this mechanism. Studies using computed tomography (CT) and electron-beam CT scanning show that CKD patients have calcification of the media in coronary arteries and even heart valves that appear to be orders of magnitude greater than that in patients without renal disease. The magnitude of the calcification is proportional to age and hyperphosphatemia and is also associated with low PTH levels and low bone turnover. It is possible that in patients with advanced kidney disease, ingested calcium cannot be deposited in bones with low turnover and, therefore, is deposited at

1	low PTH levels and low bone turnover. It is possible that in patients with advanced kidney disease, ingested calcium cannot be deposited in bones with low turnover and, therefore, is deposited at extraosseous sites, such as the vascular bed and soft tissues. It is interesting in this regard that there is also an association between osteoporosis and vascular calcification in the general population. Finally, hyperphosphatemia can induce a change in gene expression in vascular cells to an osteoblast-like profile, leading to vascular calcification and even ossification.

1	FIGuRE 335-5 Calciphylaxis. This peritoneal dialysis patient was on chronic warfarin therapy for atrial fibrillation. She noticed a small painful nodule on the abdomen that was followed by progressive skin necrosis and ulceration of the anterior abdominal wall. She was treated with hyperbaric oxygen, intravenous thiosulfate, and discon-tinuation of warfarin, with slow resolution of the ulceration.

1	Other Complications of Abnormal Mineral Metabolism Calciphylaxis (calcific uremic arteriolopathy) is a devastating condition seen almost exclusively in patients with advanced CKD. It is heralded by livedo reticularis and advances to patches of ischemic necrosis, especially on the legs, thighs, abdomen, and breasts (Fig. 335-5). Pathologically, there is evidence of vascular occlusion in association with extensive vascular and soft tissue calcification. It appears that this condition is increasing in incidence. Originally it was ascribed to severe abnormalities in calcium and phosphorus control in dialysis patients, usually associated with advanced hyperparathyroidism. However, more recently, calciphylaxis has been seen with increasing frequency in the absence of severe hyperparathyroidism. Other etiologies have been suggested, including the increased use of oral calcium as a phosphate binder. Warfarin is commonly used in hemodialysis patients, and one of the effects of warfarin therapy

1	Other etiologies have been suggested, including the increased use of oral calcium as a phosphate binder. Warfarin is commonly used in hemodialysis patients, and one of the effects of warfarin therapy is to decrease the vitamin K– dependent regeneration of matrix GLA protein. This latter protein is important in preventing vascular calcification. Thus, warfarin treatment is considered a risk factor for calciphylaxis, and if a patient develops this syndrome, this medication should be discontinued and replaced with alternative forms of anticoagulation.

1	Calcitriol exerts a direct suppressive effect on PTH secretion and also indirectly suppresses PTH secretion by raising the concentration of ionized calcium. However, calcitriol therapy may result in hypercalcemia and/or hyperphosphatemia through increased GI absorption of these minerals. Certain analogues of calcitriol are available (e.g., paricalcitol) that suppress PTH secretion with less attendant hypercalcemia. Recognition of the role of the extracellular calcium-sensing receptor has led to the development of calcimimetic agents that enhance the sensitivity of the parathyroid cell to the suppressive effect of calcium. This class of drug, which includes cinacalcet, produces a dose-dependent reduction in PTH and plasma calcium concentration in some patients.

1	Current National Kidney Foundation Kidney Disease Outcomes Quality Initiative guidelines recommend a target PTH level between 150 and 300 pg/mL, recognizing that very low PTH levels are associated with adynamic bone disease and possible consequences of fracture and ectopic calcification. Cardiovascular disease is the leading cause of morbidity and mortality in patients at every stage of CKD. The incremental risk of cardiovascular disease in those with CKD compared to the ageand sex-matched general population ranges from 10-to 200-fold, depending on the stage of CKD. Between 30 and 45% of patients reaching stage 5 CKD already have advanced cardiovascular complications. As a result, most patients with CKD succumb to cardiovascular disease (Fig. 335-6) before ever reaching stage 5 CKD. Thus, the focus of patient care in earlier CKD stages should be directed to prevention of cardiovascular complications.

1	Ischemic Vascular Disease The presence of any stage of CKD is a major risk factor for ischemic cardiovascular disease, including occlusive coronary, cerebrovascular, and peripheral vascular disease. The increased prevalence of vascular disease in CKD patients derives from both traditional (“classic”) and nontraditional (CKD-related) risk factors. Traditional risk factors include hypertension, hypervolemia, dyslipidemia, sympathetic overactivity, and hyperhomocysteinemia. The CKD-related risk factors comprise anemia, hyperphosphatemia, hyperparathyroidism, increased FGF-23, sleep apnea, and generalized inflammation. The inflammatory state associated with a reduction in kidney function is reflected in increased circulating acute-phase reactants, such as inflammatory cytokines and C-reactive protein, with a corresponding fall in the “negative acute-phase reactants,” such as serum albumin and fetuin. The inflammatory state appears to accelerate vascular occlusive disease, and low levels

1	protein, with a corresponding fall in the “negative acute-phase reactants,” such as serum albumin and fetuin. The inflammatory state appears to accelerate vascular occlusive disease, and low levels of fetuin may permit more rapid vascular calcification, especially in the face of hyperphosphatemia. Other abnormalities seen in CKD may augment

1	The optimal management of secondary hyperparathyroidism and osteitis fibrosa is prevention. Once the parathyroid gland mass is very large, it is difficult to control the disease. Careful attention should be paid to the plasma phosphate concentration in CKD patients, who should be counseled on a low-phosphate diet as well as the appropriate use of phosphate-binding agents. These Medicare Cohort, 1998–99, % are agents that are taken with meals and complex the dietary phos-0 phate to limit its GI absorption. Examples of phosphate binders are calcium acetate and calcium carbonate. A major side effect of calcium-based phosphate binders is calcium accumulation and hypercalcemia, especially in patients with low-turnover bone disease.

1	Sevelamer and lanthanum are non-calcium-containing polymers FIGuRE 335-6 U.S. Renal Data System showing increased likelihood that also function as phosphate binders; they do not predispose of dying rather than starting dialysis or reaching stage 5 chronic kidney CKD patients to hypercalcemia and may attenuate calcium deposi-disease (CKD).

1	1 , Death; 2 , ESRD; 3 , event-free. DM; diabetes mellitus. tion in the vascular bed. (Adapted from RN Foley et al: J Am Soc Nephrol 16:489-495, 2005.) myocardial ischemia, including left ventricular hypertrophy and microvascular disease. In addition, hemodialysis, with its attendant episodes of hypotension and hypovolemia, may further aggravate coronary ischemia and repeatedly stun the myocardium. Interestingly, however, the largest increment in cardiovascular mortality rate in dialysis patients is not necessarily directly associated with documented acute myocardial infarction but, instead, presents with congestive heart failure and all of its manifestations and sudden death.

1	Cardiac troponin levels are frequently elevated in CKD without evidence of acute ischemia. The elevation complicates the diagnosis of acute myocardial infarction in this population. Serial measurements may be needed, and if the level is unchanged, it is possible that there is no acute myocardial ischemia. Therefore, the trend in levels over the hours after presentation may be more informative than a single, elevated level. Interestingly, consistently elevated levels are an independent prognostic factor for adverse cardiovascular events in this population.

1	Heart Failure Abnormal cardiac function secondary to myocardial ischemia, left ventricular hypertrophy, and frank cardiomyopathy, in combination with the salt and water retention that can be seen with CKD, often results in heart failure or even pulmonary edema. Heart failure can be a consequence of diastolic or systolic dysfunction, or both. A form of “low-pressure” pulmonary edema can also occur in advanced CKD, manifesting as shortness of breath and a “bat wing” distribution of alveolar edema fluid on the chest x-ray. This finding can occur even in the absence of ECFV overload and is associated with normal or mildly elevated pulmonary capillary wedge pressure. This process has been ascribed to increased permeability of alveolar capillary membranes as a manifestation of the uremic state, and it responds to dialysis. Other CKD-related risk factors, including anemia and sleep apnea, may contribute to the risk of heart failure.

1	Hypertension and Left Ventricular Hypertrophy Hypertension is one of the most common complications of CKD. It usually develops early during the course of CKD and is associated with adverse outcomes, including the development of ventricular hypertrophy and a more rapid loss of renal function. Many studies have shown a relationship between the level of blood pressure and the rate of progression of diabetic and nondiabetic kidney disease. Left ventricular hypertrophy and dilated cardiomyopathy are among the strongest risk factors for cardiovascular morbidity and mortality in patients with CKD and are thought to be related primarily, but not exclusively, to prolonged hypertension and ECFV overload. In addition, anemia and the placement of an arteriovenous fistula for hemodialysis can generate a high cardiac output state and consequent heart failure.

1	The absence of hypertension may signify poor left ventricular function. Indeed, in epidemiologic studies of dialysis patients, low blood pressure actually carries a worse prognosis than does high blood pressure. This mechanism, in part, accounts for the “reverse causation” seen in dialysis patients, wherein the presence of traditional risk factors, such as hypertension, hyperlipidemia, and obesity, appear to portend a better prognosis. Importantly, these observations derive from cross-sectional studies of late-stage CKD patients and should not be interpreted to discourage appropriate management of these risk factors in CKD patients, especially at early stages. In contrast to the general population, it is possible that in late-stage CKD, low blood pressure, reduced body mass index, and hypolipidemia indicate the presence of an advanced malnutrition-inflammation state, with poor prognosis.

1	The use of exogenous erythropoiesis-stimulating agents can increase blood pressure and the requirement for antihypertensive drugs. Chronic ECFV overload is also a contributor to hypertension, and improvement in blood pressure can often be seen with the use of dietary sodium restriction, diuretics, and fluid removal with dialysis. Nevertheless, because of activation of the RAS and other disturbances in the balance of vasoconstrictors and vasodilators, some patients remain hypertensive despite careful attention to ECFV status.

1	The overarching goal of hypertension therapy in CKD is to prevent the extrarenal complications of high blood pressure, such as cardiovascular disease and stroke. Although a clear-cut generalizable benefit in slowing progression of CKD remains as yet unproven, the benefit for cardiac and neurologic health is compelling. In all patients with CKD, blood pressure should be controlled to levels recommended by national guideline panels. In CKD patients with diabetes or proteinuria >1 g per 24 h, blood pressure should be reduced to 130/80 mmHg, if achievable without prohibitive adverse effects. Salt restriction should be the first line of therapy. When volume management alone is not sufficient, the choice of antihypertensive agent is similar to that in the general population. ACE inhibitors and ARBs appear to slow the rate of decline of kidney function in a manner that extends beyond reduction of systemic arterial pressure and that involves correction of the intraglomerular hyperfiltration

1	ARBs appear to slow the rate of decline of kidney function in a manner that extends beyond reduction of systemic arterial pressure and that involves correction of the intraglomerular hyperfiltration and hypertension involved in progression of CKD described above. Occasionally, introduction of ACE inhibitors and ARBs can actually precipitate an episode of acute kidney injury, especially when used in combination in patients with ischemic renovascular disease. The use of ACE inhibitors and ARBs may also be complicated by the development of hyperkalemia. Often the concomitant use of a kaliuretic diuretic, such as metolazone, can improve potassium excretion in addition to improving blood pressure control. Potassium-sparing diuretics should be used with caution or avoided altogether in most patients.

1	There are many strategies available to treat the traditional and nontraditional risk factors in CKD patients. Although these have proved effective in the general population, there is little evidence for their benefit in patients with advanced CKD, especially those on dialysis. Certainly hypertension, elevated serum levels of homocysteine, and dyslipidemia promote atherosclerotic disease and are treatable complications of CKD. Renal disease complicated by nephrotic syndrome is associated with a very atherogenic lipid profile and hypercoagulability, which increases the risk of occlusive vascular disease. Because diabetes mellitus and hypertension are the two most frequent causes of advanced CKD, it is not surprising that cardiovascular disease is the most frequent cause of death in dialysis patients. The role of “inflammation” may be quantitatively more important in patients with kidney disease, and the treatment of more traditional risk factors may result in only modest success.

1	patients. The role of “inflammation” may be quantitatively more important in patients with kidney disease, and the treatment of more traditional risk factors may result in only modest success. However, modulation of traditional risk factors may be the only weapon in the therapeutic armamentarium for these patients until the nature of inflammation in CKD and its treatment are better understood.

1	Lifestyle changes, including regular exercise, should be advocated. Hyperlipidemia in patients with CKD should be managed according to national guidelines. If dietary measures are not sufficient, preferred lipid-lowering medications, such as statins, should be used. Again, the use of these agents has not been of proven benefit for patients with advanced CKD. Pericardial Disease Chest pain with respiratory accentuation, accompanied by a friction rub, is diagnostic of pericarditis. Classic electrocardiographic abnormalities include PR-interval depression and diffuse ST-segment elevation. Pericarditis can be accompanied by pericardial effusion that is seen on echocardiography and can rarely lead to tamponade. However, the pericardial effusion can be asymptomatic, and pericarditis can be seen without significant effusion.

1	Pericarditis is observed in advanced uremia, and with the advent of timely initiation of dialysis, is not as common as it once was. It is now more often observed in underdialyzed, nonadherent patients than in those starting dialysis. intolerance, the patient may have to undergo IV iron infusion. For patients on hemodialysis, IV iron can be administered during dialy

1	intolerance, the patient may have to undergo IV iron infusion. For patients on hemodialysis, IV iron can be administered during dialy Uremic pericarditis is an absolute indication for the urgent initiation of dialysis or for intensification of the dialysis prescription in those already receiving dialysis. Because of the propensity to hemorrhage in pericardial fluid, hemodialysis should be performed without heparin. A pericardial drainage procedure should be considered in patients with recurrent pericardial effusion, especially with echocardiographic signs of impending tamponade. Nonuremic causes of pericarditis and effusion include viral, malignant, tuberculous, and autoimmune etiologies. It may also be seen after myocardial infarction and as a complication of treatment with the antihypertensive drug minoxidil.

1	HEMATOLOGIC ABNORMALITIES Anemia A normocytic, normochromic anemia is observed as early as stage 3 CKD and is almost universal by stage 4. The primary cause in patients with CKD is insufficient production of erythropoietin (EPO) by the diseased kidneys. Additional factors are reviewed in Table 335-3.

1	The anemia of CKD is associated with a number of adverse pathophysiologic consequences, including decreased tissue oxygen delivery and utilization, increased cardiac output, ventricular dilation, and ventricular hypertrophy. Clinical manifestations include fatigue and diminished exercise tolerance, angina, heart failure, decreased cognition and mental acuity, and impaired host defense against infection. In addition, anemia may play a role in growth restriction in children with CKD. Although many studies in CKD patients have found that anemia and resistance to exogenous erythropoietic-stimulating agents (ESA) are associated with a poor prognosis, the relative contribution to a poor outcome of the low hematocrit itself, versus inflammation as a cause of the anemia and ESA resistance, remains unclear.

1	The availability of recombinant human ESA has been one of the most significant advances in the care of renal patients since the introduction of dialysis and renal transplantation. The routine use of these recombinant hormones has obviated the need for regular blood transfusions in severely anemic CKD patients, thus dramatically reducing the incidence of transfusion-associated infections and iron overload. Frequent blood transfusions in dialysis patients also lead to the development of alloantibodies that can sensitize the patient to donor kidney antigens and make renal transplantation more problematic.

1	Adequate bone marrow iron stores should be available before treatment with ESA is initiated. Iron supplementation is usually essential to ensure an optimal response to ESA in patients with CKD because the demand for iron by the marrow frequently exceeds the amount of iron that is immediately available for erythropoiesis (measured by percent transferrin saturation), as well as the amount in iron stores (measured by serum ferritin). For the CKD patient not yet on dialysis or the patient treated with peritoneal dialysis, oral iron supplementation should be attempted. If there is GI

1	Relative deficiency of erythropoietin Diminished red blood cell survival Bleeding diathesis Iron deficiency Hyperparathyroidism/bone marrow fibrosis Chronic inflammation Folate or vitamin B12 deficiency Hemoglobinopathy Comorbid conditions: hypo-/hyperthyroidism, pregnancy, HIV-associated disease, autoimmune disease, immunosuppressive drugs sis, keeping in mind that iron therapy can increase the susceptibility to bacterial infections. In addition to iron, an adequate supply of other major substrates and cofactors for red cell production must be ensured, including vitamin B12 and folate. Anemia resistant to recommended doses of ESA in the face of adequate iron stores may be due to some combination of the following: acute or chronic inflammation, inadequate dialysis, severe hyperparathyroidism, chronic blood loss or hemolysis, chronic infection, or malignancy. Blood transfusions increase the risk of hepatitis, iron overload, and transplant sensitization; they should be avoided unless

1	chronic blood loss or hemolysis, chronic infection, or malignancy. Blood transfusions increase the risk of hepatitis, iron overload, and transplant sensitization; they should be avoided unless the anemia fails to respond to ESA and the patient is symptomatic.

1	Randomized, controlled trials of ESA in CKD have failed to show an improvement in cardiovascular outcomes with this therapy. Indeed, there has been an indication that the use of ESA in CKD may be associated with an increased risk of stroke in those with type 2 diabetes, an increase in thromboembolic events, and perhaps a faster progression to the need for dialysis. Therefore, any benefit in terms of improvement of anemic symptoms needs to be balanced against the potential cardiovascular risk. Although further studies are needed, it is quite clear that complete normalization of the hemoglobin concentration has not been demonstrated to be of incremental benefit to CKD patients. Current practice is to target a hemoglobin concentration of 100–115 g/L.

1	Abnormal Hemostasis Patients with later stages of CKD may have a prolonged bleeding time, decreased activity of platelet factor III, abnormal platelet aggregation and adhesiveness, and impaired prothrombin consumption. Clinical manifestations include an increased tendency to bleeding and bruising, prolonged bleeding from surgical incisions, menorrhagia, and GI bleeding. Interestingly, CKD patients also have a greater susceptibility to thromboembolism, especially if they have renal disease that includes nephrotic-range proteinuria. The latter condition results in hypoalbuminemia and renal loss of anticoagulant factors, which can lead to a thrombophilic state. Abnormal bleeding time and coagulopathy in patients with renal failure may be reversed temporarily with desmopressin (DDAVP), cryoprecipitate, IV conjugated estrogens, blood transfusions, and ESA therapy. Optimal dialysis will usually correct a prolonged bleeding time.

1	Given the coexistence of bleeding disorders and a propensity to thrombosis that is unique in the CKD patient, decisions about anticoagulation that have a favorable risk-benefit profile in the general population may not be applicable to the patient with advanced CKD. One example is warfarin anticoagulation for atrial fibrillation; the decision to anticoagulate should be made on an individual basis in the CKD patient because there appears to be a greater risk of bleeding complications.

1	Certain anticoagulants, such as fractionated low-molecularweight heparin, may need to be avoided or dose-adjusted in these patients, with monitoring of factor Xa activity where available. It is often more prudent to use conventional unfractionated heparin, titrated to the measured partial thromboplastin time, in hospitalized patients requiring an alternative to warfarin anticoagulation. The new classes of oral anticoagulants are all, in part, renally eliminated and need dose adjustment in the face of decreased GFR (Chap. 143).

1	Central nervous system (CNS), peripheral, and autonomic neuropathy as well as abnormalities in muscle structure and function are all well-recognized complications of CKD. Subtle clinical manifestations of uremic neuromuscular disease usually become evident at stage 3 CKD. Early manifestations of CNS complications include mild disturbances in memory and concentration and sleep disturbance. Neuromuscular irritability, including hiccups, cramps, and twitching, becomes evident at later stages. In advanced untreated kidney failure, asterixis, myoclonus, seizures, and coma can be seen.

1	Peripheral neuropathy usually becomes clinically evident after the patient reaches stage 4 CKD, although electrophysiologic and histologic evidence occurs earlier. Initially, sensory nerves are involved more than motor, lower extremities more than upper, and distal parts of the extremities more than proximal. The “restless leg syndrome” is characterized by ill-defined sensations of sometimes debilitating discomfort in the legs and feet relieved by frequent leg movement. If dialysis is not instituted soon after onset of sensory abnormalities, motor involvement follows, including muscle weakness. Evidence of peripheral neuropathy without another cause (e.g., diabetes mellitus) is an indication for starting renal replacement therapy. Many of the complications described above will resolve with dialysis, although subtle nonspecific abnormalities may persist.

1	Uremic fetor, a urine-like odor on the breath, derives from the breakdown of urea to ammonia in saliva and is often associated with an unpleasant metallic taste (dysgeusia). Gastritis, peptic disease, and mucosal ulcerations at any level of the GI tract occur in uremic patients and can lead to abdominal pain, nausea, vomiting, and GI bleeding. These patients are also prone to constipation, which can be worsened by the administration of calcium and iron supplements. The retention of uremic toxins also leads to anorexia, nausea, and vomiting.

1	Protein restriction may be useful to decrease nausea and vomiting; however, it may put the patient at risk for malnutrition and should be carried out, if possible, in consultation with a registered dietitian specializing in the management of CKD patients. Protein-energy malnutrition, a consequence of low protein and caloric intake, is common in advanced CKD and is often an indication for initiation of renal replacement therapy. Metabolic acidosis and the activation of inflammatory cytokines can promote protein catabolism. Assessment for protein-energy malnutrition should begin at stage 3 CKD. A number of indices are useful in this assessment and include dietary history, including food diary and subjective global assessment; edema-free body weight; and measurement of urinary protein nitrogen appearance. Dual-energy x-ray absorptiometry is now widely used to estimate lean body mass versus ECFV. Adjunctive tools include clinical signs, such as skinfold thickness, mid-arm muscle

1	nitrogen appearance. Dual-energy x-ray absorptiometry is now widely used to estimate lean body mass versus ECFV. Adjunctive tools include clinical signs, such as skinfold thickness, mid-arm muscle circumference, and additional laboratory tests such as serum pre-albumin and cholesterol levels. Nutritional guidelines for patients with CKD are summarized in the “Treatment” section.

1	Glucose metabolism is impaired in CKD, as evidenced by a slowing of the rate at which blood glucose levels decline after a glucose load. However, fasting blood glucose is usually normal or only slightly elevated, and the mild glucose intolerance does not require specific therapy. Because the kidney contributes to insulin removal from the circulation, plasma levels of insulin are slightly to moderately elevated in most uremic patients, both in the fasting and postprandial states. Because of this diminished renal degradation of insulin, patients on insulin therapy may need progressive reduction in dose as their renal function worsens. Many hypoglycemic agents, including the gliptins, require dose reduction in renal failure, and some, such as metformin, are contraindicated when the GFR is less than half of normal.

1	In women with CKD, estrogen levels are low, and menstrual abnormalities, infertility, and inability to carry pregnancies to term are common. When the GFR has declined to ~40 mL/min, pregnancy is associated with a high rate of spontaneous abortion, with only ~20% of pregnancies leading to live births, and pregnancy may hasten the progression of the kidney disease itself. Women with CKD who are contemplating pregnancy should consult first with a nephrologist in conjunction with an obstetrician specializing in high-risk pregnancy. Men with CKD have reduced plasma testosterone levels, and sexual dysfunction and oligospermia may supervene. Sexual maturation may be delayed or impaired in adolescent children with CKD, even among those treated with dialysis. Many of these abnormalities improve or reverse with intensive dialysis or with suc-1819 cessful renal transplantation.

1	Abnormalities of the skin are prevalent in progressive CKD. Pruritus is quite common and one of the most vexing manifestations of the uremic state. In advanced CKD, even on dialysis, patients may become more pigmented, and this is felt to reflect the deposition of retained pigmented metabolites, or urochromes. Although many of the cutaneous abnormalities improve with dialysis, pruritus is often tenacious. The first lines of management are to rule out unrelated skin disorders, such as scabies, and to treat hyperphosphatemia, which can cause itch. Local moisturizers, mild topical glucocorticoids, oral antihistamines, and ultraviolet radiation have been reported to be helpful.

1	A skin condition unique to CKD patients called nephrogenic fibrosing dermopathy consists of progressive subcutaneous induration, especially on the arms and legs. The condition is similar to scleromyxedema and is seen very rarely in patients with CKD who have been exposed to the magnetic resonance contrast agent gadolinium. Current recommendations are that patients with CKD stage 3 (GFR 30–59 mL/min) should minimize exposure to gadolinium, and those with CKD stages 4–5 (GFR <30 mL/min) should avoid the use of gadolinium agents unless it is medically necessary. Concomitant liver disease appears to be a risk factor. However, no patient should be denied an imaging investigation that is critical to management, and under such circumstances, rapid removal of gadolinium by hemodialysis (even in patients not yet receiving renal replacement therapy) shortly after the procedure may mitigate this sometimes devastating complication.

1	INITIAL APPROACH History and Physical Examination Symptoms and overt signs of kidney disease are often subtle or absent until renal failure supervenes. Thus, the diagnosis of kidney disease often surprises patients and may be a cause of skepticism and denial. Particular aspects of the history that are germane to renal disease include a history of hypertension (which can cause CKD or more commonly be a consequence of CKD), diabetes mellitus, abnormal urinalyses, and problems with pregnancy such as preeclampsia or early pregnancy loss. A careful drug history should be elicited: patients may not volunteer use of analgesics, for example. Other drugs to consider include nonsteroidal anti-inflammatory agents, cyclooxygenase-2 (COX-2) inhibitors, antimicrobials, chemotherapeutic agents, antiretroviral agents, proton pump inhibitors, phosphate-containing bowel cathartics, and lithium. In evaluating the uremic syndrome, questions about appetite, weight loss, nausea, hiccups, peripheral edema,

1	agents, proton pump inhibitors, phosphate-containing bowel cathartics, and lithium. In evaluating the uremic syndrome, questions about appetite, weight loss, nausea, hiccups, peripheral edema, muscle cramps, pruritus, and restless legs are especially helpful. A careful family history of kidney disease, together with assessment of manifestations in other organ systems such as auditory, visual, and integumentary, may lead to the diagnosis of a heritable form of CKD (e.g., Alport or Fabry disease, cystinosis) or shared environmental exposure to nephrotoxic agents (e.g., heavy metals, aristolochic acid). It should be noted that clustering of CKD, sometimes of different etiologies, is often observed within families.

1	The physical examination should focus on blood pressure and target organ damage from hypertension. Thus, funduscopy and precordial examination (left ventricular heave, a fourth heart sound) should be carried out. Funduscopy is important in the diabetic patient, because it may show evidence of diabetic retinopathy, which is associated with nephropathy. Other physical examination manifestations of CKD include edema and sensory polyneuropathy. The finding of asterixis or a pericardial friction rub not attributable to other causes usually signifies the presence of the uremic syndrome.

1	Laboratory Investigation Laboratory studies should focus on a search for clues to an underlying causative or aggravating disease process and on the degree of renal damage and its consequences. Serum and urine protein electrophoresis, looking for multiple myeloma, should be obtained in all patients >35 years with unexplained CKD, especially if there is associated anemia and elevated, or even inappropriately 1820 normal, serum calcium concentration in the face of renal insufficiency. In the presence of glomerulonephritis, autoimmune diseases such as lupus and underlying infectious etiologies such as hepatitis B and C and HIV should be tested. Serial measurements of renal function should be obtained to determine the pace of renal deterioration and ensure that the disease is truly chronic rather than acute or subacute and hence potentially reversible. Serum concentrations of calcium, phosphorus, vitamin D, and PTH should be measured to evaluate metabolic bone disease. Hemoglobin

1	rather than acute or subacute and hence potentially reversible. Serum concentrations of calcium, phosphorus, vitamin D, and PTH should be measured to evaluate metabolic bone disease. Hemoglobin concentration, iron, vitamin B12, and folate should also be evaluated. A 24-h urine collection may be helpful, because protein excretion >300 mg may be an indication for therapy with ACE inhibitors or ARBs.

1	Imaging Studies The most useful imaging study is a renal ultrasound, which can verify the presence of two kidneys, determine if they are symmetric, provide an estimate of kidney size, and rule out renal masses and evidence of obstruction. Because it takes time for kidneys to shrink as a result of chronic disease, the finding of bilaterally small kidneys supports the diagnosis of CKD of long-standing duration, with an irreversible component of scarring. If the kidney size is normal, it is possible that the renal disease is acute or subacute. The exceptions are diabetic nephropathy (where kidney size is increased at the onset of diabetic nephropathy before CKD supervenes), amyloidosis, and HIV nephropathy, where kidney size may be normal in the face of CKD. Polycystic kidney disease that has reached some degree of renal failure will almost always present with enlarged kidneys with multiple cysts (Chap. 339). A discrepancy >1 cm in kidney length suggests either a unilateral developmental

1	reached some degree of renal failure will almost always present with enlarged kidneys with multiple cysts (Chap. 339). A discrepancy >1 cm in kidney length suggests either a unilateral developmental abnormality or disease process or renovascular disease with arterial insufficiency affecting one kidney more than the other. The diagnosis of renovascular disease can be undertaken with different techniques, including Doppler sonography, nuclear medicine studies, or CT or magnetic resonance imaging (MRI) studies. If there is a suspicion of reflux nephropathy (recurrent childhood urinary tract infection, asymmetric renal size with scars on the renal poles), a voiding cystogram may be indicated. However, in most cases, by the time the patient has CKD, the reflux has resolved, and even if still present, repair does not improve renal function. Radiographic contrast imaging studies are not particularly helpful in the investigation of CKD. Intravenous or intraarterial dye should be avoided where

1	repair does not improve renal function. Radiographic contrast imaging studies are not particularly helpful in the investigation of CKD. Intravenous or intraarterial dye should be avoided where possible in the CKD patient, especially with diabetic nephropathy, because of the risk of radiographic contrast dye–induced renal failure. When unavoidable, appropriate precautionary measures include avoidance of hypovolemia at the time of contrast exposure, minimization of the dye load, and choice of radiographic contrast preparations with the least nephrotoxic potential. Additional measures thought to attenuate contrast-induced worsening of renal function include judicious administration of sodium bicarbonate–containing solutions and N -acetylcysteine.

1	Kidney Biopsy In the patient with bilaterally small kidneys, renal biopsy is not advised because (1) it is technically difficult and has a greater likelihood of causing bleeding and other adverse consequences, (2) there is usually so much scarring that the underlying disease may not be apparent, and (3) the window of opportunity to render disease-specific therapy has passed. Other contraindications to renal biopsy include uncontrolled hypertension, active urinary tract infection, bleeding diathesis (including ongoing anticoagulation), and severe obesity. Ultrasound-guided percutaneous biopsy is the favored approach, but a surgical or laparoscopic approach can be considered, especially in the patient with a single kidney where direct visualization and control of bleeding are crucial. In the CKD patient in whom a kidney biopsy is indicated (e.g., suspicion of a concomitant or superimposed active process such as interstitial nephritis or in the face of accelerated loss of GFR), the

1	In the CKD patient in whom a kidney biopsy is indicated (e.g., suspicion of a concomitant or superimposed active process such as interstitial nephritis or in the face of accelerated loss of GFR), the bleeding time should be measured, and if increased, desmopressin should be administered immediately prior to the procedure.

1	A brief run of hemodialysis (without heparin) may also be considered prior to renal biopsy to normalize the bleeding time.

1	The most important initial diagnostic step is to distinguish newly diagnosed CKD from acute or subacute renal failure, because the latter two conditions may respond to targeted therapy. Previous measurements of serum creatinine concentration are particularly helpful in this regard. Normal values from recent months or even years suggest that the current extent of renal dysfunction could be more acute, and hence reversible, than might otherwise be appreciated. In contrast, elevated serum creatinine concentration in the past suggests that the renal disease represents a chronic process. Even if there is evidence of chronicity, there is the possibility of a superimposed acute process (e.g., ECFV depletion, urinary infection or obstruction, or nephrotoxin exposure) supervening on the chronic condition. If the history suggests multiple systemic manifestations of recent onset (e.g., fever, polyarthritis, rash), it should be assumed that renal insufficiency is part of an acute systemic

1	condition. If the history suggests multiple systemic manifestations of recent onset (e.g., fever, polyarthritis, rash), it should be assumed that renal insufficiency is part of an acute systemic illness.

1	Although renal biopsy can usually be performed in early CKD (stages 1–3), it is not always indicated. For example, in a patient with a history of type 1 diabetes mellitus for 15–20 years with retinopathy, nephrotic-range proteinuria, and absence of hematuria, the diagnosis of diabetic nephropathy is very likely and biopsy is usually not necessary. However, if there were some other finding not typical of diabetic nephropathy, such as hematuria or white blood cell casts, or absence of diabetic retinopathy, some other disease may be present and a biopsy may be indicated.

1	In the absence of a clinical diagnosis, renal biopsy may be the only recourse to establish an etiology in early-stage CKD. However, as noted above, once the CKD is advanced and the kidneys are small and scarred, there is little utility and significant risk in attempting to arrive at a specific diagnosis. Genetic testing is increasingly entering the repertoire of diagnostic tests, since the patterns of injury and kidney morphologic abnormalities often reflect overlapping causal mechanisms, whose origins can sometimes be attributed to a genetic predisposition or cause.

1	Treatments aimed at specific causes of CKD are discussed elsewhere. Among others, these include optimized glucose control in diabetes mellitus, immunosuppressive agents for glomerulonephritis, and emerging specific therapies to retard cystogenesis in polycystic kidney disease. The optimal timing of both specific and nonspecific therapy is usually well before there has been a measurable decline in GFR and certainly before CKD is established. It is helpful to measure sequentially and plot the rate of decline of GFR in all patients. Any acceleration in the rate of decline should prompt a search for superimposed acute or subacute processes that may be reversible. These include ECFV depletion, uncontrolled hypertension, urinary tract infection, new obstructive uropathy, exposure to nephrotoxic agents (such as nonsteroidal anti-inflammatory drugs [NSAIDs] or radiographic dye), and reactivation or flare of the original disease, such as lupus or vasculitis.

1	There is variation in the rate of decline of GFR among patients with CKD. However, the following interventions should be considered in an effort to stabilize or slow the decline of renal function.

1	Reducing Intraglomerular Hypertension and Proteinuria Increased intraglomerular filtration pressures and glomerular hypertrophy develop as a response to loss of nephron number from different kidney diseases. This response is maladaptive, as it promotes the ongoing decline of kidney function even if the inciting process has been treated or spontaneously resolved. Control of glomerular hypertension is important in slowing the progression of CKD. Moreover, elevated blood pressure increases proteinuria by increasing its flux across the glomerular capillaries. Conversely, the renoprotective effect of antihypertensive medications is gauged through the consequent reduction of proteinuria. Thus, the more effective a given treatment is in lowering protein excretion, the greater the subsequent impact on protection from decline in GFR. This observation is the basis for the treatment guideline establishing 130/80 mmHg as the target blood pressure in proteinuric CKD patients.

1	ACE inhibitors and ARBs inhibit the angiotensin-induced vasoconstriction of the efferent arterioles of the glomerular microcirculation. This inhibition leads to a reduction in both intraglomerular filtration pressure and proteinuria. Several controlled studies have shown that these drugs are effective in slowing the progression of renal failure in patients with advanced stages of both diabetic and nondiabetic CKD. This slowing in progression of CKD is strongly associated with the proteinuria-lowering effect. In the absence of an antiproteinuric response with either agent alone, combined treatment with both ACE inhibitors and ARBs has been considered. The combination is associated with a greater reduction in proteinuria compared to either agent alone. Insofar as reduction in proteinuria is a surrogate for improved renal outcome, the combination would appear to be advantageous. However, there is a greater incidence of acute kidney injury and adverse cardiac events from such combination

1	a surrogate for improved renal outcome, the combination would appear to be advantageous. However, there is a greater incidence of acute kidney injury and adverse cardiac events from such combination therapy. It is uncertain, therefore, whether the ACE inhibitor plus ARB therapy can be advised routinely. Adverse effects from these agents include cough and angioedema with ACE inhibitors and anaphylaxis and hyperkalemia with either class. A progressive increase in serum creatinine concentration with these agents may suggest the presence of renovascular disease within the large or small arteries. Development of these side effects may mandate the use of second-line antihypertensive agents instead of the ACE inhibitors or ARBs. Among the calcium channel blockers, diltiazem and verapamil may exhibit superior antiproteinuric and renoprotective effects compared to the dihydropyridines. At least two different categories of response can be considered: one in which progression is strongly

1	may exhibit superior antiproteinuric and renoprotective effects compared to the dihydropyridines. At least two different categories of response can be considered: one in which progression is strongly associated with systemic and intraglomerular hypertension and proteinuria (e.g., diabetic nephropathy, glomerular diseases) and in which ACE inhibitors and ARBs are likely to be the first choice; and another in which proteinuria is mild or absent initially (e.g., adult polycystic kidney disease and other tubulointerstitial diseases), where the contribution of intraglomerular hypertension is less prominent and other antihypertensive agents can be useful for control of systemic hypertension.

1	See Chap. 418.

1	MANAGING OTHER COMPLICATIONS OF CHRONIC KIDNEY DISEASE Medication Dose Adjustment Although the loading dose of most drugs is not affected by CKD because no renal elimination is used in the calculation, the maintenance doses of many drugs will need to be adjusted. For those agents in which >70% excretion is by a nonrenal route, such as hepatic elimination, dose adjustment may not be needed. Some drugs that should be avoided include metformin, meperidine, and oral hypoglycemics that are eliminated by the kidney. NSAIDs should be avoided because of the risk of further worsening of kidney function. Many antibiotics, antihypertensives, and antiarrhythmics may require a reduction in dosage or change in the dose interval. Several online Web-based databases for dose adjustment of medications according to stage of CKD or estimated GFR are available (e.g., http://www.globalrph.com/renaldosing2.htm). Nephrotoxic radiocontrast agents and gadolinium should be avoided or used according to strict

1	to stage of CKD or estimated GFR are available (e.g., http://www.globalrph.com/renaldosing2.htm). Nephrotoxic radiocontrast agents and gadolinium should be avoided or used according to strict guidelines when medically necessary as described above.

1	PREPARATION FOR RENAL REPLACEMENT THERAPY (See also Chap. 337) Temporary relief of symptoms and signs of impending uremia, such as anorexia, nausea, vomiting, lassitude, and pruritus, may sometimes be achieved with protein restriction. However, this carries a significant risk of malnutrition, and thus plans for more long-term management should be in place. Maintenance dialysis and kidney transplantation have extended the lives of hundreds of thousands of patients with CKD worldwide. Clear indications for initiation of renal replacement therapy for patients with CKD include uremic pericarditis, encephalopathy, intractable muscle cramping, anorexia, and nausea not attributable to reversible causes such as peptic ulcer disease, evidence of malnutrition, and fluid and electrolyte abnormalities, principally hyperkalemia or ECFV overload, that are refractory to other measures. Recommendations for the Optimal Time for Initiation of Renal Replacement 1821

1	Recommendations for the Optimal Time for Initiation of Renal Replacement 1821 Therapy Because of the individual variability in the severity of uremic symptoms and renal function, it is ill-advised to assign an arbitrary urea nitrogen or creatinine level to the need to start dialysis. Moreover, patients may become accustomed to chronic uremia and deny symptoms, only to find that they feel better with dialysis and realize in retrospect how poorly they were feeling before its initiation.

1	Previous studies suggested that starting dialysis before the onset of severe symptoms and signs of uremia was associated with prolongation of survival. This led to the concept of “healthy” start and is congruent with the philosophy that it is better to keep patients feeling well all along rather than allowing them to become ill with uremia before trying to return them to better health with dialysis or transplantation. Although recent studies have not confirmed an association of early-start dialysis with improved patient survival, there may be merit in this approach for some patients. On a practical level, advanced preparation may help to avoid problems with the dialysis process itself (e.g., a poorly functioning fistula for hemodialysis or malfunctioning peritoneal dialysis catheter) and, thus, preempt the morbidity associated with resorting to the insertion of temporary hemodialysis access with its attendant risks of sepsis, bleeding, thrombosis, and association with accelerated

1	and, thus, preempt the morbidity associated with resorting to the insertion of temporary hemodialysis access with its attendant risks of sepsis, bleeding, thrombosis, and association with accelerated mortality.

1	Patient Education Social, psychological, and physical preparation for the transition to renal replacement therapy and the choice of the optimal initial modality are best accomplished with a gradual approach involving a multidisciplinary team. Along with conservative measures discussed in the sections above, it is important to prepare patients with an intensive educational program, explaining the likelihood and timing of initiation of renal replacement therapy and the various forms of therapy available, and the option of nondialytic maximum conservative care. The more knowledgeable that patients are about hemodialysis (both in-center and home-based), peritoneal dialysis, and kidney transplantation, the easier and more appropriate will be their decisions. Patients who are provided with educational programs are more likely to choose home-based dialysis therapy. This approach is of societal benefit because home-based therapy is less expensive and is associated with improved quality of

1	programs are more likely to choose home-based dialysis therapy. This approach is of societal benefit because home-based therapy is less expensive and is associated with improved quality of life. The educational programs should be commenced no later than stage 4 CKD so that the patient has sufficient time and cognitive function to learn the important concepts, make informed choices, and implement preparatory measures for renal replacement therapy.

1	Exploration of social support is also important. In those who may perform home dialysis or undergo preemptive renal transplantation, early education of family members for selection and preparation of a home dialysis helper or a biologically or emotionally related potential living kidney donor should occur long before the onset of symptomatic renal failure. Kidney transplantation (Chap. 337) offers the best potential for complete rehabilitation, because dialysis replaces only a small fraction of the kidneys’ filtration function and none of the other renal functions, including endocrine and anti-inflammatory effects. Generally, kidney transplantation follows a period of dialysis treatment, although preemptive kidney transplantation (usually from a living donor) can be carried out if it is certain that the renal failure is irreversible.

1	In contrast to the natural decline and successful eradication of many devastating infectious diseases, there is rapid growth in the prevalence of metabolic and vascular disease in developing countries. Diabetes mellitus is becoming increasingly prevalent in these countries, perhaps due in part to change in dietary habits, diminished physical activity, and weight gain. Therefore, it follows that there will be a proportionate increase in vascular and renal disease. Health care agencies must plan for improved screening for early detection, prevention, and treatment plans in these nations and must start considering options for improved availability of renal replacement therapies.

1	1822 part 13 Dialysis in the treatment of renal Failure Kathleen D. Liu, Glenn M. Chertow Dialysis may be required for the treatment of either acute or chronic kidney disease. The use of continuous renal replacement therapies 336 (CRRTs) and slow low-efficiency dialysis (SLED) is specific to the management of acute renal failure and is discussed in Chap. 334. These modalities are performed continuously (CRRT) or over 6–12 h per session (SLED), in contrast to the 3–4 h of an intermittent hemodialysis session. Advantages and disadvantages of CRRT and SLED are discussed in Chap. 334. Peritoneal dialysis is rarely used in developed countries for the treatment of acute renal failure because of the increased risk of infection and (as will be discussed in more detail below) less efficient clearance per unit of time. The focus of this chapter will be on the use of peritoneal and hemodialysis for end-stage renal disease (ESRD).

1	With the widespread availability of dialysis, the lives of hundreds of thousands of patients with ESRD have been prolonged. In the United States alone, there are now approximately 615,000 patients with ESRD, the vast majority of whom require dialysis. The incidence rate for ESRD is 357 cases per million population per year. The incidence of ESRD is disproportionately higher in African Americans (940 per million population per year) as compared with white Americans (280 per million population per year). In the United States, the leading cause of ESRD is diabetes mellitus, currently accounting for nearly 45% of newly diagnosed cases of ESRD. Approximately 30% of patients have ESRD that has been attributed to hypertension, although it is unclear whether in these cases hypertension is the cause or a consequence of vascular disease or other unknown causes of kidney failure. Other prevalent causes of ESRD include glomerulonephritis, polycystic kidney disease, and obstructive uropathy.

1	Globally, mortality rates for patients with ESRD are lowest in Europe and Japan but very high in the developing world because of the limited availability of dialysis. In the United States, the mortality rate of patients on dialysis has decreased slightly but remains extremely high, with a 5-year survival rate of approximately 35–40%. Deaths are due mainly to cardiovascular diseases and infections (approximately 40 and 10% of deaths, respectively). Older age, male sex, nonblack race, diabetes mellitus, malnutrition, and underlying heart disease are important predictors of death.

1	Commonly accepted criteria for initiating patients on maintenance dialysis include the presence of uremic symptoms, the presence of hyperkalemia unresponsive to conservative measures, persistent extracellular volume expansion despite diuretic therapy, acidosis refractory to medical therapy, a bleeding diathesis, and a creatinine clearance or estimated glomerular filtration rate (GFR) below 10 mL/min per 1.73 m2 (see Chap. 335 for estimating equations). Timely referral to a nephrologist for advanced planning and creation of a dialysis access, education about ESRD treatment options, and management of the complications of advanced chronic kidney disease (CKD), including hypertension, anemia, acidosis, and secondary hyperparathyroidism, are advisable. Recent data have suggested that a sizable fraction of ESRD cases result following episodes of acute renal failure, particularly among persons with underlying CKD. Furthermore, there is no benefit to initiating dialysis preemptively at a GFR

1	fraction of ESRD cases result following episodes of acute renal failure, particularly among persons with underlying CKD. Furthermore, there is no benefit to initiating dialysis preemptively at a GFR of 10–14 mL/min per 1.73 m2 compared to initiating dialysis for symptoms of uremia.

1	In ESRD, treatment options include hemodialysis (in center or at home); peritoneal dialysis, as either continuous ambulatory peritoneal dialysis (CAPD) or continuous cyclic peritoneal dialysis (CCPD); or transplantation (Chap. 337). Although there are significant geographic variations and differences in practice patterns, hemodialysis remains the most common therapeutic modality for ESRD (>90% of patients) in the United States. In contrast to hemodialysis, peritoneal dialysis is continuous, but much less efficient, in terms of solute clearance. Although no large-scale clinical trials have been completed comparing outcomes among patients randomized to either hemodialysis or peritoneal dialysis, outcomes associated with both therapies are similar in most reports, and the decision of which modality to select is often based on personal preferences and quality-of-life considerations.

1	Hemodialysis relies on the principles of solute diffusion across a semipermeable membrane. Movement of metabolic waste products takes place down a concentration gradient from the circulation into the dialysate. The rate of diffusive transport increases in response to several factors, including the magnitude of the concentration gradient, the membrane surface area, and the mass transfer coefficient of the membrane. The latter is a function of the porosity and thickness of the membrane, the size of the solute molecule, and the conditions of flow on the two sides of the membrane. According to laws of diffusion, the larger the molecule, the slower is its rate of transfer across the membrane. A small molecule, such as urea (60 Da), undergoes substantial clearance, whereas a larger molecule, such as creatinine (113 Da), is cleared less efficiently. In addition to diffusive clearance, movement of waste products from the circulation into the dialysate may occur as a result of ultrafiltration.

1	as creatinine (113 Da), is cleared less efficiently. In addition to diffusive clearance, movement of waste products from the circulation into the dialysate may occur as a result of ultrafiltration. Convective clearance occurs because of solvent drag, with solutes being swept along with water across the semipermeable dialysis membrane.

1	There are three essential components to hemodialysis: the dialyzer, the composition and delivery of the dialysate, and the blood delivery system (Fig. 336-1). The dialyzer is a plastic chamber with the ability to perfuse blood and dialysate compartments simultaneously at very high flow rates. The hollow-fiber dialyzer is the most common in use in the United States. These dialyzers are composed of bundles of capillary tubes through which blood circulates while dialysate travels on the outside of the fiber bundle. The majority of dialyzers now manufactured in the United States are “biocompatible” synthetic membranes derived from polysulfone or related compounds (versus older cellulose “bioincompatible” membranes that activated the complement cascade). The frequency of reprocessing and reuse of hemodialyzers and blood lines varies across the world. In general, as the cost of disposable supplies has decreased, their use has increased. Formaldehyde, peracetic acid–hydrogen peroxide,

1	reuse of hemodialyzers and blood lines varies across the world. In general, as the cost of disposable supplies has decreased, their use has increased. Formaldehyde, peracetic acid–hydrogen peroxide, glutaraldehyde, and bleach have all been used as reprocessing agents.

1	The potassium concentration of dialysate may be varied from 0 to 4 mmol/L depending on the predialysis serum potassium concentration. The usual dialysate calcium concentration is 1.25 mmol/L (2.5 meq/L), although modification may be required in selected settings (e.g., higher dialysate calcium concentrations may be used in patients with hypocalcemia associated with secondary hyperparathyroidism or following parathyroidectomy). The usual dialysate sodium concentration is 136– 140 mmol/L. In patients who frequently develop hypotension during their dialysis run, “sodium modeling” to counterbalance urea-related osmolar gradients is often used. With sodium modeling, the dialysate sodium concentration is gradually lowered from the range of 145–155 mmol/L to isotonic concentrations (136–140 mmol/L) near the end of the dialysis treatment, typically declining either in steps or in a linear or exponential fashion. Higher dialysate sodium concentrations and sodium modeling may predispose

1	mmol/L) near the end of the dialysis treatment, typically declining either in steps or in a linear or exponential fashion. Higher dialysate sodium concentrations and sodium modeling may predispose patients to positive sodium balance and increased thirst; thus, these strategies to ameliorate intradialytic hypotension may be undesirable in hypertensive patients or in patients with large interdialytic weight gains. Because patients are exposed to approximately 120 L of water during each dialysis treatment, water used for the dialysate is subjected to filtration, softening, deionization, and, ultimately, reverse osmosis to remove microbiologic contaminants and dissolved ions.

1	Dialysis in the Treatment of Renal FailureChapter 336Venous Arterial Dialysate Dialysate "Delivery" system Dialysate drain Hollow fiber dialyzer Arterial line Venous line V Arteriovenous fistula Na+ Cl– K+ Acetate– Ca2+ Mg2+ Water treatment (deionization and reverse osmosis) AcidconcentrateNaBicarb NaCl Arterial pressure Venous pressure Blood flow rate Air (leak) detection Dialysate flow rate Dialysate pressure Dialysate conductivity Blood (leak) detection A FIGuRE 336-1 Schema for hemodialysis. A, artery; V, vein.

1	The blood delivery system is composed of the extracorporeal circuit and the dialysis access. The dialysis machine consists of a blood pump, dialysis solution delivery system, and various safety monitors. The blood pump moves blood from the access site, through the dialyzer, and back to the patient. The blood flow rate may range from 250–500 mL/min, depending on the type and integrity of the vascular access. Negative hydrostatic pressure on the dialysate side can be manipulated to achieve desirable fluid removal or ultrafiltration. Dialysis membranes have different ultrafiltration coefficients (i.e., mL removed/min per mmHg) so that along with hydrostatic changes, fluid removal can be varied. The dialysis solution delivery system dilutes the concentrated dialysate with water and monitors the temperature, conductivity, and flow of dialysate.

1	The fistula, graft, or catheter through which blood is obtained for hemodialysis is often referred to as a dialysis access. A native fistula created by the anastomosis of an artery to a vein (e.g., the Brescia-Cimino fistula, in which the cephalic vein is anastomosed end-to-side to the radial artery) results in arterialization of the vein. This facilitates its subsequent use in the placement of large needles (typically 15 gauge) to access the circulation. Although fistulas have the highest long-term patency rate of all dialysis access options, fistulas are created in a minority of patients in the United States. Many patients undergo placement of an arteriovenous graft (i.e., the interposition of prosthetic material, usually polytetrafluoroethylene, between an artery and a vein) or a tunneled dialysis catheter. In recent years, nephrologists, vascular surgeons, and health care policy makers in the United States have encouraged creation of arteriovenous fistulas in a larger fraction of

1	dialysis catheter. In recent years, nephrologists, vascular surgeons, and health care policy makers in the United States have encouraged creation of arteriovenous fistulas in a larger fraction of patients (the “fistula first” initiative). Unfortunately, even when created, arteriovenous fistulas may not mature sufficiently to provide reliable access to the circulation, or they may thrombose early in their development.

1	Grafts and catheters tend to be used among persons with smaller-caliber veins or persons whose veins have been damaged by repeated venipuncture, or after prolonged hospitalization. The most important complication of arteriovenous grafts is thrombosis of the graft and graft failure, due principally to intimal hyperplasia at the anastomosis between the graft and recipient vein. When grafts (or fistulas) fail, catheter-guided angioplasty can be used to dilate stenoses; monitoring of venous pressures on dialysis and of access flow, although not routinely performed, may assist in the early recognition of impending vascular access failure. In addition to an increased rate of access failure, grafts and (in particular) catheters are associated with much higher rates of infection than fistulas.

1	Intravenous large-bore catheters are often used in patients with acute and chronic kidney disease. For persons on maintenance hemodialysis, tunneled catheters (either two separate catheters or a single catheter with two lumens) are often used when arteriovenous fistulas and grafts have failed or are not feasible due to anatomic considerations. These catheters are tunneled under the skin; the tunnel reduces bacterial translocation from the skin, resulting in a lower infection rate than with nontunneled temporary catheters. Most tunneled catheters are placed in the internal jugular veins; the external jugular, femoral, and subclavian veins may also be used.

1	Nephrologists, interventional radiologists, and vascular surgeons generally prefer to avoid placement of catheters into the subclavian veins; while flow rates are usually excellent, subclavian stenosis is a frequent complication and, if present, will likely prohibit permanent vascular access (i.e., a fistula or graft) in the ipsilateral extremity. Infection rates may be higher with femoral catheters. For patients with multiple vascular access complications and no other options for permanent vascular access, tunneled catheters may be the last “lifeline” for hemodialysis. Translumbar or transhepatic approaches into the inferior vena cava may be required if the superior vena cava or other central veins draining the upper extremities are stenosed or thrombosed.

1	The hemodialysis procedure consists of pumping heparinized blood through the dialyzer at a flow rate of 300–500 mL/min, while dialysate flows in an opposite counter-current direction at 500–800 mL/ min. The efficiency of dialysis is determined by blood and dialysate flow through the dialyzer as well as dialyzer characteristics (i.e., its efficiency in removing solute). The dose of dialysis, which is currently defined as a derivation of the fractional urea clearance during a single 1824 treatment, is further governed by patient size, residual kidney function, dietary protein intake, the degree of anabolism or catabolism, and the presence of comorbid conditions. Since the landmark studies of Sargent and Gotch relating the measurement of the dose of dialysis using urea concentrations with morbidity in the National Cooperative Dialysis Study, the delivered dose of dialysis has been measured and considered as a quality assurance and improvement tool. Although the fractional removal of urea

1	in the National Cooperative Dialysis Study, the delivered dose of dialysis has been measured and considered as a quality assurance and improvement tool. Although the fractional removal of urea nitrogen and derivations thereof are considered to be the standard methods by which “adequacy of dialysis” is measured, a large multicenter randomized clinical trial (the HEMO Study) failed to show a difference in mortality associated with a large difference in urea clearance. Current targets include a urea reduction ratio (the fractional reduction in blood urea nitrogen per hemodialysis session) of >65–70% and a body water– indexed clearance × time product (KT/V) above 1.2 or 1.05, depending on whether urea concentrations are “equilibrated.” For the majority of patients with ESRD, between 9 and 12 h of dialysis are required each week, usually divided into three equal sessions. Several studies have suggested that longer hemodialysis session lengths may be beneficial (independent of urea

1	and 12 h of dialysis are required each week, usually divided into three equal sessions. Several studies have suggested that longer hemodialysis session lengths may be beneficial (independent of urea clearance), although these studies are confounded by a variety of patient characteristics, including body size and nutritional status. Hemodialysis “dose” should be individualized, and factors other than the urea nitrogen should be considered, including the adequacy of ultrafiltration or fluid removal and control of hyperkalemia, hyperphosphatemia, and metabolic acidosis. A recent randomized clinical trial (the Frequent Hemodialysis Network Trial) demonstrated improved control of hypertension and hyperphosphatemia, reduced left ventricular mass, and improved self-reported physical health with six times per week hemodialysis compared to the usual three times per week therapy. A companion trial in which frequent nocturnal hemodialysis was compared to conventional hemodialysis at home showed

1	six times per week hemodialysis compared to the usual three times per week therapy. A companion trial in which frequent nocturnal hemodialysis was compared to conventional hemodialysis at home showed no significant effect on left ventricular mass or self-reported physical health. Finally, an evaluation of the U.S. Renal Data System registry showed a significant increase in mortality and hospitalization for heart failure after the longer interdialytic interval that occurs over the dialysis “weekend.”

1	Hypotension is the most common acute complication of hemodialysis, particularly among patients with diabetes mellitus. Numerous factors appear to increase the risk of hypotension, including excessive ultrafiltration with inadequate compensatory vascular filling, impaired vasoactive or autonomic responses, osmolar shifts, overzealous use of antihypertensive agents, and reduced cardiac reserve. Patients with arteriovenous fistulas and grafts may develop high-output cardiac failure due to shunting of blood through the dialysis access; on rare occasions, this may necessitate ligation of the fistula or graft. Because of the vasodilatory and cardiodepressive effects of acetate, its use as the buffer in dialysate was once a common cause of hypotension. Since the introduction of bicarbonate-containing dialysate, dialysis-associated hypotension has become less common. The management of hypotension during dialysis consists of discontinuing ultrafiltration, the administration of 100–250 mL of

1	dialysate, dialysis-associated hypotension has become less common. The management of hypotension during dialysis consists of discontinuing ultrafiltration, the administration of 100–250 mL of isotonic saline or 10 mL of 23% saturated hypertonic saline, or administration of salt-poor albumin. Hypotension during dialysis can frequently be prevented by careful evaluation of the dry weight and by ultrafiltration modeling, such that more fluid is removed at the beginning rather than the end of the dialysis procedure. Additional maneuvers include the performance of sequential ultrafiltration followed by dialysis, cooling of the dialysate during dialysis treatment, and avoiding heavy meals during dialysis. Midodrine, an oral selective α1 adrenergic agent, has been advocated by some practitioners, although there is insufficient evidence of its safety and efficacy to support its routine use.

1	Muscle cramps during dialysis are also a common complication. The etiology of dialysis-associated cramps remains obscure. Changes in muscle perfusion because of excessively rapid volume removal (e.g., >10–12 mL/kg per hour) or targeted removal below the patient’s estimated dry weight often precipitate dialysis-associated cramps. Strategies that may be used to prevent cramps include reducing volume removal during dialysis, ultrafiltration profiling, and the use of sodium modeling (see above).

1	Anaphylactoid reactions to the dialyzer, particularly on its first use, have been reported most frequently with the bioincompatible cellulosic-containing membranes. Dialyzer reactions can be divided into two types, A and B. Type A reactions are attributed to an IgEmediated intermediate hypersensitivity reaction to ethylene oxide used in the sterilization of new dialyzers. This reaction typically occurs soon after the initiation of a treatment (within the first few minutes) and can progress to full-blown anaphylaxis if the therapy is not promptly discontinued. Treatment with steroids or epinephrine may be needed if symptoms are severe. The type B reaction consists of a symptom complex of nonspecific chest and back pain, which appears to result from complement activation and cytokine release. These symptoms typically occur several minutes into the dialysis run and typically resolve over time with continued dialysis.

1	In peritoneal dialysis, 1.5–3 L of a dextrose-containing solution is infused into the peritoneal cavity and allowed to dwell for a set period of time, usually 2–4 h. As with hemodialysis, toxic materials are removed through a combination of convective clearance generated through ultrafiltration and diffusive clearance down a concentration gradient. The clearance of solutes and water during a peritoneal dialysis exchange depends on the balance between the movement of solute and water into the peritoneal cavity versus absorption from the peritoneal cavity. The rate of diffusion diminishes with time and eventually stops when equilibration between plasma and dialysate is reached. Absorption of solutes and water from the peritoneal cavity occurs across the peritoneal membrane into the peritoneal capillary circulation and via peritoneal lymphatics into the lymphatic circulation. The rate of peritoneal solute transport varies from patient to patient and may be altered by the presence of

1	capillary circulation and via peritoneal lymphatics into the lymphatic circulation. The rate of peritoneal solute transport varies from patient to patient and may be altered by the presence of infection (peritonitis), drugs, and physical factors such as position and exercise.

1	Peritoneal dialysis may be carried out as CAPD, CCPD, or a combination of both. In CAPD, dialysate is manually infused into the peritoneal cavity and exchanged three to five times during the day. A nighttime dwell is frequently instilled at bedtime and remains in the peritoneal cavity through the night. In CCPD, exchanges are performed in an automated fashion, usually at night; the patient is connected to an automated cycler that performs a series of exchange cycles while the patient sleeps. The number of exchange cycles required to optimize peritoneal solute clearance varies by the peritoneal membrane characteristics; as with hemodialysis, solute clearance should be tracked to ensure dialysis “adequacy.”

1	Peritoneal dialysis solutions are available in volumes typically ranging from 1.5 to 3 L. The major difference between the dialysate used for peritoneal dialysis rather than hemodialysis is that the hypertonicity of peritoneal dialysis solutions drives solute and fluid removal, whereas solute removal in hemodialysis depends on concentration gradients, and fluid removal requires transmembrane pressure. Typically, dextrose at varying concentrations contributes to the hypertonicity of peritoneal dialysate. Icodextrin is a nonabsorbable carbohydrate that can be used in place of dextrose. Studies have demonstrated more efficient ultrafiltration with icodextrin than with dextrose-containing solutions. Icodextrin is typically used as the “last fill” for patients on CCPD or for the longest dwell in patients on CAPD. The most common additives to peritoneal dialysis solutions are heparin to prevent obstruction of the dialysis catheter lumen with fibrin and antibiotics during an episode of acute

1	patients on CAPD. The most common additives to peritoneal dialysis solutions are heparin to prevent obstruction of the dialysis catheter lumen with fibrin and antibiotics during an episode of acute peritonitis. Insulin may also be added in patients with diabetes mellitus.

1	Access to the peritoneal cavity is obtained through a peritoneal catheter. Catheters used for maintenance peritoneal dialysis are flexible, being made of silicone rubber with numerous side holes at the distal end. These catheters usually have two Dacron cuffs. The scarring that occurs around the cuffs anchors the catheter and seals it from bacteria tracking from the skin surface into the peritoneal cavity; it also prevents the external leakage of fluid from the peritoneal cavity. The cuffs are placed in the preperitoneal plane and ~2 cm from the skin surface.

1	The peritoneal equilibrium test is a formal evaluation of peritoneal membrane characteristics that measures the transfer rates of creatinine and glucose across the peritoneal membrane. Patients are classified as low, low–average, high–average, and high transporters. Patients with rapid equilibration (i.e., high transporters) tend to absorb more glucose and lose efficiency of ultrafiltration with long daytime dwells. High transporters also tend to lose larger quantities of albumin and other proteins across the peritoneal membrane. In general, patients with rapid transporting characteristics require more frequent, shorter dwell time exchanges, nearly always obligating use of a cycler. Slower (low and low–average) transporters tend to do well with fewer exchanges. The efficiency of solute clearance also depends on the volume of dialysate infused. Larger volumes allow for greater solute clearance, particularly with CAPD in patients with low and low– average transport characteristics.

1	As with hemodialysis, the optimal dose of peritoneal dialysis is unknown. Several observational studies have suggested that higher rates of urea and creatinine clearance (the latter generally measured in liters per week) are associated with lower mortality rates and fewer uremic complications. However, a randomized clinical trial (Adequacy of Peritoneal Dialysis in Mexico [ADEMEX]) failed to show a significant reduction in mortality or complications with a relatively large increment in urea clearance. In general, patients on peritoneal dialysis do well when they retain residual kidney function. The rates of technique failure increase with years on dialysis and have been correlated with loss of residual function to a greater extent than loss of peritoneal membrane capacity. For some patients in whom CCPD does not provide sufficient solute clearance, a hybrid approach can be adopted where one or more daytime exchanges are added to the CCPD regimen. Although this approach can enhance

1	in whom CCPD does not provide sufficient solute clearance, a hybrid approach can be adopted where one or more daytime exchanges are added to the CCPD regimen. Although this approach can enhance solute clearance and prolong a patient’s capacity to remain on peritoneal dialysis, the burden of the hybrid approach can be overwhelming.

1	The major complications of peritoneal dialysis are peritonitis, catheter-associated nonperitonitis infections, weight gain and other metabolic disturbances, and residual uremia (especially among patients with no residual kidney function).

1	Peritonitis typically develops when there has been a break in sterile technique during one or more of the exchange procedures. Peritonitis is usually defined by an elevated peritoneal fluid leukocyte count (100/μL, of which at least 50% are polymorphonuclear neutrophils); these cutoffs are lower than in spontaneous bacterial peritonitis because of the presence of dextrose in peritoneal dialysis solutions and rapid bacterial proliferation in this environment without antibiotic therapy. The clinical presentation typically consists of pain and cloudy dialysate, often with fever and other constitutional symptoms. The most common culprit organisms are gram-positive cocci, including Staphylococcus, reflecting the origin from the skin. Gram-negative rod infections are less common; fungal and mycobacterial infections can be seen in selected patients, particularly after antibacterial therapy. Most cases of peritonitis can be managed either with intraperitoneal or oral antibiotics, depending on

1	infections can be seen in selected patients, particularly after antibacterial therapy. Most cases of peritonitis can be managed either with intraperitoneal or oral antibiotics, depending on the organism; many patients with peritonitis do not require hospitalization. In cases where peritonitis is due to hydrophilic gram-negative rods (e.g., Pseudomonas sp.) or yeast, antimicrobial therapy is usually not sufficient, and catheter removal is required to ensure complete eradication of infection. Nonperitonitis catheter-associated infections (often termed tunnel infections) vary widely in severity. Some cases can be managed with local antibiotic or silver nitrate administration, whereas others are severe enough to require parenteral antibiotic therapy and catheter removal.

1	Peritoneal dialysis is associated with a variety of metabolic complications. Albumin and other proteins can be lost across the peritoneal membrane in concert with the loss of metabolic wastes. Hypoproteinemia obligates a higher dietary protein intake in order to maintain nitrogen balance. Hyperglycemia and weight gain are also common complications of peritoneal dialysis. Several hundred calories in the form of dextrose are absorbed each day, depending on the concentration 1825 employed. Peritoneal dialysis patients, particularly those with diabetes mellitus, are then prone to other complications of insulin resistance, including hypertriglyceridemia. On the positive side, the continuous nature of peritoneal dialysis usually allows for a more liberal diet, due to continuous removal of potassium and phosphorus—two major dietary components whose accumulation can be hazardous in ESRD.

1	Cardiovascular disease constitutes the major cause of death in patients with ESRD. Cardiovascular mortality and event rates are higher in dialysis patients than in patients after transplantation, although rates are extraordinarily high in both populations. The underlying cause of cardiovascular disease is unclear but may be related to shared risk factors (e.g., diabetes mellitus, hypertension, atherosclerotic and arteriosclerotic vascular disease), chronic inflammation, massive changes in extracellular volume (especially with high interdialytic weight gains), inadequate treatment of hypertension, dyslipidemia, anemia, dystrophic vascular calcification, hyperhomocysteinemia, and, perhaps, alterations in cardiovascular dynamics during the dialysis treatment. Few studies have targeted cardiovascular risk reduction in ESRD patients; none have demonstrated consistent benefit. Two clinical trials of statin agents in ESRD demonstrated significant reductions in low-density lipoprotein (LDL)

1	risk reduction in ESRD patients; none have demonstrated consistent benefit. Two clinical trials of statin agents in ESRD demonstrated significant reductions in low-density lipoprotein (LDL) cholesterol concentrations, but no significant reductions in death or cardiovascular events (Die Deutsche Diabetes Dialyse Studie [4D] and A Study to Evaluate the Use of Rosuvastatin in Subjects on Regular Hemodialysis [AURORA]). The Study of Heart and Renal Protection (SHARP), which included patients on dialysisand nondialysis-requiring CKD, showed a 17% reduction in the rate of major cardiovascular events or cardiovascular death with simvastatin-ezetimibe treatment. Most experts recommend conventional cardioprotective strategies (e.g., lipid-lowering agents, aspirin, inhibitors of the renin-angiotensin-aldosterone system, and β-adrenergic antagonists) in dialysis patients based on the patients’ cardiovascular risk profile, which appears to be increased by more than an order of magnitude relative

1	system, and β-adrenergic antagonists) in dialysis patients based on the patients’ cardiovascular risk profile, which appears to be increased by more than an order of magnitude relative to persons unaffected by kidney disease. Other complications of ESRD include a high incidence of infection, progressive debility and frailty, protein-energy malnutrition, and impaired cognitive function.

1	The incidence of ESRD is increasing worldwide with longer life expectancies and improved care of infectious and cardiovascular diseases. The management of ESRD varies widely by country and within country by region, and it is influenced by economic and other major factors. In general, peritoneal dialysis is more commonly performed in poorer countries owing to its lower expense and the high cost of establishing in-center hemodialysis units. transplantation in the treatment 337 of renal Failure Jamil Azzi, Edgar L. Milford, Mohamed H. Sayegh, Anil Chandraker

1	transplantation in the treatment 337 of renal Failure Jamil Azzi, Edgar L. Milford, Mohamed H. Sayegh, Anil Chandraker Transplantation of the human kidney is the treatment of choice for advanced chronic renal failure. Worldwide, tens of thousands of these procedures have been performed with more than 180,000 patients bearing functioning kidney transplants in the United States today. When azathioprine and prednisone initially were used as immunosuppressive drugs in the 1960s, the results with properly matched familial donors were superior to those with organs from deceased donors: 75–90% compared with 50–60% graft survival rates at 1 year. During the 1970s and 1980s, the success rate at the 1-year mark for deceased-donor Transplantation in the Treatment of Renal Failure

1	Transplantation in the Treatment of Renal Failure Deceased donor >60 years Deceased donor >50 years and hypertension and creatinine >1.5 mg/dL Deceased donor >50 years and hypertension and death caused by cerebro vascular accident (CVA) Deceased donor >50 years and death caused by CVA and creatinine >1.5 mg/dL I: Brought in dead II: Unsuccessful resuscitation III: Awaiting cardiac arrest IV: Cardiac arrest after brainstem death V: Cardiac arrest in a hospital patient aKidneys can be used for transplantation from categories II–V but are commonly only used from categories III and IV. The survival of these kidneys has not been shown to be inferior to that of deceased-donor kidneys. Note: Kidneys can be both ECD and DCD. ECD kidneys have been shown to have a poorer survival, and there is a separate shorter waiting list for ECD kidneys. They are generally used for patients for whom the benefits of being transplanted earlier outweigh the associated risks of using an ECD kidney.

1	transplants rose progressively. Currently, deceased-donor grafts have a 92% 1-year survival and living-donor grafts have a 96% 1-year survival. Although there has been improvement in long-term survival, it has not been as impressive as the short-term survival, and currently the “average” (t1/2) life expectancy of a living-donor graft is around 20 years and that of a deceased-donor graft is close to 14 years.

1	Mortality rates after transplantation are highest in the first year and are age-related: 2% for ages 18–34 years, 3% for ages 35–49 years, and 6.8% for ages ≥50–60 years. These rates compare favorably with those in the chronic dialysis population even after risk adjustments for age, diabetes, and cardiovascular status. Although the loss of kidney transplant due to acute rejection is currently rare, most allografts succumb at varying rates to a chronic process consisting of interstitial fibrosis, tubular atrophy, vasculopathy, and glomerulopathy, the pathogenesis of which is incompletely understood. Overall, transplantation returns most patients to an improved lifestyle and an improved life expectancy compared with patients on dialysis.

1	In 2011, there were more than 11,835 deceased-donor kidney transplants and 5772 living-donor transplants in the United States, with the ratio of deceased to living donors remaining stable over the last few years. The backlog of patients with end-stage renal disease (ESRD) has been increasing every year, and it always lags behind the number of available donors. As the number of patients with end-stage kidney disease increases, the demand for kidney transplants continues to increase. In 2011, there were 55,371 active adult candidates on the waiting list, and less than 18,000 patients were transplanted. This imbalance is set to worsen over the coming years with the predicted increased rates of obesity and diabetes worldwide. In an attempt to increase utilization of deceased-donor kidneys and reduce discard rates of organs, criteria for the use of so-called expanded criteria donor (ECD) kidneys and kidneys from donors after cardiac death (DCD) have been developed (Table 337-1). ECD

1	and reduce discard rates of organs, criteria for the use of so-called expanded criteria donor (ECD) kidneys and kidneys from donors after cardiac death (DCD) have been developed (Table 337-1). ECD kidneys are usually used for older patients who are expected to fare less well on dialysis.

1	The overall results of transplantation are presented in Table 337-2 as the survival of grafts and of patients. At the 1-year mark, graft survival is higher for living-donor recipients, most likely because those grafts are not subject to as much ischemic injury. The more effective drugs now in use for immunosuppression have almost equalized the risk of graft rejection in all patients for the first year. At 5 and 10 years, however, there has been a steeper decline in survival of those with deceased-donor kidneys. There are few absolute contraindications to renal transplantation. The transplant procedure is relatively noninvasive, as the organ is placed in the inguinal fossa without entering the peritoneal cavity. Recipients without perioperative complications often can be discharged from the hospital in excellent condition within 5 days of the operation.

1	Virtually all patients with ESRD who receive a transplant have a higher life expectancy than do risk-matched patients who remain on dialysis. Even though diabetic patients and older candidates have a higher mortality rate than other transplant recipients, their survival is improved with transplantation compared with those remaining on dialysis. This global benefit of transplantation as a treatment modality poses substantial ethical issues for policy makers, as the number of deceased kidneys available is far from sufficient to meet the current needs of the candidates. The current standard of care is that the candidate should have a life expectancy of >5 years to be put on a deceased organ wait list. Even for living donation, the candidate should have >5 years of life expectancy. This standard has been established because the benefits of kidney transplantation over dialysis are realized only after a perioperative period in which the mortality rate is higher in transplanted patients than

1	has been established because the benefits of kidney transplantation over dialysis are realized only after a perioperative period in which the mortality rate is higher in transplanted patients than in dialysis patients with comparable risk profiles.

1	All candidates must have a thorough risk-versus-benefit evaluation before being approved for transplantation. In particular, an aggressive approach to diagnosis of correctable coronary artery disease, presence of latent or indolent infection (HIV, hepatitis B or C, tuberculosis), and neoplasm should be a routine part of the candidate workup. Most transplant centers consider overt AIDS and active hepatitis absolute contraindications to transplantation because of the high risk of opportunistic infection. Some centers are now transplanting individuals with hepatitis and even HIV infection under strict protocols to determine whether the risks and benefits favor transplantation over dialysis.

1	Among the few absolute “immunologic” contraindications to transplantation is the presence of antibodies against the donor kidney at the time of the anticipated transplant that can cause hyperacute rejection. Those harmful antibodies include natural antibodies against the ABO blood group antigens and antibodies against human leukocyte antigen (HLA) class I (A, B, C) or class II (DR) antigens. These antibodies are routinely excluded by proper screening of the candidate’s ABO compatibility and direct cytotoxic cross-matching of candidate serum with lymphocytes of the donor. Matching for antigens of the HLA major histocompatibility gene complex (Chap. 373e) is an important criterion for selection of donors for renal allografts. Each mammalian species has a single chromosomal Mean rates OF graFt anD patIent surVIVaL FOr KIDneys transpLanteD In the unIteD states FrOM 1998 tO 2008a

1	Mean rates OF graFt anD patIent surVIVaL FOr KIDneys transpLanteD In the unIteD states FrOM 1998 tO 2008a Grafts, % Patients, % Grafts, % Patients, % Grafts, % Patients, % aAll patients transplanted are included, and the follow-up unadjusted survival data from the 1-, 5-, and 10-year periods are presented to show the attrition rates over time within the two types of organ donors. Source: Data from Summary Tables, 2009 Annual Reports, Scientific Registry of Transplant Recipients.

1	region that encodes the strong, or major, transplantation antigens, and this region on the human sixth chromosome is called HLA. HLA antigens have been classically defined by serologic techniques, but methods to define specific nucleotide sequences in genomic DNA are increasingly being used. Other “minor” antigens may play crucial roles, in addition to the ABH(O) blood groups and endothelial antigens that are not shared with lymphocytes. The Rh system is not expressed on graft tissue. Evidence for designation of HLA as the genetic region that encodes major transplantation antigens comes from the success rate in living related donor renal and bone marrow transplantation, with superior results in HLA-identical sibling pairs. Nevertheless, 5% of HLA-identical renal allografts are rejected, often within the first weeks after transplantation. These failures represent states of prior sensitization to non-HLA antigens. Non-HLA minor antigens are relatively weak when initially encountered and

1	within the first weeks after transplantation. These failures represent states of prior sensitization to non-HLA antigens. Non-HLA minor antigens are relatively weak when initially encountered and are, therefore, suppressible by conventional immunosuppressive therapy. Once priming has occurred, however, secondary responses are much more refractory to treatment.

1	Donors can be deceased or volunteer living donors. When first-degree relatives are donors, graft survival rates at 1 year are 5–7% greater than those for deceased-donor grafts. The 5-year survival rates still favor a partially matched (3/6 HLA mismatched) family donor over a randomly selected cadaver donor. In addition, living donors provide the advantage of immediate availability. For both living and deceased donors, the 5-year outcomes are poor if there is a complete (6/6) HLA mismatch. The survival rate of living unrelated renal allografts is as high as that of perfectly HLA-matched cadaver renal transplants and comparable to that of kidneys from living relatives. This outcome is probably a consequence of both short cold ischemia time and the extra care taken to document that the condition and renal function of the donor are optimal before proceeding with a living unrelated donation. It is illegal in the United States to purchase organs for transplantation.

1	Living volunteer donors should be cleared of any medical conditions that may cause morbidity and mortality after kidney transplantation. Concern has been expressed about the potential risk to a volunteer kidney donor of premature renal failure after several years of increased blood flow and hyperfiltration per nephron in the remaining kidney. There are a few reports of the development of hypertension, proteinuria, and even lesions of focal segmental sclerosis in donors over long-term follow-up. It is also desirable to consider the risk of development of type 1 diabetes mellitus in a family member who is a potential donor to a diabetic renal failure patient. Anti-insulin and anti-islet cell antibodies should be measured and glucose tolerance tests should be performed in such donors to exclude a prediabetic state. Selective renal arteriography should be performed on donors to rule out the presence of multiple or abnormal renal arteries, because the surgical procedure is difficult and

1	a prediabetic state. Selective renal arteriography should be performed on donors to rule out the presence of multiple or abnormal renal arteries, because the surgical procedure is difficult and the ischemic time of the transplanted kidney is long when there are vascular abnormalities. Transplant surgeons are now using a laparoscopic method to isolate and remove the living donor’s kidney. This operation has the advantage of less evident surgical scars, and, because there is less tissue trauma, the laparoscopic donors have a substantially shorter hospital stay and less discomfort than those who have the traditional surgery.

1	Deceased donors should be free of malignant neoplastic disease, hepatitis, and HIV due to possible transmission to the recipient, although there is increasing interest in using hepatitis C– and HIV-positive organs in previously infected recipients. Increased risk of graft failure exists when the donor is elderly or has renal failure and when the kidney has a prolonged period of ischemia and storage. In the United States, there is a coordinated national system of regulations, allocation support, and outcomes analysis for kidney transplantation called the Organ Procurement Transplant Network. It is now possible to remove deceased-donor kidneys and maintain them for up to 48 h on cold pulsatile perfusion or with simple flushing and cooling. This approach permits adequate time for typing, cross-1827 matching, transportation, and selection problems to be solved.

1	A positive cytotoxic cross-match of recipient serum with donor T lymphocytes indicates the presence of preformed donor-specific anti-HLA class I antibodies and is usually predictive of an acute vasculitic event termed hyperacute rejection. This finding, along with ABO incompatibility, represents the only absolute immunologic contraindication for kidney transplantation. Recently, more tissue typing laboratories have shifted to a flow cytometric–based cross-match assay, which detects the presence of anti-HLA antibodies that are not necessarily detected on a cytotoxic cross-match assay and may not be an absolute contraindication to transplantation. The known sources of such sensitization are blood transfusion, a prior transplant, pregnancy, and vaccination/ infection. Patients sustained by dialysis often show fluctuating antibody titers and specificity patterns. At the time of assignment of a cadaveric kidney, cross-matches are performed with at least a current serum. Previously analyzed

1	often show fluctuating antibody titers and specificity patterns. At the time of assignment of a cadaveric kidney, cross-matches are performed with at least a current serum. Previously analyzed antibody specificities and additional cross-matches are performed accordingly. Flow cytometry detects binding of anti-HLA antibodies of a candidate’s serum by a recipient’s lymphocytes. This highly sensitive test can be useful for avoidance of accelerated, and often untreatable, early graft rejection in patients receiving second or third transplants.

1	For the purposes of cross-matching, donor T lymphocytes, which express class I but not class II antigens, are used as targets for detection of anti–class I (HLA-A and -B) antibodies that are expressed on all nucleated cells. Preformed anti–class II (HLA-DR and -DQ) antibodies against the donor also carry a higher risk of graft loss, particularly in recipients who have suffered early loss of a prior kidney transplant. B lymphocytes, which express both class I and class II antigens, are used as targets in these assays. Some non-HLA antigens restricted in expression to endothelium and monocytes have been described, but clinical relevance is not well established. A series of minor histocompatibility antigens do not elicit antibodies, and sensitization to these antigens is detectable only by cytotoxic T cells, an assay too cumbersome for routine use.

1	Desensitization before transplantation by reducing the level of anti-donor antibodies using plasmapheresis and administration of pooled immunoglobulin, or both, has been useful in reducing the risk of hyperacute rejection following transplantation. Both cellular and humoral (antibody-mediated) effector mechanisms can play roles in kidney transplant rejection.

1	Cellular rejection is mediated by lymphocytes that respond to HLA antigens expressed within the organ. The CD4+ lymphocyte responds to class II (HLA-DR) incompatibility by proliferating and releasing pro-inflammatory cytokines that augment the proliferative response of the immune system. CD8+ cytotoxic lymphocyte precursors respond primarily to class I (HLA-A, -B) antigens and mature into cytotoxic effector cells that cause organ damage through direct contact and lysis of donor target cells. Full T cell activation requires not only T cell receptor binding to the alloantigens presented by self or donor HLA molecules (indirect and direct presentation, respectively), but also engaging costimulatory molecules such as CD28 on T cells and CD80 and CD86 ligands on antigen-presenting cells (Fig. 337-1). Signaling through both of these pathways induces activation of the kinase activity of calcineurin, which, in turn, activates transcription factors, leading to upregulation of multiple genes,

1	337-1). Signaling through both of these pathways induces activation of the kinase activity of calcineurin, which, in turn, activates transcription factors, leading to upregulation of multiple genes, including interleukin 2 (IL-2) and interferon gamma. IL-2 signals through the target of rapamycin (TOR) to induce cell proliferation in an autocrine fashion. There is evidence that non-HLA antigens can also play a role in renal transplant rejection episodes. Recipients who receive a kidney from an HLA-identical sibling can have rejection episodes and require maintenance immunosuppression, whereas identical twin transplants require no immunosuppression. There are documented non-HLA antigens, such as an endothelial-specific antigen system with limited polymorphism and a tubular antigen, which can act as targets of humoral or cellular rejection responses, respectively.

1	Transplantation in the Treatment of Renal Failure

1	FIGuRE 337-1 Recognition pathways for major histocompatibility complex (MHC) antigens. Graft rejection is initiated by CD4 helper T lymphocytes (TH) having antigen receptors that bind to specific complexes of peptides and MHC class II molecules on antigen-presenting cells (APC). In transplantation, in contrast to other immunologic responses, there are two sets of T cell clones involved in rejection. In the direct pathway, the class II MHC of donor allogeneic APCs is recognized by CD4 TH cells that bind to the intact MHC molecule, and class I MHC allogeneic cells are recognized by CD8 T cells. The latter generally proliferate into cytotoxic cells (TC). In the indirect pathway, the incompatible MHC molecules are processed into peptides that are presented by the self-APCs of the recipient. The indirect, but not the direct, pathway is the normal physiologic process in T cell recognition of foreign antigens. Once TH cells are activated, they proliferate and, by secretion of cytokines and

1	The indirect, but not the direct, pathway is the normal physiologic process in T cell recognition of foreign antigens. Once TH cells are activated, they proliferate and, by secretion of cytokines and direct contact, exert strong helper effects on macrophages, TC, and B cells. (From MH Sayegh, LH Turka: N Engl J Med, 338:1813, 1998. Copyright 1998, Massachusetts Medical Society. All rights reserved.)

1	Immunosuppressive therapy, as currently available, generally suppresses all immune responses, including those to bacteria, fungi, and even malignant tumors. In general, all clinically useful drugs are more selective to primary than to memory immune responses. Agents to suppress the immune response are classically divided into induction and maintenance agents and will be discussed in the following paragraphs. Those currently in clinical use are listed in Table 337-3. Induction therapy is currently given to most kidney transplant recipients in the United States at the time of transplant to reduce the risk of early acute rejection and to minimize or eliminate the use of either steroids or calcineurin inhibitors and their associated toxicities. Induction therapy consists of antibodies that could be monoclonal or polyclonal and depletional or nondepletional.

1	Depleting Agents Peripheral human lymphocytes, thymocytes, or lymphocytes from spleens or thoracic duct fistulas are injected into horses, rabbits, or goats to produce antilymphocyte serum, from which the globulin fraction is then separated, resulting in antithymocyte globulin. Those polyclonal antibodies induce lymphocyte depletion, and the immune system may take several months to recover. Monoclonal antibodies against defined lymphocyte subsets offer a more precise and standardized form of therapy. Alemtuzumab is directed to the CD52 protein, widely distributed on immune cells such as B and T cells, natural killer cells, macrophages, and some granulocytes.

1	Nondepleting Agents Another approach to more selective therapy is to target the 55-kDa alpha chain of the IL-2 receptor, which is expressed only on T cells that have been recently activated. This approach is used as prophylaxis for acute rejection in the immediate posttransplant period and is effective at decreasing the early acute rejection rate with few adverse side effects. The next step in the evolution of this therapeutic strategy, which has already been achieved in the short term in small numbers of immunologically well-matched patients, is the elimination of all maintenance immunosuppression therapy.

1	All kidney transplant recipients should receive maintenance immunosuppressive therapies except identical twins. The most frequently used combination is triple therapy with prednisone, a calcineurin inhibitor, and an antimetabolite; mammalian TOR (mTOR) inhibitors can replace one of the last two agents. More recently, the U.S. Food and Drug Administration (FDA) approved a new costimulatory blocking antibody, belatacept, as a new strategy to prevent long-term calcineurin inhibitor toxicity.

1	Antimetabolites Azathioprine, an analogue of mercaptopurine, was for two decades the keystone to immunosuppressive therapy in humans, but has given way to more effective agents. This agent can inhibit synthesis of DNA, RNA, or both. Azathioprine is administered in doses of 1.5–2 mg/kg per day. Reduction in the dose is required because of leukopenia and occasionally thrombocytopenia. Excessive amounts of azathioprine may also cause jaundice, anemia, and alopecia. If it is essential to administer allopurinol concurrently, the azathioprine dose must be reduced. Because inhibition of xanthine oxidase delays degradation, this combination is best avoided.

1	Mycophenolate mofetil or mycophenolate sodium, both of which are metabolized to mycophenolic acid, is now used in place of azathioprine in most centers. It has a similar mode of action and a mild degree of gastrointestinal toxicity but produces less bone marrow suppression. Its advantage is its increased potency in preventing or reversing rejection.

1	Steroids Glucocorticoids are important adjuncts to immunosuppressive therapy. Among all the agents employed, prednisone has effects that are easiest to assess, and in large doses it is usually effective for the reversal of rejection. In general, 200–300 mg prednisone is given immediately before or at the time of transplantation, and the dose is reduced to 30 mg within a week. The side effects of the glucocorticoids, particularly impairment of wound healing and predisposition to infection, make it desirable to taper the dose as rapidly as possible in the immediate postoperative period. Many centers now have protocols for early discontinuance or avoidance of steroids because of long-term adverse effects on bone, skin, and glucose metabolism. For treatment of acute rejection, methylprednisolone, 0.5–1 g IV, is administered immediately upon diagnosis of beginning rejection and continued once daily for 3 days. Such “pulse” doses are not effective in chronic rejection. Most patients whose

1	0.5–1 g IV, is administered immediately upon diagnosis of beginning rejection and continued once daily for 3 days. Such “pulse” doses are not effective in chronic rejection. Most patients whose renal function is stable after 6 months or a year do not require large doses of prednisone; maintenance doses of 5–10 mg/d are the rule. A major effect of steroids is preventing the release of IL-6 and IL-1 by monocytes-macrophages.

1	Calcineurin Inhibitors Cyclosporine is a fungal peptide with potent immunosuppressive activity. It acts on the calcineurin pathway to block transcription of mRNA for IL-2 and other proinflammatory cytokines, thereby inhibiting T cell proliferation. Although it works alone, cyclosporine is more effective in conjunction with glucocorticoids and mycophenolate. Clinical results with tens of thousands of renal transplants have been impressive. Among its toxic effects (nephrotoxicity, hepatotoxicity, hirsutism, tremor, gingival hyperplasia, Transplantation in the Treatment of Renal Failure Abbreviations: FKBP-12, FK506 binding protein 12; IFN, interferon; IL, interleukin; RBC, red blood cells; TGF, transforming growth factor; TNF, tumor necrosis factor; WBC, white blood cells. diabetes), only nephrotoxicity presents a serious management problem and is further discussed below.

1	diabetes), only nephrotoxicity presents a serious management problem and is further discussed below. Tacrolimus (previously called FK506) is a fungal macrolide that has the same mode of action as cyclosporine as well as a similar side effect profile; it does not, however, produce hirsutism or gingival hyperplasia. De novo diabetes mellitus is more common with tacrolimus. The drug was first used in liver transplantation and may substitute for cyclosporine entirely or as an alternative in renal patients whose rejections are poorly controlled by cyclosporine. mTOR Inhibitors Sirolimus (previously called rapamycin) is another fungal macrolide but has a different mode of action; i.e., it inhibits T cell growth factor signaling pathways, preventing the response to IL-2 and other cytokines. Sirolimus can be used in conjunction with cyclosporine or tacrolimus, or with mycophenolic acid, to avoid the use of calcineurin inhibitors.

1	Everolimus is another mTOR inhibitor with similar mechanism of action as sirolimus but with better bioavailability. Belatacept Belatacept is a fusion protein that binds costimulatory ligands (CD80 and CD86) present on antigen-presenting cells, interrupting their binding to CD28 on T cells. This inhibition leads to T cell anergy and apoptosis. Belatacept is FDA approved for kidney transplant recipients and is given monthly as an intravenous infusion.

1	Adequate hemodialysis should be performed within 48 h of surgery, and care should be taken that the serum potassium level is not markedly elevated so that intraoperative cardiac arrhythmias can be averted. The diuresis that commonly occurs postoperatively must be carefully monitored. In some instances, it may be massive, reflecting the inability of ischemic tubules to regulate sodium and water excretion; with large diureses, massive potassium losses may occur. Most chronically uremic patients have some excess of extracellular fluid, and it is useful to maintain an expanded fluid volume in the immediate postoperative period. Acute tubular necrosis (ATN) due to ischemia may cause immediate oliguria or may follow an initial short period of graft function. Recovery usually occurs within 3 weeks, although periods as long as 6 weeks have been reported. Superimposition of rejection on ATN is common, and the differential diagnosis may be difficult without a graft biopsy. Cyclosporine therapy

1	although periods as long as 6 weeks have been reported. Superimposition of rejection on ATN is common, and the differential diagnosis may be difficult without a graft biopsy. Cyclosporine therapy prolongs ATN, and some patients do not diurese until the dose is reduced drastically. Many centers avoid starting cyclosporine for the first several days, using antilymphocyte globulin (ALG) or a monoclonal antibody along with mycophenolic acid and prednisone until renal function is established. Figure 337-2 illustrates an algorithm followed by many transplant centers for early posttransplant management of recipients at high or low risk of early renal dysfunction.

1	Early diagnosis of rejection allows prompt institution of therapy to preserve renal function and prevent irreversible damage. Clinical evidence of rejection is rarely characterized by fever, swelling, and tenderness over the allograft. Rejection may present only with a rise in serum creatinine, with or without a reduction in urine volume. The focus should be on ruling out other causes of functional deterioration.

1	Doppler ultrasonography may be useful in ascertaining changes in the renal vasculature and in renal blood flow. Thrombosis of the renal vein occurs rarely; it may be reversible if it is caused by technical factors and intervention is prompt. Diagnostic ultrasound is the procedure of choice to rule out urinary obstruction or to confirm the presence of perirenal collections of urine, blood, or lymph. A rise in the serum creatinine level is a late marker of rejection, but it may be the only sign. Novel biomarkers are needed for early noninvasive detection of allograft rejection.

1	Calcineurin inhibitors (cyclosporine and tacrolimus) have an afferent arteriolar constrictor effect on the kidney and may produce permanent vascular and interstitial injury after sustained high-dose therapy. This action will lead to a deterioration in renal function difficult to distinguish from rejection without a renal biopsy. Interstitial fibrosis, isometric tubular vacuolization, and thickening of arteriolar walls are suggestive of this side effect, but not diagnostic. Hence, if no rejection is detected on the biopsy, serum creatinine may respond to a reduction in dose. However, if rejection activity is present in the biopsy, appropriate therapy is indicated. The first rejection episode is usually treated with IV administration of methylprednisolone, 500–1000 mg daily for 3 days. Failure to respond is an indication for antibody therapy, usually with antithymocyte globulin.

1	Evidence of antibody-mediated injury is present when endothelial injury and deposition of complement component c4d is detected by fluorescence labeling. This is usually accompanied by detection of the antibody in the recipient blood. The prognosis is poor, and aggressive use of plasmapheresis, immunoglobulin infusions, anti-CD20 monoclonal antibody (rituximab) to target B lymphocytes, bortezomib to target antibody-producing plasma cells, and eculizumab to inhibit complement is indicated. The typical times after transplantation when the most common opportunistic infections occur are shown in Table 337-4. Prophylaxis for cytomegalovirus (CMV) and Pneumocystis jiroveci pneumonia is given for 6–12 months after transplantation.

1	The signs and symptoms of infection may be masked or distorted. Fever without obvious cause is common, and only after days or weeks may it become apparent that it has a viral or fungal origin. Bacterial infections are most common during the first month after transplantation. Recipient High % PRA (sensitization level) Recipient PRA <10%, and

1	No response Empirical IV steroid “pulse” therapy (methylprednisolone 0.2–1.0 g/d x 3 days) Low calcineurin inhibitor level Adequate calcineurin inhibitor level Transplant dysfunction* “High risk” Antilymphocyte globulin “induction” therapy Avoid calcineurin inhibitor until kidney function is established Steroids Calcineurin inhibitor Mycophenolic acid mofetil No response Acute rejection Renal biopsy Empirical IV steroid “pulse” therapy (methylprednisolone 0.2–1 g/d x 3 days) “Low risk” Persistent renal dysfunction orDe novo transplant dysfunction* with adequate calcineurin inhibitor levels Steroids Calcineurin inhibitor Mycophenolic acid mofetil Anti-CD3 monoclonal antibody (OKT3 5 g/d x 7–10 days) FIGuRE 337-2 A typical algorithm for early posttransplant care of a kidney recipient. If any of the recipient or donor “high-risk” factors exist, more aggressive management is called for. Low-risk patients can be treated with a standard immunosuppressive regimen. Patients at higher risk of

1	of the recipient or donor “high-risk” factors exist, more aggressive management is called for. Low-risk patients can be treated with a standard immunosuppressive regimen. Patients at higher risk of rejection or early ischemic and nephrotoxic transplant dysfunction are often induced with an antilymphocyte globulin to provide more potent early immunosuppression or to spare calcineurin nephrotoxicity. *When there is early transplant dysfunction, prerenal, obstructive, and vascular causes must be ruled out by ultrasonographic examination. The panel reactive antibody (PRA) is a quantitation of how much anti-body is present in a candidate against a panel of cells representing the distribution of antigens in the donor pool.

1	the MOst COMMOn OppOrtunIstIC InFeCtIOns In renaL transpLant reCIpIents The importance of blood cultures in such patients cannot be overemphasized because systemic infection without obvious foci is common. Particularly ominous are rapidly occurring pulmonary lesions, which may result in death within 5 days of onset. When these lesions become apparent, immunosuppressive agents should be discontinued, except for maintenance doses of prednisone.

1	Aggressive diagnostic procedures, including transbronchial and open-lung biopsy, are frequently indicated. In the case of P. jiroveci (Chap. 244) infection, trimethoprim-sulfamethoxazole (TMP-SMX) is the treatment of choice; amphotericin B has been used effectively in systemic fungal infections. Prophylaxis against P. jiroveci with daily or alternate-day low-dose TMP-SMX is very effective. Involvement of the oropharynx with Candida (Chap. 240) may be treated with local nystatin. Tissue-invasive fungal infections require treatment with systemic agents such as fluconazole. Small doses (a total of 300 mg) of amphotericin given over a period of 2 weeks may be effective in fungal infections refractory to fluconazole. Macrolide antibiotics, especially ketoconazole and erythromycin, and some calcium channel blockers (diltiazem, verapamil) compete with calcineurin inhibitors for P450 catabolism and cause elevated levels of these immunosuppressive drugs. Analeptics, such as phenytoin and

1	calcium channel blockers (diltiazem, verapamil) compete with calcineurin inhibitors for P450 catabolism and cause elevated levels of these immunosuppressive drugs. Analeptics, such as phenytoin and carbamazepine, will increase catabolism to result in low levels. Aspergillus (Chap. 241), Nocardia (Chap. 199), and especially CMV (Chap. 219) infections also occur.

1	CMV is a common and dangerous DNA virus in transplant recipients. It does not generally appear until the end of the first post-transplant month. Active CMV infection is sometimes associated, or occasionally confused, with rejection episodes. Patients at highest risk for severe CMV disease are those without anti-CMV antibodies who receive a graft from a CMV antibody–positive donor (15% mortality). Valganciclovir is a cost-effective and bioavailable oral form of ganciclovir that has been proved effective in both prophylaxis and treatment of CMV disease. Early diagnosis in a febrile patient with clinical suspicion of CMV disease can be made by determining CMV viral load in the blood. A rise in IgM antibodies to CMV is also diagnostic. Culture of CMV from blood may be less sensitive. Tissue invasion of CMV is common in the gastrointestinal tract and lungs. CMV retinopathy occurs late in the course, if untreated. Treatment of active CMV disease with valganciclovir is always indicated. In

1	invasion of CMV is common in the gastrointestinal tract and lungs. CMV retinopathy occurs late in the course, if untreated. Treatment of active CMV disease with valganciclovir is always indicated. In many patients immune to CMV, viral activation can occur with major immunosuppressive regimens.

1	The polyoma group (BK, JC, SV40) is another class of DNA viruses that can become dormant in kidneys and can be activated by immunosuppression. When reactivation occurs with BK, there is a 50% chance of progressive fibrosis and loss of the graft within 1 year by the activated virus. Risk of infection is associated with the overall degree of immunosuppression rather than the individual immunosuppressive drugs used. Renal biopsy is necessary for the diagnosis. There have been variable results with leflunomide, cidofovir, and quinolone antibiotics (which are effective against polyoma helicase), but it is most important to reduce the immunosuppressive load.

1	The complications of glucocorticoid therapy are well known and include gastrointestinal bleeding, impairment of wound healing, osteoporosis, diabetes mellitus, cataract formation, and hemorrhagic pancreatitis. The treatment of unexplained jaundice in transplant patients should include cessation or reduction of immunosuppressive drugs if hepatitis or drug toxicity is suspected. Therapy in such circumstances often does not result in rejection of a graft, at least for several weeks. Acyclovir is effective in therapy for herpes simplex virus infections.

1	Although 1-year transplant survival is excellent, most recipients experience progressive decline in kidney function over time thereafter. Chronic renal transplant dysfunction can be caused by recurrent disease, hypertension, cyclosporine or tacrolimus nephrotoxicity, chronic immunologic rejection, secondary focal glomerulosclerosis, or a combination of these pathophysiologies. Chronic vascular changes with intimal proliferation and medial hypertrophy are commonly found. Control of systemic and intrarenal hypertension with angiotensinconverting enzyme (ACE) inhibitors is thought to have a beneficial influence on the rate of progression of chronic renal transplant dysfunction. Renal biopsy can distinguish subacute cellular rejection from recurrent disease or secondary focal sclerosis.

1	The incidence of tumors in patients on immunosuppressive therapy is 5–6%, or approximately 100 times greater than that in the general population in the same age range. The most common lesions are cancer of the skin and lips and carcinoma in situ of the cervix, as well as lymphomas such as non-Hodgkin’s lymphoma. The risks are increased in proportion to the total immunosuppressive load administered and the time elapsed since transplantation. Surveillance for skin and cervical cancers is necessary. Both chronic dialysis and renal transplant patients have a higher incidence of death from myocardial infarction and stroke than does the population at large, and this is particularly true in diabetic patients. 1831

1	Contributing factors are the use of glucocorticoids and sirolimus and hypertension. Recipients of renal transplants have a high prevalence of coronary artery and peripheral vascular diseases. The percentage of deaths from these causes has been slowly rising as the numbers of transplanted diabetic patients and the average age of all recipients increase. More than 50% of renal recipient mortality is attributable to cardiovascular disease. In addition to strict control of blood pressure and blood lipid levels, close monitoring of patients for indications of further medical or surgical intervention is an important part of management.

1	Hypertension may be caused by (1) native kidney disease, (2) rejection activity in the transplant, (3) renal artery stenosis if an end-to-end anastomosis was constructed with an iliac artery branch, and (4) renal calcineurin inhibitor toxicity, which may improve with reduction in dose. Whereas ACE inhibitors may be useful, calcium channel blockers are more frequently used initially. Amelioration of hypertension to the range of 120–130/70–80 mmHg should be the goal in all patients. Hypercalcemia after transplantation may indicate failure of hyper-plastic parathyroid glands to regress. Aseptic necrosis of the head of the femur is probably due to preexisting hyperparathyroidism, with aggravation by glucocorticoid treatment. With improved management of calcium and phosphorus metabolism during chronic dialysis, the incidence of parathyroid-related complications has fallen dramatically. Persistent hyperparathyroid activity may require subtotal parathyroidectomy.

1	Although most transplant patients have robust production of erythropoietin and normalization of hemoglobin, anemia is commonly seen in the posttransplant period. Often the anemia is attributable to bone marrow–suppressant immunosuppressive medications such as azathioprine, mycophenolic acid, and sirolimus. Gastrointestinal bleeding is a common side effect of high-dose and long-term steroid administration. Many transplant patients have creatinine clearances of 30–50 mL/min and can be considered in the same way as other patients with chronic renal insufficiency for anemia management, including supplemental erythropoietin. Chronic hepatitis, particularly when due to hepatitis B virus, can be a progressive, fatal disease over a decade or so. Patients who are persistently hepatitis B surface antigen–positive are at higher risk, according to some studies, but the presence of hepatitis C virus is also a concern when one embarks on a course of immunosuppression in a transplant recipient.

1	Julia B. Lewis, Eric G. Neilson

1	Two human kidneys harbor nearly 1.8 million glomerular capillary tufts. Each glomerular tuft resides within Bowman’s space. The capsule circumscribing this space is lined by parietal epithelial cells that transition into tubular epithelia forming the proximal nephron or migrate into the tuft to replenish podocytes. The glomerular capillary tuft derives from an afferent arteriole that forms a branching capillary bed embedded in mesangial matrix (Fig. 338-1). This capillary network funnels into an efferent arteriole, which passes filtered blood into cortical peritubular capillaries or medullary vasa recta that supply and exchange with a folded tubular architecture. Hence the glomerular capillary tuft, fed and drained by arterioles, represents an arteriolar portal system. Fenestrated endothelial cells resting on a glomerular basement membrane (GBM) line glomerular capillaries. Delicate foot processes extending from epithelial podocytes shroud the outer surface of these capillaries, and

1	cells resting on a glomerular basement membrane (GBM) line glomerular capillaries. Delicate foot processes extending from epithelial podocytes shroud the outer surface of these capillaries, and podocytes interconnect to each other by slit-pore membranes forming a selective filtration barrier.

1	The glomerular capillaries filter 120–180 L/d of plasma water containing various solutes for reclamation or discharge by downstream FIGuRE 338-1 Glomerular architecture. A. The glomerular capillaries form from a branching network of renal arteries, arterioles, leading to an afferent arteriole, glomerular capillary bed (tuft), and a draining efferent arteriole. (From VH Gattone II et al: Hypertension 5:8, 1983.) B. Scanning electron micrograph of podocytes that line the outer surface of the glomerular capillaries (arrow shows foot process). C. Scanning electron micrograph of the fenestrated endothelia lining the glomerular capillary. D. The various normal regions of the glomerulus on light microscopy.

1	(A–C: Courtesy of Dr. Vincent Gattone, Indiana University; with permission.) tubules. Most large proteins and all cells are excluded from filtration by a physicochemical barrier governed by pore size and negative electrostatic charge. The mechanics of filtration and reclamation are quite complicated for many solutes (Chap. 325). For example, in the case of serum albumin, the glomerulus is an imperfect barrier. Although albumin has a negative charge, which would tend to repel the negatively charged GBM, it only has a physical radius of 3.6 nm, while pores in the GBM and slit-pore membranes have a radius of 4 nm. Consequently, variable amounts of albumin inevitably cross the filtration barrier to be reclaimed by megalin and cubilin receptors along the proximal tubule. Remarkably, humans with normal nephrons excrete on average 8–10 mg of albumin in daily voided urine, approximately 20–60% of total excreted protein. This amount of albumin, and other proteins, can rise to gram quantities

1	normal nephrons excrete on average 8–10 mg of albumin in daily voided urine, approximately 20–60% of total excreted protein. This amount of albumin, and other proteins, can rise to gram quantities following glomerular injury.

1	The breadth of diseases affecting the glomerulus is expansive because the glomerular capillaries can be injured in a variety of ways, producing many different lesions. Some order to this vast subject is brought by grouping all of these diseases into a smaller number of clinical syndromes. There are many forms of glomerular disease with pathogenesis variably linked to the presence of genetic mutations, infection, toxin exposure, autoimmunity, atherosclerosis, hypertension, emboli, thrombosis, or diabetes mellitus. Even after careful study, however, the cause often remains unknown, and the lesion is called idiopathic. Specific or unique features of pathogenesis are mentioned with the description of each of the glomerular diseases later in this chapter.

1	ing familial disease or a founder effect: congenital nephrotic syn drome from mutations in NPHS1 (nephrin) and NPHS2 (podocin) affect the slit-pore membrane at birth, and TRPC6 cation channel mutations produce focal segmental glomerulosclerosis (FSGS) in adulthood; polymorphisms in the gene encoding apolipoprotein L1, APOL1, are a major risk for nearly 70% of African Americans with nondiabetic end-stage renal disease, particularly FSGS; mutations in complement factor H associate with membranoproliferative glomerulonephritis (MPGN) or atypical hemolytic uremic syndrome (aHUS), type II partial lipodystrophy from mutations in genes encoding lamin A/C, or PPARγ cause a metabolic syndrome associated with MPGN, which is sometimes accompanied by dense deposits and C3 nephritic factor; Alport’s syndrome, from mutations in the genes encoding for the α3, α4, or α5 chains of type IV collagen, produces split-basement membranes with glomerulosclerosis; and lysosomal storage diseases, such as

1	syndrome, from mutations in the genes encoding for the α3, α4, or α5 chains of type IV collagen, produces split-basement membranes with glomerulosclerosis; and lysosomal storage diseases, such as α-galactosidase A deficiency causing Fabry’s disease and N -acetylneuraminic acid hydrolase deficiency causing nephrosialidosis, produce FSGS.

1	Systemic hypertension and atherosclerosis can produce pressure stress, ischemia, or lipid oxidants that lead to chronic glomerulosclerosis. Malignant hypertension can quickly complicate glomerulosclerosis with fibrinoid necrosis of arterioles and glomeruli, thrombotic microangiopathy, and acute renal failure. Diabetic nephropathy is an acquired sclerotic injury associated with thickening of the GBM secondary to the long-standing effects of hyperglycemia, advanced glycosylation end products, and reactive oxygen species.

1	Inflammation of the glomerular capillaries is called glomerulonephritis. Most glomerular or mesangial antigens involved in immune-mediated glomerulonephritis are unknown (Fig. 338-2). Glomerular epithelial or mesangial cells may shed or express epitopes that mimic other immunogenic proteins made elsewhere in the body. Bacteria, fungi, and viruses can directly infect the kidney producing their own antigens. Autoimmune diseases like idiopathic membranous glomerulonephritis (MGN) or MPGN are confined to the kidney, whereas systemic inflammatory diseases like lupus nephritis or granulomatosis with polyangiitis (Wegener’s) spread to the kidney, causing secondary glomerular injury. Antiglomerular basement membrane disease producing Goodpasture’s syndrome primarily injures both the lung and kidney because of the narrow distribution of the α3 NC1 domain of type IV collagen that is the target antigen.

1	Local activation of Toll-like receptors on glomerular cells, deposition of immune complexes, or complement injury to glomerular structures induces mononuclear cell infiltration, which subsequently leads to an adaptive immune response attracted to the kidney by local release of chemokines. Neutrophils, macrophages, and T cells are drawn by chemokines into the glomerular tuft, where they react with antigens 1833 and epitopes on or near somatic cells or their structures, producing more cytokines and proteases that damage the mesangium, capillaries, and/or the GBM. While the adaptive immune response is similar to that of other tissues, early T cell activation plays an important role in the mechanism of glomerulonephritis. Antigens presented by class II major histocompatibility complex (MHC) molecules on macrophages and dendritic cells in conjunction with associative recognition molecules engage the CD4/8 T cell repertoire.

1	Mononuclear cells by themselves can injure the kidney, but auto immune events that damage glomeruli classically produce a humoral immune response. Poststreptococcal glomerulonephritis, lupus nephritis, and idiopathic membranous nephritis typically are associated with immune deposits along the GBM, while anti-GBM antibodies produce the linear binding of anti-GBM disease. Preformed circulating FIGuRE 338-2 The glomerulus is injured by a variety of mechanisms. A. Preformed immune deposits can precipitate from the circulation and collect along the glomerular basement membrane (GBM) in the subendothelial space or can form in situ along the subepithelial space.

1	B. Immunofluorescent staining of glomeruli with labeled anti-IgG demonstrating linear staining from a patient with anti-GBM disease or immune deposits from a patient with membranous glomerulonephritis. C. The mechanisms of glomerular injury have a complicated pathogenesis. Immune deposits and complement deposition classically draw macrophages and neutrophils into the glomerulus. T lymphocytes may follow to participate in the injury pattern as well. D. Amplification mediators as locally derived oxidants and proteases expand this inflammation, and, depending on the location of the target antigen and the genetic polymorphisms of the host, basement membranes are damaged with either endocapillary or extracapillary proliferation.

1	1834 immune complexes can precipitate along the subendothelial side of the GBM, while other immune deposits form in situ on the subepithelial side. These latter deposits accumulate when circulating autoantibodies find their antigen trapped along the subepithelial edge of the GBM. Immune deposits in the glomerular mesangium may result from the deposition of preformed circulating complexes or in situ antigen-antibody interactions. Immune deposits stimulate the release of local proteases and activate the complement cascade, producing C5–9 attack complexes. In addition, local oxidants damage glomerular structures, producing proteinuria and effacement of the podocytes. Overlapping etiologies or pathophysiologic mechanisms can produce similar glomerular lesions, suggesting that downstream molecular and cellular responses often converge toward common patterns of injury.

1	Persistent glomerulonephritis that worsens renal function is always accompanied by interstitial nephritis, renal fibrosis, and tubular atrophy (see Fig. 62e-27). What is not so obvious, however, is that renal failure in glomerulonephritis best correlates histologically with the appearance of tubulointerstitial nephritis rather than with the type of inciting glomerular injury.

1	Loss of renal function due to interstitial damage is explained hypothetically by several mechanisms. The simplest explanation is that urine flow is impeded by tubular obstruction as a result of interstitial inflammation and fibrosis. Thus, obstruction of the tubules with debris or by extrinsic compression results in aglomerular nephrons. A second mechanism suggests that interstitial changes, including interstitial edema or fibrosis, alter tubular and vascular architecture and thereby compromise the normal tubular transport of solutes and water from tubular lumen to vascular space. This failure increases the solute and water content of the tubule fluid, resulting in isosthenuria and polyuria. Adaptive mechanisms related to tubuloglomerular feedback also fail, resulting in a reduction of renin output from the juxtaglomerular apparatus trapped by interstitial inflammation. Consequently, the local vasoconstrictive influence of angiotensin II on the glomerular arterioles decreases, and

1	renin output from the juxtaglomerular apparatus trapped by interstitial inflammation. Consequently, the local vasoconstrictive influence of angiotensin II on the glomerular arterioles decreases, and filtration drops owing to a generalized decrease in arteriolar tone. A third mechanism involves changes in vascular resistance due to damage of peritubular capillaries. The cross-sectional volume of these capillaries is decreased by interstitial inflammation, edema, or fibrosis. These structural alterations in vascular resistance affect renal function through two mechanisms. First, tubular cells are very metabolically active, and, as a result, decreased perfusion leads to ischemic injury. Second, impairment of glomerular arteriolar outflow leads to increased intraglomerular hypertension in less-involved glomeruli; this selective intraglomerular hypertension aggravates and extends mesangial sclerosis and glomerulosclerosis to less-involved glomeruli. Regardless of the exact mechanism, early

1	glomeruli; this selective intraglomerular hypertension aggravates and extends mesangial sclerosis and glomerulosclerosis to less-involved glomeruli. Regardless of the exact mechanism, early acute tubulointerstitial nephritis (see Fig. 62e-27) suggests potentially recoverable renal function, whereas the development of chronic interstitial fibrosis prognosticates permanent loss (see Fig. 62e-30).

1	Persistent damage to glomerular capillaries spreads to the tubulointerstitium in association with proteinuria. There is a hypothesis that efferent arterioles leading from inflamed glomeruli carry forward inflammatory mediators, which induces downstream interstitial nephritis, resulting in fibrosis. Glomerular filtrate from injured glomerular capillaries adherent to Bowman’s capsule may also be misdirected to the periglomerular interstitium. Most nephrologists believe, however, that proteinuric glomerular filtrate forming tubular fluid is the primary route to downstream tubulointerstitial injury, although none of these hypotheses are mutually exclusive.

1	The simplest explanation for the effect of proteinuria on the development of interstitial nephritis is that increasingly severe proteinuria, carrying activated cytokines and lipoproteins producing reactive oxygen species, triggers a downstream inflammatory cascade in and around epithelial cells lining the tubular nephron. These effects induce T lymphocyte and macrophage infiltrates in the interstitial spaces along with fibrosis and tubular atrophy.

1	Tubules disaggregate following direct damage to their basement membranes, leading to epithelial-mesenchymal transitions forming more interstitial fibroblasts at the site of injury. Transforming growth factor β (TGF-β), fibroblast growth factor 2 (FGF-2), hypoxemiainducible factor 1α (HIF-1α), and platelet-derived growth factor (PDGF) are particularly active in this transition. With persistent nephritis, fibroblasts multiply and lay down tenascin and a fibronectin scaffold for the polymerization of new interstitial collagen types I/III. These events form scar tissue through a process called fibrogenesis. In experimental studies, bone morphogenetic protein 7 and hepatocyte growth factor can reverse early fibrogenesis and preserve tubular architecture. When fibroblasts outdistance their survival factors, apoptoses occurs, and the permanent renal scar becomes acellular, leading to irreversible renal failure. APPROACH TO THE PATIENT: HEMATuRIA, PROTEINuRIA, AND PYuRIA

1	Patients with glomerular disease usually have some hematuria with varying degrees of proteinuria. Hematuria is typically asymptomatic. As few as three to five red blood cells in the spun sediment from first-voided morning urine is suspicious. The diagnosis of glomerular injury can be delayed because patients will not realize they have microscopic hematuria, and only rarely with the exception of IgA nephropathy and sickle cell disease is gross hematuria present. When working up microscopic hematuria, perhaps accompanied by minimal proteinuria (<500 mg/24 h), it is important to exclude anatomic lesions, such as malignancy of the urinary tract, particularly in older men. Microscopic hematuria may also appear with the onset of benign prostatic hypertrophy, interstitial nephritis, papillary necrosis, hypercalciuria, renal stones, cystic kidney diseases, or renal vascular injury. However, when red blood cell casts (see Fig. 62e-34) or dysmorphic red blood cells are found in the sediment,

1	necrosis, hypercalciuria, renal stones, cystic kidney diseases, or renal vascular injury. However, when red blood cell casts (see Fig. 62e-34) or dysmorphic red blood cells are found in the sediment, glomerulonephritis is likely.

1	Sustained proteinuria >1–2 g/24 h is also commonly associated with glomerular disease. Patients often will not know they have proteinuria unless they become edematous or notice foaming urine on voiding. Sustained proteinuria has to be distinguished from lesser amounts of so-called benign proteinuria in the normal population (Table 338-1). This latter class of proteinuria is nonsustained, generally <1 g/24 h, and is sometimes called functional or transient proteinuria. Fever, exercise, obesity, sleep apnea, emotional stress, and congestive heart failure can explain transient proteinuria. Proteinuria only seen with upright posture is called orthostatic proteinuria and has a benign prognosis. Isolated proteinuria sustained over multiple clinic visits is found in many glomerular lesions. Proteinuria in most adults with glomerular disease is nonselective, containing albumin and a mixture of other serum proteins, whereas in children with minimal change disease, the proteinuria is selective

1	in most adults with glomerular disease is nonselective, containing albumin and a mixture of other serum proteins, whereas in children with minimal change disease, the proteinuria is selective and composed largely of albumin.

1	Some patients with inflammatory glomerular disease, such as CLINICAL SYNDROMES acute poststreptococcal glomerulonephritis or MPGN, have pyuria Various forms of glomerular injury can also be parsed into sevcharacterized by the presence of considerable numbers of leuko-eral distinct syndromes on clinical grounds (Table 338-2). These cytes. This latter finding has to be distinguished from urine infected syndromes, however, are not always mutually exclusive. There is with bacteria. an acute nephritic syndrome producing 1–2 g/24 h of proteinuria, aCan present as rapidly progressive glomerulonephritis (RPGN); sometimes called crescentic glomerulonephritis. bCan present as a malignant hypertensive crisis producing an aggressive fibrinoid necrosis in arterioles and small arteries with microangiopathic hemolytic anemia. cCan present with gross hematuria. Abbreviations: AA, amyloid A; AL, amyloid L; ANCA, antineutrophil cytoplasmic antibodies; GBM, glomerular basement membrane.

1	hematuria with red blood cell casts, pyuria, hypertension, fluid retention, and a rise in serum creatinine associated with a reduction in glomerular filtration. If glomerular inflammation develops slowly, the serum creatinine will rise gradually over many weeks, but if the serum creatinine rises quickly, particularly over a few days, acute nephritis is sometimes called rapidly progressive glomerulonephritis (RPGN); the histopathologic term crescentic glomerulonephritis is the pathologic equivalent of the clinical presentation of RPGN. When patients with RPGN present with lung hemorrhage from Goodpasture’s syndrome, antineutrophil cytoplasmic antibodies (ANCA)-associated small-vessel vasculitis, lupus erythematosus, or cryoglobulinemia, they are often diagnosed as having a pulmonary-renal syndrome. Nephrotic syndrome describes the onset of heavy proteinuria (>3.0 g/24 h), hypertension, hypercholesterolemia, hypoalbuminemia, edema/anasarca, and microscopic hematuria; if only large

1	syndrome. Nephrotic syndrome describes the onset of heavy proteinuria (>3.0 g/24 h), hypertension, hypercholesterolemia, hypoalbuminemia, edema/anasarca, and microscopic hematuria; if only large amounts of proteinuria are present without clinical manifestations, the condition is sometimes called nephrotic-range proteinuria. The glomerular filtration rate (GFR) in these patients may initially be normal or, rarely, higher than normal, but with persistent hyperfiltration and continued nephron loss, it typically declines over months to years. Patients with a basement membrane syndrome either have genetically abnormal basement membranes (Alport’s syndrome) or an autoimmune response to basement membrane collagen IV (Goodpasture’s syndrome) associated with microscopic hematuria, mild to heavy proteinuria, and hypertension with variable elevations in serum creatinine. Glomerular–vascular syndrome describes patients with vascular injury producing hematuria and moderate proteinuria. Affected

1	proteinuria, and hypertension with variable elevations in serum creatinine. Glomerular–vascular syndrome describes patients with vascular injury producing hematuria and moderate proteinuria. Affected individuals can have vasculitis, thrombotic microangiopathy, antiphospholipid syndrome, or, more commonly, a systemic disease such as atherosclerosis, cholesterol emboli, hypertension, sickle cell anemia, and autoimmunity. Infectious disease–associated syndrome is most important if one has a global perspective. Save for subacute bacterial endocarditis in the Western Hemisphere, malaria and schistosomiasis may be the most common causes of glomerulonephritis throughout the world, closely followed by HIV and chronic hepatitis B and C. These infectious diseases produce a variety of inflammatory reactions in glomerular capillaries, ranging from nephrotic syndrome to acute nephritic injury, and urinalyses that demonstrate a combination of hematuria and proteinuria.

1	These six general categories of syndromes are usually determined at the bedside with the help of a history and physical examination, blood chemistries, renal ultrasound, and urinalysis. These initial studies help frame further diagnostic workup that typically involves testing of the serum for the presence of various proteins (HIV and hepatitis B and C antigens), antibodies (anti-GBM, antiphospholipid, antistreptolysin O [ASO], anti-DNAse, antihyaluronidase, ANCA, anti-DNA, cryoglobulins, anti-HIV, and anti-hepatitis B and C antibodies) or depletion of complement components (C3 and C4). The bedside history and physical examination can also help determine whether the glomerulonephritis is isolated to the kidney (primary glomerulonephritis) or is part of a systemic disease (secondary glomerulonephritis).

1	When confronted with an abnormal urinalysis and elevated serum creatinine, with or without edema or congestive heart failure, one must consider whether the glomerulonephritis is acute or chronic. This assessment is best made by careful history (last known urinalysis or serum creatinine during pregnancy or insurance physical, evidence of infection, or use of medication or recreational drugs); the size of the kidneys on renal ultrasound examination; and how the patient feels at presentation. Chronic glomerular disease often presents with decreased kidney size. Patients who quickly develop renal failure are fatigued and weak and often have uremic symptoms associated with nausea, vomiting, fluid retention, and somnolence. Primary glomerulonephritis presenting with renal failure that has progressed slowly, however, can be remarkably asymptomatic, as are patients with acute glomerulonephritis without much loss in renal function. Once this initial information is collected, selected patients

1	slowly, however, can be remarkably asymptomatic, as are patients with acute glomerulonephritis without much loss in renal function. Once this initial information is collected, selected patients who are clinically stable, have adequate blood clotting parameters, and are willing and able to receive treatment are encouraged to have a renal biopsy.

1	A renal biopsy in the setting of glomerulonephritis quickly identifies the type of glomerular injury and often suggests a course of treatment. The biopsy is processed for light microscopy using stains for hematoxylin and eosin (H&E) to assess cellularity and architecture, periodic acid–Schiff (PAS) to stain carbohydrate moieties in the membranes of the glomerular tuft and tubules, Jones-methenamine silver to enhance basement membrane structure, Congo red for amyloid deposits, and Masson’s trichrome to identify collagen deposition and assess the degree of glomerulosclerosis and interstitial fibrosis. Biopsies are also processed for direct immunofluorescence using conjugated antibodies against IgG, IgM, and IgA to detect the presence of “lumpy-bumpy” immune deposits or “linear” IgG or IgA antibodies bound to GBM, antibodies against trapped complement proteins (C3 and C4), or specific antibodies against a relevant antigen. High-resolution electron microscopy can clarify the principal

1	antibodies bound to GBM, antibodies against trapped complement proteins (C3 and C4), or specific antibodies against a relevant antigen. High-resolution electron microscopy can clarify the principal location of immune deposits and the status of the basement membrane.

1	Each region of a renal biopsy is assessed separately. By light microscopy, glomeruli (at least 10 and ideally 20) are reviewed individually for discrete lesions; <50% involvement is considered focal, and >50% is diffuse. Injury in each glomerular tuft can be segmental, involving a portion of the tuft, or global, involving most of the glomerulus. Glomeruli having proliferative characteristics show increased cellularity. When cells in the capillary tuft proliferate, it is called endocapillary, and when cellular proliferation extends into Bowman’s space, it is called extracapillary. Synechiae are formed when epithelial podocytes attach to Bowman’s capsule in the setting of glomerular injury; crescents, which in some cases may be the extension of synechiae, develop when fibrocellular/fibrin collections fill all or part of Bowman’s space; and sclerotic glomeruli show acellular, amorphous accumulations of proteinaceous material throughout the tuft with loss of functional capillaries and

1	collections fill all or part of Bowman’s space; and sclerotic glomeruli show acellular, amorphous accumulations of proteinaceous material throughout the tuft with loss of functional capillaries and normal mesangium. Since age-related glomerulosclerosis is common in adults, one can estimate the background percentage of sclerosis by dividing the patient’s age in half and subtracting 10. Immunofluorescent and electron microscopy can detect the presence and location of subepithelial, subendothelial, or mesangial immune deposits, or reduplication or splitting of the basement membrane. In the other regions of the biopsy, the vasculature surrounding glomeruli and tubules can show angiopathy, vasculitis, the presence of fibrils, or thrombi. The tubules can be assessed for adjacency to one another; separation can be the result of edema, tubular dropout, or collagen deposition resulting from interstitial fibrosis. Interstitial fibrosis is an ominous sign of irreversibility and progression to

1	separation can be the result of edema, tubular dropout, or collagen deposition resulting from interstitial fibrosis. Interstitial fibrosis is an ominous sign of irreversibility and progression to renal failure.

1	Acute nephritic syndromes classically present with hypertension, hematuria, red blood cell casts, pyuria, and mild to moderate proteinuria. Extensive inflammatory damage to glomeruli causes a fall in GFR and eventually produces uremic symptoms with salt and water retention, leading to edema and hypertension.

1	Poststreptococcal glomerulonephritis is prototypical for acute endocapillary proliferative glomerulonephritis. The incidence of poststreptococcal glomerulonephritis has dramatically decreased in developed countries and in these locations is typically sporadic. Acute poststreptococcal glomerulonephritis in underdeveloped countries is epidemic and usually affects children between the ages of 2 and 14 years, but in developed countries is more typical in the elderly, especially in association with debilitating conditions. It is more common in males, and the familial or cohabitant incidence is as high as 40%. Skin and throat infections with particular M types of streptococci (nephritogenic strains) antedate glomerular disease; M types 47, 49, 55, 2, 60, and 57 are seen following impetigo and M types 1, 2, 4, 3, 25, 49, and 12 with pharyngitis. Poststreptococcal glomerulonephritis due to impetigo develops 2–6 weeks after skin infection and 1–3 weeks after streptococcal pharyngitis.

1	The renal biopsy in poststreptococcal glomerulonephritis demonstrates hypercellularity of mesangial and endothelial cells, glomerular infiltrates of polymorphonuclear leukocytes, granular subendothelial immune deposits of IgG, IgM, C3, C4, and C5–9, and subepithelial deposits (which appear as “humps”) (see Fig. 62e-6). (See Glomerular Schematic 1.) Poststreptococcal glomerulonephritis is an immune-mediated disease involving putative streptococcal antigens, circulating immune complexes, and activation of complement in association with 1837 cell-mediated injury. Many candidate antigens have been proposed over the years; candidates from nephritogenic streptococci of interest at the moment are: a cationic cysteine proteinase known as streptococcal pyrogenic exotoxin B (SPEB) that is generated by proteolysis of a zymogen precursor (zSPEB), and NAPlr, the nephritis-associated plasmin receptor. These two antigens have biochemical affinity for plasmin, bind as complexes facilitated by this

1	by proteolysis of a zymogen precursor (zSPEB), and NAPlr, the nephritis-associated plasmin receptor. These two antigens have biochemical affinity for plasmin, bind as complexes facilitated by this relationship, and activate the alternate complement pathway. The nephritogenic antigen, SPEB, has been demonstrated inside the subepithelial “humps” on biopsy.

1	The classic presentation is an acute nephritic picture with hematuria, pyuria, red blood cell casts, edema, hypertension, and oliguric renal failure, which may be severe enough to appear as RPGN. Systemic symptoms of headache, malaise, anorexia, and flank pain (due to swelling of the renal capsule) are reported in as many as 50% of cases. Five percent of children and 20% of adults have proteinuria in the nephrotic range. In the first week of symptoms, 90% of patients will have a depressed CH50 and decreased levels of C3 with normal levels of C4. Positive rheumatoid factor (30–40%), cryoglobulins and circulating immune complexes (60–70%), and ANCA against myeloperoxidase (10%) are also reported. Positive cultures for streptococcal infection are inconsistently present (10–70%), but increased titers of ASO (30%), anti-DNAse, (70%), or antihyaluronidase antibodies (40%) can help confirm the diagnosis. Consequently, the diagnosis of poststreptococcal glomerulonephritis rarely requires a

1	titers of ASO (30%), anti-DNAse, (70%), or antihyaluronidase antibodies (40%) can help confirm the diagnosis. Consequently, the diagnosis of poststreptococcal glomerulonephritis rarely requires a renal biopsy. A subclinical disease is reported in some series to be four to five times as common as clinical nephritis, and these latter cases are characterized by asymptomatic microscopic hematuria with low serum C3 complement levels.

1	Treatment is supportive, with control of hypertension, edema, and dialysis as needed. Antibiotic treatment for streptococcal infection should be given to all patients and their cohabitants. There is no role for immunosuppressive therapy, even in the setting of crescents. Recurrent poststreptococcal glomerulonephritis is rare despite repeated streptococcal infections. Early death is rare in children but does occur in the elderly. Overall, the prognosis is good, with permanent renal failure being very uncommon, less than 1% in children. Complete resolution of the hematuria and proteinuria in the majority of children occurs within 3–6 weeks of the onset of nephritis but 3–10% of children may have persistent microscopic hematuria, nonnephrotic proteinuria, or hypertension. The prognosis in elderly patients is worse with a high incidence of azotemia (up to 60%), nephrotic-range proteinuria, and end-stage renal disease.

1	Endocarditis-associated glomerulonephritis is typically a complication of subacute bacterial endocarditis, particularly in patients who remain untreated for a long time, have negative blood cultures, or have right-sided endocarditis. Glomerulonephritis is unusual in acute bacterial endocarditis because it takes 10–14 days to develop immune complex–mediated injury, by which time the patient has been treated, often with emergent surgery. Grossly, the kidneys in subacute bacterial endocarditis have subcapsular hemorrhages with a “flea-bitten” appearance, and microscopy on renal biopsy reveals focal proliferation around foci of necrosis associated with abundant mesangial, subendothelial, and subepithelial immune deposits of IgG, IgM, and C3. Patients who present with a clinical picture of RPGN have crescents. Embolic infarcts or septic abscesses may also be present. The pathogenesis hinges on the renal deposition of circulating immune complexes in the kidney with complement activation.

1	have crescents. Embolic infarcts or septic abscesses may also be present. The pathogenesis hinges on the renal deposition of circulating immune complexes in the kidney with complement activation. Patients present with gross or microscopic hematuria, pyuria, and mild proteinuria or, less commonly, RPGN with rapid loss of renal function. A normocytic anemia, elevated erythrocyte sedimentation rate, hypocomplementemia, high titers of rheumatoid factor, type III cryoglobulins, circulating immune complexes, and ANCAs may be present. Levels of serum creatinine may be elevated at diagnosis, but with modern therapy there is little progression to chronic renal failure. Primary treatment is eradication of the infection with 4–6 weeks of antibiotics, and if accomplished expeditiously, the prognosis for renal recovery is good. ANCA-associated vasculitis sometimes accompanies or is confused 1838 with subacute bacterial endocarditis (SBE) and should be ruled out, as the treatment is different. As

1	for renal recovery is good. ANCA-associated vasculitis sometimes accompanies or is confused 1838 with subacute bacterial endocarditis (SBE) and should be ruled out, as the treatment is different. As variants of persistent bacterial infection in blood-associated glomerulonephritis, postinfectious glomerulonephritis can occur in patients with ventriculoatrial and ventriculoperitoneal shunts; pulmonary, intraabdominal, pelvic, or cutaneous infections; and infected vascular prostheses. In developed countries, a significant proportion of cases afflict adults, especially the immunocompromised, and the predominant organism is Staphylococcus. The clinical presentation of these conditions is variable and includes proteinuria, microscopic hematuria, acute renal failure, and hypertension. Serum complement levels are low, and there may be elevated levels of C-reactive proteins, rheumatoid factor, antinuclear antibodies, and cryoglobulins. Renal lesions include membranoproliferative

1	Serum complement levels are low, and there may be elevated levels of C-reactive proteins, rheumatoid factor, antinuclear antibodies, and cryoglobulins. Renal lesions include membranoproliferative glomerulonephritis (MPGN), diffuse proliferative and exudative glomerulonephritis (DPGN), or mesangioproliferative glomerulonephritis, sometimes leading to RPGN. Treatment focuses on eradicating the infection, with most patients treated as if they have endocarditis. The prognosis is guarded.

1	Lupus nephritis is a common and serious complication of systemic lupus erythematosus (SLE) and most severe in African-American female adolescents. Thirty to 50% of patients will have clinical manifestations of renal disease at the time of diagnosis, and 60% of adults and 80% of children develop renal abnormalities at some point in the course of their disease. Lupus nephritis results from the deposition of circulating immune complexes, which activate the complement cascade leading to complement-mediated damage, leukocyte infiltration, activation of procoagulant factors, and release of various cytokines. In situ immune complex formation following glomerular binding of nuclear antigens, particularly necrotic nucleosomes, also plays a role in renal injury. The presence of antiphospholipid antibodies may also trigger a thrombotic microangiopathy in a minority of patients.

1	The clinical manifestations, course of disease, and treatment of lupus nephritis are closely linked to renal pathology. The most common clinical sign of renal disease is proteinuria, but hematuria, hypertension, varying degrees of renal failure, and active urine sediment with red blood cell casts can all be present. Although significant renal pathology can be found on biopsy even in the absence of major abnormalities in the urinalysis, most nephrologists do not biopsy patients until the urinalysis is convincingly abnormal. The extrarenal manifestations of lupus are important in establishing a firm diagnosis of systemic lupus because, while serologic abnormalities are common in lupus nephritis, they are not diagnostic. Anti-dsDNA antibodies that fix complement correlate best with the presence of renal disease. Hypocomplementemia is common in patients with acute lupus nephritis (70–90%) and declining complement levels may herald a flare. Although urinary biomarkers of lupus nephritis

1	of renal disease. Hypocomplementemia is common in patients with acute lupus nephritis (70–90%) and declining complement levels may herald a flare. Although urinary biomarkers of lupus nephritis are being identified to assist in predicting renal flares, renal biopsy is the only reliable method of identifying the morphologic variants of lupus nephritis.

1	The World Health Organization (WHO) workshop in 1974 first outlined several distinct patterns of lupus-related glomerular injury; these were modified in 1982. In 2004 the International Society of Nephrology in conjunction with the Renal Pathology Society again updated the classification. This latest version of lesions seen on biopsy (Table 338-3) best defines clinicopathologic correlations, provides valuable prognostic information, and forms the basis for modern treatment recommendations. Class I nephritis describes normal glomerular histology by any technique or normal light microscopy with minimal mesangial deposits on immunofluorescent or electron microscopy. Class II designates mesangial immune complexes with mesangial proliferation. Both class I and II lesions are typically associated with minimal renal manifestation and normal renal function; nephrotic syndrome is rare. Patients with lesions limited to the renal mesangium have an excellent prognosis and generally do not need

1	with minimal renal manifestation and normal renal function; nephrotic syndrome is rare. Patients with lesions limited to the renal mesangium have an excellent prognosis and generally do not need therapy for their lupus nephritis.

1	The subject of lupus nephritis is presented under acute nephritic syndromes because of the aggressive and important proliferative lesions seen in class III–V renal disease. Class III describes focal lesions Note: Revised in 2004 by the International Society of Nephrology-Renal Pathology Society Study Group.

1	with proliferation or scarring, often involving only a segment of the glomerulus (see Fig. 62e-12). Class III lesions have the most varied course. Hypertension, an active urinary sediment, and proteinuria are common with nephrotic-range proteinuria in 25–33% of patients. Elevated serum creatinine is present in 25% of patients. Patients with mild proliferation involving a small percentage of glomeruli respond well to therapy with steroids alone, and fewer than 5% progress to renal failure over 5 years. Patients with more severe proliferation involving a greater percentage of glomeruli have a far worse prognosis and lower remission rates. Treatment of those patients is the same as that for class IV lesions. Many nephrologists believe that class III lesions are simply an early presentation of class IV disease. Others believe severe class III disease is a discrete lesion requiring aggressive therapy. Class IV describes global, diffuse proliferative lesions involving the vast majority of

1	of class IV disease. Others believe severe class III disease is a discrete lesion requiring aggressive therapy. Class IV describes global, diffuse proliferative lesions involving the vast majority of glomeruli. Patients with class IV lesions commonly have high anti-DNA antibody titers, low serum complement, hematuria, red blood cell casts, proteinuria, hypertension, and decreased renal function; 50% of patients have nephrotic-range proteinuria. Patients with crescents on biopsy often have a rapidly progressive decline in renal function (see Fig. 62e-12). Without treatment, this aggressive lesion has the worst renal prognosis. However, if a remission—defined as a return to near-normal renal function and proteinuria ≤330 mg/dL per day—is achieved with treatment, renal outcomes are excellent. Current evidence suggests that inducing a remission with administration of high-dose steroids and either cyclophosphamide or mycophenolate mofetil for 2–6 months, followed by maintenance therapy

1	Current evidence suggests that inducing a remission with administration of high-dose steroids and either cyclophosphamide or mycophenolate mofetil for 2–6 months, followed by maintenance therapy with lower doses of steroids and mycophenolate mofetil or azathioprine, best balances the likelihood of successful remission with the side effects of therapy. There is no consensus on use of high-dose intravenous methylprednisolone versus oral prednisone, monthly intravenous cyclophosphamide versus daily oral cyclophosphamide, or other immunosuppressants such as cyclosporine, tacrolimus, rituximab, or belimumab. Nephrologists tend to avoid prolonged use of cyclophosphamide in patients of childbearing age without first banking eggs or sperm.

1	The class V lesion describes subepithelial immune deposits producing a membranous pattern; a subcategory of class V lesions is associated with proliferative lesions and is sometimes called mixed membranous and proliferative disease (see Fig. 62e-11); this category of injury is treated like class IV glomerulonephritis. Sixty percent of patients present with nephrotic syndrome or lesser amounts of proteinuria. Patients with lupus nephritis class V, like patients with idiopathic membranous nephropathy, are predisposed to renal-vein thrombosis and other thrombotic complications. A minority of patients with class V will present with hypertension and renal dysfunction. There are conflicting data on the clinical course, prognosis, and appropriate therapy for patients with class V disease, which may reflect the heterogeneity of this group of patients. Patients with severe nephrotic syndrome, elevated serum creatinine, and a progressive course will probably benefit from therapy with steroids

1	may reflect the heterogeneity of this group of patients. Patients with severe nephrotic syndrome, elevated serum creatinine, and a progressive course will probably benefit from therapy with steroids in combination with other immunosuppressive agents. Therapy with inhibitors of the renin-angiotensin system also may attenuate the proteinuria. Antiphospholipid antibodies present in lupus may result in glomerular microthromboses and complicate the course in up to 20% of lupus nephritis patients. The renal prognosis is worse even with anticoagulant therapy.

1	Patients with any of the above lesions also can transform to another lesion; hence patients often require reevaluation, including repeat renal biopsy. Lupus patients with class VI lesions have greater than 90% sclerotic glomeruli and end-stage renal disease with interstitial fibrosis. As a group, approximately 20% of patients with lupus nephritis will reach end-stage disease, requiring dialysis or transplantation. Systemic lupus tends to become quiescent once there is renal failure, perhaps due to the immunosuppressant effects of uremia. However, patients with lupus nephritis have a markedly increased mortality compared with the general population. Renal transplantation in renal failure from lupus, usually performed after approximately 6 months of inactive disease, results in allograft survival rates comparable to patients transplanted for other reasons.

1	Patients who develop autoantibodies directed against glomerular basement antigens frequently develop a glomerulonephritis termed antiglomerular basement membrane (anti-GBM) disease. When they present with lung hemorrhage and glomerulonephritis, they have a pulmonary-renal syndrome called Goodpasture’s syndrome. The target epitopes for this autoimmune disease lie in the quaternary structure of α3 NC1 domain of collagen IV. Indeed, anti-GBM disease may be considered an autoimmune “conformeropathy” that involves the perturbation of quaternary structure of the α 345NC1 hexamer. MHC-restricted T cells initiate the autoantibody response because humans are not tolerant to the epitopes created by this quaternary structure. The epitopes are normally sequestered in the collagen IV hexamer and can be exposed by infection, smoking, oxidants, or solvents. Goodpasture’s syndrome appears in two age groups: in young men in their late twenties and in men and women in their sixties and seventies.

1	can be exposed by infection, smoking, oxidants, or solvents. Goodpasture’s syndrome appears in two age groups: in young men in their late twenties and in men and women in their sixties and seventies. Disease in the younger age group is usually explosive, with hemoptysis, a sudden fall in hemoglobin, fever, dyspnea, and hematuria. Hemoptysis is largely confined to smokers, and those who present with lung hemorrhage as a group do better than older populations who have prolonged, asymptomatic renal injury; presentation with oliguria is often associated with a particularly bad outcome. The performance of an urgent kidney biopsy is important in suspected cases of Goodpasture’s syndrome to confirm the diagnosis and assess prognosis. Renal biopsies typically show focal or segmental necrosis that later, with aggressive destruction of the capillaries by cellular proliferation, leads to crescent formation in Bowman’s space (see Fig. 62e-14). As these lesions progress, there is concomitant

1	that later, with aggressive destruction of the capillaries by cellular proliferation, leads to crescent formation in Bowman’s space (see Fig. 62e-14). As these lesions progress, there is concomitant interstitial nephritis with fibrosis and tubular atrophy.

1	The presence of anti-GBM antibodies and complement is recognized on biopsy by linear immunofluorescent staining for IgG (rarely IgA). In testing serum for anti-GBM antibodies, it is particularly important that the α3 NC1 domain of collagen IV alone be used as the target. This is because nonnephritic antibodies against the α1 NC1 domain are seen in paraneoplastic syndromes and cannot be discerned from assays that use whole basement membrane fragments as the binding target. Between 10 and 15% of sera from patients with Goodpasture’s syndrome also contain ANCA antibodies against myeloperoxidase. This subset of patients has a vasculitis-associated variant, which has a surprisingly good prognosis with treatment. Prognosis at presentation is worse if there are >50% crescents on renal biopsy with advanced fibrosis, if serum creatinine is >5–6 mg/dL, if oliguria is present, or if there is a need for acute dialysis. Although frequently attempted, most of these latter patients will not respond

1	advanced fibrosis, if serum creatinine is >5–6 mg/dL, if oliguria is present, or if there is a need for acute dialysis. Although frequently attempted, most of these latter patients will not respond to plasmapheresis and steroids. Patients with advanced renal failure who present with hemoptysis should still be treated for their lung hemorrhage, as it responds to plasmapheresis and can be lifesaving. Treated patients with less severe 1839 disease typically respond to 8–10 treatments of plasmapheresis accompanied by oral prednisone and cyclophosphamide in the first 2 weeks. Kidney transplantation is possible, but because there is risk of recurrence, experience suggests that patients should wait for 6 months and until serum antibodies are undetectable.

1	Berger first described the glomerulonephritis now termed IgA nephropathy. It is classically characterized by episodic hematuria asso ciated with the deposition of IgA in the mesangium. IgA nephropathy is one of the most common forms of glomerulonephritis worldwide. There is a male preponderance, a peak incidence in the second and third decades of life, and rare familial clustering. There are geographic differences in the prevalence of IgA nephropathy, with 30% prevalence along the Asian and Pacific Rim and 20% in southern Europe, compared to a much lower prevalence in northern Europe and North America. It was initially hypothesized that variation in detection, in part, accounted for regional differences. With clinical care in nephrology becoming more uniform, this variation in prevalence more likely reflects true differences among racial and ethnic groups.

1	IgA nephropathy is predominantly a sporadic disease but susceptibility to it has been shown uncommonly to have a genetic component depending on geography and the existence of “founder effects.” Familial forms of IgA nephropathy are more common in northern Italy and eastern Kentucky. No single causal gene has been identified. Clinical and laboratory evidence suggests close similarities between Henoch-Schönlein purpura and IgA nephropathy. Henoch-Schönlein purpura is distinguished clinically from IgA nephropathy by prominent systemic symptoms, a younger age (<20 years old), preceding infection, and abdominal complaints. Deposits of IgA are also found in the glomerular mesangium in a variety of systemic diseases, including chronic liver disease, Crohn’s disease, gastrointestinal adenocarcinoma, chronic bronchiectasis, idiopathic interstitial pneumonia, dermatitis herpetiformis, mycosis fungoides, leprosy, ankylosing spondylitis, relapsing polychondritis, and Sjögren’s syndrome. IgA

1	chronic bronchiectasis, idiopathic interstitial pneumonia, dermatitis herpetiformis, mycosis fungoides, leprosy, ankylosing spondylitis, relapsing polychondritis, and Sjögren’s syndrome. IgA deposition in these entities is not usually associated with clinically significant glomerular inflammation or renal dysfunction and thus is not called IgA nephropathy.

1	IgA nephropathy is an immune complex–mediated glomerulonephritis defined by the presence of diffuse mesangial IgA deposits often associated with mesangial hypercellularity. (See Glomerular Schematic 2.) IgM, IgG, C3, or immunoglobulin light chains may be codistributed with IgA. IgA deposited in the mesangium is typically polymeric and of the IgA1 subclass, the pathogenic significance of which is not clear. Abnormalities have been described in IgA production by plasma cells, particularly secretory IgA; in IgA clearance, predominately by the liver; in mesangial IgA clearance and receptors for IgA; and in growth factor and cytokine-mediated events. Currently, however, abnormalities in the O -glycosylation of the hinge region of IgA seem to best account for the pathogenesis of sporadic IgA nephropathy. Despite the presence of elevated serum IgA levels in 20–50% of patients, IgA deposition in skin biopsies in 15–55% of patients, or elevated levels of secretory IgA and IgA-fibronectin

1	nephropathy. Despite the presence of elevated serum IgA levels in 20–50% of patients, IgA deposition in skin biopsies in 15–55% of patients, or elevated levels of secretory IgA and IgA-fibronectin complexes, a renal biopsy is necessary to confirm the diagnosis. Although the immunofluorescent pattern of IgA on renal biopsy defines IgA nephropathy in the proper clinical context, a variety of histologic lesions may be seen on light microscopy (see Fig. 62e-8), including DPGN; segmental sclerosis; and, rarely, segmental necrosis with cellular crescent formation, which typically presents as RPGN.

1	The two most common presentations of IgA nephropathy are recurrent episodes of macroscopic hematuria during or immediately following an upper respiratory infection often accompanied by proteinuria or persistent asymptomatic microscopic hematuria. Nephrotic syndrome is uncommon. Proteinuria can also first appear late in the course of the disease. Rarely patients present with acute renal failure and a rapidly progressive clinical picture. IgA nephropathy is a benign disease for the majority of patients, and 5–30% of patients may go into a complete remission, with others having hematuria but well preserved renal function. In the minority of patients who have progressive disease, progression is slow, with renal failure seen in only 25–30% of patients with IgA nephropathy over 20–25 years. This risk varies considerably among populations. Cumulatively, risk factors for the loss of renal function identified thus far account for less than 50% of the variation in observed outcome but include

1	risk varies considerably among populations. Cumulatively, risk factors for the loss of renal function identified thus far account for less than 50% of the variation in observed outcome but include the presence of hypertension or proteinuria, the absence of episodes of macroscopic hematuria, male sex, older age of onset, and extensive glomerulosclerosis or interstitial fibrosis on renal biopsy. Several analyses in large populations of patients found persistent proteinuria for 6 months or longer to have the greatest predictive power for adverse renal outcomes.

1	There is no agreement on optimal treatment. Both large studies that include patients with multiple glomerular diseases and small studies of patients with IgA nephropathy support the use of angiotensinconverting enzyme (ACE) inhibitors in patients with proteinuria or declining renal function. Tonsillectomy, steroid therapy, and fish oil have all been suggested in small studies to benefit select patients with IgA nephropathy. When presenting as RPGN, patients typically receive steroids, cytotoxic agents, and plasmapheresis.

1	A group of patients with small-vessel vasculitis (arterioles, capillaries, and venules; rarely small arteries) and glomerulonephritis have serum ANCA; the antibodies are of two types, anti-proteinase 3 (PR3) or anti-myeloperoxidase (MPO) (Chap. 385); Lamp-2 antibodies have also been reported experimentally as potentially pathogenic. ANCA are produced with the help of T cells and activate leukocytes and monocytes, which together damage the walls of small vessels. Endothelial injury also attracts more leukocytes and extends the inflammation. Granulomatosis with polyangiitis, microscopic polyangiitis, and Churg-Strauss syndrome belong to this group because they are ANCA-positive and have a pauci-immune glomerulonephritis with few immune complexes in small vessels and glomerular capillaries. Patients with any of these three diseases can have any combination of the above serum antibodies, but anti-PR3 antibodies are more common in granulomatosis with polyangiitis and anti-MPO antibodies

1	Patients with any of these three diseases can have any combination of the above serum antibodies, but anti-PR3 antibodies are more common in granulomatosis with polyangiitis and anti-MPO antibodies are more common in microscopic polyangiitis or Churg-Strauss. Although each of these diseases has some unique clinical features, most features do not predict relapse or progression, and as a group, they are generally treated in the same way. Since mortality is high without treatment, virtually all patients receive urgent treatment. Induction therapy usually includes some combination of plasmapheresis, methylprednisolone, and cyclophosphamide. Monthly “pulse” IV cyclophosphamide to induce remission of ANCA-associated vasculitis is as effective as daily oral cyclophosphamide but may be associated with increased relapses. Steroids are tapered soon after acute inflammation subsides, and patients are maintained on cyclophosphamide or azathioprine for up to a year to minimize the risk of relapse.

1	with increased relapses. Steroids are tapered soon after acute inflammation subsides, and patients are maintained on cyclophosphamide or azathioprine for up to a year to minimize the risk of relapse. Benefit with using mycophenolate mofetil or rituximab has not been proven.

1	Granulomatosis with Polyangiitis Patients with this disease classically present with fever, purulent rhinorrhea, nasal ulcers, sinus pain, polyarthralgias/arthritis, cough, hemoptysis, shortness of breath, microscopic hematuria, and 0.5–1 g/24 h of proteinuria; occasionally there may be cutaneous purpura and mononeuritis multiplex. Presentation without renal involvement is termed limited granulomatosis with polyangiitis, although some of these patients will show signs of renal injury later. Chest x-ray often reveals nodules and persistent infiltrates, sometimes with cavities. Biopsy of involved tissue will show a small-vessel vasculitis and adjacent noncaseating granulomas. Renal biopsies during active disease demonstrate segmental necrotizing glomerulonephritis without immune deposits (see Fig. 62e-13). The disease is more common in patients exposed to silica dust and those with α1-antitrypsin deficiency, which is an inhibitor of PR3. Relapse after achieving remission is common and

1	Fig. 62e-13). The disease is more common in patients exposed to silica dust and those with α1-antitrypsin deficiency, which is an inhibitor of PR3. Relapse after achieving remission is common and is more common in patients with granulomatosis with polyangiitis than the other ANCA-associated vasculitis, necessitating diligent follow-up care. Although associated with an unacceptable high mortality rate without treatment, the greatest threat to patients, especially elderly patients in the first year of therapy, is from adverse events, which are often secondary to treatment, rather than active vasculitis.

1	Microscopic Polyangiitis Clinically, these patients look somewhat similar to those with granulomatosis with polyangiitis, except they rarely have significant lung disease or destructive sinusitis. The distinction is made on biopsy, where the vasculitis in microscopic polyangiitis is without granulomas. Some patients will also have injury limited to the capillaries and venules.

1	Churg-Strauss Syndrome When small-vessel vasculitis is associated with peripheral eosinophilia, cutaneous purpura, mononeuritis, asthma, and allergic rhinitis, a diagnosis of Churg-Strauss syndrome is considered. Hypergammaglobulinemia, elevated levels of serum IgE, or the presence of rheumatoid factor sometimes accompanies the allergic state. Lung inflammation, including fleeting cough and pulmonary infiltrates, often precedes the systemic manifestations of disease by years; lung manifestations are rarely absent. A third of patients may have exudative pleural effusions associated with eosinophils. Small-vessel vasculitis and focal segmental necrotizing glomerulonephritis can be seen on renal biopsy, usually absent eosinophils or granulomas. The cause of Churg-Strauss syndrome is autoimmune, but the inciting factors are unknown.

1	MPGN is sometimes called mesangiocapillary glomerulonephritis or lobar glomerulonephritis. It is an immune-mediated glomerulonephritis characterized by thickening of the GBM with mesangioproliferative changes; 70% of patients have hypocomplementemia. MPGN is rare in African Americans, and idiopathic disease usually presents in childhood or young adulthood. MPGN is subdivided pathologically into type I, type II, and type III disease. Type I MPGN is commonly associated with persistent hepatitis C infections, autoimmune diseases like lupus or cryoglobulinemia, or neoplastic diseases (Table 338-4). Types II and III MPGN are usually idiopathic, except in patients with complement factor H deficiency, in the presence of C3 nephritic factor and/or in partial lipodystrophy producing type II disease, or complement receptor deficiency in type III disease. MPGN has been proposed to be reclassified into immunoglobulin-mediated disease (driven by the classical complement pathway) and

1	type II disease, or complement receptor deficiency in type III disease. MPGN has been proposed to be reclassified into immunoglobulin-mediated disease (driven by the classical complement pathway) and non–immunoglobulin-mediated disease (driven by the alternative complement pathway).

1	Idiopathic Subacute bacterial endocarditis Systemic lupus erythematosus Hepatitis C ± cryoglobulinemia Mixed cryoglobulinemia Hepatitis B Cancer: Lung, breast, and ovary (germinal)

1	Type I MPGN, the most proliferative of the three types, shows mesangial proliferation with lobular segmentation on renal biopsy and mesangial interposition between the capillary basement membrane and endothelial cells, producing a double contour sometimes called tram-tracking (see Fig. 62e-9). (See Glomerular Schematic 3.) Subendothelial deposits with low serum levels of C3 are typical, although 50% of patients have normal levels of C3 and occasional intramesangial deposits. Low serum C3 and a dense thickening of the GBM containing ribbons of dense deposits and C3 characterize type II MPGN, sometimes called dense deposit disease (see Fig. 62e-10). Classically, the glomerular tuft has a lobular appearance; intramesangial deposits are rarely present and subendothelial deposits are generally absent. Proliferation in type III MPGN is less common than the other two types and is often focal; mesangial interposition is rare, and subepithelial deposits can occur along widened segments of the

1	absent. Proliferation in type III MPGN is less common than the other two types and is often focal; mesangial interposition is rare, and subepithelial deposits can occur along widened segments of the GBM that appear laminated and disrupted.

1	Type I MPGN is secondary to glomerular deposition of circulating immune complexes or their in situ formation. Types II and III MPGN may be related to “nephritic factors,” which are autoantibodies that stabilize C3 convertase and allow it to activate serum C3. MPGN can also result from acquired or genetic abnormalities in the alternative complement pathway. Patients with MPGN present with proteinuria, hematuria, and pyuria (30%); systemic symptoms of fatigue and malaise that are most common in children with type I disease; or an acute nephritic picture with RPGN and a speedy deterioration in renal function in up to 25% of patients. Low serum C3 levels are common. Fifty percent of patients with MPGN develop end-stage disease 10 years after diagnosis, and 90% have renal insufficiency after 20 years. Nephrotic 1841 syndrome, hypertension, and renal insufficiency all predict poor outcome. In the presence of proteinuria, treatment with inhibitors of the renin-angiotensin system is prudent.

1	years. Nephrotic 1841 syndrome, hypertension, and renal insufficiency all predict poor outcome. In the presence of proteinuria, treatment with inhibitors of the renin-angiotensin system is prudent. Evidence for treatment with dipyridamole, Coumadin (warfarin), or cyclophosphamide is not strongly established. There is some evidence supporting the efficacy of treatment of primary MPGN with steroids, particularly in children, as well as reports of efficacy with plasma exchange and other immunosuppressive drugs. If defects in the complement pathway are found, treatment with eculizumab is of hypothetical but unproven benefit. In secondary MPGN, treating the associated infection, autoimmune disease, or neoplasms is of demonstrated benefit. In particular, pegylated interferon and ribavirin are useful in reducing viral load. Although all primary renal diseases can recur over time in transplanted renal allografts, patients with MPGN are well known to be at risk for not only a histologic

1	are useful in reducing viral load. Although all primary renal diseases can recur over time in transplanted renal allografts, patients with MPGN are well known to be at risk for not only a histologic recurrence but also a clinically significant recurrence with loss of graft function.

1	Mesangioproliferative glomerulonephritis is characterized by expansion of the mesangium, sometimes associated with mesangial hypercellularity; thin, single contoured capillary walls; and mesangial immune deposits. Clinically, it can present with varying degrees of proteinuria and, commonly, hematuria. Mesangioproliferative disease may be seen in IgA nephropathy, Plasmodium falciparum malaria, resolving postinfectious glomerulonephritis, and class II nephritis from lupus, all of which can have a similar histologic appearance. With these secondary entities excluded, the diagnosis of primary mesangioproliferative glomerulonephritis is made in less than 15% of renal biopsies. As an immune-mediated renal lesion with deposits of IgM, C1q, and C3, the clinical course is variable. Patients with isolated hematuria may have a very benign course, and those with heavy proteinuria occasionally progress to renal failure. There is little agreement on treatment, but some clinical reports suggest

1	isolated hematuria may have a very benign course, and those with heavy proteinuria occasionally progress to renal failure. There is little agreement on treatment, but some clinical reports suggest benefit from use of inhibitors of the renin-angiotensin system, steroid therapy, and even cytotoxic agents.

1	Nephrotic syndrome classically presents with heavy proteinuria, minimal hematuria, hypoalbuminemia, hypercholesterolemia, edema, and hypertension. If left undiagnosed or untreated, some of these syndromes will progressively damage enough glomeruli to cause a fall in GFR, producing renal failure. Multiple studies have noted that the higher the 24-h urine protein excretion, the more rapid is the decline in GFR.

1	Therapies for various causes of nephrotic syndrome are noted under individual disease headings below. In general, all patients with hypercholesterolemia secondary to nephrotic syndrome should be treated with lipid-lowering agents because they are at increased risk for cardiovascular disease. Edema secondary to salt and water retention can be controlled with the judicious use of diuretics, avoiding intravascular volume depletion. Venous complications secondary to the hypercoagulable state associated with nephrotic syndrome can be treated with anticoagulants. The losses of various serum binding proteins, such as thyroid-binding globulin, lead to alterations in functional tests. Lastly, proteinuria itself is hypothesized to be nephrotoxic, and treatment of proteinuria with inhibitors of the renin-angiotensin system can lower urinary protein excretion.

1	Minimal change disease (MCD), sometimes known as nil lesion, causes 70–90% of nephrotic syndrome in childhood but only 10–15% of nephrotic syndrome in adults. Minimal change disease usually presents as a primary renal disease but can be associated with several other conditions, including Hodgkin’s disease, allergies, or use of nonsteroidal anti-inflammatory agents; significant interstitial nephritis often accompanies cases associated with nonsteroidal drug use. Minimal change disease on renal biopsy shows no obvious glomerular lesion by light microscopy and is negative for deposits by immunofluorescent microscopy, or occasionally shows small amounts of IgM in the mesangium (see Fig. 62e-1). (See Glomerular Schematic 4.) Electron microscopy, however, consistently demonstrates an effacement of the foot process supporting the epithelial podocytes with weakening of slit-pore membranes. The pathophysiology of this lesion is uncertain. Most agree there is a circulating cytokine, perhaps

1	of the foot process supporting the epithelial podocytes with weakening of slit-pore membranes. The pathophysiology of this lesion is uncertain. Most agree there is a circulating cytokine, perhaps related to a T cell response that alters capillary charge and podocyte integrity. The evidence for cytokine-related immune injury is circumstantial and is suggested by the presence of preceding allergies, altered cell-mediated immunity during viral infections, and the high frequency of remissions with steroids.

1	Minimal change disease presents clinically with the abrupt onset of edema and nephrotic syndrome accompanied by acellular urinary sediment. Average urine protein excretion reported in 24 h is 10 g with severe hypoalbuminemia. Less common clinical features include hypertension (30% in children, 50% in adults), microscopic hematuria (20% in children, 33% in adults), atopy or allergic symptoms (40% in children, 30% in adults), and decreased renal function (<5% in children, 30% in adults). The appearance of acute renal failure in adults is often seen more commonly in patients with low serum albumin and intrarenal edema (nephrosarca) that is responsive to intravenous albumin and diuretics. This presentation must be distinguished from acute renal failure secondary to hypovolemia. Acute tubular necrosis and interstitial inflammation are also reported. In children, the abnormal urine principally contains albumin with minimal amounts of highermolecular-weight proteins, and is sometimes called

1	necrosis and interstitial inflammation are also reported. In children, the abnormal urine principally contains albumin with minimal amounts of highermolecular-weight proteins, and is sometimes called selective proteinuria. Although up to 30% of children have a spontaneous remission, all children today are treated with steroids; only children who are nonresponders are biopsied in this setting. Primary responders are patients who have a complete remission (<0.2 mg/24 h of proteinuria) after a single course of prednisone; steroid-dependent patients relapse as their steroid dose is tapered. Frequent relapsers have two or more relapses in the 6 months following taper, and steroid-resistant patients fail to respond to steroid therapy. Adults are not considered steroid-resistant until after 4 months of therapy. Ninety to 95% of children will develop a complete remission after 8 weeks of steroid therapy, and 80–85% of adults will achieve complete remission, but only after a longer course of

1	of therapy. Ninety to 95% of children will develop a complete remission after 8 weeks of steroid therapy, and 80–85% of adults will achieve complete remission, but only after a longer course of 20–24 weeks. Patients with steroid resistance may have FSGS on repeat biopsy. Some hypothesize that if the first renal biopsy does not have a sample of deeper corticomedullary glomeruli, then the correct diagnosis of FSGS may be missed.

1	Relapses occur in 70–75% of children after the first remission, and early relapse predicts multiple subsequent relapses, as do high levels of basal proteinuria. The frequency of relapses decreases after puberty. There is an increased risk of relapse following the rapid tapering of steroids in all groups. Relapses are less common in adults but are more resistant to subsequent therapy. Prednisone is first-line therapy, either given daily or on alternate days. Other immunosuppressive drugs, such as cyclophosphamide, chlorambucil, and mycophenolate mofetil, are saved for frequent relapsers, steroid-dependent patients, or steroid-resistant patients. Cyclosporine can induce remission, but relapse is also common when cyclosporine is withdrawn. The longterm prognosis in adults is less favorable when acute renal failure or steroid resistance occurs.

1	Focal segmental glomerulosclerosis (FSGS) refers to a pattern of renal injury characterized by segmental glomerular scars that involve some but not all glomeruli; the clinical findings of FSGS largely manifest as proteinuria. When the secondary causes of FSGS are eliminated (Table 338-5), the remaining patients are considered to have primary FSGS. The incidence of this disease is increasing, and it now represents up to one-third of cases of nephrotic syndrome in adults and one-half of cases of nephrotic syndrome in African Americans, in whom it is seen more commonly. The pathogenesis of FSGS is probably multifactorial. Possible mechanisms include a T cell–mediated circulating permeability factor, increased soluble urokinase receptor levels, TGF-β–mediated cellular proliferation and matrix synthesis, and podocyte abnormalities associated with genetic mutations. Risk polymorphisms at the APOL1 locus encoding apolipoprotein L1 expressed in podocytes substantially explain the increased

1	synthesis, and podocyte abnormalities associated with genetic mutations. Risk polymorphisms at the APOL1 locus encoding apolipoprotein L1 expressed in podocytes substantially explain the increased burden of FSGS among African Americans with or without HIV-associated disease.

1	The pathologic changes of FSGS are most prominent in glomeruli located at the corticomedullary junction (see Fig. 62e-2), so if the renal biopsy specimen is from superficial tissue, the lesions can be missed, which sometimes leads to a misdiagnosis of MCD. In addition to focal and segmental scarring, other variants have been described, including cellular lesions with endocapillary hypercellularity and heavy proteinuria; collapsing glomerulopath y (see Fig. 62e-3) with segmental or global glomerular collapse and a rapid decline in renal function; a hilar stalk lesion (see Fig. 62e-4); or the glomerular tip lesion (see Fig. 62e-5), which may have a better prognosis. (See Glomerular Schematic 5.)

1	FSGS can present with hematuria, hypertension, any level of proteinuria, or renal insufficiency. Nephrotic-range proteinuria, African-American race, and renal insufficiency are associated with a poor outcome, with 50% of patients reaching renal failure in 6–8 years. FSGS rarely remits spontaneously, but treatment-induced remission of proteinuria significantly improves prognosis. Treatment of patients with primary FSGS should include inhibitors of the renin-angiotensin system. Based on retrospective studies, patients with nephrotic-range proteinuria can be treated with steroids but respond far less often and Viruses: HIV/hepatitis B/parvovirus

1	Viruses: HIV/hepatitis B/parvovirus Drugs: Heroin/analgesics/pamidronate after a longer course of therapy than patients with MCD. Proteinuria remits in only 20–45% of patients receiving a course of steroids over 6–9 months. Limited evidence suggests the use of cyclosporine in steroid-responsive patients helps ensure remissions. Relapse frequently occurs after cessation of cyclosporine therapy, and cyclosporine itself can lead to a deterioration of renal function due to its nephrotoxic effects. A role for other agents that suppress the immune system has not been established. Primary FSGS recurs in 25–40% of patients given allografts at end-stage disease, leading to graft loss in half of those cases. The treatment of secondary FSGS typically involves treating the underlying cause and controlling proteinuria. There is no role for steroids or other immunosuppressive agents in secondary FSGS.

1	Membranous glomerulonephritis (MGN), or membranous nephropathy as it is sometimes called, accounts for approximately 30% of cases of nephrotic syndrome in adults, with a peak incidence between the ages of 30 and 50 years and a male to female ratio of 2:1. It is rare in childhood and the most common cause of nephrotic syndrome in the elderly. In 25–30% of cases, MGN is associated with a malignancy (solid tumors of the breast, lung, colon), infection (hepatitis B, malaria, schistosomiasis), or rheumatologic disorders like lupus or rarely rheumatoid arthritis (Table 338-6).

1	Uniform thickening of the basement membrane along the peripheral capillary loops is seen by light microscopy on renal biopsy (see Fig. 62e-7); this thickening needs to be distinguished from that seen in diabetes and amyloidosis. (See Glomerular Schematic 6.) Immunofluorescence demonstrates diffuse granular deposits of IgG and C3, and electron microscopy typically reveals electron-dense subepithelial deposits. While different stages (I–V) of progressive membranous lesions have been described, some published analyses indicate the degree of tubular atrophy or interstitial fibrosis is more predictive of progression than is the stage of glomerular disease. The presence of subendothelial deposits or the presence of tubuloreticular inclusions strongly points to a diagnosis of membranous lupus nephritis, which may precede the extrarenal manifestations of lupus. Work in Heyman nephritis, an animal model of MGN, suggests that glomerular lesions result from in situ formation of immune complexes

1	which may precede the extrarenal manifestations of lupus. Work in Heyman nephritis, an animal model of MGN, suggests that glomerular lesions result from in situ formation of immune complexes with megalin receptor–associated protein as the putative antigen. This antigen is not found in human podocytes. Human antibodies have been described against neutral endopeptidase expressed by podocytes in infants whose mothers lack this protein. In most adults, autoantibodies against the M-type phospholipase A2 receptor (PLA2R) circulate and bind to a conformational epitope present in the receptor on human podocytes, producing in situ deposits characteristic of idiopathic membranous nephropathy. Other renal diseases and secondary membranous nephropathy do not appear to involve such

1	Infection: Hepatitis B and C, syphilis, malaria, schistosomiasis, leprosy, Cancer: Breast, colon, lung, stomach, kidney, esophagus, neuroblastoma Drugs: Gold, mercury, penicillamine, nonsteroidal anti-inflammatory agents, probenecid Autoimmune diseases: Systemic lupus erythematosus, rheumatoid arthritis, primary biliary cirrhosis, dermatitis herpetiformis, bullous pemphigoid, myasthenia gravis, Sjögren’s syndrome, Hashimoto’s thyroiditis

1	Other systemic diseases: Fanconi’s syndrome, sickle cell anemia, diabetes, Crohn’s disease, sarcoidosis, Guillain-Barré syndrome, Weber-Christian disease, angiofollicular lymph node hyperplasia autoantibodies and levels of these autoantibodies have correlated with the severity of MGN. Eighty percent of patients with MGN present with nephrotic syndrome and nonselective proteinuria. Microscopic hematuria is seen but less commonly than in IgA nephropathy or FSGS. Spontaneous remissions occur in 20–33% of patients and often occur late in the course after years of nephrotic syndrome, which make treatment decisions difficult. One-third of patients continue to have relapsing nephrotic syndrome but maintain normal renal function, and approximately another third of patients develop renal failure or die from the complications of nephrotic syndrome. Male gender, older age, hypertension, and the persistence of proteinuria are associated with worse prognosis. Although thrombotic complications are

1	die from the complications of nephrotic syndrome. Male gender, older age, hypertension, and the persistence of proteinuria are associated with worse prognosis. Although thrombotic complications are a feature of all nephrotic syndromes, MGN has the highest reported incidences of renal vein thrombosis, pulmonary embolism, and deep vein thrombosis. Prophylactic anticoagulation is controversial but has been recommended for patients with severe or prolonged proteinuria in the absence of risk factors for bleeding.

1	In addition to the treatment of edema, dyslipidemia, and hypertension, inhibition of the renin-angiotensin system is recommended. Therapy with immunosuppressive drugs is also recommended for patients with primary MGN and persistent proteinuria (>3.0 g/24 h). The choice of immunosuppressive drugs for therapy is controversial, but current recommendations are to treat with steroids and cyclophosphamide, chlorambucil, mycophenolate mofetil, or cyclosporine. In patients who relapse or fail to respond to this therapy, the use of rituximab, an anti-CD20 antibody directed at B cells, or synthetic adrenocorticotropic hormone may be considered.

1	Diabetic nephropathy is the single most common cause of chronic renal failure in the United States, accounting for 45% of patients receiving renal replacement therapy, and is a rapidly growing problem worldwide. The dramatic increase in the number of patients with diabetic nephropathy reflects the epidemic increase in obesity, metabolic syndrome, and type 2 diabetes mellitus. Approximately 40% of patients with types 1 or 2 diabetes develop nephropathy, but due to the higher prevalence of type 2 diabetes (90%) compared to type 1 (10%), the majority of patients with diabetic nephropathy have type 2 disease. Renal lesions are more common in African-American, Native American, Polynesian, and Maori populations. Risk factors for the development of diabetic nephropathy include hyperglycemia, hypertension, dyslipidemia, smoking, a family history of diabetic nephropathy, and gene polymorphisms affecting the activity of the renin-angiotensin-aldosterone axis.

1	Within 1–2 years after the onset of clinical diabetes, morphologic changes appear in the kidney. Thickening of the GBM is a sensitive indicator for the presence of diabetes but correlates poorly with the presence or absence of clinically significant nephropathy. The composition of the GBM is altered notably with a loss of heparan sulfate moieties that form the negatively charged filtration barrier. This change results in increased filtration of serum proteins into the urine, predominately negatively charged albumin. The expansion of the mesangium due to the accumulation of extracellular matrix correlates with the clinical manifestations of diabetic nephropathy (see stages in Fig. 62e-20). This expansion in mesangial matrix is associated with the development of mesangial sclerosis. Some patients also develop eosinophilic, PAS+ nodules called nodular glomerulosclerosis or Kimmelstiel-Wilson nodules. Immunofluorescence microscopy often reveals the nonspecific deposition of IgG (at times

1	also develop eosinophilic, PAS+ nodules called nodular glomerulosclerosis or Kimmelstiel-Wilson nodules. Immunofluorescence microscopy often reveals the nonspecific deposition of IgG (at times in a linear pattern) or complement staining without immune deposits on electron microscopy. Prominent vascular changes are frequently seen with hyaline and hypertensive arteriosclerosis. This is associated with varying degrees of chronic glomerulosclerosis and tubulointerstitial changes. Renal biopsies from patients with types 1 and 2 diabetes are largely indistinguishable.

1	These pathologic changes are the result of a number of postulated factors. Multiple lines of evidence support an important role for increases in glomerular capillary pressure (intraglomerular hypertension) in alterations in renal structure and function. Direct effects of hyperglycemia on the actin cytoskeleton of renal mesangial and vascular smooth-muscle cells as well as diabetes-associated changes in circulating factors such as atrial natriuretic factor, angiotensin II, and insulin-like growth factor (IGF) may account for this. Sustained glomerular hypertension increases matrix production, alterations in the GBM with disruption in the filtration barrier (and hence proteinuria), and glomerulosclerosis. A number of factors have also been identified that alter matrix production, including the accumulation of advanced glycosylation end products, circulating factors including growth hormone, IGF-I, angiotensin II, connective tissue growth factor, TGF-β, and dyslipidemia.

1	The natural history of diabetic nephropathy in patients with types 1 and 2 diabetes is similar. However, since the onset of type 1 diabetes is readily identifiable and the onset of type 2 diabetes is not, a patient newly diagnosed with type 2 diabetes may present with advanced diabetic nephropathy. At the onset of diabetes, renal hypertrophy and glomerular hyperfiltration are present. The degree of glomerular hyperfiltration correlates with the subsequent risk of clinically significant nephropathy. In the approximately 40% of patients with diabetes who develop diabetic nephropathy, the earliest manifestation is an increase in albuminuria detected by sensitive radioimmunoassay (Table 338-1). Albuminuria in the range of 30–300 mg/24 h is called microalbuminuria. Microalbuminuria appears 5–10 years after the onset of diabetes. It is currently recommended to test patients with type 1 disease for microalbuminuria 5 years after diagnosis of diabetes and yearly thereafter and, because the

1	years after the onset of diabetes. It is currently recommended to test patients with type 1 disease for microalbuminuria 5 years after diagnosis of diabetes and yearly thereafter and, because the time of onset of type 2 diabetes is often unknown, to test type 2 patients at the time of diagnosis of diabetes and yearly thereafter.

1	Patients with small increases in albuminuria increase their levels of urinary albumin excretion, typically reaching dipstick positive levels of proteinuria (>300 mg albuminuria) 5–10 years after the onset of early albuminuria. Microalbuminuria is a potent risk factor for cardiovascular events and death in patients with type 2 diabetes. Many patients with type 2 diabetes and microalbuminuria succumb to cardiovascular events before they progress to proteinuria or renal failure. Proteinuria in frank diabetic nephropathy can be variable, ranging from 500 mg to 25 g/24 h, and is often associated with nephrotic syndrome. More than 90% of patients with type 1 diabetes and nephropathy have diabetic retinopathy, so the absence of retinopathy in type 1 patients with proteinuria should prompt consideration of a diagnosis other than diabetic nephropathy; only 60% of patients with type 2 diabetes with nephropathy have diabetic retinopathy. There is a significant correlation between the presence of

1	of a diagnosis other than diabetic nephropathy; only 60% of patients with type 2 diabetes with nephropathy have diabetic retinopathy. There is a significant correlation between the presence of retinopathy and the presence of Kimmelstiel-Wilson nodules (see Fig. 62e-20). Also, characteristically, patients with advanced diabetic nephropathy have normal to enlarged kidneys, in contrast to other glomerular diseases where kidney size is usually decreased. Using the above epidemiologic and clinical data, and in the absence of other clinical or serologic data suggesting another disease, diabetic nephropathy is usually diagnosed without a renal biopsy. After the onset of proteinuria, renal function inexorably declines, with 50% of patients reaching renal failure over another 5–10 years; thus, from the earliest stages of microalbuminuria, it usually takes 10–20 years to reach end-stage renal disease. Once renal failure appears, however, survival on dialysis is shorter for patients with

1	from the earliest stages of microalbuminuria, it usually takes 10–20 years to reach end-stage renal disease. Once renal failure appears, however, survival on dialysis is shorter for patients with diabetes compared to other dialysis patients. Survival is best for patients with type 1 diabetes who receive a transplant from a living related donor.

1	Good evidence supports the benefits of blood sugar and blood pressure control as well as inhibition of the renin-angiotensin system in retarding the progression of diabetic nephropathy. In patients with type 1 diabetes, intensive control of blood sugar clearly prevents the development or progression of diabetic nephropathy. The evidence for benefit of intensive blood glucose control in patients with type 2 diabetes is less certain, with current studies reporting conflicting results.

1	Controlling systemic blood pressure decreases renal and cardiovascular adverse events in this high-risk population. The vast majority of patients with diabetic nephropathy require three or more antihypertensive drugs to achieve this goal. Drugs that inhibit the renin-angiotensin system, independent of their effects on systemic blood pressure, have been shown in numerous large clinical trials to slow the progression of diabetic nephropathy at early (microalbuminuria) and late (proteinuria with reduced glomerular filtration) stages, independent of any effect they may have on systemic blood pressure. Since angiotensin II increases efferent arteriolar resistance and, hence, glomerular capillary pressure, one key mechanism for the efficacy of ACE inhibitors or angiotensin receptor blockers (ARBs) is reducing glomerular hypertension. Patients with type 1 diabetes for 5 years who develop albuminuria or declining renal function should be treated with ACE inhibitors. Patients with type 2

1	(ARBs) is reducing glomerular hypertension. Patients with type 1 diabetes for 5 years who develop albuminuria or declining renal function should be treated with ACE inhibitors. Patients with type 2 diabetes and microalbuminuria or proteinuria may be treated with ACE inhibitors or ARBs. Evidence suggests increased risk for cardiovascular adverse events in some patients with a combination of two drugs (ACE inhibitors, ARBs, renin inhibitors, or aldosterone antagonists) that suppress several components of the reninangiotensin system.

1	Plasma cell dyscrasias producing excess light chain immunoglobulin sometimes lead to the formation of glomerular and tubular deposits that cause heavy proteinuria and renal failure; the same is true for the accumulation of serum amyloid A protein fragments seen in several inflammatory diseases. This broad group of proteinuric patients has glomerular deposition disease.

1	Light Chain Deposition Disease The biochemical characteristics of nephrotoxic light chains produced in patients with light chain malignancies often confer a specific pattern of renal injury; that of either cast nephropathy (see Fig. 62e-17), which causes renal failure but not heavy proteinuria or amyloidosis, or light chain deposition disease (see Fig. 62e-16), which produces nephrotic syndrome with renal failure. These latter patients produce kappa light chains that do not have the biochemical features necessary to form amyloid fibrils. Instead, they self-aggregate and form granular deposits along the glomerular capillary and mesangium, tubular basement membrane, and Bowman’s capsule. When predominant in glomeruli, nephrotic syndrome develops, and about 70% of patients progress to dialysis. Light-chain deposits are not fibrillar and do not stain with Congo red, but they are easily detected with anti–light chain antibody using immunofluorescence or as granular deposits on electron

1	Light-chain deposits are not fibrillar and do not stain with Congo red, but they are easily detected with anti–light chain antibody using immunofluorescence or as granular deposits on electron microscopy. A combination of the light chain rearrangement, self-aggregating properties at neutral pH, and abnormal metabolism probably contribute to the deposition. Treatment for light chain deposition disease is treatment of the primary disease and, if possible, autologous stem cell transplantation.

1	Renal Amyloidosis Most renal amyloidosis is either the result of primary fibrillar deposits of immunoglobulin light chains known as amyloid L (AL), or secondary to fibrillar deposits of serum amyloid A (AA) protein fragments (Chap. 137). Even though both occur for different reasons, their clinicopathophysiology is quite similar and will 1845 be discussed together. Amyloid infiltrates the liver, heart, peripheral nerves, carpal tunnel, upper pharynx, and kidney, producing restrictive cardiomyopathy, hepatomegaly, macroglossia, and heavy proteinuria sometimes associated with renal vein thrombosis. In systemic AL amyloidosis, also called primary amyloidosis, light chains produced in excess by clonal plasma cell dyscrasias are made into fragments by macrophages so they can self-aggregate at acid pH. A disproportionate number of these light chains (75%) are of the lambda class. About 10% of these patients have overt myeloma with lytic bone lesions and infiltration of the bone marrow with

1	acid pH. A disproportionate number of these light chains (75%) are of the lambda class. About 10% of these patients have overt myeloma with lytic bone lesions and infiltration of the bone marrow with >30% plasma cells; nephrotic syndrome is common, and about 20% of patients progress to dialysis. AA amyloidosis is sometimes called secondary amyloidosis and also presents as nephrotic syndrome. It is due to deposition of β-pleated sheets of serum amyloid A protein, an acute phase reactant whose physiologic functions include cholesterol transport, immune cell attraction, and metalloproteases activation. Forty percent of patients with AA amyloid have rheumatoid arthritis, and another 10% have ankylosing spondylitis or psoriatic arthritis; the rest derive from other lesser causes. Less common in Western countries but more common in Mediterranean regions, particularly in Sephardic and Iraqi Jews, is familial Mediterranean fever (FMF). FMF is caused by a mutation in the gene encoding pyrin,

1	in Western countries but more common in Mediterranean regions, particularly in Sephardic and Iraqi Jews, is familial Mediterranean fever (FMF). FMF is caused by a mutation in the gene encoding pyrin, whereas Muckle-Wells syndrome, a related disorder, results from a mutation in cryopyrin; both proteins are important in the apoptosis of leukocytes early in inflammation; such proteins with pyrin domains are part of a new pathway called the inflammasome. Receptor mutations in tumor necrosis factor receptor 1 (TNFR1)-associated periodic syndrome also produce chronic inflammation and secondary amyloidosis. Fragments of serum amyloid A protein increase and self-aggregate by attaching to receptors for advanced glycation end products in the extracellular environment; nephrotic syndrome is common, and about 40–60% of patients progress to dialysis. AA and AL amyloid fibrils are detectable with Congo red or in more detail with electron microscopy (see Fig. 62e-15). Currently developed serum free

1	and about 40–60% of patients progress to dialysis. AA and AL amyloid fibrils are detectable with Congo red or in more detail with electron microscopy (see Fig. 62e-15). Currently developed serum free light chain nephelometry assays are useful in the early diagnosis and follow-up of disease progression. Biopsy of involved liver or kidney is diagnostic 90% of the time when the pretest probability is high; abdominal fat pad aspirates are positive about 70% of the time, but apparently less so when looking for AA amyloid. Amyloid deposits are distributed along blood vessels and in the mesangial regions of the kidney. The treatment for primary amyloidosis, melphalan and autologous hematopoietic stem cell transplantation, can delay the course of disease in about 30% of patients. Secondary amyloidosis is also relentless unless the primary disease can be controlled. Some new drugs in development that disrupt the formation of fibrils may be available in the future.

1	Fibrillary-Immunotactoid Glomerulopathy Fibrillary-immunotactoid glomerulopathy is a rare (<1.0% of renal biopsies), morphologically defined disease characterized by glomerular accumulation of nonbranching randomly arranged fibrils. Some classify amyloid and nonamyloid fibril-associated renal diseases all as fibrillary glomerulopathies with immunotactoid glomerulopathy reserved for nonamyloid fibrillary disease not associated with a systemic illness. Others define fibrillary glomerulonephritis as a nonamyloid fibrillary disease with fibrils 12–24 nm and immunotactoid glomerulonephritis with fibrils >30 nm. In either case, fibrillar/microtubular deposits of oligoclonal or oligotypic immunoglobulins and complement appear in the mesangium and along the glomerular capillary wall. Congo red stains are negative. The cause of this “nonamyloid” glomerulopathy is mostly idiopathic; reports of immunotactoid glomerulonephritis describe an occasional association with chronic lymphocytic leukemia

1	are negative. The cause of this “nonamyloid” glomerulopathy is mostly idiopathic; reports of immunotactoid glomerulonephritis describe an occasional association with chronic lymphocytic leukemia or B cell lymphoma. Both disorders appear in adults in the fourth decade with moderate to heavy proteinuria, hematuria, and a wide variety of histologic lesions, including DPGN, MPGN, MGN, or mesangioproliferative glomerulonephritis. Nearly half of patients develop renal failure over a few years. There is no consensus on treatment of this uncommon disorder. The disease has been reported to recur following renal transplantation in a minority of cases.

1	α5.α5.α6(IV) network appears in skin, smooth muscle, and esophagus and along Bowman’s capsule in the kidney. This switch probably occurs because the α3.α4.α5(IV) network is more resistant to proteases and ensures the structural longevity of critical tissues. When basement membranes are the target of glomerular disease, they produce moderate proteinuria, some hematuria, and progressive renal failure. Autoimmune disease where antibodies are directed against the α3 NC1 domain of collagen IV produces an anti-GBM disease often associated with RPGN and/or a pulmonary-renal syndrome called Goodpasture’s syndrome. Discussion of this disease is covered earlier in “Acute Nephritic Syndromes.”

1	Classically, patients with Alport’s syndrome develop hematuria, thinning and splitting of the GBMs, mild proteinuria (<1–2 g/24 h), which appears late in the course, followed by chronic glomerulosclerosis leading to renal failure in association with sensorineural deafness. Some patients develop lenticonus of the anterior lens capsule, “dot and fleck” retinopathy, and rarely, mental retardation or leiomyomatosis. Approximately 85% of patients with Alport’s syndrome have an X-linked inheritance of mutations in the α5(IV) collagen chain on chromosome Xq22–24. Female carriers have variable penetrance depending on the type of mutation or the degree of mosaicism created by X inactivation. Fifteen percent of patients have autosomal recessive disease of the α3(IV) or α4(IV) chains on chromosome 2q35–37. Rarely, some kindred have an autosomal dominant inheritance of dominant-negative mutations in α3(IV) or α4(IV) chains.

1	Pedigrees with the X-linked syndrome are quite variable in their rate and frequency of tissue damage leading to organ failure. Seventy percent of patients have the juvenile form with nonsense or missense mutations, reading frame shifts, or large deletions and generally develop renal failure and sensorineural deafness by age 30. Patients with splice variants, exon skipping, or missense mutations of α-helical glycines generally deteriorate after the age of 30 (adult form) with mild or late deafness. Early severe deafness, lenticonus, or proteinuria suggests a poorer prognosis. Usually females from X-linked pedigrees have only microhematuria, but up to 25% of carrier females have been reported to have more severe renal manifestations. Pedigrees with the autosomal recessive form of the disease have severe early disease in both females and males with asymptomatic parents.

1	Clinical evaluation should include a careful eye examination and hearing tests. However, the absence of extrarenal symptoms does not rule out the diagnosis. Since α5(IV) collagen is expressed in the skin, some X-linked Alport’s patients can be diagnosed with a skin biopsy revealing the lack of the α5(IV) collagen chain on immunofluorescent analysis. Patients with mutations in α3(IV) or α4(IV) require a renal biopsy. Genetic testing can be used for the diagnosis of Alport’s syndrome and the demonstration of the mode of inheritance. Early in their disease, Alport’s patients typically have thin basement membranes on renal biopsy (see Fig. 62e-19), which thicken over time into multilamellations surrounding lucent areas that often contain granules of varying density—the so-called split basement membrane. In any Alport’s kidney, there are areas of thinning mixed with splitting of the GBM. Tubules drop out, glomeruli scar, and the kidney eventually succumbs to interstitial fibrosis. All

1	membrane. In any Alport’s kidney, there are areas of thinning mixed with splitting of the GBM. Tubules drop out, glomeruli scar, and the kidney eventually succumbs to interstitial fibrosis. All affected members of a family with X-linked Alport’s syndrome should be identified and followed, including mothers of affected males. Primary treatment is control of systemic hypertension and use of ACE inhibitors to slow renal progression. Although patients who receive renal allografts usually develop anti-GBM antibodies directed toward the collagen epitopes absent in their native kidney, overt Goodpasture’s syndrome is rare and graft survival is good.

1	Thin basement membrane disease (TBMD) characterized by persistent or recurrent hematuria is not typically associated with proteinuria, hypertension, or loss of renal function or extrarenal disease. Although not all cases are familial (perhaps a founder effect), it usually presents 1847 in childhood in multiple family members and is also called benign familial hematuria. Cases of TBMD have genetic defects in type IV collagen but in contrast to Alport behave as an autosomal dominant disorder that in ~40% of families segregates with the COL(IV) α3/ COL(IV) α4 loci. Mutations in these loci can result in a spectrum of disease ranging from TBMD to autosomal dominant or recessive Alport’s. The GBM shows diffuse thinning compared to normal values for the patient’s age in otherwise normal biopsies (see Fig. 62e-19). The vast majority of patients have a benign course.

1	Patients with nail-patella syndrome develop iliac horns on the pelvis and dysplasia of the dorsal limbs involving the patella, elbows, and nails, variably associated with neural-sensory hearing impairment, glaucoma, and abnormalities of the GBM and podocytes, leading to hematuria, proteinuria, and FSGS. The syndrome is autosomal dominant, with haploinsufficiency for the LIM homeodomain transcription factor LMX1B; pedigrees are extremely variable in the penetrance for all features of the disease. LMX1B regulates the expression of genes encoding α3 and α4 chains of collagen IV, interstitial type III collagen, podocin, and CD2AP that help form the slit-pore membranes connecting podocytes. Mutations in the LIM domain region of LMX1B associate with glomerulopathy, and renal failure appears in as many as 30% of patients. Proteinuria or isolated hematuria is discovered throughout life, but usually by the third decade, and is inexplicably more common in females. On renal biopsy there is

1	in as many as 30% of patients. Proteinuria or isolated hematuria is discovered throughout life, but usually by the third decade, and is inexplicably more common in females. On renal biopsy there is lucent damage to the lamina densa of the GBM, an increase in collagen III fibrils along glomerular capillaries and in the mesangium, and damage to the slit-pore membrane, producing heavy proteinuria not unlike that seen in congenital nephrotic syndrome. Patients with renal failure do well with transplantation.

1	A variety of diseases result in classic vascular injury to the glomerular capillaries. Most of these processes also damage blood vessels elsewhere in the body. The group of diseases discussed here lead to vasculitis, renal endothelial injury, thrombosis, ischemia, and/or lipid-based occlusions.

1	Aging in the developed world is commonly associated with the occlusion of coronary and systemic blood vessels. The reasons for this include obesity, insulin resistance, smoking, hypertension, and diets rich in lipids that deposit in the arterial and arteriolar circulation, producing local inflammation and fibrosis of small blood vessels. When the renal arterial circulation is involved, the glomerular microcirculation is damaged, leading to chronic nephrosclerosis. Patients with GFRs <60 mL/min have more cardiovascular events and hospitalizations than those with higher filtration rates. Several aggressive lipid disorders can accelerate this process, but most of the time atherosclerotic progression to chronic nephrosclerosis is associated with poorly controlled hypertension. Approximately 10% of glomeruli are normally sclerotic by age 40, rising to 20% by age 60 and 30% by age 80. Serum lipid profiles in humans are greatly affected by apolipoprotein E polymorphisms; the E4 allele is

1	10% of glomeruli are normally sclerotic by age 40, rising to 20% by age 60 and 30% by age 80. Serum lipid profiles in humans are greatly affected by apolipoprotein E polymorphisms; the E4 allele is accompanied by increases in serum cholesterol and is more closely associated with atherogenic profiles in patients with renal failure. Mutations in E2 alleles, particularly in Japanese patients, produce a specific renal abnormality called lipoprotein glomerulopathy associated with glomerular lipoprotein thrombi and capillary dilation.

1	Uncontrolled systemic hypertension causes permanent damage to the kidneys in about 6% of patients with elevated blood pressure. As many as 27% of patients with end-stage kidney disease have hypertension as a primary cause. Although there is not a clear correlation between the extent or duration of hypertension and the risk of end-organ damage, hypertensive nephrosclerosis is fivefold more frequent in African Americans than whites. Risk alleles associated with APOL1, a functional gene for apolipoprotein L1 expressed in podocytes 1848 substantially explains the increased burden of end-stage renal disease among African Americans. Associated risk factors for progression to end-stage kidney disease include increased age, male gender, race, smoking, hypercholesterolemia, duration of hypertension, low birth weight, and preexisting renal injury. Kidney biopsies in patients with hypertension, microhematuria, and moderate proteinuria demonstrate arteriolosclerosis, chronic nephrosclerosis, and

1	low birth weight, and preexisting renal injury. Kidney biopsies in patients with hypertension, microhematuria, and moderate proteinuria demonstrate arteriolosclerosis, chronic nephrosclerosis, and interstitial fibrosis in the absence of immune deposits (see Fig. 62e-21). Today, based on a careful history, physical examination, urinalysis, and some serologic testing, the diagnosis of chronic nephrosclerosis is usually inferred without a biopsy. Treating hypertension is the best way to avoid progressive renal failure; most guidelines recommend lowering blood pressure to <130/80 mmHg if there is preexisting diabetes or kidney disease. In the presence of kidney disease, most patients begin antihypertensive therapy with two drugs, classically a thiazide diuretic and an ACE inhibitor; most will require three drugs. There is strong evidence in African Americans with hypertensive nephrosclerosis that therapy initiated with an ACE inhibitor can slow the rate of decline in renal function

1	will require three drugs. There is strong evidence in African Americans with hypertensive nephrosclerosis that therapy initiated with an ACE inhibitor can slow the rate of decline in renal function independent of effects on systemic blood pressure. Malignant acceleration of hypertension complicates the course of chronic nephrosclerosis, particularly in the setting of scleroderma or cocaine use (see Fig. 62e-24). The hemodynamic stress of malignant hypertension leads to fibrinoid necrosis of small blood vessels, thrombotic microangiography, a nephritic urinalysis, and acute renal failure. In the setting of renal failure, chest pain, or papilledema, the condition is treated as a hypertensive emergency. Slightly lowering the blood pressure often produces an immediate reduction in GFR that improves as the vascular injury attenuates and autoregulation of blood vessel tone is restored.

1	Aging patients with clinical complications from atherosclerosis sometimes shower cholesterol crystals into the circulation—either spontaneously or, more commonly, following an endovascular procedure with manipulation of the aorta—or with use of systemic anticoagulation. Spontaneous emboli may shower acutely or shower subacutely and somewhat more silently. Irregular emboli trapped in the microcirculation produce ischemic damage that induces an inflammatory reaction. Depending on the location of the atherosclerotic plaques releasing these cholesterol fragments, one may see cerebral transient ischemic attacks; livedo reticularis in the lower extremities; Hollenhorst plaques in the retina with visual field cuts; necrosis of the toes; and acute glomerular capillary injury leading to focal segmental glomerulosclerosis sometimes associated with hematuria, mild proteinuria, and loss of renal function, which typically progresses over a few years. Occasional patients have fever, eosinophilia,

1	glomerulosclerosis sometimes associated with hematuria, mild proteinuria, and loss of renal function, which typically progresses over a few years. Occasional patients have fever, eosinophilia, or eosinophiluria. A skin biopsy of an involved area may be diagnostic. Since tissue fixation dissolves the cholesterol, one typically sees only residual, biconvex clefts in involved vessels (see Fig. 62e-22). There is no therapy to reverse embolic occlusions, and steroids do not help. Controlling blood pressure and lipids and cessation of smoking are usually recommended for prevention.

1	Although individuals with SA-hemoglobin are usually asymptomatic, most will gradually develop hyposthenuria due to subclinical infarction of the renal medulla, thus predisposing them to volume depletion. There is an unexpectedly high prevalence of sickle trait among dialysis patients who are African American. Patients with homozygous SS-sickle cell disease develop chronic vasoocclusive disease in many organs. Polymers of deoxygenated SS-hemoglobin distort the shape of red blood cells. These cells attach to endothelia and obstruct small blood vessels, producing frequent and painful sickle cell crises over time. Vessel occlusions in the kidney produce glomerular hypertension, FSGS, interstitial nephritis, and renal infarction associated with hyposthenuria, microscopic hematuria, and even gross hematuria; some patients also present with MPGN. Renal function can be overestimated due to the increased tubular secretion of creatinine seen in many patients with SS-sickle cell. By the second

1	hematuria; some patients also present with MPGN. Renal function can be overestimated due to the increased tubular secretion of creatinine seen in many patients with SS-sickle cell. By the second or third decade of life, persistent vasoocclusive disease in the kidney leads to varying degrees of renal failure, and some patients end up on dialysis. Treatment is directed to reducing the frequency of painful crises and administering ACE inhibitors in the hope of delaying a progressive decline in renal function. In sickle cell patients undergoing renal transplantation, renal graft survival is comparable to African Americans in the general transplant population.

1	Thrombotic thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome (HUS) represent a spectrum of thrombotic microangiopathies. Thrombotic thrombocytopenic purpura and hemolytic-uremic syndrome share the general features of idiopathic thrombocytopenic purpura, hemolytic anemia, fever, renal failure, and neurologic disturbances. When patients, particularly children, have more evidence of renal injury, their condition tends to be called HUS. In adults with neurologic disease, it is considered to be TTP. In adults there is often a mixture of both, which is why they are often referred to as having TTP/ HUS. On examination of kidney tissue, there is evidence of glomerular capillary endotheliosis associated with platelet thrombi, damage to the capillary wall, and formation of fibrin material in and around glomeruli (see Fig. 62e-23). These tissue findings are similar to what is seen in preeclampsia/HELLP (hemolysis, elevated liver enzymes, and low platelet count syndrome), malignant

1	in and around glomeruli (see Fig. 62e-23). These tissue findings are similar to what is seen in preeclampsia/HELLP (hemolysis, elevated liver enzymes, and low platelet count syndrome), malignant hypertension, and the antiphospholipid syndrome. TTP/HUS is also seen in pregnancy; with the use of oral contraceptives or quinine; in renal transplant patients given OKT3 for rejection; in patients taking the calcineurin inhibitors, cyclosporine and tacrolimus, or in patients taking the antiplatelet agents, ticlopidine and clopidogrel; or following HIV infection.

1	Although there is no agreement on how much they share a final common pathophysiology, two general groups of patients are recognized: childhood HUS associated with enterohemorrhagic diarrhea and TTP/HUS in adults. Childhood HUS is caused by a toxin released by Escherichia coli 0157:H7 and occasionally by Shigella dysenteriae. This shiga toxin (verotoxin) directly injures endothelia, enterocytes, and renal cells, causing apoptosis, platelet clumping, and intravascular hemolysis by binding to the glycolipid receptors (Gb3). These receptors are more abundant along endothelia in children compared to adults. Shiga toxin also inhibits the endothelial production of ADAMTS13. In familial cases of adult TTP/HUS, there is a genetic deficiency of the ADAMTS13 metalloprotease that cleaves large multimers of von Willebrand’s factor. Absent ADAMTS13, these large multimers cause platelet clumping and intravascular hemolysis. An antibody to ADAMTS13 is found in many sporadic cases of adult TTP/HUS,

1	of von Willebrand’s factor. Absent ADAMTS13, these large multimers cause platelet clumping and intravascular hemolysis. An antibody to ADAMTS13 is found in many sporadic cases of adult TTP/HUS, but not all; many patients also have antibodies to the thrombospondin receptor on selected endothelial cells in small vessels or increased levels of plasminogen-activator inhibitor 1 (PAI-1). Some children with complement protein deficiencies express atypical HUS (aHUS), which can be treated with liver transplant. The treatment of adult TTP/HUS is daily plasmapheresis, which can be lifesaving. Plasmapheresis is given until the platelet count rises, but in relapsing patients it normally is continued well after the platelet count improves, and in resistant patients twice-daily exchange may be helpful. Most patients respond within 2 weeks of daily plasmapheresis. Since TTP/HUS often has an autoimmune basis, there is an anecdotal role in relapsing patients for using splenectomy, steroids,

1	helpful. Most patients respond within 2 weeks of daily plasmapheresis. Since TTP/HUS often has an autoimmune basis, there is an anecdotal role in relapsing patients for using splenectomy, steroids, immunosuppressive drugs, bortezomib, or rituximab, an anti-CD20 antibody. Patients with childhood HUS from infectious diarrhea are not given antibiotics, because antibiotics are thought to accelerate the release of the toxin and the diarrhea is usually self-limited. No intervention appears superior to supportive therapy in children with postdiarrheal HUS.

1	ANTIPHOSPHOLIPID ANTIBODY SYNDROME (SEE CHAP. 379) A number of infectious diseases will injure the glomerular capillaries as part of a systemic reaction producing an immune response or from direct infection of renal tissue. Evidence of this immune response is collected by glomeruli in the form of immune deposits that damage the kidney, producing moderate proteinuria and hematuria. A high prevalence of many of these infectious diseases in undeveloped countries results in infection-associated renal disease being the most common cause of glomerulonephritis in many parts of the world. Poststreptococcal Glomerulonephritis This form of glomerulonephritis is one of the classic complications of streptococcal infection. The discussion of this disease can be found earlier, in the section “Acute Nephritic Syndromes.”

1	Subacute Bacterial Endocarditis Renal injury from persistent bacteremia absent the continued presence of a foreign body, regardless of cause, is treated presumptively as if the patient has endocarditis. The discussion of this disease can be found earlier, in the section “Acute Nephritic Syndromes.”

1	Human Immunodeficiency Virus Renal disease is an important complication of HIV disease. The risk of development of end-stage renal disease is much higher in HIV-infected African Americans than in HIV-infected whites. About 50% of HIV-infected patients with kidney disease have HIV-associated nephropathy (HIVAN) on biopsy. The lesion in HIVAN is FSGS, characteristically revealing a collapsing glomerulopathy (see Fig. 62e-3) with visceral epithelial cell swelling, microcystic dilatation of renal tubules, and tubuloreticular inclusion. Renal epithelial cells express replicating HIV virus, but host immune responses also play a role in the pathogenesis. MPGN and DPGN have also been reported but more commonly in HIV-infected whites and in patients coinfected with hepatitis B or C. HIV-associated TTP has also been reported. Other renal lesions include DPGN, IgA nephropathy, and MCD. Renal biopsy may be indicated to distinguish between these lesions.

1	HIV patients with FSGS typically present with nephrotic-range proteinuria and hypoalbuminemia, but unlike patients with other etiologies for nephrotic syndrome, they do not commonly have hypertension, edema, or hyperlipidemia. Renal ultrasound also reveals large, echogenic kidneys despite the finding that renal function in some patients declines rapidly. Treatment with inhibitors of the reninangiotensin system decreases the proteinuria. Effective antiretroviral therapy benefits both the patient and the kidney and improves survival of HIV-infected patients with chronic kidney disease (CKD) or end-stage renal disease. In HIV-infected patients not yet on therapy, the presence of HIVAN is an indication to initiate therapy. Following the introduction of antiretroviral therapy, survival on dialysis for the HIV-infected patient has improved dramatically. Renal transplantations in HIV-infected patients without detectable viral loads or histories of opportunistic infections provide a better

1	for the HIV-infected patient has improved dramatically. Renal transplantations in HIV-infected patients without detectable viral loads or histories of opportunistic infections provide a better survival benefit over dialysis. Following transplantation, patient and graft survival are similar to the general transplant population despite frequent rejections.

1	Hepatitis B and C Typically infected patients present with microscopic hematuria, nonnephrotic or nephrotic-range proteinuria, and hypertension. There is a close association between hepatitis B infection and polyarteritis nodosa with vasculitis appearing generally in the first 6 months following infection. Renal manifestations include renal artery aneurysms, renal infarction, and ischemic scars. Alternatively, the hepatitis B carrier state can produce a MGN that is more common in children than adults, or MPGN that is more common in adults than in children. Renal histology is indistinguishable from idiopathic MGN or type I MPGN. Viral antigens are found in the renal deposits. There are no good treatment guidelines, but interferon α-2b and lamivudine have been used to some effect in small studies. Children have a good prognosis, with 60–65% achieving spontaneous remission within 4 years. In contrast, 30% of adults have renal insufficiency and 10% have renal failure 5 years after

1	small studies. Children have a good prognosis, with 60–65% achieving spontaneous remission within 4 years. In contrast, 30% of adults have renal insufficiency and 10% have renal failure 5 years after diagnosis.

1	Up to 30% of patients with chronic hepatitis C infection have some renal manifestations. Patients often present with type II mixed cryoglobulinemia, nephrotic syndrome, microscopic hematuria, abnormal liver function tests, depressed C3 levels, anti–hepatitis C virus (HCV) antibodies, and viral RNA in the blood. The renal lesions most commonly seen, in order of decreasing frequency, are cryoglobulinemic glomerulonephritis, MGN, and type I MPGN. Treatment with pegylated interferon and ribavirin is typical to reduce the viral load.

1	Other Viruses Other viral infections are occasionally associated with 1849 glomerular lesions, but cause and effect are not well established. These viral infections and their respective glomerular lesions include: cytomegalovirus producing MPGN; influenza and anti-GBM disease; measles-associated endocapillary proliferative glomerulonephritis, with measles antigen in the capillary loops and mesangium; parvovirus causing mild proliferative or mesangioproliferative glomerulonephritis or FSGS; mumps and mesangioproliferative glomerulonephritis; Epstein-Barr virus producing MPGN, diffuse proliferative nephritis, or IgA nephropathy; dengue hemorrhagic fever causing endocapillary proliferative glomerulonephritis; and coxsackievirus producing focal glomerulonephritis or DPGN.

1	Syphilis Secondary syphilis, with rash and constitutional symptoms, develops weeks to months after the chancre first appears and occasionally presents with the nephrotic syndrome from MGN caused by subepithelial immune deposits containing treponemal antigens. Other lesions have also rarely been described including interstitial syphilitic nephritis. The diagnosis is confirmed with nontreponemal and treponemal tests for Treponema pallidum. The renal lesion responds to treatment with penicillin or an alternative drug, if allergic. Additional testing for other sexually transmitted diseases is an important part of disease management.

1	Leprosy Despite aggressive eradication programs, approximately 400,000 new cases of leprosy appear annually worldwide. The diagnosis is best made in patients with multiple skin lesions accompanied by sensory loss in affected areas, using skin smears showing paucibacillary or multibacillary infection (WHO criteria). Leprosy is caused by infection with Mycobacterium leprae and can be classified by Ridley-Jopling criteria into various types: tuberculoid, borderline tuberculoid, mid-borderline and borderline lepromatous, and lepromatous. Renal involvement in leprosy is related to the quantity of bacilli in the body, and the kidney is one of the target organs during splanchnic localization. In some series, all cases with borderline lepromatous and lepromatous types of leprosy have various forms of renal involvement including FSGS, mesangioproliferative glomerulonephritis, or renal amyloidosis; much less common are the renal lesions of DPGN and MPGN. Treatment of the infection can cause

1	of renal involvement including FSGS, mesangioproliferative glomerulonephritis, or renal amyloidosis; much less common are the renal lesions of DPGN and MPGN. Treatment of the infection can cause remission of the renal disease.

1	Malaria There are 300–500 million incident cases of malaria each year worldwide, and the kidney is commonly involved. Glomerulonephritis is due to immune complexes containing malarial antigens that are implanted in the glomerulus. In malaria from P. falciparum, mild proteinuria is associated with subendothelial deposits, mesangial deposits, and mesangioproliferative glomerulonephritis that usually resolve with treatment. In quartan malaria from infection with Plasmodium malariae, children are more commonly affected and renal involvement is more severe. Transient proteinuria and microscopic hematuria can resolve with treatment of the infection. However, resistant nephrotic syndrome with progression to renal failure over 3–5 years does happen, as <50% of patients respond to steroid therapy. Affected patients with nephrotic syndrome have thickening of the glomerular capillary walls, with subendothelial deposits of IgG, IgM, and C3 associated with a sparse membranoproliferative lesion.

1	Affected patients with nephrotic syndrome have thickening of the glomerular capillary walls, with subendothelial deposits of IgG, IgM, and C3 associated with a sparse membranoproliferative lesion. The rare mesangioproliferative glomerulonephritis reported with Plasmodium vivax or Plasmodium ovale typically has a benign course.

1	Schistosomiasis Schistosomiasis affects more than 300 million people worldwide and primarily involves the urinary and gastrointestinal tracts. Glomerular involvement varies with the specific strain of schistosomiasis; Schistosoma mansoni is most commonly associated with clinical renal disease, and the glomerular lesions can be classified: Class I is a mesangioproliferative glomerulonephritis; class II is an extracapillary proliferative glomerulonephritis; class III is a membranoproliferative glomerulonephritis; class IV is a focal segmental glomerulonephritis; and class V is amyloidosis. Classes I–II often remit with treatment of the infection, but classes III and IV lesions are associated with IgA immune deposits and progress despite antiparasitic and/or immunosuppressive therapy.

1	Other Parasites Renal involvement with toxoplasmosis infections is rare. When it occurs, patients present with nephrotic syndrome 1850 and have a histologic picture of MPGN. Fifty percent of patients with leishmaniasis will have mild to moderate proteinuria and microscopic hematuria, but renal insufficiency is rare. Acute DPGN, MGN, and mesangioproliferative glomerulonephritis have all been observed on biopsy. Filariasis and trichinosis are caused by nematodes and are sometimes associated with glomerular injury presenting with proteinuria, hematuria, and a variety of histologic lesions that typically resolve with eradication of the infection.

1	polycystic Kidney Disease and Other Inherited Disorders of tubule growth and Development Jing Zhou, Martin R. Pollak The polycystic kidney diseases are a group of genetically heteroge-339Disorders of the Kidney and Urinary Tract neous disorders and a leading cause of kidney failure. The autosomal dominant form of polycystic kidney disease (ADPKD) is the most common life-threatening monogenic disease, affecting 12 million people worldwide. The autosomal recessive form of polycystic kidney disease (ARPKD) is rarer but affects the pediatric population. Kidney cysts are often seen in a wide range of syndromic diseases. Recent studies have shown that defects in the structure or function of the primary cilia may underlie this group of genetic diseases collectively termed ciliopathies (Table 339-1).

1	AuTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE Etiology and Pathogenesis (Fig. 339-1) ADPKD is characterized by progressive formation of epithelial-lined cysts in the kidney. Although cysts only occur in 5% of the tubules in the kidney, the enormous growth of these cysts ultimately leads to the loss of normal surrounding tissues and loss of renal function. The cellular defects in ADPKD that have been known for a long time are increased cell proliferation and fluid secretion, decreased cell differentiation, and abnormal extracellular matrix. ADPKD is caused by mutations in PKD1 and PKD2, which, respectively, code for polycystin-1 (PC1) and polycystin-2 (PC2). PC1 is a large 11-transmembrane protein that functions like a G protein–coupled receptor. PC2 is a calcium-permeable six-transmembrane protein that structurally belongs to the transient receptor potential (TRP) cation channel family. PC1 and PC2 are widely expressed in almost all tissues and organs. PC1 expression is high in

1	protein that structurally belongs to the transient receptor potential (TRP) cation channel family. PC1 and PC2 are widely expressed in almost all tissues and organs. PC1 expression is high in development and low in the adult, whereas PC2 expression is relatively constant. PC1 and PC2 are found on the primary cilium, a hair-like structure present on the apical membrane of a cell, in addition to the cell membranes and cell-cell junctions of tubular epithelial cells. Defects in the primary cilia are linked to a wide spectrum of human diseases, collectively termed ciliopathies. The most common phenotype shared by many ciliopathies is kidney cysts. PC1 and PC2 bind to each other via their respective C-terminal tails to form a receptor-channel complex and regulate each other’s function. The PC1/2 protein complex serves as a mechanosensor or chemical sensor and regulates calcium and G-protein signaling. The PC1/2 protein complex may also directly regulate a number of cellular functions

1	PC1/2 protein complex serves as a mechanosensor or chemical sensor and regulates calcium and G-protein signaling. The PC1/2 protein complex may also directly regulate a number of cellular functions including the cell cycle, the actin cytoskeleton, planar cell polarity (PCP), and cell migration. This protein complex has also been implicated in regulating a number of signaling pathways, including Wnt, mammalian target of rapamycin (mTOR), STAT3, cMET, phosphoinositide 3-kinase (PI3K)/AKT, G protein–coupled receptor (GPCR), and epidermal growth factor receptor (EGFR), as well as in the localization and activity of cystic fibrosis transmembrane conductance (CFTR). One hypothesis is

1	Mode of Renal Disease Inheritance Abnormalities Other Clinical Features Genes Renal cysts Liver, pancreatic cysts, hypertension, subarachnoid hemorrhage Oligohydramnios if severe, hypertension, ascending cholangitis, liver fibrosis In adults, gout Growth retardation, anemia (visual loss, liver fibrosis, cerebellar ataxia if associated with another syndrome) Juvenile nephronophthisis, Leber’s amaurosis Visual impairment in first year of life, pigmentary retinopathy CNS anomalies, polydactyly, congenital heart defects Obesity, polydactyly, retinitis pigmentosa, anosmia, congenital heart defects, mental retardation Oral cavity, face, and digit anomalies; CNS abnormalities; cystic kidney disease; X-linked with male lethality, primary ciliary dyskinesia Skeletal dysplasia, thoracic deformities, polydactyly, renal cysts, retinitis pigmentosa Angiomyolipomas, renal cell carcinoma, facial angiofibromas, CNS hamartomas

1	Skeletal dysplasia, thoracic deformities, polydactyly, renal cysts, retinitis pigmentosa Angiomyolipomas, renal cell carcinoma, facial angiofibromas, CNS hamartomas Renal cell carcinoma, retinal angiomas, CNS hemangioblastomas, pheochromocytomas PKD1, PKD2 MCKD1, MCKD2/UMOD NPHP1-4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, SDCCAG8, TMEM67, TTC21B NPHP1-6, SDCCAG8 GUCY2D, RPE65, LCA3-14 (including LCA10, CEP290) MKS1, TMEM216, TMEM67, CEP290, RPGRIP1L, CC2D2A, TCTN2, B9D1, B9D2, NPHP3 BBS1, 2, ARL6, BBS4,5, MKKS, BBS7, TTC8, BBS9, 10, TRIM32, BBS12, MKS1, CEP290, C2ORF86; modifiers MKS1, MKS3, CCDC28B TSC1, TSC2 Abbreviations: AD, autosomal dominant; AR, autosomal recessive; CNS, central nervous system. Extracellular signals Receptors Ca2+, Wnt, SHH, cAMP and other signaling events Defective Planar cell polarity Cell proliferation During development morphogenesis Polycystic Kidney Disease and Other Inherited Disorders of Tubule Growth and Development

1	Polycystic Kidney Disease and Other Inherited Disorders of Tubule Growth and Development FIGuRE 339-1 Scheme of the primary cilium and cystic kidney disease proteins. Left. A scheme of the primary cilium. Primary cilia share a “9+0” organization of microtubule doublets. Proteins are transported into the cilium by motor protein kinesin 2 and transported out of the cilium by dynein. The cilium is connected to the basal body through the transition zone. Middle. Topology of autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD) proteins polycystin-1, polycystin-2, and fibrocystin/ polyductin (FPC) are shown. PC1 also interacts with other proteins such as components of the BBSome and NPHP1. PC2 and FPC both interact with kinesin 2 (KIF 3A/B). Localization of disease proteins in the cilium, the transition zone, and the basal body is color coded. Right. Potential disease mechanisms due to cilium-mediated signaling events.

1	that loss of ciliary function of PC1 and PC2 leads to reduced calcium signaling and a subsequent increase of adenylyl cyclase activity and decrease of phosphodiesterase activity, which, in turn, causes increased cellular cyclic AMP (cAMP). Increased cAMP promotes protein kinase A activity, among other effectors, and, in turn, leads to cyst growth by promoting proliferation and fluid secretion of cyst-lining cells through chloride and aquaporin channels in ADPKD kidneys.

1	Genetic Considerations ADPKD is inherited as an autosomal dominant trait with complete penetrance but variable expressivity. The disease affects all ethnic groups worldwide with an estimated prevalence of 1:1000 to 1:400. Only half of the patients with ADPKD are clinically diagnosed during their lifetime. ADPKD is genetically heterogeneous. The first disease gene (PKD1) was localized to the region of the α-globin gene on chromosome 16p13 in 1985, and a second disease gene (PKD2) locus was mapped to chromosome 4q21-q23 in 1993. Mutations of PKD1 and PKD2 are responsible for ~85% and ~15% of ADPKD cases, respectively. However, patients with PKD2 mutations may be higher than 15% because they tend to have milder clinical disease and, as a result, may be underdiagnosed. Embryonic lethality of Pkd1 and Pkd2 knockout mice suggests that human homozygotes may be lethal and thus not clinically recognized. PKD1 is comprised of 46 exons occupying ~52 kb of genomic DNA. It produces an ~14-kb

1	of Pkd1 and Pkd2 knockout mice suggests that human homozygotes may be lethal and thus not clinically recognized. PKD1 is comprised of 46 exons occupying ~52 kb of genomic DNA. It produces an ~14-kb transcript that encodes PC1, a protein of ~4300 amino acids. A feature of the PKD1 gene is that the 5´ three-quarters of PKD1 have been duplicated at six other sites on chromosome 16p, and many of them produce mRNA transcripts, which provides a major challenge for genetic analysis of the duplicated region. PKD2 is a single-copy gene with 15 exons producing an ~5.3-kb mRNA transcript that encodes PC2, a protein of 968 amino acids. The presence of additional genes for ADPKD was suggested based on several families linked to neither PKD1 nor PKD2 genes. However, careful analyses have excluded the existence of a third ADPKD gene.

1	In ADPKD patients, every cell carries a germline mutant allele of either PKD1 or PKD2. However, cysts develop in only a small fraction of the nephrons. Cysts are thought to originate from clonal growth of single cells that have received a somatic “second hit” mutation in the “normal” allele of the PKD1 or PKD2 gene. Accumulating evidence in mouse models now shows that partial loss of function of the second allele of Pkd1 in a proliferative environment is sufficient for cystogenesis, suggesting that a critical amount of PKD1 is needed in a cell. Somatic inactivation of the second allele of Pkd1 in adult mice results in very slow onset of cyst development in the kidney, but a “third hit,” such as an additional genetic or epigenetic event, the inactivation of a growth-suppressor gene, the activation of a growth-promoting gene(s), or an event like renal injury that activates the developmental program, may promote rapid cyst formation.

1	Clinical Manifestations ADPKD is characterized by the progressive bilateral formation of renal cysts. Focal renal cysts are typically detected in affected subjects before 30 years of age. Hundreds to thousands of cysts are usually present in the kidneys of most patients in the fifth decade (Fig. 339-2). Enlarged kidneys can each reach a fourfold increase in length and weigh up to 20 times the normal weight. The clinical presentations of ADPKD are highly variable. Although many patients are asymptomatic until the fourth to fifth decade of life and are diagnosed by incidental discoveries of hypertension or abdominal masses, back or flank pain is a frequent symptom in ~60% of patients with ADPKD. The pain may result from renal cyst infection, hemorrhage, or nephrolithiasis. Gross hematuria resulting from cyst rupture occurs in ~40% of patients during the course of their disease, and many of them will have recurrent episodes. Flank pain and hematuria

1	FIGuRE 339-2 Photograph showing a kidney from a patient with autosomal dominant polycystic kidney disease. The kidney has been cut open to expose the parenchyma and internal aspects of cysts.

1	may coexist if the cyst that ruptures is connected with the collecting system. Proteinuria is usually a minor feature of ADPKD. Infection is the second most common cause of death for patients with ADPKD. Up to half of patients with ADPKD will have one or more episodes of renal infection during their lifetime. An infected cyst and acute pyelonephritis are the most common renal infections often due to gram-negative bacteria, which are associated with fever and flank pain, with or without bacteremia. These complications and renal insufficiency often correlate with structural abnormality of the renal parenchyma. Kidney stones occur in ~20% of patients with ADPKD. Different from the general population, more than half of the stones in patients with ADPKD are composed of uric acid, with the remainder due to calcium oxalate. Distal acidification defects, abnormal ammonium transport, low urine pH, and hypocitraturia may be important in the pathogenesis of renal stones in ADPKD. Renal cell

1	remainder due to calcium oxalate. Distal acidification defects, abnormal ammonium transport, low urine pH, and hypocitraturia may be important in the pathogenesis of renal stones in ADPKD. Renal cell carcinoma is a rare complication of ADPKD with no apparent increased frequency compared to the general population. However, in ADPKD, these tumors are more often bilateral at presentation, multicentric, and sarcomatoid in type. Radiologic imaging is often not helpful in distinguishing cyst infection and cyst hemorrhage because of their complexity. Computed tomography (CT) scan and magnetic resonance imaging (MRI) are often useful in distinguishing a malignancy from a complex cyst. Cardiovascular complications are the major cause of mortality in patients with ADPKD. Hypertension is common and typically occurs before any reduction in glomerular filtration rate (GFR). Hypertension is a risk factor for both cardiovascular and kidney disease progression in ADPKD. Notably, some normotensive

1	typically occurs before any reduction in glomerular filtration rate (GFR). Hypertension is a risk factor for both cardiovascular and kidney disease progression in ADPKD. Notably, some normotensive patients with ADPKD may also have left ventricular hypertrophy. Hypertension in ADPKD may result from the increased activation of the renin-angiotensin-aldosterone system, increased sympathetic nerve activity, and impaired endothelial cilium function-dependent relaxation of small resistant blood vessels.

1	The progression of ADPKD has striking interand intrafamilial variability. The disease can present as early as in utero, but end-stage renal disease typically occurs in late middle age. Risk factors include early diagnosis of ADPKD, hypertension, gross hematuria, multiple pregnancies, and large kidney size. Liver cysts derived from the biliary epithelia are the most common extrarenal complication. Polycystic liver disease associated with ADPKD is different from autosomal dominant polycystic liver disease (ADPLD), which is caused by mutations in at least two distinct genes (PRKCSH and SEC63) and does not progress to renal failure. Massive polycystic liver disease occurs almost exclusively in women with ADPKD, particularly those with multiple pregnancies.

1	Intracranial aneurysm (ICA) occurs four to five times more frequently in ADPKD patients than in the general population and causes high mortality. The disease gene products PC1 and PC2 may be directly responsible for defects in arterial smooth muscle cells and myofibroblasts. The focal nature and the natural history of ICA in ADPKD remain unclear. A family history of ICA is a risk factor of aneurysm rupture in ADPKD, but whether hypertension and cigarette smoking are independent risk factors is not clear. About 20–50% of patients may experience “warning headaches” preceding the index episode of subarachnoid hemorrhage due to ruptured ICA. A CT scan is generally used as the first diagnostic test. A lumbar puncture may be used to confirm the diagnosis. The role of radiologic screening for ICA in asymptomatic patients with ADPKD remains unclear. ADPKD patients with a positive family history of ICAs may undergo presymptomatic screening of ICAs by magnetic resonance angiography. Other

1	ICA in asymptomatic patients with ADPKD remains unclear. ADPKD patients with a positive family history of ICAs may undergo presymptomatic screening of ICAs by magnetic resonance angiography. Other vascular abnormalities in ADPKD patients include diffuse arterial dolichoectasias of the anterior and posterior cerebral circulation, which can predispose to arterial dissection and stroke. Mitral valve prolapse occurs in up to 30% of patients with ADPKD, and tricuspid valve prolapse is less common. Other valvular abnormalities occurring with increased frequency in ADPKD patients include insufficiency of the mitral, aortic, and tricuspid valves. Most patients are asymptomatic, but some may progress and require valve replacement. The prevalence of colonic diverticulae and abdominal wall hernias is also increased in ADPKD patients.

1	Diagnosis Diagnosis is typically made from a positive family history consistent with autosomal dominant inheritance and multiple kidney cysts bilaterally. Renal ultrasonography is often used for presymptomatic screening of at-risk subjects and for evaluation of potential living-related kidney donors from ADPKD families. The presence of at least two renal cysts (unilateral or bilateral) is sufficient for diagnosis among at-risk subjects between 15 and 29 years of age with a sensitivity of 96% and specificity of 100%. The presence of at least two cysts in each kidney and the presence at least four cysts in each kidney are required for the diagnosis of at-risk subjects age 30 to 59 years and age 60 years or older, respectively, with a sensitivity of 100% and specificity of 100%. This is because there is an increased frequency of developing simple renal cysts with age. Conversely, in subjects between age 30 and 59 years, the absence of at least two cysts in each kidney, which is

1	is because there is an increased frequency of developing simple renal cysts with age. Conversely, in subjects between age 30 and 59 years, the absence of at least two cysts in each kidney, which is associated with a false-negative rate of 0%, can be used for disease exclusion. These criteria have a lower sensitivity for patients with a PKD2 mutation because of a late onset of ADPKD2. CT scan and T2-weighted MRI, with and without contrast enhancement, are more sensitive than ultrasonography and can detect cysts of smaller size. However, a CT scan exposes the patient to radiation and radiocontrast, which may cause serious allergic reactions and nephrotoxicity in patients with renal insufficiency. T2-weighted MRI, with gadolinium as a contrast agent, has minimal renal toxicity and can detect cysts of only 2–3 mm in diameter. However, a large majority of cysts may still be below the detection level. Genetic testing by linkage analyses and mutational analyses is available for ambiguous

1	cysts of only 2–3 mm in diameter. However, a large majority of cysts may still be below the detection level. Genetic testing by linkage analyses and mutational analyses is available for ambiguous cases. Because of the large size of the PKD1 gene and the presence of multiple highly homologous pseudogenes, mutational analysis of the PKD1 gene is difficult and costly. Application of new technologies, such as paired-end next-generation sequencing with multiplexing individually bar-coded long-range polymerase chain reaction libraries, may reduce the costs and improve the sensitivity for clinical genetic testing.

1	No specific treatment to prevent cyst growth or the decline of renal function has been approved by U.S. Food and Drug Administration. Blood pressure control to a target of 140/90 mmHg is recommended according to the guidelines from the eighth report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC VIII report) for reducing cardiovascular complications in ADPKD and renal disease progression. More rigorous blood pressure control does not equal greater clinical benefits. Maintaining a target systolic blood pressure to 110 mmHg in patients with moderate or advanced disease may increase the risk of renal disease progression by reducing renal blood flow. Lipid-soluble antibiotics against common gram-negative enteric organisms, such as trimethoprim-sulfamethoxazole, quinolones, and chloramphenicol, are preferred for cyst infection because most renal cysts are not connected to glomerular filtration and antibiotics that are capable

1	trimethoprim-sulfamethoxazole, quinolones, and chloramphenicol, are preferred for cyst infection because most renal cysts are not connected to glomerular filtration and antibiotics that are capable of penetrating the cyst walls are likely to be more effective. Treatment often requires 4–6 weeks. The treatment of kidney stones in ADPKD includes standard measures such as analgesics for pain relief and hydration to ensure adequate urine flow. Management of chronic flank, back, or abdominal pain due to renal enlargement may include both pharmacologic (nonnarcotic and narcotic analgesics) and nonpharmacologic measures (transcutaneous electrical nerve stimulation, acupuncture, and biofeedback). Occasionally, surgical decompression of cysts may be necessary. More than half of ADPKD patients eventually require peritoneal dialysis, hemodialysis, or kidney transplantation. Peritoneal dialysis may not be suitable for some patients with massively enlarged polycystic kidneys due to the small

1	eventually require peritoneal dialysis, hemodialysis, or kidney transplantation. Peritoneal dialysis may not be suitable for some patients with massively enlarged polycystic kidneys due to the small intraabdominal space for efficient peritoneal exchange of fluid and solutes and increased chance of abdominal hernia and back pain. Patients with very large polycystic kidneys and recurrent renal cyst infection may require pretransplant nephrectomy or bilateral nephrectomy to accommodate the allograft and reduce the pain.

1	Specific treatment strategies for ADPKD have focused on slowing renal disease progression and lowering cardiovascular risk. For the latter, the main approach is to control blood pressure by inhibiting the renin-angiotensin-aldosterone system. The ongoing HALT PKD trial was set to evaluate the impact of intensive blockade of the renin-angiotensin-aldosterone system and levels of blood pressure control on progressive renal disease. Most approaches target the slowing of renal disease progression by inhibiting cell proliferation and fluid secretion. Several clinical trials have been conducted targeting cell proliferation, including studies on sirolimus and everolimus, inhibitors of the mTOR pathway; OPC31260 and tolvaptan, which inhibit cAMP pathways by antagonizing the activation of vasopressin V2 receptor (V2R) in collecting ducts and reduce cell proliferation by decreasing renal cAMP levels; and somatostatin analogues, which reduce cAMP levels by binding to several GPCRs. Both the V2R

1	V2 receptor (V2R) in collecting ducts and reduce cell proliferation by decreasing renal cAMP levels; and somatostatin analogues, which reduce cAMP levels by binding to several GPCRs. Both the V2R antagonists and somatostatin analogues appear to slow the decline of renal function, although with some side effects such as liver function impairment, polydipsia, and diarrhea. A combination of different growth inhibitors may enhance efficacy and reduce side effects. Additional preclinical studies in animal models include the use of inhibitors to nonreceptor tyrosine kinase Src, B-raf, cyclin-dependent kinase (CDK), transcription factors STAT3 and STAT6 (pyrimethamine and leflunomide), purinergic receptors, hepatocyte growth factor receptor, and glucosylceramide, and agonists to peroxisome proliferator activated receptor-γ (PPARγ receptors (thiazolidodinediones).

1	Genetic Considerations ARPKD is a significant hereditary renal disease in childhood, with an estimated prevalence of 1 in 20,000 live births. A carrier frequency of up to 1:70 has been reported. Mutations in a single gene, PKHD1, are responsible for all the clinical presentations of ARPKD. PKHD1, localized on human chromosome region 6p21.1-6p12.2, is one of the largest genes in the genome, occupies ~450 kb of DNA, and contains at least 86 exons. It produces multiple alternatively spliced transcripts. The largest transcript encodes fibrocystin/polyductin (FPC), which is a large receptor-like integral membrane protein of 4074 amino acids. FPC has a single transmembrane, a large N-terminal extracellular region, and a short intracellular cytoplasmic domain. FPC is localized on the primary cilia of epithelia cells of cortical and medullary collecting ducts and cholangiocytes of bile ducts, similar to polycystins and several other ciliopathy proteins. FPC is also expressed on the basal body

1	of epithelia cells of cortical and medullary collecting ducts and cholangiocytes of bile ducts, similar to polycystins and several other ciliopathy proteins. FPC is also expressed on the basal body and plasma membrane. The large extracellular domain of FPC is presumed to bind to an as yet unknown ligand(s) and is involved in cell-cell and cell-matrix interactions. FPC interacts with ADPKD protein PC2 and may also participate in regulation of the mechanosensory function of the primary cilia, calcium signaling, and PCP, suggesting a common mechanism underlying cystogenesis between ADPKD and ARPKD. FPC is also found on the centrosomes and mitotic spindle and may regulate centrosome duplication and mitotic spindle assembly during cell division. A large number of various mutations have been found throughout PKHD1 and are unique to individual families. Most patients are compound heterozygotes for PKHD1 mutations. Patients with two truncation mutations appear to have an earlier onset of the

1	throughout PKHD1 and are unique to individual families. Most patients are compound heterozygotes for PKHD1 mutations. Patients with two truncation mutations appear to have an earlier onset of the disease.

1	Clinical Features Classic ARPKD is generally diagnosed in utero or within the neonatal period and characterized by greatly enlarged echogenic kidneys in diseased fetuses. Reduced fetal urine production may contribute to oligohydramnios and pulmonary hypoplasia. About 30% of affected neonates die shortly after birth due to respiratory insufficiency. Close to 60% of mortality occurs within the first month of life. In the classic group, most patients are born with renal insufficiency and ESRD. However, infants often have a transient improvement in their GFR; death from renal insufficiency at this stage is rare. Some patients are diagnosed after the neonatal stage and form the older group. Morbidity and mortality in this group often involve systemic hypertension, progressive renal insufficiency, and liver manifestations. The hallmarks of ARPKD liver disease are biliary dysgenesis due to a primary ductal plate malformation with associated periportal fibrosis, namely congenital hepatic

1	and liver manifestations. The hallmarks of ARPKD liver disease are biliary dysgenesis due to a primary ductal plate malformation with associated periportal fibrosis, namely congenital hepatic fibrosis (CHF) and dilatation of intrahepatic bile ducts (Caroli’s disease). CHF and Caroli’s disease can then lead to portal hypertension exhibiting hepatosplenomegaly, variceal bleeding, and cholangitis. Some patients with the diagnosis of ARPKD at 1 year of age with nephromegaly exhibit slowly declining renal function over 20 years with only minimally enlarged kidneys at ESRD and markedly atrophic kidneys following renal transplantation. The slow progression of renal disease is likely due to increasing fibrosis rather than the development of cysts. Systemic hypertension is common in all ARPKD patients, even those with normal renal function.

1	Diagnosis Ultrasonography, CT, and MRI all can be used for diagnosis. Ultrasonography reveals large, echogenic kidneys with poor corticomedullary differentiation. The diagnosis can be made in utero after 24 weeks of gestation in severe cases. Macrocysts generally are not common at birth in ARPKD patients. The absence of renal cysts in either parent, particularly if they are more than 40 years of age on ultrasonography, helps distinguish ARPKD from ADPKD in older patients. Clinical, laboratory, or radiographic evidence of hepatic fibrosis, hepatic pathology demonstrating characteristic ductal plate abnormalities, family history of affected siblings, or parental consanguinity suggestive of autosomal recessive inheritance is helpful. The lack of mutational hotspots and the large and complex genomic structure of PKHD1 make molecular diagnosis difficult; however, presymptomatic screening of other at-risk members in a family with already identified ARPKD mutations is straightforward and

1	genomic structure of PKHD1 make molecular diagnosis difficult; however, presymptomatic screening of other at-risk members in a family with already identified ARPKD mutations is straightforward and inexpensive.

1	Polycystic Kidney Disease and Other Inherited Disorders of Tubule Growth and Development There is no specific therapy for ARPKD. Appropriate neonatal intensive care, blood pressure control, dialysis, and kidney transplantation increase survival into adulthood. Complications of hepatic fibrosis may necessitate liver transplantation. Patients with severe Caroli’s disease may need portosystemic shunting. Upcoming therapies may target abnormal cell signaling mechanisms, as described above for ADPKD.

1	Tuberous sclerosis (TS) is a rare autosomal dominant syndrome caused by mutations in one of two genes, TSC1, encoding hamartin, or, TSC2, encoding tuberin. Published estimates of prevalence vary widely, but it certainly occurs in less than 1:5000 births. Kidney cysts are a frequent feature of this condition, as are two other abnormalities of kidney growth, renal cell carcinoma and renal angiomyolipomas. TS is a syndrome affecting multiple organ systems. Other features of TS include benign growths in the nervous system, eyes, heart, lung, liver, and skin. Essentially all TS patients have associated skin lesions, and a large proportion of patients have neurologic and cognitive manifestations. The TSC2 gene is adjacent to PKD1 in the human genome. Some patients have deletions in their genomic DNA that inactivate these two genes. Such individuals may have manifestations of both ADPKD and TS.

1	The most common kidney finding in TS is the presence of angiomyolipomas. These growths tend to be multiple and bilateral. Although they are usually benign, they may bleed. Surgical removal is often recommended as a prophylactic measure in people with angiomyolipomas larger than 4 cm in diameter. The cysts in TS are radio-graphically similar to those seen in ADPKD. In contrast to ADPKD, there is a clearly increased risk of renal cell carcinoma in TS patients. Regular periodic imaging is recommended in TS patients with kidney involvement to screen for the development of renal cell carcinoma. Although not common, TS may lead to significant chronic kidney disease (CKD) and progress to end-stage kidney failure. Patients with TS and CKD typically have an unremarkable urine sediment and only minimal to mild amounts of proteinuria.

1	Mechanistically, the TSC1 and TSC2 gene products tuberin and hamartin interact physically. This protein complex is localized to the base of the cilia and inhibits intracellular signaling processes mediated by mTOR, leading to abnormal growth in a number of tissues. Investigation of mTOR inhibitors as therapy for TS is ongoing.

1	Von Hippel-Lindau disease (VHL) is an inherited cancer syndrome with renal manifestations. VHL is an autosomal dominant condition caused by mutations in the VHL tumor-suppressor gene. VHL is localized to the primary cilia and is necessary for the formation of primary cilia. Like many autosomal dominant cancer syndromes, VHL is recessive at the cellular level: a somatic mutation in the second VHL allele leads to loss of VHL in the cell and abnormal growth. Kidney manifestations of VHL include multiple bilateral kidney cysts and renal cell carcinomas. Kidney cysts and carcinoma affect the majority of VHL patients. Nonrenal features of VHL include pheochromocytomas, cerebellar hemangioblastomas, and retinal hemangiomas. Annual screening of the kidneys by imaging with CT or MRI is recommended for early detection of renal cell carcinomas. Increasingly, nephron-sparing surgical approaches are being used for removal of cancerous lesions in order to preserve kidney function.

1	ADPKD is by far the most common adult-onset, single-gene form of kidney disease. The large cysts that are sometimes seen in VHL and TS are similar in appearance to the cysts seen in ADPKD. A variety of other inherited disorders affecting primarily tubule and renal interstitial function can lead to CKD and eventual end-stage kidney disease in the absence of large tubule-derived cysts. Inherited diseases affecting the tubulointerstitial compartment of the kidney can lead to secondary glomerular stress and glomerulosclerosis with some degree of concomitant proteinuria. Similarly, disorders of glomerular function will typically lead to secondary interstitial fibrosis and tubule atrophy. From a clinical perspective, therefore, distinguishing between a genetic disease of the renal tubules and a disease of the glomerulus may not be easy, particularly in the absence of a gross phenotype such as large kidney cysts.

1	The medullary cystic kidney diseases (MCKD) are autosomal dominant disorders. Despite the nosology, kidney cysts are not invariably present. Older literature often grouped MCKD together with the childhood-onset disorders known as the nephronophthises, but these are distinct clinical and genetic entities. Medullary Cystic Kidney Disease Type I Patients with MCKD type I (MCKD I) have mutations in the mucin 1 gene MUC1. In contrast to MCKD type II (MCKD II) patients, individuals with MCKD I do not have elevated uric acid levels. The disease-causing MUC1 mutations that have been reported all alter a repeat region within the MUC1 gene, leading to a large “neoprotein” fragment that may lead to toxic effects on the kidney tubule.

1	Clinically, patients with MCKD I exhibit slowly progressive CKD in adulthood, with only minimal amounts of increased urine protein and occasional renal cysts seen on ultrasound examination. Kidney histology shows tubulointerstitial fibrosis and tubular atrophy. The mechanisms by which MUC1 mutations cause human kidney disease are not known.

1	Medullary Cystic Kidney Disease Type II MCKD II is caused by mutations in the UMOD gene, which encodes the protein uromodulin, also known as Tamm-Horsfall protein. Uromodulin is also found on the centrosome, the mitotic spindle, and the primary cilia; it colocalizes with nephrocystin-1 and KIF3A on the cilia. UMOD mutations also cause the conditions that have been referred to as familial juvenile hyperuricemic nephropathy (HNFJ1) and glomerulocystic kidney disease (GCKD), although it is not clear that these different names represent clearly distinct disorders. The term uromodulin-associated kidney disease (UAKD) has been suggested as a better name for MCKD II and the various other related UMOD-associated diseases. Despite the name, kidney cysts are not a common feature of MCKD II. MCKD II should be suspected clinically in patients with a family history of late-onset kidney disease, benign urine sediments, absence of significant proteinuria, and hyperuricemia. Large genome-wide

1	MCKD II should be suspected clinically in patients with a family history of late-onset kidney disease, benign urine sediments, absence of significant proteinuria, and hyperuricemia. Large genome-wide association studies have suggested that certain common noncoding sequence variants in UMOD are associated with a moderately increased risk of CKD in the general population.

1	Other Forms of Familial Tubulointerstitial Kidney Disease A small number of families have been identified with autosomal dominant tubulointerstitial kidney disease and hyperuricemia who lack UMOD mutations. Some of these families carry disease-segregating mutations in the renin gene REN. There are other families who lack mutations in UMOD, MUC1, or REN. Thus, mutations in other yet-to-be identified genes are able to produce similar interstitial kidney disease, both with and without hyperuricemia. Kidney biopsies in patients with any of the various forms of MCKD typically show interstitial fibrosis. These histologic features are not diagnostic of any particular genetic entity, and the specific diagnosis must be made by other means. Genetic tests for alterations in specific genes are increasingly available in the clinical setting.

1	Patients with autosomal dominant interstitial kidney disease, UMOD or REN mutations, or hyperuricemia and gout should be treated similarly to others with these findings, with uric acid–lowering agents, such allopurinol or febuxostat. A large and growing number of genetically distinct but related autosomal recessive disorders are referred to as nephronophthises. These should not be confused with the adult-onset autosomal dominant medullary cystic kidney diseases discussed above, despite the often confusing nomenclature seen in older medical literature. Nephronophthisis is quite rare but is nevertheless the most common inherited childhood form of kidney failure requiring kidney replacement therapy.

1	Like ADPKD and ARPKD, the various genetically heterogeneous entities that fall under the category of nephronophthisis (NPHP) are disorders of ciliary function. Mutations in a very large number of genes have been identified that lead to NPHP under an autosomal recessive pattern of inheritance. The various forms of NPHP share common features, including tubulointerstitial fibrosis, corticomedullary cysts, and progressive CKD, leading to renal failure. Proteinuria is absent or mild, and the urine sediment is not active.

1	NPHP is often divided into infantile, juvenile, and adolescent forms. The juvenile form is the most frequent and usually caused by mutations in the NPHP2 gene. The infantile form, usually caused by NPHP2 mutations, is associated with end-stage kidney failure in early childhood. Patients with the adolescent form of NPHP typically develop end-stage kidney failure in early adulthood. The products of the NPHP genes are referred to as nephrocystins. NPHP1 through NPHP16 have been reported; some are referred to by other names as well.

1	NPHP can present as an isolated finding or be part of several multiorgan syndromes. Neurologic abnormalities are present in a significant number of patients. Bone and liver abnormities are seen in some NPHP patients. Senior-Løken syndrome is defined by the presence of NPHP with retinitis pigmentosa. Joubert’s syndrome is defined by multiple neurologic findings, including hypoplasia of the cerebellar vermis. Some forms of this genetically heterogeneous syndrome include NPHP as a component.

1	The multisystem disease Bardet-Biedl syndrome (BBS) is defined clinically by a spectrum of features, including truncal obesity, cognitive impairment, retinal dystrophy, polydactyly, developmental urogenital abnormalities, and kidney cysts. The kidney phenotype is NPHP-like, with small cysts deriving from the tubules, tubulointerstitial and often secondary glomerular disease, and urine concentrating defects. There are 18 BBS genes cloned. BBS follows autosomal recessive inheritance. Like ADPKD, ARPKD, and NPHP, BBS is a disease of abnormal ciliary function. The multiple genes and gene products (nephrocystins) that are responsible for NPHP are expressed in cilia, basal bodies, and the centrosomes of kidney tubule cells. It has been hypothesized that all of the NPHP gene defects lead to a clinical phenotype by interfering with the regulation of PCP.

1	There are no specific clinical tests that define NPHP. Genetic diagnosis is possible but cumbersome because of the large number of genes that can be responsible. There are no specific therapies for NPHP. Rather, therapy is aimed at treating signs of these diseases as well as the systemic abnormalities seen with all CKDs. Chronic dialysis or kidney transplantation is eventually required for NPHP-affected individuals.

1	Karyomegalic tubulointerstitial nephritis is an exceptionally rare form of kidney disease with adult-onset progressive kidney failure. Kidney biopsy shows chronic tubulointerstitial nephritis, as well as interstitial fibrosis. This is a recessive disorder caused by inheritance of two mutant copies of the FAN1 gene. FAN1 encodes a component of a DNA repair machinery complex. Individuals with two mutant FAN1 genes are genetically sensitized to the effect of DNA damage. Kidney histology shows karyomegaly in addition to the nonspecific findings of interstitial fibrosis and tubular atrophy. Medullary sponge kidney (MSK) is often grouped together with inherited disorders of the kidney affecting tubule growth and development, although it is usually a sporadic finding rather than an inherited phenotype. MSK is caused by developmental malformation and cystic 1855 dilatation of the renal collecting ducts. The medullary cysts seen in this entity can be quite variable in size.

1	MSK is usually a benign entity. The diagnosis of MSK is often made incidentally. In the past, the diagnosis of MSK was often made by intravenous pyelography (IVP). CT scans, which have replaced IVPs for much routine kidney imaging, are not as sensitive in detecting MSK. MSK is associated with an increased frequency of calcium phosphate and calcium oxalate kidney stones. Altered flow characteristics in the kidney tubules may lead to the development of formation of a nidus for stone formation. Kidney stones in this group are treated the same as are kidney stones in the general population. MSK patients also often exhibit reduced kidney concentrating ability and an increased frequency of urinary tract infections.

1	The structural abnormalities known as the congenital abnormalities of the kidney and urinary tract (CAKUTs) are a group of etiologically and phenotypically heterogeneous disorders. Some form of CAKUT is estimated to occur in up to 1 in 500 live births. Specific abnormalities classified as part of the CAKUT spectrum include kidney hypoplasia, kidney agenesis, ureteropelvic junction obstruction, and vesicoureteral reflux. CAKUT can be the cause of clinically significant problems in both adults and children. However, it is a major contributor to kidney failure in children, accounting for more than one-third of end-stage kidney disease in this group.

1	CAKUT is typically a sporadic finding but can also cluster in families. Familial forms can be observed as parts of multisystem developmental syndromes. A growing list of specific genes have been identified, which when mutated lead to syndromic forms of CAKUT. For example, the branchio-oto-renal syndrome, characterized by developmental abnormalities in the neck, ears, and kidney, can be caused by mutations in the EYA1 and SIX1 genes. Mutations in the PAX2 transcription factor gene can cause the autosomal dominant renal coloboma syndrome, characterized by optic nerve malformations and hypoplastic kidneys. In many instances, CAKUT is caused by environmental influences rather than genetic alterations. For example, renal tubular dysgenesis, defined by altered tubule development, can be caused by prenatal exposure to angiotensin-converting enzyme inhibitors or angiotensin receptor blockers.

1	Inherited disorders of the mitochondrial genome (discussed elsewhere in this text [Chap. 85e]) commonly affect kidney function. Thirteen of the genes involved in encoding components of the mitochondrial respiratory chain are located on the mitochondrial genome that is inherited maternally. The remainder of these components is encoded by the nuclear genome. These defects of oxidative phosphorylation may affect multiple organs and tissues.

1	Neuromuscular disease is the best recognized part of this complex phenotype. Kidney disease is now recognized as a common component as well. Tubulointerstitial disease may be seen on kidney biopsy, and progression to kidney failure may occur. Glomerular involvement, manifest as proteinuria and glomerulosclerosis, can also develop. Changes in proximal tubule activity are the most common renal phenotype. Patients may have several defects in proximal tubule transport, including the Fanconi syndrome. Some patients may also have acidosis, hypophosphatemic rickets, hypercalciuria, glycosuria, and tubular proteinuria. Decreased urine concentrating ability is common. The disorders discussed above are all seen worldwide. In addition, a previously unrecognized epidemic of kidney disease is leading to very high rates of kidney failure in and near the

1	Polycystic Kidney Disease and Other Inherited Disorders of Tubule Growth and Development may be involved as well. tubulointerstitial Diseases of the Kidney Laurence H. Beck, David J. Salant Inflammation or fibrosis of the renal interstitium and atrophy of the tubular compartment are common consequences of diseases that 340Disorders of the Kidney and Urinary Tract 1856 western coast of Central America. This Mesoamerican nephropathy is particularly common in Nicaragua and El Salvador. Mesoamerican nephropathy patients do not have significant proteinuria, suggesting that this is a disease of the kidney tubules and interstitium. The cause is unknown, but some have suggested that a combination of toxic environmental factors and heat stress underlies the development of this kidney disease, which has a striking male predominance. However, the fact that, in many families, a large fraction of the men are affected with kidney disease has suggested that a strong genetic component target the

1	which has a striking male predominance. However, the fact that, in many families, a large fraction of the men are affected with kidney disease has suggested that a strong genetic component target the glomeruli or vasculature. Distinct from these secondary phenomena, however, are a group of disorders that primarily affect the tubules and interstitium, with relative sparing of the glomeruli and renal vessels. Such disorders are conveniently divided into acute and chronic tubulointerstitial nephritis (TIN) (Table 340-1).

1	Acute TIN most often presents with acute renal failure (Chap. 334). The acute nature of this group of disorders may be caused by aggressive inflammatory infiltrates that lead to tissue edema, tubular cell injury, and compromised tubular flow, or by frank obstruction of the tubules with casts, cellular debris, or crystals. There is sometimes flank pain due to distention of the renal capsule. Urinary sediment is often active with leukocytes and cellular casts, but depends on the exact nature of the disorder in question.

1	The clinical features of chronic TIN are more indolent and may manifest with disorders of tubular function, including polyuria from impaired concentrating ability (nephrogenic diabetes insipidus), defective proximal tubular reabsorption leading to features of Fanconi’s syndrome (glycosuria, phosphaturia, aminoaciduria, hypokalemia, and type II renal tubular acidosis [RTA] from bicarbonaturia), or non-anion-gap metabolic acidosis and hyperkalemia (type IV RTA) due to impaired ammoniagenesis, as well as progressive azotemia (rising creatinine and blood urea nitrogen [BUN]). There is often modest proteinuria (rarely >2 g/d) attributable to decreased tubular reabsorption of filtered proteins; however, nephrotic-range albuminuria may occur in some conditions due to the development of secondary focal segmental glomerulosclerosis (FSGS). Renal ultrasonography may reveal changes of “medical renal disease,” such as increased echogenicity of the renal parenchyma with loss of corticomedullary

1	focal segmental glomerulosclerosis (FSGS). Renal ultrasonography may reveal changes of “medical renal disease,” such as increased echogenicity of the renal parenchyma with loss of corticomedullary differentiation, prominence of the renal pyramids, and cortical scarring in some conditions. The predominant pathology in chronic TIN is interstitial fibrosis with patchy mononuclear cell infiltration and widespread tubular atrophy, luminal dilation, and thickening of tubular basement membranes. Because of the nonspecific nature of the histopathology, biopsy specimens rarely provide a specific diagnosis. Thus, diagnosis relies on careful analysis of history, drug or toxin exposure, associated symptoms, and imaging studies.

1	In 1897, Councilman reported on eight cases of acute interstitial nephritis (AIN) in the Medical and Surgical Reports of the Boston City Hospital; three as a postinfectious complication of scarlet fever and two from diphtheria. Later, he described the lesion as “an acute inflammation of the kidney characterized by cellular and fluid exudation in the interstitial tissue, accompanied by, but not dependant on, degeneration (β-lactams, sulfonamides, quinolones, vancomycin, erythromycin, linezolid, minocycline, rifampin, ethambutol, acyclovir) anti-inflammatory drugs, COX-2 inhibitors (rarely thiazides, loop diuretics, triamterene) (phenytoin, valproate, carbamazepine, phenobarbital) (proton pump inhibitors, H2 blockers, captopril, mesalazine, indinavir, allopurinol, lenalidomide) (Streptococcus, Staphylococcus, Legionella, Salmonella, Brucella, Yersinia, Corynebacterium diphtheriae) (EBV, CMV, hantavirus, polyomavirus, HIV) (Leptospira, Rickettsia, Mycoplasma, Histoplasma) nephritis with

1	Staphylococcus, Legionella, Salmonella, Brucella, Yersinia, Corynebacterium diphtheriae) (EBV, CMV, hantavirus, polyomavirus, HIV) (Leptospira, Rickettsia, Mycoplasma, Histoplasma) nephritis with uveitis (TINU) exposure to toxins or therapeutic agents • Analgesics, metals (lead, cadmium) inhibitors (cyclosporine, tacrolimus) (see Chap. 339) Cystic and Hereditary Disorders (see Chap. 339)

1	Abbreviations: CMV, cytomegalovirus; COX, cyclooxygenase; EBV, Epstein-Barr virus. of the epithelium; the exudation is not purulent in character, and the lesions may be both diffuse and focal.” Today AIN is far more often encountered as an allergic reaction to a drug (Table 340-1). Immune-mediated AIN may also occur as part of a known autoimmune syndrome, but in some cases there is no identifiable cause despite features suggestive of an immunologic etiology (Table 340-1). more than ~15% of cases of unexplained acute renal failure, this is likely a substantial underestimate of the true incidence. This is because potentially offending medications are more often identified and empirically discontinued in a patient noted to have a rising serum creatinine, without the benefit of a renal biopsy to establish the diagnosis of AIN.

1	Clinical Features The classic presentation of AIN, namely, fever, rash, peripheral eosinophilia, and oliguric renal failure occurring after 7–10 days of treatment with methicillin or another β-lactam antibiotic, is the exception rather than the rule. More often, patients are found incidentally to have a rising serum creatinine or present with symptoms attributable to acute renal failure (Chap. 334). Atypical reactions can occur, most notably nonsteroidal anti-inflammatory drug (NSAID)-induced AIN, in which fever, rash, and eosinophilia are rare, but acute renal failure with heavy proteinuria is common. A particularly severe and rapid-onset AIN may occur upon reintroduction of rifampin after a drug-free period. More insidious reactions to the agents listed in Table 340-1 may lead to progressive tubulointerstitial damage. Examples include proton pump inhibitors and, rarely, sulfonamide and 5-aminosalicylate (mesalazine and sulfasalazine) derivatives and antiretrovirals.

1	Diagnosis Finding otherwise unexplained renal failure with or without oliguria and exposure to a potentially offending agent usually points to the diagnosis. Peripheral blood eosinophilia adds supporting evidence but is present in only a minority of patients. Urinalysis reveals pyuria with white blood cell casts and hematuria. Urinary eosinophils are neither sensitive nor specific for AIN; therefore, testing is not recommended. Renal biopsy is generally not required for diagnosis but reveals extensive interstitial and tubular infiltration of leukocytes, including eosinophils.

1	Discontinuation of the offending agent often leads to reversal of the renal injury. However, depending on the duration of exposure and degree of tubular atrophy and interstitial fibrosis that has occurred, the renal damage may not be completely reversible. Glucocorticoid therapy may accelerate renal recovery, but does not appear to impact long-term renal survival. It is best reserved for those cases with severe renal failure in which dialysis is imminent or if renal function continues to deteriorate despite stopping the offending drug (Fig. 340-1 and Table 340-2).

1	Sjögren’s syndrome is a systemic autoimmune disorder that primarily targets the exocrine glands, especially the lacrimal and salivary glands, and thus results in symptoms, such as dry eyes and mouth, that constitute the “sicca syndrome” (Chap. 383). Tubulointerstitial nephritis with a predominant lymphocytic infiltrate is the most common renal manifestation of Sjögren’s syndrome and can be associated with distal RTA, nephrogenic diabetes insipidus, and moderate renal failure. Diagnosis is strongly supported by positive serologic testing for anti-Ro (SS-A) and anti-La (SS-B) antibodies. A large proportion of patients with Sjögren’s syndrome also have polyclonal hypergammaglobulinemia. Treatment

1	Tubulointerstitial Diseases of the Kidney Chapter 340Continue observation Improvement Supportive˜care˜and close observation No˝improvement˝in˝1˝week OR˝rapid˝progression Classic˝allergic˝AIN Atypical˝features Corticosteroids Renal biopsy Fibrosis Classic˝AIN Conservative Corticosteroids Immunosuppressive˜drugs Granulomatous or˝other˝immune˝IN FIGuRE 340-1 Algorithm for the treatment of allergic and other immune-mediated acute interstitial nephritis (AIN). ARF, acute renal failure; IN, interstitial nephritis. See text for immunosuppressive drugs used for refractory or relapsing AIN. (Modified from S Reddy, DJ Salant: Ren Fail 20:829, 1998.) is initially with glucocorticoids, although patients may require maintenance therapy with azathioprine or mycophenolate mofetil to prevent relapse (Fig. 340-1 and Table 340-2).

1	TINU is a systemic autoimmune disease of unknown etiology. It accounts for fewer than 5% of all cases of AIN, affects females three times more often than males, and has a median age of onset of 15 years. Its hallmark feature, in addition to a lymphocyte-predominant interstitial nephritis (Fig. 340-2), is a painful anterior uveitis, often bilateral and accompanied by blurred vision and photophobia. Diagnosis is • Drug-induced or idiopathic AIN with: Rapid progression of renal failure Diffuse infiltrates on biopsy Impending need for dialysis Delayed recovery AIN with delayed recovery (?) Abbreviations: AIN, acute interstitial nephritis; SLE, systemic lupus erythematosus; TINU, tubulointerstitial nephritis with uveitis. Source: Modified from S Reddy, DJ Salant: Ren Fail 20:829, 1998.

1	Source: Modified from S Reddy, DJ Salant: Ren Fail 20:829, 1998. FIGuRE 340-2 Acute interstitial nephritis (AIN) in a patient who presented with acute iritis, low-grade fever, erythrocyte sedimentation rate of 103, pyuria and cellular casts on urinalysis, and a newly elevated serum creatinine of 2.4 mg/dL. Both the iritis and AIN improved after intravenous methylprednisolone. This PAS-stained renal biopsy shows a mononuclear cell interstitial infiltrate (asterisks) and edema separating the tubules (T) and a normal glomerulus (G). Some of the tubules contain cellular debris and infiltrating inflammatory cells. The findings in this biopsy are indistinguishable from those that would be seen in a case of drug-induced AIN. PAS, Periodic acid–Schiff.

1	often confounded by the fact that the ocular symptoms precede or accompany the renal disease in only one-third of cases. Additional extrarenal features include fever, anorexia, weight loss, abdominal pain, and arthralgia. The presence of such symptoms as well as elevated creatinine, sterile pyuria, mild proteinuria, features of Fanconi’s syndrome, and elevated erythrocyte sedimentation rate should raise suspicion for this disorder. Serologies suggestive of the more common autoimmune diseases are usually negative, and TINU is often a diagnosis of exclusion after other causes of uveitis and renal disease, such as Sjögren’s syndrome, Behçet’s disease, sarcoidosis, and systemic lupus erythematosus, have been considered. Clinical symptoms are typically self-limited in children, but are more apt to follow a relapsing course in adults. The renal and ocular manifestations generally respond well to oral glucocorticoids, although maintenance therapy with agents such as methotrexate,

1	are more apt to follow a relapsing course in adults. The renal and ocular manifestations generally respond well to oral glucocorticoids, although maintenance therapy with agents such as methotrexate, azathioprine, or mycophenolate may be necessary to prevent relapses (Fig. 340-1 and Table 340-2).

1	An interstitial mononuclear cell inflammatory reaction often accompanies the glomerular lesion in most cases of class III or IV lupus nephritis (Chap. 338), and deposits of immune complexes can be identified in tubule basement membranes in about 50% of cases. Occasionally, however, the tubulointerstitial inflammation predominates and may manifest with azotemia and type IV RTA rather than features of glomerulonephritis.

1	Some patients may present with features of AIN but follow a protracted and relapsing course. Renal biopsy in such patients reveals a more chronic inflammatory infiltrate with granulomas and multinucleated giant cells. Most often, no associated disease or cause is found; however, some of these cases may have or subsequently develop the pulmonary, cutaneous, or other systemic manifestations of sarcoidosis such as hypercalcemia. Most patients experience some improvement in renal function if treated early with glucocorticoids before the development of significant interstitial fibrosis and tubular atrophy (Table 340-2). Other immunosuppressive agents may be required for those who relapse frequently upon steroid withdrawal. Other immunosuppressive agents may be required for those who relapse frequently upon steroid withdrawal (Fig. 340-1). Tuberculosis should be ruled out before starting treatment because this too is a rare cause of granulomatous interstitial nephritis.

1	A form of AIN characterized by a dense inflammatory infiltrate containing IgG4-expressing plasma cells can occur as a part of a syndrome known as IgG4-related systemic disease. Autoimmune pancreatitis, sclerosing cholangitis, retroperitoneal fibrosis, and a chronic sclerosing sialadenitis (mimicking Sjögren’s syndrome) may variably be present as well. Fibrotic lesions that form pseudotumors in the affected organs soon replace the initial inflammatory infiltrates and often lead to biopsy or excision for fear of true malignancy. Although the involvement of IgG4 in the pathogenesis is not understood, glucocorticoids have been successfully used as first-line treatment in this group of disorders, once they are correctly diagnosed.

1	Some patients present with typical clinical and histologic features of AIN but have no evidence of drug exposure or clinical or serologic features of an autoimmune disease. The presence in some cases of auto-antibodies to a tubular antigen, similar to that identified in rats with an induced form of interstitial nephritis, suggests that an autoimmune response may be involved. Like TINU and granulomatous interstitial nephritis, idiopathic AIN is responsive to glucocorticoid therapy but may follow a relapsing course requiring maintenance treatment with another immunosuppressive agent (Fig. 340-1 and Table 340-2).

1	AIN may also occur as a local inflammatory reaction to microbial infection (Table 340-1) and should be distinguished from acute bacterial pyelonephritis (Chap. 162). Acute bacterial pyelonephritis does not generally cause acute renal failure unless it affects both kidneys or causes septic shock. Presently, infection-associated AIN is most often seen in immunocompromised patients, particularly renal transplant recipients with reactivation of polyomavirus BK (Chaps. 169 and 337).

1	Acute renal failure may occur when crystals of various types are deposited in tubular cells and interstitium or when they obstruct tubules. Oliguric acute renal failure, often accompanied by flank pain from tubular obstruction, may occur in patients treated with sulfadiazine for toxoplasmosis, indinavir and atazanavir for HIV, and intravenous acyclovir for severe herpesvirus infections. Urinalysis reveals “sheaf of wheat” sulfonamide crystals, individual or parallel clusters of needle-shaped indinavir crystals, or red-green birefringement needle-shaped crystals of acyclovir. This adverse effect is generally precipitated by hypovolemia and is reversible with saline volume repletion and drug withdrawal. Distinct from the obstructive disease, a frank AIN from indinavir crystal deposition has also been reported.

1	Acute tubular obstruction is also the cause of oliguric renal failure in patients with acute urate nephropathy. It typically results from severe hyperuricemia from tumor lysis syndrome in patients with lymphoor myeloproliferative disorders treated with cytotoxic agents, but also may occur spontaneously before the treatment has been initiated (Chap. 331). Uric acid crystallization in the tubules and collecting system leads to partial or complete obstruction of the collecting ducts, renal pelvis, or ureter. A dense precipitate of birefringent uric acid crystals is found in the urine, usually in association with microscopic or gross hematuria. Prophylactic allopurinol reduces the risk of uric acid nephropathy but is of no benefit once tumor lysis has occurred. Once oliguria has developed, attempts to increase tubular flow and solubility of uric acid with alkaline diuresis may be of some benefit; however, emergent treatment with hemodialysis or rasburicase, a recombinant urate oxidase, is

1	to increase tubular flow and solubility of uric acid with alkaline diuresis may be of some benefit; however, emergent treatment with hemodialysis or rasburicase, a recombinant urate oxidase, is usually required to rapidly lower uric acid levels and restore renal function.

1	Calcium oxalate crystal deposition in tubular cells and interstitium may lead to permanent renal dysfunction in patients who survive ethylene glycol intoxication, in patients with enteric hyperoxaluria from ileal resection or small-bowel bypass surgery, and in patients with hereditary hyperoxaluria (Chap. 342). Acute phosphate nephropathy is an uncommon but serious complication of oral Phospho-soda used as a laxative or for bowel preparation for colonoscopy. It results from calcium phosphate crystal deposition in tubules and interstitium and occurs especially in subjects with underlying renal impairment and hypovolemia. Consequently, Phospho-soda should be avoided in patients with chronic kidney disease.

1	Patients with multiple myeloma may develop acute renal failure in the setting of hypovolemia, infection, or hypercalcemia or after exposure to NSAIDs or radiographic contrast media. The diagnosis of light chain cast nephropathy (LCCN)—commonly known as myeloma kidney—should be considered in patients who fail to recover when the precipitating factor is corrected or in any elderly patient with otherwise unexplained acute renal failure.

1	In this disorder, filtered monoclonal immunoglobulin light chains (Bence-Jones proteins) form intratubular aggregates with secreted Tamm-Horsfall protein in the distal tubule. Casts, in addition to obstructing the tubular flow in affected nephrons, incite a giant cell or foreign body reaction and can lead to tubular rupture, resulting in interstitial fibrosis (Fig. 340-3). Although LCCN generally occurs in patients with known multiple myeloma and a large plasma cell burden, the disorder should also be considered as a possible diagnosis in patients who have known monoclonal gammopathy even in the absence of frank myeloma. Filtered monoclonal light chains may also cause less pronounced renal manifestations in the absence of obstruction, due to direct toxicity to proximal tubular cells and intracellular crystal formation. This may result in isolated tubular disorders such as RTA or full Fanconi’s syndrome.

1	Diagnosis Clinical clues to the diagnosis include anemia, bone pain, hypercalcemia, and an abnormally narrow anion gap due to hypoalbuminemia and hypergammaglobulinemia. Urinary dipsticks detect albumin but not immunoglobulin light chains; however, laboratory detection of increased amounts of protein in a spot urine specimen and a negative dipstick result are highly suggestive that the urine contains Bence-Jones protein. Serum and urine should both be sent for protein electrophoresis and for immunofixation for the detection and identification of a potential monoclonal band. A sensitive method is available to detect urine and serum free light chains.

1	FIGuRE 340-3 Histologic appearance of myeloma cast nephropathy. A hematoxylin-eosin–stained kidney biopsy shows many atrophic tubules filled with eosinophilic casts (consisting of Bence-Jones protein), which are surrounded by giant cell reactions. (Courtesy of Dr. Michael N. Koss, University of Southern California Keck School of Medicine; with permission.) The goals of treatment are to correct precipitating factors such as hypovolemia and hypercalcemia, discontinue potential nephrotoxic agents, and treat the underlying plasma cell dyscrasia (Chap. 136); plasmapheresis to remove light chains is of questionable value for LCCN.

1	Interstitial infiltration by malignant B lymphocytes is a common autopsy finding in patients dying of chronic lymphocytic leukemia and non-Hodgkin’s lymphoma; however, this is usually an incidental finding. Rarely, such infiltrates may cause massive enlargement of the kidneys and oliguric acute renal failure. Although high-dose glucocorticoids and subsequent chemotherapy often result in recovery of renal function, the prognosis in such cases is generally poor.

1	Improved occupational and public health measures, together with the banning of over-the-counter phenacetin-containing analgesics, has led to a dramatic decline in the incidence of chronic interstitial nephritis (CIN) from heavy metal—particularly lead and cadmium—exposure and analgesic nephropathy in North America. Today, CIN is most often the result of renal ischemia or secondary to a primary glomerular disease (Chap. 338). Other important forms of CIN are the result of developmental anomalies or inherited diseases such as reflux nephropathy or sickle cell nephropathy and may not be recognized until adolescence or adulthood. Although it is impossible to reverse damage that has already occurred, further deterioration may be prevented or at least slowed in such cases by treating glomerular hypertension, a common denominator in the development of secondary FSGS and progressive loss of functioning nephrons. Therefore, awareness and early detection of patients at risk may prevent them

1	hypertension, a common denominator in the development of secondary FSGS and progressive loss of functioning nephrons. Therefore, awareness and early detection of patients at risk may prevent them from developing end-stage renal disease (ESRD).

1	Reflux nephropathy is the consequence of vesicoureteral reflux (VUR) or other urologic anomalies in early childhood. It was previously called chronic pyelonephritis because it was believed to result from recurrent urinary tract infections (UTIs) in childhood. VUR stems from abnormal retrograde urine flow from the bladder into one or both ureters and kidneys because of mislocated and incompetent ureterovesical valves (Fig. 340-4). Although high-pressure sterile reflux may impair normal growth of the kidneys, when coupled with recurrent UTIs in early childhood, the result is patchy interstitial scarring and tubular atrophy. Loss of functioning nephrons leads to hypertrophy of the remnant glomeruli and eventual secondary FSGS. Reflux nephropathy often goes unnoticed until early adulthood when chronic kidney disease is detected during routine evaluation or during pregnancy. Affected adults are frequently asymptomatic, but may give a history of prolonged bed-wetting or recurrent UTIs

1	when chronic kidney disease is detected during routine evaluation or during pregnancy. Affected adults are frequently asymptomatic, but may give a history of prolonged bed-wetting or recurrent UTIs during childhood, and exhibit variable renal insufficiency, hypertension, mild to moderate proteinuria, and unremarkable urine sediment. When both kidneys are affected, the disease often progresses inexorably over several years to ESRD, despite the absence of ongoing urinary infections or reflux. A single affected kidney may go undetected, except for the presence of hypertension. Renal ultrasound in adults characteristically shows asymmetric small kidneys with irregular outlines, thinned cortices, and regions of compensatory hypertrophy (Fig. 340-4).

1	Maintenance of sterile urine in childhood has been shown to limit scarring of the kidneys. Surgical reimplantation of the ureters into the bladder to restore competency is indicated in young children with persistent high-grade reflux but is ineffective and is Tubulointerstitial Diseases of the Kidney FIGuRE 340-4 Radiographs of vesicoureteral reflux (VUR) and reflux nephropathy. A. Voiding cystourethrogram in a 7-month-old baby with bilateral high-grade VUR evidenced by clubbed calyces (arrows) and dilated tortuous ureters (U) entering the bladder (B). B. Abdominal computed tomography scan (coronal plane reconstruction) in a child showing severe scarring of the lower portion of the right kidney (arrow).

1	C. Sonogram of the right kidney showing loss of parenchyma at the lower pole due to scarring (arrow) and hypertrophy of the mid-region (arrowhead). (Courtesy of Dr. George Gross, University of Maryland Medical Center; with permission.) not indicated in adolescents or adults after scarring has occurred. Aggressive control of blood pressure with an angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) and other agents is effective in reducing proteinuria and may significantly forestall further deterioration of renal function.

1	The pathogenesis and clinical manifestations of sickle cell nephropathy are described in Chap. 341. Evidence of tubular injury may be evident in childhood and early adolescence in the form of polyuria due to decreased concentrating ability or type IV renal tubular acidosis years before there is significant nephron loss and proteinuria from secondary FSGS. Early recognition of these subtle renal abnormalities or development of microalbuminuria in a child with sickle cell disease may warrant consultation with a nephrologist and/or therapy with low-dose ACEIs. Papillary necrosis may result from ischemia due to sickling of red cells in the relatively hypoxemic and hypertonic medullary vasculature and present with gross hematuria and ureteric obstruction by sloughed ischemic papillae (Table 340-3).

1	Primary glomerulopathies are often associated with damage to tubules and interstitium. This may occasionally be due to the same pathologic process affecting the glomerulus and tubulointerstitium, as is the case with immune-complex deposition in lupus nephritis. More often, however, chronic tubulointerstitial changes occur as a secondary Diabetes with urinary tract infection Abbreviation: NSAID, nonsteroidal anti-inflammatory drug.

1	Diabetes with urinary tract infection Abbreviation: NSAID, nonsteroidal anti-inflammatory drug. consequence of prolonged glomerular dysfunction. Potential mechanisms by which glomerular disease might cause tubulointerstitial injury include proteinuria-mediated damage to the epithelial cells, activation of tubular cells by cytokines and complement, or reduced peritubular blood flow leading to downstream tubulointerstitial ischemia, especially in the case of glomeruli that are globally obsolescent due to severe glomerulonephritis. It is often difficult to discern the initial cause of injury by renal biopsy in a patient who presents with advanced renal disease in this setting.

1	Analgesic nephropathy results from the long-term use of compound analgesic preparations containing phenacetin (banned in the United States since 1983), aspirin, and caffeine. In its classic form, analgesic nephropathy is characterized by renal insufficiency, papillary necrosis (Table 340-3) attributable to the presumed concentration of the drug to toxic levels in the inner medulla, and a radiographic constellation of small, scarred kidneys with papillary calcifications best appreciated by computed tomography (Fig. 340-5). Patients may also have polyuria due to impaired concentrating ability and non-anion-gap metabolic acidosis from tubular damage. Shedding of a sloughed necrotic papilla can cause gross hematuria and ureteric colic due to ureteral obstruction. Individuals with ESRD as a result of analgesic nephropathy are at increased risk of a urothelial malignancy compared to patients with other causes of renal failure. Recent cohort studies in individuals with normal baseline renal

1	of analgesic nephropathy are at increased risk of a urothelial malignancy compared to patients with other causes of renal failure. Recent cohort studies in individuals with normal baseline renal function suggest that the moderate chronic use of current analgesic preparations available in the United States, including acetaminophen and NSAIDs, does not seem to cause the constellation of findings known as analgesic nephropathy, although volume-depleted individuals and those with chronic kidney disease are at higher risk of NSAID-related renal toxicity. Nonetheless, it is recommended that heavy users of acetaminophen and NSAIDs be screened for evidence of renal disease.

1	Two seemingly unrelated forms of CIN, Chinese herbal nephropathy and Balkan endemic nephropathy, have recently been linked by the underlying etiologic agent aristolochic acid and are now collectively termed aristolochic acid nephropathy (AAN). In Chinese herbal nephropathy, first described in the early 1990s in young women tak-1861 ing traditional Chinese herbal preparations as part of a weight-loss regimen, one of the offending agents has been identified as aristolochic acid, a known carcinogen from the plant Aristolochia. Multiple Aristolochia species have been used in traditional herbal remedies for centuries and continue to be available despite official bans on their use in many countries. Molecular evidence has also implicated aristolochic acid in Balkan endemic nephropathy, a chronic tubulointerstitial nephritis found primarily in towns along the tributaries of the Danube River and first described in the 1950s. Although the exact route of exposure is not known with certainty,

1	tubulointerstitial nephritis found primarily in towns along the tributaries of the Danube River and first described in the 1950s. Although the exact route of exposure is not known with certainty, contamination of local grain preparations with the seeds of Aristolochia species seems most likely. Aristolochic acid, after prolonged exposure, produces renal interstitial fibrosis with a relative paucity of cellular infiltrates. The urine sediment is bland, with rare leukocytes and only mild proteinuria. Anemia may be disproportionately severe relative to the level of renal dysfunction. Definitive diagnosis of AAN requires two of the following three features: characteristic histology on kidney biopsy; confirmation of aristolochic acid ingestion; and detection of aristolactam-DNA adducts in kidney or urinary tract tissue. These latter lesions represent a molecular signature of aristolochic acid–derived DNA damage and often consist of characteristic A:T-to-T:A transversions. Due to this

1	kidney or urinary tract tissue. These latter lesions represent a molecular signature of aristolochic acid–derived DNA damage and often consist of characteristic A:T-to-T:A transversions. Due to this mutagenic activity, AAN is associated with a very high incidence of upper urinary tract urothelial cancers, with risk related to cumulative dose. Surveillance with computed tomography, ureteroscopy, and urine cytology is warranted, and consideration should be given to bilateral nephroureterectomy once a patient has reached ESRD.

1	FIGuRE 340-5 Radiologic appearance of analgesic nephropathy. A noncontrast computed tomography scan shows an atrophic left kidney with papillary calcifications in a garland pattern. (Reprinted by permission from Macmillan Publishers, Ltd., MM Elseviers et al: Kidney International 48:1316, 1995.) Karyomegalic interstitial nephritis is an unusual form of slowly progressive chronic kidney disease with mild proteinuria, interstitial fibrosis, tubular atrophy, and oddly enlarged nuclei of proximal tubular epithelial cells. It has been linked to mutations in FAN1, a nuclease involved in DNA repair, which may render carriers of the mutation susceptible to environmental DNA-damaging agents.

1	The use of lithium salts for the treatment of manic-depressive illness may have several renal sequelae, the most common of which is nephrogenic diabetes insipidus manifesting as polyuria and polydipsia. Lithium accumulates in principal cells of the collecting duct by entering through the epithelial sodium channel (ENaC), where it inhibits glycogen synthase kinase 3β and downregulates vasopressin-regulated aquaporin water channels. Less frequently, chronic tubulointerstitial nephritis develops after prolonged (>10–20 years) lithium use and is most likely to occur in patients who have experienced repeated episodes of toxic lithium levels. Findings on renal biopsy include interstitial fibrosis and tubular atrophy that are out of proportion to the degree of glomerulosclerosis or vascular disease, a sparse lymphocytic infiltrate, and small cysts or dilation of the distal tubule and collecting duct that are highly characteristic of this disorder. The degree of interstitial fibrosis

1	disease, a sparse lymphocytic infiltrate, and small cysts or dilation of the distal tubule and collecting duct that are highly characteristic of this disorder. The degree of interstitial fibrosis correlates with both duration and cumulative dose of lithium. Individuals with lithium-associated nephropathy are typically asymptomatic, with minimal proteinuria, few urinary leukocytes, and normal blood pressure. Some patients develop more severe proteinuria due to secondary FSGS, which may contribute to further loss of renal function.

1	Renal function should be followed regularly in patients taking lithium, and caution should be exercised in patients with underlying renal disease. The use of amiloride to inhibit lithium entry via ENaC has been effective to prevent and treat lithium-induced nephrogenic diabetes insipidus, but it is not clear if it will prevent lithium-induced CIN. Once lithium-associated nephropathy is detected, the discontinuation of lithium in attempt to forestall further renal deterioration can be problematic, as lithium is an effective mood stabilizer that is often incompletely substituted by other agents. Furthermore, despite discontinuation of lithium, chronic

1	Tubulointerstitial Diseases of the Kidney 1862 renal disease in such patients is often irreversible and can slowly progress to ESRD. The most prudent approach is to monitor lithium levels frequently and adjust dosing to avoid toxic levels (preferably <1 meq/L). This is especially important because lithium is cleared less effectively as renal function declines. In patients who develop significant proteinuria, ACEI or ARB treatment should be initiated.

1	The calcineurin inhibitor (CNI) immunosuppressive agents cyclosporine and tacrolimus can cause both acute and chronic renal injury. Acute forms can result from vascular causes such as vasoconstriction or the development of thrombotic microangiopathy, or can be due to a toxic tubulopathy. Chronic CNI-induced renal injury is typically seen in solid organ (including heart-lung and liver) transplant recipients and manifests with a slow but irreversible reduction of glomerular filtration rate, with mild proteinuria and arterial hypertension. Hyperkalemia is a relatively common complication and is caused, in part, by tubular resistance to aldosterone. The histologic changes in renal tissue include patchy interstitial fibrosis and tubular atrophy, often in a “striped” pattern. In addition, the intrarenal vasculature often demonstrates hyalinosis, and focal glomerulosclerosis can be present as well. Similar changes may occur in patients receiving CNIs for autoimmune diseases, although the

1	intrarenal vasculature often demonstrates hyalinosis, and focal glomerulosclerosis can be present as well. Similar changes may occur in patients receiving CNIs for autoimmune diseases, although the doses are generally lower than those used for organ transplantation. Dose reduction or CNI avoidance appears to mitigate the chronic tubulointerstitial changes, but may increase the risk of rejection and graft loss.

1	Heavy metals, such as lead or cadmium, can lead to a chronic tubulointerstitial process after prolonged exposure. The disease entity is no longer commonly diagnosed, because such heavy metal exposure has been greatly reduced due to the known health risks from lead and the consequent removal of lead from most commercial products and fuels. Nonetheless, occupational exposure is possible in workers involved in the manufacture or destruction of batteries, removal of lead paint, or manufacture of alloys and electrical equipment (cadmium) in countries where industrial regulation is less stringent. In addition, ingestion of moonshine whiskey distilled in lead-tainted containers has been one of the more frequent sources of lead exposure.

1	Early signs of chronic lead intoxication are attributable to proximal tubule dysfunction, particularly hyperuricemia as a result of diminished urate secretion. The triad of “saturnine gout,” hypertension, and renal insufficiency should prompt a practitioner to ask specifically about lead exposure. Unfortunately, evaluating lead burden is not as straightforward as ordering a blood test; the preferred methods involve measuring urinary lead after infusion of a chelating agent or by radiographic fluoroscopy of bone. Several recent studies have shown an association between chronic low-level lead exposure and decreased renal function, although either of these two factors may have been the primary event. In those patients who have CIN of unclear origin and an elevated total body lead burden, repeated treatments of lead chelation therapy have been shown to slow the decline in renal function.

1	Disorders leading to excessively high or low levels of certain electrolytes and products of metabolism can also lead to chronic kidney disease if untreated. The constellation of pathologic findings that represent gouty nephropathy are very uncommon nowadays and are more of historical interest than clinical importance, as gout is typically well managed with allopurinol and other agents. However, there is emerging evidence that hyperuricemia is an independent risk factor for the development of chronic kidney disease, perhaps through endothelial damage. The complex interactions of hyperuricemia, hypertension, and renal failure are still incompletely understood.

1	Presently, gouty nephropathy is most likely to be encountered in patients with severe tophaceous gout and prolonged hyperuricemia from a hereditary disorder of purine metabolism (Chap. 431e). This should be distinguished from juvenile hyperuricemic nephropathy, a form of medullary cystic kidney disease caused by mutations in uromodulin (UMOD) (Chap. 339). Histologically, the distinctive feature of gouty nephropathy is the presence of crystalline deposits of uric acid and monosodium urate salts in the kidney parenchyma. These deposits not only cause intrarenal obstruction but also incite an inflammatory response, leading to lymphocytic infiltration, foreign-body giant cell reaction, and eventual fibrosis, especially in the medullary and papillary regions of the kidney. Since patients with gout frequently suffer from hypertension and hyperlipidemia, degenerative changes of the renal arterioles may constitute a striking feature of the histologic abnormality, out of proportion to the

1	gout frequently suffer from hypertension and hyperlipidemia, degenerative changes of the renal arterioles may constitute a striking feature of the histologic abnormality, out of proportion to the other morphologic defects. Clinically, gouty nephropathy is an insidious cause of chronic kidney disease. Early in its course, glomerular filtration rate may be near normal, often despite morphologic changes in medullary and cortical interstitium, proteinuria, and diminished urinary concentrating ability. Treatment with allopurinol and urine alkalinization is generally effective in preventing uric acid nephrolithiasis and the consequences of recurrent kidney stones; however, gouty nephropathy may be intractable to such measures. Furthermore, the use of allopurinol in asymptomatic hyperuricemia has not been consistently shown to improve renal function.

1	(See also Chap. 424) Chronic hypercalcemia, as occurs in primary hyperparathyroidism, sarcoidosis, multiple myeloma, vitamin D intoxication, or metastatic bone disease, can cause tubulointerstitial disease and progressive renal failure. The earliest lesion is a focal degenerative change in renal epithelia, primarily in collecting ducts, distal tubules, and loops of Henle. Tubular cell necrosis leads to nephron obstruction and stasis of intrarenal urine, favoring local precipitation of calcium salts and infection. Dilation and atrophy of tubules eventually occur, as do interstitial fibrosis, mononuclear leukocyte infiltration, and interstitial calcium deposition (nephrocalcinosis). Calcium deposition may also occur in glomeruli and the walls of renal arterioles.

1	Clinically, the most striking defect is an inability to maximally concentrate the urine, due to reduced collecting duct responsiveness to arginine vasopressin and defective transport of sodium and chloride in the loop of Henle. Reductions in both glomerular filtration rate and renal blood flow can occur, both in acute and in prolonged hypercalcemia. Eventually, uncontrolled hypercalcemia leads to severe tubulointerstitial damage and overt renal failure. Abdominal x-rays may demonstrate nephrocalcinosis as well as nephrolithiasis, the latter due to the hypercalciuria that often accompanies hypercalcemia. Treatment consists of reducing the serum calcium concentration toward normal and correcting the primary abnormality of calcium metabolism (Chap. 424). Renal dysfunction of acute hypercalcemia may be completely reversible. Gradual progressive renal insufficiency related to chronic hypercalcemia, however, may not improve even with correction of the calcium disorder.

1	Patients with prolonged and severe hypokalemia from chronic laxative or diuretic abuse, surreptitious vomiting, or primary aldosteronism may develop a reversible tubular lesion characterized by vacuolar degeneration of proximal and distal tubular cells. Eventually, tubular atrophy and cystic dilation accompanied by interstitial fibrosis may ensue, leading to irreversible chronic kidney disease. Timely correction of the hypokalemia will prevent further progression, but persistent hypokalemia can cause ESRD.

1	The causes of acute and chronic interstitial nephritis vary widely across the globe. Analgesic nephropathy continues to be seen in countries where phenacetin-containing compound analgesic preparations are readily available. Adulterants in unregulated herbal and traditional medicaments pose a threat of toxic interstitial nephritis, as exemplified by aristolochic acid contamination of herbal slimming preparations. Contamination of food sources with toxins, such as the recent outbreak of nephrolithiasis and acute renal failure from melamine contamination of infant milk formula, poses a continuing risk. Large-scale exposure to aristolochic acid remains prevalent in many Asian countries where traditional herbal medicine use is common. Although industrial exposure to lead and cadmium has largely disappeared as a cause of chronic interstitial nephritis in developed nations, it remains a risk for nephrotoxicity in countries where such exposure is less well controlled. New endemic forms of

1	disappeared as a cause of chronic interstitial nephritis in developed nations, it remains a risk for nephrotoxicity in countries where such exposure is less well controlled. New endemic forms of chronic kidney disease continue to be described, such as the nephropathy found among Pacific coastal plantation workers in Central America, which may be related to repetitive heat exposure and fluid losses.

1	Vascular Injury to the Kidney Nelson Leung, Stephen C. Textor The renal circulation is complex and is characterized by a highly perfused arteriolar network, reaching cortical glomerular structures adjacent to lower-flow vasa recta that descend into medullary seg-ments. Disorders of the larger vessels, including renal artery stenosis 341 and atheroembolic disease, are discussed elsewhere (Chap. 354). This chapter examines primary disorders of the renal microvessels, many of which are associated with thrombosis and hemolysis.

1	Thrombotic microangiopathy (TMA) is characterized by injured endothelial cells that are thickened, swollen, or detached mainly from arterioles and capillaries. Platelet and hyaline thrombi causing partial or complete occlusion are integral to the histopathology of TMA. TMA is usually the result of microangiopathic hemolytic anemia (MAHA), with its typical features of thrombocytopenia and schistocytes. In the kidney, TMA is characterized by swollen endocapillary cells (endotheliosis), fibrin thrombi, platelet plugs, arterial intimal fibrosis, and a membranoproliferative pattern. Fibrin thrombi may extend into the arteriolar vascular pole, producing glomerular collapse and at times cortical necrosis. In kidneys that recover from acute TMA, secondary focal segmental glomerulosclerosis may be seen. Diseases associated with this lesion include thrombotic thrombocytopenic purpura (TTP), hemolytic-uremic syndrome (HUS), malignant hypertension, scleroderma renal crisis, antiphospholipid

1	be seen. Diseases associated with this lesion include thrombotic thrombocytopenic purpura (TTP), hemolytic-uremic syndrome (HUS), malignant hypertension, scleroderma renal crisis, antiphospholipid syndrome, preeclampsia/HELLP (hemolysis, elevated liver enzymes, low platelet count) syndrome, HIV infection, and radiation nephropathy.

1	HUS and TTP are the prototypes for MAHA. Historically, HUS and TTP were distinguished mainly by their clinical and epidemiologic differences. TTP develops more commonly in adults and was thought to include neurologic involvement more often. HUS occurs more commonly in children, particularly when associated with hemorrhagic diarrhea. However, atypical HUS (aHUS) can first appear in adulthood, and better testing has revealed that neurologic involvement is as common in HUS as in TTP. Accordingly, HUS and TTP now should be differentiated and treated according to their specific pathophysiologic features. Hemolytic-uremic Syndrome HUS is loosely defined by the presence of MAHA and renal impairment. At least four variants are recognized. The most common is Shiga toxin–producing Escherichia coli (STEC) HUS, which is also known as D+HUS or enterohemorrhagic

1	E. coli (EHEC) HUS. Most cases involve children <5 years of age, but adults also are susceptible, as evidenced by a 2011 outbreak in northern Europe. Diarrhea, often bloody, precedes MAHA within 1 week in >80% of cases. Abdominal pain, cramping, and vomiting 1863 are frequent, whereas fever is typically absent. Neurologic symptoms, including dysphasia, hyperreflexia, blurred vision, memory deficits, encephalopathy, perseveration, and agraphia, often develop, especially in adults. Seizures and cerebral infarction can occur in severe cases. STEC HUS is caused by the Shiga toxins (Stx1 and Stx2), which are also referred to as verotoxins. These toxins are produced by certain strains of E. coli and Shigella dysenteriae. In the United States and Europe, the most common STEC strain is O157:H7, but HUS due to other strains (O157/H−, O111:H−, O26:H11/H−, O145:H28, and

1	O104:H4) has occurred. After entry into the circulation, Shiga toxin binds to the glycolipid surface receptor globotriaosylceramide (Gb3), which is richly expressed on cells of the renal microvasculature. Upon binding, the toxin enters the cells, inducing inflammatory cytokines (interleukin 8 [IL-8], monocyte chemotactic protein 1 [MCP-1], and stromal cell–derived factor 1 [SDF-1]) and chemokine receptors (CXCR4 and CXCR7); this action results in platelet aggregation and the microangiopathic process. Streptococcus pneumoniae can also cause HUS. Certain strains produce a neuraminidase that cleaves the N-acetylneuraminic acid moieties covering the Thomsen-Friedenreich antigen on platelets and endothelial cells. Exposure of this normally cryptic antigen to preformed IgM results in severe MAHA.

1	Atypical HUS is the result of congenital complement dysregulation. The affected patients have the low C3 and normal C4 levels characteristic of alternative pathway activation. Factor H deficiency, the most common defect, has been linked to families with aHUS. Factor H competes with factor B to prevent the formation of C3bBb and acts as a cofactor for factor I, which proteolytically degrades C3b. More than 70 mutations of the factor H gene have been identified. Most are missense mutations that produce abnormalities in the C-terminus region, affecting its binding to C3b but not its concentration. Other mutations result in low levels or the complete absence of the protein. Deficiencies in other complement-regulatory proteins, such as factor I, factor B, membrane cofactor protein (CD46), C3, complement factor H–related protein 1 (CFHR1), CFHR3, CFHR5, and thrombomodulin, have also been reported. Finally, an autoimmune variant of aHUS has been discovered. DEAP (deficient for CFHR protein

1	complement factor H–related protein 1 (CFHR1), CFHR3, CFHR5, and thrombomodulin, have also been reported. Finally, an autoimmune variant of aHUS has been discovered. DEAP (deficient for CFHR protein and positive for factor H autoantibody) HUS occurs when an autoantibody to factor H is formed. DEAP HUS is often associated with a deletion of an 84-kb fragment of the chromosome that encodes for CFHR1 and CFHR3. The autoantibody blocks the binding of factor H to C3b and surface-bound C3 convertase.

1	Thrombotic Thrombocytopenic Purpura Traditionally, TTP is characterized by the pentad: MAHA, thrombocytopenia, neurologic symptoms, fever, and renal failure. The pathophysiology of TTP involves the accumulation of ultra-large multimers of von Willebrand factor as a result of the absence or markedly decreased activity (<5–10%) of the plasma protease ADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13. These ultra-large multimers form clots and shear erythrocytes, resulting in MAHA; however, the absence of ADAMTS13 alone may not itself produce TTP. Often, an additional trigger (such as infection, surgery, pancreatitis, or pregnancy) is required to initiate clinical TTP.

1	Data from the Oklahoma TTP/HUS Registry suggest an incidence rate of 11.3 cases/106 patients in the United States. The median age of onset is 40 years. The incidence is more than nine times higher among blacks than among non-blacks. Like that of systemic lupus erythematosus, the incidence of TTP is nearly three times higher among women than among men. If untreated, TTP has a mortality rate exceeding 90%. Even with modern therapy, 20% of patients die within the first month from complications of microvascular thrombosis.

1	The classic form of TTP is idiopathic TTP, which is usually the result of a deficiency in ADAMTS13. While TTP had traditionally been associated with infection, malignancy, and intense inflammation (e.g., pancreatitis), ADAMTS13 activity usually is not decreased in these conditions. In idiopathic TTP, the formation of an autoantibody to ADAMTS13 (IgG or IgM) either increases its clearance or inhibits its activity. Upshaw-Schülman syndrome is a hereditary condition characterized by congenital deficiency of ADAMTS13. TTP in these

1	Vascular Injury to the Kidney 1864 patients can start within the first weeks of life but in some instances may not present until the patient is several years of age. Both environmental and genetic factors are thought to influence the development of TTP. Plasma transfusion is an effective strategy for prevention and treatment. Drug-induced TMA is a recognized complication of treatment with some chemotherapeutic agents, immunosuppressive agents, antiplatelet agents, and quinine. Two different mechanisms have been described. Endothelial damage (pathologically similar to that in HUS) is the main cause of the TMA that develops in association with chemotherapeutic agents (e.g., mitomycin C, gemcitabine) and immunosuppressive agents (cyclosporine, tacrolimus, and sirolimus). This process is usually dose-dependent. Alternatively, TMA may develop as a result of drug-induced autoantibodies. This form is less likely to be dose-dependent and can, in fact, occur after a single dose in patients

1	dose-dependent. Alternatively, TMA may develop as a result of drug-induced autoantibodies. This form is less likely to be dose-dependent and can, in fact, occur after a single dose in patients with previous exposure. Ticlopidine produces TTP by inducing an autoantibody to ADAMTS13, but ADAMTS13 deficiency is found in fewer than half of patients with clopidogrel-associated TTP. Quinine appears to induce autoantibodies to granulocytes, lymphocytes, endothelial cells, and platelet glycoprotein IbB/IX or IIb/IIIa complexes, but not to ADAMTS13. Quinine-associated TTP is more common among women. TMA has been reported with drugs that inhibit vascular endothelial growth factor, such as bevacizumab; the mechanism is not completely understood.

1	Treatment should be based on pathophysiology. Autoantibodymediated TTP and DEAP HUS respond to plasma exchange or plasmapheresis. In addition to removing the autoantibodies, plasma exchange with fresh-frozen plasma replaces ADAMTS13. Twice-daily plasma exchanges with administration of vincristine and rituximab may be effective in refractory cases. Plasma infusion is usually sufficient to replace the ADAMTS13 in Upshaw-Schülman syndrome. Plasma exchange should be considered if larger volumes are necessary. Drug-induced TMA secondary to endothelial damage typically does not respond to plasma exchange and is treated primarily by discontinuing use of the agent and providing supportive care. Similarly, STEC HUS should be treated with supportive measures. Plasma exchange has not been found to be effective. Antimotility agents and antibiotics increase the incidence of HUS among children, but azithromycin was recently found to decrease the duration of bacterial shedding by adults. Eculizumab

1	effective. Antimotility agents and antibiotics increase the incidence of HUS among children, but azithromycin was recently found to decrease the duration of bacterial shedding by adults. Eculizumab is a monoclonal antibody to C5 that is approved for use in aHUS, for which ongoing therapy may be necessary. Plasma infusion/exchange may play a role in aHUS by replacing complement-regulatory proteins. Antibiotics and washed red cells should be given in neuraminidase-associated HUS, and plasmapheresis may be helpful. However, plasma and whole-blood transfusion should be avoided since these products contain IgM, which may exacerbate MAHA. Finally, combined factor H and ADAMTS13 deficiency have been reported. The affected patients are generally less responsive to plasma infusion, a result illustrating the complexity of the management of these cases.

1	HSCT-TMA develops after HSCT, with an incidence of 8.2%. Etiologic factors include conditioning regimens, immunosuppression, infections, and graft-versus-host disease. Other risk factors include female sex and human leukocyte antigen (HLA)–mismatched donor grafts. HSCT-TMA usually occurs within the first 100 days of HSCT. Table 341-1 lists definitions of HSCT-TMA currently used for clinical trials. Diagnosis may be difficult since thrombocytopenia, anemia, and renal insufficiency are common after HSCT. HSCTTMA carries a high mortality rate (75% within 3 months). The majority of patients have >5% ADAMTS13 activity, and plasma exchange is beneficial in <50% of patients. Discontinuation of calcineurin inhibitors and substitution with daclizumab (antibody to >4% schistocytes in the blood RBC fragmentation and at least 2 schistocytes per high-power field De novo, prolonged, or progressive Concurrent increase in LDH concenthrombocytopenia tration above baseline

1	De novo, prolonged, or progressive Concurrent increase in LDH concenthrombocytopenia tration above baseline Note: These features underscore the need to identify pathways of hemolysis and thrombocytopenia that accompany deterioration of kidney function. Abbreviations: LDH, lactate dehydrogenase; RBC, red blood cell. the IL-2 receptor) are recommended. Treatment with rituximab and defibrotide may also be helpful.

1	Abbreviations: LDH, lactate dehydrogenase; RBC, red blood cell. the IL-2 receptor) are recommended. Treatment with rituximab and defibrotide may also be helpful. HIV-related TMA is a complication encountered mainly before widespread use of highly active antiretroviral therapy. It is seen in patients with advanced AIDS and low CD4+ T cell counts although it can be the first manifestation of HIV infection. The presence of MAHA, thrombocytopenia, and renal failure are suggestive, but renal biopsy is required for diagnosis since other renal diseases are also associated with HIV infection. Thrombocytopenia may prohibit renal biopsy in some patients. The mechanism of injury is unclear, although HIV can induce apoptosis in endothelial cells. ADAMTS13 activity is not reduced in these patients. Cytomegalovirus co-infection may also be a risk factor. Effective antiviral therapy is key, while plasma exchange should be limited to patients who have evidence of TTP.

1	Either local or total body irradiation can produce microangiopathic injury. The kidney is one of the most radiosensitive organs, and injury can result with as little as 4–5 Gy. Such injury is characterized by renal insufficiency, proteinuria, and hypertension usually developing ≥6 months after radiation exposure. Renal biopsy reveals classic TMA with damage to glomerular, tubular, and vascular cells, but systemic evidence of MAHA is uncommon. Because of its high incidence after allogeneic HSCT, radiation nephropathy is often referred to as bone marrow transplant nephropathy. No specific therapy is available, although observational evidence supports renin-angiotensin system blockade.

1	Kidney involvement is common (up to 52%) in patients with widespread scleroderma, with 20% of cases resulting directly from scleroderma renal crisis. Other renal manifestations in scleroderma include transient (prerenal) or medication-related forms of acute kidney injury (e.g., associated with D-penicillamine, nonsteroidal anti-inflammatory drugs, or cyclosporine). Scleroderma renal crisis occurs in 12% of patients with diffuse systemic sclerosis but in only 2% of those with limited systemic sclerosis. Scleroderma renal crisis is the most severe manifestation of renal involvement, and is characterized by accelerated hypertension, a rapid decline in renal function, nephrotic proteinuria, and hematuria. Retinopathy and encephalopathy may accompany the hypertension. Salt and water retention with microvascular injury can lead to pulmonary edema. Cardiac manifestations, including myocarditis, pericarditis, and arrhythmias, denote an especially poor prognosis. Although MAHA is present in

1	with microvascular injury can lead to pulmonary edema. Cardiac manifestations, including myocarditis, pericarditis, and arrhythmias, denote an especially poor prognosis. Although MAHA is present in more half of patients, coagulopathy is rare.

1	The renal lesion in scleroderma renal crisis is characterized by arcuate artery intimal and medial proliferation with luminal narrowing.

1	This lesion is described as “onion-skinning” and can be accompanied by glomerular collapse due to reduced blood flow. Histologically, scleroderma renal crisis is indistinguishable from malignant hypertension, with which it can coexist. Fibrinoid necrosis and thrombosis are common. Before the availability of angiotensin-converting enzyme (ACE) inhibitors, the mortality rate for scleroderma renal crisis was >90% at 1 month. Introduction of renin-angiotensin system blockade has lowered the mortality rate to 30% at 3 years. Nearly two-thirds of patients with scleroderma renal crisis may require dialysis support, with recovery of renal function in 50% (median time, 1 year). Glomerulonephritis and vasculitis associated with antineutrophil cytoplasmic antibodies and systemic lupus erythematosus have been described in patients with scleroderma. An association has been found with a speckled pattern of antinuclear antibodies and with antibodies to RNA polymerases I and III. Anti-U3-RNP may

1	have been described in patients with scleroderma. An association has been found with a speckled pattern of antinuclear antibodies and with antibodies to RNA polymerases I and III. Anti-U3-RNP may identify young patients at risk for scleroderma renal crisis. Anticentromere antibody, in contrast, is a negative predictor of this disorder. Because of the overlap between scleroderma renal crisis and other autoimmune disorders, a renal biopsy is recommended for patients with atypical renal involvement, especially if hypertension is absent.

1	Treatment with ACE inhibition is the first-line therapy unless contraindicated. The goal of therapy is to reduce systolic and diastolic blood pressure by 20 mmHg and 10 mmHg, respectively, every 24 h until blood pressure is normal. Additional antihypertensive therapy may be given once the dose of drug for ACE inhibition is maximized. Both ACE inhibitors and angiotensin II receptor antagonists are effective, although data suggest that treatment with ACE inhibitors is superior. ACE inhibition alone does not prevent scleroderma renal crisis, but it does reduce the impact of hypertension. Intravenous iloprost has been used in Europe for blood pressure management and improvement of renal perfusion. Kidney transplantation is not recommended for 2 years after the start of dialysis since delayed recovery may occur.

1	Antiphospholipid syndrome (Chap. 379) can be either primary or secondary to systemic lupus erythematosus. It is characterized by a predisposition to systemic thrombosis (arterial and venous) and fetal morbidity mediated by antiphospholipid antibodies—mainly anticardiolipin antibodies (IgG, IgM, or IgA), lupus anticoagulant, or anti-β-2 glycoprotein I antibodies (antiβ2GPI). Patients with both anticardiolipin antibodies and antiβ2GPI appear to have the highest risk of thrombosis. The vascular compartment within the kidney is the main site of renal involvement. Arteriosclerosis is commonly present in the arcuate and intralobular arteries. In the intralobular arteries, fibrous intimal hyperplasia characterized by intimal thickening secondary to intense myofibroblastic intimal cellular proliferation with extracellular matrix deposition is frequently seen along with onion-skinning. Arterial and arteriolar fibrous and fibrocellular occlusions are present in more than two-thirds of biopsy

1	with extracellular matrix deposition is frequently seen along with onion-skinning. Arterial and arteriolar fibrous and fibrocellular occlusions are present in more than two-thirds of biopsy samples. Cortical necrosis and focal cortical atrophy may result from vascular occlusion. TMA is commonly present in renal biopsies, although signs of MAHA and platelet consumption are usually absent. TMA is especially common in the catastrophic variant of antiphospholipid syndrome. In patients with secondary antiphospholipid syndrome, other glomerulopathies may be present, including membranous nephropathy, minimal change disease, focal segmental glomerulosclerosis, and pauci-immune crescentic glomerulonephritis.

1	Large vessels can be involved in antiphospholipid syndrome and may form the proximal nidus near the ostium for thrombosis of the renal artery. Renal vein thrombosis can occur and should be suspected in patients with lupus anticoagulant who develop nephroticrange proteinuria. Progression to end-stage renal disease can occur, and a thrombosis may form in the vascular access and the renal allografts. Hypertension is common. Treatment entails lifelong anticoagulation. Glucocorticoids may be beneficial in accelerated hypertension. Immunosuppression and plasma exchange may be helpful for catastrophic episodes of antiphospholipid syndrome but by themselves do not reduce recurrent thrombosis.

1	HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome is a dangerous complication of pregnancy associated with microvascular injury. Occurring in 0.2–0.9% of all pregnancies and in 10–20% of women with severe preeclampsia, this syndrome carries a mortality rate of 7.4–34%. Most commonly developing in the third trimester, 10% of cases occur before week 27 and 30% post-partum. Although a strong association exists between HELLP syndrome and preeclampsia, nearly 20% of cases are not preceded by recognized preeclampsia. Risk factors include abnormal placentation, family history, and elevated levels of fetal mRNA for FLT1 (vascular endothelial growth factor receptor 1) and endoglin. Patients with HELLP syndrome have higher levels of inflammatory markers (C-reactive protein, IL-1Ra, and IL-6) and soluble HLA-DR than do those with preeclampsia alone.

1	Renal failure occurs in half of patients with HELLP syndrome, although the etiology is not well understood. Limited data suggest that renal failure is the result of both preeclampsia and acute tubular necrosis. Renal histologic findings are those of TMA with endothelial cell swelling and occlusion of the capillary lumens, but luminal thrombi are typically absent. However, thrombi become more common in severe eclampsia and HELLP syndrome. Although renal failure is common, the organ that defines this syndrome is the liver. Subcapsular hepatic hematomas sometimes produce spontaneous rupture of the liver and can be life-threatening. Neurologic complications such as cerebral infarction, cerebral and brainstem hemorrhage, and cerebral edema are other potentially life-threatening complications. Nonfatal complications include placental abruption, permanent vision loss due to Purtscher-like (hemorrhagic and vaso-occlusive vasculopathy) retinopathy, pulmonary edema, bleeding, and fetal demise.

1	Many features are shared by HELLP syndrome and MAHA. Diagnosis of HELLP syndrome is complicated by the fact that aHUS and TTP also can be triggered by pregnancy. Patients with antiphospholipid syndrome also have an elevated risk of HELLP syndrome. A history of MAHA before pregnancy is of diagnostic value. Serum levels of ADAMTS13 activity are reduced (by 30–60%) in HELLP syndrome but not to the levels seen in TTP (<5%). Determination of the ratio of lactate dehydrogenase to aspartate aminotransferase may be helpful; this ratio is 13:1 in patients with HELLP syndrome and preeclampsia as opposed to 29:1 in patients without preeclampsia. Other markers, such as antithrombin III (decreased in HELLP syndrome but not in TTP) and D-dimer (elevated in HELLP syndrome but not in TTP), may also be useful. HELLP syndrome usually resolves spontaneously after delivery, although a small percentage of HELLP cases occur post-partum. Glucocorticoids may decrease inflammatory markers, although two

1	be useful. HELLP syndrome usually resolves spontaneously after delivery, although a small percentage of HELLP cases occur post-partum. Glucocorticoids may decrease inflammatory markers, although two randomized controlled trials failed to show much benefit. Plasma exchange should be considered if hemolysis is refractory to glucocorticoids and/or delivery, especially if TTP has not been ruled out.

1	Renal complications in sickle cell disease result from occlusion of the vasa recta in the renal medulla. The low partial pressure of oxygen and high osmolarity predispose to hemoglobin S polymerization and erythrocyte sickling. Sequelae include hyposthenuria, hematuria, and papillary necrosis (which can also occur in sickle trait). The kidney responds by increases in blood flow and glomerular filtration rate mediated by prostaglandins. This dependence on prostaglandins may explain the greater reduction of glomerular filtration rate by nonsteroidal anti-inflammatory drugs in these patients than in others. The glomeruli are typically enlarged. Intracapillary fragmentation and phagocytosis of sickled erythrocytes are thought to be responsible for the membranoproliferative glomerulonephritis–like lesion, and focal segmental glomerulosclerosis is seen in more advanced cases. Proteinuria is present in 20–30%, and nephrotic-range proteinuria is associated with progression to renal failure.

1	lesion, and focal segmental glomerulosclerosis is seen in more advanced cases. Proteinuria is present in 20–30%, and nephrotic-range proteinuria is associated with progression to renal failure. ACE inhibitors reduce proteinuria, although data are lacking on prevention of renal failure. Patients with sickle cell disease are also more prone to acute renal failure. The cause is thought to reflect microvascular occlusion associated with nontraumatic rhabdomyolysis, high fever, infection, and

1	Vascular Injury to the Kidney 1866 generalized sickling. Chronic kidney disease is present in 12–20% of patients. Despite the frequency of renal disease, hypertension is uncommon in patients with sickle cell disease.

1	Renal vein thrombosis either can present with flank pain, tenderness, hematuria, rapid decline in renal function, and proteinuria or can be silent. Occasionally, renal vein thrombosis is identified during a workup for pulmonary embolism. The left renal vein is more commonly involved, and two-thirds of cases are bilateral. Etiologies can be divided into three broad categories: endothelial damage, venous stasis, and hypercoagulability. Homocystinuria, endovascular intervention, and surgery can produce vascular endothelial damage. Dehydration, which is more common among male patients, is a common cause of stasis in the pediatric population. Stasis also can result from compression and kinking of the renal veins from retroperitoneal processes such as retroperitoneal fibrosis and abdominal neoplasms. Thrombosis can occur throughout the renal circulation, including the renal veins, with antiphospholipid antibody syndrome. Renal vein thrombosis can also be secondary to nephrotic syndrome,

1	Thrombosis can occur throughout the renal circulation, including the renal veins, with antiphospholipid antibody syndrome. Renal vein thrombosis can also be secondary to nephrotic syndrome, particularly membranous nephropathy. Other hypercoagulable states less commonly associated with renal vein thrombosis include proteins C and S, antithrombin deficiency, factor V Leiden, disseminated malignancy, and oral contraceptives. Severe nephrotic syndrome may also predispose patients to renal vein thrombosis.

1	Diagnostic screening can be performed with Doppler ultrasonography, which is more sensitive than ultrasonography alone. CT angiography is nearly 100% sensitive. Magnetic resonance angiography is another option but is more expensive. Treatment for renal vein thrombosis consists of anticoagulation and therapy for the underlying cause. Endovascular thrombolysis may be considered in severe cases. Occasionally, nephrectomy may be undertaken for life-threatening complications. Vena caval filters are often used to prevent migration of thrombi.

1	nephrolithiasis Gary C. Curhan Nephrolithiasis, or kidney stone disease, is a common, painful, and costly condition. Each year, billions of dollars are spent on nephrolithiasis-related activity, with the majority of expenditures on surgical treatment of existing stones. While a stone may form due to 342 crystallization of lithogenic factors in the upper urinary tract, it can subsequently move into the ureter and cause renal colic. Although nephrolithiasis is rarely fatal, patients who have had renal colic report that it is the worst pain they have ever experienced. The evidence on which to base clinical recommendations is not as strong as desired; nonetheless, most experts agree that the recurrence of most, if not all, types of stones can be prevented with careful evaluation and targeted recommendations. Preventive treatment may be lifelong; therefore, an in-depth understanding of this condition must inform the implementation of tailored interventions that are most appropriate for and

1	Preventive treatment may be lifelong; therefore, an in-depth understanding of this condition must inform the implementation of tailored interventions that are most appropriate for and acceptable to the patient.

1	There are various types of kidney stones. It is clinically important to identify the stone type, which informs prognosis and selection of the optimal preventive regimen. Calcium oxalate stones are most common (~75%); next, in order, are calcium phosphate (~15%), uric acid (~8%), struvite (~1%), and cystine (<1%) stones. Many stones are a mixture of crystal types (e.g., calcium oxalate and calcium phosphate) and also contain protein in the stone matrix. Rarely, stones are composed of medications, such as acyclovir, indinavir, and triamterene. Infectious stones, if not appropriately treated, can have devastating consequences and lead to end-stage renal disease. Consideration should be given to teaching practitioners strategies to prevent stone recurrence and its related morbidity.

1	Nephrolithiasis is a global disease. Data suggest an increasing prevalence, likely due to Westernization of lifestyle habits (e.g., dietary changes, increasing body mass index). National Health and Nutrition Examination Survey data for 2007–2010 indicate that up to 19% of men and 9% of women will develop at least one stone during their lifetime. The prevalence is ~50% lower among black individuals than among whites. The incidence of nephrolithiasis (i.e., the rate at which previously unaffected individuals develop their first stone) also varies by age, sex, and race. Among white men, the peak annual incidence is ~3.5 cases/1000 at age 40 and declines to ~2 cases/1000 by age 70. Among white women in their thirties, the annual incidence is ~2.5 cases/1000; the figure decreases to ~1.5/1000 at age 50 and beyond. In addition to the medical costs associated with nephrolithiasis, this condition also has a substantial economic impact, as those affected are often of working age. Once an

1	at age 50 and beyond. In addition to the medical costs associated with nephrolithiasis, this condition also has a substantial economic impact, as those affected are often of working age. Once an individual has had a stone, the prevention of a recurrence is essential. Published recurrence rates vary by the definitions and diagnostic methods used. Some reports have relied on symptomatic events, while others have been based on imaging. Most experts agree that radiographic evidence of a second stone should be considered to represent a recurrence, even if the stone has not yet caused symptoms.

1	Nephrolithiasis is a systemic disorder. Several conditions predispose to stone formation, including gastrointestinal malabsorption (e.g., Crohn’s disease, gastric bypass surgery), primary hyperparathyroidism, obesity, type 2 diabetes mellitus, and distal renal tubular acidosis. A number of other medical conditions are more likely to be present in individuals with a history of nephrolithiasis, including hypertension, gout, cholelithiasis, reduced bone mineral density, and chronic kidney disease.

1	Individuals with medullary sponge kidney (MSK), a condition designated by an anatomic description, often have metabolic abnormalities, such as higher levels of urine calcium and lower levels of urine citrate, and are more likely to form calcium phosphate stones. As intravenous urography is now rarely used, the diagnosis of MSK has become less frequent. Fortunately, the diagnosis of MSK does not change either the evaluation or the treatment recommendations; thus, it is not essential in pursuing the diagnosis of nephrolithiasis. Although nephrolithiasis does not directly cause upper urinary tract infections (UTIs), a UTI in the setting of an obstructing stone is a urologic emergency (“pus under pressure”) and requires urgent intervention to reestablish drainage.

1	In the consideration of the processes involved in crystal formation, it is helpful to view urine as a complex solution. A clinically useful concept is supersaturation (the point at which the concentration product exceeds the solubility product). However, even though the urine in most individuals is supersaturated with respect to one or more types of crystals, the presence of inhibitors of crystallization prevents the majority of the population from continuously forming stones. The most clinically important inhibitor of calcium-containing stones is urine citrate. While supersaturation is a calculated value (rather than being directly measured) and does not perfectly predict stone formation, it is a useful guide as it integrates the multiple factors that are measured in a 24-h urine collection.

1	Recent studies have changed the paradigm for the site of initiation of stone formation. Renal biopsies of stone formers have revealed calcium phosphate in the renal interstitium. It is hypothesized that this calcium phosphate extends down to the papilla and erodes through the papillary epithelium, where it provides a site for deposition of calcium oxalate and calcium phosphate crystals. The majority of calcium oxalate stones grow on calcium phosphate at the tip of the renal papilla (Randall’s plaque). Thus, the process of stone formation may begin years before a clinically detectable stone is identified. The processes involved in interstitial deposition are under active investigation. Risk factors for nephrolithiasis can be categorized as dietary, nondietary, or urinary.These risk factors vary by stone type and by clinical characteristics.

1	Risk factors for nephrolithiasis can be categorized as dietary, nondietary, or urinary.These risk factors vary by stone type and by clinical characteristics. Dietary Risk Factors Patients who develop stones often change their diet; therefore, studies that retrospectively assess diet may be hampered by recall bias. Some studies have examined the relation between diet and changes in the lithogenic composition of the urine, often using calculated supersaturation. However, the composition of the urine does not perfectly predict risk, and not all components that modify risk are included in the calculation of supersaturation. Thus, dietary associations are best investigated by prospective studies that examine actual stone formation as the outcome. Dietary factors that are associated with an increased risk of nephrolithiasis include animal protein, oxalate, sodium, sucrose, and fructose. Dietary factors associated with a lower risk include calcium, potassium, and phytate.

1	CALCIUM The role of dietary calcium deserves special attention. Although in the past dietary calcium had been suspected of increasing the risk of stone disease, several prospective observational studies and a randomized controlled trial have demonstrated that higher dietary calcium intake is related to a lower risk of stone formation. The reduction in risk associated with higher calcium intake may be due to a reduction in intestinal absorption of dietary oxalate that results in lower urine oxalate. Low calcium intake is contraindicated as it increases the risk of stone formation and may contribute to lower bone density in stone formers. Despite similar bioavailability, supplemental calcium may increase the risk of stone formation. The discrepancy between the risks from dietary calcium and calcium supplements may be due to the timing of supplemental calcium intake or to higher total calcium consumption leading to higher urinary calcium excretion.

1	OXALATE Urinary oxalate is derived from both endogenous production and absorption of dietary oxalate. Owing to its low and often variable bioavailability, much of the oxalate in food may not be readily absorbed. However, absorption may be higher in stone formers. Although observational studies demonstrate that dietary oxalate is only a weak risk factor for stone formation, urinary oxalate is a strong risk factor for calcium oxalate stone formation, and efforts to avoid high oxalate intake should thus be beneficial.

1	OTHER NUTRIENTS Several other nutrients have been studied and implicated in stone formation. Higher intake of animal protein may lead to increased excretion of calcium and uric acid as well as to decreased urinary excretion of citrate, all of which increase the risk of stone formation. Higher sodium and sucrose intake increases calcium excretion independent of calcium intake. Higher potassium intake decreases calcium excretion, and many potassium-rich foods increase urinary citrate excretion due to their alkali content. Other dietary factors that have been inconsistently associated with lower stone risk include magnesium and phytate.

1	Vitamin C supplements are associated with an increased risk of calcium oxalate stone formation, possibly because of raised levels of oxalate in urine. Thus, calcium oxalate stone formers should be advised to avoid vitamin C supplements. Although high doses of supplemental vitamin B6 may be beneficial in selected patients with type 1 primary hyperoxaluria, the benefit of supplemental vitamin B6 in other patients is uncertain.

1	FLUIDS AND BEVERAGES The risk of stone formation increases as urine volume decreases. When the urine output is less than 1 L/d, the risk of stone formation more than doubles. Fluid intake is the main determinant of urine volume, and the importance of fluid intake in preventing stone formation has been demonstrated in observational studies and in a randomized controlled trial. Observational studies have found that coffee, tea, beer, and wine are associated with a reduced risk of stone formation. Sugar-sweetened carbonated beverage consumption may 1867 increase risk. Nondietary Risk Factors Age, race, body size, and environment are important risk factors for nephrolithiasis. The incidence of stone disease is highest in middle-aged white men, but stones can form in infants as well as in the elderly. There is geographic variability, with the highest prevalence in the southeastern United States. Weight gain increases the risk of stone formation, and the increasing prevalence of

1	as in the elderly. There is geographic variability, with the highest prevalence in the southeastern United States. Weight gain increases the risk of stone formation, and the increasing prevalence of nephrolithiasis in the United States may be due in part to the increasing prevalence of obesity. Environmental and occupational influences that may lead to lower urine volume, such as working in a hot environment or lack of ready access to water or a bathroom, are important considerations.

1	urinary Risk Factors • URINE VOLUME As mentioned above, lower urine volume results in increased concentrations of lithogenic factors and is a common and readily modifiable risk factor. A randomized trial has demonstrated the effectiveness of elevated fluid intake in increasing urine volume and reducing the risk of stone recurrence.

1	URINE CALCIUM Higher urine calcium excretion increases the likelihood of formation of calcium oxalate and calcium phosphate stones. While the term hypercalciuria is often used, there is no widely accepted cutoff that distinguishes between normal and abnormal urine calcium excretion. In fact, the relation between urine calcium and stone risk appears to be continuous; thus the use of an arbitrary threshold should be avoided. Levels of urine calcium excretion are higher in individuals with a history of nephrolithiasis; however, the mechanisms remain poorly understood. Greater gastrointestinal calcium absorption is one important contributor, and greater bone turnover (with a resultant reduction in bone mineral density) may be another. Primary renal calcium loss, with lower serum calcium concentrations and elevated serum levels of parathyroid hormone (PTH) (and a normal 25-hydroxy vitamin D level), is rare.

1	URINE OXALATE Higher urine oxalate excretion increases the likelihood of calcium oxalate stone formation. As for urine calcium, no definition for “abnormal” urine oxalate excretion is widely accepted. Given that the relation between urine oxalate and stone risk is continuous, simple dichotomization of urine oxalate excretion is not helpful in assessing risk. The two sources of urine oxalate are endogenous generation and dietary intake. Dietary oxalate is the major contributor and also the source that can be modified. Notably, higher dietary calcium intake reduces gastrointestinal oxalate absorption and thereby reduces urine oxalate.

1	URINE CITRATE Urine citrate is a natural inhibitor of calcium-containing stones; thus, lower urine citrate excretion increases the risk of stone formation. Citrate reabsorption is influenced by the intracellular pH of proximal tubular cells. Metabolic acidosis will lead to a reduction in citrate excretion by increasing reabsorption of filtered citrate. However, a notable proportion of patients have lower urine citrate for reasons that remain unclear. URINE URIC ACID Higher urine levels of uric acid—a risk factor for uric acid stone formation—are found in individuals with excess purine consumption and rare genetic conditions that lead to overproduction of uric acid. This characteristic does not appear to be associated with the risk of calcium oxalate stone formation.

1	URINE pH Urine pH influences the solubility of some crystal types. Uric acid stones form only when the urine pH is consistently ≤5.5 or lower, whereas calcium phosphate stones are more likely to form when the urine pH is ≥6.5 or higher. Cystine is more soluble at higher urine pH. Calcium oxalate stones are not influenced by urine pH. Genetic Risk Factors The risk of nephrolithiasis is more than twofold greater in individuals with a family history of stone dis ease. This association is likely due to a combination of genetic predisposition and similar environmental exposures. While a number of monogenic disorders cause nephrolithiasis, the genetic contributors to common forms of stone disease remain to be determined.

1	1868 The two most common and well-characterized rare monogenic disorders that lead to stone formation are primary hyperoxaluria and cystinuria. Primary hyperoxaluria is an autosomal recessive disorder that causes excessive endogenous oxalate generation by the liver, with consequent calcium oxalate stone formation and crystal deposition in organs. Intraparenchymal calcium oxalate deposition in the kidney can eventually lead to renal failure. Cystinuria is an autosomal recessive disorder that causes abnormal reabsorption of filtered dibasic amino acids. The excessive urinary excretion of cystine, which is poorly soluble, leads to cystine stone formation. Cystine stones are visible on plain radiographs and often manifest as staghorn calculi or multiple bilateral stones. Repeat episodes of obstruction and instrumentation can cause chronic renal impairment. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: At present, there are no widely accepted, evidence-based guidelines for the evaluation and treatment of nephrolithiasis. However, there are standard approaches to patients with acute and chronic presentations that can reasonably guide the clinical evaluation. It typically requires weeks to months (and often much longer) for a kidney stone to grow to a clinically detectable size. Although the passage of a stone is a dramatic event, stone formation and growth are characteristically clinically silent. A stone can remain asymptomatic in the kidney for years or even decades before signs (e.g., hematuria) or symptoms (e.g., pain) become apparent. Thus, it is important to remember that the onset of symptoms, typically attributable to a stone moving into the ureter, does not provide insight into when the stone actually formed. The factors that induce stone movement are unknown.

1	Clinical Presentation and Differential Diagnosis There are two common presentations for individuals with an acute stone event: renal colic and painless gross hematuria. Renal colic is a misnomer because pain typically does not subside completely; rather, it varies in intensity. When a stone moves into the ureter, the discomfort often begins with a sudden onset of unilateral flank pain. The intensity of the pain can increase rapidly, and there are no alleviating factors. This pain, which is accompanied often by nausea and occasionally by vomiting, may radiate, depending on the location of the stone. If the stone lodges in the upper part of the ureter, pain may radiate anteriorly; if the stone is in the lower part of the ureter, pain can radiate to the ipsilateral testicle in men or the ipsilateral labium in women. Occasionally, a patient has gross hematuria without pain.

1	Other diagnoses may be confused with acute renal colic. If the stone is lodged at the right ureteral pelvic junction, symptoms may mimic those of acute cholecystitis. If the stone blocks the ureter as it crosses over the right pelvic brim, symptoms may mimic acute appendicitis, whereas blockage at the left pelvic brim may be confused with acute diverticulitis. If the stone lodges in the ureter at the ureterovesical junction, the patient may experience urinary urgency and frequency. In female patients, the latter symptoms may lead to an incorrect diagnosis of bacterial cystitis; the urine will contain red and white blood cells, but the urine culture will be negative. An obstructing stone with proximal infection may present as acute pyelonephritis. A UTI in the setting of ureteral obstruction is a medical emergency that requires immediate restoration of drainage by placement of either a ureteral stent or a percutaneous nephrostomy tube. Other conditions to consider in the differential

1	is a medical emergency that requires immediate restoration of drainage by placement of either a ureteral stent or a percutaneous nephrostomy tube. Other conditions to consider in the differential diagnosis include muscular or skeletal pain, herpes zoster, duodenal ulcer, abdominal aortic aneurysm, gynecologic conditions, ureteral stricture, and ureteral obstruction by materials other than a stone, such as a blood clot or sloughed papilla. Extraluminal processes can lead to ureteral compression and obstruction; however, because of the gradual onset, these conditions do not typically present with renal colic.

1	Diagnosis and Intervention Serum chemistry findings are typically normal, but the white blood cell count may be elevated. Examination of the urine sediment will usually reveal red and white blood cells and occasionally crystals (Fig. 342-1). The absence of hematuria does not exclude a stone, particularly when urine flow is completely obstructed by a stone.

1	The diagnosis is often made on the basis of the history, physical examination, and urinalysis. Thus, it may not be necessary to wait for radiographic confirmation before treating the symptoms. The diagnosis is confirmed by an appropriate imaging study—preferably helical CT, which is highly sensitive, allows visualization of uric acid stones (traditionally considered “radiolucent”), and is able to avoid radiocontrast (Fig. 342-2). Helical CT detects stones as small as 1 mm that may be missed by other imaging modalities. Typically, helical CT reveals a ureteral stone or evidence of recent passage (e.g., perinephric stranding or hydronephrosis), whereas a plain abdominal radiograph (kidney/ureter/bladder, or KUB) can miss a stone in the ureter or kidney, even if it is radiopaque, and does not provide information on obstruction. Abdominal ultrasound offers the advantage of avoiding radiation and provides

1	FIGuRE 342-1 Urine sediment from a patient with calcium oxalate stones (left) and a patient with cystine stones (right). Calcium oxalate dihydrate crystals are bipyramidally shaped, and cystine crystals are hexagonal. (Left panel image courtesy of Dr. John Lieske, Mayo Clinic.) FIGuRE 342-2 Coronal noncontrast CT image from a patient who presented with left-sided renal colic. An obstructing calculus, present in the distal left ureter at the level of S1, measures 10 mm in maximal dimension. There is severe left hydroureteronephrosis and associated left perinephric fat stranding. In addition, there is a non-obstructing 6-mm left renal calculus in the interpolar region. (Image courtesy of Dr. Stuart Silverman, Brigham and Women’s Hospital.) information on hydronephrosis, but it is not as sensitive as CT and images only the kidney and possibly the proximal segment of the ureter; thus most ureteral stones are not detectable by ultrasound.

1	Many patients who experience their first episode of colic seek emergent medical care. Randomized trials have demonstrated that parenterally administered nonsteroidal anti-inflammatory drugs (such as ketorolac) are just as effective as opioids in relieving symptoms and have fewer side effects. Excessive fluid administration has not been shown to be beneficial; therefore, the goal should be to maintain euvolemia. If the pain can be adequately controlled and the patient is able to take fluids orally, hospitalization can be avoided. Use of an alpha-blocker may increase the rate of spontaneous stone passage.

1	Urologic intervention should be postponed unless there is evidence of UTI, a low probability of spontaneous stone passage (e.g., a stone measuring ≥6 mm or an anatomic abnormality), or intractable pain. A ureteral stent may be placed cystoscopically, but this procedure typically requires general anesthesia, and the stent can be quite uncomfortable, may cause gross hematuria, and may increase the risk of UTI.

1	If an intervention is indicated, the selection of the most appropriate intervention is determined by the size, location, and composition of the stone; the urinary tract anatomy; and the experience of the urologist. Extracorporeal shockwave lithotripsy, the least invasive option, uses shock waves generated outside the body to fragment the stone. An endourologic approach can remove a stone by basket extraction or laser fragmentation. For large upper-tract stones, percutaneous nephrostolithotomy has the highest likelihood of rendering the patient stone-free. Advances in urologic approaches and instruments have nearly eliminated the need for open surgical procedures such as ureterolithotomy or pyelolithotomy.

1	Evaluation for Stone Prevention More than half of first-time stone formers will have a recurrence within 10 years. A careful evaluation is indicated to identify predisposing factors, which can then be modified to reduce the risk of new stone formation. It is appropriate to proceed with an evaluation even after the first stone because recurrences are common and are usually preventable with inexpensive lifestyle modifications or other treatments.

1	History A detailed history, obtained from the patient and from a thorough review of medical records, should include the number and frequency of episodes (distinguishing stone passage from stone formation) and previous imaging studies, interventions, evaluations, and treatments. Inquiries about the patient’s medical history should cover UTIs, bariatric surgery, gout, hypertension, and diabetes mellitus. A family history of stone disease may reveal a genetic predisposition. A complete list of current prescription and over-the-counter medications as well as vitamin and mineral supplements is essential. The review of systems should focus on identifying possible etiologic factors related to low urine volume (e.g., high insensible losses; low fluid intake) and gastrointestinal malabsorption as well as on ascertaining how frequently the patient voids during the day and overnight.

1	A large body of compelling evidence has demonstrated the important role of diet in stone disease. Thus, the dietary history should encompass information on usual dietary habits (meals and snacks), calcium intake, consumption of high-oxalate foods (spinach, rhubarb, potatoes), and fluid intake (including specific beverages typically consumed). Physical Examination The physical examination should assess weight, blood pressure, costovertebral angle tenderness, and lower-extremity edema as well as signs of other systemic conditions such as primary hyperparathyroidism and gout.

1	Laboratory Evaluation If not recently measured, the following serum levels should be determined: electrolytes (to uncover hypokalemia or renal tubular acidosis), creatinine, calcium, and uric acid. The PTH level should be measured if indicated by high-normal or elevated serum and urine calcium concentrations. Often, 25-hydroxy vitamin D is measured in concert with PTH to investigate the possible role of secondarily elevated PTH levels in the setting of vitamin D insufficiency. The urinalysis, including examination of the sediment, can provide useful information. In individuals with asymptomatic residual renal stones, red and white blood cells are frequently present in urine. If there is concern about the possibility of an infection, a urine culture should be performed. The sediment may also reveal crystals (Fig. 342-1), which may help identify the stone type and also provide prognostic information, as crystalluria is a strong risk factor for new stone formation.

1	The results from 24-h urine collections serve as the cornerstone on which therapeutic recommendations are based. Recommendations on lifestyle modification should be deferred until urine collection is complete. As a baseline assessment, patients should collect at least two 24-h urine samples while consuming their usual diet and usual volume of fluid. The following factors should be measured: total volume, calcium, oxalate, citrate, uric acid, sodium, potassium, phosphorus, pH, and creatinine. When available, the calculated supersaturation is also informative. There is substantial day-to-day variability in the 24-h excretion of many relevant factors; therefore, obtaining values from two collections is important before committing a patient to long-term lifestyle changes or medication. The interpretation of the 24-h urine results should take into account that the collections are usually performed on a weekend day when the patient is staying at home; an individual’s habits may differ

1	interpretation of the 24-h urine results should take into account that the collections are usually performed on a weekend day when the patient is staying at home; an individual’s habits may differ dramatically (beneficially or detrimentally) at work or outside the home. Specialized testing, such as calcium loading or restriction, is not recommended as it does not influence clinical recommendations.

1	Stone composition analysis is essential if a stone or fragment is available; patients should be encouraged to retrieve passed stones. The stone type cannot be determined with certainty from 24-h urine results. Imaging The “gold standard” diagnostic test is helical CT without contrast. If not already performed during an acute episode, a CT should be considered to definitively establish the baseline stone burden. A suboptimal imaging study may not detect a residual stone that, if subsequently passed, would be mistaken for a new stone. In this instance, the preventive medical regimen might be unnecessarily changed as the result of a preexisting stone.

1	Recommendations for follow-up imaging should be tailored to the individual patient. While CT provides the best information, the radiation dose is substantially higher than from other modalities; therefore, CT should be performed only if the results will lead to a change in clinical recommendations. Although they are less sensitive, renal ultrasound or a KUB exam are typically used to minimize radiation exposure, with recognition of the limitations.

1	Prevention of New Stone Formation Recommendations for preventing stone formation depend on the stone type and the results of metabolic evaluation. After remediable secondary causes of stone formation (e.g., primary hyperparathyroidism) are excluded, the focus should turn to modification of the urine composition to reduce the risk of new stone formation. The urinary constituents are continuous variables, and the associated risk is continuous; thus, there is no definitive threshold. Dichotomization into “normal” and “abnormal” can be misleading and should be avoided.

1	For all stone types, consistently diluted urine reduces the likelihood of crystal formation. The urine volume should be at least 2 L/d. Because of differences in insensible fluid losses and fluid intake from food sources, the required total fluid intake will vary from person to person. Rather than specify how much to drink, it is more helpful to educate patients about how much more they need to drink in light of their 24-h urine volume. For example, if the daily urine volume is 1.5 L, then the patient should be advised to drink at least 0.5 L more per day in order to increase the urine volume to the goal of 2 L/day. RECOMMENDATIONS FOR SPECIFIC STONE TYPES Calcium Oxalate Risk factors for calcium oxalate stones include higher urine calcium, higher urine oxalate, and lower urine citrate. This stone type is insensitive to pH in the physiologic range.

1	Individuals with higher urine calcium excretion tend to absorb a higher percentage of ingested calcium. Nevertheless, dietary calcium restriction is not beneficial and, in fact, is likely to be harmful (see “Dietary Risk Factors,” above). In a randomized trial in men with high urine calcium and recurrent calcium oxalate stones, a diet containing 1200 mg of calcium and a low intake of sodium and animal protein significantly reduced subsequent stone formation from that with a low-calcium diet (400 mg/d). Excessive calcium intake (>1200 mg/d) should be avoided.

1	A thiazide diuretic, in doses higher than those used to treat hypertension, can substantially lower urine calcium excretion. Several randomized controlled trials have demonstrated that thiazide diuretics can reduce calcium oxalate stone recurrence by ~50%. When a thiazide is prescribed, dietary sodium restriction is essential to obtain the desired reduction in urinary calcium excretion. While bisphosphonates may reduce urine calcium excretion in some individuals, there are no data on whether this class of medication can reduce stone formation; therefore, bisphosphonates cannot be recommended solely for stone prevention at present. A reduction in urine oxalate will in turn reduce the supersaturation of calcium oxalate. In patients with the common form of nephrolithiasis, avoiding high-dose vitamin C supplements is the only known strategy that reduces endogenous oxalate production.

1	Oxalate is a metabolic end product; therefore, any dietary oxalate that is absorbed will be excreted in the urine. Reducing absorption of exogenous oxalate involves two approaches. First, the avoidance of foods that contain high amounts of oxalate, such as spinach, rhubarb, and potatoes, is prudent. However, extreme oxalate restriction has not been demonstrated to reduce stone recurrence and could be harmful to overall health, given other health benefits of many foods that are erroneously considered to be high in oxalate. Controversy exists regarding the most clinically relevant measure of the oxalate content of foods (e.g., bioavailability). Notably, the absorption of oxalate is reduced by higher calcium intake; therefore, individuals with higher-than-desired urinary oxalate should be counseled to consume adequate calcium. Oxalate absorption can be influenced by the intestinal microbiota, depending on the presence of oxalate-degrading bacteria. Currently, however, there are no

1	be counseled to consume adequate calcium. Oxalate absorption can be influenced by the intestinal microbiota, depending on the presence of oxalate-degrading bacteria. Currently, however, there are no available therapies to alter the microbiota that beneficially affect urinary oxalate excretion over the long term.

1	Citrate is a natural inhibitor of calcium oxalate and calcium phosphate stones. Higher-level consumption of foods rich in alkali (i.e., fruits and vegetables) can increase urine citrate. For patients with lower urine citrate in whom dietary modification does not adequately increase urine citrate, the addition of supplemental alkali (typically potassium citrate) will lead to an increase in urinary citrate excretion. Sodium salts, such as sodium bicarbonate, while successful in raising urine citrate, are typically avoided due to the adverse effects of sodium on urine calcium excretion.

1	Past reports suggested that higher levels of urine uric acid may increase the risk of calcium oxalate stones, but more recent studies do not support this association. However, allopurinol reduced stone recurrence in one randomized controlled trial in patients with calcium oxalate stones and high urine uric acid levels. The lack of association between urine uric acid level and calcium oxalate stones suggests that a different mechanism underlies the observed beneficial effect of allopurinol. Additional dietary modifications may be beneficial in reducing stone recurrence. Restriction of nondairy animal protein (e.g., meat, chicken, seafood) is a reasonable approach and may result in higher excretion of citrate and lower excretion of calcium. In addition, reducing sodium intake to <2.5 g/d may decrease urinary excretion of calcium. Sucrose and fructose intake should be minimized.

1	For adherence to a dietary pattern that is more manageable for patients than manipulating individual nutrients, the DASH (Dietary Approaches to Stop Hypertension) diet provides an appropriate and readily available option. Randomized trials have conclusively shown the DASH diet to reduce blood pressure. At present, only data from observational studies are available, but these demonstrate a strong and consistent inverse association between the DASH diet and risk of stone formation. Calcium Phosphate Calcium phosphate stones share risk factors with calcium oxalate stones, including higher concentrations of urine calcium and lower concentrations of urine citrate, but additional factors deserve attention. Higher urine phosphate levels and higher urine pH (typically ≥6.5) are associated with an increased likelihood of calcium phosphate stone formation. Calcium phosphate stones are more common in patients with distal renal tubular acidosis and primary hyperparathyroidism.

1	There are no randomized trials on which to base preventive recommendations for calcium phosphate stone formers, so the interventions are focused on modification of the recognized risk factors. Thiazide diuretics (with sodium restriction) may be used to reduce urine calcium, as described above for calcium oxalate stones. In patients with low urine citrate levels, alkali supplements (e.g., potassium citrate) may be used to increase these concentrations. However, the urine pH of these patients should be monitored carefully because supplemental alkali can raise urine pH, thereby potentially increasing the risk of stone formation. Reduction of dietary phosphate may be beneficial by reducing urine phosphate excretion.

1	uric Acid The two main risk factors for uric acid stones are persistently low urine pH and higher uric acid excretion. Urine pH is the predominant influence on uric acid solubility; therefore, the mainstay of prevention of uric acid stone formation entails increasing urine pH. While acidifying the urine is not easily done, alkalinizing the urine can be readily achieved by increasing the intake of foods rich in alkali (e.g., fruits and vegetables) and reducing the intake of foods that produce acid (e.g., animal flesh). If necessary, supplementation with bicarbonate or citrate salts (preferably potassium citrate) can be used to reach the recommended pH goal of 6 to 7 throughout the day and night.

1	Urine uric acid excretion is determined by uric acid generation. Uric acid is the end product of purine metabolism; thus reduced consumption of purine-containing foods can lower urine uric acid excretion. It is noteworthy that the serum uric acid level is dependent on the fractional excretion of uric acid and therefore does not provide information on urine uric acid excretion. For example, an individual with high uric acid generation and concurrent high fractional excretion of uric acid will have high urine uric acid excretion with a normal (or even low) serum uric acid level. If alkalinization of the urine alone is not successful and if dietary modifications do not reduce urine uric acid sufficiently, then the use of a xanthine oxidase inhibitor, such as allopurinol or febuxostat, can reduce urine uric acid excretion by 40–50%.

1	Cystine Cystine excretion is not easily modified. Long-term dietary cystine restriction is not feasible and is unlikely to be successful; thus the focus for cystine stone prevention is on increasing cystine solubility. This goal may be achieved by treatment with medication that covalently binds to cystine (tiopronin and penicillamine) and a medication that raises urine pH. Tiopronin is the preferred choice due to its better adverse event profile. The preferred alkalinizing agent is potassium citrate as sodium salts may increase cystine excretion. As with all stone types, and especially in patients with cystinuria, maintaining a high urine volume is an essential component of the preventive regimen.

1	Struvite Struvite stones, also known as infection stones or triple-phosphate stones, form only when the upper urinary tract is infected with urease-producing bacteria such as Proteus mirabilis, Klebsiella pneumoniae, or Providencia species. Urease produced by these bacteria hydrolyzes urea and may elevate the urine pH to a supraphysiologic level (>8.0). Struvite stones may grow quickly and fill the renal pelvis (staghorn calculi). Struvite stones require complete removal by a urologist. New stone formation can be avoided by the prevention of UTIs. In patients with recurrent upper UTIs (e.g., some individuals with surgically altered urinary drainage or spinal cord injury), the urease inhibitor acetohydroxamic acid can be considered; however, this agent should be used with caution because of potential side effects.

1	In general, the preventive regimens described above do not cure the underlying pathophysiologic process. Thus these recommendations typically need to be followed for the patient’s lifetime, and it is essential to tailor recommendations in a way that is acceptable to the patient. Because the memory of the acute stone event fades and patients often return to old habits (e.g., insufficient fluid intake), long-term follow-up is important to ensure that the preventive regimen has been implemented and has resulted in the desired reduction in the risk of new stone formation.

1	Follow-up imaging should be planned thoughtfully. Many patients with recurrent episodes of renal colic that lead to emergency room visits often undergo repeat CT studies. While CT does provide the best information, the radiation dose is substantially higher than that with plain abdominal radiography (KUB). Small stones may be missed by KUB, and ultrasound has a limited ability to determine size and number of stones. Minimizing radiation exposure should be a goal of the long-term follow-up plan and must be balanced against the gain in diagnostic information.

1	urinary tract Obstruction Julian L. Seifter Obstruction to the flow of urine, with attendant stasis and elevation in urinary tract pressure, impairs renal and urinary conduit functions and is a common cause of acute and chronic kidney disease (obstruc-tive nephropathy). With early relief of obstruction, the defects in func-343 tion usually disappear completely. However, chronic obstruction may produce permanent loss of renal mass (renal atrophy) and excretory capability, as well as enhanced susceptibility to local infection and stone formation. Early diagnosis and prompt therapy are, therefore, 1871 essential to minimize the otherwise devastating effects of obstruction on kidney structure and function.

1	Obstruction to urine flow can result from intrinsic or extrinsic mechanical blockade as well as from functional defects not associated with fixed occlusion of the urinary drainage system. Mechanical obstruction can occur at any level of the urinary tract, from the renal calyces to the external urethral meatus. Normal points of narrowing, such as the ureteropelvic and ureterovesical junctions, bladder neck, and urethral meatus, are common sites of obstruction. When obstruction is above the level of the bladder, unilateral dilatation of the ureter (hydroureter) and renal pyelocalyceal system (hydronephrosis) occurs; lesions at or below the level of the bladder cause bilateral involvement.

1	Common forms of obstruction are listed in Table 343-1. Childhood causes include congenital malformations, such as narrowing of the ureteropelvic junction and abnormal insertion of the ureter into the bladder, the most common cause. Vesicoureteral reflux in the absence of urinary tract infection or bladder neck obstruction often resolves with age. Reinsertion of the ureter into the bladder is indicated if reflux is severe and unlikely to improve spontaneously, if renal function deteriorates, or if urinary tract infections recur despite chronic antimicrobial therapy. Vesicoureteral reflux may cause prenatal hydronephrosis and, if severe, can lead to recurrent urinary infections and renal scarring in childhood. Posterior urethral valves are the most common cause of bilateral hydronephrosis in boys. In adults, urinary tract obstruction (UTO) is due mainly to acquired defects. Pelvic tumors, calculi, and urethral stricture predominate. Ligation of, or injury to, the ureter during pelvic or

1	boys. In adults, urinary tract obstruction (UTO) is due mainly to acquired defects. Pelvic tumors, calculi, and urethral stricture predominate. Ligation of, or injury to, the ureter during pelvic or colonic surgery can lead to hydronephrosis which, if unilateral, may remain undetected. Obstructive uropathy may also result from extrinsic neoplastic (carcinoma of

1	Tumor Cancer of prostate Infection Calculi Cancer of bladder Trauma Pregnant uterus Carcinoma of cervix, Trauma colon

1	Retroperitoneal fibrosis Aortic aneurysm Trauma Uterine leiomyomata Carcinoma of uterus, prostate, bladder, colon, rectum Lymphoma Pelvic inflammatory disease, 1872 cervix or colon) or inflammatory disorders. Lymphomas and pelvic or colonic neoplasms with retroperitoneal involvement are causes of ureteral obstruction. As many as 50% of men over 40 years old may have lower urinary tract symptoms associated with benign prostatic hypertrophy, but these symptoms may occur without bladder outlet obstruction. Functional impairment of urine flow occurs when voiding is altered by abnormal pontine or sacral centers of micturition control. It may be asymptomatic or associated with lower urinary tract symptoms such as frequency, urgency, urge and postmicturition incontinence, nocturia, straining to void, slow stream, hesitancy, or a feeling of incomplete emptying. A history should be sought for trauma, back injury, surgery, diabetes, neurologic or psychiatric conditions, and medications. Causes

1	void, slow stream, hesitancy, or a feeling of incomplete emptying. A history should be sought for trauma, back injury, surgery, diabetes, neurologic or psychiatric conditions, and medications. Causes include neurogenic bladder, often with adynamic ureter, and vesicoureteral reflux. Reflux in children may result in severe unilateral or bilateral hydroureter and hydronephrosis. Urinary retention may be the consequence of α-adrenergic and anticholinergic agents, as well as opiates. Hydronephrosis in pregnancy is due to relaxational effects of progesterone on smooth muscle of the renal pelvis, as well as ureteral compression by the enlarged uterus. Diagnostic tools to identify anatomic obstruction include urinary flow measurements and a postvoid residual. Cystourethroscopy and urodynamic studies may be reserved for the symptomatic patient to assess the filling phase (cystometry), pressure-volume relationship of the bladder, bladder compliance, and capacity. Pressure-flow analysis

1	studies may be reserved for the symptomatic patient to assess the filling phase (cystometry), pressure-volume relationship of the bladder, bladder compliance, and capacity. Pressure-flow analysis evaluates bladder contractility and bladder outlet resistance during voiding. Bladder obstruction is characterized by high pressures in women, whereas in men, a diagnosis of bladder outlet obstruction is based on flow rate and voiding pressures. A voiding cystourethrogram may be useful in evaluating incomplete emptying and bladder neck and urethral pathology.

1	The pathophysiology and clinical features of UTO are summarized in Table 343-2. Pain, the symptom that most commonly leads to medical attention, is due to distention of the collecting system or renal capsule. Pain severity is influenced more by the rate at which distention develops than by the degree of distention. Acute supravesical obstruction, as from a stone lodged in a ureter (Chap. 342), is associated with excruciating pain, known as renal colic. This pain often radiates to the ↑ Reabsorption of Na+, urea, water Vasodilator prostaglandins, nitric oxide ↓↓ GFR AVP-insensitive polyuria ↓ Concentrating ↑ Vasoconstrictor Natriuresis ability prostaglandins Hyperkalemic, hyperchlo- production ↓ Transport functions for Na+, K+, H+ Release of Obstruction Abbreviations: AVP, arginine vasopressin; GFR, glomerular filtration rate.

1	Release of Obstruction Abbreviations: AVP, arginine vasopressin; GFR, glomerular filtration rate. lower abdomen, testes, or labia. By contrast, more insidious causes of obstruction, such as chronic narrowing of the ureteropelvic junction, may produce little or no pain and yet result in total destruction of the affected kidney. Flank pain that occurs only with micturition is pathognomonic of vesicoureteral reflux. Obstruction of urine flow results in an increase in hydrostatic pressures proximal to the site of obstruction. It is this buildup of pressure that leads to the accompanying pain, the distention of the collecting system in the kidney, and elevated intratubular pressures that initiate tubular dysfunction. As the increased hydrostatic pressure is expressed in the urinary space of the glomeruli, further filtration decreases or stops completely.

1	Azotemia develops when overall excretory function is impaired, often in the setting of bladder outlet obstruction, bilateral renal pelvic or ureteric obstruction, or unilateral disease in a patient with a solitary functioning kidney. Complete bilateral obstruction should be suspected when acute renal failure is accompanied by anuria. Any patient with renal failure otherwise unexplained, or with a history of nephrolithiasis, hematuria, diabetes mellitus, prostatic enlargement, pelvic surgery, trauma, or tumor should be evaluated for UTO.

1	In the acute setting, partial, bilateral obstruction may mimic prerenal azotemia with concentrated urine and sodium retention. However, with more prolonged obstruction, symptoms of polyuria and nocturia commonly accompany partial UTO and result from diminished renal concentrating ability. Impairment of transcellular salt reabsorption in the proximal tubule, medullary thick ascending limb of Henle, and collecting duct cells is due to downregulation of transport proteins including the Na+, K+ adenosine triphosphatase (ATPase), NaK2Cl cotransporter (NKCC) in the thick ascending limb, and the epithelial Na+ channel (ENaC) in collecting duct cells. Consequences include failure to produce urine free of salt (natriuresis) and loss of medullary hypertonicity producing a urinary concentrating defect. In addition to direct effects on renal transport mechanisms, increased prostaglandin E2 (PGE2) (due to induction of cyclooxygenase-2 [COX-2]), angiotensin II (with its downregulation of Na+

1	defect. In addition to direct effects on renal transport mechanisms, increased prostaglandin E2 (PGE2) (due to induction of cyclooxygenase-2 [COX-2]), angiotensin II (with its downregulation of Na+ transporters), and atrial or B-type natriuretic peptides (ANP or BNP) (due to volume expansion in the azotemic patient) contribute to the decreased salt reabsorption along the nephron.

1	Dysregulation of aquaporin-2 water channels in the collecting duct contributes to the polyuria. The defect usually does not improve with administration of vasopressin and is therefore a form of acquired nephrogenic diabetes insipidus. Wide fluctuations in urine output in a patient with azotemia should always raise the possibility of intermittent or partial UTO. If fluid intake is inadequate, severe dehydration and hypernatremia may develop. However, as with other causes of poor renal function, excesses of salt and water intake may result in edema and hyponatremia.

1	Partial bilateral UTO often results in acquired distal renal tubular acidosis, hyperkalemia, and renal salt wasting. The H+-ATPase, situated on the apical membrane of the intercalated cells of the collecting duct, is critical for distal H+ secretion. The trafficking of intracellular H+ pumps from the cytoplasm to the cell membrane is disrupted in UTO. The decreased function of the ENaC, in the apical membrane of neighboring collecting duct principal cells, contributes to decreased Na+ reabsorption (salt-wasting), decreased electronegativity of the tubule lumen, and therefore decreased K+ secretion via K+ channels (hyperkalemia) and H+ secretion via the H+-ATPases (distal renal tubular acidosis [RTA]). Proximal tubule ammoniagenesis, important to the elimination of H+ as NH4+, is impaired. These defects in tubule function are often accompanied by renal tubulointerstitial damage. Azotemia with hyperkalemia and metabolic acidosis should prompt consideration of UTO.

1	The renal interstitium becomes edematous and infiltrated with mononuclear inflammatory cells early in UTO. Later, interstitial fibrosis and atrophy of the papillae and medulla occur and precede these processes in the cortex. The increase in angiotensin II noted in UTO contributes to the inflammatory response and fibroblast accumulation through mechanisms involving profibrotic cytokines. With time, this process leads to chronic kidney damage. FIGuRE 343-1 Diagnostic approach for urinary tract obstruction in unexplained renal failure. CT, computed tomography.

1	FIGuRE 343-1 Diagnostic approach for urinary tract obstruction in unexplained renal failure. CT, computed tomography. UTO must always be considered in patients with urinary tract infections or urolithiasis. Urinary stasis encourages the growth of organisms. Urea-splitting bacteria are associated with magnesium ammonium phosphate (struvite) calculi. Hypertension is frequent in acute and sub-acute unilateral obstruction and is usually a consequence of increased release of renin by the involved kidney. Chronic kidney disease from bilateral UTO, often associated with extracellular volume expansion, may result in significant hypertension. Erythrocytosis, an infrequent complication of obstructive uropathy, is secondary to increased erythropoietin production.

1	A history of difficulty in voiding, pain, infection, or change in urinary volume is common. Evidence for distention of the kidney or urinary bladder can often be obtained by palpation and percussion of the abdomen. A careful rectal and genital examination may reveal enlargement or nodularity of the prostate, abnormal rectal sphincter tone, or a rectal or pelvic mass.

1	Urinalysis may reveal hematuria, pyuria, and bacteriuria. The urine sediment is often normal, even when obstruction leads to marked azotemia and extensive structural damage. An abdominal scout film may detect nephrocalcinosis or a radiopaque stone. As indicated in Fig. 343-1, if UTO is suspected, a bladder catheter should be inserted. Abdominal ultrasonography should be performed to evaluate renal and bladder size, as well as pyelocalyceal contour. Ultrasonography is approximately 90% specific and sensitive for detection of hydronephrosis. False-positive results are associated with diuresis, renal cysts, or the presence of an extrarenal pelvis, a normal congenital variant. Congenital ureteropelvic junction (UPJ) obstruction may be mistaken for renal cystic disease. Hydronephrosis may be absent on ultrasound when obstruction is less than 48 h in duration or associated with volume contraction, staghorn calculi, retroperitoneal fibrosis, or infiltrative renal disease. Duplex Doppler

1	be absent on ultrasound when obstruction is less than 48 h in duration or associated with volume contraction, staghorn calculi, retroperitoneal fibrosis, or infiltrative renal disease. Duplex Doppler ultrasonography may detect an increased resistive index in urinary obstruction.

1	Recent advances in technology have led to alternatives and have largely replaced the once standard intravenous urogram in the further evaluation of UTO. The high-resolution multidetector row computed tomography (CT) scan in particular has advantages of visualizing the retroperitoneum, as well as identifying both intrinsic and extrinsic sites of obstruction. Noncontrast CT scans improve visualization of the urinary tract in the patient with renal impairment and are safer for patients at risk for contrast nephropathy. Magnetic resonance urography is a promising technique but, at this time, not superior to the CT scan and carries the risk of certain gadolinium agents in patients with renal insufficiency, i.e., nephrogenic systemic fibrosis. The intravenous urogram may define the site of obstruction and demonstrate dilatation of the calyces, renal pelvis, and ureter above the obstruction. The ureter may be tortuous in chronic obstruction. Radionuclide scans are able to give differential

1	and demonstrate dilatation of the calyces, renal pelvis, and ureter above the obstruction. The ureter may be tortuous in chronic obstruction. Radionuclide scans are able to give differential renal function but give less anatomic detail than CT or intravenous urography (IVU).

1	To facilitate visualization of a suspected lesion in a ureter or renal pelvis, retrograde or antegrade urography should be attempted. These procedures do not carry risk of contrast-induced acute renal failure in patients with renal insufficiency. The retrograde approach involves catheterization of the involved ureter under cystoscopic control, whereas the antegrade technique necessitates percutaneous placement of a catheter into the renal pelvis. Although the antegrade approach may provide immediate decompression of a unilateral obstructing lesion, many urologists initially attempt the retrograde approach unless the catheterization is unsuccessful. Voiding cystourethrography is of value in the diagnosis of vesicoureteral reflux and bladder neck and urethral obstructions. Postvoiding films reveal residual urine. Endoscopic visualization by the urologist often permits precise identification of lesions involving the urethra, prostate, bladder, and ureteral orifices.

1	UTO complicated by infection requires immediate relief of obstruction to prevent development of generalized sepsis and progressive renal damage. Sepsis necessitates prompt urologic intervention. Drainage may be achieved by nephrostomy, ureterostomy, or ureteral, urethral, or suprapubic catheterization. Prolonged antibiotic treatment may be necessary. Chronic or recurrent infections in a 1874 poorly functioning obstructed kidney may necessitate nephrectomy. When infection is not present, surgery is often delayed until acid-base, fluid, and electrolyte status is restored. Nevertheless, the site of obstruction should be ascertained as soon as feasible. Elective relief of obstruction is usually recommended in patients with urinary retention, recurrent urinary tract infections, persistent pain, or progressive loss of renal function. Benign prostatic hypertrophy may be treated medically with α-adrenergic blockers and 5α-reductase inhibitors. Functional obstruction secondary to neurogenic

1	or progressive loss of renal function. Benign prostatic hypertrophy may be treated medically with α-adrenergic blockers and 5α-reductase inhibitors. Functional obstruction secondary to neurogenic bladder may be decreased with the combination of frequent voiding and cholinergic drugs.

1	With relief of obstruction, the prognosis regarding return of renal function depends largely on whether irreversible renal damage has occurred. When obstruction is not relieved, the course will depend mainly on whether the obstruction is complete or incomplete and bilateral or unilateral, as well as whether or not urinary tract infection is also present. Complete obstruction with infection can lead to total destruction of the kidney within days. Partial return of glomerular filtration rate may follow relief of complete obstruction of 1 and 2 weeks’ duration, but after 8 weeks of obstruction, recovery is unlikely. In the absence of definitive evidence of irreversibility, every effort should be made to decompress the obstruction in the hope of restoring renal function at least partially. A renal radionuclide scan, performed after a prolonged period of decompression, may be used to predict the reversibility of renal dysfunction.

1	Relief of bilateral, but not unilateral, complete obstruction commonly results in polyuria, which may be massive. The urine is usually hypo-tonic and may contain large amounts of sodium chloride, potassium, phosphate, and magnesium. The natriuresis is due in part to the normal correction of extracellular volume expansion, the increase in natriuretic factors accumulated during the period of renal failure, and depressed salt and water reabsorption when urine flow is reestablished. The retained urea is excreted with improved GFR, resulting in an osmotic diuresis which increases the urine volume of electrolyte-free water. In the majority of patients, this diuresis results in the appropriate excretion of the excesses of retained salt and water. When extracellular volume and composition return to normal, the diuresis usually abates spontaneously. Occasionally, iatrogenic expansion of extracellular volume is responsible for, or sustains, the diuresis observed in the postobstructive period.

1	to normal, the diuresis usually abates spontaneously. Occasionally, iatrogenic expansion of extracellular volume is responsible for, or sustains, the diuresis observed in the postobstructive period. Replacement with intravenous fluids in amounts less than urinary losses usually prevents this complication. More aggressive fluid management is required in the setting of hypovolemia, hypotension, or disturbances in serum electrolyte concentrations.

1	The loss of electrolyte-free water with urea may result in hypernatremia. Serum and urine sodium and osmolal concentrations should guide the use of appropriate intravenous replacement. Often replacement with 0.45% saline is required. Relief of obstruction may be followed by urinary salt and water losses severe enough to provoke profound dehydration and vascular collapse. In these patients, decreased tubule reabsorptive capacity is probably responsible for the marked diuresis. Appropriate therapy in such patients includes intravenous administration of salt-containing solutions to replace sodium and volume deficits.

1	Approach to the Patient with Gastrointestinal Disease William L. Hasler, Chung Owyang ANATOMIC CONSIDERATIONS The gastrointestinal (GI) tract extends from the mouth to the anus 344 SEC Tion 1 and is composed of several organs with distinct functions. Specialized independently controlled thickened sphincters that assist in gut compartmentalization separate the organs. The gut wall is organized into well-defined layers that contribute to functional activities in each region. The mucosa is a barrier to luminal contents or a site for transfer of fluids or nutrients. Gut smooth muscle in association with the enteric nervous system mediates propulsion from one region to the next. Many GI organs possess a serosal layer that provides a supportive foundation but that also permits external input.

1	Interactions with other organ systems serve the needs both of the gut and the body. Pancreaticobiliary conduits deliver bile and enzymes into the duodenum. A rich vascular supply is modulated by GI tract activity. Lymphatic channels assist in gut immune activities. Intrinsic gut wall nerves provide the basic controls for propulsion and fluid regulation. Extrinsic neural input provides volitional or involuntary control to degrees that are specific for each gut region.

1	The GI tract serves two main functions—assimilating nutrients and eliminating waste. The gut anatomy is organized to serve these functions. In the mouth, food is processed, mixed with salivary amylase, and delivered to the gut lumen. The esophagus propels the bolus into the stomach; the lower esophageal sphincter prevents oral reflux of gastric contents. The esophageal mucosa has a protective squamous histology, which does not permit significant diffusion or absorption. Propulsive esophageal activities are exclusively aboral and coordinate with relaxation of the upper and lower esophageal sphincters on swallowing.

1	The stomach furthers food preparation by triturating and mixing the bolus with pepsin and acid. Gastric acid also sterilizes the upper gut. The proximal stomach serves a storage function by relaxing to accommodate the meal. The distal stomach exhibits phasic contractions that propel solid food residue against the pylorus, where it is repeatedly propelled proximally for further mixing before it is emptied into the duodenum. Finally, the stomach secretes intrinsic factor for vitamin B12 absorption.

1	The small intestine serves most of the nutrient absorptive function of the gut. The intestinal mucosa exhibits villus architecture to provide maximal surface area for absorption and is endowed with specialized enzymes and transporters. Triturated food from the stomach mixes with pancreatic juice and bile in the duodenum to facilitate digestion. Pancreatic juice contains the main enzymes for carbohydrate, protein, and fat digestion as well as bicarbonate to optimize the pH for activation of these enzymes. Bile secreted by the liver and stored in the gallbladder is essential for intestinal lipid digestion. The proximal intestine is optimized for rapid absorption of nutrient breakdown products and most minerals, whereas the ileum is better suited for absorption of vitamin B12 and bile acids. The small intestine also aids in waste elimination. Bile contains by-products of erythrocyte degradation, toxins, metabolized and unmetabolized medications, and cholesterol. Motor function of the

1	The small intestine also aids in waste elimination. Bile contains by-products of erythrocyte degradation, toxins, metabolized and unmetabolized medications, and cholesterol. Motor function of the small intestine delivers indigestible food residue and sloughed enterocytes into the colon for further processing. The small intestine terminates in the ileocecal junction, a sphincteric structure that prevents coloileal reflux and maintains small-intestinal sterility.

1	The colon prepares the waste material for controlled evacuation. The colonic mucosa dehydrates the stool, decreasing daily fecal volumes from 1000–1500 mL delivered from the ileum to 100–200 mL expelled from the rectum. The colonic lumen possesses a dense bacterial colonization that ferments undigested carbohydrates and short-chain fatty acids. Whereas transit times in the esophagus are on the order of seconds and times in the stomach and small intestine range from minutes to a few hours, propagation through the colon takes more than 1 day in most individuals. Colonic motor patterns exhibit a to-and-fro character that facilitates slow fecal desiccation. The proximal colon serves to mix and absorb fluid, while the distal colon exhibits peristaltic contractions and mass actions that function to expel the stool. The colon terminates in the anus, a structure with volitional and involuntary controls to permit retention of the fecal bolus until it can be released in a socially convenient

1	to expel the stool. The colon terminates in the anus, a structure with volitional and involuntary controls to permit retention of the fecal bolus until it can be released in a socially convenient setting.

1	GI function is modified by influences outside of the gut. Unlike other organ systems, the gut is in continuity with the outside environment. Thus, protective mechanisms are vigilant against deleterious effects of foods, medications, toxins, and infectious organisms. Mucosal immune mechanisms include chronic lymphocyte and plasma cell populations in the epithelial layer and lamina propria backed up by lymph node chains to prevent noxious agents from entering the circulation. Antimicrobial peptides secreted by Paneth cells in the intestine further contribute to the defense mechanisms against pathogens in the lumen. All substances absorbed into the bloodstream are filtered through the liver via the portal venous circulation. In the liver, many drugs and toxins are detoxified by a variety of mechanisms. Although intrinsic nerves control most basic gut activities, extrinsic neural input modulates many functions. Two activities under voluntary control are swallowing and defecation. Many

1	of mechanisms. Although intrinsic nerves control most basic gut activities, extrinsic neural input modulates many functions. Two activities under voluntary control are swallowing and defecation. Many normal GI reflexes involve extrinsic vagus or splanchnic nerve pathways. The brain-gut axis further alters function in regions not under volitional regulation. As an example, stress has potent effects on gut motor, secretory, and sensory functions.

1	GI diseases develop as a result of abnormalities within or outside of the gut and range in severity from those that produce mild symptoms and no long-term morbidity to those with intractable symptoms or adverse outcomes. Diseases may be localized to one organ or exhibit diffuse involvement at many sites. GI diseases are manifestations of alterations in nutrient assimilation or waste evacuation or in the activities supporting these main functions.

1	GI diseases are manifestations of alterations in nutrient assimilation or waste evacuation or in the activities supporting these main functions. Impaired Digestion and Absorption Diseases of the stomach, intestine, biliary tree, and pancreas can disrupt digestion and absorption. The most common intestinal maldigestion syndrome, lactase deficiency, produces gas and diarrhea after ingestion of dairy products and has no adverse outcomes. Other intestinal enzyme deficiencies produce similar symptoms after ingestion of other simple sugars. Conversely, celiac disease, bacterial overgrowth, infectious enteritis, Crohn’s ileitis, and radiation damage, which affect digestion and/or absorption more diffusely, produce anemia, dehydration, electrolyte disorders, or malnutrition. Gastric hypersecretory conditions such as Zollinger-Ellison

1	Approach to the Patient with Gastrointestinal Disease 1876 syndrome damage the intestinal mucosa, impair pancreatic enzyme activation, and accelerate transit due to excess gastric acid. Biliary obstruction from stricture or neoplasm impairs fat digestion. Impaired pancreatic enzyme release in chronic pancreatitis or pancreatic cancer decreases intraluminal digestion and can lead to malnutrition.

1	Altered Secretion Selected GI diseases result from dysregulation of gut secretion. Gastric acid hypersecretion occurs in Zollinger-Ellison syndrome, G cell hyperplasia, retained antrum syndrome, and some individuals with duodenal ulcers. Conversely, patients with atrophic gastritis or pernicious anemia release little or no gastric acid. Inflammatory and infectious small-intestinal and colonic diseases produce fluid loss through impaired absorption or enhanced secretion. Common intestinal and colonic hypersecretory conditions cause diarrhea and include acute bacterial or viral infection, chronic Giardia or cryptosporidia infections, small-intestinal bacterial overgrowth, bile salt diarrhea, microscopic colitis, diabetic diarrhea, and abuse of certain laxatives. Less common causes include large colonic villus adenomas and endocrine neoplasias with tumor overproduction of secretagogue transmitters like vasoactive intestinal polypeptide.

1	Altered Gut Transit Impaired gut transit may be secondary to mechanical obstruction. Esophageal occlusion often results from acid-induced stricture or neoplasm. Gastric outlet obstruction develops from peptic ulcer disease or gastric cancer. Small-intestinal obstruction most commonly results from adhesions but may also occur with Crohn’s disease, radiationor drug-induced strictures, and less likely malignancy. The most common cause of colonic obstruction is colon cancer, although inflammatory strictures develop in patients with inflammatory bowel disease, after certain infections such as diverticulitis, or with some drugs.

1	Retardation of propulsion also develops from disordered motor function. Achalasia is characterized by impaired esophageal body peristalsis and incomplete lower esophageal sphincter relaxation. Gastroparesis is the symptomatic delay in gastric emptying of meals due to impaired gastric motility. Intestinal pseudoobstruction causes marked delays in small-bowel transit due to enteric nerve or intestinal smooth-muscle injury. Slow-transit constipation is produced by diffusely impaired colonic propulsion. Constipation also is produced by outlet abnormalities such as rectal prolapse, intussusception, or dyssynergia—a failure of anal or puborectalis relaxation upon attempted defecation.

1	Disorders of rapid propulsion are less common than those with delayed transit. Rapid gastric emptying occurs in postvagotomy dumping syndrome, with gastric hypersecretion, and in some cases of functional dyspepsia and cyclic vomiting syndrome. Exaggerated intestinal or colonic motor patterns may be responsible for diarrhea in irritable bowel syndrome. Accelerated transit with hyperdefecation is noted in hyperthyroidism.

1	Immune Dysregulation Many inflammatory GI conditions are consequences of altered gut immune function. The mucosal inflammation of celiac disease results from dietary ingestion of gluten-containing grains. Some patients with food allergy also exhibit altered immune populations. Eosinophilic esophagitis and eosinophilic gastroenteritis are inflammatory disorders with prominent mucosal eosinophils. Ulcerative colitis and Crohn’s disease are disorders of uncertain etiology that produce mucosal injury primarily in the lower gut. The microscopic colitides, lymphocytic and collagenous colitis, exhibit colonic subepithelial infiltrates without visible mucosal damage. Bacterial, viral, and protozoal organisms may produce ileitis or colitis in selected patient populations.

1	Impaired Gut Blood Flow Different GI regions are at variable risk for ischemic damage from impaired blood flow. Rare cases of gastroparesis result from blockage of the celiac and superior mesenteric arteries. More commonly encountered are intestinal and colonic ischemia that are consequences of arterial embolus, arterial thrombosis, venous thrombosis, or hypoperfusion from dehydration, sepsis, hemorrhage, or reduced cardiac output. These may produce mucosal injury, hemorrhage, or even perforation. Chronic ischemia may result in intestinal stricture. Some cases of radiation enterocolitis exhibit reduced mucosal blood flow.

1	Neoplastic Degeneration All GI regions are susceptible to malignant degeneration to varying degrees. In the United States, colorectal cancer is most common and usually presents after age 50 years. Worldwide, gastric cancer is prevalent especially in certain Asian regions. Esophageal cancer develops with chronic acid reflux or after an extensive alcohol or tobacco use history. Small-intestinal neoplasms are rare and occur with underlying inflammatory disease. Anal cancers arise after prior anal infection or inflammation. Pancreatic and biliary cancers elicit severe pain, weight loss, and jaundice and have poor prognoses. Hepatocellular carcinoma usually arises in the setting of chronic viral hepatitis or cirrhosis secondary to other causes. Most GI cancers exhibit carcinomatous histology; however, lymphomas and other cell types also are observed.

1	Disorders Without Obvious Organic Abnormalities The most common GI disorders show no abnormalities on biochemical or structural testing and include irritable bowel syndrome, functional dyspepsia, functional chest pain, and functional heartburn. These disorders exhibit altered gut motor function; however, the pathogenic relevance of these abnormalities is uncertain. Exaggerated visceral sensory responses to noxious stimulation may cause discomfort in these disorders. Symptoms in other patients result from altered processing of visceral pain sensations in the central nervous system. Functional bowel patients with severe symptoms may exhibit significant emotional disturbances on psychometric testing. Subtle immunologic defects may contribute to functional symptoms as well.

1	Genetic Influences Although many GI diseases result from environmental factors, others exhibit hereditary components. Family members of inflammatory bowel disease patients show a genetic predisposition to disease development themselves. Colonic and esophageal malignancies arise in certain inherited disorders. Rare genetic dysmotility syndromes are described. Familial clustering is even observed in the functional bowel disorders, although this may be secondary learned familial illness behavior rather than a true hereditary factor. The most common GI symptoms are abdominal pain, heartburn, nausea and vomiting, altered bowel habits, GI bleeding, and jaundice (Table 344-1). Others are dysphagia, anorexia, weight loss, fatigue, and extraintestinal symptoms.

1	Abdominal Pain Abdominal pain results from GI disease and extra-intestinal conditions involving the genitourinary tract, abdominal wall, thorax, or spine. Visceral pain generally is midline in location and vague in character, whereas parietal pain is localized and precisely described. Common inflammatory diseases with pain include peptic ulcer, appendicitis, diverticulitis, inflammatory bowel disease, and infectious enterocolitis. Other intraabdominal causes of pain include gallstone disease and pancreatitis. Noninflammatory visceral sources include mesenteric ischemia and neoplasia. The most common causes of abdominal pain are irritable bowel syndrome and functional dyspepsia.

1	Heartburn Heartburn, a burning substernal sensation, is reported intermittently by at least 40% of the population. Classically, heartburn is felt to result from excess gastroesophageal reflux of acid. However, some cases exhibit normal esophageal acid exposure and may result from reflux of nonacidic material or heightened sensitivity of esophageal mucosal nerves. Nausea and Vomiting Nausea and vomiting are caused by GI diseases, medications, toxins, acute and chronic infection, endocrine disorders, labyrinthine conditions, and central nervous system disease. The best-characterized GI etiologies relate to mechanical obstruction of the upper gut; however, disorders of propulsion including gastroparesis and intestinal pseudoobstruction also elicit prominent symptoms. Nausea and vomiting also are commonly reported by patients with irritable bowel syndrome and functional disorders of the upper gut (including chronic idiopathic nausea and functional vomiting).

1	Altered Bowel Habits Altered bowel habits are common complaints of patients with GI disease. Constipation is reported as infrequent defecation, straining with defecation, passage of hard stools, or a sense of incomplete fecal evacuation. Causes of constipation include obstruction, motor disorders of the colon, medications, and endocrine diseases such as hypothyroidism and hyperparathyroidism. Diarrhea is reported as frequent defecation, passage of loose or watery stools, fecal urgency, or a similar sense of incomplete evacuation. The differential diagnosis of diarrhea is broad and includes infections, inflammatory causes, malabsorption, and medications. Irritable bowel syndrome produces constipation, diarrhea, or an alternating bowel pattern. Fecal mucus is common in irritable bowel syndrome, whereas pus characterizes inflammatory disease. Steatorrhea develops with malabsorption.

1	GI Bleeding Hemorrhage may develop from any gut organ. Most commonly, upper GI bleeding presents with melena or hematemesis, whereas lower GI bleeding produces passage of bright red or maroon stools. However, briskly bleeding upper sites can elicit voluminous red rectal bleeding, whereas slowly bleeding ascending colon sites may produce melena. Chronic slow GI bleeding may present with iron deficiency anemia. The most common upper GI causes of bleeding are ulcer disease, gastroduodenitis, and esophagitis. Other etiologies include portal hypertensive causes, malignancy, tears across the gastroesophageal junction, and vascular lesions. The most prevalent lower GI sources of hemorrhage include hemorrhoids, anal fissures, diverticula, ischemic colitis, and arteriovenous malformations. Other causes include neoplasm, inflammatory bowel disease, infectious colitis, drug-induced colitis, and other vascular lesions.

1	Jaundice Jaundice results from prehepatic, intrahepatic, or post-hepatic disease. Posthepatic causes of jaundice include biliary diseases, such as choledocholithiasis, acute cholangitis, primary sclerosing cholangitis, other strictures, and neoplasm, and pancreatic disorders, such as acute and chronic pancreatitis, stricture, and malignancy.

1	Other Symptoms Other symptoms are manifestations of GI disease. Dysphagia, odynophagia, and unexplained chest pain suggest esophageal disease. A globus sensation is reported with esophagopharyngeal conditions, but also occurs with functional GI disorders. Weight loss, anorexia, and fatigue are nonspecific symptoms of neoplastic, inflammatory, gut motility, pancreatic, small-bowel mucosal, and psychiatric conditions. Fever is reported with inflammatory illness, but malignancies also evoke febrile responses. GI disorders also produce extraintestinal symptoms. Inflammatory bowel disease is associated with hepatobiliary dysfunction, skin and eye lesions, and arthritis. Celiac disease may present with dermatitis herpetiformis. Jaundice can produce pruritus. Conversely, systemic diseases can have GI consequences. Systemic lupus may cause gut ischemia, presenting with pain or bleeding. Overwhelming stress or severe burns may lead to gastric ulcer formation.

1	Evaluation of the patient with GI disease begins with a careful history and examination. Subsequent investigation with a variety of tools designed to test gut structure or function are indicated in selected cases. Some patients exhibit normal findings on diagnostic testing. In these individuals, validated symptom profiles are used to confidently diagnose a functional bowel disorder.

1	The history of the patient with suspected GI disease has several components. Symptom timing suggests specific etiologies. Symptoms of short duration commonly result from acute infection, toxin exposure, or abrupt inflammation or ischemia. Long-standing symptoms point to underlying chronic inflammatory or neoplastic conditions or functional bowel disorders. Symptoms from mechanical obstruction, ischemia, inflammatory bowel disease, and functional bowel disorders are worsened by meals. Conversely, ulcer symptoms may be relieved by eating or antacids. Symptom patterns and duration may suggest underlying etiologies. Ulcer pain occurs at intermittent intervals lasting weeks to months, whereas biliary colic has a sudden onset and lasts up to several hours. Pain from acute inflammation as with acute pancreatitis is severe and persists for days to weeks. Meals elicit diarrhea in some cases of inflammatory bowel disease and irritable bowel syndrome. Defecation relieves discomfort in

1	as with acute pancreatitis is severe and persists for days to weeks. Meals elicit diarrhea in some cases of inflammatory bowel disease and irritable bowel syndrome. Defecation relieves discomfort in inflammatory bowel disease and irritable bowel syndrome. Functional bowel disorders are exacerbated by stress. Sudden awakening from sound sleep suggests organic rather than functional disease. Diarrhea from malabsorption usually improves with fasting, whereas secretory diarrhea persists without oral intake.

1	Symptom relation to other factors narrows the list of diagnostic possibilities. Obstructive symptoms with prior abdominal surgery raise concern for adhesions, whereas loose stools after gastrectomy or gallbladder excision suggest dumping syndrome or postcholecystectomy diarrhea. Symptom onset after travel prompts a search for enteric infection. Medications may produce pain, altered bowel habits, or GI bleeding. Lower GI bleeding likely results from neoplasms, diverticula, or vascular lesions in an older person and from anorectal abnormalities or inflammatory bowel disease in a younger individual. Celiac disease is prevalent in people of northern European descent, whereas inflammatory bowel disease is more common in certain Jewish populations. A sexual history may raise concern for sexually transmitted diseases or immunodeficiency.

1	For more than two decades, working groups have been convened to devise symptom criteria to improve the confident diagnosis of functional bowel disorders and to minimize the numbers of unnecessary diagnostic tests performed. The most widely accepted symptom-based Approach to the Patient with Gastrointestinal Disease 1878 criteria are the Rome criteria. When tested against findings of structural investigations, the Rome criteria exhibit diagnostic specificities exceeding 90% for many of the functional bowel disorders.

1	The physical exam complements information from the history. Abnormal vital signs provide diagnostic clues and determine the need for acute intervention. Fever suggests inflammation or neoplasm. Orthostasis is found with significant blood loss, dehydration, sepsis, or autonomic neuropathy. Skin, eye, or joint findings may point to specific diagnoses. Neck exam with swallowing assessment evaluates dysphagia. Cardiopulmonary disease may present with abdominal pain or nausea; thus lung and cardiac exams are important. Pelvic examination tests for a gynecologic source of abdominal pain. Rectal exam may detect blood, indicating gut mucosal injury or neoplasm or a palpable inflammatory mass in appendicitis. Metabolic conditions and gut motor disorders have associated peripheral neuropathy.

1	Inspection of the abdomen may reveal distention from obstruction, tumor, or ascites or vascular abnormalities with liver disease. Ecchymoses develop with severe pancreatitis. Auscultation can detect bruits or friction rubs from vascular disease or hepatic tumors. Loss of bowel sounds signifies ileus, whereas high-pitched, hyperactive sounds characterize intestinal obstruction. Percussion assesses liver size and can detect shifting dullness from ascites. Palpation assesses for hepatosplenomegaly as well as neoplastic or inflammatory masses. Abdominal exam is helpful in evaluating unexplained pain. Intestinal ischemia elicits severe pain but little tenderness. Patients with visceral pain may exhibit generalized discomfort, whereas those with parietal pain or peritonitis have directed pain, often with involuntary guarding, rigidity, or rebound. Patients with musculoskeletal abdominal wall pain may note tenderness exacerbated by Valsalva or straight-leg lift maneuvers.

1	Laboratory, radiographic, and functional tests can assist in diagnosis of suspected GI disease. The GI tract also is amenable to internal evaluation with upper and lower endoscopy and to examination of luminal contents. Histopathologic exams of GI tissues complement these tests.

1	Laboratory Selected laboratory tests facilitate the diagnosis of GI disease. Iron-deficiency anemia suggests mucosal blood loss, whereas vitamin B12 deficiency results from small-intestinal, gastric, or pancreatic disease. Either also can result from inadequate oral intake. Leukocytosis and increased sedimentation rates and C-reactive protein levels are found in inflammatory conditions, whereas leukopenia is seen in viremic illness. Severe vomiting or diarrhea elicits electrolyte disturbances, acid-base abnormalities, and elevated blood urea nitrogen. Pancreaticobiliary or liver disease is suggested by elevated pancreatic or liver chemistries. Thyroid chemistries, cortisol, and calcium levels are obtained to exclude endocrinologic causes of GI symptoms. Pregnancy testing is considered for women with unexplained nausea. Serologic tests can screen for celiac disease, inflammatory bowel disease, rheumatologic diseases like lupus or scleroderma, and paraneoplastic dysmotility syndromes.

1	women with unexplained nausea. Serologic tests can screen for celiac disease, inflammatory bowel disease, rheumatologic diseases like lupus or scleroderma, and paraneoplastic dysmotility syndromes. Hormone levels are obtained for suspected endocrine neoplasia. Intraabdominal malignancies produce other tumor markers including the carcinoembryonic antigen CA 19-9 and α-fetoprotein. Blood testing also monitors medication therapy in some diseases, as with thiopurine metabolite levels in inflammatory bowel disease. Other body fluids are sampled under certain circumstances. Ascitic fluid is analyzed for infection, malignancy, or findings of portal hypertension. Cerebrospinal fluid is obtained for suspected central nervous system causes of vomiting. Urine samples screen for carcinoid, porphyria, and heavy metal intoxication.

1	Luminal Contents Luminal contents can be examined for diagnostic clues. Stool samples are cultured for bacterial pathogens, examined for leukocytes and parasites, or tested for Giardia antigen. Duodenal aspirates can be examined for parasites or cultured for bacterial overgrowth. Fecal fat is quantified in possible malabsorption. Stool electrolytes can be measured in diarrheal conditions. Laxative screens are done when laxative abuse is suspected. Gastric acid is quantified to rule out Zollinger-Ellison syndrome. Esophageal pH testing is done for refractory symptoms of acid reflux, whereas impedance techniques assess for nonacidic reflux. Pancreatic juice is analyzed for enzyme or bicarbonate content to exclude pancreatic exocrine insufficiency.

1	Endoscopy The gut is accessible with endoscopy, which can provide the diagnosis of the causes of bleeding, pain, nausea and vomiting, weight loss, altered bowel function, and fever. Table 344-2 lists the most common indications for the major endoscopic procedures. Upper endoscopy evaluates the esophagus, stomach, and duodenum, whereas colonoscopy assesses the colon and distal ileum. Upper endoscopy is advocated as the initial structural test performed in patients with suspected ulcer disease, esophagitis, neoplasm, malabsorption, and Barrett’s metaplasia because of its ability to directly visualize as well as biopsy the abnormality. Colonoscopy is the procedure of choice for colon cancer screening and surveillance as well as diagnosis of colitis secondary to infection, ischemia, radiation, and inflammatory bowel disease. Sigmoidoscopy examines the colon up to the splenic flexure and is currently used to exclude distal colonic inflammation or obstruction in young patients not at

1	and inflammatory bowel disease. Sigmoidoscopy examines the colon up to the splenic flexure and is currently used to exclude distal colonic inflammation or obstruction in young patients not at significant risk for colon cancer. For elusive GI bleeding secondary to arteriovenous malformations or superficial ulcers, small-intestinal examination is performed with push enteroscopy, capsule endoscopy, or double-balloon enteroscopy. Capsule endoscopy also can visualize small-intestinal Crohn’s disease in individuals with negative barium radiography. Endoscopic retrograde cholangiopancreaticography (ERCP) provides diagnoses of pancreatic and biliary disease. Endoscopic ultrasound is useful for evaluating extent of disease in GI malignancy as well as exclusion of choledocholithiasis, evaluation of pancreatitis, drainage of pancreatic pseudocysts, and assessment of anal continuity.

1	Radiography/Nuclear Medicine Radiographic tests evaluate diseases of the gut and extraluminal structures. Oral or rectal contrast agents like barium provide mucosal definition from the esophagus to the rectum. Contrast radiography also assesses gut transit and pelvic floor dysfunction. Barium swallow is the initial procedure for evaluation of dysphagia to exclude subtle rings or strictures and assess for achalasia, whereas small-bowel contrast radiology reliably diagnoses intestinal tumors and Crohn’s ileitis. Contrast enemas are performed when colonoscopy is unsuccessful or contraindicated. Ultrasound and computed tomography (CT) evaluate regions not accessible by endoscopy or contrast studies, including the liver, pancreas, gallbladder, kidneys, and retroperitoneum. These tests are useful for diagnosis of mass lesions, fluid collections, organ enlargement, and, in the case of ultrasound, gallstones. CT and magnetic resonance (MR) colonography are being evaluated as alternatives to

1	for diagnosis of mass lesions, fluid collections, organ enlargement, and, in the case of ultrasound, gallstones. CT and magnetic resonance (MR) colonography are being evaluated as alternatives to colonoscopy for colon cancer screening. MR imaging assesses the pancreaticobiliary ducts to exclude neoplasm, stones, and sclerosing cholangitis, and the liver to characterize benign and malignant tumors. Specialized CT or MR enterography can assess intensity of inflammatory bowel disease. Angiography excludes mesenteric ischemia and determines spread of malignancy. Angiographic techniques also access the biliary tree in obstructive jaundice. CT and MR techniques can be used to screen for mesenteric occlusion, thereby limiting exposure to angiographic dyes. Positron emission tomography can facilitate distinguishing malignant from benign disease in several organ systems.

1	Scintigraphy both evaluates structural abnormalities and quantifies luminal transit. Radionuclide bleeding scans localize bleeding sites in patients with brisk hemorrhage so that therapy with endoscopy, angiography, or surgery may be directed. Radiolabeled leukocyte scans can search for intraabdominal abscesses not visualized on CT. Biliary scintigraphy is complementary to ultrasound in the assessment of cholecystitis. Scintigraphy to quantify esophageal and gastric emptying is well established, whereas techniques to measure small-intestinal or colonic transit are less widely used. Histopathology Gut mucosal biopsies obtained at endoscopy evaluate for inflammatory, infectious, and neoplastic disease. Deep rectal biopsies assist with diagnosis of Hirschsprung’s disease or amyloid. Liver

1	Approach to the Patient with Gastrointestinal Disease biopsy is indicated in cases with abnormal liver chemistries, in unexplained jaundice, following liver transplant to exclude rejection, and to characterize the degree of inflammation in patients with chronic viral hepatitis prior to initiating antiviral therapy. Biopsies obtained during CT or ultrasound can evaluate for other intraabdominal conditions not accessible by endoscopy.

1	Functional Testing Tests of gut function provide important data when structural testing is nondiagnostic. In addition to gastric acid and pancreatic function testing, functional testing of motor activity is provided by manometric techniques. Esophageal manometry is useful for suspected achalasia, whereas small-intestinal manometry tests for pseudoobstruction. A wireless motility capsule is now available to measure transit and contractile activity in the stomach, small intestine, and colon in a single test. Anorectal manometry with balloon expulsion testing is used for unexplained incontinence or constipation from outlet dysfunction. Anorectal manometry and electromyography also assess anal function in fecal incontinence. Biliary manometry tests for sphincter of Oddi dysfunction with unexplained biliary pain. Measurement of breath hydrogen while fasting and after oral monoor oligosaccharide challenge can screen for carbohydrate intolerance and small-intestinal bacterial overgrowth.

1	Management options for the patient with GI disease depend on the cause of symptoms. Available treatments include modifications in dietary intake, medications, interventional endoscopy or radiology techniques, surgery, and therapies directed to external influences.

1	Dietary modifications for GI disease include treatments that only reduce symptoms, therapies that correct pathologic defects, and measures that replace normal food intake with enteral or parenteral formulations. Changes that improve symptoms but do not reverse an organic abnormality include lactose restriction for lactase deficiency, liquid meals in gastroparesis, carbohydrate restrictions with dumping syndrome, and low-FODMAP (fermentable oligodi-monosaccharides and polyols) diets in irritable bowel syndrome. The gluten-free diet for celiac disease exemplifies a modification that serves as primary therapy to reduce mucosal inflammation. Enteral medium-chain triglycerides replace normal fats in short-gut syndrome or severe ileal disease. Perfusion of liquid meals through a gastrostomy is performed in those who cannot swallow safely. Enteral feeding through a jejunostomy is considered for gastric dysmotility syndromes that preclude feeding into the stomach. Intravenous

1	gastrostomy is performed in those who cannot swallow safely. Enteral feeding through a jejunostomy is considered for gastric dysmotility syndromes that preclude feeding into the stomach. Intravenous hyperalimentation is used for individuals with generalized gut malfunction who cannot tolerate or who cannot be sustained with enteral nutrition.

1	Several medications are available to treat GI diseases. Considerable health care resources are expended on over-the-counter remedies. Many prescription drug classes are offered as short-term or continuous therapy of GI illness. A plethora of alternative treatments have gained popularity in GI conditions for which traditional therapies provide incomplete relief.

1	Over-the-Counter Agents Over-the-counter agents are reserved for mild GI symptoms. Antacids and histamine H2 antagonists decrease symptoms in gastroesophageal reflux and dyspepsia, whereas antiflatulents and adsorbents reduce gaseous symptoms. More potent acid inhibitors such as proton pump inhibitors are now available over the counter for treatment of chronic gastroesophageal reflux disease (GERD). Fiber supplements, stool softeners, enemas, and laxatives are used for constipation. Laxatives are categorized as stimulants, osmotic agents (including isotonic preparations containing polyethylene glycol), and poorly absorbed sugars. Nonprescription antidiarrheal agents include bismuth subsalicylate, kaolin-pectin combinations, and loperamide. Supplemental enzymes include lactase pills for lactose intolerance and bacterial α-galactosidase to treat excess gas. In general, use of a nonprescription preparation for more than a short time for chronic persistent symptoms should be supervised by

1	intolerance and bacterial α-galactosidase to treat excess gas. In general, use of a nonprescription preparation for more than a short time for chronic persistent symptoms should be supervised by a health care provider.

1	1880 Prescription Drugs Prescription drugs for GI diseases are a major focus of attention from pharmaceutical companies. Potent acid suppressants, including drugs that inhibit the proton pump, are advocated for acid reflux when over-the-counter preparations are inadequate. Cytoprotective agents rarely are used for upper gut ulcers. Prokinetic drugs stimulate GI propulsion in gastroparesis and pseudoobstruction. Prosecretory drugs are prescribed for constipation refractory to other agents. Prescription antidiarrheals include opiate drugs, anticholinergic antispasmodics, tricyclics, bile acid binders, and serotonin antagonists. Antispasmodics and antidepressants also are useful for functional abdominal pain, whereas narcotics are used for pain control in organic conditions such as disseminated malignancy and chronic pancreatitis. Antiemetics in several classes reduce nausea and vomiting. Potent pancreatic enzymes decrease malabsorption and pain from pancreatic disease. Antisecretory

1	malignancy and chronic pancreatitis. Antiemetics in several classes reduce nausea and vomiting. Potent pancreatic enzymes decrease malabsorption and pain from pancreatic disease. Antisecretory drugs such as the somatostatin analogue octreotide treat hypersecretory states. Antibiotics treat ulcer disease secondary to Helicobacter pylori, infectious diarrhea, diverticulitis, intestinal bacterial overgrowth, and Crohn’s disease. Some cases of irritable bowel syndrome (especially those with diarrhea) respond to nonabsorbable antibiotic therapy. Anti-inflammatory and immunosuppressive drugs are used in ulcerative colitis, Crohn’s disease, microscopic colitis, refractory celiac disease, and gut vasculitis. Chemotherapy with or without radiotherapy is offered for GI malignancies. Most GI carcinomas respond poorly to such therapy, whereas lymphomas may be cured with such intervention.

1	Alternative Therapies Alternative treatments are marketed to treat selected GI symptoms. Ginger, acupressure, and acustimulation have been advocated for nausea, whereas pyridoxine has been investigated for nausea of first-trimester pregnancy. Probiotics containing active bacterial cultures are used as adjuncts in some cases of infectious diarrhea and irritable bowel syndrome. Probiotics that selectively nourish benign commensal bacteria may ultimately show benefit in functional disorders as well. Low-potency pancreatic enzyme preparations are sold as general digestive aids but have little evidence to support their efficacy.

1	Simple luminal interventions are commonly performed for GI diseases. Nasogastric tube suction decompresses the upper gut in ileus or mechanical obstruction. Nasogastric lavage of saline or water in the patient with upper GI hemorrhage determines the rate of bleeding and helps evacuate blood prior to endoscopy. Enteral feedings can be initiated through a nasogastric or nasoenteric tube. Enemas relieve fecal impaction or assist in gas evacuation in acute colonic pseudoobstruction. A rectal tube can be left in place to vent the distal colon in colonic pseudoobstruction and other colonic distention disorders.

1	In addition to its diagnostic role, endoscopy has therapeutic capabilities in certain settings. Cautery techniques can stop hemorrhage from ulcers, vascular malformations, and tumors. Injection with vasoconstrictor substances or sclerosants is used for bleeding ulcers, vascular malformations, varices, and hemorrhoids. Endoscopic encirclement of varices and hemorrhoids with constricting bands stops hemorrhage from these sites, whereas endoscopically placed clips can occlude arterial bleeding sites. Endoscopy can remove polyps or debulk lumen-narrowing malignancies. Endoscopic mucosal resection and radiofrequency techniques can remove or ablate some cases of Barrett’s esophagus with dysplasia. Endoscopic sphincterotomy of the ampulla of Vater relieves symptoms of choledocholithiasis. Obstructions of the gut lumen and pancreaticobiliary tree are relieved by endoscopic dilatation or placement of plastic or expandable metal stents. In cases of acute colonic pseudoobstruction, colonoscopy

1	of the gut lumen and pancreaticobiliary tree are relieved by endoscopic dilatation or placement of plastic or expandable metal stents. In cases of acute colonic pseudoobstruction, colonoscopy is used to withdraw luminal gas. Finally, endoscopy is commonly used to insert feeding tubes.

1	Radiologic measures also are useful in GI disease. Angiographic embolization or vasoconstriction decreases bleeding from sites not amenable to endoscopic intervention. Dilatation or stenting with fluoroscopic guidance relieves luminal strictures. Contrast enemas can reduce volvulus and evacuate air in acute colonic pseudoobstruction. CT and ultrasound help drain abdominal fluid collections, in many cases obviating the need for surgery. Percutaneous transhepatic cholangiography relieves biliary obstruction when ERCP is contraindicated. Lithotripsy can fragment gallstones in patients who are not candidates for surgery. In some instances, radiologic approaches offer advantages over endoscopy for gastroenterostomy placement. Finally, central venous catheters for parenteral nutrition may be placed using radiographic techniques.

1	Surgery is performed to cure disease, control symptoms without cure, maintain nutrition, or palliate unresectable neoplasm. Medication-unresponsive ulcerative colitis, diverticulitis, cholecystitis, appendicitis, and intraabdominal abscess are curable with surgery, whereas only symptom control without cure is possible with Crohn’s disease. Surgery is mandated for ulcer complications such as bleeding, obstruction, or perforation and intestinal obstructions that persist after conservative care. Fundoplication of the gastroesophageal junction is performed for severe ulcerative esophagitis and drug-refractory symptomatic acid reflux. Achalasia responds to operations to relieve lower esophageal sphincter pressure. Operations for motor disorders have been introduced including implanted electrical stimulators for gastroparesis and electrical devices and artificial sphincters for fecal incontinence. Surgery may be needed to place a jejunostomy for long-term enteral feedings. The threshold for

1	stimulators for gastroparesis and electrical devices and artificial sphincters for fecal incontinence. Surgery may be needed to place a jejunostomy for long-term enteral feedings. The threshold for performing surgery depends on the clinical setting. In all cases, the benefits of operation must be weighed against the potential for postoperative complications.

1	In some conditions, GI symptoms respond to treatments directed outside the gut. Psychological therapies including psychotherapy, behavior modification, hypnosis, and biofeedback have shown efficacy in functional bowel disorders. Patients with significant psychological dysfunction and those with little response to treatments targeting the gut are likely to benefit from this form of therapy. Louis Michel Wong Kee Song, Mark Topazian Gastrointestinal endoscopy has been attempted for over 200 years, but the introduction of semirigid gastroscopes in the middle of the twentieth century marked the dawn of the modern endoscopic era. Since then, rapid advances in endoscopic technology have led to dramatic changes in the diagnosis and treatment of many digestive diseases. Innovative endoscopic devices and new endoscopic treatment modalities continue to expand the use of endoscopy in patient care.

1	Current flexible endoscopes provide an electronic video image generated by a charge-coupled device in the tip of the endoscope. Operator controls permit deflection of the endoscope tip; fiberoptic bundles or light-emitting diodes bring light to the tip of the endoscope; and working channels allow washing, suctioning, and the passage of instruments. Progressive changes in the diameter and stiffness of endoscopes have improved the ease and patient tolerance of endoscopy. FIGURE 345-1 Duodenal ulcers. A. Ulcer with a clean base. B. Ulcer with a visible vessel (arrow) in a patient with recent hemorrhage.

1	Upper endoscopy, also referred to as esophagogastroduodenoscopy (EGD), is performed by passing a flexible endoscope through the mouth into the esophagus, stomach, and duodenum. The procedure is the best method for examining the upper gastrointestinal mucosa. While the upper gastrointestinal radiographic series has similar accuracy for diagnosis of duodenal ulcer (Fig. 345-1), EGD is superior for detection of gastric ulcers (Fig. 345-2) and flat mucosal lesions such as Barrett’s esophagus (Fig. 345-3), and it permits directed biopsy and endoscopic therapy. Intravenous conscious sedation is given to most patients in the United States to ease the anxiety and discomfort of the procedure, although in many countries EGD is routinely performed with topical pharyngeal anesthesia only. Patient tolerance of unsedated EGD is improved by the use of an ultrathin, 5-mm diameter endoscope that can be passed transorally or transnasally.

1	Colonoscopy is performed by passing a flexible colonoscope through the anal canal into the rectum and colon. The cecum is reached in >95% of cases, and the terminal ileum can often be examined. Colonoscopy is the gold standard for imaging the colonic mucosa. Colonoscopy has greater sensitivity than barium enema for colitis (Fig. 345-4), polyps (Fig. 345-5), and cancer (Fig. 345-6). Computed tomography (CT) colonography is an emerging technology that rivals the accuracy of colonoscopy for detection of some polyps and cancer, although it may not be sensitive for the detection of flat lesions, such as serrated polyps (Fig. 345-7). Conscious sedation is usually given before colonoscopy in the United States, although a willing patient and a skilled examiner can complete the procedure without sedation in many cases. Flexible sigmoidoscopy is similar to colonoscopy, but visualizes only the rectum and a variable portion of the left colon, typically to 60 cm

1	Flexible sigmoidoscopy is similar to colonoscopy, but visualizes only the rectum and a variable portion of the left colon, typically to 60 cm FIGURE 345-2 Gastric ulcers. A. Benign gastric ulcer. B. Malignant gastric ulcer involving greater curvature of stomach. from the anal verge. This procedure causes abdominal cramping, but 1881 it is brief and is usually performed without sedation. Flexible sigmoidoscopy is primarily used for evaluation of diarrhea and rectal outlet bleeding.

1	Three endoscopic techniques are currently used to evaluate the small intestine, most often in patients presenting with presumed small-bowel bleeding. For capsule endoscopy, the patient swallows a disposable capsule that contains a complementary metal oxide silicon (CMOS) chip camera. Color still images (Fig. 345-8) are transmitted wirelessly to an external receiver at several frames per second until the capsule’s battery is exhausted or it is passed into the toilet. Capsule endoscopy enables visualization of the small-bowel mucosa beyond the reach of a conventional endoscope and, at present, is solely a diagnostic procedure.

1	Push enteroscopy is performed with a long endoscope similar in design to an upper endoscope. The enteroscope is pushed down the small bowel, sometimes with the help of a stiffening overtube that extends from the mouth to the small intestine. The proximal to mid-jejunum is usually reached, and the instrument channel of the endoscope allows for biopsy or endoscopic therapy. Deeper insertion into the small bowel can be accomplished by single-or double-balloon enteroscopy or spiral enteroscopy (Fig. 345-9). These instruments enable pleating of the small intestine onto an over-tube (see Video 346e-1). With balloon-assisted enteroscopy, the entire intestinal tract can be visualized in some patients when both the oral and anal routes of insertion are used. Biopsies and endoscopic therapy can be performed throughout the visualized small bowel (Fig. 345-10).

1	During ERCP a side-viewing endoscope is passed through the mouth to the duodenum, the ampulla of Vater is identified and cannulated with a thin plastic catheter, and radiographic contrast material is injected into the bile duct and pancreatic duct under fluoroscopic guidance (Fig. 345-11). When indicated, the sphincter of Oddi can be opened using the technique of endoscopic sphincterotomy (Fig. 345-12). Stones can be retrieved from the ducts (see Video 346e-15), biopsies can be performed, strictures can be dilated and/or stented (Fig. 345-13), and ductal leaks can be stented (Fig. 345-14). ERCP is often performed for therapy but remains important in diagnosis, especially for sphincter of Oddi dysfunction and for tissue sampling of ductal strictures.

1	EUS utilizes high-frequency ultrasound transducers incorporated into the tip of a flexible endoscope. Ultrasound images are obtained of the gut wall and adjacent organs, vessels, and lymph nodes. By sacrificing depth of ultrasound penetration and bringing the ultrasound transducer close to the area of interest via endoscopy, high-resolution images are obtained. EUS provides the most accurate preoperative local staging of esophageal, pancreatic, and rectal malignancies (Fig. 345-15), although it does not detect most distant metastases. EUS is also useful for diagnosis of bile duct stones, gallbladder disease, submucosal gastrointestinal lesions, and chronic pancreatitis. Fine-needle aspirates and core biopsies of masses and lymph nodes in the posterior mediastinum, abdomen, pancreas, retroperitoneum, and pelvis can be obtained under EUS guidance (Fig. 345-16). EUS-guided therapeutic procedures are increasingly performed, including drainage of abscesses, pseudocysts, and pancreatic

1	and pelvis can be obtained under EUS guidance (Fig. 345-16). EUS-guided therapeutic procedures are increasingly performed, including drainage of abscesses, pseudocysts, and pancreatic necrosis into the gut lumen (see Video 346e-2), celiac plexus neurolysis for treatment of pancreatic pain, ethanol ablation of pancreatic neuroendocrine tumors, treatment of gastrointestinal hemorrhage, and drainage of obstructed biliary and pancreatic ducts.

1	NOTES is an evolving collection of endoscopic methods that entail passage of an endoscope or its accessories into or through the wall of the gastrointestinal tract to perform diagnostic or therapeutic FIGURE 345-3 Barrett’s esophagus. A. Pink tongues of Barrett’s mucosa extending proximally from the gastroesophageal junction. B. Barrett’s esophagus with a suspicious nodule (arrow) identified during endoscopic surveillance. C. Histologic finding of intramucosal adenocarcinoma in the endoscopically resected nodule. Tumor extends into the esophageal submucosa (arrow). D. Barrett’s esophagus with locally advanced adenocarcinoma. FIGURE 345-5 Colonic polyps. A. Pedunculated colon polyp on a thick stalk covered with normal mucosa (arrow). B. Sessile rectal polyp.

1	FIGURE 345-5 Colonic polyps. A. Pedunculated colon polyp on a thick stalk covered with normal mucosa (arrow). B. Sessile rectal polyp. FIGURE 345-4 Causes of colitis. A. Chronic ulcerative colitis with diffuse ulcerations and exudates. B. Severe Crohn’s colitis with deep ulcers. C. Pseudomembranous colitis with yellow, adherent pseudo-membranes. D. Ischemic colitis with patchy mucosal edema, subepithelial hemorrhage, and cyanosis. FIGURE 345-6 Colon adenocarcinoma growing into the lumen. FIGURE 345-7 Flat serrated polyp in the cecum. A. Appearance of the lesion under conventional white-light imaging. B. Mucosal patterns and boundary of the lesion enhanced with narrow band imaging. C. Submucosal lifting of the lesion with dye (methylene blue) injection prior to resection.

1	interventions. Some NOTES procedures, such as percutaneous endoscopic gastrostomy (PEG) or endoscopic necrosectomy of pancreatic necrosis, are well-established clinical procedures (see Video 346e-2); others, such as per-oral endoscopic myotomy (POEM) and endoscopic full-thickness resection of gastrointestinal mural lesions (Fig. 345-17, see Video 346e-3), are emerging as viable clinical therapeutic options; and still others, such as endoscopic appendectomy, cholecystectomy, and tubal ligation, are in development, and their ultimate clinical application is presently unclear. NOTES is currently an area of intense innovation and endoscopic research. FIGURE 345-8 Capsule endoscopy image of jejunal vascular ectasia.

1	FIGURE 345-8 Capsule endoscopy image of jejunal vascular ectasia. Endoscopic mucosal resection (EMR) (see Video 346e-4) and endoscopic submucosal dissection (ESD) (Fig. 345-18, see Video 346e-5) are two commonly used techniques for the resection of benign and early-stage malignant gastrointestinal neoplasms. In addition to providing larger specimens for more accurate histopathologic assessment and diagnosis, these techniques can be potentially FIGURE 345-9 Radiograph of a double-balloon enteroscope in the small intestine. FIGURE 345-10 Nonsteroidal anti-inflammatory drug (NSAID)–induced proximal ileal stricture diagnosed by double-balloon endoscopy. A. Ileal stricture causing obstructive symptoms. B. Balloon dilatation of the ileal stricture. C. Appearance of stricture after dilatation.

1	curative for certain dysplastic lesions and focal intramucosal carcinomas involving the esophagus, stomach, and colon. Several devices are also available for closure of EMR and ESD defects, as well as gastrointestinal fistulas and perforations. Endoscopic clips deployed through the working channel of an endoscope have been used for many years to treat bleeding lesions, but the development of more robust over-thescope clips has facilitated endoscopic closure of gastrointestinal fistulas and perforations not previously amenable to endoscopic therapy (see Video 346e-6). Endoscopic suturing is also feasible, and the technique can be used to close perforations and large defects (Fig. 345-19, see Video 346e-7), anastomotic leaks, and fistulas. Other potential indications for endoscopic suturing include stent fixation to prevent its migration (Fig. 345-20), and endoscopic bariatric procedures. These technologies are likely to have an expanding role in patient care.

1	Medications used during conscious sedation may cause respiratory depression or allergic reactions. All endoscopic procedures carry some risk of bleeding and gastrointestinal perforation. The risk is small with diagnostic upper endoscopy and colonoscopy (<1:1000 procedures), but ranges from 0.5 to 5% when therapeutic procedures, such as EMR and ESD, control of hemorrhage, or stricture dilatation, are performed. Bleeding and perforation are rare adverse events with flexible sigmoidoscopy. The risk of adverse events for diagnostic EUS (without needle aspiration) is similar to that for diagnostic upper endoscopy. FIGURE 345-11 Endoscopic retrograde cholangiopancreatogra-phy (ERCP) for bile duct stones with cholangitis. A. Faceted bile duct stones are demonstrated in the common bile duct. B. After endoscopic sphincterotomy, the stones are extracted with a Dormia basket. A small abscess communicates with the left hepatic duct.

1	Infectious complications are uncommon with most endoscopic procedures. Some procedures carry a higher incidence of postprocedure bacteremia, and prophylactic antibiotics may be indicated (Table 345-1). Management of antithrombotic agents prior to endoscopic procedures should take into account the procedural risk of hemorrhage, the agent, and the patient condition, as summarized in Table 345-2. ERCP carries additional risks. Pancreatitis occurs in about 5% of patients undergoing the procedure and in up to 30% of patients with sphincter of Oddi dysfunction. Young anicteric patients with normal ducts are at increased risk. Post-ERCP pancreatitis is usually mild and self-limited, but may result in prolonged hospitalization, surgery, diabetes, or death when severe. Bleeding occurs in 1% of endoscopic sphincterotomies. Ascending cholangitis, pseudocyst infection, retroperitoneal perforation, and abscess formation may occur as a result of ERCP.

1	Percutaneous gastrostomy tube placement during EGD is associated with a 10–15% incidence of adverse events, most often wound infections. Fasciitis, pneumonia, bleeding, buried bumper syndrome, and colonic injury may result from gastrostomy tube placement. FIGURE 345-12 Endoscopic sphincterotomy. A. A normal-appearing ampulla of Vater. B. Sphincterotomy is performed with electrocautery. C. Bile duct stones are extracted with a balloon catheter. D. Final appearance of the sphincterotomy.

1	C. Bile duct stones are extracted with a balloon catheter. D. Final appearance of the sphincterotomy. FIGURE 345-13 Endoscopic diagnosis, staging, and palliation of hilar cholangiocarcinoma. A. Endoscopic retrograde cholangiopancreatography (ERCP) in a patient with obstructive jaundice demonstrates a malignant-appearing stricture of the biliary confluence extending into the left and right intrahepatic ducts. B. Intraductal ultrasound of the biliary stricture demonstrates marked bile duct wall thickening due to tumor (T) with partial encasement of the hepatic artery (arrow). C. Intraductal biopsy obtained during ERCP demonstrates malignant cells infiltrating the submucosa of the bile duct wall (arrow). D. Endoscopic placement of bilateral self-expanding metal stents (arrow) relieves the biliary obstruction. GB, gallbladder. (Image C courtesy of Dr. Thomas Smyrk; with permission.)

1	C courtesy of Dr. Thomas Smyrk; with permission.) Endoscopy is an important diagnostic and therapeutic technique for patients with acute gastrointestinal hemorrhage. Although gastrointestinal bleeding stops spontaneously in most cases, some patients will have persistent or recurrent hemorrhage that may be life-threatening. Clinical predictors of rebleeding help identify patients most likely to benefit from urgent endoscopy and endoscopic, angiographic, or surgical hemostasis. FIGURE 345-14 Bile leak (arrow) from a duct of Luschka after lapa-roscopic cholecystectomy. Contrast leaks from a small right intrahe-patic duct into the gallbladder fossa and then flows into the pigtail of a percutaneous drainage catheter.

1	Initial Evaluation The initial evaluation of the bleeding patient focuses on the severity of hemorrhage as reflected by the postural vital signs, the frequency of hematemesis or melena, and (in some cases) findings on nasogastric lavage. Decreases in hematocrit and hemoglobin lag behind the clinical course and are not reliable gauges of the magnitude of acute bleeding. This initial evaluation, completed well before the bleeding source is confidently identified, guides immediate supportive care of the patient, triage to the ward or intensive care unit, and timing of endoscopy. The severity of the initial hemorrhage is the most important indication for urgent endoscopy, since a large initial bleed increases the likelihood of ongoing or recurrent bleeding. Patients with resting hypotension or orthostatic change in vital signs, repeated hematemesis, or bloody nasogastric aspirate that does not clear with large-volume lavage, or those requiring blood transfusions, should be considered for

1	orthostatic change in vital signs, repeated hematemesis, or bloody nasogastric aspirate that does not clear with large-volume lavage, or those requiring blood transfusions, should be considered for urgent endoscopy. In addition, patients with cirrhosis, coagulopathy, or respiratory or renal failure and those over 70 years of age are more likely to have significant rebleeding.

1	FIGURE 345-15 Local staging of gastrointestinal cancers with endoscopic ultrasound. In each example, the white arrowhead marks the primary tumor and the black arrow indicates the muscularis propria of the intestinal wall. A. T1 gastric cancer. The tumor does not invade the mp. B. T2 esophageal cancer. The tumor invades the muscularis propria. C. T3 esophageal cancer. The tumor extends through the muscularis propria into the surrounding tissue and focally abuts the aorta. AO, aorta. FIGURE 345-16 Endoscopic ultrasound (EUS)–guided fine-needle aspiration (FNA). A. Ultrasound image of a 22-gauge needle passed through the duodenal wall and positioned in a hypoechoic pancreatic head mass. B. Micrograph of aspirated malignant cells. (Image B courtesy of Dr. Michael R. Henry; with permission.)

1	Bedside evaluation also suggests an upper or lower gastrointestinal source of bleeding in most patients. Over 90% of patients with melena are bleeding proximal to the ligament of Treitz, and about 85% of patients with hematochezia are bleeding from the colon. Melena can result from bleeding in the small bowel or right colon, especially in older patients with slow colonic transit. Conversely, some patients with massive hematochezia may be bleeding from an upper gastrointestinal source, such as a gastric Dieulafoy lesion or duodenal ulcer, with rapid intestinal transit. Early upper endoscopy should be considered in such patients.

1	Endoscopy should be performed after the patient has been resuscitated with intravenous fluids and transfusions, as necessary. Marked coagulopathy or thrombocytopenia is usually treated before endoscopy, since correction of these abnormalities may lead to resolution of bleeding, and techniques for endoscopic hemostasis are limited in such patients. Metabolic derangements should also be addressed. Tracheal intubation for airway protection should be considered before upper endoscopy in patients with repeated recent hematemesis, encephalopathy, and suspected variceal hemorrhage.

1	Most patients with significant hematochezia can undergo colonoscopy after a rapid colonic purge with a polyethylene glycol solution; the preparation fluid may be administered via a nasogastric tube. Colonoscopy has a higher diagnostic yield than radionuclide bleeding scans or angiography in lower gastrointestinal bleeding, and endoscopic therapy can be applied in some cases. In a minority of cases, endoscopic assessment is hindered by poor visualization due to persistent vigorous bleeding with recurrent hemodynamic instability, and other techniques (such as angiography or emergent subtotal colectomy) must be employed. In such patients, massive bleeding originating from an upper gastrointestinal source should also be considered and excluded by upper endoscopy. The anal and rectal mucosa should be visualized endoscopically early in the course of massive rectal bleeding, because bleeding lesions in or close to the anal canal may be identified that are amenable to endoscopic or surgical

1	be visualized endoscopically early in the course of massive rectal bleeding, because bleeding lesions in or close to the anal canal may be identified that are amenable to endoscopic or surgical transanal hemostatic techniques.

1	Peptic Ulcer The endoscopic appearance of peptic ulcers provides useful prognostic information and guides the need for endoscopic therapy in patients with acute hemorrhage (Fig. 345-21). A clean-based ulcer is associated with a low risk (3–5%) of rebleeding; patients with melena and a clean-based ulcer are often discharged home from the emergency FIGURE 345-17 Endoscopic full-thickness resection of a gastrointestinal stromal tumor. A. Subepithelial lesion in the proximal stomach. B. Hypoechoic lesion arising from the fourth layer (muscularis propria) at endoscopic ultrasound. C. Full-thickness resection defect. D. Closure of defect using an over-the-scope clip. FIGURE 345-18 Endoscopic submucosal dissection. A. Large flat distal rectal adenoma with central lobulation. B. Marking the periphery of the lesion with coagulation dots. C. Rectal defect following endoscopic submucosal dissection. D. Specimen resected en bloc.

1	FIGURE 345-19 Closure of large defect using an endoscopic suturing device. A. Ulcerated inflammatory fibroid polyp in the antrum. B. Large defect following endoscopic submucosal dissection of the lesion. C. Closure of the defect using endoscopic sutures (arrows). D. Resected specimen. FIGURE 345-20 Prevention of stent migration using endoscopic sutures. A. Esophagogastric anastomotic stricture refractory to balloon dilation. B. Temporary placement of covered esophageal stent. C. Endoscopic suturing device to anchor stent to esophageal wall. D. Stent fixation with endoscopic sutures (arrows). Periprocedural Antibiotic Patient Condition Procedure Contemplated Goal of Prophylaxis Prophylaxis All cardiac conditions Any endoscopic procedure Prevention of infective endocarditis Not indicated Bile duct obstruction in the absence ERCP with complete drainage Prevention of cholangitis Not recommended of cholangitis

1	Bile duct obstruction in the absence ERCP with complete drainage Prevention of cholangitis Not recommended of cholangitis Bile duct obstruction in absence of ERCP with anticipated incomplete Prevention of cholangitis Recommended; continue antibiotics cholangitis drainage (e.g., sclerosing cholangitis, after the procedure hilar strictures) Sterile pancreatic fluid collection (e.g., ERCP Prevention of cyst infection Recommended; continue antibiotics pseudocyst, necrosis), which commu after the procedure nicates with pancreatic duct Sterile pancreatic fluid collection Transmural drainage Prevention of cyst infection Recommended Solid lesion along upper GI tract EUS-FNA Prevention of local infection Not recommendeda Solid lesion along lower GI tract EUS-FNA Prevention of local infection Insufficient data to make firm recommendationb Cystic lesions along GI tract (including EUS-FNA Prevention of cyst infection Recommended mediastinum)

1	All patients Percutaneous endoscopic feeding Prevention of peristomal infection Recommended tube placement Cirrhosis with acute GI bleeding Required for all such patients, regard-Prevention of infectious complications Recommended, upon admissionc less of endoscopic procedures and reduction of mortality

1	Synthetic vascular graft and other Any endoscopic procedure Prevention of graft and device Not recommendedd nonvalvular cardiovascular devices infection Prosthetic joints Any endoscopic procedure Prevention of septic arthritis Not recommendede aLow rates of bacteremia and local infection. bEndoscopists may choose on a case-by-case basis. cRisk for bacterial infection associated with cirrhosis and GI bleeding is well established. dNo reported cases of infection associated with endoscopy. eVery low risk of infection. Abbreviations: ERCP, endoscopic retrograde cholangiopancreatography; EUS-FNA, endoscopic ultrasound–fine-needle aspiration; GI, gastrointestinal. Source: Adapted from S Banerjee et al: Gastrointest Endosc 67:719, 2008; with permission from Elsevier.

1	Interval Between Last Dose Drug of Procedure Management and Procedure Comments aLow-risk endoscopic procedures include esophagogastroduodenoscopy (EGD) or colonoscopy with or without biopsy, endoscopic ultrasound (EUS) without fine-needle aspiration (FNA), and endoscopic retrograde cholangiopancreatography (ERCP) with stent exchange. bHigh-risk endoscopic procedures include EGD or colonoscopy with dilation, polypectomy, or thermal ablation; percutaneous endoscopic gastrostomy; EUS with FNA; and ERCP with sphincterotomy or pseudocyst drainage. cBridging therapy with heparin may be considered for patients discontinuing warfarin who are at high risk for thromboembolism, including those with mitral valve replacement or aortic valve replacement with other risk factors; those with nonvalvular atrial fibrillation with a history of stroke, embolic event, cardiac thrombus, or CHADS2 score ≥4; and those with venous thromboembolism within the past 3 months or severe underlying thrombophilia.

1	Source: TH Baron et al: N Engl J Med 368:2113, 2013; MA Anderson et al: Gastrointest Endosc 70:1060, 2009; MJ Zuckerman et al: Gastrointest Endosc 61:189, 2005. FIGURE 345-21 Stigmata of hemorrhage in peptic ulcers. A. Gastric antral ulcer with a clean base. B. Duodenal ulcer with flat pigmented spots (arrows). C. Duodenal ulcer with a dense adherent clot. D. Gastric ulcer with a pigmented protuberance/visible vessel. E. Duodenal ulcer with active spurting (arrow). FIGURE 345-22 Endoscopic hemostasis of ulcer bleeding. A. Pyloric channel ulcer with visible vessel (arrow). B. Ulcer hemostasis with placement of an over-the-scope clip.

1	FIGURE 345-22 Endoscopic hemostasis of ulcer bleeding. A. Pyloric channel ulcer with visible vessel (arrow). B. Ulcer hemostasis with placement of an over-the-scope clip. room or endoscopy suite if they are young, reliable, and otherwise healthy. Flat pigmented spots and adherent clots covering the ulcer base have a 10% and 20% risk of rebleeding, respectively. Endoscopic therapy is often considered for an ulcer with an adherent clot. When a fibrin plug is seen protruding from a vessel wall in the base of an ulcer (so-called sentinel clot or visible vessel), the risk of rebleeding from the ulcer is 40%. This finding generally leads to endoscopic therapy to decrease the rebleeding rate. Occasionally, active spurting from an ulcer is seen, with >90% risk of ongoing bleeding without therapy.

1	Endoscopic therapy of ulcers with high-risk stigmata typically lowers the rebleeding rate to 5–10%. Several hemostatic techniques are available, including injection of epinephrine or a sclerosant into and around the vessel, “coaptive coagulation” of the vessel in the base of the ulcer using a thermal probe that is pressed against the site of bleeding, placement of hemoclips (Fig. 345-22), or a combination of these modalities (see Video 346e-8). In conjunction with endoscopic therapy, the administration of a proton pump inhibitor decreases the risk of rebleeding and improves patient outcome.

1	Varices Two complementary strategies guide therapy of bleeding varices: local treatment of the bleeding varices and treatment of the underlying portal hypertension. Local therapies, including endoscopic variceal band ligation, endoscopic variceal sclerotherapy, and balloon tamponade with a Sengstaken-Blakemore tube, effectively control acute hemorrhage in most patients, although therapies that decrease portal pressure (pharmacologic treatment, surgical shunts, or radiologically placed intrahepatic portosystemic shunts) also play an important role. Endoscopic variceal ligation (EVL) is indicated for the prevention of a first bleed (primary prophylaxis) from large esophageal varices (Figs. 345-23 and 24), particularly in patients in whom beta blockers are contraindicated or not tolerated. EVL is also the preferred endoscopic FIGURE 345-23 Esophageal varices.

1	FIGURE 345-23 Esophageal varices. therapy for control of active esophageal variceal bleeding and for subsequent eradication of esophageal varices (secondary prophylaxis). During EVL, a varix is suctioned into a cap fitted on the end of the endoscope, and a rubber band is released from the cap, ligating the varix (Fig. 345-24, see Video 346e-9). EVL controls acute hemorrhage in up to 90% of patients. Complications of EVL, such as postbanding ulcer bleeding and esophageal stenosis, are uncommon. Endoscopic variceal sclerotherapy (EVS) involves the injection of a sclerosing, thrombogenic solution into or next to esophageal varices. EVS also controls acute hemorrhage in most patients, but it is generally used as salvage therapy when band ligation fails because of its higher complication rate compared to EVL. These techniques are used when varices are actively bleeding during endoscopy or (more commonly) when FIGURE 345-24 Endoscopic band ligation of esophageal varices.

1	FIGURE 345-24 Endoscopic band ligation of esophageal varices. A. Large esophageal varices with stigmata of recent bleeding. B. Band ligation of varices. FIGURE 345-25 Gastric varices. A. Large gastric fundal varices. B. Stigmata of recent bleeding from the same gastric varices (arrow). varices are the only identifiable cause of acute hemorrhage. Bleeding from large gastric fundic varices (Fig. 345-25) is best treated with endoscopic cyanoacrylate (“glue”) injection (see Video 346e-10), because EVL or EVS of these varices is associated with a high rebleeding rate. Complications of cyanoacrylate injection include infection and glue embolization to other organs, such as the lungs, brain, and spleen.

1	After treatment of the acute hemorrhage, an elective course of endoscopic therapy can be undertaken with the goal of eradicating esophageal varices and preventing rebleeding months to years later. However, this chronic therapy is less successful, preventing long-term rebleeding in ~50% of patients. Pharmacologic therapies that Mallory-Weiss Tear A Mallory-Weiss tear is a linear mucosal rent 1891 near or across the gastroesophageal junction that is often associated with retching or vomiting (Fig. 345-27). When the tear disrupts a submucosal arteriole, brisk hemorrhage may result. Endoscopy is the best method of diagnosis, and an actively bleeding tear can be treated endoscopically with epinephrine injection, coaptive coagulation, band ligation, or hemoclips (see Video 346e-12). Unlike peptic ulcer, a Mallory-Weiss tear with a nonbleeding sentinel clot in its base rarely rebleeds and thus does not necessitate endoscopic therapy.

1	Vascular Ectasias Vascular ectasias are flat mucosal vascular anomalies that are best diagnosed by endoscopy. They usually cause slow intestinal blood loss and occur either in a sporadic fashion or in a well-defined pattern of distribution (e.g., gastric antral vascular ectasia [GAVE] or “watermelon stomach”) (Fig. 345-28). Cecal vascular ectasias, GAVE, and radiation-induced rectal ectasias are often responsive to local endoscopic ablative therapy, such as argon plasma coagulation (see Video 346e-13). Patients with diffuse small-bowel vascular ectasias (associated with chronic renal failure and with hereditary hemor rhagic telangiectasia) may continue to bleed despite endoscopic treatment of easily accessible lesions by conventional endoscopy. These patients may benefit from deep enteroscopy with endoscopic therapy, pharmacologic treatment with octreotide or estrogen/progesterone therapy, or intraoperative enteroscopy.

1	Colonic Diverticula Diverticula form where nutrient arteries penetrate the muscular wall of the colon en route to the colonic mucosa (Fig. 345-29). The artery found in the base of a diverticulum may bleed, causing painless and impressive hematochezia. Colonoscopy is indicated in patients with hematochezia and suspected diverticular hemorrhage, because other causes of bleeding (such as vascular ectasias, colitis, and colon cancer) must be excluded. In addition, an actively bleeding diverticulum may be seen and treated during colonoscopy (Fig. 345-30, see Video 346e-14). Endoscopy is useful for evaluation and treatment of some forms of gastrointestinal obstruction. An important exception is small-bowel obstruction due to surgical adhesions, which is generally not diagnosed or treated endoscopically. Esophageal, gastroduodenal, and colonic obstruction or pseudoobstruction can all be diagnosed and often managed endoscopically.

1	Esophageal obstruction by impacted food (Fig. 345-31) or an ingested foreign body is a potentially life-threatening event and represents an endoscopic emergency. Left untreated, the patient may develop esophageal ulceration, ischemia, and perforation. Patients with persistent esophageal obstruction often have hypersalivation and are usually decrease portal pressure have similar efficacy, and the two modalities may be combined.

1	Dieulafoy’s Lesion This lesion, also called persistent caliber artery, is a large-caliber arteriole that runs immediately beneath the gastrointestinal mucosa and bleeds through a pinpoint mucosal erosion (Fig. 345-26). Dieulafoy’s lesion is seen most commonly on the lesser curvature of the proximal stomach, causes impressive arterial hemorrhage, and may be difficult to diagnose; it is often recognized only after repeated endoscopy for recurrent bleeding. Endoscopic therapy, such as thermal coagulation or band ligation, is typically effective for control of bleeding and ablation of the underlying vessel once the lesion has been identified AB (see Video 346e-11). Rescue therapies, such as angio-FIGURE 345-26 Dieulafoy’s lesion. A. Actively spurting jejunal Dieulafoy’s lesion.

1	graphic embolization or surgical oversewing, are con-There is no underlying mucosal lesion. B. Histology of a gastric Dieulafoy’s lesion. A sidered in situations where endoscopic therapy has persistent caliber artery (arrows) is present in the gastric submucosa, immediately failed. beneath the mucosa. FIGURE 345-27 Mallory-Weiss tear at the gastroesophageal junction.

1	FIGURE 345-27 Mallory-Weiss tear at the gastroesophageal junction. unable to swallow water; endoscopy is generally the best initial test in such patients, because endoscopic removal of the obstructing material is usually possible, and the presence of an underlying esophageal pathology can often be determined. Radiographs of the chest and neck should be considered before endoscopy in patients with fever, obstruction for ≥24 h, or ingestion of a sharp object, such as a fishbone. Radiographic contrast studies interfere with subsequent endoscopy and are not advisable in most patients with a clinical picture of esophageal obstruction. Sips of a carbonated beverage, sublingual nifedipine or nitrates, or intravenous glucagon may resolve an esophageal food impaction, but in most patients, an underlying web, ring, or stricture is present and endoscopic removal of the obstructing food bolus is necessary.

1	Gastric Outlet Obstruction Obstruction of the gastric outlet is commonly caused by gastric, duodenal, or pancreatic malignancy or chronic peptic ulceration with stenosis of the pylorus (Fig. 345-32). Patients vomit partially digested food many hours after eating. Gastric decompression with a nasogastric tube and subsequent lavage for removal of retained material is the first step in treatment. The diagnosis can then be confirmed with a saline load test, if desired. Endoscopy is useful for diagnosis and treatment. Patients with benign pyloric stenosis may be treated with endoscopic balloon dilatation of the pylorus, and a course of endoscopic dilatation results in long-term relief of symptoms in about 50% of patients. Malignant gastric outlet obstruction can be relieved with endoscopically placed expandable stents in patients with inoperable malignancy (Fig. 345-33).

1	Colonic Obstruction and Pseudoobstruction These both present with abdominal distention and discomfort; tympany; and a dilated, air-filled

1	FIGURE 345-28 Gastrointestinal vascular ectasias. A. Gastric antral vascular ectasia (“watermelon window of approximately 24 h during stomach”) characterized by stripes of prominent flat or raised vascular ectasias. B. Cecal vascular ectasias. which biliary drainage should be estab-C. Radiation-induced vascular ectasias of the rectum in a patient previously treated for prostate cancer. lished, typically by ERCP. Undue delay jaundice, abdominal pain, and fever is present in about 70% of patients with ascending cholangitis and biliary sepsis. These patients are managed initially with fluid resuscitation and intravenous antibiotics. Abdominal ultrasound is often performed to assess for gallbladder stones and bile duct dilation. However, the bile duct may not be dilated early in the course of acute biliary obstruction. Medical management usually improves the patient’s clinical status, providing a colon on plain abdominal radiography. The radiographic appearance can be characteristic of a

1	acute biliary obstruction. Medical management usually improves the patient’s clinical status, providing a colon on plain abdominal radiography. The radiographic appearance can be characteristic of a particular condition, such as sigmoid volvulus (Fig. 345-34). Both structural obstruction and pseudoobstruction may lead to colonic perforation if left untreated. Acute colonic pseudoobstruction is a form of colonic ileus that is usually attributable to electrolyte disorders, narcotic and anticholinergic medications, immobility (as after surgery), and retroperitoneal hemorrhage or mass. Multiple causative factors are often present. Colonoscopy, water-soluble contrast enema, or CT may be used to assess for an obstructing lesion and differentiate obstruction from pseudoobstruction. One of these diagnostic studies should be strongly considered if the patient does not have clear risk factors for pseudoobstruction, if radiographs do not show air in the rectum, or if the patient fails to improve

1	studies should be strongly considered if the patient does not have clear risk factors for pseudoobstruction, if radiographs do not show air in the rectum, or if the patient fails to improve when underlying causes of pseudoobstruction have been addressed. The risk of cecal perforation in pseudoobstruction rises when the cecal diameter exceeds 12 cm, and decompression of the colon may be achieved using intravenous neostigmine or via colonoscopic decompression (Fig. 345-35). Most patients should receive a trial of conservative therapy (with correction of electrolyte disorders, removal of offending medications, and increased mobilization) before undergoing an invasive decompressive procedure for colonic pseudoobstruction.

1	Colonic obstruction is an indication for urgent intervention. In the past, emergent diverting colostomy was usually performed with a subsequent second operation after bowel preparation to treat the underlying cause of obstruction. Colonoscopic placement of an expandable stent is now a widely used alternative that can relieve malignant colonic obstruction without emergency surgery and permit bowel preparation for an elective one-stage operation (Fig. 345-36, see Video 346e-15).

1	The steady, severe pain that occurs when a gallstone acutely obstructs the common bile duct often brings patients to a hospital. The diagnosis of a ductal stone is suspected when the patient is jaundiced or when serum liver tests or pancreatic enzyme levels are elevated; it is confirmed by EUS, magnetic resonance cholangiography (MRCP), or direct cholangiography (performed endoscopically, percutaneously, or during surgery). ERCP is currently the primary means of diagnosing and treating common bile duct stones in most hospitals in the United States (Figs. 345-11 and 345-12). Bile Duct Imaging Whereas transabdominal ultrasound diagnoses only a minority of bile duct stones, MRCP and EUS are >90% accurate and have an important role in diagnosis. Examples of these modalities are shown in Fig. 345-37.

1	If the suspicion for a bile duct stone is high and urgent treatment is required (as in a patient with obstructive jaundice and biliary sepsis), ERCP is the procedure of choice, because it remains the gold standard for diagnosis and allows for immediate treatment (see Video 346e-16). If a persistent bile duct stone is relatively unlikely (as in a patient with gallstone pancreatitis), ERCP may be supplanted by less invasive imaging techniques, such as EUS, MRCP, or intraoperative cholangiography performed during cholecystectomy, sparing patients the risk and discomfort of ERCP. Ascending Cholangitis Charcot’s triad of FIGURE 345-29 Colonic diverticula. FIGURE 345-30 Diverticular hemorrhage. A. Actively bleeding sigmoid diverticulum. B. Hemostasis achieved using endoscopic clips. FIGURE 345-31 Esophageal food (meat) impaction. FIGURE 345-32 Gastric outlet obstruction due to pyloric stenosis.

1	FIGURE 345-31 Esophageal food (meat) impaction. FIGURE 345-32 Gastric outlet obstruction due to pyloric stenosis. A. Sequela of nonsteroidal anti-inflammatory drug (NSAID)–induced ulcer disease with severe stenosis of the pylorus (arrow). B. Balloon dilation of the stenosis. C. Appearance of pyloric ring after dilation. can result in recrudescence of overt sepsis and increased morbidity and mortality rates. In addition to Charcot’s triad, the additional presence of shock and confusion (Reynolds’s pentad) is associated with high mortality rate and should prompt urgent intervention to restore biliary drainage.

1	Gallstone Pancreatitis Gallstones may cause acute pancreatitis as they pass through the ampulla of Vater. The occurrence of gallstone pancreatitis usually implies passage of a stone into the duodenum, and only about 20% of patients harbor a persistent stone in the ampulla or the common bile duct. Retained stones are more common in patients with jaundice, rising serum liver tests following hospitalization, severe pancreatitis, or superimposed ascending cholangitis. Urgent ERCP decreases the morbidity rate of gallstone pancreatitis in a subset of patients with retained bile duct stones. It is unclear FIGURE 345-33 Biliary and duodenal self-expanding metal stents (SEMS) for obstruction caused by pancreatic cancer. A. Endoscopic retrograde cholangiopancreatography (ERCP) demonstrates a distal bile duct stricture (arrow). B. A biliary SEMS is placed. C. Contrast injection demonstrates a duodenal stricture (arrow). D. Biliary and duodenal SEMS in place.

1	C. Contrast injection demonstrates a duodenal stricture (arrow). D. Biliary and duodenal SEMS in place. whether the benefit of ERCP is mainly attributable to treatment and prevention of ascending cholangitis or to relief of pancreatic ductal obstruction. ERCP is warranted early in the course of gallstone pancreatitis if ascending cholangitis is suspected, especially in a jaundiced patient. Urgent ERCP may also benefit patients predicted to have severe pancreatitis using a clinical index of severity, such as the Glasgow or Ranson score. Because the benefit of ERCP is limited to patients with a retained bile duct stone, a strategy of initial MRCP or EUS for diagnosis decreases the utilization of ERCP in gallstone pancreatitis and improves clinical outcomes by limiting the occurrence of ERCP-related adverse events. FIGURE 345-34 Sigmoid volvulus with the characteristic radiologic appearance of a “bent inner tube.”

1	FIGURE 345-34 Sigmoid volvulus with the characteristic radiologic appearance of a “bent inner tube.” FIGURE 345-35 Acute colonic pseudoobstruction. A. Acute colonic dilatation occurring in a patient soon after knee surgery. B. Colonoscopic placement of decompression tube with marked improvement in colonic dilatation.

1	B. Colonoscopic placement of decompression tube with marked improvement in colonic dilatation. Dyspepsia is a chronic or recurrent burning discomfort or pain in the upper abdomen that may be caused by diverse processes such as gastroesophageal reflux, peptic ulcer disease, and “nonulcer dyspepsia,” a heterogeneous category that includes disorders of motility, sensation, and somatization. Gastric and esophageal malignancies are less common causes of dyspepsia. Careful history-taking allows accurate differential diagnosis of dyspepsia in only about half of patients. In the remainder, endoscopy can be a useful diagnostic tool, especially in patients whose symptoms are not resolved by an empirical trial of symptomatic treatment. Endoscopy should be performed at the outset in patients with dyspepsia and alarm features, such as weight loss or iron-deficiency anemia.

1	When classic symptoms of gastroesophageal reflux are present, such as water brash and substernal heartburn, presumptive diagnosis and empirical treatment are often sufficient. Endoscopy is a sensitive test for diagnosis of esophagitis (Fig. 345-38), but will miss nonerosive reflux disease (NERD) because some patients have symptomatic reflux without esophagitis. The most sensitive test for diagnosis of GERD is 24-h ambulatory pH monitoring. Endoscopy is indicated in patients with reflux symptoms refractory to antisecretory therapy; in those with alarm symptoms, such as dysphagia, weight loss, or gastrointestinal bleeding; and in those with recurrent dyspepsia after treatment that is not clearly due to reflux on clinical grounds alone. Endoscopy should be considered in patients with long-standing (≥10 years) GERD, because they have a sixfold increased risk of harboring Barrett’s esophagus compared to a patient with <1 year of reflux symptoms. Patients with Barrett’s esophagus (Fig.

1	(≥10 years) GERD, because they have a sixfold increased risk of harboring Barrett’s esophagus compared to a patient with <1 year of reflux symptoms. Patients with Barrett’s esophagus (Fig. 345-3) generally undergo a surveillance program of periodic endoscopy with biopsies to detect dysplasia or early carcinoma.

1	Barrett’s Esophagus Barrett’s esophagus is specialized columnar metaplasia that replaces the normal squamous mucosa of the distal esophagus in some persons with GERD. Barrett’s epithelium is a major risk factor for adenocarcinoma of the esophagus and is readily detected endoscopically, due to proximal displacement of the squamocolumnar junction (Fig. 345-3). A screening EGD for Barrett’s esophagus should be considered in patients with a chronic (≥10 year) history of GERD symptoms. Endoscopic biopsy is the gold standard for confirmation of Barrett’s esophagus and for dysplasia or cancer arising in Barrett’s mucosa. FIGURE 345-36 Obstructing colonic carcinoma. A. Colonic adenocarcinoma causing marked luminal narrowing of the distal transverse colon. B. Endoscopic placement of a self-expandable metal stent. C. Radiograph of expanded stent across the obstructing tumor with a residual waist (arrow).

1	Peptic ulcer classically causes epigastric gnawing or burning, often occurring nocturnally and promptly relieved by food or antacids. Although endoscopy is the most sensitive diagnostic test for peptic ulcer, it is not a cost-effective strategy in young patients with ulcer-like dyspeptic symptoms unless endoscopy is available at low cost. Patients with suspected peptic ulcer should be evaluated for Helicobacter pylori infection. Serology (past or present infection), urea breath testing (current infection), and stool tests are noninvasive and less costly than endoscopy with biopsy. Patients with alarm symptoms and those with persistent symptoms despite treatment should undergo endoscopy to exclude gastric malignancy and other etiologies.

1	Nonulcer dyspepsia may be associated with bloating and, unlike peptic ulcer, tends not to remit and recur. Most patients describe marginal relief on acid-reducing, prokinetic, or anti-Helicobacter therapy, and are referred for endoscopy to exclude a refractory ulcer and assess for other causes. Although endoscopy is useful for excluding other diagnoses, its impact on the treatment of patients with nonulcer dyspepsia is limited.

1	About 50% of patients presenting with difficulty swallowing have a mechanical obstruction; the remainder has a motility disorder, such as achalasia or diffuse esophageal spasm. Careful history-taking often points to a presumptive diagnosis and leads to the appropriate use of diagnostic tests. Esophageal strictures (Fig. 345-39) typically cause progressive dysphagia, first for solids, then for liquids; motility disorders often cause intermittent dysphagia for both solids and liquids. Some underlying disorders have characteristic historic features: Schatzki’s ring (Fig. 345-40) causes episodic dysphagia for solids, typically at the beginning of a meal; oropharyngeal motor disorders typically present with difficulty initiating deglutition (transfer dysphagia) and nasal reflux or coughing with swallowing; and achalasia may cause nocturnal regurgitation of undigested food.

1	When mechanical obstruction is suspected, endoscopy is a useful initial diagnostic test, because it permits immediate biopsy and/or dilatation of strictures, masses, or rings. The presence of linear furrows and multiple corrugated rings throughout a narrowed esophagus (feline esophagus) should raise suspicion for eosinophilic esophagitis, an increasingly recognized cause for recurrent dysphagia and food impaction (Fig. 345-41). Blind or forceful passage of an endoscope may lead to perforation in a patient with stenosis of the cervical esophagus or a Zenker’s diverticulum, but gentle passage of an endoscope under direct visual guidance is reasonably safe. Endoscopy can miss a subtle stricture or ring in some patients. When transfer dysphagia is evident or an esophageal motility disorder is suspected, esophageal radiography and/or a video-swallow study

1	When transfer dysphagia is evident or an esophageal motility disorder is suspected, esophageal radiography and/or a video-swallow study FIGURE 345-37 Methods of bile duct imaging. Arrows mark bile duct stones. Arrowheads indicate the common bile duct, and the asterisk marks the portal vein. A. Endoscopic ultrasound (EUS). B. Magnetic resonance cholangiopancreatography (MRCP). C. Helical computed tomography (CT).

1	(see Video 346e-3). In general, endoscopic techniques offer the advantage of a minimally invasive approach to treatment, but rely on other imaging techniques (such as CT, magnetic resonance imaging [MRI], positron emission tomography [PET], and EUS) to exclude distant metastases or locally advanced disease better treated by surgery or other modalities. The decision to treat an early-stage gastrointestinal malignancy endoscopically is often made in collaboration with a surgeon and/or oncologist. Endoscopic palliation of gastrointestinal malignancies relieves symptoms and in many cases prolongs survival. Malignant obstruction can be relieved by endoscopic stent placement (Figs. 345-13, 345-33, and 345-36; see Video 346e-15), and malignant gastrointestinal bleeding can often be palliated endoscopically as well. EUS-guided celiac plexus neurolysis may relieve pancreatic cancer pain.

1	Iron-deficiency anemia may be attributed to poor iron absorption (as in celiac sprue) or, more commonly, chronic blood loss. Intestinal bleeding should be strongly suspected in men and postmenopausal women with iron-deficiency anemia, and colonoscopy is indicated in such patients, even in the absence of detectable occult blood in the stool. Approximately 30% will have large colonic polyps, 10% will have colorectal cancer, and a few additional patients will have colonic vascular lesions. When a FIGURE 345-38 Causes of esophagitis. A. Severe reflux esophagitis with mucosal convincing source of blood loss is not found in the colon, ulceration and friability. B. Cytomegalovirus esophagitis. C. Herpes simplex virus esophagitis with target-type shallow ulcerations. D. Candida esophagitis with white plaques adherent to the esophageal mucosa.

1	are the best initial diagnostic tests. The oropharyngeal swallowing mechanism, esophageal peristalsis, and the lower esophageal sphincter can all be assessed. In some disorders, subsequent esophageal manometry may also be important for diagnosis.

1	Endoscopy plays an important role in the treatment of gastrointestinal malignancies. Early-stage malignancies limited to the superficial layers of the gastrointestinal mucosa may be resected using the techniques of endoscopic mucosal resection (EMR) (see Video 346e-4) or endoscopic submucosal dissection (ESD) (see Video 346e-5). Photodynamic therapy (PDT) and radiofrequency ablation (RFA) are effective modalities for ablative treatment of high-grade dysplasia and intramucosal cancer in Barrett’s esophagus. Gastrointestinal stromal tumors can be removed en bloc by endoscopic full-thickness resection no lesion is found, duodenal biopsies should be obtained to exclude sprue (Fig. 345-42). Small-bowel evaluation with capsule endoscopy (Fig. 345-43), CT or magnetic resonance (MR) enterography, or balloon-assisted enteroscopy may be appropriate if both EGD and colonoscopy are unrevealing.

1	Tests for occult blood in the stool detect hemoglobin or the heme moiety and are most sensitive for colonic blood loss, although they will also detect larger amounts of upper gastrointestinal bleeding. Patients over age 50 with occult blood in normal-appearing stool should undergo colonoscopy to diagnose or exclude colorectal neoplasia. The diagnostic yield is lower than in iron-deficiency anemia. Whether upper endoscopy is also indicated depends on the patient’s symptoms.

1	The small intestine may be the source of chronic intestinal bleeding, especially if colonoscopy and upper endoscopy are not diagnostic. The utility of small-bowel evaluation varies with the clinical setting and is most important in patients in whom bleeding causes chronic or recurrent anemia. In contrast to the low diagnostic yield of small-bowel radiography, positive findings on capsule endoscopy are seen in 50–70% of patients with suspected small intestinal bleeding. The most common finding is mucosal vascular ectasias. CT or MR enterography accurately detects small-bowel masses and inflammation and is also useful for initial small-bowel evaluation. Deep enteroscopy may follow capsule endoscopy for biopsy of lesions or to provide specific therapy, such as argon plasma coagulation of vascular ectasias (Fig. 345-44). FIGURE 345-39 Peptic esophageal stricture associated with esophagitis. FIGURE 345-40 Schatzki’s ring at the gastroesophageal junction.

1	FIGURE 345-39 Peptic esophageal stricture associated with esophagitis. FIGURE 345-40 Schatzki’s ring at the gastroesophageal junction. FIGURE 345-41 Eosinophilic esophagitis with multiple circular rings of the esophagus creating a corrugated appearance, and an impacted grape at the narrowed esophagogastric junction. The diag-nosis requires biopsy with histologic finding of > 15–20 eosinophils per high-power field. The majority of colon cancers develop from preexisting colonic adenomas, and colorectal cancer can be largely prevented by the detection and removal of adenomatous polyps (see Video 346e-17). The choice of screening strategy for an asymptomatic person depends on personal and family history. Individuals with inflammatory bowel disease, a history of colorectal polyps or cancer, family members with adenomatous polyps or cancer, or certain familial cancer syndromes (Fig. 345-45)

1	FIGURE 345-43 Capsule endoscopy images of a mildly scalloped jejunal fold (left) and an ileal tumor (right) in a patient with celiac sprue. (Images courtesy of Dr. Elizabeth Rajan; with permission.) FIGURE 345-44 A. Mid-jejunal vascular ectasia identified by double-balloon endoscopy. B. Ablation of vascular ectasia with argon plasma coagulation. FIGURE 345-42 Scalloped duodenal folds in a patient with celiac sprue. FIGURE 345-45 Innumerable colon polyps of various sizes in a patient with familial adenomatous polyposis syndrome. Asymptomatic individuals ≥50 years of age (≥45 Colonoscopy every 10 yearsa Preferred cancer prevention strategy years of age for African Americans) Annual fecal immunochemical test (FIT) or fecal Cancer detection strategy; fails to detect most pol-occult blood test (FOBT), multiple take-home yps; colonoscopy if results are positive specimen cards Computed tomography (CT) colonography every Colonoscopy if results are positive 5 years

1	Computed tomography (CT) colonography every Colonoscopy if results are positive 5 years Flexible sigmoidoscopy every 5 years Fails to detect proximal colon polyps and cancers Personal History of Polyps or Colorectal Cancer aAssumes good colonic preparation and complete exam to cecum. bHigh-risk adenoma: any adenoma ≥1 cm in size or containing high-grade dysplasia or villous features. Abbreviations: CRC, colorectal cancer; FAP, familial adenomatous polyposis; HNPCC, hereditary nonpolyposis colorectal cancer. Source: Adapted from DA Lieberman et al: Gastroenterology 143:844, 2012; B Levin et al: CA Cancer J Clin 58:130, 2008; American Cancer Society Guidelines (http://www.cancer.org/ cancer/colonandrectumcancer/moreinformation/colonandrectumcancerearlydetection/colorectal-cancer-early-detection-acs-recommendations), accessed November 15, 2013. are at increased risk for colorectal cancer. An individual without these factors is generally considered at average risk.

1	Screening strategies are summarized in Table 345-3. Although stool tests for occult blood have been shown to decrease mortality rate from colorectal cancer, they do not detect some cancers and many polyps, and direct visualization of the colon is a more effective screening strategy. Either sigmoidoscopy or colonoscopy may be used for cancer screening in asymptomatic average-risk individuals. The use of sigmoidoscopy was based on the historical finding that the majority of colorectal cancers occurred in the rectum and left colon and that patients with right-sided colon cancers had left-sided polyps. Over the past several decades, however, the distribution of colon cancers has changed in the United States, with proportionally fewer rectal and left-sided cancers than in the past. Large American studies of colonoscopy for screening of average-risk individuals show that cancers are roughly equally distributed between left and right colon and half of patients with right-sided lesions have

1	studies of colonoscopy for screening of average-risk individuals show that cancers are roughly equally distributed between left and right colon and half of patients with right-sided lesions have no polyps in the left colon. Visualization of the entire colon thus appears to be the optimal strategy for colorectal cancer screening and prevention.

1	Virtual colonoscopy (VC) is a radiologic technique that images the colon with CT following rectal insufflation of the colonic lumen. Computer rendering of CT images generates an electronic display of FIGURE 345-47 Ulcerated ileal carcinoid tumor. FIGURE 345-46 Virtual colonoscopy image of a colon polyp (arrow). (Image courtesy of Dr. Jeff Fidler; with permission.) a virtual “flight” along the colonic lumen, simulating colonoscopy (Fig. 345-46). Comparative studies of virtual and routine colonoscopy have shown conflicting results, but technical refinements have improved the performance characteristics of VC. The use of VC for colorectal cancer screening may become more widespread in the future, particularly at institutions with demonstrated skill with this technique. Findings detected during virtual colonoscopy often require subsequent conventional colonoscopy for confirmation and treatment.

1	Most cases of diarrhea are acute, self-limited, and due to infections or medication. Chronic diarrhea (lasting >6 weeks) is more often due to a primary inflammatory, malabsorptive, or motility disorder; is less likely to resolve spontaneously; and generally requires diagnostic evaluation. Patients with chronic diarrhea or severe, unexplained acute diarrhea often undergo endoscopy if stool tests for pathogens are unrevealing. The choice of endoscopic testing depends on the clinical setting.

1	Patients with colonic symptoms and findings such as bloody diarrhea, tenesmus, fever, or leukocytes in stool generally undergo sigmoidoscopy or colonoscopy to assess for colitis (Fig. 345-4). Sigmoidoscopy is an appropriate initial test in most patients. Conversely, patients with symptoms and findings suggesting small-bowel disease, such as large-volume watery stools, substantial weight loss, and malabsorption of iron, calcium, or fat, may undergo upper endoscopy with duodenal aspirates for assessment of bacterial overgrowth and biopsies for assessment of mucosal diseases, such as celiac sprue.

1	Many patients with chronic diarrhea do not fit either of these patterns. In the setting of a long-standing history of alternating constipation and diarrhea dating to early adulthood, without findings such as blood in the stool or anemia, a diagnosis of irritable bowel syndrome may be made without direct visualization of the bowel. Steatorrhea and upper abdominal pain may prompt evaluation of the pancreas rather than the gut. Patients whose chronic diarrhea is not easily categorized often undergo initial colonoscopy to examine the entire colon and terminal ileum for inflammatory or neoplastic disease (Fig. 345-47).

1	Bright red blood passed with or on formed brown stool usually has a rectal, anal, or distal sigmoid source (Fig. 345-48). Patients with even trivial amounts of hematochezia should be investigated with flexible sigmoidoscopy and anoscopy to exclude polyps or cancers in the distal colon. Patients reporting red blood on the toilet tissue only, without blood in the toilet or on the stool, are generally bleeding from a lesion in the anal canal. Careful external inspection, digital examination, and proctoscopy with anoscopy are sufficient for diagnosis in most cases.

1	About 20% of patients with pancreatitis have no identified cause after routine clinical investigation (including a review of medication and alcohol use, measurement of serum triglyceride and calcium levels, abdominal ultrasonography, and CT). Endoscopic assessment leads to a specific diagnosis in the majority of such patients, often altering clinical management. Endoscopic investigation is particularly appropriate if the patient has had more than one episode of pancreatitis.

1	Microlithiasis, or the presence of microscopic crystals in bile, is a leading cause of previously unexplained acute pancreatitis and is sometimes seen during abdominal ultrasonography as layering sludge or flecks of floating, echogenic material in the gallbladder. Gallbladder bile can be obtained for microscopic analysis by administering a cholecystokinin analogue during endoscopy, causing contraction of the gallbladder. Bile is suctioned from the duodenum as it drains from the papilla, and the darkest fraction is examined for cholesterol crystals or bilirubinate granules. The combination of EUS of the gallbladder and bile microscopy is probably the most sensitive means of diagnosing microlithiasis. FIGURE 345-48 Internal hemorrhoids with bleeding (arrow) as seen on a retroflexed view of the rectum.

1	FIGURE 345-48 Internal hemorrhoids with bleeding (arrow) as seen on a retroflexed view of the rectum. 1900 Previously undetected chronic pancreatitis, pancreatic malignancy, or pancreas divisum may be diagnosed by either ERCP or EUS. Sphincter of Oddi dysfunction or stenosis is a potential cause for pancreatitis and can be diagnosed by manometric studies performed during ERCP. Autoimmune pancreatitis may require EUS-guided pancreatic biopsy for histologic diagnosis. Severe pancreatitis often results in pancreatic fluid collections. Both pseudocysts and areas of walled-off pancreatic necrosis can be drained into the stomach or duodenum endoscopically, using transpapillary and transmural endoscopic techniques. Pancreatic necrosis can be treated by direct endoscopic necrosectomy (see Video 346e-2).

1	Local staging of esophageal, gastric, pancreatic, bile duct, and rectal cancers can be obtained with EUS (Fig. 345-15). EUS with fine-needle aspiration (Fig. 345-16) currently provides the most accurate preoperative assessment of local tumor and nodal staging, but it does not detect most distant metastases. Details of the local tumor stage can guide treatment decisions including resectability and need for neoadjuvant therapy. EUS with transesophageal needle biopsy may also be used to assess the presence of non-small-cell lung cancer in mediastinal nodes. Direct scheduling of endoscopic procedures by primary care physicians without preceding gastroenterology consultation, or open-access endoscopy, is common. When the indications for endoscopy are clear-cut and appropriate, the procedural risks are low, and the patient understands what to expect, open-access endoscopy streamlines patient care and decreases costs.

1	Patients referred for open-access endoscopy should have a recent history, physical examination, and medication review. A copy of such an evaluation should be available when the patient comes to the endoscopy suite. Patients with unstable cardiovascular or respiratory conditions should not be referred directly for open-access endoscopy. Patients with particular conditions and undergoing certain procedures should be prescribed prophylactic antibiotics prior to endoscopy (Table 345-1). In addition, patients taking anticoagulants and/or anti-platelet drugs may require adjustment of these agents before endoscopy based on the procedure risk for bleeding and condition risk for a thromboembolic event (Table 345-2).

1	Common indications for open-access EGD include dyspepsia resistant to a trial of appropriate therapy; dysphagia; gastrointestinal bleeding; and persistent anorexia or early satiety. Open-access colonoscopy is often requested in men or postmenopausal women with iron-deficiency anemia, in patients over age 50 with occult blood in the stool, in patients with a previous history of colorectal adenomatous polyps or cancer, and for colorectal cancer screening. Flexible sigmoidoscopy is commonly performed as an open-access procedure.

1	When patients are referred for open-access colonoscopy, the primary care provider may need to choose a colonic preparation. Commonly used oral preparations include polyethylene glycol lavage solution, with or without citric acid. A “split-dose” regimen improves the quality of colonic preparation. Sodium phosphate purgatives may cause fluid and electrolyte abnormalities and renal toxicity, especially in patients with renal failure or congestive heart failure and those over 70 years of age.

1	Video Atlas of Gastrointestinal Endoscopy Louis Michel Wong Kee Song, Mark Topazian Gastrointestinal endoscopy is an increasingly important method for diagnosis and treatment of disease. This atlas demonstrates endoscopic 346e findings in a variety of gastrointestinal infectious, inflammatory, vascular, and neoplastic conditions. Cancer screening and prevention are common indications for gastrointestinal endoscopy, and the premalignant conditions of Barrett’s esophagus and colonic polyps are illustrated. Endoscopic treatment modalities for gastrointestinal bleeding, polyps, and biliary stones are demonstrated in video clips. The images shown in this atlas are also found in Chap. 345 of the book. Video 346e-1 Methods of deep enteroscopy. (Animations courtesy of Dr. Mark Stark and Dr. Jonathan Leighton; with permission.)

1	Video 346e-1 Methods of deep enteroscopy. (Animations courtesy of Dr. Mark Stark and Dr. Jonathan Leighton; with permission.) Video 346e-2 Pancreatic necrosis treated by transduodenal endoscopic drainage and necrosectomy. Video 346e-3 Endoscopic full-thickness resection of a gastric subepithelial lesion. Video 346e-4 Endoscopic submucosal dissection of a large rectal adenoma. Video 346e-5 Over-the-scope clip closure of a spontaneous 346e-1 esophageal perforation. Video 346e-6 Endoscopic suturing for stent fixation. Video 346e-7 Actively bleeding duodenal ulcer treated with dilute epinephrine injection, thermal probe application, and hemoclips. (Video courtesy of Dr. Navtej Buttar; with permission.)

1	(Video courtesy of Dr. Navtej Buttar; with permission.) Video 346e-8 Actively bleeding esophageal varices treated with endoscopic band ligation. Video 346e-9 Large, bleeding gastric varix treated with endoscopic cyanoacrylate injection. Video 346e-10 Dieulafoy’s lesion treated endoscopically. Video 346e-11 Bleeding Mallory-Weiss tear treated with hemoclip placement. Video 346e-12 Radiation proctopathy treated with argon plasma coagulation. Video 346e-13 Actively bleeding colonic diverticulum treated with dilute epinephrine injection and band ligation. Video 346e-14 Stent placement for palliation of malignant colonic obstruction. Video 346e-15 Bile duct stones removed after endoscopic sphincterotomy. Video 346e-16 Barrett’s esophagus with high-grade dysplasia treated with endoscopic mucosal resection. Video 346e-17 Pedunculated and sessile colonic polyps removed with snare cautery during colonoscopy. CHAPTER 346e Video Atlas of Gastrointestinal Endoscopy

1	CHAPTER 346e Video Atlas of Gastrointestinal Endoscopy Diseases of the Esophagus Peter J. Kahrilas, Ikuo Hirano ESOPHAGEAL STRUCTURE AND FUNCTION The esophagus is a hollow, muscular tube coursing through the pos-terior mediastinum joining the hypopharynx to the stomach with a 347 sphincter at each end. It functions to transport food and fluid between these ends, otherwise remaining empty. The physiology of swallowing, esophageal motility, and oral and pharyngeal dysphagia are described in Chap. 53. Esophageal diseases can be manifested by impaired function or pain. Key functional impairments are swallowing disorders and excessive gastroesophageal reflux. Pain, sometimes indistinguishable from cardiac chest pain, can result from inflammation, infection, dysmotility, or neoplasm.

1	The clinical history remains central to the evaluation of esophageal symptoms. A thoughtfully obtained history will often expedite management. Important details include weight gain or loss, gastrointestinal bleeding, dietary habits including the timing of meals, smoking, and alcohol consumption. The major esophageal symptoms are heartburn, regurgitation, chest pain, dysphagia, odynophagia, and globus sensation.

1	Heartburn (pyrosis), the most common esophageal symptom, is characterized by a discomfort or burning sensation behind the sternum that arises from the epigastrium and may radiate toward the neck. Heartburn is an intermittent symptom, most commonly experienced after eating, during exercise, and while lying recumbent. The discomfort is relieved with drinking water or antacid but can occur frequently interfering with normal activities including sleep. The association between heartburn and gastroesophageal reflux disease (GERD) is so strong that empirical therapy for GERD has become accepted management. However, the term “heartburn” is often misused and/or referred to with other terms such as “indigestion” or “repeating,” making it important to clarify the intended meaning.

1	Regurgitation is the effortless return of food or fluid into the pharynx without nausea or retching. Patients report a sour or burning fluid in the throat or mouth that may also contain undigested food particles. Bending, belching, or maneuvers that increase intraabdominal pressure can provoke regurgitation. A clinician needs to discriminate among regurgitation, vomiting, and rumination. Vomiting is preceded by nausea and accompanied by retching. Rumination is a behavior in which recently swallowed food is regurgitated and then reswallowed repetitively for up to an hour. Although there is some linkage between rumination and mental deficiency, the behavior is also exhibited by unimpaired individuals who sometimes even find it pleasurable.

1	Chest pain is a common esophageal symptom with characteristics similar to cardiac pain, sometimes making this distinction difficult. Esophageal pain is usually experienced as a pressure type sensation in the mid chest, radiating to the mid back, arms, or jaws. The similarity to cardiac pain is likely because the two organs share a nerve plexus and the nerve endings in the esophageal wall have poor discriminative ability among stimuli. Esophageal distention or even chemostimulation (e.g., with acid) will often be perceived as chest pain. Gastroesophageal reflux is the most common cause of esophageal chest pain.

1	Esophageal dysphagia (Chap. 53) is often described as a feeling of food “sticking” or even lodging in the chest. Important distinctions are between uniquely solid food dysphagia as opposed to liquid and solid, episodic versus constant dysphagia, and progressive versus static dysphagia. If the dysphagia is for liquids as well as solid food, it suggests a motility disorder such as achalasia. Conversely, uniquely solid food dysphagia is suggestive of a stricture, ring, or tumor. Of note, a patient’s localization of food hang-up in the esophagus is notoriously imprecise. Approximately 30% of distal esophageal obstructions are perceived as cervical dysphagia. In such instances, the absence of concomitant symptoms generally associated with oropharyngeal dysphagia such as aspiration, nasopharyngeal regurgitation, cough, drooling, or obvious neuromuscular compromise should suggest an esophageal etiology.

1	Odynophagia is pain either caused by or exacerbated by swallowing. Although typically considered distinct from dysphagia, odynophagia may manifest concurrently with dysphagia. Odynophagia is more common with pill or infectious esophagitis than with reflux esophagi-tis and should prompt a search for these entities. When odynophagia does occur in GERD, it is likely related to an esophageal ulcer or deep erosion. Globus sensation, alternatively labeled “globus hystericus,” is the perception of a lump or fullness in the throat that is felt irrespective of swallowing. Although such patients are frequently referred for an evaluation of dysphagia, globus sensation is often relieved by the act of swallowing. As implied by its alternative name (globus hystericus), globus sensation often occurs in the setting of anxiety or obsessive-compulsive disorders. Clinical experience teaches that it is often attributable to GERD.

1	Water brash is excessive salivation resulting from a vagal reflex triggered by acidification of the esophageal mucosa. This is not a common symptom. Afflicted individuals will describe the unpleasant sensation of the mouth rapidly filling with salty thin fluid, often in the setting of concomitant heartburn.

1	Endoscopy, also known as esophagogastroduodenoscopy (EGD), is the most useful test for the evaluation of the proximal gastrointestinal tract. Modern instruments produce high-quality, color images of the esophageal, gastric, and duodenal lumen. Endoscopes also have an instrumentation channel through which biopsy forceps, injection catheters for local delivery of therapeutic agents, balloon dilators, or hemostatic devices can be used. The key advantages of endoscopy over barium radiography are: (1) increased sensitivity for the detection of mucosal lesions, (2) vastly increased sensitivity 1901 for the detection of abnormalities mainly identifiable by color such as Barrett’s metaplasia or vascular lesions, (3) the ability to obtain biopsy specimens for histologic examination of suspected abnormalities, and (4) the ability to dilate strictures during the examination. The main disadvantages of endoscopy are cost and the utilization of sedatives or anesthetics.

1	Contrast radiography of the esophagus, stomach, and duodenum can demonstrate reflux of the contrast media, hiatal hernia, mucosal granularity, erosions, ulcerations, and strictures. The sensitivity of radiography compared with endoscopy for detecting reflux esophagitis reportedly ranges from 22–95%, with higher grades of esophagitis (i.e., ulceration or stricture) exhibiting greater detection rates. Conversely, the sensitivity of barium radiography for detecting esophageal strictures is greater than that of endoscopy, especially when the study is done in conjunction with barium-soaked bread or a 13-mm barium tablet. Barium studies also provide an assessment of esophageal function and morphology that may be undetected on endoscopy. Tracheoesophageal fistula, altered postsurgical anatomy, and extrinsic esophageal compression are conditions where radiographic imaging complements endoscopic assessment. Hypopharyngeal pathology and disorders of the cricopharyngeus muscle are better

1	and extrinsic esophageal compression are conditions where radiographic imaging complements endoscopic assessment. Hypopharyngeal pathology and disorders of the cricopharyngeus muscle are better appreciated on radiographic examination than with endoscopy, particularly with rapid sequence or video fluoroscopic recording. The major shortcoming of barium radiography is that it rarely obviates the need for endoscopy. Either a positive or a negative study is usually followed by an endoscopic evaluation either to obtain biopsies, provide therapy, or clarify findings in the case of a positive examination or to add a level of certainty in the case of a negative one.

1	Endoscopic ultrasound (EUS) instruments combine an endoscope with an ultrasound transducer to create a transmural image of the tissue surrounding the endoscope tip. The key advantage of EUS over alternative radiologic imaging techniques is much greater resolution attributable to the proximity of the ultrasound transducer to the area being examined. Available devices can provide either radial imaging (360-degree, cross-sectional) or a curved linear image that can guide fine-needle aspiration of imaged structures such as lymph nodes or tumors. Major esophageal applications of EUS are to stage esophageal cancer, to evaluate dysplasia in Barrett’s esophagus, and to assess submucosal lesions.

1	Esophageal manometry, or motility testing, entails positioning a pressure-sensing catheter within the esophagus and then observing the contractility following test swallows. The upper and lower esophageal sphincters appear as zones of high pressure that relax on swallowing, while the intersphincteric esophagus exhibits peristaltic contractions. Manometry is used to diagnose motility disorders (achalasia, diffuse esophageal spasm) and to assess peristaltic integrity prior to the surgery for reflux disease. Technologic advances have enhanced esophageal manometry as high-resolution esophageal pressure topography (Fig. 347-1). Manometry can also be combined with intraluminal impedance monitoring. Impedance recordings use a catheter with a series of paired electrodes. Esophageal luminal contents in contact with the electrodes decrease (liquid) or increase (air) the impedance signal, allowing detection of anterograde or retrograde esophageal bolus transit.

1	GERD is often diagnosed in the absence of endoscopic esophagitis, which would otherwise define the disease. This occurs in the settings of partially treated disease, an abnormally sensitive esophageal mucosa, or without obvious explanation. In such instances, reflux

1	Diseases of the Esophagus testing can demonstrate excessive esophageal exposure to refluxed gastric juice, the physiologic abnormality of GERD. This can be done by ambulatory 24to 48-h esophageal pH recording using either a wireless pH-sensitive transmitter that is anchored to the esophageal mucosa or a transnasally positioned wire electrode with the tip stationed in the distal esophagus. Either way, the outcome is expressed as the percentage of the day that the pH was less than 4 (indicative of recent acid reflux), with values exceeding 5% indicative of GERD. Reflux testing is useful with atypical symptoms or an inexplicably poor response to therapy. Intraluminal impedance monitoring can be added to pH monitoring to detect reflux events irrespective of whether or not they are acidic, potentially increasing the sensitivity of the study. Hiatus hernia is a herniation of viscera, most commonly the stomach,

1	Hiatus hernia is a herniation of viscera, most commonly the stomach, FIGURE 347-1 High-resolution esophageal pressure topography (right) and conventional manometry (left) of a normal swallow. E, esophageal body; LES, lower esophageal sphincter; UES, upper esophageal sphincter.

1	E, esophageal body; LES, lower esophageal sphincter; UES, upper esophageal sphincter. A lower esophageal mucosal ring, also called a B ring, is a thin membranous narrowing at the squamocolumnar mucosal junction (Fig. 347-2). Its origin is unknown, but B rings are demonstrable in about 10–15% of the general population and are usually asymptomatic. When the lumen diameter is less than 13 mm, distal rings are usually associated with episodic solid food dysphagia and are called Schatzki rings. Patients typically present older than 40 years, consistent with an acquired rather than congenital origin. Schatzki ring is one of the most common causes of intermittent food impaction, also known as “steakhouse syndrome” because meat is a typical instigator. Symptomatic rings are easily treated by dilation.

1	Web-like constrictions higher in the esophagus can be of congenital or inflammatory origin. Asymptomatic cervical esophageal webs are demonstrated in about 10% of people and typically originate along the anterior aspect of the esophagus. When circumferential, they can into the mediastinum through the esophageal hiatus of the diaphragm. esophagusFour types of hiatus hernia are distinguished with type I, or sliding hiatal hernia, comprising at least 95% of the overall total. A sliding hiatal hernia is one in which the gastroesophageal junction and gastric cardia translocate cephalad as a result of weakening of the phrenoesophageal ligament attaching the gastroesophageal junction to the diaphragm at the hiatus and dilatation of the diaphragmatic hiatus. The incidence of sliding hernia increases with age. True to its name, sliding hernias enlarge with increased intraabdominal pressure, swallowing, and respiration. Conceptually, sliding hernias are the result of wear and tear: increased

1	with age. True to its name, sliding hernias enlarge with increased intraabdominal pressure, swallowing, and respiration. Conceptually, sliding hernias are the result of wear and tear: increased intraabdominal pressure from abdominal obesity, pregnancy, etc., along with hereditary factors predisposing to the condition. The main significance of sliding hernias is the propensity of affected individuals to have GERD.

1	Types II, III, and IV hiatal hernias are all subtypes of paraesophageal hernia in which the herniation into the mediastinum includes a visceral structure other than the gastric cardia. With type II and III paraesophageal hernias, the gastric fundus also herniates with the distinction being that in type II, the gastroesophageal junction remains fixed at the hiatus, whereas type III is a combined sliding and paraesophageal hernia. With type IV hiatal hernias, viscera other than the stomach herniate into the mediastinum, most commonly the colon. With type II and III paraesophageal hernias, the stomach inverts as it herniates and large paraesophageal hernias can lead to an upside down stomach, gastric volvulus, and even strangulation of the stomach. Because of this risk, surgical repair is often advocated for large paraesophageal hernias. FIGURE 347-2 Radiographic anatomy of the gastroesophageal junction.

1	FIGURE 347-2 Radiographic anatomy of the gastroesophageal junction. FIGURE 347-3 Examples of small (A) and large (B, C) Zenker’s diverticula arising from Killian’s triangle in the distal hypopharynx. Smaller diverticula are evident only during the swallow, whereas larger ones retain food and fluid. cause intermittent dysphagia to solids similar to Schatzki rings and are similarly treated with dilatation. The combination of symptomatic proximal esophageal webs and iron-deficiency anemia in middle-aged women constitutes Plummer-Vinson syndrome.

1	Esophageal diverticula are categorized by location with the most common being epiphrenic, hypopharyngeal (Zenker’s), and midesophageal. Epiphrenic and Zenker’s diverticula are false diverticula involving herniation of the mucosa and submucosa through the muscular layer of the esophagus. These lesions result from increased intraluminal pressure associated with distal obstruction. In the case of Zenker’s, the obstruction is a stenotic cricopharyngeus muscle (upper esophageal sphincter), and the hypopharyngeal herniation most commonly occurs in an area of natural weakness proximal to the cricopharyngeus known as Killian’s triangle (Fig. 347-3). Small Zenker’s diverticula are usually asymptomatic, but when they enlarge sufficiently to retain food and saliva they can be associated with dysphagia, halitosis, and aspiration. Treatment is by surgical diverticulectomy and cricopharyngeal myotomy or a marsupialization procedure in which an endoscopic stapling device is used to divide the

1	halitosis, and aspiration. Treatment is by surgical diverticulectomy and cricopharyngeal myotomy or a marsupialization procedure in which an endoscopic stapling device is used to divide the cricopharyngeus.

1	Epiphrenic diverticula are usually associated with achalasia or a distal esophageal stricture. Midesophageal diverticula may be caused by traction from adjacent inflammation (classically tuberculosis) in which case they are true diverticula involving all layers of the esophageal wall, or by pulsion associated with esophageal motor disorders. Midesophageal and epiphrenic diverticula are usually asymptomatic until they enlarge sufficiently to retain food and cause dysphagia and regurgitation. Symptoms attributable to the diverticula tend to correlate more with the underlying esophageal disorder than the size of the diverticula. Large diverticula can be removed surgically, usually in conjunction with a myotomy if the underlying cause is achalasia. Diffuse intramural esophageal diverticulosis is a rare entity that results from dilatation of the excretory ducts of submucosal esophageal glands (Fig. 347-4). Esophageal candidiasis and proximal esophageal strictures are commonly found in

1	is a rare entity that results from dilatation of the excretory ducts of submucosal esophageal glands (Fig. 347-4). Esophageal candidiasis and proximal esophageal strictures are commonly found in association with this disorder.

1	FIGURE 347-4 Intramural esophageal pseudodiverticulosis associ-ated with chronic obstruction. Invaginations of contrast into the esophageal wall outline deep esophageal glands.

1	Esophageal cancer occurs in about 4.5:100,000 people in the United emphasize both the rarity and lethality of esophageal cancer. OneStates with the associated mortality being only slightly less at notable trend is the shift of dominant esophageal cancer type from 4.4:100,000. It is about 10 times less common than colorectal can-squamous cell to adenocarcinoma, strongly linked to reflux disease cer but kills about one-quarter as many patients. These statistics and Barrett’s metaplasia. Other distinctions between cell types are the 1904 predilection for adenocarcinoma to affect the distal esophagus in white males and squamous cell to affect the more proximal esophagus in black males with the added risk factors of smoking, alcohol consumption, caustic injury, and human papilloma virus infection (Chap. 109). The typical presentation of esophageal cancer is of progressive solid food dysphagia and weight loss. Associated symptoms may include odynophagia, iron deficiency, and, with

1	infection (Chap. 109). The typical presentation of esophageal cancer is of progressive solid food dysphagia and weight loss. Associated symptoms may include odynophagia, iron deficiency, and, with midesophageal tumors, hoarseness from left recurrent laryngeal nerve injury. Generally, these are indications of locally invasive or even metastatic disease manifest by tracheoesophageal fistulas and vocal cord paralysis. Even when detected as a small lesion, esophageal cancer has poor survival because of the abundant esophageal lymphatics leading to regional lymph node metastases. Benign esophageal tumors are uncommon and usually discovered incidentally. In decreasing frequency of occurrence, cell types include leiomyoma, fibrovascular polyps, squamous papilloma, granular cell tumors, lipomas, neurofibromas, and inflammatory fibroid polyps. These generally become symptomatic only when they are associated with dysphagia and merit removal only under the same circumstances.

1	The most common congenital esophageal anomaly is esophageal atresia, occurring in about 1 in 5000 live births. Atresia can occur in several permutations, the common denominator being developmental failure of fusion between the proximal and distal esophagus associated with a tracheoesophageal fistula, most commonly with the distal segment excluded. Alternatively, there can be an H-type configuration in which esophageal fusion has occurred, but with a tracheoesophageal fistula. Esophageal atresia is usually recognized and corrected surgically within the first few days of life. Later life complications include dysphagia from anastomotic strictures or absent peristalsis and reflux, which can be severe. Less common developmental anomalies include congenital esophageal stenosis, webs, and duplications.

1	Dysphagia can also result from congenital abnormalities that cause extrinsic compression of the esophagus. In dysphagia lusoria, the esophagus is compressed by an aberrant right subclavian artery arising from the descending aorta and passing behind the esophagus. Alternatively vascular rings may surround and constrict the esophagus. Heterotopic gastric mucosa, also known as an esophageal inlet patch, is a focus of gastric type epithelium in the proximal cervical esophagus; the estimated prevalence is 4.5%. The inlet patch is thought to result from incomplete replacement of embryonic columnar epithelium with squamous epithelium. The majority of inlet patches are asymptomatic, but acid production can occur as most contain fundic type gastric epithelium with parietal cells.

1	Esophageal motility disorders are diseases attributable to esophageal neuromuscular dysfunction commonly associated with dysphagia, chest pain, or heartburn. The major entities are achalasia, diffuse esophageal spasm (DES), and GERD. Motility disorders can also be secondary to broader disease processes as is the case with pseudoachalasia, Chagas’ disease, and scleroderma. Not included in this discussion are diseases affecting the pharynx and proximal esophagus, the impairment of which is almost always part of a more global neuromuscular disease process.

1	Achalasia is a rare disease caused by loss of ganglion cells within the esophageal myenteric plexus with a population incidence of about 1:100,000 and usually presenting between age 25 and 60. With longstanding disease, aganglionosis is noted. The disease involves both excitatory (cholinergic) and inhibitory (nitric oxide) ganglionic neurons. Functionally, inhibitory neurons mediate deglutitive lower esophageal sphincter (LES) relaxation and the sequential propagation of peristalsis. Their absence leads to impaired deglutitive LES relaxation and absent peristalsis. Increasing evidence suggests that the ultimate cause of ganglion cell degeneration in achalasia is an autoimmune process attributable to a latent infection with human herpes simplex virus 1 combined with genetic susceptibility.

1	Long-standing achalasia is characterized by progressive dilatation and sigmoid deformity of the esophagus with hypertrophy of the LES. Clinical manifestations may include dysphagia, regurgitation, chest pain, and weight loss. Most patients report solid and liquid food dysphagia. Regurgitation occurs when food, fluid, and secretions are retained in the dilated esophagus. Patients with advanced achalasia are at risk for bronchitis, pneumonia, or lung abscess from chronic regurgitation and aspiration. Chest pain is frequent early in the course of achalasia, thought to result from esophageal spasm. Patients describe a squeezing, pressure-like retrosternal pain, sometimes radiating to the neck, arms, jaw, and back. Paradoxically, some patients complain of heartburn that may be a chest pain equivalent. Treatment of achalasia is less effective in relieving chest pain than it is in relieving dysphagia or regurgitation.

1	The differential diagnosis of achalasia includes DES, Chagas’ disease, and pseudoachalasia. Chagas’ disease is endemic in areas of central Brazil, Venezuela, and northern Argentina and spread by the bite of the reduviid (kissing) bug that transmits the protozoan, Trypanosoma cruzi. The chronic phase of the disease develops years after infection and results from destruction of autonomic ganglion cells throughout the body, including the heart, gut, urinary tract, and respiratory tract. Tumor infiltration, most commonly seen with carcinoma in the gastric fundus or distal esophagus, can mimic idiopathic achalasia. The resultant “pseudoachalasia” accounts for up to 5% of suspected cases and is more likely with advanced age, abrupt onset of symptoms (<1 year), and weight loss. Hence, endoscopy is a necessary part of the evaluation of achalasia. When the clinical suspicion for pseudoachalasia is high and endoscopy nondiagnostic, computed tomography (CT) scanning or EUS may be of value.

1	is a necessary part of the evaluation of achalasia. When the clinical suspicion for pseudoachalasia is high and endoscopy nondiagnostic, computed tomography (CT) scanning or EUS may be of value. Rarely, pseudoachalasia can result from a paraneoplastic syndrome with circulating antineuronal antibodies.

1	Achalasia is diagnosed by barium swallow x-ray and/or esophageal manometry; endoscopy has a relatively minor role other than to exclude pseudoachalasia. The barium swallow x-ray appearance is of a dilated esophagus with poor emptying, an air-fluid level, and tapering at the LES giving it a beak-like appearance (Fig. 347-5). Occasionally, an epiphrenic diverticulum is observed. In long-standing achalasia, the esophagus may assume a sigmoid configuration. The diagnostic criteria for achalasia with esophageal manometry are impaired LES relaxation and absent peristalsis. High-resolution manometry has somewhat advanced this diagnosis; three subtypes of achalasia are differentiated based on the pattern of pressurization in the nonperistaltic esophagus (Fig. 347-6). Because manometry identifies early disease before esophageal dilatation and food retention, it is the most sensitive diagnostic test.

1	There is no known way of preventing or reversing achalasia. Therapy is directed at reducing LES pressure so that gravity and esophageal pressurization promote esophageal emptying. Peristalsis rarely, if ever, recovers. However, in many instances, remnants of peristalsis masked by esophageal pressurization and dilatation prior to therapy are demonstrable following effective treatment. LES pressure can be reduced by pharmacologic therapy, pneumatic balloon dilatation, or surgical myotomy. No large, controlled trials of the therapeutic alternatives exist, and the optimal approach is debated. Pharmacologic therapies are relatively ineffective but are often used as temporizing therapies. Nitrates or calcium channel blockers are administered before eating, advising caution because of their effects on blood pressure. Botulinum toxin, injected into the LES under endoscopic guidance, inhibits acetylcholine release from nerve endings and improves dysphagia in about 66% of cases for at least 6

1	on blood pressure. Botulinum toxin, injected into the LES under endoscopic guidance, inhibits acetylcholine release from nerve endings and improves dysphagia in about 66% of cases for at least 6 months. Sildenafil and alternative phosphodiesterase inhibitors effectively decrease LES pressure, but practicalities limit their clinical use in achalasia.

1	FIGURE 347-5 Achalasia with esophageal dilatation, tapering at the gastroesophageal junction, and an air-fluid level within the esophagus. The example on the left shows sigmoid deformity with very advanced disease.

1	The only durable therapies for achalasia are pneumatic dilatation and Heller myotomy. Pneumatic dilatation, with a reported efficacy ranging from 32–98%, is an endoscopic technique using a noncompliant, cylindrical balloon dilator positioned across the LES and inflated to a diameter of 3–4 cm. The major complication is perforation with a reported incidence of 0.5–5%. The most common surgical procedure for achalasia is laparoscopic Heller myotomy, usually performed in conjunction with an antireflux procedure (partial fundoplication); good to excellent results are reported in 62–100% of cases. A European randomized controlled trial demonstrated an equivalent response rate of approximately 90% for both pneumatic dilation and laparoscopic Heller myotomy at 2-year follow-up. Occasionally, patients with advanced disease fail to respond to pneumatic dilatation or Heller myotomy. In such refractory cases, esophageal resection with gastric pull-up or interposition of a segment of transverse

1	with advanced disease fail to respond to pneumatic dilatation or Heller myotomy. In such refractory cases, esophageal resection with gastric pull-up or interposition of a segment of transverse colon may be the only option other than gastrostomy feeding.

1	An endoscopic approach to LES myotomy has been introduced, referred to as per oral esophageal myotomy. This technique involves the creation of a tunnel within the esophageal wall through which the circular muscle of the LES and distal esophagus are transected with electrocautery. Short-term studies of efficacy have been favorable. Potential advantages over the conventional laparoscopic approach include avoidance of surgical disruption of the diaphragmatic hiatus and more rapid recovery. In untreated or inadequately treated achalasia, esophageal dilatation predisposes to stasis esophagitis. Prolonged stasis esophagitis is the likely explanation for the association between achalasia and esophageal squamous cell cancer. Tumors develop after years of achalasia, usually in the setting of a greatly dilated esophagus with the overall squamous cell cancer risk increased 17-fold compared to controls.

1	DES is manifested by episodes of dysphagia and chest pain attributable to abnormal esophageal contractions with normal deglutitive LES relaxation. Beyond that, there is little consensus. The pathophysiology and natural history of DES are ill defined. Radiographically, DES has been characterized by tertiary contractions or a “corkscrew esophagus” (Fig. 347-7), but in many instances, these abnormalities are actually indicative of achalasia. Manometrically, a variety of defining features have been proposed including uncoordinated (“spastic”) activity in the distal esophagus, spontaneous and repetitive contractions, or high-amplitude and prolonged contractions. The current consensus, derived from high-resolution manometry studies, is to define spasm by the occurrence of contractions in the distal esophagus with short latency relative to the time of the pharyngeal contraction, a dysfunction indicative of impairment of inhibitory myenteric plexus neurons. A. Classic achalasia 1905

1	A. Classic achalasia 1905 B. Achalasia with compression C. Spastic achalasia FIGURE 347-6 Three subtypes of achalasia: classic (A), with esophageal compression (B), and spastic achalasia (C) imaged with pressure topography. All are characterized by impaired lower esophageal sphincter (LES) relaxation and absent peristalsis. However, classic achalasia has minimal pressurization of the esophageal body, whereas substantial fluid pressurization is observed in achalasia with esophageal compression, and spastic esophageal contractions are observed with spastic achalasia. When defined in this restrictive fashion (Fig. 347-8), DES is actually much less common than achalasia.

1	When defined in this restrictive fashion (Fig. 347-8), DES is actually much less common than achalasia. Esophageal chest pain closely mimics angina pectoris. Features suggesting esophageal pain include pain that is nonexertional, prolonged, interrupts sleep, meal-related, relieved with antacids, and accompanied by heartburn, dysphagia, or regurgitation. However, all of these features exhibit overlap with cardiac pain, which still must be the primary consideration. Furthermore, even within the spectrum of esophageal diseases, both chest pain and dysphagia are also characteristic of peptic or infectious esophagitis. Only after these more common entities have been excluded by evaluation and/or treatment should a diagnosis of DES be pursued.

1	Diseases of the Esophagus 1906 peristalsis, hypertensive LES) that are insufficient to diagnose either achalasia or DES. These findings are of unclear significance. Reflux and psychiatric diagnoses, particularly anxiety and depression, are common among such individuals. A lower visceral pain threshold and symptoms of irritable bowel syndrome are noted in more than half of such patients. Consequently, therapy for these individuals should either target the most common esophageal disorder, GERD, or more global conditions such as depression or somatization neurosis that are found to be coexistent.

1	The current conception of GERD is to encompass a family of conditions with the commonality that they are caused by gastroesophageal reflux resulting in either troublesome symptoms or an array of potential esophageal and extraesophageal manifestations. It is estimated that 15% of adults in the United States are affected by GERD, although such estimates are based only on population studies of self-reported chronic heartburn. With respect to the esophagus, the spectrum of injury includes esophagitis, stricture, Barrett’s esophagus, and adenocarcinoma (Fig. 347-9). Of particular concern is the rising incidence of esophageal adenocarcinoma, an epidemiologic trend that parallels the increasing incidence of GERD. There were about 8000 incident cases of esophageal adenocarcinoma in the United States in 2013 (half of all esophageal cancers); it is estimated that this disease burden has increased twoto sixfold in the last 20 years.

1	Although the defining criteria are in flux, DES is diagnosed by manom- etry. Endoscopy is useful to identify alternative structural and inflamma-The best-defined subset of GERD patients, albeit a minority overall,tory lesions that may cause chest pain. Radiographically, a “corkscrew have esophagitis. Esophagitis occurs when refluxed gastric acid andesophagus,” “rosary bead esophagus,” pseudodiverticula, or curling can pepsin cause necrosis of the esophageal mucosa causing erosions andbe indicative of DES, but these are also found with spastic achalasia.

1	ulcers. Note that some degree of gastroesophageal reflux is normal,Given these vagaries of defining DES, and the resultant heterogeneity of physiologically intertwined with the mechanism of belching (tranpatients identified for inclusion in therapeutic trials, it is not surprising sient LES relaxation), but esophagitis results from excessive reflux,that trial results have been disappointing. Only small, uncontrolled trials often accompanied by impaired clearance of the refluxed gastric juice.

1	exist, reporting response to nitrates, calcium channel blockers, hydrala-Restricting reflux to that which is physiologically intended depends on zine, botulinum toxin, and anxiolytics. The only controlled trial showing the anatomic and physiologic integrity of the esophagogastric junction,efficacy was with an anxiolytic. Surgical therapy (long myotomy or even a complex sphincter comprised of both the LES and the surroundingesophagectomy) should be considered only with severe weight loss or crural diaphragm. Three dominant mechanisms of esophagogastricunbearable pain. These indications are extremely rare.

1	junction incompetence are recognized: (1) transient LES relaxations NONSPECIFIC MANOMETRIC FINDINGS (a vagovagal reflex in which LES relaxation is elicited by gastric dis-Manometric studies done to evaluate chest pain and/or dysphagia tention), (2) LES hypotension, or (3) anatomic distortion of the often report minor abnormalities (e.g., hypertensive or hypotensive esophagogastric junction inclusive of hiatus hernia. Of note, the third factor, esophagogastric junction anatomic disruption, is both significant unto itself mmHg and also because it interacts with the first two mechanisms. Transient LES relaxations

1	Jackhammer esophagus Diffuse esophageal spasm account for about 90% of reflux in normal subjects or GERD patients without hiatus hernia, but patients with hiatus hernia have a more heterogeneous mechanistic profile. Factors tending to exacerbate reflux regardless of mechanism are abdominal obesity, pregnancy, gastric hypersecretory states, delayed gastric emptying, disruption of esophageal peristalsis, and gluttony. After acid reflux, peristalsis returns the refluxed fluid to the stomach and acid clearance is completed by titration of the residual acid by bicarbonate contained in swallowed saliva. Consequently, two causes of prolonged acid clearance are impaired

1	Normal latency with hypercontractility Short latency, premature contraction peristalsis and reduced salivation. Impaired FIGURE 347-8 Esophageal pressure topography of the two major variants of esophageal peristaltic emptying can be attributable spasm: jackhammer esophagus (left) and diffuse esophageal spasm (right). Jackhammer to disrupted peristalsis or superimposed esophagus is defined by the extraordinarily vigorous and repetitive contractions with normal reflux associated with a hiatal hernia. With peristaltic onset and normal latency of the contraction. Diffuse esophageal spasm is similar but superimposed reflux, fluid retained within primarily defined by a short latency (premature) contraction. a sliding hiatal hernia refluxes back into the

1	FIGURE 347-7 Diffuse esophageal spasm. The characteristic “cork-screw” esophagus results from spastic contraction of the circular muscle in the esophageal wall; more precisely, this is actually a helical array of muscle. These findings are also seen with spastic achalasia. Latency= 3.5 s a peptic stricture or adenocarcinoma, each of which 1907 benefits from early detection and/or specific therapy.

1	Latency= 3.5 s a peptic stricture or adenocarcinoma, each of which 1907 benefits from early detection and/or specific therapy. Extraesophageal syndromes with an established association to GERD include chronic cough, laryngitis, asthma, and dental erosions. A multitude of other conditions including pharyngitis, chronic bronchitis, pulmonary fibrosis, chronic sinusitis, cardiac arrhythmias, sleep apnea, and recurrent aspiration pneumonia have proposed associations with GERD. However, in both cases, it is important to emphasize the word association as opposed to causation. In many instances, the disorders likely coexist because of shared pathogenetic mechanisms rather than strict causality. Potential mechanisms for extraesophageal Erosive esophagitis Esophageal stricture with chronic

1	Erosive esophagitis Esophageal stricture with chronic GERD manifestations are either regurgitation with direct contact between the refluxate and supraesophageal structures or via a vagovagal reflex wherein reflux activation of esophageal afferent nerves triggers efferent vagal reflexes such as bronchospasm, cough, or arrhythmias.

1	Although generally quite characteristic, symptoms from GERD need to be distinguished from symptoms related to infectious, pill, or eosinophilic esophagitis, peptic ulcer disease, dyspepsia, biliary colic, coronary artery disease, and esophageal motility disorders. It is especially important that coronary artery disease be given early consideration because of its potentially lethal implications. The remaining elements of the CD differential diagnosis can be addressed by endoscopy, upper gastrointestinal series, or biliary tract ultraso-FIGURE 347-9 Endoscopic appearance of (A) peptic esophagitis, (B) a peptic stricture, nography as appropriate. The distinction among eti (C) Barrett’s metaplasia, and (D) adenocarcinoma developing within an area of Barrett’s ologies of esophagitis is usually easily made by endos- Diseases of the Esophagus esophagus. esophagus during swallow-related LES relaxation, a phenomenon that does not normally occur.

1	Inherent in the pathophysiologic model of GERD is that gastric juice is harmful to the esophageal epithelium. However, gastric acid hypersecretion is usually not a dominant factor in the development of esophagitis. An obvious exception is with Zollinger-Ellison syndrome, which is associated with severe esophagitis in about 50% of patients. Another caveat is with chronic Helicobacter pylori gastritis, which may have a protective effect by inducing atrophic gastritis with concomitant hypoacidity. Pepsin, bile, and pancreatic enzymes within gastric secretions can also injure the esophageal epithelium, but their noxious properties are either lessened without an acidic environment or dependent on acidity for activation. Bile warrants attention because it persists in refluxate despite acid-suppressing medications. Bile can transverse the cell membrane, imparting severe cellular injury in a weakly acidic environment, and has also been invoked as a cofactor in the pathogenesis of Barrett’s

1	medications. Bile can transverse the cell membrane, imparting severe cellular injury in a weakly acidic environment, and has also been invoked as a cofactor in the pathogenesis of Barrett’s metaplasia and adenocarcinoma. Hence, the causticity of gastric refluxate extends beyond hydrochloric acid.

1	Heartburn and regurgitation are the typical symptoms of GERD. Somewhat less common are dysphagia and chest pain. In each case, multiple potential mechanisms for symptom genesis operate that extend beyond the basic concepts of mucosal erosion and activation of afferent sensory nerves. Specifically, hypersensitivity and functional pain are increasingly recognized as cofactors. Nonetheless, the dominant clinical strategy is empirical treatment with acid inhibitors, reserving further evaluation for those who fail to respond. Important exceptions to this are patients with chest pain or persistent dysphagia, each of which may be indicative of more morbid conditions. With chest pain, cardiac disease must be carefully considered. In the case of persistent dysphagia, chronic reflux can lead to the development of copy with mucosal biopsies, which are necessary tion. In terms of endoscopic appearance, infectious esophagitis is diffuse and tends to involve the proximal esophagus far more

1	to the development of copy with mucosal biopsies, which are necessary tion. In terms of endoscopic appearance, infectious esophagitis is diffuse and tends to involve the proximal esophagus far more frequently than does reflux esophagitis. The ulcerations seen in peptic esophagitis are usually solitary and distal, whereas infectious ulcerations are punctate and diffuse. Eosinophilic esophagitis characteristically exhibits multiple esophageal rings, linear furrows, or white punctate exudate. Esophageal ulcerations from pill esophagitis are usually singular and deep at points of luminal narrowing, especially near the carina, with sparing of the distal esophagus.

1	The complications of GERD are related to chronic esophagitis (bleeding and stricture) and the relationship between GERD and esophageal adenocarcinoma. However, both esophagitis and peptic strictures have become increasingly rare in the era of potent antisecretory medications. Conversely, the most severe histologic consequence of GERD is Barrett’s metaplasia with the associated risk of esophageal adenocarcinoma, and the incidence of these lesions has increased, not decreased, in the era of potent acid suppression. Barrett’s metaplasia, endoscopically recognized by tongues of reddish mucosa extending proximally from the gastroesophageal junction (Fig. 347-9) or histopathologically by the finding of specialized columnar metaplasia, is associated with a substantially increased risk for development of esophageal adenocarcinoma.

1	Barrett’s metaplasia can progress to adenocarcinoma through the intermediate stages of lowand high-grade dysplasia (Fig. 347-10). Owing to this risk, areas of Barrett’s and especially any included areas of mucosal irregularity should be extensively biopsied. The rate of cancer development is estimated at 0.1–0.3% per year, but vagaries in definitional criteria and of the extent of Barrett’s metaplasia requisite to establish the diagnosis have contributed to variability and inconsistency in this risk assessment. The group at greatest risk is obese white males in their sixth decade of life. However, despite common practice, the utility of endoscopic screening and surveillance programs intended FIGURE 347-10 Histopathology of Barrett’s metaplasia and Barrett’s with high-grade dysplasia. H&E, hematoxylin and eosin.

1	FIGURE 347-10 Histopathology of Barrett’s metaplasia and Barrett’s with high-grade dysplasia. H&E, hematoxylin and eosin. to control the adenocarcinoma risk has not been established. Also of note, no high-level evidence confirms that aggressive antisecretory therapy or antireflux surgery causes regression of Barrett’s esophagus or prevents adenocarcinoma.

1	Although the management of Barrett’s esophagus remains controversial, the finding of dysplasia in Barrett’s, particularly high-grade dysplasia, mandates further intervention. In addition to the high rate of progression to adenocarcinoma, there is also a high prevalence of unrecognized coexisting cancer with high-grade dysplasia. Nonetheless, treatment remains controversial. Esophagectomy, intensive endoscopic surveillance, and mucosal ablation have all been advocated. Currently, esophagectomy is the gold standard treatment for high-grade dysplasia in an otherwise healthy patient with minimal surgical risk. However, esophagectomy has a mortality ranging from 3–10%, along with substantial morbidity. That, along with increasing evidence of the effectiveness of endoscopic therapy with purpose-built radiofrequency ablation devices, has led many to favor this therapy as a preferable management strategy.

1	Lifestyle modifications are routinely advocated as GERD therapy. Broadly speaking, these fall into three categories: (1) avoidance of foods that reduce LES pressure, making them “refluxogenic” (these commonly include fatty foods, alcohol, spearmint, peppermint, tomato-based foods, and possibly coffee and tea); (2) avoidance of acidic foods that are inherently irritating; and (3) adoption of behaviors to minimize reflux and/or heartburn. In general, minimal evidence supports the efficacy of these measures. However, clinical experience dictates that subsets of patients are benefitted by specific recommendations, based on their unique history and symptom profile. A patient with sleep disturbance from nighttime heartburn is likely to benefit from elevation of the head of the bed and avoidance of eating before retiring, but those recommendations are superfluous for a patient without nighttime symptoms. The most broadly applicable recommendation is for weight reduction. Even though the

1	of eating before retiring, but those recommendations are superfluous for a patient without nighttime symptoms. The most broadly applicable recommendation is for weight reduction. Even though the benefit with respect to reflux cannot be assured, the strong epidemiologic relationship between body mass index and GERD and the secondary health gains of weight reduction are beyond dispute.

1	The dominant pharmacologic approach to GERD management is with inhibitors of gastric acid secretion, and abundant data support the effectiveness of this approach. Pharmacologically reducing the acidity of gastric juice does not prevent reflux, but it ameliorates reflux symptoms and allows esophagitis to heal. The hierarchy of effectiveness among pharmaceuticals parallels their antisecretory potency. Proton pump inhibitors (PPIs) are more efficacious than histamine2 receptor antagonists (H2RAs), and both are superior to placebo. No major differences exist among PPIs, and only modest gain is achieved by increased dosage.

1	Paradoxically, the perceived frequency and severity of heartburn correlate poorly with the presence or severity of esophagitis. When GERD treatments are assessed in terms of resolving heartburn, both efficacy and differences among pharmaceuticals are less clear-cut than with the objective of healing esophagitis. Although the same overall hierarchy of effectiveness exists, observed efficacy rates are lower and vary widely, likely reflecting patient heterogeneity.

1	Reflux symptoms tend to be chronic, irrespective of esophagitis. Thus, a common management strategy is indefinite treatment with PPIs or H2RAs as necessary for symptom control. The side effects of PPI therapy are generally minimal. Vitamin B12 and iron absorption may be compromised and susceptibility to enteric infections, particularly Clostridium difficile colitis, increased with treatment. Population studies have also suggested a slight increased risk of bone fracture with chronic PPI use suggesting an impairment of calcium absorption, but prospective studies have failed to corroborate this. Nonetheless, as with any medication, PPI dosage should be minimized to that necessary for the clinical indication.

1	Laparoscopic Nissen fundoplication, wherein the proximal stomach is wrapped around the distal esophagus to create an antireflux barrier, is a surgical alternative to the management of chronic GERD. Just as with PPI therapy, evidence on the utility of fundoplication is strongest for treating esophagitis, and controlled trials suggest similar efficacy to PPI therapy. However, the benefits of fundoplication must be weighed against potential deleterious effects, including surgical morbidity and mortality, postoperative dysphagia, failure or breakdown requiring reoperation, an inability to belch, and increased bloating, flatulence, and bowel symptoms after surgery.

1	Eosinophilic esophagitis (EoE) is increasingly recognized in adults and children around the world. Current prevalence estimates identified 4–6 cases per 10,000 with a predilection for white males. The increasing prevalence of EoE is attributable to a combination of an increasing incidence and a growing recognition of the condition. There is also an incompletely understood, but important, overlap between EoE and GERD that confuses diagnosis of the disease.

1	EoE is diagnosed based on the combination of typical esophageal symptoms and esophageal mucosal biopsies demonstrating squamous epithelial eosinophil-predominant inflammation. Alternative etiologies of esophageal eosinophilia include GERD, drug hypersensitivity, connective tissue disorders, hypereosinophilic syndrome, and infection. Current evidence indicates that EoE is an immunologic disorder induced by antigen sensitization in susceptible individuals. Dietary factors play an important role in both the pathogenesis and treatment of EoE. Aeroallergens may also contribute, but the evidence is weaker. The natural history of EoE is unclear, but an increased risk of esophageal stricture development paralleling the duration of untreated disease has been noted. Diseases of the Esophagus FIGURE 347-11 Endoscopic features of (A) eosinophilic esophagitis (EoE), (B) Candida esophagitis, (C) giant ulcer associated with HIV, (D) and a Schatzki ring.

1	EoE should be strongly considered in children and adults with dysphagia and esophageal food impactions. In preadolescent children, symptom presentations of EoE include chest or abdominal pain, nausea, vomiting, and food aversion. Other symptoms in adults may include atypical chest pain and heartburn, particularly heartburn that is refractory to PPI therapy. An atopic history of food allergy, asthma, eczema, or allergic rhinitis is present in the majority of patients. Peripheral blood eosinophilia is demonstrable in up to 50% of patients, but the specificity of this finding is problematic in the setting of concomitant atopy. The characteristic endoscopic esophageal findings are loss of vascular markings (edema), multiple esophageal rings, longitudinally oriented furrows, and punctate exudate (Fig. 347-11). Histologic confirmation is made with the demonstration of esophageal mucosal eosinophilia (greatest density ±15 eosinophils per high-power field) (Fig. 347-12). Complications of EoE

1	(Fig. 347-11). Histologic confirmation is made with the demonstration of esophageal mucosal eosinophilia (greatest density ±15 eosinophils per high-power field) (Fig. 347-12). Complications of EoE include esophageal stricture, narrow-caliber esophagus, food impaction, and esophageal perforation.

1	The goals of EoE management are symptom control and the prevention of complications. Once esophageal eosinophilia is demonstrated, patients typically undergo a trial of PPI therapy as a practical means of excluding a contribution of GERD to the esophageal mucosal inflammation. PPI-responsive esophageal eosinophilia, characterized by elimination of mucosal eosinophilia, occurs in 30–50% of cases of suspected EoE. Patients with persistent symptoms and eosinophilic inflammation following PPI therapy are subsequently considered for EoE treatments such as elimination diets or swallowed topical glucocorticoids. Elemental formula diets are a highly effective therapy that have primarily been studied in children but are limited by palatability. Notably, allergy testing by means of either serum IgE or skin prick testing has demonstrated poor sensitivity and specificity in the identification of foods that incite the esophageal inflammatory response. Allergy testing combining skin prick and atopy

1	or skin prick testing has demonstrated poor sensitivity and specificity in the identification of foods that incite the esophageal inflammatory response. Allergy testing combining skin prick and atopy patch testing has been effective in children with EoE, but additional validation is needed. Empiric elimination of common food allergies (milk, wheat, egg, soy, nuts, and seafood) followed by systematic reintroduction has been an effective diet therapy in both children and adults with EoE. The intent of the elimination diet approach is the identification of a single food trigger or a small number of food triggers. Swallowed, topical glucocorticoids (fluticasone propionate or budesonide) are highly effective, but recurrence of disease is common following the cessation of therapy. Systemic glucocorticoids are reserved for severely afflicted patients refractory to less morbid treatments. Esophageal dilation is very effective at relieving dysphagia in patients with fibrostenosis. Dilation

1	are reserved for severely afflicted patients refractory to less morbid treatments. Esophageal dilation is very effective at relieving dysphagia in patients with fibrostenosis. Dilation should be approached conservatively because of the risk of deep, esophageal mural laceration or perforation in the stiff-walled esophagus that is characteristic of the disease.

1	FIGURE 347-12 Histopathology of eosinophilic esophagitis (EoE) showing infiltration of the esophageal squamous epithelium with eosinophils. Additional features of basal cell hyperplasia and lamina propria fibrosis are present. Eosinophilic inflammation can also be seen with gastroesophageal reflux disease.

1	With the increased use of immunosuppression for organ transplantation as well as chronic inflammatory diseases and chemotherapy along with the AIDS epidemic, infections with Candida species, herpesvirus, and cytomegalovirus (CMV) have become relatively common. Although rare, infectious esophagitis also occurs among the nonimmunocompromised, with herpes simplex and Candida albicans being the most common pathogens. Among AIDS patients, infectious esophagitis becomes more common as the CD4 count declines; cases are rare with a CD4 count >200 and common when <100. HIV itself may also be associated with a self-limited syndrome of acute esophageal ulceration with oral ulcers and a maculopapular skin rash at the time of seroconversion. Additionally, some patients with advanced disease have deep, persistent esophageal ulcers treated with oral glucocorticoids or thalidomide. However, with the widespread use of protease inhibitors, a reduction in these HIV complications has been noted.

1	Regardless of the infectious agent, odynophagia is a characteristic symptom of infectious esophagitis; dysphagia, chest pain, and hemorrhage are also common. Odynophagia is uncommon with reflux esophagitis, so its presence should always raise suspicion of an alternative etiology.

1	1910 CANDIDA ESOPHAGITIS Candida is normally found in the throat, but can become pathogenic and produce esophagitis in a compromised host; C. albicans is most common. Candida esophagitis also occurs with esophageal stasis secondary to esophageal motor disorders and diverticula. Patients complain of odynophagia and dysphagia. If oral thrush is present, empirical therapy is appropriate, but co-infection is common, and persistent symptoms should lead to prompt endoscopy with biopsy, which is the most useful diagnostic evaluation. Candida esophagitis has a characteristic appearance of white plaques with friability. Rarely, Candida esophagitis is complicated by bleeding, perforation, stricture, or systemic invasion. Oral fluconazole (200–400 mg on the first day, followed by 100–200 mg daily) for 14–21 days is the preferred treatment. Patients refractory to fluconazole may respond to itraconazole, voriconazole, or posaconazole. Alternatively, poorly responsive patients or those who cannot

1	for 14–21 days is the preferred treatment. Patients refractory to fluconazole may respond to itraconazole, voriconazole, or posaconazole. Alternatively, poorly responsive patients or those who cannot swallow medications can be treated with an intravenous echinocandin (caspofungin 50 mg daily for 7–21 days).

1	Herpes simplex virus type 1 or 2 may cause esophagitis. Vesicles on the nose and lips may coexist and are suggestive of a herpetic etiology. Varicella-zoster virus can also cause esophagitis in children with chickenpox or adults with zoster. The characteristic endoscopic findings are vesicles and small, punched-out ulcerations. Because herpes simplex infections are limited to squamous epithelium, biopsies from the ulcer margins are most likely to reveal the characteristic ground-glass nuclei, eosinophilic Cowdry’s type A inclusion bodies, and giant cells. Culture or polymerase chain reaction (PCR) assays are helpful to identify acyclovir-resistant strains. Acyclovir (200 mg orally five times a day for 7–10 days) can be used for immunocompetent hosts, although the disease is typically self-limited after a 1to 2-week period in such patients. Immunocompromised patients are treated with acyclovir (400 mg orally five times a day for 14–21 days), famciclovir (500 mg orally three times a

1	after a 1to 2-week period in such patients. Immunocompromised patients are treated with acyclovir (400 mg orally five times a day for 14–21 days), famciclovir (500 mg orally three times a day), or valacyclovir (1 g orally three times a day). In patients with severe odynophagia, intravenous acyclovir, 5 mg/kg every 8 h for 7–14 days, reduces this morbidity.

1	CMV esophagitis occurs primarily in immunocompromised patients, particularly organ transplant recipients. CMV is usually activated from a latent stage. Endoscopically, CMV lesions appear as serpiginous ulcers in an otherwise normal mucosa, particularly in the distal esophagus. Biopsies from the ulcer bases have the greatest diagnostic yield for finding the pathognomonic large nuclear or cytoplasmic inclusion bodies. Immunohistology with monoclonal antibodies to CMV and in situ hybridization tests are useful for early diagnosis. Data on therapy for CMV esophagitis are limited. Treatment studies of CMV gastrointestinal disease have demonstrated effectiveness of both ganciclovir (5 mg/kg every 12 h intravenously) and foscarnet (90 mg/kg every 12 h intravenously). Valganciclovir (900 mg two times a day), an oral formulation of ganciclovir, can also be used. Therapy is continued until healing, which may take 3–6 weeks. Maintenance therapy may be needed for patients with relapsing disease.

1	Most cases of esophageal perforation are from instrumentation of the esophagus or trauma. Alternatively, forceful vomiting or retching can lead to spontaneous rupture at the gastroesophageal junction (Boerhaave’s syndrome). More rarely, corrosive esophagitis or neoplasms lead to perforation. Instrument perforation from endoscopy or nasogastric tube placement typically occurs in the hypopharynx or at the gastroesophageal junction. Perforation may also occur at the site of a stricture in the setting of endoscopic food disimpaction or esophageal dilation. Esophageal perforation causes pleuritic retrosternal pain that can be associated with pneumomediastinum and subcutaneous emphysema. Mediastinitis is a major complication of esophageal perforation, and prompt recognition is key to optimizing outcome. CT of the chest is most sensitive in detecting mediastinal air. Esophageal perforation is confirmed by a contrast swallow, usually Gastrografin followed by thin barium. Treatment includes

1	outcome. CT of the chest is most sensitive in detecting mediastinal air. Esophageal perforation is confirmed by a contrast swallow, usually Gastrografin followed by thin barium. Treatment includes nasogastric suction and parenteral broad-spectrum antibiotics with prompt surgical drainage and repair in noncontained leaks. Conservative therapy with NPO status and antibiotics without surgery may be appropriate in cases of contained perforation that are detected early. Endoscopic clipping or stent placement may be indicated in nonoperated iatrogenic perforations or nonoperable cases such as perforated tumors.

1	Vomiting, retching, or vigorous coughing can cause a nontransmural tear at the gastroesophageal junction that is a common cause of upper gastrointestinal bleeding. Most patients present with hematemesis. Antecedent vomiting is anticipated but not always evident. Bleeding usually abates spontaneously, but protracted bleeding may respond to local epinephrine or cauterization therapy, endoscopic clipping, or angiographic embolization. Surgery is rarely needed.

1	Radiation esophagitis can complicate treatment for thoracic cancers, especially breast and lung, with the risk proportional to radiation dosage. Radiosensitizing drugs such as doxorubicin, bleomycin, cyclophosphamide, and cisplatin also increase the risk. Dysphagia and odynophagia may last weeks to months after therapy. The esophageal mucosa becomes erythematous, edematous, and friable. Submucosal fibrosis and degenerative tissue changes and stricturing may occur years after the radiation exposure. Radiation exposure in excess of 5000 cGy has been associated with increased risk of esophageal stricture. Treatment for acute radiation esophagitis is supportive. Chronic strictures are managed with esophageal dilation.

1	Caustic esophageal injury from ingestion of alkali or, less commonly, acid can be accidental or from attempted suicide. Absence of oral injury does not exclude possible esophageal involvement. Thus, early endoscopic evaluation is recommended to assess and grade the injury to the esophageal mucosa. Severe corrosive injury may lead to esophageal perforation, bleeding, stricture, and death. Glucocorticoids have not been shown to improve the clinical outcome of acute corrosive esophagitis and are not recommended. Healing of more severe grades of caustic injury is commonly associated with severe stricture formation and often requires repeated dilatation.

1	Pill-induced esophagitis occurs when a swallowed pill fails to traverses the entire esophagus and lodges within the lumen. Generally, this is attributed to poor “pill taking habits”: inadequate liquid with the pill or lying down immediately after taking a pill. The most common location for the pill to lodge is in the mid-esophagus near the crossing of the aorta or carina. Extrinsic compression from these structures halts the movement of the pill or capsule. Since initially reported in 1970, more than 1000 cases of pill esophagitis have been reported, suggesting that this is not an unusual occurrence. A wide variety of medications are implicated with the most common being doxycycline, tetracycline, quinidine, phenytoin, potassium chloride, ferrous sulfate, nonsteroidal anti-inflammatory drugs (NSAIDs), and bisphosphonates. However, virtually any pill can result in pill esophagitis if taken carelessly.

1	Typical symptoms of pill esophagitis are the sudden onset of chest pain and odynophagia. Characteristically, the pain will develop over a period of hours or will awaken the individual from sleep. A classic history in the setting of ingestion of recognized pill offenders obviates the need for diagnostic testing in most patients. When endoscopy is performed, localized ulceration or inflammation is evident. Histologically, acute inflammation is typical. Chest CT imaging will sometimes reveal esophageal thickening consistent with transmural inflammation. Although the condition usually resolves within days to weeks, symptoms may persist for months and stricture can develop in severe cases. No specific therapy is known to hasten the healing process, but antisecretory medications are frequently prescribed to remove concomitant reflux as an aggravating factor. When healing results in stricture formation, dilation is indicated.

1	Food or foreign bodies may lodge in the esophagus causing complete obstruction, which in turn can cause an inability to handle secretions (foaming at the mouth) and severe chest pain. Food impaction may occur due to stricture, carcinoma, Schatzki ring, eosinophilic esophagitis, or simply inattentive eating. If it does not spontaneously resolve, impacted food can be dislodged endoscopically. Use of meat tenderizer enzymes to facilitate passage of a meat bolus is discouraged because of potential esophageal injury. Glucagon (1 mg IV) is sometimes tried before endoscopic dislodgement. After emergent treatment, patients should be evaluated for potential causes of the impaction with treatment rendered as indicated.

1	Scleroderma esophagus (hypotensive LES and absent esophageal peristalsis) was initially described as a manifestation of scleroderma or other collagen vascular diseases and thought to be specific for these disorders. However, this nomenclature subsequently proved unfortunate and has been discarded because an estimated half of qualifying patients do not have an identifiable systemic disease, and reflux disease is often the only identifiable association. When scleroderma esophagus occurs as a manifestation of a collagen vascular disease, the histopathologic findings are of infiltration and destruction of the esophageal muscularis propria with collagen deposition and fibrosis. The pathogenesis of absent peristalsis and LES hypo-tension in the absence of a collagen vascular disease is unknown. Regardless of the underlying cause, the manometric abnormalities predispose patients to severe GERD due to inadequate LES barrier function combined with poor esophageal clearance of refluxed acid.

1	Regardless of the underlying cause, the manometric abnormalities predispose patients to severe GERD due to inadequate LES barrier function combined with poor esophageal clearance of refluxed acid. Dysphagia may also be manifest but is generally mild and alleviated by eating in an upright position and using liquids to facilitate solid emptying.

1	A host of dermatologic disorders (pemphigus vulgaris, bullous pemphigoid, cicatricial pemphigoid, Behçet’s syndrome, and epidermolysis bullosa) can affect the oropharynx and esophagus, particularly the proximal esophagus with blisters, bullae, webs, and strictures. Glucocorticoid treatment is usually effective. Erosive lichen planus, Stevens-Johnson syndrome, and graft-versus-host disease can also involve the esophagus. Esophageal dilatation may be necessary to treat strictures.

1	meals is a symptom complex associated with peptic ulcer disease (PUD). An ulcer is defined as disruption of the mucosal integrity of the stomach and/or duodenum leading to a local defect or excavation due to active inflammation. Ulcers occur within the stomach and/ or duodenum and are often chronic in nature. Acid peptic disorders are very common in the United States, with 4 million individuals (new cases and recurrences) affected per year. Lifetime prevalence of PUD in the United States is ~12% in men and 10% in women. PUD significantly affects quality of life by impairing overall patient well-1911 being and contributing substantially to work absenteeism. Moreover, an estimated 15,000 deaths per year occur as a consequence of complicated PUD. The financial impact of these common disorders has been substantial, with an estimated burden on direct and indirect health care costs of ~$6 billion per year in the United States, with $3 billion spent on hospitalizations, $2 billion on

1	has been substantial, with an estimated burden on direct and indirect health care costs of ~$6 billion per year in the United States, with $3 billion spent on hospitalizations, $2 billion on physician office visits, and $1 billion in decreased productivity and days lost from work.

1	Despite the constant attack on the gastroduodenal mucosa by a host of noxious agents (acid, pepsin, bile acids, pancreatic enzymes, drugs, and bacteria), integrity is maintained by an intricate system that provides mucosal defense and repair. Gastric Anatomy The gastric epithelial lining consists of rugae that contain microscopic gastric pits, each branching into four or five gastric glands made up of highly specialized epithelial cells. The makeup of gastric glands varies with their anatomic location. Glands within the gastric cardia comprise <5% of the gastric gland area and contain mucous and endocrine cells. The 75% of gastric glands are found within the oxyntic mucosa and contain mucous neck, parietal, chief, endocrine, enterochromaffin, and enterochromaffin-like (ECL) cells (Fig. 348-1). Pyloric glands contain mucous and endocrine cells (including gastrin cells) and are found in the antrum.

1	The parietal cell, also known as the oxyntic cell, is usually found in the neck, or isthmus, or in the oxyntic gland. The resting, or unstimulated, parietal cell has prominent cytoplasmic tubulovesicles and intracellular canaliculi containing short microvilli along its apical surface (Fig. 348-2). H+,K+-adenosine triphosphatase (ATPase) is expressed in the tubulovesicle membrane; upon cell stimulation, this membrane, along with apical membranes, transforms into a dense network of apical intracellular canaliculi containing long microvilli. Acid secretion, a FIGURE 348-1 Diagrammatic representation of the oxyntic gastric gland. (Adapted from S Ito, RJ Winchester: J Cell Biol 16:541, 1963. doi:10.1083/jcb.16.3.541. © 1963 Ito and Winchester.) HCl H+,K+–ATPase KCl

1	HCl H+,K+–ATPase KCl FIGURE 348-2 Gastric parietal cell undergoing transformation after secretagogue-mediated stimulation. cAMP, cyclic adenosine mono-phosphate. (Adapted from SJ Hersey, G Sachs: Physiol Rev 75:155, 1995.) process requiring high energy, occurs at the apical canalicular surface. Numerous mitochondria (30–40% of total cell volume) generate the energy required for secretion. Gastroduodenal Mucosal Defense The gastric epithelium is under constant assault by a series of endogenous noxious factors, including hydrochloric acid (HCl), pepsinogen/pepsin, and bile salts. In addition, a steady flow of exogenous substances such as medications, alcohol, and bacteria encounter the gastric mucosa. A highly intricate biologic system is in place to provide defense from mucosal injury and to repair any injury that may occur.

1	The mucosal defense system can be envisioned as a three-level barrier, composed of preepithelial, epithelial, and subepithelial elements (Fig. 348-3). The first line of defense is a mucus-bicarbonatephospholipid layer, which serves as a physicochemical barrier to multiple molecules, including hydrogen ions. Mucus is secreted in a regulated fashion by gastroduodenal surface epithelial cells. It consists primarily of water (95%) and a mixture of phospholipids and glycoproteins (mucin). The mucous gel functions as a nonstirred water layer impeding diffusion of ions and molecules such as pepsin. Bicarbonate, secreted in a regulated manner by surface epithelial cells of the gastroduodenal mucosa into the mucous gel, forms a pH gradient ranging from 1 to 2 at the gastric luminal surface and reaching 6 to 7 along the epithelial cell surface.

1	Surface epithelial cells provide the next line of defense through several factors, including mucus production, epithelial cell ionic transporters that maintain intracellular pH and bicarbonate production, and intracellular tight junctions. Surface epithelial cells generate heat shock proteins that prevent protein denaturation and protect cells from certain factors such as increased temperature, cytotoxic agents, or oxidative stress. Epithelial cells also generate trefoil factor family peptides and cathelicidins, which also play a role in surface cell protection and regeneration. If the preepithelial barrier were breached, gastric epithelial cells bordering a site of injury can migrate to restore a damaged region (restitution). This process occurs independent of cell division and requires uninterrupted blood flow and an alkaline pH in the surrounding environment. Several growth factors, including epidermal growth factor (EGF), transforming growth factor (TGF) α, and basic fibroblast

1	uninterrupted blood flow and an alkaline pH in the surrounding environment. Several growth factors, including epidermal growth factor (EGF), transforming growth factor (TGF) α, and basic fibroblast growth factor (FGF), modulate the process of restitution. Larger defects that are not effectively repaired by restitution require cell proliferation. Epithelial cell regeneration is regulated by prostaglandins and growth factors such as EGF and TGF-α. In tandem with epithelial cell renewal, formation of new vessels (angiogenesis) within the injured microvascular bed occurs. Both FGF and vascular endothelial growth factor (VEGF) are important in regulating angiogenesis in the gastric mucosa.

1	An elaborate microvascular system within the gastric submucosal layer is the key component of the subepithelial defense/repair system, providing HCO3−, which neutralizes the acid generated by the parietal cell. Moreover, this microcirculatory bed provides an adequate supply of micronutrients and oxygen while removing toxic metabolic by-products.

1	Prostaglandins play a central role in gastric epithelial defense/ repair (Fig. 348-4). The gastric mucosa contains abundant levels of prostaglandins that regulate the release of mucosal bicarbonate and mucus, inhibit parietal cell secretion, and are important in maintaining mucosal blood flow and epithelial cell restitution. Prostaglandins are derived from esterified arachidonic acid, which is formed from phospholipids (cell membrane) by the action of phospholipase A2. A key enzyme that controls the rate-limiting step in prostaglandin synthesis is cyclooxygenase (COX), which is present in two isoforms (COX-1, COX-2), each having distinct characteristics regarding structure, tissue distribution, and expression. COX-1 is expressed in a host of tissues, including the stomach, platelets, kidneys, and endothelial cells. This isoform is expressed in a constitutive manner and plays an important role in maintaining the integrity of renal function, platelet aggregation, and gastrointestinal

1	and endothelial cells. This isoform is expressed in a constitutive manner and plays an important role in maintaining the integrity of renal function, platelet aggregation, and gastrointestinal (GI) mucosal integrity. In contrast, the expression of COX-2 is inducible by inflammatory stimuli, and it is expressed in macrophages, leukocytes, fibroblasts, and synovial cells. The beneficial effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on tissue inflammation are due to inhibition of COX-2; the toxicity of these drugs (e.g., GI mucosal ulceration and renal dysfunction) is related to inhibition of the COX-1 isoform. The highly COX-2–selective NSAIDs have the potential to provide the beneficial effect of decreasing tissue inflammation while minimizing toxicity in the GI tract. Selective COX-2 inhibitors have had adverse effects on the cardiovascular system, leading to increased risk of myocardial infarction. Therefore, the

1	U.S. Food and Drug Administration (FDA) has removed two of these agents (valdecoxib and rofecoxib) from the market (see below). Nitric oxide (NO) is important in the maintenance of gastric mucosal integrity. The key enzyme NO synthase is constitutively expressed in the mucosa and contributes to cytoprotection by stimulating gastric mucus, increasing mucosal blood flow, and maintaining epithelial cell barrier function. The central nervous system (CNS) and hormonal factors also play a role in regulating mucosal defense through multiple pathways (Fig. 348-3).

1	Physiology of Gastric Secretion Hydrochloric acid and pepsinogen are the two principal gastric secretory products capable of inducing mucosal injury. Gastric acid and pepsinogen play a physiologic role in protein digestion; absorption of iron, calcium, magnesium, and vitamin B12; and killing ingested bacteria. Acid secretion should be viewed as occurring under basal and stimulated conditions. Basal acid production occurs in a circadian pattern, with highest levels occurring during the night and lowest levels during the morning hours. Cholinergic input via the vagus nerve and histaminergic input from local gastric sources are the principal contributors to basal acid secretion. Stimulated gastric acid secretion occurs primarily in three phases based on the site where the signal originates (cephalic, gastric, and intestinal). Sight, smell, and taste of food are the components of the cephalic phase, which stimulates gastric secretion via the vagus nerve. The gastric phase is activated

1	(cephalic, gastric, and intestinal). Sight, smell, and taste of food are the components of the cephalic phase, which stimulates gastric secretion via the vagus nerve. The gastric phase is activated once food enters the stomach. This component of secretion is driven by nutrients (amino acids and amines) that directly stimulate the G cell to release gastrin, which in turn activates the parietal cell via direct and indirect mechanisms. Distention of the stomach wall also leads to gastrin release and acid production. The last phase of gastric acid secretion is initiated as food enters the intestine and is mediated by luminal distention and nutrient assimilation. A series of pathways that inhibit gastric acid production are also set into motion during these phases. The GI hormone somatostatin is released from endocrine cells found in the gastric mucosa (D cells) in response to HCl. Somatostatin can inhibit acid production by both direct (parietal cell) and indirect mechanisms (decreased

1	is released from endocrine cells found in the gastric mucosa (D cells) in response to HCl. Somatostatin can inhibit acid production by both direct (parietal cell) and indirect mechanisms (decreased histamine release from ECL cells and gastrin release from G cells). Additional neural (central and peripheral) and humoral (amylin, atrial natriuretic peptide [ANP], cholecystokinin, ghrelin, interleukin 11 [IL-11], obestatin, secretin, and serotonin) factors play a role in counterbalancing acid secretion. Under physiologic circumstances, these phases occur simultaneously. Ghrelin, the appetite-regulating hormone expressed in Gr cells in the

1	FIGURE 348-3 Components involved in providing gastroduodenal mucosal defense and repair. CCK, cholecystokinin; CRF, corticotropinreleasing factor; EGF, epidermal growth factor; HCl, hydrochloride; IGF, insulin-like growth factor; TGFα, transforming growth factor α; TRF, thyrotropin releasing factor. (Modified and updated from Tarnawski A. Cellular and molecular mechanisms of mucosal defense and repair. In: Yoshikawa T, Arakawa T. Bioregulation and Its Disorders in the Gastrointestinal Tract. Tokyo, Japan: Blackwell Science, 1998:3–17.) stomach, may increase gastric acid secretion through stimulation of histamine release from ECL cells, but this remains to be confirmed.

1	The acid-secreting parietal cell is located in the oxyntic gland, adjacent to other cellular elements (ECL cell, D cell) important in the gastric secretory process (Fig. 348-5). This unique cell also secretes intrinsic factor (IF) and IL-11. The parietal cell expresses receptors for several stimulants of acid secretion, including histamine (H2), gastrin (cholecystokinin B/gastrin receptor), and acetylcholine (muscarinic, M3). Binding of histamine to the H2 receptor leads to activation of adenylate cyclase and an increase in cyclic adenosine monophosphate (AMP). Activation of the gastrin and muscarinic receptors results in activation of the protein kinase C/phosphoinositide signaling pathway. Each of these signaling pathways in turn regulates a series of downstream kinase cascades that control the acid-secreting pump, H+,K+-ATPase. The discovery that different ligands and their corresponding receptors lead to activation of different signaling pathways explains the potentiation of acid

1	the acid-secreting pump, H+,K+-ATPase. The discovery that different ligands and their corresponding receptors lead to activation of different signaling pathways explains the potentiation of acid secretion that occurs when histamine and gastrin or acetylcholine are combined. More importantly, this observation explains why blocking one receptor type (H2) decreases acid secretion stimulated by agents that activate a different pathway (gastrin, acetylcholine). Parietal cells also express receptors for ligands that inhibit acid production (prostaglandins, somatostatin, and EGF). Histamine also stimulates gastric acid secretion indirectly by activating the histamine H3 receptor on D-cells, which inhibits somatostatin release.

1	The enzyme H+,K+-ATPase is responsible for generating the large concentration of H+. It is a membrane-bound protein that consists of two subunits, α and β. The active catalytic site is found within the α subunit; the function of the β subunit is unclear. This enzyme uses the 1914 Membrane phospholipids Arachidonic acid Phospholipase A2 Stomach Kidney Platelets Endothelium TXA2, PGI2, PGE2 Gastrointestinal mucosal integrity Platelet aggregation Renal function PGI2, PGE2 Inflammation Mitogenesis Bone formation Other functions? Macrophages Leukocytes Fibroblasts Endothelium COX-1 housekeeping COX-2 inflammation

1	Gastric ulcers In contrast to DUs, GUs can represent a malignancy and should be biopsied upon discovery. Benign GUs are most often found distal to the junction between the antrum and the acid secretory mucosa. Benign GUs are quite rare in the gastric fundus and are histologically similar to DUs. Benign GUs associated with H. pylori are also associated with antral gastritis. In contrast, NSAID-related GUs are not accompanied by chronic active gastritis but may instead have evidence of a chemical gastropathy, typified by foveolar hyperplasia, edema of the lamina propria, and epithelial regeneration in the absence of H. pylori. Extension of smooth-muscle fibers into the upper portions of the mucosa, where they are not typically found, may also occur.

1	Pathophysiology • DuoDenal ulcers H. pylori and NSAID-induced injury account for the majority of DUs. Many acid secretory abnormalities have been described in DU patients. Of these, average basal and nocturnal gastric acid secretion appears to be increased in DU patients as compared to controls; however, the level of overlap between DU patients and control subjects is substantial. The reason for this altered secretory process is unclear, but H. pylori infection may contribute. Bicarbonate secretion is significantly decreased in the duodenal bulb of patients with an active DU as compared to control subjects. H. pylori infection may also play a role in this process (see below).

1	H. pylori infection may also play a role in this process (see below). Gastric ulcers As in DUs, the majority of GUs can be attributed to either H. pylori or NSAID-induced mucosal damage. GUs that occur in the prepyloric area or those in the body associated with a DU or a duodenal scar are similar in pathogenesis to DUs. Gastric acid output (basal and stimulated) tends to be normal or decreased in GU patients. When GUs develop in the presence of minimal acid levels, impairment of mucosal defense factors may be present. GUs have been classified based on their location: Type I occur in the gastric body and tend to be associated with low gastric acid production; type II occur in the antrum and gastric acid can vary from low to normal; type III occur within 3 cm of the pylorus and are commonly accompanied by DUs and normal or high gastric acid production; and type IV are found in the cardia and are associated with low gastric acid production.

1	H.pylori anD aciD peptic DisorDers Gastric infection with the bacterium H. pylori accounts for the majority of PUD (Chap. 188). This organism also plays a role in the development of gastric mucosa-associated lymphoid tissue (MALT) lymphoma and gastric adenocarcinoma. Although the entire genome of H. pylori has been sequenced, it is still not clear how this organism, which resides in the stomach, causes ulceration in the duodenum, or whether its eradication will lead to a decrease in gastric cancer.

1	the bacterium The bacterium, initially named Campylobacter pyloridis, is a gram-negative microaerophilic rod found most commonly in the deeper portions of the mucous gel coating the gastric mucosa or between the mucous layer and the gastric epithelium. It may attach to gastric epithelium but under normal circumstances does not appear to invade cells. It is strategically designed to live within the aggressive environment of the stomach. It is S-shaped (~0.5–3 μm in size) and contains multiple sheathed flagella. Initially, H. pylori resides in the antrum but, over time, migrates toward the more proximal segments of the stomach. The organism is capable of transforming into a coccoid form, which represents a dormant state that may facilitate survival in adverse conditions. The genome of H. pylori (1.65 million base pairs) encodes ~1500 proteins. Among this multitude of proteins there are factors that are essential determinants of H. pylori–mediated pathogenesis and colonization such as

1	pylori (1.65 million base pairs) encodes ~1500 proteins. Among this multitude of proteins there are factors that are essential determinants of H. pylori–mediated pathogenesis and colonization such as the outer membrane protein (Hop proteins), urease, and the vacuolating cytotoxin (Vac A). Moreover, the majority of H. pylori strains contain a genomic fragment that encodes the cag pathogenicity island (cag-PAI). Several of the genes that make up cag-PAI encode components of a type IV secretion island that translocates Cag A into host cells. Once in the cell, Cag A activates a series of cellular events important in cell growth and cytokine production. H. pylori also has extensive genetic diversity that in turn enhances its ability to promote disease. The first step in infection by H. pylori is dependent on the bacteria’s motility and its ability to produce urease. Urease produces ammonia from urea, an essential 1915 step in alkalinizing the surrounding pH. Additional bacterial factors

1	is dependent on the bacteria’s motility and its ability to produce urease. Urease produces ammonia from urea, an essential 1915 step in alkalinizing the surrounding pH. Additional bacterial factors include catalase, lipase, adhesins, platelet-activating factor, and pic B (induces cytokines). Multiple strains of H. pylori exist and are characterized by their ability to express several of these factors (Cag A, Vac A, etc.). It is possible that the different diseases related to H. pylori infection can be attributed to different strains of the organism with distinct pathogenic features.

1	epidemiology The prevalence of H. pylori varies throughout the world and depends largely on the overall standard of living in the region. In developing parts of the world, 80% of the population may be infected by the age of 20, whereas the prevalence is 20–50% in industrialized countries. In contrast, in the United States this organism is rare in childhood. The overall prevalence of H. pylori in the United States is ~30%, with individuals born before 1950 having a higher rate of infection than those born later. About 10% of Americans <30 years of age are colonized with the bacteria. The rate of infection with H. pylori in industrialized countries has decreased substantially in recent decades.

1	The steady increase in the prevalence of H. pylori noted with increasing age is due primarily to a cohort effect, reflecting higher transmission during a period in which the earlier cohorts were children. It has been calculated through mathematical models that improved sanitation during the latter half of the nineteenth century dramatically decreased transmission of H. pylori. Moreover, with the present rate of intervention, the organism will be ultimately eliminated from the United States. Two factors that predispose to higher colonization rates include poor socioeconomic status and less education. These factors, not race, are responsible for the rate of H. pylori infection in blacks and Hispanic Americans being double the rate seen in whites of comparable age. Other risk factors for H. pylori infection are (1) birth or residence in a developing country, (2) domestic crowding, (3) unsanitary living conditions, (4) unclean food or water, and (5) exposure to gastric contents of an

1	H. pylori infection are (1) birth or residence in a developing country, (2) domestic crowding, (3) unsanitary living conditions, (4) unclean food or water, and (5) exposure to gastric contents of an infected individual.

1	Transmission of H. pylori occurs from person to person, following an oral-oral or fecal-oral route. The risk of H. pylori infection is declining in developing countries. The rate of infection in the United States has fallen by >50% when compared to 30 years ago. pathophysiology H. pylori infection is virtually always associated with a chronic active gastritis, but only 10–15% of infected individuals develop frank peptic ulceration. The basis for this difference is unknown, but is likely due to a combination of host and bacterial factors some of which are outlined below. Initial studies suggested that >90% of all DUs were associated with H. pylori, but H. pylori is present in only 30–60% of individuals with GUs and 50–70% of patients with DUs. The pathophysiology of ulcers not associated with H. pylori or NSAID ingestion (or the rare Zollinger-Ellison syndrome [ZES]) is becoming more relevant as the incidence of H. pylori is dropping, particularly in the Western world (see below).

1	The particular end result of H. pylori infection (gastritis, PUD, gastric MALT lymphoma, gastric cancer) is determined by a complex interplay between bacterial and host factors (Fig. 348-6). 1. Bacterial factors: H. pylori is able to facilitate gastric residence, induce mucosal injury, and avoid host defense. Different strains of

1	H. pylori produce different virulence factors. A specific region of the bacterial genome, the pathogenicity island (cag-PAI), encodes the virulence factors Cag A and pic B. Vac A also contributes to pathogenicity, although it is not encoded within the pathogenicity island. These virulence factors, in conjunction with additional bacterial constituents, can cause mucosal damage, in part through their ability to target the host immune cells. For example, Vac A targets human CD4 T cells, inhibiting their proliferation and in addition can disrupt normal function of B cells, CD8 T cells, macrophages, and mast cells. Multiple studies have demonstrated that H. pylori strains that are cag-PAI positive are associated with a higher risk of PUD, premalignant gastric lesions, and gastric cancer than are strains that lack the cag-PAI. In addition, H. pylori may directly inhibit parietal cell H+,K+-ATPase activity through a

1	Bacterial factors Structure Adhesins Porins Enzymes (urease, vac A, cag A, etc.) Host factors Duration Location Inflammatory response Genetics?? FIGURE 348-6 Outline of the bacterial and host factors important in determining H. pylori–induced gastrointestinal disease. MALT, mucosal-associated lymphoid tissue.

1	FIGURE 348-6 Outline of the bacterial and host factors important in determining H. pylori–induced gastrointestinal disease. MALT, mucosal-associated lymphoid tissue. Cag A–dependent mechanism, leading in part to the low acid production observed after acute infection with the organism. Urease, which allows the bacteria to reside in the acidic stomach, generates NH3, which can damage epithelial cells. The bacteria produce surface factors that are chemotactic for neutrophils and monocytes, which in turn contribute to epithelial cell injury (see below). H. pylori makes proteases and phospholipases that break down the glycoprotein lipid complex of the mucous gel, thus reducing the efficacy of this first line of mucosal defense. H. pylori expresses adhesins (OMPs like BabA), which facilitate attachment of the bacteria to gastric epithelial cells. Although lipopolysaccharide (LPS) of gram-negative bacteria often plays an important role in the infection,

1	H. pylori LPS has low immunologic activity compared to that of other organisms. It may promote a smoldering chronic inflammation.

1	2. Host factors: Studies in twins suggest that there may be genetic predisposition to acquire H. pylori. The inflammatory response to H. pylori includes recruitment of neutrophils, lymphocytes (T and B), macrophages, and plasma cells. The pathogen leads to local injury by binding to class II major histocompatibility complex (MHC) molecules expressed on gastric epithelial cells, leading to cell death (apoptosis). Moreover, bacterial strains that encode cag-PAI can introduce Cag A into the host cells, leading to further cell injury and activation of cellular pathways involved in cytokine production and repression of tumor-suppressor genes. Elevated concentrations of multiple cytokines are found in the gastric epithelium of H. pylori–infected individuals, including interleukin (IL) 1α/β, IL-2, IL-6, IL-8, tumor necrosis factor (TNF) α, and interferon (IFN) γ. H. pylori infection also leads to both a mucosal and a systemic humoral response, which does not lead to eradication of the

1	IL-2, IL-6, IL-8, tumor necrosis factor (TNF) α, and interferon (IFN) γ. H. pylori infection also leads to both a mucosal and a systemic humoral response, which does not lead to eradication of the bacteria but further compounds epithelial cell injury. Additional mechanisms by which H. pylori may cause epithelial cell injury include (1) activated neutrophil-mediated production of reactive oxygen or nitrogen species and enhanced epithelial cell turnover and (2) apoptosis related to interaction with T cells (T helper 1, or TH1, cells) and IFN-γ. Finally, the human stomach can be colonized by a host of commensal organisms that may affect the likelihood of

1	H. pylori–mediated mucosal injury. The reason for H. pylori–mediated duodenal ulceration remains unclear. Studies suggest that H. pylori associated with duodenal ulceration may be more virulent. In addition, certain specific bacterial factors such as the DU-promoting gene A (dupA), may be associated with the development of DUs. Another potential contributing factor is that gastric metaplasia in the duodenum of DU patients, which may be due to high acid exposure (see below), permits H. pylori to bind to it and produce local injury secondary to the host response. Another hypothesis is that H. pylori antral infection could lead to increased

1	Vagus Acetylcholine Histamine Histamine H, K ATPase Tubulovesicles Somatostatin Somatostatin ECL cell Gastrin ECL cell D cell Canaliculus Blood vessel Gastrin D cell G cell ANTRUMSomatostatin + + – – + + + – FIGURE 348-7 Summary of potential mechanisms by which H. pylori may lead to gastric secretory abnormalities. D, somatostatin cell; ECL, enterochromaffin-like cell; G, G cell. (Adapted from J Calam et al: Gastroenterology 113:543, 1997.) acid production, increased duodenal acid, and mucosal injury. Basal and stimulated (meal, gastrin-releasing peptide [GRP]) gastrin release are increased in H. pylori–infected individuals, and somatostatinsecreting D cells may be decreased. H. pylori infection might induce increased acid secretion through both direct and indirect actions of H. pylori and proinflammatory cytokines (IL-8, TNF, and IL-1) on G, D, and parietal cells (Fig. 348-7). GUs, in contrast, are associated with

1	H. pylori and proinflammatory cytokines (IL-8, TNF, and IL-1) on G, D, and parietal cells (Fig. 348-7). GUs, in contrast, are associated with H. pylori–induced pangastritis and normal or low gastric acid secretion. H. pylori infection has also been associated with decreased duodenal mucosal bicarbonate production. Data supporting and contradicting each of these interesting theories have been demonstrated. Thus, the mechanism by which H. pylori infection of the stomach leads to duodenal ulceration remains to be established. In summary, the final effect of H. pylori on the GI tract is variable and determined by microbial and host factors. The type and distribution of gastritis correlate with the ultimate gastric and duodenal pathology observed. Specifically, the presence of antral-predominant gastritis is associated with DU formation; gastritis involving primarily the corpus predisposes to the development of GUs, gastric atrophy, and ultimately gastric carcinoma (Fig. 348-8).

1	nsaiD-inDuceD Disease epidemiology NSAIDs represent a group of the most commonly used medications in the United States. More than 30 billion over-thecounter tablets and over 100 million prescriptions are sold yearly in the United States alone. In fact, after the introduction of COX-2 inhibitors in the year 2000, the number of prescriptions written for NSAIDs was >111 million at a cost of $4.8 billion. Side effects and complications due to NSAIDs are considered the most common drug-related toxicities in the United States. The spectrum of NSAID-induced morbidity ranges from nausea and dyspepsia (prevalence reported as high as 50–60%) to a serious GI complication such as endoscopy-documented peptic ulceration (15–30% of individuals taking NSAIDs regularly) complicated by bleeding or perforation in as many as 1.5% of users per year. It is estimated that NSAID-induced GI bleeding accounts for 60,000–120,000 hospital admissions per year, and deaths related to NSAID-induced toxicity may be

1	in as many as 1.5% of users per year. It is estimated that NSAID-induced GI bleeding accounts for 60,000–120,000 hospital admissions per year, and deaths related to NSAID-induced toxicity may be as high as 16,000 per year in the United States. Approximately 4–5% of patients develop symptomatic ulcers within 1 year. Unfortunately, dyspeptic symptoms do not correlate with NSAID-induced pathology. Over 80% of patients with serious NSAID-related complications did not have preceding dyspepsia.

1	High level of acid production Duodenal ulcer Normal gastric H. pylori H. pylori H.pylori Low level of acid production Gastric cancer FIGURE 348-8 Natural history of H. pylori infection. MALT, mucosalassociated lymphoid tissue. (Used with permission from S Suerbaum, P Michetti: N Engl J Med 347:1175, 2002.)

1	In view of the lack of warning signs, it is important to identify patients who are at increased risk for morbidity and mortality related to NSAID usage. Even 75 mg/d of aspirin may lead to serious GI ulceration; thus, no dose of NSAID is completely safe. In fact, the incidence of mucosal injury (ulcers and erosions) in patients taking low-dose aspirin (75–325 mg) has been estimated to range from as low as 8% to as high as 60%. It appears that H. pylori infection increases the risk of PUD-associated GI bleeding in chronic users of low-dose aspirin. Established risk factors include advanced age, history of ulcer, concomitant use of glucocorticoids, high-dose NSAIDs, multiple NSAIDs, concomitant use of anticoagulants, clopidogrel, and serious or multisystem disease. Possible risk factors include concomitant infection with H. pylori, cigarette smoking, and alcohol consumption.

1	pathophysiology Prostaglandins play a critical role in maintaining gastroduodenal mucosal integrity and repair. It therefore follows that interruption of prostaglandin synthesis can impair mucosal defense and repair, thus facilitating mucosal injury via a systemic mechanism. Animal studies have demonstrated that neutrophil adherence to the gastric microcirculation plays an essential role in the initiation of NSAID-induced mucosal injury. A summary of the pathogenetic pathways by which systemically administered NSAIDs may lead to mucosal injury is shown in Fig. 348-9. Single nucleotide polymorphisms (SNPs) have been found in several genes, including those encoding certain subtypes of cytochrome P450 (see below), interleukin-1β (IL-1β), angiotensinogen (AGT), and an organic ion transporting polypeptide (SLCO1B1), but these findings need confirmation in larger scale studies.

1	Injury to the mucosa also occurs as a result of the topical encounter with NSAIDs. Aspirin and many NSAIDs are weak acids that remain in a nonionized lipophilic form when found within the acid environment of the stomach. Under these conditions, NSAIDs migrate across lipid membranes of epithelial cells, leading to cell injury once trapped intracellularly in an ionized form. Topical NSAIDs can also alter the surface mucous layer, permitting back diffusion of H+ and pepsin, leading to further epithelial cell damage. Moreover, enteric-coated or buffered preparations are also associated with risk of peptic ulceration.

1	The interplay between H. pylori and NSAIDs in the pathogenesis of PUD is complex. Meta-analysis supports the conclusion that each of these aggressive factors is independent and synergistic risk factors for PUD and its complications such as GI bleeding. For example, eradication of H. pylori reduces the likelihood of GI complications in high-risk individuals to levels observed in individuals with average risk of NSAID-induced complications.

1	Endothelial effects • Stasis IschemiaULCER EROSIONS HEALING (spontaneous or therapeutic) Epithelial effects (due to prostaglandin depletion) • ˜HCl secretion • °Mucin secretion • °HCO3 – secretion • °Surface active phospholipid secretion • °Epithelial cell proliferation • Direct toxicity “ion trapping” Acid FIGURE 348-9 Mechanisms by which nonsteroidal anti-inflammatory drugs may induce mucosal injury. (Adapted from J Scheiman et al: J Clin Outcomes Management 3:23, 1996. Copyright 2003 Turner White Communications, Inc., www.turner-white.com. Used with permission.) patHoGenetic factors unrelateD to H. pylori anD nsaiDs in aciD peptic Disease Cigarette smoking has been implicated in the pathogenesis of PUD. Not only have smokers been found to have ulcers more frequently than do nonsmokers, but smoking appears to decrease healing rates, impair response to therapy, and increase ulcer-related complications such as perforation. The mechanism responsible for increased ulcer diathesis in

1	but smoking appears to decrease healing rates, impair response to therapy, and increase ulcer-related complications such as perforation. The mechanism responsible for increased ulcer diathesis in smokers is unknown. Theories have included altered gastric emptying, decreased proximal duodenal bicarbonate production, increased risk for H. pylori infection, and cigarette-induced generation of noxious mucosal free radicals. Genetic predisposition may play a role in ulcer development. First-degree relatives of DU patients are three times as likely to develop an ulcer; however, the potential role of H. pylori infection in contacts is a major consideration. Increased frequencies of blood group O and of the nonsecretor status have also been implicated as genetic risk factors for peptic diathesis. However, H. pylori preferentially binds to group O antigens. Additional genetic factors have been postulated to predispose certain individuals to developing PUD and/or upper GI bleeding.

1	diathesis. However, H. pylori preferentially binds to group O antigens. Additional genetic factors have been postulated to predispose certain individuals to developing PUD and/or upper GI bleeding. Specifically, genes encoding the NSAID-metabolizing enzymes cytochrome P450 2C9 and 2C8 (CYP2C9 and CYP2C8) are potential susceptibility genes for NSAID-induced PUD, but unfortunately, the studies have not been consistent in demonstrating this association. In a United Kingdom study, the CYP2C19*17 gain-of-function polymorphism was associated with PUD in a Caucasian cohort, irrespective of ulcer etiology. These findings need to be confirmed in broader studies. Psychological stress has been thought to contribute to PUD, but studies examining the role of psychological factors in its pathogenesis have generated conflicting results. Although PUD is associated with certain personality traits (neuroticism), these same traits are also present in individuals with nonulcer dyspepsia (NUD) and other

1	have generated conflicting results. Although PUD is associated with certain personality traits (neuroticism), these same traits are also present in individuals with nonulcer dyspepsia (NUD) and other functional and organic disorders.

1	Diet has also been thought to play a role in peptic diseases. Certain foods and beverages can cause dyspepsia, but no convincing studies indicate an association between ulcer formation and a specific diet. Specific chronic disorders have been shown to have a strong association with PUD: (1) advanced age, (2) chronic pulmonary disease, (3) chronic renal failure, (4) cirrhosis, (5) nephrolithiasis, (6) α1antitrypsin deficiency, and (7) systemic mastocytosis. Disorders with a possible association are (1) hyperparathyroidism, (2) coronary artery disease, (3) polycythemia vera, (4) chronic pancreatitis, (5) former alcohol use, (6) obesity, (7) African-American race, and (8) three or more doctor visits in a year. Multiple factors play a role in the pathogenesis of PUD. The two predominant causes are H. pylori infection and NSAID ingestion. PUD CAuSES of ulCERS noT CAuSED By HELICOBACTER PYLORI AnD nSAiDs Pathogenesis of Non-Hp and Non-NSAID Ulcer Disease

1	CAuSES of ulCERS noT CAuSED By HELICOBACTER PYLORI AnD nSAiDs Pathogenesis of Non-Hp and Non-NSAID Ulcer Disease Bisphosphonates Chemotherapy Clopidogrel Crack cocaine Glucocorticoids (when combined with NSAIDs) Mycophenolate mofetil Potassium chloride Duodenal obstruction (e.g., annular pancreas) Idiopathic hypersecretory state Abbreviations: Hp, H. pylori; NSAIDs, nonsteroidal anti-inflammatory drugs. not related to H. pylori or NSAIDs is increasing. Other less common causes of PUD are shown in Table 348-1. These etiologic agents should be considered as the incidence of H. pylori is decreasing. Independent of the inciting or injurious agent, peptic ulcers develop as a result of an imbalance between mucosal protection/repair and aggressive factors. Gastric acid plays an important role in mucosal injury.

1	CLINICAL FEATURES History Abdominal pain is common to many GI disorders, including DU and GU, but has a poor predictive value for the presence of either DU or GU. Up to 10% of patients with NSAID-induced mucosal disease can present with a complication (bleeding, perforation, and obstruction) without antecedent symptoms. Despite this poor correlation, a careful history and physical examination are essential components of the approach to a patient suspected of having peptic ulcers.

1	Epigastric pain described as a burning or gnawing discomfort can be present in both DU and GU. The discomfort is also described as an ill-defined, aching sensation or as hunger pain. The typical pain pattern in DU occurs 90 minutes to 3 hours after a meal and is frequently relieved by antacids or food. Pain that awakes the patient from sleep (between midnight and 3 A.M.) is the most discriminating symptom, with two-thirds of DU patients describing this complaint. Unfortunately, this symptom is also present in one-third of patients with NUD (see below). Elderly patients are less likely to have abdominal pain as a manifestation of PUD and may instead present with a complication such as ulcer bleeding or perforation. The pain pattern in GU patients may be different from that in DU patients, where discomfort may actually be precipitated by food. Nausea and weight loss occur more commonly in GU patients. Endoscopy detects ulcers in <30% of patients who have dyspepsia.

1	The mechanism for development of abdominal pain in ulcer patients is unknown. Several possible explanations include acid-induced activation of chemical receptors in the duodenum, enhanced duodenal sensitivity to bile acids and pepsin, or altered gastroduodenal motility. Variation in the intensity or distribution of the abdominal pain, as well as the onset of associated symptoms such as nausea and/or vomiting, may be indicative of an ulcer complication. Dyspepsia that becomes constant, is no longer relieved by food or antacids, or radiates to the back may indicate a penetrating ulcer (pancreas). Sudden onset of severe, generalized abdominal pain may indicate perforation. Pain worsening with meals, nausea, and vomiting of undigested food suggest gastric outlet obstruction. Tarry stools or coffee-ground emesis indicate bleeding.

1	Physical examination Epigastric tenderness is the most frequent finding in patients with GU or DU. Pain may be found to the right of the midline in 20% of patients. Unfortunately, the predictive value of this finding is rather low. Physical examination is critically important for discovering evidence of ulcer complication. Tachycardia and orthostasis suggest dehydration secondary to vomiting or active GI blood loss. A severely tender, board-like abdomen suggests a perforation. Presence of a succussion splash indicates retained fluid in the stomach, suggesting gastric outlet obstruction.

1	PUD-Related Complications • Gastrointestinal bleeDinG GI bleeding is the most common complication observed in PUD. Bleeding is estimated to occur in 19.4–57 per 100,000 individuals in a general population or in approximately 15% of patients. Bleeding and complications of ulcer disease occur more often in individuals >60 years of age. The 30-day mortality rate is as high as 5–10%. The higher incidence in the elderly is likely due to the increased use of NSAIDs in this group. In addition, up to 80% of the mortality in PUD-related bleeding is due to nonbleeding causes such as multiorgan failure (24%), pulmonary complications (24%), and malignancy (34%). Up to 20% of patients with ulcer-related hemorrhage bleed without any preceding warning signs or symptoms.

1	Up to 20% of patients with ulcer-related hemorrhage bleed without any preceding warning signs or symptoms. perforation The second most common ulcer-related complication is perforation, being reported in as many as 6–7% of PUD patients with an estimated 30-day mortality of over 20%. As in the case of bleeding, the incidence of perforation in the elderly appears to be increasing secondary to increased use of NSAIDs. Penetration is a form of perforation in which the ulcer bed tunnels into an adjacent organ. DUs tend to penetrate posteriorly into the pancreas, leading to pancreatitis, whereas GUs tend to penetrate into the left hepatic lobe. Gastrocolic fistulas associated with GUs have also been described.

1	Gastric outlet obstruction Gastric outlet obstruction is the least common ulcer-related complication, occurring in 1–2% of patients. A patient may have relative obstruction secondary to ulcer-related inflammation and edema in the peripyloric region. This process often resolves with ulcer healing. A fixed, mechanical obstruction secondary to scar formation in the peripyloric areas is also possible. The latter requires endoscopic (balloon dilation) or surgical intervention. Signs and symptoms relative to mechanical obstruction may develop insidiously. New onset of early satiety, nausea, vomiting, increase of postprandial abdominal pain, and weight loss should make gastric outlet obstruction a possible diagnosis.

1	Differential Diagnosis The list of GI and non-GI disorders that can mimic ulceration of the stomach or duodenum is quite extensive. The most commonly encountered diagnosis among patients seen for upper abdominal discomfort is NUD. NUD, also known as functional dyspepsia or essential dyspepsia, refers to a group of heterogeneous disorders typified by upper abdominal pain without the presence of an ulcer. Dyspepsia has been reported to occur in up to 30% of the U.S. population. Up to 60% of patients seeking medical care for dyspepsia have a negative diagnostic evaluation. The etiology of NUD is not established, and the potential role of H. pylori in NUD remains controversial. Several additional disease processes that may present with “ulcerlike” symptoms include proximal GI tumors, gastroesophageal reflux, vascular disease, pancreaticobiliary disease (biliary colic, chronic pancreatitis), and gastroduodenal Crohn’s disease.

1	Diagnostic Evaluation In view of the poor predictive value of abdominal pain for the presence of a gastroduodenal ulcer and the multiple disease processes that can mimic this disease, the clinician is often confronted with having to establish the presence of an ulcer. Documentation of an ulcer requires either a radiographic (barium study) or an endoscopic procedure. However, a large percentage of patients with symptoms suggestive of an ulcer have NUD; testing for H. pylori and antibiotic therapy (see below) is appropriate for individuals who are otherwise healthy and <45 years of age, before embarking on a diagnostic evaluation (Chap. 54). FIGURE 348-10 Barium study demonstrating (A) a benign duodenal ulcer and (B) a benign gastric ulcer.

1	Barium studies of the proximal GI tract are still occasionally used as a first test for documenting an ulcer. The sensitivity of older single-contrast barium meals for detecting a DU is as high as 80%, with a double-contrast study providing detection rates as high as 90%. Sensitivity for detection is decreased in small ulcers (<0.5 cm), with presence of previous scarring, or in postoperative patients. A DU appears as a well-demarcated crater, most often seen in the bulb (Fig. 348-10A). A GU may represent benign or malignant disease. Typically, a benign GU also appears as a discrete crater with radiating mucosal folds originating from the ulcer margin (Fig. 348-10B). Ulcers >3 cm in size or those associated with a mass are more often malignant. Unfortunately, up to 8% of GUs that appear to be benign by radiographic appearance are malignant by endoscopy or surgery. Radiographic studies that show a GU must be followed by endoscopy and biopsy.

1	Endoscopy provides the most sensitive and specific approach for examining the upper GI tract (Fig. 34811). In addition to permitting direct visualization of the mucosa, endoscopy facilitates photographic documentation of a mucosal defect and tissue biopsy to rule out malignancy (GU) or H. pylori. Endoscopic examination is particularly helpful in identifying lesions too small to detect by radiographic examination, for evaluation of atypical radiographic abnormalities, or to determine if an ulcer is a source of blood loss. Although the methods for diagnosing H. pylori are outlined in Chap. 181, a brief summary will be included here (Table 348-2). Several biopsy urease tests have been developed (PyloriTek, CLOtest, Hpfast, Pronto Dry) that have a sensitivity and specificity of >90–95%. Several noninvasive methods for detecting this organism have been developed. Three types of studies routinely used include serologic testing, the 13Cor 14C-urea breath test, and the fecal

1	H. pylori (Hp) antigen test. A urinary Hp antigen test, as well as a refined monoclonal antibody stool antigen test, appears promising. Occasionally, specialized testing such as serum gastrin and gastric acid analysis or sham feeding may be needed in individuals with complicated or refractory PUD (see “Zollinger-Ellison Syndrome [ZES],” below). Screening for aspirin or NSAIDs (blood or urine) may also be necessary in refractory H. pylori–negative PUD patients. Before the discovery of H. pylori, the therapy of PUD was centered on the old dictum by Schwartz of “no acid, no ulcer.” Although acid secretion is still important in the pathogenesis of PUD, eradication of H. pylori and therapy/prevention of NSAID-induced disease is the mainstay of treatment. A summary of commonly used drugs for treatment of acid peptic disorders is shown in Table 348-3.

1	ACID-NEUTRALIZING/INHIBITORY DRUGS Antacids Before we understood the important role of histamine in stimulating parietal cell activity, neutralization of secreted acid with antacids constituted the main form of therapy for peptic FIGURE 348-11 Endoscopy demonstrating (A) a benign duodenal ulcer and (B) a benign gastric ulcer. TABlE 348-2 TESTS foR DETECTion of H. PYLORI Sensitivity/ Test Specificity, % Comments Rapid urease 80–95/95–100 Simple, false negative with recent use of PPIs, antibiotics, or bismuth compounds Culture —/— Time-consuming, expensive, dependent on experience; allows determination of antibiotic susceptibility Serology >80/>90 Inexpensive, convenient; not useful for early follow-up Urea breath test >90/>90 Simple, rapid; useful for early follow-up; false negatives with recent therapy (see rapid urease test); exposure to low-dose radiation with 14C test Stool antigen >90/>90 Inexpensive, convenient Abbreviation: PPIs, proton pump inhibitors.

1	Stool antigen >90/>90 Inexpensive, convenient Abbreviation: PPIs, proton pump inhibitors. ulcers. They are now rarely, if ever, used as the primary therapeutic agent but instead are often used by patients for symptomatic relief of dyspepsia. The most commonly used agents are mixtures of aluminum hydroxide and magnesium hydroxide. Aluminum hydroxide can produce constipation and phosphate depletion; magnesium hydroxide may cause loose stools. Many of the commonly used antacids (e.g., Maalox, Mylanta) have a combination of both aluminum and magnesium hydroxide in order to avoid these side effects. The magnesium-containing preparation should not be used in chronic renal failure patients because of possible hypermagnesemia, and aluminum may cause chronic neurotoxicity in these patients.

1	Calcium carbonate and sodium bicarbonate are potent antacids with varying levels of potential problems. The long-term use of calcium carbonate (converts to calcium chloride in the stomach) can lead to milk-alkali syndrome (hypercalcemia, hyperphosphatemia with possible renal calcinosis and progression to renal insufficiency). Sodium bicarbonate may induce systemic alkalosis. H2 Receptor Antagonists Four of these agents are presently available (cimetidine, ranitidine, famotidine, and nizatidine), and their structures share homology with histamine. Although each has different potency, all will significantly inhibit basal and stimulated acid secretion to comparable levels when used at therapeutic doses. Moreover, similar ulcer-healing rates are achieved with each drug when used at the correct dosage. Presently, this class of drug is often used for treatment of active ulcers (4–6 weeks) in combination with antibiotics directed at eradicating H. pylori (see below).

1	Cimetidine was the first H2 receptor antagonist used for the treatment of acid peptic disorders. The initial recommended dosing profile for cimetidine was 300 mg qid. Subsequent studies have documented the efficacy of using 800 mg at bedtime for treatment of active ulcer, with healing rates approaching 80% at 4 weeks. Cimetidine may have weak antiandrogenic side effects resulting in reversible gynecomastia and impotence, primarily in patients receiving high doses for prolonged periods of time (months to years, as in ZES). In view of cimetidine’s ability to inhibit cytochrome P450, careful monitoring of drugs such as warfarin, phenytoin, and theophylline is indicated with long-term usage. Other rare reversible adverse effects reported with cimetidine include confusion and elevated levels of serum aminotransferases, creatinine, and serum prolactin. Ranitidine, famotidine, and nizatidine are more potent H2 receptor antagonists than cimetidine. Each can be used once a day at bedtime for

1	of serum aminotransferases, creatinine, and serum prolactin. Ranitidine, famotidine, and nizatidine are more potent H2 receptor antagonists than cimetidine. Each can be used once a day at bedtime for ulcer prevention, which was commonly done before the discovery of H. pylori and the development of proton pump inhibitors (PPIs). Patients may develop tolerance to H2 blockers, a rare event with PPIs (see below). Comparable nighttime dosing regimens are ranitidine 300 mg, famotidine 40 mg, and nizatidine 300 mg.

1	Additional rare, reversible systemic toxicities reported with H2 receptor antagonists include pancytopenia, neutropenia, anemia, and thrombocytopenia, with a prevalence rate varying from 0.01– 0.2%. Cimetidine and ranitidine (to a lesser extent) can bind to hepatic cytochrome P450; famotidine and nizatidine do not.

1	Proton Pump (H+,K+-ATPase) Inhibitors Omeprazole, esomeprazole, lansoprazole, rabeprazole, and pantoprazole are substituted benzimidazole derivatives that covalently bind and irreversibly inhibit H+,K+-ATPase. Esomeprazole, one of the newest members of this drug class, is the S-enantiomer of omeprazole, which is a racemic mixture of both Sand R-optical isomers. The R-isomer of lansoprazole, dexlansoprazole, is the most recent PPI approved for clinical use. Its reported advantage is a dual delayed-release system, aimed at improving treatment of gastroesophageal reflux disease (GERD). These are the most potent acid inhibitory agents available. Omeprazole and lansoprazole are the PPIs that have been used for the longest time. Both are acid-labile and are administered as enteric-coated granules in a sustained-release capsule that dissolves within the small intestine at a pH of 6. Lansoprazole is available in an orally disintegrating tablet that can be taken with or without water, an

1	in a sustained-release capsule that dissolves within the small intestine at a pH of 6. Lansoprazole is available in an orally disintegrating tablet that can be taken with or without water, an advantage for individuals who have significant dysphagia. Absorption kinetics are similar to the capsule. In addition, a lansoprazole-naproxen combination preparation that has been made available is targeted at decreasing NSAID-related GI injury (see below). Omeprazole is available as nonenteric-coated granules mixed with sodium bicarbonate in a powder form that can be administered orally or via gastric tube. The sodium bicarbonate has two purposes: to protect the omeprazole from acid degradation and to promote rapid gastric alkalinization and subsequent proton pump activation, which facilitates rapid action of the PPI. Pantoprazole and rabeprazole are available as enteric-coated tablets. Pantoprazole is also available as a parenteral formulation for intravenous use. These agents are lipophilic

1	action of the PPI. Pantoprazole and rabeprazole are available as enteric-coated tablets. Pantoprazole is also available as a parenteral formulation for intravenous use. These agents are lipophilic compounds; upon entering the parietal cell, they are protonated and trapped within the acid environment of the tubulovesicular and canalicular system. These agents potently inhibit all phases of gastric acid secretion. Onset of action is rapid, with a maximum acid inhibitory effect between 2 and 6 h after administration and duration of inhibition lasting up to 72–96 h. With repeated daily dosing, progressive acid inhibitory effects are observed, with basal and secretagogue-stimulated acid production being inhibited by >95% after 1 week of therapy. The half-life of PPIs is ~18 h; thus, it can take between 2 and 5 days for gastric acid secretion to return to normal levels once these drugs have been discontinued. Because the pumps need to be activated for these agents to be effective, their

1	take between 2 and 5 days for gastric acid secretion to return to normal levels once these drugs have been discontinued. Because the pumps need to be activated for these agents to be effective, their efficacy is maximized if they are administered before a meal (except for the immediate-release formulation of omeprazole) (e.g., in the morning before breakfast). Mild to moderate hypergastrinemia has been observed in patients taking these drugs. Carcinoid tumors developed in some animals given the drugs preclinically; however, extensive experience has failed to demonstrate gastric carcinoid tumor development in humans. Serum gastrin levels return to normal levels within 1–2 weeks after drug cessation. Rebound gastric acid hypersecretion has been described in H. pylori–negative individuals after discontinuation of PPIs. It occurs even after relatively short-term usage (2 months) and may last for up to 2 months after the PPI has been discontinued. The mechanism involves gastrininduced

1	after discontinuation of PPIs. It occurs even after relatively short-term usage (2 months) and may last for up to 2 months after the PPI has been discontinued. The mechanism involves gastrininduced hyperplasia and hypertrophy of histamine-secreting ECL cells. The clinical relevance of this observation is that individuals may have worsening symptoms of GERD or dyspepsia upon stopping the PPI. Gradual tapering of the PPI and switching to an H2 receptor antagonist may prevent this from occurring. H. pylori– induced inflammation and concomitant decrease in acid production may explain why this does not occur in H. pylori–positive patients. IF production is also inhibited, but vitamin B12-deficiency anemia is uncommon, probably because of the large stores of the vitamin. As with any agent that leads to significant hypochlorhydria, PPIs may interfere with absorption of drugs such as ketoconazole, ampicillin, iron, and digoxin. Hepatic cytochrome P450 can be inhibited by the earlier PPIs

1	leads to significant hypochlorhydria, PPIs may interfere with absorption of drugs such as ketoconazole, ampicillin, iron, and digoxin. Hepatic cytochrome P450 can be inhibited by the earlier PPIs (omeprazole, lansoprazole). Rabeprazole, pantoprazole, and esomeprazole do not appear to interact significantly with drugs metabolized by the cytochrome P450 system. The overall clinical significance of this observation is not definitely established. Caution should be taken when using theophylline, warfarin, diazepam, atazanavir, and phenytoin concomitantly with PPIs. Long-term acid suppression, especially with PPIs, has been associated with a higher incidence of community-acquired pneumonia as well as community and hospital acquired Clostridium difficile–associated disease. These observations require confirmation but should alert the practitioner to take caution when recommending these agents for long-term use, especially in elderly patients at risk for developing pneumonia or C. difficile

1	confirmation but should alert the practitioner to take caution when recommending these agents for long-term use, especially in elderly patients at risk for developing pneumonia or C. difficile infection. A population-based study revealed that long-term use of PPIs was associated with the development of hip fractures in older women. The absolute risk of fracture remained low despite an observed increase associated with the dose and duration of acid suppression. The mechanism for this observation is not clear, and this finding must be confirmed before making broad recommendations regarding the discontinuation of these agents in patients who benefit from them. Long-term use of PPIs has also been implicated in the development of iron and magnesium deficiency, but here again, the studies are limited and inconclusive. PPIs may exert a negative effect on the antiplatelet effect of clopidogrel. Although the evidence is mixed and inconclusive, a small increase in mortality and readmission rate

1	and inconclusive. PPIs may exert a negative effect on the antiplatelet effect of clopidogrel. Although the evidence is mixed and inconclusive, a small increase in mortality and readmission rate for coronary events was seen in patients receiving a PPI while on clopidogrel in earlier studies. Subsequently, three meta-analyses reported an inverse correlation between clopidogrel and PPI use; therefore, the influence of this drug interaction on mortality is not clearly established. The mechanism involves the competition of the PPI and clopidogrel with the same cytochrome P450 (CYP2C19). Whether this is a class effect of PPIs is unclear; there appears to be at least a theoretical advantage of pantoprazole over the other PPIs, but this has not been confirmed. This drug interaction is particularly relevant in light of the common use of aspirin and clopidogrel for prevention of coronary events and the efficacy of PPIs in preventing GI bleeding in these patients. The FDA has made several

1	relevant in light of the common use of aspirin and clopidogrel for prevention of coronary events and the efficacy of PPIs in preventing GI bleeding in these patients. The FDA has made several recommendations while awaiting further evidence to clarify the impact of PPI therapy on clopidogrel use. Health care providers should continue to prescribe clopidogrel to patients who require it and 1921 should reevaluate the need for starting or continuing treatment with a PPI. From a practical standpoint, additional recommendations to consider include the following: Patients taking clopidogrel with aspirin, especially with other GI risk factors for bleeding, should receive GI protective therapy. Although high-dose H2 blockers have been considered an option, these do not appear to be as effective as PPIs. If PPIs are to be given, some have recommended that there be a 12-h separation between administration of the PPI and clopidogrel to minimize competition of the two agents with the involved cyto

1	PPIs. If PPIs are to be given, some have recommended that there be a 12-h separation between administration of the PPI and clopidogrel to minimize competition of the two agents with the involved cyto chrome P450. One option is to give the PPI 30 min before breakfast and the clopidogrel at bedtime. Insufficient data are available to firmly recommend one PPI over another. Patients 65 years of age or older have a higher risk for some of the long-term side effects of PPIs highlighted above, in part due to the higher prevalence of concomitant chronic diseases. It is therefore important to carefully select individuals, especially among the elderly, who need long-term PPI therapy and discontinue it in those individuals who do not need it.

1	Two new formulations of acid inhibitory agents are being developed. Tenatoprazole is a PPI containing an imidazopyridine ring instead of a benzimidazole ring, which promotes irreversible proton pump inhibition. This agent has a longer half-life than the other PPIs and may be beneficial for inhibiting nocturnal acid secretion, which has significant relevance in GERD. A second new class of agents is the potassium-competitive acid pump antagonists (P-CABs). These compounds inhibit gastric acid secretion via potassium competitive binding of the H+,K+-ATPase.

1	CYTOPROTECTIVE AGENTS Sucralfate Sucralfate is a complex sucrose salt in which the hydroxyl groups have been substituted by aluminum hydroxide and sulfate. This compound is insoluble in water and becomes a viscous paste within the stomach and duodenum, binding primarily to sites of active ulceration. Sucralfate may act by several mechanisms: serving as a physicochemical barrier, promoting a trophic action by binding growth factors such as EGF, enhancing prostaglandin synthesis, stimulating mucus and bicarbonate secretion, and enhancing mucosal defense and repair. Toxicity from this drug is rare, with constipation being most common (2–3%). It should be avoided in patients with chronic renal insufficiency to prevent aluminum-induced neurotoxicity. Hypophosphatemia and gastric bezoar formation have also been reported rarely. Standard dosing of sucralfate is 1 g qid.

1	Bismuth-Containing Preparations Sir William Osler considered bismuth-containing compounds the drug of choice for treating PUD. The resurgence in the use of these agents is due to their effect against H. pylori. Colloidal bismuth subcitrate (CBS) and bismuth subsalicylate (BSS, Pepto-Bismol) are the most widely used preparations. The mechanism by which these agents induce ulcer healing is unclear. Adverse effects with short-term use include black stools, constipation, and darkening of the tongue. Long-term use with high doses, especially with the avidly absorbed CBS, may lead to neurotoxicity. These compounds are commonly used as one of the agents in an anti-H. pylori regimen (see below).

1	Prostaglandin Analogues In view of their central role in maintaining mucosal integrity and repair, stable prostaglandin analogues were developed for the treatment of PUD. The mechanism by which this rapidly absorbed drug provides its therapeutic effect is through enhancement of mucosal defense and repair. The most common toxicity noted with this drug is diarrhea (10–30% incidence). Other major toxicities include uterine bleeding and contractions; misoprostol is contraindicated in women who may be pregnant, and women of childbearing age must be made clearly aware of this potential drug toxicity. The standard therapeutic dose is 200 μg qid. Miscellaneous Drugs A number of drugs including anticholinergic agents and tricyclic antidepressants were used for treating acid 1922 peptic disorders, but in light of their toxicity and the development of potent antisecretory agents, these are rarely, if ever, used today. THERAPY OF H. PYLORI

1	THERAPY OF H. PYLORI The physician’s goal in treating PUD is to provide relief of symptoms (pain or dyspepsia), promote ulcer healing, and ultimately prevent ulcer recurrence and complications. The greatest influence of understanding the role of H. pylori in peptic disease has been the ability to prevent recurrence. Documented eradication of H. pylori in patients with PUD is associated with a dramatic decrease in ulcer recurrence to <10–20% as compared to 59% in GU patients and 67% in DU patients when the organism is not eliminated. Eradication of the organism may lead to diminished recurrent ulcer bleeding. The effect of its eradication on ulcer perforation is unclear.

1	Extensive effort has been made in determining who of the many individuals with H. pylori infection should be treated. The common conclusion arrived at by multiple consensus conferences around the world is that H. pylori should be eradicated in patients with documented PUD. This holds true independent of time of presentation (first episode or not), severity of symptoms, presence of confounding factors such as ingestion of NSAIDs, or whether the ulcer is in remission. Some have advocated treating patients with a history of documented PUD who are found to be H. pylori–positive by serology or breath testing. Over one-half of patients with gastric MALT lymphoma experience complete remission of the tumor in response to H. pylori eradication. The Maastricht IV/Florence Consensus Report recommends a test-and-treat approach for patients with uninvestigated dyspepsia if the local incidence of H. pylori is greater than 20%. In addition, recommendations from this consensus report include testing

1	a test-and-treat approach for patients with uninvestigated dyspepsia if the local incidence of H. pylori is greater than 20%. In addition, recommendations from this consensus report include testing and eradicating H. pylori in patients who will be using NSAIDs (including low-dose aspirin) on a long-term basis, especially if there is a prior history of PUD. These individuals will require continued PPI treatment as well as eradication treatment, because eradication of the organism alone does not eliminate the risk of gastroduodenal ulcers in patients already receiving long-term NSAIDs. Treating patients with NUD to prevent gastric cancer or patients with GERD requiring long-term acid suppression remains controversial. Guidelines from the American College of Gastroenterology suggest eradication of H. pylori in patients who have undergone resection of early gastric cancer. The Maastricht IV/Florence Consensus Report also evaluated H. pylori treatment in gastric cancer prevention and

1	of H. pylori in patients who have undergone resection of early gastric cancer. The Maastricht IV/Florence Consensus Report also evaluated H. pylori treatment in gastric cancer prevention and recommends that eradication should be considered in the following situations: first-degree relatives of family members with gastric cancer; patients with previous gastric neoplasm treated by endoscopic or subtotal resection; individuals with a risk of gastritis (severe pangastritis or body-predominant gastritis) or severe atrophy; patients with gastric acid inhibition for more than 1 year; individuals with strong environmental risk factors for gastric cancer (heavy smoking; high exposure to dust, coal, quartz, or cement; and/or work in quarries); and H. pylori–positive patients with a fear of gastric cancer.

1	Multiple drugs have been evaluated in the therapy of H. pylori. No single agent is effective in eradicating the organism. Combination therapy for 14 days provides the greatest efficacy, although regimens based on sequential administration of antibiotics also appear promising (see below). A shorter administration course (7–10 days), although attractive, has not proved as successful as the 14-day regimens. The agents used with the greatest frequency include amoxicillin, metronidazole, tetracycline, clarithromycin, and bismuth compounds.

1	Suggested treatment regimens for H. pylori are outlined in Table 348-4. Choice of a particular regimen will be influenced by several factors, including efficacy, patient tolerance, existing antibiotic resistance, and cost of the drugs. The aim for initial eradication rates should be 85–90%. Dual therapy (PPI plus amoxicillin, PPI plus clarithromycin, ranitidine bismuth citrate [Tritec] plus clarithromycin) is not recommended in view of studies demonstrating eradication rates of <80–85%. The combination of bismuth, metronidazole, and tetracycline was the first triple regimen found effective against TABlE 348-4 REGimEnS RECommEnDED foR ERADiCATion of H. PYLORI infECTion aAlternative: use prepacked Helidac (see text). bAlternative: use prepacked Prevpac (see text). cUse either metronidazole or amoxicillin, not both.

1	H. pylori. The combination of two antibiotics plus either a PPI, H2 blocker, or bismuth compound has comparable success rates. Addition of acid suppression assists in providing early symptom relief and enhances bacterial eradication.

1	Triple therapy, although effective, has several drawbacks, including the potential for poor patient compliance and drug-induced side effects. Compliance is being addressed by simplifying the regimens so that patients can take the medications twice a day. Simpler (dual therapy) and shorter regimens (7 and 10 days) are not as effective as triple therapy for 14 days. Two anti-H. pylori regimens are available in prepackaged formulation: Prevpac (lansoprazole, clarithromycin, and amoxicillin) and Helidac (BSS, tetracycline, and metronidazole). The contents of the Prevpac are to be taken twice per day for 14 days, whereas Helidac constituents are taken four times per day with an antisecretory agent (PPI or H2 blocker), also for at least 14 days. Clarithromycin-based triple therapy should be avoided in settings where H. pylori resistance to this agent exceeds 15–20%.

1	Side effects have been reported in up to 20–30% of patients on triple therapy. Bismuth may cause black stools, constipation, or darkening of the tongue. The most feared complication with amoxicillin is pseudomembranous colitis, but this occurs in <1–2% of patients. Amoxicillin can also lead to antibiotic-associated diarrhea, nausea, vomiting, skin rash, and allergic reaction. Concomitant use of probiotics may ameliorate some of the antibiotic side effects (see below). Tetracycline has been reported to cause rashes and, very rarely, hepatotoxicity and anaphylaxis.

1	One important concern with treating patients who may not need therapy is the potential for development of antibiotic-resistant strains. The incidence and type of antibiotic-resistant H. pylori strains vary worldwide. Strains resistant to metronidazole, clarithromycin, amoxicillin, and tetracycline have been described, with the latter two being uncommon. Antibiotic-resistant strains are the most common cause for treatment failure in compliant patients. Unfortunately, in vitro resistance does not predict outcome in patients. Culture and sensitivity testing of H. pylori is not performed routinely. Although resistance to metronidazole has been found in as many as 30% of isolates in North America and 80% in developing countries, triple therapy is effective in eradicating the organism in >50% of patients infected with a resistant strain. Clarithromycin resistance is seen in 13% of individuals in the United States, with resistance to amoxicillin being <1% and resistance to both metronidazole

1	patients infected with a resistant strain. Clarithromycin resistance is seen in 13% of individuals in the United States, with resistance to amoxicillin being <1% and resistance to both metronidazole and clarithromycin in the 5% range.

1	Failure of H. pylori eradication with triple therapy in a compliant patient is usually due to infection with a resistant organism. Quadruple therapy (Table 348-4), where clarithromycin is substituted for metronidazole (or vice versa), should be the next step. The combination of pantoprazole, amoxicillin, and rifabutin for 10 days has also been used successfully (86% cure rate) in patients infected with resistant strains. Additional regimens considered for second-line therapy include levofloxacin-based triple therapy (levofloxacin, amoxicillin, PPI) for 10 days and furazolidone-based triple therapy (furazolidone, amoxicillin, PPI) for 14 days. Unfortunately, there is no universally accepted treatment regimen recommended for patients who have failed two courses of antibiotics. If eradication is still not achieved in a compliant patient, then culture and sensitivity of the organism should be considered. Additional factors that may lower eradication rates include the patient’s country of

1	is still not achieved in a compliant patient, then culture and sensitivity of the organism should be considered. Additional factors that may lower eradication rates include the patient’s country of origin (higher in Northeast Asia than other parts of Asia or Europe) and cigarette smoking. In addition, meta-analysis suggests that even the most effective regimens (quadruple therapy including PPI, bismuth, tetracycline, and metronidazole and triple therapy including PPI, clarithromycin, and amoxicillin) may have suboptimal eradication rates (<80%), thus demonstrating the need for the development of more efficacious treatments.

1	In view of the observation that 15–25% of patients treated with first-line therapy may still remain infected with the organism, new approaches to treatment have been explored. One promising approach is sequential therapy. Regimens examined consist of 5 days of amoxicillin and a PPI, followed by an additional 5 days of PPI plus tinidazole and clarithromycin or levofloxacin. One promising regimen that has the benefit of being shorter in duration, easier to take, and less expensive is 5 days of concomitant therapy (PPI twice daily, amoxicillin 1 g twice daily, levofloxacin 500 mg twice daily, and tinidazole 500 mg twice daily). Initial studies have demonstrated eradication rates of >90% with good patient tolerance. Confirmation of these findings and applicability of this approach in the United States are needed, although some experts are recommending abandoning clarithromycin-based triple therapy in the United States for the concomitant therapy or the alternative sequential therapies

1	States are needed, although some experts are recommending abandoning clarithromycin-based triple therapy in the United States for the concomitant therapy or the alternative sequential therapies highlighted above.

1	Innovative non–antibiotic-mediated approaches have been explored in an effort to improve eradication rates of H. pylori. Pretreatment of patients with N-acetylcysteine as a mucolytic agent to destroy the H. pylori biofilm and therefore impair antibiotic resistance has been examined, but more studies are needed to confirm the applicability of this approach. In vitro studies suggest that certain probiotics like Lactobacillus or its metabolites can inhibit H. pylori. Administration of probiotics has been attempted in several clinical studies in an effort to maximize antibiotic-mediated eradication with varying results. Overall, it appears that the use of certain probiotics, such as Lactobacillus spp., Saccharomyces spp., Bifidobacterium spp., and Bacillus clausii, did not alter eradication rates but importantly decreased antibiotic-associated side effects including nausea, dysgeusia, diarrhea, and abdominal discomfort/pain, resulting in enhanced tolerability of H. pylori therapies.

1	rates but importantly decreased antibiotic-associated side effects including nausea, dysgeusia, diarrhea, and abdominal discomfort/pain, resulting in enhanced tolerability of H. pylori therapies. Additional studies are needed to confirm the potential benefits of probiotics in this setting.

1	Reinfection after successful eradication of H. pylori is rare in the United States (<1% per year). If recurrent infection occurs within the first 6 months after completing therapy, the most likely explanation is recrudescence as opposed to reinfection. Medical intervention for NSAID-related mucosal injury includes treatment of an active ulcer and primary prevention of future injury. Recommendations for the treatment and primary prevention of NSAID-related mucosal injury are listed in Table 348-5. Ideally, the injurious agent should be stopped as the first step in the therapy of an active NSAID-induced ulcer. If that is possible, then treatment with one of the acid inhibitory agents (H2 blockers, PPIs) is indicated. Cessation of NSAIDs is not always possible because of the patient’s H. pylori infection Eradication if active ulcer present or there is a past history of peptic ulcer disease

1	H. pylori infection Eradication if active ulcer present or there is a past history of peptic ulcer disease Abbreviations: COX-2, isoenzyme of cyclooxygenase; NSAID, nonsteroidal anti-inflammatory drug; PPI, proton pump inhibitor. severe underlying disease. Only PPIs can heal GUs or DUs, independent of whether NSAIDs are discontinued.

1	The approach to primary prevention has included avoiding the agent, using the lowest possible dose of the agent, using NSAIDs that are theoretically less injurious, using newer topical NSAID preparations, and/or using concomitant medical therapy to prevent NSAID-induced injury. Several nonselective NSAIDs that are associated with a lower likelihood of GI toxicity include diclofenac, aceclofenac, and ibuprofen, although the beneficial effect may be eliminated if higher dosages of the agents are used. Primary prevention of NSAID-induced ulceration can be accomplished by misoprostol (200 μg qid) or a PPI. High-dose H2 blockers (famotidine, 40 mg bid) have also shown some promise in preventing endoscopically documented ulcers, although PPIs are superior. The highly selective COX-2 inhibitors, celecoxib and rofecoxib, are 100 times more selective inhibitors of COX-2 than standard NSAIDs, leading to gastric or duodenal mucosal injury that is comparable to placebo; their utilization led to

1	celecoxib and rofecoxib, are 100 times more selective inhibitors of COX-2 than standard NSAIDs, leading to gastric or duodenal mucosal injury that is comparable to placebo; their utilization led to an increase in cardiovascular events and withdrawal from the market. Additional caution was engendered when the CLASS study demonstrated that the advantage of celecoxib in preventing GI complications was offset when low-dose aspirin was used simultaneously. Therefore, gastric protection therapy is required in individuals taking COX-2 inhibitors and aspirin prophylaxis. Finally, much of the work demonstrating the benefit of COX-2 inhibitors and PPIs on GI injury has been performed in individuals of average risk; it is unclear if the same level of benefit will be achieved in high-risk patients. For example, concomitant use of warfarin and a COX-2 inhibitor was associated with rates of GI bleeding similar to those observed in patients taking nonselective NSAIDs. A combination of factors,

1	For example, concomitant use of warfarin and a COX-2 inhibitor was associated with rates of GI bleeding similar to those observed in patients taking nonselective NSAIDs. A combination of factors, including withdrawal of the majority of COX-2 inhibitors from the market, the observation that low-dose aspirin appears to diminish the beneficial effect of COX-2 selective inhibitors, and the growing use of aspirin for prophylaxis of cardiovascular events, have significantly altered the approach to gastric protective therapy during the use of NSAIDs. A set of guidelines for the approach to the use of NSAIDs was published by the American College of Gastroenterology and is shown in Table 348-6. Individuals who are not at risk for cardiovascular events, do not use aspirin, and are without risk for GI complications can receive nonselective NSAIDs without gastric protection. In those without cardiovascular risk factors but with a high potential risk (prior GI bleeding or multiple GI risk factors)

1	complications can receive nonselective NSAIDs without gastric protection. In those without cardiovascular risk factors but with a high potential risk (prior GI bleeding or multiple GI risk factors) for NSAID-induced GI toxicity, cautious use of a selective COX-2 inhibitor and co-therapy with misoprostol or high-dose PPI are recommended. Individuals at moderate GI risk without cardiac risk factors can be treated with a COX-2 inhibitor alone or with a nonselective NSAID with misoprostol or a PPI. Individuals with cardiovascular risk factors, who require low-dose aspirin and have low potential for NSAID-induced toxicity, should be considered for a non-NSAID agent or use of a traditional NSAID in combination with gastric protection, if warranted. Finally, individuals with cardiovascular and GI risks who require aspirin must be considered for non-NSAID therapy, but if that is not an option, then gastric protection with any type of NSAID must be considered. Any patient, regardless

1	Abbreviations: CV, cardiovascular; GI, gastrointestinal; NSAID, nonsteroidal anti-inflammatory drug; PPI, proton pump inhibitor. Source: Adapted from AM Fendrick: Am J Manag Care 10:740, 2004. Reproduced with permission of INTELLISPHERE, LLC via Copyright Clearance Center.

1	of risk status, who is being considered for long-term traditional NSAID therapy, should also be considered for H. pylori testing and treatment if positive. Assuring the use of GI protective agents with NSAIDs is difficult, even in high-risk patients. This is in part due to underprescribing of the appropriate protective agent; other times the difficulty is related to patient compliance. The latter may be due to patients forgetting to take multiple pills or preferring not to take the extra pill, especially if they have no GI symptoms. Several NSAID gastroprotective-containing combination pills are now commercially available, including double-dose famotidine with ibuprofen, diclofenac with misoprostol, and naproxen with esomeprazole. Although initial studies suggested improved compliance and a cost advantage when taking these combination drugs, their clinical benefit over the use of separate pills has not been established. Efforts continue toward developing safer NSAIDs, including

1	and a cost advantage when taking these combination drugs, their clinical benefit over the use of separate pills has not been established. Efforts continue toward developing safer NSAIDs, including NO–releasing NSAIDs, hydrogen sulfide–releasing NSAIDs, dual COX/5-LOX inhibitors, NSAID prodrugs, or agents that can effectively sequester unbound NSAIDs without interfering with their efficacy.

1	APPROACH AND THERAPY: SUMMARY Controversy continues regarding the best approach to the patient who presents with dyspepsia (Chap. 54). The discovery of H. pylori and its role in pathogenesis of ulcers has added a new variable to the equation. Previously, if a patient <50 years of age presented with dyspepsia and without alarming signs or symptoms suggestive of an ulcer complication or malignancy, an empirical therapeutic trial with acid suppression was commonly recommended. Although this approach is practiced by some today, an approach presently gaining approval for the treatment of patients with dyspepsia is outlined in Fig. 348-12. The referral to a gastroenterologist is for the potential need of endoscopy and subsequent evaluation and treatment if the endoscopy is negative.

1	Once an ulcer (GU or DU) is documented, the main issue at stake is whether H. pylori or an NSAID is involved. With H. pylori present, independent of the NSAID status, triple therapy is recommended for 14 days, followed by continued acid-suppressing drugs (H2 receptor antagonist or PPIs) for a total of 4–6 weeks. Selection of patients for documentation of H. pylori eradication (organisms gone at least 4 weeks after completing antibiotics) is an area of some debate. The test of choice for documenting eradication is the laboratory-based validated monoclonal stool antigen test or a urea breath test (UBT). The patient must be off antisecretory agents when being tested for eradication of H. pylori with UBT or stool antigen. Serologic testing is not useful for the purpose of documenting eradication because antibody titers fall slowly and often do not become undetectable. Two approaches toward documentation of eradication exist: (1) Test for eradication only in individuals with a complicated

1	because antibody titers fall slowly and often do not become undetectable. Two approaches toward documentation of eradication exist: (1) Test for eradication only in individuals with a complicated course or in individuals who are frail or with multisystem disease who would do poorly with an ulcer recurrence, and (2) test all patients for >40 y/o Alarm Symptoms New-Onset Dyspepsia – Noninvasive Hp testing Confirm eradication UBT + Anti-Hp therapy Empiric trial H2 blocker 4 weeks after therapy or – Refer to gastroenterologist + Symptoms remain or recur Exclude by history GERD, biliary pain, IBS, aerophagia, medication-related

1	FIGURE 348-12 Overview of new-onset dyspepsia. GERD, gastroesophageal reflux disease; Hp, Helicobacter pylori; IBS, irritable bowel syndrome; UBT, urea breath test. (Adapted from BS Anand and DY Graham: Endoscopy 31:215, 1999.) successful eradication. Some recommend that patients with complicated ulcer disease, or who are frail, should be treated with longterm acid suppression, thus making documentation of H. pylori eradication a moot point. In view of this discrepancy in practice, it would be best to discuss with the patient the different options available.

1	Several issues differentiate the approach to a GU versus a DU. GUs, especially of the body and fundus, have the potential of being malignant. Multiple biopsies of a GU should be taken initially; even if these are negative for neoplasm, repeat endoscopy to document healing at 8–12 weeks should be performed, with biopsy if the ulcer is still present. About 70% of GUs eventually found to be malignant undergo significant (usually incomplete) healing. Repeat endoscopy is warranted in patients with DU if symptoms persist despite medical therapy or a complication is suspected.

1	The majority (>90%) of GUs and DUs heal with the conventional therapy outlined above. A GU that fails to heal after 12 weeks and a DU that does not heal after 8 weeks of therapy should be considered refractory. Once poor compliance and persistent H. pylori infection have been excluded, NSAID use, either inadvertent or surreptitious, must be excluded. In addition, cigarette smoking must be eliminated. For a GU, malignancy must be meticulously excluded. Next, consideration should be given to a gastric acid hypersecretory state such as ZES (see “Zollinger-Ellison Syndrome,” below) or the idiopathic form, which can be excluded with gastric acid analysis. Although a subset of patients have gastric acid hypersecretion of unclear etiology as a contributing factor to refractory ulcers, ZES should be excluded with a fasting gastrin or secretin stimulation test (see below). More than 90% of refractory ulcers (either DUs or GUs) heal after 8 weeks of treatment with higher doses of PPI

1	ZES should be excluded with a fasting gastrin or secretin stimulation test (see below). More than 90% of refractory ulcers (either DUs or GUs) heal after 8 weeks of treatment with higher doses of PPI (omeprazole, 40 mg/d; lansoprazole 30–60 mg/d). This higher dose is also effective in maintaining remission. Surgical intervention may be a consideration at this point; however, other rare causes of refractory ulcers must be excluded before recommending surgery. Rare etiologies of refractory ulcers that may be diagnosed by gastric or duodenal biopsies include ischemia, Crohn’s disease, amyloidosis, sarcoidosis, lymphoma, eosinophilic gastroenteritis, or infection (cytomegalovirus [CMV], tuberculosis, or syphilis).

1	Surgical intervention in PUD can be viewed as being either elective, for treatment of medically refractory disease, or as urgent/ emergent, for the treatment of an ulcer-related complication. The development of pharmacologic and endoscopic approaches for the treatment of peptic disease and its complications has led to a substantial decrease in the number of operations needed for this disorder with a drop of over 90% for elective ulcer surgery over the last four decades. Refractory ulcers are an exceedingly rare occurrence. Surgery is more often required for treatment of an ulcer-related complication.

1	Hemorrhage is the most common ulcer-related complication, occurring in ~15–25% of patients. Bleeding may occur in any age group but is most often seen in older patients (sixth decade or beyond). The majority of patients stop bleeding spontaneously, but endoscopic therapy (Chap. 345) is necessary in some. Parenterally and orally administered PPIs also decrease ulcer rebleeding in patients who have undergone endoscopic therapy. Patients unresponsive or refractory to endoscopic intervention will require surgery (~5% of transfusion-requiring patients). Free peritoneal perforation occurs in ~2–3% of DU patients. As in the case of bleeding, up to 10% of these patients will not have antecedent ulcer symptoms. Concomitant bleeding may occur in up to 10% of patients with perforation, with mortality being increased substantially. Peptic ulcer can also penetrate into adjacent organs, especially with a posterior DU, which can penetrate into the pancreas, colon, liver, or biliary tree.

1	Pyloric channel ulcers or DUs can lead to gastric outlet obstruction in ~2–3% of patients. This can result from chronic scarring or from impaired motility due to inflammation and/or edema with pylorospasm. Patients may present with early satiety, nausea, vomiting of undigested food, and weight loss. Conservative management with nasogastric suction, intravenous hydration/nutrition, and antisecretory agents is indicated for 7–10 days with the hope that a functional obstruction will reverse. If a mechanical obstruction persists, endoscopic intervention with balloon dilation may be effective. Surgery should be considered if all else fails.

1	Surgical treatment was originally designed to decrease gastric acid secretion. Operations most commonly performed include (1) vagotomy and drainage (by pyloroplasty, gastroduodenostomy, or gastrojejunostomy), (2) highly selective vagotomy (which does not require a drainage procedure), and (3) vagotomy with antrectomy. The specific procedure performed is dictated by the underlying circumstances: elective versus emergency, the degree and extent of duodenal ulceration, the etiology of the ulcer (H. pylori, NSAIDs, malignancy), and the expertise of the surgeon. Moreover, the trend has been toward a dramatic decrease in the need for surgery for treatment of refractory PUD, and when needed, minimally invasive and anatomy-preserving operations are preferred.

1	Vagotomy is a component of each of these procedures and is aimed at decreasing acid secretion through ablating cholinergic input to the stomach. Unfortunately, both truncal and selective vagotomy (preserves the celiac and hepatic branches) result in gastric atony despite successful reduction of both basal acid output (BAO; decreased by 85%) and maximal acid output (MAO; decreased by 50%). Drainage through pyloroplasty or gastroduodenostomy is required in an effort to compensate for the vagotomy-induced gastric motility disorder. This procedure has an intermediate complication rate and a 10% ulcer recurrence rate. To minimize gastric dysmotility, highly selective vagotomy (also known as parietal cell, super-selective, or proximal vagotomy) was developed. Only the vagal fibers innervating the portion of the stomach that contains parietal cells is transected, thus leaving fibers important for regulating gastric motility intact. Although this procedure leads to an immediate decrease in

1	the portion of the stomach that contains parietal cells is transected, thus leaving fibers important for regulating gastric motility intact. Although this procedure leads to an immediate decrease in both BAO and stimulated acid output, acid secretion recovers over time. By the end of the first postoperative year, basal and stimulated acid output are ~30 and 50%, respectively, of preoperative levels. Ulcer recurrence rates are higher 1925 with highly selective vagotomy (≥10%), although the overall complication rates are the lowest of the three procedures.

1	The procedure that provides the lowest rates of ulcer recurrence (1%) but has the highest complication rate is vagotomy (truncal or selective) in combination with antrectomy. Antrectomy is aimed at eliminating an additional stimulant of gastric acid secretion, gastrin. Two principal types of reanastomoses are used after antrectomy: gastroduodenostomy (Billroth I) or gastrojejunostomy (Billroth II) (Fig. 348-13). Although Billroth I is often preferred over II, severe duodenal inflammation or scarring may preclude its performance. Prospective, randomized studies confirm that partial gastrectomy followed by Roux-en-Y reconstruction leads to a significantly better clinical, endoscopic, and histologic outcome than Billroth II reconstruction.

1	Of these procedures, highly selective vagotomy may be the one of choice in the elective setting, except in situations where ulcer recurrence rates are high (prepyloric ulcers and those refractory to medical therapy). Selection of vagotomy and antrectomy may be more appropriate in these circumstances. These procedures have been traditionally performed by standard laparotomy. The advent of laparoscopic surgery has led several surgical teams to successfully perform highly selective vagotomy, truncal vagotomy/pyloroplasty, and truncal vagotomy/antrectomy through this approach. An increase in the number of laparoscopic procedures for treatment of PUD has occurred. Laparoscopic repair of perforated peptic ulcers is safe, feasible for the experienced surgeon and is associated with decreased postoperative pain, although it does take longer than an open approach. Moreover, no difference between the two approaches is noted in postoperative complications or length of hospital stay.

1	Specific Operations for Gastric Ulcers The location and the presence of a concomitant DU dictate the operative procedure performed for a GU. Antrectomy (including the ulcer) with a Billroth I FIGURE 348-13 Schematic representation of Billroth I and II procedures. 1926 anastomosis is the treatment of choice for an antral ulcer. Vagotomy is performed only if a DU is present. Although ulcer excision with vagotomy and drainage procedure has been proposed, the higher incidence of ulcer recurrence makes this a less desirable approach. Ulcers located near the esophagogastric junction may require a more radical approach, a subtotal gastrectomy with a Roux-en-Y esophagogastrojejunostomy (Csendes’ procedure). A less aggressive approach, including antrectomy, intraoperative ulcer biopsy, and vagotomy (Kelling-Madlener procedure), may be indicated in fragile patients with a high GU. Ulcer recurrence approaches 30% with this procedure.

1	Surgery-Related Complications Complications seen after surgery for PUD are related primarily to the extent of the anatomic modification performed. Minimal alteration (highly selective vagotomy) is associated with higher rates of ulcer recurrence and less GI disturbance. More aggressive surgical procedures have a lower rate of ulcer recurrence but a greater incidence of GI dysfunction. Overall, morbidity and mortality related to these procedures are quite low. Morbidity associated with vagotomy and antrectomy or pyloroplasty is ≤5%, with mortality ~1%. Highly selective vagotomy has lower morbidity and mortality rates of 1 and 0.3%, respectively. In addition to the potential early consequences of any intraabdominal procedure (bleeding, infection, thromboembolism), gastroparesis, duodenal stump leak, and efferent loop obstruction can be observed.

1	recurrent ulceration The risk of ulcer recurrence is directly related to the procedure performed. Ulcers that recur after partial gastric resection tend to develop at the anastomosis (stomal or marginal ulcer). Epigastric abdominal pain is the most frequent presenting complaint (>90%). Severity and duration of pain tend to be more progressive than observed with DUs before surgery.

1	Ulcers may recur for several reasons, including incomplete vagotomy, inadequate drainage, retained antrum, and, less likely, persistent or recurrent H. pylori infection. ZES should have been excluded preoperatively. Surreptitious use of NSAIDs is an important reason for recurrent ulcers after surgery, especially if the initial procedure was done for an NSAID-induced ulcer. Once H. pylori and NSAIDs have been excluded as etiologic factors, the question of incomplete vagotomy or retained gastric antrum should be explored. For the latter, fasting plasma gastrin levels should be determined. If elevated, retained antrum or ZES (see below) should be considered. Incomplete vagotomy can be ruled out by gastric acid analysis coupled with sham feeding. In this test, gastric acid output is measured while the patient sees, smells, and chews a meal (without swallowing). The cephalic phase of gastric secretion, which is mediated by the vagus, is being assessed with this study. An increase in

1	while the patient sees, smells, and chews a meal (without swallowing). The cephalic phase of gastric secretion, which is mediated by the vagus, is being assessed with this study. An increase in gastric acid output in response to sham feeding is evidence that the vagus nerve is intact. A rise in serum pancreatic polypeptide >50% within 30 min of sham feeding is also suggestive of an intact vagus nerve.

1	Medical therapy with H2 blockers will heal postoperative ulceration in 70–90% of patients. The efficacy of PPIs has not been fully assessed in this group, but one may anticipate greater rates of ulcer healing compared to those obtained with H2 blockers. Repeat operation (complete vagotomy, partial gastrectomy) may be required in a small subgroup of patients who have not responded to aggressive medical management.

1	afferent loop synDromes Although rarely seen today as a result of the decrease in the performance of Billroth II anastomosis, two types of afferent loop syndrome can occur in patients who have undergone this type of partial gastric resection. The more common of the two is bacterial overgrowth in the afferent limb secondary to stasis. Patients may experience postprandial abdominal pain, bloating, and diarrhea with concomitant malabsorption of fats and vitamin B12. Cases refractory to antibiotics may require surgical revision of the loop. The less common afferent loop syndrome can present with severe abdominal pain and bloating that occur 20–60 min after meals. Pain is often followed by nausea and vomiting of bile-containing material. The pain and bloating may improve after emesis. The cause of this clinical picture is theorized to be incomplete drainage of bile and pancreatic secretions from an afferent loop that is partially obstructed. Cases refractory to dietary measures may need

1	of this clinical picture is theorized to be incomplete drainage of bile and pancreatic secretions from an afferent loop that is partially obstructed. Cases refractory to dietary measures may need surgical revision or conversion of the Billroth II anastomosis to a Roux-en-Y gastrojejunostomy.

1	DumpinG synDrome Dumping syndrome consists of a series of vasomotor and GI signs and symptoms and occurs in patients who have undergone vagotomy and drainage (especially Billroth procedures). Two phases of dumping, early and late, can occur. Early dumping takes place 15–30 min after meals and consists of crampy abdominal discomfort, nausea, diarrhea, belching, tachycardia, palpitations, diaphoresis, light-headedness, and, rarely, syncope. These signs and symptoms arise from the rapid emptying of hyperosmolar gastric contents into the small intestine, resulting in a fluid shift into the gut lumen with plasma volume contraction and acute intestinal distention. Release of vasoactive GI hormones (vasoactive intestinal polypeptide, neurotensin, motilin) is also theorized to play a role in early dumping.

1	The late phase of dumping typically occurs 90 min to 3 h after meals. Vasomotor symptoms (light-headedness, diaphoresis, palpitations, tachycardia, and syncope) predominate during this phase. This component of dumping is thought to be secondary to hypoglycemia from excessive insulin release. Dumping syndrome is most noticeable after meals rich in simple carbohydrates (especially sucrose) and high osmolarity. Ingestion of large amounts of fluids may also contribute. Up to 50% of postvagotomy and drainage patients will experience dumping syndrome to some degree early on. Signs and symptoms often improve with time, but a severe protracted picture can occur in up to 1% of patients.

1	Dietary modification is the cornerstone of therapy for patients with dumping syndrome. Small, multiple (six) meals devoid of simple carbohydrates coupled with elimination of liquids during meals is important. Antidiarrheals and anticholinergic agents are complementary to diet. Guar and pectin, which increase the viscosity of intraluminal contents, may be beneficial in more symptomatic individuals. Acarbose, an α-glucosidase inhibitor that delays digestion of ingested carbohydrates, has also been shown to be beneficial in the treatment of the late phases of dumping. The somatostatin analogue octreotide has been successful in diet-refractory cases. This drug is administered subcutaneously (50 μg tid), titrated according to clinical response. A long-acting depot formulation of octreotide can be administered once every 28 days and provides symptom relief comparable to the short-acting agent. In addition, patient weight gain and quality of life appear to be superior with the long-acting

1	be administered once every 28 days and provides symptom relief comparable to the short-acting agent. In addition, patient weight gain and quality of life appear to be superior with the long-acting form.

1	postvaGotomy DiarrHea Up to 10% of patients may seek medical attention for the treatment of postvagotomy diarrhea. This complication is most commonly observed after truncal vagotomy, which is rarely performed today. Patients may complain of intermittent diarrhea that occurs typically 1–2 h after meals. Occasionally the symptoms may be severe and relentless. This is due to a motility disorder from interruption of the vagal fibers supplying the luminal gut. Other contributing factors may include decreased absorption of nutrients (see below), increased excretion of bile acids, and release of luminal factors that promote secretion. Diphenoxylate or loperamide is often useful in symptom control. The bile salt–binding agent cholestyramine may be helpful in severe cases. Surgical reversal of a 10-cm segment of jejunum may yield a substantial improvement in bowel frequency in a subset of patients.

1	bile reflux GastropatHy A subset of post–partial gastrectomy patients who present with abdominal pain, early satiety, nausea, and vomiting will have mucosal erythema of the gastric remnant as the only finding. Histologic examination of the gastric mucosa reveals minimal inflammation but the presence of epithelial cell injury. This clinical picture is categorized as bile or alkaline reflux gastropathy/gastritis. Although reflux of bile is implicated as the reason for this disorder, the mechanism is unknown. Prokinetic agents, cholestyramine, and sucralfate have been somewhat effective treatments. Severe refractory symptoms may require using either nuclear scanning with 99mTc-HIDA to document reflux or an alkaline challenge test, where 0.1 N NaOH is infused into the stomach in an effort to reproduce the patient’s symptoms. Surgical diversion of pancreaticobiliary secretions away from the gastric remnant with a Roux-en-Y gastrojejunostomy consisting of a long (50–60 cm) Roux limb has

1	to reproduce the patient’s symptoms. Surgical diversion of pancreaticobiliary secretions away from the gastric remnant with a Roux-en-Y gastrojejunostomy consisting of a long (50–60 cm) Roux limb has been used in severe cases. Bilious vomiting improves, but early satiety and bloating may persist in up to 50% of patients.

1	malDiGestion anD malabsorption Weight loss can be observed in up to 60% of patients after partial gastric resection. Patients can experience a 10% loss of body weight, which stabilizes 3 months postoperatively. A significant component of this weight reduction is due to decreased oral intake. However, mild steatorrhea can also develop. Reasons for maldigestion/malabsorption include decreased gastric acid production, rapid gastric emptying, decreased food dispersion in the stomach, reduced luminal bile concentration, reduced pancreatic secretory response to feeding, and rapid intestinal transit. Decreased serum vitamin B12 levels can be observed after partial gastrectomy. This is usually not due to deficiency of IF, since a minimal amount of parietal cells (source of IF) are removed during antrectomy. Reduced vitamin B12 may be due to competition for the vitamin by bacterial overgrowth or inability to split the vitamin from its protein-bound source due to hypochlorhydria.

1	Iron-deficiency anemia may be a consequence of impaired absorption of dietary iron in patients with a Billroth II gastrojejunostomy. Absorption of iron salts is normal in these individuals; thus, a favorable response to oral iron supplementation can be anticipated. Folate deficiency with concomitant anemia can also develop in these patients. This deficiency may be secondary to decreased absorption or diminished oral intake.

1	Malabsorption of vitamin D and calcium resulting in osteoporosis and osteomalacia is common after partial gastrectomy and gastrojejunostomy (Billroth II). Osteomalacia can occur as a late complication in up to 25% of post–partial gastrectomy patients. Bone fractures occur twice as commonly in men after gastric surgery as in a control population. It may take years before x-ray findings demonstrate diminished bone density. Elevated alkaline phosphatase, reduced serum calcium, bone pain, and pathologic fractures may be seen in patients with osteomalacia. The high incidence of these abnormalities in this subgroup of patients justifies treating them with vitamin D and calcium supplementation indefinitely. Therapy is especially important in females. Copper deficiency has also been reported in patients undergoing surgeries that bypass the duodenum, where copper is primarily absorbed. Patients may present with a rare syndrome that includes ataxia, myelopathy, and peripheral neuropathy.

1	Gastric aDenocarcinoma The incidence of adenocarcinoma in the gastric stump is increased 15 years after resection. Some have reported a fourto fivefold increase in gastric cancer 20–25 years after resection. The pathogenesis is unclear but may involve alkaline reflux, bacterial proliferation, or hypochlorhydria. The role of endoscopic screening is not clear, and most guidelines do not support its use. aDDitional complications Reflux esophagitis and a higher incidence of gallstones and cholecystitis have been reported to patients undergoing subtotal gastrectomy. The latter is thought to be due to decreased gallbladder contractility associated with vagotomy and bypass of the duodenum, leading to decreased postprandial release of cholecystokinin.

1	Severe peptic ulcer diathesis secondary to gastric acid hypersecretion due to unregulated gastrin release from a non-β cell endocrine tumor (gastrinoma) defines the components of ZES. Initially, ZES was typified by aggressive and refractory ulceration in which total gastrec-1927 tomy provided the only chance for enhancing survival. Today it can be cured by surgical resection in up to 40% of patients.

1	Epidemiology The incidence of ZES varies from 0.1–1% of individuals presenting with PUD. Males are more commonly affected than females, and the majority of patients are diagnosed between ages 30 and 50. Gastrinomas are classified into sporadic tumors (more common) and those associated with multiple endocrine neoplasia (MEN) type 1 (see below). The widespread availability and use of PPIs has led to a decreased patient referral for gastrinoma evaluation, delay in diagnosis, and an increase in false-positive diagnoses of ZES. In fact, diagnosis may be delayed for 6 or more years after symptoms consistent with ZES are displayed.

1	Pathophysiology Hypergastrinemia originating from an autonomous neoplasm is the driving force responsible for the clinical manifestations in ZES. Gastrin stimulates acid secretion through gastrin receptors on parietal cells and by inducing histamine release from ECL cells. Gastrin also has a trophic action on gastric epithelial cells. Longstanding hypergastrinemia leads to markedly increased gastric acid secretion through both parietal cell stimulation and increased parietal cell mass. The increased gastric acid output leads to peptic ulcer diathesis, erosive esophagitis, and diarrhea.

1	Tumor Distribution Although early studies suggested that the vast majority of gastrinomas occurred within the pancreas, a significant number of these lesions are extrapancreatic. Over 80% of these tumors are found within the hypothetical gastrinoma triangle (confluence of the cystic and common bile ducts superiorly, junction of the second and third portions of the duodenum inferiorly, and junction of the neck and body of the pancreas medially). Duodenal tumors constitute the most common nonpancreatic lesion; between 50 and 75% of gastrinomas are found here. Duodenal tumors are smaller, slower growing, and less likely to metastasize than pancreatic lesions. Less common extrapancreatic sites include stomach, bones, ovaries, heart, liver, and lymph nodes. More than 60% of tumors are considered malignant, with up to 30–50% of patients having multiple lesions or metastatic disease at presentation. Histologically, gastrin-producing cells appear well-differentiated, expressing markers

1	malignant, with up to 30–50% of patients having multiple lesions or metastatic disease at presentation. Histologically, gastrin-producing cells appear well-differentiated, expressing markers typically found in endocrine neoplasms (chromogranin, neuron-specific enolase).

1	Clinical Manifestations Gastric acid hypersecretion is responsible for the signs and symptoms observed in patients with ZES. Peptic ulcer is the most common clinical manifestation, occurring in >90% of gastrinoma patients. Initial presentation and ulcer location (duodenal bulb) may be indistinguishable from common PUD. Clinical situations that should create suspicion of gastrinoma are ulcers in unusual locations (second part of the duodenum and beyond), ulcers refractory to standard medical therapy, ulcer recurrence after acid-reducing surgery, ulcers presenting with frank complications (bleeding, obstruction, and perforation), or ulcers in the absence of H. pylori or NSAID ingestion. Symptoms of esophageal origin are present in up to two-thirds of patients with ZES, with a spectrum ranging from mild esophagitis to frank ulceration with stricture and Barrett’s mucosa.

1	Diarrhea, the next most common clinical manifestation, is found in up to 50% of patients. Although diarrhea often occurs concomitantly with acid peptic disease, it may also occur independent of an ulcer. Etiology of the diarrhea is multifactorial, resulting from marked volume overload to the small bowel, pancreatic enzyme inactivation by acid, and damage of the intestinal epithelial surface by acid. The epithelial damage can lead to a mild degree of maldigestion and malabsorption of nutrients. The diarrhea may also have a secretory component due to the direct stimulatory effect of gastrin on enterocytes or the co-secretion of additional hormones from the tumor such as vasoactive intestinal peptide.

1	Gastrinomas can develop in the presence of MEN 1 syndrome (Chaps. 113 and 408) in ~25% of patients. This autosomal dominant disorder involves primarily three organ sites: the parathyroid glands (80–90%), pancreas (40–80%), and pituitary gland (30–60%). The 1928 syndrome is caused by inactivating mutations of the MEN1 tumor suppressor gene found on the long arm of chromosome 11q13. The gene encodes for Menin, which has an important role in DNA replication and transcriptional regulation. A genetic diagnosis is obtained by sequencing of the MEN1 gene, which can reveal mutations in 70–90% of typical MEN 1 cases. A family may have an unknown mutation, making a genetic diagnosis impossible, and therefore certain individuals will require a clinical diagnosis, which is determined by whether a patient has tumors in two of the three endocrine organs (parathyroid, pancreas/duodenum, or pituitary) or has a family history of MEN 1 and one of the endocrine organ tumors. In view of the stimulatory

1	has tumors in two of the three endocrine organs (parathyroid, pancreas/duodenum, or pituitary) or has a family history of MEN 1 and one of the endocrine organ tumors. In view of the stimulatory effect of calcium on gastric secretion, the hyperparathyroidism and hypercalcemia seen in MEN 1 patients may have a direct effect on ulcer disease. Resolution of hypercalcemia by parathyroidectomy reduces gastrin and gastric acid output in gastrinoma patients. An additional distinguishing feature in ZES patients with MEN 1 is the higher incidence of gastric carcinoid tumor development (as compared to patients with sporadic gastrinomas). ZES presents and is diagnosed earlier in MEN 1 patients, and they have a more indolent course as compared to patients with sporadic gastrinoma. Gastrinomas tend to be smaller, multiple, and located in the duodenal wall more often than is seen in patients with sporadic ZES. Establishing the diagnosis of MEN 1 is critical in order to provide genetic counseling to

1	be smaller, multiple, and located in the duodenal wall more often than is seen in patients with sporadic ZES. Establishing the diagnosis of MEN 1 is critical in order to provide genetic counseling to the patient and his or her family and also to determine the recommended surgical approach.

1	Diagnosis Biochemical measurements of gastrin and acid secretion in patients suspected of ZES play an important role is establishing this rare diagnosis. Often, patients suspected of having ZES will be treated with a PPI in an effort to ameliorate symptoms and decrease the likelihood of possible acid-related complications. The presence of the PPI, which will lower acid secretion and potentially elevate fasting gastrin levels in normal individuals, will make the diagnostic approach in these individuals somewhat difficult. Significant morbidity related to peptic diathesis has been described when stopping PPIs in gastrinoma patients; therefore, a systematic approach in stopping these agents is warranted (see below). The first step in the evaluation of a patient suspected of having ZES is to obtain a fasting gastrin level. A list of clinical scenarios that should arouse suspicion regarding this diagnosis is shown in Table 348-7. Fasting gastrin levels obtained using a dependable assay are

1	a fasting gastrin level. A list of clinical scenarios that should arouse suspicion regarding this diagnosis is shown in Table 348-7. Fasting gastrin levels obtained using a dependable assay are usually <150 pg/mL. A normal fasting gastrin, on two separate occasions, especially if the patient is on a PPI, virtually excludes this diagnosis. Virtually all gastrinoma patients will have a gastrin level >150–200 pg/mL. Measurement of fasting gastrin should be repeated to confirm the clinical suspicion. Some of the commercial biochemical assays used for measuring serum gastrin may be inaccurate. Variable specificity of the antibodies used have led to both false-positive and false-negative fasting gastrin levels, placing in jeopardy the ability to make an accurate diagnosis of ZES.

1	Multiple processes can lead to an elevated fasting gastrin level, the most frequent of which are gastric hypochlorhydria and achlorhydria, with or without pernicious anemia. Gastric acid induces feedback inhibition of gastrin release. A decrease in acid production will Ulcers in unusual locations; associated with severe esophagitis; resistant to therapy with frequent recurrences; in the absence of nonsteroidal anti-inflammatory drug ingestion or H. pylori infection

1	Family history of pancreatic islet, pituitary, or parathyroid tumor subsequently lead to failure of the feedback inhibitory pathway, resulting in net hypergastrinemia. Gastrin levels will thus be high in patients using antisecretory agents for the treatment of acid peptic disorders and dyspepsia. H. pylori infection can also cause hypergastrinemia. Additional causes of elevated gastrin include retained gastric antrum; G cell hyperplasia; gastric outlet obstruction; renal insufficiency; massive small-bowel obstruction; and conditions such as rheumatoid arthritis, vitiligo, diabetes mellitus, and pheochromocytoma. Although a fasting gastrin >10 times normal is highly suggestive of ZES, two-thirds of patients will have fasting gastrin levels that overlap with levels found in the more common disorders outlined above, especially if a PPI is being taken by the patient. The effect of the PPI on gastrin levels and acid secretion will linger several days after stopping the PPI; therefore, it

1	disorders outlined above, especially if a PPI is being taken by the patient. The effect of the PPI on gastrin levels and acid secretion will linger several days after stopping the PPI; therefore, it should be stopped for a minimum of 7 days before testing. During this period, the patient should be placed on a histamine H2 antagonist, such as famotidine, twice to three times per day. Although this type of agent has a short-term effect on gastrin and acid secretion, it needs to be stopped 24 h before repeating fasting gastrin levels or performing some the tests highlighted below. The patient may take antacids for the final day, stopping them approximately 12 h before testing is performed. Heightened awareness of complications related to gastric acid hypersecretion during the period of PPI cessation is critical.

1	The next step in establishing a biochemical diagnosis of gastrinoma is to assess acid secretion. Nothing further needs to be done if decreased acid output in the absence of a PPI is observed. A pH can be measured on gastric fluid obtained either during endoscopy or through nasogastric aspiration; a pH <3 is suggestive of a gastrinoma, but a pH >3 is not helpful in excluding the diagnosis. In those situations where the pH is >3, formal gastric acid analysis should be performed if available. Normal BAO in nongastric surgery patients is typically <5 meq/h. A BAO >15 meq/h in the presence of hypergastrinemia is considered pathognomonic of ZES, but up to 12% of patients with common PUD may have elevated BAO to a lesser degree that can overlap with levels seen in ZES patients. In an effort to improve the sensitivity and specificity of gastric secretory studies, a BAO/MAO ratio was established using pentagastrin infusion as a way to maximally stimulate acid production, with a BAO/ MAO ratio

1	the sensitivity and specificity of gastric secretory studies, a BAO/MAO ratio was established using pentagastrin infusion as a way to maximally stimulate acid production, with a BAO/ MAO ratio >0.6 being highly suggestive of ZES. Pentagastrin is no longer available in the United States, making measurement of MAO virtually impossible. An endoscopic method for measuring gastric acid output has been developed but requires further validation.

1	Gastrin provocative tests have been developed in an effort to differentiate between the causes of hypergastrinemia and are especially helpful in patients with indeterminate acid secretory studies. The tests are the secretin stimulation test and the calcium infusion study. The most sensitive and specific gastrin provocative test for the diagnosis of gastrinoma is the secretin study. An increase in gastrin of ≥120 pg within 15 min of secretin injection has a sensitivity and specificity of >90% for ZES. PPI-induced hypochlorhydria or achlorhydria may lead to a false-positive secretin test; thus, this agent must be stopped for 1 week before testing.

1	The calcium infusion study is less sensitive and specific than the secretin test, which, coupled with it being a more cumbersome study with greater potential for adverse effects, relegates it to rare utilization in the cases where the patient’s clinical characteristics are highly suggestive of ZES but the secretin stimulation is inconclusive.

1	Tumor Localization Once the biochemical diagnosis of gastrinoma has been confirmed, the tumor must be located. Multiple imaging studies have been used in an effort to enhance tumor localization (Table 348-8). The broad range of sensitivity is due to the variable success rates achieved by the different investigative groups. Endoscopic ultrasound (EUS) permits imaging of the pancreas with a high degree of resolution (<5 mm). This modality is particularly helpful in excluding small neoplasms within the pancreas and in assessing the presence of surrounding lymph nodes and vascular involvement, but it is not very sensitive for finding duodenal lesions. Several types of endocrine tumors express cell-surface receptors for somatostatin. This permits the localization of gastrinomas by measuring the uptake of the stable somatostatin analogue111 In-pentreotide (OctreoScan) with sensitivity and specificity rates of >85%. Sensitivity, %

1	Sensitivity, % Abbreviations: CT, computed tomography; EUS, endoscopic ultrasonography; MRI, magnetic resonance imaging; N/A, not applicable; OctreoScan, imaging with 111In-pentreotide; SASI, selective arterial secretin injection.

1	Up to 50% of patients have metastatic disease at diagnosis. Success in controlling gastric acid hypersecretion has shifted the emphasis of therapy toward providing a surgical cure. Detecting the primary tumor and excluding metastatic disease are critical in view of this paradigm shift. Once a biochemical diagnosis has been confirmed, the patient should first undergo an abdominal computed tomography (CT) scan, magnetic resonance imaging (MRI), or OctreoScan (depending on availability) to exclude metastatic disease. In addition, the positron emitter 68Ga has been used to label somatostatin analogues for positron emission tomography (PET) with some success. In addition, hybrid scanners combining CT scan with PET scan are also available in certain specialized centers. Once metastatic disease has been excluded, an experienced endocrine surgeon may opt for exploratory laparotomy with intraoperative ultrasound or transillumination. In other centers, careful examination of the peripancreatic

1	has been excluded, an experienced endocrine surgeon may opt for exploratory laparotomy with intraoperative ultrasound or transillumination. In other centers, careful examination of the peripancreatic area with EUS, accompanied by endoscopic exploration of the duodenum for primary tumors, will be performed before surgery. Selective arterial secretin injection may be a useful adjuvant for localizing tumors in a subset of patients. The extent of the diagnostic and surgical approach must be carefully balanced with the patient’s overall physiologic condition and the natural history of a slow-growing gastrinoma.

1	Treatment of functional endocrine tumors is directed at ameliorating the signs and symptoms related to hormone overproduction, curative resection of the neoplasm, and attempts to control tumor growth in metastatic disease.

1	PPIs are the treatment of choice and have decreased the need for total gastrectomy. Initial PPI doses tend to be higher than those used for treatment of GERD or PUD. The initial dose of omeprazole, lansoprazole, rabeprazole, or esomeprazole should be in the range of 60 mg in divided doses in a 24-h period. Dosing can be adjusted to achieve a BAO <10 meq/h (at the drug trough) in surgery-naive patients and to <5 meq/h in individuals who have previously undergone an acid-reducing operation. Although the somatostatin analogue has inhibitory effects on gastrin release from receptor-bearing tumors and inhibits gastric acid secretion to some extent, PPIs have the advantage of reducing parietal cell activity to a greater degree. Despite this, octreotide may be considered as adjunctive therapy to the PPI in patients with tumors that express somatostatin receptors and have peptic symptoms that are difficult to control with high-dose PPI.

1	The ultimate goal of surgery would be to provide a definitive cure. Improved understanding of tumor distribution has led to immediate cure rates as high as 60% with 10-year disease-free intervals as high as 34% in sporadic gastrinoma patients undergoing surgery. A positive outcome is highly dependent on the experience of the surgical team treating these rare tumors. Surgical therapy of gastrinoma patients with MEN 1 remains controversial because of the difficulty in rendering these patients disease-free with surgery. In contrast to the encouraging postoperative results observed in patients with spo-1929 radic disease, only 6% of MEN 1 patients are disease free 5 years after an operation. Moreover, in contrast to patients with sporadic ZES, the clinical course of MEN 1 patients is benign and rarely leads to disease-related mortality, recommending that early surgery be deferred. Some groups suggest surgery only if a clearly identifiable, nonmetastatic lesion is documented by structural

1	rarely leads to disease-related mortality, recommending that early surgery be deferred. Some groups suggest surgery only if a clearly identifiable, nonmetastatic lesion is documented by structural studies. Others advocate a more aggressive approach, where all patients free of hepatic metastasis are explored and all detected tumors in the duodenum are resected; this is followed by enucleation of lesions in the pan creatic head, with a distal pancreatectomy to follow. The outcome of the two approaches has not been clearly defined. Laparoscopic surgical interventions may provide attractive approaches in the future but currently seem to be of some limited benefit in patients with gastrinoma because a significant percentage of the tumors may be extrapancreatic and difficult to localize with a laparoscopic approach. Finally, patients selected for surgery should be individuals whose health status would lead them to tolerate a more aggressive operation and obtain the long-term benefits from

1	laparoscopic approach. Finally, patients selected for surgery should be individuals whose health status would lead them to tolerate a more aggressive operation and obtain the long-term benefits from such aggressive surgery, which are often witnessed after 10 years.

1	Therapy of metastatic endocrine tumors in general remains suboptimal; gastrinomas are no exception. In light of the observation that in many instances tumor growth is indolent and that many individuals with metastatic disease remain relatively stable for significant periods of time, many advocate not instituting systemic tumor-targeted therapy until evidence of tumor progression or refractory symptoms not controlled with PPIs are noted. Medical approaches, including biological therapy (IFN-α, long-acting somatostatin analogues, peptide receptor radionuclides), systemic chemotherapy (streptozotocin, 5-fluorouracil, and doxorubicin), and hepatic artery embolization, may lead to significant toxicity without a substantial improvement in overall survival. 111In-pentetreotide has been used in the therapy of metastatic neuroendocrine tumors; further studies are needed. Several novel therapies are being explored, including radiofrequency ablation or cryoablation of liver lesions and use of

1	the therapy of metastatic neuroendocrine tumors; further studies are needed. Several novel therapies are being explored, including radiofrequency ablation or cryoablation of liver lesions and use of agents that block the vascular endothelial growth receptor pathway (bevacizumab, sunitinib) or the mammalian target of rapamycin (Chap. 113).

1	Surgical approaches, including debulking surgery and liver transplantation for hepatic metastasis, have also produced limited benefit. The overall 5and 10-year survival rates for gastrinoma patients are 62–75% and 47–53%, respectively. Individuals with the entire tumor resected or those with a negative laparotomy have 5and 10-year survival rates >90%. Patients with incompletely resected tumors have 5and 10-year survival rates of 43% and 25%, respectively. Patients with hepatic metastasis have <20% survival at 5 years. Favorable prognostic indicators include primary duodenal wall tumors, isolated lymph node tumor, the presence of MEN 1, and undetectable tumor upon surgical exploration. Poor outcome is seen in patients with shorter disease duration; higher gastrin levels (>10,000 pg/mL); large pancreatic primary tumors (>3 cm); metastatic disease to lymph nodes, liver, and bone; and Cushing’s syndrome. Rapid growth of hepatic metastases is also predictive of poor outcome.

1	Patients suffering from shock, sepsis, massive burns, severe trauma, or head injury can develop acute erosive gastric mucosal changes or frank ulceration with bleeding. Classified as stress-induced gastritis or ulcers, injury is most commonly observed in the acid-producing (fundus and body) portions of the stomach. The most common presentation is GI bleeding, which is usually minimal but can occasionally be life threatening. Respiratory failure requiring mechanical ventilation and underlying coagulopathy are risk factors for bleeding, which tends to occur 48–72 h after the acute injury or insult.

1	Histologically, stress injury does not contain inflammation or H. pylori; thus, “gastritis” is a misnomer. Although elevated gastric acid secretion may be noted in patients with stress ulceration after head 1930 trauma (Cushing’s ulcer) and severe burns (Curling’s ulcer), mucosal ischemia, breakdown of the normal protective barriers of the stomach, systemic release of cytokines, poor GI motility, and oxidative stress also play an important role in the pathogenesis. Acid must contribute to injury in view of the significant drop in bleeding noted when acid inhibitors are used as prophylaxis for stress gastritis. Improvement in the general management of intensive care unit patients has led to a significant decrease in the incidence of GI bleeding due to stress ulceration. The estimated decrease in bleeding is from 20–30% to <5%. This improvement has led to some debate regarding the need for prophylactic therapy. The high mortality associated with stress-induced clinically important GI

1	in bleeding is from 20–30% to <5%. This improvement has led to some debate regarding the need for prophylactic therapy. The high mortality associated with stress-induced clinically important GI bleeding (>40%) and the limited benefit of medical (endoscopic, angiographic) and surgical therapy in a patient with hemodynamically compromising bleeding associated with stress ulcer/gastritis support the use of preventive measures in high-risk patients (mechanically ventilated, coagulopathy, multiorgan failure, or severe burns). Maintenance of gastric pH >3.5 with continuous infusion of H2 blockers or liquid antacids administered every 2–3 h are viable options. Tolerance to the H2 blocker is likely to develop; thus, careful monitoring of the gastric pH and dose adjustment are important if H2 blockers are used. Sucralfate slurry (1 g every 4–6 h) has also been somewhat successful but requires a gastric tube and may lead to constipation and aluminum toxicity. Sucralfate use in endotracheal

1	blockers are used. Sucralfate slurry (1 g every 4–6 h) has also been somewhat successful but requires a gastric tube and may lead to constipation and aluminum toxicity. Sucralfate use in endotracheal intubated patients has also been associated with aspiration pneumonia. Meta-analysis comparing H2 blockers with PPIs for the prevention of stress-associated clinically important and overt GI bleeding demonstrates superiority of the latter without increasing the risk of nosocomial infections, increasing mortality, or prolonging intensive care unit length of stay. Therefore, PPIs are the treatment of choice for stress prophylaxis. Oral PPI is the best option if the patient can tolerate enteral administration. Pantoprazole is available as an intravenous formulation for individuals in whom enteral administration is not possible. If bleeding occurs despite these measures, endoscopy, intra-arterial vasopressin, and embolization are options. If all else fails, then surgery should be considered.

1	administration is not possible. If bleeding occurs despite these measures, endoscopy, intra-arterial vasopressin, and embolization are options. If all else fails, then surgery should be considered. Although vagotomy and antrectomy may be used, the better approach would be a total gastrectomy, which has an exceedingly high mortality rate in this setting.

1	The term gastritis should be reserved for histologically documented inflammation of the gastric mucosa. Gastritis is not the mucosal erythema seen during endoscopy and is not interchangeable with “dyspepsia.” The etiologic factors leading to gastritis are broad and heterogeneous. Gastritis has been classified based on time course (acute vs chronic), histologic features, and anatomic distribution or proposed pathogenic mechanism (Table 348-9). The correlation between the histologic findings of gastritis, the clinical picture of abdominal pain or dyspepsia, and endoscopic findings noted on gross inspection of the gastric mucosa is poor. Therefore, there is no typical clinical manifestation of gastritis.

1	Acute Gastritis The most common causes of acute gastritis are infectious. Acute infection with H. pylori induces gastritis. However, H. pylori acute gastritis has not been extensively studied. It is reported as presenting with sudden onset of epigastric pain, nausea, and vomiting, and limited mucosal histologic studies demonstrate a marked infiltrate of neutrophils with edema and hyperemia. If not treated, this picture will evolve into one of chronic gastritis. Hypochlorhydria lasting for up to 1 year may follow acute H. pylori infection.

1	Bacterial infection of the stomach or phlegmonous gastritis is a rare, potentially life-threatening disorder characterized by marked and diffuse acute inflammatory infiltrates of the entire gastric wall, at times accompanied by necrosis. Elderly individuals, alcoholics, and AIDS patients may be affected. Potential iatrogenic causes include polypectomy and mucosal injection with India ink. Organisms associated with this entity include streptococci, staphylococci, Escherichia coli, Proteus, and Haemophilus species. Failure of supportive measures and antibiotics may result in gastrectomy. ClASSifiCATion of GASTRiTiS I. Acute gastritis A. Acute H. pylori infection B. Other acute Infectious gastritides 1. Bacterial (other than H. pylori) 2. H. heilmannii 3. 4. 5. 6. 7. 8. II. A. Type A: Autoimmune, body-predominant B. Type B: H. pylori–related, antral-predominant C. III. Uncommon forms of gastritis A. B. C. D. E. F.

1	H. heilmannii 3. 4. 5. 6. 7. 8. II. A. Type A: Autoimmune, body-predominant B. Type B: H. pylori–related, antral-predominant C. III. Uncommon forms of gastritis A. B. C. D. E. F. Other types of infectious gastritis may occur in immunocompromised individuals such as AIDS patients. Examples include herpetic (herpes simplex) or CMV gastritis. The histologic finding of intra-nuclear inclusions would be observed in the latter.

1	Chronic Gastritis Chronic gastritis is identified histologically by an inflammatory cell infiltrate consisting primarily of lymphocytes and plasma cells, with very scant neutrophil involvement. Distribution of the inflammation may be patchy, initially involving superficial and glandular portions of the gastric mucosa. This picture may progress to more severe glandular destruction, with atrophy and metaplasia. Chronic gastritis has been classified according to histologic characteristics. These include superficial atrophic changes and gastric atrophy. The association of atrophic gastritis with the development of gastric cancer has led to the development of endoscopic and serologic markers of severity. Some of these include gross inspection and classification of mucosal abnormalities during standard endoscopy, magnification endoscopy, endoscopy with narrow band imaging and/or autofluorescence imaging, and measurement of several serum biomarkers including pepsinogen I and II levels,

1	standard endoscopy, magnification endoscopy, endoscopy with narrow band imaging and/or autofluorescence imaging, and measurement of several serum biomarkers including pepsinogen I and II levels, gastrin-17, and anti-H. pylori serologies. The clinical utility of these tools is currently being explored.

1	The early phase of chronic gastritis is superficial gastritis. The inflammatory changes are limited to the lamina propria of the surface mucosa, with edema and cellular infiltrates separating intact gastric glands. The next stage is atrophic gastritis. The inflammatory infiltrate extends deeper into the mucosa, with progressive distortion and destruction of the glands. The final stage of chronic gastritis is gastric atrophy. Glandular structures are lost, and there is a paucity of inflammatory infiltrates. Endoscopically, the mucosa may be substantially thin, permitting clear visualization of the underlying blood vessels.

1	Gastric glands may undergo morphologic transformation in chronic gastritis. Intestinal metaplasia denotes the conversion of gastric glands to a small intestinal phenotype with small-bowel mucosal glands containing goblet cells. The metaplastic changes may vary in distribution from patchy to fairly extensive gastric involvement. Intestinal metaplasia is an important predisposing factor for gastric cancer (Chap. 109). Chronic gastritis is also classified according to the predominant site of involvement. Type A refers to the body-predominant form (autoimmune), and type B is the antral-predominant form (H. pylori–related). This classification is artificial in view of the difficulty in distinguishing between these two entities. The term AB gastritis has been used to refer to a mixed antral/body picture.

1	This classification is artificial in view of the difficulty in distinguishing between these two entities. The term AB gastritis has been used to refer to a mixed antral/body picture. type a Gastritis The less common of the two forms involves primarily the fundus and body, with antral sparing. Traditionally, this form of gastritis has been associated with pernicious anemia (Chap. 128) in the presence of circulating antibodies against parietal cells and IF; thus, it is also called autoimmune gastritis. H. pylori infection can lead to a similar distribution of gastritis. The characteristics of an autoimmune picture are not always present.

1	Antibodies to parietal cells have been detected in >90% of patients with pernicious anemia and in up to 50% of patients with type A gastritis. The parietal cell antibody is directed against H+,K+-ATPase. T cells are also implicated in the injury pattern of this form of gastritis. A subset of patients infected with H. pylori develop antibodies against H+,K+-ATPase, potentially leading to the atrophic gastritis pattern seen in some patients infected with this organism. The mechanism is thought to involve molecular mimicry between H. pylori LPS and H+,K+-ATPase.

1	Parietal cell antibodies and atrophic gastritis are observed in family members of patients with pernicious anemia. These antibodies are observed in up to 20% of individuals over age 60 and in ~20% of patients with vitiligo and Addison’s disease. About one-half of patients with pernicious anemia have antibodies to thyroid antigens, and about 30% of patients with thyroid disease have circulating antiparietal cell antibodies. Anti-IF antibodies are more specific than parietal cell antibodies for type A gastritis, being present in ~40% of patients with pernicious anemia. Another parameter consistent with this form of gastritis being autoimmune in origin is the higher incidence of specific familial histocompatibility haplotypes such as HLA-B8 and HLA-DR3.

1	The parietal cell–containing gastric gland is preferentially targeted in this form of gastritis, and achlorhydria results. Parietal cells are the source of IF, the lack of which will lead to vitamin B12 deficiency and its sequelae (megaloblastic anemia, neurologic dysfunction). Gastric acid plays an important role in feedback inhibition of gastrin release from G cells. Achlorhydria, coupled with relative sparing of the antral mucosa (site of G cells), leads to hypergastrinemia. Gastrin levels can be markedly elevated (>500 pg/mL) in patients with pernicious anemia. ECL cell hyperplasia with frank development of gastric carcinoid tumors may result from gastrin trophic effects. Hypergastrinemia and achlorhydria may also be seen in nonpernicious anemia–associated type A gastritis.

1	type b Gastritis Type B, or antral-predominant, gastritis is the more common form of chronic gastritis. H. pylori infection is the cause of this entity. Although described as “antral-predominant,” this is likely a misnomer in view of studies documenting the progression of the inflammatory process toward the body and fundus of infected individuals. The conversion to a pangastritis is time-dependent and estimated to require 15–20 years. This form of gastritis increases with age, being present in up to 100% of persons over age 70. Histology improves after

1	H. pylori eradication. The number of H. pylori organisms decreases dramatically with progression to gastric atrophy, and the degree of inflammation correlates with the level of these organisms. Early on, with antral-predominant findings, the quantity of H. pylori is highest and a dense chronic inflammatory infiltrate of the lamina propria is noted, accompanied by epithelial cell infiltration with polymorphonuclear leukocytes (Fig. 348-14).

1	Multifocal atrophic gastritis, gastric atrophy with subsequent metaplasia, has been observed in chronic H. pylori–induced gastritis. This may ultimately lead to development of gastric adenocarcinoma (Fig. 348-8; Chap. 109). H. pylori infection is now considered an independent risk factor for gastric cancer. Worldwide epidemiologic studies have documented a higher incidence of H. pylori infection in patients with adenocarcinoma of the stomach as compared to control subjects. Seropositivity for H. pylori is associated with a three-to sixfold increased risk of gastric cancer. This risk may be as high as ninefold after adjusting for the inaccuracy of serologic testing in the elderly. The mechanism by which H. pylori infection leads to cancer

1	FIGURE 348-14 Chronic gastritis and H. pylori organisms. Steiner silver stain of superficial gastric mucosa showing abundant darkly stained microorganisms layered over the apical portion of the surface epithelium. Note that there is no tissue invasion. is unknown, but it appears to be related to the chronic inflammation induced by the organism. Eradication of H. pylori as a general preventative measure for gastric cancer is being evaluated but is not yet recommended.

1	Infection with H. pylori is also associated with development of a low-grade B cell lymphoma, gastric MALT lymphoma (Chap. 134). The chronic T cell stimulation caused by the infection leads to production of cytokines that promote the B cell tumor. The tumor should be initially staged with a CT scan of the abdomen and EUS. Tumor growth remains dependent on the presence of H. pylori, and its eradication is often associated with complete regression of the tumor. The tumor may take more than a year to regress after treating the infection. Such patients should be followed by EUS every 2–3 months. If the tumor is stable or decreasing in size, no other therapy is necessary. If the tumor grows, it may have become a high-grade B cell lymphoma. When the tumor becomes a high-grade aggressive lymphoma histologically, it loses responsiveness to H. pylori eradication.

1	Treatment in chronic gastritis is aimed at the sequelae and not the underlying inflammation. Patients with pernicious anemia will require parenteral vitamin B12 supplementation on a long-term basis. Eradication of H. pylori is often recommended even if PUD or a low-grade MALT lymphoma is not present.

1	Miscellaneous Forms of Gastritis Lymphocytic gastritis is characterized histologically by intense infiltration of the surface epithelium with lymphocytes. The infiltrative process is primarily in the body of the stomach and consists of mature T cells and plasmacytes. The etiology of this form of chronic gastritis is unknown. It has been described in patients with celiac sprue, but whether there is a common factor associating these two entities is unknown. No specific symptoms suggest lymphocytic gastritis. A subgroup of patients have thickened folds noted on endoscopy. These folds are often capped by small nodules that contain a central depression or erosion; this form of the disease is called varioliform gastritis. H. pylori probably plays no significant role in lymphocytic gastritis. Therapy with glucocorticoids or sodium cromoglycate has obtained unclear results.

1	Marked eosinophilic infiltration involving any layer of the stomach (mucosa, muscularis propria, and serosa) is characteristic of eosinophilic gastritis. Affected individuals will often have circulating eosinophilia with clinical manifestation of systemic allergy. Involvement may 1932 range from isolated gastric disease to diffuse eosinophilic gastroenteritis. Antral involvement predominates, with prominent edematous folds being observed on endoscopy. These prominent antral folds can lead to outlet obstruction. Patients can present with epigastric discomfort, nausea, and vomiting. Treatment with glucocorticoids has been successful. Several systemic disorders may be associated with granulomatous gastritis. Gastric involvement has been observed in Crohn’s disease. Involvement may range from granulomatous infiltrates noted only on gastric biopsies to frank ulceration and stricture formation. Gastric Crohn’s disease usually occurs in the presence of small-intestinal disease. Several rare

1	granulomatous infiltrates noted only on gastric biopsies to frank ulceration and stricture formation. Gastric Crohn’s disease usually occurs in the presence of small-intestinal disease. Several rare infectious processes can lead to granulomatous gastritis, including histoplasmosis, candidiasis, syphilis, and tuberculosis. Other unusual causes of this form of gastritis include sarcoidosis, idiopathic granulomatous gastritis, and eosinophilic granulomas involving the stomach. Establishing the specific etiologic agent in this form of gastritis can be difficult, at times requiring repeat endoscopy with biopsy and cytology. Occasionally, a surgically obtained full-thickness biopsy of the stomach may be required to exclude malignancy. Russell body gastritis (RBG) is a mucosal lesion of unknown etiology that has a pseudotumoral endoscopic appearance. Histologically, it is defined by the presence of numerous plasma cells containing Russell bodies (RBs) that express kappa and lambda light

1	etiology that has a pseudotumoral endoscopic appearance. Histologically, it is defined by the presence of numerous plasma cells containing Russell bodies (RBs) that express kappa and lambda light chains. Only 10 cases have been reported, and 7 of these have been associated with H. pylori infection. The lesion can be confused with a neoplastic process, but it is benign in nature, and the natural history of the lesion is not known. There have been cases of resolution of the lesion when H. pylori was eradicated.

1	Ménétrier’s disease (MD) is a very rare gastropathy characterized by large, tortuous mucosal folds. MD has an average age of onset of 40–60 years with a male predominance. The differential diagnosis of large gastric folds includes ZES, malignancy (lymphoma, infiltrating carcinoma), infectious etiologies (CMV, histoplasmosis, syphilis, tuberculosis), gastritis polyposa profunda, and infiltrative disorders such as sarcoidosis. MD is most commonly confused with large or multiple gastric polyps (prolonged PPI use) or familial polyposis syndromes. The mucosal folds in MD are often most prominent in the body and fundus, sparing the antrum. Histologically, massive foveolar hyperplasia (hyperplasia of surface and glandular mucous cells) and a marked reduction in oxyntic glands and parietal cells and chief cells are noted. This hyperplasia produces the prominent folds observed. The pits of the gastric glands elongate and may become extremely dilated and tortuous. Although the lamina propria

1	and chief cells are noted. This hyperplasia produces the prominent folds observed. The pits of the gastric glands elongate and may become extremely dilated and tortuous. Although the lamina propria may contain a mild chronic inflammatory infiltrate including eosinophils and plasma cells, MD is not considered a form of gastritis. The etiology of this unusual clinical picture in children is often CMV, but the etiology in adults is unknown. Overexpression of the growth factor TGF-α has been demonstrated in patients with MD. The overexpression of TGF-α in turn results in overstimulation of the epidermal growth factor receptor (EGFR) pathway and increased proliferation of mucus cells, resulting in the observed foveolar hyperplasia.

1	The clinical presentation in adults is usually insidious and progressive. Epigastric pain, nausea, vomiting, anorexia, peripheral edema, and weight loss are signs and symptoms in patients with MD. Occult GI bleeding may occur, but overt bleeding is unusual and, when present, is due to superficial mucosal erosions. In fact, bleeding is more often seen in one of the common mimics of MD, gastric polyposis. Twenty to 100% of patients (depending on time of presentation) develop a protein-losing gastropathy due to hypersecretion of gastric mucus accompanied by hypoalbuminemia and edema. Gastric acid secretion is usually reduced or absent because of the decreased parietal cells. Large gastric folds are readily detectable by either radiographic (barium meal) or endoscopic methods. Endoscopy with deep mucosal biopsy, preferably full thickness with a snare technique, is required to establish the diagnosis and exclude other entities that may present similarly. A nondiagnostic biopsy may lead to

1	deep mucosal biopsy, preferably full thickness with a snare technique, is required to establish the diagnosis and exclude other entities that may present similarly. A nondiagnostic biopsy may lead to a surgically obtained full-thickness biopsy to exclude malignancy. Although MD is considered premalignant by some, the risk of neoplastic progression is not defined. Complete blood count, serum gastrin, serum albumin, CMV and H. pylori serology, and pH testing of gastric aspirate during endoscopy should be included as part of the initial evaluation of patients with large gastric folds.

1	Medical therapy with anticholinergic agents, prostaglandins, PPIs, prednisone, somatostatin analogues (octreotide) and H2 receptor antagonists yields varying results. Ulcers should be treated with a standard approach. The discovery that MD is associated with over-stimulation of the EGFR pathway has led to the successful use of the EGF inhibitory antibody, cetuximab, in these patients. Specifically, four of seven patients who completed a 1-month trial with this agent demonstrated near complete histologic remission and improvement in symptoms. Cetuximab is now considered the first-line treatment for MD, leaving total gastrectomy for severe disease with persistent and substantial protein loss despite therapy with this agent. Disorders of Absorption Henry J. Binder

1	Disorders of Absorption Henry J. Binder Disorders of absorption constitute a broad spectrum of conditions with multiple etiologies and varied clinical manifestations. Almost all of these clinical problems are associated with diminished intestinal absorption of one or more dietary nutrients and are often referred to as the malabsorption syndrome. This term is not ideal as it represents a pathophysiologic state, does not provide an etiologic explanation for the underlying problem, and should not be considered an adequate final diagnosis. The only clinical conditions in which absorption is increased are hemochromatosis and Wilson’s disease, in which absorption of iron and copper, respectively, is elevated.

1	Most malabsorption syndromes are associated with steatorrhea, an increase in stool fat excretion to >6% of dietary fat intake. Some malabsorption disorders are not associated with steatorrhea: primary lactase deficiency, a congenital absence of the small-intestinal brush border disaccharidase enzyme lactase, is associated with lactose “malabsorption,” and pernicious anemia is associated with a marked decrease in intestinal absorption of cobalamin (vitamin B12) due to an absence of gastric parietal-cell intrinsic factor, which is required for cobalamin absorption.

1	Disorders of absorption must be included in the differential diagnosis of diarrhea (Chap. 55). First, diarrhea is frequently associated with and/or is a consequence of the diminished absorption of one or more dietary nutrients. The diarrhea may be secondary either to the intestinal process that is responsible for the steatorrhea or to steatorrhea per se. Thus, celiac disease (see below) is associated with both extensive morphologic changes in the small-intestinal mucosa and reduced absorption of several dietary nutrients; in contrast, the diarrhea of steatorrhea is the result of the effect of nonabsorbed dietary fatty acids on intestinal (usually colonic) ion transport. For example, oleic acid and ricinoleic acid (a bacterially hydroxylated fatty acid that is also the active ingredient in castor oil, a widely used laxative) induce active colonic Cl ion secretion, most likely secondary to increasing intracellular Ca. In addition, diarrhea per se may result in mild steatorrhea (<11 g of

1	castor oil, a widely used laxative) induce active colonic Cl ion secretion, most likely secondary to increasing intracellular Ca. In addition, diarrhea per se may result in mild steatorrhea (<11 g of fat excretion while on a 100-g fat diet). Second, most patients will indicate that they have diarrhea, not that they have fat malabsorption. Third, many intestinal disorders that have diarrhea as a prominent symptom (e.g., ulcerative colitis, traveler’s diarrhea secondary to an enterotoxin produced by Escherichia coli) do not necessarily have diminished absorption of any dietary nutrient.

1	Diarrhea as a symptom (i.e., when the term is used by patients to describe their bowel movement pattern) may reflect a decrease in stool consistency, an increase in stool volume, an increase in number of bowel movements, or any combination of these three changes. In contrast, diarrhea as a sign is a quantitative increase in stool water or weight of >200–225 mL or g per 24 h when a Western-type diet is consumed. Individuals consuming a diet with higher fiber content may normally have a stool weight of up to 400 g/24 h. Thus, the clinician must clarify what an individual patient means by diarrhea. Some 10% of patients referred to gastroenterologists for further evaluation of unexplained diarrhea do not have an increase in stool water when this variable is determined quantitatively. Such patients may have small, frequent, somewhat loose bowel movements with stool urgency that is indicative of proctitis but do not have an increase in stool weight or volume.

1	It is also critical to establish whether a patient’s diarrhea is secondary to diminished absorption of one or more dietary nutrients rather than being due to smalland/or large-intestinal fluid and electrolyte secretion. The former has often been termed osmotic diarrhea, while the latter has been referred to as secretory diarrhea. Unfortunately, both secretory and osmotic elements can be present simultaneously in the same disorder; thus, this distinction is not always precise. Nonetheless, two studies—determination of stool electrolytes and observation of the effect of a fast on stool output—can help make this distinction.

1	The demonstration of the effect of prolonged (>24 h) fasting on stool output can suggest that a dietary nutrient is responsible for the individual’s diarrhea. Secretory diarrhea associated with enterotoxininduced traveler’s diarrhea would not be affected by prolonged fasting, as enterotoxin-induced stimulation of intestinal fluid and electrolyte secretion is not altered by eating. In contrast, diarrhea secondary to lactose malabsorption in primary lactase deficiency would undoubtedly cease during a prolonged fast. Thus, a substantial decrease in stool output by a fasting patient during quantitative stool collection lasting at least 24 h is presumptive evidence that the diarrhea is related to malabsorption of a dietary nutrient. The persistence of stool output during fasting indicates that the diarrhea is likely secretory and that its cause is not a dietary nutrient. Either a luminal (e.g., E. coli enterotoxin) or a circulating (e.g., vasoactive intestinal peptide) secretagogue could

1	the diarrhea is likely secretory and that its cause is not a dietary nutrient. Either a luminal (e.g., E. coli enterotoxin) or a circulating (e.g., vasoactive intestinal peptide) secretagogue could be responsible for unaltered persistence of a patient’s diarrhea during a prolonged fast. The observed effects of fasting can be compared and correlated with stool electrolyte and osmolality determinations.

1	Measurement of stool electrolytes and osmolality requires comparison of Na+ and K+ concentrations in liquid stool with the osmolality of the stool in order to determine the presence or absence of a so-called stool osmotic gap. The following formula is used:

1	The cation concentrations are doubled to estimate stool anion concentrations. The presence of a significant osmotic gap suggests the presence in stool water of a substance (or substances) other than Na/K/anions that is presumably responsible for the patient’s diarrhea. Originally, stool osmolality was measured, but it is almost invariably greater than the required 290–300 mosmol/kg H2O, reflecting bacterial degradation of nonabsorbed carbohydrate either immediately before defecation or in the stool jar while specimen awaits chemical analysis, even when the stool is refrigerated. As a result, the stool osmolality should be assumed to be 300 mosmol/ kg H2O. A low stool osmolality (<290 mosmol/kg H2O) reflects the addition of either dilute urine or water, indicating either collection of urine and stool together or so-called factitious diarrhea, a form of Münchausen’s syndrome. When the calculated difference in the formula above is >50, an osmotic gap exists; its presence suggests that

1	and stool together or so-called factitious diarrhea, a form of Münchausen’s syndrome. When the calculated difference in the formula above is >50, an osmotic gap exists; its presence suggests that the diarrhea is due to a nonabsorbed dietary nutrient—e.g., a fatty acid and/or a carbohydrate. When this difference is <25, it is presumed that a dietary nutrient is not responsible for the diarrhea. Since elements of both osmotic diarrhea (i.e., due to malabsorption of a dietary nutrient) and secretory diarrhea may be present, this distinction at times is less clear-cut at the bedside than when used as a teaching example. Ideally, the presence of an osmotic gap will be associated with a marked decrease in stool output during a prolonged 1933 fast, while an osmotic gap will likely be absent in an individual whose stool output is not reduced substantially during a period of fasting.

1	The lengths of the small intestine and the colon are ~300 cm and ~80 cm, respectively. However, the effective functional surface area is ~600-fold greater than that of a hollow tube as a result of folds, villi (in the small intestine), and microvilli. The functional surface area of the small intestine is somewhat greater than that of a doubles tennis court. In addition to nutrient digestion and absorption, the intestinal epithelia have several other functions: 1. Barrier and immune defense. The intestine is exposed to a large number of potential antigens and enteric and invasive microorganisms, and it is extremely effective at preventing the entry of almost all of these agents. The intestinal mucosa also synthesizes and secretes secretory IgA. 2.

1	2. Fluid and electrolyte absorption and secretion. The intestine absorbs ~7–8 L of fluid daily, a volume comprising dietary fluid intake (1–2 L/d) and salivary, gastric, pancreatic, biliary, and intestinal fluid (6–7 L/d). Several stimuli, especially bacteria and bacterial enterotoxins, induce fluid and electrolyte secretion that may lead to diarrhea (Chap. 160). 3. Synthesis and secretion of several proteins. The intestinal mucosa is a major site for the production of proteins, including apolipoproteins. 4. Production of several bioactive amines and peptides. The intestine is one of the largest endocrine organs in the body and produces several amines (e.g., 5-hydroxytryptophan) and peptides that serve as paracrine and hormonal mediators of intestinal function.

1	The small and large intestines are distinct anatomically (villi are present in the small intestine but are absent in the colon) and functionally (nutrient digestion and absorption take place in the small intestine but not in the colon). No precise anatomic characteristics separate duodenum, jejunum, and ileum, although certain nutrients are absorbed exclusively in specific areas of the small intestine. However, villous cells in the small intestine (surface epithelial cells in the colon) and crypt cells have distinct anatomic and functional characteristics. Intestinal epithelial cells are continuously renewed; new proliferating epithelial cells at the base of the crypt migrate over 48–72 h to the tip of the villus (or surface of the colon), where they exist as well-developed epithelial cells with digestive and absorptive function. This high rate of cell turnover explains the relatively rapid resolution of diarrhea and other digestive-tract side effects during chemotherapy as new cells

1	with digestive and absorptive function. This high rate of cell turnover explains the relatively rapid resolution of diarrhea and other digestive-tract side effects during chemotherapy as new cells not exposed to these toxic agents are produced. Equally important is the paradigm of separation of villous/surface cell and crypt cell functions. Digestive hydrolytic enzymes are present primarily in the brush border of villous epithelial cells. Absorptive and secretory functions are also separate: villous/surface cells are primarily, but not exclusively, the site for absorptive function, while secretory function is located in crypts of both the small and large intestines.

1	Nutrients, minerals, and vitamins are absorbed by one or more active-transport mechanisms. These mechanisms are energy dependent and are mediated by membrane transport proteins. These processes will result in the net movement of a substance against or in the absence of an electrochemical concentration gradient. Intestinal absorption of amino acids and monosaccharides (e.g., glucose) is also a specialized form of active transport—secondary active transport. The movement of actively transported nutrients against a concentration gradient is Na+ dependent and is due to a Na+ gradient across the apical membrane. The Na+ gradient is maintained by Na+,K+-adenosine triphosphatase (ATPase), the so-called Na+ pump located on the basolateral membrane, which extrudes Na+ and maintains low intracellular [Na] as well as the Na+ gradient across the apical membrane. As a result, active glucose absorption and glucose-stimulated Na+ absorption require both the apical membrane transport protein SGLT1

1	[Na] as well as the Na+ gradient across the apical membrane. As a result, active glucose absorption and glucose-stimulated Na+ absorption require both the apical membrane transport protein SGLT1 and the basolateral Na+,K+-ATPase. In addition to exhibiting Na+ for its absorption, glucose stimulates Na+ and fluid absorption; this effect is

1	Disorders of Absorption 1934 the physiologic basis of oral rehydration therapy for the treatment of diarrhea (Chap. 55). The mechanisms of intestinal fluid and electrolyte absorption and secretion are discussed in Chap. 55. Although the intestinal epithelial cells are crucial mediators of absorption and of ion and water flow, the several cell types in the lamina propria (e.g., mast cells, macrophages, myofibroblasts) and the enteric nervous system interact with the epithelium to regulate mucosal cell function. Intestinal function results from the integrated responses and interactions of intestinal epithelial cells and intestinal muscle.

1	Bile acids are not present in the diet but are synthesized in the liver by a series of enzymatic steps that also represent cholesterol catabolism. Indeed, interruption of the enterohepatic circulation of bile acids can reduce serum cholesterol levels by 10% before a new steady state is established. Bile acids are either primary or secondary. Primary bile acids are synthesized in the liver from cholesterol, and secondary bile acids are synthesized from primary bile acids in the intestine by colonic bacterial enzymes. The two primary bile acids in humans are cholic acid and chenodeoxycholic acid; the two most abundant secondary bile acids are deoxycholic acid and lithocholic acid. The liver synthesizes ~500 mg of bile acids daily; the bile acids are conjugated to either taurine or glycine (to form tauroconjugated and glycoconjugated bile acids, respectively) and are secreted into the duodenum in bile. The primary functions of bile acids are (1) to promote bile flow, (2) to solubilize

1	(to form tauroconjugated and glycoconjugated bile acids, respectively) and are secreted into the duodenum in bile. The primary functions of bile acids are (1) to promote bile flow, (2) to solubilize cholesterol and phospholipid in the gallbladder by mixed micelle formation, and (3) to enhance dietary lipid digestion and absorption by forming mixed micelles in the proximal small intestine.

1	Bile acids are primarily absorbed by an active, Na+-dependent process that takes place exclusively in the ileum; to a lesser extent, they are absorbed by non-carrier-mediated transport processes in the jejunum, ileum, and colon. Conjugated bile acids that enter the colon are deconjugated by colonic bacterial enzymes. The unconjugated bile acids are rapidly absorbed by nonionic diffusion. Colonic bacterial enzymes also dehydroxylate bile acids to secondary bile acids.

1	Bile acids absorbed from the intestine return to the liver via the portal vein and are then re-secreted (Fig. 349-1). Bile acid synthesis is largely autoregulated by 7α-hydroxylase, the initial enzyme in cholesterol degradation. A decrease in the volume of bile acids returning to the liver from the intestine is associated with an increase in bile acid synthesis/cholesterol catabolism, which helps keep the bile-acid pool size relatively constant. However, the capacity to increase bile acid synthesis is limited to ~2to 2.5-fold (see below). The bile-acid pool size is ~4 g. The pool is circulated via the enterohepatic circulation about twice during each meal, or six to eight times during a 24-h period. A relatively small quantity of bile acids is not absorbed and is excreted in stool daily; this fecal loss is matched by hepatic bile-acid synthesis.

1	Defects in any of the steps in enterohepatic circulation of bile acids can result in a decrease in the duodenal concentration of conjugated bile acids and consequently in the development of steatorrhea. Thus, steatorrhea can be caused by abnormalities in bile acid synthesis and excretion, their physical state in the intestinal lumen, and reabsorption (Table 349-1). Synthesis Decreased bile acid synthesis and steatorrhea have been demonstrated in chronic liver disease, but steatorrhea often is not a major component of illness in these patients.

1	Synthesis Decreased bile acid synthesis and steatorrhea have been demonstrated in chronic liver disease, but steatorrhea often is not a major component of illness in these patients. Secretion Although bile acid secretion may be reduced or absent in biliary obstruction, steatorrhea is rarely a significant medical problem in these patients. In contrast, primary biliary cirrhosis represents a defect in canalicular excretion of organic anions, including bile acids, and not infrequently is associated with steatorrhea and its consequences (e.g., chronic bone disease). Thus, the osteopenia/osteomalacia and other chronic bone abnormalities often present in patients with primary biliary cirrhosis and other cholestatic syndromes are secondary to steatorrhea that then leads to calcium and vitamin D malabsorption as well as to the effects of cholestasis (e.g., bile acids and inflammatory cytokines). 0.5 g synthesized Bile acid pool size 4.0 g IleumNa0.5 gBile acidsexcreted per day COLON

1	0.5 g synthesized Bile acid pool size 4.0 g IleumNa0.5 gBile acidsexcreted per day COLON FIGURE 349-1 Schematic representation of the enterohepatic circulation of bile acids. Bile acid synthesis is cholesterol catabolism and occurs in the liver. Bile acids are secreted in bile and are stored in the gallbladder between meals and at night. Food in the duodenum induces the release of cholecystokinin, a potent stimulus for gallbladder contraction resulting in bile acid entry into the duodenum. Bile acids are primarily absorbed via an Na-dependent transport process that is located only in the ileum. A relatively small quantity of bile acids (~500 mg) is not absorbed in a 24-h period and is lost in stool. Fecal bile acid losses are matched by bile acid synthesis. The bile acid pool (the total amount of bile acids in the body) is ~4 g and is circulated twice during each meal or six to eight times in a 24-h period.

1	Maintenance of Conjugated Bile Acids In bacterial overgrowth syndromes associated with diarrhea, steatorrhea, and macrocytic anemia, a colonic type of bacterial flora is increased in the small intestine. Steatorrhea is primarily a result of the decrease in conjugated bile acids secondary to their deconjugation by colonic-type bacteria. Two complementary explanations account for the resulting impairment of micelle formation: (1) Unconjugated bile acids are rapidly absorbed in the jejunum by nonionic diffusion, and the result is a reduced concentration of duodenal bile acids. (2) The critical micellar concentration (CMC) of unconjugated bile acids is higher than that of conjugated bile acids; therefore, unconjugated bile acids are less effective than conjugated bile acids in micelle formation.

1	Reabsorption Ileal dysfunction caused by either Crohn’s disease or surgical resection results in a decrease in bile acid reabsorption in the ileum and an increase in the delivery of bile acids to the large intestine. The resulting clinical consequences—diarrhea with or without steatorrhea—are determined by the degree of ileal dysfunction and the response of the enterohepatic circulation to bile acid losses (Table 349-2). Patients with limited ileal disease or resection often have diarrhea but not steatorrhea. The diarrhea, a result of stimulation of active Cl secretion by bile acids in the colon, has been called bile acid diarrhea or choleretic enteropathy and responds promptly to cholestyramine, an anion-binding resin. Steatorrhea does not develop because hepatic synthesis of bile acids increases to compensate for the rate of fecal bile-acid losses, resulting in maintenance of both the bile-acid pool size and the intraduodenal concentrations of bile acids. In contrast, patients with

1	increases to compensate for the rate of fecal bile-acid losses, resulting in maintenance of both the bile-acid pool size and the intraduodenal concentrations of bile acids. In contrast, patients with greater degrees of ileal disease and/or resection often have diarrhea and steatorrhea that do not respond to cholestyramine. In this situation, ileal disease is also associated with increased volumes of bile acids entering the colon; however, hepatic synthesis can no longer increase sufficiently to maintain the bile-acid pool size. As a consequence, the intraduodenal concentration of bile acids is reduced to less than the CMC, and the result is impaired micelle formation and steatorrhea. This second situation is often called fatty acid diarrhea. Cholestyramine may not be effective (and may even exacerbate the diarrhea by further depleting the intraduodenal bile-acid concentration); however, a low-fat diet to reduce fatty acid entry into the colon can be effective. Two clinical

1	(and may even exacerbate the diarrhea by further depleting the intraduodenal bile-acid concentration); however, a low-fat diet to reduce fatty acid entry into the colon can be effective. Two clinical features—the length of the ileal section removed and the degree of steatorrhea—can predict whether an individual patient will respond to cholestyramine. Unfortunately, these predictors are imperfect, and a therapeutic trial of cholestyramine is often necessary to establish whether an individual patient will benefit from cholestyramine. Table 349-2 contrasts the characteristics of bile acid diarrhea (small ileal dysfunction) and fatty acid diarrhea (large ileal dysfunction).

1	Bile acid diarrhea can also occur in the absence of ileal inflammation and/or resection and is characterized by an abnormal 75SeHCAT retention study and reduced ileal release of fibroblast growth factor 19, a negative regulator of bile acid synthesis, with a consequent increase in bile acid synthesis and secretion that exceeds ileal bile-acid absorption. The diarrhea in these patients also responds to cholestyramine.

1	Steatorrhea is caused by one or more defects in the digestion and absorption of dietary fat. The average intake of dietary fat in the United States is ~120–150 g/d, and fat absorption is linear to dietary fat intake. The total load of fat presented to the small intestine is considerably greater, as substantial amounts of lipid are secreted in bile each day (see “Enterohepatic Circulation of Bile Acids,” above). Three types of fatty acids compose fats: long-chain fatty acids (LCFAs), medium-chain fatty acids (MCFAs), and short-chain fatty acids (SCFAs) (Table 349-3). Dietary fat is exclusively composed of long-chain triglycerides (LCTs)—i.e., glycerol that is bound via ester linkages to three LCFAs. While the majority of dietary LCFAs have carbon chain lengths of 16 or 18, all fatty acids of carbon chain length >12 are metabolized in the same manner; saturated and unsaturated fatty acids are handled identically.

1	Assimilation of dietary lipid requires three integrated processes: (1) an intraluminal, or digestive, phase; (2) a mucosal, or absorptive, phase; and (3) a delivery, or postabsorptive, phase. An abnormality at any site involved in these processes can cause steatorrhea (Table 349-4). Therefore, it is essential that any patient with steatorrhea be evaluated to identify the specific physiologic defect in overall lipid digestion/ absorption, as therapy will be determined by the specific etiology.

1	The digestive phase has two components, lipolysis and micelle formation. Although dietary lipid is in the form of LCTs, the intestinal mucosa does not absorb triglycerides; they must first be hydrolyzed (Fig. 349-2). The initial step in lipid digestion is the formation of emulsions of finely dispersed lipid, which is accomplished by mastication and gastric contractions. Lipolysis, the hydrolysis of triglycerides to free fatty acids, monoglycerides, and glycerol by lipase, is initiated in the stomach by lingual and gastric lipases that have a pH optimum of 4.5–6.0. About 20–30% of total lipolysis occurs in the stomach. Lipolysis is completed in the duodenum and jejunum by pancreatic lipase, which is inactivated by a pH <7.0. Pancreatic lipolysis is greatly enhanced by the presence of a second pancreatic enzyme, colipase, which facilitates the movement of lipase to the triglyceride.

1	Impaired lipolysis can lead to steatorrhea and can occur in the presence of pancreatic insufficiency due to chronic pancreatitis in adults or cystic fibrosis in children and adolescents. Normal lipolysis can be maintained by ~5% of maximal pancreatic lipase secretion; thus, steatorrhea is a late manifestation of these disorders. A reduction in intra-duodenal pH can also result in altered lipolysis, as pancreatic lipase is inactivated at pH <7. Thus, ~15% of patients who have gastrinoma (Chap. 348), with substantial increases in gastric acid secretion from ectopic production of gastrin (usually from an islet cell adenoma), have diarrhea, and some have steatorrhea believed to be secondary to acid inactivation of pancreatic lipase. Similarly, patients who have chronic pancreatitis (with reduced lipase secretion) often have a decrease in pancreatic bicarbonate secretion, which will also result in a lowering of intraduodenal pH and inactivation of endogenous pancreatic lipase or of

1	reduced lipase secretion) often have a decrease in pancreatic bicarbonate secretion, which will also result in a lowering of intraduodenal pH and inactivation of endogenous pancreatic lipase or of therapeutically administered lipase.

1	Overlying the microvillus membrane of the small intestine is the so-called unstirred water layer, a relatively stagnant aqueous phase that must be traversed by the products of lipolysis that are primarily water insoluble. Water-soluble mixed micelles provide a mechanism Disorders of Absorption 1936 Pancreas Liver Jejunal Mucosa Lymphatics lymphangiectasia) may also be associated with steatorrhea as well as protein loss (see below). Steatorrhea can result from defects at any of the with Bile Acid several steps in lipid digestion/absorption. The mechanism of lipid digestion/absorption outlined above is limited to dietary lipid, which is almost exclusively in the form of LCTs (Table for utilization 349-3). Medium-chain triglycerides (MCTs), composed of fatty acids with carbon chain lengths Phospholipid of 8–12, are present in large amounts in coconut oil and are used as a nutritional supplement.

1	Phospholipid of 8–12, are present in large amounts in coconut oil and are used as a nutritional supplement. formation different from that involved in LCT digestion and absorption; at one time, MCTs held promise as an important treatment for steatorrhea of almost

1	formation different from that involved in LCT digestion and absorption; at one time, MCTs held promise as an important treatment for steatorrhea of almost FIGURE 349-2 Schematic representation of lipid digestion and absorption. Dietary all etiologies. Unfortunately, they have been less lipid is in the form of long-chain triglycerides. The overall process can be divided into (1) therapeutically effective than expected because, a digestive phase that includes both lipolysis and micelle formation requiring pancreatic for reasons that are not completely understood, their use often is not associated with an increase lipase and conjugated bile acids, respectively, in the duodenum; (2) an absorptive phase for mucosal uptake and re-esterification; and (3) a postabsorptive phase that includes chy-in body weight. lomicron formation and exit from the intestinal epithelial cell via lymphatics. (Courtesy of In contrast to LCTs, MCTs do not require

1	John M. Dietschy, MD; with permission.) by which the water-insoluble products of lipolysis can reach the luminal plasma membrane of villous epithelial cells—the site for lipid absorption. Mixed micelles are molecular aggregates composed of fatty acids, monoglycerides, phospholipids, cholesterol, and conjugated bile acids. These mixed micelles are formed when the concentration of conjugated bile acids is greater than its CMC, which differs among the several bile acids present in the small-intestinal lumen. Conjugated bile acids, synthesized in the liver and excreted into the duodenum in bile, are regulated by the enterohepatic circulation (see above). Steatorrhea can result from impaired movement of fatty acids across the unstirred aqueous fluid layer in two situations: (1) an increase in the relative thickness of the unstirred water layer that occurs in bacterial overgrowth syndromes (see below) secondary to functional stasis (e.g., scleroderma); and (2) a decrease in the duodenal

1	in the relative thickness of the unstirred water layer that occurs in bacterial overgrowth syndromes (see below) secondary to functional stasis (e.g., scleroderma); and (2) a decrease in the duodenal concentration of conjugated bile acids below the CMC, resulting in impaired micelle formation. Thus, steatorrhea can be caused by one or more defects in the enterohepatic circulation of bile acids.

1	Uptake and re-esterification constitute the absorptive phase of lipid digestion/absorption. Although passive diffusion has been thought to be responsible, a carrier-mediated process may mediate fatty acid and monoglyceride uptake. Regardless of the uptake process, fatty acids and monoglycerides are re-esterified by a series of enzymatic steps in the endoplasmic reticulum to form triglycerides, in which lipid exits from the intestinal epithelial cell. Impaired lipid absorption as a result of mucosal inflammation (e.g., celiac disease) and/or intestinal resection can also lead to steatorrhea.

1	The re-esterified triglycerides require the formation of chylomicrons to permit their exit from the small-intestinal epithelial cell and their delivery to the liver via the lymphatics. Chylomicrons are composed of β-lipoprotein and contain triglycerides, cholesterol, cholesterol esters, and phospholipids and enter the lymphatics, not the portal vein. Defects in the postabsorptive phase of lipid digestion/absorption can also result in steatorrhea, but these disorders are uncommon. Abetalipoproteinemia, or acanthocytosis, is a rare disorder of impaired synthesis of β-lipoprotein associated with abnormal erythrocytes (acanthocytes), neurologic problems, and steatorrhea (Chap. 421). Lipolysis, micelle formation, and lipid uptake are all normal in patients with abetalipoproteinemia, but the re-esterified triglyceride cannot exit the epithelial cell because of the failure to produce chylomicrons. Small-intestinal biopsy samples obtained from these rare patients in the postprandial state

1	re-esterified triglyceride cannot exit the epithelial cell because of the failure to produce chylomicrons. Small-intestinal biopsy samples obtained from these rare patients in the postprandial state reveal lipid-laden small-intestinal epithelial cells that become perfectly normal in appearance after a 72to 96-h fast. Similarly, abnormalities of intestinal lymphatics (e.g., intestinal pancreatic lipolysis as they can be absorbed intact by the intestinal epithelial cell. Further, micelle formation is not necessary for the absorp tion of MCTs (or MCFAs, if hydrolyzed by pancreatic lipase). MCTs are absorbed more efficiently than LCTs for the following reasons: (1) The rate of absorption is greater for MCTs than for LCFAs. (2) After absorption, MCFAs are not reesterified. (3) After absorption, MCTs are hydrolyzed to MCFAs.

1	(4) MCTs do not require chylomicron formation to exit intestinal epithelial cells. (5) The route of MCT exit is via the portal vein and not via lymphatics. Thus, the absorption of MCTs is greater than that of LCTs in pancreatic insufficiency, conditions with reduced intraduodenal bile acid concentrations, small-intestinal mucosal disease, abetalipoproteinemia, and intestinal lymphangiectasia.

1	SCFAs are not dietary lipids but are synthesized by colonic bacterial enzymes from nonabsorbed carbohydrate and are the anions present at the highest concentration in stool (80–130 mM). The SCFAs in stool are primarily acetate, propionate, and butyrate, whose carbon chain lengths are 2, 3, and 4, respectively. Butyrate is the primary nutrient for colonic epithelial cells, and its deficiency can be associated with one or more colitides. SCFAs conserve calories and carbohydrate: carbohydrates that are not completely absorbed in the small intestine will not be absorbed in the large intestine because of the absence of both disaccharidases and SGLT1, the transport protein that mediates monosaccharide absorption. In contrast, SCFAs are rapidly absorbed and stimulate colonic NaCl and fluid absorption. Most antibiotic-associated diarrhea not caused by Clostridium difficile is due to antibiotic suppression of the colonic microbiota, with a resulting decrease in SCFA production. As C. difficile

1	Most antibiotic-associated diarrhea not caused by Clostridium difficile is due to antibiotic suppression of the colonic microbiota, with a resulting decrease in SCFA production. As C. difficile accounts for only ~15–20% of all antibiotic-associated diarrhea, a relative decrease in colonic production of SCFA is likely the cause of most antibiotic-associated diarrhea.

1	The clinical manifestations of steatorrhea are a consequence both of the underlying disorder responsible for its development and of steatorrhea per se. Depending on the degree of steatorrhea and the level of dietary intake, significant fat malabsorption may lead to weight loss. Steatorrhea per se can be responsible for diarrhea; if the primary cause of the steatorrhea has not been identified, a low-fat diet can often ameliorate the diarrhea by decreasing fecal fat excretion. Steatorrhea is commonly associated with fat-soluble vitamin deficiency, which requires replacement with water-soluble preparations of these vitamins.

1	Disorders of absorption may also be associated with malabsorption of other dietary nutrients— most often carbohydrates—with or without a decrease in dietary lipid digestion and absorption. Therefore, knowledge of the mechanisms of digestion and absorption of carbohydrates, proteins, and other minerals and vitamins is useful in the evaluation of patients with altered intestinal nutrient absorption. Ethnic Group Prevalence of Lactase Deficiency, % Source: From FJ Simoons: Am J Dig Dis 23:963, 1978. Carbohydrates in the diet are present in the form of starch, disaccharides (sucrose and lactose), and glucose. Carbohydrates are absorbed only in the small intestine and only in the form of monosaccharides. Therefore, before their absorption, starch and disaccharides must first be digested by pancreatic amylase and intestinal brush border disaccharidases to monosaccharides. Monosaccharide absorption occurs by a Na-dependent process mediated by the brush border transport protein SGLT1.

1	Lactose malabsorption is the only clinically important disorder of carbohydrate absorption. Lactose, the disaccharide present in milk, requires digestion by brush border lactase to its two constituent monosaccharides, glucose and galactose. Lactase is present in almost all species in the postnatal period but then disappears throughout the animal kingdom, except in humans. Lactase activity persists in many individuals throughout life. Two different types of lactase deficiency exist—primary and secondary. In primary lactase deficiency, a genetically determined decrease or absence of lactase is noted, while all other aspects of both intestinal absorption and brush border enzymes are normal. In a number of nonwhite groups, primary lactase deficiency is common in adulthood. In fact, Northern European and North American whites are the only groups to maintain small-intestinal lactase activity throughout adult life. Table 349-5 presents the incidence of primary lactase deficiency in several

1	European and North American whites are the only groups to maintain small-intestinal lactase activity throughout adult life. Table 349-5 presents the incidence of primary lactase deficiency in several ethnic groups. Lactase persistence in adults is an abnormality due to a defect in the regulation of its maturation. In contrast, secondary lactase deficiency occurs in association with small-intestinal mucosal disease, with abnormalities in both structure and function of other brush border enzymes and transport processes. Secondary lactase deficiency is often seen in celiac disease.

1	As lactose digestion is rate-limiting compared to glucose/galactose absorption, lactase deficiency is associated with significant lactose malabsorption. Some individuals with lactose malabsorption develop symptoms such as diarrhea, abdominal pain, cramps, and/or flatus. Most individuals with primary lactase deficiency do not have symptoms. Since lactose intolerance may be associated with symptoms suggestive of irritable bowel syndrome, persistence of such symptoms in an individual who exhibits lactose intolerance while on a strict lactose-free diet suggests that the person’s symptoms were related to irritable bowel syndrome. The development of symptoms of lactose intolerance is related to several factors: 1. Amount of lactose in the diet. 2.

1	The development of symptoms of lactose intolerance is related to several factors: 1. Amount of lactose in the diet. 2. Rate of gastric emptying. Symptoms are more likely when gastric emptying is rapid than when it is slower. Therefore, skim milk is more likely to be associated with symptoms of lactose intolerance than whole milk, as the rate of gastric emptying after skim milk intake is more rapid. Similarly, diarrhea following subtotal gastrectomy is often a result of lactose intolerance, as gastric emptying is accelerated in patients with a gastrojejunostomy. 3. Small-intestinal transit time. Although the small and large intestines both contribute to the development of symptoms, many symptoms of lactase deficiency are related to the interaction of colonic bacteria and nonabsorbed lactose. More rapid small-intes-1937 tinal transit makes symptoms more likely.

1	4. Colonic compensation by production of SCFAs from nonabsorbed lactose. Reduced levels of colonic microflora, which can follow antibiotic use, are associated with increased symptoms after lactose ingestion, especially in a lactase-deficient individual.

1	Glucose-galactose or monosaccharide malabsorption may also be associated with diarrhea and is due to a congenital absence of SGLT1. Diarrhea develops when individuals with this disorder ingest carbo hydrates that contain actively transported monosaccharides (e.g., glucose, galactose) but not when they ingest monosaccharides that are not actively transported (e.g., fructose). Fructose is absorbed by the brush border transport protein GLUT 5, a facilitated diffusion process that is not Na-dependent and is distinct from SGLT1. In contrast, some individuals develop diarrhea as a result of the consumption of large quantities of sorbitol, a sugar used in diabetic candy; sorbitol is only minimally absorbed because of the absence of an intestinal absorptive transport mechanism for this sugar.

1	Protein is present in food almost exclusively as polypeptides and requires extensive hydrolysis to diand tripeptides and amino acids before absorption. Proteolysis occurs in both the stomach and the small intestine; it is mediated by pepsin, which is secreted as pepsinogen by gastric chief cells, and by trypsinogen and other peptidases from pancreatic acinar cells. The proenzymes pepsinogen and trypsinogen must be activated to pepsin (by pepsin at a pH <5) and to trypsin (by the intestinal brush border enzyme enterokinase and subsequently by trypsin), respectively. Proteins are absorbed by separate transport systems for diand tripeptides and for different types of amino acids—e.g., neutral and dibasic. Alterations in either protein or amino acid digestion and absorption are rarely observed clinically, even in the presence of extensive small-intestinal mucosal inflammation. However, three rare genetic disorders involve protein digestion/absorption: (1) Enterokinase deficiency is due to

1	clinically, even in the presence of extensive small-intestinal mucosal inflammation. However, three rare genetic disorders involve protein digestion/absorption: (1) Enterokinase deficiency is due to an absence of the brush border enzyme that converts the proenzyme trypsinogen to trypsin and is associated with diarrhea, growth retardation, and hypoproteinemia. (2) Hartnup’s syndrome, a defect in neutral amino acid transport, is characterized by a pellagra-like rash and neuropsychiatric symptoms. (3) Cystinuria, a defect in dibasic amino acid transport, is associated with renal calculi and chronic pancreatitis.

1	APPROACH TO THE PATIENT: The clues provided by the history, symptoms, and initial preliminary observations will serve to limit extensive, ill-focused, and expensive laboratory and imaging studies. For example, a clinician evaluating a patient who has symptoms suggestive of malabsorption and who has recently undergone extensive small-intestinal resection for mesenteric ischemia should direct the initial assessment almost exclusively to defining whether a short-bowel syndrome might explain the entire clinical picture. Similarly, the development of a pattern of bowel movements suggestive of steatorrhea in a patient with long-standing alcohol abuse and chronic pancreatitis should prompt an assessment of pancreatic exocrine function.

1	The classic picture of malabsorption is rarely seen today in most parts of the United States. As a consequence, diseases with malabsorption must be suspected in individuals who have less severe symptoms and signs and subtle evidence of the altered absorption of only a single nutrient rather than obvious evidence of the malabsorption of multiple nutrients. Although diarrhea can be caused by changes in fluid and electrolyte movement in either the small or the large intestine, dietary nutrients are absorbed almost exclusively in the small intestine. Disorders of Absorption

1	Disorders of Absorption Therefore, the demonstration of diminished absorption of a dietary nutrient provides unequivocal evidence for small-intestinal disease, although colonic dysfunction may also be present (e.g., Crohn’s disease may involve both the small and large intestines). Dietary nutrient absorption may be segmental or diffuse along the small intestine and is site specific. Thus, for example, calcium, iron, and folic acid are exclusively absorbed by active-transport processes in the proximal small intestine, especially the duodenum; in contrast, the active-transport mechanisms for both cobalamin and bile acids are operative only in the ileum. Therefore, in an individual who years previously has had an intestinal resection, the details of which are not presently available, a presentation with evidence of calcium, folic acid, and/or iron malabsorption but without cobalamin deficiency makes it likely that the duodenum and proximal jejunum, but not the ileum, were resected.

1	Some nutrients—e.g., glucose, amino acids, and lipids—are absorbed throughout the small intestine, although their rate of absorption is greater in the proximal than in the distal segments. However, after segmental resection of the small intestine, the remaining segments undergo both morphologic and functional “adaptation” to enhance absorption. Such adaptation is secondary to the presence of luminal nutrients and hormonal stimuli and may not be complete in humans for several months after resection. Adaptation is critical for the survival of individuals who have undergone massive resection of the small intestine and/or colon.

1	Establishing the diagnosis of steatorrhea and identifying its specific cause are often quite difficult. The “gold standard” remains a timed, quantitative stool-fat determination. From a practical standpoint, stool collections are invariably difficult and often incomplete, as nobody wants to handle stool. A qualitative test—Sudan III staining—has long been available to document an increase in stool fat. This test is rapid and inexpensive but, as a qualitative test, does not establish the degree of fat malabsorption and is best used as a preliminary screening study. Many of the blood, breath, and isotopic tests that have been developed (1) do not directly measure fat absorption; (2) exhibit excellent sensitivity when steatorrhea is obvious and severe but poor sensitivity when steatorrhea is mild (e.g., assays for stool chymotrypsin and elastase, which can potentially distinguish pancreatic from nonpancreatic etiologies of steatorrhea); or (3) have not survived the transition from the

1	is mild (e.g., assays for stool chymotrypsin and elastase, which can potentially distinguish pancreatic from nonpancreatic etiologies of steatorrhea); or (3) have not survived the transition from the research laboratory to commercial application.

1	Nevertheless, routine laboratory studies (i.e., complete blood count, prothrombin time, serum protein determination, alkaline phosphatase) may suggest dietary nutrient depletion, especially deficiencies of iron, folate, cobalamin, and vitamins D and K. Additional studies include measurement of serum carotene, cholesterol, albumin, iron, folate, and cobalamin levels. The serum carotene level can also be reduced if the patient’s dietary intake of leafy vegetables is poor.

1	If steatorrhea and/or altered absorption of other nutrients is suspected, then history, clinical observations, and laboratory testing can help detect deficiency of a nutrient, especially of a fat-soluble vitamin (A, D, E, or K). Thus, evidence of metabolic bone disease with elevated alkaline phosphatase concentrations and/or reduced serum calcium levels suggests vitamin D malabsorption. A deficiency of vitamin K is suggested by an elevated prothrombin time in an individual without liver disease who is not taking anticoagulants. Macrocytic anemia leads to an evaluation for possible cobalamin or folic acid malabsorption. Iron-deficiency anemia in the absence of occult bleeding from the gastrointestinal tract in either a male patient or a nonmenstruating female patient requires an evaluation for iron malabsorption and the exclusion of celiac disease, as iron is absorbed exclusively in the proximal small intestine.

1	At times, however, a timed (72-h) quantitative stool collection, preferably while the patient is on a defined diet, must be undertaken in order to determine stool fat content and establish the diagnosis of steatorrhea. The presence of steatorrhea then requires further assessment to identify the pathophysiologic process(es) responsible for the defect in dietary lipid digestion/absorption (Table 349-4). Other studies include the Schilling test (Chap. 350e), the D-xylose test, duodenal mucosal biopsy, small-intestinal radiologic examination, and tests of pancreatic exocrine function. This test (Chap. 350e) is performed to determine the cause of cobalamin malabsorption. An understanding of the physiology and pathophysiology of cobalamin absorption is very valuable, enhancing comprehension of aspects of gastric, pancreatic, and ileal function. Unfortunately, the Schilling test has not been available commercially in the United States for the past few years.

1	The urinary d-xylose test for carbohydrate absorption provides an assessment of proximal small-intestinal mucosal function. d-Xylose, a pentose, is absorbed almost exclusively in the proximal small intestine. The d-xylose test is usually performed by administering 25 g of d-xylose and collecting urine for 5 h. An abnormal test (excretion of <4.5 g) primarily reflects duodenal/jejunal mucosal disease. The d-xylose test can also be abnormal in patients with blind loop syndrome (as a consequence primarily of an abnormal intestinal mucosa) and, as a false-positive study, in patients with large collections of fluid in a third space (i.e., ascites, pleural fluid). The ease of obtaining a mucosal biopsy of the small intestine by endoscopy and the false-negative rate of the d-xylose test have led to its diminished use. When small-intestinal mucosal disease is suspected, a small-intestinal mucosal biopsy should be performed.

1	Radiologic examination of the small intestine using barium contrast (small-bowel series or study) can provide important information in the evaluation of the patient with presumed or suspected malabsorption. This study is most often performed in conjunction with an examination of the esophagus, stomach, and duodenal bulb. Because insufficient barium is given to the patient to permit an adequate examination of the small-intestinal mucosa, especially in the ileum, many gastrointestinal radiologists alter the procedure by performing either a small-bowel series in which a large amount of barium is given by mouth, without concurrent examination of the esophagus and stomach, or an enteroclysis study in which a large amount of barium is introduced into the duodenum via a fluoroscopically placed tube. In addition, many of the diagnostic features initially described by radiologists to denote the presence of small-intestinal disease (e.g., flocculation, segmentation) are rarely seen with current

1	In addition, many of the diagnostic features initially described by radiologists to denote the presence of small-intestinal disease (e.g., flocculation, segmentation) are rarely seen with current barium suspensions. Nonetheless, in skilled hands, barium contrast examination of the small intestine can yield important information. For example, with extensive mucosal disease, intestinal dilation can be seen as a dilution of barium from increased intestinal fluid secretion (Fig. 349-3). A normal barium contrast study does not exclude the possibility of small-intestinal disease. However, a small-bowel series remains useful in the search for anatomic abnormalities, such as strictures and fistulas (as in Crohn’s disease) or blind loop syndrome (e.g., multiple jejunal diverticula) and to define the extent of a previous surgical resection. Other imaging studies that assess the integrity of small-intestinal morphology are CT enterography and magnetic resonance enterography. Capsule endoscopy

1	the extent of a previous surgical resection. Other imaging studies that assess the integrity of small-intestinal morphology are CT enterography and magnetic resonance enterography. Capsule endoscopy and double-balloon enteroscopy are other useful aids in the diagnostic assessment of small-intestinal pathology.

1	A small-intestinal mucosal biopsy is essential in the evaluation of a patient with documented steatorrhea or chronic diarrhea (i.e., that lasting >3 weeks) (Chap. 55). The ready availability of endoscopic equipment to examine the stomach and duodenum has led to its almost uniform use as the preferred method of obtaining Disorders of Absorption FIGURE 349-3 Barium contrast small-intestinal radiologic examinations. A. Normal individual. B. Celiac sprue. C. Jejunal diverticulosis. D. Crohn’s disease. (Courtesy of Morton Burrell, MD, Yale University; with permission.) histologic material from the proximal small-intestinal mucosa. The primary indications for a small-intestinal biopsy are evaluation of a patient (1) either with documented or suspected steatorrhea or with chronic diarrhea, and (2) with diffuse or focal abnormalities of the small intestine defined on a small-intestinal series. Lesions seen on small-bowel biopsy can be classified into three categories (Table 349-6): 1.

1	Diffuse, specific lesions. Relatively few diseases associated with altered nutrient absorption have specific histopathologic abnormalities on small-intestinal mucosal biopsy, and these diseases are uncommon. Whipple’s disease is characterized by the presence of periodic acid–Schiff (PAS)–positive macrophages in the lamina propria; the bacilli that are also present may require electron microscopic examination for identification (Fig. 349-4). Abetalipoproteinemia is characterized by a normal mucosal appearance except for the presence of mucosal absorptive cells that contain lipid postprandially and disappear after a prolonged period of either fat-free intake or fasting. Immune globulin deficiency is associated with a variety of histopathologic findings on small-intestinal mucosal biopsy. The characteristic feature is the absence of or substantial reduction in the number of plasma cells in the lamina propria; the mucosal architecture may be either perfectly normal or flat (i.e., villous

1	characteristic feature is the absence of or substantial reduction in the number of plasma cells in the lamina propria; the mucosal architecture may be either perfectly normal or flat (i.e., villous atrophy). As patients with immune globulin deficiency are often infected with Giardia lamblia, Giardia trophozoites may also be seen in the biopsy.

1	2.

1	Patchy, specific lesions. Several diseases feature an abnormal small-intestinal mucosa with a patchy distribution. As a result, biopsy samples obtained randomly or in the absence of endoscopically visualized abnormalities may not reveal diagnostic features. Intestinal lymphoma can at times be diagnosed on mucosal biopsy by the identification of malignant lymphoma cells in the lamina propria and submucosa (Chap. 134). Dilated lymphatics in the submucosa and sometimes in the lamina propria indicate lymphangiectasia associated with hypoproteinemia secondary to protein loss into the intestine. Eosinophilic gastroenteritis comprises a heterogeneous group of disorders with a spectrum of presentations and symptoms, with an eosinophilic infiltrate of the lamina propria, and with or without peripheral eosinophilia. The patchy nature of the infiltrate and its presence in the sub-mucosa often lead to an absence of histopathologic findings on mucosal biopsy. As the involvement of the duodenum in

1	eosinophilia. The patchy nature of the infiltrate and its presence in the sub-mucosa often lead to an absence of histopathologic findings on mucosal biopsy. As the involvement of the duodenum in Crohn’s disease is also submucosal and not necessarily continuous, mucosal biopsies are not the most direct approach to the diagnosis of duodenal Crohn’s disease (Chap. 351). Amyloid deposition can be identified by Congo Red staining in some patients with amyloidosis involving the duodenum (Chap. 136).

1	3. Diffuse, nonspecific lesions. Celiac disease presents with a characteristic mucosal appearance on duodenal/proximal jejunal mucosal biopsy that is not diagnostic of the disease. The diagnosis of celiac disease is established by clinical, histologic, and immunologic responses to a gluten-free diet. Tropical sprue (see below) is associated with histologic findings similar to those of celiac disease after a tropical or subtropical exposure but does not respond to gluten restriction; most often symptoms improve with antibiotics and folate administration.

1	Several microorganisms can be identified in small-intestinal biopsy samples, establishing a correct diagnosis. At times, the biopsy is performed specifically to diagnose infection (e.g., Whipple’s disease or giardiasis). In most other instances, the infection is detected incidentally during the workup for diarrhea or other abdominal symptoms. Many of these infections occur in immunocompromised patients with diarrhea; the etiologic agents include Cryptosporidium, Isospora belli, microsporidia, Cyclospora, Toxoplasma, cytomegalovirus, adenovirus, Mycobacterium aviumintracellulare, and G. lamblia. In immunocompromised patients, when Candida, Aspergillus, Cryptococcus, or Histoplasma organisms are seen on duodenal biopsy, their presence generally reflects systemic infection. Apart from Whipple’s disease and infections in the immunocompromised host, small-bowel biopsy is seldom used as the primary mode of diagnosis of infection. Even giardiasis is more easily diagnosed by stool antigen

1	disease and infections in the immunocompromised host, small-bowel biopsy is seldom used as the primary mode of diagnosis of infection. Even giardiasis is more easily diagnosed by stool antigen studies and/or duodenal aspiration than by duodenal biopsy.

1	Patients with steatorrhea require assessment of pancreatic exocrine function, which is often abnormal in chronic pancreatitis. The secretin test that collects pancreatic secretions by duodenal intubation following intravenous administration of secretin is the only test that directly measures pancreatic exocrine function but is available only at a few specialized centers. Endoscopic approaches (endoscopic retrograde cholangiopancreatography, endoscopic ultrasound) provide an excellent assessment of pancreatic duct anatomy but do not assess exocrine function (Chap. 370). Table 349-7 summarizes the results of the d-xylose test, the Schilling test, and small-intestinal mucosal biopsy in patients with steatorrhea of various etiologies.

1	Celiac disease is a common cause of malabsorption of one or more nutrients. Although celiac disease was originally consid ered largely a disease of white individuals, especially persons of European descent, recent observations have established that it is a common disease with protean manifestations, a worldwide distribution, and an estimated incidence in the United States that is as high as 1 in 113 people. Its incidence has increased over the past 50 years. Celiac disease has had several other names, including nontropical sprue, celiac sprue, adult celiac disease, and gluten-sensitive enteropathy. The etiology of celiac disease is not known, but environmental, immunologic, and genetic factors are important. Celiac disease is considered an “iceberg” disease. A small number of individuals have classical symptoms and manifestations related to nutrient malabsorption along with a varied natural history; the onset of symptoms can occur at all points from the first year of life through the

1	have classical symptoms and manifestations related to nutrient malabsorption along with a varied natural history; the onset of symptoms can occur at all points from the first year of life through the eighth decade. A much larger number of individuals have “atypical celiac disease”, with manifestations that are not obviously related to intestinal malabsorption (e.g., anemia, osteopenia, infertility, and neurologic symptoms). Finally, an even larger number of persons have “silent celiac disease”; they are essentially asymptomatic despite abnormal small-intestinal histopathology and serologies (see below).

1	The hallmark of celiac disease is an abnormal small-intestinal biopsy (Fig. 349-4) and the response of the condition (including symptoms and histologic changes on small-intestinal biopsy) to the elimination of gluten from the diet. The histologic changes have a proximal-to-distal intestinal distribution of severity, which probably reflects the exposure of the intestinal mucosa to varied amounts of dietary gluten. The symptoms do not necessarily correlate with histologic changes, especially as many newly diagnosed patients with celiac disease may be asymptomatic or only minimally symptomatic (often with no gastrointestinal symptoms).

1	The symptoms of celiac disease may appear with the introduction of cereals into an infant’s diet, although spontaneous remissions often occur during the second decade of life that may be either permanent or followed by the reappearance of symptoms over several years. Alternatively, the symptoms of celiac disease may first become evident at almost any age throughout adulthood. In many patients, frequent spontaneous remissions and exacerbations occur. The symptoms range from significant malabsorption of multiple nutrients, with diarrhea, steatorrhea, weight loss, and the consequences of nutrient depletion (i.e., anemia and metabolic bone disease), to the absence of gastrointestinal symptoms despite evidence of the depletion of a single nutrient (e.g., iron or folate deficiency, osteomalacia, edema from protein loss). Asymptomatic relatives of patients with celiac disease have been identified as having this disease either by small-intestinal biopsy or by serologic studies (e.g.,

1	edema from protein loss). Asymptomatic relatives of patients with celiac disease have been identified as having this disease either by small-intestinal biopsy or by serologic studies (e.g., antiendomysial antibodies, tissue transglutaminase [tTG], deamidated gliadin peptide). The availability of these “celiac serologies” has led to a substantial increase in the frequency of diagnosis of celiac disease, and the diagnosis is now being made primarily in patients without “classic” symptoms but with atypical and subclinical presentations.

1	Etiology The etiology of celiac disease is not known, but environmental, immunologic, and genetic factors all appear to contribute to the disease. One environmental factor is the clear association of the disease with gliadin, a component of gluten that is present in wheat, barley, and rye. In addition to the role of gluten restriction in treatment, the instillation of gluten into both the normal-appearing rectum and the distal ileum of patients with celiac disease results in morphologic changes within hours. An immunologic component in the pathogenesis of celiac disease is critical and involves both adaptive and innate immune responses. Serum Disorders of Absorption

1	FIGURE 349-4 Small-intestinal mucosal biopsies. A. Normal individual. B. Untreated celiac sprue. C. Treated celiac sprue. D. Intestinal lymphangiectasia. E. Whipple’s disease. F. Lymphoma. G. Giardiasis. (Courtesy of Marie Robert, MD, Yale University; with permission.) antibodies—IgA antigliadin, antiendomysial, and anti-tTG antibodies— true prevalence of celiac disease in the general population. A 4-week are present, but it is not known whether such antibodies are primary or course of treatment with prednisolone induces a remission in a patient secondary to the tissue damage. The presence of antiendomysial anti-with celiac disease who continues to eat gluten and converts the “flat” body is 90–95% sensitive and 90–95% specific; the antigen recognized by abnormal duodenal biopsy to a more normal-appearing one. In addiantiendomysial antibody is tTG, which deaminates gliadin, which is pre-tion, gliadin peptides interact with gliadin-specific T cells that mediate sented to HLA-DQ2 or

1	normal-appearing one. In addiantiendomysial antibody is tTG, which deaminates gliadin, which is pre-tion, gliadin peptides interact with gliadin-specific T cells that mediate sented to HLA-DQ2 or HLA-DQ8 (see below). Antibody studies are fre-tissue injury and induce the release of one or more cytokines (e.g., interquently used to identify patients with celiac disease; patients with these feron γ) that cause tissue injury. antibodies should undergo duodenal biopsy. This autoantibody has not

1	Genetic factor(s) are also involved in celiac disease. The incibeen linked to a pathogenetic mechanism (or mechanisms) responsible dence of symptomatic celiac disease varies widely in different for celiac disease. Nonetheless, this antibody is useful in establishing the population groups (high among whites, low among blacks and 1942 Asians) and is 10% among first-degree relatives of celiac disease patients. However, serologic studies provide clear evidence that celiac disease is present worldwide. Furthermore, all patients with celiac disease express the HLA-DQ2 or HLA-DQ8 allele, although only a minority of people expressing DQ2/DQ8 have celiac disease. Absence of DQ2/DQ8 excludes the diagnosis of celiac disease.

1	Diagnosis A small-intestinal biopsy is required to establish a diagnosis of celiac disease (Fig. 349-4). A biopsy should be performed when patients have symptoms and laboratory findings suggestive of nutrient malabsorption and/or deficiency as well as a positive tTG antibody test. Since the presentation of celiac disease is often subtle, without overt evidence of malabsorption or nutrient deficiency, a relatively low threshold for biopsy performance is important. It is more prudent to perform a biopsy than another test of intestinal absorption that can never completely exclude or establish this diagnosis.

1	The diagnosis of celiac disease requires the detection of characteristic histologic changes on small-intestinal biopsy together with a prompt clinical and histologic response after the institution of a gluten-free diet. If IgA antiendomysial or tTG antibodies have been detected in serologic studies, they too should disappear after a gluten-free diet is started. With the increase in the number of patients diagnosed with celiac disease (mostly by serologic studies), the spectrum of histologic changes seen on duodenal biopsy has increased and includes findings that are not as severe as the classic changes shown in Fig. 349-4. The classic changes seen on duodenal/jejunal biopsy are restricted to the mucosa and include (1) an increase in the number of intraepithelial lymphocytes; (2) absence or a reduced height of villi, which causes a flat appearance with increased crypt cell proliferation resulting in crypt hyperplasia and loss of villous structure, with consequent villous, but not

1	or a reduced height of villi, which causes a flat appearance with increased crypt cell proliferation resulting in crypt hyperplasia and loss of villous structure, with consequent villous, but not mucosal, atrophy; (3) a cuboidal appearance and nuclei that are no longer oriented basally in surface epithelial cells; and (4) increased numbers of lymphocytes and plasma cells in the lamina propria (Fig. 349-4B). Although these features are characteristic of celiac disease, they are not diagnostic because a similar appearance can develop in tropical sprue, eosinophilic enteritis, and milk-protein intolerance in children and occasionally in lymphoma, bacterial overgrowth, Crohn’s disease, and gastrinoma with acid hypersecretion. However, a characteristic histologic appearance that reverts toward normal after the initiation of a gluten-free diet establishes the diagnosis of celiac disease (Fig. 349-4C). Readministration of gluten, with or without an additional small-intestinal biopsy, is not

1	normal after the initiation of a gluten-free diet establishes the diagnosis of celiac disease (Fig. 349-4C). Readministration of gluten, with or without an additional small-intestinal biopsy, is not necessary.

1	A number of patients exhibit gluten sensitivity; i.e., they have gastrointestinal symptoms that respond to gluten restriction but do not have celiac disease. The basis for such gluten sensitivity is not known.

1	Failure to Respond to Gluten Restriction The most common cause of persistent symptoms in a patient who fulfills all the criteria for the diagnosis of celiac disease is continued intake of gluten. Gluten is ubiquitous, and a significant effort must be made to exclude all gluten from the diet. Use of rice flour in place of wheat flour is very helpful, and several support groups provide important aid to patients with celiac disease and to their families. More than 90% of patients who have the characteristic findings of celiac disease respond to complete dietary gluten restriction. The remainder constitute a heterogeneous group (whose condition is often called refractory celiac disease or refractory sprue) that includes some patients who (1) respond to restriction of other dietary protein (e.g., soy); (2) respond to glucocorticoid treatment; (3) are “temporary” (i.e., whose clinical and morphologic findings disappear after several months or years); or (4) fail to respond to all measures

1	soy); (2) respond to glucocorticoid treatment; (3) are “temporary” (i.e., whose clinical and morphologic findings disappear after several months or years); or (4) fail to respond to all measures and have a fatal outcome, with or without documented complications of celiac disease, such as the development of intestinal T cell lymphoma or autoimmune enteropathy.

1	Therapeutic approaches that do not include a gluten-free diet are being developed and include the use of peptidases to inactivate toxic gliadin peptides and of small molecules to block toxic peptide uptake across intestinal tight junctions. Mechanism of Diarrhea The diarrhea in celiac disease has several pathogenetic mechanisms. Diarrhea may be secondary to (1) steatorrhea, which is primarily a result of changes in jejunal mucosal function; (2) secondary lactase deficiency, a consequence of changes in jejunal brush border enzymatic function; (3) bile acid malabsorption resulting in bile acid–induced fluid secretion in the colon (in cases with more extensive disease involving the ileum); and (4) endogenous fluid secretion resulting from crypt hyperplasia. Celiac disease patients with more severe involvement may improve temporarily with dietary lactose and fat restriction while awaiting the full effects of total gluten restriction, which constitutes primary therapy.

1	Associated Diseases Celiac disease is associated with dermatitis herpetiformis (DH), but this association has not been explained. Patients with DH have characteristic papulovesicular lesions that respond to dapsone. Almost all patients with DH have histologic changes in the small intestine consistent with celiac disease, although usually much milder and less diffuse in distribution. Most patients with DH have mild or no gastrointestinal symptoms. In contrast, relatively few patients with celiac disease have DH. Celiac disease is also associated with diabetes mellitus type 1, IgA deficiency, Down syndrome, and Turner’s syndrome. The clinical importance of the association with diabetes is that, although severe watery diarrhea without evidence of malabsorption is most often diagnosed as “diabetic diarrhea” (Chap. 417), assay of antiendomysial antibodies and/or a small-intestinal biopsy must be considered to exclude celiac disease.

1	Complications The most important complication of celiac disease is the development of cancer. The incidences of both gastrointestinal and nongastrointestinal neoplasms as well as intestinal lymphoma are elevated among patients with celiac disease. For unexplained reasons, the frequency of lymphoma in patients with celiac disease is higher in Ireland and the United Kingdom than in the United States. The possibility of lymphoma must be considered whenever a patient with celiac disease who has previously done well on a gluten-free diet is no longer responsive to gluten restriction or a patient who presents with clinical and histologic features consistent with celiac disease does not respond to a gluten-free diet. Other complications of celiac disease include the development of intestinal ulceration independent of lymphoma and so-called refractory sprue (see above) and collagenous sprue. In collagenous sprue, a layer of collagen-like material is present beneath the basement membrane;

1	ulceration independent of lymphoma and so-called refractory sprue (see above) and collagenous sprue. In collagenous sprue, a layer of collagen-like material is present beneath the basement membrane; patients with collagenous sprue generally do not respond to a gluten-free diet and often have a poor prognosis.

1	Tropical sprue is a poorly understood syndrome that affects and is manifested by chronic diarrhea, steatorrhea, weight loss, and nutritional deficiencies, including those of both folate and cobalamin. This disease affects 5–10% of the population in some tropical areas. Chronic diarrhea in a tropical environment is most often caused by infectious agents, including G. lamblia, Yersinia enterocolitica, C. difficile, Cryptosporidium parvum, and Cyclospora cayetanensis. Tropical sprue should not be entertained as a possible diagnosis until the presence of cysts and trophozoites has been excluded in three stool samples. Chronic infections of the gastrointestinal tract and diarrhea in patients with or without AIDS are discussed in Chaps. 160, 161, and 226.

1	The small-intestinal mucosa of individuals living in tropical areas is not identical to that of individuals who reside in temperate climates. In residents of tropical areas, biopsies reveal a mild alteration of villous architecture with a modest increase in mononuclear cells in the lamina propria, which on occasion can be as severe as that seen in celiac disease. These changes are observed both in native residents and in expatriates living in tropical regions and are usually associated with mild decreases in absorptive function, but they revert to “normal” when an individual moves or returns to a temperate area. Some have suggested that the changes seen in tropical enteropathy and in tropical sprue represent different ends of the spectrum of a single entity, but convincing evidence to support this concept is lacking.

1	Etiology Because tropical sprue responds to antibiotics, the consensus is that it may be caused by one or more infectious agents. Nonetheless, the etiology and pathogenesis of tropical sprue are uncertain. First, its occurrence is not evenly distributed in all tropical areas; rather, it is found in specific locations, including southern India, the Philippines, and several Caribbean islands (e.g., Puerto Rico, Haiti), but is rarely observed in Africa, Jamaica, or Southeast Asia. Second, an occasional individual does not develop symptoms of tropical sprue until long after having left an endemic area. For this reason, celiac disease (often referred to as celiac sprue) was originally called nontropical sprue to distinguish it from tropical sprue. Third, multiple microorganisms have been identified in jejunal aspirates, with relatively little consistency among studies. Klebsiella pneumoniae, Enterobacter cloacae, and E. coli have been implicated in some studies of tropical sprue, while

1	in jejunal aspirates, with relatively little consistency among studies. Klebsiella pneumoniae, Enterobacter cloacae, and E. coli have been implicated in some studies of tropical sprue, while other studies have favored a role for a toxin produced by one or more of these bacteria. Fourth, the incidence of tropical sprue appears to have decreased substantially during the past two or three decades, perhaps in relation to improved sanitation in many tropical countries during this time. Some have speculated that the reduced occurrence is attributable to the wider use of antibiotics in acute diarrhea, especially in travelers to tropical areas from temperate countries. Fifth, the role of folic acid deficiency in the pathogenesis of tropical sprue requires clarification. Folic acid is absorbed exclusively in the duodenum and proximal jejunum, and most patients with tropical sprue have evidence of folate malabsorption and depletion. Although folate deficiency can cause changes in

1	absorbed exclusively in the duodenum and proximal jejunum, and most patients with tropical sprue have evidence of folate malabsorption and depletion. Although folate deficiency can cause changes in small-intestinal mucosa that are corrected by folate replacement, several earlier studies reporting that tropical sprue could be cured by folic acid did not provide an explanation for the “insult” that was initially responsible for folate malabsorption.

1	The clinical pattern of tropical sprue varies in different areas of the world (e.g., India vs. Puerto Rico). Not infrequently, individuals in southern India initially report the occurrence of acute enteritis before the development of steatorrhea and malabsorption. In contrast, in Puerto Rico, a more insidious onset of symptoms and a more dramatic response to antibiotics are seen than in some other locations. Tropical sprue in different areas of the world may not be the same disease, and similar clinical entities may have different etiologies.

1	Diagnosis The diagnosis of tropical sprue is best based on an abnormal small-intestinal mucosal biopsy in an individual with chronic diarrhea and evidence of malabsorption who is either residing or has recently lived in a tropical country. The small-intestinal biopsy in tropical sprue does not reveal pathognomonic features but resembles, and can often be indistinguishable from, that seen in celiac disease (Fig. 349-4). The biopsy sample in tropical sprue has less villous architectural alteration and more mononuclear cell infiltrate in the lamina propria. In contrast to those of celiac disease, the histologic features of tropical sprue manifest with a similar degree of severity throughout the small intestine, and a gluten-free diet does not result in either clinical or histologic improvement in tropical sprue.

1	Broad-spectrum antibiotics and folic acid are most often curative, especially if the patient leaves the tropical area and does not return. Tetracycline should be used for up to 6 months and may be associated with improvement within 1–2 weeks. Folic acid alone induces hematologic remission as well as improvement in appetite, weight gain, and some morphologic changes in small-intestinal biopsy. Because of marked folate deficiency, folic acid is most often given together with antibiotics.

1	Short-bowel syndrome is a descriptive term for the myriad clinical problems that follow resection of various lengths of small intestine or, on rare occasions, are congenital (e.g., microvillous inclusion disease). 1943 The factors that determine both the type and degree of symptoms include (1) the specific segment (jejunum vs. ileum) resected, (2) the length of the resected segment, (3) the integrity of the ileocecal valve, whether any large intestine has also been removed, (5) residual disease in the remaining small and/or large intestine (e.g., Crohn’s disease, mesenteric artery disease), and (6) the degree of adaptation in the remaining intestine. Short-bowel syndrome can occur in persons of any age, from neonates to the elderly.

1	Three different situations in adults mandate intestinal resection: mesenteric vascular disease, including atherosclerosis, thrombotic phenomena, and vasculitides; (2) primary mucosal and submucosal disease (e.g., Crohn’s disease); and (3) operations without preexisting small-intestinal disease (e.g., after trauma).

1	After resection of the small intestine, the residual intestine undergoes adaptation of both structure and function that may last for up to 6–12 months. Continued intake of dietary nutrients and calories is required to stimulate adaptation via direct contact with the intestinal mucosa, the release of one or more intestinal hormones, and pancreatic and biliary secretions. Thus, enteral nutrition with calorie administration must be maintained, especially in the early postoperative period, even if an extensive intestinal resection requiring parenteral nutrition (PN) has been performed. The subsequent ability of such patients to absorb nutrients will not be known for several months, until adaptation is complete.

1	Multiple factors besides the absence of intestinal mucosa (required for lipid, fluid, and electrolyte absorption) contribute to diarrhea and steatorrhea in these patients. Removal of the ileum, and especially the ileocecal valve, is often associated with more severe diarrhea than jejunal resection. Without part or all of the ileum, diarrhea can be caused by an increase in bile acids entering the colon; these acids stimulate colonic fluid and electrolyte secretion. Absence of the ileocecal valve is also associated with a decrease in intestinal transit time and bacterial overgrowth from the colon. The presence of the colon (or a major portion) is associated with substantially less diarrhea and a lower likelihood of intestinal failure (an inability to maintain nutrition without parenteral support) as a result of fermentation of nonabsorbed carbohydrates to SCFAs. The latter are absorbed in the colon and stimulate Na and water absorption, improving overall fluid balance. Lactose

1	support) as a result of fermentation of nonabsorbed carbohydrates to SCFAs. The latter are absorbed in the colon and stimulate Na and water absorption, improving overall fluid balance. Lactose intolerance as a result of the removal of lactase-containing mucosa as well as gastric hypersecretion may also contribute to the diarrhea.

1	In addition to diarrhea and/or steatorrhea, a range of nonintestinal symptoms is observed in some patients. The frequency of renal calcium oxalate calculi increases significantly in patients with a small-intestinal resection and an intact colon; this greater frequency is due to an increase in oxalate absorption by the large intestine, with subsequent enteric hyperoxaluria. Two possible mechanisms for the increase in oxalate absorption in the colon have been suggested: (1) increased bile acids and fatty acids that augment colonic mucosal permeability, resulting in enhanced oxalate absorption; and (2) increased fatty acids that bind calcium, resulting in an enhanced amount of soluble oxalate that is then absorbed. Since oxalate is high in relatively few foods (e.g., spinach, rhubarb, tea), dietary restrictions alone do not constitute adequate treatment. Cholestyramine (an anion-binding resin) and calcium have proved useful in reducing hyperoxaluria. Similarly, an increase in cholesterol

1	restrictions alone do not constitute adequate treatment. Cholestyramine (an anion-binding resin) and calcium have proved useful in reducing hyperoxaluria. Similarly, an increase in cholesterol gallstones is related to a decrease in the bile-acid pool size, which results in the generation of cholesterol supersaturation in gallbladder bile. Gastric hypersecretion of acid occurs in many patients after large resections of the small intestine. The etiology is unclear but may be related to either reduced hormonal inhibition of acid secretion or increased gastrin levels due to reduced small-intestinal catabolism of circulating gastrin. The resulting gastric acid secretion may be an important factor contributing to diarrhea and steatorrhea. A reduced pH in the duodenum can inactivate pancreatic lipase and/or precipitate duodenal bile acids, thereby increasing steatorrhea, and an increase in gastric secretion can create a volume overload relative to the reduced small-intestinal absorptive

1	lipase and/or precipitate duodenal bile acids, thereby increasing steatorrhea, and an increase in gastric secretion can create a volume overload relative to the reduced small-intestinal absorptive capacity. Inhibition of gastric acid secretion with proton pump inhibitors can help reduce diarrhea and steatorrhea, but only for the first 6 months.

1	Disorders of Absorption duodenal afferent loop following subtotal gastrectomy and gastrojejunostomy; (4) a bypass of the intestine (e.g., a jejunoileal bypass for

1	Treatment of short-bowel syndrome depends on the severity of symptoms and on whether the individual is able to maintain caloric and electrolyte balance with oral intake alone. Initial treatment includes judicious use of opiates (including codeine) to reduce stool output and to establish an effective diet. If the colon is in situ, the initial diet should be low in fat and high in carbohydrate in order to minimize diarrhea from fatty acid stimulation of colonic fluid secretion. MCTs (see Table 349-3), a low-lactose diet, and various soluble fiber–containing diets should also be tried. In the absence of an ileocecal valve, possible bacterial overgrowth must be considered and treated. If gastric acid hypersecretion is contributing to diarrhea and steatorrhea, a proton pump inhibitor may be helpful. Usually none of these therapeutic approaches provides an instant solution, but each can contribute to the reduction of disabling diarrhea.

1	The patient’s vitamin and mineral status must also be monitored; replacement therapy should be initiated if indicated. Fat-soluble vitamins, folate, cobalamin, calcium, iron, magnesium, and zinc are the most critical factors to monitor on a regular basis. If these approaches are not successful, home PN is an established therapy that can be maintained for many years. Small-intestinal transplantation is becoming established as a possible approach for individuals with extensive intestinal resection who cannot be maintained without PN—i.e., those with intestinal failure. A recombinant analogue of glucagon-like peptide 2 (GLP-2; teduglutide) is approved for use in patients with PN-dependent short-bowel syndrome on the basis of its ability to increase intestinal growth and improve absorption.

1	Bacterial overgrowth syndromes comprise a group of disorders with diarrhea, steatorrhea, and macrocytic anemia whose common feature is the proliferation of colonic-type bacteria within the small intestine. This bacterial proliferation is due to stasis caused by impaired peristalsis (functional stasis), changes in intestinal anatomy (anatomic stasis), or direct communication between the small and large intestine. These conditions have also been referred to as stagnant bowel syndrome or blind loop syndrome.

1	Pathogenesis The manifestations of bacterial overgrowth syndromes are a direct consequence of the presence of increased amounts of a colonic-type bacterial flora, such as E. coli or Bacteroides, in the small intestine. Macrocytic anemia is due to cobalamin—not folate—deficiency. Most bacteria require cobalamin for growth, and increasing concentrations of bacteria use up the relatively small amounts of dietary cobalamin. Steatorrhea is due to impaired micelle formation as a consequence of a reduced intraduodenal concentration of conjugated bile acids and the presence of unconjugated bile acids. Certain bacteria, including Bacteroides, deconjugate conjugated bile acids to unconjugated bile acids. Unconjugated bile acids are absorbed more rapidly than conjugated bile acids; as a result, the intraduodenal concentration of bile acids is reduced. In addition, the CMC of unconjugated bile acids is higher than that of conjugated bile acids, and the result is a decrease in micelle formation.

1	intraduodenal concentration of bile acids is reduced. In addition, the CMC of unconjugated bile acids is higher than that of conjugated bile acids, and the result is a decrease in micelle formation. Diarrhea is due, at least in part, to steatorrhea, when it is present. However, some patients manifest diarrhea without steatorrhea, and it is assumed that the colonic-type bacteria in these patients are producing one or more bacterial enterotoxins that are responsible for fluid secretion and diarrhea.

1	Etiology The etiology of these different disorders is bacterial proliferation in the small-intestinal lumen secondary to anatomic or functional stasis or to a communication between the relatively sterile small intestine and the colon, with its high levels of aerobic and anaerobic bacteria. Several examples of anatomic stasis have been identified: (1) one or more diverticula (both duodenal and jejunal) (Fig. 294-3C); (2) fistulas and strictures related to Crohn’s disease (Fig. 349-3D); (3) a proximal obesity); and (5) dilation at the site of a previous intestinal anastomosis. These anatomic derangements are often associated with the presence of a segment (or segments) of intestine out of continuity of propagated peristalsis, with consequent stasis and bacterial proliferation. Bacterial overgrowth syndromes can also occur in the absence of an anatomic blind loop when functional stasis is present. Impaired peristalsis and bacterial overgrowth in the absence of a blind loop occur in

1	overgrowth syndromes can also occur in the absence of an anatomic blind loop when functional stasis is present. Impaired peristalsis and bacterial overgrowth in the absence of a blind loop occur in scleroderma, where motility abnormalities exist in both the esophagus and the small intestine (Chap. 382). Functional stasis and bacterial overgrowth can also develop in association with diabetes mellitus and in the small intestine when a direct connection exists between the small and large intestines, including an ileocolonic resection, or occasionally after an enterocolic anastomosis that permits entry of bacteria into the small intestine as a result of bypassing the ileocecal valve.

1	Diagnosis The diagnosis may be suspected from the combination of a low serum cobalamin level and an elevated serum folate level, as enteric bacteria frequently produce folate compounds that are absorbed in the duodenum. Ideally, the bacterial overgrowth syndromes are diagnosed by the demonstration of increased levels of aerobic and/or anaerobic colonic-type bacteria in a jejunal aspirate obtained by intubation. However, this specialized test is rarely available. Breath hydrogen testing with administration of lactulose (a nondigestible disaccharide) has also been used to detect bacterial overgrowth. The Schilling test can diagnose bacterial overgrowth (see Chap. 350e) but is not available routinely. Often the diagnosis is suspected clinically and confirmed by the response to treatment.

1	Primary treatment should be directed, if at all possible, to the surgical correction of an anatomic blind loop. In the absence of functional stasis, it is important to define the anatomic relationships responsible for stasis and bacterial overgrowth. For example, bacterial overgrowth secondary to strictures, one or more diverticula, or a proximal afferent loop can potentially be cured by surgical correction of the anatomic state. In contrast, the functional stasis of scleroderma or certain anatomic stasis states (e.g., multiple jejunal diverticula) cannot be corrected surgically, and these conditions should be treated with broad-spectrum antibiotics. Tetracycline used to be the initial drug of choice; because of increasing resistance, however, other antibiotics, such as metronidazole, amoxicillin/clavulanic acid, rifaximin and cephalosporins, have been employed. The antibiotic should be given for ~3 weeks or until symptoms remit. Although the natural history of these conditions is

1	amoxicillin/clavulanic acid, rifaximin and cephalosporins, have been employed. The antibiotic should be given for ~3 weeks or until symptoms remit. Although the natural history of these conditions is chronic, antibiotics should not be given continuously. Symptoms usually remit within 2–3 weeks of initial antibiotic therapy. Treatment need not be repeated until symptoms recur. For frequent recurrences, several treatment strategies exist, but the use of antibiotics for 1 week per month, whether or not symptoms are present, is often most effective.

1	Unfortunately, therapy for bacterial overgrowth syndromes is largely empirical, with an absence of clinical trials on which to base rational decisions regarding antibiotic choice, treatment duration, and/or the best approach to therapy for recurrences. Bacterial overgrowth may also occur as a component of another chronic disease, such as Crohn’s disease, radiation enteritis, or short-bowel syndrome. Treatment of the bacterial overgrowth in these settings will not cure the underlying problem but may be very important in ameliorating a subset of clinical problems that are related to bacterial overgrowth.

1	Whipple’s disease is a chronic multisystemic disease associated with diarrhea, steatorrhea, weight loss, arthralgia, and central nervous system (CNS) and cardiac problems; it is caused by the bacterium Tropheryma whipplei. Until the identification of T. whipplei by polymerase chain reaction, the hallmark of Whipple’s disease had been the presence of PAS-positive macrophages in the small intestine (Fig. 349-4E) and other organs with evidence of disease. Etiology T. whipplei, a small (50–500 nm) gram-positive bacillus in the group Actinobacteria, has low virulence but high infectivity. Symptoms of Whipple’s disease are relatively minimal compared to the bacterial burden in multiple tissues.

1	Clinical presentation The onset of Whipple’s disease is insidious and is characterized by diarrhea, steatorrhea, abdominal pain, weight loss, migratory large-joint arthropathy, and fever as well as ophthalmologic and CNS symptoms. Dementia is a relatively late symptom and an extremely poor prognostic sign, especially in patients who experience relapse after the induction of a remission with antibiotics. For unexplained reasons, the disease occurs primarily in middle-aged white men. The steatorrhea in these patients is generally believed to be secondary to both small-intestinal mucosal injury and lymphatic obstruction due to the increased number of PAS-positive macrophages in the lamina propria of the small intestine.

1	Diagnosis The diagnosis of Whipple’s disease is suggested by a multisystemic disease in a patient with diarrhea and steatorrhea. Tissue biopsy of the small intestine and/or other organs that may be involved (e.g., liver, lymph nodes, heart, eyes, CNS, or synovial membranes), given the patient’s symptoms, is the primary approach. The presence of PAS-positive macrophages containing the characteristic small bacilli is suggestive of this diagnosis. However, T. whipplei–containing macrophages can be confused with PAS-positive macrophages containing M. avium complex, which may be a cause of diarrhea in AIDS. The presence of the T. whipplei bacillus outside of macrophages is a more important indicator of active disease than is their presence within the macrophages. T. whipplei has now been successfully grown in culture.

1	The treatment for Whipple’s disease is prolonged use of antibiotics. The current regimen of choice is double-strength trimethoprimsulfamethoxazole for ~1 year. PAS-positive macrophages can persist after successful treatment, and the presence of bacilli outside of macrophages is indicative of persistent infection or an early sign of recurrence. Recurrence of disease activity, especially with dementia, is an extremely poor prognostic sign and requires an antibiotic that crosses the blood-brain barrier. If trimethoprim-sulfamethoxazole is not tolerated, chloramphenicol is an appropriate second choice.

1	Protein-losing enteropathy is not a specific disease but rather a group of gastrointestinal and nongastrointestinal disorders with hypoproteinemia and edema in the absence of either proteinuria or defects in protein synthesis (e.g., chronic liver disease). These diseases are characterized by excess protein loss into the gastrointestinal tract. Normally, ~10% of total protein catabolism occurs via the gastrointestinal tract. Evidence of increased protein loss into the gastrointestinal tract is found in more than 65 different diseases, which can be classified into three groups: (1) mucosal ulceration, such that the protein loss primarily represents exudation across damaged mucosa (e.g., ulcerative colitis, gastrointestinal carcinomas, and peptic ulcer); (2) nonulcerated mucosa, but with evidence of mucosal damage so that the protein loss represents loss across epithelia with altered permeability (e.g., celiac disease and Ménétrier’s disease in the small intestine and stomach,

1	with evidence of mucosal damage so that the protein loss represents loss across epithelia with altered permeability (e.g., celiac disease and Ménétrier’s disease in the small intestine and stomach, respectively); and (3) lymphatic dysfunction, representing either primary lymphatic disease or lymphatic disease secondary to partial lymphatic obstruction that may occur as a result of enlarged lymph nodes or cardiac disease.

1	Diagnosis The diagnosis of protein-losing enteropathy is suggested 1945 by peripheral edema and low serum albumin and globulin levels in the absence of renal and hepatic disease. An individual with protein-losing enteropathy only rarely has selective loss of only albumin or only globulins. Therefore, marked reduction of serum albumin with normal serum globulins should not initiate an evaluation for protein-losing enteropathy but should suggest renal and/or hepatic disease. Likewise, reduced serum globulins with normal serum albumin levels are more likely a result of reduced globulin synthesis rather than enhanced globulin loss into the intestine. An increase in protein loss into the gastrointestinal tract has been documented by the administration of one of several radiolabeled proteins and its quantitation in stool during a 24or 48-h period. Unfortunately, none of these radiolabeled proteins is available for routine clinical use. α1-Antitrypsin, a protein that accounts for ~4% of

1	and its quantitation in stool during a 24or 48-h period. Unfortunately, none of these radiolabeled proteins is available for routine clinical use. α1-Antitrypsin, a protein that accounts for ~4% of total serum proteins and is resistant to proteolysis, can be used to detect enhanced rates of serum protein loss into the intestinal tract but cannot be used to assess gastric protein loss because of its degradation in an acid milieu. α1-Antitrypsin clearance is measured by determining stool volume as well as both stool and plasma α1-antitrypsin concentrations. In addition to the loss of protein via abnormal and distended lymphatics, peripheral lymphocytes may be lost via lymphatics, with consequent relative lymphopenia. Thus, lymphopenia in a patient with hypoproteinemia indicates increased loss of protein into the gastrointestinal tract.

1	Patients with increased protein loss into the gastrointestinal tract from lymphatic obstruction often have steatorrhea and diarrhea. The steatorrhea is a result of altered lymphatic flow as lipid-containing chylomicrons exit from intestinal epithelial cells via intestinal lymphatics (Table 349-4; Fig. 349-4). In the absence of mechanical or anatomic lymphatic obstruction, intrinsic intestinal lymphatic dysfunction— with or without lymphatic dysfunction in the peripheral extremities—has been designated intestinal lymphangiectasia. Similarly, ~50% of individuals with intrinsic peripheral lymphatic disease (Milroy’s disease) also have intestinal lymphangiectasia and hypoproteinemia. Other than steatorrhea and enhanced protein loss into the gastrointestinal tract, all other aspects of intestinal absorptive function are normal in intestinal lymphangiectasia.

1	Other Causes Patients who appear to have idiopathic protein-losing enteropathy without evidence of gastrointestinal disease should be examined for cardiac disease—especially right-sided valvular disease and chronic pericarditis (Chaps. 284 and 288). On occasion, hypoproteinemia can be the only presenting manifestation in these two types of heart disease. Ménétrier’s disease (also called hypertrophic gastropathy) is an uncommon entity that involves the body and fundus of the stomach and is characterized by large gastric folds, reduced gastric acid secretion, and, at times, enhanced protein loss into the stomach.

1	As excess protein loss into the gastrointestinal tract is most often secondary to a specific disease, treatment should be directed primarily to the underlying disease process and not to the hypoproteinemia. For example, if significant hypoproteinemia with resulting peripheral edema is secondary to celiac disease or ulcerative colitis, a gluten-free diet and mesalamine, respectively, would be the initial therapy. When enhanced protein loss is secondary to lymphatic obstruction, it is critical to establish the nature of this obstruction. Identification of mesenteric nodes or lymphoma may be possible by imaging studies. Similarly, it is important to exclude cardiac disease as a cause of protein-losing enteropathy, either by echosonography or, on occasion, by a right-heart catheterization.

1	The increased protein loss that occurs in intestinal lymphangiectasia is a result of distended lymphatics associated with lipid malabsorption. The hypoproteinemia is treated with a low-fat diet and the administration of MCTs (Table 349-3), which do not exit from the intestinal epithelial cells via lymphatics but are delivered to the body via the portal vein. Disorders of Absorption taBle 349-8 ClassifiCation of malaBsorPtion syndromes Deficiency or inactivation of pancreatic lipase Exocrine pancreatic insufficiency Pancreatic insufficiency—congenital or acquired Gastrinoma—acid inactivation of lipasea Cholestatic liver disease Bacterial overgrowth in small intestine: Interrupted enterohepatic circulation of bile salts

1	Crohn’s diseasea Drugs (binding or precipitating bile salts)—neomycin, cholestyramine, calcium carbonate Impaired mucosal absorption/mucosal loss or defect Intestinal resection or bypassa Inflammation, infiltration, or infection: Crohn’s diseasea Celiac disease Amyloidosis Collagenous sprue Sclerodermaa Whipple’s diseasea Lymphomaa Radiation enteritisa Eosinophilic enteritis Folate and vitamin B12 deficiency Mastocytosis Infections—giardiasis Tropical sprue Graft versus host disease Genetic disorders Disaccharidase deficiency Agammaglobulinemia Abetalipoproteinemia Hartnup’s disease Cystinuria Impaired nutrient delivery to and/or from intestine: Lymphatic obstruction Circulatory disorders Lymphomaa Congestive heart failure Lymphangiectasia Constrictive pericarditis Mesenteric artery atherosclerosis Vasculitis Endocrine and metabolic disorders Diabetesa Hypoparathyroidism Adrenal insufficiency Hyperthyroidism Carcinoid syndrome aMalabsorption caused by more than one mechanism.

1	Weight loss/malnutrition Anorexia, malabsorption of nutrients Diarrhea Impaired absorption or secretion of water and electrolytes; colonic fluid secretion secondary to unabsorbed dihydroxy bile acids and fatty acids Flatus Bacterial fermentation of unabsorbed carbohydrate Glossitis, cheilosis, stomatitis Deficiency of iron, vitamin B12, folate, and vitamin A Abdominal pain Bowel distention or inflammation, pancreatitis Bone pain Calcium, vitamin D malabsorption, protein deficiency, osteoporosis Tetany, paresthesia Calcium and magnesium malabsorption Weakness Anemia, electrolyte depletion Azotemia, hypotension Fluid and electrolyte depletion Amenorrhea, decreased libido Protein depletion, decreased calories, Anemia Impaired absorption of iron, folate, vitamin B12 Bleeding Vitamin K malabsorption, Dermatitis Deficiency of vitamin A, zinc, and essential

1	Anemia Impaired absorption of iron, folate, vitamin B12 Bleeding Vitamin K malabsorption, Dermatitis Deficiency of vitamin A, zinc, and essential The many conditions that can produce malabsorption are classified by their pathophysiology in Table 349-8. The pathophysiology of the various clinical manifestations of malabsorption is summarized in Table 349-9. The Schilling Test Henry J. Binder The Schilling test is performed to determine the cause of cobala-min malabsorption. Unfortunately, this test has not been available commercially in the United States for the last few years. Since an under-standing of the physiology and pathophysiology of cobalamin absorp-350e

1	CHAPTER 350e The Schilling Test tion is very valuable in enhancing one’s understanding of aspects of gastric, pancreatic, and ileal function, discussion of the Schilling test is provided as supplemental information to Chap. 349. Because cobalamin absorption requires multiple steps, including gastric, pancreatic, and ileal processes, the Schilling test also can be used to assess the integrity of the organs involved in those processes (Chap. 128).

1	Cobalamin is present primarily in meat. Except in strict vegans, dietary cobalamin deficiency is exceedingly uncommon. Dietary cobalamin is bound in the stomach to a glycoprotein called R-binder protein, which is synthesized in both the stomach and the salivary glands. This cobalamin–R binder complex is formed in the acid milieu of the stomach. Cobalamin absorption has an absolute requirement for intrinsic factor, another glycoprotein synthesized and released by gastric parietal cells, to promote its uptake by specific cobalamin receptors on the brush border of ileal enterocytes. Pancreatic protease enzymes split the cobalamin–R binder complex to release cobalamin in the proximal small intestine, where cobalamin then is bound by intrinsic factor. As a consequence, cobalamin absorption may be abnormal in the following conditions: 1.

1	As a consequence, cobalamin absorption may be abnormal in the following conditions: 1. Pernicious anemia. In this disease, immunologically mediated atrophy of gastric parietal cells leads to an absence of both gastric acid and intrinsic factor secretion. 2. Chronic pancreatitis can result from a deficiency of pancreatic proteases to split the cobalamin–R binder complex. Although 50% of patients with chronic pancreatitis reportedly have an abnormal Schilling test that is corrected by pancreatic enzyme replacement, cobalamin-responsive macrocytic anemia in chronic pancreatitis is extremely rare. Although this probably reflects a difference in the digestion/absorption of cobalamin in food versus that in a crystalline form, the Schilling test still can be used to assess pancreatic exocrine function. 3.

1	3. Achlorhydria is the absence of hydrochloric acid; intrinsic factor is also secreted with acid which is responsible for splitting cobalamin away from the proteins in food to which it is bound. Up to one-third of individuals >60 years of age have marginal vitamin B12 absorption because of an inability to release cobalamin from food; these people have no defects in the absorption of crystalline 350e-1 vitamin B12. 4. Bacterial overgrowth syndromes, which are most often secondary to stasis in the small intestine, lead to bacterial utilization of cobalamin (often referred to as stagnant bowel syndrome; see below). 5. Ileal dysfunction (as a result of either inflammation or prior intestinal resection) is due to impaired function of the mechanism of cobalamin–intrinsic factor uptake by ileal intestinal epithelial cells.

1	In the Schilling test, 58Co-labeled cobalamin is administered orally, and urine is collected for 24 h. The test is dependent on normal renal and bladder function. Urinary excretion of cobalamin reflects cobalamin absorption, provided that intrahepatic binding sites for cobalamin are fully occupied. To ensure saturation of these binding sites so that all absorbed radiolabeled cobalamin will be excreted in urine, 1 mg of cobalamin is administered intramuscularly 1 h after ingestion of the radiolabeled cobalamin. The Schilling test may yield an abnormal result (usually defined as <10% excretion in 24 h) in pernicious anemia, chronic pancreatitis, blind loop syndrome, and ileal disease (Table 350e-1). Therefore, whenever an abnormal Schilling result is obtained, 58Co-labeled cobalamin should be administered on another occasion, this time bound to intrinsic factor, with pancreatic enzymes, or after a 5-day course of antibiotic treatment (often with tetracycline). A variation of the

1	should be administered on another occasion, this time bound to intrinsic factor, with pancreatic enzymes, or after a 5-day course of antibiotic treatment (often with tetracycline). A variation of the Schilling test can detect failure to split cobalamin from food proteins. The labeled cobalamin is cooked together with a scrambled egg and administered orally. People with achlorydria excrete <10% of the labeled cobalamin in the urine. In addition to establishing the etiology for cobalamin deficiency, the Schilling test can help delineate the pathologic process responsible for steatorrhea by assessing ileal, pancreatic, and small-intestinal luminal function. Unfortunately, the Schilling test is performed infrequently because of the unavailability of human intrinsic factor.

1	With After 58Co-Labeled With Intrinsic Pancreatic 5 Days of Cobalamin Factor Enzymes Antibiotics inflammatory Bowel Disease Sonia Friedman, Richard S. Blumberg Inflammatory bowel disease (IBD) is an immune-mediated chronic intestinal condition. Ulcerative colitis (UC) and Crohn’s disease (CD) are the two major types of IBD. GLOBAL CONSIDERATIONS: EPIDEMIOLOGY 351

1	The incidence and prevalence of IBD are highest in Westernized nations, with UC incidence estimates ranging from 0.6 to 24.3 per 100,000 in Europe, 0 to 19.2 per 100,000 in North America, and 0.1 to 6.3 per 100,000 in the Middle East and Asia and CD estimates ranging from 0.3 to 12.7 per 100,000 in Europe, 0 to 20.2 per 100,000 in North America, and 0.04 to 5.0 per 100,000 in the Middle East and Asia (Table 351-1). For prevalence rates, the UC estimates range from 4.9 to 505 per 100,000 in Europe, 37.5 to 248.6 per 100,000 in North America, and 4.9 to 168.3 per 100,000 in the Middle East and Asia, and the CD estimates range from 0.6 to 322 per 100,000 in Europe, 16.7 to 318.5 per 100,000 in North America, and 0.88 to 67.9 per 100,000 in Asia and the Middle East. The highest reported incidence rates are in Canada (19.2 per 100,000 for UC and 20.2 per 100,000 for CD), with approximately 0.6% of the Canadian population having IBD. Countries in the Pacific, including New Zealand and

1	incidence rates are in Canada (19.2 per 100,000 for UC and 20.2 per 100,000 for CD), with approximately 0.6% of the Canadian population having IBD. Countries in the Pacific, including New Zealand and Australia, which share many possible environmental risk factors and similar genetic background as northwest Europe and North America, have high incidence rates of IBD.

1	In countries that are becoming more Westernized, including China, South Korea, India, Lebanon, Iran, Thailand, and countries in the French West Indies and North Africa, IBD appears to be emerging, emphasizing the importance of environmental factors in disease pathogenesis. In Japan, the prevalence of CD has risen rapidly from 2.9 cases per 100,000 in 1986 to 13.5 per 100,000 in 1998, whereas in South Korea, the prevalence of UC has quadrupled from 7.6 per 100,000 in 1997 to 30.9 per 100,000 in 2005. In Hong Kong, the prevalence of UC almost tripled from 2.3 in 1997 to 6.3 per 100,000 over a 9-year period. In Singapore, the prevalence of CD increased from 1.3 in 1990 to 7.2 per 100,000 in 2004. In China the number of cases of UC has increased by fourfold between 1981–1990 and 1991–2000. Increasing immigration to Western societies also has an impact on the incidence and prevalence of IBD. The prevalence of UC among southern Asians who immigrated to the United Kingdom (UK) was

1	Increasing immigration to Western societies also has an impact on the incidence and prevalence of IBD. The prevalence of UC among southern Asians who immigrated to the United Kingdom (UK) was Incidence (North 0–19.2 per 100,000 0–20.2 per 100,000 America) per person-years Age of onset Second to fourth decades Second to fourth and seventh to ninth decades and seventh decades to ninth decades Abbreviation: IBD, inflammatory bowel disease. higher in comparison to the European UK population (17 cases per 1947 100,000 persons vs 7 per 100,000). Spanish patients who emigrated within Europe, but not those who immigrated to Latin America, developed IBD more frequently than controls. Individuals who have immigrated to Westernized countries and then returned to their country of birth also continue to demonstrate an increased risk of developing IBD.

1	Peak incidence of UC and CD is in the second to fourth decades, with 78% of CD studies and 51% of UC studies reporting the highest incidence among those age 20–29 years old. A second modest rise in incidence occurs between the seventh and ninth decades of life. The female-to-male ratio ranges from 0.51 to 1.58 for UC studies and 0.34 to 1.65 for CD studies, suggesting that the diagnosis of IBD is not gender specific. The greatest incidence of IBD is among white and Jewish people, but the incidence of IBD in Hispanic and Asian people is increasing, as noted above. Urban areas have a higher prevalence of IBD than rural areas, and high socioeconomic classes have a higher prevalence than lower socioeconomic classes.

1	Epidemiologic studies have identified a number of potential envi ronmental factors that are associated with disease risk (Fig. 351-1). In Caucasian populations, smoking is an important risk factor in IBD with opposite effects on UC (odds ratio [OR] 0.58) and CD (OR 1.76), whereas in other ethnic groups with different genetic susceptibility, smoking may play a lesser role. There is a protective effect of previous appendectomy with confirmed appendicitis (reduction of 13–26%), particularly at a young age, on the development of UC across different geographical regions and populations. There is a modest association with the development of CD. Oral contraceptive use is associated with the risk of CD (OR 1.4). The association between oral contraceptive use and UC is limited to women with a history of smoking. There is an association between antibiotic use and the development of childhood IBD with children who received one or more dispensations of antibiotics during the first year of life

1	of smoking. There is an association between antibiotic use and the development of childhood IBD with children who received one or more dispensations of antibiotics during the first year of life having a 2.9-fold increase in the risk of developing IBD during childhood. Breastfeeding may also protect against the development of IBD. These factors are consistent with the rapid increase in IBD incidence recently noted during the first decade of life. Infectious gastroenteritis with pathogens (e.g., Salmonella, Shigella, Campylobacter spp., Clostridium difficile) increases IBD risk by twoto threefold. Diets high in animal protein, sugars, sweets, oils, fish and shellfish, and dietary fat, especially ω-6 fatty acids, and low in ω-3 fatty acids have been implicated in increasing the risk of IBD.

1	IBD is a familial disease in 5–10% of patients (Fig. 351-2). Some of these patients may exhibit early-onset disease during the first decade of life and, in CD, a concordance of anatomic site and clinical type within families. In the remainder of patients, IBD is observed in the absence of a family history (i.e., sporadic disease). If a patient has IBD, the lifetime risk that a first-degree relative will be affected is ∼10%. If two parents have IBD, each child has a 36% chance of being affected. In twin studies, 38–58% of monozygotic twins are concordant for CD and 6–18% are concordant for UC, whereas 4% of dizygotic twins are concordant for CD and 0–2% are concordant for UC in Swedish and Danish cohorts. The risks of developing IBD are higher in first-degree relatives of Jewish versus non-Jewish patients: 7.8% versus 5.2% for CD and 4.5% versus 1.6% for UC. GLOBAL CONSIDERATIONS: IBD PHENOTYPES

1	GLOBAL CONSIDERATIONS: IBD PHENOTYPES There are racial differences in IBD location and behavior that may reflect underlying genetic variations and have important implications for diagnosis and management of disease. For example, African-American patients are more likely than non-Hispanic whites to develop esophagogastroduodenal CD, colorectal disease, and perianal disease and are less likely to have ileal involvement. They are also at higher risk for uveitis and sacroiliitis. Hispanics have a higher prevalence of perianal CD and erythema nodosum and a more proximal extent of disease. Fistulizing CD has been reported in nearly one-third of Hispanic patients, up to one-quarter of African-American patients, and up to one-half of Asian patients. Both African-American

1	Stress Microbial flora Enteropathogens Antibiotics Diet, hygiene NSAIDs, smoking Diet, hygiene Immune dysregulation IEC XBP1 NOD2 ATG16L1 TLR4 XBP1 DLG5 ECM1 ITLN1 SLC22A5 DMBT1 PTGER4 IL23R, IL12B, JAK2, STAT3, CCR6, NOD2,TLR4, CARD9, IRF5, ATG16L1, IRGM, LRRK2 TNFSF15,TNFRSF6B TNFAIP3, PTPN2/22 NLRP3, IL18RAP ICOSL,ARPC2, STAT3, IL10 FIGURE 351-1 Pathogenesis of inflammatory bowel disease (IBD). In IBD, the tridirectional relationship between the commensal flora (microbiota), intestinal epithelial cells (IEC), and mucosal immune system is dysregulated, leading to chronic inflammation. Each of these three factors is affected by genetic and environmental factors that determine risk for the disease. NSAIDs, nonsteroidal anti-inflammatory drugs.

1	(Adapted from A Kaser et al: Annu Rev Immunol 28:573, 2010.) and Hispanic CD patients, but not UC patients, had a lower prevalence of family history of IBD than their white counterparts. There are few data on all aspects of disease in Hispanics, in the incidence and prevalence of IBD in African Americans, and in Asians with IBD outside Asia. These ethnic variations implicate the importance of different genetic and/or environmental factors in the pathogenesis of this disorder. Environment Undiagnosedinfections?Early onset Genetics

1	Environment Undiagnosedinfections?Early onset Genetics FIGURE 351-2 A model for the syndromic nature of inflammatory bowel disease. Genetic and environmental factors variably influence the development and phenotypic manifestations of IBD. At the one extreme, IBD is a exemplified as a simple Mendelian disorder as observed in “early-onset IBD” due to single gene defects such as IL10, IL10RA, and IL10RB; and at the other extreme, it may be exemplified by as yet to be described emerging infectious diseases. (Adapted from A Kaser et al: Dig Dis 28:395, 2010.)

1	Under physiologic conditions, homeostasis normally exists between the commensal microbiota, epithelial cells that line the interior of the intestines (intestinal epithelial cells [IECs]) and immune cells within the tissues (Fig. 351-1). A consensus hypothesis is that each of these three major host compartments that function together as an integrated “supraorganism” (microbiota, IECs, and immune cells) are affected by specific environmental (e.g., smoking, antibiotics, enteropathogens) and genetic factors that, in a susceptible host, cumulatively and interactively disrupt homeostasis, which in so doing culminates in a chronic state of dysregulated inflammation; that is IBD. Although chronic activation of the mucosal immune system may represent an appropriate response to an infectious agent, a search for such an agent has thus far been unrewarding in IBD. As such, IBD is currently considered an inappropriate immune response to the endogenous (autocthonous) commensal microbiota within

1	a search for such an agent has thus far been unrewarding in IBD. As such, IBD is currently considered an inappropriate immune response to the endogenous (autocthonous) commensal microbiota within the intestines, with or without some component of autoimmunity. Importantly, the normal, uninflamed intestines contain a large number of immune cells that are in a unique state of activation, in which the gut is restrained from full immunologic responses to the commensal microbiota and dietary antigens by very powerful regulatory pathways that function within the immune system (e.g., T regulatory cells that express the FoxP3 transcription factor and suppress inflammation). During the course of infections or other environmental stimuli in the normal host, full activation of the gut-associated lymphoid tissues occurs but is rapidly superseded by dampening of the immune response and tissue repair. In IBD such processes may not be regulated normally.

1	The genetic underpinning of IBD is known from its occurrence in the context of several genetic syndromes and the development of severe, refractory IBD in early life in the setting of single gene defects that affect the immune system (Table 351-2). In addition, IBD has a familial origin in at least 10% of afflicted individuals (Fig. 351-2). In the majority of patients, IBD is considered to be a polygenic disorder that gives rise to multiple clinical subgroups within UC and CD. A variety of genetic approaches including candidate gene studies, linkage analysis, and genome-wide association studies (GWASs) that focus on the identification of disease-associated, single-nucleotide polymorphisms (SNPs) within the human genome and, more recently, whole-genome sequencing have elucidated many of the genetic factors that affect risk for these diseases. GWASs have, to date, identified 163 genetic loci with 100 of these loci observed to be associated with both disease phenotypes (Table 351-3). The

1	genetic factors that affect risk for these diseases. GWASs have, to date, identified 163 genetic loci with 100 of these loci observed to be associated with both disease phenotypes (Table 351-3). The remainder are specific for either CD (30 loci) or UC (20 loci). These genetic similarities account for the overlapping immunopathogenesis and consequently epidemiologic observations of both diseases in the same families and similarities in response to therapies. Because the specific causal variants for each identified gene or locus are largely unknown, it is not clear whether the similarities in the genetic risk factors associated with CD and UC that are observed are shared at structural or functional levels. The risk conferred by each identified gene or locus is unequal and generally small, such that only ∼20% of the genetic variance is considered to be explained by the current genetic information. Further, many of the genetic risk factors identified are also observed to be associated

1	such that only ∼20% of the genetic variance is considered to be explained by the current genetic information. Further, many of the genetic risk factors identified are also observed to be associated with risk for other immune-mediated diseases, suggesting that related immunogenetic pathways are involved in the pathogenesis of multiple different disorders accounting for the common responsiveness to similar types of biologic therapies (e.g., anti–tumor necrosis factor therapies) and possibly the simultaneous occurrence of these disorders. The diseases and the genetic risk factors that are shared with IBD include rheumatoid arthritis (TNFAIP3), psoriasis (IL23R, IL12B), ankylosing spondylitis (IL23R), type 1 diabetes mellitus (IL10, PTPN2), asthma (ORMDL3), and systemic lupus erythematosus (TNFAIP3, IL10) among others.

1	The genetic factors defined to date that are recognized to mediate risk for IBD have highlighted the importance of several common mechanisms of disease (Table 351-3). These include the following: those genes that are associated with fundamental cell biologic Abbreviations: CD, Crohn’s disease; IBD, inflammatory bowel disease; IL, interleukin; UC, ulcerative colitis.

1	processes such as endoplasmic reticulum (ER) and metabolic stress 1949 (e.g., XBP1, ORMDL3, OCTN), which serve to regulate the secretory activity of cells involved in responses to the commensal microbiota such as Paneth and goblet cells and the manner in which intestinal cells respond to the metabolic products of bacteria; those associated with innate immunity and autophagy (e.g., NOD2, ATG16L1, IRGM, JAK2, STAT3) that function in innate immune cells (both parenchymal and hematopoietic) to respond to and effectively clear bacteria, mycobacteria, and viruses; those that are associated with the regulation of adaptive immunity (e.g., IL23R, IL12B, IL10, PTPN2), which regulate the balance between inflammatory and anti-inflammatory (regulatory) cytokines; and, finally, those that are involved in the development and resolution of inflammation (e.g., MST1, CCR6, TNFAIP3, PTGER4) and ultimately leukocyte recruitment and inflammatory mediator production. Some of these loci are associated with

1	in the development and resolution of inflammation (e.g., MST1, CCR6, TNFAIP3, PTGER4) and ultimately leukocyte recruitment and inflammatory mediator production. Some of these loci are associated with specific subtypes of disease such as the association between NOD2 polymorphisms and fibrostenosing CD or ATG16L1 and fistulizing disease, especially within the ileum. However, the clinical utility of these genetic risk factors for the diagnosis or determination of prognosis and therapeutic responses remains to be defined.

1	The endogenous commensal microbiota within the intestines plays a central role in the pathogenesis of IBD. Humans are born sterile and acquire their commensal microbiota initially from the mother during egress through the birth canal and subsequently from environmental sources. A stable configuration of up to 1000 species of bacteria that achieves a biomass of approximately 1012 colony-forming units per gram of feces is achieved by 3 years of age, which likely persists into adult life, with each individual human possessing a unique combination of species. In addition, the intestines contain other microbial life forms including archae, viruses, and protists. The microbiota is thus considered as a critical and sustaining component of the organism. The establishment and maintenance of the intestinal microbiota composition and function is under the control of host (e.g., immune and epithelial responses), environmental (e.g., diet and antibiotics), and likely genetic (e.g., NOD2) factors

1	microbiota composition and function is under the control of host (e.g., immune and epithelial responses), environmental (e.g., diet and antibiotics), and likely genetic (e.g., NOD2) factors (Fig. 351-1). In turn, the microbiota, through its structural components and metabolic activity, has major influences on the epithelial and immune function of the host, which, through epigenetic effects, may have durable consequences. During early life when the commensal microbiota is being established, these microbial effects on the host may be particularly important in determining later life risk for IBD. Specific components of the microbiota can promote or protect from disease. The commensal microbiota in patients with both UC and CD is demonstrably different from nonafflicted individuals, a state of dysbiosis, suggesting the presence of microorganisms that drive disease (e.g., Proteobacteria such as enteroinvasive and adherent Escherichia coli) and to which the immune response is directed

1	of dysbiosis, suggesting the presence of microorganisms that drive disease (e.g., Proteobacteria such as enteroinvasive and adherent Escherichia coli) and to which the immune response is directed and/or the loss of microorganisms that hinder inflammation (e.g., Firmicutes such as Faecalibacterium prausnitzii). Many of the changes in the commensal microbiota occur as a consequence of the inflammation. In addition, agents that alter the intestinal microbiota such as metronidazole, ciprofloxacin, and elemental diets, may improve CD. CD may also respond to fecal diversion, demonstrating the ability of luminal contents to exacerbate disease.

1	The mucosal immune system is normally unreactive to luminal contents due to oral (mucosal) tolerance. When soluble antigens are administered orally rather than subcutaneously or intramuscularly, antigen-specific nonresponsiveness is induced. Multiple mechanisms are involved in the induction of oral tolerance and include deletion or anergy of antigen-reactive T cells or induction of CD4+ T cells that suppress gut inflammation (e.g., T regulatory cells expressing the FoxP3 transcription factor) that secrete anti-inflammatory cytokines such as interleukin (IL) 10, IL-35, and transforming growth factor β (TGF-β). Oral tolerance may be responsible for the lack of immune responsiveness to dietary antigens and the commensal microbiota + 5q33 IL12B Interleukin 12B IL-12 p40 chain of IL-12/IL-23 ++ 18p11 PTPN2 Protein tyrosine phosphatase, nonreceptor type 2 T cell regulation + Inflammation 3p21 MST1 Macrophage stimulating 1 Macrophage activation ++ 5p13 PTGER4 Prostaglandin E receptor 4 PGE2

1	18p11 PTPN2 Protein tyrosine phosphatase, nonreceptor type 2 T cell regulation + Inflammation 3p21 MST1 Macrophage stimulating 1 Macrophage activation ++ 5p13 PTGER4 Prostaglandin E receptor 4 PGE2 receptor ++ 6q23 TNFAIP3 Tumor necrosis factor, alpha-induced protein 3 (A20) Toll-like receptor regulation + 6q27 CCR6 Chemokine (C-C motif ) receptor 6 Dendritic cell migration +

1	Abbreviations: CD, Crohn’s disease; ER, endoplasmic reticulum; GTPase, guanosine triphosphatase; IL, interleukin; PGE2, prostaglandin E2; UC, ulcerative colitis. Source: Adapted from A Kaser et al: Ann Rev Immunol 28:573, 2010; B Khor et al: Nature 474:307, 2011; and L Jostins et al: Nature 491:119, 2012. in the intestinal lumen. In IBD this suppression of inflammation is altered, leading to uncontrolled inflammation. The mechanisms of this regulated immune suppression are incompletely known.

1	Gene knockout (–/–) or transgenic (Tg) mouse models of IBD, which include those that are directed at genes demonstrated to be associated with risk for the human disease, have revealed that deleting specific cytokines (e.g., IL-2, IL-10, TGF-β) or their receptors, deleting molecules associated with T cell antigen recognition (e.g., T cell antigen receptors), or interfering with IEC barrier function and the regulation of responses to commensal bacteria (e.g., XBP1, N-cadherin, mucus glycoprotein, or nuclear factor-κB [NF-κB]) leads to spontaneous colitis or enteritis. In the majority of circumstances, intestinal inflammation in these animal models requires the presence of the commensal microbiota. Thus, a variety of specific alterations can lead to immune activation by commensal microbiota and inflammation directed at the intestines in mice. How these relate to human IBD remains to be defined, but they are consistent with inappropriate responses of the genetically susceptible host to

1	and inflammation directed at the intestines in mice. How these relate to human IBD remains to be defined, but they are consistent with inappropriate responses of the genetically susceptible host to the commensal microbiota.

1	In both UC and CD, an inflammatory pathway thus likely emerges from the genetic predisposition that is associated with inappropriate innate immune and epithelial sensing and reactivity to commensal bacteria that secrete inflammatory mediators together with inadequate regulatory pathways that lead to activated CD4+ and CD8+ T cells within the epithelium and lamina propria that altogether secrete excessive quantities of inflammatory cytokines relative to anti-inflammatory cytokines. Some cytokines activate other inflammatory cells (macrophages and B cells), and others act indirectly to recruit other lymphocytes, inflammatory leukocytes, and mononuclear cells from the bloodstream into the gut through interactions between homing receptors on leukocytes (e.g., α4β7 integrin) and addressins on vascular endothelium (e.g., MadCAM1). Consistent with this, neutralization of tumor necrosis factor (TNF) or α4β7 integrin demonstrate therapeutic efficacy in IBD. CD4+ T helper (TH) cells that

1	on vascular endothelium (e.g., MadCAM1). Consistent with this, neutralization of tumor necrosis factor (TNF) or α4β7 integrin demonstrate therapeutic efficacy in IBD. CD4+ T helper (TH) cells that promote inflammation are of three major types, all of which may be associated with colitis in animal models and perhaps humans: TH1 cells (secrete interferon [IFN] γ), TH2 cells (secrete IL-4, IL-5, IL-13), and TH17 cells (secrete IL-17, IL-21). TH1 cells induce transmural granulomatous inflammation that resembles CD; TH2 cells, and related natural killer T cells that secrete IL-13, induce superficial mucosal inflammation resembling UC in animal models; and TH17 cells may be responsible for neutrophilic recruitment. However, neutralization of the cytokines produced by these cells, such as IFN-γ or IL-17, has yet to show efficacy in therapeutic trials. Each of these T cell subsets cross-regulate each other. The TH1 cytokine pathway is initiated by IL-12, a key cytokine in the pathogenesis of

1	or IL-17, has yet to show efficacy in therapeutic trials. Each of these T cell subsets cross-regulate each other. The TH1 cytokine pathway is initiated by IL-12, a key cytokine in the pathogenesis of experimental models of mucosal inflammation. IL-4 and IL-23, together with IL-6 and TGF-β, induce TH2 and TH17 cells, respectively, and IL-23 inhibits the suppressive function of regulatory T cells. Activated macrophages secrete TNF and IL-6. These characteristics of the immune response in IBD explain the beneficial therapeutic effects of antibodies to block proinflammatory cytokines or the signaling by their receptors (e.g., anti-TNF, anti-IL-12, anti-IL-23, anti-IL-6, or Janus kinase [JAK] inhibitors) or molecules associated with leukocyte recruitment (e.g., anti-α4β7), or the use of cytokines that inhibit inflammation and promote regulatory T cells (e.g., IL-10) or promote intestinal barrier function and may be beneficial to humans with intestinal inflammation.

1	Once initiated in IBD by abnormal innate immune sensing of bacteria by parenchymal cells (e.g., IECs) and hematopoietic cells (e.g., dendritic cells), the immune inflammatory response is perpetuated by T cell activation. A sequential cascade of inflammatory mediators extends the response; each step is a potential target for therapy. Inflammatory cytokines such as IL-1, IL-6, and TNF have diverse effects on tissues. They promote fibrogenesis, collagen production, activation of tissue metalloproteinases, and the production of other inflammatory mediators; they also activate the coagulation cascade in local blood vessels (e.g., increased production of von Willebrand’s factor). These cytokines are normally produced in response to infection but are usually turned off or inhibited at the appropriate time to limit tissue damage. In IBD their activity is not regulated, resulting in an imbalance between the proinflammatory and anti-inflammatory mediators. Therapies such as the 5-aminosalicylic

1	time to limit tissue damage. In IBD their activity is not regulated, resulting in an imbalance between the proinflammatory and anti-inflammatory mediators. Therapies such as the 5-aminosalicylic acid (5-ASA) compounds and glucocorticoids are potent inhibitors of these inflammatory mediators through inhibition of transcription factors such as NF-κB that regulate their expression.

1	ULCERATIVE COLITIS: MACROSCOPIC FEATURES

1	UC is a mucosal disease that usually involves the rectum and extends proximally to involve all or part of the colon. About 40–50% of patients have disease limited to the rectum and rectosigmoid, 30–40% have disease extending beyond the sigmoid but not involving the whole colon, and 20% have a total colitis. Proximal spread occurs in continuity without areas of uninvolved mucosa. When the whole colon is involved, the inflammation extends 2–3 cm into the terminal ileum in 10–20% of patients. The endoscopic changes of backwash ileitis are superficial and mild and are of little clinical significance. Although variations in macroscopic activity may suggest skip areas, biopsies from normal-appearing mucosa are usually abnormal. Thus, it is important to obtain multiple biopsies from apparently uninvolved mucosa, whether proximal or distal, during endoscopy. One caveat is that effective medical therapy can change the appearance of the mucosa such that either skip areas or the entire colon can

1	mucosa, whether proximal or distal, during endoscopy. One caveat is that effective medical therapy can change the appearance of the mucosa such that either skip areas or the entire colon can be microscopically normal.

1	With mild inflammation, the mucosa is erythematous and has a fine granular surface that resembles sandpaper. In more severe disease, the mucosa is hemorrhagic, edematous, and ulcerated (Fig. 351-3). In long-standing disease, inflammatory polyps (pseudopolyps) may be present as a result of epithelial regeneration. The mucosa may appear normal in remission, but in patients with many years of disease it appears atrophic and featureless, and the entire colon becomes narrowed and shortened. Patients with fulminant disease can develop a toxic colitis or megacolon where the bowel wall thins and the mucosa is severely ulcerated; this may lead to perforation. ULCERATIVE COLITIS: MICROSCOPIC FEATURES

1	ULCERATIVE COLITIS: MICROSCOPIC FEATURES Histologic findings correlate well with the endoscopic appearance and clinical course of UC. The process is limited to the mucosa and superficial submucosa, with deeper layers unaffected except in fulminant disease. In UC, two major histologic features suggest chronicity and help distinguish it from infectious or acute self-limited colitis. First, the crypt architecture of the colon is distorted; crypts may be bifid and reduced in number, often with a gap between the crypt bases and the muscularis mucosae. Second, some patients have basal plasma cells and multiple basal lymphoid aggregates. Mucosal vascular congestion, with edema and focal hemorrhage, and an inflammatory cell infiltrate of neutrophils, lymphocytes, plasma cells, and macrophages may be present.

1	FIGURE 351-4 Medium-power view of colonic mucosa in ulcerative colitis showing diffuse mixed inflammation, basal lymphoplasmacytosis, crypt atrophy and irregularity, and superficial erosion. These features are typical of chronic active ulcerative colitis. (Courtesy of Dr. R. Odze, Division of Gastrointestinal Pathology, Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts; with permission.) The neutrophils invade the epithelium, usually in the crypts, giving rise to cryptitis and, ultimately, to crypt abscesses (Fig. 351-4). Ileal changes in patients with backwash ileitis include villous atrophy and crypt regeneration with increased inflammation, increased neutrophil and mononuclear inflammation in the lamina propria, and patchy cryptitis and crypt abscesses. CROHN’S DISEASE: MACROSCOPIC FEATURES

1	CROHN’S DISEASE: MACROSCOPIC FEATURES CD can affect any part of the gastrointestinal (GI) tract from the mouth to the anus. Some 30–40% of patients have small bowel disease alone, 40–55% have disease involving both the small and large intestines, and 15–25% have colitis alone. In the 75% of patients with small intestinal disease, the terminal ileum is involved in 90%. Unlike UC, which almost always involves the rectum, the rectum is often spared in CD. CD is segmental with skip areas in the midst of diseased intestine (Fig. 351-5). Perirectal fistulas, fissures, abscesses, and anal stenosis are present in one-third of patients with CD, particularly those with

1	FIGURE 351-3 Ulcerative colitis. Diffuse (nonsegmental) mucosal disease, with broad areas of ulceration. The bowel wall is not thick-ened, and there is no cobblestoning. (Courtesy of Dr. R. Odze, Division of Gastrointestinal Pathology, Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts; with permission.) FIGURE 351-5 Crohn’s disease of the colon showing thickening of the wall, with stenosis, linear serpiginous ulcers and cobbleston-ing of the mucosa. (Courtesy of Dr. R Odze, Division of Gastrointestinal Pathology, Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts; with permission.)

1	FIGURE 351-6 Medium-power view of Crohn’s colitis showing mixed acute and chronic inflammation, crypt atrophy, and multiple small epithelioid granulomas in the mucosa. (Courtesy of Dr. R Odze, Division of Gastrointestinal Pathology, Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts; with permission.) colonic involvement. Rarely, CD may also involve the liver and the pancreas. Unlike UC, CD is a transmural process. Endoscopically, aphthous or small superficial ulcerations characterize mild disease; in more active disease, stellate ulcerations fuse longitudinally and transversely to demarcate islands of mucosa that frequently are histologically normal. This “cobblestone” appearance is characteristic of CD, both endoscopically and by barium radiography. As in UC, pseudopolyps can form in CD.

1	Active CD is characterized by focal inflammation and formation of fistula tracts, which resolve by fibrosis and stricturing of the bowel. The bowel wall thickens and becomes narrowed and fibrotic, leading to chronic, recurrent bowel obstructions. Projections of thickened mesentery encase the bowel (“creeping fat”), and serosal and mesenteric inflammation promotes adhesions and fistula formation. CROHN’S DISEASE: MICROSCOPIC FEATURES

1	CROHN’S DISEASE: MICROSCOPIC FEATURES The earliest lesions are aphthoid ulcerations and focal crypt abscesses with loose aggregations of macrophages, which form noncaseating granulomas in all layers of the bowel wall (Fig. 351-6). Granulomas can be seen in lymph nodes, mesentery, peritoneum, liver, and pancreas. Although granulomas are a pathognomonic feature of CD, they are rarely found on mucosal biopsies. Surgical resection reveals granulomas in about one-half of cases. Other histologic features of CD include submucosal or subserosal lymphoid aggregates, particularly away from areas of ulceration, gross and microscopic skip areas, and transmural inflammation that is accompanied by fissures that penetrate deeply into the bowel wall and sometimes form fistulous tracts or local abscesses.

1	ULCERATIVE COLITIS Signs and Symptoms The major symptoms of UC are diarrhea, rectal bleeding, tenesmus, passage of mucus, and crampy abdominal pain. The severity of symptoms correlates with the extent of disease. Although UC can present acutely, symptoms usually have been present for weeks to months. Occasionally, diarrhea and bleeding are so intermittent and mild that the patient does not seek medical attention. Patients with proctitis usually pass fresh blood or blood-stained mucus, either mixed with stool or streaked onto the surface of a normal or hard stool. They also have tenesmus, or urgency with a feeling of incomplete evacuation, but rarely have abdominal pain. With proctitis or proctosigmoiditis, proximal transit slows, which may account for the constipation commonly seen in patients with distal disease.

1	When the disease extends beyond the rectum, blood is usually mixed with stool or grossly bloody diarrhea may be noted. Colonic motility is altered by inflammation with rapid transit through the inflamed intestine. When the disease is severe, patients pass a liquid stool containing blood, pus, and fecal matter. Diarrhea is often nocturnal and/or postprandial. Although severe pain is not a prominent symptom, some patients with active disease may experience vague lower abdominal discomfort or mild central abdominal cramping. Severe cramping and abdominal pain can occur with severe attacks of the disease. Other symptoms in moderate to severe disease include anorexia, nausea, vomiting, fever, and weight loss.

1	Physical signs of proctitis include a tender anal canal and blood on rectal examination. With more extensive disease, patients have tenderness to palpation directly over the colon. Patients with a toxic colitis have severe pain and bleeding, and those with megacolon have hepatic tympany. Both may have signs of peritonitis if a perforation has occurred. The classification of disease activity is shown in Table 351-4.

1	Laboratory, Endoscopic, and Radiographic Features Active disease can be associated with a rise in acute-phase reactants (C-reactive protein [CRP]), platelet count, and erythrocyte sedimentation rate (ESR), and a decrease in hemoglobin. Fecal lactoferrin is a highly sensitive and specific marker for detecting intestinal inflammation. Fecal calprotectin levels correlate well with histologic inflammation, predict relapses, and detect pouchitis. Both fecal lactoferrin and calprotectin are becoming an integral part of IBD management and are used frequently to rule out active inflammation versus symptoms of irritable bowel or bacterial overgrowth. In severely ill patients, the serum albumin level will fall rather quickly. Leukocytosis may be present but is not a specific indicator of disease activity. Proctitis or proctosigmoiditis rarely causes a rise in CRP. Diagnosis relies on the patient’s history; clinical symptoms; negative stool examination for bacteria, C. difficile toxin, and ova

1	Proctitis or proctosigmoiditis rarely causes a rise in CRP. Diagnosis relies on the patient’s history; clinical symptoms; negative stool examination for bacteria, C. difficile toxin, and ova and parasites; sigmoidoscopic appearance (see Fig. 345-4A); and histology of rectal or colonic biopsy specimens.

1	Sigmoidoscopy is used to assess disease activity and is usually performed before treatment. If the patient is not having an acute flare, colonoscopy is used to assess disease extent and activity (Fig. 351-7). Endoscopically mild disease is characterized by erythema, decreased vascular pattern, and mild friability. Moderate disease is characterized by marked erythema, absent vascular pattern, friability and erosions, and severe disease by spontaneous bleeding and ulcerations. Histologic features change more slowly than clinical features but can also be used to grade disease activity.

1	The earliest radiologic change of UC seen on single-contrast barium enema is a fine mucosal granularity. With increasing severity, the mucosa becomes thickened, and superficial ulcers are seen. Deep ulcerations can appear as “collar-button” ulcers, which indicate that the ulceration has penetrated the mucosa. Haustral folds may be normal in mild disease, but as activity progresses they become edematous and thickened. Loss of haustration can occur, especially in patients with long-standing disease. In addition, the colon becomes shortened and narrowed. Polyps in the colon may be postinflammatory polyps or pseudopolyps, adenomatous polyps, or carcinoma. FIGURE 351-7 Colonoscopy with acute ulcerative colitis: severe colon inflammation with erythema, friability, and exudates. (Courtesy of Dr. M. Hamilton, Gastroenterology Division, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts; with permission.)

1	Computed tomography (CT) scanning or magnetic resonance imaging (MRI) is not as helpful as endoscopy in making the diagnosis of UC, but typical findings include mild mural thickening (<1.5 cm), inhomogeneous wall density, absence of small bowel thickening, increased perirectal and presacral fat, target appearance of the rectum, and adenopathy.

1	Complications Only 15% of patients with UC present initially with catastrophic illness. Massive hemorrhage occurs with severe attacks of disease in 1% of patients, and treatment for the disease usually stops the bleeding. However, if a patient requires 6–8 units of blood within 24–48 h, colectomy is indicated. Toxic megacolon is defined as a transverse or right colon with a diameter of >6 cm, with loss of haustration in patients with severe attacks of UC. It occurs in about 5% of attacks and can be triggered by electrolyte abnormalities and narcotics. About 50% of acute dilations will resolve with medical therapy alone, but urgent colectomy is required for those that do not improve. Perforation is the most dangerous of the local complications, and the physical signs of peritonitis may not be obvious, especially if the patient is receiving glucocorticoids. Although perforation is rare, the mortality rate for perforation complicating a toxic megacolon is about 15%. In addition, patients

1	be obvious, especially if the patient is receiving glucocorticoids. Although perforation is rare, the mortality rate for perforation complicating a toxic megacolon is about 15%. In addition, patients can develop a toxic colitis and such severe ulcerations that the bowel may perforate without first dilating.

1	Strictures occur in 5–10% of patients and are always a concern in UC because of the possibility of underlying neoplasia. Although benign strictures can form from the inflammation and fibrosis of UC, strictures that are impassable with the colonoscope should be presumed malignant until proven otherwise. A stricture that prevents passage of the colonoscope is an indication for surgery. UC patients occasionally develop anal fissures, perianal abscesses, or hemorrhoids, but the occurrence of extensive perianal lesions should suggest CD. CROHN’S DISEASE Signs and Symptoms Although CD usually presents as acute or chronic bowel inflammation, the inflammatory process evolves toward one of two patterns of disease: a fibrostenotic obstructing pattern or a penetrating fistulous pattern, each with different treatments and prognoses. The site of disease influences the clinical manifestations.

1	ileocolitis Because the most common site of inflammation is the terminal ileum, the usual presentation of ileocolitis is a chronic history of recurrent episodes of right lower quadrant pain and diarrhea. Sometimes the initial presentation mimics acute appendicitis with 1953 pronounced right lower quadrant pain, a palpable mass, fever, and leukocytosis. Pain is usually colicky; it precedes and is relieved by defecation. A low-grade fever is usually noted. High-spiking fever suggests intraabdominal abscess formation. Weight loss is common—typically 10–20% of body weight—and develops as a consequence of diarrhea, anorexia, and fear of eating. An inflammatory mass may be palpated in the right lower quadrant of the abdomen. The mass is composed of inflamed bowel, adherent and indurated mesentery, and enlarged abdominal lymph nodes.

1	An inflammatory mass may be palpated in the right lower quadrant of the abdomen. The mass is composed of inflamed bowel, adherent and indurated mesentery, and enlarged abdominal lymph nodes. Extension of the mass can cause obstruction of the right ureter or bladder inflammation, manifested by dysuria and fever. Edema, bowel wall thickening, and fibrosis of the bowel wall within the mass account for the radiographic “string sign” of a narrowed intestinal lumen.

1	Bowel obstruction may take several forms. In the early stages of disease, bowel wall edema and spasm produce intermittent obstructive manifestations and increasing symptoms of postprandial pain. Over several years, persistent inflammation gradually progresses to fibrostenotic narrowing and stricture. Diarrhea will decrease and be replaced by chronic bowel obstruction. Acute episodes of obstruction occur as well, precipitated by bowel inflammation and spasm or sometimes by impaction of undigested food or medication. These episodes usually resolve with intravenous fluids and gastric decompression.

1	Severe inflammation of the ileocecal region may lead to localized wall thinning, with microperforation and fistula formation to the adjacent bowel, the skin, or the urinary bladder, or to an abscess cavity in the mesentery. Enterovesical fistulas typically present as dysuria or recurrent bladder infections or, less commonly, as pneumaturia or fecaluria. Enterocutaneous fistulas follow tissue planes of least resistance, usually draining through abdominal surgical scars. Enterovaginal fistulas are rare and present as dyspareunia or as a feculent or foul-smelling, often painful vaginal discharge. They are unlikely to develop without a prior hysterectomy.

1	JeJunoileitis Extensive inflammatory disease is associated with a loss of digestive and absorptive surface, resulting in malabsorption and steatorrhea. Nutritional deficiencies can also result from poor intake and enteric losses of protein and other nutrients. Intestinal malabsorption can cause anemia, hypoalbuminemia, hypocalcemia, hypomagnesemia, coagulopathy, and hyperoxaluria with nephrolithiasis in patients with an intact colon. Many patients need to take oral and often intravenous iron. Vertebral fractures are caused by a combination of vitamin D deficiency, hypocalcemia, and prolonged glucocorticoid use. Pellagra from niacin deficiency can occur in extensive small-bowel disease, and malabsorption of vitamin B12 can lead to megaloblastic anemia and neurologic symptoms. Other important nutrients to measure and replete if low are folate and vitamins A, E, and K. Levels of minerals such as zinc, selenium, copper, and magnesium are often low in patients with extensive small-bowel

1	nutrients to measure and replete if low are folate and vitamins A, E, and K. Levels of minerals such as zinc, selenium, copper, and magnesium are often low in patients with extensive small-bowel inflammation or resections, and these should be repleted as well. Most patients should take a daily multivitamin, calcium, and vitamin D supplements.

1	Diarrhea is characteristic of active disease; its causes include (1) bacterial overgrowth in obstructive stasis or fistulization, (2) bile-acid malabsorption due to a diseased or resected terminal ileum, and (3) intestinal inflammation with decreased water absorption and increased secretion of electrolytes. colitis anD perianal Disease Patients with colitis present with low-grade fevers, malaise, diarrhea, crampy abdominal pain, and sometimes hematochezia. Gross bleeding is not as common as in UC and appears in about one-half of patients with exclusively colonic disease. Only 1–2% bleed massively. Pain is caused by passage of fecal material through narrowed and inflamed segments of the large bowel. Decreased rectal compliance is another cause for diarrhea in Crohn’s colitis patients. Toxic megacolon is rare but may be seen with severe inflammation and short duration disease.

1	Stricturing can occur in the colon in 4–16% of patients and produce symptoms of bowel obstruction. If the endoscopist is unable to traverse a stricture in Crohn’s colitis, surgical resection should be considered, especially if the patient has symptoms of chronic obstruction. 1954 Colonic disease may fistulize into the stomach or duodenum, causing feculent vomiting, or to the proximal or mid-small bowel, causing malabsorption by “short circuiting” and bacterial overgrowth. Ten percent of women with Crohn’s colitis will develop a rectovaginal fistula. Perianal disease affects about one-third of patients with Crohn’s colitis and is manifested by incontinence, large hemorrhoidal tags, anal strictures, anorectal fistulae, and perirectal abscesses. Not all patients with perianal fistula will have endoscopic evidence of colonic inflammation.

1	GastroDuoDenal Disease Symptoms and signs of upper GI tract disease include nausea, vomiting, and epigastric pain. Patients usually have an Helicobacter pylori–negative gastritis. The second portion of the duodenum is more commonly involved than the bulb. Fistulas involving the stomach or duodenum arise from the small or large bowel and do not necessarily signify the presence of upper GI tract involvement. Patients with advanced gastroduodenal CD may develop a chronic gastric outlet obstruction. Laboratory, Endoscopic, and Radiographic Features Laboratory abnormalities include elevated ESR and CRP. In more severe disease, findings include hypoalbuminemia, anemia, and leukocytosis.

1	Endoscopic features of CD include rectal sparing, aphthous ulcerations, fistulas, and skip lesions. Colonoscopy allows examination and biopsy of mass lesions or strictures and biopsy of the terminal ileum. Upper endoscopy is useful in diagnosing gastroduodenal involvement in patients with upper tract symptoms. Ileal or colonic strictures may be dilated with balloons introduced through the colonoscope. Strictures ≤4 cm and those at anastomotic sites respond better to endoscopic dilation. The perforation rate is as high as 10%. Most endoscopists dilate only fibrotic strictures and not those associated with active inflammation. Wireless capsule endoscopy (WCE) allows direct visualization of the entire small-bowel mucosa (Fig. 351-8). The diagnostic yield of detecting lesions suggestive of active CD is higher with WCE than CT or magnetic resonance (MR) enterography or small-bowel series. WCE cannot be used in the setting of a small-bowel stricture. Capsule retention occurs in <1% of

1	of active CD is higher with WCE than CT or magnetic resonance (MR) enterography or small-bowel series. WCE cannot be used in the setting of a small-bowel stricture. Capsule retention occurs in <1% of patients with suspected CD, but retention rates of 4–6% are seen in patients with established CD. It is helpful to give the patient with CD a patency capsule, which is made of barium and starts to dissolve 30 h after ingestion. An abdominal x-ray can be taken at around 30 h after ingestion to see if the capsule is still present in the small bowel, which would indicate a stricture.

1	FIGURE 351-8 Wireless capsule endoscopy image in a patient with Crohn’s disease of the ileum shows ulcerations and narrow-ing of the intestinal lumen. (Courtesy of Dr. S. Reddy, Gastroenterology Division, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts; with permission.) In CD, early radiographic findings in the small bowel include thickened folds and aphthous ulcerations. “Cobblestoning” from longitudinal and transverse ulcerations most frequently involves the small bowel. In more advanced disease, strictures, fistulas, inflammatory masses, and abscesses may be detected. The earliest macroscopic findings of colonic CD are aphthous ulcers. These small ulcers are often multiple and separated by normal intervening mucosa. As the disease progresses, aphthous ulcers become enlarged, deeper, and occasionally connected to one another, forming longitudinal stellate, serpiginous, and linear ulcers (see Fig. 345-4B).

1	The transmural inflammation of CD leads to decreased luminal diameter and limited distensibility. As ulcers progress deeper, they can lead to fistula formation. The radiographic “string sign” represents long areas of circumferential inflammation and fibrosis, resulting in long segments of luminal narrowing. The segmental nature of CD results in wide gaps of normal or dilated bowel between involved segments.

1	Both CT and MRI of the small bowel can be performed by enterography (CTE or MRE), using oral and IV contrast, as well as enteroclysis. Although institutional preference guides technique selection, CTE and MRE tend to be preferred over enteroclysis due to ease and patient preference. Although CTE, MRE, and small-bowel follow-through (SBFT) have been shown to be equally accurate in the identification of active small-bowel inflammation, CTE and MRE have been shown to be superior to SBFT in the detection of extraluminal complications, including fistulas, sinus tracts, and abscesses. Currently, the use of CT scans is more common than MRI due to institutional availability and expertise. However, MRI is thought to offer superior soft tissue contrast and has the added advantage of avoiding radiation exposure changes (Figs. 351-9 and 351-10). The lack of ionizing radiation is particularly appealing in younger patients and when monitoring response to therapy where serial images will be

1	radiation exposure changes (Figs. 351-9 and 351-10). The lack of ionizing radiation is particularly appealing in younger patients and when monitoring response to therapy where serial images will be obtained. Either CTE or MRE is the first-line test for the evaluation of suspected CD and its complications. Pelvic MRI is superior to CT for demonstrating pelvic lesions such as ischiorectal abscesses and perianal fistulae (Fig. 351-11).

1	Complications Because CD is a transmural process, serosal adhesions develop that provide direct pathways for fistula formation and reduce the incidence of free perforation. Perforation occurs in 1–2% of patients, usually in the ileum but occasionally in the jejunum or as a complication of toxic megacolon. The peritonitis of free perforation, especially colonic, may be fatal. Intraabdominal and pelvic abscesses occur in 10–30% of patients with CD at some time in the course of their illness. CT-guided percutaneous drainage of the abscess is standard therapy. Despite adequate drainage, most patients need resection of the offending bowel segment. Percutaneous drainage has an especially high failure rate in abdominal wall abscesses. Systemic glucocorticoid therapy increases the risk of intraabdominal and pelvic abscesses in CD patients who have never had an operation. Other complications include intestinal obstruction in 40%, massive hemorrhage, malabsorption, and severe perianal disease.

1	Serologic Markers Patients with CD show a wide variation in the way they present and progress over time. Some patients present with mild disease activity and do well with generally safe and mild medications, but many others exhibit more severe disease and can develop serious complications that will require surgery. Current and developing biologic therapies can help halt progression of disease and give patients with moderate to severe CD a better quality of life. There are potential risks of biologic therapies such as infection and malignancy, and it would be optimal to determine at the time of diagnosis which patients will require more aggressive medical therapy. This same argument holds true for UC patients as well. Subsets of patients with differing immune responses to microbial antigens have been described, and serology is often tested for

1	Subsets of patients with differing immune responses to microbial antigens have been described, and serology is often tested for FIGURE 351-9 A coronal magnetic resonance image was obtained using a half Fourier single-shot T2-weighted acquisition with fat saturation in a 27-year-old pregnant (23 weeks’ gestation) woman. The patient had Crohn’s disease and was maintained on 6-mercaptopurine and prednisone. She presented with abdominal pain, distension, vomiting, and small-bowel obstruction. The image reveals a 7to 10-cm long stricture at the terminal ileum (white arrows) causing obstruction and significant dilatation of the proximal small bowel (white asterisk). A fetus is seen in the uterus (dashed white arrows). (Courtesy of Drs. J. F. B. Chick and

1	P. B. Shyn, Abdominal Imaging and Intervention, Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; with permission.) perinuclear antineutrophil cytoplasmic antibodies (pANCAs) and anti-Saccharomyces cerevisiae antibodies (ASCAs). Unfortunately, these serologic markers are only marginally useful in helping to make the diagnosis of UC or CD and in predicting the course of disease. For success in diagnosing IBD and in differentiating between CD and UC, the efficacy of these serologic tests depends on the prevalence of IBD in a specific population. pANCA positivity is found in about 60–70% of UC patients and 5–10% of CD patients; 5–15% of first-degree relatives of UC patients are pANCA positive, whereas only 2–3% of the general population is pANCA positive. Sixty to 70% of CD patients, 10–15% of UC patients, and up to 5% of non-IBD controls are ASCA positive. In a patient population with a combined prevalence of UC and CD of 62%,

1	is pANCA positive. Sixty to 70% of CD patients, 10–15% of UC patients, and up to 5% of non-IBD controls are ASCA positive. In a patient population with a combined prevalence of UC and CD of 62%, pANCA/ASCA serology showed a sensitivity of 64% and a specificity of 94%. Positive and negative predictive values (PPVs and NPVs) for pANCA/ASCA also vary based on the prevalence of IBD in a given population. For the patient population with a prevalence of IBD of 62%, the PPV is 94%, and the NPV is 63%.

1	Other serologic tests include antibodies to Escherichia coli outer membrane porin protein C (OmpC), which is found in 55% of CD patients; antibodies to I2, a homologue of the bacterial transcription factor families from a Pseudomonas fluorescens–associated sequence that is found in 50–54% of CD patients; and anti-flagellin (anti-CBir1) antibodies, which have been identified in approximately 50% of CD patients. Children with CD positive for all four immune responses (ASCA+, OmpC+, I2+, and anti-Cbir1+) may have more aggressive disease and a shorter time to progression to internal perforating

1	Children with CD positive for all four immune responses (ASCA+, OmpC+, I2+, and anti-Cbir1+) may have more aggressive disease and a shorter time to progression to internal perforating FIGURE 351-10 A coronal balanced, steady-state, free precession, T2-weighted image with fat saturation was obtained in a 32-year-old man with Crohn’s disease and prior episodes of bowel obstruction, fistulas, and abscesses. He was being treated with 6-mercaptopurine and presented with abdominal distention and diarrhea. The image demonstrates a new gastrocolic fistula (solid white arrows). Multifocal involvement of the small bowel and terminal ileum is also present (dashed white arrows). (Courtesy of Drs. J. F. B. Chick and P. B. Shyn, Abdominal Imaging and Intervention, Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; with permission.)

1	FIGURE 351-11 Axial T2-weighted magnetic resonance image obtained in a 37-year-old man with Crohn’s disease shows a linear fluid-filled perianal fistula (arrow) in the right ischioanal fossa. (Courtesy of Dr. K. Mortele, Gastrointestinal Radiology, Department of Radiology, Brigham and Women’s Hospital, Boston, Massachusetts; with permission.) 1956 and/or stricturing disease. However, larger prospective studies in both children and adults have not yet been performed and compared to CRP or other markers. Clinical factors described at diagnosis are more helpful than serologies at predicting the natural history of CD. The initial requirements for glucocorticoid use, an age at diagnosis below 40 years and the presence of perianal disease at diagnosis, have been shown to be independently associated with subsequent disabling CD after 5 years. Except in special circumstances (such as before consideration of an ileoanal pouch anastomosis [IPAA] in a patient with indeterminate colitis),

1	associated with subsequent disabling CD after 5 years. Except in special circumstances (such as before consideration of an ileoanal pouch anastomosis [IPAA] in a patient with indeterminate colitis), serologic markers have only minimal clinical utility.

1	UC and CD have similar features to many other diseases. In the absence of a key diagnostic test, a combination of features is used (Table 351-5). Once a diagnosis of IBD is made, distinguishing between UC and CD is impossible initially in up to 15% of cases. These are termed indeterminate colitis. Fortunately, in most cases, the true nature of the underlying colitis becomes evident later in the course of the patient’s disease. Approximately 5% (range 1–20%) of colon resection specimens are difficult to classify as either UC or CD because they exhibit overlapping histologic features.

1	Infections of the small intestines and colon can mimic CD or UC. They may be bacterial, fungal, viral, or protozoal in origin (Table 351-6). Campylobacter colitis can mimic the endoscopic appearance of severe UC and can cause a relapse of established UC. Salmonella can cause watery or bloody diarrhea, nausea, and vomiting. Shigellosis causes watery diarrhea, abdominal pain, and fever followed by rectal tenesmus and by the passage of blood and mucus per rectum. All three are usually self-limited, but 1% of patients infected with Salmonella become asymptomatic carriers. Yersinia enterocolitica infection occurs mainly in the terminal ileum and causes mucosal ulceration, neutrophil invasion, and thickening of the ileal wall. Other bacterial infections that may mimic IBD include C. difficile, which presents with watery TABlE 351-5 DiffEREnT CliniCAl, EnDoSCoPiC, AnD RADioGRAPHiC fEATuRES Abbreviation: IBD, inflammatory bowel disease; NSAIDs, nonsteroidal anti-inflammatory drugs.

1	TABlE 351-5 DiffEREnT CliniCAl, EnDoSCoPiC, AnD RADioGRAPHiC fEATuRES Abbreviation: IBD, inflammatory bowel disease; NSAIDs, nonsteroidal anti-inflammatory drugs. diarrhea, tenesmus, nausea, and vomiting; and E. coli, three categories of which can cause colitis. These are enterohemorrhagic, enteroinvasive, and enteroadherent E. coli, all of which can cause bloody diarrhea and abdominal tenderness. Diagnosis of bacterial colitis is made by sending stool specimens for bacterial culture and C. difficile toxin analysis. Gonorrhea, Chlamydia, and syphilis can also cause proctitis.

1	GI involvement with mycobacterial infection occurs primarily in the immunosuppressed patient but may occur in patients with normal immunity. Distal ileal and cecal involvement predominates, and patients present with symptoms of small-bowel obstruction and a tender abdominal mass. The diagnosis is made most directly by colonoscopy with biopsy and culture. Mycobacterium avium-intracellulare complex infection occurs in advanced stages of HIV infection and in other immunocompromised states; it usually manifests as a systemic infection with diarrhea, abdominal pain, weight loss, fever, and malabsorption. Diagnosis is established by acid-fast smear and culture of mucosal biopsies.

1	Although most of the patients with viral colitis are immunosuppressed, cytomegalovirus (CMV) and herpes simplex proctitis may occur in immunocompetent individuals. CMV occurs most commonly in the esophagus, colon, and rectum but may also involve the small intestine. Symptoms include abdominal pain, bloody diarrhea, fever, and weight loss. With severe disease, necrosis and perforation can occur. Diagnosis is made by identification of characteristic intranuclear inclusions in mucosal cells on biopsy. Herpes simplex infection of the GI tract is limited to the oropharynx, anorectum, and perianal areas. Symptoms include anorectal pain, tenesmus, constipation, inguinal adenopathy, difficulty with urinary voiding, and sacral paresthesias. Diagnosis is made by rectal biopsy with identification of characteristic cellular inclusions and viral culture. HIV itself can cause diarrhea, nausea, vomiting, and anorexia. Small intestinal biopsies show partial villous atrophy; small bowel bacterial

1	of characteristic cellular inclusions and viral culture. HIV itself can cause diarrhea, nausea, vomiting, and anorexia. Small intestinal biopsies show partial villous atrophy; small bowel bacterial overgrowth and fat malabsorption may also be noted.

1	Protozoan parasites include Isospora belli, which can cause a self-limited infection in healthy hosts but causes a chronic profuse, watery diarrhea, and weight loss in AIDS patients. Entamoeba histolytica or related species infect about 10% of the world’s population; symptoms include abdominal pain, tenesmus, frequent loose stools containing blood and mucus, and abdominal tenderness. Colonoscopy reveals focal punctate ulcers with normal intervening mucosa; diagnosis is made by biopsy or serum amebic antibodies. Fulminant amebic colitis is rare but has a mortality rate of >50%. Other parasitic infections that may mimic IBD include hookworm (Necator americanus), whipworm (Trichuris trichiura), and Strongyloides stercoralis. In severely immunocompromised patients, Candida or Aspergillus can be identified in the submucosa. Disseminated histoplasmosis can involve the ileocecal area.

1	Diverticulitis can be confused with CD clinically and radiographically. Both diseases cause fever, abdominal pain, tender abdominal mass, leukocytosis, elevated ESR, partial obstruction, and fistulas. Perianal disease or ileitis on small-bowel series favors the diagnosis of CD. Significant endoscopic mucosal abnormalities are more likely in CD than in diverticulitis. Endoscopic or clinical recurrence following segmental resection favors CD. Diverticular-associated colitis is similar to CD, but mucosal abnormalities are limited to the sigmoid and descending colon.

1	Ischemic colitis is commonly confused with IBD. The ischemic process can be chronic and diffuse, as in UC, or segmental, as in CD. Colonic inflammation due to ischemia may resolve quickly or may persist and result in transmural scarring and stricture formation. Ischemic bowel disease should be considered in the elderly following abdominal aortic aneurysm repair or when a patient has a hyper-coagulable state or a severe cardiac or peripheral vascular disorder. Patients usually present with sudden onset of left lower quadrant pain, urgency to defecate, and the passage of bright red blood per rectum. Endoscopic examination often demonstrates a normal-appearing rectum and a sharp transition to an area of inflammation in the descending colon and splenic flexure.

1	The effects of radiotherapy on the GI tract can be difficult to distinguish from IBD. Acute symptoms can occur within 1–2 weeks of starting radiotherapy. When the rectum and sigmoid are irradiated, patients develop bloody, mucoid diarrhea and tenesmus, as in distal UC. With small-bowel involvement, diarrhea is common. Late symptoms include malabsorption and weight loss. Stricturing with obstruction and bacterial overgrowth may occur. Fistulas can penetrate the bladder, vagina, or abdominal wall. Flexible sigmoidoscopy reveals mucosal granularity, friability, numerous telangiectasias, and occasionally discrete ulcerations. Biopsy can be diagnostic.

1	Solitary rectal ulcer syndrome is uncommon and can be confused with IBD. It occurs in persons of all ages and may be caused by impaired evacuation and failure of relaxation of the puborectalis muscle. Single or multiple ulcerations may arise from anal sphincter overactivity, higher intrarectal pressures during defecation, and digital removal of stool. Patients complain of constipation with straining and pass blood and mucus per rectum. Other symptoms include abdominal pain, diarrhea, tenesmus, and perineal pain. Ulceration as large as 5 cm in diameter is usually seen anteriorly or anterior-laterally 3–15 cm from the anal verge. Biopsies can be diagnostic.

1	Several types of colitis are associated with nonsteroidal anti-inflammatory drugs (NSAIDs), including de novo colitis, reactivation of IBD, and proctitis caused by use of suppositories. Most patients with NSAID-related colitis present with diarrhea and abdominal pain, and complications include stricture, bleeding, obstruction, perforation, and fistulization. Withdrawal of these agents is crucial, and in cases of reactivated IBD, standard therapies are indicated.

1	There are complications of two drugs used in a hospital setting that mimic IBD. The first is ipilimumab, a drug that targets cytotoxic T lymphocyte antigen 4 (CTLA-4) and reverses T cell inhibition and is used to treat metastatic melanoma; ipilimumab has an incidence of IBD in 0.0017 cases per 100 person-years. Ipilimumab-induced colitis is typically treated with glucocorticoids or infliximab. The 1957 second is mycophenolate mofetil (MMF), an immunosuppressive agent commonly used to prevent posttransplant rejection. The colitis associated with MMF is common and can occur in more than one-third of patients taking the drug. Treatment is dose reduction or cessation of the drug.

1	Two atypical colitides—collagenous colitis and lymphocytic colitis— have completely normal endoscopic appearances. Collagenous colitis has two main histologic components: increased subepithelial collagen deposition and colitis with increased intraepithelial lymphocytes. The female to male ratio is 9:1, and most patients present in the sixth or seventh decades of life. The main symptom is chronic watery diarrhea. Treatments range from sulfasalazine or mesalamine and diphenoxylate/atropine (Lomotil) to bismuth to budesonide to prednisone or azathioprine/6-mercaptopurine for refractory disease. Risk factors include smoking; use of NSAIDs, proton pump inhibitors, or beta blockers; and a history of autoimmune disease.

1	Lymphocytic colitis has features similar to collagenous colitis, including age at onset and clinical presentation, but it has almost equal incidence in men and women and no subepithelial collagen deposition on pathologic section. However, intraepithelial lymphocytes are increased. Use of sertraline (but not beta blockers) is an additional risk factor. The frequency of celiac disease is increased in lymphocytic colitis and ranges from 9 to 27%. Celiac disease should be excluded in all patients with lymphocytic colitis, particularly if diarrhea does not respond to conventional therapy. Treatment is similar to that of collagenous colitis with the exception of a gluten-free diet for those who have celiac disease.

1	Diversion colitis is an inflammatory process that arises in segments of the large intestine that are excluded from the fecal stream. It usually occurs in patients with ileostomy or colostomy when a mucus fistula or a Hartmann’s pouch has been created. Clinically, patients have mucus or bloody discharge from the rectum. Erythema, granularity, friability, and, in more severe cases, ulceration can be seen on endoscopy. Histopathology shows areas of active inflammation with foci of cryptitis and crypt abscesses. Crypt architecture is normal, which differentiates it from UC. It may be impossible to distinguish from CD. Short-chain fatty acid enemas may help in diversion colitis, but the definitive therapy is surgical reanastomosis. Up to one-third of IBD patients have at least one extraintestinal disease manifestation.

1	Up to one-third of IBD patients have at least one extraintestinal disease manifestation. Erythema nodosum (EN) occurs in up to 15% of CD patients and 10% of UC patients. Attacks usually correlate with bowel activity; skin lesions develop after the onset of bowel symptoms, and patients frequently have concomitant active peripheral arthritis. The lesions of EN are hot, red, tender nodules measuring 1–5 cm in diameter and are found on the anterior surface of the lower legs, ankles, calves, thighs, and arms. Therapy is directed toward the underlying bowel disease.

1	Pyoderma gangrenosum (PG) is seen in 1–12% of UC patients and less commonly in Crohn’s colitis. Although it usually presents after the diagnosis of IBD, PG may occur years before the onset of bowel symptoms, run a course independent of the bowel disease, respond poorly to colectomy, and even develop years after proctocolectomy. It is usually associated with severe disease. Lesions are commonly found on the dorsal surface of the feet and legs but may occur on the arms, chest, stoma, and even the face. PG usually begins as a pustule and then spreads concentrically to rapidly undermine healthy skin. Lesions then ulcerate, with violaceous edges surrounded by a margin of erythema. Centrally, they contain necrotic tissue with blood and exudates. Lesions may be single or multiple and grow as large as 30 cm. They are sometimes very difficult to treat and often require IV antibiotics, IV glucocorticoids, dapsone, azathioprine, thalidomide, IV cyclosporine, or infliximab.

1	1958 Other dermatologic manifestations include pyoderma vegetans, which occurs in intertriginous areas; pyostomatitis vegetans, which involves the mucous membranes; Sweet syndrome, a neutrophilic dermatosis; and metastatic CD, a rare disorder defined by cutaneous granuloma formation. Psoriasis affects 5–10% of patients with IBD and is unrelated to bowel activity consistent with the potential shared immunogenetic basis of these diseases. Perianal skin tags are found in 75–80% of patients with CD, especially those with colon involvement. Oral mucosal lesions, seen often in CD and rarely in UC, include aphthous stomatitis and “cobblestone” lesions of the buccal mucosa.

1	Peripheral arthritis develops in 15–20% of IBD patients, is more common in CD, and worsens with exacerbations of bowel activity. It is asymmetric, polyarticular, and migratory and most often affects large joints of the upper and lower extremities. Treatment is directed at reducing bowel inflammation. In severe UC, colectomy frequently cures the arthritis. Ankylosing spondylitis (AS) occurs in about 10% of IBD patients and is more common in CD than UC. About two-thirds of IBD patients with AS express the HLA-B27 antigen. The AS activity is not related to bowel activity and does not remit with glucocorticoids or colectomy. It most often affects the spine and pelvis, producing symptoms of diffuse low-back pain, buttock pain, and morning stiffness. The course is continuous and progressive, leading to permanent skeletal damage and deformity. Anti-TNF therapy reduces spinal inflammation and improves functional status and quality of life.

1	Sacroiliitis is symmetric, occurs equally in UC and CD, is often asymptomatic, does not correlate with bowel activity, and does not always progress to AS. Other rheumatic manifestations include hyper-trophic osteoarthropathy, pelvic/femoral osteomyelitis, and relapsing polychondritis. The incidence of ocular complications in IBD patients is 1–10%. The most common are conjunctivitis, anterior uveitis/iritis, and episcleritis. Uveitis is associated with both UC and Crohn’s colitis, may be found during periods of remission, and may develop in patients following bowel resection. Symptoms include ocular pain, photophobia, blurred vision, and headache. Prompt intervention, sometimes with systemic glucocorticoids, is required to prevent scarring and visual impairment. Episcleritis is a benign disorder that presents with symptoms of mild ocular burning. It occurs in 3–4% of IBD patients, more commonly in Crohn’s colitis, and is treated with topical glucocorticoids.

1	Hepatic steatosis is detectable in about one-half of the abnormal liver biopsies from patients with CD and UC; patients usually present with hepatomegaly. Fatty liver usually results from a combination of chronic debilitating illness, malnutrition, and glucocorticoid therapy. Cholelithiasis occurs in 10–35% of CD patients with ileitis or ileal resection. Gallstone formation is caused by malabsorption of bile acids, resulting in depletion of the bile salt pool and the secretion of lithogenic bile.

1	Primary sclerosing cholangitis (PSC) is a disorder characterized by both intrahepatic and extrahepatic bile duct inflammation and fibrosis, frequently leading to biliary cirrhosis and hepatic failure; approximately 5% of patients with UC have PSC, but 50–75% of patients with PSC have IBD. PSC occurs less often in patients with CD. Although it can be recognized after the diagnosis of IBD, PSC can be detected earlier or even years after proctocolectomy. Consistent with this, the immunogenetic basis for PSC appears to be overlapping but distinct from UC based on GWAS, although both IBD and PSC are commonly pANCA positive. Most patients have no symptoms at the time of diagnosis; when symptoms are present, they consist of fatigue, jaundice, abdominal pain, fever, anorexia, and malaise. The traditional gold standard diagnostic test is endoscopic retrograde cholangiopancreatography (ERCP), but magnetic resonance cholangiopancreatography (MRCP) is also sensitive and specific. MRCP is

1	The traditional gold standard diagnostic test is endoscopic retrograde cholangiopancreatography (ERCP), but magnetic resonance cholangiopancreatography (MRCP) is also sensitive and specific. MRCP is reasonable as an initial diagnostic test in children and can visualize irregularities, multifocal strictures, and dilatations of all levels of the biliary tree. In patients with PSC, both ERCP and MRCP demonstrate multiple bile duct strictures alternating with relatively normal segments.

1	The bile acid ursodeoxycholic acid (ursodiol) may reduce alkaline phosphatase and serum aminotransferase levels, but histologic improvement has been marginal. High doses (25–30 mg/kg per day) may decrease the risk of colorectal dysplasia and cancer in patients with UC and PSC. Endoscopic stenting may be palliative for cholestasis secondary to bile duct obstruction. Patients with symptomatic disease develop cirrhosis and liver failure over 5–10 years and eventually require liver transplantation. PSC patients have a 10–15% lifetime risk of developing cholangiocarcinoma and then cannot be transplanted. Patients with IBD and PSC are at increased risk of colon cancer and should be surveyed yearly by colonoscopy and biopsy.

1	In addition, cholangiography is normal in a small percentage of patients who have a variant of PSC known as small duct primary sclerosing cholangitis. This variant (sometimes referred to as “pericholangitis”) is probably a form of PSC involving small-caliber bile ducts. It has similar biochemical and histologic features to classic PSC. It appears to have a significantly better prognosis than classic PSC, although it may evolve into classic PSC. Granulomatous hepatitis and hepatic amyloidosis are much rarer extraintestinal manifestations of IBD.

1	The most frequent genitourinary complications are calculi, ureteral obstruction, and ileal bladder fistulas. The highest frequency of nephrolithiasis (10–20%) occurs in patients with CD following small bowel resection. Calcium oxalate stones develop secondary to hyperoxaluria, which results from increased absorption of dietary oxalate. Normally, dietary calcium combines with luminal oxalate to form insoluble calcium oxalate, which is eliminated in the stool. In patients with ileal dysfunction, however, nonabsorbed fatty acids bind calcium and leave oxalate unbound. The unbound oxalate is then delivered to the colon, where it is readily absorbed, especially in the presence of inflammation.

1	Low bone mass occurs in 3–30% of IBD patients. The risk is increased by glucocorticoids, cyclosporine, methotrexate, and total parenteral nutrition (TPN). Malabsorption and inflammation mediated by IL-1, IL-6, TNF, and other inflammatory mediators also contribute to low bone density. An increased incidence of hip, spine, wrist, and rib fractures has been noted: 36% in CD and 45% in UC. The absolute risk of an osteoporotic fracture is about 1% per person per year. Fracture rates, particularly in the spine and hip, are highest among the elderly (age >60). One study noted an OR of 1.72 for vertebral fracture and an OR of 1.59 for hip fracture. The disease severity predicted the risk of a fracture. Only 13% of IBD patients who had a fracture were on any kind of antifracture treatment. Up to 20% of bone mass can be lost per year with chronic glucocorticoid use. The effect is dosage-dependent. Budesonide may also suppress the pituitary-adrenal axis and thus carries a risk of causing

1	Up to 20% of bone mass can be lost per year with chronic glucocorticoid use. The effect is dosage-dependent. Budesonide may also suppress the pituitary-adrenal axis and thus carries a risk of causing osteoporosis.

1	Osteonecrosis is characterized by death of osteocytes and adipocytes and eventual bone collapse. The pain is aggravated by motion and swelling of the joints. It affects the hips more often than knees and shoulders, and in one series, 4.3% of patients developed osteonecrosis within 6 months of starting glucocorticoids. Diagnosis is made by bone scan or MRI, and treatment consists of pain control, cord decompression, osteotomy, and joint replacement.

1	Patients with IBD have an increased risk of both venous and arterial thrombosis even if the disease is not active. Factors responsible for the hypercoagulable state have included abnormalities of the platelet-endothelial interaction, hyperhomocysteinemia, alterations in the coagulation cascade, impaired fibrinolysis, involvement of tissue factor-bearing microvesicles, disruption of the normal coagulation system by autoantibodies, and a genetic predisposition. A spectrum of vasculitides involving small, medium, and large vessels has also been observed.

1	More common cardiopulmonary manifestations include endocarditis, myocarditis, pleuropericarditis, and interstitial lung disease. A secondary or reactive amyloidosis can occur in patients with long-standing IBD, especially in patients with CD. Amyloid material is deposited systemically and can cause diarrhea, constipation, and renal failure. The renal disease can be successfully treated with colchicine. Pancreatitis is a rare extraintestinal manifestation of IBD and results from duodenal fistulas; ampullary CD; gallstones; PSC; drugs such as 6-mercaptopurine, azathioprine, or, very rarely, 5-ASA agents; autoimmune pancreatitis; and primary CD of the pancreas.

1	The mainstay of therapy for mild to moderate UC is sulfasalazine and the other 5-ASA agents. These agents are effective at inducing and maintaining remission in UC. They may have a limited role in inducing remission in CD but no clear role in maintenance of CD. Newer sulfa-free aminosalicylate preparations deliver increased amounts of the pharmacologically active ingredient of sulfasalazine (5-ASA, mesalamine) to the site of active bowel disease while limiting systemic toxicity. Peroxisome proliferator activated receptor γ (PPAR-γ) may mediate 5-ASA therapeutic action by decreasing nuclear localization of NF-κB. Sulfa-free aminosalicylate formulations include alternative azo-bonded carriers, 5-ASA dimers, and delayed-release and controlled-release preparations. Each has the same efficacy as sulfasalazine when equimolar concentrations are used.

1	Sulfasalazine was originally developed to deliver both antibacterial (sulfapyridine) and anti-inflammatory (5-ASA) therapy into the connective tissues of joints and the colonic mucosa. The molecular structure provides a convenient delivery system to the colon by allowing the intact molecule to pass through the small intestine after only partial absorption and to be broken down in the colon by bacterial azo reductases that cleave the azo bond linking the sulfa and 5-ASA moieties. Sulfasalazine is effective treatment for mild to moderate UC and is occasionally used in Crohn’s colitis, but its high rate of side effects limits its use. Although sulfasalazine is more effective at higher doses, at 6 or 8 g/d up to 30% of patients experience allergic reactions or intolerable side effects such as headache, anorexia, nausea, and vomiting that are attributable to the sulfapyridine moiety. Hypersensitivity reactions, independent of sulfapyridine levels, include rash, fever, hepatitis,

1	such as headache, anorexia, nausea, and vomiting that are attributable to the sulfapyridine moiety. Hypersensitivity reactions, independent of sulfapyridine levels, include rash, fever, hepatitis, agranulocytosis, hypersensitivity pneumonitis, pancreatitis, worsening of colitis, and reversible sperm abnormalities. Sulfasalazine can also impair folate absorption, and patients should be given folic acid supplements.

1	Balsalazide contains an azo bond binding mesalamine to the car-1959 rier molecule 4-aminobenzoyl-β-alanine; it is effective in the colon. Olsalazine is composed of two 5-ASA radicals linked by an azo bond, which is split in the colon by bacterial reduction, and two 5-ASA molecules are released. Olsalazine is similar in effectiveness to sulfasalazine in treating UC, but up to 17% of patients experience nonbloody diarrhea caused by increased secretion of fluid in the small bowel.

1	Delzicol and Asacol HD (high dose) are enteric-coated forms of mesalamine with the 5-ASA being released at pH >7. They disinte grate with complete breakup of the tablet occurring in many different parts of the gut ranging from the small intestine to the splenic flexure; they have increased gastric residence when taken with a meal. Asacol has recently been discontinued and replaced with Delzicol, which lacks dibutyl phthalate (DBP), an inactive ingredient in Asacol’s enteric coating. DBP has been associated with adverse effects on the male reproductive system in animals at very high doses. Asacol HD with the same chemical in its coating is still on the market, but the human doses of DBP are within acceptable limits of toxicity.

1	Lialda is a once-a-day formulation of mesalamine (Multi-Matrix System [MMX]) designed to release mesalamine in the colon. The MMX technology incorporates mesalamine into a lipophilic matrix within a hydrophilic matrix encapsulated in a polymer resistant to degradation at a low pH (<7) to delay release throughout the colon. The safety profile appears to be comparable to other 5-ASA formulations. Apriso is a formulation containing encapsulated mesalamine granules that delivers mesalamine to the terminal ileum and colon via a proprietary extended-release mechanism (Intellicor). The outer coating (Eudragit L) dissolves at a pH >6. In addition, there is a polymer matrix core that aids in sustained release throughout the colon. Because Lialda and Apriso are given once daily, an anticipated benefit is improved compliance compared with two to four daily doses required for other mesalamine preparations.

1	Pentasa is another mesalamine formulation that uses an ethylcellulose coating to allow water absorption into small beads containing the mesalamine. Water dissolves the 5-ASA, which then diffuses out of the bead into the lumen. Disintegration of the capsule occurs in the stomach. The microspheres then disperse throughout the entire GI tract from the small intestine through the distal colon in both fasted and fed conditions. Salofalk® Granu-Stix, an unencapsulated version of mesalamine, has been in use in Europe for induction and maintenance of remission for several years.

1	Appropriate doses of the 5-ASA compounds are shown in Table 351-7. Some 50–75% of patients with mild to moderate UC improve when treated with 5-ASA doses equivalent to 2 g/d of 1960 mesalamine; the dose response continues up to at least 4.8 g/d. As a general rule, 5-ASA agents act within 2–4 weeks. 5-ASA doses equivalent to 1.5–4 g/d of mesalamine maintain remission in 50–75% of patients with UC. More common side effects of the 5-ASA medications include headaches, nausea, hair loss, and abdominal pain. Rare side effects of the 5-ASA medications include renal impairment, hematuria, pancreatitis, and paradoxical worsening of colitis. Renal function tests and urinalysis should be checked yearly. Topical Rowasa enemas are composed of mesalamine and are effective in mild-to-moderate distal UC. Clinical response occurs in up to 80% of UC patients with colitis distal to the splenic flexure. Combination therapy with mesalamine in both oral and enema form is more effective than either

1	UC. Clinical response occurs in up to 80% of UC patients with colitis distal to the splenic flexure. Combination therapy with mesalamine in both oral and enema form is more effective than either treatment alone for both distal and extensive UC. Canasa suppositories composed of mesalamine are effective in treating proctitis.

1	The majority of patients with moderate to severe UC benefit from oral or parenteral glucocorticoids. Prednisone is usually started at doses of 40–60 mg/d for active UC that is unresponsive to 5-ASA therapy. Parenteral glucocorticoids may be administered as hydro-cortisone, 300 mg/d, or methylprednisolone, 40–60 mg/d. A new glucocorticoid for UC, budesonide (Uceris), is released entirely in the colon and has minimal to no glucocorticoid side effects. The dose is 9 mg/d for 8 weeks, and no taper is required. Topically applied glucocorticoids are also beneficial for distal colitis and may serve as an adjunct in those who have rectal involvement plus more proximal disease. Hydrocortisone enemas or foam may control active disease, although they have no proven role as maintenance therapy. These glucocorticoids are significantly absorbed from the rectum and can lead to adrenal suppression with prolonged administration. Topical 5-ASA therapy is more effective than topical steroid therapy in

1	glucocorticoids are significantly absorbed from the rectum and can lead to adrenal suppression with prolonged administration. Topical 5-ASA therapy is more effective than topical steroid therapy in the treatment of distal UC.

1	Glucocorticoids are also effective for treatment of moderate to severe CD and induce a 60–70% remission rate compared to a 30% placebo response. The systemic effects of standard glucocorticoid formulations have led to the development of more potent formulations that are less well-absorbed and have increased first-pass metabolism. Controlled ileal-release budesonide has been nearly equal to prednisone for ileocolonic CD with fewer glucocorticoid side effects. Budesonide is used for 2–3 months at a dose of 9 mg/d, and then tapered. Budesonide 6 mg/d is effective in reducing relapse rates at 3–6 months but not at 12 months in CD patients with a medically induced remission.

1	Glucocorticoids play no role in maintenance therapy in either UC or CD. Once clinical remission has been induced, they should be tapered according to the clinical activity, normally at a rate of no more than 5 mg/week. They can usually be tapered to 20 mg/d within 4–5 weeks but often take several months to be discontinued altogether. The side effects are numerous, including fluid retention, abdominal striae, fat redistribution, hyperglycemia, subcapsular cataracts, osteonecrosis, osteoporosis, myopathy, emotional disturbances, and withdrawal symptoms. Most of these side effects, aside from osteonecrosis, are related to the dose and duration of therapy. Antibiotics have no role in the treatment of active or quiescent UC. However, pouchitis, which occurs in about a third of UC patients after colectomy and IPAA, usually responds to treatment with metronidazole and/or ciprofloxacin.

1	Metronidazole is effective in active inflammatory, fistulous, and perianal CD and may prevent recurrence after ileal resection. The most effective dose is 15–20 mg/kg per day in three divided doses; it is usually continued for several months. Common side effects include nausea, metallic taste, and disulfiram-like reaction. Peripheral neuropathy can occur with prolonged administration (several months) and on rare occasions is permanent despite discontinuation. Ciprofloxacin (500 mg bid) is also beneficial for inflammatory, perianal, and fistulous CD but has been associated with Achilles tendinitis and rupture. Both ciprofloxacin and metronidazole antibiotics can be used as first-line drugs for short periods of time in active inflammatory, fistulizing, and perianal CD.

1	Azathioprine and 6-mercaptopurine (6-MP) are purine analogues commonly employed in the management of glucocorticoid-dependent IBD. Azathioprine is rapidly absorbed and converted to 6-MP, which is then metabolized to the active end product, thioinosinic acid, an inhibitor of purine ribonucleotide synthesis and cell proliferation. These agents also inhibit the immune response. Efficacy can be seen as early as 3–4 weeks but can take up to 4–6 months. Adherence can be monitored by measuring the levels of 6-thioguanine and 6-methylmercaptopurine, end products of 6-MP metabolism. Azathioprine (2–3 mg/kg per day) and 6-MP (1–1.5 mg/kg per day) have been used successfully as glucocorticoid-sparing agents in up to two-thirds of UC and CD patients previously unable to be weaned from glucocorticoids. They are also used as maintenance therapy in UC and CD and for treating active perianal disease and fistulas in CD. In addition, 6-MP or azathioprine is effective for postoperative prophylaxis of

1	They are also used as maintenance therapy in UC and CD and for treating active perianal disease and fistulas in CD. In addition, 6-MP or azathioprine is effective for postoperative prophylaxis of CD.

1	Although azathioprine and 6-MP are usually well tolerated, pancreatitis occurs in 3–4% of patients, typically presents within the first few weeks of therapy, and is completely reversible when the drug is stopped. Other side effects include nausea, fever, rash, and hepatitis. Bone marrow suppression (particularly leukopenia) is dose-related and often delayed, necessitating regular monitoring of the complete blood cell count (CBC). Additionally, 1 in 300 individuals lacks thiopurine methyltransferase, the enzyme responsible for drug metabolism to inactive end-products (6-methylmercaptopurine); an additional 11% of the population are heterozygotes with intermediate enzyme activity. Both are at increased risk of toxicity because of increased accumulation of active 6-thioguanine metabolites. Although 6-thioguanine and 6-methylmercaptopurine levels can be followed to determine correct drug dosing and reduce toxicity, weight-based dosing is an acceptable alternative. CBCs and liver function

1	6-thioguanine and 6-methylmercaptopurine levels can be followed to determine correct drug dosing and reduce toxicity, weight-based dosing is an acceptable alternative. CBCs and liver function tests should be monitored frequently regardless of dosing strategy. IBD patients treated with azathioprine/6-MP are at approximately a fourfold increased risk of developing a lymphoma. This increased risk could be a result of the medications, the underlying disease, or both.

1	Methotrexate (MTX) inhibits dihydrofolate reductase, resulting in impaired DNA synthesis. Additional anti-inflammatory properties may be related to decreased IL-1 production. Intramuscular (IM) or subcutaneous (SC) MTX (25 mg/week) is effective in inducing remission and reducing glucocorticoid dosage; 15 mg/week is effective in maintaining remission in active CD. Potential toxicities include leukopenia and hepatic fibrosis, necessitating periodic evaluation of CBCs and liver enzymes. The role of liver biopsy in patients on long-term MTX is uncertain but is probably limited to those with increased liver enzymes. Hypersensitivity pneumonitis is a rare but serious complication of therapy.

1	Cyclosporine (CSA) is a lipophilic peptide with inhibitory effects on both the cellular and humoral immune systems. CSA blocks the production of IL-2 by T helper lymphocytes. CSA binds to cyclophilin, and this complex inhibits calcineurin, a cytoplasmic phosphatase enzyme involved in the activation of T cells. CSA also indirectly inhibits B cell function by blocking helper T cells. CSA has a more rapid onset of action than 6-MP and azathioprine. CSA is most effective when given at 2–4 mg/kg per day IV in severe UC that is refractory to IV glucocorticoids, with 82% of patients responding. CSA can be an alternative to colectomy. The long-term success of oral CSA is not as dramatic, but if patients are started on 6-MP or azathioprine at the time of hospital discharge, remission can be maintained. For the 2 mg/kg dose, levels as measured by monoclonal radioimmunoassay or by the high-performance liquid chromatography assay should be maintained between 150 and 350 ng/mL.

1	CSA may cause significant toxicity; renal function should be monitored frequently. Hypertension, gingival hyperplasia, hypertrichosis, paresthesias, tremors, headaches, and electrolyte abnormalities are common side effects. Creatinine elevation calls for dose reduction or discontinuation. Seizures may also complicate therapy, especially if the patient is hypomagnesemic or if serum cholesterol levels are <3.1 mmol/L (<120 mg/dL). Opportunistic infections, most notably Pneumocystis carinii pneumonia, may occur with combination immunosuppressive treatment; prophylaxis should be given. Major adverse events occurred in 15% of patients in one large study, including nephrotoxicity not responding to dose adjustment, serious infections, seizures, anaphylaxis, and death of two patients. This high incidence suggests that vigorous monitoring by experienced clinicians at tertiary care centers may be required. To compare IV cyclosporine versus infliximab, a large trial was conducted in Europe by

1	incidence suggests that vigorous monitoring by experienced clinicians at tertiary care centers may be required. To compare IV cyclosporine versus infliximab, a large trial was conducted in Europe by the GETAID group. The results indicated identical 7-day response rates between cyclosporine 2 mg/kg (with doses adjusted for levels of 150–250 ng/mL) and infliximab 5 mg/kg, with both groups achieving response rates of 85%. Serious infections occurred in 5 of 55 cyclosporine patients and 4 of 56 infliximab patients. Response rates were similar in the two groups at day 98 among patients treated with oral cyclosporine versus infliximab at the usual induction dose and maintenance dose regimen (40% and 46%, respectively). In light of data showing equal efficacy of CSA and infliximab in severe UC, more physicians are relying on infliximab rather than CSA in these patients.

1	Tacrolimus is a macrolide antibiotic with immunomodulatory properties similar to CSA. It is 100 times as potent as CSA and is not dependent on bile or mucosal integrity for absorption. These pharmacologic properties enable tacrolimus to have good oral absorption despite proximal small bowel Crohn’s involvement. It has shown efficacy in children with refractory IBD and in adults with extensive involvement of the small bowel. It is also effective in adults with glucocorticoid-dependent or refractory UC and CD as well as refractory fistulizing CD.

1	Biologic therapy was traditionally reserved for moderately to severely ill patients with CD who had failed other therapies. However, it is now commonly given as an initial therapy for patients with moderate to severe CD in order to prevent future disease complications. Patients who respond to biologic therapies enjoy an improvement in clinical symptoms; a better quality of life; less disability, fatigue, and depression; and fewer surgeries and hospitalizations.

1	Anti-TNF Therapies The first biologic therapy approved for CD was infliximab, a chimeric IgG1 antibody against TNF-α, which is now also approved for treatment of moderately to severely active UC. Of active CD patients refractory to glucocorticoids, 6-MP, or 5-ASA, 65% will respond to IV infliximab (5 mg/kg); one-third will enter complete remission. The ACCENT I (A Crohn’s Disease Clinical Trial Evaluating Infliximab in a New Long-Term Treatment Regimen) study showed that of the patients who experience an initial response, 40% will maintain remission for at least 1 year with repeated infusions of infliximab every 8 weeks. Infliximab is also effective in CD patients with refractory perianal and enterocutaneous fistulas, with the ACCENT II trial showing a 68% response rate (50% reduction in fistula drainage) and a 50% complete remission rate. Reinfusion, typically every 8 weeks, is necessary to continue therapeutic benefits in many patients.

1	The SONIC (Study of Biologic and Immunomodulator-Naive Patients with Crohn’s Disease) trial compared infliximab plus azathioprine, infliximab alone, and azathioprine alone in immunomodulatorand biologic-naive patients with moderate to severe CD. At 1 year, the infliximab plus azathioprine group had a glucocorticoid-free remission rate of 46% compared with 35% for infliximab alone and 1961 24% for azathioprine alone. There was also complete mucosal healing at week 26 with the combined approach relative to either infliximab or azathioprine alone (44% vs 30% vs 17%). The adverse events were equal between groups.

1	Two large trials of infliximab in moderate to severe UC also showed efficacy with a response rate of 37–49%, with about one-fifth of patients maintaining remission after 54 weeks. Dosing for UC and CD are identical, with induction dosing at 0, 2, and 6 weeks and every 8 weeks thereafter. There is a similar study to SONIC in patients with moderate to severe UC. After 16 weeks of therapy, UC patients taking azathioprine plus infliximab had a glucocorticoidfree remission rate of 40% compared to 24% (article now published) and 22% of those on azathioprine and infliximab alone, respectively. This is even further evidence for “top-down” or more aggressive therapy for both moderate to severe CD and UC.

1	Adalimumab is a recombinant human monoclonal IgG1 antibody containing only human peptide sequences and is injected subcu taneously. Adalimumab binds TNF and neutralizes its function by blocking the interaction between TNF and its cell-surface receptor. Therefore, it seems to have a similar mechanism of action to infliximab but with less immunogenicity. Adalimumab has been approved for treatment of moderate to severe CD. CHARM (Crohn’s Trial of the Fully Human Adalimumab for Remission Maintenance) is an adalimumab maintenance study in patients who responded to adalimumab induction therapy. About 50% of the patients in this trial were previously treated with infliximab. Remission rates ranged from 42–48% of infliximab-naïve patients at 1 year compared with remission rates of 31–34% in patients who had previously received infliximab. Another trial showed a remission rate of 21% at 4 weeks in patients who had initially responded to and then failed infliximab. In clinical practice, the

1	who had previously received infliximab. Another trial showed a remission rate of 21% at 4 weeks in patients who had initially responded to and then failed infliximab. In clinical practice, the remission rate in patients taking adalimumab increases with a dose increase to 40 mg weekly instead of every other week. Adalimumab is now also approved for the treatment of moderately to severely active UC.

1	Certolizumab pegol is a pegylated form of an anti-TNF Fab portion of an antibody administered SC once monthly. SC certolizumab pegol was effective for induction of clinical response in patients with active inflammatory CD. In the PRECISE II (Pegylated Antibody Fragment Evaluation in Crohn’s Disease) trial of maintenance therapy with certolizumab in patients who responded to certolizumab induction, the results were similar to the CHARM trial. At week 26, the subgroup of patients who were infliximab naïve had a response of 69% as compared to 44% in patients who had previously received infliximab. Golimumab is another fully human IgG1 antibody against TNF-α and is currently approved for the treatment of moderately to severely active UC. All of the patients in the golimumab trial were infliximab-naive. Like adalimumab and certolizumab, golimumab is injected SC.

1	Side Effects of Anti-TNF Therapies • Development of antiboDies The development of antibodies to infliximab (ATIs) is associated with an increased risk of infusion reactions and a decreased response to treatment. Current practice does not include giving on-demand or episodic infusions in contrast to periodic (every 8 week) infusions because patients are most likely to develop ATIs. ATIs are generally present when the quality of response or the response duration to infliximab infusion decreases. Decreasing the dosing intervals or increasing the dosage to 10 mg/kg may restore the efficacy. There are commercial assays for both infliximab and adalimumab antibodies and trough levels to determine optimal dosing. If a patient has high ATIs and a low trough level of infliximab, it is best to switch to another anti-TNF therapy. Most acute infusion reactions and serum sickness can be managed with glucocorticoids and antihistamines. Some reactions can be serious and would necessitate a change in

1	to another anti-TNF therapy. Most acute infusion reactions and serum sickness can be managed with glucocorticoids and antihistamines. Some reactions can be serious and would necessitate a change in therapy, especially if a patient has ATIs.

1	non-HoDGkin’s lympHoma (nHl) The baseline risk of NHL in CD patients is 2:10,000, which is slightly higher than in the general population. 1962 Azathioprine and/or 6-MP therapy increases the risk to about 4:10,000. The highest risk for thiopurine-associated NHL is in patients over 65 years old, with a moderate risk in those between the ages of 50 and 65. Anti-TNF therapy increases the risk to approximately 6:10,000.

1	Hepatosplenic t cell lympHoma (Hstcl) HSTCL is a nearly universally fatal lymphoma in patients with or without CD. In patients with CD, events reported to the Food and Drug Administration Adverse Event Reporting System (FDA AERS) and search of PubMed and Embase published case reports demonstrate a total of 37 unique cases. Eighty-six percent of the patients were male, with a median age of 26 years. Patients had CD for a mean of 10 years before the diagnosis of HSTCL. Thirty-six cases had used either 6-MP or azathioprine, and 28 cases had used infliximab. Of these 28 cases, 27 had also used 6-MP or azathioprine. The other case had a history of both infliximab and adalimumab exposure.

1	skin lesions New-onset psoriasiform skin lesions develop in nearly 5% of IBD patients treated with anti-TNF therapy. Most often, these can be treated topically, and rarely, anti-TNF therapy must be decreased, switched, or stopped. The risk of melanoma is increased almost twofold with anti-TNF and not thiopurine use. The risk of nonmelanoma skin cancer is increased with thiopurines and biologics, especially with 1 year of follow-up or greater. Patients on these medications should have a skin check at least once a year.

1	infections All of the anti-TNF drugs are associated with an increased risk of infections, particularly reactivation of latent tuberculosis and opportunistic fungal infections including disseminated histoplasmosis and coccidioidomycosis. It is recommended that patients have a purified protein derivative (PPD) or a QuantiFERON-TB gold test as well as a chest x-ray before initiation of anti-TNF therapy. Patients over 65 have a higher rate of infections and death on infliximab or adalimumab than those younger than 65 years of age.

1	otHer Acute liver injury due to reactivation of hepatitis B virus and to autoimmune effects and cholestasis has been reported. Rarely, infliximab and the other anti-TNF drugs have been associated with optic neuritis, seizures, new onset or exacerbation of clinical symptoms, and radiographic evidence of central nervous system demyelinating disorders, including multiple sclerosis. They may exacerbate symptoms in patients with New York Heart Association functional class III/IV heart failure. anti-inteGrins Integrins are expressed on the cell surface of leukocytes and serve as mediators of leukocyte adhesion to vascular endothelium. α4-Integrin along with its β1 or β7 subunit interact with endothelial ligands termed adhesion molecules. Interaction between α4β7 and mucosal addressin cellular adhesion molecule (MAdCAM-1) is important in lymphocyte trafficking to gut mucosa.

1	Natalizumab is a recombinant humanized IgG4 antibody against α4-integrin that has been shown to be effective in induction and maintenance of patients with CD. It has been approved since February 2008 for the treatment of patients with CD refractory or intolerant to anti-TNF therapy. The rates of response and remission at 3 months are about 60% and 40%, respectively, with a sustained remission rate of about 40% at 36 weeks.

1	One case of progressive multifocal leukoencephalopathy (PML) after eight infusions of natalizumab was observed among 1043 patients in the clinical trials for CD, and two patients developed PML in the multiple sclerosis (MS) trials after a median of 120 weeks. There were 410 postmarketing cases of PML, 408 in MS and 2 in CD. The most important risk factor for development of PML is exposure to the John Cunningham (JC) polyomavirus, seen in 50–55% of the adult population. The other two risk factors for development of PML are longer duration of treatment, especially beyond 2 years, and prior treatment with an immunosuppressant medication. Patients with all three risk factors have an estimated risk of 11:1000.

1	The FDA approved a commercial enzyme-linked immunosorbent assay (ELISA) kit to assay anti-JC viral antibodies (Stratify JCV Antibody ELISA; Focus Diagnostics, Cypress, CA) in early 2012. The test is 99% accurate in stratifying risk of PML. It is recommended that all patients be tested prior to initiating natalizumab therapy. JC virus serologies are then measured every 6 months because 1–2% of patients will seroconvert yearly. All patients taking natalizumab and their providers must be enrolled in the TOUCH (Tysabri Outreach Unified Commitment for Health) pharmacovigilance program. Natalizumab is administered IV, 300 mg every 4 weeks. Labeling requirements mandate that it not be used in combination with any immunosuppressant medications.

1	Vedolizumab, another leukocyte trafficking inhibitor, is indicated for patients who have had an inadequate response or lost response to, or were intolerant of a TNF blocker or immunomodulator; or had an inadequate response or were intolerant to, or demonstrated dependence on glucocorticoids. It is an option for patients who are JC antibody positive since it does not cross the blood-brain barrier. Vedolizumab is a monoclonal antibody directed against α4β7 integrin specifically and has the ability to convey gut-selective immunosuppression. Ustekinumab, a fully human IgG1 monoclonal antibody, blocks the biologic activity of IL-12 and IL-23 through their common p40 subunit by inhibiting the interaction of these cytokines with their receptors on T cells, natural killer cells, and antigen presenting cells. It shows efficacy in moderate to severe CD in clinical trials.

1	Tofacitinib is an oral inhibitor of Janus kinases 1, 3, and, to a lesser extent, 2. It is expected to block signaling involving common gamma chain–containing cytokines including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. These cytokines are integral to lymphocyte activation, function, and proliferation. It is effective in moderate to severe UC in clinical trials.

1	Dietary antigens may stimulate the mucosal immune response. Patients with active CD respond to bowel rest, along with TPN. Bowel rest and TPN are as effective as glucocorticoids at inducing remission of active CD but are not effective as maintenance therapy. Enteral nutrition in the form of elemental or peptide-based preparations is also as effective as glucocorticoids or TPN, but these diets are not palatable. Enteral diets may provide the small intestine with nutrients vital to cell growth and do not have the complications of TPN. In contrast to CD, dietary intervention does not reduce inflammation in UC. Standard medical management of UC and CD is shown in Fig. 351-12.

1	SURGICAL THERAPY Ulcerative Colitis Nearly one-half of patients with extensive chronic UC undergo surgery within the first 10 years of their illness. The indications for surgery are listed in Table 351-8. Morbidity is about 20% for elective, 30% for urgent, and 40% for emergency proctocolectomy. The risks are primarily hemorrhage, contamination and sepsis, and neural injury. The operation of choice is an ileoanal J pouch anastomosis (IPAA).

1	Because UC is a mucosal disease, the rectal mucosa can be dissected and removed down to the dentate line of the anus or about 2 cm proximal to this landmark. The ileum is fashioned into a pouch that serves as a neorectum. This ileal pouch is then sutured circumferentially to the anus in an end-to-end fashion. If performed carefully, this operation preserves the anal sphincter and maintains continence. The overall operative morbidity is 10%, with the major complication being bowel obstruction. Pouch failure necessitating conversion to permanent ileostomy occurs in 5–10% of patients. Some inflamed rectal mucosa is usually left behind, and thus endoscopic surveillance is necessary. Primary dysplasia of the ileal mucosa of the pouch has occurred rarely.

1	Patients with IPAA usually have about 6–10 bowel movements a day. On validated quality-of-life indices, they report better performance in sports and sexual activities than ileostomy patients. The most frequent complication of IPAA is pouchitis in about 30–50% FIGURE 351-12 Medical management of inflammatory bowel disease. 5-ASA, 5-aminosalicylic acid; CD, Crohn’s disease; UC, ulcerative colitis.

1	FIGURE 351-12 Medical management of inflammatory bowel disease. 5-ASA, 5-aminosalicylic acid; CD, Crohn’s disease; UC, ulcerative colitis. of patients with UC. This syndrome consists of increased stool frequency, watery stools, cramping, urgency, nocturnal leakage of stool, arthralgias, malaise, and fever. Pouch biopsies may distinguish true pouchitis from underlying CD. Although pouchitis usually responds to antibiotics, 3–5% of patients remain refractory and may require glucocorticoids, immunomodulators, anti-TNF therapy, or even pouch removal. A highly concentrated probiotic preparation with four strains of Lactobacillus, three strains of Bifidobacterium, and one strain of Streptococcus salivarius can prevent the recurrence of pouchitis when taken daily.

1	Intractable disease Small Intestine Fulminant disease Stricture and obstruction Toxic megacolon unresponsive to medical therapy Colonic perforation Massive hemorrhage Massive colonic hemorrhage Refractory fistula Extracolonic disease Abscess Colonic obstruction Colon and rectum Colon cancer prophylaxis Intractable disease Colon dysplasia or cancer Fulminant disease Perianal disease unresponsive to medical therapy Refractory fistula Colonic obstruction Cancer prophylaxis Colon dysplasia or cancer Crohn’s Disease Most patients with CD require at least one operation in their lifetime. The need for surgery is related to duration of disease and the site of involvement. Patients with small-bowel disease have an 80% chance of requiring surgery. Those with colitis alone have a 50% chance. Surgery is an option only when medical treatment has failed or complications dictate its necessity. The indications for surgery are shown in Table 351-8.

1	small intestinal Disease Because CD is chronic and recurrent, with no clear surgical cure, as little intestine as possible is resected. Current surgical alternatives for treatment of obstructing CD include resection of the diseased segment and strictureplasty. Surgical resection of the diseased segment is the most frequently performed operation, and in most cases, primary anastomosis can be done to restore continuity. If much of the small bowel has already been resected and the strictures are short, with intervening areas of normal mucosa, strictureplasties should be done to avoid a functionally insufficient length of bowel. The strictured area of intestine is incised longitudinally and the incision sutured transversely, thus widening the narrowed area. Complications of strictureplasty include prolonged ileus, hemorrhage, fistula, abscess, leak, and restricture.

1	There is evidence that mesalamine, nitroimidazole antibiotics, 6-MP/azathioprine, infliximab, and adalimumab are all superior to placebo for the prevention of postoperative recurrence of CD. Mesalamine is the least effective, and the side effects of the nitroimidazole antibiotics limit their use. Risk factors for early recurrence of disease include cigarette smoking, penetrating disease (internal fistulas, abscesses, or other evidence of penetration through the wall of the bowel), early recurrence since a previous surgery, multiple surgeries, and a young age at the time of the first surgery. Aggressive postoperative treatment with 6-MP/ 1964 azathioprine, infliximab, or adalimumab should be considered for this group of patients. It is also recommended to evaluate for endoscopic recurrence of CD via a colonoscopy, if possible, 6 months after surgery.

1	colorectal Disease A greater percentage of patients with Crohn’s colitis require surgery for intractability, fulminant disease, and anorectal disease. Several alternatives are available, ranging from the use of a temporary loop ileostomy to resection of segments of diseased colon or even the entire colon and rectum. For patients with segmental involvement, segmental colon resection with primary anastomosis can be performed. In 20–25% of patients with extensive colitis, the rectum is spared sufficiently to consider rectal preservation. Most surgeons believe that an IPAA is contraindicated in CD due to the high incidence of pouch failure. A diverting colostomy may help heal severe perianal disease or rectovaginal fistulas, but disease almost always recurs with reanastomosis. These patients often require a total proctocolectomy and ileostomy.

1	Patients with quiescent UC and CD have normal fertility rates; the fallopian tubes can be scarred by the inflammatory process of CD, especially on the right side because of the proximity of the terminal ileum. In addition, perirectal, perineal, and rectovaginal abscesses and fistulae can result in dyspareunia. Infertility in men can be caused by sulfasalazine but reverses when treatment is stopped. In women who have an ileoanal J pouch anastomosis, most studies show that the fertility rate is reduced to about 50–80% of normal. This is due to scarring or occlusion of the fallopian tubes secondary to pelvic inflammation.

1	In mild or quiescent UC and CD, fetal outcome is nearly normal. Spontaneous abortions, stillbirths, and developmental defects are increased with increased disease activity, not medications. The courses of CD and UC during pregnancy mostly correlate with disease activity at the time of conception. Patients should be in remission for 6 months before conceiving. Most CD patients can deliver vaginally, but cesarean delivery may be the preferred route of delivery for patients with anorectal and perirectal abscesses and fistulas to reduce the likelihood of fistulas developing or extending into the episiotomy scar. Unless they desire multiple children, UC patients with an IPAA should consider a cesarean delivery due to an increased risk of future fecal incontinence.

1	Sulfasalazine, Lialda, Apriso, Delzicol, and balsalazide are safe for use in pregnancy and nursing with the caveat that additional folate supplementation must be given with sulfasalazine. Asacol HD and olsalazine are considered by the FDA to be class C agents in pregnancy and thus not recommended. Topical 5-ASA agents are also safe during pregnancy and nursing. Glucocorticoids are generally safe for use during pregnancy and are indicated for patients with moderate to severe disease activity. The amount of glucocorticoids received by the nursing infant is minimal. The safest antibiotics to use for CD in pregnancy for short periods of time (weeks, not months) are ampicillin and cephalosporins. Metronidazole can be used in the second or third trimester. Ciprofloxacin causes cartilage lesions in immature animals and should be avoided because of the absence of data on its effects on growth and development in humans.

1	6-MP and azathioprine pose minimal or no risk during pregnancy, but experience is limited. If the patient cannot be weaned from the drug or has an exacerbation that requires 6-MP/azathioprine during pregnancy, she should continue the drug with informed consent. Breast milk has been shown to contain negligible levels of 6-MP/azathioprine when measured in a limited number of patients. Little data exist on CSA in pregnancy. In a small number of patients with severe IBD treated with IV CSA during pregnancy, 80% of pregnancies were successfully completed without development of renal toxicity or congenital malformations. However, because of the lack of data, CSA should probably be avoided unless the patient would otherwise require surgery.

1	MTX is contraindicated in pregnancy and nursing. In a large prospective study, no increased risk of stillbirths, miscarriages, or spontaneous abortions was seen with infliximab, adalimumab, or certolizumab, which are all class B drugs. Infliximab and adalimumab are IgG1 antibodies and are actively transported across the placenta in the late second and third trimester. Infants can have serum levels of both infliximab and adalimumab up to 7 months of age, and live vaccines should be avoided during this time. Certolizumab crosses the placenta by passive diffusion, and infant serum and cord blood levels are minimal. The anti-TNF drugs are relatively safe in nursing. Miniscule levels of both infliximab and adalimumab, but not certolizumab, have been reported in breast milk. These levels are of no clinical significance. It is recommended that drugs not be switched during pregnancy unless necessitated by the medical condition of the IBD. Natalizumab is considered as a class C drug because

1	of no clinical significance. It is recommended that drugs not be switched during pregnancy unless necessitated by the medical condition of the IBD. Natalizumab is considered as a class C drug because there is limited data in pregnancy.

1	Surgery in UC should be performed only for emergency indications, including severe hemorrhage, perforation, and megacolon refractory to medical therapy. Total colectomy and ileostomy carry a 50% risk of postoperative spontaneous abortion. Fetal mortality is also high in CD requiring surgery. Patients with IPAAs have increased nighttime stool frequency during pregnancy that resolves postpartum. Transient small-bowel obstruction or ileus has been noted in up to 8% of patients with ileostomies. Patients with long-standing UC are at increased risk for developing colonic epithelial dysplasia and carcinoma (Fig. 351-13).

1	Patients with long-standing UC are at increased risk for developing colonic epithelial dysplasia and carcinoma (Fig. 351-13). The risk of neoplasia in chronic UC increases with duration and extent of disease. From one large meta-analysis, the risk of cancer in patients with UC is estimated at 2% after 10 years, 8% after 20 years, and 18% after 30 years of disease. Data from a 30-year surveillance program in the United Kingdom calculated the risk of colorectal cancer to be 7.7% at 20 years and 15.8% at 30 years of disease. The rates of colon cancer are higher than in the general population, and colonoscopic surveillance is the standard of care.

1	Annual or biennial colonoscopy with multiple biopsies is recommended for patients with >8–10 years of extensive colitis (greater than one-third of the colon involved) or 12–15 years of proctosigmoiditis (less than one-third but more than just the rectum) and has been widely used to screen and survey for subsequent dysplasia and carcinoma. Risk factors for cancer in UC include long-duration disease, extensive disease, family history of colon cancer, PSC, a colon stricture, and the presence of postinflammatory pseudopolyps on colonoscopy.

1	Risk factors for developing cancer in Crohn’s colitis are long-duration and extensive disease, bypassed colon segments, colon strictures, PSC, and family history of colon cancer. The cancer risks in CD and UC are probably equivalent for similar extent and duration of disease. In the CESAME study, a prospective observational cohort of IBD patients in France, the standardized incidence ratios of colorectal cancer were 2.2 for all IBD patients (95% confidence interval [CI], 1.5–3.0; p < .001) and 7.0 for patients with long-standing extensive colitis (both Crohn’s and UC) (95% CI, 4.4–10.5; p < .001). Thus, the same endoscopic surveillance strategy used for UC is recommended for patients with chronic Crohn’s colitis. A pediatric colonoscope can be used to pass narrow strictures in CD patients, but surgery should be considered in symptomatic patients with impassable strictures.

1	FIGURE 351-13 Medium-power view of low-grade dysplasia in a patient with chronic ulcerative colitis. Low-grade dysplastic crypts are interspersed among regenerating crypts. (Courtesy of Dr. R. Odze, Division of Gastrointestinal Pathology, Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts; with permission.) Dysplasia can be flat or polypoid. If flat high-grade dysplasia is encountered on colonoscopic surveillance, the usual treatment is colectomy for UC and either colectomy or segmental resection for CD. If flat low-grade dysplasia is found (Fig. 351-13), most investigators recommend immediate colectomy. Adenomas may occur coincidently in UC and CD patients with chronic colitis and can be removed endoscopically provided that biopsies of the surrounding mucosa are free of dysplasia. High-definition and high-magnification colonoscopes and dye sprays have increased the rate of dysplasia detection.

1	IBD patients are also at greater risk for other malignancies. Patients with CD may have an increased risk of non-Hodgkin’s lymphoma, leukemia, and myelodysplastic syndromes. Severe, chronic, complicated perianal disease in CD patients may be associated with an increased risk of cancer in the lower rectum and anal canal (squamous cell cancers). Although the absolute risk of small-bowel adenocarcinoma in CD is low (2.2% at 25 years in one study), patients with long-standing, extensive, small-bowel disease should consider screening.

1	Irritable bowel syndrome (IBS) is a functional bowel disorder characterized by abdominal pain or discomfort and altered bowel habits in the absence of detectable structural abnormalities. No clear diagnostic markers exist for IBS; thus the diagnosis of the disorder is based on clinical presentation. In 2006, the Rome II criteria for the diagnosis of IBS were revised (Table 352-1). Throughout the world, about 10–20% of adults and adolescents have symptoms consistent with IBS, and most studies show a female predominance. IBS symptoms tend to come and go over time and often overlap with other functional disorders such as fibromyalgia, headache, backache, and genitourinary symptoms. Severity of symptoms varies and can significantly impair quality of life, resulting in high health care costs. Advances in basic, mechanistic, and clinical investigations have improved our understanding of this disorder and its physiologic and psychosocial determinants. Altered

1	Recurrent abdominal pain or discomfortb at least 3 days per month in the last 3 months associated with two or more of the following: 1. Improvement with defecation 2. Onset associated with a change in frequency of stool 3. Onset associated with a change in form (appearance) of stool aCriteria fulfilled for the last 3 months with symptom onset at least 6 months prior to diagnosis. bDiscomfort means an uncomfortable sensation not described as pain. In pathophysiology research and clinical trials, a pain/discomfort frequency of at least 2 days a week during screening evaluation is required for subject eligibility. Source: Adapted from GF Longstreth et al: Gastroenterology 130:1480, 2006.

1	Source: Adapted from GF Longstreth et al: Gastroenterology 130:1480, 2006. gastrointestinal (GI) motility, visceral hyperalgesia, disturbance of 1965 brain-gut interaction, abnormal central processing, autonomic and hormonal events, genetic and environmental factors, and psychosocial disturbances are variably involved, depending on the individual. This progress may result in improved methods of treatment.

1	IBS is a disorder that affects all ages, although most patients have their first symptoms before age 45. Older individuals have a lower reporting frequency. Women are diagnosed with IBS two to three times as often as men and make up 80% of the population with severe IBS. As indicated in Table 352-1, pain or abdominal discomfort is a key symptom for the diagnosis of IBS. These symptoms should be improved with defecation and/or have their onset associated with a change in frequency or form of stool. Painless diarrhea or constipation does not fulfill the diagnostic criteria to be classified as IBS. Supportive symptoms that are not part of the diagnostic criteria include defecation straining, urgency or a feeling of incomplete bowel movement, passing mucus, and bloating.

1	Abdominal Pain According to the current IBS diagnostic criteria, abdominal pain or discomfort is a prerequisite clinical feature of IBS. Abdominal pain in IBS is highly variable in intensity and location. It is frequently episodic and crampy, but it may be superimposed on a background of constant ache. Pain may be mild enough to be ignored or it may interfere with daily activities. Despite this, malnutrition due to inadequate caloric intake is exceedingly rare with IBS. Sleep deprivation is also unusual because abdominal pain is almost uniformly present only during waking hours. However, patients with severe IBS frequently wake repeatedly during the night; thus, nocturnal pain is a poor discriminating factor between organic and functional bowel disease. Pain is often exacerbated by eating or emotional stress and improved by passage of flatus or stools. In addition, female patients with IBS commonly report worsening symptoms during the premenstrual and menstrual phases.

1	Altered Bowel Habits Alteration in bowel habits is the most consistent clinical feature in IBS. The most common pattern is constipation alternating with diarrhea, usually with one of these symptoms predominating. At first, constipation may be episodic, but eventually it becomes continuous and increasingly intractable to treatment with laxatives. Stools are usually hard with narrowed caliber, possibly reflecting excessive dehydration caused by prolonged colonic retention and spasm. Most patients also experience a sense of incomplete evacuation, thus leading to repeated attempts at defecation in a short time span. Patients whose predominant symptom is constipation may have weeks or months of constipation interrupted with brief periods of diarrhea. In other patients, diarrhea may be the predominant symptom. Diarrhea resulting from IBS usually consists of small volumes of loose stools. Most patients have stool volumes of <200 mL. Nocturnal diarrhea does not occur in IBS. Diarrhea may be

1	symptom. Diarrhea resulting from IBS usually consists of small volumes of loose stools. Most patients have stool volumes of <200 mL. Nocturnal diarrhea does not occur in IBS. Diarrhea may be aggravated by emotional stress or eating. Stool may be accompanied by passage of large amounts of mucus. Bleeding is not a feature of IBS unless hemorrhoids are present, and malabsorption or weight loss does not occur.

1	Bowel pattern subtypes are highly unstable. In a patient population with ~33% prevalence rates of IBS-diarrhea predominant (IBSD), IBS-constipation predominant (IBS-C), and IBS-mixed (IBS-M) forms, 75% of patients change subtypes and 29% switch between IBS-C and IBS-D over 1 year. The heterogeneity and variable natural history of bowel habits in IBS increase the difficulty of conducting pathophysiology studies and clinical trials.

1	Gas and Flatulence Patients with IBS frequently complain of abdominal distention and increased belching or flatulence, all of which they attribute to increased gas. Although some patients with these symptoms actually may have a larger amount of gas, quantitative measurements reveal that most patients who complain of increased gas generate no more than a normal amount of intestinal gas. Most IBS patients have impaired transit and tolerance of intestinal gas loads. In addition, patients with IBS tend to reflux gas from the distal to the more proximal intestine, which may explain the belching.

1	1966 Some patients with bloating may also experience visible distention with increase in abdominal girth. Both symptoms are more common among female patients and in those with higher overall Somatic Symptom Checklist scores. IBS patients who experienced bloating alone have been shown to have lower thresholds for pain and desire to defecate compared to those with concomitant distention irrespective of bowel habit. When patients were grouped according to sensory threshold, hyposensitive individuals had distention significantly more than those with hypersensitivity and this was observed more in the constipation subgroup. This suggests that the pathogenesis of bloating and distention may not be the same.

1	Upper Gastrointestinal Symptoms Between 25 and 50% of patients with IBS complain of dyspepsia, heartburn, nausea, and vomiting. This suggests that other areas of the gut apart from the colon may be involved. Prolonged ambulant recordings of small-bowel motility in patients with IBS show a high incidence of abnormalities in the small bowel during the diurnal (waking) period; nocturnal motor patterns are not different from those of healthy controls. The overlap between dyspepsia and IBS is great. The prevalence of IBS is higher among patients with dyspepsia (31.7%) than among those who reported no symptoms of dyspepsia (7.9%). Conversely, among patients with IBS, 55.6% reported symptoms of dyspepsia. In addition, the functional abdominal symptoms can change over time. Those with predominant dyspepsia or IBS can flux between the two. Although the prevalence of functional gastrointestinal disorders is stable over time, the turnover in symptom status is high. Many episodes of symptom

1	dyspepsia or IBS can flux between the two. Although the prevalence of functional gastrointestinal disorders is stable over time, the turnover in symptom status is high. Many episodes of symptom disappearance are due to subjects changing symptoms rather than total symptom resolution. Thus it is conceivable that functional dyspepsia and IBS are two manifestations of a single, more extensive digestive system disorder. Furthermore, IBS symptoms are prevalent in noncardiac chest pain patients, suggesting overlap with other functional gut disorders.

1	The pathogenesis of IBS is poorly understood, although roles of abnormal gut motor and sensory activity, central neural dysfunction, psychological disturbances, mucosal inflammation, stress, and luminal factors have been proposed.

1	Gastrointestinal Motor Abnormalities Studies of colonic myoelectrical and motor activity under unstimulated conditions have not shown consistent abnormalities in IBS. In contrast, colonic motor abnormalities are more prominent under stimulated conditions in IBS. IBS patients may exhibit increased rectosigmoid motor activity for up to 3 h after eating. Similarly, inflation of rectal balloons both in IBS-D and IBS-C patients leads to marked and prolonged distention-evoked contractile activity. Recordings from the transverse, descending, and sigmoid colon showed that the motility index and peak amplitude of high-amplitude propagating contractions (HAPCs) in diarrhea-prone IBS patients were greatly increased compared to those in healthy subjects and were associated with rapid colonic transit and accompanied by abdominal pain.

1	Visceral Hypersensitivity As with studies of motor activity, IBS patients frequently exhibit exaggerated sensory responses to visceral stimulation. The frequency of perceptions of food intolerance is at least twofold more common than in the general population. Postprandial pain has been temporally related to entry of the food bolus into the cecum in 74% of patients. On the other hand, prolonged fasting in IBS patients is often associated with significant improvement in symptoms. Rectal balloon inflation produces nonpainful and painful sensations at lower volumes in IBS patients than in healthy controls without altering rectal tension, suggestive of visceral afferent dysfunction in IBS. Similar studies show gastric and esophageal hypersensitivity in patients with nonulcer dyspepsia and noncardiac chest pain, raising the possibility that these conditions have a similar pathophysiologic basis. Lipids lower the thresholds for the first sensation of gas, discomfort, and pain in IBS

1	and noncardiac chest pain, raising the possibility that these conditions have a similar pathophysiologic basis. Lipids lower the thresholds for the first sensation of gas, discomfort, and pain in IBS patients. Hence, postprandial symptoms in IBS patients may be explained in part by a nutrient-dependent exaggerated sensory component of the gastrocolonic response. In contrast to enhanced gut sensitivity, IBS patients do not exhibit heightened sensitivity

1	Abbreviation: CNS, central nervous system. elsewhere in the body. Thus, the afferent pathway disturbances in IBS appear to be selective for visceral innervation with sparing of somatic pathways. The mechanisms responsible for visceral hypersensitivity are still under investigation. It has been proposed that these exaggerated responses may be due to (1) increased end-organ sensitivity with recruitment of “silent” nociceptors; (2) spinal hyperexcitability with activation of nitric oxide and possibly other neurotransmitters; (3) endogenous (cortical and brainstem) modulation of caudad nociceptive transmission; and (4) over time, the possible development of longterm hyperalgesia due to development of neuroplasticity, resulting in permanent or semipermanent changes in neural responses to chronic or recurrent visceral stimulation (Table 352-2).

1	Central Neural Dysregulation The role of central nervous system (CNS) factors in the pathogenesis of IBS is strongly suggested by the clinical association of emotional disorders and stress with symptom exacerbation and the therapeutic response to therapies that act on cerebral cortical sites. Functional brain imaging studies such as magnetic resonance imaging (MRI) have shown that in response to distal colonic stimulation, the mid-cingulate cortex—a brain region concerned with attention processes and response selection—shows greater activation in IBS patients. Modulation of this region is associated with changes in the subjective unpleasantness of pain. In addition, IBS patients also show preferential activation of the prefrontal lobe, which contains a vigilance network within the brain that increases alertness. These may represent a form of cerebral dysfunction leading to the increased perception of visceral pain.

1	Abnormal Psychological Features Abnormal psychiatric features are recorded in up to 80% of IBS patients, especially in referral centers; however, no single psychiatric diagnosis predominates. Most of these patients demonstrated exaggerated symptoms in response to visceral distention, and this abnormality persists even after exclusion of psychological factors. Psychological factors influence pain thresholds in IBS patients, as stress alters sensory thresholds. An association between prior sexual or physical abuse and development of IBS has been reported. Abuse is associated with greater pain reporting, psychological distress, and poor health outcome. Brain functional MRI studies show greater activation of the posterior and middle dorsal cingulate cortex, which is implicated in affect processing in IBS patients with a past history of sexual abuse.

1	Thus, patients with IBS frequently demonstrate increased motor reactivity of the colon and small bowel to a variety of stimuli and altered visceral sensation associated with lowered sensation thresholds. These may result from CNS–enteric nervous system dysregulation (Fig. 352-1).

1	Postinfectious IBS IBS may be induced by GI infection. In an investigation of 544 patients with confirmed bacterial gastroenteritis, one-quarter developed IBS subsequently. Conversely, about a third of IBS patients experienced an acute “gastroenteritis-like” illness at the onset of their chronic IBS symptomatology. This group of “postinfective” IBS occurs more commonly in females and affects younger rather than older patients. Risk factors for developing postinfectious IBS include, in order of importance, prolonged duration of initial illness, toxicity of infecting bacterial strain, smoking, mucosal markers of inflammation, female gender, depression, hypochondriasis, and adverse life events in the preceding 3 months. Age older than 60 years might protect against postinfectious IBS, whereas treatment with antibiotics has been CNS Antidepressants, Psychological Cognitive

1	CNS Antidepressants, Psychological Cognitive Psychotherapy, behavioral therapy, hypnotherapy, Somatization-disorder management Antispasmodics, Antidiarrheals, Dietary modification, Fiber supplements, Newer gut serotonin modulators Heightened sensorimotor activity FIGURE 352-1 Therapeutic targets for irritable bowel syndrome. Patients with mild to moderate symptoms usually have intermittent symptoms that correlate with altered gut physiology. Treatments include gut-acting pharmacologic agents such as antispasmodics, antidiarrheals, fiber supplements, and gut serotonin modulators. Patients who have severe symptoms usually have constant pain and psychosocial difficulties. This group of patients is best managed with antidepressants and other psychosocial treatments. CNS, central nervous system; ENS, enteric nervous system.

1	associated with increased risk. The microbes involved in the initial infection are Campylobacter, Salmonella, and Shigella. Those patients with Campylobacter infection who are toxin-positive are more likely to develop postinfective IBS. Increased rectal mucosal enteroendocrine cells, T lymphocytes, and increased gut permeability are acute changes following Campylobacter enteritis that could persist for more than a year and may contribute to postinfective IBS.

1	Immune Activation and Mucosal Inflammation Some patients with IBS display persistent signs of low-grade mucosal inflammation with activated lymphocytes, mast cells, and enhanced expression of proinflammatory cytokines. These abnormalities may contribute to abnormal epithelial secretion and visceral hypersensitivity. There is increasing evidence that some members of the superfamily of transient receptor potential (TRP) cation channels such as TRPV1 (vanilloid) channels are central to the initiation and persistence of visceral hypersensitivity. Mucosal inflammation can lead to increased expression of TRPV1 in the enteric nervous system. Enhanced expression of TRPV1 channels in the sensory neurons of the gut has been observed in IBS, and such expression appears to correlate with visceral hypersensitivity and abdominal pain. Interestingly, clinical studies have also shown increased intestinal permeability in patients with IBS-D. Psychological stress and anxiety can increase the release of

1	and abdominal pain. Interestingly, clinical studies have also shown increased intestinal permeability in patients with IBS-D. Psychological stress and anxiety can increase the release of proinflammatory cytokine, and this in turn may alter intestinal permeability. This provides a functional link between psychological stress, immune activation, and symptom generation in patients with IBS.

1	Altered Gut Flora A high prevalence of small intestinal bacterial overgrowth in IBS patients has been noted based on positive lactulose hydrogen breath test. This finding, however, has been challenged by a number of other studies that found no increased incidence of bacterial overgrowth based on jejunal aspirate culture. Abnormal H2 breath test can occur because of small-bowel rapid transit and may lead to 1967 erroneous interpretation. Hence, the role of testing for small intestinal bacterial overgrowth in IBS patients remains unclear.

1	Studies using culture-independent approaches such as 16S rRNA gene-based analysis found significant differences between the molecular profile of the fecal microbiota of IBS patients and that of healthy subjects. IBS patients had decreased proportions of the genera Bifidobacterium and Lactobacillus and increased ratios of Firmicutes:Bacteroidetes. It has been speculated that these changes may be related to stress and diet. A temporary reduction in lactobacilli has been reported in animal models of early-life stress. On the other hand, Firmicutes is the dominant phylum in adults consuming a diet high in animal fat and protein. However, it is still unclear whether such changes in fecal microbiota are causal, consequential, or merely the result of constipation and diarrhea. In addition, the stability of the change in the microbiota needs to be determined.

1	Abnormal Serotonin Pathways The serotonin (5-HT)-containing enterochromaffin cells in the colon are increased in a subset of IBS-D patients compared to healthy individuals or patients with ulcerative colitis. Furthermore, postprandial plasma 5-HT levels were significantly higher in this group of patients compared to healthy controls. Because serotonin plays an important role in the regulation of GI motility and visceral perception, the increased release of serotonin may contribute to the postprandial symptoms of these patients and provides a rationale for the use of serotonin antagonists in the treatment of this disorder. APPROACH TO THE PATIENT:

1	Because IBS is a disorder for which no pathognomonic abnormalities have been identified, its diagnosis relies on recognition of positive clinical features and elimination of other organic diseases. Symptom-based criteria have been developed for the purpose of differentiating patients with IBS from those with organic diseases. These include the Manning, Rome I, Rome II, and Rome III criteria (Table 352-1). The diagnostic values of these criteria are shown in Table 352-3. In a validation study, Rome III performed less well than either the Rome I and II criteria and all criteria studied to date showed positive predictive values of <50%, which underscores the need for developing diagnostic strategies for IBS that are more cost-effective than the current approaches. A careful history and physical examination are frequently helpful in establishing the diagnosis. Clinical features suggestive of IBS include the following: recurrence of lower abdominal pain with altered bowel habits over a

1	examination are frequently helpful in establishing the diagnosis. Clinical features suggestive of IBS include the following: recurrence of lower abdominal pain with altered bowel habits over a period of time without progressive deterioration, onset of symptoms during periods of stress or emotional upset, absence of other systemic symptoms such as fever and weight loss, and small-volume stool without any evidence of blood.

1	On the other hand, the appearance of the disorder for the first time in old age, progressive course from time of onset, persistent diarrhea after a 48-h fast, and presence of nocturnal diarrhea or steatorrheal stools argue against the diagnosis of IBS. SEnSiTiViTy, SPECifiCiTy, PoSiTiVE AnD nEGATiVE PREDiCTiVE VAluES, AnD PoSiTiVE AnD nEGATiVE likEliHooD RATioS foR THE RomE AnD mAnninG CRiTERiA foR iRRiTABlE BoWEl SynDRomEa

1	Because the major symptoms of IBS—abdominal pain, abdominal bloating, and alteration in bowel habits—are common complaints of many GI organic disorders, the list of differential diagnoses is a long one. The quality, location, and timing of pain may be helpful to suggest specific disorders. Pain due to IBS that occurs in the epigastric or periumbilical area must be differentiated from biliary tract disease, peptic ulcer disorders, intestinal ischemia, and carcinoma of the stomach and pancreas. If pain occurs mainly in the lower abdomen, the possibility of diverticular disease of the colon, inflammatory bowel disease (including ulcerative colitis and Crohn’s disease), and carcinoma of the colon must be considered. Postprandial pain accompanied by bloating, nausea, and vomiting suggests gastroparesis or partial intestinal obstruction. Intestinal infestation with Giardia lamblia or other parasites may cause similar symptoms. When diarrhea is the major complaint, the possibility of lactase

1	or partial intestinal obstruction. Intestinal infestation with Giardia lamblia or other parasites may cause similar symptoms. When diarrhea is the major complaint, the possibility of lactase deficiency, laxative abuse, malabsorption, celiac sprue, hyperthyroidism, inflammatory bowel disease, and infectious diarrhea must be ruled out. On the other hand, constipation may be a side effect of many different drugs, such as anticholinergic, antihypertensive, and antidepressant medications. Endocrinopathies such as hypothyroidism and hypoparathyroidism must also be considered in the differential diagnosis of constipation, particularly if other systemic signs or symptoms of these endocrinopathies are present. In addition, acute intermittent porphyria and lead poisoning may present in a fashion similar to IBS, with painful constipation as the major complaint. These possibilities are suspected on the basis of their clinical presentations and are confirmed by appropriate serum and urine tests.

1	Few tests are required for patients who have typical IBS symptoms and no alarm features. Unnecessary investigations may be costly and even harmful. The American Gastroenterological Association has delineated factors to be considered when determining the aggressiveness of the diagnostic evaluation. These include the duration of symptoms, the change in symptoms over time, the age and sex of the patient, the referral status of the patient, prior diagnostic studies, a family history of colorectal malignancy, and the degree of psychosocial dysfunction. Thus, a younger individual with mild symptoms requires a minimal diagnostic evaluation, while an older person or an individual with rapidly progressive symptoms should undergo a more thorough exclusion of organic disease. Most patients should have a complete blood count and sigmoidoscopic examination; in addition, stool specimens should be examined for ova and parasites in those who have diarrhea. In patients with persistent diarrhea not

1	have a complete blood count and sigmoidoscopic examination; in addition, stool specimens should be examined for ova and parasites in those who have diarrhea. In patients with persistent diarrhea not responding to simple antidiarrheal agents, a sigmoid colon biopsy should be performed to rule out microscopic colitis. In those age >40 years, an air-contrast barium enema or colonoscopy should also be performed. If the main symptoms are diarrhea and increased gas, the possibility of lactase deficiency should be ruled out with a hydrogen breath test or with evaluation after a 3-week lactose-free diet. Some patients with IBS-D may have undiagnosed celiac sprue. Because the symptoms of celiac sprue respond to a gluten-free diet, testing for celiac sprue in IBS may prevent years of morbidity and attendant expense. Decision-analysis studies show that serology testing for celiac sprue in patients with IBS-D has an acceptable cost when the prevalence of celiac sprue is >1% and is the dominant

1	attendant expense. Decision-analysis studies show that serology testing for celiac sprue in patients with IBS-D has an acceptable cost when the prevalence of celiac sprue is >1% and is the dominant strategy when the prevalence is >8%. In patients with concurrent symptoms of dyspepsia, upper GI radiographs or esophagogastroduodenoscopy may be advisable. In patients with postprandial right upper quadrant pain, an ultrasonogram of the gallbladder should be obtained. Laboratory features that argue against IBS include evidence of anemia, elevated sedimentation rate, presence of leukocytes or blood in stool, and stool volume >200–300 mL/d. These findings would necessitate other diagnostic considerations.

1	Reassurance and careful explanation of the functional nature of the disorder and of how to avoid obvious food precipitants are important first steps in patient counseling and dietary change. Occasionally, a meticulous dietary history may reveal substances (such as coffee, disaccharides, legumes, and cabbage) that aggravate symptoms. Excessive fructose and artificial sweeteners, such as sorbitol or mannitol, may cause diarrhea, bloating, cramping, or flatulence. As a therapeutic trial, patients should be encouraged to eliminate any foodstuffs that appear to produce symptoms. However patients should avoid nutritionally depleted diets. A diet low in fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) (Table 352-4) has been shown to be helpful in IBS patients. FODMAPs are poorly absorbed by the small intestine and fermented by bacteria in the colon to produce gas and osmotically active carbohydrates. Clinical studies demonstrate that in IBS patients,

1	FODMAPs are poorly absorbed by the small intestine and fermented by bacteria in the colon to produce gas and osmotically active carbohydrates. Clinical studies demonstrate that in IBS patients, ingestion of FODMAPs such as lactose, fructose, or sorbitol, alone or in combination, produce gut symptoms such as gas and diarrhea. On the other hand, a randomized controlled study showed that a diet low in FODMAPs reduced symptoms in IBS patients. This

1	Inulin, FOS Sorbitol, mannitol, maltitol, xylitol, isomalt Abbreviations: FODMAPs, fermentable oligosaccharides, disaccharides, monosaccharides, and polyols; FOS, fructo-oligosaccharides Source: Adapted from PR Gibson et al: Am J Gastroenterol 107:657, 2012. approach may be used in diarrhea-predominant IBS patients with severe gas and bloating. Durable adherence can be expected in up to 75% of patients.

1	Stool-Bulking Agents High-fiber diets and bulking agents, such as bran or hydrophilic colloid, are frequently used in treating IBS. The water-holding action of fibers may contribute to increased stool bulk because of the ability of fiber to increase fecal output of bacteria. Fiber also speeds up colonic transit in most persons. In diarrhea-prone patients, whole-colonic transit is faster than average; however, dietary fiber can delay transit. Furthermore, because of their hydrophilic properties, stool-bulking agents bind water and thus prevent both excessive hydration and dehydration of stool. The latter observation may explain the clinical experience that a high-fiber diet relieves diarrhea in some IBS patients. Fiber supplementation with psyllium has been shown to reduce perception of rectal distention, indicating that fiber may have a positive effect on visceral afferent function.

1	The beneficial effects of dietary fiber on colonic physiology suggest that dietary fiber should be an effective treatment for IBS patients, but controlled trials of dietary fiber have produced variable results. This is not surprising since IBS is a heterogeneous disorder, with some patients being constipated and other having predominant diarrhea. Most investigations report increases in stool weight, decreases in colonic transit times, and improvement in constipation. Others have noted benefits in patients with alternating diarrhea and constipation, pain, and bloating. However, most studies observe no responses in patients with diarrheaor pain-predominant IBS. It is possible that different fiber preparations may have dissimilar effects on selected symptoms in IBS. A cross-over comparison of different fiber preparations found that psyllium produced greater improvements in stool pattern and abdominal pain than bran. Furthermore, psyllium preparations tend to produce less bloating and

1	of different fiber preparations found that psyllium produced greater improvements in stool pattern and abdominal pain than bran. Furthermore, psyllium preparations tend to produce less bloating and distention. Despite the equivocal data regarding efficacy, most gastroenterologists consider stool-bulking agents worth trying in patients with IBS-C. Fiber should be started at a nominal dose and slowly titrated up as tolerated over the course of several weeks to a targeted dose of 20–30 g of total dietary and supplementary fiber per day. Even when used judiciously, fiber can exacerbate bloating, flatulence, constipation, and diarrhea.

1	Antispasmodics Clinicians have observed that anticholinergic drugs may provide temporary relief for symptoms such as painful cramps related to intestinal spasm. Although controlled clinical trials have produced mixed results, evidence generally supports beneficial effects of anticholinergic drugs for pain. A meta-analysis of 26 double-blind clinical trials of antispasmodic agents in IBS reported better global improvement (62%) and abdominal pain reductions (64%) compared to placebo (35% and 45%, respectively), suggesting efficacy in some patients. The drugs are most effective when prescribed in anticipation of predictable pain. Physiologic studies demonstrate that anticholinergic drugs inhibit the gastrocolic reflex; hence, postprandial pain is best managed by giving antispasmodics 30 min before meals so that effective blood levels are achieved shortly before the anticipated onset of pain. Most anticholinergics contain natural belladonna alkaloids, which may cause xerostomia, urinary

1	before meals so that effective blood levels are achieved shortly before the anticipated onset of pain. Most anticholinergics contain natural belladonna alkaloids, which may cause xerostomia, urinary hesitancy and retention, blurred vision, and drowsiness. They should be used in the elderly with caution. Some physicians prefer to use synthetic anticholinergics such as dicyclomine that have less effect on mucous membrane secretions and produce fewer undesirable side effects.

1	Antidiarrheal Agents Peripherally acting opiate-based agents are the initial therapy of choice for IBS-D. Physiologic studies demonstrate increases in segmenting colonic contractions, delays in fecal transit, increases in anal pressures, and reductions in rectal perception with these drugs. When diarrhea is severe, especially in the painless diarrhea variant of IBS, small doses of loperamide, 2–4 mg every 4–6 h up to a maximum of 12 g/d, can be prescribed. These agents are less addictive than paregoric, codeine, or tincture of opium. In general, the intestines do not become tolerant of the antidiarrheal effect of opiates, and increasing doses are not required to maintain antidiarrheal potency. These agents are most useful if taken before anticipated 1969 stressful events that are known to cause diarrhea. However, not infrequently, a high dose of loperamide may cause cramping because of increases in segmenting colonic contractions. Another antidiarrheal agent that may be used in IBS

1	to cause diarrhea. However, not infrequently, a high dose of loperamide may cause cramping because of increases in segmenting colonic contractions. Another antidiarrheal agent that may be used in IBS patients is the bile acid binder cholestyramine resin.

1	Antidepressant Drugs In addition to their mood-elevating effects, antidepressant medications have several physiologic effects that suggest they may be beneficial in IBS. In IBS-D patients, the tricyclic antidepressant imipramine slows jejunal migrating motor complex transit propagation and delays orocecal and whole-gut transit, indicative of a motor inhibitory effect. Some studies also suggest that tricyclic agents may alter visceral afferent neural function. A number of studies indicate that tricyclic antidepressants may be effective in some IBS patients. In a 2-month study of desipramine, abdominal pain improved in 86% of patients compared to 59% given placebo. Another study of desipramine in 28 IBS patients showed improvement in stool frequency, diarrhea, pain, and depression.

1	When stratified according to the predominant symptoms, improvements were observed in IBS-D patients, with no improvement being noted in IBS-C patients. The beneficial effects of the tricyclic compounds in the treatment of IBS appear to be independent of their effects on depression. The therapeutic benefits for the bowel symptoms occur faster and at a lower dosage. The efficacy of antidepressant agents in other chemical classes in the management of IBS is less well evaluated. In contrast to tricyclic agents, the selective serotonin reuptake inhibitor (SSRI) paroxetine accelerates orocecal transit, raising the possibility that this drug class may be useful in IBS-C patients. The SSRI citalopram blunts perception of rectal distention and reduces the magnitude of the gastrocolonic response in healthy volunteers. A small placebo-controlled study of citalopram in IBS patients reported reductions in pain. However, these findings could not be confirmed in another randomized controlled trial

1	healthy volunteers. A small placebo-controlled study of citalopram in IBS patients reported reductions in pain. However, these findings could not be confirmed in another randomized controlled trial that showed that citalopram at 20 mg/d for 4 weeks was not superior to placebo in treating nondepressed IBS patients. Hence, the efficacy of SSRIs in the treatment of IBS needs further confirmation.

1	Antiflatulence Therapy The management of excessive gas is seldom satisfactory, except when there is obvious aerophagia or disaccharidase deficiency. Patients should be advised to eat slowly and not chew gum or drink carbonated beverages. Bloating may decrease if an associated gut syndrome such as IBS or constipation is improved. If bloating is accompanied by diarrhea and worsens after ingesting dairy products, fresh fruits, vegetables, or juices, further investigation or a dietary exclusion trial may be worthwhile. Avoiding flatogenic foods, exercising, losing excess weight, and taking activated charcoal are safe but unproven remedies. Data regarding the use of surfactants such as simethicone are conflicting. Antibiotics may help in a subgroup of IBS patients with predominant symptoms of bloating. Beano, an over-the-counter oral β-glycosidase solution, may reduce rectal passage of gas without decreasing bloating and pain. Pancreatic enzymes reduce bloating, gas, and fullness during

1	bloating. Beano, an over-the-counter oral β-glycosidase solution, may reduce rectal passage of gas without decreasing bloating and pain. Pancreatic enzymes reduce bloating, gas, and fullness during and after high-calorie, high-fat meal ingestion.

1	Modulation of Gut Flora Antibiotic treatment benefits a subset of IBS patients. In a double-blind, randomized, placebo-controlled study, neomycin dosed at 500 mg twice daily for 10 days was more effective than placebo at improving symptom scores among IBS patients. The nonabsorbed oral antibiotic rifaximin is the most thoroughly studied antibiotic for the treatment of IBS. In a double-blind, placebo-controlled study, patients receiving rifaximin at a dose of 550 mg two times daily for 2 weeks experienced substantial improvement of global IBS symptoms over placebo. Rifaximin is the only antibiotic with demonstrated sustained benefit beyond therapy cessation in IBS patients. The drug has a favorable safety and tolerability profile compared with systemic antibiotics. A systematic review and meta-analysis of five studies of IBS patients found that rifaximin is more effective than placebo for global symptoms and bloating (odds ratio 1.57) with a number needed to treat (NNT) of 1970 10.2.

1	meta-analysis of five studies of IBS patients found that rifaximin is more effective than placebo for global symptoms and bloating (odds ratio 1.57) with a number needed to treat (NNT) of 1970 10.2. The modest therapeutic gain was similar to that yielded by other current available therapies for IBS. However, currently there are still insufficient data to recommend routine use of this antibiotic in the treatment of IBS. Because altered colonic flora may contribute to the pathogenesis of IBS, this has led to great interest in using probiotics to naturally alter the flora. A meta-analysis of 10 probiotic studies in IBS patients found significant relief of pain and bloating with the use of Bifidobacterium breve, B. longum, and Lactobacillus acidophilus species compared to placebo. However, there was no change in stool frequency or consistency. Large-scale studies of well-phenotyped IBS patients are needed to establish the efficacy of these probiotics.

1	Serotonin Receptor Agonist and Antagonists Serotonin receptor antagonists have been evaluated as therapies for IBS-D. Serotonin acting on 5-HT3 receptors enhances the sensitivity of afferent neurons projecting from the gut. In humans, a 5-HT3 receptor antagonist such as alosetron reduces perception of painful visceral stimulation in IBS. It also induces rectal relaxation, increases rectal compliance, and delays colonic transit. Meta-analysis of 14 randomized controlled trials of alosetron or cilansetron showed that these antagonists are more effective than placebo in achieving global improvement in IBS symptoms and relief of abdominal pain and discomfort. These agents are more likely to cause constipation in IBS patients with diarrhea alternating with constipation. Also, 0.2% of patients using 5-HT3 antagonists developed ischemic colitis versus none in the control group. In postrelease surveillance, 84 cases of ischemic colitis were observed, including 44 cases that required surgery

1	using 5-HT3 antagonists developed ischemic colitis versus none in the control group. In postrelease surveillance, 84 cases of ischemic colitis were observed, including 44 cases that required surgery and 4 deaths. As a consequence, the medication was voluntarily withdrawn by the manufacturer in 2000. Alosetron has been reintroduced under a new risk-management program where patients have to sign a patient-physician agreement. This has significantly limited its usage.

1	Novel 5-HT4 receptor agonists such as tegaserod exhibit prokinetic activity by stimulating peristalsis. In IBS patients with constipation, tegaserod accelerated intestinal and ascending colon transit. Clinical trials involving >4000 IBS-C patients reported reductions in discomfort and improvements in constipation and bloating, compared to placebo. Diarrhea is the major side effect. However, tegaserod has been withdrawn from the market; a meta-analysis revealed an increase in serious cardiovascular events.

1	Chloride Channel Activators Lubiprostone is a bicyclic fatty acid that stimulates chloride channels in the apical membrane of intestinal epithelial cells. Chloride secretion induces passive movement of sodium and water into the bowel lumen and improves bowel function. Oral lubiprostone was effective in the treatment of patients with constipation-predominant IBS in large phase II and phase III randomized, double-blinded, placebo-controlled multicenter trials. Responses were significantly greater in patients receiving lubiprostone 8 μg twice daily for 3 months than in those receiving placebo. In general, the drug was quite well tolerated. The major side effects are nausea and diarrhea. Lubiprostone is a new class of compounds for treatment of chronic constipation with or without IBS.

1	Guanylate Cyclase-C Agonist Linaclotide is a minimally absorbed 14-amino-acid peptide guanylate cyclase-C (GC-C) agonist that binds to and activates GC-C on the luminal surface of intestinal epithelium. Activation of GC-C results in generation of cyclic guanosine monophosphate (cGMP), which triggers secretion of fluid, sodium, and bicarbonate. In animal models, linaclotide accelerates GI transit and reduces visceral nociception. The analgesic action of linaclotide appears to be mediated by cGMP acting on afferent pain fibers innervating the GI tract. A phase III, double-blind, controlled trial showed that linaclotide, 290 μg given once daily, significantly improved abdominal pain, bloating, and spontaneous bowel movement. The only significant side effect was diarrhea, which occurred in 4.5% of the patients. The drug has been approved for treatment of constipation in IBS-C patients. Correlations with gut physiology +++ ++ +

1	The treatment strategy of IBS depends on the severity of the disorder (Table 352-5). Most IBS patients have mild symptoms. They are usually cared for in primary care practices, have little or no psychosocial difficulties, and do not seek health care often. Treatment usually involves education, reassurance, and dietary/lifestyle changes. A smaller portion have moderate symptoms that are usually intermittent and correlate with altered gut physiology, e.g., worsened with eating or stress and relieved by defecation. For IBS-D patients, treatments include gut-acting pharmacologic agents such as antispasmodics, antidiarrheals, bile acid binders, and the newer gut serotonin modulators (Table 352-6). In IBS-C patients, increased fiber intake and the use of osmotic agents such as polyethylene glycol may achieve satisfactory results. For patients with more severe constipation, a chloride channel opener (lubiprostone) or GC-C agonist (linaclotide) may be considered. For IBS patients with

1	glycol may achieve satisfactory results. For patients with more severe constipation, a chloride channel opener (lubiprostone) or GC-C agonist (linaclotide) may be considered. For IBS patients with predominant gas and bloating, a low-FODMAP diet may provide significant relief. Some patients may benefit from probiotics and rifaximin treatment. A small proportion of IBS patients have severe and refractory symptoms, are usually seen in referral centers, and frequently have constant pain and psychosocial difficulties (Fig. 352-1). This group of patients is best managed with antidepressants and other psychological treatments (Table 352-6).

1	aAvailable only in the United States. Abbreviation: FODMAP, fermentable oligosaccharides, disaccharides, monosaccharides, and polyols. Source: Adapted from GF Longstreth et al: Gastroenterology 130:1480, 2006. Diverticular Disease and Common Anorectal Disorders Rizwan Ahmed, Susan L. Gearhart DIVERTICULAR DISEASE Incidence and Epidemiology In the United States, diverticulo-353 sis affects 70% of the population above the age of 80.

1	Fortunately, only 20% of patients with diverticulosis develop symptomatic disease, 1–2% require hospitalization, and <1% will require surgery. Diverticular disease has become the fifth most costly gastrointestinal disorder in the United States. Previously overlooked, the majority of patients with diverticular disease report a lower health-related quality of life and more depression as compared to matched controls, thus adding to health care costs. Formerly, diverticular disease was confined to developed countries; however, with the adoption of westernized diets in underdeveloped countries, diverticulosis is on the rise across the globe. Immigrants to the United States develop diverticular disease at the same rate as U.S. natives. Although the prevalence among females and males is similar, males tend to present at a younger age. The mean age at presentation of the disease is 59 years and is now shifting to affect younger populations.

1	Anatomy and Pathophysiology Two types of diverticula occur in the intestine: true and false (or pseudo diverticula). A true diverticulum is a saclike herniation of the entire bowel wall, whereas a pseudo diverticulum involves only a protrusion of the mucosa and submucosa through the muscularis propria of the colon (Fig. 353-1). The type of diverticulum affecting the colon is the pseudodiverticulum. Diverticula commonly affect the left and sigmoid colon; the rectum is always spared. However, in Asian populations, 70% of diverticula are seen in the right colon and cecum as well. Diverticulitis is inflammation of a diverticulum. Previous understanding of the pathogenesis of diverticulosis attributed a low-fiber diet as the sole culprit, and onset of diverticulitis would occur acutely when these diverticula become obstructed. However, evidence now suggests that the pathogenesis is more complex and multifactorial. The diverticula occur at the point where the nutrient artery, or vasa recti,

1	diverticula become obstructed. However, evidence now suggests that the pathogenesis is more complex and multifactorial. The diverticula occur at the point where the nutrient artery, or vasa recti, penetrates through the muscularis propria, resulting in a break in the integrity of the colonic wall. This anatomic restriction may be a result of the relative high-pressure zone within the muscular sigmoid colon. Thus, higher-amplitude contractions combined with constipated, high-fat-content stool within the sigmoid lumen in an area of weakness in the colonic wall results in the creation of these diverticula. Consequently, the vasa recti is either compressed or eroded, leading to either perforation or bleeding. Chronic low-grade inflammation is thought to play a key role. Furthermore, better understanding of the gut microbiota suggests that dysbiosis is an important aspect of disease.

1	Presentation, Evaluation, and Management of Diverticular Bleeding Hemorrhage from a colonic diverticulum is the most common cause of hematochezia in patients >60 years, yet only 20% of patients with diverticulosis will have gastrointestinal bleeding. Patients at increased risk for bleeding tend to be hypertensive, have atherosclerosis, and regularly use aspirin and nonsteroidal anti-inflammatory agents. Most bleeds are self-limited and stop spontaneously with bowel rest. The lifetime risk of rebleeding is 25%.

1	Initial localization of diverticular bleeding may include colonoscopy, multiplanar computed tomography (CT) angiogram, or nuclear medicine tagged red cell scan. If the patient is stable, ongoing bleeding is best managed by angiography. If mesenteric angiography can localize the bleeding site, the vessel can be occluded successfully with a coil in 80% of cases. The patient can then be followed closely with repetitive colonoscopy, if necessary, looking for evidence of colonic ischemia. Alternatively, a segmental resection of the colon can be undertaken to eliminate the risk of further bleeding. This may be advantageous in patients on chronic anticoagulation. However, with highly selective coil embolization, the rate of colonic ischemia is <10% and the risk of FIGURE 353-1 Gross and microscopic view of sigmoid diverticular disease. Arrows mark an inflamed diverticulum with the diverticular wall made up only of mucosa.

1	FIGURE 353-1 Gross and microscopic view of sigmoid diverticular disease. Arrows mark an inflamed diverticulum with the diverticular wall made up only of mucosa. acute rebleeding is <25%. Long-term results (40 months) indicate that more than 50% of patients with acute diverticular bleeds treated with highly selective angiography have had definitive treatment. As another alternative, a selective infusion of vasopressin can be given to stop the hemorrhage, although this has been associated with significant complications, including myocardial infarction and intestinal ischemia. Furthermore, bleeding recurs in 50% of patients once the infusion is stopped.

1	If the patient is unstable or has had a 6-unit bleed within 24 h, current recommendations are that surgery should be performed. If the bleeding has been localized, a segmental resection can be performed. If the site of bleeding has not been definitively identified, a subtotal colectomy may be required. In patients without severe comorbidities, surgical resection can be performed with a primary anastomosis. A higher anastomotic leak rate has been reported in patients who received >10 units of blood.

1	Presentation, Evaluation, and Staging of Diverticulitis Acute uncomplicated diverticulitis characteristically presents with fever, anorexia, left lower quadrant abdominal pain, and obstipation (Table 353-1). In <25% of cases, patients may present with generalized peritonitis indicating the presence of a diverticular perforation. If a pericolonic abscess has formed, the patient may have abdominal distention and signs of localized peritonitis. Laboratory investigations will demonstrate a leukocytosis. Rarely, a patient may present with an air-fluid level in the left lower quadrant on plain abdominal film. This is a giant diverticulum of the sigmoid colon and is managed with resection to avoid impending perforation.

1	The diagnosis of diverticulitis is best made on CT with the following findings: sigmoid diverticula, thickened colonic wall >4 mm, and inflammation within the periodic fat ± the collection of contrast material or fluid. In 16% of patients, an abdominal abscess may be present. Symptoms of irritable bowel syndrome (Chap. 352) may mimic those of diverticulitis. Therefore, suspected diverticulitis that does not meet CT criteria or is not associated with a leukocytosis or fever is not diverticular disease. Other conditions that can mimic diverticular disease include an ovarian cyst, endometriosis, acute appendicitis, and pelvic inflammatory disease.

1	Although the benefit of colonoscopy in the evaluation of patients with diverticular disease has been called into question, its use is still considered important in the exclusion of colorectal cancer. The parallel epidemiology of colorectal cancer and diverticular disease provides enough concern for an endoscopic evaluation before operative management. Therefore, a colonoscopy should be performed ~6 weeks after an attack of diverticular disease.

1	Complicated diverticular disease is defined as diverticular disease associated with an abscess or perforation and less commonly with a fistula (Table 353-1). Perforated diverticular disease is staged using the Hinchey classification system (Fig. 353-2). This staging system was developed to predict outcomes following the surgical management of complicated diverticular disease. In complicated diverticular disease with fistula formation, common locations include cutaneous, vaginal, or vesicle fistulas. These conditions present with either passage of stool through the skin or vagina or the presence of air in the urinary stream (pneumaturia). Colovaginal fistulas are more common in women who have undergone a hysterectomy.

1	Asymptomatic diverticular disease discovered on imaging studies or at the time of colonoscopy is best managed by diet alterations. Patients should be instructed to eat a fiber-enriched diet that includes 30 g of fiber each day. Supplementary fiber products such as Metamucil, Fibercon, or Citrucel are useful. The incidence of complicated diverticular disease appears to be increased in patients who smoke. Therefore, patients should be encouraged to refrain from smoking. The historical recommendation to avoid eating nuts is not based on more than anecdotal data.

1	Symptomatic uncomplicated diverticular disease with confirmation of inflammation and infection within the colon should be treated initially with antibiotics and bowel rest. Nearly 75% of patients hospitalized for acute diverticulitis will respond to nonoperative treatment with a suitable antimicrobial regimen. The current recommended antimicrobial coverage is trimethoprim/ sulfamethoxazole or ciprofloxacin and metronidazole targeting aerobic gram-negative rods and anaerobic bacteria. Unfortunately, these agents do not cover enterococci, and the addition of ampicillin

1	FIGURE 353-2 Hinchey classification of diverticulitis. Stage I: Perforated diverticulitis with a confined paracolic abscess. Stage II: Perforated diverticulitis that has closed spontaneously with distant abscess formation. Stage III: Noncommunicating perforated diverticulitis with fecal peritonitis (the diverticular neck is closed off, and therefore, contrast will not freely expel on radiographic images). Stage IV: Perforation and free communication with the peritoneum, resulting in fecal peritonitis. to this regimen for nonresponders is recommended. Alternatively, single-agent therapy with a third-generation penicillin such as IV piperacillin or oral penicillin/clavulanic acid may be effective. The usual course of antibiotics is 7–10 days, although this length of time is being investigated. Patients should remain on a limited diet until their pain resolves.

1	Once the acute attack has resolved, the mainstay medical management of diverticular disease to prevent symptoms has evolved. Newer directions are targeted at colonic inflammation and dysbiosis. Diverticular disease is now considered a functional bowel disorder associated with low-grade inflammation. Therefore, the use of anti-inflammatory medications such as mesalazine has become popular. Patients treated with mesalazine have a decreased recurrence of symptomatic disease. Randomized trials of anti-inflammatory medications are ongoing.

1	Treatment strategies targeting dysbiosis in diverticular disease are also beneficial. Use of the polymerase chain reaction (PCR) on stool specimens from consumers of a high-fiber diet has shown different bacterial content than stool of consumers of a low-fiber, high-fat diet. Probiotics are being increasingly used by gastroenterologists for multiple bowel disorders and have been shown to prevent recurrence of diverticulitis. Specifically probiotics containing Lactobacillus acidophilus and Bifidobacterium strains have been shown to be beneficial. Furthermore, rifaximin (a poorly absorbed broad-spectrum antibiotic), when compared to fiber alone, is associated with 30% less frequent recurrent symptoms from uncomplicated diverticular disease.

1	Preoperative risk factors influencing postoperative mortality rates include higher American Society of Anesthesiologists (ASA) physical status class (Table 353-2) and preexisting organ failure. In patients who are low risk (ASA P1 and P2), surgical therapy can be offered to those who do not rapidly improve on medical therapy. For uncomplicated diverticular disease, medical therapy can be continued beyond two attacks without an increased risk of perforation requiring a AmERiCAn SoCiETy of AnESTHESioloGiSTS PHySiCAl STATuS ClASSifiCATion SySTEm colostomy. However, patients on immunosuppressive therapy, in chronic renal failure, or with a collagen-vascular disease have a fivefold greater risk of perforation during recurrent attacks. Surgical therapy is indicated in all low-surgical-risk patients with complicated diverticular disease.

1	The goals of surgical management of diverticular disease include controlling sepsis, eliminating complications such as fistula or obstruction, removing the diseased colonic segment, and restoring intestinal continuity. These goals must be obtained while minimizing morbidity rate, length of hospitalization, and cost in addition to maximizing survival and quality of life. Table 353-3 lists the operations most commonly indicated based on the Hinchey classification and the predicted morbidity and mortality rates. Surgical objectives include removal of the diseased sigmoid down to the rectosigmoid junction. Failure to do this may result in recurrent disease. The current options for uncomplicated diverticular disease include an open sigmoid resection or a laparoscopic sigmoid resection. The benefits of laparoscopic resection over open surgical techniques include early discharge (by at least 1 day), less narcotic use, less postoperative complications, and an earlier return to work.

1	The options for the surgical management of complicated diverticular disease (Fig. 353-3) include the following: (1) proximal diversion of the fecal stream with an ileostomy or colostomy and sutured omental patch with drainage, (2) resection with colostomy and mucous fistula or closure of distal bowel with formation of a Hartmann’s pouch, (3) resection with anastomosis (coloproctostomy), or (4) resection with anastomosis and diversion (coloproctostomy with loop ileostomy or colostomy). Laparoscopic techniques have been used for complicated diverticular disease; however, higher conversion rates to open techniques have been reported.

1	Patients with Hinchey stages I and II disease are managed with percutaneous drainage followed by resection with anastomosis about 6 weeks later. Current guidelines put forth by the American Society of Colon and Rectal Surgeons suggest, in addition to antibiotic therapy, CT-guided percutaneous drainage of diverticular abscesses that are greater than 3 cm and have a well-defined wall. Abscesses that are less than 3 cm may resolve with antibiotic therapy alone. Contraindications to percutaneous drainage are no percutaneous access route, pneumoperitoneum, and fecal peritonitis. I Resection with primary 3.8 22 anastomosis without diverting stoma II Resection with primary 3.8 30 anastomosis +/− diversion III Hartmann’s procedure vs — 0 vs. 6 mortality diverting colostomy and omental pedal graft IV Hartmann’s procedure vs — 6 vs. 2 mortality diverting colostomy and omental pedicle graft

1	III Hartmann’s procedure vs — 0 vs. 6 mortality diverting colostomy and omental pedal graft IV Hartmann’s procedure vs — 6 vs. 2 mortality diverting colostomy and omental pedicle graft FIGURE 353-3 Methods of surgical management of complicated diverticular disease. (1) Drainage, omental pedicle graft, and proxi-mal diversion. (2) Hartmann’s procedure. (3) Sigmoid resection with coloproctostomy. (4) Sigmoid resection with coloproctostomy and proximal diversion.

1	Urgent operative intervention is undertaken if patients develop generalized peritonitis, and most will need to be managed with a Hartmann’s procedure (resection of the sigmoid colon with end colostomy and rectal stump). In selected cases, nonoperative therapy may be considered. In one nonrandomized study, nonoperative management of isolated paracolic abscesses (Hinchey stage I) was associated with only a 20% recurrence rate at 2 years. More than 80% of patients with distant abscesses (Hinchey stage II) required surgical resection for recurrent symptoms.

1	Hinchey stage III disease is managed with a Hartmann’s procedure or with primary anastomosis and proximal diversion. If the patient has significant comorbidities, making operative intervention risky, a limited procedure including intraoperative peritoneal lavage (irrigation), omental patch to the oversewn perforation, and proximal diversion of the fecal stream with either an ileostomy or transverse colostomy can be performed. No anastomosis of any type should be attempted in Hinchey stage IV disease. A limited approach to these patients is associated with a decreased mortality rate.

1	Recurrent Symptoms Recurrent abdominal symptoms following surgical resection for diverticular disease occur in 10% of patients. Recurrent diverticular disease develops in patients following inadequate surgical resection. A retained segment of diseased rectosigmoid colon is associated with twice the incidence of recurrence. The presence of irritable bowel syndrome may also cause recurrence of initial symptoms. Patients undergoing surgical resection for presumed diverticulitis and symptoms of chronic abdominal cramping and irregular loose bowel movements consistent with irritable bowel syndrome have poorer functional outcomes.

1	RECTAL PROLAPSE (PROCIDENTIA) Incidence and Epidemiology Rectal prolapse is six times more common in women than in men. The incidence of rectal prolapse peaks in women >60 years. Women with rectal prolapse have a higher incidence 1974 of associated pelvic floor disorders including urinary incontinence, rectocele, cystocele, and enterocele. About 20% of children with rectal prolapse will have cystic fibrosis. All children presenting with prolapse should undergo a sweat chloride test. Less common associations include Ehlers-Danlos syndrome, solitary rectal ulcer syndrome, congenital hypothyroidism, Hirschsprung’s disease, dementia, mental retardation, and schizophrenia.

1	Anatomy and Pathophysiology Rectal prolapse (procidentia) is a circumferential, full-thickness protrusion of the rectal wall through the anal orifice. It is often associated with a redundant sigmoid colon, pelvic laxity, and a deep rectovaginal septum (pouch of Douglas). Initially, rectal prolapse was felt to be the result of early internal rectal intussusception, which occurs in the upper to mid rectum. This was considered to be the first step in an inevitable progression to full-thickness external prolapse. However, only 1 of 38 patients with internal prolapse followed for >5 years developed full-thickness prolapse. Others have suggested that full-thickness prolapse is the result of damage to the nerve supply to the pelvic floor muscles or pudendal nerves from repeated stretching with straining to defecate. Damage to the pudendal nerves would weaken the pelvic floor muscles, including the external anal sphincter muscles. Bilateral pudendal nerve injury is more significantly

1	with straining to defecate. Damage to the pudendal nerves would weaken the pelvic floor muscles, including the external anal sphincter muscles. Bilateral pudendal nerve injury is more significantly associated with prolapse and incontinence than unilateral injury.

1	Presentation and Evaluation In external prolapse, the majority of patient complaints include anal mass, bleeding per rectum, and poor perianal hygiene. Prolapse of the rectum usually occurs following defecation and will spontaneously reduce or require the patient to manually reduce the prolapse. Constipation occurs in ~30–67% of patients with rectal prolapse. Differing degrees of fecal incontinence occur in 50–70% of patients. Patients with internal rectal prolapse will present with symptoms of both constipation and incontinence. Other associated findings include outlet obstruction (anismus) in 30%, colonic inertia in 10%, and solitary rectal ulcer syndrome in 12%.

1	Office evaluation is best performed after the patient has been given an enema, which enables the prolapse to protrude. An important distinction should be made between full-thickness rectal prolapse and isolated mucosal prolapse associated with hemorrhoidal disease (Fig. 353-4). Mucosal prolapse is known for radial grooves rather than circumferential folds around the anus and is due to increased laxity of the connective tissue between the submucosa and underlying muscle of the anal canal. The evaluation of prolapse should also include cystoproctography and colonoscopy. These examinations evaluate for associated pelvic floor disorders and rule out a malignancy or a polyp as the lead point for prolapse. If rectal prolapse is associated with chronic constipation, the patient should undergo a defecating proctogram and a sitzmark study. This will evaluate for the presence of anismus or colonic inertia. Anismus is the result of attempting to defecate against a closed pelvic floor and is also

1	proctogram and a sitzmark study. This will evaluate for the presence of anismus or colonic inertia. Anismus is the result of attempting to defecate against a closed pelvic floor and is also known as nonrelaxing puborectalis. This can be seen when straightening of the rectum fails to occur on fluoroscopy while the patient is attempting to defecate. In colonic inertia, a sitzmark study will demonstrate retention of >20% of markers on abdominal x-ray 5 days after swallowing. For patients with fecal incontinence, endoanal ultrasound and manometric evaluation, including pudendal nerve testing of their anal sphincter muscles, may be performed before surgery for prolapse (see “Fecal Incontinence,” below).

1	The medical approach to the management of rectal prolapse is limited and includes stool-bulking agents or fiber supplementation to ease the process of evacuation. Surgical correction of rectal prolapse is the mainstay of therapy. Two approaches are commonly considered, transabdominal and transperineal. Transabdominal approaches have been associated with lower recurrence rates, but some patients with significant comorbidities are better served by a transperineal approach. Common transperineal approaches include a transanal proctectomy (Altmeier procedure), mucosal proctectomy (Delorme FIGURE 353-4 Degrees of rectal prolapse. Mucosal prolapse only (A, B, sagittal view). Full-thickness prolapse associated with redundant rectosigmoid and deep pouch of Douglas (C, D, sagittal view).

1	procedure), or placement of a Tirsch wire encircling the anus. The goal of the transperineal approach is to remove the redundant rectosigmoid colon. Common transabdominal approaches include presacral suture or mesh rectopexy (Ripstein) with (Frykman-Goldberg) or without resection of the redundant sigmoid. Colon resection, in general, is reserved for patients with constipation and outlet obstruction. Ventral rectopexy is an effective method of abdominal repair of full-thickness prolapse that does not require sigmoid resection (see description below). This repair may have improved functional results over other abdominal repairs. Transabdominal procedures can be performed effectively with laparoscopic and, more recently, robotic techniques without increased incidence of recurrence. The goal of the transabdominal approach is to restore normal anatomy by removing redundant bowel and reattaching the supportive tissue of the rectum to the presacral fascia. The final alternative is abdominal

1	of the transabdominal approach is to restore normal anatomy by removing redundant bowel and reattaching the supportive tissue of the rectum to the presacral fascia. The final alternative is abdominal proctectomy with end-sigmoid colostomy. If total colonic inertia is present, as defined by a history of constipation and a positive sitzmark study, a subtotal colectomy with an ileosigmoid or rectal anastomosis may be required at the time of rectopexy.

1	Previously, the presence of internal rectal prolapse identified on imaging studies has been considered a nonsurgical disorder and biofeedback was recommended. However, only one-third of patients will have successful resolution of symptoms from biofeedback. Two surgical procedures more effective than biofeedback are the Stapled Transanal Rectal Resection (STARR) and the Laparoscopic Ventral Rectopexy (LVR). The STARR procedure (Fig. 353-5) is performed through the anus in patients with internal prolapse. A circular stapling device is inserted through the anus; the internal prolapse is identified and ligated with the stapling device. LVR (Fig. 353-6) is performed through an abdominal approach. An opening in the peritoneum is created on the left side of the recto-sigmoid junction, and this opening continues down anterior on the rectum into the pouch of Douglas. No rectal mobilization is performed, thus avoiding any autonomic nerve injury. Mesh is secured to the anterior and lateral

1	opening continues down anterior on the rectum into the pouch of Douglas. No rectal mobilization is performed, thus avoiding any autonomic nerve injury. Mesh is secured to the anterior and lateral portion of the rectum, the vaginal fornix, and the sacral promontory, allowing for closure of the rectovaginal septum and correction of the internal prolapse. In both procedures, recurrence at 1 year was low (<10%) and symptoms improved in more than three-fourths of patients.

1	FIGURE 353-5 Stapled transanal rectal resection. Schematic of placement of the circular stapling device. FECAL INCONTINENCE Incidence and Epidemiology Fecal incontinence is the involuntary passage of fecal material for at least 1 month in an individual with a developmental age of at least 4 years. The prevalence of fecal incontinence in the United States is 0.5–11%. The majority of patients are women and

1	FIGURE 353-6 Laparoscopic ventral rectopexy (LVR). To reduce the internal prolapse and close any rectovaginal septal defect, the pouch of Douglas is opened and mesh is secured to the anterolateral rec-tum, vaginal fornix, and sacrum. (From A D’Hoore et al: Br J Surg 91:1500, 2004.) • Myopathies, above the age of 65. A higher incidence of incontinence is seen among parous women. One-half of patients with fecal incontinence also suffer from urinary incontinence. The majority of incontinence is a result of obstetric injury to the pelvic floor, either while carrying a fetus or during the delivery. An anatomic sphincter defect may occur in up to 32% of women following childbirth regardless of visible damage to the perineum. Risk factors at the time of delivery include prolonged labor, the use of forceps, and the need for an episiotomy. Symptoms of incontinence can present after two or more decades following obstetric injury. Medical conditions known to contribute to the development of

1	use of forceps, and the need for an episiotomy. Symptoms of incontinence can present after two or more decades following obstetric injury. Medical conditions known to contribute to the development of fecal incontinence are listed in Table 353-4.

1	Anatomy and Pathophysiology The anal sphincter complex is made up of the internal and external anal sphincter. The internal sphincter is smooth muscle and a continuation of the circular fibers of the rectal wall. It is innervated by the intestinal myenteric plexus and is therefore not under voluntary control. The external anal sphincter is formed in continuation with the levator ani muscles and is under voluntary control. The pudendal nerve supplies motor innervation to the external anal sphincter. Obstetric injury may result in tearing of the muscle fibers anteriorly at the time of the delivery. This results in an obvious anterior defect on endoanal ultrasound. Injury may also be the result of stretching of the pudendal nerves during pregnancy or delivery of the fetus through the birth canal.

1	Presentation and Evaluation Patients may suffer with varying degrees of fecal incontinence. Minor incontinence includes incontinence to flatus and occasional seepage of liquid stool. Major incontinence is frequent inability to control solid waste. As a result of fecal incontinence, patients suffer from poor perianal hygiene. Beyond the immediate problems associated with fecal incontinence, these patients are often withdrawn and suffer from depression. For this reason, quality-of-life measures are an important component in the evaluation of patients with fecal incontinence.

1	The evaluation of fecal incontinence should include a thorough history and physical exam including digital rectal examination (DRE). Weak sphincter tone on DRE and loss of the “anal wink” reflex (S1-level control) may indicate a neurogenic dysfunction. Perianal scars may represent surgical injury. Other studies helpful in the diagnosis of fecal incontinence include anal manometry, pudendal nerve terminal motor latency (PNTML), and endoanal ultrasound. Centers that care for patients with fecal incontinence will have an anorectal physiology laboratory that uses standardized methods of evaluating anorectal physiology. Anorectal manometry (ARM) measures resting and squeeze pressures within the anal canal using an intraluminal water-perfused catheter. Current methods of ARM include use of a three-dimensional, high-resolution system with a 12-catheter perfusion system, which allows physiologic delineation of anatomic abnormalities.

1	1976 Pudendal nerve studies evaluate the function of the nerves innervating the anal canal using a finger electrode placed in the anal canal. Stretch injuries to these nerves will result in a delayed response of the sphincter muscle to a stimulus, indicating a prolonged latency. Finally, endoanal ultrasound will evaluate the extent of the injury to the sphincter muscles before surgical repair. Unfortunately, all of these investigations are user-dependent, and very few studies demonstrate that these studies predict outcome following an intervention. Magnetic resonance imaging (MRI) has been used, but its routine use for imaging in fecal incontinence is not well established. Rarely does a pelvic floor disorder exist alone. The majority of patients with fecal incontinence will have some degree of urinary incontinence. Similarly, fecal incontinence is a part of the spectrum of pelvic organ prolapse. For this reason, patients may present with symptoms of obstructed defecation as well as

1	of urinary incontinence. Similarly, fecal incontinence is a part of the spectrum of pelvic organ prolapse. For this reason, patients may present with symptoms of obstructed defecation as well as fecal incontinence. Careful evaluation including dynamic MRI or cinedefecography should be performed to search for other associated defects. Surgical repair of incontinence without attention to other associated defects may decrease the success of the repair.

1	Medical management of fecal incontinence includes strategies to bulk up the stool, which help in increasing fecal sensation. These include fiber supplementation, loperamide, diphenoxylate, and bile acid binders. These agents harden the stool and delay frequency of bowel movements and are helpful in patients with minimal to mild symptoms. Furthermore, patients can be offered a form of physical therapy called biofeedback. This therapy helps strengthen the external sphincter muscle while training the patient to relax with defecation to avoid unnecessary straining and further injury to the sphincter muscles. Biofeedback has had variable success and is dependent on the motivation of the patient. At a minimum, biofeedback is risk free and safe. Most patients will have some improvement. For this reason, it should be incorporated into the initial recommendation to all patients with fecal incontinence.

1	The “gold standard” for the treatment of fecal incontinence with an isolated sphincter defect has been the overlapping sphincteroplasty. The external anal sphincter muscle and scar tissue as well as any identifiable internal sphincter muscle are dissected free from the surrounding adipose and connective tissue and then an overlapping repair is performed in an attempt to rebuild the muscular ring and restore its function. Long-term results following overlapping sphincteroplasty show about a 50% failure rate over 5 years. Poorer outcome has been seen in patients with prolonged pudendal nerve terminal motor latency.

1	Sacral neuromodulation, collagen-enhancing injectables, radio-frequency therapy, and the artificial bowel sphincter are other options. Sacral nerve stimulation and the artificial bowel sphincter are both adaptations of procedures developed for the management of urinary incontinence. Sacral nerve stimulation is ideally suited for patients with intact but weak anal sphincters. A temporary nerve stimulator is placed on the third sacral nerve. If there is at least a 50% improvement in symptoms, a permanent nerve stimulator is placed under the skin. The artificial bowel sphincter is a cuff and reservoir apparatus that allows for manual inflation of a cuff placed around the anus, increasing anal tone. This allows the patient to manually close off the anal canal until defecation is necessary. Long-term results for sacral stimulation have been promising, with nearly 80% of patients having a reduction in incontinence episodes by at least 50%. This reduction has been sustainable in studies out

1	results for sacral stimulation have been promising, with nearly 80% of patients having a reduction in incontinence episodes by at least 50%. This reduction has been sustainable in studies out to 5 years. Unfortunately, the artificial bowel sphincter has been associated with a 30% infection rate. Accordingly, implantation is performed less often.

1	Collagen-enhancing injectables have been around for several years. The largest open trial involved 115 incontinent patients treated with nonanimal stabilized hyaluronic acid (NASHA/DX) gel. In this study, patients underwent injections of NASHA/DX (Solesta) into the anal mucosa and were followed for 12 months. The results were promising, with over 50% achieving greater than 50% reduction in incontinence episodes, and these results were sustainable up to 2 years. This method is another less invasive therapy for patients with fecal incontinence. Radiofrequency energy delivery to the anal canal in patients with fecal incontinence aids in the development and restructuring of collagen fibers and provides tensile strength to the sphincter muscles. The radiofrequency is delivered as an office procedure with sedation. The results have been variable, with 20–50% of patients having a sustained reduction in incontinence episodes for 5 years.

1	Finally, the use of stem cells to increase the bulk of the sphincter muscles is currently being tested. Stem cells can be harvested from the patient’s own muscle, grown, and then implanted into their sphincter complex. Concern for cost and the need for an additional procedure dampen enthusiasm. Trial results are awaited. HEMORRHOIDAL DISEASE Incidence and Epidemiology Symptomatic hemorrhoids affect >1 million individuals in the Western world per year. The prevalence of hemorrhoidal disease is not selective for age or sex. However, age is known to be a risk factor. The prevalence of hemorrhoidal disease is less in underdeveloped countries. The typical low-fiber, high-fat Western diet is associated with constipation and straining and the development of symptomatic hemorrhoids.

1	Anatomy and Pathophysiology Hemorrhoidal cushions are a normal part of the anal canal. The vascular structures contained within this tissue aid in continence by preventing damage to the sphincter muscle. Three main hemorrhoidal complexes traverse the anal canal—the left lateral, the right anterior, and the right posterior. Engorgement and straining lead to prolapse of this tissue into the anal canal. Over time, the anatomic support system of the hemorrhoidal complex weakens, exposing this tissue to the outside of the anal canal where it is susceptible to injury. Hemorrhoids are commonly classified as external or internal. External hemorrhoids originate below the dentate line and are covered with squamous epithelium and are associated with an internal component. External hemorrhoids are painful when thrombosed. Internal hemorrhoids originate above the dentate line and are covered with mucosa and transitional zone epithelium and represent majority of hemorrhoids. The standard

1	are painful when thrombosed. Internal hemorrhoids originate above the dentate line and are covered with mucosa and transitional zone epithelium and represent majority of hemorrhoids. The standard classification of hemorrhoidal disease is based on the progression of the disease from their normal internal location to the prolapsing external position (Table 353-5).

1	Presentation and Evaluation Patients commonly present to a physician for two reasons: bleeding and protrusion. Pain is less common than with fissures and, if present, is described as a dull ache from engorgement of the hemorrhoidal tissue. Severe pain may indicate a thrombosed hemorrhoid. Hemorrhoidal bleeding is described as painless bright red blood seen either in the toilet or upon wiping. Occasional patients can present with significant bleeding, which may be a cause of anemia; however, the presence of a colonic neoplasm must be ruled out in anemic patients. Patients who present with a protruding mass complain about inability to maintain perianal hygiene and are often concerned about the presence of a malignancy.

1	The diagnosis of hemorrhoidal disease is made on physical examination. Inspection of the perianal region for evidence of thrombosis or excoriation is performed, followed by a careful digital examination. Anoscopy is performed paying particular attention to the known position of hemorrhoidal disease. The patient is asked to strain. If this is difficult for the patient, the maneuver can be performed while sitting on a toilet. The physician is notified when the tissue prolapses. It is important to differentiate the circumferential appearance of a full-thickness rectal prolapse from the radial nature of prolapsing hemorrhoids (see “Rectal Prolapse,” above). The stage and location of the hemorrhoidal complexes are defined.

1	The treatment for bleeding hemorrhoids is based on the stage of the disease (Table 353-5). In all patients with bleeding, the possibility of other causes must be considered. In young patients without a family history of colorectal cancer, the hemorrhoidal disease may be treated first and a colonoscopic examination performed if the bleeding continues. Older patients who have not had colorectal cancer screening should undergo colonoscopy or flexible sigmoidoscopy.

1	With rare exceptions, the acutely thrombosed hemorrhoid can be excised within the first 72 h by performing an elliptical excision. Sitz baths, fiber, and stool softeners are prescribed. Additional therapy for bleeding hemorrhoids includes the office procedures of banding and sclerotherapy. Sensation begins at the dentate line; therefore, banding or sclerotherapy can be performed without discomfort in the office. Bands are placed around the engorged tissue, causing ischemia and fibrosis. This aids in fixing the tissue proximally in the anal canal. Patients may complain of a dull ache for 24 h following band application. During sclerotherapy, 1–2 mL of a sclerosant (usually sodium tetradecyl sulfate) is injected using a 25-gauge needle into the submucosa of the hemorrhoidal complex. Care must be taken not to inject the anal canal circumferentially, or stenosis may occur.

1	For surgical management of hemorrhoidal disease, excisional hemorrhoidectomy, transhemorrhoidal dearterialization (THD), or stapled hemorrhoidectomy (“the procedure for prolapse or hemorrhoids” [PPH]) is the procedure of choice. All surgical methods of management are equally effective in the treatment of symptomatic thirdand fourth-degree hemorrhoids. However, because the sutured hemorrhoidectomy involves the removal of redundant tissue down to the anal verge, unpleasant anal skin tags are removed as well. The stapled hemorrhoidectomy is associated with less discomfort; however, this procedure does not remove anal skin tags. THD uses ultrasound guidance to ligate the blood supply to the anal tissue, hence reducing hemorrhoidal engorgement. No procedures on hemorrhoids should be done in patients who are immunocompromised or who have active proctitis. Furthermore, emergent hemorrhoidectomy for bleeding hemorrhoids is associated with a higher complication rate.

1	Acute complications associated with the treatment of hemorrhoids include pain, infection, recurrent bleeding, and urinary retention. Care should be taken to place bands properly and to avoid overhydration in patients undergoing operative hemorrhoidectomy. Late complications include fecal incontinence as a result of injury to the sphincter during the dissection. Anal stenosis may develop from overzealous excision, with loss of mucosal skin bridges for reepithelialization. Finally, an ectropion (prolapse of rectal mucosa from the anal canal) may develop. Patients with an ectropion complain of a “wet” anus as a result of inability to prevent soiling once the rectal mucosa is exposed below the dentate line. FIGURE 353-7 Common locations of anorectal abscess (left) and fistula in ano (right).

1	FIGURE 353-7 Common locations of anorectal abscess (left) and fistula in ano (right). ANORECTAL ABSCESS Incidence and Epidemiology The development of a perianal abscess is more common in men than women by a ratio of 3:1. The peak incidence is in the third to fifth decade of life. Perianal pain associated with the presence of an abscess accounts for 15% of office visits to a colorectal surgeon. The disease is more prevalent in immunocompromised patients such as those with diabetes, hematologic disorders, or inflammatory bowel disease and persons who are HIV positive. These disorders should be considered in patients with recurrent perianal infections.

1	Anatomy and Pathophysiology An anorectal abscess is an abnormal fluid-containing cavity in the anorectal region. Anorectal abscess results from an infection involving the glands surrounding the anal canal. Normally, these glands release mucus into the anal canal, which aids in defecation. When stool accidentally enters the anal glands, the glands become infected and an abscess develops. Anorectal abscesses are perianal in 40–50% of patients, ischiorectal in 20–25%, intersphincteric in 2–5%, and supralevator in 2.5% (Fig. 353-7).

1	Presentation and Evaluation Perianal pain and fever are the hallmarks of an abscess. Patients may have difficulty voiding and have blood in the stool. A prostatic abscess may present with similar complaints, including dysuria. Patients with a prostatic abscess will often have a history of recurrent sexually transmitted diseases. On physical examination, a large fluctuant area is usually readily visible. Routine laboratory evaluation shows an elevated white blood cell count. Diagnostic procedures are rarely necessary unless evaluating a recurrent abscess. A CT scan or MRI has an accuracy of 80% in determining incomplete drainage. If there is a concern about the presence of inflammatory bowel disease, a rigid or flexible sigmoidoscopic examination may be done at the time of drainage to evaluate for inflammation within the rectosigmoid region. A more complete evaluation for Crohn’s disease would include a full colonoscopy and small-bowel series.

1	As with all abscesses, the “gold standard” is drainage. Office drainage of an uncomplicated anorectal abscess may suffice. A small incision close to the anal verge is made, and a Mallenkot drain is advanced into the abscess cavity. For patients who have a complicated abscess or who are diabetic or immunocompromised, drainage should be performed in an operating room under anesthesia. These patients are at greater risk for developing necrotizing fasciitis. There is limited role of antibiotics in management of anorectal abscesses. The antibiotics are only warranted in patients who are 1978 immunocompromised or have prosthetic heart valves, artificial joints, diabetes, or inflammatory bowel disease.

1	FISTULA IN ANO Incidence and Epidemiology The incidence and prevalence of fistulating perianal disease parallels the incidence of anorectal abscess, estimating to be 1 in 10,000 individuals. Some 30–40% of abscesses will give rise to fistula in ano. Although the majority of the fistulas are cryptoglandular in origin, 10% are associated with IBD, tuberculosis, malignancy, and radiation. Anatomy and Pathophysiology A fistula in ano is defined as a communication of an abscess cavity with an identifiable internal opening within the anal canal. This identifiable opening is most commonly located at the dentate line where the anal glands enter the anal canal. Patients experiencing continuous drainage following the treatment of a perianal abscess likely have a fistula in ano. These fistulas are classified by their relationship to the anal sphincter muscles, with 70% being intersphincteric, 23% transsphincteric, 5% suprasphincteric, and 2% extrasphincteric (Fig. 353-7).

1	Presentation and Evaluation A patient with a fistula in ano will complain of constant drainage from the perianal region associated with a firm mass. The drainage may increase with defecation. Perianal hygiene is difficult to maintain. Examination under anesthesia is the best way to evaluate a fistula. At the time of the examination, anoscopy is performed to look for an internal opening. Diluted hydrogen peroxide will aid in identifying such an opening. In lieu of anesthesia, MRI with an endoanal coil will also identify tracts in 80% of the cases. After drainage of an abscess with insertion of a Mallenkot catheter, a fistulagram through the catheter can be obtained in search of an occult fistula tract. Goodsall’s rule states that a posterior external fistula will enter the anal canal in the posterior midline, whereas an anterior fistula will enter at the nearest crypt. A fistula exiting >3 cm from the anal verge may have a complicated upward extension and may not obey Goodsall’s rule.

1	A newly diagnosed draining fistula is best managed with placement of a seton, a vessel loop or silk tie placed through the fistula tract, which maintains the tract open and quiets down the surrounding inflammation that occurs from repeated blockage of the tract. Once the inflammation is less, the exact relationship of the fistula tract to the anal sphincters can be ascertained. A simple fistulotomy can be performed for intersphincteric and low (less than one-third of the muscle) transsphincteric fistulas without compromising continence. For a higher transsphincteric fistula, an anorectal advancement flap in combination with a drainage catheter or fibrin glue may be used. Very long (>2 cm) and narrow tracts respond better to fibrin glue than shorter tracts. Simple ligation of the internal fistula tract (LIFT procedure) has also been used in the management of simple fistula with good success.

1	Patients should be maintained on stool-bulking agents, nonnarcotic pain medication, and sitz baths following surgery for a fistula. Early complications from these procedures include urinary retention and bleeding. Later complications are rare (<10%) and include temporary and permanent incontinence. Recurrence is 0–18% following fistulotomy and 20–30% following anorectal advancement flap and the LIFT procedure, ANAL FISSURE Incidence and Epidemiology Anal fissures occur at all ages but are more common in the third through the fifth decades. A fissure is the most common cause of rectal bleeding in infancy. The prevalence is equal in males and females. It is associated with constipation, diarrhea, infectious etiologies, perianal trauma, and Crohn’s disease.

1	Anatomy and Pathophysiology Trauma to the anal canal occurs following defecation. This injury occurs in the anterior or, more commonly, the posterior anal canal. Irritation caused by the trauma to the anal canal results in an increased resting pressure of the internal sphincter. The blood supply to the sphincter and anal mucosa enters laterally. Therefore, increased anal sphincter tone results in a relative ischemia in the region of the fissure and leads to poor healing of the anal injury. A fissure that is not in the posterior or anterior position should raise suspicion for other causes, including tuberculosis, syphilis, Crohn’s disease, and malignancy.

1	Presentation and Evaluation A fissure can be easily diagnosed on history alone. The classic complaint is pain, which is strongly associated with defecation and is relentless. The bright red bleeding that can be associated with a fissure is less extensive than that associated with hemorrhoids. On examination, most fissures are located in either the posterior or anterior position. A lateral fissure is worrisome because it may have a less benign nature, and systemic disorders should be ruled out. A chronic fissure is indicated by the presence of a hypertrophied anal papilla at the proximal end of the fissure and a sentinel pile or skin tag at the distal end. Often the circular fibers of the hypertrophied internal sphincter are visible within the base of the fissure. If anal manometry is performed, elevation in anal resting pressure and a sawtooth deformity with paradoxical contractions of the sphincter muscles are pathognomonic.

1	The management of the acute fissure is conservative. Stool softeners for those with constipation, increased dietary fiber, topical anesthetics, glucocorticoids, and sitz baths are prescribed and will heal 60–90% of fissures. Chronic fissures are those present for >6 weeks. These can be treated with modalities aimed at decreasing the anal canal resting pressure including nifedipine or nitroglycerin ointment applied three times a day and botulinum toxin type A, up to 20 units, injected into the internal sphincter on each side of the fissure. Surgical management includes anal dilatation and lateral internal sphincterotomy. Usually, one-third of the internal sphincter muscle is divided; it is easily identified because it is hypertrophied. Recurrence rates from medical therapy are higher, but this is offset by a risk of incontinence following sphincterotomy. Lateral internal sphincterotomy may lead to incontinence more commonly in women.

1	We would like to thank Cory Sandore for providing some illustrations for this chapter. Gregory Bulkley, MD, contributed to this chapter in an earlier edition and some of that material has been retained here. Rizwan Ahmed, Mahmoud Malas Intestinal ischemia occurs when splanchnic perfusion fails to meet the metabolic demands of the intestines, resulting in ischemic tissue injury. Mesenteric ischemia affects 2–3 people per 100,000, and the incidence of mesenteric ischemia is bound to increase in the aging population. Delay in diagnosis and management results in a high mortality, and prompt interventions may be life-saving. Intestinal ischemia is further classified based on etiology, which dictates management: (1) arterioocclusive mesenteric ischemia, (2) nonocclusive mesenteric ischemia, and (3) mesenteric venous thrombosis.

1	Risk factors for arterioocclusive mesenteric ischemia are generally acute in onset and include atrial fibrillation, recent myocardial infarction, valvular heart disease, and recent cardiac or vascular catheterization, all of which result in embolic clots reaching the mesenteric circulation. Nonocclusive mesenteric ischemia, also known as “intestinal angina,” is generally more insidious and most often seen in the aging population affected by atherosclerotic disease. Patients with chronic atherosclerotic disease could also suffer an acute insult from emboli leading to complete occlusion. Nonocclusive mesenteric ischemia is also seen in patients receiving high-dose vasopressor infusions, patients with cardiogenic or septic shock, and patients with cocaine overdose. Nonocclusive mesenteric ischemia is the most prevalent gastrointestinal disease complicating cardiovascular surgery. The incidence of ischemic colitis following elective aortic repair is 5–9%, and the incidence triples in

1	ischemia is the most prevalent gastrointestinal disease complicating cardiovascular surgery. The incidence of ischemic colitis following elective aortic repair is 5–9%, and the incidence triples in patients following emergent repair. Mesenteric venous thrombosis is less common and is associated with the presence of a hypercoagulable state including protein C or S deficiency, antithrombin III deficiency, polycythemia vera, and carcinoma.

1	The blood supply to the intestines is depicted in Fig. 354-1. To prevent ischemic injury, extensive collateralization occurs between major mesenteric trunks and branches of the mesenteric arcades. Collateral vessels within the small bowel are numerous and meet within the duodenum and the bed of the pancreas. Collateral vessels within the colon meet at the splenic flexure and descending/sigmoid colon. These areas, which are inherently at risk for decreased blood flow, are known as Griffiths’ point and Sudeck’s point, respectively, and are the most common locations for colonic ischemia (Fig. 354-1, shaded areas). The splanchnic circulation can receive up to 30% of the cardiac output. Protective responses to prevent intestinal ischemia include abundant Left Phrenic a. Aorta

1	Left Phrenic a. Aorta Right Phrenic a. Marginal a. Arch of Riolan IIAPancreatico-duodenal a. Griffiths’ point Sudeck’s point SMAIMAHemorrhoidal aa. Superior Middle Inferior Celiac trunk Splenic a. FIGURE 354-1 Blood supply to the intestines includes the celiac artery, superior mesenteric artery (SMA), inferior mesenteric artery (IMA), and branches of the internal iliac artery (IIA). Griffiths’ and Sudeck’s points, indicated by shaded areas, are watershed areas with-in the colonic blood supply and common locations for ischemia. collateralization, autoregulation of blood flow, and the ability to 1979 increase oxygen extraction from the blood.

1	collateralization, autoregulation of blood flow, and the ability to 1979 increase oxygen extraction from the blood. Occlusive ischemia is a result of disruption of blood flow by an embolus or progressive thrombosis in a major artery supplying the intestine. Emboli originate from the heart in more than 75% of cases and lodge preferentially in the superior mesenteric artery just distal to the origin of the middle colic artery. Progressive thrombosis of at least two of the major vessels supplying the intestine is required for the development of chronic intestinal angina. Nonocclusive ischemia is disproportionate mesenteric vasoconstriction (arteriolar vasospasm) in response to a severe physiologic stress such as shock. If left untreated, early mucosal stress ulceration will progress to full-thickness injury. Even in the early stages of ischemia, there is translocation of bacteria across the intestinal mucosa, resulting in bacteremia that can lead to sepsis.

1	PRESENTATION, EVALUATION, AND MANAGEMENT Intestinal ischemia remains one of the most challenging diagnoses. The mortality rate is greater than 50%. The most significant indicator of survival is the timeliness of diagnosis and treatment. An overview of diagnosis and management of each form of intestinal ischemia is given in Table 354-1.

1	Acute mesenteric ischemia resulting from arterial embolus or thrombosis presents with severe acute, nonremitting abdominal pain strikingly out of proportion to the physical findings. Associated symptoms may include nausea and vomiting, transient diarrhea, anorexia, and bloody stools. With the exception of minimal abdominal distention and hypoactive bowel sounds, early abdominal examination is unimpressive. Later findings will demonstrate peritonitis and cardiovascular collapse. In the evaluation of acute intestinal ischemia, routine laboratory tests should be obtained, including complete blood count, serum chemistry, coagulation profile, arterial blood gas, amylase, lipase, lactic acid, blood type and cross match, and cardiac enzymes. Regardless of the need for urgent surgery, emergent admission to a monitored bed or intensive care unit is recommended for resuscitation and further evaluation. If the diagnosis of intestinal ischemia is being considered, consultation with a surgical

1	admission to a monitored bed or intensive care unit is recommended for resuscitation and further evaluation. If the diagnosis of intestinal ischemia is being considered, consultation with a surgical service is necessary. Often the decision to operate is made on a high index of suspicion from the history and physical exam despite normal laboratory findings.

1	Other diagnostic modalities that may be useful in diagnosis but should not delay surgical therapy include electrocardiogram (ECG), echocardiogram, abdominal radiographs, computed tomography (CT), and mesenteric angiography. More recently, mesentery duplex scanning and visible light spectroscopy during colonoscopy have been demonstrated to be beneficial. The ECG may demonstrate an arrhythmia, indicating the possible source of the emboli. A plain abdominal film may show evidence of free intraperitoneal air, indicating a perforated viscus and the need for emergent exploration. Earlier features of intestinal ischemia seen on abdominal radiographs include bowel-wall edema, known as “thumbprinting.” If the ischemia progresses, air can be seen within the bowel wall (pneumatosis intestinalis) and within the portal venous system. Other features include calcifications of the aorta and its tributaries, indicating atherosclerotic disease. With the administration of oral and IV contrast, dynamic

1	within the portal venous system. Other features include calcifications of the aorta and its tributaries, indicating atherosclerotic disease. With the administration of oral and IV contrast, dynamic CT angiography with three-dimensional reconstruction is a highly sensitive test for intestinal ischemia. In acute embolic disease, mesenteric angiography is best performed intraoperatively. A mesenteric duplex scan demonstrating a high peak velocity of flow in the superior mesenteric artery (SMA) is associated with an approximately 80% positive predictive value of mesenteric ischemia. More significantly, a negative duplex scan virtually precludes the diagnosis of mesenteric ischemia. Duplex imaging serves as a screening test; further investigations with angiography are needed. The biggest limitation of duplex scanning is body habitus; in obese patients, imaging is poor yield. However, in patients with chronic disease, “food fear” often leads to a decreased appetite and therefore less

1	limitation of duplex scanning is body habitus; in obese patients, imaging is poor yield. However, in patients with chronic disease, “food fear” often leads to a decreased appetite and therefore less abdominal fat, and duplex imaging is very high yield. The endoscopic techniques using visible light spectroscopy can be used in the diagnosis of chronic ischemia. When suspecting mesenteric ischemia involving the colon, performing an endoscopy to evaluate up to the splenic

1	Treatment of Treatment of Systemic Condition Key to Early Diagnosis Underlying Cause Treatment of Specific Lesion Consequence 1. 2. Angiography with venous phase Vasospasm: Angiography Hypoperfusion: Spiral CT or colonoscopy Endovascular approach: thrombolysis, angioplasty and stenting Source: Modified from GB Bulkley, in JL Cameron (ed): Current Surgical Therapy, 2nd ed. Toronto, BC Decker, 1986. flexure is high yield. This is often an excellent diagnostic tool in patients with chronic renal insufficiency who cannot tolerate IV contrast.

1	The “gold standard” for the diagnosis of acute arterial occlusive disease is angiography, and management is laparotomy. Surgical exploration should not be delayed if suspicion of acute occlusive mesenteric ischemia is high or evidence of clinical deterioration or frank peritonitis is present. The goal of operative exploration is to resect compromised bowel and restore blood supply. The entire length of the small and large bowel beginning at the ligament of Treitz should be evaluated. The pattern of intestinal ischemia may indicate the level of arterial occlusion. In the case of SMA occlusion where the embolus usually lies just proximal to the origin of the middle colic artery, the proximal jejunum is often spared while the remainder of the small bowel to the transverse colon will be ischemic. The surgical management of acute mesenteric ischemia of the small bowel is embolectomy via arteriotomy; a small incision is made in the artery through which the clot is retrieved. Another way to

1	The surgical management of acute mesenteric ischemia of the small bowel is embolectomy via arteriotomy; a small incision is made in the artery through which the clot is retrieved. Another way to manage acute thrombosis is thrombolysis therapy and angioplasty, with stent placement. However, this approach is more commonly applied to treat chronic mesenteric ischemia. If this is unsuccessful, a bypass from the aorta or iliac artery to the SMA is performed.

1	Nonocclusive or vasospastic mesenteric ischemia presents with generalized abdominal pain, anorexia, bloody stools, and abdominal distention. Often these patients are obtunded, and physical findings may not assist in the diagnosis. The presence of a leukocytosis, metabolic acidosis, elevated amylase or creatinine phosphokinase levels, and/or lactic acidosis is useful in support of the diagnosis of advanced intestinal ischemia; however, these markers may not be indicative of either reversible ischemia or frank necrosis. Investigational markers for intestinal ischemia include D-dimer, glutathione S-transferase, platelet-activating factor (PAF), and mucosal pH monitoring. Regardless of the need for urgent surgery, emergent admission to a monitored bed or intensive care unit is recommended for resuscitation and further evaluation. Early manifestations of intestinal ischemia include fluid sequestration within the bowel wall leading to a loss of interstitial volume. Aggressive fluid

1	for resuscitation and further evaluation. Early manifestations of intestinal ischemia include fluid sequestration within the bowel wall leading to a loss of interstitial volume. Aggressive fluid resuscitation may be necessary. To optimize oxygen delivery, nasal O2 and blood transfusions may be given. Broad-spectrum antibiotics should be given to provide sufficient coverage for enteric pathogens, including gram-negative and anaerobic organisms. Frequent monitoring of the patient’s vital signs, urine output, blood gases, and lactate levels is paramount, as is frequent abdominal examination. All vasoconstricting agents should be avoided; fluid resuscitation is the intervention of choice to maintain hemodynamics.

1	If ischemic colitis is a concern, colonoscopy should be performed to assess the integrity of the colon mucosa. Visualization of the rectosigmoid region may demonstrate decreased mucosal integrity, associated more commonly with nonocclusive mesenteric ischemia, or, on occasion, occlusive disease as a result of acute loss of inferior mesenteric arterial flow following aortic surgery. Ischemia of the colonic mucosa is graded as mild with minimal mucosal erythema or as moderate with pale mucosal ulcerations and evidence of extension to the muscular layer of the bowel wall. Severe ischemic colitis presents with severe ulcerations resulting in black or green discoloration of the mucosa, consistent with full-thickness bowel-wall necrosis. The degree of reversibility can be predicted from the mucosal findings: mild erythema is nearly 100% reversible, moderate is approximately 50% reversible, and frank necrosis is simply dead bowel. Follow-up colonoscopy can be performed to rule out

1	the mucosal findings: mild erythema is nearly 100% reversible, moderate is approximately 50% reversible, and frank necrosis is simply dead bowel. Follow-up colonoscopy can be performed to rule out progression of ischemic colitis.

1	Laparotomy for nonocclusive mesenteric ischemia is warranted for signs of peritonitis or worsening endoscopic findings and if the patient’s condition does not improve with aggressive resuscitation. Ischemic colitis is optimally treated with resection of the ischemic bowel and formation of a proximal stoma. Primary anastomosis should not be performed in patients with acute intestinal ischemia.

1	Patients with mesenteric venous thrombosis may present with a gradual or sudden onset. Symptoms include vague abdominal pain, nausea, and vomiting. Examination findings include abdominal distention with mild to moderate tenderness and signs of dehydration. The diagnosis of mesenteric thrombosis is frequently made on abdominal spiral CT with oral and IV contrast. Findings on CT angiography with venous phase include bowel-wall thickening and ascites. Intravenous contrast will demonstrate a delayed arterial phase and clot within the superior mesenteric vein. The goal of management is to optimize hemodynamics and correct electrolyte abnormalities with massive fluid resuscitation. Intravenous antibiotics as well as anticoagulation should be initiated. If laparotomy is performed and 1981 mesenteric venous thrombosis is suspected, heparin anticoagulation is Acute intestinal obstruction immediately initiated, and compromised bowel is resected. Of all acute

1	Danny O. Jacobs intestinal disorders, mesenteric venous insufficiency is associated with the best prognosis.

1	Chronic intestinal ischemia presents with intestinal angina or postprandial abdominal pain associated with need for increased blood flow to the intestine following meals. Patients report abdominal cramping and pain following ingestion of a meal. Weight loss and chronic diarrhea may also be noted. Abdominal pain without weight loss is not chronic mesenteric angina. Physical examination will often reveal a malnourished patient with an abdominal bruit as well as other manifestations of atherosclerosis. Duplex ultrasound evaluation of the mesenteric vessels has gained in popularity. It is important to perform the test fasting because the presence of increased bowel gas prevents adequate visualization of flow disturbances within the vessels or the lack of a vasodilation response to feeding during the test. This tool is frequently used as a screening test for patients with symptoms suggestive of chronic mesenteric ischemia. The gold standard for confirmation of mesenteric arterial occlusion

1	the test. This tool is frequently used as a screening test for patients with symptoms suggestive of chronic mesenteric ischemia. The gold standard for confirmation of mesenteric arterial occlusion is mesenteric angiography. Evaluation with mesenteric angiography allows for identification and possible intervention for the treatment of atherosclerosis within the vessel lumen and will also evaluate the patency of remaining mesenteric vessels. The use of mesenteric angiography may be limited in the presence of renal failure or contrast allergy. Magnetic resonance angiography is an alternative if the administration of contrast dye is contraindicated.

1	The management of chronic intestinal ischemia includes medical management of atherosclerotic disease by exercise, cessation of smoking, and antiplatelet and lipid-lowering medications. A full cardiac evaluation should be performed before intervention on chronic mesenteric ischemia. Newer endovascular procedures may avoid an operative intervention in selected patient populations. Angioplasty with endovascular stenting in the treatment of chronic mesenteric ischemia is associated with an 80% long-term success rate. In patients requiring surgical exploration, the approach used is determined by findings of the mesenteric angiogram. The entire length of the small and large bowel should be evaluated, beginning at the ligament of Treitz. Restoration of blood flow at the time of laparotomy is accomplished with mesenteric vessel endarterectomy or bypass.

1	Determination of intestinal viability intraoperatively in patients with suspected intestinal ischemia can be challenging. After revascularization, the bowel wall should be observed for return of a pink color and peristalsis. Palpation of major arterial mesenteric vessels can be performed, as well as applying a Doppler flowmeter to the antimesenteric border of the bowel wall, but neither is a definitive indicator of viability. In equivocal cases, 1 g of IV sodium fluorescein is administered, and the pattern of bowel reperfusion is observed under ultraviolet illumination with a standard (3600 A) Wood’s lamp. An area of nonfluorescence >5 mm in diameter suggests nonviability. If doubt persists, reexploration performed 24–48 h following surgery will allow demarcation of nonviable bowel. Primary intestinal anastomosis in patients with ischemic bowel is always worrisome; thus, delayed bowel reconstruction and reanastomosis should be deferred to the time of second-look laparotomy.

1	We thank Cory Sandore for providing the illustration for this chapter. Susan Gearhart contributed to this chapter in the 18th edition. Morbidity and mortality from acute intestinal obstruction have been decreasing over the past several decades. Nevertheless, the diagnosis can still be challenging, and the type of complications that patients suffer has not changed significantly. The extent of mechanical obstruction is typically described as partial, high-grade, or complete—generally correlating with the risk of complications and the urgency with which the underlying disease process must be addressed. Obstruction is also commonly described as being either “simple” or, alternatively, “strangulated” if vascular insufficiency and intestinal ischemia are evident.

1	Acute intestinal obstruction occurs either mechanically from blockage or from intestinal dysmotility when there is no blockage. In the latter instance, the abnormality is described as being functional. Mechanical bowel obstruction may be caused by extrinsic processes, intrinsic abnormalities of the bowel wall, or intraluminal abnormalities (Table 355-1). Within each of these broad categories are many diseases that can impede intestinal propulsion. Intrinsic diseases that can cause intestinal obstruction are usually congenital, inflammatory, neoplastic, or traumatic in origin, although intussusception and radiation injury can also be etiologic. Primary small-bowel cancers rarely cause acute obstruction.

1	Acute intestinal obstruction accounts for approximately 1–3 % of all hospitalizations and a quarter of all urgent or emergent general surgery admissions. Approximately 80% of cases involve the small bowel, and about one-third of these patients show evidence of significant ischemia. The mortality rate for patients with strangulation who are operated on within 24–30 h of the onset of symptoms is approximately 8% but triples shortly thereafter. Extrinsic diseases most commonly cause mechanical obstruction of the small intestine. In the United States and Europe, almost all cases are caused by postoperative adhesions (>50%), carcinomatosis, or herniation of the anterior abdominal wall. Carcinomatosis most often originates from the ovary, pancreas, stomach, or colon, although rarely, metastasis from distant organs like the breast and skin can occur. Adhesions are responsible for >90% of cases of early postoperative obstruction that require intervention.

1	Operations of the lower abdomen, including appendectomy and colorectal and gynecologic procedures, are especially likely to create Adhesions (especially due to previous abdominal surgery), internal or external hernias, neoplasms (including carcinomatosis and extraintestinal malignancies, mostly commonly ovarian), endometriosis or intraperitoneal abscesses, and idiopathic sclerosis Congenital (e.g., malrotation, atresia, stenosis, intestinal duplication, cyst formation, and congenital bands—the latter rarely in adults) Inflammation (e.g., inflammatory bowel disease, especially Crohn’s disease, but also diverticulitis, radiation, tuberculosis, lymphogranuloma venereum, and schistosomiasis) Neoplasia (note: primary small-bowel cancer is rare; obstructive colon cancer may mimic small-bowel obstruction if the ileocecal valve is incompetent) Traumatic (e.g., hematoma formation, anastomotic strictures)

1	Traumatic (e.g., hematoma formation, anastomotic strictures) Other, including intussusception (where the lead point is typically a polyp or tumor in adults), volvulus, obstruction of duodenum by superior mesenteric artery, radiation or ischemic injury, and aganglionosis, which is Hirschsprung’s disease Bezoars, feces, foreign bodies including inspissated barium, gallstones (entering the lumen via a cholecystoenteric fistula), enteroliths adhesions that can cause bowel obstruction (Table 355-2). Overall, small-bowel obstruction is slightly more common in women. The risk of internal herniation is increased by abdominal procedures such as laparoscopic or open Roux-en-Y gastric bypass. Although laparoscopic procedures may generate fewer postoperative adhesions compared with open surgery, the risk of obstructive adhesion formation is not eliminated.

1	In many patients who are successfully treated for adhesive small-bowel obstruction, obstruction will recur. The rate varies according to how patients were initially managed. Approximately 20% of patients who were treated conservatively and between 5 and 30% of patients who were managed operatively will require readmission within 10 years.

1	Volvulus, which occurs when bowel twists on its mesenteric axis, can cause partial or complete obstruction and vascular insufficiency. The sigmoid colon is most commonly affected, accounting for approximately two-thirds of all cases of volvulus and 4% of all cases of large-bowel obstruction. The cecum and terminal ileum can also volvulize, or the cecum alone may be involved as a cecal bascule. Risk factors include institutionalization, the presence of neuropsychiatric conditions requiring psychotropic medication, chronic constipation, and aging; patients typically present in their seventies or eighties. Colonic volvulus is more common in Eastern Europe, Russia, and Africa than it is in the United States. It is rare for adhesions or hernias to obstruct the colon. Cancer of the descending colon and rectum is responsible for approximately two-thirds of all cases, followed by diverticulitis and volvulus.

1	Functional obstruction, also known as ileus and pseudo-obstruction, is present when dysmotility prevents intestinal contents from being propelled distally and no mechanical blockage exists. Ileus that occurs after intraabdominal surgery is the most commonly identified form of functional bowel obstruction, although there are many other causes (Table 355-3). Although postoperative ileus is most often transient, Intraabdominal procedures, lumbar spinal injuries, or surgical procedures on the lumbar spine and pelvis Metabolic or electrolyte abnormalities, especially hypokalemia and hypo magnesemia, but also hyponatremia, uremia, and severe hyperglycemia Drugs such as opiates, antihistamines, and some psychotropic (e.g., haloperi dol, tricyclic antidepressants) and anticholinergic agents Intraoperative radiation (likely due to muscle damage) Ileus secondary to hereditary or acquired visceral myopathies and neuropa

1	Intraoperative radiation (likely due to muscle damage) Ileus secondary to hereditary or acquired visceral myopathies and neuropa Some collagen vascular diseases such as lupus erythematosus or scleroderma it is the most common reason why hospital discharge is delayed. Pseudo-obstruction of the colon, also known as Ogilvie’s syndrome, is a relatively rare disease. Some patients with Ogilvie’s syndrome have colonic dysmotility due to abnormalities of their autonomic nervous system that may be inherited.

1	The manifestations of acute intestinal obstruction depend on the nature of the underlying disease process, its location, and changes in blood flow (Fig. 355-1). Increased intestinal contractility, which occurs proximally and distal to the obstruction, is a characteristic response. Subsequently, intestinal peristalsis slows as the intestine or stomach proximal to the point of obstruction dilates and fills with gastrointestinal secretions and swallowed air. Although swallowed air is the primary contributor to intestinal distension, intraluminal air may also accumulate from fermentation, local carbon dioxide production, and altered gaseous diffusion.

1	Intraluminal dilation also increases intraluminal pressure. When luminal pressure exceeds venous pressure, venous and lymphatic drainage is impeded. Edema ensues, and the bowel wall proximal to the site of blockage may become hypoxemic. Epithelial necrosis can be identified within 12 h of obstruction. Ultimately, arterial blood supply may become so compromised that full-thickness ischemia, necrosis, and perforation result. Stasis increases the bacteria counts within the jejunum and ileum. The most commonly cultured intraluminal organisms are Escherichia coli, Streptococcus faecalis, and Klebsiella, which may also be recovered from mesenteric lymph nodes and other more distant sites.

1	Other manifestations depend on the degree of hypovolemia, the patient’s metabolic response, and the presence or absence of associated intestinal ischemia. Inflammatory edema eventually increases the production of reactive oxygen species and activates neutrophils and macrophages, which accumulate within the bowel wall. Their accumulation, along with changes in innate immunity, disrupts secretory and neuromotor processes. Dehydration is caused by loss of the normal intestinal absorptive capacity as well as fluid accumulation in the gastric or intestinal wall and intraperitoneally.

1	Anorexia and emesis tend to exacerbate intravascular volume depletion. In the worst case scenario that is most commonly identified after distal obstruction, emesis leads to losses of gastric potassium, hydrogen, and chloride, while dehydration stimulates proximal renal tubule bicarbonate reabsorption. Intraperitoneal fluid accumulation, especially in patients with severe distal bowel obstruction, may increase intraabdominal pressure enough to elevate the diaphragm and inhibit respiration and to impede systemic venous return and promote vascular instability. Severe hemodynamic compromise may elicit a systemic inflammatory response and generalized microvascular leakage.

1	Closed-loop obstruction results when the proximal and distal openings of a given bowel segment are both occluded, e.g., due to volvulus or a hernia. It is the most common precursor for strangulation, but not every closed loop strangulates. The risk of vascular insufficiency, systemic inflammation, hemodynamic compromise, and irreversible intestinal ischemia is much greater in patients with closed-loop obstruction. Pathologic changes may occur more rapidly, and emergency intervention is indicated. Irreversible bowel ischemia progresses to transmural necrosis even if the obstruction is relieved. It is also important to remember that patients with high-grade distal colonic obstruction who have competent ileocecal valves may present with closed-loop obstruction. In the latter instance, the cecum may progressively dilate such that ischemic necrosis results in cecal perforation. This risk is generally greatest when the cecal diameter exceeds 12 cm, as informed by Laplace’s law. Patients

1	cecum may progressively dilate such that ischemic necrosis results in cecal perforation. This risk is generally greatest when the cecal diameter exceeds 12 cm, as informed by Laplace’s law. Patients with distal colonic obstruction whose ileocecal valves are incompetent tend to present later in the course of disease and mimic patients with distal small-bowel obstruction.

1	Even though the presenting signs and symptoms can be misleading, many patients with acute obstruction can be accurately diagnosed after a thorough history and physical examination is performed. Early Point of obstruction from extrinsic, intrinsic, or intraluminal disease Patients with distal obstruction may still discharge intraluminal contents FIGURE 355-1 Pathophysiologic changes of small-bowel obstruction. recognition allows earlier treatment that decreases the risk of progression or other excess morbidity. Small-bowel obstruction with strangulation can be especially difficult to diagnosis promptly.

1	recognition allows earlier treatment that decreases the risk of progression or other excess morbidity. Small-bowel obstruction with strangulation can be especially difficult to diagnosis promptly. The cardinal signs are colicky abdominal pain, abdominal distention, emesis, and obstipation. More intraluminal fluid accumulates in patients with distal obstruction, which typically leads to greater distention, more discomfort, and delayed emesis. This emesis is feculent when there is bacterial overgrowth. Patients with more proximal obstruction commonly present with less abdominal distention but more pronounced vomiting. Elements of the history that might be helpful include any prior history of surgery, including herniorrhaphy, as well as any history of cancer or inflammatory bowel disease.

1	Most patients, even with simple obstruction, appear to be critically ill. Many may be oliguric, hypotensive, and tachycardic because of severe intravascular volume depletion. Fever is worrisome for strangulation or systemic inflammatory changes. Bowel sounds and bowel functional activity are notoriously difficult to interpret. Classically, many patients with early small-bowel obstruction will have high-pitched, “musical” tinkling bowel sounds and peristaltic “rushes” known as borborygmi. Later in the course of disease, the bowel sounds may be absent or hypoactive as peristaltic activity decreases. This is in contrast to the common findings in patients with ileus or pseudo-obstruction where bowel sounds are typically absent or hypoactive from the beginning. Lastly, patients with partial blockage may continue to pass flatus and stool, and those with complete blockage may evacuate bowel contents present downstream beyond their obstruction.

1	All surgical incisions should be examined. The presence of a tender abdominal or groin mass strongly suggests that an incarcerated hernia may be the cause of obstruction. The presence of tenderness Collapsed bowel distal to obstruction should increase the concern about the presence of complications such as ischemia, necrosis, or peritonitis. Severe pain with localization or signs of peritoneal irritation is suspicious for strangulated or closed-loop obstruction. It is important to remember that the discomfort may be out of proportion to physical findings mimicking the complaints of patients with acute mesenteric ischemia. Every patient should have a rectal examination. Patients with colonic volvulus present with the classic manifestations of closed-loop obstruction: severe abdominal pain, vomiting, and obstipation. Asymmetrical abdominal distension and a tympanic mass may be evident.

1	Patients with ileus or pseudo-obstruction may have signs and symptoms similar to those of bowel obstruction. Although abdominal distention is present, colicky abdominal pain is typically absent, and patients may not have nausea or emesis. Ongoing, regular discharge of stool or flatus can sometimes help distinguish patients with ileus from those with complete mechanical bowel obstruction.

1	Laboratory testing should include a complete blood count and serum electrolyte and creatinine measurements. Serial assessments are often useful. Mild hemoconcentration and slight elevation of the white blood cell count commonly occur after simple bowel obstruction. Emesis and dehydration may cause hypokalemia, hypochloremia, elevated blood urea nitrogen–to–creatinine ratios, and metabolic alkalosis. Patients may be hyponatremic on admission because many have attempted to rehydrate themselves with hypotonic fluids. The presence of guaiac-positive stools and iron-deficiency anemia are strongly suggestive of malignancy.

1	Higher white blood cell counts with the presence of immature forms or the presence of metabolic acidosis are worrisome for severe volume 1984 depletion or ischemic necrosis and sepsis. At this time, there are no laboratory tests that are especially useful for identifying the presence of simple or strangulated obstruction, although increases in serum D-lactate, creatine kinase bb isoenzymes, or intestinal fatty acid binding protein levels may be suggestive of the latter. In all cases, when considering diagnostic imaging, the key is not to delay surgical consultation and operative intervention when the patient’s signs or symptoms strongly suggest that high-grade or complete obstruction or bowel compromise is present. Plain films of the abdomen, which must include upright or cross-table lateral views, can be completed quickly and may confirm the clinical suspicion 60% of the time. Interpretation immediately after operation is difficult. A “staircasing” pattern of dilated air and

1	lateral views, can be completed quickly and may confirm the clinical suspicion 60% of the time. Interpretation immediately after operation is difficult. A “staircasing” pattern of dilated air and fluid-filled small-bowel loops >2.5 cm in diameter with little or no air seen in the colon are classical findings in patients with small-bowel obstruction, although findings may be equivocal in some patients with documented disease. Little bowel gas appears in patients with proximal bowel obstruction or in patients whose intestinal lumens are filled with fluid. Upright plain films of the abdomen of patients with large-bowel obstruction typically show colon dilatation. Small-bowel air-fluid levels will not be obvious if the ileocecal valve is competent. Although it can be difficult to distinguish from ileus, small-bowel obstruction is more likely when air-fluid levels are seen without significant colonic distension. Free air suggests that perforation has occurred in patients who have not

1	from ileus, small-bowel obstruction is more likely when air-fluid levels are seen without significant colonic distension. Free air suggests that perforation has occurred in patients who have not recently undergone surgical procedures. Radiopaque foreign bodies or enteroliths may be visualized. A gas-filled, “coffee bean”–shaped dilated shadow may be seen in patients with volvulus. More sophisticated imaging can be beneficial when the diagnosis is unclear. Magnetic resonance imaging has been used to diagnose small-bowel obstruction, but it is more expensive and, typically, provides less spatial resolution. Ultrasonographic evaluations are especially difficult to interpret but may be sensitive and appropriate studies to evaluate patients who are pregnant or for whom x-ray exposure is otherwise contraindicated or inappropriate. Computed tomography (CT) is the most commonly used imaging modality. Its sensitivity for detecting bowel obstruction is approximately 95% (78–100%) in patients

1	contraindicated or inappropriate. Computed tomography (CT) is the most commonly used imaging modality. Its sensitivity for detecting bowel obstruction is approximately 95% (78–100%) in patients with high-grade obstruction, with a specificity of 96% and an accuracy of ≥95%. Its accuracy in diagnosing closed-loop obstruction is much lower (60%). Examples of some CT images are reproduced in Fig. 355-2. It may also provide useful information regarding location or identify particular circumstances where surgical intervention is needed urgently. Patients who have evidence of contrast appearing within the cecum within 4–24 h of oral administration can be expected to improve with high sensitivity and specificity (~95% each). For example, contrast studies may demonstrate a “bird’s beak,” a “c-loop,” or “whorl” deformity on CT imaging at the site where twisting obstructs the lumen when a colonic volvulus is present. CT imaging with enteral and IV contrast can also identify ischemia. Altered

1	or “whorl” deformity on CT imaging at the site where twisting obstructs the lumen when a colonic volvulus is present. CT imaging with enteral and IV contrast can also identify ischemia. Altered bowel wall enhancement is the most specific early finding, but its sensitivity is low. Mesenteric venous gas, pneumoperitoneum, and pneumatosis intestinalis are late findings indicating the presence of bowel necrosis. CT scanning after a water-soluble contrast enema may help distinguish ileus or pseudo-obstruction from distal large-bowel obstruction in patients who present with evidence of small-bowel and colonic distention. CT enteroclysis can accurately identify neoplasia as a cause of bowel obstruction. Contrast enemas or colonoscopies are almost always needed to identify causes of acute colonic obstruction. Barium studies are generally contraindicated in patients with firm evidence of complete or high-grade bowel obstruction, especially when they present acutely. Barium should never be

1	obstruction. Barium studies are generally contraindicated in patients with firm evidence of complete or high-grade bowel obstruction, especially when they present acutely. Barium should never be given orally to a patient with possible obstruction until that diagnosis has been excluded. In every other case, such investigations should only be performed in exceptional circumstances and with great caution because patients with significant obstruction may develop barium concretions as an additional source of blockage and some who would have otherwise recovered will require operative intervention. Barium opacification also renders cross-sectional imaging studies or angiography uninterpretable.

1	FIGURE 355-2 Computed tomography with oral and intravenous contrast demonstrating (A) evidence of small-bowel dilatation with air-fluid levels consistent with a small-bowel obstruction; (B) a partial small-bowel obstruction from an incarcerated ventral hernia (arrow); and (C) decompressed bowel seen distal to the hernia (arrow). (From W Silen: Acute intestinal obstruction, in DL Longo et al [eds]: Harrison’s Principles of Internal Medicine, 18th ed. New York, McGraw-Hill, 2012.)

1	An improved understanding of the pathophysiology of bowel obstruction and the importance of fluid resuscitation, electrolyte repletion, intestinal decompression, and the selected use of antibiotics have likely contributed to a reduction in the mortality from acute bowel obstruction. Every patient should be stabilized as quickly as possible. Nasogastric tube suction decompresses the stomach, minimizes further distention from swallowed air, improves patient comfort, and reduces the risk of aspiration. Urine output should be assessed using a Foley catheter. In some cases, for example, in patients with cardiac disease, central venous pressures should be monitored. The use of antibiotics is controversial, although prophylactic administration is warranted if surgery is required. Complete bowel obstruction is an indication for intervention. Stenting may be possible and warranted for some patients with high-grade obstruction due to unresectable stage IV malignancy. Stenting may also allow

1	obstruction is an indication for intervention. Stenting may be possible and warranted for some patients with high-grade obstruction due to unresectable stage IV malignancy. Stenting may also allow elective mechanical bowel preparation before surgery is undertaken. Because treatment options are so variable, it is helpful to make as precise a diagnosis as possible preoperatively.

1	Patients with ileus are treated supportively with intravenous fluids and nasogastric decompression while any underlying pathology is treated. Pharmacologic therapy is not yet proven to be efficacious or cost-effective. However, peripherally active μ-opioid receptor antagonists (e.g., alvimopan and methylnaltrexone) may accelerate gastrointestinal recovery in some patients who have undergone abdominal surgery.

1	Neostigmine is an acetylcholinsterase inhibitor that increases cholinergic (parasympathetic) activity, which can stimulate colonic motility. Some studies have shown it to be moderately effective in alleviating acute colonic pseudo-obstruction. It is the most common therapeutic approach and can be used once it is certain that there is no mechanical obstruction. Cardiac monitoring is required, and atropine should be immediately available. Intravenous administration induces defecation and flatus within 10 min in the majority of patients who will respond. Sympathetic blockade by epidural anesthesia can successfully ameliorate pseudo-obstruction in some patients.

1	Patients with sigmoid volvulus can often be decompressed using a flexible tube inserted through a rigid proctoscope or using a flexible sigmoidoscope. Successful decompression results in sudden release of gas and fluid with evidence of decreased abdominal distension and allows definitive correction to be scheduled electively. Cecal volvulus most often requires laparotomy or laparoscopic correction.

1	Approximately 60–80% of selected patients with mechanical bowel obstruction can be successfully treated conservatively. Indeed, most cases of radiation-induced obstruction should also be managed nonoperatively if possible. In most circumstances, early consultation with a general surgeon is prudent when there is concern about strangulation obstruction or other abnormality that needs to be addressed urgently. Deterioration signifies a need for intervention. At this time, the decision as to whether the patient can continue to be treated nonoperatively can only be based on clinical judgment, although, as described earlier, imaging studies can sometimes be helpful. The frequency of major complications after operation ranges from 12 to 47%, with greater risk being attributed to resection therapies and the patient’s overall health. Risk is increased for patients with American Society of Anesthesiologists (ASA) class III or higher.

1	At operation, dilation proximal to the site of blockage with distal collapse is a defining feature of bowel obstruction. Intraoperative strategies depend on the underlying problem and range from lysis of adhesions to resection with or without diverting ostomy to primary resection with anastomosis. Resection is warranted when there is concern about the bowel’s viability after the obstructive process is relieved. Laparoscopic approaches can be useful for patients with early obstruction when extensive adhesions are not expected to be present. Some patients with high-grade obstruction secondary to malignant disease that is not amendable to resection 1985 will benefit from bypass procedures.

1	Primary resection is prudent. Careful manual reduction of any involved bowel may limit the amount of intestine that needs to be removed. A proximal ostomy may be required if unprepped colon is involved. Only 60% of patients with gallstone ileus obstruct in the ileum. The most common site of intestinal obstruction in patients with gallstone “ileus” is the ileum (60% of patients). The gallstone enters the intestinal tract most often via a cholecystoduodenal fistula. It can usually be removed by operative enterolithotomy. Addressing the gallbladder disease during urgent or emergent surgery is not recommended.

1	Early postoperative mechanical bowel obstruction is that which occurs within the first 6 weeks of operation. Most are partial and can be expected to resolve spontaneously. It tends to respond and behave differently from classic mechanical bowel obstruction and may be very difficult to distinguish from postoperative ileus. A higher index of suspicion for a definitive site of obstruction is warranted for patients who undergo laparoscopic surgical procedures. Patients who first had ileus and then subsequently develop obstructive symptoms after an initial return of normal bowel function are more likely to have true postoperative small-bowel obstruction. The longer it takes for a patient’s obstructive symptoms to resolve after hospitalization, the more likely the patient is to require surgical intervention. The wisdom and expertise of Dr. William Silen are gratefully acknowledged. Danny O. Jacobs

1	The wisdom and expertise of Dr. William Silen are gratefully acknowledged. Danny O. Jacobs Appendicitis occurs more frequently in Westernized societies. Although its incidence is decreasing for uncertain reasons, acute appendicitis remains the most common emergency general surgical disease affecting the abdomen, with a rate of approximately 100 per 100,000 person-years in Europe and the Americas or about 11 cases per 10,000 people annually. Approximately 9% of men and 7% of women will experience an episode during their lifetime. Appendicitis occurs most commonly in 10to 19-year-olds, although the average age at diagnosis appears to be gradually increasing, as is the frequency of the disease in African Americans, Asians, and Native Americans. Overall, 70% of patients are less than 30 years old and most are men; the male-to-female ratio is 1.4:1.

1	One of the more common complications and most important causes of excess morbidity and mortality is perforation, whether it is contained and localized or unconstrained within the peritoneal cavity. In contrast to the trend observed for appendicitis and appendectomy, the incidence of perforated appendicitis (~20 cases per 100,000 person-years) is increasing. The explanation for this phenomenon is unknown. Approximately 20% of all patients have evidence of perforation at presentation, but the percentage risk is much higher in patients under 5 or over 65 years of age.

1	1986 PATHOGENESIS OF APPENDICITIS AND APPENDICEAL PERFORATION Appendicitis was first described in 1886 by Reginald Fitz. Its etiology is still not completely understood. Fecaliths, incompletely digested food residue, lymphoid hyperplasia, intraluminal scarring, tumors, bacteria, viruses, and inflammatory bowel disease have all been associated with inflammation of the appendix and appendicitis. Although not proven, obstruction of the appendiceal lumen is believed to be an important step in the development of appendicitis. In some cases, obstruction leads to bacterial overgrowth and luminal distension, with an increase in intraluminal pressure that can inhibit the flow of lymph and blood in some cases. Then, vascular thrombosis and ischemic necrosis with perforation of the distal appendix may occur. Any perforation that occurs near the base of the appendix should raise concerns about another disease process. Most patients who will perforate do so before they are evaluated by surgeons.

1	may occur. Any perforation that occurs near the base of the appendix should raise concerns about another disease process. Most patients who will perforate do so before they are evaluated by surgeons. Appendiceal fecaliths (or appendicoliths) are found in approximately 50% of patients with gangrenous appendicitis who perforate but are rarely identified in those who have simple disease. As mentioned earlier, the incidence of perforated, but not simple, appendicitis is increasing. The rate of perforated and nonperforated appendicitis is correlated in men but not in women. Together these observations suggest that the underlying pathophysiologic processes are different and that simple appendicitis does not always progress to perforation. Furthermore, some cases of simple acute appendicitis may resolve spontaneously or with antibiotic therapy, and recurrent disease is remotely possible. The relative frequency of these events is unknown. When perforation occurs, the resultant leak may be

1	may resolve spontaneously or with antibiotic therapy, and recurrent disease is remotely possible. The relative frequency of these events is unknown. When perforation occurs, the resultant leak may be contained by the omentum or other surrounding tissues to form an abscess. Free perforation normally causes severe peritonitis. These patients may also develop infective suppurative thrombosis of the portal vein and its tributaries along with intrahepatic abscesses. The prognosis of the very unfortunate patients who develop this dreaded complication is very poor.

1	More refined approaches to diagnosis, supportive care, and surgical intervention are likely responsible for the remarkable decrease in the risk of mortality from simple appendicitis to currently less than 1%. Nevertheless, it is still important to identify patients who might have appendicitis as early as possible to minimize their risk of developing complications. Patients who have had symptoms for more than 48 h are more likely to perforate. Appendicitis should be included in the differential diagnosis of abdominal pain for every patient in any age group unless it is certain that the organ has been previously removed (Table 356-1).

1	Appendicitis should be included in the differential diagnosis of abdominal pain for every patient in any age group unless it is certain that the organ has been previously removed (Table 356-1). The appendix’s anatomical location, which varies, directly influences how the patient presents for care. Where the appendix can be “found” ranges from local differences in how the appendiceal body and tip lie relative to its attachment to the cecum (Figs. 356-1 and 356-2), to where the appendix is actually situated in the peritoneal cavity—for example, from its typical location in the right lower quadrant, to the pelvis, right flank, right upper quadrant (as may be observed during Ruptured ovarian cyst or other cystic Hepatitis disease of the ovaries Kidney disease, including Small-bowel obstruction nephrolithiasis 0.5% FIGURE 356-1 Regional anatomical variations of the appendix.

1	Kidney disease, including Small-bowel obstruction nephrolithiasis 0.5% FIGURE 356-1 Regional anatomical variations of the appendix. pregnancy), or even the left side of the abdomen for patients with malrotation or who have severely redundant colons. Because the differential diagnosis of appendicitis is so extensive, deciding if a patient has appendicitis can be difficult (Table 356-2). Soliciting an appropriate history requires detecting symptoms that might suggest alternative diagnoses. Patients with appendicitis may not have any abdominal discomfort early in the disease process. Furthermore, many patients may not present with the classically described history or physical findings.

1	What is the classic history? Nonspecific complaints occur first. Patients may notice changes in bowel habits or malaise and vague, perhaps intermittent, crampy, abdominal pain in the epigastric or periumbilical region. The pain subsequently migrates to the right lower quadrant over 12–24 h, where it is sharper and can be definitively localized as transmural inflammation when the appendix irritates the parietal peritoneum. Parietal peritoneal irritation may be associated with local muscle rigidity and stiffness. Patients with appendicitis FIGURE 356-2 Locations of the appendix and cecum.

1	FIGURE 356-2 Locations of the appendix and cecum. Migration of pain to right lower 50–60% quadrant will most often observe that their nausea, if present, followed the development of abdominal pain, which can help distinguish them from patients with gastroenteritis, for example, where nausea occurs first. Emesis, if present, also occurs after the onset of pain and is typically mild and scant. Thus, timing of the onset of symptoms and the characteristics of the patient’s pain and any associated findings must be rigorously assessed. Anorexia is so common that the diagnosis of appendicitis should be questioned in its absence.

1	Arriving at the correct diagnosis is even more challenging when the appendix is not located in the right lower quadrant, in women of childbearing age, and in the very young or elderly. Because the differential diagnosis of appendicitis is so broad, often the key question to answer expeditiously is whether the patient has appendicitis or some other condition that requires immediate operative intervention. A major concern is that the likelihood of a delay in diagnosis is greater if the appendix is unusually positioned. All patients should undergo a rectal examination. An inflamed appendix located behind the cecum or below the pelvic brim may prompt very little tenderness of the anterior abdominal wall.

1	Patients with pelvic appendicitis are more likely to present with dysuria, urinary frequency, diarrhea, or tenesmus. They may only experience pain in the suprapubic region on palpation or on rectal or pelvic examination. A pelvic examination in women is mandatory to rule out conditions affecting urogynecologic organs that can cause abdominal pain and mimic appendicitis such as pelvic inflammatory disease, ectopic pregnancy, and ovarian torsion. The relative frequencies of some presenting signs are displayed in Table 356-3. Patients with simple appendicitis normally only appear mildly ill with a pulse and temperature that are usually only slightly above normal. The provider should be concerned about other disease processes beside appendicitis or the presence of complications such as perforation, phlegmon, or abscess formation if the temperature is >38.3°C (~101°F) and if there are rigors.

1	Patients with appendicitis will be found to lie quite still to avoid peritoneal irritation caused by movement, and some will report discomfort caused by a bumpy car ride on the way to the hospital or clinic, coughing, sneezing, or other actions that replicate a Valsalva maneuver. The entire abdomen should be examined systematically starting in an area where the patient does not report discomfort if possible. Classically, maximal tenderness is identified in the right lower quadrant at or near McBurney’s point, which is located approximately one-third of the way along a line originating at the anterior iliac spine and running to the umbilicus. Gentle pressure in the left lower quadrant may elicit pain in the right lower quadrant if the appendix is located there. This is Rovsing’s sign (Table 356-4). Evidence of parietal peritoneal irritation is often best elicited by gentle abdominal percussion, jiggling the patient’s gurney or bed, or mildly bumping the feet.

1	Atypical presentation and pain patterns are common, especially in the very old or the very young. Diagnosing appendicitis in children can be especially challenging because they tend to respond so dramatically to stimulation and obtaining an accurate history may be difficult. In addition, it is important to remember that the smaller omentum found in children may be less likely to wall off an appendiceal perforation. Observing the child in a quiet surrounding may be helpful.

1	Signs and symptoms of appendicitis can be subtle in the elderly who may not react as vigorously to appendicitis as younger people. Pain, if noticed, may be minimal and have originated in the right lower quadrant or, otherwise, where the appendix is located. It may never have been noticed to be intermittent, or there may only be significant discomfort with deep palpation. Nausea, anorexia, and emesis may be the predominant complaints. The rare patient may even present with signs and symptoms of distal bowel obstruction secondary to appendiceal inflammation and phlegmon or abscess formation.

1	Laboratory testing does not identify patients with appendicitis but can help the clinician work through the differential diagnosis. The white blood cell count is only mildly to moderately elevated in approximately 70% of patients with simple appendicitis (with a leukocytosis of 10,000–18,000 cells/μL). A “left shift” toward immature polymorphonuclear leukocytes is present in >95% of cases. A sickle cell preparation may be prudent to obtain in those of African, Spanish, Mediterranean, or Indian ancestry. Serum amylase and lipase levels should be measured.

1	Urinalysis is indicated to help exclude genitourinary conditions that may mimic acute appendicitis, but a few red or white blood cells may be present as a nonspecific finding. However, an inflamed appendix that abuts the ureter or bladder may cause sterile pyuria or hematuria. Every woman of childbearing age should have a pregnancy test. Cervical cultures are indicated if pelvic inflammatory disease is suspected. Anemia and guaiac-positive stools should raise concern about the presence of other diseases or complications such as cancer.

1	Plain films of the abdomen are rarely helpful and so are not routinely obtained unless the clinician is worried about other conditions such as intestinal obstruction, perforated viscus, or ureterolithiasis. Less than 5% of patients will present with an opaque fecalith in the right lower quadrant. The presence of a fecalith is not diagnostic of appendicitis, although its presence in an appropriate location where the patient complains of pain is suggestive. The effectiveness of ultrasonography as a tool to diagnosis appendicitis is highly operator dependent. Even in very skilled hands, the appendix may not be visualized. Its overall sensitivity is 0.86, with a FIGURE 356-3 Computed tomography with oral and intravenous contrast of acute appendicitis. There is thickening of the wall of the appendix and periappendiceal stranding (arrow).

1	FIGURE 356-3 Computed tomography with oral and intravenous contrast of acute appendicitis. There is thickening of the wall of the appendix and periappendiceal stranding (arrow). specificity of 0.81. Ultrasonography, especially intravaginal techniques, appears to be most useful for identifying pelvic pathology in women. Ultrasonographic findings suggesting the presence of appendicitis include wall thickening, an increased appendiceal diameter, and the presence of free fluid.

1	The sensitivity and specificity of computed tomography (CT) are 0.94 and 0.95, respectively. Thus, CT imaging, given its high negative predictive value, may be helpful if the diagnosis is in doubt, although studies performed early in the course of disease may not have any typical radiographic findings. Suggestive findings on CT examination include dilatation >6 mm with wall thickening, a lumen that does not fill with enteric contrast, and fatty tissue stranding or air surrounding the appendix, which suggests inflammation (Figs. 356-3 and 356-4). The presence of luminal air or contrast is not consistent with a diagnosis of appendicitis. Furthermore, nonvisualization of the appendix is a nonspecific finding that should not be used to rule out the presence of appendiceal or periappendiceal inflammation.

1	Appendicitis in the most common extrauterine general surgical emergency observed during pregnancy. Early symptoms of appendicitis such as nausea and anorexia may be overlooked. Diagnosing appendicitis in pregnant patients may be especially difficult because as the uterus enlarges the appendix may be pushed higher along the right flank even to the right upper quadrant or because the gravid uterus may obscure typical physical findings. Ultrasonography may facilitate early diagnosis. A high index of suspicion is required because of the effects of unrecognized and untreated appendicitis on the fetus. For example, the fetal mortality rate is four times greater (from 5 to 20%) in patients with perforation. FIGURE 356-4 Appendiceal fecalith (arrow).

1	FIGURE 356-4 Appendiceal fecalith (arrow). Immunocompromised patients may present with only mild tenderness and may have many other disease processes in their differential diagnosis, including atypical infections from mycobacteria, Cytomegalovirus, or other fungi. Enterocolitis is a concern and may be present in patients who present with abdominal pain, fever, and neutropenia due to chemotherapy. CT imaging may be very helpful, although it is important not to be overly cautious and delay operative intervention for those patients who are believed to have appendicitis.

1	In the absence of contraindications, a patient who has a strongly suggestive medical history and physical examination with supportive laboratory findings should undergo appendectomy urgently. In this instance, imaging studies are not required. In patients in whom the evaluation is suggestive but not convincing, imaging and further study are appropriate. Pelvic ultrasonography is indicated in women of childbearing age. Thereafter, CT may accurately indicate the presence of appendicitis or other intraabdominal processes that warrant intervention. Whenever the diagnosis is uncertain, it is prudent to observe the patient and repeat the abdominal examination over 6–8 h. Any evidence of progression is an indication for operation. Narcotics can be given to patients with severe discomfort, especially if the first abdominal examination is completed before drugs are administered.

1	All patients should be fully prepared for surgery and have any fluid and electrolyte abnormalities corrected. Either laparoscopic or open appendectomy is a satisfactory choice for patients with uncomplicated appendicitis. Management of those who present with a mass representing a phlegmon or abscess can be more difficult. Such patients are best served by treatment with broad-spectrum antibiotics, drainage if there is an abscess >3 cm in diameter, and parenteral fluids and bowel rest if they appear to respond to conservative management. The appendix can then be more safely removed 6–12 weeks later when inflammation has diminished.

1	Laparoscopic appendectomy now accounts for approximately 60% of all appendectomies. Laparoscopic appendectomy is associated with less postoperative pain and, possibly, a shorter length of stay and faster return to normal activity. Patients who undergo laparoscopic appendectomy also appear to have fewer wound infections, although the risk of intraabdominal abscess formation may be higher. A laparoscopic approach may also be useful when the exact diagnosis is uncertain, yet direct visualization and exploration of the abdomen are needed. A laparoscopic approach may also facilitate exposure in those who are very obese. A thorough examination of the abdomen is indicated if the appendix appears normal at operation, which can be expected to occur in up to 15–20% of cases.

1	Absent complications, most patients can be discharged within 24–40 h of operation. The most common postoperative complications are fever and leukocytosis. Continuation of these findings beyond 5 days should raise concern for the presence of an intraabdominal abscess. The mortality rate for uncomplicated, nonperforated appendicitis is 0.1–0.5%, which approximates the overall risk of general anesthesia. The mortality rate for perforated appendicitis or other complicated disease is much higher, ranging from 3% overall to a high as 15% in the elderly. Approach to the Patient with liver Disease Marc G. Ghany, Jay H. Hoofnagle A diagnosis of liver disease usually can be made accurately by careful elicitation of the patient’s history, physical examination, and appli-357 SECTion 2 Approach to the Patient with Liver Disease

1	Approach to the Patient with Liver Disease Acute peritonitis, or inflammation of the visceral and parietal peritoneum, is most often but not always infectious in origin, resulting from perforation of a hollow viscus. This is called secondary peritonitis, as opposed to primary or spontaneous peritonitis, when a specific intraabdominal source cannot be identified. In either instance, the inflammation can be localized or diffuse.

1	Infective organisms may contaminate the peritoneal cavity after spillage from a hollow viscus, because of a penetrating wound of the abdominal wall, or because of the introduction of a foreign object like a peritoneal dialysis catheter or port that becomes infected. Secondary peritonitis most commonly results from perforation of the appendix, colonic diverticuli, or the stomach and duodenum. It may also occur as a complication of bowel infarction or incarceration, cancer, inflammatory bowel disease, and intestinal obstruction or volvulus. Conditions that may cause secondary bacterial peritonitis and their mechanisms are listed in Table 356-5. Over 90% of the cases of primary or spontaneous bacterial peritonitis occur in patients with ascites or hypoproteinemia (<1 g/L).

1	cation of a few laboratory tests. In some circumstances, radiologic examinations are helpful or, indeed, diagnostic. Liver biopsy is considered the criterion standard in evaluation of liver disease but is now needed less for diagnosis than for grading and staging of disease. This chapter provides an introduction to diagnosis and management of liver disease, briefly reviewing the structure and function of the liver; the major clinical manifestations of liver disease; and the use of clinical Perforation or Leakage of Other Organs Biliary leakage (e.g., after liver biopsy) Cholecystitis Intraperitoneal bleeding Pancreatitis Salpingitis Traumatic or other rupture of urinary bladder Loss of peritoneal integrity Iatrogenic (e.g., postoperative foreign body) Perinephric abscess Peritoneal dialysis or other

1	Salpingitis Traumatic or other rupture of urinary bladder Loss of peritoneal integrity Iatrogenic (e.g., postoperative foreign body) Perinephric abscess Peritoneal dialysis or other Aseptic peritonitis is most commonly caused by the abnormal pres-1989 ence of physiologic fluids like gastric juice, bile, pancreatic enzymes, blood, or urine. It can also be caused by the effects of normally sterile foreign bodies like surgical sponges or instruments. More rarely, it occurs as a complication of systemic diseases like lupus erythematosus, porphyria, and familial Mediterranean fever. The chemical irritation caused by stomach acid and activated pancreatic enzymes is extreme and secondary bacterial infection may occur.

1	The cardinal signs and symptoms of peritonitis are acute, typically severe, abdominal pain with tenderness and fever. How the patient’s complaints of pain are manifested depends on their overall physical health and whether the inflammation is diffuse or localized. Elderly and immunosuppressed patients may not respond as aggressively to the irritation. Diffuse, generalized peritonitis is most often recognized as diffuse abdominal tenderness with local guarding, rigidity, and other evidence of parietal peritoneal irritation. Physical findings may only be identified in a specific region of the abdomen if the intraperitoneal inflammatory process is limited or otherwise contained as may occur in patients with uncomplicated appendicitis or diverticulitis. Bowel sounds are usually absent to hypoactive.

1	Most patients present with tachycardia and signs of volume depletion with hypotension. Laboratory testing typically reveals a significant leukocytosis, and patients may be severely acidotic. Radiographic studies may show dilatation of the bowel and associated bowel wall edema. Free air, or other evidence of leakage, requires attention and could represent a surgical emergency. In stable patients in whom ascites is present, diagnostic paracentesis is indicated, where the fluid is tested for protein and lactate dehydrogenase and the cell count is measured.

1	Whereas mortality rates can be less than 10% for reasonably healthy patients with relatively uncomplicated, localized peritonitis, mortality rates >40% have been reported for the elderly or immunocompromised. Successful treatment depends on correcting any electrolyte abnormalities, restoration of fluid volume and stabilization of the cardiovascular system, appropriate antibiotic therapy, and surgical correction of any underlying abnormalities. The wisdom and expertise of Dr. William Silen is gratefully acknowledged in this updated chapter on acute appendicitis and peritonitis. history, physical examination, laboratory tests, imaging studies, and liver biopsy.

1	history, physical examination, laboratory tests, imaging studies, and liver biopsy. The liver is the largest organ of the body, weighing 1–1.5 kg and representing 1.5–2.5% of the lean body mass. The size and shape of the liver vary and generally match the general body shape—long and lean or squat and square. This organ is located in the right upper quadrant of the abdomen under the right lower rib cage against the diaphragm and projects for a variable extent into the left upper quadrant. It is held in place by ligamentous attachments to the diaphragm, peritoneum, great vessels, and upper gastrointestinal organs. The liver receives a dual blood supply; ~20% of the blood flow is oxygen-rich blood from the hepatic artery, and 80% is nutrient-rich blood from the portal vein arising from the stomach, intestines, pancreas, and spleen.

1	1990 The majority of cells in the liver are hepatocytes, which constitute two-thirds of the organ’s mass. The remaining cell types are Kupffer cells (members of the reticuloendothelial system), stellate (Ito or fat-storing) cells, endothelial and blood vessel cells, bile ductular cells, and cells of supporting structures. Viewed by light microscopy, the liver appears to be organized in lobules, with portal areas at the periphery and central veins in the center of each lobule. However, from a functional point of view, the liver is organized into acini, with both hepatic arterial and portal venous blood entering the acinus from the portal areas (zone 1) and then flowing through the sinusoids to the terminal hepatic veins (zone 3); the intervening hepatocytes constitute zone 2. The advantage of viewing the acinus as the physiologic unit of the liver is that this perspective helps to explain the morphologic patterns and zonality of many vascular and biliary diseases not explained by the

1	of viewing the acinus as the physiologic unit of the liver is that this perspective helps to explain the morphologic patterns and zonality of many vascular and biliary diseases not explained by the lobular arrangement. Portal areas of the liver consist of small veins, arteries, bile ducts, and lymphatics organized in a loose stroma of supporting matrix and small amounts of collagen. Blood flowing into the portal areas is distributed through the sinusoids, passing from zone 1 to zone 3 of the acinus and draining into the terminal hepatic veins (“central veins”). Secreted bile flows in the opposite direction—i.e., in a counter-current pattern from zone 3 to zone 1. The sinusoids are lined by unique endothelial cells that have prominent fenestrae of variable sizes, allowing the free flow of plasma but not of cellular elements. The plasma is thus in direct contact with hepatocytes in the subendothelial space of Disse. Hepatocytes have distinct polarity. The basolateral side of the

1	of plasma but not of cellular elements. The plasma is thus in direct contact with hepatocytes in the subendothelial space of Disse. Hepatocytes have distinct polarity. The basolateral side of the hepatocyte lines the space of Disse and is richly lined with microvilli; it exhibits endocytotic and pinocytotic activity, with passive and active uptake of nutrients, proteins, and other molecules. The apical pole of the hepatocyte forms the canalicular membranes through which bile components are secreted. The canaliculi of hepatocytes form a fine network, which fuses into the bile ductular elements near the portal areas. Kupffer cells usually lie within the sinusoidal vascular space and represent the largest group of fixed macrophages in the body. The stellate cells are located in the space of Disse but are not usually prominent unless activated, when they produce collagen and matrix. Red blood cells stay in the sinusoidal space as blood flows through the lobules, but white blood cells can

1	Disse but are not usually prominent unless activated, when they produce collagen and matrix. Red blood cells stay in the sinusoidal space as blood flows through the lobules, but white blood cells can migrate through or around endothelial cells into the space of Disse and from there to portal areas, where they can return to the circulation through lymphatics. Hepatocytes perform numerous and vital roles in maintaining homeostasis and health. These functions include the synthesis of most essential serum proteins (albumin, carrier proteins, coagulation factors, many hormonal and growth factors), the production of bile and its carriers (bile acids, cholesterol, lecithin, phospholipids), the regulation of nutrients (glucose, glycogen, lipids, cholesterol, amino acids), and the metabolism and conjugation of lipophilic compounds (bilirubin, anions, cations, drugs) for excretion in the bile or urine. Measurement of these activities to assess liver function is complicated by the multiplicity

1	conjugation of lipophilic compounds (bilirubin, anions, cations, drugs) for excretion in the bile or urine. Measurement of these activities to assess liver function is complicated by the multiplicity and variability of these functions. The most commonly used liver “function” tests are measurements of serum bilirubin, serum albumin, and prothrombin time. The serum bilirubin level is a measure of hepatic conjugation and excretion; the serum albumin level and prothrombin time are measures of protein synthesis. Abnormalities of bilirubin, albumin, and prothrombin time are typical of hepatic dysfunction. Frank liver failure is incompatible with life, and the functions of the liver are too complex and diverse to be sub-served by a mechanical pump; a dialysis membrane; or a concoction of infused hormones, proteins, and growth factors.

1	While there are many causes of liver disease (Table 357-1), these disorders generally present clinically in a few distinct patterns and are usually classified as hepatocellular, cholestatic (obstructive), or mixed. In hepatocellular diseases (such as viral hepatitis and alcoholic liver disease), features of liver injury, inflammation, and necrosis predominate. In cholestatic diseases (such as gallstone or malignant obstruction, primary biliary cirrhosis, and some drug-induced liver Gilbert’s syndrome Crigler-Najjar syndrome, types I and II Dubin-Johnson syndrome Rotor syndrome nucleosis] herpesvirus, adenovirus hepatitis) Cryptogenic hepatitis Progressive familial intrahepatic cholestasis, types I–III Others (galactosemia, tyrosinemia, cystic fibrosis, Newman-Pick disease, Gaucher’s disease)

1	Progressive familial intrahepatic cholestasis, types I–III Others (galactosemia, tyrosinemia, cystic fibrosis, Newman-Pick disease, Gaucher’s disease) Acute fatty liver of pregnancy diseases), features of inhibition of bile flow predominate. In a mixed pattern, features of both hepatocellular and cholestatic injury are present (such as in cholestatic forms of viral hepatitis and many drug-induced liver diseases). The pattern of onset and prominence of symptoms can rapidly suggest a diagnosis, particularly if major risk factors are considered, such as the age and sex of the patient and a history of exposure or risk behaviors.

1	Typical presenting symptoms of liver disease include jaundice, fatigue, itching, right-upper-quadrant pain, nausea, poor appetite, abdominal distention, and intestinal bleeding. At present, however, many patients are diagnosed with liver disease who have no symptoms and who have been found to have abnormalities in biochemical liver tests as a part of a routine physical examination or screening for blood Jaundice of sepsis Total parenteral nutrition–induced jaundice Cholestasis of pregnancy Cholangitis and cholecystitis Extrahepatic biliary obstruction (stone, stricture, cancer) Biliary atresia Caroli’s disease Cryptosporidiosis Hepatocellular patterns (isoniazid, acetaminophen) Mixed patterns (sulfonamides, phenytoin)

1	Hepatocellular patterns (isoniazid, acetaminophen) Mixed patterns (sulfonamides, phenytoin) Microand macrovesicular steatosis (methotrexate, fialuridine) donation or for insurance or employment. The wide availability of batteries of liver tests makes it relatively simple to demonstrate the presence of liver injury as well as to rule it out in someone in whom liver disease is suspected.

1	Evaluation of patients with liver disease should be directed at (1) establishing the etiologic diagnosis, (2) estimating disease severity (grading), and (3) establishing the disease stage (staging). Diagnosis should focus on the category of disease (hepatocellular, cholestatic, or mixed injury) as well as on the specific etiologic diagnosis. Grading refers to assessment of the severity or activity of disease—active or inactive as well as mild, moderate, or severe. Staging refers to estimation of the point in the course of the natural history of the disease, whether early or late; or precirrhotic, cirrhotic, or end-stage. This chapter introduces general, salient concepts in the evaluation of patients with liver disease that help lead to the diagnoses discussed in subsequent chapters.

1	The clinical history should focus on the symptoms of liver disease— their nature, patterns of onset, and progression—and on potential risk factors for liver disease. The manifestations of liver disease include constitutional symptoms such as fatigue, weakness, nausea, poor appetite, and malaise and the more liver-specific symptoms of jaundice, dark urine, light stools, itching, abdominal pain, and bloating. Symptoms can also suggest the presence of cirrhosis, end-stage liver disease, or complications of cirrhosis such as portal hypertension. Generally, the constellation of symptoms and their patterns of onset rather than a specific symptom points to an etiology.

1	Fatigue is the most common and most characteristic symptom of liver disease. It is variously described as lethargy, weakness, listlessness, malaise, increased need for sleep, lack of stamina, and poor energy. The fatigue of liver disease typically arises after activity or exercise and is rarely present or severe after adequate rest; i.e., it is “afternoon” rather than “morning” fatigue. Fatigue in liver disease is often intermittent and variable in severity from hour to hour and day to day. In some patients, it may not be clear whether fatigue is due to the liver disease or to other problems such as stress, anxiety, sleep disturbance, or a concurrent illness.

1	Nausea occurs with more severe liver disease and may accompany fatigue or be provoked by smelling food odors or eating fatty foods. Vomiting can occur but is rarely persistent or prominent. Poor appetite with weight loss occurs frequently in acute liver disease but is rare in chronic disease except when cirrhosis is present and advanced. Diarrhea is uncommon in liver disease except with severe jaundice, in which a lack of bile acids reaching the intestine can lead to steatorrhea. Right-upper-quadrant discomfort or ache (“liver pain”) occurs in many liver diseases and is usually marked by tenderness over the liver area. The pain arises from stretching or irritation of Glisson’s capsule, which surrounds the liver and is rich in nerve endings. Severe pain is most typical of gallbladder disease, liver abscess, and severe veno-occlusive disease but is also an occasional accompaniment of acute hepatitis.

1	Itching occurs with acute liver disease, appearing early in obstructive jaundice (from biliary obstruction or drug-induced cholestasis) and somewhat later in hepatocellular disease (acute hepatitis). Itching also occurs in chronic liver diseases—typically the cholestatic forms such as primary biliary cirrhosis and sclerosing cholangitis, in which it is often the presenting symptom, preceding the onset of jaundice. However, itching can occur in any liver disease, particularly once cirrhosis develops.

1	Jaundice is the hallmark symptom of liver disease and perhaps the most reliable marker of severity. Patients usually report darkening of the urine before they notice scleral icterus. Jaundice is rarely detectable with a bilirubin level <43 μmol/L (2.5 mg/dL). With severe cholestasis, there will also be lightening of the color of the stools and steatorrhea. Jaundice without dark urine usually indicates indirect (unconjugated) hyperbilirubinemia and is typical of hemolytic anemia and the genetic disorders of bilirubin conjugation, the common and benign form being Gilbert’s syndrome and the rare and severe form being Crigler-Najjar syndrome. Gilbert’s syndrome affects up to 5% of the 1991 general population; the jaundice in this condition is more noticeable after fasting and with stress.

1	Major risk factors for liver disease that should be sought in the clinical history include details of alcohol use, medication use (including herbal compounds, birth control pills, and over-the-counter medications), personal habits, sexual activity, travel, exposure to jaundiced or other high-risk persons, injection drug use, recent surgery, remote or recent transfusion of blood or blood products, occupation, accidental exposure to blood or needlestick, and familial history of liver disease.

1	For assessing the risk of viral hepatitis, a careful history of sexual activity is of particular importance and should include the number of lifetime sexual partners and, for men, a history of having sex with men. Sexual exposure is a common mode of spread of hepatitis B but is rare for hepatitis C. A family history of hepatitis, liver disease, and liver cancer is also important. Maternal-infant transmission occurs with both hepatitis B and C. Vertical spread of hepatitis B can now be prevented by passive and active immunization of the infant at birth. Vertical spread of hepatitis C is uncommon, but there are no reliable means of prevention. Transmission is more common among HIV-coinfected mothers and is also linked to prolonged and difficult labor and delivery, early rupture of membranes, and internal fetal monitoring. A history of injection drug use, even in the remote past, is of great importance in assessing the risk for hepatitis B and C. Injection drug use is now the single most

1	and internal fetal monitoring. A history of injection drug use, even in the remote past, is of great importance in assessing the risk for hepatitis B and C. Injection drug use is now the single most common risk factor for hepatitis C. Transfusion with blood or blood products is no longer an important risk factor for acute viral hepatitis. However, blood transfusions received before the introduction of sensitive enzyme immunoassays for antibody to hepatitis C virus in 1992 is an important risk factor for chronic hepatitis C. Blood transfusion before 1986, when screening for antibody to hepatitis B core antigen was introduced, is also a risk factor for hepatitis

1	B. Travel to a developing area of the world, exposure to persons with jaundice, and exposure to young children in day-care centers are risk factors for hepatitis A. Tattooing and body piercing (for hepatitis B and C) and eating shellfish (for hepatitis A) are frequently mentioned but are actually types of exposure that quite rarely lead to the acquisition of hepatitis. Hepatitis E is one of the more common causes of jaundice in Asia and Africa but is uncommon in developed nations.

1	Hepatitis E is one of the more common causes of jaundice in Asia and Africa but is uncommon in developed nations. Recently, non-travel-related (autochthonous) cases of hepatitis E have been described in developed countries, including the United States. These cases appear to be due to strains of hepatitis E virus that are endemic in swine and some wild animals (genotypes 3 and 4). While occasional cases are associated with eating raw or undercooked pork or game (deer and wild boars), most cases of hepatitis E occur without known exposure, predominantly in elderly man without typical risk factors for viral hepatitis. Hepatitis E infection can become chronic in immunosuppressed individuals (such as transplant recipients, patients receiving chemotherapy, or patients with HIV infection), in whom it presents with abnormal serum enzymes in the absence of markers of hepatitis B or C.

1	A history of alcohol intake is important in assessing the cause of liver disease and also in planning management and recommendations. In the United States, for example, at least 70% of adults drink alcohol to some degree, but significant alcohol intake is less common; in population-based surveys, only 5% of individuals have more than two drinks per day, the average drink representing 11–15 g of alcohol. Alcohol consumption associated with an increased rate of alcoholic liver disease is probably more than two drinks (22–30 g) per day in women and three drinks (33–45 g) in men. Most patients with alcoholic cirrhosis have a much higher daily intake and have drunk excessively for ≥10 years before onset of liver disease. In assessing alcohol intake, the history should also focus on whether alcohol abuse or dependence is present. Alcoholism is usually defined by the behavioral patterns and consequences of alcohol intake, not by the amount. Abuse is defined by a repetitive pattern of

1	alcohol abuse or dependence is present. Alcoholism is usually defined by the behavioral patterns and consequences of alcohol intake, not by the amount. Abuse is defined by a repetitive pattern of drinking alcohol that has adverse effects on social, family, occupational, or health status. Dependence is defined by alcohol-seeking behavior, despite its adverse effects. Many

1	Approach to the Patient with Liver Disease Have you ever felt you ought to cut down on your drinking? A Have people annoyed you by criticizing your drinking? G Have you ever felt guilty or bad about your drinking? E Have you ever had a drink first thing in the morning to steady your nerves or get rid of a hangover (eye-opener)? aOne “yes” response should raise suspicion of an alcohol use problem, and more than one is a strong indication of abuse or dependence. alcoholics demonstrate both dependence and abuse, and dependence is considered the more serious and advanced form of alcoholism. A clinically helpful approach to diagnosis of alcohol dependence and abuse is the use of the CAGE questionnaire (Table 357-2), which is recommended for all medical history-taking.

1	Family history can be helpful in assessing liver disease. Familial causes of liver disease include Wilson’s disease; hemochromatosis and α1 antitrypsin deficiency; and the more uncommon inherited pediatric liver diseases—i.e., familial intrahepatic cholestasis, benign recurrent intrahepatic cholestasis, and Alagille syndrome. Onset of severe liver disease in childhood or adolescence in conjunction with a family history of liver disease or neuropsychiatric disturbance should lead to investigation for Wilson’s disease. A family history of cirrhosis, diabetes, or endocrine failure and the appearance of liver disease in adulthood suggests hemochromatosis and should prompt investigation of iron status. Abnormal iron studies in adult patients warrant genotyping of the HFE gene for the C282Y and H63D mutations typical of genetic hemochromatosis. In children and adolescents with iron overload, other non-HFE causes of hemochromatosis should be sought. A family history of emphysema should

1	and H63D mutations typical of genetic hemochromatosis. In children and adolescents with iron overload, other non-HFE causes of hemochromatosis should be sought. A family history of emphysema should provoke investigation of α1 anti-trypsin levels and, if levels are low, for protease inhibitor (Pi) genotype.

1	The physical examination rarely uncovers evidence of liver dysfunction in a patient without symptoms or laboratory findings, nor are most signs of liver disease specific to one diagnosis. Thus, the physical examination complements rather than replaces the need for other diagnostic approaches. In many patients, the physical examination is normal unless the disease is acute or severe and advanced. Nevertheless, the physical examination is important in that it can yield the first evidence of hepatic failure, portal hypertension, and liver decompensation. In addition, the physical examination can reveal signs—related either to risk factors or to associated diseases or findings—that point to a specific diagnosis.

1	Typical physical findings in liver disease are icterus, hepatomegaly, hepatic tenderness, splenomegaly, spider angiomata, palmar erythema, and excoriations. Signs of advanced disease include muscle wasting, ascites, edema, dilated abdominal veins, hepatic fetor, asterixis, mental confusion, stupor, and coma. In male patients with cirrhosis, particularly that related to alcohol use, signs of hyperestrogenemia such as gynecomastia, testicular atrophy, and loss of male-pattern hair distribution may be found. Icterus is best appreciated when the sclera is inspected under natural light. In fair-skinned individuals, a yellow tinge to the skin may be obvious. In dark-skinned individuals, examination of the mucous membranes below the tongue can demonstrate jaundice. Jaundice is rarely detectable if the serum bilirubin level is <43 μmol/L (2.5 mg/dL) but may remain detectable below this level during recovery from jaundice (because of protein and tissue binding of conjugated bilirubin).

1	Spider angiomata and palmar erythema occur in both acute and chronic liver disease; these manifestations may be especially prominent in persons with cirrhosis but can develop in normal individuals and are frequently found during pregnancy. Spider angiomata are superficial, tortuous arterioles and—unlike simple telangiectases— typically fill from the center outward. Spider angiomata occur only on the arms, face, and upper torso; they can be pulsatile and may be difficult to detect in dark-skinned individuals.

1	Hepatomegaly is not a highly reliable sign of liver disease because of variability in the liver’s size and shape and the physical impediments to assessment of liver size by percussion and palpation. Marked hepatomegaly is typical of cirrhosis, veno-occlusive disease, infiltrative disorders such as amyloidosis, metastatic or primary cancers of the liver, and alcoholic hepatitis. Careful assessment of the liver edge may also reveal unusual firmness, irregularity of the surface, or frank nodules. Perhaps the most reliable physical finding in the liver examination is hepatic tenderness. Discomfort when the liver is touched or pressed upon should be carefully sought with percussive comparison of the right and left upper quadrants. Splenomegaly, which occurs in many medical conditions, can be a subtle but significant physical finding in liver disease. The availability of ultrasound methods for assessment of the spleen allows confirmation of the physical finding.

1	Signs of advanced liver disease include muscle wasting and weight loss as well as hepatomegaly, bruising, ascites, and edema. Ascites is best appreciated by attempts to detect shifting dullness by careful percussion. Ultrasound examination will confirm the finding of ascites in equivocal cases. Peripheral edema can occur with or without ascites. In patients with advanced liver disease, other factors frequently contribute to edema formation, including hypoalbuminemia, venous insufficiency, heart failure, and medications.

1	Hepatic failure is defined as the occurrence of signs or symptoms of hepatic encephalopathy in a person with severe acute or chronic liver disease. The first signs of hepatic encephalopathy can be subtle and nonspecific—change in sleep patterns, change in personality, irritability, and mental dullness. Thereafter, confusion, disorientation, stupor, and eventually coma supervene. In acute liver failure, excitability and mania may be present. Physical findings include asterixis and flapping tremors of the body and tongue. Fetor hepaticus refers to the slightly sweet, ammoniacal odor that can develop in patients with liver failure, particularly if there is portal-venous shunting of blood around the liver. Other causes of coma and disorientation should be excluded, mainly electrolyte imbalances, sedative use, and renal or respiratory failure. The appearance of hepatic encephalopathy during acute hepatitis is the major criterion for diagnosis of fulminant hepatitis and indicates a poor

1	sedative use, and renal or respiratory failure. The appearance of hepatic encephalopathy during acute hepatitis is the major criterion for diagnosis of fulminant hepatitis and indicates a poor prognosis. In chronic liver disease, encephalopathy is usually triggered by a medical complication such as gastrointestinal bleeding, over-diuresis, uremia, dehydration, electrolyte imbalance, infection, constipation, or use of narcotic analgesics.

1	A helpful measure of hepatic encephalopathy is a careful mental-status examination and use of the trail-making test, which consists of a series of 25 numbered circles that the patient is asked to connect as rapidly as possible using a pencil. The normal range for the connect-the-dot test is 15–30 sec; it is considerably longer in patients with early hepatic encephalopathy. Other tests include drawing of abstract objects or comparison of a signature to previous examples. More sophisticated testing—e.g., with electroencephalography and visual evoked potentials—can detect mild forms of encephalopathy but are rarely clinically useful.

1	Other signs of advanced liver disease include umbilical hernia from ascites, hydrothorax, prominent veins over the abdomen, and caput medusa, a condition that consists of collateral veins radiating from the umbilicus and results from recanulation of the umbilical vein. Widened pulse pressure and signs of a hyperdynamic circulation can occur in patients with cirrhosis as a result of fluid and sodium retention, increased cardiac output, and reduced peripheral resistance. Patients with long-standing cirrhosis and portal hypertension are prone to develop the hepatopulmonary syndrome, which is defined by the triad of liver disease, hypoxemia, and pulmonary arteriovenous shunting. The hepatopulmonary syndrome is characterized by platypnea and orthodeoxia: shortness of breath and oxygen desaturation that occur paradoxically upon the assumption of an upright position. Measurement of oxygen saturation by pulse oximetry is a reliable screening test for hepatopulmonary syndrome.

1	Several skin disorders and changes are common in liver disease. Hyperpigmentation is typical of advanced chronic cholestatic diseases such as primary biliary cirrhosis and sclerosing cholangitis. In these same conditions, xanthelasma and tendon xanthomata occur as a result of retention and high serum levels of lipids and cholesterol. Slate-gray pigmentation of the skin is also seen with hemochromatosis if iron levels are high for a prolonged period. Mucocutaneous vasculitis with palpable purpura, especially on the lower extremities, is typical of cryoglobulinemia of chronic hepatitis C but can also occur in chronic hepatitis B.

1	Some physical signs point to specific liver diseases. Kayser-Fleischer rings occur in Wilson’s disease and consist of a golden-brown copper pigment deposited in Descemet’s membrane at the periphery of the cornea; they are best seen by slit-lamp examination. Dupuytren contracture and parotid enlargement are suggestive of chronic alcoholism and alcoholic liver disease. In metastatic liver disease or primary hepatocellular carcinoma, signs of cachexia and wasting as well as firm hepatomegaly and a hepatic bruit may be prominent.

1	The major causes of liver disease and key diagnostic features are outlined in Table 357-3, and an algorithm for evaluation of the patient with suspected liver disease is shown in Fig. 357-1. Specifics of diagnosis are discussed in later chapters. The most common causes of acute liver disease are viral hepatitis (particularly hepatitis A, B, and C), drug-induced liver injury, cholangitis, and alcoholic liver disease. Liver biopsy usually is not needed in the diagnosis and management of acute liver disease, exceptions being situations where the diagnosis remains unclear despite thorough clinical and laboratory investigation. Liver biopsy can be helpful in diagnosing drug-induced liver disease and acute alcoholic hepatitis.

1	The most common causes of chronic liver disease, in general order of frequency, are chronic hepatitis C, alcoholic liver disease, nonalcoholic steatohepatitis, chronic hepatitis B, autoimmune hepatitis, sclerosing cholangitis, primary biliary cirrhosis, hemochromatosis, and Wilson’s disease. Hepatitis E virus is a rare cause of chronic hepatitis, with cases occurring mostly in persons who are immunosuppressed or immunodeficient. Strict diagnostic criteria have not been developed for most

1	Hepatitis C Anti-HCV and HCV RNA Hepatitis D (delta) HBsAg and anti-HDV Hepatitis E Anti-HEV IgM and HEV RNA Autoimmune hepatitis ANA or SMA, elevated IgG levels, and com patible histology Primary biliary cirrhosis Mitochondrial antibody, elevated IgM levels, and compatible histology Primary sclerosing cholangitis P-ANCA, cholangiography Drug-induced liver disease History of drug ingestion Alcoholic liver disease History of excessive alcohol intake and compatible histology Nonalcoholic steatohepatitis Ultrasound or CT evidence of fatty liver and compatible histology α1 Antitrypsin disease Reduced α1 antitrypsin levels, phenotype PiZZ or PiSZ Wilson’s disease Decreased serum ceruloplasmin and increased urinary copper; increased hepatic copper level Hemochromatosis Elevated iron saturation and serum ferritin; genetic testing for HFE gene mutations Hepatocellular cancer Elevated α-fetoprotein level (to >500 ng/mL); ultrasound or CT image of mass Abbreviations: HAV, HBV, HCV, HDV, HEV:

1	and serum ferritin; genetic testing for HFE gene mutations Hepatocellular cancer Elevated α-fetoprotein level (to >500 ng/mL); ultrasound or CT image of mass Abbreviations: HAV, HBV, HCV, HDV, HEV: hepatitis A, B, C, D, E virus; HBsAg, hepatitis B surface antigen; anti-HBc, antibody to hepatitis B core (antigen); HBeAg, hepatitis B e antigen; ANA, antinuclear antibody; SMA, smooth-muscle antibody; P-ANCA, peripheral antineutrophil cytoplasmic antibody.

1	liver diseases, but liver biopsy plays an important role in the diagnosis 1993 of autoimmune hepatitis, primary biliary cirrhosis, nonalcoholic and alcoholic steatohepatitis, and Wilson’s disease (with a quantitative hepatic copper level in the last instance).

1	Laboratory Testing Diagnosis of liver disease is greatly aided by the availability of reliable and sensitive tests of liver injury and function. A typical battery of blood tests used for initial assessment of liver disease includes measurement of levels of serum alanine and aspartate aminotransferases, alkaline phosphatase, direct and total serum bilirubin and albumin, and prothrombin time. The pattern of abnormalities generally points to hepatocellular versus cholestatic liver disease and helps determine whether the disease is acute or chronic and whether cirrhosis and hepatic failure are present. On the basis of these results, further testing over time may be necessary. Other laboratory tests may be helpful, such as γ-glutamyl transpeptidase to define whether alkaline phosphatase elevations are due to liver disease; hepatitis serology to define the type of viral hepatitis; and autoimmune markers to diagnose primary biliary cirrhosis (antimitochondrial antibody), scleros ing

1	elevations are due to liver disease; hepatitis serology to define the type of viral hepatitis; and autoimmune markers to diagnose primary biliary cirrhosis (antimitochondrial antibody), scleros ing cholangitis (peripheral antineutrophil cytoplasmic antibody), and autoimmune hepatitis (antinuclear, smooth-muscle, and liver-kidney microsomal antibody). A simple delineation of laboratory abnormalities and common liver diseases is given in Table 357-3.

1	The use and interpretation of liver function tests are summarized in Chap. 358.

1	Diagnostic Imaging Great advances have been made in hepatobiliary imaging, although no method is adequately accurate in demonstrating underlying cirrhosis. Of the many modalities available for imaging the liver, ultrasound, CT, and MRI are the most commonly employed and are complementary to one another. In general, ultrasound and CT are highly sensitive for detecting biliary duct dilation and are the first-line options for investigating cases of suspected obstructive jaundice. All three modalities can detect a fatty liver, which appears bright on imaging studies. Modifications of CT and MRI can be used to quantify liver fat, and this information may ultimately be valuable in monitoring therapy in patients with fatty liver disease. Magnetic resonance cholangiopancreatography (MRCP) and endoscopic retrograde cholangiopancreatography (ERCP) are the procedures of choice for visualization of the biliary tree. MRCP offers several advantages over ERCP: there is no need for contrast media or

1	retrograde cholangiopancreatography (ERCP) are the procedures of choice for visualization of the biliary tree. MRCP offers several advantages over ERCP: there is no need for contrast media or ionizing radiation, images can be acquired faster, the procedure is less operator dependent, and it carries no risk of pancreatitis. MRCP is superior to ultrasound and CT for detecting choledocholithiasis but is less specific. MRCP is useful in the diagnosis of bile duct obstruction and congenital biliary abnormalities, but ERCP is more valuable in evaluating ampullary lesions and primary sclerosing cholangitis. ERCP permits biopsy, direct visualization of the ampulla and common bile duct, and intraductal ultrasonography. It also provides several therapeutic options in patients with obstructive jaundice, such as sphincterotomy, stone extraction, and placement of nasobiliary catheters and biliary stents. Doppler ultrasound and MRI are used to assess hepatic vasculature and hemodynamics and to

1	such as sphincterotomy, stone extraction, and placement of nasobiliary catheters and biliary stents. Doppler ultrasound and MRI are used to assess hepatic vasculature and hemodynamics and to monitor surgically or radiologically placed vascular shunts, including transjugular intrahepatic portosystemic shunts. Multidetector or spiral CT and MRI with contrast-enhancement are the procedures of choice for the identification and evaluation of hepatic masses, the staging of liver tumors, and preoperative assessment. With regard to mass lesions, the sensitivity of hepatic imaging continues to increase; unfortunately, specificity remains a problem, and often two and sometimes three studies are needed before a diagnosis can be reached. Recently, ultrasound transient elastography has been approved for the measurement of hepatic stiffness—providing an indirect assessment of cirrhosis; this technique can eliminate the need for liver biopsy if the only indication is the assessment of disease stage.

1	the measurement of hepatic stiffness—providing an indirect assessment of cirrhosis; this technique can eliminate the need for liver biopsy if the only indication is the assessment of disease stage. Magnetic resonance elastography is now undergoing evaluation for its ability to detect different degrees of hepatic fibrosis. Studies are ongoing to determine whether hepatic elastography is an appropriate means of monitoring fibrosis and disease progression. Finally, interventional radiologic techniques allow the biopsy of solitary lesions, the radiofrequency

1	Approach to the Patient with Liver Disease Suspected Liver Disease Abnormal liver tests Acute < 6 months Chronic > 6 months Diagnostic evaluation 1. IgM Anti-HAV 2. HBsAg 3. IgM Anti-HBc 4. Anti-HCV 5. ANA, SMA 6. Monospot, heterophile 7. Ceruloplasmin 8. Alcohol history 9. Drug history Diagnostic evaluation 1. AMA 2. Drug history 3. Ultrasound/MRI 4. MRCP/ERCP Liver biopsy in acute liver disease: Reserved for patients in whom the diagnosis remains unclear despite medical evaluation Liver biopsy in chronic liver disease: Often valuable for diagnosis as well as staging and grading liver disease Diagnostic evaluation 1. HBsAg 2. Anti-HCV 3. Fe saturation, ferritin 4. Ceruloplasmin 5. ˜1AT 6. ANA, SMA 7. Ultrasound 8. Alcohol history Diagnostic evaluation 1. Drug history 2. AMA 3. P-ANCA 4. Ultrasound 5. MRCP/ERCP Hepatitic: °°ALT Mixed: ˛ALT, ˛AlkP Cholestatic: °°AlkP, °°gGT, ˛ALT Hepatitic: °°ALT Mixed: ˛ALT, ˛AlkP Cholestatic: °°AlkP, °°gGT, ˛ALT

1	FIGURE 357-1 Algorithm for evaluation of abnormal liver tests. For patients with suspected liver disease, an appropriate approach to evaluation is initial routine liver testing—e.g., measurement of serum bilirubin, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AlkP). These results (sometimes complemented by testing of γ-glutamyl transpeptidase; gGT) will establish whether the pattern of abnormalities is hepatic, cholestatic, or mixed. In addition, the duration of symptoms or abnormalities will indicate whether the disease is acute or chronic. If the disease is acute and if history, laboratory tests, and imaging studies do not reveal a diagnosis, liver biopsy is appropriate to help establish the diagnosis. If the disease is chronic, liver biopsy can be helpful not only for diagnosis but also for grading of the activity and staging the progression of disease. This approach is generally applicable to patients without immune

1	liver biopsy can be helpful not only for diagnosis but also for grading of the activity and staging the progression of disease. This approach is generally applicable to patients without immune deficiency. In patients with HIV infection or recipients of bone marrow or solid organ transplants, the diagnostic evaluation should also include evaluation for opportunistic infections (e.g., with adenovirus, cytomegalovirus, Coccidioides, hepatitis E virus) as well as for vascular and immunologic conditions (veno-occlusive disease, graft-versus-host disease). HAV, hepatitis A virus; HCV, hepatitis C virus; HBsAg, hepatitis B surface antigen; anti-HBc, antibody to hepatitis B core (antigen); ANA, antinuclear antibody; SMA, smooth-muscle antibody; MRCP, magnetic resonance cholangiopancreatography; ERCP, endoscopic retrograde cholangiopancreatography; α1 AT, α1 antitrypsin; AMA; antimitochondrial antibody; P-ANCA, peripheral antineutrophil cytoplasmic antibody.

1	assessment of fibrosis. In the future, noninvasive means of assessing disease activity (batteries of blood tests) and fibrosis (elastography and fibrosis markers) may replace liver biopsy for the staging and grading of disease.

1	Grading refers to an assessment of the severity or activity of liver disease, whether acute or chronic; active or inactive; and mild, moderate, or severe. Liver biopsy is the most accurate means of assessing severity, particularly in chronic liver disease. Serum aminotransferase levels serve as convenient and noninvasive markers for disease activity but do not always reliably reflect disease severity. Thus, normal serum aminotransferase levels in patients with hepatitis B surface antigen in serum may indicate the inactive carrier state or may reflect mild chronic hepatitis B or hepatitis B with fluctuating disease activity. Serum testing for hepatitis B e antigen and hepatitis B virus DNA can help sort out these different patterns, but these markers can also fluctuate and change over time. Similarly, in chronic hepatitis C, serum aminotransferase levels can be normal despite moderate disease activity. Finally, in both alcoholic and nonalcoholic steatohepatitis, aminotransferase levels

1	Similarly, in chronic hepatitis C, serum aminotransferase levels can be normal despite moderate disease activity. Finally, in both alcoholic and nonalcoholic steatohepatitis, aminotransferase levels are quite unreliable in reflecting severity. In these conditions, liver biopsy is helpful in guiding management and identifying appropriate therapy, particularly if treatment is difficult, prolonged, and expensive, as is often the case in chronic viral hepatitis. Of the several well-verified numerical scales for grading activity in chronic liver disease, the most commonly used are the histology activity index and the Ishak histology scale.

1	Liver biopsy is also the most accurate means of assessing stage of disease as early or advanced, precirrhotic, and cirrhotic. Staging of disease pertains largely to chronic liver diseases in which progression to cirrhosis and end-stage disease can occur but may require years or decades. Clinical features, biochemical tests, and hepatic imaging studies are helpful in assessing stage but generally become abnormal only in the middle to late stages of cirrhosis. Noninvasive tests that suggest advanced fibrosis include mild elevations of bilirubin, prolongation of prothrombin time, slight decreases in serum albumin, and mild thrombocytopenia (which is often the first indication of worsening fibrosis). Combinations of blood test results have been used to create models for predicting advanced liver disease, but these models are not reliable enough to use on a regular basis and only separate advanced from early disease. Recently, elastography and noninvasive breath tests using 13C-labeled

1	liver disease, but these models are not reliable enough to use on a regular basis and only separate advanced from early disease. Recently, elastography and noninvasive breath tests using 13C-labeled compounds have been proposed as a means of detecting early stages of fibrosis and liver dysfunction, but their reliability and reproducibility remain to be proven. Thus, at present, mild to moderate stages of hepatic fibrosis are detectable only by liver biopsy.

1	ablation and chemoembolization of cancerous lesions, the insertion of In the assessment of stage, the degree of fibrosis is usually used as the drains into hepatic abscesses, the measurement of portal pressure, and quantitative measure. The amount of fibrosis is generally staged on a the creation of vascular shunts in patients with portal hypertension. scale of 0 to 4+ (Metavir scale) or 0 to 6+ (Ishak scale). The importance Which modality to use depends on factors such as availability, cost, of staging relates primarily to prognosis and to optimal management and experience of the radiologist with each technique. of complications. Patients with cirrhosis are candidates for screening and surveillance for esophageal varices and hepatocellular carcinoma. Liver Biopsy Liver biopsy remains the criterion standard in the evalu-Patients without advanced fibrosis need not undergo screening. ation of patients with liver disease, particularly chronic liver disease. Cirrhosis can also be staged

1	criterion standard in the evalu-Patients without advanced fibrosis need not undergo screening. ation of patients with liver disease, particularly chronic liver disease. Cirrhosis can also be staged clinically. A reliable staging system Liver biopsy is necessary for diagnosis in selected instances but is more is the modified Child-Pugh classification, with a scoring system of often useful for assessment of the severity (grade) and stage of liver 5–15: scores of 5 and 6 represent Child-Pugh class A (consistent damage, prediction of prognosis, and monitoring of the response to with “compensated cirrhosis”), scores of 7–9 represent class B, and treatment. The size of the liver biopsy sample is an important deter-scores of 10–15 represent class C (Table 357-4). This scoring system minant of reliability; a length of 1.5–2 cm is necessary for accurate was initially devised to stratify patients into risk groups before portal mg/dL <2.0 2.0–3.0 >3.0 g/dL >3.5 3.0–3.5 <3.0

1	INRa <1.7 1.7–2.3 >2.3 aInternational normalized ratio. Note: The Child-Pugh score is calculated by adding the scores for the five factors and can range from 5 to 15. The resulting Child-Pugh class can be A (a score of 5–6), B (7–9), or C (≥10). Decompensation indicates cirrhosis, with a Child-Pugh score of ≥7 (class B). This level has been the accepted criterion for listing a patient for liver transplantation.

1	decompressive surgery. The Child-Pugh score is a reasonably reliable predictor of survival in many liver diseases and predicts the likelihood of major complications of cirrhosis, such as bleeding from varices and spontaneous bacterial peritonitis. This classification scheme was used to assess prognosis in cirrhosis and to provide standard criteria for listing a patient as a candidate for liver transplantation (Child-Pugh class B). Recently, the Child-Pugh system has been replaced by the Model for End-Stage Liver Disease (MELD) system for the latter purpose. The MELD score is a prospectively derived system designed to predict the prognosis of patients with liver disease and portal hypertension. This score is calculated from three noninvasive variables: the prothrombin time expressed as the international normalized ratio (INR), the serum bilirubin level, and the serum creatinine concentration. (http://optn .transplant.hrsa.gov/resources/MeldPeldCalculator.asp?index=98).

1	The MELD system provides a more objective means of assessing disease severity and has less center-to-center variation than the Child-Pugh score as well as a wider range of values. MELD is currently used to establish priority listing for liver transplantation in the United States. A similar system, PELD (pediatric end-stage liver disease), is based on bilirubin, INR, serum albumin, age, and nutritional status and is used for children <12 years of age. Thus, liver biopsy is helpful not only in diagnosis but also in management of chronic liver disease and assessment of prognosis. Because liver biopsy is an invasive procedure and not without complications, it should be used only when it will contribute materially to decisions about management and therapy.

1	Specifics on the management of different forms of acute or chronic liver disease are supplied in subsequent chapters, but certain issues are applicable to any patient with liver disease. These issues include advice regarding alcohol use, medication use, vaccination, and surveillance for complications of liver disease. Alcohol should be used sparingly, if at all, by patients with liver disease. Abstinence from alcohol should be encouraged for all patients with alcohol-related liver disease, patients with cirrhosis, and patients receiving interferon-based therapy for hepatitis B or C. With regard to vaccinations, all patients with liver disease should receive hepatitis A vaccine, and those with risk factors should receive hepatitis B vaccine as well. Influenza and pneumococcal vaccination should also be encouraged, with adherence to the recommendations of the Centers for Disease Control and Prevention. Patients with liver disease should exercise caution in using any medications other

1	should also be encouraged, with adherence to the recommendations of the Centers for Disease Control and Prevention. Patients with liver disease should exercise caution in using any medications other than those that are most necessary. Drug-induced hepatotoxicity can mimic many forms of liver disease and can cause exacerbations of chronic hepatitis and cirrhosis; drugs should be suspected in any situation in which the cause of exacerbation is unknown. Finally, consideration should be given to surveillance for complications of 1995 chronic liver disease such as variceal hemorrhage and hepatocellular carcinoma. Cirrhosis warrants upper endoscopy to assess the presence of varices, and the patient should receive chronic therapy with beta blockers or should be offered endoscopic obliteration if large varices are found. Moreover, cirrhosis warrants screening and long-term surveillance for development of hepatocellular carcinoma. While the optimal regimen for such surveillance has not been

1	if large varices are found. Moreover, cirrhosis warrants screening and long-term surveillance for development of hepatocellular carcinoma. While the optimal regimen for such surveillance has not been established, an appropriate approach is ultrasound of the liver at 6to 12-month intervals.

1	Evaluation of liver function Daniel S. Pratt Several biochemical tests are useful in the evaluation and management of patients with hepatic dysfunction. These tests can be used to (1) detect the presence of liver disease, (2) distinguish among different types of liver disorders, (3) gauge the extent of known liver damage, and (4) follow the response to treatment. Liver tests have shortcomings. They can be normal in patients with serious liver disease and abnormal in patients with diseases that do not affect the liver. Liver tests rarely suggest a specific diagnosis; rather, they suggest a general category of liver disease, such as hepatocellular or cholestatic, which then further directs the evaluation.

1	The liver carries out thousands of biochemical functions, most of which cannot be easily measured by blood tests. Laboratory tests measure only a limited number of these functions. In fact, many tests, such as the aminotransferases or alkaline phosphatase, do not measure liver function at all. Rather, they detect liver cell damage or interference with bile flow. Thus, no one test enables the clinician to accurately assess the liver’s total functional capacity.

1	To increase both the sensitivity and the specificity of laboratory tests in the detection of liver disease, it is best to use them as a battery. Tests usually employed in clinical practice include the bilirubin, aminotransferases, alkaline phosphatase, albumin, and prothrombin time tests. When more than one of these tests provide abnormal findings or the findings are persistently abnormal on serial determinations, the probability of liver disease is high. When all test results are normal, the probability of missing occult liver disease is low. When evaluating patients with liver disorders, it is helpful to group these tests into general categories as outlined below.

1	TESTS BASED ON DETOXIFICATION AND EXCRETORY FUNCTIONS Serum Bilirubin (See also Chap. 58) Bilirubin, a breakdown product of the porphyrin ring of heme-containing proteins, is found in the blood in two fractions—conjugated and unconjugated. The unconjugated fraction, also termed the indirect fraction, is insoluble in water and is bound to albumin in the blood. The conjugated (direct) bilirubin fraction is water soluble and can therefore be excreted by the kidney. When measured by modifications of the original van den Bergh method, normal values of total serum bilirubin are reported between 1 and 1.5 mg/dL with 95% of a normal population falling between 0.2 and 0.9 mg/dL. If the direct-acting fraction is less than 15% of the total, the bilirubin can be considered to all be indirect. The most frequently reported upper limit of normal for conjugated bilirubin is 0.3 mg/dL.

1	Elevation of the unconjugated fraction of bilirubin is rarely due to liver disease. An isolated elevation of unconjugated bilirubin is seen primarily in hemolytic disorders and in a number of genetic conditions such as Crigler-Najjar and Gilbert’s syndromes (Chap. 58). Isolated unconjugated hyperbilirubinemia (bilirubin elevated but <15% direct) should prompt a workup for hemolysis (Fig. 358-1). In the absence of hemolysis, an isolated, unconjugated hyperbilirubinemia in an otherwise healthy patient can be attributed to Gilbert’s syndrome, and no further evaluation is required. Evaluation of Liver Function

1	Review drug list Hepatitis C antibody Hepatitis B surface Ag Iron, TIBC, ferritin ANA, SPEP Ceruloplasmin (if patient < 40) Ultrasound to look for fatty liver <15% Direct Gilbert’s syndrome Isolated elevation of the bilirubin Hepatocellular pattern (see Table 358-1) W/U negative W/U negative W/U negative Dilated ducts W/U positive Isolated elevation of the alkaline phosphatase Cholestatic pattern (see Table 358-1) Consider liver biopsy ERCP/Liver Bx CT/MRCP/ERCP Liver Bx Ducts not dilated Dilated ducts AMA positive AMA negative Alkaline phos. of liver origin Alkaline phos. of bone origin Bone Eval Ducts not dilated and/or AMA positive MRCP Evaluation for hemolysis Dubin-Johnson or Rotor syndrome Hemolysis Fractionate bilirubin >15% Direct Check AMA Review drugs Ultrasound Liver Tests Fractionate the alkaline phosphatase or check GGT or 5' nucleotidase to assess origin of alkaline phosphatase Ultrasound Review drug list Check AMA Liver biopsy R/O Celiac disease Consider other

1	Fractionate the alkaline phosphatase or check GGT or 5' nucleotidase to assess origin of alkaline phosphatase Ultrasound Review drug list Check AMA Liver biopsy R/O Celiac disease Consider other nonhepatic cause

1	FIGURE 358-1 Algorithm for the evaluation of chronically abnormal liver tests. AMA, antimitochondrial antibody; ANA, antinuclear antibody; Bx, biopsy; CT, computed tomography; ERCP, endoscopic retrograde cholangiopancreatography; GGT, γ glutamyl transpeptidase; MRCP, magnetic resonance cholangiopancreatography; R/O, rule out; SPEP, serum protein electrophoresis; TIBC, total iron-binding capacity; W/U, workup.

1	In contrast, conjugated hyperbilirubinemia almost always implies liver or biliary tract disease. The rate-limiting step in bilirubin metabolism is not conjugation of bilirubin, but rather the transport of conjugated bilirubin into the bile canaliculi. Thus, elevation of the conjugated fraction may be seen in any type of liver disease. In most liver diseases, both conjugated and unconjugated fractions of the bilirubin tend to be elevated. Except in the presence of a purely unconjugated hyperbilirubinemia, fractionation of the bilirubin is rarely helpful in determining the cause of jaundice.

1	Although the degree of elevation of the serum bilirubin has not been critically assessed as a prognostic marker, it is important in a number of conditions. In viral hepatitis, the higher the serum bilirubin, the greater is the hepatocellular damage. Total serum bilirubin correlates with poor outcomes in alcoholic hepatitis. It is also a critical component of the Model for End-Stage Liver Disease (MELD) score, a tool used to estimate survival of patients with end-stage liver disease and assess operative risk of patients with cirrhosis. An elevated total serum bilirubin in patients with drug-induced liver disease indicates more severe injury.

1	Urine Bilirubin Unconjugated bilirubin always binds to albumin in the serum and is not filtered by the kidney. Therefore, any bilirubin found in the urine is conjugated bilirubin; the presence of bilirubinuria implies the presence of liver disease. A urine dipstick test can theoretically give the same information as fractionation of the serum bilirubin. This test is almost 100% accurate. Phenothiazines may give a false-positive reading with the Ictotest tablet. In patients recovering from jaundice, the urine bilirubin clears prior to the serum bilirubin.

1	Blood Ammonia Ammonia is produced in the body during normal protein metabolism and by intestinal bacteria, primarily those in the colon. The liver plays a role in the detoxification of ammonia by converting it to urea, which is excreted by the kidneys. Striated muscle also plays a role in detoxification of ammonia, where it is combined with glutamic acid to form glutamine. Patients with advanced liver disease typically have significant muscle wasting, which likely contributes to hyperammonemia in these patients. Some physicians use the blood ammonia for detecting encephalopathy or for monitoring hepatic synthetic function, although its use for either of these indications has problems. There is very poor correlation between either the presence or the severity of acute encephalopathy and elevation of blood ammonia; it can be occasionally useful for identifying occult liver disease in patients with mental status changes. There is also a poor correlation of the blood serum ammonia and

1	elevation of blood ammonia; it can be occasionally useful for identifying occult liver disease in patients with mental status changes. There is also a poor correlation of the blood serum ammonia and hepatic function. The ammonia can be elevated in patients with severe portal hypertension and portal blood shunting around the liver even in the presence of normal or near-normal hepatic function. Elevated arterial ammonia levels have been shown to correlate with outcome in fulminant hepatic failure.

1	Serum Enzymes The liver contains thousands of enzymes, some of which are also present in the serum in very low concentrations. These enzymes have no known function in the serum and behave like other serum proteins. They are distributed in the plasma and in interstitial fluid and have characteristic half-lives, which are usually measured in days. Very little is known about the catabolism of serum enzymes, although they are probably cleared by cells in the reticuloendothelial system. The elevation of a given enzyme activity in the serum is thought to primarily reflect its increased rate of entrance into serum from damaged liver cells. Serum enzyme tests can be grouped into three categories: (1) enzymes whose elevation in serum reflects damage to hepatocytes, (2) enzymes whose elevation in serum reflects cholestasis, and (3) enzyme tests that do not fit precisely into either pattern.

1	enzymes tHat reflect DamaGe to Hepatocytes The aminotransferases (transaminases) are sensitive indicators of liver cell injury and are most helpful in recognizing acute hepatocellular diseases such as hepatitis. They include aspartate aminotransferase (AST) and alanine aminotransferase (ALT). AST is found in the liver, cardiac muscle, skeletal muscle, kidneys, brain, pancreas, lungs, leukocytes, and erythrocytes in decreasing order of concentration. ALT is found primarily in the liver and is therefore a more specific indicator of liver injury. The aminotransferases are normally present in the serum in low concentrations. These enzymes are released into the blood in greater amounts when there is damage to the liver cell membrane resulting in increased permeability. Liver cell necrosis is not required for the release of the aminotransferases, and there is a poor correlation between the degree of liver cell damage and the level of the aminotransferases. Thus, the absolute elevation of

1	not required for the release of the aminotransferases, and there is a poor correlation between the degree of liver cell damage and the level of the aminotransferases. Thus, the absolute elevation of the aminotransferases is of no prognostic significance in acute hepatocellular disorders.

1	The normal range for aminotransferases varies widely among laboratories, but generally ranges from 10–40 IU/L. The interlaboratory variation in normal range is due to technical reasons; no reference standards exist to establish upper limits of normal for ALT and AST. Some have recommended revisions of normal limits of the aminotransferases to adjust for sex and body mass index, but others have noted the potential costs and unclear benefits of implementing this change.

1	Any type of liver cell injury can cause modest elevations in the serum aminotransferases. Levels of up to 300 IU/L are nonspecific and may be found in any type of liver disorder. Minimal ALT elevations in asymptomatic blood donors rarely indicate severe liver disease; studies have shown that fatty liver disease is the most likely explanation. Striking elevations—i.e., aminotransferases >1000 IU/L—occur almost exclusively in disorders associated with extensive hepatocellular injury such as (1) viral hepatitis, (2) ischemic liver injury (prolonged hypo-tension or acute heart failure), or (3) toxinor drug-induced liver injury.

1	The pattern of the aminotransferase elevation can be helpful diagnostically. In most acute hepatocellular disorders, the ALT is higher than or equal to the AST. Whereas the AST:ALT ratio is typically <1 in patients with chronic viral hepatitis and nonalcoholic fatty liver disease, a number of groups have noted that as cirrhosis develops, this ratio rises to >1. An AST:ALT ratio >2:1 is suggestive, whereas a ratio >3:1 is highly suggestive, of alcoholic liver disease. The AST in alcoholic liver disease is rarely >300 IU/L, and the ALT is often normal. A low level of ALT in the serum is due to an alcohol-induced deficiency of pyridoxal phosphate.

1	The aminotransferases are usually not greatly elevated in obstructive jaundice. One notable exception occurs during the acute phase of biliary obstruction caused by the passage of a gallstone into the common bile duct. In this setting, the aminotransferases can briefly be in the 1000– 2000 IU/L range. However, aminotransferase levels decrease quickly, and the liver function tests rapidly evolve into those typical of cholestasis.

1	enzymes tHat reflect cHolestasis The activities of three enzymes— alkaline phosphatase, 5ʹ-nucleotidase, and γ-glutamyl transpeptidase (GGT)—are usually elevated in cholestasis. Alkaline phosphatase and 5ʹ-nucleotidase are found in or near the bile canalicular membrane of 1997 hepatocytes, whereas GGT is located in the endoplasmic reticulum and in bile duct epithelial cells. Reflecting its more diffuse localization in the liver, GGT elevation in serum is less specific for cholestasis than are elevations of alkaline phosphatase or 5ʹ-nucleotidase. Some have advocated the use of GGT to identify patients with occult alcohol use. Its lack of specificity makes its use in this setting questionable.

1	The normal serum alkaline phosphatase consists of many distinct isoenzymes found in the liver; bone; placenta; and, less commonly, small intestine. Patients over age 60 can have a mildly elevated alkaline phosphatase (1–1.5 times normal), whereas individuals with blood types O and B can have an elevation of the serum alkaline phosphatase after eating a fatty meal due to the influx of intestinal alkaline phosphatase into the blood. It is also nonpathologically elevated in children and adolescents undergoing rapid bone growth because of bone alkaline phosphatase, and late in normal pregnancies due to the influx of placental alkaline phosphatase.

1	Elevation of liver-derived alkaline phosphatase is not totally specific for cholestasis, and a less than threefold elevation can be seen in almost any type of liver disease. Alkaline phosphatase elevations greater than four times normal occur primarily in patients with cholestatic liver disorders, infiltrative liver diseases such as cancer and amyloidosis, and bone conditions characterized by rapid bone turnover (e.g., Paget’s disease). In bone diseases, the elevation is due to increased amounts of the bone isoenzymes. In liver diseases, the elevation is almost always due to increased amounts of the liver isoenzyme.

1	If an elevated serum alkaline phosphatase is the only abnormal finding in an apparently healthy person, or if the degree of elevation is higher than expected in the clinical setting, identification of the source of elevated isoenzymes is helpful (Fig. 358-1). This problem can be approached in two ways. First, and most precise, is the fractionation of the alkaline phosphatase by electrophoresis. The second, best substantiated, and most available approach involves the measurement of serum 5′-nucleotidase or GGT. These enzymes are rarely elevated in conditions other than liver disease.

1	In the absence of jaundice or elevated aminotransferases, an elevated alkaline phosphatase of liver origin often, but not always, suggests early cholestasis and, less often, hepatic infiltration by tumor or granulomata. Other conditions that cause isolated elevations of the alkaline phosphatase include Hodgkin’s disease, diabetes, hyperthyroidism, congestive heart failure, amyloidosis, and inflammatory bowel disease.

1	The level of serum alkaline phosphatase elevation is not helpful in distinguishing between intrahepatic and extrahepatic cholestasis. There is essentially no difference among the values found in obstructive jaundice due to cancer, common duct stone, sclerosing cholangitis, or bile duct stricture. Values are similarly increased in patients with intrahepatic cholestasis due to drug-induced hepatitis; primary biliary cirrhosis; rejection of transplanted livers; and, rarely, alcohol-induced steatohepatitis. Values are also greatly elevated in hepatobiliary disorders seen in patients with AIDS (e.g., AIDS cholangiopathy due to cytomegalovirus or cryptosporidial infection and tuberculosis with hepatic involvement).

1	TESTS THAT MEASURE BIOSYNTHETIC FUNCTION OF THE LIVER Serum Albumin Serum albumin is synthesized exclusively by hepatocytes. Serum albumin has a long half-life: 18–20 days, with ~4% degraded per day. Because of this slow turnover, the serum albumin is not a good indicator of acute or mild hepatic dysfunction; only minimal changes in the serum albumin are seen in acute liver conditions such as viral hepatitis, drug-related hepatotoxicity, and obstructive jaundice. In hepatitis, albumin levels <3 g/dL should raise the possibility of chronic liver disease. Hypoalbuminemia is more common in chronic liver disorders such as cirrhosis and usually reflects severe liver damage and decreased albumin synthesis. One exception is the patient with ascites in whom synthesis may be normal or even increased, but levels are low because of the increased volume of distribution. However, hypoalbuminemia is not specific for liver disease and may occur in protein malnutrition of any cause, as well as

1	but levels are low because of the increased volume of distribution. However, hypoalbuminemia is not specific for liver disease and may occur in protein malnutrition of any cause, as well as protein-losing enteropathies, nephrotic syndrome, and chronic infections that are

1	Evaluation of Liver Function 1998 associated with prolonged increases in levels of serum interleukin 1 and/or tumor necrosis factor, cytokines that inhibit albumin synthesis. Serum albumin should not be measured for screening in patients in whom there is no suspicion of liver disease. A general medical clinic study of consecutive patients in whom no indications were present for albumin measurement showed that although 12% of patients had abnormal test results, the finding was of clinical importance in only 0.4%.

1	Serum Globulins Serum globulins are a group of proteins made up of γ globulins (immunoglobulins) produced by B lymphocytes and α and β globulins produced primarily in hepatocytes. γ globulins are increased in chronic liver disease, such as chronic hepatitis and cirrhosis. In cirrhosis, the increased serum γ globulin concentration is due to the increased synthesis of antibodies, some of which are directed against intestinal bacteria. This occurs because the cirrhotic liver fails to clear bacterial antigens that normally reach the liver through the hepatic circulation. Increases in the concentration of specific isotypes of γ globulins are often helpful in the recognition of certain chronic liver diseases. Diffuse polyclonal increases in IgG levels are common in autoimmune hepatitis; increases >100% should alert the clinician to this possibility. Increases in the IgM levels are common in primary biliary cirrhosis, whereas increases in the IgA levels occur in alcoholic liver disease.

1	With the exception of factor VIII, which is produced by vascular endothelial cells, the blood clotting factors are made exclusively in hepatocytes. Their serum half-lives are much shorter than albumin, ranging from 6 h for factor VII to 5 days for fibrinogen. Because of their rapid turnover, measurement of the clotting factors is the single best acute measure of hepatic synthetic function and helpful in both diagnosis and assessing the prognosis of acute parenchymal liver disease. Useful for this purpose is the serum prothrombin time, which collectively measures factors II, V, VII, and X. Biosynthesis of factors II, VII, IX, and X depends on vitamin K. The international normalized ratio (INR) is used to express the degree of anticoagulation on warfarin therapy. The INR standardizes prothrombin time measurement according to the characteristics of the thromboplastin reagent used in a particular lab, which is expressed as an International Sensitivity Index (ISI); the ISI is then used in

1	time measurement according to the characteristics of the thromboplastin reagent used in a particular lab, which is expressed as an International Sensitivity Index (ISI); the ISI is then used in calculating the INR.

1	The prothrombin time may be elevated in hepatitis and cirrhosis as well as in disorders that lead to vitamin K deficiency such as obstructive jaundice or fat malabsorption of any kind. Marked prolongation of the prothrombin time, >5 s above control and not corrected by parenteral vitamin K administration, is a poor prognostic sign in acute viral hepatitis and other acute and chronic liver diseases. The INR, along with the total serum bilirubin and creatinine, are components of the MELD score, which is used as a measure of hepatic decompensation and to allocate organs for liver transplantation. Although tests may direct the physician to a category of liver disease, additional radiologic testing and procedures are often necessary to make the proper diagnosis, as shown in Fig. 358-1. The most commonly used ancillary tests are reviewed here, as are the noninvasive tests available for assessing hepatic fibrosis.

1	Percutaneous Liver Biopsy Percutaneous biopsy of the liver is a safe procedure that can be easily performed at the bedside with local anesthesia and ultrasound guidance. Liver biopsy is of proven value in the following situations: (1) hepatocellular disease of uncertain cause, (2) prolonged hepatitis with the possibility of autoimmune hepatitis, (3) unexplained hepatomegaly, (4) unexplained splenomegaly, (5) hepatic filling defects by radiologic imaging, (6) fever of unknown origin, (7) and staging of malignant lymphoma. Liver biopsy is most accurate in disorders causing diffuse changes throughout the liver and is subject to sampling error in focal infiltrative disorders such as hepatic metastases. Liver biopsy should not be the initial procedure in the diagnosis of cholestasis. The biliary tree should first be assessed for signs of obstruction. Contraindications to performing a percutaneous liver biopsy include significant ascites and prolonged INR. Under these circumstances, the

1	tree should first be assessed for signs of obstruction. Contraindications to performing a percutaneous liver biopsy include significant ascites and prolonged INR. Under these circumstances, the biopsy can be performed via the transjugular approach.

1	Type of Disorder Bilirubin Aminotransferases Alkaline Phosphatase Albumin Prothrombin Time Acute hepatocellular necrosis (viral and drug hepatitis, hepatotoxins, acute heart failure) Alcoholic hepatitis, cirrhosis Infiltrative diseases (tumor, granulomata); partial bile duct obstruction Normal to 86 μmol/L (5 mg/dL) 85% due to indirect fractions Elevated, often >500 IU, ALT > AST Elevated, but usually <300 IU AST:ALT >2 suggests alcoholic hepatitis or cirrhosis Normal to moderate elevation Normal to slight elevation Normal to <3× normal elevation Normal to <3× normal elevation Normal to <3× normal elevation Elevated, often >4× normal elevation Elevated, often >4× normal elevation Fractionate, or confirm liver origin with 5’nucleotidase or γ glutamyl transpeptidase Normal Usually normal. If >5× above control and not corrected by parenteral vitamin K, suggests poor prognosis Fails to correct with parenteral vitamin K Fails to correct with parenteral vitamin K

1	Normal Usually normal. If >5× above control and not corrected by parenteral vitamin K, suggests poor prognosis Fails to correct with parenteral vitamin K Fails to correct with parenteral vitamin K Normal, unless chronic Normal If prolonged, will correct with parenteral vitamin K Noninvasive Tests to Detect Hepatic Fibrosis Although liver biopsy 1999 is the standard for the assessment of hepatic fibrosis, noninva-The Hyperbilirubinemiassive measures of hepatic fibrosis have been developed and show

1	Allan W. Wolkoff promise. These measures include multiparameter tests aimed at detecting and staging the degree of hepatic fibrosis and imaging techniques. FibroTest (marketed as FibroSure in the United States) is the best evaluated of the multiparameter blood tests. The test incorporates haptoglobin, bilirubin, GGT, apolipoprotein A-I, and α2-macroglobulin and has been found to have high positive and negative predictive values for diagnosing advanced fibrosis in patients with chronic hepatitis C, chronic hepatitis B, and alcoholic liver disease and patients taking methotrexate for psoriasis. Transient elastography (TE), marketed as FibroScan, and magnetic resonance elastography (MRE) both have gained U.S. Food and Drug Administration approval for use in the management of patients with liver disease. TE uses ultrasound waves to measure hepatic stiffness noninvasively. TE has been shown to be accurate for identifying advanced fibrosis in patients with chronic hepatitis C, primary

1	liver disease. TE uses ultrasound waves to measure hepatic stiffness noninvasively. TE has been shown to be accurate for identifying advanced fibrosis in patients with chronic hepatitis C, primary biliary cirrhosis, hemochromatosis, nonalcoholic fatty liver disease, and recurrent chronic hepatitis after liver transplantation. MRE has been found to be superior to TE for staging liver fibrosis in patients with a variety of chronic liver diseases, but requires access to a magnetic resonance imaging scanner.

1	Ultrasonography Ultrasonography is the first diagnostic test to use in patients whose liver tests suggest cholestasis, to look for the presence of a dilated intrahepatic or extrahepatic biliary tree or to identify gallstones. In addition, it shows space-occupying lesions within the liver, enables the clinician to distinguish between cystic and solid masses, and helps direct percutaneous biopsies. Ultrasound with Doppler imaging can detect the patency of the portal vein, hepatic artery, and hepatic veins and determine the direction of blood flow. This is the first test ordered in patients suspected of having Budd-Chiari syndrome.

1	As previously noted, the best way to increase the sensitivity and specificity of laboratory tests in the detection of liver disease is to employ a battery of tests that includes the aminotransferases, alkaline phosphatase, bilirubin, albumin, and prothrombin time along with the judicious use of the other tests described in this chapter. Table 358-1 shows how patterns of liver tests can lead the clinician to a category of disease that will direct further evaluation. However, it is important to remember that no single set of liver tests will necessarily provide a diagnosis. It is often necessary to repeat these tests on several occasions over days to weeks for a diagnostic pattern to emerge. Figure 358-1 is an algorithm for the evaluation of chronically abnormal liver tests.

1	The tests and principles presented in this chapter are appli cable worldwide. The causes of liver test abnormalities vary according to region. In developing nations, infectious diseases are more commonly the etiology of abnormal serum liver tests than in developed nations. This chapter represents a revised version of a chapter in previous editions of Harrison’s in which Marshall M. Kaplan was a co-author. The details of bilirubin metabolism are presented in Chap. 58. However, the hyperbilirubinemias are best understood in terms of perturbations of specific aspects of bilirubin metabolism and transport, and these will be briefly reviewed here as depicted in Fig. 359-1.

1	Bilirubin is the end product of heme degradation. Some 70–90% of bilirubin is derived from degradation of the hemoglobin of senescent red blood cells. Bilirubin produced in the periphery is transported to the liver within the plasma, where, due to its insolubility in aqueous solutions, it is tightly bound to albumin. Under normal circumstances, bilirubin is removed from the circulation rapidly and efficiently by hepatocytes. Transfer of bilirubin from blood to bile involves four distinct but interrelated steps (Fig. 359-1). 1. Hepatocellular uptake: Uptake of bilirubin by the hepatocyte has carrier-mediated kinetics. Although a number of candidate bilirubin transporters have been proposed, the actual transporter remains elusive. 2. Intracellular binding: Within the hepatocyte, bilirubin is kept in solution by binding as a nonsubstrate ligand to several of the glutathione-S-transferases, formerly called ligandins. 3.

1	2. Intracellular binding: Within the hepatocyte, bilirubin is kept in solution by binding as a nonsubstrate ligand to several of the glutathione-S-transferases, formerly called ligandins. 3. Conjugation: Bilirubin is conjugated with one or two glucuronic acid moieties by a specific UDP-glucuronosyltransferase to form bilirubin mono-and diglucuronide, respectively. Conjugation disrupts the internal hydrogen bonding that limits aqueous solubility of bilirubin, and the resulting glucuronide conjugates are highly soluble in water. Conjugation is obligatory for excretion of bilirubin across the bile canalicular membrane into bile. The UDP-glucuronosyltransferases have been classified into gene families based on the degree of homology among the mRNAs for the various isoforms. Those that conjugate bilirubin and certain

1	FIGURE 359-1 Hepatocellular bilirubin transport. Albumin-bound bilirubin in sinusoidal blood passes through endothelial cell fenestrae to reach the hepatocyte surface, entering the cell by both facilitated and simple diffusional processes. Within the cell, it is bound to glutathione-S-transferases and conjugated by bilirubin-UDP-glucuronosyltransferase (UGT1A1) to monoand diglucuronides, which are actively transported across the canalicular membrane into the bile. In addition to this direct excretion of bilirubin glucuronides, a portion are transported into the portal circulation by MRP3 and subjected to reuptake into the hepatocyte by OATP1B1 and OATP1B3. ALB, albumin; BDG, bilirubin diglucuronide; BMG, bilirubin monoglucuronide; BT, proposed bilirubin transporter; GST, glutathione-S-transferase; MRP2 and MRP3, multidrug resistance–associated proteins 2 and 3; OATP1B1 and OATP1B3, organic anion transport proteins 1B1 and 1B3; UCB, unconjugated bilirubin; UGT1A1,

1	glutathione-S-transferase; MRP2 and MRP3, multidrug resistance–associated proteins 2 and 3; OATP1B1 and OATP1B3, organic anion transport proteins 1B1 and 1B3; UCB, unconjugated bilirubin; UGT1A1, bilirubin-UDP-glucuronosyltransferase.

1	The Hyperbilirubinemias 3˜ hyperbilirubinemia (e.g., Crigler-Najjar syndrome, type I [CN-I]). Unconjugated Variable (Substrate Specific) First Exons Common Exons bilirubin that reaches the gut is partly reabsorbed, amplifying any underlying hyperbilirubinemia. Recent reports suggest that oral administration of calcium phosphate with or without the lipase inhibitor orlistat may be an efficient means to interrupt bilirubin enterohe patic cycling to reduce serum bilirubin levels in this situation. Although orli- TATA Box stat administration for 4–6 weeks to 16 patients with Crigler-Najjar syndrome

1	TATA Box stat administration for 4–6 weeks to 16 patients with Crigler-Najjar syndrome FIGURE 359-2 Structural organization of the human UGT1 gene complex. This large complex was associated with a 10–20% decrease on chromosome 2 contains at least 13 substrate-specific first exons (A1, A2, etc.). Since four of these in serum bilirubin in 7 patients, the cost are pseudogenes, nine UGT1 isoforms with differing substrate specificities are expressed. Each exon and side effects (i.e., diarrhea) may obvi 1 has its own promoter and encodes the amino-terminal substrate-specific ∼286 amino acids of the ate the small benefit achievable with this various UGT1-encoded isoforms, and common exons 2–5 that encode the 245 carboxyl-terminal treatment.

1	amino acids common to all of the isoforms. mRNAs for specific isoforms are assembled by splicing a particular first exon such as the bilirubin-specific exon A1 to exons 2 to 5. The resulting message Renal Excretion of Bilirubin Conjugates encodes a complete enzyme, in this particular case bilirubin-UDP-glucuronosyltransferase (UGT1A1). Unconjugated bilirubin is not excreted Mutations in a first exon affect only a single isoform. Those in exons 2–5 affect all enzymes encoded in urine, as it is too tightly bound to by the UGT1 complex. albumin for effective glomerular filtra tion and there is no tubular mechanism for its renal secretion. In contrast, the bilirubin conjugates are readily filtered other substrates have been designated the UGT1 family. These at the glomerulus and can appear in urine in disorders characterizedare expressed from a single gene complex by alternative promoter by increased bilirubin conjugates in the circulation.usage. This gene complex contains multiple

1	in urine in disorders characterizedare expressed from a single gene complex by alternative promoter by increased bilirubin conjugates in the circulation.usage. This gene complex contains multiple substrate-specific first exons, designated A1, A2, etc. (Fig. 359-2), each with its own DISORDERS OF BILIRUBIN METABOLISM LEADING promoter and each encoding the amino-terminal half of a specific TO UNCONJUGATED HYPERBILIRUBINEMIA isoform. In addition, there are four common exons (exons 2–5) that encode the shared carboxyl-terminal half of all of the UGT1 isoforms. The various first exons encode the specific aglycone Hemolysis Increased destruction of erythrocytes leads to increased substrate binding sites for each isoform, while the shared exons bilirubin turnover and unconjugated hyperbilirubinemia; the hyper-encode the binding site for the sugar donor, UDP-glucuronic bilirubinemia is usually modest in the presence of normal liver funcacid, and the transmembrane domain. Exon A1 and the four

1	the hyper-encode the binding site for the sugar donor, UDP-glucuronic bilirubinemia is usually modest in the presence of normal liver funcacid, and the transmembrane domain. Exon A1 and the four tion. In particular, the bone marrow is only capable of a sustained common exons, collectively designated the UGT1A1 gene (Fig. eightfold increase in erythrocyte production in response to a hemo359-2), encode the physiologically critical enzyme bilirubin-UDP-lytic stress. Therefore, hemolysis alone cannot result in a sustained glucuronosyltransferase (UGT1A1). A functional corollary of the hyperbilirubinemia of more than ∼68 μmol/L (4 mg/dL). Higher values organization of the UGT1 gene is that a mutation in one of the imply concomitant hepatic dysfunction. When hemolysis is the only first exons will affect only a single enzyme isoform. By contrast, a abnormality in an otherwise healthy individual, the result is a purely mutation in exons 2–5 will alter all isoforms encoded by the UGT1

1	exons will affect only a single enzyme isoform. By contrast, a abnormality in an otherwise healthy individual, the result is a purely mutation in exons 2–5 will alter all isoforms encoded by the UGT1 unconjugated hyperbilirubinemia, with the direct-reacting fraction as gene complex. measured in a typical clinical laboratory being ≤15% of the total serum 4. Biliary excretion: It has been thought until recently that biliru-bilirubin. In the presence of systemic disease, which may include a bin monoand diglucuronides are excreted directly across the degree of hepatic dysfunction, hemolysis may produce a component canalicular plasma membrane into the bile canaliculus by an ATP-of conjugated hyperbilirubinemia in addition to an elevated uncondependent transport process mediated by a canalicular membrane jugated bilirubin concentration. Prolonged hemolysis may lead to the protein called multidrug resistance–associated protein 2 (MRP2). precipitation of bilirubin salts within the gallbladder

1	membrane jugated bilirubin concentration. Prolonged hemolysis may lead to the protein called multidrug resistance–associated protein 2 (MRP2). precipitation of bilirubin salts within the gallbladder or biliary tree, Mutations of MRP2 result in the Dubin-Johnson syndrome (see resulting in the formation of gallstones in which bilirubin, rather than below). However, studies in patients with Rotor syndrome (see cholesterol, is the major component. Such pigment stones may lead to below) indicate that after formation, a portion of the glucuro-acute or chronic cholecystitis, biliary obstruction, or any other biliary nides are transported into the portal circulation by a sinusoidal tract consequence of calculous disease. membrane protein called multidrug resistance–associated protein

1	Ineffective Erythropoiesis During erythroid maturation, small amounts 3 (MRP3) and subjected to reuptake into the hepatocyte by the of hemoglobin may be lost at the time of nuclear extrusion, and a fracsinusoidal membrane uptake transporters organic anion transport tion of developing erythroid cells is destroyed within the marrow. protein 1B1 (OATP1B1) and OATP1B3.

1	protein 1B1 (OATP1B1) and OATP1B3. These processes normally account for a small proportion of bilirubin that is produced. In various disorders, including thalassemia major, megaloblastic anemias due to folate or vitamin B12 deficiency, congeni- tal erythropoietic porphyria, lead poisoning, and various congenitalBilirubin in the Gut Following secretion into bile, conjugated bilirubin and acquired dyserythropoietic anemias, the fraction of total bilirubinreaches the duodenum and passes down the gastrointestinal tract with-production derived from ineffective erythropoiesis is increased, reach-out reabsorption by the intestinal mucosa. An appreciable fraction is ing as much as 70% of the total. This may be sufficient to produceconverted by bacterial metabolism in the gut to the water-soluble col-modest degrees of unconjugated hyperbilirubinemia.

1	orless compound urobilinogen. Urobilinogen undergoes enterohepatic cycling. Urobilinogen not taken up by the liver reaches the systemic Miscellaneous Degradation of the hemoglobin of extravascular colleccirculation, from which some is cleared by the kidneys. Unconjugated tions of erythrocytes, such as those seen in massive tissue infarctions bilirubin ordinarily does not reach the gut except in neonates or, by ill-or large hematomas, may lead transiently to unconjugated hyperbilidefined alternative pathways, in the presence of severe unconjugated rubinemia.

1	DECREASED HEPATIC BILIRUBIN CLEARANCE Decreased Hepatic Uptake Decreased hepatic bilirubin uptake is believed to contribute to the unconjugated hyperbilirubinemia of Gilbert syndrome (GS), although the molecular basis for this finding remains unclear (see below). Several drugs, including flavaspidic acid, novobiocin, and rifampin, as well as various cholecystographic contrast agents, have been reported to inhibit bilirubin uptake. The resulting unconjugated hyperbilirubinemia resolves with cessation of the medication.

1	Impaired Conjugation • pHysioloGic neonatal JaunDice Bilirubin produced by the fetus is cleared by the placenta and eliminated by the maternal liver. Immediately after birth, the neonatal liver must assume responsibility for bilirubin clearance and excretion. However, many hepatic physiologic processes are incompletely developed at birth. Levels of UGT1A1 are low, and alternative excretory pathways allow passage of unconjugated bilirubin into the gut. Since the intestinal flora that convert bilirubin to urobilinogen are also undeveloped, an enterohepatic circulation of unconjugated bilirubin ensues. As a consequence, most neonates develop mild unconjugated hyperbilirubinemia between days 2 and 5 after birth. Peak levels are typically <85–170 μmol/L (5–10 mg/dL) and decline to normal adult concentrations within 2 weeks, as mechanisms required for bilirubin disposition mature. Prematurity, often associated with more profound immaturity of hepatic function and hemolysis, can result in

1	concentrations within 2 weeks, as mechanisms required for bilirubin disposition mature. Prematurity, often associated with more profound immaturity of hepatic function and hemolysis, can result in higher levels of unconjugated hyperbilirubinemia. A rapidly rising unconjugated bilirubin concentration, or absolute levels >340 μmol/L (20 mg/dL), puts the infant at risk for bilirubin encephalopathy, or kernicterus. Under these circumstances, bilirubin crosses an immature blood-brain barrier and precipitates in the basal ganglia and other areas of the brain. The consequences range from appreciable neurologic deficits to death. Treatment options include phototherapy, which converts bilirubin into water-soluble photoisomers that are excreted directly into bile, and exchange transfusion. The canalicular mechanisms responsible for bilirubin excretion are also immature at birth, and their maturation may lag behind that of UGT1A1; this can lead to transient conjugated neonatal

1	The canalicular mechanisms responsible for bilirubin excretion are also immature at birth, and their maturation may lag behind that of UGT1A1; this can lead to transient conjugated neonatal hyperbilirubinemia, especially in infants with hemolysis.

1	acquireD conJuGation Defects A modest reduction in bilirubin conjugating capacity may be observed in advanced hepatitis or cirrhosis. However, in this setting, conjugation is better preserved than other aspects of bilirubin disposition, such as canalicular excretion. Various drugs, including pregnanediol, novobiocin, chloramphenicol, and gentamicin, may produce unconjugated hyperbilirubinemia by inhibiting UGT1A1 activity. Bilirubin conjugation may be inhibited by certain fatty acids that are present in breast milk but not serum of mothers whose infants have excessive neonatal hyperbilirubinemia (breast milk jaundice). Alternatively, there may be increased entero-2001 hepatic circulation of bilirubin in these infants. A recent study has correlated epidermal growth factor (EGF) content of breast milk with elevated bilirubin levels in these infants; however, a cause-and-effect relationship remains to be established. The pathogenesis of breast milk jaundice appears to differ from that of

1	milk with elevated bilirubin levels in these infants; however, a cause-and-effect relationship remains to be established. The pathogenesis of breast milk jaundice appears to differ from that of transient familial neonatal hyperbilirubinemia (Lucey-Driscoll syndrome), in which there is a UGT1A1 inhibitor in maternal serum.

1	Three familial disorders characterized by differing degrees of unconjugated hyperbilirubinemia have long been recognized. The defining clinical features of each are described below (Table 359-1). While these disorders have been recognized for decades to reflect differing degrees of deficiency in the ability to conjugate bilirubin, recent advances in the molecular biology of the UGT1 gene complex have elucidated their interrelationships and clarified previously puzzling features.

1	Crigler-Najjar Syndrome, Type I CN-I is characterized by striking uncon jugated hyperbilirubinemia of about 340–765 μmol/L (20–45 mg/dL) that appears in the neonatal period and persists for life. Other conventional hepatic biochemical tests such as serum aminotransferases and alkaline phosphatase are normal, and there is no evidence of hemolysis. Hepatic histology is also essentially normal except for the occasional presence of bile plugs within canaliculi. Bilirubin glucuronides are virtually absent from the bile, and there is no detectable constitutive expression of UGT1A1 activity in hepatic tissue. Neither UGT1A1 activity nor the serum bilirubin concentration responds to administration of phenobarbital or other enzyme inducers. In the absence of conjugation, unconjugated bilirubin accumulates in plasma, from which it is eliminated very slowly by alternative pathways that include direct passage into the bile and small intestine. These account for the small amounts of urobilinogen

1	in plasma, from which it is eliminated very slowly by alternative pathways that include direct passage into the bile and small intestine. These account for the small amounts of urobilinogen found in feces. No bilirubin is found in the urine. First described in 1952, the disorder is rare (estimated prevalence, 0.6–1.0 per million). Many patients are from geographically or socially isolated communities in which consanguinity is common, and pedigree analyses show an autosomal recessive pattern of inheritance. The majority of patients (type IA) exhibit defects in the glucuronide conjugation of a spectrum of substrates in addition to bilirubin, including various drugs and other xenobiotics. These individuals have mutations in one of the common exons (2–5) of the UGT1 gene (Fig. 359-2). In a smaller subset (type IB), the defect is limited largely to bilirubin conjugation, and the causative mutation is in the bilirubin-specific exon A1. Estrogen glucuronidation is mediated by UGT1A1 and is

1	subset (type IB), the defect is limited largely to bilirubin conjugation, and the causative mutation is in the bilirubin-specific exon A1. Estrogen glucuronidation is mediated by UGT1A1 and is defective in all CN-I patients. More than 30 different genetic lesions of UGT1A1 responsible for CN-I have been identified,

1	The Hyperbilirubinemias 2002 including deletions, insertions, alterations in intron splice donor and acceptor sites, exon skipping, and point mutations that introduce premature stop codons or alter critical amino acids. Their common feature is that they all encode proteins with absent or, at most, traces of bilirubin-UDP-glucuronosyltransferase enzymatic activity. Prior to the availability of phototherapy, most patients with CN-I died of bilirubin encephalopathy (kernicterus) in infancy or early childhood. A few lived as long as early adult life without overt neurologic damage, although more subtle testing usually indicated mild but progressive brain damage. In the absence of liver transplantation, death eventually supervened from late-onset bilirubin encephalopathy, which often followed a nonspecific febrile illness. Although isolated hepatocyte transplantation has been used in a small number of cases of CN-I, early liver transplantation (Chap. 368) remains the best hope to prevent

1	a nonspecific febrile illness. Although isolated hepatocyte transplantation has been used in a small number of cases of CN-I, early liver transplantation (Chap. 368) remains the best hope to prevent brain injury and death.

1	Crigler-Najjar Syndrome, Type II (CN-II) This condition was recognized as a distinct entity in 1962 and is characterized by marked unconjugated hyperbilirubinemia in the absence of abnormalities of other conventional hepatic biochemical tests, hepatic histology, or hemolysis. It differs from CN-I in several specific ways (Table 359-1): (1) Although there is considerable overlap, average bilirubin concentrations are lower in CN-II; (2) accordingly, CN-II is only infrequently associated with kernicterus; (3) bile is deeply colored, and bilirubin glucuronides are present, with a striking, characteristic increase in the proportion of monoglucuronides; (4) UGT1A1 in liver is usually present at reduced levels (typically ≤10% of normal) but may be undetectable by older, less sensitive assays; and (5) while typically detected in infancy, hyperbilirubinemia was not recognized in some cases until later in life and, in one instance, at age 34. As with CN-I, most CN-II cases exhibit abnormalities

1	(5) while typically detected in infancy, hyperbilirubinemia was not recognized in some cases until later in life and, in one instance, at age 34. As with CN-I, most CN-II cases exhibit abnormalities in the conjugation of other compounds, such as salicylamide and menthol, but in some instances, the defect appears limited to bilirubin. Reduction of serum bilirubin concentrations by >25% in response to enzyme inducers such as phenobarbital distinguishes CN-II from CN-I, although this response may not be elicited in early infancy and often is not accompanied by measurable UGT1A1 induction. Bilirubin concentrations during phenobarbital administration do not return to normal but are typically in the range of 51–86 μmol/L (3–5 mg/dL). Although the incidence of kernicterus in CN-II is low, instances have occurred, not only in infants but also in adolescents and adults, often in the setting of an intercurrent illness, fasting, or another factor that temporarily raises the serum bilirubin

1	have occurred, not only in infants but also in adolescents and adults, often in the setting of an intercurrent illness, fasting, or another factor that temporarily raises the serum bilirubin concentration above baseline and reduces serum albumin levels. For this reason, phenobarbital therapy is widely recommended, a single bedtime dose often sufficing to maintain clinically safe serum bilirubin concentrations.

1	Over 77 different mutations in the UGT1 gene have been identified as causing CN-I or CN-II. It was found that missense mutations are more common in CN-II patients, as would be expected in this less severe phenotype. Their common feature is that they encode for a bilirubin-UDP-glucuronosyltransferase with markedly reduced, but detectable, enzymatic activity. The spectrum of residual enzyme activity explains the spectrum of phenotypic severity of the resulting hyperbilirubinemia. Molecular analysis has established that a large majority of CN-II patients are either homozygotes or compound heterozygotes for CN-II mutations and that individuals carrying one mutated and one entirely normal allele have normal bilirubin concentrations.

1	Gilbert Syndrome (GS) This syndrome is characterized by mild unconjugated hyperbilirubinemia, normal values for standard hepatic biochemical tests, and normal hepatic histology other than a modest increase of lipofuscin pigment in some patients. Serum bilirubin concentrations are most often <51 μmol/L (<3 mg/dL), although both higher and lower values are frequent. The clinical spectrum of hyperbilirubinemia fades into that of CN-II at serum bilirubin concentrations of 86–136 μmol/L (5–8 mg/dL). At the other end of the scale, the distinction between mild cases of GS and a normal state is often blurred. Bilirubin concentrations may fluctuate substantially in any given individual, and at least 25% of patients will exhibit temporarily normal values during prolonged follow-up. More elevated values are associated with stress, fatigue, alcohol use, reduced caloric intake, and intercurrent illness, while increased caloric intake or administration of enzyme-inducing agents produces lower

1	values are associated with stress, fatigue, alcohol use, reduced caloric intake, and intercurrent illness, while increased caloric intake or administration of enzyme-inducing agents produces lower bilirubin levels. GS is most often diagnosed at or shortly after puberty or in adult life during routine examinations that include multichannel biochemical analyses. UGT1A1 activity is typically reduced to 10–35% of normal, and bile pigments exhibit a characteristic increase in bilirubin monoglucuronides. Studies of radiobilirubin kinetics indicate that hepatic bilirubin clearance is reduced to an average of one-third of normal. Administration of phenobarbital normalizes both the serum bilirubin concentration and hepatic bilirubin clearance; however, failure of UGT1A1 activity to improve in many such instances suggests the possible coexistence of an additional defect. Compartmental analysis of bilirubin kinetic data suggests that GS patients have a defect in bilirubin uptake as well as in

1	such instances suggests the possible coexistence of an additional defect. Compartmental analysis of bilirubin kinetic data suggests that GS patients have a defect in bilirubin uptake as well as in conjugation. Defect(s) in the hepatic uptake of other organic anions that at least partially share an uptake mechanism with bilirubin, such as sulfobromophthalein and indocyanine green (ICG), are observed in a minority of patients. The metabolism and transport of bile acids that do not utilize the bilirubin uptake mechanism are normal. The magnitude of changes in the serum bilirubin concentration induced by provocation tests such as 48 hours of fasting or the IV administration of nicotinic acid have been reported to be of help in separating GS patients from normal individuals. Other studies dispute this assertion. Moreover, on theoretical grounds, the results of such studies should provide no more information than simple measurements of the baseline serum bilirubin concentration. Family

1	dispute this assertion. Moreover, on theoretical grounds, the results of such studies should provide no more information than simple measurements of the baseline serum bilirubin concentration. Family studies indicate that GS and hereditary hemolytic anemias such as hereditary spherocytosis, glucose-6-phosphate dehydrogenase deficiency, and β-thalassemia trait sort independently. Reports of hemolysis in up to 50% of GS patients are believed to reflect better case finding, since patients with both GS and hemolysis have higher bilirubin concentrations, and are more likely to be jaundiced, than patients with either defect alone.

1	GS is common, with many series placing its prevalence at ≥8%. Males predominate over females by reported ratios ranging from 1.5:1 to >7:1. However, these ratios may have a large artifactual component since normal males have higher mean bilirubin levels than normal females, but the diagnosis of GS is often based on comparison to normal ranges established in men. The high prevalence of GS in the general population may explain the reported frequency of mild unconjugated hyperbilirubinemia in liver transplant recipients. The disposition of most xenobiotics metabolized by glucuronidation appears to be normal in GS, as is oxidative drug metabolism in the majority of reported studies. The principal exception is the metabolism of the anti-tumor agent irinotecan (CPT-11), whose active metabolite (SN-38) is glucuronidated specifically by bilirubin-UDP-glucuronosyltransferase. Administration of CPT-11 to patients with GS has resulted in several toxicities, including intractable diarrhea and

1	(SN-38) is glucuronidated specifically by bilirubin-UDP-glucuronosyltransferase. Administration of CPT-11 to patients with GS has resulted in several toxicities, including intractable diarrhea and myelosuppression. Some reports also suggest abnormal disposition of menthol, estradiol benzoate, acetaminophen, tolbutamide, and rifamycin SV. Although some of these studies have been disputed, and there have been no reports of clinical complications from use of these agents in GS, prudence should be exercised in prescribing them, or any agents metabolized primarily by glucuronidation, in this condition. It should also be noted that the HIV protease inhibitors indinavir and atazanavir (Chap. 226) can inhibit UGT1A1, resulting in hyperbilirubinemia that is most pronounced in patients with preexisting GS.

1	Most older pedigree studies of GS were consistent with autosomal dominant inheritance with variable expressivity. However, studies of the UGT1 gene in GS have indicated a variety of molecular genetic bases for the phenotypic picture and several different patterns of inheritance. Studies in Europe and the United States found that nearly all patients had normal coding regions for UGT1A1 but were homozygous for the insertion of an extra TA (i.e., A[TA]7TAA rather than A[TA]6TAA) in the promoter region of the first exon. This appeared to be necessary, but not sufficient, for clinically expressed GS, since 15% of normal controls were also homozygous for this variant. While normal by standard criteria, these individuals had somewhat higher bilirubin concentrations than the rest of the controls studied. Heterozygotes for this abnormality had bilirubin concentrations identical to those homozygous for the normal A[TA]6TAA allele. The prevalence of the A[TA]7TAA allele in a general Western

1	studied. Heterozygotes for this abnormality had bilirubin concentrations identical to those homozygous for the normal A[TA]6TAA allele. The prevalence of the A[TA]7TAA allele in a general Western population is 30%, in which case 9% would be homozygotes. This is slightly higher than the prevalence of GS based on purely phenotypic parameters. It was suggested that additional variables, such as mild hemolysis or a defect in bilirubin uptake, might be among the factors enhancing phenotypic expression of the defect.

1	Phenotypic expression of GS due solely to the A[TA]7TAA promoter abnormality is inherited as an autosomal recessive trait. A number of CN-II kindreds have been identified in whom there is also an allele containing a normal coding region but the A[TA]7TAA promoter abnormality. CN-II heterozygotes who have the A[TA]6TAA promoter are phenotypically normal, whereas those with the A[TA]7TAA promoter express the phenotypic picture of GS. GS in such kindreds may also result from homozygosity for the A[TA]7TAA promoter abnormality. Seven different missense mutations in the UGT1 gene that reportedly cause GS with dominant inheritance have been found in Japanese individuals. Another Japanese patient with mild unconjugated hyperbilirubinemia was homozygous for a missense mutation in exon 5. GS in her family appeared to be recessive. Missense mutations causing GS have not been reported outside of certain Asian populations.

1	In hyperbilirubinemia due to acquired liver disease (e.g., acute hepatitis, common bile duct stone), there are usually elevations in the serum concentrations of both conjugated and unconjugated bilirubin. Although biliary tract obstruction or hepatocellular cholestatic injury may present on occasion with a predominantly conjugated hyperbilirubinemia, it is generally not possible to differentiate intrahepatic from extrahepatic causes of jaundice based on the serum levels or relative proportions of unconjugated and conjugated bilirubin. The major reason for determining the amounts of conjugated and unconjugated bilirubin in the serum is for the initial differentiation of hepatic parenchymal and obstructive disorders (mixed conjugated and unconjugated hyperbilirubinemia) from the inheritable and hemolytic disorders discussed above that are associated with unconjugated hyperbilirubinemia.

1	FAMILIAL DEFECTS IN HEPATIC EXCRETORY FUNCTION Dubin-Johnson Syndrome (DJS) This benign, relatively rare disorder is characterized by low-grade, predominantly conjugated hyperbilirubinemia (Table 359-2). Total bilirubin concentrations are typically between 34 and 85 μmol/L (2 and 5 mg/dL) but on occasion can be in the normal range or as high as 340–430 μmol/L (20–25 mg/dL) and can fluctuate widely in any given patient. The degree of hyperbiliru-2003 binemia may be increased by intercurrent illness, oral contraceptive use, and pregnancy. Because the hyperbilirubinemia is due to a predominant rise in conjugated bilirubin, bilirubinuria is characteristically present. Aside from elevated serum bilirubin levels, other routine laboratory tests are normal. Physical examination is usually normal except for jaundice, although an occasional patient may have hepatosplenomegaly.

1	Patients with DJS are usually asymptomatic, although some may have vague constitutional symptoms. These latter patients have usually undergone extensive and often unnecessary diagnostic examinations for unexplained jaundice and have high levels of anxiety. In women, the condition may be subclinical until the patient becomes pregnant or receives oral contraceptives, at which time chemical hyperbilirubinemia becomes frank jaundice. Even in these situations, other routine liver function tests, including serum alkaline phosphatase and transaminase activities, are normal. A cardinal feature of DJS is the accumulation in the lysosomes of centrilobular hepatocytes of dark, coarsely granular pigment. As a result, the liver may be grossly black in appearance. This pigment is thought to be derived from epinephrine metabolites that are not excreted normally. The pigment may disappear during bouts of viral hepatitis, only to reaccumulate slowly after recovery.

1	Biliary excretion of a number of anionic compounds is compromised in DJS. These include various cholecystographic agents, as well as sulfobromophthalein (Bromsulphalein, BSP), a synthetic dye formerly used in a test of liver function. In this test, the rate of disappearance of BSP from plasma was determined following bolus IV administration. BSP is conjugated with glutathione in the hepatocyte; the resulting conjugate is normally excreted rapidly into the bile canaliculus. Patients with DJS exhibit characteristic rises in plasma concentrations at 90 minutes after injection, due to reflux of conjugated BSP into the circulation from the hepatocyte. Dyes such as ICG that are taken up by hepatocytes but are not further metabolized prior to biliary excretion do not show this reflux phenomenon. Continuous BSP infusion studies suggest a reduction in the time to maximum plasma concentration (t max) for biliary excretion. Bile acid disposition, including hepatocellular uptake and biliary

1	Continuous BSP infusion studies suggest a reduction in the time to maximum plasma concentration (t max) for biliary excretion. Bile acid disposition, including hepatocellular uptake and biliary excretion, is normal in DJS. These patients have normal serum and biliary bile acid concentrations and do not have pruritus.

1	By analogy with findings in several mutant rat strains, the selective defect in biliary excretion of bilirubin conjugates and certain other classes of organic compounds, but not of bile acids, that characterizes DJS in humans was found to reflect defective expression of MRP2, an ATP-dependent canalicular membrane transporter. Several different mutations in the MRP2 gene produce the Dubin-Johnson phenotype, which has an autosomal recessive pattern of inheritance. Although The Hyperbilirubinemias Abbreviations: BRIC, benign recurrent intrahepatic cholestasis; BSEP, bile salt excretory protein; DJS, Dubin-Johnson syndrome; γ-GT, γ-glutamyltransferase; MRP2, multidrug resistance– associated protein 2; OATP1A/1B, organic anion transport proteins 1B1 and 1B3; PFIC, progressive familial intrahepatic cholestasis; ↑↑, increased.

1	2004 MRP2 is undoubtedly important in the biliary excretion of conjugated bilirubin, the fact that this pigment is still excreted in the absence of MRP2 suggests that other, as yet uncharacterized, transport proteins may serve in a secondary role in this process. Patients with DJS also have a diagnostic abnormality in urinary coproporphyrin excretion. There are two naturally occurring coproporphyrin isomers, I and III. Normally, ∼75% of the coproporphyrin in urine is isomer III. In urine from DJS patients, total coproporphyrin content is normal, but >80% is isomer I. Heterozygotes for the syndrome show an intermediate pattern. The molecular basis for this phenomenon remains unclear.

1	Rotor Syndrome This benign, autosomal recessive disorder is clinically similar to DJS (Table 359-2), although it is seen even less frequently. A major phenotypic difference is that the liver in patients with Rotor syndrome has no increased pigmentation and appears totally normal. The only abnormality in routine laboratory tests is an elevation of total serum bilirubin, due to a predominant rise in conjugated bilirubin. This is accompanied by bilirubinuria. Several additional features differentiate Rotor syndrome from DJS. In Rotor syndrome, the gallbladder is usually visualized on oral cholecystography, in contrast to the nonvisualization that is typical of DJS. The pattern of urinary coproporphyrin excretion also differs. The pattern in Rotor syndrome resembles that of many acquired disorders of hepatobiliary function, in which coproporphyrin I, the major coproporphyrin isomer in bile, refluxes from the hepatocyte back into the circulation and is excreted in urine. Thus, total

1	disorders of hepatobiliary function, in which coproporphyrin I, the major coproporphyrin isomer in bile, refluxes from the hepatocyte back into the circulation and is excreted in urine. Thus, total urinary coproporphyrin excretion is substantially increased in Rotor syndrome, in contrast to the normal levels seen in DJS. Although the fraction of coproporphyrin I in urine is elevated, it is usually <70% of the total, compared with ≥80% in DJS. The disorders also can be distinguished by their patterns of BSP excretion. Although clearance of BSP from plasma is delayed in Rotor syndrome, there is no reflux of conjugated BSP back into the circulation as seen in DJS. Kinetic analysis of plasma BSP infusion studies suggests the presence of a defect in intrahepatocellular storage of this compound. This has never been demonstrated directly. Recent studies indicate that the molecular basis of Rotor syndrome results from simultaneous deficiency of the plasma membrane transporters OATP1B1 and

1	This has never been demonstrated directly. Recent studies indicate that the molecular basis of Rotor syndrome results from simultaneous deficiency of the plasma membrane transporters OATP1B1 and OATP1B3. This results in reduced reuptake of conjugated bilirubin that has been pumped out of the cell into the portal circulation by MRP3 (Fig. 359-1).

1	Benign Recurrent Intrahepatic Cholestasis (BRIC) This rare disorder is characterized by recurrent attacks of pruritus and jaundice. The typical episode begins with mild malaise and elevations in serum aminotransferase levels, followed rapidly by rises in alkaline phosphatase and conjugated bilirubin and onset of jaundice and itching. The first one or two episodes may be misdiagnosed as acute viral hepatitis. The cholestatic episodes, which may begin in childhood or adulthood, can vary in duration from several weeks to months, followed by a complete clinical and biochemical resolution. Intervals between attacks may vary from several months to years. Between episodes, physical examination is normal, as are serum levels of bile acids, bilirubin, transaminases, and alkaline phosphatase. The disorder is familial and has an autosomal recessive pattern of inheritance. BRIC is considered a benign disorder in that it does not lead to cirrhosis or end-stage liver disease. However, the episodes

1	is familial and has an autosomal recessive pattern of inheritance. BRIC is considered a benign disorder in that it does not lead to cirrhosis or end-stage liver disease. However, the episodes of jaundice and pruritus can be prolonged and debilitating, and some patients have undergone liver transplantation to relieve the intractable and disabling symptoms. Treatment during the cholestatic episodes is symptomatic; there is no specific treatment to prevent or shorten the occurrence of episodes.

1	A gene termed FIC1 was recently identified and found to be mutated in patients with BRIC. Curiously, this gene is expressed strongly in the small intestine but only weakly in the liver. The protein encoded by FIC1 shows little similarity to those that have been shown to play a role in bile canalicular excretion of various compounds. Rather, it appears to be a member of a P-type ATPase family that transports aminophospholipids from the outer to the inner leaflet of a variety of cell membranes. Its relationship to the pathobiology of this disorder remains unclear. A second phenotypically identical form of BRIC, termed BRIC type 2, has been described resulting from mutations in the bile salt excretory protein (BSEP), the protein that is defective in progressive familial intrahepatic cholestasis type 2 (Table 359-2). How some mutations in this protein result in the episodic BRIC phenotype is unknown.

1	Progressive Familial Intrahepatic Cholestasis (FIC) This name is applied to three phenotypically related syndromes (Table 359-2). Progressive FIC type 1 (Byler disease) presents in early infancy as cholestasis that may be initially episodic. However, in contrast to BRIC, Byler disease progresses to malnutrition, growth retardation, and end-stage liver disease during childhood. This disorder is also a consequence of a FIC1 mutation. The functional relationship of the FIC1 protein to the pathogenesis of cholestasis in these disorders is unknown. Two other types of progressive FIC (types 2 and 3) have been described. Progressive FIC type 2 is associated with a mutation in the protein originally named sister of p-glycoprotein, now known as bile salt excretory protein, which is the major bile canalicular exporter of bile acids. As noted above, some mutations of this protein are associated with BRIC type 2, rather than the progressive FIC type 2 phenotype. Progressive FIC type 3 has been

1	canalicular exporter of bile acids. As noted above, some mutations of this protein are associated with BRIC type 2, rather than the progressive FIC type 2 phenotype. Progressive FIC type 3 has been associated with a mutation of MDR3, a protein that is essential for normal hepatocellular excretion of phospholipids across the bile canaliculus. Although all three types of progressive FIC have similar clinical phenotypes, only type 3 is associated with high serum levels of γ-glutamyltransferase activity. In contrast, activity of this enzyme is normal or only mildly elevated in symptomatic BRIC and progressive FIC types 1 and 2.

1	Jules L. Dienstag Acute viral hepatitis is a systemic infection affecting the liver predominantly. Almost all cases of acute viral hepatitis are caused by one of five viral agents: hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the HBV-associated delta agent or hepatitis D virus (HDV), and hepatitis E virus (HEV). All these human hepatitis viruses are RNA viruses, except for hepatitis B, which is a DNA virus but replicates like a retrovirus. Although these agents can be distinguished by their molecular and antigenic properties, all types of viral hepatitis produce clinically similar illnesses. These range from asymptomatic and inapparent to fulminant and fatal acute infections common to all types, on the one hand, and from subclinical persistent infections to rapidly progressive chronic liver disease with cirrhosis and even hepatocellular carcinoma, common to the bloodborne types (HBV, HCV, and HDV), on the other.

1	VIROLOGY AND ETIOLOGY Hepatitis A HAV is a nonenveloped 27-nm, heat-, acid-, and ether-resistant RNA virus in the Hepatovirus genus of the picornavirus family (Fig. 360-1). Its virion contains four capsid polypeptides, designated VP1 to VP4, which are cleaved posttranslationally from the polyprotein product of a 7500-nucleotide genome. Inactivation of viral activity can be achieved by boiling for 1 min, by contact with formaldehyde and chlorine, or by ultraviolet irradiation. Despite nucleotide sequence variation of up to 20% among isolates of HAV, and despite the recognition of four genotypes affecting humans, all strains of this virus are immunologically indistinguishable and belong to one serotype. Hepatitis A has an incubation period of ~4 weeks. Its replication is limited to the liver, but the virus is present in the liver, bile, stools, and blood during the late incubation period and acute preicteric/ presymptomatic phase of illness. Despite slightly longer persistence of virus

1	but the virus is present in the liver, bile, stools, and blood during the late incubation period and acute preicteric/ presymptomatic phase of illness. Despite slightly longer persistence of virus in the liver, fecal shedding, viremia, and infectivity diminish rapidly once jaundice becomes apparent. HAV can be cultivated reproducibly in vitro.

1	template, hepadnaviruses rely on reverse 2005 transcription (effected by the DNA polymerase) of minus-strand DNA from a “pregenomic” RNA intermediate. Then plus-strand DNA is transcribed from the minus-strand DNA template by the DNA-dependent DNA polymerase and converted in the hepatocyte nucleus to a covalently closed circular DNA, which serves as a template for messenger RNA and pregenomic RNA. Viral proteins are translated by the messenger RNA, and the proteins and genome are packaged into FIGURE 360-1 Electron micrographs of hepatitis A virus particles and serum from a patient virions and secreted from the hepatocyte. with hepatitis B. Left: 27-nm hepatitis A virus particles purified from stool of a patient with acute

1	with hepatitis B. Left: 27-nm hepatitis A virus particles purified from stool of a patient with acute Although HBV is difficult to cultivate hepatitis A and aggregated by antibody to hepatitis A virus. Right: Concentrated serum from a in vitro in the conventional sense from patient with hepatitis B, demonstrating the 42-nm virions, tubular forms, and spherical 22-nm clinical material, several cell lines have particles of hepatitis B surface antigen. 132,000×. (Hepatitis D resembles 42-nm virions of hepatitis been transfected with HBV DNA. Such B but is smaller, 35–37 nm; hepatitis E resembles hepatitis A virus but is slightly larger, 32–34 nm; hepatitis C has been visualized as a 55-nm particle.)

1	B but is smaller, 35–37 nm; hepatitis E resembles hepatitis A virus but is slightly larger, 32–34 nm; hepatitis C has been visualized as a 55-nm particle.) Antibodies to HAV (anti-HAV) can be detected during acute illness when serum aminotransferase activity is elevated and fecal HAV shedding is still occurring. This early antibody response is predominantly of the IgM class and persists for several (~3) months, rarely for 6–12 months. During convalescence, however, anti-HAV of the IgG class becomes the predominant antibody (Fig. 360-2). Therefore, the diagnosis of hepatitis A is made during acute illness by demonstrating anti-HAV of the IgM class. After acute illness, anti-HAV of the IgG class remains detectable indefinitely, and patients with serum anti-HAV are immune to reinfection. Neutralizing antibody activity parallels the appearance of anti-HAV, and the IgG anti-HAV present in immune globulin accounts for the protection it affords against HAV infection.

1	Hepatitis B HBV is a DNA virus with a remarkably compact genomic structure; despite its small, circular, 3200-bp size, HBV DNA codes for four sets of viral products with a complex, multiparticle structure. HBV achieves its genomic economy by relying on an efficient strategy of encoding proteins from four overlapping genes: S, C, P, and X (Fig. 360-3), as detailed below. Once thought to be unique among viruses, HBV is now recognized as one of a family of animal viruses, hepadnaviruses (hepatotropic DNA viruses), and is classified as hepadnavirus type 1. Similar viruses infect certain species of woodchucks, ground and tree squirrels, and Pekin ducks, to mention the most carefully characterized. Like HBV, all have the same distinctive three morphologic forms, have counterparts to the envelope and nucleocapsid virus antigens of HBV, replicate in the liver but exist in extrahepatic sites, contain their own endogenous DNA polymerase, have partially double-strand and partially single-strand

1	and nucleocapsid virus antigens of HBV, replicate in the liver but exist in extrahepatic sites, contain their own endogenous DNA polymerase, have partially double-strand and partially single-strand genomes, are associated with acute and chronic hepatitis and hepatocellular carcinoma, and rely on a replicative strategy unique among DNA viruses but typical of retroviruses. Instead of DNA replication directly from a DNA of hepatitis A virus (HAV). ALT, alanine aminotransferase.

1	tion of the intact virus and its component proteins. viral proteins anD particles Of the three particulate forms of HBV (Table 360-1), the most numerous are the 22-nm particles, which appear as spherical or long filamentous forms; these are antigenically indistinguishable from the outer surface or envelope protein of HBV and are thought to represent excess viral envelope protein. Outnumbered in serum by a factor of 100 or 1000 to 1 compared with the spheres and tubules are large, 42-nm, double-shelled spherical particles, which represent the intact hepatitis B virion (Fig. 360-1). The envelope protein expressed on the outer surface of the virion and on the smaller spherical and tubular structures is referred to as hepatitis B surface antigen (HBsAg). The concentration of HBsAg and virus particles in the blood may reach 500 μg/mL and 10 trillion particles per milliliter, respectively. The envelope protein, HBsAg, is the product of the S gene of HBV.

1	Envelope HBsAg subdeterminants include a common group-reactive antigen, a, shared by all HBsAg isolates and one of several subtype-specific antigens—d or y, w or r —as well as other specificities. Hepatitis B isolates fall into one of at least eight subtypes and ten

1	FIGURE 360-3 Compact genomic structure of hepatitis B virus (HBV). This structure, with overlapping genes, permits HBV to code for multiple proteins. The S gene codes for the “major” envelope protein, HBsAg. Pre-S1 and pre-S2, upstream of S, combine with S to code for two larger proteins, “middle” protein, the product of pre-S2 + S, and “large” protein, the product of pre-S1 + pre-S2 + S. The largest gene, P, codes for DNA polymerase. The C gene codes for two nucleocapsid proteins, HBeAg, a soluble, secreted protein (initiation from the pre-C region of the gene), and HBcAg, the intracellular core protein (initiation after pre-C). The X gene codes for HBxAg, which can transactivate the transcription of cellular and viral genes; its clinical relevance is not known, but it may contribute to carcinogenesis by binding to p53. Hepatitis Virus Type Particle, nm Morphology Genomea Classification Antigen(s) Antibodies Remarks

1	Hepatitis Virus Type Particle, nm Morphology Genomea Classification Antigen(s) Antibodies Remarks HAV 27 Icosahedral 7.5-kb RNA, Hepatovirus HAV Anti-HAV Early fecal shedding Diagnosis: IgM anti-HAV nonenveloped linear, ss, + Previous infection: IgG anti-HAV HCV Approx. Enveloped 50–80 HDV 35–37 Enveloped hybrid particle with HBsAg coat and HDV core HEV 32–34 Nonenveloped icosahedral 3.2-kb DNA, Hepadnavirus HBsAg Anti-HBs circular, ss/ds 9.4-kb RNA, Hepacivirus HCV Anti-HCV linear, ss, + 1.7-kb RNA, Resembles HBsAg Anti-HBs circular, ss, – viroids and HDAg Anti-HDV plant satellite viruses (genus Deltavirus) 7.6-kb RNA, Hepevirus HEV antigen Anti-HEV linear, ss, + Bloodborne virus; carrier state Acute diagnosis: HBsAg, IgM anti-HBc Chronic diagnosis: IgG anti-HBc, HBsAg Markers of replication: HBeAg, HBV DNA Liver, lymphocytes, other organs

1	Acute diagnosis: HBsAg, IgM anti-HBc Chronic diagnosis: IgG anti-HBc, HBsAg Markers of replication: HBeAg, HBV DNA Liver, lymphocytes, other organs Nucleocapsid contains DNA and DNA polymerase; present in hepatocyte nucleus; HBcAg does not circulate; HBeAg (soluble, nonparticulate) and HBV DNA circulate—correlate with infectivity and complete virions HBsAg detectable in >95% of patients with acute hepatitis B; found in serum, body fluids, hepatocyte cytoplasm; anti-HBs appears following infection—protective antibody Bloodborne agent, formerly labeled non-A, non-B hepatitis Acute diagnosis: anti-HCV (C33c, C22-3, NS5), HCV RNA Chronic diagnosis: anti-HCV (C100-3, C33c, C223, NS5) and HCV RNA; cytoplasmic location in hepatocytes Defective RNA virus, requires helper function of HBV (hepadnaviruses); HDV antigen (HDAg) present in hepatocyte nucleus Diagnosis: anti-HDV, HDV RNA; HBV/HDV co-infection—IgM anti-HBc and anti-HDV; HDV superinfection—IgG anti-HBc and anti-HDV

1	Diagnosis: anti-HDV, HDV RNA; HBV/HDV co-infection—IgM anti-HBc and anti-HDV; HDV superinfection—IgG anti-HBc and anti-HDV Agent of enterically transmitted hepatitis; rare in United States; occurs in Asia, Mediterranean countries, Central America Diagnosis: IgM/IgG anti-HEV (assays not routinely available); virus in stool, bile, hepatocyte cytoplasm ass, single-strand; ss/ds, partially single-strand, partially double-strand; −, minus-strand; +, plus-strand. Note: See text for abbreviations.

1	Note: See text for abbreviations. genotypes (A–J). Geographic distribution of genotypes and subtypes varies; genotypes A (corresponding to subtype adw) and D (ayw) predominate in the United States and Europe, whereas genotypes B (adw) and C (adr) predominate in Asia. Clinical course and outcome are independent of subtype, but genotype B appears to be associated with less rapidly progressive liver disease and cirrhosis and a lower likelihood, or delayed appearance, of hepatocellular carcinoma than genotype C or D. Patients with genotype A are more likely to clear circulating viremia and to achieve HBeAg and HBsAg seroconversion, both spontaneously and in response to antiviral therapy. In addition, “precore” mutations are favored by certain genotypes (see below).

1	Upstream of the S gene are the pre-S genes (Fig. 360-3), which code for pre-S gene products, including receptors on the HBV surface for polymerized human serum albumin and for hepatocyte membrane proteins. The pre-S region actually consists of both pre-S1 and pre-S2. Depending on where translation is initiated, three potential HBsAg gene products are synthesized. The protein product of the S gene is HBsAg (major protein), the product of the S region plus the adjacent pre-S2 region is the middle protein, and the product of the pre-S1 plus pre-S2 plus S regions is the large protein. Compared with the smaller spherical and tubular particles of HBV, complete 42-nm virions are enriched in the large protein. Both pre-S proteins and their respective antibodies can be detected during HBV infection, and the period of pre-S antigenemia appears to coincide with other markers of virus replication, as detailed below; however, pre-S proteins have little clinical relevance and are not included in

1	and the period of pre-S antigenemia appears to coincide with other markers of virus replication, as detailed below; however, pre-S proteins have little clinical relevance and are not included in routine serologic testing repertoires.

1	The intact 42-nm virion contains a 27-nm nucleocapsid core particle. Nucleocapsid proteins are coded for by the C gene. The antigen expressed on the surface of the nucleocapsid core is hepatitis B core antigen (HBcAg), and its corresponding antibody is anti-HBc. A third HBV antigen is hepatitis B e antigen (HBeAg), a soluble, nonparticulate, nucleocapsid protein that is immunologically distinct from intact HBcAg but is a product of the same C gene. The C gene has two initiation codons, a precore and a core region (Fig. 360-3). If translation is initiated at the precore region, the protein product is HBeAg, which has a signal peptide that binds it to the smooth endoplasmic reticulum, the secretory apparatus of the cell, leading to its secretion into the circulation. If translation begins at the core region, HBcAg is the protein product; it has no signal peptide, it is not secreted, but it assembles into nucleocapsid particles, which bind to and incorporate RNA, and which, ultimately,

1	at the core region, HBcAg is the protein product; it has no signal peptide, it is not secreted, but it assembles into nucleocapsid particles, which bind to and incorporate RNA, and which, ultimately, contain HBV DNA. Also packaged within the nucleocapsid core is a DNA polymerase, which directs replication and repair of HBV DNA. When packaging within viral proteins is complete, synthesis of the incomplete plus strand stops; this accounts for the single-strand gap and for differences in the size of the gap. HBcAg particles remain in the hepatocyte, where they are readily detectable by immunohistochemical staining and are exported after encapsidation by an envelope of HBsAg. Therefore, naked core particles do not circulate in the serum. The secreted nucleocapsid protein, HBeAg, provides a convenient, readily detectable, qualitative marker of HBV replication and relative infectivity.

1	HBsAg-positive serum containing HBeAg is more likely to be highly infectious and to be associated with the presence of hepatitis B virions (and detectable HBV DNA, see below) than HBeAg-negative or anti-HBe-positive serum. For example, HBsAg-positive mothers who are HBeAg-positive almost invariably (>90%) transmit hepatitis B infection to their offspring, whereas HBsAg-positive mothers with anti-HBe rarely (10–15%) infect their offspring. Early during the course of acute hepatitis B, HBeAg appears transiently; its disappearance may be a harbinger of clinical improvement and resolution of infection. Persistence of HBeAg in serum beyond the first 3 months of acute infection may be predictive of the development of chronic infection, and the presence of HBeAg during chronic hepatitis B tends to be associated with ongoing viral replication, infectivity, and inflammatory liver injury (except during the early decades after perinatally acquired HBV infection; see below).

1	The third and largest of the HBV genes, the P gene (Fig. 360-3), codes for HBV DNA polymerase; as noted above, this enzyme has both DNA-dependent DNA polymerase and RNA-dependent reverse transcriptase activities. The fourth gene, X, codes for a small, non-particulate protein, hepatitis B x antigen (HBxAg), that is capable of transactivating the transcription of both viral and cellular genes (Fig. 360-3). In the cytoplasm, HBxAg effects calcium release (possibly from mitochondria), which activates signal-transduction pathways that lead to stimulation of HBV reverse transcription and HBV DNA replication. Such transactivation may enhance the replication of HBV, leading to the clinical association observed between the expression of HBxAg and antibodies to it in patients with severe chronic hepatitis and hepatocellular carcinoma. The transactivating activity can enhance the transcription and replication of other viruses besides HBV, such as HIV. Cellular processes transactivated by X

1	hepatitis and hepatocellular carcinoma. The transactivating activity can enhance the transcription and replication of other viruses besides HBV, such as HIV. Cellular processes transactivated by X include the human interferon γ gene and class I major histocompatibility genes; potentially, these effects could contribute to enhanced susceptibility of HBV-infected hepatocytes to cytolytic T cells. The expression of X can also induce programmed cell death (apoptosis). The clinical relevance of HBxAg is limited, however, and testing for it is not part of routine clinical practice.

1	seroloGic anD viroloGic markers After a person is infected with HBV, the first virologic marker detectable in serum within 1–12 weeks, usually between 8 and 12 weeks, is HBsAg (Fig. 360-4). Circulating HBsAg precedes elevations of serum aminotransferase activity and clinical symptoms by 2–6 weeks and remains detectable during the entire icteric or symptomatic phase of acute hepatitis B and beyond. In typical cases, HBsAg becomes undetectable 1–2 months after the onset of jaundice and rarely persists beyond 6 months. After HBsAg disappears, antibody to HBsAg (anti-HBs) becomes detectable in serum and remains detectable indefinitely thereafter. Because HBcAg is intracellular and, when in the serum, sequestered within an HBsAg coat, naked core particles do not circulate in serum, and therefore, HBcAg is not detectable routinely in the serum of patients with HBV infection. By contrast, anti-HBc is readily demonstrable in serum, beginning within the first 1–2 weeks after the appearance of

1	HBcAg is not detectable routinely in the serum of patients with HBV infection. By contrast, anti-HBc is readily demonstrable in serum, beginning within the first 1–2 weeks after the appearance of HBsAg and preceding detectable levels of anti-HBs by weeks to months.

1	of acute hepatitis B. ALT, alanine aminotransferase.

1	Because variability exists in the time of appearance of anti-HBs after 2007 HBV infection, occasionally a gap of several weeks or longer may separate the disappearance of HBsAg and the appearance of anti-HBs. During this “gap” or “window” period, anti-HBc may represent the only serologic evidence of current or recent HBV infection, and blood containing anti-HBc in the absence of HBsAg and anti-HBs has been implicated in transfusion-associated hepatitis B. In part because the sensitivity of immunoassays for HBsAg and anti-HBs has increased, however, this window period is rarely encountered. In some persons, years after HBV infection, anti-HBc may persist in the circulation longer than anti-HBs. Therefore, isolated anti-HBc does not necessarily indicate active virus replication; most instances of isolated anti-HBc represent hepatitis B infection in the remote past. Rarely, however, isolated anti-HBc represents low-level hepatitis B viremia, with HBsAg below the detection threshold, and,

1	of isolated anti-HBc represent hepatitis B infection in the remote past. Rarely, however, isolated anti-HBc represents low-level hepatitis B viremia, with HBsAg below the detection threshold, and, occasionally, isolated anti-HBc represents a cross-reacting or false-positive immunologic specificity. Recent and remote HBV infections can be distinguished by determination of the immunoglobulin class of anti-HBc. Anti-HBc of the IgM class (IgM anti-HBc) predominates during the first 6 months after acute infection, whereas IgG anti-HBc is the predominant class of anti-HBc beyond 6 months. Therefore, patients with current or recent acute hepatitis B, including those in the anti-HBc window, have IgM anti-HBc in their serum. In patients who have recovered from hepatitis B in the remote past as well as those with chronic HBV infection, anti-HBc is predominantly of the IgG class. Infrequently, in ≤1–5% of patients with acute HBV infection, levels of HBsAg are too low to be detected; in such

1	well as those with chronic HBV infection, anti-HBc is predominantly of the IgG class. Infrequently, in ≤1–5% of patients with acute HBV infection, levels of HBsAg are too low to be detected; in such cases, the presence of IgM anti-HBc establishes the diagnosis of acute hepatitis B. When isolated anti-HBc occurs in the rare patient with chronic hepatitis B whose HBsAg level is below the sensitivity threshold of contemporary immunoassays (a low-level carrier), anti-HBc is of the IgG class. Generally, in persons who have recovered from hepatitis B, anti-HBs and anti-HBc persist indefinitely.

1	The temporal association between the appearance of anti-HBs and resolution of HBV infection as well as the observation that persons with anti-HBs in serum are protected against reinfection with HBV suggests that anti-HBs is the protective antibody. Therefore, strategies for prevention of HBV infection are based on providing susceptible persons with circulating anti-HBs (see below). Occasionally, in ~10% of patients with chronic hepatitis B, low-level, low-affinity anti-HBs can be detected. This antibody is directed against a subtype determinant different from that represented by the patient’s HBsAg; its presence is thought to reflect the stimulation of a related clone of antibody-forming cells, but it has no clinical relevance and does not signal imminent clearance of hepatitis B. These patients with HBsAg and such nonneutralizing anti-HBs should be categorized as having chronic HBV infection.

1	The other readily detectable serologic marker of HBV infection, HBeAg, appears concurrently with or shortly after HBsAg. Its appearance coincides temporally with high levels of virus replication and reflects the presence of circulating intact virions and detectable HBV DNA (with the notable exception of patients with precore mutations who cannot synthesize HBeAg—see “Molecular Variants”). Pre-S1 and pre-S2 proteins are also expressed during periods of peak replication, but assays for these gene products are not routinely available. In self-limited HBV infections, HBeAg becomes undetectable shortly after peak elevations in aminotransferase activity, before the disappearance of HBsAg, and anti-HBe then becomes detectable, coinciding with a period of relatively lower infectivity (Fig. 360-4). Because markers of HBV replication appear transiently during acute infection, testing for such markers is of little clinical utility in typical cases of acute HBV infection. In contrast, markers of

1	Because markers of HBV replication appear transiently during acute infection, testing for such markers is of little clinical utility in typical cases of acute HBV infection. In contrast, markers of HBV replication provide valuable information in patients with protracted infections.

1	Departing from the pattern typical of acute HBV infections, in chronic HBV infection, HBsAg remains detectable beyond 6 months, anti-HBc is primarily of the IgG class, and anti-HBs is either undetectable or detectable at low levels (see “Laboratory Features”) (Fig. 360-5). During early chronic HBV infection, HBV DNA can be detected both in serum and in hepatocyte nuclei, where it is present in free or episomal form. This relatively highly replicative stage of HBV infection is type chronic hepatitis B. HBeAg and hepatitis B virus (HBV) DNA can be detected in serum during the relatively replicative phase of chronic infection, which is associated with infectivity and liver injury. Seroconversion from the replicative phase to the relatively nonreplicative phase occurs at a rate of ~10% per year and is heralded by an acute hepatitis–like elevation of alanine aminotransferase (ALT) activity; during the nonreplicative phase, infectivity and liver injury are limited. In HBeAg-negative chronic

1	and is heralded by an acute hepatitis–like elevation of alanine aminotransferase (ALT) activity; during the nonreplicative phase, infectivity and liver injury are limited. In HBeAg-negative chronic hepatitis B associated with mutations in the precore region of the HBV genome, replicative chronic hepatitis B occurs in the absence of HBeAg.

1	the time of maximal infectivity and liver injury; HBeAg is a qualitative marker and HBV DNA a quantitative marker of this replicative phase, during which all three forms of HBV circulate, including intact virions. Over time, the relatively replicative phase of chronic HBV infection gives way to a relatively nonreplicative phase. This occurs at a rate of ~10% per year and is accompanied by seroconversion from HBeAg to anti-HBe. In many cases, this seroconversion coincides with a transient, usually mild, acute hepatitis-like elevation in aminotransferase activity, believed to reflect cell-mediated immune clearance of virus-infected hepatocytes. In the nonreplicative phase of chronic infection, when HBV DNA is demonstrable in hepatocyte nuclei, it tends to be integrated into the host genome. In this phase, only spherical and tubular forms of HBV, not intact virions, circulate, and liver injury tends to subside. Most such patients would be characterized as inactive HBV carriers. In

1	In this phase, only spherical and tubular forms of HBV, not intact virions, circulate, and liver injury tends to subside. Most such patients would be characterized as inactive HBV carriers. In reality, the designations replicative and nonreplicative are only relative; even in the so-called nonreplicative phase, HBV replication can be detected at levels of approximately ≤103 virions with highly sensitive amplification probes such as the polymerase chain reaction (PCR); below this replication threshold, liver injury and infectivity of HBV are limited to negligible. Still, the distinctions are pathophysiologically and clinically meaningful. Occasionally, nonreplicative HBV infection converts back to replicative infection. Such spontaneous reactivations are accompanied by reexpression of HBeAg and HBV DNA, and sometimes of IgM anti-HBc, as well as by exacerbations of liver injury. Because high-titer IgM anti-HBc can reappear during acute exacerbations of chronic hepatitis B, relying on

1	and HBV DNA, and sometimes of IgM anti-HBc, as well as by exacerbations of liver injury. Because high-titer IgM anti-HBc can reappear during acute exacerbations of chronic hepatitis B, relying on IgM anti-HBc versus IgG anti-HBc to distinguish between acute and chronic hepatitis B infection, respectively, may not always be reliable; in such cases, patient history is invaluable in helping to distinguish de novo acute hepatitis B infection from acute exacerbation of chronic hepatitis B infection.

1	molecular variants Variation occurs throughout the HBV genome, and clinical isolates of HBV that do not express typical viral proteins have been attributed to mutations in individual or even multiple gene locations. For example, variants have been described that lack nucleocapsid proteins (commonly), envelope proteins (very rarely), or both. Two categories of naturally occurring HBV variants have attracted the most attention. One of these was identified initially in Mediterranean countries among patients with severe chronic HBV infection and detectable HBV DNA but with anti-HBe instead of HBeAg. These patients were found to be infected with an HBV mutant that contained an alteration in the precore region rendering the virus incapable of encoding HBeAg. Although several potential mutation sites exist in the pre-C region, the region of the C gene necessary for the expression of HBeAg (see “Virology and Etiology”), the most commonly encountered in such patients is a single base

1	mutation sites exist in the pre-C region, the region of the C gene necessary for the expression of HBeAg (see “Virology and Etiology”), the most commonly encountered in such patients is a single base substitution, from G to A in the second to last codon of the pre-C gene at nucleotide 1896. This substitution results in the replacement of the TGG tryptophan codon by a stop codon (TAG), which prevents the translation of HBeAg. Another mutation, in the core-promoter region, prevents transcription of the coding region for HBeAg and yields an HBeAgnegative phenotype. Patients with such mutations in the precore region and who are unable to secrete HBeAg may have severe liver disease that progresses more rapidly to cirrhosis, or alternatively, they are identified clinically later in the course of the natural history of chronic hepatitis B, when the disease is more advanced. Both “wild-type” HBV and precore-mutant HBV can coexist in the same patient, or mutant HBV may arise late during

1	of the natural history of chronic hepatitis B, when the disease is more advanced. Both “wild-type” HBV and precore-mutant HBV can coexist in the same patient, or mutant HBV may arise late during wild-type HBV infection. In addition, clusters of fulminant hepatitis B in Israel and Japan were attributed to common-source infection with a precore mutant. Fulminant hepatitis B in North America and western Europe, however, occurs in patients infected with wild-type HBV, in the absence of precore mutants, and both precore mutants and other mutations throughout the HBV genome occur commonly, even in patients with typical, self-limited, milder forms of HBV infection. HBeAg-negative chronic hepatitis with mutations in the precore region is now the most frequently encountered form of hepatitis B in Mediterranean countries and in Europe. In the United States, where HBV genotype A (less prone to G1896A mutation) is prevalent, precore-mutant HBV is much less common; however, as a result of

1	B in Mediterranean countries and in Europe. In the United States, where HBV genotype A (less prone to G1896A mutation) is prevalent, precore-mutant HBV is much less common; however, as a result of immigration from Asia and Europe, the proportion of HBeAg-negative hepatitis B–infected individuals has increased in the United States, and they now represent approximately 30–40% of patients with chronic hepatitis B. Characteristic of such HBeAg-negative chronic hepatitis B are lower levels of HBV DNA (usually ≤105 IU/mL) and one of several patterns of aminotransferase activity—persistent elevations, periodic fluctuations above the normal range, and periodic fluctuations between the normal and elevated range.

1	The second important category of HBV mutants consists of escape mutants, in which a single amino acid substitution, from glycine to arginine, occurs at position 145 of the immunodominant a determinant common to all HBsAg subtypes. This HBsAg alteration leads to a critical conformational change that results in a loss of neutralizing activity by anti-HBs. This specific HBV/a mutant has been observed in two situations, active and passive immunization, in which humoral immunologic pressure may favor evolutionary change (“escape”) in the virus— in a small number of hepatitis B vaccine recipients who acquired HBV infection despite the prior appearance of neutralizing anti-HBs and in HBV-infected liver transplant recipients treated with a high-potency human monoclonal anti-HBs preparation. Although such mutants have not been recognized frequently, their existence raises a concern that may complicate vaccination strategies and serologic diagnosis.

1	Different types of mutations emerge during antiviral therapy of chronic hepatitis B with nucleoside analogues; such “YMDD” and similar mutations in the polymerase motif of HBV are described in Chap. 362. extraHepatic sites Hepatitis B antigens and HBV DNA have been identified in extrahepatic sites, including lymph nodes, bone marrow, circulating lymphocytes, spleen, and pancreas. Although the virus does not appear to be associated with tissue injury in any of these extrahepatic sites, its presence in these “remote” reservoirs has been invoked (but is not necessary) to explain the recurrence of HBV infection after orthotopic liver transplantation. The clinical relevance of such extra-hepatic HBV is limited.

1	Hepatitis D The delta hepatitis agent, or HDV, the only member of the genus Deltavirus, is a defective RNA virus that co-infects with and requires the helper function of HBV (or other hepadnaviruses) for its replication and expression. Slightly smaller than HBV, HDV is a formalin-sensitive, 35to 37-nm virus with a hybrid structure. Its nucleocapsid expresses HDV antigen (HDAg), which bears no antigenic homology with any of the HBV antigens, and contains the virus genome. The HDV core is “encapsidated” by an outer envelope of HBsAg, indistinguishable from that of HBV except in its relative compositions of major, middle, and large HBsAg component proteins. The genome is a small, 1700-nucleotide, circular, single-strand RNA of negative polarity that is nonhomologous with HBV DNA (except for a small area of the polymerase gene) but that has features and the rolling circle model of replication common to genomes of plant satellite viruses or viroids. HDV RNA contains many areas of internal

1	a small area of the polymerase gene) but that has features and the rolling circle model of replication common to genomes of plant satellite viruses or viroids. HDV RNA contains many areas of internal complementarity; therefore, it can fold on itself by internal base pairing to form an unusual, very stable, rodlike structure that contains a very stable, self-cleaving and self-ligating ribozyme. HDV RNA requires host RNA polymerase II for its replication in the hepatocyte nucleus via RNA-directed RNA synthesis by transcription of genomic RNA to a complementary antigenomic (plus strand) RNA; the antigenomic RNA, in turn, serves as a template for subsequent genomic RNA synthesis effected by host RNA polymerase I. HDV RNA has only one open reading frame, and HDAg, a product of the antigenomic strand, is the only known HDV protein; HDAg exists in two forms: a small, 195-amino-acid species, which plays a role in facilitating HDV RNA replication, and a large, 214-amino-acid species, which

1	strand, is the only known HDV protein; HDAg exists in two forms: a small, 195-amino-acid species, which plays a role in facilitating HDV RNA replication, and a large, 214-amino-acid species, which appears to suppress replication but is required for assembly of the antigen into virions. HDV antigens have been shown to bind directly to RNA polymerase II, resulting in stimulation of transcription. Although complete hepatitis D virions and liver injury require the cooperative helper function of HBV, intracellular replication of HDV RNA can occur without HBV. Genomic heterogeneity among HDV isolates has been described; however, pathophysiologic and clinical consequences of this genetic diversity have not been recognized. The clinical spectrum of hepatitis D is common to all eight genotypes identified, the predominant of which is genotype 1.

1	HDV can either infect a person simultaneously with HBV (coinfection) or superinfect a person already infected with HBV (superinfection); when HDV infection is transmitted from a donor with one HBsAg subtype to an HBsAg-positive recipient with a different subtype, HDV assumes the HBsAg subtype of the recipient, rather than the donor. Because HDV relies absolutely on HBV, the duration of HDV infection is determined by the duration of (and cannot outlast) HBV infection. HDV replication tends to suppress HBV replication; therefore, patients with hepatitis D tend to have lower levels of HBV replication. HDV antigen is expressed primarily in hepatocyte nuclei and is occasionally detectable in serum. During acute HDV infection, anti-HDV of the IgM class predominates, and 30–40 days may elapse after symptoms appear before anti-HDV can be detected. In self-limited infec-500 virus polyprotein of ~3000 amino acids, which is cleaved after transla-2009 tion to yield 10 viral proteins. The 5′ end

1	symptoms appear before anti-HDV can be detected. In self-limited infec-500 virus polyprotein of ~3000 amino acids, which is cleaved after transla-2009 tion to yield 10 viral proteins. The 5′ end of the genome consists of an untranslated region (containing an internal ribosomal entry site, IRES) adjacent to the genes for three structural proteins, the nucleocapsid core protein, C, and two structural envelope glycoproteins, E1 and E2. The 5′ untranslated region and core gene are highly conserved among genotypes, but the envelope proteins are coded for by the hypervariable region, which varies from isolate to isolate and may allow the virus to evade host immunologic containment directed at accessible virus-envelope proteins. The 3′ end of the genome also includes an untranslated region and contains the genes for seven nonstructural (NS) proteins, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. p7 is a membrane ion channel protein necessary for efficient assembly and release of HCV. The NS2

1	contains the genes for seven nonstructural (NS) proteins, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. p7 is a membrane ion channel protein necessary for efficient assembly and release of HCV. The NS2 cysteine protease cleaves NS3 from NS2, and the NS3-4A serine protease cleaves all the downstream proteins from the polyprotein. Important NS proteins involved in virus replication include the NS3 helicase; NS3-4A serine protease; the multifunctional membrane-associated phosphoprotein NS5A, an essential component of the viral replication membranous web (along with NS4B); and the

1	NS5B RNA-dependent RNA polymerase (Fig. 360-6). Because HCV does not replicate via a DNA intermediate, it does not integrate into the host genome. Because HCV tends to circulate in relatively low titer, 103−107 virions/mL, visualization of the 50to 80-nm virus particles remains difficult. Still, the replication rate of HCV is very high, 1012 virions per day; its half-life is 2.7 h. The chimpanzee is a helpful but cumbersome animal model. Although a robust, reproducible, small animal model is lacking, HCV replication has been documented in an immunodeficient mouse model containing explants of human liver and in transgenic mouse and rat models. Although in vitro replication is difficult, replicons in hepatocellular carcinoma–derived cell lines support replication of genetically manipulated, truncated, or full-length HCV RNA (but not intact virions); infectious pseudotyped retroviral HCV particles have been shown to yield functioning envelope proteins. In 2005, complete replication of

1	truncated, or full-length HCV RNA (but not intact virions); infectious pseudotyped retroviral HCV particles have been shown to yield functioning envelope proteins. In 2005, complete replication of HCV and intact 55-nm virions were described in cell culture systems. HCV entry into the hepatocyte occurs via the nonliver-specific CD81 receptor and the liver-specific tight junction protein claudin-1. A growing list of additional host receptors to which HCV binds on cell entry includes occludin, low-density lipoprotein receptors, glycosaminoglycans, scavenger receptor B1, and epidermal growth factor receptor, among others. Relying on the same assembly and secretion pathway as low-density and very-low-density lipoproteins, HCV is a lipoviroparticle and masquerades as a lipoprotein, which may limit its visibility to the adaptive immune system and which may explain its ability to evade immune containment and clearance. After viral entry and uncoating, translation is initiated by the IRES on

1	its visibility to the adaptive immune system and which may explain its ability to evade immune containment and clearance. After viral entry and uncoating, translation is initiated by the IRES on the endoplasmic reticulum tion, anti-HDV is low-titer and transient, AA rarely remaining detectable beyond the Helicase

1	Envelope Serine RNA-dependent clearance of HBsAg and HDV antigen. Core glycoproteins protease RNA polymerase antigen in the liver and HDV RNA in serum and liver can be detected during HDV replication. Conserved Hypervariable

1	Hepatitis C Hepatitis C virus, which, FIGURE 360-6 Organization of the hepatitis C virus genome and its associated, 3000-aminobefore its identification was labeled acid (AA) proteins. The three structural genes at the 5’ end are the core region, C, which codes “non-A, non-B hepatitis,” is a linear, sin-for the nucleocapsid, and the envelope regions, E1 and E2, which code for envelope glycoproteins. gle-strand, positive-sense, 9600-nucleo-The 5’ untranslated region and the C region are highly conserved among isolates, whereas the tide RNA virus, the genome of which envelope domain E2 contains the hypervariable region. At the 3’ end are seven nonstructural (NS) is similar in organization to that of fla-regions—p7, a membrane protein adjacent to the structural proteins that appears to function as an viviruses and pestiviruses; HCV is the ion channel; NS2, which codes for a cysteine protease; NS3, which codes for a serine protease and only member of the genus Hepacivirus an RNA helicase;

1	as an viviruses and pestiviruses; HCV is the ion channel; NS2, which codes for a cysteine protease; NS3, which codes for a serine protease and only member of the genus Hepacivirus an RNA helicase; NS4 and NS4B; NS5A, a multifunctional membrane-associated phosphoprotein, in the family Flaviviridae. The HCV an essential component of the viral replication membranous web; and NS5B, which codes for an genome contains a single, large open RNA-dependent RNA polymerase. After translation of the entire polyprotein, individual proteins are reading frame (gene) that codes for a cleaved by both host and viral proteases.

1	In chronic HDV infection, anti-HDV circulates in high titer, and both IgM and IgG anti-HDV can be detected. HDV 5' 3'C E1 E2 NS2 NS3 NS4B NS5A NS5B 2010 membrane, and the HCV polyprotein is cleaved during translation and posttranslationally by host cellular proteases as well as HCV NS2-3 and NS3-4A proteases. Host cofactors involved in HCV replication include cyclophilin A, which binds to NS5A and yields conformational changes required for viral replication, and liver-specific host microRNA miR-122. At least six distinct major genotypes (and a minor genotype 7), as well as >50 subtypes within genotypes, of HCV have been identified by nucleotide sequencing. Genotypes differ from one another in sequence homology by ≥30%, and subtypes differ by approximately 20%. Because divergence of HCV isolates within a genotype or subtype and within the same host may vary insufficiently to define a distinct genotype, these intragenotypic differences are referred to as quasispecies and differ in

1	isolates within a genotype or subtype and within the same host may vary insufficiently to define a distinct genotype, these intragenotypic differences are referred to as quasispecies and differ in sequence homology by only a few percent. The genotypic and quasispecies diversity of HCV, resulting from its high mutation rate, interferes with effective humoral immunity. Neutralizing antibodies to HCV have been demonstrated, but they tend to be short lived, and HCV infection does not induce lasting immunity against reinfection with different virus isolates or even the same virus isolate. Thus, neither heterologous nor homologous immunity appears to develop commonly after acute HCV infection. Some HCV genotypes are distributed worldwide, whereas others are more geographically confined (see “Epidemiology and Global Features”). In addition, differences exist among genotypes in responsiveness to antiviral therapy but not in pathogenicity or clinical progression (except for genotype 3, in

1	“Epidemiology and Global Features”). In addition, differences exist among genotypes in responsiveness to antiviral therapy but not in pathogenicity or clinical progression (except for genotype 3, in which hepatic steatosis and clinical progression are more likely). Currently available, third-generation immunoassays, which incorporate proteins from the core, NS3, and NS5 regions, detect anti-HCV antibodies during acute infection. The most sensitive indicator of HCV infection is the presence of HCV RNA, which requires molecular amplification by PCR or transcription-mediated amplification (TMA) (Fig. 360-7). To allow standardization of the quantification of HCV RNA among laboratories and commercial assays, HCV RNA is reported as international units (IUs) per milliliter; quantitative assays with a broad dynamic range are available that allow detection of HCV RNA with a sensitivity as low as 5 IU/mL. HCV RNA can be detected within a few days of exposure to HCV—well before the appearance of

1	a broad dynamic range are available that allow detection of HCV RNA with a sensitivity as low as 5 IU/mL. HCV RNA can be detected within a few days of exposure to HCV—well before the appearance of anti-HCV—and tends to persist for the duration of HCV infection. Application of sensitive molecular probes for HCV RNA has revealed the presence of replicative HCV in peripheral blood lymphocytes of infected persons; however, as is the case for HBV in lymphocytes, the clinical relevance of HCV lymphocyte infection is not known.

1	Hepatitis E Previously labeled epidemic or enterically transmitted non-A, non-B hepatitis, HEV is an enterically transmitted virus that causes clinically apparent hepatitis primarily in India, Asia, Africa, and Central America; in those geographic areas, HEV is the most common cause of acute hepatitis; one-third of the global population appears to have been infected. This agent, with epidemiologic features resembling those of hepatitis A, is a 27to 34-nm, nonenveloped, HAV-like virus with a 7200-nucleotide, single-strand, positive-sense RNA genome. HEV has three open reading frames (ORF) (genes), the largest of FIGURE 360-7 Scheme of typical laboratory features during acute hepatitis C progressing to chronicity. Hepatitis C virus (HCV) RNA is the first detectable event, preceding alanine aminotransferase (ALT) elevation and the appearance of anti-HCV.

1	which, ORF1, encodes nonstructural proteins involved in virus replication. A middle-sized gene, ORF2, encodes the nucleocapsid protein, the major nonstructural protein, and the smallest, ORF3, encodes a structural protein whose function remains undetermined. All HEV isolates appear to belong to a single serotype, despite genomic heterogeneity of up to 25% and the existence of five genotypes, only four of which have been detected in humans; genotypes 1 and 2 appear to be more virulent, whereas genotypes 3 and 4 are more attenuated and account for subclinical infections. Contributing to the perpetuation of this virus are animal reservoirs, most notably in swine. No genomic or antigenic homology, however, exists between HEV and HAV or other picornaviruses; and HEV, although resembling caliciviruses, is sufficiently distinct from any known agent to merit its own classification as a unique genus, Hepevirus, within the family Hepeviridae. The virus has been detected in stool, bile, and

1	is sufficiently distinct from any known agent to merit its own classification as a unique genus, Hepevirus, within the family Hepeviridae. The virus has been detected in stool, bile, and liver and is excreted in the stool during the late incubation period. Both IgM anti-HEV during early acute infection and IgG anti-HEV predominating after the first 3 months can be detected. Currently, availability and reliability of serologic/virologic testing for HEV infection is limited but can be done in specialized laboratories (e.g., the Centers for Disease Control and Prevention).

1	Under ordinary circumstances, none of the hepatitis viruses is known to be directly cytopathic to hepatocytes. Evidence suggests that the clinical manifestations and outcomes after acute liver injury associated with viral hepatitis are determined by the immunologic responses of the host. Among the viral hepatitides, the immunopathogenesis of hepatitis B and C has been studied most extensively.

1	Hepatitis B For HBV, the existence of inactive hepatitis B carriers with normal liver histology and function suggests that the virus is not directly cytopathic. The fact that patients with defects in cellular immune competence are more likely to remain chronically infected rather than to clear HBV supports the role of cellular immune responses in the pathogenesis of hepatitis B–related liver injury. The model that has the most experimental support involves cytolytic T cells sensitized specifically to recognize host and hepatitis B viral antigens on the liver cell surface. Nucleocapsid proteins (HBcAg and possibly HBeAg), present on the cell membrane in minute quantities, are the viral target antigens that, with host antigens, invite cytolytic T cells to destroy HBV-infected hepatocytes. Differences in the robustness and broad polyclonality of CD8+ cytolytic T cell responsiveness; in the level of HBV-specific helper CD4+ T cells; in attenuation, depletion, and exhaustion of

1	Differences in the robustness and broad polyclonality of CD8+ cytolytic T cell responsiveness; in the level of HBV-specific helper CD4+ T cells; in attenuation, depletion, and exhaustion of virus-specific T cells; in viral T cell epitope escape mutations that allow the virus to evade T cell containment; and in the elaboration of antiviral cytokines by T cells have been invoked to explain differences in outcomes between those who recover after acute hepatitis and those who progress to chronic hepatitis, or between those with mild and those with severe (fulminant) acute HBV infection.

1	Although a robust cytolytic T cell response occurs and eliminates virus-infected liver cells during acute hepatitis B, >90% of HBV DNA has been found in experimentally infected chimpanzees to disappear from the liver and blood before maximal T cell infiltration of the liver and before most of the biochemical and histologic evidence of liver injury. This observation suggests that components of the innate immune system and inflammatory cytokines, independent of cytopathic antiviral mechanisms, participate in the early immune response to HBV infection; this effect has been shown to represent elimination of HBV replicative intermediates from the cytoplasm and covalently closed circular viral DNA from the nucleus of infected hepatocytes. In turn, the innate immune response to HBV infection is mediated largely by natural killer (NK) cell cytotoxicity, activated by immunosuppressive cytokines (e.g., interleukin [IL] 10 and transforming growth factor [TGF] β), reduced signals from inhibitory

1	mediated largely by natural killer (NK) cell cytotoxicity, activated by immunosuppressive cytokines (e.g., interleukin [IL] 10 and transforming growth factor [TGF] β), reduced signals from inhibitory receptor expression (e.g., major histocompatibility complex), or increased signals from activating receptor expression on infected hepatocytes. In addition, NK cells reduce helper CD4+ cells, which results in reduced CD8+ cells and exhaustion of the virus-specific T cell response to HBV infection. Ultimately, HBV-HLA-specific cytolytic T cell responses of the adaptive immune system are felt to be responsible for recovery from HBV infection.

1	Debate continues over the relative importance of viral and host factors in the pathogenesis of HBV-associated liver injury and its outcome. As noted above, precore genetic mutants of HBV have been associated with the more severe outcomes of HBV infection (severe chronic and fulminant hepatitis), suggesting that, under certain circumstances, relative pathogenicity is a property of the virus, not the host. The fact that concomitant HDV and HBV infections are associated with more severe liver injury than HBV infection alone and the fact that cells transfected in vitro with the gene for HDV antigen express HDV antigen and then become necrotic in the absence of any immunologic influences are also consistent with a viral effect on pathogenicity. Similarly, in patients who undergo liver transplantation for end-stage chronic hepatitis B, occasionally, rapidly progressive liver injury appears in the new liver. This clinical pattern is associated with an unusual histologic pattern in the new

1	for end-stage chronic hepatitis B, occasionally, rapidly progressive liver injury appears in the new liver. This clinical pattern is associated with an unusual histologic pattern in the new liver, fibrosing cholestatic hepatitis, which, ultrastructurally, appears to represent a choking of the cell with overwhelming quantities of HBsAg. This observation suggests that, under the influence of the potent immunosuppressive agents required to prevent allograft rejection, HBV may have a direct cytopathic effect on liver cells, independent of the immune system.

1	Although the precise mechanism of liver injury in HBV infection remains elusive, studies of nucleocapsid proteins have shed light on the profound immunologic tolerance to HBV of babies born to mothers with highly replicative (HBeAg-positive), chronic HBV infection. In HBeAg-expressing transgenic mice, in utero exposure to HBeAg, which is sufficiently small to traverse the placenta, induces T cell tolerance to both nucleocapsid proteins. This, in turn, may explain why, when infection occurs so early in life, immunologic clearance does not occur, and protracted, lifelong infection ensues.

1	An important distinction should be drawn between HBV infection acquired at birth, common in endemic areas, such as East Asia, and infection acquired in adulthood, common in the West. Infection in the neonatal period is associated with the acquisition of high-level immunologic tolerance to HBV and absence of an acute hepatitis illness, but the almost invariable establishment of chronic, often lifelong infection. Neonatally acquired HBV infection can culminate decades later in cirrhosis and hepatocellular carcinoma (see “Complications and Sequelae”). In contrast, when HBV infection is acquired during adolescence or early adulthood, the host immune response to HBV-infected hepatocytes tends to be robust, an acute hepatitis-like illness is the rule, and failure to recover is the exception. After adulthood-acquired infection, chronicity is uncommon, and the risk of hepatocellular carcinoma is very low. Based on these observations, some authorities categorize HBV infection into an

1	After adulthood-acquired infection, chronicity is uncommon, and the risk of hepatocellular carcinoma is very low. Based on these observations, some authorities categorize HBV infection into an “immunotolerant” phase, an “immunoreactive” phase, and an “inactive” phase. This somewhat simplistic formulation does not apply at all to the typical adult in the West with self-limited acute hepatitis B, in whom no period of immunologic tolerance occurs. Even among those with neonatally acquired HBV infection, in whom immunologic tolerance is established definitively, intermittent bursts of hepatic necroinflammatory activity punctuate the early decades of life during which liver injury appears to be quiescent (labeled by some as the “immunotolerant” phase). In addition, even when clinically apparent liver injury and progressive fibrosis emerge during later decades (the so-called immunoreactive, or immunointolerant, phase), the level of immunologic tolerance to HBV remains substantial. More

1	liver injury and progressive fibrosis emerge during later decades (the so-called immunoreactive, or immunointolerant, phase), the level of immunologic tolerance to HBV remains substantial. More accurately, in patients with neonatally acquired HBV infection, a dynamic equilibrium exists between tolerance and intolerance, the outcome of which determines the clinical expression of chronic infection. Persons infected as neonates tend to have a relatively higher level of immunologic tolerance during the early decades of life and a relatively lower level (but only rarely a loss) of tolerance in the later decades of life.

1	Hepatitis C Cell-mediated immune responses and elaboration by T cells of antiviral cytokines contribute to the multicellular innate and adaptive immune responses involved in the containment of infection and pathogenesis of liver injury associated with hepatitis C. The fact that HCV is so efficient in evading these immune mechanisms is a testament to its highly evolved ability to disrupt host immune responses at multiple levels. After exposure to HCV, the host cell identifies viral 2011 product motifs (pattern recognition receptors) that distinguish the virus from “self,” resulting in the elaboration of interferons and other cytokines that result in activation of innate and adaptive immune responses. Intrahepatic HLA class I restricted cytolytic T cells directed at nucleocapsid, envelope, and nonstructural viral protein antigens have been demonstrated in patients with chronic hepatitis C; however, such virus-specific cytolytic T cell responses do not correlate adequately with the

1	and nonstructural viral protein antigens have been demonstrated in patients with chronic hepatitis C; however, such virus-specific cytolytic T cell responses do not correlate adequately with the degree of liver injury or with recovery. Yet, a consensus has emerged supporting a role in the pathogenesis of HCV-associated liver injury of virus-activated CD4+ helper T cells that stimulate, via the cytokines they elaborate, HCV-specific CD8+ cytotoxic T cells. These responses appear to be more robust (higher in number, more diverse in viral antigen specificity, more functionally effective, and more long lasting) in those who recover from HCV than in those who have chronic infection. Contributing to chronic infection are a CD4+ proliferative defect that results in rapid contraction of CD4+ responses, mutations in CD8+ T cell–targeted viral epitopes that allow HCV to escape immune-mediated clearance, and upregulation of inhibi tory receptors on functionally impaired, exhausted T cells.

1	responses, mutations in CD8+ T cell–targeted viral epitopes that allow HCV to escape immune-mediated clearance, and upregulation of inhibi tory receptors on functionally impaired, exhausted T cells. Although attention has focused on adaptive immunity, HCV proteins have been shown to interfere with innate immunity by resulting in blocking of type 1 interferon responses and inhibition of interferon signaling and effector molecules in the interferon signaling cascade. Several HLA alleles have been linked with self-limited hepatitis C, the most convincing of which is the CC haplotype of the IL28B gene, which codes for interferon λ3, a component of innate immune antiviral defense. The IL28B association is even stronger when combined with HLA class II DQB1*03:01. The link between non-CC IL28B polymorphisms and failure to clear HCV infection has been explained by a chromosome 19q13.13 frameshift variant upstream of IL28B, the ΔG polymorphism of which creates an ORF in a novel interferon gene

1	and failure to clear HCV infection has been explained by a chromosome 19q13.13 frameshift variant upstream of IL28B, the ΔG polymorphism of which creates an ORF in a novel interferon gene (IFN-λ4) associated with impaired HCV clearance. Also shown to contribute to limiting HCV infection are NK cells of the innate immune system that function when HLA class I molecules required for successful adaptive immunity are underexpressed. Both peripheral and intrahepatic NK cell cytotoxicity are dysfunctional in persistent HCV infection. Adding to the complexity of the immune response, HCV core, NS4B, and NS5B have been shown to suppress the immunoregulatory nuclear factor (NF)-κB pathway, resulting in reduced antiapoptotic proteins and a resultant increased vulnerability to tumor necrosis factor (TNF) α–mediated cell death. Patients with hepatitis C and unfavorable (non-CC, associated with reduced HCV clearance) IL28B alleles have been shown to have depressed NK cell/innate immune function. Of

1	α–mediated cell death. Patients with hepatitis C and unfavorable (non-CC, associated with reduced HCV clearance) IL28B alleles have been shown to have depressed NK cell/innate immune function. Of note, the emergence of substantial viral quasispecies diversity and HCV sequence variation allow the virus to evade attempts by the host to contain HCV infection by both humoral and cellular immunity.

1	Finally, cross-reactivity between viral antigens (HCV NS3 and NS5A) and host autoantigens (cytochrome P450 2D6) has been invoked to explain the association between hepatitis C and a subset of patients with autoimmune hepatitis and antibodies to liver-kidney microsomal (LKM) antigen (anti-LKM) (Chap. 362). Immune complex–mediated tissue damage appears to play a pathogenetic role in the extrahepatic manifestations of acute hepatitis B. The occasional prodromal serum sickness–like syndrome observed in acute hepatitis B appears to be related to the deposition in tissue blood vessel walls of HBsAg-anti-HBs circulating immune complexes, leading to activation of the complement system and depressed serum complement levels.

1	In patients with chronic hepatitis B, other types of immune-complex disease may be seen. Glomerulonephritis with the nephrotic syndrome is observed occasionally; HBsAg, immunoglobulin, and C3 deposition has been found in the glomerular basement membrane. Whereas generalized vasculitis (polyarteritis nodosa) develops in considerably fewer than 1% of patients with chronic HBV infection, 20–30% of patients with polyarteritis nodosa have HBsAg in serum (Chap. 385). In these patients, the affected smalland medium-size arterioles contain 2012 HBsAg, immunoglobulins, and complement components. Another extrahepatic manifestation of viral hepatitis, essential mixed cryoglobulinemia (EMC), was reported initially to be associated with hepatitis

1	B. The disorder is characterized clinically by arthritis, cutaneous vasculitis (palpable purpura), and occasionally, glomerulonephritis and serologically by the presence of circulating cryoprecipitable immune complexes of more than one immunoglobulin class (Chaps. 338 and 385). Many patients with this syndrome have chronic liver disease, but the association with HBV infection is limited; instead, a substantial proportion has chronic HCV infection, with circulating immune complexes containing HCV RNA. Immune-complex glomerulonephritis is another recognized extrahepatic manifestation of chronic hepatitis C.

1	The typical morphologic lesions of all types of viral hepatitis are similar and consist of panlobular infiltration with mononuclear cells, hepatic cell necrosis, hyperplasia of Kupffer cells, and variable degrees of cholestasis. Hepatic cell regeneration is present, as evidenced by numerous mitotic figures, multinucleated cells, and “rosette” or “pseudoacinar” formation. The mononuclear infiltration consists primarily of small lymphocytes, although plasma cells and eosinophils occasionally are present. Liver cell damage consists of hepatic cell degeneration and necrosis, cell dropout, ballooning of cells, and acidophilic degeneration of hepatocytes (forming so-called Councilman or apoptotic bodies). Large hepatocytes with a ground-glass appearance of the cytoplasm may be seen in chronic but not in acute HBV infection; these cells contain HBsAg and can be identified histochemically with orcein or aldehyde fuchsin. In uncomplicated viral hepatitis, the reticulin framework is preserved.

1	In hepatitis C, the histologic lesion is often remarkable for a relative paucity of inflammation, a marked increase in activation of sinusoidal lining cells, lymphoid aggregates, the presence of fat (more frequent in genotype 3 and linked to increased fibrosis), and, occasionally, bile duct lesions in which biliary epithelial cells appear to be piled up without interruption of the basement membrane. Occasionally, microvesicular steatosis occurs in hepatitis D. In hepatitis E, a common histologic feature is marked cholestasis. A cholestatic variant of slowly resolving acute hepatitis A also has been described.

1	A more severe histologic lesion, bridging hepatic necrosis, also termed subacute or confluent necrosis or interface hepatitis, is observed occasionally in acute hepatitis. “Bridging” between lobules results from large areas of hepatic cell dropout, with collapse of the reticulin framework. Characteristically, the bridge consists of condensed reticulum, inflammatory debris, and degenerating liver cells that span adjacent portal areas, portal to central veins, or central vein to central vein. This lesion had been thought to have prognostic significance; in many of the originally described patients with this lesion, a subacute course terminated in death within several weeks to months, or severe chronic hepatitis and cirrhosis developed; however, the association between bridging necrosis and a poor prognosis in patients with acute hepatitis has not been upheld. Therefore, although demonstration of this lesion in patients with chronic hepatitis has prognostic significance (Chap. 362), its

1	a poor prognosis in patients with acute hepatitis has not been upheld. Therefore, although demonstration of this lesion in patients with chronic hepatitis has prognostic significance (Chap. 362), its demonstration during acute hepatitis is less meaningful, and liver biopsies to identify this lesion are no longer undertaken routinely in patients with acute hepatitis. In massive hepatic necrosis (fulminant hepatitis, “acute yellow atrophy”), the striking feature at postmortem examination is the finding of a small, shrunken, soft liver. Histologic examination reveals massive necrosis and dropout of liver cells of most lobules with extensive collapse and condensation of the reticulin framework. When histologic documentation is required in the management of fulminant or very severe hepatitis, a biopsy can be done by the angiographically guided transjugular route, which permits the performance of this invasive procedure in the presence of severe coagulopathy.

1	Immunohistochemical and electron-microscopic studies have localized HBsAg to the cytoplasm and plasma membrane of infected liver cells. In contrast, HBcAg predominates in the nucleus, but, occasionally, scant amounts are also seen in the cytoplasm and on the cell membrane. HDV antigen is localized to the hepatocyte nucleus, whereas HAV, HCV, and HEV antigens are localized to the cytoplasm. Before the availability of serologic tests for hepatitis viruses, all viral hepatitis cases were labeled either as “infectious” or “serum” hepatitis. Modes of transmission overlap, however, and a clear distinction among the different types of viral hepatitis cannot be made solely on the basis of clinical or epidemiologic features (Table 360-2). The most accurate means to distinguish the various types of viral hepatitis involves specific serologic testing.

1	Hepatitis A This agent is transmitted almost exclusively by the fecal-oral route. Person-to-person spread of HAV is enhanced by poor personal hygiene and overcrowding; large outbreaks as well as sporadic cases have been traced to contaminated food, water, milk, frozen raspberries and strawberries, green onions imported from Mexico, and shellfish. Intrafamily and intrainstitutional spread are also common. Early epidemiologic observations supported a predilection for hepatitis A to occur in late fall and early winter. In temperate zones, epidemic waves have been recorded every 5–20 years as new segments of nonimmune population appeared; however, in developed countries, the incidence of hepatitis A has been declining, presumably as a function of improved sanitation, and these cyclic patterns are no longer observed. No HAV carrier state has been identified after acute hepatitis A; perpetuation of the virus in nature depends presumably on nonepidemic, inapparent subclinical infection,

1	are no longer observed. No HAV carrier state has been identified after acute hepatitis A; perpetuation of the virus in nature depends presumably on nonepidemic, inapparent subclinical infection, ingestion of contaminated food or water in, or imported from, endemic areas, and/or contamination linked to environmental reservoirs.

1	In the general population, anti-HAV, a marker for previous HAV infection, increases in prevalence as a function of increasing age and of decreasing socioeconomic status. In the 1970s, serologic evidence of prior hepatitis A infection occurred in ~40% of urban populations in the United States, most of whose members never recalled having had a symptomatic case of hepatitis. In subsequent decades, however, the prevalence of anti-HAV has been declining in the United States. In developing countries, exposure, infection, and subsequent immunity are almost universal in childhood. As the frequency of subclinical childhood infections declines in developed countries, a susceptible cohort of adults emerges. Hepatitis A tends to be more symptomatic in adults; therefore, paradoxically, as the frequency of HAV infection declines, the likelihood of clinically apparent, even severe, HAV illnesses increases in the susceptible adult population. Travel to endemic areas is a common source of infection

1	of HAV infection declines, the likelihood of clinically apparent, even severe, HAV illnesses increases in the susceptible adult population. Travel to endemic areas is a common source of infection for adults from nonendemic areas. More recently recognized epidemiologic foci of HAV infection include child care centers, neonatal intensive care units, promiscuous men who have sex with men, injection drug users, and unvaccinated close contacts of newly arrived international adopted children, most of whom emanate from countries with intermediate-to-high hepatitis A endemicity. Although hepatitis A is rarely bloodborne, several outbreaks have been recognized in recipients of clotting-factor concentrates. In the United States, the introduction of hepatitis A vaccination programs among children from high-incidence states has resulted in a >70% reduction in the annual incidence of new HAV infections and has shifted the burden of new infections from children to young adults. In the most recent,

1	high-incidence states has resulted in a >70% reduction in the annual incidence of new HAV infections and has shifted the burden of new infections from children to young adults. In the most recent, 1999–2006 U.S. Public Health Service National Health and Nutrition Examination Survey (NHANES), the prevalence of anti-HAV in the U.S. population was 35%, representing (compared to the 1988–1994 survey) a stable frequency of infection and natural immunity in adults >19 years old but an increase in vaccine-induced immunity for children age 6–19 years.

1	Hepatitis B Percutaneous inoculation has long been recognized as a major route of hepatitis B transmission, but the outmoded designation “serum hepatitis” is an inaccurate label for the epidemiologic spectrum of HBV infection. As detailed below, most of the hepatitis transmitted by blood transfusion is not caused by HBV; moreover, in approximately two-thirds of patients with acute type B hepatitis, no history of an identifiable percutaneous exposure can be elicited. We now recognize that many cases of hepatitis B result from less obvious modes of nonpercutaneous or covert percutaneous transmission. HBsAg has been identified in almost every body fluid from aPrimarily with HIV co-infection and high-level viremia in index case; risk ~5%. bUp to 5% in acute HBV/HDV co-infection; up to 20% in HDV superinfection of chronic HBV infection. c Varies considerably throughout the world and in subpopulations within countries; see text. dIn acute HBV/HDV co-infection, the frequency of chronicity is

1	superinfection of chronic HBV infection. c Varies considerably throughout the world and in subpopulations within countries; see text. dIn acute HBV/HDV co-infection, the frequency of chronicity is the same as that for HBV; in HDV superinfection, chronicity is invariable. e10–20% in pregnant women. f Except as observed in immunosuppressed liver allograft recipients or other immunosuppressed hosts. gCommon in Mediterranean countries; rare in North America and western Europe. hAnecdotal reports and retrospective studies suggest that pegylated interferon and/or ribavirin are effective in treating chronic hepatitis E, observed in immunocompromised persons.

1	Abbreviation: HBIG, hepatitis B immunoglobulin. See text for other abbreviations. infected persons, and at least some of these body fluids—most notably semen and saliva—are infectious, albeit less so than serum, when administered percutaneously or nonpercutaneously to experimental animals. Among the nonpercutaneous modes of HBV transmission, oral ingestion has been documented as a potential but inefficient route of exposure. By contrast, the two nonpercutaneous routes considered to have the greatest impact are intimate (especially sexual) contact and perinatal transmission.

1	In sub-Saharan Africa, intimate contact among toddlers is considered instrumental in contributing to the maintenance of the high frequency of hepatitis B in the population. Perinatal transmission occurs primarily in infants born to mothers with chronic hepatitis B or (rarely) mothers with acute hepatitis B during the third trimester of pregnancy or during the early postpartum period. Perinatal transmission is uncommon in North America and western Europe but occurs with great frequency and is the most important mode of HBV perpetuation in East Asia and developing countries. Although the precise mode of perinatal transmission is unknown, and although ~10% of infections may be acquired in utero, epidemiologic evidence suggests that most infections occur approximately at the time of delivery and are not related to breast-feeding. The likelihood of perinatal transmission of HBV correlates with the presence of HBeAg and high-level viral replication; 90% of HBeAg-positive mothers but only

1	and are not related to breast-feeding. The likelihood of perinatal transmission of HBV correlates with the presence of HBeAg and high-level viral replication; 90% of HBeAg-positive mothers but only 10–15% of anti-HBe-positive mothers transmit HBV infection to their offspring. In most cases, acute infection in the neonate is clinically asymptomatic, but the child is very likely to remain chronically infected.

1	The >350–400 million HBsAg carriers in the world constitute the main reservoir of hepatitis B in human beings. Whereas serum HBsAg is infrequent (0.1–0.5%) in normal populations in the United States and western Europe, a prevalence of up to 5–20% has been found in East Asia and in some tropical countries; in persons with Down’s syndrome, lepromatous leprosy, leukemia, Hodgkin’s disease, or polyarteritis nodosa; in patients with chronic renal disease on hemodialysis; and in injection drug users.

1	Other groups with high rates of HBV infection include spouses of acutely infected persons; sexually promiscuous persons (especially promiscuous men who have sex with men); health care workers exposed to blood; persons who require repeated transfusions especially with pooled blood-product concentrates (e.g., hemophiliacs); residents and staff of custodial institutions for the developmentally handicapped; prisoners; and, to a lesser extent, family members of chronically infected patients. In volunteer blood donors, the prevalence of anti-HBs, a reflection of previous HBV infection, ranges from 5–10%, but the prevalence is higher in lower socioeconomic strata, older age groups, and per-sons—including those mentioned above—exposed to blood products. Because of highly sensitive virologic screening of donor blood, the risk of acquiring HBV infection from a blood transfusion is 1 in 230,000.

1	Prevalence of infection, modes of transmission, and human behavior conspire to mold geographically different epidemiologic patterns of HBV infection. In East Asia and Africa, hepatitis B, a disease of the newborn and young children, is perpetuated by a cycle of maternal-neonatal spread. In North America and western Europe, hepatitis B is primarily a disease of adolescence and early adulthood, the time of life when intimate sexual contact and recreational and occupational percutaneous exposures tend to occur. To some degree, however, this dichotomy between high-prevalence and low-prevalence geographic regions has been minimized by immigration from high-prevalence to low-prevalence areas. The introduction of hepatitis B vaccine in the early 1980s and adoption of universal childhood vaccination policies in many countries resulted in a dramatic, ~90% decline in the incidence of new HBV infections in those countries as well as in the dire

1	Persons born in countries/regions with a high (≥8%) and intermediate (≥2%) prevalence of HBV infection including immigrants and adopted children and including persons born in the United States who were not vaccinated as infants and whose parents emigrated from areas of high HBV endemicity Household and sexual contacts of persons with hepatitis B Babies born to HBsAg-positive mothers Persons with multiple sexual contacts or a history of sexually transmitted disease Men who have sex with men Inmates of correctional facilities Persons with elevated alanine or aspartate aminotransferase levels Persons with HCV or HIV infection Persons who are the source of blood or body fluids that would be an indication for postexposure prophylaxis (e.g., needlestick, mucosal exposure, sexual assault) consequences of chronic infection, including hepatocellular carcinoma. Populations and groups for whom HBV infection screening is recommended are listed in Table 360-3.

1	Hepatitis D Infection with HDV has a worldwide distribution, but two epidemiologic patterns exist. In Mediterranean countries (northern Africa, southern Europe, the Middle East), HDV infection is endemic among those with hepatitis B, and the disease is transmitted predominantly by nonpercutaneous means, especially close personal contact. In nonendemic areas, such as the United States and northern Europe, HDV infection is confined to persons exposed frequently to blood and blood products, primarily injection drug users and hemophiliacs. HDV infection can be introduced into a population through drug users or by migration of persons from endemic to nonendemic areas. Thus, patterns of population migration and human behavior facilitating percutaneous contact play important roles in the introduction and amplification of HDV infection. Occasionally, the migrating epidemiology of hepatitis D is expressed in explosive outbreaks of severe hepatitis, such as those that have occurred in remote

1	and amplification of HDV infection. Occasionally, the migrating epidemiology of hepatitis D is expressed in explosive outbreaks of severe hepatitis, such as those that have occurred in remote South American villages as well as in urban centers in the United States. Ultimately, such outbreaks of hepatitis D—either of co-infections with acute hepatitis B or of superinfections in those already infected with HBV—may blur the distinctions between endemic and nonendemic areas. On a global scale, HDV infection declined at the end of the 1990s. Even in Italy, an HDV-endemic area, public health measures introduced to control HBV infection resulted during the 1990s in a 1.5%/year reduction in the prevalence of HDV infection. Still, the frequency of HDV infection during the first decade of the twenty-first century has not fallen below levels reached during the 1990s; the reservoir has been sustained by survivors infected during 1970–1980 and recent immigrants from still-endemic to less-endemic

1	century has not fallen below levels reached during the 1990s; the reservoir has been sustained by survivors infected during 1970–1980 and recent immigrants from still-endemic to less-endemic countries.

1	Hepatitis C Routine screening of blood donors for HBsAg and the elimination of commercial blood sources in the early 1970s reduced the frequency of, but did not eliminate, transfusion-associated hepatitis. During the 1970s, the likelihood of acquiring hepatitis after transfusion of voluntarily donated, HBsAg-screened blood was ~10% per patient (up to 0.9% per unit transfused); 90–95% of these cases were classified, based on serologic exclusion of hepatitis A and B, as “non-A, non-B” hepatitis. For patients requiring transfusion of pooled products, such as clotting factor concentrates, the risk was even higher, up to 20–30%.

1	During the 1980s, voluntary self-exclusion of blood donors with risk factors for AIDS and then the introduction of donor screening for anti-HIV reduced further the likelihood of transfusion-associated hepatitis to <5%. During the late 1980s and early 1990s, the introduction first of “surrogate” screening tests for non-A, non-B hepatitis (alanine aminotransferase [ALT] and anti-HBc, both shown to identify blood donors with a higher likelihood of transmitting non-A, non-B hepatitis to recipients) and, subsequently, after the discovery of HCV, first-generation immunoassays for anti-HCV reduced the frequency of transfusion-associated hepatitis even further. A prospective analysis of transfusion-associated hepatitis conducted between 1986 and 1990 showed that the frequency of transfusion-associated hepatitis at one urban university hospital fell from a baseline of 3.8% per patient (0.45% per unit transfused) to 1.5% per patient (0.19% per unit) after the introduction of surrogate testing

1	hepatitis at one urban university hospital fell from a baseline of 3.8% per patient (0.45% per unit transfused) to 1.5% per patient (0.19% per unit) after the introduction of surrogate testing and to 0.6% per patient (0.03% per unit) after the introduction of first-generation anti-HCV assays. The introduction of second-generation anti-HCV assays reduced the frequency of transfusion-associated hepatitis C to almost imperceptible levels—1 in 100,000—and these gains were reinforced by the application of third-generation anti-HCV assays and of automated PCR testing of donated blood for HCV RNA, which has resulted in a reduction in the risk of transfusion-associated HCV infection to 1 in 2.3 million transfusions.

1	In addition to being transmitted by transfusion, hepatitis C can be transmitted by other percutaneous routes, such as injection drug use. In addition, this virus can be transmitted by occupational exposure to blood, and the likelihood of infection is increased in hemodialysis units. Although the frequency of transfusion-associated hepatitis C fell as a result of blood-donor screening, the overall frequency of hepatitis C remained the same until the early 1990s, when the overall frequency fell by 80%, in parallel with a reduction in the number of new cases in injection drug users. After the exclusion of anti-HCV-positive plasma units from the donor pool, rare, sporadic instances have occurred of hepatitis C among recipients of immunoglobulin (Ig) preparations for intravenous (but not intramuscular) use.

1	Serologic evidence for HCV infection occurs in 90% of patients with a history of transfusion-associated hepatitis (almost all occurring before 1992, when second-generation HCV screening tests were introduced); hemophiliacs and others treated with clotting factors; injection drug users; 60–70% of patients with sporadic “non-A, non-B” hepatitis who lack identifiable risk factors; 0.5% of volunteer blood donors; and, in a survey conducted in the United States between 1999 and 2002, 1.6% of the general population in the United States, which translates into 4.1 million persons (3.2 million with viremia), the majority of whom are unaware of their infections. Moreover, such population surveys do not include higher-risk groups such as incarcerated prisoners and active injection drug users, indicating that the actual prevalence is even higher. Comparable frequencies of HCV infection occur in most countries around the world, with 170 million persons infected worldwide, but extraordinarily high

1	that the actual prevalence is even higher. Comparable frequencies of HCV infection occur in most countries around the world, with 170 million persons infected worldwide, but extraordinarily high prevalences of HCV infection occur in certain countries such as Egypt, where >20% of the population (as high as 50% in persons born prior to 1960) in some cities is infected. The high frequency in Egypt is attributable to contaminated equipment used for medical procedures and unsafe injection practices in the 1950s to 1980s (during a campaign to eradicate schistosomiasis with intravenous tartar emetic). In the United States, African Americans and Mexican Americans have higher frequencies of HCV infection than whites. Between 1988 and 1994, 30to 40-year-old adult males had the highest prevalence of HCV infection; however, in a survey conducted between 1999 and 2002, the peak age decile had shifted to those age 40–49 years; an increase in hepatitis C–related mortality has paralleled this secular

1	HCV infection; however, in a survey conducted between 1999 and 2002, the peak age decile had shifted to those age 40–49 years; an increase in hepatitis C–related mortality has paralleled this secular trend, increasing since 1995 predominantly in the 45to 65-year age group. Thus, despite an 80% reduction in new HCV infections during the 1990s, the prevalence of HCV infection in the population was sustained by an aging cohort that had acquired their infections three to four decades earlier, during the 1960s and 1970s, as a result predominantly of self-inoculation with recreational drugs. As death resulting from HIV infection fell after 1999, age-adjusted mortality associated with HCV infection surpassed that of HIV infection in 2007; >70% of HCV-associated deaths occurred in the “baby boomer” cohort born between 1945 and 1965. Compared to the 1.6% prevalence of HCV infection in the population at large, the prevalence in the 1945–1965 birth cohort was 3.2%, representing three-quarters of

1	cohort born between 1945 and 1965. Compared to the 1.6% prevalence of HCV infection in the population at large, the prevalence in the 1945–1965 birth cohort was 3.2%, representing three-quarters of all infected persons. Therefore, in 2012, the Centers for Disease Control and Prevention recommended that all persons born between 1945 and 1965 be screened for hepatitis C, without ascertainment of risk, a recommendation shown to be cost-effective and predicted to identify 800,000 infected persons. Because of the availability of highly effective antiviral therapy, such screening would have the potential to avert 200,000 cases of cirrhosis and 47,000 cases of hepatocellular carcinoma and to prevent 120,000 hepatitis-related deaths.

1	Hepatitis C accounts for 40% of chronic liver disease, is the most frequent indication for liver transplantation, and is estimated to account for 8000–10,000 deaths per year in the United States. The distribution of HCV genotypes varies in different parts of the world. Worldwide, genotype 1 is the most common. In the United States, genotype 1 accounts for 70% of HCV infections, whereas genotypes 2 and 3 account for the remaining 30%; among African Americans, the frequency of genotype 1 is even higher (i.e., 90%). Genotype 4 predominates in Egypt; genotype 5 is localized to South Africa, genotype 6 to Hong Kong, and genotype 7 to Central Africa. Most asymptomatic blood donors found to have anti-HCV and ~20–30% of persons with reported cases of acute hepatitis C do not fall into a recognized risk group; however, many such blood donors do recall risk-associated behaviors when questioned carefully.

1	As a bloodborne infection, HCV potentially can be transmitted sexually and perinatally; however, both of these modes of transmission are inefficient for hepatitis C. Although 10–15% of patients with acute hepatitis C report having potential sexual sources of infection, most studies have failed to identify sexual transmission of this agent. The chances of sexual and perinatal transmission have been estimated to be ~5% but shown in a prospective study to be only 1% between monogamous sexual partners, well below comparable rates for HIV and HBV infections. Moreover, sexual transmission appears to be confined to such subgroups as persons with multiple sexual partners and sexually transmitted diseases. Breast-feeding does not increase the risk of HCV infection between an infected mother and her infant. Infection of health workers is not dramatically higher than among the general population; however, health workers are more likely to acquire HCV infection through accidental needle

1	and her infant. Infection of health workers is not dramatically higher than among the general population; however, health workers are more likely to acquire HCV infection through accidental needle punctures, the efficiency of which is ~3%. Infection of household contacts is rare as well.

1	Besides persons born between 1945 and 1965, other groups with an increased frequency of HCV infection are listed in Table 360-4. In immunosuppressed individuals, levels of anti-HCV may be undetectable, and a diagnosis may require testing for HCV RNA. Although new acute cases of hepatitis C are rare, newly diagnosed cases are common among otherwise healthy persons who experimented briefly with injection drugs, as noted above, three or four decades earlier. Such instances usually remain unrecognized for years, until unearthed by laboratory screening for routine medical examinations, insurance applications, and attempted blood donation. Although, overall, the annual incidence of new HCV infections has continued to fall, the rate of new infections has been increasing since 2002 in a new cohort

1	Persons born between 1945 and 1965 Persons who have ever used injection drugs Persons with HIV infection Hemophiliacs treated with clotting factor concentrates prior to 1987 Persons who have ever undergone long-term hemodialysis Persons with unexplained elevations of aminotransferase levels Transfusion or transplantation recipients prior to July 1992 Recipients of blood or organs from a donor found to be positive for hepatitis C Children born to women with hepatitis C Health care, public safety, and emergency medical personnel following needle injury or mucosal exposure to HCV-contaminated blood Sexual partners of persons with hepatitis C infection of young injection drug users, age 15–24 years (accounting for more 2015 than two-thirds of all acute cases), who, unlike older cohorts, had not learned to take precautions to prevent bloodborne infections.

1	Hepatitis E This type of hepatitis, identified in India, Asia, Africa, the Middle East, and Central America, resembles hepatitis A in its primarily enteric mode of spread. The commonly recognized cases occur after contamination of water supplies such as after monsoon flooding, but sporadic, isolated cases occur. An epidemiologic feature that distinguishes HEV from other enteric agents is the rarity of secondary person-to-person spread from infected persons to their close contacts. Large waterborne outbreaks in endemic areas are linked to genotypes 1 and 2, arise in populations that are immune to HAV, favor young adults, and account for antibody prevalences of 30–80%. In nonendemic areas of the world, such as the United States, clinically apparent acute hepatitis E is extremely rare; however, during the 1988–1994 NHANES survey conducted by the U.S. Public Health Service, the prevalence of anti-HEV was 21%, reflecting subclinical infections, infection with genotypes 3 and 4,

1	rare; however, during the 1988–1994 NHANES survey conducted by the U.S. Public Health Service, the prevalence of anti-HEV was 21%, reflecting subclinical infections, infection with genotypes 3 and 4, predominantly in older males (>60 years). In nonendemic areas, HEV accounts hardly at all for cases of sporadic hepatitis; however, cases imported from endemic areas have been found in the United States. Evidence supports a zoonotic reservoir for HEV primarily in swine, which may account for the mostly subclinical infections in nonendemic areas.

1	CLINICAL AND LABORATORY FEATURES Symptoms and Signs Acute viral hepatitis occurs after an incubation period that varies according to the responsible agent. Generally, incubation periods for hepatitis A range from 15–45 days (mean, 4 weeks), for hepatitis B and D from 30–180 days (mean, 8–12 weeks), for hepatitis C from 15–160 days (mean, 7 weeks), and for hepatitis E from 14–60 days (mean, 5–6 weeks). The prodromal symptoms of acute viral hepatitis are systemic and quite variable. Constitutional symptoms of anorexia, nausea and vomiting, fatigue, malaise, arthralgias, myalgias, headache, photophobia, pharyngitis, cough, and coryza may precede the onset of jaundice by 1–2 weeks. The nausea, vomiting, and anorexia are frequently associated with alterations in olfaction and taste. A low-grade fever between 38° and 39°C (100°–102°F) is more often present in hepatitis A and E than in hepatitis B or C, except when hepatitis B is heralded by a serum sickness–like syndrome; rarely, a fever of

1	fever between 38° and 39°C (100°–102°F) is more often present in hepatitis A and E than in hepatitis B or C, except when hepatitis B is heralded by a serum sickness–like syndrome; rarely, a fever of 39.5°–40°C (103°–104°F) may accompany the constitutional symptoms. Dark urine and clay-colored stools may be noticed by the patient from 1–5 days before the onset of clinical jaundice.

1	With the onset of clinical jaundice, the constitutional prodromal symptoms usually diminish, but in some patients, mild weight loss (2.5–5 kg) is common and may continue during the entire icteric phase. The liver becomes enlarged and tender and may be associated with right upper quadrant pain and discomfort. Infrequently, patients present with a cholestatic picture, suggesting extrahepatic biliary obstruction. Splenomegaly and cervical adenopathy are present in 10–20% of patients with acute hepatitis. Rarely, a few spider angiomas appear during the icteric phase and disappear during convalescence. During the recovery phase, constitutional symptoms disappear, but usually some liver enlargement and abnormalities in liver biochemical tests are still evident. The duration of the posticteric phase is variable, ranging from 2–12 weeks, and is usually more prolonged in acute hepatitis B and C. Complete clinical and biochemical recovery is to be expected 1–2 months after all cases of

1	phase is variable, ranging from 2–12 weeks, and is usually more prolonged in acute hepatitis B and C. Complete clinical and biochemical recovery is to be expected 1–2 months after all cases of hepatitis A and E and 3–4 months after the onset of jaundice in three-quarters of uncomplicated, self-limited cases of hepatitis B and C (among healthy adults, acute hepatitis B is self-limited in 95–99%, whereas hepatitis C is self-limited in only ~15%). In the remainder, biochemical recovery may be delayed. A substantial proportion of patients with viral hepatitis never become icteric.

1	Infection with HDV can occur in the presence of acute or chronic HBV infection; the duration of HBV infection determines the duration of HDV infection. When acute HDV and HBV infection occur simultaneously, clinical and biochemical features may be indistinguishable from those of HBV infection alone, although occasionally they are 2016 more severe. As opposed to patients with acute HBV infection, patients with chronic HBV infection can support HDV replication indefinitely, as when acute HDV infection occurs in the presence of a nonresolving acute HBV infection or, more commonly, when acute hepatitis D is superimposed on underlying chronic hepatitis B. In such cases, the HDV superinfection appears as a clinical exacerbation or an episode resembling acute viral hepatitis in someone already chronically infected with HBV. Superinfection with HDV in a patient with chronic hepatitis B often leads to clinical deterioration (see below). In addition to superinfections with other hepatitis

1	chronically infected with HBV. Superinfection with HDV in a patient with chronic hepatitis B often leads to clinical deterioration (see below). In addition to superinfections with other hepatitis agents, acute hepatitis-like clinical events in persons with chronic hepatitis B may accompany spontaneous HBeAg to anti-HBe seroconversion or spontaneous reactivation (i.e., reversion from relatively nonreplicative to replicative infection). Such reactivations can occur as well in therapeutically immunosuppressed patients with chronic HBV infection when cytotoxic/immunosuppressive drugs are withdrawn; in these cases, restoration of immune competence is thought to allow resumption of previously checked cell-mediated immune cytolysis of HBV-infected hepatocytes. Occasionally, acute clinical exacerbations of chronic hepatitis B may represent the emergence of a precore mutant (see “Virology and Etiology”), and the subsequent course in such patients may be characterized by periodic exacerbations.

1	of chronic hepatitis B may represent the emergence of a precore mutant (see “Virology and Etiology”), and the subsequent course in such patients may be characterized by periodic exacerbations. Cytotoxic chemotherapy can lead to reactivation of chronic hepatitis C as well, and anti-TNF-α therapy can lead to reactivation of both hepatitis B and C.

1	Laboratory Features The serum aminotransferases aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (previously designated SGOT and SGPT) increase to a variable degree during the prodromal phase of acute viral hepatitis and precede the rise in bilirubin level (Figs. 360-2 and 360-4). The level of these enzymes, however, does not correlate well with the degree of liver cell damage. Peak levels vary from 400–4000 IU or more; these levels are usually reached at the time the patient is clinically icteric and diminish progressively during the recovery phase of acute hepatitis. The diagnosis of anicteric hepatitis is based on clinical features and on aminotransferase elevations.

1	Jaundice is usually visible in the sclera or skin when the serum bilirubin value is >43 μmol/L (2.5 mg/dL). When jaundice appears, the serum bilirubin typically rises to levels ranging from 85–340 μmol/L (5–20 mg/dL). The serum bilirubin may continue to rise despite falling serum aminotransferase levels. In most instances, the total bilirubin is equally divided between the conjugated and unconjugated fractions. Bilirubin levels >340 μmol/L (20 mg/dL) extending and persisting late into the course of viral hepatitis are more likely to be associated with severe disease. In certain patients with underlying hemolytic anemia, however, such as glucose-6-phosphate dehydrogenase deficiency and sickle cell anemia, a high serum bilirubin level is common, resulting from superimposed hemolysis. In such patients, bilirubin levels >513 μmol/L (30 mg/dL) have been observed and are not necessarily associated with a poor prognosis.

1	Neutropenia and lymphopenia are transient and are followed by a relative lymphocytosis. Atypical lymphocytes (varying between 2 and 20%) are common during the acute phase. Measurement of the prothrombin time (PT) is important in patients with acute viral hepatitis, because a prolonged value may reflect a severe hepatic synthetic defect, signify extensive hepatocellular necrosis, and indicate a worse prognosis. Occasionally, a prolonged PT may occur with only mild increases in the serum bilirubin and aminotransferase levels. Prolonged nausea and vomiting, inadequate carbohydrate intake, and poor hepatic glycogen reserves may contribute to hypoglycemia noted occasionally in patients with severe viral hepatitis. Serum alkaline phosphatase may be normal or only mildly elevated, whereas a fall in serum albumin is uncommon in uncomplicated acute viral hepatitis. In some patients, mild and transient steatorrhea has been noted, as well as slight microscopic hematuria and minimal proteinuria.

1	A diffuse but mild elevation of the γ globulin fraction is common during acute viral hepatitis. Serum IgG and IgM levels are elevated in about one-third of patients during the acute phase of viral hepatitis, but the serum IgM level is elevated more characteristically during acute hepatitis A. During the acute phase of viral hepatitis, antibodies to smooth muscle and other cell constituents may be present, and low titers of rheumatoid factor, nuclear antibody, and heterophile antibody can also be found occasionally. In hepatitis C and D, antibodies to LKM may occur; however, the species of LKM antibodies in the two types of hepatitis are different from each other as well as from the LKM antibody species characteristic of autoimmune hepatitis type 2 (Chap. 362). The autoantibodies in viral hepatitis are nonspecific and can also be associated with other viral and systemic diseases. In contrast, virus-specific antibodies, which appear during and after hepatitis virus infection, are

1	viral hepatitis are nonspecific and can also be associated with other viral and systemic diseases. In contrast, virus-specific antibodies, which appear during and after hepatitis virus infection, are serologic markers of diagnostic importance.

1	As described above, serologic tests are available routinely with which to establish a diagnosis of hepatitis A, B, D, and C. Tests for fecal or serum HAV are not routinely available. Therefore, a diagnosis of hepatitis A is based on detection of IgM anti-HAV during acute illness (Fig. 360-2). Rheumatoid factor can give rise to false-positive results in this test. A diagnosis of HBV infection can usually be made by detection of HBsAg in serum. Infrequently, levels of HBsAg are too low to be detected during acute HBV infection, even with contemporary, highly sensitive immunoassays. In such cases, the diagnosis can be established by the presence of IgM anti-HBc.

1	The titer of HBsAg bears little relation to the severity of clinical disease. Indeed, an inverse correlation exists between the serum concentration of HBsAg and the degree of liver cell damage. For example, titers are highest in immunosuppressed patients, lower in patients with chronic liver disease (but higher in mild chronic than in severe chronic hepatitis), and very low in patients with acute fulminant hepatitis. These observations suggest that, in hepatitis B, the degree of liver cell damage and the clinical course are related to variations in the patient’s immune response to HBV rather than to the amount of circulating HBsAg. In immunocompetent persons, however, a correlation exists between markers of HBV replication and liver injury (see below).

1	Another important serologic marker in patients with hepatitis B is HBeAg. Its principal clinical usefulness is as an indicator of relative infectivity. Because HBeAg is invariably present during early acute hepatitis B, HBeAg testing is indicated primarily in chronic infection. In patients with hepatitis B surface antigenemia of unknown duration (e.g., blood donors found to be HBsAg-positive) testing for IgM anti-HBc may be useful to distinguish between acute or recent infection (IgM anti-HBc-positive) and chronic HBV infection (IgM antiHBc-negative, IgG anti-HBc-positive). A false-positive test for IgM anti-HBc may be encountered in patients with high-titer rheumatoid factor. Also, IgM anti-HBc may be reexpressed during acute reactivation of chronic hepatitis B.

1	Anti-HBs is rarely detectable in the presence of HBsAg in patients with acute hepatitis B, but 10–20% of persons with chronic HBV infection may harbor low-level anti-HBs. This antibody is directed not against the common group determinant, a, but against the heterotypic subtype determinant (e.g., HBsAg of subtype ad with anti-HBs of subtype y). In most cases, this serologic pattern cannot be attributed to infection with two different HBV subtypes, and the presence of this antibody is not a harbinger of imminent HBsAg clearance. When such antibody is detected, its presence is of no recognized clinical significance (see “Virology and Etiology”).

1	After immunization with hepatitis B vaccine, which consists of HBsAg alone, anti-HBs is the only serologic marker to appear. The commonly encountered serologic patterns of hepatitis B and their interpretations are summarized in Table 360-5. Tests for the detection of HBV DNA in liver and serum are now available. Like HBeAg, serum HBV DNA is an indicator of HBV replication, but tests for HBV DNA are more sensitive and quantitative. First-generation hybridization assays for HBV DNA had a sensitivity of 105−106 virions/mL, a relative threshold below which infectivity and liver injury are limited and HBeAg is usually undetectable. Currently, testing for HBV DNA has shifted from insensitive hybridization assays to amplification assays (e.g., the PCR-based assay, which can detect as few as 10 or 100 virions/mL); among the commercially available PCR assays, the most useful are those with the highest sensitivity (5–10 IU/mL) and the largest dynamic range (100–109 IU/mL). With increased

1	10 or 100 virions/mL); among the commercially available PCR assays, the most useful are those with the highest sensitivity (5–10 IU/mL) and the largest dynamic range (100–109 IU/mL). With increased sensitivity, fied. The other involves target ampli + − IgM + − Acute hepatitis B, high infectivitya fication (i.e., synthesis of multiple − IgG + − Chronic hepatitis B, high infectivity copies of the viral genome) by PCR − IgG − + 1. Late acute or chronic hepatitis B, low infectivity or TMA, in which the viral RNA is 2. HBeAg-negative (“precore-mutant”) hepatitis B reverse transcribed to complemen(chronic or, rarely, acute) tary DNA and then amplified by + + + +/− +/− 1. HBsAg of one subtype and heterotypic anti-HBs repeated cycles of DNA synthesis. (common) Both can be used as quantitative 2. Process of seroconversion from HBsAg to anti-assays and a measurement of relative HBs (rare) “viral load”; PCR and TMA, with − − IgM +/− +/− 1. Acute hepatitis Ba a sensitivity of 10–102 IU/mL, are

1	Process of seroconversion from HBsAg to anti-assays and a measurement of relative HBs (rare) “viral load”; PCR and TMA, with − − IgM +/− +/− 1. Acute hepatitis Ba a sensitivity of 10–102 IU/mL, are more sensitive than bDNA, with a 2. Anti-HBc “window” sensitivity of 103 IU/mL; assays are − − IgG − +/− 1. Low-level hepatitis B carrier available with a wide dynamic range 2. Hepatitis B in remote past (10–107 IU/mL). Determination of

1	HCV RNA level is not a reliable − + − − − 1. Immunization with HBsAg (after vaccination) marker of disease severity or progno 2. Hepatitis B in the remote past (?) sis but is helpful in predicting relative 3. False-positive aIgM anti-HBc may reappear during acute reactivation of chronic hepatitis B. Note: See text for abbreviations.

1	amplification assays remain reactive well below the current 103–104 IU/ mL threshold for infectivity and liver injury. These markers are useful in following the course of HBV replication in patients with chronic hepatitis B receiving antiviral chemotherapy (Chap. 362). Except for the early decades of life after perinatally acquired HBV infection (see above), in immunocompetent adults with chronic hepatitis B, a general correlation exists between the level of HBV replication, as reflected by the level of serum HBV DNA, and the degree of liver injury. High-serum HBV DNA levels, increased expression of viral antigens, and necroinflammatory activity in the liver go hand in hand unless immunosuppression interferes with cytolytic T cell responses to virus-infected cells; reduction of HBV replication with antiviral drugs tends to be accompanied by an improvement in liver histology. Among patients with chronic hepatitis B, high levels of HBV DNA increase the risk of cirrhosis, hepatic

1	replication with antiviral drugs tends to be accompanied by an improvement in liver histology. Among patients with chronic hepatitis B, high levels of HBV DNA increase the risk of cirrhosis, hepatic decompensation, and hepatocellular carcinoma (see “Complications and Sequelae”).

1	In patients with hepatitis C, an episodic pattern of aminotransferase elevation is common. A specific serologic diagnosis of hepatitis C can be made by demonstrating the presence in serum of anti-HCV. When contemporary immunoassays are used, anti-HCV can be detected in acute hepatitis C during the initial phase of elevated aminotransferase activity and remains detectable after recovery (rare) and during chronic infection (common). Nonspecificity can confound immunoassays for anti-HCV, especially in persons with a low prior probability of infection, such as volunteer blood donors, or in persons with circulating rheumatoid factor, which can bind nonspecifically to assay reagents; testing for HCV RNA can be used in such settings to distinguish between true-positive and false-positive anti-HCV determinations. Assays for HCV RNA are the most sensitive tests for HCV infection and represent the “gold standard” in establishing a diagnosis of hepatitis C. HCV RNA can be detected even before

1	determinations. Assays for HCV RNA are the most sensitive tests for HCV infection and represent the “gold standard” in establishing a diagnosis of hepatitis C. HCV RNA can be detected even before acute elevation of aminotransferase activity and before the appearance of anti-HCV in patients with acute hepatitis C. In addition, HCV RNA remains detectable indefinitely, continuously in most but intermittently in some, in patients with chronic hepatitis C (detectable as well in some persons with normal liver tests, i.e., inactive carriers). In the very small minority of patients with hepatitis C who lack anti-HCV, a diagnosis can be supported by detection of HCV RNA. If all these tests are negative and the patient has a well-characterized case of hepatitis after percutaneous exposure to blood or blood products, a diagnosis of hepatitis caused by an unidentified agent can be entertained.

1	Amplification techniques are required to detect HCV RNA, and two types are available. One is a branched-chain complementary DNA (bDNA) assay, in which the detection signal (a colorimetrically responsiveness to antiviral therapy. The same is true for determinations of HCV genotype (Chap. 362). A proportion of patients with hepatitis C have isolated anti-HBc in their blood, a reflection of a common risk in certain populations of exposure to multiple bloodborne hepatitis agents. The anti-HBc in such cases is almost invariably of the IgG class and usually represents HBV infection in the remote past (HBV DNA undetectable); it rarely represents current HBV infection with low-level virus carriage.

1	The presence of HDV infection can be identified by demonstrating intrahepatic HDV antigen or, more practically, an anti-HDV seroconversion (a rise in titer of anti-HDV or de novo appearance of anti-HDV). Circulating HDV antigen, also diagnostic of acute infection, is detectable only briefly, if at all. Because anti-HDV is often undetectable once HBsAg disappears, retrospective serodiagnosis of acute self-limited, simultaneous HBV and HDV infection is difficult. Early diagnosis of acute infection may be hampered by a delay of up to 30–40 days in the appearance of anti-HDV.

1	When a patient presents with acute hepatitis and has HBsAg and anti-HDV in serum, determination of the class of anti-HBc is helpful in establishing the relationship between infection with HBV and HDV. Although IgM anti-HBc does not distinguish absolutely between acute and chronic HBV infection, its presence is a reliable indicator of recent infection and its absence a reliable indicator of infection in the remote past. In simultaneous acute HBV and HDV infections, IgM anti-HBc will be detectable, whereas in acute HDV infection superimposed on chronic HBV infection, anti-HBc will be of the IgG class. Tests for the presence of HDV RNA are useful for determining the presence of ongoing HDV replication and relative infectivity.

1	The serologic/virologic course of events during acute hepatitis E is entirely analogous to that of acute hepatitis A, with brief fecal shedding of virus and viremia and an early IgM anti-HEV response that predominates during approximately the first 3 months but is eclipsed thereafter by long-lasting IgG anti-HEV. Diagnostic tests of varying reliability for hepatitis E are commercially available but used routinely primarily outside the United States; in the United States, diagnostic serologic/virologic assays can be performed at the Centers for Disease Control and Prevention or other specialized reference laboratories. Liver biopsy is rarely necessary or indicated in acute viral hepatitis, except when the diagnosis is questionable or when clinical evidence suggests a diagnosis of chronic hepatitis.

1	Liver biopsy is rarely necessary or indicated in acute viral hepatitis, except when the diagnosis is questionable or when clinical evidence suggests a diagnosis of chronic hepatitis. A diagnostic algorithm can be applied in the evaluation of cases of acute viral hepatitis. A patient with acute hepatitis should undergo four serologic tests, HBsAg, IgM anti-HAV, IgM anti-HBc, and anti-HCV (Table 360-6). The presence of HBsAg, with or without IgM anti-HBc, represents HBV infection. If IgM anti-HBc is present, the HBV infection is considered acute; if IgM anti-HBc is absent, the HBV infection is considered chronic. A diagnosis of acute hepatitis B can be made in Serologic Tests of Patient’s Serum Note: See text for abbreviations.

1	Serologic Tests of Patient’s Serum Note: See text for abbreviations. the absence of HBsAg when IgM anti-HBc is detectable. A diagnosis of acute hepatitis A is based on the presence of IgM anti-HAV. If IgM anti-HAV coexists with HBsAg, a diagnosis of simultaneous HAV and HBV infections can be made; if IgM anti-HBc (with or without HBsAg) is detectable, the patient has simultaneous acute hepatitis A and B, and if IgM anti-HBc is undetectable, the patient has acute hepatitis A superimposed on chronic HBV infection. The presence of anti-HCV supports a diagnosis of acute hepatitis C. Occasionally, testing for HCV RNA or repeat anti-HCV testing later during the illness is necessary to establish the diagnosis. Absence of all serologic markers is consistent with a diagnosis of “non-A, non-B, non-C” hepatitis, if the epidemiologic setting is appropriate.

1	In patients with chronic hepatitis, initial testing should consist of HBsAg and anti-HCV. Anti-HCV supports and HCV RNA testing establishes the diagnosis of chronic hepatitis C. If a serologic diagnosis of chronic hepatitis B is made, testing for HBeAg and anti-HBe is indicated to evaluate relative infectivity. Testing for HBV DNA in such patients provides a more quantitative and sensitive measure of the level of virus replication and, therefore, is very helpful during antiviral therapy (Chap. 362). In patients with chronic hepatitis B and normal aminotransferase activity in the absence of HBeAg, serial testing over time is often required to distinguish between inactive carriage and HBeAg-negative chronic hepatitis B with fluctuating virologic and necroinflammatory activity. In persons with hepatitis B, testing for anti-HDV is useful in those with severe and fulminant disease, with severe chronic disease, with chronic hepatitis B and acute hepatitis-like exacerbations, with frequent

1	hepatitis B, testing for anti-HDV is useful in those with severe and fulminant disease, with severe chronic disease, with chronic hepatitis B and acute hepatitis-like exacerbations, with frequent percutaneous exposures, and from areas where HDV infection is endemic.

1	Virtually all previously healthy patients with hepatitis A recover completely with no clinical sequelae. Similarly, in acute hepatitis B, 95–99% of previously healthy adults have a favorable course and recover completely. Certain clinical and laboratory features, however, suggest a more complicated and protracted course. Patients of advanced age and with serious underlying medical disorders may have a prolonged course and are more likely to experience severe hepatitis. Initial presenting features such as ascites, peripheral edema, and symptoms of hepatic encephalopathy suggest a poorer prognosis. In addition, a prolonged PT, low serum albumin level, hypoglycemia, and very high serum bilirubin values suggest severe hepatocellular disease. Patients with these clinical and laboratory features deserve prompt hospital admission. The case fatality rate in hepatitis A and B is very low (~0.1%) but is increased by advanced age and underlying debilitating disorders. Among patients ill enough

1	deserve prompt hospital admission. The case fatality rate in hepatitis A and B is very low (~0.1%) but is increased by advanced age and underlying debilitating disorders. Among patients ill enough to be hospitalized for acute hepatitis B, the fatality rate is 1%. Hepatitis C is less severe during the acute phase than hepatitis B and is more likely to be anicteric; fatalities are rare, but the precise case fatality rate is not known. In outbreaks of waterborne hepatitis E in India and Asia, the case fatality rate is 1–2% and up to 10–20% in pregnant women. Contributing to fulminant hepatitis E in endemic countries are instances of acute hepatitis E superimposed on underlying chronic liver disease (“acute-on-chronic” liver disease). Patients with simultaneous acute hepatitis B and hepatitis D do not necessarily experience a higher mortality rate than do patients with acute hepatitis B alone; however, in several outbreaks of acute simultaneous HBV and HDV infection among injection drug

1	D do not necessarily experience a higher mortality rate than do patients with acute hepatitis B alone; however, in several outbreaks of acute simultaneous HBV and HDV infection among injection drug users, the case fatality rate was ~5%. When HDV superinfection occurs in a person with chronic hepatitis B, the likelihood of fulminant hepatitis and death is increased substantially. Although the case fatality rate for hepatitis D is not known definitively, in outbreaks of severe HDV superinfection in isolated populations with a high hepatitis B carrier rate, a mortality rate >20% has been recorded.

1	A small proportion of patients with hepatitis A experience relapsing hepatitis weeks to months after apparent recovery from acute hepatitis. Relapses are characterized by recurrence of symptoms, aminotransferase elevations, occasionally jaundice, and fecal excretion of HAV. Another unusual variant of acute hepatitis A is cholestatic hepatitis, characterized by protracted cholestatic jaundice and pruritus. Rarely, liver test abnormalities persist for many months, even up to a year. Even when these complications occur, hepatitis A remains self-limited and does not progress to chronic liver disease. During the prodromal phase of acute hepatitis B, a serum sickness–like syndrome characterized by arthralgia or arthritis, rash, angioedema, and rarely, hematuria and proteinuria may develop in 5–10% of patients. This syndrome occurs before the onset of clinical jaundice, and these patients are often diagnosed erroneously as having rheumatologic diseases. The diagnosis can be established by

1	5–10% of patients. This syndrome occurs before the onset of clinical jaundice, and these patients are often diagnosed erroneously as having rheumatologic diseases. The diagnosis can be established by measuring serum aminotransferase levels, which are almost invariably elevated, and serum HBsAg. As noted above, EMC is an immune-complex disease that can complicate chronic hepatitis C and is part of a spectrum of B cell lymphoproliferative disorders, which, in rare instances, can evolve to B cell lymphoma (Chap. 134). Attention has been drawn as well to associations between hepatitis C and such cutaneous disorders as porphyria cutanea tarda and lichen planus. A mechanism for these associations is unknown. Finally, related to the reliance of HCV on lipoprotein secretion and assembly pathways and on interactions of HCV with glucose metabolism, HCV infection may be complicated by hepatic steatosis, hypercholesterolemia, insulin resistance (and other manifestations of the metabolic

1	pathways and on interactions of HCV with glucose metabolism, HCV infection may be complicated by hepatic steatosis, hypercholesterolemia, insulin resistance (and other manifestations of the metabolic syndrome), and type 2 diabetes mellitus; both hepatic steatosis and insulin resistance appear to accelerate hepatic fibrosis and blunt responsiveness to antiviral therapy (Chap. 362).

1	The most feared complication of viral hepatitis is fulminant hepatitis (massive hepatic necrosis); fortunately, this is a rare event. Fulminant hepatitis is seen primarily in hepatitis B, D, and E, but rare fulminant cases of hepatitis A occur primarily in older adults and in persons with underlying chronic liver disease, including, according to some reports, chronic hepatitis B and C. Hepatitis B accounts for >50% of fulminant cases of viral hepatitis, a sizable proportion of which are associated with HDV infection and another proportion with underlying chronic hepatitis C. Fulminant hepatitis is hardly ever seen in hepatitis C, but hepatitis E, as noted above, can be complicated by fatal fulminant hepatitis in 1–2% of all cases and in up to 20% of cases in pregnant women. Patients usually present with signs and symptoms of encephalopathy that may evolve to deep coma. The liver is usually small and the PT excessively prolonged. The combination of rapidly shrinking liver size, rapidly

1	present with signs and symptoms of encephalopathy that may evolve to deep coma. The liver is usually small and the PT excessively prolonged. The combination of rapidly shrinking liver size, rapidly rising bilirubin level, and marked prolongation of the PT, even as aminotransferase levels fall, together with clinical signs of confusion, disorientation, somnolence, ascites, and edema, indicates that the patient has hepatic failure with encephalopathy. Cerebral edema is common; brainstem compression, gastrointestinal bleeding, sepsis, respiratory failure, cardiovascular collapse, and renal failure are terminal events. The mortality rate is exceedingly high (>80% in patients with deep coma), but patients who survive may have a complete biochemical and histologic recovery. If a donor liver can be located in time, liver transplantation may be life-saving in patients with fulminant hepatitis (Chap. 368).

1	Documenting the disappearance of HBsAg after apparent clinical recovery from acute hepatitis B is particularly important. Before laboratory methods were available to distinguish between acute hepatitis and acute hepatitis-like exacerbations (spontaneous reactivations) of chronic hepatitis B, observations suggested that ~10% of previously healthy patients remained HBsAg-positive for >6 months after the onset of clinically apparent acute hepatitis B. One-half of these persons cleared the antigen from their circulations during the next several years, but the other 5% remained chronically HBsAg-positive. More recent observations suggest that the true rate of chronic infection after clinically apparent acute hepatitis B is as low as 1% in normal, immunocompetent, young adults. Earlier, higher estimates may have been confounded by inadvertent inclusion of acute exacerbations in chronically infected patients; these patients, chronically HBsAg-positive before exacerbation, were unlikely to

1	estimates may have been confounded by inadvertent inclusion of acute exacerbations in chronically infected patients; these patients, chronically HBsAg-positive before exacerbation, were unlikely to seroconvert to HBsAg-negative thereafter. Whether the rate of chronicity is 10% or 1%, such patients have IgG anti-HBc in serum; anti-HBs is either undetected or detected at low titer against the opposite subtype specificity of the antigen (see “Laboratory Features”). These patients may (1) be inactive carriers; (2) have low-grade, mild chronic hepatitis; or (3) have moderate to severe chronic hepatitis with or without cirrhosis. The likelihood of remaining chronically infected after acute HBV infection is especially high among neonates, persons with Down’s syndrome, chronically hemodialyzed patients, and immunosuppressed patients, including persons with HIV infection.

1	Chronic hepatitis is an important late complication of acute hepatitis B occurring in a small proportion of patients with acute disease but more common in those who present with chronic infection without having experienced an acute illness, as occurs typically after neonatal infection or after infection in an immunosuppressed host (Chap. 362). The following clinical and laboratory features suggest progression of acute hepatitis to chronic hepatitis: (1) lack of complete resolution of clinical symptoms of anorexia, weight loss, fatigue, and the persistence of hepatomegaly; (2) the presence of bridging/interface or multilobular hepatic necrosis on liver biopsy during protracted, severe acute viral hepatitis; (3) failure of the serum aminotransferase, bilirubin, and globulin levels to return to normal within 6–12 months after the acute illness; and (4) the persistence of HBeAg for >3 months or HBsAg for >6 months after acute hepatitis.

1	Although acute hepatitis D infection does not increase the likelihood of chronicity of simultaneous acute hepatitis B, hepatitis D has the potential for contributing to the severity of chronic hepatitis B. Hepatitis D superinfection can transform inactive or mild chronic hepatitis B into severe, progressive chronic hepatitis and cirrhosis; it also can accelerate the course of chronic hepatitis B. Some HDV superinfections in patients with chronic hepatitis B lead to fulminant hepatitis. As defined in longitudinal studies over three decades, the annual rates of cirrhosis and hepatocellular carcinoma in patients with chronic hepatitis D are 4% and 2.8%, respectively. Although HDV and HBV infections are associated with severe liver disease, mild hepatitis and even inactive carriage have been identified in some patients, and the disease may become indolent beyond the early years of infection.

1	After acute HCV infection, the likelihood of remaining chronically infected approaches 85–90%. Although many patients with chronic hepatitis C have no symptoms, cirrhosis may develop in as many as 20% within 10–20 years of acute illness; in some series of cases reported by referral centers, cirrhosis has been reported in as many as 50% of patients with chronic hepatitis C. Although chronic hepatitis C accounts for at least 40% of cases of chronic liver disease and of patients undergoing liver transplantation for end-stage liver disease in the United States and Europe, in the majority of patients with chronic hepatitis C, morbidity and mortality are limited during the initial 20 years after the onset of infection. Progression of chronic hepatitis C may be influenced by advanced age of acquisition, long duration of infection, immunosuppression, coexisting excessive alcohol use, concomitant hepatic steatosis, other hepatitis virus infection, or HIV co-infection. In fact, instances of

1	long duration of infection, immunosuppression, coexisting excessive alcohol use, concomitant hepatic steatosis, other hepatitis virus infection, or HIV co-infection. In fact, instances of severe and rapidly progressive chronic hepatitis B and C are being recognized with increasing frequency in patients with HIV infection (Chap. 226). In contrast, neither HAV nor HEV causes chronic liver disease in immunocompetent hosts; 2019 however, cases of chronic hepatitis E have been observed in immunosuppressed organ-transplant recipients, persons receiving cytotoxic chemotherapy, and persons with HIV infection.

1	Rare complications of viral hepatitis include pancreatitis, myocarditis, atypical pneumonia, aplastic anemia, transverse myelitis, and peripheral neuropathy. Persons with chronic hepatitis B, particularly those infected in infancy or early childhood and especially those with HBeAg and/or high-level HBV DNA, have an enhanced risk of hepatocellular carcinoma. The risk of hepatocellular carcinoma is increased as well in patients with chronic hepatitis C, almost exclusively in patients with cirrhosis, and almost always after at least several decades, usually after three decades of disease (Chap. 111). In children, hepatitis B may present rarely with anicteric hepatitis, a nonpruritic papular rash of the face, buttocks, and limbs, and lymphadenopathy (papular acrodermatitis of childhood or Gianotti-Crosti syndrome). Rarely, autoimmune hepatitis (Chap. 362) can be triggered by a bout of otherwise self-limited acute hepatitis, as reported after acute hepatitis A, B, and C.

1	Viral diseases such as infectious mononucleosis; those due to cytomegalovirus, herpes simplex, and coxsackieviruses; and toxoplasmosis may share certain clinical features with viral hepatitis and cause elevations in serum aminotransferase and, less commonly, in serum bilirubin levels. Tests such as the differential heterophile and serologic tests for these agents may be helpful in the differential diagnosis if HBsAg, anti-HBc, IgM anti-HAV, and anti-HCV determinations are negative. Aminotransferase elevations can accompany almost any systemic viral infection; other rare causes of liver injury confused with viral hepatitis are infections with Leptospira, Candida, Brucella, Mycobacteria, and Pneumocystis. A complete drug history is particularly important because many drugs and certain anesthetic agents can produce a picture of either acute hepatitis or cholestasis (Chap. 361). Equally important is a past history of unexplained “repeated episodes” of acute hepatitis. This history should

1	agents can produce a picture of either acute hepatitis or cholestasis (Chap. 361). Equally important is a past history of unexplained “repeated episodes” of acute hepatitis. This history should alert the physician to the possibility that the underlying disorder is chronic hepatitis. Alcoholic hepatitis must also be considered, but usually the serum aminotransferase levels are not as markedly elevated, and other stigmata of alcoholism may be present. The finding on liver biopsy of fatty infiltration, a neutrophilic inflammatory reaction, and “alcoholic hyaline” would be consistent with alcohol-induced rather than viral liver injury. Because acute hepatitis may present with right upper quadrant abdominal pain, nausea and vomiting, fever, and icterus, it is often confused with acute cholecystitis, common duct stone, or ascending cholangitis. Patients with acute viral hepatitis may tolerate surgery poorly; therefore, it is important to exclude this diagnosis, and in confusing cases, a

1	common duct stone, or ascending cholangitis. Patients with acute viral hepatitis may tolerate surgery poorly; therefore, it is important to exclude this diagnosis, and in confusing cases, a percutaneous liver biopsy may be necessary before laparotomy. Viral hepatitis in the elderly is often misdiagnosed as obstructive jaundice resulting from a common duct stone or carcinoma of the pancreas. Because acute hepatitis in the elderly may be quite severe and the operative mortality high, a thorough evaluation including biochemical tests, radiographic studies of the biliary tree, and even liver biopsy may be necessary to exclude primary parenchymal liver disease. Another clinical constellation that may mimic acute hepatitis is right ventricular failure with passive hepatic congestion or hypoperfusion syndromes, such as those associated with shock, severe hypotension, and severe left ventricular failure. Also included in this general category is any disorder that interferes with venous return

1	syndromes, such as those associated with shock, severe hypotension, and severe left ventricular failure. Also included in this general category is any disorder that interferes with venous return to the heart, such as right atrial myxoma, constrictive pericarditis, hepatic vein occlusion (Budd-Chiari syndrome), or venoocclusive disease. Clinical features are usually sufficient to distinguish among these vascular disorders and viral hepatitis. Acute fatty liver of pregnancy, cholestasis of pregnancy, eclampsia, and the HELLP (h emolysis, e levated l iver tests, and l ow p latelets) syndrome can be confused with viral hepatitis during pregnancy. Very rarely, malignancies metastatic to the liver can mimic acute or even fulminant viral hepatitis. Occasionally, genetic or metabolic liver disorders (e.g., Wilson’s disease, α1 antitrypsin deficiency) and nonalcoholic fatty liver disease are confused with acute viral hepatitis.

1	In hepatitis B, among previously healthy adults who present with clinically apparent acute hepatitis, recovery occurs in ~99%; therefore, antiviral therapy is not likely to improve the rate of recovery and is not required. In rare instances of severe acute hepatitis B, treatment with a nucleoside analogue at oral doses used to treat chronic hepatitis B (Chap. 362) has been attempted successfully. Although clinical trials have not been done to establish the efficacy or duration of this approach, most authorities would recommend institution of antiviral therapy with a nucleoside analogue (entecavir or tenofovir, the most potent and least resistance-prone agents) for severe, but not mild–moderate, acute hepatitis B. Treatment should continue until 3 months after HBsAg seroconversion or 6 months after HBeAg seroconversion.

1	In typical cases of acute hepatitis C, recovery is rare, progression to chronic hepatitis is the rule, and meta-analyses of small clinical trials suggest that antiviral therapy with interferon α monotherapy (3 million units SC three times a week) is beneficial, reducing the rate of chronicity considerably by inducing sustained responses in 30–70% of patients. In a German multicenter study of 44 patients with acute symptomatic hepatitis C, initiation of intensive interferon α therapy (5 million units SC daily for 4 weeks, then three times a week for another 20 weeks) within an average of 3 months after infection resulted in a sustained virologic response rate of 98%. Although treatment of acute hepatitis C is recommended, the optimum regimen, duration of therapy, and time to initiate therapy remain to be determined. Many authorities now opt for a 24-week course (beginning within 2–3 months after onset) of long-acting pegylated interferon plus the nucleoside analogue ribavirin, although

1	remain to be determined. Many authorities now opt for a 24-week course (beginning within 2–3 months after onset) of long-acting pegylated interferon plus the nucleoside analogue ribavirin, although the value of adding ribavirin has not been demonstrated (see Chap. 362 for doses). Patients with jaundice and women are more likely to recover from acute hepatitis C, and now that genetic markers associated with spontaneous recovery (IL28B CC haplotype) versus persistence (non-CC haplotypes) have been defined, such genetic testing can help determine the need for and immediacy of treating acute hepatitis C—maintaining a high threshold for treating patients with CC and a very low threshold for early intervention in patients with non-CC genotypes. Protease inhibitor–based antiviral therapy with telaprevir or boceprevir, now approved for chronic hepatitis C, genotype 1 (Chap. 362), has not been approved for acute hepatitis C. Moreover, given the high efficacy of pegylated interferon–based

1	telaprevir or boceprevir, now approved for chronic hepatitis C, genotype 1 (Chap. 362), has not been approved for acute hepatitis C. Moreover, given the high efficacy of pegylated interferon–based therapy for acute hepatitis C, in all likelihood, the addition of a protease inhibitor would add costs and side effects without incremental efficacy. When, however, after 2014, all-oral, brief-duration, low-resistance antiviral regimens replace the current standard of care, the new approaches will be applied to acute hepatitis C and, potentially (pending the outcome of clinical trials), could even be used immediately after exposure (e.g., occupational) to prevent infection and the onset of hepatitis. Because of the marked reduction over the past two decades in the frequency of acute hepatitis C, opportunities to identify and treat patients with acute hepatitis C are rare, except in injection drug users and health workers who sustain hepatitis C–contaminated needle sticks. After such

1	C, opportunities to identify and treat patients with acute hepatitis C are rare, except in injection drug users and health workers who sustain hepatitis C–contaminated needle sticks. After such occupational accidents, when monitoring for ALT elevations and the presence of HCV RNA identifies acute hepatitis C (risk only ~3%), therapy should be initiated.

1	Notwithstanding these specific therapeutic considerations, in most cases of typical acute viral hepatitis, specific treatment generally is not necessary. Although hospitalization may be required for clinically severe illness, most patients do not require hospital care. Forced and prolonged bed rest is not essential for full recovery, but many patients will feel better with restricted physical activity. A high-calorie diet is desirable, and because many patients may experience nausea late in the day, the major caloric intake is best tolerated in the morning. Intravenous feeding is necessary in the acute stage if the patient has persistent vomiting and cannot maintain oral intake. Drugs capable of producing adverse reactions such as cholestasis and drugs metabolized by the liver should be avoided. If severe pruritus is present, the use of the bile salt-sequestering resin cholestyramine is helpful. Glucocorticoid therapy has no value in acute viral hepatitis, even in severe cases

1	be avoided. If severe pruritus is present, the use of the bile salt-sequestering resin cholestyramine is helpful. Glucocorticoid therapy has no value in acute viral hepatitis, even in severe cases associated with bridging necrosis, and may be deleterious, even increasing the risk of chronicity (e.g., of acute hepatitis B).

1	Physical isolation of patients with hepatitis to a single room and bathroom is rarely necessary except in the case of fecal incontinence for hepatitis A and E or uncontrolled, voluminous bleeding for hepatitis B (with or without concomitant hepatitis D) and hepatitis C. Because most patients hospitalized with hepatitis A excrete little, if any, HAV, the likelihood of HAV transmission from these patients during their hospitalization is low. Therefore, burdensome enteric precautions are no longer recommended. Although gloves should be worn when the bed pans or fecal material of patients with hepatitis A are handled, these precautions do not represent a departure from sensible procedure and contemporary universal precautions for all hospitalized patients. For patients with hepatitis B and hepatitis C, emphasis should be placed on blood precautions (i.e., avoiding direct, ungloved hand contact with blood and other body fluids). Enteric precautions are unnecessary. The importance of simple

1	C, emphasis should be placed on blood precautions (i.e., avoiding direct, ungloved hand contact with blood and other body fluids). Enteric precautions are unnecessary. The importance of simple hygienic precautions such as hand washing cannot be overemphasized. Universal precautions that have been adopted for all patients apply to patients with viral hepatitis. Hospitalized patients may be discharged following substantial symptomatic improvement, a significant downward trend in the serum aminotransferase and bilirubin values, and a return to normal of the PT. Mild aminotransferase elevations should not be considered contraindications to the gradual resumption of normal activity.

1	In fulminant hepatitis, the goal of therapy is to support the patient by maintenance of fluid balance, support of circulation and respiration, control of bleeding, correction of hypoglycemia, and treatment of other complications of the comatose state in anticipation of liver regeneration and repair. Protein intake should be restricted, and oral lactulose or neomycin administered. Glucocorticoid therapy has been shown in controlled trials to be ineffective. Likewise, exchange transfusion, plasmapheresis, human cross-circulation, porcine liver cross-perfusion, hemoperfusion, and extracorporeal liver-assist devices have not been proven to enhance survival. Meticulous intensive care that includes prophylactic antibiotic coverage is the one factor that does appear to improve survival. Orthotopic liver transplantation is resorted to with increasing frequency, with excellent results, in patients with fulminant hepatitis (Chap. 368).

1	Because application of therapy for acute viral hepatitis is limited and because antiviral therapy for chronic viral hepatitis is cumbersome, costly, and not effective in all patients (Chap. 362), emphasis is placed on prevention through immunization. The prophylactic approach differs for each of the types of viral hepatitis. In the past, immunoprophylaxis relied exclusively on passive immunization with antibody-containing globulin preparations purified by cold ethanol fractionation from the plasma of hundreds of normal donors. Currently, for hepatitis A, B, and E, active immunization with vaccines is the preferable approach to prevention.

1	Hepatitis A Both passive immunization with IG and active immunization with killed vaccines are available. All preparations of IG contain anti-HAV concentrations sufficient to be protective. When administered before exposure or during the early incubation period, IG is effective in preventing clinically apparent hepatitis A. For postexposure prophylaxis of intimate contacts (household, sexual, institutional) of persons with hepatitis A, the administration of 0.02 mL/kg is recommended as early after exposure as possible; it may be effective even when administered as late as 2 weeks after exposure. Prophylaxis is not necessary for those who have already received hepatitis A vaccine, for casual contacts (office, factory, school, or hospital), for most elderly persons, who are very likely to be immune, or for those known to have anti-HAV in their serum. In day care centers, recognition of hepatitis A in children or staff should provide a stimulus for immunoprophylaxis in the center and in

1	immune, or for those known to have anti-HAV in their serum. In day care centers, recognition of hepatitis A in children or staff should provide a stimulus for immunoprophylaxis in the center and in the children’s family members. By the time most common-source outbreaks of hepatitis A are recognized, it is usually too late in the incubation period for IG to be effective; however, prophylaxis may limit the frequency of secondary cases. For travelers to tropical countries, developing countries, and other areas outside standard tourist routes, IG prophylaxis had been recommended before a vaccine became available. When such travel lasted <3 months, 0.02 mL/ kg was given; for longer travel or residence in these areas, a dose of 0.06 mL/kg every 4–6 months was recommended. Administration of plasma-derived globulin is safe; all contemporary lots of IG are subjected to viral inactivation steps and must be free of HCV RNA as determined by PCR testing. Administration of IM lots of IG has not

1	globulin is safe; all contemporary lots of IG are subjected to viral inactivation steps and must be free of HCV RNA as determined by PCR testing. Administration of IM lots of IG has not been associated with transmission of HBV, HCV, or HIV.

1	Formalin-inactivated vaccines made from strains of HAV attenuated in tissue culture have been shown to be safe, immunogenic, and effective in preventing hepatitis A. Hepatitis A vaccines are approved for use in persons who are at least 1 year old and appear to provide adequate protection beginning 4 weeks after a primary inoculation. If it can be given within 4 weeks of an expected exposure, such as by travel to an endemic area, hepatitis A vaccine is the preferred approach to pre-exposure immunoprophylaxis. If travel is more imminent, IG (0.02 mL/kg) should be administered at a different injection site, along with the first dose of vaccine. Because vaccination provides long-lasting protection (protective levels of anti-HAV should last 20 years after vaccination), persons whose risk will be sustained (e.g., frequent travelers or those remaining in endemic areas for prolonged periods) should be vaccinated, and vaccine should supplant the need for repeated IG injections. Shortly after

1	be sustained (e.g., frequent travelers or those remaining in endemic areas for prolonged periods) should be vaccinated, and vaccine should supplant the need for repeated IG injections. Shortly after its introduction, hepatitis A vaccine was recommended for children living in communities with a high incidence of HAV infection; in 1999, this recommendation was extended to include all children living in states, counties, and communities with high rates of HAV infection. As of 2006, the Advisory Committee on Immunization Practices of the U.S. Public Health Service recommended routine hepatitis A vaccination of all children. Other groups considered to be at increased risk for HAV infection and who are candidates for hepatitis A vaccination include military personnel, populations with cyclic outbreaks of hepatitis A (e.g., Alaskan natives), employees of day care centers, primate handlers, laboratory workers exposed to hepatitis A or fecal specimens, and patients with chronic liver disease.

1	of hepatitis A (e.g., Alaskan natives), employees of day care centers, primate handlers, laboratory workers exposed to hepatitis A or fecal specimens, and patients with chronic liver disease. Because of an increased risk of fulminant hepatitis A—observed in some experiences but not confirmed in others—among patients with chronic hepatitis C, patients with chronic hepatitis C are candidates for hepatitis A vaccination, as are persons with chronic hepatitis B. Other populations whose recognized risk of hepatitis A is increased should be vaccinated, including men who have sex with men, injection drug users, persons with clotting disorders who require frequent administration of clotting-factor concentrates, persons traveling from the United States to countries with high or intermediate hepatitis A endemicity, postexposure prophylaxis for contacts of persons with hepatitis A, and household members and other close contacts of adopted children arriving from countries with high and moderate

1	A endemicity, postexposure prophylaxis for contacts of persons with hepatitis A, and household members and other close contacts of adopted children arriving from countries with high and moderate hepatitis A endemicity. Recommendations for dose and frequency differ for the two approved vaccine preparations (Table 360-7); all injections are IM. Hepatitis A vaccine has been reported to be effective in preventing secondary household and day care center–associated cases of acute hepatitis A. Because the vaccine 1–18 2 25 units (0.5 mL) 0, 6–18 ≥19 2 50 units (1 mL) 0, 6–18 aA combination of this hepatitis A vaccine and hepatitis B vaccine, TWINRIX, is licensed for simultaneous protection against both of these viruses among adults (age ≥18 years). Each 1-mL dose contains 720 ELU of hepatitis A vaccine and 20 μg of hepatitis B vaccine. These doses are recommended at months 0, 1, and 6. bEnzyme-linked immunoassay units.

1	Abbreviation: ELU, enzyme-linked immunoassay unit. provides long-lasting protection and is simpler to use, in 2006, the 2021 Immunization Practices Advisory Committee of the U.S. Public Health Service favored hepatitis A vaccine to IG for postexposure prophylaxis of healthy persons age 2–40 years; for younger or older persons, for immunosuppressed patients, and for patients with chronic liver disease, IG should continue to be used. In the United States, reported mortality resulting from hepatitis A declined in parallel with hepatitis A vaccine– associated reductions in the annual incidence of new infections.

1	Hepatitis B Until 1982, prevention of hepatitis B was based on passive immunoprophylaxis either with standard Ig, containing modest levels of anti-HBs, or hepatitis B immunoglobulin (HBIG), containing high-titer anti-HBs. The efficacy of standard IG has never been established and remains questionable; even the efficacy of HBIG, demonstrated in several clinical trials, has been challenged, and its contribution appears to be in reducing the frequency of clinical illness, not in preventing infection. The first vaccine for active immunization, introduced in 1982, was prepared from purified, noninfectious 22-nm spherical

1	HBsAg particles derived from the plasma of healthy HBsAg carriers. In 1987, the plasma-derived vaccine was supplanted by a genetically engineered vaccine derived from recombinant yeast. The latter vaccine consists of HBsAg particles that are nonglycosylated but are otherwise indistinguishable from natural HBsAg; two recombinant vaccines are licensed for use in the United States. Current recommendations can be divided into those for pre-exposure and postexposure prophylaxis.

1	For pre-exposure prophylaxis against hepatitis B in settings of frequent exposure (health workers exposed to blood; first-responder public safety workers; hemodialysis patients and staff; residents and staff of custodial institutions for the developmentally handicapped; injection drug users; inmates of long-term correctional facilities; persons with multiple sexual partners or who have had a sexually transmitted disease; men who have sex with men; persons such as hemophiliacs who require long-term, high-volume therapy with blood derivatives; household and sexual contacts of persons with chronic HBV infection; persons living in or traveling extensively in endemic areas; unvaccinated children under the age of 18; unvaccinated children who are Alaskan natives, Pacific Islanders, or residents in households of first-generation immigrants from endemic countries; persons born in countries with a prevalence of HBV infection ≥2%; patients with chronic liver disease; persons <age 60 with

1	in households of first-generation immigrants from endemic countries; persons born in countries with a prevalence of HBV infection ≥2%; patients with chronic liver disease; persons <age 60 with diabetes mellitus [those ≥60 at the discretion of their physicians]; persons with end-stage renal disease; persons with HIV infection), three IM (deltoid, not gluteal) injections of hepatitis B vaccine are recommended at 0, 1, and 6 months (other, optional schedules are summarized in Table 360-8). Pregnancy is not a contraindication to vaccination. In areas of low HBV endemicity such as the United States, despite the availability of safe and effective hepatitis B vaccines, a strategy of vaccinating persons in high-risk groups was not effective. The incidence of new hepatitis B cases continued to increase in the United States after the introduction of vaccines; <10% of all targeted persons in high-risk groups were actually vaccinated, and ~30% of persons with sporadic acute hepatitis B did not

1	in the United States after the introduction of vaccines; <10% of all targeted persons in high-risk groups were actually vaccinated, and ~30% of persons with sporadic acute hepatitis B did not fall into any highrisk-group category. Therefore, to have an impact on the frequency of HBV infection in an area of low endemicity such as the United States, universal hepatitis B vaccination in childhood has been recommended. For unvaccinated children born after the implementation of universal infant vaccination, vaccination during early adolescence, at age 11–12 years, was recommended, and this recommendation has been extended to include all unvaccinated children age 0–19 years. In HBVhyperendemic areas (e.g., Asia), universal vaccination of children has resulted in a marked 10to 15-year decline in hepatitis B and its complications, including hepatocellular carcinoma.

1	The two available recombinant hepatitis B vaccines are comparable, one containing 10 μg of HBsAg (Recombivax-HB) and the other containing 20 μg of HBsAg (Engerix-B), and recommended doses for each injection vary for the two preparations (Table 360-8). Combinations of hepatitis B vaccine with other childhood vaccines are available as well (Table 360-8). For unvaccinated persons sustaining an exposure to HBV, post-exposure prophylaxis with a combination of HBIG (for rapid

1	For unvaccinated persons sustaining an exposure to HBV, post-exposure prophylaxis with a combination of HBIG (for rapid No. of Target Group Doses Dose Schedule, months detectable anti-HBs or immunocompetent persons who sustain percutaneous HBsAg-positive inoculations after losing detectable antibody. Specifically, for hemodialysis patients, annual anti-HBs testing is recommended after vaccination; booster doses are recommended when anti-HBs levels fall to <10 mIU/mL. As noted above, for persons at risk of both hepatitis A and B, a combined vaccine is available containing 720 enzyme-linked immunoassay units (ELUs) of inactivated HAV and 20 μg of recombinant HBsAg (at 0, 1, and 6 months).

1	Hepatitis D Infection with hepatitis D can be prevented by vaccinating susceptible persons with hepatitis B vaccine. No product is available for immunoprophylaxis to prevent HDV superinfection in HBsAg carriers; for them, avoidance of percutaneous exposures and limitation of intimate contact with persons who have HDV infection are recommended.

1	Hepatitis C IG is ineffective in preventing hepatitis C and is no longer recommended for postexposure prophylaxis in cases of perinatal, needle stick, or sexual exposure. Although prototype vaccines that induce antibodies to HCV envelope proteins have been developed, currently, hepatitis C vaccination is not feasible practically. Genotype and quasispecies viral heterogeneity as well as rapid evasion of neutralizing antibodies by this rapidly mutating virus, conspire to render HCV a difficult target for immunoprophylaxis with a vaccine. Prevention of transfusion-associated hepatitis C has been accomplished by the following successively introduced measures: exclusion of commercial blood donors and reliance on a volunteer blood supply; screening donor blood with surrogate markers such as ALT (no longer recommended) and anti-HBc, markers that identify segments of the blood donor population with an increased risk of bloodborne infections; exclusion of blood donors in high-risk groups for

1	(no longer recommended) and anti-HBc, markers that identify segments of the blood donor population with an increased risk of bloodborne infections; exclusion of blood donors in high-risk groups for AIDS and the introduction of anti-HIV screening tests; and progressively sensitive serologic and virologic screening tests for HCV infection.

1	In the absence of active or passive immunization, prevention of hepatitis C includes behavior changes and precautions to limit exposures to infected persons. Recommendations designed to identify patients with clinically inapparent hepatitis as candidates for medical management have as a secondary benefit the identification of persons whose contacts could be at risk of becoming infected. A so-called look-back program has been recommended to identify persons who were transfused before 1992 with blood from a donor found subsequently to have hepatitis C. In addition, anti-HCV testing is recommended for persons born between 1945 and 1965, anyone who received a blood transfusion or a transplanted organ before the introduction of second-generation screening tests in 1992, those who ever used injection drugs (or took other illicit drugs by noninjection routes), chronically hemodialyzed patients, persons with clotting disorders who received clotting factors made before 1987 from pooled blood

1	drugs (or took other illicit drugs by noninjection routes), chronically hemodialyzed patients, persons with clotting disorders who received clotting factors made before 1987 from pooled blood products, persons with elevated aminotransferase levels, health workers exposed to HCV-positive blood or contaminated needles, recipients of blood or organs from a donor found to be positive for hepatitis C, persons with HIV infection, health care and public safety personnel following a needle stick or other nonpercutaneous exposure to HCV-infected material, sexual partners of persons with hepatitis C, and children born to HCV-positive mothers (Table 360-4).

1	For stable, monogamous sexual partners, sexual transmission of hepatitis C is unlikely, and sexual barrier precautions are not recommended. For persons with multiple sexual partners or with sexually transmitted diseases, the risk of sexual transmission of hepatitis C is increased, and barrier precautions (latex condoms) are recommended. A person with hepatitis C should avoid sharing such items as razors, toothbrushes, and nail clippers with sexual partners and family members. No special precautions are recommended for babies born to mothers with hepatitis C, and breast-feeding does not have to be restricted. Hepatitis E Whether IG prevents hepatitis E remains undetermined. Safe and effective recombinant genotype 1 vaccines, which protect against other genotypes as well, have been developed and are available in endemic areas but not in the United States.

1	Infants, children (<1–10 years) 5 μg (0.5 mL) 5 μg (0.5 mL) 5 μg (0.5 mL) 40 μg (4 mL) 0, 1–2, 4–6 0–2, 1–4, 4–6 or 0, 12, 24 or 0, 1, 2, 12 0, 4–6 (age 11–15) 0–2, 1–4, 4–6 0, 1, 6 0, 1, 6

1	Infants, children 10 μg (0.5 mL) 10 μg (0.5 mL) 20 μg (1 mL) 10 μg (0.5 mL) 40 μg (2 mL) 0, 1–2, 4–6 or 0, 1, 2, 12 0, 1–2, 4–6 or 0, 12, 24 or 0, 1, 2, 12 0–2, 1–4, 4–6 or 0, 1, 2, 12 0, 1, 2, 6 0, 1, 2, 6 aThis manufacturer produces a licensed combination of hepatitis B vaccine and vaccines against Haemophilus influenzae type b and Neisseria meningitides, Comvax, for use in infants and young children. Please consult product insert for dose and schedule. bThis group also includes other immunocompromised persons. cThis manufacturer produces two licensed combination hepatitis B vaccines: (1) Twinrix, recombinant hepatitis B vaccine plus inactivated hepatitis A vaccine, is licensed for simultaneous protection against both of these viruses among adults (age ≥18 years). Each 1-mL dose contains 720 ELU of hepatitis A vaccine and 20 μg of hepatitis B vaccine. These doses are recommended at months 0, 1, and 6. (2) Pediatrix, recombinant hepatitis B vaccine plus diphtheria and tetanus toxoid,

1	720 ELU of hepatitis A vaccine and 20 μg of hepatitis B vaccine. These doses are recommended at months 0, 1, and 6. (2) Pediatrix, recombinant hepatitis B vaccine plus diphtheria and tetanus toxoid, pertussis, and inactivated poliovirus, is licensed for use in infants and young children. Please consult product insert for doses and schedules.

1	achievement of high-titer circulating anti-HBs) and hepatitis B vaccine (for achievement of long-lasting immunity as well as its apparent efficacy in attenuating clinical illness after exposure) is recommended. For perinatal exposure of infants born to HBsAg-positive mothers, a single dose of HBIG, 0.5 mL, should be administered IM in the thigh immediately after birth, followed by a complete course of three injections of recombinant hepatitis B vaccine (see doses above) to be started within the first 12 h of life. For those experiencing a direct percutaneous inoculation or transmucosal exposure to HBsAg-positive blood or body fluids (e.g., accidental needle stick, other mucosal penetration, or ingestion), a single IM dose of HBIG, 0.06 mL/kg, administered as soon after exposure as possible, is followed by a complete course of hepatitis B vaccine to begin within the first week. For those exposed by sexual contact to a patient with acute hepatitis B, a single IM dose of HBIG, 0.06

1	is followed by a complete course of hepatitis B vaccine to begin within the first week. For those exposed by sexual contact to a patient with acute hepatitis B, a single IM dose of HBIG, 0.06 mL/kg, should be given within 14 days of exposure, to be followed by a complete course of hepatitis B vaccine. When both HBIG and hepatitis B vaccine are recommended, they may be given at the same time but at separate sites. Testing adults for anti-HBs after a course of vaccine is advisable to document the acquisition of immunity, but, because hepatitis B vaccine immunogenicity is nearly universal in infants, postvaccination anti-HBs testing of children is not recommended.

1	The precise duration of protection afforded by hepatitis B vaccine is unknown; however, ~80–90% of immunocompetent adult vaccinees retain protective levels of anti-HBs for at least 5 years, and 60–80% for 10 years, and protective antibody has been documented to last for at least two decades after vaccination in infancy. Thereafter and even after anti-HBs becomes undetectable, protection persists against clinical hepatitis B, hepatitis B surface antigenemia, and chronic HBV infection. Currently, booster immunizations are not recommended routinely, except in immunosuppressed persons who have lost dence for hepatotoxicity detected during clinical trials for drug development is the most common reason for failure of compounds to reach approval status. Drug-induced liver injury requires careful history taking to identify unrecognized exposure to chemicals used in work or at home, drugs taken by prescription or bought over the counter, and herbal or dietary supplement medicines. Hepatotoxic

1	history taking to identify unrecognized exposure to chemicals used in work or at home, drugs taken by prescription or bought over the counter, and herbal or dietary supplement medicines. Hepatotoxic drugs can injure the hepatocyte directly, e.g., via a free-radical or metabolic intermediate that causes peroxidation of membrane lipids and that results in liver cell injury. Alternatively, a drug or its metabolite may activate components of the innate or adaptive immune system, stimulate apoptotic pathways, or initiate damage to bile excretory pathways (Fig. 361-1). Interference with bile canalicular pumps can allow endogenous bile acids, which can injure the liver, to accumulate. Such secondary injury, in turn, may lead to necrosis of hepatocytes; injure bile ducts, producing cholestasis; or block pathways of lipid movement, inhibit protein synthesis, or impair mitochondrial oxidation of fatty acids, resulting in lactic acidosis and intracellular triglyceride accumulation (expressed

1	or block pathways of lipid movement, inhibit protein synthesis, or impair mitochondrial oxidation of fatty acids, resulting in lactic acidosis and intracellular triglyceride accumulation (expressed histologically as microvesicular steatosis). In other instances, drug metabolites sensitize hepatocytes to toxic cytokines. The differences observed between susceptible and nonsusceptible drug recipients may be attributable to HLA haplotypes that determine binding of drug-related haptens on the cell surface as well as to polymorphisms in elaboration of competing, protective cytokines, as has been suggested for acetaminophen hepatotoxicity (see below). Immune mechanisms may include cytotoxic lymphocytes or antibody-mediated cellular cytotoxicity. In addition, a role has been shown for activation of nuclear transporters, such as the constitutive androstane receptor (CAR) or, more recently, the pregnane X receptor (PXR), in the induction of drug hepatotoxicity.

1	Toxic and Drug-induced Hepatitis William M. Lee, Jules L. Dienstag Liver injury is a possible consequence of ingestion of any xenobiotic, including industrial toxins, pharmacologic agents, and complementary and alternative medications (CAMs). Among patients with acute liver failure, drug-induced liver injury is the most common cause, and evi-361 Most drugs, which are water-insoluble, undergo a series of metabolic steps, culminating in a water-soluble form appropriate for renal or biliary excretion. This process begins with oxidation or methylation mediated initially by the microsomal mixed-function oxygenases, cytochrome P450 (phase I reaction), followed by glucuronidation or sulfation (phase II reaction) or inactivation by glutathione. Most drug hepatotoxicity is mediated by a phase I toxic metabolite, but glutathione depletion, precluding inactivation of harmful compounds by glutathione S-transferase, can contribute as well.

1	In general, two major types of chemical hepatotoxicity have been recognized: (1) direct toxic and (2) idiosyncratic. As shown in Table 361-1, direct toxic hepatitis occurs with predictable regularity in individuals exposed to the offending agent and is dose-dependent. The latent period between exposure and liver injury is usually short (often several hours), although clinical manifestations may be delayed for 24–48 h. Agents producing toxic hepatitis are generally systemic poisons or are converted in the liver to toxic metabolites. The direct hepatotoxins result in morphologic abnormalities that are reasonably characteristic and reproducible for each toxin. For example, carbon tetrachloride and trichloroethylene characteristically produce a centrilobular zonal necrosis, whereas yellow phosphorus poisoning typically results in periportal injury. The hepatotoxic octapeptides of Amanita phalloides usually produce massive hepatic necrosis; the lethal dose of the toxin is ~10 mg, the

1	phosphorus poisoning typically results in periportal injury. The hepatotoxic octapeptides of Amanita phalloides usually produce massive hepatic necrosis; the lethal dose of the toxin is ~10 mg, the amount found in a single deathcap mushroom. Liver injury, which is often only one facet of the toxicity produced by the direct hepatotoxins, may go unrecognized until jaundice appears.

1	In idiosyncratic drug reactions, the occurrence of hepatitis is usually 2023 infrequent (1 in 103–105 patients) and unpredictable; the response is not as clearly dose-dependent as is injury associated with direct hepatotoxins, and liver injury may occur at any time during or shortly after exposure to the drug. That said, recent data suggest that most agents causing idiosyncratic toxicity are given at a daily dose exceeding 100 mg, suggesting a role for dose—drugs with low potency must be given in higher doses that engender greater chances for “off-target” effects. Adding to the difficulty of predicting or identifying idiosyncratic drug hepatotoxicity is the occurrence of mild, transient, nonprogressive serum aminotransferase elevations that resolve with continued drug use. Such “adaptation,” the mechanism of which is unknown, is well recognized for drugs such as isoniazid, valproate, phenytoin, and HMG-CoA reductase inhibitors (statins). Extrahepatic manifestations of

1	Such “adaptation,” the mechanism of which is unknown, is well recognized for drugs such as isoniazid, valproate, phenytoin, and HMG-CoA reductase inhibitors (statins). Extrahepatic manifestations of hypersensitivity, such as rash, arthralgias, fever, leukocytosis, and eosinophilia, occur in about one-quarter of patients with idiosyncratic hepatotoxic drug reactions but are characteristic for certain drugs and not others. Both primary immunologic injury and direct hepatotoxicity related to idiosyncratic differences in generation of toxic metabolites have been invoked to explain idiosyncratic drug reactions. The most current data appear to implicate the adaptive immune system responding to the formation of immune stimulatory compounds resulting from phase I metabolic activation of the offending drug. Differences in host susceptibility may result from varying kinetics of toxic metabolite generation and genetic polymorphisms in downstream drug-metabolizing pathways or cytokine activation;

1	drug. Differences in host susceptibility may result from varying kinetics of toxic metabolite generation and genetic polymorphisms in downstream drug-metabolizing pathways or cytokine activation; in addition, certain HLA haplotypes have been associated with hepatotoxicity of certain drugs such as amoxicillin-clavulanate and flucloxacillin. Occasionally, however, the clinical features of an allergic reaction (prominent tissue eosinophilia, autoantibodies, etc.) are difficult to ignore and suggest activation of IgE pathways. A few instances of drug hepatotoxicity are observed to be associated with autoantibodies, including a class of antibodies to liver-kidney microsomes, anti-LKM2, directed against a cytochrome P450 enzyme.

1	Idiosyncratic reactions lead to a morphologic pattern that is more variable than those produced by direct toxins; a single agent is often capable of causing a variety of lesions, although certain patterns tend to predominate. Depending on the agent involved, idiosyncratic hepatitis may result in a clinical and morphologic picture indistinguishable from that of viral hepatitis (e.g., isoniazid or ciprofloxacin). So-called hepatocellular injury is the most common form, featuring spotty necrosis in the liver lobule with a predominantly lymphocytic infiltrate resembling that observed in acute hepatitis A, B, or C. Drug-induced cholestasis ranges from mild to increasingly severe: (1) bland cholestasis with limited hepatocellular injury (e.g., estrogens, 17,α-substituted androgens); (2) inflammatory cholestasis (e.g., amoxicillin-clavulanic acid [the most frequently implicated antibiotic among cases of drug-induced liver injury], oxacillin, erythromycin estolate); (3) sclerosing cholangitis

1	cholestasis (e.g., amoxicillin-clavulanic acid [the most frequently implicated antibiotic among cases of drug-induced liver injury], oxacillin, erythromycin estolate); (3) sclerosing cholangitis (e.g., after intrahepatic infusion of the chemotherapeutic agent floxuridine for hepatic metastases from a primary colonic carcinoma); and (4) disappearance of bile ducts, “ductopenic” cholestasis, similar to that observed in chronic rejection (Chap. 368) following liver transplantation (e.g., carbamazepine, levofloxacin). Cholestasis may result from binding of drugs to canalicular membrane transporters, accumulation of toxic bile acids resulting from canalicular pump failure, or genetic defects in canalicular transporter proteins. Clinically, the distinction between a hepatocellular and a cholestatic reaction is indicated by the R value, the ratio of alanine aminotransferase (ALT) to alkaline phosphatase values, both expressed as multiples of the upper limit of normal. An R value of >5.0 is

1	reaction is indicated by the R value, the ratio of alanine aminotransferase (ALT) to alkaline phosphatase values, both expressed as multiples of the upper limit of normal. An R value of >5.0 is associated with hepatocellular injury, R <2.0 with cholestatic injury, and R between 2.0 and 5.0 with mixed hepatocellular-cholestatic injury.

1	Morphologic alterations may also include bridging hepatic necrosis (e.g., methyldopa) or, infrequently, hepatic granulomas (e.g., sulfonamides). Some drugs result in macrovesicular or microvesicular steatosis or steatohepatitis, which, in some cases, has been linked to mitochondrial dysfunction and lipid peroxidation. Severe hepatotoxicity associated with steatohepatitis, most likely a result of mitochondrial toxicity, is being recognized with increasing frequency among patients receiving antiretroviral therapy with reverse transcriptase inhibitors for HIV infection (e.g., zidovudine, didanosine), although 2024 Six Mechanisms of Liver Injury Inhibition of Caspase ˜-oxidation, respiration, or both Caspase Caspase Cytolytic Mitochondrion TNF-° receptor, T cell Fas A. Rupture of cell membrane. D. Drug adducts targeted by CTLs/cytokines. B. Injury of bile canaliculus (disruption of transport pumps). E. Activation of apoptotic pathway by TNF°/Fas.

1	A. Rupture of cell membrane. D. Drug adducts targeted by CTLs/cytokines. B. Injury of bile canaliculus (disruption of transport pumps). E. Activation of apoptotic pathway by TNF°/Fas. C. P-450-drug covalent binding (drug adducts). F. Inhibition of mitochondrial function.

1	FIGURE 361-1 Potential mechanisms of drug-induced liver injury. The normal hepatocyte may be affected adversely by drugs through (A) disruption of intracellular calcium homeostasis that leads to the disassembly of actin fibrils at the surface of the hepatocyte, resulting in blebbing of the cell membrane, rupture, and cell lysis; (B) disruption of actin filaments next to the canaliculus (the specialized portion of the cell responsible for bile excretion), leading to loss of villous processes and interruption of transport pumps such as multidrug resistance–associated protein 3 (MRP3), which, in turn, prevents the excretion of bilirubin and other organic compounds; (C) covalent binding of the heme-containing cytochrome P450 enzyme to the drug, thus creating nonfunctioning adducts; (D) migration of these enzyme-drug adducts to the cell surface in vesicles to serve as target immunogens for cytolytic attack by T cells, stimulating an immune response involving cytolytic T cells and

1	migration of these enzyme-drug adducts to the cell surface in vesicles to serve as target immunogens for cytolytic attack by T cells, stimulating an immune response involving cytolytic T cells and cytokines; (E) activation of apoptotic pathways by tumor necrosis factor α (TNF-α) receptor or Fas (DD denotes death domain), triggering the cascade of intercellular caspases, resulting in programmed cell death; or (F) inhibition of mitochondrial function by a dual effect on both β-oxidation and the respiratory-chain enzymes, leading to failure of free fatty acid metabolism, a lack of aerobic respiration, and accumulation of lactate and reactive oxygen species (which may disrupt mitochondrial DNA). Toxic metabolites excreted in bile may damage bile-duct epithelium (not shown). CTLs, cytolytic T lymphocytes. (Reproduced from WM Lee: Drug-induced hepatotoxicity. N Engl J Med 349:474, 2003, with permission.)

1	Latent period Arthralgia, fever, rash, eosinophilia Necrosis, fatty Centrilobular aThe drugs listed are typical examples.

1	many of these drugs have been withdrawn because of such hepatotoxicity (Chap. 226). Generally, such mitochondrial hepatotoxicity of these antiretroviral agents is reversible, but dramatic, nonreversible hepatotoxicity associated with mitochondrial injury (inhibition of DNA polymerase γ) was the cause of acute liver failure encountered during early clinical trials of now-abandoned fialuridine, a fluorinated pyrimidine analogue with potent antiviral activity against hepatitis B virus. Another potential target for idiosyncratic drug hepatotoxicity is sinusoidal lining cells; when these are injured, such as by high-dose chemotherapeutic agents (e.g., cyclophosphamide, melphalan, busulfan) administered prior to bone marrow transplantation, venoocclusive disease can result. Nodular regenerative hyperplasia, a subtle form of portal hypertension, may also result from vascular injury to portal venous endothelium following systemic chemotherapy, such as with oxaliplatin, as part of adjuvant

1	hyperplasia, a subtle form of portal hypertension, may also result from vascular injury to portal venous endothelium following systemic chemotherapy, such as with oxaliplatin, as part of adjuvant treatment for colon cancer.

1	Not all adverse hepatic drug reactions can be classified as either toxic or idiosyncratic. For example, oral contraceptives, which combine estrogenic and progestational compounds, may result in impairment of hepatic tests and, occasionally, jaundice; however, they do not produce necrosis or fatty change, manifestations of hypersensitivity are generally absent, and susceptibility to the development of oral contraceptive–induced cholestasis appears to be genetically determined. Such estrogen-induced cholestasis is more common in women with cholestasis of pregnancy, a disorder linked to genetic defects in multi-drug resistance–associated canalicular transporter proteins.

1	Any idiosyncratic reaction that occurs in <1:10,000 recipients will go unrecognized in most clinical trials, which involve only several thousand recipients. The U.S. Food and Drug Administration (FDA) and pharmaceutical companies have learned to look for even subtle indications of serious toxicity and monitor regularly the number of trial subjects in whom any aminotransferase elevations develop, as a possible surrogate for more serious toxicity. Even more valid as a predictor of severe hepatotoxicity is the occurrence of jaundice in patients enrolled in a clinical drug trial, so called “Hy’s Law,” named after Hyman Zimmerman, one of the pioneers of the field of drug hepatotoxicity. He recognized that, if jaundice occurred during a phase III trial, more serious liver injury was likely, with a 10:1 ratio between cases of jaundice and liver failure—10 patients with jaundice to 1 patient with acute liver failure. Thus, the finding of such Hy’s Law cases during drug development often

1	with a 10:1 ratio between cases of jaundice and liver failure—10 patients with jaundice to 1 patient with acute liver failure. Thus, the finding of such Hy’s Law cases during drug development often portends failure of approval, particularly if any of the subjects sustains a bad outcome. Troglitazone, a peroxisome proliferator-activated receptor γ agonist, was the first in its class of thiazolidinedione insulin-sensitizing agents. Although in retrospect, Hy’s Law cases of jaundice had occurred during phase III trials, no instances of liver failure were recognized until well after the drug was introduced, underlining the importance of postmarketing surveillance in identifying toxic drugs and in leading to their withdrawal from use. Fortunately, such hepatotoxicity is not characteristic of the second-generation thiazolidinedione insulin-sensitizing agents rosiglitazone and pioglitazone; in clinical trials, the frequency of aminotransferase elevations in patients treated with these

1	of the second-generation thiazolidinedione insulin-sensitizing agents rosiglitazone and pioglitazone; in clinical trials, the frequency of aminotransferase elevations in patients treated with these medications did not differ from that in placebo recipients, and isolated reports of liver injury among recipients are extremely rare.

1	Proving that an episode of liver injury is caused by a drug is difficult in many cases. Drug-induced liver injury is nearly always a presumptive diagnosis, and many other disorders produce a similar clinicopathologic picture. Thus, causality may be difficult to establish and requires several separate supportive assessment variables to lead to a high level of certainty, including temporal association (time of onset, time to resolution), clinical-biochemical features, type of injury (hepatocellular versus cholestatic), extrahepatic features, likelihood that a given agent is to blame based on its past record, and exclusion of other potential causes. Scoring systems such as the Roussel-Uclaf Causality Assessment Method (RUCAM) yield residual uncertainty and have not been adopted widely. Currently, the U.S. Drug-Induced Liver Injury Network (DILIN) relies on a structured expert opinion process requiring detailed data on each case and a comprehensive review by three experts who arrive at a

1	the U.S. Drug-Induced Liver Injury Network (DILIN) relies on a structured expert opinion process requiring detailed data on each case and a comprehensive review by three experts who arrive at a consensus on a five-degree scale of likelihood (definite, highly likely, probable, possible, unlikely); however, this approach is not practical for routine clinical application.

1	Generally, drug hepatotoxicity is not more frequent in persons with underlying chronic liver disease, although the severity of the outcome may be amplified. Reported exceptions include hepatotoxicity of aspirin, methotrexate, isoniazid (only in certain experiences), antiretroviral therapy for HIV infection, and certain drugs such as conditioning regimens for bone marrow transplantation in the presence of hepatitis C.

1	Treatment is largely supportive, except in acetaminophen hepatotoxicity (see below). In patients with fulminant hepatitis resulting from drug hepatotoxicity, liver transplantation may be lifesaving (Chap. 368). Withdrawal of the suspected agent is indicated at the first sign of an adverse reaction. A number of studies have suggested that lethal outcomes follow continued use of an agent in the face of symptoms and signs of liver injury. In the case of the direct toxins, liver involvement should not divert attention from renal or other organ involvement, which may also threaten survival. A number of agents are occasionally used but are of questionable value: glucocorticoids for drug hepatotoxicity with allergic features, silibinin for hepatotoxic mushroom poisoning, and ursodeoxycholic acid for cholestatic drug hepatotoxicity have never been shown to be effective and are not recommended.

1	In Table 361-2, several classes of chemical agents are listed together with examples of the pattern of liver injury produced by them. Certain drugs appear to be responsible for the development of chronic as well as acute hepatic injury. For example, nitrofurantoin, minocycline, hydralazine, and methyldopa have been associated with moderate to severe chronic hepatitis with autoimmune features. Methotrexate, tamoxifen, and amiodarone have been implicated in the development of cirrhosis. Portal hypertension in the absence of cirrhosis may result from alterations in hepatic architecture produced by vitamin A or arsenic intoxication, industrial exposure to vinyl chloride, or Principal Morphologic Change Class of Agent Example

1	Calcium channel blocker Cholinesterase inhibitor Diuretic Laxative Norepinephrine-reuptake inhibitor Oral hypoglycemic Antibiotic Antibacterial Antifungal Antihistamine Immunosuppressive Lipid-lowering Analgesic Hydrocarbon Metal Mushroom Solvent Antiarrhythmic Antibiotic Anticonvulsant Anti-inflammatory Xanthine oxidase inhibitor Chemotherapeutic Methyl testosterone, many other body-building supplements Erythromycin estolate, nitrofurantoin, rifampin, amoxicillin-clavulanic acid, oxacillin Carbamazepine Duloxetine, mirtazapine, tricyclic antidepressants Sulindac Clopidogrel Irbesartan, fosinopril Methimazole Nifedipine, verapamil Cyclosporine Ezetimibe Anabolic steroids, busulfan, tamoxifen, irinotecan, cytarabine, temozolomide Norethynodrel with mestranol Chlorpropamide Chlorpromazineb Amiodarone Tetracycline (high-dose, IV) Valproic acid Dideoxynucleosides (e.g., zidovudine), protease inhibitors (e.g., indinavir, ritonavir) Asparaginase, methotrexate, tamoxifen Halothane, fluothane

1	Tetracycline (high-dose, IV) Valproic acid Dideoxynucleosides (e.g., zidovudine), protease inhibitors (e.g., indinavir, ritonavir) Asparaginase, methotrexate, tamoxifen Halothane, fluothane Flutamide Isoniazid,c rifampicin, nitrofurantoin, telithromycin, minocycline,d pyrazinamide, trovafloxacine Phenytoin, carbamazepine, valproic acid, phenobarbital Iproniazid, amitriptyline, trazodone, venlafaxine, fluoxetine, paroxetine, duloxetine, sertraline, nefazodonee Ketoconazole, fluconazole, itraconazole Methyldopa,c captopril, enalapril, lisinopril, losartan Ibuprofen, indomethacin, diclofenac, sulindac, bromfenac Risperidone Zidovudine, didanosine, stavudine, nevirapine, ritonavir, indinavir, tipranavir, zalcitabine Nifedipine, verapamil, diltiazem Tacrine Chlorothiazide Oxyphenisatinc,e Atomoxetine Troglitazone,e acarbose Amoxicillin-clavulanic acid, trimethoprim-sulfamethoxazole Clindamycin Terbinafine Cyproheptadine Azathioprine Nicotinic acid, lovastatin, ezetimibe Acetaminophen

1	Atomoxetine Troglitazone,e acarbose Amoxicillin-clavulanic acid, trimethoprim-sulfamethoxazole Clindamycin Terbinafine Cyproheptadine Azathioprine Nicotinic acid, lovastatin, ezetimibe Acetaminophen Carbon tetrachloride Yellow phosphorus

1	Dimethylformamide Quinidine, diltiazem Sulfonamides Carbamazepine Phenylbutazone Allopurinol Oxaliplatin, melphalan aSeveral agents cause more than one type of liver lesion and appear under more than one category. bRarely associated with primary biliary cirrhosis–like lesion. cOccasionally associated with chronic hepatitis or bridging hepatic necrosis or cirrhosis. dAssociated with an autoimmune hepatitis–like syndrome. eWithdrawn from use because of severe hepatotoxicity.

1	administration of thorium dioxide. The latter three agents have also been associated with angiosarcoma of the liver. Oral contraceptives have been implicated in the development of hepatic adenoma and, rarely, hepatocellular carcinoma and hepatic vein occlusion (Budd-Chiari syndrome). Another unusual lesion, peliosis hepatis (blood cysts of the liver), has been observed in some patients treated with anabolic or contraceptive steroids. The existence of these hepatic disorders expands the spectrum of liver injury induced by chemical agents and emphasizes the need for a thorough drug history in all patients with liver dysfunction. A helpful LiverTox website that contains up-to-date information on drug-induced liver injury is available through the National Institute of Diabetes and Digestive and Kidney Diseases and the National Library of Medicine (www.livertox.nih.gov). The following are patterns of adverse hepatic reactions for some prototypic agents.

1	Acetaminophen represents the most prevalent cause of acute liver failure in the Western world; up to 72% of patients with acetaminophen hepatotoxicity in Scandinavia—somewhat lower frequencies in the United Kingdom and the United States—progress to encephalopathy and coagulopathy. Acetaminophen causes dose-related centrilobular hepatic necrosis after single-time-point ingestions, as intentional self-harm, or over extended periods, as unintentional overdoses, when multiple drug preparations or inappropriate drug amounts are used daily for several days, e.g., for relief of pain or fever. In these instances, 8 g/d, twice the daily recommended maximum dose, over several days can readily lead to liver failure. Use of opioid-acetaminophen combinations appears to be particularly harmful, because habituation to the opioid may occur with a gradual increase in opioid-acetaminophen combination dosing over days or weeks. A single dose of 10–15 g, occasionally less, may produce clinical evidence

1	habituation to the opioid may occur with a gradual increase in opioid-acetaminophen combination dosing over days or weeks. A single dose of 10–15 g, occasionally less, may produce clinical evidence of liver injury. Fatal fulminant disease is usually (although not invariably) associated with ingestion of ≥25 g. Blood levels of acetaminophen correlate with severity of hepatic injury (levels >300 μg/mL 4 h after ingestion are predictive of the development of severe damage; levels <150 μg/mL suggest that hepatic injury is highly unlikely). Nausea, vomiting, diarrhea, abdominal pain, and shock are early manifestations occurring 4–12 h after ingestion. Then 24–48 h later, when these features are abating, hepatic injury becomes apparent. Maximal abnormalities and hepatic failure are evident 3–5 days after ingestion, and aminotransferase levels exceeding 10,000 IU/L are not uncommon (i.e., levels far exceeding those in patients with viral hepatitis). Renal failure and myocardial injury may be

1	after ingestion, and aminotransferase levels exceeding 10,000 IU/L are not uncommon (i.e., levels far exceeding those in patients with viral hepatitis). Renal failure and myocardial injury may be present. Whether or not a clear history of overdose can be elicited, clinical suspicion of acetaminophen hepatotoxicity should be raised by the presence of the extremely high aminotransferase levels in association with low bilirubin levels that are characteristic of this hyperacute injury. This biochemical signature should trigger further questioning of the subject if possible; however, denial or altered mentation may confound diagnostic efforts. In this setting, a presumptive diagnosis is reasonable, and the proven antidote, N-acetylcysteine— both safe and presumed to be effective even when injury has already begun to evolve—should be instituted.

1	Acetaminophen is metabolized predominantly by a phase II reaction to innocuous sulfate and glucuronide metabolites; however, a small proportion of acetaminophen is metabolized by a phase I reaction to a hepatotoxic metabolite formed from the parent compound by the cytochrome P450 CYP2E1. This metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), is detoxified by binding to “hepatoprotective” glutathione to become harmless, water-soluble mercapturic acid, which undergoes renal excretion. When excessive amounts of NAPQI are formed, or when glutathione levels are low, glutathione levels are depleted and overwhelmed, permitting covalent binding to nucleophilic hepatocyte macromolecules forming acetaminophen-protein “adducts.” These adducts, which can be measured in serum by high-performance liquid chromatography, hold promise as diagnostic markers of acetaminophen hepatotoxicity, and a point-of-care assay for acetaminophen-Cys adducts is under development. The binding of acetaminophen to

1	chromatography, hold promise as diagnostic markers of acetaminophen hepatotoxicity, and a point-of-care assay for acetaminophen-Cys adducts is under development. The binding of acetaminophen to hepatocyte macromolecules is believed to lead to hepatocyte necrosis; the precise sequence and mechanism are 2027 unknown. Hepatic injury may be potentiated by prior administration of alcohol, phenobarbital, isoniazid, or other drugs; by conditions that stimulate the mixed-function oxidase system; or by conditions such as starvation (including inability to maintain oral intake during severe febrile illnesses) that reduce hepatic glutathione levels. Alcohol induces cytochrome P450 CYP2E1; consequently, increased levels of the toxic metabolite NAPQI may be produced in chronic alcoholics after acetaminophen ingestion, but the role of alcohol in potentiating acute acetaminophen injury is still debated. Alcohol also suppresses hepatic glutathione production. Therefore, in chronic alcoholics, the

1	ingestion, but the role of alcohol in potentiating acute acetaminophen injury is still debated. Alcohol also suppresses hepatic glutathione production. Therefore, in chronic alcoholics, the toxic dose of acetaminophen may be as low as 2 g, and alcoholic patients should be warned specifically about the dangers of even standard doses of this commonly used drug. In a 2006 study, aminotransferase elevations were identified in 31–44% of normal subjects treated for 14 days with the maximal recommended dose of acetaminophen, 4 g daily (administered alone or as part of an acetaminophen-opioid combination); because these changes were transient and never associated with bilirubin elevation, the clinical relevance of these findings remains to be determined. Although underlying hepatitis C virus (HCV) infection was found to be associated with an increased risk of acute liver injury in patients hospitalized for acetaminophen overdose, generally, in patients with nonalcoholic liver disease,

1	(HCV) infection was found to be associated with an increased risk of acute liver injury in patients hospitalized for acetaminophen overdose, generally, in patients with nonalcoholic liver disease, acetaminophen taken in recommended doses is well tolerated. Acetaminophen use in cirrhotic patients has not been associated with hepatic decompensation. On the other hand, because of the link between acetaminophen use and liver injury, and because of the limited safety margin between safe and toxic doses, the FDA has recommended that the daily dose of acetaminophen be reduced from 4 g to 3 g (even lower for persons with chronic alcohol use), that all acetaminophen-containing products be labeled prominently as containing acetaminophen, and that the potential for liver injury be prominent in the packaging of acetaminophen and acetaminophen-containing products. Within opioid combination products, the limit for the acetaminophen component has been lowered to 325 mg per tablet.

1	Treatment includes gastric lavage, supportive measures, and oral administration of activated charcoal or cholestyramine to prevent absorption of residual drug. Neither charcoal nor cholestyramine appears to be effective if given >30 min after acetaminophen ingestion; if they are used, the stomach lavage should be done before other agents are administered orally. The chances of possible, probable, and high-risk hepatotoxicity can be derived from a nomogram plot (Fig. 361-2), readily available in emergency departments, as a function of measuring acetaminophen plasma levels 8 h after ingestion. In patients with high acetaminophen blood levels (>200 μg/mL measured at 4 h or >100 μg/mL at 8 h after ingestion), the administration of N-acetylcysteine reduces the severity of hepatic necrosis. This agent provides sulfhydryl donor groups to replete glutathione, which is required to render harmless toxic metabolites that would otherwise bind covalently via sulfhydryl linkages to cell proteins,

1	agent provides sulfhydryl donor groups to replete glutathione, which is required to render harmless toxic metabolites that would otherwise bind covalently via sulfhydryl linkages to cell proteins, resulting in the formation of drug metabolite-protein adducts. Therapy should be begun within 8 h of ingestion but may be at least partially effective when given as late as 24–36 h after overdose. Later administration of sulfhydryl compounds is of uncertain value. Routine use of N-acetylcysteine has substantially reduced the occurrence of fatal acetaminophen hepatotoxicity. N-acetylcysteine may be given orally but is more commonly used as an IV solution, with a loading dose of 140 mg/kg over 1 h, followed by 70 mg/kg every 4 h for 15–20 doses. Whenever a patient with potential acetaminophen hepatotoxicity is encountered, a local poison control center should be contacted. Treatment can be stopped when plasma acetaminophen levels indicate that the risk of liver damage is low. If signs of

1	hepatotoxicity is encountered, a local poison control center should be contacted. Treatment can be stopped when plasma acetaminophen levels indicate that the risk of liver damage is low. If signs of hepatic failure (e.g., progressive jaundice, coagulopathy, confusion) occur despite N-acetylcysteine therapy for acetaminophen hepatotoxicity, liver transplantation may be the only option. Early arterial blood lactate levels among such patients with acute liver failure

1	FIGURE 361-2 Nomogram to define risk of acetaminophen hepatotoxicity according to initial plasma acetaminophen concentration. (After BH Rumack, H Matthew: Pediatrics 55:871, 1975.) may distinguish patients highly likely to require liver transplantation (lactate levels >3.5 mmol/L) from those likely to survive without liver replacement. Acute renal injury occurs in nearly 75% of patients with severe acetaminophen injury but is virtually always self-limited. Survivors of acute acetaminophen overdose rarely, if ever, have ongoing liver injury or sequelae.

1	Isoniazid (INH) remains central to most antituberculous prophylactic and therapeutic regimens, despite its long-standing recognition as a hepatotoxin. In 10% of patients treated with INH, elevated serum aminotransferase levels develop during the first few weeks of therapy; however, these elevations in most cases are self-limited, mild (values for ALT <200 IU/L), and resolve despite continued drug use. This adaptive response allows continuation of the agent if symptoms and progressive enzyme elevations do not follow the initial elevations. Acute hepatocellular drug-induced liver injury secondary to INH is evident with a variable latency period up to 6 months and is more frequent in alcoholics and patients taking certain other medications, such as barbiturates, rifampin, and pyrazinamide. If the clinical threshold of encephalopathy is reached, severe hepatic injury is likely to be fatal or to require liver transplantation. Liver biopsy reveals morphologic changes similar to those of

1	If the clinical threshold of encephalopathy is reached, severe hepatic injury is likely to be fatal or to require liver transplantation. Liver biopsy reveals morphologic changes similar to those of viral hepatitis or bridging hepatic necrosis. Substantial liver injury appears to be age-related, increasing substantially after age 35; the highest frequency is in patients over age 50, and the lowest is in patients under the age of 20. Even for patients >50 years of age monitored carefully during therapy, hepatotoxicity occurs in only ~2%, well below the risk estimate derived from earlier experiences. Fever, rash, eosinophilia, and other manifestations of drug allergy are distinctly unusual. Recently, antibodies to INH have been detected in INH recipients, but a link to causality of liver injury remains unclear. A clinical picture resembling chronic hepatitis has been observed in a few patients. Many public health programs that require INH prophylaxis for a positive tuberculin skin test

1	remains unclear. A clinical picture resembling chronic hepatitis has been observed in a few patients. Many public health programs that require INH prophylaxis for a positive tuberculin skin test or Quantiferon test include monthly monitoring of aminotransferase levels, although this practice has been called into question. Even more effective in limiting serious outcomes may be encouraging patients to be alert for symptoms such as nausea, fatigue, or jaundice, because most fatalities occur in the setting of continued INH use despite clinically apparent illness.

1	Sodium valproate, an anticonvulsant useful in the treatment of petit mal and other seizure disorders, has been associated with the development of severe hepatic toxicity and, rarely, fatalities, predominantly in children but also in adults. Among children listed as candidates for liver transplantation, valproate is the most common antiepileptic drug implicated. Asymptomatic elevations of serum aminotransferase levels have been recognized in as many as 45% of treated patients. These “adaptive” changes, however, appear to have no clinical importance, because major hepatotoxicity is not seen in the majority of patients despite continuation of drug therapy. In the rare patients in whom jaundice, encephalopathy, and evidence of hepatic failure are found, examination of liver tissue reveals microvesicular fat and bridging hepatic necrosis, predominantly in the centrilobular zone. Bile duct injury may also be apparent. Most likely, sodium valproate is not directly hepatotoxic, but its

1	microvesicular fat and bridging hepatic necrosis, predominantly in the centrilobular zone. Bile duct injury may also be apparent. Most likely, sodium valproate is not directly hepatotoxic, but its metabolite, 4-pentenoic acid, may be responsible for hepatic injury. Valproate hepatotoxicity is more common in persons with mitochondrial enzyme deficiencies and may be ameliorated by IV administration of carnitine, which valproate therapy can deplete. Recently, valproate toxicity has been linked to HLA haplotypes (DR4 and B*1502) and to mutations in mitochondrial DNA polymerase gamma 1.

1	This commonly used antibiotic for urinary tract infections may cause an acute hepatitis leading to fatal outcome or, more frequently, chronic hepatitis of varying severity but indistinguishable from autoimmune chronic hepatitis. These two scenarios may reflect the frequent use and reuse of the drug for treatment of recurrent cystitis in women. Although most toxic agents manifest injury within 6 months of first ingestion, nitrofurantoin may have a longer latency period, in part perhaps because of its intermittent, recurrent use. Autoantibodies to nuclear components, smooth muscle, and mitochondria are seen and may subside after resolution of infection; however, glucocorticoid or other immunosuppressive medication may be necessary to resolve the autoimmune injury, and cirrhosis may be seen in cases that are not recognized quickly. Interstitial pulmonary fibrosis presenting as chronic cough and dyspnea may be present and resolve slowly with medication withdrawal. Histologic findings are

1	in cases that are not recognized quickly. Interstitial pulmonary fibrosis presenting as chronic cough and dyspnea may be present and resolve slowly with medication withdrawal. Histologic findings are identical to those of autoimmune hepatitis. A similar disease pattern can be observed with minocycline that is used repeatedly for the treatment of acne in teenagers as well as with hydralazine and alpha methyldopa.

1	Currently, the most common agent implicated as causing drug-induced liver injury in the United States and in Europe is amoxicillinclavulanate (most frequent brand name: Augmentin). This medication causes a very specific syndrome of mixed or primarily cholestatic injury. Because hepatotoxicity may follow amoxicillin-clavulanate therapy after a relatively long latency period, the liver injury may begin to manifest at the time of drug withdrawal or after the drug has been withdrawn. The high prevalence of hepatotoxicity reflects in part the very frequent use of this drug for respiratory tract infections, including community-acquired pneumonia. The mechanism of hepatotoxicity is unclear, but the liver injury is thought to be caused by amoxicillin toxicity that is potentiated in some way by clavulanate, which itself appears not to be toxic. Symptoms include nausea, anorexia, fatigue, and jaundice—which may be prolonged—with pruritus. Rash is quite uncommon. On occasion,

1	in some way by clavulanate, which itself appears not to be toxic. Symptoms include nausea, anorexia, fatigue, and jaundice—which may be prolonged—with pruritus. Rash is quite uncommon. On occasion, amoxicillin-clavulanate, like other cholestatic hepatotoxic drugs, causes permanent injury to small bile ducts, leading to the so-called “vanishing bile duct syndrome.” In vanishing bile duct syndrome, initially, liver injury is minimal except for severe cholestasis; however, over time, histologic evidence of bile duct abnormalities is replaced by a paucity and eventual absence of discernible ducts on subsequent biopsies.

1	Phenytoin, formerly diphenylhydantoin, a mainstay in the treatment of seizure disorders, has been associated in rare instances with the development of severe hepatitis-like liver injury leading to fulminant hepatic failure. In many patients, the hepatitis is associated with striking fever, lymphadenopathy, rash (Stevens-Johnson syndrome or exfoliative dermatitis), leukocytosis, and eosinophilia, suggesting an immunologically mediated hypersensitivity mechanism. Despite these observations, evidence suggests that metabolic idiosyncrasy may be responsible for hepatic injury. In the liver, phenytoin is converted by cytochrome P450 to metabolites, including the highly reactive electrophilic arene oxides. These metabolites are normally metabolized further by epoxide hydrolases. A defect (genetic or acquired) in epoxide hydrolase activity could permit covalent binding of arene oxides to hepatic macromolecules, thereby leading to hepatic injury. Hepatic injury is usually manifest within the

1	or acquired) in epoxide hydrolase activity could permit covalent binding of arene oxides to hepatic macromolecules, thereby leading to hepatic injury. Hepatic injury is usually manifest within the first 2 months after beginning phenytoin therapy. With the exception of an abundance of eosinophils in the liver, the clinical, biochemical, and histologic picture resembles that of viral hepatitis. In rare instances, bile duct injury may be the salient feature of phenytoin hepatotoxicity, with striking features of intrahepatic cholestasis. Asymptomatic elevations of aminotransferase and alkaline phosphatase levels have been observed in a sizable proportion of patients receiving long-term phenytoin therapy. These liver changes are believed by some authorities to represent the potent hepatic enzyme-inducing properties of phenytoin and are accompanied histologically by swelling of hepatocytes in the absence of necroinflammatory activity or evidence of chronic liver disease.

1	Therapy with this potent antiarrhythmic drug is accompanied in 15–50% of patients by modest elevations of serum aminotransferase levels that may remain stable or diminish despite continuation of the drug. Such abnormalities may appear days to many months after beginning therapy. A proportion of those with elevated aminotransferase levels have detectable hepatomegaly, and clinically important liver disease develops in <5% of patients. Features that represent a direct effect of the drug on the liver and that are common to the majority of long-term recipients are ultrastructural phospholipidosis, unaccompanied by clinical liver disease, and interference with hepatic mixed-function oxidase metabolism of other drugs. The cationic amphiphilic drug and its major metabolite desethylamiodarone accumulate in hepatocyte lysosomes and mitochondria and in bile duct epithelium. The relatively common elevations in aminotransferase levels are also considered a predictable, dose-dependent, direct

1	accumulate in hepatocyte lysosomes and mitochondria and in bile duct epithelium. The relatively common elevations in aminotransferase levels are also considered a predictable, dose-dependent, direct hepatotoxic effect. On the other hand, in the rare patient with clinically apparent, symptomatic liver disease, liver injury resembling that seen in alcoholic liver disease is observed. The so-called pseudoalcoholic liver injury can range from steatosis to alcoholic hepatitis–like neutrophilic infiltration and Mallory’s hyaline to cirrhosis. Electron-microscopic demonstration of phospholipid-laden lysosomal lamellar bodies can help to distinguish amiodarone hepatotoxicity from typical alcoholic hepatitis. This category of liver injury appears to be a metabolic idiosyncrasy that allows hepatotoxic metabolites to be generated. Rarely, an acute idiosyncratic hepatocellular injury resembling viral hepatitis or cholestatic hepatitis occurs. Hepatic granulomas have occasionally been observed.

1	metabolites to be generated. Rarely, an acute idiosyncratic hepatocellular injury resembling viral hepatitis or cholestatic hepatitis occurs. Hepatic granulomas have occasionally been observed. Because amiodarone has a long half-life, liver injury may persist for months after the drug is stopped.

1	The most important adverse effect associated with erythromycin, more common in children than adults, is the infrequent occurrence of a cholestatic reaction. Although most of these reactions have been associated with erythromycin estolate, other erythromycins may also be responsible. The reaction usually begins during the first 2 or 3 weeks of therapy and includes nausea, vomiting, fever, right upper quadrant abdominal pain, jaundice, leukocytosis, and moderately elevated aminotransferase and alkaline phosphatase levels. The clinical picture 2029 can resemble acute cholecystitis or bacterial cholangitis. Liver biopsy reveals variable cholestasis; portal inflammation comprising lymphocytes, polymorphonuclear leukocytes, and eosinophils; and scattered foci of hepatocyte necrosis. Symptoms and laboratory findings usually subside within a few days of drug withdrawal, and evidence of chronic liver disease has not been found on follow-up. The precise mechanism remains ill-defined.

1	The administration of oral contraceptive combinations of estrogenic and progestational steroids leads to intrahepatic cholestasis with pruritus and jaundice in a small number of patients weeks to months after taking these agents. Especially susceptible seem to be patients with recurrent idiopathic jaundice of pregnancy, severe pruritus of pregnancy, or a family history of these disorders. With the exception of liver biochemical tests, laboratory studies are normal, and extrahepatic manifestations of hypersensitivity are absent. Liver biopsy reveals cholestasis with bile plugs in dilated canaliculi and striking bilirubin stain ing of liver cells. In contrast to chlorpromazine-induced cholestasis, portal inflammation is absent. The lesion is reversible on withdrawal of the agent. The two steroid components appear to act synergistically on hepatic function, although the estrogen may be primarily responsible. Oral contraceptives are contraindicated in patients with a history of recurrent

1	components appear to act synergistically on hepatic function, although the estrogen may be primarily responsible. Oral contraceptives are contraindicated in patients with a history of recurrent jaundice of pregnancy. Primarily benign, but rarely malignant, neoplasms of the liver, hepatic vein occlusion, and peripheral sinusoidal dilatation have also been associated with oral contraceptive therapy. Focal nodular hyperplasia of the liver is not more frequent among users of oral contraceptives.

1	The most common form of liver injury caused by complementary and alternative medications is the profound cholestasis associated with anabolic steroids used by body builders. Unregulated agents sold in gyms and health food stores as diet supplements, which are taken by athletes to improve their performance, may contain anabolic steroids. Jaundice in a young male that is accompanied by a cholestatic, rather than a hepatitic, laboratory profile almost invariably will turn out to be caused by the use of one of a variety of androgen congeners. Such agents have the potential to injure bile transport pumps and to cause intense cholestasis; the time to onset is variable, and resolution, which is the rule, may require many weeks to months. Initially, anorexia, nausea, and malaise may occur, followed by pruritus in some but not all patients. Serum aminotransferase levels are usually <100 IU/L and serum alkaline phosphatase levels are generally moderately elevated with bilirubin levels

1	followed by pruritus in some but not all patients. Serum aminotransferase levels are usually <100 IU/L and serum alkaline phosphatase levels are generally moderately elevated with bilirubin levels frequently exceeding 342 μmol/L (20 mg/dL). Examination of liver tissue reveals cholestasis without substantial inflammation or necrosis. Anabolic steroids have also been used by prescription to treat bone marrow failure. In this setting, hepatic sinusoidal dilatation and peliosis hepatis have been reported in rare patients, as have hepatic adenomas and hepatocellular carcinoma.

1	This antibiotic combination is used routinely for urinary tract infections in immunocompetent persons and for prophylaxis against and therapy of Pneumocystis carinii pneumonia in immunosuppressed persons (transplant recipients, patients with AIDS). With its increasing use, its occasional hepatotoxicity is being recognized with growing frequency. Its likelihood is unpredictable, but when it occurs, trimethoprimsulfamethoxazole hepatotoxicity follows a relatively uniform latency period of several weeks and is often accompanied by eosinophilia, rash, and other features of a hypersensitivity reaction. Biochemically and histologically, acute hepatocellular necrosis predominates, but cholestatic features are quite frequent. Occasionally, cholestasis without necrosis occurs, and, very rarely, a severe cholangiolytic pattern of liver injury is observed. In most cases, liver injury is self-limited, but rare fatalities have been recorded. The hepatotoxicity is attributable to the

1	rarely, a severe cholangiolytic pattern of liver injury is observed. In most cases, liver injury is self-limited, but rare fatalities have been recorded. The hepatotoxicity is attributable to the sulfamethoxazole component of the drug and is similar in features to 2030 that seen with other sulfonamides; tissue eosinophilia and granulomas may be seen. The risk of trimethoprim-sulfamethoxazole hepatotoxicity is increased in persons with HIV infection.

1	Between 1 and 2% of patients taking lovastatin, simvastatin, pravastatin, fluvastatin, or one of the newer statin drugs for the treatment of hypercholesterolemia experience asymptomatic, reversible elevations (>threefold) of aminotransferase activity. Acute hepatitis-like histologic changes, centrilobular necrosis, and centrilobular cholestasis have been described in a very small number of cases. In a larger proportion, minor aminotransferase elevations appear during the first several weeks of therapy. Careful laboratory monitoring can distinguish between patients with minor, transitory changes, who may continue therapy and those with more profound and sustained abnormalities, who should discontinue therapy. Because clinically meaningful aminotransferase elevations are so rare after statin use and do not differ in meta-analyses from the frequency of such laboratory abnormalities in placebo recipients, a panel of liver experts recommended to the National Lipid Association’s Safety Task

1	use and do not differ in meta-analyses from the frequency of such laboratory abnormalities in placebo recipients, a panel of liver experts recommended to the National Lipid Association’s Safety Task Force that liver test monitoring was not necessary in patients treated with statins and that statin therapy need not be discontinued in patients found to have asymptomatic isolated aminotransferase elevations during therapy. Statin hepatotoxicity is not increased in patients with chronic hepatitis C, hepatic steatosis, or other underlying liver diseases, and statins can be used safely in these patients.

1	TOTAL PARENTERAL NUTRITION (STEATOSIS, CHOLESTASIS)

1	Total parenteral nutrition (TPN) is often complicated by cholestatic hepatitis attributable to steatosis, cholestasis, or gallstones (or gallbladder sludge). Steatosis or steatohepatitis may result from the excess carbohydrate calories in these nutritional supplements and is the predominant form of TPN-associated liver disorder in adults. The frequency of this complication has been reduced substantially by the introduction of balanced TPN formulas that rely on lipid as an alternative caloric source. Cholestasis and cholelithiasis, caused by the absence of stimulation of bile flow and secretion resulting from the lack of oral intake, is the predominant form of TPN-associated liver disease in infants, especially in premature neonates. Often, cholestasis in such neonates is multifactorial, contributed to by other factors such as sepsis, hypoxemia, and hypotension; occasionally, TPN-induced cholestasis in neonates culminates in chronic liver disease and liver failure. When TPN-associated

1	to by other factors such as sepsis, hypoxemia, and hypotension; occasionally, TPN-induced cholestasis in neonates culminates in chronic liver disease and liver failure. When TPN-associated liver test abnormalities occur in adults, balancing the TPN formula with more lipid is the intervention of first recourse. In infants with TPN-associated cholestasis, the addition of oral feeding may ameliorate the problem. Therapeutic interventions suggested, but not shown, to be of proven benefit, include cholecystokinin, ursodeoxycholic acid, S -adenosyl methionine, and taurine.

1	ALTERNATIVE AND COMPLEMENTARY MEDICINES (IDIOSYNCRATIC HEPATITIS, STEATOSIS)

1	Herbal medications that are of scientifically unproven efficacy and that lack prospective safety oversight by regulatory agencies currently account for more than 20% of drug-induced liver injury in the United States. Besides anabolic steroids, the most common category of dietary or herbal products is weight loss agents. Included among the herbal remedies associated with toxic hepatitis are Jin Bu Huan, xiao-chai-hutang, germander, chaparral, senna, mistletoe, skullcap, gentian, comfrey (containing pyrrolizidine alkaloids), ma huang, bee pollen, valerian root, pennyroyal oil, kava, celandine, Impila (Callilepis laureola), LipoKinetix, Hydroxycut, herbal nutritional supplements, and herbal teas containing Camellia sinensis (green tea extract). Well characterized are the acute hepatitis-like histologic lesions following Jin Bu Huan use: focal hepatocellular necrosis, mixed mononuclear portal tract infiltration, coagulative necrosis, apoptotic hepatocyte degeneration, tissue eosinophilia,

1	histologic lesions following Jin Bu Huan use: focal hepatocellular necrosis, mixed mononuclear portal tract infiltration, coagulative necrosis, apoptotic hepatocyte degeneration, tissue eosinophilia, and microvesicular steatosis. Megadoses of vitamin A can injure the liver, as can pyrrolizidine alkaloids, which often contaminate Chinese herbal preparations and can cause a venoocclusive injury leading to sinusoidal hepatic vein obstruction. Because some alternative medicines induce toxicity via active metabolites, alcohol and drugs that stimulate cytochrome P450 enzymes may enhance the toxicity of some of these products. Conversely, some alternative medicines also stimulate cytochrome P450 and may result in or amplify the toxicity of recognized drug hepatotoxins. Given the widespread use of such poorly defined herbal preparations, hepatotoxicity is likely to be encountered with increasing frequency; therefore, a drug history in patients with acute and chronic liver disease should

1	use of such poorly defined herbal preparations, hepatotoxicity is likely to be encountered with increasing frequency; therefore, a drug history in patients with acute and chronic liver disease should include use of “alternative medicines” and other nonprescription preparations sold in so-called health food stores.

1	HIGHLY ACTIVE ANTIRETROVIRAL THERAPY (HAART) FOR HIV INFECTION (MITOCHONDRIAL TOXIC, IDIOSYNCRATIC, STEATOSIS; HEPATOCELLULAR, CHOLESTATIC, AND MIXED)

1	The recognition of drug hepatotoxicity in persons with HIV infection is complicated in this population by the many alternative causes of liver injury (chronic viral hepatitis, fatty infiltration, infiltrative disorders, mycobacterial infection, etc.), but drug hepatotoxicity associated with HAART is an emerging and common type of liver injury in HIV-infected persons (Chap. 226). Although no one antiviral agent is recognized as a potent hepatotoxin, combination regimens including reverse transcriptase and protease inhibitors cause hepatotoxicity in ~10% of treated patients. Implicated most frequently are combinations including nucleoside analogue reverse transcriptase inhibitors zidovudine, didanosine, and, to a lesser extent, stavudine; protease inhibitors ritonavir and indinavir (and amprenavir when used together with ritonavir), as well as tipranavir; and nonnucleoside reverse transcriptase inhibitors nevirapine and, to a lesser extent, efavirenz. These drugs cause predominantly

1	amprenavir when used together with ritonavir), as well as tipranavir; and nonnucleoside reverse transcriptase inhibitors nevirapine and, to a lesser extent, efavirenz. These drugs cause predominantly hepatocellular injury but cholestatic injury as well, and prolonged (>6 months) use of reverse transcriptase inhibitors has been associated with mitochondrial injury, steatosis, and lactic acidosis. Indirect hyperbilirubinemia, resulting from direct inhibition of bilirubin-conjugating activity by UDP-glucuronosyltransferase, usually without elevation of aminotransferase or alkaline phosphatase activities, occurs in ~10% of patients treated with the protease inhibitor indinavir. Distinguishing the impact of HAART hepatotoxicity in patients with HIV and hepatitis virus co-infection is made challenging by the following: (1) both chronic hepatitis B and hepatitis C can affect the natural history of HIV infection and the response to HAART, and (2) HAART can have an impact on chronic viral

1	challenging by the following: (1) both chronic hepatitis B and hepatitis C can affect the natural history of HIV infection and the response to HAART, and (2) HAART can have an impact on chronic viral hepatitis. For example, immunologic reconstitution with HAART can result in immunologically mediated liver-cell injury in patients with chronic hepatitis B co-infection if treatment with an antiviral agent for hepatitis B (e.g., the nucleoside analogue lamivudine) is withdrawn or if nucleoside analogue resistance emerges. Infection with HIV, especially with low CD4+ T cell counts, has been reported to increase the rate of hepatic fibrosis associated with chronic hepatitis C, and HAART therapy can increase levels of serum aminotransferases and HCV RNA in patients with hepatitis C co-infection. Didanosine or stavudine should not be used with ribavirin in patients with HIV/HCV co-infection because of an increased risk of severe mitochondrial toxicity and lactic acidosis.

1	Kurt J. Isselbacher, MD, contributed to this chapter in previous editions of Harrison’s. Type of Hepatitis Diagnostic Test(s) Autoantibodies Therapy Jules L. Dienstag

1	Chronic hepatitis represents a series of liver disorders of varying causes and severity in which hepatic inflammation and necrosis continue for at least 6 months. Milder forms are nonprogressive or only slowly progressive, while more severe forms may be associated with scarring and architectural reorganization, which, when advanced, lead ultimately to cirrhosis. Several categories of chronic hepatitis have been recognized. These include chronic viral hepatitis, drug-induced chronic hepatitis (Chap. 361), and autoimmune chronic hepatitis. In many cases, clinical and laboratory features are insufficient to allow assignment into one of these three categories; these “idiopathic” cases are also believed to represent autoimmune chronic hepatitis. Finally, clinical and laboratory features of chronic hepatitis are observed occasionally in patients with such hereditary/metabolic disorders as Wilson’s disease (copper overload), α1 antitrypsin deficiency (Chaps. 365 and 429), and nonalcoholic

1	hepatitis are observed occasionally in patients with such hereditary/metabolic disorders as Wilson’s disease (copper overload), α1 antitrypsin deficiency (Chaps. 365 and 429), and nonalcoholic fatty liver disease (Chap. 367e) and even occasionally in patients with alcoholic liver injury (Chap. 363). Although all types of chronic hepatitis share certain clinical, laboratory, and histopathologic features, chronic viral and chronic autoimmune hepatitis are sufficiently distinct to merit separate discussions. For discussion of acute hepatitis, see Chap. 360.

1	Common to all forms of chronic hepatitis are histopathologic distinctions based on localization and extent of liver injury. These vary from the milder forms, previously labeled chronic persistent hepatitis and chronic lobular hepatitis, to the more severe form, formerly called chronic active hepatitis. When first defined, these designations were believed to have prognostic implications, which were not corroborated by subsequent observations. Categorization of chronic hepatitis based primarily on histopathologic features has been replaced by a more informative classification based on a combination of clinical, serologic, and histologic variables. Classification of chronic hepatitis is based on (1) its cause; (2) its histologic activity, or grade; and (3) its degree of progression, or stage. Thus, neither clinical features alone nor histologic features—requiring liver biopsy—alone are sufficient to characterize and distinguish among the several categories of chronic hepatitis.

1	Clinical and serologic features allow the establishment of a diagnosis of chronic viral hepatitis, caused by hepatitis B, hepatitis B plus D, or hepatitis C; autoimmune hepatitis, including several subcategories, I and II (perhaps III), based on serologic distinctions; drug-associated chronic hepatitis; and a category of unknown cause, or cryptogenic chronic hepatitis (Table 362-1). These are addressed in more detail below.

1	Grade, a histologic assessment of necroinflammatory activity, is based on examination of the liver biopsy. An assessment of important histologic features includes the degree of periportal necrosis and the disruption of the limiting plate of periportal hepatocytes by inflammatory cells (so-called piecemeal necrosis or interface hepatitis); the degree of confluent necrosis that links or forms bridges between vascular structures— between portal tract and portal tract or even more important bridges between portal tract and central vein—referred to as bridging necrosis; the degree of hepatocyte degeneration and focal necrosis within the lobule; and the degree of portal inflammation. Several scoring systems that take these histologic features into account have been devised, and the most popular are the histologic activity index (HAI), used commonly in the United States, and the METAVIR score, used in Europe (Table 362-2). Based on the presence and degree of these features of histologic

1	are the histologic activity index (HAI), used commonly in the United States, and the METAVIR score, used in Europe (Table 362-2). Based on the presence and degree of these features of histologic activity, chronic hepatitis can be graded as mild, moderate, or severe.

1	aAntibodies to liver-kidney microsomes type 1 (autoimmune hepatitis type II and some cases of hepatitis C). bAdministered as a triple-drug combination with PEG IFN and ribavirin. Between the writing and publication of this chapter, two additional drugs were approved for hepatitis C, simeprevir and sofosbuvir (see www.hcvguidelines.org). cEarly clinical trials suggested benefit of IFN-α therapy; PEG IFN-α is as effective, if not more so, and has supplanted standard IFN-α. dAntinuclear antibody (autoimmune hepatitis type I). eAntibodies to soluble liver antigen (autoimmune hepatitis type III). Abbreviations: HBc, hepatitis B core; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; HDV, hepatitis D virus; IFN-α, interferon α; IgG, immunoglobulin G; LKM, liver-kidney microsome; PEG IFN-α, pegylated interferon α; SLA, soluble liver antigen.

1	The stage of chronic hepatitis, which reflects the level of progression of the disease, is based on the degree of hepatic fibrosis. When fibrosis is so extensive that fibrous septa surround parenchymal nodules and alter the normal architecture of the liver lobule, the histologic lesion is defined as cirrhosis. Staging is based on the degree of fibrosis as categorized on a numerical scale from 0−6 (HAI) or 0−4 (METAVIR) (Table 362-2). Several noninvasive approaches have been introduced to provide approximations of hepatic histologic stage, including serum biomarkers of fibrosis and imaging determinations of liver elasticity.

1	Both the enterically transmitted forms of viral hepatitis, hepatitis A and E, are self-limited and do not cause chronic hepatitis (rare reports notwithstanding in which acute hepatitis A serves as a trigger for the onset of autoimmune hepatitis in genetically susceptible patients or in which hepatitis E (Chap. 360) can cause chronic liver disease in immunosuppressed hosts, e.g., after liver transplantation). In contrast, the entire clinicopathologic spectrum of chronic hepatitis occurs in patients with chronic viral hepatitis B and C as well as in patients with chronic hepatitis D superimposed on chronic hepatitis B.

1	The likelihood of chronicity after acute hepatitis B varies as a function of age. Infection at birth is associated with clinically silent acute infection but a 90% chance of chronic infection, whereas infection in young adulthood in immunocompetent persons is typically associated with clinically apparent acute hepatitis but a risk of chronicity of only approximately 1%. Most cases of chronic hepatitis B among adults, however, occur in patients who never had a recognized episode of clinically apparent acute viral hepatitis. The degree of liver injury (grade) in patients with chronic hepatitis B is variable, ranging from none in inactive carriers to mild to moderate to severe. Among adults

1	The relatively replicative phase is characterized by the presence in the serum of HBeAg and HBV DNA levels well in excess of 103−104 IU/mL, sometimes exceedingHistologic Feature Severity Score Severity Score 109 IU/mL; by the presence in the liver of detectable Necroinflammatory Activity (grade) intrahepatocyte nucleocapsid antigens (primarily hepa-Periportal necrosis, None 0 None 0 titis B core antigen [HBcAg]); by high infectivity; and including piecemeal Mild 1 Mild 1 by accompanying liver injury. In contrast, the relativelynecrosis and/or nonreplicative phase is characterized by the absence of the conventional serum marker of HBV replication (HBeAg), the appearance of anti-HBe, levels of HBV DNA below a threshold of ~103 IU/mL, the absence ofBridging Yes intrahepatocytic HBcAg, limited infectivity, and mini-

1	DNA below a threshold of ~103 IU/mL, the absence ofBridging Yes intrahepatocytic HBcAg, limited infectivity, and mini- No mal liver injury. Patients in the replicative phase tend Intralobular Confluent —None 0 None or mild 0 to have more severe chronic hepatitis, whereas those necrosis —Focal 1 Moderate 1 in the nonreplicative phase tend to have minimal or mild chronic hepatitis or to be inactive hepatitis B car riers. The likelihood in a patient with HBeAg-reactive chronic hepatitis B of converting spontaneously from relatively replicative to nonreplicative infection is approximately 10% per year. Distinctions in HBV replication and in histologic category, however, do not always coincide. In patients with HBeAg-reactive chronic HBV infection, especially when acquired at birth or in early childhood, as recognized commonly in Asian countries, a dichotomy is common between very high levels of HBV replication during the early decades of life (when the level of host tolerance of

1	HBV is relatively high) and negligible levels of liver injury. Yet despite the relatively immediate, apparently benign nature of liver disease for many decades in this population, in the middle decades, activation of liver injury emerges as relative tolerance of the host to HBV declines, and these patients with childhood-acquired HBV infection are ultimately at increased risk later in life of cirrhosis, hepatocellular carcinoma (HCC) F0 (Chap. 111), and liver-related death. A discussion Portal fibrosis—some F1 of the pathogenesis of liver injury in patients with Portal fibrosis—most F1 chronic hepatitis B appears in Chap. 360. Bridging fibrosis—few HBeAg-negative chronic hepatitis B (i.e., chronic HBV infection with active virus replication, readily detectable HBV DNA

1	F1 chronic hepatitis B appears in Chap. 360. Bridging fibrosis—few HBeAg-negative chronic hepatitis B (i.e., chronic HBV infection with active virus replication, readily detectable HBV DNA F4 but without HBeAg [anti-HBe-reactive]), is more com- 4 Mediterranean and European countries and in Asia aIshak K, Baptista A, Bianchi L, et al: Histologic grading and staging of chronic hepatitis. J Hepatol 22:696, (and, correspondingly, in HBV genotypes other than A).1995. bBedossa P, Poynard T, French METAVIR Cooperative Study Group: An algorithm for grading activity in

1	Compared to patients with HBeAg-reactive chronic chronic hepatitis C. Hepatology 24:289, 1996. cNecroinflammatory grade: A0 = none; A1 = mild; A2 = moderate; A3 = severe. hepatitis B, patients with HBeAg-negative chronic hepatitis B have levels of HBV DNA that are several orders of magnitude lower (no more than 105−106 IU/mL) than those observed in the HBeAg-reactive subset. Most such with chronic hepatitis B, histologic features are of prognostic impor-cases represent precore or core-promoter mutations acquired late in the tance. In one long-term study of patients with chronic hepatitis B, natural history of the disease (mostly early-life onset; age range 40−55 investigators found a 5-year survival rate of 97% for patients with mild years, older than that for HBeAg-reactive chronic hepatitis B); these chronic hepatitis, 86% for patients with moderate to severe chronic mutations prevent translation of HBeAg from the precore component of hepatitis, and only 55% for patients with

1	hepatitis B); these chronic hepatitis, 86% for patients with moderate to severe chronic mutations prevent translation of HBeAg from the precore component of hepatitis, and only 55% for patients with chronic hepatitis and postne-the HBV genome (precore mutants) or are characterized by downregucrotic cirrhosis. The 15-year survival in these cohorts was 77%, 66%, lated transcription of precore mRNA (core-promoter mutants; Chap. and 40%, respectively. On the other hand, more recent observations 360). Although their levels of HBV DNA tend to be lower than among do not allow us to be so sanguine about the prognosis in patients with patients with HBeAg-reactive chronic hepatitis B, patients with HBeAgmild chronic hepatitis; among such patients followed for 1−13 years, negative chronic hepatitis B can have progressive liver injury (compliprogression to more severe chronic hepatitis and cirrhosis has been cated by cirrhosis and HCC) and experience episodic reactivation of observed in more than

1	can have progressive liver injury (compliprogression to more severe chronic hepatitis and cirrhosis has been cated by cirrhosis and HCC) and experience episodic reactivation of observed in more than a quarter of cases. liver disease reflected in fluctuating levels of aminotransferase activity More important to consider than histology alone in patients with (“flares”). The biochemical and histologic activity of HBeAg-negative chronic hepatitis B is the degree of hepatitis B virus (HBV) replication. disease tends to correlate closely with levels of HBV replication, unlike As reviewed in Chap. 360, chronic HBV infection can occur in the the case mentioned above of Asian patients with HBeAg-reactive presence or absence of serum hepatitis B e antigen (HBeAg), and gen-chronic hepatitis B during the early decades of their HBV infection. An erally, for both HBeAg-reactive and HBeAg-negative chronic hepatitis important point worth reiterating is the observation that the level of B, the level

1	the early decades of their HBV infection. An erally, for both HBeAg-reactive and HBeAg-negative chronic hepatitis important point worth reiterating is the observation that the level of B, the level of HBV DNA correlates with the level of liver injury and HBV replication is the most important risk factor for the ultimate develrisk of progression. In HBeAg-reactive chronic hepatitis B, two phases opment of cirrhosis and HCC in both HBeAg-reactive and HBeAghave been recognized based on the relative level of HBV replication. negative patients. Although levels of HBV DNA are lower and more readily suppressed by therapy to undetectable levels in HBeAg-negative (compared to HBeAg-reactive) chronic hepatitis B, achieving sustained responses that permit discontinuation of antiviral therapy is less likely in HBeAg-negative patients (see below). Inactive carriers are patients with circulating hepatitis B surface antigen (HBsAg), normal serum aminotransferase levels, undetectable HBeAg, and

1	less likely in HBeAg-negative patients (see below). Inactive carriers are patients with circulating hepatitis B surface antigen (HBsAg), normal serum aminotransferase levels, undetectable HBeAg, and levels of HBV DNA that are either undetectable or present at a threshold of ≤103 IU/mL. This serologic profile can occur not only in inactive carriers but also in patients with HBeAg-negative chronic hepatitis B during periods of relative inactivity; distinguishing between the two requires sequential biochemical and virologic monitoring over many months.

1	The spectrum of clinical features of chronic hepatitis B is broad, ranging from asymptomatic infection to debilitating disease or even end-stage, fatal hepatic failure. As noted above, the onset of the disease tends to be insidious in most patients, with the exception of the very few in whom chronic disease follows failure of resolution of clinically apparent acute hepatitis B. The clinical and laboratory features associated with progression from acute to chronic hepatitis B are discussed in Chap. 360.

1	Fatigue is a common symptom, and persistent or intermittent jaundice is a common feature in severe or advanced cases. Intermittent deepening of jaundice and recurrence of malaise and anorexia, as well as worsening fatigue, are reminiscent of acute hepatitis; such exacerbations may occur spontaneously, often coinciding with evidence of virologic reactivation; may lead to progressive liver injury; and, when superimposed on well-established cirrhosis, may cause hepatic decompensation. Complications of cirrhosis occur in end-stage chronic hepatitis and include ascites, edema, bleeding gastroesophageal varices, hepatic encephalopathy, coagulopathy, or hypersplenism. Occasionally, these complications bring the patient to initial clinical attention. Extrahepatic complications of chronic hepatitis B, similar to those seen during the prodromal phase of acute hepatitis B, are associated with deposition of circulating hepatitis B antigen–antibody immune complexes. These include arthralgias and

1	B, similar to those seen during the prodromal phase of acute hepatitis B, are associated with deposition of circulating hepatitis B antigen–antibody immune complexes. These include arthralgias and arthritis, which are common, and the more rare purpuric cutaneous lesions (leukocytoclastic vasculitis), immune-complex glomerulonephritis, and generalized vasculitis (polyarteritis nodosa) (Chaps. 360 and 385).

1	Laboratory features of chronic hepatitis B do not distinguish adequately between histologically mild and severe hepatitis. Aminotransferase elevations tend to be modest for chronic hepatitis B but may fluctuate in the range of 100−1000 units. As is true for acute viral hepatitis B, alanine aminotransferase (ALT) tends to be more elevated than aspartate aminotransferase (AST); however, once cirrhosis is established, AST tends to exceed ALT. Levels of alkaline phosphatase activity tend to be normal or only marginally elevated. In severe cases, moderate elevations in serum bilirubin (51.3−171 μmol/L [3−10 mg/ dL]) occur. Hypoalbuminemia and prolongation of the prothrombin time occur in severe or end-stage cases. Hyperglobulinemia and detectable circulating autoantibodies are distinctly absent in chronic hepatitis B (in contrast to autoimmune hepatitis). Viral markers of chronic HBV infection are discussed in Chap. 360.

1	Although progression to cirrhosis is more likely in severe than in mild or moderate chronic hepatitis B, all forms of chronic hepatitis B can be progressive, and progression occurs primarily in patients with active HBV replication. Moreover, in populations of patients with chronic hepatitis B who are at risk for HCC (Chap. 111), the risk is highest for those with continued, high-level HBV replication and lower for persons in whom initially high-level HBV DNA falls spontaneously over time. Therefore, management of chronic hepatitis B is directed at suppressing the level of virus replication. Although clinical trials tend to focus on clinical endpoints achieved over 1−2 years (e.g., suppression of HBV DNA to undetectable levels, loss of HBeAg/HBsAg, improvement in histology, normalization of ALT), these short-term gains translate into reductions in the risk of clinical progression, hepatic decompensation, and death. To date, seven drugs have been approved for treatment of chronic

1	of ALT), these short-term gains translate into reductions in the risk of clinical progression, hepatic decompensation, and death. To date, seven drugs have been approved for treatment of chronic hepatitis B: injectable interferon (IFN) α; pegylated interferon (long-acting IFN 2033 bound to polyethylene glycol, PEG [PEG IFN]); and the oral agents lamivudine, adefovir dipivoxil, entecavir, telbivudine, and tenofovir.

1	Antiviral therapy for hepatitis B has evolved rapidly since the mid1990s, as has the sensitivity of tests for HBV DNA. When IFN and lamivudine were evaluated in clinical trials, HBV DNA was measured by insensitive hybridization assays with detection thresholds of 105−106 virions/mL; when adefovir, entecavir, telbivudine, tenofovir, and PEG IFN were studied in clinical trials, HBV DNA was measured by sensitive amplification assays (polymerase chain reaction [PCR]) with detec tion thresholds of 101−103 viral copies/mL or IU/mL. Recognition of these distinctions is helpful when comparing results of clinical trials that established the efficacy of these therapies (reviewed below in chronological order of publication of these efficacy trials).

1	IFN-α was the first approved therapy for chronic hepatitis B. Although it is no longer used to treat hepatitis B, standard IFN is important historically, having provided important lessons about antiviral therapy in general. For immunocompetent adults with HBeAg-reactive chronic hepatitis B (who tend to have high-level HBV DNA [>105−106 virions/mL] and histologic evidence of chronic hepatitis on liver biopsy), a 16-week course of IFN given subcutaneously at a daily dose of 5 million units, or three times a week at a dose of 10 million units, results in a loss of HBeAg and hybridization-detectable HBV DNA (i.e., a reduction to levels below 105−106 virions/mL) in ~30% of patients, with a concomitant improvement in liver histology. Seroconversion from HBeAg to anti-HBe occurred in approximately 20%, and, in early trials, approximately 8% lost HBsAg. Successful IFN therapy and seroconversion are often accompanied by an acute hepatitis-like elevation in aminotransferase activity, which has

1	20%, and, in early trials, approximately 8% lost HBsAg. Successful IFN therapy and seroconversion are often accompanied by an acute hepatitis-like elevation in aminotransferase activity, which has been postulated to result from enhanced cytolytic T cell clearance of HBV-infected hepatocytes. Relapse after successful therapy is rare (1 or 2%). The likelihood of responding to IFN is higher in patients with lower levels of HBV DNA and substantial elevations of ALT. Although children can respond as well as adults, IFN therapy has not been effective in very young children infected at birth. Similarly, IFN therapy has not been effective in immunosuppressed persons, Asian patients with neonatal acquisition of infection and minimal-to-mild ALT elevations, or patients with decompensated chronic hepatitis B (in whom such therapy can actually be detrimental, sometimes precipitating decompensation, often associated with severe adverse effects). Among patients with HBeAg loss during therapy,

1	hepatitis B (in whom such therapy can actually be detrimental, sometimes precipitating decompensation, often associated with severe adverse effects). Among patients with HBeAg loss during therapy, long-term follow-up has demonstrated that 80% experience eventual loss of HBsAg (i.e., all serologic markers of infection, and normalization of ALT over a 9-year posttreatment period). In addition, improved long-term and complication-free survival as well as a reduction in the frequency of HCC have been documented among IFN responders, supporting the conclusion that successful antiviral therapy improves the natural history of chronic hepatitis B.

1	Initial trials of brief-duration IFN therapy in patients with HBeAgnegative chronic hepatitis B were disappointing, suppressing HBV replication transiently during therapy but almost never resulting in sustained antiviral responses. In subsequent IFN trials among patients with HBeAg-negative chronic hepatitis B, however, more protracted courses, lasting up to 1.5 years, have been reported to result in sustained remissions documented to last for several years, with suppressed HBV DNA and aminotransferase activity, in ~20%. Complications of IFN therapy include systemic “flu-like” symptoms; marrow suppression; emotional lability (irritability, depression, anxiety); autoimmune reactions (especially autoimmune thyroiditis); and miscellaneous side effects such as alopecia, rashes, diarrhea, and numbness and tingling of the extremities. With the possible exception of autoimmune thyroiditis, all these side effects are reversible upon dose lowering or cessation of therapy.

1	Although no longer competitive with the newer generation of antivirals, IFN did represent the first successful antiviral approach and set a standard against which to measure subsequent drugs in the achievement of durable virologic, serologic, biochemical, and 2034 histologic responses; consolidation of virologic and biochemical benefit in the ensuing years after therapy; and improvement in the natural history of chronic hepatitis B. Standard IFN has been supplanted by long-acting PEG IFN (see below), and IFN nonresponders are now treated with one of the newer oral nucleoside analogues.

1	The first of the nucleoside analogues to be approved, the dideoxynucleoside lamivudine inhibits reverse transcriptase activity of both HIV and HBV and is a potent and effective agent for patients with chronic hepatitis B. Although generally superseded by newer, more potent agents, lamivudine is still used in regions of the world where newer agents are not yet approved are or not affordable. In clinical trials among patients with HBeAg-reactive chronic hepatitis B, lamivudine therapy at daily doses of 100 mg for 48−52 weeks suppressed HBV DNA by a median of approximately 5.5 log10 copies/ mL and to undetectable levels, as measured by PCR amplification assays, in approximately 40% of patients. Therapy was associated with HBeAg loss in 32−33%; HBeAg seroconversion (i.e., conversion from HBeAg-reactive to anti-HBe-reactive) in 16−21%; normalization of ALT in 40−75%; improvement in histology in 50−60%; retardation in fibrosis in 20−30%; and prevention of progression to cirrhosis. HBeAg

1	to anti-HBe-reactive) in 16−21%; normalization of ALT in 40−75%; improvement in histology in 50−60%; retardation in fibrosis in 20−30%; and prevention of progression to cirrhosis. HBeAg responses can occur even in subgroups who are resistant to IFN (e.g., those with high-level HBV DNA) or who failed in the past to respond to it. As is true for IFN therapy of chronic hepatitis B, patients with near-normal ALT activity tend not to experience HBeAg responses (despite suppression of HBV DNA), and those with ALT levels exceeding 5 × the upper limit of normal can expect 1-year HBeAg seroconversion rates of 50−60%. Generally, HBeAg seroconversions are confined to patients who achieve suppression of HBV DNA to <104 copies/mL (equivalent to ~103 IU/mL). Lamivudine-associated HBeAg responses are accompanied by a posttreatment HBsAg seroconversion rate comparable to that seen after IFN-induced HBeAg responses. Among Western patients who undergo HBeAg responses during a year-long course of

1	are accompanied by a posttreatment HBsAg seroconversion rate comparable to that seen after IFN-induced HBeAg responses. Among Western patients who undergo HBeAg responses during a year-long course of therapy and in whom the response is sustained for 4−6 months after cessation of therapy, the response is durable thereafter in the vast majority (>80%); therefore, the achievement of an HBeAg response represents a viable stopping point in therapy. Reduced durability has been reported in Asian patients; therefore, to support the durability of HBeAg responses, patients should receive a period of consolidation therapy of ≥6 months in Western patients and ≥1 year in Asian patients after HBeAg seroconversion. Close posttreatment monitoring is necessary to identify HBV reactivation promptly and to resume therapy. If HBeAg is unaffected by lamivudine therapy, the current approach is to continue therapy until an HBeAg response occurs, but long-term therapy may be required to suppress HBV

1	and to resume therapy. If HBeAg is unaffected by lamivudine therapy, the current approach is to continue therapy until an HBeAg response occurs, but long-term therapy may be required to suppress HBV replication and, in turn, limit liver injury; HBeAg seroconversions can increase to a level of 50% after 5 years of therapy. Histologic improvement continues to accrue with therapy beyond the first year; after a cumulative course of 3 years of lamivudine therapy, necroinflammatory activity is reduced in the majority of patients, and even cirrhosis has been shown to regress to precirrhotic stages in as many as three-quarters of patients.

1	Losses of HBsAg have been few during the first year of lamivudine therapy, and this observation had been cited as an advantage of IFN-based over lamivudine therapy; however, in head-to-head comparisons between standard IFN and lamivudine monotherapy, HBsAg losses were rare in both groups. Trials in which lamivudine and IFN were administered in combination failed to show a benefit of combination therapy over lamivudine monotherapy for either treatment-naïve patients or prior IFN nonresponders.

1	In patients with HBeAg-negative chronic hepatitis B (i.e., in those with precore and core-promoter HBV mutations), 1 year of lamivudine therapy results in HBV DNA suppression and normalization of ALT in three-quarters of patients and in histologic improvement in approximately two-thirds. Therapy has been shown to suppress HBV DNA by approximately 4.5 log10 copies/mL (baseline HBV DNA levels are lower than in patients with HBeAg-reactive hepatitis B) and to undetectable levels in approximately 70%, as measured by sensitive PCR amplification assays. Lacking HBeAg at the outset, patients with HBeAg-negative chronic hepatitis B cannot achieve an HBeAg response—a stopping point in HBeAg-reactive patients; almost invariably, when therapy is discontinued, reactivation is the rule. Therefore, these patients require long-term therapy; with successive years, the proportion with suppressed HBV DNA and normal ALT increases.

1	Clinical and laboratory side effects of lamivudine are negligible and indistinguishable from those observed in placebo recipients. Still, lamivudine doses should be reduced in patients with reduced creatinine clearance. During lamivudine therapy, transient ALT elevations, resembling those seen during IFN therapy and during spontaneous HBeAg-to-anti-HBe seroconversions, occur in one-fourth of patients. These ALT elevations may result from restored cytolytic T cell activation permitted by suppression of HBV replication. Similar ALT elevations, however, occur at an identical frequency in placebo recipients, but ALT elevations associated with HBeAg seroconversion are confined to lamivudine-treated patients. When therapy is stopped after a year of therapy, twoto threefold ALT elevations occur in 20−30% of lamivudine-treated patients, representing renewed liver-cell injury as HBV replication returns. Although these posttreatment flares are almost always transient and mild, rare severe

1	in 20−30% of lamivudine-treated patients, representing renewed liver-cell injury as HBV replication returns. Although these posttreatment flares are almost always transient and mild, rare severe exacerbations, especially in cirrhotic patients, have been observed, mandating close and careful clinical and virologic monitoring after discontinuation of treatment. Many authorities caution against discontinuing therapy in patients with cirrhosis, in whom posttreatment flares could precipitate decompensation.

1	Long-term monotherapy with lamivudine is associated with methionine-to-valine (M204V) or methionine-to-isoleucine (M204I) mutations, primarily at amino acid 204 in the tyrosine-methionineaspartate-aspartate (YMDD) motif of HBV DNA polymerase, analogous to mutations that occur in HIV-infected patients treated with this drug. During a year of therapy, YMDD mutations occur in 15−30% of patients; the frequency increases with each year of therapy, reaching 70% at year 5. Ultimately, patients with YMDD mutants experience degradation of clinical, biochemical, and histologic responses; therefore, if treatment is begun with lamivudine monotherapy, the emergence of lamivudine resistance, reflected clinically by a breakthrough from suppressed levels of HBV DNA and ALT, is managed by adding another antiviral to which YMDD variants are sensitive (e.g., adefovir, tenofovir; see below).

1	Currently, although lamivudine is very safe and still used widely in other parts of the world, in the United States and Europe, lamivudine has been eclipsed by more potent antivirals that have superior resistance profiles (see below); it is no longer recommended as first-line therapy. Still, as the first successful oral antiviral agent for use in hepatitis B, lamivudine has provided proof of the concept that polymerase inhibitors can achieve virologic, serologic, biochemical, and histologic benefits. In addition, lamivudine has been shown to be effective in the treatment of patients with decompensated hepatitis B (for whom IFN is contraindicated), in some of whom decompensation can be reversed. Moreover, among patients with cirrhosis or advanced fibrosis, lamivudine has been shown to be effective in reducing the risk of progression to hepatic decompensation and, marginally, the risk of HCC. In the half decade following the introduction in the United States of lamivudine therapy for

1	effective in reducing the risk of progression to hepatic decompensation and, marginally, the risk of HCC. In the half decade following the introduction in the United States of lamivudine therapy for hepatitis B, referral of patients with HBV-associated end-stage liver disease for liver transplantation was reduced by ~30%, supporting further the beneficial impact of oral antiviral therapy on the natural history of chronic hepatitis B.

1	Because lamivudine monotherapy can result universally in the rapid emergence of YMDD variants in persons with HIV infection, patients with chronic hepatitis B should be tested for anti-HIV prior to therapy; if HIV infection is identified, lamivudine monotherapy at the HBV daily dose of 100 mg is contraindicated. These patients should be treated for both HIV and HBV with an HIV drug regimen that includes or is supplemented by at least two drugs active against HBV; antiretroviral therapy (ART) often contains two drugs with antiviral activity against HBV (e.g., tenofovir and emtricitabine), but if lamivudine is part of the regimen, the daily dose should be 300 mg (Chap. 226). The safety of lamivudine during pregnancy has not been established; however, the drug is not teratogenic in rodents and has been used safely in pregnant women with HIV infection and with HBV infection. Limited data even suggest that administration of lamivudine during the last months of pregnancy to mothers with

1	and has been used safely in pregnant women with HIV infection and with HBV infection. Limited data even suggest that administration of lamivudine during the last months of pregnancy to mothers with high-level hepatitis B viremia (≥108 IU/mL) can reduce the likelihood of perinatal transmission of hepatitis B.

1	At an oral daily dose of 10 mg, the acyclic nucleotide analogue adefovir dipivoxil, the prodrug of adefovir, reduces HBV DNA by approximately 3.5−4 log10 copies/mL and is equally effective in treatmentnaïve patients and IFN nonresponders. In HBeAg-reactive chronic hepatitis B, a 48-week course of adefovir dipivoxil was shown to achieve histologic improvement (and reduce the progression of fibrosis) and normalization of ALT in just over one-half of patients, HBeAg seroconversion in 12%, HBeAg loss in 23%, and suppression to an undetectable level of HBV DNA in 13−21%, as measured by PCR. Similar to IFN and lamivudine, adefovir dipivoxil is more likely to achieve an HBeAg response in patients with high baseline ALT (e.g., among adefovir-treated patients with ALT level >5 × the upper limit of normal), and HBeAg seroconversions occurred in 25%. The durability of adefovir-induced HBeAg responses is high (91% in one study); therefore, HBeAg response can be relied upon as a stopping point for

1	normal), and HBeAg seroconversions occurred in 25%. The durability of adefovir-induced HBeAg responses is high (91% in one study); therefore, HBeAg response can be relied upon as a stopping point for adefovir therapy, after a period of consolidation therapy, as outlined above. Although data on the impact of additional therapy beyond 1 year are limited, biochemical, serologic, and virologic outcomes improve progressively as therapy is continued.

1	In patients with HBeAg-negative chronic hepatitis B, a 48-week course of 10 mg/d of adefovir dipivoxil resulted in histologic improvement in two-thirds, normalization of ALT in three-fourths, and suppression of HBV DNA to PCR-undetectable levels in one-half to two-thirds. As was true for lamivudine, because HBeAg responses— a potential stopping point—cannot be achieved in this group, reactivation is the rule when adefovir therapy is discontinued, and indefinite, long-term therapy is required. Treatment beyond the first year consolidates the gain of the first year; after 5 years of therapy, improvement in hepatic inflammation and regression of fibrosis were observed in three-fourths of patients, ALT was normal in 70%, and HBV DNA was undetectable in almost 70%. In one study, stopping adefovir after 5 years was followed by sustained suppression of HBV DNA and ALT, but most HBeAg-negative patients are treated indefinitely unless HBsAg loss, albeit very rare, is achieved.

1	Adefovir contains a flexible acyclic linker instead of the L-nucleoside ring of lamivudine, avoiding steric hindrance by mutated amino acids. In addition, the molecular structure of phosphorylated adefovir is very similar to that of its natural substrate; therefore, mutations to adefovir would also affect binding of the natural substrate, dATP. Hypothetically, these are among the reasons that resistance to adefovir dipivoxil is much less likely than resistance to lamivudine; no resistance was encountered in 1 year of clinical trial therapy. In subsequent years, however, adefovir resistance begins to emerge (asparagine to threonine at amino acid 236 [N236T] and alanine to valine or threonine at amino acid 181 [A181V/T], primarily), occurring in 2.5% after 2 years, but in 29% after 5 years of therapy (reported in HBeAg-negative patients). Among patients co-infected with HBV and HIV and who have normal CD4+ T cell counts, adefovir dipivoxil is effective in suppressing HBV dramatically

1	of therapy (reported in HBeAg-negative patients). Among patients co-infected with HBV and HIV and who have normal CD4+ T cell counts, adefovir dipivoxil is effective in suppressing HBV dramatically (by 5 logs10 in one study). Moreover, adefovir dipivoxil is effective in lamivudine-resistant, YMDD-mutant HBV and can be used when such lamivudine-induced variants emerge. When lamivudine resistance occurs, adding adefovir (i.e., maintaining lamivudine to preempt the emergence of adefovir resistance) is superior to switching to adefovir. Almost invariably, patients with adefovir-mutant HBV respond to lamivudine (or newer agents, such as entecavir, see below). When, in the past, adefovir had been evaluated as therapy for HIV infection, doses of 60−120 mg were required to suppress HIV, and, at these doses, the drug was nephro-2035 toxic. Even at 30 mg/d, creatinine elevations of 44 μmol/L (0.5 mg/ dL) occurred in 10% of patients; however, at the HBV-effective dose of 10 mg, such elevations

1	these doses, the drug was nephro-2035 toxic. Even at 30 mg/d, creatinine elevations of 44 μmol/L (0.5 mg/ dL) occurred in 10% of patients; however, at the HBV-effective dose of 10 mg, such elevations of creatinine are rarely encountered. If any nephrotoxicity does occur, it rarely appears before 6−8 months of therapy. Although renal tubular injury is a rare potential side effect, and although creatinine monitoring is recommended during treatment, the therapeutic index of adefovir dipivoxil is high, and the nephrotoxicity observed in clinical trials at higher doses was reversible. For patients with underlying renal disease, frequency of administration of adefovir dipivoxil should be reduced to every 48 h for creatinine clearances of 30−49 mL/min; to every 72 h for creatinine clearances of 10−29 mL/min; and once a week, following dialysis, for patients undergoing hemodialysis. Adefovir dipivoxil is very well tolerated, and ALT elevations during and after withdrawal of therapy are

1	of 10−29 mL/min; and once a week, following dialysis, for patients undergoing hemodialysis. Adefovir dipivoxil is very well tolerated, and ALT elevations during and after withdrawal of therapy are similar to those observed and described above in clinical trials of lamivudine. An advantage of adefovir is its relatively favorable resistance profile; however, it is not as potent as the other approved oral agents, it does not suppress HBV DNA as rapidly or as uniformly as the others, it is the least likely of all agents to result in HBeAg seroconversion, and 20−50% of patients fail to suppress HBV DNA by 2 log10 (“primary nonresponders”). For these reasons, adefovir, which has been supplanted in both treatment-naïve and lamivudine-resistant patients by the more potent, less resistance-prone nucleotide analogue tenofovir (see below), is no longer recommended as first-line therapy.

1	After long-acting PEG IFN was shown to be effective in the treatment of hepatitis C (see below), this more convenient drug was evaluated in the treatment of chronic hepatitis B. Once-a-week PEG IFN is more effective than the more frequently administered, standard IFN, and several large-scale trials of PEG IFN versus oral nucleoside analogues have been conducted among patients with HBeAg-reactive and HBeAg-negative chronic hepatitis B.

1	In HBeAg-reactive chronic hepatitis B, two large-scale studies were done. One study evaluated PEG IFN-α 2b (100 μg weekly for 32 weeks, then 50 μg weekly for another 20 weeks for a total of 52 weeks, with a comparison arm of combination PEG IFN with oral lamivudine) in 307 subjects. The other study involved PEG IFN-α 2a (180 μg weekly for 48 weeks) in 814 primarily Asian patients, three-fourths of whom had ALT ≥2 × the upper limit of normal, with comparison arms of lamivudine monotherapy and combination PEG IFN plus lamivudine. At the end of therapy (48−52 weeks) in the PEG IFN monotherapy arms, HBeAg loss occurred in approximately 30%, HBeAg seroconversion in 22−27%, undetectable HBV DNA (<400 copies/mL by PCR) in 10−25%, normal ALT in 34−39%, and a mean reduction in HBV DNA of 2 log10 copies/mL (PEG IFN-α 2b) to 4.5 log10 copies/mL (PEG IFN-α 2a). Six months after completing PEG IFN monotherapy in these trials, HBeAg losses were present in approximately 35%, HBeAg seroconversion in

1	(PEG IFN-α 2b) to 4.5 log10 copies/mL (PEG IFN-α 2a). Six months after completing PEG IFN monotherapy in these trials, HBeAg losses were present in approximately 35%, HBeAg seroconversion in approximately 30%, undetectable HBV DNA in 7−14%, normal ALT in 32−41%, and a mean reduction in HBV DNA of 2−2.4 log10 copies/mL. Although the combination of PEG IFN and lamivudine was superior at the end of therapy in one or more serologic, virologic, or biochemical outcomes, neither the combination arm (in both studies) nor the lamivudine monotherapy arm (in the PEG IFN-α 2a trial) demonstrated any benefit compared to the PEG IFN monotherapy arms 6 months after therapy. Moreover, HBsAg seroconversion occurred in 3−7% of PEG IFN recipients (with or without lamivudine); some of these seroconversions were identified by the end of therapy, but many were identified during the posttreatment follow-up period. The likelihood of HBeAg loss in PEG IFN−treated HBeAg-reactive patients is associated with HBV

1	identified by the end of therapy, but many were identified during the posttreatment follow-up period. The likelihood of HBeAg loss in PEG IFN−treated HBeAg-reactive patients is associated with HBV genotype A > B > C > D (shown for PEG IFN-α2b but not for α-2a).

1	Based on these results, some authorities concluded that PEG IFN monotherapy should be the first-line therapy of choice in HBeAgreactive chronic hepatitis B; however, this conclusion has been challenged. Although a finite, 1-year course of PEG IFN results in a higher 2036 rate of sustained response (6 months after treatment) than is achieved with oral nucleoside/nucleotide analogue therapy, the comparison is confounded by the fact that oral agents are not discontinued at the end of 1 year. Instead, taken orally and free of side effects, therapy with oral agents is extended indefinitely or until after the occurrence of an HBeAg response. The rate of HBeAg responses after 2 years of oral-agent nucleoside analogue therapy is at least as high as, if not higher than, that achieved with PEG IFN after 1 year; favoring oral agents is the absence of injections, difficult-to-tolerate side effects, and laboratory monitoring as well as lower direct and indirect medical care costs and

1	with PEG IFN after 1 year; favoring oral agents is the absence of injections, difficult-to-tolerate side effects, and laboratory monitoring as well as lower direct and indirect medical care costs and inconvenience. The association of HBsAg responses with PEG IFN therapy occurs in such a small proportion of patients that subjecting everyone to PEG IFN for the marginal gain of HBsAg responses during or immediately after therapy in such a very small minority is questionable. Moreover, HBsAg responses occur in a comparable proportion of patients treated with early-generation nucleoside/nucleotide analogues in the years after therapy, and, with the newer, more potent nucleoside analogues, the frequency of HBsAg loss during the first year of therapy equals that of PEG IFN and is exceeded during year 2 and beyond (see below). Of course, resistance is not an issue during PEG IFN therapy, but the risk of resistance is much lower with new agents (≤1% up to 3−6 years in previously

1	during year 2 and beyond (see below). Of course, resistance is not an issue during PEG IFN therapy, but the risk of resistance is much lower with new agents (≤1% up to 3−6 years in previously treatment-naïve, entecavirtreated and tenofovir-treated patients; see below). Finally, the level of HBV DNA inhibition that can be achieved with the newer agents, and even with lamivudine, exceeds that which can be achieved with PEG IFN, in some cases by several orders of magnitude. In HBeAg-negative chronic hepatitis B, a trial of PEG IFN-α 2a (180 μg weekly for 48 weeks versus comparison arms of lamivudine monotherapy and of combination therapy) in 564 patients showed that PEG IFN monotherapy resulted at the end of therapy in suppression of HBV DNA by a mean of 4.1 log10 copies/mL, undetectable HBV DNA (<400 copies/mL by PCR) in 63%, normal ALT in 38%, and loss of HBsAg in 4%. Although lamivudine monotherapy and combination lamivudine−PEG IFN therapy were both superior to PEG IFN at the end of

1	DNA (<400 copies/mL by PCR) in 63%, normal ALT in 38%, and loss of HBsAg in 4%. Although lamivudine monotherapy and combination lamivudine−PEG IFN therapy were both superior to PEG IFN at the end of therapy, no advantage of lamivudine monotherapy or combination therapy was apparent over PEG IFN monotherapy 6 months after therapy—suppression of HBV DNA by a mean of 2.3 log10 copies/mL, undetectable HBV DNA in 19%, and normal ALT in 59%. In subjects involved in this trial followed for up to 5 years, among the two-thirds followed who had been treated initially with PEG IFN, 17% maintained HBV DNA suppression to <400 copies/mL, but ALT remained normal in only 22%; HBsAg loss increased gradually to 12%. Among the half followed who had been treated initially with lamivudine monotherapy, HBV DNA remained <400 copies/mL in 7% and ALT normal in 16%; by year 5, 3.5% had lost HBsAg. As was the case for standard IFN therapy in HBeAg-negative patients, only a small proportion maintained

1	HBV DNA remained <400 copies/mL in 7% and ALT normal in 16%; by year 5, 3.5% had lost HBsAg. As was the case for standard IFN therapy in HBeAg-negative patients, only a small proportion maintained responsiveness after completion of PEG IFN therapy, raising questions about the relative value of a finite period of PEG IFN, versus a longer course with a potent, low-resistance oral nucleoside analogue in these patients. Moreover, the value of PEG IFN for HBeAg-negative chronic hepatitis B has not been confirmed. In the only other controlled clinical trial of PEG IFN for HBeAg-negative chronic hepatitis B, the hepatitis C regimen of PEG IFN plus ribavirin was compared to PEG IFN monotherapy. In this trial, HBV DNA suppression (<400 copies/mL) occurred in only 7.5% of the two groups combined, and no study subject lost HBsAg. In patients treated with PEG IFN, HBeAg and HBsAg responses have been associated with IL28B genotype CC, the favorable genotype identified in trials of PEG IFN for

1	and no study subject lost HBsAg. In patients treated with PEG IFN, HBeAg and HBsAg responses have been associated with IL28B genotype CC, the favorable genotype identified in trials of PEG IFN for chronic hepatitis C. Also, reductions in quantitative HBsAg levels have been shown to correlate with and to be predictive of responsiveness to PEG IFN in chronic hepatitis B. If HBsAg levels fail to fall within the first 12–24 weeks or to reach <20,000 IU/mL by week 24, PEG IFN therapy is unlikely to be effective and should be discontinued.

1	Entecavir, an oral cyclopentyl guanosine analogue polymerase inhibitor, appears to be the most potent of the HBV antivirals and is just as well tolerated as lamivudine. In a 709-subject clinical trial among HBeAg-reactive patients, oral entecavir, 0.5 mg daily, was compared to lamivudine, 100 mg daily. At 48 weeks, entecavir was superior to lamivudine in suppression of HBV DNA (mean 6.9 versus 5.5 log10 copies/mL), percentage with undetectable HBV DNA (<300 copies/mL by PCR; 67% versus 36%), histologic improvement (≥2-point improvement in necroinflammatory HAI score; 72% versus 62%), and normal ALT (68% versus 60%). The two treatments were indistinguishable in percentage with HBeAg loss (22% versus 20%) and seroconversion (21% versus 18%). Among patients treated with entecavir for 96 weeks, HBV DNA was undetectable cumulatively in 80% (versus 39% for lamivudine), and HBeAg seroconversions had occurred in 31% (versus 26% for lamivudine). After 3–6 years of entecavir, HBeAg

1	for 96 weeks, HBV DNA was undetectable cumulatively in 80% (versus 39% for lamivudine), and HBeAg seroconversions had occurred in 31% (versus 26% for lamivudine). After 3–6 years of entecavir, HBeAg seroconversions have been observed in 39–44% and HBsAg loss in 5–6%. Similarly, in a 638-subject clinical trial among HBeAg-negative patients, at week 48, oral entecavir, 0.5 mg daily, was superior to lamivudine, 100 mg daily, in suppression of HBV DNA (mean 5.0 versus 4.5 log10 copies/mL) and in percentage with undetectable HBV DNA (90% versus 72%), histologic improvement (70% versus 61%), and normal ALT (78% versus 71%). No resistance mutations were encountered in previously treatment-naïve, entecavir-treated patients during 96 weeks of therapy, and in a cohort of subjects treated for up to 6 years, resistance emerged in only 1.2%. Entecavir-induced HBeAg seroconversions are as durable as those achieved with other antivirals. Its high barrier to resistance coupled with its high potency

1	6 years, resistance emerged in only 1.2%. Entecavir-induced HBeAg seroconversions are as durable as those achieved with other antivirals. Its high barrier to resistance coupled with its high potency renders entecavir a first-line drug for patients with chronic hepatitis B.

1	Entecavir is also effective against lamivudine-resistant HBV infection. In a trial of 286 lamivudine-resistant patients, entecavir, at a higher daily dose of 1 mg, was superior to lamivudine, as measured at week 48, in achieving suppression of HBV DNA (mean 5.1 versus 0.48 log10 copies/mL), undetectable HBV DNA (72% versus 19%), normal ALT (61% versus 15%), HBeAg loss (10% versus 3%), and HBeAg seroconversion (8% versus 3%). In this population of lamivudine-experienced patients, however, entecavir resistance emerged in 7% at 48 weeks. Although entecavir resistance requires both a YMDD mutation and a second mutation at one of several other sites (e.g., T184A, S202G/I, or M250V), resistance to entecavir in lamivudine-resistant chronic hepatitis B has been recorded to increase progressively to 43% at 4 years; therefore, entecavir is not as attractive a choice as adefovir or tenofovir for patients with lamivudine-resistant hepatitis B.

1	In clinical trials, entecavir has an excellent safety profile; in addition, on-treatment and posttreatment ALT flares are relatively uncommon and relatively mild in entecavir-treated patients. Doses should be reduced for patients with reduced creatinine clearance. Entecavir does have low-level antiviral activity against HIV and cannot be used as monotherapy to treat HBV infection in HIV/HBV co-infected persons.

1	Telbivudine, a cytosine analogue, is similar in efficacy to entecavir but slightly less potent in suppressing HBV DNA (a slightly less profound median 6.4 log10 reduction in HBeAg-reactive disease and a similar 5.2 log10 reduction in HBeAg-negative disease). In its registration trial, telbivudine at an oral daily dose of 600 mg suppressed HBV DNA to <300 copies/mL in 60% of HBeAg-positive and 88% of HBeAg-negative patients, reduced ALT to normal in 77% of HBeAg-positive and 74% of HBeAg-negative patients, and improved histology in 65% of HBeAg-positive and 67% of HBeAgnegative patients. Although resistance to telbivudine (M204I, not M204V, mutations) was less frequent than resistance to lamivudine at the end of 1 year, resistance mutations after 2 years of treatment occurred in up to 22%. Generally well tolerated, telbivudine has been associated with a low frequency of asymptomatic creatine kinase elevations and with a very low frequency of peripheral neuropathy; frequency of

1	to 22%. Generally well tolerated, telbivudine has been associated with a low frequency of asymptomatic creatine kinase elevations and with a very low frequency of peripheral neuropathy; frequency of administration should be reduced for patients with impaired creatinine clearance. Its excellent potency notwithstanding, the inferior resistance profile of telbivudine has limited its appeal; telbivudine is neither recommended as first-line therapy nor widely used.

1	Tenofovir disoproxil fumarate, an acyclic nucleotide analogue and potent antiretroviral agent used to treat HIV infection, is similar to adefovir but more potent in suppressing HBV DNA and inducing HBeAg responses; it is highly active against both wild-type and lamivudineresistant HBV and active in patients whose response to adefovir is slow and/or limited. At an oral once-daily dose of 300 mg for 48 weeks, tenofovir suppressed HBV DNA by 6.2 log10 (to undetectable levels [<400 copies/mL] in 76%) in HBeAg-positive patients and by 4.6 log10 (to undetectable levels in 93%) in HBeAg-negative patients; reduced ALT to normal in 68% of HBeAg-positive and 76% of HBeAg-negative patients; and improved histology in 74% of HBeAg-positive and 72% of HBeAg-negative patients. In HBeAg-positive patients, HBeAg seroconversions occurred in 21% by the end of year 1, 27% by year 2, 34% by year 3, and 40% by year 5 of tenofovir treatment; HBsAg loss occurred in 3% by the end of year 1 and 6% at year 2,

1	HBeAg seroconversions occurred in 21% by the end of year 1, 27% by year 2, 34% by year 3, and 40% by year 5 of tenofovir treatment; HBsAg loss occurred in 3% by the end of year 1 and 6% at year 2, and 8% by year 5. After 5 years of tenofovir therapy, 87% of patients experienced histologic 2037 improvement, including reduction in fibrosis score (51%) and regression of cirrhosis (71%). The 5-year safety (negligible renal toxicity, in 1%, and mild reduction in bone density, in ~0.5%) and resistance profiles (none recorded through 5 years) of tenofovir are very favorable as well; therefore, tenofovir has supplanted adefovir both as first-line therapy for chronic hepatitis B and as add-on therapy for lamivudineresistant chronic hepatitis B. Frequency of tenofovir administration should be reduced for patients with impaired creatinine clearance.

1	A comparison of the six antiviral therapies in current use appears in Table 362-3; their relative potencies in suppressing HBV DNA are shown in Fig. 362-1. Although the combination of lamivudine and PEG IFN suppresses HBV DNA more profoundly during therapy than does monotherapy ComPARiSon of PEGylATED inTERfERon (PEG ifn), lAmiVuDinE, ADEfoViR, EnTECAViR, TElBiVuDinE, AnD TEnofoViR THERAPy foR CHRoniC HEPATiTiS Ba Route of administration Subcutaneous Oral Oral Oral Oral Oral injection Duration of therapyc 48–52 weeks ≥52 weeks ≥48 weeks ≥48 weeks ≥52 weeks ≥48 weeks HBeAg seroconversion 1 yr Rx 18–20% 16–21% 12% 21% 22% 21% >1 yr Rx NA up to 50% @ 5 yrs 43% @ 3 yrsd 31% @ 2 yrs 30% @ 2 yrs 40% @ 5 yrs (mean copies/mL) HBeAg-reactive 4.5 5.5 median 3.5–5 6.9 6.4 6.2 HBeAg-negative 4.1 4.4–4.7 median 3.5–3.9 5.0 5.2 4.6 HBV DNA PCR negative (<300–400 copies/mL; <1000 copies/mL for adefovir) at end of yr 1

1	HBsAg loss yr 1 3–4% ≤1% 0% 2% <1% 3% >yr 1 12% 5 yr after No data 5% at yr 5 6% at yr 6 No data 8% at yr 5 1 yr of Rx Histologic improvement (≥2 point reduction in HAI) at yr 1 HBeAg-reactive 38% 6 months 49–62% 53–68% 72% 65% 74% after HBeAg-negative 48% 6 months 61–66% 64% 70% 67% 72% after Viral resistance None 15–30% @ 1 yr None @ 1 yr ≤1% @ 1 yre Up to 5% @ yr 1 0% @ yr 1 70% @ 5 yrs 29% @ 5 yrs 1.2% @ 6 yrse Up to 22% @ yr 2 0% through yr 5 Pregnancy category C Cf C CBB Cost (US$) for 1 yr ~$18,000 ~$2,500 ~$6,500 ~$8,700g ~$6,000 ~$6,000 aGenerally, these comparisons are based on data on each drug tested individually versus placebo in registration clinical trials; because, with rare exception, these comparisons are not based on head-to-head testing of these drugs, relative advantages and disadvantages should be interpreted cautiously. bAlthough standard interferon α administered daily or three times a week is approved as therapy for chronic hepatitis B, it has been supplanted

1	and disadvantages should be interpreted cautiously. bAlthough standard interferon α administered daily or three times a week is approved as therapy for chronic hepatitis B, it has been supplanted by PEG IFN, which is administered once a week and is more effective. Standard interferon has no advantages over PEG IFN. cDuration of therapy in clinical efficacy trials; use in clinical practice may vary. dBecause of a computer-generated randomization error that resulted in misallocation of drug versus placebo during the second year of clinical trial treatment, the frequency of HBeAg seroconversion beyond the first year is an estimate (Kaplan-Meier analysis) based on the small subset in whom adefovir was administered correctly. e7% during a year of therapy (43% at year 4) in lamivudine-resistant patients. fDespite its Class C designation, lamivudine has an extensive pregnancy safety record in women with HIV/AIDS. gApproximately $17,400 for lamivudine-refractory patients.

1	Abbreviations: ALT, alanine aminotransferase; HAI, histologic activity index; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; NA, not applicable; PEG IFN, pegylated interferon; PCR, polymerase chain reaction; Rx, therapy; yr, year. FIGURE 362-1 Relative potency of antiviral drugs for hepatitis B, as reflected by median log10 HBV DNA reduction in HBeAgpositive chronic hepatitis B. These data are from individual reports of large, randomized controlled registration trials that were the basis for approval of the drugs. In most instances, these data do not represent direct comparisons among the drugs, because study populations were different, baseline patient variables were not always uniform, and the sensitivity and dynamic range of the HBV DNA assays used in the trials varied. ADV, adefovir dipivoxil; ETV, entecavir; LAM, lamivudine; PEG IFN, pegylated interferon α2a; TBV, telbivudine; TDF, tenofovir disoproxil fumarate.

1	with either drug alone (and is much less likely to be associated with lamivudine resistance), this combination used for a year is no better than a year of PEG IFN in achieving sustained responses. To date, combinations of oral nucleoside/nucleotide agents have not achieved an enhancement in virologic, serologic, or biochemical efficacy over that achieved by the more potent of the combined drugs given individually. In a 2-year trial of combination entecavir and tenofovir versus entecavir monotherapy, for a small subgroup of patients with very high HBV DNA levels (≥108 IU/mL), a reduction in HBV DNA to <50 IU/mL was higher in the combination group (79% versus 62%); however, no differences in HBeAg responses or any other endpoint were observed between the combination-therapy and monotherapy groups, even in the high-HBV DNA subgroup. On the other hand, combining agents that are not cross-resistant (e.g., lamivudine and adefovir or tenofovir) has the potential to reduce the risk or perhaps

1	even in the high-HBV DNA subgroup. On the other hand, combining agents that are not cross-resistant (e.g., lamivudine and adefovir or tenofovir) has the potential to reduce the risk or perhaps even to preempt entirely the emergence of drug resistance. In the future, the treatment paradigm may shift from the current approach of sequential monotherapy to preemptive combination therapy, perhaps not for all patients but for subsets (e.g., patients with very high levels of HBV DNA, immunosuppressed patients); however, designing and executing clinical trials that demonstrate superior efficacy and resistance profile of combination therapy over monotherapy with entecavir or tenofovir will remain challenging.

1	In addition to the seven approved antiviral drugs for hepatitis B, emtricitabine, a fluorinated cytosine analogue very similar to lamivudine in structure, efficacy, and resistance profile, offers no advantage over lamivudine. A combination of emtricitabine and tenofovir is approved for the treatment of HIV infection and is an appealing combination therapy for hepatitis B, especially for lamivudine-resistant disease; however, neither emtricitabine nor the combination is approved yet for hepatitis B. Several initially promising antiviral agents have been abandoned because of toxicity (e.g., clevudine, which was linked to myopathy during its clinical development). Because direct-acting antivirals have been so successful in the management of chronic hepatitis B, more unconventional approaches— e.g., immunologic (e.g., toll receptor agonists) or genetic manipulation (e.g., RNA interference—gene silencing—to reduce HBV DNA transcription)—are not likely to be competitive, unless they can be

1	e.g., immunologic (e.g., toll receptor agonists) or genetic manipulation (e.g., RNA interference—gene silencing—to reduce HBV DNA transcription)—are not likely to be competitive, unless they can be shown to go beyond current antivirals in achieving recovery (HBsAg seroconversion) from HBV infection. Finally, initial emphasis in the development of antiviral therapy for hepatitis B was placed on monotherapy; whether combination regimens will yield additive or synergistic efficacy remains to be determined.

1	Several learned societies and groups of expert physicians have issued treatment recommendations for patients with chronic hepatitis B; the most authoritative and updated (and free of financial support by pharmaceutical companies) are those of the American Association for the Study of Liver Diseases (AASLD) and of the European Association for the Study of the Liver (EASL). Although the recommendations differ slightly, a consensus has emerged on most of the important points (Table 362-4). No treatment is recommended or available for inactive “nonreplicative” hepatitis B carriers (undetectable HBeAg with normal ALT and HBV DNA ≤103 IU/mL documented serially over time). In patients with detectable HBeAg and HBV DNA levels >2 × 104 IU/mL, treatment is recommended by the AASLD for those with ALT levels above 2 × the upper limit of normal. (The EASL recommends treatment in HBeAg-positive patients for HBV DNA levels >2 × 103 IU/mL and ALT above the upper limit of normal.) For HBeAg-positive

1	levels above 2 × the upper limit of normal. (The EASL recommends treatment in HBeAg-positive patients for HBV DNA levels >2 × 103 IU/mL and ALT above the upper limit of normal.) For HBeAg-positive patients with ALT ≤2 × the upper limit of normal, in whom sustained responses are not likely and who would require multiyear therapy, antiviral therapy is not recommended currently. This pattern is common during the early decades of life among Asian patients infected at birth; even in this group, therapy would be considered for those >40 years of age, ALT persistently at the high end of the twofold range, and/ or with a family history of HCC, especially if the liver biopsy shows moderate to severe necroinflammatory activity or fibrosis. In this group, when, eventually, ALT becomes elevated later in life, antiviral therapy should be instituted. For patients with HBeAg-negative chronic hepatitis B, ALT >2 × the upper limit of normal (above the upper limit of normal according to EASL), and HBV

1	in life, antiviral therapy should be instituted. For patients with HBeAg-negative chronic hepatitis B, ALT >2 × the upper limit of normal (above the upper limit of normal according to EASL), and HBV DNA >2 × 103 IU/ mL, antiviral therapy is recommended. If HBV DNA is >2 × 103 IU/mL and ALT is 1 to >2 × the upper limit of normal, liver biopsy should be considered to help in arriving at a decision to treat if substantial liver injury is present (treatment in this subset would be recommended according to EASL guidelines, because ALT is elevated).

1	For patients with compensated cirrhosis, because antiviral therapy has been shown to retard clinical progression, treatment is recommended regardless of HBeAg status and ALT as long as HBV DNA is detectable at >2 × 103 IU/mL (detectable at any level according to the EASL); monitoring without therapy is recommended for those with HBV DNA <2 × 103 IU/mL, unless ALT is elevated. For patients with decompensated cirrhosis, treatment is recommended regardless of serologic and biochemical status, as long as HBV DNA is detectable. Patients with decompensated cirrhosis should be evaluated as candidates for liver transplantation.

1	Among the seven available drugs for hepatitis B, PEG IFN has supplanted standard IFN, entecavir has supplanted lamivudine, and tenofovir has supplanted adefovir. PEG IFN, entecavir, or tenofovir is recommended as first-line therapy (Table 362-3). PEG IFN requires finite-duration therapy, achieves the highest rate of HBeAg responses after a year of therapy, and does not support viral mutations, but it requires subcutaneous injections and is associated with inconvenience, more intensive clinical and laboratory monitoring, and intolerability. Oral nucleoside analogues require long-term therapy in most patients, and when used alone, lamivudine and telbivudine foster the emergence of viral mutations, adefovir somewhat less so, and entecavir (except in lamivudine-experienced patients) and tenofovir rarely at all. Oral agents do not require injections or cumbersome laboratory monitoring, are very well tolerated, lead to improved histology in 50−90% of patients, suppress HBV DNA more

1	tenofovir rarely at all. Oral agents do not require injections or cumbersome laboratory monitoring, are very well tolerated, lead to improved histology in 50−90% of patients, suppress HBV DNA more profoundly than PEG IFN, and are effective even in patients who fail to respond to IFN-based therapy. Although oral agents are less likely to result in HBeAg responses during the first year of therapy, as compared to PEG IFN, treatment with oral agents tends to be extended beyond the first year and, by the end of the second year,

1	Chronic hepatitis >103 1 to >2 × ULNd Consider liver biopsy; treath if biopsy shows moderate to severe inflammation or fibrosis Chronic hepatitis >104 >2 × ULNd Treath,i Cirrhosis compensated >2 × 103 < or > ULN Treate with oral agents, not PEG IFN <2 × 103 < or > ULN Treath with oral agentsg, not PEG IFN; refer for liver transplantation Undetectable aBased on practice guidelines of the American Association for the Study of Liver Diseases (AASLD). Except as indicated in footnotes, these guidelines are similar to those issued by the European Association for the Study of the Liver (EASL). bLiver disease tends to be mild or inactive clinically; most such patients do not undergo liver biopsy. cThis pattern is common during early decades of life in Asian patients infected at birth. dAccording to the EASL guidelines, treat if HBV DNA is >2 × 103 IU/mL and ALT >ULN. eOne of the potent oral drugs with a high barrier to resistance (entecavir or tenofovir) or PEG IFN can be used as first-line

1	to the EASL guidelines, treat if HBV DNA is >2 × 103 IU/mL and ALT >ULN. eOne of the potent oral drugs with a high barrier to resistance (entecavir or tenofovir) or PEG IFN can be used as first-line therapy (see text). These oral agents, but not PEG IFN, should be used for interferon-refractory/ intolerant and immunocompromised patients. PEG IFN is administered weekly by subcutaneous injection for a year; the oral agents are administered daily for at least a year and continued indefinitely or until at least 6 months after HBeAg seroconversion. fAccording to EASL guidelines, patients with compensated cirrhosis and detectable HBV DNA at any level, even with normal ALT, are candidates for therapy. Most authorities would treat indefinitely, even in HBeAg-positive disease after HBeAg seroconversion. gBecause the emergence of resistance can lead to loss of antiviral benefit and further deterioration in decompensated cirrhosis, a low-resistance regimen is recommended—entecavir or tenofovir

1	gBecause the emergence of resistance can lead to loss of antiviral benefit and further deterioration in decompensated cirrhosis, a low-resistance regimen is recommended—entecavir or tenofovir monotherapy or combination therapy with the more resistance-prone lamivudine (or telbivudine) plus adefovir. Therapy should be instituted urgently. hBecause HBeAg seroconversion is not an option, the goal of therapy is to suppress HBV DNA and maintain a normal ALT. PEG IFN is administered by subcutaneous injection weekly for a year; caution is warranted in relying on a 6-month posttreatment interval to define a sustained response, because the majority of such responses are lost thereafter. Oral agents, entecavir or tenofovir, are administered daily, usually indefinitely or until, as very rarely occurs, virologic and biochemical responses are accompanied by HBsAg seroconversion. iFor older patients and those with advanced fibrosis, consider lowering the HBV DNA threshold to >2 × 103 IU/mL.

1	Abbreviations: AASLD, American Association for the Study of Liver Diseases; ALT, alanine aminotransferase; EASL, European Association for the Study of the Liver; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; PEG IFN, pegylated interferon; ULN, upper limit of normal.

1	yields HBeAg responses (and even HBsAg responses) comparable in frequency to those achieved after 1 year of PEG IFN (and without the associated side effects) (Table 362-5). Although adefovir and tenofovir are safe, creatinine monitoring is recommended. Substantial experience with lamivudine during pregnancy (see above) has identified no teratogenicity. Although interferons do not appear to cause congenital anomalies, interferons have antiproliferative properties and should be avoided during pregnancy. Adefovir during pregnancy has not been associated with birth defects; however, there may be an increased risk of spontaneous abortion. Data on the safety of entecavir during pregnancy have not been published. Sufficient data in animals and limited data in humans suggest that telbivudine and tenofovir can be used safely during pregnancy. In general, except perhaps for lamivudine, and until additional data become available, the other antivirals for hepatitis B should be avoided or used

1	tenofovir can be used safely during pregnancy. In general, except perhaps for lamivudine, and until additional data become available, the other antivirals for hepatitis B should be avoided or used with extreme caution during pregnancy.

1	As noted above, some physicians prefer to begin with PEG IFN, while other physicians and patients prefer oral agents as first-line therapy. For patients with decompensated cirrhosis, the emergence of resistance can result in further deterioration and loss of antiviral effectiveness. Therefore, in this patient subset, the threshold for relying on therapy with a very favorable resistance profile (e.g., entecavir or tenofovir) or on combination therapy is low. PEG IFN should not be used in patients with compensated or decompensated cirrhosis.

1	For patients with end-stage chronic hepatitis B who undergo liver transplantation, reinfection of the new liver is almost universal in the absence of antiviral therapy. The majority of patients become high-level viremic carriers with minimal liver injury. Before the availability of antiviral therapy, an unpredictable proportion experienced severe hepatitis B−related liver injury, sometimes a fulminant-like hepatitis and sometimes a rapid recapitulation of the original severe chronic hepatitis B (Chap. 360). Currently, however, prevention of recurrent hepatitis B after liver transplantation has been achieved definitively by combining hepatitis B immune globulin with one of the oral nucleoside or nucleotide analogues (Chap. 368); preliminary data suggest that the newer, more potent, and less resistance-prone oral agents may be used instead of hepatitis B immune globulin for posttransplantation therapy.

1	Patients with HBV-HIV co-infection can have progressive HBV-associated liver disease and, occasionally, a severe exacerbation of hepatitis B resulting from immunologic reconstitution following ART. Lamivudine should never be used as monotherapy in patients with HBV-HIV infection because HIV resistance emerges rapidly to both viruses. Adefovir has been used successfully to treat chronic hepatitis B in HBV-HIV co-infected patients but is no longer considered a first-line agent for HBV. Entecavir has low-level activity against HIV and can result in selection of HIV resistance; therefore, it should be avoided in HBV-HIV co-infection. Tenofovir and the combination of tenofovir and emtricitabine in one pill are approved therapies for HIV and represent excellent choices for treating HBV infection in HBV-HIV co-infected patients. Generally, even for HBVHIV co-infected patients who do not yet meet treatment criteria for HIV infection, treating for both HBV and HIV is recommended.

1	Patients with chronic hepatitis B who undergo cytotoxic chemotherapy for treatment of malignancies as well as patients treated with immunosuppressive, anticytokine, or antitumor necrosis factor therapies experience enhanced HBV replication and viral expression on hepatocyte membranes during chemotherapy coupled with suppression of cellular immunity. When chemotherapy is withdrawn, such patients are at risk for reactivation of hepatitis B, often severe and occasionally fatal. Such rebound reactivation represents restoration of cytolytic T cell function against a target organ enriched in HBV expression. Preemptive treatment with lamivudine prior to the initiation of chemotherapy has been shown to reduce the risk of such reactivation. The newer, more potent oral antiviral agents are with chronic hepatitis C (see below). The clinical and laboratory features of chronic HDV infection are summarized in Chap. 360. Administration Weekly injection Daily, orally

1	Administration Weekly injection Daily, orally Tolerability Poorly toler-Well tolerated, limited moniated, intensive toring monitoring Duration of therapy Finite 48 weeks ≥1 year, indefinite in most patients Maximum mean HBV DNA 4.5 log 6.9 log During 1 year of therapy ~30% ~20% During >1 year of Not applicable 30% (year 2) to up to 50% therapy During 1 year of therapy 3–4% 0–3% During >1 year of therapy Not applicable 3–8% @ 5 years of therapy After 1 year of therapy– 12% @ 5 years 3.5% @ 5 years HBeAg-negative Antiviral resistance None Lamivudine: ~30% year 1, ~70% year 5 Adefovir: 0% year 1, ~30% Telbivudine: up to 4% year 1, 22% year 2 Entecavir: ≤1.2% through year 6 Tenofovir: 0% through year 5 Use in cirrhosis, transplanta-No Yes tion, immunosuppressed Cost, 1 year of therapy ++++ + to ++ Abbreviations: HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; PEG IFN, pegylated interferon.

1	Abbreviations: HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; PEG IFN, pegylated interferon. even more effective in preventing hepatitis B reactivation and with a lower risk of antiviral drug resistance. The optimal duration of antiviral therapy after completion of chemotherapy is not known, but a suggested approach is 6 months for inactive hepatitis B carriers and longer-duration therapy in patients with baseline HBV DNA levels >2 × 103 IU/mL, until standard clinical endpoints are met (Table 362-4).

1	Chronic hepatitis D virus (HDV) may follow acute co-infection with HBV but at a rate no higher than the rate of chronicity of acute hepatitis B. That is, although HDV co-infection can increase the severity of acute hepatitis B, HDV does not increase the likelihood of progression to chronic hepatitis B. When, however, HDV superinfection occurs in a person who is already chronically infected with HBV, long-term HDV infection is the rule, and a worsening of the liver disease is the expected consequence. Except for severity, chronic hepatitis B plus D has similar clinical and laboratory features to those seen in chronic hepatitis B alone. Relatively severe and progressive chronic hepatitis, with or without cirrhosis, is the rule, and mild chronic hepatitis is the exception. Occasionally, however, mild hepatitis or even, rarely, inactive carriage occurs in patients with chronic hepatitis B plus D, and the disease may become indolent after several years of infection. A distinguishing

1	however, mild hepatitis or even, rarely, inactive carriage occurs in patients with chronic hepatitis B plus D, and the disease may become indolent after several years of infection. A distinguishing serologic feature of chronic hepatitis D is the presence in the circulation of antibodies to liver-kidney microsomes (anti-LKM); however, the anti-LKM seen in hepatitis D, anti-LKM3, are directed against uridine diphosphate glucuronosyltransferase and are distinct from anti-LKM1 seen in patients with autoimmune hepatitis and in a subset of patients

1	Management is not well defined. Glucocorticoids are ineffective and are not used. Preliminary experimental trials of IFN-α suggested that conventional doses and durations of therapy lower levels of HDV RNA and aminotransferase activity only transiently during treatment but have no impact on the natural history of the disease. In contrast, high-dose IFN-α (9 million units three times a week) for 12 months may be associated with a sustained loss of HDV replication and clinical improvement in up to 50% of patients. Moreover, the beneficial impact of treatment has been observed to persist for 15 years and to be associated with a reduction in grade of hepatic necrosis and inflammation, reversion of advanced fibrosis (improved stage), and clearance of HDV RNA in some patients. A suggested approach to therapy has been high-dose, long-term IFN for at least a year and, in responders, extension of therapy until HDV RNA and HBsAg clearance. PEG IFN has also been shown to be effective in the

1	to therapy has been high-dose, long-term IFN for at least a year and, in responders, extension of therapy until HDV RNA and HBsAg clearance. PEG IFN has also been shown to be effective in the treatment of chronic hepatitis D (e.g., after 48 weeks of therapy, associated with undetectable HDV RNA, durable for at least 24 posttreatment weeks, in a quarter of patients) and is a more convenient replacement for standard IFN. None of the nucleoside analogue antiviral agents for hepatitis B are effective in hepatitis D. In patients with end-stage liver disease secondary to chronic hepatitis D, liver transplantation has been effective. If hepatitis D recurs in the new liver without the expression of hepatitis B (an unusual serologic profile in immunocompetent persons but common in transplant patients), liver injury is limited. In fact, the outcome of transplantation for chronic hepatitis D is superior to that for chronic hepatitis B; in such patients, combination hepatitis B immune globulin

1	liver injury is limited. In fact, the outcome of transplantation for chronic hepatitis D is superior to that for chronic hepatitis B; in such patients, combination hepatitis B immune globulin and nucleoside analogue therapy for hepatitis B is indicated (Chap. 368).

1	Regardless of the epidemiologic mode of acquisition of hepatitis C virus (HCV) infection, chronic hepatitis follows acute hepatitis C in 50−70% of cases; chronic infection is common even in those with a return to normal in aminotransferase levels after acute hepatitis C, adding up to an 85% likelihood of chronic HCV infection after acute hepatitis C. Few clues had emerged to explain host differences associated with chronic infection until recently, when variation in a single nucleotide polymorphism (SNP) on chromosome 19, IL28B (which codes for IFN-λ3), was identified that distinguished between responders and nonresponders to antiviral therapy (see below). The same variants correlated with spontaneous resolution after acute infection: 53% in genotype C/C, 30% in genotype C/T, but only 23% in genotype T/T. The association with HCV clearance after acute infection is even stronger when IL28B haplotype is combined with haplotype G/G of an SNP near HLA class II DBQ1*03:01.

1	In patients with chronic hepatitis C followed for 20 years, progression to cirrhosis occurs in about 20−25%. Such is the case even for patients with relatively clinically mild chronic hepatitis, including those without symptoms, with only modest elevations of aminotransferase activity, and with mild chronic hepatitis on liver biopsy. Even in cohorts of well-compensated patients with chronic hepatitis C referred for clinical research trials (no complications of chronic liver disease and with normal hepatic synthetic function), the prevalence of cirrhosis may be as high as 50%. Most cases of hepatitis C are identified initially in asymptomatic patients who have no history of acute hepatitis C (e.g., those discovered while attempting to donate blood, while undergoing lab testing as part of an application for life insurance, or as a result of routine laboratory tests). The source of HCV infection in many of these cases is not defined, although a long-forgotten percutaneous exposure (e.g.,

1	application for life insurance, or as a result of routine laboratory tests). The source of HCV infection in many of these cases is not defined, although a long-forgotten percutaneous exposure (e.g., injection drug use) in the remote past can be elicited in a substantial proportion and probably accounts for most infections; most of these infections were acquired in the 1960s and 1970s, coming to clinical attention decades later.

1	Approximately one-third of patients with chronic hepatitis C have normal or near-normal aminotransferase activity; although one-third to one-half of these patients have chronic hepatitis on liver biopsy, the grade of liver injury and stage of fibrosis tend to be mild in the vast majority. In some cases, more severe liver injury has been reported— even, rarely, cirrhosis, most likely the result of previous histologic activity. Among patients with persistent normal aminotransferase activity sustained over ≥5−10 years, histologic progression has been shown to be rare; however, approximately one-fourth of patients with normal aminotransferase activity experience subsequent aminotransferase elevations, and histologic injury can be progressive once abnormal biochemical activity resumes. Therefore, continued clinical monitoring is indicated, even for patients with normal aminotransferase activity.

1	Despite this substantial rate of progression of chronic hepatitis C, and despite the fact that liver failure can result from end-stage chronic hepatitis C, the long-term prognosis for chronic hepatitis C in a majority of patients is relatively benign. Mortality over 10−20 years among patients with transfusion-associated chronic hepatitis C has been shown not to differ from mortality in a matched population of transfused patients in whom hepatitis C did not develop. Although death in the hepatitis group is more likely to result from liver failure, and although hepatic decompensation may occur in ~15% of such patients over the course of a decade, the majority (almost 60%) of patients remain asymptomatic and well compensated, with no clinical sequelae of chronic liver disease. Overall, chronic hepatitis C tends to be very slowly and insidiously progressive, if at all, in the vast majority of patients, whereas in approximately one-fourth of cases, chronic hepatitis C will progress

1	chronic hepatitis C tends to be very slowly and insidiously progressive, if at all, in the vast majority of patients, whereas in approximately one-fourth of cases, chronic hepatitis C will progress eventually to end-stage cirrhosis. In fact, because HCV infection is so prevalent, and because a proportion of patients progress inexorably to end-stage liver disease, hepatitis C is the most frequent indication for liver transplantation (Chap. 368). In the United States, hepatitis C accounts for up to 40% of all chronic liver disease, and, as of 2007, mortality caused by hepatitis C surpassed that associated with HIV/AIDS. Moreover, because the prevalence of HCV infection is so much higher in the “baby boomer” cohort borne between 1945 and 1965, three-quarters of the mortality associated with hepatitis C occurs in this age cohort. Referral bias may account for the more severe outcomes described in cohorts of patients reported from tertiary care centers (20-year progression of ≥20%) versus

1	hepatitis C occurs in this age cohort. Referral bias may account for the more severe outcomes described in cohorts of patients reported from tertiary care centers (20-year progression of ≥20%) versus the more benign outcomes in cohorts of patients monitored from initial bloodproduct-associated acute hepatitis or identified in community settings (20-year progression of only 4−7%). Still unexplained, however, are the wide ranges in reported progression to cirrhosis, from 2% over 17 years in a population of women with hepatitis C infection acquired from contaminated anti-D immune globulin to 30% over ≤11 years in recipients of contaminated intravenous immune globulin.

1	Progression of liver disease in patients with chronic hepatitis C has been reported to be more likely in patients with older age, longer duration of infection, advanced histologic stage and grade, more complex quasispecies diversity, increased hepatic iron, concomitant other liver disorders (alcoholic liver disease, chronic hepatitis B, hemochromatosis, α1 antitrypsin deficiency, and steatohepatitis), HIV infection, and obesity. Among these variables, however, duration of infection appears to be one of the most important, and some of the others probably reflect disease duration to some extent (e.g., quasispecies diversity, hepatic iron accumulation). No other epidemiologic or clinical features of chronic hepatitis C (e.g., severity of acute hepatitis, level of aminotransferase activity, level of HCV RNA, presence or absence of jaundice during acute hepatitis) are predictive of eventual outcome. Despite the relatively benign nature of chronic hepatitis C over time in many patients,

1	level of HCV RNA, presence or absence of jaundice during acute hepatitis) are predictive of eventual outcome. Despite the relatively benign nature of chronic hepatitis C over time in many patients, cirrhosis following chronic hepatitis C has been associated with the late development, after several decades, of HCC (Chap. 111); the annual rate of HCC in cirrhotic patients with hepatitis C is 1−4%, occurring primarily in patients who have had HCV infection for 30 years or more.

1	Perhaps the best prognostic indicator in chronic hepatitis C is liver histology; the rate of hepatic fibrosis may be slow, moderate, or rapid. Patients with mild necrosis and inflammation as well as those with limited fibrosis have an excellent prognosis and limited progression to cirrhosis. In contrast, among patients with moderate to severe necroinflammatory activity or fibrosis, including septal or bridging 2041 fibrosis, progression to cirrhosis is highly likely over the course of 10−20 years. The pace of fibrosis progression may be accelerated by such factors as concomitant HIV infection, other causes of liver disease, excessive alcohol use, and hepatic steatosis. Among patients with compensated cirrhosis associated with hepatitis C, the 10-year survival rate is close to 80%; mortality occurs at a rate of 2−6% per year; decompensation at a rate of 4−5% per year; and, as noted above, HCC at a rate of 1−4% per year. A discussion of the pathogenesis of liver injury in patients with

1	occurs at a rate of 2−6% per year; decompensation at a rate of 4−5% per year; and, as noted above, HCC at a rate of 1−4% per year. A discussion of the pathogenesis of liver injury in patients with chronic hepatitis C appears in Chap. 360. Clinical features of chronic hepatitis C are similar to those described above for chronic hepatitis B. Generally, fatigue is the most common symptom; jaundice is rare. Immune complex−mediated extrahepatic complications of chronic hepatitis C are less common than in chronic hepatitis B (despite the fact that assays for immune complexes are often positive in patients with chronic hepatitis C), with the exception of essential mixed cryoglobulinemia (Chap. 360), which is linked to cutaneous vasculitis and membranoproliferative glomerulonephritis as well as lymphoproliferative disorders such as B-cell lymphoma and unexplained monoclonal gammopathy. In addition, chronic hepatitis C has been associated with extrahepatic complications unrelated to

1	well as lymphoproliferative disorders such as B-cell lymphoma and unexplained monoclonal gammopathy. In addition, chronic hepatitis C has been associated with extrahepatic complications unrelated to immune-complex injury. These include Sjögren’s syndrome, lichen planus, porphyria cutanea tarda, type 2 diabetes mellitus, and the metabolic syndrome (including insulin resistance and steatohepatitis).

1	Laboratory features of chronic hepatitis C are similar to those in patients with chronic hepatitis B, but aminotransferase levels tend to fluctuate more (the characteristic episodic pattern of aminotransferase activity) and to be lower, especially in patients with long-standing disease. An interesting and occasionally confusing finding in patients with chronic hepatitis C is the presence of autoantibodies. Rarely, patients with autoimmune hepatitis (see below) and hyperglobulinemia have false-positive immunoassays for anti-HCV. On the other hand, some patients with serologically confirmable chronic hepatitis C have circulating anti-LKM. These antibodies are anti-LKM1, as seen in patients with autoimmune hepatitis type 2 (see below), and are directed against a 33-amino-acid sequence of cytochrome P450 IID6. The occurrence of anti-LKM1 in some patients with chronic hepatitis C may result from the partial sequence homology between the epitope recognized by anti-LKM1 and two segments of

1	P450 IID6. The occurrence of anti-LKM1 in some patients with chronic hepatitis C may result from the partial sequence homology between the epitope recognized by anti-LKM1 and two segments of the HCV polyprotein. In addition, the presence of this autoantibody in some patients with chronic hepatitis C suggests that autoimmunity may be playing a role in the pathogenesis of chronic hepatitis C.

1	Histopathologic features of chronic hepatitis C, especially those that distinguish hepatitis C from hepatitis B, are described in Chap. 360.

1	Therapy for chronic hepatitis C has evolved substantially in the two decades since IFN-α was introduced for this indication. The therapeutic armamentarium has grown to include PEG IFN with ribavirin and, in 2011, the introduction of protease inhibitors telaprevir and boceprevir used in combination with PEG IFN and ribavirin in patients with HCV genotype 1. When first approved, IFN-α was administered via subcutaneous injection three times a week for 6 months but achieved a sustained virologic response (SVR) (Fig. 362-2) (a reduction of HCV RNA to undetectable levels by PCR when measured ≥6 months after completion of therapy) below 10%. Doubling the duration of therapy—but not increasing the dose or changing IFN preparations—increased the SVR rate to ~20%, and addition to the regimen of daily ribavirin, an oral guanosine nucleoside, increased the SVR rate to 40%. When used alone, ribavirin is ineffective and does not reduce HCV RNA levels appreciably, but ribavirin enhances the efficacy

1	ribavirin, an oral guanosine nucleoside, increased the SVR rate to 40%. When used alone, ribavirin is ineffective and does not reduce HCV RNA levels appreciably, but ribavirin enhances the efficacy of IFN by reducing the likelihood of virologic relapse after the achievement of an end-treatment response (Fig. 362-2) (response measured during, and maintained to the end of, treatment). Proposed mechanisms to explain the role of ribavirin include subtle direct reduction of HCV replication, inhibition of host inosine monophosphate dehydrogenase activity (and associated depletion –8–4–2 0 4 8 12162024324048526072 Weeks after start of therapy

1	FIGURE 362-2 Classification of virologic responses based on outcomes during and after a 48-week course of pegylated interferon (PEG IFN) plus ribavirin antiviral therapy in patients with hepatitis C, genotype 1 or 4 (for genotype 2 or 3, the course would be 24 weeks). Nonresponders can be classified as null responders (hepatitis C virus [HCV] RNA reduction of <2 log10 IU/mL) or partial responders (HCV RNA reduction ≥2 log10 IU/mL but not suppressed to undetectable) by week 24 of therapy. In responders, HCV RNA can become undetectable, as shown with sensitive amplification assays, within 4 weeks (RVR, rapid virologic response); can be reduced by ≥2 log10 IU/mL within 12 weeks (early virologic response, EVR; if HCV RNA is undetectable at 12 weeks, the designation is “complete” EVR); or at the end of therapy, 48 weeks (ETR, end-treatment response). In responders, if HCV RNA remains undetectable for 24 weeks after ETR, week 72, the patient has a sustained virologic response (SVR), but if

1	end of therapy, 48 weeks (ETR, end-treatment response). In responders, if HCV RNA remains undetectable for 24 weeks after ETR, week 72, the patient has a sustained virologic response (SVR), but if HCV RNA becomes detectable again, the patient is considered to have relapsed. In patients treated with protease inhibitor–based therapy, several additional milestones are monitored: (1) among boceprevirtreated patients, the level of HCV RNA reduction (>1 log10 or ≥1 log10 IU/mL) during the 4-week PEG IFN–ribavirin lead-in phase; (2) during boceprevir therapy, undetectable HCV RNA at week 8 (week 4 of triple-drug therapy; RVR); and (3) among telaprevir-treated patients, undetectable HCV RNA at week 4 and 12 (extended RVR). (Reproduced with permission, courtesy of Marc G. Ghany, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health and the American Association for the Study of Liver Diseases. Hepatology 49:1335, 2009.) of guanosine pools), immune

1	of Diabetes and Digestive and Kidney Diseases, National Institutes of Health and the American Association for the Study of Liver Diseases. Hepatology 49:1335, 2009.) of guanosine pools), immune modulation, induction of virologic mutational catastrophe, and enhancement of IFN-stimulated gene expression. IFN therapy results in activation of the JAK-STAT signal transduction pathway, which culminates in the intracellular elaboration of genes and their protein products that have antiviral properties. Hepatitis C proteins inhibit JAK-STAT signaling at several steps along the pathway, and exogenous IFN restores expression of IFN-stimulated genes and their antiviral effects.

1	Treatment with the combination of PEG IFN and ribavirin increased responsiveness (frequency of SVR) to as high as 55% overall, to >40% in genotypes 1 and 4, and to >80% in genotypes 2 and 3. Still, many important lessons about antiviral therapy for chronic hepatitis C were learned from the experience with IFN monotherapy and combination IFN-ribavirin therapy. Even in the absence of biochemical and virologic responses, histologic improvement occurs in approximately three-fourths of all treated patients. In chronic hepatitis C, unlike the case in hepatitis B, responses to therapy are not accompanied by transient, acute hepatitis-like aminotransferase elevations. Instead, ALT levels fall precipitously during therapy. Up to 90% of virologic responses are achieved within the first 12 weeks of therapy; responses thereafter are rare. Most relapses occur within the first 12 weeks after treatment; therefore, an SVR at week 12 posttreatment is roughly equivalent to a 24-week SVR. SVRs are very

1	therapy; responses thereafter are rare. Most relapses occur within the first 12 weeks after treatment; therefore, an SVR at week 12 posttreatment is roughly equivalent to a 24-week SVR. SVRs are very durable; normal ALT, improved histology, and absence of HCV RNA in serum and liver have been documented a decade after successful therapy, and “relapses” 2 years after sustained responses are almost unheard of. Thus, an SVR to antiviral therapy of chronic hepatitis C is tantamount to a cure.

1	Patient variables that tend to correlate with sustained virologic responsiveness to IFN-based therapy include favorable genotype (genotypes 2 and 3 as opposed to genotypes 1 and 4); low baseline HCV RNA level (<2 million copies/mL, which is equivalent to <800,000 IU/mL, the current convention of quantitation); histologically mild hepatitis and minimal fibrosis; age <40; female gender; and absence of obesity, insulin resistance, and type 2 diabetes mellitus. Patients with cirrhosis can respond, but they are less likely to do so. For patients treated with combination IFNribavirin, therapy for those with genotype 1 should last a full 48 weeks, whereas in those with genotypes 2 and 3, a 24-week course of therapy suffices (although refined tailoring of treatment duration may be indicated based on rapidity of response or associated cofactors, see below). The response rate in African Americans is disappointingly low for reasons that are not fully understood. Potentially contributing to, but

1	on rapidity of response or associated cofactors, see below). The response rate in African Americans is disappointingly low for reasons that are not fully understood. Potentially contributing to, but not explaining entirely, low responsiveness in African Americans are a higher proportion with genotype 1, slower early viral kinetics during therapy, impaired HCV-specific immunity, and recently recognized host genetic differences in IL28B alleles, described below. The response rate in Latino patients is also low, despite the fact that the frequency of the favorable IL28B C allele is as common in Hispanic patients as in whites. Moreover, the likelihood of a sustained response is best if adherence to the treatment regimen is high (i.e., if patients receive ≥80% of the IFN and ribavirin doses and if they continue treatment for ≥80% of the anticipated duration of therapy). Other variables reported to correlate with increased responsiveness include brief duration of infection, low HCV

1	doses and if they continue treatment for ≥80% of the anticipated duration of therapy). Other variables reported to correlate with increased responsiveness include brief duration of infection, low HCV quasispecies diversity, immunocompetence, absence of hepatic steatosis, and low liver iron levels. High levels of HCV RNA, more histologically advanced liver disease, and high quasispecies diversity all go hand in hand with advanced duration of infection, which is one of the most important clinical variables determining IFN responsiveness. The ironic fact, then, is that patients whose disease is least likely to progress are the ones most likely to respond to IFN and vice versa.

1	Genetic changes in the virus may explain differences in treatment responsiveness in some patients (e.g., among patients with genotype 1b, responsiveness to IFN is enhanced in those with amino-acid-substitution mutations in the nonstructural protein 5A gene). As described above in the discussion of spontaneous recovery from acute hepatitis C, IFN gene variants discovered recently in genome-wide association studies have been shown to have a substantial impact on responsiveness of patients with genotype 1 to antiviral therapy. In studies of patients treated with PEG IFN and ribavirin, variants of the IL28B SNP that code for IFN-λ3 (a type III IFN, the receptors for which are more discretely distributed than IFN-α receptors and more concentrated in hepatocytes) correlate significantly with responsiveness. Patients homozygous for the C allele at this locus have the highest frequency of achieving an SVR (~80%), those homozygous for the T allele at this locus are least likely to achieve an

1	Patients homozygous for the C allele at this locus have the highest frequency of achieving an SVR (~80%), those homozygous for the T allele at this locus are least likely to achieve an SVR (~25%), and those heterozygous at this locus (C/T) have an intermediate level of responsiveness (SVRs in ~35%). The fact that C/C is common in whites of European ancestry and even more so in Japanese persons but rare in African Americans helps explain the differences in observed responsiveness among these population groups.

1	Side effects of IFN therapy are described above in the section on treatment of chronic hepatitis B. The most pronounced side effect of ribavirin therapy is hemolysis; a reduction in hemoglobin of up to 2−3 g or in hematocrit of 5−10% can be anticipated. A small, unpredictable proportion of patients experience profound, brisk hemolysis, resulting in symptomatic anemia; therefore, close monitoring of blood counts is crucial, and ribavirin should be avoided in patients with anemia or hemoglobinopathies and in patients with coronary artery disease or cerebrovascular disease, in whom anemia can precipitate an ischemic event. When symptomatic anemia occurs, ribavirin dose reductions or addition of erythropoietin to boost red blood cell levels may be required; erythropoietin has been shown to improve patients’ quality of life but not the likelihood of achieving an SVR. If ribavirin is stopped during therapy, SVR rates fall, but responsiveness can be maintained as long as ribavirin is not

1	improve patients’ quality of life but not the likelihood of achieving an SVR. If ribavirin is stopped during therapy, SVR rates fall, but responsiveness can be maintained as long as ribavirin is not stopped and the total ribavirin dose exceeds 60% of the planned dose. In addition, ribavirin, which is excreted renally, should not be used in patients with renal insufficiency; the drug is teratogenic, precluding its use during pregnancy and mandating the scrupulous use of efficient contraception during therapy (IFNs, too, because of their antiproliferative properties, are contraindicated during pregnancy).

1	Ribavirin can also cause nasal and chest congestion, pruritus, and precipitation of gout. Combination IFN-ribavirin therapy is more difficult to tolerate than IFN monotherapy. In one large clinical trial of combination therapy versus monotherapy, among those in the 1-year treatment group, 21% of the combination group (but only 14% of the monotherapy group) had to discontinue treatment, whereas 26% of the combination group (but only 9% of the mono-therapy group) required dose reductions.

1	Studies of viral kinetics have shown that despite a virion half-life in serum of only 2−3 h, the level of HCV is maintained by a high replication rate of 1012 hepatitis C virions per day. IFN-α blocks virion production or release with an efficacy that increases with increasing drug doses; moreover, the calculated death rate for infected cells during IFN therapy is inversely related to level of HCV RNA; patients with the most rapid death rate of infected hepatocytes are more likely to achieve undetectable HCV RNA at 3 months; in practice, failure to achieve an early virologic response (EVR), a ≥2-log10 reduction in HCV RNA by week 12, predicts failure to experience a subsequent SVR. Similarly, patients in whom HCV RNA becomes undetectable within 4 weeks (i.e., who achieve a rapid virologic response [RVR]) have a very high likelihood of achieving an SVR (Fig. 362-2). Therefore, to achieve rapid viral clearance from serum and the liver, high-dose induction therapy has been advocated. In

1	response [RVR]) have a very high likelihood of achieving an SVR (Fig. 362-2). Therefore, to achieve rapid viral clearance from serum and the liver, high-dose induction therapy has been advocated. In practice, however, high-dose induction with IFN-based therapy has not yielded higher sustained response rates.

1	For the treatment of chronic hepatitis C, standard IFNs were supplanted by PEG IFNs. These have elimination times up to sevenfold longer than standard IFNs (i.e., a substantially longer half-life) and achieve prolonged concentrations, permitting administration once (rather than three times) a week. Instead of the frequent drug peaks (linked to side effects) and troughs (when drug is absent) associated with frequent administration of short-acting IFNs, administration of PEG IFNs results in drug concentrations that are more stable and sustained over time. Once-a-week PEG IFN monotherapy is twice as effective as monotherapy with its standard IFN counterpart, approaches the efficacy of combination standard IFN plus ribavirin, and is as well tolerated as standard IFNs, without more difficult-to-manage thrombocytopenia and leukopenia than standard IFNs. For most of the decade prior to 2011, when protease inhibitors were introduced for HCV genotype 1 (see below), the standard of care was a

1	thrombocytopenia and leukopenia than standard IFNs. For most of the decade prior to 2011, when protease inhibitors were introduced for HCV genotype 1 (see below), the standard of care was a combination of PEG IFN plus ribavirin for all HCV genotypes.

1	Two PEG IFNs are available: PEG IFN-α2b and -α2a. PEG IFN-α2b consists of a 12-kD, linear PEG molecule bound to IFN-α2b, whereas PEG IFN-α2a consists of a larger, 40-kD, branched PEG molecule bound to IFN-α2a; because of its larger size and smaller volume of extravascular distribution, PEG IFN-α2a can be given at a uniform dose independent of weight, whereas the dose of the smaller PEG IFN-α2b, which has a much wider volume distribution, must be weight-based (Table 362-6). In the registration trial for PEG IFN-α2b plus ribavirin, the best regimen was 48 weeks of 1.5 μg/kg of PEG IFN once a week plus 800 mg of ribavirin daily. A post hoc analysis suggested that weight-based dosing of ribavirin would have been more effective than the fixed 800-mg dose used in the study (a broader dose/weight range was approved subsequently; see below). In the first registration trial for PEG IFN-α2a plus ribavirin, the best regimen was 48 weeks of 180 μg of PEG IFN plus 1000 mg (for patients <75 kg) to

1	range was approved subsequently; see below). In the first registration trial for PEG IFN-α2a plus ribavirin, the best regimen was 48 weeks of 180 μg of PEG IFN plus 1000 mg (for patients <75 kg) to 1200 mg (for patients ≥75 kg) of ribavirin. SVRs of 54 and 56% were reported in these two studies, respectively. A subsequent aIn the registration trial for PEG IFN-α2b plus ribavirin, the optimal regimen was 1.5 μg of PEG IFN plus 800 mg of ribavirin; however, a post hoc analysis of this study suggested that higher ribavirin doses are better. In subsequent trials of PEG IFN-α2b with ribavirin in patients with genotype 1, the following daily ribavirin doses have been validated: 800 mg for patients weighing <65 kg, 1000 mg for patients weighing >65–85 kg, 1200 for patients weighing >85–105 kg, and 1400 mg for patients weighing >105 kg. b1000 mg for patients weighing <75 kg; 1200 mg for patients weighing ≥75 kg. cIn the registration trial for PEG IFN-α2b plus ribavirin, all patients were

1	and 1400 mg for patients weighing >105 kg. b1000 mg for patients weighing <75 kg; 1200 mg for patients weighing ≥75 kg. cIn the registration trial for PEG IFN-α2b plus ribavirin, all patients were treated for 48 weeks; however, data from other trials of standard interferons and the other PEG IFN demonstrated that 24 weeks suffices for patients with genotypes 2 and 3. For patients with genotype 3 who have advanced fibrosis/cirrhosis and/or high-level HCV RNA, a full 48 weeks is preferable. dAttempts to compare the two PEG IFN preparations based on the results of registration clinical trials are confounded by differences between trials of the two agents in methodologic details (different ribavirin doses, different methods for recording depression, and other side effects) and study-population composition (different proportion with bridging fibrosis/ cirrhosis, proportion from the United States versus international, mean weight, proportion with genotype 1, and proportion with high-level

1	composition (different proportion with bridging fibrosis/ cirrhosis, proportion from the United States versus international, mean weight, proportion with genotype 1, and proportion with high-level HCV RNA). In the head-to-head comparison of the two PEG IFN preparations in the IDEAL trial reported in 2009, the two drugs were comparable in tolerability and efficacy. PEG IFN-α2b was administered at a weekly weight-based dose of 1.0 μg/kg or 1.5 μg/kg, and PEG IFN-α2a at a weekly fixed dose of 180 μg. For PEG IFN-α2b, daily ribavirin weight-based doses ranged between 800 and 1400 mg based on weight criteria (see footnote a, above), whereas for PEG IFN-α2a, daily ribavirin weight-based doses ranged between 1000 and 1200 mg (see footnote b, above). For the two PEG IFN-α2b study arms, ribavirin dose reductions for ribavirin-associated adverse effects were done in 200to 400-mg decrements; for PEG IFN-α2a, the ribavirin dose was reduced to 600 mg for intolerability. Sustained virologic

1	dose reductions for ribavirin-associated adverse effects were done in 200to 400-mg decrements; for PEG IFN-α2a, the ribavirin dose was reduced to 600 mg for intolerability. Sustained virologic responses occurred in 38.0% of the low-dose PEG IFN-α2b group, 39.8% of the standard, full-dose PEG IFN-α2b group, and 40.9% of the PEG IFN-α2a group.

1	Abbreviations: HCV RNA, hepatitis C virus RNA; PEG, polyethylene glycol. study of PEG IFN-α2a plus ribavirin showed that, for patients with genotypes 2 and 3, a duration of 24 weeks and a ribavirin dose of 800 mg were sufficient. Among the three studies, for patients in the optimal treatment arm, SVR rates for patients with genotype 1 were 42−51%, and for patients with genotypes 2 and 3, rates were 76−82%. Between genotypes 2 and 3, genotype 3 is somewhat more refractory, and some authorities would extend therapy for a full 48 weeks in patients with genotype 3, especially if they have advanced hepatic fibrosis or cirrhosis and/or high-level HCV RNA.

1	In the initial registration trials for combination PEG IFN plus ribavirin, both combination PEG IFN regimens were compared to standard IFN-α2b plus ribavirin. Side effects of the combination PEG IFN-α2b regimen were comparable to those for the combination standard IFN regimen; however, when the combination PEG IFN-α2a regimen was compared with the combination standard IFN-α2b regimen, flu-like symptoms and depression were less common in the combination PEG IFN group. Although ascertainment of side effects differed between studies of the two drugs, when each was tested against standard IFN-α2b plus ribavirin, combination PEG IFN-α2a plus ribavirin appeared to be better tolerated. In a head-to-head trial of the two PEG IFNs (the IDEAL trial), the two PEG IFNs were found to be comparable in efficacy (achievement of SVR) and tolerability, although headache, nausea, fever, myalgia, depression, and drug 2044 discontinuation for any reason were less frequent in patients treated with PEG

1	in efficacy (achievement of SVR) and tolerability, although headache, nausea, fever, myalgia, depression, and drug 2044 discontinuation for any reason were less frequent in patients treated with PEG IFN-α2a than standard-dose PEG IFN-α2b. In contrast, neutropenia and rash were more frequent in patients treated with PEG IFN-α2a than standard-dose PEG IFN-α2b. In two subsequent head-to-head trials and a systematic review of randomized trials, PEG IFN-α2a was more effective than PEG IFN-α2b (SVR in genotypes 1–4: 48–55% versus 32–40%, respectively). In trials of PEG IFN-α2b among patients with HCV genotype 1, a broader range of weight-based daily ribavirin doses has been validated: 800 mg for weight <65 kg, 1000 mg for weight 65−85 kg, 1200 mg for weight >85−105 kg, and 1400 mg for weight >105 kg. Recommended doses for the two PEG IFNs plus ribavirin and other comparisons between the two therapies are shown in Table 362-6. Until the 2011 introduction of protease inhibitors, unless

1	>105 kg. Recommended doses for the two PEG IFNs plus ribavirin and other comparisons between the two therapies are shown in Table 362-6. Until the 2011 introduction of protease inhibitors, unless ribavirin was contraindicated (see above), combination PEG IFN plus ribavirin was the recommended course of therapy—24 weeks for genotypes 2 and 3 and 48 weeks for genotype 1. For patients with genotypes 1 and 4, the standard of care now includes protease inhibitors or other direct-acting antiviral agents (see below); however, PEG IFN– ribavirin remained the standard of care for patients with genotypes 2 and 3 until late 2013. For patients treated with combination PEG IFN–ribavirin, measurement of quantitative HCV RNA levels at 12 weeks is helpful in guiding therapy; if a 2-log10 drop in HCV RNA has not been achieved by this time, chances for an SVR are negligible, and additional therapy is futile. If the 12-week HCV RNA has fallen by 2 log10 (EVR), the chances for an SVR at the end of

1	RNA has not been achieved by this time, chances for an SVR are negligible, and additional therapy is futile. If the 12-week HCV RNA has fallen by 2 log10 (EVR), the chances for an SVR at the end of therapy are approximately two-thirds; if the 12-week HCV RNA is undetectable (“complete” EVR), the chances for an SVR exceed 80% (Fig. 362-2). Because absence of an EVR is such a strong predictor of the absence of an ultimate SVR, therapy is discontinued for failure to achieve a 12-week 2-log10 drop in HCV RNA (EVR). Studies have suggested that the frequency of an SVR to PEG IFN– ribavirin therapy can be increased in patients with baseline variables weighing against a response (e.g., HCV RNA >8 × 105 IU/mL, weight >85 kg) by raising the dose of PEG IFN (e.g., to as high as 270 μg of PEG IFN-α2a) and/or the dose of ribavirin to as high as 1600 mg daily (if tolerated or supplemented by erythropoietin) or by tailoring treatment based on viral response to prolong the duration of viral clearance

1	and/or the dose of ribavirin to as high as 1600 mg daily (if tolerated or supplemented by erythropoietin) or by tailoring treatment based on viral response to prolong the duration of viral clearance before discontinuing therapy, i.e., extending therapy from 48 to 72 weeks for patients with genotype 1 and a slow virologic response (i.e., those whose HCV RNA has not fallen rapidly to undetectable levels within 4 weeks [absence of RVR]). Tailoring therapy based on the kinetics of HCV RNA reduction has also been applied to abbreviating the duration of therapy in patients with genotype 1 (and 4). The results of several clinical trials suggest that, in patients with genotype 1 (and 4) who have a 4-week RVR (which occurs in ≤20%), especially in the subset with low baseline HCV RNA, 24 weeks of therapy with PEG IFN and weight-based ribavirin suffices, yielding SVR rates of ~90% and comparable to those achieved in this cohort with 48 weeks of therapy. Although initial reports suggested that,

1	therapy with PEG IFN and weight-based ribavirin suffices, yielding SVR rates of ~90% and comparable to those achieved in this cohort with 48 weeks of therapy. Although initial reports suggested that, for patients with genotype 2 and (somewhat less so) genotype 3, in rapid virologic responders with undetectable HCV RNA at week 4, the total duration of therapy required to achieve an SVR could be as short as 12−16 weeks, a very sizable, definitive subsequent trial showed that relapse is increased if treatment duration is curtailed and that a full 24 weeks is superior for these genotypes (except for the minority with very low baseline levels of HCV RNA). Persons with chronic HCV infection have been shown to suffer increased liver-related mortality. On the other hand, successful antiviral therapy of chronic hepatitis C resulting in an SVR has been shown to improve survival (and to reduce the need for liver transplantation), to lower the risk of liver failure and liver-related death and

1	of chronic hepatitis C resulting in an SVR has been shown to improve survival (and to reduce the need for liver transplantation), to lower the risk of liver failure and liver-related death and all-cause deaths, to slow the progression of chronic hepatitis C, and to reverse fibrosis and even cirrhosis. Although successful treatment reduces mortality in cirrhotic patients (and those with advanced fibrosis) and reduces the likelihood of HCC, the risk of liver-related death and HCC persists, albeit at a much reduced level, necessitating continued clinical monitoring and cancer surveillance after SVR in cirrhotics. On the other hand, in the absence of an SVR, routine-dose/duration IFN-based therapy does not reduce the risk of HCC. Similarly, for nonresponders to PEG IFN–ribavirin therapy, three trials of long-term maintenance therapy with PEG IFN have shown no benefit in reducing the risk of histologic progression or clinical decompensation, including the development of HCC. For PEG

1	three trials of long-term maintenance therapy with PEG IFN have shown no benefit in reducing the risk of histologic progression or clinical decompensation, including the development of HCC. For PEG IFN–ribavirin nonresponders who have had a full, adequate course of therapy, the benefit of retreatment—with higher doses or a longer course of the original PEG IFN regimen or the alternative PEG IFN regimen or with a different type of IFN preparation (e.g., consensus IFN)—is marginal at best. Fortunately, such non-responders can now be retreated with protease inhibitor-based therapy (see following).

1	The HCV RNA genome encodes a single polyprotein, which is cleaved during and after translation by host and viral-encoded proteases. One protease involved in the cleavage of the viral polyprotein is an NS3-4A viral protein that has serine protease activity. Telaprevir and boceprevir are serine protease inhibitors that target NS3-4A. In 2011, telaprevir and boceprevir used in combination with PEG IFN and ribavirin were approved by the U.S. Food and Drug Administration (FDA) for the treatment of hepatitis C genotype 1 in adults with stable liver disease, both in patients who have not been treated before or who have failed previous treatment. Because the presently available HCV protease inhibitors have not been studied comprehensively in patients with genotypes other than 1, their use in these populations is not recommended.

1	Because resistance develops rapidly, both telaprevir and boceprevir must be used in combination with a PEG IFN and ribavirin-based regimen and should never be used alone. Ribavirin in particular appears to reduce relapse rates significantly in protease inhibitor– based regimens, such that those who cannot take or are intolerant to ribavirin are unlikely to benefit from the addition of these agents. All current telaprevir and boceprevir regimens consist of periods of triple therapy (protease inhibitor plus PEG IFN plus ribavirin) and periods of dual therapy (PEG IFN plus ribavirin). Telaprevir regimens begin with 12 weeks of triple therapy followed by dual therapy of a duration based on HCV RNA status at weeks 4 and 12 (“responseguided therapy”) and prior treatment status. Boceprevir-based regimens consist of a 4-week lead-in period of dual (PEG IFN–ribavirin) therapy followed by triple therapy and, in some instances, a further extension of dual therapy, with duration of

1	regimens consist of a 4-week lead-in period of dual (PEG IFN–ribavirin) therapy followed by triple therapy and, in some instances, a further extension of dual therapy, with duration of response-guided therapy based on HCV RNA status at weeks 4, 8, and 24 and prior treatment status (Table 362-7).

1	For patients with HCV genotype 1, protease inhibitors have significantly improved the frequency of RVRs and SVRs as compared to PEG IFN plus ribavirin alone. In treatment-naïve patients treated with telaprevir, an SVR was seen in up to 79% of patients who received 12 weeks of triple therapy followed by 12–36 weeks of dual therapy, and among those with EVRs (undetectable HCV RNA at weeks 4 and 12) and response-guided therapy stopped at week 24 (12 weeks of triple therapy, then 12 weeks of dual therapy), the rate of SVRs was 83–89% (92% in a subsequent study). In studies with boceprevir in treatment-naïve patients, SVRs were seen in 59–66% of patients, and among those with undetectable HCV RNA at 8 weeks, the SVR rate increased to 86–88%.

1	Protease inhibitors have also been studied in patients previously treated unsuccessfully with PEG IFN plus ribavirin. In studies with telaprevir, SVRs were seen in 83–88% of patients who had a previous relapse, 54–59% of partial responders (HCV RNA reduced by ≥2 log10 IU/mL but not to undetectable levels), and 29–33% of null responders (HCV RNA reduced by <2 log10 IU/mL). With boceprevir, SVRs occurred in 75% of prior relapsers and in 40–52% of previous partial responders; response rates in null responders are similar to those achieved with telaprevir-based therapy. In a substantial proportion of protease inhibitor nonresponders, resistance-associated variants can be identified, but these variants are not archived, and wild-type HCV reemerges in almost all cases within 1.5 to 2 years. SVRs to these protease inhibitors are highest in prior relapsers and

1	Detectable HCV RNA (with or without elevated ALT) Portal/bridging fibrosis or moderate to severe hepatitis on liver biopsy (the necessity of a pretreatment biopsy is no longer embraced universally) Indications for IFN/ribavirin-based therapy apply to adults as well as to children age 2–17, in whom treatment may be considered at reduced weight-based doses (see product inserts); protease inhibitors are not recommended for children age <18 years Relapsers, partial responders, or nonresponders after a previous course of standard IFN monotherapy or combination standard IFN/ribavirin therapy or PEG IFN/ ribavirin A course of PEG IFN/ribavirin plus protease inhibitor as below Genotypes 2, 3, 4

1	A course of PEG IFN/ribavirin plus protease inhibitor as below Genotypes 2, 3, 4 Relapsers after a previous course of standard IFN monotherapy or combination standard IFN/ribavirin therapy A course of PEG IFN plus ribavirin Nonresponders to a previous course of standard IFN monotherapy or combination standard IFN/ribavirin therapy A course of PEG IFN plus ribavirin—more likely to achieve a sustained virologic response in white patients without previous ribavirin therapy, with low baseline HCV RNA levels, with a ≥2-log10 reduction in HCV RNA during previous therapy, with genotypes 2 and 3, and without reduction in ribavirin dose Children (age <18 years)—protease inhibitors not recommended. Age >70 (in protease inhibitor trials, telaprevir trials included patients age 18–70; boceprevir trials included patients >18 years of age [no upper age cutoff ]) Mild hepatitis on liver biopsy Persons with severe renal insufficiency (require reduced doses of PEG IFN and ribavirin)

1	Cutaneous vasculitis and glomerulonephritis associated with chronic hepatitis C Decompensated cirrhosis (except, perhaps, in transplantation centers with experience in graded escalation, low-dose treatment to achieve undetectable HCV RNA prior to transplantation; results are mixed) Pregnancy (teratogenicity of ribavirin) Contraindications to use of antiviral medications PEG IFN-α2a 180 μg weekly plus weight-based ribavirin 1000 mg/d (<75 kg) to 1200 mg/d (≥75 kg) or PEG IFN-α2b 1.5 μg/kg weekly plus weight-based ribavirin 800 mg/d (≤65 kg), 1000 mg/d (>65–85 kg), 1200 mg/d (>85–105 kg), or 1400 mg/d (>105 kg) Plus response-guided therapy with a protease inhibitor consisting of either: Boceprevir 800 mg three times daily with food started after a lead-in treatment of 4 weeks with PEG IFN–ribavirin • Patients with undetectable HCV RNA at 8 and 24 weeks should receive triple-drug therapy (PEG IFN, ribavirin, boceprevir) through week 28 (4 weeks of PEG

1	IFN–ribavirin then 24 weeks of triple-drug therapy). If HCV RNA is detectable at 4 weeks, continuing therapy through 48 weeks (4 weeks of PEG IFN–ribavirin then 44 weeks of triple-drug therapy) may increase the sustained response rate. • Patients with detectable HCV RNA at 8 weeks and undetectable at 24 weeks should receive triple-drug therapy (PEG IFN, ribavirin, boceprevir) through week 36 (4 weeks of PEG IFN–ribavirin then 32 weeks of triple-drug therapy) followed by a return to PEG IFN–ribavirin for 12 more weeks, for a total treatment duration of 48 weeks. • Patients with cirrhosis who are treatment-naive and have undetectable HCV RNA at weeks 8 and 24 should continue triple-drug therapy (PEG IFN, ribavirin, boceprevir) through 48 weeks (4 weeks of PEG IFN–ribavirin then 44 weeks of triple-drug therapy). • Stopping rules for futility: HCV RNA ≥100 IU/mL at week 12 or any detectable HCV RNA at week 24

1	• Stopping rules for futility: HCV RNA ≥100 IU/mL at week 12 or any detectable HCV RNA at week 24 Telaprevir 750 mg three times daily with fatty food started at the beginning of therapy without a PEG IFN–ribavirin lead-in with undetectable HCV RNA at 4 and 12 weeks should receive triple-drug therapy (PEG IFN, ribavirin, telaprevir) for 12 weeks then PEG IFN and ribavirin for another 12 weeks, for a total of 24 weeks. with detectable HCV RNA at 4 or 12 weeks and undetectable at 24 weeks should receive triple-drug therapy (PEG IFN, ribavirin, telaprevir) for 12 weeks, then PEG IFN–ribavirin for another 36 weeks, for a total treatment duration of 48 weeks. • Patients with cirrhosis who are treatment-naïve and have undetectable HCV RNA at 4 and 12 weeks should receive triple-drug therapy for 12 weeks then PEG IFN–ribavirin for another 36 weeks, for a total treatment duration of 48 weeks.

1	IFN–ribavirin for another 36 weeks, for a total treatment duration of 48 weeks. • Stopping rules for futility: HCV RNA >1000 IU/mL at week 4 or 12 or any detectable HCV RNA at week 24

1	• Stopping rules for futility: HCV RNA >1000 IU/mL at week 4 or 12 or any detectable HCV RNA at week 24 PEG IFN-α2a 180 μg weekly plus weight-based ribavirin 1000 mg/d (<75 kg) to 1200 mg/d (≥75 kg) or PEG IFN-α2b 1.5 μg/kg weekly plus weight-based ribavirin 800 mg/d (≤65kg), 1000 mg/d (>65–85 kg), 1200 mg/d (>85–105 kg), or 1400 mg/d (>105 kg) Plus a protease inhibitor consisting of either: Response-guided therapy with boceprevir 800 mg three times daily with food started after a lead-in treatment of 4 weeks with PEG IFN–ribavirin • For prior relapsers and partial responders (HCV RNA reduction of ≥2 log10 during previous therapy), follow the response-guided algorithm below; for prior null responders (HCV RNA reduction <2 log10 during previous therapy), a full 48-week course of therapy (4-week PEG IFN–ribavirin lead-in, followed by 44 weeks of triple-drug therapy [PEG IFN, ribavirin, boceprevir]) is recommended.

1	• Patients with undetectable HCV RNA at 8 and 24 weeks should receive triple-drug therapy (PEG IFN, ribavirin, boceprevir) through week 36 (4 weeks of PEG IFN–ribavirin then 32 weeks of triple-drug therapy). If HCV RNA is detectable at 4 weeks, continuing therapy through 48 weeks (4 weeks of PEG IFN–ribavirin then 44 weeks of triple therapy) may increase the sustained response rate. • Patients with detectable HCV RNA at 8 weeks and undetectable at week 24 should receive triple-drug therapy (PEG IFN, ribavirin, boceprevir) through week 36 (4 weeks of PEG IFN–ribavirin then 32 weeks of triple-drug therapy), followed by a return to PEG IFN–ribavirin for 12 more weeks, for a total treatment duration of 48 weeks. with cirrhosis who are treatment-experienced and have undetectable HCV RNA at weeks 8 and 24 should continue triple-drug therapy (PEG IFN, ribavirin, boceprevir) through 48 weeks (4 weeks of PEG IFN–ribavirin then 44 weeks of triple-drug therapy).

1	rules for futility: HCV RNA ≥100 IU/mL at week 12 or any detectable HCV RNA at week 24 Telaprevir 750 mg three times daily with fatty food started at the beginning of therapy without a PEG IFN–ribavirin lead-in and without a response-guided approach, i.e., all patients receive a full 48-week course, independent of early responsiveness. prior relapsers, follow guidelines for treatment-naïve patients above. partial responders and null responders should receive triple-drug therapy (PEG IFN, ribavirin, telaprevir) for 12 weeks then PEG IFN and ribavirin for another 36 weeks, for a total of 48 weeks. • Stopping rules for futility: HCV RNA >1000 IU/mL at week 4 or 12 or any detectable HCV RNA at week 24

1	• Stopping rules for futility: HCV RNA >1000 IU/mL at week 4 or 12 or any detectable HCV RNA at week 24 HCV genotype 1 but protease inhibitors unavailable or contraindicated: 48 weeks of therapy PEG IFN-α2a 180 μg weekly plus weight-based ribavirin 1000 mg/d (<75 kg) to 1200 mg/d (≥75 kg) or PEG IFN-α2b 1.5 μg/kg weekly plus weight-based ribavirin 800 mg/d (≤65 kg), 1000 mg/d (>65–85 kg), 1200 mg/d (>85–105 kg), or 1400 mg/d (>105 kg) HCV genotype 4: 48 weeks of PEG IFN–ribavirin therapy PEG IFN-α2a 180 μg weekly plus weight-based ribavirin 1000 mg/d (<75 kg) to 1200 mg/d (≥75 kg) or PEG IFN-α2b 1.5 μg/kg weekly plus weight-based ribavirin 800 mg/d (≤65 kg), 1000 mg/d (>65–85 kg), 1200 mg/d (>85–105 kg), or 1400 mg/d (>105 kg) Treatment should be discontinued in patients who do not achieve an early virologic response at week 12. Patients who do achieve an early virologic response should be retested at week 24, and treatment should be discontinued if HCV RNA remains detectable.

1	Patients who do achieve an early virologic response should be retested at week 24, and treatment should be discontinued if HCV RNA remains detectable. HCV genotypes 2 and 3: 24 weeks of therapy PEG IFN-α2b 1.5 μg/kg weekly plus ribavirin 800 mg/d (for patients with genotype 3 who have advanced fibrosis and/or high-level HCV RNA, a full 48 weeks of therapy may be preferable)

1	For HCV/HIV co-infected patients: 48 weeks, regardless of genotype, of weekly PEG IFN-α2a (180 μg) or PEG IFN-α2b (1.5 μg/kg) plus a daily ribavirin dose of at least 600–800 mg, up to full weight-based 1000–1400 mg dosing if tolerated. Protease inhibitors may be used for genotype 1; however, because of potential drug-drug interactions between HCV protease inhibitors and HIV antiretroviral drugs, HCV protease inhibitors should be used cautiously in HCV/HIV co-infected patients. If protease inhibitors are used, a full 48-week course is recommended without response-guided therapy. For boceprevir, 4 weeks of PEG IFN–ribavirin lead-in, followed by 44 weeks of triple-drug therapy (PEG IFN, ribavirin, boceprevir). For telaprevir, 12 weeks of triple-drug therapy (PEG IFN, ribavirin, telaprevir), followed by 36 weeks of PEG IFN–ribavirin therapy. Stopping rules for futility are as noted above. Features Associated with Reduced Responsiveness

1	Single nucleotide polymorphism (SNP) T allele (as opposed to C allele) at IL28B locus Genotype 1a (compared to 1b) High-level HCV RNA (>800,000 IU/mL)b Advanced fibrosis (bridging fibrosis, cirrhosis) Long-duration disease Age >40b High HCV quasispecies diversity Immunosuppression African-American ethnicity Latino ethnicity Obesity Hepatic steatosis Insulin resistance, type 2 diabetes mellitusb Reduced adherence (lower drug doses and reduced duration of therapy) For boceprevir, <1 log10 reduction in HCV RNA during 4-week PEG IFN–ribavirin lead-in For protease inhibitor therapy, absence of extended rapid virologic response (eRVR), i.e., detectable HCV RNA, at weeks 4 and 12 for telaprevir; at weeks 8 and 24 for boceprevir aAs this chapter was going to press, two additional drugs were approved for hepatitis C, simeprevir and sofosbuvir. Rapidly evolving new recommendations are supplanting the recommendations in this table; for up-to-date treatment recommendations, please see

1	were approved for hepatitis C, simeprevir and sofosbuvir. Rapidly evolving new recommendations are supplanting the recommendations in this table; for up-to-date treatment recommendations, please see www.hcvguidelines.org. bLess influential in patients treated with protease inhibitors.

1	Abbreviations: ALT, alanine aminotransferase; HCV, hepatitis C virus; IFN, interferon; PEG IFN, pegylated interferon; IU, international units (1 IU/mL is equivalent to ~2.5 copies/mL).

1	treatment-naïve patients (white > black ethnicity), lower in prior PEG IFN–ribavirin lead-in therapy. Age and HCV RNA level are less partial responders, lower still in prior null responders, and lowest in influential and insulin resistance is noninfluential on response to cirrhotic prior null responders (Fig. 362-3). Responses to protease these antiviral agents. inhibitor triple-drug regimens are higher in patients with IL28B C Both protease inhibitors have potential toxicities. Telaprevir is than non-C genotypes, HCV genotype 1b than genotype 1a, less associated with a severe, generalized (trunk and extremities), often advanced than more advanced fibrosis stage, whites than blacks, confluent, maculopapular, pruritic rash in ~6% of treated patients. lower body mass index (BMI) than elevated BMI, and, for boceprevir, Other common side effects include pruritus, rectal burning, nausea, achievement of a >1 log10 HCV RNA reduction during 4 weeks of diarrhea, fatigue, and anemia, which may

1	BMI, and, for boceprevir, Other common side effects include pruritus, rectal burning, nausea, achievement of a >1 log10 HCV RNA reduction during 4 weeks of diarrhea, fatigue, and anemia, which may be relatively refractory, % with sustained virologic response

1	FIGURE 362-3 Maximal efficacy (sustained virologic responses, SVR) of telaprevir (blue bars) and boceprevir (yellow bars) reported in phase III clinical trials. (Figure created using data from Bacon BR et al: N Engl J Med 364:1207, 2011; Jacobson IM et al: N Engl J Med 364:2405, 2011; Poordad F et al: N Engl J Med 364:1195, 2011; Zeuzem S et al: N Engl J Med 364:2417, 2011; Vierling JM et al: Hepatology 54 [Suppl 1]:796A-797A, 2011; Ghany MG et al: Hepatology 54:1433, 2011.) occasionally requiring transfusion. Complete blood counts should be obtained at baseline and then at 2, 4, 8, and 12 weeks after starting telaprevir. Anemia can occur in half of boceprevir-treated patients, as can neutropenia in up to 30% and thrombocytopenia in 3–4%. Complete blood counts should be obtained at baseline and then at 4, 8, and 12 weeks after starting boceprevir. Other side effects of boceprevir include fatigue, nausea, headache, dysgeusia (altered or unpleasant taste), dry mouth, vomiting, and

1	and then at 4, 8, and 12 weeks after starting boceprevir. Other side effects of boceprevir include fatigue, nausea, headache, dysgeusia (altered or unpleasant taste), dry mouth, vomiting, and diarrhea.

1	Use of protease inhibitors is further complicated by numerous drug-drug interactions. As telaprevir and boceprevir are both eliminated by and inhibit CYP3A4, these agents should not be administered with other medications that induce CYP3A4 or are dependent on CYP3A4 for elimination. Care should be taken to examine for any potential interactions between protease inhibitors and other medications the patient may be taking, because serious adverse events can occur. A convenient website is available to check for such drug-drug interactions (www.hep-druginteractions.org).

1	Prior to therapy, HCV genotype should be determined, because the genotype dictates the duration of therapy and potentially the agents to be used. PEG IFN plus ribavirin represents the foundation of treatment for all HCV genotypes; patients infected with genotype 1 should also receive a protease inhibitor (telaprevir or boceprevir) when these are available and not contraindicated (Table 362-7). For chronic HCV genotype 1 infection, the AASLD and EASL published treatment guidelines in 2011 reflecting FDA-approved indications for the new protease inhibitors, and in 2012, United Kingdom and French consensus guidelines were published. For treatment-naïve patients and prior relapsers, response-guided therapy with telaprevir or boceprevir is recommended. For telaprevir, the regimen consists of 12 weeks of triple therapy, followed by 12 or 36 weeks of PEG IFN–ribavirin consolidation, depending on whether extended RVR milestones (HCV RNA undetectable at weeks 4 and 12) are met.

1	*As this chapter was going to press, two additional antiviral drugs, a second-generation protease inhibitor simeprevir and nucleoside analogue polymerase inhibitor sofosbuvir were approved for the treatment of hepatitis C. Simerprevir, which is effective for genotype 1, must be administered, like first-generation protease inhibitors, for 12 weeks with PEG IFN and ribavirin, followed by another 12 weeks of PEG IFN and ribavirin (no response-guided therapy). Sofosbuvir, the more convenient and broadly applicable of the two new drugs, must be administered with PEG IFN and ribavirin but for only 12 weeks in patients with genotyes 1, 4-6; for patients with genotypes 2 and 3, PEG IFN is not required. Sofosbuvir plus ribavirin are administered for 12 weeks in genotype 2 and for 24 weeks in genotype 3. Antiviral therapy is evolving very rapidly; by the end of 2014, all-oral, interferon-free combinations (e.g., sofosbuvir plus the NS5a inhibitor ledipasvir) will supplant earlier treatment

1	3. Antiviral therapy is evolving very rapidly; by the end of 2014, all-oral, interferon-free combinations (e.g., sofosbuvir plus the NS5a inhibitor ledipasvir) will supplant earlier treatment regimens. For updated treatment recommendations, please consult www.hcvguidelines.org.

1	For boceprevir, the regimen consists of a 4-week PEG IFN–ribavirin 2047 lead-in period, followed by 24–32 weeks of triple-drug therapy, depending on whether HCV RNA milestones (undetectable at weeks 8 and 24) are met; if HCV RNA is detectable at week 8 but undetectable at week 24, after 36 weeks of therapy (4-week PEG IFN–ribavirin lead-in plus 32 weeks of triple-drug therapy), an additional 12 weeks of PEG IFN–ribavirin consolidation is recommended. For prior partial and null responders, a full 48-week course of telaprevir (no lead-in period, no response-guided therapy) is recommended; for boceprevir, a 4-week PEG IFN–ribavirin lead-in period is followed by response-guided therapy (32 weeks of triple-drug therapy if HCV RNA is undetectable at weeks 8 and 24 or, if HCV RNA is still detectable at week 8 [but undetectable at week 24], 32 weeks of triple-drug therapy followed by 12 weeks of PEG IFN–ribavirin consolidation). For cirrhotic patients (and for any boceprevir-treated patient

1	at week 8 [but undetectable at week 24], 32 weeks of triple-drug therapy followed by 12 weeks of PEG IFN–ribavirin consolidation). For cirrhotic patients (and for any boceprevir-treated patient whose HCV RNA does not fall by >1 log10 by week 4), a full 48-week course without response-guided therapy should be considered.

1	Monitoring of HCV plasma RNA is crucial in assessing response to therapy. The goal of treatment is to eradicate HCV RNA, which is predicted by the absence of HCV RNA by PCR 6 months after stopping treatment (SVR). When therapy relied on PEG IFN and ribavirin, failure to achieve a 2-log10 drop in HCV RNA by week 12 of therapy (EVR) rendered it unlikely that further therapy would result in an SVR. When PEG IFN and ribavirin are part of a protease inhibitor regimen, HCV RNA should be measured at baseline and at weeks 4, 8 (for boceprevir), 12, and 24 to assess response to treatment and to aid in decisions regarding treatment duration (response-guided therapy), as well as 12 and 24 weeks after therapy. Stopping rules are important to prevent the emergence of resistance; if HCV RNA is >1000 IU/ mL at 4 or 12 weeks of telaprevir (or still detectable at week 24), or if HCV RNA is ≥100 IU/mL at week 12 of boceprevir (or detectable at week 24), all treatment should be stopped.

1	Patients with chronic hepatitis C who have detectable HCV RNA in serum, whether or not aminotransferase levels are increased, and chronic hepatitis of at least moderate grade and stage (portal or bridging fibrosis) are candidates for antiviral therapy with PEG IFN plus ribavirin. Most authorities recommend 800 mg of ribavirin for patients with genotypes 2 and 3 for both types of PEG IFN and weight-based 1000−1200 mg (when used with PEG IFN-α2a) or 800−1400 mg (when used with PEG IFN-α2b) ribavirin for patients with genotype 1 (and 4), unless ribavirin is contraindicated (Table 362-7). These PEG IFN and ribavirin doses are used with protease inhibitors for patients with genotype 1 (Table 362-7). Although patients with persistently normal ALT activity tend to progress histologically very slowly or not at all, they respond to antiviral therapy just as well as do patients with elevated ALT levels; therefore, although observation without therapy is an option, such patients are potential

1	slowly or not at all, they respond to antiviral therapy just as well as do patients with elevated ALT levels; therefore, although observation without therapy is an option, such patients are potential candidates for antiviral therapy. As noted above, therapy with IFN has been shown to improve survival and complication-free survival and to slow progression of (and to reverse) fibrosis.

1	HCV genotype determines the duration of PEG IFN and ribavirin therapy: 24 weeks for those with genotypes 2 and 3 and 48 weeks for patients with genotypes 4 and 1 (in patients for whom protease inhibitors are not available or contraindicated). For patients with genotype 4, treatment should be discontinued in patients who do not achieve an EVR at week 12. For patients with genotypes 2 and 3, a full, 24-week course is most effective, although the duration may be reduced to 12−16 weeks for patients with genotype 2, a low baseline level of viremia, and an RVR, especially to be considered for patients who tolerate therapy poorly. Also, consideration should be given to increasing the duration of therapy to 48 weeks for patients with genotype 3 who have advanced fibrosis and/or a high baseline level of viremia. As noted above, the absence of a 2-log10 drop in HCV RNA at week 12 (EVR) weighs heavily against the likelihood of an SVR; therefore, measuring HCV RNA at 12 weeks is recommended

1	level of viremia. As noted above, the absence of a 2-log10 drop in HCV RNA at week 12 (EVR) weighs heavily against the likelihood of an SVR; therefore, measuring HCV RNA at 12 weeks is recommended routinely (Fig. 362-2), and therapy can be discontinued if an EVR is not achieved. Among patients with genotype 4 who achieve an 2048 EVR (≥2-log10 HCV RNA reduction) but in whom HCV RNA remains detectable at week 24, an SVR is unlikely, and therapy can be discontinued. Although response rates are lower in patients with certain pretreatment variables, selection for treatment should not be based on symptoms, genotype, HCV RNA level, mode of acquisition of hepatitis C, or advanced hepatic fibrosis. Patients with cirrhosis can respond and should not be excluded as candidates for therapy. For patients being treated with telaprevir and boceprevir, treating physicians should explain the negative impact of non-C IL28B genotype and advanced fibrosis on outcome. Patients who have relapsed after, or

1	being treated with telaprevir and boceprevir, treating physicians should explain the negative impact of non-C IL28B genotype and advanced fibrosis on outcome. Patients who have relapsed after, or failed to respond to (Fig. 362-2), a course of IFN monotherapy are potential candidates for retreatment with PEG IFN plus ribavirin (i.e., a more effective treatment regimen is required), and this approach remains current for patients with genotypes 2, 3, or 4; however, for patients with genotype 1, combination protease inhibitor/PEG IFN/ribavirin therapy is indicated. For patients with genotypes 2, 3, or 4 who were nonresponders to a prior course of IFN monotherapy, retreatment with IFN monotherapy or combination IFN plus ribavirin therapy is unlikely to achieve an SVR; however, a trial of combination PEG IFN plus ribavirin may be worthwhile, although an SVR is the outcome in <15−20% of patients. SVRs to retreatment of nonresponders are more frequent in those who had never received ribavirin

1	PEG IFN plus ribavirin may be worthwhile, although an SVR is the outcome in <15−20% of patients. SVRs to retreatment of nonresponders are more frequent in those who had never received ribavirin in the past, those with genotypes 2 and 3, those with low pretreatment HCV RNA levels, and noncirrhotics, but less frequent in African Americans, those who failed to achieve a substantial reduction in HCV RNA during their previous course of therapy (null responders, Fig. 362-2), and those who required ribavirin dose reductions. Potential approaches to improving responsiveness to PEG IFN–ribavirin in prior nonresponders include longer duration of treatment; higher doses of PEG IFN, ribavirin, or both; and switching to a different IFN preparation; however, as noted above, none of these approaches achieves more than a marginal benefit. Treatment with a protease inhibitor–based regimen should be pursued in patients with genotype 1 who have relapsed after or not responded to prior treatment with IFN

1	more than a marginal benefit. Treatment with a protease inhibitor–based regimen should be pursued in patients with genotype 1 who have relapsed after or not responded to prior treatment with IFN monotherapy or PEG IFN plus ribavirin, unless these protease inhibitors are not available or contraindicated (Table 362-7). Early PEG IFN treatment is indicated for persons with acute hepatitis C; ribavirin, which is used frequently in such instances, has not been shown to improve efficacy over that of PEG IFN alone, and the new protease inhibitors have not been approved for acute hepatitis C (Chap. 360). In patients with biochemically and histologically mild chronic hepatitis C, the rate of progression is slow, and monitoring without therapy is an option; however, such patients respond just as well to combination PEG IFN plus ribavirin therapy or triple-drug, protease-based therapy (for genotype 1) as those with elevated ALT and more histologically severe hepatitis. Therefore, therapy for

1	well to combination PEG IFN plus ribavirin therapy or triple-drug, protease-based therapy (for genotype 1) as those with elevated ALT and more histologically severe hepatitis. Therefore, therapy for these patients should be considered and the decision made based on such factors as patient motivation, genotype, stage of fibrosis, age, and comorbid conditions. A pretreatment liver biopsy to assess histologic grade and stage provides substantial information about progression of hepatitis C in the past, has prognostic value for future progression, and can identify such histologic factors as steatosis and stage of fibrosis, which can influence responsiveness to therapy. As therapy has improved for patients with a broad range of histologic severity, and as noninvasive laboratory markers and imaging correlates of fibrosis have gained popularity, some authorities, especially in Europe, place less value on, and do not recommend, pretreatment liver biopsies. On the other hand, serum markers of

1	correlates of fibrosis have gained popularity, some authorities, especially in Europe, place less value on, and do not recommend, pretreatment liver biopsies. On the other hand, serum markers of fibrosis are not considered sufficiently accurate, and histologic findings provide important prognostic information to physician and patient. Therefore, although the contemporary role of a pretreatment liver biopsy commands less of a consensus, a pretreatment liver biopsy still provides useful information and should be considered. Patients with compensated cirrhosis can respond to therapy, although their likelihood of a sustained response is lower than in noncirrhotics; moreover, survival has been shown to improve after successful antiviral therapy in cirrhotics. Similarly, although several retrospective studies have suggested that antiviral therapy in cirrhotics with chronic hepatitis C, independent of treatment outcome per se, reduces the frequency of HCC, less advanced disease in the

1	retrospective studies have suggested that antiviral therapy in cirrhotics with chronic hepatitis C, independent of treatment outcome per se, reduces the frequency of HCC, less advanced disease in the treated cirrhotics, not treatment itself (i.e., lead-time bias), may have accounted for the reduced frequency of HCC observed in the treated cohorts in these reports; prospective studies to address this question have failed to demonstrate benefit, unless an SVR is achieved. Patients with decompensated cirrhosis are not candidates for IFN-based antiviral therapy but should be referred for liver transplantation. Some liver transplantation centers have evaluated progressively escalated, low-dose antiviral therapy in an attempt to eradicate hepatitis C viremia prior to transplantation; however, such therapy has been shown to reduce but not to prevent the risk of HCV reinfection after transplantation. After liver transplantation for end-stage liver disease caused by hepatitis C, recurrent

1	such therapy has been shown to reduce but not to prevent the risk of HCV reinfection after transplantation. After liver transplantation for end-stage liver disease caused by hepatitis C, recurrent hepatitis C is the rule, and the pace of disease progression is more accelerated than in immunocompetent patients (Chap. 368). Current therapy with PEG IFN and ribavirin after liver transplantation is unsatisfactory in most patients, but attempts to minimize immunosuppression are beneficial. Early experience with protease inhibitor–based therapy is encouraging, but inhibition of CYP3A4 by protease inhibitors can lead to markedly increased levels of immunosuppressive calcineurin inhibitors (especially tacrolimus), which requires intensive monitoring and can be very challenging. The cutaneous and renal vasculitis of HCV-associated essential mixed cryoglobulinemia (Chap. 360) may respond to antiviral therapy, but sustained responses are rare after discontinuation of therapy; therefore,

1	and renal vasculitis of HCV-associated essential mixed cryoglobulinemia (Chap. 360) may respond to antiviral therapy, but sustained responses are rare after discontinuation of therapy; therefore, prolonged, perhaps indefinite, therapy (as reported with IFN-based therapy) is recommended in this group (no indication for prolonged protease inhibitor therapy exists currently). Anecdotal reports suggest that antiviral therapy may be effective in porphyria cutanea tarda or lichen planus associated with hepatitis C.

1	In patients with HCV/HIV co-infection, hepatitis C is more progressive and severe than in HCV-monoinfected patients. Although patients with HCV/HIV co-infection respond to antiviral therapy for hepatitis C, they do not respond as well as patients with HCV infection alone. Four large national and international trials of antiviral therapy among patients with HCV/HIV co-infection have shown that PEG IFN (both α2a and α2b) plus ribavirin (daily doses ranging from flat-dosed 600−800 mg to weight-based 1000/1200 mg) is superior to standard IFN regimens; however, SVR rates were lower than in HCV-monoinfected patients, ranging from 14 to 38% for patients with genotypes 1 and 4 and from 44 to 73% for patients with genotypes 2 and 3. In the three largest trials, all patients, including those with genotypes 2 and 3, were treated for a full 48 weeks. In addition, tolerability of therapy was lower than in HCV-monoinfected patients; therapy was discontinued because of side effects in 12−39% of

1	genotypes 2 and 3, were treated for a full 48 weeks. In addition, tolerability of therapy was lower than in HCV-monoinfected patients; therapy was discontinued because of side effects in 12−39% of patients in these clinical trials. Based on these trials, weekly PEG IFN plus daily ribavirin at a daily dose of at least 600−800 mg, up to full weight-based doses, at doses recommended for HCV-monoinfected patients, if tolerated, is recommended for a full 48 weeks, regardless of genotype. An alternative recommendation for ribavirin doses was issued by a European Consensus Conference and consisted of standard, weight-based 1000−1200 mg for genotypes 1 and 4, but 800 mg for genotypes 2 and 3. A head-to-head trial of combination PEG IFN–ribavirin therapy in HCV/HIV co-infection demonstrated statistically indistinguishable efficacy of the two types of PEG IFN, despite a small advantage for PEG IFN-α2a: For PEG IFN-α2b and -α2a, SVRs occurred in 28% versus 32%, respectively, of patients with

1	indistinguishable efficacy of the two types of PEG IFN, despite a small advantage for PEG IFN-α2a: For PEG IFN-α2b and -α2a, SVRs occurred in 28% versus 32%, respectively, of patients with genotypes 1 and 4 and in 62% versus 71%, respectively, of patients with genotypes 2 and 3.

1	Although data are limited and recommendations pending, protease inhibitors may be used for genotype 1; however, because of potential drug-drug interactions between HCV protease inhibitors and HIV antiretroviral drugs (especially in ritonavir-boosted HIV protease inhibitors), HCV protease inhibitors should be used cautiously in HCV-HIV co-infected patients. If protease inhibitors are used, a full 48-week course is recommended without response-guided therapy: for boceprevir, 4 weeks of PEG IFN–ribavirin lead-in, followed by 44 weeks of triple-drug therapy (PEG IFN, ribavirin, boceprevir), and for telaprevir, 12 weeks of triple-drug therapy (PEG IFN, ribavirin, telaprevir), followed by 36 weeks of PEG IFN–ribavirin therapy. In preliminary trials among HIV-HCV co-infected patients, telaprevirbased triple-drug therapy (independent of whether they were receiving antiretroviral therapy [no antiretroviral drugs, efavirenztenofovir-emtricitabine, or ritonavir-boosted

1	patients, telaprevirbased triple-drug therapy (independent of whether they were receiving antiretroviral therapy [no antiretroviral drugs, efavirenztenofovir-emtricitabine, or ritonavir-boosted atazanavir-tenofoviremtricitabine or lamivudine]) resulted in an SVR in 28 of 38 patients (74%), compared with 10 of 22 control patients (45%) treated with PEG IFN–ribavirin (60 study subjects); boceprevir-based triple-drug therapy (all were also receiving antiretroviral therapy) resulted in an SVR in 40 of 64 patients (63%), compared with 10 of 34 control patients (29%) treated with PEG IFN–ribavirin (98 study subjects). Thus, for the prior standard of care, PEG IFN plus ribavirin, although the likelihood of an SVR is lower for HIV-HCV co-infected patients than for HCV-monoinfected patients, for protease inhibitor–based regimens, rates of SVR are comparable in HIV-HCV co-infected and HCV-monoinfected patients.

1	In HCV/HIV-infected patients, ribavirin can potentiate the toxicity of didanosine (e.g., lactic acidosis) and the lipoatrophy of stavudine, and zidovudine can exacerbate ribavirin-associated hemolytic anemia; therefore, these drug combinations should be avoided.

1	Patients with a history of injection drug use and alcoholism can be treated successfully for chronic hepatitis C, preferably in conjunction with drug and alcohol treatment programs. Because ribavirin is excreted renally, patients with end-stage renal disease, including those undergoing dialysis (which does not clear ribavirin), are not ideal candidates for ribavirin therapy. Rare reports suggest that reduced-dose ribavirin can be used, but the frequency of anemia is very high, and data on efficacy are limited. If patients with renal failure (glomerular filtration rate <60 mL/min) are treated, the PEG IFNα2a dose should be reduced from 180 to 135 μg weekly and the PEG IFN-α2b dose reduced from 1.5 to 1 μg/kg weekly; similarly, the daily ribavirin dose in this population should be reduced to 200−800 mg (but not used or used cautiously at very low doses) if hemodialysis is required. Neither the optimal regimen nor the efficacy of therapy is well established in this population.

1	To date, attempts to develop better-tolerated ribavirin successors or improved types of IFN-α or longer acting IFNs than PEG IFN have not been successful. The demonstration that responsiveness to antiviral therapy is influenced by genetic variation in IL28B, which codes for IFN-λ (as noted above), raises the possibility that IFN-λ might be an effective or even more effective IFN for treating hepatitis C; early trials are in progress, but elevations of aminotransferase levels in treated subjects have raised concerns and delayed development. Beyond telaprevir and boceprevir, other direct antivirals that target HCV polymerase, protease, or NS5A (a membrane phosphoprotein component of the viral replication complex) are being investigated, as well as agents that can target host-encoded proteins. Among the novel antivirals are drugs with improved pharmacokinetic and resistance profiles, less treatment complexity, pangenotypic activity, fewer side effects, and fewer drug-drug interactions.*

1	Among the novel antivirals are drugs with improved pharmacokinetic and resistance profiles, less treatment complexity, pangenotypic activity, fewer side effects, and fewer drug-drug interactions.* The pace of successful trials of all-oral regimens has accelerated. All-oral combinations of a second-generation protease inhibitor (asunaprevir) plus an NS5A inhibitor (daclatasvir); of a uridine nucleoside polymerase inhibitor (sofosbuvir)* plus ribavirin; of a polymerase inhibitor (sofosbuvir) plus an NS5A inhibitor (ledipasvir or daclatasvir) and ribavirin; and of combinations of a ritonavirboosted protease inhibitor (ABT-450) plus a nonnucleoside polymerase inhibitor (ABT-333) plus an NS5A inhibitor (ABT-267) with or without ribavirin have been studied in clinical trials. Several of these drug combinations have achieved SVR rates exceeding 90%, even approaching 100%, for both treatment-naïve and treatment-experienced patients (including patients who failed to respond to first-generation

1	combinations have achieved SVR rates exceeding 90%, even approaching 100%, for both treatment-naïve and treatment-experienced patients (including patients who failed to respond to first-generation protease inhibitors), across all HCV genotypes and independent of host IL28B genotype, and with treatment durations of 12–24 weeks or even shorter (8 weeks). Potentially, as early as 2014 or 2015, such combinations of direct antiviral agents will be used in drug cocktails that may replace IFN-based regimens 2049 entirely.

1	Less advanced is development of inhibitors of host proteins, such as oral, nonimmunosuppressive inhibitors of cyclophilin A (which interacts with NS5A during HCV replication) and subcutaneous antisense antagonists of host liver-expressed micro-RNA-122 (which promotes HCV replication). Given the accelerated progress of all-oral, short-treatment-duration, high-efficacy, direct-acting antivirals, these alternative approaches may not be practical or competitive.

1	Autoimmune hepatitis is a chronic disorder characterized by continuing hepatocellular necrosis and inflammation, usually with fibrosis, which can progress to cirrhosis and liver failure. When fulfilling criteria of severity, this type of chronic hepatitis, when untreated, may have a 6-month mortality of as high as 40%. Based on contemporary estimates of the natural history of autoimmune hepatitis, the 10-year survival is 80−98% for treated and 67% for untreated patients. The prominence of extrahepatic features of autoimmunity and seroimmunologic abnormalities in this disorder supports an autoimmune process in its pathogenesis; this concept is reflected in the prior labels lupoid and plasma cell hepatitis. Autoantibodies and other typical features of autoimmunity, however, do not occur in all cases; among the broader categories of “idiopathic” or cryptogenic chronic hepatitis, many, perhaps the majority, are probably autoimmune in origin. Cases in which hepatotropic viruses,

1	occur in all cases; among the broader categories of “idiopathic” or cryptogenic chronic hepatitis, many, perhaps the majority, are probably autoimmune in origin. Cases in which hepatotropic viruses, metabolic/genetic derangements (including nonalcoholic fatty liver disease), and hepatotoxic drugs have been excluded represent a spectrum of heterogeneous liver disorders of unknown cause, a proportion of which are most likely autoimmune hepatitis.

1	The weight of evidence suggests that the progressive liver injury in patients with autoimmune hepatitis is the result of a cell-mediated immunologic attack directed against liver cells. In all likelihood, predisposition to autoimmunity is inherited, whereas the liver specificity of this injury is triggered by environmental (e.g., chemical, drug [e.g., minocycline], or viral) factors. For example, patients have been described in whom apparently self-limited cases of acute hepatitis A, B, or C led to autoimmune hepatitis, presumably because of genetic susceptibility or predisposition. Evidence to support an autoimmune pathogenesis in this type of hepatitis includes the following: (1) In the liver, the histopathologic lesions are composed predominantly of cytotoxic T cells and plasma cells; (2) circulating autoantibodies (nuclear, smooth muscle, thyroid, etc.; see below), rheumatoid factor, and hyperglobulinemia are common; (3) other autoimmune disorders— such as thyroiditis, rheumatoid

1	(2) circulating autoantibodies (nuclear, smooth muscle, thyroid, etc.; see below), rheumatoid factor, and hyperglobulinemia are common; (3) other autoimmune disorders— such as thyroiditis, rheumatoid arthritis, autoimmune hemolytic anemia, ulcerative colitis, membranoproliferative glomerulonephritis, juvenile diabetes mellitus, celiac disease, and Sjögren’s syndrome— occur with increased frequency in patients and in their relatives who have autoimmune hepatitis; (4) histocompatibility haplotypes associated with autoimmune diseases, such as HLA-B1, -B8, -DR3, and -DR4 as well as extended haplotype DRB1*0301 and DRB1*0401 alleles, are common in patients with autoimmune hepatitis; and (5) this type of chronic hepatitis is responsive to glucocorticoid/immunosuppressive therapy, effective in a variety of autoimmune disorders.

1	Cellular immune mechanisms appear to be important in the pathogenesis of autoimmune hepatitis. In vitro studies have suggested that in patients with this disorder, CD4+ T lymphocytes are capable of becoming sensitized to hepatocyte membrane proteins and of destroying liver cells. Molecular mimicry by cross-reacting antigens that contain epitopes similar to liver antigens is postulated to activate these T cells, which infiltrate, and result in injury to, the liver. Abnormalities of immunoregulatory control over cytotoxic lymphocytes (impaired regulatory CD4+CD25+ T cell influences) may play a role as well. Studies of genetic predisposition to autoimmune hepatitis demonstrate that certain haplotypes are associated with the disorder, as enumerated above, 2050 as are polymorphisms in cytotoxic T lymphocyte antigens (CTLA-4) and tumor necrosis factor α (TNFA*2). The precise triggering factors, genetic influences, and cytotoxic and immunoregulatory mechanisms involved in this type of liver

1	T lymphocyte antigens (CTLA-4) and tumor necrosis factor α (TNFA*2). The precise triggering factors, genetic influences, and cytotoxic and immunoregulatory mechanisms involved in this type of liver injury remain incompletely defined. Intriguing clues into the pathogenesis of autoimmune hepatitis come from the observation that circulating autoantibodies are prevalent in patients with this disorder. Among the autoantibodies described in these patients are antibodies to nuclei (so-called antinuclear antibodies [ANAs], primarily in a homogeneous pattern) and smooth muscle (so-called anti-smooth-muscle antibodies, directed at actin, vimentin, and skeletin), antibodies to F-actin, antibodies to liver-kidney microsomes (anti-LKM, see below), antibodies to “soluble liver antigen” (directed against a uracil-guanine-adenine transfer RNA suppressor protein), antibodies to α-actinin, and antibodies to the liver-specific asialoglycoprotein receptor (or “hepatic lectin”) and other hepatocyte

1	against a uracil-guanine-adenine transfer RNA suppressor protein), antibodies to α-actinin, and antibodies to the liver-specific asialoglycoprotein receptor (or “hepatic lectin”) and other hepatocyte membrane proteins. Although some of these provide helpful diagnostic markers, their involvement in the pathogenesis of autoimmune hepatitis has not been established. Humoral immune mechanisms have been shown to play a role in the extrahepatic manifestations of autoimmune and idiopathic hepatitis. Arthralgias, arthritis, cutaneous vasculitis, and glomerulonephritis occurring in patients with autoimmune hepatitis appear to be mediated by the deposition of circulating immune complexes in affected tissue vessels, followed by complement activation, inflammation, and tissue injury. While specific viral antigen-antibody complexes can be identified in acute and chronic viral hepatitis, the nature of the immune complexes in autoimmune hepatitis has not been defined.

1	Many of the clinical features of autoimmune hepatitis are similar to those described for chronic viral hepatitis. The onset of disease may be insidious or abrupt; the disease may present initially like, and be confused with, acute viral hepatitis; a history of recurrent bouts of what had been labeled acute hepatitis is not uncommon. In approximately a quarter of patients, the diagnosis is made in the absence of symptoms, based on abnormal liver laboratory tests. A subset of patients with autoimmune hepatitis has distinct features. Such patients are predominantly young to middle-aged women with marked hyperglobulinemia and high-titer circulating ANAs. This is the group with positive lupus erythematosus (LE) preparations (initially labeled “lupoid “ hepatitis) in whom other autoimmune features are common. Fatigue, malaise, anorexia, amenorrhea, acne, arthralgias, and jaundice are common. Occasionally, arthritis, maculopapular eruptions (including cutaneous vasculitis), erythema nodosum,

1	are common. Fatigue, malaise, anorexia, amenorrhea, acne, arthralgias, and jaundice are common. Occasionally, arthritis, maculopapular eruptions (including cutaneous vasculitis), erythema nodosum, colitis, pleurisy, pericarditis, anemia, azotemia, and sicca syndrome (keratoconjunctivitis, xerostomia) occur. In some patients, complications of cirrhosis, such as ascites and edema (associated with portal hypertension and hypoalbuminemia), encephalopathy, hypersplenism, coagulopathy, or variceal bleeding may bring the patient to initial medical attention.

1	The course of autoimmune hepatitis may be variable. In patients with mild disease or limited histologic lesions (e.g., piecemeal necrosis without bridging), progression to cirrhosis is limited, but, even in this subset, clinical monitoring is important to identify progression; up to half left untreated can progress to cirrhosis over the course of 15 years. In North America, cirrhosis at presentation is more common in African Americans than in whites. In those with severe symptomatic autoimmune hepatitis (aminotransferase levels >10 times normal, marked hyperglobulinemia, “aggressive” histologic lesions—bridging necrosis or multilobular collapse, cirrhosis), the 6-month mortality without therapy may be as high as 40%. Such severe disease accounts for only 20% of cases; the natural history of milder disease is variable, often accentuated by spontaneous remissions and exacerbations. Especially poor prognostic signs include the presence histologically of multilobular collapse at the time

1	of milder disease is variable, often accentuated by spontaneous remissions and exacerbations. Especially poor prognostic signs include the presence histologically of multilobular collapse at the time of initial presentation and failure of serum bilirubin to improve after 2 weeks of therapy. Death may result from hepatic failure, hepatic coma, other complications of cirrhosis (e.g., variceal hemorrhage), and intercurrent infection. In patients with established cirrhosis, HCC may be a late complication (Chap. 111) but occurs less frequently than in cirrhosis associated with viral hepatitis.

1	Laboratory features of autoimmune hepatitis are similar to those seen in chronic viral hepatitis. Liver biochemical tests are invariably abnormal but may not correlate with the clinical severity or histopathologic features in individual cases. Many patients with autoimmune hepatitis have normal serum bilirubin, alkaline phosphatase, and globulin levels with only minimal aminotransferase elevations. Serum AST and ALT levels are increased and fluctuate in the range of 100−1000 units. In severe cases, the serum bilirubin level is moderately elevated (51−171 μmol/L [3−10 mg/dL]). Hypoalbuminemia occurs in patients with very active or advanced disease. Serum alkaline phosphatase levels may be moderately elevated or near normal. In a small proportion of patients, marked elevations of alkaline phosphatase activity occur; in such patients, clinical and laboratory features overlap with those of primary biliary cirrhosis (Chap. 365). The prothrombin time is often prolonged, particularly late in

1	activity occur; in such patients, clinical and laboratory features overlap with those of primary biliary cirrhosis (Chap. 365). The prothrombin time is often prolonged, particularly late in the disease or during active phases.

1	Hypergammaglobulinemia (>2.5 g/dL) is common in autoimmune hepatitis, as is the presence of rheumatoid factor. As noted above, circulating autoantibodies are also prevalent, most characteristically ANAs in a homogeneous staining pattern. Smooth-muscle antibodies are less specific, seen just as frequently in chronic viral hepatitis. Because of the high levels of globulins achieved in the circulation of some patients with autoimmune hepatitis, occasionally the globulins may bind nonspecifically in solid-phase binding immunoassays for viral antibodies. This has been recognized most commonly in tests for antibodies to hepatitis C virus, as noted above. In fact, studies of auto-antibodies in autoimmune hepatitis have led to the recognition of new categories of autoimmune hepatitis. Type I autoimmune hepatitis is the classic syndrome prevalent in North America and northern Europe occurring in young women, associated with marked hyperglobulinemia, lupoid features, circulating ANAs, and

1	autoimmune hepatitis is the classic syndrome prevalent in North America and northern Europe occurring in young women, associated with marked hyperglobulinemia, lupoid features, circulating ANAs, and HLA-DR3 or HLA-DR4 (especially B8-DRB1*03). Also associated with type I autoimmune hepatitis are autoantibodies against actin and atypical perinuclear antineutrophilic cytoplasmic antibodies (pANCA).

1	Type II autoimmune hepatitis, often seen in children, more common in Mediterranean populations, and linked to HLA-DRB1 and HLA-DQB1 haplotypes, is associated not with ANA but with anti-LKM. Actually, anti-LKM represent a heterogeneous group of antibodies. In type II autoimmune hepatitis, the antibody is anti-LKM1, directed against cytochrome P450 2D6. This is the same anti-LKM seen in some patients with chronic hepatitis C. Anti-LKM2 is seen in drug-induced hepatitis, and anti-LKM3 (directed against uridine diphosphate glucuronyltransferases) is seen in patients with chronic hepatitis D. Another autoantibody observed in type II autoimmune hepatitis is directed against liver cytosol formiminotransferase cyclodeaminase (anti-liver cytosol 1). More controversial is whether or not a third category of autoimmune hepatitis exists, type III autoimmune hepatitis. These patients lack ANA and anti-LKM1 but have circulating antibodies to soluble liver antigen. Most of these patients are women

1	of autoimmune hepatitis exists, type III autoimmune hepatitis. These patients lack ANA and anti-LKM1 but have circulating antibodies to soluble liver antigen. Most of these patients are women and have clinical features similar to, perhaps more severe than, those of patients with type I autoimmune hepatitis. Type III autoimmune hepatitis does not appear to represent a distinct category but, instead, is part of the spectrum of type I autoimmune hepatitis; this subcategory has not been adopted by a consensus of international experts.

1	Liver biopsy abnormalities are similar to those described for chronic viral hepatitis. Expanding portal tracts and extending beyond the plate of periportal hepatocytes into the parenchyma (designated interface hepatitis or piecemeal necrosis) is a mononuclear cell infiltrate that, in autoimmune hepatitis, may include the presence of plasma cells. Necroinflammatory activity characterizes the lobular parenchyma, and evidence of hepatocellular regeneration is reflected by “rosette” formation, the occurrence of thickened liver cell plates, and regenerative “pseudolobules.” Septal fibrosis, bridging fibrosis, and cirrhosis are frequent. In patients with early autoimmune hepatitis presenting as an acute-hepatitis-like illness, lobular and centrilobular (as opposed to the more common periportal) necrosis has been reported. Bile duct injury and granulomas are uncommon; however, a subgroup of patients with autoimmune hepatitis has histologic, biochemical, and serologic features overlapping

1	necrosis has been reported. Bile duct injury and granulomas are uncommon; however, a subgroup of patients with autoimmune hepatitis has histologic, biochemical, and serologic features overlapping those of primary biliary cirrhosis (Chap. 365).

1	An international group has suggested a set of criteria for establishing a diagnosis of autoimmune hepatitis. Exclusion of liver disease caused by genetic disorders, viral hepatitis, drug hepatotoxicity, and alcohol are linked with such inclusive diagnostic criteria as hyperglobulinemia, autoantibodies, and characteristic histologic features. This international group has also suggested a comprehensive diagnostic scoring system that, rarely required for typical cases, may be helpful when typical features are not present. Factors that weigh in favor of the diagnosis include female gender; predominant aminotransferase elevation; presence and level of globulin elevation; presence of nuclear, smooth muscle, LKM1, and other autoantibodies; concurrent other autoimmune diseases; characteristic histologic features (interface hepatitis, plasma cells, rosettes); HLA-DR3 or -DR4 markers; and response to treatment (see below). A more simplified, more specific scoring system relies on four

1	histologic features (interface hepatitis, plasma cells, rosettes); HLA-DR3 or -DR4 markers; and response to treatment (see below). A more simplified, more specific scoring system relies on four variables: autoantibodies, serum IgG level, typical or compatible histologic features, and absence of viral hepatitis markers. Weighing against the diagnosis are predominant alkaline phosphatase elevation, mitochondrial antibodies, markers of viral hepatitis, history of hepatotoxic drugs or excessive alcohol, histologic evidence of bile duct injury, or such atypical histologic features as fatty infiltration, iron overload, and viral inclusions.

1	Early during the course of chronic hepatitis, autoimmune hepatitis may resemble typical acute viral hepatitis (Chap. 360). Without histologic assessment, severe chronic hepatitis cannot be readily distinguished based on clinical or biochemical criteria from mild chronic hepatitis. In adolescence, Wilson’s disease (Chaps. 365 and 429) may present with features of chronic hepatitis long before neurologic manifestations become apparent and before the formation of Kayser-Fleischer rings (copper deposition in Descemet’s membrane in the periphery of the cornea). In this age group, serum ceruloplasmin and serum and urinary copper determinations plus measurement of liver copper levels establish the correct diagnosis. Postnecrotic or cryptogenic cirrhosis and primary biliary cirrhosis (Chap. 365) share clinical features with autoimmune hepatitis, and both alcoholic hepatitis (Chap. 363) and nonalcoholic steatohepatitis (Chap. 367e) may present with many features common to autoimmune hepatitis;

1	clinical features with autoimmune hepatitis, and both alcoholic hepatitis (Chap. 363) and nonalcoholic steatohepatitis (Chap. 367e) may present with many features common to autoimmune hepatitis; historic, biochemical, serologic, and histologic assessments are usually sufficient to allow these entities to be distinguished from autoimmune hepatitis. Of course, the distinction between autoimmune and chronic viral hepatitis is not always straightforward, especially when viral antibodies occur in patients with autoimmune disease or when autoantibodies occur in patients with viral disease. Furthermore, the presence of extrahepatic features such as arthritis, cutaneous vasculitis, or pleuritis—not to mention the presence of circulating autoantibodies—may cause confusion with rheumatologic disorders such as rheumatoid arthritis and systemic lupus erythematosus. The existence of clinical and biochemical features of progressive necroinflammatory liver disease distinguishes chronic hepatitis

1	such as rheumatoid arthritis and systemic lupus erythematosus. The existence of clinical and biochemical features of progressive necroinflammatory liver disease distinguishes chronic hepatitis from these other disorders, which are not associated with severe liver disease. Rarely, hepatic venous outflow obstruction (Budd-Chiari syndrome) may present with features suggestive of autoimmune hepatitis, but painful hepatomegaly, ascites, and vascular imaging provide distinguishing diagnostic clues. Other diagnostic considerations would include celiac disease and ischemic liver disease, which would be readily distinguishable by clinical and laboratory features from autoimmune hepatitis.

1	Finally, occasionally, features of autoimmune hepatitis overlap with features of autoimmune biliary disorders such as primary biliary cirrhosis, primary sclerosing cholangitis (Chaps. 365 and 369), or, even more rarely, mitochondrial antibody-negative autoimmune cholangitis. Such overlap syndromes are difficult to categorize, and often response to therapy may be the distinguishing factor that establishes the diagnosis.

1	The mainstay of management in autoimmune hepatitis is glucocorticoid therapy. Several controlled clinical trials have documented that such therapy leads to symptomatic, clinical, biochemical, and histologic improvement as well as increased survival. A therapeutic response can be expected in up to 80% of patients. Unfortunately, therapy has not been shown in clinical trials to prevent ultimate progression to cirrhosis; however, instances of reversal of fibrosis and cirrhosis have been reported in patients responding to treatment, and rapid treatment responses within 1 year do translate into a reduction in progression to cirrhosis. Although some advocate the use of prednisolone (the hepatic metabolite of prednisone), prednisone is just as effective and is favored by most authorities. Therapy may be initiated at 20 mg/d, but a popular regimen in the United States relies on an initiation dose of 60 mg/d. This high dose is tapered successively over the course of a month down to a

1	Therapy may be initiated at 20 mg/d, but a popular regimen in the United States relies on an initiation dose of 60 mg/d. This high dose is tapered successively over the course of a month down to a maintenance level of 20 mg/d. An alternative, but equally effective, approach is to begin with half the prednisone dose (30 mg/d) along with azathioprine (50 mg/d). With azathioprine maintained at 50 mg/d, the prednisone dose is tapered over the course of a month down to a maintenance level of 10 mg/d. The advantage of the combination approach is a reduction, over the span of an 18-month course of therapy, in serious, life-threatening complications of steroid therapy (e.g., cushingoid features, hypertension, diabetes, osteoporosis) from 66% down to under 20%. Genetic analysis for thiopurine S-methyltransferase allelic variants does not correlate with azathioprine-associated cytopenias or efficacy and is not assessed routinely in patients with autoimmune hepatitis. In combination regimens,

1	allelic variants does not correlate with azathioprine-associated cytopenias or efficacy and is not assessed routinely in patients with autoimmune hepatitis. In combination regimens, 6-mercaptopurine may be substituted for its prodrug azathioprine, but this is rarely required. Azathioprine alone, however, is not effective in achieving remission, nor is alternate-day glucocorticoid therapy. Limited experience with budesonide in noncirrhotic patients suggests that this steroid side effect−sparing drug may be effective. Although therapy has been shown to be effective for severe autoimmune hepatitis (AST ≥10 × the upper limit of normal or ≥5 × the upper limit of normal in conjunction with serum globulin greater than or equal to twice normal; bridging necrosis or multilobular necrosis on liver biopsy; presence of symptoms), therapy is not indicated for mild forms of chronic hepatitis, and the efficacy of therapy in mild or asymptomatic autoimmune hepatitis has not been established.

1	Improvement of fatigue, anorexia, malaise, and jaundice tends to occur within days to several weeks; biochemical improvement occurs over the course of several weeks to months, with a fall in serum bilirubin and globulin levels and an increase in serum albumin. Serum aminotransferase levels usually drop promptly, but improvements in AST and ALT alone do not appear to be reliable markers of recovery in individual patients; histologic improvement, characterized by a decrease in mononuclear infiltration and in hepatocellular necrosis, may be delayed for 6−24 months. Still, if interpreted cautiously, aminotransferase levels are valuable indicators of relative disease activity, and many authorities do not advocate for serial liver biopsies to assess therapeutic success or to guide decisions to alter or stop therapy. Rapidity of response is more common in older patients (≥69 years) and those with HLA DBR1*04; although rapid responders may progress less slowly to cirrhosis and liver

1	to alter or stop therapy. Rapidity of response is more common in older patients (≥69 years) and those with HLA DBR1*04; although rapid responders may progress less slowly to cirrhosis and liver transplantation, they are no less likely than slower responders to relapse after therapy. Therapy should continue for at least 12−18 months. After tapering and cessation of therapy, the likelihood of relapse is at least 50%, even if posttreatment histology has improved to show mild chronic hepatitis, and the majority of patients require therapy at maintenance doses indefinitely. Continuing azathioprine alone (2 mg/kg body weight daily) after cessation of prednisone therapy has been shown to reduce the frequency of relapse. Long-term maintenance with low-dose prednisone (≤10 mg daily) has also been shown to keep autoimmune hepatitis in check, but maintenance azathioprine is more effective in maintaining remission.

1	2052 In medically refractory cases, an attempt should be made to intensify treatment with high-dose glucocorticoid monotherapy (60 mg daily) or combination glucocorticoid (30 mg daily) plus high-dose azathioprine (150 mg daily) therapy. After a month, doses of prednisone can be reduced by 10 mg a month, and doses of azathioprine can be reduced by 50 mg a month toward ultimate, conventional maintenance doses. Patients refractory to this regimen may be treated with cyclosporine, tacrolimus, or mycophenolate mofetil; however, to date, only limited anecdotal reports support these approaches. If medical therapy fails, or when chronic hepatitis progresses to cirrhosis and is associated with life-threatening complications of liver decompensation, liver transplantation is the only recourse (Chap. 368); failure of the bilirubin to improve after 2 weeks of therapy should prompt early consideration of the patient for liver transplantation. Recurrence of autoimmune hepatitis in the new liver

1	368); failure of the bilirubin to improve after 2 weeks of therapy should prompt early consideration of the patient for liver transplantation. Recurrence of autoimmune hepatitis in the new liver occurs rarely in most experiences but in as many as 35−40% of cases in others. Like all patients with chronic liver disease, patients with autoimmune hepatitis should be vaccinated against hepatitis A and B, ideally before immunosuppressive therapy is begun, if practical.

1	Disorders of the Gastrointestinal System Kurt J. Isselbacher, MD, contributed to this chapter in previous editions of Harrison’s. Mark E. Mailliard, Michael F. Sorrell

1	Chronic and excessive alcohol ingestion is one of the major causes of liver disease. The pathology of alcoholic liver disease consists of three major lesions, with the progressive injury rarely existing in a pure form: (1) fatty liver, (2) alcoholic hepatitis, and (3) cirrhosis. Fatty liver is present in >90% of daily as well as binge drinkers. A much smaller percentage of heavy drinkers will progress to alcoholic hepatitis, thought to be a precursor to cirrhosis. The prognosis of severe alcoholic liver disease is dismal; the mortality of patients with alcoholic hepatitis concurrent with cirrhosis is nearly 60% at 4 years. Although alcohol is considered a direct hepatotoxin, only between 10 and 20% of alcoholics will develop alcoholic hepatitis. The explanation for this apparent paradox is unclear but involves the complex interaction of facilitating factors, such as drinking patterns, diet, obesity, and gender. There are no diagnostic tools that can predict individual susceptibility

1	is unclear but involves the complex interaction of facilitating factors, such as drinking patterns, diet, obesity, and gender. There are no diagnostic tools that can predict individual susceptibility to alcoholic liver disease.

1	Alcohol is the world’s third largest risk factor for disease bur den. The harmful use of alcohol results in 2.5 million deaths each year. Most of the mortality attributed to alcohol is secondary to cirrhosis. Mortality from cirrhosis is declining in most Western countries, concurrent with a reduction in alcohol consumption, with the exceptions of the United Kingdom, Russia, Romania, and Hungary. These increases in cirrhosis and its complications are closely correlated with increased volume of alcohol consumed per capita population and are regardless of gender.

1	Quantity and duration of alcohol intake are the most important risk factors involved in the development of alcoholic liver disease (Table 363-1). The roles of beverage type(s), i.e. wine, beer, or spirits, and pattern of drinking (daily versus binge drinking) are less clear. Progress beyond the fatty liver stage seems to require additional risk factors that remain incompletely defined. Although there are genetic predispositions for alcoholism (Chap. 467), gender is a strong determinant for alcoholic liver disease. Women are more susceptible to alcoholic liver injury when compared to men. They develop advanced liver disease with substantially less alcohol intake. In general, the time it takes to develop liver disease is directly related to the amount of alcohol consumed. It is useful in estimating alcohol consumption to understand that one beer, four ounces of wine, or one ounce of 80% spirits all contain ∼12 g of alcohol. The threshold for developing alcoholic liver disease is higher

1	alcohol consumption to understand that one beer, four ounces of wine, or one ounce of 80% spirits all contain ∼12 g of alcohol. The threshold for developing alcoholic liver disease is higher in men, while women are at increased risk for developing similar degrees of liver injury by consuming significantly less. Gender-dependent differences result from poorly understood effects of estrogen, proportion of body fat, and the gastric metabolism of alcohol. Obesity, a high-fat diet, and the protective effect of coffee have been postulated to play a part in the development of the pathogenic process.

1	Chronic infection with hepatitis C virus (HCV) (Chap. 362) is an important comorbidity in the progression of alcoholic liver disease to cirrhosis in chronic and excessive drinkers. Even moderate alcohol intake of 20–50 g/d increases the risk of cirrhosis and hepatocellular cancer in HCV-infected individuals. Patients with both alcoholic liver injury and HCV infection develop decompensated liver disease at a younger age and have poorer overall survival. Increased liver iron stores and, rarely, porphyria cutanea tarda can occur as a consequence of the overlapping injurious processes secondary to alcohol abuse and HCV infection. In addition, alcohol intake of >50 g/d by HCV-infected patients decreases the efficacy of interferon-based antiviral therapy.

1	The pathogenesis of alcoholic liver injury is unclear. The present conceptual foundation is that alcohol acts as a direct hepatotoxin and that malnutrition does not have a major role. Ingestion of alcohol initiates an inflammatory cascade by its metabolism to acetaldehyde, resulting in a variety of metabolic responses. Steatosis from lipogenesis, fatty acid synthesis, and depression of fatty acid oxidation appears secondary to effects on sterol regulatory transcription factor and peroxisome proliferator-activated receptor α (PPAR-α). Intestinal-derived endotoxin initiates a pathogenic process through toll-like receptor 4 and tumor necrosis factor α (TNF-α) that facilitates hepatocyte apoptosis and necrosis. The cell injury and endotoxin release initiated by ethanol and its metabolites also activate innate and adaptive immunity pathways releasing proinflammatory cytokines (e.g., TNF-α), chemokines, and proliferation of T and B cells. The production of toxic protein-aldehyde adducts,

1	activate innate and adaptive immunity pathways releasing proinflammatory cytokines (e.g., TNF-α), chemokines, and proliferation of T and B cells. The production of toxic protein-aldehyde adducts, generation of reducing equivalents, and oxidative stress also contribute to the liver injury. Hepatocyte injury and impaired regeneration following chronic alcohol ingestion are ultimately associated with stellate cell activation and collagen production, which are key events in fibrogenesis. The resulting fibrosis from continuing alcohol use determines the architectural derangement of the liver and associated pathophysiology.

1	The liver has a limited repertoire in response to injury. Fatty liver is the initial and most common histologic response to hepatotoxic stimuli, including excessive alcohol ingestion. The accumulation of fat within the perivenular hepatocytes coincides with the location of alcohol dehydrogenase, the major enzyme responsible for alcohol metabolism. Continuing alcohol ingestion results in fat accumulation throughout the entire hepatic lobule. Despite extensive fatty change and distortion of the hepatocytes with macrovesicular fat, the cessation of drinking results in normalization of hepatic architecture and fat content. Alcoholic fatty liver has traditionally been regarded as entirely benign, but similar to the spectrum of nonalcoholic fatty liver disease (Chap. 367e), the appearance of steatohepatitis and certain pathologic features such as giant mitochondria, perivenular fibrosis, and macrovesicular fat may be associated with progressive liver injury.

1	The transition between fatty liver and the development of alcoholic hepatitis is blurred. The hallmark of alcoholic hepatitis is hepatocyte injury characterized by ballooning degeneration, spotty necrosis, polymorphonuclear infiltrate, and fibrosis in the perivenular and perisinusoidal space of Disse. Mallory-Denk bodies are often present in florid cases but are neither specific nor necessary to establish the diagnosis. Alcoholic hepatitis is thought to be a precursor to the development of cirrhosis. However, like fatty liver, it is potentially reversible with cessation of drinking. Cirrhosis is present in up to 50% of patients with biopsy-proven alcoholic hepatitis, and its regression is uncertain, even with abstention.

1	The clinical manifestations of alcoholic fatty liver are subtle and characteristically detected as a consequence of the patient’s visit for a seemingly unrelated matter. Previously unsuspected hepatomegaly is often the only clinical finding. Occasionally, patients with fatty liver will present with right upper quadrant discomfort, nausea, and, rarely, jaundice. Differentiation of alcoholic fatty liver from nonalcoholic fatty liver is difficult unless an accurate drinking history is ascertained. In every instance where liver disease is present, a thoughtful and sensitive drinking history should be obtained. Standard, validated questions accurately detect alcohol-related problems (Chap. 467). Alcoholic hepatitis is associated with a wide gamut of clinical features. Fever, spider nevi, jaundice, and abdominal pain simulating an acute abdomen represent the extreme end of the spectrum, while many patients will be entirely asymptomatic. Portal hypertension, ascites, or variceal bleeding can

1	and abdominal pain simulating an acute abdomen represent the extreme end of the spectrum, while many patients will be entirely asymptomatic. Portal hypertension, ascites, or variceal bleeding can occur in the absence of cirrhosis. Recognition of the clinical features of alcoholic hepatitis is central to the initiation of an effective and appropriate diagnostic and therapeutic strategy. It is important to recognize that patients with alcoholic cirrhosis often exhibit clinical features identical to other causes of cirrhosis.

1	Patients with alcoholic liver disease are often identified through routine screening tests. The typical laboratory abnormalities seen in fatty liver are nonspecific and include modest elevations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and γ-glutamyl transpeptidase (GGTP), often accompanied by hypertriglyceridemia and hyperbilirubinemia. In alcoholic hepatitis and in contrast to other causes of fatty liver, AST and ALT are usually elevated twoto sevenfold. They are rarely >400 IU, and the AST/ALT ratio is >1 (Table 363-2). Hyperbilirubinemia is accompanied by modest increases in the alkaline phosphatase level. Derangement in hepatocyte synthetic function indicates more serious disease. Hypoalbuminemia and coagulopathy are common in advanced liver injury. Ultrasonography is useful in detecting fatty infiltration of the liver and determining liver size. The demonstration by ultrasound of portal vein flow reversal, ascites, and intraabdominal venous

1	Ultrasonography is useful in detecting fatty infiltration of the liver and determining liver size. The demonstration by ultrasound of portal vein flow reversal, ascites, and intraabdominal venous collaterals indicates serious liver injury with less potential for complete reversal.

1	AST Increased twoto sevenfold, <400 IU/L, greater than ALT ALT Increased twoto sevenfold, <400 IU/L GGTP Not specific to alcohol, easily inducible, elevated in all forms of fatty liver Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGTP, γ-glutamyl transpeptidase.

1	Critically ill patients with alcoholic hepatitis have short-term (30-day) mortality rates >50%. Severe alcoholic hepatitis is heralded by coagulopathy (prothrombin time increased >5 s), anemia, serum albumin concentrations <25 g/L (2.5 mg/dL), serum bilirubin levels >137 μmol/L (8 mg/dL), renal failure, and ascites. A discriminant function calculated as 4.6 X (the prolongation of the prothrombin time above control [seconds]) + serum bilirubin (mg/dL) can identify patients with a poor prognosis (discriminant function >32). A Model for End-Stage Liver Disease (MELD) score (Chap. 368) ≥21 also is associated with significant mortality in alcoholic hepatitis. The presence of ascites, variceal hemorrhage, deep encephalopathy, or hepatorenal syndrome predicts a dismal prognosis. The pathologic stage of the injury can be helpful in predicting prognosis. Liver biopsy should be performed whenever possible to establish the diagnosis and to guide the therapeutic decisions.

1	Complete abstinence from alcohol is the cornerstone in the treatment of alcoholic liver disease. Improved survival and the potential for reversal of histologic injury regardless of the initial clinical presentation are associated with total avoidance of alcohol ingestion. Referral of patients to experienced alcohol counselors and/or alcohol treatment programs should be routine in the management of patients with alcoholic liver disease. Attention should be directed to the nutritional and psychosocial states during the evaluation and treatment periods. Because of data suggesting that the pathogenic mechanisms in alcoholic hepatitis involve cytokine release and the perpetuation of injury by immunologic processes, glucocorticoids have been extensively evaluated in the treatment of alcoholic hepatitis. Patients with severe alcoholic hepatitis, defined as a discriminant function >32 or MELD >20, should be given prednisone, 40 mg/d, or prednisolone, 32 mg/d, for 4 weeks, followed by a

1	hepatitis. Patients with severe alcoholic hepatitis, defined as a discriminant function >32 or MELD >20, should be given prednisone, 40 mg/d, or prednisolone, 32 mg/d, for 4 weeks, followed by a steroid taper (Fig. 363-1). Exclusion criteria include active gastrointestinal bleeding, renal failure, or pancreatitis. Women with encephalopathy from severe alcoholic hepatitis may be particularly good candidates for glucocorticoids. A Lille score >0.45, at http://www.lillemodel.com, uses pretreatment variables plus the change in total bilirubin at day 7 of glucocorticoids to identify patients unresponsive to therapy.

1	The role of TNF-α expression and receptor activity in alcoholic liver injury has led to an examination of TNF inhibition as an alternative to glucocorticoids for severe alcoholic hepatitis. The nonspecific TNF inhibitor, pentoxifylline, demonstrated improved survival in the therapy of severe alcoholic hepatitis, primarily due to a decrease in hepatorenal syndrome (Fig. 363-2). Monoclonal antibodies that neutralize serum TNF-α should not be used in alcoholic hepatitis because of studies reporting increased deaths secondary to infection and renal failure.

1	Liver transplantation is an accepted indication for treatment in selected and motivated patients with end-stage cirrhosis. Outcomes are equal or superior to other indications for transplantation. In general, transplant candidacy should be reevaluated after a defined 0 7 14 21 84.6 3.4% p = .001 65.1 4.8% 28 nonalcoholic fatty liver Diseases and nonalcoholic Steatohepatitis Manal F. Abdelmalek, Anna Mae Diehl INCIDENCE, PREVALENCE, AND NATURAL HISTORY Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in many parts of the world, including the United States. 364

1	FIGURE 363-1 Effect of glucocorticoid therapy of severe alcoholic hepatitis on short-term survival: the result of a meta-analysis of individual data from three studies. Prednisolone, solid line; placebo, dotted line. (Adapted from P Mathurin et al: J Hepatol 36:480, 2002, with permission from Elsevier Science.) period of sobriety. Patients presenting with alcoholic hepatitis have been largely excluded from transplant candidacy because of the perceived risk of increased surgical mortality and high rates of recidivism following transplantation. Recently, a European multidisciplinary group has reported excellent long-term transplant outcomes in highly selected patients with florid alcoholic hepatitis. General application of transplantation in such patients must await confirmatory outcomes by others. Cumulative survival, %

1	Cumulative survival, % Alcoholic Hepatitis Alcohol abstinence Nutritional support Treatment options Preferred Alternative Discriminant function ˜ 32 or MELD ˜ 21 (with absence of co-morbidity) Prednisolone 32 mg p.o. daily for 4 weeks, then taper for 4 weeks Pentoxifylline 400 mg p.o. TID for 4 weeks FIGURE 363-2 Treatment algorithm for alcoholic hepatitis. As identified by a calculated discriminant function >32 (see text), patients with severe alcoholic hepatitis, without the presence of gastrointestinal bleeding or infection, would be candidates for either glucocorticoids or pentoxifylline administration.

1	Population-based abdominal imaging studies have demonstrated fatty liver in at least 25% of American adults. Because the vast majority of these subjects deny hazardous levels of alcohol consumption (defined as greater than one drink per day in women or two drinks per day in men), they are considered to have NAFLD. NAFLD is strongly associated with overweight/obesity and insulin resistance. However, it can also occur in lean individuals and is particularly common in those with a paucity of adipose depots (i.e., lipodystrophy). Ethnic/racial factors also appear to influence liver fat accumulation; the documented prevalence of NAFLD is lowest in African Americans (~25%), highest in Americans of Hispanic ancestry (~50%), and intermediate in American whites (~33%).

1	NAFLD encompasses a spectrum of liver pathology with different clinical prognoses. The simple accumulation of triglyceride within hepatocytes (hepatic steatosis) is on the most clinically benign extreme of the spectrum. On the opposite, most clinically ominous extreme, are cirrhosis (Chap. 365) and primary liver cancer (Chap. 111). The risk of developing cirrhosis is extremely low in individuals with chronic hepatic steatosis, but increases as steatosis becomes complicated by histologically conspicuous hepatocyte death and inflammation (i.e., nonalcoholic steatohepatitis [NASH]). NASH itself is also a heterogeneous condition; sometimes it improves to steatosis or normal histology, sometimes it remains relatively stable for years, but sometimes it results in progressive accumulation of fibrous scar that eventuates in cirrhosis. Once NAFLD-related cirrhosis develops, the annual incidence of primary liver cancer is 1%.

1	Abdominal imaging is not able to determine which individuals with NAFLD have associated liver cell death and inflammation (i.e., NASH), and specific blood tests to diagnose NASH are not yet available. However, population-based studies that have used elevated serum ALT as a marker of liver injury indicate that about 6–8% of American adults have serum ALT elevations that cannot be explained by excessive alcohol consumption, other known causes of fatty liver disease (Table 364-1), viral hepatitis, or drug-induced or congenital liver diseases. Because the prevalence of such “cryptogenic” ALT elevations increases with body mass index, it is presumed that they are due to NASH. Hence, at any given point in time, NASH is present in about 25% of individuals who have NAFLD (i.e., about 6% of the general

1	U.S. adult population has NASH). Smaller cross-sectional studies in which liver biopsies have been performed on NASH patients at tertiary referral centers consistently demonstrate advanced fibrosis or cirrhosis in about 25% of those cohorts. By extrapolation, therefore, cirrhosis develops in about 6% of individuals with NAFLD (i.e., in about 1.5–2% of the general U.S. population). The risk for advanced liver fibrosis is highest in individuals with NASH who are older than 45–50 years of age and overweight/obese or afflicted with type 2 diabetes.

1	To put these data in perspective, it is helpful to recall that the prevalence of hepatitis C–related cirrhosis in the United States is about 0.5%. Thus, NAFLD-related cirrhosis is about three to four times more common than cirrhosis caused by chronic hepatitis C infection. Consistent with these data, experts have predicted that NAFLD will surpass hepatitis C as the leading indication for liver transplantation in the United States within the next decade. Similar to cirrhosis caused by other liver diseases, cirrhosis caused by NAFLD increases the risk for primary liver cancer. Both hepatocellular carcinoma and intrahepatic cholangiocarcinoma (ICC) have also been reported to occur in NAFLD patients without cirrhosis, suggesting that NAFLD per se may be a premalignant condition. NAFLD, NASH, and NAFLD-related cirrhosis are not limited to adults. All have been well documented in children. As in adults, obesity and insulin resistance are the main risk

1	AlTERnATiVE CAuSES of HEPATiC STEAToSiS errors of metabolism exposure to petrochemical fatty liver of pregnancy syndrome (hemolytic anemia, elevated liver enzymes, low platelet count) factors for pediatric NAFLD. Thus, the rising incidence and prevalence of childhood obesity suggests that NAFLD is likely to become an even greater contributor to society’s burden of liver disease in the future.

1	The mechanisms underlying the pathogenesis and progression of NAFLD are not entirely clear. The best-understood mechanisms pertain to hepatic steatosis. This is proven to result when hepatocyte mechanisms for triglyceride synthesis (e.g., lipid uptake and de novo lipogenesis) overwhelm mechanisms for triglyceride disposal (e.g., degradative metabolism and lipoprotein export), leading to accumulation of fat (i.e., triglyceride) within hepatocytes. Obesity stimulates hepatocyte triglyceride accumulation by altering the intestinal microbiota to enhance both energy harvest from dietary sources and intestinal permeability. Reduced intestinal barrier function increases hepatic exposure to gut-derived products, which stimulate liver cells to generate inflammatory mediators that inhibit insulin actions. Obese adipose depots also produce excessive soluble factors (adipokines) that inhibit tissue insulin sensitivity. Insulin resistance promotes hyperglycemia. This drives the pancreas to produce

1	Obese adipose depots also produce excessive soluble factors (adipokines) that inhibit tissue insulin sensitivity. Insulin resistance promotes hyperglycemia. This drives the pancreas to produce more insulin to maintain glucose homeostasis. However, hyperinsulinemia also promotes lipid uptake, fat synthesis, and fat storage. The net result is hepatic triglyceride accumulation (i.e., steatosis).

1	Triglyceride per se is not hepatotoxic. However, its precursors (e.g., fatty acids and diacylglycerols) and metabolic by-products (e.g., reactive oxygen species) may damage hepatocytes, leading to hepatocyte lipotoxicity. Lipotoxicity also triggers the generation of other factors (e.g., inflammatory cytokines, hormonal mediators) that deregulate systems that normally maintain hepatocyte viability. The net result is increased hepatocyte death. Dying hepatocytes, in turn, release various factors that trigger wound healing responses that aim to replace (regenerate) lost hepatocytes. Such repair involves transient 2055 expansion of other cell types, such as myofibroblasts and progenitor cells, that make and degrade matrix, remodel the vasculature, and generate replacement hepatocytes, as well as the recruitment of immune cells that release factors that modulate liver injury and repair. NASH is the morphologic manifestation of lipotoxicity and resultant wound healing responses. Because the

1	as the recruitment of immune cells that release factors that modulate liver injury and repair. NASH is the morphologic manifestation of lipotoxicity and resultant wound healing responses. Because the severity and duration of lipotoxic liver injury dictate the intensity and duration of repair, the histologic features and outcomes of NASH are variable. Cirrhosis and liver cancer are potential outcomes of chronic NASH. Cirrhosis results from futile repair, i.e., progressive accumulation of wound healing cells, fibrous matrix, and abnormal vasculature (scarring), rather than efficient reconstruction/regeneration of healthy hepatic parenchyma. Primary liver cancers develop when malignantly transformed liver cells escape mechanisms that normally control regenerative growth. The mechanisms responsible for futile repair (cirrhosis) and liver carcinogenesis are not well understood. Because normal liver regeneration is a very complex process, there are multiple opportunities for deregulation

1	for futile repair (cirrhosis) and liver carcinogenesis are not well understood. Because normal liver regeneration is a very complex process, there are multiple opportunities for deregulation and, thus, pathogenic heterogeneity. To date, this heterogeneity has confounded development of both diagnostic tests and treatments for defective/deregulated liver repair (i.e., cirrhosis and cancer). Hence, current strategies focus on circumventing misrepair by preventing and/or reducing lipotoxic liver injury.

1	Diagnosing NAFLD requires demonstration of increased liver fat in the absence of hazardous levels of alcohol consumption. Thresholds for potentially dangerous alcohol ingestion have been set at more than one drink per day in women and two drinks per day in men based on epidemiologic evidence that the prevalence of serum aminotransferase elevations increases when alcohol consumption habitually exceeds these levels. In those studies, one drink was defined as having 10 g of ethanol and, thus, is equivalent to one can of beer, 4 ounces of wine, or 1.5 ounces (one shot) of distilled spirits. Other causes of liver fat accumulation (particularly exposure to certain drugs; Table 364-2) and liver injury (e.g., viral hepatitis, autoimmune liver disease, iron or copper overload, α1 antitrypsin deficiency) must also be excluded. Thus, establishing the diagnosis of NAFLD does not require invasive testing: it can be accomplished by history and physical examination, liver imaging (ultrasound is an

1	must also be excluded. Thus, establishing the diagnosis of NAFLD does not require invasive testing: it can be accomplished by history and physical examination, liver imaging (ultrasound is an acceptable first-line test; computed tomography [CT] or magnetic resonance imaging [MRI] enhances sensitivity for liver fat detection but adds expense), and blood tests to exclude other liver diseases. It is important to emphasize that the liver may not be enlarged, and serum aminotransferases and liver function tests (e.g., bilirubin, albumin, prothrombin time) may be completely normal, in individuals with NAFLD. Because there is yet no one specific blood test for NAFLD, confidence in the diagnosis of NAFLD is increased by identification of NAFLD risk factors. The latter include increased body mass index, insulin resistance/type 2 diabetes mellitus, and other parameters indicative of the metabolic syndrome (e.g., systemic hypertension, dyslipidemia, hyperuricemia/gout, cardiovascular disease;

1	index, insulin resistance/type 2 diabetes mellitus, and other parameters indicative of the metabolic syndrome (e.g., systemic hypertension, dyslipidemia, hyperuricemia/gout, cardiovascular disease; Chap. 422) in the patient or family members.

1	Establishing the severity of NAFLD-related liver injury and related scarring (i.e., staging NAFLD) is more difficult than simply diagnosing NAFLD. Staging is critically important, however, because it is necessary to define prognosis and thereby determine treatment recommendations. The goal of staging is to distinguish patients with NASH from those with simple steatosis and to identify which of the NASH patients have advanced fibrosis. The 10-year probability of developing liver-related morbidity or mortality in steatosis is negligible, and hence, this subgroup of NAFLD patients tends to be managed conservatively (see below). In contrast, more intensive follow-up and therapy are justified in NASH patients, and the subgroup with advanced fibrosis merits the most intensive scrutiny and intervention because their 10-year risk of liver-related morbidity and mortality is clearly increased.

1	Staging approaches can be separated into noninvasive testing (i.e., blood testing, physical examination, and imaging) and invasive approaches (i.e., liver biopsy). Blood test evidence of hepatic dysfunction (e.g., hyperbilirubinemia, hypoalbuminemia, prothrombin time earths of low atomic number • 4,4’-Diethylaminoethoxyhexesterol prolongation) or portal hypertension (e.g., thrombocytopenia) and stigmata of portal hypertension on physical examination (e.g., spider angiomata, palmar erythema, splenomegaly, ascites, clubbing, encephalopathy) suggest a diagnosis of advanced NAFLD. Currently, however, liver biopsy is the gold standard for establishing the severity of liver injury and fibrosis because it is both more sensitive and specific than these other tests for establishing NAFLD severity. Although invasive, liver biopsy is seldom complicated by serious adverse sequelae such as significant bleeding, pain, or inadvertent puncture of other organs and thus is relatively safe. However,

1	Although invasive, liver biopsy is seldom complicated by serious adverse sequelae such as significant bleeding, pain, or inadvertent puncture of other organs and thus is relatively safe. However, biopsy suffers from potential sampling error unless tissue cores of 2 cm or longer are acquired. Also, examination of tissue at a single point in time is not reliable for determining whether the pathologic processes are progressing or regressing. The risk of serial liver biopsies within short time intervals is generally deemed as unacceptable outside of research studies. These limitations of liver biopsy have stimulated efforts to develop noninvasive approaches to stage NAFLD. As is true for many other types of chronic liver disease, in NAFLD the levels of serum aminotransferases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) do not reliably reflect the severity of liver cell injury, extent of liver cell death, or related liver inflammation and fibrosis. Thus, they are

1	[AST] and alanine aminotransferase [ALT]) do not reliably reflect the severity of liver cell injury, extent of liver cell death, or related liver inflammation and fibrosis. Thus, they are imperfect for determining which individuals with NAFLD have NASH. This has stimulated research to identify superior markers of liver injury. Serum levels of keratin 8 and keratin 18 appear to be promising surrogates. Keratins 8 and 18 (K8/18) are epithelial cytoskeletal proteins that undergo cleavage during programmed cell death (apoptosis). Both cleaved and full-length K8/18 are released into the blood as hepatocytes die, and studies suggest that serum levels of K8/18 differentiate individuals with NASH from those with simple steatosis or normal livers more reliably than do serum aminotransferase levels. Moreover, K8/18 levels appear to parallel the severity of liver fibrosis, with higher levels marking individuals who are likely to have worse scarring (i.e., advanced liver fibrosis or cirrhosis).

1	Moreover, K8/18 levels appear to parallel the severity of liver fibrosis, with higher levels marking individuals who are likely to have worse scarring (i.e., advanced liver fibrosis or cirrhosis). While promising, testing for K8/18 has not yet become standard clinical practice. Other blood tests and imaging approaches that quantify liver fibrosis are also being developed. Recently, the U.S. Food and Drug Administration (FDA) approved an ultrasound-based test that measures liver stiffness as a surrogate marker of fibrosis (FibroScan®) (Chap. 358). This new tool will likely be used serially to monitor fibrosis progression and regression in NAFLD patients. Studies that compare the receiver operator characteristics of K8/18 plus FibroScan® versus liver biopsy for monitoring NAFLD evolution are forthcoming.

1	Most subjects with NAFLD are asymptomatic. The diagnosis is often made when abnormal liver aminotransferases or features of fatty liver are noted during an evaluation performed for other reasons. NAFLD may also be diagnosed during the workup of vague right upper quadrant abdominal pain, hepatomegaly, or an abnormal-appearing liver at time of abdominal surgery. Obesity is present in 50–90% of subjects. Most patients with NAFLD also have other features of the metabolic syndrome (Chap. 422). Some have subtle stigmata of chronic liver disease, such as spider angiomata, palmer erythema, or splenomegaly. In a small minority of patients with advanced NAFLD, complications of end-stage liver disease (e.g., jaundice, features of portal hypertension such as ascites or variceal hemorrhage) may be the initial findings.

1	The association of NAFLD with obesity, diabetes, hypertriglyceridemia, hypertension, and cardiovascular disease is well known. Other associations include chronic fatigue, mood alterations, obstructive sleep apnea, thyroid dysfunction, and chronic pain syndrome. NAFLD is an independent risk factor for metabolic syndrome (Chap. 422). Longitudinal studies suggest that patients with NASH are at twoto threefold increased risk for the development of metabolic syndrome. Similarly, studies have shown that patients with NASH have a higher risk for the development of hypertension and diabetes mellitus. The presence of NAFLD is also independently associated with endothelial dysfunction, increased carotid intimal thickness, and the number of plaques in carotid and coronary arteries. Such data indicate that NAFLD has many deleterious effects on health in general.

1	Treatment of NAFLD can be divided into three components: (1) specific therapy of NAFLD-related liver disease; (2) treatment of NAFLD-associated comorbidities; and (3) treatment of the complications of advanced NAFLD. The subsequent discussion focuses on specific therapies for NAFLD, with some mention of their impact on major NAFLD comorbidities (insulin resistance/diabetes, obesity, and dyslipidemia). Treatment of the complications of advanced NAFLD involves management of the complications of cirrhosis and portal hypertension, including primary liver cancers. Approaches to accomplish these objectives are similar to those used in other chronic liver diseases and are covered elsewhere in the textbook (Chaps. 365 and 111).

1	At present, there are no FDA-approved therapies for the treatment of NAFLD. Thus, the current approach to NAFLD management focuses on treatment to improve the risk factors for NASH (i.e., obesity, insulin resistance, metabolic syndrome, dyslipidemia). Based on our understanding of the natural history of NAFLD, only patients with NASH or those with features of hepatic fibrosis on liver biopsy are considered currently for targeted pharmacologic therapies. This approach may change as our understanding of disease pathophysiology improves and potential targets of therapy evolve.

1	Diet and Exercise Lifestyle changes and dietary modification are the foundation for NAFLD treatment. Many studies indicate that lifestyle modification can improve serum aminotransferases and hepatic steatosis, with loss of at least 3–5% of body weight improving steatosis, but greater weight loss (up to 10%) necessary to improve steatohepatitis. The benefits of different dietary macronutrient contents (e.g., low-carbohydrate vs low-fat diets, saturated vs unsaturated fat diets) and different intensities of calorie restriction appear to be comparable. In adults with NAFLD, exercise regimens that improve fitness may be sufficient to reduce hepatic steatosis, but their impact on other aspects of liver histology remains unknown. Unfortunately, most NAFLD patients are unable to achieve sustained weight loss. Although pharmacologic therapies such as orlistat, topiramate, and phentermine to facilitate weight loss are available, their role in the treatment of NAFLD remains experimental.

1	Pharmacologic Therapies Several drug therapies have been tried in both research and clinical settings. No agent has yet been approved by the FDA for the treatment of NAFLD. Hence, this remains an area of active research. Because NAFLD is strongly associated with the metabolic syndrome and type 2 diabetes (Chaps. 417 and 418), the efficacy of various insulin-sensitizing agents has been examined. Metformin, an agent that mainly improves hepatic insulin sensitivity, has been evaluated in several small, open-label studies in adults and a recent larger, prospectively randomized trial in children (dubbed the TONIC study). Although several of the adult NASH studies suggested improvements in aminotransferases and/or liver histology, metformin did not improve liver histology in the TONIC study of children with NASH. Thus, it is not currently recommended as a treatment for NASH. Uncontrolled open-label studies have also investigated thiazolidinediones (pioglitazone and rosiglitazone) in adults

1	children with NASH. Thus, it is not currently recommended as a treatment for NASH. Uncontrolled open-label studies have also investigated thiazolidinediones (pioglitazone and rosiglitazone) in adults with NASH. This class of drugs is known to improve systemic insulin resistance. Both pioglitazone and rosiglitazone reduced aminotransferases and improved some of the histologic features of NASH in small, uncontrolled studies. A large, National Institutes of Health–sponsored, randomized placebo-controlled clinical trial, the PIVENs Study (Pioglitazone vs Vitamin E vs Placebo for the Treatment of 247 Nondiabetic Adults with NASH), demonstrated that resolution of histologic NASH occurred more often in subjects treated with pioglitazone (30 mg/d) than with placebo for 18 months (47 vs 21%, p = .001). However, many subjects in the pioglitazone group gained weight, and liver fibrosis did not improve. Also, it should be noted that the longterm safety and efficacy of thiazolidinediones in

1	p = .001). However, many subjects in the pioglitazone group gained weight, and liver fibrosis did not improve. Also, it should be noted that the longterm safety and efficacy of thiazolidinediones in patients with NASH has not been established. Five-year follow-up of subjects treated with rosiglitazone demonstrated no reduction in liver fibrosis, and rosiglitazone has been associated with increased long-term risk for cardiovascular mortality. Hence, it is not recommended as a treatment for NAFLD. Pioglitazone may be safer because in a recent large meta-analysis it was associated with reduced overall morality, myocardial infarction, and stroke. However, caution must be exercised when considering its use in patients with impaired myocardial function.

1	Antioxidants have also been evaluated for the treatment of NAFLD because oxidant stress is thought to contribute to the pathogenesis of NASH. Vitamin E, an inexpensive yet potent antioxidant, has been examined in several small pediatric and adult studies with varying results. In all of those studies, vitamin E was well tolerated, and most showed modest improvements in aminotransferase levels, radiographic features of hepatic steatosis, and/or histologic features of NASH. Vitamin E (800 IU/d) was also compared to placebo in the PIVENs and TONIC studies. In PIVENs, vitamin E was the only agent that achieved the predetermined primary endpoint (i.e., improvement in steatohepatitis, lobular inflammation, and steatosis score, without an increase in the fibrosis score). This endpoint was met in 43% of patients in the vitamin E group (p = .001 vs placebo), 34% in the pioglitazone group (p = .04 vs placebo), and 19% in the placebo group. Vitamin E also improved NASH histology in pediatric

1	in 43% of patients in the vitamin E group (p = .001 vs placebo), 34% in the pioglitazone group (p = .04 vs placebo), and 19% in the placebo group. Vitamin E also improved NASH histology in pediatric patients with NASH (TONIC trial). However, a recent population-based study suggested that chronic vitamin E therapy may increase the risk for cardiovascular mortality. Thus, vitamin E should only be considered as a first-line pharmacotherapy for nondiabetic NASH patients. Also, given its potentially negative effects on cardiovascular health, caution should be exercised until the risk-to-benefit ratio and long-term therapeutic efficacy of vitamin E are better defined. Ursodeoxycholic acid (a bile acid that improves certain cholestatic liver diseases) and betaine (metabolite of choline that raises SAM levels and decreases cellular oxidative damage) offer no histologic benefit over placebo in patients with NASH. Experimental evidence to support the use of omega-3 fatty acids in NAFLD exists;

1	SAM levels and decreases cellular oxidative damage) offer no histologic benefit over placebo in patients with NASH. Experimental evidence to support the use of omega-3 fatty acids in NAFLD exists; however, a recent large, multicenter, placebo-controlled study failed to demonstrate a histologic benefit. Other pharmacotherapies are also being evaluated in NAFLD (e.g., probiotics, farnesoid X receptor agonists, anticytokine agents, glucagon-like peptide agonists, dipeptidyl IV antagonists); however, sufficient data do not yet exist to 2057 justify their use as NASH treatments in standard clinical practice.

1	Statins are an important class of agents to treat dyslipidemia and decrease cardiovascular risk. There is no evidence to suggest that statins cause liver failure in patients with any chronic liver disease, including NAFLD. The incidence of liver enzyme elevations in NAFLD patients taking statins is also no different than that of healthy controls or patients with other chronic liver diseases. Moreover, several studies have suggested that statins may improve aminotransferases and histology in patients with NASH. Yet, there is continued reluctance to use statins in patients with NAFLD. The lack of evidence that statins harm the liver in NAFLD patients, combined with the increase risk for cardiovascular morbidity and mortality in NAFLD patients, warrants the use of statins to treat dyslipidemia in patients with NAFLD/NASH.

1	Bariatric Surgery Although interest in bariatric surgery as a treatment for NAFLD exists, a recently published Cochrane review concluded that lack of randomized clinical trials or adequate clinical studies prevents definitive assessment of benefits and harms of bariatric surgery as a treatment for NASH. Most studies of bariatric surgery have shown that bariatric surgery is generally safe in individuals with well-compensated chronic liver disease and improves hepatic steatosis and necroinflammation (i.e., features of NAFLD/NASH); however, effects on hepatic fibrosis have been variable. Concern lingers because some of the largest prospective studies suggest that hepatic fibrosis might progress after bariatric surgery. Thus, the Cochrane review deemed it premature to recommend bariatric surgery as a primary treatment for NASH. There is also general agreement that patients with NAFLD-related cirrhosis and portal hypertension should be excluded as candidates for bariatric surgery. However,

1	as a primary treatment for NASH. There is also general agreement that patients with NAFLD-related cirrhosis and portal hypertension should be excluded as candidates for bariatric surgery. However, given growing evidence for the benefits of bariatric surgery on metabolic syndrome complications in individuals with refractory obesity, it is not contraindicated in otherwise eligible patients with NAFLD or NASH.

1	Liver Transplantation Patients with NAFLD in whom end-stage liver disease develops should be evaluated for liver transplantation (Chap. 368). The outcomes of liver transplantation in well-selected patients with NAFLD are generally good, but comorbid medical conditions associated with NAFLD, such as diabetes mellitus, obesity, and cardiovascular disease, often limit transplant candidacy. NAFLD may recur after liver transplantation. The risk factors for recurrent or de novo NAFLD after liver transplantation are multifactorial and include hypertriglyceridemia, obesity, diabetes mellitus, and immunosuppressive therapies, particularly glucocorticoids.

1	The epidemic of obesity is now a global and accelerating phe nomenon. Worldwide, there are over 1 billion overweight adults, of whom at least 300 million are obese. In the wake of the obesity epidemic follow numerous comorbidities, including NAFLD. NAFLD is the most common liver disease identified in Western countries and the fastest rising form of chronic liver disease worldwide. Present understanding of NAFLD natural history is based mainly on studies in whites who became overweight/obese and developed the metabolic syndrome in adulthood. The impact of the global childhood obesity epidemic on NAFLD pathogenesis/progression is unknown. Emerging evidence demonstrates that advanced NAFLD, including cirrhosis and primary liver cancer, can occur in children, prompting concerns that childhood-onset NAFLD might follow a more aggressive course than typical adult-acquired NAFLD. Some of the most populated parts of the world are in the midst of industrial revolutions, and certain

1	childhood-onset NAFLD might follow a more aggressive course than typical adult-acquired NAFLD. Some of the most populated parts of the world are in the midst of industrial revolutions, and certain environmental pollutants seem to exacerbate NAFLD. Some studies also suggest that the risk for NASH and NAFLD-related cirrhosis may be higher in certain ethnic groups such as Asians, certain Hispanics, and Native Americans and lower in others such as African Americans, compared with whites. Although all of these variables confound efforts to predict the net impact of this obesity-related liver disease on global health, it seems likely that NAFLD will remain a major cause of chronic liver disease worldwide for the foreseeable future.

1	2058 Cirrhosis and its Complications Bruce R. Bacon Cirrhosis is a condition that is defined histopathologically and has a variety of clinical manifestations and complications, some of which can be life-threatening. In the past, it has been thought that cirrhosis was never reversible; however, it has become apparent that when the 365 underlying insult that has caused the cirrhosis has been removed, there can be reversal of fibrosis. This is most apparent with the successful treatment of chronic hepatitis C; however, reversal of fibrosis is also seen in patients with hemochromatosis who have been successfully treated and in patients with alcoholic liver disease who have discontinued alcohol use.

1	Regardless of the cause of cirrhosis, the pathologic features consist of the development of fibrosis to the point that there is architectural distortion with the formation of regenerative nodules. This results in a decrease in hepatocellular mass, and thus function, and an alteration of blood flow. The induction of fibrosis occurs with activation of hepatic stellate cells, resulting in the formation of increased amounts of collagen and other components of the extracellular matrix.

1	Clinical features of cirrhosis are the result of pathologic changes and mirror the severity of the liver disease. Most hepatic pathologists provide an assessment of grading and staging when evaluating liver biopsy samples. These grading and staging schemes vary between disease states and have been developed for most conditions, including chronic viral hepatitis, nonalcoholic fatty liver disease, and primary biliary cirrhosis. Advanced fibrosis usually includes bridging fibrosis with nodularity designated as stage 3 and cirrhosis designated as stage 4. Patients who have cirrhosis have varying degrees of compensated liver function, and clinicians need to differentiate between those who have stable, compensated cirrhosis and those who have decompensated cirrhosis. Patients who have developed complications of their liver disease and have become decompensated should be considered for liver transplantation. Many of the complications of cirrhosis will require specific therapy. Portal

1	complications of their liver disease and have become decompensated should be considered for liver transplantation. Many of the complications of cirrhosis will require specific therapy. Portal hypertension is a significant complicating feature of decompensated cirrhosis and is responsible for the development of ascites and bleeding from esophagogastric varices, two complications that signify decompensated cirrhosis. Loss of hepatocellular function results in jaundice, coagulation disorders, and hypoalbuminemia and contributes to the causes of portosystemic encephalopathy. The complications of cirrhosis are basically the same regardless of the etiology. Nonetheless, it is useful to classify patients by the cause of their liver disease (Table 365-1); patients can be divided into broad groups with alcoholic cirrhosis, cirrhosis due to chronic viral hepatitis, biliary cirrhosis, and other, less common causes such as cardiac cirrhosis, cryptogenic cirrhosis, and other miscellaneous causes.

1	Excessive chronic alcohol use can cause several different types of chronic liver disease, including alcoholic fatty liver, alcoholic hepatitis, and alcoholic cirrhosis. Furthermore, use of excessive alcohol can CAuSES of CiRRHoSiS contribute to liver damage in patients with other liver diseases, such as hepatitis C, hemochromatosis, and fatty liver disease related to obesity. Chronic alcohol use can produce fibrosis in the absence of accompanying inflammation and/or necrosis. Fibrosis can be centrilobular, pericellular, or periportal. When fibrosis reaches a certain degree, there is disruption of the normal liver architecture and replacement of liver cells by regenerative nodules. In alcoholic cirrhosis, the nodules are usually <3 mm in diameter; this form of cirrhosis is referred to as micronodular. With cessation of alcohol use, larger nodules may form, resulting in a mixed micronodular and macronodular cirrhosis.

1	Pathogenesis Alcohol is the most commonly used drug in the United States, and more than two-thirds of adults drink alcohol each year. Thirty percent have had a binge within the past month, and over 7% of adults regularly consume more than two drinks per day. Unfortunately, more than 14 million adults in the United States meet the diagnostic criteria for alcohol abuse or dependence. In the United States, chronic liver disease is the tenth most common cause of death in adults, and alcoholic cirrhosis accounts for approximately 40% of deaths due to cirrhosis.

1	Ethanol is mainly absorbed by the small intestine and, to a lesser degree, through the stomach. Gastric alcohol dehydrogenase (ADH) initiates alcohol metabolism. Three enzyme systems account for metabolism of alcohol in the liver. These include cytosolic ADH, the microsomal ethanol oxidizing system (MEOS), and peroxisomal catalase. The majority of ethanol oxidation occurs via ADH to form acetaldehyde, which is a highly reactive molecule that may have multiple effects. Ultimately, acetaldehyde is metabolized to acetate by aldehyde dehydrogenase (ALDH). Intake of ethanol increases intracellular accumulation of triglycerides by increasing fatty acid uptake and by reducing fatty acid oxidation and lipoprotein secretion. Protein synthesis, glycosylation, and secretion are impaired. Oxidative damage to hepatocyte membranes occurs due to the formation of reactive oxygen species; acetaldehyde is a highly reactive molecule that combines with proteins to form protein-acetaldehyde adducts. These

1	to hepatocyte membranes occurs due to the formation of reactive oxygen species; acetaldehyde is a highly reactive molecule that combines with proteins to form protein-acetaldehyde adducts. These adducts may interfere with specific enzyme activities, including microtubular formation and hepatic protein trafficking. With acetaldehyde-mediated hepatocyte damage, certain reactive oxygen species can result in Kupffer cell activation. As a result, profibrogenic cytokines are produced that initiate and perpetuate stellate cell activation, with the resultant production of excess collagen and extracellular matrix. Connective tissue appears in both periportal and pericentral zones and eventually connects portal triads with central veins forming regenerative nodules. Hepatocyte loss occurs, and with increased collagen production and deposition, together with continuing hepatocyte destruction, the liver contracts and shrinks in size. This process generally takes from years to decades to occur and

1	increased collagen production and deposition, together with continuing hepatocyte destruction, the liver contracts and shrinks in size. This process generally takes from years to decades to occur and requires repeated insults.

1	Clinical Features The diagnosis of alcoholic liver disease requires an accurate history regarding both amount and duration of alcohol consumption. Patients with alcoholic liver disease can present with nonspecific symptoms such as vague right upper quadrant abdominal pain, fever, nausea and vomiting, diarrhea, anorexia, and malaise. Alternatively, they may present with more specific complications of chronic liver disease, including ascites, edema, or upper gastrointestinal (GI) hemorrhage. Many cases present incidentally at the time of autopsy or elective surgery. Other clinical manifestations include the development of jaundice or encephalopathy. The abrupt onset of any of these complications may be the first event prompting the patient to seek medical attention. Other patients may be identified in the course of an evaluation of routine laboratory studies that are found to be abnormal. On physical examination, the liver and spleen may be enlarged, with the liver edge being firm and

1	in the course of an evaluation of routine laboratory studies that are found to be abnormal. On physical examination, the liver and spleen may be enlarged, with the liver edge being firm and nodular. Other frequent findings include scleral icterus, palmar erythema (Fig. 365-1), spider angiomas (Fig. 365-2), parotid gland enlargement, digital clubbing, muscle wasting, or the development of edema and ascites. Men may have decreased body hair and gynecomastia as well as testicular atrophy, which may be a consequence of hormonal abnormalities or a direct toxic effect of alcohol on the testes. In women with advanced alcoholic cirrhosis, menstrual irregularities usually occur, and some women may be amenorrheic. These changes are often reversible following cessation of alcohol.

1	FIGURE 365-1 Palmar erythema. This figure shows palmar erythema in a patient with alcoholic cirrhosis. The erythema is peripheral over the palm with central pallor.

1	Laboratory tests may be completely normal in patients with early compensated alcoholic cirrhosis. Alternatively, in advanced liver disease, many abnormalities usually are present. Patients may be anemic either from chronic GI blood loss, nutritional deficiencies, or hypersplenism related to portal hypertension, or as a direct suppressive effect of alcohol on the bone marrow. A unique form of hemolytic anemia (with spur cells and acanthocytes) called Zieve’s syndrome can occur in patients with severe alcoholic hepatitis. Platelet counts are often reduced early in the disease, reflective of portal hypertension with hypersplenism. Serum total bilirubin can be normal or elevated with advanced disease. Direct bilirubin is frequently mildly elevated in patients with a normal total bilirubin, but the abnormality typically progresses as the disease worsens. Prothrombin times are often prolonged and usually do not respond to administration of parenteral vitamin K. Serum sodium levels are

1	but the abnormality typically progresses as the disease worsens. Prothrombin times are often prolonged and usually do not respond to administration of parenteral vitamin K. Serum sodium levels are usually normal unless patients have ascites and then can be depressed, largely due to ingestion of excess free water. Serum alanine and aspartate aminotransferases (ALT, AST) are typically elevated, particularly in patients who continue to drink, with AST levels being higher than ALT levels, usually by a 2:1 ratio.

1	Diagnosis Patients who have any of the above-mentioned clinical features, physical examination findings, or laboratory studies should be considered to have alcoholic liver disease. The diagnosis, however, requires accurate knowledge that the patient is continuing to use and abuse alcohol. Furthermore, other forms of chronic liver disease (e.g., 2059 chronic viral hepatitis or metabolic or autoimmune liver diseases) must be considered or ruled out, or if present, an estimate of relative causality along with the alcohol use should be determined. Liver biopsy can be helpful to confirm a diagnosis, but generally when patients present with alcoholic hepatitis and are still drinking, liver biopsy is withheld until abstinence has been maintained for at least 6 months to determine residual, nonreversible disease.

1	FIGURE 365-2 Spider angioma. This figure shows a spider angioma in a patient with hepatitis C cirrhosis. With release of central compres-sion, the arteriole fills from the center and spreads out peripherally. In patients who have had complications of cirrhosis and who continue to drink, there is a <50% 5-year survival. In contrast, in patients who are able to remain abstinent, the prognosis is significantly improved. In patients with advanced liver disease, the prognosis remains poor; however, in individuals who are able to remain abstinent, liver transplantation is a viable option.

1	Abstinence is the cornerstone of therapy for patients with alcoholic liver disease. In addition, patients require good nutrition and longterm medical supervision to manage underlying complications that may develop. Complications such as the development of ascites and edema, variceal hemorrhage, or portosystemic encephalopathy all require specific management and treatment. Glucocorticoids are occasionally used in patients with severe alcoholic hepatitis in the absence of infection. Survival has been shown to improve in certain studies. Treatment is restricted to patients with a discriminant function (DF) value of >32. The DF is calculated as the serum total bilirubin plus the difference in the patient’s prothrombin time compared to control (in seconds) multiplied by 4.6. In patients for whom this value is >32, there is improved survival at 28 days with the use of glucocorticoids.

1	Other therapies that have been used include oral pentoxifylline, which decreases the production of tumor necrosis factor α (TNF-α) and other proinflammatory cytokines. In contrast to glucocorticoids, with which complications can occur, pentoxifylline is relatively easy to administer and has few, if any, side effects. A variety of nutritional therapies have been tried with either parenteral or enteral feedings; however, it is unclear whether any of these modalities have significantly improved survival. Recent studies have used parenterally administered inhibitors of TNF-α such as infliximab or etanercept. Early results have shown no adverse events; however, there was no clear-cut improvement in survival. Anabolic steroids, propylthiouracil, antioxidants, colchicine, and penicillamine have all been used but do not show clear-cut benefits and are not recommended.

1	As mentioned above, the cornerstone to treatment is cessation of alcohol use. Recent experience with medications that reduce craving for alcohol, such as acamprosate calcium, has been favorable. Patients may take other necessary medications even in the presence of cirrhosis. Acetaminophen use is often discouraged in patients with liver disease; however, if no more than 2 g of acetaminophen per day are consumed, there generally are no problems.

1	Of patients exposed to the hepatitis C virus (HCV), approximately 80% develop chronic hepatitis C, and of those, about 20–30% will develop cirrhosis over 20–30 years. Many of these patients have had concomitant alcohol use, and the true incidence of cirrhosis due to hepatitis C alone is unknown. Nonetheless, this represents a significant number of patients. It is expected that an even higher percentage will go on to develop cirrhosis over longer periods of time. In the United States, approximately 5 to 6 million people have been exposed to HCV, with about 4 million who are chronically viremic. Worldwide, about 170 million individuals have hepatitis C, with some areas of the world (e.g., Egypt) having up to 15% of the population infected. HCV is a noncytopathic virus, and liver damage is probably immune-mediated. Progression of liver disease due to chronic 2060 hepatitis C is characterized by portal-based fibrosis with bridging fibrosis and nodularity developing, ultimately culminating

1	immune-mediated. Progression of liver disease due to chronic 2060 hepatitis C is characterized by portal-based fibrosis with bridging fibrosis and nodularity developing, ultimately culminating in the development of cirrhosis. In cirrhosis due to chronic hepatitis C, the liver is small and shrunken with characteristic features of a mixed microand macronodular cirrhosis seen on liver biopsy. In addition to the increased fibrosis that is seen in cirrhosis due to hepatitis C, an inflammatory infiltrate is found in portal areas with interface hepatitis and occasionally some lobular hepatocellular injury and inflammation. In patients with HCV genotype 3, steatosis is often present. Similar findings are seen in patients with cirrhosis due to chronic hepatitis B. Of adult patients exposed to hepatitis B, about 5% develop chronic hepatitis B, and about 20% of those patients will go on to develop cirrhosis. Special stains for hepatitis B core (HBc) and hepatitis B surface (HBs) antigen will be

1	B, about 5% develop chronic hepatitis B, and about 20% of those patients will go on to develop cirrhosis. Special stains for hepatitis B core (HBc) and hepatitis B surface (HBs) antigen will be positive, and ground-glass hepatocytes signifying hepatitis B surface antigen (HBsAg) may be present. In the United States, there are about 2 million carriers of hepatitis B, whereas in other parts of the world where hepatitis B virus (HBV) is endemic (i.e., Asia, Southeast Asia, sub-Saharan Africa), up to 15% of the population may be infected, having acquired the infection vertically at the time of birth. Thus, over 300–400 million individuals are thought to have hepatitis B worldwide. Approximately 25% of these individuals may ultimately develop cirrhosis.

1	Clinical Features and Diagnosis Patients with cirrhosis due to either chronic hepatitis C or B can present with the usual symptoms and signs of chronic liver disease. Fatigue, malaise, vague right upper quadrant pain, and laboratory abnormalities are frequent presenting features. Diagnosis requires a thorough laboratory evaluation, including quantitative HCV RNA testing and analysis for HCV genotype, or hepatitis B serologies to include HBsAg, anti-HBs, HBeAg (hepatitis B e antigen), anti-HBe, and quantitative HBV DNA levels.

1	Management of complications of cirrhosis revolves around specific therapy for treatment of whatever complications occur (e.g., esophageal variceal hemorrhage, development of ascites and edema, or encephalopathy). In patients with chronic hepatitis B, numerous studies have shown beneficial effects of antiviral therapy, which is effective at viral suppression, as evidenced by reducing aminotransferase levels and HBV DNA levels, and improving histology by reducing inflammation and fibrosis. Several clinical trials and case series have demonstrated that patients with decompensated liver disease can become compensated with the use of antiviral therapy directed against hepatitis B. Currently available therapy includes lamivudine, adefovir, telbivudine, entecavir, and tenofovir. Interferon α can also be used for treating hepatitis B, but it should not be used in cirrhotics.

1	Treatment of patients with cirrhosis due to hepatitis C is a little more difficult because the side effects of pegylated interferon and ribavirin therapy are often difficult to manage. Dose-limiting cytopenias (platelets, white blood cells, red blood cells) or severe side effects can result in discontinuation of treatment. Nonetheless, if patients can tolerate treatment, and if it is successful, the benefit is great and disease progression is reduced. Recent studies have shown that if platelets are <100,000, albumin is <3.5 g/dL, and Model for End-Stage Liver Disease (MELD) score is >10, the risk of severe complications of interferon-based antiviral therapy is significant. Recent approval of Direct Acting Antivirals (DAAs) has led to improved efficacy of treatment with regimens that are safe and well tolerated.

1	Other causes of posthepatitic cirrhosis include autoimmune hepatitis and cirrhosis due to nonalcoholic steatohepatitis. Many patients with autoimmune hepatitis (AIH) present with cirrhosis that is already established. Typically, these patients will not benefit from immunosuppressive therapy with glucocorticoids or azathioprine because the AIH is “burned out.” In this situation, liver biopsy does not show a significant inflammatory infiltrate. Diagnosis in this setting requires positive autoimmune markers such as antinuclear antibody (ANA) or anti-smooth-muscle antibody (ASMA). When patients with AIH present with cirrhosis and active inflammation accompanied by elevated liver enzymes, there can be considerable benefit from the use of immunosuppressive therapy.

1	Patients with nonalcoholic steatohepatitis are increasingly being found to have progressed to cirrhosis. With the epidemic of obesity that continues in Western countries, more and more patients are identified with nonalcoholic fatty liver disease (Chap. 364). Of these, a significant subset has nonalcoholic steatohepatitis and can progress to increased fibrosis and cirrhosis. Over the past several years, it has been increasingly recognized that many patients who were thought to have cryptogenic cirrhosis in fact have nonalcoholic steatohepatitis. As their cirrhosis progresses, they become catabolic and then lose the telltale signs of steatosis seen on biopsy. Management of complications of cirrhosis due to either AIH or nonalcoholic steatohepatitis is similar to that for other forms of cirrhosis.

1	Biliary cirrhosis has pathologic features that are different from either alcoholic cirrhosis or posthepatitic cirrhosis, yet the manifestations of end-stage liver disease are the same. Cholestatic liver disease may result from necroinflammatory lesions, congenital or metabolic processes, or external bile duct compression. Thus, two broad categories reflect the anatomic sites of abnormal bile retention: intrahepatic and extrahepatic. The distinction is important for obvious therapeutic reasons. Extrahepatic obstruction may benefit from surgical or endoscopic biliary tract decompression, whereas intrahepatic cholestatic processes will not improve with such interventions and require a different approach.

1	The major causes of chronic cholestatic syndromes are primary biliary cirrhosis (PBC), autoimmune cholangitis (AIC), primary sclerosing cholangitis (PSC), and idiopathic adulthood ductopenia. These syndromes are usually clinically distinguished from each other by antibody testing, cholangiographic findings, and clinical presentation. However, they all share the histopathologic features of chronic cholestasis, such as cholate stasis; copper deposition; xanthomatous transformation of hepatocytes; and irregular, so-called biliary fibrosis. In addition, there may be chronic portal inflammation, interface activity, and chronic lobular inflammation. Ductopenia is a result of this progressive disease as patients develop cirrhosis.

1	PBC is seen in about 100–200 individuals per million, with a strong female preponderance and a median age of around 50 years at the time of diagnosis. The cause of PBC is unknown; it is characterized by portal inflammation and necrosis of cholangiocytes in smalland medium-sized bile ducts. Cholestatic features prevail, and biliary cirrhosis is characterized by an elevated bilirubin level and progressive liver failure. Liver transplantation is the treatment of choice for patients with decompensated cirrhosis due to PBC. A variety of therapies have been proposed, but ursodeoxycholic acid (UDCA) is the only approved treatment that has some degree of efficacy by slowing the rate of progression of the disease.

1	Antimitochondrial antibodies (AMA) are present in about 90% of patients with PBC. These autoantibodies recognize intermitochondrial membrane proteins that are enzymes of the pyruvate dehydrogenase complex (PDC), the branched-chain 2-oxoacid dehydrogenase complex, and the 2-oxogluterate dehydrogenase complex. Most relate to pyruvate dehydrogenase. These autoantibodies are not pathogenic but rather are useful markers for making a diagnosis of PBC.

1	Pathology Histopathologic analyses of liver biopsies of patients with PBC have resulted in identifying four distinct stages of the disease as it progresses. The earliest lesion is termed chronic nonsuppurative destructive cholangitis and is a necrotizing inflammatory process of the portal tracts. Medium and small bile ducts are infiltrated with lymphocytes and undergo duct destruction. Mild fibrosis and sometimes bile stasis can occur. With progression, the inflammatory infiltrate becomes less prominent, but the number of bile ducts is reduced and there is proliferation of smaller bile ductules. Increased fibrosis ensues with the expansion of periportal fibrosis to bridging fibrosis. Finally, cirrhosis, which may be micronodular or macronodular, develops.

1	Clinical Features Currently, most patients with PBC are diagnosed well before the end-stage manifestations of the disease are present, and, as such, most patients are actually asymptomatic. When symptoms are present, they most prominently include a significant degree of fatigue out of proportion to what would be expected for either the severity of the liver disease or the age of the patient. Pruritus is seen in approximately 50% of patients at the time of diagnosis, and it can be debilitating. It might be intermittent and usually is most bothersome in the evening. In some patients, pruritus can develop toward the end of pregnancy, and there are examples of patients having been diagnosed with cholestasis of pregnancy rather than PBC. Pruritus that presents prior to the development of jaundice indicates severe disease and a poor prognosis.

1	Physical examination can show jaundice and other complications of chronic liver disease, including hepatomegaly, splenomegaly, ascites, and edema. Other features that are unique to PBC include hyperpigmentation, xanthelasma, and xanthomata, which are related to the altered cholesterol metabolism seen in this disease. Hyperpigmentation is evident on the trunk and the arms and is seen in areas of exfoliation and lichenification associated with progressive scratching related to the pruritus. Bone pain resulting from osteopenia or osteoporosis is occasionally seen at the time of diagnosis.

1	Laboratory Findings Laboratory findings in PBC show cholestatic liver enzyme abnormalities with an elevation in γ-glutamyl transpeptidase and alkaline phosphatase (ALP) along with mild elevations in aminotransferases (ALT and AST). Immunoglobulins, particularly IgM, are typically increased. Hyperbilirubinemia usually is seen once cirrhosis has developed. Thrombocytopenia, leukopenia, and anemia may be seen in patients with portal hypertension and hypersplenism. Liver biopsy shows characteristic features as described above and should be evident to any experienced hepatopathologist. Up to 10% of patients with characteristic PBC will have features of AIH as well and are defined as having “overlap” syndrome. These patients are usually treated as PBC patients and may progress to cirrhosis with the same frequency as typical PBC patients. Some patients require immunosuppressive medications as well.

1	Diagnosis PBC should be considered in patients with chronic cholestatic liver enzyme abnormalities. It is most often seen in middle-aged women. AMA testing may be negative, and it should be remembered that as many as 10% of patients with PBC may be AMA-negative. Liver biopsy is most important in this setting of AMA-negative PBC. In patients who are AMA-negative with cholestatic liver enzymes, PSC should be ruled out by way of cholangiography.

1	Treatment of the typical manifestations of cirrhosis are no different for PBC than for other forms of cirrhosis. UDCA has been shown to improve both biochemical and histologic features of the disease. Improvement is greatest when therapy is initiated early; the likelihood of significant improvement with UDCA is low in patients with PBC who present with manifestations of cirrhosis. UDCA is given in doses of 13–15 mg/kg per day; the medication is usually well-tolerated, although some patients have worsening pruritus with initiation of therapy. A small proportion of patients may have diarrhea or headache as a side effect of the drug. UDCA has been shown to slow the rate of progression of PBC, but it does not reverse or cure the disease. Patients with PBC require long-term follow-up by a physician experienced with the disease. Certain patients may need to be considered for liver transplantation should their liver disease 2061 decompensate.

1	The main symptoms of PBC are fatigue and pruritus, and symptom management is important. Several therapies have been tried for treatment of fatigue, but none of them have been successful; frequent naps should be encouraged. Pruritus is treated with antihistamines, narcotic receptor antagonists (naltrexone), and rifampin. Cholestyramine, a bile salt–sequestering agent, has been helpful in some patients but is somewhat tedious and difficult to take. Plasmapheresis has been used rarely in patients with severe intractable pruritus. There is an increased incidence of osteopenia and osteoporosis in patients with cholestatic liver disease, and bone density testing should be performed. Treatment with a bisphosphonate should be instituted when bone disease is identified.

1	As in PBC, the cause of PSC remains unknown. PSC is a chronic cholestatic syndrome that is characterized by diffuse inflammation and fibrosis involving the entire biliary tree, resulting in chronic cholestasis. This pathologic process ultimately results in obliteration of both the intraand extrahepatic biliary tree, leading to biliary cirrhosis, portal hypertension, and liver failure. The cause of PSC remains unknown despite extensive investigation into various mechanisms related to bacterial and viral infections, toxins, genetic predisposition, and immunologic mechanisms, all of which have been postulated to contribute to the pathogenesis and progression of this syndrome.

1	Pathologic changes that can occur in PSC show bile duct proliferation as well as ductopenia and fibrous cholangitis (pericholangitis). Often, liver biopsy changes in PSC are not pathognomonic, and establishing the diagnosis of PSC must involve imaging of the biliary tree. Periductal fibrosis is occasionally seen on biopsy specimens and can be quite helpful in making the diagnosis. As the disease progresses, biliary cirrhosis is the final, end-stage manifestation of PSC. Clinical Features The usual clinical features of PSC are those found in cholestatic liver disease, with fatigue, pruritus, steatorrhea, deficiencies of fat-soluble vitamins, and the associated consequences. As in PBC, the fatigue is profound and nonspecific. Pruritus can often be debilitating and is related to the cholestasis. The severity of pruritus does not correlate with the severity of the disease. Metabolic bone disease, as seen in PBC, can occur with PSC and should be treated (see above).

1	Laboratory Findings Patients with PSC typically are identified in the course of an evaluation of abnormal liver enzymes. Most patients have at least a twofold increase in ALP and may have elevated aminotransferases as well. Albumin levels may be decreased, and prothrombin times are prolonged in a substantial proportion of patients at the time of diagnosis. Some degree of correction of a prolonged prothrombin time may occur with parenteral vitamin K. A small subset of patients have aminotransferase elevations greater than five times the upper limit of normal and may have features of AIH on biopsy. These individuals are thought to have an overlap syndrome between PSC and AIH. Autoantibodies are frequently positive in patients with the overlap syndrome but are typically negative in patients who only have PSC. One autoantibody, the perinuclear antineutrophil cytoplasmic antibody (p-ANCA), is positive in about 65% of patients with PSC. Over 50% of patients with PSC also have ulcerative

1	who only have PSC. One autoantibody, the perinuclear antineutrophil cytoplasmic antibody (p-ANCA), is positive in about 65% of patients with PSC. Over 50% of patients with PSC also have ulcerative colitis (UC); accordingly, once a diagnosis of PSC is established, colonoscopy should be performed to look for evidence of UC.

1	Diagnosis The definitive diagnosis of PSC requires cholangiographic imaging. Over the last several years, magnetic resonance imaging (MRI) with magnetic resonance cholangiopancreatography (MRCP) has been used as the imaging technique of choice for initial evaluation. Once patients are screened in this manner, some investigators feel that endoscopic retrograde cholangiopancreatography (ERCP) should also be performed to be certain whether or not a dominant stricture is present. Typical cholangiographic findings in PSC are multifocal stricturing and beading involving both the intrahepatic and extrahepatic biliary tree. However, although involvement may be of 2062 the intrahepatic bile ducts alone or of the extrahepatic bile ducts alone, more commonly, both are involved. These strictures are typically short and with intervening segments of normal or slightly dilated bile ducts that are distributed diffusely, producing the classic beaded appearance. The gallbladder and cystic duct can be

1	typically short and with intervening segments of normal or slightly dilated bile ducts that are distributed diffusely, producing the classic beaded appearance. The gallbladder and cystic duct can be involved in up to 15% of cases. Patients with high-grade, diffuse stricturing of the intrahepatic bile ducts have an overall poor prognosis. Gradually, biliary cirrhosis develops, and patients will progress to decompensated liver disease with all the manifestations of ascites, esophageal variceal hemorrhage, and encephalopathy.

1	There is no specific proven treatment for PSC. A recently completed study of high-dose (20 mg/kg per day) UDCA was found to be harmful. Some clinicians use UDCA at “PBC dosages” of 13–15 mg/ kg per day with anecdotal improvement. Endoscopic dilatation of dominant strictures can be helpful, but the ultimate treatment is liver transplantation. A dreaded complication of PSC is the development of cholangiocarcinoma, which is a relative contraindication to liver transplantation. Symptoms of pruritus are common, and the approach is as mentioned previously for this problem in patients with PBC (see above). Definition Patients with long-standing right-sided congestive heart failure may develop chronic liver injury and cardiac cirrhosis. This is an increasingly uncommon, if not rare, cause of chronic liver disease given the advances made in the care of patients with heart failure.

1	Etiology and Pathology In the case of long-term right-sided heart failure, there is an elevated venous pressure transmitted via the inferior vena cava and hepatic veins to the sinusoids of the liver, which become dilated and engorged with blood. The liver becomes enlarged and swollen, and with long-term passive congestion and relative ischemia due to poor circulation, centrilobular hepatocytes can become necrotic, leading to pericentral fibrosis. This fibrotic pattern can extend to the periphery of the lobule outward until a unique pattern of fibrosis causing cirrhosis can occur. Clinical Features Patients typically have signs of congestive heart failure and will manifest an enlarged firm liver on physical examination. ALP levels are characteristically elevated, and aminotransferases may be normal or slightly increased with AST usually higher than ALT. It is unlikely that patients will develop variceal hemorrhage or encephalopathy.

1	Diagnosis The diagnosis is usually made in someone with clear-cut cardiac disease who has an elevated ALP and an enlarged liver. Liver biopsy shows a pattern of fibrosis that can be recognized by an experienced hepatopathologist. Differentiation from Budd-Chiari syndrome (BCS) can be made by seeing extravasation of red blood cells in BCS, but not in cardiac hepatopathy. Venoocclusive disease can also affect hepatic outflow and has characteristic features on liver biopsy. Venoocclusive disease can be seen under the circumstances of conditioning for bone marrow transplant with radiation and chemotherapy; it can also be seen with the ingestion of certain herbal teas as well as pyrrolizidine alkaloids. This is typically seen in Caribbean countries and rarely in the United States. Treatment is based on management of the underlying cardiac disease.

1	There are several other less common causes of chronic liver disease that can progress to cirrhosis. These include inherited metabolic liver diseases such as hemochromatosis, Wilson’s disease, α1 antitrypsin (α1AT) deficiency, and cystic fibrosis. For all of these disorders, the manifestations of cirrhosis are similar, with some minor variations, to those seen in other patients with other causes of cirrhosis.

1	Hemochromatosis is an inherited disorder of iron metabolism that results in a progressive increase in hepatic iron deposition, which, over time, can lead to a portal-based fibrosis progressing to cirrhosis, liver failure, and hepatocellular cancer. While the frequency of hemochromatosis is relatively common, with genetic susceptibility occurring in 1 in 250 individuals, the frequency of end-stage manifestations due to the disease is relatively low, and fewer than 5% of those patients who are genotypically susceptible will go on to develop severe liver disease from hemochromatosis. Diagnosis is made with serum iron studies showing an elevated transferrin saturation and an elevated ferritin level, along with abnormalities identified by HFE mutation analysis. Treatment is straightforward, with regular therapeutic phlebotomy.

1	Wilson’s disease is an inherited disorder of copper homeostasis with failure to excrete excess amounts of copper, leading to an accumulation in the liver. This disorder is relatively uncommon, affecting 1 in 30,000 individuals. Wilson’s disease typically affects adolescents and young adults. Prompt diagnosis before end-stage manifestations become irreversible can lead to significant clinical improvement. Diagnosis requires determination of ceruloplasmin levels, which are low; 24-h urine copper levels, which are elevated; typical physical examination findings, including Kayser-Fleischer corneal rings; and characteristic liver biopsy findings. Treatment consists of copper-chelating medications.

1	α1AT deficiency results from an inherited disorder that causes abnormal folding of the α1AT protein, resulting in failure of secretion of that protein from the liver. It is unknown how the retained protein leads to liver disease. Patients with α1AT deficiency at greatest risk for developing chronic liver disease have the ZZ phenotype, but only about 10–20% of such individuals will develop chronic liver disease. Diagnosis is made by determining α1AT levels and phenotype. Characteristic periodic acid–Schiff (PAS)-positive, diastase-resistant globules are seen on liver biopsy. The only effective treatment is liver transplantation, which is curative. Cystic fibrosis is an uncommon inherited disorder affecting whites of northern European descent. A biliary-type cirrhosis can occur, and some patients derive benefit from the chronic use of UDCA.

1	Cystic fibrosis is an uncommon inherited disorder affecting whites of northern European descent. A biliary-type cirrhosis can occur, and some patients derive benefit from the chronic use of UDCA. The clinical course of patients with advanced cirrhosis is often complicated by a number of important sequelae that can occur regardless of the underlying cause of the liver disease. These include portal hypertension and its consequences of gastroesophageal variceal hemorrhage, splenomegaly, ascites, hepatic encephalopathy, spontaneous bacterial peritonitis (SBP), hepatorenal syndrome, and hepatocellular carcinoma (Table 365-2).

1	Portal hypertension is defined as the elevation of the hepatic venous pressure gradient (HVPG) to >5 mmHg. Portal hypertension is caused by a combination of two simultaneously occurring hemodynamic processes: (1) increased intrahepatic resistance to the passage of blood flow through the liver due to cirrhosis and regenerative nodules, and (2) increased splanchnic blood flow secondary to vasodilation within the splanchnic vascular bed. Portal hypertension is directly responsible for the two major complications of cirrhosis: variceal hemorrhage and ascites. Variceal hemorrhage is an immediate life-threatening problem with a 20–30% mortality rate associated with each episode of bleeding. The portal venous system normally drains blood from the stomach, intestines, spleen, pancreas, and gallbladder, and the portal vein is formed by the confluence of the superior mesenteric and splenic veins. Deoxygenated blood from the small bowel drains into the superior mesenteric vein along with blood

1	and the portal vein is formed by the confluence of the superior mesenteric and splenic veins. Deoxygenated blood from the small bowel drains into the superior mesenteric vein along with blood from the head of the pancreas, the ascending colon, and part of the transverse colon. Conversely, the splenic vein drains the spleen and the pancreas and is joined by the inferior mesenteric vein, which brings blood from the transverse and descending colon as well as from the superior two-thirds of the rectum. Thus, the portal vein normally receives blood from almost the entire GI tract.

1	The causes of portal hypertension are usually subcategorized as prehepatic, intrahepatic, and posthepatic (Table 365-3). Prehepatic causes of portal hypertension are those affecting the portal venous system before it enters the liver; they include portal vein thrombosis and splenic vein thrombosis. Posthepatic causes encompass those affecting the hepatic veins and venous drainage to the heart; they include BCS, venoocclusive disease, and chronic right-sided cardiac congestion. Intrahepatic causes account for over 95% of cases of portal hypertension and are represented by the major forms of cirrhosis. Intrahepatic causes of portal hypertension can be further subdivided into presinusoidal, sinusoidal, and postsinusoidal causes. Postsinusoidal causes include venoocclusive disease, whereas presinusoidal causes include congenital hepatic fibrosis and schistosomiasis. Sinusoidal causes are related to cirrhosis from various causes.

1	Cirrhosis is the most common cause of portal hypertension in the United States, and clinically significant portal hypertension is present in >60% of patients with cirrhosis. Portal vein obstruction may be idiopathic or can occur in association with cirrhosis or with infection, pancreatitis, or abdominal trauma. Coagulation disorders that can lead to the development of portal vein thrombosis include polycythemia vera; essential thrombocytosis; deficiencies in protein C, protein S, antithrombin 3, and factor V Leiden; and abnormalities in the gene-regulating prothrombin production. Some patients may have a subclinical myeloproliferative disorder. Clinical Features The three primary complications of portal hypertension are gastroesophageal varices with hemorrhage, ascites, and

1	Clinical Features The three primary complications of portal hypertension are gastroesophageal varices with hemorrhage, ascites, and ClASSifiCATion of PoRTAl HyPERTEnSion hypersplenism. Thus, patients may present with upper GI bleeding, 2063 which, on endoscopy, is found to be due to esophageal or gastric varices; with the development of ascites along with peripheral edema; or with an enlarged spleen with associated reduction in platelets and white blood cells on routine laboratory testing.

1	esopHaGeal varices Over the last decade, it has become common practice to screen known cirrhotics with endoscopy to look for esophageal varices. Such screening studies have shown that approximately one-third of patients with histologically confirmed cirrhosis have varices. Approximately 5–15% of cirrhotics per year develop varices, and it is estimated that the majority of patients with cirrhosis will develop varices over their lifetimes. Furthermore, it is anticipated that roughly one-third of patients with varices will develop bleeding. Several factors predict the risk of bleeding, including the severity of cirrhosis (Child’s class, MELD score); the height of wedged-hepatic vein pressure; the size of the varix; the location of the varix; and certain endoscopic stigmata, including red wale signs, hematocystic spots, diffuse erythema, bluish color, cherry red spots, or white-nipple spots. Patients with tense ascites are also at increased risk for bleeding from varices.

1	Diagnosis In patients with cirrhosis who are being followed chronically, the development of portal hypertension is usually revealed by the presence of thrombocytopenia; the appearance of an enlarged spleen; or the development of ascites, encephalopathy, and/or esophageal varices with or without bleeding. In previously undiagnosed patients, any of these features should prompt further evaluation to determine the presence of portal hypertension and liver disease. Varices should be identified by endoscopy. Abdominal imaging, either by computed tomography (CT) or MRI, can be helpful in demonstrating a nodular liver and in finding changes of portal hypertension with intraabdominal collateral circulation. If necessary, interventional radiologic procedures can be performed to determine wedged and free hepatic vein pressures that will allow for the calculation of a wedged-to-free gradient, which is equivalent to the portal pressure. The average normal wedged-to-free gradient is 5 mmHg, and

1	and free hepatic vein pressures that will allow for the calculation of a wedged-to-free gradient, which is equivalent to the portal pressure. The average normal wedged-to-free gradient is 5 mmHg, and patients with a gradient >12 mmHg are at risk for variceal hemorrhage.

1	Treatment for variceal hemorrhage as a complication of portal hypertension is divided into two main categories: (1) primary prophylaxis and (2) prevention of rebleeding once there has been an initial variceal hemorrhage. Primary prophylaxis requires routine screening by endoscopy of all patients with cirrhosis. Once varices that are at increased risk for bleeding are identified, primary prophylaxis can be achieved either through nonselective beta blockade or by variceal band ligation. Numerous placebo-controlled clinical trials of either propranolol or nadolol have been reported in the literature. The most rigorous studies were those that only included patients with significantly enlarged varices or with hepatic vein pressure gradients >12 mmHg. Patients treated with beta blockers have a lower risk of variceal hemorrhage than those treated with placebo over 1 and 2 years of follow-up. There is also a decrease in mortality related to variceal hemorrhage. Unfortunately, overall survival

1	risk of variceal hemorrhage than those treated with placebo over 1 and 2 years of follow-up. There is also a decrease in mortality related to variceal hemorrhage. Unfortunately, overall survival was improved in only one study. Further studies have demonstrated that the degree of reduction of portal pressure is a significant feature to determine success of therapy. Therefore, it has been suggested that repeat measurements of hepatic vein pressure gradients may be used to guide pharmacologic therapy; however, this may be cost-prohibitive. Several studies have evaluated variceal band ligation and variceal sclerotherapy as methods for providing primary prophylaxis.

1	Endoscopic variceal ligation (EVL) has achieved a level of success and comfort with most gastroenterologists who see patients with these complications of portal hypertension. Thus, in patients with cirrhosis who are screened for portal hypertension and are found to have large varices, it is recommended that they receive either beta blockade or primary prophylaxis with EVL.

1	2064 The approach to patients once they have had a variceal bleed is first to treat the acute bleed, which can be life-threatening, and then to prevent further bleeding. Prevention of further bleeding is usually accomplished with repeated variceal band ligation until varices are obliterated. Treatment of acute bleeding requires both fluid and blood-product replacement as well as prevention of subsequent bleeding with EVL. The medical management of acute variceal hemorrhage includes the use of vasoconstricting agents, usually somatostatin or octreotide. Vasopressin was used in the past but is no longer commonly used. Balloon tamponade (Sengstaken-Blakemore tube or Minnesota tube) can be used in patients who cannot get endoscopic therapy immediately or who need stabilization prior to endoscopic therapy. Control of bleeding can be achieved in the vast majority of cases; however, bleeding recurs in the majority of patients if definitive endoscopic therapy has not been instituted.

1	endoscopic therapy. Control of bleeding can be achieved in the vast majority of cases; however, bleeding recurs in the majority of patients if definitive endoscopic therapy has not been instituted. Octreotide, a direct splanchnic vasoconstrictor, is given at dosages of 50–100 µg/h by continuous infusion. Endoscopic intervention is used as first-line treatment to control bleeding acutely. Some endoscopists will use variceal injection therapy (sclerotherapy) as initial therapy, particularly when bleeding is vigorous. Variceal band ligation is used to control acute bleeding in over 90% of cases and should be repeated until obliteration of all varices is accomplished. When esophageal varices extend into the proximal stomach, band ligation is less successful. In these situations, when bleeding continues from gastric varices, consideration for a transjugular intrahepatic portosystemic shunt (TIPS) should be made. This technique creates a portosystemic shunt by a percutaneous approach using

1	continues from gastric varices, consideration for a transjugular intrahepatic portosystemic shunt (TIPS) should be made. This technique creates a portosystemic shunt by a percutaneous approach using an expandable metal stent, which is advanced under angiographic guidance to the hepatic veins and then through the substance of the liver to create a direct portocaval shunt. This offers an alternative to surgery for acute decompression of portal hypertension. Encephalopathy can occur in as many as 20% of patients after TIPS and is particularly problematic in elderly patients and in patients with preexisting encephalopathy. TIPS should be reserved for individuals who fail endoscopic or medical management or who are poor surgical risks. TIPS can sometimes be used as a bridge to transplantation. Surgical esophageal transsection is a procedure that is rarely used and generally is associated with a poor outcome.

1	PREVENTION OF RECURRENT BLEEDING (FIG. 365-3) Once patients have had an acute bleed and have been managed successfully, attention should be paid to preventing recurrent bleeding. This usually requires repeated variceal band ligation until varices are obliterated. Beta blockade may be of adjunctive benefit in patients who are having recurrent variceal band ligation; however, once varices have been obliterated, the need for beta blockade is lessened. Despite successful variceal obliteration, many patients will still have portal hypertensive gastropathy from which bleeding can occur. Nonselective beta blockade may be helpful to prevent further bleeding from portal hypertensive gastropathy once varices have been obliterated. Portosystemic shunt surgery is less commonly performed with the advent of TIPS; nonetheless, this procedure should be considered for patients with good hepatic synthetic function who could benefit by having portal decompressive surgery.

1	Congestive splenomegaly is common in patients with portal hypertension. Clinical features include the presence of an enlarged spleen on physical examination and the development of thrombocytopenia and leukopenia in patients who have cirrhosis. Some patients will have fairly significant left-sided and left upper quadrant abdominal pain related to an enlarged and engorged spleen. Splenomegaly itself usually requires no specific treatment, although splenectomy can be successfully performed under very special circumstances. Hypersplenism with the development of thrombocytopenia is a common feature of patients with cirrhosis and is usually the first indication of portal hypertension. FIGURE 365-3 Management of recurrent variceal hemorrhage.

1	FIGURE 365-3 Management of recurrent variceal hemorrhage. This algorithm describes an approach to management of patients who have recurrent bleeding from esophageal varices. Initial therapy is generally with endoscopic therapy often supplemented by pharmacologic therapy. With control of bleeding, a decision needs to be made as to whether patients should go on to a surgical shunt or TIPS (if they are Child’s class A) and be considered for transplant, or if they should have TIPS and be considered for transplant (if they are Child’s class B or C). TIPS, transjugular intrahepatic portosystemic shunt. ASCITES Definition Ascites is the accumulation of fluid within the peritoneal cavity. Overwhelmingly, the most common cause of ascites is portal hypertension related to cirrhosis; however, clinicians should remember that malignant or infectious causes of ascites can be present as well, and careful differentiation of these other causes are obviously important for patient care.

1	Pathogenesis The presence of portal hypertension contributes to the development of ascites in patients who have cirrhosis (Fig. 365-4). There is an increase in intrahepatic resistance, causing increased portal pressure, but there is also vasodilation of the splanchnic arterial system, which, in turn, results in an increase in portal venous inflow. Both of these abnormalities result in increased production of splanchnic lymph. Vasodilating factors such as nitric oxide are responsible for the vasodilatory effect. These hemodynamic changes result in sodium retention by causing activation of the renin-angiotensin-aldosterone system with the development of hyperaldosteronism. The renal effects of increased aldosterone leading to sodium retention also contribute to the development of ascites. Sodium retention causes fluid accumulation and expansion of the extracellular fluid volume, which results in the formation of peripheral edema and ascites. Sodium retention is the consequence of a

1	Sodium retention causes fluid accumulation and expansion of the extracellular fluid volume, which results in the formation of peripheral edema and ascites. Sodium retention is the consequence of a homeostatic response caused by underfilling of the arterial circulation secondary to arterial vasodilation in the splanchnic vascular bed. Because the retained fluid is constantly leaking out of the intravascular compartment into the peritoneal cavity, the sensation of vascular filling is not achieved, and the process continues. Hypoalbuminemia and reduced plasma oncotic pressure also contribute to the loss of fluid from the vascular compartment into the ˜ Splanchnic pressure Arterial underfilling Formation of ascites Lymph formation Activation of vasoconstrictors and antinatriuretic factors* Sodium retention Plasma volume expansion FIGURE 365-4 Development of ascites in cirrhosis. This flow diagram illustrates the importance of portal hypertension with splanchnic vasodilation in the

1	Sodium retention Plasma volume expansion FIGURE 365-4 Development of ascites in cirrhosis. This flow diagram illustrates the importance of portal hypertension with splanchnic vasodilation in the development of ascites. *Antinatriuretic factors include the renin-angiotensin-aldosterone system and the sympathetic nervous system.

1	peritoneal cavity. Hypoalbuminemia is due to decreased synthetic function in a cirrhotic liver. Clinical Features Patients typically note an increase in abdominal girth that is often accompanied by the development of peripheral edema. The development of ascites is often insidious, and it is surprising that some patients wait so long and become so distended before seeking medical attention. Patients usually have at least 1–2 L of fluid in the abdomen before they are aware that there is an increase. If ascitic fluid is massive, respiratory function can be compromised, and patients will complain of shortness of breath. Hepatic hydrothorax may also occur in this setting, contributing to respiratory symptoms. Patients with massive ascites are often malnourished and have muscle wasting and excessive fatigue and weakness.

1	Diagnosis Diagnosis of ascites is by physical examination and is often aided by abdominal imaging. Patients will have bulging flanks, may have a fluid wave, or may have the presence of shifting dullness. This is determined by taking patients from a supine position to lying on either their left or right side and noting the movement of the dullness to percussion. Subtle amounts of ascites can be detected by ultrasound or CT scanning. Hepatic hydrothorax is more common on the right side and implicates a rent in the diaphragm with free flow of ascitic fluid into the thoracic cavity.

1	When patients present with ascites for the first time, it is recommended that a diagnostic paracentesis be performed to characterize the fluid. This should include the determination of total protein and albumin content, blood cell counts with differential, and cultures. In the appropriate setting, amylase may be measured and cytology performed. In patients with cirrhosis, the protein concentration of the ascitic fluid is quite low, with the majority of patients having an ascitic fluid protein concentration <1 g/dL. The development of the serum ascites-to-albumin gradient (SAAG) has replaced the description of exudative or transudative fluid. When the gradient between the serum albumin level and the ascitic fluid albumin level is >1.1 g/dL, the cause of the ascites is most likely due to portal hypertension; this is usually in the setting of cirrhosis. When the gradient is <1.1 g/dL, infectious or malignant causes of ascites should be considered. When levels of ascitic fluid proteins

1	portal hypertension; this is usually in the setting of cirrhosis. When the gradient is <1.1 g/dL, infectious or malignant causes of ascites should be considered. When levels of ascitic fluid proteins are very low, patients are at increased risk for developing SBP. A high level of red blood cells in the ascitic fluid signifies a traumatic tap or perhaps a hepatocellular cancer or a ruptured omental varix. When the absolute level of polymorphonuclear leukocytes is >250/µL, the question of ascitic fluid infection should be strongly considered. Ascitic fluid cultures should be obtained using bedside inoculation of culture media.

1	Patients with small amounts of ascites can usually be managed with dietary sodium restriction alone. Most average diets in the United States contain 6–8 g of sodium per day, and if patients eat at restaurants or fast-food outlets, the amount of sodium in their diet can exceed this amount. Thus, it is often extremely difficult to get patients to change their dietary habits to ingest <2 g of sodium per day, which is the recommended amount. Patients are frequently surprised to realize how much sodium is in the standard U.S. diet; thus, it is important to make educational pamphlets available to the patient. Often, a simple recommendation is to eat fresh or frozen foods, avoiding canned or processed foods, which are usually preserved with sodium. When a moderate amount of ascites is present, diuretic therapy is usually necessary. Traditionally, spironolactone at 100–200 mg/d as a single dose is started, and furosemide may be added at 40–80 mg/d, particularly in patients who have peripheral

1	therapy is usually necessary. Traditionally, spironolactone at 100–200 mg/d as a single dose is started, and furosemide may be added at 40–80 mg/d, particularly in patients who have peripheral edema. In patients who have never received diuretics before, the failure of the above-mentioned dosages suggests that they are not being compliant with a low-sodium diet. If compliance is confirmed and ascitic fluid is not being mobilized, spironolactone can be increased to 400–600 mg/d and furosemide increased to 120–160 mg/d. If ascites is still present with these dosages of diuretics in patients who are compliant with a low-sodium diet, then they are defined as having refractory ascites, and alternative treatment modalities including repeated large-volume paracentesis or a TIPS procedure should be considered (Fig. 365-5). Recent studies have shown that TIPS, while managing the ascites, does not improve survival in these patients. Unfortunately, TIPS is often associated with an increased

1	be considered (Fig. 365-5). Recent studies have shown that TIPS, while managing the ascites, does not improve survival in these patients. Unfortunately, TIPS is often associated with an increased frequency of hepatic encephalopathy and must be considered carefully on a case-by-case basis. The prognosis for patients with cirrhosis with ascites is poor, and some studies have shown that <50% of patients survive 2 years after the onset of ascites. Thus, there should be consideration for liver transplantation in patients with the onset of ascites.

1	SBP is a common and severe complication of ascites characterized by spontaneous infection of the ascitic fluid without an intraabdominal source. In patients with cirrhosis and ascites severe enough for hospitalization, SBP can occur in up to 30% of individuals and can have a 25% in-hospital mortality rate. Bacterial translocation is the presumed mechanism for development of SBP, with gut flora traversing the intestine FIGURE 365-5 Treatment of refractory ascites. In patients who develop azotemia in the course of receiving diuretics in the management of their ascites, some will require repeated large-volume paracentesis (LVP), some may be considered for transjugular intrahepatic portosystemic shunt (TIPS), and some would be good candidates for liver transplantation. These decisions are all individualized.

1	2066 into mesenteric lymph nodes, leading to bacteremia and seeding of the ascitic fluid. The most common organisms are Escherichia coli and other gut bacteria; however, gram-positive bacteria, including Streptococcus viridans, Staphylococcus aureus, and Enterococcus sp., can also be found. If more than two organisms are identified, secondary bacterial peritonitis due to a perforated viscus should be considered. The diagnosis of SBP is made when the fluid sample has an absolute neutrophil count >250/μL. Bedside cultures should be obtained when ascitic fluid is tapped. Patients with ascites may present with fever, altered mental status, elevated white blood cell count, and abdominal pain or discomfort, or they may present without any of these features. Therefore, it is necessary to have a high degree of clinical suspicion, and peritoneal taps are important for making the diagnosis. Treatment is with a second-generation cephalosporin, with cefotaxime being the most commonly used

1	have a high degree of clinical suspicion, and peritoneal taps are important for making the diagnosis. Treatment is with a second-generation cephalosporin, with cefotaxime being the most commonly used antibiotic. In patients with variceal hemorrhage, the frequency of SBP is significantly increased, and prophylaxis against SBP is recommended when a patient presents with upper GI bleeding. Furthermore, in patients who have had an episode(s) of SBP and recovered, once-weekly administration of antibiotics is used as prophylaxis for recurrent SBP.

1	The hepatorenal syndrome (HRS) is a form of functional renal failure without renal pathology that occurs in about 10% of patients with advanced cirrhosis or acute liver failure. There are marked disturbances in the arterial renal circulation in patients with HRS; these include an increase in vascular resistance accompanied by a reduction in systemic vascular resistance. The reason for renal vasoconstriction is most likely multifactorial and is poorly understood. The diagnosis is made usually in the presence of a large amount of ascites in patients who have a stepwise progressive increase in creatinine. Type 1 HRS is characterized by a progressive impairment in renal function and a significant reduction in creatinine clearance within 1–2 weeks of presentation. Type 2 HRS is characterized by a reduction in glomerular filtration rate with an elevation of serum creatinine level, but it is fairly stable and is associated with a better outcome than that of type 1 HRS.

1	HRS is often seen in patients with refractory ascites and requires exclusion of other causes of acute renal failure. Treatment has, unfortunately, been difficult, and in the past, dopamine or prostaglandin analogues were used as renal vasodilating medications. Carefully performed studies have failed to show clear-cut benefit from these therapeutic approaches. Currently, patients are treated with midodrine, an α-agonist, along with octreotide and intravenous albumin. The best therapy for HRS is liver transplantation; recovery of renal function is typical in this setting. In patients with either type 1 or type 2 HRS, the prognosis is poor unless transplant can be achieved within a short period of time.

1	Portosystemic encephalopathy is a serious complication of chronic liver disease and is broadly defined as an alteration in mental status and cognitive function occurring in the presence of liver failure. In acute liver injury with fulminant hepatic failure, the development of encephalopathy is a requirement for a diagnosis of fulminant failure. Encephalopathy is much more commonly seen in patients with chronic liver disease. Gut-derived neurotoxins that are not removed by the liver because of vascular shunting and decreased hepatic mass get to the brain and cause the symptoms that we know of as hepatic encephalopathy. Ammonia levels are typically elevated in patients with hepatic encephalopathy, but the correlation between severity of liver disease and height of ammonia levels is often poor, and most hepatologists do not rely on ammonia levels to make a diagnosis. Other compounds and metabolites that may contribute to the development of encephalopathy include certain false

1	is often poor, and most hepatologists do not rely on ammonia levels to make a diagnosis. Other compounds and metabolites that may contribute to the development of encephalopathy include certain false neurotransmitters and mercaptans.

1	Clinical Features In acute liver failure, changes in mental status can occur within weeks to months. Brain edema can be seen in these patients, with severe encephalopathy associated with swelling of the gray matter. Cerebral herniation is a feared complication of brain edema in acute liver failure, and treatment is meant to decrease edema with mannitol and judicious use of intravenous fluids.

1	In patients with cirrhosis, encephalopathy is often found as a result of certain precipitating events such as hypokalemia, infection, an increased dietary protein load, or electrolyte disturbances. Patients may be confused or exhibit a change in personality. They may actually be quite violent and difficult to manage; alternatively, patients may be very sleepy and difficult to rouse. Because precipitating events are so commonly found, they should be sought carefully. If patients have ascites, this should be tapped to rule out infection. Evidence of GI bleeding should be sought, and patients should be appropriately hydrated. Electrolytes should be measured and abnormalities corrected. In patients presenting with encephalopathy, asterixis is often present. Asterixis can be elicited by having patients extend their arms and bend their wrists back. In this maneuver, patients who are encephalopathic have a “liver flap”—i.e., a sudden forward movement of the wrist. This requires patients to

1	patients extend their arms and bend their wrists back. In this maneuver, patients who are encephalopathic have a “liver flap”—i.e., a sudden forward movement of the wrist. This requires patients to be able to cooperate with the examiner and obviously cannot be elicited in patients who are severely encephalopathic or in hepatic coma.

1	The diagnosis of hepatic encephalopathy is clinical and requires an experienced clinician to recognize and put together all of the various features. Often when patients have encephalopathy for the first time, they are unaware of what is transpiring, but once they have been through the experience for the first time, they can identify when this is developing in subsequent situations and can often self-medicate to impair the development or worsening of encephalopathy.

1	Treatment is multifactorial and includes management of the above-mentioned precipitating factors. Sometimes hydration and correction of electrolyte imbalance are all that is necessary. In the past, restriction of dietary protein was considered for patients with encephalopathy; however, the negative impact of that maneuver on overall nutrition is thought to outweigh the benefit when treating encephalopathy, and it is thus discouraged. There may be some benefit to replacing animal-based protein with vegetable-based protein in some patients with encephalopathy that is difficult to manage. The mainstay of treatment for encephalopathy, in addition to correcting precipitating factors, is to use lactulose, a nonabsorbable disaccharide, which results in colonic acidification. Catharsis ensues, contributing to the elimination of nitrogenous products in the gut that are responsible for the development of encephalopathy. The goal of lactulose therapy is to promote 2–3 soft stools per day.

1	contributing to the elimination of nitrogenous products in the gut that are responsible for the development of encephalopathy. The goal of lactulose therapy is to promote 2–3 soft stools per day. Patients are asked to titrate their amount of ingested lactulose to achieve the desired effect. Poorly absorbed antibiotics are often used as adjunctive therapies for patients who have had a difficult time with lactulose. The alternating administration of neomycin and metronidazole has commonly been used to reduce the individual side effects of each: neomycin for renal insufficiency and ototoxicity and metronidazole for peripheral neuropathy. More recently, rifaximin at 550 mg twice daily has been very effective in treating encephalopathy without the known side effects of neomycin or metronidazole. Zinc supplementation is sometimes helpful in patients with encephalopathy and is relatively harmless. The development of encephalopathy in patients with chronic liver disease is a poor prognostic

1	Zinc supplementation is sometimes helpful in patients with encephalopathy and is relatively harmless. The development of encephalopathy in patients with chronic liver disease is a poor prognostic sign, but this complication can be managed in the vast majority of patients.

1	Because the liver is principally involved in the regulation of protein and energy metabolism in the body, it is not surprising that patients with advanced liver disease are commonly malnourished. Once patients become cirrhotic, they are more catabolic, and muscle protein is metabolized. There are multiple factors that contribute to the malnutrition of cirrhosis, including poor dietary intake, alterations in gut nutrient absorption, and alterations in protein metabolism. Dietary supplementation for patients with cirrhosis is helpful in preventing patients from becoming catabolic.

1	Coagulopathy is almost universal in patients with cirrhosis. There is decreased synthesis of clotting factors and impaired clearance of anticoagulants. In addition, patients may have thrombocytopenia from hypersplenism due to portal hypertension. Vitamin K–dependent clotting factors are factors II, VII, IX, and X. Vitamin K requires biliary excretion for its subsequent absorption; thus, in patients with chronic cholestatic syndromes, vitamin K absorption is frequently diminished. Intravenous or intramuscular vitamin K can quickly correct this abnormality. More commonly, the synthesis of vitamin K–dependent clot ting factors is diminished because of a decrease in hepatic mass, and, under these circumstances, administration of parenteral vitamin K does not improve the clotting factors or the prothrombin time. Platelet function is often abnormal in patients with chronic liver disease, in addition to decreases in platelet levels due to hypersplenism.

1	Osteoporosis is common in patients with chronic cholestatic liver disease because of malabsorption of vitamin D and decreased calcium ingestion. The rate of bone resorption exceeds that of new bone formation in patients with cirrhosis, resulting in bone loss. Dual x-ray absorptiometry (DEXA) is a useful method for determining osteoporosis or osteopenia in patients with chronic liver disease. When a DEXA scan shows decreased bone mass, treatment should be administered with bisphosphonates that are effective at inhibiting resorption of bone and efficacious in the treatment of osteoporosis. Numerous hematologic manifestations of cirrhosis are present, including anemia from a variety of causes including hypersplenism, hemolysis, iron deficiency, and perhaps folate deficiency from malnutrition. Macrocytosis is a common abnormality in red blood cell morphology seen in patients with chronic liver disease, and neutropenia may be seen as a result of hypersplenism.

1	and/or nuclei, viral inclusion bodies, copper or iron deposition, other 366e-1 Atlas of Liver Biopsies inclusion bodies) and others involving the portal tracts (e.g., the Jules L. Dienstag, Atul K. Bhan the border of periportal hepatocytes in chronic hepatitis C, autoim- Although clinical and laboratory features yield clues to the extent of inflammatory processes (disease grade), the degree of scarring and architectural distortion (disease stage), and the nature of the disease process, the liver biopsy is felt to represent the gold standard for assessing the degree of liver injury and fibrosis. Examination of liver histology provides not only a basis for quantitative scoring of disease activity and progression but also a wealth of qualitative information that can direct and inform diagnosis and management.

1	A normal liver lobule consists of portal (zone 1), lobular (midzonal or zone 2), and central (zone 3) zones. The portal tract contains the hepatic artery (HA) and portal vein (PV), which represent the dual vascular supply to the liver, as well as the bile duct (BD). The lobular area contains cords of liver cells surrounded by vascular sinusoids, and the central zone consists of the central vein (CV), the terminal branch of the hepatic vein (see figure below).

1	Included in this atlas of liver biopsies are examples of common morphologic features of acute and chronic liver disorders, some involving the lobular areas (e.g., the lobular inflammatory changes of acute hepatitis, apoptotic hepatocyte degeneration in acute and chronic hepatitis, virus antigen localization in hepatocyte cytoplasm mune hepatitis, and liver allograft rejection) or centrizonal areas (e.g., acute acetaminophen hepatotoxicity). Other histologic features of importance include hepatic steatosis (observed in alcoholic liver injury, nonalcoholic fatty liver disorders, and metabolic disorders–including mitochondrial injury–and in patients with chronic viral hepatitis); injury of bile ducts in the portal tract, an important diagnostic hallmark of primary biliary cirrhosis, primary sclerosing cholangitis, and liver allograft rejection; cholestasis in intrahepatic or extrahepatic biliary obstruction or in infiltrative disorders; ductular proliferation in the setting of marked

1	sclerosing cholangitis, and liver allograft rejection; cholestasis in intrahepatic or extrahepatic biliary obstruction or in infiltrative disorders; ductular proliferation in the setting of marked hepatocellular necrosis; plasma cell infiltration common in autoimmune hepatitis; portal inflammation affecting portal veins (“endothelialitis”) in liver allograft rejection; and mild-to-severe fibrosis, in varying distribution and pattern, as a consequence of liver injury common to many disorders. (All magnifications reflect the objective lens used.)

1	Figure 366e-2 Acute hepatitis, higher magnification, showing lobu-lar inflammation, hepatocellular ballooning, and acidophilic bodies (arrows) (H&E, 20×). CHAPTER 366e Atlas of Liver Biopsies Figure 366e-1 Acute hepatitis with lobular inflammation and hepa-tocellular ballooning (hematoxylin and eosin [H&E], 10×). Figure 366e-3 Chronic hepatitis C with portal lymphoid infiltrate and lymphoid follicle containing germinal center (H&E, 10×). PART 14 Disorders of the Gastrointestinal System Figure 366e-4 Chronic hepatitis C with portal and lobular inflam-mation and steatosis (H&E, 10×). Figure 366e-7 Chronic hepatitis B with hepatocellular cytoplasmic staining for hepatitis B surface antigen (immunoperoxidase, 20×). Figure 366e-5 Chronic hepatitis C with portal inflammation and interface hepatitis (erosion of the limiting plate of periportal hepato-cytes by infiltrating mononuclear cells) (H&E, 20×).

1	Figure 366e-5 Chronic hepatitis C with portal inflammation and interface hepatitis (erosion of the limiting plate of periportal hepato-cytes by infiltrating mononuclear cells) (H&E, 20×). Figure 366e-8 Chronic hepatitis B with hepatocellular nuclear stain-ing for hepatitis B core antigen (immunoperoxidase, 20×). Figure 366e-6 Lobular inflammation with acidophilic body (apop-totic body) surrounded by lymphoid cells (H&E, 40×). Figure 366e-9 Autoimmune hepatitis with portal and lobular inflammation, interface hepatitis, and cholestasis (H&E, 10×). Figure 366e-10 Autoimmune hepatitis, higher magnification, showing dense plasma cell infiltrate in the portal and periportal regions (H&E, 40×). Figure 366e-13 Cirrhosis with architectural alteration resulting from fibrosis and nodular hepatocellular regeneration (Masson trichrome, 2×).

1	Figure 366e-13 Cirrhosis with architectural alteration resulting from fibrosis and nodular hepatocellular regeneration (Masson trichrome, 2×). Figure 366e-11 Primary biliary cirrhosis with degenerating bile duct epithelium (“florid ductular lesion”) (arrow) surrounded by epi-thelioid granulomatous reaction and lymphoplasmacytic infiltrate (H&E, 40×). Figure 366e-14 Acute cellular rejection of orthotopic liver allograft demonstrating a mixed inflammatory cell infiltrate (lymphoid cells, eosinophils, neutrophils) of the portal tract as well as endothelialitis of the portal vein (arrow) and bile duct injury (H&E, 10×). Figure 366e-15 Liver allograft with cytomegalovirus infection showing hepatocytes with nuclear inclusions (arrows) surrounded by a neutrophilic and lymphoid infiltrate (H&E, 10×). Figure 366e-12 Chronic hepatitis C with bridging fibrosis (arrow) (Masson trichrome, 10×). CHAPTER 366e Atlas of Liver Biopsies PART 14 Disorders of the Gastrointestinal System

1	Figure 366e-12 Chronic hepatitis C with bridging fibrosis (arrow) (Masson trichrome, 10×). CHAPTER 366e Atlas of Liver Biopsies PART 14 Disorders of the Gastrointestinal System Figure 366e-16 Combined acetaminophen hepatotoxicity and alcoholic liver injury with extensive centrilobular areas of necrosis (H&E, 4×). Figure 366e-19 α1 Antitrypsin deficiency with higher magnification of PAS-positive, diastase-resistant globules (PAS, 40×). Figure 366e-17 Combined acetaminophen hepatotoxicity and alcoholic liver injury at higher magnification showing necrotic cen-trilobular area with Mallory bodies (H&E 20×). Figure 366e-20 Cirrhosis secondary to hemochromatosis with hepatocellular carcinoma; brown hemosiderin pigment (iron) is pres-ent in the cirrhotic liver, while the hepatocellular carcinoma nodules are hemosiderin-free (H&E, 4×).

1	Figure 366e-18 α1 Antitrypsin deficiency with cytoplasmic periodic acid–Schiff (PAS)-positive, diastase-resistant globules in many hepato-cytes, predominantly at the periphery of a cirrhotic nodule (PAS, 20×). Figure 366e-21 Cirrhosis secondary to hemochromatosis with hepatocellular carcinoma at higher magnification, demonstrating nodules of large malignant cells with highly disorganized architecture (H&E, 10×). Figure 366e-22 Hemochromatosis with iron stain demonstrating extensive iron deposition and characteristic pattern of pericanalicular distribution of iron (iron stain, 10×). Figure 366e-25 Primary sclerosing cholangitis showing peripheral cholestasis (green) and cytoplasmic red granular staining of hepatocytes for copper (rhodamine copper stain, 20×). Figure 366e-23 Primary sclerosing cholangitis showing cirrhosis and periductular fibrosis (Masson trichrome, 4×).

1	Figure 366e-23 Primary sclerosing cholangitis showing cirrhosis and periductular fibrosis (Masson trichrome, 4×). Figure 366e-26 Nonalcoholic steatohepatitis (NASH) showing steatosis, ballooned hepatocytes, and Mallory bodies with surrounding polymorphonuclear leukocytes (arrow) (H&E, 20×). Figure 366e-24 Primary sclerosing cholangitis showing the extra-hepatic bile duct (in a liver explant obtained at the time of hepatec-tomy for orthotopic liver transplantation) with marked mural chronic inflammation and fibrosis as well as peribiliary glands (H&E, 2×). Figure 366e-27 Nonalcoholic steatohepatitis (NASH) showing steatosis with perisinusoidal and pericellular fibrosis (H&E, 20×). CHAPTER 366e Atlas of Liver Biopsies PART 14 Disorders of the Gastrointestinal System

1	CHAPTER 366e Atlas of Liver Biopsies PART 14 Disorders of the Gastrointestinal System Figure 366e-28 Acute hepatitis with submassive hepatic necrosis Figure 366e-29 Wilson’s disease showing cirrhosis, extensive col-with marked parenchymal collapse, remnant islands of surviving lapse, and ductular reaction in a teenager with an acute presentation hepatocytes, and a marked ductular reaction (H&E, 10×). (H&E, 4×). Figure 366e-30 Wilson’s disease showing extensive hepatocyte cytoplasmic red granular staining for copper in a cirrhotic nodule (rhodanine copper stain, 20×).

1	Figure 366e-30 Wilson’s disease showing extensive hepatocyte cytoplasmic red granular staining for copper in a cirrhotic nodule (rhodanine copper stain, 20×). Genetic, Metabolic, and Infiltrative Diseases Affecting the Liver Bruce R. Bacon There are a number of disorders of the liver that fit within the 367e categories of genetic, metabolic, and infiltrative disorders (Table 367e-1). Inherited disorders include hemochromatosis, Wilson’s disease, α1 antitrypsin (α1AT) deficiency, and cystic fibrosis (CF). Hemochromatosis is the most common inherited disorder affecting white populations, with the genetic susceptibility for the disease being identified in 1 in 250 individuals. Over the past 15 years, it has become increasingly apparent that nonalcoholic fatty liver disease (NAFLD) is the most common cause of elevated liver enzymes found in the U.S. population. This disorder is discussed in greater detail in Chap. 364. Infiltrative disorders of the liver are relatively rare.

1	GENETIC LIVER DISEASES Hereditary Hemochromatosis Hereditary hemochromatosis (HH) is a common inherited disorder of iron metabolism (Chap. 428). Our knowledge of the disease and its phenotypic expression has changed since 1996, when the gene for HH, called HFE, was identified, allowing for genetic testing for the two major mutations (C282Y and H63D) that are responsible for HFE-related HH. Subsequently, several additional genes/proteins involved in the regulation of iron homeostasis have been identified, contributing to a better understanding of cellular iron uptake and release and the characterization of additional causes of inherited iron overload (Table 367e-2).

1	Most patients with HH are asymptomatic; however, when patients present with symptoms, they are frequently nonspecific and include weakness, fatigue, lethargy, and weight loss. Specific, organ-related symptoms include abdominal pain, arthralgias, and symptoms and signs of chronic liver disease. Increasingly, most patients are now identified before they have symptoms, either through family studies or from the performance of screening iron studies. Several prospective population studies have shown that C282Y homozygosity is found in about 1 in 250 individuals of northern European descent, with the heterozygote frequency seen in approximately 1 in 10 individuals. It is important to consider HH in patients who present with the symptoms TAbLE 367e-1 GEnETIC, METAboLIC, AnD InfILTRATIvE DIsEAsEs AffECTInG THE LIvER Abbreviations: HAMP, hepcidin; HJV, hemojuvelin; TfR2, transferrin receptor 2.

1	and signs known to occur in established HH. When confronted with abnormal serum iron studies, clinicians should not wait for typical symptoms or findings of HH to appear before considering the diagnosis. However, once the diagnosis of HH is considered, either by an evaluation of abnormal screening iron studies in the context of family studies, in a patient with an abnormal genetic test, or in the evaluation of a patient with any of the typical symptoms (Table 367e-3) or clinical findings (Table 367e-4), definitive diagnosis is relatively straightforward. Transferrin saturation (serum iron divided by total iron-binding capacity [TIBC] or transferrin, times 100%) and ferritin levels should be obtained. Both of these will be elevated in a symptomatic patient. It must be remembered that ferritin is an acute-phase reactant and can be elevated in a number of other inflammatory disorders, such as rheumatoid arthritis, or in various neoplastic diseases, such as lymphoma or other cancers.

1	is an acute-phase reactant and can be elevated in a number of other inflammatory disorders, such as rheumatoid arthritis, or in various neoplastic diseases, such as lymphoma or other cancers. Also, serum ferritin is elevated in a majority of patients with nonalcoholic steatohepatitis (NASH), hepatitis C, and alcoholic liver disease in the absence of iron overload.

1	At present, if patients have an elevated transferrin saturation or ferritin level, genetic testing should be performed; if they are a C282Y homozygote or a compound heterozygote (C282Y/H63D), the diagnosis is confirmed. If liver enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST]) are elevated or the ferritin is >1000 μg/L, the patient should be considered for liver biopsy because there is an increased frequency of advanced fibrosis in these individuals. If liver biopsy is performed, iron deposition is found in a periportal distribution with a periportal to pericentral gradient; iron is found predominantly in parenchymal cells, and Kupffer cells are spared. CHAPTER 367e Genetic, Metabolic, and Infiltrative Diseases Affecting the Liver

1	CHAPTER 367e Genetic, Metabolic, and Infiltrative Diseases Affecting the Liver Hepatomegaly 60–85 Cirrhosis 50–95 Skin pigmentation 40–80 Arthritis (second, third metacarpophalangeal joints) 40–60 Clinical diabetes 10–60 Splenomegaly 10–40 Loss of body hair 10–30 Testicular atrophy 10–30 Dilated cardiomyopathy 0–30 Treatment of HH is relatively straightforward with weekly phlebotomy aimed to reduce iron stores, recognizing that each unit of blood contains 200–250 mg of iron. If patients are diagnosed and treated before the development of hepatic fibrosis, all complications of the disease can be avoided. Maintenance phlebotomy is required in most patients and usually can be achieved with 1 unit of blood removed every 2–3 months. Family studies should be performed with transferrin saturation, ferritin, and genetic testing offered to all first-degree relatives.

1	Wilson’s Disease Wilson’s disease is an inherited disorder of copper homeostasis first described in 1912 (Chap. 429). The Wilson’s disease gene was discovered in 1993, with the identification of ATP7B. This P-type ATPase is involved in copper transport and is necessary for the export of copper from the hepatocyte. Thus, in patients with mutations in ATP7B, copper is retained in the liver, leading to increased copper storage and ultimately liver disease as a result.

1	The clinical presentation of Wilson’s disease is variable and includes chronic hepatitis, hepatic steatosis, and cirrhosis in adolescents and young adults. Neurologic manifestations indicate that liver disease is present and include speech disorders and various movement disorders. Diagnosis includes the demonstration of a reduced ceruloplasmin level, increased urinary excretion of copper, the presence of Kayser-Fleischer rings in the corneas of the eyes, and an elevated hepatic copper level, in the appropriate clinical setting. The genetic diagnosis of Wilson’s disease is difficult because >500 mutations in ATP7B have been described with different degrees of frequency and penetration in certain populations. PART 14 Disorders of the Gastrointestinal System

1	PART 14 Disorders of the Gastrointestinal System Treatment consists of copper-chelating medications such as D-penicillamine and trientine. A role for zinc acetate has also been established. Medical treatment is lifelong, and severe relapses leading to liver failure and death can occur with cessation of therapy. Liver transplantation is curative with respect to the underlying metabolic defect and restores the normal phenotype with respect to copper homeostasis.

1	α1 Antitrypsin Deficiency α1AT deficiency was first described in the late 1960s in patients with severe pulmonary disease. α1AT is a 52-kDa glycoprotein produced in hepatocytes, phagocytes, and epithelial cells in the lungs, which inhibits serine proteases, primarily neutrophil elastase. In α1AT deficiency, increased amounts of neutrophil elastase can result in progressive lung injury from degradation of elastin, leading to premature emphysema. In the 1970s, α1AT deficiency was discovered as a cause of neonatal liver disease, so-called “neonatal hepatitis.” It is now known to be a cause of liver disease in infancy, early childhood, and adolescence, and in adults.

1	In α1AT deficiency, variants in the proteinase inhibitor (Pi) gene located on chromosome 14 alter α1AT structure, interfering with hepatocellular export. Aggregated, deformed polymers of α1AT accumulate in the hepatocyte endoplasmic reticulum. There are over 75 different α1AT variants. Conventional nomenclature identifies normal variants as PiMM; these individuals have normal blood levels of α1AT. The most common abnormal variants are called S and Z. Individuals homozygous for the Z mutation (PiZZ) have low levels of α1AT (about 15% of normal), and these patients are susceptible to liver and/or lung disease, yet only a proportion (about 25%) of PiZZ patients develop disease manifestations. Null variants have undetectable levels of α1AT and are susceptible to premature lung disease.

1	α1AT deficiency has been identified in all populations; however, the disorder is most common in patients of northern European and Iberian descent. The disorder affects about 1 in 1500 to 2000 individuals in North America. The natural history of α1AT deficiency is quite variable because many individuals with the PiZZ variant never develop disease, whereas others can develop childhood cirrhosis leading to liver transplantation. In adults, the diagnosis often comes in the course of evaluation of patients with abnormal liver test abnormalities or in a workup for cirrhosis. A hint to diagnosis may be coexistent lung disease at a relatively young age or a family history of liver and/or lung disease. Patients may have symptoms of pulmonary disease with cough and dyspnea. Liver disease may be asymptomatic other than fatigue, or patients may present with complications of decompensated liver disease.

1	Diagnosis of α1AT deficiency is confirmed by blood tests showing reduced levels of serum α1AT, accompanied by Pi determinations. Most patients with liver disease have either PiZZ or PiSZ; occasionally, patients with PiMZ have reduced levels of α1AT, but they usually do not have a low enough level to cause disease. Liver biopsy is often performed to determine stage of hepatic fibrosis and shows characteristic PAS-positive, diastase-resistant globules in the periphery of the hepatic lobule.

1	Treatment of α1AT deficiency is usually nonspecific and supportive. For patients with liver involvement, other sources of liver injury, such as alcohol, should be avoided. Evidence for other liver diseases (e.g., viral hepatitis B and C, hemochromatosis, NAFLD) should be sought and treated if possible. Smoking can worsen lung disease progression in α1AT deficiency and should be discontinued. Patients with lung disease may be eligible to receive infusions of α1AT, which has been shown to halt further damage to the lungs. If liver disease becomes decompensated, transplantation should be pursued and is curative. Following transplant, patients express the Pi phenotype of the donor. Finally, risk of hepatocellular carcinoma is significantly increased in patients with cirrhosis due to α1AT deficiency.

1	Cystic Fibrosis CF should also be considered as an inherited form of chronic liver disease, although the principal manifestations of CF include chronic lung disease and pancreatic insufficiency (Chap. 313). A small percentage of patients with CF who survive to adulthood have a form of biliary cirrhosis characterized by cholestatic liver enzyme abnormalities and the development of chronic liver disease. Ursodeoxycholic acid is occasionally helpful in improving liver test abnormalities and in reducing symptoms. The disease is slowly progressive. METABOLIC LIVER DISEASES Nonalcoholic Fatty Liver Disease NAFLD and NASH are common liver diseases causing abnormal liver test results and progressing to cirrhosis. NAFLD and NASH are discussed in detail in Chap. 364.

1	Lipid Storage Diseases There are a number of rare lipid storage diseases that involve the liver, including the inherited disorders of Gaucher’s disease and Niemann-Pick disease (Chap. 433e). Other rare disorders include abetalipoproteinemia, Tangier disease, Fabry’s disease, and types I and V hyperlipoproteinemia (Table 367e–5). Hepatomegaly is present due to increased fat deposition, and increased glycogen is found in the liver.

1	Porphyrias The porphyrias are a group of metabolic disorders in which there are defects in the biosynthesis of heme necessary for incorporation into numerous hemoproteins such as hemoglobin, myoglobin, catalase, and the cytochromes (Chap. 430). Porphyrias can present as either acute or chronic diseases, with the acute disorder causing recurring bouts of abdominal pain, and the chronic disorders characterized by painful skin lesions. Porphyria cutanea tarda (PCT) is the most commonly encountered porphyria. Patients present with characteristic vesicular lesions on sun-exposed areas of the skin, principally the dorsum of the hands, the tips of the ears, or the cheeks. About 40% of patients with PCT have mutations in the gene for hemochromatosis (HFE), and ~50% have hepatitis C; thus, iron studies and HFE mutation analysis as well as hepatitis C testing should be considered in all patients who present with PCT. PCT is also associated with excess alcohol use and some medications, most

1	studies and HFE mutation analysis as well as hepatitis C testing should be considered in all patients who present with PCT. PCT is also associated with excess alcohol use and some medications, most notably estrogens.

1	The mainstay of treatment of PCT is iron reduction by therapeutic phlebotomy, which is successful in reversing the skin lesions in the majority of patients. If hepatitis C is present, this should be treated as well. Acute intermittent porphyria presents with abdominal pain, with the diagnosis made by avoidance of certain precipitating factors such as starvation or certain diets. Intravenous heme as hematin has been used for treatment. INFILTRATIVE DISORDERS Amyloidosis Amyloidosis is a metabolic storage disease that results from deposition of insoluble proteins that are aberrantly folded and assembled and then deposited in a variety of tissues (Chap. 136).

1	Amyloidosis is divided into two types, primary and secondary, based 367e-3 on the broad concepts of association with myeloma (primary) or chronic inflammatory illnesses (secondary). The disease is generally considered rare, although, in certain disease states or in certain populations, it can be more common. For example, when associated with familial Mediterranean fever, it is seen in high frequency in Sephardic Jews and Armenians living in Armenia and less frequently in Ashkenazi Jews, Turks, and Arabs. Amyloidosis frequently affects patients suffering from tuberculosis and leprosy and can be seen in upwards of 10–15% of patients with ankylosing spondylitis, rheumatoid arthritis, or Crohn’s disease. In one surgical pathology series, amyloid was found in <1% of cases. The liver is commonly involved in cases of systemic amyloidosis, but it is frequently not clinically apparent and only documented at autopsy. Pathologic findings in the liver include positive staining with the Congo red

1	involved in cases of systemic amyloidosis, but it is frequently not clinically apparent and only documented at autopsy. Pathologic findings in the liver include positive staining with the Congo red histochemical stain where there is an apple-green birefringence noted under polarizing light.

1	Granulomas Granulomas are frequently found in the liver when patients are being evaluated for cholestatic liver enzyme abnormalities. Granulomas can be seen in primary biliary cirrhosis, but there are other characteristic clinical (e.g., pruritus, fatigue) and laboratory findings (cholestatic liver tests, antimitochondrial antibody) that allow for a definitive diagnosis of that disorder. Granulomatous infiltration can also be seen as the principal hepatic manifestation of sarcoidosis, and this is the most common presentation of hepatic granulomas (Chap. 390). The vast majority of these patients do not require any specific treatment other than what would normally be used for treatment of their sarcoidosis. A small subset, however, can develop a particularly bothersome desmoplastic reaction with a significant increase in fibrosis, which can progress to cirrhosis and liver failure. These patients may require treatment with immunosuppressive therapy and may require liver transplantation.

1	with a significant increase in fibrosis, which can progress to cirrhosis and liver failure. These patients may require treatment with immunosuppressive therapy and may require liver transplantation. In patients who have granulomas in the liver not associated with sarcoidosis, treatment is rarely needed.

1	Diagnosis requires liver biopsy, and it is important to establish a diagnosis so that a cause for the elevated liver enzymes is carefully identified. Some medications can cause granulomatous infiltration of the liver, the most notable of which is allopurinol. Lymphoma Involvement of the liver with lymphoma can sometimes be with bulky mass lesions but can also be as a difficult-to-diagnose infiltrative disorder that does not show any characteristic findings on abdominal imaging studies (Chap. 134). Patients may present with severe liver disease, jaundice, hypoalbuminemia, mild to moderately elevated aminotransferases, and an elevated alkaline phosphatase. A liver biopsy is required for diagnosis and should be considered when routine blood testing does not lead to a diagnosis of the liver dysfunction. CHAPTER 367e Genetic, Metabolic, and Infiltrative Diseases Affecting the Liver

1	Liver TransplantationCHAPTER 368liver Transplantation Raymond T. Chung, Jules L. Dienstag Liver transplantation—the replacement of the native, diseased liver by a normal organ (allograft)—has matured from an experimental pro-cedure reserved for desperately ill patients to an accepted, lifesaving operation applied more optimally in the natural history of end-stage 368 liver disease. The preferred and technically most advanced approach is orthotopic transplantation, in which the native organ is removed and the donor organ is inserted in the same anatomic location. Pioneered in the 1960s by Thomas Starzl at the University of Colorado and, later, at the University of Pittsburgh and by Roy Calne in Cambridge, England, liver transplantation is now performed routinely worldwide. Success measured as 1-year survival has improved from ~30% in the 1970s to >90% today. These improved prospects for prolonged survival resulted from refinements in operative technique, improvements in organ

1	measured as 1-year survival has improved from ~30% in the 1970s to >90% today. These improved prospects for prolonged survival resulted from refinements in operative technique, improvements in organ procurement and preservation, advances in immunosuppressive therapy, and, perhaps most influentially, more enlightened patient selection and timing. Despite the perioperative morbidity and mortality, the technical and management challenges of the procedure, and its costs, liver transplantation has become the approach of choice for selected patients whose chronic or acute liver disease is progressive, life-threatening, and unresponsive to medical therapy. Based on the current level of success, the number of liver transplants has continued to grow each year; in 2012, 6256 patients received liver allografts in the United States. Still, the demand for new livers continues to outpace availability; as of mid-2013, 15,806 patients in the United States were on a waiting list for a donor liver. In

1	allografts in the United States. Still, the demand for new livers continues to outpace availability; as of mid-2013, 15,806 patients in the United States were on a waiting list for a donor liver. In response to this drastic shortage of donor organs, many transplantation centers supplement cadaver-organ liver transplantation with living-donor transplantation.

1	Potential candidates for liver transplantation are children and adults who, in the absence of contraindications (see below), suffer from severe, irreversible liver disease for which alternative medical or surgical treatments have been exhausted or are unavailable. Timing of the operation is of critical importance. Indeed, improved timing and better patient selection are felt to have contributed more to the increased 2068 success of liver transplantation in the 1980s and beyond than all the impressive technical and immunologic advances combined. Although the disease should be advanced, and although opportunities for spontaneous or medically induced stabilization or recovery should be allowed, the procedure should be done sufficiently early to give the surgical procedure a fair chance for success. Ideally, transplantation should be considered in patients with end-stage liver disease who are experiencing or have experienced a life-threatening complication of hepatic decompensation or

1	success. Ideally, transplantation should be considered in patients with end-stage liver disease who are experiencing or have experienced a life-threatening complication of hepatic decompensation or whose quality of life has deteriorated to unacceptable levels. Although patients with well-compensated cirrhosis can survive for many years, many patients with quasi-stable chronic liver disease have much more advanced disease than may be apparent. As discussed below, the better the status of the patient prior to transplantation, the higher will be its anticipated success rate. The decision about when to transplant is complex and requires the combined judgment of an experienced team of hepatologists, transplant surgeons, anesthesiologists, and specialists in support services, not to mention the well-informed consent of the patient and the patient’s family.

1	Indications for transplantation in children are listed in Table 368-1. The most common is biliary atresia. Inherited or genetic disorders of metabolism associated with liver failure constitute another major indication for transplantation in children and adolescents. In Crigler-Najjar disease type I and in certain hereditary disorders of the urea cycle and of amino acid or lactate-pyruvate metabolism, transplantation may be the only way to prevent impending deterioration of central nervous system function, despite the fact that the native liver is structurally normal. Combined heart and liver transplantation has yielded dramatic improvement in cardiac function and in cholesterol levels in children with homozygous familial hypercholesterolemia; combined liver and kidney transplantation has been successful in patients with primary hyperoxaluria type I. In hemophiliacs with transfusion-associated hepatitis and liver failure, liver transplantation has been associated with recovery of

1	has been successful in patients with primary hyperoxaluria type I. In hemophiliacs with transfusion-associated hepatitis and liver failure, liver transplantation has been associated with recovery of normal factor VIII synthesis.

1	Liver transplantation is indicated for end-stage cirrhosis of all causes (Table 368-1). In sclerosing cholangitis and Caroli’s disease (multiple cystic dilatations of the intrahepatic biliary tree), recurrent infections and sepsis associated with inflammatory and fibrotic obstruction of the biliary tree may be an indication for transplantation. Because prior biliary surgery complicates and is a relative contraindication for liver transplantation, surgical diversion of the biliary tree has been all but abandoned for patients with sclerosing cholangitis. In patients who

1	Biliary atresia Primary biliary cirrhosis Neonatal hepatitis Secondary biliary cirrhosis Congenital hepatic fibrosis Primary sclerosing cholangitis Alagille’s syndromeα Autoimmune hepatitis Byler’s diseaseb Caroli’s diseasec α1-Antitrypsin deficiency Cryptogenic cirrhosis Inherited disorders of metabolism Chronic hepatitis with cirrhosis αArteriohepatic dysplasia, with paucity of bile ducts, and congenital malformations, including pulmonary stenosis. bIntrahepatic cholestasis, progressive liver failure, and mental and growth retardation. cMultiple cystic dilatations of the intrahepatic biliary tree.

1	undergo transplantation for hepatic vein thrombosis (Budd-Chiari syndrome), postoperative anticoagulation is essential; underlying myeloproliferative disorders may have to be treated but are not a contraindication to liver transplantation. If a donor organ can be located quickly, before life-threatening complications—including cerebral edema—set in, patients with acute liver failure are candidates for liver transplantation. Routine candidates for liver transplantation are patients with alcoholic cirrhosis, chronic viral hepatitis, and primary hepatocellular malignancies. Although all three of these categories are considered to be high risk, liver transplantation can be offered to carefully selected patients. Currently, chronic hepatitis C and alcoholic liver disease are the most common indications for liver transplantation, accounting for over 40% of all adult candidates who undergo the procedure. Patients with alcoholic cirrhosis can be considered as candidates for transplantation if

1	for liver transplantation, accounting for over 40% of all adult candidates who undergo the procedure. Patients with alcoholic cirrhosis can be considered as candidates for transplantation if they meet strict criteria for abstinence and reform; however, these criteria still do not prevent recidivism in up to a quarter of cases. In highly selected cases in a limited number of centers, transplantation for severe acute alcoholic hepatitis has been performed with success; however, because patients with acute alcoholic hepatitis are still actively using alcohol, and because continued alcohol abuse remains a concern, acute alcoholic hepatitis is not a routine indication for liver transplantation. Patients with chronic hepatitis C have early allograft and patient survival comparable to those of other subsets of patients after transplantation; however, reinfection in the donor organ is universal, recurrent hepatitis C is insidiously progressive, allograft cirrhosis develops in 20–30% at 5

1	of other subsets of patients after transplantation; however, reinfection in the donor organ is universal, recurrent hepatitis C is insidiously progressive, allograft cirrhosis develops in 20–30% at 5 years, and cirrhosis and late organ failure occur at a higher frequency beyond 5 years. With the introduction of highly effective direct acting antiviral agents targeting HCV, it is expected that allograft outcomes will improve significantly in the coming years. In patients with chronic hepatitis B, in the absence of measures to prevent recurrent hepatitis B, survival after transplantation is reduced by approximately 10–20%; however, prophylactic use of hepatitis B immune globulin (HBIg) during and after transplantation increases the success of transplantation to a level comparable to that seen in patients with nonviral causes of liver decompensation. Specific oral antiviral drugs (e.g., entecavir, tenofovir disoproxil fumarate) (Chap. 362) can be used both for prophylaxis against and for

1	in patients with nonviral causes of liver decompensation. Specific oral antiviral drugs (e.g., entecavir, tenofovir disoproxil fumarate) (Chap. 362) can be used both for prophylaxis against and for treatment of recurrent hepatitis B, facilitating further the management of patients undergoing liver transplantation for end-stage hepatitis B; most transplantation centers rely on antiviral drugs with or without HBIg to manage patients with hepatitis B. Issues of disease recurrence are discussed in more detail below. Patients with nonmetastatic primary hepatobiliary tumors—primary hepatocellular carcinoma (HCC), cholangiocarcinoma, hepatoblastoma, angiosarcoma, epithelioid hemangioendothelioma, and multiple or massive hepatic adenomata—have undergone liver transplantation; however, for some hepatobiliary malignancies, overall survival is significantly lower than that for other categories of liver disease. Most transplantation centers have reported 5-year recurrence-free survival rates in

1	malignancies, overall survival is significantly lower than that for other categories of liver disease. Most transplantation centers have reported 5-year recurrence-free survival rates in patients with unresectable HCC for single tumors <5 cm in diameter or for three or fewer lesions all <3 cm comparable to those seen in patients undergoing transplantation for nonmalignant indications. Consequently, liver transplantation is currently restricted to patients whose hepatic malignancies meet these criteria. Expanded criteria for patients with HCC continue to be evaluated. Because the likelihood of recurrent cholangiocarcinoma is very high, only highly selected patients with limited disease are being evaluated for transplantation after intensive chemotherapy and radiation.

1	Absolute contraindications for transplantation include life-threatening systemic diseases, uncontrolled extrahepatic bacterial or fungal infections, preexisting advanced cardiovascular or pulmonary disease, multiple uncorrectable life-threatening congenital anomalies, metastatic malignancy, and active drug or alcohol abuse (Table 368–2). Because carefully selected patients in their sixties and even seventies have undergone transplantation successfully, advanced age per se is no longer considered an absolute contraindication; however, in older patients a more thorough preoperative evaluation should be undertaken to Active, untreated sepsis Prior extensive hepatobiliary surgery Uncorrectable, life-limiting congeni-Portal vein thrombosis tal anomalies Active substance or alcohol abuse Renal failure not attributable to liver disease Metastatic malignancy to the liver Severe malnutrition/wasting AIDS HIV seropositivity with failure to control HIV viremia or CD4 <100/μL

1	Severe hypoxemia secondary to right-to-left intrapulmonary shunts (PO2 <50 mmHg) exclude ischemic cardiac disease and other comorbid conditions. Advanced age (>70 years), however, should be considered a relative contraindication—that is, a factor to be taken into account with other relative contraindications. Other relative contraindications include portal vein thrombosis, HIV infection, preexisting renal disease not associated with liver disease (which may prompt consideration of combined liver and kidney transplantation), intrahepatic or biliary sepsis, severe hypoxemia (Po2 <50 mmHg) resulting from right-toleft intrapulmonary shunts, portopulmonary hypertension with high mean pulmonary artery pressures (>35 mmHg), previous extensive hepatobiliary surgery, any uncontrolled serious psychiatric disorder, and lack of sufficient social supports. Any one of these relative contraindications is insufficient in and of itself to preclude transplantation. For example, the problem of portal

1	disorder, and lack of sufficient social supports. Any one of these relative contraindications is insufficient in and of itself to preclude transplantation. For example, the problem of portal vein thrombosis can be overcome by constructing a graft from the donor liver portal vein to the recipient’s superior mesenteric vein. Now that highly active antiretroviral therapy has dramatically improved the survival of persons with HIV infection (Chap. 226), and because end-stage liver disease caused by chronic hepatitis C and B has emerged as a serious source of morbidity and mortality in the HIV-infected population, liver transplantation has now been performed successfully in selected HIV-positive persons who have excellent control of HIV infection. Selected patients with CD4± T cell counts >100/μL and with pharmacologic suppression of HIV viremia have undergone transplantation for end-stage liver disease. HIV-infected persons who have received liver allografts for end-stage liver disease

1	and with pharmacologic suppression of HIV viremia have undergone transplantation for end-stage liver disease. HIV-infected persons who have received liver allografts for end-stage liver disease resulting from chronic hepatitis B have experienced survival rates compared to those of HIV-negative persons undergoing transplantation for the same indication. In contrast, recurrent hepatitis C virus (HCV) in the allograft has limited long-term success in persons with HCV-related end-stage liver disease. Again, it is expected that the availability of direct acting antiviral agents targeting HCV, will significantly improve allograft outcomes.

1	Cadaver donor livers for transplantation are procured primarily from victims of head trauma. Organs from brain-dead donors up to age 60 are acceptable if the following criteria are met: hemodynamic stability, adequate oxygenation, absence of bacterial or fungal infection, absence 2069 of abdominal trauma, absence of hepatic dysfunction, and serologic exclusion of hepatitis B (HBV) and C viruses and HIV. Occasionally, organs from donors with hepatitis B and C are used (e.g., for recipients with prior hepatitis B and C, respectively). Organs from donors with antibodies to hepatitis B core antigen (anti-HBc) can also be used when the need is especially urgent, and recipients of these organs are treated prophylactically with antiviral drugs. Cardiovascular and respiratory functions are maintained artificially until the liver can be removed. Transplantation of organs procured from deceased donors who have succumbed to cardiac death can be performed successfully under selected

1	are maintained artificially until the liver can be removed. Transplantation of organs procured from deceased donors who have succumbed to cardiac death can be performed successfully under selected circumstances, when ischemic time is minimized and liver histology preserved. Compatibility in ABO blood group and organ size between donor and recipient are important considerations in donor selection; however, ABO-incompatible, split liver, or reduced-donor-organ transplants can be performed in emergencies or marked donor scarcity. Tissue typing for human leukocyte antigen (HLA) matching is not required, and preformed cytotoxic HLA antibodies do not preclude liver transplantation. Following perfusion with cold electrolyte solution, the donor liver is removed and packed in ice. The use of University of Wisconsin (UW) solution, rich in lactobionate and raffinose, has permitted the extension of cold ischemic time up to 20 h; however, 12 h may be a more reasonable limit. Improved techniques

1	of Wisconsin (UW) solution, rich in lactobionate and raffinose, has permitted the extension of cold ischemic time up to 20 h; however, 12 h may be a more reasonable limit. Improved techniques for harvesting multiple organs from the same donor have increased the availability of donor livers, but the availability of donor livers is far outstripped by the demand. Currently in the United States, all donor livers are distributed through a nationwide organ-sharing network (United Network for Organ Sharing [UNOS]) designed to allocate available organs based on regional considerations and recipient acuity. Recipients who have the highest disease severity generally have the highest priority, but allocation strategies that balance highest urgency against best outcomes continue to evolve to distribute cadaver organs most effectively. Allocation based on the Child-Turcotte-Pugh (CTP) score, which uses five clinical variables (encephalopathy stage, ascites, bilirubin, albumin, and prothrombin

1	cadaver organs most effectively. Allocation based on the Child-Turcotte-Pugh (CTP) score, which uses five clinical variables (encephalopathy stage, ascites, bilirubin, albumin, and prothrombin time) and waiting time, has been replaced by allocation based on urgency alone, calculated by the Model for End-Stage Liver Disease (MELD) score. The MELD score is based on a mathematical model that includes bilirubin, creatinine, and prothrombin time expressed as international normalized ratio (INR) (Table 368-3). Neither waiting time (except as a tie breaker between two potential recipients with the same MELD scores) nor posttransplantation outcome is taken into account, but use of the MELD score

1	Status 1 Fulminant hepatic failure (including primary graft nonfunction and hepatic artery thrombosis within 7 days after transplantation as well as acute decompensated Wilson’s disease)α The Model for End-Stage Liver Disease (MELD) score, on a continuous scale,b determines allocation of the remainder of donor organs. This model is based on the following calculation: 3.78 × loge bilirubin (mg/100 mL) ± 11.2 × loge international normalized ratio (INR) ± 9.57 × loge creatinine (mg/100 mL) ± 6.43 (v 0 for alcoholic and cholestatic liver disease, × 1 for all other types of liver disease).c,d,e Online calculators to determine MELD scores are available, such as the following: http://optn.transplant.hrsa.gov/resources/professionalresources .asp?index=9.

1	aFor children <18 years of age, status 1 includes acute or chronic liver failure plus hospitalization in an intensive care unit or inborn errors of metabolism. Status 1 is retained for those persons with fulminant hepatic failure and supersedes the MELD score. bThe MELD scale is continuous, with 34 levels ranging between 6 and 40. Donor organs usually do not become available unless the MELD score exceeds 20. cPatients with stage T2 hepatocellular carcinoma receive 22 disease-specific points. dCreatinine is included because renal function is a validated predictor of survival in patients with liver disease. For adults undergoing dialysis twice a week, the creatinine in the equation is set to 4 mg/100 mL. eFor children <18 years of age, the Pediatric End-Stage Liver Disease (PELD) scale is used. This scale is based on albumin, bilirubin, INR, growth failure, and age. Status 1 is retained.

1	2070 has been shown to reduce waiting list mortality, to reduce waiting time prior to transplantation, to be the best predictor of pretransplantation mortality, to satisfy the prevailing view that medical need should be the decisive determinant, and to eliminate both the subjectivity inherent in the CTP scoring system (presence and degree of ascites and hepatic encephalopathy) and the differences in waiting times among different regions of the country. Recent data indicate that liver recipients with MELD scores <15 experienced higher posttransplantation mortality rates than similarly classified patients who remained on the wait list. This observation led to the modification of UNOS policy to allocate donor organs to candidates with MELD scores exceeding 15 within the local or regional procurement organization before offering the organ to local patients whose scores are <15. In addition, serum sodium, another important predictor of survival in liver transplantation candidates, is taken

1	organization before offering the organ to local patients whose scores are <15. In addition, serum sodium, another important predictor of survival in liver transplantation candidates, is taken into consideration in allocating donor livers. The highest priority (status 1) continues to be reserved for patients with fulminant hepatic failure or primary graft nonfunction. Because candidates for liver transplantation who have HCC may not be sufficiently decompensated to compete for donor organs based on urgency criteria alone, and because protracted waiting for cadaver donor organs often results in tumor growth beyond acceptable limits for transplantation, such patients are assigned disease-specific MELD points (Table 368–3). Other disease-specific MELD exceptions include portopulmonary hypertension, hepatopulmonary syndrome, familial amyloid polyneuropathy, primary hyperoxaluria (necessitating liver-kidney transplantation), cystic fibrosis liver disease, and highly selected cases of hilar

1	hepatopulmonary syndrome, familial amyloid polyneuropathy, primary hyperoxaluria (necessitating liver-kidney transplantation), cystic fibrosis liver disease, and highly selected cases of hilar cholangiocarcinoma.

1	Occasionally, especially for liver transplantation in children, one cadaver organ can be split between two recipients (one adult and one child). A more viable alternative, transplantation of the right lobe of the liver from a healthy adult donor into an adult recipient, has gained increased popularity. Living donor transplantation of the left lobe (left lateral segment), introduced in the early 1990s to alleviate the extreme shortage of donor organs for small children, accounts currently for approximately one-third of all liver transplantation procedures in children. Driven by the shortage of cadaver organs, living donor transplantation involving the more sizable right lobe is being considered with increasing frequency in adults; however, living donor liver transplantation cannot be expected to solve the donor organ shortage; 246 such procedures were done in 2012, representing only about 4% of all liver transplant operations done in the United States.

1	Living donor transplantation can reduce waiting time and coldischemia time; is done under elective, rather than emergency, circumstances; and may be lifesaving in recipients who cannot afford to wait for a cadaver donor. The downside, of course, is the risk to the healthy donor (a mean of 10 weeks of medical disability; biliary complications in ~5%; postoperative complications such as wound infection, small-bowel obstruction, and incisional hernias in 9–19%; and even, in 0.2–0.4%, death) as well as the increased frequency of biliary (15–32%) and vascular (10%) complications in the recipient. Potential donors must participate voluntarily without coercion, and transplantation teams should go to great lengths to exclude subtle coercive or inappropriate psychological factors as well as outline carefully to both donor and recipient the potential benefits and risks of the procedure. Donors for the procedure should be 18–60 years old; have a compatible blood type with the recipient; have no

1	carefully to both donor and recipient the potential benefits and risks of the procedure. Donors for the procedure should be 18–60 years old; have a compatible blood type with the recipient; have no chronic medical problems or history of major abdominal surgery; be related genetically or emotionally to the recipient; and pass an exhaustive series of clinical, biochemical, and serologic evaluations to unearth disqualifying medical disorders. The recipient should meet the same UNOS criteria for liver transplantation as recipients of a cadaver donor allograft. Comprehensive outcome data on adult-to-adult living donor liver transplantation are being collected (www.nih-a2all.org).

1	Removal of the recipient’s native liver is technically difficult, particularly in the presence of portal hypertension with its associated collateral circulation and extensive varices and especially in the presence of scarring from previous abdominal operations. The combination of portal hypertension and coagulopathy (elevated prothrombin time and thrombocytopenia) may translate into large blood product transfusion requirements. After the portal vein and infrahepatic and suprahepatic inferior vena cavae are dissected, the hepatic artery and common bile duct are dissected. Then the native liver is removed and the donor organ inserted. During the anhepatic phase, coagulopathy, hypoglycemia, hypocalcemia, and hypothermia are encountered and must be managed by the anesthesiology team. Caval, portal vein, hepatic artery, and bile duct anastomoses are performed in succession, the last by end-to-end suturing of the donor and recipient common bile ducts (Fig. 368-1) or by choledochojejunostomy

1	vein, hepatic artery, and bile duct anastomoses are performed in succession, the last by end-to-end suturing of the donor and recipient common bile ducts (Fig. 368-1) or by choledochojejunostomy to a Roux-en-Y loop if the recipient common bile duct cannot be used for reconstruction (e.g., in sclerosing cholangitis). A typical transplant operation lasts 8 h, with a range of 6–18 h. Because of excessive bleeding, large volumes of blood, blood products, and volume expanders may be required during surgery; however, blood requirements have fallen sharply with improvements in surgical technique, blood-salvage interventions, and experience.

1	FIGURE 368-1 The anastomoses in orthotopic liver transplanta-tion. The anastomoses are performed in the following sequence: (1) suprahepatic and infrahepatic vena cava, (2) portal vein, (3) hepatic artery, and (4) common bile duct-to-duct anastomosis. (Adapted from JL Dienstag, AB Cosimi: N Engl J Med 367:1483, 2012.)

1	As noted above, emerging alternatives to orthotopic liver transplantation include split-liver grafts, in which one donor organ is divided and inserted into two recipients; and living donor procedures, in which part of the left (for children), the left (for children or small adults), or the right (for adults) lobe of the liver is harvested from a living donor for transplantation into the recipient. In the adult procedure, once the right lobe is removed from the donor, the donor right hepatic vein is anastomosed to the recipient right hepatic vein remnant, followed by donor-to-recipient anastomoses of the portal vein and then the hepatic artery. Finally, the biliary anastomosis is performed, duct-to-duct if practical or via Roux-en-Y anastomosis. Heterotopic liver transplantation, in which the donor liver is inserted without removal of the native liver, has met with very limited success and acceptance, except in a very small number of centers. In attempts to support desperately ill

1	the donor liver is inserted without removal of the native liver, has met with very limited success and acceptance, except in a very small number of centers. In attempts to support desperately ill patients until a suitable donor organ can be identified, several transplantation centers are studying extracorporeal perfusion with bioartificial liver cartridges constructed from hepatocytes bound to hollow fiber systems and used as temporary hepatic-assist devices, but their efficacy remains to be established. Areas of research with the potential to overcome the shortage of donor organs include hepatocyte transplantation and xenotransplantation with genetically modified organs of nonhuman origin (e.g., swine).

1	The introduction in 1980 of cyclosporine as an immunosuppressive agent contributed substantially to the improvement in survival after liver transplantation. Cyclosporine, a calcineurin inhibitor, blocks early activation of T cells and is specific for T cell functions that result from the interaction of the T cell with its receptor and that involve the cal-cium-dependent signal transduction pathway. As a result, the activity of cyclosporine leads to inhibition of lymphokine gene activation, blocking interleukins 2, 3, and 4, tumor necrosis factor a, and other lymphokines. Cyclosporine also inhibits B cell functions. This process occurs without affecting rapidly dividing cells in the bone marrow, which may account for the reduced frequency of posttransplantation systemic infections. The most common and important side effect of cyclosporine therapy is nephrotoxicity. Cyclosporine causes dose-dependent renal tubular injury and direct renal artery vasospasm. Following renal function is

1	most common and important side effect of cyclosporine therapy is nephrotoxicity. Cyclosporine causes dose-dependent renal tubular injury and direct renal artery vasospasm. Following renal function is therefore important in monitoring cyclosporine therapy, perhaps even a more reliable indicator than blood levels of the drug. Nephrotoxicity is reversible and can be managed by dose reduction. Other adverse effects of cyclosporine therapy include hypertension, hyperkalemia, tremor, hirsutism, glucose intolerance, and gingival hyperplasia.

1	Tacrolimus, a macrolide lactone antibiotic isolated from a Japanese soil fungus, Streptomyces tsukubaensis, has the same mechanism of action as cyclosporine but is 10–100 times more potent. Initially applied as “rescue” therapy for patients in whom rejection occurred despite the use of cyclosporine, tacrolimus was shown to be associated with a reduced frequency of acute, refractory, and chronic rejection. Although patient and graft survival are the same with these two drugs, the advantage of tacrolimus in minimizing episodes of rejection, reducing the need for additional glucocorticoid doses, and reducing the likelihood of bacterial and cytomegalovirus (CMV) infection has simplified the management of patients undergoing liver transplantation. In addition, the oral absorption of tacrolimus is more predictable than that of cyclosporine, especially during the early postoperative period when T-tube drainage interferes with the enterohepatic circulation of cyclosporine. As a result, in

1	is more predictable than that of cyclosporine, especially during the early postoperative period when T-tube drainage interferes with the enterohepatic circulation of cyclosporine. As a result, in most transplantation centers, tacrolimus has now supplanted cyclosporine for primary immunosuppression, and many centers rely on oral rather than IV administration from the outset. For transplantation centers that prefer cyclosporine, a better-absorbed microemulsion preparation is available.

1	Although more potent than cyclosporine, tacrolimus is also more toxic and more likely to be discontinued for adverse events. The toxicity of tacrolimus is similar to that of cyclosporine; nephrotoxicity and neurotoxicity are the most commonly encountered adverse effects, and neurotoxicity (tremor, seizures, hallucinations, psychoses, coma) is more likely and more severe in tacrolimus-treated patients. Both drugs can cause diabetes mellitus, but tacrolimus does not cause hirsutism or gingival hyperplasia. Because of overlapping toxicity between cyclosporine and tacrolimus, especially nephrotoxicity, and because tacrolimus reduces cyclosporine clearance, these two drugs should not be used together. Because 99% of tacrolimus is metabolized by the liver, hepatic dysfunction reduces its clearance; in primary graft nonfunction (when, for technical reasons or because of ischemic damage prior to its insertion, the allograft is defective and does not function normally from the outset),

1	clearance; in primary graft nonfunction (when, for technical reasons or because of ischemic damage prior to its insertion, the allograft is defective and does not function normally from the outset), tacrolimus doses have to be reduced substantially, especially in children. Both cyclosporine and tacrolimus are metabolized by the cytochrome P450 IIIA system, and, therefore, drugs that induce cytochrome P450 (e.g., phenytoin, phenobarbital, carbamazepine, rifampin) reduce available levels of cyclosporine and tacrolimus; and drugs that inhibit cytochrome P450 (e.g., erythromycin, fluconazole, ketoconazole, clotrimazole, itraconazole, verapamil, diltiazem, danazol, metoclopramide, the HIV protease inhibitor ritonavir, and the HCV protease inhibitors telaprevir and boceprevir) increase cyclosporine and tacrolimus blood levels. Indeed, itraconazole is used occasionally to help boost tacrolimus levels. Like azathioprine, cyclosporine and tacrolimus appear to be associated with 2071 a risk of

1	and tacrolimus blood levels. Indeed, itraconazole is used occasionally to help boost tacrolimus levels. Like azathioprine, cyclosporine and tacrolimus appear to be associated with 2071 a risk of lymphoproliferative malignancies (see below), which may occur earlier after cyclosporine or tacrolimus than after azathioprine therapy. Because of these side effects, combinations of cyclosporine or tacrolimus with prednisone and an antimetabolite (azathioprine or mycophenolic acid, see below)—all at reduced doses—are preferable regimens for immunosuppressive therapy.

1	Mycophenolic acid, a nonnucleoside purine metabolism inhibitor derived as a fermentation product from several Penicillium species, is another immunosuppressive drug being used for patients undergo ing liver transplantation. Mycophenolate has been shown to be better than azathioprine, when used with other standard immunosuppressive drugs, in preventing rejection after renal transplantation and has been adopted widely as well for use in liver transplantation. The most common adverse effects of mycophenolate are bone marrow suppression and gastrointestinal complaints.

1	In patients with pretransplantation renal dysfunction or renal deterioration that occurs intraoperatively or immediately postoperatively, tacrolimus or cyclosporine therapy may not be practical; under these circumstances, induction or maintenance of immunosuppression with antithymocyte globulin (ATG, thymoglobulin) or monoclonal antibodies to T cells, OKT3, may be appropriate. Therapy with these agents has been especially effective in reversing acute rejection in the posttransplantation period and is the standard treatment for acute rejection that fails to respond to methylprednisolone boluses. Available data support the use of thymoglobulin induction to delay calcineurin inhibitor use and its attendant nephrotoxicity. IV infusions of thymoglobulin may be complicated by fever and chills, which can be ameliorated by premedication with antipyretics and a low dose of glucocorticoids. Infusions of OKT3 may be complicated by fever, chills, and diarrhea, or by pulmonary edema, which can be

1	which can be ameliorated by premedication with antipyretics and a low dose of glucocorticoids. Infusions of OKT3 may be complicated by fever, chills, and diarrhea, or by pulmonary edema, which can be fatal. Because OKT3 is such a potent immunosuppressive agent, its use is also more likely to be complicated by opportunistic infection or lymphoproliferative disorders; therefore, because of the availability of alternative immunosuppressive drugs, OKT3 is now used sparingly.

1	Sirolimus, an inhibitor of the mammalian target of rapamycin (mTOR), blocks later events in T cell activation, is approved for use in kidney transplantation, but is not approved for use in liver transplant recipients because of the reported association with an increased frequency of hepatic artery thrombosis in the first month posttransplantation. In patients with calcineurin inhibitor–related nephrotoxicity, conversion to sirolimus has been demonstrated to be effective in preventing rejection with accompanying improvements in renal function. Because of its profound antiproliferative effects, sirolimus has also been suggested to be a useful immunosuppressive agent in patients with a prior or current history of malignancy, such as HCC. Side effects include hyperlipidemia, peripheral edema, oral ulcers, and interstitial pneumonitis. Everolimus is a hydroxyethyl derivative of sirolimus that, when used in conjunction with low-dose tacrolimus, also provides successful protection against

1	oral ulcers, and interstitial pneumonitis. Everolimus is a hydroxyethyl derivative of sirolimus that, when used in conjunction with low-dose tacrolimus, also provides successful protection against acute rejection, with decreased renal impairment compared to that associated with standard tacrolimus dosing. Everolimus and sirolimus share a similar adverse events profile; therefore, neither of these agents is approved for routine use in liver allograft recipients.

1	The most important principle of immunosuppression is that the ideal approach strikes a balance between immunosuppression and immunologic competence. In general, given sufficient immunosuppression, acute liver allograft rejection is nearly always reversible. On one hand, incompletely treated acute rejection predisposes to the development of chronic rejection, which can threaten graft survival. On the other hand, if the cumulative dose of immunosuppressive therapy is too large, the patient may succumb to opportunistic infection. In hepatitis C, pulse glucocorticoids or OKT3 use accelerate recurrent allograft hepatitis. Further complicating matters, acute rejection can be difficult to distinguish histologically from recurrent hepatitis

1	C. Therefore, immunosuppressive drugs must be used judiciously, with strict attention to the infectious consequences of such therapy and careful confirmation of the diagnosis of acute rejection. In this vein, efforts have been made to minimize the use of glucocorticoids, a 2072 mainstay of immunosuppressive regimens, and steroid-free immunosuppression can be achieved in some instances. Patients who undergo liver transplantation for autoimmune diseases such as primary biliary cirrhosis, autoimmune hepatitis, and primary sclerosing cholangitis are less likely to achieve freedom from glucocorticoids.

1	Complications of liver transplantation can be divided into nonhepatic and hepatic categories (Tables 368-4 and 368-5). In addition, both immediate postoperative and late complications are encountered. As a rule, patients who undergo liver transplantation have been chronically ill for protracted periods and may be malnourished and wasted. The impact of such chronic illness and the multisystem failure that accompanies liver failure continue to require attention in the postoperative period. Because of the massive fluid losses and fluid shifts that occur during the operation, patients may remain fluid-overloaded during the immediate postoperative period, straining cardiovascular reserve; this effect can be amplified in the face of transient renal dysfunction and pulmonary capillary vascular permeability. Continuous monitoring of cardiovascular and pulmonary function, measures to maintain the integrity of the intravascular compartment and to treat extravascular volume overload, and

1	permeability. Continuous monitoring of cardiovascular and pulmonary function, measures to maintain the integrity of the intravascular compartment and to treat extravascular volume overload, and scrupulous attention to potential sources and sites of infection are of paramount importance. Cardiovascular instability may also result from the electrolyte imbalance that may accompany reperfusion of the donor liver as well as from restoration of systemic vascular resistance following implantation. Pulmonary function may be compromised further by paralysis of the right hemidiaphragm associated with phrenic nerve injury. The hyperdynamic state with increased cardiac output that is characteristic of patients with liver failure reverses rapidly after successful liver transplantation.

1	Other immediate management issues include renal dysfunction. Prerenal azotemia, acute kidney injury associated with hypoperfusion (acute tubular necrosis), and renal toxicity caused by antibiotics, ↓ Renal blood flow secondary to ↑ intraabdominal pressure Hematologic Anemia secondary to gastrointestinal and/or intraabdominal bleeding Hemolytic anemia, aplastic anemia Thrombocytopenia Infection Bacterial: early, common postoperative infections Fungal/parasitic: late, opportunistic infections Viral: late, opportunistic infections, recurrent hepatitis Diseases of donor Infectious Malignant

1	Fungal/parasitic: late, opportunistic infections Viral: late, opportunistic infections, recurrent hepatitis Diseases of donor Infectious Malignant Hepatic Dysfunction Unique to Liver Transplantation tacrolimus, or cyclosporine are encountered frequently in the postoperative period, sometimes necessitating dialysis. Hemolytic-uremic syndrome can be associated with cyclosporine, tacrolimus, or OKT3. Occasionally, postoperative intraperitoneal bleeding may be sufficient to increase intraabdominal pressure, which, in turn, may reduce renal blood flow; this effect is rapidly reversible when abdominal distention is relieved by exploratory laparotomy to identify and ligate the bleeding site and to remove intraperitoneal clot.

1	Anemia may also result from acute upper gastrointestinal bleeding or from transient hemolytic anemia, which may be autoimmune, especially when blood group O livers are transplanted into blood group A or B recipients. This autoimmune hemolytic anemia is mediated by donor intrahepatic lymphocytes that recognize red blood cell A or B antigens on recipient erythrocytes. Transient in nature, this process resolves once the donor liver is repopulated by recipient bone marrow–derived lymphocytes; the hemolysis can be treated by transfusing blood group O red blood cells and/or by administering higher doses of glucocorticoids. Transient thrombocytopenia is also commonly encountered. Aplastic anemia, a late occurrence, is rare but has been reported in almost 30% of patients who underwent liver transplantation for acute, severe hepatitis of unknown cause.

1	Bacterial, fungal, or viral infections are common and may be life-threatening postoperatively. Early after transplant surgery, common postoperative infections predominate—pneumonia, wound infections, infected intraabdominal collections, urinary tract infections, and IV line infections—rather than opportunistic infections; these infections may involve the biliary tree and liver as well. Beyond the first postoperative month, the toll of immunosuppression becomes evident, and opportunistic infections—CMV, herpes viruses, fungal infections (Aspergillus, Candida, cryptococcal disease), mycobacterial infections, parasitic infections (Pneumocystis, Toxoplasma), bacterial infections (Nocardia, Legionella, Listeria)—predominate. Rarely, early infections represent those transmitted with the donor liver, either infections present in the donor or infections acquired during procurement processing. De novo viral hepatitis infections acquired from the donor organ or, almost unheard of now, from

1	liver, either infections present in the donor or infections acquired during procurement processing. De novo viral hepatitis infections acquired from the donor organ or, almost unheard of now, from transfused blood products occur after typical incubation periods for these agents (well beyond the first month). Obviously, infections in an immunosuppressed host demand early recognition and prompt management; prophylactic antibiotic therapy is administered routinely in the immediate postoperative period. Use of sulfamethoxazole with trimethoprim reduces the incidence of postoperative Pneumocystis carinii pneumonia. Antiviral prophylaxis for CMV with ganciclovir should be administered in patients at high risk (e.g., when a CMV-seropositive donor organ is implanted into a CMVseronegative recipient).

1	Neuropsychiatric complications include seizures (commonly associated with cyclosporine and tacrolimus toxicity), metabolic encephalopathy, depression, and difficult psychosocial adjustment. Rarely, diseases are transmitted by the allograft from the donor to the recipient. In addition to viral and bacterial infections, malignancies of donor origin have occurred. Posttransplantation lymphoproliferative disorders, especially B cell lymphoma, are a recognized complication associated with immunosuppressive drugs such as azathioprine, tacrolimus, and cyclosporine (see above). Epstein-Barr virus has been shown to play a contributory role in some of these tumors, which may regress when immunosuppressive therapy is reduced. De novo neoplasms appear at increased frequency after liver transplantation, particularly squamous cell carcinomas of the skin. Routine screening should be performed.

1	Long-term complications after liver transplantation attributable primarily to immunosuppressive medications include diabetes mellitus and osteoporosis (associated with glucocorticoids and calcineurin inhibitors) as well as hypertension, hyperlipidemia, and chronic renal insufficiency (associated with cyclosporine and tacrolimus). Monitoring and treating these disorders are routine components of posttransplantation care; in some cases, they respond to changes in immunosuppressive regimen, while in others, specific treatment of the disorder is introduced. Data from a large U.S. database showed that the prevalence of renal failure was 18% at year 5 and 25% at year 10 after liver transplantation. Similarly, the high frequency of diabetes, hypertension, hyperlipidemia, obesity, and the metabolic syndrome renders patients susceptible to cardiovascular disease after liver transplantation; although hepatic complications account for most of the mortality after liver transplantation, renal

1	syndrome renders patients susceptible to cardiovascular disease after liver transplantation; although hepatic complications account for most of the mortality after liver transplantation, renal failure and cardiovascular disease are the other leading causes of late mortality after liver transplantation.

1	Hepatic dysfunction after liver transplantation is similar to the hepatic complications encountered after major abdominal and cardiothoracic surgery; however, in addition, hepatic complications include primary graft failure, vascular compromise, failure or stricture of the biliary anastomoses, and rejection. As in nontransplantation surgery, postoperative jaundice may result from prehepatic, intrahepatic, and posthepatic sources. Prehepatic sources represent the massive hemoglobin pigment load from transfusions, hemolysis, hematomas, ecchymoses, and other collections of blood. Early intrahepatic liver injury includes effects of hepatotoxic drugs and anesthesia; hypoperfusion injury associated with hypotension, sepsis, and shock; and benign postoperative cholestasis. Late intrahepatic sources of liver injury include exacerbation of primary disease. Posthepatic sources of hepatic dysfunction include biliary obstruction and reduced renal clearance of conjugated bilirubin. Hepatic

1	sources of liver injury include exacerbation of primary disease. Posthepatic sources of hepatic dysfunction include biliary obstruction and reduced renal clearance of conjugated bilirubin. Hepatic complications unique to liver transplantation include primary graft failure associated with ischemic injury to the organ during harvesting; vascular compromise associated with thrombosis or stenosis of the portal vein or hepatic artery anastomoses; vascular anastomotic leak; stenosis, obstruction, or leakage of the anastomosed common bile duct; recurrence of primary hepatic disorder (see below); and rejection.

1	Despite the use of immunosuppressive drugs, rejection of the transplanted liver still occurs in a proportion of patients, beginning 1–2 weeks after surgery. Clinical signs suggesting rejection are fever, right upper quadrant pain, and reduced bile pigment and volume. Leukocytosis may occur, but the most reliable indicators are increases in serum bilirubin and aminotransferase levels. Because these tests lack specificity, distinguishing among rejection, biliary obstruction, primary graft nonfunction, vascular compromise, viral hepatitis, CMV infection, drug hepatotoxicity, and recurrent primary disease may be difficult. Radiographic visualization of the biliary tree and/or percutaneous liver biopsy often help to establish the correct diagnosis. 2073 Morphologic features of acute rejection include a mixed portal cellular infiltrate, bile duct injury, and/or endothelial inflammation (“endothelialitis”); some of these findings are reminiscent of graft-versus-host disease, primary biliary

1	include a mixed portal cellular infiltrate, bile duct injury, and/or endothelial inflammation (“endothelialitis”); some of these findings are reminiscent of graft-versus-host disease, primary biliary cirrhosis, or recurrent allograft hepatitis C. As soon as transplant rejection is suspected, treatment consists of IV methylprednisolone in repeated boluses; if this fails to abort rejection, many centers use thymoglobulin or OKT3. Caution should be exercised when managing acute rejection with pulse glucocorticoids or OKT3 in patients with HCV infection, because of the high risk of triggering recurrent allograft hepatitis C.

1	Chronic rejection is a relatively rare outcome that can follow repeated bouts of acute rejection or that occurs unrelated to preceding rejection episodes. Morphologically, chronic rejection is characterized by progressive cholestasis, focal parenchymal necrosis, mononuclear infiltration, vascular lesions (intimal fibrosis, subintimal foam cells, fibrinoid necrosis), and fibrosis. This process may be reflected as ductopenia—the vanishing bile duct syndrome, which is more common in patients undergoing liver transplantation for autoimmune liver disease. Reversibility of chronic rejection is limited; in patients with therapy-resistant chronic rejection, retransplantation has yielded encouraging results.

1	The survival rate for patients undergoing liver transplantation has improved steadily since 1983. One-year survival rates have increased from ~70% in the early 1980s to 85–90% from 2003 to the present time. Currently, the 5-year survival rate exceeds 60%. An important observation is the relationship between clinical status before transplantation and outcome. For patients who undergo liver transplantation when their level of compensation is high (e.g., still working or only partially disabled), a 1-year survival rate of >85% is common. For those whose level of decompensation mandates continuous in-hospital care prior to transplantation, the 1-year survival rate is ~70%, whereas for those who are so decompensated that they require life support in an intensive care unit, the 1-year survival rate is ~50%. Since the adoption by UNOS in 2002 of the MELD system for organ allocation, posttransplantation survival has been found to be affected adversely for candidates with MELD scores >25,

1	rate is ~50%. Since the adoption by UNOS in 2002 of the MELD system for organ allocation, posttransplantation survival has been found to be affected adversely for candidates with MELD scores >25, considered high disease severity. Thus, irrespective of allocation scheme, high disease severity before transplantation corresponds to diminished posttransplantation survival. Another important distinction in survival has been drawn between highand low-risk patient categories. For patients who do not fit any “high-risk” designations, 1-year and 5-year survival rates of 85 and 80%, respectively, have been recorded. In contrast, among patients in high-risk categories—cancer, fulminant hepatitis, age >65, concurrent renal failure, respirator dependence, portal vein thrombosis, and history of a portacaval shunt or multiple right upper quadrant operations—survival statistics fall into the range of 60% at 1 year and 35% at 5 years. Survival after retransplantation for primary graft non-function is

1	shunt or multiple right upper quadrant operations—survival statistics fall into the range of 60% at 1 year and 35% at 5 years. Survival after retransplantation for primary graft non-function is ~50%. Causes of failure of liver transplantation vary with time. Failures within the first 3 months result primarily from technical complications, postoperative infections, and hemorrhage. Transplant failures after the first 3 months are more likely to result from infection, rejection, or recurrent disease (such as malignancy or viral hepatitis).

1	Features of autoimmune hepatitis, primary sclerosing cholangitis, and primary biliary cirrhosis overlap with those of rejection or post-transplantation bile duct injury. Whether autoimmune hepatitis and sclerosing cholangitis recur after liver transplantation is controversial; data supporting recurrent autoimmune hepatitis (in up to one-third of patients in some series) are more convincing than those supporting recurrent sclerosing cholangitis. Similarly, reports of recurrent primary biliary cirrhosis after liver transplantation have appeared; however, the histologic features of primary biliary cirrhosis and chronic rejection are virtually indistinguishable and occur as frequently in patients with 2074 primary biliary cirrhosis as in patients undergoing transplantation for other reasons. The presence of a florid inflammatory bile duct lesion is highly suggestive of the recurrence of primary biliary cirrhosis, but even this lesion can be observed in acute rejection. Hereditary

1	The presence of a florid inflammatory bile duct lesion is highly suggestive of the recurrence of primary biliary cirrhosis, but even this lesion can be observed in acute rejection. Hereditary disorders such as Wilson’s disease and α1-antitrypsin deficiency have not recurred after liver transplantation; however, recurrence of disordered iron metabolism has been observed in some patients with hemochromatosis. Hepatic vein thrombosis (Budd-Chiari syndrome) may recur; this can be minimized by treating underlying myeloproliferative disorders and by anticoagulation. Because cholangiocarcinoma recurs almost invariably, few centers now offer transplantation to such patients; however, a few highly selected patients with operatively confirmed stage I or II cholangiocarcinoma who undergo liver transplantation combined with neoadjuvant chemoradiation may experience excellent outcomes. In patients with intrahepatic HCC who meet criteria for transplantation, 1and 5-year survivals are similar to

1	combined with neoadjuvant chemoradiation may experience excellent outcomes. In patients with intrahepatic HCC who meet criteria for transplantation, 1and 5-year survivals are similar to those observed in patients undergoing liver transplantation for nonmalignant disease. Finally, metabolic disorders such as nonalcoholic steatohepatitis recur frequently, especially if the underlying metabolic predisposition is not altered. The metabolic syndrome occurs commonly after liver transplantation as a result of recurrent nonalcoholic fatty liver, immunosuppressive medications, and/or, in patients with hepatitis C related to the impact of HCV infection on insulin resistance, diabetes and fatty liver. Hepatitis A can recur after transplantation for fulminant hepatitis A, but such acute reinfection has no serious clinical sequelae. In fulminant hepatitis B, recurrence is not the rule; however, in the absence of any prophylactic measures, hepatitis B usually recurs after transplantation for

1	has no serious clinical sequelae. In fulminant hepatitis B, recurrence is not the rule; however, in the absence of any prophylactic measures, hepatitis B usually recurs after transplantation for end-stage chronic hepatitis B. Before the introduction of prophylactic antiviral therapy, immunosuppressive therapy sufficient to prevent allograft rejection led inevitably to marked increases in hepatitis B viremia, regardless of pretransplantation levels. Overall graft and patient survival were poor, and some patients experienced a rapid recapitulation of severe injury—severe chronic hepatitis or even fulminant hepatitis—after transplantation. Also recognized in the era before availability of antiviral regimens was fibrosing cholestatic hepatitis, rapidly progressive liver injury associated with marked hyperbilirubinemia, substantial prolongation of the prothrombin time (both out of proportion to relatively modest elevations of aminotransferase activity), and rapidly progressive liver

1	with marked hyperbilirubinemia, substantial prolongation of the prothrombin time (both out of proportion to relatively modest elevations of aminotransferase activity), and rapidly progressive liver failure. This lesion has been suggested to represent a “choking off” of the hepatocyte by an overwhelming density of HBV proteins. Complications such as sepsis and pancreatitis were also observed more frequently in patients undergoing liver transplantation for hepatitis B prior to the introduction of antiviral therapy. The introduction of long-term prophylaxis with HBIg revolutionized liver transplantation for chronic hepatitis B. Preoperative hepatitis B vaccination, preoperative or postoperative interferon (IFN) therapy, or short-term (≤2 months) HBIg prophylaxis has not been shown to be effective, but a retrospective analysis of data from several hundred European patients followed for 3 years after transplantation has shown that long-term (≥6 months) prophylaxis with HBIg is associated

1	but a retrospective analysis of data from several hundred European patients followed for 3 years after transplantation has shown that long-term (≥6 months) prophylaxis with HBIg is associated with a lowering of the risk of HBV reinfection from ~75 to 35% and a reduction in mortality from ~50 to 20%. As a result of long-term HBIg use following liver transplantation for chronic hepatitis B, similar improvements in outcome have been observed in the United States, with 1-year survival rates between 75% and 90%. Currently, with HBIg prophylaxis, the outcome of liver transplantation for chronic hepatitis B is indistinguishable from that for chronic liver disease unassociated with chronic hepatitis B; essentially, medical concerns regarding liver transplantation for chronic hepatitis B have been eliminated. Passive immunoprophylaxis with HBIg is begun during the anhepatic stage of surgery, repeated daily for the first 6 postoperative days, and then continued with infusions that are given

1	eliminated. Passive immunoprophylaxis with HBIg is begun during the anhepatic stage of surgery, repeated daily for the first 6 postoperative days, and then continued with infusions that are given either at regular intervals of 4–6 weeks or, alternatively, when anti-hepatitis B surface (HBs) levels fall below a threshold of 100 mIU/mL. The current approach in most centers is to continue HBIg indefinitely, which can add approximately $20,000 per year to the cost of care; some centers are evaluating regimens that shift to less frequent administration or to IM administration in the late posttransplantation period or, in low-risk patients, maintenance with antiviral therapy (see below) alone. Still, “breakthrough” HBV infection occasionally occurs.

1	Further improving the outcome of liver transplantation for chronic hepatitis B is the current availability of such antiviral drugs as lamivudine, adefovir, entecavir, and tenofovir disoproxil fumarate (Chap. 362). When these drugs are administered to patients with decompensated liver disease, a proportion improve sufficiently to postpone imminent liver transplantation. In addition, antiviral therapy can be used to prevent recurrence of HBV infection when administered prior to transplantation; to treat hepatitis B that recurs after transplantation, including in patients who break through HBIg prophylaxis; and to reverse the course of otherwise fatal fibrosing cholestatic hepatitis. Clinical trials have shown that lamivudine antiviral therapy reduces the level of HBV replication substantially, sometimes even resulting in clearance of hepatitis B surface antigen (HBsAg); reduces alanine aminotransferase (ALT) levels; and improves histologic features of necrosis and inflammation.

1	sometimes even resulting in clearance of hepatitis B surface antigen (HBsAg); reduces alanine aminotransferase (ALT) levels; and improves histologic features of necrosis and inflammation. Long-term use of lamivudine is safe and effective, but after several months, a proportion of patients become resistant to lamivudine, resulting from YMDD (tyrosine-methionine-aspartate-aspartate) mutations in the HBV polymerase motif (Chap. 362). In approximately one-half of such resistant patients, hepatic deterioration may ensue. Fortunately, adefovir and tenofovir disoproxil fumarate are available as well and can be used to treat lamivudine-associated YMDD variants, effectively “rescuing” patients experiencing hepatic decompensation after lamivudine breakthrough. Currently, most liver transplantation centers combine HBIg plus lamivudine, adefovir, entecavir, or tenofovir disoproxil fumarate. In low-risk patients with no detectable hepatitis B viremia at the time of transplantation, a number of

1	centers combine HBIg plus lamivudine, adefovir, entecavir, or tenofovir disoproxil fumarate. In low-risk patients with no detectable hepatitis B viremia at the time of transplantation, a number of clinical trials have suggested that antiviral prophylaxis can suffice, without HBIg or with a finite duration of HBIg, to prevent recurrent HBV infection of the allograft. Antiviral prophylactic approaches applied to patients undergoing liver transplantation for chronic hepatitis B are being used as well for patients without hepatitis B who receive organs from donors with antibody to hepatitis B core antigen (anti-HBc). Patients who undergo liver transplantation for chronic hepatitis B plus D are less likely to experience recurrent liver injury than patients undergoing liver transplantation for hepatitis B alone; still, such co-infected patients would also be offered standard posttransplantation prophylactic therapy for hepatitis B.

1	Accounting for up to 40% of all liver transplantation procedures, the most common indication for liver transplantation is end-stage liver disease resulting from chronic hepatitis C. Recurrence of HCV infection after liver transplantation can be documented in almost every patient. The clinical consequences of recurrent hepatitis C are limited during the first 5 years after transplantation. Nonetheless, despite the relative clinical benignity of recurrent hepatitis C in the early years after liver transplantation, and despite the negligible impact on patient survival during these early years, histologic studies have documented the presence of moderate to severe chronic hepatitis in more than one-half of all patients and cirrhosis in ~20% at 5 years. Allograft cirrhosis is even more common, occurring in up to two-thirds of patients at 5 years, if moderate hepatitis is detected in a 1-year liver biopsy. Not surprisingly, then, for patients undergoing liver transplantation for hepatitis C,

1	in up to two-thirds of patients at 5 years, if moderate hepatitis is detected in a 1-year liver biopsy. Not surprisingly, then, for patients undergoing liver transplantation for hepatitis C, allograft and patient survival are diminished substantially between 5 and 10 years after transplantation.

1	In a proportion of patients, even during the early posttransplantation period, recurrent hepatitis C may be sufficiently severe biochemically and histologically to merit antiviral therapy. Treatment with pegylated IFN can suppress HCV-associated liver injury but rarely leads to sustained benefit. Sustained virologic responses are the exception, and reduced tolerability is often dose-limiting. Preemptive combination antiviral therapy with pegylated IFN and the nucleoside analogue ribavirin immediately after transplantation does not appear to provide any advantage over therapy introduced after clinical hepatitis has occurred. Similarly, although IFN-based antiviral therapy is not recommended for patients with decompensated liver disease, some centers have experimented with pretransplantation antiviral therapy in an attempt to eradicate HCV replication prior to transplantation; preliminary results are promising, but IFN treatment of patients with end-stage liver disease can lead to

1	antiviral therapy in an attempt to eradicate HCV replication prior to transplantation; preliminary results are promising, but IFN treatment of patients with end-stage liver disease can lead to worsening of hepatic decompensation, and HCV infection has recurred after transplantation in some of these recipients. Trials of hepatitis C immune globulin preparations to prevent recurrent hepatitis C after liver transplantation have not been successful. Similarly, a trial of a high-dose monoclonal antibody to the HCV E2 envelope glycoprotein delayed but did not prevent reappearance of viremia.

1	Although the current standard-of-care treatment of allograft hepatitis C is pegylated IFN and ribavirin, in a number of studies, the safety and efficacy of the addition of the approved HCV protease inhibitors telaprevir or boceprevir to pegylated IFN and ribavirin in genotype 1–infected patients with recurrent hepatitis C have been examined. Because of the profound inhibitory effects of the HCV protease inhibitors on the metabolism of the calcineurin inhibitors (increasing cyclosporine levels almost 5-fold and tacrolimus levels 70-fold), calcineurin inhibitor doses must be reduced to safe levels in these patients. In one multicenter study, treatment with a telapreviror boceprevir-based triple-drug regimen (with pegylated IFN and ribavirin) achieved rates of HCV clearance similar to those achieved in patients with chronic hepatitis C who had not undergone transplantation. Unfortunately, tolerability of these protease inhibitor–based regimens remains problematic in this population,

1	achieved in patients with chronic hepatitis C who had not undergone transplantation. Unfortunately, tolerability of these protease inhibitor–based regimens remains problematic in this population, particularly in persons with allograft cirrhosis, in whom the frequency of hepatic decompensation is increased. The approval of several new direct-acting antiviral (DAA) agents and of IFN-free DAA regimens against HCV will have a major impact on the management and outcome of both pretransplantation and post-transplantation HCV infection. Such therapeutic approaches (1) permit the clearance of viremia in a substantial proportion of decompensated cirrhotics, thereby preventing recurrent allograft infection and, possibly, even improving the clinical status of these patients, delaying or obviating the need for liver replacement; and (2) achieve sustained virologic responses in a much higher proportion of persons with allograft HCV infection, because of improvements in antiviral treatment efficacy

1	need for liver replacement; and (2) achieve sustained virologic responses in a much higher proportion of persons with allograft HCV infection, because of improvements in antiviral treatment efficacy and tolerability.

1	A small number of allograft recipients succumb to early HCV-associated liver injury, and a syndrome reminiscent of fibrosing cholestatic hepatitis (see above) has been observed rarely. Because patients with more episodes of rejection receive more immunosuppressive therapy, and because immunosuppressive therapy enhances HCV replication, patients with severe or multiple episodes of rejection are more likely to experience early recurrence of hepatitis C after transplantation. Both high viral levels and older donor age have been linked to recurrent HCV-induced liver disease and to earlier disease recurrence after transplantation.

1	Patients who undergo liver transplantation for end-stage alcoholic cirrhosis are at risk of resorting to drinking again after transplantation, a potential source of recurrent alcoholic liver injury. Currently, alcoholic liver disease is one of the more common indications for liver transplantation, accounting for 20–25% of all liver transplantation procedures, and most transplantation centers screen candidates carefully for predictors of continued abstinence. Recidivism is more likely in patients whose sobriety prior to transplantation was <6 months. For abstinent patients with alcoholic cirrhosis, liver transplantation can be undertaken successfully, with outcomes comparable to those for other categories of patients with chronic liver disease, when coordinated by a team approach that includes substance abuse counseling.

1	Full rehabilitation is achieved in the majority of patients who survive the early postoperative months and escape chronic rejection or unmanageable infection. Psychosocial maladjustment interferes with medical compliance in a small number of patients, but most manage to adhere to immunosuppressive regimens, which must be continued indefinitely. In one study, 85% of patients who survived their transplant operations returned to gainful activities. In fact, some women have conceived and carried pregnancies to term after transplantation without demonstrable injury to their infants. Diseases of the Gallbladder and Bile Ducts Norton J. Greenberger, Gustav Paumgartner

1	Diseases of the Gallbladder and Bile Ducts Norton J. Greenberger, Gustav Paumgartner Bile formed in the hepatic lobules is secreted into a complex network of canaliculi, small bile ductules, and larger bile ducts that run with lymphatics and branches of the portal vein and hepatic artery in portal tracts situated between hepatic lobules. These interlobular bile ducts coalesce to form larger septal bile ducts that join to form the right and left hepatic ducts, which in turn, unite to form the common hepatic duct. The common hepatic duct is joined by the cystic duct of the gallbladder to form the common bile duct (CBD), which enters the duodenum (often after joining the main pancreatic duct) through the ampulla of Vater.

1	Hepatic bile is an isotonic fluid with an electrolyte composition resembling blood plasma. The electrolyte composition of gallbladder bile differs from that of hepatic bile because most of the inorganic anions, chloride and bicarbonate, have been removed by reabsorption across the gallbladder epithelium. As a result of water reabsorption, total solute concentration of bile increases from 3–4 g/dL in hepatic bile to 10–15 g/dL in gallbladder bile. Major solute components of bile by moles percent include bile acids (80%), lecithin and traces of other phospholipids (16%), and unesterified cholesterol (4.0%). In the lithogenic state, the cholesterol value can be as high as 8–10%. Other constituents include conjugated bilirubin; proteins (all immunoglobulins, albumin, metabolites of hormones, and other proteins metabolized in the liver); electrolytes; mucus; and, often, drugs and their metabolites.

1	The total daily basal secretion of hepatic bile is ∼500–600 mL. Many substances taken up or synthesized by the hepatocyte are secreted into the bile canaliculi. The canalicular membrane forms microvilli and is associated with microfilaments of actin, microtubules, and other contractile elements. Prior to their secretion into the bile, many substances are taken up into the hepatocyte, while others, such as phospholipids, a portion of primary bile acids, and some cholesterol, are synthesized de novo in the hepatocyte. Three mechanisms are important in regulating bile flow: (1) active transport of bile acids from hepatocytes into the bile canaliculi, (2) active transport of other organic anions, and (3) cholangiocellular secretion. The last is a secretin-mediated and cyclic AMP–dependent mechanism that results in the secretion of a sodiumand bicarbonate-rich fluid into the bile ducts.

1	Active vectorial secretion of biliary constituents from the portal blood into the bile canaliculi is driven by a set of polarized transport systems at the basolateral (sinusoidal) and the canalicular apical plasma membrane domains of the hepatocyte. Two sinusoidal bile salt uptake systems have been cloned in humans, the Na+/taurocholate cotransporter (NTCP, SLC10A1) and the organic anion–transporting proteins (OATPs), which also transport a large variety of non-bile salt organic anions. Several ATP-dependent canalicular transport systems, “export pumps,” (ATP-binding cassette transport proteins, also known as ABC transporters) have been identified, the most important of which are: the bile salt export pump (BSEP, ABCB11); the anionic conjugate export pump (MRP2, ABCC2), which mediates the canalicular excretion of various amphiphilic conjugates formed by phase II conjugation (e.g., bilirubin monoand diglucuronides and drugs); the multidrug export pump (MDR1, ABCB1) for hydrophobic

1	the canalicular excretion of various amphiphilic conjugates formed by phase II conjugation (e.g., bilirubin monoand diglucuronides and drugs); the multidrug export pump (MDR1, ABCB1) for hydrophobic cationic compounds; and the phospholipid export pump (MDR3, ABCB4). Two hemitransporters ABCG5/G8, functioning as a couple, constitute the canalicular cholesterol and phytosterol transporter. F1C1 (ATP8B1) is an aminophospholipid transferase (“flippase”) essential for maintaining the lipid asymmetry of the canalicular membrane. The canalicular membrane also contains ATP-independent transport systems such as the Cl/HCO3 anion exchanger isoform 2 (AE2, SLC4A2)

1	Diseases of the Gallbladder and Bile Ducts 2076 for canalicular bicarbonate secretion. For most of these transporters, genetic defects have been identified that are associated with various forms of cholestasis or defects of biliary excretion. F1C1 is defective in progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1) and results in ablation of all other ATP-dependent transporter functions. BSEP is defective in PFIC2 and BRIC2. Mutations of MRP2 (ABCC2) cause the Dubin-Johnson syndrome, an inherited form of conjugated hyperbilirubinemia (Chap. 359). A defective MDR3 (ABCB4) results in PFIC3. ABCG5/G8, the canalicular half transporters for cholesterol and other neutral sterols, are defective in sitosterolemia. The cystic fibrosis trans-membrane regulator (CFTR, ABCC7) located on bile duct epithelial cells but not on canalicular membranes is defective in cystic fibrosis, which is associated with impaired cholangiocellular

1	trans-membrane regulator (CFTR, ABCC7) located on bile duct epithelial cells but not on canalicular membranes is defective in cystic fibrosis, which is associated with impaired cholangiocellular pH regulation during ductular bile formation and chronic cholestatic liver disease, occasionally resulting in biliary cirrhosis.

1	The primary bile acids, cholic acid and chenodeoxycholic acid (CDCA), are synthesized from cholesterol in the liver, conjugated with glycine or taurine, and secreted into the bile. Secondary bile acids, including deoxycholate and lithocholate, are formed in the colon as bacterial metabolites of the primary bile acids. However, lithocholic acid is much less efficiently absorbed from the colon than deoxycholic acid. Another secondary bile acid, found in low concentration, is ursodeoxycholic acid (UDCA), a stereoisomer of CDCA. In healthy subjects, the ratio of glycine to taurine conjugates in bile is ∼3:1.

1	Bile acids are detergent-like molecules that in aqueous solutions and above a critical concentration of about 2 mM form molecular aggregates called micelles. Cholesterol alone is sparingly soluble in aqueous environments, and its solubility in bile depends on both the total lipid concentration and the relative molar percentages of bile acids and lecithin. Normal ratios of these constituents favor the formation of solubilizing mixed micelles, while abnormal ratios promote the precipitation of cholesterol crystals in bile via an intermediate liquid crystal phase. In addition to facilitating the biliary excretion of cholesterol, bile acids facilitate the normal intestinal absorption of dietary fats, mainly cholesterol and fat-soluble vitamins, via a micellar transport mechanism (Chap. 349). Bile acids also serve as a major physiologic driving force for hepatic bile flow and aid in water and electrolyte transport in the small bowel and colon.

1	Bile acids are efficiently conserved under normal conditions. Unconjugated, and to a lesser degree also conjugated, bile acids are absorbed by passive diffusion along the entire gut. Quantitatively much more important for bile salt recirculation, however, is the active transport mechanism for conjugated bile acids in the distal ileum (Chap. 349). The reabsorbed bile acids enter the portal bloodstream and are taken up rapidly by hepatocytes, reconjugated, and resecreted into bile (enterohepatic circulation).

1	The normal bile acid pool size is approximately 2–4 g. During digestion of a meal, the bile acid pool undergoes at least one or more enterohepatic cycles, depending on the size and composition of the meal. Normally, the bile acid pool circulates ∼5–10 times daily. Intestinal reabsorption of the pool is about 95% efficient; therefore, fecal loss of bile acids is in the range of 0.2–0.4 g/d. In the steady state, this fecal loss is compensated by an equal daily synthesis of bile acids by the liver, and, thus, the size of the bile acid pool is maintained. Bile acids in the intestine release fibroblast growth factor 19 (FGF19) into the circulation, which is transported to the liver where it suppresses synthesis of bile acids from cholesterol by inhibiting the rate-limiting enzyme cytochrome P450 7A1 (CYP7A1) and also promotes gallbladder relaxation. While the loss of bile salts in stool is usually matched by increased hepatic synthesis, the maximum rate of synthesis is ∼5 g/d, which may be

1	7A1 (CYP7A1) and also promotes gallbladder relaxation. While the loss of bile salts in stool is usually matched by increased hepatic synthesis, the maximum rate of synthesis is ∼5 g/d, which may be insufficient to replete the bile acid pool size when there is pronounced impairment of intestinal bile salt reabsorption.

1	The expression of ABC transporters in the enterohepatic circulation and of the rate-limiting enzymes of bile acid and cholesterol synthesis are regulated in a coordinated fashion by nuclear receptors, which are ligand-activated transcription factors. The hepatic BSEP (ABCB11) is upregulated by the farnesoid X receptor (FXR), a bile acid sensor that also represses bile acid synthesis. The expression of the cholesterol transporter, ABCG5/G8, is upregulated by the liver X receptor (LXR), which is an oxysterol sensor.

1	In the fasting state, the sphincter of Oddi offers a high-pressure zone of resistance to bile flow from the CBD into the duodenum. Its tonic contraction serves to (1) prevent reflux of duodenal contents into the pancreatic and bile ducts and (2) promote filling of the gallbladder. The major factor controlling the evacuation of the gallbladder is the peptide hormone cholecystokinin (CCK), which is released from the duodenal mucosa in response to the ingestion of fats and amino acids. CCK produces (1) powerful contraction of the gallbladder, (2) decreased resistance of the sphincter of Oddi, and (3) enhanced flow of biliary contents into the duodenum.

1	Hepatic bile is “concentrated” within the gallbladder by energy-dependent transmucosal absorption of water and electrolytes. Almost the entire bile acid pool may be sequestered in the gallbladder following an overnight fast for delivery into the duodenum with the first meal of the day. The normal capacity of the gallbladder is ∼30 mL of bile. Anomalies of the biliary tract are not uncommon and include abnormalities in number, size, and shape (e.g., agenesis of the gallbladder, duplications, rudimentary or oversized “giant” gallbladders, and diverticula). Phrygian cap is a clinically innocuous entity in which a partial or complete septum (or fold) separates the fundus from the body. Anomalies of position or suspension are not uncommon and include left-sided gallbladder, intrahepatic gallbladder, retrodisplacement of the gallbladder, and “floating” gallbladder. The latter condition predisposes to acute torsion, volvulus, or herniation of the gallbladder.

1	GALLSTONES Epidemiology and Pathogenesis Gallstones are quite prevalent in most Western countries. Gallstone formation increases after age 50. In the United States, the third National Health and Nutrition Examination Survey (NHANES III) has revealed an overall prevalence of gallstones of 7.9% in men and 16.6% in women. The prevalence was high in Mexican Americans (8.9% in men, 26.7% in women), intermediate for non-Hispanic whites (8.6% in men, 16.6% in women), and low for African Americans (5.3% in men, 13.9% in women).

1	Gallstones are formed because of abnormal bile composition. They are divided into two major types: cholesterol stones and pigment stones. Cholesterol stones account for more than 90% of all gallstones in Western industrialized countries. Cholesterol gallstones usually contain >50% cholesterol monohydrate plus an admixture of calcium salts, bile pigments, proteins, and fatty acids. Pigment stones are composed primarily of calcium bilirubinate; they contain <20% cholesterol and are classified into “black” and “brown” types, the latter forming secondary to chronic biliary infection.

1	cHolesterol stones anD biliary sluDGe Cholesterol is essentially water insoluble and requires aqueous dispersion into either micelles or vesicles, both of which require the presence of a second lipid to solubilize the cholesterol. Cholesterol and phospholipids are secreted into bile as unilamellar bilayered vesicles, which are converted into mixed micelles consisting of bile acids, phospholipids, and cholesterol by the action of bile acids. If there is an excess of cholesterol in relation to phospholipids and bile acids, unstable, cholesterol-rich vesicles remain, which aggregate into large multilamellar vesicles from which cholesterol crystals precipitate (Fig. 369-1). There are several important mechanisms in the formation of lithogenic (stone-forming) bile. The most important is increased biliary secretion of cholesterol. This may occur in association with obesity, the metabolic syndrome, high-caloric and cholesterol-rich diets, I. II. Supersaturation III. Nucleation

1	I. II. Supersaturation III. Nucleation IV. Microstone FIGURE 369-1 Scheme showing pathogenesis of cholesterol gallstone formation. Conditions or factors that increase the ratio of cholesterol to bile acids and phospholipids (lecithin) favor gallstone formation. ABCB4, ATP-binding cassette transporter; ABCG5/8, ATP-binding cassette (ABC) transporter G5/G8; CYP7A1, cytochrome P450 7A1; MDR3, multidrug resistance protein 3, also called phospholipid export pump.

1	or drugs (e.g., clofibrate) and may result from increased activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of hepatic cholesterol synthesis, and increased hepatic uptake of cholesterol from blood. In patients with gallstones, dietary cholesterol increases biliary cholesterol secretion. This does not occur in non-gallstone patients on high-cholesterol diets. In addition to environmental factors such as high-caloric and cholesterol-rich diets, genetic factors play an important role in gallstone disease. A large study of symptomatic gallstones in Swedish twins provided strong evidence for a role of genetic factors in gallstone pathogenesis. Genetic factors accounted for 25%, shared environmental factors for 13%, and individual environmental factors for 62% of the phenotypic variation among monozygotic twins. A single nucleotide polymorphism of the gene encoding the hepatic cholesterol transporter ABCG5/G8 has been found in 21% of patients with

1	for 62% of the phenotypic variation among monozygotic twins. A single nucleotide polymorphism of the gene encoding the hepatic cholesterol transporter ABCG5/G8 has been found in 21% of patients with gallstones, but only in 9% of the general population. It is thought to cause a gain of function of the cholesterol transporter and to contribute to cholesterol hypersecretion. A high prevalence of gallstones is found among first-degree relatives of gallstone carriers and in certain ethnic populations such as American Indians, Chilean Indians, and Chilean Hispanics. A common genetic trait has been identified for some of these populations by mitochondrial DNA analysis. In some patients, impaired hepatic conversion of cholesterol to bile acids may also occur, resulting in an increase of the lithogenic cholesterol/bile acid ratio. Although most cholesterol stones have a polygenic basis, there are rare monogenic (Mendelian) causes.

1	Recently, a mutation in the CYP7A1 gene has been described that 2077 results in a deficiency of the enzyme cholesterol 7-hydroxylase, which catalyzes the initial step in cholesterol catabolism and bile acid synthesis. The homozygous state is associated with hypercholesterolemia and gallstones. Because the phenotype is expressed in the heterozygote state, mutations in the CYP7A1 gene may contribute to the susceptibility to cholesterol gallstone disease in the population. Mutations in the MDR3 (ABCB4) gene, which encodes the phospholipid export pump in the canalicular membrane of the hepatocyte, may cause defective phospholipid secretion into bile, resulting in cholesterol supersaturation of bile and formation of cholesterol gallstones in the gallbladder and in the bile ducts. Thus, an excess of biliary cholesterol in relation to bile acids and phospholipids is primarily due to hypersecretion of cholesterol, but hyposecretion of bile acids or phospholipids may contribute. An additional

1	of biliary cholesterol in relation to bile acids and phospholipids is primarily due to hypersecretion of cholesterol, but hyposecretion of bile acids or phospholipids may contribute. An additional disturbance of bile acid metabolism that is likely to contribute to supersaturation of bile with cholesterol is enhanced conversion of cholic acid to deoxycholic acid, with replacement of the cholic acid pool by an expanded deoxycholic acid pool. It may result from enhanced dehydroxylation of cholic acid and increased absorption of newly formed deoxycholic acid. An increased deoxycholate secretion is associated with hypersecretion of cholesterol into bile.

1	While supersaturation of bile with cholesterol is an important prerequisite for gallstone formation, it is generally not sufficient by itself to produce cholesterol precipitation in vivo. Most individuals with supersaturated bile do not develop stones because the time required for cholesterol crystals to nucleate and grow is longer than the time bile remains in the gallbladder.

1	An important mechanism is nucleation of cholesterol monohydrate crystals, which is greatly accelerated in human lithogenic bile. Accelerated nucleation of cholesterol monohydrate in bile may be due to either an excess of pronucleating factors or a deficiency of antinucleating factors. Mucin and certain nonmucin glycoproteins, principally immunoglobulins, appear to be pronucleating factors, while apolipoproteins A-I and A-II and other glycoproteins appear to be antinucleating factors. Pigment particles may possibly play a role as nucleating factors. In a genome-wide analysis of serum bilirubin levels, the uridine diphosphate-glucuronyltransferase 1A1 (UGT1A1) Gilbert’s syndrome gene variant was associated with the presence of gallstone disease. Because most gallstones associated with the UGT1A1 variant were cholesterol stones, this finding points to the role of pigment particles in the pathogenesis of gallbladder stones. Cholesterol mono-hydrate crystal nucleation and crystal growth

1	UGT1A1 variant were cholesterol stones, this finding points to the role of pigment particles in the pathogenesis of gallbladder stones. Cholesterol mono-hydrate crystal nucleation and crystal growth probably occur within the mucin gel layer. Vesicle fusion leads to liquid crystals, which, in turn, nucleate into solid cholesterol monohydrate crystals. Continued growth of the crystals occurs by direct nucleation of cholesterol molecules from supersaturated unilamellar or multilamellar biliary vesicles.

1	A third important mechanism in cholesterol gallstone formation is gallbladder hypomotility. If the gallbladder emptied all supersaturated or crystal-containing bile completely, stones would not be able to grow. A high percentage of patients with gallstones exhibit abnormalities of gallbladder emptying. Ultrasonographic studies show that gallstone patients display an increased gallbladder volume during fasting and also after a test meal (residual volume) and that fractional emptying after gallbladder stimulation is decreased. The incidence of gallstones is increased in conditions associated with infrequent or impaired gallbladder emptying such as fasting, parenteral nutrition, or pregnancy and in patients using drugs that inhibit gallbladder motility.

1	Biliary sludge is a thick, mucous material that, upon microscopic examination, reveals lecithin-cholesterol liquid crystals, cholesterol monohydrate crystals, calcium bilirubinate, and mucin gels. Biliary sludge typically forms a crescent-like layer in the most dependent portion of the gallbladder and is recognized by characteristic echoes on ultrasonography (see below). The presence of biliary sludge implies two abnormalities: (1) the normal balance between gallbladder mucin secretion and elimination has become deranged, and (2) nucleation of biliary solutes has occurred. That biliary sludge may be a precursor form of gallstone disease is evident from several observations. In one study, 96 patients with gallbladder sludge were followed prospectively by serial ultrasound studies. In 18%, biliary sludge disappeared and did

1	CHAPTER 369 Diseases of the Gallbladder and Bile Ducts 2078 not recur for at least 2 years. In 60%, biliary sludge disappeared and reappeared; in 14%, gallstones (8% asymptomatic, 6% symptomatic) developed; and in 6%, severe biliary pain with or without acute pancreatitis occurred. In 12 patients, cholecystectomies were performed, 6 for gallstone-associated biliary pain and 3 in symptomatic patients with sludge but without gallstones who had prior attacks of pancreatitis; the latter did not recur after cholecystectomy. It should be emphasized that biliary sludge can develop with disorders that cause gallbladder hypomotility; i.e., surgery, burns, total parenteral nutrition, pregnancy, and oral contraceptives—all of which are associated with gallstone formation. However, the presence of biliary sludge implies supersaturation of bile with either cholesterol or calcium bilirubinate. Two other conditions are associated with cholesterol-stone or biliary-sludge formation: pregnancy and

1	biliary sludge implies supersaturation of bile with either cholesterol or calcium bilirubinate. Two other conditions are associated with cholesterol-stone or biliary-sludge formation: pregnancy and rapid weight reduction through a very-low-calorie diet. There appear to be two key changes during pregnancy that contribute to a “cholelithogenic state”: (1) a marked increase in cholesterol saturation of bile during the third trimester and (2) sluggish gallbladder contraction in response to a standard meal, resulting in impaired gallbladder emptying. That these changes are related to pregnancy per se is supported by several studies that show reversal of these abnormalities quite rapidly after delivery. During pregnancy, gallbladder sludge develops in 20–30% of women and gallstones in 5–12%. Although biliary sludge is a common finding during pregnancy, it is usually asymptomatic and often resolves spontaneously after delivery. Gallstones, which are less common than sludge and frequently

1	Although biliary sludge is a common finding during pregnancy, it is usually asymptomatic and often resolves spontaneously after delivery. Gallstones, which are less common than sludge and frequently associated with biliary colic, may also disappear after delivery because of spontaneous dissolution related to bile becoming unsaturated with cholesterol postpartum. Approximately 10–20% of persons with rapid weight reduction achieved through very-low-calorie dieting develop gallstones. In a study involving 600 patients who completed a 3-month, 520-kcal/d diet, UDCA in a dosage of 600 mg/d proved highly effective in preventing gallstone formation; gallstones developed in only 3% of UDCA recipients, compared to 28% of placebo-treated patients. In obese patients treated by gastric banding, 500 mg/d of UDCA reduced the risk of gallstone formation from 30% to 8% within a follow-up of 6 months. To summarize, cholesterol gallstone disease occurs because of several defects, which include (1) bile

1	of UDCA reduced the risk of gallstone formation from 30% to 8% within a follow-up of 6 months. To summarize, cholesterol gallstone disease occurs because of several defects, which include (1) bile supersaturation with cholesterol, (2) nucleation of cholesterol monohydrate with subsequent crystal retention and stone growth, and (3) abnormal gallbladder motor function with delayed emptying and stasis. Other important factors known to predispose to cholesterol-stone formation are summarized in Table 369-1.

1	piGment stones Black pigment stones are composed of either pure calcium bilirubinate or polymer-like complexes with calcium and mucin glycoproteins. They are more common in patients who have chronic hemolytic states (with increased conjugated bilirubin in bile), liver cirrhosis, Gilbert’s syndrome, or cystic fibrosis. Gallbladder stones in patients with ileal diseases, ileal resection, or ileal bypass generally are also black pigment stones. Enterohepatic recycling of bilirubin in ileal disease states contributes to their pathogenesis. Brown pigment stones are composed of calcium salts of unconjugated bilirubin with varying amounts of cholesterol and protein. They are caused by the presence of increased amounts of unconjugated, insoluble bilirubin in bile that precipitates to form stones. Deconjugation of an excess of soluble bilirubin monoand diglucuronides may be mediated by endogenous β-glucuronidase but may also occur by spontaneous hydrolysis. Sometimes, the enzyme is also

1	Deconjugation of an excess of soluble bilirubin monoand diglucuronides may be mediated by endogenous β-glucuronidase but may also occur by spontaneous hydrolysis. Sometimes, the enzyme is also produced when bile is chronically infected by bacteria, and such stones are brown. Pigment stone formation is frequent in Asia and is often associated with infections in the gallbladder and biliary tree (Table 369-1).

1	Diagnosis Procedures of potential use in the diagnosis of cholelithiasis and other diseases of the gallbladder are detailed in Table 369-2. Ultrasonography of the gallbladder is very accurate in the identification of cholelithiasis and has replaced oral cholecystography (Fig. 369-2A). Stones as small as 1.5 mm in diameter may be confidently identified provided that firm criteria are used (e.g., acoustic “shadowing” of opacities that are within the gallbladder lumen and that change with the patient’s position [by gravity]). In major medical centers, the false-negative and 1. Demographic/genetic factors: Prevalence highest in North American Indians, Chilean Indians, and Chilean Hispanics, greater in Northern Europe and North America than in Asia, lowest in Japan; familial disposition; hereditary aspects 2. Obesity, metabolic syndrome: Normal bile acid pool and secretion but increased biliary secretion of cholesterol 3.

1	Obesity, metabolic syndrome: Normal bile acid pool and secretion but increased biliary secretion of cholesterol 3. Weight loss: Mobilization of tissue cholesterol leads to increased biliary cholesterol secretion while enterohepatic circulation of bile acids is decreased 4. a. Estrogens stimulate hepatic lipoprotein receptors, increase uptake of dietary cholesterol, and increase biliary cholesterol secretion b. Natural estrogens, other estrogens, and oral contraceptives lead to decreased bile salt secretion and decreased conversion of cholesterol to cholesteryl esters 5. Pregnancy: Impaired gallbladder emptying caused by progesterone combined with the influence of estrogens, which increase biliary cholesterol secretion 6. Increasing age: Increased biliary secretion of cholesterol, decreased size of bile acid pool, decreased secretion of bile salts 7. Gallbladder hypomotility leading to stasis and formation of sludge a. b. c. d. Drugs such as octreotide 8.

1	Gallbladder hypomotility leading to stasis and formation of sludge a. b. c. d. Drugs such as octreotide 8. Clofibrate therapy: Increased biliary secretion of cholesterol 9. a. b. Genetic defect of the CYP7A1 gene 10. Decreased phospholipid secretion: Genetic defect of the MDR3 gene 11. a. High-calorie, high-fat diet b. 1. Demographic/genetic factors: Asia, rural setting 2. 3. 4. 5. 6. Chronic biliary tract infection, parasite infections 7. 8. Ileal disease, ileal resection or bypass false-positive rates for ultrasound in gallstone patients are ∼2–4%. Biliary sludge is material of low echogenic activity that typically forms a layer in the most dependent position of the gallbladder. This layer shifts with postural changes but fails to produce acoustic shadowing; these two characteristics distinguish sludges from gallstones. Ultrasound can also be used to assess the emptying function of the gallbladder.

1	The plain abdominal film may detect gallstones containing sufficient calcium to be radiopaque (10–15% of cholesterol and ∼50% of pigment stones). Plain radiography may also be of use in the diagnosis of emphysematous cholecystitis, porcelain gallbladder, limey bile, and gallstone ileus. Oral cholecystography (OCG) has historically been a useful procedure for the diagnosis of gallstones but has been replaced by ultrasound and is regarded as obsolete. It may be used to assess the patency of the cystic duct and gallbladder emptying function. Further, OCG can also delineate the size and number of gallstones and determine whether they are calcified.

1	Radiopharmaceuticals such as 99mTc-labeled N-substituted iminodiacetic acids (HIDA, DIDA, DISIDA, etc.) are rapidly extracted from the blood and are excreted into the biliary tree in high concentration even in the presence of mild to moderate serum bilirubin elevations. Failure to image the gallbladder in the presence of biliary ductal visualization may indicate cystic duct obstruction, acute or chronic cholecystitis, or surgical absence of the organ. Such scans have some application in the diagnosis of acute cholecystitis.

1	Symptoms of Gallstone Disease Gallstones usually produce symptoms by causing inflammation or obstruction following their migration into the cystic duct or CBD. The most specific and characteristic symptom of gallstone disease is biliary colic that is a constant and often long-lasting pain (see below). Obstruction of the cystic duct or CBD by a stone produces increased intraluminal pressure and distention of the viscus that cannot be relieved by repetitive biliary contractions. The resultant visceral pain is characteristically a severe, steady ache or fullness in the epigastrium or right upper quadrant (RUQ) of the abdomen with frequent radiation to the interscapular area, right scapula, or shoulder.

1	Biliary colic begins quite suddenly and may persist with severe intensity for 30 min to 5 h, subsiding gradually or rapidly. It is steady rather than intermittent, as would be suggested by the word colic, which must be regarded as a misnomer, although it is in widespread use. An episode of biliary pain persisting beyond 5 h should raise the suspicion of acute cholecystitis (see below). Nausea and vomiting frequently accompany episodes of biliary pain. An elevated level of serum bilirubin and/or alkaline phosphatase suggests a common duct stone. Fever or chills (rigors) with biliary pain usually imply a complication, i.e., cholecystitis, pancreatitis, or cholangitis. Complaints of short-lasting, vague epigastric fullness, dyspepsia, eructation, or flatulence, especially following a fatty meal, should not be confused with biliary pain. Such symptoms are frequently elicited from patients with or without gallstone disease Diseases of the Gallbladder and Bile Ducts

1	Diseases of the Gallbladder and Bile Ducts FIGURE 369-2 Examples of ultrasound and radiologic studies of the biliary tract. A. An ultrasound study showing a distended gallbladder (GB) containing a single large stone (arrow), which casts an acoustic shadow. B. Endoscopic retrograde cholangiopancreatogram (ERCP) showing normal biliary tract anatomy. In addition to the endoscope and large vertical gallbladder filled with contrast dye, the common hepatic duct (CHD), common bile duct (CBD), and pancreatic duct (PD) are shown. The arrow points to the ampulla of Vater. C. Endoscopic retrograde cholangiogram (ERC) showing choledocholithiasis. The biliary tract is dilated and contains multiple radiolucent calculi. D. ERCP showing sclerosing cholangitis. The common bile duct shows areas that are strictured and narrowed.

1	2080 but are not specific for biliary calculi. Biliary colic may be precipitated by eating a fatty meal, by consumption of a large meal following a period of prolonged fasting, or by eating a normal meal; it is frequently nocturnal, occurring within a few hours of retiring.

1	Natural History Gallstone disease discovered in an asymptomatic patient or in a patient whose symptoms are not referable to cholelithiasis is a common clinical problem. Sixty to 80% of persons with asymptomatic gallstones remain asymptomatic over follow-up periods of up to 25 years. The probability of developing symptoms within 5 years after diagnosis is 2–4% per year and decreases in the years thereafter to 1–2%. The yearly incidence of complications is about 0.1–0.3%. Patients remaining asymptomatic for 15 years were found to be unlikely to develop symptoms during further follow-up, and most patients who did develop complications from their gallstones experienced prior warning symptoms. Similar conclusions apply to diabetic patients with silent gallstones. Decision analysis has suggested that (1) the cumulative risk of death due to gallstone disease while on expectant management is small, and (2) prophylactic cholecystectomy is not warranted.

1	Complications requiring cholecystectomy are much more common in gallstone patients who have developed symptoms of biliary pain. Patients found to have gallstones at a young age are more likely to develop symptoms from cholelithiasis than are patients >60 years at the time of initial diagnosis. Patients with diabetes mellitus and gallstones may be somewhat more susceptible to septic complications, but the magnitude of risk of septic biliary complications in diabetic patients is incompletely defined.

1	In asymptomatic gallstone patients, the risk of developing symptoms or complications requiring surgery is quite small (see above). Thus, a recommendation for cholecystectomy in a patient with gallstones should probably be based on assessment of three factors: (1) the presence of symptoms that are frequent enough or severe enough to interfere with the patient’s general routine; the presence of a prior complication of gallstone disease, i.e., history of acute cholecystitis, pancreatitis, gallstone fistula, etc.; or the presence of an underlying condition predisposing the patient to increased risk of gallstone complications (e.g., calcified or porcelain gallbladder and/or a previous attack of acute cholecystitis regardless of current symptomatic status). Patients with very large gallstones (>3 cm in diameter) and patients harboring gallstones in a congenitally anomalous gallbladder might also be considered for prophylactic cholecystectomy. Although young age is a worrisome factor in

1	(>3 cm in diameter) and patients harboring gallstones in a congenitally anomalous gallbladder might also be considered for prophylactic cholecystectomy. Although young age is a worrisome factor in asymptomatic gallstone patients, few authorities would now recommend routine cholecystectomy in all young patients with silent stones. Laparoscopic cholecystectomy is a minimal-access approach for the removal of the gallbladder together with its stones. Its advantages include a markedly shortened hospital stay, minimal disability, and decreased cost, and it is the procedure of choice for most patients referred for elective cholecystectomy.

1	From several studies involving >4000 patients undergoing laparoscopic cholecystectomy, the following key points emerge: complications develop in ∼4% of patients, (2) conversion to laparotomy occurs in 5%, (3) the death rate is remarkably low (i.e., <0.1%), and (4) the rate of bile duct injuries is low (i.e., 0.2–0.6%) and comparable with open cholecystectomy. These data indicate why laparoscopic cholecystectomy has become the “gold standard” for treating symptomatic cholelithiasis.

1	In carefully selected patients with a functioning gallbladder and with radiolucent stones <10 mm in diameter, complete dissolution can be achieved in ∼50% of patients within 6 months to 2 years. For good results within a reasonable time period, this therapy should be limited to radiolucent stones smaller than 5 mm in diameter. The dose of UDCA should be 10–15 mg/kg per day. Stones larger than 10 mm in size rarely dissolve. Pigment stones are not responsive to UDCA therapy. Probably ≤10% of patients with symptomatic cholelithiasis are candidates for such treatment. However, in addition to the vexing problem of recurrent stones (30–50% over 3–5 years of follow-up), there is also the factor of taking an expensive drug for up to 2 years. The advantages and success of laparoscopic cholecystectomy have largely reduced the role of gallstone dissolution to patients who wish to avoid or are not candidates for elective cholecystectomy. However, patients with cholesterol gallstone disease who

1	have largely reduced the role of gallstone dissolution to patients who wish to avoid or are not candidates for elective cholecystectomy. However, patients with cholesterol gallstone disease who develop recurrent choledocholithiasis after cholecystectomy should be on long-term treatment with UDCA.

1	ACUTE AND CHRONIC CHOLECYSTITIS Acute Cholecystitis Acute inflammation of the gallbladder wall usually follows obstruction of the cystic duct by a stone. Inflammatory response can be evoked by three factors: (1) mechanical inflammation produced by increased intraluminal pressure and distention with resulting ischemia of the gallbladder mucosa and wall, (2) chemical inflammation caused by the release of lysolecithin (due to the action of phospholipase on lecithin in bile) and other local tissue factors, and (3) bacterial inflammation, which may play a role in 50–85% of patients with acute cholecystitis. The organisms most frequently isolated by culture of gallbladder bile in these patients include Escherichia coli, Klebsiella spp., Streptococcus spp., and Clostridium spp.

1	Acute cholecystitis often begins as an attack of biliary pain that progressively worsens. Approximately 60–70% of patients report having experienced prior attacks that resolved spontaneously. As the episode progresses, however, the pain of acute cholecystitis becomes more generalized in the right upper abdomen. As with biliary colic, the pain of cholecystitis may radiate to the interscapular area, right scapula, or shoulder. Peritoneal signs of inflammation such as increased pain with jarring or on deep respiration may be apparent. The patient is anorectic and often nauseated. Vomiting is relatively common and may produce symptoms and signs of vascular and extracellular volume depletion. Jaundice is unusual early in the course of acute cholecystitis but may occur when edematous inflammatory changes involve the bile ducts and surrounding lymph nodes.

1	A low-grade fever is characteristically present, but shaking chills or rigors are not uncommon. The RUQ of the abdomen is almost invariably tender to palpation. An enlarged, tense gallbladder is palpable in 25–50% of patients. Deep inspiration or cough during subcostal palpation of the RUQ usually produces increased pain and inspiratory arrest (Murphy’s sign). Localized rebound tenderness in the RUQ is common, as are abdominal distention and hypoactive bowel sounds from paralytic ileus, but generalized peritoneal signs and abdominal rigidity are usually lacking, in the absence of perforation.

1	The diagnosis of acute cholecystitis is usually made on the basis of a characteristic history and physical examination. The triad of sudden onset of RUQ tenderness, fever, and leukocytosis is highly suggestive. Typically, leukocytosis in the range of 10,000–15,000 cells per microliter with a left shift on differential count is found. The serum bilirubin is mildly elevated (<85.5 μmol/L [5 mg/dL]) in fewer than half of patients, whereas about one-fourth have modest elevations in serum aminotransferases (usually less than a fivefold elevation). Ultrasound will demonstrate calculi in 90–95% of cases and is useful for detection of signs of gallbladder inflammation including thickening of the wall, pericholecystic fluid, and dilatation of the bile duct. The radionuclide (e.g., HIDA) biliary scan may be confirmatory if bile duct imaging is seen without visualization of the gallbladder.

1	Approximately 75% of patients treated medically have remission of acute symptoms within 2–7 days following hospitalization. In 25%, however, a complication of acute cholecystitis will occur despite conservative treatment (see below). In this setting, prompt surgical intervention is required. Of the 75% of patients with acute cholecystitis who undergo remission of symptoms, ∼25% will experience a recurrence of cholecystitis within 1 year, and 60% will have at least one recurrent bout within 6 years. In view of the natural history of the disease, acute cholecystitis is best treated by early surgery whenever possible.

1	Mirizzi’s syndrome is a rare complication in which a gallstone becomes impacted in the cystic duct or neck of the gallbladder causing compression of the CBD, resulting in CBD obstruction and jaundice. Ultrasound shows gallstone(s) lying outside the hepatic duct. Endoscopic retrograde cholangiopancreatography (ERCP) (Fig. 369-2B), percutaneous transhepatic cholangiography (PTC), or magnetic resonance cholangiopancreatography (MRCP) will usually demonstrate the characteristic extrinsic compression of the CBD. Surgery consists of removing the cystic duct, diseased gallbladder, and the impacted stone. The preoperative diagnosis of Mirizzi’s syndrome is important to avoid CBD injury.

1	acalculous cHolecystitis In 5–10% of patients with acute cholecystitis, calculi obstructing the cystic duct are not found at surgery. In >50% of such cases, an underlying explanation for acalculous inflammation is not found. An increased risk for the development of acalculous cholecystitis is especially associated with serious trauma or burns, with the postpartum period following prolonged labor, and with orthopedic and other nonbiliary major surgical operations in the postoperative period. It may possibly complicate periods of prolonged parenteral hyperalimentation. For some of these cases, biliary sludge in the cystic duct may be responsible. Other precipitating factors include vasculitis, obstructing adenocarcinoma of the gallbladder, diabetes mellitus, torsion of the gallbladder, “unusual” bacterial infections of the gallbladder (e.g., Leptospira, Streptococcus, Salmonella, or Vibrio cholerae), and parasitic infestation of the gallbladder. Acalculous cholecystitis may also be seen

1	bacterial infections of the gallbladder (e.g., Leptospira, Streptococcus, Salmonella, or Vibrio cholerae), and parasitic infestation of the gallbladder. Acalculous cholecystitis may also be seen with a variety of other systemic disease processes (e.g., sarcoidosis, cardiovascular disease, tuberculosis, syphilis, actinomycosis).

1	Although the clinical manifestations of acalculous cholecystitis are indistinguishable from those of calculous cholecystitis, the setting of acute gallbladder inflammation complicating severe underlying illness is characteristic of acalculous disease. Ultrasound or computed tomography (CT) examinations demonstrating a large, tense, static gallbladder without stones and with evidence of poor emptying over a prolonged period may be diagnostically useful in some cases. The complication rate for acalculous cholecystitis exceeds that for calculous cholecystitis. Successful management of acute acalculous cholecystitis appears to depend primarily on early diagnosis and surgical intervention, with meticulous attention to postoperative care.

1	acalculous cHolecystopatHy Disordered motility of the gallbladder can produce recurrent biliary pain in patients without gallstones. Infusion of an octapeptide of CCK can be used to measure the gallbladder ejection fraction during cholescintigraphy. The surgical findings have included abnormalities such as chronic cholecystitis, gallbladder muscle hypertrophy, and/or a markedly narrowed cystic duct. Some of these patients may well have had antecedent gallbladder disease. The following criteria can be used to identify patients with acalculous cholecystopathy: (1) recurrent episodes of typical RUQ pain characteristic of biliary tract pain, (2) abnormal CCK cholescintigraphy demonstrating a gallbladder ejection fraction of <40%, and (3) infusion of CCK reproducing the patient’s pain. An additional clue would be the identification of a large gallbladder on ultrasound examination. Finally, it should be noted that sphincter of Oddi dysfunction can also give rise to recurrent RUQ pain and

1	clue would be the identification of a large gallbladder on ultrasound examination. Finally, it should be noted that sphincter of Oddi dysfunction can also give rise to recurrent RUQ pain and CCK-scintigraphic abnormalities.

1	empHysematous cHolecystitis So-called emphysematous cholecystitis is thought to begin with acute cholecystitis (calculous or acalculous) followed by ischemia or gangrene of the gallbladder wall and infection by gas-producing organisms. Bacteria most frequently cultured in this setting include anaerobes, such as Clostridium welchii or Clostridium perfringens, and aerobes, such as E. coli. This condition occurs most frequently in elderly men and in patients with diabetes mellitus. The clinical manifestations are essentially indistinguishable from those of nongaseous cholecystitis. The diagnosis is usually made on plain abdominal film by finding gas within the gallbladder lumen, dissecting within the gallbladder wall to form a gaseous ring, or in the pericholecystic tissues. The morbidity and mortality rates with emphysematous cholecystitis are considerable. Prompt surgical intervention coupled with appropriate antibiotics is mandatory.

1	Chronic Cholecystitis Chronic inflammation of the gallbladder wall 2081 is almost always associated with the presence of gallstones and is thought to result from repeated bouts of subacute or acute cholecystitis or from persistent mechanical irritation of the gallbladder wall by gallstones. The presence of bacteria in the bile occurs in >25% of patients with chronic cholecystitis. The presence of infected bile in a patient with chronic cholecystitis undergoing elective cholecystectomy probably adds little to the operative risk. Chronic cholecystitis may be asymptomatic for years, may progress to symptomatic gallbladder disease or to acute cholecystitis, or may present with complications (see below).

1	Complications of Cholecystitis • empyema anD HyDrops Empyema of the gallbladder usually results from progression of acute cholecystitis with persistent cystic duct obstruction to superinfection of the stagnant bile with a pus-forming bacterial organism. The clinical picture resembles that of cholangitis with high fever; severe RUQ pain; marked leukocytosis; and often, prostration. Empyema of the gallbladder carries a high risk of gram-negative sepsis and/or perforation. Emergency surgical intervention with proper antibiotic coverage is required as soon as the diagnosis is suspected.

1	Hydrops or mucocele of the gallbladder may also result from prolonged obstruction of the cystic duct, usually by a large solitary calculus. In this instance, the obstructed gallbladder lumen is progressively distended, over a period of time, by mucus (mucocele) or by a clear transudate (hydrops) produced by mucosal epithelial cells. A visible, easily palpable, nontender mass sometimes extending from the RUQ into the right iliac fossa may be found on physical examination. The patient with hydrops of the gallbladder frequently remains asymptomatic, although chronic RUQ pain may also occur. Cholecystectomy is indicated, because empyema, perforation, or gangrene may complicate the condition.

1	GanGrene anD perforation Gangrene of the gallbladder results from ischemia of the wall and patchy or complete tissue necrosis. Underlying conditions often include marked distention of the gallbladder, vasculitis, diabetes mellitus, empyema, or torsion resulting in arterial occlusion. Gangrene usually predisposes to perforation of the gallbladder, but perforation may also occur in chronic cholecystitis without premonitory warning symptoms. Localized perforations are usually contained by the omentum or by adhesions produced by recurrent inflammation of the gallbladder. Bacterial superinfection of the walled-off gallbladder contents results in abscess formation. Most patients are best treated with cholecystectomy, but some seriously ill patients may be managed with cholecystostomy and drainage of the abscess. Free perforation is less common but is associated with a mortality rate of ∼30%. Such patients may experience a sudden transient relief of RUQ pain as the distended gallbladder

1	of the abscess. Free perforation is less common but is associated with a mortality rate of ∼30%. Such patients may experience a sudden transient relief of RUQ pain as the distended gallbladder decompresses; this is followed by signs of generalized peritonitis.

1	fistula formation anD Gallstone ileus Fistula formation into an adjacent organ adherent to the gallbladder wall may result from inflammation and adhesion formation. Fistulas into the duodenum are most common, followed in frequency by those involving the hepatic flexure of the colon, stomach or jejunum, abdominal wall, and renal pelvis. Clinically “silent” biliary-enteric fistulas occurring as a complication of acute cholecystitis have been found in up to 5% of patients undergoing cholecystectomy. Asymptomatic cholecystoenteric fistulas may sometimes be diagnosed by finding gas in the biliary tree on plain abdominal films. Barium contrast studies or endoscopy of the upper gastrointestinal tract or colon may demonstrate the fistula. Treatment in the symptomatic patient usually consists of cholecystectomy, CBD exploration, and closure of the fistulous tract.

1	Gallstone ileus refers to mechanical intestinal obstruction resulting from the passage of a large gallstone into the bowel lumen. The stone customarily enters the duodenum through a cholecystoenteric fistula at that level. The site of obstruction by the impacted gallstone is usually at the ileocecal valve, provided that the more proximal small bowel is of normal caliber. The majority of patients do not give a history of either prior biliary tract symptoms or complaints suggestive of acute cholecystitis or fistula formation. Large stones, >2.5 cm in diameter, are

1	Diseases of the Gallbladder and Bile Ducts 2082 thought to predispose to fistula formation by gradual erosion through the gallbladder fundus. Diagnostic confirmation may occasionally be found on the plain abdominal film (e.g., small-intestinal obstruction with gas in the biliary tree and a calcified, ectopic gallstone) or following an upper gastrointestinal series (cholecystoduodenal fistula with small-bowel obstruction at the ileocecal valve). Laparotomy with stone extraction (or propulsion into the colon) remains the procedure of choice to relieve obstruction. Evacuation of large stones within the gallbladder should also be performed. In general, the gallbladder and its attachment to the intestines should be left alone.

1	limey (milk of calcium) bile anD porcelain GallblaDDer Calcium salts in the lumen of the gallbladder in sufficient concentration may produce calcium precipitation and diffuse, hazy opacification of bile or a layering effect on plain abdominal roentgenography. This so-called limey bile, or milk of calcium bile, is usually clinically innocuous, but cholecystectomy is recommended, especially when it occurs in a hydropic gallbladder. In the entity called porcelain gallbladder, calcium salt deposition within the wall of a chronically inflamed gallbladder may be detected on the plain abdominal film. Cholecystectomy is advised in all patients with porcelain gallbladder because in a high percentage of cases this finding appears to be associated with the development of carcinoma of the gallbladder.

1	Although surgical intervention remains the mainstay of therapy for acute cholecystitis and its complications, a period of in-hospital stabilization may be required before cholecystectomy. Oral intake is eliminated, nasogastric suction may be indicated, and extracellular volume depletion and electrolyte abnormalities are repaired. Meperidine or nonsteroidal anti-inflammatory drugs (NSAIDs) are usually employed for analgesia because they may produce less spasm of the sphincter of Oddi than drugs such as morphine. Intravenous antibiotic therapy is usually indicated in patients with severe acute cholecystitis, even though bacterial superinfection of bile may not have occurred in the early stages of the inflammatory process. Antibiotic therapy is guided by the most common organisms likely to be present, which are E. coli, Klebsiella spp., and Streptococcus spp. Effective antibiotics include ureidopenicillins such as piperacillin or mezlocillin, ampicillin sulbactam, ciprofloxacin,

1	to be present, which are E. coli, Klebsiella spp., and Streptococcus spp. Effective antibiotics include ureidopenicillins such as piperacillin or mezlocillin, ampicillin sulbactam, ciprofloxacin, moxifloxacin, and third-generation cephalosporins. Anaerobic coverage by a drug such as metronidazole should be added if gangrenous or emphysematous cholecystitis is suspected. Imipenem and meropenem represent potent parenteral antibiotics that cover the whole spectrum of bacteria causing ascending cholangitis. They should, however, be reserved for the most severe, life-threatening infections when other regimens have failed (Chap. 186). Postoperative complications of wound infection, abscess formation, and sepsis are reduced in antibiotic-treated patients.

1	The optimal timing of surgical intervention in patients with acute cholecystitis depends on stabilization of the patient. The clear trend is toward earlier surgery, and this is due in part to requirements for shorter hospital stays. Urgent (emergency) cholecystectomy or cholecystostomy is probably appropriate in most patients in whom a complication of acute cholecystitis such as empyema, emphysematous cholecystitis, or perforation is suspected or confirmed. Patients with uncomplicated acute cholecystitis should undergo early elective laparoscopic cholecystectomy, ideally within 48–72 h after diagnosis. The complication rate is not increased in patients undergoing early as opposed to delayed (>6 weeks after diagnosis) cholecystectomy. Delayed surgical intervention is probably best reserved for (1) patients in whom the overall medical condition imposes an unacceptable risk for early surgery and (2) patients in whom the diagnosis of acute cholecystitis is in doubt. Thus, early

1	reserved for (1) patients in whom the overall medical condition imposes an unacceptable risk for early surgery and (2) patients in whom the diagnosis of acute cholecystitis is in doubt. Thus, early cholecystectomy (within 72 h) is the treatment of choice for most patients with acute cholecystitis. Mortality figures for emergency cholecystectomy in most centers range from 1–3%, whereas the mortality risk for early elective cholecystectomy is ∼0.5% in patients under age 60. Of course, the operative risks increase with age-related diseases of other organ systems and with the presence of longor short-term complications of gallbladder disease. Seriously ill or debilitated patients with cholecystitis may be managed with cholecystostomy and tube drainage of the gallbladder. Elective cholecystectomy may then be done at a later date.

1	Postcholecystectomy Complications Early complications following cholecystectomy include atelectasis and other pulmonary disorders, abscess formation (often subphrenic), external or internal hemorrhage, biliary-enteric fistula, and bile leaks. Jaundice may indicate absorption of bile from an intraabdominal collection following a biliary leak or mechanical obstruction of the CBD by retained calculi, intraductal blood clots, or extrinsic compression.

1	Overall, cholecystectomy is a very successful operation that provides total or near-total relief of preoperative symptoms in 75–90% of patients. The most common cause of persistent postcholecystectomy symptoms is an overlooked symptomatic nonbiliary disorder (e.g., reflux esophagitis, peptic ulceration, pancreatitis, or—most often— irritable bowel syndrome). In a small percentage of patients, however, a disorder of the extrahepatic bile ducts may result in persistent symptomatology. These so-called postcholecystectomy syndromes may be due to (1) biliary strictures, (2) retained biliary calculi, (3) cystic duct stump syndrome, (4) stenosis or dyskinesia of the sphincter of Oddi, or (5) bile salt–induced diarrhea or gastritis.

1	cystic Duct stump synDrome In the absence of cholangiographically demonstrable retained stones, symptoms resembling biliary pain or cholecystitis in the postcholecystectomy patient have frequently been attributed to disease in a long (>1 cm) cystic duct remnant (cystic duct stump syndrome). Careful analysis, however, reveals that postcholecystectomy complaints are attributable to other causes in almost all patients in whom the symptom complex was originally thought to result from the existence of a long cystic duct stump. Accordingly, considerable care should be taken to investigate the possible role of other factors in the production of postcholecystectomy symptoms before attributing them to cystic duct stump syndrome.

1	papillary Dysfunction, papillary stenosis, spasm of tHe spHincter of oDDi, anD biliary Dyskinesia Symptoms of biliary colic accompanied by signs of recurrent, intermittent biliary obstruction may be produced by acalculous cholecystopathy, papillary stenosis, papillary dysfunction, spasm of the sphincter of Oddi, and biliary dyskinesia. Papillary stenosis is thought to result from acute or chronic inflammation of the papilla of Vater or from glandular hyperplasia of the papillary segment. Five criteria have been used to define papillary stenosis: upper abdominal pain, usually RUQ or epigastric; (2) abnormal liver tests; (3) dilatation of the CBD upon ERCP examination; (4) delayed (>45 min) drainage of contrast material from the duct; and increased basal pressure of the sphincter of Oddi, a finding that may be of only minor significance. An alternative to ERCP is magnetic resonance cholangiography (MRC) if ERCP and/or biliary manometry are either unavailable or not feasible. After

1	a finding that may be of only minor significance. An alternative to ERCP is magnetic resonance cholangiography (MRC) if ERCP and/or biliary manometry are either unavailable or not feasible. After exclusion of acalculous cholecystopathy, treatment consists of endoscopic or surgical sphincteroplasty to ensure wide patency of the distal portions of both the bile and pancreatic ducts. The greater the number of the preceding criteria present, the greater is the likelihood that a patient does have a degree of papillary stenosis sufficient to justify correction. The factors usually considered as indications for sphincterotomy include (1) prolonged duration of symptoms, (2) lack of response to symptomatic treatment, presence of severe disability, and (4) the patient’s choice of sphincterotomy over surgery (given a clear understanding on his or her part of the risks involved in both procedures).

1	Criteria for diagnosing dyskinesia of the sphincter of Oddi are even more controversial than those for papillary stenosis. Proposed mechanisms include spasm of the sphincter, denervation sensitivity resulting in hypertonicity, and abnormalities of the sequencing or frequency rates of sphincteric-contraction waves. When thorough evaluation has failed to demonstrate another cause for the pain, and when cholangiographic and manometric criteria suggest a diagnosis of biliary dyskinesia, medical treatment with nitrites or anticholinergics to attempt pharmacologic relaxation of the sphincter has been proposed. Endoscopic biliary sphincterotomy (EBS) or surgical sphincteroplasty may be indicated in patients who fail to respond to a 2to 3-month trial of medical therapy, especially if basal sphincter of Oddi pressures are elevated. EBS has become the procedure of choice for removing bile duct stones and for other biliary and pancreatic problems.

1	bile salt–inDuceD DiarrHea anD Gastritis Postcholecystectomy patients may develop symptoms of dyspepsia, which have been attributed to duodenogastric reflux of bile. However, firm data linking these symptoms to bile gastritis after surgical removal of the gallbladder are lacking. Cholecystectomy induces persistent changes in gut transit, and these changes effect a noticeable modification of bowel habits. Cholecystectomy shortens gut transit time by accelerating passage of the fecal bolus through the colon with marked acceleration in the right colon, thus causing an increase in colonic bile acid output and a shift in bile acid composition toward the more diarrheagenic secondary bile acids, i.e. deoxycholic acid. Diarrhea that is severe enough, i.e., three or more watery movements per day, can be classified as postcholecystectomy diarrhea, and this occurs in 5–10% of patients undergoing elective cholecystectomy. Treatment with bile acid–sequestering agents such as cholestyramine or

1	can be classified as postcholecystectomy diarrhea, and this occurs in 5–10% of patients undergoing elective cholecystectomy. Treatment with bile acid–sequestering agents such as cholestyramine or colestipol is often effective in ameliorating troublesome diarrhea.

1	The term hyperplastic cholecystoses is used to denote a group of disorders of the gallbladder characterized by excessive proliferation of normal tissue components. Adenomyomatosis is characterized by a benign proliferation of gallbladder surface epithelium with glandlike formations, extramural sinuses, transverse strictures, and/or fundal nodule (“adenoma” or “adenomyoma”) formation. Cholesterolosis is characterized by abnormal deposition of lipid, especially cholesteryl esters, within macrophages in the lamina propria of the gallbladder wall. In its diffuse form (“strawberry gallbladder”), the gallbladder mucosa is brick red and speckled with bright yellow flecks of lipid. The localized form shows solitary or multiple “cholesterol polyps” studding the gallbladder wall. Cholesterol stones of the gallbladder are found in nearly half the cases. Cholecystectomy is indicated in both adenomyomatosis and cholesterolosis when symptomatic or when cholelithiasis is present.

1	The prevalence of gallbladder polyps in the adult population is ∼5%, with a marked male predominance. Few significant changes have been found over a 5-year period in asymptomatic patients with gallbladder polyps <10 mm in diameter. Cholecystectomy is recommended in symptomatic patients, as well as in asymptomatic patients >50 years of age, or in those whose polyps are >10 mm in diameter or associated with gallstones or polyp growth on serial ultrasonography.

1	CONGENITAL ANOMALIES Biliary Atresia and Hypoplasia Atretic and hypoplastic lesions of the extrahepatic and large intrahepatic bile ducts are the most common biliary anomalies of clinical relevance encountered in infancy. The clinical picture is one of severe obstructive jaundice during the first month of life, with pale stools. When biliary atresia is suspected on the basis of clinical, laboratory, and imaging findings, the diagnosis is confirmed by surgical exploration and operative cholangiography. Approximately 10% of cases of biliary atresia are treatable with Roux-en-Y choledochojejunostomy, with the Kasai procedure (hepatic portoenterostomy) being attempted in the remainder in an effort to restore some bile flow. Most patients, even those having successful biliary-enteric anastomoses, eventually develop chronic cholangitis, extensive hepatic fibrosis, and portal hypertension.

1	Choledochal Cysts Cystic dilatation may involve the free portion of the 2083 CBD, i.e., choledochal cyst, or may present as diverticulum formation in the intraduodenal segment. In the latter situation, chronic reflux of pancreatic juice into the biliary tree can produce inflammation and stenosis of the extrahepatic bile ducts leading to cholangitis or biliary obstruction. Because the process may be gradual, ∼50% of patients present with onset of symptoms after age 10. The diagnosis may be made by ultrasound, abdominal CT, MRC, or cholangiography. Only one-third of patients show the classic triad of abdominal pain, jaundice, and an abdominal mass. Ultrasonographic detection of a cyst separate from the gallbladder should suggest the diagnosis of choledochal cyst, which can be confirmed by demonstrating the entrance of extrahepatic bile ducts into the cyst. Surgical treatment involves excision of the “cyst” and biliary-enteric anastomosis. Patients with choledochal cysts are at increased

1	the entrance of extrahepatic bile ducts into the cyst. Surgical treatment involves excision of the “cyst” and biliary-enteric anastomosis. Patients with choledochal cysts are at increased risk for the subsequent development of cholangiocarcinoma.

1	Congenital Biliary Ectasia Dilatation of intrahepatic bile ducts may involve either the major intrahepatic radicles (Caroli’s disease), the interand intralobular ducts (congenital hepatic fibrosis), or both. In Caroli’s disease, clinical manifestations include recurrent cholangitis, abscess formation in and around the affected ducts, and, often, brown pigment gallstone formation within portions of ectatic intrahepatic biliary radicles. Ultrasound, MRC, and CT are of great diagnostic value in demonstrating cystic dilatation of the intrahepatic bile ducts. Treatment with ongoing antibiotic therapy is usually undertaken in an effort to limit the frequency and severity of recurrent bouts of cholangitis. Progression to secondary biliary cirrhosis with portal hypertension, extrahepatic biliary obstruction, cholangiocarcinoma, or recurrent episodes of sepsis with hepatic abscess formation is common.

1	CHOLEDOCHOLITHIASIS Pathophysiology and Clinical Manifestations Passage of gallstones into the CBD occurs in ∼10–15% of patients with cholelithiasis. The incidence of common duct stones increases with increasing age of the patient, so that up to 25% of elderly patients may have calculi in the common duct at the time of cholecystectomy. Undetected duct stones are left behind in ∼1–5% of cholecystectomy patients. The overwhelming majority of bile duct stones are cholesterol stones formed in the gallbladder, which then migrate into the extrahepatic biliary tree through the cystic duct. Primary calculi arising de novo in the ducts are usually brown pigment stones developing in patients with (1) hepatobiliary parasitism or chronic, recurrent cholangitis; (2) congenital anomalies of the bile ducts (especially Caroli’s disease); (3) dilated, sclerosed, or strictured ducts; or (4) an MDR3 (ABCB4) gene defect leading to impaired biliary phospholipids secretion (low phospholipid–associated

1	ducts (especially Caroli’s disease); (3) dilated, sclerosed, or strictured ducts; or (4) an MDR3 (ABCB4) gene defect leading to impaired biliary phospholipids secretion (low phospholipid–associated cholesterol cholelithiasis). Common duct stones may remain asymptomatic for years, may pass spontaneously into the duodenum, or (most often) may present with biliary colic or a complication.

1	Complications • cHolanGitis Cholangitis may be acute or chronic, and symptoms result from inflammation, which usually is caused by at least partial obstruction to the flow of bile. Bacteria are present on bile culture in ∼75% of patients with acute cholangitis early in the symptomatic course. The characteristic presentation of acute cholangitis involves biliary pain, jaundice, and spiking fevers with chills (Charcot’s triad). Blood cultures are frequently positive, and leukocytosis is typical. Nonsuppurative acute cholangitis is most common and may respond relatively rapidly to supportive measures and to treatment with antibiotics. In suppurative acute cholangitis, however, the presence of pus under pressure in a completely obstructed ductal system leads to symptoms of severe toxicity—mental confusion, bacteremia, and septic shock. Response to antibiotics alone in this setting is relatively poor, multiple hepatic abscesses are often present, and the mortality rate approaches 100%

1	confusion, bacteremia, and septic shock. Response to antibiotics alone in this setting is relatively poor, multiple hepatic abscesses are often present, and the mortality rate approaches 100% unless prompt endoscopic or surgical relief of the obstruction and drainage of infected bile are carried out. Endoscopic management of bacterial cholangitis is as effective as surgical intervention. ERCP with endoscopic sphincterotomy is safe and the preferred initial procedure for both establishing a definitive diagnosis and providing effective therapy.

1	Diseases of the Gallbladder and Bile Ducts 2084 obstructive JaunDice Gradual obstruction of the CBD over a period of weeks or months usually leads to initial manifestations of jaundice or pruritus without associated symptoms of biliary colic or cholangitis. Painless jaundice may occur in patients with choledocholithiasis, but is much more characteristic of biliary obstruction secondary to malignancy of the head of the pancreas, bile ducts, or ampulla of Vater. In patients whose obstruction is secondary to choledocholithiasis, associated chronic calculous cholecystitis is very common, and the gallbladder in this setting may be unable to distend. The absence of a palpable gallbladder in most patients with biliary obstruction from duct stones is the basis for Courvoisier’s law, i.e., that the presence of a palpably enlarged gallbladder suggests that the biliary obstruction is secondary to an underlying malignancy rather than to calculous disease. Biliary obstruction causes progressive

1	the presence of a palpably enlarged gallbladder suggests that the biliary obstruction is secondary to an underlying malignancy rather than to calculous disease. Biliary obstruction causes progressive dilatation of the intra-hepatic bile ducts as intrabiliary pressures rise. Hepatic bile flow is suppressed, and reabsorption and regurgitation of conjugated bilirubin into the bloodstream lead to jaundice accompanied by dark urine (bilirubinuria) and light-colored (acholic) stools. CBD stones should be suspected in any patient with cholecystitis whose serum bilirubin level is >85.5 μmol/L (5 mg/dL). The maximum bilirubin level is seldom >256.5 μmol/L (15.0 mg/dL) in patients with choledocholithiasis unless concomitant hepatic or renal disease or another factor leading to marked hyperbilirubinemia exists. Serum bilirubin levels ≥342.0 μmol/L (20 mg/dL) should suggest the possibility of neoplastic obstruction. The serum alkaline phosphatase level is almost always elevated in biliary

1	exists. Serum bilirubin levels ≥342.0 μmol/L (20 mg/dL) should suggest the possibility of neoplastic obstruction. The serum alkaline phosphatase level is almost always elevated in biliary obstruction. A rise in alkaline phosphatase often precedes clinical jaundice and may be the only abnormality in routine liver function tests. There may be a twoto tenfold elevation of serum aminotransferases, especially in association with acute obstruction. Following relief of the obstructing process, serum aminotransferase elevations usually return rapidly to normal, while the serum bilirubin level may take 1–2 weeks to return to normal. The alkaline phosphatase level usually falls slowly, lagging behind the decrease in serum bilirubin.

1	pancreatitis The most common associated entity discovered in patients with nonalcoholic acute pancreatitis is biliary tract disease. Biochemical evidence of pancreatic inflammation complicates acute cholecystitis in 15% of cases and choledocholithiasis in >30%, and the common factor appears to be the passage of gallstones through the common duct. Coexisting pancreatitis should be suspected in patients with symptoms of cholecystitis who develop (1) back pain or pain to the left of the abdominal midline, (2) prolonged vomiting with paralytic ileus, or (3) a pleural effusion, especially on the left side. Surgical treatment of gallstone disease is usually associated with resolution of the pancreatitis.

1	seconDary biliary cirrHosis Secondary biliary cirrhosis may complicate prolonged or intermittent duct obstruction with or without recurrent cholangitis. Although this complication may be seen in patients with choledocholithiasis, it is more common in cases of prolonged obstruction from stricture or neoplasm. Once established, secondary biliary cirrhosis may be progressive even after correction of the obstructing process, and increasingly severe hepatic cirrhosis may lead to portal hypertension or to hepatic failure and death. Prolonged biliary obstruction may also be associated with clinically relevant deficiencies of the fat-soluble vitamins A, D, E, and K.

1	Diagnosis and Treatment The diagnosis of choledocholithiasis is usually made by cholangiography (Table 369-3), either preoperatively by endoscopic retrograde cholangiogram (ERC) (Fig. 369-2C) or MRCP or intraoperatively at the time of cholecystectomy. As many as 15% of patients undergoing cholecystectomy will prove to have CBD stones. When CBD stones are suspected prior to laparoscopic cholecystectomy, preoperative ERCP with endoscopic papillotomy and stone extraction is the preferred approach. It not only provides stone clearance but also defines the anatomy of the biliary tree in relationship to the cystic duct. CBD stones should be suspected in gallstone patients who have any of the following risk factors: (1) a history of jaundice or pancreatitis, (2) abnormal tests of liver function, and (3) ultrasonographic or MRCP evidence of a dilated CBD or stones in the duct. Alternatively, if intraoperative cholangiography reveals retained stones, postoperative ERCP can be carried out. The

1	and (3) ultrasonographic or MRCP evidence of a dilated CBD or stones in the duct. Alternatively, if intraoperative cholangiography reveals retained stones, postoperative ERCP can be carried out. The need for preoperative ERCP is expected to decrease further as laparoscopic techniques for bile duct exploration improve.

1	The widespread use of laparoscopic cholecystectomy and ERCP has decreased the incidence of complicated biliary tract disease and the need for choledocholithotomy and T-tube drainage of the bile ducts. EBS followed by spontaneous passage or stone extraction is the treatment of choice in the management of patients with common duct stones, especially in elderly or poor-risk patients. TRAUMA, STRICTURES, AND HEMOBILIA

1	Most benign strictures of the extrahepatic bile ducts result from surgical trauma and occur in about 1 in 500 cholecystectomies. Strictures may present with bile leak or abscess formation in the immediate postoperative period or with biliary obstruction or cholangitis as long as 2 years or more following the inciting trauma. The diagnosis is established by percutaneous or endoscopic cholangiography. Endoscopic brushing of biliary strictures may be helpful in establishing the nature of the lesion and is more accurate than bile cytology alone. When positive exfoliative cytology is obtained, the diagnosis of a neoplastic stricture is established. This procedure is especially important in patients with primary sclerosing cholangitis (PSC) who are predisposed to the development of cholangiocarcinomas. Successful operative correction of non-PSC bile duct strictures by a skillful surgeon with duct-to-bowel anastomosis is usually possible, although mortality rates from surgical complications,

1	Successful operative correction of non-PSC bile duct strictures by a skillful surgeon with duct-to-bowel anastomosis is usually possible, although mortality rates from surgical complications, recurrent cholangitis, or secondary biliary cirrhosis are high.

1	Hemobilia may follow traumatic or operative injury to the liver or bile ducts, intraductal rupture of a hepatic abscess or aneurysm of the hepatic artery, biliary or hepatic tumor hemorrhage, or mechanical complications of choledocholithiasis or hepatobiliary parasitism. Diagnostic procedures such as liver biopsy, PTC, and transhepatic biliary drainage catheter placement may also be complicated by hemobilia. Patients often present with a classic triad of biliary pain, obstructive jaundice, and melena or occult blood in the stools. The diagnosis is sometimes made by cholangiographic evidence of blood clot in the biliary tree, but selective angiographic verification may be required. Although minor episodes of hemobilia may resolve without operative intervention, surgical ligation of the bleeding vessel is frequently required.

1	Partial or complete biliary obstruction may be produced by extrinsic compression of the ducts. The most common cause of this form of obstructive jaundice is carcinoma of the head of the pancreas. Biliary obstruction may also occur as a complication of either acute or chronic pancreatitis or involvement of lymph nodes in the porta hepatis by lymphoma or metastatic carcinoma. The latter should be distinguished from cholestasis resulting from massive replacement of the liver by tumor.

1	Infestation of the biliary tract by adult helminths or their ova may produce a chronic, recurrent pyogenic cholangitis with or without multiple hepatic abscesses, ductal stones, or biliary obstruction. This condition is relatively rare but does occur in inhabitants of southern China and elsewhere in Southeast Asia. The organisms most commonly involved are trematodes or flukes, including Clonorchis sinensis, Opisthorchis viverrini or Opisthorchis felineus, and Fasciola hepatica. The biliary tract also may be involved by intraductal migration of adult Ascaris lumbricoides from the duodenum or by intrabiliary rupture of hydatid cysts of the liver produced by Echinococcus spp. The diagnosis is made by cholangiography and the presence of characteristic ova on stool examination. When obstruction is present, the treatment of choice is laparotomy under antibiotic coverage, with common duct exploration and a biliary drainage procedure.

1	Primary or idiopathic sclerosing cholangitis is characterized by a progressive, inflammatory, sclerosing, and obliterative process Diseases of the Gallbladder and Bile Ducts Most sensitive method to detect ampullary stones Abbreviations: CBD, common bile duct; ERCP, endoscopic retrograde cholangiopancreatography; GB, gallbladder; HBUS, hepatobiliary ultrasound. affecting the extrahepatic and/or the intrahepatic bile ducts. The disorder occurs up to 75% in association with inflammatory bowel disease, especially ulcerative colitis. It may also be associated with autoimmune pancreatitis; multifocal fibrosclerosis syndromes such as retroperitoneal, mediastinal, and/or periureteral fibrosis; Riedel’s struma; or pseudotumor of the orbit.

1	Immunoglobulin G4 (IgG4)–associated cholangitis is a recently described biliary disease of unknown etiology that presents with biochemical and cholangiographic features indistinguishable from PSC, is often associated with autoimmune pancreatitis and other fibrosing conditions, and is characterized by elevated serum IgG4 and infiltration of IgG4-positive plasma cells in bile ducts and liver tissue. In contrast to PSC, it is not associated with inflammatory bowel disease and should be suspected if associated with increased serum IgG4 and unexplained pancreatic disease. Glucocorticoids are regarded as the initial treatment of choice. Relapse is common after steroid withdrawal, especially with proximal strictures. Long-term treatment with glucocorticoids and/or azathioprine may be needed after relapse or for inadequate response (Chap. 371).

1	Patients with primary sclerosing cholangitis often present with signs and symptoms of chronic or intermittent biliary obstruction: RUQ abdominal pain, pruritus, jaundice, or acute cholangitis. Late in the course, complete biliary obstruction, secondary biliary cirrhosis, hepatic failure, or portal hypertension with bleeding varices may occur. The diagnosis is usually established by finding multifocal, diffusely distributed strictures with intervening segments of normal or dilated ducts, producing a beaded appearance on cholangiography (Fig. 369-2D). The cholangiographic techniques of choice in suspected 2086 cases are MRCP and ERCP. When a diagnosis of sclerosing cholangitis has been established, a search for associated diseases, especially for chronic inflammatory bowel disease, should be carried out. A recent study describes the natural history and outcome for 305 patients of Swedish descent with primary sclerosing cholangitis; 134 (44%) of the patients were asymptomatic at the time

1	carried out. A recent study describes the natural history and outcome for 305 patients of Swedish descent with primary sclerosing cholangitis; 134 (44%) of the patients were asymptomatic at the time of diagnosis and, not surprisingly, had a significantly higher survival rate. The independent predictors of a bad prognosis were advanced age, serum bilirubin concentration, and liver histologic changes. Cholangiocarcinoma was found in 24 patients (8%). Inflammatory bowel disease was closely associated with primary sclerosing cholangitis and had a prevalence of 81% in this study population. Small duct PSC is defined by the presence of chronic cholestasis and hepatic histology consistent with PSC but with normal findings on cholangiography. Small duct PSC is found in ∼5% of patients with PSC and may represent an earlier stage of PSC associated with a significantly better long-term prognosis. However, such patients may progress to classic PSC and/or end-stage liver disease with consequent

1	and may represent an earlier stage of PSC associated with a significantly better long-term prognosis. However, such patients may progress to classic PSC and/or end-stage liver disease with consequent necessity of liver transplantation. In patients with AIDS, cholangiopancreatography may demonstrate a broad range of biliary tract changes as well as pancreatic duct obstruction and occasionally pancreatitis (Chap. 226). Further, biliary tract lesions in AIDS include infection and cholangiopancreatographic changes similar to those of PSC. Changes noted include: (1) diffuse involvement of intrahepatic bile ducts alone, (2) involvement of both intraand extrahepatic bile ducts, (3) ampullary stenosis, (4) stricture of the intrapancreatic portion of the CBD, and (5) pancreatic duct involvement. Associated infectious organisms include Cryptosporidium, Mycobacterium avium-intracellulare, cytomegalovirus, Microsporidia, and Isospora. In addition, acalculous cholecystitis occurs in up to 10% of

1	Associated infectious organisms include Cryptosporidium, Mycobacterium avium-intracellulare, cytomegalovirus, Microsporidia, and Isospora. In addition, acalculous cholecystitis occurs in up to 10% of patients. ERCP sphincterotomy, while not without risk, provides significant pain reduction in patients with AIDS-associated papillary stenosis. Secondary sclerosing cholangitis may occur as a long-term complication of choledocholithiasis, cholangiocarcinoma, operative or traumatic biliary injury, or contiguous inflammatory processes.

1	Therapy with cholestyramine may help control symptoms of pruritus, and antibiotics are useful when cholangitis complicates the clinical picture. Vitamin D and calcium supplementation may help prevent the loss of bone mass frequently seen in patients with chronic cholestasis. Glucocorticoids, methotrexate, and cyclosporine have not been shown to be efficacious in PSC. UDCA in high dosage (20 mg/kg) improves serum liver tests, but an effect on survival has not been documented. In cases where high-grade biliary obstruction (dominant strictures) has occurred, balloon dilatation or stenting may be appropriate. Only rarely is surgical intervention indicated. Efforts at biliary-enteric anastomosis or stent placement may, however, be complicated by recurrent cholangitis and further progression of the stenosing process. The prognosis is unfavorable, with a median survival of 9–12 years following the diagnosis, regardless of therapy. Four variables (age, serum bilirubin level, histologic stage,

1	the stenosing process. The prognosis is unfavorable, with a median survival of 9–12 years following the diagnosis, regardless of therapy. Four variables (age, serum bilirubin level, histologic stage, and splenomegaly) predict survival in patients with PSC and serve as the basis for a risk score. PSC is one of the most common indications for liver transplantation.

1	Approach to the Patient with Pancreatic Disease Darwin L. Conwell, Norton J. Greenberger, Peter A. Banks SEC Tion 3 DiSoRDERS of THE PAnCREAS As emphasized in Chap. 371, the etiologies as well as clinical manifestations of pancreatitis are quite varied. Although it is well-appreciated that pancreatitis is frequently secondary to biliary tract disease and alcohol abuse, it can also be caused by drugs, genetic mutations, trauma, and viral infections and is associated with metabolic and connective tissue disorders. In ~30% of patients with acute pancreatitis and 25–40% of patients with chronic pancreatitis, the etiology initially can be obscure.

1	The incidence of acute pancreatitis is about 5–35/100,000 new cases per year worldwide, with a mortality rate of about 3%. The incidence of chronic pancreatitis is about 4–8 new cases per 100,000 per year with a prevalence of 26–42 cases per 100,000. The number of patients admitted to the hospital who suffer with both acute and chronic pancreatitis in the United States is largely increasing and is now estimated to be 274,119 for acute pancreatitis and 19,724 for chronic pancreatitis. Acute pancreatitis is now the most common gastrointestinal diagnosis requiring hospitalization in the United States. Acute and chronic pancreatic disease costs an estimated 3 billion dollars annually in health care expenditures. These numbers may underestimate the true incidence and prevalence, because non–alcohol-induced pancreatitis has been largely ignored. At autopsy, the prevalence of chronic pancreatitis ranges from 0.04 to 5%.

1	The diagnosis of acute pancreatitis is generally clearly defined based on a combination of laboratory, imaging, and clinical symptoms. The diagnosis of chronic pancreatitis, especially in mild disease, is hampered by the relative inaccessibility of the pancreas to direct examination and the nonspecificity of the abdominal pain associated with chronic pancreatitis. Many patients with chronic pancreatitis do not have elevated blood amylase or lipase levels. Some patients with chronic pancreatitis develop signs and symptoms of pancreatic exocrine insufficiency, and thus, objective evidence for pancreatic disease can be demonstrated. However, there is a very large reservoir of pancreatic exocrine function. More than 90% of the pancreas must be damaged before maldigestion of fat and protein is manifested. Noninvasive, indirect tests of pancreatic exocrine function (fecal elastase) are much more likely to give abnormal results in patients with obvious advanced pancreatic disease (i.e.,

1	is manifested. Noninvasive, indirect tests of pancreatic exocrine function (fecal elastase) are much more likely to give abnormal results in patients with obvious advanced pancreatic disease (i.e., pancreatic calcification, steatorrhea, or diabetes mellitus) than in patients with occult disease. Invasive, direct tests of pancreatic secretory function (secretin tests) are the most sensitive and specific tests to detect early chronic pancreatic disease when imaging is equivocal or normal.

1	Several tests have proved of value in the evaluation of pancreatic disease. Examples of specific tests and their usefulness in the diagnosis of acute and chronic pancreatitis are summarized in Table 370-1 and Fig. 370-1. At some institutions, pancreatic function tests are available and performed if the diagnosis of chronic pancreatic disease remains a possibility after noninvasive tests (ultrasound, computed tomography [CT], magnetic resonance cholangiopancreatography [MRCP]) or invasive tests (endoscopic retrograde cholangiopancreatography [ERCP], endoscopic ultrasonography [EUS]) have given normal or Serum lipase Pancreatic inflammation leads to increased serum enzyme Enzyme measurement of choice for diagnosis of acute pancreatitis levels 1. Serum Pancreatic inflammation leads to increased serum enzyme Simple; reliable if test results are three times the upper limit of levels normal 2. Urine Renal clearance of amylase is increased in acute pancreatitis Infrequently used 3.

1	Urine Renal clearance of amylase is increased in acute pancreatitis Infrequently used 3. Ascitic fluid Disruption of gland or main pancreatic duct leads to Can help establish source of ascites; false positives occur with 4. Pleural fluid Exudative pleural effusion with pancreatitis False positives occur with carcinoma of the lung and esophageal perforation Studies Pertaining to Pancreatic Structure 1. Plain film of the abdomen 2. 3. 4. 5. 6. 7. Pancreatic biopsy with US or CT guidance Can be abnormal in acute and chronic pancreatitis Can provide information on edema, inflammation, calcification, pseudocysts, and mass lesions Permits detailed visualization of pancreas and surrounding structures, pancreatic fluid collection, pseudocyst; assessment of necrosis or interstitial disease Three-dimensional imaging has been used to produce very good images of the pancreatic-biliary ductal system by a noninvasive technique

1	Three-dimensional imaging has been used to produce very good images of the pancreatic-biliary ductal system by a noninvasive technique High-frequency transducer used with EUS can produce very high-resolution images and depict changes in the pancreatic duct and parenchyma with great detail Cannulation of pancreatic and common bile duct permits visualization of pancreatic-biliary ductal system Percutaneous aspiration biopsy of mass-forming lesions of the pancreas Approach to the Patient with Pancreatic Disease Tests of Exocrine Pancreatic Function Simple, noninvasive; sequential studies quite feasible; useful in diagnosis of gallstones; pancreas visualization limited by interference from overlying bowel gas Useful in the diagnosis of pancreatic calcification, dilated pancreatic ducts, and pancreatic tumors; may not be able to distinguish between inflammatory and neoplastic mass lesions Has replaced ERCP as a diagnostic test; noninvasive

1	Has replaced ERCP as a diagnostic test; noninvasive Can be used to assess gallstones, chronic pancreatitis, and pancreatic carcinoma High diagnostic yield; laparotomy avoided; can be done with EUS for the evaluation of chronic pancreatitis, autoimmune pancreatitis, and pancreatic carcinoma Direct stimulation of the pancreas with analysis of duodenal contents 1. Secretin test Secretin leads to increased output of pancreatic juice and HCO3–; pancreatic secretory response is related to the functional mass of pancreatic tissue 2. Measurement of intraluminal digestion products 1. Quantitative stool fat Lack of lipolytic enzymes brings about impaired fat determination digestion Measurement of pancreatic enzymes in feces 1. Elastase Pancreatic secretion of proteolytic enzymes; not degraded inconclusive results. In this regard, tests using direct stimulation of the pancreas with secretin are the most sensitive.

1	Pancreatic Enzymes in Body Fluids The serum amylase and lipase levels are widely used as screening tests for acute pancreatitis in the patient with acute abdominal pain or back pain. Values greater than three times the upper limit of normal in combination with epigastric pain strongly suggest the diagnosis if gut perforation or infarction is excluded. In acute pancreatitis, the serum amylase and lipase are usually elevated within 24 h of onset and remain so for 3–7 days. Levels usually return to normal within 7 days unless there is pancreatic ductal disruption, ductal obstruction, or pseudocyst formation. Approximately 85% of patients with acute pancreatitis have a threefold or greater elevated serum lipase and amylase levels. The values may be

1	Sensitive enough to detect occult disease; involves duodenal intubation and fluoroscopic placement of gastroduodenal tube; poorly defined normal enzyme response; overlap in chronic pancreatitis; large secretory reserve capacity of the pancreas; currently done at only a few medical centers Sensitive enough to detect occult disease; high negative predictive value; avoids intubation and fluoroscopy; requires sedation Reliable reference standard for defining severity of malabsorption; does not distinguish between maldigestion and malabsorption

1	Reliable reference standard for defining severity of malabsorption; does not distinguish between maldigestion and malabsorption Diagnostic accuracy best if value is <100 mg/g performed on a solid stool normal if (1) there is a delay (of 2–5 days) before blood samples are obtained, (2) the underlying disorder is chronic pancreatitis rather than acute pancreatitis, or (3) hypertriglyceridemia is present. Patients with hypertriglyceridemia and proven pancreatitis have been found to have spuriously low levels of amylase and perhaps lipase activity. In the absence of objective evidence of pancreatitis by abdominal ultrasound, CT scan, MRCP, or EUS, mild to moderate elevations of amylase and/ or lipase are not helpful in making a diagnosis of chronic pancreatitis.

1	The serum amylase can be elevated in other conditions (Table 370-2), in part because the enzyme is found in many organs. In addition to the pancreas and salivary glands, small quantities of amylase are found in the tissues of the fallopian tubes, lung, thyroid, and tonsils and can be produced by various tumors (carcinomas of the lung, esophagus, breast, and ovary). Isoamylase determinations do not accurately Clinical signs and symptoms suggestive of chronic pancreatic disease: abdominal pain, weight loss, steatorrhea, malabsorption, history of alcohol abuse, recurrent pancreatitis, fatty-food intolerance Perform history, physical exam, review of laboratory studies, consider fecal elastase measurement Initial imaging modality CP Diagnostic criteria: calcifications in combination with atrophy and/or dilated duct Inconclusive or nondiagnostic results; continue to Step 2 MRI/MRCP with secretin enhancement (sMRCP)

1	CP Diagnostic criteria: calcifications in combination with atrophy and/or dilated duct Inconclusive or nondiagnostic results; continue to Step 2 MRI/MRCP with secretin enhancement (sMRCP) CP Diagnostic criteria: Cambridge Class III, dilated duct, atrophy of gland, fillings defects in duct suggestive of stones Inconclusive or nondiagnostic results; continue to Step 3 Endoscopic Ultrasound (EUS) with quantification of parenchymal and ductal criteria CP Diagnostic criteria: ˜5 EUS CP criteria Inconclusive or nondiagnostic results; continue to Step 4 CP Diagnostic criteria: peak [bicarbonate] <80 meq/L Step 4 • Diagnostic criteria met; no further imaging needed Inconclusive or nondiagnostic results; continue to Step 5 Note: Consider combining ePFT with EUS CP Diagnostic criteria: Cambridge III, Dilated main pancreatic duct and greater than 3 dilated side branch Inconclusive or nondiagnostic results require monitoring of signs and symptoms and repeat testing in 6 months–1 year

1	Inconclusive or nondiagnostic results require monitoring of signs and symptoms and repeat testing in 6 months–1 year FIGURE 370-1 A stepwise diagnostic approach to the patient with suspected chronic pancreatitis (CP). Endoscopic ultrasonography (EUS) and magnetic resonance cholangiopancreatography (sMRCP/MRCP) are appropriate diagnostic alternatives to endoscopic retrograde cholangiopancreatography (ERCP). CT, computed tomography. distinguish elevated blood amylase levels due to bona fide pancreatitis from elevated blood amylase levels due to a nonpancreatic source of amylase, especially when the blood amylase level is only moderately elevated. In patients with unexplained hyperamylasemia, measurement of macroamylase can avoid numerous tests in patients with this rare disorder.

1	Elevation of ascitic fluid amylase occurs in acute pancreatitis as well as in (1) ascites due to disruption of the main pancreatic duct or a leaking pseudocyst and (2) other abdominal disorders that simulate pancreatitis (e.g., intestinal obstruction, intestinal infarction, or perforated peptic ulcer). Elevation of pleural fluid amylase can occur in acute pancreatitis, chronic pancreatitis, carcinoma of the lung, and esophageal perforation. Lipase is the single best enzyme to measure for the diagnosis of acute pancreatitis. No single blood test is reliable for the diagnosis of acute pancreatitis in patients with renal failure. Pancreatic enzyme elevations are usually less than three times the upper limit of normal. Determining whether a patient with renal failure and abdominal pain has pancreatitis remains a difficult clinical problem. One study found that serum amylase levels were elevated in patients with renal dysfunction only when creatinine clearance was <0.8 mL/s (<50 mL/min).

1	remains a difficult clinical problem. One study found that serum amylase levels were elevated in patients with renal dysfunction only when creatinine clearance was <0.8 mL/s (<50 mL/min). In such patients, the serum amylase level was invariably <500 IU/L in the absence of objective evidence of acute pancreatitis. In that study, serum lipase and trypsin levels paralleled serum amylase values. With these limitations in mind, the recommended screening test for acute pancreatitis in renal disease is serum lipase.

1	Studies Pertaining to Pancreatic Structure • raDioloGic tests Plain films of the abdomen, which once provided useful information in patients with acute and chronic pancreatitis, have been superseded by other more detailed imaging procedures (ultrasound, EUS, CT, MRCP).

1	Ultrasonography (US) can provide important information in patients with acute pancreatitis, chronic pancreatitis, pseudocysts, and pancreatic carcinoma. Echographic appearances can indicate the presence of edema, inflammation, and calcification (not obvious on plain films of the abdomen), as well as pseudocysts, mass lesions, and gallstones. In acute pancreatitis, the pancreas is characteristically enlarged. In pancreatic pseudocyst, the usual appearance is primarily that of smooth, round fluid collection. Pancreatic carcinoma distorts the usual landmarks, and mass lesions >3.0 cm are usually detected as localized, solid lesions. US is often the initial investigation for most patients with suspected pancreatic disease. However, obesity and excess smalland large-bowel gas can interfere with pancreatic imaging by US studies. Computed tomography (CT) is the best imaging study for initial evaluation of a suspected pancreatic disorder and for the assessment of I. Pancreatitis A. Acute

1	Computed tomography (CT) is the best imaging study for initial evaluation of a suspected pancreatic disorder and for the assessment of I. Pancreatitis A. Acute B. Chronic: ductal obstruction C. Complications of pancreatitis 1. 2. 3. II. Pancreatic trauma III. Pancreatic carcinoma I. Renal insufficiency II. A. B. C. D. III. A. Carcinoma of the lung, esophagus, breast, or ovary IV. V. VI. Diabetic ketoacidosis VII. Pregnancy VIII. Renal transplantation IX. X. Drugs: opiates I. Biliary tract disease: cholecystitis, choledocholithiasis II. A. B. C. D. E. F.

1	complications of acute and chronic pancreatitis. It is especially useful in the detection of pancreatic and peripancreatic acute fluid collections, fluid-containing lesions such as pseudocysts, walled-off necrosis, calcium deposits (see Chap. 371, Figs. 371-1, 371-2, and 371-4), and pancreatic neoplasms. Acute pancreatitis is characterized by (1) enlargement of the pancreatic outline, (2) distortion of the pancreatic contour, and/or (3) a pancreatic fluid that has a different attenuation coefficient than normal pancreas. Oral, water-soluble contrast agents are used to opacify the stomach and duodenum during CT scans; this strategy permits more precise delineation of various organs as well as mass lesions. Dynamic CT (using rapid IV administration of contrast) is useful in estimating the extent of pancreatic necrosis and in predicting morbidity and mortality. CT provides clear images much more rapidly and essentially negates artifact caused by patient movement. If acute pancreatitis is

1	of pancreatic necrosis and in predicting morbidity and mortality. CT provides clear images much more rapidly and essentially negates artifact caused by patient movement. If acute pancreatitis is confirmed with serology and physical examination findings, CT scan in the first 3 days is not recommended to avoid overuse and minimize costs.

1	Endoscopic ultrasonography (EUS) produces high-resolution images of the pancreatic parenchyma and pancreatic duct with a transducer fixed to an endoscope that can be directed onto the surface of the pancreas through the stomach or duodenum. EUS and MRCP have largely replaced ERCP for diagnostic purposes in many centers. EUS allows one to obtain information about the pancreatic duct as well as the parenchyma and has few procedure-related complications associated with it, in contrast to the 5–10% of post-ERCP pancreatitis observed. EUS is also helpful in detecting common bile duct stones in acute pancreatitis. Pancreatic masses can also be biopsied via EUS in cases with suspected pancreas cancer, and one can deliver nerve-blocking agents through EUS fine-needle injection in patients suffering from pancreatic pain from chronic pancreatitis or cancer. EUS has been studied as a diagnostic modality for chronic pancreatitis. Criteria for abnormalities on EUS in severe chronic pancreatic

1	from pancreatic pain from chronic pancreatitis or cancer. EUS has been studied as a diagnostic modality for chronic pancreatitis. Criteria for abnormalities on EUS in severe chronic pancreatic disease have been developed. There is general agreement that the presence of five or more of the nine criteria listed in Table 370-3 is highly predictive of chronic pancreatitis. Recent studies comparing EUS and ERCP to the secretin test in patients with unexplained abdominal pain suspected of having chronic pancreatitis show similar diagnostic accuracy in detecting early changes of chronic pancreatitis. The exact role of EUS versus CT, ERCP, or function testing in the early diagnosis of chronic pancreatitis has yet to be clearly defined.

1	Magnetic resonance imaging (MRI) and magnetic resonance cholangiopancreatography (MRCP) are now being used to view the bile ducts, pancreatic duct, and the pancreas parenchyma in both acute pancreatitis and chronic pancreatitis. For diagnostic imaging in chronic pancreatitis, non-breath-holding and three-dimensional turbo spin-echo techniques are being used to produce superb MRCP images. The main pancreatic duct and common bile duct can be seen well, but there is still a question as to whether changes can be detected consistently in the secondary ducts. The secondary ducts are not visualized in a normal pancreas. Secretin-enhanced MRCP is currently under investigation but is emerging as a method to better evaluate ductal changes. In anteroposterior imaging, T2 imaging of fluid collections can differentiate necrotic debris from fluid in suspected walled-off necrosis, and T1 imaging can diagnose hemorrhage in suspected pseudoaneurysm rupture.

1	Both EUS and MRCP have largely replaced ERCP in the diagnostic evaluation of pancreatic disease. As these techniques become more refined, especially with the administration of secretin, they may well be the diagnostic tests of choice to evaluate the pancreatic duct. ERCP is still needed for treatment of bile duct and pancreatic duct lesions. ERCP is primarily of therapeutic value after CT, EUS, or MRCP has detected abnormalities requiring invasive endoscopic treatment. ERCP can also be helpful at clarification of equivocal findings discovered with other imaging techniques (see Chap. 371, Fig. 371-1). Pancreatic carcinoma is characterized by stenosis or obstruction of either the pancreatic duct or the common bile duct; both ductal systems are often abnormal (double-duct sign). In chronic pancreatitis, ERCP abnormalities in the main pancreatic duct and side branches have been outlined by the Cambridge classification. The presence of ductal stenosis and irregularity can make it difficult

1	ERCP abnormalities in the main pancreatic duct and side branches have been outlined by the Cambridge classification. The presence of ductal stenosis and irregularity can make it difficult to distinguish chronic pancreatitis from carcinoma. It is important to be aware that ERCP changes interpreted as indicating chronic pancreatitis actually may be due to the effects of aging on the pancreatic duct or sequelae of a recent attack of acute pancreatitis. Although aging may cause impressive ductal alterations, it does not affect the results of pancreatic function tests (i.e., the secretin test). Elevated serum amylase levels after ERCP have been reported in the majority of patients, and clinical pancreatitis in 5–10% of patients. Recent data suggest that pancreatic duct stenting and rectal indomethacin can decrease the incidence of ERCP-induced pancreatitis. ERCP should rarely be done for diagnostic purposes and should especially be avoided in high-risk patients.

1	pancreatic biopsy witH raDioloGic GuiDance Percutaneous aspiration biopsy or a trucut biopsy of a pancreatic mass often distinguishes a pancreatic inflammatory mass from a pancreatic neoplasm. Approach to the Patient with Pancreatic Disease Acute and Chronic Pancreatitis Darwin L. Conwell, Peter Banks, Norton J. Greenberger BIOCHEMISTRY AND PHYSIOLOGY OF PANCREATIC EXOCRINE SECRETION GENERAL CONSIDERATIONS 371 2090 TESTS OF EXOCRINE PANCREATIC FUNCTION Pancreatic function tests (Table 370-1) can be divided into the following: 1. Direct stimulation of the pancreas by IV infusion of secretin followed by collection and measurement of duodenal contents

1	The secretin test, used to detect diffuse pancreatic disease, is based on the physiologic principle that the pancreatic secretory response is directly related to the functional mass of pancreatic tissue. In the standard assay, secretin is given IV in a dose of 0.2 mg/kg of synthetic human secretin as a bolus. Normal values for the standard secretin test are (1) volume output >2 mL/kg per hour, (2) bicarbonate (HCO3 -) concentration >80 mmol/L, and (3) HCO3 output >10 mmol/L in 1 h. The most reproducible measurement, giving the highest level of discrimination between normal subjects and patients with chronic pancreatic exocrine insufficiency, appears to be the maximal bicarbonate concentration. A cutoff point below 80 mmol/L is considered abnormal and suggestive of abnormal secretory function that is most commonly observed in early chronic pancreatitis.

1	There may be a dissociation between the results of the secretin test and other tests of absorptive function. For example, patients with chronic pancreatitis often have abnormally low outputs of HCO3 -after secretin but have normal fecal fat excretion. Thus the secretin test measures the secretory capacity of ductular epithelium, whereas fecal fat excretion indirectly reflects intraluminal lipolytic activity. Steatorrhea does not occur until intraluminal levels of lipase are markedly reduced, underscoring the fact that only small amounts of enzymes are necessary for intraluminal digestive activities. It must be emphasized that an abnormal secretin test result suggests only that chronic pancreatic damage is present. 2. Measurement of fecal pancreatic enzymes such as elastase

1	2. Measurement of fecal pancreatic enzymes such as elastase Measurement of intraluminal digestion products (i.e., undigested muscle fibers, stool fat, and fecal nitrogen) is discussed in Chap. 349. The amount of human elastase in stool reflects the pancreatic output of this proteolytic enzyme. Decreased elastase-1 activity (FE-1) in stool is an excellent test to detect severe pancreatic exocrine insufficiency (PEI) in patients with chronic pancreatitis and cystic fibrosis. FE-1 levels >200 mg/g are normal; levels of 100–200 mg/g are considered mild, and levels <100 mg/g are severe for PEI. Although the test is simple and noninvasive, it can give false-positive results and has a low sensitivity. Fecal levels <50 mg/g are definitive for PEI provided that the stool specimen is solid. Tests useful in the diagnosis of exocrine pancreatic insufficiency and the differential diagnosis of malabsorption are also discussed in Chaps. 349 and 371.

1	Tests useful in the diagnosis of exocrine pancreatic insufficiency and the differential diagnosis of malabsorption are also discussed in Chaps. 349 and 371. The pancreas secretes 1500–3000 mL of isosmotic alkaline (pH >8) fluid per day containing about 20 enzymes. The pancreatic secretions provide the enzymes and bicarbonate needed to affect the major digestive activity of the gastrointestinal tract and provide an optimal pH for the function of these enzymes.

1	The exocrine pancreas is influenced by intimately interacting hormonal and neural systems. Gastric acid is the stimulus for the release of secretin from the duodenal mucosa (S cells), which stimulates the secretion of water and electrolytes from pancreatic ductal cells. Release of cholecystokinin (CCK) from the duodenal and proximal jejunal mucosa (Ito cells) is largely triggered by long-chain fatty acids, essential amino acids (tryptophan, phenylalanine, valine, methionine), and gastric acid itself. CCK evokes an enzyme-rich secretion from acinar cells in the pancreas. The parasympathetic nervous system (via the vagus nerve) exerts significant control over pancreatic secretion. Secretion evoked by secretin and CCK depends on permissive roles of vagal afferent and efferent pathways. This is particularly true for enzyme secretion, whereas water and bicarbonate secretions are heavily dependent on the hormonal effects of secretin and to a lesser extent CCK. Also, vagal stimulation

1	This is particularly true for enzyme secretion, whereas water and bicarbonate secretions are heavily dependent on the hormonal effects of secretin and to a lesser extent CCK. Also, vagal stimulation affects the release of vasoactive intestinal peptide (VIP), a secretin agonist. Pancreatic exocrine secretion is also influenced by inhibitory neuropeptides such as somatostatin, pancreatic polypeptide, peptide YY, neuropeptide Y, enkephalin, pancreastatin, calcitonin gene–related peptides, glucagon, and galanin. Although pancreatic polypeptide and peptide YY may act primarily on nerves outside the pancreas, somatostatin acts at multiple sites. Nitric oxide (NO) is also an important neurotransmitter.

1	Bicarbonate is the ion of primary physiologic importance within pancreatic secretion. The ductal cells secrete bicarbonate predominantly derived from plasma (93%) more than from intracellular metabolism (7%). Bicarbonate enters the duct lumen through the sodium bicarbonate cotransporter with depolarization caused by chloride efflux through the cystic fibrosis transmembrane conductance regulator (CFTR). Secretin and VIP bind at the basolateral surface and cause an increase in secondary messenger intracellular cyclic AMP, and act on the apical surface of the ductal cells opening the CFTR in promoting secretion. CCK, acting as a neuromodulator, markedly potentiates the stimulatory effects of secretin. Acetylcholine also plays an important role in ductal cell secretion. Intraluminal bicarbonate secreted from the ductal cells helps neutralize gastric acid and creates the appropriate pH for the activity of pancreatic enzymes and bile salts on ingested food.

1	The acinar cell is highly compartmentalized and is concerned with the secretion of pancreatic enzymes. Proteins synthesized by the rough endoplasmic reticulum are processed in the Golgi and then targeted to the appropriate site, whether that be zymogen granules, lysosomes, or other cell compartments. The zymogen granules migrate to the apical region of the acinar cell awaiting the appropriate neural or hormonal stimulatory response. The pancreas secretes amylolytic, lipolytic, and proteolytic enzymes into the duct lumen. Amylolytic enzymes, such as amylase, hydrolyze starch to oligosaccharides and to the disaccharide maltose. The lipolytic enzymes include lipase, phospholipase A2, and cholesterol esterase. Bile salts inhibit lipase in isolation, but colipase, another constituent of pancreatic secretion, binds to lipase and prevents this inhibition. Bile salts activate phospholipase A and cholesterol esterase. Proteolytic enzymes include endopeptidases (trypsin, chymotrypsin), which

1	secretion, binds to lipase and prevents this inhibition. Bile salts activate phospholipase A and cholesterol esterase. Proteolytic enzymes include endopeptidases (trypsin, chymotrypsin), which act on internal peptide bonds of proteins and polypeptides; exopeptidases (carboxypeptidases, aminopeptidases), which act on the free carboxyland amino-terminal ends of peptides, respectively; and elastase. The proteolytic enzymes are secreted as inactive zymogen precursors. Ribonucleases (deoxyribonucleases, ribonuclease) are also secreted. Enterokinase, an enzyme found in the duodenal mucosa, cleaves the lysine-isoleucine bond of trypsinogen to form trypsin. Trypsin then activates the other proteolytic zymogens and phospholipase A2 in a cascade phenomenon. All pancreatic enzymes have pH optima in the alkaline range. The nervous system initiates pancreatic enzyme secretion. The neurologic stimulation is cholinergic, involving extrinsic innervation by the vagus nerve and subsequent innervation

1	the alkaline range. The nervous system initiates pancreatic enzyme secretion. The neurologic stimulation is cholinergic, involving extrinsic innervation by the vagus nerve and subsequent innervation by intrapancreatic cholinergic nerves. The stimulatory neurotransmitters are acetylcholine and gastrin-releasing peptides. These neurotransmitters activate calcium-dependent secondary messenger systems, resulting in the release of zymogens into the pancreas duct. VIP is present in intrapancreatic nerves and potentiates the effect of acetylcholine. In contrast to other species, there are no CCK receptors on acinar cells in humans. CCK in physiologic concentrations stimulates pancreatic secretion by stimulating afferent vagal and intrapancreatic nerves.

1	Autodigestion of the pancreas is prevented by (1) the packaging of pancreatic proteases in precursor (proenzyme) form, (2) intracellular calcium homeostasis (low intracellular calcium in the cytosol of the acinar cell promotes the destruction of spontaneously activated trypsin), (3) acid-base balance, and (4) the synthesis of protective protease inhibitors (pancreatic secretory trypsin inhibitor [PSTI] or SPINK1), which can bind and inactivate about 20% of intracellular trypsin activity. Chymotrypsin C can also lyse and inactivate trypsin. These protease inhibitors are found in the acinar cell, the pancreatic secretions, and the α1and α2-globulin fractions of plasma. Loss of any of these four protective mechanisms leads to premature enzyme activation, autodigestion, and acute pancreatitis.

1	Pancreatic enzyme secretion is controlled, at least in part, by a negative feedback mechanism induced by the presence of active serine proteases in the duodenum. To illustrate, perfusion of the duodenal lumen with phenylalanine (stimulates early digestion) causes a prompt increase in plasma CCK levels as well as increased secretion of chymotrypsin and other pancreatic enzymes. However, simultaneous perfusion with trypsin (stimulates late digestion) blunts both responses. Conversely, perfusion of the duodenal lumen with protease inhibitors actually leads to enzyme hypersecretion. The available evidence supports the concept that the duodenum contains a peptide called CCK-releasing factor (CCK-RF) that is involved in stimulating CCK release. It appears that serine proteases inhibit pancreatic secretion by inactivating a CCK-releasing peptide in the lumen of the small intestine. Thus, the integrative result of both bicarbonate and enzyme secretion depends on a feedback process for both

1	secretion by inactivating a CCK-releasing peptide in the lumen of the small intestine. Thus, the integrative result of both bicarbonate and enzyme secretion depends on a feedback process for both bicarbonate and pancreatic enzymes. Acidification of the duodenum releases secretin, which stimulates vagal and other neural pathways to activate pancreatic duct cells, which secrete bicarbonate. This bicarbonate then neutralizes the duodenal acid, and the feedback loop is completed. Dietary proteins bind proteases, thereby leading to an increase in free CCK-RF. CCK is then released into the blood in physiologic concentrations, acting primarily through the neural pathways (vagal-vagal). This leads to acetylcholinemediated pancreatic enzyme secretion. Proteases continue to be secreted from the pancreas until the protein within the duodenum is digested. At this point, pancreatic protease secretion is reduced to basic levels, thus completing this step in the feedback process.

1	Recent U.S. estimates from the National Inpatient Sample report that acute pancreatitis is the most common inpatient principal gastrointestinal diagnosis. The incidence of acute pancreatitis also varies in different countries and depends on cause (e.g., alcohol, gallstones, metabolic factors, drugs [Table 371-1]). The annual incidence ranges from 13–45/100,000 persons. Acute pancreatitis results in >250,000 hospitalizations per year. The median length of hospital stay is 4 days, with a median hospital cost of $6,096 and a mortality of 1%. The estimated cost annually approaches $2.6 billion. Hospitalization rates increase with age, are 88% higher among blacks, and are higher among males than females. The age-adjusted rate of hospital discharges with an acute pancreatitis diagnosis increased 62% between 1988 and 2004. From 2000 to 2009, the rate increased 30%. Thus, acute pancreatitis is increasing and is a significant burden on health care costs and resource utilization.

1	There are many causes of acute pancreatitis (Table 371-1), but the mechanisms by which these conditions trigger pancreatic inflammation have not been fully elucidated. Gallstones continue to be the leading cause of acute pancreatitis in most series (30–60%). The risk Gallstones (including microlithiasis) Alcohol (acute and chronic alcoholism) Hypertriglyceridemia Endoscopic retrograde cholangiopancreatography (ERCP), especially after Drugs (azathioprine, 6-mercaptopurine, sulfonamides, estrogens, tetracycline, valproic acid, anti-HIV medications, 5-aminosalicylic acid [5-ASA]) Cancer of the pancreas Infections (mumps, coxsackievirus, cytomegalovirus, echovirus, parasites) Autoimmune (e.g., type 1 and type 2) Causes to Consider in Patients with Recurrent Bouts of Acute Pancreatitis Without an Obvious Etiology Occult disease of the biliary tree or pancreatic ducts, especially microlithiasis, biliary sludge Metabolic: Hypertriglyceridemia, hypercalcemia

1	Anatomic: Pancreas divisum of acute pancreatitis in patients with at least one gallstone <5 mm in diameter is fourfold greater than that in patients with larger stones. Alcohol is the second most common cause, responsible for 15–30% of cases in the United States. The incidence of pancreatitis in alcoholics is surprisingly low (5/100,000), indicating that in addition to the amount of alcohol ingested, other factors affect a person’s susceptibility to pancreatic injury such as cigarette smoking. Acute pancreatitis occurs in 5–10% of patients following endoscopic retrograde cholangiopancreatography (ERCP). Use of a prophylactic pancreatic duct stent and rectal nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to reduce pancreatitis after ERCP. Risk factors for post-ERCP pancreatitis include minor papilla sphincterotomy, sphincter of Oddi dysfunction, prior history of post-ERCP pancreatitis, age <60 years, >2 contrast injections into the pancreatic duct, and endoscopic trainee

1	include minor papilla sphincterotomy, sphincter of Oddi dysfunction, prior history of post-ERCP pancreatitis, age <60 years, >2 contrast injections into the pancreatic duct, and endoscopic trainee involvement.

1	Hypertriglyceridemia is the cause of acute pancreatitis in 1.3–3.8% of cases; serum triglyceride levels are usually >11.3 mmol/L (>1000 mg/dL). Most patients with hypertriglyceridemia, when subsequently examined, show evidence of an underlying derangement in lipid metabolism, probably unrelated to pancreatitis. Such patients are prone to recurrent episodes of pancreatitis. Any factor (e.g., drugs or alcohol) that causes an abrupt increase in serum triglycerides can precipitate a bout of acute pancreatitis. Patients with a deficiency of apolipoprotein CII have an increased incidence of pancreatitis; apolipoprotein CII 2092 activates lipoprotein lipase, which is important in clearing chylomicrons from the bloodstream. Patients with diabetes mellitus who have developed ketoacidosis and patients who are on certain medications such as oral contraceptives may also develop high triglyceride levels. Approximately 0.1–2% of cases of acute pancreatitis are drug related. Drugs cause pancreatitis

1	who are on certain medications such as oral contraceptives may also develop high triglyceride levels. Approximately 0.1–2% of cases of acute pancreatitis are drug related. Drugs cause pancreatitis either by a hypersensitivity reaction or by the generation of a toxic metabolite, although in some cases, it is not clear which of these mechanisms is operative (Table 371-1). Pathologically, acute pancreatitis varies from interstitial pancreatitis (pancreas blood supply maintained), which is generally self-limited to necrotizing pancreatitis (pancreas blood supply interrupted), in which the extent of necrosis may correlate with the severity of the attack and its systemic complications. Autodigestion is a currently accepted pathogenic theory; according to this theory, pancreatitis results when proteolytic enzymes (e.g., trypsinogen, chymotrypsinogen, proelastase, and lipolytic enzymes such as phospholipase A2) are activated in the pancreas acinar cell rather than in the intestinal lumen. A

1	proteolytic enzymes (e.g., trypsinogen, chymotrypsinogen, proelastase, and lipolytic enzymes such as phospholipase A2) are activated in the pancreas acinar cell rather than in the intestinal lumen. A number of factors (e.g., endotoxins, exotoxins, viral infections, ischemia, oxidative stress, lysosomal calcium, and direct trauma) are believed to facilitate premature activation of trypsin. Activated proteolytic enzymes, especially trypsin, not only digest pancreatic and peripancreatic tissues but also can activate other enzymes, such as elastase and phospholipase A2. Spontaneous activation of trypsin also can occur.

1	Several recent studies have suggested that pancreatitis is a disease that evolves in three phases. The initial phase is characterized by intrapancreatic digestive enzyme activation and acinar cell injury. Trypsin activation appears to be mediated by lysosomal hydrolases such as cathepsin B that become colocalized with digestive enzymes in intracellular organelles; it is currently believed that acinar cell injury is the consequence of trypsin activation. The second phase of pancreatitis involves the activation, chemoattraction, and sequestration of leukocytes and macrophages in the pancreas, resulting in an enhanced intrapancreatic inflammatory reaction. Neutrophil depletion induced by prior administration of an antineutrophil serum has been shown to reduce the severity of experimentally induced pancreatitis. There is also evidence to support the concept that neutrophils can activate trypsinogen. Thus, intrapancreatic acinar cell activation of trypsinogen could be a two-step process

1	induced pancreatitis. There is also evidence to support the concept that neutrophils can activate trypsinogen. Thus, intrapancreatic acinar cell activation of trypsinogen could be a two-step process (i.e., an early neutrophil-independent and a later neutrophil-dependent phase). The third phase of pancreatitis is due to the effects of activated proteolytic enzymes and cytokines, released by the inflamed pancreas, on distant organs. Activated proteolytic enzymes, especially trypsin, not only digest pancreatic and peripancreatic tissues but also activate other enzymes such as elastase and phospholipase A2. The active enzymes and cytokines then digest cellular membranes and cause proteolysis, edema, interstitial hemorrhage, vascular damage, coagulation necrosis, fat necrosis, and parenchymal cell necrosis. Cellular injury and death result in the liberation of bradykinin peptides, vasoactive substances, and histamine that can produce vasodilation, increased vascular permeability, and edema

1	necrosis. Cellular injury and death result in the liberation of bradykinin peptides, vasoactive substances, and histamine that can produce vasodilation, increased vascular permeability, and edema with profound effects on many organs. The systemic inflammatory response syndrome (SIRS) and acute respiratory distress syndrome (ARDS), as well as multiorgan failure, may occur as a result of this cascade of local and distant effects.

1	A number of genetic factors can increase the susceptibility and/or modify the severity of pancreatic injury in acute pancreatitis, recurrent pancreatitis, and chronic pancreatitis. All of the major genetic susceptibility factors center on the control of trypsin activity within the pancreatic acinar cell, in part because they were identified as candidate genes linked to intrapancreatic trypsin control. Five genetic variants have been identified as being associated with susceptibility to pancreatitis. The genes that have been identified include (1) cationic trypsinogen gene (PRSS1), (2) pancreatic secretory trypsin inhibitor (SPINK1), (3) the cystic fibrosis transmembrane conductance regulator gene (CFTR), (4) the chymotrypsin C gene (CTRC), and (5) the calcium-sensing receptor (CASR). Investigations of other genetic variants are currently under way, and new genes will be added to this list in the future. Multiple medical, ethical, and psychological issues arise when these genes are

1	of other genetic variants are currently under way, and new genes will be added to this list in the future. Multiple medical, ethical, and psychological issues arise when these genes are discovered, and referral to genetic counselors is recommended.

1	APPROACH TO THE PATIENT: Abdominal pain is the major symptom of acute pancreatitis. Pain may vary from a mild discomfort to severe, constant, and incapacitating distress. Characteristically, the pain, which is steady and boring in character, is located in the epigastrium and periumbilical region, and may radiate to the back, chest, flanks, and lower abdomen. Nausea, vomiting, and abdominal distention due to gastric and intestinal hypomotility and chemical peritonitis are also frequent complaints.

1	Physical examination frequently reveals a distressed and anxious patient. Low-grade fever, tachycardia, and hypotension are fairly common. Shock is not unusual and may result from (1) hypovolemia secondary to exudation of blood and plasma proteins into the retroperitoneal space; (2) increased formation and release of kinin peptides, which cause vasodilation and increased vascular permeability; and (3) systemic effects of proteolytic and lipolytic enzymes released into the circulation. Jaundice occurs infrequently; when present, it usually is due to edema of the head of the pancreas with compression of the intrapancreatic portion of the common bile duct or passage of a biliary stone or sludge. Erythematous skin nodules due to subcutaneous fat necrosis may rarely occur. In 10–20% of patients, there are pulmonary findings, including basilar rales, atelectasis, and pleural effusion, the latter most frequently left sided. Abdominal tenderness and muscle rigidity are present to a variable

1	there are pulmonary findings, including basilar rales, atelectasis, and pleural effusion, the latter most frequently left sided. Abdominal tenderness and muscle rigidity are present to a variable degree, but compared with the intense pain, these signs may be less impressive. Bowel sounds are usually diminished or absent. An enlarged pancreas from acute fluid collection, walled off necrosis, or a pseudocyst may be palpable in the upper abdomen later in the course of the disease (i.e., 4–6 weeks). A faint blue discoloration around the umbilicus (Cullen’s sign) may occur as the result of hemoperitoneum, and a blue-red-purple or green-brown discoloration of the flanks (Turner’s sign) reflects tissue catabolism of hemoglobin from severe necrotizing pancreatitis with hemorrhage.

1	Serum amylase and lipase values threefold or more above normal virtually clinch the diagnosis if gut perforation, ischemia, and infarction are excluded. Serum lipase is the preferred test. However, it should be noted that there is no correlation between the severity of pancreatitis and the degree of serum lipase and amylase elevations. After 3–7 days, even with continuing evidence of pancreatitis, total serum amylase values tend to return toward normal. However, pancreatic isoamylase and lipase levels may remain elevated for 7–14 days. It should be recognized that amylase elevations in serum and urine occur in many conditions other than pancreatitis (see Chap. 370, Table 370-2). Importantly, patients with acidemia (arterial pH ≤7.32) may have spurious elevations in serum amylase. This finding explains why patients with diabetic ketoacidosis may have marked elevations in serum amylase without any other evidence of acute pancreatitis. Serum lipase activity increases in parallel with

1	finding explains why patients with diabetic ketoacidosis may have marked elevations in serum amylase without any other evidence of acute pancreatitis. Serum lipase activity increases in parallel with amylase activity and is more specific than amylase. A serum lipase measurement can be instrumental in differentiating a pancreatic or nonpancreatic cause for hyperamylasemia. Leukocytosis (15,000–20,000 leukocytes/μL) occurs frequently. Patients with more severe disease may show hemoconcentration with hematocrit values >44% and/or prerenal azotemia with a blood urea nitrogen (BUN) level >22 mg/dL resulting from loss of plasma into the retroperitoneal space and peritoneal cavity.

1	Hemoconcentration may be the harbinger of more severe disease (i.e., pancreatic necrosis), whereas azotemia is a significant risk factor for mortality. Hyperglycemia is common and is due to multiple factors, including decreased insulin release, increased glucagon release, and Acute inflammation of the pancreatic parenchyma and peripancreatic tissues, but without recognizable tissue necrosis Inflammation associated with pancreatic parenchymal necrosis and/or peripancreatic necrosis Peripancreatic fluid associated with interstitial edematous pancreatitis with no associated peripancreatic necrosis. This term applies only to areas of peripancreatic fluid seen within the first 4 weeks after onset of interstitial edematous pancreatitis and without the features of a pseudocyst.

1	An encapsulated collection of fluid with a well-defined inflammatory wall usually outside the pancreas with minimal or no necrosis. This entity usually occurs >4 weeks after onset of interstitial edematous pancreatitis. A collection containing variable amounts of both fluid and necrosis associated with necrotizing pancreatitis; the necrosis can involve the pancreatic parenchyma and/or the peripancreatic tissues. A mature, encapsulated collection of pancreatic and/or peripancreatic necrosis that has developed a well-defined inflammatory wall. WON usually occurs >4 weeks after onset of necrotizing pancreatitis. No findings of peripancreatic necrosis Lack of pancreatic parenchymal enhancement by IV contrast agent and/or presence of findings of peripancreatic necrosis (see below—ANC and WON) Occurs in the setting of interstitial edematous pancreatitis Homogeneous collection with fluid density No definable wall encapsulating the collection

1	Occurs in the setting of interstitial edematous pancreatitis Homogeneous collection with fluid density No definable wall encapsulating the collection Adjacent to pancreas (no intra-pancreatic extension) Well circumscribed, usually round or oval Well-defined wall; that is, completely encapsulated Maturation usually requires >4 weeks after onset of acute pancreatitis; occurs after interstitial edematous pancreatitis Occurs only in the setting of acute necrotizing pancreatitis Heterogeneous and nonliquid density of varying degrees in different locations (some appear homogeneous early in their course) No definable wall encapsulating the collection Heterogeneous with liquid and nonliquid density with varying degrees of loculations (some may appear homogeneous) Well-defined wall; that is, completely encapsulated Maturation usually requires 4 weeks after onset of acute necrotizing pancreatitis Source: Modified from P Banks et al: Gut 62:102, 2013.

1	an increased output of adrenal glucocorticoids and catecholamines. Hypocalcemia occurs in ~25% of patients, and its pathogenesis is incompletely understood. Although earlier studies suggested that the response of the parathyroid gland to a decrease in serum calcium is impaired, subsequent observations have failed to confirm this phenomenon. Intraperitoneal saponification of calcium by fatty acids in areas of fat necrosis occurs occasionally, with large amounts (up to 6.0 g) dissolved or suspended in ascitic fluid. Such “soap formation” may also be significant in patients with pancreatitis, mild hypocalcemia, and little or no obvious ascites. Hyperbilirubinemia (serum bilirubin >68 2093 mmoL or >4.0 mg/dL) occurs in ~10% of patients. However, jaundice is transient, and serum bilirubin levels return to normal in 4–7 days. Serum alkaline phosphatase and aspartate aminotransferase levels are also transiently elevated, and they parallel serum bilirubin values and may point to

1	levels return to normal in 4–7 days. Serum alkaline phosphatase and aspartate aminotransferase levels are also transiently elevated, and they parallel serum bilirubin values and may point to gallbladder-related disease or inflammation in the pancreatic head. Hypertriglyceridemia occurs in 5–10% of patients, and serum amylase levels in these individuals are often spuriously normal (Chap. 370). Approximately 5–10% of patients have hypoxemia (arterial PO2 ≤60 mmHg), which may herald the onset of ARDS. Finally, the electrocardiogram is occasionally abnormal in acute pancreatitis with ST-segment and T-wave abnormalities simulating myocardial ischemia.

1	An abdominal ultrasound is recommended in the emergency ward as the initial diagnostic imaging modality and is most useful to evaluate for gallstone disease and the pancreatic head. The revised Atlanta criteria have clearly outlined the morphologic features of acute pancreatitis on computed tomography (CT) scan as follows: (1) interstitial pancreatitis, (2) necrotizing pancreatitis, (3) acute pancreatic fluid collection, (4) pancreatic pseudocyst, (5) acute necrotic collection (ANC), and (6) walled-off pancreatic necrosis (WON) (Table 371-2 and Fig. 371-1). Radiologic studies useful in the diagnosis of acute pancreatitis are discussed in Chap. 370 and listed in Table 370-1.

1	Any severe acute pain in the abdomen or back should suggest the possibility of acute pancreatitis. The diagnosis is established by two of the following three criteria: (1) typical abdominal pain in the epigastrium that may radiate to the back, (2) threefold or greater elevation in serum lipase and/or amylase, and (3) confirmatory findings of acute pancreatitis on cross-sectional abdominal imaging. Patients also have associated nausea, emesis, fever, tachycardia, and abnormal findings on abdominal examination. Laboratory studies may reveal leukocytosis, hypocalcemia, and hyperglycemia. Although not required for diagnosis, markers of severity may include hemoconcentration (hematocrit >44%), admission azotemia (BUN >22 mg/dL), SIRS, and signs of organ failure (Table 371-3).

1	The differential diagnosis should include the following disorders: perforated viscus, especially peptic ulcer; (2) acute cholecystitis and biliary colic; (3) acute intestinal obstruction; (4) mesenteric vascular occlusion; (5) renal colic; (6) inferior myocardial infarction; (7) dissecting aortic aneurysm; (8) connective tissue disorders with vasculitis; pneumonia; and (10) diabetic ketoacidosis. It may be difficult to differentiate acute cholecystitis from acute pancreatitis, because an elevated serum amylase may be found in both disorders. Pain of biliary tract origin is more right sided or epigastric than periumbilical or left upper quadrant and can be more severe; ileus is usually absent. Ultrasound is helpful in establishing the diagnosis of cholelithiasis and cholecystitis. Intestinal obstruction due to mechanical factors can be differentiated from pancreatitis by the history of crescendo-decrescendo pain, findings on abdominal examination, and CT of the abdomen showing changes

1	obstruction due to mechanical factors can be differentiated from pancreatitis by the history of crescendo-decrescendo pain, findings on abdominal examination, and CT of the abdomen showing changes characteristic of mechanical obstruction. Acute mesenteric vascular occlusion is usually suspected in elderly debilitated patients with brisk leukocytosis, abdominal distention, and bloody diarrhea, confirmed by CT or magnetic resonance angiography. Vasculitides secondary to systemic lupus erythematosus and polyarteritis nodosa may be confused with pancreatitis, especially because pancreatitis may develop as a complication of these diseases. Diabetic ketoacidosis is often accompanied by abdominal pain and elevated total serum amylase levels, thus closely mimicking acute pancreatitis. However, the serum lipase level is not elevated in diabetic ketoacidosis.

1	CLINICAL COURSE, DEFINITIONS, AND CLASSIFICATIONS The Revised Atlanta Classification (1) defines phases of acute pancreatitis, (2) defines severity of acute pancreatitis, and (3) clarifies imaging definitions as outlined below. Phases of Acute Pancreatitis Two phases of acute pancreatitis have been defined, early (<2 weeks) and late (>2 weeks), which primarily

1	FIGURE 371-1 Acute pancreatitis: computed tomography (CT) evolution. A. Contrast-enhanced CT scan of the abdomen performed on admission for a patient with clinical and biochemical parameters suggestive of acute pancreatitis. Note the abnormal enhancement of the pancreatic parenchyma (arrow) suggestive of interstitial pancreatitis. B. Contrast-enhanced CT scan of the abdomen performed on the same patient 6 days later for persistent fever and systemic inflammatory response syndrome. The pancreas now demonstrates significant areas of nonenhancement consistent with development of necrosis, particularly in the body and neck region (arrow). Note that an early CT scan obtained within the first 48 h of hospitalization may underestimate or miss necrosis. C. Contrast-enhanced CT scan of the abdomen performed on the same patient 2 months after the initial episode of acute pancreatitis. CT now demonstrates evidence of a fluid collection consistent with walled-off pancreatic necrosis (arrow).

1	performed on the same patient 2 months after the initial episode of acute pancreatitis. CT now demonstrates evidence of a fluid collection consistent with walled-off pancreatic necrosis (arrow). (Courtesy of Dr. KJ Mortele, Brigham and Women’s Hospital; with permission.) describes the hospital course of the disease. In the early phase of acute pancreatitis, which lasts 1–2 weeks, severity is defined by clinical parameters rather than morphologic findings. Most patients exhibit SIRS, and if this persists, patients are predisposed to organ failure. Three organ systems should be assessed to define organ failure: respiratory, cardiovascular, and renal. Organ failure is defined as a score of 2 or more for one of these three organ systems using the modified Marshall scoring system. Persistent organ failure (>48 h) is the most important clinical finding in regard to severity of the acute pancreatitis • Obesity,

1	Markers of Severity at Admission or Within 24 h by presence of 2 or more criteria: blood cell count >12,000/μL, <4000/μL, or 10% bands SIRS: ≥2 of 4 present • Cardiovascular: systolic BP <90 mmHg, heart rate >130 beats/min • Pulmonary: PaO2 <60 mmHg • Renal: serum creatinine >2.0 mg% Markers of Severity During Hospitalization Abbreviations: APACHE II, Acute Physiology and Chronic Health Evaluation II; BMI, body mass index; BISAP, Bedside Index of Severity in Acute Pancreatitis; BP, blood pressure; BUN, blood urea nitrogen; SIRS, systemic inflammatory response syndrome. episode. Organ failure that affects more than one organ is considered multisystem organ failure. CT imaging is usually not needed or recommended during the first 48 h of admission in acute pancreatitis.

1	The late phase is characterized by a protracted course of illness and may require imaging to evaluate for local complications. The important clinical parameter of severity, as in the early phase, is persistent organ failure. These patients may require supportive measures such as renal dialysis, ventilator support, or need for supplemental nutrition via the nasojejunal or parenteral route. The radiographic feature of greatest importance to recognize in this phase is the development of necrotizing pancreatitis on CT imaging. Necrosis generally prolongs hospitalization and, if infected, may require operative, endoscopic, or percutaneous intervention.

1	Severity of Acute Pancreatitis Three severity classifications have also been defined: mild, moderately severe, and severe. Mild acute pancreatitis is without local complications or organ failure. Most patients with interstitial acute pancreatitis have mild pancreatitis. In mild acute pancreatitis, the disease is self-limited and subsides spontaneously, usually within 3–7 days after treatment is instituted. Oral intake can be resumed if the patient is hungry, has normal bowel function, and is without nausea and vomiting. Typically, a clear or full liquid diet has been recommended for the initial meal; however, a low-fat solid diet is a reasonable choice following recovery from mild acute pancreatitis.

1	Moderately severe acute pancreatitis is characterized by transient organ failure (resolves in <48 h) or local or systemic complications in the absence of persistent organ failure. These patients may or may not have necrosis, but may develop a local complication such as a fluid collection that requires a prolonged hospitalization greater than 1 week. Severe acute pancreatitis is characterized by persistent organ failure (>48 h). Organ failure can be single or multiple. A CT scan or magnetic resonance imaging (MRI) should be obtained to assess for necrosis and/or complications. If a local complication is encountered, management is dictated by clinical symptoms, evidence of infection, maturity of fluid collection, and clinical stability of the patient. Prophylactic antibiotics are not recommended.

1	Imaging in Acute Pancreatitis Two types of pancreatitis are recognized on imaging as interstitial or necrotizing based on pancreatic perfusion. CT imaging is best evaluated 3–5 days into hospitalization when patients are not responding to supportive care to look for local complications such as necrosis. Recent studies report the overutilization of CT imaging in acute pancreatitis and its inability to be better than clinical judgment in the early days of acute pancreatitis management. The revised criteria also outline the terminology for local complications and fluid collections along with a CT imaging template to guide reporting of findings. Local morphologic features are summarized in Table 371-1. Interstitial pancreatitis occurs in 90–95% of admissions for acute pancreatitis and is characterized by diffuse gland enlargement, homogenous contrast enhancement, and mild inflammatory changes or peripancreatic stranding. Symptoms generally resolve with a week of hospitalization.

1	is characterized by diffuse gland enlargement, homogenous contrast enhancement, and mild inflammatory changes or peripancreatic stranding. Symptoms generally resolve with a week of hospitalization. Necrotizing pancreatitis occurs in 5–10% of acute pancreatitis admissions and does not evolve until several days of hospitalization. It is characterized by lack of pancreatic parenchymal enhancement by intravenous contrast agent and/ or presence of findings of peripancreatic necrosis. According to the revised Atlanta criteria, the natural history of pancreatic and peripancreatic necrosis is variable because it may remain solid or liquefy, remain sterile or become infected, and persist or disappear over time. CT identification of local complications, particularly necrosis, is critical in patients who are not responding to therapy because patients with infected and sterile necrosis are at greatest risk of mortality (Figs. 371-1B, 3712, and 371-3). The median prevalence of organ failure is 54%

1	who are not responding to therapy because patients with infected and sterile necrosis are at greatest risk of mortality (Figs. 371-1B, 3712, and 371-3). The median prevalence of organ failure is 54% in necrotizing pancreatitis. The prevalence of organ failure is perhaps slightly higher in infected versus sterile necrosis. With single-organ system failure, the mortality is 3–10% but increases to 47% with multisystem organ failure.

1	We will briefly describe the management of patients with acute pancreatitis from the time of diagnosis in the emergency ward to ongoing hospital admission and, finally, to time of discharge, highlighting salient features based on severity and complications. It is important to note that 85–90% of cases of acute pancreatitis are self-limited and subside spontaneously, usually within 3–7 days after initiation of treatment, and do not exhibit organ failure or local complications. The management of acute pancreatitis begins in the emergency ward. After a diagnosis has been

1	FIGURE 371-2 A. Acute necrotizing pancreatitis: computed tomography (CT) scan. Contrast-enhanced CT scan showing acute pancreatitis with necrosis. Arrow shows partially enhancing body/tail of pancreas surrounded by fluid with decreased enhancement in the neck/body of the pancreas. B. Acute fluid collection: CT scan. Contrast-enhanced CT scan showing fluid collection in the retroperitoneum (arrow) compressing the air-filled stomach arising from the pancreas in a patient with asparaginase-induced acute necrotizing pancreatitis. C. Walled-off pancreatic necrosis: CT scan. CT scan showing marked walled-off necrosis of the pancreas and peripancreatic area (arrow) in a patient with necrotizing pancreatitis. Addendum: In past years, both of these CT findings (Figs. 371-2B and 371-2C ) would have been misinterpreted as pseudocysts. D. Spiral CT showing a pseudo-cyst (small arrow) with a pseudoaneurysm (light area in pseudocyst). Note the demonstration of the main pancreatic duct (big arrow),

1	been misinterpreted as pseudocysts. D. Spiral CT showing a pseudo-cyst (small arrow) with a pseudoaneurysm (light area in pseudocyst). Note the demonstration of the main pancreatic duct (big arrow), even though this duct is minimally dilated by endoscopic retrograde cholangiopancreatography. (A, B, C, courtesy of Dr. KJ Mortele, Brigham and Women’s Hospital; D, courtesy of Dr. PR Ros, Brigham and Women’s Hospital; with permission.)

1	FIGURE 371-3 A. Pancreaticopleural fistula: pancreatic duct leak on endoscopic retrograde cholangiopancreatography. Pancreatic duct leak (arrow) demonstrated at the time of retrograde pancreatogram in a patient with acute exacerbation of alcohol-induced acute or chronic pancreatitis. B. Pancreaticopleural fistula: computed tomography (CT) scan. Contrast-enhanced CT scan (coronal view) with arrows showing fistula tract from pancreatic duct disruption in the pancreatic pleural fistula. C. Pancreaticopleural fistula: chest x-ray. Large pleural effusion in the left hemithorax from a disrupted pancreatic duct. Analysis of pleural fluid revealed elevated amylase concentration. (Courtesy of Dr. KJ Mortele, Brigham and Women’s Hospital; with permission.) 2096 confirmed, aggressive fluid resuscitation is initiated, intravenous analgesics are administered, severity is assessed, and a search for etiologies that may impact acute care is begun. Patients who do not respond to aggressive fluid

1	is initiated, intravenous analgesics are administered, severity is assessed, and a search for etiologies that may impact acute care is begun. Patients who do not respond to aggressive fluid resuscitation in the emergency ward should be considered for admission to a step-down or intensive care unit for aggressive fluid resuscitation, hemodynamic monitoring, and management of necrosis or organ failure.

1	Fluid Resuscitation and Monitoring Response to Therapy The most important treatment intervention for acute pancreatitis is safe, aggressive intravenous fluid resuscitation. The patient is made NPO to rest the pancreas and is given intravenous narcotic analgesics to control abdominal pain and supplemental oxygen (2 L) via nasal cannula.

1	Intravenous fluids of lactated Ringer’s or normal saline are initially bolused at 15–20 cc/kg (1050–1400 mL), followed by 3 mg/kg per hour (200–250 mL/h), to maintain urine output >0.5 cc/kg per hour. Serial bedside evaluations are required every 6–8 h to assess vital signs, oxygen saturation, and change in physical examination. Lactated Ringer’s solution has been shown to decrease systemic inflammation and may be a better crystalloid than normal saline. A targeted resuscitation strategy with measurement of hematocrit and BUN every 8–12 h is recommended to ensure adequacy of fluid resuscitation and monitor response to therapy, noting less aggressive resuscitation strategy may be needed in milder forms of pancreatitis. A rising BUN during hospitalization is not only associated with inadequate hydration but also higher in-hospital mortality.

1	A decrease in hematocrit and BUN during the first 12–24 h is strong evidence that sufficient fluids are being administered. Serial measurements and bedside assessment for fluid overload are continued, and fluid rates are maintained at the current rate. Adjustments in fluid resuscitation may be required in patients with cardiac, pulmonary, or renal disease. A rise in hematocrit or BUN during serial measurement should be treated with a repeat volume challenge with a 2-L crystalloid bolus followed by increasing the fluid rate by 1.5 mg/kg per hour. If the BUN or hematocrit fails to respond (i.e., remains elevated or does not decrease) to this bolus challenge and increase in fluid rate, consideration of transfer to an intensive care unit is strongly recommended for hemodynamic monitoring.

1	Assessment of Severity and Hospital Triage Severity of acute pancreatitis should be determined in the emergency ward to assist in patient triage to a regular hospital ward or step-down unit or direct admission to an intensive care unit. The Bedside Index of Severity in Acute Pancreatitis (BISAP) incorporates five clinical and laboratory parameters obtained within the first 24 h of hospitalization (Table 371-3)—BUN >25 mg/dL, impaired mental status (Glasgow coma score <15), SIRS, age >60 years, and pleural effusion on radiography—that can be useful in assessing severity. Presence of three or more of these factors was associated with substantially increased risk for in-hospital mortality among patients with acute pancreatitis. In addition, an elevated hematocrit >44% and admission BUN >22 mg/dL are also associated with more severe acute pancreatitis. Incorporating these indices with the overall patient response to initial fluid resuscitation in the emergency ward can be useful at

1	>22 mg/dL are also associated with more severe acute pancreatitis. Incorporating these indices with the overall patient response to initial fluid resuscitation in the emergency ward can be useful at triaging patients to the appropriate hospital acute care setting.

1	In general, patients with lower BISAP scores, hematocrits, and admission BUNs tend to respond to initial management and are triaged to a regular hospital ward for ongoing care. If SIRS is not present at 24 h, the patient is unlikely to develop organ failure or necrosis. Therefore, patients with persistent SIRS at 24 h or underlying comorbid illnesses (e.g., chronic obstructive pulmonary disease, congestive heart failure) should be considered for a step-down unit setting if available. Patients with higher BISAP scores and elevations in hematocrit and admission BUN that do not respond to initial fluid resuscitation and exhibit evidence of respiratory failure, hypotension, or organ failure should be considered for direct admission to an intensive care unit.

1	Special Considerations Based on Etiology A careful history, review of medications, selected laboratory studies (liver profile, serum triglycerides, serum calcium), and an abdominal ultrasound are recommended in the emergency ward to assess for etiologies that may impact acute management. An abdominal ultrasound is the initial imaging modality of choice and will evaluate the gallbladder and common duct and assess the pancreatic head. Gallstone pancreatitis Patients with evidence of ascending cholangitis (rising white blood cell count, increasing liver enzymes) should undergo ERCP within 24–48 h of admission. Patients with gallstone pancreatitis are at increased risk of recurrence, and consideration should be given to performing a cholecystectomy during the same admission or within 4–6 weeks of discharge. An alternative for patients who are not surgical candidates would be to perform an endoscopic biliary sphincterotomy before discharge.

1	HypertriGlyceriDemia Serum triglycerides >1000 mg/dL are associated with acute pancreatitis. Initial therapy may include insulin, heparin, or plasmapheresis. Outpatient therapies include control of diabetes if present, administration of lipid-lowering agents, weight loss, and avoidance of drugs that elevate lipid levels. Other potential etiologies that may impact acute hospital care include hypercalcemia, autoimmune pancreatitis, post-ERCP pancreatitis, and drug-induced pancreatitis. Treatment of hyperparathyroidism or malignancy is effective at reducing serum calcium. Autoimmune pancreatitis is responsive to glucocorticoid administration. Pancreatic duct stenting and rectal indomethacin administration are effective at decreasing pancreatitis after ERCP. Drugs that cause pancreatitis should be discontinued. Multiple drugs have been implicated, but only about 30 have been challenged (Class 1A) and found to be causative.

1	Nutritional Therapy A low-fat solid diet can be administered to subjects with mild acute pancreatitis after the abdominal pain has resolved. Enteral nutrition should be considered 2–3 days after admission in subjects with more severe pancreatitis instead of total parenteral nutrition (TPN). Enteral feeding maintains gut barrier integrity, limits bacterial translocation, is less expensive, and has fewer complications than TPN. The choice of gastric versus nasojejunal enteral feeding is currently under investigation.

1	Management of Local Complications (Table 371-4) Patients exhibiting signs of clinical deterioration despite aggressive fluid resuscitation and hemodynamic monitoring should be assessed for local complications, which may include necrosis, pseudocyst formation, pancreas duct disruption, peripancreatic vascular complications, and extrapancreatic infections. A multidisciplinary team approach is recommended including gastroenterology, surgery, interventional radiology, and intensive care specialists, and consideration should also be made for transfer to a pancreas center.

1	necrosis The management of necrosis requires a multidisciplinary team approach. Percutaneous aspiration of necrosis with Gram stain and culture should be performed if there are ongoing signs of possible pancreatic infection such as sustained leukocytosis, fever, or organ failure. There is currently no role for prophylactic antibiotics in necrotizing pancreatitis. It is reasonable to start broad-spectrum antibiotics in a patient who appears septic while awaiting the results of Gram stain and cultures. If cultures are negative, the antibiotics should be discontinued to minimize the risk of developing opportunistic or fungal superinfection. Repeated fine-needle aspiration and Gram stain with culture of pancreatic necrosis may be done every 5–7 days in the presence of persistent fever. Repeated CT or MRI imaging should also be considered with any change in clinical course to monitor for complications (e.g., thromboses, hemorrhage, abdominal compartment syndrome).

1	In general, sterile necrosis is most often managed conservatively unless complications arise. Once a diagnosis of infected necrosis is established and an organism identified, targeted antibiotics should be instituted. Pancreatic debridement (necrosectomy) should be considered for definitive management of infected necrosis, but clinical decisions are generally influenced by response to antibiotic treatment and overall clinical condition. Symptomatic local complications as outlined in the revised Atlanta criteria may require definitive therapy. Walled-off necrosis Pancreatic fluid collections Pancreatic pseudocyst Disruption of main pancreatic duct or secondary branches Pancreatic ascites Involvement of contiguous organs by necrotizing pancreatitis Thrombosis of blood vessels (splenic vein, portal vein) Pancreatic enteric fistula Bowel infarction Obstructive jaundice Hemorrhagic pancreatic necrosis with erosion into major blood vessels

1	Hemorrhagic pancreatic necrosis with erosion into major blood vessels Portal vein thrombosis, splenic vein thrombosis, variceal hemorrhage Miscellaneous (mediastinum, pleura, nervous system)

1	Miscellaneous (mediastinum, pleura, nervous system) A step-up approach (percutaneous or endoscopic transgastric drainage followed, if necessary, by open necrosectomy) has been successfully reported by some pancreatic centers. One-third of the patients successfully treated with the step-up approach did not require major abdominal surgery. A recent randomized trial reported advantages to an initial endoscopic approach compared to an initial surgical 2097 necrosectomy approach in select patients requiring intervention for symptomatic WON. Taken together, a more conservative approach to the management of infected pancreatic necrosis has evolved over the years under the close supervision of a multidisciplinary team. If conservative therapy can be safely implemented for 4–6 weeks, to allow the pancreatic collections to resolve or “wall-off,” surgical or endoscopic intervention is generally much safer and better tolerated by the patient.

1	pseuDocyst The incidence of pseudocyst is low, and most acute col lections resolve over time. Less than 10% of patients have persistent fluid collections after 6 weeks that would meet the definition of a pseudocyst. Only symptomatic collections should be drained with surgery or endoscopy or by percutaneous route. pancreatic Duct Disruption Pancreatic duct disruption may present with symptoms of increasing abdominal pain or shortness of breath in the setting of an enlarging fluid collection. Diagnosis can be confirmed on magnetic resonance cholangiopancreatography (MRCP) or ERCP. Placement of a bridging pancreatic stent for at least 6 weeks is >90% effective at resolving the leak. Nonbridging stents are less effective.

1	Placement of a bridging pancreatic stent for at least 6 weeks is >90% effective at resolving the leak. Nonbridging stents are less effective. perivascular complications Perivascular complications may include splenic vein thrombosis with gastric varices and pseudoaneurysms. Gastric varices bleed less than 5% of the time. Life-threatening bleeding from a ruptured pseudoaneurysm can be diagnosed and treated with mesenteric angiography and embolization. extrapancreatic infections Hospital-acquired infections occur in up to 20% of patients with acute pancreatitis. Patients should be continually monitored for the development pneumonia, urinary tract infection, and line infection. Continued culturing of urine, monitoring of chest x-rays, and routine changing of intravenous lines are important during hospitalization.

1	Follow-Up Care Hospitalizations for moderately severe and severe acute pancreatitis can last weeks to months and often involve a period of intensive care unit admission and outpatient rehabilitation or subacute nursing care. Follow-up evaluation should assess for development of diabetes, exocrine insufficiency, recurrent cholangitis, or development of infected fluid collections. As mentioned previously, cholecystectomy should be performed within 4–6 weeks of discharge if possible for patients with uncomplicated gallstone pancreatitis.

1	Approximately 25% of patients who have had an attack of acute pancreatitis have a recurrence. The two most common etiologic factors are alcohol and cholelithiasis. In patients with recurrent pancreatitis without an obvious cause, the differential diagnosis should encompass occult biliary tract disease including microlithiasis, hypertriglyceridemia, drugs, pancreatic cancer, pancreas divisum, and cystic fibrosis (Table 371-1). In one series of 31 patients diagnosed initially as having idiopathic or recurrent acute pancreatitis, 23 were found to have occult gallstone disease. Thus, approximately two-thirds of patients with recurrent acute pancreatitis without an obvious cause actually have occult gallstone disease due to microlithiasis. Genetic defects as in hereditary pancreatitis and cystic fibrosis mutations can result in recurrent pancreatitis. Other diseases of the biliary tree and pancreatic ducts that can cause acute pancreatitis include choledochocele; ampullary tumors; pancreas

1	fibrosis mutations can result in recurrent pancreatitis. Other diseases of the biliary tree and pancreatic ducts that can cause acute pancreatitis include choledochocele; ampullary tumors; pancreas divisum; and pancreatic duct stones, stricture, and tumor. Approximately 2–4% of patients with pancreatic carcinoma present with acute pancreatitis.

1	The incidence of acute pancreatitis is increased in patients with AIDS for two reasons: (1) the high incidence of infections involving the pancreas such as infections with cytomegalovirus, Cryptosporidium, and the Mycobacterium avium complex; and (2) the frequent use by patients with AIDS of medications such as didanosine, pentamidine, 2098 trimethoprim-sulfamethoxazole, and protease inhibitors. Incidence has been markedly reduced due to advances in therapy (Chap. 226).

1	Chronic pancreatitis is a disease process characterized by irreversible damage to the pancreas as distinct from the reversible changes noted in acute pancreatitis (Table 371-4). The events that initiate and then perpetuate the inflammatory process in the pancreas are becoming more clearly understood. Irrespective of the mechanism of injury, it is becoming apparent that stellate cell activation that results in cytokine expression and production of extracellular matrix proteins cause acute and chronic inflammation and collagen deposition in the pancreas. Thus, the condition is defined by the presence of histologic abnormalities, including chronic inflammation, fibrosis, and progressive destruction of both exocrine and eventually endocrine tissue (atrophy). A number of etiologies have been associated with chronic pancreatitis resulting in the cardinal manifestations of the disease such as abdominal pain, steatorrhea, weight loss, and diabetes mellitus (Table 371-5).

1	Although alcohol has been believed to be the primary cause of chronic pancreatitis, other factors contribute to the disease because not all heavy consumers of alcohol develop pancreatic disease. There is also a strong association between smoking and chronic pancreatitis. Cigarette smoke leads to an increased susceptibility to pancreatic auto-digestion and predisposes to dysregulation of duct cell CFTR function. CHRoniC PAnCREATiTiS AnD PAnCREATiC ExoCRinE inSuffiCiEnCy: TiGAR-o ClASSifiCATion SySTEm Alcoholic Tobacco smoking Hypercalcemia Hyperlipidemia Chronic renal failure Medications—phenacetin abuse Toxins—organotin compounds (e.g., dibutylin dichloride, DBTC) Pancreas divisum Duct obstruction (e.g., tumor) Preampullary duodenal wall cysts Posttraumatic pancreatic duct scars Abbreviations: DBTC, dibutylin dichloride; TIGAR-O, toxic-metabolic, idiopathic, genetic, autoimmune, recurrent and severe acute pancreatitis, obstructive.

1	Abbreviations: DBTC, dibutylin dichloride; TIGAR-O, toxic-metabolic, idiopathic, genetic, autoimmune, recurrent and severe acute pancreatitis, obstructive. Smoking is an independent, dose-dependent risk factor for chronic pancreatitis and recurrent acute pancreatitis. Both continued alcohol and smoking exposure are associated with pancreatic fibrosis, calcifications, and progression of disease

1	Recent characterization of pancreatic stellate cells (PSCs) has added insight into the underlying cellular responses behind development of chronic pancreatitis. Specifically, PSCs are believed to play a role in maintaining normal pancreatic architecture that can shift toward fibrogenesis in the case of chronic pancreatitis. The sentinel acute pancreatitis event (SAPE) hypothesis uniformly describes the events in the pathogenesis of chronic pancreatitis. It is believed that alcohol or additional stimuli lead to matrix metalloproteinase–mediated destruction of normal collagen in pancreatic parenchyma, which later allows for pancreatic remodeling. Proinflammatory cytokines, tumor necrosis factor α (TNF-α), interleukin 1 (IL-1), and interleukin 6 (IL-6), as well as oxidant complexes, are able to induce PSC activity with subsequent new collagen synthesis. In addition to being stimulated by cytokines, oxidants, or growth factors, PSCs also possess transforming growth factor β

1	are able to induce PSC activity with subsequent new collagen synthesis. In addition to being stimulated by cytokines, oxidants, or growth factors, PSCs also possess transforming growth factor β (TGF-β)–mediated self-activating autocrine pathways that may explain disease progression in chronic pancreatitis even after removal of noxious stimuli.

1	Among adults in the United States, alcoholism is the most common cause of clinically apparent chronic pancreatitis, whereas cystic fibrosis is the most frequent cause in children. As many as 25% of adults in the United States with chronic pancreatitis have the idiopathic form. Recent investigations have indicated that up to 15% of patients with idiopathic pancreatitis may have pancreatitis due to genetic defects (Table 371-5).

1	Whitcomb and associates studied several large families with hereditary chronic pancreatitis and were able to identify a genetic defect that affects the gene encoding for trypsinogen. Several additional defects of this gene have also been described. The defect prevents the destruction of prematurely activated trypsin and allows it to be resistant to the intracellular protective effect of trypsin inhibitor. It is hypothesized that this continual activation of digestive enzymes within the gland leads to acute injury and, finally, chronic pancreatitis. Since the initial discovery of the PRSS1 mutation defect, other genetic diseases have been detected (Table 371-5).

1	Several other groups of investigators have documented mutations of CFTR. This gene functions as a cyclic AMP–regulated chloride channel. In patients with cystic fibrosis, the high concentration of macromolecules can block the pancreatic ducts. It must be appreciated, however, that there is a great deal of heterogeneity in relationship to the CFTR gene defect. More than 1000 putative mutations of the CFTR gene have been identified. Attempts to elucidate the relationship between the genotype and pancreatic manifestations have been hampered by the number of mutations. The ability to detect CFTR mutations has led to the recognition that the clinical spectrum of the disease is broader than previously thought. Two studies have clarified the association between mutations of the CFTR gene and another monosymptomatic form of cystic fibrosis (i.e., chronic pancreatitis). It is estimated that in patients with idiopathic pancreatitis, the frequency of a single CFTR mutation is 11 times the

1	another monosymptomatic form of cystic fibrosis (i.e., chronic pancreatitis). It is estimated that in patients with idiopathic pancreatitis, the frequency of a single CFTR mutation is 11 times the expected frequency and the frequency of two mutant alleles is 80 times the expected frequency. In these studies, the patients were adults when the diagnosis of pancreatitis was made; none had any clinical evidence of pulmonary disease, and sweat test results were not diagnostic of cystic fibrosis. The prevalence of such mutations is unclear, and further studies are certainly needed. In addition, the therapeutic and prognostic implication of these findings with respect to managing pancreatitis remains to be determined. Long-term follow-up of affected patients is needed. CFTR mutations are common in the general population. It is unclear whether the CFTR mutation alone can lead to pancreatitis as an autosomal recessive disease. A study evaluated 39 patients with idiopathic chronic pancreatitis

1	in the general population. It is unclear whether the CFTR mutation alone can lead to pancreatitis as an autosomal recessive disease. A study evaluated 39 patients with idiopathic chronic pancreatitis to assess the risk associated with these mutations. Patients with two CFTR mutations (compound heterozygotes) demonstrated CFTR function at a level between that seen in typical cystic fibrosis and cystic fibrosis carriers and had a 40-fold increased risk of pancreatitis. The presence of an N34S SPINK1 mutation increased the risk 20-fold. A combination of two CFTR mutations and an N34S SPINK1 mutation increased the risk of pancreatitis 900-fold. Knowledge of the genetic defects and downstream alterations in protein expression has led to the development of novel genetic therapy in cystic fibrosis children that potentiates the CFTR channel resulting in improvement in lung function, quality of life, and weight gain. Table 371-5 lists recognized causes of chronic pancreatitis and pancreatic

1	children that potentiates the CFTR channel resulting in improvement in lung function, quality of life, and weight gain. Table 371-5 lists recognized causes of chronic pancreatitis and pancreatic exocrine insufficiency.

1	Autoimmune pancreatitis (AIP) is an uncommon disorder of presumed autoimmune causation with characteristic laboratory, histologic, and morphologic findings. In type 1 AIP, the pancreas is involved as part of an IgG4 systemic disease (Chap. 391e) and meets HISORt criteria as defined below. The characteristic pancreatic histopathologic findings include lymphoplasmacytic infiltrate, storiform fibrosis, and abundant IgG4 cells. AIP type 2 is histologically confirmed idiopathic duct centric pancreatitis with granulocytic infiltration of the duct wall (termed GEL), but without IgG4 positive cells and systemic involvement. Although AIP was initially described as a primary pancreatic disorder, it is now recognized that it is associated with other disorders of presumed autoimmune etiology, and this has been termed IgG4 systemic disease (Chap. 391e). The clinical features include IgG4-associated cholangitis, rheumatoid arthritis, Sjögren’s syndrome, ulcerative colitis, mediastinal fibrosis and

1	has been termed IgG4 systemic disease (Chap. 391e). The clinical features include IgG4-associated cholangitis, rheumatoid arthritis, Sjögren’s syndrome, ulcerative colitis, mediastinal fibrosis and adenopathy, autoimmune thyroiditis, tubulointerstitial nephritis, retroperitoneal fibrosis, chronic periaortitis, chronic sclerosing sialadenitis, and Mikulicz’s disease. Mild symptoms, usually abdominal pain, and recurrent acute pancreatitis are unusual. Furthermore, AIP is not a common cause of idiopathic recurrent pancreatitis.

1	Weight loss and new onset of diabetes may also occur. An obstructive pattern on liver tests is common (i.e., disproportionately elevated serum alkaline phosphatase and minimally elevated serum aminotransferases). Elevated serum levels of IgG4 provide a marker for the disease, particularly in Western populations. Serum IgG4 normally accounts for only 5–6% of the total IgG4 in healthy patients but is elevated to values >280 mg/dL in those with AIP. CT scans reveal abnormalities in the majority of patients and include diffuse enlargement, focal enlargement, and a distinct enlargement at the head of the pancreas. ERCP or MRCP reveals strictures in the bile duct in more than one-third of patients with AIP; these may include common bile duct strictures, intrahepatic bile duct strictures, or proximal bile duct strictures, with accompanying narrowing of the pancreatic portion of the bile duct. This has been termed autoimmune IgG4 cholangitis. Characteristic histologic findings include

1	or proximal bile duct strictures, with accompanying narrowing of the pancreatic portion of the bile duct. This has been termed autoimmune IgG4 cholangitis. Characteristic histologic findings include extensive lymphoplasmacytic infiltrates

1	CliniCAl fEATuRES of AuToimmunE PAnCREATiTiS (AiP) • Mild symptoms, usually abdominal pain, but without frequent attacks of swelling and enlargement of the pancreas of patients present with either obstructive jaundice or a “mass” in the head of the pancreas mimicking carcinoma irregular narrowing of the pancreatic duct (MRCP or ERCP) levels of serum gamma globulins, especially IgG4 of other autoantibodies (ANA), rheumatoid factor (RF) occur with other autoimmune diseases: Sjögren’s syndrome, primary sclerosing cholangitis, ulcerative colitis, rheumatoid arthritis • Extrapancreatic bile duct changes such as stricture of the common bile • Glucocorticoids are effective in alleviating symptoms, decreasing size of the pancreas, and reversing histopathologic changes Abbreviations: ERCP, endoscopic retrograde cholangiopancreatography; MRCP, magnetic resonance cholangiopancreatography.

1	Abbreviations: ERCP, endoscopic retrograde cholangiopancreatography; MRCP, magnetic resonance cholangiopancreatography. with dense fibrosis around pancreatic ducts, as well as a lymphoplas-2099 macytic infiltration, resulting in an obliterative phlebitis. The Mayo Clinic HISORt criteria indicate that AIP can be diagnosed by the presence of at least two of the following: (1) histology; (2) imaging; (3) serology (elevated serum IgG4 levels); (4) other organ involvement; and (5) response to glucocorticoid therapy, with improvement in pancreatic and extrapancreatic manifestations.

1	Glucocorticoids have shown efficacy in alleviating symptoms, decreasing the size of the pancreas, and reversing histopathologic features in patients with AIP. Patients may respond dramatically to glucocorticoid therapy within a 2to 4-week period. Prednisone is usually administered at an initial dose of 40 mg/d for 4 weeks followed by a taper of the daily dosage by 5 mg/wk based on monitoring of clinical parameters. Relief of symptoms, serial changes in abdominal imaging of the pancreas and bile ducts, decreased serum γ-globulin and IgG4 levels, and improvements in liver tests are parameters to follow. A poor response to glucocorticoids over a 2to 4-week period should raise suspicion of pancreatic cancer or other forms of chronic pancreatitis. A recent multicenter international report reviewed 1064 patients with

1	AIP. Clinical remission was achieved in 99% of type I and 92% of type II AIP patients with steroids. However, disease relapse occurred in 31% of type I and 9% of type II AIP patients. For treatment of disease relapse in type 1 AIP, glucocorticoids were successful in 201 of 295 (68%) patients, and azathioprine was successful in 52 of 58 patients (85%). A small number of patients responded favorably to 6-mercaptapurine, rituximab, cyclosporine, and cyclophosphamide. Types 1 and 2 AIP are highly responsive to initial glucocorticoid treatment. Relapse is common in type 1 patients, especially those with biliary tract strictures. Most relapses occur after glucocorticoids are discontinued. Patients with refractory symptoms and strictures generally require immunomodulator therapy as noted above. Appearance of interval cancers following a diagnosis of AIP is uncommon.

1	Clinical Features of Chronic Pancreatitis Patients with chronic pancreatitis seek medical attention predominantly because of two symptoms: abdominal pain or maldigestion and weight loss. The abdominal pain may be quite variable in location, severity, and frequency. The pain can be constant or intermittent with frequent pain-free intervals. Eating may exacerbate the pain, leading to a fear of eating with consequent weight loss. The spectrum of abdominal pain ranges from mild to quite severe, with narcotic dependence as a frequent consequence. Maldigestion is manifested as chronic diarrhea, steatorrhea, weight loss, and fatigue. Patients with chronic abdominal pain may or may not progress to maldigestion, and ~20% of patients will present with symptoms of maldigestion without a history of abdominal pain. Patients with chronic pancreatitis have significant morbidity and mortality and use appreciable amounts of societal resources. Despite steatorrhea, clinically apparent deficiencies of

1	abdominal pain. Patients with chronic pancreatitis have significant morbidity and mortality and use appreciable amounts of societal resources. Despite steatorrhea, clinically apparent deficiencies of fat-soluble vitamins are surprisingly uncommon. Physical findings in these patients are usually unimpressive, so that there is a disparity between the severity of abdominal pain and the physical signs that usually consist of some mild tenderness.

1	The diagnosis of early or mild chronic pancreatitis can be challenging because there is no biomarker for the disease. In contrast to acute pancreatitis, the serum amylase and lipase levels are usually not strikingly elevated in chronic pancreatitis. Elevation of serum bilirubin and alkaline phosphatase may indicate cholestasis secondary to common bile duct stricture caused by chronic inflammation. Many patients have impaired glucose tolerance with elevated fasting blood glucose levels. The fecal elastase-1 and small-bowel biopsy are useful in the evaluation of patients with suspected pancreatic steatorrhea. The fecal elastase level will be abnormal and small-bowel histology will be normal in such patients. A decrease of fecal elastase level to <100 μg per gram of stool strongly suggests severe pancreatic exocrine insufficiency.

1	The radiographic evaluation of a patient with suspected chronic pancreatitis usually proceeds from a noninvasive to more invasive approach. Abdominal CT imaging (Fig. 371-4A,B) is the initial modality of choice, followed by MRI (Fig. 371-4C), endoscopic ultrasound, and pancreas function testing. In addition to excluding a

1	FIGURE 371-4 A. Chronic pancreatitis and pancreatic calculi: computed tomography (CT) scan. In this contrast-enhanced CT scan of the abdomen, there is evidence of an atrophic pancreas with multiple calcifications and stones in the parenchyma and dilated pancreatic duct (arrow). B. In this contrast-enhanced CT scan of the abdomen, there is evidence of an atrophic pancreas with multiple calcifications (arrows). Note the markedly dilated pancreatic duct seen in this section through the body and tail (open arrows). C. Chronic pancreatitis on magnetic resonance cholangiopancreatography (MRCP): dilated duct with filling defects. Gadolinium-enhanced magnetic resonance imaging/MRCP reveals a dilated pancreatic duct (arrow) in chronic pancreatitis with multiple filling defects suggestive of pancreatic duct calculi. (A, C, courtesy of Dr. KJ Mortele, Brigham and Women’s Hospital; with permission.) pseudocyst and pancreatic cancer, CT may show calcification, dilated ducts, or an atrophic

1	pancreatic duct calculi. (A, C, courtesy of Dr. KJ Mortele, Brigham and Women’s Hospital; with permission.) pseudocyst and pancreatic cancer, CT may show calcification, dilated ducts, or an atrophic pancreas. Although abdominal CT scanning and MRCP greatly aid in the diagnosis of pancreatic disease, the diagnostic test with the best sensitivity and specificity is the hormone stimulation test using secretin. The secretin test becomes abnormal when ≥60% of the pancreatic exocrine function has been lost. This usually correlates well with the onset of chronic abdominal pain. The role of endoscopic ultrasonography (EUS) in diagnosing early chronic pancreatitis is still being defined. A total of nine endosonographic features have been described in chronic pancreatitis. The presence of five or more features is considered diagnostic of chronic pancreatitis. EUS is not a sensitive enough test for detecting early chronic pancreatitis alone (Chap. 370) and may show positive features in patients

1	features is considered diagnostic of chronic pancreatitis. EUS is not a sensitive enough test for detecting early chronic pancreatitis alone (Chap. 370) and may show positive features in patients who have dyspepsia or even normal aging individuals. Recent data suggest that EUS can be combined with endoscopic pancreatic function testing (EUS-ePFT) during a single endoscopy to screen for chronic pancreatitis in patients with chronic abdominal pain. Diffuse calcifications noted on plain film of the abdomen usually indicate significant damage to the pancreas and are pathognomic for chronic pancreatitis (Fig. 371-4A). Although alcohol is by far the most common cause of pancreatic calcification, such calcification may also be noted in hereditary pancreatitis, post-traumatic pancreatitis, hypercalcemic pancreatitis, idiopathic chronic pancreatitis, and tropical pancreatitis.

1	Complications of Chronic Pancreatitis The complications of chronic pancreatitis are protean and are listed in Table 371-7. Although most patients have impaired glucose tolerance, diabetic ketoacidosis and diabetic coma are uncommon. Likewise, end-organ damage (retinopathy, neuropathy, nephropathy) is also uncommon. A nondiabetic retinopathy may be due to either vitamin A and/or zinc deficiency. Gastrointestinal bleeding may occur from peptic ulceration, gastritis, a pseudocyst eroding into the duodenum, arterial bleeding into the pancreatic duct (hemosuccus pancreaticus), or ruptured varices secondary to splenic vein thrombosis due to chronic inflammation of the tail of the pancreas. Jaundice, cholestasis, and biliary cirrhosis may occur from the chronic inflammatory reaction around the intrapancreatic portion of the common bile duct. Twenty years after the diagnosis of calcific chronic pancreatitis, the cumulative risk of pancreatic carcinoma is 4%. Patients with hereditary

1	the intrapancreatic portion of the common bile duct. Twenty years after the diagnosis of calcific chronic pancreatitis, the cumulative risk of pancreatic carcinoma is 4%. Patients with hereditary pancreatitis are at a 10-fold higher risk for pancreatic cancer.

1	The treatment of steatorrhea with pancreatic enzymes is straightforward even though complete correction of steatorrhea is unusual. Enzyme therapy usually brings diarrhea under control and restores absorption of fat to an acceptable level and affects weight gain. Thus, pancreatic enzyme replacement has been the cornerstone of therapy. In treating steatorrhea, it is important to use a potent pancreatic formulation that will deliver sufficient lipase into the duodenum to correct maldigestion and decrease steatorrhea. In an attempt to standardize the enzyme activity, potency, and bioavailability, the U.S. Food and Drug Administration (FDA) required that all pancreas enzyme drugs in the United States obtain a New Drug Application (NDA) by April 2008. Table 371-8 lists frequently used formulations, but availability will be based on compliance with the FDA mandate. Recent data suggest that dosages up to ComPliCATionS of CHRoniC PAnCREATiTiS

1	ComPliCATionS of CHRoniC PAnCREATiTiS Enzyme Content/Unit Dose, U.S. Pharmacopeia Units Note: The FDA has mandated all enzyme manufacturers to submit New Drug Applications (NDAs) for all pancreatic extract drug products after reviewing data that showed substantial variations among currently marketed products. Numerous manufacturers have investigations under way to seek FDA approval for the treatment of exocrine pancreatic insufficiency due to cystic fibrosis or other conditions under the new guidelines for this class of drugs (www.fda.gov). 80,000–100,000 units of lipase taken during the meal may be necessary to normalize nutritional parameters in malnourished chronic pancreatitis patients, and some may require acid suppression with proton pump inhibitors. The management of pain in patients with chronic pancreatitis is problematic.

1	The management of pain in patients with chronic pancreatitis is problematic. Recent meta-analyses have shown no consistent benefit of enzyme therapy at reducing pain in chronic pancreatitis. In some patients with idiopathic chronic pancreatitis, conventional nonenteric-coated enzyme preparations containing high concentrations of serine proteases may relieve mild abdominal pain or discomfort. The pain relief experienced by these patients actually may be due to improvements in the dyspepsia from maldigestion. Gastroparesis is also quite common in patients with chronic pancreatitis. It is important to recognize and treat with prokinetic drugs because treatment with enzymes may fail simply because gastric dysmotility is interfering with the delivery of enzymes into the upper intestine. A recent prospective study reported that pregabalin can improve pain in chronic pancreatitis and lower pain medication requirement.

1	Endoscopic treatment of chronic pancreatitis pain may involve 2101 sphincterotomy, stenting, stone extraction, and drainage of a pancreatic pseudocyst. Therapy directed to the pancreatic duct would seem to be most appropriate in the setting of a dominant stricture, especially if a ductal stone has led to obstruction. The use of endoscopic stenting for patients with chronic pain, but without a dominant stricture, has not been subjected to any controlled trials. It is now appreciated that significant complications can occur from stenting (i.e., bleeding, cholangitis, stent migration, pancreatitis, and stent clogging). In patients with large-duct disease usually from alcohol-induced chronic pancreatitis, ductal decompression with surgical therapy has been the therapy of choice. Among such patients, 80% seem to obtain immediate relief; however, at the end of 3 years, one-half of the patients have recurrence of pain. Two randomized prospective trials comparing endoscopic to surgical

1	patients, 80% seem to obtain immediate relief; however, at the end of 3 years, one-half of the patients have recurrence of pain. Two randomized prospective trials comparing endoscopic to surgical therapy for chronic pancreatitis demonstrated that surgical therapy was superior to endoscopy at decreasing pain and improving quality of life in selected patients with dilated ducts and abdominal pain. This would suggest that chronic pancreatitis patients with dilated ducts and pain should be considered for surgical intervention. The role of preoperative stenting prior to surgery as a predictor of response has yet to be proven.

1	A Whipple procedure, total pancreatectomy, and autologous islet cell transplantation have been used in selected patients with chronic pancreatitis and abdominal pain refractory to conventional therapy. The patients who have benefited the most from total pancreatectomy have chronic pancreatitis without prior pancreatic surgery or evidence of islet cell insufficiency. The role of this procedure remains to be fully defined but may be an option in lieu of ductal decompression surgery or pancreatic resection in patients with intractable, painful small-duct disease, particularly as the standard surgical procedures tend to decrease islet cell yield. Celiac plexus block has not resulted in long-lasting pain relief.

1	Hereditary pancreatitis is a rare disease that is similar to chronic pancreatitis except for an early age of onset and evidence of hereditary factors. A genomewide search using genetic linkage analysis identified the hereditary pancreatitis gene on chromosome 7. Mutations in ion codons 29 (exon 2) and 122 (exon 3) of the cationic trypsinogen gene cause autosomal dominant forms of hereditary pancreatitis. The codon 122 mutations lead to a substitution of the corresponding arginine with another amino acid, usually histidine. This substitution, when it occurs, eliminates a fail-safe trypsin self-destruction site necessary to eliminate trypsin that is prematurely activated within the acinar cell. These patients have recurring attacks of severe abdominal pain that may last from a few days to a few weeks. The serum amylase and lipase levels may be elevated during acute attacks but are usually normal. Patients frequently develop pancreatic calcification, diabetes mellitus, and steatorrhea;

1	a few weeks. The serum amylase and lipase levels may be elevated during acute attacks but are usually normal. Patients frequently develop pancreatic calcification, diabetes mellitus, and steatorrhea; in addition, they have an increased incidence of pancreatic carcinoma, with the cumulative incidence being as high as 40% by age 70 years. A recent natural history study of hereditary pancreatitis in more than 200 patients from France reported that abdominal pain started in childhood at age 10 years, steatorrhea developed at age 29 years, diabetes at age 38 years, and pancreatic carcinoma at age 55 years. Such patients often require surgical ductal decompression for pain relief. Abdominal complaints in relatives of patients with hereditary pancreatitis should raise the question of pancreatic disease.

1	PSTI, or SPINK1, is a 56-amino-acid peptide that specifically inhibits trypsin by physically blocking its active site. SPINK1 acts as the first line of defense against prematurely activated trypsinogen in the acinar cell. Recently, it has been shown that the frequency of SPINK1 mutations in patients with idiopathic chronic pancreatitis is markedly increased, suggesting that these mutations may be associated with pancreatitis. Pancreatic endocrine tumors are discussed in Chap. 113.

1	When the ventral pancreatic anlage fails to migrate correctly to make contact with the dorsal anlage, the result may be a ring of pancreatic tissue encircling the duodenum. Such an annular pancreas may cause intestinal obstruction in the neonate or the adult. Symptoms of postprandial fullness, epigastric pain, nausea, and vomiting may be present for years before the diagnosis is entertained. The radiographic findings are symmetric dilation of the proximal duodenum with bulging of the recesses on either side of the annular band, effacement but not destruction of the duodenal mucosa, accentuation of the findings in the right anterior oblique position, and lack of change on repeated examinations. The differential diagnosis should include duodenal webs, tumors of the pancreas or duodenum, postbulbar peptic ulcer, regional enteritis, and adhesions. Patients with annular pancreas have an increased incidence of pancreatitis and peptic ulcer. Because of these and other potential

1	duodenum, postbulbar peptic ulcer, regional enteritis, and adhesions. Patients with annular pancreas have an increased incidence of pancreatitis and peptic ulcer. Because of these and other potential complications, the treatment is surgical even if the condition has been present for years. Retrocolic duodenojejeunostomy is the procedure of choice, although some surgeons advocate Billroth II gastrectomy, gastroenterostomy, and vagotomy.

1	Pancreas divisum is present in 7–10% of the population and occurs when the embryologic ventral and dorsal pancreatic anlagen fail to fuse, so that pancreatic drainage is accomplished mainly through the accessory papilla. Pancreas divisum is the most common congenital anatomic variant of the human pancreas. Current evidence indicates that this anomaly does not predispose to the development of pancreatitis in the great majority of patients who harbor it. However, the combination of pancreas divisum and a small accessory orifice could result in dorsal duct obstruction. The challenge is to identify this subset of patients with dorsal duct pathology. Cannulation of the dorsal duct by ERCP is not as easily done as is cannulation of the ventral duct. Patients with pancreatitis and pancreas divisum demonstrated by MRCP or ERCP should be treated with conservative measures. In many of these patients, pancreatitis is idiopathic and unrelated to the pancreas divisum. Endoscopic or surgical

1	divisum demonstrated by MRCP or ERCP should be treated with conservative measures. In many of these patients, pancreatitis is idiopathic and unrelated to the pancreas divisum. Endoscopic or surgical intervention is indicated only if pancreatitis recurs and no other cause can be found. If marked dilation of the dorsal duct can be demonstrated, surgical ductal decompression should be performed. It should be stressed that the ERCP/MRCP appearance of pancreas divisum (i.e., a small-caliber ventral duct with an arborizing pattern) may be mistaken as representing an obstructed main pancreatic duct secondary to a mass lesion.

1	In macroamylasemia, amylase circulates in the blood in a polymer form too large to be easily excreted by the kidney. Patients with this condition demonstrate an elevated serum amylase value and a low urinary amylase value. The presence of macroamylase can be documented by chromatography of the serum. The prevalence of macroamylasemia is 1.5% of the nonalcoholic general adult hospital population. Usually macroamylasemia is an incidental finding and is not related to disease of the pancreas or other organs. Macrolipasemia has now been documented in a few patients with cirrhosis or non-Hodgkin’s lymphoma. In these patients, the pancreas appeared normal on ultrasound and CT examination. Lipase was shown to be complexed with immunoglobulin A. Thus, the possibility of both macroamylasemia and macrolipasemia should be considered in patients with elevated blood levels of these enzymes.

1	This chapter represents a revised version of chapters by Drs. Norton J. Greenberger, Phillip P. Toskes, and Bechien Wu that were in previous editions of Harrison’s. Introduction to the Immune System Barton F. Haynes, Kelly A. Soderberg, Anthony S. Fauci 372e Sec tIon 1 the Immune SyStem In health and dISeaSe ParT 15: Immune-mediated, Inflammatory, and Rheumatologic Disorders Adaptive immune system—recently evolved system of immune responsesmediated by TandBlymphocytes.Immuneresponsesby these cells are based on specific antigen recognition by clonotypic receptors that are products of genes that rearrange during development and throughout the life of the organism. Additional cells of the adaptive immune system include various types of antigen-presenting cells.

1	Antibody—B cell–produced molecules encoded by genes that rearrange during B cell development consisting of immunoglobulin heavy and light chains that together form the central component of the B cell receptor for antigen. Antibody can exist as B cell–surface antigen-recognition molecules or as secreted molecules in plasma and other body fluids. Antigens—foreign or self-molecules that are recognized by the adaptive and innate immune systems resulting in immune cell triggering, T cell activation, and/or B cell antibody production. Antimicrobial peptides—small peptides <100 amino acids in length that are produced by cells of the innate immune system and have anti-infectious agent activity.

1	Antimicrobial peptides—small peptides <100 amino acids in length that are produced by cells of the innate immune system and have anti-infectious agent activity. Apoptosis—the process of programmed cell death whereby signaling through various “death receptors” on the surface of cells (e.g., tumor necrosis factor [TNF] receptors, CD95) leads to a signaling cascade that involves activation of the caspase family of molecules and leads to DNA cleavage and cell death. Apoptosis, which does not lead to induction of inordinate inflammation, is to be contrasted with cell necrosis, which does lead to induction of inflammatory responses. Autoimmune diseases—diseases such as systemic lupus erythematosus and rheumatoid arthritis in which cells of the adaptive immune system such as autoreactive T and B cells become over-reactive and produce self-reactive T cell and antibody responses.

1	Autoinflammatory diseases—hereditary disorders such as hereditary periodic fevers (HPFs) characterized by recurrent episodes of severe inflammation and fever due to mutations in controls of the innate inflammatory response, i.e., the inflammasome (see below and Table 372e-6). Patients with HPFs also have rashes and serosal and joint inflammation, and some can have neurologic symptoms. Autoinflammatory diseases are different from autoimmune diseases in that evidence for activation of adaptive immune cells such as autoreactive B cells is not present. B cell receptor for antigen—complex of surface molecules that rearrange during postnatal B cell development, made up of surface immunoglobulin (Ig) and associated Ig αβ chain molecules that recognize nominal antigen via Ig heavy-and light-chain variable regions, and signal the B cell to terminally differentiate to make antigen-specific antibody.

1	B lymphocytes—bone marrow-derived or bursal-equivalent lymphocytes that express surface immunoglobulin (the B cell receptor for antigen) and secrete specific antibody after interaction with antigen. CD classification of human lymphocyte differentiation antigens—the development of monoclonal antibody technology led to the discovery of a large number of new leukocyte surface molecules. In 1982, the First International Workshop on Leukocyte Differentiation Antigens was held to establish a nomenclature for cell-surface molecules of human leukocytes. From this and subsequent leukocyte differentiation workshops has come the cluster of differentiation (CD) classification of leukocyte antigens. Chemokines—soluble molecules that direct and determine immune cell movement and circulation pathways.

1	Chemokines—soluble molecules that direct and determine immune cell movement and circulation pathways. Complement—cascading series of plasma enzymes and effector proteins whose function is to lyse pathogens and/or target them to be phagocytized by neutrophils and monocyte/macrophage lineage cells of the reticuloendothelial system. Co-stimulatory molecules—molecules of antigen-presenting cells (such as B7-1 and B7-2 or CD40) that lead to T cell activation when bound by ligands on activated T cells (such as CD28 or CD40 ligand). Cytokines—soluble proteins that interact with specific cellular receptors that are involved in the regulation of the growth and activation of immune cells and mediate normal and pathologic inflammatory and immune responses.

1	Dendritic cells—myeloid and/or lymphoid lineage antigen-presenting cells of the adaptive immune system. Immature dendritic cells, or dendritic cell precursors, are key components of the innate immune system by responding to infections with production of high levels of cytokines. Dendritic cells are key initiators both of innate immune responses via cytokine production and of adaptive immune responses via presentation of antigen to T lymphocytes. Ig Fc receptors—receptors found on the surface of certain cells including B cells, natural killer cells, macrophages, neutrophils, and mast cells. Fc receptors bind to antibodies that have attached to invading pathogen-infected cells. They stimulate cytotoxic cells to destroy microbe-infected cells through a mechanism known as antibody-dependent cell-mediated cytotoxicity (ADCC). Examples of important Fc receptors include CD16 (FcγRIIIa), CD23 (FcεR), CD32 (FcγRII), CD64 (FcγRI), and CD89 (FcαR).

1	Inflammasome—large cytoplasmic complexes of intracellular proteins that link the sensing of microbial products and cellular stress to the proteolytic activation of interleukin (IL)-1β and IL-18 inflammatory cytokines. Activation of molecules in the inflammasome is a key step in the response of the innate immune system for intracellular recognition of microbial and other danger signals in both health and pathologic states. Innate immune system—ancient immune recognition system of host cells bearing germline-encoded pattern recognition receptors that recognize pathogens and trigger a variety of mechanisms of pathogen elimination. Cells of the innate immune system include natural killer cell lymphocytes, monocytes/macrophages, dendritic cells, neutrophils, basophils, eosinophils, tissue mast cells, and epithelial cells.

1	Large granular lymphocytes—lymphocytes of the innate immune system with azurophilic cytotoxic granules that have natural killer cell activitycapableofkillingforeignandhostcellswithfewornoself-major histocompatibility complex(MHC) class I molecules. Natural killer (NK) cells—large granular lymphocytes that kill target cells expressing few or no human leukocyte antigen (HLA) class I molecules, such as malignantly transformed cells and virally infectedcells.NKcellsexpressreceptorsthatinhibitkillercellfunction when self-MHC class I is present. Introduction to the Immune System NK T cells—innate-like lymphocytes that use an invariant T cell receptor (TCR)-α chain combined with a limited set of TCR-β chains and coexpress receptors commonly found on NK cells. NK T cells recognize lipid antigens of bacterial, viral, fungal, and protozoal infectious agents.

1	Pathogen-associated molecular patterns (PAMPs)—Invariant molecular structures expressed by large groups of microorganisms that arerecognized by host cellular pattern recognition receptors in the mediation of innate immunity. Pattern recognition receptors (PRRs)—germline-encoded receptors expressedbycellsoftheinnateimmunesystemthatrecognizePAMPs. Polyreactive natural antibodies—preexistinglow-affinity antibodies produced by B cells that cross-react with multiple antigens and are available at the time of infection to bind to and coat the invading pathogen and harness innate responses to slow the infection until anadaptivehigh-affinityprotectiveantibodyresponsecanbemade. T cell exhaustion—state of T cells when the persistence of antigen disrupts memory T cell function, resulting in defects in memory T cell responses. Most frequently occurs in malignancies and in chronic viral infections such as HIV-1 and hepatitis C.

1	T cell receptor (TCR) for antigen—complex of surface molecules that rearrange during postnatal T cell development made up of clonotypicTCR-αand-βchainsthatareassociatedwiththeCD3complex composed of invariant γ, δ, ε, ζ, and η chains. TCR-α and -β chains recognize peptide fragments of protein antigen physically bound in antigen-presentingcell MHC class I or II molecules, leading to signaling via the CD3 complex to mediate effector functions. T follicular helper T cells (Tfh)—CD4 T cells in B cell follicle germinal centers that produce IL-4 and IL-21 and drive B cell development and affinity maturation in peripheral lymphoid tissues such as lymph node and spleen. TH17 T cells—CD4 T cells that secrete IL-17, IL-22, and IL-26 and play roles in autoimmune inflammatory disorders as well as defend against bacterial and fungal pathogens.

1	TH17 T cells—CD4 T cells that secrete IL-17, IL-22, and IL-26 and play roles in autoimmune inflammatory disorders as well as defend against bacterial and fungal pathogens. T lymphocytes—thymus-derived lymphocytes that mediate adaptive cellular immune responses including T helper, T regulatory, and cytotoxic T lymphocyte effector cell functions. Tolerance—B and T cell nonresponsiveness to antigens that results from encounter with foreign or self-antigens by B and T lymphocytes in the absence of expression of antigen-presenting cell co-stimulatory molecules. Tolerance to antigens may be induced and maintained by multiple mechanisms either centrally (in the thymus for T cells or bone marrow for B cells) or peripherally at sites throughout the peripheral immune system.

1	The human immune system has evolved over millions of years from both invertebrate and vertebrate organisms to develop sophisticated defense mechanisms to protect the host from microbes and their virulence factors. The normal immune system has three key properties: a highly diverse repertoire of antigen receptors that enables recognition of a nearly infinite range of pathogens; immune memory, to mount rapid recall immune responses; and immunologic tolerance, to avoid immune damage to normal self-tissues. From invertebrates, humans have inherited the innate immune system, an ancient defense system that uses germline-encoded proteins to recognize pathogens. Cells of the innate immune system, such as macrophages, dendritic cells, and NK lymphocytes, recognize PAMPs that are highly conserved among manymicrobesanduseadiversesetofPRRmolecules.Importantcomponents of the recognition of microbes by the innate immune system include recognition by germline-encoded host molecules, recognition of

1	among manymicrobesanduseadiversesetofPRRmolecules.Importantcomponents of the recognition of microbes by the innate immune system include recognition by germline-encoded host molecules, recognition of key microbe virulence factors but not recognition of self-molecules, and nonrecognition of benign foreign molecules or microbes. Upon contact with pathogens, macrophages and NK cells may kill pathogens directlyor,inconcertwithdendriticcells,mayactivateaseriesofevents that both slow the infection and recruit the more recently evolved arm of the human immune system, the adaptive immune system.

1	Adaptive immunity is found only in vertebrates and is based on the generation of antigen receptors on T and B lymphocytes by gene rearrangements, such that individual T or B cells express unique antigen receptors on their surface capable of specifically recognizing diverse antigens of the myriad infectious agents in the environment. Coupled with finely tuned specific recognition mechanisms that maintain tolerance (nonreactivity) to self-antigens, T and B lymphocytes bring both specificity and immune memory to vertebrate host defenses. This chapter describes the cellular components, key molecules (Table 372e-1), and mechanisms thatmakeup theinnateand adaptive immune systems and describes how adaptive immunity is recruited to the defense of the host by innate immune responses. An appreciation of the cellular and molecular bases of innate and adaptive immune responses is critical to understanding the pathogenesis of inflammatory, autoimmune, infectious, and immunodeficiency diseases.

1	All multicellular organisms, including humans, have developed the use of a limited number of surface and intracellular germline-encoded molecules that recognize large groups of pathogens. Because of the myriad human pathogens, host molecules of the human innate immune system sense “danger signals” and either recognize PAMPs, the common molecular structures shared by many pathogens, or recognize host cell molecules produced in response to infection such as heat shock proteins and fragments of the extracellular matrix. PAMPs must be conserved structures vital to pathogen virulence and survival, such as bacterial endotoxin, so that pathogens cannot mutate molecules of PAMPs to evade human innate immune responses. PRRs are host proteins of the innate immune system that recognize PAMPs as hostdangersignalmolecules (Tables 372e-2 and 372e-3).Thus,recognition of pathogen molecules by hematopoietic and nonhematopoietic cell types leads to activation/production of the complement cascade,

1	hostdangersignalmolecules (Tables 372e-2 and 372e-3).Thus,recognition of pathogen molecules by hematopoietic and nonhematopoietic cell types leads to activation/production of the complement cascade, cytokines,andantimicrobial peptides aseffectormolecules.Inaddition, pathogen PAMPs as host danger signal molecules activate dendritic cells to mature and to express molecules on the dendritic cell surface that optimize antigen presentation to respond to foreign antigens.

1	Major PRR families of proteins include transmembrane proteins, such as the Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), and cytoplasmic proteins, such as the retinoic acid–inducible gene (RIG)-1-like receptors (RLRs) and NOD-like receptors (NLRs) (Table 372e-3). A major group of PRR collagenous glycoproteins with C-type lectin domains are termed collectins and include the serum protein mannose-binding lectin (MBL). MBL and other collectins, as well as two other protein families—the pentraxins (such as C-reactive protein and serum amyloid P) and macrophage scavenger receptors—all have the property of opsonizing (coating) bacteria for phagocytosis by macrophages and can also activate the complement cascade to lyse bacteria. Integrins are cell-surface adhesion molecules that affect attachment between cells and the extracellular matrix and mediate signal transduction that reflects the chemical composition of the cell environment. For example, integrins signal after cells

1	affect attachment between cells and the extracellular matrix and mediate signal transduction that reflects the chemical composition of the cell environment. For example, integrins signal after cells bind bacterial lipopolysaccharide (LPS) and activate phagocytic cells to ingest pathogens.

1	There are multiple connections between the innate and adaptive immune systems; these include (1) a plasma protein, LPS-binding protein, that binds and transfers LPS to the macrophage LPS receptor, CD14; (2) a human family of proteins called Toll-like receptor proteins (TLRs), some of which are associated with CD14, bind LPS, and signal epithelial cells, dendritic cells, and macrophages to produce cytokines andupregulatecell-surfacemolecules thatsignaltheinitiationofadaptive immune responses (Fig. 372e-1, Tables 372e-3 and 372e-4), and (3) families of intracellular microbial sensors called NLRs and RLRs. Proteins intheTollfamily can beexpressedonmacrophages,dendritic cells, and B cells as well as on a variety of nonhematopoietic cell types, including respiratory epithelial cells. Eleven TLRs have been identified in humans, and 13 TLRs have been identified in mice (Tables 372e-4 and 372e-5). Upon ligation, TLRs activate a series of intracellular events that lead to the killing of

1	TLRs have been identified in humans, and 13 TLRs have been identified in mice (Tables 372e-4 and 372e-5). Upon ligation, TLRs activate a series of intracellular events that lead to the killing of bacteria-and viral-infected cells as well as to the recruitment and ultimate activation of antigen-specific T and B lymphocytes (Fig. 372e-1). Importantly, signaling by massive

1	Introduction to the Immune System Molecular Mass, (Other Names) Family kDa Distribution Ligand(s) Function CD64 (FcγRI) Ig 45–55 Macrophages and Fc portion of IgG Mediates phagocytosis and ADCC monocytes CD80 (B7-1, BB1) Ig 60 CD86 (B7-2, B70) Ig 80 CD89 (FCαR) Ig 55–100 CD95 (APO-1, Fas) TNFR 43 Activated B and T, MP, DC Subset B, DC, EC, activated T, thymic epithelium Neutrophils, eosinophils, monocytes, and MP Activated T and B Activated T Activated CD4+ T, subset CD8+ T, NK, M, basophil B, T, TfH CD28, CD152 CD28, CD152 Fc portion of IgG Fas ligand CD80, CD86 CD40 PD-L1, PD-L2 Co-regulator of T cell activation; signaling through CD28 stimulates and through CD152 inhibits T cell activation Co-regulator of T cell activation; signaling through CD28 stimulates and through CD152 inhibits T cell activation Mediates phagocytosis and ADCC of IgA-coated pathogens Co-stimulatory for T cell activation, B cell proliferation and differentiation

1	Abbreviations: ADCC, antibody-dependent cell-mediated cytotoxicity; CTLA, cytotoxic T lymphocyte–associated protein; DC, dendritic cells; EBV, Epstein-Barr virus; EC, endothelial cells; ECM, extracellular matrix; Fcγ RIII, low-affinity IgG receptor isoform A; FDC, follicular dendritic cells; G, granulocytes; GC, germinal center; GPI, glycosyl phosphatidylinositol; HTA, human thymocyte antigen; Ig, immunoglobulin; IgG, immunoglobulin G; LCA, leukocyte common antigen; LPS, lipopolysaccharide; MHC-I, major histocompatibility complex class I; MP, macrophages; Mr, relative molecular mass; NK, natural killer cells; P, platelets; PBT, peripheral blood T cells; PD-1, programmed cell death-1; PI, phosphatidylinositol; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; PTP, protein tyrosine phosphatase; TCR, T cell receptor; TfH, T follicular helper cells; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor. For an expanded list of cluster of differentiation (CD) human

1	phosphatase; TCR, T cell receptor; TfH, T follicular helper cells; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor. For an expanded list of cluster of differentiation (CD) human antigens, see Harrison’s Online at http://www.accessmedicine.com; and for a full list of CD human antigens from the most recent Human Workshop on Leukocyte Differentiation Antigens (VII), see http://mpr.nci.nih.gov/prow/.

1	Source: Compiled from T Kishimoto et al (eds): Leukocyte Typing VI. New York: Garland Publishing, 1997; R Brines et al: Immunology Today 18S:1, 1997; and S Shaw (ed): Protein reviews on the Web. http://mpr.nci.nih.gov/prow/. Abbreviation: NK, natural killer. Toll-like receptors (TLRs), C-type lectin receptors (CLRs), retinoic acid– inducible gene (RIG)-1-like receptors (RLRs), and NOD-like receptors (NLRs) α-Defensins, β-defensins, cathelin, protegrin, granulysin, histatin, secretory leukoprotease inhibitor, and probiotics Macrophages, dendritic cells, NK cells, NK-T cells, neutrophils, eosinophils, mast cells, basophils, and epithelial cells Classic and alternative complement pathway, and proteins that bind complement components

1	Classic and alternative complement pathway, and proteins that bind complement components Autocrine, paracrine, endocrine cytokines that mediate host defense and inflammation, as well as recruit, direct, and regulate adaptive immune responses amounts of LPS through TLR4 leads to the release of large amounts of cytokines that mediate LPS-induced shock. Mutations in TLR4 proteins in mice protect from LPS shock, and TLR mutations in humans protect from LPS-induced inflammatory diseases such as LPS-induced asthma (Fig. 372e-1). Twootherfamiliesofcytoplasmic PRRsarethe NLRsand the RLRs. These families, unlike the TLRs, are composed primarily of soluble intracellular proteins that scan host cell cytoplasm for intracellular pathogens (Tables 372e-2 and 372e-3).

1	Theintracellularmicrobialsensors,NLRs,aftertriggering,formlarge cytoplasmic complexes termed inflammasomes, which are aggregates of molecules including NOD-like receptor pyrin (NLRP) proteins that are members of the NLR family (Table 372e-3). Inflammasomes activate inflammatorycaspasesandIL-1βinthepresenceofnonbacterialdanger signals (cell stress) and bacterial PAMPs. Mutations in inflammasome proteins can lead to chronic inflammation in a group of periodic febrile diseases called autoinflammatory syndromes (Table 372e-6). Cells of the innate immune system and their roles in the first line of host defense are listed in Table 372e-5. Equally important as their roles in the mediation of innate immune responses are the roles that each cell type plays in recruiting T and B lymphocytes of the adaptive immune system to engage in specific pathogen responses.

1	Monocytes-Macrophages Monocytes arise from precursor cells within bone marrow (Fig. 372e-2) and circulate with a half-life ranging from 1 to 3 days. Monocytes leave the peripheral circulation via capillaries and migration into a vast extravascular cellular pool. Tissue macrophages arise from monocytes that have migrated out of the circulation and by in situ proliferation of macrophage precursors in tissue. Common locations where tissue macrophages (and certain of their specialized forms) are found are lymph node, spleen, bone marrow, perivascular connective tissue, serous cavities such as the peritoneum, pleura, skin connective tissue, lung (alveolar macrophages), liver (Kupffer cells), bone (osteoclasts), central nervous system (microglia cells), and synovium (type A lining cells).

1	In general, monocytes-macrophages are on the first line of defense associated with innate immunity and ingest and destroy microorganisms through the release of toxic products such as hydrogen peroxide (H2O2) and nitric oxide (NO). Inflammatory mediators produced by macrophages attract additional effector cells such as neutrophils to the site of infection. Macrophage mediators include prostaglandins; leukotrienes; platelet activating factor; cytokines such as IL-1, TNF-α, IL-6, and IL-12; and chemokines (Tables 372e-7 to 372e-9).

1	Although monocytes-macrophages were originally thought to be the major antigen-presenting cells (APCs) of the immune system, it is now clear that cell types called dendritic cells are the most potent and effective APCs in the body (see below). Monocytesmacrophages mediate innate immune effector functions such as destruction of antibody-coated bacteria, tumor cells, or even normal hematopoietic cells in certain types of autoimmune cytopenias. Monocytes-macrophages ingest bacteria or are infected by viruses, and in doing so, they frequently undergo programmed cell death or apoptosis. Macrophages that are infected by intracellular infectious agents are recognized by dendritic cells as infected and apoptotic cells and are phagocytosed by dendritic cells. In this manner, dendritic cells “cross-present” infectious agent antigens of macrophages

1	Abbreviations: CLR, C-type lectin receptors; dsRNA, double-strand RNA; iE-DAP, D-glutamyl-meso-diaminopimelic acid moiety; LGP2, Laboratory of Genetics and Physiology 2 protein encoded by the gene DHX58; MDA5, melanoma differentiation-associated protein 5; MDP, MurNAc-L-Ala-D-isoGln, also known as muramyl dipeptide; MINCLE, macrophage-inducible C-type lectin; NLR, NOD-like receptor; NOD, NOTCH protein domain; RIG, retinoic acid–inducible gene; RLR, RIG-like receptors; TLR, Toll-like receptor. Source: Adapted from O Takeuchi, S Akira: Cell 140:805, 2010, with permission. Introduction to the Immune System

1	Source: Adapted from O Takeuchi, S Akira: Cell 140:805, 2010, with permission. Introduction to the Immune System FIGUrE 372e-1 Overview of major TLR signaling pathways. All TLRs signal through MyD88, with the exception of TLR3. TLR4 and the TLR2 subfamily (TLR1, TLR2, TLR6) also engage TIRAP. TLR3 signals through TRIF. TRIF is also used in conjunction with TRAM in the TLR4-MyD88independent pathway. Dashed arrows indicate translocation into the nucleus. dsRNA, double-strand RNA; IFN, interferon; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinases; NF-κB, nuclear factor-κB; ssRNA, single-strand RNA; TLR, Toll-like receptor. (Adapted from D van Duin et al: Trends Immunol 27:49, 2006, with permission.) TLRs TLR2 (heterodimer with TLR1 or 6) Env of HIV; core protein of HCV; components of Mycobacterium tuberculosis; Helicobacter pylori, Lewis Ag Muramyl dipeptide of peptidoglycan of bacteria

1	TLRs TLR2 (heterodimer with TLR1 or 6) Env of HIV; core protein of HCV; components of Mycobacterium tuberculosis; Helicobacter pylori, Lewis Ag Muramyl dipeptide of peptidoglycan of bacteria Mannosylated lipoarabinomannans from bacillus Calmette-Guérin and M. tuberculosis Low IL-12p70 TH1 High IL-10 TH2 IL-6 T regulatory IL-12p70 TH1 IFN-α IL-6 High IL-12p70 TH1 Intermediate IL-10 IL-6 High IL-12p70 TH1 Low IL-12p70 TH2 High IL-12p70 TH1 IFN-α IL-6 High IL-12p70 TH1 Low IL-10 IL-6 IFN-α ?? ?? H. pylori, Lewis Ag TH2 Suppresses IL-12p70 T regulatory Suppresses TLR signaling in DCs Induces IL-10 in DCs Weak T cell response (tolerogenic?) Suppresses IL-12 and TLR signaling in DCs Weak T cell response? (tolerogenic?)

1	Abbreviations: CpG, sequences in DNA recognized by TLR-9; DC, dendritic cell; DC-SIGN, DC-specific C-type lectin; dsRNA, double-strand RNA; HCV, hepatitis C; HIV, human immunodeficiency virus; LPS, lipopolysaccharide; MALP, macrophage-activating lipopeptide; NOD, NOTCH protein domain; ssRNA, single-strand RNA; TH1, helper T cell; TH2, helper T cell; TLR, Toll-like receptor. Source: B Pulendran: J Immunol 174:2457, 2005. Copyright 2005 The American Association of Immunologists, Inc., with permission. to T cells. Activated macrophages can also mediate antigen-nonspecific lytic activity and eliminate cell types such as tumor cells in the absence of antibody. This activity is largely mediated by cytokines (i.e., TNF-α and IL-1). Monocytes-macrophages express lineage-specific molecules (e.g., the cell-surface LPS receptor, CD14) as well as surface receptors for a number of molecules, including the Fc region of IgG, activated complement components, and various cytokines (Table 372e-7).

1	Dendritic Cells Human dendritic cells (DCs) contain several subsets, including myeloid DCs and plasmacytoid DCs. Myeloid DCs can differentiate into either macrophages-monocytes or tissue-specific DCs. In contrast to myeloid DCs, plasmacytoid DCs are inefficient APCs but are potent producers of type I interferon (IFN) (e.g., IFN-α) in response to viral infections. The maturation of DCs is regulated through cell-to-cell contact and soluble factors, and DCs attract immune effectors through secretion of chemokines. When DCs come in contact with bacterial products, viral proteins, or host proteins released as danger signals from distressed host cells (Figs. 372e-2 and 372e-3), infectious agent molecules bind to various TLRs and activate DCs to release cytokines and chemokines that drive cells of the innate immune system to become activated to respond to the invading organism, and recruit T and B cells of the adaptive immune system to respond. Plasmacytoid DCs produce antiviral IFN-α that

1	of the innate immune system to become activated to respond to the invading organism, and recruit T and B cells of the adaptive immune system to respond. Plasmacytoid DCs produce antiviral IFN-α that activates NK cell killing of pathogen-infected cells; IFN-α also activates T cells to mature into antipathogen cytotoxic (killer) T cells. Following contact with pathogens, both plasmacytoid and myeloid DCs produce chemokines that attract helper and cytotoxic T cells, B cells, polymorphonuclear cells, and naïve and memory T cells as well as regulatory T cells to ultimately dampen the immune response once the pathogen is controlled. TLR engagement on DCs upregulates MHC class II, B7-1 (CD80), and B7-2 (CD86), which enhance DC-specific antigen presentation and induce cytokine production (Table 372e-7). Thus, DCs are important bridges between early (innate) and later (adaptive) immunity. DCs also modulate and determinethetypes ofimmuneresponsesinducedbypathogensviatheTLRs

1	production (Table 372e-7). Thus, DCs are important bridges between early (innate) and later (adaptive) immunity. DCs also modulate and determinethetypes ofimmuneresponsesinducedbypathogensviatheTLRs expressedonDCs(TLR7–9onplasmacytoidDCs,TLR4onmonocytoid DCs) and via the TLR adapter proteins that are induced to associate with TLRs (Fig. 372e-1, Table 372e-4). In addition, other PRRs, such as C-type lectins, NLRs, and mannose receptors, upon ligation by pathogenproducts,activatecellsoftheadaptiveimmunesystemand,likeTLR stimulation, by a variety of factors, determine the type and quality of the adaptive immune response that is triggered (Table 372e-4).

1	Large Granular Lymphocytes/Natural Killer Cells Large granular lymphocytes (LGLs) or NK cells account for ~5–15% of peripheral blood lymphocytes.NK cells are nonadherent, nonphagocytic cells withlarge azurophilic cytoplasmic granules. NK cells express surface receptors for the Fc portion of IgG (FcR) (CD16) and for NCAM-I (CD56), and many NK cells express T lineage markers, particularly CD8, and proliferatein response toIL-2. NK cellsarise in both bonemarrow and thymic microenvironments.

1	Functionally, NK cells share features with both monocytes-macrophages and neutrophils in that they mediate both ADCC and NK cell activity.ADCCisthebindingofanopsonized(antibody-coated)target celltoanFcreceptor-bearingeffectorcellviatheFcregionofantibody, resulting in lysis of the target by the effector cell. NK cell cytotoxicity is the nonimmune (i.e., effector cell never having had previous contact with the target), MHC-unrestricted, non-antibody-mediated killing of target cells, which are usually malignant cell types, transplanted foreign cells, or virus-infected cells. Thus, NK cell cytotoxicity may play Abbreviations: IL-4, IL-5, IL-6, IL-10, and IL-12, interleukin 4, 5, 6, 10, and 12, respectively; MHC, major histocompatibility complex: LPS, lipopolysaccharide; PAMP, pathogen-associated molecular patterns; TH, helper T cell; TNF-α, tumor necrosis factor-alpha.

1	Source: Adapted from R Medzhitov, CA Janeway: Curr Opinion Immunol 9:4, 1997. Copyright 1997, with permission from Elsevier. an important role in immune surveillance and destruction of malig-to uninfected, nonmalignant self-cells; however, they are activated nant and virus-infected host cells. NK cell hyporesponsiveness is also to attack malignant and virally infected cells (Fig. 372e-4). Recent observed in patients with Chédiak-Higashi syndrome, an autosomal evidence suggests that NK cells, although not possessing rearranging recessive disease associated with fusion of cytoplasmic granules and immune recognition genes, may be able to mediate recall for NK cell defective degranulation of neutrophil lysosomes. responses to viruses and for immune responses such as contact hyper-

1	NK cells have a variety of surface receptors that have inhibitory sensitivity. or activating functions and belong to two structural families. These Some NK cells express CD3 and invariant TCR-α chains and are families include the immunoglobulin superfamily and the lectin-like termed NK T cells. TCRs of NK T cells recognize lipid molecules type II transmembrane proteins. NK immunoglobulin superfamily of intracellular bacteria when presented in the context of CD1d receptors include the killer cell immunoglobulin-like activating or molecules on APCs. Upon activation, NK T cells secrete effector inhibitory receptors (KIRs), many of which have been shown to have cytokines such as IL-4 and IFN-γ. This mode of recognition of intra-HLA class I ligands. The KIRs are made up proteins with either two cellular bacteria such as Listeria monocytogenes and Mycobacterium (KIR2D) or three (KIR3D) extracellular immunoglobulin domains tuberculosis by NK T cells leads to induction of activation of DCs

1	two cellular bacteria such as Listeria monocytogenes and Mycobacterium (KIR2D) or three (KIR3D) extracellular immunoglobulin domains tuberculosis by NK T cells leads to induction of activation of DCs (D). Moreover, their nomenclature designates their function as either and is thought to be an important innate defense mechanism against inhibitory KIRs with a long (L) cytoplasmic tail and immunoreceptor these organisms. tyrosine-based inhibitory motif (ITIM) (KIRDL) or activating KIRs The receptors for the Fc portion of IgG (FcγRs) are present on NK with a short (S) cytoplasmic tail (KIRDS). NK cell inactivation by KIRs cells, B cells, macrophages, neutrophils, and mast cells and mediate isacentralmechanismtopreventdamagetonormalhostcells.Genetic interactions of IgG with antibody-coated target cells, such as virally studies have demonstrated the association of KIRs with viral infection infected cells. Antibody-NK interaction via antibody Fc and NK cell outcome and autoimmune disease

1	target cells, such as virally studies have demonstrated the association of KIRs with viral infection infected cells. Antibody-NK interaction via antibody Fc and NK cell outcome and autoimmune disease (Table 372e-10). FcR links the adaptive and innate immune systems and regulates the

1	In addition to the KIRs, a second set of immunoglobulin superfam-mediation of IgG antibody effector functions such as ADCC. There ily receptors includes the natural cytotoxicity receptors (NCRs), which are both activation and inhibitory FcγRs. Activation FcRs, such as include NKp46, NKp30, and NKp44. These receptors help to mediate FcγRI (CD64), FcγRII (CD32), and FcγRII (CD64), are character-NK cell activation against target cells. The ligands to which NCRs bind ized by the presence of an immunoreceptor tyrosine-based activating on target cells have been recently recognized to be comprised of mol-motif (ITAM) sequence, whereas inhibitory FcRs, such as FcγRIIb, ecules of pathogens such as influenza, vaccinia, and malaria as well as contain an immunoreceptor tyrosine-based inhibitory motif (ITIM) host molecules expressed on tumor cells. sequence. There is evidence that dysregulation in IgG-FcγR interac-

1	NK cell signaling is, therefore, a highly coordinated series of inhib-tions plays a role in arthritis, multiple sclerosis, and systemic lupus iting and activating signals that prevent NK cells from responding erythematosus. Introduction to the Immune System

1	Introduction to the Immune System Neutrophils, Eosinophils, and Basophils Granulocytes are present in nearly all forms of inflammation and are amplifiers and effectors of innate immune responses (Figs. 372e-2 and 372e-3). Unchecked accumulation and activation of granulocytes can lead to host tissue damage, as seen in neutrophil-and eosinophil-mediated systemic necrotizing vasculitis. Granulocytes are derived from stem cells in bone marrow. Each type of granulocyte (neutrophil, eosinophil, or basophil) is derived from a different subclass of progenitor cell that is stimulated to proliferate by colony-stimulating factors (Table 372e-7). During terminal maturation of granulocytes, class-specific nuclear morphology and cytoplasmic granules appear that allow for histologic identification of granulocyte type.

1	Neutrophils express Fc receptor IIIa for IgG (CD16) as well as receptors for activated complement components (C3b or CD35). Upon interaction of neutrophils with antibody-coated (opsonized) bacteria or immune complexes, azurophilic granules (containing myeloperoxidase, lysozyme, elastase, and other enzymes) and specific granules (containing lactoferrin, lysozyme, collagenase, and other enzymes) are released, and microbicidal superoxide radicals (O2 -) are generatedattheneutrophilsurface.Thegenerationofsuperoxideleads to inflammation by direct injury to tissue and by alteration of macromolecules such as collagen and DNA.

1	Eosinophils express Fc receptor II for IgG (CD32) and are potent cytotoxic effector cells for various parasitic organisms. In Nippostrongylus brasiliensis helminth infection, eosinophils are important cytotoxic effector cells for removal of these parasites. Key to regulation of eosinophil cytotoxicitytoN. brasiliensis wormsareantigen-specificThelpercellsthat produce IL-4, thus providing an example of regulation of innate immune responsesbyadaptiveimmunityantigen-specificTcells.Intracytoplasmic contents of eosinophils, such as major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin, are capable of directly damaging tissues and may be responsible in part for the organ system dysfunction in the hypereosinophilic syndromes (Chap. 80). Because the eosinophil granule contains anti-inflammatory types of enzymes (histaminase, arylsulfatase, phospholipase D), eosinophils may homeostatically downregulate or terminate ongoing inflammatory responses.

1	Basophils and tissue mast cells are potent reservoirs of cytokines suchasIL-4andcanrespondtobacteriaandviruseswithantipathogen cytokine production through multiple TLRs expressed on their surface. Mast cells and basophils can also mediate immunity through the binding of antipathogen antibodies. This is a particularly important host defense mechanism against parasitic diseases. Basophils express high-affinity surface receptors for IgE (FcεRII) (CD23) and, upon cross-linking of basophil-bound IgE by antigen, can release histamine, eosinophil chemotactic factor of anaphylaxis, and neutral protease— all mediators of allergic immediate (anaphylaxis) hypersensitivity responses (Table 372e-11). In addition, basophils express surface receptors for activated complement components (C3a, C5a), through IL-12 antigen presentation Myeloid dendritic cell IL-1,IL-6 phagocytosis of microbes

1	IL-12 antigen presentation Myeloid dendritic cell IL-1,IL-6 phagocytosis of microbes CD8+ cytotoxic T cell CD4+, CD8+ regulatory cells TH1 TH2 TH0 TR IL-12 IL-4 IFN-°intracellular microbes IL-4,IL-5 extracellular microbes Phagocytosis of microbes; secretion of inflammatory products Neutrophilic granulocyte FIGUrE 372e-2 Schematic model of intercellular interactions of adaptive immune system cells.

1	In this figure, the arrows denote that cells develop from precursor cells or produce cytokines or antibodies; lines ending with bars indicate suppressive intercellular interactions. Stem cells differentiate into either T cells, antigen-presenting dendritic cells, natural killer cells, macrophages, granulocytes, or B cells. Foreign antigen is processed by dendritic cells, and peptide fragments of foreign antigen are presented to CD4+ and/or CD8+ T cells. CD8+ T cell activation leads to induction of cytotoxic T lymphocyte (CTL) or killer T cell generation, as well as induction of cytokine-producing CD8+ cytotoxic T cells. For antibody production against the same antigen, active antigen is bound to sIg within the B cell receptor complex and drives B cell maturation into plasma cells that secrete Ig. TH1 or TH2 CD4+ T cells producing interleukin (IL) 4, IL-5, or interferon (IFN) γ regulate the Ig class switching and determine the type of antibody produced. TH17 cells secrete IL-17, IL-22,

1	Ig. TH1 or TH2 CD4+ T cells producing interleukin (IL) 4, IL-5, or interferon (IFN) γ regulate the Ig class switching and determine the type of antibody produced. TH17 cells secrete IL-17, IL-22, IL-26, which contribute to host defense against extracellular bacteria and fungi, particularly at mucosal surfaces. CD4+, CD25+ T regulatory cells produce IL-10 and downregulate T and B cell responses once the microbe has been eliminated. GM-CSF, granulocyte-macrophage colony-stimulating factor; TNF, tumor necrosis factor.

1	surveillance of HLA class I– negative cells (malignant and virus-infected cells) Introduction to the Immune System which mediator release can be directly affected. Thus, basophils, like most cells of the immune system, can be activated in the service of host defense against pathogens, or they can be activated for mediation release and cause pathogenic responses in allergic and inflammatory diseases. For further discussion of tissue mast cells, see Chap. 376.

1	The Complement System Thecomplementsystem,animportantsoluble componentoftheinnateimmunesystem,isaseriesofplasmaenzymes, regulatory proteins, and proteins that are activated in a cascading fashion, resulting in cell lysis. There are four pathways of the complement system: the classic activation pathway activated by antigen/antibody immune complexes, the mannose-binding lectin (MBL) (a serum collectin; Table 372e-3) activation pathway activated by microbes with terminal mannose groups, the alternative activation pathway activated by microbes or tumor cells, and the terminal pathway that is common to the first three pathways and leads to the membrane attack complex that lyses cells (Fig. 372e-5). The series of enzymes of the complement system are serine proteases.

1	Activation of the classic complement pathway via immune complex binding to C1q links the innate and adaptive immune systems via specific antibodyintheimmunecomplex.The alternative complement activation pathway is antibody-independent and is activated by binding of C3 directly to pathogens and “altered self” such as tumor cells. In the renal glomerular inflammatory disease IgA nephropathy, IgA activates the alternative complement pathway and causes glomerular damage and decreased renal function. Activation of the classic complement pathway via C1, C4, and C2 and activation of the alternative pathway via factor D, C3, and factor B both lead to cleavage and activation of C3. C3 activation fragments, when bound to target surfaces such as bacteria and other foreign antigens, are critical for opsonization (coating by antibody and complement) in preparation for phagocytosis. The MBL pathway substitutes MBL-associated serine proteases (MASPs) 1 and 2 for C1q, C1r, and C1s to activate C4. The

1	(coating by antibody and complement) in preparation for phagocytosis. The MBL pathway substitutes MBL-associated serine proteases (MASPs) 1 and 2 for C1q, C1r, and C1s to activate C4. The MBL activation pathway is activated by mannose on the surface of bacteria and viruses.

1	The three pathways of complement activation all converge on the final common terminal pathway. C3 cleavage by each pathway results in activation of C5, C6, C7, C8, and C9, resulting in the membrane attack complex that physically inserts into the membranes of target cells or bacteria and lyses them. Thus, complement activation is a critical component of innate immunity for responding to microbial infection. The functional consequences of complement activation by the three initiating pathways and the terminal pathway are shown in Fig. 372e-5. In general the cleavage products of complement components facilitate microbe or damaged cell clearance (C1q, C4, C3), promote activation and enhancement of inflammation (anaphylatoxins, C3a, C5a), and promote microbe or opsonized cell lysis (membrane attack complex).

1	Cytokinesare soluble proteinsproducedbya wide varietyof cell types (Tables 372e-7 to 372e-9). They are critical for both normal innate and adaptive immune responses, and their expression may be perturbed in most immune, inflammatory, and infectious disease states. IL-1α, β Type I IL-1r, Type II IL-1r IL-2r α, β, common γ IL-3r, common β IL-4r α, common γ IL-5r α, common γ IL-6r, gp130 IL-7r α, common γ CXCR1, CXCR2 IL-9r α, common γ IL-10r IL-11r α, gp130 IL-15r α, common γ, IL2r β CD4 Monocytes/macrophages, B cells, fibroblasts, most epithelial cells including thymic epithelium, endothelial cells T cells, NK cells, mast cells T cells, mast cells, basophils T cells, mast cells, eosinophils Monocytes-macrophages, B cells, fibroblasts, most epithelium including thymic epithelium, endothelial cells Bone marrow, thymic epithelial cells Monocytes-macrophages, T cells, neutrophils, fibroblasts, endothelial cells, epithelial cells

1	Bone marrow, thymic epithelial cells Monocytes-macrophages, T cells, neutrophils, fibroblasts, endothelial cells, epithelial cells Monocytes-macrophages, T cells, B cells, keratinocytes, mast cells Activated macrophages, dendritic cells, neutrophils Monocytes-macrophages, epithelial cells, fibroblasts Mast cells, eosinophils, CD8+ T cells, respiratory epithelium Keratinocytes, macrophages DC, T cells Macrophages, other cell types All cells T cells, B cells, NK cells, monocytes-macrophages Monocytes-macrophages, mast cells, eosinophils, bone marrow progenitors T cells, B cells, NK cells, monocytes-macrophages, neutrophils, eosinophils, endothelial cells, fibroblasts Eosinophils, basophils, murine B cells T cells, B cells, epithelial cells, hepatocytes, monocytesmacrophages T cells, B cells, bone marrow cells Neutrophils, T cells, monocytes-macrophages, endothelial cells, basophils Bone marrow progenitors, B cells, T cells, mast cells

1	T cells, B cells, bone marrow cells Neutrophils, T cells, monocytes-macrophages, endothelial cells, basophils Bone marrow progenitors, B cells, T cells, mast cells Monocytes-macrophages, T cells, B cells, NK cells, mast cells Megakaryocytes, B cells, hepatocytes T cells, NK cells Monocytes-macrophages, B cells, endothelial cells, keratinocytes T cells, NK cells CD4+ T cells, monocytesmacrophages, eosinophils Fibroblasts, endothelium, epithelium, macrophages T cells, B cells, NK cells T cells Upregulates adhesion molecule expression, neutrophil and macrophage emigration, mimics shock, fever, upregulates hepatic acute-phase protein production, facilitates hematopoiesis Promotes T cell activation and proliferation, B cell growth, NK cell proliferation and activation, enhanced monocyte/macrophage cytolytic activity

1	Promotes T cell activation and proliferation, B cell growth, NK cell proliferation and activation, enhanced monocyte/macrophage cytolytic activity Stimulates TH2 helper T cell differentiation and proliferation; stimulates B cell Ig class switch to IgG1 and IgE anti-inflammatory action on T cells, monocytes; produced by T follicular helper cells in B cell germinal centers that stimulate B cell maturation. Induces acute-phase protein production, T and B cell differentiation and growth, myeloma cell growth, and osteoclast growth and activation Differentiates B, T, and NK cell precursors, activates T and NK cells Induces neutrophil, monocyte, and T cell migration, induces neutrophil adherence to endothelial cells and histamine release from basophils, and stimulates angiogenesis; suppresses proliferation of hepatic precursors Induces mast cell proliferation and function, synergizes with IL-4 in IgG and IgE production and T cell growth, activation, and differentiation

1	Induces mast cell proliferation and function, synergizes with IL-4 in IgG and IgE production and T cell growth, activation, and differentiation Inhibits macrophage proinflammatory cytokine production, downregulates cytokine class II antigen and B7-1 and B7-2 expression, inhibits differentiation of TH1 helper T cells, inhibits NK cell function, stimulates mast cell proliferation and function, B cell activation, and differentiation Induces megakaryocyte colony formation and maturation, enhances antibody responses, stimulates acute-phase protein production Induces TH1 T helper cell formation and lymphokine-activated killer cell formation; increases CD8+ CTL cytolytic activity; ↓IL-17, ↑IFN-γ Upregulates VCAM-1 and C-C chemokine expression on endothelial cells and B cell activation and differentiation, and inhibits macrophage proinflammatory cytokine production Induces B cell proliferation, inhibits antibody secretion, and expands selected B cell subgroups

1	Induces B cell proliferation, inhibits antibody secretion, and expands selected B cell subgroups Promotes T cell activation and proliferation, angiogenesis, and NK cells Promotes chemoattraction of CD4+ T cells, monocytes, and eosinophils; inhibits HIV replication; inhibits T cell activation through CD3/T cell receptor Upregulates IFN-γ production, enhances NK cell cytotoxicity Downregulates NK cell–activating molecules, NKG2D/DAP10; produced by T follicular helper cells in B cell germinal centers that stimulate B cell maturation. Opposite effects of IL-12 (↑IL-17, ↑IFN-γ) TNFrI, TNFrII TNFrI, TNFrII LTβR G-CSFr; gp130 GM-CSFr, common β Type I, II, III TGF-β receptor CCR1, CCR2 Macrophages, CD4 T cells, mast cells TH1, TH17 T cells, synovial cells T cells, NK cells Monocytes-macrophages, mast cells, basophils, eosinophils, NK cells, B cells, T cells, keratinocytes, fibroblasts, thymic epithelial cells T cells, B cells

1	T cells, NK cells Monocytes-macrophages, mast cells, basophils, eosinophils, NK cells, B cells, T cells, keratinocytes, fibroblasts, thymic epithelial cells T cells, B cells Monocytes-macrophages, fibroblasts, endothelial cells, thymic epithelial cells, stromal cells T cells, monocytes-macrophages, fibroblasts, endothelial cells, thymic epithelial cells Fibroblasts, endothelial cells, monocytes-macrophages, T cells, B cells, epithelial cells including thymic epithelium Activated T cells, bone marrow stromal cells, thymic epithelium Activated monocytesmacrophages and T cells, bone marrow stromal cells, some breast carcinoma cell lines, myeloma cells NK cells, mast cells, double-negative thymocytes, activated CD8+ T cells Fibroblasts, smooth-muscle cells, activated PBMCs Fibroblasts, activated PBMCs Nonhematopoietic cells such as fibroblasts Fibroblasts, endothelium, epithelium, macrophages Myeloid cells, endothelial cells

1	Fibroblasts, activated PBMCs Nonhematopoietic cells such as fibroblasts Fibroblasts, endothelium, epithelium, macrophages Myeloid cells, endothelial cells Monocytes-macrophages, neutrophils, eosinophils, fibroblasts, endothelial cells Megakaryocytes, monocytes, hepatocytes, possibly lymphocyte subpopulations Neurons, hepatocytes, monocytes-macrophages, adipocytes, alveolar epithelial cells, embryonic stem cells, melanocytes, endothelial cells, fibroblasts, myeloma cells Embryonic stem cells, myeloid and lymphoid precursors, mast cells T cells, NK cells Monocytes-macrophages, NK cells, memory T cells, basophils Monocytes-macrophages, T cells, eosinophils, basophils, NK cells Promotes wound healing Promotes antiviral activity; stimulates T cell, macrophage, and NK cell activity; direct antitumor effects; upregulates MHC class I antigen expression; used therapeutically in viral and autoimmune conditions

1	Antiviral activity; stimulates T cell, macrophage, and NK cell activity; direct antitumor effects; upregulates MHC class I antigen expression; used therapeutically in viral and autoimmune conditions Regulates macrophage and NK cell activations; stimulates immunoglobulin secretion by B cells; induction of class II histocompatibility antigens; TH1 T cell differentiation Fever, anorexia, shock, capillary leak syndrome, enhanced leukocyte cytotoxicity, enhanced NK cell function, acute phase protein synthesis, proinflammatory cytokine induction Cell cytotoxicity, lymph node and spleen development Cell cytotoxicity, normal lymph node development Regulates myelopoiesis; enhances survival and function of neutrophils; clinical use in reversing neutropenia after cytotoxic chemotherapy

1	Regulates myelopoiesis; enhances survival and function of neutrophils; clinical use in reversing neutropenia after cytotoxic chemotherapy Regulates myelopoiesis; enhances macrophage bactericidal and tumoricidal activity; mediator of dendritic cell maturation and function; upregulates NK cell function; clinical use in reversing neutropenia after cytotoxic chemotherapy Induces hepatic acute-phase protein production; stimulates macrophage differentiation; promotes growth of myeloma cells and hematopoietic progenitors; stimulates thrombopoiesis Induces hepatic acute-phase protein production; stimulates macrophage differentiation; promotes growth of myeloma cells and hematopoietic progenitors; stimulates thrombopoiesis; stimulates growth of Kaposi’s sarcoma cells Stimulates hematopoietic progenitor cell growth, mast cell growth; promotes embryonic stem cell migration

1	Stimulates hematopoietic progenitor cell growth, mast cell growth; promotes embryonic stem cell migration Downregulates T cell, macrophage, and granulocyte responses; stimulates synthesis of matrix proteins; stimulates angiogenesis Chemoattractant for lymphocytes; only known chemokine of C class Chemoattractant for monocytes, activated memory T cells, and NK cells; induces granule release from CD8+ T cells and NK cells; potent histamine-releasing factor for basophils; suppresses proliferation of hematopoietic precursors; regulates monocyte protease production Chemoattractant for monocytes, memory and naïve T cells, eosinophils, ?NK cells; activates basophils and eosinophils; regulates monocyte protease production Introduction to the Immune System MIG SDF-1 CCR1, CCR2 CCR2, CCR3 CCR3 CCR4 CCR4 CCR1, CCR5 CCR5 CCR1, CCR2, CCR5 CXCR3 CXCR4 Fibroblasts, activated PBMCs Lung, colon, small intestinal epithelial cells, activated endothelial cells

1	CCR2, CCR3 CCR3 CCR4 CCR4 CCR1, CCR5 CCR5 CCR1, CCR2, CCR5 CXCR3 CXCR4 Fibroblasts, activated PBMCs Lung, colon, small intestinal epithelial cells, activated endothelial cells Pulmonary epithelial cells, heart Thymus, dendritic cells, activated T cells Monocytes-macrophages, dendritic cells, thymus Monocytes-macrophages, T cells Monocytes-macrophages, T cells Monocytes-macrophages, T cells, fibroblasts, eosinophils Dendritic cells, fetal liver cells, activated T cells Thymus, lymph node, appendix Thymic epithelial cells, lymph node, appendix, and spleen Dendritic cells, thymus, liver, small intestine Activated granulocytes, monocyte-macrophages, and epithelial cells Monocytes-macrophages, T cells, fibroblasts, endothelial cells, epithelial cells Monocytes-macrophages, T cells, fibroblasts Monocytes-macrophages, T cells, eosinophils, basophils, NK cells, dendritic cells Monocytes-macrophages, T cells, eosinophils, basophils Eosinophils, basophils T cells, NK cells

1	Monocytes-macrophages, T cells, eosinophils, basophils, NK cells, dendritic cells Monocytes-macrophages, T cells, eosinophils, basophils Eosinophils, basophils T cells, NK cells Monocytes-macrophages, T cells, dendritic cells, NK cells, eosinophils, basophils Monocytes-macrophages, T cells, NK cells, dendritic cells Monocytes-macrophages, T cells, NK cells, dendritic cells, eosinophils, basophils T cells, B cells Monocytes-macrophages, T cells T cells, monocytesmacrophages, dendritic cells Neutrophils, epithelial cells, ?endothelial cells Neutrophils and ?endothelial cells Neutrophils, basophils Activated T cells, tumor-infiltrating lymphocytes, ?endothelial cells, ?NK cells Activated T cells, tumor-infiltrating lymphocytes T cells, dendritic cells, ?basophils, ?endothelial cells Chemoattractant for monocytes, memory and naïve T cells, dendritic cells, eosinophils, ?NK cells; activates basophils and eosinophils; regulates monocyte protease production

1	Chemoattractant for monocytes, memory and naïve T cells, dendritic cells, eosinophils, ?NK cells; activates basophils and eosinophils; regulates monocyte protease production Chemoattractant for monocytes, T cells, eosinophils, and basophils Potent chemoattractant for eosinophils and basophils; induces allergic airways disease; acts in concert with IL-5 to activate eosinophils; antibodies to eotaxin inhibit airway inflammation Chemoattractant for activated T cells; inhibits infection with T cell tropic HIV Chemoattractant for monocytes, T cells, dendritic cells, and NK cells, and weak chemoattractant for eosinophils and basophils; activates NK cell function; suppresses proliferation of hematopoietic precursors; necessary for myocarditis associated with coxsackievirus infection; inhibits infection with monocytotropic HIV Chemoattractant for monocytes, T cells, and NK cells; activates NK cell function; inhibits infection with monocytotropic HIV

1	Chemoattractant for monocytes, T cells, and NK cells; activates NK cell function; inhibits infection with monocytotropic HIV Chemoattractant for monocytes-macrophages, CD4+, CD45Ro+ T cells, CD8+ T cells, NK cells, eosinophils, and basophils; induces histamine release from basophils; inhibits infections with monocytotropic HIV May have a role in induction of immune responses Thymic dendritic cell-derived cytokine, possibly involved in T cell development Neutrophil chemoattractant and activator; mitogenic for some melanoma cell lines; suppresses proliferation of hematopoietic precursors; angiogenic activity IFN-γ-inducible protein that is a chemoattractant for T cells; suppresses proliferation of hematopoietic precursors IFN-γ-inducible protein that is a chemoattractant for T cells; suppresses proliferation of hematopoietic precursors

1	IFN-γ-inducible protein that is a chemoattractant for T cells; suppresses proliferation of hematopoietic precursors Low-potency, high-efficacy T cell chemoattractant; required for B-lymphocyte development; prevents infection of CD4+, CXCR4+ cells by T cell tropic HIV

1	Abbreviations: B7-1, CD80; B7-2, CD86; CCR, CC-type chemokine receptor; CXCR, CXC-type chemokine receptor; DC-CK, dendritic cell chemokine; EBV, Epstein-Barr virus; ELC, EB11 ligand chemokine (MIP-1b); G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; GRP, growth-related peptide; HSV, herpes simplex virus; IFN, interferon; Ig, immunoglobulin; IL, interleukin; IP-10, IFN-γ-inducible protein-10; LARC, liverand activation-regulated chemokine; LIF, leukemia inhibitory factor; MCP, monocyte chemotactic protein; M-CSF, macrophage colony-stimulating factor; MDC, macrophage-derived chemokine; MGSA, melanoma growth-stimulating activity; MHC, major histocompatibility complex; MIG, monokine induced by IFN-γ; MIP, macrophage inflammatory protein; NAP, neutrophil-activating protein; NK, natural killer; OSM, oncostatin M; PARC, pulmonaryand activation-regulated chemokine; PBMC, peripheral blood mononuclear cells; PF, platelet factor; RANTES,

1	NAP, neutrophil-activating protein; NK, natural killer; OSM, oncostatin M; PARC, pulmonaryand activation-regulated chemokine; PBMC, peripheral blood mononuclear cells; PF, platelet factor; RANTES, regulated on activation, normally T cell–expressed and –secreted; SCF, stem cell factor; SDF, stromal cell–derived factor; SLC, secondary lymphoid tissue chemokine; TARC, thymusand activation-regulated chemokine; TCA, T cell activation protein; TECK, thymus expressed chemokine; TGF, transforming growth factor; TH1 and TH2, helper T cell subsets; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule.

1	Source: Data from JS Sundy et al: Appendix B, in Inflammation, Basic Principles and Clinical Correlates, 3rd ed, J Gallin and R Snyderman (eds). Philadelphia, Lippincott Williams and Wilkins, 1999. taBle 372e-8 cc, cXc1, cX3, c1, and Xc famIlIeS of chemokIneS and chemokIne receptorS CXCR3-A CXCL9 (MIG), CXCL10 (IP-10), CXCL11 Type 1 helper cells, mast cells, mesangial Inflammatory skin disease, multiple sclerosis, transplant (I-TAC) cells rejection

1	Abbreviations: BCA-1, B-cell chemoattractant 1; COPD, chronic obstructive pulmonary disease; CTACK, cutaneous T cell–attracting chemokine; ELC, Epstein-Barr I1-ligand chemokine; ENA, epithelial cell–derived neutrophil-activating peptide; GCP, granulocyte chemotactic protein; GRO, growth-regulated oncogene; HCC, hemofiltrate chemokine; IP-10, interferon inducible 10; I-TAC, interferon-inducible T cell alpha chemoattractant; LARC, liverand activation-regulated chemokine; MCP, monocyte chemoattractant protein; MDC, macrophage-derived chemokine; MEC, mammary-enriched chemokine; MIG, monokine induced by interferon-γ; MIP, macrophage inflammatory protein; PF, platelet factor; SDF, stromal cell–derived factor; SLC, secondary lymphoid-tissue chemokine; SR-PSOX, scavenger receptor for phosphatidylserine-containing oxidized lipids; TARC, thymusand activation-regulated chemokine; TECK, thymus-expressed chemokine; TH2, type 2 helper T cells.

1	Source: From IF Charo, RM Ranshohoff: N Engl J Med 354:610, 2006, with permission. Copyright Massachusetts Medical Society. All rights reserved. Introduction to the Immune System Hematopoietins IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15, IL-16, IL-17, IL-21, IL-23, EPO, LIF, GM-CSF, G-CSF, OSM, CNTF, GH, and TPO TNF-α, LT-α, LT-β, CD40L, CD30L, CD27L, 4-1BBL, OX40, OPG, and FasL IL-1α, IL-1β, IL-1ra, IL-18, bFGF, aFGF, and ECGF PDGF PDGF A, PDGF B, and M-CSF TGF-β TGF-β and BMPs (1, 2, 4, etc.) C-X-C chemokines IL-8, Gro-α/β/γ, NAP-2, ENA78, GCP-2, PF4, CTAP-3, MIG, and IP-10 C-C chemokines MCP-1, MCP-2, MCP-3, MIP-1α, MIP-1β, RANTES

1	Abbreviations: aFGF, acidic fibroblast growth factor; 4-1BBL, 401 BB ligand; bFGF, basic fibroblast growth factor; BMP, bone marrow morphogenetic proteins; C-C, cysteinecysteine; CD, cluster of differentiation; CNTF, ciliary neurotrophic factor; CTAP, connective tissue–activating peptide; C-X-C, cysteine-x-cysteine; ECGF, endothelial cell growth factor; EPO, erythropoietin; FasL, Fas ligand; GCP-2, granulocyte chemotactic protein 2; G-CSF, granulocyte colony-stimulating factor; GH, growth hormone; GM-CSF, granulocytemacrophage colony-stimulating factor; Gro, growth-related gene products; IFN, interferon; IL, interleukin; IP, interferon-γ inducible protein; LIF, leukemia inhibitory factor; LT, lymphotoxin; MCP, monocyte chemoattractant; M-CSF, macrophage colony-stimulating factor; MIG, monokine induced by interferon-γ; MIP, macrophage inflammatory protein; NAP-2, neutrophil activating protein 2; OPG, osteoprotegerin; OSM, oncostatin M; PDGF, platelet-derived growth factor; PF, platelet

1	induced by interferon-γ; MIP, macrophage inflammatory protein; NAP-2, neutrophil activating protein 2; OPG, osteoprotegerin; OSM, oncostatin M; PDGF, platelet-derived growth factor; PF, platelet factor; R, receptor; RANTES, regulated on activation, normal T cell–expressed and –secreted; TGF, transforming growth factor; TNF, tumor necrosis factor; TPO, thyroperoxidase.

1	Cytokines are involved in the regulation of the growth, development, and activation of immune system cells and in the mediation of the inflammatory response. In general, cytokines are characterized by considerable redundancy; different cytokines have similar functions. In addition, many cytokines are pleiotropic in that they are capable of acting on many different cell types. This pleiotropism results from the expression on multiple cell types of receptors for the same cytokine (see below),leadingtothe formation of“cytokine networks.”The action of cytokines may be (1) autocrine when the target cell is the same cell that secretes the cytokine, (2) paracrine when the target cell is nearby, and (3) endocrine when the cytokine is secreted into the circulation and acts distal to the source.

1	Cytokines have been named based on presumed targets or based on presumed functions. Those cytokines that are thought to primarily target leukocytes have been named interleukins (IL-1, -2, -3, etc.). Many cytokines that were originally described as having a certain function have retained those names (e.g., granulocyte colony-stimulating factor [G-CSF]). Cytokines belong in general to three major structural families: the hematopoietin family; the TNF, IL-1, platelet-derived growth factor (PDGF), and transforming growth factor (TGF) β families; and the CXC and C-C chemokine families (Table 372e-9). Chemokines are cytokines that regulate cell movement and trafficking; they act through G protein-coupled receptors and have a distinctive three-dimensional structure. IL-8istheonlychemokinethatearlyonwasnamedanIL(Table372e-7). Activation of TH1 CD4+ T cells Activation of TH2 CD4+ T cells IL-2, IFN-˜, IL-3

1	Activation of TH1 CD4+ T cells Activation of TH2 CD4+ T cells IL-2, IFN-˜, IL-3 IL-3, IL-4, IL-5, IL-6, TNF-°, TNF-˛, GM-CSF IL-10, IL-13 Inhibition Inhibition of TH2 of TH1 responses type responses Eosinophil Mast cell B cell IgM, Induce CD8+ B cell IgG Macrophage basophil G, A, and E cytotoxic antibody activation Kill parasites Regulation Direct antibody killing Kill microbe Opsonize Kill opsonized of vascular of microbes and infected cells microbes for microbes permeability; opsonize for microbial phagocytosis allergic responses; phagocytosis protective responses to bacteria, viruses, and parasitic infections

1	FIGUrE 372e-3 CD4+ helper T1 (TH1) cells and TH2 T cells secrete distinct but overlapping sets of cytokines. TH1 CD4+ cells are frequently activated in immune and inflammatory reactions against intracellular bacteria or viruses, whereas TH2 CD4+ cells are frequently activated for certain types of antibody production against parasites and extracellular encapsulated bacteria; they are also activated in allergic diseases. GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor. (Adapted from S Romagnani: CD4 effector cells, in Inflammation: Basic Principles and Clinical Correlates, 3rd ed, J Gallin, R Snyderman [eds]. Philadelphia, Lippincott Williams & Wilkins, 1999, p 177; with permission.) Introduction to the Immune System Spondylarthritides Increased KIR3DL2 expression May contribute to disease pathology

1	Introduction to the Immune System Spondylarthritides Increased KIR3DL2 expression May contribute to disease pathology Interaction HLA-B27 homodi-May contribute to mers with KIR3DL1/KIR3DL2; disease pathogenesis independent of peptide patients with extraarticular may have different with respect to KIR Behçet’s disease Altered KIR3DL1 expression Associated with severe eye disease Type 1 diabetes KIR2DS2; HLA-C1 and no Increased disease HLA-C2, no HLA-Bw4 progression Preeclampsia KIR2DL1 with fewer KIR2DS Increased disease (mother); HLA-C2 (fetus) progression Abbreviations: HCV, hepatitis C virus; HLA, human leukocyte antigen; HPV, human papillomavirus; IDDM, insulin-dependent diabetes mellitus; KIR, killer cell immunoglobulin-like receptor. Source: Adapted from R Diaz-Pena et al: Adv Exp Med Biol 649:286, 2009.

1	Source: Adapted from R Diaz-Pena et al: Adv Exp Med Biol 649:286, 2009. In general, cytokines exert their effects by influencing gene activation that results in cellular activation, growth, differentiation, functional cell-surface molecule expression, and cellular effector function. In this regard, cytokines can have dramatic effects on the regulation of immune responses and the pathogenesis of a variety of diseases. Indeed, T cells have been categorized on the basis of the pattern of cytokines that they secrete, which results in either humoral immune response (TH2) or cell-mediated immune response (TH1). A third type of T helper cell is the TH17 cell that contributes to host defense against extracellular bacteria and fungi, particularly at mucosal sites (Fig. 372e-2).

1	Cytokine receptors can be grouped into five general families based on similarities in their extracellular amino acid sequences and conserved structural domains. The immunoglobulin (Ig) superfamily represents a large number of cell-surface and secreted proteins. The IL-1 receptors (type 1, type 2) are examples of cytokine receptors with extracellular Ig domains. The hallmark of the hematopoietic growth factor (type 1) receptor family is that the extracellular regions of each receptor contain two conserved motifs. One motif, located at the N terminus, is rich in cysteine residues. The other motif is located at the C terminus proximal to the transmembrane region and comprises five amino acid residues, Outcome determined by balance of signals

1	Outcome determined by balance of signals FIGUrE 372e-4 Encounters between NK cells: potential targets and possible outcomes. The amount of activating and inhibitory receptors on the NK cells and the amount of ligands on the target cell, as well as the qualitative differences in the signals transduced, determine the extent of the NK response. A. When target cells have no HLA class I or activating ligands, NK cells cannot kill target cells.

1	B. When target cells bear self-HLA, NK cells cannot kill targets. C. When target cells are pathogen infected and have downregulated HLA and express activating ligands, NK cells kill target cells. D. When NK cells encounter targets with both self-HLA and activating receptors, then the level of target killing is determined by the balance of inhibitory and activating signals to the NK cell. HLA, human leukocyte antigen; NK, natural killer. (Adapted from L Lanier: Annu Rev Immunol 23:225, 2005; reproduced with permission from Annual Reviews Inc. Copyright 2011 by Annual Reviews Inc.) Slow reacting substance of anaphylaxis (SRSA) (leukotriene C4, D4, E4) Eosinophil chemotactic factor of anaphylaxis (ECF-A) Neutrophil chemotactic factor (NCF) Leukotactic activity (leukotriene B4) Heparin Basophil kallikrein of anaphylaxis Smooth-muscle contraction, increased vascular permeability Chemotactic attraction of eosinophils

1	Smooth-muscle contraction, increased vascular permeability Chemotactic attraction of eosinophils Activates platelets to secrete serotonin and other mediators: smooth-muscle contraction; induces vascular permeability Chemotactic attraction of neutrophils Chemotactic attraction of neutrophils Anticoagulant Cleaves kininogen to form bradykinin Clearance of

1	FIGUrE 372e-5 The four pathways and the effector mechanisms of the complement system. Dashed arrows indicate the functions of pathway components. (After BJ Morley, MJ Walport: The Complement Facts Books. London, Academic Press, 2000, Chap. 2; with permission. Copyright Academic Press, London, 2000.) tryptophan-serine-X-tryptophan-serine (WSXWS). This family can be grouped on the basis of the number of receptor subunits they have and on the utilization of shared subunits. A number of cytokine receptors, i.e., IL-6, IL-11, IL-12, and leukemia inhibitory factor, are paired with gp130. There is also a common 150-kDa subunit shared by IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors. The gamma chain (γc) of the IL-2 receptor is common to the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors. Thus, the specific cytokine receptor is responsible for ligand-specific binding, whereas the subunits such as gp130, the 150-kDa subunit, and γcare important in signal

1	IL-9, and IL-15 receptors. Thus, the specific cytokine receptor is responsible for ligand-specific binding, whereas the subunits such as gp130, the 150-kDa subunit, and γcare important in signal transduction. The γcgene is on the X chromosome, and mutations in the γ cprotein result in the X-linked form of severe combined immune deficiency syndrome (X-SCID) (Chap. 374).

1	The members of the interferon (type II) receptor family include the receptors for IFN-γ and -β, which share a similar 210-amino-acid binding domain with conserved cysteine pairs at both the amino and carboxy termini. The members of the TNF (type III) receptor family share a common binding domain composed of repeated cysteine-rich regions. Members of this family include the p55 and p75 receptors for TNF (TNF-R1 and TNF-R2, respectively); CD40 antigen, which is an important B cell-surface marker involved in immunoglobulin isotype switching; fas/Apo-1, whose triggering induces apoptosis; CD27 and CD30, which are found on activated T cells and B cells; and nerve growth factor receptor.

1	The common motif for the seven transmembrane helix family was originally found in receptors linked to GTP-binding proteins. This family includes receptors for chemokines (Table 372e-8), β-adrenergic receptors,and retinalrhodopsin. Itisimportanttonote thattwomembers of the chemokine receptor family, CXC chemokine receptor type 4 (CXCR4) and β chemokine receptor type 5 (CCR5), have been found to serve as the two major co-receptors for binding and entry of HIV into CD4-expressing host cells (Chap. 226).

1	Significant advances have been made in defining the signaling pathways through which cytokines exert their intracellular effects. The Janus family of protein tyrosine kinases (JAK) is a critical element involved in signaling via the hematopoietin receptors. Four JAK kinases, JAK1, JAK2, JAK3, and Tyk2, preferentially bind different cytokine receptor subunits. Cytokine binding to its receptor brings the cytokine receptor subunits into apposition and allows a pair of JAKs to transphosphorylate and activate one another. The JAKs then phosphorylate the receptor on the tyrosine residues and allow signaling molecules to bind to the receptor, whereby the signaling molecules become phosphorylated. Signaling molecules bind the receptor because they have domains (SH2, or src homology 2 domains) that can bind phosphorylated tyrosine residues. There are a number of these important signaling molecules that bind the receptor, such as the adapter molecule SHC, which can couple the receptor to the

1	can bind phosphorylated tyrosine residues. There are a number of these important signaling molecules that bind the receptor, such as the adapter molecule SHC, which can couple the receptor to the activation of the mitogen-activated protein kinase pathway. In addition, an important class of substrate of the JAKs is the signal transducers and activators of transcription (STAT) family of transcription factors. STATs have SH2 domains that enable them to bind to phosphorylated receptors, where they are then phosphorylated by the JAKs. It appears that different STATs have specificity for different receptor subunits. The STATs then dissociate from the receptor and translocate to the nucleus, bind to DNA motifs that they recognize, and regulate gene expression. The STATs preferentially bind DNA motifs that are slightly different from one another and thereby control transcription of specific genes. The importance of this pathway is particularly relevant to lymphoid development. Mutations of

1	motifs that are slightly different from one another and thereby control transcription of specific genes. The importance of this pathway is particularly relevant to lymphoid development. Mutations of JAK3 itself also result in a disorder identical to X-SCID; however, because JAK3 is found on chromosome 19 and not on the X chromosome, JAK3 deficiency occurs in boys and girls (Chap. 374).

1	Adaptive immunity is characterized by antigen-specific responses to a foreign antigen or pathogen. A key feature of adaptive immunity is that following the initial contact with antigen (immunologic priming), subsequent antigen exposure leads to more rapid and vigorous immune responses (immunologic memory). The adaptive immune system consists of dual limbs of cellular and humoral immunity. The principal effectors of cellular immunity are T lymphocytes, whereas the principal effectors of humoral immunity are B lymphocytes. Both B and T lymphocytes derive from a common stem cell (Fig. 372e-6).

1	The proportion and distribution of immunocompetent cells in various tissues reflect cell traffic, homing patterns, and functional capabilities. Bone marrow is the major site of maturation of B cells, monocytes-macrophages, DCs, and granulocytes and contains pluripotent stem cells that, under the influenceofvarious colony-stimulating factors, are capable of giving rise to all hematopoietic cell types. T cell precursors also arise from hematopoietic stem cells and home to the thymus for maturation. Mature T lymphocytes, B lymphocytes,monocytes, and DCs enter the circulation and home to peripheral lymphoid organs (lymph nodes, spleen) and mucosal surface-associated lymphoid tissue (gut, genitourinary, and respiratory tracts) as well as the skin and mucous membranes and await activation by foreign antigen.

1	T Cells The pool of effector T cells is established in the thymus early in life and is maintained throughout life both by new T cell production in the thymus and by antigen-driven expansion of virgin peripheral T cells into “memory” T cells that reside in peripheral lymphoid organs. The thymus exports ~2% of the total number of thymocytes per day throughout life, with the total number of daily thymic emigrants decreasing by ~3% per year during the first four decades of life.

1	Mature T lymphocytes constitute 70–80% of normal peripheral blood lymphocytes (only 2% of the total-body lymphocytes are containedinperipheralblood),90%ofthoracicductlymphocytes,30–40% of lymph node cells, and 20–30% of spleen lymphoid cells. In lymph nodes, T cells occupy deep paracortical areas around B cell germinal centers, and in the spleen, they are located in periarteriolar areas of whitepulp(Chap. 79).Tcellsaretheprimaryeffectorsofcell-mediated immunity, withsubsetsofT cellsmaturing into CD8+ cytotoxic Tcells capable of lysis of virus-infected or foreign cells (short-lived effector T cells) and CD4+ T cells capable of T cell help for CD8+ T cell and B cell development. Two populations of long-lived memory T cells are triggered by infections: effector memory and central memory T cells. CD7 CD2 cCD3, TCR°˛ CD1 °,˛ Germ line °,˛ Germ line °Germ line ˛-V-DJ Rearranged CD4, CD8 CD7 CD2 cCD3, TCR°˛ CD4 CD7 CD2 cCD3, TCR°˛ CD8 cCD3, TCR˝˙ CD8

1	CD7 CD2 cCD3, TCR°˛ CD1 °,˛ Germ line °,˛ Germ line °Germ line ˛-V-DJ Rearranged CD4, CD8 CD7 CD2 cCD3, TCR°˛ CD4 CD7 CD2 cCD3, TCR°˛ CD8 cCD3, TCR˝˙ CD8 Surface Ig Absent Absent ˆ H-chain at ˆ H-chain in IgM expressed IgD and IgM surface as cytoplasm on cell surface made from part of pre-˛ and at surface FIGUrE 372e-6 Development stages of T and B cells. Elements of the developing T and B cell receptor for antigen are shown schematically. The classification into the various stages of B cell development is primarily defined by rearrangement of the immunoglobulin (Ig) heavy (H) and light (L) chain genes and by the absence or presence of specific surface markers. The classification of stages of T cell development is primarily defined by cell-surface marker protein expression (sCD3, surface CD3 expression; cCD3, cytoplasmic CD3 expression; TCR, T cell receptor).

1	(Adapted from CA Janeway et al [eds]: Immunobiology. The Immune Systemic Health and Disease, 4th ed. New York, Garland, 1999; with permission.) Introduction to the Immune System Effector memory T cells reside in nonlymphoid organs and respond rapidly to repeated pathogenic infections with cytokine production and cytotoxic functions to kill virus-infected cells. Central memory T cells home to lymphoid organs where they replenish long-and short-lived and effector memory T cells as needed.

1	In general, CD4+ T cells are the primary regulatory cells of T and B lymphocyte and monocyte function by the production of cytokines and by direct cell contact (Fig. 372e-2). In addition, T cells regulate erythroid cell maturation in bone marrow and, through cell contact (CD40 ligand), have an important role in activation of B cells and induction of Ig isotype switching. Considerable evidence now exists that colonization of the gut by commensal bacteria (the gut microbiome) is responsible for expansion of the peripheral CD4+ T cell compartment in normal children and adults. Human T cells express cell-surface proteins that mark stages of intrathymic T cell maturation or identify specific functional subpopulations of mature T cells. Many of these molecules mediate or participate in important T cell functions (Table 372e-1, Fig. 372e-6).

1	The earliest identifiable T cell precursors in bone marrow are CD34+ pro-T cells (i.e., cells in which TCR genes are neither rearranged nor expressed). In the thymus, CD34+ T cell precursors begin cytoplasmic (c) synthesis of components of the CD3 complex of TCR-associated molecules (Fig. 372e-6). Within T cell precursors, TCR for antigen gene rearrangement yields two T cell lineages, expressing either TCR-αβ chains or TCR-γδ chains. T cells expressing the TCR-αβ chains constitute the majority of peripheral T cells in blood, lymph node, and spleen and terminally differentiate into either CD4+ or CD8+ cells. Cells expressing TCR-γδ chains circulate as a minor population in blood; their functions, although not fully understood, have been postulated to be those of immune surveillance at epithelial surfaces and cellular defenses against mycobacterial organisms and other intracellular bacteria through recognition of bacterial lipids.

1	In the thymus, the recognition of self-peptides on thymic epithelial cells, thymic macrophages, and DCs plays an important role in shaping the T cell repertoire to recognize foreign antigen (positive selection) and in eliminating highly autoreactive T cells (negative selection). As immature cortical thymocytes begin to express surface TCR for antigen, autoreactive thymocytes are destroyed (negative selection), thymocytes with TCRs capable of interacting with foreign antigen peptidesin thecontext of self-MHC antigens are activatedanddevelop to maturity (positive selection), and thymocytes with TCRs that are incapable of binding to self-MHC antigens die of attrition (no selection). Mature thymocytes that are positively selected are either CD4+ helper T cells or MHC class II–restricted cytotoxic (killer) T cells, or they are CD8+ T cells destined to become MHC class I–restricted cytotoxic T cells. MHC class I– or class II–restricted means that T cells recognize antigen peptide fragments

1	(killer) T cells, or they are CD8+ T cells destined to become MHC class I–restricted cytotoxic T cells. MHC class I– or class II–restricted means that T cells recognize antigen peptide fragments only when they are presented in the antigen-recognition site of a class I or class II MHC molecule, respectively (Chap. 373e).

1	After thymocyte maturation and selection, CD4 and CD8 thymocytes leave the thymus and migrate to the peripheral immune system. The thymus continues to be a contributor to the peripheral immune system well into adult life, both normally and when the peripheral T cell pool is damaged, such as occurs in AIDS and cancer chemotherapy.

1	MOLECULAR BASIS OF T CELL RECOGNITION OF ANTIGEN The TCR for antigen is a complex of molecules consisting of an antigen-binding heterodimer of either αβ or γδ chains noncovalently linked with five CD3 subunits (γ, δ, ε, ζ, and η) (Fig. 372e-7). The CD3 ζ chains are either disulfide-linked homodimers (CD3-ζ2) or disulfide-linked heterodimers composed of one ζ chain and one η chain. TCR-αβ or TCR-γδ molecules must be associated with CD3 molecules to be inserted into the T cell-surface membrane, TCRα being paired with TCR-β and TCR-γ being paired with TCR-δ. Molecules of the CD3 complex mediate transduction of T cell activation signals via TCRs, whereas TCR-α and -β or -γ and -δ molecules combine to form the TCR antigen-binding site.

1	The α, β, γ, and δ TCR for antigen molecules have amino acid sequence homology and structural similarities to immunoglobulin heavy and light chains and are members of the immunoglobulin gene superfamily of molecules. The genes encoding TCR molecules are encoded as clusters of gene segments that rearrange during the course of T cell maturation. This creates an efficient and compact mechanism for housing the diversity requirements of antigen receptor molecules. The TCR-α chain is on chromosome 14 and consists of a series of V (variable), J (joining), and C (constant) regions. The TCR-β chain is on chromosome 7 and consists of multiple V, D (diversity), J, and C TCR-β loci. The TCR-γ chain is on chromosome 7, and the TCR-δ chain is in the middle of the TCR-α locus on chromosome 14. Thus, molecules of the TCR for antigen have constant (framework) and variable regions, and the gene segments encoding the α, β, γ, and δ chains of these molecules are recombined and selected in the thymus,

1	molecules of the TCR for antigen have constant (framework) and variable regions, and the gene segments encoding the α, β, γ, and δ chains of these molecules are recombined and selected in the thymus, culminating in synthesis of the completed molecule. In both T and B cell precursors (see below), DNA rearrangements of antigen receptor genes involve the same enzymes, recombinase activating gene (RAG) 1 and RAG2, both DNA-dependent protein kinases.

1	TCR diversity is created by the different V, D, and J segments that are possible for each receptor chain by themany permutations of V, D, and J segment combinations, by “N-region diversification” due to the addition of nucleotides at the junction of rearranged gene segments, and by the pairing of individual chains to form a TCR dimer. As T cells mature in the thymus, the repertoire of antigen-reactive T cells is modified by selection processes that eliminate many autoreactive T cells,enhancetheproliferationofcellsthatfunctionappropriatelywith self-MHC molecules and antigen, and allow T cells with nonproductive TCR rearrangements to die. TCR-αβ cells do not recognize native protein or carbohydrate anti-gens.Instead,Tcells recognizeonly short(~9–13amino acids) peptide

1	TCR-αβ cells do not recognize native protein or carbohydrate anti-gens.Instead,Tcells recognizeonly short(~9–13amino acids) peptide PtdIns (4,5)P3 Lipid raft CD3 TCR InsP3 DAG PKC RASGRP Activation of downstream effectors such as NFkB, AP1, and NFAT to induce specific gene transcription leading to cell proliferation and differentiation Release of Ca2+ Translocation of NFAT to the nucleus ZAP-70 Integrin activation MAPK activation Cytoskeletal reorganization RAS SOS GRB2LAT PLC˛GADS HPK1 NCK ADAP ITK LCK VAV1 LFA-1 CD2CD28

1	FIGUrE 372e-7 Signaling through the T cell receptor. Activation signals are mediated via immunoreceptor tyrosine-based activation (ITAM) sequences in LAT and CD3 chains (blue bars) that bind to enzymes and transduce activation signals to the nucleus via the indicated intracellular activation pathways. Ligation of the T cell receptor (TCR) by MHC complexed with antigen results in sequential activation of LCK and γ-chainassociated protein kinase of 70 kDa (ZAP-70). ZAP-70 phosphorylates several downstream targets, including LAT (linker for activation of T cells) and SLP76 (SCR homology 2 [SH2] domain-containing leukocyte protein of 76 kDa). SLP76 is recruited to membrane-bound LAT through its constitutive interaction with GADS (GRB2-related adaptor protein). Together, SLP76 and LAT nucleate a multimolecular signaling complex, which induces a host of downstream responses, including calcium flux, mitogen-activated protein kinase (MAPK) activation, integrin activation, and cytoskeletal

1	a multimolecular signaling complex, which induces a host of downstream responses, including calcium flux, mitogen-activated protein kinase (MAPK) activation, integrin activation, and cytoskeletal reorganization. APC, antigen-presenting cell. (Adapted from GA Koretzky et al: Nat Rev Immunol 6:67, 2006; with permission from Macmillan Publishers Ltd. Copyright 2006.) fragments derived from protein antigens taken up or produced in APCs. Foreign antigens may be taken up by endocytosis into acidified intracellular vesicles or by phagocytosis and degraded into small peptides that associate with MHC class II molecules (exogenous antigen-presentation pathway). Other foreign antigens arise endogenously in thecytosol(suchasfromreplicatingviruses)andarebrokendowninto small peptides that associate with MHC class I molecules (endogenous antigen-presenting pathway). Thus, APCs proteolytically degrade foreign proteins and display peptide fragments embedded in the MHC class I or II antigen-recognition

1	MHC class I molecules (endogenous antigen-presenting pathway). Thus, APCs proteolytically degrade foreign proteins and display peptide fragments embedded in the MHC class I or II antigen-recognition site on the MHC molecule surface, where foreign peptide fragments are available to bind to TCR-αβ or TCR-γδ chains of reactive T cells. CD4 molecules act as adhesives and, by direct binding to MHC class II (DR, DQ, or DP) molecules, stabilize the interaction of TCR with peptide antigen (Fig. 372e-7). Similarly, CD8 molecules also act as adhesives to stabilize the TCR-antigen interaction by direct CD8 molecule binding to MHC class I (A, B, or C) molecules.

1	Antigens that arise in the cytosol and are processed via the endogenous antigen-presentation pathway are cleaved into small peptides by a complex of proteases called the proteasome. From the proteasome, antigen peptide fragments are transported from the cytosol into the lumen of the endoplasmic reticulum by a heterodimeric complex termed transporters associated with antigen processing, or TAP proteins. There, MHC class I molecules in the endoplasmic reticulum membrane physically associate with processed cytosolic peptides. Following peptide association with class I molecules, peptide-class I complexes are exported to the Golgi apparatus, and then to the cell surface, for recognition by CD8+ T cells.

1	Antigens taken up from the extracellular space via endocytosis into intracellular acidified vesicles are degraded by vesicle proteases into peptide fragments. Intracellular vesicles containing MHC class II molecules fuse with peptide-containing vesicles, thus allowing peptide fragmentsto physically bindto MHCclass IImolecules.Peptide-MHC class II complexes are then transported to the cell surface for recognition by CD4+ T cells (Chap. 373e).

1	Whereas it is generally agreed that the TCR-αβ receptor recognizes peptide antigens in the context of MHC class I or class II molecules, lipids in the cell wall of intracellular bacteria such as M. tuberculosis can also be presented to a wide variety of T cells, including subsets of TCR-γδ T cells, and a subset of CD8+ TCR-αβ T cells. Importantly, bacterial lipid antigens are not presented in the context of MHC class I orIImolecules,butratherarepresentedinthecontextofMHC-related CD1 molecules. Some γδ T cells that recognize lipid antigens via CD1 molecules have very restricted TCR usage, do not need antigen priming to respond to bacterial lipids, and may actually be a form of innate rather than acquired immunity to intracellular bacteria.

1	Just as foreign antigens are degraded and their peptide fragments presented in the context of MHC class I or class II molecules on APCs, endogenous self-proteins also are degraded, and self-peptide fragments are presented to T cells in the context of MHC class I or class II molecules on APCs. In peripheral lymphoid organs, there are T cells that are capable of recognizing self-protein fragments but normally are anergic or tolerant, i.e., nonresponsive to self-antigenic stimulation, due to lack of self-antigen upregulating APC co-stimulatory molecules such as B7-1 (CD80) and B7-2 (CD86) (see below).

1	Once engagement of mature T cell TCR by foreign peptide occurs in the context of self-MHC class I or class II molecules, binding of non-antigen-specific adhesion ligand pairs such as CD54-CD11/CD18 and CD58-CD2 stabilizes MHC peptide-TCR binding, and the expression of these adhesion molecules is upregulated (Fig. 372e-7). Once antigen ligation of the TCR occurs, the T cell membrane is partitioned into lipid membrane microdomains, or lipid rafts, that coalesce the key signaling molecules TCR/CD3 complex, CD28, CD2, LAT (linker for activation of T cells), intracellular activated (dephosphorylated) src family protein tyrosine kinases (PTKs), and the key CD3ζ-associated protein-70 (ZAP-70) PTK (Fig. 372e-7). Importantly, during T cell activation, the CD45 molecule, with protein tyrosine phosphatase activity, is partitioned away from the TCR complex to allow activating phosphorylation events to occur. The coalescence of signaling molecules of activated T lymphocytes in microdomains has

1	activity, is partitioned away from the TCR complex to allow activating phosphorylation events to occur. The coalescence of signaling molecules of activated T lymphocytes in microdomains has suggested that T cell-APC interactions can be considered immunologic synapses, analogous in function to neuronal synapses.

1	After TCR-MHC binding is stabilized, activation signals are transmitted through the cell to the nucleus and lead to the expression of geneproductsimportant inmediatingthewidediversityofTcellfunctions such as the secretion of IL-2. The TCR does not have intrinsic signaling activity but is linked to a variety of signaling pathways via immunoreceptor tyrosine-based activation motifs (ITAMs) expressed on the various CD3 chains that bind to proteins that mediate signal transduction. Each of the pathways results in the activation of particular transcription factors that control the expression of cytokine and cytokine receptor genes. Thus, antigen-MHC binding to the TCR induces the activation of the src family of PTKs, fyn and lck (lck is associated with CD4 or CD8 co-stimulatory molecules); phosphorylation of CD3ζ chain; activation of the related tyrosine kinases ZAP-70 and syk; and downstream activation of the calcium-dependent calcineurin pathway, the ras pathway, and the protein kinase C

1	of CD3ζ chain; activation of the related tyrosine kinases ZAP-70 and syk; and downstream activation of the calcium-dependent calcineurin pathway, the ras pathway, and the protein kinase C pathway. Each of these pathways leads to activation of specific families of transcription factors (including NF-AT, fos and jun, and rel/NF-κB) that form heteromultimers capable of inducing expression of IL-2, IL-2 receptor, IL-4, TNF-α, and other T cell mediators.

1	In addition to the signals delivered to the T cell from the TCR complex and CD4 and CD8, molecules on the T cell, such as CD28 and inducible co-stimulator (ICOS), and molecules on DCs, such as B7-1 (CD80) and B7-2 (CD86), also deliver important co-stimulatory signals that upregulate T cell cytokine production and are essential for T cell activation. If signaling through CD28 or ICOS does not occur, or if CD28 is blocked, the T cell becomes anergic rather than activated (see “Immune Tolerance and Autoimmunity” below). CTLA-4 (CD152) is similar to CD28 in its ability to bind CD80 and CD86. Unlike CD28, CTLA-4 transmits an inhibitory signal to T cells, acting as an off switch.

1	T CELL EXHAUSTION IN VIRAL INFECTIONS AND CANCER In chronic viral infections such as HIV-1, hepatitis C virus, and hepatitis B virus and in chronic malignancies, the persistence of antigen disrupts memory T cell function, resulting in defects in memory T cell responses. This has been defined as T cell exhaustion and is associated with T cell programmed cell death protein 1 (PD-1) (CD279) expression. Exhausted T cells have compromised proliferation and lose the ability to produce effector molecules, like IL-2, TNF-α, and IFN-γ. PD-1 downregulates T cell responses and is associated with T cell exhaustion and disease progression. For this reason, inhibition of T cell PD-1 activity to enhance effectorTcellfunction isbeing explored as atargetforimmunotherapy in both viral infections and certain malignancies.

1	T CELL SUPERANTIGENS Conventional antigens bind to MHC class I or II molecules in the groove of the αβ heterodimer and bind to T cells via the V regions of the TCR-α and -β chains. In contrast, superantigens bind directly to the lateral portion of the TCR-β chain and MHC class II β chain and stimulate T cells based solely on the Vβ gene segment used independent of the D, J, and Vα sequences present. Superantigens are protein molecules capable of activating up to 20% of the peripheral T cell pool, whereas conventional antigens activate <1 in 10,000 T cells. T cell superantigens include staphylococcal enterotoxins and other bacterial products. Superantigen stimulation of human peripheral T cells occurs in the clinical setting of staphylococcal toxic shock syndrome, leading to massive overproduction of T cell cytokines that leads to hypotension and shock (Chap. 172).

1	B CELLS Mature B cells constitute 10–15% of human peripheral blood lymphocytes, 20–30% of lymph node cells, 50% of splenic lymphocytes, and ~10% of bone marrow lymphocytes. B cells express on their surface intramembrane immunoglobulin (Ig) molecules that function as B cell receptors (BCRs) for antigen in a complex of Ig-associated α and β signaling molecules with properties similar to those described in T cells (Fig. 372e-8). Unlike T cells, which recognize only processed peptide fragments of conventional antigens embedded in the notches of MHC class I and class II antigens of APCs, B cells are capable of Introduction to the Immune System Fab region Light chain BCR SLP65 Release of Ca2+ Activation of downstream effectors Ig˜Ig°PtdIns(4,S)P3 InsP3 a b MAPK activation Cytoskeletal reorganization RAS PLC˛NCK VAV1 LYN SOS GRB2 DAG PKC˜RASGRP BTK SYK

1	FIGUrE 372e-8 B cell receptor (BCR) activation results in the sequential activation of protein tyrosine kinases, which results in the formation of a signaling complex and activation of downstream pathways as shown. Whereas SLP76 is recruited to the membrane through GADS and LAT, the mechanism of SLP65 recruitment is unclear. Studies have indicated two mechanisms: (a) direct binding by the SH2 domain of SLP65 to immunoglobulin (Ig) of the BCR complex or (b) membrane recruitment through a leucine zipper in the amino terminus of SLP65 and an unknown binding partner. ADAP, adhesionand degranulation-promoting adaptor protein; AP1, activator protein 1; BTK, Bruton’s tyrosine kinase; DAG, diacylglycerol; GRB2, growth factor receptor-bound protein 2; HPK1, hematopoietic progenitor kinase 1; InsP3, inositol-1,4,5-trisphosphate; ITK, interleukin-2-inducible T cell kinase; NCK, noncatalytic region of tyrosine kinase; NF-B, nuclear factor B; PKC, protein kinase C; PLC, phospholipase C;

1	1; InsP3, inositol-1,4,5-trisphosphate; ITK, interleukin-2-inducible T cell kinase; NCK, noncatalytic region of tyrosine kinase; NF-B, nuclear factor B; PKC, protein kinase C; PLC, phospholipase C; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; RASGRP, RAS guanyl-releasing protein; SOS, son of sevenless homologue; SYK, spleen tyrosine kinase. (Adapted from GA Koretzky et al: Nat Rev Immunol 6:67, 2006; with permission from Macmillan Publishers Ltd. Copyright 2006.) recognizingand proliferating towhole unprocessed native antigens via antigen binding to B cell–surface Ig (sIg) receptors. B cells also express surface receptors for the Fc region of IgG molecules (CD32) as well as receptors for activated complement components (C3d or CD21, C3b or CD35). The primary function of B cells is to produce antibodies. B cells also serve as APCs and are highly efficient at antigen processing. Their antigen-presenting function is enhanced by a variety of cytokines. Mature B cells are derived

1	to produce antibodies. B cells also serve as APCs and are highly efficient at antigen processing. Their antigen-presenting function is enhanced by a variety of cytokines. Mature B cells are derived from bone marrow precursor cells that arise continuously throughout life (Fig. 372e-6).

1	B lymphocyte development can be separated into antigen-independent and antigen-dependent phases. Antigen-independent B cell development occurs in primary lymphoid organs and includes all stages of B cell maturation up to the sIg+ mature B cell. Antigen-dependent B cell maturation is driven by the interaction of antigen with the mature B cell sIg, leading to memory B cell induction, Ig class switching, and plasma cell formation. Antigen-dependent stages of B cell maturation occur in secondary lymphoid organs, including lymph node, spleen, and gut Peyer’s patches. In contrast to the T cell repertoire that is generated intrathymically before contact with foreign antigen, the repertoire of B cells expressing diverse antigen-reactive sites is modified by further alteration of Ig genes after stimulation by antigen—a process called somatic hypermutation—that occurs in lymph node germinal centers.

1	DuringBcelldevelopment,diversityoftheantigen-bindingvariable region of Ig is generated by an ordered set of Ig gene rearrangements that are similar to the rearrangements undergone by TCR α, β, γ, and δ genes. For the heavy chain, there is first a rearrangement of D segments to J segments, followed by a second rearrangement between a V gene segment and the newly formed D-J sequence; the C segment is aligned to the V-D-J complex to yield a functional Ig heavy chain gene (V-D-J-C). During later stages, a functional κ or γ light chain gene is generated by rearrangement of a V segment to a J segment, ultimately yielding an intact Ig molecule composed of heavy and light chains.

1	The process of Ig gene rearrangement is regulated and results in a single antibody specificity produced by each B cell, with each Ig molecule comprising one type of heavy chain and one type of light chain. Although each B cell contains two copies of Ig light and heavy chain genes, only one gene of each type is productively rearranged and expressed in each B cell, a process termed allelic exclusion.

1	Thereare~300Vκgenes and5 Jκgenes,resultinginthe pairingofVκ and Jκ genes to create >1500 different kappa light chain combinations. There are ~70 Vλgenes and 4Jλgenes for >280 different lambda light chain combinations. The number of distinct light chains that can be generated is increased by somatic mutations within the V and J genes, thus creating large numbers of possible specificities from a limited amount of germline genetic information. As noted above, in heavy chain Ig gene rearrangement, the VH domain is created by the joining of three types of germline genes called VH, DH, and JH, thus allowing for even greater diversity in the variable region of heavy chains than of light chains.

1	The most immature B cell precursors (early pro-B cells) lack cytoplasmic Ig (cIg) andsIg (Fig.372e-6). Thelarge pre-Bcellis marked by theacquisitionofthesurfacepre-BCRcomposedofμheavy (H)chains and a pre-B light chain, termed ψLC. ψLC is a surrogate light chain receptor encoded by the nonrearranged V pre-B and the γ5 light chain locus (the pre-BCR). Pro-and pre-B cells are driven to proliferate and mature by signals from bone marrow stroma—in particular, IL-7. Light chain rearrangement occurs in the small pre-B cell stage such that the full BCR is expressed at the immature B cell stage. Immature B cells have rearranged Ig light chain genes and express sIgM. As immature B cells develop into mature B cells, sIgD is expressed as well as sIgM. At this point, B lineage development in bone marrow is complete,andBcellsexitintotheperipheralcirculationandmigrateto secondary lymphoid organs to encounter specific antigens.

1	Random rearrangements of Ig genes occasionally generate self-reactive antibodies, and mechanisms must be in place to correct these mistakes. One such mechanism is BCR editing, whereby autoreactive BCRs are mutated to not react with self-antigens. If receptor editing is unsuccessful in eliminating autoreactive B cells, then autoreactive B cellsundergonegativeselectioninthebonemarrowthroughinduction of apoptosis after BCR engagement of self-antigen.

1	After leaving the bone marrow, B cells populate peripheral B cell sites, such as lymph node and spleen, and await contact with foreign antigens that react with each B cell’s clonotypic receptor. Antigen-driven B cell activation occurs through the BCR, and a process known as somatic hypermutation takes place whereby point mutations in rearranged H-and L-genes give rise to mutant sIg molecules, some of which bind antigen better than the original sIg molecules. Somatic hypermutation, therefore, is a process whereby memory B cells in peripheral lymph organs have the best binding, or the highest-affinity antibodies. This overall process of generating the best antibodies is called affinity maturation of antibody.

1	Lymphocytes that synthesize IgG, IgA, and IgE are derived from sIgM+, sIgD+ mature B cells. Ig class switching occurs in lymph node and other peripheral lymphoid tissue germinal centers. CD40 on B cells and CD40 ligand on T cells constitute a critical co-stimulatory receptor-ligand pair of immune-stimulatory molecules. Pairs of CD40+ B cells and CD40 ligand+ T cells bind and drive B cell Ig class switching via T cell-produced cytokines such as IL-4 and TGF-β. IL-1, -2, -4, -5, and -6 synergize to drive mature B cells to proliferate and differentiate into Ig-secreting cells.

1	IL-1, -2, -4, -5, and -6 synergize to drive mature B cells to proliferate and differentiate into Ig-secreting cells. Humoral Mediators of adaptive Immunity: Immunoglobulins Immunoglobulins are the products of differentiated B cells and mediate the humoral arm of the immune response. The primary functions of antibodies are to bind specifically to antigen and bring about the inactivation or removal of the offending toxin, microbe, parasite, or other foreign substance from thebody.ThestructuralbasisofIg moleculefunctionandIggeneorganization has provided insight into the role of antibodies in normal protective immunity, pathologic immune-mediated damage by immune complexes, and autoantibody formation against host determinants.

1	All immunoglobulins have the basic structure of two heavy and two light chains (Fig. 372e-8). Immunoglobulin isotype (i.e., G, M, A, D, E) is determined by the type of Ig heavy chain present. IgG and IgA isotypes can be divided further into subclasses (G1, G2, G3, G4, and A1, A2) based on specific antigenic determinants on Ig heavy chains. The characteristics of human immunoglobulins are outlined in Table 372e-12. The four chains are covalently linked by disulfide bonds. Each chain is made up of a V region and C regions (also called domains), themselves made up of units of ~110 amino acids. Light chains have one variable (VL) and one constant (CL) unit; heavy chains have one variable unit (VH) and three or four constant (CH) units, depending on isotype. As the name suggests, the constant, or C, regions of Ig molecules are made up of homologous sequences and share the same primary structure as all other Ig chains of the same isotype and subclass. Constant regions are involved in

1	or C, regions of Ig molecules are made up of homologous sequences and share the same primary structure as all other Ig chains of the same isotype and subclass. Constant regions are involved in biologic functions of Ig molecules. The CH2 domain of IgG and the CH4 units of IgM are involved with the binding of the C1q portion of C1 during complement activation. The CH region at the carboxy-terminal end of the IgG molecule, the Fc region, binds to surface Fc receptors (CD16, CD32, CD64) of macrophages, DCs, NK cells, B cells, neutrophils, and eosinophils. The Fc of IgA binds to FcαR (CD89), and the Fc of IgE binds to FcεR (CD23).

1	Variable regions (VL and VH) constitute the antibody-binding (Fab) region of the molecule. Within the VL and VH regions are hypervariable regions (extreme sequence variability) that constitute the antigen-binding site unique to each Ig molecule. The idiotype is defined as the specific region of the Fab portion of the Ig molecule to which antigen Introduction to the Immune System taBle 372e 12 phySIcal, chemIcal, and BIologIc propertIeS of human ImmunogloBulInS Source: After L Carayannopoulos, JD Capra, in WE Paul (ed): Fundamental Immunology, 3rd ed. New York, Raven, 1993; with permission. binds. Antibodies againstthe idiotypeportion of an antibody molecule are called anti-idiotype antibodies. The formation of such antibodies in vivo during a normal B cell antibody response may generate a negative (or “off") signal to B cells to terminate antibody production.

1	IgG constitutes ~75–85% of total serum immunoglobulin. The four IgG subclasses are numbered in order of their level in serum, IgG1 being found in greatest amounts and IgG4 the least. IgG subclasses have clinical relevance in their varying ability to bind macrophage and neutrophil Fc receptors and to activate complement (Table 372e-12). Moreover, selective deficiencies of certain IgG subclasses give rise to clinical syndromes in which the patient is inordinately susceptible to bacterial infections. IgG antibodies are frequently the predominant antibody made after rechallenge of the host with antigen (secondary antibody response).

1	IgM antibodies normally circulate as a 950-kDa pentamer with 160-kDa bivalent monomers joined by a molecule called the J chain,a 15-kDa nonimmunoglobulin molecule that also effects polymerization of IgA molecules. IgM is the first immunoglobulin to appear in the immune response (primary antibody response) and is the initial type ofantibodymadebyneonates.MembraneIgMinthemonomericform also functions as a major antigen receptor on the surface of mature B cells (Table 372e-12). IgM is an important component of immune complexes in autoimmune diseases. For example, IgM antibodies against IgG molecules (rheumatoid factors) are present in high titers in rheumatoid arthritis, other collagen diseases, and some infectious diseases (subacute bacterial endocarditis).

1	IgAconstitutesonly7–15%oftotalserumimmunoglobulinbutisthe predominant class of immunoglobulin in secretions. IgA in secretions (tears, saliva, nasal secretions, gastrointestinal tract fluid, and human milk) is in the form of secretory IgA (sIgA), a polymer consisting of two IgA monomers, a joining molecule, again called the J chain, and a glycoprotein called the secretory protein. Of the two IgA subclasses, IgA1 is primarily found in serum, whereas IgA2 is more prevalent in secretions. IgA fixes complement via the alternative complement pathway and has potent antiviral activity in humans by prevention of virus binding to respiratory and gastrointestinal epithelial cells.

1	IgD is found in minute quantities in serum and, together with IgM, is a major receptor for antigen on the naïve B cell surface. IgE, which is present in serum in very low concentrations, is the major class of immunoglobulin involved in arming mast cells and basophils by binding to these cells via the Fc region. Antigen cross-linking of IgE molecules on basophil and mastcellsurfacesresultsinrelease of mediators of the immediate hypersensitivity (allergic) response (Table 372e-12). The net result of activation of the humoral (B cell) and cellular (T cell) arms of the adaptive immune system by foreign antigen is the elimination of antigen directly by specific effector T cells or in concert with specific antibody. Figure 372e-2 is a simplified schematic diagram of theTandBcellresponsesindicatingsomeofthesecellularinteractions.

1	The expression of adaptive immune cell function is the result of a complex series of immunoregulatory events that occur in phases. Both T and B lymphocytes mediate immune functions, and each of these cell types, when given appropriate signals, passes through stages, from activation and induction through proliferation, differentiation, and ultimately effector functions. The effector function expressed may be at the end point of a response, such as secretion of antibody by a differentiated plasma cell, or it might serve a regulatory function that modulates other functions, such as is seen with CD4+ and CD8+ T lymphocytes that modulate both differentiation of B cells and activation of CD8+ cytotoxic T cells.

1	CD4 helper T cells can be subdivided on the basis of cytokines produced (Fig. 372e-2). Activated TH1-type helper T cells secrete IL-2, IFN-γ,IL-3,TNF-a,GM-CSF,andTNF-β,whereasactivatedTH2-type helper T cells secrete IL-3, -4, -5, -6, -10, and -13. TH1 CD4+ T cells, throughelaborationofIFN-γ,haveacentralroleinmediatingintracellular killing by a variety of pathogens. TH1 CD4+ T cells also provide T cell help for generation of cytotoxic T cells and some types of opsonizing antibody, and they generally respond to antigens that lead to delayed hypersensitivity types of immune responses for many intracellular viruses and bacteria (such as HIV or M. tuberculosis). In contrast, TH2 cells have a primary role in regulatory humoral immunity and isotype switching. TH2 cells, through production of IL-4 and IL-10, have a regulatory role in limiting proinflammatory responses mediated by TH1 cells (Fig. 372e-2). In addition, TH2 CD4+ T cells provide help to B cells for specific Ig production and

1	IL-4 and IL-10, have a regulatory role in limiting proinflammatory responses mediated by TH1 cells (Fig. 372e-2). In addition, TH2 CD4+ T cells provide help to B cells for specific Ig production and respond to antigens that require high antibody levels for foreign antigen elimination (extracellular encapsulated bacteria such as Streptococcus pneumoniae and certain parasite infections). A new subset of the TH family has been described, termedTH17,characterizedascellsthatsecretecytokinessuchasIL-17, -22, and -26. TH17 cells have been shown to play a role in autoimmune inflammatory disorders in addition to defense against extracellular bacteria and fungi, particularly at mucosal surfaces. In summary, the typeofTcellresponsegeneratedinanimmuneresponseisdetermined by the microbe PAMPs presented to the DCs, the TLRs on the DCs that become activated, the types of DCs that are activated, and the cytokines that are produced (Table 372e-4). Commonly, myeloid DCs produce IL-12 and activate TH1 T

1	to the DCs, the TLRs on the DCs that become activated, the types of DCs that are activated, and the cytokines that are produced (Table 372e-4). Commonly, myeloid DCs produce IL-12 and activate TH1 T cell responses that result in IFN-γ and cytotoxic T cell induction, and plasmacytoid DCs produce IFN-α and lead to TH2 responses that result in IL-4 production and enhanced antibody responses.

1	As shown in Figs. 372e-2 and 372e-3, upon activation by DCs, T cell subsets that produce IL-2, IL-3, IFN-γ, and/or IL-4, -5, -6, -10, and -13 are generated and exert positive and negative influences on effector T and B cells. For B cells, trophic effects are mediated by a variety of cytokines, particularly T cell–derived IL-3, -4, -5, and -6, that act at sequential stages of B cell maturation, resulting in B cell proliferation, differentiation, and ultimately antibody secretion. For cytotoxic T cells, trophic factors include inducer T cell secretion of IL-2, IFN-γ, and IL-12.

1	An important type of immunomodulatory T cell that controls immune responses is CD4+ and CD8+ T regulatory cells. These cells constitutively express the α chain of theIL-2receptor(CD25), produce large amounts of IL-10, and can suppress both T and B cell responses. T regulatory cells are induced by immature DCs and play key roles in maintaining tolerance to self-antigens in the periphery. Loss of T regulatory cells is the cause of organ-specific autoimmune disease in mice such as autoimmune thyroiditis, adrenalitis, and oophoritis (see “Immune Tolerance and Autoimmunity” below). T regulatory cells also play key roles in controlling the magnitude and duration of immune responses to microbes. Normally, after the initial immune response to a microbe has eliminated the invader, T regulatory cells are activated to suppress the antimicrobe response and prevent host injury. Some microbes have adapted to induce T regulatory cell activation at the site of infection to promote parasite infection

1	are activated to suppress the antimicrobe response and prevent host injury. Some microbes have adapted to induce T regulatory cell activation at the site of infection to promote parasite infection and survival. In Leishmania infection, the parasite locally induces T regulatory cell accumulation at skin infection sites that dampens anti-Leishmania T cell responses and prevents elimination of the parasite. It is thought that many chronic infections such as by M. tuberculosis are associated with abnormal T regulatory cell activation that prevents elimination of the microbe.

1	Although B cells recognize native antigen via B cell–surface Ig receptors, B cells require T cell help to produce high-affinity antibody of multiple isotypes that are the most effective in eliminating foreign antigen. This T cell dependence likely functions in the regulation of B cell responses and in protection against excessive autoantibody production. T cell–B cell interactions that lead to high-affinity antibody production require (1) processing of native antigen by B cells and expression of peptide fragments on the B cell surface for presentation to TH cells, (2) the ligation of B cells by both the TCR complex and the CD40 ligand, (3) induction of the process termed antibody isotype switching in antigen-specific B cell clones, and (4) induction of the process of affinity maturation of antibody in the germinal centers of B cell follicles of lymph node and spleen.

1	Naïve B cells express cell-surface IgD and IgM, and initial contact of naïve B cells with antigen is via binding of native antigen to B cell– surfaceIgM.Tcellcytokines,releasedfollowingTH2cellcontactwithB cellsorbya“bystander”effect,inducechangesinIggeneconformation that promote recombination of Ig genes. These events then result in the “switching” of expression of heavy chain exons in a triggered B cell, leading to the secretion of IgG, IgA, or, in some cases, IgE antibody with the same V region antigen specificity as the original IgM antibody, for response to a wide variety of extracellular bacteria, protozoa, and helminths. CD40 ligand expression by activated T cells is critical for induction of B cell antibody isotype switching and for B cell responsiveness to cytokines. Patients with mutations in T cell CD40 ligand haveB cellsthat areunableto undergoisotypeswitching,resulting in lack of memory B cell generation and the immunodeficiency syndrome of X-linked hyper-IgM syndrome

1	with mutations in T cell CD40 ligand haveB cellsthat areunableto undergoisotypeswitching,resulting in lack of memory B cell generation and the immunodeficiency syndrome of X-linked hyper-IgM syndrome (Chap. 374).

1	Immune tolerance is defined as the absence of activation of pathogenic autoreactivity. Autoimmune diseases are syndromes caused by theactivation of T or B cells or both, with no evidence of other causes such as infections or malignancies (Chap. 377e). Once thought to be mutually exclusive, immune tolerance and autoimmunity are now both recognized to be present normally in health; when abnormal, they represent extremes from the normal state. For example, it is now known that low levels of autoreactivity of T and B cells with self-antigens in the periphery are critical to their survival. Similarly, low levels of autoreactivity and thymocyte recognition of self-antigens in the thymus are the mechanisms whereby (1) normal T cells are positively selected to survive and leave the thymus to respond to foreign microbes in the periphery and (2) T cells highly reactive to self-antigens are negatively selected and die to prevent overly self-reactive T cells from getting into the periphery

1	respond to foreign microbes in the periphery and (2) T cells highly reactive to self-antigens are negatively selected and die to prevent overly self-reactive T cells from getting into the periphery (central tolerance). However, not all self-antigens are expressed in the thymus to delete highly self-reactive T cells, and there are mechanisms for peripheral tolerance induction of T cells as well. Unlike the presentation of microbial antigens by mature DCs, the presentation of self-antigens by immature DCs neither activates nor matures the DCs to express high levels of co-stimulatory molecules such as B7-1 (CD80) or B7-2 (CD86). When peripheral T cells are stimulated by DCs expressing self-antigens in the context of HLA molecules, sufficient stimulation of T cells occurs to keep them alive, but otherwise they remain anergic, or nonresponsive, until they contact a DC with high levels of co-stimulatory molecules expressing microbial antigens. In the latter setting, normal T cells then

1	but otherwise they remain anergic, or nonresponsive, until they contact a DC with high levels of co-stimulatory molecules expressing microbial antigens. In the latter setting, normal T cells then become activated to respond to the microbe. If B cells have high-self-reactivity BCRs, they normally undergo either deletion in the bone marrow or receptoreditingtoexpress a less autoreactivereceptor.Althoughmany autoimmune diseases are characterized by abnormal or pathogenic autoantibody production (Table 372e-13), most autoimmune diseases are caused by a combination of excess T and B cell reactivity.

1	Multiple factors contribute to the genesis of clinical autoimmune disease syndromes, including genetic susceptibility (Table 372e-13), environmental immune stimulants such as drugs (e.g., procainamide andphenytoin[Dilantin]withdrug-inducedsystemiclupuserythematosus), infectious agent triggers (such as Epstein-Barr virus and auto-antibody production against red blood cells and platelets), and loss of T regulatory cells (leading to thyroiditis, adrenalitis, and oophoritis). Immunity at Mucosal Surfaces Mucosa covering the respiratory, digestive, and urogenital tracts; the eye conjunctiva; the inner ear; and the ducts of all exocrine glands contain cells of the innate and adaptive mucosal immune system that protect these surfaces against pathogens. In the healthy adult, mucosa-associated lymphoid tissue (MALT) contains 80% of all immune cells within the body and constitutes the largest mammalian lymphoid organ system.

1	MALT has three main functions: (1) to protect the mucous membranes from invasive pathogens; (2) to prevent uptake of foreign antigens from food, commensal organisms, and airborne pathogens and particulate matter; and (3) to prevent pathologic immune responses from foreign antigens if they do cross the mucosal barriers of the body (Fig. 372e-9).

1	MALT is a compartmentalized system of immune cells that functions independently from systemic immune organs. Whereas the systemic immune organs are essentially sterile under normal conditions and respond vigorously to pathogens, MALT immune cells are continuously bathed in foreign proteins and commensal bacteria, and they must select those pathogenic antigens that must be eliminated. MALT contains anatomically defined foci of immune cells in the intestine, tonsil, appendix, and peribronchial areas that are inductive sites for mucosal immune responses. From these sites, immune T and B cells migrate to effector sites in mucosal parenchyma and exocrine glands where mucosal immune cells eliminate pathogen-infected cells. In addition to mucosal immune responses, all mucosal sites have strong mechanical and chemical barriers and cleansing functions to repel pathogens.

1	Key components of MALT include specialized epithelial cells called “membrane” or “M” cells that take up antigens and deliver them to DCs or other APCs. Effector cells in MALT include B cells producing antipathogen neutralizing antibodies of secretory IgA as well as IgG isotype, T cells producing similar cytokines as in systemic immune system response, and T helper and cytotoxic T cells that respond to pathogen-infected cells. Secretory IgA is produced in amounts of >50 mg/kg of body weight per 24h and functions toinhibitbacterialadhesion, inhibit macromolecule absorption in the gut, neutralize viruses, and enhance antigen elimination in tissue through binding to IgA and receptor-mediated transport of immune complexes through epithelial cells.

1	Recent studies have demonstrated the importance of commensal gut and other mucosal bacteria to the health of the human immune system. Normal commensal flora induces anti-inflammatory events in the gut and protects epithelial cells from pathogens through TLRs and other PRR signaling. When the gut is depleted of normal commensal flora, the immune system becomes abnormal, with loss of TH1 T cell function. Restoration of the normal gut flora can reestablish the balance in T helper cell ratios characteristic of the normal immune system. When the gut barrier is intact, either antigens do not transverse the gut epithelium or, when pathogens are present, a self-limited, protectiveMALTimmuneresponse eliminates the pathogen(Fig.372e-9). However, when the gut barrier breaks down, immune responses to commensal flora antigens can cause inflammatory bowel diseases such as Crohn’s disease and, perhaps, ulcerative colitis (Fig. 372e-9) (Chap. 351). Uncontrolled MALT immune responses to food antigens,

1	commensal flora antigens can cause inflammatory bowel diseases such as Crohn’s disease and, perhaps, ulcerative colitis (Fig. 372e-9) (Chap. 351). Uncontrolled MALT immune responses to food antigens, such as gluten, can cause celiac disease (Chap. 351).

1	The process of apoptosis (programmed cell death) plays a crucial role in regulating normal immune responses to antigen. In general, a wide variety of stimuli trigger one of several apoptotic pathways to eliminate microbe-infected cells, eliminate cells with damaged DNA, or eliminate activated immune cells that are no longer needed (Fig. 372e-10). The largest known family of “death receptors” is the TNF receptor (TNF-R) family (TNFR1,TNF-R2,Fas[CD95],deathreceptor3[DR3],deathreceptor4[DR4; TNF-related apoptosis-including ligand receptor 1, or TRAIL-R1], and death receptor 5 [DR5, TRAIL-R2]); their ligands are all in the TNF-α family. Binding of ligands to these death receptors leads to a signaling cascade that involves activation of the caspase family of molecules that leads to DNA cleavage and cell death. Two other pathways of programmed cell death involve nuclear p53 in the elimination of cells with abnormal DNA and mitochondrial cytochrome c to induce cell death in damaged cells

1	and cell death. Two other pathways of programmed cell death involve nuclear p53 in the elimination of cells with abnormal DNA and mitochondrial cytochrome c to induce cell death in damaged cells (Fig. 372e-10). A number of human diseases have now been described that result from, or are associated with, mutated apoptosis genes (Table 372e-14). These include mutations in the Fas and Fas ligand genes in autoimmune and lymphoproliferation syndromes, and multiple associations of mutations in genes in the apoptotic pathway with malignant syndromes.

1	Several responses by the host innate and adaptive immune systems to foreign microbes culminate in rapid and efficient elimination of microbes. In these scenarios, the classic weapons of the adaptive immunesystem(Tcells,Bcells)interfacewithcells(macrophages,DCs, NK cells, neutrophils, eosinophils, basophils) and soluble products Introduction to the Immune System

1	Autoantigen Autoimmune Diseases Autoantigen Autoimmune Diseases Cellor Organ-Specific Autoimmunity Acetylcholine receptor Myasthenia gravis Insulin receptor Type B insulin resistance, acanthosis, systemic lupus erythematosus (SLE) Actin Chronic active hepatitis, primary biliary cirrhosis Intrinsic factor type 1 Pernicious anemia Adenine nucleotide translator (ANT) Dilated cardiomyopathy, myocarditis Leukocyte function-associated antigen (LFA-1) Treatment-resistant Lyme arthritis β-Adrenoreceptor Dilated cardiomyopathy Aromatic L-amino acid decarboxylase Autoimmune polyendocrine syndrome type 1 (APS-1) Myelin-associated glycoprotein (MAG) Polyneuropathy Asialoglycoprotein receptor Autoimmune hepatitis Myelin-basic protein Multiple sclerosis, demyelinating diseases Bactericidal/permeability-increasing protein (Bpi) Cystic fibrosis vasculitides Myelin oligodendrocyte glycoprotein (MOG) Multiple sclerosis Calcium-sensing receptor Acquired hypoparathyroidism Myosin Rheumatic fever

1	protein (Bpi) Cystic fibrosis vasculitides Myelin oligodendrocyte glycoprotein (MOG) Multiple sclerosis Calcium-sensing receptor Acquired hypoparathyroidism Myosin Rheumatic fever Cholesterol side-chain cleavage enzyme (CYPlla) Autoimmune polyglandular syndrome-1 p-80-Collin Atopic dermatitis Collagen type IV-α3-chain Goodpasture’s syndrome Pyruvate dehydrogenase complex-E2 (PDC-E2) Primary biliary cirrhosis Cytochrome P450 2D6 (CYP2D6) Autoimmune hepatitis Desmin Crohn’s disease, coronary artery disease Sodium iodide symporter (NIS) Graves’ disease, autoimmune hypothyroidism Desmoglein 1 Pemphigus foliaceus Desmoglein 3 Pemphigus vulgaris SOX-10 Vitiligo F-actin Autoimmune hepatitis Thyroid and eye muscle shared protein Thyroid-associated ophthalmopathy GM gangliosides Guillain-Barré syndrome Glutamate decarboxylase (GAD65) Type 1 diabetes, stiff-person syndrome Thyroglobulin Autoimmune thyroiditis Glutamate receptor (GLUR) Rasmussen encephalitis Thyroid peroxidase Autoimmune

1	syndrome Glutamate decarboxylase (GAD65) Type 1 diabetes, stiff-person syndrome Thyroglobulin Autoimmune thyroiditis Glutamate receptor (GLUR) Rasmussen encephalitis Thyroid peroxidase Autoimmune Hashimoto’s thyroiditis H/K ATPase Autoimmune gastritis Thyrotropin receptor Graves’ disease 17-α-Hydroxylase (CYP17) Autoimmune polyglandular syndrome-1 Tissue transglutaminase Celiac disease 21-Hydroxylase (CYP21) Addison’s disease Transcription coactivator p75 Atopic dermatitis IA-2 (ICA512) Type 1 diabetes Tryptophan hydroxylase Autoimmune polyglandular syndrome-1 Insulin Type 1 diabetes, insulin hypoglycemic syndrome (Hirata’s disease) Tyrosinase Tyrosine hydroxylase Vitiligo, metastatic melanoma Autoimmune polyglandular syndrome-1 Systemic Autoimmunity ACTH ACTH deficiency Histone H2A-H2B-DNA Systemic lupus erythematosus Aminoacyl-tRNA histidyl synthetase Myositis, dermatomyositis IgE receptor Chronic idiopathic urticaria Aminoacyl-tRNA synthetase (several) Polymyositis, dermatomyositis

1	Systemic lupus erythematosus Aminoacyl-tRNA histidyl synthetase Myositis, dermatomyositis IgE receptor Chronic idiopathic urticaria Aminoacyl-tRNA synthetase (several) Polymyositis, dermatomyositis Keratin Rheumatoid arthritis Cardiolipin Systemic lupus erythematosus, antiphospholipid syndrome Ku-DNA-protein kinase Systemic lupus erythematosus Carbonic anhydrase II Systemic lupus erythematosus, Sjögren’s syndrome, systemic sclerosis Ku-nucleoprotein La phosphoprotein (La 55-B) Connective tissue syndrome Sjögren’s syndrome Collagen (multiple types) Rheumatoid arthritis, systemic lupus erythematosus, progressive systemic sclerosis Myeloperoxidase Necrotizing and crescentic glomerulo-nephritis (NCGN), systemic vasculitis Centromere-associated proteins Systemic sclerosis Proteinase 3 (PR3) Granulomatosis with polyangiitis (Wegener’s), Churg-Strauss syndrome DNA-dependent nucleoside-stimulated ATPase Dermatomyositis RNA polymerase I–III (RNP) Systemic sclerosis, systemic lupus

1	3 (PR3) Granulomatosis with polyangiitis (Wegener’s), Churg-Strauss syndrome DNA-dependent nucleoside-stimulated ATPase Dermatomyositis RNA polymerase I–III (RNP) Systemic sclerosis, systemic lupus erythematosus Fibrillarin Scleroderma Signal recognition protein (SRP54) Polymyositis Fibronectin Systemic lupus erythematosus, rheumatoid arthritis, morphea Topoisomerase-1 (Scl-70) Scleroderma, Raynaud’s syndrome Glucose-6-phosphate isomerase Rheumatoid arthritis Tublin Chronic liver disease, visceral leishmaniasis β2-Glycoprotein I (B2-GPI) Primary antiphospholipid syndrome Golgin (95, 97, 160, 180) Heat shock protein Sjögren’s syndrome, systemic lupus erythematosus, rheumatoid arthritis Various immune-related disorders Vimentin Systemic autoimmune disease Hemidesmosomal protein 180 Bullous pemphigoid, herpes gestationis, cicatricial pemphigoid taBle 372e 13 recomBInant or purIfIed autoantIgenS recognIzed By autoantIBodIeS aSSocIated wIth human autoImmune dISorderS

1	Autoantigen Autoimmune Diseases Autoantigen Autoimmune Diseases Plasma Protein and Cytokine Autoimmunity C1 inhibitor Autoimmune C1 deficiency Glycoprotein IIb/IIIg and Ib/IX Autoimmune thrombocytopenia purpura C1q Systemic lupus erythematosus, mem-brane proliferative glomerulonephritis (MPGN) IgA Immunodeficiency associated with systemic lupus erythematosus, perni-cious anemia, thyroiditis, Sjögren’s syn-drome, and chronic active hepatitis Cytokines (IL-1α, IL-1β, IL-6, IL-10, LIF) Rheumatoid arthritis, systemic sclerosis, normal subjects Factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, thrombin vWF Prolonged coagulation time Oxidized LDL (OxLDL) Atherosclerosis Cancer and Paraneoplastic Autoimmunity Amphiphysin Neuropathy, small-cell lung cancer p62 (IGF-II mRNA-binding protein) Hepatocellular carcinoma (China) Cyclin B1 Hepatocellular carcinoma Recoverin Cancer-associated retinopathy DNA topoisomerase II Liver cancer Ri protein Paraneoplastic opsoclonus

1	protein) Hepatocellular carcinoma (China) Cyclin B1 Hepatocellular carcinoma Recoverin Cancer-associated retinopathy DNA topoisomerase II Liver cancer Ri protein Paraneoplastic opsoclonus myoclonus ataxia Desmoplakin Paraneoplastic pemphigus Gephyrin Paraneoplastic stiff-person syndrome βIV spectrin Lower motor neuron syndrome Hu proteins Paraneoplastic encephalomyelitis Synaptotagmin Lambert-Eaton myasthenic syndrome Neuronal nicotinic acetylcholine receptor Subacute autonomic neuropathy, cancer Voltage-gated calcium channels Lambert-Eaton myasthenic syndrome p53 Cancer, systemic lupus erythematosus Yo protein Paraneoplastic cerebellar degeneration taBle 372e 13 recomBInant or purIfIed autoantIgenS recognIzed By autoantIBodIeS aSSocIated wIth human autoImmune dISorderS (CONTINUED)

1	Source: From A Lernmark et al: J Clin Invest 108:1091, 2001; with permission. FIGUrE 372e-9 Increased epithelial permeability may be important in the development of chronic gut T cell–mediated inflammation. CD4 T cells activated by gut antigens in Peyer’s patches migrate to the lamina propria (LP). In healthy individuals, these cells die by apoptosis. Increased epithelial permeability may allow sufficient antigen to enter the LP to trigger T cell activation, breaking tolerance mediated by immunosuppressive cytokines and perhaps T regulatory cells. Proinflammatory cytokines then further increase epithelial permeability, setting up a vicious cycle of chronic inflammation. (From TT MacDonald et al: Science 307:1920, 2005; with permission.) Introduction to the Immune System (FAS, TNF, TRAIL) (˜ Radiation)

1	Introduction to the Immune System (FAS, TNF, TRAIL) (˜ Radiation) Death ligand Death Receptor Oxygen radicals DNA injury BIM, PUMA, other BH3-only proteins BCL-XL-BCL2 BCL2-BCL-XL ? BAX FADD Capsase 8 BAK c-FLIP SMAC/DIABLO SMAC/DIABLOCaspase 3 Caspase 9 activation Substrate cleavage ApoptosisApoptosis IAPS IAPS BID APAF1 Cytochrome cCytochrome ctBID

1	FIGUrE 372e-10 Pathways of cellular apoptosis. There are two major pathways of apoptosis: the death-receptor pathway, which is mediated by activation of death receptors, and the BCL2-regulated mitochondrial pathway, which is mediated by noxious stimuli that ultimately lead to mitochondrial injury. Ligation of death receptors recruits the adaptor protein FAS-associated death domain (FADD). FADD in turn recruits caspase 8, which ultimately activates caspase 3, the key “executioner” caspase. Cellular FLICE-inhibitory protein (c-FLIP) can either inhibit or potentiate binding of FADD and caspase 8, depending on its concentration. In the intrinsic pathway, proapoptotic BH3 proteins are activated by noxious stimuli, which interact with and inhibit antiapoptotic BCL2 or BCL-XL. Thus, BAX and BAK are free to induce mitochondrial permeabilization with release of cytochrome c, which ultimately results in the activation of caspase 9 through the apoptosome. Caspase 9 then activates caspase 3.

1	BAK are free to induce mitochondrial permeabilization with release of cytochrome c, which ultimately results in the activation of caspase 9 through the apoptosome. Caspase 9 then activates caspase 3. SMAC/DIABLO is also released after mitochondrial permeabilization and acts to block the action of inhibitors of apoptosis protein (IAPs), which inhibit caspase activation. There is potential cross-talk between the two pathways, which is mediated by the truncated form of BID (tBID) that is produced by caspase 8–mediated BID cleavage; tBID acts to inhibit the BCL2-BCL-XL pathway and to activate BAX and BAK. There is debate (indicated by the question mark) as to whether proapoptotic BH3 molecules (e.g., BIM and PUMA) act directly on BAX and BAK to induce mitochondrial permeability or whether they act only on BCL2-BCL-XL. APAF1, apoptotic protease-activating factor 1; BH3, BCL homologue; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand. (From RS Hotchkiss et al: N Engl

1	act only on BCL2-BCL-XL. APAF1, apoptotic protease-activating factor 1; BH3, BCL homologue; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand. (From RS Hotchkiss et al: N Engl J Med 361:1570, 2009; with permission.) of antigen particles by phagocytosis (by macrophages or neutrophils) or by direct cytotoxic mechanisms (involving macrophages, neutrophils, DCs, and lymphocytes). Under normal circumstances, orderly progression of host defenses through these phases results in a well-controlled immune and inflammatory response that protects the hostfromtheoffendingantigen.However,dysfunction of any of the host defense systems can damage host tissue and produce clinical disease. Furthermore, for certain pathogens or antigens, the normal immune response itself mightcontribute substantially tothetissuedamage. For example, the immune and inflammatory response in the brain to certain pathogens such as M. tuberculosis may be responsible for much of the morbidity rate of

1	substantially tothetissuedamage. For example, the immune and inflammatory response in the brain to certain pathogens such as M. tuberculosis may be responsible for much of the morbidity rate of this disease in that organ system (Chap. 202). In addition, the morbidity rate associated with certain pneumonias such as that caused by Pneumocystis jiroveci may be associated more with inflammatory infiltrates than with the tissue-destructive effects of the microorganism itself (Chap. 244).

1	Molecular Basis of Lymphocyte–Endothelial Cell Interactions

1	The control of lymphocyte circulatory patterns between the bloodstream and peripheral lymphoid organs operates at the level of lymphocyte–endothelial cell interactions to control the specificity of lymphocyte subset entry into organs. Similarly, lymphocyte–endothelial cell interactions regulate the entry of lymphocytes into inflamed tissue. Adhesion molecule expression on lymphocytes and endothelial cells regulates the retention and subsequent egress of lymphocytes within tissue sites of antigenic stimulation, delaying cell exit from tissue and preventingreentryintothe circulatinglymphocyte pool (Fig. 372e-11). All types of lymphocyte migration begin with lymphocyte attachment to specialized regions of vessels, termed high endothelial venules (HEVs). An important concept is that adhesion molecules do not generally bind their ligand until a conformational change (ligand activation) occurs in the adhesion molecule that allows ligand binding. Induction of a conformation-dependent

1	molecules do not generally bind their ligand until a conformational change (ligand activation) occurs in the adhesion molecule that allows ligand binding. Induction of a conformation-dependent determinant on an adhesion molecule can be accomplished by cytokines or via ligation of other adhesion molecules on the cell.

1	The first stage of lymphocyte–endothelial cell interactions, attachment and rolling, occurs when lymphocytes leave the stream of flowing blood cells in a postcapillary venule and roll along venule endothelial cells (Fig. 372e-11). Lymphocyte rolling is mediated by the l-selectin molecule (LECAM-1, LAM-1, CD62L) and slows cell transit time through venules, allowing time for activation of adherent cells.

1	The second stage of lymphocyte–endothelial cell interactions, firm adhesion with activation-dependent stable arrest,requiresstimulation oflymphocytesbychemoattractants or by endothelial cell–derived cytokines. Cytokines thought to participate in adherent cell activation include members of the IL-8 family, platelet-activation factor, leukotriene B4, and C5a. In addition, HEVs express che (microbial peptides, pentraxins, complement and coagulation systems) mokines, SLC (CCL21) and ELC (CCL19), which participate in this of the innate immune system (Chaps. 80 and 376). process. Following activation by chemoattractants, lymphocytes shed

1	Therearefivegeneralphasesofhostdefenses:(1)migrationofleuko-l-selectin from the cell surface and upregulate cell CD11b/18 (MACcytestositesofantigenlocalization;(2)antigen-nonspecificrecognition 1) or CD11a/18 (LFA-1) molecules, resulting in firm attachment of of pathogens by macrophages and other cells and systems of the innate lymphocytes to HEVs. immune system; (3) specific recognition of foreign antigens mediated Lymphocyte homing to peripheral lymph nodes involves adhesion byTandBlymphocytes;(4)amplificationoftheinflammatoryresponse of l-selectin to glycoprotein HEV ligands collectively referred to as with recruitment of specific and nonspecific effector cells by comple-peripheral node addressin (PNAd), whereas homing of lymphocytes ment components, cytokines, kinins, arachidonic acid metabolites, and to intestine Peyer’s patches primarily involves adhesion of the a4β7 mast cell–basophil products; and (5) macrophage, neutrophil, and lym-integrin to mucosal addressin cell adhesion

1	metabolites, and to intestine Peyer’s patches primarily involves adhesion of the a4β7 mast cell–basophil products; and (5) macrophage, neutrophil, and lym-integrin to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) phocyte participation in destruction of antigen with ultimate removal on the Peyer’s patch HEVs. However, for migration to mucosal aMany autoimmune diseases are associated with a myriad of major histocompatibility complex gene allele (HLA) types. They are presented here as examples. Abbreviation: MALT, mucosa-associated lymphoid tissue. Source: Adapted from L Mullauer: Mutat Res 488:211, 2001 and A Davidson, B Diamond: N Engl J Med 345:340, 2001.

1	Introduction to the Immune System Peyer’s patch lymphoid aggregates, naïve lymphocytes primarily use l-selectin, whereas memory lymphocytes use a4β7 integrin. a4β1 integrin (CD49d/CD29, VLA-4)–VCAM-1 interactions are important in the initial interaction of memory lymphocytes with HEVs of multiple organs in sites of inflammation (Table 372e-15).

1	The third stage of leukocyte emigration in HEVs is sticking and arrest. Sticking of the lymphocyte to endothelial cells and arrest at the site of sticking are mediated predominantly by ligation of a1β2 integrin LFA-1 to the integrin ligand ICAM-1 on HEVs. Whereas the first three stages of lymphocyte attachment to HEVs take only a few seconds, the fourth stage of lymphocyte emigration, transendothelial migration,takes ~10min.Althoughthemolecularmechanismsthatcontrol lymphocyte transendothelial migration are not fully characterized, the HEV CD44 molecule and molecules of the HEV glycocalyx (extracellular matrix) are thought to play important regulatory roles in this process (Fig. 372e-11). Finally, expression of matrix metalloproteases 1. Tethering and rolling 2. Chemokine signal 3. Arrest 4. Polarization and diapedesis 5. Junctional rearrangement 6. Proteolysis 8. DC migration to draining LN 7. Interstitial migration Cytokine-stimulated parenchymal cell Damaged or inflamed tissue

1	4. Polarization and diapedesis 5. Junctional rearrangement 6. Proteolysis 8. DC migration to draining LN 7. Interstitial migration Cytokine-stimulated parenchymal cell Damaged or inflamed tissue Basement membrane Lymph vessel DC Blood vesssel lumen

1	ECM with GAG chemoattractants

1	FIGUrE 372e-11 Key migration steps of immune cells at sites of inflammation. Inflammation due to tissue damage or infection induces the release of cytokines (not shown) and inflammatory chemoattractants (red arrowheads) from distressed stromal cells and “professional” sentinels, such as mast cells and macrophages (not shown). The inflammatory signals induce upregulation of endothelial selectins and immunoglobulin “superfamily” members, particularly ICAM-1 and/or VCAM-1. Chemoattractants, particularly chemokines, are produced by or translocated across venular endothelial cells (red arrow) and are displayed in the lumen to rolling leukocytes. Those leukocytes that express the appropriate set of trafficking molecules undergo a multistep adhesion cascade (steps 1–3) and then polarize and move by diapedesis across the venular wall (steps 4 and 5). Diapedesis involves transient disassembly of endothelial junctions and penetration through the underlying basement membrane (step 6). Once in

1	by diapedesis across the venular wall (steps 4 and 5). Diapedesis involves transient disassembly of endothelial junctions and penetration through the underlying basement membrane (step 6). Once in the extravascular (interstitial) space, the migrating cell uses different integrins to gain “footholds” on collagen fibers and other ECM molecules, such as laminin and fibronectin, and on inflammation-induced ICAM-1 on the surface of parenchymal cells (step 7). The migrating cell receives guidance cues from distinct sets of chemoattractants, particularly chemokines, which may be immobilized on glycosaminoglycans (GAG) that “decorate” many ECM molecules and stromal cells. Inflammatory signals also induce tissue dendritic cells (DCs) to undergo maturation. Once DCs process material from damaged tissues and invading pathogens, they upregulate CCR7, which allows them to enter draining lymph vessels that express the CCR7 ligand CCL21 (and CCL19). In lymph nodes (LNs), these antigen-loaded mature

1	and invading pathogens, they upregulate CCR7, which allows them to enter draining lymph vessels that express the CCR7 ligand CCL21 (and CCL19). In lymph nodes (LNs), these antigen-loaded mature DCs activate naïve T cells and expand pools of effector lymphocytes, which enter the blood and migrate back to the site of inflammation. T cells in tissue also use this CCR7-dependent route to migrate from peripheral sites to draining lymph nodes through afferent lymphatics. (Adapted from AD Luster et al: Nat Immunol 6:1182, 2005; with permission from Macmillan Publishers Ltd. Copyright 2005.) capable of digesting the subendothelial basement membrane, rich in nonfibrillar collagen, appears to be required for the penetration of lymphoid cells into the extravascular sites.

1	AbnormalinductionofHEVformationanduseofthemoleculesdiscussed above have been implicated in the induction and maintenance of inflammation in a number of chronic inflammatory diseases. In animalmodelsoftype1diabetesmellitus,MAdCAM-1andGlyCAM-1 have been shown to be highly expressed on HEVs in inflamed pancreatic islets, and treatment of these animals with inhibitors of l-selectin and a4 integrin function blocked the development of type 1 diabetes mellitus (Chap. 417). A similar role for abnormal induction of the adhesion molecules of lymphocyte emigration has been suggested in rheumatoid arthritis (Chap. 380), Hashimoto’s thyroiditis (Chap. 405), Graves’ disease (Chap. 405), multiple sclerosis (Chap. 458), Crohn’s disease (Chap. 351), and ulcerative colitis (Chap. 351).

1	Immune-Complex Formation Clearance of antigen by immune-complex formation between antigen, complement, and antibody is a highly effective mechanism of host defense. However, depending on the level of immune complexes formed and their physicochemical properties, immune complexes may or may not result in host and foreign cell damage. After antigen exposure, certain types of soluble antigen-antibody complexes freely circulate and, if not cleared by the reticuloendothelial system, can be deposited in blood vessel walls and in other tissues such as renal glomeruli and cause vasculitis or glomerulonephritis syndromes (Chaps. 338 and 385). Deficiencies of early complement components are associated with inefficient clearance of immune complexes and immune complex mediated tissue damage in autoimmune syndromes, whereas deficiencies of the later complement components are associated with susceptibility to recurrent Neisseria infections (Table 372e-16).

1	Disease Key Effector Cell L-Selectin, Ligand GPCR Integrina aVarious β1 integrins have been linked in different ways in basal lamina and interstitial migration of distinct cell types and inflammatory settings. LFA-1 VLA-4, LFA-1 VLA-4, LFA-1 VLA-4, LFA-1 bIn some settings, Mac-1 has been linked to transmigration. cCD44 can act in concert with VLA-4 in particular models of leukocyte arrest. dTH2 cells require VAP-1 to traffic to inflamed liver. Source: From AD Luster et al: Nat Immunol 6:1182, 2005; with permission from Macmillan Publishers Ltd. Copyright 2005. Introduction to the Immune System Clq, Clr, Cls, C4 C2 C1 inhibitor Immune-complex syndromes,a pyogenic infections Immune-complex syndromes,a few with pyogenic infections Rare immune-complex disease, few with pyogenic infections

1	Clq, Clr, Cls, C4 C2 C1 inhibitor Immune-complex syndromes,a pyogenic infections Immune-complex syndromes,a few with pyogenic infections Rare immune-complex disease, few with pyogenic infections Immune-complex syndromes,a pyogenic infections Pyogenic infections Neisseria infections Pyogenic infections Hemolytic-uremic syndrome aImmune-complex syndromes include systemic lupus erythematosus (SLE) and SLE-like syndromes, glomerulonephritis, and vasculitis syndromes. Source: After JA Schifferli, DK Peters: Lancet 322:957, 1983. Copyright 1983, with permission from Elsevier.

1	Immediate-Type Hypersensitivity Helper T cells that drive antiallergen IgE responses are usually TH2-type inducer T cells that secrete IL-4, IL-5, IL-6, and IL-10. Mast cells and basophils have high-affinity receptors for the Fc portion of IgE (FcRI), and cell-bound antiallergen IgE effectively “arms” basophils and mast cells. Mediator release is triggered by antigen (allergen) interaction with Fc receptor-bound IgE, and the mediators released are responsible for the pathophysiologic changes of allergic diseases (Table 372e-11). Mediators released from mast cells and basophils can be divided into three broad functional types: (1) those that increase vascular permeability and contract smooth muscle (histamine, platelet-activating factor, SRS-A, BK-A), (2) those that are chemotactic for or activate other inflammatory cells (ECF-A, NCF, leukotriene B4), and (3) those that modulate the release of other mediators (BK-A, platelet-activating factor) (Chap. 376).

1	Cytotoxic reactions of antibody In this type of immunologic injury, complement-fixing (C1-binding) antibodies against normal or foreign cells or tissues (IgM, IgG1, IgG2, IgG3) bind complement via the classic pathway and initiate a sequence of events similar to that initiated by immune-complex deposition, resulting in cell lysis or tissue injury. Examples of antibody-mediated cytotoxic reactions include red cell lysis in transfusion reactions, Goodpasture’s syndrome with anti– glomerular basement membrane antibody formation, and pemphigus vulgaris withantiepidermalantibodiesinducingblisteringskindisease.

1	Classic Delayed-Type Hypersensitivity reactions Inflammatory reactions initiated by mononuclear leukocytes and not by antibody alone have been termed delayed-type hypersensitivity reactions. The term delayed has been used to contrast a secondary cellular response that appears 48–72 h after antigen exposure with an immediate hypersensitivity response generally seen within 12 h of antigen challenge and initiated by basophil mediator release or preformed antibody. For example, in an individual previously infected with M. tuberculosis organisms, intradermal placement of tuberculin purified protein derivative as a skin test challenge results in an indurated area of skin at 48–72 h, indicating previous exposure to tuberculosis.

1	The cellular events that result in classic delayed-type hypersensitivity responses are centered on T cells (predominantly, although not exclusively, IFN-γ, IL-2, and TNF-α-secreting TH1-type helper T cells) and macrophages. Recently NK cells have been suggested to play a major role in the form of delayed hypersensitivity that occurs following skin contact with immunogens. First, local immune and inflammatory responses at the site of foreign antigen upregulate endothelial cell adhesion molecule expression, promoting the accumulation of lymphocytes at the tissue site. In the general schemes outlined in Figs. 372e-2 and 372e-3, antigen is processed by DCs and presented to small numbers of CD4+ T cells expressing a TCR specific for the antigen. IL-12 produced by APCs induces T cells to produce IFN-γ (TH1 response). Macrophages frequently undergo epithelioid cell transformation and fuse to form multinucleated giant cells in response to IFN-γ. This type of mononuclear cell infiltrate is

1	IFN-γ (TH1 response). Macrophages frequently undergo epithelioid cell transformation and fuse to form multinucleated giant cells in response to IFN-γ. This type of mononuclear cell infiltrate is termed granulomatous inflammation. Examples of diseases in which delayed-type hypersensitivity plays a major role are fungal infections (histoplasmosis; Chap. 236), mycobacterial infections (tuberculosis, leprosy; Chaps. 202 and 203), chlamydial infections (lymphogranuloma venereum; Chap. 213), helminth infections (schistosomiasis; Chap. 259), reactions to toxins (berylliosis; Chap. 311), and hypersensitivity reactions to organic dusts (hypersensitivity pneumonitis; Chap. 310). In addition, delayed-type hypersensitivity responses play important roles in tissue damage in autoimmune diseases such as rheumatoid arthritis, temporal arteritis, and granulomatosis with polyangiitis (Wegener’s) (Chaps. 380 and 385).

1	Clinical assessment of immunity requires investigation of the four major components of the immune system that participate in host defense and in the pathogenesis of autoimmune diseases: (1) humoral immunity (B cells); (2) cell-mediated immunity (T cells, monocytes); (3) phagocytic cells of the reticuloendothelial system (macrophages), as well as polymorphonuclear leukocytes; and (4) complement. Clinical problems that require an evaluation of immunity include chronic infections, recurrent infections, unusual infecting agents, and certain autoimmune syndromes. The type of clinical syndrome under evaluation can provide information regarding possible immune defects (Chap. 374). Defects in cellular immunity generally result in viral, mycobacterial, and fungal infections. An extreme example of deficiency in cellular immunity is AIDS (Chap. 226). Antibody deficiencies result in recurrent bacterial infections, frequently with organisms such as S. pneumoniae and Haemophilus influenzae

1	of deficiency in cellular immunity is AIDS (Chap. 226). Antibody deficiencies result in recurrent bacterial infections, frequently with organisms such as S. pneumoniae and Haemophilus influenzae (Chap. 374). Disorders of phagocyte function are frequently manifested by recurrent skin infections, often due to Staphylococcus aureus (Chap. 80). Finally, deficiencies of early and late complement components are associated with autoimmune phenomena and recurrent Neisseria infections (Table 372e-16). For further discussion of useful initial screening tests of immune function, see Chap. 374.

1	Many therapies for autoimmune and inflammatory diseases involve the use of nonspecific immune-modulating or immunosuppressive agents such as glucocorticoids or cytotoxic drugs. The goal of development of new treatments for immune-mediated diseases is to design ways to specifically interrupt pathologic immune responses, leaving nonpathologic immune responses intact. Novel ways to interrupt pathologic immune responses that are under investigation include the use of anti-inflammatory cytokines or specific cytokine inhibitors as anti-inflammatory agents, the use of monoclonal antibodies against T or B lymphocytes as therapeutic agents, the use of intravenous Ig for certain infections and immune complex–mediated diseases, the use of specific cytokines to reconstitute components of the immune system, and bone marrow transplantation to replace the pathogenic immune system with a more normal immune system (Chaps. 80, 374, and 226). In particular, the use of a monoclonal antibody to B cells

1	system, and bone marrow transplantation to replace the pathogenic immune system with a more normal immune system (Chaps. 80, 374, and 226). In particular, the use of a monoclonal antibody to B cells (rituximab, anti-CD20 MAb) is approved in the United States for the treatment of non-Hodgkin’s lymphoma (Chap. 134) and, in combination with methotrexate, for treatment of adult patients with severe rheumatoid arthritis resistant to TNF-α inhibitors (Chap. 380). The U.S. Food and Drug Administration (FDA) approved the use of CTLA-4 antibodies in 2010 to block T cell anergy for use in cancer immunotherapy, and it was the first agent to demonstrate survival benefit in patients with advanced melanoma. Early-stage clinical trials have now shown that PD-1 blockade to reverse T cell exhaustion can induce tumor regression.

1	Cell-based therapies have been studied for many years, including ex vivo activation of NK cells for reinfusion into patients with malignancies and DC therapy of ex vivo priming of DCs for enhanced presentationofcancerantigens,withreinfusionofprimedDCsintothepatient. One such strategy for DC therapy has been approved by the FDA for treatment of advanced prostate cancer.

1	Cytokines and Cytokine Inhibitors Several TNF inhibitors are used as biological therapies in the treatment of rheumatoid arthritis; these include monoclonal antibodies, TNF-R Fc fusion proteins, and Fab fragments. Use of anti-TNF-α antibody therapies such as adalimumab, infliximab, and golimumab has resulted in clinical improvement in patients with these diseases and has opened the way for targeting TNF-α to treat other severe forms of autoimmune and/or inflammatory disease. Blockage of TNF-α has been effective in rheumatoid arthritis, psoriasis, Crohn’s disease, and ankylosing spondylitis. Other cytokine inhibitors are recombinant soluble TNF-α receptor (R) fused to human Ig and anakinra (soluble IL-1 receptor antagonist, or IL-1ra). The treatment of autoinflammatory syndromes (Table 372e-6) with recombinant IL-1 receptor antagonist can prevent symptoms in these syndromes, because the overproduction of IL-1β is a hallmark of these diseases.

1	TNF-αR-Fc fusion protein (etanercept) and IL-1ra act to inhibit the activity of pathogenic cytokines in rheumatoid arthritis, i.e., TNF-α and IL-1, respectively. Similarly, anti-IL-6, IFN-β, and IL-11 act to inhibit pathogenic proinflammatory cytokines. Anti-IL-6 (tocilizumab) inhibits IL-6 activity, whereas IFN-β and IL-11 decrease IL-1 and TNF-α production. Of particular note has been the successful use of IFN-γ in the treatment of the phagocytic cell defect in chronic granulomatous disease (Chap. 80).

1	Monoclonal antibodies to T and B Cells The OKT3 MAb against human T cells has been used for several years as a T cell-specific immunosuppressive agent that can substitute for horse antithymocyte globulin (ATG) in the treatment of solid organ transplant rejection. OKT3 produces fewer allergic reactions than ATG but does induce human anti-mouse Ig antibody—thus limiting its use. Anti-CD4 MAb therapy has been used in trials to treat patients with rheumatoid arthritis. While inducing profound immunosuppression, anti-CD4 MAb treatment also induces susceptibility to severe infections. Treatment of patients with a MAb against the T cell molecule CD40 ligand (CD154) is under investigation to induce tolerance to organ transplants, with promising results reported in animal studies. Monoclonal antibodies to the CD25 (IL-2a) receptor (basiliximab) are being used for treatment of graft-versus-host disease in bone marrow transplantation, and anti-CD20 MAb (rituximab) is used to treat hematologic

1	to the CD25 (IL-2a) receptor (basiliximab) are being used for treatment of graft-versus-host disease in bone marrow transplantation, and anti-CD20 MAb (rituximab) is used to treat hematologic neoplasms, autoimmune diseases, kidney transplant rejection, and rheumatoid arthritis. The anti-IgE monoclonal antibody (omalizumab) is used for blocking antigen-specific IgE that causes hay fever and allergic rhinitis (Chap. 376); however, side effects of anti-IgE include increased risk of anaphylaxis. Studies have shown that TH17 cells, in addition to TH1, are mediators of inflammation in Crohn’s disease, and anti–IL-12/ IL-23p40 antibody therapy has been studied as a treatment.

1	It is important to realize the potential risks of these immunosuppressive monoclonal antibodies. Natalizumab is a humanized IgG antibody against an a4 integrin that inhibits leukocyte migration into tissues and has been approved for treatment of multiple sclerosis in the United States. Both it and anti-CD20 (rituximab) have been associated with the onset of progressive multifocal leukoencephalopathy (PML)—a serious and usually fatal CNS infection caused by JC polyomavirus. Efalizumab, a humanized IgG monoclonal antibody previously approved for treatment of plaque psoriasis, has now been taken off the market due to reactivation of JC virus leading to fatal PML. Thus, use of any currently approved immunosuppressant immunotherapies should be undertaken with caution and with careful monitoring of patients according to FDA guidelines.

1	Tolerance Induction Specific immunotherapy has moved into a new erawiththe introduction of solubleCTLA-4 protein intoclinical trials. Use of this molecule to block T cell activation via TCR/CD28 ligation during organ or bone marrow transplantation has showed promising results in animals and in early human clinical trials. Specifically, treatment of bone marrow with CTLA-4 protein reduces rejection of the graft in HLA-mismatched bone marrow transplantation. In addition, promising results with soluble CTLA-4 have been reported in the downmodulation of autoimmune T cell responses in the treatment of psoriasis; and it is being studied for treatment of systemic lupus erythematosus (Chap. 378).

1	Intravenous Immunoglobulin (IVIg) IVIg has been used successfully to block reticuloendothelial cell function and immune complex clearance in various immune cytopenias such as immune thrombocytopenia (Chap. 140). In addition, IVIg is useful for prevention of tissue damage in certain inflammatory syndromes such as Kawasaki disease (Chap. 385) and as Ig replacement therapy for certain types of immunoglobulin deficiencies (Chap. 374). In addition, controlled clinical trials support the useof IVIginselectedpatientswith graft-versus-host disease, multiple sclerosis, myasthenia gravis, Guillain-Barré syndrome, and chronic demyelinating polyneuropathy.

1	Stem Cell Transplantation Hematopoietic stem cell transplantation (SCT) is now being comprehensively studied to treat several autoimmune diseases, including systemic lupus erythematosus, multiple sclerosis, and scleroderma. The goal of immune reconstitution in autoimmune disease syndromes is to replace a dysfunctional immune system with a normally reactive immune cell repertoire. Preliminary results in patients with scleroderma and lupus have showed encouraging results. Controlledclinicaltrialsinthesethreediseasesarenowbeinglaunched in the United States and Europe to compare the toxicity and efficacy of conventional immunosuppression therapy with that of myeloablative autologous SCT. Recently, SCT was used in the setting of HIV-1 infection. HIV-1 infection of CD4+ T cells requires the presence of surface CD4 receptor and the chemokine receptor 5 (CCR5) co-receptor. Studies have demonstrated that patients who are homozygous for a 32-bp deletion in the CCR5 allele do not express CD4+ T

1	of surface CD4 receptor and the chemokine receptor 5 (CCR5) co-receptor. Studies have demonstrated that patients who are homozygous for a 32-bp deletion in the CCR5 allele do not express CD4+ T cell CCR5 and thus are resistant to HIV-1 infection with HIV-1 strains that use this co-receptor. Stem cells from a homozygous CCR5 delta32 donor were transplanted to an HIV-infected patient following standard conditioning for such transplants, and the patient has maintained longterm control of the virus without antiretrovirals. Thus, a number of recentinsightsintoimmune system function havespawned anewfield of interventional immunotherapy and have enhanced the prospect for development of more specific and nontoxic therapies for immune and inflammatory diseases.

1	Introduction to the Immune System the Major histocompatibility Complex Gerald T. Nepom THE HLA COMPLEX AND ITS PRODUCTS The human major histocompatibility complex (MHC), commonly 373e called the human leukocyte antigen (HLA) complex, is a 4-megabase (Mb) region on chromosome 6 (6p21.3) that is densely packed with expressed genes. The best known of these genes are the HLA class I and class II genes, whose products are critical for immunologic specificity and transplantation histocompatibility, and they play a major role in susceptibility to a number of autoimmune diseases. Many other genes in the HLA region are also essential to the innate and antigen-specific functioning of the immune system. The HLA region shows extensive conservation with the MHC of other mammals in terms of genomic organization, gene sequence, and protein structure and function. The HLA class I genes are located in a 2-Mb stretch of DNA at the telomeric end of the HLA region (Fig. 373e-1). The classic (MHC class

1	gene sequence, and protein structure and function. The HLA class I genes are located in a 2-Mb stretch of DNA at the telomeric end of the HLA region (Fig. 373e-1). The classic (MHC class Ia) HLA-A, -B, and -C loci, the products of which are integral par-ticipants in the immune response to intracellular infections, tumors, and allografts, are expressed in all nucleated cells and are highly poly-morphic in the population. Polymorphism refers to a high degree of allelic variation within a genetic locus that leads to extensive variation between different individuals expressing different alleles. More than 2000 alleles at HLA-A, nearly 3000 alleles at HLA-B, and more than 1700 at HLA-C have been identified in different human populations, making this the most highly polymorphic segment known within the human genome. Each of the alleles at these loci encodes a heavy chain (also called an α chain) that associates noncovalently with the nonpoly-morphic light chain β2-microglobulin, encoded on

1	the human genome. Each of the alleles at these loci encodes a heavy chain (also called an α chain) that associates noncovalently with the nonpoly-morphic light chain β2-microglobulin, encoded on chromosome 15. The nomenclature of HLA genes and their products is based on a revised World Health Organization (WHO) nomenclature, in which alleles are given a single designation that indicates locus, allotype, and sequence-based subtype. For example, HLA-A*02:01 indicates subtype 1 of a group of alleles that encode HLA-A2 molecules. Subtypes that differ from each other at the nucleotide but not the amino acid sequence level are designated by an extra numeral (e.g., HLA-B*07:02:01 and HLA-B*07:02:02 are two variants of HLA-B*07:02, both encoding the same HLA-B7 molecule). The nomenclature of class II genes, discussed below, is made more complicated by the fact that both chains of a class II molecule are encoded by closely linked HLA-encoded loci, each of which may be polymorphic, and by the

1	II genes, discussed below, is made more complicated by the fact that both chains of a class II molecule are encoded by closely linked HLA-encoded loci, each of which may be polymorphic, and by the presence of differing numbers of isotypic DRB loci in different and provides inhibitory signals to both NK cells and T cells, presumably in the service of maintaining maternofetal tol-erance. Pathologic expression in cancer and infections may also deliver a simi-lar inhibitory immunologic function; 16 HLA-G alleles have been identified. The protein product of HLA-F is found mainly intracellularly, and the function of this locus, which encodes four alleles but has multiple transcriptional varia-tions, remains largely unknown. Additional class I–like genes have been identified, some HLA-linked and some encoded on other chromosomes, that show only distant homology to the class Ia and Ib molecules but share the three-dimensional class I structure.

1	genes have been identified, some HLA-linked and some encoded on other chromosomes, that show only distant homology to the class Ia and Ib molecules but share the three-dimensional class I structure. 0RPS18RXRBDPB2DNADMALMP2TAP1DPB1COLIIA2MICABCTUBBEAGFMOGHFEMICCPOU5F1TCF19DPA2DPA1LMP7DQB1DRB1DRB2DRAC4BLTAMICBC4ACYP21BTAP250010001500200020003000350040002500DMBDOBDQA2RAGELPAATBfC2CK11˜DQB2DQA1DRB3FIgURE 373e-1 Physical map of the HLA region, showing the class I and class II loci, other immu-nologically important loci, and a sampling of other genes mapped to this region. Gene orientation is indicated by arrowheads. Scale is in kilobase (kb). The approximate genetic distance from DP to A is 3.2 cM. This includes 0.8 cM between A and B (including 0.2 cM between C and B), 0.4–0.8 cM between B and DR-DQ, and 1.6–2.0 cM between DR-DQ and DP.

1	individuals. It has become clear that accurate HLA genotyping requires DNA sequence analysis, and the identification of alleles at the DNA sequence level has contributed greatly to the understanding of the role of HLA molecules as peptide-binding ligands, to the analysis of associations of HLA alleles with certain diseases, to the study of the population genetics of HLA, and to a clearer understanding of the contribution of HLA differences to allograft rejection and graft-versushost disease. Current databases of HLA class I and class II sequences can be accessed by the Internet (e.g., from the IMGT/HLA Database, http://www.ebi.ac.uk/imgt/hla), and frequent updates of HLA gene lists are published in several journals.

1	The biologic significance of this MHC genetic diversity, resulting in extreme variation in the human population, is evident from the perspective of the structure of MHC molecules. As shown in Fig. 373e-2, the MHC class I and class II genes encode MHC molecules that bind small peptides, and together this complex (pMHC; peptide-MHC) forms the ligand for recognition by T lymphocytes, through the antigen-specific T cell receptor (TCR). There is a direct link between the genetic variation and this structural interaction: The allelic changes in genetic sequence result in diversification of the peptide-binding capabilities of each MHC molecule and in differences for specific TCR binding. Thus, different pMHC complexes bind different antigens and are targets for recognition by different T cells.

1	The class I MHC and class II MHC structures, shown in Fig. 373e-2 B, C, are structurally closely related; however, there are a few key differences. While both bind peptides and present them to T cells, the binding pockets have different shapes, which influence the types of immune responses that result (discussed below). In addition, there are structural contact sites for T cell molecules known as CD8 and CD4, expressed on the class I or class II membrane-proximal domains, respectively. This ensures that when peptide antigens are presented by class I molecules, the responding T cells are predominantly of the CD8 class, and similarly, that T cells responding to class II pMHC complexes are predominantly CD4.

1	The nonclassic, or class Ib, MHC molecules, HLA-E, -F, and -G, are much less polymorphic than MHC Ia and appear to have distinct functions. The HLA-E molecule has a peptide repertoire displaying signal peptides cleaved from classic MHC class I molecules and is the major self-recognition target for the natural killer (NK) cell–inhibitory receptors NKG2A or NKG2C paired with CD94 (see below and Chap. 372e). This appears to be a function of immune surveillance, because loss of MHC class I signal peptides serves as a surrogate marker for injured or infected cells, leading to release of the inhibitory signal and subsequent activation of NK cells. HLA-E can also bind and present peptides to CD8 T cells, albeit with a limited scope, as only three HLA-E alleles are known. HLA-G is expressed mainly in stem cells and in extravillous trophoblasts, the fetal cell population directly in contact with maternal tissues. It binds a wide array of peptides, is expressed inLTB six different alternatively

1	in stem cells and in extravillous trophoblasts, the fetal cell population directly in contact with maternal tissues. It binds a wide array of peptides, is expressed inLTB six different alternatively spliced forms,

1	Chapter 373e The Major Histocompatibility Complex FIgURE 373e-2 A. The trimolecular complex of TCR (top), MHC molecule (bottom), and a bound peptide form the structural determinants of specific antigen recognition. Other panels (B and C) show the domain structure of MHC class I (B) and class II (C) molecules. The α1 and α2 domains of class I and the α1 and β1 domains of class II form a β-sheet platform that forms the floor of the peptide-binding groove, and α helices that form the sides of the groove. The α3 (B) and β2 domains (C) project from the cell surface and form the contact sites for CD8 and CD4, respectively. (Adapted from EL Reinherz et al: Science 286:1913, 1999; and C Janeway et al: Immunobiology Bookshelf, 2nd ed. Garland Publishing, New York, 1997; with permission.)

1	Those on chromosome 6p21 include MIC-A and MIC-B, which are encoded centromeric to HLA-B, and HLA-HFE, located 3 to 4 cM (centi-Morgan) telomeric of HLA-F. MIC-A and MIC-B do not bind peptide but are expressed on gut and other epithelium in a stress-inducible manner and serve as activation signals for certain γδ T cells, NK cells, CD8 T cells, and activated macrophages, acting through the activating NKG2D receptors. Ninety-one MIC-A and 40 MIC-B alleles are known, and additional diversification comes from variable alanine repeat sequences in the transmembrane domain. Due to this structural diversity, MIC-A can be recognized as a foreign tissue target during organ transplantation, contributing to graft failure. HLA-HFE encodes the gene defective in hereditary hemochromatosis (Chap. 428). Among the non-HLA, class I–like genes, CD1 refers to a family of molecules that present glycolipids or other nonpeptide ligands to certain T cells, including T cells with NK activity; FcRn binds IgG

1	Among the non-HLA, class I–like genes, CD1 refers to a family of molecules that present glycolipids or other nonpeptide ligands to certain T cells, including T cells with NK activity; FcRn binds IgG within lysosomes and protects it from catabolism (Chap. 372e); and Zn-α2-glycoprotein 1 binds a nonpeptide ligand and promotes catabolism of triglycerides in adipose tissue. Like the HLA-A, -B, -C, -E, -F, and -G heavy chains, each of which forms a heterodimer with β2-microglobulin (Fig. 373e-2), the class I–like molecules, HLA-HFE, FcRn, and CD1 also bind to β2-microglobulin, but MIC-A, MIC-B, and Zn-α2-glycoprotein 1 do not.

1	The HLA class II region is also illustrated in Fig. 373e-1. Multiple class II genes are arrayed within the centromeric 1 Mb of the HLA region, forming distinct haplotypes. A haplotype refers to an array of alleles at polymorphic loci along a chromosomal segment. Multiple class II genes are present on a single haplotype, clustered into three major subregions: HLA-DR, -DQ, and -DP. Each of these subregions contains at least one functional alpha (A) locus and one functional beta (B) locus. Together these encode proteins that form the α and β polypeptide chains of a mature class II HLA molecule. Thus, the DRA and DRB genes encode an HLA-DR molecule; DQA and DQB genes encode HLA-DQ molecules; and DPA and DPB genes encode HLA-DP molecules. There are several DRB genes (DRB1, DRB2, DRB3, etc.), so that two expressed DR molecules are encoded on most haplotypes by combining the α-chain product of the DRA gene with separate β chains. More than 1000 alleles have been identified at the HLA-DRB1

1	so that two expressed DR molecules are encoded on most haplotypes by combining the α-chain product of the DRA gene with separate β chains. More than 1000 alleles have been identified at the HLA-DRB1 locus, with most of the variation occurring within limited segments encoding residues that interact with antigens. Detailed analysis of sequences and population distribution of these alleles strongly suggest that this diversity is actively selected by environmental pressures associated with pathogen diversity. In the DQ region, both DQA1 and DQB1 are polymorphic, with 50 DQA1 alleles and over 300 DQB1 alleles. The current nomenclature is largely analogous to that discussed above for class I, using the convention “locus * allele.”

1	In addition to allelic polymorphism, products of different DQA alleles can, with some limitations, pair with products of different DQB alleles through both cis and trans pairing to create combinatorial complexity and expand the number of expressed class II molecules. Because of the enormous allelic diversity in the general population, most individuals are heterozygous at all of the class I and class II loci. Thus, most individuals express six classic class I molecules (two each of HLA-A, -B, and -C) and many class II molecules—two DP, two to four DR, and multiple DQ (both cis and trans dimers).

1	In addition to the class I and class II genes themselves, there are numerous genes interspersed among the HLA loci that have interesting and important immunologic functions. Our current concept of the function of MHC genes now encompasses many of these additional genes, some of which are also highly polymorphic. Indeed, direct comparison of the complete DNA sequences for eight of the entire 4-Mb MHC regions from different haplotypes show >44,000 nucleotide variations, encoding an extremely high potential for biologic diversity, and at least 97 genes located in this region are known to have coding region sequence variation. Specific examples include the TAP and LMP genes, as discussed in more detail below, which encode molecules that participate in intermediate steps in the HLA class I biosynthetic pathway. Another set of HLA genes, DMA and DMB, perform an analogous function for the class II pathway. These genes encode an intracellular molecule that facilitates the proper complexing of

1	pathway. Another set of HLA genes, DMA and DMB, perform an analogous function for the class II pathway. These genes encode an intracellular molecule that facilitates the proper complexing of HLA class II molecules with antigen (see below). The HLA class III region is a name given to a cluster of genes between the class I and class II complexes, which includes genes for the two closely related cytokines tumor necrosis factor (TNF)-α and lymphotoxin (TNF-β); the complement components C2, C4, and Bf; heat shock protein (HSP) 70; and the enzyme 21-hydroxylase.

1	The class I genes HLA-A, -B, and -C are expressed in all nucleated cells, although generally to a higher degree on leukocytes than on nonleukocytes. In contrast, the class II genes show a more restricted distribution: HLA-DR and HLA-DP genes are constitutively expressed on most cells of the myeloid cell lineage, whereas all three class II gene families (HLA-DR, -DQ, and -DP) are inducible by certain stimuli provided by inflammatory cytokines such as interferon γ. Within the lymphoid lineage, expression of these class II genes is constitutive on B cells and inducible on human T cells. Most endothelial and epithelial cells in the body, including the vascular endothelium and the intestinal epithelium, are also inducible for class II gene expression, and some cells show specialized expression, such as HLA-DQA2 and HLA-DQB2 on Langerhans cells. While somatic tissues normally express only class I and not class II genes, during times of local inflammation, they are recruited by cytokine

1	such as HLA-DQA2 and HLA-DQB2 on Langerhans cells. While somatic tissues normally express only class I and not class II genes, during times of local inflammation, they are recruited by cytokine stimuli to express class II genes as well, thereby becoming active participants in ongoing immune responses. Class II expression is controlled largely at the transcriptional level through a conserved set of promoter elements that interact with a protein known as CIITA. Cytokine-mediated induction of CIITA is a principal method by which tissue-specific expression of HLA gene expression is controlled. Other HLA genes involved in the immune response, such as TAP and LMP, are also susceptible to upregulation by signals such as interferon γ.

1	In addition to extensive polymorphism at the class I and class II loci, another characteristic feature of the HLA complex is linkage disequilibrium. This is formally defined as a deviation from Hardy-Weinberg equilibrium for alleles at linked loci. This is reflected in the very low recombination rates between certain loci within the HLA complex. For example, recombination between DR and DQ loci is almost never observed in family studies, and characteristic haplotypes with particular arrays of DR and DQ alleles are found in every population. Similarly, the complement components C2, C4, and Bf are almost invariably inherited together, and the alleles at these loci are found in characteristic haplotypes. In contrast, there is a recombinational hotspot between DQ and DP, which are separated by 1–2 cM of genetic distance, despite their close physical proximity. Certain extended haplotypes encompassing the interval from DQ into the class I region are commonly found, the most notable being

1	by 1–2 cM of genetic distance, despite their close physical proximity. Certain extended haplotypes encompassing the interval from DQ into the class I region are commonly found, the most notable being the haplotype DR3-B8-A1, which is found, in whole or in part, in 10–30% of northern European whites. It has been hypothesized that selective pressures may maintain linkage disequilibrium in HLA, but this remains to be determined. As discussed below under HLA and immunologic disease, one consequence of the phenomenon of linkage disequilibrium has been the resulting difficulty in assigning HLA-disease associations to a single allele at a single locus.

1	Class I and class II molecules display a distinctive structural architecture, which contains specialized functional domains responsible for the unique genetic and immunologic properties of the HLA complex. The principal known function of both class I and class II HLA molecules is to bind antigenic peptides in order to present antigen to an appropriate T cell. The ability of a particular peptide to satisfactorily bind to an individual HLA molecule is a direct function of the molecular fit between the amino acid residues on the peptide with respect to the amino acid residues of the HLA molecule. The bound peptide forms a tertiary structure called the MHC-peptide complex, which communicates with T lymphocytes through binding to the TCR molecule. The first site of TCR-MHC-peptide interaction in the life of a T cell occurs in the thymus, where self-peptides are presented to developing thymocytes by MHC molecules expressed on thymic epithelium and hematopoietically derived

1	interaction in the life of a T cell occurs in the thymus, where self-peptides are presented to developing thymocytes by MHC molecules expressed on thymic epithelium and hematopoietically derived antigen-presenting cells, which are primarily responsible for positive and negative selection, respectively (Chap. 372e). Thus, the population of MHC–T cell complexes expressed in the thymus shapes the TCR repertoire. Mature T cells encounter MHC molecules in the periphery both in the maintenance of tolerance (Chap. 377e) and in the initiation of immune responses. The MHCpeptide-TCR interaction is the central event in the initiation of most antigen-specific immune responses, since it is the structural determinant of the specificity. For potentially immunogenetic peptides, the ability of a given peptide to be generated and bound by an HLA molecule is a primary feature of whether or not an immune response to that peptide can be generated, and the repertoire of peptides that a particular

1	given peptide to be generated and bound by an HLA molecule is a primary feature of whether or not an immune response to that peptide can be generated, and the repertoire of peptides that a particular individual’s HLA molecules can bind exerts a major influence over the specificity of that individual’s immune response.

1	When a TCR molecule binds to an HLA-peptide complex, it forms 373e-3 intermolecular contacts with both the antigenic peptide and with the HLA molecule itself. The outcome of this recognition event depends on the density and duration of the binding interaction, accounting for a dual specificity requirement for activation of the T cell. That is, the TCR must be specific both for the antigenic peptide and for the HLA molecule. The polymorphic nature of the presenting molecules, and the influence that this exerts on the peptide repertoire of each molecule, results in the phenomenon of MHC restriction of the T cell specificity for a given peptide. The binding of CD8 or CD4 molecules to the class I or class II molecule, respectively, also contributes to the interaction between T cell and the HLA-peptide complex, by providing for the selective activation of the appropriate T cell.

1	(Fig. 373e-2B) As noted above, MHC class I molecules provide a cell-surface display of peptides derived from intracellular proteins, and they also provide the signal for self-recognition by NK cells. Surface-expressed class I molecules consist of an MHC-encoded 44-kD glycoprotein heavy chain, a non-MHC-encoded 12-kD light chain β2-microglobulin, and an antigenic peptide, typically 8–11 amino acids in length and derived from intracellularly produced protein. The heavy chain displays a prominent peptide-binding groove. In HLA-A and -B molecules, the groove is ∼3 nm in length by 1.2 nm in maximumwidth (30 Å × 12 Å), whereas it is apparently somewhat wider in HLA-C. Antigenic peptides are noncovalently bound in an extended conformation within the peptide-binding groove, with both Nand C-terminal ends anchored in pockets within the groove (A and F pockets, respectively) and, in many cases, with a prominent kink, or arch, approximately one-third of the way from the N-terminus that elevates

1	ends anchored in pockets within the groove (A and F pockets, respectively) and, in many cases, with a prominent kink, or arch, approximately one-third of the way from the N-terminus that elevates the peptide main chain off the floor of the groove.

1	A remarkable property of peptide binding by MHC molecules is the ability to form highly stable complexes with a wide array of peptide sequences. This is accomplished by a combination of peptide sequence– independent and peptide sequence–dependent bonding. The former consists of hydrogen bond and van der Waals interactions between conserved residues in the peptide-binding groove and charged or polar atoms along the peptide backbone. The latter is dependent upon the six side pockets that are formed by the irregular surface produced by protrusion of amino acid side chains from within the binding groove. The side chains lining the pockets interact with some of the peptide side chains. The sequence polymorphism among different class I alleles and isotypes predominantly affects the residues that line these pockets, and the interactions of these residues with peptide residues constitute the sequence-dependent bonding that confers a particular sequence “motif” on the range of peptides that

1	line these pockets, and the interactions of these residues with peptide residues constitute the sequence-dependent bonding that confers a particular sequence “motif” on the range of peptides that can bind any given MHC molecule.

1	(Fig. 373e-3A) The biosynthesis of the classic MHC class I molecules reflects their role in presenting endogenous peptides. The heavy chain is cotranslationally inserted into the membrane of the endoplasmic reticulum (ER), where it becomes glycosylated and associates sequentially with the chaperone proteins calnexin and ERp57. It then forms a complex with β2-microglobulin, and this complex associates with the chaperone calreticulin and the MHC-encoded molecule tapasin, which physically links the class I complex to TAP, the MHC-encoded transporter associated with antigen processing. Meanwhile, peptides generated within the cytosol from intracellular proteins by the multisubunit, multicatalytic proteasome complex are actively transported into the ER by TAP, where they are trimmed by enzymes known as ER aminopeptidases. At this point, peptides with appropriate sequence complementarity bind specific class I molecules to form complete, folded heavy chain–β2microglobulin–peptide trimer

1	known as ER aminopeptidases. At this point, peptides with appropriate sequence complementarity bind specific class I molecules to form complete, folded heavy chain–β2microglobulin–peptide trimer complexes. These are transported rapidly from the ER, through the cis-and trans-Golgi where the N-linked oligosaccharide is further processed, and thence to the cell surface.

1	Most of the peptides transported by TAP are produced in the cytosol by proteolytic cleavage of intracellular proteins by the multisubunit, multicatalytic proteasome, and inhibitors of the proteasome dramatically reduce expression of class I–presented antigenic Chapter 373e The Major Histocompatibility Complex To cell surface Endoglycosidase H resistant Asn-linked oligosaccharide Endoglycosidase H sensitive Asn-linked oligosaccharide Degradation? alternative to calnexin Peptide trimming gp96TAP-independent peptides PDI Golgi ER lumen HSP70? HC/ribosome ERp57 HSP90?

1	FIgURE 373e-3 Biosynthesis of class I (A) and class II (B) molecules. A. Nascent heavy chain (HC) becomes associated with β2-microglobulin (β2m) and peptide through interactions with a series of chaperones. Peptides generated by the proteasome are transported into the endoplasmic reticulum (ER) by TAP. Peptides undergo N-terminal trimming in the ER and become associated with chaperones, including gp96 and PDI. Once peptide binds to HC-β2m, the HC-β2m-peptide trimeric complex exits the ER and is transported by the secretory pathway to the cell surface. In the Golgi, the N-linked oligosaccharide undergoes maturation, with addition of sialic acid residues. Molecules are not necessarily drawn to scale. B. Pathway of HLA class II molecule assembly and antigen processing. After transport through the Golgi and post-Golgi compartment, the class II–invariant chain complex moves to an acidic endosome, where the invariant chain is proteolytically cleaved into fragments and displaced by antigenic

1	the Golgi and post-Golgi compartment, the class II–invariant chain complex moves to an acidic endosome, where the invariant chain is proteolytically cleaved into fragments and displaced by antigenic peptides, facilitated by interactions with the DMA-DMB chaperone protein. This class II molecule–peptide complex is then transported to the cell surface.

1	peptides. A thiol-dependent oxidoreductase ERp57, which mediates disulfide bond rearrangements, also appears to play an important role in folding the class I–peptide complex into a stable multicomponent molecule. The MHC-encoded proteasome subunits LMP2 and LMP7 may influence the spectrum of peptides produced but are not essential for proteasome function.

1	CLASS I FUNCTION Peptide Antigen Presentation On any given cell, a class I molecule occurs in 100,000–200,000 copies and binds several hundred to several thousand distinct peptide species. The vast majority of these peptides are self-peptides to which the host immune system is tolerant by one or more of the mechanisms that maintain tolerance (e.g., clonal deletion in the thymus or clonal anergy or clonal ignorance in the periphery [Chaps. 372e and 377e]). However, class I molecules bearing foreign peptides expressed in a permissive immunologic context activate CD8 T cells, which, if naïve, will then differentiate into cytolytic T lymphocytes (CTLs). These T cells and their progeny, through their αβ TCRs, are then capable of Fas/CD95and/or perforin-mediated cytotoxicity and/or cytokine secretion (Chap. 372e) upon further encounter with the class I–peptide combination that originally activated it, or other structurally related class I–peptide complexes. As alluded to above, this

1	secretion (Chap. 372e) upon further encounter with the class I–peptide combination that originally activated it, or other structurally related class I–peptide complexes. As alluded to above, this phenomenon by which T cells recognize foreign antigens in the context of specific MHC alleles is termed MHC restriction, and the specific MHC molecule is termed the restriction element. The most common source of foreign peptides presented by class I molecules is viral infection, in the course of which peptides from viral proteins enter the class I pathway. The generation of a strong CTL response that destroys virally infected cells represents an important antigen-specific defense against many viral infections (Chap. 372e). In the case of some viral infections—hepatitis B, for example—CTL-induced target cell apoptosis is thought to be a more important mechanism of tissue damage than any direct cytopathic effect of the virus itself. The importance of the class I pathway in the defense against

1	target cell apoptosis is thought to be a more important mechanism of tissue damage than any direct cytopathic effect of the virus itself. The importance of the class I pathway in the defense against viral infection is underscored by the identification of a number of viral products that interfere with the normal class I biosynthetic pathway and thus block the immunogenetic expression of viral antigens.

1	Other examples of intracellularly generated peptides that can be presented by class I molecules in an immunogenic manner include peptides derived from nonviral intracellular infectious agents (e.g., Listeria, Plasmodium), tumor antigens, minor histocompatibility antigens, and certain autoantigens. There are also situations in which cell surface–expressed class I molecules are thought to acquire and present exogenously derived peptides.

1	HLA Class I Receptors and NK Cell Recognition (Chap. 372e) NK cells, which play an important role in innate immune responses, are activated to cytotoxicity and cytokine secretion by contact with cells that lack MHC class I expression, and NK cell activation is inhibited by cells that express MHC class I. In humans, the recognition of class I molecules by NK cells is carried out by three classes of receptor families, the killer cell–inhibitory cell receptor (KIR) family, the leukocyte Ig-like receptor (LIR) family, and the CD94/NKG2 family. The KIR family, also called CD158, is encoded on chromosome 19q13.4. KIR gene nomenclature is based on the number of domains (2D or 3D) and the presence of long (L) or short (S) cytoplasmic domains. The KIR2DL1 and S1 molecules primarily recognize alleles of HLA-C, which possess a lysine at position 80 (HLA-Cw2, -4, -5, and -6), whereas the KIR2DL2/S2 and KIR2DL3/S3 families primarily recognize alleles of HLA-C with asparagine at this position

1	alleles of HLA-C, which possess a lysine at position 80 (HLA-Cw2, -4, -5, and -6), whereas the KIR2DL2/S2 and KIR2DL3/S3 families primarily recognize alleles of HLA-C with asparagine at this position (HLA-Cw1, -3, -7, and -8). The KIR3DL1 and S1 molecules predominantly recognize HLA-B alleles that fall into the HLA-Bw4 class determined by residues 77–83 in the α1 domain of the heavy chain, whereas the KIR3DL2 molecule is an inhibitory receptor for HLA-A*03. One of the KIR products, KIR2DL4, is known to be an activating receptor for HLA-G. The most common KIR haplotype in whites contains one activating KIR and six inhibitory KIR genes, although there is a great deal of diversity in the population, with >100 different combinations. It appears that most individuals have at least one inhibitory KIR for a self-HLA class I molecule, providing a structural basis for NK cell target specificity, which helps prevent NK cells from attacking normal cells. The importance of KIR-HLA interactions to

1	KIR for a self-HLA class I molecule, providing a structural basis for NK cell target specificity, which helps prevent NK cells from attacking normal cells. The importance of KIR-HLA interactions to many immune responses is illustrated by studies associating KIR3DL1 or S1 with multiple sclerosis (Chap. 458), an autoimmune disease, but also with partial protection against HIV (Chap. 226), in both cases consistent with a role for HLA-KIR mediated NK activation. Studies also show an association of KIR2DS1 with protection from relapse following allogeneic bone marrow transplantation in acute myeloid leukemia when these inhibitory receptors in the donors do not recognize the recipient HLA-C.

1	The LIR gene family (CD85, also called ILT) is encoded centromeric of the KIR locus on 19q13.4, and it encodes a variety of inhibitory immunoglobulin-like receptors expressed on many lymphocyte and other hematopoietic lineages. Interaction of LIR-1 (ILT2) with NK or T cells inhibits activation and cytotoxicity, mediated by many different HLA class I molecules, including HLA-G. HLA-F also appears to interact with LIR molecules, although the functional context for this is not understood.

1	The third family of NK receptors for HLA is encoded in the NK complex on chromosome 12p12.3-13.1 and consists of CD94 and five NKG2 genes, A/B, C, E/H, D, and F. These molecules are C-type (calcium-binding) lectins, and most function as disulfide-bonded heterodimers between CD94 and one of the NKG2 glycoproteins. The principal ligand of CD94/NKG2A receptors is the HLA-E molecule, complexed to a peptide derived from the signal sequence of classic HLA class I molecules and HLA-G. Thus, analogous to the way in which KIR receptors recognize HLA-C, the NKG2 receptor monitors self–class I expression, albeit indirectly through peptide recognition in the context of HLA-E. NKG2C, -E, and -H appear to have similar specificities but act as activating receptors. NKG2D is expressed as a homodimer and functions as an activating receptor expressed on NK cells, γδ TCR T cells, and activated CD8 T cells. When complexed with an adaptor called DAP10, NKG2D recognizes MIC-A and MIC-B molecules and

1	and functions as an activating receptor expressed on NK cells, γδ TCR T cells, and activated CD8 T cells. When complexed with an adaptor called DAP10, NKG2D recognizes MIC-A and MIC-B molecules and activates the cytolytic response. NKG2D also binds a class of molecules known as ULBP, structurally related to class I molecules but not encoded in the MHC. The function of NK cells in immune responses is discussed in Chap. 372e.

1	(Fig. 373e-2C) A specialized functional architecture similar to that of the class I molecules can be seen in the example of a class II molecule depicted in Fig. 373e-2C, with an antigen-binding cleft arrayed above a supporting scaffold that extends the cleft toward the external cellular environment. However, in contrast to the HLA class I molecular structure, β2-microglobulin is not associated with class II molecules. Rather, the class II molecule is a heterodimer, composed of a 29-kD α chain and a 34-kD β chain. The amino-terminal domains of each chain form the antigen-binding elements that, like the class I molecule, cradle a bound peptide in a groove bounded by extended α-helical loops, one encoded by the A (α chain) gene and one by the B (β chain) gene. Like the class I groove, the class II antigen-binding groove is punctuated by pockets that contact the side chains of amino acid residues of the bound peptide, but unlike the class I groove, it is open at both ends. Therefore,

1	class II antigen-binding groove is punctuated by pockets that contact the side chains of amino acid residues of the bound peptide, but unlike the class I groove, it is open at both ends. Therefore, peptides bound by class II molecules vary greatly in length, since both the Nand C-terminal ends of the peptides can extend through the open ends of this groove. Approximately 11 amino acids 373e-5 within the bound peptide form intimate contacts with the class II molecule itself, with backbone hydrogen bonds and specific side chain interactions combining to provide, respectively, stability and specificity to the binding (Fig. 373e-4).

1	The genetic polymorphisms that distinguish different class II genes correspond to changes in the amino acid composition of the class II molecule, and these variable sites are clustered predominantly around the pocket structures within the antigen-binding groove. As with class I, this is a critically important feature of the class II molecule, which explains how genetically different individuals have functionally different HLA molecules.

1	(Fig. 373e-3B) The intracellular assembly of class II molecules occurs within a specialized compartmentalized pathway that differs dramatically from the class I pathway described above. As illustrated in Fig. 373e-3B, the class II molecule assembles in the ER in association with a chaperone molecule, known as the invariant chain. The invariant chain performs at least two roles. First, it binds to the class II molecule and blocks the peptide-binding groove, thus preventing antigenic peptides from binding. This role of the invariant chain appears to account for one of the important differences between class I and class II MHC pathways, since it can explain why class I molecules present endogenous peptides from proteins newly synthesized in the ER but class II molecules generally do not. Second, the invariant chain contains molecular localization signals that direct the class II molecule to traffic into post-Golgi compartments known as endosomes, which develop into specialized acidic

1	Second, the invariant chain contains molecular localization signals that direct the class II molecule to traffic into post-Golgi compartments known as endosomes, which develop into specialized acidic compartments where proteases cleave the invariant chain, and antigenic peptides can now occupy the class II groove. The specificity and tissue distribution of these proteases appear to be an important way in which the immune system regulates access to the peptide-binding groove and T cells become exposed to specific self-antigens. Differences in protease expression in the thymus and in the periphery may in part determine which specific peptide sequences comprise the peripheral repertoire for T cell recognition. It is at this stage in the intracellular pathway, after cleavage of the invariant chain, that the MHC-encoded DM molecule catalytically facilitates the exchange of peptides within the class II groove to help optimize the specificity and stability of the MHC-peptide complex.

1	Once this MHC-peptide complex is deposited in the outer cell membrane, it becomes the target for T cell recognition via a specific TCR expressed on lymphocytes. Because the endosome environment contains internalized proteins retrieved from the extracellular environment, the class II–peptide complex often contains bound antigens that were originally derived from extracellular proteins. In this way, the class II peptide–loading pathway provides a mechanism for immune surveillance of the extracellular space. This appears to be an important feature that permits the class II molecule to bind foreign peptides, distinct from the endogenous pathway of class I–mediated presentation.

1	The development of modern clinical transplantation in the decades since the 1950s provided a major impetus for elucidation of the HLA system, as allograft survival is highest when donor and recipient are HLA-identical. Although many molecular events participate in transplantation rejection, allogeneic differences at class I and class II loci play a major role. Class I molecules can promote T cell responses in several different ways. In the cases of allografts in which the host and donor are mismatched at one or more class I loci, host T cells can be activated by classic direct alloreactivity, in which the antigen receptors on the host T cells react with the foreign class I molecule expressed on the allograft. In this situation, the response of any given TCR may be dominated by the allogeneic MHC molecule, the peptide bound to it, or some combination of the two. Another type of host anti-graft T cell response involves the uptake and processing of donor MHC antigens by host

1	the allogeneic MHC molecule, the peptide bound to it, or some combination of the two. Another type of host anti-graft T cell response involves the uptake and processing of donor MHC antigens by host antigen-presenting cells and the subsequent presentation of the resulting peptides by host MHC molecules. This mechanism is termed indirect alloreactivity.

1	Chapter 373e The Major Histocompatibility Complex

1	P1 Pro P3 Pro P5 Pro P6 Glu P8 Pro P9 Tyr P7 LeuP4 Gln P2 Phe Arg-˜52Tyr-˜22Asn-˜11Asn-˜62Asn-˜69Ser-°30Tyr-˜9Trp-°61Lys-°71Tyr-°9Asn-°82His-°81BCFIgURE 373e-4 Specific intermolecular interactions determine peptide binding to MHC class II molecules. A short peptide sequence derived from alpha-gliadin (A) is accommodated within the MHC class II binding groove by specific interactions between peptide side chains (the P1–P9 residues illustrated in B) and corresponding pockets in the MHC class II structure. The latter are determined by the genetic polymor-phisms of the MHC gene, in this case encoding an HLA-DQ2 molecule (C). This shows the extensive hydrogen bond and salt bridge network, which tightly constrains the pMHC complex and presents the complex of antigen and restriction element for CD4 T cell recognition. (From C Kim et al: Structural basis for HLA-DQ2-mediated presentation of gluten epitopes in celiac disease. Proc Natl Acad Sci USA 101:4175, 2004.)

1	In the case of class I molecules on allografts that are shared by the host and the donor, a host T cell response may still be triggered because of peptides that are presented by the class I molecules of the graft but not of the host. The most common basis for the existence of these endogenous antigen peptides, called minor histocompatibility antigens, is a genetic difference between donor and host at a non-MHC locus encoding the structural gene for the protein from which the peptide is derived. These loci are termed minor histocompatibility loci, and nonidentical individuals typically differ at many such loci. CD4 T cells react to analogous class II variation, both direct and indirect, and class II differences alone are sufficient to drive allograft rejection.

1	It has long been postulated that infectious agents provide the driving force for the allelic diversification seen in the HLA system. An important corollary of this hypothesis is that resistance to specific pathogens may differ between individuals, based on HLA genotype. Observations of specific HLA genes associated with resistance to malaria or dengue fever, persistence of hepatitis B, and to disease progression in HIV infection are consistent with this model. For example, failure to clear persistent hepatitis B or C viral infection may reflect the inability of particular HLA molecules to present viral antigens effectively to T cells. Similarly, both protective and susceptible HLA allelic associations have been described for human papilloma virus–associated cervical neoplasia, implicating the MHC as an influence in mediating viral clearance in this form of cancer.

1	Pathogen diversity is probably also the major selective pressure favoring HLA heterozygosity. The extraordinary scope of HLA allelic diversity increases the likelihood that most new pathogens will be recognized by some HLA molecules, helping to ensure immune fitness to the host. However, another consequence of diversification is that some alleles may become capable of recognition of “innocent bystander” molecules, including drugs, environmental molecules, and tissue-derived self-antigens. In a few instances, single HLA alleles display a strong selectivity for binding of a particular agent that accounts for a genetically determined response: Hypersensitivity to abacavir, an antiretroviral therapeutic, is directly linked to binding of abacavir in the antigen-binding pockets of HLA-B*57:01, where it is buried underneath antigenic peptides and distorts the landscape, changing T cell recognition specificity; an adverse drug reaction to abacavir is more than 500 times more likely to occur

1	where it is buried underneath antigenic peptides and distorts the landscape, changing T cell recognition specificity; an adverse drug reaction to abacavir is more than 500 times more likely to occur in persons with HLA-B*57:01 than in individuals without this HLA allele. Another example is chronic beryllium toxicity, which is linked to binding of beryllium by HLA-DP molecules with a specific glutamic acid polymorphic residue on the class II beta chain. Even in the case of more complex diseases, particular HLA alleles are strongly associated with certain inappropriate immune-mediated disease states, particularly for some common autoimmune disorders (Chap. 377e). By comparing allele frequencies in patients with any particular disease and in control populations, >100 such associations have been identified, some of which are listed in Table 373e-1. The strength of genetic association is reflected in the term relative risk, which is a statistical odds ratio representing the risk of disease

1	identified, some of which are listed in Table 373e-1. The strength of genetic association is reflected in the term relative risk, which is a statistical odds ratio representing the risk of disease for an individual carrying a particular genetic marker compared with the risk for individuals in that population without that marker. The nomenclature shown in Table 373e-1 reflects both the HLA serotype (e.g., DR3, DR4) and the HLA genotype (e.g., DRB1*03:01, DRB1*04:01). It very likely the class I and class II alleles themselves are the true susceptibility alleles for most of these associations. However, because of the extremely strong linkage disequilibrium between the DR and DQ loci, in some cases it has been difficult to determine the specific locus or combination of class II loci involved. In some cases, the susceptibility gene may be one of the HLA-linked genes located near the class I or class II region, but not the HLA gene itself, and in other cases, the susceptibility gene may be

1	In some cases, the susceptibility gene may be one of the HLA-linked genes located near the class I or class II region, but not the HLA gene itself, and in other cases, the susceptibility gene may be a non-HLA gene such as TNF-α, which is nearby. Indeed, since linkage disequilibrium of some haplotypes extends across large segments of the MHC region, it is quite possible that combinations of genes may account for the particular associations of HLA haplotypes with disease. For example, on some haplotypes associated with rheumatoid arthritis, both HLA-DRB1 alleles and a particular polymorphism associated with the TNF locus may be contributory to disease risk. Other candidates for similar epistatic effects include the IKBL gene and the MICA locus, potentially in combination with classic HLA class II risk alleles.

1	As might be predicted from the known function of the class I and class II gene products, almost all of the diseases associated with specific HLA alleles have an immunologic component to their pathogenesis. The recent development of soluble HLA-peptide recombinant molecules as biological probes of T cell function, often in multivalent complexes referred to as “MHC tetramers,” represents an opportunity to use HLA genetic associations to develop biomarkers for detection of early disease progression. However, it should be stressed that even the strong HLA associations with disease (those associations with relative risk of ≥10) implicate normal, rather than defective, alleles. Most individuals who carry these susceptibility genes do not express the associated disease; in this way, the particular HLA gene is permissive for disease but requires other environmental (e.g., the presence of specific antigens) or genetic factors for full penetrance. In each case studied, even in diseases with

1	HLA gene is permissive for disease but requires other environmental (e.g., the presence of specific antigens) or genetic factors for full penetrance. In each case studied, even in diseases with very strong HLA associations, the concordance of disease in mono-zygotic twins is higher than in HLA-identical dizygotic twins or other sibling pairs, indicating that non-HLA genes contribute to susceptibility and can significantly modify the risk attributable to HLA.

1	Another group of diseases is genetically linked to HLA, not because of the immunologic function of HLA alleles but rather because they are caused by autosomal dominant or recessive abnormal alleles at loci that happen to reside in or near the HLA region. Examples of these are 21-hydroxylase deficiency (Chap. 406), hemochromatosis (Chap. 428), and spinocerebellar ataxia (Chap. 452).

1	Although the associations of human disease with particular HLA alleles or haplotypes predominantly involve the class II region, there are also several prominent disease associations with class I alleles. These include the association of Behçet’s disease (Chap. 387) with HLA-B51, psoriasis vulgaris (Chap. 71) with HLA-Cw6, and, most notably, the spondyloarthritides (Chap. 384) with HLA-B27. Twenty-five HLA-B locus alleles, designated HLA-B*27:01–B*27:25, encode the family of 373e-7 B27 class I molecules. All of the subtypes share a common B pocket in the peptide-binding groove—a deep, negatively charged pocket that shows a strong preference for binding the arginine side chain. In addition, B27 is among the most negatively charged of HLA class I heavy chains, and the overall preference is for positively charged peptides. HLA-B*27:05 is the predominant subtype in whites and most other non-Asian populations, and this subtype is very highly associated with ankylosing spondylitis (AS)

1	is for positively charged peptides. HLA-B*27:05 is the predominant subtype in whites and most other non-Asian populations, and this subtype is very highly associated with ankylosing spondylitis (AS) (Chap. 384), both in its idiopathic form and in association with chronic inflammatory bowel disease or psoriasis vulgaris. It is also associated with reactive arthritis (ReA) (Chap. 384), with other idiopathic forms of peripheral arthritis (undifferentiated spondyloarthropathy), and with recurrent acute anterior uveitis. B27 is found in 50–90% of individuals with these conditions, compared with a prevalence of ∼7% in North American whites.

1	It can be concluded that the B27 molecule itself is involved in disease pathogenesis, based on strong evidence from clinical epidemiology and on the occurrence of a spondyloarthropathy-like disease in HLA-B27 transgenic rats. The association of B27 with these diseases may derive from the specificity of a particular peptide or family of peptides bound to B27 or through another mechanism that is independent of the peptide specificity of B27. In particular, HLA-B27 has been shown to form heavy chain homodimers, utilizing the cysteine residue at position 67 of the B57 α chain, in the absence of β2-microglobulin. These homodimers are expressed on the surface of lymphocytes and monocytes from patients with AS, and receptors including KIR3DL1, KIR3DL2, and ILT4 (LILRB2) are capable of binding to them, promoting the activation and survival of cells expressing these receptors. Alternatively, this dimerization “misfolding” of B27 may initiate an intracellular stress signaling response, called

1	them, promoting the activation and survival of cells expressing these receptors. Alternatively, this dimerization “misfolding” of B27 may initiate an intracellular stress signaling response, called the unfolded protein response (UPR), capable of modulating immune cell function, possibly in enthesial-resident T cells that act as sensors of damage and environmental stress.

1	As can be seen in Table 373e-1, the majority of associations of HLA and disease are with class II alleles. Several diseases have complex HLA genetic associations.

1	Celiac Disease In the case of celiac disease (Chap. 349), it is probable that the HLA-DQ genes are the primary basis for the disease association. HLA-DQ genes present on both the celiac-associated DR3 and DR7 haplotypes include the DQB1*02:01 gene, and further detailed studies have documented a specific class II αβ dimer encoded by the DQA1*05:01 and DQB1*02:01 genes, which appears to account for most of the HLA genetic contribution to celiac disease susceptibility. This specific HLA association with celiac disease may have a straightforward explanation: Peptides derived from the wheat gluten component gliaden are bound to the molecule encoded by DQA1*05:01 and DQB1*02:01 and presented to T cells. Gliaden-derived peptides that are implicated in this immune activation bind the DQ class II dimer best when the peptide contains a glutamine to glutamic acid substitution. It has been proposed that tissue transglutaminase, an enzyme present at increased levels in the intestinal cells of

1	II dimer best when the peptide contains a glutamine to glutamic acid substitution. It has been proposed that tissue transglutaminase, an enzyme present at increased levels in the intestinal cells of celiac patients, converts glutamine to glutamic acid in gliadin, creating peptides that are capable of being bound by the DQ2 molecule and presented to T cells.

1	Pemphigus Vulgaris In the case of pemphigus vulgaris (Chap. 73), there are two HLA genes associated with disease, DRB1*04:02 and DQB1*05:03. Peptides derived from desmoglein-3, an epidermal autoantigen, bind to the DRB1*04:02and DQB1*05:03-encoded HLA molecules, and this combination of specific peptide binding and disease-associated class II molecule is sufficient to stimulate desmogleinspecific T cells. A bullous pemphigoid clinical variant, not involving desmoglein recognition, has been found to be associated with HLADQB1*03:01. Juvenile Arthritis Pauciarticular juvenile arthritis (Chap. 380) is an autoimmune disease associated with genes at the DRB1 locus and also with genes at the DPB1 locus. Patients with both DPB1*02:01 and a Chapter 373e The Major Histocompatibility Complex Juvenile arthritis, pauciarticular

1	DRB1*04:01, –04, –05 +++ ++ related haplotypes that carry a different DQB1 gene are not. However, the relative risk associated with inheritance of this gene can be modified, depending on other HLA genes present either on the same or a second haplotype. For example, the presence of a DR2-positive haplotype containing a DQB1*06:02 gene is associated with decreased risk. This gene, DQB1*06:02, is considered “protective” for type 1 diabetes. Even some DRB1 genes that can occur on the same haplotype as DQB1*03:02 may modulate risk, so that individuals with the DR4 haplotype that contains DRB1*04:03 are less susceptible to type 1 diabetes than individuals with other DR4-DQB1*03:02 haplotypes. There are some characteristic structural features of the diabetes-associated DQ molecule encoded by DQB1*03:02, particularly the capability for binding peptides that have negatively charged amino acids near their C-termini. This may indicate a role for specific antigenic peptides or T cell interactions

1	particularly the capability for binding peptides that have negatively charged amino acids near their C-termini. This may indicate a role for specific antigenic peptides or T cell interactions in the immune response to islet-associated proteins.

1	Although the presence of a DR3 haplotype in combination with the DR4-DQB1*0302 haplotype is a very high-risk combination for diabetes susceptibility, the specific gene on the DR3 haplotype that is responsible for this synergy is not yet identified. Rheumatoid Arthritis The HLA genes associated with rheumatoid arthritis (RA) (Chap. 380) encode a distinctive sequence of amino acids from codons 67–74 of the DRβ molecule: RA-associated class II molecules carry the sequence LeuLeuGluGlnArgArgAlaAla or LeuLeuGluGlnLysArgAlaAla in this region, whereas non-RA-associated genes carry one or more differences in this region. These residues form a portion of the molecule that lies in the middle of the α-helical portion of the DRB1encoded class II molecule, termed the shared DQA1*05:01 DQB1*02:01 DRB1*04:02 DQB1*05:03 DQB1*03:01 aStrong negative association, i.e., genetic association with protection from diabetes. Abbreviation: GBM, glomerular basement membrane.

1	DQA1*05:01 DQB1*02:01 DRB1*04:02 DQB1*05:03 DQB1*03:01 aStrong negative association, i.e., genetic association with protection from diabetes. Abbreviation: GBM, glomerular basement membrane. DRB1 susceptibility allele (usually DRB1*08 or -*05) have a higher relative risk than expected from the additive effect of those genes alone. In juvenile patients with rheumatoid factor–positive polyarticular disease, heterozygotes carrying both DRB1*04:01 and -*04:04 have a relative risk >100, reflecting an apparent synergy in individuals inheriting both of these susceptibility genes. Type 1 Diabetes Mellitus Type 1 (autoimmune) diabetes mellitus (Chap. 417) is associated with MHC genes on more than one haplotype. The presence of both the DR3 and DR4 haplotypes in one individual confers a twentyfold increased risk for type 1 diabetes; the strongest single association is with DQB1*03:02, and all haplotypes that carry a DQB1*03:02 gene are associated with type 1 diabetes, whereas + epitope.

1	Multiple sclerosis DR2 DRB1*15:01 + The highest risk for susceptibility to RA comes in individuals who carry both a DR2 DRB5*01:01 ++ DRB1*04:01 and DRB1*04:04 gene. These DR4-positive RA-associated alleles with the shared epitope are most frequent among patients with more severe, erosive disease. ++++ Several mechanisms have been proposed that ++ link the shared epitope to immune reactivity in RA. This portion of the class II molecule may allow preferential binding of an arthritogenic peptide, it may favor the expansion of a type of self-reactive T lymphocyte, or it may itself form part of the pMHC ligand recognized by TCR that initiates synovial tissue recognition.

1	As noted above, HLA molecules play a key role in the selection and establishment of the antigen-specific T cell repertoire and a major role in the subsequent activation of those T cells during the initiation of an immune response. Precise genetic polymorphisms characteristic of individual alleles dictate the specificity of these interactions and thereby instruct and guide antigen-specific immune events. These same genetically determined pathways are therefore implicated in disease pathogenesis when specific HLA genes are responsible for autoimmune disease susceptibility.

1	The fate of developing T cells within the thymus is determined by the affinity of interaction between TCR and HLA molecules bearing self-peptides, and thus the particular HLA types of each individual control the precise specificity of the T cell repertoire (Chap. 372e). The primary basis for HLA-associated disease susceptibility may well lie within this thymic maturation pathway. The positive selection of potentially autoreactive T cells, based on the presence of specific HLA susceptibility genes, may establish the threshold for disease risk in a particular individual.

1	At the time of onset of a subsequent immune response, the primary role of the HLA molecule is to bind peptide and present it to antigen-specific T cells. The HLA complex can therefore be viewed as encoding genetic determinants of precise immunologic activation events. Antigenic peptides that bind particular HLA molecules are capable of stimulating T cell immune responses; peptides that do not bind are not presented to T cells and are not immunogenic. This genetic control of the immune response is mediated by the polymorphic sites within the HLA antigen–binding groove that interact with the bound peptides. In autoimmune and immune-mediated diseases, it is likely that specific tissue antigens that are targets for pathogenic lymphocytes are complexed with the HLA molecules encoded by specific susceptibility 373e-9 alleles. In autoimmune diseases with an infectious etiology, it is likely that immune responses to peptides derived from the initiating pathogen are bound and presented by

1	susceptibility 373e-9 alleles. In autoimmune diseases with an infectious etiology, it is likely that immune responses to peptides derived from the initiating pathogen are bound and presented by particular HLA molecules to activate T lymphocytes that play a triggering or contributory role in disease pathogenesis. The concept that early events in disease initiation are triggered by specific HLA-peptide complexes offers some prospects for therapeutic intervention, since it may be possible to design compounds that interfere with the formation or function of specific HLA-peptide– TCR interactions.

1	When considering mechanisms of HLA associations with immune response and disease, it is well to remember that just as HLA genetics are complex, so are the mechanisms likely to be heterogeneous. Immune-mediated disease is a multistep process in which one of the HLA-associated functions is to establish a repertoire of potentially reactive T cells, whereas another HLA-associated function is to provide the essential peptide-binding specificity for T cell recognition. For diseases with multiple HLA genetic associations, it is possible that both of these interactions occur and synergize to advance an accelerated pathway of disease. Chapter 373e The Major Histocompatibility Complex 2103 SeC tIOn 1 the IMMune SySteM In health anD DISeaSe ParT 15: Immune-Mediated, Inflammatory, and Rheumatologic Disorders

1	Immunity is intrinsic to life and an important tool in the fight for survival against pathogenic microorganisms. The human immune system can be divided into two major components: the innate immune system and the adaptive immune system (Chap. 372e). The innate immune system provides the rapid triggering of inflammatory responses based ontherecognition(atthecellsurfaceorwithincells)ofeithermolecules expressed by microorganisms or molecules that serve as “danger signals” released by cells under attack. These receptor/ligand interactions trigger signaling events that ultimately lead to inflammation. Virtually all cell lineages (not just immune cells) are involved in innate immune responses; however, myeloid cells (i.e., neutrophils and macrophages) play a major role because of their phagocytic capacity. The adaptive immune system operates by clonal recognition of antigens followed by a dramatic expansion of antigen-reactive cells and execution of an immune effector program. Most of the

1	capacity. The adaptive immune system operates by clonal recognition of antigens followed by a dramatic expansion of antigen-reactive cells and execution of an immune effector program. Most of the effector cells die off rapidly, whereas memory cells persist. Although both T and B lymphocytes recognizedistinctchemicalmoietiesandexecutedistinctadaptiveimmune responses, the latter is largely dependent on the former in generating long-lived humoral immunity. Adaptive responses utilize components of the innate immune system; for example, the antigen-presentation capabilities of dendritic cells help to determine the type of effector response. Not surprisingly, immune responses are controlled by a series of regulatory mechanisms.

1	Hundreds of gene products have been characterized as effectors or mediators of the immune system (Chap. 372e). Whenever the expression or function of one of these products is genetically impaired (provided the function is nonredundant), a primary immunodeficiency (PID) occurs.

1	PIDs are genetic diseases with primarily Mendelian inheritance. More than 250 conditions have now been described, and deleterious mutations in approximately 210 genes have been identified. The overall prevalence of PIDs has been estimated in various countries at 5 per 100,000 individuals; however, given the difficulty in diagnosing these rare and complex diseases, this figure is probably an underestimate. PIDscaninvolveallpossibleaspectsofimmuneresponses,frominnate throughadaptive, cell differentiation,andeffector functionandregulation. For the sake of clarity, PIDs should be classified according to (1) the arm of the immune system that is defective and (2) the mechanism of the defect (when known). Table 374-1 classifies the most prevalent PIDs according to this manner of classification; however, one should bear in mind that the classification of PIDs sometimes involves arbitrary decisions because of overlap and, in some cases, lack of data.

1	TheconsequencesofPIDsvarywidelyasafunctionofthemolecules thataredefective.Thisconcepttranslatesintomultiplelevelsofvulnerability to infection by pathogenic and opportunistic microorganisms, ranging from extremely broad (as in severe combined immunodeficiency [SCID]) to narrowly restricted to a single microorganism (as in Mendelian susceptibility to mycobacterial disease [MSMD]). The locations of the sites of infection and the causal microorganisms involved will thus help physicians arrive at proper diagnoses. PIDs can also lead to immunopathologic responses such as allergy (as in Wiskott-Aldrich syndrome), lymphoproliferation, and autoimmunity. A combination infections, and opportunistic infections are generally suggestive of impaired T cell immunity. Skin infec-

1	Deficiencies of the Innate Immune System • Phagocytic cells: immune defects (such as chronic granulomatous dis -Impaired production: severe congenital neutropenia (SCN) ease); however, they may also appear in the autosomal -Asplenia dominanthyper-IgEsyndrome. Table 374-2 summarizes adhesion: leukocyte adhesion deficiency (LAD) the laboratory tests that are most frequently used todiag killing: chronic granulomatous disease (CGD) nose a PID. More specific tests (notably genetic tests) are then used to make a definitive diagnosis. • Innate immunity receptors and signal transduction: The PIDs discussed below have been grouped together according to the affected cells and the mecha -Mendelian susceptibility to mycobacterial disease nisms involved (Table 374-1, Fig. 374-1). • Complement deficiencies: -Classical, alternative, and lectin pathways PrIMarY IMMUNODEFICIENCIES OF THE INNaTE IMMUNE SYSTEM

1	• Complement deficiencies: -Classical, alternative, and lectin pathways PrIMarY IMMUNODEFICIENCIES OF THE INNaTE IMMUNE SYSTEM Deficiencies of the Adaptive Immune System PIDs of the innate immune system are relatively rare and • T lymphocytes: account for approximately 10% of all PIDs.

1	Severe congenital neutropenia (SCN) consists of a group -Impaired survival, migration, Combined immunodeficiencies of inherited diseases that are characterized by severely impaired neutrophil counts (<500 polymorphonuclear DOCK8 deficiency leukocytes [PMN]/μL of blood). The condition is usually CD40 ligand deficiency manifested from birth. SCN may also be cyclic (with a Wiskott-Aldrich syndrome 3-week periodicity), and other neutropenia syndromes can also be intermittent. Although the most frequent inheritance pattern for SCN is autosomal dominant, • B lymphocytes: also exist. Bacterial infections at the interface between the body and the external milieu (e.g., the orifices, wounds, Common variable immunodeficiency (CVID) and the respiratory tract) are common manifestations. IgA deficiency Bacterial infections can rapidly progress through soft Regulatory Defects tissue and are followed by dissemination in the bloodstream. Severe visceral fungal infections can also ensue.

1	• Innate immunity Autoinflammatory syndromes (outside the scope of this chapter) The absence of pus is a hallmark of this condition. Diagnosis of SCN requires examination of the bone marrow. Most SCNs are associated with a block in granu- lopoiesis at the promyelocytic stage (Fig. 374-1). SCN has multiple etiologies, and to date, mutations in 11 differ- Autoimmunity and inflammatory diseases (IPEX, ent genes have been identified. Most of these mutations result in isolated SCN, whereas others are syndromic Abbreviations: APECED, autoimmune polyendocrinopathy candidiasis ectodermal dysplasia; AR, auto (Chap. 80). The most frequent forms of SCN are caused somal recessive; IPEX, immunodysregulation polyendocrinopathy enteropathy X-linked syndrome; XL, by the premature cell death of granulocyte precursors, as X-linked.

1	X-linked. observed in deficiencies of GFI1, HAX1, and elastase 2 (ELANE), with the latter accounting for 50% of SCN sufof recurrent infections, inflammation, and autoimmunity can be ferers. Certain ELANE mutations cause cyclic neutropenia syndrome. observed in a number of PIDs, thus creating obvious therapeutic chal-A gain-of-function mutation in the WASP gene (see the section on lenges. Finally, some PIDs increase the risk of cancer, notably but not “Wiskott-Aldrich syndrome” below) causes X-linked SCN, which is exclusively lymphocytic cancers, e.g., lymphoma. also associated with monocytopenia.

1	As mentioned above, SCN exposes the patient to life-threatening, disseminated bacterial and fungal infections. Treatment requires care- ful hygiene measures, notably in infants. Later in life, special oral and The most frequent symptom prompting the diagnosis of a PID is the dental care is essential, along with the prevention of bacterial infection presence of recurrent or unusually severe infections. As mentioned by prophylactic administration of trimethoprim/sulfamethoxazole. above, recurrent allergic or autoimmune manifestations may also Subcutaneousinjectionofthecytokinegranulocytecolony-stimulating alert the physician to a possible diagnosis of PID. In such cases, a factor (G-CSF) usually improves neutrophil development and thus detailed account of the subject’s personal and family medical his-prevents infection in most SCN diseases. However, there are two tory should be obtained. It is of the utmost importance to gather as caveats: (1) a few cases of SCN with ELANE mutation are

1	his-prevents infection in most SCN diseases. However, there are two tory should be obtained. It is of the utmost importance to gather as caveats: (1) a few cases of SCN with ELANE mutation are refractorymuch medical information as possible on relatives and up to several to G-CSF and may require curative treatment via allogeneic hematogenerations of ancestors. In addition to the obvious focus on primary poietic stem cell transplantation (HSCT); and (2) a subset of G-CSFsymptoms, the clinical examination should evaluate the size of lym-treated patients carrying ELANE mutations are at a greater risk of phoid organs and, when appropriate, look for the characteristic signs developing acute myelogenous leukemia associated (in most cases)ofanumberofcomplexsyndromesthatmaybeassociatedwithaPID. withsomaticgain-of-functionmutationsoftheG-CSFreceptorgene.

1	The performance of laboratory tests should be guided to some extent by the clinical findings. Infections of the respiratory tract aSPLENIa (bronchi, sinuses) mostly suggest a defective antibody response. In Primary failure of the development of a spleen is an extremely rare general, invasive bacterial infections can result from complement diseasethatcanbeeithersyndromic(inIvemarksyndrome)orisolated deficiencies, signaling defects of innate immune responses, asplenia, with an autosomal dominant expression; in the latter case, mutations or defective antibody responses. Viral infections, recurrent Candida in the ribosomal protein SA gene were recently found. Due to the aNormal counts vary with age. For example, the lymphocyte count is between 3000 and 9000/μL of blood below the age of 3 months and between 1500 and 2500/μL in adults.

1	Abbreviations: ID, immunodeficiency; LAD, leukocyte adhesion deficiency; PMNs, polymorphonuclear leukocytes; SCID, severe combined immunodeficiency; WAS, Wiskott-Aldrich syndrome. absence of natural filtration of microbes in the blood, asplenia predisposes affected individuals to fulminant infections by encapsulated bacteria. Although most infections occur in the first years of life, cases may also arise in adulthood. The diagnosis is confirmed by abdominal ultrasonography and the detection of Howell-Jolly bodies in red blood cells. Effective prophylactic measures (twice-daily oral penicillin and appropriate vaccination programs) usually prevent fatal outcomes.

1	Recently an immunodeficiency combining monocytopenia and dendritic and lymphoid (B and natural killer [NK]) cell deficiency (DCML), also called monocytopenia with nontuberculous mycobacterial infections (mono-MAC), has been described as a consequence of a dominantmutationinthegene GATA2,atranscriptionfactorinvolved in hematopoiesis. This condition also predisposes to lymphedema, Neutrophil Phagocytosis Phagocytosis Killing ROS production Killing ROS production Adhesion Migration HSC CMP MB SCN WHIM LAD CGD CGD MSMD GM prog. Mono blast Pro mono Pro myelo. myelo. Monocyte Dendritic cells Tissue macrophages Bone Marrow Blood Tissue GATA2 deficiency

1	FIGUrE 374-1 Differentiation of phagocytic cells and related primary immunodeficiencies (PIDs). Hematopoietic stem cells (HSCs) differentiate into common myeloid progenitors (CMPs) and then granulocyte-monocyte progenitors (GM-prog.), which, in turn, differentiate into neutrophils (MB: myeloblasts; Promyelo: promyelocytes; myelo: myelocytes) or monocytes (monoblasts and promonocytes). Upon activation, neutrophils adhere to the vascular endothelium, transmigrate, and phagocytose the targets. Reactive oxygen species (ROS) are delivered to the microorganism-containing phagosomes. Macrophages in tissues kill using the same mechanism. Following activation by interferon γ (not shown here), macrophages can be armed to kill intracellular pathogens such as mycobacteria. For sake of simplicity, not all cell differentiation stages are shown. The abbreviations for PIDs are contained in boxes placed at corresponding stages of the pathway. CGD, chronic granulomatous diseases; GATA2, zinc finger

1	not all cell differentiation stages are shown. The abbreviations for PIDs are contained in boxes placed at corresponding stages of the pathway. CGD, chronic granulomatous diseases; GATA2, zinc finger transcription factor; LAD, leukocyte adhesion deficiencies; MSMD, Mendelian susceptibility to mycobacterial disease; SCN, severe congenital neutropenia; WHIM, warts, hypogammaglobulinemia, infections, and myelokathexis.

1	2106 myelodysplasia, and acute myeloid leukemia. Infections (bacterial and viral) are life-threatening, thus indicating, together with the malignant risk, HSCT. Leukocyte adhesion deficiency (LAD) consists of three autosomal recessive conditions (LAD I,II, andIII) (Chap. 80). The mostfrequent condition (LAD I) is caused by mutations in the β2 integrin gene; followingleukocyteactivation,β2integrins mediate adhesion toinflamed endothelium expressing cognate ligands. LAD III results from a defect in a regulatory protein (kindlin, also known as Fermt 3) involved in activating the ligand affinity of β2 integrins. The extremely rare LAD II condition is the end result of a defect in selectin-mediated leukocyte rolling that occurs prior to β2 integrin binding. There is a primary defect in fucose transporter such that oligosaccharide selectin ligands are missing in this syndromic condition.

1	Given that neutrophils are not able to reach infected tissues, LAD renders the individual susceptible to bacterial and fungal infections in a way that is similar to that of patients with SCN. LAD also causes impaired wound healing and delayed loss of the umbilical cord. A diagnosis can be suspected in cases of pus-free skin/tissue infections and massive hyperleukocytosis (>30,000/μL) in the blood (mostly granulocytes). Patients with LAD III also develop bleeding because the β2 integrin in platelets is not functional. Use of immunofluorescence and functional assays to detect β2 integrin can help form a diagnosis. Severe forms of LAD may require HSCT, although gene therapy is also now being considered. Neutrophil-specific granule deficiency (a very rare condition caused by a mutation in the gene for transcription factor C/EBPα) results in a condition that is clinically similar to LAD.

1	Chronicgranulomatousdiseases(CGDs)arecharacterizedbyimpaired phagocytic killingofmicroorganismsby neutrophils and macrophages (Chap. 80). The incidence is approximately 1 per 200,000 live births. About 70% of cases are associated with X-linked recessive inheritance versusautosomalinheritanceintheremaining30%.CGDcausesdeeptissue bacterial and fungal abscesses in macrophage-rich organs such as the lymph nodes, liver, and lungs. Recurrent skin infections (such as folliculitis) are common and can promptan early diagnosis of CGD. The infectious agents are typically catalase-positive bacteria (such as Staphylococcus aureus and Serratia marcescens) but also include Burkholderia cepacia, pathogenic mycobacteria (in certain regions of the world), and fungi (mainly filamentous molds, such as Aspergillus).

1	CGD is caused by defective production of reactive oxygen species (ROS) in the phagolysosome membrane following phagocytosis of microorganisms. It results from the lack of a component of NADPH oxidase (gp91phox or p22phox) or of the associated adapter/activating proteins (p47phox, p67phox, or p40phox) that mediate the transport of electrons into the phagolysosome for creating ROS by interaction with O2. Under normal circumstances, these ROS either directly kill engulfed microorganisms or enable the rise in pH needed to activate the phagosomal proteases that contribute to microbial killing. Diagnosisof CGDisbased onassays ofROSproductionin neutrophils and monocytes (Table 374-2). As its name suggests, CGD is also a granulomatous disease. Macrophage-rich granulomas can often arise in the liver, spleen, and other organs. These are sterile granulomas that cause disease by obstruction (bladder, pylorus, etc.) or inflammation (colitis, restrictive lung disease).

1	The management of infections in patients with CGD can be a complex process. The treatment of bacterial infections is generally based on combination therapy with antibiotics that are able to penetrate into cells. The treatment of fungal infections requires aggressive, long-term use of antifungals. Inflammatory/granulomatous lesions are usually steroid-sensitive; however, glucocorticoids often contribute to the spread of infections. Hence, there is strong need for new therapeutic options in what is still a poorly understood disease.

1	The treatment of CGD mostly relies on preventing infections. It has been unambiguously demonstrated that prophylactic usage of trimethoprim/sulfamethoxazole is both well tolerated and highly effective inreducingtheriskofbacterialinfection.Dailyadministrationofazole derivatives (notably itraconazole) also reduces the frequency of fungal complications. Ithaslongbeen suggested thatinterferon γ administration is helpful, although medical experts continue to disagree over this controversial issue. Patients may do reasonably well with prophylaxis and careful management. However, other patients develop severe and persistent fungal infections and/or chronic inflammatory complications that ultimately require HSCT. The latter is an established curative approach for CGD; however, the risk-versus-benefit ratio must be carefullyassessedonacase-by-casebasis.Genetherapyapproachesare also being evaluated.

1	This group of diseases is characterized by a defect in the interleukin-12 (IL-12)–interferon (IFN) γ axis (including IL-12p40, IL-12 receptor [R] β1, IFN-γ R1 and R2, STAT1, IRF8 and ISG515 deficiencies), which ultimately leads to impaired IFN-γ-dependent macrophage activation. Both recessive and dominant inheritance modes have been observed. The hallmark of this PID is a specific and narrow vulnerability to tuberculous and nontuberculous mycobacteria. The most severe phenotype (as observed in complete IFN-γ receptor deficiency) is characterized by disseminated infection that can be fatal even when aggressive and appropriate antimycobacterial therapy is applied. In addition to mycobacterial infections, MSMD patients (and particularly those with an IL-12/IL-12 R deficiency) are prone to developing Salmonella infections. Although MSMDs are very rare, they should be considered in any patient with persistent mycobacterial infection. Treatment with IFN-γ may efficiently bypass an

1	to developing Salmonella infections. Although MSMDs are very rare, they should be considered in any patient with persistent mycobacterial infection. Treatment with IFN-γ may efficiently bypass an IL-12/IL-12R deficiency.

1	In a certain group of patients with early-onset, invasive Streptococcus pneumoniae infections or (less frequently) Staphylococcus aureus or other pyogenic infections, conventional screening for PIDs does not identify the cause of the defect in host defense. It has been established that these patients carry recessive mutations in genes that encode essential adaptor molecules (IRAK4 and MYD88) involved in the signaling pathways of the majority of known Toll-like receptors (TLRs) (Chap. 372e). Remarkably, susceptibility to infection appears to decrease after the first few years of life—perhaps an indication that adaptive immunity (once triggered by an initial microbial challenge) is then able to prevent recurrent infections.

1	Certain TLRs (TLR-3, -7, -8, and -9) are involved in the recognition of RNA and DNA and usually become engaged during viral infections. Very specific susceptibility to herpes simplex encephalitis has been described in patients with a deficiency in Unc93b (a molecule associated with TLR-3, -7, -8, and -9 required for correct subcellular localization), TLR-3, or associated signaling molecules TRIF, TBK1, and TRAF3, resulting in defective type I IFN production. The fact that no other TLR deficiencies have been found—despite extensive screening of patients with unexplained, recurrent infections—strongly suggests that these receptors are functionally redundant. Hypomorphic mutations in NEMO/IKK-γ (a member of the NF-κB complex, which is activated downstream of TLR receptors) lead to a complex, variable immunodeficiency and a number of associated features. Susceptibility to both invasive, pyogenic infections and mycobacteria may be observed in this particular setting.

1	The complement system is composed of a complex cascade of plasma proteins (Chap. 372e) that leads to the deposition of C3b fragments on the surface of particles and the formation of immune complexes that can culminate in the activation of a lytic complex at the bacterial surface. C3 cleavage can be mediated via three pathways: the classic, alternate, and lectin pathways. C3b coats particles as part of the opsonization process that facilitates phagocytosis following binding to cognate receptors. A deficiency in any component of the classic pathway (C1q, C1r, C1s, C4, and C2) can predispose an individual to bacterial infections that are tissue-invasive or that occur in the respiratory tract. Likewise, a C3 deficiency or a deficiency in factor I (a protein that regulates C3 consumption, thus leading to a C3 deficiency due to its absence) also results in the same type of vulnerability to infection. It has recently been reported that a very rare deficiency in ficolin-3 predisposes affected

1	leading to a C3 deficiency due to its absence) also results in the same type of vulnerability to infection. It has recently been reported that a very rare deficiency in ficolin-3 predisposes affected individuals to bacterial infections. Deficiencies in the alternative pathway (factors D and properdin) are associated with the occurrence of invasive Neisseria infections.

1	Lastly, deficiencies of any complement component involved in the lytic phase (C5, C6, C7, C8, and, to a lesser extent, C9) predispose affected individuals to systemic infection by Neisseria. This is explainedbythecriticalroleofcomplementinthelysisofthethickcell wall possessed by this class of bacteria. Diagnosisof acomplementdeficiencyrelies primarilyon testingthe status oftheclassicandalternatepathway via functional assays,i.e., the CH50andAP50tests,respectively.Wheneitherpathwayisprofoundly impaired, determination of the status of the relevant components in that pathway enables a precise diagnosis. Appropriate vaccinations and daily administration of oral penicillin are efficient means of preventing recurrent infections. It is noteworthy that several complement deficiencies (in the classic pathway and the lytic phase) may also predispose affected individuals to autoimmune diseases (notably systemic lupus erythematosus; Chap. 378).

1	T LYMPHOCYTE DEFICIENCIES (TaBLE 374-1, FIGS. 374-2 aND 374-3)

1	Given the central role of T lymphocytes in adaptive immune responses (Chap. 372e), PIDs involving T cells generallyhaveseverepathologicconsequences;this explains the poor overall prognosis and the need for early diagnosis and the early intervention with appropriate therapy. Several differentiation pathways of T cell effectors have been described, one or all of which may be affected by a given PID (Fig. 374-2). Follicular helper CD4+ T cells in germinal centers are required for T-dependent antibody production, including the generation of Ig class-switched, high-affinity antibodies. CD4+ TH1 cells provide cytokine-dependent (mostly IFN-γ-dependent) help to macrophages for intracellular killing of various microorganisms, including mycobacteria and Salmonella. CD4+ TH2 cells produce IL-4, IL-5, and IL-13 and thus recruit and activate eosinophils and other cells required to fight helminth infections. CD4+ TH17 cells produce IL-17 and IL-22 cytokines that recruit neutrophils to the skin and

1	and IL-13 and thus recruit and activate eosinophils and other cells required to fight helminth infections. CD4+ TH17 cells produce IL-17 and IL-22 cytokines that recruit neutrophils to the skin and lungs to fight bacterial and fungal infections. Cytotoxic CD8+ T cells can kill infected cells, notably in the context of viral infections. In addition, certain T cell deficiencies predispose affected individuals to Pneumocystis jiroveci lung infections early in life and to chronic gut/biliary duct/liver infections by Cryptosporidium and related genera later on in life. Lastly, naturally occurring or induced regulatory T cells are essential for controlling inflammation (notably reactivity to commensal bacteria in the gut) and autoimmunity. The role of other T cell subsets with limited T cell receptor (TCR) diversity (such as γδTCR T cells or natural killer T [NKT] cells) in PIDs is less well known; however, these subsets can be defective in certain PIDs, and this finding can sometimes

1	receptor (TCR) diversity (such as γδTCR T cells or natural killer T [NKT] cells) in PIDs is less well known; however, these subsets can be defective in certain PIDs, and this finding can sometimes contribute to the diagnosis (e.g., NKT cell deficiency in X-linked proliferative syndrome). T cell deficiencies account for approximately 20% of all cases of PID.

1	Severe Combined Immunodeficiencies Severe combined immunodefi-2107 ciencies (SCIDs) constitute a group of rare PIDs characterized by a profound block in T cell development and thus the complete absence of these cells. The developmental block is always the consequence of an intrinsic deficiency. The incidence of SCID is estimated to be 1 in 50,000 live births. Given the severity of the T cell deficiency, clinical consequences occur early in life (usually within 3 to 6 months of birth). The most frequent clinical manifestations are recurrent oral candidiasis, failure to thrive, and protracted diarrhea and/or acute interstitial pneumonitis caused by Pneumocystis jiroveci (although the latter can also be observed in the first year of life in children with B cell deficiencies). Severe viral infections or invasive bacterial infections can also occur. Patients may also experience complications related to infections caused by live vaccines (notably bacille Calmette-Guérin [BCG]) that may lead

1	or invasive bacterial infections can also occur. Patients may also experience complications related to infections caused by live vaccines (notably bacille Calmette-Guérin [BCG]) that may lead not only to local and regional infection but also to disseminated infection manifested by fever, splenomegaly, and skin and lytic bone lesions. A scaly skin eruption can be observed in a context of maternal T cell engraftment (see below). A diagnosis of SCID can be suspected based on the patient’s clinical history and, possibly, a family history of deaths in very young children (suggestive of either X-linked or recessive inheritance). Lymphocytopenia is strongly suggestive of SCIDinmorethan90%ofcases (Table 316-2).Theabsenceofathymic shadow on a chest x-ray can also be suggestive of SCID. An accurate diagnosis relies on precise determination of the number of circulating T, B, and NK lymphocytes and their subsets. T cell lymphopenia may be masked in some patients by the presence of maternal T

1	diagnosis relies on precise determination of the number of circulating T, B, and NK lymphocytes and their subsets. T cell lymphopenia may be masked in some patients by the presence of maternal T cells ˜IFN, etc...

1	HLH Cytotoxicity Tc ˜IFN, TNF, etc... TH1 ˜IFN, etc... MSMD TH2 IL4, etc... Tyk2 IL17, IL17F, IL17RA STAT1 gof TFh IL21, etc... Treg IL10, TGF°, etc... IL4, cytotoxicity NKT ˜IFN, etc... XLP Myeloid (SAP, XIAP)

1	FIGUrE 374-2 T cell differentiation, effector pathways, and related primary immunodeficiencies (PIDs). Hematopoietic stem cells (HSCs) differentiate into common lymphoid progenitors (CLPs), which, in turn, give rise to the T cell precursors that migrate to the thymus. The development of CD4+ and CD8+ T cells is shown. Known T cell effector pathways are indicated, i.e., γδ cells, cytotoxic T cells (Tc), TH1, TH2, TH17, TFh (follicular helper) CD4 effector T cells, regulatory T cells (Treg), and natural killer T cells (NKTs); abbreviations for PIDs are contained in boxes. Vertical bars indicate a complete deficiency; broken bars a partial deficiency. SCID, severe combined immunodeficiency; ZAP-70, zeta-associated protein deficiency; MHCII, major histocompatibility complex class II deficiency; TAP, TAP1 and TAP2 deficiencies; Orai1, STIM1 deficiencies; HLH, hematopoietic lymphohistiocytosis; MSMD, Mendelian susceptibility to mycobacterial disease; Tyk2, DOCK8, autosomal recessive form of

1	TAP, TAP1 and TAP2 deficiencies; Orai1, STIM1 deficiencies; HLH, hematopoietic lymphohistiocytosis; MSMD, Mendelian susceptibility to mycobacterial disease; Tyk2, DOCK8, autosomal recessive form of hyper-IgE syndrome; STAT3, autosomal dominant form of hyper-IgE syndrome; IL17F, IL17RA, STAT1 (gof: gain of function), CMC (chronic mucocutaneous candidiasis), CD40L, ICOS, SAP deficiencies; IPEX, immunodysregulation polyendocrinopathy enteropathy X-linked syndrome; XLP, X-linked proliferative syndromes.

1	2108 Prevention of cell apoptosis DNA replication (purine metabolism) ADA ˜c cytokine-dependent signal ˜c, JAK-3, IL7R˛ Pre TCR/TCR signalling CD45, CD3˝, ˙, ˆ V(D)J recombination Rag-1/-2, Artemis, DNA PKcs, DNA L4, Cernunnos Myeloid compartment Cell survival Adenylate kinase 2 ThymusNK CD8 CD4 B CLP HSC account for 20–30% of SCID cases and result from mutations in genes encoding proteins that mediate the recombination of V(D)J gene elements in T and B cell antigen receptor genes (required for the generation of diversity in antigen recognition). The main deficiencies involve RAG-1, RAG-2, DNA-dependent protein kinase, and Artemis. A less severe (albeit variable) immunologic phenotype can result from other deficiencies in the same pathway, i.e., DNA ligase 4 and Cernunnos deficiencies. Given that these latter factors are involved in DNA repair, these deficiencies also cause developmental defects.

1	Defective (pre-)t cell receptor signaling in the thymus A selective T cell defect can be caused by a series of rare deficiencies in molecules involved in signaling via the pre-TCR or the TCR. These include deficiencies in CD3 subunits associated with the (pre-)TCR (i.e., CD3δ, ε, and ζ) and CD45.

1	FIGUrE 374-3 T cell differentiation and severe combined immunodeficiencies (SCIDs). The vertical bars indicate the five mechanisms currently known to lead to SCID. The names of deficient reticular Dysgenesis Reticular dysgeneproteins are indicated in the boxes adjacent to the vertical bars. A broken line means that deficien-sis is an extremely rare form of SCID that cy is partial or involves only some of the indicated immunodeficiencies. ADA, adenosine deaminase causes T and NK deficiencies with severe deficiency; CLPs, common lymphoid progenitors; DNAL4, DNA ligase 4; HSCs, hematopoietic stem neutropenia and sensorineural deafness. cells; NKs, natural killer cells; TCR, T cell receptor. It results from an adenylate kinase 2 deficiency. (derivedfrommaternal-fetalbloodtransfers)thatcannotbeeliminated. Patients with SCID require appropriate care with aggressive anti-Although counts are usually low (<500/μL of blood), higher maternal infective therapies, immunoglobulin replacement, and

1	Patients with SCID require appropriate care with aggressive anti-Although counts are usually low (<500/μL of blood), higher maternal infective therapies, immunoglobulin replacement, and (when neces-T cell counts may, under some circumstances, initially mask the pres-sary) parenteral nutrition support. In most cases, curative treatment ence of SCID. Thus, screening for maternal cells by using adequate relies on HSCT. Today, HSCT provides a very high curative potential genetic markers should be performed whenever necessary. Inheritance for SCID patients who are otherwise in reasonably good condition. patternanalysisandlymphocytephenotypingcandiscriminatebetween In this regard, neonatal screening, based on quantification of T cell various forms of SCID and provide guidance in the choice of accurate receptor excision circles (TRECs) on a Guthrie card sample, is being molecular diagnostic tests (see below). To date, five distinct causative developed. Gene therapy has been found to be

1	of accurate receptor excision circles (TRECs) on a Guthrie card sample, is being molecular diagnostic tests (see below). To date, five distinct causative developed. Gene therapy has been found to be successful for cases of mechanismsforSCID(Fig.374-3)havebeenidentified: X-linkedSCID(γcdeficiency)andSCIDcausedbyanADAdeficiency, although toxicity has become an issue in the treatment of the former disease that may now be overcome by use of newly generated vectors.

1	Deficiency The most frequent SCID phenotype (accounting for Lastly, a third option for the treatment of ADA deficiency consists of 40–50% of all cases) is the absence of both T and NK cells. This out- enzyme substitution with a pegylated enzyme.

1	come results from a deficiency in either the common γ chain (γc) receptor that is shared by several cytokine receptors (the IL-2, -4, -7, Thymic Defects A profound T cell defect can also result from faulty -9, -15, and -21 receptors) or Jak-associated kinase (JAK) 3 that binds development of the thymus, as is most often observed in rare cases to the cytoplasmic portion of the γc chain receptor and induces signal of DiGeorge syndrome—a relatively common condition leading to a transduction following cytokine binding. The former form of SCID constellation of developmental defects. In approximately 1% of such (γc deficiency) has an X-linked inheritance mode, while the second is cases, the thymus is completely absent, leading to virtually no mature autosomal recessive. A lack of the IL-7Rα chain (which, together with T cells. However, expansion of oligoclonal T cells can occur and is γc, forms the IL-7 receptor) induces a selective T cell deficiency. associated with skin lesions. Diagnosis

1	(which, together with T cells. However, expansion of oligoclonal T cells can occur and is γc, forms the IL-7 receptor) induces a selective T cell deficiency. associated with skin lesions. Diagnosis (using immunofluorescence in situ hybridization) is based on the identification of a hemizygouspurine metabolism Deficiency Ten to 20% of SCID patients exhibit deletion in the long arm of chromosome 22. To recover the capability a deficiency in adenosine deaminase (ADA), an enzyme of purine for T cell differentiation, these cases require a thymic graft. CHARGE metabolism that deaminates adenosine (ado) and deoxyadenosine (colobomaoftheeye,heartanomaly,choanal atresia,retardation, geni(dAdo). An ADA deficiency results in the accumulation of ado and tal and ear anomalies) syndrome (CHD7 deficiency) is a less frequent dAdo metabolites that induce premature cell death of lymphocyte pro-cause of impaired thymus development. Lastly, the very rare “nude”

1	and ear anomalies) syndrome (CHD7 deficiency) is a less frequent dAdo metabolites that induce premature cell death of lymphocyte pro-cause of impaired thymus development. Lastly, the very rare “nude” genitors.TheconditionresultsintheabsenceofBandNKlymphocytes defectischaracterizedbytheabsenceofbothhairandthethymus. as well as T cells. The clinical expression of complete ADA deficiency

1	Omenn Syndrome Omenn syndrome consists of a subset of T cell defi typically occurs very early in life. Since ADA is a ubiquitous enzyme, ciencies that present with a unique phenotype, including early-onset its deficiency can also cause bone dysplasia with abnormal costochonerythrodermia, alopecia, hepatosplenomegaly, and failure to thrive. dral junctions and metaphyses (found in 50% of cases) and neurologic These patients usually display T cell lymphocytosis, eosinophilia, and defects. The very rare purine nucleoside phosphorylase (PNP) defilow B cell counts. It has been found that the T cells of these patients ciency causes a profound although incomplete T cell deficiency that is exhibit a low TCR heterogeneity. This peculiar syndrome is the con-often associated with severe neurologic impairments.

1	sequence of hypomorphic mutations in genes usually associated with Defective rearrangements of t anD b cell receptors A series of SCID SCID, i.e., RAG-1, RAG-2, or (less frequently) Artemis or IL-7Rα. conditions are characterized by a selective deficiency in T and B The impaired homeostasis of differentiating T cells thus causes this lymphocytes with autosomal recessive inheritance. These conditions immune system–associated disease. These patients are very fragile, requiringsimultaneousanti-infectivetherapy,nutritionalsupport,and immunosuppression. HSCT provides a curative approach.

1	Functional T Cell Defects (Fig. 374-2) AsubsetofTcellPIDswithautosomal inheritance is characterized by partially preserved T cell differentiation but defective activation resulting in abnormal effector function. There are many causes of these defects, but all lead to susceptibility to viral and opportunistic infections, chronic diarrhea, and failure to thrive, with onset during childhood. Careful phenotyping and in vitro functional assays are required to identify these diseases, the best characterized of which are the following. Zeta-associateD protein 70 (Zap70) Deficiency Zeta-associated protein 70 (ZAP70) is recruited to the TCR following antigen recognition. A ZAP70 deficiency leads typically to an almost complete absence of CD8+ T cells; CD4+ T cells arepresent but cannot be activated in vitro by TCR stimulation.

1	calcium signaling Defects A small number of patients have been reported who exhibit a profound defect in in vitro T and B cell activation as a result of defective antigen receptor-mediated Ca2+ influx. This defect is caused by a mutation in the calcium channel gene (ORAI-) or its activator (STIM-1). It is noteworthy that these patients are also prone to autoimmune manifestations (blood cytopenias) and exhibit a nonprogressive muscle disease. human leukocyte antigen (hla) class ii Deficiency Defective expression of HLAclassIImoleculesisthehallmarkofagroupoffourrecessivegenetic defects all of which affect molecules (RFX5, RFXAP, RFXANK, and CIITA) involved in the transactivation of the genes coding for HLA class

1	II. As a result, low but variable CD4+ T cell counts are observed in addition to defective antigen-specific T and B cell responses. These patients are particularly susceptible to herpesvirus, adenovirus, and enterovirus infections and chronic gut/liver Cryptosporidium infections. hla class i Deficiency Defective expression of molecules involved in antigen presentation by HLA class I molecules (i.e., TAP-1, TAP-2, and Tapasin) leads to reduced CD8+ T cell counts, loss of HLA class I antigen expression, and a particular phenotype consisting of chronic obstructive pulmonary disease and severe vasculitis.

1	other Defects A variety of other T cell PIDs have been described, some of which are associated with a precise molecular defect (e.g., IL-2-inducible T cell kinase [ITK] deficiency, IL-21 receptor deficiency, CARD11 deficiency). These conditions are also characterized by profound vulnerability to infections, such as severe Epstein-Barr virus (EBV)–induced B cell proliferation and autoimmune disorders in ITK deficiency. Milder phenotypes are associated with CD8 and CD3γ deficiencies. HSCTisindicatedformostofthesediseases,althoughtheprognosisis worse than in SCID because many patients are chronically infected at the time of diagnosis. Fairly aggressive immunosuppression and myeloablation may be necessary to achieve engraftment of allogeneic stem cells.

1	T Cell Primary Immunodeficiencies with DNa repair Defects This is a group of PIDs characterized by a combination of T and B cell defects of variable intensity, together with a number of nonimmunologic features resulting from DNA fragility. The autosomal recessive disorder ataxia-telangiectasia (AT) is the most frequently encountered condition in this group. It has an incidence of 1:40,000 live births and causes B cell defects (low IgA, IgG2 deficiency, and low antibody production), which often require immunoglobulin replacement. AT is associated with a progressive T cell immunodeficiency. As the name suggests, the hallmark features of AT are telangiectasia and cerebellar ataxia. The latter manifestations may not be detectable before the age of 3–4 years, so that AT should be considered in young children with IgA deficiency and recurrent and problematic infections. Diagnosis is based on a cytogenetic analysis showing excessive chromosomal rearrangements (mostly affecting chromosomes 7

1	young children with IgA deficiency and recurrent and problematic infections. Diagnosis is based on a cytogenetic analysis showing excessive chromosomal rearrangements (mostly affecting chromosomes 7 and 14) in lymphocytes. AT is caused by a mutation in the gene encoding the ATM protein—a kinase that plays an important role in the detection and repair of DNA lesions (or cell death if the lesions are too numerous) by triggering several different pathways. Overall, AT is a progressive disease that 2109 carries a very high risk of lymphoma, leukemia, and (during adulthood) carcinomas. A variant of AT (“AT-like disease”) is caused by mutation in the MRE11 gene.

1	Nijmegen breakage syndrome (NBS)isalesscommonconditionthat also results from chromosome instability (with the same cytogenetic abnormalities as in AT). NBS is characterized by a severe T and B cell combined immune deficiency with autosomal recessive inheritance. Individuals with NBS exhibit microcephaly and a bird-like face, but have neither ataxia nor telangiectasia. The risk of malignancies is very high. NBS results from a deficiency in nibrin (NBSI, a protein associatedwithMRE11 and Rad50thatisinvolved incheckingDNAlesions) caused by hypomorphic mutations.

1	Severe forms of dyskeratosis congenita (also known as Hoyeraal-Hreidarsson syndrome) combine a progressive immunodeficiency thatcan alsoincludean absence ofB and NK lymphocytes,progressive bone marrow failure, microcephaly, in utero growth retardation, and gastrointestinal disease. The disease can be X-linked or, more rarely, autosomal recessive. It is caused by the mutation of genes encoding telomere maintenance proteins, including dyskerin (DKC1).

1	Finally, immunodeficiency with centromeric and facial anomalies (ICF) is a complex syndrome of autosomal recessive inheritance that variably combines a mild T cell immune deficiency with a more severe B cell immune deficiency, coarse face, digestive disease, and mild mental retardation. A diagnostic feature is the detection by cytogenetic analysis of multiradial aspects in multiple chromosomes (most frequently 1, 9, and 16) corresponding to an abnormal DNA structure secondary to defective DNA methylation. It is the consequence of a deficiency in the DNA methyltransferase DNMT3B, or ZBTB24.

1	T Cell Primary Immunodeficiencies with Hyper-IgE Several T cell PIDs are associated with elevated serum IgE levels (as in Omenn syndrome). A condition sometimes referred to as autosomal recessive hyper-IgE syndrome is notably characterized by recurrent bacterial infections in the skin and respiratory tract and severe skin and mucosal infections by pox viruses and human papillomaviruses, together with severe allergic manifestations. T and B lymphocyte counts are low. Mutations in the DOCK8 genehavebeenfoundinmostofthesepatients.Thiscondition is an indication for HSCT. A very rare, related condition with autosomal recessive inheritance that causes a similar susceptibility to infection with various microbes (see above), including mycobacteria, reportedly results from a deficiency in Tyk-2, a JAK family kinase involved in the signaling of many different cytokine receptors.

1	autosomal Dominant Hyper-IgE Syndrome This unique condition, the autosomal dominant hyper-IgE syndrome, is usually diagnosed by the combination of recurrent skin and lung infections that can be complicatedbypneumatoceles.Infectionsarecausedbypyogenicbacteriaand fungi. Several other manifestations characterize hyper-IgE syndrome, including facial dysmorphy, defective loss of primary teeth, hyperextensibility, scoliosis, and osteoporosis. Elevated serum IgE levels are typical of this syndrome. Defective TH17 effector responses have been shown toaccount at leastin part forthe specific patternsof susceptibility to particular microbes. This condition is caused by a heterozygous (dominant) mutation in the gene encoding the transcription factor STAT3 that is required in a number of signaling pathways following binding of cytokine to cytokine receptors (such as that of IL-6 and the IL-6 receptor). It also results in partially defective antibody production because of defective IL-21R

1	pathways following binding of cytokine to cytokine receptors (such as that of IL-6 and the IL-6 receptor). It also results in partially defective antibody production because of defective IL-21R signaling. Hence, immunoglobulin substitution can be considered as prophylaxis of bacterial infections.

1	Cartilage Hair Hypoplasia The autosomal recessive cartilage hair hypoplasia (CHH) disease is characterized by short-limb dwarfism, metaphyseal dysostosis, and sparse hair, together with a combined T and B cell PID of extremely variable intensity (ranging from quasi-SCID to no clinically significant immune defects). The condition can predispose to erythroblastopenia, autoimmunity, and tumors. It is caused by mutations in the RMRP gene for a noncoding ribosome-associated RNA.

1	FIGUrE 374-4 B cell differentiation and related primary immunodeficiencies (PIDs). Hematopoietic stem cells (HSCs) differentiate into common lymphoid progenitors (CLPs), which give rise to pre-B cells. The B cell differentiation pathway goes through the pre–B cell stage (expression of the μ heavy chain and surrogate light chain), the immature B cell stage (expression of surface IgM), and the mature B cell stage (expression of surface IgM and IgD). The main phenotypic characteristics of these cells are indicated. In lymphoid organs, B cells can differentiate into plasma cells and produce IgM or undergo (in germinal centers) Ig class switch recombination (CSR) and somatic mutation of the variable region of V genes (SHM) that enable selection of high-affinity antibodies. These B cells produce antibodies of various isotypes and generate memory B cells. PIDs are indicated in the purple boxes. CVID, common variable immunodeficiency.

1	CD40 Ligand and CD40 Deficiencies Hyper-IgM syndrome (HIGM) is a glomerulonephritis, skin and visceral vasculitis (including brain well-known PID that is usually classified as a B cell immune deficiency vasculitis), erythema nodosum, and arthritis. Another possible conse(see Fig. 374-4 and below). It results from defective immunoglobulin quence of WAS is lymphoma, which may be virally induced (e.g., by classswitchrecombination(CSR)ingerminalcentersandleadsto EBVorKaposi’ssarcoma–associatedherpesvirus).Thrombocytopenia profound deficiency in production of IgG, IgA, and IgE (although IgM can be severe and compounded by the peripheral destruction of production is maintained). Approximately half of HIGM sufferers are platelets associated with autoimmune disorders. Hypomorphic mutaalso prone to opportunistic infections, e.g., interstitial pneumonitis tions usually lead to milder outcomes that are generally limited to caused by Pneumocystis jiroveci (in young children), protracted

1	prone to opportunistic infections, e.g., interstitial pneumonitis tions usually lead to milder outcomes that are generally limited to caused by Pneumocystis jiroveci (in young children), protracted diar-thrombocytopenia. It is noteworthy that even patients with “isolated” rhea and cholangitis caused by Cryptosporidium, and infection of the X-linked thrombocytopenia can develop severe autoimmune disease brain with Toxoplasma gondii. or lymphoma later in life. The immunologic workup is not very

1	In the majority of cases, this condition has an X-linked inheritance informative; there can be a relative CD8+ T cell deficiency, frequently and is caused by a deficiency in CD40 ligand (L). CD40L induces sig-accompanied by low serum IgM levels and decreased antigen-specific naling events in B cells that are necessary for both CSR and adequate antibody responses. A typical feature is reduced-sized platelets on a activation of other CD40-expressing cells that are involved in innate blood smear. Diagnosis is based on intracellular immunofluorescence immune responses against the above-mentioned microorganisms. analysisofWASprotein(WASp)expressioninbloodcells.WASpreg-More rarely, the condition is caused by a deficiency in CD40 itself. The ulates the actin cytoskeleton and thus plays an important role in many poorer prognosis of CD40L and CD40 deficiencies (relative to most lymphocyte functions, including cell adhesion and migration and the other HIGM conditions) implies that (1) thorough

1	role in many poorer prognosis of CD40L and CD40 deficiencies (relative to most lymphocyte functions, including cell adhesion and migration and the other HIGM conditions) implies that (1) thorough investigations have formation of synapses between antigen-presenting and target cells. tobeperformedinallcasesofHIGMand(2)potentiallycurativeHSCT Predispositiontoautoimmunedisordersisinpartrelatedtodefective should be discussed on a case-by-case basis for this group of patients. regulatory T cells. The treatment of WAS should match the severity of disease expression. Prophylactic antibiotics, immunoglobulin G (IgG) Wiskott-aldrich Syndrome Wiskott-Aldrich syndrome (WAS) is a com-supplementation, and careful topical treatment of eczema are indiplex, recessive, X-linked disease with an incidence of approximately 1 cated. Although splenectomy improves platelet count in a majority of in 200,000 live births. It is caused by mutations in the WASP gene that cases, this intervention is associated

1	of approximately 1 cated. Although splenectomy improves platelet count in a majority of in 200,000 live births. It is caused by mutations in the WASP gene that cases, this intervention is associated with a significant risk of infection affect not only T lymphocytes but also the other lymphocyte subsets, (both before and after HSCT). Allogeneic HSCT is curative, with fairly dendritic cells, and platelets. WAS is typically characterized by the fol-good results overall. Gene therapy trials are also under way. A similar lowing clinical manifestations: recurrent bacterial infections, eczema, condition has been reported in a girl with a deficiency in the Wiskottand bleeding caused by thrombocytopenia. However, these mani-Aldrich interacting protein (WIP). festations are highly variable—mostly as a consequence of the many A few other complex PIDs are worth mentioning. Sp110 deficiency different WASP mutations that have been observed. Null mutations causes a T cell PID with liver

1	as a consequence of the many A few other complex PIDs are worth mentioning. Sp110 deficiency different WASP mutations that have been observed. Null mutations causes a T cell PID with liver venoocclusive disease and hypogammapredispose affected individuals to invasive and bronchopulmonary globulinemia.Chronic mucocutaneous candidiasis (CMC)isaheterogeinfections, viral infections, severe eczema, and autoimmune manifes-neous disease, considering the different inheritance patterns that have tations. The latter include autoantibody-mediated blood cytopenia, been observed. In some cases, chronic candidiasis is associated with late-onset bronchopulmonary infections, bronchiectasis, and brain aneurysms. Moderate forms of CMC are related to autoimmunity and AIRE deficiency (see below). In this setting, predisposition to Candida infection is associated with the detection of autoantibodies to TH17 cytokines. Recently, deficiencies in IL-17F and IL-17 receptor A and, above all, gain-of-function

1	predisposition to Candida infection is associated with the detection of autoantibodies to TH17 cytokines. Recently, deficiencies in IL-17F and IL-17 receptor A and, above all, gain-of-function mutations in STAT1 have been found to be associated with CMC. In all cases, CMC is related to defective TH17 function. Innate immunodeficiency in CARD9 also predisposes to chronic invasive fungal infection.

1	B LYMPHOCYTE DEFICIENCIES (TaBLE 374-1, FIG. 374-4)

1	Deficiencies that predominantly affect B lymphocytes are the most frequent PIDs and account for 60–70% of all cases. B lymphocytes make antibodies. Pentameric IgMs are found in the vascular compartment and are also secreted at mucosal surfaces. IgG antibodies diffuse freely into extravascular spaces, whereas IgA antibodies are produced and secreted predominantly from mucosa-associated lymphoid tissues. Although Ig isotypes have distinct effector functions, including Fc receptor–mediated and (indirectly) C3 receptor– dependent phagocytosis of microorganisms, they share the ability to recognize and neutralize a given pathogen. Defective antibody production therefore allows the establishment of invasive, pyogenic bacterialinfectionsaswellasrecurrentsinusandpulmonaryinfections (mostly caused by Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and, less frequently, gram-negative bacteria). If left untreated, recurrent bronchial infections lead to bronchiectasis and,

1	Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and, less frequently, gram-negative bacteria). If left untreated, recurrent bronchial infections lead to bronchiectasis and, ultimately, cor pulmonale and death. Parasitic infections such as caused by Giardia lamblia and bacterial infections caused by Helicobacter and Campylobacter of the gut are also observed. A complete lack of antibody production (namely agammaglobulinemia) can also predispose affected individuals to severe, chronic, disseminated enteroviral infections causing meningoencephalitis, hepatitis, and a dermatomyositis-like disease.

1	Even with the most profound of B cell deficiencies, infections rarely occur before the age of 6 months; this is because of transient protection provided by the transplacental passage of immunoglobulins during the last trimester of pregnancy. Conversely, a genetically nonimmunodeficient child born to a mother with hypogammaglobulinemia is, in the absence of maternal Ig substitution, usually prone to severe bacterial infections in utero and for several months after birth.

1	Diagnosis of B cell PIDs relies on the determination of serum Ig levels (Table 316-2). Determination of antibody production following immunization with tetanus toxoid vaccine or nonconjugated pneumococcal polysaccharide antigens can also help diagnose more subtle deficiencies. Another useful test is B cell phenotype determination in switched μ−δ− CD27+ and nonswitched memory B cells (μ+δ+ CD27+). In agammaglobulinemic patients, examination of bone marrow B cell precursors (Fig. 374-4) can help obtain a precise diagnosis and guide the choice of genetic tests.

1	agammaglobulinemia Agammaglobulinemia is characterized by a profounddefectinBcelldevelopment(<1%ofthenormalBcellblood count). In most patients, very low residual Ig isotypes can be detected in the serum. In 85% of cases, agammaglobulinemia is caused by a mutation in the BTK gene that is located on the X chromosome. The BTK gene product is a kinase that participates in (pre) B cell receptor signaling. When the kinase is defective, there is a block (albeit a leaky one) at the pre-B to B cell stage (Fig. 374-4). Detection of BTK by intracellular immunofluorescence of monocytes, and lack thereof in patients with X-linked agammaglobulinemia, is a useful diagnostic test. Not all of the mutations in BTK result in agammaglobulinemia, since some patients have a milder form of hypogammaglobulinemia and low but detectable B cell counts. These cases should not be confused with common variable immunodeficiency (CVID, see below). About 10% of agammaglobulinemia cases are caused by alterations in

1	and low but detectable B cell counts. These cases should not be confused with common variable immunodeficiency (CVID, see below). About 10% of agammaglobulinemia cases are caused by alterations in genes encoding elements of the pre-B cell receptor, i.e., the μ heavy chain, the λ5 surrogate light chain, Igα or Igβ, the scaffold protein BLNK, and the p85 α subunit of phosphatidylinositol 3 phosphate kinase (P13K). In 5% of cases, the defect is unknown. It is noteworthy that agammaglobulinemia can be observed in patients with ICF syndrome, despite the presence of normal peripheral B cell counts. 2111 Lastly, agammaglobulinemia can be a manifestation of a myelodysplastic syndrome (associated or not with neutropenia). Treatment of agammaglobulinemic patients is based on immunoglobulin replacement (see below). Profound hypogammaglobulinemia is also observed in adults, in association with thymoma.

1	Hyper-IgM (HIGM) Syndromes HIGM is a rare B cell PID characterized by defective Ig CSR. It results in very low serum levels of IgG and IgA and elevated or normal serum IgM levels. The clinical severity is similar to that seen in agammaglobulinemia, although chronic lung disease and sinusitis are less frequent and enteroviral infections are uncommon. As discussed above, a diagnosis of HIGM involves screening for an X-linked CD40L deficiency and an autosomal recessive CD40 deficiency, which affect both B and T cells. In 50% of cases affecting only B cells, these isolated HIGM syndromes result from mutations in the gene encodingactivation-induceddeaminase,theproteinthatinducesCSRin B cell germinal centers. These patients usually have enlarged lymphoid organs. In the other 50% of cases, the etiology is unknown (except for rareUNGandPMS2deficiencies).Furthermore,IgM-mediatedautoimmunity and lymphomas can occur in HIGM syndrome. It is noteworthy that HIGM can result from fetal rubella

1	the etiology is unknown (except for rareUNGandPMS2deficiencies).Furthermore,IgM-mediatedautoimmunity and lymphomas can occur in HIGM syndrome. It is noteworthy that HIGM can result from fetal rubella syndrome or can be a predominant immunologic feature of other PIDs, such as the immunodeficiency associated with ectodermic anhydrotic hypoplasia X-linked NEMO deficiency and the combined T and B cell PIDs caused by DNA repair defects such as AT and Cernunnos deficiency.

1	Common Variable Immunodeficiency (CVID) CVID is an ill-defined condi tion characterized by low serum levels of one or more Ig isotypes. Its prevalence is estimated to be 1 in 20,000. The condition is recognized predominantly in adults, although clinical manifestations can occur earlier in life. Hypogammaglobulinemia is associated with at least partially defective antibody production in response to vaccine antigens. B lymphocyte counts are often normal but can be low. Besides infections, CVID patientsmaydevelop lymphoproliferation (splenomegaly),granulomatouslesions,colitis,antibody-mediatedautoimmunedisease,and lymphomas.Afamilyhistoryisfoundin10%ofcases.Aclear-cutdominant inheritance pattern is found in some families, whereas recessive inheritance is observed more rarely. In most cases, no molecular cause canbeidentified.AsmallnumberofpatientsinGermanywerefoundto carry mutations in the ICOS gene encoding a T cell-membrane protein

1	inheritance is observed more rarely. In most cases, no molecular cause canbeidentified.AsmallnumberofpatientsinGermanywerefoundto carry mutations in the ICOS gene encoding a T cell-membrane protein thatcontributestoBcellactivationandsurvival.In10%ofpatientswith CVID, monoallelic or biallelic mutations of the gene encoding TACI (a member of the tumor necrosis factor [TNF] receptor family that is expressed on B cells) have been found. In fact, heterozygous TACI mutations correspond to a genetic susceptibility factor, since similar heterozygous mutations are found in 1% of controls. The BAFF receptor was found to be defective in a kindred with CVID, although not all individuals carrying the mutation have CVID.

1	Recently a group of patients with hypogammaglobulinemia and lymphoproliferation were shown to exhibit dominant gain of function mutations in the PIK3CD gene encoding the p110δ form of P13 kinase. A diagnosis of CVID should be made after excluding the presence of hypomorphic mutations associated with agammaglobulinemia or more subtle T cell defects; this is particularly the case in children. It is possible that many cases of CVID result from a constellation of factors, rather than a single genetic defect. Recently, rare cases of hypogammaglobulinemia were found to be associated with CD19 and CD81 deficiencies. These patients have B cells that can be identified by typing for other B cell markers. Hypogammaglobulinemia can be associated with neutropenia and lymphopenia in the warts, hypogammaglobulinemia, infections, and myelokathexis syndrome (WHIM) caused by dominant gain-of-function mutation of CXCR4, resulting in cell retention in the bone marrow.

1	Selective Ig Isotype Deficiencies IgA deficiency and CVID represent polar ends of a clinical spectrum due to the same underlying gene defect(s) in a large subset of these patients. IgA deficiency is the most common PID; it can be found in 1 in every 600 individuals. It is asymptomatic in most cases; however, individuals may present 2112 with increased numbers of acute and chronic respiratory infections that may lead to bronchiectasis. In addition, over their lifetime, these patients experience an increased susceptibility to drug allergies, atopic disorders, and autoimmune diseases. Symptomatic IgA deficiency is probably related to CVID, since it can be found in relatives of patients with CVID. Furthermore, IgA deficiency may progress to CVID. It is thus important to assess serum Ig levels in IgA-deficient patients (especially when infections occur frequently) in order to detect changes that should prompt the initiation of immunoglobulin replacement. Selective IgG2 (+G4) deficiency

1	in IgA-deficient patients (especially when infections occur frequently) in order to detect changes that should prompt the initiation of immunoglobulin replacement. Selective IgG2 (+G4) deficiency (which in some cases may be associated with IgA deficiency) can also result in recurrent sinopulmonaryinfectionsandshouldthusbespecificallysoughtinthis clinical setting. These conditions are ill-defined and often transient duringchildhood.Apathophysiologicexplanationhasnotbeenfound.

1	Selective antibody Deficiency to Polysaccharide antigens Some patients with normal serum Ig levels are prone to S. pneumoniae and H. influenzae infections of the respiratory tract. Defective production of antibodies against polysaccharide antigens (such as those in the S. pneumoniae cell wall) can be observed and is probably causative. This condition may correspond to a defect in marginal zone B cells, a B cell subpopulation involved in T-independent antibody responses.

1	Immunoglobulin replacement IgG antibodies have a half-life of 21–28 days. Thus, injection of plasma-derived polyclonal IgG containing a myriad of high-affinity antibodies can provide protection against disease-causing microorganisms in patients with defective IgG antibody production. This form of therapy should not be based on laboratory data alone (i.e., IgG and/or antibody deficiency) but should be guided by the occurrence or not of infections; otherwise, patients might be subjected to unjustified IgG infusions. Immunoglobulin replacement can be performed by IV or subcutaneous routes. In the former case, injections have to be repeated every 3–4 weeks, with a residual target level of 800 mg/mL in patients who had very low IgG levels prior to therapy. Subcutaneous injections are typically performed once a week, although the frequency can be adjusted on a case-by-case basis. A trough level of 800 mg/mL is desirable. Whatever the mode of administration, the main goal is to reduce the

1	performed once a week, although the frequency can be adjusted on a case-by-case basis. A trough level of 800 mg/mL is desirable. Whatever the mode of administration, the main goal is to reduce the frequency of the respiratory tract infections and prevent chronic lung and sinus disease. The two routes appear to be equally safe and efficacious, and so the choice should be left to the preference of the patient.

1	In patients with chronic lung disease, chest physical therapy with good pulmonary toilet and the cyclic use of antibiotics are also needed. Immunoglobulinreplacementiswelltoleratedbymostpatients,although the selection of the best-tolerated Ig preparation may be necessary in certain cases. Since IgG preparations contain a small proportion of IgAs, caution should be taken in patients with residual antibody production capacity and a complete IgA deficiency, as these subjects may develop anti-IgA antibodies that can trigger anaphylactic shock. These patients should be treated with IgA-free IgG preparations. Immunoglobulin replacement is a lifelong therapy; its rationale and procedures have to be fully understood and mastered by the patient and his or her family in order to guarantee the strict observance required for efficacy.

1	An increasing number of PIDs have been found to cause homeostatic dysregulation of the immune system, either alone or in association with increased vulnerability to infections. Defects of this type affecting the innate immune system and autoinflammatory syndromes will not be covered in this chapter. However, three specific entities (hemophagocytic lymphohistiocytosis, lymphoproliferation, and autoimmunity) will be described below. Hemophagocytic lymphohistiocytosis (HLH) is characterized by an unremitting activation of CD8+ T lymphocytes and macrophages that leads to organ damage (notably in the liver, bone marrow, and central nervous system). This syndrome results from a broad set of inherited diseases, all of which impair T and NK lymphocyte cytotoxicity. The manifestations of HLH are often induced by a viral infection. EBV is the most frequent trigger. In severe forms of HLH, disease onset may start during the first year of life or even (in rare cases) at birth.

1	Diagnosis reliesonthe identification of thecharacteristic symptoms ofHLH(fever,hepatosplenomegaly,edema,neurologicdiseases,blood cytopenia, increased liver enzymes, hypofibrinogenemia, high triglyceride levels, elevated markers of T cell activation, and hemophagocytic features in the bone marrow or cerebrospinal fluid). Functional assays of postactivation cytotoxic granule exocytosis (CD107 fluorescence at the cell membrane) can suggest genetically determined HLH. The conditions can be classified into three subsets: 1. Familial HLH with autosomal recessive inheritance, including perforin deficiency (30% of cases) that can be recognized by assessing intracellular perforin expression; Munc13-4 deficiency (30% of cases); syntaxin 11 deficiency (10% of cases); Munc18-2 deficiency (20% of cases); and a few residual cases that lack a known molecular defect. 2.

1	2. HLH with partial albinism. Three conditions combine HLH and abnormal pigmentation, where hair examination can help in the diagnosis: Chédiak-Higashi syndrome, Griscelli syndrome, and Hermansky Pudlak syndrome type II. Chédiak-Higashi syndrome is also characterized by the presence of giant lysosomes within leukocytes (Chap. 80), in addition to a primary neurologic disorder with slow progression of symptoms over time. 3.

1	3. X-linked proliferative syndrome (XLP) is characterized in most patients by the induction of HLH following EBV infection, while other patients develop progressive hypogammaglobulinemia similar to what is observed in CVID and/or certain lymphomas. XLP is caused by a mutation in the SH2DIA gene that encodes the adaptor protein SAP (associated with a SLAM family receptor). Several immunologic abnormalities have been described, including low 2B4-mediated NK cell cytotoxicity, impaired differentiation of NKT cells, defective antigen-induced T cell death, and defective T cell helper activity for B cells. A related disorder (XLP2) has recently been described. It is also X-linked and induces HLH (frequently after EBV infection), although the clinical manifestation may be less pronounced. The condition is associated with a deficiency of the antiapoptotic molecule XIAP. The pathophysiology of XLP2 and its relationship to XLP1 remain unclear.

1	HLH is a life-threatening complication. The treatment of this condition requires aggressive immunosuppression with either the cytotoxic agent etoposide or anti–T cell antibodies. Once remission has been achieved, HSCT should be performed, since it provides the only curative form of therapy. Autoimmune lymphoproliferative syndrome (ALPS) is characterized by nonmalignant T and B lymphoproliferation causing splenomegaly and enlargedlymphnodes;70%ofpatientsalsodisplayautoimmunemanifestations such as autoimmune cytopenias, Guillain-Barré syndrome, uveitis, and hepatitis (Chaps. 79 and 372e). A hallmark of ALPS is the presence of CD4–CD8– TCRαβ+ T cells (2–50%) in the blood of affectedindividuals.HypergammaglobulinemiainvolvingIgGandIgA is also frequently observed. The syndrome is caused by a defect in Fas-mediated apoptosis of lymphocytes, which can thus accumulate and mediate autoimmunity. Furthermore, ALPS can lead to malignancies.

1	MostpatientscarryaheterozygousmutationinthegeneencodingFas that is characterized by dominant inheritance and variable penetrance, depending on the nature of the mutation. A rare and severe form of the disease with early onset can be observed in patients carrying a biallelic mutationofFas,whichprofoundlyimpairstheprotein’sexpressionand/ or function. Fas-ligand, caspase 10, caspase 8, and neuroblastoma RAS viral oncogene homologue (NRAS) mutations have also been reported in a few cases of ALPS. Many cases of ALPS have not been precisely delineated at the molecular level. A B cell–predominant ALPS has recently been found associated with a protein kinase Cδ gene mutation. Treatment ofALPS isessentially basedon the useof proapoptoticdrugs, which need to be carefully administered in order to avoid toxicity. CoLITIS, AUToIMMUNITY, ANd PRIMARY IMMUNodEFICIENCIES

1	Several PIDs (most of which are T cell–related) can cause severe gut inflammation. The prototypic example is immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX), characterized by a widespread inflammatory enteropathy, food intolerance, skin rashes, autoimmune cytopenias, and diabetes. The syndrome is caused by loss-of-function mutations in the gene encoding the transcription factorFOXP3,whichisrequiredfortheacquisitionofeffectorfunction by regulatory T cells. In most cases of IPEX, CD4+CD25+ regulatory T cells are absent from the blood. This condition has a poor prognosis and requires aggressive immunosuppression. The only possible curative approach is allogeneic HSCT. IPEX-like syndromes that lack a FOXP3 mutation have also been described. In some cases, a CD25 deficiency has been found. Defective CD25 expression also impairs regulatory cell expansion/function. This functional T cell deficiency means that CD25-deficient patients are also at increased risk of

1	has been found. Defective CD25 expression also impairs regulatory cell expansion/function. This functional T cell deficiency means that CD25-deficient patients are also at increased risk of opportunistic infections. It is noteworthy that abnormalities in regulatory T cells have been described in other PID settings, such as in Omenn syndrome, STAT5b deficiency, STIM1 (Ca flux) deficiency, and WAS; these abnormalities mayaccount (at least in part) for theoccurrence of inflammation and autoimmunity. The autoimmune features observed in a small fraction of patients with DiGeorge syndrome may have the same cause. Recently, severe inflammatory gut disease has been described in patients with a deficiency in the IL-10 receptor or IL-10.

1	A distinct autoimmune entity is observed in autoimmune polyendocrinopathy candidiasis ectodermal dysplasia (APECED) syndrome, which is characterized by autosomal recessive inheritance. It consists of multiple autoimmune manifestations that can affect solid organs in general and endocrine glands in particular. Mild, chronic Candida infection is often associated with this syndrome. The condition is due to mutations in the autoimmune regulator (AIRE) gene and results in impaired thymic expression of self-antigens by medullary epithelial cells and impaired negative selection of self-reactive T cells that leads to autoimmune manifestations. A combination of hypogammaglobulinemia, autoantibody production, cold-induced urticaria or skin granulomas, or autoinflammation hasbeenreported, andhasbeentermedthe PLCβ2-associated antibody deficiency and immune dysregulation (PLAID or APLAID).

1	The variety and complexity of the clinical manifestations of the many different PIDs strongly indicate that it is important to raise awareness of these diseases. Indeed, early diagnosis is essential for establishing an appropriate therapeutic regimen. Hence, patients with suspected PIDs must always be referred to experienced clinical centers that are able to perform appropriate molecular and genetic tests. A precise molecular 2113 diagnosisis notonlynecessary for initiating the mostsuitabletreatment, but is also important for genetic counseling and prenatal diagnosis.

1	One pitfall that may hamper diagnosis is the high variability that is associated with many PIDs. Variable disease expression can result from the differing consequences of various mutations associated with a givencondition,asexemplifiedbyWASand,toalesserextent,X-linked agammaglobulinemia (XLA). There can also be effects of modifier genes (as also suspected in XLA) and environmental factors such as EBVinfectionthatcanbethemaintriggerofdiseaseinXLPconditions. Furthermore, it has recently been established that somatic mutations in an affected gene can attenuate the phenotype of a number of T cell PIDs. This has been described for ADA deficiency, X-linked SCID, RAG deficiencies, NF-κB essential modulator (NEMO) deficiency, and, most frequently, WAS. In contrast, somatic mutations can create disease states analogous to PID, as reported for ALPS. Lastly, cytokineneutralizing autoantibodies can mimic a PID, as shown for IFN-γ.

1	ManyaspectsofthepathophysiologyofPIDsare still unknown, and thedisease-causinggenemutationshavenotbeenidentifiedinallcases (as illustrated by CVID and IgA deficiency). However, our medical understanding of PIDs has now reached the stage where scientifically based approaches to the diagnosis and treatment of these diseases can be implemented. immunodeficiency (PID) has been described as one facet of a more complex disease setting. It is essential to consider associated diseases when a PID is identified as the primary manifestation and, conversely, not neglect the potentially harmful consequences of a PID that could be masked by other manifestations of a particular syndrome. Below is a short description of these syndromes in which the PID is classified according to the arm of the immune system that is affected. 1. Primary Immunodeficiencies of the Innate Immune System

1	Below is a short description of these syndromes in which the PID is classified according to the arm of the immune system that is affected. 1. Primary Immunodeficiencies of the Innate Immune System Several severe congenital neutropenia (SCN) syndromes can be associated with malformations. The recently described SCN disease caused by glucose-6-phosphatase deficiency (G6PC3) can be associated with heart and urogenital malformations. The related glycogenesis Ib disease combines SCN with hypoglycemia and hepatosplenomegaly. Some HAX-1 gene mutations lead to neurocognitive impairments as well as SCN. Barth syndrome combines SCN with cardiomyopathy. Lastly, Shwachman syndrome is a known autosomal recessive entity (caused by mutation of the SBDS gene) in which the defect in granulopoiesis can extend to the other hematopoietic lineages; short stature, bone metaphyseal dysplasia, and exocrine pancreatic insufficiency are known hallmarks of this condition.

1	Syndromic asplenia combines the risk of infection with heart defects and situs inversus. Leukocyte adhesion deficiency (LAD) type II includes growth retardation and impairment of cognitive development. A few patients with X-linked chronic granulomatous disease present with a contiguous gene deletion syndrome that can include the McLeod phenotype, which is characterized by anemia, acanthocytosis, and a severe risk of immune reaction against donor red cells because the patient’s red cells do not express the Kell antigen. The McLeod phenotype also can result in a neurologic disease. X-linked nuclear factor-κB (NF-κB) essential modulator (NEMO) deficiency provokes not only a variable set of deficiencies of both innate and adaptive immunity but also mild osteopetrosis, lymphedema, and, more frequently, anhydrotic ectodermal dysplasia, dysmorphic facies, and abnormal conical teeth. The last finding is often helpful in the diagnosis of that condition.

1	2. Primary Immunodeficiencies of the Adaptive Immune System T cell primary immunodeficiencies. Reticular dysgenesis, a rare severe combined immunodeficiency (SCID) characterized by T lymphopenia and agranulocytosis, can cause sensorineural deafness. Coronin A deficiency is another SCID variant that can be associated with behavioral disorders because the Coronin A gene is located in a genome area known to have been deleted in some patients with this disorder. The lack of enzymes of purine metabolism (adenosine deaminase and purine nucleoside phosphorylase) provokes not only profound T cell lymphocytopenia but also neurologic impairment, including dysautonomia and abnormalities of cognitive development of variable intensity, in many patients. The neurologic impairment can persist after hematopoietic stem cell transplantation (HSCT). Mild chondrodysplasia is a common finding in adenosine deaminase (ADA) deficiency and, indeed, can help the physician arrive at a final diagnosis.

1	Primary thymic defects. DiGeorge syndrome is a complex embryopathy that is caused by hemizygous interstitial deletion of chromosome 22, leading to multiple developmental defects, including conotruncal defects, hypoparathyroidism, and dys-375e-1 morphic syndrome. Although a profound T cell immunodeficiency is rare in DiGeorge syndrome (∼1% of cases), failure to recognize this feature is likely to have a fatal outcome. Similarly, some forms of the related CHARGE syndrome (mutation of the CHD7 gene) also cause a profound T cell immunodeficiency. c) T cell primary immunodeficiencies related to calcium influx defects. Recently, rare T cell PIDs were found to be caused by defective store-operated entry of calcium ions into T and B lymphocytes after antigen stimulation. These defects (caused by ORA-1 and STIM-1 deficiencies) also lead to anhydrotic ectodermal dysplasia, abnormal teeth, and, above all, a nonprogressive muscle disease characterized by excessive fatigue.

1	d) DNA repair defects. Several genetic defects impair DNA repair pathways. Many lead to combined T and B lymphocyte PIDs in a syndromal setting of varying complexity. The most common is ataxia-telangiectasia (AT), an autosomal recessive disorder with an incidence of 1 in 40,000 live births; AT causes a B cell immunodeficiency (low IgA, IgG2 deficiency, and low antibody production) that often requires immunoglobulin replacement therapy. AT is associated with a progressive T cell immunodeficiency. As the condition’s name suggests, the hallmark features are telangiectasia and cerebellar ataxia. These manifestations may not be detectable before age 3–4 years, and so AT should be considered in young children with IgA deficiency and problematic infections. Diagnosis is based on a cytogenetic analysis showing excessive chromosomal rearrangements (mostly affecting chromosomes 7 and 14) in lymphocytes. AT is caused by mutation of the gene encoding the ATM protein, a kinase that plays a major

1	showing excessive chromosomal rearrangements (mostly affecting chromosomes 7 and 14) in lymphocytes. AT is caused by mutation of the gene encoding the ATM protein, a kinase that plays a major role in the detection of DNA lesions and the organization of DNA repair (or cell death if the lesions are too numerous) by triggering several different pathways. Overall, AT is a progressive disease that carries a very high risk of lymphoma, leukemia, and (during adulthood) carcinomas. A variant of AT (AT-like disease) is caused by mutation of the MRE11 gene. Nijmegen breakage syndrome (NBS) is a less common condition that also results from chromosome instability (and the same cytogenetic abnormalities as in AT). It is characterized by a severe T and B cell combined immunodeficiency with autosomal recessive inheritance. Subjects with NBS exhibit microcephaly and a birdlike face but neither ataxia nor telangiectasia. The risk of malignancies is also very high. Nijmegen breakage syndrome results

1	recessive inheritance. Subjects with NBS exhibit microcephaly and a birdlike face but neither ataxia nor telangiectasia. The risk of malignancies is also very high. Nijmegen breakage syndrome results from a deficiency in Nibrin (NBS1, a protein associated with MRE11 and Rad50 that is involved in checking DNA lesions) caused by hypomorphic mutations. Severe forms of dyskeratosis congenita (also known as Hoyeraal-Hreidersson syndrome) combine a progressive immunodeficiency that can include an absence of B and natural killer cell (NK) lymphocytes, progressive bone marrow failure, microcephaly, in utero growth retardation, and gut disease. The disease can be X-linked or, more rarely, autosomal recessive. It is caused by the mutation of genes encoding telomere maintenance proteins, including dyskerin (DKC1). Bloom syndrome (helicase deficiency) combines a typical dysmorphic syndrome with growth retardation, skin lesions, and a mild immunodeficiency that also can be found in some patients

1	dyskerin (DKC1). Bloom syndrome (helicase deficiency) combines a typical dysmorphic syndrome with growth retardation, skin lesions, and a mild immunodeficiency that also can be found in some patients with Fanconi’s anemia. Rare forms of combined T and B cell immunodeficiencies with autosomal recessive inheritance are associated in more complex syndromes with microcephaly, failure to grow, and a variable dysmorphic syndrome. These disorders are caused by mutation of the genes that encode DNA ligase 4 and Cernunnos (XLF), both of which are members of the nonhomologous end-joining DNA repair pathway.

1	The Vici syndrome combines callosal agenesis, cataracts, cardiomyopathy, hyperpigmentation, and a combined immunodeficiency. It is caused by biallelic EPG5 gene mutation that results in defective autophagy. CHAPTER 375e Primary Immunodeficiencies Associated with (or Secondary to) Other Diseases Lastly, immunodeficiency, centromere instability, and facial anomalies (ICF) syndrome is a complex autosomal recessive syndrome that variably combines a mild T cell immunodeficiency and a more severe B cell immunodeficiency with a coarse face, intestinal disease, and mild mental retardation. A cytogenetic diagnostic feature is the presence of multiradial chromosomes (most frequently chromosomes 1, 9, and 16) caused by DNA defective methylation. The syndrome is a result of either DNA methyltransferase DNMT3B deficiency or ZBTB24 deficiency.

1	Growth hormone insensitivity syndrome (Laron dwarfism) with combined primary immunodeficiency. Mutations in the STAT5b gene, which encodes a transcription factor involved in signaling downstream of the growth hormone receptor and the interleukin 2 (IL-2) receptor, lead to susceptibility to infection because of a partial, functional T cell immunodeficiency associated with autoimmune manifestations. The autoimmune manifestations probably result from defective generation/activation of regulatory T cells.

1	Hyper-IgE syndrome (autosomal dominant form). Hyper-IgE syndrome is a complex disorder that combines skin infections, inflammation, and susceptibility to bacterial and fungal infections of skin and lungs, often with pneumatoceles, with characteristic syndromic signs such as facial dysmorphy, defective loss of primary teeth, hyperextensibility, scoliosis, and osteoporosis. Elevated serum IgE levels are typical of hyper-IgE syndrome. The recently reported defects in TH17 effector responses account, at least in part, for the vulnerability to specific infections. This condition is caused by heterozygous (dominant) mutation of the gene encoding the transcription factor STAT3, which is required in a number of signaling pathways downstream of cytokine/cytokine receptor interactions (notably for IL-6 and IL21).

1	Primary immunodeficiencies with bone disease. The autosomal recessive cartilage hair hypoplasia (CHH) disease is characterized by short-limb dwarfism, metaphyseal dysostosis, and sparse hair, together with a combined T and B cell PID of variable intensity, ranging from quasi-SCID to an absence of clinically significant immunodeficiency. The condition can predispose to erythroblastopenia, autoimmunity, and tumors. It is caused by mutations in the RMRP gene for a noncoding ribosome-associated RNA. Schimke immunoosseous dysplasia is a rare autosomal recessive condition characterized by severe T and B cell immunodeficiency with spondyloepiphyseal dysplasia, growth retardation, and kidney and vascular diseases. It is the consequence of mutations in the SMARCAL1 gene. The function of the gene product may be related to DNA repair.

1	Venoocclusive disease with immunodeficiency (VODI syndrome) is a rare autosomal recessive condition predominantly found in populations originating from Lebanon. It combines severe hepatic venoocclusive disease with usually mild T cell immunodeficiency and panhypogammaglobulinemia. It is caused by a deficiency in a nuclear protein, Sp110. 3.

1	3. B Cell Primary Immunodeficiencies Hypogammaglobulinemia can be associated with chromosomal defects such as trisomy 18 and Jacobsen syndrome (hemizygous deletion of part of the long arm of chromosome 11). A rare biallelic deficiency of the mismatch repair protein PMS2 leads to a partial deficiency in Ig class switch recombination in patients at a very high risk of cancer in general and colon carcinomas and lymphomas in particular. Transcobalamin deficiency disturbs vitamin B12 transport and therefore impairs hematopoiesis. Hypogammaglobulinemia is easily corrected by vitamin B12 administration and can be a characteristic of this very rare disorder. 4.

1	4. Primary Immunodeficiencies Affecting Regulatory Pathways Several inherited disorders that lead to hemophagocytic lymphohistiocytosis (HLH) also have features that are important in terms of both diagnosis and prognosis. Three of these disorders—Griscelli syndrome, Chédiak-Higashi syndrome, and the Hermansky-Pudlak type II syndrome—are characterized by partial albinism and silvery hair appearance that can facilitate diagnosis. Hermansky-Pudlak type II also can be a bleeding disorder if platelet aggregation is defective. Chédiak-Higashi syndrome also is characterized by an early-onset progressive neurologic disorder with impaired cognitive development and motor and sensory deficiencies, culminating in a generalized encephalopathy. The encephalopathy is not prevented or arrested by allogeneic HSCT even when the HLH risk is controlled. 5. Primary Immunodeficiencies Associated with Other Conditions

1	5. Primary Immunodeficiencies Associated with Other Conditions Predisposition to infection, notably severe disseminated opportunistic infections including nontuberculous mycobacterial infections, can be associated with autoantibodies against interferon γ as observed in Asia. A number of conditions can cause PIDs indirectly. For example, hypercatabolism in patients with Steinert’s disease may cause hypogammaglobulinemia. Intestinal lymphangiectasia that includes both immunoglobulin and naive T cell loss and can expose the patient to a significant infectious risk. Urinary IgG loss may result from severe nephritic syndromes. A number of drugs, including antimalarials, captopril, penicillamine, phenytoin, and sulfasalazine, can induce predominantly IgA hypogammaglobulinemia in (probably predisposed) adults.

1	One also should also consider (1) diseases that are not thought to be PIDs but include the occurrence of recurrent infections and (2) genetic defects of the immune system that lead to other clinical manifestations. A very good example of the first group is cystic fibrosis (CF). Despite having a functionally normal immune system, patients with CF develop protracted bacterial respiratory tract infections, notably Pseudomonas aeruginosa colonization. This bacterium can incapacitate innate immune responses and cause unremitting inflammation that further facilitates infection. An example of the second group is primary alveolar proteinosis, which is caused by a defect in surfactant clearance by alveolar macrophages. The condition results from mutation of the gene encoding the granulocyte-macrophage colony-stimulating factor receptor α. Allergies, Anaphylaxis, and Systemic Mastocytosis allergies, anaphylaxis, and 376 Systemic Mastocytosis Joshua A. Boyce, K. Frank Austen

1	Allergies, Anaphylaxis, and Systemic Mastocytosis allergies, anaphylaxis, and 376 Systemic Mastocytosis Joshua A. Boyce, K. Frank Austen The term atopy implies a tendency to manifest asthma, rhinitis, urticaria, and atopic dermatitis alone or in combination, in association with the presence of allergen-specific IgE. However, individuals without an atopic background may also develop hypersensitivity reactions, particularly urticaria and anaphylaxis, associated with the presence of IgE. Inasmuch as the mast cell is a key effector cell in allergic rhinitis and asthma, and the dominant effector in urticaria, anaphylaxis, and systemic mastocytosis, its developmental biology, activation pathway, product profile, and target tissues will be considered in the introduction to these clinical disorders.

1	The binding of IgE to human mast cells and basophils, a process termed sensitization, prepares these cells for subsequent antigen-specific activation. The high-affinity Fc receptor for IgE, designated FcεRI, is composed of one α, one β, and two disulfide-linked γ chains, which together cross the plasma membrane seven times. The α chain is responsible for IgE binding, and the β and γ chains provide for signal transduction that follows the aggregation of the sensitized tetrameric receptors by polymeric antigen. The binding of IgE stabilizes the α chain at the plasma membrane, thus increasing the density of FcεRI receptors at the cell surface while sensitizing the cell for effector responses. This accounts for the correlation between serum IgE levels and the numbers of FcεRI receptors detected on circulating basophils.

1	2114 Signal transduction is initiated through the action of a Src family– related tyrosine kinase termed Lyn that is constitutively associated with the β chain. Lyn transphosphorylates the canonical immunoreceptor tyrosine-based activation motifs (ITAMs) of the β and γ chains of the receptor, resulting in recruitment of more active Lyn to the β chain and of Syk tyrosine kinase. The phosphorylated tyrosines in the ITAMs function as binding sites for the tandem src homology two (SH2) domains within Syk. Syk activates not only phospholipase Cγ, which associates with the linker of activated T cells at the plasma membrane, but also phosphatidylinositol 3-kinase to provide phosphatidylinositol-3,4,5-trisphosphate, which allows membrane targeting of the Tec family kinase Btk and its activation by Lyn. In addition, the Src family tyrosine kinase Fyn becomes activated after aggregation of IgE receptors and phosphorylates the adapter protein Gab2 that enhances activation of phosphatidylinositol

1	Lyn. In addition, the Src family tyrosine kinase Fyn becomes activated after aggregation of IgE receptors and phosphorylates the adapter protein Gab2 that enhances activation of phosphatidylinositol 3-kinase. Indeed, this additional input is essential for mast cell activation, but it can be partially inhibited by Lyn, indicating that the extent of mast cell activation is in part regulated by the interplay between these Src family kinases. Activated phospholipase Cγ cleaves phospholipid membrane substrates to provide inositol-1,4,5-trisphosphate (IP3) and 1,2-diacylglycerols (1,2-DAGs) so as to mobilize intracellular calcium and activate protein kinase C, respectively. The subsequent opening of calcium-regulated activated channels provides the sustained elevations of intracellular calcium required to recruit the mitogen-activated protein kinases, ERK, JNK, and p38 (serine/threonine kinases), which provide cascades to augment arachidonic acid release and to mediate nuclear translocation

1	to recruit the mitogen-activated protein kinases, ERK, JNK, and p38 (serine/threonine kinases), which provide cascades to augment arachidonic acid release and to mediate nuclear translocation of transcription factors for various cytokines. The calcium ion–dependent activation of phospholipases cleaves membrane phospholipids to generate lysophospholipids, which, like 1,2-DAG, may facilitate the fusion of the secretory granule perigranular membrane with the cell membrane, a step that releases the membrane-free granules containing the preformed mediators of mast cell effects. The secretory granule of the human mas t cell has a crystalline structure, unlike mast cells of lower species. IgE-dependent cell activation results in solubilization and swelling of the granule contents within the first minute of receptor perturbation; this reaction is followed by the ordering of intermediate filaments about the swollen granule, movement of the granule toward the cell surface, and fusion of the

1	minute of receptor perturbation; this reaction is followed by the ordering of intermediate filaments about the swollen granule, movement of the granule toward the cell surface, and fusion of the perigranular membrane with that of other granules and with the plasmalemma to form extracellular channels for mediator release while maintaining cell viability. In addition to exocytosis, aggregation of FcεRI initiates two other pathways for generation of bioactiveproducts, namely, lipid mediators and cytokines. The biochemical steps involved in expression of such cytokines as tumor necrosis factor α (TNF-α), interleukin (IL) 1, IL-6, IL-4, IL-5, IL-13, granulocyte-macrophage colony-stimulating factor (GM-CSF), and others, including an array of chemokines, have not been specifically defined for mast cells. Inhibition studies of cytokine production (IL-1β, TNF-α, and IL-6) in mouse mast cells with cyclosporine or FK506 reveal binding to the ligand-specific immunophilin and attenuation of the

1	mast cells. Inhibition studies of cytokine production (IL-1β, TNF-α, and IL-6) in mouse mast cells with cyclosporine or FK506 reveal binding to the ligand-specific immunophilin and attenuation of the calcium ion-and calmodulin-dependent serine/ threonine phosphatase, calcineurin. Lipid mediator generation (Fig. 376-1) involves translocation of calcium ion–dependent cytosolic phospholipase A2 to the outer nuclear membrane, with subsequent release of arachidonic acid for metabolic processing by the distinct prostanoid and leukotriene pathways. The constitutiveprostaglandinendoperoxide synthase-1(PGHS-1/cyclooxygenase-1)andthedenovoinduciblePGHS-2(cyclooxygenase-2)convert releasedarachidonicacidtothesequentialintermediates,prostaglandins G2 and H2. The glutathione-dependent hematopoietic prostaglandin D2 (PGD2) synthase then converts PGH2 to PGD2, the predominant mast cell prostanoid. The PGD2 receptor DP1 is expressed by platelets and epithelial cells, whereas DP2 is expressed by TH2

1	D2 (PGD2) synthase then converts PGH2 to PGD2, the predominant mast cell prostanoid. The PGD2 receptor DP1 is expressed by platelets and epithelial cells, whereas DP2 is expressed by TH2 lymphocytes, eosinophils, and basophils. Mast cells also generate thromboxane A2 (TXA2), a short lived but powerful mediator that induces bronchoconstriction and platelet activation through the T prostanoid (TP) receptor. For leukotriene biosynthesis, the released arachidonic acid is metabolized by 5-lipoxygenase (5-LO) in the presence of an integral nuclear membrane protein, 5-LO activating protein (FLAP). The calcium

1	LTC,4 LTD4 FIGUrE 376-1 Pathways for biosynthesis and release of membrane-derived lipid mediators from mast cells. In the 5-lipoxygenase pathway, leukotriene A4 (LTA4) is the intermediate from which the terminal-pathway enzymes generate the distinct final products, leukotriene C (LTC ) and leukotriene B (LTB ), which leave the cell by separate saturable transport systems. Gamma glutamyl transpeptidase and a dipeptidase then cleave glutamic acid and glycine from LTC to form LTD and LTE , respectively. The major mast cell product of the cyclooxygenase system is PGD2.

1	ion–dependent translocation of 5-LO to the nuclear membrane converts the arachidonic acid to the sequential intermediates, 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene (LT) A4. LTA4 is conjugated with reduced glutathione by LTC4 synthase, an integral nuclear membrane protein homologous to FLAP. Intracellular LTC4 is released by a carrier-specific export step for extracellular metabolism to the additional cysteinyl leukotrienes, LTD4 and LTE4, by the sequential removal of glutamic acid and glycine. Alternatively, cytosolic LTA4 hydrolase converts some LTA4 to the dihydroxy leukotriene LTB4, whichalsoundergoesspecificexport.TworeceptorsforLTB4,BLT1and BLT2, mediate chemotaxis of human neutrophils. Two receptors for the cysteinyl leukotrienes, CysLT1 and CysLT2, are present on smooth muscle of the airways and the microvasculature and on hematopoietic cells such as macrophages, eosinophils, and mast cells. Whereas the CysLT1 receptor has a preference for LTD4 and is

1	on smooth muscle of the airways and the microvasculature and on hematopoietic cells such as macrophages, eosinophils, and mast cells. Whereas the CysLT1 receptor has a preference for LTD4 and is blocked by the receptor antagonists in clinical use, the CysLT2 receptor is equally responsive to LTD4 and LTC4, is unaffected by these antagonists, and is a negative regulatorofthefunctionoftheCysLT1receptor.LTD4,actingatCysLT1 receptors, is the most potent known bronchoconstrictor, whereas LTE4 induces a vascular leak and mediates the recruitment of eosinophils to the bronchial mucosa. Studies in gene-deleted mice indicate the existence of additional receptors for LTE4. The lysophospholipid formed during the release of arachidonic acid from 1-O-alkyl-2-acyl-snglyceryl-3-phosphorylcholine can be acetylated in the second position to form platelet-activating factor (PAF). Serum levels of PAF correlated positively with the severity of anaphylaxis to peanut in a recent study, whereas the levels

1	acetylated in the second position to form platelet-activating factor (PAF). Serum levels of PAF correlated positively with the severity of anaphylaxis to peanut in a recent study, whereas the levels of PAF acetyl hydrolase (a PAF-degrading enzyme) were inversely related to the same outcome.

1	Unlike most other cells of bone marrow origin, mast cells circulate as committed progenitors lacking their characteristic secretory granules. These committed progenitors express c-kit, the receptor for stem cell factor (SCF). Unlike most other lineages, they retain and increase c-kit expression with maturation. The SCF interaction with c-kit is an absolute requirement for the development of constitutive tissue mast cells residing in skin and connective tissue sites and for the accumulation of mast cells at mucosal surfaces during TH2-type immune responses. Several T cell-derived cytokines (IL-3, IL-4, IL-5, and IL-9) can potentiate SCF-dependent mast cell proliferation and/ or survival in vitro in mice and humans. Indeed, mast cells are absent from the intestinal mucosa in clinical T cell deficiencies, but are present in the submucosa. Based on the immunodetection of secretory granuleneutralproteases, mastcells inthelungparenchymaandintestinal mucosa selectively express tryptase, and

1	deficiencies, but are present in the submucosa. Based on the immunodetection of secretory granuleneutralproteases, mastcells inthelungparenchymaandintestinal mucosa selectively express tryptase, and those in the intestinal and airway submucosa, perivascular spaces, skin, lymph nodes, and breast parenchyma express tryptase, chymase, and carboxypeptidase A (CPA). In the mucosal epithelium of severe asthmatics, mast cells can express tryptase and CPA without chymase. The secretory granules of mast cells selectively positive for tryptase exhibit closed scrolls with a periodicitysuggestiveofacrystalline structureby electronmicroscopy, whereasthesecretorygranulesofmastcellswithmultipleproteasesare scroll-poor, with an amorphous or lattice-like appearance.

1	Mast cells are distributed at cutaneous and mucosal surfaces and in submucosal tissues about venules and could influence the entry of foreign substances by their rapid response capability (Fig. 376-2). Upon stimulus-specific activation and secretory granule exocytosis, histamineandacid hydrolasesaresolubilized,whereastheneutral proteases, which are cationic, remain largely bound to the anionic proteoglycans, heparin and chondroitin sulfate E, with which they function as a complex. Histamine and the various lipid mediators (PGD2, LTC4/ D4/E4, PAF) alter venular permeability, thereby allowing influx of plasma proteins such as complement and immunoglobulins, whereas LTB4 mediates leukocyte–endothelial cell adhesion and subsequent directed migration (chemotaxis). The accumulation of leukocytes and plasma opsonins facilitates defense of the microenvironment. The inflammatory response can also be detrimental, as in asthma, where the smooth-muscle constrictor activity of the cysteinyl

1	and plasma opsonins facilitates defense of the microenvironment. The inflammatory response can also be detrimental, as in asthma, where the smooth-muscle constrictor activity of the cysteinyl leukotrienes is evident and much more potent than that of histamine.

1	The cellular component of the mast cell–mediated inflammatory response is augmented and sustained by cytokines and chemokines. IgE-dependent activation of human skin mast cells in situ elicits TNF-α production and release, which in turn induces endothelial cell responses favoring leukocyte adhesion. Similarly, activation of purified human lung mast cells or cord blood–derived cultured mast cells in vitro results in substantial production of proinflammatory (TNF-α) and immunomodulatory cytokines (IL-4, IL-5, IL-13) and Allergies, Anaphylaxis, and Systemic Mastocytosis Leukocyte responses tion and on the cytokine microenvironment pro- • Adherence vided by the antigen-presenting dendritic cells, LTC4 with IL-4 directing a TH2 subset, interferon (IFN) • Mast cell proliferation γ a TH1 profile, and IL-6 with transforming • Eosinophil activation growth factor β (TGF-β) a TH17 subset. Allergens

1	Proteoglycans • Globopentaosylceramide production that facilitate the immune response by direct initiation of cytokine generation from innate cell types such as basophils, mast cells, eosinophils, • Activation of matrix and others. The TH2 response is associated with activation of specific B cells that can also pres • Activation of coagulation cascade • GM-CSF • Augmented venular permeability for antibody production. Synthesis and release Activated mast cell • IL-13 • Leukocyte adherence into the plasma of allergen-specific IgE results in sensitization of FcεR1-bearing cells such as mast • TNF–° cells and basophils, which become activated on exposure to the specific allergen. In certain dis-FIGUrE 376-2 Bioactive mediators of three categories generated by IgE-dependent eases, including those associated with atopy, the activation of murine mast cells can elicit common but sequential target cell effects lead-monocyte and eosinophil populations can express ing to acute and sustained

1	those associated with atopy, the activation of murine mast cells can elicit common but sequential target cell effects lead-monocyte and eosinophil populations can express ing to acute and sustained inflammatory responses. GM-CSF, granulocyte-macrophage a trimeric FcεR1, which lacks the β chain, and yet colony-stimulating factor; IL, interleukin; IFN, interferon; LT, leukotriene; PAF, platelet-respond to its aggregation. An additional recently activating factor; PGD2, prostaglandin D2; TNF, tumor necrosis factor. recognized class of c-kit-expressing innate cells chemokines. Bronchial biopsy specimens from patients with asthma 2115 reveal that mast cells are immunohistochemically positive for IL-4 and IL-5, but that the predominant localization of IL-4, IL-5, and GM-CSF is to T cells, defined as TH2 by this profile. IL-4 modulates the T cell phenotype to the TH2 subtype, determines the isotype switch to IgE (as does IL-13), and upregulates FcεRI-mediated expression of cytokines by mast

1	as TH2 by this profile. IL-4 modulates the T cell phenotype to the TH2 subtype, determines the isotype switch to IgE (as does IL-13), and upregulates FcεRI-mediated expression of cytokines by mast cells based on in vitro studies.

1	An immediateandlate cellular phase ofallergicinflammation can be induced in the skin, nose, or lung of some allergic humans with local allergen challenge. The immediate phase in the nose involves pruritus and waterydischarge; in the lung, it involves bronchospasm and mucus secretion; and in the skin, it involves a wheal-and-flare response with pruritus. The reduced nasal patency, reduced pulmonary function, or erythema with swelling at the skin site in a late-phase response at 6–8 h is associated with biopsy findings of infiltrating and activated TH2 cells, eosinophils, basophils, and some neutrophils. The progression from early mast cell activation to late cellular infiltration has been used as an experimentalsurrogateofrhinitisorasthma.However,inasthma,there is an intrinsic hyperreactivity of the airways independent of the associated inflammation. Moreover, early-and late-phase responses (at least in the lung) are far more sensitive to blockade of IgE-dependent mast cell activation

1	of the airways independent of the associated inflammation. Moreover, early-and late-phase responses (at least in the lung) are far more sensitive to blockade of IgE-dependent mast cell activation (or actions of histamine and cysteinyl leukotrienes) than are spontaneous or virally induced asthma exacerbations.

1	Consideration of the mechanism of immediate-type hypersensitivity diseases in the human has focused largely on the IgE-dependent recognition of otherwise innocuous substances. A region of chromosome 5 (5q23-31) contains genes implicated in the control of IgE levels including IL-4 and IL-13, as well as IL-3 and IL-9, which are involved in mucosal mast cell hyperplasia, and IL-5 and GM-CSF, which are central to eosinophil development and their enhanced tissue viability. Genes with linkage to the specific IgE response to particular allergens include those encoding the major histocompatibility complex (MHC) and certain chains of the T cell receptor (TCR-αδ). The complexity of atopy and the associated diseases includes susceptibility, severity, and therapeutic responses, each of which is among the separate variables modulated by both innate and adaptive immune stimuli.

1	The induction of allergic disease requires sensitization of a predisposed individual to a specific allergen. The greatest propensity for the development of atopic allergy occurs in childhood and early adolescence. The allergen is processed by antigen-presenting cells of the monocytic lineage (particularly dendritic cells) located throughout the body at surfaces that contact the outside environment, such as the nose, lungs, eyes, skin, and intestine. These antigen-presenting cells presentthe epitope-bearing peptidesvia their MHC to T helper cells and their subsets. The T cell response depends both on cognate recogni- 2116 (termed nuocytes, natural helper cells, or group 2 innate lymphoid cells) can generate large quantities of IL-5 and IL-13 during antihelminth responses, are prominent in nasal polyps from humans, and could well contribute to inflammation in allergic diseases.

1	Life-threatening anaphylactic responses of sensitized humans occur within minutes after systemic exposure to specific antigen. They are manifested by respiratory distress due to laryngeal edema and/or intensebronchospasm,oftenfollowedbyvascularcollapse,orbyshock without antecedent respiratory difficulty. Cutaneous manifestations exemplified by pruritus and urticaria with or without angioedema are characteristic of such systemic anaphylactic reactions. Gastrointestinal manifestations include nausea, vomiting, crampy abdominal pain, and diarrhea.

1	There is no convincingevidence that age,sex, race,orgeographiclocation predisposes a human to anaphylaxis except through exposure to specific immunogens. According to most studies, atopy does not predispose individualsto anaphylaxisfrom penicillin therapy orvenomof a stinging insect but is a risk factor for allergens in food or latex. Risk factors for a poor outcome, however, include older age, use of beta blockers, and the presence of preexisting asthma. Severe hymenoptera anaphylaxis (generally with prominent hypotension) can be a presenting feature of underlying systemic mastocytosis. Additionally, some individuals suffering from recurrent episodes of idiopathic anaphylaxispossess morphologically aberrant mast cellsintheir bone marrow that express a mutant, constitutively active form of c-kit, even without evidence of frank mastocytosis.

1	The materials capable of eliciting the systemic anaphylactic reaction in humans include the following: heterologous proteins in the form of hormones (insulin, vasopressin, parathormone); enzymes (trypsin, chymotrypsin, penicillinase, streptokinase); pollen extracts (ragweed, grass, trees); nonpollen allergen extracts (dust mites, dander of cats, dogs, horses, and laboratory animals); food (peanuts, milk, eggs, seafood, nuts, grains, beans, gelatin in capsules); monoclonal antibodies; occupation-related products (latex rubber products); Hymenoptera venom (yellow jacket, yellow and white-faced hornets, paper wasp, honey bee, imported fire ants); polysaccharides such as dextran and thiomersal as a vaccine preservative; drugs such as protamine; antibiotics (penicillins, cephalosporins, amphotericin B, nitrofurantoin, quinolones); chemotherapy agents (carboplatin, paclitaxel, doxorubicin); local anesthetics (procaine, lidocaine); muscle relaxants (suxamethonium, gallamine, pancuronium);

1	B, nitrofurantoin, quinolones); chemotherapy agents (carboplatin, paclitaxel, doxorubicin); local anesthetics (procaine, lidocaine); muscle relaxants (suxamethonium, gallamine, pancuronium); vitamins (thiamine, folic acid); diagnostic agents (sodium dehydrocholate, sulfobromophthalein); biologics (omalizumab, rituximab, etanercept); and occupation-related chemicals (ethylene oxide). Drugs function as haptens that form immunogenic conjugates with host proteins. The conjugating hapten may be the parent compound, a nonenzymatically derived storage product, or a metabolite formed in the host. Recombinant biologics can also induce the formation of IgE against the proteins or against glycosylated structures that serve as immunogens. Most recently, outbreaks of anaphylaxis to the anti-epidermal growth factor antibody cetuximab were reported in association with elevated titers of serum IgE to alpha-1,3-galactose, an oligosaccharide found on certain nonprimate proteins. Alpha-galactose

1	growth factor antibody cetuximab were reported in association with elevated titers of serum IgE to alpha-1,3-galactose, an oligosaccharide found on certain nonprimate proteins. Alpha-galactose antibodies also account for some episodes of delayed anaphylaxis to beef, lamb, and pork.

1	Individuals differ in the time of appearance of symptoms and signs, butthehallmarkoftheanaphylacticreactionistheonsetofsomemanifestation within seconds to minutes after introduction of the antigen (with the exception of alpha-galactose allergy), generally by injection or less commonly by ingestion. There may be upper or lower airway obstruction or both. Laryngeal edema may be experienced as a “lump” in the throat, hoarseness, or stridor, whereas bronchial obstruction is associated with a feeling of tightness in the chest and/or audible wheezing. Patients with asthma are predisposed to severe involvement of the lower airways and increased mortality. Flushing with diffuse erythema and a feeling of warmth may occur. A characteristic feature is the eruption of well-circumscribed, discrete cutaneous wheals with erythematous,raised, serpiginous borders and blanched centers. These urticarial eruptions are intensely pruritic and may be localized or disseminated. They may coalesce to form

1	cutaneous wheals with erythematous,raised, serpiginous borders and blanched centers. These urticarial eruptions are intensely pruritic and may be localized or disseminated. They may coalesce to form giant hives, and they seldom persistbeyond48h.Alocalized,nonpitting,deeperedematouscutaneousprocess, angioedema,may also bepresent. It maybeasymptomatic or cause a burning or stinging sensation. Angioedema of the bowel wall may cause sufficient intravascular volume depletion to precipitate cardiovascular collapse.

1	In fatal cases with clinical bronchial obstruction, the lungs show marked hyperinflation on gross and microscopic examination. The microscopic findings in the bronchi, however, are limited to luminal secretions, peribronchial congestion, submucosal edema, and eosinophilic infiltration,andthe acute emphysema isattributedtointractable bronchospasm that subsides with death. The angioedema resulting in death by mechanical obstruction occurs in the epiglottis and larynx, but the process also is evident in the hypopharynx and to some extent in the trachea. On microscopic examination, there is wide separation of the collagen fibers and the glandular elements; vascular congestion andeosinophilicinfiltrationalsoarepresent.Patientsdyingofvascular collapse without antecedent hypoxia from respiratory insufficiency have visceral congestion with a presumptive loss of intravascular fluid volume. The associated electrocardiographic abnormalities, with or without infarction, in some patients may

1	insufficiency have visceral congestion with a presumptive loss of intravascular fluid volume. The associated electrocardiographic abnormalities, with or without infarction, in some patients may reflect a primary cardiac event mediated by mast cells (which are prominent near the coronary vessels) or may be secondary to a critical reduction in blood volume.

1	The angioedematous and urticarial manifestations of anaphylaxis have been attributed to the release of endogenous histamine. A role for the cysteinyl leukotrienes in causing marked bronchiolar constriction seems likely. Vascular collapse without respiratory distress in response to experimental challenge with the sting of a hymenopteran was associated with marked and prolonged elevations in blood histamine and intravascular coagulation and kinin generation. The finding that patients with systemic mastocytosis and episodic vascular collapse excrete large amounts of PGD2 metabolites in addition to histamine suggests that PGD2 is also of importance in the hypotensive anaphylactic reactions. As noted, serum PAF levels correlate with severity of anaphylaxis and are inversely proportional to the constitutive level of the acetylhydrolase involved in PAF inactivation. The actions of the array of mast cell–derived mediators are likely additive or synergistic at the target tissues.

1	Thediagnosisofananaphylacticreactiondependsonahistoryrevealing the onset of symptoms and signs within minutes after the responsible material is encountered. It is appropriate to rule out a complement-mediated immune complex reaction, an idiosyncratic response to a nonsteroidal anti-inflammatory drug (NSAID), or the direct effect of certain drugs or diagnostic agents on mast cells. Intravenous administration of a chemical mast cell–degranulating agent, including opiate derivatives and radiographic contrast media, may elicit generalized urticaria, angioedema, and a sensation of retrosternal oppression with or without clinically detectable bronchoconstriction or hypotension. In the transfusion anaphylactic reaction that occurs in patients with IgA deficiency, the responsible specificity resides in IgG or IgE anti-IgA; the mechanism of the reaction mediated by IgG anti-IgA is presumed to be complement activation with secondary mast cell participation.

1	The presence of specific IgE in the blood of patients with systemic anaphylaxis was demonstrated historically by passive transfer of the serum intradermally into a normal recipient, followed 24 h later by antigen challenge into the same site, with subsequent development of a wheal and flare (the Prausnitz-Küstner reaction). In current clinical practice, immunoassays using purified or recombinant antigens can demonstrate the presence of specific IgE in the serum of patients with anaphylactic reactions, and skin testing may be performed after the patient has recovered to elicit a local wheal and flare in response to the putative antigen. Elevations of tryptase levels in serum implicate mast cell activation in a systemic reaction and are particularly informative for anaphylaxis with episodes of hypotension during general anesthesia or when there has been a fatal outcome. However, because of the short half-life of tryptase, elevated levels are best detected within 4 h of a systemic

1	of hypotension during general anesthesia or when there has been a fatal outcome. However, because of the short half-life of tryptase, elevated levels are best detected within 4 h of a systemic reaction. Moreover, anaphylactic reactions to foods characteristically are not associated with elevations in serum tryptase.

1	Early recognition of an anaphylactic reaction is mandatory, since death can occur within minutes to hours after the first symptoms. Mild symptoms such as pruritus and urticaria can be controlled by administration of 0.3–0.5 mL of 1:1000 (1 mg/mL) epinephrine SC or IM, with repeated doses as required at 5to 20-min intervals for a severe reaction. The failure to use epinephrine within the first 20 min of symptoms is a risk factor for poor outcome in studies of anaphylaxis to food. If the antigenic material was injected into an extremity, the rate of absorption may be reduced by prompt application of a tourniquet proximal to the reaction site, administration of 0.2 mL of 1:1000 epinephrine into the site, and removal without compression of an insect stinger, if present. An IV infusion should be initiated to provide a route for administration of 2.5 mL epinephrine, diluted 1:10,000, at 5to 10-min intervals, volume expanders such as normal saline, and vasopressor agents such as dopamine if

1	be initiated to provide a route for administration of 2.5 mL epinephrine, diluted 1:10,000, at 5to 10-min intervals, volume expanders such as normal saline, and vasopressor agents such as dopamine if intractable hypotension occurs. Replacement of intravascular volume due to postcapillary venular leakage may require several liters of saline. Epinephrine provides both αand β-adrenergic effects, resulting in vasoconstriction, bronchial smooth-muscle relaxation, and attenuation of enhanced venular permeability. Oxygen alone via a nasal catheter or with nebulized albuterol may be helpful, but either endotracheal intubation or a tracheostomy is mandatory for oxygen delivery if progressive hypoxia develops. Ancillary agents such as the antihistamine diphenhydramine, 50–100 mg IM or IV, and aminophylline, 0.25–0.5 g IV, are appropriate for urticaria-angioedema and bronchospasm, respectively. Intravenous glucocorticoids, 0.5–1 mg/kg of methylprednisolone, are not effective for the acute event

1	0.25–0.5 g IV, are appropriate for urticaria-angioedema and bronchospasm, respectively. Intravenous glucocorticoids, 0.5–1 mg/kg of methylprednisolone, are not effective for the acute event but may alleviate later recurrence of bronchospasm, hypotension, or urticaria.

1	Prevention of anaphylaxis must take into account the sensitivity of the individual, the dose and character of the diagnostic or therapeutic agent, and the effect of the route of administration on the rate of absorption. Beta blockers are relatively contraindicated in persons at risk for anaphylactic reactions, especially those sensitive to Hymenoptera venom or those undergoing immunotherapy for respiratory system allergy. Ifthereis a definitehistory of a past anaphylactic reaction to a medication, it is advisable to select a structurally unrelated agent. A knowledge of cross-reactivity among agents is critical since, for example, cephalosporins have a cross-reactive ring structure with the penicillins. When skin testing, a prick or scratch skin test should precede an intradermal test, since the latter has a higher risk of causing anaphylaxis. These tests should be performed before the administration of certain materials that are likely to elicit anaphylactic reactions, such as

1	since the latter has a higher risk of causing anaphylaxis. These tests should be performed before the administration of certain materials that are likely to elicit anaphylactic reactions, such as allergenic extracts. Skin testing for antibiotics or chemotherapeutic agents should be performed only on patients with a positive clinical history consistent with an IgE-mediated reaction and in imminent need of the antibiotic in question; skin testing is of no value for non-IgE-mediated eruptions. With regard to penicillin, two-thirds ofpatientswithapositivereactionhistory and positiveskintests to benzylpenicilloyl-polylysine (BPL) and/or the minor determinant mixture (MDM) of benzylpenicillin products experience allergic reactions with treatment, and these reactions are almost uniformly of the anaphylactic type in those patients with minor determinant reactivity. Even patients without a history of previous clinical reactions have a 2–6% incidence of positive skin tests to the two test

1	the anaphylactic type in those patients with minor determinant reactivity. Even patients without a history of previous clinical reactions have a 2–6% incidence of positive skin tests to the two test materials, and about 3 per 1000 with a negative history experience anaphylaxis with 2117 therapy, with a mortality of about 1 per 100,000.

1	If an agent carrying a risk of eliciting an anaphylactic response is required because a non-cross-reactive alternative is not available, desensitization can be performed with most antibiotics and other classes of therapeutic agents by the IV, SC, or PO route. Typically, graded quantities of the drug are given by the selected route starting below the threshold dose for an adverse reaction and then doubling each dose until a therapeutic dosage is achieved. Due to the risk of systemic anaphylaxis during the course of desensitization, such a procedure should be performed under the supervision of a specialist and in a setting in which resuscitation equipment is at hand and an IV line is in place. Once a desensitized state is achieved, it is critical to continue administration of the therapeutic agent at regular intervals throughout the treatment period to prevent the reestablishment of a significant pool of sensitized cells.

1	A different form of protection involves the development of blocking antibody of the IgG class, which protects against Hymenoptera venom– induced anaphylaxis by interacting with antigen so that less reaches the sensitized tissue mast cells. The maximal risk for systemic anaphylactic reactions in persons with Hymenoptera sensitivity occurs in association withacurrentlypositiveskintest.Althoughthereislittlecross-reactivity between honey bee and yellow jacket venoms, there is a high degree of cross-reactivity between yellow jacket venom and the rest of the vespid venoms (yellow or white-faced hornets and wasps). Prevention involves modification of outdoor activities to exclude bare feet, wearing perfumed toiletries, eating in areas attractive to insects, clipping hedges or grass, and hauling away trash or fallen fruit. As with each anaphylactic sensitivity, the individual should wear an informational bracelet and have immediate access to an unexpired autoinjectable epinephrine kit. Venom

1	away trash or fallen fruit. As with each anaphylactic sensitivity, the individual should wear an informational bracelet and have immediate access to an unexpired autoinjectable epinephrine kit. Venom immunotherapy for 5 years can induce a state of resistance to sting reactions that is independent of serum levels of specific IgG or IgE. Forchildren under the ageof 10 with a systemic reaction limitedto skin, the likelihood of progression to more serious respiratory or vascular manifestations is low, and thus immunotherapy is not recommended.

1	Urticaria and angioedema may appear separately or together as cutaneous manifestations of localized nonpitting edema; a similar process may occur at mucosal surfaces of the upper respiratory or gastrointestinal tract. Urticaria involves only the superficial portion of the dermis, presenting as well-circumscribed wheals with erythematous raised serpiginous borders and blanched centers that may coalesce to become giant wheals. Angioedema is a well-demarcated localized edema involving the deeper layers of the skin, including the subcutaneous tissue, and can also involve the bowel wall. Recurrent episodes of urticaria and/or angioedema of less than 6 weeks’ duration are considered acute, whereas attacks persisting beyond this period are designated chronic.

1	Urticaria and angioedema probably occur more frequently than reported because of the evanescent, self-limited nature of such eruptions,whichseldomrequiremedicalattentionwhenlimitedtotheskin. Although persons in any age group may experience acute or chronic urticaria and/or angioedema, these lesions increase in frequency after adolescence, with the highest incidence occurring in persons in the third decade of life; indeed, one survey of college students indicated that 15–20% had experienced a pruritic wheal reaction.

1	Theclassification ofurticaria-angioedema presentedin Table 376-1 focuses on the different mechanisms for eliciting clinical disease and can be useful for differential diagnosis; nonetheless, most cases of chronic urticaria are idiopathic. Urticaria and/or angioedema occurring during the appropriate season in patients with seasonal respiratory allergy or as a result of exposure to animals or molds is attributed to inhalation or physical contact with pollens, animal dander, and mold spores, respectively. However, urticaria and angioedema secondary to inhalation are relatively uncommon compared to urticaria and

1	Allergies, Anaphylaxis, and Systemic Mastocytosis 1. IgE-dependent a. Specific antigen sensitivity (pollens, foods, drugs, fungi, molds, Hymenoptera venom, helminths) b. Physical: dermographism, cold, solar, pressure, cholinergic c. Autoimmune 2. Bradykinin-mediated a. Hereditary angioedema: C1 inhibitor deficiency: null (type 1) and dysfunctional (type 2); mutated factor XII (type 3) b. Acquired angioedema: C1 inhibitor deficiency: anti-idiotype and anti-C1 inhibitor c. Angiotensin-converting enzyme inhibitors 3. Complement-mediated a. Necrotizing vasculitis b. Serum sickness c. Reactions to blood products 4. Nonimmunologic a. Direct mast cell–releasing agents (opiates, antibiotics, curare, D-tubocurarine, radiocontrast media) b. Agents that alter arachidonic acid metabolism (aspirin and nonsteroidal anti-inflammatory agents, azo dyes, and benzoates) 5. Idiopathic angioedema elicited by ingestion of fresh fruits, shellfish, fish, milk products, chocolate, legumes including peanuts,

1	anti-inflammatory agents, azo dyes, and benzoates) 5. Idiopathic angioedema elicited by ingestion of fresh fruits, shellfish, fish, milk products, chocolate, legumes including peanuts, and various drugs that may elicit not only the anaphylactic syndrome with prominent gastrointestinal complaints but also urticaria alone.

1	Additional etiologies include physical stimuli such as cold, heat, solar rays, exercise, and mechanical irritation. The physical urticarias can be distinguished by the precipitating event and other aspects of the clinical presentation. Dermographism, which occurs in 1–4% of the population, is defined by the appearance of a linear wheal at the site of a brisk stroke with a firm object or by any configuration appropriate to the eliciting event (Fig. 376-3). Dermographism has a prevalence that peaks in the second to third decades. It is not influenced by atopy and has a duration generally of <5 years. Pressure urticaria, which often accompanies chronic idiopathic urticaria, presents in response to a sustained stimulus such as a shoulder strap or belt, running (feet), or manual labor (hands). Cholinergic urticaria is distinctive in that the pruritic wheals are of small size (1–2 mm) and are surrounded by a large area of erythema; attacks are precipitated by fever, a hot bath or shower, or

1	Cholinergic urticaria is distinctive in that the pruritic wheals are of small size (1–2 mm) and are surrounded by a large area of erythema; attacks are precipitated by fever, a hot bath or shower, or exercise and are presumptively attributed to a rise in core

1	FIGUrE 376-3 Dermographic urticarial lesion inducedbystrokingtheforearmlightlywiththeedgeofatongueblade.Thephotograph,takenafter2minutes,demonstratesaprominentwheal-and-flarereactionintheshapeofanX.(From LA Goldsmith et al [eds]: Fitzpatrick’s Dermatology in General Medicine, 8th ed. New York, McGraw-Hill, 2012. Photograph pro-vided by Allen P. Kaplan, MD, Medical University of South Carolina.) body temperature. Exercise-induced anaphylaxis can be precipitated by exertion alone or can be dependent on prior food ingestion. There is an association with the presence of IgE specific for α-5 gliadin, a component of wheat. The clinical presentation can be limited to flushing, erythema, and pruritic urticaria but may progress to angioedema of the face, oropharynx, larynx, or intestine or to vascular collapse; it is distinguished from cholinergic urticaria by presenting with wheals of conventional size and by not occurring with fever or a hot bath. Cold urticaria is local at body areas

1	vascular collapse; it is distinguished from cholinergic urticaria by presenting with wheals of conventional size and by not occurring with fever or a hot bath. Cold urticaria is local at body areas exposed to low ambient temperature or cold objects but can progress to vascular collapse with immersion in cold water (swimming). Solar urticaria is subdivided into six groups by the response to specific portions of the light spectrum. Vibratory angioedema may occur after years of occupational exposure or can be idiopathic; it may be accompanied by cholinergic urticaria. Other rare formsofphysicalallergy,alwaysdefinedbystimulus-specificelicitation, include local heat urticaria, aquagenic urticaria fromcontactwithwater of anytemperature (sometimesassociatedwithpolycythemiavera),and contact urticaria from direct interaction with some chemical substance.

1	Angioedema without urticaria due to the generation of bradykinin occurs with C1 inhibitor (C1INH) deficiency that may be inborn as an autosomal dominant characteristic or may be acquired through the appearance of an autoantibody. The angiotensin-converting enzyme (ACE) inhibitors can provoke a similar clinical presentation in 0.1– 0.5% of hypertensive patients due to attenuated degradation of bradykinin. The urticaria and angioedema associated with classic serum sickness or with hypocomplementemic cutaneous necrotizing angiitis are believed to be immune-complex diseases. The drug reactions to mast cell granule–releasing agents and to NSAIDs may be systemic, resembling anaphylaxis, or limited to cutaneous sites.

1	Urticarial eruptions are distinctly pruritic, may involve any area of the body from the scalp to the soles of the feet, and appear in crops of 12-to 36-h duration, with old lesions fading as new ones appear. Most of the physical urticarias (cold, cholinergic, dermatographism) are an exception, with individual lesions lasting less than 2 h. The most common sites for urticaria are the extremities and face, with angioedema often being periorbital and in the lips. Although self-limited in duration, angioedemaoftheupper respiratorytractmaybe life-threateningdueto laryngealobstruction,whereasgastrointestinalinvolvementmaypresent with abdominal colic, with or without nausea and vomiting, and may result in unnecessary surgical intervention. No residual discoloration occurs with either urticaria or angioedema unless there is an underlying vasculiticprocessleadingtosuperimposedextravasationoferythrocytes.

1	The pathology is characterized by edema of the superficial dermis in urticaria and of the subcutaneous tissue and deep dermis in angioedema. Collagen bundles in affected areas are widely separated, and the venules are sometimes dilated. Any perivenular infiltrate consists of lymphocytes,monocytes,eosinophils, and neutrophils that are present in varying combination and numbers.

1	Perhaps the best-studied example of IgE-and mast cell–mediated urticaria and angioedema is cold urticaria. Cryoglobulins or cold agglutinins are present in up to 5% of these patients. Immersion of an extremity in an ice bath precipitates angioedema of the distal portion with urticaria at the air interface within minutes of the challenge. Histologic studies reveal marked mast cell degranulation with associatededema of the dermis and subcutaneous tissues. Thehistamine level in the plasma of venous effluent of the cold-challenged and angioedematous extremity is markedly increased, but no such increase appears in the plasma of effluent of the contralateral normal extremity. Elevated levels of histamine have been found in the plasma of venous effluent and in the fluid of suction blisters at experimentally induced lesional sites in patients with dermographism, pressure urticaria, vibratory angioedema, light urticaria, and heat urticaria. By ultrastructural analysis, the pattern of mast cell

1	induced lesional sites in patients with dermographism, pressure urticaria, vibratory angioedema, light urticaria, and heat urticaria. By ultrastructural analysis, the pattern of mast cell degranulation in cold urticaria resembles an IgE-mediated response with solubilization of granule contents, fusion of the perigranular and cell membranes, and discharge of granule contents, whereas in a dermographic lesion, there is additional superimposed zonal (piecemeal) degranulation. There are several reports of resolution of cold urticaria by treatment with monoclonal anti-human IgE (omalizumab). Elevations of plasma histamine levels with biopsy-proven mast cell degranulation have also been demonstrated with generalized attacks of cholinergic urticaria and exercise-related anaphylaxis precipitated experimentally in subjects exercising on a treadmill while wearing a wet suit; however, only subjects with cholinergic urticaria have a concomitant decrease in pulmonary function.

1	Up to 40% of patients with chronic urticaria have an autoimmune cause for their disease including autoantibodies to IgE (5–10%) or, more commonly, to the α chain of FcεRI (35–45%). In these patients, autologous serum injected into their own skin can induce a wheal-and-flare reaction involving mast cell activation. The presence of these antibodies can alsoberecognized by their capacityto releasehistamine or induce activation markers such as CD63 or CD203 on basophils. An association with antibodies to microsomal peroxidase and/or thyroglobulin has been observed often with clinically significant Hashimoto’s thyroiditis. In vitro studies reveal that these autoantibodies can mediate basophil degranulation with enhancement by serum as a source of the anaphylatoxic fragment, C5a.

1	Hereditary angioedema is an autosomal dominant disease due to a deficiency of C1INH (type 1) in about 85% of patients and to a dysfunctional protein (type 2) in the remainder. A third type of hereditary angioedema has been described in which C1INH function is normal, and the causal lesion is a mutant form of factor XII, which leads to generation of excessive bradykinin. In the acquired form of C1INH deficiency,thereisexcessiveconsumptiondueeithertoimmunecomplexes formedbetweenanti-idiotypicantibodyandmonoclonalIgGpresented by B cell lymphomas or to an autoantibody directed to C1INH. C1INH blocks the catalytic function of activated factor XII (Hageman factor) and of kallikrein, as well as the C1r/C1s components of C1. During clinical attacks of angioedema, C1INH-deficient patients have elevated plasma levels of bradykinin, particularly in the venous effluent of an involvedextremity,andreducedlevelsofprekallikreinandhigh-molecular-weight kininogen, from which bradykinin is cleaved. The

1	plasma levels of bradykinin, particularly in the venous effluent of an involvedextremity,andreducedlevelsofprekallikreinandhigh-molecular-weight kininogen, from which bradykinin is cleaved. The parallel decline in the complement substrates C4 and C2 reflects the action of activated C1 during such attacks. Mice with targeted disruption of the geneforC1INHexhibitachronicincreaseinvascularpermeability.The pathobiology isaggravated byadministrationof anACE inhibitor (captopril) and is attenuated by breeding the C1INH null strain to a bradykinin2receptor(Bk2R)nullstrain.AsACEisalsodescribedaskininase II, the use of blockers results in impaired bradykinin degradation and explains the angioedema that occurs idiosyncratically in hypertensive patients with a normal C1INH. Bradykinin-mediated angioedema, whether caused by ACE inhibitors or by C1INH deficiency, is noteworthy for the conspicuous absence of concomitant urticaria.

1	The rapid onset and self-limited nature of urticarial and angioedematous eruptions are distinguishing features. Additional characteristics are the occurrence of the urticarial crops in various stages of evolution and the asymmetric distribution of the angioedema. Urticaria and/or angioedema involving IgE-dependent mechanisms are often appreciatedby historicconsiderationsimplicating specific allergens orphysical stimuli, by seasonal incidence, and by exposure to certain environments. Direct reproduction of the lesion with physical stimuli is particularly valuable because it so often establishes the cause of the lesion. Thediagnosisofanenvironmentalallergenbasedontheclinicalhistory can be confirmed by skin testing or assay for allergen-specific IgE in serum. IgE-mediated urticaria and/or angioedema may or may not be associated with anelevationoftotal IgEorwithperipheral eosinophilia. Fever, leukocytosis, and an elevated sedimentation rate are absent.

1	The classification of urticarial and angioedematous states presented in Table 376-1 in terms of possible mechanisms necessarily includes some differential diagnostic points. Hypocomplementemia is not observed in IgE-mediated mast cell disease and may reflect either an acquired abnormality generally attributed to the formation of immune complexes or a genetic or acquired deficiency of C1INH. Chronic recurrent urticaria, generally in females, associated with arthralgias, an elevated sedimentation rate, and normo-or hypocomplementemia suggests an underlying cutaneous necrotizing angiitis. Vasculitic urti-2119 cariatypicallypersistslongerthan 72h,whereasconventionalurticaria often has a duration of 12–36 h. Confirmation depends on a biopsy that reveals cellular infiltration, nuclear debris, and fibrinoid necrosis of the venules. The same pathobiologic process accounts for the urticariainassociationwith suchdiseasesassystemiclupus erythematosus or viral hepatitis with or without associated

1	fibrinoid necrosis of the venules. The same pathobiologic process accounts for the urticariainassociationwith suchdiseasesassystemiclupus erythematosus or viral hepatitis with or without associated arteritis. Serum sickness per se or a similar clinical entity due to drugs includes not only urticaria but also pyrexia, lymphadenopathy, myalgia, and arthralgia or arthritis. Urticarial reactions to blood products or intravenous administration of immunoglobulin are defined by the event andgenerally are not progressive unless the recipient is IgA-deficient in the former case or the reagent is aggregated in the latter.

1	The diagnosis of hereditary angioedema is suggested not only by family history but also by the lack of pruritus and of urticarial lesions, the prominence of recurrent gastrointestinal attacks of colic, and episodes of laryngeal edema. Laboratory diagnosis depends on demonstrating a deficiency of C1INH antigen (type 1) or a nonfunctional protein (type 2) by a catalytic inhibition assay. While levels of C1 are normal, its substrates, C4 and C2, are chronically depleted and fall further during attacks due to the activation of additional C1. Patients with the acquired forms of C1INH deficiency have the same clinical manifestations but differ in the lack of a familial element. Furthermore, their sera exhibit a reduction of C1 function and C1q protein as well as C1INH, C4, and C2. Inborn C1INH deficiency and ACE inhibitor–elicited angioedema are associated with elevated levels of bradykinin. Lastly, type 3 hereditary angioedema is associated with normal levels of complement proteins.

1	Urticaria and angioedema are distinct from contact sensitivity, a vesicular eruption that progresses to chronic thickening of the skin with continued allergenic exposure. They also differ from atopic dermatitis, a condition that may present as erythema, edema, papules, vesiculation, and oozing proceeding to a subacute and chronic stage in which vesiculation is less marked or absent and scaling, fissuring, and lichenification predominate in a distribution that characteristically involves the flexor surfaces. In cutaneous mastocytosis, the reddish brown macules and papules, characteristic of urticaria pigmentosa, urticate with pruritus upon trauma; and in systemic mastocytosis, without or with urticaria pigmentosa, there is episodic systemic flushing with or without urtication but no angioedema.

1	Identification and subsequent elimination of the etiologic factor(s) provide the most satisfactory therapeutic program; this approach is feasible to varying degrees with IgE-mediated reactions to allergens or physical stimuli. For most forms of urticaria, H1 antihistamines such as chlorpheniramine or diphenhydramine effectively attenuate both urtication and pruritus, but because of their side effects, nonsedating agents such as loratadine, desloratadine, and fexofenadine, or low-sedating agents such as cetirizine or levocetirizine generally are used first. Cyproheptadine in dosages beginning at 8 mg and ranging up to 32 mg daily and especially hydroxyzine in dosages beginning at 40 mg and ranging up to 200 mg daily have proven effective when H1 antihistamines fail. The addition of an H2 antagonist such as cimetidine, ranitidine, or famotidine in conventional dosages may add benefit when H1 antihistamines are inadequate. Doxepin, a dibenzoxepin tricyclic compound with both H1 and H2

1	antagonist such as cimetidine, ranitidine, or famotidine in conventional dosages may add benefit when H1 antihistamines are inadequate. Doxepin, a dibenzoxepin tricyclic compound with both H1 and H2 receptor antagonist activity, is yet another alternative. A CysLT1 receptor antagonist such as montelukast, 10 mg/d, or zafirlukast, 20 mg twice a day, can be important add-on therapy.

1	Topical glucocorticoids are of no value, and systemic glucocorticoids are generally avoided in idiopathic, allergen-induced, or physical urticarias due to their long-term toxicity. Systemic glucocorticoids are useful in the management of patients with pressure urticaria, vasculitic urticaria (especially with eosinophil prominence), idiopathic angioedema with or without urticaria, or chronic urticaria that responds poorly to conventional treatment. With persistent vasculitic urticaria, hydroxychloroquine, dapsone, or colchicine may be added to the regimen after hydroxyzine and before or

1	Allergies, Anaphylaxis, and Systemic Mastocytosis 2120 along with systemic glucocorticoids. Cyclosporine can be efficacious for patients with chronic idiopathic or chronic autoimmune urticaria that is severe and poorly responsive to other modalities and/or where a glucocorticoid requirement is excessive. For chronic urticaria induced by autoantibody activation of mast cells and basophils or cold urticaria, monoclonal anti-IgE antibodies such as omalizumab may be considered. The therapy of inborn C1INH deficiency has been simplified by the finding that attenuated androgens correct the biochemical defect and afford prophylactic protection; their efficacy is attributed to production by the normal gene of an amount of functional C1INH sufficient to control the spontaneous activation of C1. The antifibrinolytic agent ε-aminocaproic acid may be used for preoperative prophylaxis but is contraindicated in patients with thrombotic tendencies or ischemia due to arterial atherosclerosis.

1	C1. The antifibrinolytic agent ε-aminocaproic acid may be used for preoperative prophylaxis but is contraindicated in patients with thrombotic tendencies or ischemia due to arterial atherosclerosis. Infusion of isolated C1INH protein may be used for prophylaxis or treatment of an acute attack; a bradykinin 2 receptor antagonist and ecallantide, a kallikrein inhibitor, which are administered SC, are each being assessed for amelioration of attacks. Treatment of the underlying hematologic malignancy is indicated for acquired C1INH deficiency.

1	Systemic mastocytosis is defined by a clonal expansion of mast cells that in most instances is indolent and nonmalignant. The mast cell expansion is generally recognized only in bone marrow and in the normal peripheral distribution sites of the cells, such as skin, gastrointestinal mucosa, liver, and spleen. Mastocytosis occurs at any age and has a slight preponderanceinmales. Theprevalence of systemicmastocytosis is not known, a familial occurrence is rare, and atopy is not increased.

1	A consensus classification formastocytosis recognizescutaneousmastocytosis with variants and four systemic forms (Table 376-2). Cutaneous mastocytosis is the most common diagnosis in children, whereas the form designated as indolent systemic mastocytosis (ISM) accounts for the majority of adult patients; it implies that there is no evidence of an associated hematologic disorder, liver disease, or lymphadenopathy and is not known to alter life expectancy. In systemic mastocytosis associated with clonal hematologic non–mast cell lineage disease (SM-AHNMD), the prognosis is determined by the nature of the associated disorder, which can range from dysmyelopoiesis to leukemia. In aggressive systemic mastocytosis (ASM), mast cell infiltration/proliferation in multiple organs such as liver, spleen, gut, and/or bone results in a poor prognosis; a subset of patients with this form has prominent eosinophilia with hepatosplenomegaly and lymphadenopathy. Mast cell leukemia (MCL) is the rarest

1	gut, and/or bone results in a poor prognosis; a subset of patients with this form has prominent eosinophilia with hepatosplenomegaly and lymphadenopathy. Mast cell leukemia (MCL) is the rarest form of the disease and is invariably fatal at present; the peripheral blood contains circulating, metachromatically staining,

1	Variants: plaque form, nodular form; telangiectasia macularis Solitary mastocytoma of skin Systemic mastocytosis with an associated clonal hematologic non–mast cell lineage disease (SM-AHNMD) Source: Modified from SH Swerdlow et al (eds): World Health Organization Classification of Tumors: Pathology and Genetics in Tumors of Hematopoietic and Lymphoid Tissues. Lyon, IARC Press, 2008. atypical mast cells. An aleukemic form of MCL is recognized without circulating mast cells when the percentage ofhigh-grade immature mast cells in bone marrow smears exceeds 20% in a nonspicular area. Mast cell sarcoma and extracutaneous mastocytomas are rare solid mast cell tumors with malignant and benign features, respectively.

1	A point mutation of A to T at codon 816 of c-kit that causes an aspartic acid to valine substitution is found in multiple cell lineages in patients with mastocytosis, resulting in a somatic gain-in-function mutation. This substitution, as well as other rare mutations of c-kit, is characteristic of patients with all forms of systemic mastocytosis but is also present in some children with cutaneous mastocytosis, as might be anticipated because mast cells are of bone marrow lineage. The prognosis for patients with cutaneous mastocytosis and for almost all with ISM is a normal life expectancy, whereas that for patients with SM-AHNMD is determined by a non–mast cell component. ASM and MCL carry a poorer prognosis. In infants and children with cutaneous manifestations, namely, urticaria pigmentosa or bullous lesions, visceral involvement is usually lacking, and resolution is common.

1	The clinical manifestations of systemic mastocytosis, distinct from a leukemic complication, are due to tissue occupancy by the mast cell mass, the tissue response to that mass, and the release of bioactive substances acting at both local and distal sites. The pharmacologically induced manifestations are pruritus, flushing, palpitations and vascular collapse, gastric distress, lower abdominal crampy pain, and recurrent headache. The increase in local cell burden is evidenced by the lesions of urticaria pigmentosa at skin sites and may be a direct local cause of bone pain and/or malabsorption. Mast cell–mediated fibrotic changes occur in liver, spleen, and bone marrow but not in gastrointestinal tissue or skin. Immunofluorescent analysis of bone marrow and skin lesions in ISM and of spleen, lymph node, and skin inASMhasrevealedonlyonemastcellphenotype,namely,scroll-poor cells expressing tryptase, chymase, and CPA.

1	The cutaneous lesions of urticaria pigmentosa are reddish-brown macules or papules that respond to trauma with urtication and erythema (Darier’s sign). The apparent incidence of these lesions is ≥80% in patients with ISM and <50% in those with SM-AHNMD or ASM. Approximately 1% of patients with ISM have skin lesions that appear as tan-brown macules with striking patchy erythema and associated telangiectasia (telangiectasia macularis eruptiva perstans). In the upper gastrointestinal tract, gastritis and peptic ulcer are significant problems. In the lower intestinal tract, the occurrence of diarrhea and abdominal pain is attributed to increased motility due to mast cell mediators; this problem can be aggravated by malabsorption, which can also cause secondary nutritional insufficiency and osteomalacia. The periportal fibrosis associated with mast cell infiltration and a prominence of eosinophils may lead to portal hypertension and ascites. In some patients, flushing and recurrent

1	and osteomalacia. The periportal fibrosis associated with mast cell infiltration and a prominence of eosinophils may lead to portal hypertension and ascites. In some patients, flushing and recurrent vascular collapse are markedly aggravated by an idiosyncratic response to a minimal dosage of NSAIDs. The neuropsychiatric disturbances are clinically most evident as impaired recent memory, decreased attention span, and “migraine-like” headaches. Patients may experience exacerbation of a specific clinical sign or symptom with alcohol ingestion, temperature changes, stress, use of mast cell–interactive narcotics, or ingestion of NSAIDs.

1	Although the diagnosis of mastocytosis is generally suspected on the basis of the clinical history and physical findings, and can be supported by laboratory procedures, it can be established only by a tissue diagnosis. By convention, the diagnosis of systemic mastocytosis depends heavily on bone marrow biopsy to meet the criteria of one major plus one minor or three minor findings (Table 376-3). The bone marrow provides the major criterion by revealing aggregates of mast cells, often in paratrabecular and perivascular locations with lymphocytes and eosinophils, as well as the minor criteria of an abnormal mast cell morphology, an aberrant mast cell membrane immunophenotype, or a codon 816 mutation in any cell type. A serum total tryptase level Major: Multifocal dense infiltrates of mast cells in bone marrow or other extracutaneous tissues with confirmation by immunodetection of tryptase or metachromasia

1	Major: Multifocal dense infiltrates of mast cells in bone marrow or other extracutaneous tissues with confirmation by immunodetection of tryptase or metachromasia Minor: Abnormal mast cell morphology with a spindle shape and/or multilobed or eccentric nucleus Aberrant mast cell surface phenotype with expression of CD25 (IL-2 receptor) and CD2 in addition to C117 (c-kit) Detection of codon 816 mutation in peripheral blood cells, bone marrow cells, or lesional tissue aDiagnosis requires either the major criterion and one minor criterion or three minor criteria.

1	Detection of codon 816 mutation in peripheral blood cells, bone marrow cells, or lesional tissue aDiagnosis requires either the major criterion and one minor criterion or three minor criteria. and/ora24-hurinecollectionformeasurementofhistamine,histamine metabolites,ormetabolitesofPGD2arenoninvasiveapproachestoconsiderbeforebonemarrowbiopsy.Thepro-β andαformsof tryptaseare elevated in more than one-half of patients with systemic mastocytosis and provide a minor criterion; the fully processed (“mature”) β form is increased in patients undergoing an anaphylactic reaction. Additional studies directed by the presentation include a bone densitometry, bone scan, or skeletal survey; contrast studies of the upper gastrointestinal tract with small-bowel follow-through, computed tomography scan, or endoscopy; and a neuropsychiatric evaluation. Osteoporosis is increased in mastocytosis and may lead to pathologic fractures.

1	The differential diagnosis requires the exclusion of other flushing disorders. The 24-h urine assessment of 5-hydroxy-indoleacetic acid and metanephrines should exclude a carcinoid tumor or a pheochromocytoma. Some patients presenting with recurrent mast cell activation symptoms without an obvious increase in mast cell burden in skin or bone marrow have been shown to carry aberrant mast cells with clonality markers of D816C c-kit mutation or surface CD25 expression. Most patients with recurrent anaphylaxis, including the idiopathic group, present with angioedema and/or wheezing, which are not manifestations of systemic mastocytosis.

1	The management of systemic mastocytosis uses a stepwise and symptom/sign–directed approach that includes an H1 antihistamine for flushing and pruritus, an H2 antihistamine or proton pump inhibitor for gastric acid hypersecretion, oral cromolyn sodium for diarrhea and abdominal pain, and aspirin for severe flushing with or without associated vascular collapse, despite use of H1 and H2 antihistamines, to block biosynthesis of PGD2. Systemic glucocorticoids appear to alleviate the malabsorption. Mast cell cytoreductive therapy consisting of IFN-α or cladribine is generally reserved for advanced, nonindolent variants of systemic mastocytosis. Their efficacy in ASM is variable, perhaps because of dosage limitations due to side effects. Chemotherapy is appropriate for the frank leukemias. A self-injectable epinephrine prescription is recommended for most patients due to increased incidence of anaphylaxis. Although c-kit is a receptor tyrosine kinase, the gain-in-function mutation of codon

1	epinephrine prescription is recommended for most patients due to increased incidence of anaphylaxis. Although c-kit is a receptor tyrosine kinase, the gain-in-function mutation of codon 816 is not susceptible to inhibition by imatinib mesylate.

1	Allergic rhinitis is characterized by sneezing; rhinorrhea; obstruction of the nasal passages; conjunctival, nasal, and pharyngeal itching; and lacrimation, all occurring in a temporal relationship to allergen exposure. Although commonly seasonal due to elicitation by airborne pollens, it can be perennial in an environment of chronic exposure to house dust mites, animal danders, or insect products. In North America, the incidence of allergic rhinitis is about 7%. The overall prevalence in North America is nearly 20%, with the peak prevalence of nearly 40% occurring in childhood and adolescence.

1	Allergic rhinitis generally occurs in atopic individuals, often in association with atopic dermatitis, food allergy, urticaria, and/or asthma (Chap. 309). Up to 40% of patients with rhinitis manifest asthma, whereas ∼70% of individuals with asthma experience rhinitis. Symptoms generally appear before the fourth decade of life and tend to diminish gradually with aging, although complete spontaneous remissions are uncommon. A relatively small number of weeds that depend on wind rather than insects for pollination, as well as grasses and some trees, produce sufficient quantities of pollen suitable for wide distribution by air currents to elicit seasonal allergic rhinitis. The dates of pollination of these species generally vary little from year to yearinaparticularlocalebutmaybequitedifferentinanotherclimate. In the temperate areas of North America, trees typically pollinate from March through May, grasses in June and early July, and ragweed from mid-August to early October. Molds, which

1	In the temperate areas of North America, trees typically pollinate from March through May, grasses in June and early July, and ragweed from mid-August to early October. Molds, which are widespread in nature becausetheyoccurinsoilordecayingorganicmatter,propagatespores in a pattern that depends on climatic conditions. Perennial allergic rhinitis occurs in response to allergens that are present throughout the year, including animal dander, cockroach-derived proteins, mold spores, or dust mites such as Dermatophagoides farinae and Dermatophagoides pteronyssinus. Dust mites are scavengers of human skin and excrete cysteine protease allergens in their feces. In up to one-half of patients with perennial rhinitis, no clear-cut allergen can be demonstrated as causative. The ability of many allergens to cause rhinitisratherthan lower respiratory tract symptoms (particularlypollens) may be attributed to their large size, 10–100 μm, and retention within the nose.

1	Episodic rhinorrhea, sneezing, obstruction of the nasal passages with lacrimation, and pruritus of the conjunctiva, nasal mucosa, and oropharynx are the hallmarks of allergic rhinitis. The nasal mucosa is pale and boggy, the conjunctiva congested and edematous, and the pharynx generally unremarkable. Swelling of the turbinates and mucous membranes with obstruction of the sinus ostia and eustachian tubes precipitates secondary infections of the sinuses and middle ear, respectively. Nasal polyps, representing mucosal protrusions containing edema fluid with variable numbers of eosinophils and degranulated mast cells, can increase obstructive symptoms and can concurrently arise within the nasopharynx or sinuses. However, atopy is not a risk factor for nasal polyps, which instead may occur in the setting of the aspirin-intolerant triad of rhinosinusitis and asthma and in patients with chronic staphylococcal colonization, which produces superantigens leading to an intense TH2 inflammatory

1	the setting of the aspirin-intolerant triad of rhinosinusitis and asthma and in patients with chronic staphylococcal colonization, which produces superantigens leading to an intense TH2 inflammatory response.

1	The nose presents a large mucosal surface area through the folds of the turbinates and serves to adjust the temperature and moisture content of inhaled air and to filter out particulate materials >10 μm in size by impingement in a mucous blanket; ciliary action moves the entrapped particles toward the pharynx. Entrapment of pollen and digestion of the outer coat by mucosal enzymes such as lysozymes release protein allergens generally of 10,000–40,000 molecular weight. The initial interaction occurs between the allergen and intraepithelial mast cells and then proceeds to involve deeper perivenular mast cells, both of which are sensitized with specific IgE. During the symptomatic season when the mucosae are already swollen and hyperemic, there is enhanced adverse reactivity to the seasonal pollen. Biopsy specimens of nasal mucosa during seasonal rhinitis show submucosal edema with infiltration byeosinophils, along with somebasophilsand neutrophils.

1	The mucosal surface fluid contains IgA that is present because of its secretorypieceandalsoIgE,whichapparentlyarrivesbydiffusionfrom plasma cells in proximity to mucosal surfaces. IgE fixes to mucosal and submucosal mast cells, and the intensity of the clinical response to inhaled allergens is quantitatively related to the naturally occurring pollen dose. In sensitive individuals, the introduction of allergen into thenoseisassociatedwithsneezing,“stuffiness,”anddischarge,andthe fluid contains histamine, PGD2, and leukotrienes. Thus the mast cells of the nasal mucosa and submucosa generate and release mediators Allergies, Anaphylaxis, and Systemic Mastocytosis 2122 through IgE-dependent reactions that are capable of producing tissue edema and eosinophilic infiltration.

1	The diagnosis of seasonal allergic rhinitis depends largely on an accurate history of occurrence coincident with the pollination of the offending weeds, grasses, or trees. The continuous character of perennial allergic rhinitis due to contamination of the home or place of work makes historic analysis difficult, but there may be variability in symptoms that can be related to exposure to animal dander, dust mite and/or cockroach allergens, fungal spores, or work-related allergens such as latex. Patients with perennial rhinitis commonly develop the problem in adult life, and manifest nasal congestion and a postnasal discharge, often associated with thickening of the sinus membranes demonstrated by radiography. Perennial nonallergic rhinitis with eosinophilia syndrome (NARES) occurs in the middle decades of life and is characterized by nasal obstruction, anosmia, chronic sinusitis, andfrequentaspirinintolerance.Theterm vasomotor rhinitis or perennial nonallergic rhinitis designates a

1	middle decades of life and is characterized by nasal obstruction, anosmia, chronic sinusitis, andfrequentaspirinintolerance.Theterm vasomotor rhinitis or perennial nonallergic rhinitis designates a condition of enhanced reactivity of the nasopharynx in which a symptom complex resembling perennial allergic rhinitis occurs with nonspecific stimuli, including chemical odors, temperature and humidity variations, and position changes but occurs without tissue eosinophilia or an allergic etiology. Other entitiestobeexcludedarestructuralabnormalitiesofthenasopharynx; exposure to irritants; gustatory rhinitis associated with cholinergic activation that occurs while eating or ingesting alcohol; hypothyroidism; upper respiratorytractinfection;pregnancywith prominent nasal mucosal edema; prolonged topical use of α-adrenergic agents in the form of nose drops (rhinitis medicamentosa); and the use of certain therapeutic agents such as rauwolfia, β-adrenergic antagonists, estrogens, progesterone,

1	topical use of α-adrenergic agents in the form of nose drops (rhinitis medicamentosa); and the use of certain therapeutic agents such as rauwolfia, β-adrenergic antagonists, estrogens, progesterone, ACE inhibitors, aspirin and other NSAIDS, and drugs for erectile dysfunction (phosphodiesterase-5 inhibitors).

1	The nasal secretions of allergic patients are rich in eosinophils, and a modest peripheral eosinophilia is a common feature. Local or systemic neutrophilia implies infection. Total serum IgE is frequently elevated, but the demonstration of immunologic specificity for IgE is critical to an etiologic diagnosis. A skin test by the intracutaneous route (punctureorprick) withthe allergensofinterestprovidesa rapid and reliable approach to identifying allergen-specific IgE that has sensitized cutaneous mast cells. A positive intracutaneous skin test with 1:10–1:20 weight/volume of extract has a high predictive value for the presence of allergy. An intradermal test with a 1:500–1:1000 dilution of 0.05 mL may follow if indicated by history when the intracutaneous test is negative, but while more sensitive, it is less reliable due to the reactivity of some asymptomatic individuals at the test dose. Skin testing by the intracutaneous route for food allergens can be supportive of the clinical

1	sensitive, it is less reliable due to the reactivity of some asymptomatic individuals at the test dose. Skin testing by the intracutaneous route for food allergens can be supportive of the clinical history. A double-blind, placebo-controlled challenge may document a food allergy, but such a procedure does bear the risk of an anaphylactic reaction. An elimination diet is safer but is tedious and less definitive. Food allergy is uncommon as a cause of allergic rhinitis.

1	Newer methodology for detecting total IgE, including the development of enzyme-linked immunosorbent assays (ELISA) employing anti-IgE bound to either a solid-phase or a liquid-phase particle, provides rapid and cost-effective determinations. Measurements of specific anti-IgE in serum are obtained by its binding to an allergen and quantitation by subsequent uptake of labeled anti-IgE. As compared to the skin test, the assay of specific IgE in serum is less sensitive but has high specificity.

1	Avoidance of exposure to the offending allergen is the most effective means of controlling allergic diseases; removal of pets from the home toavoidanimaldanders,utilizationofair-filtrationdevicestominimize the concentrations of airborne pollens, elimination of cockroach-derived proteins by chemical destruction of the pest and careful food storage, travel to areas where the allergen is not being generated, and even a change of domicile to eliminate a mold spore problem may be necessary. Control of dust mites by allergen avoidance includes use of plastic-lined covers for mattresses, pillows, and comforters; using a filter-equipped vacuum cleaner; washing bedding and clothes at temperatures>54.5°C(above130°F);andeliminationofcarpetsanddrapes.

1	Although allergen avoidance is the most cost-effective means of managing allergic rhinitis, treatment with pharmacologic agents represents the standard approach to seasonal or perennial allergic rhinitis. Oral H1 antihistamines are effective for nasopharyngeal itching, sneezing, and watery rhinorrhea and for such ocular manifestations as itching, tearing, and erythema, but they are less efficacious for the nasal congestion. The older antihistamines are sedating, and they induce psychomotor impairment, including reduced eye-hand coordination and impaired automobile driving skills. Their anticholinergic (muscarinic) effects include visual disturbance, urinary retention, and constipation. Because the newer H1 antihistamines such as fexofenadine, loratadine, desloratadine, cetirizine, levocetirizine, olopatadine, bilastine, and azelastine are less lipophilic and more H1 selective, their ability to cross the blood-brain barrier is reduced, and thus their sedating and anticholinergic side

1	olopatadine, bilastine, and azelastine are less lipophilic and more H1 selective, their ability to cross the blood-brain barrier is reduced, and thus their sedating and anticholinergic side effects are minimized. These newer antihistamines do not differ appreciably in efficacy for relief of rhinitis and/or sneezing. Azelastine nasal spray may benefit individuals with nonallergic vasomotor rhinitis, but it has an adverse effect of dysgeusia (taste perversion) in some patients. Because antihistamines have little effect on congestion, α-adrenergic agents such as phenylephrine or oxymetazoline are generally used topically to alleviate nasal congestion and obstruction. However, the duration of their efficacy is limited because of rebound rhinitis (i.e., 7-to 14-day use can lead to rhinitis medicamentosa) and such systemic responses as hypertension. Oral α-adrenergic agonist decongestants containing pseudoephedrine are standard for the management of nasal congestion, generally in

1	medicamentosa) and such systemic responses as hypertension. Oral α-adrenergic agonist decongestants containing pseudoephedrine are standard for the management of nasal congestion, generally in combination with an antihistamine. While oral antihistamines typically reduce nasal and ocular symptoms by about one-third, pseudoephedrine must be added to achieve a similar reduction in nasal congestion. These pseudoephedrine combination products can cause insomnia and are precluded from use in patients with narrow angle glaucoma, urinary retention, severe hypertension, marked coronary artery disease, or a first-trimester pregnancy. The CysLT1 blocker montelukast is approved for treatment of both seasonal and perennial rhinitis, and it reduces both nasal and ocular symptoms by about 20%. Cromolyn sodium, a nasal spray, is essentially without side effects and is used prophylactically on a continuous basis during the season. Intranasal high-potency glucocorticoids are the most potent drugs

1	sodium, a nasal spray, is essentially without side effects and is used prophylactically on a continuous basis during the season. Intranasal high-potency glucocorticoids are the most potent drugs available for the relief of established rhinitis, seasonal or perennial, and are effective in relieving nasal congestion. They provide efficacy with substantially reduced side effects as compared with this same class of agent administered orally. Their most frequent side effect is local irritation, with Candida overgrowth being a rare occurrence. The currently available intranasal glucocorticoids— beclomethasone, flunisolide, triamcinolone, budesonide, fluticasone propionate, fluticasone furoate, ciclesonide, and mometasone furoate—are equally effective for nasal symptom relief, including nasal congestion; these agents all achieve up to 70% overall symptom relief with some variation in the time period for onset of benefit. Topical ipratropium is an anticholinergic agent effective in reducing

1	these agents all achieve up to 70% overall symptom relief with some variation in the time period for onset of benefit. Topical ipratropium is an anticholinergic agent effective in reducing rhinorrhea, including that in patients with perennial symptoms, and it can be additionally efficacious when combined with intranasal glucocorticoids. Local treatment with cromolyn sodium is effective in treating mild allergic conjunctivitis. Topical antihistamines such as olopatadine, azelastine, ketotifen, or epinastine administered to the eye provide rapid relief of itching and redness and are more effective than oral antihistamines.

1	Immunotherapy, often termed hyposensitization, consists of repeated subcutaneous injections of gradually increasing concentrations of the allergen(s) considered to be specifically responsible for the symptom complex. Controlled studies of ragweed, grass, dust mite, and cat dander allergens administered for treatment of allergic rhinitis have demonstrated at least partial relief of symptoms and

1	Intranasal glucocorticoids Past history of allergic rhinitis Treat as allergic rhinitis Wet or sneezy Exclude foreign body and anatomic defect Non-allergic rhinitis No specific allergen identified If negative Topical intranasal antihistamines or oral decongestants No past history of allergic rhinitis Treat as infection (viral vs bacterial) Chronic Anatomic defects, polyps, foreign body, and sinusitis Consider GERD assessment Exclude medication-induced rhinitis Acute Infectious symptoms Present Present Absent Absent Treat medically Consider immune deficiency evaluation if chronic sinusitis Assess for asthma and/or refer to ENT Allergy evaluation History/skin test or blood test for allergen-specific IgE Assess for asthma Oral or intranasal antihistamines, decongestants, intranasal cromolyn, or CysLT1 receptor antagonist Intranasal glucocorticoids (+ antihistamines/decongestants if required and/or + CysLT1 receptor antagonist) Consider nasal saline Allergic rhinitis Specific allergen

1	or CysLT1 receptor antagonist Intranasal glucocorticoids (+ antihistamines/decongestants if required and/or + CysLT1 receptor antagonist) Consider nasal saline Allergic rhinitis Specific allergen identified Oral glucocorticoids (brief: 3-7 days) If associated with severe asthma, consider omalizumabIpratropium bromide Immunotherapy Blocked nose Oral decongestants

1	FIGUrE 376-4 Algorithm for the diagnosis and management of rhinitis. ENT, ear, nose, and throat; GERD, gastroesophageal reflux disease.

1	Allergies, Anaphylaxis, and Systemic Mastocytosis 2124 signs. The duration of such immunotherapy is –5 years, with discontinuation being based on minimal symptoms over two consecutive seasons of exposure to the allergen. Clinical benefit appears related to the administration of a high dose of relevant allergen, advancing from weekly to monthly intervals. Patients should remain at the treatment site for at least 20 minutes after allergen administration so that any anaphylactic conseuence can be managed. Local reactions with erythema and induration are not uncommon and may persist for 1– days. Immunotherapy is contraindicated in patients with significant cardiovascular disease or unstable asthma and should be conducted with particular caution in any patient reuiring adrenergic blocking therapy because of the difficulty in managing an anaphylactic complication. The response to immunotherapy is associated with a complex of cellular and humoral effects that includes a modulation in T cell

1	because of the difficulty in managing an anaphylactic complication. The response to immunotherapy is associated with a complex of cellular and humoral effects that includes a modulation in T cell cytokine production. Immunotherapy should be reserved for clearly documented seasonal or perennial rhinitis that is clinically related to defined allergen exposure with confirmation by the presence of allergen-specific IgE. Systemic treatment with a monoclonal antibody to IgE (omalizumab) that blocks mast cell and basophil sensitization has efficacy for allergic rhinitis and can be used with immunotherapy to enhance safety and efficacy. However, current approval is only for treatment of patients with persistent allergic asthma not controlled by inhaled glucocorticoid therapy. A seuence for the management of allergic or perennial rhinitis based on an allergen-specific diagnosis and stepwise management as reuired for symptom control would include the following(1) identification of the offending

1	of allergic or perennial rhinitis based on an allergen-specific diagnosis and stepwise management as reuired for symptom control would include the following(1) identification of the offending allergen(s) by history with confirmation of the presence of allergen-specific IgE by skin test andor serum assay(2) avoidance of the offending allergenand () medical management in a stepwise fashion (Fig. 376-4). Mild intermittent symptoms of allergic rhinitis are treated with oral antihistamines, oral CysLT1 receptor antagonists, intranasal antihistamines, or intranasal cromolyn prophylaxis. Moderate to more severe allergic rhinitis is managed with intranasal glucocorticoids plus oral antihistamines, oral CysLT1 receptor antagonists, or antihistamine-decongestant combinations. Persistent allergic rhinitis reuiring the daily use of intranasal glucocorticoids with add-on interventions such as oral antihistamines, decongestant combinations, or topical ipratropium merits consideration of

1	allergic rhinitis reuiring the daily use of intranasal glucocorticoids with add-on interventions such as oral antihistamines, decongestant combinations, or topical ipratropium merits consideration of allergen-specific immunotherapy. Even a brief course of oral prednisone can be indicated for rapid relief of severe allergic rhinitis symptoms.

1	autoimmunity and autoimmune Diseases Betty Diamond, Peter E. Lipsky One of the central features of the immune system is the capacity to mount an inflammatory response to potentially harmful foreign mate-377e rials while avoiding damage to self-tissues. Whereas recognition of self plays an important role in shaping the repertoires of immune receptors on both T and B cells and in clearing apoptotic and other tissue debris from sites throughout the body, the development of potentially harmful immune responses to self-antigens is, in general, prohibited. The essential feature of an autoimmune disease is that tissue injury is caused by the immunologic reaction of the organism against its own tissues. Autoimmunity, on the other hand, refers merely to the presence of antibodies or T lymphocytes that react with self-antigens and does not necessarily imply that the self-reactivity has pathogenic consequences. Autoimmunity is present in all individuals; however, autoimmune disease occurs only

1	that react with self-antigens and does not necessarily imply that the self-reactivity has pathogenic consequences. Autoimmunity is present in all individuals; however, autoimmune disease occurs only in those individuals in whom the breakdown of one or more of the basic mechanisms regulating immune tolerance results in self-reactivity that can cause tissue damage.

1	Autoimmunity is seen in normal individuals, with a higher frequency among normal older people. Polyreactive autoantibodies that recognize many host antigens are present throughout life. Expression of these autoantibodies may be increased after some inciting events. These antibodies are usually of the IgM heavy chain isotype and are encoded by nonmutated germline immunoglobulin variable region genes. When autoimmunity is induced by an inciting event, such as infection or tissue damage from trauma or ischemia, the autoreactivity is in general self-limited. When such autoimmunity does persist, however, pathology may or may not result. Even in the presence of organ pathology, it may be difficult to determine whether the damage is mediated by autoreactivity. After an inciting event, the development of self-reactivity may be the consequence of an ongoing pathologic process, may be nonpathogenic, or may contribute to tissue inflammation and damage. Individuals with autoimmune disease may

1	of self-reactivity may be the consequence of an ongoing pathologic process, may be nonpathogenic, or may contribute to tissue inflammation and damage. Individuals with autoimmune disease may have numerous autoantibodies, only some or even none of which may be pathogenic. Patients with systemic sclerosis may have a wide array of antinuclear antibodies that are important in disease classification but are not clearly pathogenic; patients with pemphigus may also exhibit a wide array of autoantibodies, only one of which (antibody to desmoglein) is known to be pathogenic.

1	Since Ehrlich first postulated the existence of mechanisms to prevent the generation of self-reactivity in the early1900s, ideas concerning the nature of this prohibition have developed in parallel with a progressive increase in understanding of the immune system. Burnet’s clonal selection theory included the idea that interaction of lymphoid cells with their specific antigens during fetal or early postnatal life would lead to elimination of such “forbidden clones.” This idea was refuted, however, when it was shown that autoimmune diseases could be induced in experimental animals by simple immunization procedures, that autoantigen-binding cells could be demonstrated easily in the circulation of normal individuals, and that self-limited autoimmune phenomena frequently developed after tissue damage from infection or trauma. These observations indicated that clones of cells capable of responding to autoantigens were present in the repertoire of antigen-reactive cells in normal adults and

1	damage from infection or trauma. These observations indicated that clones of cells capable of responding to autoantigens were present in the repertoire of antigen-reactive cells in normal adults and suggested that mechanisms in addition to clonal deletion were responsible for preventing their activation.

1	Currently, three general processes are thought to be involved in the maintenance of selective unresponsiveness to autoantigens (Table 377e-1): (1) sequestration of self-antigens, rendering them inaccessible to the immune system; (2) specific unresponsiveness (tolerance or anergy) of relevant T or B cells; and (3) limitation of potential reactivity by regulatory mechanisms. Derangements of these normal 1. Sequestration of self-antigens 2. Generation and maintenance of tolerance a. Central deletion of autoreactive lymphocytes b. Peripheral anergy of autoreactive lymphocytes c. 3. Regulatory mechanisms a. b. c. d. e.

1	processes may predispose to the development of autoimmunity (Table 377e-2). In general, these abnormal responses require both an exogenous trigger, such as bacterial or viral infection or cigarette smoking, and the presence of endogenous abnormalities in the cells of the immune system. Microbial superantigens, such as staphylococcal protein A and staphylococcal enterotoxins, are substances that can stimulate a broad range of T and B cells through specific interactions with selected families of immune receptors, irrespective of their antigen specificity. If autoantigen-reactive T and/or B cells express these receptors, autoimmunity may develop. Alternatively, molecular mimicry or cross-reactivity between a microbial product and a self-antigen may lead to activation of autoreactive lymphocytes. One of the best examples of autoreactivity and autoimmune disease resulting from molecular mimicry is rheumatic fever, in which antibodies to the M protein of streptococci cross-react with

1	One of the best examples of autoreactivity and autoimmune disease resulting from molecular mimicry is rheumatic fever, in which antibodies to the M protein of streptococci cross-react with myosin, laminin, and other matrix proteins as well as with neuronal antigens. Deposition of these autoantibodies in the heart initiates an inflammatory response, whereas their penetration into the brain can result in Sydenham’s chorea. Molecular mimicry between microbial proteins and host tissues has been reported in type 1 diabetes mellitus, rheumatoid arthritis, celiac disease, and multiple sclerosis. It is presumed that infectious agents may be able to overcome self-tolerance because they possess pathogen-associated molecular patterns (PAMPs). These molecules (e.g., bacterial endotoxin, RNA, or DNA) exert adjuvant-like effects on the immune system by interacting with Toll-like receptors (TLRs) and other pattern recognition receptors (PRRs)

1	MeChanisMs of autoiMMunity I. Exogenous A. Molecular mimicry B. Superantigenic stimulation C. Microbial and tissue damage–associated adjuvanticity II. A. 1. Loss of immunologic privilege 2. Presentation of novel or cryptic epitopes (epitope spreading) 3. Alteration of self-antigen 4. Enhanced function of antigen-presenting cells a. b. B. 1. 2. C. Increased B cell function 1. 2. D. Apoptotic defects or defects in clearance of apoptotic material E. Cytokine imbalance F. Altered immunoregulation

1	b. B. 1. 2. C. Increased B cell function 1. 2. D. Apoptotic defects or defects in clearance of apoptotic material E. Cytokine imbalance F. Altered immunoregulation G. Endocrine abnormalities that increase the immunogenicity and immunostimulatory capacity of the microbial material. The adjuvants activate dendritic cells through TLRs, which in turn stimulate the activation of previously quiescent lymphocytes that recognize both microbial antigens and self-antigens. Similarly, cellular and tissue damage due to the release of damage-associated molecular patterns (DAMPs), including DNA, RNA nucleosomes, and other tissue debris, may activate cells of the inflammatory and immune systems through engagement of the same array of PRRs.

1	Endogenous derangements of the immune system may also contribute to the loss of immunologic tolerance to self-antigens and the development of autoimmunity (Table 377e-2). Some autoantigens reside in immunologically privileged sites, such as the brain or the anterior chamber of the eye. These sites are characterized by the inability of engrafted tissue to elicit immune responses. Immunologic privilege results from a number of events, including the limited entry of proteins from those sites into lymphatics, the local production of immunosuppressive cytokines such as transforming growth factor β, and the local expression of molecules (including Fas ligand) that can induce apoptosis of activated T cells. Lymphoid cells remain in a state of immunologic ignorance (neither activated nor anergized) with regard to proteins expressed uniquely in immunologically privileged sites. If the privileged site is damaged by trauma or inflammation or if T cells are activated elsewhere, proteins expressed

1	with regard to proteins expressed uniquely in immunologically privileged sites. If the privileged site is damaged by trauma or inflammation or if T cells are activated elsewhere, proteins expressed at this site can become immunogenic and also be the targets of immunologic assault. In multiple sclerosis and sympathetic ophthalmia, for example, antigens uniquely expressed in the brain and eye, respectively, become the target of activated T cells.

1	Alterations in antigen presentation may also contribute to autoimmunity. Peptide determinants (epitopes) of a self-antigen that are not routinely presented to lymphocytes may be recognized as a result of altered proteolytic processing of the molecule and the ensuing presentation of novel peptides (cryptic epitopes). When B cells rather than dendritic cells present self-antigen, they may also present cryptic epitopes that can activate autoreactive T cells. These cryptic epitopes will not previously have been available to effect the silencing of autoreactive lymphocytes. Furthermore, once there is immunologic recognition of one protein component of a multimolecular complex, reactivity may be induced to other components of the complex after internalization and presentation of all molecules within the complex (epitope spreading). Finally, inflammation, environmental agents, drug exposure, or normal senescence may cause a post-transitional alteration in proteins, resulting in the

1	within the complex (epitope spreading). Finally, inflammation, environmental agents, drug exposure, or normal senescence may cause a post-transitional alteration in proteins, resulting in the generation of immune responses that cross-react with normal self-proteins. For example, the induction and/or release of protein arginine deiminase enzymes results in the conversion of arginine residues to citrullines in a variety of proteins, thereby altering their capacity to induce immune responses. Production of antibodies to citrullinated proteins has been observed in rheumatoid arthritis and chronic lung disease as well as in normal smokers and may contribute to organ pathology. Alterations in the availability and presentation of autoantigens may be important components of immunoreactivity in certain models of organ-specific autoimmune diseases. In addition, these factors may be relevant to an understanding of the pathogenesis of various drug-induced autoimmune conditions. However, the

1	in certain models of organ-specific autoimmune diseases. In addition, these factors may be relevant to an understanding of the pathogenesis of various drug-induced autoimmune conditions. However, the diversity of autoreactivity manifesting in non-organ-specific systemic autoimmune diseases suggests that these conditions may result from a more general activation of the immune system rather than from an alteration in individual self-antigens.

1	Many autoimmune diseases are characterized by the presence of antibodies that react with apoptotic material. Defects in the clearance of apoptotic material have been shown to elicit auto-immunity and autoimmune disease in a number of animal models. Moreover, such defects have been found in patients with systemic lupus erythematosus (SLE). Apoptotic debris that is not cleared quickly by the immune system can function as endogenous ligands for a number of PRRs on dendritic cells and B cells. Under such circumstances, dendritic cells and/or B cells are activated, and an immune response to apoptotic debris can develop. In addition, the presence of extracellular apoptotic material within germinal centers of secondary lymphoid organs in patients with SLE may facilitate the direct activation of autoimmune B cell clones or may function to select such clones during immune responses.

1	Studies in a number of experimental models have suggested that intense stimulation of T lymphocytes can produce nonspecific signals that bypass the need for antigen-specific helper T cells and lead to polyclonal B cell activation with the formation of multiple autoantibodies. For example, antinuclear, antierythrocyte, and antilymphocyte antibodies are produced during the chronic graft-versus-host reaction. In addition, true autoimmune diseases, including autoimmune hemolytic anemia and immune complex–mediated glomerulonephritis, can be induced in this manner. While such diffuse activation of helper T cell activity clearly can cause autoimmunity, nonspecific stimulation of B lymphocytes can also lead to the production of autoantibodies. Thus, the administration of polyclonal B cell activators, such as bacterial endotoxin, to normal mice leads to the production of a number of autoantibodies, including those to DNA and IgG (rheumatoid factor). A variety of genetic modifications resulting

1	such as bacterial endotoxin, to normal mice leads to the production of a number of autoantibodies, including those to DNA and IgG (rheumatoid factor). A variety of genetic modifications resulting in hyperresponsiveness of B cells also can lead to the production of autoantibodies and, in animals of appropriate genetic background, a lupus-like syndrome. Moreover, excess B cell activating factor (BAFF), a B cell survival factor, can cause T cell–independent B cell activation and the development of autoimmunity. SLE can also be induced in mice through exuberant dendritic cell activation, through a redundancy of TLR7 on the Y chromosome (as in BXSB-Yaa mice), or through exposure to CpG, a ligand for TLR9. The ensuing induction of inflammatory mediators can cause a switch from the production of nonpathogenic IgM auto-antibodies to the production of pathogenic IgG autoantibodies in the absence of antigen-specific T cell help. Aberrant selection of the B or T cell repertoire at the time of

1	of nonpathogenic IgM auto-antibodies to the production of pathogenic IgG autoantibodies in the absence of antigen-specific T cell help. Aberrant selection of the B or T cell repertoire at the time of antigen receptor expression can also predispose to autoimmunity. For example, B cell immunodeficiency caused by an absence of the B cell receptor–associated kinase (Bruton’s tyrosine kinase) leads to X-linked agammaglobulinemia. This syndrome is characterized by reduced B cell numbers. This leads to high levels of BAFF which alter B cell selection and lead to greater survival of autoreactive B cells. Likewise, negative selection of autoreactive T cells in the thymus requires expression of the autoimmune regulator (AIRE) gene that enables the expression of tissue-specific proteins in thymic medullary epithelial cells. Peptides from these proteins are expressed in the context of major histocompatibility complex (MHC) molecules and mediate the central deletion of autoreactive T cells. The

1	medullary epithelial cells. Peptides from these proteins are expressed in the context of major histocompatibility complex (MHC) molecules and mediate the central deletion of autoreactive T cells. The absence of AIRE gene expression leads to a failure of negative selection of autoreactive cells, autoantibody production, and severe inflammatory destruction of multiple organs. Individuals deficient in AIRE gene expression develop autoimmune polyendocrinopathy–candidiasis– ectodermal dystrophy (APECED).

1	Primary alterations in the activity of T and/or B cells, cytokine imbalances, or defective immunoregulatory circuits may also contribute to the emergence of autoimmunity. Diminished production of tumor necrosis factor (TNF) and interleukin (IL) 10 has been reported to be associated with the development of autoimmunity. Overproduction or therapeutic administration of type 1 interferon has also been associated with autoimmunity. Overexpression of costimulatory molecules on T cells similarly can lead to autoantibody production.

1	Autoimmunity may also result from an abnormality of immunoregulatory mechanisms. Observations made in both human autoimmune disease and animal models suggest that defects in the generation and expression of regulatory T cell (Treg) activity may allow the production of autoimmunity. It has recently been appreciated that the IPEX (immunodysregulation, polyendocrinopathy, enteropathy X-linked) syndrome results from the failure to express the FOXP3 gene, which encodes a molecule critical in the differentiation of Tregs. Administration of normal Tregs or of factors derived from them can prevent the development of autoimmune disease in rodent models of autoimmunity, and allogeneic stem cell transplantation ameliorates human IPEX. Abnormalities in the function of Tregs have been noted in a number of human autoimmune diseases, including rheumatoid arthritis and SLE, although it remains uncertain whether these functional abnormalities are causative or are secondary to inflammation. One of the

1	of human autoimmune diseases, including rheumatoid arthritis and SLE, although it remains uncertain whether these functional abnormalities are causative or are secondary to inflammation. One of the mechanisms by which Tregs control immune/inflammatory responses is by the production of the cytokine IL-10. In this regard, children with a deficiency in the expression of IL-10 or the IL-10 receptor develop inflammatory bowel disease that mimics Crohn’s disease and that can be cured by allogeneic stem cell transplantation. Finally, recent data indicate that B cells may also exert regulatory function, largely through the production of IL-10. Deficiency of IL-10producing regulatory B cells can prolong the course of multiple sclerosis in an animal model, and such cells are thought to be functionally diminished in human SLE.

1	It should be apparent that no single mechanism can explain all the varied manifestations of autoimmunity or autoimmune disease. Furthermore, genetic evaluation has shown that convergence of a number of abnormalities is often required for the induction of an autoimmune disease. Additional factors that appear to be important determinants in the induction of autoimmunity include age, sex (many autoimmune diseases are far more common in women), exposure to infectious agents, and environmental contacts. How all of these disparate factors affect the capacity to develop self-reactivity is currently being investigated intensively.

1	Evidence in humans that there are susceptibility genes for auto of twins. Studies in type 1 diabetes mellitus, rheumatoid arthritis, multiple sclerosis, and SLE have shown that ~15–30% of pairs of mono-zygotic twins show disease concordance, whereas the figure is <5% for dizygotic twins. The occurrence of different autoimmune diseases within the same family has suggested that certain susceptibility genes may predispose to a variety of autoimmune diseases. Genome-wide association studies have begun to identify polymorphisms in individual genes that are associated with specific autoimmune diseases. More than 50 genetic polymorphisms associated with one or more autoimmune diseases have been identified to date. It is notable that some genes are associated with multiple autoimmune diseases, whereas others are specifically associated with only one autoimmune condition. Moreover, recent genetic evidence suggests that clusters of genetic risk factors can commonly be found in groups of

1	whereas others are specifically associated with only one autoimmune condition. Moreover, recent genetic evidence suggests that clusters of genetic risk factors can commonly be found in groups of autoimmune diseases. Four general clusters have been identified: one group of 6 genetic polymorphisms most frequently associated with Crohn’s disease, psoriasis, and multiple sclerosis; a second cluster of 8 polymor years, genome-wide association studies have demonstrated a variety of 377e-3 other genes that are involved in human autoimmune diseases. Most genes individually confer a relatively low risk for autoimmune diseases and are found in normal individuals. No gene has been identified that is essential for autoimmune diseases. In addition to this evidence from humans, certain inbred mouse strains reproducibly develop specific spontaneous or experimentally induced autoimmune diseases, whereas others do not. These findings have led to an extensive search for genes that determine

1	mouse strains reproducibly develop specific spontaneous or experimentally induced autoimmune diseases, whereas others do not. These findings have led to an extensive search for genes that determine susceptibility to autoimmune disease and for genes that might be protective.

1	The strongest consistent association for susceptibility to autoimmune disease is with particular MHC alleles. It has been suggested that the association of MHC genotype with autoimmune disease relates to differences in the ability of different allelic variations of MHC molecules to present autoantigenic peptides to autoreactive T cells. An alternative hypothesis involves the role of MHC alleles in shaping the T cell receptor repertoire during T cell ontogeny in the thymus. In addition, specific MHC gene products may themselves be the source of peptides that can be recognized by T cells. Cross-reactivity between such MHC peptides and peptides derived from proteins produced by common microbes may trigger autoimmunity by molecular mimicry. However, MHC genotype alone does not determine the development of autoimmunity. Identical twins are far more likely to develop the same autoimmune disease than MHC-identical nontwin siblings; this observation suggests that genetic factors other than

1	development of autoimmunity. Identical twins are far more likely to develop the same autoimmune disease than MHC-identical nontwin siblings; this observation suggests that genetic factors other than the MHC affect disease susceptibility. Studies of the genetics of type 1 diabetes mellitus, SLE, rheumatoid arthritis, and multiple sclerosis in humans and mice have identified several independently segregating disease susceptibility loci in addition to the MHC. Genes that encode molecules of the innate immune response are also involved in autoimmunity. In humans, inherited homozygous deficiency of the early proteins of the classic pathway of complement (C1q, C4, or C2) as well as genes involved in the type 1 interferon pathway are very strongly associated with the development of SLE.

1	The mechanisms of tissue injury in autoimmune diseases can be divided into antibody-mediated and cell-mediated processes. Representative examples are listed in Table 377e-3.

1	phisms most strongly associated with celiac disease, rheumatoid arthritis, and SLE; a third cluster of 7 polymorphisms most Effector Mechanism Target Disease strongly associated with type 1 diabetes, mul-Autoantibody Blocking or inactivation tiple sclerosis, and rheumatoid arthritis; and a fourth cluster of more than 12 polymorphisms most strongly associated with type 1 diabetes, rheumatoid arthritis, celiac disease, Crohn’s disease, and SLE. These results imply that autoimmune diseases with widely differ- ent clinical presentations and patterns of organ involvement could involve similar immunopathogenic pathways. For example, the same allele of the gene encoding PTPN22 is associated with multiple autoimmune dis- eases. Its product is a phosphatase expressed by a variety of hematopoietic cells that down- regulates antigen receptor–mediated stimulation of T and B cells. The risk allele is associated with type 1 diabetes mellitus, rheumatoid arthritis, and SLE in some populations. The

1	cells that down- regulates antigen receptor–mediated stimulation of T and B cells. The risk allele is associated with type 1 diabetes mellitus, rheumatoid arthritis, and SLE in some populations. The explanation for the association of this polymorphism with autoimmune dis cellular cytotoxicity ishes antigen receptor signaling during lym-T cells Cytokine production phocyte development, permitting escape of autoreactive clones or decreased activation-ease is uncertain, but it is likely that it dimin-

1	Cellular cytotoxicity α Chain of the nicotinic Insulin receptor Intrinsic factor TSH receptor (LATS) Proteinase-3 (ANCA) Epidermal cadherin Desmoglein 3 α3 Chain of collagen IV Immunoglobulin Platelet GpIIb:IIIa Rh antigens, I antigen Thyroid peroxidase, thyroglobulin ? ? Myasthenia gravis Insulin-resistant diabetes mellitus Pernicious anemia Graves’ disease Granulomatosis with polyangiitis Pemphigus vulgaris Rheumatoid arthritis, multiple sclerosis, type 1 diabetes mellitus induced apoptosis of autoantigen-reactive Abbreviations: ANCA, antineutrophil cytoplasmic antibody; LATS, long-acting thyroid stimulator; TSH, thyroid-stimulating lymphocytes in the periphery. In recent hormone. The pathogenicity of autoantibodies can be mediated through several mechanisms, including opsonization of soluble factors or cells, activation of an inflammatory cascade via the complement system, and interference with the physiologic function of soluble molecules or cells.

1	In autoimmune thrombocytopenic purpura, opsonization of platelets targets them for elimination by phagocytes. Likewise, in autoimmune hemolytic anemia, binding of immunoglobulin to red cell membranes leads to phagocytosis and lysis of the opsonized cell. Goodpasture’s syndrome, a disease characterized by lung hemorrhage and severe glomerulonephritis, represents an example of antibody binding leading to local activation of complement and neutrophil accumulation and activation. The autoantibody in this disease binds to the α3 chain of type IV collagen in the basement membrane. In SLE, activation of the complement cascade at sites of immunoglobulin deposition in renal glomeruli is considered to be a major mechanism of renal damage. Moreover, the DNAand RNA-containing immune complexes in SLE activate TLR9 and TLR7, respectively, in dendritic cells and promote a proinflammatory, immunogenic milieu conducive to amplification of the autoimmune response.

1	Autoantibodies can also interfere with normal physiologic functions of cells or soluble factors. Autoantibodies to hormone receptors can lead to stimulation of cells or to inhibition of cell function through interference with receptor signaling. For example, long-acting thyroid stimulators—autoantibodies that bind to the receptor for thyroid-stimulating hormone (TSH)—are present in Graves’ disease and function as agonists, causing the thyroid to respond as if there were an excess of TSH. Alternatively, antibodies to the insulin receptor can cause insulin-resistant diabetes mellitus through receptor blockade. In myasthenia gravis, autoantibodies to the acetylcholine receptor can be detected in 85–90% of patients and are responsible for muscle weakness. The exact location of the antigenic epitope, the valence and affinity of the antibody, and perhaps other characteristics determine whether activation or blockade results from antibody binding.

1	Antiphospholipid antibodies are associated with thromboembolic events in primary and secondary antiphospholipid syndrome and have also been associated with fetal wastage. The major antibody is directed to the phospholipid–β2-glycoprotein I complex and appears to exert a procoagulant effect. In pemphigus vulgaris, autoantibodies bind to desmoglein 3, a component of the epidermal cell desmosome, and play a role in the induction of the disease. These antibodies exert their pathologic effect by disrupting cell–cell junctions through stimulation of the production of epithelial proteases, with consequent blister formation. Cytoplasmic antineutrophil cytoplasmic antibody (c-ANCA), found in granulomatosis with polyangiitis, is an antibody to an intracellular antigen, the 29-kDa serine protease (proteinase-3). In vitro experiments have shown that IgG anti-c-ANCA causes cellular activation and degranulation of primed neutrophils.

1	It is important to note that autoantibodies of a given specificity may cause disease only in genetically susceptible hosts, as has been shown in experimental models of myasthenia gravis, SLE, rheumatic fever, and rheumatoid arthritis. Furthermore, once organ damage is initiated, new inflammatory cascades are initiated that can sustain and amplify the autoimmune process. Finally, some autoantibodies seem to be markers for disease but have, as yet, no known pathogenic potential. Manifestations of autoimmunity are found in a large number of pathologic conditions. However, their presence does not necessarily imply that the pathologic process is an autoimmune disease. A number of attempts to establish formal criteria for the classification of diseases as autoimmune have been made, but none is universally accepted. One set of criteria is shown in Table 377e-4; however, this scheme should be viewed merely as a guide in consideration of the problem.

1	To classify a disease as autoimmune, it is necessary to demonstrate that the immune response to a self-antigen causes the observed pathology. Initially, the detection of antibodies to the affected tissue in the serum of patients suffering from various diseases was taken as evidence that these diseases had an autoimmune basis. However, such huMan autoiMMune Disease: presuMptive eviDenCe for iMMunoLogiC pathogenesis 1. Presence of autoantibodies or evidence of cellular reactivity to self 2. Documentation of relevant autoantibody or lymphocytic infiltrate in the pathologic lesion 3. a. b. c. 1. 2. 3. Association with other evidence of autoimmunity 4. No evidence of infection or other obvious cause autoantibodies are also found when tissue damage is caused by trauma or infection and in these cases are secondary to tissue damage. Thus, autoimmunity must be shown to be pathogenic before a disease is categorized as autoimmune.

1	To confirm autoantibody pathogenicity, it may be possible to transfer disease to experimental animals by the administration of autoantibodies from a patient, with the subsequent development of pathology in the recipient similar to that seen in the patient. This scenario has been documented, for example, in Graves’ disease. Some autoimmune diseases can be transferred from mother to fetus and are observed in the newborn babies of diseased mothers. The symptoms of the disease in the newborn usually disappear as the levels of maternal antibody decrease. An exception, however, is congenital heart block, in which damage to the developing conducting system of the heart follows in utero transfer of anti-Ro antibody from the mother to the fetus. This antibody transfer can result in a permanent developmental defect in the heart.

1	In most situations, the critical factors that determine when the development of autoimmunity results in autoimmune disease have not been delineated. The relationship of autoimmunity to the development of autoimmune disease may be associated with the fine specificity of the antibodies or T cells or their specific effector capabilities. In many circumstances, a mechanistic understanding of the pathogenic potential of autoantibodies has not been established. In some autoimmune diseases, biased production of cytokines by helper T (TH) cells may play a role in pathogenesis. In this regard, T cells can differentiate into specialized effector cells that predominantly produce interferon γ (TH1), IL-4 (TH2), or IL-17 (TH17) or that provide help to B cells (T follicular helper, TFH) (Chap. 372e). TH1 cells facilitate macrophage activation and classic cell-mediated immunity, whereas TH2 cells are thought to have regulatory functions and are involved in the resolution of normal immune responses

1	TH1 cells facilitate macrophage activation and classic cell-mediated immunity, whereas TH2 cells are thought to have regulatory functions and are involved in the resolution of normal immune responses as well as in the development of responses to a variety of parasites. TH17 cells produce a number of inflammatory cytokines, including IL-17 and IL-22, and seem to be prominently involved in host resistance to certain fungal infections.

1	cells help B cells by constitutively producing IL-21. In a number of autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, type 1 diabetes mellitus, and Crohn’s disease, there appears to be biased differentiation of TH1 and TH17 cells, with resultant organ damage. Studies suggest an accentuated differentiation of TH17 cells associated with animal models of inflammatory arthritis, whereas increased differentiation of TFH cells has been associated with animal models of SLE. The spectrum of autoimmune diseases ranges from conditions specifically affecting a single organ to systemic disorders that involve many organs (Table 377e-5). Hashimoto’s autoimmune thyroiditis is an example of an organ-specific autoimmune disease (Chap. 405). In this

1	Systemic lupus erythematosus Granulomatosis with polyangiitis disorder, a specific lesion in the thyroid is associated with infiltration of mononuclear cells and damage to follicular cells. Antibody to thyroid constituents can be demonstrated in nearly all cases. Other organ-or tissue-specific autoimmune disorders include pemphigus vulgaris, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpasture’s syndrome, myasthenia gravis, and sympathetic ophthalmia. One important feature of some organ-specific autoimmune diseases is the tendency for overlap, such that an individual with one specific syndrome is more likely to develop a second syndrome. For example, there is a high incidence of pernicious anemia in individuals with autoimmune thyroiditis. More striking is the tendency for individuals with an organ-specific autoimmune disease to develop multiple other manifestations of autoimmunity without the development of associated organ pathology. Thus, as many as 50% of

1	for individuals with an organ-specific autoimmune disease to develop multiple other manifestations of autoimmunity without the development of associated organ pathology. Thus, as many as 50% of individuals with pernicious anemia have non-cross-reacting antibodies to thyroid constituents, whereas patients with myasthenia gravis may develop antinuclear antibodies, antithyroid antibodies, rheumatoid factor, antilymphocyte antibodies, and polyclonal hypergammaglobulinemia. Part of the explanation may relate to the genetic elements shared by individuals with these different diseases.

1	Systemic autoimmune diseases differ from organ-specific diseases in that pathologic lesions are found in multiple diverse organs and tissues. The hallmark of these conditions is the demonstration of associated relevant autoimmune manifestations that are likely to have an etiologic role in organ pathology. SLE represents the prototype of these 377e-5 disorders because of its abundant autoimmune manifestations. SLE is a disease of protean manifestations that characteristically involves the kidneys, joints, skin, serosal surfaces, blood vessels, and central nervous system (Chap. 378). The disease is associated with a vast array of autoantibodies whose production appears to be a part of a generalized hyperreactivity of the humoral immune system. Other features of SLE include generalized B cell hyperresponsiveness and polyclonal hypergammaglobulinemia. Current evidence suggests that both hypoand hyperresponsiveness to antigen can lead to survival and activation of autoreactive B cells in

1	hyperresponsiveness and polyclonal hypergammaglobulinemia. Current evidence suggests that both hypoand hyperresponsiveness to antigen can lead to survival and activation of autoreactive B cells in SLE. The autoantibodies in SLE are thought to arise as part of an accentuated T cell–dependent B cell response since most pathogenic anti-DNA autoantibodies exhibit evidence of extensive somatic hypermutation.

1	Treatment of autoimmune diseases can focus on suppressing the induction of autoimmunity, restoring normal regulatory mechanisms, or inhibiting the effector mechanisms. To decrease the number or function of autoreactive cells, immunosuppressive or ablative therapies are most commonly used. In recent years, cytokine blockade has been demonstrated to be effective in preventing immune activation in some diseases or in inhibiting the extensive inflammatory effector mechanisms characteristic of these diseases. New therapies have also been developed to target lymphoid cells more specifically by blocking a costimulatory signal needed for T or B cell activation, by blocking the migratory capacity of lymphocytes, or by eliminating the effector T cells or B cells. The efficacy of these therapies in some diseases—e.g., SLE (belimumab), rheumatoid arthritis (TNF neutralization, IL-6 receptor blockade, CD28 competition, B cell depletion, IL-1 competition), psoriasis (IL-12/23 depletion, TNF

1	in some diseases—e.g., SLE (belimumab), rheumatoid arthritis (TNF neutralization, IL-6 receptor blockade, CD28 competition, B cell depletion, IL-1 competition), psoriasis (IL-12/23 depletion, TNF neutralization), and inflammatory bowel disease (TNF neutralization, IL-12 neutralization)—has been demonstrated. One major advance in inhibiting effector mechanisms has been the introduction of cytokine blockade that appears to limit organ damage in some diseases, including rheumatoid arthritis, inflammatory bowel disease, psoriasis, and the spondyloarthritides. Small molecules that block cytokine signaling pathways have recently been introduced into the clinic. Biologicals that interface with T cell activation (CTLA-4Ig) or delete B cells (anti-CD20 antibody) have recently been approved for the treatment of rheumatoid arthritis. Therapies that prevent target-organ damage or support target-organ function remain important in the management of autoimmune disease.

1	Immune-Mediated, Inflammatory, and Rheumatologic Disorders Systemic lupus erythematosus Bevra Hannahs Hahn DEFINITION aND PrEVaLENCE Systemiclupuserythematosus(SLE)isanautoimmunediseaseinwhichorgansandcellsundergodamageinitiallymediatedbytissue-bindingautoantibodiesandimmunecomplexes.Inmostpatients,autoantibod-378 iesarepresent forafewyears beforethefirst clinicalsymptom appears. Ninety percent of patients are women of child-bearing years; people of all genders, ages, and ethnic groups are susceptible. Prevalence of SLE in the United States is 20 to 150 per 100,000 women depending on race and gender; highest prevalence is in African-American and Afro-Caribbean women, and lowest prevalence is in white men.

1	The proposed pathogenic mechanisms of SLE are illustrated in Fig. 378-1. Interactions between susceptibility genes and environmental factors result in abnormal immune responses, which vary between different patients. Those responses may include (1) activation of innate immunity (dendritic cells, monocyte/macrophages) by CpG DNA, DNA in immune complexes, viral DNA or RNA, and RNA in RNA/ protein self-antigens; (2) lowered activation thresholds and abnormal activation pathways in adaptive immunity cells (mature T and B lymphocytes);(3)ineffectiveregulatoryCD4+andCD8+Tcells,Bcells,and myeloid-derived suppressor cells; and (4) reduced clearance of immune complexesandapoptoticcells.Self-antigens(nucleosomalDNA/protein; RNA/protein in Sm, Ro, and La; phospholipids) are recognized by the immune system in surface blebs of apoptotic cells; thus autoantigens, autoantibodies, and immune complexes persist for prolonged periods of time, allowing inflammation and disease to develop. Immune cell

1	system in surface blebs of apoptotic cells; thus autoantigens, autoantibodies, and immune complexes persist for prolonged periods of time, allowing inflammation and disease to develop. Immune cell activation is accompanied by increased secretion of proinflammatory type1and2interferons(IFNs),tumornecrosisfactorα(TNF-α),interleukin (IL) 17 and B cell-maturation/survival cytokines B lymphocyte stimulator (BLyS/BAFF), and IL-10. Upregulation of genes induced by 1.Genes

1	Renal Failure Arthritis Artherosclerosis Leukopenia EPIGENETIC CHANGES Pulm fibrosisCNS dz(DNA hypomethylation, 3.Autoantibodies Stroke miRNA) Clotting Etc. Etc. ?Infection Others EBV

1	FIGUrE 378-1 Pathogenesis of systemic lupus erythematosus (SLE). Genes confirmed in more than one genome-wide association analysis in northern European whites (several confirmed in Asians as well) as increasing susceptibility to SLE or lupus nephritis are listed (reviewed in SG Guerra et al: Arthritis Res Ther 14:211, 2012). Gene-environment interactions (reviewed in KH Costenbader et al: Autoimmune Rev 11:604, 2012) result in abnormal immune responses that generate pathogenic autoantibodies and immune complexes that deposit in tissue, activate complement, cause inflammation, and over time lead to irreversible organ damage (reviewed in GC Tsokos: N Engl J Med 365:2110, 2011; and BH Hahn, in DJ Wallace, BH Hahn [eds]: Dubois’ Lupus Erythematosus and Related Syndromes, 8th ed. New York, Elsevier, 2013). Ag, antigen; C1q, complement system; C3, complement component; CNS, central nervous system; DC, dendritic cell; EBV, Epstein-Barr virus; HLA, human leukocyte antigen; FcR, immunoglobulin

1	2013). Ag, antigen; C1q, complement system; C3, complement component; CNS, central nervous system; DC, dendritic cell; EBV, Epstein-Barr virus; HLA, human leukocyte antigen; FcR, immunoglobulin Fc-binding receptor; IL, interleukin; MCP, monocyte chemotactic protein; PTPN, phosphotyrosine phosphatase; UV, ultraviolet.

1	IFNsisa genetic “signature”in peripheral blood cells of50–60% ofSLE patients. Decreased production of other cytokines also contributes to SLE: lupus T and natural killer (NK) cells fail to produce enough IL-2 and transforming growth factor beta (TGF-β) to induce and sustain regulatory CD4+ and CD8+ T cells. The result of these abnormalities is sustained production of autoantibodies (referred to in Fig. 378-1 and described in Table 378-1) and immune complexes; pathogenic subsets bind target tissues, with activation of complement, leading to release of cytokines, chemokines, vasoactive peptides, oxidants, and proteolytic enzymes. This results in activation of multiple tissue cells (endothelial cells, tissue-fixed macrophages, mesangial cells, podocytes, renal tubular epithelial cells) and influx into target tissues of T and B cells, monocyte/macrophages, and dendritic cells. In the setting of chronic inflammation, accumulation of growth factors and products of chronic oxidation

1	and influx into target tissues of T and B cells, monocyte/macrophages, and dendritic cells. In the setting of chronic inflammation, accumulation of growth factors and products of chronic oxidation contribute to irreversible tissue damage, including fibrosis/ sclerosis in glomeruli, arteries, lungs, and other tissues.

1	SLE is a multigenic disease. Rare single-gene defects confer high hazard ratios (HRs) for SLE (5 to 25), including homozygous deficiencies of early components of complement (C1q,r,s; C2; C4) and a mutation in TREX1 on the X chromosome. In most genetically susceptible individuals, normal alleles of multiple genes each contribute a small amount to abnormal immune/inflammation/tissue damage responses;ifenoughpredisposingvariationsarepresent,diseaseresults. Approximately 45 predisposing genes (examples listed in Fig. 378-1) have been identified in recent genome-wide association studies in different racial groups. Individually, they confer an HR for SLE of 1.5–3 and account for approximately 18% of disease susceptibility, suggesting that environmental exposures and epigenetics play major roles. Predisposing, antigen-presenting human leukocyte antigen (HLA) molecules are most commonly found, in multiple ethnic groups (HLA DRB1 *0301 and *1501, as well as multiple genes across the major

1	Predisposing, antigen-presenting human leukocyte antigen (HLA) molecules are most commonly found, in multiple ethnic groups (HLA DRB1 *0301 and *1501, as well as multiple genes across the major histocompatibility complex (MHC) 120-gene region). Other genetic factors in whites include innate immunity pathway gene polymorphisms, especially associated with IFN-α (STAT4, IRF5, IRAK1, TNFAIP3, PTPN22), genes in lymphocyte signaling pathways (PTPN22, PDCD-1, Ox40L, BANK-1, LYN, BLK), genes that affect clearance of apoptotic cellsor immune complexes (C1q, FCRGIIA, FCRGIIIA, CRP, ITGAM), genes that influence neutrophil adherence (ITGAM), and genes that influenceDNArepair(TREX-1).Somepolymorphismsinfluenceclinical manifestations; such as single nucleotide polymorphisms (SNPs) of STAT4 that associate with severe disease, anti-DNA, nephritis, and antiphospholipid syndrome, and an allele of FCGRIIA encoding a receptor that binds immune complexes poorly and predisposes to nephritis. Some gene

1	with severe disease, anti-DNA, nephritis, and antiphospholipid syndrome, and an allele of FCGRIIA encoding a receptor that binds immune complexes poorly and predisposes to nephritis. Some gene effects are in promoter regions (e.g., IL-10), and others are conferred by copy numbers (e.g., C4A). In addition to genome-encoded susceptibility and protective genes, the influence of certain microRNAs (miRNAs) on gene transcription, as well as posttranscriptional epigenetic modification of DNA (which is hypomethylated in T cells of SLE patients), probably play major roles in disease susceptibility.

1	Some gene polymorphisms contribute to several autoimmune diseases, such as STAT4 and CTLA4. All of these gene polymorphisms/ transcription/epigenetic combinations influence immune responses to the external and internal environment; when such responses are too high and/or too prolonged and/or inadequately regulated, autoimmune disease results. Female sex is permissive for SLE with evidence for hormone effects, genes on the X chromosome, and epigenetic differences between genders playing a role. Females of many mammalian species make higher antibody responses than males. Women exposed to Antibody Prevalence, % Antigen Recognized Clinical Utility Protein complexed to 6 species of nuclear U1 RNA Protein complexed to U1 RNA Protein complexed to hY RNA, primarily 60 kDa and 52 kDa 47-kDa protein complexed to hY RNA Histones associated with DNA (in nucleosome, chromatin) Phospholipids, β2 glycoprotein 1 (β2G1) cofactor, prothrombin

1	Phospholipids, β2 glycoprotein 1 (β2G1) cofactor, prothrombin High titers are SLE-specific and in some patients correlate with disease activity, nephritis, vasculitis Not specific for SLE; high titers associated with syndromes that have overlap features of several rheumatic syndromes including SLE; more common in blacks than whites Not specific for SLE; associated with sicca syndrome, predisposes to subacute cutaneous lupus, and to neonatal lupus with congenital heart block; associated with decreased risk for nephritis Usually associated with anti-Ro; associated with decreased risk for nephritis Three tests available—ELISAs for cardiolipin and β2G1, sensitive prothrombin time (DRVVT); predisposes to clotting, fetal loss, thrombocytopenia Measured as direct Coombs test; a small proportion develops overt hemolysis Associated with thrombocytopenia, but sensitivity and specificity are not good; this is not a useful clinical test

1	Measured as direct Coombs test; a small proportion develops overt hemolysis Associated with thrombocytopenia, but sensitivity and specificity are not good; this is not a useful clinical test In some series, a positive test in CSF correlates with active CNS lupus In some series, a positive test in serum correlates with depression or psychosis due to CNS lupus Abbreviations: CNS, central nervous system; CSF, cerebrospinal fluid; DRVVT, dilute Russell viper venom time; ELISA, enzyme-linked immunosorbent assay.

1	Abbreviations: CNS, central nervous system; CSF, cerebrospinal fluid; DRVVT, dilute Russell viper venom time; ELISA, enzyme-linked immunosorbent assay. estrogen-containingoralcontraceptivesorhormonereplacementhave an increased risk of developing SLE (1.2-to 2-fold). Estradiol binds to receptors on T and B lymphocytes, increasing activation and survival of those cells, thus favoring prolonged immune responses. Genes on theXchromosomethatinfluenceSLE,suchas TREX-1,mayplayarole in gender predisposition, possibly because some genes on the second X in females are not silent. People with XXY karyotype (Klinefelter’s syndrome) have a significantly increased risk for SLE.

1	Several environmental stimuli may influence SLE (Fig. 378-1). Exposure to ultraviolet light causes flares of SLE in approximately 70% of patients, possibly by increasing apoptosis in skin cells or by altering DNA and intracellular proteins to make them antigenic. Some infections induce normal immune responses that involve certain T and B cells that recognize self-antigens; such cells are not appropriately regulated, and autoantibody production occurs. Most SLE patients have autoantibodies for 3 years or more before the first symptoms of disease, suggesting that regulation controls the degree of autoimmunity for years before quantities and qualities of autoantibodies and pathogenic B and T cells cause clinical disease. Epstein-Barr virus (EBV) may be one infectious agent that can trigger SLE in susceptible individuals. Children and adults with SLE are more likely to be infected by EBV than age-, sex-, and ethnicity-matched controls. EBV contains amino acid sequences that mimic

1	SLE in susceptible individuals. Children and adults with SLE are more likely to be infected by EBV than age-, sex-, and ethnicity-matched controls. EBV contains amino acid sequences that mimic sequences on human spliceosomes (RNA/protein antigens) often recognized by autoantibodies in people with SLE. Current tobacco smoking increases risk for SLE (odds ratio [OR] 1.5). Prolonged occupational exposure to silica (e.g., inhalation of soap powder dust or soil in farming activities) increases risk (OR 4.3) in African-American women. Thus, interplay between genetic susceptibility, environment, gender, and abnormal immune responses results in autoimmunity (Chap. 377e).

1	In SLE, biopsies of affected skin show deposition of Ig at the dermalepidermaljunction(DEJ),injurytobasalkeratinocytes,andinflammation dominated by T lymphocytes in the DEJ and around blood vessels and dermal appendages. Clinically unaffected skin may also show Ig deposition at the DEJ.

1	In renal biopsies, the pattern and severity of injury are important in diagnosisandinselectingthebesttherapy.Mostrecentclinicalstudiesof lupusnephritishaveusedtheInternationalSocietyofNephrology(ISN) and the Renal Pathology Society (RPS) classification (Table 378-2). In the ISN/RPS classification, the addition of “a” for active and “c” for chronic changes gives physicians information regarding the potential reversibility of disease. The system focuses on glomerular disease, although the presence of tubular interstitial and vascular disease is important to clinical outcomes. In general, class III and IV disease, as well as class V accompanied by III or IV disease, should be treated with aggressiveimmunosuppressionifpossible,becausethereisahighriskfor end-stage renal disease (ESRD) if patients are untreated or undertreated. Incontrast,treatmentforlupusnephritisisnotrecommendedinpatients with class I or II disease or with extensive irreversible changes. In the recent Systemic Lupus

1	patients are untreated or undertreated. Incontrast,treatmentforlupusnephritisisnotrecommendedinpatients with class I or II disease or with extensive irreversible changes. In the recent Systemic Lupus International Collaborating Clinic (SLICC) criteria for classification of SLE, a diagnosis can be established on the basis of renal histology without meeting additional criteria (Table 378-3).

1	Histologic abnormalities in blood vessels may also determine therapy. Patterns of vasculitis are not specific for SLE but may indicate activedisease:leukocytoclasticvasculitisismostcommon (Chap. 385). Lymph node biopsies are usually performed to rule out infection or malignancies.InSLE,theyshownonspecificdiffusechronicinflammation.

1	ThediagnosisofSLEisbasedoncharacteristicclinicalfeaturesandautoantibodies. Current criteria for classification are listed in Table 378-3, and an algorithm for diagnosis and initial therapy is shown in Fig. 378-2. The criteria are intended for confirming the diagnosis of SLE in patients included in studies; the author uses them in individual patients for estimating the probability that a disease is SLE. Any combination of four or more criteria, with at least one in the clinical and one in the immunologic category, well documented at any time during an individual’s history, makes it likely that the patient has SLE. (Specificity and sensitivity are ~93% and ~92%, respectively.) In many patients, criteria accrue over time. Antinuclear antibodies (ANA) are positive in >98% of patients during the course of disease; repeated negative tests by immunofluorescent methods suggest that the diagnosis is not SLE, unless other autoantibodies are present (Fig. 378-2). High-titer IgG antibodies to

1	the course of disease; repeated negative tests by immunofluorescent methods suggest that the diagnosis is not SLE, unless other autoantibodies are present (Fig. 378-2). High-titer IgG antibodies to double-stranded DNA and antibodies to the Sm antigen are both specific for SLE and, therefore, favor the diagnosis in the presence of compatible clinical manifestations. The

1	Class I: Minimal Mesangial Lupus Nephritis Normal glomeruli by light microscopy, but mesangial immune deposits by immunofluorescence. Class II: Mesangial Proliferative Lupus Nephritis Purely mesangial hypercellularity of any degree or mesangial matrix expansion by light microscopy, with mesangial immune deposits. A few isolated subepithelial or subendothelial deposits may be visible by immunofluorescence or electron microscopy, but not by light microscopy. Class III: Focal Lupus Nephritis Active or inactive focal, segmental or global endo-or extracapillary glomerulonephritis involving ≤50% of all glomeruli, typically with focal subendothelial immune deposits, with or without mesangial alterations. Class III (A): Active lesions—focal proliferative lupus nephritis Class III (A/C): Active and chronic lesions—focal proliferative and sclerosing lupus nephritis Class III (C): Chronic inactive lesions with glomerular scars—focal sclerosing lupus nephritis

1	Class III (A/C): Active and chronic lesions—focal proliferative and sclerosing lupus nephritis Class III (C): Chronic inactive lesions with glomerular scars—focal sclerosing lupus nephritis Class IV: Diffuse Lupus Nephritis Active or inactive diffuse, segmental or global endo-or extracapillary glomerulonephritis involving ≥50% of all glomeruli, typically with diffuse subendothelial immune deposits, with or without mesangial alterations. This class is divided into diffuse segmental (IV-S) lupus nephritis when ≥50% of the involved glomeruli have segmental lesions, and diffuse global (IV-G) lupus nephritis when ≥50% of the involved glomeruli have global lesions. Segmental is defined as a glomerular lesion that involves less than one-half of the glomerular tuft. This class includes cases with diffuse wire loop deposits but with little or no glomerular proliferation. Class IV-S (A): Active lesions—diffuse segmental proliferative lupus nephritis

1	Class IV-S (A): Active lesions—diffuse segmental proliferative lupus nephritis Class IV-G (A): Active lesions—diffuse global proliferative lupus nephritis Class IV-S (A/C): Active and chronic lesions—diffuse segmental proliferative and sclerosing lupus nephritis Class IV-G (A/C): Active and chronic lesions—diffuse global proliferative and sclerosing lupus nephritis Class IV-S (C): Chronic inactive lesions with scars—diffuse segmental sclerosing lupus nephritis Class IV-G (C): Chronic inactive lesions with scars—diffuse global sclerosing lupus nephritis Class V: Membranous Lupus Nephritis Global or segmental subepithelial immune deposits or their morphologic sequelae by light microscopy and by immunofluorescence or electron microscopy, with or without mesangial alterations. Class V lupus nephritis may occur in combination with class III or IV, in which case both will be diagnosed. Class V lupus nephritis may show advanced sclerosis.

1	Class VI: Advanced Sclerotic Lupus Nephritis ≥90% of glomeruli globally sclerosed without residual activity. Note: Indicate and grade (mild, moderate, severe) tubular atrophy, interstitial inflammation and fibrosis, and severity of arteriosclerosis or other vascular lesions. Source: JJ Weening et al: Kidney Int 65:521, 2004. Reprinted by permission from Macmillan Publishers Ltd., Copyright 2004. presence in an individual of multiple autoantibodies without clinical symptoms should not be considered diagnostic for SLE, although such persons are at increased risk. When a diagnosis of SLE is made, it is important to establish the severity and potential reversibility of the illness and to estimate the possible consequences of various therapeutic interventions. In the following paragraphs, descriptions of some disease manifestations begin with relatively mild problems and progress to those more life-threatening.

1	At its onset, SLE may involve one or several organ systems; over time, additional manifestations may occur (Tables 378-3 and 378-4). Most of the autoantibodies characteristic of each person are present at the time clinical manifestations appear (Tables 378-1 and 378-3). Severity aRenal biopsy read as systemic lupus qualifies for classification as SLE even if none of the other above features are present. Interpretation: Presence of any 4 criteria (must have at least 1 in each category) qualifies patient to be classified as having SLE with 93% specificity and 92% sensitivity. Abbreviations: ANA, antinuclear antibody; Cr, creatinine; LE, lupus erythematosus; Prot, protein. Source: M Petri et al: Arthritis Rheum 64:2677, 2012. Because these criteria are new, currently ongoing clinical studies use prior American College of Rheumatology Criteria; see EM Tan et al: Arthritis Rheum 25:1271, 1982; update MC Hochberg: Arthritis Rheum 40:1725, 1997.

1	of SLE varies from mild and intermittent to severe and fulminant. Approximately 85% of patients have either continuing active disease (while being treated) or one or more flares of active disease annually. Permanent complete remissions (absence of symptoms with no treatment) are rare. Systemic symptoms, particularly fatigue and myalgias/ arthralgias, arepresentmostofthetime.Severesystemic illnessrequiring glucocorticoid therapy can occur with fever, prostration, weight loss, and anemia with or without other organ-targeted manifestations.

1	Most people with SLE have intermittent polyarthritis, varying from mild to disabling, characterized by soft tissue swelling and tenderness in joints and/or tendons, most commonly in hands, wrists, and knees. Joint deformities (hands and feet) develop in only 10%. Erosions on joint x-rays are rare but can be identified by ultrasound in almost half of patients. Some individuals have rheumatoid-like arthritis with erosions and fulfill criteria for both RA and SLE (“rhupus”); they may be coded as having both diseases. If pain persists in a single joint, such as knee, shoulder, or hip, a diagnosis of ischemic necrosis of bone should be considered, particularly if there are no other manifestations of active SLE because its prevalence is increased in SLE, especially in patients treated with systemic glucocorticoids. Myositis with clinical muscle weakness, elevated creatine kinase levels, positive magnetic resonance imaging (MRI)scan,andmusclenecrosis and inflammation on biopsy can occur,

1	glucocorticoids. Myositis with clinical muscle weakness, elevated creatine kinase levels, positive magnetic resonance imaging (MRI)scan,andmusclenecrosis and inflammation on biopsy can occur, although most patients have myalgias without frank myositis. Glucocorticoid therapies (commonly) and antimalarial therapies (rarely) can cause muscle weakness; these adverse effects must be distinguished from active inflammatory disease.

1	Lupus dermatitis can be classified as acute, subacute, or chronic, and there are many different types of lesions encompassed within these groups. Discoid lupus erythematosus (DLE) is the most common Diagnosis: Symptom complex suggestive of SLE Order laboratory tests: ANA, CBC, platelets, urinalysis

1	Diagnosis: Symptom complex suggestive of SLE Order laboratory tests: ANA, CBC, platelets, urinalysis All tests normal symptoms subside All tests normal symptoms persist ANA positive Not SLE Not SLE Treatment Repeat ANA, add anti-dsDNA, anti-Ro All negative Some positive Definite SLE (˜4 criteria, Table 378-3) Possible SLE (<4 criteria, Table 378-3) Not lifeor organ-threatening Lifeor organ-threatening Quality of life: Acceptable Quality of life: Not acceptable High-dose glucocorticoids, usually with addition of second agent Conservative manage-ment (Table 378-5) Conservative treatment plus low-dose glucocorticoids Consider belimumab Mycophenolate mofetil (or myfortic acid) Cyclophosphamide Low or high dose Do not exceed 6 months of Rx After response, d/c cyclophosphamide; maintain with mycophenolate or azathioprine No response Response Taper dose of all agents, especially glucocorticoids Belimumab, Rituximab, calcineurin inhibitors, or experimental therapies

1	FIGUrE 378-2 Algorithm for diagnosis and initial therapy of systemic lupus erythematosus (SLE). For guidelines on management of lupus and lupus nephritis, see BH Hahn et al: Arthritis Care Res (Hoboken) 64:797, 2012; GK Bertsias et al: Ann Rheum Dis 71:1771, 2012; and G Bertsias et al: Ann Rheum Dis 67:195, 2008. For details on mycophenolate and cyclophosphamide induction and maintenance therapies, see L Henderson et al: Cochrane Database Syst Rev 12:CD002922, 2012; Z Touma et al: J Rheumatol 38:69, 2011; EM Ginzler et al: Arthritis Rheum 62:211, 2010; FA Houssiau et al: Ann Rheum Dis 69:61, 2010; and MA Dooley et al: N Engl J Med 365:1886, 2011. For belimumab in treatment, see BH Hahn: N Eng J Med 368:1528, 2013. For rituximab, see L Lightstone: Lupus 22:390, 2013; and BH Rovin et al: Arthritis Rheum 64:1215, 2012. ANA, antinuclear antibodies; CBC, complete blood count.

1	extensor surfaces of the arms. Worsening of this rash often accompanies flare of systemic disease. Subacute cutaneous lupus erythematosus (SCLE) consists of scaly red patches similar topsoriasis,orcircularflatred-rimmedlesions. Patients with these manifestations are exquisitely photosensitive; most have antibodies to Ro (SS-A). Other SLE rashes include recurring urticaria, lichen planus-like dermatitis, bullae, and panniculitis (“lupus profundus”). Rashes can be minor or severe; they may be the major disease manifestation. Small ulcerations on the oral or nasal mucosa are common in SLE; the lesions resemble aphthous ulcers.

1	Nephritis is usually the most serious manifestation of SLE, particularly because nephritis and infection are the leading causes of mortality in the first decade of disease. Because nephritis is asymptomatic in most lupus patients, urinalysis should be ordered in any person suspected of having SLE. The classification of lupus nephritis is primarily histologic (see “Pathology,” above, and Table 378-2). Renal biopsy is recommended for every SLE patient with any clinical evidence of nephritis; results are used to plan current andnear-futuretherapies.Patients withdangerous proliferative forms of glomerular damage (ISN III and IV) usually have microscopic hematuria and proteinuria (>500 mg per 24 h); approximately one-half develop nephrotic syndrome, and most develop hypertension. If diffuse proliferative glomerulonephritis (DPGN) is inadequately treated, virtually all patients develop ESRD within 2 years of diagnosis. Therefore, aggressive

1	and most develop hypertension. If diffuse proliferative glomerulonephritis (DPGN) is inadequately treated, virtually all patients develop ESRD within 2 years of diagnosis. Therefore, aggressive immunosuppressionisindicated(usuallysystemicglucocorticoids plus a cytotoxic drug), unless damage is irreversible (Fig. 378-2, Table 378-5). African Americans are more likely to develop ESRD than are whites, even with the most current therapies. Overall in the United States, ~20% ofindividualswithlupusDPGNdieordevelop ESRD within 10 years of diagnosis. Such individuals require aggressive control of SLE and of the complications of renal disease and of therapy. Approximately 20% of SLE patients with proteinuria (usually nephrotic) have membranous glomerular changes without proliferative changes on renal biopsy. Their outcome is better than for those with DPGN, but patients with class V and nephrotic range proteinuria should be treated in the same way as those with classes III or IV proliferative

1	biopsy. Their outcome is better than for those with DPGN, but patients with class V and nephrotic range proteinuria should be treated in the same way as those with classes III or IV proliferative disease. Lupus nephritis tends to be an ongoing disease, with flares requiring re-treatment chronic dermatitis in lupus; lesions are roughly circular with slightly or increased treatment over many years. For most people with lupus raised, scaly hyperpigmented erythematous rims and depigmented, nephritis, accelerated atherosclerosis becomes important after several atrophic centers in which all dermal appendages are permanently years of disease; attention must be given to control of systemic inflamdestroyed. Lesions can be disfiguring, particularly on the face and mation, blood pressure, hyperlipidemia, and hyperglycemia. scalp. Treatment consists primarily of topical or locally injected glucocorticoids and systemic antimalarials. Only 5% of people with NErVOUS SYSTEM MaNIFESTaTIONS DLE have

1	and hyperglycemia. scalp. Treatment consists primarily of topical or locally injected glucocorticoids and systemic antimalarials. Only 5% of people with NErVOUS SYSTEM MaNIFESTaTIONS DLE have SLE (although half have positive ANA); however, among There are many central nervous system (CNS) and peripheral nerindividuals with SLE, as many as 20% have DLE. The most common vous system manifestations of SLE; in some patients, these are the acute SLE rash is a photosensitive, slightly raised erythema, occasion-major cause of morbidity and mortality. It is useful to approach ally scaly, on the face (particularly the cheeks and nose—the “butter-this diagnostically by asking first whether the symptoms result from fly” rash), ears, chin, V region of the neck and chest, upper back, and SLE or another condition (such as infection in immunosuppressed

1	Manifestation Prevalence, % aNumbers indicate percentage of patients who have the manifestation at some time during the course of illness. Abbreviations: ARDS, acute respiratory distress syndrome; TIA, transient ischemic attack.

1	individuals or side effects of therapies). If symptoms are related to 2129 SLE, it should be determined whether they are caused by a diffuse process (requiring immunosuppression) or vascular occlusive disease (requiring anticoagulation). The most common manifestation of diffuse CNS lupus is cognitive dysfunction, including difficulties with memory and reasoning. Headaches are also common. When excruciating, they often indicate SLE flare; when milder, they are difficult to distinguish from migraine or tension headaches. Seizures of any type may be caused by lupus; treatment often requires both antiseizure and immunosuppressive therapies. Psychosis can be the dominant manifestation of SLE; it must be distinguished from glucocorticoid-induced psychosis. The latter usually occurs in the first weeks of glucocorticoid therapy, at daily doses of ≥40 mg of prednisone or equivalent; psychosis resolves over several days after glucocorticoids are decreased or stopped. Myelopathy is not rare and

1	weeks of glucocorticoid therapy, at daily doses of ≥40 mg of prednisone or equivalent; psychosis resolves over several days after glucocorticoids are decreased or stopped. Myelopathy is not rare and is often disabling; rapid initiation ofimmunosuppressivetherapystartingwithhigh-doseglucocorticoids is standard of care.

1	The prevalence of transient ischemic attacks, strokes, and myocardial infarctions is increased in patients with SLE. These vascular events are increased in, but not exclusive to, SLE patients with antibodies to phospholipids (antiphospholipid antibodies), which are associated with hypercoagulability and acute thrombotic events (Chap. 379). Chronic SLE with or without antiphospholipid antibodies is associated withacceleratedatherosclerosis.Ischemiainthebraincanbecausedby focal occlusion (either noninflammatory or associated with vasculitis) or by embolization from carotid artery plaque or from fibrinous vegetations of Libman-Sacks endocarditis. Appropriate tests for antiphospholipid antibodies (see below) and for sources of emboli should be ordered in such patients to estimate the need for, intensity of, and duration of anti-inflammatory and/or anticoagulant therapies. In SLE, myocardial infarctions are primarily manifestations of accelerated atherosclerosis. The increased risk for

1	for, intensity of, and duration of anti-inflammatory and/or anticoagulant therapies. In SLE, myocardial infarctions are primarily manifestations of accelerated atherosclerosis. The increased risk for vascular events is three-to tenfold overall, and is highest in women <49 years old. Characteristics associated with increased risk for atherosclerosis include older age, hypertension, dyslipidemia, dysfunctional proinflammatory high-density lipoproteins, repeated high scores for disease activity, high cumulative or daily doses of glucocorticoids, and high levels of homocysteine. When it is most likely that an event results from clotting, long-term anticoagulation is the therapy of choice. Two processes can occur at once—vasculitis plus bland vascular occlusions—in which case it is appropriate to treat with anticoagulation plus immunosuppression. Statin therapies reduce levels of low-density lipoproteins (LDL) in SLE patients; reduction of cardiac events by statins has been shown in SLE

1	to treat with anticoagulation plus immunosuppression. Statin therapies reduce levels of low-density lipoproteins (LDL) in SLE patients; reduction of cardiac events by statins has been shown in SLE patients with renal transplants but not in other SLE cohorts to date.

1	ThemostcommonpulmonarymanifestationofSLEispleuritiswithor without pleural effusion. This manifestation, when mild, may respond to treatment with nonsteroidal anti-inflammatory drugs (NSAIDs); when more severe, patients require a brief course of glucocorticoid therapy. Pulmonary infiltrates also occur as a manifestation of active SLE and are difficult to distinguish from infection on imaging studies. Life-threatening pulmonary manifestations include interstitial inflammation leading to fibrosis, shrinking lung syndrome, and intraalveolar hemorrhage; all of these probably require early aggressive immunosuppressive therapy as well as supportive care.

1	Pericarditis is the most frequent cardiac manifestation; it usually responds to anti-inflammatory therapy and infrequently leads to tamponade.Moreseriouscardiacmanifestationsare myocarditis andfibrinous endocarditis of Libman-Sacks. The endocardial involvement can lead to valvular insufficiencies, most commonly of the mitral or aortic valves, or to embolic events. It has not been proven that glucocorticoid or other immunosuppressive therapies lead to improvement of lupus myocarditis or endocarditis, but it is usual practice to administer a trial of high-dose steroids along with appropriate supportive therapy for NSAIDs, salicylates (Ecotrina and St. Joseph’s aspirina approved by FDA for use in SLE) Methotrexate (for dermatitis, arthritis) Glucocorticoids, orala (several specific brands are approved by FDA for use in SLE) Methylprednisolone sodium succinate, IVa (FDA approved for lupus nephritis) Rituximab (for patients resistant to above therapies)

1	Methylprednisolone sodium succinate, IVa (FDA approved for lupus nephritis) Rituximab (for patients resistant to above therapies) Doses toward upper limit of recommended range usually required Mid potency for face; mid to high potency for other areas 10–25 mg once a week, PO or SC, with folic acid; decrease dose if CrCl <60 mL/min Prednisone, prednisolone: 0.5–1 mg/ kg per day for severe SLE 0.07–0.3 mg/kg per day or qod for milder disease For severe disease, 1 g IV qd × 3 days Low dose (for whites of northern European backgrounds): 500 mg every 2 weeks for 6 doses, then begin maintenance with MMF or AZA. High dose: 7–25 mg/kg q month × 6; consider mesna administration with dose 1.5–3 mg/kg per day; decrease dose for CrCl <25 mL/min MMF: 2–3 g/d PO for induction therapy, 1–2 g/d for maintenance therapy; max 1 g bid if CrCl <25 mL/min

1	MMF: 2–3 g/d PO for induction therapy, 1–2 g/d for maintenance therapy; max 1 g bid if CrCl <25 mL/min MPA: 360–1080 mg bid; caution if CrCl <25 mL/min 2–3 mg/kg per day PO for induction; 1–2 mg/kg per day for maintenance; decrease frequency of dose if CrCl <50 mL/min 10 mg/kg IV wks 0, 2, and 4, then monthly aIndicates medication is approved for use in SLE by the U.S. Food and Drug Administration. A2R/ACE inhibitors, glucocorticoids, fluconazole, methotrexate, thiazides Acitretin, leflunomide, NSAIDs and salicylates, penicillins, probenecid, sulfonamides, trimethoprim A2R/ACE antagonists, antiarrhythmics class III, cyclosporine, NSAIDs and salicylates, phenothiazines, phenytoins, quinolones, rifampin, risperidone, thiazides, sulfonylureas, warfarin Allopurinol, bone marrow suppressants, colony-stimulating factors, doxorubicin, rituximab, succinylcholine, zidovudine

1	Allopurinol, bone marrow suppressants, colony-stimulating factors, doxorubicin, rituximab, succinylcholine, zidovudine Acyclovir, antacids, azathioprine, bile acid-binding resins, ganciclovir, iron, salts, probenecid, oral contraceptives ACE inhibitors, allopurinol, bone marrow suppressants, interferons, mycophenolate mofetil, rituximab, warfarin, zidovudine IVIg NSAIDs: Higher incidence of aseptic meningitis, elevated liver enzymes, decreased renal function, vasculitis of skin; entire class, especially COX-2specific inhibitors, may increase risk for myocardial infarction Salicylates: ototoxicity, tinnitus Both: GI events and symptoms, allergic reactions, dermatitis, dizziness, acute renal failure, edema, hypertension Atrophy of skin, contact dermatitis, folliculitis, hypopigmentation, infection

1	Both: GI events and symptoms, allergic reactions, dermatitis, dizziness, acute renal failure, edema, hypertension Atrophy of skin, contact dermatitis, folliculitis, hypopigmentation, infection Retinal damage, agranulocytosis, aplastic anemia, ataxia, cardiomyopathy, dizziness, myopathy, ototoxicity, peripheral neuropathy, pigmentation of skin, seizures, thrombocytopenia. Quinacrine usually causes diffuse yellow skin coloration. Acne, menstrual irregularities, high serum levels of testosterone Anemia, bone marrow suppression, leukopenia, thrombocytopenia, hepatotoxicity, nephrotoxicity, infections, neurotoxicity, pulmonary fibrosis, pneumonitis, severe dermatitis, seizures. Infection, VZV infection, hypertension, hyperglycemia, hypokalemia, acne, allergic reactions, anxiety, aseptic necrosis of bone, cushingoid changes, CHF, fragile skin, insomnia, menstrual irregularities, mood swings, osteoporosis, psychosis

1	Infection, VZV infection, bone marrow suppression, leukopenia, anemia, thrombocytopenia, hemorrhagic cystitis (less with IV), carcinoma of the bladder, alopecia, nausea, diarrhea, malaise, malignancy, ovarian and testicular failure. Ovarian failure is probably not a problem with low dose. Infection, leukopenia, anemia, thrombocytopenia, lymphoma, lymphoproliferative disorders, malignancy, alopecia, cough, diarrhea, fever, GI symptoms, headache, hypertension, hypercholesterolemia, hypokalemia, insomnia, peripheral edema, elevated liver enzymes, tremor, rash Infection, VZV infection, bone marrow suppression, leukopenia, anemia, thrombocytopenia, pancreatitis, hepatotoxicity, malignancy, alopecia, fever, flulike illness, GI symptoms Infusion reactions, allergy, infections probable Infection (including PML), infusion reactions, headache, arrhythmias, allergic responses bIndicates the medication has been used with glucocorticoids in the trials showing efficacy.

1	Infection (including PML), infusion reactions, headache, arrhythmias, allergic responses bIndicates the medication has been used with glucocorticoids in the trials showing efficacy. Abbreviations: A2R, angiotensin II receptor; ACE, angiotensin-converting enzyme; CHF, congestive heart failure; CrCl, creatinine clearance; FDA, U.S. Food and Drug Administration; GI, gastrointestinal; IVIg, intravenous immunoglobulin; NSAIDs, nonsteroidal anti-inflammatory drugs; PML, progressive multifocal leukoencephalopathy; SLE, systemic lupus erythematosus; SPF, sun protection factor; VZV, varicella-zoster virus. heartfailure,arrhythmia,orembolicevents.Asdiscussedabove,patients with SLE are at increased risk for myocardial infarction, usually due to accelerated atherosclerosis, which probably results from immune attack, chronic inflammation, and/or chronic oxidative damage to arteries.

1	The most frequent hematologic manifestation of SLE is anemia, usually normochromic normocytic, reflecting chronic illness. Hemolysis can be rapid in onset and severe, requiring high-dose glucocorticoid therapy, which is effective in most patients. Leukopenia is also common and almost always consists of lymphopenia, not granulocytopenia; lymphopenia rarely predisposes to infections and by itself usually does not requiretherapy.Thrombocytopenia maybe a recurring prob-lem.If platelet counts are >40,000/μL and abnormal bleeding is absent, therapy may not be required. High-dose glucocorticoid therapy (e.g., 1 mg/kg per day of prednisone or equivalent) is usually effective for the first few episodes of severe thrombocytopenia. Recurring or prolonged hemolytic anemia or thrombocytopenia, or disease requiring an unacceptably high dose of daily glucocorticoids, should be treated with an additional strategy (see “Management of Systemic Lupus Erythematosus” below).

1	Nausea, sometimes with vomiting, and diarrhea can be manifestations of an SLE flare, as can diffuse abdominal pain probably caused by autoimmune peritonitis and/or intestinal vasculitis. Increases in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are common when SLE is active. These manifestations usually improve promptly during systemic glucocorticoid therapy. Vasculitis involving the intestine may be life-threatening; perforations, ischemia, bleeding, and sepsis are frequent complications. Aggressive immunosuppressive therapy with high-doseglucocorticoidsis recommendedfor short-term control;evidenceofrecurrenceis anindicationforadditionaltherapies.

1	Siccasyndrome(Sjögren’ssyndrome; Chap. 383)andnonspecificconjunctivitis are common in SLE and rarely threaten vision. In contrast, retinal vasculitis and optic neuritis are serious manifestations: blindness can develop over days to weeks. Aggressive immunosuppression is recommended, although there arenocontrolledtrialstoprove effectiveness. Complications of systemic and intraorbital glucocorticoid therapy include cataracts (common) and glaucoma. Laboratory tests serve (1) to establish or rule out the diagnosis; (2) to followthe courseof disease,particularly to suggest that a flare is occurring or organ damage is developing; and (3) to identify adverse effects of therapies.

1	Diagnostically, the most important autoantibodies to detect are ANA because the test is positive in >95% of patients, usually at the onset of symptoms. A few patients develop ANA within 1 year of symptom onset; repeated testing may thus be useful. ANA tests using immunofluorescent methods are more reliable than enzyme-linked immunosorbent assays (ELISAs) and/or bead assays, which have less specificity. ANA-negative lupus exists but is rare in adults and is usually associated with other autoantibodies (anti-Ro or anti-DNA). High-titer IgG antibodies to double-stranded DNA (dsDNA) (but not to single-stranded DNA) are specific for SLE. ELISA and immunofluorescent reactions of sera with the dsDNA in the flagellate Crithidia luciliae have ~60% sensitivity for SLE; identification of high-avidity anti-dsDNA in the Farr assay is not as sensitive but may correlate better with risk for nephritis. Titers of anti-dsDNA vary over time. In some patients, increases in quantities of anti-dsDNA herald

1	anti-dsDNA in the Farr assay is not as sensitive but may correlate better with risk for nephritis. Titers of anti-dsDNA vary over time. In some patients, increases in quantities of anti-dsDNA herald a flare, particularly of nephritis or vasculitis, especially when associated with declining levels of C3 or C4 complement. Antibodies to Sm are also specific for SLE and assist in diagnosis; anti-Sm antibodies do not usually correlate with disease activity or clinical manifestations.

1	Antiphospholipid antibodies are not specific for SLE, but their pres-2131 ence fulfills one classification criterion, and they identify patients at increased risk for venous or arterial clotting, thrombocytopenia, and fetal loss. There are three widely accepted tests that measure different antibodies (anticardiolipin, anti-β2-glycoprotein, and the lupus anticoagulant). ELISA is used for anticardiolipin and anti-β2-glycoprotein (both internationally standardized with good reproducibility); a sensitive phospholipid-based activated prothrombin time such as the dilute Russell venom viper test is used to identify the lupus anticoagulant. The higher the titers of IgG anticardiolipin (>40 IU is considered high), and the greater the number of different antiphospholipid antibodies that are detected, the greater is the risk for a clinical episode of clotting. Quantities of antiphospholipid antibodies may vary markedly over time; repeated testing is justified if clinical manifestations of the

1	the greater is the risk for a clinical episode of clotting. Quantities of antiphospholipid antibodies may vary markedly over time; repeated testing is justified if clinical manifestations of the antiphospholipid syndrome (APS) appear (Chap. 379). To classify a patient as having APS, with or without SLE, by international criteria requires the presence of one or more clotting episodes and/or repeated fetal losses plus at least two positive tests for antiphospholipid anti bodies, at least 12 weeks apart; however, many patients with APS do not meet these stringent criteria, which are intended for inclusion of patients into studies.

1	An additional autoantibody test with predictive value (not used for diagnosis) detects anti-Ro/SS-A, which indicates increased risk for neonatallupus,siccasyndrome,andSCLE.Womenwithchild-bearing potential and SLE should be screened for antiphospholipid antibodies and anti-Ro, because both antibodies have the potential to cause fetal harm. Screening testsforcomplete blood count,platelet count,and urinalysis may detect abnormalities that contribute to the diagnosis and influence management decisions.

1	It is useful to follow tests that indicate the status of organ involvement known to be present during SLE flares. These might include urinalysis for hematuria and proteinuria, hemoglobin levels, platelet counts, and serum levels of creatinine or albumin. There is great interest in identification of additional markers of disease activity. Candidates include levels of anti-DNA and anti-C1q antibodies, several components of complement (C3 is most widely available), activated complement products (including those that bind to the C4d receptor on erythrocytes), IFN-inducible gene expression in peripheral blood cells, serum levels of BLyS (B lymphocyte stimulator, also called BAFF), and urinary levels of TNF-like weak inducer of apoptosis (TWEAK), neutrophil gelatinase-associated lipocalin (NGAL), or monocyte chemotactic protein1(MCP-1).Noneisuniformlyagreeduponasareliableindicator of flare or of response to therapeutic interventions. It is likely that a panelof multiple proteins willbe

1	or monocyte chemotactic protein1(MCP-1).Noneisuniformlyagreeduponasareliableindicator of flare or of response to therapeutic interventions. It is likely that a panelof multiple proteins willbe developed topredictbothimpending flare and response to recently instituted therapies. For now, the physician should determine for each patient whether certain laboratory test changes predict flare. If so, altering therapy in response to these changes may be advisable (30 mg of prednisone daily for 2 weeks has been shown to prevent flares in patients with rising anti-DNA plus falling complement). In addition, given the increased prevalence of atherosclerosis in SLE, it is advisable to follow the recommendations of the National Cholesterol Education Program for testing and treatment, including scoring of SLE as an independent risk factor, similar to diabetes mellitus.

1	There is no cure for SLE, and complete sustained remissions are rare. Therefore, the physician should plan to induce remissions of acute flares and then maintain improvements with strategies that suppress symptoms to an acceptable level and prevent organ damage. Usually patients will endure some adverse effects of medications. Therapeutic choices depend on (1) whether disease manifestations are life-threatening or likelytocause organ damage,justifyingaggressive therapies;(2)whether 2132 manifestations are potentially reversible; and (3) the best approaches to preventingcomplicationsofdiseaseanditstreatments.Therapies,doses, and adverse effects are listed in Table 378-5.

1	Among patients with fatigue, pain, and autoantibodies indicative of SLE, but without major organ involvement, management can be directed to suppression of symptoms. Analgesics and antimalarials are mainstays. NSAIDs are useful analgesics/anti-inflammatories, particularly for arthritis/arthralgias. However, two major issues indicate caution in using NSAIDs. First, SLE patients compared with the general populationareatincreasedriskforNSAID-inducedasepticmeningitis, elevated serum transaminases, hypertension, and renal dysfunction. Second, all NSAIDs, particularly those that inhibit cyclooxygenase-2 specifically,mayincreaseriskformyocardialinfarction.Acetaminophen to control pain may be a good strategy, but NSAIDs are more effective in some patients. The relative hazards of NSAIDs compared with low-dose glucocorticoid therapy have not been established. Antimalarials (hydroxychloroquine, chloroquine, and quinacrine) often reduce dermatitis, arthritis, and fatigue. A randomized,

1	compared with low-dose glucocorticoid therapy have not been established. Antimalarials (hydroxychloroquine, chloroquine, and quinacrine) often reduce dermatitis, arthritis, and fatigue. A randomized, placebo-controlled, prospective trial has shown that withdrawal of hydroxychloroquine results inincreasednumbersofdiseaseflares;hydroxychloroquinealsoreduces accrual of tissue damage, including renal damage, over time. Because of potential retinal toxicity, patients receiving antimalarials should undergo ophthalmologic examinations annually. A placebo-controlled prospective trial suggests that administration of dehydroepiandrosterone may reduce disease activity. If quality of life is inadequate despite these conservative measures, treatment with low doses of systemic glucocorticoids may be necessary. The clinician may also considertreatmentwithbelimumab(anti-BLyS)inthesepatients,although published clinical trials enrolled patients who had failed to respond to conservative therapies. Lupus

1	The clinician may also considertreatmentwithbelimumab(anti-BLyS)inthesepatients,although published clinical trials enrolled patients who had failed to respond to conservative therapies. Lupus dermatitis should be managed with topical sunscreens, antimalarials, topical glucocorticoids, and/or tacrolimus, and if severe or unresponsive, systemic glucocorticoids with or without mycophenolate mofetil.

1	LIFE-THrEaTENING SLE: PrOLIFEraTIVE FOrMS OF LUPUS NEPHrITIS

1	Guidelines for management of lupus nephritis have been published recently by the American College of Rheumatology and the European LeagueAgainstRheumatism(encompassedandreferencedinFig. 378-2 andTable378-5).Themainstayoftreatmentforanyinflammatorylifethreatening or organ-threatening manifestations of SLE is systemic glucocorticoids(0.5–1mg/kgperdayPOor500–1000mgofmethylprednisolone sodium succinate IV daily for 3 days followed by 0.5–1 mg/kg of daily prednisone or equivalent). Evidence that glucocorticoid therapy is life-saving comes from retrospective studies from the predialysis era; survival was significantly better in people with DPGN treated with high-dose daily glucocorticoids (40–60 mg of prednisone daily for 4–6 months) versus lower doses. Currently, high doses are recommended for much shorter periods; recent trials of interventions for severe SLE use 4–6 weeks of 0.5–1 mg/kg per day of prednisone or equivalent. Thereafter, doses are tapered as rapidly as the clinical

1	for much shorter periods; recent trials of interventions for severe SLE use 4–6 weeks of 0.5–1 mg/kg per day of prednisone or equivalent. Thereafter, doses are tapered as rapidly as the clinical situation permits, usually to a maintenance dose ranging from 5 to 10 mg of prednisone or equivalent per day. Most patients with an episode of severe SLE require many years of maintenance therapy with low-dose glucocorticoids, which can be increased to prevent or treat disease flares. Frequent attempts to gradually reduce the glucocorticoid requirement are recommended because virtually everyone develops important adverse effects (Table 378-5). High-quality clinical studies regarding initiating therapy for severe, active SLE with IV pulses of high-dose glucocorticoids are not available. Most recent clinical trials in lupus nephritis have initiated therapy with high-dose IV glucocorticoid pulses (500–1000 mg daily for 3–5 days). This approach must be tempered by safety considerations, such as

1	clinical trials in lupus nephritis have initiated therapy with high-dose IV glucocorticoid pulses (500–1000 mg daily for 3–5 days). This approach must be tempered by safety considerations, such as the presence of conditions adversely affected by glucocorticoids (e.g., infection, hyperglycemia, hypertension, osteoporosis).

1	Cytotoxic/immunosuppressive agents added to glucocorticoids are recommended to treat serious SLE. Almost all prospective controlled trials in SLE involvingcytotoxicagentshavebeen conducted incombinationwithglucocorticoidsinpatientswithlupusnephritis.Therefore, the following recommendations apply to treatment of nephritis. Either cyclophosphamide (an alkylating agent) or mycophenolate mofetil (a relatively lymphocyte-specific inhibitor of inosine monophosphatase andthereforeofpurine synthesis)isan acceptablechoiceforinduction of improvement in severely ill patients; azathioprine (a purine analogue and cycle-specific antimetabolite) may be effective but is slower to influence response and associated with more flares. In patients whose renal biopsies show ISN grade III or IV disease, early treatment with combinations of glucocorticoids and cyclophosphamide reduces progression to ESRD and death. Shorter-term studies with glucocorticoids plus mycophenolate mofetil (prospective randomized

1	treatment with combinations of glucocorticoids and cyclophosphamide reduces progression to ESRD and death. Shorter-term studies with glucocorticoids plus mycophenolate mofetil (prospective randomized trials of 6 months, follow-up studies of 36 months) show that this regimen is similar to cyclophosphamide in achieving improvement. Comparisons are complicated by effects of race, since higher proportions of African Americans (and other non-Asian, nonwhite races) respond to mycophenolate than to cyclophosphamide, whereas similar proportions of whitesandAsiansrespondtoeachdrug.Regardingtoxicity,diarrheais more common with mycophenolate mofetil; amenorrhea, leukopenia, and nausea are more common with cyclophosphamide. Importantly, rates of severe infections and death are similar in meta-analyses. Two different regimens of IV cyclophosphamide are available. For white patients with northern European backgrounds, low doses of cyclophosphamide (500 mg every 2 weeks for six total doses, followed

1	Two different regimens of IV cyclophosphamide are available. For white patients with northern European backgrounds, low doses of cyclophosphamide (500 mg every 2 weeks for six total doses, followed by azathioprine or mycophenolate maintenance) are as effective as standard high doses, with less toxicity. Ten-year follow-up has shown no differences between the high-dose and low-dose groups (death or ESRD in 9–20% of patients in each group). The majority of the European patients were white; it is not clear whether the data apply to U.S. populations. High-dose cyclophosphamide (500–1000 mg/m2 body surface area given monthly IV for 6 months, followed by azathioprine or mycophenolate maintenance) is an acceptable approach for patients with severe nephritis (e.g., multiple cellular crescents and/or fibrinoid necrosis on renal biopsy, or rapidly progressive glomerulonephritis). Cyclophosphamide and mycophenolate responses begin 3–16 weeks after treatment is initiated, whereas glucocorticoid

1	fibrinoid necrosis on renal biopsy, or rapidly progressive glomerulonephritis). Cyclophosphamide and mycophenolate responses begin 3–16 weeks after treatment is initiated, whereas glucocorticoid responses may begin within 24 h.

1	For maintenance therapy, mycophenolate and azathioprine probably are similar in efficacy and toxicity; both are safer than cyclophosphamide. In a recently published multicenter study, mycophenolate was superior to azathioprine in maintaining renal function and survival in patients who responded to induction therapy with either cyclophosphamide or mycophenolate. The incidence of ovarian failure, a common effect of high-dose cyclophosphamide therapy (but probably not of low-dose therapy), can be reduced by treatment with a gonadotropin-releasing hormone agonist (e.g., leuprolide 3.75 mg intramuscularly) prior to each monthly cyclophosphamide dose. Patients with high serum creatinine levels (e.g., ≥265 μmol/L [≥3.0 mg/dL]) many months in duration and high chronicity scores on renal biopsy are not likely to respond to any of these therapies. In general, it may be better to induce improvement in an African-American or Hispanic patient with proliferative glomerulonephritis with

1	biopsy are not likely to respond to any of these therapies. In general, it may be better to induce improvement in an African-American or Hispanic patient with proliferative glomerulonephritis with mycophenolate mofetil (2–3 g daily) rather than cyclophosphamide, with the option to switch if no evidence of response is detectable after 3–6 months of treatment. For whites and Asians, induction with either mycophenolate mofetil or cyclophosphamide is acceptable. Cyclophosphamide may be discontinued when it is clear that a patient is improving. The number of SLE flares is reduced by maintenance therapy with mycophenolate mofetil (1.5–2 g daily) or azathioprine (1–2.5 mg/kg per day). Both cyclophosphamide and mycophenolate mofetil are potentially teratogenic; patients should be off either medication for at least 3 months before attempting to conceive. Azathioprine can be used if necessary to control active SLE in patients who are pregnant. If azathioprine is used either for induction or

1	for at least 3 months before attempting to conceive. Azathioprine can be used if necessary to control active SLE in patients who are pregnant. If azathioprine is used either for induction or maintenance therapy, patients may be prescreened for homozygous deficiency of the TMPT enzyme (which is required to metabolize the 6-mercaptopurine product of azathioprine) because they are at higher risk for bone marrow suppression.

1	Good improvement occurs in ~80% of lupus nephritis patients receiving either cyclophosphamide or mycophenolate at 1–2 years of follow-up. However, in some studies, at least 50% of these individuals have flares of nephritis over the next 5 years, and re-treatment is required; such individuals are more likely to progress to ESRD. Long-term outcome of lupus nephritis to most interventions is better in whites than in African Americans. Methotrexate (a folinic acid antagonist) may have a role in the treatment of arthritis and dermatitis but probably not in nephritis or other life-threatening disease. Small controlled trials (in Asia) of leflunomide, a relatively lymphocyte-specific pyrimidine antagonist licensed for use in rheumatoid arthritis, have suggested it can suppress disease activity in some SLE patients. Cyclosporine and tacrolimus, which inhibit production of IL-2 and T lymphocyte functions, have not been studied in prospective controlled trials in SLE in the United States;

1	in some SLE patients. Cyclosporine and tacrolimus, which inhibit production of IL-2 and T lymphocyte functions, have not been studied in prospective controlled trials in SLE in the United States; several studies in Asia have shown they are effective in lupus nephritis. Because they have potential nephrotoxicity but little bone marrow toxicity, the author uses them for periods of a few months in patients with steroid-resistant cytopenias of SLE or in steroid-resistant patients who have developed bone marrow suppression from standard cytotoxic agents.

1	Use of biologics directed against B cells for active SLE is under intense study. Use of anti-CD20 (rituximab), particularly in patients with SLE who are resistant to the more standard combination therapies discussed above, is controversial. Several open trials have shown efficacy in a majority of such patients, both for nephritis and for extrarenal lupus. However, recent prospective placebo-controlled randomized trials, one in renal and one in nonrenal SLE, did not show a difference between anti-CD20 and placebo when added to standard combination therapies. In contrast, recent trials of standard therapy plus belimumab (anti-BLyS, which binds soluble BLyS/BAFF, which is required for maturation of naïve and transitional B cells to plasma cellsandmemory Bcells) showed improvement in51%ofSLEpatients compared to 36% of those on placebo; these differences were statistically significant. The U.S. Food and Drug Administration (FDA) has approved belimumab for treatment of seropositive patients

1	compared to 36% of those on placebo; these differences were statistically significant. The U.S. Food and Drug Administration (FDA) has approved belimumab for treatment of seropositive patients with SLE who have failed standard treatments. The belimumab trial did not includepatientswithactivenephritisorCNSdisease.Posthocanalyses have shown that the SLE patient most likely to respond to belimumab has fairly robust clinical activity (a Systemic Lupus Erythematosus Disease Activity Index [SLEDAI] score of ≥10), positive anti-DNA, and low serum complement. SLEDAI is a widely used measure of SLE diseaseactivity;scores>3reflectclinicallyactivedisease.Atthistime,it is useful to add belimumab to the therapeutic armamentarium in SLE, and it is clear that some patients benefit. However, its role in management of lupus nephritis is not yet known.

1	SPECIaL CONDITIONS IN SLE THaT MaY rEQUIrE aDDITIONaL Or DIFFErENT THEraPIES Crescentic Lupus Nephritis The presence of cellular or fibrotic crescents in glomeruli with proliferative glomerulonephritis indicates a worse prognosis than in patients without this feature. There are no large prospective multinational controlled trials showing efficacy of cyclophosphamide, mycophenolate, cyclosporine, or tacrolimus in such cases. Most authorities currently recommend that high-dose cyclophosphamide is the induction therapy of choice, in addition to high-dose glucocorticoids. One prospective trial from China showed superiority of mycophenolate to cyclophosphamide.

1	Membranous Lupus Nephritis Most SLE patients with membranous (INS-V) nephritis also have proliferativechanges and should be treated for proliferative disease. However, some have pure membranous changes. Treatment for this group is less well defined. Some authorities do not recommend immunosuppression unless proteinuria is in the nephrotic range (although treatment with angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers is recommended). In those patients, recent prospective controlled trials suggest that alternate-day glucocorticoids plus cyclophosphamide or mycophenolate mofetil or cyclosporine are all effective in the majority of patients in reducing proteinuria. It is more controversial whether they 2133 preserve renal function over the long term.

1	Pregnancy and Lupus Fertility rates for men and women with SLE are probably normal. However, rate of fetal loss is increased (approximately two-to threefold) in women with SLE. Fetal demise is higher in mothers with high disease activity, antiphospholipid antibodies, and/or active nephritis. Suppression of disease activity can be achieved by administration of systemic glucocorticoids. A placental enzyme, 11-β-dehydrogenase 2, deactivates glucocorticoids; it is more effective in deactivating prednisone and prednisolone than the fluorinated glucocorticoids dexamethasone and betamethasone. Glucocorticoids are listed by the FDA as pregnancy category A (no evidence of teratogenicity in human studies); cyclosporine, tacrolimus, and rituximab are listed as category C (may be teratogenic in animals but no good evidence in humans); azathioprine, hydroxychloroquine, mycophenolate mofetil, and cyclophosphamide are category D (there is evidence of teratogenicity in humans, but benefits might

1	but no good evidence in humans); azathioprine, hydroxychloroquine, mycophenolate mofetil, and cyclophosphamide are category D (there is evidence of teratogenicity in humans, but benefits might outweigh risks in certain situations); and methotrexate is category X (risks outweigh benefits).

1	Therefore, active SLE in pregnant women should be controlled with hydroxychloroquine and, if necessary, prednisone/prednisolone at the lowest effective doses for the shortest time required. Azathioprine may be added if these treatments do not suppress disease activity. Adverse effects of prenatal glucocorticoid exposure (primarily betamethasone) on offspring may include low birth weight, developmental abnormalities in the CNS, and predilection toward adult metabolic syndrome. It is likely that each of these glucocorticoids and immunosuppressive medications gets into breast milk, at least in low levels; patients should consider not breastfeeding if they need therapy for SLE. In SLE patients with antiphospholipid antibodies (on at least two occasions) and prior fetal losses, treatment with heparin (usually low-molecular-weight) plus low-dose aspirin has been shown in prospective controlled trials to increase significantly the proportion of live births; however, a recent

1	treatment with heparin (usually low-molecular-weight) plus low-dose aspirin has been shown in prospective controlled trials to increase significantly the proportion of live births; however, a recent prospectivetrialshowednodifferencesinfetaloutcomesinwomentakingaspirincomparedtothosetakingaspirinpluslow-molecular-weight heparin.Anadditionalpotential problemfor the fetusis the presenceof antibodies to Ro, sometimes associated with neonatal lupus consisting of rash and congenital heart block with or without cardiomyopathy. The cardiac manifestations can be life-threatening; therefore the presence of anti-Ro requires vigilant monitoring of fetal heart rates with prompt intervention (delivery if possible) if distress occurs. Recent evidence shows that hydroxychloroquine treatment of an anti-Ro-positive mother whose infant develops congenital heart block significantly reduces the chance that subsequent fetuses will develop heart block. There is some evidence that dexamethasone treatment of

1	mother whose infant develops congenital heart block significantly reduces the chance that subsequent fetuses will develop heart block. There is some evidence that dexamethasone treatment of a mother in whom first-or second-degree heart block is detected in utero may sometimes prevent progression of heart block. Women with SLE usuallytolerate pregnancy without disease flares. However, a small proportion develops severe flares requiring aggressive glucocorticoid therapy or early delivery. Poor maternal outcomes are highest in women with active nephritisor irreversibleorgandamagein kidneys, brain,orheart.

1	Lupus and antiphospholipid Syndrome (aPS) Patients with SLE who have venous or arterial clotting and/or repeated fetal losses and at least two positive tests for antiphospholipid antibodies have APS and should be managed with long-term anticoagulation (Chap. 379). A target international normalized ratio (INR) of 2.0–2.5 is recommended for patients with one episode of venous clotting; an INR of 3.0–3.5 is recommended for patients with recurring clots or arterial clotting, particularly in the CNS. Recommendations are based on both retrospective and prospective studies of posttreatment clotting events and adverse effects from anticoagulation.

1	Microvascular Thrombotic Crisis (Thrombotic Thrombocytopenic Purpura, Hemolytic-Uremic Syndrome) This syndrome of hemolysis, thrombocytopenia, and microvascular thrombosis in kidneys, brain, and other tissues carries a high mortality rate and occurs most commonly in young individuals with lupus nephritis. The most useful laboratory tests are identification of schistocytes on peripheral blood smears, elevated serum levels of lactate dehydrogenase, and antibodies to 2134 ADAMS13. Plasma exchange or extensive plasmapheresis is usually life-saving; most authorities recommend concomitant glucocorticoid therapy; there is no evidence that cytotoxic drugs are effective.

1	Lupus Dermatitis Patients with any form of lupus dermatitis should minimize exposure to ultraviolet light, using appropriate clothing and sunscreens with a sun protection factor of at least 30. Topical glucocorticoids and antimalarials (such as hydroxychloroquine) are effective in reducing lesion severity in most patients and are relatively safe. Systemictreatmentwithretinoicacidisausefulstrategyinpatientswith inadequate improvement on topical glucocorticoids and antimalarials; adverse effects are potentially severe (particularly fetal abnormalities), and there are stringent reporting requirements for its use in the United States. Extensive, pruritic, bullous, or ulcerating dermatitides usually improve promptly after institution of systemic glucocorticoids; tapering may be accompanied by flare of lesions, thus necessitating use of a second medication such as hydroxychloroquine, retinoids, or cytotoxic medications such as methotrexate, azathioprine, or mycophenolate mofetil. In

1	by flare of lesions, thus necessitating use of a second medication such as hydroxychloroquine, retinoids, or cytotoxic medications such as methotrexate, azathioprine, or mycophenolate mofetil. In therapy-resistant lupus dermatitis there are reports of success with topical tacrolimus (caution must be exerted because of the possible increased risk for malignancies) or with systemic dapsone or thalidomide (the extreme danger of fetal deformities from thalidomide requires permission from and supervision by the supplier).

1	Prevention of complications of SLE and its therapy include providing appropriatevaccinations(theadministrationofinfluenzaandpneumococcal vaccines has been studied in patients with SLE; flare rates are similar to those receiving placebo) and suppressing recurrent urinary tract infections. Vaccination with attenuated live viruses is generally discouragedin patients who are immunosuppressed.Strategiesto prevent osteoporosis should be initiated in most patients likely to require long-term glucocorticoid therapy and/or with other predisposing factors. Postmenopausal women can be protected from steroid-induced osteoporosis with either bisphosphonates or denosumab. Safety of long-term use of these strategies in premenopausal women is not well established. Control of hypertension and appropriate prevention strategies for atherosclerosis, including monitoring and treatment of dyslipidemias, management of hyperglycemia, and management of obesity, are recommended.

1	Studies of highly targeted experimental therapies for SLE are in progress. They include targeting (1) activated B lymphocytes with anti-CD22 or TACI-Ig, (2) inhibition of IFN-α, (3) inhibition of B/T cell second signal coactivation with CTLA-Ig, (4) inhibition of innate immune activation via TLR7 or TLR7 and 9, (5) induction of regulatory T cells with peptides from immunoglobulins or autoantigens; (6) suppression of T cells, B cells, and monocyte/macrophages with laquinimod; and (7) inhibition of lymphocyte activation by blockade of Jak/Stat. A few studies have used vigorous untargeted immunosuppression with high-dose cyclophosphamide plus anti-T cell strategies, with rescue by transplantation of autologous hematopoietic stem cells forthetreatmentofsevereandrefractory SLE. OneU.S.report showed an estimated mortality rate over 5 years of 15% and sustained remission in 50%. It is hoped that in the next edition of this text, we will be able to recommend more effective and less toxic

1	showed an estimated mortality rate over 5 years of 15% and sustained remission in 50%. It is hoped that in the next edition of this text, we will be able to recommend more effective and less toxic approaches to treatment of SLE based on some of these strategies.

1	PaTIENT OUTCOMES, PrOGNOSIS, aND SUrVIVaL

1	Survival in patients with SLE in the United States, Canada, Europe, and China is approximately 95% at 5 years, 90% at 10 years, and 78% at 20 years. In the United States, African Americans and Hispanic Americans with a mestizo heritage have a worse prognosis than whites, whereas Africans in Africa and Hispanic Americans with a Puerto Rican origin do not. The relative importance of gene mixtures and environmental differences accounting for ethnic differences is not known. Poor prognosis (~50% mortality in 10 years) in most series is associated with (at the time of diagnosis) high serum creatinine levels (>124 μmol/L [>1.4 mg/dL]), hypertension, nephrotic syndrome (24-h urine protein excretion >2.6 g), anemia (hemoglobin <124 g/L [<12.4 g/dL]), hypoalbuminemia, hypocomplementemia, antiphospholipid antibodies, male sex, ethnicity (African American, Hispanic with mestizo heritage), and low socioeconomic status. Data regarding outcomes in SLE patients with renal transplants show mixed

1	antibodies, male sex, ethnicity (African American, Hispanic with mestizo heritage), and low socioeconomic status. Data regarding outcomes in SLE patients with renal transplants show mixed results: some series show a twofold increase in graft rejection compared to patients with other causes of ESRD, whereas others show no differences. Overall patient survival is comparable (85% at 2 years). Lupus nephritis occurs in approximately 10% of transplanted kidneys. Disability in patients with SLE is common due primarily to chronic fatigue, arthritis, and pain, as well as renal disease. As many as 25% of patients may experience remissions, sometimes for a few years, but these are rarely permanent. The leading causes of death in the first decade of disease are systemic disease activity, renal failure, and infections; subsequently, thromboembolic events become increasingly frequent causes of mortality.

1	This is a syndrome of positive ANA associated with symptoms such as fever, malaise, arthritis or intense arthralgias/myalgias, serositis, and/ or rash. The syndrome appears during therapy with certain medications and biologic agents, is predominant in whites, has less female predilection than SLE, rarely involves kidneys or brain, is rarely associated with anti-dsDNA, is commonly associated with antibodies to histones, and usually resolves over several weeks after discontinuation of the offending medication. The list of substances that can induce lupus-like disease is long. Among the most frequent are the antiarrhythmics procainamide, disopyramide, and propafenone; the anti-hypertensive hydralazine; several angiotensin-converting enzyme inhibitors and beta blockers; the antithyroid propylthiouracil; the antipsychotics chlorpromazine and lithium; the anticonvulsants carbamazepine and phenytoin; the antibiotics isoniazid, minocycline, and nitrofurantoin (Macrodantin); the antirheumatic

1	the antipsychotics chlorpromazine and lithium; the anticonvulsants carbamazepine and phenytoin; the antibiotics isoniazid, minocycline, and nitrofurantoin (Macrodantin); the antirheumatic sulfasalazine; the diuretic hydrochlorothiazide; the antihyperlipidemics lovastatin and simvastatin; and IFNs and TNF inhibitors. ANA usually appears before symptoms; however, many of the medications mentioned above induce ANA in patients who never develop symptoms of drug-induced lupus. It is appropriate to test for ANA at the first hint of relevant symptoms and to use test results to help decide whether to withdraw the suspect agent.

1	379 Haralampos M. Moutsopoulos, Panayiotis G. Vlachoyiannopoulos

1	Antiphospholipid syndrome (APS) is an autoantibody-mediated acquired thrombophilia characterized by recurrent arterial or venous thrombosis and/or pregnancy morbidity. The major autoantibodies detected in the patient’s sera are directed against phospholipid (PL)binding plasma proteins, mainly against a 43-kDa plasma apolipoproteinknownasβ2glycoproteinI(β2GPI)andprothrombin.Theplasma concentrationofβ2GPI is 50–200 μg/mL. β2GPI consists of326 amino acids arranged in five domains (I through V). Domain V forms a positively charged patch, suitable to interact with negatively charged PL. In plasma, β2GPI has a circular conformation with domain V binding to andconcealingthe Bcellepitopes lying on domainI. Another groupof antibodies termed lupus anticoagulant (LA) elongate clotting times in vitro; this elongation is not corrected by adding normal plasma to the detection system (Table 379-1). Patients with APS often possess antibodies recognizing Treponema pallidum PL/cholesterol complexes,

1	this elongation is not corrected by adding normal plasma to the detection system (Table 379-1). Patients with APS often possess antibodies recognizing Treponema pallidum PL/cholesterol complexes, which are detected as biologic false-positive serologic tests for syphilis

1	Lupus anticoagulant Activated partial thromboplastin time (aPTT) Antibodies recognize β2GPI or prothrombin (PT) and elongate aPTT, implying that they (LA) interfere with the generation of thrombin by prothrombin. Prolongation of the clotting Kaolin clotting time (KCT) times is an in vitro phenomenon, and LA induces thromboses in vivo. Abbreviations: APL, antiphospholipid syndrome; β2GPI, β2 glycoprotein I; PL, phospholipid. (BFP-STS) and Venereal Disease Research Laboratory (VDRL) tests. APS may occur alone (primary) or in association with any other autoimmune disease (secondary). Catastrophic APS (CAPS) is defined as a rapidly progressive thromboembolic disease involving simultaneously three or more organs, organ systems, or tissues leading to corresponding functional defects.

1	Anti-PL (aPL)-binding plasma protein antibodies occur in 1–5% of the general population. Their prevalence increases with age; however, it is questionable whether they induce thrombotic events in elderly individuals. One-third of patients with systemic lupus erythematosus (SLE) (Chap. 378) possess these antibodies, whereas their prevalence in other autoimmune connective tissue disorders, such as systemic sclerosis (scleroderma), Sjögren’s syndrome, dermatomyositis, rheumatoid arthritis, and early undifferentiated connective tissue disease, ranges from 6 to 15%. One-third of aPL-positive individuals experience thrombotic events or pregnancy morbidity.

1	The trigger for the induction of antibodies to PL-binding proteins is not known. However infections, oxidative stress, major physical stresses such as surgery, and discontinuation of anticoagulant treatment may induce the exacerbation of the disease. Experimental data have shown that these phenomena are induced via (1) conformational changes of β2GPI either complexed with microbial antigens or dimerization through interaction with endothelial cell surface receptor annexin 2/TLR4, the platelet receptors apolipoprotein E receptor 2′ (apoER2′) and/or GPIb/IX/V receptor, and/or the chemokine platelet factor 4 (PF4); or (2) impaired defensive mechanisms such as reduced generation of endothelial nitric oxide synthase. Adherence of β2GPI to apoER2′, GPIb/IX/V receptor, and/or PF4 induces activation of endothelial cells, platelets, and monocytes. This process activates downstream pathways such as p38 mitogen-activated protein (p38 MAP) kinase and nuclear factor (NF)-κB, leading to the

1	activation of endothelial cells, platelets, and monocytes. This process activates downstream pathways such as p38 mitogen-activated protein (p38 MAP) kinase and nuclear factor (NF)-κB, leading to the following events: secretion of proinflammatory cytokines, such as interleukin (IL) 1, IL-6, and IL-8; the expression of adhesion molecules; inhibition of cell-surface plasminogen activation; and expression of tissue factor. The above events change the phenotype of these cells to a prothrombotic form. In addition, anti-β2GPI antibodies induce fetal injury in mice through complement activation, as shown by the evidence that C4-deficient mice were protected from fetal injury.

1	Clinical manifestations represent mainly a direct or indirect expression of venous or arterial thrombosis and/or pregnancy morbidity (Table 379-2). Clinical features associated with venous thrombosis are superficial and deep vein thrombosis, cerebral venous thrombosis, signs and symptoms of intracranial hypertension, retinal vein thrombosis, pulmonary emboli, pulmonary arterial hypertension, and Budd-Chiari syndrome. Livedo reticularis consists of a mottled reticular vascular pattern that appears as a lace-like, purplish discoloration of the skin. It is probably caused by swelling of the venules owing to obstruction of capillaries by thrombi. This clinical manifestation correlates with vascular lesions such as those in the central nervous system as well as aseptic bone necrosis. Arterial thrombosis is manifested as migraines, cognitive dysfunction, transient ischemic attacks, stroke, myocardial infarction, arterial thrombosis of upper and lower thrombosis Leg ulcers and/or digital

1	thrombosis is manifested as migraines, cognitive dysfunction, transient ischemic attacks, stroke, myocardial infarction, arterial thrombosis of upper and lower thrombosis Leg ulcers and/or digital gangrene 9 Arterial thrombosis in the extremities 7 Retinal artery thrombosis/amaurosis fugax 7 Ischemia of visceral organs or avascular necrosis of bone 6 Multi-infarct dementia 3

1	Neurologic Manifestations of Uncertain Etiology Migraine 20 Epilepsy 7 Chorea 1 Cerebellar ataxia 1 Transverse myelopathy 0.5 Renal Manifestations Due to Various Reasons (Renal Artery/Renal Vein/Glomerular Thrombosis, Fibrous Intima Hyperplasia) 3 39 Arthritis 27 Obstetric Manifestations (Referred to the Number of Pregnancies) Preeclampsia 10 Eclampsia

1	Fetal Manifestations (Referred to the Number of Pregnancies) 2136 extremities, ischemic leg ulcers, digital gangrene, avascular necrosis of bone, retinal artery occlusion leading to painless transient vision loss, renal artery stenosis, and glomerular lesions, as well as infarcts of spleen, pancreas, and adrenals. Libman-Sacks endocarditis consists of very small vegetations, histologically characterized by organized platelet-fibrin microthrombi surrounded by growing fibroblasts and macrophages. Glomerular lesions are manifested with hypertension, mildly elevated serum creatinine levels, proteinuria, and mild hematuria. Histologically, these lesions are characterized in an acute phase by thrombotic microangiopathy involving glomerularcapillaries, and in a chronic phasewith fibrousintima hyperplasia, fibrous and/or fibrocellular occlusions of arterioles, and focal cortical atrophy (Table 379-2). PrematureatherosclerosishasbeenrecognizedasararefeatureofAPS. Coombs-positive hemolytic

1	hyperplasia, fibrous and/or fibrocellular occlusions of arterioles, and focal cortical atrophy (Table 379-2). PrematureatherosclerosishasbeenrecognizedasararefeatureofAPS. Coombs-positive hemolytic anemia and thrombocytopenia are laboratory findings associated with APS. Discontinuation of therapy, major surgery, infection, and trauma may trigger CAPS.

1	ThediagnosisofAPSshouldbeseriouslyconsideredincasesofthrombosis, cerebral vascular accidents in individuals younger than 55 years of age, or pregnancy morbidity in the presence of livedo reticularis or thrombocytopenia. In these cases, aPL antibodies should be measured. The presence of at least one clinical and one laboratory criterion ensures the diagnosis even in the presence of other causes of thrombophilia. Clinical criteria include: (1) vascular thrombosis defined as one ormoreclinicalepisodesofarterial,venous,orsmallvesselthrombosis in any tissue or organ; and (2) pregnancy morbidity, defined as (a) one or more unexplained deaths of a morphologically normal fetus at or beyond the tenth week of gestation; (b) one or more premature births of a morphologically normal neonate before the thirty-fourth week of gestation because of eclampsia, severe preeclampsia, or placental insufficiency; or (c) three or more unexplained consecutive spontaneous abortions before the tenth week of

1	the thirty-fourth week of gestation because of eclampsia, severe preeclampsia, or placental insufficiency; or (c) three or more unexplained consecutive spontaneous abortions before the tenth week of gestation. Laboratory criteria include (1) LA, (2) anticardiolipin (aCL), and/or (3) anti-β2GPI antibodies, at intermediate or high titers on two occasions, 12 weeks apart.

1	Differential diagnosis is based on the exclusion of other inherited or acquiredcausesofthrombophilia (Chap. 141),Coombs-positivehemolytic anemia (Chap. 129), and thrombocytopenia (Chap. 140). Livedo reticularis with or without a painful ulceration on the lower extremities also may be a manifestation of disorders affecting (1) the vascular wall, such as polyarteritis nodosa, SLE, cryoglobulinemia, and lymphomas; or (2) the vascular lumen, such as myeloproliferative disorders, atherosclerosis, hypercholesterolemia, or other causes of thrombophilia.

1	After the first thrombotic event, APS patients should be placed on warfarin for life, aiming to achieve an international normalized ratio (INR) ranging from 2.5 to 3.5, alone or in combination with 80 mg of aspirin daily. Pregnancy morbidity is prevented by a combination of heparin with aspirin 80 mg daily. IV immunoglobulin (IVIg) 400 mg/ kg every day for 5 days may also prevent abortions, whereas glucocorticoids are ineffective. Patients with aPL in the absence of any clinical event who are simultaneously positive for aCL, anti-β2GPI, and LA or have SLE are at risk to develop thrombotic events and can be protected by aspirin 80 mg daily.

1	Some patients with APS and patients with CAPS have recurrent thrombotic events despite appropriate anticoagulation. In these cases, IVIg 400 mg/kg every day for 5 days may be of benefit. Patients with CAPS, who are treated in the intensive care unit, are unable to receive warfarin; in this situation, therapeutic doses of low-molecular-weight heparin should be administered. In cases of heparin-induced thrombocytopenia and thrombosis syndrome, inhibitors of phospholipid-bound activated factor X (FXa), such as fondaparinux 7.5 mg SC daily or rivaroxaban 10 mg PO daily, are effective. The above drugs are administered by fixed doses and do not require close monitoring; their safety during the first trimester of pregnancy has not been clearly established. Ankoor Shah, E. William St. Clair

1	Ankoor Shah, E. William St. Clair Rheumatoid arthritis (RA) is a chronic inflammatory disease of unknown etiology marked by a symmetric, peripheral polyarthritis. It is the most common form of chronic inflammatory arthritis and often results in joint damage and physical disability. Because it is a systemic disease, RA may result in a variety of extraarticular manifestations, including fatigue, subcutaneous nodules, lung involvement, pericarditis, peripheral neuropathy, vasculitis, and hematologic abnormalities.

1	Insights gained by a wealth of basic and clinical research over the past two decades have revolutionized the contemporary paradigms for the diagnosis and management of RA. Serum antibodies to cyclic citrullinated peptides (anti-CCPs) are routinely used along with rheumatoidfactorasabiomarkerofdiagnosticandprognosticsignificance. Advances in imaging modalities have improved our ability to detect jointinflammationanddestructioninRA.ThescienceofRAhastaken a major leap forward with the identification of new disease-related genes and further deciphering of the molecular pathways of disease pathogenesis. The relative importance of these different mechanisms hasbeenhighlighted by the observedbenefitsofthenewclassofhighly targeted biologic and small-molecule therapies. Despite these gains, incomplete understanding of the initiating pathogenic pathways of RA remains a sizable barrier to its cure and prevention.

1	The last two decades have witnessed a remarkable improvement in the outcomes of RA. The historic descriptions of crippling arthritis are currently encounteredmuch less frequently.Much of thisprogress can be traced to the expanded therapeutic armamentarium and the adoption of early treatment intervention. The shift in treatment strategy dictates a new mind-set for primary care practitioners—namely, one that demands early referral of patients with inflammatory arthritis to a rheumatologist for prompt diagnosis and initiation of therapy. Only then will patients achieve their best outcomes.

1	The incidence of RA increases between 25 and 55 years of age, after which it plateaus until the age of 75 and then decreases. The presenting symptoms of RA typically result from inflammation of the joints, tendons, and bursae. Patients often complain of early morning joint stiffness lasting more than 1 h that eases with physical activity. The earliest involved joints are typically the small joints of the hands and feet. The initial pattern of joint involvement may be monoarticular, oligoarticular (≤4 joints), or polyarticular (>5 joints), usually in a symmetric distribution. Some patients with inflammatory arthritis will present with too few affected joints to be classified as having RA— so-calledundifferentiatedinflammatoryarthritis.Thosewithanundifferentiated arthritis who are most likely to be diagnosed later with RA have a higher number of tender and swollen joints, test positive for serum rheumatoid factor (RF) or anti-CCP antibodies, and have higher scores for physical

1	most likely to be diagnosed later with RA have a higher number of tender and swollen joints, test positive for serum rheumatoid factor (RF) or anti-CCP antibodies, and have higher scores for physical disability.

1	Once the disease process of RA is established, the wrists, metacarpophalangeal (MCP), and proximal interphalangeal (PIP) joints stand out as the most frequently involved joints (Fig. 380-1). Distal interphalangeal(DIP)jointinvolvementmayoccurinRA,butitusuallyisa manifestation of coexistent osteoarthritis. Flexor tendon tenosynovitis is a frequent hallmark of RA and leads to decreased range of motion, reduced grip strength, and “trigger” fingers. Progressive destruction of the joints and soft tissues may lead to chronic, irreversible deformities. Ulnar deviation results from subluxation of the MCP joints, with subluxation of the proximal phalanx to the volar side of the hand. Hyperextension of the PIP joint with flexion of the DIP joint (“swanneck deformity”), flexion of the PIP joint with hyperextension of the DIP joint (“boutonnière deformity”), and subluxation of the first MCP jointwithhyperextensionofthefirstinterphalangeal(IP)joint(“Z-line

1	FIGUrE 380-1 Metacarpophalangeal and proximal interphalangeal joint swelling in rheumatoid arthritis. (Courtesy of the American College of Rheumatology Image Bank.) Ocular: Keratoconjunctivitis sicca, episcleritis, scleritis Neurologic: Cervical myelopathy Hematologic: Anemia of

1	Ocular: Keratoconjunctivitis sicca, episcleritis, scleritis Neurologic: Cervical myelopathy Hematologic: Anemia of Oral: Xerostomia, periodontitis chronic disease, neutropenia, splenomegaly, Felty’s syndrome, large granular lymphocyte leukemia, lymphoma deformity”) also may result from damage to the tendons, joint capsule, 2137 and other soft tissues in these small joints. Inflammation about the ulnar styloid and tenosynovitis of the extensor carpi ulnaris may cause subluxation of the distal ulna, resulting in a “piano-key movement” of the ulnar styloid. Although metatarsophalangeal (MTP) joint involvement in the feet is an early feature of disease, chronic inflammation of the ankle and midtarsal regions usually comes later and may lead to pes planovalgus (“flat feet”). Large joints, including the knees and shoulders, are often affected in established disease, although these joints may remain asymptomatic for many years after onset.

1	Atlantoaxial involvement of the cervical spine is clinically noteworthy because of its potential to cause compressive myelopathy and neurologic dysfunction. Neurologic manifestations are rarely a presenting sign or symptom of atlantoaxial disease, but they may evolve over time withprogressive instability of C1 on C2.Theprevalenceof atlantoaxial subluxation has been declining in recent years, and occurs now in less than 10% of patients. Unlike the spondyloarthritides (Chap. 384), RA rarely affects the thoracic and lumbar spine. Radiographic abnormali ties of the temporomandibular joint occur commonly in patients with RA, but they are generally not associated with significant symptoms or functional impairment.

1	Extraarticular manifestations may develop during the clinical course of RA, even prior to the onset of arthritis (Fig. 380-2). Patients most likely to develop extraarticular disease have a history of smoking, have early onset of significant physical disability, and test positive for serum RF. Subcutaneous nodules, secondary Sjögren’s syndrome, pulmonary nodules, and anemia are among the most frequently observed Pulmonary: Pleural effusions, pulmonary nodules, interstitial lung disease, pulmonary vasculitis, organizing pneumonia Cardiac: Pericarditis, ischemic heart disease, myocarditis, cardiomyopathy, arrhythmia, mitral regurgitation Renal: Membranous nephropathy, secondary GI: Vasculitis amyloidosis Skeletal: Osteoporosis Endocrine: Hypoandrogenism Skin: Rheumatoid nodules, purpura, pyoderma gangrenosum FIGUrE 380-2 Extraarticular manifestations of rheumatoid arthritis.

1	Skeletal: Osteoporosis Endocrine: Hypoandrogenism Skin: Rheumatoid nodules, purpura, pyoderma gangrenosum FIGUrE 380-2 Extraarticular manifestations of rheumatoid arthritis. 2138 extraarticular manifestations. Recent studies have shown a decrease in the incidence and severity of at least some extraarticular manifestations, particularly Felty’s syndrome and vasculitis. The most common systemic and extraarticular features of RA are described in more detail in the sections below. These signs and symptoms include weight loss, fever, fatigue, malaise, depression, and in the most severe cases, cachexia; they generally reflect a high degree of inflammation and may even precede the onset of joint symptoms. In general, the presence of a fever of >38.3°C (101°F) at any time during the clinical course should raise suspicion of systemic vasculitis (see below) or infection.

1	Subcutaneous nodules occur in 30–40% of patients and more commonly in those with the highest levels of disease activity, the disease-related shared epitope (see below), a positive test for serum RF, and radiographic evidence of joint erosions. When palpated, the nodules aregenerally firm; nontender; andadherent toperiosteum, tendons, or bursae; developing in areas of the skeleton subject to repeated trauma or irritation such as the forearm, sacral prominences, and Achilles tendon. They may also occur in the lungs, pleura, pericardium, and peritoneum.Nodules are typicallybenign,although they canbe associated with infection, ulceration, and gangrene. Secondary Sjögren’s syndrome (Chap. 383) is defined by the presence of either keratoconjunctivitis sicca (dry eyes) or xerostomia (dry mouth) in association with another connective tissue disease, such as RA. Approximately 10% of patients with RA have secondary Sjögren’s syndrome.

1	Pleuritis, the most common pulmonary manifestation of RA, may produce pleuritic chest pain and dyspnea, as well as a pleural friction rub and effusion. Pleural effusions tend to be exudative with increased numbers of monocytes and neutrophils. Interstitial lung disease (ILD) may also occur in patients with RA and is heralded by symptoms of dry cough and progressive shortness of breath. ILD can be associated with cigarette smoking and is generally found in patients with higher disease activity, although it may be diagnosed in up to 3.5% of patients prior to the onset of joint symptoms. Diagnosis is readily made by high-resolution chest computed tomography (CT) scan. Pulmonary function testing shows a restrictive pattern (e.g., reduced total lung capacity) with a reduced diffusing capacity for carbon monoxide (DLCO). The presence of ILD confers a poor prognosis. The prognosis is not quite as poor as that of idiopathic pulmonary fibrosis (e.g., usual interstitial pneumonitis) because ILD

1	carbon monoxide (DLCO). The presence of ILD confers a poor prognosis. The prognosis is not quite as poor as that of idiopathic pulmonary fibrosis (e.g., usual interstitial pneumonitis) because ILD secondary to RA responds more favorably than idiopathic ILD to immunosuppressive therapy (Chap. 315). Pulmonary nodules may be solitary or multiple. Caplan’s syndrome is a rare subset of pulmonary nodulosis characterized by the development of nodules and pneumoconiosis following silica exposure. Other less common pulmonary findings include respiratory bronchiolitis and bronchiectasis.

1	The most frequent site of cardiac involvement in RA is the pericardium. However, clinical manifestations of pericarditis occur in less than 10% of patients with RA despite the fact that pericardial involvement may be detected in nearly one-half of the these patients by echocardiogram or autopsy studies. Cardiomyopathy, another clinically important manifestation of RA, may result from necrotizing or granulomatous myocarditis, coronary artery disease, or diastolic dysfunction. This involvement too may be subclinical and only identified by echocardiography or cardiac magnetic resonance imaging (MRI). Rarely, the heart muscle may contain rheumatoid nodules or be infiltrated with amyloid. Mitral regurgitation is the most common valvular abnormality in RA, occurring at a higher frequency than the general population.

1	Rheumatoid vasculitis (Chap. 385) typically occurs in patients with long-standing disease, a positive test for serum RF, and hypocomplementemia. The overall incidence has decreased significantly in the last decade to be less than 1% of patients. The cutaneous signs vary and include petechiae, purpura, digital infarcts, gangrene, livedo reticularis, and in severe cases large, painful lower extremity ulcerations. Vasculitic ulcers, which may be difficult to distinguish from those caused by venous insufficiency, may be treated successfully with immunosuppressive agents (requiring cytotoxic treatment in severe cases) as well as skin grafting. Sensorimotor polyneuropathies, such as mononeuritis multiplex, may occur in association with systemic rheumatoid vasculitis.

1	A normochromic, normocytic anemia often develops in patients with RA and is the most common hematologic abnormality. The degree of anemia parallels the degree of inflammation, correlating with the levels of serum C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Platelet counts may also be elevated in RA as an acute-phase reactant. Immune-mediated thrombocytopenia is rare in this disease.

1	Felty’s syndrome is defined by the clinical triad of neutropenia, splenomegaly, and nodular RA and is seen in less than 1% of patients, although its incidence appears to be declining in the face of more aggressive treatment of the joint disease. It typically occurs in the late stages of severe RA and is more common in whites than other racial groups. T cell large granular lymphocyte leukemia (T-LGL) may have a similar clinical presentation and often occurs in association with RA. T-LGL is characterized by a chronic, indolent clonal growth of LGL cells, leading to neutropenia and splenomegaly. As opposed to Felty’s syndrome, T-LGL may develop early in the course of RA. Leukopenia apart from these disorders is uncommon and most often due to drug therapy.

1	Large cohort studies have shown a two-to fourfold increased risk of lymphoma in RA patients compared with the general population. The most common histopathologic type of lymphoma is a diffuse large B cell lymphoma. The risk of developing lymphoma increases if the patient has high levels of disease activity or Felty’s syndrome. In addition to extraarticular manifestations, several conditions associated with RA contribute to disease morbidity and mortality rates. They are worthy of mention because they affect chronic disease management.

1	Cardiovascular Disease The most common cause of death in patients with RA is cardiovascular disease. The incidence of coronary artery disease and carotid atherosclerosis is higher in RA patients than in the general population even when controlling for traditional cardiac risk factors, such as hypertension, obesity, hypercholesterolemia, diabetes, and cigarette smoking. Furthermore, congestive heart failure (including both systolic and diastolic dysfunction) occurs at anapproximately twofold higher rate in RA than in the general population. The presence of elevated serum inflammatory markers appears to confer an increased risk of cardiovascular disease in this population.

1	Osteoporosis Osteoporosis is more common in patients with RA than an age-and sex-matched population, with prevalence rates of 20–30%. The inflammatory milieu of the joint probably spills over into the rest of the body and promotes generalized bone loss by activating osteoclasts.Chronicuseofglucocorticoidsanddisability-relatedimmobility also contributes to osteoporosis. Hip fractures are more likely to occur in patients withRA and are significant predictors of increased disability and mortality rate in this disease.

1	Hypoandrogenism Men and postmenopausal women with RA have lower mean serum testosterone, luteinizing hormone (LH), and dehydroepiandrosterone (DHEA) levels than control populations. It has thus been hypothesized that hypoandrogenism may play a role in the pathogenesis of RA or arise as a consequence of the chronic inflammatory response. It is also important to realize that patients receiving chronicglucocorticoidtherapymaydevelophypoandrogenismowingto inhibition of LH and follicle-stimulating hormone (FSH) secretion from the pituitary gland. Because low testosterone levels may lead to osteoporosis, men with hypoandrogenism should be considered for androgen replacement therapy.

1	RA affects approximately 0.5–1% of the adult population worldwide. There is evidence that the overall incidence of RA has been decreasing in recent decades, whereas the prevalence has remained the same because individuals with RA are living longer. The incidence and prevalence of RA varies based on geographic location, both globally and among certain ethnic groups within a country (Fig. 380-3). For example, the Native American Yakima, Pima, and Chippewa tribes of North America have reported prevalence rates in some studies of nearly 7%. In contrast, many population studies from Africa and Asia show lower prevalence rates for RA in the range of 0.2–0.4%.

1	Like many other autoimmune diseases, RA occurs more commonly infemalesthaninmales,witha2–3:1ratio.Interestingly,studiesofRA from some of the Latin American and African countries show an even greater predominance of disease in females compared to males, with ratios of 6–8:1. Given this preponderance of females, various theories have been proposed to explain the possible role of estrogen in disease pathogenesis. Most of the theories center on the role of estrogens in enhancing the immune response. For example, some experimental studies have shown that estrogen can stimulate production of tumor necrosis factor a (TNF-α), a major cytokine in the pathogenesis of RA. It has been recognized for over 30 years that genetic factors contribute to the occurrence of RA as well as to its severity. The likelihood that a first-degree relative of a patient will share the diagnosis of RA is 2–10 times greater than in the general population. European ancestry: HLA-DRB1:

1	European ancestry: HLA-DRB1: PTPN22: European STAT4: North American TNFAIP3: North American TRAFI/CF: North American CTLA4: European Asian ancestry: HLA-DRB1: *0401 (East Asian) *0405 *0901 (Japanese, Malaysian, Korean) Other: CD40 There remains, however, some uncertainty in the extent to which 2139 geneticsplaysaroleinthecausativemechanismsofRA.Althoughtwin studies imply that genetic factors may explain up to 60% of the occurrence of RA, the more commonly stated estimate falls in the range of 10–25%. The estimate of genetic influence may vary across studies due to gene–environment interactions.

1	The alleles known to confer the greatest risk of RA are located within the major histocompatibility complex (MHC). It has been estimated that one-third of the genetic risk for RA resides within this locus. Most, but probably not all, of this risk is associated with allelic variation in the HLA-DRB1 gene, which encodes the MHC II β-chain molecule. The disease-associated HLA-DRB1 alleles share an amino acid sequence at positions 70–74 in the third hypervariable regions of the HLA-DR β-chain, termed the shared epitope (SE). Carriership of the SE alleles is associated with production of anti-CCP antibodies and worse disease outcomes. Some of these HLA-DRB1 alleles bestow a high risk of disease (*0401), whereas others confer a more moderate risk (*0101, *0404, *1001, and *0901). Additionally, there is regional variation. In Greece, for example, where RA tends to be milder than in western European countries, RA susceptibility has been associated with the *0101 SE allele. By comparison, the

1	there is regional variation. In Greece, for example, where RA tends to be milder than in western European countries, RA susceptibility has been associated with the *0101 SE allele. By comparison, the *0401 or *0404 alleles are found in approximately 50–70% of northern Europeans and are the predominant risk alleles in this group. The most common disease susceptibility SE alleles in Asians, namely the Japanese, Koreans, and Chinese, are *0405 and *0901. Lastly, disease susceptibility of Native American populations such as the Pima and Tlingit Indians, where the prevalence of RA can be as high as 7%, is associated with the SE allele *1042.TheriskofRAconferredbytheseSEallelesislessinAfricanand Hispanic Americans than in individuals of European ancestry.

1	Genome-wide association studies (GWAS) have made possible the identification of several non-MHC-related genes that contribute to RA susceptibility. GWAS are based on the detection of single-nucleotide polymorphisms (SNPs), which allow for examination of the genetic architecture of complex diseases such as RA. There are approximately 10 million common SNPs within a human genome consisting of 3 billion base pairs. As a rule, GWAS identify only common variants, namely, those with a frequency of more than 5% in the general population. Overall, several themes have emerged from GWAS in RA. First, the non-MHC loci identified as risk alleles for RA have only a modest US: 0.7-1.3% Brazil: 0.4-1.4% Jamaica: 1.9-2.2% UK: 0.8-1.1% South Africa: 2.5-3.6% Lesotho: 1.7-4.5% Saudi Arabia: 0.1-0.2% Java: 0.1-0.2% Liberia: 2-3% Norway: 0.4% Spain: 0.2-0.8% Greece: 0.3-1% Bulgaria: 0.2-0.6% Iraq: 0.4-1.5% India: 0.1-0.4% Japan: 0.2-0.3% Hong Kong: 0.1-0.5%

1	FIGUrE 380-3 Global prevalence rates of rheumatoid arthritis (RA) with genetic associations. Listed are the major genetic alleles associated with RA. Although human leukocyte antigen (HLA)-DRB1 mutations are found globally, some alleles have been associated with RA in only certain ethnic groups.

1	2140 effect on risk; they also contribute to the risk for developing other autoimmune diseases, such as type 1 diabetes mellitus, systemic lupus erythematosus, and multiple sclerosis. Second, although most of the non-HLA associations are described in patients with anti-CCP antibody-positive disease, there are several risk loci that are unique to anti-CCP antibody-negative disease. Third, risk alleles vary among ethnic groups. And fourth, the risk loci mostly reside in genes encoding proteins involved in the regulation of the immune response. However, the risk alleles identified by GWAS only account at present for approximately 5% of the genetic risk, suggesting that rare variants or other classes of DNA variants, such as variants in copy number, maybeyetfoundthatsignificantlycontributetotheoverallriskmodel. Recently, imputation of SNP data from a GWAS meta-analysis shows amino acid substitutions in the MHC locus independently associated with the risk forRAareatposition11,

1	Recently, imputation of SNP data from a GWAS meta-analysis shows amino acid substitutions in the MHC locus independently associated with the risk forRAareatposition11, 71,and74inHLA-DRβ1, position 9 of HLA-B, andposition 9of HLA-DPβ1.Theamino acidsat position 11, 71, and 74 are located in the antigen-binding grove of the HLA-DRβ1 molecule, highlighting positions 71 and 74 that form part of the original shared epitope. Among the best examples of the non-MHC genes contributing to the risk of RA is the gene encoding protein tyrosine phosphatase non-receptor 22 (PTPN22). This gene varies in frequency among patients from different parts of Europe (e.g., 3–10%), but is absent in patients of East Asian ancestry. PTPN22 encodes lymphoid tyrosine phosphatase, a protein that regulates T and B cell function. Inheritance of the risk allele for PTPN22 produces a gain-of-function in the protein that is hypothesized to result in the abnormal thymic selection of auto-reactive T and B cells and

1	cell function. Inheritance of the risk allele for PTPN22 produces a gain-of-function in the protein that is hypothesized to result in the abnormal thymic selection of auto-reactive T and B cells and appears to be associated exclusively with anti-CCP-positive disease. The peptidyl arginine deiminase type IV (PADI4) gene is another risk allele that encodes an enzyme involved in the conversion of arginine to citrulline and is postulated to play a role in the development of antibodies to citrullinated antigens. A polymorphism in PADI4 has been associated with RA only in Asian populations. Epigeneticsis the study of heritabletraits thataffect gene expression but do not modify DNA sequence. It may provide a link between environmental exposure and predisposition to disease. The best-studied mechanismsinclude posttranslational histone modifications and DNA methylation. Although studies of epigenetic phenomena are limited, DNA methylation patterns have been shown to differ between RA patients

1	posttranslational histone modifications and DNA methylation. Although studies of epigenetic phenomena are limited, DNA methylation patterns have been shown to differ between RA patients and healthy controls, as well as patients with osteoarthritis.

1	In addition to genetic predisposition, a host of environmental factors have been implicated in the pathogenesis of RA. The most reproducible of these environmental links is cigarette smoking. Numerous cohort and case control studies have demonstrated that smoking confers a relative risk for developing RA of 1.5–3.5. In particular, women who smoke cigarettes have a nearly 2.5 times greater risk of RA, a risk that persistseven 15years after smoking cessation.A twin whosmokes willhavea significantlyhigher risk forRAthanhisor hermonozygotic co-twin, theoretically with the same genetic risk, who does not smoke. Interestingly, the risk from smoking is almost exclusively related to RF and anti-CCP antibody-positive disease. However, it has not been shown that smoking cessation, while having many health benefits, improves disease activity.

1	Researchers began to aggressively seek an infectious etiology for RA after the discovery in 1931 that sera from patients with this disease could agglutinate strains of streptococci. Certain viruses such as Epstein-Barr virus (EBV) have garnered the most interestover the past 30 years given their ubiquity, ability to persist for many years in the host, and frequent association with arthritic complaints. For example, titers of IgG antibodies against EBV antigens in the peripheral blood and saliva are significantly higher in patients with RA than the general population. EBV DNA has also been found in synovial fluid and synovial cells of RA patients. Because the evidence for these links is largely circumstantial, it has not been possible to directly implicate infection as a causative factor in RA.

1	RA affects the synovial tissue and underlying cartilage and bone. The synovial membrane, which covers most articular surfaces, tendon sheaths, and bursae, normally is a thin layer of connective tissue. In joints, it faces the bone and cartilage, bridging the opposing bony surfaces and inserting at periosteal regions close to the articular cartilage. It consists primarily of two cell types—type A synoviocytes (macrophage-derived) and type B synoviocytes (fibroblast-derived). The synovial fibroblasts are the most abundant and produce the structural components of joints, including collagen, fibronectin, and laminin, as well as other extracellular constituents of the synovial matrix. The sublining layer consists of blood vessels and a sparse population of mononuclear cells within a loose networkof connective tissue. Synovial fluid, an ultrafiltrate of blood, diffuses through the subsynovial lining tissue across the synovial membrane and into the joint cavity. Its main constituents are

1	connective tissue. Synovial fluid, an ultrafiltrate of blood, diffuses through the subsynovial lining tissue across the synovial membrane and into the joint cavity. Its main constituents are hyaluronan and lubricin. Hyaluronan is a glycosaminoglycan that contributes to the viscous nature of synovial fluid, which along with lubricin, lubricates the surface of the articular cartilage.

1	The pathologic hallmarks of RA are synovial inflammation and proliferation, focal bone erosions, and thinning of articular cartilage. Chronic inflammation leads to synovial lining hyperplasia and the formation of pannus, a thickened cellular membrane containing fibroblast-like synoviocytes and granulation-reactive fibrovascular tissue that invades the underlying cartilage and bone. The inflammatory infiltrateismadeupofnolessthansixcelltypes:Tcells,Bcells,plasma cells, dendritic cells, mast cells, and, to a lesser extent, granulocytes. The T cells comprise 30–50% of the infiltrate, with the other cells accounting for the remainder. The topographical organization of these cells is complex and may vary among individuals with RA. Most often, the lymphocytes are diffusely organized among the tissue resident cells; however, in some cases, the B cells, T cells, and dendritic cells may form higher levels of organization, such as lymphoid follicles and germinal center–like structures. Growth

1	tissue resident cells; however, in some cases, the B cells, T cells, and dendritic cells may form higher levels of organization, such as lymphoid follicles and germinal center–like structures. Growth factors secreted by synovial fibroblasts and macrophages promote the formation of new blood vessels in the synovial sublining that supply the increasing demands for oxygenation and nutrition required by the infiltrating leukocytes and expanding synovial tissue.

1	The structural damage to the mineralized cartilage and subchondral bone is mediated by the osteoclast. Osteoclasts are multinucleated giant cells that can be identified by their expression of CD68, tartrateresistant acid phosphatase, cathepsin K, and the calcitonin receptor. They appear at the pannus-bone interface where they eventually form resorption lacunae. These lesions typically localize where the synovial membraneinsertsintotheperiostealsurfaceattheedgesofbonesclose to the rim of articular cartilage and at the attachment sitesof ligaments and tendon sheaths. This process most likely explains why bone erosions usually develop at the radial sites of the MCP joints juxtaposed to the insertion sites of the tendons, collateral ligaments, and synovial membrane. Another form of bone loss is periarticular osteopenia that occursin jointswithactive inflammation. It is associatedwithsubstantial thinning of the bony trabeculae along the metaphyses of bones, and likely results from

1	loss is periarticular osteopenia that occursin jointswithactive inflammation. It is associatedwithsubstantial thinning of the bony trabeculae along the metaphyses of bones, and likely results from inflammation of the bone marrow cavity. These lesions can be visualized on MRI scans, where they appear as signal alterations in the bone marrow adjacent to inflamed joints. Their signal characteristics show they are water-rich with a low fat content and are consistent with highly vascularized inflammatory tissue. These bone marrow lesions are often the forerunner of bone erosions.

1	The cortical bone layer that separates the bone marrow from the invading pannus is relatively thin and susceptible to penetration by the inflamed synovium. The bone marrow lesions seen on MRI scans are associated with an endosteal bone response characterized by the accumulation of osteoblasts and deposition of osteoid. Thus, in recent years, the concept of joint pathology in RA has been extended to include the bone marrow cavity. Finally, generalized osteoporosis, which results in the thinning of trabecular bone throughout the body, is a third form of bone loss found in patients with RA.

1	Articular cartilage is an avascular tissue comprised of a specialized matrix of collagens, proteoglycans, and other proteins. It is organized in four distinct regions (superficial, middle, deep, and calcified cartilage zones)—chondrocytes constitute the unique cellular component in these layers. Originally, cartilage was considered to be an inert tissue, but it is now known to be a highly responsive tissue that reacts to inflammatory mediators and mechanical factors, which in turn, alter the balance between cartilage anabolism and catabolism. In RA, the initial areas of cartilage degradation are juxtaposed to the synovial pannus. The cartilage matrix is characterized by a generalized loss of proteoglycan, most evident in the superficial zones adjacent to the synovial fluid. Degradation of cartilage may also take place in the perichondrocytic zone and in regions adjacent to the subchondral bone.

1	The pathogenic mechanisms of synovial inflammation are likely to result from a complex interplay of genetic, environmental, and immunologic factors that produces dysregulation of the immune system and a breakdown in self-tolerance (Fig. 380-4). Precisely what triggers these initiating events and what genetic and environmental factors disrupt the immune system remains a mystery. However, a detailed molecular picture is emerging of the mechanisms underlying the chronic inflammatory response and the destruction of the articular cartilage and bone.

1	In RA, the preclinical stage appears to be characterized by a breakdown in self-tolerance. This idea is supported by the finding that autoantibodies, such as RF and anti-CCP antibodies, may be found in sera from patients many years before clinical disease can be detected. However, the antigenic targets of anti-CCP antibodies and RF are not restricted to the joint, and their role in disease pathogenesis remains speculative.Anti-CCPantibodiesaredirectedagainstdeaminatedpeptides, which result from posttranslational modification by the enzyme PADI4. They recognize citrulline-containing regions of several different matrix proteins, including filaggrin, keratin, fibrinogen, and vimentin, and are present at higher levels in the joint fluid compared to the serum. Other autoantibodies have been found in a minority of patients with RA, but they also occur in the setting of other types of arthritis.They bind toadiversearrayofautoantigens,includingtypeII collagen, human cartilage gp-39, aggrecan,

1	in a minority of patients with RA, but they also occur in the setting of other types of arthritis.They bind toadiversearrayofautoantigens,includingtypeII collagen, human cartilage gp-39, aggrecan, calpastatin, BiP (immunoglobulin binding protein), and glucose-6-phosphate isomerase.

1	In theory, environmental stimulants may synergize with other factors to bring about inflammation in RA. People who smoke display higher citrullination of proteins in bronchoalveolar fluid than those who do not smoke. Thus, it has been speculated that long-term exposure to tobacco smoke might induce citrullination of cellular proteins in the lung and stimulate the expression of a neoepitope capable of inducing self-reactivity, which in turns, leads to formation of immune complexes and joint inflammation. Exposure to silicone dust and mineraloil,whichhasadjuvanteffects,hasalso beenlinkedtoanincreased risk for anti-CCP antibody-positive RA.

1	How might microbes or their products be involved in the initiating events of RA? The immune system is alerted to the presence of microbial infections through Toll-like receptors (TLRs). There are 10 TLRsinhumansthatrecognizeavarietyofmicrobialproducts,including bacterial cell-surface lipopolysaccharides and heat-shock proteins (TLR4), lipoproteins (TLR2), double-strand RNA viruses (TLR3), and unmethylated CpG DNA from bacteria (TLR9). TLR2, -3, and -4 are abundantly expressed by synovial fibroblasts in early RA and, when bound by their ligands, upregulate production of proinflammatory cytokines. Although such events could amplify inflammatory pathways in RA, a specific role for TLRs in disease pathogenesis has not been elucidated.

1	The pathogenesis of RA is built upon the concept that self-reactive T cells drive the chronic inflammatory response. In theory, self-reactive T cells might arise in RA from abnormal central (thymic) selection due to defects in DNA repair leading to an imbalance of T cell death and life, or defects in the cell signaling apparatus lowering the threshold for T cell activation. Similarly, abnormal selection of the T cell repertoire in the periphery might lead to a breakdown in T cell tolerance. The support for these theories comes mainly from studies of arthritis in mouse models. It has not been shown that patients with RA have 2141 abnormal thymic selection of T cells or defective apoptotic pathways regulating cell death. At least some antigen stimulation inside the joint seems likely, owing to the fact that T cells in the synovium express a cell-surface phenotype indicating prior antigen exposure and show evidence of clonal expansion. Of interest, peripheral blood T cells from patients

1	to the fact that T cells in the synovium express a cell-surface phenotype indicating prior antigen exposure and show evidence of clonal expansion. Of interest, peripheral blood T cells from patients with RA have been shown to display a fingerprint of premature aging that mostly affects inexperienced naïve T cells. In these studies, the most glaring findings have been the loss of telomeric sequencesandadecreaseinthethymicoutputofnewTcells.Although intriguing, it is not clear how generalized T cell abnormalities might provoke a systemic disease dominated by synovitis.

1	There is substantial evidence supporting a role for CD4+ T cells in the pathogenesis of RA. First, the co-receptor CD4 on the surface of T cells binds to invariant sites on MHC class II molecules, stabilizing the MHC-peptide–T cell receptor complex during T cell activation. Because the SE on MHCclass IImolecules is arisk factor for RA, itfollows that CD4+ T cell activation may play a role in the pathogenesis of thisdisease.Second,CD4+memoryTcellsareenrichedinthesynovial tissue from patients with RA and can be implicated through “guilt by association.” Third, CD4+ T cells have been shown to be important in the initiation of arthritis in animal models. Fourth, some, but not all, T cell–directed therapies have shown clinical efficacy in this disease. Taken together, these lines of evidence suggest that CD4+ T cells play an important role in orchestrating the chronic inflammatory response in RA. However, other cell types, such as CD8+ T cells, natural killer (NK)cells, andBcellsarepresent

1	that CD4+ T cells play an important role in orchestrating the chronic inflammatory response in RA. However, other cell types, such as CD8+ T cells, natural killer (NK)cells, andBcellsarepresent insynovialtissueandmayalsoinflu ence pathogenic responses.

1	In the rheumatoid joint, by mechanisms of cell-cell contact and release of soluble mediators, activated T cells stimulate macrophages and fibroblast-like synoviocytes to generate proinflammatory mediators and proteases that drive the synovial inflammatory response and destroy the cartilage and bone. CD4+ T cell activation is dependent on two signals: (1) T cell receptor binding to peptide-MHC on antigen-presenting cells; and (2) CD28 binding to CD80/86 on antigen-presenting cells. CD4+ T cells also provide help to B cells, which in turn, produce antibodies that may promote further inflammation in the joint. The previous T cell–centric model for the pathogenesis of RA was based on a TH1-driven paradigm, which came from studies indicating that CD4+ T helper (TH) cells differentiated into TH1 and TH2 subsets, each with their distinctive cytokine profiles. TH1 cells were found to mainly produce interferon γ (IFN-γ), lymphotoxin β, and TNF-α, whereas TH2 cells predominately secreted

1	TH1 and TH2 subsets, each with their distinctive cytokine profiles. TH1 cells were found to mainly produce interferon γ (IFN-γ), lymphotoxin β, and TNF-α, whereas TH2 cells predominately secreted interleukin (IL)-4, IL-5, IL-6, IL-10, and IL-13. The recent discovery of another subset of TH cells, namely the TH17 lineage, has revolutionized our concepts concerning the pathogenesis of RA. In humans, naïve T cells are induced to differentiate into TH17 cells by exposure to transforming growth factor β (TGF-β), IL-1, IL-6, and IL-23. Upon activation, TH17 cells secrete a variety of proinflammatory mediators such as IL-17, IL-21, IL-22, TNF-α, IL-26, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Substantial evidence now exists from both animal models and humans that IL-17 plays an important role not only in promoting joint inflammation, but also in destroying cartilage and subchondral bone

1	The immune system has evolved mechanisms to counterbalance the potential harmful immune-mediated inflammatory responses provoked by infectious agents and other triggers. Among these negative regulators are regulatory T (Treg) cells, which are produced in the thymus and induced in the periphery to suppress immune-mediated inflammation. They are characterized by the surface expression of CD25 and the transcription factor forkhead box P3 (FOXP3) and orchestrate dominant tolerance through contact with other immune cells and secretion of inhibitory cytokines, such as TGF-β, IL-10, and IL-35. T reg cells appear to be heterogeneous and capable of suppressing distinct classes (TH1, TH2, TH17) of the immune response. In RA, the data that Treg numbers are deficient compared to normal healthy controls are contradictory and inconclusive. Although some experimental evidence suggests that Treg suppressive activity is lost due to dysfunctional expression of cytotoxic T lymphocyte antigen 4

1	IFN-˜, TNF-° IL-17A, IL-17F, lymphotoxin-˛ TNF-°, IL-6, GM-CSF +++St CD40 Pre-OB Wnt Dkk-1 IFN-˜, IL17 IL15, GM-CSF, TNF-°RF, Anti-CCP Ab IL-6, IL-8 IL-1, IL-6, IL-18 MMP OPG RANK-L RANK FGF, TGF-˛TH OC OB B M SF Teff

1	FIGUrE 380-4 Pathophysiologic mechanisms of inflammation and joint destruction. Genetic predisposition along with environmental factors may trigger the development of rheumatoid arthritis (RA), with subsequent synovial T cell activation. CD4+ T cells become activated by antigen-presenting cells (APCs) through interactions between the T cell receptor and class II major histocompatibility complex (MHC)-peptide antigen (signal 1) with co-stimulation through the CD28-CD80/86 pathway, as well as other pathways (signal 2). In theory, ligands binding Toll-like receptors (TLRs) may further stimulate activation of APCs inside the joint. Synovial CD4+ T cells differentiate into TH1 and TH17 cells, each with their distinctive cytokine profile. CD4+ TH cells in turn activate B cells, some of which are destined to differentiate into autoantibody-producing plasma cells. Immune complexes, possibly comprised of rheumatoid factors (RFs) and anti–cyclic citrullinated peptides (CCP) antibodies, may form

1	destined to differentiate into autoantibody-producing plasma cells. Immune complexes, possibly comprised of rheumatoid factors (RFs) and anti–cyclic citrullinated peptides (CCP) antibodies, may form inside the joint, activating the complement pathway and amplifying inflammation. T effector cells stimulate synovial macrophages (M) and fibroblasts (SF) to secrete proinflammatory mediators, among which is tumor necrosis factor α (TNF-α). TNF-α upregulates adhesion molecules on endothelial cells, promoting leukocyte influx into the joint. It also stimulates the production of other inflammatory mediators, such as interleukin 1 (IL-1), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). TNF-α has a critically important function in regulating the balance between bone destruction and formation. It upregulates the expression of dickkopf-1 (DKK-1), which can then internalize Wnt receptors on osteoblast precursors. Wnt is a soluble mediator that promotes osteoblastogenesis and

1	and formation. It upregulates the expression of dickkopf-1 (DKK-1), which can then internalize Wnt receptors on osteoblast precursors. Wnt is a soluble mediator that promotes osteoblastogenesis and bone formation. In RA, bone formation is inhibited through the Wnt pathway, presumably due to the action of elevated levels of DKK-1. In addition to inhibiting bone formation, TNF-α stimulates osteoclastogenesis. However, it is not sufficient by itself to induce the differentiation of osteoclast precursors (Pre-OC) into activated osteoclasts capable of eroding bone. Osteoclast differentiation requires the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB (RANK) ligand (RANKL), which binds to RANK on the surface of Pre-OC. Inside the joint, RANKL is mainly derived from stromal cells, synovial fibroblasts, and T cells. Osteoprotegerin (OPG) acts as a decoy receptor for RANKL, thereby inhibiting osteoclastogenesis and bone loss. FGF,

1	RANKL is mainly derived from stromal cells, synovial fibroblasts, and T cells. Osteoprotegerin (OPG) acts as a decoy receptor for RANKL, thereby inhibiting osteoclastogenesis and bone loss. FGF, fibroblast growth factor; IFN, interferon; TGF, transforming growth factor.

1	(CTLA-4), the nature of Treg defects in RA, if they exist, remains unclear. Cytokines, chemokines, antibodies, and endogenous danger signals bind to receptors on the surface of immune cells and stimulate a cascade of intracellular signaling events that can amplify the inflammatory response. Signaling molecules and their binding partners in these pathways are the target of small-molecule drugs designed to interfere with signal transduction and block these reinforcing inflammatory loops. Examples of signal molecules in these critical inflammatory pathways include Janus kinase (JAK)/signal transducers and activators of transcription (STAT), spleen tyrosine kinase (Syk), mitogen-activated protein kinases (MAPKs), and nuclear factor-κB (NF-κB). These pathways exhibit significant cross-talk and are found in many cell types. Some signal transducers, such as JAK3, are primarily expressed in hematopoieticcellsandplayanimportantroleintheinflammatoryresponseinRA.

1	Activated B cells are also important players in the chronic inflammatory response. B cells give rise to plasma cells, which in turn, produce antibodies, including RF and anti-CCP antibodies. RFs may form large immune complexes inside the joint that contribute to the pathogenic process by fixing complement and promoting the release of proinflammatory chemokines and chemoattractants. In mouse models of arthritis, RF-containing immune complexes and anti-CCPcontaining immune complexes synergize with other mechanisms to exacerbate the synovial inflammatory response.

1	RA is often considered to be a macrophage-driven disease because this cell type is the predominant source of proinflammatory cytokines inside the joint. Key proinflammatory cytokines released by synovial macrophagesincludeTNF-α,IL-1,IL-6,IL-12,IL-15,IL-18,andIL-23. Synovial fibroblasts, the other major cell type in this microenvironment, produce the cytokines IL-1 and IL-6 as well as TNF-α. TNF-α is a pivotal cytokine in the pathobiology of synovial inflammation. It upregulates adhesion molecules on endothelial cells, promoting the influx of leukocytes into the synovial microenvironment; activates synovial fibroblasts; stimulates angiogenesis; promotes pain receptor sensitizing pathways; and drives osteoclastogenesis. Fibroblasts secrete matrix metalloproteinases (MMPs) as well as other proteases that are chiefly responsible for the breakdown of articular cartilage.

1	Osteoclast activation at the site of the pannus is closely tied to the presence of focal bone erosion. Receptor activator of nuclear factor-κB ligand (RANKL) is expressed by stromal cells, synovial fibroblasts, and T cells. Upon binding to its receptor RANK on osteoclast progenitors, RANKL stimulates osteoclast differentiation and bone resorption. RANKL activity is regulated by osteoprotegerin (OPG), a decoy receptor of RANKL that blocks osteoclast formation. Monocytic cells in the synovium serve as the precursors of osteoclasts and, when exposed to macrophage colony-stimulating factor (M-CSF) and RANKL, fuse to formpolykaryonstermed preosteoclasts.Theseprecursorcells undergo further differentiation into osteoclasts with the characteristic ruffled membrane. Cytokines such as TNF-α, IL-1, IL-6, and IL-17 increase the expression of RANKL in the joint and thus promote osteoclastogenesis. Osteoclasts also secrete cathepsin K, which is a cysteine protease that degrades the bone matrix by

1	IL-6, and IL-17 increase the expression of RANKL in the joint and thus promote osteoclastogenesis. Osteoclasts also secrete cathepsin K, which is a cysteine protease that degrades the bone matrix by cleaving collagen. Stimulation of osteoclasts also contributes to generalized bone loss and osteoporosis.

1	Increased bone loss is only part of the story in RA, as decreased bone formation plays a crucial role in bone remodeling at sites of inflammation. Recent evidence shows that inflammation suppresses boneformation.TheproinflammatorycytokineTNF-αplaysakeyrole in actively suppressing bone formation by enhancing the expression of dickkopf-1 (DKK-1). DKK-1 is an important inhibitor of the Wnt pathway, which acts to promote osteoblast differentiation and bone formation. The Wnt system is a family of soluble glycoproteins that bind to cell-surface receptors known as frizzled (fz) and low-density lipoprotein (LDL) receptor–related proteins (LRPs) and promote cell growth. In animal models, increased levels of DKK-1 are associated with decreased bone formation, whereas inhibition of DKK-1 protects against structural damage in the joint. Wnt proteins also induce the formation of OPG and thereby shut down bone resorption, emphasizing their key role in tightly regulating the balance between bone

1	against structural damage in the joint. Wnt proteins also induce the formation of OPG and thereby shut down bone resorption, emphasizing their key role in tightly regulating the balance between bone resorption and formation.

1	The clinical diagnosis of RA is largely based on signs and symptoms of a chronic inflammatory arthritis, with laboratory and radiographic results providing important supplemental information. In 2010, a collaborative effort between the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) revised the 1987 ACR classification criteria for RA in an effort to improve early diagnosis with the goal of identifying patients who would benefit from early introduction of disease-modifying therapy (Table 380-1). Application of the newly revised criteria yields a score of 0–10, with a score of ≥ 6 fulfilling the requirements for definite RA. The new classification criteria differ in severalwaysfromtheoldercriteriaset.Thenewcriteriaincludeapositive testforserumanti-CCPantibodies(alsotermedACPA,anti-citrullinated peptide antibodies) as an item, which carries greater specificity for the diagnosis of RA than a positive test for RF. The newer classification criteria

1	peptide antibodies) as an item, which carries greater specificity for the diagnosis of RA than a positive test for RF. The newer classification criteria also do not take into account whether the patient has rheumatoid nodules orradiographic jointdamagebecause these findingsoccurrarely inearlyRA.Itisimportanttoemphasizethatthenew2010ACR-EULAR criteria are “classification criteria” as opposed to “diagnostic criteria” and serve todistinguishpatientsatthe onsetof disease who haveahigh likelihood of evolution to chronic disease with persistent synovitis and joint damage. The presence of radiographic joint erosions or subcutaneous nodules may inform the diagnosis in the later stages of the disease.

1	Patients with systemic inflammatory diseases such as RA will often present with elevated nonspecific inflammatory markers such as an ESRorCRP.DetectionofserumRFandanti-CCPantibodiesisimportant in differentiating RA from other polyarticular diseases, although RF lacks diagnostic specificity and may be found in association with other chronic inflammatory diseases in which arthritis figures in the clinical manifestations.

1	IgM, IgG, and IgA isotypes of RF occur in sera from patients with RA, although the IgM isotype is the one most frequently measured by commercial laboratories. Serum IgM RF has been found in 75–80% of patientswithRA;therefore,anegativeresultdoesnotexcludethepresence of this disease. It is also found in other connective tissue diseases, such as primary Sjögren’s syndrome, systemic lupus erythematosus, and type II mixed essential cryoglobulinemia, as well as chronic infections such as subacute bacterial endocarditis and hepatitis B and C. Serum RF may also be detected in 1–5% of the healthy population. Note: These criteria are aimed at classification of newly presenting patients who have at least one joint with definite clinical synovitis that is not better explained by another disease. A score of ≥6 fulfills requirements for definite RA.

1	Abbreviations: ACPA, anti-citrullinated peptide antibodies; CCP, cyclic citrullinated peptides; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; IP, interphalangeal joint; MCP, metacarpophalangeal joint; MTP, metatarsophalangeal joint; PIP, proximal interphalangeal joint; RF, rheumatoid factor; ULN, upper limit of normal. Source: D Aletaha et al: Arthritis Rheum 62:2569, 2010.

1	Source: D Aletaha et al: Arthritis Rheum 62:2569, 2010. 2144 The presence of serum anti-CCP antibodies has about the same sensitivityasserumRFforthediagnosisofRA.However,itsdiagnostic specificity approaches 95%, so a positive test for anti-CCP antibodies in the setting of an early inflammatory arthritis is useful for distinguishing RA from other forms of arthritis. There is some incremental value in testing for the presence of both RF and anti-CCP, as some patientswithRAarepositiveforRF butnegativefor anti-CCP and visa versa. The presence of RF or anti-CCP antibodies also has prognostic significance, with anti-CCP antibodies showing the most value for predicting worse outcomes.

1	Typically,synovialfluidfrompatientswithRAreflectsaninflammatory state. Synovialfluidwhitebloodcell (WBC)countscanvarywidely, but generally range between 5000 and 50,000 WBC/μL compared to <2000 WBC/μL for a noninflammatory condition such as osteoarthritis. In contrast to the synovial tissue, the overwhelming cell type in the synovial fluid is the neutrophil. Clinically, the analysis of synovial fluid is most useful for confirming an inflammatory arthritis (as opposed to osteoarthritis), while at the same time excluding infection or a crystal-induced arthritis such as gout or pseudogout (Chap. 395).

1	Joint imaging is a valuable tool not only for diagnosing RA, but also for tracking progression of any joint damage. Plain x-ray is the most common imaging modality, but it is limited to visualization of the bony structures and inferences about the state of the articular cartilage based on the amount of narrowing of the joint space. MRI and ultrasound techniques offer the added value of detecting changes in the soft tissues such as synovitis, tenosynovitis, and effusions as well as greater sensitivity for identifying bony abnormalities. Plain radio-graphs are usually relied upon in clinical practice for the purpose of diagnosis and monitoring of affected joints. However, in selected cases, MRI and ultrasound can provide additional diagnostic information that may guide clinical decision making. Musculoskeletal ultrasound with power Doppler is increasingly used in rheumatology clinical practice for detecting synovitis and bone erosion.

1	Plain radiography Classically in RA, the initial radiographic finding is periarticular osteopenia. Practically speaking, however, this finding is difficult to appreciate on plain films and, in particular, on the newer digitalized x-rays. Other findings on plain radiographs include soft tissue swelling, symmetric joint space loss, and subchondral erosions, most frequently in the wrists and hands (MCPs and PIPs) and the feet (MTPs). In the feet, the lateral aspect of the fifth MTP is often targeted first, but other MTP joints may be involved at the same time. X-ray imaging of advanced RA may reveal signs of severe destruction, including joint subluxation and collapse (Fig. 380-5). FIGUrE 380-5 X-ray demonstrating progression of erosions ontheproximalinterphalangealjoint.(Courtesy of the American College of Rheumatology.)

1	FIGUrE 380-5 X-ray demonstrating progression of erosions ontheproximalinterphalangealjoint.(Courtesy of the American College of Rheumatology.) MrI MRI offers the greatest sensitivity for detecting synovitis and joint effusions, as well as early bone and bone marrow changes. These soft tissue abnormalities often occur before osseous changes are noted on x-ray. Presence of bone marrow edemahasbeen recognizedto be an early sign of inflammatory joint disease and can predict the subsequent development of erosions on plain radiographs as well as MRI scans. Cost and availability of MRI are the main factors limiting its routine clinical use.

1	Ultrasound Ultrasound, including power color Doppler, has the ability to detect more erosions than plain radiography, especially in easily accessible joints. It can also reliably detect synovitis, including increased joint vascularity indicative of inflammation. The usefulness of ultrasound is dependent on the experience of the sonographer; however, it does offer the advantages of portability, lack of radiation, and low expense relative to MRI, factors that make it attractive as a clinical tool.

1	The natural history of RA is complex and affected by a number of factors including age of onset, gender, genotype, phenotype (i.e., extraarticular manifestations or variants of RA), and comorbid conditions, which make for a truly heterogeneous disease. There is no simple way to predict the clinical course. It is important to realize that as many as 10% of patients with inflammatory arthritis fulfilling ACR classification criteria for RA will undergo a spontaneous remission within 6 months (particularly seronegative patients). However, the vast majority of patients will exhibit a pattern of persistent and progressive disease activity that waxes and wanes in intensity over time. A minority of patients will show intermittent and recurrent explosive attacks of inflammatory arthritis interspersed with periods of disease quiescence. Finally, an aggressive form of RA may occur in an unfortunate few with inexorable progression of severe erosive joint disease, although this highly destructive

1	with periods of disease quiescence. Finally, an aggressive form of RA may occur in an unfortunate few with inexorable progression of severe erosive joint disease, although this highly destructive course is less common in the modern treatment era of biologics.

1	Disability, as measured by the Health Assessment Questionnaire (HAQ), shows gradual worsening of disability over time in the face of poorly controlled disease activity and disease progression. Disability may result from both a disease activity–related component that is potentially reversible with therapy and a joint damage–related component owing to the cumulative and largely irreversible effects of cartilage and bone breakdown. Early in the course of disease, the extent of joint inflammation is the primary determinant of disability, while in the later stages of disease, the amount of joint damage is the dominant contributing factor. Previous studies have shown that more than one-half of patients with RA are unable to work 10 years after the onset of their disease; however, increased employability and less work absenteeismhasbeen reported recently with the useofnewer therapies and earlier treatment intervention.

1	The overall mortality rate in RA is two times greater than the general population, with ischemic heart disease being the most common cause of death followed by infection. Median life expectancy is shortened by an average of 7 years for men and 3 years for women compared to control populations. Patients at higher risk for shortened survival are those with systemic extraarticular involvement, low functional capacity, low socioeconomic status, low education, and chronic prednisone use.

1	The amount of clinical disease activity in patients with RA reflects the overall burden of inflammation and is the variable most influencing treatment decisions. Joint inflammation is the main driver of joint damage and is the most important cause of functional disability in the early stages of disease. Several composite indices have been developed to assess clinical disease activity. The ACR 20, 50, and 70 improvement criteria (which corresponds to a 20%, 50%, and 70% improvement, respectively, in joint counts, physician/patient assessment of disease severity, pain scale, serum levels of acute-phase reactants [ESR or CRP], and a functional assessment of disability using a self-administered patient questionnaire) are a composite index with a dichotomous response variable. The ACR improvement criteria are commonly used in clinical trials as an endpoint for comparing the proportion of responders between treatment groups. In contrast, the Disease Activity Score (DAS), Simplified Disease

1	criteria are commonly used in clinical trials as an endpoint for comparing the proportion of responders between treatment groups. In contrast, the Disease Activity Score (DAS), Simplified Disease Activity Index (SDAI), and the Clinical Disease Activity Index (CDAI) are continuous measures of disease activity. These scales are increasingly used in clinical practice for tracking disease status and, in particular, for documenting treatment response.

1	Several developments during the past two decades have changed the therapeutic landscape in RA. They include (1) the emergence of methotrexate as the disease-modifying antirheumatic drug (DMARD) of first choice for the treatment of early RA; (2) the development of novel highly efficacious biologicals that can be used alone or in combination with methotrexate; and (3) the proven superiority of combination DMARD regimens over methotrexate alone. The medications used for the treatment of RA may be divided into broad categories: nonsteroidal anti-inflammatory drugs (NSAIDs); glucocorticoids, such as prednisone and methylprednisolone; conventional DMARDs; and biologic DMARDs (Table 380-2). Although disease for some patients with RA is managed adequately with a single DMARD, such as methotrexate, the situation in most cases demands the use of a combination DMARD regimen that may vary in its components over the treatment course depending on fluctuations in disease activity and emergence of

1	the situation in most cases demands the use of a combination DMARD regimen that may vary in its components over the treatment course depending on fluctuations in disease activity and emergence of drug-related toxicities and comorbidities.

1	NSAIDs were formerly viewed as the core of all other RA therapy, but they are now considered to be adjunctive therapy for management of symptoms uncontrolled by other measures. NSAIDs exhibit both analgesic and anti-inflammatory properties. The anti-inflammatory effects of NSAIDs derive from their ability to nonselectively inhibit cyclooxygenase (COX)-1 and COX-2. Although the results of clinical trials suggest NSAIDs are roughly equivalent in their efficacy, experience suggests that some individuals may preferentially respond to a particular NSAID. Chronic use should be minimized due to the possibility of side effects, including gastritis and peptic ulcer disease as well as impairment of renal function.

1	Glucocorticoids may serve in several ways to control disease activity in RA. First, they may be administered in low to moderate doses to achieve rapid disease control before the onset of fully effective DMARD therapy, which often takes several weeks or even months. Second, a 1to 2-week burst of glucocorticoids may be prescribed for the management of acute disease flares, with dose and duration guided by the severity of the exacerbation. Chronic administration of low doses (5–10 mg/d) of prednisone (or its equivalent) may also be warranted to control disease activity in patients with an inadequate response to DMARD therapy. Low-dose prednisone therapy has been shown in prospective studies to retard radiographic progression of joint disease; however, the benefits of this approach must be carefully weighed against the risks. Best practices minimize chronic use of low-dose prednisone therapy owing to the risk of osteoporosis and other long-term complications; however, the use of chronic

1	weighed against the risks. Best practices minimize chronic use of low-dose prednisone therapy owing to the risk of osteoporosis and other long-term complications; however, the use of chronic prednisone therapy is unavoidable in many cases. High-dose glucocorticoids may be necessary for treatment of severe extraarticular manifestations of RA, such as ILD. Finally, if a patient exhibits one or a few actively inflamed joints, the clinician may consider intraarticular injection of an intermediate-acting glucocorticoid such as triamcinolone acetonide. This approach may allow for rapid control of inflammation in the setting of a limited number of affected joints. Caution must be exercised to appropriately exclude joint infection, as it often mimics an RA flare.

1	Osteoporosis ranks as an important long-term complication of chronic prednisone use. The ACR recommends primary prevention of glucocorticoid-induced osteoporosis with a bisphosphonate in any patient receiving 5 mg/d or more of prednisone for greater than 2145 3 months. Although prednisone use is known to increase the risk of peptic ulcer disease, especially with concomitant NSAID use, no evidence-based guidelines have been published regarding the use of gastrointestinal ulcer prophylaxis in this situation.

1	DMARDs are so named because of their ability to slow or prevent structural progression of RA. The conventional DMARDs include hydroxychloroquine, sulfasalazine, methotrexate, and leflunomide; they exhibit a delayed onset of action of approximately 6–12 weeks. Methotrexate is the DMARD of choice for the treatment of RA and is the anchor drug for most combination therapies. It was approved for the treatment of RA in 1986 and remains the benchmark for the efficacy and safety of new disease-modifying therapies. At the dosages used for the treatment of RA, methotrexate has been shown to stimulate adenosine release from cells, producing an anti-inflammatory effect. The clinical efficacy of leflunomide, an inhibitor of pyrimidine synthesis, appears similar to that of methotrexate; it has been shown in well-designed trials to be effective for the treatment of RA as monotherapy or in combination with methotrexate and other DMARDs.

1	Although similar to the other DMARDs in its slow onset of action, hydroxychloroquine has not been shown to delay radiographic progression of disease and thus is not considered to be a true DMARD. In clinical practice, hydroxychloroquine is generally used for treatment of early, mild disease or as adjunctive therapy in combination with other DMARDs. Sulfasalazine is used in a similar manner and has been shown in randomized, controlled trials to reduce radiographic progression of disease. Minocycline, gold salts, penicillamine, azathioprine, and cyclosporine have all been used for the treatment of RA with varying degrees of success; however, they are used sparingly now due to their inconsistent clinical efficacy or unfavorable toxicity profile.

1	Biologic DMARDs have revolutionized the treatment of RA over the past decade (Table 380-2). They are protein therapeutics designed mostly to target cytokines and cell-surface molecules. The TNF inhibitors were the first biologicals approved for the treatment of RA. Anakinra, an IL-1 receptor antagonist, was approved shortly thereafter; however, its benefits have proved to be relatively modest compared with the other biologicals and is rarely used for the treatment of RA with the availability of other more effective agents. Abatacept, rituximab, and tocilizumab are the newest members of this class.

1	anti-TNF agents The development of TNF inhibitors was originally spurred by the experimental finding that TNF is a critical upstream mediator of joint inflammation. Currently, five agents that inhibit TNF-α are approved for the treatment of RA. There are three different anti-TNF monoclonal antibodies. Infliximab is a chimeric (part mouse and human) monoclonal antibody, whereas adalimumab and golimumab are humanized monoclonal antibodies. Certolizumab pegol is a pegylated Fc-free fragment of a humanized monoclonal antibody with binding specificity for TNF-α. Lastly, etanercept is a soluble fusion protein comprising the TNF receptor 2 in covalent linkage with the Fc portion of IgG1. All of the TNF inhibitors have been shown in randomized controlled clinical trials to reduce the signs and symptoms of RA, slow radiographic progression of joint damage, and improve physical function and quality of life. Anti-TNF drugs are typically used in combination with background methotrexate therapy.

1	of RA, slow radiographic progression of joint damage, and improve physical function and quality of life. Anti-TNF drugs are typically used in combination with background methotrexate therapy. This combination regimen, which affords maximal benefit in many cases, is often the next step for treatment of patients with an inadequate response to methotrexate therapy. Etanercept, adalimumab, certolizumab pegol, and golimumab have also been approved for use as monotherapy.

1	Anti-TNF agents should be avoided in patients with active infection or a history of hypersensitivity to these agents and are contraindicated in patients with chronic hepatitis B infection or class III/IV congestive Rituximab 200–400 mg/d orally (≤6.5 mg/kg) Initial: 500 mg orally twice daily Maintenance: 1000–1500 mg twice daily Folic acid 1 mg/d to reduce toxicities Infliximab: 3 mg/kg IV at weeks 0, 2, 6, then every 8 weeks. May increase dose up to 10 mg/kg every 4 weeks Etanercept: 50 mg SQ weekly, or 25 mg SQ biweekly Adalimumab: 40 mg SQ every other week Golimumab: 50 mg SQ monthly Certolizumab: 400 mg SQ weeks 0, 2, 4, then 200 mg every other week Weight based: <60 kg: 500 mg 60–100 kg: 750 mg >100 kg: 1000 mg IV dose at weeks 0, 2, and 4, and then every 4 weeks 1000 mg IV × 2, days 0 and 14 Premedicate with methylprednisolone 100 mg to decrease infusion reaction Risk bacterial, fungal infections Reactivation of latent TB Risk bacterial, viral infections

1	Premedicate with methylprednisolone 100 mg to decrease infusion reaction Risk bacterial, fungal infections Reactivation of latent TB Risk bacterial, viral infections Risk bacterial, viral infections Reactivation of latent TB Neutropenia Risk bacterial, viral infections Infusion reaction CBC, LFTs G6PD level CBC, LFTs CBC, LFTs Viral hepatitis panela CBC every 2–4 weeks for first 3 months, then every 3 months CBC, creatinine, CBC, creatinine, LFTs every 2–3 months CBC every month for 3 months, then every 4 months for 1 year Risk of infection Risk of infection Upper respiratory PPD skin test CBC, LFTs, and lipids at tract infections aViral hepatitis panel: hepatitis B surface antigen, hepatitis C viral antibody. Abbreviations: CBC, complete blood count; DMARDs, disease-modifying antirheumatic drugs; G6PD, glucose-6-phosphate dehydrogenase; IV, intravenous; LFTs, liver function tests; PPD, purified protein derivative; SQ, subcutaneous; TB, tuberculosis.

1	heart failure. The major concern is the increased risk for infection, including serious bacterial infections, opportunistic fungal infection, and reactivation of latent tuberculosis. For this reason, all patients are screened for latent tuberculosis according to national guidelines prior to starting anti-TNF therapy (Chap. 202). In the United States, patients are skin tested using an intradermal injection of purified protein derivative (PPD); individuals with skin reactions of more than 5 mm are presumed to have had previous exposure to tuberculosis and are evaluated for active disease and treated accordingly. The QuantiFERON IFN-γ release assay may also be used in selected circumstances to screen for previous exposure to tuberculosis.

1	anakinra Anakinra, the recombinant form of the naturally occurring IL-1 receptor antagonist. Although anakinra has seen limited use for the treatment of RA, it has enjoyed a resurgence of late as an effective therapy of some rare inherited syndromes dependent on IL-1 production, including neonatal-onset inflammatory disease, Muckle-Wells syndrome, and familial cold urticaria, as well as systemic juvenile-onset inflammatory arthritis and adult-onset Still’s disease. Anakinra should not be combined with an anti-TNF drug due to the high rate of serious infections as observed with this regimen in a clinical trial.

1	abatacept Abatacept is a soluble fusion protein consisting of the extracellular domain of human CTLA-4 linked to the modified portion of human IgG. It inhibits the co-stimulation of T cells by blocking CD28-CD80/86 interactions and may also inhibit the function of antigen-presenting cells by reverse signaling through CD80 and CD86. Abatacept has been shown in clinical trials to reduce disease activity, slow radiographic progression of damage, and improve functional disability. Many patients receive abatacept in combination with methotrexate or another DMARD such as leflunomide. Abatacept therapy has been associated with an increased risk of infection.

1	rituximab Rituximab is a chimeric monoclonal antibody directed against CD20, a cell-surface molecule expressed by most mature B lymphocytes. It works by depleting B cells, which in turn, leads to a reduction in the inflammatory response by unknown mechanisms. These mechanisms may include a reduction in autoantibodies, inhibition of T cell activation, and alteration of cytokine production. Rituximab has been approved for the treatment of refractory RA in combination with methotrexate and has been shown to be more effective for patients with seropositive than seronegative disease. Rituximab therapy has been associated with mild to moderate infusion reactions as well as an increased risk of infection. Notably, there have been isolated reports of a potentially lethal brain disorder, progressive multifocal leukoencephalopathy (PML), in association with rituximab therapy, although the absolute risk of this complication appears to be very low in patients with RA. Most of these cases have

1	multifocal leukoencephalopathy (PML), in association with rituximab therapy, although the absolute risk of this complication appears to be very low in patients with RA. Most of these cases have occurred on a background of previous or current exposure to other potent immunosuppressive drugs.

1	Tocilizumab Tocilizumab is a humanized monoclonal antibody directed against the membrane and soluble forms of the IL-6 receptor. IL-6 is a proinflammatory cytokine implicated in the pathogenesis of RA, with detrimental effects on both joint inflammation and damage. IL-6 binding to its receptor activates intracellular signaling pathways that affect the acute-phase response, cytokine production, and osteoclast activation. Clinical trials attest to the clinical efficacy of tocilizumab therapy for RA, both as monotherapy and in combination with methotrexate and other DMARDs. Tocilizumab has been associated with an increased risk of infection, neutropenia, and thrombocytopenia; however, the hematologic abnormalities appear to be reversible upon stopping the drug. In addition, this agent has been shown to increase LDL cholesterol; however, it is not known as yet if this effect on lipid levels increases the risk for development of atherosclerotic disease.

1	Because some patients do not adequately respond to conventional DMARDS or biologic therapy, other therapeutic targets have been investigated to fill this gap. Recently, drug development in RA has focused attention on the intracellular signaling pathways that transduce the positive signals of cytokines and other inflammatory mediators that create the positive feedback loops in the immune response. These synthetic DMARDs aim to provide the same efficacy as biological therapies in an oral formulation.

1	Tofacitinib Tofacitinib is a small-molecule inhibitor that primarily inhibits JAK1 and JAK3, which mediate signaling of the receptors for the common γ-chain-related cytokines IL-2, -4, -7, -9, -15, and -21 as well as IFN-γ and IL-6. These cytokines all play roles in promoting T and B cell activation as well as inflammation. Tofacitinib, an oral agent, has been shown in randomized, placebo-controlled clinical trials to improve the signs and symptoms of RA significantly over placebo. Major adverse events include elevated serum transaminases indicative of liver injury, neutropenia, increased cholesterol levels, and elevation in serum creatinine. Its use is also associated with an increased risk of infections. Tofacitinib can be used as mono-therapy or in combination with methotrexate. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: The original treatment pyramid for RA is now considered to be obsolete and has evolved into a new strategy that focuses on several goals: (1) early, aggressive therapy to prevent jointdamageanddisability;(2) frequent modification of therapy withutilization of combination therapy where appropriate; (3) individualization of therapy in an attempt to maximize response and minimize side effects; and (4) achieving, whenever possible, remission of clinical disease activity. A considerable amount of evidence supports this intensive treatment approach.

1	As mentioned earlier, methotrexate is the DMARD of first choice for initial treatment of moderate to severe RA. Failure to achieve adequate improvement with methotrexate therapy calls for a change in DMARD therapy, usually transition to an effective combination regimen. Effective combinations include: methotrexate, sulfasalazine, and hydroxychloroquine (oral triple therapy); methotrexate and leflunomide; and methotrexate plus a biological. The combination of methotrexate and an anti-TNF agent, for example, has been shown in randomized, controlled trials to be superior to methotrexate alone not only for reducing signs and symptoms of disease, but also for retarding the progression of structural joint damage. Predicting which patients will ultimately show radiologic joint damage is imprecise at best, although some factors such as an elevated serum level of acute-phase reactants, high burden of joint inflammation, and the presence of erosive disease are associated with increased

1	imprecise at best, although some factors such as an elevated serum level of acute-phase reactants, high burden of joint inflammation, and the presence of erosive disease are associated with increased likelihood of developing structural injury.

1	In 2012 a joint task force of the ACR and EULAR updated the treatment guidelines for RA. They do make a distinction between patients with early RA (<6 months of disease duration) and patients with established RA. These guidelines highlight the need to switch or add DMARD therapy after 3 months of worsening or persistent moderate/high disease activity. If disease still persists after 3 months of intense DMARD therapy, addition of a biologic agent is warranted. Treatment with a biologic agent or aggressive combination DMARD therapy was also recommended as initial therapy in certain patients with high disease activity and poor prognosis. However, it has not been clearly established that this more intensive initial approach is superior to starting with methotrexate alone and, in the absence of an inadequate therapeutic response, moving rapidly to combination therapy.

1	Some patients may not respond to an anti-TNF drug or may be intolerant of its side effects. Initial responders to an anti-TNF agent that later worsen may benefit from switching to another anti-TNF agent. The 2012 guidelines recommend that with loss or lack of effectiveness of anti-TNF after 3 months, one should switch to another anti-TNF or non-TNF biologic agent. In patients with high diseaseactivityandaserious adverseeventfromananti-TNFagent, a non-TNF drug should be used. Studies have also shown that oral triple therapy (hydroxychloroquine, methotrexate, and sulfasalazine) is a reasonable first step for the treatment of early RA, including its use as a step-up strategy where treatment is initiated with methotrexate alone and then combined at 6 months with hydroxychloroquine and sulfasalazine if the disease is not adequately controlled.

1	A clinical state defined as low disease activity or remission is the optimal goal of therapy, although most patients never achieve remission despite every effort to achieve it. Composite indices, such as the Disease Activity Score-28 (DAS-28), are useful for classifying states of low disease activity and remission; however, they are imperfect tools due to the limitations of the clinical joint examination in which low-grade synovitis may escape detection. Complete remission has been stringently defined as the total absence of all articular and extraarticular inflammation and immunologic activity related to RA. However, evidence for this state can be difficult to demonstrate in clinical practice. In an effort to standardize and simplify the definition of remission for clinical trials, the ACR and EULARdevelopedtwoprovisionaloperationaldefinitionsofremissioninRA (Table 380-3).A patientmaybe consideredinremission if he or she (1) meets all of the clinical and laboratory criteria listed in

1	and EULARdevelopedtwoprovisionaloperationaldefinitionsofremissioninRA (Table 380-3).A patientmaybe consideredinremission if he or she (1) meets all of the clinical and laboratory criteria listed in Table 380-3 or (2) has a composite SDAI score of <3.3. The SDAI is calculated by taking the sum of a tender joint and swollen joint count (using 28 joints), patient global assessment (0–10 scale), physicianglobalassessment (0–10scale), and CRP (inmg/dL). This definition of remission does not take into account the possibility of subclinical synovitis or that damage alone may produce a tender or swollen joint. Ignoring the semantics of these definitions, the aforementioned remission criteria are nonetheless useful for setting

1	At any time point, patient must satisfy all of the following: At any time point, patient must have a Simplified Disease Activity Index score of ≤3.3 Source: Adapted from DT Felson et al: Arthritis Rheum 63:573, 2011. a level of disease control that will likely result in minimal or no progression of structural damage and disability. All patients should receive a prescription for exercise and physical activity. Dynamic strength training, community-based comprehensive physical therapy, and physical-activity coaching (emphasizing 30 min of moderately intensive activity most days a week) have all been shown to improve muscle strength and perceived health status. Foot orthotics for painful valgus deformity decrease foot pain and resulting disability and functional limitations. Judicious use of wrist splints can also decrease pain; however, their benefits may be offset by decreased dexterity and a variable effect on grip strength.

1	Surgical procedures may improve pain and disability in RA—most notably the hands, wrists, and feet, typically after the failure of medical therapy with varying degrees of reported long-term success. For large joints, such as the knee, hip, shoulder, or elbow, total joint arthroplasty is an option for advanced joint disease. A few surgical options exist for dealing with the smaller hand joints. Silicone implants are the most common prosthetic for MCP arthroplasty and are generally implanted in patients with severe decreased arc of motion, marked flexion contractures, MCP joint pain with radiographic abnormalities, and severe ulnar drift. Arthrodesis and total wrist arthroplasty are reserved for patients with severe disease who have substantial pain and functional impairment. These two procedures appear to have equal efficacy in terms of pain control and patient satisfaction. Numerous surgical options exist for correction of hallux valgus in the forefoot, including arthrodesis and

1	procedures appear to have equal efficacy in terms of pain control and patient satisfaction. Numerous surgical options exist for correction of hallux valgus in the forefoot, including arthrodesis and arthroplasty, as well as primarily arthrodesis for refractory hindfoot pain.

1	OTHEr MaNaGEMENT CONSIDEraTIONS Pregnancy Up to 75% of female RA patients will note overall improvement in symptoms during pregnancy, but often will flare after delivery. Flares during pregnancy are generally treated with low doses of prednisone; hydroxychloroquine and sulfasalazine are probably the safest DMARDs to use during pregnancy. Methotrexate and leflunomide therapy are contraindicated during pregnancy due to their teratogenicity in animals and humans. The experience with biologic agents has been insufficient to make specific recommendations for their use during pregnancy. Most rheumatologists avoid their use in this setting; however, exceptions are considered depending on the circumstances.

1	Elderly Patients RA presents in up to one-third of patients after the ageof60;however,olderindividualsmayreceivelessaggressivetreatment due to concerns about increased risks of drug toxicity. Studies suggest that conventional DMARDs and biologic agents are equally effective and safe in younger and older patients. Due to comorbidities, many elderly patients have an increased risk of infection. Aging alsoleadstoagradualdeclineinrenalfunctionthatmayraisetherisk for side effects from NSAIDs and some DMARDS, such as methotrexate. Renal function must be taken into consideration before prescribing methotrexate, which is mostly cleared by the kidneys. To reduce the risks of side effects, methotrexate doses may need to be adjusted downward for the drop in renal function that usually comes with the seventh and eighth decades of life. Methotrexate is usually not prescribed for patients with a serum creatinine greater Jonathan R. Carapetis than 2 mg/dL.

1	Developing countries are finding an increase in the incidence of noncommunicable, chronic diseases such as diabetes, car diovascular disease, and RA in the face of ongoing poverty, rampant infectious disease, and poor access to modern health care facilities. In theseareas,patientstend tohavea greater delay indiagnosis and limited access to specialists, and thus greater disease activity and disability at presentation. In addition, infection risk remains a significant issue for the treatment of RA in developing countries because of the immunosuppression associated with the use of glucocorticoids and most DMARDs. For example, in some developing countries, patients undergoing treatment for RA have a substantial increase in the incidence of tuberculosis, which demands the implementation of far more comprehensive screening practices and liberal useofisoniazidprophylaxisthanindevelopedcountries.Theincreased prevalence of hepatitis B and C, as well as human immunodeficiency virus (HIV), in

1	more comprehensive screening practices and liberal useofisoniazidprophylaxisthanindevelopedcountries.Theincreased prevalence of hepatitis B and C, as well as human immunodeficiency virus (HIV), in these developing countries also poses challenges. Reactivation of viral hepatitis has been observed in association with some of the DMARDs, such as rituximab. Also, reduced access to antiretroviral therapy may limit the control of HIV infection and therefore the choice of DMARD therapies.

1	Despite these challenges, one should attempt to initiate early treatment of RA in the developing countries with the resources at hand. Sulfasalazine and methotrexate are all reasonably accessible throughout the world where they can be used as both monotherapy and in combination with other drugs. The use of biologic agents is increasing in the developedcountriesaswellasinotherareasaroundtheworld,although theiruseislimitedbyhighcost;nationalprotocolsrestricttheiruse,and concerns remain about the risk for opportunistic infections.

1	Improved understanding of the pathogenesis of RA and its treatment has dramatically revolutionized the management of this disease. The outcomes of patients with RA are vastly superior to those of the prebiologic modifier era; more patients than in years past are able to avoid significant disability and continue working, albeit with some job modifications in many cases. The need for early and aggressive treatment of RA as well as frequent follow-up visits for monitoring of drug therapy has implications for our health care system. Primary care physicians and rheumatologists must be prepared to work together as a team to reach the ambitious goals of best practice. In many settings, rheumatologists have reengineered their practice in a way that places high priority on consultations for any new patient with early inflammatory arthritis.

1	The therapeutic regimens for RA are becoming increasingly complex with the rapidly expanding therapeutic armamentarium. Patients receiving these therapies must be carefully monitored by both the primary care physician and the rheumatologist to minimize the risk of side effects and identify quickly any complications of chronic immunosuppression. Also, prevention and treatment of RA-associated conditionssuchasischemicheartdiseaseandosteoporosiswilllikelybenefit from a team approach owing to the value of multidisciplinary care.

1	Research will continue to search for new therapies with superior efficacyandsafetyprofiles and investigatetreatmentstrategiesthatcan bring the disease under control more rapidly and nearer to remission. However, prevention and cure of RA will likely require new breakthroughs in our understanding of disease pathogenesis. These insights may come from genetic studies illuminating critical pathways in the mechanisms of joint inflammation. Equally ambitious is the lofty goal of biomarker discovery that will open the door to personalized medicine for the care of patients with RA. Acute rheumatic fever (ARF) is a multisystem disease resulting from an autoimmune reaction to infection with group A streptococcus. Although many parts of the body may be affected, almost all of the manifestations resolve completely. The major exception is cardiac valvular damage (rheumatic heart disease [RHD]), which may persist after the other features have disappeared.

1	ARF and RHD are diseases of poverty. They were common in all countries until the early twentieth century, when their inci dence began to decline in industrialized nations. This decline was largely attributable to improved living conditions—particularly less crowded housing and better hygiene—which resulted in reduced transmission of group A streptococci. The introduction of antibiotics and improved systems of medical care had a supplemental effect.

1	The virtual disappearance of ARF and reduction in the incidence of RHDin industrialized countries during thetwentieth centuryunfortunately was not replicated in developing countries, where these diseases continue unabated. RHD is the mostcommon cause of heartdisease in children in developing countries and is a major cause of mortality and morbidity in adults as well. It has been estimated that between 15 and 19 million people worldwide are affected by RHD, with approximately one-quarter of a million deaths occurring each year. Some 95% of ARF cases and RHD deaths now occur in developing countries, with particularly high rates in sub-Saharan Africa, Pacific nations, Australasia, and South and Central Asia. The pathogenetic pathway from exposure to group A streptococcus followed by pharyngeal infection and subsequent development of ARF, ARF recurrences, and development of RHD and its complications is associated with a range of risk factors and, therefore, potential interventions at each

1	infection and subsequent development of ARF, ARF recurrences, and development of RHD and its complications is associated with a range of risk factors and, therefore, potential interventions at each point (Fig. 381-1). In affluent countries, many of these risk factors are well controlled, and where needed, interventions are in place. Unfortunately, the greatest burden of disease is found in developing countries, most of which do not have the resources, capacity, and/or interestto tacklethis multifaceted disease. In particular, almost none of the developing countries has a coordinated, register-based RHD control program, which is proven to be cost effective in reducing the burden of RHD. Enhancing awareness of RHD and mobilizing resources for its control in developing countries are issues requiring international attention.

1	ARF is mainly a disease of children age 5–14 years. Initial episodes become less common in older adolescents and young adults and are rare in persons age >30 years. By contrast, recurrent episodes of ARF remain relatively common in adolescents and young adults. This pattern contrasts with the prevalence of RHD, which peaks between 25 and40years.ThereisnocleargenderassociationforARF,butRHD more commonly affects females, sometimes up to twice as frequently as males. Based on currently available evidence, ARF is exclusively caused by infection of the upper respiratory tract with group A streptococci (Chap. 173). Although classically, certain M-serotypes (particularly types 1, 3, 5, 6, 14, 18, 19, 24, 27, and 29) were associated with ARF, in high-incidence regions, it is now thought that any strain of group A streptococcus has the potential to cause ARF. The potential role of skin infection and of groups C and G streptococci is currently being investigated.

1	Exposure to Group A streptococcal *Role of streptococcal skin infection unclear FIGUrE 381-1 Pathogenetic pathway for acute rheumatic fever and rheumatic heart disease, with associated risk factors and opportunities for intervention at each step. Interventions in parentheses are either unproven or currently unavailable.

1	Approximately 3–6% of any population may be susceptible to ARF, and this proportion does not vary dramatically between populations. Findings of familial clustering of cases and concordance in mono-zygotic twins—particularly for chorea—confirm that susceptibility to ARF is an inherited characteristic, with 44% concordance in mono-zygotic twins compared to 12% in dizygotic twins, and heritability more recently estimated at 60%. Most evidence for host factors focuses on immunologic determinants. Some human leukocyte antigen (HLA) class II alleles, particularly HLA-DR7 and HLA-DR4, appear to be associated with susceptibility, whereas other class II alleles have been associated with protection (HLA-DR5, HLA-DR6, HLA-DR51, HLA-DR52, and HLA-DQ). Associations have also been described with polymorphisms at the tumor necrosis factor α locus (TNF-α-308 and TNF-α-238), high levels of circulating mannose-binding lectin, and Toll-like receptors.

1	The most widely accepted theory of rheumatic fever pathogenesis is based on the concept of molecular mimicry, whereby an immune response targeted at streptococcal antigens (mainly thought to be on the M protein and the N-acetylglucosamine of group A streptococcal carbohydrate) also recognizes human tissues. In this model, cross-reactive antibodies bind to endothelial cells on the heart valve, leading to activation of the adhesion molecule VCAM-1, with resulting recruitment of activated lymphocytes and lysis of endothelial cells in the presence of complement. The latter leads to release of peptides including laminin, keratin, and tropomyosin, which, in turn, activates cross-reactive T cells that invade the heart, amplifying the damage and causing epitope spreading. An alternative hypothesis proposes that the initial damage is due to streptococcal invasion of epithelial surfaces, with binding of M protein to type IV collagen RHD allowing it to become immunogenic, but not through the

1	proposes that the initial damage is due to streptococcal invasion of epithelial surfaces, with binding of M protein to type IV collagen RHD allowing it to become immunogenic, but not through the mechanism of molecular mimicry.

1	There is a latent period of ~3 weeks (1–5 weeks) between the precipitating group A streptococcal infection and the appearance of the clinical features of ARF. The exceptions are chorea and indolent carditis, which may follow prolonged latent periods lasting up to 6 months. Although many patients report a prior sore throat, the preceding group A streptococcal infection is commonly subclinical; in these cases, it can only be confirmed using streptococcal antibody testing. The most common clinical features are polyarthritis (present in 60–75% of cases) and carditis (50–60%). The prevalence of chorea in ARF varies substantially between populations, ranging from <2 to 30%. Erythema marginatum and subcutaneous nodules are now rare, being found in <5% of cases. Up to 60% of patients with ARF progress to RHD. The endocardium, pericardium, or myocardium may be affected. Valvular damage is the hallmark of rheumatic carditis. The mitral valve is almost always

1	FIGUrE 381-2 Transthoracic echocardiographic image from a 5-year-old boy with chronic rheumatic heart disease. This diastolic image demonstrates leaflet thickening, restriction of the anterior mitral valve leaflet tip and doming of the body of the leaflet toward the inter-ventricular septum. This appearance (marked by the arrowhead) is commonly described as a “hockey stick” or an “elbow” deformity. AV, aortic valve; LA, left atrium; LV, left ventricle; MV, mitral valve; RV, right ventricle. (Courtesy of Dr. Bo Remenyi, Department of Paediatric and Congenital Cardiac Services, Starship Children’s Hospital, Auckland, New Zealand.) affected, sometimes together with the aortic valve; isolated aortic valve involvement is rare. Damage to the pulmonary or tricuspid valves is usually secondary to increased pulmonary pressures resulting from left-sided valvular disease. Early valvular damage leads to regurgitation. Over ensuing years, usually as a result of recurrent episodes, leaflet

1	to increased pulmonary pressures resulting from left-sided valvular disease. Early valvular damage leads to regurgitation. Over ensuing years, usually as a result of recurrent episodes, leaflet thickening, scarring, calcification, and valvular stenosis may develop (Fig. 381-2). See Videos 381-1 and 381-2 on the DVD. Therefore, the characteristic manifestation of carditis in previously unaffected individuals is mitral regurgitation, sometimes accompanied by aortic regurgitation. Myocardial inflammation may affect electrical conduction pathways, leading to P-R interval prolongation (first-degree atrioventricular block or rarely higher level block) and softening of the first heart sound.

1	People with RHD are often asymptomatic for many years before their valvular disease progresses to cause cardiac failure. Moreover, particularly in resource-poor settings, the diagnosis of ARF is often not made, so children, adolescents, and young adults may have RHD but not know it. These cases can be diagnosed using echocardiography; auscultation is poorly sensitive and specific for RHD diagnosis in asymptomatic patients. Echocardiographic screening of school-aged children in populations with high rates of RHD is becoming more widespread and has been facilitated by improving technologies in portable echocardiography and the availability of consensus guidelines for the diagnosis of RHD on echocardiography (Table 381-1). Although a diagnosis of definite RHD on screening echocardiography should lead to commencement of secondary prophylaxis, the clinical significance of borderline RHD has yet to be determined.

1	The most common form of joint involvement in ARF is arthritis, i.e., objective evidence of inflammation, with hot, swollen, red, and/or tender joints, and involvement of more than one joint (i.e., polyarthritis). Polyarthritis is typically migratory, moving from one joint to another over a period of hours. ARF almost always affects the large joints— most commonly the knees, ankles, hips, and elbows—and is asymmetric. The pain is severe and usually disabling until anti-inflammatory medication is commenced. Definite RHD (either A, B, C, or D): Pathologic MR and at least two morphologic features of RHD of the mitral valve MS mean gradient ≥4 mmHg (note: congenital MV anomalies must be excluded) Pathologic AR and at least two morphologic features of RHD of the AV (note: bicuspid AV and dilated aortic root must be excluded) Borderline disease of both the MV and AV Borderline RHD (either A, B, or C): At least two morphologic features of RHD of the MV without pathologic MR or MS

1	Borderline disease of both the MV and AV Borderline RHD (either A, B, or C): At least two morphologic features of RHD of the MV without pathologic MR or MS Normal Echocardiographic Findings (all of A, B, C, and D): An isolated morphologic feature of RHD of the MV (e.g., valvular thickening), without any associated pathologic stenosis or regurgitation Morphologic feature of RHD of the AV (e.g., valvular thickening), without any associated pathologic stenosis or regurgitation Definitions of Pathologic Regurgitation and Morphologic Features of RHD: Pathologic MR: All of the following: seen in two views; in at least one view, jet length 2 cm; peak velocity ≥3 m/s; pansystolic jet in at least one envelope Pathologic AR: All of the following: seen in two views; in at least one view, jet length ≥1 cm; peak velocity ≥3 m/s; pandiastolic jet in at least one envelope

1	Pathologic AR: All of the following: seen in two views; in at least one view, jet length ≥1 cm; peak velocity ≥3 m/s; pandiastolic jet in at least one envelope Morphologic features of RHD in MV: anterior MV leaflet thickening ≥3 mm (age specific); chordal thickening; restricted leaflet motion; excessive leaflet tip motion during systole Morphologic features of RHD in AV: irregular or focal thickening; coaptation defect; restricted leaflet motion; prolapse aFor criteria in individuals >20 years of age, see source document. Abbreviations: AR, aortic regurgitation; AV, aortic valve; MR, mitral regurgitation; MS, mitral stenosis; MV, mitral valve. Source: Adapted from Remenyi B et al: World Heart Federation criteria for echocardiographic diagnosis of rheumatic heart disease-an evidence-based guideline. Nat Rev Cardiol 9:297–309, 2012.

1	Source: Adapted from Remenyi B et al: World Heart Federation criteria for echocardiographic diagnosis of rheumatic heart disease-an evidence-based guideline. Nat Rev Cardiol 9:297–309, 2012. Less severe joint involvement is also relatively common and has been recognized as a potential major manifestation in high-risk populations in diagnostic guidelines from Australia, but at the time of writing, this was not reflected in the Jones criteria. Arthralgia without objective joint inflammation usually affects large joints in the same migratory pattern as polyarthritis. In some populations, aseptic monoarthritis may be a presenting feature of ARF, which may, in turn, result from early commencement of anti-inflammatory medication before the typical migratory pattern is established.

1	The joint manifestations of ARF are highly responsive to salicylates and other nonsteroidal anti-inflammatory drugs (NSAIDs). Indeed, joint involvement that persists for more than 1 or 2 days after starting salicylates is unlikely to be due to ARF.

1	Sydenham’s chorea commonly occurs in the absence of other manifestations, follows a prolonged latent period after group A streptococcal infection, and is found mainly in females. The choreiform movements affect particularly the head (causing characteristic darting movements of the tongue) and the upper limbs (Chap. 448). They may be generalized or restrictedtoonesideofthebody(hemi-chorea).Inmildcases,choreamay be evident only on careful examination, whereas in the most severe cases, the affected individuals are unable to perform activities of daily living. Thereisoftenassociatedemotionallabilityorobsessive-compulsivetraits, 2152 which may last longer than the choreiform movements (which usually resolve within 6 weeks but sometimes may take up to 6 months).

1	The classic rash of ARF is erythema marginatum (Chap. 24), which begins as pink macules that clear centrally, leaving a serpiginous, spreading edge. The rash is evanescent, appearing and disappearing before the examiner’s eyes. It occurs usually on the trunk, sometimes on the limbs, but almost never on the face. Subcutaneous nodules occur as painless, small (0.5–2 cm), mobile lumps beneath the skin overlying bony prominences, particularly of the hands, feet, elbows, occiput, and occasionally the vertebrae. They are a delayed manifestation, appearing 2–3 weeks after the onset of disease, last for just a few days up to 3 weeks, and are commonly associated with carditis. Fever occurs in most cases of ARF, although rarely in cases of pure chorea. Although high-grade fever (≥39°C) is the rule, lower grade temperature elevations are not uncommon. Elevated acute-phase reactants are also present in most cases.

1	With the exception of chorea and low-grade carditis, both of which may become manifest many months later, evidence of a preceding group A streptococcal infection is essential in making the diagnosis of ARF. Because most cases do not have a positive throat swab culture or rapidantigen test,serologicevidenceisusuallyneeded. Themost common serologic tests are the anti-streptolysin O (ASO) and anti-DNase B (ADB) titers. Where possible, age-specific reference ranges should be determined in a local population of healthy people without a recent group A streptococcal infection.

1	Because there is no definitive test, the diagnosis of ARF relies on the presence of a combination of typical clinical features together with evidence of the precipitating group A streptococcal infection, and the exclusion of otherdiagnoses. ThisuncertaintyledDr. T. DuckettJones in 1944 to develop a set of criteria (subsequently known as the Jones criteria)toaidinthediagnosis.Atthetimeofwriting,theJonescriteria were undergoing revision but had not yet been released. The existing diagnostic guideline is a World Health Organization update of the 1992JonesCriteria (Table 381-2),thoughitshouldbenotedthatother guidelines, including those from Australia and New Zealand, suggest more sensitive criteria for making the diagnosis in patients from settings or populations at high risk of ARF.

1	Patients with possible ARF should be followed closely to ensure that the diagnosis is confirmed, treatment of heart failure and other symptoms is undertaken, and preventive measures including commencement of secondary prophylaxis, inclusion on an ARF registry, and health education are commenced. Echocardiography should be performed on all possible cases to aid in making the diagnosis and to determine the severity at baseline of any carditis. Other tests that should be performed are listed in Table 381-3. There is no treatment for ARF that has been proven to alter the likelihood of developing, or the severity of, RHD. With the exception of treatment of heart failure, which may be life-saving in cases of severe carditis, the treatment of ARF is symptomatic.

1	All patients with ARF should receive antibiotics sufficient to treat the precipitating group A streptococcal infection (Chap. 173). Penicillin is the drug of choice and can be given orally (as phenoxymethyl penicillin, 500 mg [250 mg for children ≤27 kg] PO twice daily, or amoxicillin, 50 mg/kg [maximum, 1 g] daily, for 10 days) or as a 2002–2003 WOrlD health OrganIzatIOn CrIterIa fOr the DIagnOSIS Of rheuMatIC feVer anD rheuMatIC heart DISeaSe (BaSeD On the 1992 reVISeD jOneS CrIterIa) Primary episode of rheumatic fevera Two major or one major and two Recurrent attack of rheumatic fever in Two major or one major and two matic heart disease of preceding group A streptococcal Recurrent attack of rheumatic fever in Two minor manifestations plus evia patient with established rheumatic dence of preceding group A streptoheart diseaseb coccal infectionc Rheumatic chorea Other major manifestations or evidence of group A streptococcal

1	Rheumatic chorea Other major manifestations or evidence of group A streptococcal Chronic valve lesions of rheumatic Do not require any other criteria to heart disease (patients presenting for be diagnosed as having rheumatic the first time with pure mitral steno-heart disease sis or mixed mitral valve disease and/ or aortic valve disease)d Minor manifestations Clinical: fever, polyarthralgia Laboratory: elevated erythrocyte sedimentation rate or leukocyte counte Electrocardiogram: prolonged P-R interval Supporting evidence of a preceding Elevated or rising anti-streptolysin streptococcal infection within the last O or other streptococcal antibody, or 45 days A positive throat culture, or

1	Rapid antigen test for group A streptococcus, or aPatients may present with polyarthritis (or with only polyarthralgia or monoarthritis) and with several (three or more) other minor manifestations, together with evidence of recent group A streptococcal infection. Some of these cases may later turn out to be rheumatic fever. It is prudent to consider them as cases of “probable rheumatic fever” (once other diagnoses are excluded) and advise regular secondary prophylaxis. Such patients require close follow-up and regular examination of the heart. This cautious approach is particularly suitable for patients in vulnerable age groups in high-incidence settings. bInfective endocarditis should be excluded. cSome patients with recurrent attacks may not fulfill these criteria. dCongenital heart disease should be excluded. e1992 Revised Jones criteria do not include elevated leukocyte count as a laboratory minor manifestation (but do include elevated C-reactive protein), and do not include

1	disease should be excluded. e1992 Revised Jones criteria do not include elevated leukocyte count as a laboratory minor manifestation (but do include elevated C-reactive protein), and do not include recent scarlet fever as supporting evidence of a recent streptococcal infection.

1	Source: Reprinted with permission from WHO Expert Consultation on Rheumatic Fever and Rheumatic Heart Disease (2001: Geneva, Switzerland): Rheumatic Fever and Rheumatic Heart Disease: Report of a WHO Expert Consultation (WHO Tech Rep Ser, 923). Geneva, World Health Organization, 2004. single dose of 1.2 million units (600,000 units for children ≤27 kg) IM benzathine penicillin G. These may be used for the treatment of arthritis, arthralgia, and fever, once the diagnosis is confirmed. They are of no proven value in the treatment of carditis or chorea. Aspirin is the drug of choice, delivered at a dose of 50–60 mg/kg per day, up to a maximum of 80–100 mg/kg per day (4–8 g/d in adults) in four to five divided doses. At higher doses, the patient should be monitored for symptoms of salicylate toxicity such as nausea, vomiting, or tinnitus; if symptoms appear, lower doses should be used. When the acute

1	White blood cell count Erythrocyte sedimentation rate C-reactive protein Blood cultures if febrile Electrocardiogram (if prolonged P-R interval or other rhythm abnormality, repeat in 2 weeks and again at 2 months if still abnormal) Chest x-ray if clinical or echocardiographic evidence of carditis Echocardiogram (consider repeating after 1 month if negative) Throat swab (preferably before giving antibiotics)—culture for group A streptococcus Antistreptococcal serology: both anti-streptolysin O and anti-DNase B titers, if available (repeat 10–14 days later if first test not confirmatory) Tests for Alternative Diagnoses, Depending on Clinical Features Copper, ceruloplasmin, antinuclear antibody, drug screen for choreiform movements Serology and autoimmune markers for arboviral, autoimmune, or reactive arthritis Source: Reprinted with permission from Menzies School of Health Research.

1	Serology and autoimmune markers for arboviral, autoimmune, or reactive arthritis Source: Reprinted with permission from Menzies School of Health Research. symptoms are substantially resolved, usually within the first 2 weeks, patients on higher doses can have the dose reduced to 50–60 mg/kg per day for a further 2–4 weeks. Fever, joint manifestations, and elevated acute-phase reactants sometimes recur up to 3 weeks after the medication is discontinued. This does not indicate a recurrence and can be managed by recommencing salicylates for a brief period. Naproxen at a dose of 10–20 mg/kg per day is a suitable alternative to aspirin and has the advantage of twice-daily dosing.

1	CONGESTIVE HEarT FaILUrE Glucocorticoids The use of glucocorticoids in ARF remains controversial. Two meta-analyses have failed to demonstrate a benefit of glucocorticoids compared to placebo or salicylates in improving the shortor longer term outcome of carditis. However, the studies included in these meta-analyses all took place >40 years ago and did not use medications in common usage today. Many clinicians treat cases of severe carditis (causing heart failure) with glucocorticoids in the belief that they may reduce the acute inflammation and result in more rapid resolution of failure. However, the potential benefits of this treatment should be balanced against the possible adverse effects. If used, prednisone or prednisolone is recommended at a dose of 1–2 mg/kg per day (maximum, 80 mg), usually for a few days or up to a maximum of 3 weeks. See Chap. 280.

1	See Chap. 280. Traditional recommendations for long-term bed rest, once the cornerstone of management, are no longer widely practiced. Instead, bed rest should be prescribed as needed while arthritis and arthralgia are present and for patients with heart failure. Once symptoms are well controlled, gradual mobilization can commence as tolerated.

1	Medications to control the abnormal movements do not alter the duration or outcome of chorea. Milder cases can usually be managed by providing a calm environment. In patients with severe chorea, carbamazepine or sodium valproate is preferred to haloperidol. A response may not be seen for 1–2 weeks, and medication should be continued for 1–2 weeks after symptoms subside. There is recent evidence that corticosteroids are effective and lead to more rapid 2153 symptom reduction in chorea. They should be considered in severe or refractory cases. Prednisone or prednisolone may be commenced at 0.5 mg/kg daily, with weaning as early as possible, preferably after 1 week if symptoms are reduced, although slower weaning or temporary dose escalation may be required if symptoms worsen.

1	Small studies have suggested that IVIg may lead to more rapid resolution of chorea but have shown no benefit on the shortor long-term outcome of carditis in ARF without chorea. In the absence of better data, IVIg is not recommended except in cases of severe chorea refractory to other treatments. Untreated, ARF lasts on average 12 weeks. With treatment, patients are usually discharged from hospital within 1–2 weeks. Inflammatory markers should be monitored every 1–2 weeks until they have normalized (usually within 4–6 weeks), and an echocardiogram should be performed after 1 month to determine if there has been progression of carditis. Cases with more severe carditis need close clinical and echocardiographic monitoring in the longer term.

1	Once the acute episode has resolved, the priority in management is to ensure long-term clinical follow-up and adherence to a regimen of secondary prophylaxis. Patients should be entered onto the local ARF registry (if present) and contact made with primary care practitioners to ensure a plan for follow-up and administration of secondary prophylaxis before the patient is discharged. Patients and their families should also be educated about their disease, emphasizing the importance of adherence to secondary prophylaxis. Ideally, primary prevention would entail elimination of the major risk factors for streptococcal infection, particularly overcrowded housing. This is difficult to achieve in most places where ARF is common.

1	Therefore, the mainstay of primary prevention for ARF remains primary prophylaxis (i.e., the timely and complete treatment of group A streptococcal sore throat with antibiotics). If commenced within 9 days of sore throat onset, a course of penicillin (as outlined above for treatment of ARF) will prevent almost all cases of ARF that would otherwisehavedeveloped.InsettingswhereARFandRHDarecommonbut microbiologic diagnosis of group A streptococcal pharyngitis is not available, such as in resource-poor countries, primary care guidelines often recommend that all patients with sore throat be treated with penicillin or, alternatively, that a clinical algorithm be used to identify patients with a higher likelihood of group A streptococcal pharyngitis. Although imperfect, such approaches recognize the importance of ARF prevention at the expense of overtreating many cases of sore throat that are not caused by group A streptococcus.

1	The mainstay of controlling ARF and RHD is secondary prevention. Because patients with ARF are at dramatically higher risk than the generalpopulationofdevelopingafurtherepisodeofARFafteragroupA streptococcal infection, they should receive long-term penicillin prophylaxis to prevent recurrences. The best antibiotic for secondary prophylaxis is benzathine penicillin G (1.2 million units, or 600,000 units if ≤27 kg) delivered every 4 weeks. It can be given every 3 weeks, or even every 2 weeks, to persons considered to be at particularly high risk, although in settings where good compliance with an every- 4-week dosing schedule can be achieved, more frequent dosing is rarely needed. Oral penicillin V (250 mg) can be given twice daily instead but is less effective than benzathine penicillin G. Penicillin-allergic patients can receive erythromycin (250 mg) twice daily.

1	The duration of secondary prophylaxis is determined by many factors, in particular the duration since the last episode of ARF Category of Patient Duration of Prophylaxis Rheumatic fever without carditis For 5 years after the last attack or 21 years of age (whichever is longer) Rheumatic fever with carditis but no For 10 years after the last attack, or residual valvular disease 21 years of age (whichever is longer) Rheumatic fever with persistent val-For 10 years after the last attack, or vular disease, evident clinically or on 40 years of age (whichever is longer); echocardiography sometimes lifelong prophylaxis aThese are only recommendations and must be modified by individual circumstances as warranted. Note that some organizations recommend a minimum of 10 years of prophylaxis after the most recent episode, or until 21 years of age (whichever is longer), regardless of the presence of carditis with the initial episode.

1	Source: Adapted from AHA Scientific Statement Prevention of Rheumatic Fever and Diagnosis and Treatment of Acute Streptococcal Pharyngitis. Circulation 119:1541, 2009. (recurrences become less likely with increasing time), age (recurrences are less likely with increasing age), and the severity of RHD (if severe, it may be prudent to avoid even a very small risk of recurrence because of the potentially serious consequences) (Table 381-4). Secondary prophylaxisisbestdeliveredaspartofacoordinatedRHDcontrolprogram,basedaroundaregistryofpatients.Registriesimprovetheability to follow patients and identify those who default from prophylaxis and to institute strategies to improve adherence.

1	VIDEO 381-1a Transthoracic echocardiographic images of a 9-year-old girl with first episode of acute rheumatic fever. Images demonstrate the typical echocardiographic findings of acute rheumatic carditis. The valve leaflets are relatively thin and highly mobile. The failure of coaptation of the mitral valve leaflets is the result of chordal elongation and annular dilatation. The mitral valve regurgitation is moderate with a typical posterolaterally directed regurgitant jet of rheumatic carditis. A. Acute rheumatic carditis (apical four-chamber view echocardiogram).

1	VIDEO 381-1B Transthoracic echocardiographic images of a 9-year-old girl with first episode of acute rheumatic fever. Images demonstrate the typical echocardiographic findings of acute rheumatic carditis. The valve leaflets are relatively thin and highly mobile. The failure of coaptation of the mitral valve leaflets is the result of chordal elongation and annular dilatation. The mitral valve regurgitation is moderate with a typical posterolaterally directed regurgitant jet of rheumatic carditis. B. Acute rheumatic carditis (apical four-chamber view color Doppler echocardiogram).

1	VIDEO 381-1C Transthoracic echocardiographic images of a 9-year-old girl with first episode of acute rheumatic fever. Images demonstrate the typical echocardiographic findings of acute rheumatic carditis. The valve leaflets are relatively thin and highly mobile. The failure of coaptation of the mitral valve leaflets is the result of chordal elongation and annular dilatation. The mitral valve regurgitation is moderate with a typical posterolaterally directed regurgitant jet of rheumatic carditis. C. Acute rheumatic carditis (parasternal long-axis view echocardiogram).

1	VIDEO 381-1D Transthoracic echocardiographic images of a 9-year-old girl with first episode of acute rheumatic fever. Images demonstrate the typical echocardiographic findings of acute rheumatic carditis. The valve leaflets are relatively thin and highly mobile. The failure of coaptation of the mitral valve leaflets is the result of chordal elongation and annular dilatation. The mitral valve regurgitation is moderate with a typical posterolaterally directed regurgitant jet of rheumatic carditis. D. Acute rheumatic carditis (parasternal long-axis view color Doppler echocardiogram). VIDEO 381-2a Transthoracic echocardiographic images are from a 5-year-old boy with chronic rheumatic heart disease with severe mitral valve regurgitation and moderate mitral valve stenosis.

1	VIDEO 381-2a Transthoracic echocardiographic images are from a 5-year-old boy with chronic rheumatic heart disease with severe mitral valve regurgitation and moderate mitral valve stenosis. Images demonstrate the typical echocardiographic findings in advanced chronic rheumatic heart disease. Both the anterior and posterior mitral valve leaflets are markedly thickened. During diastole, the motion of the anterior mitral valve leaflet tip is restricted with doming of the body of the leaflet toward the interventricular septum. This appearance is commonly described as a “hockey stick” or an “elbow” deformity. A. Chronic rheumatic heart disease (parasternal long-axis view).

1	A. Chronic rheumatic heart disease (parasternal long-axis view). VIDEO 381-2B Transthoracic echocardiographic images are from a 5-year-old boy with chronic rheumatic heart disease with severe mitral valve regurgitation and moderate mitral valve stenosis. Images demonstrate the typical echocardiographic findings in advanced chronic rheumatic heart disease. Both the anterior and posterior mitral valve leaflets are markedly thickened. During diastole, the motion of the anterior mitral valve leaflet tip is restricted with doming of the body of the leaflet toward the interventricular septum. This appearance is commonly described as a “hockey stick” or an “elbow” deformity. B. Chronic rheumatic heart disease (apical two-chamber view echocardiogram).

1	Systemic sclerosis (SSc) is an uncommon connective tissue disorder characterized by multisystem involvement, heterogeneous clinical manifestations, a chronic and often progressive course, and significant disability and mortality. Multiple genes contribute to disease susceptibility; however, environmental exposures are likely to play a major role in causing SSc. The early stage of the disease is associated with prominent inflammatory features. Over time, functional and structural alterations in multiple vascular beds and progressive visceral organ dysfunction due to fibrosis dominate the clinical picture. Although thickened skin (scleroderma) isthe distinguishing hallmark of SSc, skin induration can occur in localized forms of scleroderma and other disorders (Table 382-1). Patients with SSc can be broadly Guttate (plaque) morphea, diffuse (pansclerotic) morphea, bullous morphea Linear scleroderma, coup de sabre, hemifacial atrophy

1	Guttate (plaque) morphea, diffuse (pansclerotic) morphea, bullous morphea Linear scleroderma, coup de sabre, hemifacial atrophy Stiff skin syndrome Diabetic scleredema and scleredema of Buschke Scleromyxedema (papular mucinosis) Chronic graft-versus-host disease Diffuse fasciitis with eosinophilia (Shulman’s disease, eosinophilic fasciitis) Chemically induced and drug-associated scleroderma-like conditions Vinyl chloride–induced disease Eosinophilia-myalgia syndrome (associated with L-tryptophan) Nephrogenic systemic fibrosis (associated with gadolinium) Skin involvement Indolent onset. Limited to fingers, distal to elbows, face; slow progression phenomenon ment, sometimes by years; may be associated with critical ischemia in the digits Interstitial lung Slowly progressive, disease generally mild Rapid onset. Diffuse: fingers, extremities, face, trunk; rapid progression Onset coincident with skin involvement; critical ischemia less common

1	Onset coincident with skin involvement; critical ischemia less common Severe arthralgia, carpal tunnel syndrome, tendon friction rubs

1	Frequent, early onset and progression, can be severe grouped into diffuse cutaneous andlimitedcutaneous subsets defined by the pattern of skin involvement, as well as clinical and laboratory features (Table 382-2). Diffuse cutaneous SSc (dcSSc) is associated with extensive skin induration, starting in the fingers and ascending from distal to proximal limbs and the trunk. These patients often have early interstitial lung disease and acute renal involvement. In contrast, in patients with limited cutaneous SSc (lcSSc), Raynaud’s phenomenon may precede other manifestations of SSc by years. In these patients, skin involvement remains limited to the fingers (sclerodactyly), distal limbs, and face, and the trunk is not affected. The constellation of calcinosis cutis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia, seen in some lcSSc patients, is termed the CREST syndrome. Visceral organ involvement in lcSSc tends to show insidious progression, and pulmonary

1	dysmotility, sclerodactyly, and telangiectasia, seen in some lcSSc patients, is termed the CREST syndrome. Visceral organ involvement in lcSSc tends to show insidious progression, and pulmonary arterial hypertension (PAH), interstitial lung disease, hypothyroidism, and primary biliary cirrhosis may occur as late complications. In some patients, Raynaud’s phenomenon and other characteristic features of SSc occur in the absence of skin thickening. This syndrome has been termed SSc sine scleroderma.

1	SSc is an acquired sporadic disease with a worldwide distribution and affecting all races. In the United States, the incidence is estimated at 9–19 cases per million per year. The only community-based survey of SSc yielded a prevalence of 286 cases per million. There are an estimated 100,000 cases in the United States, although this number may be significantly higher if patients who do not meet strict classification criteria are also included. Rates of SSc in England, Australia, and Japan appear to be lower. Age, gender, and ethnicity are important in disease susceptibility. In common with other connective tissue diseases, SSc shows a strong female predominance (4.6:1), which is most pronounced in the childbearing years and declines after menopause. Although SSc can present at any age, the peak age of onset for both limited and diffuse cutaneous forms is 30–50 years. The incidence is higher in blacks than whites, and disease onset occurs at an earlier age. Furthermore, blacks with SSc

1	peak age of onset for both limited and diffuse cutaneous forms is 30–50 years. The incidence is higher in blacks than whites, and disease onset occurs at an earlier age. Furthermore, blacks with SSc are more likely to have diffuse cutaneous disease associated with interstitial lung involvement and a worse prognosis.

1	In general, SSc shows modest heritability, and the genetic associations identified to date make only a small contribution to disease susceptibility. Concordance rates for SSc are low (4.7%) in monozygotic twins, although concordance for antinuclear antibody (ANA) positivity is significantly higher. On the other hand, evidence for genetic contribution to disease susceptibility is provided by the observation that 1.6% of SSc patients have a first-degree relative with SSc, a prevalence rate markedly increased compared to the general population. The risk of Raynaud’s phenomenon, interstitial lung disease, and other autoimmune diseases, including systemic lupus erythematosus (SLE) (Chap. 378), rheumatoid arthritis (Chap. 380), andautoimmunethyroiditis(Chap. 405),isalsoincreased.Approaches to study therole of geneticsin SScuse candidategenesingle nucleotide polymorphism (SNP) analysis and genome-wide association studies (GWASs). Candidate gene studies in SSc have shown associations with

1	study therole of geneticsin SScuse candidategenesingle nucleotide polymorphism (SNP) analysis and genome-wide association studies (GWASs). Candidate gene studies in SSc have shown associations with multiple gene variants, many related to B and T lymphocyte activation and signaling (BANK1, BLK, CD247, CSK, IRAK1, IL2RA, PTPN22, and TNIP1). IRAK1, which codes for a gene involved in both innate and adaptive immunity, is the first X-linked gene associated with SSc and may contribute to female predominance. Other gene variants associatedwithSScareinvolved ininnateimmunityand the interferon pathways (IRF5, IRF7, STAT4, TNFAIP3, and TLR2). In addition, candidate gene studies and GWAS both identified association with genes in the major histocompatibility complex (MHC), including NOTCH4 and PSORSC1. In addition to disease susceptibility, some of these geneticloci are associated with particularSSc diseasemanifestations or serologic subsets, including interstitial lung disease (ILD) (CTGF,

1	In addition to disease susceptibility, some of these geneticloci are associated with particularSSc diseasemanifestations or serologic subsets, including interstitial lung disease (ILD) (CTGF, CD226), PAH (TNIP1), and scleroderma renal crisis (HLA-DRB1*). Although the functional consequences of these gene variants are currentlynotwellunderstood,theymayresultinalteredimmunefunction, leading to increased susceptibility to autoimmunity and inflammation. Of note, many of the genetic variants associated with SSc are also seen in other autoimmune disorders, including SLE, rheumatoid arthritis, and psoriasis, suggesting common pathways shared among these conditions. The genetic associations identified to date only explain a fraction of the heritability of SSc, and GWASs, fine mapping, and resequencing of DNA regions of interest to identify additional genetic susceptibility factors in SSc, particularly rare variants, are currently ongoing.

1	Given the relatively modest geneticcontribution to diseasesusceptibility, environmental factors, such as infectious agents, intestinal microbiota, and occupational, dietary, and drug exposures, are likely to play amajor roleincausingSSc.Patientswith SSc showevidenceofchronic infection of lesional tissue with Epstein-Barr virus (EBV). They also have increased antibodies to human cytomegalovirus (hCMV), and anti–topoisomerase I (Scl-70) autoantibodies recognize hCMV-associated antigenic epitopes, suggesting molecular mimicry as a possible mechanistic link between hCMV infection and SSc. An epidemic of a novel syndrome with features suggestive of SSc occurred in Spain in the 1980s. The outbreak, termed toxic oil syndrome, was linked to contaminated rapeseed oils used for cooking. Another epidemic outbreak, termed eosinophilia-myalgia syndrome (EMS), occurred a decade later andwaslinkedtotheconsumptionof l-tryptophan-containingdietary supplements. Although both of these novel

1	Another epidemic outbreak, termed eosinophilia-myalgia syndrome (EMS), occurred a decade later andwaslinkedtotheconsumptionof l-tryptophan-containingdietary supplements. Although both of these novel toxic-epidemic syndromes were characterized by scleroderma-like chronic skin changes and variable visceral organ involvement, they were associated with clinical, pathologic, and laboratory features distinguishing them from SSc. Occupational exposures tentatively linked with SSc include silica dust in miners, polyvinyl chloride, epoxy resins, and aromatic hydrocarbons including toluene and trichloroethylene. Drugs implicated in SSc-like illnesses include bleomycin, pentazocine, and cocaine, and appetite suppressants linked with pulmonary hypertension. Although case reports and series describing SSc in women with silicone breast implants had raisedconcern regarding a possiblecausal roleofsilicone 2156 in SSc, large-scale epidemiologic investigations found no evidence of increased prevalence

1	SSc in women with silicone breast implants had raisedconcern regarding a possiblecausal roleofsilicone 2156 in SSc, large-scale epidemiologic investigations found no evidence of increased prevalence of SSc.

1	The following three cardinal pathophysiologic processes account for theproteanclinicalmanifestationsofSSc:(1)diffusemicroangiopathy, (2) inflammation and autoimmunity, and (3) visceral and vascular fibrosis in multiple organs (Fig. 382-1). Autoimmunity and altered vascular reactivity are early manifestations. Complex and dynamic interplay between these processes initiates and then amplifies the fibrotic process.

1	No single animal model of SSc fully reproduces the three cardinal processes that underlie the pathogenesis, but some recapitulate selected aspects of the human disease, including fibrosis, microvascular involvement, and autoimmunity. Tight-skin mice (Tsk1) develop spontaneous skin thickening due to a mutation in the fibrillin-1 gene. The mutant fibrillin-1 protein disrupts extracellular matrix assembly and causes aberrant activation of transforming growth factor β (TGF-β). Fibrillin-1 mutations are associated with Marfan’s disease as well as the stiff skin syndrome but not SSc. Skin and lung fibrosis can be induced in mice by injection of bleomycin, HOCl, or double-stranded RNA or by transplantation of human leukocyte antigen (HLA)–mismatched bone marrow or spleen cells. Targeted genetic modifications in mice give rise to new disease models for dissecting the pathogenetic roles of individual molecules, cell types, and networks. For example, mice lacking Smad3, an intracellular TGF-β

1	modifications in mice give rise to new disease models for dissecting the pathogenetic roles of individual molecules, cell types, and networks. For example, mice lacking Smad3, an intracellular TGF-β signal transducer, adiponectin, or the nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ or overexpressing Wnt10b

1	Vascular Injury Endothelial cell activation Platelet activation; ROS Leukocyte Recruitment CD4+ CD8+ T cells Activated monocytes/macrophages Activated B cells; innate immunity Th2 Cytokines TGF-˜, CTGF, PDGF Chemokines Fibroblast Activation Fibrocyte differentiation Myofibroblast differentiation Impaired mesencymal cell Epithelial cell/endothelial cell to mesenchyme transaction apoptosis Collagen, connective tissue accumulation Extracellular matrix reorganization, contraction Impaired matrix degradation Autoantibodies Endothelin-1 Obliterative Vasculopathy Tissue Hypoxia

1	FIGUrE 382-1 Initial vascular injury in a genetically susceptible individual leads to functional and structural vascular alterations, inflammation, and autoimmunity. The inflammatory and immune responses initiate and sustain fibroblast activation and differentiation, resulting in pathologic fibrogenesis and irreversible tissue damage. Vascular damage results in tissue ischemia that further contributes to progressive fibrosis and atrophy. CTGF, connective tissue growth factor; PDGF, platelet-derived growth factor; TGF-β, transforming growth factor β. or adiponectin were either resistant or hypersensitive to chemically induced experimental scleroderma. These mouse models have potential utility in preclinical evaluation of potential therapies.

1	Involvement of small blood vessels in SSc affects multiple vascular beds and has important clinical sequelae including Raynaud’s phenomenon, ischemic digital ulcers, scleroderma renal crisis, and PAH. Raynaud’s phenomenon,anearlydiseasemanifestation,ischaracterizedbyanaltered blood-flow response to cold challenge. This initially reversible functional abnormality is associated with autonomic and peripheral nervous system alterations, including impaired production of the neuropeptide calcitonin gene-related peptide from sensory afferent nerves and heightened sensitivity of α2-adrenergic receptorsonvascular smooth-muscle cells. Isolated (primary) Raynaud’s phenomenon is extremely common and generally benign and nonprogressive. In contrast, SSc-associated Raynaud’s phenomenon is often progressive and complicated by irreversible structural changes, culminating in ischemic digital ulcers and loss of digits. Viruses, vascularcytotoxicfactors, thrombogenicmicroparticles, complementand

1	progressive and complicated by irreversible structural changes, culminating in ischemic digital ulcers and loss of digits. Viruses, vascularcytotoxicfactors, thrombogenicmicroparticles, complementand autoantibodiestophospholipids,β2glycoproteinI(β2GPI),andendothelialcellsaresuspectedtriggersofendothelialcellinjuryinSSc.Endothelial injury results in dysregulated production of endothelium-derived vasodilatory(nitricoxideandprostacyclin)andvasoconstricting(endothelin-1) substances, as well as increased expression of intercellular adhesion molecule1(ICAM-1)andothersurfaceadhesionmolecules.Microvessels show enhanced permeability and transendothelial leukocyte diapedesis, abnormal activation of coagulation cascades, elevated thrombin production, and impaired fibrinolysis. Spontaneous platelet aggregation causes release of serotonin, platelet-derived growth factor (PDGF), and platelet alphagranulesincludingthromboxane,apotentvasoconstrictor.Smoothmuscle cell–like myointimal cells

1	platelet aggregation causes release of serotonin, platelet-derived growth factor (PDGF), and platelet alphagranulesincludingthromboxane,apotentvasoconstrictor.Smoothmuscle cell–like myointimal cells proliferate, the basement membrane is thickenedand reduplicated,and fibrosis oftheadventitial layers develops. The vasculopathic process affects capillaries, as well as arterioles, and even large vessels in many organs, resulting in reduced blood flow and tissue ischemia. Progressive luminal occlusion due to intimal and medial hypertrophy, combined with persistent endothelial cell damage and adventitial fibrosis, establish a vicious cycle that culminates in the striking absence of blood vessels (rarefaction) in late-stage disease. Recurrent ischemia-reperfusiongeneratesreactiveoxygenspecies(ROS)thatfurther damage the endothelium through peroxidation of membrane lipids. Paradoxically, the process of revascularization that normally reestablishes blood flow to ischemic tissue is defective in

1	damage the endothelium through peroxidation of membrane lipids. Paradoxically, the process of revascularization that normally reestablishes blood flow to ischemic tissue is defective in SSc despite elevated levels of vascular endothelial growth factor (VEGF) and other angiogenic factors. Moreover, the number of bone marrow–derived circulating endothelial progenitor cells is reduced. Thus, widespread capillary loss, obliterative vasculopathy of small and medium-sized arteries, and failure to repair damaged vessels are hallmarks of SSc.

1	IMMUNE DYSrEGULaTION Cellular Immunity The following observations highlight the autoimmune nature of SSc: presence of circulating autoantibodies; familial clustering of SSc with other autoimmune diseases; detection of immune cells, including T cells with oligoclonal antigen receptors, in target organs; elevated circulating levels and spontaneous secretion from blood mononuclear cells of inflammatory cytokines and chemokines such as interleukin (IL) 1, IL-4, IL-10, IL-17, IL-33, CCL2, and CXCL4; and the association with variants in genes functionally implicated in immune responses. Genetic studies in SSc reveal strong and consistent associations with major histocompatibility locus alleles, as well as non-HLA-linked genes encoding mediators of both adaptive and innate immune responses (CD247, STAT4, IRF5, CD226, and TNFSF4). In early SSc, mononuclear inflammatory cell infiltrates comprised of activated T cells, monocytes/macrophages, and dendritic cells can be seen in skin, lungs, and

1	STAT4, IRF5, CD226, and TNFSF4). In early SSc, mononuclear inflammatory cell infiltrates comprised of activated T cells, monocytes/macrophages, and dendritic cells can be seen in skin, lungs, and other affected organs prior to appearance of fibrosis or vascular damage. Dendritic cells and T cells can often be found in close proximity to activated fibroblasts and myofibroblasts. Tissue-infiltrating T cells express CD45 and HLA-DR activation markers and display restricted T cell receptor signatures indicative of oligoclonal expansion in response to (unknown) antigen.

1	Abbreviations: dcSSc, diffuse cutaneous SSc; GAVE, gastric antral vascular ectasia; ILD, interstitial lung disease; lcSSc, limited cutaneous SSc; MCTD, mixed connective tissue disease; PAH, pulmonary arterial hypertension; SLE, systemic lupus erythematosus.

1	Circulating T cells have elevated levels of chemokine receptors and α1 integrin adhesion molecules, accounting for their enhanced binding to endothelium and to fibroblasts. Endothelial cells express ICAM-1 and otheradhesionmoleculesthatfacilitateleukocytediapedesis.Activated macrophages and T cells show a TH2-polarized type 2 immune response driven by dendritic cells and thymic stromal lymphopoietin. TH2 cytokines such as IL-4 and IL-13 induce fibroblast activation and alternate M2 macrophage polarization, whereas the TH1 cytokine interferon γ (IFN-γ) blocks cytokine-mediated fibroblast activation. Alternately activated M2 macrophages produce TGF-β and promote fibrosis. Although the frequency of circulating regulatory T cells that enforce immune tolerance is elevated in SSc, their immunosuppressive function is defective. Molecular characterization of SSc skin biopsies using DNA microarrays identifies a subset showing markedly elevated expression of inflammation-associated genes,

1	function is defective. Molecular characterization of SSc skin biopsies using DNA microarrays identifies a subset showing markedly elevated expression of inflammation-associated genes, particularly chemokines andtheirreceptors,interferonresponsegenes,andmediators of innate immunity. Evidence of activated innate immunity and toll-like receptor signaling, indicative of activation by type 1 interferon produced by plasmacytoid dendritic cells, is prominent in peripheral blood cells.

1	Humoral autoimmunity Circulating ANAs can be detected in virtually all patients with SSc. In addition, a number of SSc-specific autoantibodies have been described. These SSc-specific antibodies show strong association with distinct disease endophenotypes (Table 382-3). While most are directed against intracellular proteins associated with cell proliferation, such as topoisomerase I and RNA polymerases I, II, and III, others are directed against cell-surface antigens, receptors, or secreted proteins. Autoantibodieshaveclinicalutilityasdiagnosticandprognosticbiomarkers in SSc, and some, such as antibodies directed against the angiotensin II receptor or the PDGF receptor, may have a direct pathogenic role.

1	A variety of mechanisms have been proposed for the development of autoantibodies in SSc. Proteolytic cleavage, increased expression, or altered subcellular localization of certain cellular proteins in SSc could lead to their recognition as neoepitopes by the immune system, resulting in breakdown of immune tolerance. B cells are implicated in both the autoimmune and fibrotic process in SSc. In addition to antibody production, B cells also present antigen, secrete IL-6 and TGF-β, and modulate T cell and dendritic cell function.

1	Fibrosis affecting multiple organs, a distinguishing feature of SSc, is characterized by progressive replacement of normal tissue architecture with dense, stiff, and acellular connective tissue. Fibrosis characteristically follows, and is thought to be a consequence of, inflammation, autoimmunity,andmicrovasculardamage.Fibroblastsaremesenchymal 2157 cells responsible for maintaining the functional and structural integrity of connective tissue. Upon activation by TGF-β and other extracellular cues, fibroblasts proliferate; migrate; secrete collagens, growth factors, chemokines, and cytokines; and transdifferentiate into contractile myofibroblasts. Under normal conditions,these fibroticresponses constitute self-limited physiologic remodeling necessary for tissue repair and regeneration. When these responses become sustained and amplified, pathologic fibrosis results. Autocrine stimulatory signaling by endogenously produced TGF-β and fibrotic mediators such as hypoxia, ROS, thrombin, Wnt

1	these responses become sustained and amplified, pathologic fibrosis results. Autocrine stimulatory signaling by endogenously produced TGF-β and fibrotic mediators such as hypoxia, ROS, thrombin, Wnt ligands, connective tissue growth factor (CTGF), PDGF, lysophosphatidicacid,endothelin-1,mechanicalforces,andendogenous ligands for toll-like receptors are responsible for maintaining sustained fibroblast activation underlying progressive fibrosis in SSc.

1	In addition to tissue-resident fibroblasts and transformation of epithelial cells into fibroblasts, bone marrow–derived circulating mesenchymal progenitor cells also contribute to fibrosis. The factors that regulate the differentiation of mesenchymal progenitor cells and their trafficking from the circulation into lesional tissue are unknown. Epithelial and endothelial cells, mesenchymal progenitor cells, and tissuefibroblastscandifferentiateintosmooth-muscle-likemyofibroblasts. Although myofibroblasts can be detected transiently during normal wound healing, they persist in fibrotic tissue, possibly due to resistance toapoptosis,andcontributetoscarformationviaproductionofcollagen and TGF-β and contraction of the surrounding extracellular matrix.

1	Explanted SSc fibroblasts may display an abnormally activated phenotype ex vivo, with variably increased rates of collagen gene tran scription, spontaneous ROS generation, and constitutive expression of alpha smooth-muscle actin stress fibers. The persistence of the “sclerodermaphenotype”ofthesecellsduringtheirserialpassageinvitromay reflect autocrine TGF-β stimulatory loops, deregulated microRNA expressions, histone acetylation, and other epigenetic modifications.

1	The distinguishing pathologic hallmark of SSc is the combination of widespread capillary loss and obliterative microangiopathy, together with fibrosis in the skin and internal organs. In early disease, perivascular inflammatory cell infiltrates composed of T lymphocytes, monocytes/macrophages, plasma cells, mast cells, and occasionally B cells may be detected in multiple organs. A bland noninflammatory obliterative vasculopathy as a late finding is prominent in the heart, lungs, kidneys, and intestinal tract. Fibrosis is found in the skin, lungs, gastrointestinal tract, heart, tendon sheaths, perifascicular tissue surrounding skeletal muscle, and some endocrine organs. In these tissues, accumulation of collagens, fibronectin, proteoglycans, tenascin, cartilage oligomeric matrix protein (COMP), and other structural macromolecules progressively disrupts normal architecture, resulting in impaired function of affected organs.

1	In the skin, fibrosis causes dermal expansion and obliteration of the hair follicles, eccrine glands, and other appendages (Fig. 382-2A). Collagen fiber accumulation is most prominent in the reticular dermis, and the fibrotic process invades the subjacent adipose layer with entrapment of adipocytes. With disease progression, the intradermal adipose layer is diminished and may completely disappear. The epidermis is atrophic, and the rete pegs are effaced.

1	Patchy infiltration of the alveolar walls with T lymphocytes, macrophages, and eosinophils occurs in early disease. With progression, interstitial fibrosis and vascular damage dominate the pathologic picture, often coexisting within the same lesions in patients with dcSSc. Pulmonaryfibrosis is characterizedbyexpansionofthe alveolar interstitium, with accumulation of collagen and other matrix proteins. The most common histologic pattern in SSc-associated ILD is nonspecific interstitial pneumonia (NSIP), distinct from the usual interstitial pneumonia (UIP) pattern characteristically seen in patients with FIGUrE 382-2 Pathologic findings in systemic sclerosis (SSc).

1	FIGUrE 382-2 Pathologic findings in systemic sclerosis (SSc). A. Left panel: The skin is thickened due the marked expansion of the dermis. Inset, higher magnification showing thick hyalinized collagen bundles replace skin appendages. Right panel: Inflammation in the reticular dermis. Mononuclear inflammatory cells infiltrating the dermis and intradermal adipose tissue. B. Early interstitial lung disease. Diffuse fibrosis of the alveolar septae and a chronic inflammatory cell infiltrate. Trichrome stain. C. Pulmonary arterial obliterative vasculopathy. Striking intimal hyperplasia and narrowing of the lumen of a small pulmonary artery, with minimal interstitial fibrosis, in a patient with limited cutaneous SSc.

1	idiopathic pulmonary fibrosis (Fig. 382-2B). Progressive thickening of the alveolar septae results in obliteration of the airspaces and loss of pulmonary blood vessels. This process impairs gas exchange and contributes to pulmonary hypertension. Intimal thickening of the pulmonary arteries, best seen with elastin stain, underlies pulmonary hypertension (Fig. 382-2C) and, at autopsy, is often associated with multiple pulmonary emboli and evidence of myocardial fibrosis.

1	Pathologicchangescanbefoundatanylevelfromthemouthtotherectum. The lower esophagus shows prominent atrophy of the muscular layers and characteristic vascular lesions; striated muscle in the upper third of the esophagus is generally spared. Replacement of the normal intestinal tract architecture results in diminished peristaltic activity, withgastroesophagealreflux,dysmotility, andsmall-bowel obstruction. Chronic reflux is associated with esophageal inflammation, ulcerations, and stricture formation and may lead to Barrett’s metaplasia. In the kidneys, vascular lesions affecting the interlobular and arcuate arteries predominate. Chronic renal ischemia is associated with shrunken glomeruli. Acute scleroderma renal crisis is associated with a classic thrombotic microangiopathic pathology: reduplication of elastic lamina, marked intimal proliferation, and narrowing of the lumen in small renal arteries, commonly accompanied by thrombosis and hemolysis.

1	The heart is frequently affected, with prominent involvement of the myocardium and pericardium. The characteristic arteriolar lesions are concentric intimal hypertrophy and luminal narrowing, accompanied by contraction band necrosis reflecting ischemia-reperfusion injury andmyocardialfibrosis. Fibrosisof the conduction systemis common, especially at the sinoatrial node. Despite the prominent role of ischemia in SSc, the frequency of atherosclerotic coronary artery disease is comparable to the general population. Synovitis may be found in early SSc; however, with disease progression, the synovium becomes fibrotic. Fibrosis of tendon sheaths and fascia produces palpable and sometimes audible tendon friction rubs. Inflammation and, in later stages, atrophy and fibrosis of the muscles are common findings. Fibrosis of the thyroid gland and of the minor salivary glands may be seen.

1	Virtually every organ can be clinically affected (Table 382-4). Most patients with SSc can be classified as lcSSc or dcSSc (Table 382-2). Although stratification of SSc patients into diffuse and limited cutaneous subsets is useful, disease expression is far more complex, and several distinct endophenotypes exist within each subset. For example, 10–15% of patients with lcSSc develop PAH without significant ILD. Other patients have systemic features of SSc without appreciable skin involvement (SSc sine scleroderma). Unique clinical phenotypes of SSc associate with specific autoantibodies (Table 382-3). Patients with “overlap” have typical SSc features coexisting with clinical and laboratory evidence of another autoimmune disease such as polymyositis, Sjögren’s syndrome, polyarthritis, autoimmune liver disease, or SLE. aApproximately 10% of limited cutaneous SSc patients have SSc sine scleroderma.

1	The initial presentation is quite different in the diffuse and the limited cutaneous forms of the disease. In dcSSc, the interval between Raynaud’s phenomenon and onset of other disease manifestations is typically brief (weeks to months). Soft tissue swelling and intense pruritus are signs of the early inflammatory “edematous” phase. The fingers, hands, distal limbs, and face are usually affected first. Diffuse skin hyperpigmentation and carpal tunnel syndrome can occur. Arthralgias, muscle weakness, fatigue, and decreased joint mobility are common. During the ensuing weeks to months, the inflammatory edematous phase evolves into the “fibrotic” phase, with skin induration associated with hair loss, reduced production of skin oils, and a decline in sweating capacity. Progressive flexion contractures of the fingers ensue. The wrists, elbows, shoulders, hip girdles, knees, and ankles become stiff due to fibrosis of the supporting joint structures. While advancing skin involvement is the

1	of the fingers ensue. The wrists, elbows, shoulders, hip girdles, knees, and ankles become stiff due to fibrosis of the supporting joint structures. While advancing skin involvement is the most visible manifestation of early dcSSc, important and frequently clinically silent internal organ involvementdevelopsduringthisstage.Theinitial4yearsfromdisease onset is the period of rapidly evolving pulmonary and renal damage. If organ failure does not occur during this period, the systemic process may stabilize.

1	Compared to dcSSc, the course of lcSSc is characteristically more indolent. In these patients, the interval between Raynaud’s phenomenon and onset of manifestations such as gastroesophageal reflux, cutaneous telangiectasia, or soft tissue calcifications can be several years. On the other hand, scleroderma renal crisis and severe pulmonary fibrosis are uncommon in lcSSc. Clinically evident cardiac involvement and PAH develop in more than 15%. Overlap with keratoconjunctivitis sicca, polyarthritis, cutaneous vasculitis, and biliary cirrhosis is seen in some patients with lcSSc.

1	Raynaud’s phenomenon, the most frequent extracutaneous complication of SSc, is characterized by episodes of reversible vasoconstriction in the fingers and toes. Vasoconstriction may also affect the tip of the nose and earlobes. Attacks are triggered by a decrease in temperature, as well as emotional stress and vibration. Typical attacks start with pallor, followed by cyanosis of variable duration. Hyperemia ensues spontaneously or with rewarming of the digit. The progression of the three color phases reflects the underlying vasoconstriction, ischemia, and reperfusion.

1	Asmuch as3–5%ofthegeneralpopulationhas Raynaud’sphenomenon. In the absence of signs or symptoms of an underlying condition, Raynaud’s phenomenon is classified as primary and represents an exaggerated physiologic response to cold. Secondary Raynaud’s phenomenon can occur as a complication of SSc and other connective tissue diseases, hematologic and endocrine conditions, and occupational disorders, and can complicate the use of beta blockers and anticancer drugs such as cisplatin and bleomycin. Distinguishing primary versus secondary Raynaud’s phenomenon presents a diagnostic challenge. Primary Raynaud’s phenomenon is supported by the following: absence of an underlying cause; a family history of Raynaud’s phenomenon; absence of digital tissue necrosis, ulceration, or gangrene; and a negative ANA test. Secondary Raynaud’s phenomenon tends to develop at an older age (>30 years), is clinically more severe (episodes more frequent, prolonged, and painful), and is frequently associated with

1	ANA test. Secondary Raynaud’s phenomenon tends to develop at an older age (>30 years), is clinically more severe (episodes more frequent, prolonged, and painful), and is frequently associated with ischemic digital ulcers and loss of digits (Fig. 382-3). Nailfold capillaroscopy, where the cutaneous capillaries at the nail bed are viewed under a drop of grade B immersion oil using a low-power stereoscopic microscope, can be helpful in the evaluation of Raynaud’s phenomenon.Primary Raynaud’s phenomenonisassociated withnormal capillaries that appear as regularly spaced parallel vascular loops, whereas in patients with Raynaud’s associated with SSc and other connective tissue diseases, nailfold capillaries are distorted with widened and irregular loops, dilated lumen, and areas of vascular “dropout.” In addition to digits, cold-induced episodic Raynaud’s-like vasospasm has been documented in the pulmonary, renal, gastrointestinal, and coronary circulations in SSc.

1	While early-stage SSc is associated with edematous skin changes, skin thickening is the hallmark that distinguishes SSc from other connective tissue diseases. The distribution of skin thickening is invariably symmetric and bilateral. It typically starts in the fingers and then characteristically advances from distal to proximal extremities in an ascending fashion. The involved skin is firm, coarse, and thickened, and the extremities and trunk may be darkly pigmented. In some patients, diffuse tanning in the absence of sun exposure is a very early manifestation of skin involvement. In dark-skinned patients, vitiligolike hypopigmentation may occur. Because pigment loss spares the perifollicular areas, the skin may have a “salt-and-pepper” appearance, most prominently on the scalp, upper back, and chest. Dermal sclerosis due to collagen accumulation obliterates hair follicles, sweat glands, and eccrine and sebaceous glands, resulting in hair loss, decreased sweating, and dry skin.

1	back, and chest. Dermal sclerosis due to collagen accumulation obliterates hair follicles, sweat glands, and eccrine and sebaceous glands, resulting in hair loss, decreased sweating, and dry skin. Transverse creases on the dorsum of the fingers disappear (Fig. 382-4). Fixed flexion contractures of the fingers cause reduced hand mobility and lead to muscle atrophy. Skin thickening in combination with fibrosis of the subjacent tendons accounts for contractures of the wrists, elbows, and knees. Thick ridges at the neck due

1	FIGUrE 382-4 Sclerodactyly. Noteskinindurationonthefingers,andfixedflexioncontracturesattheproximalinterphalangealjointsinapatientwithlimitedcutaneoussystemicsclerosis(SSc). FIGUrE 382-3 Digital necrosis. Sharply demarcated necrosis of the fingertip in a patient with limited cutaneous systemic sclerosis (SSc) associated with severe Raynaud’s phenomenon. FIGUrE 382-5 Cutaneous vascular changes. A. Capillary changes at the nailfold in a patient with limited cutaneous systemic sclerosis (lcSSc). B. Telangiectasia on the face.

1	FIGUrE 382-5 Cutaneous vascular changes. A. Capillary changes at the nailfold in a patient with limited cutaneous systemic sclerosis (lcSSc). B. Telangiectasia on the face. to firm adherence of skin to the underlying platysma muscle interfere with neck extension. The face assumes a characteristic “mauskopf” appearance with taut and shiny skin, loss of wrinkles, and occasionally an expressionless facies due to reduced mobility of the eyelids, cheeks, andmouth.Thinningofthelipswithaccentuationofthecentralincisor teeth and fine wrinkles (radial furrowing) around the mouth complete the picture. Reduced oral aperture (microstomia) interferes with eating and oral hygiene. The nose assumes a pinched, beak-like appearance.

1	In established SSc, the skin is firmly bound to the subcutaneous fat (tethering) and undergoes thinning and atrophy. Telangiectasias are dilated skin capillaries 2–20 mm in diameter frequently seen in lcSSc. These lesions, reminiscent of hereditary hemorrhagic telangiectasia, are prominent on the face, hands, lips, and oral mucosa (Fig. 382-5).A greater number of telangiectasias correlates with the extent of microvascular complications,includingPAH.Breakdown ofatrophicskinleadsto chronic ulcerations at the extensor surfaces of the proximal interphalangealjoints,thevolarpadsofthefingertips,andbonyprominencessuchas the elbows and malleoli. Ulcers are painful and may become secondarily infected, resulting in osteomyelitis. Healing of ischemic fingertip ulcerations leaves characteristic fixed digital “pits.” Loss of soft tissue at the fingertipsdue to ischemiais frequentandmaybeassociated withstriking resorption of the terminal phalanges (acro-osteolysis) (Fig. 382-6).

1	Calcium deposits (calcinosis) in the skin and soft tissues occur in patients with lcSSc who are positive for anticentromere antibodies. The deposits, varying in size from tiny punctate lesions to large conglomerate masses, are composed of calcium hydroxyapatite crystals and can be readily visualized on plain x-rays. Frequent locations include the finger pads, palms, extensor surfaces of the forearms, and the olecranon and prepatellar bursae (Fig. 382-7). They may occasionally ulcerate through the overlying skin, producing drainage of chalky white material, pain, and local inflammation. Paraspinal soft tissue calcifications may cause neurologic complications. FIGUrE 382-6 Acro-osteolysis. Notedissolutionofterminalphalan-gesinapatientwithlong-standinglimitedcutaneoussystemicsclero-sis(lcSSc)andRaynaud’sphenomenon.

1	FIGUrE 382-6 Acro-osteolysis. Notedissolutionofterminalphalan-gesinapatientwithlong-standinglimitedcutaneoussystemicsclero-sis(lcSSc)andRaynaud’sphenomenon. Pulmonary involvement is frequent in SSc and is the leading cause of death. The two principal forms are ILD and pulmonary vascular disease. Patients with SSc frequently develop some degree of both complications. Less frequent pulmonary manifestations include aspiration pneumonitis complicating chronic gastroesophageal reflux, pulmonary hemorrhage due to endobronchial telangiectasia, obliterative bronchiolitis, pleural reactions, restrictive ventilatory defect due to chest wall fibrosis, spontaneous pneumothorax, and drug-induced lung toxicity. The incidence of lung cancer is increased.

1	Interstitial Lung Disease (ILD) Evidence of ILD can be found in up to 90% of patients with SSc at autopsy and 85% by thin-section high-resolution computed tomography (HRCT). In contrast, clinically significant ILD develops in 16–43%; the frequency varies depending on the detection method used. Risk factors include male gender, African-American race, diffuse skin involvement, severe gastroesophageal reflux, and the presence of topoisomerase I autoantibodies, as well as a low forced vital capacity (FVC) or single-breath diffusing capacity of the lung for carbon monoxide (DLco) at initial presentation. In these patients, the most rapid progression in lung disease occurs early in FIGUrE 382-7 Calcinosis cutis. Notelargecalcificdepositbreakingthroughtheskininapatientwithlimitedcutaneoussystemicsclerosis(lcSSc).

1	FIGUrE 382-7 Calcinosis cutis. Notelargecalcificdepositbreakingthroughtheskininapatientwithlimitedcutaneoussystemicsclerosis(lcSSc). FIGUrE 382-8 HRCT images of the chest from patients with systemic sclerosis. Top panel: Early interstitial lung disease. Mild changes with sub pleural reticulations and ground glass opacities in the lower lobes of the lung. Patient in supine position. Bottom panel: Extensive lung fibrosis with ground glass opacities, coarse reticular honeycombing, and traction bronchiectasis. (Courtesy of Rishi Agrawal, MD.) the course of the disease (within the first 3 years), when the FVC can decline by 30% per year.

1	Pulmonary involvement can remain asymptomatic until it is advanced. The most common presenting respiratory symptoms— exertional dyspnea, fatigue, and reduced exercise tolerance—are subtle and slowly progressive. A chronic dry cough may be present. Physical examination may reveal “Velcro” crackles at the lung bases. Pulmonary function testing (PFT) is a sensitive method for detecting early pulmonary involvement. The most common abnormalities are reductions in FVC, total lung capacity (TLC), and DLco. A reduction in DLcothatis significantly out of proportion to the reduction in lung volumes should raise suspicion for pulmonary vascular disease, but may also be due to anemia. Oxygen desaturation with exercise is common.

1	Chest radiography can rule out infection and other causes of pulmonary involvement, but compared to HRCT, it is relatively insensitive for detection of early ILD. HRCT shows subpleural reticular linear opacities and ground-glass opacifications, predominantly in the lower lobes, even in asymptomatic patients (Fig. 382-8). Additional HRCT findings include mediastinal lymphadenopathy, pulmonary nodules, traction bronchiectasis, and uncommonly, honeycomb changes. The extent of pulmonary interstitial changes on HRCT is a predictor of mortality in SSc. Bronchoalveolar lavage (BAL) can demonstrate inflammation in the lower respiratory tract and may be useful for ruling out infection. Although an elevated proportion of neutrophils (>2%) and/or eosinophils (>3%) in the BAL fluid is correlated with more extensive lung disease on HRCT and is associated with more rapid decline in FVC and reduced survival, BAL is not useful for identifying reversible alveolitis. Lung biopsy is indicated only in

1	more extensive lung disease on HRCT and is associated with more rapid decline in FVC and reduced survival, BAL is not useful for identifying reversible alveolitis. Lung biopsy is indicated only in patients with atypical findings on chest radiographs and should be thoracoscopically guided. The histologic pattern on lung biopsy may predict theriskofprogressionofILD.ThemostcommonpatterninSSc,NSIP, carries a better prognosis than UIP.

1	Pulmonary arterial Hypertension (PaH) PAH, defined as a mean pulmonary arterial pressure ≥25 mmHg with a pulmonary capillary wedge pressure≤15 mmHg, developsinapproximately15%ofpatientswithSSc and can occur in association with ILD or as an isolated abnormality. The natural history of SSc-associated PAH is variable, but in many patients, it follows a downhill course with development of right heart failure. The 2161 median survival of SSc patients with untreated PAH is 1 year following diagnosis. Risk factors for PAH include limited cutaneous disease, older age at disease onset, severe Raynaud’s phenomenon, and the presence of antibodies to centromere, U1-RNP, U3-RNP (fibrillarin), and B23.

1	The initial symptom of PAH is typically exertional dyspnea and reduced exercise capacity. With disease progression, angina, exertional near-syncope, and symptoms and signs of right-sided heart failure appear. Physical examination may show tachypnea, a prominent split S2heartsound,palpablerightventricularheave,elevatedjugularvenous pressure, and dependent edema. Doppler echocardiography provides a noninvasive method for estimating the pulmonary arterial pressure. In light of the poor prognosis of untreated PAH, all SSc patients should be screened for its presence at initial evaluation. Echocardiographic estimates of pulmonary arterial systolic pressures >40 mmHg at rest suggest PAH. Pulmonary function testing may show a reduced DLcoin isolation or out of proportion with the severity of restriction. Right heart catheterization is the gold standard for diagnosing PAH. Because echocardiography can result in over-or underestimation of pulmonary arterial pressures in SSc, cardiac

1	of restriction. Right heart catheterization is the gold standard for diagnosing PAH. Because echocardiography can result in over-or underestimation of pulmonary arterial pressures in SSc, cardiac catheterization is always required to confirm the presence of PAH; accurately assess its severity, including thedegreeofrightheartdysfunction;andruleoutvenoocclusivedisease and other cardiac causes of pulmonary hypertension. Yearly echocardiographic screening for PAH is recommended in most patients with SSc; an isolated decline in DLcomay also be indicative of developing PAH. Serum levels of brain natriuretic peptide (BNP) and N-terminal pro-BNPcorrelatewith the presenceandseverity of PAHinSSc,aswell as survival. While BNP measurements can be useful in screening for PAH and in monitoring the response to treatment, elevated BNP levels are not specific for PAH and also occur in other forms of right and left heart disease. The prognosis of SSc-associated PAH is worse, and treatment response

1	response to treatment, elevated BNP levels are not specific for PAH and also occur in other forms of right and left heart disease. The prognosis of SSc-associated PAH is worse, and treatment response poorer, than in idiopathic PAH

1	The gastrointestinal tract is affected in up to 90% of SSc patients with both limited and diffuse cutaneous forms of the disease. The pathologic features of atrophy of smooth muscle, intact mucosa, and obliterative small-vessel vasculopathy are similar throughout the length of the gastrointestinal tract.

1	Upper Gastrointestinal Tract Involvement Oropharyngeal manifestations due to a combination of xerostomia, reduced oral aperture, periodontal disease, and resorption of the mandibular condyles are frequent and cause much distress. The frenulum of the tongue may be shortened. Most patients have symptoms of gastroesophageal reflux disease (GERD): heartburn, regurgitation, and dysphagia. A combination of reduced lower esophageal sphincter pressure resulting in gastroesophageal reflux, impaired esophageal clearance of refluxed gastric contents due to diminished motility in the distal two-thirds of the esophagus, and delayed gastric emptying accounts for GERD. Manometry shows abnormal upper intestinal motility in most patients with SSc. Extraesophageal manifestations of GERD include hoarseness, chronic cough, and aspiration pneumonitis, which may aggravate underlying ILD. Chest computed tomography (CT) scan characteristically shows a dilated esophagus with intraluminal air. Endoscopy may be

1	chronic cough, and aspiration pneumonitis, which may aggravate underlying ILD. Chest computed tomography (CT) scan characteristically shows a dilated esophagus with intraluminal air. Endoscopy may be necessary to rule out opportunistic infections with Candida, herpes virus, and cytomegalovirus.Severeerosiveesophagitismay befoundonendoscopy in patients with minimal symptoms. Esophageal strictures and Barrett’s esophagus may complicate chronic GERD. Because Barrett’s esophagus isassociatedwithanincreasedriskofadenocarcinoma,SScpatientswith Barrett’s esophagus need periodic endoscopy and esophageal biopsy.

1	Gastroparesis with early satiety, abdominal distention, and aggravated reflux symptoms is common. The presence and severity of gastroparesis can be assessed by radionuclide gastric emptying studies. Gastric antral vascular ectasia (GAVE) in the antrum may occur. These subepithelial lesions, reflecting the diffuse small-vessel vasculopathy of SSc, are described as “watermelon stomach” due to their endoscopic appearance. Patients with GAVE can have recurrent episodes of gastrointestinal bleeding, resulting in chronic unexplained anemia.

1	2162 Lower Gastrointestinal Tract Involvement Impaired intestinal motility may result in malabsorptionand chronic diarrheasecondarytobacterial overgrowth. Fat and protein malabsorption and vitamin B12 and vitamin D deficiency ensue, sometimes culminating in severe malnutrition. Disturbed intestinal motor function can also cause intestinal pseudo-obstruction, with symptoms of nausea and abdominal distension that are indistinguishable from those of delayed gastric emptying. Patients present with recurrent episodes of acute abdominal pain, nausea, and vomiting. Radiographic studies show acute intestinal obstruction, and the major diagnostic challenge is to differentiate pseudoobstruction, which responds to supportive care and intravenous nutritional supplementation, from mechanical obstruction. Colonic involvement may cause severe constipation, fecal incontinence, gastrointestinal bleeding from telangiectasia, and rectal prolapse. In late-stage SSc, wide-mouth sacculations or diverticula

1	Colonic involvement may cause severe constipation, fecal incontinence, gastrointestinal bleeding from telangiectasia, and rectal prolapse. In late-stage SSc, wide-mouth sacculations or diverticula occur in the colon, occasionally causing perforation and bleeding. An occasional radiologic finding is pneumatosis cystoides intestinalis due to airtrappinginthe bowelwall thatmayrarelyrupture andcausebenign pneumoperitoneum. Although the liver is rarely affected, primary biliary cirrhosis may coexist with SSc.

1	rENaL INVOLVEMENT: SCLErODErMa rENaL CrISIS

1	Scleroderma renal crisis occurs in 10–15% of patients and generally within4yearsoftheonsetofthedisease.Priortotheadventofangiotensin-converting enzyme (ACE) inhibitors, short-term survival in scleroderma renalcrisiswas <10%. The pathogenesis involves obliterative vasculopathy and luminal narrowing of the renal arcuate and interlobular arteries. Progressive reduction in renal blood flow, aggravated by vasospasm, leads to juxtaglomerular hyperplasia, increased renin secretion, and activation of angiotensin, with further renal vasoconstriction resulting in a vicious cycle that culminates in accelerated hypertension. Risk factors for scleroderma renal crisis include African-American race, male gender, and dcSSc with extensive and progressive skin involvement. Up to 50% of patients with scleroderma renal crisis have anti-RNA polymeraseIIIantibodies.Palpabletendonfrictionrubs,pericardialeffusion, new unexplained anemia, and thrombocytopenia may be harbingers of impending scleroderma renal

1	renal crisis have anti-RNA polymeraseIIIantibodies.Palpabletendonfrictionrubs,pericardialeffusion, new unexplained anemia, and thrombocytopenia may be harbingers of impending scleroderma renal crisis. High-risk patients with early SSc should be counseled to check their blood pressure daily. Patients with lcSSc or anticentromere antibodies rarely develop scleroderma renal crisis. Because there is an association between glucocorticoid use and scleroderma renal crisis, prednisone should be used in high-risk SSc patients only when absolutely required and at low doses (<10 mg/d).

1	Patients characteristically present with accelerated hypertension and progressive oliguric renal insufficiency. However, approximately 10% of patients with scleroderma renal crisis present with normal blood pressure. Normotensive renal crisis is generally associated with a poor outcome.Headache,blurred vision, and congestiveheartfailure mayaccompanyelevationofbloodpressure.Urinalysistypicallyshows mild proteinuria, granular casts, and microscopic hematuria; thrombocytopenia and microangiopathic hemolysis with fragmented red blood cells can be seen. Progressive oliguric renal failure over several days generally follows. In some cases, scleroderma renal crisis is misdiagnosed as thrombotic thrombocytopenic purpura or other forms of thrombotic microangiopathy. In these cases, a renal biopsy may be of some benefit. In addition, biopsy findings of vascular thrombosis and glomerular ischemic collapse predict poor renal outcomes. Oliguria or a creatinine >3 mg/dL at presentation predicts

1	may be of some benefit. In addition, biopsy findings of vascular thrombosis and glomerular ischemic collapse predict poor renal outcomes. Oliguria or a creatinine >3 mg/dL at presentation predicts poor outcome, with permanent hemodialysis and high mortality. Rarely, crescentic glomerulonephritis occurs in the setting of SSc and may be associated with myeloperoxidase-specific antineutrophil cytoplasmic antibodies. Membranous glomerulonephritis may occur in patients treated with d-penicillamine. Asymptomatic renal function impairment occurs in up to half of SSc patients. Such subclinical renal involvement is associated with other vascular manifestations of SSc and rarely progresses.

1	Although it is often silent, cardiac involvement in SSc is frequently detected when patients are screened with sensitive diagnostic tools. Clinically evident cardiac involvement is associated with poor outcomes. Cardiac disease in SSc may be primary or secondary to PAH, ILD, or renal involvement. It occurs more frequently in patients with dcSSc than in those with lcSSc and generally develops within 3 yearsoftheonsetofskinthickening.Clinicallyevidentcardiacinvolvement in SSc is a poor prognostic factor. The endocardium, myocardium, and pericardium may each be affected separately or together. Manifestations of pericardial involvement include acute pericarditis, pericardial effusions, constrictive pericarditis, and cardiac tamponade. Conduction system fibrosis occurs commonly and may be silent or manifested by atrial and ventricular tachycardias or heart block. Recurrent vasospasm and ischemia-reperfusion injury contribute to myocardial fibrosis, resulting in asymptomatic systolic or

1	or manifested by atrial and ventricular tachycardias or heart block. Recurrent vasospasm and ischemia-reperfusion injury contribute to myocardial fibrosis, resulting in asymptomatic systolic or diastolic left ventricular dysfunction that may progress to overt heart failure. Systemic and pulmonary hypertension and lung and renal involvement may alsoimpact ontheheart. Despitethepresenceof widespread obliterative vasculopathy, the frequency of clinical or pathologic epicardial coronary artery disease in SSc is not increased. While conventional echocardiography has low sensitivity for detecting SSc preclinical heart involvement, newer modalities such as tissue Doppler echocardiography(TDE),cardiacmagneticresonanceimaging(cMRI), thallium perfusion, and nuclear imaging (single photon emission CT [SPECT]) reveal a high prevalence of abnormal myocardial function or perfusion in SSc patients. The serum level of N-terminal pro-BNP, a ventricular hormone, is a marker for PAH in SSc, but may also

1	[SPECT]) reveal a high prevalence of abnormal myocardial function or perfusion in SSc patients. The serum level of N-terminal pro-BNP, a ventricular hormone, is a marker for PAH in SSc, but may also have utility as a marker of primary cardiac involvement.

1	Carpal tunnel syndrome occurs frequently and may be a presenting manifestation. Generalized arthralgia and stiffness are prominent in early disease. Mobility of small and large joints is progressively impaired, especially in dcSSc. Most commonly affected are the hands. Contractures develop at the proximal interphalangeal joints and wrists. Large joint contractures can be accompanied by tendon friction rubs, characterized by leathery crepitation that can be heard or palpated upon passivemovement, thataredue to extensive fibrosis and adhesion of the tendon sheaths and fascial planes at the affected joint. Presence of tendon friction rubs is associated with increased risk for renal and cardiac complications and reduced survival. True joint inflammation is uncommon; however, occasional patients develop erosive polyarthritis in the hands. Muscle weakness is common and may indicate deconditioning, disuse atrophy, and malnutrition. Less commonly, inflammatory myositis indistinguishable from

1	develop erosive polyarthritis in the hands. Muscle weakness is common and may indicate deconditioning, disuse atrophy, and malnutrition. Less commonly, inflammatory myositis indistinguishable from idiopathic polymyositis may occur. A chronic noninflammatory myopathy characterized by atrophy and fibrosis in the absence of elevated muscle enzyme levels can be seen in late-stage SSc. Bone resorption occurs most commonly in the terminal phalanges, where it causes loss of the distal tufts (acroosteolysis)(Fig.382-5). Resorption of themandibular condylescanlead to bite difficulties. Osteolysis can also affect the ribs and distal clavicles.

1	Many SSc patients develop dry eyes and dry mouth (sicca complex). Biopsy of the minor salivary glands shows fibrosis rather than focal lymphocytic infiltration characteristic of primary Sjögren’s syndrome (Chap. 383). Hypothyroidismis commonand generally due tofibrosis of the thyroid gland. The frequency of macrovascular involvement, including peripheral vascular and coronary artery disease, may be increased. Whereas the central nervous system is generally spared, sensory trigeminal neuropathy due to fibrosis or vasculopathy can occur,presentingwithgradualonsetofpainandnumbness.Pregnancy in women with SSc may be associated with an increased rate of adverse fetal outcomes. Furthermore, cardiopulmonary involvement may worsen during pregnancy, and new onset of scleroderma renal crisis has been described. Erectile dysfunction is frequent in men with SSc and may be the initial disease manifestation. Inability to attain or maintain penile erection is due to vascular insufficiency and

1	has been described. Erectile dysfunction is frequent in men with SSc and may be the initial disease manifestation. Inability to attain or maintain penile erection is due to vascular insufficiency and fibrosis.

1	Malignancy in SSc Epidemiologic studies indicate an increased risk of cancer in SSc. Lung cancer and esophageal adenocarcinoma typically occur in the setting of long-standing ILD or gastroesophageal reflux diseaseandmaybecausedbychronicinflammationandrepair.Incontrast, breast, lung, and ovarian carcinomas and lymphomas tend to occur in close temporal association with the clinical onset of SSc, particularly in patients who have autoantibodies to RNA polymerase III. In these cases, SSc may represent a paraneoplastic syndrome triggered by the anti-tumor immune response.

1	A mild normocytic or microcytic anemia is frequent in patients with SSc and may indicate gastrointestinal bleeding caused by GAVE or chronic esophagitis. Macrocytic anemia may be caused by folate and vitamin B12 deficiency due to small-bowel bacterial overgrowth and malabsorption or by drugs such as methotrexate or alkylating agents. Microangiopathic hemolytic anemia caused by mechanical fragmentation of red blood cells during their passage through microvessels coated withfibrin orplateletthrombi isahallmark of the thromboticmicroangiopathy associated with scleroderma renal crisis. Thrombocytopenia and leukopenia may indicate drug toxicity. In contrast to other connective tissue diseases, the erythrocyte sedimentation rate (ESR) is generally normal; an elevation may signal coexisting myositis or malignancy.

1	Antinuclear autoantibodies are present in almost all patients with SSc andcanbedetectedatdiseaseonset.Autoantibodiesagainsttopoisomerase I(Scl-70)andcentromerearemutuallyexclusiveandquitespecificforSSc (Table 382-3).Topoisomerase Iantibodies aredetectedin 31%ofpatients with dcSSc, but in only 13% of patients with lcSSc. They are associated withincreasedriskofILDandpooroutcomes.Anticentromereantibodies are detected in 38% of patients with lcSSc, but in only 2% of patients with dcSSc and rarely in patients with Raynaud’s phenomenon and Sjögren’s syndrome. Anticentromere antibodies in SSc are associated with PAH, but only infrequently with significant cardiac or renal involvement or ILD. Nucleolar immunofluorescence pattern on serologic testing reflects antibodies to U3-RNP (fibrillarin), Th/To, or PM/Scl, whereas a speckled immunofluorescencepattern indicates antibodies to RNA polymerase III. Although antibodies to β2GPI occur in antiphospholipid antibody syndrome

1	(fibrillarin), Th/To, or PM/Scl, whereas a speckled immunofluorescencepattern indicates antibodies to RNA polymerase III. Although antibodies to β2GPI occur in antiphospholipid antibody syndrome andarenotspecificforSSc,theirpresenceinSScisassociatedwith an increased risk of ischemic lesions in the fingers.

1	DIaGNOSIS, STaGING, aND MONITOrING

1	ThediagnosisofSScismadeprimarilyonclinicalgroundsandisgenerally straightforward in patients with established disease. The presence of skin induration,withacharacteristicsymmetricdistributionpatternassociated with typical visceral organ manifestations, establishes the diagnosis with a highdegreeofcertainty.AlthoughtheconditionslistedinTable382-1can beassociatedwithskininduration,thedistributionpatternofskinlesions, together with the absence of Raynaud’s phenomenon or typical visceral organ manifestations or SSc-specific autoantibodies, differentiates these conditions from SSc. Occasionally, full-thickness biopsy of the skin is requiredforestablishingthediagnosisofscleredema, scleromyxedema,or nephrogenic systemic fibrosis. In lcSSc, a history of antecedent Raynaud’s phenomenon and gastroesophageal reflux symptoms, coupled with the presenceofsclerodactylyandcapillarychangesonnailfoldcapillaroscopy, often in combinations with cutaneous telangiectasia and calcinosis, helps

1	and gastroesophageal reflux symptoms, coupled with the presenceofsclerodactylyandcapillarychangesonnailfoldcapillaroscopy, often in combinations with cutaneous telangiectasia and calcinosis, helps toestablishthediagnosis.Thefindingofdigitaltippittingscarsandradiologic evidence of pulmonary fibrosis in the lower lobes are particularly helpful diagnostically. Primary Raynaud’s phenomenon is a common benign condition that must be differentiated from early or limited SSc. Nailfoldmicroscopyisparticularlyhelpfulinthissituation,becauseinprimaryRaynaud’sphenomenon,thenailfoldcapillariesarenormal,whereas in SSc, capillary abnormalities, as well as serum autoantibodies, can be detected even before other disease manifestations.

1	EstablishingthediagnosisofSScatanearlystageofthediseasemaybe a challenge. In dcSSc, initial symptoms are often nonspecific and relate to inflammation. Patients complain of fatigue, swelling, aching, and stiffness, and Raynaud’s phenomenon may initially be absent. Physical examinationmayrevealdiffuseupperextremityedemaandpuffyfingers. Patientsatthisstagearesometimesdiagnosedasearlyrheumatoidarthritis, SLE, myositis, or, most commonly, undifferentiated connective tissue disease. Within weeks to months, Raynaud’s phenomenon and characteristic clinical features appear accompanied by advancing induration of the skin. The presence of antinuclear and SSc-specific autoantibodies provides a high degree of diagnostic specificity. Raynaud’s phenomenon with fingertip ulcerations or other evidence of digital ischemia, coupled 2163 with telangiectasia, distal esophageal dysmotility, unexplained ILD or PAH, or accelerated hypertension with renal failure in the absence of clinically evident skin

1	of digital ischemia, coupled 2163 with telangiectasia, distal esophageal dysmotility, unexplained ILD or PAH, or accelerated hypertension with renal failure in the absence of clinically evident skin induration, suggests the diagnosis of SSc sine scleroderma. These patients may have anticentromere antibodies.

1	OVErVIEW: MaNaGEMENT PrINCIPLES To date, no therapy has been shown to significantly alter the natural history of SSc. In contrast, multiple interventions are highly effective in alleviating the symptoms, slowing the progression of the cumulative organ damage, and reducing disability. A significant reduction in disease-related mortality has been noted during the past 25 years. In light of the marked heterogeneity in disease manifestations, organ complications, and natural history, treatment must be tailored to each individual patient’s unique needs.

1	A thorough investigation should be undertaken at baseline. Optimal management incorporates the following principles (Table 382-5): prompt and accurate diagnosis; classification and risk stratification based on clinical and laboratory evaluation; early recognition of organ-based complications and assessment of their extent, severity, and likelihood of deterioration; regular monitoring for disease progression, activity, new complications, and response to therapy; adjusting therapy; and continuing patient education. In order to minimize irreversible organ damage, the management of life-threatening complications must be proactive, with regular screening and initiation of appropriate intervention at the earliest possible opportunity. In light of the complex and multisystemic nature of the SSc, a team-based management approach integrating multiple specialists should be pursued whenever possible. Most patients are treated with combinations of drugs that impact different aspects of the

1	SSc, a team-based management approach integrating multiple specialists should be pursued whenever possible. Most patients are treated with combinations of drugs that impact different aspects of the disease. We encourage patients to become familiar with the spectrum of potential complications and understand therapeutic options and natural history, and empower them to partner with their treating physicians. This requires a long-term relationship between patient and physician, with ongoing counseling and encouragement.

1	DISEaSE-MODIFYING THEraPY: IMMUNOSUPPrESSIVE aGENTS Immunosuppressive agents used in the treatment of other autoimmune or connective tissue diseases have generally shown modest or no benefit in SSc. Glucocorticoids may alleviate stiffness and aching in early-stage dcSSc but do not influence the progression of skin or internal organ involvement, and their use is associated with an increased risk of scleroderma renal crisis. Therefore, glucocorticoids should be given only when absolutely necessary, at the lowest dose possible, and for brief periods only. The use of cyclophosphamide has been extensively studied in light of its efficacy in the treatment of vasculitis (Chap. 385), SLE (Chap. 378), and other autoimmune diseases (Chap. 377e).

1	Both oral and intermittent IV cyclophosphamide were shown to reduce the progression of SSc-associated ILD, with stabilization and, rarely, modest improvement of pulmonary function and HRCT findings after 1 year of treatment. Improvement in respiratory symptoms and skin induration was also noted. These beneficial effects wane upon discontinuation of therapy. The benefits of cyclophosphamide need to be balanced against its potential toxicity, including bone marrow suppression, opportunistic infections, hemorrhagic cystitis and bladder cancer, premature ovarian failure, and late secondary malignancies. early and accurate diagnosis. internal organ involvement. clinical disease stage and activity. individualized therapy to each patient’s unique needs. treatment response, and adjust therapy as needed; monitor for disease progression and new complications.

1	2164 Methotrexate was associated with a modest skin improvement in small studies. Mycophenolate mofetil treatment was associated with improved skin induration in uncontrolled studies and was generally well tolerated. Small studies support the use of rituximab in SSc patients with skin involvement and ILD. The use of cyclosporine, azathioprine, extracorporeal photopheresis, thalidomide, rapamycin, imatinib, and IV immunoglobulin is currently not well supported by the literature. Intensive immune ablation using a conditioning regimen of high-dose chemotherapy with or without irradiation, followed by autologous stem cell reconstitution, has resulted in durable disease remission in some cases and is undergoing evaluation in randomized clinical trials. In light of its potential morbidity and mortality, as well as significant cost, autologous stem cell transplantation in SSc is still considered experimental.

1	antifibrotic Therapy Because widespread tissue fibrosis in SSc causes progressive organ damage, drugs that interfere with the fibrotic process represent a rational therapeutic approach. D-Penicillamine has been extensively used as an antifibrotic agent. In retrospective studies, D-penicillamine stabilized and improved skin induration, prevented new internal organ involvement, and improved survival. However, a randomized controlled clinical trial in early active SSc found no difference in the extent of skin involvement between patients treated with standard-dose (750 mg/d) or very low-dose (125 mg every other day) D-penicillamine. Recent clinical trials show benefit of pirfenidone and of nintedanib in patients with idiopathic pulmonary fibrosis, with significant slowing of the loss of lung function. Whether these two new drugs will have comparable efficacy in the treatment of SSc-associated lung disease is still under investigation.

1	Vascular Therapy The goal of therapy is to control Raynaud’s phenomenon, prevent the development and enhance the healing of ischemic complications, and slow the progression of obliterative vasculopathy. Patients should dress warmly, minimize cold exposure or stress, and avoid drugs that precipitate or exacerbate vasospastic episodes. Some patients with Raynaud’s may respond to biofeedback therapy. Extended-release dihydropyridine calcium channel blockers such as nifedipine, amlodipine, or diltiazem can ameliorate Raynaud’s phenomenon, but their use is often limited by side effects (palpitations, dependent edema, worsening gastroesophageal reflux). While ACE inhibitors do not reduce the frequency or severity of episodes, angiotensin II receptor blockers such as losartan are effective and generally well tolerated. Patients with Raynaud’s phenomenon unresponsive to these therapies may require the addition of α1-adrenergic receptor blockers (e.g., prazosin), 5-phosphodiesterase inhibitors

1	well tolerated. Patients with Raynaud’s phenomenon unresponsive to these therapies may require the addition of α1-adrenergic receptor blockers (e.g., prazosin), 5-phosphodiesterase inhibitors (e.g., sildenafil), serotonin reuptake inhibitors (e.g., fluoxetine), topical nitroglycerine, and intermittent infusions of IV prostaglandins. Low-dose aspirin and dipyridamole prevent platelet aggregation and may have a role as adjunctive agents. In patients with ischemic ulcers, the endothelin-1 receptor antagonist bosentan reduces the risk of new ulcers. Digital sympathectomy and local injections of botulinum type A (Botox) into the digits are options in patients with severe ischemia and impending loss of the digits. Empirical long-term therapy with statins and antioxidants may retard the progression of vascular damage and obliteration. Vasodilators such as ACE inhibitors, calcium channel blockers, and endothelin receptor blockers may also improve myocardial perfusion and left ventricular

1	of vascular damage and obliteration. Vasodilators such as ACE inhibitors, calcium channel blockers, and endothelin receptor blockers may also improve myocardial perfusion and left ventricular function.

1	TrEaTMENT OF GaSTrOINTESTINaL COMPLICaTIONS Because oral problems including decreased oral aperture, decreased saliva production, gum recession and periodontal disease leading to teeth loss are common, regular dental care is recommended. Gastroesophageal reflux is very common and may occur in the absence of symptoms; therefore all patients with SSc should be treated. Patients should be instructed to elevate the head of the bed, eat frequent small meals, and avoid oral intake before bedtime. Proton pump inhibitors reduce acid reflux and may need to be given in relatively high doses. Prokinetic agents such as domperidone may be helpful, especially if delayed gastric emptying is present. Episodic gastrointestinal bleeding from gastric antral vascular ectasia (watermelon stomach) may be amenable to treatment with endoscopic laser photocoagulation, although recurrence can occur. Bacterial overgrowth due to small-bowel dysmotility causes abdominal bloating and diarrhea and may lead to

1	amenable to treatment with endoscopic laser photocoagulation, although recurrence can occur. Bacterial overgrowth due to small-bowel dysmotility causes abdominal bloating and diarrhea and may lead to malabsorption and severe malnutrition. Treatment with short courses of rotating broad-spectrum antibiotics such as metronidazole, erythromycin, and tetracycline can eradicate bacterial overgrowth. Parenteral hyperalimentation is indicated if malnutrition develops. Chronic hypomotility of the small bowel may respond to octreotide, but pseudo-obstruction is difficult to treat. Fecal incontinence, a frequently underreported complication of SSc, may respond to anti-diarrheal medication and biofeedback therapy.

1	TrEaTMENT OF PULMONarY arTErIaL HYPErTENSION (PaH) In patients with SSc, PAH carries an extremely poor prognosis and accounts for 30% of deaths. Because PAH is asymptomatic until advanced, patients with SSc should be screened for its presence at initial evaluation, and on a yearly basis thereafter. Treatment is generally started with an oral endothelin-1 receptor antagonist such as bosentan or a phosphodiesterase type 5 inhibitor such as sildenafil. Patients may also require diuretics and digoxin when appropriate. If hypoxemia is documented, supplemental oxygen should be prescribed in order to avoid hypoxia-induced secondary pulmonary vasoconstriction. Prostacyclin analogues such as epoprostenol or treprostinil can be given by continuous IV or SC infusion, or via intermittent nebulized inhalations. Combination therapy with different classes of agents, such as an endothelin-1 antagonist and a phosphodiesterase inhibitor, is often necessary. Lung transplantation remains an option for

1	inhalations. Combination therapy with different classes of agents, such as an endothelin-1 antagonist and a phosphodiesterase inhibitor, is often necessary. Lung transplantation remains an option for selected patients who fail medical therapy.

1	TrEaTMENT OF rENaL CrISIS Scleroderma renal crisis is a medical emergency. Since the outcome is largely determined by the extent of renal damage present at the time that aggressive therapy is initiated, prompt recognition of impending or early scleroderma renal crisis is essential, and efforts should be made to avoid its occurrence. High-risk SSc patients with early disease, extensive and progressive skin involvement, tendon friction rubs, and anti-RNA polymerase III antibodies should be instructed to monitor their blood pressure daily and report significant alterations immediately. Potentially nephrotoxic drugs should be avoided, and glucocorticoids should be used only when absolutely necessary and at low doses. Patients presenting with scleroderma renal crisis should be immediately hospitalized. Once other causes of renal disease are excluded, treatment should be started promptly with titration of short-acting ACE inhibitors, with the goal of achieving rapid normalization of the

1	Once other causes of renal disease are excluded, treatment should be started promptly with titration of short-acting ACE inhibitors, with the goal of achieving rapid normalization of the blood pressure. In patients with hypertension persisting despite ACE inhibitor therapy, addition of angiotensin II receptor blockers, calcium channel blockers, and direct renin inhibitors should be considered. Anecdotal evidence indicates responses to endothelin-1 receptor blockers and prostacyclins. Up to two-thirds of patients with scleroderma renal crisis go on to dialysis. The outcome of scleroderma renal crisis is worse in patients with antibodies to topoisomerase I compared to those with antibodies to RNA polymerase III. Substantial renal recovery can occur following scleroderma renal crisis, and dialysis can be discontinued, in 30–50% of the patients. Kidney transplantation is appropriate for those unable to discontinue dialysis after 2 years. Survival of transplanted SSc patients is comparable

1	can be discontinued, in 30–50% of the patients. Kidney transplantation is appropriate for those unable to discontinue dialysis after 2 years. Survival of transplanted SSc patients is comparable to that of patients with other connective tissue diseases, and recurrence of renal crisis is rare.

1	SKIN CarE Because skin involvement in SSc is never life-threatening and because it stabilizes and may even regress spontaneously, over time, the management of SSc should not be dictated by its cutaneous manifestations. The inflammatory symptoms of early skin involvement can be controlled with antihistamines and cautious short-term use of low-dose glucocorticoids (<5 mg/d of prednisone). Retrospective studies have shown that D-penicillamine reduced the extent and progression of skin induration; however, these benefits could not be substantiated in a controlled prospective trial. Cyclophosphamide and methotrexate have modest effects on skin induration. Because the skin is dry, the use of hydrophilic ointments and bath oils is encouraged. Regular skin massage is helpful. Telangiectasia may present a cosmetic problem, especially on the face. Treatment with pulsed dye laser may have short-term benefit. Ischemic digital ulcers should be protected by occlusive dressing to promote healing and

1	a cosmetic problem, especially on the face. Treatment with pulsed dye laser may have short-term benefit. Ischemic digital ulcers should be protected by occlusive dressing to promote healing and prevent infection. Infected skin ulcers are treated with topical antibiotics. Surgical debridement may be indicated. No therapy has been shown to be effective in preventing the formation of calcific soft tissue deposits or promoting their dissolution.

1	TrEaTMENT OF MUSCULOSKELETaL COMPLICaTIONS Arthralgia and joint stiffness are common and distressing manifestations most prominent in early-stage disease. Short courses of nonsteroidal anti-inflammatory agents, weekly methotrexate, and cautious use of low-dose corticosteroids may alleviate these symptoms. Physical and occupational therapy can be effective for maintaining musculoskeletal function and improving long-term outcomes. The natural history of SSc is highly variable and difficult to predict, especially in early stages of the disease, when the specific subset— diffuse or limited cutaneous form—is not clear. Patient with dcSSc tend to have a more rapidly progressive course and worse prognosis than those with lcSSc.

1	In dcSSc, inflammatory symptoms such as fatigue, edema, arthralgia, and pruritus tend to subside, and the extent of skin thickening reaches a plateau at 2–4 years after disease onset, followed by slow regression.Itisduringtheearlyedematous/inflammatorystage,generally lasting <3years, that important visceralorganinvolvement occurs. While existing visceral organ involvement, such as pulmonaryfibrosis, may progress even after skin involvement peaks, new organ involvement is rare. Scleroderma renal crisis almost invariably occurs within the first 4 years of disease. In late-stage disease (>6 years), the skin is usually soft and atrophic. Skin regression characteristically occurs in an order that is the reverse of initial involvement, with softening on the trunks followed by proximal and finally distal extremities; however, sclerodactyly and finger contractures generally persist. Relapse or recurrence of skin thickening after the peak of skin involvement has been

1	by proximal and finally distal extremities; however, sclerodactyly and finger contractures generally persist. Relapse or recurrence of skin thickening after the peak of skin involvement has been reachedisuncommon.Patientswith lcSScfollow aclinicalcourse that is markedly different than that of dcSSc. Raynaud’s phenomenon typically precedes other disease manifestations by years or even decades. Visceral organ complications such as PAH and ILD generally develop late and progress slowly.

1	SSc confers a substantial increase in the risk of premature death. Age-and gender-adjusted mortality rates are fivefold to eightfold higher compared to the general population, and more than half of all patients with SSc die from their disease. In one population-based study of SSc, the median survival was 11 years. In patients with dcSSc, 5-and 10-year survival rates are 70% and 55%, respectively, whereas in patients with lcSSc, 5-and 10-year survival rates are 90% and 75%, respectively. The prognosis correlates with the extent of skin involvement, which itself is a surrogate for visceral organ involvement. Major causes of death are PAH, pulmonary fibrosis, gastrointestinal involvement, and cardiac disease. Scleroderma renal crisis is associated with a 30% 3-year mortality. Lung cancer and excess cardiovascular deaths also contribute to increased mortality. Markers of poor prognosis include male gender, African-American race, older age at disease onset, extensive skin thickening with

1	excess cardiovascular deaths also contribute to increased mortality. Markers of poor prognosis include male gender, African-American race, older age at disease onset, extensive skin thickening with truncal involvement, palpable tendon friction rubs, and evidence of significant or progressive visceral organ involvement. Laboratory predictors of increased mortality at initial evaluation include an elevated ESR, anemia, proteinuria and anti–topoisomerase I antibodies. In one study, SSc 2165 patients with extensive skin involvement, lung vital capacity <55% predicted, significant gastrointestinal involvement (pseudoobstruction ormalabsorption), evidenceof cardiac involvement(arrhythmias or congestive heart failure), or scleroderma renal crisis had a cumulative 9-year survival <40%. The severity of PAH is strongly associated with mortality, and SSc patients who had a mean pulmonary arterial pressure ≥45 mmHg had a 33% 3-year survival. The advent of ACE inhibitors in scleroderma renal

1	of PAH is strongly associated with mortality, and SSc patients who had a mean pulmonary arterial pressure ≥45 mmHg had a 33% 3-year survival. The advent of ACE inhibitors in scleroderma renal crisis had a dramatic impact on survival, increasing from <10% at 1 year in the pre–ACE inhibitor era to >70% 3-year survival at the present time. Moreover, 10-year survival inSSc has improvedfrom<60%inthe 1970s to >66–78%inthe 1990s, a trend that reflects both earlier detection and better management of complications.

1	The term scleroderma is commonly used to describe a group of localizedskindisorders(Table382-1).Theseoccurmorecommonlyinchildren than in adults. In contrast to SSc, localized scleroderma is rarely complicated by Raynaud’s phenomenon or significant internal organ involvement. Morphea presents as solitary or multiple circular patches of thickened skin or, rarely, as widespread induration (generalized or pansclerotic morphea); the fingers are spared. Linear scleroderma— streaks of thickened skin, typically in one or both lower extremities— may affect the subcutaneous tissues, leading to fibrosis and atrophy of supporting structures, muscle, and bone. In children, the growth of affected long bones can be retarded. When linear scleroderma lesions cross joints, significant contractures can develop.

1	Patients who have lcSSc coexisting with features of SLE, polymyositis, and rheumatoid arthritis may have mixed connective tissue disease (MCTD). This overlap syndrome is generally associated with the presence of high titers of autoantibodies to U1-RNP. The characteristic initial presentation is Raynaud’s phenomenon associated with puffy fingers and myalgia. Gradually, lcSSc features of sclerodactyly, calcinosis, and cutaneous telangiectasia develop. Skin rashes suggestive of SLE (malar rash, photosensitivity) or of dermatomyositis (heliotrope rash on the eyelids, erythematous rash on the knuckles) occur. Arthralgia is common, and some patients develop erosive polyarthritis. Pulmonary fibrosis and isolated or secondary PAH may develop. Other manifestations include esophageal dysmotility, pericarditis, Sjögren’s syndrome, and renal disease, especially membranous glomerulonephritis. Laboratory evaluation indicates features of inflammation with elevated ESR and hypergammaglobulinemia.

1	pericarditis, Sjögren’s syndrome, and renal disease, especially membranous glomerulonephritis. Laboratory evaluation indicates features of inflammation with elevated ESR and hypergammaglobulinemia. While anti-U1RNP antibodies are detected in the serum in high titers, SSc-specific autoantibodies are not found. In contrast to SSc, patients withMCTDoften show a good responseto treatmentwithglucocorticoids, and the long-term prognosis is better than that of SSc. Whether MCTD is a truly distinct entity or is, rather, a subset of SLE or SSc remains controversial.

1	Eosinophilic fasciitis is a rare idiopathic disorder associated with induration of the skin that generally develops rapidly. Adults are primarily affected. The skin has a coarse cobblestone “peau d’orange” appearance. In contrast to SSc, internal organ involvement is rare, and Raynaud’s phenomenon and SSc-associated autoantibodies are absent. Furthermore, skin involvement spares the fingers. Full-thickness excisionalbiopsyofthelesionalskinrevealsfibrosisofthesubcutaneousfasciaandisgenerallyrequiredfordiagnosis.Inflammationandeosinophil infiltration in the fascia are variably present. In the acute phase of the illness, peripheral blood eosinophilia may be prominent. MRI appears to be a sensitive tool for the diagnosis of eosinophilic fasciitis. In some patients, eosinophilic fasciitis occurs in association with, or preceding, myelodysplastic syndromes or multiple myeloma. Treatment with glucocorticoids leads to prompt resolution of the eosinophilia. In contrast, skinchanges generally

1	in association with, or preceding, myelodysplastic syndromes or multiple myeloma. Treatment with glucocorticoids leads to prompt resolution of the eosinophilia. In contrast, skinchanges generally show slow and variable improvement. Theprognosis of patients with eosinophilic fasciitis is good.

1	2166 DEFINITION, INCIDENCE, aND PrEVaLENCE Haralampos M. Moutsopoulos, Athanasios G. Tzioufas Sjögren’s syndrome is a chronic, slowly progressive autoimmune disease characterized by lymphocytic infiltration of the exocrine glands result ing in xerostomia and dry eyes. Approximately one-third of patients present with systemic manifestations; a small but significant number of patients develop malignant lymphoma. The disease presents alone (primary Sjögren’s syndrome) or in association with other autoimmune rheumatic diseases (secondary Sjögren’s syndrome) (Table 383-1). Middle-aged women (female-to-male ratio, 9:1) are primarily affected, although Sjögren’s syndrome may occur at any age, including childhood. The prevalence of primary Sjögren’s syndrome is ~0.5–1%, while30% of patients with autoimmune rheumatic diseases suffer from secondary Sjögren’s syndrome.

1	Sjögren’s syndrome is characterized by both lymphocytic infiltration of the exocrine glands and B lymphocyte hyperreactivity. An oligomonoclonal B cell process, which is characterized by cryoprecipitable monoclonal immunoglobulins (IgMκ) with rheumatoid factor activity, is evident in up to 25% of patients. Sera from patients with Sjögren’s syndrome often contain auto-antibodies to non-organ-specific antigens such as immunoglobulins (rheumatoid factors) and extractable nuclear and cytoplasmic antigens (Ro/SS-A, La/SS-B). Ro/SS-A autoantigen consists of two polypeptides (52 and 60 kDa, respectively) in conjunction with cytoplasmic RNAs, whereas the 48-kDa La/SS-B protein is bound to RNA III polymerase transcripts. Autoantibodies to Ro/SS-A and La/SS-B antigens are usually detected at the time of diagnosis and are associated with earlier diseaseonset,longerdiseaseduration,salivaryglandenlargement, and more intense lymphocytic infiltration of minor salivary glands.

1	The major infiltrating cells in the affected exocrine glands are activated T and B lymphocytes. T cells predominate in mild lesions, whereas B cells are dominant in more severe lesions. Macrophages and dendritic cells also are found. The number of macrophages positive for interleukin (IL) 18 has been shown to correlate with parotid gland enlargement and low levels of the C4 component of complement, both of which are adverse predictors for lymphoma development.

1	Ductal andacinarepithelial cells appeartoplay a significant rolein the initiation and perpetuation of autoimmune injury. These cells (1) express classIImajorhistocompatibilitycomplex(MHC)molecules,costimulatory molecules, and abberant expression of intracellular autoantigens on cell membranes and thus are able to provide signals essential for lymphocytic activation; (2) inappropriately produce proinflammatory cytokines and lymphoattractant chemokines necessary for sustaining the autoimmune lesion and allowing progression to more sophisticated ectopic germinal center formation, which occurs in one-fifth of patients; and (3) express functional receptors of innate immunity, particularly Toll-like receptors (TLRs) 3, 7, and 9, that may account for the perpetuation of the autoimmuneresponse.

1	Both infiltrating T and B cells have a tendency to be resistant to apoptosis. Levels of B cell–activating factor (BAFF) have been found to be elevated in patients with Sjögren’s syndrome, especially those with hypergammaglobulinemia,andprobably accounts for thisantiapoptotic effect. Glandular epithelial cells seem to have an active role in the productionofBAFF, whichmaybe expressedandsecretedafterstimulation with type I interferon as well as with viral or synthetic double-stranded RNA. The triggering factor for epithelial activation appears to be a persistent enteroviral infection (possibly with coxsackievirus strains). Type I and type II interferon signatures have been described in ductal epithelial cells and T cells,respectively;their detection impliesthat interferons exert direct and cross-regulating effects on the pathogenic process.

1	A defect in cholinergic activity mediated through the M3 receptor and redistribution of the water-channel protein aquaporin 5, both leadingtoneuroepithelialdysfunctionanddiminishedglandularsecretions, have been proposed. Molecularanalysisofhumanleukocyteantigen(HLA)classIIgenes has revealed that Sjögren’s syndrome, regardless of the patient’s ethnic origin, is highly associated with the HLA DQA1*0501 allele. Genome-wide association studies have disclosed an increased prevalence of single-nucleotidepolymorphismsin genes of IRF-5 and STAT-4, which participate in the activation of the type I interferon pathway. The majority of patients with Sjögren’s syndrome have symptoms related to diminished lacrimal and salivary gland function. In most patients, the primary syndrome runs a slow and benign course. The initial manifestations can be mucosal or nonspecific dryness, and 8–10 years may elapse from the initial symptoms to full-blown development of the disease.

1	The principal oral symptom of Sjögren’s syndrome is dryness (xerostomia). Patients report difficulty in swallowing dry food, an inabilitytospeakcontinuously,aburningsensation,anincreaseindental caries, and problems in wearing complete dentures. Physical examination shows a dry, erythematous, sticky oral mucosa. There is atrophy of the filiform papillae on the dorsum of the tongue, and saliva from the major glands is either not expressible or cloudy. Enlargement of the parotid or other major salivary glands occurs in two-thirds of patients with primary Sjögren’s syndrome but is uncommon in those with the secondary syndrome. Diagnostic tests include sialometry, sialography, and scintigraphy. Newer imaging techniques, including ultrasound, MRI, and magnetic resonance sialography of the major salivary glands, are also being used. Biopsy of the labial minor salivary gland permits histopathologic confirmation of focal lymphocytic infiltrates.

1	Ocular involvement is the other major manifestation of Sjögren’s syndrome. Patients usually describe a sandy or gritty feeling under the eyelids. Other symptoms include burning, accumulation of secretions in thick strands at the inner canthi, decreased tearing, redness, itching, eye fatigue, and increased photosensitivity. These symptoms, which define keratoconjunctivitis sicca, are attributed to the destruction of corneal and bulbar conjunctival epithelium. Diagnostic evaluation of keratoconjunctivitis sicca includes measurement of tear flow by SchirmerItestanddeterminationoftearcomposition,withassessment of tear breakup time or tear lysozyme content. Slit-lamp examination ofthecorneaandconjunctivaafterrosebengalstainingrevealspunctuate corneal ulcerations and attached filaments of corneal epithelium.

1	Involvement of other exocrine glands, which occurs less frequently, includes a decrease in mucous gland secretions of the upper and lower respiratorytree,resultingin drynose,throat, andtrachea(xerotrachea). In addition, diminished secretion of the exocrine glands of the gastrointestinal tract leads to esophageal mucosal atrophy, atrophic gastritis, andsubclinicalpancreatitis.Dyspareuniaduetodrynessoftheexternal genitalia and dry skin also may occur. Extraglandular (systemic) manifestations are seen in one-third of patients with Sjögren’s syndrome (Table 383-2) but are very rare in patients whose Sjögren’s syndrome is associated with rheumatoid aMucosa-associated lymphoid tissue.

1	arthritis. Patients with primary Sjögren’s syndrome more often report easy fatigability, low-grade fever, Raynaud’s phenomenon, myalgias, and arthralgias.Mostpatientswithprimary Sjögren’ssyndromeexperience at least one episode of non-erosive arthritis during the course of their disease. Manifestations of pulmonary involvement are frequently evident histologically but are rarely important clinically. Dry cough is themajormanifestationthatisattributedtosmallairwaydisease. Renal involvement includes interstitial nephritis, clinically manifested by hyposthenuria and renal tubular dysfunction with or without acidosis. Untreated acidosis may lead to nephrocalcinosis. Glomerulonephritis is a rare finding that occurs in patients with mixed cryoglobulinemia or with systemic lupus erythematosus overlapping with Sjögren’s syndrome. Vasculitis affects small and medium-sized vessels. The most common clinical features are purpura, recurrent urticaria, skin ulcerations, glomerulonephritis, and

1	overlapping with Sjögren’s syndrome. Vasculitis affects small and medium-sized vessels. The most common clinical features are purpura, recurrent urticaria, skin ulcerations, glomerulonephritis, and mononeuritis multiplex.

1	Different autoantibodies may determine the clinical expression of the disease. Patients positive for anticentromere autoantibody present with a clinical picture similar to that of limited scleroderma (Chap. 382). Antimitochondrial antibodies may connote liver involvement in the form of primary biliary cirrhosis (Chap. 369). Autoantibodies to 21-hydroxylate have recently been described in almost 20% of patients; their presence is associated with a blunted adrenal response. Centralnervoussysteminvolvementisrarelyrecognized.Afewcases ofmyelitisassociatedwithantibodytoaquaporin4havebeendescribed.

1	Centralnervoussysteminvolvementisrarelyrecognized.Afewcases ofmyelitisassociatedwithantibodytoaquaporin4havebeendescribed. Lymphoma is a well-known manifestation of Sjögren’s syndrome that usually presents later in the illness. Persistent parotid gland enlargement, purpura, leukopenia, cryoglobulinemia, low C4 complement levels, and ectopic germinal centers in minor salivary gland biopsy samples are manifestationssuggestingthedevelopmentoflymphoma.Itisinteresting thatthesameriskfactorsaccountforglomerulonephritisandlymphoma and that these risk factors are the ones that confer increased mortality risk. Most lymphomas are extranodal, low-grade marginal-zone B cell lymphomas and are usually detected incidentally during evaluation of the labial biopsy. The affected lymph nodes are usually peripheral. Survival rates are decreased in patients with B symptoms, lymph node mass >7 cm in diameter, and high or intermediate histologic grade.

1	Routine laboratory tests in Sjögren’s syndrome reveal mild normochromic, normocytic anemia. An elevated erythrocyte sedimentation rate is found in ~70% of patients.

1	Primary Sjögren’s syndrome is diagnosed if (1) the patient presents with eye and/or mouth dryness, (2) eye tests disclose keratoconjunctivitis sicca, (3) mouth evaluation reveals the classic manifestations of the syndrome, and/or(4) the patient’s serum reacts with Ro/SS-A and/or La/SS-B auto-antigens. Labial biopsy is needed when the diagnosis is uncertain or to rule out other conditions that may cause dry mouth or eyes or parotid glandenlargement (Tables 383-3 and 383-4). Validated diagnosticcriteriahavebeenestablishedbyaEuropeanstudyandhavenowbeenfurther improvedbyaEuropean-Americanstudygroup(Table 383-5).Hepatitis C virus infection should be ruled out since, apart from serologic tests, the clinicopathologic picture is almost identical to that of Sjögren’s syndrome. Enlargement of major salivary glands, particularly in seronegative patients, should raise the suspicion of IgG4-related syndrome, which may present also as chronic pancreatitis, interstitial nephritis, retroperitoneal

1	salivary glands, particularly in seronegative patients, should raise the suspicion of IgG4-related syndrome, which may present also as chronic pancreatitis, interstitial nephritis, retroperitoneal fibrosis, and aortitis.

1	Treatment of Sjögren’s syndrome is aimed at symptom relief and limitation of the damaging local effects of chronic xerostomia and keratoconjunctivitis sicca through substitution for or stimulation of the missing secretions (Fig. 383-1). Lack of autoantibodies to Ro/SS-A and/or La/SS-B Lymphoid infiltrates of salivary glands by CD8+ T lymphocytes Association with Presence of autoantibodies Lymphoid infiltrates of salivary glands by CD4+ T lymphocytes Association with Lack of autoantibodies to Ro/SS-A and/or La/SS-B 2168 taBle 383-5 reVISeD InternatIOnal ClaSSIfICatIOn CrIterIa fOr Sjögren’S SynDrOMea,b,c I. Ocular symptoms: a positive response to at least one of three validated questions. 1. Have you had daily, persistent, troublesome dry eyes for more than 3 months? 2. Do you have a recurrent sensation of sand or gravel in the eyes? 3. Do you use tear substitutes more than three times a day?

1	II. Oral symptoms: a positive response to at least one of three validated questions. 1. Have you had a daily feeling of dry mouth for more than 3 months? 2. Have you had recurrent or persistently swollen salivary glands as an adult? 3. Do you frequently drink liquids to aid in swallowing dry foods? III. Ocular signs: objective evidence of ocular involvement defined as a positive result to at least one of the following two tests: 1. Shirmer’s I test, performed without anesthesia (≤5 mm in 5 min) 2. Rose Bengal score or other ocular dye score (≥4 according to van Bijsterveld’s scoring system) IV. Histopathology: In minor salivary glands focal lymphocytic sialoadenitis, with a focus score ≥1. V. Salivary gland involvement: objective evidence of salivary gland involvement defined by a positive result to at least one of the following diagnostic tests: 1. Unstimulated whole salivary flow (≤1.5 mL in 15 min) 2. Parotid sialography 3. Salivary scintigraphy

1	VI. Antibodies in the serum to Ro/SS-A or La/SS-B antigens, or both. aExclusion criteria: past head and neck radiation treatment, hepatitis C infection, AIDS, preexisting lymphoma, sarcoidosis, graft-versus-host disease, use of anticholinergic drugs. bPrimary Sjögren’s syndrome: any four of the six items, as long as item IV (histopathology) or VI (serology) is positive; or any three of the four objective-criteria items (III, IV, V, VI). cIn patients with a potentially associated disease (e.g., another well-defined connective tissue disease), the presence of item I or item II plus any two from among items III, IV, and V may be considered indicative of secondary Sjögren’s syndrome. Source: From C Vitali et al: Ann Rheum Dis 61:554, 2002. ©2002 with permission from BMJ Publishing Group Ltd.

1	Avoid Smoking areas, windy, low humidity environment, drugs with anticholinergic action, diuretics Lubrication Artificial tears without preservatives, bicarbonate-buffered electrolyte solutions Oral hygiene after each meal Topical application of fluoride Lubrication Water Local stimulation Sugar-free, flavored lozenges or gum Systemic stimulation As for dry eyes Oral candidiasis Topical nystatin or clotrimazole lozenges Apply Local wet heat Treat superinfection Antibiotics, analgesics Persistent, hard Rule out lymphoma Local stimulation Cyclic adenosine mono-phosphate, cyclosporine 2% olive solution Systemic stimulation Pilocarpine (5 mg thrice daily orally); cevimeline (30 mg thrice daily orally) Severe dry eyes Nasolacrimal duct occlusion (temporary or permanent); soft contact lenses; corneal transplantation Glandular manifestations Extraglandular manifestations Parotid gland enlargement Dry mouth Dry eyes Arthritis Cold protection: gloves Raynaud’s phenomenon Bicarbonate

1	lenses; corneal transplantation Glandular manifestations Extraglandular manifestations Parotid gland enlargement Dry mouth Dry eyes Arthritis Cold protection: gloves Raynaud’s phenomenon Bicarbonate replacement Renal tubular acidosis CHOP + anti-CD20 Vasculitis Standard treatment Lymphoma Hydroxychloroquine (200–400 mg/d) or Methotrexate (0.2–0.3 mg/kg body weight weekly) plus Prednisolone (<10 mg daily orally)

1	FIGUrE 383-1 Treatment algorithm for Sjögren’s syndrome. CHOP, cyclophosphamide, adriamycin (hydroxydaunorubicin), vincristine (oncovin), and prednisone. To replace deficient tears, several ophthalmic preparations are readily available (hydroxypropyl methylcellulose; polyvinyl alcohol; 0.5% methylcellulose; Hypo Tears). If corneal ulcerations are present, eye patching and boric acid ointments are recommended. Certain drugs that may decrease lacrimal and salivary secretions, such as diuretics, antihypertensive drugs, anticholinergics, and antidepressants, should be avoided. For xerostomia, the best replacement is water. Propionic acid gels may be used to treat vaginal dryness. To stimulate secretions, orally administered pilocarpine (5 mg thrice daily) or cevimeline (30 mg thrice daily) appears to improve sicca manifestations, and both are well tolerated. Hydroxychloroquine (200 mg) is helpful for arthralgias and mild arthritis.

1	Patients with renal tubular acidosis should receive sodium bicarbonate by mouth (0.5–2 mmol/kg in four divided doses). Glucocorticoids (1 mg/kg per day) and/or immunosuppressive agents (e.g., cyclophosphamide) are indicated only for the treatment of systemic vasculitis. Anti–tumor necrosis factor agents are ineffective. Monoclonal antibody to CD20 appears to be effective in patients with systemic disease, particularly in those with vasculitis, arthritis, and fatigability. Combination of anti-CD-20 with a classic CHOP regimen (cyclosporine, adriamycin [hydroxydaunorubicin], vincristine [oncovin], and prednisone) leads to increased survival rates among patients with high-grade lymphomas. the Spondyloarthritides Joel D. Taurog, John D. Carter

1	the Spondyloarthritides Joel D. Taurog, John D. Carter The spondyloarthritides are a group of overlapping disorders that share certain clinical features and genetic associations. These disorders include ankylosing spondylitis (AS), reactive arthritis, psoriatic arthritis and spondylitis, enteropathic arthritis and spondylitis, juvenile-onset spondyloarthritis (SpA), and undifferentiated SpA. The similarities in clinical manifestations and genetic predisposition suggest that these disorders share pathogenic mechanisms.

1	AS is an inflammatory disorder of unknown cause that primarily affectstheaxial skeleton;peripheral jointsandextraarticular structures are also frequently involved. The disease usually begins in the second or third decade; male-to-female prevalence is between 2:1 and 3:1. The term axial spondyloarthritis is coming into common use, supported by criteria formulated in 2009 (Table 384-1). This classification includes both definite AS and early stages that do not yet meet classical criteria for AS, but it probably also includes other conditions with a different natural history. AS shows a striking correlation with the histocompatibility antigen HLA-B27 and occurs worldwide roughly in proportion to the prevalence of B27 (Chap. 373e). In North American whites, the prevalence of B27 is 7%, whereas it is 90% in patients with AS, independent of disease severity.

1	In population surveys, AS is present in 1–6% of adults inheriting B27, whereas the prevalence is 10–30% among B27+ adult first-degree relatives of AS probands. Concordance rate in identical twins is about 65%. Susceptibility to AS is determined largely by genetic factors, with B27 comprising less than one-half of the genetic component. Genome-wide single-nucleotide polymorphism (SNP) analysis has identified over 30 additional susceptibility alleles. Inflammatory back paind on MRI highly suggestive of itis according to modified New York criteriac Good response to NSAIDsh

1	Inflammatory back paind on MRI highly suggestive of itis according to modified New York criteriac Good response to NSAIDsh Family history of SpAi aSensitivity 83%, specificity 84%. The imaging arm (sacroiliitis) alone has a sensitivity of 66% and a specificity of 97%. bBone marrow edema and/or osteitis on short tau inversion recovery (STIR) or gadolinium-enhanced T1 image. cBilateral grade ≥2 or unilateral grade 3 or 4. dSee text for criteria. ePast or present, diagnosed by a physician. fPast or present pain or tenderness on examination at calcaneus insertion of Achilles tendon or plantar fascia. gPast or present, confirmed by an ophthalmologist. hSubstantial relief of back pain at 24–48 h after a full dose of NSAID. iFirst-or second-degree relatives with ankylosing spondylitis (AS), psoriasis, uveitis, reactive arthritis (ReA), or inflammatory bowel disease (IBD). jAfter exclusion of other causes of elevated CRP.

1	Abbreviations: ASAS, Assessment of Spondyloarthritis International Society; CRP, C-reactive protein; MRI, magnetic resonance imaging; NSAIDs, nonsteroidal anti- inflammatory drugs; SpA, spondyloarthritis. Source: From M Rudwaleit et al: Ann Rheum Dis 68:777, 2009. Copyright 2009, with permission from BMJ Publishing Group Ltd. Sacroiliitis is often the earliest manifestation of AS. Knowledge of its pathology comes from both biopsy and autopsy studies that cover a range of disease durations. Synovitis and myxoid marrow represent the earliest changes, followed by pannus and subchondral granulation tissue. Marrow edema, enthesitis, and chondroid differentiation are also found. Macrophages, T cells, plasma cells, and osteoclasts are prevalent. Eventually the eroded joint margins are gradually replaced by fibrocartilage regeneration and then by ossification. The joint may become totally obliterated.

1	In the spine, the specimens studied have either been surgically resected in advanced disease or taken from autopsies. There is inflammatory granulation tissue in the paravertebral connective tissue at the junction of annulus fibrosus and vertebral bone, and in some cases along the entire outer annulus. The outer annular fibers are eroded and eventually replaced by bone, forming the beginning of a syndesmophyte, which then grows by continued endochondral ossification, ultimately bridging the adjacent vertebral bodies. Ascending progression of this process leads to the “bamboo spine.” Other lesions in the spine include diffuse osteoporosis (loss of trabecular bone despite accretion of periosteal bone), erosion of vertebral bodies at the disk margin, “squaring” or “barreling” of vertebrae, and inflammation and destruction of the disk-bone border. Inflammatory arthritis of the apophyseal (facet) joints is common, with synovitis, inflammation at the bony attachment of the joint capsule,

1	and inflammation and destruction of the disk-bone border. Inflammatory arthritis of the apophyseal (facet) joints is common, with synovitis, inflammation at the bony attachment of the joint capsule, and subchondral bone marrow granulation tissue. Erosion of joint cartilage by pannus is often followed by bony ankylosis. This may precede formation of syndesmophytes bridging the adjacent disks. Bone mineral density is diminished in the spine and proximal femur early in the course of the disease.

1	Peripheral synovitis in AS shows marked vascularity, which is also evident as tortuous macrovascularity seen during arthroscopy. Lining layer hyperplasia, lymphoid infiltration, and pannus formation are also found. Central cartilaginous erosions caused by proliferation of subchondral granulation tissue are common. It should be

1	The Spondyloarthritides 2170 emphasized that the characteristics of peripheral arthritis in AS and other forms of SpA are similar, and distinct from those of rheumatoid arthritis. Inflammation in the fibrocartilaginous enthesis, the region where a tendon, ligament, or joint capsule attaches to bone, is a characteristic lesion in AS and other SpAs, both at axial and peripheral sites. Enthesitis is associated with prominent edema of the adjacent bone marrow and is often characterized by erosive lesions that eventually undergo ossification. Subclinical intestinal inflammation has been found in the colon or distal ileum in a majority of patients with SpA. The histology is described below under “Enteropathic Arthritis.”

1	The pathogenesis of AS is immune-mediated, but there is little direct evidence for antigen-specific autoimmunity, and there is evidence to suggest more of an autoinflammatory pathogenesis. Uncertainty remains regarding the primary site of disease initiation. The dramatic responseofthediseasetotherapeuticblockadeoftumornecrosisfactor α (TNF-α) indicates that this cytokine plays a central role in the immunopathogenesis of AS. Other genes related to TNF pathways show association with AS, including TNFRSF1A, LTBR, and TBKBP1. More recent evidence strongly implicates the interleukin (IL) 23/IL-17 cytokine pathway in AS pathogenesis. At least five genes in this pathway show association with AS, including IL23R, PTER4, IL12B, CARD9, and TYK2. All of these genes are also associated with inflammatory bowel disease (IBD), and three of them are associated with psoriasis. Serum levels of IL-23 and IL-17 are elevated in AS patients. Mice expressing high levels of IL-23 show spontaneous

1	inflammatory bowel disease (IBD), and three of them are associated with psoriasis. Serum levels of IL-23 and IL-17 are elevated in AS patients. Mice expressing high levels of IL-23 show spontaneous infiltration in the entheses of CD3+CD4–CD8– cells bearing IL-23 receptors and producing IL-17 and IL-22. This finding suggests the possibility that site-specific innate immune cells may play a critical role in the anatomic specificity of the lesions. Mast cells and, to a lesser extent, neutrophils appear to be the major IL-17-producing cells in peripheral arthritis, whereas neutrophils producing IL-17 are prominent in apophyseal joints. High levels of circulating γδ T cells expressing IL-23 receptors and producing IL-17 have been found in AS patients.

1	Other associated genes encode other cytokines or cytokine receptors (IL6R, IL1R1, IL1R2, IL7R, IL27), transcription factors involved in the differentiation of immune cells (RUNX3, EOMES, BACH2, NKX2-3, TBX21), or other molecules involved in activation or regulation of immuneorinflammatoryresponses(FCGR2A,ZMIZ1,NOS2,ICOSLG).

1	The inflamed sacroiliac joint is infiltrated with CD4+ and CD8+ T cells and macrophages and shows high levels of TNF-α, particularly early in the disease. Abundant transforming growth factor β (TGFβ) has been found in more advanced lesions. Peripheral synovitis in AS and the other spondyloarthritides is characterized by neutrophils, macrophages expressing CD68 and CD163, CD4+ and CD8+ T cells, and B cells. There is prominent staining for intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), matrix metalloproteinase 3 (MMP-3), and myeloid-related proteins 8 and 14 (MRP-8 and MRP-14). Unlike rheumatoid arthritis (RA) synovium, citrullinatedproteinsandcartilagegp39peptide–majorhistocompatibility complexes (MHCs) are absent. However, citrullinated proteins can be seen in the circulation.

1	No specific event or exogenous agent that triggers the onset of disease has been identified, although overlapping features with reactive arthritis and IBD and the involvement of the IL-23/IL-17 pathway suggest that enteric bacteria may play a role, and microdamage from mechanical stress at enthesial sites has also been implicated.

1	It is firmly established that HLA-B27 plays a direct role in AS pathogenesis,butitspreciseroleatthemolecularlevelremainsunresolved.Rats transgenic for HLA-B27 develop arthritis and spondylitis, and this is unaffectedbytheabsenceofCD8.Itthusappearsthatclassicalpeptideantigen presentation to CD8+ T cells may not be the primary disease mechanism. However,theassociationofASwithERAP1,whichstronglyinfluencesthe MHC class I peptide repertoire, is only found in B27+ patients, and this suggests that peptide binding to B27 is nonetheless important. The pairs of ERAP1 alleles found in AS patients show diminished peptidase activity, compared with those found in healthy controls. The B27 heavy chain has an unusual tendency to misfold, a process that may be proinflammatory. Geneticandfunctionalstudiesinhumanshavesuggestedarolefornatural killer (NK) cells in AS, possibly through interaction with B27 heavy chain homodimers. SpA-prone B27 rats show defective dendritic cell function and share with AS

1	killer (NK) cells in AS, possibly through interaction with B27 heavy chain homodimers. SpA-prone B27 rats show defective dendritic cell function and share with AS patients a characteristic “reverse interferon” gene expression signature in antigen-presenting cells.

1	New bone formation in AS appears to be largely based on enchondral bone formation and occurs only in the periosteal compartment. It correlates with lack of regulation of the Wnt signaling pathway, which controls the differentiation of mesenchymal cells into osteophytes, by the inhibitors DKK-1 and sclerostin. Indirect evidence and data from animal models also implicate bone morphogenic proteins, hedgehog proteins, and prostaglandin E2. There is sharp controversy as to whether vertebral new bone formation in AS is a sequela of inflammation or whether it arises independently of inflammation. The second hypothesis is based on the observation that syndesmophyte formation is not suppressed by anti-TNF-α therapy that potently suppresses inflammation. TNF-α is also a known inducer of DKK-1, which inhibits bone formation. Recent magnetic resonance imaging (MRI) studies suggest that it is vertebral inflammatory lesions that undergo metaplasia to fat (increased T1-weighted signal) that are the

1	inhibits bone formation. Recent magnetic resonance imaging (MRI) studies suggest that it is vertebral inflammatory lesions that undergo metaplasia to fat (increased T1-weighted signal) that are the predominant site of subsequent syndesmophytes despite anti-TNF-α therapy, whereas early acute inflammatory lesions resolve. A recent study suggested that the rate of syndesmophyte formation decreases after >4 years of anti-TNF-α therapy.

1	The symptoms of the disease are usually first noticed in late adolescence or early adulthood; the median age in Western countries is approximately 23 years. In 5% of patients, symptoms begin after age 40. The initial symptom is usually dull pain, insidious in onset, felt deep in the lower lumbar or gluteal region, accompanied by low-back morning stiffness of up to a few hours’ duration that improves with activityandreturnsfollowinginactivity.Withinafewmonths,thepain has usually become persistent and bilateral. Nocturnal exacerbation of pain often forces the patient to rise and move around.

1	In some patients, bony tenderness (presumably reflecting enthesitis or osteitis) may accompany back pain or stiffness, whereas in others it may be the predominant complaint. Common sites include the costosternal junctions, spinous processes, iliac crests, greater trochanters, ischial tuberosities, tibial tubercles, and heels. Hip and shoulder (“root” joint) arthritis is considered part of the axial disease. Hip arthritis occurs in 25–35% of patients. Shoulder arthritis is much less common. Severe isolated hip arthritis or bony chest pain may be the presenting complaint, and symptomatic hip disease can dominate the clinical picture. Arthritis of peripheral joints other than the hips and shoulders, usually asymmetric, occurs in up to 30% of patients. Neck pain and stiffness from involvement of the cervical spine are usually relatively late manifestations but are occasionally dominant symptoms. Rare patients, particularly in the older age group, present with predominantly constitutional

1	of the cervical spine are usually relatively late manifestations but are occasionally dominant symptoms. Rare patients, particularly in the older age group, present with predominantly constitutional symptoms.

1	AS often has a juvenile onset in developing countries. Peripheral arthritis and enthesitis usually predominate, with axial symptoms supervening in late adolescence. Initially, physical findings mirror the inflammatory process. The most specific findings involve loss of spinal mobility, with limitation of anterior and lateral flexion and extension of the lumbar spine and of chest expansion. Limitation of motion is usually out of proportion to the degree of bony ankylosis and is thought to possibly reflect muscle spasm secondary to pain and inflammation. Pain in the sacroiliac joints maybeelicitedeitherwithdirectpressureorwithstressonthejoints.In addition,there is commonlytenderness uponpalpationofthe posterior spinous processes and other sites of symptomatic bony tenderness.

1	ThemodifiedSchobertestisausefulmeasureoflumbarspineflexion. The patient stands erect, with heels together, and marks are made on the spineat the lumbosacral junction(identified by ahorizontalline between theposterosuperioriliacspines)and10cmabove.Thepatientthenbends forward maximally with knees fully extended, and the distance between the two marks is measured. This distance increases by ≥5 cm in the case ofnormalmobilityandby<4cminthecaseofdecreasedmobility.Chest expansion is measured as the difference between maximal inspiration andmaximalforcedexpirationinthefourthintercostalspaceinmalesor just below the breasts in females, with the patient’s hands resting on or just behind the head. Normal chest expansion is ≥5 cm. Lateral bending measures the distance the patient’s middle finger travels down the leg with maximal lateral bending. Normal is >10 cm.

1	Limitation or pain with motion of the hips or shoulders is usually present if these joints are involved. It should be emphasized that early in the course of mild cases, symptoms may be subtle and nonspecific, and the physical examination may be unrevealing.

1	The course of the disease is extremely variable, ranging from the individual with mild stiffness and normal radiographs to the patient with a totally fused spine and severe bilateral hip arthritis, accompanied by severe peripheral arthritis and extraarticular manifestations. Pain tends to be persistent early in the disease and intermittent later, with alternating exacerbations and quiescent periods. In a typical severe untreated case with progression of the spondylitis to syndesmophyte formation, the patient’s posture undergoes characteristic changes, with obliterated lumbar lordosis, buttock atrophy, and accentuated thoracic kyphosis. There may be a forward stoop of the neck or flexion contractures at the hips, compensated by flexion at the knees. Disease progression can be estimated clinically from loss of height, limitation of chest expansion and spinal flexion, and occiput-to-wall distance.Occasionalindividualsareencounteredwithadvanced deformities who report having never had

1	clinically from loss of height, limitation of chest expansion and spinal flexion, and occiput-to-wall distance.Occasionalindividualsareencounteredwithadvanced deformities who report having never had significant symptoms.

1	The factors most predictive of radiographic progression (see below) are the presence of existing syndesmophytes, high inflammatory makers, and smoking. In some but not all studies, onset of AS in adolescence and early hip involvement correlate with a worse prognosis. In women, AS tends to progress less frequently to total spinal ankylosis, although there may be an increased prevalence of isolated cervical ankylosis and peripheral arthritis. In industrialized countries, peripheral arthritis (distal to hips and shoulders) occurs in less than one-half ofpatientswithAS,usuallyasalatemanifestation,whereasindeveloping countries, the prevalence is much higher, with onset typically early in the disease course. Pregnancy has no consistent effect on AS, with symptoms improving, remaining the same, or deteriorating in one-third of pregnant patients, respectively.

1	The most serious complication of the spinal disease is spinal fracture, which can occur with even minor trauma to the rigid, osteoporotic spine. The lower cervical spine is most commonly involved. These fractures are often displaced and cause spinal cord injury. A recent survey suggested a >10% lifetime risk of fracture. Occasionally, fracture through a diskovertebral junction and adjacent neural arch, termed pseudoarthrosis, most common in the thoracolumbar spine, can be an unrecognized source of persistent localized pain and/or neurologicdysfunction.Wedgingofthoracicvertebraeiscommonand correlates with accentuated kyphosis.

1	The most common extraarticular manifestation is acute anterior uveitis, which occurs in up to 40% of patients and can antedate the spondylitis.Attacksaretypicallyunilateral,causingpain,photophobia, and increased lacrimation. These tend to recur, often in the opposite eye. Cataracts and secondary glaucoma are not uncommon sequelae. Up to 60% of patients with AS have inflammation in the colon or ileum. This is usually asymptomatic, but frank IBD occurs in 5–10% of patients with AS (see “Enteropathic Arthritis,” below). About 10% of patients meeting criteria for AS have psoriasis (see “Psoriatic Arthritis,” below). Aortic insufficiency, sometimes leading to congestive heart failure, occurs in a small percentage of patients, occasionally early.Third-degreeheartblockmayoccuraloneortogetherwithaortic insufficiency. Subclinical pulmonary lesions and cardiac dysfunction mayberelativelycommon.Caudaequinasyndromeandupperpulmonary lobe fibrosis are rare late complications. Retroperitoneal

1	insufficiency. Subclinical pulmonary lesions and cardiac dysfunction mayberelativelycommon.Caudaequinasyndromeandupperpulmonary lobe fibrosis are rare late complications. Retroperitoneal fibrosis is a rare associated condition. Prostatitis has been reported to have an increased prevalence. Amyloidosis is rare (Chap. 137).

1	Several validated measures of disease activity and functional outcome are in widespread use in the study and management of AS, particularly the Bath Ankylosing Spondylitis Disease Activity Index 2171 (BASDAI) and the Ankylosing Spondylitis Disease Activity Score (ASDAS), both measures of disease activity; the Bath Ankylosing SpondylitisFunctionalIndex(BASFI),ameasureoflimitationinactivities of daily living; and several measures of radiographic changes. The Harris hip score, although not specific for AS, can be helpful. Despite persistence of the disease, most patients remain gainfully employed. Some but not all studies of survival in AS have suggested that AS shortens life span, compared with the general population. Mortality attributable to AS is largely the result of spinal trauma, aortic insufficiency, respiratory failure, amyloid nephropathy, or complications of therapy such as upper gastrointestinal hemorrhage. The impact of anti-TNF therapy on outcome and mortality is not yet

1	insufficiency, respiratory failure, amyloid nephropathy, or complications of therapy such as upper gastrointestinal hemorrhage. The impact of anti-TNF therapy on outcome and mortality is not yet known, except for significantly improved work productivity.

1	No laboratory test is diagnostic of AS. In most ethnic groups, HLAB27 is present in 80–90% of patients. Erythrocyte sedimentation rate (ESR)andC-reactiveprotein (CRP) are often,but notalways, elevated. Mildanemia maybe present. Patients with severe disease may show an elevatedalkalinephosphataselevel.ElevatedserumIgAlevelsarecommon. Rheumatoid factor, anti-cyclic citrullinated peptide (CCP), and antinuclear antibodies (ANAs) are largely absent unless caused by a coexistentdisease, although ANAs may appear with anti-TNF therapy. Circulating levels of CD8+ T cells tend to be low, and serum matrix metalloproteinase 3 levels correlate with disease activity. Synovial fluid from peripheral joints in AS is nonspecifically inflammatory. In cases with restriction of chest wall motion, decreased vital capacity and increased functional residual capacity are common, but airflow is normal and ventilatory function is usually well maintained.

1	Radiographically demonstrable sacroiliitis, usually symmetric, is eventually present in AS. The earliest changes by standard radiography are blurring of the cortical margins of the subchondral bone, followed by erosions and sclerosis. Progression of the erosions leads to “pseudowidening” of the joint space; as fibrous and then bony ankylosis supervene, the joints may become obliterated. In the lumbar spine, progression of the disease leads to straightening, caused by loss of lordosis, and reactive sclerosis, caused by osteitis ofthe anterior cornersof the vertebral bodies with subsequent erosion, leadingto“squaring”oreven“barreling”ofoneormorevertebralbodies. Progressive ossification leads to eventual formation of marginal syndesmophytes, visible on plain films as bony bridges connecting successive vertebral bodies anteriorly and laterally.

1	Years may elapse before unequivocal sacroiliac abnormalities are evident on plain radiographs, and consequently, MRI is being increasingly used in diagnosing AS. Active sacroiliitis is best visualized by dynamic MRI with fat saturation, either T2-weighed turbo spin-echo sequence or short tau inversion recovery (STIR) with high resolution, or T1-weighted images with contrast enhancement. These techniques sensitively identify early intraarticular inflammation, cartilage changes, and underlying bone marrow edema in sacroiliitis (Fig. 384-1). They are also highly sensitive for evaluation of acute and chronic spinal changes (Fig. 384-2). Reduced bone mineral density can be detected by dual-energy x-ray absorptiometry of the femoral neck and the lumbar spine. Use of a lateral projection of the L3 vertebral body can prevent falsely elevated readings related to spinal ossification.

1	It is important to establish the diagnosis of early AS before the development of irreversible deformity. This goal presents a challenge for several reasons: (1) Back pain is very common, but AS is much less common; (2) an early presumptive diagnosis often relies on clinical grounds requiring considerable expertise; and (3) young individuals with symptoms of AS often do not seek medical care. The widely used modified New York criteria (1984) are based on the presence of definite radiographic sacroiliitis and are too insensitive in early The Spondyloarthritides

1	The Spondyloarthritides FIGUrE 384-1 Early sacroiliitis in a patient with ankylosing spondylitis, indicatedbyprominentedemainthejuxtaarticularbonemarrow(asterisks),synoviumandjointcapsule(thin arrow),andinterosseousligaments(thick arrow)onashorttauinversionrecovery(STIR)magneticresonanceimage.(From M Bollow et al: Zeitschrift f Rheumatologie 58:61, 1999. Reproduced with permission.) or mild cases. In 2009, new criteria for axial SpA were proposed by the Assessment of Spondyloarthritis International Society (ASAS) (Table 384-1). They are applicable to individuals with ≥3 months of back pain with age of onset <45 years old. Active inflammation of the sacroiliac joints as determined by dynamic MRI is considered equivalent to definite radiographic sacroiliitis (see below).

1	AS must be differentiated from numerous other causes of low-back pain, some far more common than AS. To qualify as the criterion for inflammatory back pain of axial SpA (Table 384-1), the chronic (≥3 months) back pain should have four or more of the following characteristic features: (1) age of onset <40 years old; (2) insidious onset; (3) improvement with exercise; (4) no improvement with rest; and (5)painatnightwithimprovementupongettingup.Othercommon features of inflammatory back pain include morning stiffness >30 min, awakening from back pain during only the second half of the night, and alternating buttock pain. In clinical decision-making, all of these features are additive. The most common causes of back pain other than AS are primarily mechanical or degenerative rather than primarily inflammatory and tend not to show clustering of these features.

1	Less-common metabolic, infectious, and malignant causes of back pain must also be differentiated from AS, including infectious spondylitis, spondylodiskitis, and sacroiliitis, and primary or metastatic tumor. Ochronosis can produce a phenotype that is clinically and radiographically similar to AS. Calcification and ossification of paraspinous ligaments occur in diffuse idiopathic skeletal hyperostosis (DISH), which occurs in the middle-aged and elderly and is usually not symptomatic. Ligamentous calcification gives the appearance of “flowing wax” on the anterior bodies of the vertebrae. Intervertebral disk spaces are preserved, and sacroiliac and apophyseal joints appear normal, helping to differentiate DISH from spondylosis and from AS, respectively.

1	All management of AS should include an exercise program designed to maintain posture and range of motion. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the first line of pharmacologic therapy for AS. These agents reduce pain and tenderness and increase mobility in many patients with AS. There is mounting evidence that continuous high-dose NSAID therapy slows radiographic progression, particularly in patients who are at higher risk for progression. However, many patients with AS have continued symptoms despite NSAID therapy and are likely to benefit from anti-TNF-α therapy. Patients with AS treated with infliximab (chimeric human/mouse anti-TNF-α monoclonal antibody), etanercept (soluble p75 TNF-α receptor–IgG fusion protein), adalimumab, or golimumab (human anti-TNF-α monoclonal antibodies, or certolizumab pegol [humanized mouse anti-TNF-α monoclonal antibody]) have shown rapid, profound, and sustained reductions in all clinical and laboratory measures of disease activity. In a

1	or certolizumab pegol [humanized mouse anti-TNF-α monoclonal antibody]) have shown rapid, profound, and sustained reductions in all clinical and laboratory measures of disease activity. In a good response, there is significant improvement in both objective and subjective indicators of disease activity and function, including morning stiffness, pain, spinal mobility, peripheral joint swelling, CRP, and ESR. MRI studies indicate substantial resolution of bone marrow edema, enthesitis, and joint effusions in the sacroiliac joints, spine, and peripheral joints (Fig. 384-2). Similar results have been obtained in large randomized controlled trials of all four agents and many open-label studies. About one-half of the patients achieve a ≥50% reduction in the BASDAI. The response tends to be stable over time, and partial or full remissions are common. Predictors of the best responses include younger age, shorter disease duration, higher baseline inflammatory markers, and lower baseline

1	stable over time, and partial or full remissions are common. Predictors of the best responses include younger age, shorter disease duration, higher baseline inflammatory markers, and lower baseline functional disability. Nonetheless, some patients with long-standing disease and even spinal ankylosis can obtain significant benefit. Increased bone mineral density is found as early as 24 weeks after onset of therapy. There is evidence that anti-TNF therapy does not prevent syndesmophyte formation, although this may apply mainly during the early years of therapy. A mechanism for this has been proposed based on the observation that TNF-α inhibits new bone formation by upregulating DKK-1, a negative regulator of the wingless (Wnt) signaling pathway that promotes osteoblast activity. Serum DKK-1 levels are inappropriately low in AS patients and are also suppressed by anti-TNF therapy.

1	FIGUrE 384-2 Spinal inflammation (spondylodiskitis) in a patient with ankylosing spondylitis and its dramatic response to treatment with infliximab. Gadolinium-enhanced T1-weighted magnetic resonance images, with fat saturation, at baseline and after 24 weeks of infliximab therapy. (From J Braun et al: Arthritis Rheum 54:1646, 2006.) Infliximab is given intravenously, 3–5 mg/kg body weight, and then repeated 2 weeks later, again 6 weeks later, and then at 8-week intervals. Etanercept is given by subcutaneous injection, 50 mg once weekly. Adalimumab is given by subcutaneous injection, 40 mg biweekly. Golimumab is given by subcutaneous injection, 50 or 100 mg every 4 weeks. Certolizumab pegol is given by subcutaneous injection, 400 mg every 4 weeks.

1	Although these potent immunosuppressive agents have thus far been relatively safe, patients are at increased risk for serious infections, including disseminated tuberculosis. Hypersensitivity infusion or injection site reactions are not uncommon. Cases of anti-TNF-induced psoriasis have been increasingly recognized. Rare cases of systemic lupus erythematosus–related disease have been reported, as have hematologic disorders such as pancytopenia, demyelinating disorders, exacerbation of congestive heart failure, and severe liver disease. The overall incidence of malignancy does not appear to be increased in AS patients treated with anti-TNF therapy, but isolated cases of hematologic malignancy have occurred shortly after the start of treatment.

1	Because of the expense, potentially serious side effects, and unknown long-term effects of these agents, their use should be restricted to patients with a definite diagnosis and active disease (BASDAI ≥4 out of 10 and expert opinion) that is inadequately responsive to therapy with at least two different NSAIDs. Before initiation of anti-TNF therapy, all patients should be tested for tuberculin (TB) reactivity, and reactors (≥5 mm on PPD testing or a positive quantiferon test) should be treated with anti-TB agents. Contraindications include active infection or high risk of infection; malignancy or premalignancy; and history of systemic lupus erythematosus, multiple sclerosis, or related autoimmunity. Pregnancy and breast-feeding are relative contraindications. Continuation beyond 12 weeks of therapy requires either a 50% reduction in BASDAI or absolute reduction of ≥2 out of 10, and favorable expert opinion. Switching to a second anti-TNF agent may be effective, especially if there was

1	therapy requires either a 50% reduction in BASDAI or absolute reduction of ≥2 out of 10, and favorable expert opinion. Switching to a second anti-TNF agent may be effective, especially if there was a response to the first that was lost rather than primary failure. Sulfasalazine, in doses of 2–3 g/d, has been shown to be of modest benefit, primarily for peripheral arthritis. A therapeutic trial of this agent should precede any use of anti-TNF agents in patients with predominantly peripheral arthritis. Methotrexate, although widely used, has not been shown to be of benefit in AS, nor has any therapeutic role for gold or oral glucocorticoids been documented. Potential benefit in AS has been reported for thalidomide, 200 mg/d, perhaps acting through inhibition of TNF-α.

1	Ustekinumab (anti-IL-12/23) and secukinumab (anti-IL-17) monoclonal antibodies have shown promising efficacy in clinical trials, but have not yet been approved for use in AS. The most common indication for surgery in patients with AS is severe hip joint arthritis, the pain and stiffness of which are usually dramatically relieved by total hip arthroplasty. Rare patients may benefit from surgical correction of extreme flexion deformities of the spine or of atlantoaxial subluxation. Attacks of uveitis are usually managed effectively with local glucocorticoid administration in conjunction with mydriatic agents, although systemic glucocorticoids, immunosuppressive drugs, or anti-TNF therapy may be required. TNF inhibitors reduce the 2173 frequency of attacks of uveitis in patients with AS, although cases of new or recurrent uveitis after use of a TNF inhibitor have been observed, especially with etanercept.

1	Coexistent cardiac disease may require pacemaker implantation and/or aortic valve replacement. Management of axial osteoporosis is at present similar to that used for primary osteoporosis, since data specific for AS are not available. Reactive arthritis (ReA) refers to acute nonpurulent arthritis complicating an infection elsewhere in the body. In recent years, the term has been used primarily to refer to SpA following enteric or urogenital infections. Other forms of reactive and infection-related arthritis not associated with B27 and showing a spectrum of clinical features different from SpA, such as Lyme disease and rheumatic fever, are discussed in Chaps. 210 and 381.

1	The association of acute arthritis with episodes of diarrhea or urethritis hasbeenrecognizedforcenturies.AlargenumberofcasesduringWorld Wars I and II focused attention on the triad of arthritis, urethritis, and conjunctivitis, often with additional mucocutaneous lesions, which becamewidely knownby eponyms that are now ofhistoric interestonly.

1	The identification of bacterial species capable of triggering the clinical syndrome and the finding that many patients possess the B27 antigen led to the unifying concept of ReA as a clinical syndrome triggered by specific etiologic agents in a genetically susceptible host. A similar spectrumofclinicalmanifestations can betriggeredby enteric infection with any of several Shigella, Salmonella, Yersinia, and Campylobacter species; by genital infection with Chlamydia trachomatis; and by other agents as well. The triad of arthritis, urethritis, and conjunctivitis represents a small part of the spectrum of the clinical manifestations of ReA and only a minority of patients present with this “classic triad” of symptoms. Although emerging data suggest that asymptomatic Chlamydia trachomatis infections might trigger ReA, for the purposes of this chapter, the use of the term ReA will be restricted to those cases of SpA in which there is at least presumptive evidence for a related symptomatic

1	might trigger ReA, for the purposes of this chapter, the use of the term ReA will be restricted to those cases of SpA in which there is at least presumptive evidence for a related symptomatic antecedent infection. Patients with clinical features of ReA who lack evidence of an antecedent infection will be considered to have undifferentiated spondyloarthritis, discussed below.

1	InitialreportsmayhaveoverestimatedtheassociationofReAwithHLAB27, since 60–85% of patients who developed ReA triggered by Shigella, Yersinia, or Chlamydia were B27-positive. However, other studies demonstratedalowerprevalenceofB27inReAtriggeredbySalmonella,with one study suggesting no association in Campylobacter-induced ReA. Several more recent community-based or common-source epidemic studies demonstrated that the prevalence of B27 in ReA was below 50%. The most common age range is 18–40 years, but ReA can occur rarely in children and occasionally in older adults.

1	TheattackrateofpostentericReAgenerallyrangesfrom1%toabout 30%dependingonthestudyandcausativeorganism,whereastheattack rate of postchlamydial ReA is about 4–8%. The gender ratio in ReA following enteric infection is nearly 1:1, whereas venereally acquired ReA occurs mainly in men. The overall prevalence and incidence of ReA are difficult to assess because of the lack of validated diagnostic criteria, variable prevalence and arthritogenic potential of the triggering infectious agents, and inconstant genetic susceptibility factors in different populations. In Scandinavia, an annual incidence of 10–28:100,000 has been reported. Thespondyloarthritides were formerly almostunknown in sub-Saharan Africa. However, ReA and other peripheral SpAs have now become the most common rheumatic diseases in Africans in the wake of the AIDS epidemic, without association to B27, which is very rare in these populations. ReA is often the first manifestation of HIV infection and often remits with disease

1	in Africans in the wake of the AIDS epidemic, without association to B27, which is very rare in these populations. ReA is often the first manifestation of HIV infection and often remits with disease progression. In contrast,

1	The Spondyloarthritides 2174 WesternwhitepatientswithHIVandSpAareusuallyB27-positive,and the arthritis flares as AIDS advances. Synovial histology is similar to that of other SpAs. Enthesitis shows increased vascularity and macrophage infiltration of fibrocartilage. Microscopic histopathologic evidence of inflammation mimicking IBD has routinely been demonstrated in the colon and ileum of patients withpostentericReAand lesscommonly inpostvenereal ReA. The skin lesions of keratoderma blennorrhagica, associated mainly withvenereallyacquired ReA,arehistologically indistinguishable from pustular psoriatic lesions.

1	The bacteria identified as definitive triggers of ReA include several Salmonella spp., Shigella spp., Yersinia enterocolitica, Yersinia pseudo-tuberculosis, Campylobacter jejuni, and Chlamydia trachomatis. These triggering microbes are gram-negative bacteria with a lipopolysaccharide component to their cell walls. All four Shigella species (Shigella sonnei, Shigella boydii, Shigella flexneri, and Shigella dysenteriae) have been implicated in cases of ReA, with S. flexneri and S. sonnei being the most common. After Salmonella infection, individuals of Caucasian descent may bemore likely than thoseof Asian descent todevelop ReA.Children may be less susceptible to ReA caused by Salmonella and Campylobacter. Yersinia species in Europe and Scandinavia may have greater arthritogenic potential than in other parts of the world, and C. trachomatis appears to be a common cause worldwide. The ocular serovars of C. trachomatis appear to be particularly, perhaps uniquely, arthritogenic.

1	There is also evidence implicating Clostridium difficile, Campylobacter coli, certain toxigenic Escherichia coli, and possibly Ureaplasma urealyticum and Mycoplasma genitalium as potential triggers of ReA. Chlamydia pneumoniae is another trigger of ReA, albeit far less common than C. trachomatis. There have also been numerous isolated reports of acute arthritis preceded by other bacterial, viral, or parasitic infections, and even following intravesicular bacillus Calmette-Guérin (BCG) treatment for bladder cancer.

1	It has not been determined whether ReA occurs by the same pathogenic mechanism following infection with each of these microorganisms, nor has the mechanism been elucidated in the case of any one of the known bacterial triggers. Most, if not all, of the organisms well established to be triggers share a capacity to attack mucosal surfaces, to invade host cells, and to survive intracellularly. Antigens from Chlamydia, Yersinia, Salmonella, and Shigella have been shown to be present in the synovium and/or synovial fluid leukocytes of patients with ReA for long periods following the acute attack. In ReA triggered by Y. enterocolitica, bacterial lipopolysaccharide (LPS) and heat-shock protein antigens have been found in peripheral blood cells years after the triggering infection. Yersinia DNA and C. trachomatis DNA and RNA have been detected in synovial tissue from ReA patients, suggesting the presence of viable organisms despite uniform failure to culture the organism from these specimens.

1	C. trachomatis DNA and RNA have been detected in synovial tissue from ReA patients, suggesting the presence of viable organisms despite uniform failure to culture the organism from these specimens. In C. trachomatis–induced ReA specifically, the bacterial load in synovial tissue of patients with remitting disease is lower than that of active disease, but mRNAs encoding proinflammatory proteins are equal to or higher than those of active disease. The specificity of these findings is unclear, however, since chromosomal bacterial DNA and 16S rRNA from a very wide variety of bacteria have also been found in synovium in other rheumatic diseases, albeit less frequently. In several older studies, synovial T cells that specifically responded to antigens of the inciting organism were reported and characterized as predominantly CD4+ with a TH2 or T regulatory phenotype. More recent work has documented high levels of IL-17 in ReA synovial fluid, but the source has not been identified. HLA-B27

1	characterized as predominantly CD4+ with a TH2 or T regulatory phenotype. More recent work has documented high levels of IL-17 in ReA synovial fluid, but the source has not been identified. HLA-B27 seems to be associated with more severe and chronic forms of the “classic triad” of ReA, but its pathogenic role remains to bedetermined. HLA-B27 significantly prolongs the intracellular survival of Y. enterocolitica and Salmonella enteritidis in human and mouse cell lines. Prolonged intracellular bacterial survival, promoted by B27, other factors, or both, maypermit traffickingof infectedleukocytes fromthe site ofprimaryinfection tojoints,whereaninnate and/oradaptiveimmune response to persistent bacterial antigens may then promote arthritis.

1	The clinical manifestations of ReA constitute a spectrum that ranges from an isolated, transient monoarthritis or enthesitis to severe multisystem disease. A careful history will usually elicit evidence of an antecedent infection 1–4 weeks before onset of symptoms of the reactive disease, particularly in postenteric ReA. However, in a sizable minority, no clinical or laboratory evidence of an antecedent infection can be found, particularly in the case of postchlamydial ReA. In cases of presumed venereally acquired reactive disease, there is often a history of a recent new sexual partner, even without laboratory evidence of infection.

1	Constitutional symptoms are common, including fatigue, malaise, fever, and weight loss. The musculoskeletal symptoms are usually acute in onset. Arthritis is usually asymmetric and additive, with involvement of new joints occurring over a few days to 1–2 weeks. The joints of the lower extremities, especially the knee, ankle, and subtalar, metatarsophalangeal, and toe interphalangeal joints, are most commonly involved, but the wrist and fingers can be involved as well. The arthritis is usually quite painful, and tense joint effusions are not uncommon, especially in the knee. Dactylitis, or “sausage digit,” a diffuse swelling of a solitary finger or toe, is a distinctive feature of ReA andotherperipheralspondyloarthritidesbutcanbeseeninpolyarticular gout and sarcoidosis. Tendinitis and fasciitis are particularly characteristic lesions, producing pain at multiple insertion sites (entheses), especially the Achilles insertion, the plantar fascia, and sites along the axial skeleton. Spinal,

1	are particularly characteristic lesions, producing pain at multiple insertion sites (entheses), especially the Achilles insertion, the plantar fascia, and sites along the axial skeleton. Spinal, low-back, and buttock pain are quite common and may be caused by insertional inflammation, muscle spasm, acute sacroiliitis, or, presumably, arthritis in intervertebral joints.

1	Urogenital lesions may occur throughout the course of the disease. In males, urethritis may be marked or relatively asymptomatic and may be either an accompaniment of the triggering infection or a result ofthereactivephaseofthedisease;interestingly,itoccursinbothpostvenereal and postenteric ReA. Prostatitis is also common. Similarly, in females, cervicitis or salpingitis may be caused either by the infectious trigger or by the sterile reactive process. Ocular disease is common, ranging from transient, asymptomatic conjunctivitis to an aggressive anterior uveitis that occasionally proves refractory to treatment and may result in blindness.

1	Mucocutaneous lesions are frequent. Oral ulcers tend to be superficial,transient, andoftenasymptomatic. The characteristicskin lesions, keratoderma blennorrhagica, consist of vesicles and/or pustules that become hyperkeratotic, ultimately forming a crust before disappearing. They are most common on the palms and soles but may occur elsewhere as well. In patients with HIV infection, these lesions are often extremely severe and extensive, sometimes dominating the clinical picture (Chap. 226). Lesions may occur on the glans penis, termed circinate balanitis; these consist of vesicles that quickly rupture to form painless superficial erosions, which in circumcised individuals can form crusts similar to those of keratoderma blennorrhagica. Nail changes are common and consist of onycholysis, distal yellowish discoloration, and/or heaped-up hyperkeratosis.

1	Less-frequent or rare manifestations of ReA include cardiac conduction defects, aortic insufficiency, central or peripheral nervous system lesions, and pleuropulmonary infiltrates. Arthritis typically persists for 3–5 months, but more chronic courses do occur. Chronic joint symptoms persist in about 15% of patients and in up to 60% of patients in hospital-based series, but these tend to be less severe than in the acute stage. Recurrences of the acute syndrome are also common. Work disability or forced change in occupation is common in those with persistent joint symptoms. Chronic heel pain is often particularly distressing. Low-back pain, sacroiliitis, and frank AS are also common sequelae. In most studies, HLA-B27–positive patients have shown a worse outcome than B27-negative patients. Patients with Yersinia-or Salmonella-induced arthritis have less chronic disease than those whose initial episode follows epidemic shigellosis.

1	The ESR and acute-phase reactants are usually elevated during the acute phase of the disease, often markedly so. Mild anemia may be present. Synovial fluid is nonspecifically inflammatory. In most ethnic groups, 30–50% of the patients are B27-positive. The triggering infection usually does not persist at the site of primary mucosal infection through the time of onset of the reactive disease, but it may be possible to culture the organism, e.g., in the case of Yersinia-or Chlamydia-induced disease. Serologic evidence of exposure to one of the causative organisms with elevation of antibodies is nonspecific and is of questionable utility. Polymerase chain reaction (PCR) for chlamydial DNA in first-voided urine specimens may have high sensitivity in the acute stage but is less useful with chronic disease.

1	In early or mild disease, radiographic changes may be absent or confined to juxtaarticular osteoporosis. With long-standing persistent disease, radiographic features share those of psoriatic arthritis; marginal erosions and loss of joint space can be seen in affected joints. Periostitis with reactive new bone formation is characteristic, as in all the SpAs. Spurs at the insertion of the plantar fascia are common. Sacroiliitis and spondylitis may be seen as late sequelae. Sacroiliitis ismorecommonlyasymmetricthaninAS,andspondylitis,ratherthan ascending symmetrically, can begin anywhere along the lumbar spine. The syndesmophytes are described as nonmarginal; they are coarse, asymmetric, and “comma”-shaped, arising from the middle of a vertebral body, a pattern less commonly seen in primary AS. Progression to spinal fusion is uncommon.

1	ReA is a clinical diagnosis with no definitively diagnostic laboratory test or radiographic finding. The diagnosis should be entertained in any patient with an acute inflammatory, asymmetric, additive arthritis or tendinitis. The evaluation should include questioning regarding possible triggering events such as an episode of diarrhea or dysuria. On physical examination, attention must be paid to the distribution of the joint and tendon involvement and to possible sitesof extraarticular involvement, such as the eyes, mucous membranes, skin, nails, and genitalia. Synovial fluid analysis may be helpful in excluding septic or crystal-induced arthritis. Culture, serology, or molecularmethods may help to identify a triggering infection, but they cannot be relied upon.

1	Although typingfor B27 has low negative predictive value in ReA, it may have prognostic significance in terms of severity, chronicity, and the propensity for spondylitis and uveitis. Furthermore, if positive, it can be helpful diagnostically in atypical cases. HIV testing is often indicated and may be necessary in order to select appropriate therapy.

1	It is important to differentiate ReA from disseminated gonococcal disease (Chap. 181), both of which can be venereally acquired and associated with urethritis. Unlike ReA, gonococcal arthritis and tenosynovitis tend to involve both upper and lower extremities equally, spare the axial skeleton, and be associated with characteristic vesicular skin lesions. A positive gonococcal culture from the urethra or cervix does not exclude a diagnosis of ReA; however, culturing gonococci from blood, skin lesion, or synovium establishes the diagnosis of disseminated gonococcal disease. PCR assay for Neisseria gonorrhoeae and C. trachomatis may be helpful. Occasionally, only a therapeutic trial of antibiotics can distinguish the two.

1	ReA shares many features in common with psoriatic arthropathy. However, psoriatic arthritis is usually gradual in onset; the arthritis tends to affect primarily the upper extremities; there is less associated periarthritis; and there are usually noassociated mouth ulcers,urethritis, or bowel symptoms. Most patients with ReA benefit to some degree from high-dose NSAIDs, although acute symptoms are rarely completely ameliorated, and some patients fail to respond at all. Indomethacin, 75–150 mg/d in divided doses, is the initial treatment of choice, but other NSAIDs may be tried.

1	Prompt, appropriate antibiotic treatment of acute chlamydial 2175 urethritis or enteric infection may prevent the emergence of ReA, but is not universally successful. Data regarding the potential benefit of antibiotic therapy that is initiated after onset of arthritis are conflicting, but several trials suggest no benefit. One long-term follow-up study suggested that although antibiotic therapy had no effect on the acute episode of ReA, it helped prevent subsequent chronic SpA. Another such study failed to demonstrate any longterm benefit. A promising recent double-blind placebo-controlled study assessing combination antibiotics showed that a majority of patients with chronic ReA due to Chlamydia benefited significantly from a 6-month course of rifampin 300 mg daily plus azithromycin 500 mg daily for 5 days, then twice weekly, or 6 months of rifampin 300 mg daily plus doxycycline 100 mg twice daily. The possibility remains that acute Chlamydia-induced ReA might respond more favorably

1	mg daily for 5 days, then twice weekly, or 6 months of rifampin 300 mg daily plus doxycycline 100 mg twice daily. The possibility remains that acute Chlamydia-induced ReA might respond more favorably to antibiotic therapy than the postenteric variety.

1	Multicenter trials have suggested that sulfasalazine, up to 3 g/d in divided doses, may be beneficial to patients with persistent ReA.1 Patients with persistent disease may respond to azathioprine, 1–2 mg/ kg per day, or to methotrexate, up to 20 mg per week; however, these therapeutic regimens have never formally been studied. Although no controlled trials of anti-TNF-α in ReA have been reported, anecdotal evidence supports the use of these agents in severe chronic cases, although lack of response has also been observed.1

1	Tendinitis and other enthesitic lesions may benefit from intralesional glucocorticoids. Uveitis may require aggressive treatment to prevent serious sequelae (see above). Skin lesions ordinarily require only symptomatic topical treatment. In patients with HIV infection and ReA, many of whom have severe skin lesions, the skin lesions in particular respond to antiretroviral therapy. Cardiac complications are managed conventionally; management of neurologic complications is symptomatic. Comprehensive management includes counseling of patients in the avoidance of sexually transmitted disease and exposure to enteropathogens, as well as appropriate use of physical therapy, vocational counseling, and continued surveillance for long-term complications such as AS. Patients with a history of ReA are at increased risk for recurrent attacks following repeated exposures.

1	Psoriatic arthritis (PsA) refers to an inflammatory musculoskeletal disease that has both autoimmune and autoinflammatory features characteristically occurring in individuals with psoriasis. The association between arthritis and psoriasis was noted in the nineteenth century. In the 1960s, on the basis of epidemiologic and clinical studies, it became clear that unlike RA, the arthritis associated with psoriasis was usually seronegative, often involved the distal interphalangeal (DIP) joints of the fingers and the spine and sacroiliac joints, had distinctive radiographic features, and showed considerable familial aggregation. In the 1970s, PsA was included in the broader category of the spondyloarthritides because of features similar to those of AS and ReA.

1	Estimates of the prevalence of PsA among individuals with psoriasis range from 5 to 42%. The prevalence of PsA appears to be increasing in parallelwithdisease awareness;recent datausingscreeningtools suggest that 20% or more of patients with psoriasis have undiagnosed PsA. The durationandseverityofpsoriasis increaseone’s likelihood ofdeveloping PsA. In white populations, psoriasis is estimated to have a prevalence of 1Azathioprine, methotrexate, sulfasalazine, pamidronate, and thalidomide have not been approved for this purpose by the U.S. Food and Drug Administration at the time of publication.

1	The Spondyloarthritides 2176 1–3%. Psoriasis and PsA are less common in other races in the absence of HIV infection, and the prevalence of PsA in individuals with psoriasis may be less common. First-degree relatives of PsA patients have an elevated risk for psoriasis, for PsA itself, and for other forms of SpA. Of patientswithpsoriasis,upto30%haveanaffectedfirst-degreerelative.In monozygotic twins, the reported concordance for psoriasis varies from 35 to 72%, and for PsA from 10 to 30%. A variety of HLA associations have been found. The HLA-Cw*0602 gene is directly associated with psoriasis,particularlyfamilialjuvenile-onset(typeI)psoriasis.HLA-B27 is associated with psoriatic spondylitis (see below). HLA-DR7, -DQ3, and -B57 areassociated with PsA becauseof linkage disequilibrium with Cw6. Other associations with PsA include HLA-B13, -B37, -B38, -B39, -C12, and -DR4. A recent genome-wide scan found association of both psoriasisandPsAwithapolymorphismattheHCP5locuscloselylinked to

1	Cw6. Other associations with PsA include HLA-B13, -B37, -B38, -B39, -C12, and -DR4. A recent genome-wide scan found association of both psoriasisandPsAwithapolymorphismattheHCP5locuscloselylinked to HLA-B, and also to IL-23R, IL-12B (chromosome 5q31), IL-13, and several other chromosomal regions. Certain genetic loci are associated with PsA but not psoriasis, e.g., RUNX3 and IL-13.

1	The inflamed synovium in PsA resembles that of RA, although with somewhat less hyperplasia and cellularity than in RA. As noted with AS above, the synovial vascular pattern in PsA is generally greater and more tortuous than in RA, independent of disease duration. Some studies have indicated a higher tendency to synovial fibrosis in PsA. Unlike RA, PsA shows prominent enthesitis, with histology similar to that of the other spondyloarthritides.

1	PsA is almost certainly immune-mediated and probably shares pathogenic mechanisms with psoriasis. PsA synovium is characterized by lining layer hyperplasia; diffuse infiltration with T cells, B cells, macrophages, and NK receptor–expressing cells, with upregulation of leukocyte homing receptors; and neutrophil proliferation with angiogenesis. Clonally expanded T cell subpopulations are frequent and have been demonstrated both in the synovium and the skin. Plasmacytoid dendritic cells are thought to play a key role in psoriasis, and there is some evidence for their participation in PsA. There is abundant synovial overexpression of proinflammatory cytokines, and synovial tissue staining has identified an overexpression of monocyte-derived cytokines, such as myeloid-related protein (S100A8/A9). Interferon γ, TNF-α, and IL-1β, -2, -6, -8, -10, -12, -13, and -15 are found in PsA synovium or synovial fluid. TH17-derived cytokines are important in PsA, given the genetic association with

1	Interferon γ, TNF-α, and IL-1β, -2, -6, -8, -10, -12, -13, and -15 are found in PsA synovium or synovial fluid. TH17-derived cytokines are important in PsA, given the genetic association with genes in the IL-12/IL-23 axis and the therapeutic response to an antibody to the shared IL-12/23 p40 subunit (see below). TH17 cells have been identified from the dermal extracts of psoriatic lesions and the synovial fluid of PsA patients. The majority of these CD4+ IL-17+ T cells are of memory phenotype (CD4RO[+]CD45RA[–]CD11a[+]). Consistent with the extensive bone remodeling in PsA, patients with PsA have been found to have a marked increase in osteoclastic precursors in peripheral blood and upregulation of receptor activator of nuclear factor κβ ligand (RANKL) in the synovial lining layer. Increased serum levels of TNF-α, RANKL, leptin, and omentin positively correlate with these osteoclastic precursors.

1	In 60–70% of cases, psoriasis precedes joint disease. In 15–20% of cases, the two manifestations appear within 1 year of each other. In about 15–20% of cases, the arthritis precedes the onset of psoriasis and can present a diagnostic challenge. The frequency in men and women is almost equal, although the frequency of disease patterns differs somewhat in the two sexes. The disease can begin in childhood or late in life but typically begins in the fourth or fifth decade, at an average age of 37 years. The spectrum of arthropathy associated with psoriasis is quite broad. Many classification schemes have been proposed. In the original scheme of Wright and Moll, five patterns are described: (1) arthritis of the DIP joints; (2) asymmetric oligoarthritis; (3) symmetric polyarthritis similar to RA; (4) axial involvement (spine and sacroiliac joints); and (5) arthritis mutilans, a highly destructive form of FIGUrE 384-3 Characteristic lesions of psoriatic arthritis.

1	FIGUrE 384-3 Characteristic lesions of psoriatic arthritis. Inflammation is prominent in the distal interphalangeal joints (left 5th, 4th, 2nd; right 2nd, 3rd, and 5th) and proximal interphalangeal joints (left 2nd, right 2nd, 4th, and 5th). There is dactylitis in the left 2nd finger and thumb, with pronounced telescoping of the left 2nd finger. Nail dystrophy (hyperkeratosis and onycholysis) affects each of the fingers except the left 3rd finger, the only finger without arthritis. (Courtesy of Donald Raddatz, MD; with permission.) disease. These patterns frequently coexist, and the pattern that persists chronically often differs from thatofthe initialpresentation. A simpler scheme in recent use contains three patterns: oligoarthritis, polyarthritis, and axial arthritis.

1	Nail changes in the fingers or toes occur in up to 90% of patients with PsA, compared with 40% of psoriatic patients without arthritis, and pustular psoriasis is said to be associated with more severe arthritis. Several articular features distinguish PsA from other joint disorders;such hallmark featuresinclude dactylitis and enthesitis.Dactylitis occurs in >30%; enthesitis and tenosynovitis are also common and are probably present in most patients, although often not appreciated on physical examination. Shortening of digits because of underlying osteolysis is particularly characteristic of PsA (Fig. 384-3), and there is a much greater tendency than in RA for both fibrous and bony ankylosis of small joints. Rapid ankylosis of one or more proximal interphalangeal (PIP) joints early in the course of disease is not uncommon. Back and neck pain and stiffness are also common in PsA.

1	Arthropathy confined to the DIP joints occurs in about 5% of cases. Accompanying nail changes in the affected digits are almost always present. These joints are also often affected in the other patterns of PsA. Approximately 30% of patients have asymmetric oligoarthritis. This pattern commonly involves a knee or another large joint with a few small joints in the fingers or toes, often with dactylitis. Symmetric polyarthritis occurs in about 40% of PsA patients at presentation. It may be indistinguishable from RA in terms of the joints involved, but other features characteristic of PsA are usually also present. Almost any peripheral joint can be involved. Axial arthropathy without peripheral involvement is found in about 5% of PsA patients. It may be clinically indistinguishable from idiopathic AS, although more neck involvement and less thoracolumbar spinal involvement are characteristic,andnail changesarenot found inidiopathic AS.Asmall percentage of PsA patients have arthritis

1	idiopathic AS, although more neck involvement and less thoracolumbar spinal involvement are characteristic,andnail changesarenot found inidiopathic AS.Asmall percentage of PsA patients have arthritis mutilans, in which there can bewidespreadshorteningofdigits(“telescoping”),sometimescoexisting with ankylosis and contractures in other digits.

1	Six patterns of nail involvement are identified: pitting, horizontal ridging, onycholysis, yellowish discoloration of the nail margins, dystrophic hyperkeratosis, and combinations of these findings. Other extraarticular manifestations of the spondyloarthritides are common. Eye involvement, either conjunctivitis or uveitis, is reported in 7–33% of PsA patients. Unlike the uveitis associated with AS, the uveitis in PsA is more often bilateral, chronic, and/or posterior. Aortic valve insufficiency has been found in <4% of patients, usually after longstanding disease.

1	Widely varying estimates of clinical outcome have been reported in PsA. At its worst, severe PsA with arthritis mutilans is potentially at least as crippling and ultimately fatal as severe RA. Unlike RA, however, many patients with PsA experience temporary remissions. Overall, erosive disease develops in the majority of patients, progressive disease with deformity and disability is common, and in some largepublishedseries,mortalitywasfoundtobesignificantlyincreased compared with the general population. There appears to be a greater incidence of cardiovascular death in psoriatic disease. The psoriasis and associated arthropathy seen with HIV infection both tend to be severe and can occur in populations with very little psoriasis in noninfected individuals. Severe enthesopathy, dactylitis, and rapidly progressive joint destruction are seen, but axial involvement is very rare. This condition is prevented by or responds well to antiretroviral therapy.

1	Thereare nolaboratorytestsdiagnostic ofPsA.ESRandCRPareoften elevated.Asmallpercentageofpatientsmayhavelowtitersofrheumatoid factor or antinuclear antibodies. About 10% of patients have anti-CCP antibodies. Uric acid may be elevated in the presence of extensive psoriasis. HLA-B27 is found in 50–70% of patients with axial disease, but in ≤20% of patients with only peripheral joint involvement.

1	The peripheral and axial arthropathies in PsA show a number of radiographic features that distinguish them from RA and AS, respectively. Characteristics of peripheral PsA include DIP involvement, including the classic “pencil-in-cup” deformity; marginal erosions with adjacent bony proliferation (“whiskering”); small-joint ankylosis; osteolysis of phalangeal and metacarpal bone, with telescoping of digits; and periostitis and proliferative new bone at sites of enthesitis. Characteristics of axial PsA include asymmetric sacroiliitis. When compared with idiopathic AS, axial PsA manifests less zygapophyseal joint arthritis; nonmarginal, bulky, “comma”-shaped syndesmophytes that tend to be fewer and less symmetric and delicate than the marginal syndesmophytes of AS; fluffy hyperperiostosis on anterior vertebral bodies; severe cervical spine involvement, with a tendency to atlantoaxial subluxation but relative sparing of the thoracolumbar

1	syndesmophytes of AS; fluffy hyperperiostosis on anterior vertebral bodies; severe cervical spine involvement, with a tendency to atlantoaxial subluxation but relative sparing of the thoracolumbar spine;andparavertebralossification.UltrasoundandMRIbothreadily demonstrate enthesitis and tendon sheath effusions that can be difficult to assess on physical examination. A recent MRI study of 68 PsA patients found sacroiliitis in 35%, unrelated to B27 but correlated with restricted spinal movement.

1	Classification criteria for PsA were published in 2006 (Classification of Psoriatic Arthritis [CASPAR] criteria) that have been widely accepted (Table 384-2). The sensitivity and specificity of these criteria exceed 90%, and they are useful for early diagnosis. The the CaSpar (ClASSIfICatIOn CrIterIa fOr PSOrIatIC ARthrItIS) CrIterIaa

1	To meet the CASPAR criteria, a patient must have inflammatory articular disease (joint, spine, or entheseal) with ≥3 points from any of the following five categories: 1. Evidence of current psoriasis,b, c a personal history of psoriasis, or a family history of psoriasisd 2. Typical psoriatic nail dystrophye observed on current physical examination 3. A negative test result for rheumatoid factor 4. Either current dactylitisf or a history of dactylitis recorded by a rheumatologist 5. Radiographic evidence of juxtaarticular new bone formationg in the hand or foot aSpecificity of 99% and sensitivity of 91%. bCurrent psoriasis is assigned 2 points; all other features are assigned 1 point. cPsoriatic skin or scalp disease present at the time of examination, as judged by a rheumatologist or dermatologist. dHistory of psoriasis in a first-or second-degree relative. eOnycholysis, pitting, or hyperkeratosis. fSwelling of an entire digit. gIll-defined ossification near joint margins, excluding

1	of psoriasis in a first-or second-degree relative. eOnycholysis, pitting, or hyperkeratosis. fSwelling of an entire digit. gIll-defined ossification near joint margins, excluding osteophyte formation.

1	Source: From W Taylor et al: Arthritis Rheum, 54:2665, 2006.

1	criteria are based on the history, presence of psoriasis, characteristic 2177 peripheral or spinal joint symptoms, signs, and imaging. Diagnosis can be challenging when the arthritis precedes psoriasis, the psoriasis is undiagnosed or obscure, or the joint involvement closely resembles another form of arthritis. A high index of suspicion is needed in any patient with an undiagnosed inflammatory arthropathy. The history should include inquiry about psoriasis in the patient and family members. Patients should be asked to disrobe for the physical examination, and psoriasiform lesions should be sought in the scalp, ears, umbilicus, and gluteal folds in addition to more accessible sites; the finger and toe nails should also be carefully examined. Axial symptoms or signs, dactylitis, enthesitis, ankylosis, the pattern of joint involvement, and characteristic radiographic changes can be helpful clues. The differential diagnosis includes all other forms of arthritis, which can occur

1	ankylosis, the pattern of joint involvement, and characteristic radiographic changes can be helpful clues. The differential diagnosis includes all other forms of arthritis, which can occur coincidentally in individuals with psoriasis. The differential diagnosis of isolated DIP involvement is short. Osteoarthritis (Heberden’s nodes) is usually not inflammatory; gout involving more than one DIP joint often involves other sites and may be accompanied by tophi; the very rare entity multicentric reticulohistiocytosis involves other joints and has characteristic small pearly periungual skin nodules; and the uncommon entity inflammatory osteoarthritis, like the others, lacks the nail changes of PsA. Radiography can be helpful in all of these cases and in distinguishing between psoriatic spondylitis and idiopathic AS. A history of trauma to an affected joint preceding the onset of arthritis is said to occur more frequently in PsA than in other types of arthritis, perhaps reflecting the

1	and idiopathic AS. A history of trauma to an affected joint preceding the onset of arthritis is said to occur more frequently in PsA than in other types of arthritis, perhaps reflecting the Koebner phenomenon in which psoriatic skin lesions can arise at sites of the skin trauma.

1	Ideally, coordinated therapy is directed at both the skin and joints in PsA. As described above for AS, use of the anti-TNF-α agents has revolutionized the treatment of PsA. Prompt and dramatic resolution of both arthritis and skin lesions has been observed in large, randomized controlled trials of etanercept, infliximab, adalimumab, and golimumab. Many of the responding patients had long-standing disease that was resistant to all previous therapy, as well as extensive skin disease. The clinical response is often more dramatic than in RA, and delay of disease progression has been demonstrated radiographically. The potential additive effect of methotrexate to anti-TNF-α agents in PsA remains uncertain. As noted above, anti-TNF therapy, paradoxically, has been reported to trigger exacerbation or de novo appearance of psoriasis, typically the palmoplantar pustular variety. In some cases, the therapy can nevertheless be continued.

1	Ustekinumab, a monoclonal antibody to the shared IL-23/ IL-12p40 subunit, is an efficacious treatment for psoriasis and has shown promise in PsA in clinical trials. Other newer drugs that have shown efficacy for both psoriasis and PsA include the anti-IL-17 pathway agents, such as secukinumab and brodalumab, and an oral phosphodiesterase-4 inhibitor, apremilast. Data on the oral Jak inhibitor, tofacitinib, has been very limited but promising.

1	Other treatment for PsA has been based on drugs that have efficacy in RA and/or in psoriasis. Until recently, controlled clinical trial data on methotrexate in doses of 15–25 mg/week and sulfasalazine (usually given in doses of 2–3 g/d) suggesting clinical efficacy have been somewhat limited, but neither regimen effectively halts progression of erosive joint disease. A recent double-blind trial assessing methotrexate 15 mg weekly in PsA demonstrated no benefit to the joint-based inflammation, but improvement was seen in patient and assessor global scores and skin scores. Other agents with efficacy in psoriasis reported to benefit PsA are cyclosporine, retinoic acid derivatives, and psoralens plus ultraviolet A light (PUVA). There is controversy regarding the efficacy in PsA of gold and antimalarials, which have been widely used in RA. The pyrimidine synthetase inhibitor leflunomide has been shown in a randomized controlled trial to be beneficial in both psoriasis and PsA.

1	The Spondyloarthritides 2178 All of these treatments require careful monitoring. Immunosuppressive therapy may be used cautiously in HIV-associated PsA if the HIV infection is well controlled.

1	Many patients, usually young adults, present with some features of one or more of the spondyloarthritides discussed above. Until recently, these patients were said to have undifferentiated spondylo-arthritis, or simply spondyloarthritis, as defined by the 1991 European SpondyloarthropathyStudyGroupcriteria.Forexample,apatientmay present with inflammatory synovitis of one knee, Achilles tendinitis, and dactylitis of one digit. Some of these patients may have ReA in which the triggering infection remains clinically silent. In some other cases, the patient subsequently develops IBD or psoriasis, or the process eventually meets criteria for AS. This diagnosis of undifferentiated SpA was also commonly applied to patients with inflammatory back pain, who did meet modified New York criteria for AS. Most of these would now be classified under the new category of axial SpA (Table 384-1).

1	Comparable to the classification criteria for axial symptoms, the ASAS has recently formulated criteria for peripheral SpA. This is intended to exclude patients with axial symptoms and thus to divide the universe of patients with SpA into axial and exclusively peripheral subsets. These criteria are shown in Table 384-3. Approximately one-half of the patients with undifferentiated SpA are HLA-B27-positive, and thus the absence of B27 is not useful in establishing or excluding the diagnosis. In familial cases, which are much more frequently B27-positive, there is often eventual progression to classical AS.

1	In juvenile-onset SpA, which begins between ages 7 and 16, most commonly in boys (60–80%), an asymmetric, predominantly lower-extremity oligoarthritis and enthesitis without extraarticular features is the typical mode of presentation. The prevalence of B27 in this condition, which has been termed the seronegative enthesopathy and arthropathy (SEA) syndrome, is approximately 80%. Many, but not all, of these patients go on to develop AS in late adolescence or adulthood. Management of undifferentiated SpA is similar to that of the other spondyloarthritides. Response to anti-TNF-α therapy has been documented, and this therapy is indicated in severe, persistent cases not responsive to other treatment. Current pediatric textbooks and journals should be consulted for information on management of juvenile-onset SpA. A relationship between arthritis and IBD was observed in the 1930s. The relationship was further defined by the epidemiologic studies

1	A relationship between arthritis and IBD was observed in the 1930s. The relationship was further defined by the epidemiologic studies One or more of the following: disease or ulcerative colitis OR two or more of the following: aSensitivity 79.5%, specificity 83.3% bPeripheral arthritis, usually predominantly lower limb and/or asymmetric. Source: M Rudawaleit et al: Ann Rheum Dis 70:25, 2011. in the 1950s and 1960s and included in the concept of the spondyloarthritides in the 1970s.

1	Source: M Rudawaleit et al: Ann Rheum Dis 70:25, 2011. in the 1950s and 1960s and included in the concept of the spondyloarthritides in the 1970s. Both of the common forms of IBD, ulcerative colitis (UC) and Crohn’s disease (CD) (Chap. 351), are associated with SpA. UC and CD both have an estimated prevalence of 0.05–0.1%, and the incidence of each is thought to have increased in recent decades. AS and peripheral arthritis are both associated with UC and CD. Wide variations have been reported in the estimated frequencies of these associations. In recent series, AS was diagnosed in 1–10%, and peripheral arthritis in 10–50% of patients with IBD. Inflammatory back pain and enthesopathy are common, and many patients have sacroiliitis on imaging studies.

1	The prevalence of UC or CD in patients with AS is thought to be 5–10%. However, investigation of unselected SpA patients by ileocolonoscopy has revealed that from one-third to two-thirds of patients with AS have subclinical intestinal inflammation that is evident either macroscopically or histologically. These lesions have also been found in patients with undifferentiated SpA or ReA (both enterically and urogenitally acquired).

1	Both UC and CD have a tendency to familial aggregation, more so for CD. HLA associations have been weak and inconsistent. HLA-B27 is found in up to 70% of patients with IBD and AS, but in ≤15% of patients with IBD and peripheral arthritis or IBD alone. Three alleles of the NOD2/CARD15 gene on chromosome 16 have been found in approximately one-half of patients with CD. These alleles are not associated with the spondyloarthritides per se. However, they are found significantly more often in (1) CD patients with sacroiliitis than in those without sacroiliitis, and (2) SpA patients with chronic inflammatory gut lesions than in those with normal gut histology. These associations are independent of HLA-B27. In addition to NOD2, over 100 other genes have been found to be associated with CD, UC, or both. Around 20 of these are also associated with AS.

1	Available data for IBD-associated peripheral arthritis suggest a synovial histology similar to other spondyloarthritides. Association with arthropathydoesnotaffecttheguthistologyofUCorCD(Chap. 351). The subclinical inflammatory lesions in the colon and distal ileum associated with SpA have been classified as either acute or chronic. The former resemble acute bacterial enteritis, with largely intact architecture and neutrophilic infiltration in the lamina propria. The latter resemble the lesionsof CD,withdistortionof villi andcrypts,aphthoid ulceration, and mononuclear cell infiltration in the lamina propria.

1	Both IBD and SpA are immune-mediated, but the specific pathogenic mechanisms are poorly understood, and the connection between the two is obscure. The shared genetics evidently reflects shared pathogenic mechanisms. A number of rodent models showing various immune perturbations manifest both IBD and arthritis. Several lines of evidence indicate trafficking of leukocytes between the gut and the joint. Mucosal leukocytes from IBD patients have been shown to bind avidly to synovial vasculature through several different adhesion molecules. Macrophages expressing CD163 are prominent in the inflammatory lesions of both gut and synovium in the spondyloarthritides.

1	AS associated with IBD is clinically indistinguishable from idiopathic AS. It runs a course independent of the bowel disease, and in some patients, it precedes the onset of IBD, sometimes by many years. Peripheral arthritis may also begin before onset of overt bowel disease. The spectrum of peripheral arthritis includes acute self-limited attacks of oligoarthritis that often coincide with relapses of IBD, and more chronic and symmetric polyarticular arthritis that runs a course independent of IBD activity. The patterns of joint involvement are similar in UC and CD. In general, erosions and deformities are infrequent in IBD-associated peripheral arthritis, and joint surgery is infrequently required.IsolateddestructivehiparthritisisararecomplicationofCD, apparently distinct from osteonecrosis and septic arthritis. Dactylitis and enthesopathy are occasionally found. In addition to the ∼20% of IBD patients with SpA, a comparable percentage have arthralgias or fibromyalgia symptoms.

1	Other extraintestinal manifestations of IBD are seen in addition to arthropathy, including uveitis, pyoderma gangrenosum, erythema nodosum, and finger clubbing, all somewhat more commonly in CD than UC. The uveitis shares the features described above for PsAassociated uveitis. Laboratory findings reflect the inflammatory and metabolic manifestations of IBD. Joint fluid is usually at least mildly inflammatory. Of patients with AS and IBD,30–70% carry theHLA-B27gene, compared with >85% of patients with AS alone and 50–70% of those with AS and psoriasis. Hence, definite or probable AS in a B27-negative individual in the absence of psoriasis should prompt a search for occult IBD. Radiographic changes in the axial skeleton are the same as in uncomplicated AS. Erosions are uncommon in peripheral arthritis but may occur, particularly in the metatarsophalangeal joints. Isolated destructive hip disease has been described.

1	Diarrhea and arthritis are both common conditions that can coexist for a variety of reasons. When etiopathogenically related, ReA and IBD-associated arthritis are the most common causes. Rare causes include celiac disease, blind loop syndromes, and Whipple’s disease. In most cases, diagnosis depends on investigation of the bowel disease.

1	Treatment of CD has been improved by therapy with anti-TNF agents. Infliximab, adalimumab, and certolizumab pegol are effective for induction and maintenance of clinical remission in CD, and infliximab has been shown to be effective in fistulizing CD. IBD-associated arthritis also responds to these agents. Other treatment for IBD, including sulfasalazine and related drugs, systemic glucocorticoids, and immunosuppressive drugs, is also usually of benefit for associated peripheral arthritis. NSAIDs are generally helpful and well tolerated, but they can precipitate flares of IBD. As noted above for psoriasis, rare cases of IBD, either CD or UC, have apparently been precipitated by anti-TNF therapy, usually etanercept, given for any of several rheumatic diseases.

1	The syndrome of synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) is characterized by a variety of skin and musculoskeletal manifestations. Dermatologic manifestations include palmoplantar pustulosis, acne conglobata, acne fulminans, and hidradenitis suppurativa. The main musculoskeletal findings are sternoclavicular and spinal hyperostosis, chronic recurrent foci of sterile osteomyelitis, and axial or peripheral arthritis. Cases with one or a few manifestations are probably the rule. The ESR is usually elevated, sometimes dramatically. In some cases, bacteria, most often Propionibacterium acnes, have been cultured from bone biopsy specimens and occasionally othersites. IBD was coexistentin 8%of patients in onelarge series. B27 is not associated. Either bone scan or computed tomography scan is helpful diagnostically. An MRI report described characteristic vertebral body corner cortical erosions in 12 of 12 patients. High-dose NSAIDsmay provide relieffrom bone pain. A

1	tomography scan is helpful diagnostically. An MRI report described characteristic vertebral body corner cortical erosions in 12 of 12 patients. High-dose NSAIDsmay provide relieffrom bone pain. A number of uncontrolled series and case reports describe successful therapy with pamidronate or other bisphosphonates. Response to anti-TNF-α therapy has also been observed, although in a few cases this has been associated with a flare of skin manifestations. Successful prolonged antibiotic therapy has also been reported. Recent reports suggest a possible autoinflammatory pathogenesis and successful treatment with the IL-1 receptor antagonist anakinra.

1	385 2179 the Vasculitis Syndromes Carol A. Langford, Anthony S. Fauci Vasculitis is a clinicopathologic process characterizedby inflammation of and damage to blood vessels. The vessel lumen is usually compromised, and this is associated with ischemia of the tissues supplied by the involved vessel. A broad and heterogeneous group of syndromes may result from this process, since any type, size, and location of blood vessel may be involved. Vasculitis and its consequences may be the primary or sole manifestation of a disease; alternatively, vasculitis may be a secondary component of another disease. Vasculitis may be confined to a single organ, such as the skin, or it may simultaneously involve several organ systems.

1	A major feature of the vasculitic syndromes as a group is the fact that there is a great deal of heterogeneity at the same time as there is considerable overlap among them. This heterogeneity and overlap in addition to a lack of understanding of the pathogenesis of these syndromes have been major impediments to the development of a coherent classification system for these diseases. Table 385-1 lists the major vasculitis syndromes. The distinguishing and overlapping features of these syndromes are discussed below.

1	Generally, most of the vasculitic syndromes are assumed to be mediated at least in part by immunopathogenic mechanisms that occur in response to certain antigenic stimuli. However, evidence supporting this hypothesis is for the most part indirect and may reflect epiphenomena as opposed to true causality. Furthermore, it is unknown why some individuals might develop vasculitis in response to certain antigenic stimuli, whereas others do not. It is likely that a number of factors are involved in the ultimate expression of a vasculitic syndrome. These include the genetic predisposition, environmental exposures, and the regulatory mechanisms associated with immune response to certain antigens. Although immune complex formation, antineutrophil cytoplasmic antibodies (ANCA), and pathogenic T lymphocyte responses (Table 385-2) have been among the prominent hypothesized Granulomatosis with polyangiitis Vasculitis associated with probable (Wegener’s) etiology

1	Granulomatosis with polyangiitis Vasculitis associated with probable (Wegener’s) etiology Eosinophilic granulomatosis with Hepatitis C virus–associated polyangiitis (Churg-Strauss) cryoglobulinemic vasculitis Polyarteritis nodosa Vasculitis associated with systemic Isolated aortitis Source: Adapted from JC Jennette et al: Arthritis Rheum 65:1, 2013. The Vasculitis Syndromes Production of antineutrophilic cytoplasmic antibodies Granulomatosis with polyangiitis (Wegener’s) Eosinophilic granulomatosis with polyangiitis (Churg-Strauss) Granulomatosis with polyangiitis (Wegener’s) Eosinophilic granulomatosis with polyangiitis (Churg-Strauss) Source: Adapted from MC Sneller, AS Fauci: Med Clin North Am 81:221, 1997. mechanisms, it is likely that the pathogenesis of individual forms of vasculitis is complex and varied.

1	Deposition of immune complexes was the first and most widely accepted pathogenic mechanismof vasculitis.However, the causal role of immune complexes has not been clearly established in most of the vasculitic syndromes. Circulating immune complexes need not result in deposition of the complexes in blood vessels with ensuing vasculitis, and many patients with active vasculitis do not have demonstrable circulating or deposited immune complexes. The actual antigen contained in the immunecomplexhas only rarely been identifiedin vasculitic syndromes. In this regard, hepatitis Bantigenhas been identified inboth the circulating and deposited immune complexes in a subset of patients who have features of a systemic vasculitis, most notably in polyarteritis nodosa (see “Polyarteritis Nodosa”). Cryoglobulinemic vasculitis is strongly associated with hepatitis C virus infection; hepatitis C virions and hepatitis C virus antigen-antibody complexes have been identified in the cryoprecipitates of

1	vasculitis is strongly associated with hepatitis C virus infection; hepatitis C virions and hepatitis C virus antigen-antibody complexes have been identified in the cryoprecipitates of these patients (see “Cryoglobulinemic Vasculitis”).

1	The mechanisms of tissue damage in immune complex–mediated vasculitis resemble those described for serum sickness. In this model, antigen-antibody complexes are formed in antigen excess and are deposited in vessel walls whose permeability has been increased by vasoactive amines such as histamine, bradykinin, and leukotrienes released from platelets or from mast cells as a result of IgE-triggered mechanisms. The deposition of complexes results in activation of complement components, particularly C5a, which is strongly chemotactic for neutrophils. These cells then infiltrate the vessel wall, phagocytose the immune complexes, and release their intracytoplasmic enzymes, which damage the vessel wall. As the process becomes subacute or chronic, mononuclear cells infiltrate the vessel wall. The common denominator of the resulting syndrome is compromise of the vessel lumen with ischemic changes in the tissues supplied by the involved vessel. Several variables may explain why only certain

1	The common denominator of the resulting syndrome is compromise of the vessel lumen with ischemic changes in the tissues supplied by the involved vessel. Several variables may explain why only certain types of immune complexes cause vasculitis and why only certain vessels are affected in individual patients. These include the ability of the reticuloendothelial system to clear circulating complexes from the blood, the size and physicochemical properties of immune complexes, the relative degree of turbulence of blood flow, the intravascular hydrostatic pressure in differentvessels,andthepreexistingintegrityofthevesselendothelium.

1	ANCA are antibodies directed against certain proteins in the cytoplasmic granules of neutrophils and monocytes. These autoantibodies are present in a high percentage of patients with active granulomatosis with polyangiitis (Wegener’s) and microscopic polyangiitis, and in a lower percentage of patients with eosinophilic granulomatosis with polyangiitis (Churg-Strauss). Because these diseases share the presence of ANCA and small-vessel vasculitis, some investigators have come to refer to them collectively as “ANCA-associated vasculitis.” However,asthesediseasespossessuniqueclinicalphenotypesinwhich ANCAmaybeabsent,itremainsouropinionthatgranulomatosiswith polyangiitis (Wegener’s), microscopic polyangiitis, and eosinophilic granulomatosis with polyangiitis (Churg-Strauss) should continue to be viewed as separate entities.

1	There are two major categories of ANCA based on different targets for the antibodies. The terminology of cytoplasmic ANCA (cANCA) refers to the diffuse, granular cytoplasmic staining pattern observed by immunofluorescence microscopy when serum antibodies bind to indicator neutrophils. Proteinase-3, a 29-kDa neutral serine proteinase present in neutrophil azurophilic granules, is the major cANCA antigen. More than 90% of patients with typical active granulomatosis with polyangiitis (Wegener’s) have detectable antibodies to proteinase-3 (see below). The terminology of perinuclear ANCA (pANCA) refers to the more localized perinuclear or nuclear staining pattern of the indicator neutrophils. The major target for pANCA is the enzyme myeloperoxidase; other targets that can produce a pANCA pattern of staining include elastase, cathepsin G, lactoferrin, lysozyme, and bactericidal/permeability-increasing protein. However, only antibodies to myeloperoxidase have been convincingly associated

1	of staining include elastase, cathepsin G, lactoferrin, lysozyme, and bactericidal/permeability-increasing protein. However, only antibodies to myeloperoxidase have been convincingly associated with vasculitis. Antimyeloperoxidase antibodies have been reported to occur in variable percentages of patients with microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis (Churg-Strauss), isolated necrotizing crescentic glomerulonephritis, and granulomatosis with polyangiitis (Wegener’s) (see below). A pANCA pattern of staining that is not due to antimyeloperoxidase antibodies has been associated with nonvasculitic entities such as rheumatic and nonrheumatic autoimmune diseases, inflammatory bowel disease, certain drugs, and infections such as endocarditis and bacterial airway infections in patients with cystic fibrosis.

1	It is unclear why patients with these vasculitis syndromes develop antibodies to myeloperoxidase or proteinase-3 or what role these antibodies play in disease pathogenesis. There are a number of in vitro observations that suggest possible mechanisms whereby these antibodies can contribute to the pathogenesis of the vasculitis syndromes. Proteinase-3 and myeloperoxidase reside in the azurophilic granules and lysosomes of resting neutrophils and monocytes, where they are apparently inaccessible to serum antibodies. However, when neutrophils or monocytes are primed by tumor necrosis factor α (TNF-α) or interleukin 1 (IL-1), proteinase-3 and myeloperoxidase translocate to the cell membrane, where they can interact with extracellular ANCA. The neutrophils then degranulate and produce reactive oxygen species that can cause tissue damage. Furthermore, ANCA-activated neutrophils can adhere to and kill endothelial cells in vitro. Activation of neutrophils and monocytes by ANCA also induces the

1	species that can cause tissue damage. Furthermore, ANCA-activated neutrophils can adhere to and kill endothelial cells in vitro. Activation of neutrophils and monocytes by ANCA also induces the release of proinflammatory cytokines such as IL-1 and IL-8. Adoptive transfer experiments in genetically engineered mice provide further evidence for a direct pathogenic role of ANCA in vivo. In contradiction, however, a number of clinical and laboratory observations argue against a primary pathogenic role for ANCA. Patients may have active granulomatosis with polyangiitis (Wegener’s) in the absence of ANCA; the absolute height of the antibody titers does not correlate well with disease activity; and patients with granulomatosis with polyangiitis (Wegener’s) in remission may continue to have high antiproteinase-3 (cANCA) titers for years (see below).

1	The histopathologic feature of granulomatous vasculitis has provided evidence to support a role of pathogenic T lymphocyte responses and cell-mediated immune injury. Vascular endothelial cells can express HLA class II molecules following activation by cytokines such as interferon (IFN) γ. This allows these cells to participate in immunologic reactions such as interaction with CD4+ T lymphocytes in a manner similar to antigen-presenting macrophages. Endothelial cells can secrete IL-1, which may activate T lymphocytes and initiate or propagate in situ immunologic processes within the blood vessel. In addition, IL-1 and TNF-α are potent inducers of endothelial-leukocyte adhesion molecule 1 (ELAM-1) and vascular cell adhesion molecule 1 (VCAM-1), which may enhance the adhesion of leukocytes to endothelial cells in the blood vessel wall. APPROACH TO THE PATIENT: general principles of Diagnosis

1	The diagnosis of vasculitis is often considered in any patient with an unexplained systemic illness. However, there are certain clinical abnormalities that when present alone or in combination should suggest a diagnosis of vasculitis. These include palpable purpura, pulmonary infiltrates and microscopic hematuria, chronic inflammatory sinusitis, mononeuritis multiplex, unexplained ischemic events,andglomerulonephritiswithevidenceofmultisystemdisease. A number of nonvasculitic diseases may also produce some or all of these abnormalities. Thus, the first step in the workup of a patient with suspected vasculitis is to exclude other diseases that produce clinical manifestations that can mimic vasculitis (Table 385-3). It is particularly important to exclude infectious diseases with features that overlap those of vasculitis, especially if the patient’s clinical condition is deteriorating rapidly and empirical immunosuppressive treatment is being contemplated.

1	Once diseases that mimic vasculitis have been excluded, the workup should follow a series of progressive steps that establish the diagnosis of vasculitis and determine, where possible, the category of the vasculitis syndrome (Fig. 385-1). This approach is of considerable importance since several of the vasculitis syndromes requireaggressivetherapywithglucocorticoidsandotherimmunosuppressive agents, whereas other syndromes usually resolve spontaneously and require symptomatic treatment only. The definitive

1	Properly categorize to a specific vasculitis syndrome Determine pattern and extent of disease Presentation of patient with suspected vasculitis Clinical findings Laboratory workup Establish diagnosis Biopsy Angiogram where appropriate Look for offending antigen Look for underlying disease Characteristic syndrome (i.e., granulomatosis with polyangiitis [Wegener’s] PAN, Takayasu arteritis) Treat vasculitis Remove antigen Syndrome resolves Treat underlying disease No further action Treat vasculitis Yes No Yes No Yes No FIGUrE 385-1 Algorithm for the approach to a patient with suspected diagnosis of vasculitis. PAN, polyarteritis nodosa.

1	FIGUrE 385-1 Algorithm for the approach to a patient with suspected diagnosis of vasculitis. PAN, polyarteritis nodosa. diagnosis of vasculitis is usually made based on biopsy of involved tissue. The yield of “blind” biopsies of organs with no subjective or objective evidence of involvement is very low and should be avoided. When syndromes such as polyarteritis nodosa, Takayasu arteritis, or primary central nervous system (CNS) vasculitis are suspected, arteriogram of organs with suspected involvement should be performed.

1	GENEraL PrINCIPLES OF TrEaTMENT Once a diagnosis of vasculitis has been established, a decision regarding therapeutic strategy must be made (Fig. 385-1). If an offending antigen that precipitates the vasculitis is recognized, the antigen should be removed where possible. If the vasculitis is associated with an underlying disease such as an infection, neoplasm, or connective tissue disease, the underlying disease should be treated. If the syndrome represents a primary vasculitic disease, treatment should be initiated according to the category of the vasculitis syndrome. Specific therapeutic regimens are discussed below for the individual vasculitis syndromes; however, certain general principles regarding therapy should be considered. Decisions regarding treatment should be based on the use of regimens for which there has been published literature supporting efficacy for that particular vasculitic disease. Since the potential toxic side effects of certain therapeutic regimens may be

1	use of regimens for which there has been published literature supporting efficacy for that particular vasculitic disease. Since the potential toxic side effects of certain therapeutic regimens may be substantial, the risk-versus-benefit ratio of any therapeutic approach should be weighed carefully. On the one hand, glucocorticoids and/or other immunosuppressive agents should be instituted immediately in diseases where irreversible organ system dysfunction and high morbidity and mortality rates have been clearly established. Granulomatosis with polyangiitis (Wegener’s) is the prototype of a severe systemic vasculitis requiring such a therapeutic approach (see below). On the other hand, when feasible, aggressive therapy should be avoidedforvasculitic manifestations that rarely result in irreversible organ system dysfunction and that usually do not respond to such therapy. For example, isolated

1	The Vasculitis Syndromes idiopathic cutaneous vasculitis usually resolves with symptomatic treatment, and prolonged courses of glucocorticoids uncommonly result in clinical benefit. Cytotoxic agents have not proved to be beneficial in idiopathic cutaneous vasculitis, and their toxic side effects generally outweigh any potential beneficial effects. Glucocorticoids should be initiated in those systemic vasculitides that cannot be specifically categorized or for which there is no established standard therapy; or other immunosuppressive therapy should be added in these diseases only if an adequate response does not result or if remission can only be achieved and maintainedwith an unacceptably toxic regimen of glucocorticoids. When remission isachieved,oneshouldcontinuallyattempttotaperglucocorticoids and discontinue when possible. When using other immunosuppressive regimens, one should base the choice of agent upon the available therapeutic data supporting efficacy in that disease, the

1	and discontinue when possible. When using other immunosuppressive regimens, one should base the choice of agent upon the available therapeutic data supporting efficacy in that disease, the site and severity of organ involvement, and the toxicity profile of the drug.

1	Physicians should be thoroughly aware of the toxic side effects of therapeutic agents employed that can include both acute and long-term complications (Table 385-4). Morbidity and mortality can occur as a result of treatment, and strategies to monitor for and prevent toxicity represent an essential part of patient care. Glucocorticoids are an important part of treatment for most vasculitides but are associated with substantial toxicities. Monitoring and prevention of glucocorticoid-induced bone loss are important in all patients. With the use of daily cyclophosphamide, strategies are particularly important and are directed toward minimization of bladder toxicity and prevention of leukopenia. Instructing the patient to take cyclophosphamide all at once in the morning with a large amount of fluid throughout the day in order to maintain a dilute urine can reduce the risk of bladder injury. Bladder cancer can occur several years after discontinuation of cyclophosphamide therapy;

1	amount of fluid throughout the day in order to maintain a dilute urine can reduce the risk of bladder injury. Bladder cancer can occur several years after discontinuation of cyclophosphamide therapy; therefore, monitoring for bladder cancer should continue indefinitely in patients who have received cyclophosphamide. Bone marrow suppression is an important toxicity of cyclophosphamide and can be observed during glucocorticoid tapering or over time, even after periods of stable measurements. Monitoring of the complete bloodcountevery1–2weeks foraslong asthepatient receives cyclophosphamide can effectively prevent cytopenias. Maintaining the white blood cell (WBC) count at >3000/μL and the neutrophil count at >1500/μL is essential to reduce the risk of life-threatening infections.

1	Methotrexate and azathioprine are also associated with bone marrow suppression, and complete blood counts should be obtained every 1–2 weeks for the first 1–2 months after their initiation and once a month thereafter. To lessen toxicity, methotrexate is often given together with folic acid, 1 mg daily, or folinic acid, 5–10 mg once a week 24 h following methotrexate. Prior to initiation of azathioprine, thiopurine methyltransferase (TPMT), an enzyme involved in the metabolism of azathioprine, should be assayed because inadequate levels may result in severe cytopenia.

1	Infection represents a significant toxicity for all vasculitis patients treated with immunosuppressive therapy. Infections with Pneumocystis jiroveci and certain fungi can be seen even in the face of WBCs that are within normal limits, particularly in patients receiving glucocorticoids. All vasculitis patients who are receiving daily glucocorticoids in combination with another immunosuppressive agent should receive trimethoprim-sulfamethoxazole (TMP-SMX) or another prophylactic therapy to prevent P. jiroveci infection. Finally, it should be emphasized that each patient is unique and requires individual decision-making. The above outline should serve as a framework to guide therapeutic approaches; however, flexibility should be practiced in order to provide maximal therapeutic efficacy with minimal toxic side effects in each patient.

1	Granulomatosis with polyangiitis (Wegener’s) isadistinctclinicopathologicentity characterizedbygranulomatous vasculitisofthe upperand lower respiratory tracts together with glomerulonephritis. In addition, variable degrees of disseminated vasculitis involving both small arteries and veins may occur. Granulomatosis with polyangiitis (Wegener’s) is an uncommon disease with an estimated prevalence of 3 per 100,000. It is extremely rare in blacks compared with whites; the male-to-female ratio is 1:1. The disease can be seen at any age; ∼15% of patients are <19 years of age, butonlyrarelydoesthediseaseoccur beforeadolescence;themeanage of onset is ∼40 years.

1	The histopathologic hallmarks of granulomatosis with polyangiitis (Wegener’s) are necrotizing vasculitis of small arteries and veins together with granuloma formation, which may be either intravascular or extravascular (Fig. 385-2). Lung involvement typically appears as multiple, bilateral, nodular cavitary infiltrates (Fig. 385-3), which on biopsy almost invariably reveal the typical necrotizing granulomatous vasculitis. Upper airway lesions, particularly those in the sinuses and nasopharynx, typically reveal inflammation, necrosis, and granuloma formation, with or without vasculitis. In its earliest form, renal involvement is characterized by a focal and segmental glomerulitis that may evolve into a rapidly progressive Osteoporosis Cataracts Glaucoma Diabetes mellitus Electrolyte abnormalities Metabolic abnormalities Suppression of inflammatory and immune responses leading to opportunistic

1	Osteoporosis Cataracts Glaucoma Diabetes mellitus Electrolyte abnormalities Metabolic abnormalities Suppression of inflammatory and immune responses leading to opportunistic Growth suppression in children Hypertension Avascular necrosis of bone Myopathy Alterations in mood Psychosis Pseudotumor cerebri Peptic ulcer diathesis Pancreatitis Gastrointestinal intolerance Pneumonitis Stomatitis Teratogenicity Bone marrow suppression Opportunistic infections Hepatotoxicity (may lead to fibrosis or

1	Gastrointestinal intolerance Pneumonitis Stomatitis Teratogenicity Bone marrow suppression Opportunistic infections Hepatotoxicity (may lead to fibrosis or FIGUrE 385-2 Lung histology in granulomatosis with polyangiitis (Wegener’s). Thisareaofgeographicnecrosishasaserpiginousbor-derofhistiocytesandgiantcellssurroundingacentralnecroticzone.Vasculitisisalsopresentwithneutrophilsandlymphocytesinfiltratingthewallofasmallarteriole(upper right).(Courtesy of William D. Travis, MD; with permission.) crescentic glomerulonephritis. Granuloma formation is only rarely seen on renal biopsy. In contrast to other forms of glomerulonephritis, evidence of immune complex deposition is not found in the renal lesion of granulomatosis with polyangiitis (Wegener’s). In addition to theclassictriadofdiseaseofthe upperandlowerrespiratorytracts and kidney, virtually any organ can be involved with vasculitis, granuloma, or both.

1	The immunopathogenesis of this disease is unclear, although the involvement of upper airways and lungswith granulomatous vasculitis suggests an aberrant cell-mediated immune response to an exogenous orevenendogenousantigenthatentersthroughorresidesintheupper airway. Chronic nasal carriage of Staphylococcus aureus has been reported to be associated with a higher relapse rate of granulomatosis with polyangiitis (Wegener’s); however, there is no evidence for a role of this organism in the pathogenesis of the disease.

1	Peripheral blood mononuclear cells obtained from patients with granulomatosis with polyangiitis (Wegener’s) manifest increased secretion of IFN-γ but not of IL-4, IL-5, or IL-10 compared to normal controls. In addition, TNF-α production from peripheral blood mononuclear cells and CD4+ T cells is elevated. Furthermore, monocytes from patients with granulomatosis with polyangiitis (Wegener’s) produce increasedamounts ofIL-12.Thesefindingsindicate anunbal-2183 anced TH1-type T cell cytokine pattern in this disease that may have pathogenic and perhaps ultimately therapeutic implications. FIGUrE 385-3 Computed tomography scan of a patient with granulomatosis with polyangiitis (Wegener’s). Thepatientdevel-opedmultiple,bilateral,andcavitaryinfiltrates. A high percentage of patients with granulomatosis with polyangiitis (Wegener’s) develop ANCA, and these autoantibodies may play a role in the pathogenesis of this disease (see above).

1	A high percentage of patients with granulomatosis with polyangiitis (Wegener’s) develop ANCA, and these autoantibodies may play a role in the pathogenesis of this disease (see above). Involvement of the upper airways occurs in 95% of patients with granulomatosis with polyangiitis (Wegener’s). Patients often present with severe upper respiratory tract findings such as paranasal sinus pain and drainage and purulent or bloody nasal discharge, with or without nasal mucosal ulceration (Table 385-5). Nasal septal perforation may follow, leading to saddle nose deformity. Serous otitis media may occur as a result of eustachian tube blockage. Subglottic tracheal stenosis resulting from active disease or scarring occurs in ∼16% of patients and may result in severe airway obstruction. aFewer than 1% had parotid, pulmonary artery, breast, or lower genitourinary (urethra, cervix, vagina, testicular) involvement. Source: GS Hoffman et al: Ann Intern Med 116:488, 1992.

1	The Vasculitis Syndromes 2184 Pulmonary involvement may be manifested as asymptomatic infiltrates or may be clinically expressed as cough, hemoptysis, dyspnea, and chest discomfort. It is present in 85–90% of patients. Endobronchial disease, either in its active form or as a result of fibrous scarring, may lead to obstruction with atelectasis. Eye involvement (52% of patients) may range from a mild conjunctivitis to dacryocystitis, episcleritis, scleritis, granulomatous sclerouveitis, ciliary vessel vasculitis, and retroorbital mass lesions leading to proptosis. Skin lesions (46% of patients) appear as papules, vesicles, palpable purpura, ulcers, or subcutaneous nodules; biopsy reveals vasculitis, granuloma, or both. Cardiac involvement (8% of patients) manifests as pericarditis, coronary vasculitis, or, rarely, cardiomyopathy. Nervous system manifestations (23% ofpatients) includecranial neuritis,mononeuritis multiplex, or, rarely, cerebral vasculitis and/or granuloma. Renal disease

1	vasculitis, or, rarely, cardiomyopathy. Nervous system manifestations (23% ofpatients) includecranial neuritis,mononeuritis multiplex, or, rarely, cerebral vasculitis and/or granuloma. Renal disease (77% of patients) generally dominates the clinical picture and, if left untreated, accounts directly or indirectly for most of the mortality rate in this disease. Although it may smolder in some cases as a mildglomerulitis withproteinuria, hematuria, andredblood cell casts, it is clear that once clinically detectable renal functional impairment occurs, rapidly progressive renal failure usually ensues unless appropriate treatment is instituted. While the disease is active, most patients have nonspecific symptoms and signs such as malaise, weakness, arthralgias, anorexia, and weight loss. Fever may indicate activity of the underlying disease but more often reflects secondary infection, usually of the upper airway. Characteristic laboratory findings include a markedly elevated erythrocyte

1	may indicate activity of the underlying disease but more often reflects secondary infection, usually of the upper airway. Characteristic laboratory findings include a markedly elevated erythrocyte sedimentation rate (ESR), mild anemia and leukocytosis, mild hypergammaglobulinemia (particularly of the IgA class), and mildly elevated rheumatoid factor. Thrombocytosis may be seen as an acute-phase reactant. Approximately 90% of patients with active granulomatosis with polyangiitis (Wegener’s) have a positive antiproteinase-3 ANCA. However, in the absence of active disease, the sensitivity drops to ∼60–70%. A small percentage of patients with granulomatosis with polyangiitis (Wegener’s) may have antimyeloperoxidase rather than antiproteinase-3 antibodies, and up to 20% may lack ANCA. Patients with granulomatosis with polyangiitis (Wegener’s) have been found to have an increased incidence of venous thrombotic events. Although routine anticoagulation for all patients is not recommended, a

1	with granulomatosis with polyangiitis (Wegener’s) have been found to have an increased incidence of venous thrombotic events. Although routine anticoagulation for all patients is not recommended, a heightened awareness for any clinical features suggestive of deep venous thrombosis or pulmonary emboli is warranted.

1	The diagnosis of granulomatosis with polyangiitis (Wegener’s) is made by the demonstration of necrotizing granulomatous vasculitis on tissue biopsy in a patient with compatible clinical features. Pulmonary tissue offers the highest diagnostic yield, almost invariably revealing granulomatous vasculitis. Biopsy of upper airway tissue usually reveals granulomatous inflammation with necrosis but may not show vasculitis. Renal biopsy can confirm the presence of pauci-immune glomerulonephritis. The specificity of a positive antiproteinase-3 ANCA for granulomatosis with polyangiitis (Wegener’s) is very high, especially if active glomerulonephritisispresent.However, the presenceof ANCAshould be adjunctive and, with rare exceptions, should not substitute for a tissue diagnosis. False-positive ANCA titers have been reported in certain infectious and neoplastic diseases.

1	In its typical presentation, the clinicopathologic complex of granulomatosis with polyangiitis (Wegener’s) usually provides ready differentiation from other disorders. However, if all the typical features are not present at once, it needs to be differentiated from the other vasculitides, antiglomerular basement membrane disease (Goodpasture’s syndrome) (Chap. 338), relapsing polychondritis (Chap. 389), tumors of the upper airway or lung, and infectious diseases such as histoplasmosis (Chap. 236), mucocutaneous leishmaniasis (Chap. 251), and rhinoscleroma (Chap. 44) as well as noninfectious granulomatous diseases.

1	Of particular note is the differentiation from other midline destructive diseases. These diseases lead to extreme tissue destruction and mutilation localized to the midline upper airway structures including the sinuses; erosion through the skin of the face commonly occurs, a feature that is extremely rare in granulomatosis with polyangiitis (Wegener’s). Although blood vessels may be involved in the intense inflammatory reaction and necrosis, primary vasculitis is not seen. Upper airway neoplasms and specifically extranodal natural killer (NK)/T cell lymphoma (nasal type) are important causes of midline destructive disease. These lesions are diagnosed based on histology, which reveals polymorphous atypical lymphoid cells with an NK cell immunophenotype, typically Epstein-Barr virus (Chap. 134). Such cases are treated based on their degree of dissemination, and localized lesions have responded to irradiation. Upper airway lesions should never be irradiated in granulomatosis with

1	(Chap. 134). Such cases are treated based on their degree of dissemination, and localized lesions have responded to irradiation. Upper airway lesions should never be irradiated in granulomatosis with polyangiitis (Wegener’s). Cocaine-induced tissue injury can be another important mimic of granulomatosis with polyangiitis (Wegener’s) in patients who present with isolated midline destructive disease. ANCA that target human neutrophil elastase can be found in patients with cocaine-induced midline destructive lesions and can complicate the differentiation from granulomatosis with polyangiitis (Wegener’s). This has been further confounded by the high frequency of levamisole adulteration of cocaine, which can result in cutaneous infarction and serologic changes that may mimic vasculitis. Granulocytopenia is a common finding in levamisole-induced disease that would not be associated with granulomatosis with polyangiitis (Wegener’s).

1	Granulomatosis with polyangiitis (Wegener’s) must also be differentiated from lymphomatoid granulomatosis, which is an Epstein-Barr virus–positive B cell proliferation that is associated with an exuberant T cell reaction. Lymphomatoid granulomatosis is characterized by lung,skin, CNS,andkidneyinvolvementinwhich atypicallymphocytoid and plasmacytoid cells infiltrate nonlymphoid tissue in an angioinvasive manner. In this regard, it clearly differs from granulomatosis with polyangiitis (Wegener’s) in that it is not an inflammatory vasculitis in the classic sense but an angiocentric perivascular infiltration of atypical mononuclear cells. Up to 50% of patients may develop a true malignant lymphoma.

1	Prior to the introduction of effective therapy, granulomatosis with polyangiitis (Wegener’s) was universally fatal within a few months of diagnosis. Glucocorticoids alone led to some symptomatic improvement, with little effect on the ultimate course of the disease. The development of treatment with cyclophosphamide dramatically changed patient outcome such that marked improvement was seen in >90% of patients, complete remission in 75% of patients, and 5-year patient survival was seen in over 80%.

1	Despite the ability to successfully induce remission, 50–70% of remissions are later associated with one or more relapses. The determination of relapse should be based on objective evidence of disease activity, taking care to rule out other features that may have a similar appearance such as infection, medication toxicity, or chronic disease sequelae. The ANCA titer can be misleading and should not be used to assess disease activity. Many patients who achieve remission continue to have elevated titers for years. Results from a large prospective study found that increases in ANCA were not associated with relapse and that only 43% relapsed within 1 year of an increase in ANCA levels. Thus, a rise in ANCA by itself is not a harbinger of immediate disease relapse and should not lead to reinstitution or increase in immunosuppressive therapy. Reinduction of remission after relapse is almost always achieved; however, a high percentage of patients ultimately have some degree of damage from

1	or increase in immunosuppressive therapy. Reinduction of remission after relapse is almost always achieved; however, a high percentage of patients ultimately have some degree of damage from irreversible features of their disease, such as varying degrees of renal insufficiency, hearing loss, tracheal stenosis, saddle nose deformity, and chronically impaired sinus function. Patients who developed irreversible renal failure but who achieved subsequent remission have undergone successful renal transplantation.

1	Because long-term cyclophosphamide is associated with substantial toxicity, approaches have been developed that seek to minimize the duration of exposure to cyclophosphamide while still taking advantage of its efficacy for severe disease. Treatment of granulomatosis with polyangiitis (Wegener’s) is currently viewed as having two phases: induction, where active disease is put into remission, followed by maintenance. The decision regarding which agents to use for induction and maintenance is based on disease severity together with individual patient factors that include contraindication, relapse history, and comorbidities.

1	For patients with severe disease, daily cyclophosphamide combined with glucocorticoids has been repeatedly proved to effectively induce remission and prolong survival. At the initiation of therapy, glucocorticoids are usually given as prednisone, 1 mg/kg per day for the first month, followed by gradual tapering on an alternate-day or daily schedule with discontinuation after ∼6–9 months.

1	Cyclophosphamide is given in doses of 2 mg/kg per day orally, but as it is renally eliminated, dosage reduction should be considered in patients with renal insufficiency. Some reports have indicated therapeutic success with less frequent and severe toxic side effects using IV cyclophosphamide. In a recent randomized trial, IV cyclophosphamide 15 mg/kg, three infusions given every 2 weeks, then every 3 weeks thereafter, was compared to cyclophosphamide 2 mg/kg daily given for 3 months followed by 1.5 mg/kg daily. Although IV cyclophosphamide was found to have a comparable rate of remission with a lower cumulative cyclophosphamide dose and occurrence of leukopenia, the use of a consolidation phase and an insufficient frequency of blood count monitoring may have negatively influenced the results in those who received daily cyclophosphamide. Of note in this study was that relapse occurred in 19% of those who received IV cyclophosphamide as compared to 9% who received daily oral

1	the results in those who received daily cyclophosphamide. Of note in this study was that relapse occurred in 19% of those who received IV cyclophosphamide as compared to 9% who received daily oral administration. We continue to strongly favor daily cyclophosphamide with utilization of blood count monitoring every 1–2 weeks (as discussed above) and limiting the duration of induction exposure to 3–6 months.

1	In patients with imminently life-threatening disease, such as rapidly progressive glomerulonephritis with a creatinine greater than 4.0 mg/dL or pulmonary hemorrhage requiring mechanical ventilation, a regimen of daily cyclophosphamide and glucocorticoids is the treatment of choice to induce remission. Adjunctive plasmapheresis was found to further improve renal recovery in a study of patients with rapidly progressive glomerulonephritis who had a creatinine of greater than 5.8 mg/dL.

1	After 3–6 months of induction treatment, cyclophosphamide should be stopped and switched to another agent for remission maintenance. The agents with which there has been the greatest published experience are methotrexate and azathioprine. Methotrexate is administered orally or subcutaneously starting at a dosage of 0.3 mg/kg as a single weekly dose, not to exceed 15 mg/week. If the treatment is well tolerated after 1–2 weeks, the dosage should be increased by 2.5 mg weekly up to a dosage of 20–25 mg/week and maintained at that level. Azathioprine, 2 mg/kg per day, has also proved effective in maintaining remission following induction with daily cyclophosphamide. In a randomized trial comparing methotrexate to azathioprine for remission maintenance, comparable rates of toxicity and relapse were seen. Therefore, the choice of agent is often based on toxicity profile, because methotrexate cannot be given to patients with renal insufficiency or chronic liver disease, as well as on other

1	were seen. Therefore, the choice of agent is often based on toxicity profile, because methotrexate cannot be given to patients with renal insufficiency or chronic liver disease, as well as on other individual patient factors. In patients who are unable to receive methotrexate or azathioprine or who have relapsed through such treatment, mycophenolate mofetil, 1000 mg twice a day, may also sustain remission following cyclophosphamide induction.

1	The optimal duration of maintenance therapy is uncertain. In the absence of toxicity, maintenance therapy is usually given for a minimum of 2 years past remission, after which time consideration can be given for tapering over a 6–12 month period until discontinuation. Patients with significant organ damage or a history of relapse may benefit from longer-term continuation of a maintenance agent.

1	Rituximab is a chimeric monoclonal antibody directed against CD20 present on normal and malignant B lymphocytes that is U.S. Food and Drug Administration (FDA) approved for the treatment of granulomatosis with polyangiitis (Wegener’s) and microscopic polyangiitis. In two randomized trials that enrolled ANCA-positive patients with severe active granulomatosis with polyangiitis (Wegener’s) or microscopic polyangiitis, rituximab 375 mg/m2 once a week for 4 weeks in combination with glucocorticoids was found to be as effective as cyclophosphamide with glucocorticoids for inducing disease remission. In the trial, which also enrolled patients with relapsing disease, rituximab was found to be statistically superior to cyclophosphamide.

1	Although the data support that rituximab is effective for remission induction of severe active granulomatosis with polyangiitis (Wegener’s) or microscopic polyangiitis, there remain a number of ongoing questions regarding rituximab that must be considered in weighing its use in the individual patient. The optimal approach to remission maintenance after treatment with rituximab remains unclear, as does whether this should include conventional maintenance agents such as methotrexate or azathioprine versus scheduled retreatment with rituximab. In addition, there are no long-term safety data with rituximab in granulomatosis with polyangiitis (Wegener’s) or microscopic polyangiitis.

1	Although rituximab does not have the bladder toxicity or infertility concerns, as can occur with cyclophosphamide, in both of the randomized trials, the rate of adverse events was similar in the rituximab and cyclophosphamide arms. Serious side effects of rituximab include infusion reactions, severe mucocutaneous reactions, and rare reports of progressive multifocal leukoencephalopathy. Because rituximab can bring about reactivation of hepatitis B, all patients should undergo hepatitis screening prior to treatment with rituximab. Etanercept, a dimeric fusion protein containing the 75-kDa TNF receptor bound to human IgG1, was not found to sustain remission when used adjunctively to standard therapy and should not be used in the treatment of granulomatosis with polyangiitis (Wegener’s).

1	For selected patients whose disease is not immediately life threatening, methotrexate together with glucocorticoids given at the dosages described above may be considered as an alternative for induction therapy, which is then continued for maintenance. Although certain reports have indicated that TMP-SMX may be of benefit in the treatment of granulomatosis with polyangiitis (Wegener’s) isolated to the sinonasal tissues, it should never be used alone to treat active granulomatosis with polyangiitis (Wegener’s) outside of the upper airway such as in patients with renal or pulmonary disease. In a study examining the effect of TMP-SMX on relapse, decreased relapses were shown only with regard to upper airway disease, and no differences in major organ relapses were observed.

1	Not all manifestations of granulomatosis with polyangiitis (Wegener’s) require or respond to immunosuppressive therapy. In managing non–major organ disease, such as that isolated to the sinus, joints, or skin, the risks of treatment should be carefully weighed against the benefits. Treatment with cyclophosphamide is rarely if ever justified for the treatment of isolated sinus disease in granulomatosis with polyangiitis (Wegener’s). Although patients with non–major organ disease may be effectively treated without immunosuppressive therapy, these individuals must be monitored closely for the development of disease activity affecting the lungs, The Vasculitis Syndromes 2186 kidneys, or other major organs. Subglottic stenosis and endobronchial stenosis are examples of disease manifestations that do not typically respond to systemic immunosuppressive treatment.

1	The term microscopic polyarteritis was introduced into the literature by Davson in 1948 in recognition of the presence of glomerulonephritis in patients with polyarteritis nodosa. In 1992, the Chapel Hill Consensus Conference on the Nomenclature of Systemic Vasculitis adopted the term microscopic polyangiitis to connote a necrotizing vasculitis with few or no immune complexes affecting small vessels (capillaries, venules, or arterioles). Glomerulonephritis is very common in microscopic polyangiitis, and pulmonary capillaritis often occurs. The absence of granulomatous inflammation in microscopic polyangiitis is said to differentiate it from granulomatosis with polyangiitis (Wegener’s). The incidence of microscopic polyangiitis has not yet been reliably established due to its previous inclusion aspart of polyarteritis nodosa. The mean age of onset is ∼57 years of age, and males are slightly more frequently affected than females.

1	The vasculitis seen in microscopic polyangiitis has a predilection to involve capillaries and venules in addition to small and medium-sized arteries. Immunohistochemical staining reveals a paucity of immunoglobulin deposition in the vascular lesion of microscopic polyangiitis, suggesting that immune-complex formation does not play a role in the pathogenesis of this syndrome. The renal lesion seen in microscopic polyangiitis is identical to that of granulomatosis with polyangiitis (Wegener’s). Like granulomatosis with polyangiitis (Wegener’s), microscopic polyangiitis is highly associated with the presence of ANCA, which may play a role in pathogenesis of this syndrome (see above).

1	Because of its predilection to involve the small vessels, microscopic polyangiitis and granulomatosis with polyangiitis (Wegener’s) share similar clinical features. Disease onset may be gradual, with initial symptoms of fever, weight loss, and musculoskeletal pain; however, it is often acute. Glomerulonephritis occurs in at least 79% of patients and can be rapidly progressive, leading to renal failure. Hemoptysis may be the first symptom of alveolar hemorrhage, which occurs in 12% of patients. Other manifestations include mononeuritis multiplex and gastrointestinal tract and cutaneous vasculitis. Upper airway disease and pulmonary nodules are not typically found in microscopic polyangiitis and, if present, suggest granulomatosis with polyangiitis (Wegener’s).

1	Features of inflammation may be seen, including an elevated ESR, anemia, leukocytosis, and thrombocytosis. ANCA are present in 75% of patients with microscopic polyangiitis, with antimyeloperoxidase antibodies being the predominant ANCA associated with this disease. The diagnosis is based on histologic evidence of vasculitis or pauciimmune glomerulonephritis in a patient with compatible clinical features of multisystem disease. Although microscopic polyangiitis is strongly ANCA-associated, no studies have as yet established the sensitivity and specificity of ANCA in this disease.

1	The 5-year survival rate for patients with treated microscopic polyangiitis is 74%, with disease-related mortality occurring from alveolar hemorrhage or gastrointestinal, cardiac, or renal disease. Studies on treatment have come from trials that have included patients with granulomatosis with polyangiitis (Wegener’s) or microscopic polyangiitis. Currently, the treatment approach for microscopic polyangiitis is the same as is used for granulomatosis with polyangiitis (Wegener’s) (see “Granulomatosis with Polyangiitis [Wegener’s]” for a detailed description of this therapeutic regimen), and patients with immediately life-threatening disease should be treated with the combination of prednisone and daily cyclophosphamide or rituximab. Disease relapse has been observed in at least 34% of patients. Treatment for such relapses would be similar to that used at the time of initial presentation and based on site and severity of disease.

1	Eosinophilic granulomatosis with polyangiitis (Churg-Strauss) was described in 1951 by Churg and Strauss and is characterized by asthma, peripheral and tissue eosinophilia, extravascular granuloma formation, and vasculitis of multiple organ systems. Eosinophilic granulomatosis with polyangiitis (Churg-Strauss) is an uncommon disease with an estimated annual incidence of 1–3 per million. The disease can occur at any age with the possible exception of infants. The mean age of onset is 48 years, with a female-to-male ratio of 1.2:1.

1	The necrotizing vasculitis of eosinophilic granulomatosis with polyangiitis (Churg-Strauss) involves small and medium-sized muscular arteries,capillaries,veins,andvenules.Acharacteristichistopathologic feature of eosinophilic granulomatosis with polyangiitis (Churg-Strauss) is granulomatous reactions that may be present in the tissues or even within the walls of the vessels themselves. These are usually associated with infiltration of the tissues with eosinophils.This process can occur in any organ in the body; lung involvement is predominant, with skin, cardiovascular system, kidney, peripheral nervous system, and gastrointestinal tract also commonly involved. Although the precise pathogenesis of this disease is uncertain, its strong association with asthma and its clinicopathologic manifestations, including eosinophilia, granuloma, and vasculitis, point to aberrant immunologic phenomena.

1	Patients with eosinophilic granulomatosis with polyangiitis (Churg-Strauss) often exhibit nonspecific manifestations such as fever, malaise, anorexia, and weight loss, which are characteristic of a multisystem disease. The pulmonary findings in eosinophilic granulomatosis with polyangiitis (Churg-Strauss) clearly dominate the clinical picture with severe asthmatic attacks and the presence of pulmonary infiltrates. Mononeuritis multiplex is the second most common manifestation and occurs in up to 72% of patients. Allergic rhinitis and sinusitis develop in up to 61% of patients and are often observed early in the course of disease. Clinically recognizable heart disease occurs in ∼14% of patients and is an important cause of mortality. Skin lesions occur in ∼51% of patients and include purpura in addition to cutaneous and subcutaneous nodules. The renal disease in eosinophilic granulomatosis with polyangiitis (Churg-Strauss) is less common and generally less severe than that of

1	purpura in addition to cutaneous and subcutaneous nodules. The renal disease in eosinophilic granulomatosis with polyangiitis (Churg-Strauss) is less common and generally less severe than that of granulomatosis with polyangiitis and microscopic polyangiitis.

1	The characteristic laboratory finding in virtually all patients with eosinophilic granulomatosis with polyangiitis (Churg-Strauss) is a striking eosinophilia, which reaches levels >1000 cells/μL in >80% of patients. Evidence of inflammation as evidenced by elevated ESR, fibrinogen, or α2-globulins can be found in 81% of patients. The other laboratory findings reflect the organ systems involved. Approximately 48% of patients with eosinophilic granulomatosis with polyangiitis (Churg-Strauss) have circulating ANCA that is usually antimyeloperoxidase.

1	Although the diagnosis of eosinophilic granulomatosis with polyangiitis (Churg-Strauss) is optimally made by biopsy in a patient with the characteristic clinical manifestations (see above), histologic confirmation can be challenging because the pathognomonic features often do not occur simultaneously. In order to be diagnosed with eosinophilic granulomatosis with polyangiitis (Churg-Strauss), a patient should have evidence of asthma, peripheral blood eosinophilia, and clinical features consistent with vasculitis. The prognosis of untreated eosinophilic granulomatosis with polyangiitis (Churg-Strauss) is poor, with a reported 5-year survival of 25%. With treatment, prognosis is favorable, with one study finding a 78-month actuarial survival rate of 72%. Myocardial involvement is the most frequent cause of death and is responsible for 39% of patient mortality. Echocardiography should be performed in all newly diagnosed patients because this may influence therapeutic decisions.

1	Glucocorticoids alone appear to be effective in many patients. Dosage tapering is often limited by asthma, and many patients require low-dose prednisone for persistent asthma many years after clinical recovery from vasculitis. In glucocorticoid failure or in patients who present with fulminant multisystem disease, particularly cardiac involvement, the treatment of choice is a combined regimen of daily cyclophosphamide and prednisone (see “Granulomatosis with Polyangiitis [Wegener’s]” for a detailed description of this therapeutic regimen). Recent studies of mepolizumab (anti-IL-5 antibody) in eosinophilic granulomatosis with polyangiitis (Churg-Strauss) have been encouraging, but this treatment requires further investigation.

1	Polyarteritis nodosa was described in 1866 by Kussmaul and Maier. It is a multisystem, necrotizing vasculitis of small and medium-sized muscular arteries in which involvement of the renal and visceral arteries is characteristic. Polyarteritis nodosa does not involve pulmonary arteries, although bronchial vessels may be involved; granulomas, significant eosinophilia, and an allergic diathesis are not observed. It is difficult to establish an accurate incidence of polyarteritis nodosa becausepreviousreportshave included polyarteritisnodosa andmicroscopic polyangiitis as well as other related vasculitides. Polyarteritis nodosa, as currently defined, is felt to be a very uncommon disease.

1	Thevascularlesion in polyarteritisnodosa isa necrotizinginflammation of small and medium-sized muscular arteries. The lesions are segmental and tend to involve bifurcations and branchings of arteries. They may spread circumferentially to involve adjacent veins. However, involvement of venules is not seen in polyarteritis nodosa and, if present, suggests microscopic polyangiitis (see below). In the acute stages of disease, polymorphonuclear neutrophils infiltrate all layers of the vessel wall and perivascular areas, which results in intimal proliferation and degeneration of the vessel wall. Mononuclear cells infiltrate the area as the lesions progress to the subacute and chronic stages. Fibrinoid necrosis of the vessels ensues with compromise of the lumen, thrombosis, infarction of the tissues supplied by the involved vessel, and, in some cases, hemorrhage. Asthelesionsheal,thereis collagendeposition,whichmay leadtofurtherocclusion ofthevessel lumen.Aneurysmaldilations upto 1 cm in

1	tissues supplied by the involved vessel, and, in some cases, hemorrhage. Asthelesionsheal,thereis collagendeposition,whichmay leadtofurtherocclusion ofthevessel lumen.Aneurysmaldilations upto 1 cm in size along the involved arteries are characteristic of polyarteritis nodosa. Granulomas and substantial eosinophilia with eosinophilic tissue infiltrationsarenotcharacteristically foundand suggesteosinophilic granulomatosis with polyangiitis (Churg-Strauss) (see above).

1	Multiple organ systems are involved, and the clinicopathologic 2187 findings reflect the degree and location of vessel involvement and the resulting ischemic changes. As mentioned above, pulmonary arteries are not involved in polyarteritis nodosa, and bronchial artery involvement is uncommon. The pathology in the kidney in classic polyarteritis nodosa is that of arteritis without glomerulonephritis. In patients with significant hypertension, typical pathologic features of glomerulosclerosis may be seen. In addition, pathologic sequelae of hypertension may be found elsewhere in the body.

1	The presence of a polyarteritis nodosa–like vasculitis in patients with hepatitis B together with the isolation of circulating immune complexes composed of hepatitis B antigen and immunoglobulin, and the demonstration by immunofluorescence of hepatitis B antigen, IgM, and complement in the blood vessel walls, strongly suggest the role of immunologic phenomena in the pathogenesis of this disease. A polyarteritis nodosa–like vasculitis has also been reported in patients with hepatitis C. Hairy cell leukemia can be associated with polyarteritis nodosa; the pathogenic mechanisms of this association are unclear.

1	Nonspecific signs and symptoms are the hallmarks of polyarteritis nodosa. Fever, weight loss, and malaise are present in over one-half of cases. Patients usually present with vague symptoms such as weakness, malaise, headache,abdominalpain,and myalgias that can rapidly progress to a fulminant illness. Specific complaints related to the vascular involvement within a particular organ system may also dominate the presenting clinical picture as well as the entire course of the illness (Table 385-6). In polyarteritis nodosa, renal involvement most commonly manifests as hypertension, renal insufficiency, or hemorrhage due to microaneurysms.

1	There are no diagnostic serologic tests for polyarteritis nodosa. In >75% of patients, the leukocyte count is elevated with a predominance of neutrophils. Eosinophilia is seen only rarely and, when present at high levels, suggests the diagnosis of eosinophilic granulomatosis with polyangiitis (Churg-Strauss). The anemia of chronic disease may be seen, and an elevated ESR is almost always present. Other common laboratory findings reflect the particular organ involved. Hypergammaglobulinemia may be present, and all patients should be screened for hepatitis B and C. Antibodies against myeloperoxidase or proteinase-3 (ANCA) are rarely found in patients with polyarteritis nodosa. The diagnosis of polyarteritis nodosa is based on the demonstration of characteristic findings of vasculitis on biopsy material of involved Source: From TR Cupps, AS Fauci: The Vasculitides. Philadelphia, Saunders, 1981.

1	Source: From TR Cupps, AS Fauci: The Vasculitides. Philadelphia, Saunders, 1981. The Vasculitis Syndromes 2188 organs. In the absence of easily accessible tissue for biopsy, the arteriographic demonstration of involved vessels, particularly in the form of aneurysms of small and medium-sized arteries in the renal, hepatic, and visceral vasculature, is sufficient to make the diagnosis. This should consist of a catheter-directed dye arteriogram because magnetic resonance and computed tomography arteriograms do not have sufficient resolution at the current time to visualize the vessels affected in polyarteritis nodosa. Aneurysms of vessels are not pathognomonic of polyarteritis nodosa; furthermore, aneurysms need not always be present, and arteriographic findings may be limited to stenotic segments and obliteration of vessels. Biopsy of symptomatic organs such as nodular skin lesions, painful testes, and nerve/muscle provides the highest diagnostic yields.

1	The prognosis of untreated polyarteritis nodosa is extremely poor, with a reported 5-year survival rate between 10 and 20%. Death usually results from gastrointestinal complications, particularly bowel infarcts and perforation, and cardiovascular causes. Intractable hypertension often compounds dysfunction in other organ systems, such as the kidneys, heart, and CNS, leading to additional late morbidity and mortality in polyarteritis nodosa. With the introduction of treatment, survival rate has increased substantially. Favorable therapeutic results have been reported in polyarteritis nodosa with the combination of prednisone and cyclophosphamide (see “Granulomatosis with Polyangiitis [Wegener’s]” for a detailed description of this therapeutic regimen). In less severe cases of polyarteritis nodosa, glucocorticoids alone have resulted in disease remission. In patients with hepatitis B who have a polyarteritis nodosa–like vasculitis, antiviral therapy represents an important part of

1	nodosa, glucocorticoids alone have resulted in disease remission. In patients with hepatitis B who have a polyarteritis nodosa–like vasculitis, antiviral therapy represents an important part of therapy and has been used in combination with glucocorticoids and plasma exchange. Careful attention to the treatment of hypertension can lessen the acute and late morbidity and mortality rates associated with renal, cardiac, and CNS complications of polyarteritis nodosa. Following successful treatment, relapse of polyarteritis nodosa has been estimated to occur in 10–20% of patients.

1	Giant cell arteritis, historically referred to as temporal arteritis, is an inflammation of medium-and large-sized arteries. It characteristically involves one or more branches of the carotid artery, particularly the temporal artery. However, itis a systemic diseasethat caninvolve arteries in multiple locations, particularly the aorta and its main branches.

1	Giant cell arteritis is closely associated with polymyalgia rheumatica, which is characterized by stiffness, aching, and pain in the muscles of the neck, shoulders, lower back, hips, and thighs. Most commonly, polymyalgia rheumatica occurs in isolation, but it may be seen in 40–50% of patients with giant cell arteritis. In addition, ∼10–20% of patients who initially present with features of isolated polymyalgia rheumatica later go on to develop giant cell arteritis. This strong clinical association together with data from pathophysiologic studies has increasingly supported that giant cell arteritis and polymyalgia rheumatica represent differing clinical spectrums of a single disease process.

1	Giant cell arteritis occurs almost exclusively in individuals >50 years. It is more common in women than in men and is rare in blacks. The incidence of giant cell arteritis varies widely in different studies and in different geographic regions. A high incidence has been found in Scandinavia and in regions of the United States with large Scandinavian populations, compared to a lower incidence in southern Europe. The annual incidence rates in individuals ≥50 years range from 6.9 to 32.8 per 100,000 population. Familial aggregation has been reported, as has an association with HLA-DR4. In addition, genetic linkage studies have demonstrated an association of giant cell arteritis with alleles at the HLA-DRB1 locus, particularly HLA-DRB1*04 variants. In Olmsted County, Minnesota, the annual incidence of polymyalgia rheumatica in individuals ≥50 years is 58.7 per 100,000 population.

1	Although the temporal artery is most frequently involved in giant cell arteritis, patients often have a systemic vasculitis of multiple mediumandlarge-sizedarteries,whichmaygoundetected.Histopathologically, the disease is a panarteritis with inflammatory mononuclear cell infiltrates within the vessel wall with frequent giant cell formation. There is proliferation of the intima and fragmentation of the internal elastic lamina. Pathophysiologic findings in organs result from the ischemia related to the involved vessels.

1	Experimental data support that giant cell arteritis is an antigen-driven disease in which activated T lymphocytes, macrophages, and dendritic cells play a critical role in the disease pathogenesis. Sequence analysis of the T cell receptor of tissue-infiltrating T cells in lesions of giant cell arteritis indicates restricted clonal expansion, suggesting the presence of an antigen residing in the arterial wall. Giant cell arteritis is believed to be initiated in the adventitia where CD4+ T cells enter through the vasa vasorum, become activated, and orchestrate macrophage differentiation. T cells recruited to vasculitic lesions in patients with giant cell arteritis produce predominantly IL-2 and IFN-γ, and the latter has been suggested to be involved in the progression to overt arteritis. Recent data demonstrate that at least two separate lineages of CD4 T cells–-IFN-γ-producing TH1 cells and IL-17-producing TH17 cells—participate in vascular inflammation and may have differing levels of

1	data demonstrate that at least two separate lineages of CD4 T cells–-IFN-γ-producing TH1 cells and IL-17-producing TH17 cells—participate in vascular inflammation and may have differing levels of responsiveness to glucocorticoids.

1	Giant cell arteritis is most commonly characterized clinically by the complex of fever, anemia, high ESR, and headaches in a patient over the age of 50 years. Other phenotypic manifestations include features of systemic inflammation including malaise, fatigue, anorexia, weight loss, sweats, arthralgias, polymyalgia rheumatica, or large-vessel disease.

1	In patients with involvement of the cranial arteries, headache is the predominant symptom and may be associated with a tender, thickened, or nodular artery, which may pulsate early in the disease but may become occluded later. Scalp pain and claudication of the jaw and tongue may occur. A well-recognized and dreaded complication of giant cell arteritis, particularly in untreated patients, is ischemic optic neuropathy, which may lead to serious visual symptoms, even sudden blindness in some patients. However, most patients have complaints relating to the head or eyes before visual loss. Attention to such symptoms with institution of appropriate therapy (see below) will usually avoid this complication. Other cranial ischemic complications include strokes and scalp or tongue infarction.

1	Up to one-third of patients can have large-vessel disease that can be the primary presentation of giant cell arteritis or can emerge at a later point in patients who have had previous cranial arteritis features or polymyalgia rheumatica. Manifestations of large-vessel disease can include subclavian artery stenosis that can present as arm claudication or aortic aneurysms involving the thoracic and to a lesser degree the abdominal aorta, which carry risks of rupture or dissection. Characteristic laboratory findings in addition to the elevated ESR include a normochromic or slightly hypochromic anemia. Liver function abnormalities are common, particularly increased alkaline phosphatase levels. Increased levels of IgG and complement have been reported. Levels of enzymes indicative of muscle damage such as serum creatine kinase are not elevated.

1	The diagnosis of giant cell arteritis and its associated clinicopathologic syndromecanoftenbesuggestedclinicallybythedemonstrationofthecomplex of fever, anemia, and high ESR with or without symptoms of polymyalgia rheumatica in a patient >50 years. The diagnosis is confirmed by biopsy of the temporal artery. Since involvement of the vessel may be segmental, positive yield is increased by obtaining a biopsy segment of 3–5 cm together with serial sectioning of biopsy specimens. Ultrasonography of the temporal artery has been reported to be helpful in diagnosis. A temporal artery biopsy should be obtained as quickly as possible in the setting of ocular signs and symptoms, and under these circumstances, therapyshouldnotbedelayedpendingabiopsy.Inthisregard,ithasbeen reported that temporal artery biopsies may show vasculitis even after ∼14 days of glucocorticoid therapy. A dramatic clinical response to a trial of glucocorticoid therapy can further support the diagnosis.

1	Large-vessel disease may be suggested by symptoms and findings on physical examination such as diminished pulses or bruits. It is confirmed by vascular imaging, most commonly through magnetic resonance or computed tomography. Isolated polymyalgia rheumatica is a clinical diagnosis made by the presence of typical symptoms of stiffness, aching, and pain in the muscles of the hip and shoulder girdle, an increased ESR, the absence of clinical featuressuggestiveofgiantcellarteritis, and aprompt therapeutic response to low-dose prednisone. Acute disease-related mortality directly from giant cell arteritis is very uncommon, with fatalities occurring from cerebrovascular events or myocardial infarction. However, patients are at risk of late mortality from aortic aneurysm rupture or dissection as patients with giant cell arteritis are 18 times more likely to develop thoracic aortic aneurysms than the general population.

1	The goals of treatment in giant cell arteritis are to reduce symptoms and, most importantly, to prevent visual loss. The treatment approach for cranial and large-vessel disease in giant cell arteritis is currently the same. Giant cell arteritis and its associated symptoms are exquisitely sensitive to glucocorticoid therapy. Treatment should begin with prednisone, 40–60 mg/d for ∼1 month, followed by a gradual tapering. When ocular signs and symptoms occur, consideration should be given for the use of methylprednisolone 1000 mg daily for 3 days to protect remaining vision. Although the optimal duration of glucocorticoid therapy has not been established, most series have found that patients require treatment for ≥2 years. Symptom recurrence during prednisone tapering develops in 60–85% of patients with giant cell arteritis, requiring a dosage increase. The ESR can serve as a useful indicator of inflammatory disease activity in monitoring and tapering therapy and can be used to judge the

1	with giant cell arteritis, requiring a dosage increase. The ESR can serve as a useful indicator of inflammatory disease activity in monitoring and tapering therapy and can be used to judge the pace of the tapering schedule. However, minor increases in the ESR can occur as glucocorticoids are being tapered and do not necessarily reflect an exacerbation of arteritis, particularly if the patient remains symptom-free. Under these circumstances, the tapering should continue with caution. Glucocorticoid toxicity occurs in 35–65% of patients and represents an important cause of patient morbidity. Aspirin 81 mg daily has been found to reduce the occurrence of cranial ischemic complications in giant cell arteritis and should be given in addition to glucocorticoids in patients who do not have contraindications. The use of weekly methotrexate as a glucocorticoid-sparing agent has been examined in two randomized placebo-controlled trials that reached conflicting conclusions. Infliximab, a

1	contraindications. The use of weekly methotrexate as a glucocorticoid-sparing agent has been examined in two randomized placebo-controlled trials that reached conflicting conclusions. Infliximab, a monoclonal antibody to TNF, was studied in a randomized trial and was not found to provide benefit. Recent reports have shown favorable response of giant cell arteritis to tocilizumab (antiIL-6 receptor), but this treatment requires further study before use in clinical practice.

1	Patients with isolated polymyalgia rheumatica respond promptly to prednisone, which can be started at a lower dose of 10–20 mg/d. Similar to giant cell arteritis, the ESR can serve as a useful indicator in monitoring and prednisone reduction. Recurrent polymyalgia symptoms develop in the majority of patients during prednisone tapering. One study of weekly methotrexate found that the use of this drug reduced the prednisone dose on average by only 1 mg and did not decrease prednisone-related side effects. A randomized trial in polymyalgia rheumatica did not find infliximab to lessen relapse or glucocorticoid requirements. Takayasu arteritis is an inflammatory and stenotic disease of medium-and large-sized arteries characterized by a strong predilection for the aortic arch and its branches.

1	Takayasu arteritis is an inflammatory and stenotic disease of medium-and large-sized arteries characterized by a strong predilection for the aortic arch and its branches. Takayasu arteritis is an uncommon disease with an estimated annual incidence rate of 1.2–2.6 cases per million. It is most prevalent in adolescent girls and young women. Although it is more common in Asia, it is neither racially nor geographically restricted.

1	The disease involves medium-and large-sized arteries, with a strong predilection for the aortic arch and its branches; the pulmonary artery may also be involved. The most commonly affected arteries seen by arteriography are listed in Table 385-7. The involvement of the major branches of the aorta is much more marked at their origin than distally. The disease is a panarteritis with inflammatory mononuclear cell infiltrates and occasionally giant cells. There are marked intimal proliferation and fibrosis, scarring and vascularization of the media, and disruption and degeneration of the elastic lamina. Narrowing of the lumen occurs with or without thrombosis. The vasa vasorum are frequently involved. Pathologic changes in various organs reflect the compromise of blood flow through the involved vessels.

1	Immunopathogenic mechanisms, the precise nature of which is uncertain,aresuspected inthisdisease.Aswith severalof thevasculitis syndromes, circulating immune complexes have been demonstrated, but their pathogenic significance is unclear. Takayasu arteritis is a systemic disease with generalized as well as vascular symptoms.Thegeneralizedsymptomsincludemalaise,fever,nightsweats, arthralgias,anorexia,andweightloss,whichmayoccurmonthsbeforevesselinvolvementisapparent.Thesesymptomsmaymergeintothoserelated to vascular compromise and organ ischemia. Pulses are commonly absent in the involved vessels, particularly the subclavian artery. The frequency of arteriographic abnormalities and the potentially associated clinical Percentage of The Vasculitis Syndromes Pulmonary 10–40 Coronary <10 Arm claudication, Raynaud’s phenomenon Visual changes, syncope, transient ischemic attacks, stroke Abdominal pain, nausea, vomiting

1	The Vasculitis Syndromes Pulmonary 10–40 Coronary <10 Arm claudication, Raynaud’s phenomenon Visual changes, syncope, transient ischemic attacks, stroke Abdominal pain, nausea, vomiting Hypertension, renal failure Aortic insufficiency, congestive heart failure Visual changes, dizziness Abdominal pain, nausea, vomiting Abdominal pain, nausea, vomit ing Leg claudication Atypical chest pain, dyspnea Chest pain, myocardial infarc aArteriographic lesions at these locations are usually asymptomatic but may potentially cause these symptoms. Source: G Kerr et al: Ann Intern Med 120:919, 1994. 2190 manifestationsarelistedinTable385-7.Hypertensionoccursin32–93%of patients and contributes to renal, cardiac, and cerebral injury. Characteristic laboratory findings include an elevated ESR, mild anemia, and elevated immunoglobulin levels.

1	The diagnosis of Takayasu arteritis should be suspected strongly in a young woman who develops a decrease or absence of peripheral pulses, discrepancies in blood pressure, and arterial bruits. The diagnosis is confirmed by the characteristic pattern on arteriography, which includes irregular vessel walls, stenosis, poststenotic dilation, aneurysm formation, occlusion, and evidence of increased collateral circulation. Complete aortic arteriography by catheter-directed dye arteriographyormagneticresonancearteriographyshouldbeobtained in order to fully delineate the distribution and degree of arterial disease. Histopathologic demonstration of vessel wall inflammation that is predominantly lymphocytic with granuloma formation and giant cells involving the media and adventitia adds confirmatory data; however,tissueisrarelyreadilyavailableforexamination.IgG4-relateddisease is a potential cause of aortitis and periaortitis that is histologically differentiated from Takayasu arteritis by a

1	data; however,tissueisrarelyreadilyavailableforexamination.IgG4-relateddisease is a potential cause of aortitis and periaortitis that is histologically differentiated from Takayasu arteritis by a dense lymphoplasmacytic infiltrate rich in IgG4-positive plasma cells, a storiform pattern of fibrosis, and obliterative phlebitis.

1	The long-term outcome of patients with Takayasu arteritis has varied widely between studies. Although two North American reports found overall survival to be ≥94%, the 5-year mortality rate from other studies has ranged from 0 to 35%. Disease-related mortality most often occurs from congestive heart failure, cerebrovascular events, myocardial infarction, aneurysm rupture, or renal failure. Even in the absence of life-threatening disease, Takayasu arteritis can be associated with significant morbidity. The course of the disease is variable, and although spontaneous remissions may occur, Takayasu arteritis is most often chronic and relapsing. Although glucocorticoid therapy in doses of 40–60 mg prednisone per day alleviates symptoms, there are no convincing studies that indicate that it increases survival. The combination of glucocorticoid therapy for acute signs and symptoms and an aggressive surgical and/or arterioplastic approach to stenosed vessels has markedly improved outcome and

1	survival. The combination of glucocorticoid therapy for acute signs and symptoms and an aggressive surgical and/or arterioplastic approach to stenosed vessels has markedly improved outcome and decreased morbidity by lessening the risk of stroke, correcting hypertension due to renal artery stenosis, and improving blood flow to ischemic viscera and limbs. Unless it is urgently required, surgical correction of stenosed arteries should be undertaken only when the vascular inflammatory process is well controlled with medical therapy. In individuals who are refractory to or unable to taper glucocorticoids, methotrexate in doses up to 25 mg per week has yielded encouraging results. Preliminary results with anti-TNF therapies have been encouraging, but will require further study through randomized trials to determine efficacy.

1	IgA vasculitis (Henoch-Schönlein) is a small-vessel vasculitis characterized by palpable purpura (most commonly distributed over the buttocks and lower extremities), arthralgias, gastrointestinal signs and symptoms, and glomerulonephritis. IgA vasculitis (Henoch-Schönlein) is usually seen in children; most patients range in age from 4 to 7 years; however, the disease may also be seen in infants and adults. It is not a rare disease; in one series it accounted for between 5 and 24 admissions per year at a pediatric hospital. The male-to-female ratio is 1.5:1. A seasonal variation with a peak incidence in spring has been noted.

1	The presumptive pathogenic mechanism for IgA (Henoch-Schönlein) vasculitis is immune-complex deposition. A number of inciting antigens have been suggested including upper respiratory tract infections, various drugs, foods, insect bites, and immunizations. IgA is the antibody class most often seen in the immune complexes and has been demonstrated in the renal biopsies of these patients.

1	In pediatric patients, palpable purpura is seen in virtually all patients; most patients develop polyarthralgias in the absence of frank arthritis. Gastrointestinal involvement, which is seen in almost 70% of pediatric patients, is characterized by colicky abdominal pain usually associated with nausea, vomiting, diarrhea, or constipation and is frequently accompanied by the passage of blood and mucus per rectum; bowel intussusception may occur. Renal involvement occurs in 10–50% of patients and is usually characterized by mild glomerulonephritis leadingtoproteinuriaandmicroscopic hematuria, with redblood cell casts in the majority of patients (Chap. 338); it usually resolves spontaneously without therapy. Rarely, a progressive glomerulonephritis will develop. In adults, presenting symptoms are most frequently related to the skin and joints, while initial complaints related to the gut are less common. Although certain studies have found that renal disease is more frequentandmore severe

1	are most frequently related to the skin and joints, while initial complaints related to the gut are less common. Although certain studies have found that renal disease is more frequentandmore severe inadults,thishasnot beenaconsistent finding. However, the course of renal disease in adults may be more insidious and thus requires close follow-up. Myocardial involvement can occur in adults but is rare in children.

1	Laboratory studies generally show amildleukocytosis, a normalplateletcount,andoccasionallyeosinophilia.Serumcomplementcomponents are normal, and IgA levels are elevated in about one-half of patients. ThediagnosisofIgAvasculitis(Henoch-Schönlein)isbasedonclinical signsandsymptoms.Skinbiopsyspecimencanbeusefulinconfirming leukocytoclastic vasculitis with IgA and C3 deposition by immunofluorescence. Renal biopsy is rarely needed for diagnosis but may provide prognostic information in some patients.

1	The prognosis of IgA vasculitis (Henoch-Schönlein) is excellent. Mortality is exceedingly rare, and 1–5% of children progress to end-stage renal disease. Most patients recover completely, and some do not require therapy. Treatment is similar for adults and children. When glucocorticoid therapy is required, prednisone, in doses of 1 mg/kg per day and tapered according to clinical response, has been shown to be useful in decreasing tissue edema, arthralgias, and abdominal discomfort; however, it has not proved beneficial in the treatment of skin or renal disease and does not appear to shorten the duration of active disease or lessen the chance of recurrence. Patients with rapidly progressive glomerulonephritis have been anecdotally reported to benefit from intensive plasma exchange combined with cytotoxic drugs. Disease recurrences have been reported in 10–40% of patients.

1	Cryoglobulins are cold-precipitable monoclonal or polyclonal immunoglobulins.Cryoglobulinemiamaybeassociatedwithasystemicvasculitis characterized by palpable purpura, arthralgias, weakness, neuropathy, and glomerulonephritis. Although this can be observed in association with a variety of underlying disorders including multiple myeloma, lymphoproliferative disorders, connective tissue diseases, infection, and liverdisease,inmanyinstancesitappearstobeidiopathic.Becauseofthe apparent absence of an underlying disease and the presence of cryoprecipitate containing oligoclonal/polyclonal immunoglobulins, this entity was referred to as essential mixed cryoglobulinemia. Since the discovery of hepatitis C, it has been established that the vast majority of patients who were considered to have essential mixed cryoglobulinemia have cryoglobulinemic vasculitis related to hepatitis C infection.

1	The incidence of cryoglobulinemic vasculitis has not been established. It has been estimated, however, that 5% of patients with chronic hepatitis C will develop cryoglobulinemic vasculitis. Skin biopsies in cryoglobulinemic vasculitis reveal an inflammatory infiltrate surrounding and involving blood vessel walls, with fibrinoid necrosis, endothelial cell hyperplasia, and hemorrhage. Deposition of immunoglobulin and complement is common. Abnormalities of uninvolved skin including basement membrane alterations and deposits in vessel walls may be found. Membranoproliferative glomerulonephritis is responsible for 80% of all renal lesions in cryoglobulinemicvasculitis.

1	TheassociationbetweenhepatitisCandcryoglobulinemicvasculitis has been supported by the high frequency of documented hepatitis C infection, the presence of hepatitis C RNA and anti–hepatitis C antibodies in serum cryoprecipitates, evidence of hepatitis C antigens in vasculitic skin lesions, and the effectiveness of antiviral therapy (see below). Current evidence suggests that in the majority of cases, cryoglobulinemic vasculitis occurs when an aberrant immune response to hepatitisC infectionleads totheformationofimmunecomplexesconsisting of hepatitis C antigens, polyclonal hepatitis C–specific IgG, and monoclonal IgM rheumatoid factor. The deposition of these immune complexes in blood vessel walls triggers an inflammatory cascade that results in cryoglobulinemic vasculitis.

1	The most common clinical manifestations of cryoglobulinemic vasculitis are cutaneous vasculitis, arthritis, peripheral neuropathy, and glomerulonephritis. Renal disease develops in 10–30% of patients. Life-threatening rapidly progressive glomerulonephritis or vasculitis of the CNS, gastrointestinal tract, or heart occurs infrequently. The presence of circulating cryoprecipitates is the fundamental finding in cryoglobulinemic vasculitis. Rheumatoid factor is almost always found and may be a useful clue to the disease when cryoglobulins are not detected. Hypocomplementemia occurs in 90% of patients. An elevated ESR and anemia occur frequently. Evidence for hepatitis C infection must be sought in all patients by testing for hepatitis C antibodies and hepatitis C RNA.

1	Acute mortality directly from cryoglobulinemic vasculitis is uncommon, but the presence of glomerulonephritis is a poor prognostic sign for overall outcome. In such patients, 15% progress to end-stage renal disease, with 40% later experiencing fatal cardiovascular disease, infection, or liver failure. As indicated above, the majority of cases are associated with hepatitis C infection. In such patients, treatment with antiviral therapy (Chap. 360) can prove beneficial and should be considered first-line therapy for hepatitis C–associated cryoglobulinemic vasculitis. Clinical improvement with antiviral therapy is dependent on the virologic response. Patients who clear hepatitis C from the blood have objective improvement in their vasculitis along with significant reductions in levels of circulating cryoglobulins, IgM, and rheumatoid factor. However, substantial portions of patients with hepatitis C do not have a sustained virologic response to such therapy, and the vasculitis typically

1	cryoglobulins, IgM, and rheumatoid factor. However, substantial portions of patients with hepatitis C do not have a sustained virologic response to such therapy, and the vasculitis typically relapses with the return of viremia. While transient improvement can be observed with glucocorticoids, a complete response is seen in only 7% of patients. Plasmapheresis and cytotoxic agents have been used in anecdotal reports. These observations have not been confirmed, and such therapies carry significant risks. Randomized trials with rituximab (anti-CD20) in hepatitis C–associated cryoglobulinemic vasculitis have provided evidence of benefit such that this agent should be considered in patients with active vasculitis either in combination with antiviral therapy or alone in patients who have relapsed through, are intolerant to, or have con-2191 traindications to antiviral agents.

1	The potential for vasculitis to affect single organs has become increasingly recognized.This has beendefinedasvasculitisin arteriesorveins of any size in a single organ that has no features that indicate that it is a limited expression of a systemic vasculitis. Examples include isolated aortitis, testicular vasculitis, vasculitis of the breast, isolated cutaneous vasculitis, and primary CNS vasculitis. In some instances, this may be discovered at the time of surgery such as orchiectomy for a testicular mass where there is concern for neoplasm that is found instead to be vasculitis. Some patients originally diagnosed with single-organ vasculitis may later develop additional manifestations of a more systemic disease. In instances where there is no evidence of systemic vasculitis and the affected organ has been removed in its entirety, the patient may be followed closely without immunosuppressive therapy. In other instances, such as primary CNS vasculitis or some patients with isolated

1	organ has been removed in its entirety, the patient may be followed closely without immunosuppressive therapy. In other instances, such as primary CNS vasculitis or some patients with isolated cutaneous vasculitis, medical intervention is warranted.

1	Theterm cutaneous vasculitis isdefinedbroadlyasinflammationofthe blood vessels of the dermis. Due to its heterogeneity, cutaneous vasculitis has been described by a variety of terms including hypersensitivity vasculitis and cutaneous leukocytoclastic angiitis. However, cutaneous vasculitis is not one specific disease but a manifestation that can be seen in a variety of settings. In >70% of cases, cutaneous vasculitis occurs either as part of a primary systemic vasculitis or as a secondary vasculitis related to an inciting agent or an underlying disease (see “Secondary Vasculitis,” below). In the remaining 30% of cases, cutaneous vasculitis occurs idiopathically. Cutaneous vasculitis represents the most commonly encountered vasculitis in clinical practice. The exact incidence of idiopathic cutaneous vasculitis has not been determined due to the predilection for cutaneous vasculitis to be associated with an underlying process and the variability of its clinical course.

1	The typical histopathologic feature of cutaneous vasculitis is the presence of vasculitis of small vessels. Postcapillary venules are the most commonly involved vessels; capillaries and arterioles may be involved less frequently. This vasculitis is characterized by a leukocytoclasis,a term that refers to the nuclear debris remaining from the neutrophils that have infiltrated in and around the vessels during the acute stages. In the subacute or chronic stages, mononuclear cells predominate; in certain subgroups, eosinophilic infiltration is seen. Erythrocytes often extravasate from the involved vessels, leading to palpable purpura. Cutaneous arteritis can also occur, which involves slightly larger-sized vessels within the dermis.

1	The hallmark of idiopathic cutaneous vasculitis is the predominance of skin involvement. Skin lesions may appear typically as palpable purpura; however, other cutaneous manifestations of the vasculitis may occur, including macules, papules, vesicles, bullae, subcutaneous nodules, ulcers, and recurrent or chronic urticaria. The skin lesions may be pruritic or even quite painful, with a burning or stinging sensation. Lesions most commonly occur in the lower extremities in ambulatory patients or in the sacral area in bedridden patients due to the effects of hydrostatic forces on the postcapillary venules. Edema may accompany certain lesions, and hyperpigmentation often occurs in areas of recurrent or chronic lesions. There are no specific laboratory tests diagnostic of idiopathic cutaneous vasculitis. A mild leukocytosis with or without eosinophilia is characteristic, as is an elevated ESR. Laboratory studies should be

1	The Vasculitis Syndromes 2192 aimed toward ruling out features to suggest an underlying disease or a systemic vasculitis. The diagnosis of cutaneous vasculitis is made by the demonstration of vasculitis on biopsy. An important diagnostic principle in patients with cutaneous vasculitis is to search for an etiology of the vasculitis—be it an exogenous agent, such as a drug or an infection, or an endogenous condition, such as an underlying disease (Fig. 385-1). In addition, a careful physical and laboratory examination should be performed to rule out the possibility of systemic vasculitis. This should start with the least invasive diagnostic approach and proceed to the more invasive only if clinically indicated.

1	When an antigenic stimulus is recognized as the precipitating factor in the cutaneous vasculitis, it should be removed; if this is a microbe, appropriate antimicrobial therapy should be instituted. If the vasculitis is associated with another underlying disease, treatment of the latter often results in resolution of the former. In situations where disease is apparently self-limited, no therapy, except possibly symptomatic therapy, is indicated. When cutaneous vasculitis persists and when there is no evidence of an inciting agent, an associated disease, or an underlying systemic vasculitis, the decision to treat should be based on weighing the balance between the degree of symptoms and the risk of treatment. Some cases of idiopathic cutaneous vasculitis resolve spontaneously, whereas others remit and relapse. In patients with persistent vasculitis, a variety of therapeutic regimens have been tried with variable results. In general, the treatment of idiopathic cutaneous vasculitis has

1	remit and relapse. In patients with persistent vasculitis, a variety of therapeutic regimens have been tried with variable results. In general, the treatment of idiopathic cutaneous vasculitis has not been satisfactory. Fortunately, since the disease is generally limited to the skin, this lack of consistent response to therapy usually does not lead to a life-threatening situation. Agents with which there have been anecdotal reports of success include dapsone, colchicine, hydroxychloroquine, and nonsteroidal anti-inflammatory agents. Glucocorticoids are often used in the treatment of idiopathic cutaneous vasculitis. Therapy is usually instituted as prednisone, 1 mg/kg per day, with rapid tapering where possible, either directly to discontinuation or by conversion to an alternate-day regimen followed by ultimate discontinuation. In cases that prove refractory to glucocorticoids, a trial of a cytotoxic agent may be indicated. Patients with chronic vasculitis isolated to cutaneous venules

1	followed by ultimate discontinuation. In cases that prove refractory to glucocorticoids, a trial of a cytotoxic agent may be indicated. Patients with chronic vasculitis isolated to cutaneous venules rarely respond dramatically to any therapeutic regimen, and cytotoxic agents should be used only as a last resort in these patients. Methotrexate and azathioprine have been used in such situations in anecdotal reports. Although cyclophosphamide is the most effective therapy for the systemic vasculitides, it should almost never be used for idiopathic cutaneous vasculitis because of the potential toxicity.

1	Primary CNS vasculitis is an uncommon clinicopathologic entity characterized by vasculitis restricted to the vessels of the CNS without otherapparentsystemicvasculitis.Theinflammatoryprocessisusually composed of mononuclear cell infiltrates with or without granuloma formation.

1	Patients may present with headaches, altered mental function, and focal neurologic defects. Systemic symptoms are generally absent. Devastating neurologic abnormalities may occur depending on the extent of vessel involvement. The diagnosis can be suggested by abnormal magnetic resonance imaging of the brain, an abnormal lumbar puncture, and/or demonstration of characteristic vessel abnormalities on arteriography (Fig. 385-4), but it is confirmed by biopsy of the brainparenchymaandleptomeninges.Intheabsenceofabrainbiopsy, care should be taken not to misinterpret as true primary vasculitis arteriographic abnormalities that might actually be related to another cause. An important entity in the differential diagnosis is reversible cerebral vasoconstrictive syndrome, which typically presents with “thunderclap” headache and is associated with arteriographic abnormalities that mimic primary CNS vasculitis that are reversible. Other diagnostic considerations include infection,

1	presents with “thunderclap” headache and is associated with arteriographic abnormalities that mimic primary CNS vasculitis that are reversible. Other diagnostic considerations include infection, atherosclerosis, emboli, connective tissuedisease,sarcoidosis,malignancy,anddrug-associated causes. The prognosis of granulomatous primary CNS vasculitis is poor; however, some reports indicate that glucocorticoid therapy, alone or together with cyclophosphamide administered as described above, has induced clinical remissions.

1	FIGUrE 385-4 Cerebral arteriogram from a 32-year-old male with primary central nervous system vasculitis. Dramaticbeading(arrow)typicalofvasculitisisseen. Behçet’s disease is a clinicopathologic entity characterized by recurrent episodes of oral and genital ulcers, iritis, and cutaneous lesions. The underlying pathologic process is a leukocytoclastic venulitis, although vessels of any size and in any organ can be involved. This disorder is described in detail in Chap. 387. Cogan’s syndrome is characterized by interstitial keratitis together with vestibuloauditory symptoms. It may be associated with a systemic vasculitis, particularly aortitis with involvement of the aortic valve. Glucocorticoids are the mainstay of treatment. Initiation of treatment as early as possible after the onset of hearing loss improves the likelihood of a favorable outcome.

1	Kawasaki disease is an acute, febrile, multisystem disease of children. Some 80% of cases occur prior to the age of 5, with the peak incidence occurring at ≤2 years. It is characterized by nonsuppurative cervical adenitis and changes in the skin and mucous membranes such as edema; congested conjunctivae; erythema of the oral cavity, lips, and palms; and desquamation of the skin of the fingertips. Although the disease is generally benign and self-limited, it is associated with coronary artery aneurysms in ∼25% of cases, with an overall case fatality rate of 0.5–2.8%. These complications usually occur between the third and fourth weeks of illness during the convalescent stage. Vasculitis of the coronary arteries is seen in almost all the fatal cases that have been autopsied. There is typical intimal proliferation and infiltration of the vesselwallwithmononuclearcells.Beadlikeaneurysmsandthromboses may be seen along the artery. Other manifestations include pericarditis, myocarditis,

1	intimal proliferation and infiltration of the vesselwallwithmononuclearcells.Beadlikeaneurysmsandthromboses may be seen along the artery. Other manifestations include pericarditis, myocarditis, myocardial ischemia and infarction, and cardiomegaly.

1	Apart from the up to 2.8% of patients who develop fatal complications, the prognosis of this disease for uneventful recovery is excellent. High-dose IV γ-globulin (2 g/kg as a single infusion over 10 h) together with aspirin (100 mg/kg per day for 14 days followed by 3–5 mg/kg per day for several weeks) have been shown to be effective in reducingtheprevalenceofcoronaryarteryabnormalitieswhenadministered early in the course of the disease. Surgery may be necessary for Kawasaki disease patients who have giant coronary artery aneurysms or other coronary complications. Surgical treatment most commonly includes thromboendarterectomy, thrombus clearing, aneurysmal reconstruction, and coronary artery bypass grafting.

1	Somepatientswithsystemicvasculitismanifestclinicopathologiccharacteristics that do not fit precisely into any specific disease but have overlappingfeaturesofdifferentvasculitides.Activesystemicvasculitis in such settings has the same potential for causing irreversible organ system damage as when it occurs in one of the defined syndromes listed in Table 385-1. The diagnostic and therapeutic considerations as well as the prognosis for these patients depend on the sites and severity of active vasculitis. Patients with vasculitis that could potentially cause irreversible damage to a major organ system should be treated as described under “Granulomatosis with Polyangiitis (Wegener’s).”

1	Vasculitis associated with drug reactions usually presents as palpable purpura that may be generalized or limited to the lower extremities or other dependent areas; however, urticarial lesions, ulcers, and hemorrhagic blisters may also occur (Chap. 74). Signs and symptoms may be limited to the skin, although systemic manifestations such as fever, malaise, and polyarthralgias may occur. Although the skin is the predominant organ involved, systemic vasculitis may result from drug reactions. Drugs that have been implicated in vasculitis include allopurinol, thiazides, gold, sulfonamides, phenytoin, and penicillin (Chap. 74).

1	An increasing number of drugs have been reported to cause vasculitis associated with antimyeloperoxidase ANCA. Of these, the best evidenceof causality existsforhydralazineand propylthiouracil.Theclinical manifestations in ANCA-positive drug-induced vasculitis can range from cutaneous lesions to glomerulonephritis and pulmonary hemorrhage. Outside of drug discontinuation, treatment should be based on the severity of the vasculitis. Patients with immediately life-threatening small-vessel vasculitis should initially be treated with glucocorticoids and cyclophosphamide as described for granulomatosis with polyangiitis(Wegener’s).Followingclinicalimprovement,considerationmaybe given for tapering such agents along a more rapid schedule.

1	These reactions are characterized by the occurrence of fever, urticaria, polyarthralgias, and lymphadenopathy 7–10 days after primary exposure and 2–4 days after secondary exposure to a heterologous protein (classicserumsickness)oranonproteindrugsuchaspenicillinorsulfa (serum sickness–like reaction). Most of the manifestations are not due to a vasculitis; however, occasional patients will have typical cutaneous venulitis that may progress rarely to a systemic vasculitis. Certain infections may directly trigger an inflammatory vasculitic process. For example, rickettsias can invade and proliferate in the endothelial cells of small blood vessels causing a vasculitis (Chap. 211). In addition, the inflammatory response around blood vessels associated 21 with certain systemic fungal diseases such as histoplasmosis (Chap.

1	236) may mimic a primary vasculitic process. A leukocytoclastic vasculitis predominantly involving the skin with occasional involvement of other organ systems may be a minor component of many other infections. These include subacute bacterial endocarditis, Epstein-Barr virus infection, HIV infection, and a number of other infections. Vasculitis can be associated with certain malignancies, particularly lymphoid or reticuloendothelial neoplasms. Leukocytoclastic venulitis confinedtotheskinisthemostcommonfinding;however,widespread systemic vasculitis may occur. Of particular note is the association of hairy cell leukemia (Chap. 134) with polyarteritis nodosa.

1	A number of connective tissue diseases have vasculitis as a secondary manifestation of the underlying primary process. Foremost among these are systemic lupus erythematosus (Chap. 378), rheumatoid arthritis (Chap. 380), inflammatory myositis (Chap. 388), relapsing polychondritis (Chap. 389), and Sjren’s syndrome (Chap. 383). The most common form of vasculitis in these conditions is the small-vessel venulitis isolated to the skin. However, certain patients may develop a fulminant systemic necrotizing vasculitis. Secondary vasculitis has also been observed in association with ulcerative colitis, congenital deficiencies of various complement components, sarcoidosis, primary biliary cirrhosis, ≥1-antitrypsin deficiency, and intestinal bypass surgery. The Vasculitis Syndromes

1	The Vasculitis Syndromes Atlas of the Vasculitic Syndromes Carol A. Langford, Anthony S. Fauci Diagnosis of the vasculitic syndromes is usually based on characteristic histologic or arteriographic findings in a patient who has clinically 386e compatible features. The images provided in this atlas highlight some of the characteristic histologic and radiographic findings that may be seen in the vasculitic diseases. These images demonstrate the importance that tissue histology may have in securing the diagnosis of vasculitis, the utility of diagnostic imaging in the vasculitic diseases, and the improvements in the care of vasculitis patients that have resulted from radiologic innovations.

1	Tissue biopsies represent vital information in many patients with a suspected vasculitic syndrome, not only in confirming the presence of vasculitis and other characteristic histologic features, but also in ruling out other diseases that can have similar clinical presentations. The determination of where biopsies should be performed is based on the presence of clinical disease in an affected organ, the likelihood of a positive diagnostic yield from data contained in the published literature, and the risk of performing a biopsy in an affected site. Common sites where biopsies may be performed include the lung, kidney, and skin. Other sites such as sural nerve, brain, testicle, and gastrointestinal tissues may also demonstrate features of vasculitis and be appropriate locations for biopsy when clinically affected.

1	Surgical biopsies of radiographically abnormal pulmonary parenchyma have a diagnostic yield of 90% in patients with granulomatosis with polyangiitis (Wegener’s) and play an important role in ruling out infection or malignancy. The yield of lung biopsies is highly associated with amount of tissue that can be obtained, and transbronchial biopsies, while less invasive, have a yield of only 7%. Lung biopsies also play an important role in microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis (Churg-Strauss), and any vasculitic disease where an immunosuppressed patient has pulmonary disease that is suspected to be an infection.

1	Kidney biopsy findings of a focal, segmental, crescentic, necrotizing glomerulonephritis with few to no immune complexes (pauci-immune glomerulonephritis) are characteristic in patients with granulomatosis with polyangiitis (Wegener’s), microscopic polyangiitis, or eosinophilic granulomatosis with polyangiitis (Churg-Strauss), who have active renal disease. These findings not only distinguish these entities from other causes of glomerulonephritis, but can also confirm the presence of active glomerulonephritis that requires treatment. As a result, renal biopsies can also be helpful to guide management decisions in these diseases when an established patient has worsening renal function and an inactive or equivocal urine sediment. Cryoglobulinemic vasculitis and IgA vasculitis (Henoch-Schlein) are other vasculitides 386e-1 where renal involvement may occur and where biopsy may be important in diagnosis or prognosis.

1	Biopsies of the skin are commonly performed and are well tolerated. Because not all purpuric or ulcerative lesions are due to vasculitis, skin biopsy plays an important role to confirm the presence of vasculitis as the cause of the manifestation. Cutaneous vasculitis represents the most common vasculitic feature that affects people and can be seen in a broad spectrum of settings including infections, medications, malignancies, and connective tissue diseases. As a result, for systemic vasculitides that will require aggressive immunosuppressive treatment, a skin biopsy may not represent sufficient evidence to secure the diagnosis.

1	Diagnostic imaging represents a critical assessment tool in patients who are known or suspected to have a systemic vasculitic disease. Imaging contributes unique information about the patient that, when taken together with the history, physical examination, and laboratory determinations, can guide the differential diagnosis and the subsequent assessment or treatment plan. A diverse range of imaging techniques is used in the assessment of vasculitis including plain radiography, ultrasonography, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography, and catheter-directed dye arteriography. These procedures have specific utilities that can allow differing perspectives on the spectrum and severity of vasculitis.

1	For vasculitic diseases that involve the largeor medium-sized blood vessels, arteriography provides information regarding blood vessel stenoses or aneurysms that can support the diagnosis. Catheter-directed dye arteriography provides information on central blood pressure and offers the most precise detail regarding vessel lumen dimensions but carries risks related to dye exposure and the invasive nature of the procedure. Advancements in magnetic resonance (MR) and CT arteriography have brought about noninvasive options to view the lumen and vessel wall, thus enhancing the ability to perform serial studies for patient monitoring in large-vessel vasculitis. However, in patients suspected to have a medium-vessel vasculitis such as polyarteritis nodosa, catheter-directed dye arteriography should still be performed because MR and CT arteriograms do not currently have sufficient resolution to visualize arteries of this size.

1	Although vasculitis involving the small blood vessels cannot be directly visualized, diagnostic imaging plays an essential role in detecting tissue injury that occurs as result of blood vessel and tissue inflammation. In granulomatosis with polyangiitis (Wegener’s), 80% of patients may have pulmonary involvement during their disease course. Chest imaging should be obtained whenever active disease is suspected, because up to one-third of patients with radiographic abnormalities are asymptomatic. Pulmonary imaging is also important to detect complications of vasculitis therapy such as opportunistic pneumonias and medication-related pneumonitis. CHAPTER 386e Atlas of the Vasculitic Syndromes Figure 386e-1 Bilateral nodular infiltrates seen on computed tomography of the chest in a 40-year-old woman with granulomatosis with polyangiitis (Wegener’s).

1	Figure 386e-1 Bilateral nodular infiltrates seen on computed tomography of the chest in a 40-year-old woman with granulomatosis with polyangiitis (Wegener’s). Figure 386e-2 Computed tomography of the chest in two patients with granulomatosis with polyangiitis (Wegener’s) demonstrating (A) single and (B) multiple cavitary lung lesions. Figure 386e-3 Bilateral ground-glass infiltrates due to alveolar hemorrhage from pulmonary capillaritis as seen in the same patient by (A) chest radiograph and (B) computed tomography. This manifestation can occur in granulomatosis with polyangiitis (Wegener’s) or microscopic polyangiitis. Figure 386e-5 Computed tomography of the orbits in a patient with granulomatosis with polyangiitis (Wegener’s) who presented with right-eye proptosis. The image demonstrates inflammatory tissue extending from the ethmoid sinus through the lamina papyracea and filling the orbital space.

1	the right upper lobe due to bacterial pneumonia in an immunosuppressed patient with granulomatosis with polyangiitis (Wegener’s). Collapse of the left upper lobe secondary to endobronchial stenosis from granulomatosis with polyangiitis (Wegener’s) also is seen on this image. Figure 386e-6 Computed tomography of the sinuses in two patients with granulomatosis with polyangiitis (Wegener’s). (A) Mucosal thickening of the bilateral maxillary sinuses and a perforation of the nasal septum. (B) Osteitis with obliteration of the left maxillary sinus in a patient with long-standing sinus disease. Figure 386e-7 Computed tomography of the chest demonstrat-ing a large pericardial effusion in a patient with eosinophilic granulo-matosis with polyangiitis (Churg-Strauss). Cardiac involvement is an important cause of morbidity and mortality in eosinophilic granulo-matosis with polyangiitis and can include myocarditis, endocarditis, and pericarditis. CHAPTER 386e Atlas of the Vasculitic Syndromes

1	CHAPTER 386e Atlas of the Vasculitic Syndromes Figure 386e-8 Arteriogram of a 40-year-old man with polyarteri-tis nodosa demonstrating microaneurysms in the hepatic circulation. Figure 386e-9 Arteriogram of a 19-year-old man with polyarteri-tis nodosa demonstrating multiple microaneurysms in the renal cir-culation. The patient presented with headache and severe hyperten-sion that was due to medium-vessel vasculitis affecting the kidney. Figure 386e-11 Upper-extremity arteriogram demonstrating a long stenotic lesion of the axillary artery in a 75-year-old female with giant cell arteritis. Figure 386e-10 Cerebral arteriogram demonstrating beading along branches of the internal carotid artery in a patient with primary central nervous system vasculitis.

1	Figure 386e-10 Cerebral arteriogram demonstrating beading along branches of the internal carotid artery in a patient with primary central nervous system vasculitis. Figure 386e-12 Magnetic resonance imaging demonstrating extensive aneurysmal disease of the thoracic aorta in an 80-year-old female. The patient had been diagnosed with biopsy-proven giant cell arteritis 10 years prior to presenting with this aneurysm. Figure 386e-13 Arteriogram of the aortic arch demonstrating complete occlusion of the left common carotid artery just after its origin from the aorta. This 20-year-old female presented with syncope and was subsequently diagnosed with Takayasu arteritis. Figure 386e-14 Arteriogram demonstrating stenosis of the abdominal aorta in a 25-year-old female with Takayasu arteritis. Figure 386e-15 Arteriogram of the hand demonstrating arterial skip lesions and vessel cutoffs in a patient with cryoglobulinemic vas-culitis due to multiple myeloma.

1	Figure 386e-15 Arteriogram of the hand demonstrating arterial skip lesions and vessel cutoffs in a patient with cryoglobulinemic vas-culitis due to multiple myeloma. Figure 386e-16 Lung histology in granulomatosis with polyangi-itis (Wegener’s). This lung biopsy shows areas of geographic necrosis with a border of histiocytes and giant cells. There is also vasculitis with neutrophils, lymphocytes, and giant cells infiltrating the wall of an artery. CHAPTER 386e Atlas of the Vasculitic Syndromes Figure 386e-17 Lung histology in microscopic polyangiitis. This lung biopsy demonstrates hemorrhage in the alveolar spaces due to capillaritis in a patient with microscopic polyangiitis. Similar find-ings can also be seen in granulomatosis with polyangiitis (Wegener’s) and less commonly in eosinophilic granulomatosis with polyangiitis (Churg-Strauss).

1	Figure 386e-18 Kidney biopsy in granulomatosis with polyangiitis (Wegener’s). This renal biopsy shows a crescentic and necrotizing glomerulonephritis. These findings were focal and segmental with normal and scarred glomeruli also being found in the biopsy. By immunofluorescence and electron microscopy, no immune deposits were present, indicative of a pauci-immune glomerulonephritis. Similar findings can also be seen in microscopic polyangiitis and eosinophilic granulomatosis with polyangiitis (Churg-Strauss). Figure 386e-19 Sural nerve biopsy in polyarteritis nodosa. This sural nerve biopsy was performed in a patient with polyarteritis nodo-sa, who had presented with a mononeuritis multiplex. Neutrophils are seen infiltrating all layers of this medium-sized vessel, which resulted in vessel occlusion and nerve infraction.

1	Figure 386e-20 Temporal artery biopsy in giant cell arteritis. This temporal artery biopsy demonstrates a panmural infiltration of mono-nuclear cells and lymphocytes that are particularly seen in the media and adventitia. Scattered giant cells are also present. Figure 386e-21 Cutaneous vasculitis. This skin biopsy reveals two arterioles beneath the dermis with a neutrophilic inflammatory infiltrate in and around the vessel wall with leukocytoclasis (nuclear debris). While such features are diagnostic of vasculitis, they can be seen in a variety of settings and are not specific for any single disease.

1	Figure 386e-22 Granulomatous primary central nervous system vasculitis. This brain biopsy demonstrates a medium-sized artery with granulomatous inflammation present within the vessel wall indicative of a granulomatous vasculitis. This patient presented with progressive headache, clinical and radiographic features of a stroke, and had arteriographic features consistent with vasculitis. Because no evidence of vasculitis could be found outside of the brain, this was consistent with granulomatous primary central nervous system vasculitis. Haralampos M. Moutsopoulos DEFINITION, INCIDENCE, aND PrEVaLENCE Behçet’s syndrome is a multisystem disorder presenting with recurrent oral and genital ulcerations as well as ocular involvement. The diagnosis is clinical and based on internationally agreed diagnostic criteria (Table 387-1).

1	ThesyndromeaffectsyoungmalesandfemalesfromtheMediterranean region, the Middle East, and the Far East, suggesting a link with the ancient Silk Route. Males and females are affected equally, but males often have more severe disease. Blacks are very infrequently affected.

1	The etiology and pathogenesis of this syndrome remain obscure. The disease appears to be in the crossroads of autoinflammatory and autoimmune disorders. The main pathologic lesion is systemic perivasculitis with early neutrophil infiltration and endothelial swelling. In some patients, diffuse inflammatory disease, involving all layers of large vesselsandresultingtoformationofpseudoaneurysms,suggestsvasculitis of vasa vasorum. Apart from activated neutrophils, increased numbers of infiltrating TH1, TH17, cytotoxic CD8+, and γδ T cells are observed, suggesting a link between innate and adaptive autoreactive immune response. Circulating autoantibodies against α-enolase of endothelial cells, selenium binding protein, and anti-Saccharomyces cerevisiae antibodies have been observed, but their pathogenic role remains unclear. A recent genome-wide association study confirmed the known association of Behçet’s syndrome with HLA-B*51 and identified a second, independent association within the

1	pathogenic role remains unclear. A recent genome-wide association study confirmed the known association of Behçet’s syndrome with HLA-B*51 and identified a second, independent association within the major histocompatibility complex (MHC) class I region. In addition, an association with interleukin (IL) 10 and the IL-23R–IL-12RB2 locus was also observed. Interestingly, the disease-associated IL-10 variant was correlated with diminished mRNA expression and low protein production.

1	The recurrent aphthous ulcerations are a sine qua non for the diagnosis. The ulcers are usually painful, are shallow or deep with a central yellowish necrotic base, appear singly or in crops, and are located anywhere in the oral cavity. Small ulcers, less than 10 mm in diameter, are seen in 85% of patients, whereas large or herpetiform lesions are less frequent. The ulcers persist for 1–2 weeks and subside without leaving scars. The genital ulcers are less common but more specific, are painful, do not affect the glans penis or urethra, and produce scrotal scars. Skin involvement is observed in 80% of patients and includes folliculitis, erythema nodosum,an acne-like exanthem,and, infrequently, vasculitis, Sweet syndrome, and pyoderma gangrenosum. Nonspecific skin inflammatory reactivity to any scratches or intradermal saline injection (pathergy test) is a common and specific manifestation.

1	Eye involvement with scarring and bilateral panuveitis is the most dreadedcomplication,sinceitoccasionallyprogressesrapidlytoblindness. Theeyedisease,occurring in50% ofpatients,is usuallypresentat the onsetbut may alsodevelop within thefirst few years. Inaddition to iritis, posterior uveitis, retinal vessel occlusions, and optic neuritis can be seen in some patients with the syndrome. Nondeforming arthritis or arthralgias are seen in 50% of patients and affect the knees and ankles. Recurrent oral ulceration plus two of the following:

1	Nondeforming arthritis or arthralgias are seen in 50% of patients and affect the knees and ankles. Recurrent oral ulceration plus two of the following: Superficial or deep peripheral vein thrombosis is seen in 30% of patients. Pulmonary emboli are a rare complication. The superior vena cava is obstructed occasionally, producing a dramatic clinical picture. Arterial involvement occurs in less than 5% of patients and presents with aortitis or peripheral arterial aneurysm and arterial thrombosis. Pulmonary artery vasculitis presenting with dyspnea, cough,chestpain,hemoptysis,andinfiltratesonchestroentgenograms has been reported in 5% of patients and should be differentiated from thromboembolic disease since it warrants anti-inflammatory and not thrombolytic therapy.

1	Neurologic involvement (5–10%) appears mainly in the parenchymal form (80%); it is associated with brainstem involvement and has a serious prognosis (central nervous system [CNS]-Behçet’s syndrome). IL-6 is persistently raised in cerebrospinal fluid of these patients. Cerebral venous thrombosis is most frequently observed in the superiorsagittalandtransversesinusesandisassociatedwithheadacheand increased intracranial pressure. Magnetic resonance imaging (MRI) and/or proton magnetic resonance spectroscopy (MRS) are very sensitive and should be employed if CNS-Behçet’s syndrome is suspected. Gastrointestinal involvement is seen more frequently in patients from Japan and consists of mucosal ulcerations of the gut, resembling Crohn’s disease. Epididymitis is seen in 5% of patients, whereas amyloidosis of AA type and glomerulonephritis are uncommon.

1	Epididymitis is seen in 5% of patients, whereas amyloidosis of AA type and glomerulonephritis are uncommon. Laboratory findings are mainly nonspecific indices of inflammation, such as leukocytosis and elevated erythrocyte sedimentation rate, as well as C-reactive protein levels. The severity of the syndrome usually abates with time. Apart from the patients with CNS-Behçet’s syndrome and major vessel disease, the life expectancy seems to be normal and the only serious complication is blindness.

1	Mucous membrane involvement may respond to topical glucocorticoids in the form of mouthwash or paste. In more serious cases, thalidomide (100 mg/d) is effective. Thrombophlebitis is treated with aspirin, 325 mg/d. Colchicine can be beneficial for the mucocutaneous manifestations and arthritis. Uveitis and CNS-Behçet’s syndrome require systemic glucocorticoid therapy (prednisone, 1 mg/kg per day) and azathioprine (2–3 mg/kg per day). Cyclosporine (5 mg/kg) has been used for sight-threatening uveitis, alone or in combination with azathioprine. Pulse doses of cyclophosphamide are useful early in the course of the disease for pulmonary or peripheral arterial aneurysms. Anti–tumor necrosis factor therapy is recommended in panuveitis refractory to immunosuppressives. Administration of this therapy improves visual acuity in more than two-thirds of patients. polymyositis, Dermatomyositis, 388 and Inclusion Body Myositis Marinos C. Dalakas

1	polymyositis, Dermatomyositis, 388 and Inclusion Body Myositis Marinos C. Dalakas The inflammatory myopathies represent the largest group of acquired and potentially treatable causes of skeletal muscle weakness. They are classified into three major groups: polymyositis (PM), dermatomyositis (DM), and inclusion body myositis (IBM). The prevalence of the inflammatory myopathies is estimated at 1 in 100,000. PM as a stand-alone entity is a rare disease. DM affects both children and adults and women more often than men. IBM is three times more frequent in men than in women, more common in whites than blacks, and is most likely to affect persons age >50 years.

1	Thesedisorderspresentas progressiveandsymmetricmuscleweakness except for IBM, which can have an asymmetric pattern. Patients usually report increasing difficulty with everyday tasks requiring the use of proximal muscles, such as getting up from a chair, climbing steps, stepping onto a curb, lifting objects, or combing hair. Fine-motor movements that depend on the strength of distal muscles, such as buttoning a shirt, sewing, knitting, or writing, are affected only late in the course of PM and DM, but fairly early in IBM. Falling is common in IBM because of early involvement of the quadriceps muscle, with buckling of the knees. Ocular muscles are spared, even in advanced, untreated cases; if these muscles are affected, the diagnosis of inflammatory myopathy should be questioned. Facial muscles are unaffectedinPMandDM,butmildfacialmuscleweaknessiscommon in patients with IBM. In all forms of inflammatory myopathy, pharyngeal and neck-flexor muscles are often involved, causing dysphagia

1	are unaffectedinPMandDM,butmildfacialmuscleweaknessiscommon in patients with IBM. In all forms of inflammatory myopathy, pharyngeal and neck-flexor muscles are often involved, causing dysphagia or difficulty in holding up the head (head drop). In advanced and rarely in acute cases, respiratory muscles may also be affected. Severe weakness, if untreated, is almost always associated with muscle wasting. Sensation remains normal. The tendon reflexes are preserved but may be absent in severely weakened or atrophied muscles, especially in IBM, where atrophy of the quadriceps and the distal muscles is common. Myalgia and muscle tenderness may occur in a small number of patients, usually early in the disease, and particularly in DM associated with connective tissue disorders. Weakness in PM and DM progresses subacutely over a period of weeks or months and rarely acutely; by contrast, IBM progresses very slowly, over years, simulating a late-life muscular dystrophy (Chap. 462e) or slowly

1	and DM progresses subacutely over a period of weeks or months and rarely acutely; by contrast, IBM progresses very slowly, over years, simulating a late-life muscular dystrophy (Chap. 462e) or slowly progressive motor neuron disorder (Chap. 452).

1	aSystemic lupus erythematosus, rheumatoid arthritis, Sjögren’s syndrome, systemic sclerosis, mixed connective tissue disease. bCrohn’s disease, vasculitis, sarcoidosis, primary biliary cirrhosis, adult celiac disease, chronic graft-versus-host disease, discoid lupus, ankylosing spondylitis, Behçet’s syndrome, myasthenia gravis, acne fulminans, dermatitis herpetiformis, psoriasis, Hashimoto’s disease, granulomatous diseases, agammaglobulinemia, monoclonal gammopathy, hypereosinophilic syndrome, Lyme disease, Kawasaki disease, autoimmune thrombocytopenia, hypergammaglobulinemic purpura, hereditary complement deficiency, IgA deficiency. cHIV (human immunodeficiency virus) and HTLV-1 (human T cell lymphotropic virus type 1). dDrugs include penicillamine (dermatomyositis and polymyositis), zidovudine (polymyositis), statins (necrotizing, toxic, or autoimmune myositis), and contaminated tryptophan (dermatomyositis-like illness). Other myotoxic drugs may cause myopathy but not an

1	zidovudine (polymyositis), statins (necrotizing, toxic, or autoimmune myositis), and contaminated tryptophan (dermatomyositis-like illness). Other myotoxic drugs may cause myopathy but not an inflammatory myopathy (see text for details). eParasites (protozoa, cestodes, nematodes), tropical and bacterial myositis (pyomyositis).

1	Polymyositis The actual onset of PM is often not easily determined, 2195 and patients typically delay seeking medical advice for several weeks or even months. This is in contrast to DM, in which the rash facilitates early recognition (see below). PM mimics many other myopathies and is a diagnosis of exclusion. It is a subacute inflammatory myopathy affecting adults, and rarely children, who do not have any of the following: rash, involvement of the extraocular and facial muscles, family history of a neuromuscular disease, history of exposure to myotoxic drugs or toxins, endocrinopathy, neurogenic disease, muscular dystrophy, biochemical muscle disorder (deficiency of a muscle enzyme), or IBM as excluded by muscle biopsy analysis (see below). As an isolated entity, PM is a rare (and overdiagnosed) disorder; more commonly, PM occurs in association with a systemic autoimmune or connective tissue disease or with a known viral or bacterial infection. Drugs, especially D-penicillamine,

1	disorder; more commonly, PM occurs in association with a systemic autoimmune or connective tissue disease or with a known viral or bacterial infection. Drugs, especially D-penicillamine, statins, or zidovudine (AZT), may also trigger an inflammatory myopathy similar to PM.

1	Dermatomyositis DM is a distinctive entity identified by a character istic rash accompanying, or more often preceding, muscle weakness. The rash may consist of a blue-purple discoloration on the upper eyelids with edema (heliotrope rash; see Fig. 73-3), a flat red rash on the face and upper trunk, and erythema of the knuckles with a raised violaceous scaly eruption (Gottron’s sign; see Fig. 73-4). The erythematous rash can also occur on other body surfaces, including the knees, elbows, malleoli, neck and anterior chest (often in a V sign), or back and shoulders (shawl sign), and may worsen after sun exposure. In some patients, the rash is pruritic, especially on the scalp, chest, and back. Dilated capillary loops at the base of the fingernails are also characteristic. The cuticles may be irregular, thickened, and distorted, and the lateral and palmar areas of the fingers may become rough and cracked, with irregular, “dirty” horizontal lines, resembling mechanic’s hands. The weakness

1	irregular, thickened, and distorted, and the lateral and palmar areas of the fingers may become rough and cracked, with irregular, “dirty” horizontal lines, resembling mechanic’s hands. The weakness can be mild, moderate, or severe enough to lead to quadriparesis. At times, the muscle strength appears normal, hence the term dermatomyositis sine myositis. When muscle biopsy is performed in such cases, however, significant perivascular and perimysial inflammation is often seen.

1	DM usually occurs alone but may overlap with scleroderma and mixed connective tissue disease. Fasciitis and thickening of the skin, similar to that seen in chronic cases of DM, have occurred in patients with the eosinophilia-myalgia syndrome associated with the ingestion of contaminated l-tryptophan.

1	Inclusion Body Myositis In patients ≥50 years of age, IBM is the most common of the inflammatory myopathies. It is often misdiagnosed as PM and is suspected only later when a patient with presumed PM does not respond to therapy. Weakness and atrophy of the distal muscles, especially foot extensors and deep finger flexors, occur in almost all cases of IBM and may be a clue to early diagnosis. Some patients present with falls because their knees collapse due to early quadriceps weakness. Others present with weakness in the small muscles of the hands, especially finger flexors, and complain of inability to hold objectssuchasgolfclubsorperformtaskssuchasturningkeysortying knots. On occasion, the weakness and accompanying atrophy can be asymmetric and selectively involve the quadriceps, iliopsoas, triceps, biceps, and finger flexors, resembling a lower motor neuron disease. Dysphagia is common, occurring in up to 60% of IBM patients, and may lead to episodes of choking. Sensory examination

1	triceps, biceps, and finger flexors, resembling a lower motor neuron disease. Dysphagia is common, occurring in up to 60% of IBM patients, and may lead to episodes of choking. Sensory examination is generally normal; some patients have mildly diminished vibratory sensation at the ankles that presumably is age-related. The pattern of distal weakness, which superficially resembles motor neuron or peripheral nerve disease, results from the myopathic process affecting distal muscles selectively. Disease progression is slow but steady, and most patients require an assistive device such as cane, walker, or wheelchair within several years of onset.

1	In at least 20% of cases, IBM is associated with systemic autoimmune or connective tissue diseases. Familial aggregation of typical IBM may occur; such cases have been designated as familial inflammatory IBM. This disorder is distinct from hereditary inclusion body myopathy (h-IBM), which describes a heterogeneous group of recessive, Polymyositis, Dermatomyositis, and Inclusion Body Myositis 2196 and less frequently dominant, inherited syndromes; the h-IBMs are noninflammatory myopathies. A subset of h-IBM that spares the quadriceps muscles has emerged as a distinct entity. This disorder, originally described in Iranian Jews and now seen in many ethnic groups,islinkedtochromosome9p1andresultsfrommutationsinthe UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene. aSSOCIaTED CLINICaL FINDINGS Extramuscular Manifestations These may be present to a varying degree in patients with PM or DM, and include: 1.

1	aSSOCIaTED CLINICaL FINDINGS Extramuscular Manifestations These may be present to a varying degree in patients with PM or DM, and include: 1. Systemic symptoms, such as fever, malaise, weight loss, arthralgia, and Raynaud’s phenomenon, especially when inflammatory myopathy is associated with a connective tissue disorder. 2. Joint contractures, mostly in DM and especially in children. 3. Dysphagia and gastrointestinal symptoms, due to involvement of oropharyngeal striated muscles and upper esophagus, especially in DM and IBM. 4. Cardiac disturbances, including atrioventricular conduction defects, tachyarrhythmias, dilated cardiomyopathy, a low ejection fraction,andcongestiveheartfailure,whichmayrarelyoccureither from the disease itself or from hypertension associated with longterm use of glucocorticoids. 5.

1	5. Pulmonary dysfunction, due to weakness of the thoracic muscles, interstitial lung disease, or drug-induced pneumonitis (e.g., from methotrexate), which may cause dyspnea, nonproductive cough, and aspiration pneumonia. Interstitial lung disease may precede myopathyoroccurearlyinthediseaseanddevelopsinupto10%of patients with PM or DM, most of whom have antibodies to t-RNA synthetases, as described below. 6. Subcutaneous calcifications, in DM, sometimes extruding on the skin and causing ulcerations and infections. 7. Arthralgias, synovitis, or deforming arthropathy with subluxation intheinterphalangealjoints,whichcan occurinsome patientswith DM and PM who have Jo-1 antibodies (see below).

1	association with Malignancies Although all the inflammatory myopathies can have a chance association with malignant lesions, especially in older age groups, the incidence of malignant conditions appears to be specifically increased only in patients with DM and not in those with PM or IBM. The most common tumors associated with DM are ovarian cancer, breast cancer, melanoma, colon cancer, and non-Hodgkin’s lymphoma. The extent of the search that should be conducted for an occult neoplasm in adults with DM depends on the clinical circumstances. Tumors in these patients are usually uncovered by abnormal findings in the medical history and physical examination and not through an extensive blind search. The weight of evidence argues against performing expensive, invasive, and nondirected tumor searches. A complete annual physical examination with pelvic, breast (mammogram, if indicated), and rectal examinations (with colonoscopy according to age and family history); urinalysis; complete

1	searches. A complete annual physical examination with pelvic, breast (mammogram, if indicated), and rectal examinations (with colonoscopy according to age and family history); urinalysis; complete blood count; blood chemistry tests; and a chest film should suffice in most cases. In Asians, nasopharyngeal cancer is common, and a careful examination of ears, nose, and throat is indicated. If malignancy is clinically suspected, screening with a whole-body positron emission tomography (PET) scan should be considered.

1	Overlap Syndromes These describe the association of inflammatory myopathies with connective tissue diseases. A well-characterized overlap syndrome occurs in patients with DM who also have manifestations of systemic sclerosis or mixed connective tissue disease, such as sclerotic thickening of the dermis, contractures, esophageal hypo-motility, microangiopathy, and calcium deposits (Table 388-1). By contrast, signs of rheumatoid arthritis, systemic lupus erythematosus, or Sjögren’s syndrome are very rare in patients with DM. Patients with theoverlapsyndromeofDMandsystemicsclerosismayhaveaspecific antinuclear antibody, the anti-PM/Scl, directed against a nucleolarprotein complex.

1	An autoimmune etiology of the inflammatory myopathies is indirectly supported by an association with other autoimmune or connective tissue diseases; the presence of various autoantibodies; an association with specific major histocompatibility complex (MHC) genes; demonstration of T cell–mediated myocytotoxicity or complement-mediated microangiopathy; and a response to immunotherapy.

1	autoantibodies and Immunogenetics Various autoantibodies against nuclear antigens (antinuclear antibodies) and cytoplasmic antigens are found in up to 30% of patients with inflammatory myopathies. The antibodies to cytoplasmic antigens are directed against ribonucleoproteins involved in protein synthesis (antisynthetases) or translational transport (anti-signal-recognition particles). The antibody directed against the histidyl-transfer RNA synthetase, called anti-Jo-1, accounts for 75% of all the antisynthetases and is clinically useful because up to 80% of patients with anti-Jo-1 antibodies have interstitial lung disease. Some patients with the anti-Jo-1 antibody also have Raynaud’s phenomenon, nonerosive arthritis, and the MHC molecules DR3 and DRw52. DR3 haplotypes (molecular designation DRB1*0301, DQB1*0201) occur in up to 75% of patients with PM and IBM, whereas in juvenile DM, there is an increased frequency of DQA1*0501 (Chap. 373e). Antibodies against the cytosolic

1	designation DRB1*0301, DQB1*0201) occur in up to 75% of patients with PM and IBM, whereas in juvenile DM, there is an increased frequency of DQA1*0501 (Chap. 373e). Antibodies against the cytosolic 5′-nucleotidase 1A, an enzyme abundantly expressed in muscle and thought to be involved in DNA degradation and repair, have been detected in one-third of IBM patients. Although the pathogenic significance of these antibodies is still unknown, they highlight the presence of an immune response, as discussed below.

1	Immunopathologic Mechanisms In DM, humoral immune mechanisms are implicated, resulting in a microangiopathy and muscle ischemia (Fig. 388-1). Endomysial inflammatory infiltrates are composed of B cells located in proximity to CD4 T cells, plasmacytoid dendritic cells, and macrophages; there is a relative absence of lymphocytic invasion of nonnecrotic muscle fibers. Activation of the complement C5b-9 membranolytic attack complex is thought to be a critical early event that triggers release of proinflammatory cytokines and chemokines, induces expression of vascular cell adhesion molecule (VCAM) 1 and intercellular adhesion molecule (ICAM) 1 on endothelial cells, and facilitates migration of activated lymphoid cells to the perimysial and endomysial spaces. Necrosis of the endothelial cells, reduced numbers of endomysial capillaries, ischemia, and muscle-fiber destruction resembling microinfarcts occur. The remaining capillaries often have dilated lumens in response to the ischemic

1	reduced numbers of endomysial capillaries, ischemia, and muscle-fiber destruction resembling microinfarcts occur. The remaining capillaries often have dilated lumens in response to the ischemic process. Larger intramuscular blood vessels may also be affected in the same pattern. Residual perifascicular atrophy reflects the endofascicular hypoperfusion that is prominent in the periphery of muscle fascicles. Increased expression of type I interferon–inducible proteins is also noted in these regions.

1	By contrast, in PM and IBM, a mechanism of T cell–mediated cytotoxicity is likely. CD8 T cells, along with macrophages, initially surround and eventually invade and destroy healthy, nonnecrotic muscle fibers that aberrantly express class I MHC molecules. MHC-I expression, absent from the sarcolemma of normal muscle fibers, is probably induced by cytokines secreted by activated T cells and macrophages. The CD8/MHC-I complex is characteristic of PM and IBM; its detection can aid in confirming the histologic diagnosis of PM, as discussed below. The cytotoxic CD8 T cells contain perforin and granzymegranulesdirectedtowardthesurfaceofthemusclefibersand capable of inducing myonecrosis. Analysis of T cell receptor molecules expressed by the infiltrating CD8 cells has revealed clonal expansion and conserved sequences in the antigen-binding region, both suggesting an antigen-driven T cell response. Whether the putative antigens are endogenous (e.g., muscle) or exogenous (e.g., viral) sequences

1	conserved sequences in the antigen-binding region, both suggesting an antigen-driven T cell response. Whether the putative antigens are endogenous (e.g., muscle) or exogenous (e.g., viral) sequences is unknown. Viruses have not been identified within the muscle fibers. Co-stimulatory molecules and their counterreceptors, which are fundamental for T cell activation and antigen recognition, are strongly upregulated in PM and IBM. As noted above, the possibility that B cells and the humoral immune system might also play a role in IBM is suggested by the identification of anti-cytosolic 5′-nucleotidase

1	Molecular mimicry, tumors, viruses? NO, TNF-° STAT-1, Chemokines, Cathepsin, TGF-˜

1	FIGUrE 388-1 Immunopathogenesis of dermatomyositis. Activation of complement, possibly by autoantibodies (Y), against endothelial cells and formation of C3 via the classic or alternative pathway. Activated C3 leads to formation of C3b, C3bNEO, and membrane attack complexes (MAC), which are deposited in and around the endothelial cell wall of the endomysial capillaries. Deposition of MAC leads to destruction of capillaries, ischemia, or microinfarcts, most prominent in the periphery of the fascicles, and perifascicular atrophy. B cells, plasmacytoid dendritic cells, CD4 T cells, and macrophages traffic from the circulation to the muscle. Endothelial expression of vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) is induced by cytokines released by the mononuclear cells. Integrins, specifically very late activation antigen (VLA)-4 and lymphocyte function–associated antigen (LFA)-1, bind VCAM and ICAM and promote T cell and macrophage infiltration of

1	cells. Integrins, specifically very late activation antigen (VLA)-4 and lymphocyte function–associated antigen (LFA)-1, bind VCAM and ICAM and promote T cell and macrophage infiltration of muscle through the endothelial cell wall.

1	Polymyositis, Dermatomyositis, and Inclusion Body Myositis 1A antibodies in some patients. Key molecules involved in T cell– mediated cytotoxicity are depicted in Fig. 388-2.

1	The role of Nonimmune Factors in IBM In IBM, the presence of Congo red–positive amyloid deposits within some vacuolated muscle fibers and abnormal mitochondria with cytochrome oxidase–negative fibers suggest that, in addition to the autoimmune component, there is also a degenerative process. Similar to Alzheimer’s disease, the intracellular amyloiddepositsinIBMareimmunoreactiveagainstamyloidprecursor protein (APP), β-amyloid, chymotrypsin, apolipoprotein E, presenilin, ubiquitin, and phosphorylated tau, but it is unclear whether these deposits, which are also seen in other vacuolar myopathies, are directly pathogenic orrepresentsecondary phenomena. The sameis true for the mitochondrialabnormalities,whichmayalsobesecondarytotheeffects of aging or a bystander effect of upregulated cytokines. Expression of cytokines and upregulation of MHC class I by the muscle fibers may cause an endoplasmic reticulum stress response resulting in intracellular accumulation of stressor molecules or

1	Expression of cytokines and upregulation of MHC class I by the muscle fibers may cause an endoplasmic reticulum stress response resulting in intracellular accumulation of stressor molecules or misfolded glycoproteins and activation of nuclear factor κB (NF-κB), leading to further cytokine activation.

1	association with Viral Infections and the role of retroviruses Several viruses, including coxsackieviruses, influenza, paramyxoviruses, mumps, cytomegalovirus, and Epstein-Barr virus, have been indirectly associated with myositis. For the coxsackieviruses, an autoimmune myositis triggered by molecular mimicry has been 2197 proposed because of structural homology between histidyl-transfer RNA synthetase that is the target of the Jo-1 antibody (see above) and genomic RNA of an animal picornavirus, the encephalomyocarditisvirus.Sensitivepolymerasechainreaction(PCR) studies, however, have repeatedly failed to confirm the presence of such viruses in muscle biopsies.

1	The best evidence of a viral connection in PM and IBM is with the retroviruses. Some individuals infected with HIV or with human T cell lympho-tropicvirus1(HTLV-1)developPMorIBM;asimilar disorder has been described in nonhuman primates infected with the simian immunodeficiency virus. The inflammatory myopathy may occur as the initial manifestation of a retroviral infection, or myositis may develop later in the disease course. Retroviralantigenshavebeendetectedonlyinoccasional endomysial macrophages and not within the muscle fibers themselves, suggesting that persistent infection and viral replication within the muscle does not occur. Histologic findings are identical to retroviral-negative PM or IBM. The infiltrating T cells in the muscle have a clonal bias and a number ofthemareretroviral-specific.Thisdisordershould be distinguished from a toxic myopathy related to long-term therapy with AZT, characterized by fatigue, myalgia, mild muscle weakness, and mild elevation of creatine

1	be distinguished from a toxic myopathy related to long-term therapy with AZT, characterized by fatigue, myalgia, mild muscle weakness, and mild elevation of creatine kinase (CK). AZT-induced myopathy, which generally improves when the drug is discontinued, is a mitochondrial disorder characterized histologically by “ragged-red” fibers. AZTinhibitsγ-DNApolymerase, anenzymefound solely in the mitochondrial matrix.

1	Inadequate data exist with respect to pos sible differences in the prevalence of the inflammatory myopathies in various parts of the world. PM appears to be reported more often in Asia and southern Europe, whereas IBM seems tobemorefrequentlyrecognizedinNorthAmerica, northern Europe, and Australia. Whether this represents differences in diagnostic methods and dis easeawarenessortruediseaseprevalenceremainsunclear.Pyomyositis andparasiticmyositisareclearlymorecommoninthetropics,whereas HIV-associated PM and IBM are more commonly encountered in areas endemic for HIV. In patients from Asia, nasopharyngeal cancer appears to be a malignancy more commonly associated with DM, necessitating the need to specifically search for these tumors in this population. The clinical picture of the typical skin rash and proximal or diffuse muscle weakness has few causes other than DM. However, proximal muscle weakness without skin involvement can be due to many conditions other than PM or IBM.

1	Subacute or Chronic Progressive Muscle Weakness This may be due to denervating conditions such as the spinal muscular atrophies or amyotrophic lateral sclerosis (Chap. 452). In addition to the muscle weakness, upper motor neuron signs in the latter and signs of denervation detected by electromyography (EMG) aid in the diagnosis. The muscular dystrophies (Chap. 462e) may be additional considerations; however, these disorders usually develop over years rather than weeks or months and rarely present after the age of 30 years. It may be difficult, even with a muscle biopsy, to distinguish chronic PM from a rapidly advancing muscular dystrophy. This is particularly true of facioscapulohumeral muscular dystrophy, dysferlin myopathy, and

1	VCAM-1 TCR CD28 CTLA-4 LFA-1 Chemokines (MCP-1, Mig, IP-10) Infection? Co-stimulation Clonal expansion Cytokines Systemic immune compartment MHC TCR Macrophage Antigen Integrins LFA-4 CD8 CD8CD8 ICAM-1 MHC-I MMP-9 MMP-9 MMP-2 IFN-˜IFN-˜TFN-°TNF-°IL-1, 2 IL-1, 2 MMPs Perforin Necrosis Endoplasmic reticulum BB1 ˛2m Calnexin MHC-I TAP Ag (virus, muscle peptide) CD8 2198 with very high levels of serum CK (often in the thousands), painful muscle cramps, rhabdomyolysis, and myoglobinuria, it may be due to a necrotizing autoimmunemyositis,asdiscussedbelow,aviralinfection or a metabolic disorder such as myophosphorylase deficiency, or carnitine palmitoyltransferase deficiency (Chap. 462e).Several animal parasites,including protozoa (Toxoplasma, Trypanosoma), cestodes (cysticerci), and nematodes (trichinae), may produce a focal or diffuse inflammatory myopathy known as parasitic polymyositis.Staphylococcus aureus,Yersinia, Streptococcus, or anaerobic bacteria may produce a suppurative

1	(trichinae), may produce a focal or diffuse inflammatory myopathy known as parasitic polymyositis.Staphylococcus aureus,Yersinia, Streptococcus, or anaerobic bacteria may produce a suppurative myositis, known as tropical polymyositis, or pyomyositis. Pyomyositis, previously rare in the west,isnowoccasionallyseeninAIDSpatients.Other bacteria, such as Borrelia burgdorferi (Lyme disease) and Legionella pneumophila (Legionnaire’s disease), may infrequently cause myositis. Patients with periodic paralysis experience recurrent episodes of acute muscle weakness without pain, always beginning in childhood. Chronic alcoholics may develop painful myopathy with myoglobinuria after a bout of heavy drinking. Acute painless muscle weakness with myoglobinuria may occur with prolonged hypokalemia, or hypophosphatemia and hypomagnesemia, usually in chronic alcoholics or in patients on nasogastric suction receiving parenteral hyperalimentation.

1	Myofasciitis This distinctive inflammatory disor-FIGUrE 388-2 Cell-mediated mechanisms of muscle damage in polymyositis (PM) der affecting muscle and fascia presents as diffuse and inclusion body myositis (IBM). Antigen-specific CD8 cells are expanded in the myalgias, skin induration, fatigue, and mild muscle periphery, cross the endothelial barrier, and bind directly to muscle fibers via T cell recep-weakness; mild elevations of serum CK are usually tor (TCR) molecules that recognize aberrantly expressed major histocompatibility com-present. The most common form is eosinophilic plex (MHC)-I. Engagement of co-stimulatory molecules (BB1 and ICOSL) with their ligands myofasciitischaracterizedbyperipheralbloodeosin(CD28, CTLA-4, and ICOS), along with ICAM-1/LFA-1, stabilize the CD8–muscle fiber inter-ophiliaandeosinophilicinfiltratesinthe endomysial action. Metalloproteinases (MMPs) facilitate the migration of T cells and their attachment tissue. In some patients, the eosinophilic

1	fiber inter-ophiliaandeosinophilicinfiltratesinthe endomysial action. Metalloproteinases (MMPs) facilitate the migration of T cells and their attachment tissue. In some patients, the eosinophilic myositis/ to the muscle surface. Muscle fiber necrosis occurs via perforin granules released by the fasciitis occurs in the context of parasitic infections, autoaggressive T cells. A direct myocytotoxic effect exerted by the cytokines interferon vasculitis, mixed connective tissue disease, hypereo(IFN) γ, interleukin (IL) 1, or tumor necrosis factor (TNF) α may also play a role. Death of sinophilic syndrome, or toxic exposures (e.g., toxic the muscle fiber is mediated by necrosis. MHC class I molecules consist of a heavy chain oil syndrome, contaminated l-tryptophan) or with and a light chain (β2 microglobulin [β2m]) complexed with an antigenic peptide that is mutations in the calpain gene. A distinct subset of transported into the endoplasmic reticulum by TAP proteins (Chap. 373e).

1	chain (β2 microglobulin [β2m]) complexed with an antigenic peptide that is mutations in the calpain gene. A distinct subset of transported into the endoplasmic reticulum by TAP proteins (Chap. 373e). myofasciitis is characterized by pronounced infiltra tion of the connective tissue around the muscle by sheets of periodic acid–Schiff-positive macrophages the dystrophinopathies where inflammatory cell infiltration is often and occasional CD8 T cells (macrophagic myofasciitis or inflammatory found early in the disease. Such doubtful cases should always be given myositis with abundant macrophages [IMAM]). A focal form of this anadequatetrialofglucocorticoidtherapyandundergogenetictesting disorder,limitedtositesofpreviousvaccinations,administeredmonths to exclude muscular dystrophy. Identification of the MHC/CD8 lesion or years earlier, has been linked to an aluminum-containing substrate by muscle biopsy is helpful to identify cases of PM. Some metabolic in vaccines. This disorder, which

1	of the MHC/CD8 lesion or years earlier, has been linked to an aluminum-containing substrate by muscle biopsy is helpful to identify cases of PM. Some metabolic in vaccines. This disorder, which to date has not been observed outside myopathies,including glycogenstoragedisease dueto myophosphory-of France, responds to glucocorticoid therapy, and the overall prognosis lase or acid maltase deficiency, lipid storage myopathies due to carni-seems favorable. tine deficiency, and mitochondrial diseases produce weakness that is often associated with other characteristic clinical signs; diagnosis rests

1	Necrotizing autoimmune Myositis This is an increasingly recognized upon histochemical and biochemical studies of the muscle biopsy. entitythathasdistinctfeatures,eventhoughitisoftenlabeledasPM.It The endocrine myopathies such as those due to hypercorticosteroidpresents as an acute or subacute onset of symmetric muscle weakness; ism, hyper-and hypothyroidism, and hyper-and hypoparathyroidism CK is typically extremely high. The weakness can be severe. Coexisting require the appropriate laboratory investigations for diagnosis. Muscle interstitial lung disease and cardiomyopathy may be present. The dis-wasting in patients with an underlying neoplasm may be due to disuse, order may develop after a viral infection, in association with cancer, cachexia, or rarely a paraneoplastic neuromyopathy (Chap. 122).

1	or in patients taking statins when the myopathy continues to worsenDiseases of the neuromuscular junction, including myasthenia after statin withdrawal. Some patients have antibodies against signal gravis or the Lambert-Eaton myasthenic syndrome, cause fatiguing recognition particle (SRP) or against the 3-hydroxy-3-methylglutarylweakness that also affects ocular and other cranial muscles (Chap. coenzyme A reductase (HMGCR), a 100-kDa protein considered the 461). Repetitive nerve stimulation and single-fiber EMG studies aid pharmacologic target of statins. The muscle biopsy demonstratesin diagnosis.

1	necrotic fibers infiltrated by macrophages but only rare, if any, T cell acute Muscle Weakness Thismaybecausedbyanacuteneuropathysuch infiltrates.MuscleMHC-IexpressionisonlyslightlyandfocallyupregasGuillain-Barrésyndrome (Chap. 460),transversemyelitis (Chap. 456), ulated. The capillaries may be swollen with hyalinization, thickening a neurotoxin (Chap. 462e), or a neurotropic viral infection such as polio-of the capillary wall, and deposition of complement. Most patients myelitisorWestNilevirus(Chap. 164).Whenacuteweaknessisassociated respondtoimmunotherapy,butsomeareresistant.

1	Hyperacute Necrotizing Fasciitis/Myositis (Flesh-Eating Disease) This a fulminant infectious disease, seen most often in the tropics or in conditions with poor hygiene, characterized by widespread necrosis of the superficial fascia and muscle of a limb; if the scrotum, perineum, and abdominal wall are affected, the condition is referred to as Fournier’s gangrene. It may be caused by group A β-hemolytic Streptococcus, methicillin-sensitive S. aureus, Pseudomonas aeruginosa, Vibrio vulnificus, clostridial species (gas gangrene; Chap. 179), or polymicrobial infection with anaerobes and facultative bacteria (Chap. 201); toxins from these bacteria may act as superantigens (Chap. 372e). The port of bacterial entry is usually a trivial cut or skin abrasion, and the source is contactwithcarriersoftheorganism.Individualswithdiabetesmellitus, immunodeficiency states, or systemic illnesses such as liver failure are most susceptible. Systemic varicella is a predisposing factor in children.

1	Thediseasepresentswithswelling,pain,andrednessintheinvolved area followed by a rapid tissue necrosis of fascia and muscle that progresses at an estimated rate of 3 cm/h. Emergency debridement, antibiotics, IV immunoglobulin (IVIg), and even hyperbaric oxygen have been recommended. In progressive or advanced cases, amputation of the affected limb may be necessary to avoid a fatal outcome.

1	Drug-Induced Myopathies d-Penicillamine, procainamide, and statins may produce a true myositis resembling PM or necrotizing myositis. A DM-like illness has been associated with the contaminated preparations of l-tryptophan. As noted above, AZT causes a mitochondrial myopathy. Other drugs may elicit a toxic noninflammatory myopathy that is histologically different from DM, PM, or IBM. These include cholesterol-lowering agents such as clofibrate, lovastatin, simvastatin, or pravastatin, especially when combined with cyclosporine, amiodarone, or gemfibrozil. Mild statin-induced myopathic symptoms (such as myalgia, fatigue, or asymptomatic elevations of CK) are self-limited and usually improve after discontinuation of the drug. In rare patients, however, muscle weakness continues to progress even after the statin is withdrawn; in these cases, a diagnostic muscle biopsy is indicated and search for antibodies to HMGCR is suggested; if histologic evidence of PM or necrotizing myositis is

1	even after the statin is withdrawn; in these cases, a diagnostic muscle biopsy is indicated and search for antibodies to HMGCR is suggested; if histologic evidence of PM or necrotizing myositis is present, immunotherapy should be initiated. Rhabdomyolysis and myoglobinuria have been rarely associated with amphotericin B, ε-aminocaproic acid, fenfluramine, heroin, and phencyclidine. The use of amiodarone, chloroquine, colchicine, carbimazole, emetine, etretinate, and ipecac syrup; chronic laxative or licoriceuseresultinginhypokalemia;andglucocorticoidorgrowthhormone administration have also been associated with myopathic muscle weakness. Some neuromuscular blocking agents such as pancuronium, in combination with glucocorticoids, may cause an acute critical illness myopathy. A careful drug history is essential for diagnosis of these drug-induced myopathies, which do not require immunosuppressive therapy except when an autoimmune myopathy has been triggered, as 2199 noted above.

1	“Weakness” due to Muscle Pain and Muscle Tenderness A number of conditions including polymyalgia rheumatica (Chap. 385) and arthritic disorders of adjacent joints may enter into the differential diagnosis of inflammatory myopathy, even though they do not cause myositis. The muscle biopsy is either normal or discloses type II muscle fiber atrophy. Patients with fibrositis and fibromyalgia (Chap. 396) complain of focal or diffuse muscle tenderness, fatigue, and aching, which is sometimes poorly differentiated from joint pain. Some patients, however, have muscle tenderness, painful muscles on movement, and signs suggestive of a collagen vascular disorder, such as an increased erythrocyte sedimentation rate, C-reactive protein, antinuclear antibody, or rheumatoid factor, along with modest elevation of the serum CK and aldolase. They demonstrate a “break-away” pattern of weaknesswithdifficultysustainingeffortbutnottruemuscleweakness.

1	antibody, or rheumatoid factor, along with modest elevation of the serum CK and aldolase. They demonstrate a “break-away” pattern of weaknesswithdifficultysustainingeffortbutnottruemuscleweakness. Themusclebiopsyisusuallynormalornonspecific.Manysuchpatients show some response to nonsteroidal anti-inflammatory agents or glucocorticoids, although most continue to have indolent complaints. An indolent fasciitis in the setting of an ill-defined connective tissue disorder may be at times present, and these patients should not be labeled as having a psychosomatic disorder. Chronic fatigue syndrome, which may follow a viral infection, can present with debilitating fatigue, sore throat, painful lymphadenopathy, myalgia, arthralgia, sleep disorder, and headache (Chap. 464e). These patients do not have muscle weakness, and the muscle biopsy is normal.

1	The clinically suspected diagnosis of PM, DM, IBM, or necrotizing myositis is confirmed by analysis of serum muscle enzymes, EMG findings, and muscle biopsy (Table 388-2). The most sensitive enzyme is CK, which in active disease can be elevated as much as 50-fold. Although the CK level usually parallels disease activity, it can be normal in some patients with active IBM or DM, especially when associated with a connective tissue disease. The CK is always elevated in patients with active PM. Along with the CK, the serum glutamic-oxaloacetic and glutamate pyruvate transaminases, lactate dehydrogenase, and aldolase may be elevated.

1	Needle EMG shows myopathic potentials characterized by short-duration, low-amplitude polyphasic units on voluntary activation and increased spontaneous activity with fibrillations, complex repetitive discharges, and positive sharp waves. Mixed potentials (polyphasic units of short and long duration) indicating a chronic process and muscle fiber regeneration are often present in IBM. These EMG findings are not diagnostic of an inflammatory myopathy but are useful

1	Polymyositis, Dermatomyositis, and Inclusion Body Myositis aMyopathic muscle weakness, affecting proximal muscles more than distal ones and sparing eye and facial muscles, is characterized by a subacute onset (weeks to months) and rapid progression in patients who have no family history of neuromuscular disease, no endocrinopathy, no exposure to myotoxic drugs or toxins, and no biochemical muscle disease (excluded on the basis of muscle biopsy findings). bIn some cases with the typical rash, the muscle strength is seemingly normal (dermatomyositis sine myositis); these patients often have new onset of easy fatigue and reduced endurance. Careful muscle testing may reveal mild muscle weakness. cSee text for details. dAn adequate trial of prednisone or other immunosuppressive drugs is warranted in probable cases. If, in retrospect, the disease is unresponsive to therapy, another muscle biopsy should be considered to exclude other diseases or possible evolution in inclusion body myositis.

1	in probable cases. If, in retrospect, the disease is unresponsive to therapy, another muscle biopsy should be considered to exclude other diseases or possible evolution in inclusion body myositis. eIf the muscle biopsy does not contain vacuolated fibers but shows chronic myopathy with hypertrophic fibers, primary inflammation with the CD8/MHC-I complex, and cytochrome oxygenase–negative fibers, the diagnosis is probable inclusion body myositis. fIf rash is absent but muscle biopsy findings are characteristic of dermatomyositis, the diagnosis is probable dermatomyositis.

1	FIGUrE 388-3 Cross-section of a muscle biopsy from a patient with polymyositis demonstrates scattered inflammatory foci with lymphocytes invading or surrounding muscle fibers. Note lack of chronic myopathic features (increased connective tissue, atrophic or hypertrophic fibers) as seen in inclusion body myositis. to identify the presence of active or chronic myopathy and to exclude neurogenic disorders. Magnetic resonance imaging (MRI) is not routinely used for the diagnosis of PM, DM, or IBM. However, it may provide information or guide the location of the muscle biopsy in certain clinical settings.

1	Muscle biopsy—despite occasional variability in demonstrating all of the typical pathologic findings—is the most sensitive and specific test for establishing the diagnosis of inflammatory myopathy and for excluding other neuromuscular diseases. Inflammation is the histologic hallmark for these diseases; however, additional features are characteristic of each subtype (Figs. 388-3, 388-4, and 388-5).

1	In PM the inflammation is primary, a term used to indicate that the inflammation is not reactive and the T cell infiltrates, located primarily withinthemusclefascicles(endomysially),surroundindividual,healthy muscle fibers and result in phagocytosis and necrosis (Fig. 388-3). The MHC-I molecule is ubiquitously expressed on the sarcolemma, even in fibers not invaded by CD8+ cells. The CD8/MHC-I lesion is characteristic and useful to confirm or establish the diagnosis and to exclude disorders with secondary, nonspecific, inflammation, such as insomemusculardystrophies.Whenthediseaseischronic,connective tissue is increased and may react positively with alkaline phosphatase. In necrotizing myositis, there are abundant necrotic fibers invaded or surrounded by macrophages, but no lymphocytic infiltrates or MHC-I expression beyond the necrotic fibers.

1	FIGUrE 388-4 Cross-section of a muscle biopsy from a patient with dermatomyositis demonstratesatrophyofthefibersattheperipheryofthefascicle(perifascicularatrophy). In DM the endomysial inflammation is predominantly perivascular or in the interfascicular septae and around—rather than within—the muscle fascicles (Fig. 388-4). The intramuscular blood vessels show endothelial hyperplasia with tubuloreticular profiles, fibrin thrombi, and obliteration of capillaries. The muscle fibers undergo necrosis, degeneration, and phagocytosis, often in groups involving a portion of a muscle fasciculus in a wedge-like shape or at the periphery of the fascicle, due to microinfarcts within the muscle. This results in perifascicular atrophy, characterized by 2–10 layers of atrophic fibers at the periphery of the fascicles. The presence of perifascicular atrophy is diagnostic of DM, even in the absence of inflammation.

1	In IBM (Fig. 388-5), there is endomysial inflammation with T cells invading MHC-I-expressing nonvacuolated muscle fibers; basophilic granular deposits distributed around the edge of slit-like vacuoles (rimmed vacuoles); loss of fibers, replaced by fat and connective tissue, hypertrophic fibers, and angulated or round fibers; rare eosinophilic cytoplasmic inclusions; abnormal mitochondria characterized by the presence of ragged-red fibers or cytochrome oxidase–negative fibers; andamyloiddepositswithinornexttothevacuolesbestvisualizedwith crystal violet or Congo-red staining viewed with fluorescent optics. Electron microscopy demonstrates filamentous inclusions in the vicinity of the rimmed vacuoles. In at least 15% of patients with the typical clinical phenotype of IBM, there is brisk inflammation in the muscle biopsy but no vacuoles or amyloid deposits, leading to an erroneous diagnosis of PM. Such patients are often referred to as having “clinical IBM.” Close clinicopathologic

1	inflammation in the muscle biopsy but no vacuoles or amyloid deposits, leading to an erroneous diagnosis of PM. Such patients are often referred to as having “clinical IBM.” Close clinicopathologic correlations are therefore essential; if uncertain, a repeat muscle biopsy from another site is often helpful.

1	The goal of therapy is to improve muscle strength, thereby improving function in activities of daily living, and ameliorate the extra-muscular manifestations (rash, dysphagia, dyspnea, fever). When strength improves, the serum CK falls concurrently; however, the reverse is not always true. Unfortunately, there is a common tendency to “chase” or treat the CK level instead of the muscle weakness, a practice that has led to prolonged and unnecessary use of immunosuppressive drugs and erroneous assessment of their efficacy. It is prudent to discontinue these drugs if, after an adequate trial, there is no objective improvement in muscle strength whether or not CK levels are reduced. Agents used in the treatment of PM and DM include the following: 1. Glucocorticoids. Oral prednisone is the initial treatment of choice; the effectiveness and side effects of this therapy determine the future need for stronger immunosuppressive drugs. High-dose prednisone, at least 1 mg/kg per day, is initiated

1	treatment of choice; the effectiveness and side effects of this therapy determine the future need for stronger immunosuppressive drugs. High-dose prednisone, at least 1 mg/kg per day, is initiated as early in the disease as possible. After 3–4 weeks, prednisone is tapered slowly over a period of 10 weeks to 1 mg/kg every other day. If there is evidence of efficacy and no serious side effects, the dosage is then further reduced by 5 or 10 mg every 3–4 weeks until the lowest possible dose that controls the disease is reached. The efficacy of prednisone is determined by an objective increase in muscle strength and activities of daily living, which almost always occurs by the third month of therapy. A feeling of increased energy or a reduction of the CK level without a concomitant increase in muscle strength is not a reliable sign of improvement. If prednisone provides no objective benefit after ~3 months of high-dose therapy, the disease is probably unresponsive to the drug and tapering

1	muscle strength is not a reliable sign of improvement. If prednisone provides no objective benefit after ~3 months of high-dose therapy, the disease is probably unresponsive to the drug and tapering should be accelerated while the next-in-line immunosuppressive drug is started. Although controlled trials have not been performed, almost all patients with true PM or DM respond to glucocorticoids to some degree and for some period of time; in general, DM responds better than PM.

1	The long-term use of prednisone may cause increased weakness associated with a normal or unchanged CK level; this effect is referred to as steroid myopathy. In a patient who previously FIGUrE 388-5 Cross-sections of a muscle biopsy from a patient with inclusion body myositis demonstratethetypicalfeaturesofvacuoleswithlymphocyticinfiltratessurroundingnonvacuolatedornecroticfibers(A),tinyendomysialdepositsofamyloidvisualizedwithcrystalviolet(B),cytochromeoxidase–negativefibers,indicativeofmitochondrialdysfunction(C),andubiquitousmajorhistocompatibilitycomplexclassIexpressionattheperipheryofallfibers(D).

1	Polymyositis, Dermatomyositis, and Inclusion Body Myositis responded to high doses of prednisone, the development of new weakness may be related to steroid myopathy or to disease activity that either will respond to a higher dose of glucocorticoids or has become glucocorticoid-resistant. In uncertain cases, the prednisone dosage can be steadily increased or decreased as desired: the cause of the weakness is usually evident in 2–8 weeks. 2. Other immunosuppressive drugs. Approximately 75% of patients ultimately require additional treatment. This occurs when a patient fails to respond adequately to glucocorticoids after a 3-month trial, the patient becomes glucocorticoid-resistant, glucocorticoid-related side effects appear, attempts to lower the prednisone dose repeatedly result in a new relapse, or rapidly progressive disease with evolving severe weakness and respiratory failure develops.

1	The following drugs are commonly used but have never been tested in controlled studies: (1) Azathioprine is well tolerated, has few side effects, and appears to be as effective for longterm therapy as other drugs. The dose is up to 3 mg/kg daily. (2) Methotrexate has a faster onset of action than azathioprine. It is given orally starting at 7.5 mg weekly for the first 3 weeks (2.5 mg every 12 h for 3 doses), with gradual dose escalation by 2.5 mg per week to a total of 25 mg weekly. A rare side effect is methotrexate pneumonitis, which can be difficult to distinguish from the interstitial lung disease of the primary myopathy associated with Jo-1 antibodies (described above). (3) Mycophenolate mofetil also has a faster onset of action than azathioprine. At doses up to 2.5 or 3 g/d in two divided doses, it is well tolerated for long-term use. (4) Monoclonal anti-CD20 antibody (rituximab) has been shown in a small uncontrolled series to benefit patients with DM and PM, but a controlled

1	divided doses, it is well tolerated for long-term use. (4) Monoclonal anti-CD20 antibody (rituximab) has been shown in a small uncontrolled series to benefit patients with DM and PM, but a controlled study did not show differences between patients randomized 8 weeks apart. (5) Cyclosporine has inconsistent and mild benefit. (6) Cyclophosphamide (0.5–1 g/m2 IV monthly for 6 months) has limited success and significant toxicity.

1	(7) Tacrolimus (formerly known as Fk506) has been effective in some difficult cases of PM especially with interstitial lung disease. 3. Immunomodulation. In a controlled trial of patients with refractory DM, IVIg improved not only strength and rash but also the underlying immunopathology. The benefit is often short-lived (≤8 weeks), and repeated infusions every 6–8 weeks are generally required to maintain improvement. A dose of 2 g/kg divided over 2–5 days per course is recommended. Uncontrolled observations suggest that IVIg may also be beneficial for patients with PM. Neither plasmapheresis nor leukapheresis appears to be effective in PM and DM.

1	The following sequential empirical approach to the treatment of PM and DM is suggested: Step 1: high-dose prednisone; Step 2: azathioprine, mycophenolate, or methotrexate for steroid-sparing effect; Step 3: IVIg; Step 4: a trial, with guarded optimism, of one of the following agents, chosen according to the patient’s age, degree of disability, tolerance, experience with the drug, and general health: rituximab, cyclosporine, cyclophosphamide, or tacrolimus. Patients with interstitial lung disease may benefit from aggressive treatment with cyclophosphamide or tacrolimus.

1	Immune-Mediated, Inflammatory, and Rheumatologic Disorders relapsing polychondritis Carol A. Langford Relapsingpolychondritisisanuncommondisorderofunknowncausecharacterizedbyinflammationofcartilagepredominantlyaffectingtheears,nose,andlaryngotracheobronchialtree.Othermanifesta-tionsincludescleritis,neurosensoryhearingloss,polyarthritis,cardiac389 2202 A patient with presumed PM who has not responded to any form of immunotherapy most likely has IBM or another disease, usually a metabolic myopathy, a muscular dystrophy, a drug-induced myopathy, or an endocrinopathy. In these cases, a repeat muscle biopsy and a renewed search for another cause of the myopathy is indicated. Calcinosis, a manifestation of DM, is difficult to treat; however, new calcium deposits may be prevented if the primary disease responds to the available therapies. Bisphosphonates, aluminum hydroxide, probenecid, colchicine, low doses of warfarin, calcium blockers, and surgical excision have all been tried without

1	disease responds to the available therapies. Bisphosphonates, aluminum hydroxide, probenecid, colchicine, low doses of warfarin, calcium blockers, and surgical excision have all been tried without success. IBM is generally resistant to immunosuppressive therapies. Prednisone together with azathioprine or methotrexate is often tried for a few months in newly diagnosed patients, although results are generally disappointing. Because occasional patients may feel subjectively weaker after these drugs are discontinued, some clinicians prefer to maintain these patients on low-dose, every-otherday prednisone along with mycophenolate in an effort to slow disease progression, even though there is no objective evidence or controlled study to support this practice. In two controlled studies of IVIg in IBM, minimal benefit in up to 30% of patients was found; the strength gains, however, were not of sufficient magnitude to justify its routine use. Another trial of IVIg combined with prednisone was

1	IBM, minimal benefit in up to 30% of patients was found; the strength gains, however, were not of sufficient magnitude to justify its routine use. Another trial of IVIg combined with prednisone was ineffective. Nonetheless, some experts believe that a 2to 3-month trial with IVIg may be reasonable for selected patients with IBM who experience rapid progression of muscle weakness or choking episodes due to worsening dysphagia.

1	The 5-year survival rate for treated patients with PM and DM is ~95%, and the 10-yearsurvivalrateis 84%; deathisusuallyduetopulmonary, cardiac, or other systemic complications. The prognosis is worse for patients who are severely affected at presentation, when initial treatment is delayed, and in cases with severe dysphagia or respiratory difficulties. Older patients and those with associated cancer also have a worse prognosis. DM responds more favorably to therapy than PM and thus has a better prognosis. Most patients improve with therapy, and many make a full functional recovery, which is often sustained with maintenance therapy. Up to 30% may be left with some residual muscle weakness. Relapses may occur at any time.

1	IBM has the least favorable prognosis of the inflammatory myopathies. Most patients will require the use of an assistive device such as a cane, walker, or wheelchair within 5–10 years of onset. In general, the oldertheageofonsetinIBM,themorerapidlyprogressiveisthecourse.

1	abnormalities, skin lesions, and glomerulonephritis. Relapsing polychondritis has been estimated to have an incidence of 3.5 per million population per year. The peak age of onset is between the ages of 40 and 50 years, but relapsing polychondritis may affect children and the elderly. It is found in all races, and both sexes are equally affected. No familial tendency is apparent. A significantly higher frequency of HLA-DR4 has been found in patients with relapsing polychondritis than in healthy individuals. A predominant subtype allele(s) of HLA-DR4 was not found. Approximately 30% of patients with relapsing polychondritis will have another rheumatologic disorder, the most frequent being systemic vasculitis, followed by rheumatoid arthritis, and systemic lupus erythematosus (SLE). Nonrheumatic aSystemic vasculitis is the most common association, followed by rheumatoid arthritis and systemic lupus erythematosus. Source: Modified from CJ Michet et al: Ann Intern Med 104:74, 1986.

1	Source: Modified from CJ Michet et al: Ann Intern Med 104:74, 1986. disorders associated with relapsing polychondritis include Hashimoto’s thyroiditis, primary biliary cirrhosis, and myelodysplastic syndrome (Table 389-1). In most cases, these disorders antedate the appearance of relapsing polychondritis, usually by months or years; however, in other instances, the onset of relapsing polychondritis can accompany disease presentation.

1	The earliest abnormality of hyaline and elastic cartilage noted histologically is a focal or diffuse loss of basophilic staining indicating depletion of proteoglycan from the cartilage matrix. Inflammatory infiltrates are foundadjacenttoinvolvedcartilageandconsistpredominantlyofmononuclear cells and occasional plasma cells. In acute disease, polymorphonuclear white cells may also be present. Destruction of cartilage begins at the outer edges and advances centrally. There is lacunar breakdown and loss of chondrocytes. Degenerating cartilage is replaced by granulationtissueandlaterbyfibrosis andfocal areasofcalcification.Smallloci of cartilage regeneration may be present. Immunofluorescence studies have shown immunoglobulins and complement at sites of involvement. Extracellular granular material observed in the degenerating cartilage matrix by electron microscopy has been interpreted to be enzymes, immunoglobulins, or proteoglycans.

1	Immunologic mechanisms play a role in the pathogenesis of relapsing polychondritis. The accumulating data strongly suggest that both humoral and cell-mediated immunity play an important role in the pathogenesis of relapsing polychondritis. Immunoglobulin and complement deposits are found at sites of inflammation. In addition, antibodies to type II collagen and to matrilin-1 and immune complexes are detected in the sera of some patients. The possibility that an immune response to type II collagen may be important in the pathogenesis is supported experimentally by the occurrence of auricular chondritis in rats immunized with type II collagen. Antibodies to type II collagen are found in the sera of these animals, and immune deposits are detected at sites of ear inflammation. Humoral immune responses to type IX and type XI collagen, matrilin-1, and cartilage oligomeric matrix protein have been demonstrated in some patients. In a study, rats immunized with matrilin-1 were found to develop

1	to type IX and type XI collagen, matrilin-1, and cartilage oligomeric matrix protein have been demonstrated in some patients. In a study, rats immunized with matrilin-1 were found to develop severe inspiratory stridor and swelling of the nasal septum. The rats had severe inflammation with erosions of the involved cartilage, which was characterized by increased numbers of CD4+ and CD8+ T cells in the lesions. The cartilage of the joints and ear pinna was not involved. All had IgG antibodies to matrilin-1. Matrilin-1 is a noncollagenous protein present in the extracellular matrix in cartilage. It is present in high concentrations in the trachea and is also present in the nasal septum but not in articular cartilage. A subsequent study demonstrated serum anti-matrilin-1 antibodies in approximately 13% of patients withrelapsingpolychondritis;approximately70%ofthesepatientshad respiratory symptoms. Cell-mediated immunity may also be operative in causing tissue injury, since lymphocyte

1	13% of patients withrelapsingpolychondritis;approximately70%ofthesepatientshad respiratory symptoms. Cell-mediated immunity may also be operative in causing tissue injury, since lymphocyte transformation can be demonstrated when lymphocytes of patients are exposed to cartilage extracts. T cells specific for type II collagen have been found in some

1	Frequency, % Source: Modified from PD Kent et al: Curr Opin Rheumatol 16:56, 2004. patients, and CD4+ T cells have been observed at sites of cartilage inflammation. The onset of relapsing polychondritis is frequently abrupt, with the appearance of one or two sites of cartilaginous inflammation. The pattern of cartilaginous involvement and the frequency of episodes vary widely among patients. Noncartilaginous presentations may also occur. Systemic inflammatory features such as fever, fatigue, and weight loss occur and may precede the clinical signs of relapsing polychondritis by several weeks. Relapsing polychondritis may go unrecognized for several months or even years in patients who only initially manifest intermittent joint pain and/or swelling, or who have unexplained eye inflammation, hearing loss, valvular heart disease, or pulmonary symptoms.

1	Auricular chondritis is the most frequent presenting manifestation of relapsing polychondritis, occurring in 40% of patients and eventually affecting about 85% of patients (Table 389-2). One or both ears are involved,eithersequentiallyor simultaneously. Patientsexperience the sudden onset of pain, tenderness, and swelling of the cartilaginous portion of the ear (Fig. 389-1). This typically involves the pinna of the ears, sparing the earlobes because they do not contain cartilage. The overlying skin has a beefy red or violaceous color. Prolonged or recurrentepisodesleadtocartilagedestructionandresultinaflabbyor

1	FIGUrE 389-1 Left. The pinna is erythematous, swollen, and tender. Not shown is the ear lobule that is spared as there is no underlying cartilage. Right. The pinna is thickened and deformed. The destruction of the underlying cartilage results in a floppy ear. (Reprinted from the Clinical Slide Collection on the Rheumatic Diseases, ©1991, 1995, 1997, 1998, 1999. Used by permission of the American College of Rheumatology.) FIGUrE 389-2 Saddle nose results from destruction and collapse of the nasal cartilage. (Reprinted from the Clinical Slide Collection on the Rheumatic Diseases, ©1991, 1995, 1997, 1998, 1999. Used by permission of the American College of Rheumatology.) droopy ear. Swelling may close off the eustachian tube or the external auditory meatus, either of which can impair hearing. Inflammation of the internal auditory artery or its cochlear branch produces hearing loss, vertigo, ataxia, nausea, and vomiting. Vertigo is almost always accompanied by hearing loss.

1	Approximately 61% of patients will develop nasal involvement, with 21% having this at the time of presentation. Patients may experience nasal stuffiness, rhinorrhea, and epistaxis. The bridge of the nose and surrounding tissue become red, swollen, and tender and may collapse, producing a saddle nose deformity (Fig. 389-2). In some patients, nasal deformity develops insidiously without overt inflammation. Saddle nose is observed more frequently in younger patients, especially in women.

1	Joint involvement is the presenting manifestation in relapsing polychondritis in approximately one-third of patients and may be present for several months before other features appear. Eventually, more than one-half of the patients will have arthralgias or arthritis. The arthritis is usually asymmetric and oligo-or polyarticular, and it involves both large and small peripheral joints. An episode of arthritis lasts from a few days to several weeks and resolves spontaneously without joint erosion or deformity. Attacks of arthritis may not be temporally related to other manifestations of relapsing polychondritis. Joint fluid has been reported to be noninflammatory. In addition to peripheral joints, inflammation may involve the costochondral, sternomanubrial, and sternoclavicular cartilages. Destruction of these cartilages may result in a pectus excavatum deformity or even a flail anterior chest wall.

1	Eye manifestations occur in more than one-half of patients and include conjunctivitis, episcleritis, scleritis, iritis, uveitis, and keratitis. Ocularinflammationcanbesevereandvisuallythreatening.Othermanifestations include eyelid and periorbital edema, proptosis, optic neuritis, extraocular muscle palsies, retinal vasculitis, and renal vein occlusion. Laryngotracheobronchial involvement occurs in ∼50% of patients and is among the most serious manifestations of relapsing polychondritis. Symptoms include hoarseness, a nonproductive cough, and tenderness over the larynx and proximal trachea. Mucosal edema, strictures, and/or collapse of laryngeal or tracheal cartilage may cause stridor and life-threatening airway obstruction necessitating tracheostomy. Involvement can extend into the lower airways resulting in tracheobronchomalacia. Collapse of cartilagein bronchileadsto pneumonia and, when extensive, to respiratory insufficiency.

1	Cardiac valvular regurgitation occurs in about 5–10% of patients and is due to progressive dilation of the valvular ring or to destruction 2204 of the valve cusps. Aortic regurgitation occurs in about 7% of patients, with the mitral and other heart valves being affected less often. Other cardiac manifestations include pericarditis, myocarditis, coronary vasculitis, and conduction abnormalities. Aneurysms of the proximal, thoracic, or abdominal aorta may occur even in the absence of active chondritis and occasionally rupture. Renal disease occurs in about 10% of patients. The most common renal lesions include mesangial expansion or segmental necrotizing glomerulonephritis, which have been reported to have small amounts of electron-dense deposits in the mesangium where there is also faint depositionofC3and/orIgGorIgM.TubulointerstitialdiseaseandIgA nephropathy have also been reported. Approximately 25% of patients have skin lesions, which can include purpura, erythema nodosum, erythema

1	nephropathy have also been reported. Approximately 25% of patients have skin lesions, which can include purpura, erythema nodosum, erythema multiforme, angioedema/urticaria, livedo reticularis, and panniculitis. Features of vasculitis are seeninup to25%ofpatientsandcanaffect any size vessel. Large vessel vasculitis may present with aortic aneurysms, and medium vessel disease may affect the coronary, hepatic, mesenteric, or renal arteries or vessel supplying nerves. Skin vessel disease and involvement of the postcapillary venules can also occur. A variety of primary vasculitides have also been reported to occur in association with relapsing polychondritis (Chap. 385). One specific overlap is the “MAGIC” syndrome (mouth and genital ulcers with inflamed cartilage) in which patients present with features of both relapsing polychondritis and Behçet’s disease (Chap. 387).

1	There are no laboratory features that are diagnostic for relapsing polychondritis. Mild leukocytosis and normocytic, normochromic anemia are often present. Eosinophilia is observed in 10% of patients. The erythrocyte sedimentation rate and C-reactive protein are usually elevated. Rheumatoid factor and antinuclear antibody tests are occasionally positive in low titers, and complement levels are normal. Antibodies to type II collagen are present in fewer than one-half of the patients and are not specific. Circulating immune complexes may be detected, especially in patients with early active disease. Elevated levels of γ globulin may be present. Antineutrophil cytoplasmic antibodies (ANCA), either cytoplasmic (cANCA) or perinuclear (pANCA), are found in some patients with active disease. However, on target antigen–specific testing, there are only occasional reports of positive myeloperoxidase-ANCA, and proteinase 3-ANCA are very rarely found in relapsing polychondritis.

1	The upper and lower airways can be evaluated by imaging techniques such as computed tomography and magnetic resonance imaging (MRI). Bronchoscopy provides direct visualization of the airways but can be a high-risk procedure in patients with airway compromise. Pulmonary function testing with flow-volume loops can show inspiratory and/or expiratory obstruction. Imaging can also be useful to detect extracartilaginous disease. The chest film may show widening of the ascending or descending aorta due to an aneurysm, and cardiomegaly when aortic insufficiency is present. MRI can assess aortic aneurysmal dilatation. Electrocardiography and echocardiography can be useful in further evaluating for cardiac features of disease.

1	Diagnosis is based on recognition of the typical clinical features. Biopsies of the involved cartilage from the ear, nose, or respiratory tract will confirm the diagnosis but are only necessary when clinical features are not typical. Diagnostic criteria were suggested in 1976 by McAdam et al and modified by Damiani and Levine in 1979. These criteria continue to be generally used in clinical practice. McAdam et al proposed the following: (1) recurrent chondritis of both auricles; (2) nonerosive inflammatory arthritis; (3) chondritis of nasal cartilage; (4) inflammation of ocular structures, including conjunctivitis, keratitis, scleritis/episcleritis, and/or uveitis; (5) chondritis of the laryngeal and/or tracheal cartilages; and (6) cochlear and/or vestibular damage manifested by neurosensory hearing loss, tinnitus, and/or vertigo. The diagnosis is certain when three or more of these features are present along with a positive biopsy from the ear, nasal, or respiratory cartilage.

1	hearing loss, tinnitus, and/or vertigo. The diagnosis is certain when three or more of these features are present along with a positive biopsy from the ear, nasal, or respiratory cartilage. Damiani and Levine later suggested that the diagnosis could be made when one or more of the above features and a positive biopsy were present, when two or more separate sites of cartilage inflammation were present that responded to glucocorticoids or dapsone, or when three or more of the above features were present.

1	The differential diagnosis of relapsing polychondritis is centered around its sites of clinical involvement. Patients with granulomatosis with polyangiitis (Wegener’s) may have a saddle nose and tracheal involvement but can be distinguished by the primary inflammation occurringinthe mucosaatthesesites,theabsenceofauricularinvolvement, and the presence of pulmonary parenchymal disease. Patients with Cogan’s syndrome have interstitial keratitis and vestibular and auditory abnormalities, but this syndrome does not involve the respiratory tract or ears. Reactive arthritis may initially resemble relapsing polychondritis because of oligoarticular arthritis and eye involvement, but it is distinguished in time by the appearance of urethritis and typical mucocutaneous lesions and the absence of nose or ear cartilage involvement. Rheumatoid arthritis may initially suggest relapsing polychondritis because of arthritis and eye inflammation. The arthritis in rheumatoid arthritis, however, is

1	of nose or ear cartilage involvement. Rheumatoid arthritis may initially suggest relapsing polychondritis because of arthritis and eye inflammation. The arthritis in rheumatoid arthritis, however, is erosive and symmetric. In addition, rheumatoid factor titers are usually high compared with those in relapsingpolychondritis,andanti-cycliccitrullinatedpeptideisusually notseen.Bacterialinfectionofthepinnamaybemistakenforrelapsing polychondritis but differs by usually involving only one ear, including the earlobe. Auricular cartilage may also be damaged by trauma or frostbite. Nasal destructive disease and auricular abnormalities can also be seen in patients using cocaine adulterated with levamisole. Ear involvement in this setting differs from relapsing polychondritis by typically manifesting as purpuric plaques with necrosis extending to the pinna, which does not contain cartilage.

1	In patients with active chondritis, prednisone, 40–60 mg/d, is often effective in suppressing disease activity; it is tapered gradually once disease is controlled. In some patients, prednisone can be stopped, whereas in others, low doses in the range of 5–10 mg/d are required for continued suppression of disease. Dapsone 50–100 mg/d has been effective for cartilage inflammation and joint features in some patients. Other immunosuppressive drugs such as cyclophosphamide, methotrexate, azathioprine, or cyclosporine should be reserved for patients who have severe organ-threatening disease, fail to respond to prednisone, or require high doses to control disease activity. Patients with significant ocular inflammation often require intraocular glucocorticoids as well as high doses of prednisone. There are a small number of reports on the use of tumor necrosis factor antagonists, rituximab (anti-CD20), and tocilizumab (anti-interleukin 6 receptor), which are too few in number to assess

1	There are a small number of reports on the use of tumor necrosis factor antagonists, rituximab (anti-CD20), and tocilizumab (anti-interleukin 6 receptor), which are too few in number to assess efficacy. Heart valve replacement or repair of an aortic aneurysm may be necessary. When airway obstruction is severe, tracheostomy is required. Stents may be necessary in patients with tracheobronchial collapse.

1	PaTIENT OUTCOME, PrOGNOSIS, aND SUrVIVaL The course of relapsing polychondritis is highly variable. Some patients experience inflammatory episodes lasting from a few days to several weeks that then subside spontaneously or with treatment. Attacks may recur at intervals varying from weeks to months. In other patients, the disease has a chronic, smoldering course that may be severe.Inonestudy,the5-yearestimatedsurvivalratewas74%andthe 10-year survival rate was 55%. In contrast to earlier series, only about one-half of the deaths could be attributed to relapsing polychondritis or complications of treatment. Airway complications accounted for only 10% of all fatalities. In general, patients with more widespread disease have a worse prognosis. This chapter represents a revised version of the text authored by Dr. Bruce

1	This chapter represents a revised version of the text authored by Dr. Bruce C. Gilliland that appeared in previous editions of Harrison’s Principles of Internal Medicine. Dr. Gilliland passed away on February 17, 2007, and had been a contributor to Harrison’s since the 11th edition.

1	Sarcoidosis Robert P. Baughman, Elyse E. Lower DEFINITION Sarcoidosisisaninflammatorydiseasecharacterizedbythepres-enceofnoncaseatinggranulomas.Thediseaseisoftenmultisystemandrequiresthepresenceofinvolvementintwoormoreorgansfor390 a specific diagnosis. The finding of granulomas is not specific for sarcoidosis, and other conditions known to cause granulomas must be ruled out. These conditions include mycobacterial and fungal infections, malignancy, and environmental agents such as beryllium. Although sarcoidosis can affect virtually every organ of the body, the lung is most commonly affected. Other organs commonly affected are the liver, skin, and eye. The clinical outcome of sarcoidosis varies, with remission occurring in over one-half of patients within a few years of diagnosis; however, the remaining patients may develop a chronic disease that lasts for decades.

1	Despite multiple investigations, the cause of sarcoidosis remains unknown. Currently, the most likely etiology is an infectious or noninfectious environmentalagent that triggers aninflammatory response in a genetically susceptible host. Among the possible infectious agents, careful studies have shown a much higher incidence of Propionibacter acnes in the lymph nodes of sarcoidosis patients compared to controls. An animal model has shown that P. acnes can induce a granulomatous response in mice similar to sarcoidosis. Others have demonstrated the presence of a mycobacterial protein (Mycobacterium tuberculosis catalase-peroxidase [mKatG]) in the granulomas of some sarcoidosis patients. This protein is very resistant to degradation and may represent the persistent antigen in sarcoidosis. Immune response to this and other mycobacterial proteins has been documented by another laboratory. These studies suggest that a mycobacterium similar to

1	M. tuberculosis could be responsible for sarcoidosis. The mechanism exposure/infectionwithsuchagentshasbeenthefocusofotherstudies. Environmental exposures to insecticides and mold have been associated with anincreasedrisk for disease. In addition, healthcareworkers appeartohaveanincreasedrisk.Also,sarcoidosisinadonororganhas occurred after transplantation into a sarcoidosis patient. Some authors have suggested that sarcoidosis is not due to a single agent but represents a particular host response to multiple agents. Some studies have been able to correlate the environmental exposures to genetic markers. These studies have supported the hypothesis that a genetically susceptible host is a key factor in the disease. INCIDENCE, PrEVaLENCE, aND GLOBaL IMPaCT

1	INCIDENCE, PrEVaLENCE, aND GLOBaL IMPaCT Sarcoidosis is seen worldwide, with the highest prevalence reported in the Nordic population. In the United States, the disease has been reported more commonly in African Americans than whites, with the ratio of African Americans to whites ranging from 3:1 to 17:0. Women appear to be slightly more susceptible than men. The higher incidence in African Americans may have been influenced by the fact that African Americans seem to develop more extensive and chronic pulmonary disease. Because most sarcoidosis clinics are run by pulmonologists, a selection bias may have occurred. Worldwide, the prevalence of the disease varies from 20–60 per 100,000 for many groups such as Japanese, Italians, and American whites. Higher ratesoccurinIrelandandNordiccountries.Inonecloselyobservedcommunity in Sweden, the lifetime risk for developing sarcoidosis was 3%.

1	Sarcoidosis often occurs in young, otherwise healthy adults. It is uncommon to diagnose the disease in someone under age 18. However, it has become clear that a second peak in incidence develops around age 60. In a study of >700 newly diagnosed sarcoidosis patients in the United States, one-half of the patients were ≥40 years at the time of diagnosis. Although most cases of sarcoidosis are sporadic, a familial form of 2205 the disease exists. At least 5% of patients with sarcoidosis will have a family member with sarcoidosis. Sarcoidosis patients who are Irish or African American seem to have a two to three times higher rate of familial disease.

1	The granuloma is the pathologic hallmark of sarcoidosis. A distinct feature of sarcoidosis is the local accumulation of inflammatory cells. Extensive studies in the lung using bronchoalveolar lavage (BAL) have demonstrated that the initial inflammatory response is an influx of T helper cells. In addition, there is an accumulation of activated monocytes. Figure 390-1 is a proposed model for sarcoidosis. Using the HLA-CD4 complex, antigen-presenting cells present an unknown antigen to the helper T cell. Studies have clarified that specific HLA haplotypes such as HLA-DRB1*1101 are associated with an increased risk for developing sarcoidosis. In addition, different HLA haplotypes are associated with different clinical outcomes.

1	The macrophage/helper T cell cluster leads to activation with the increased release of several cytokines. These include interleukin (IL)-2 released from the T cell and interferon γ and tumor necrosis factor (TNF) released by the macrophage. The T cell is a necessary part of the initial inflammatory response. In advanced, untreated HIV infection, patients who lack helper T cells rarely develop sarcoidosis. In contrast, several reports confirm that sarcoidosis becomes unmasked as HIV-infected individuals receive antiretroviral therapy, with subsequent restoration of their immune system. In contrast, treatment of established pulmonary sarcoidosis with cyclosporine, a drug that downregulates helper T cell responses, seems to have little impact on sarcoidosis.

1	The granulomatous response of sarcoidosis can resolve with or without therapy. However, in at least 20% of patients with sarcoidosis, a chronic form of the disease develops. This persistent form of the disease is associated with the secretion of high levels of IL-8. Also, studies have reported that patients with this chronic form of disease release excessive amounts of TNF in areas of inflammation. Specific FIGUrE 390-1 Schematic representation of initial events of sarcoidosis. The antigen-presenting cell and helper T cell complex leads to the release of multiple cytokines. This forms a granuloma. Over time, the granuloma may resolve or lead to chronic disease, including fibrosis. APC, antigen-presenting cell; HLA, human leukocyte antigen; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

1	2206 gene signatures have been associated with more severe disease, such as cardiac, neurologic, and fibrotic pulmonary disease. At diagnosis the natural history of the disease may be difficult to predict. One form of the disease, Löfgren’s syndrome, consists of erythema nodosum and hilar adenopathy on chest roentgenogram. In some cases, periarticular arthritis may be identified without erythema nodosum. Löfgren’s syndrome is associated with a good prognosis, with >90% of patients experiencing disease resolution within 2 years. Recent studies have demonstrated that the HLA-DRB1*03 was found in two-thirds of Scandinavian patients with Löfgren’s syndrome. More than 95% of those patients who were HLA-DRB1*03 positive had resolution of their disease within 2 years, whereas nearly one-half of the remaining patients had disease for more than 2 years. It remains to be determined whether these observations can be applied to a non-Scandinavian population.

1	The presentation of sarcoidosis ranges from patients who are asymptomatic to those with organ failure. It is unclear how often sarcoidosis is asymptomatic. In countries where routine chest roentgenogram screening is performed, 20–30% of pulmonary cases are detected in asymptomatic individuals. The inability to screen for other asymptomatic forms of the disease would suggest that as many as one-third of sarcoidosis patients are asymptomatic. Respiratory complaints including cough and dyspnea are the most common presenting symptoms. In many cases, the patient presents with a 2-to 4-week history of these symptoms. Unfortunately, due to the nonspecific nature of pulmonary symptoms, the patient may see physicians for up to a year before a diagnosis is confirmed. For these patients, the diagnosis of sarcoidosis is usually only suggested when a chest roentgenogram is performed.

1	Symptoms related to cutaneous and ocular disease are the next two most common complaints. Skin lesions are often nonspecific. However, because these lesions are readily observed, the patient and treating physician are often led to a diagnosis. In contrast to patients with pulmonary disease, patients with cutaneous lesions are more likely to be diagnosed within 6 months of symptoms. Nonspecific constitutional symptoms include fatigue, fever, night sweats, and weight loss. Fatigue is perhaps the most common constitutionalsymptomthataffectsthesepatients.Givenitsinsidiousnature, patients are usually not aware of the association with their sarcoidosis until their disease resolves.

1	The overall incidence of sarcoidosis at the time of diagnosis and eventual common organ involvement are summarized in Table 390-1. Over time, skin, eye, and neurologic involvement seem more apparent. In the United States, the frequency of specific organ involvement appears to be affected by age, race, and gender. For example, eye disease is more common among African Americans. Under the age of 40, itoccursmorefrequentlyin women.However,inthosediagnosedover the age of 40, eye disease is more common in men. Presentation, %b Follow-Up, %c aPatients could have more than one organ involved. bFrom ACCESS study of 736 patients evaluated within 6 months of diagnosis. cFrom follow-up of 1024 sarcoidosis patients seen at the University of Cincinnati Interstitial Lung Disease and Sarcoidosis Clinic from 2002–2006. FIGUrE 390-2 Posterior-anterior chest roentgenogram demon-stratingbilateralhilaradenopathy,stage1disease.

1	FIGUrE 390-2 Posterior-anterior chest roentgenogram demon-stratingbilateralhilaradenopathy,stage1disease. Lung involvement occurs in >90% of sarcoidosis patients. The most commonly used method for detecting lung disease is still the chest roentgenogram. Figure 390-2 illustrates the chest roentgenogram from a sarcoidosis patient with bilateral hilar adenopathy. Although the computed tomography (CT) scan has changed the diagnostic approach to interstitial lung disease, the CT scan is not usually considered a monitoring tool for patients with sarcoidosis. Figure 390-3 demonstrates some of the characteristic CT features, including peribronchial thickening and reticular nodular changes, which are predominantly subpleural. The peribronchial thickening seen on CT scan seems to explain the high yield of granulomas from bronchial biopsies performed for diagnosis.

1	Although the CT scan is more sensitive, the standard scoring system described by Scadding in 1961 for chest roentgenograms remains the preferred method of characterizing the chest involvement. Stage 1 is hilar adenopathy alone (Fig. 390-2), often with right paratracheal involvement. Stage 2 is a combination of adenopathy plus infiltrates, whereas stage 3 reveals infiltrates alone. Stage 4 consists of fibrosis. Usually the infiltrates in sarcoidosis are predominantly an upper lobe process. Only in a few noninfectious diseases is an upper lobe predominance noted. In addition to sarcoidosis, the differential diagnosis of upper lobe disease includes hypersensitivity pneumonitis, silicosis, and Langerhans cell histiocytosis. For infectious diseases, tuberculosis and Pneumocystis pneumonia can often present as upper lobe diseases. FIGUrE 390-3 High-resolution computed tomography scan of chest demonstratingpatchyreticularnodularity,includingareasofconfluence.

1	FIGUrE 390-3 High-resolution computed tomography scan of chest demonstratingpatchyreticularnodularity,includingareasofconfluence. Lung volumes, mechanics, and diffusion are all useful in evaluating interstitial lung diseases such as sarcoidosis. The diffusion of carbon monoxide (DLCO) is the most sensitive test for an interstitial lung disease. Reduced lung volumes are a reflection of the restrictive lung disease seen in sarcoidosis. However, a third of the patients presenting with sarcoidosis still have lung volumes within the normal range, despite abnormal chest roentgenograms and dyspnea.

1	Approximately one-half of sarcoidosis patients present with obstructive disease, reflected by a reduced ratio of forced vital capacity expired in 1 second (FEV1/FVC). Cough is a very common symptom. Airway involvement causing varying degrees of obstruction underlies the cough in most sarcoidosis patients. Airway hyperreactivity, as determined by methacholine challenge, will be positive in some of these patients. A few patients with cough will respond to traditional bronchodilators as the only form of treatment. In some cases, high-dose inhaled glucocorticoids alone are useful. Airway obstruction can be due to large airway stenosis, which can become fibrotic and unresponsive to anti-inflammatory therapy.

1	Pulmonary arterial hypertension is reported in at least 5% of sarcoidosis patients. Either direct vascular involvement or the consequence of fibrotic changes in the lung can lead to pulmonary arterial hypertension. In sarcoidosis patients with end-stage fibrosis awaiting lung transplant, 70% will have pulmonary arterial hypertension. This is a much higher incidence than that reported for other fibrotic lung diseases. In less advanced, but still symptomatic, patients, pulmonary arterial hypertension has been noted in up to 50% of thecases. Because sarcoidosis-associated pulmonary arterial hypertension may respond to therapy, evaluation for this should be considered in persistently dyspneic patients.

1	Skin involvement is eventually identified in over a third of patients with sarcoidosis. The classic cutaneous lesions include erythema nodosum, maculopapular lesions, hyper-and hypopigmentation, keloid formation, and subcutaneous nodules. A specific complex of involvement of the bridge of the nose, the area beneath the eyes, and the cheeks is referred to as lupus pernio (Fig. 390-4) and is diagnostic for a chronic form of sarcoidosis. FIGUrE 390-4 Chronic inflammatory lesions aroundnose,eyes,andcheeks,referredtoaslupuspernio. FIGUrE 390-5 Maculopapular lesions on the trunk ofasarcoidosispatient.

1	FIGUrE 390-4 Chronic inflammatory lesions aroundnose,eyes,andcheeks,referredtoaslupuspernio. FIGUrE 390-5 Maculopapular lesions on the trunk ofasarcoidosispatient. In contrast, erythema nodosum is a transient rash that can be seen in association with hilar adenopathy and uveitis (Löfgren’s syndrome). Erythema nodosum is more common in women and in certain self- described demographic groups including whites and Puerto Ricans. In the United States, the other manifestations of skin sarcoidosis, especially lupus pernio, are more common in African Americans than whites.

1	The maculopapular lesions from sarcoidosis are the most common chronic form of the disease (Fig. 390-5). These are often overlooked by the patient and physician, because they are chronic and not painful. Initially, these lesions are usually purplish papules and are often indurated. They can become confluent and infiltrate large areas of the skin. With treatment, the color and induration may fade. Because these lesions are caused by noncaseating granulomas, the diagnosis of sarcoidosis can be readily made by a skin biopsy.

1	The frequency of ocular manifestations for sarcoidosis varies depending on race. In Japan, >70% of sarcoidosis patients develop ocular disease, whereas in the United States only 30% have eye disease, with problems more common in African Americans than whites. Although the most common manifestation is an anterior uveitis, over a quarter ofpatientswill have inflammationat the posteriorof theeye, including retinitis and pars planitis. Although symptoms such as photophobia, blurred vision, and increased tearing can occur, some asymptomatic patients still have active inflammation. Initially asymptomatic patients with ocular sarcoidosis can eventually develop blindness. Therefore, it is recommended that all patients with sarcoidosis receive a dedicated ophthalmologic examination. Sicca is seen in over one-half of the chronic sarcoidosis patients. Dry eyes appear to be a reflection of prior lacrimal gland disease. Although the patient may no longer have active inflammation, the dry eyes may

1	in over one-half of the chronic sarcoidosis patients. Dry eyes appear to be a reflection of prior lacrimal gland disease. Although the patient may no longer have active inflammation, the dry eyes may require natural tears or other lubricants.

1	Using biopsies to detect granulomatous disease, liver involvement can be identified in over one-half of sarcoidosis patients. However, using liver function studies, only 20–30% of patients will have evidence of liver involvement. The most common abnormality of liver function is an elevation of the alkaline phosphatase level, consistent with an obstructive pattern. In addition, elevated transaminase levels can occur. An elevated bilirubin level is a marker for more advanced liver disease. Overall, only 5% of sarcoidosis patients have sufficient symptoms from their liver disease to require specific therapy. Although symptoms can be due to hepatomegaly, more frequently symptoms 2208 result from extensive intrahepatic cholestasis leading to portal hypertension. In this case, ascites and esophageal varices can occur. It is rare that a sarcoidosis patient will require a liver transplant, because even the patient with cirrhosis due to sarcoidosis can respond to systemic therapy. On a

1	esophageal varices can occur. It is rare that a sarcoidosis patient will require a liver transplant, because even the patient with cirrhosis due to sarcoidosis can respond to systemic therapy. On a cautionary note, patients with both sarcoidosis and hepatitis C should avoid therapy with interferon a because of its association with the development or worsening of granulomatous disease.

1	One or more bone marrow manifestations can be identified in many sarcoidosis patients. The most common hematologic problem is lymphopenia, which is a reflection of sequestration of the lymphocytes into the areas of inflammation. Anemia occurs in 20% of patients, and leukopenia is less common. Bone marrow examination will reveal granulomas in about a third of patients. Although splenomegaly can be detected in 5–10% of patients, splenic biopsy reveals granulomas in 60% of patients. The CT scan can be relatively specific for sarcoidosis involvement of the spleen (Fig. 390-6). Both bone marrow and spleen involvement are more common in African Americans than whites. Although these manifestations alone are rarely an indication for therapy, on rare occasion, splenectomy may be indicated for massive symptomatic splenomegaly or profound pancytopenia. Nonthoracic lymphadenopathy can occur in up to 20% of patients.

1	Hypercalcemia and/or hypercalciuria occurs in about 10% of sarcoidosis patients. It is more common in whites than African Americans and in men. The mechanism of abnormal calcium metabolism is increased production of 1,25-dihydroxyvitamin D by the granuloma itself. The 1,25-dihydroxyvitamin D causes increased intestinal absorption of calcium, leading to hypercalcemia with a suppressed parathyroid hormone (PTH) level (Chap. 424). Increased exogenous vitaminDfromdietorsunlightexposuremayexacerbatethisproblem. Serumcalciumshould be determinedaspartoftheinitialevaluationof all sarcoidosis patients, and a repeat determination may be useful during the summer months with increased sun exposure. In patients with a history of renal calculi, a 24-h urine calcium measurement should be obtained. If a sarcoidosis patient with a history of renal calculi is to be placed on calcium supplements, a follow-up 24-h urine calcium level should be measured.

1	Direct kidney involvement occurs in <5% of sarcoidosis patients. It is associated with granulomas in the kidney itself and can lead to nephritis. FIGUrE 390-6 Computed tomography scan of the abdomen afteroralandintravenouscontrast.Thestomachiscompressedbytheenlargedspleen.Withinthespleen,areasofhypo-andhyperdensityareidentified. However, hypercalcemia is the most likely cause of sarcoidosisassociated renal disease. In 1–2% of sarcoidosis patients, acute renal failure may develop as a result of hypercalcemia. Successful treatment of hypercalcemia with glucocorticoids and other therapies often improves but usually does not totally resolve the renal dysfunction.

1	Neurologic disease is reported in 5–10% of sarcoidosis patients and appears to be of equal frequency across all ethnic groups. Any part of the central or peripheral nervous system can be affected. The presence of granulomatous inflammationis often visible on magnetic resonance imaging (MRI) studies. The MRI with gadolinium enhancement may demonstratespace-occupyinglesions, but theMRIcanbe negative due tosmalllesionsortheeffectofsystemictherapyinreducingtheinflammation. The cerebral spinal fluid (CSF) findings include lymphocytic meningitis with a mild increase in protein. The CSF glucose is usually normal but can be low. Certain areas of the nervous system are more commonly affected in neurosarcoidosis. These include cranial nerve involvement, basilar meningitis, myelopathy, and anterior hypothalamic disease with associated diabetes insipidus (Chap. 404). Seizures and cognitive changes also occur. Of the cranial nerves, seventh nerve paralysis can be transient and mistaken for Bell’s

1	hypothalamic disease with associated diabetes insipidus (Chap. 404). Seizures and cognitive changes also occur. Of the cranial nerves, seventh nerve paralysis can be transient and mistaken for Bell’s palsy (idiopathic seventh nerve paralysis). Because this form of neurosarcoidosis often resolves within weeks and may not recur, it may have occurred prior toadefinitivediagnosis of sarcoidosis.Optic neuritisisanother cranial nerve manifestation of sarcoidosis. This manifestation is more chronic and usually requires long-term systemic therapy. It can be associated with both anterior and posterior uveitis. Differentiating between neurosarcoidosis and multiple sclerosis can be difficult at times. Optic neuritis can occur in both diseases. In some patients with sarcoidosis, multiple enhancing white matter abnormalities may be detected by MRI, suggesting multiple sclerosis. In such cases, the presence of meningeal enhancement or hypothalamic involvement suggests neurosarcoidosis, as does

1	white matter abnormalities may be detected by MRI, suggesting multiple sclerosis. In such cases, the presence of meningeal enhancement or hypothalamic involvement suggests neurosarcoidosis, as does evidence of extraneurologic disease such as pulmonary or skin involvement, which also suggests sarcoidosis. Because the response of neurosarcoidosis to glucocorticoids and cytotoxic therapy is different from that of multiple sclerosis, differentiating between these disease entities is important.

1	The presence of cardiac involvement is influenced by race. Although over a quarter of Japanese sarcoidosis patients develop cardiac disease, only 5% of sarcoidosis patients in the United States and Europe develop symptomatic cardiac disease. However, there is no apparent racial predilection between whites and African Americans. Cardiac disease,whichusually presentsas either congestiveheartfailureor cardiac arrhythmias, results from infiltration of the heart muscle by granulomas. Diffuse granulomatous involvement of the heart muscle can lead to profound dysfunction with left ventricular ejection fractions below 10%. Even in this situation, improvement in the ejection fraction can occur with systemic therapy. Arrhythmias can also occur with diffuse infiltration or with more patchy cardiac involvement. If the atrioventricular (AV) node is infiltrated, heart block can occur, which can be detected by routine electrocardiography. Ventricular arrhythmias and sudden death due to ventricular

1	involvement. If the atrioventricular (AV) node is infiltrated, heart block can occur, which can be detected by routine electrocardiography. Ventricular arrhythmias and sudden death due to ventricular tachycardia are common causes of death. Arrhythmias are best detected using 24-h ambulatory monitoring, and electrophysiology studies may be negative. Other screening tests for cardiac disease include routine electrocardiography and echocardiography. The confirmation of cardiac sarcoidosis is usually performed with either MRI or positron emission tomography (PET) scanning. Because ventricular arrhythmias are usually multifocal due to patchy multiple granulomas in the heart, ablation therapy is not useful. Patients with significant ventricular arrhythmias should be considered for an implanted defibrillator, which appears to have reduced the rate of death in cardiac sarcoidosis. Although systemic therapy can be useful in treating the arrhythmias, patients may still have malignant

1	implanted defibrillator, which appears to have reduced the rate of death in cardiac sarcoidosis. Although systemic therapy can be useful in treating the arrhythmias, patients may still have malignant arrhythmias up to 6 months after starting successful treatment, and the risk for recurrent arrhythmias occurs whenever medications are tapered.

1	FIGUrE 390-7 Positron emission tomography and computed tomography scan merged demonstratingincreasedactivityinspleen,ribs,andspineofpatientwithsarcoidosis. Direct granulomatous involvement of bone and muscle can be documented by radiography (x-ray, MRI, PET scan [Fig. 390-7], or gallium scan) or confirmed by biopsy in about 10% of sarcoidosis patients. However, alargerpercentageofsarcoidosis patients complain of myalgias and arthralgias. These complaints are similar to those reported by patients with other inflammatory diseases, including chronic infections such as mononucleosis. Fatigue associated with sarcoidosis may be overwhelming formany patients. Recentstudies have demonstrated a link between fatigue and small peripheral nerve fiber disease in sarcoidosis.

1	Although sarcoidosis can affect any organ of the body, rarely does it involve the breast, testes, ovary, or stomach. Because of the rarity of involvement, a mass in one of these areas requires a biopsy to rule out other diseases including cancer. For example, in a study of breast problems in female sarcoidosis patients, a breast lesion was morelikely to be a granuloma from sarcoidosis than from breast cancer. However, findings on the physical examination or mammogram cannot reliably differentiate between these lesions. More importantly, as women with sarcoidosis age, breast cancer becomes more common. Therefore, it is recommended that routine screening including mammography be performed along with other imaging studies (ultrasound, MRI) or biopsy as clinically indicated.

1	Sarcoidosis is usually a self-limited, non-life-threatening disease. However, organ-threatening disease can occur. These complications can include blindness, paraplegia, or renal failure. Death from sarcoidosis occurs in about 5% of patients seen in sarcoidosis referral clinics. The usual causes of death related to sarcoidosis are from lung, cardiac, neurologic, or liver involvement. In respiratory failure, an elevation of the right atrial pressure is a poor prognostic finding. Lung complications can also include infections such as mycetoma, which can subsequently lead to massive bleeding. In addition, the use of immu-2209 nosuppressive agents can increase the incidence of serious infections.

1	The chest roentgenogram remains the most commonly used tool to assess lung involvement in sarcoidosis. As noted above, the chest roentgenogram classifies involvement into four stages, with stages 1 and 2having hilarand paratrachealadenopathy.TheCT scan hasbeen used increasingly in evaluating interstitial lung disease. In sarcoidosis, the presence of adenopathy and a nodular infiltrate is not specific for sarcoidosis. Adenopathy up to 2 cm can be seen in other inflammatory lungdiseasessuchasidiopathicpulmonary fibrosis.However,adenopathy >2 cm in the short axis supports the diagnosis of sarcoidosis over other interstitial lung diseases.

1	The PET scan has increasingly replaced gallium-67 scanning to identify areas of granulomatous disease in the chest and other parts of the body (Fig. 390-7). Both tests can be used to identify potential areas for biopsy. Cardiac PET scanning has also proved useful in assessing cardiac sarcoidosis. The identification of hypermetabolic activity may be due to the granulomas from sarcoidosis and not to disseminated malignancy. MRI has also proved useful in the assessment of extrapulmonary sarcoidosis. Gadolinium enhancement has been demonstrated in areas of inflammation in the brain, heart, and bone. MRI scans may detect asymptomatic lesions. Like PET scan, MRI changes appear similar to those seen with malignancy and infection. In some cases, biopsy may be necessary to determine the cause of the radiologic abnormality.

1	Serum levels of angiotensin-converting enzyme (ACE) can be helpful in the diagnosis of sarcoidosis. However, the test has somewhat low sensitivity and specificity. Elevated levels of ACE are reported in 60% of patients with acute disease and only 20% of patients with chronic disease. Although there are several causes for mild elevation of ACE, including diabetes, elevations of >50% of the upper limit of normal are seen in only a few conditions including sarcoidosis, leprosy, Gaucher’s disease, hyperthyroidism, and disseminated granulomatous infections such as miliary tuberculosis. Because the ACE level is determined by a biologic assay, the concurrent use of an ACE inhibitor such as lisinopril will lead to a very low ACE level.

1	The diagnosis of sarcoidosis requires both compatible clinical features and pathologic findings. Because the cause of sarcoidosis remains elusive, the diagnosis cannot be made with 100% certainty. Nevertheless, the diagnosis can be made with reasonable certainty based on history and physical features along with laboratory and pathologic findings. Patients are usually evaluated for possible sarcoidosis based on two scenarios (Fig. 390-8). In the first scenario, a patient may undergo a biopsy revealing a noncaseating granuloma in either a pulmonary or an extrapulmonaryorgan. If theclinicalpresentationisconsistentwith sarcoidosis and there is no alternative cause for the granulomas identified, then the patient is felt to have sarcoidosis.

1	In the second scenario, signs or symptoms suggesting sarcoidosis such as the presence of bilateral adenopathy may be present in an otherwise asymptomatic patient or a patient with uveitis or a rash consistentwithsarcoidosis.Atthispoint,adiagnosticprocedureshouldbe performed. For the patient with a compatible skin lesion, a skin biopsy should be considered. Other biopsies to consider could include liver, extrathoracic lymph node, or muscle. In some cases, a biopsy of the affected organ may not be easy to perform (such as a brain or spinal cord lesion). In other cases, such as an endomyocardial biopsy, the likelihood of a positive biopsy is low. Because of the high rate of pulmonaryinvolvement in thesecases, the lung may be easierto approach by bronchoscopy. During the bronchoscopy, a transbronchial biopsy, bronchial biopsy, or transbronchial needle aspirate can be performed. The endobronchial ultrasonography-guided (EBUS) transbronchial needle aspirate can assist in diagnosing

1	a transbronchial biopsy, bronchial biopsy, or transbronchial needle aspirate can be performed. The endobronchial ultrasonography-guided (EBUS) transbronchial needle aspirate can assist in diagnosing sarcoidosis in patients with mediastinaladenopathy(stage1or2radiographicpulmonarydisease), whereas transbronchial biopsy has a higher diagnostic yield for those nodosum. The presence of one or more of these features in a patient suspected of having sarcoid osis increases the probability of sarcoidosis. The Kviem-Siltzbach procedure is a specific diagnostic test for sarcoidosis. An intradermal Features suggesting sarcoidosis: injection of specially prepared tissue derived from Consistent chest roentgenogram (adenopathy) the spleen of a known sarcoidosis patient is biop-Consistent skin lesions: lupus pernio, erythema nodosum, sied 4–6 weeks after injection. If noncaseating maculopapular lesions granulomas are seen, this is highly specific for the

1	Uveitis, optic neuritis, hypercalcemia, hypercalciuria, diagnosis of sarcoidosis. Unfortunately, there is no commercially available Kviem-Siltzbach reagent, and some locally prepared batches have lower specificity. Thus, this test is of historic interest and is rarely used in current clinical practice. Because the diagnosis of sarcoidosis can never be certain, over time other features may arise that lead to an alternative diagnosis. Conversely, evidence for new organ involvement may eventually confirm the diagnosis of sarcoidosis. The risk of death or loss of organ function remains

1	The risk of death or loss of organ function remains Features highly consistent with sarcoidosis: low in sarcoidosis. Poor outcomes usually occur in patients who present with advanced disease in whom treatment seems to have little impact. In these cases, irreversible fibrotic changes have frequently occurred. Over the past 20 years, the reported mortality from sarcoidosis has increased in the United States and England. Whether this is due to heightened awareness of the chronic nature of this disease or to other factors such as more FIGUrE 390-8 Proposed approach to management of patient with possible sarcoid osis. Presence of one or more of these features supports the diagnosis of sarcoidosis: uve remains unclear. itis, optic neuritis, hypercalcemia, hypercalciuria, seventh cranial nerve paralysis, diabetes For the majority of patients, initial presentation insipidus. ACE, angiotensin-converting enzyme; BAL, bronchoalveolar lavage.

1	For the majority of patients, initial presentation insipidus. ACE, angiotensin-converting enzyme; BAL, bronchoalveolar lavage. occurs during the granulomatous phase of the disease as depicted in Fig. 390-1. It is clear that with only parenchymal lung disease (stage 3). These tests are comple-many patients resolve their disease within 2–5 years. These patients mentary and may be performed together. are felt to have acute, self-limiting sarcoidosis. However, there is a If the biopsy reveals granulomas, an alternative diagnosis such as form of the disease that does not resolve within the first 2–5 years.

1	infection or malignancy must be excluded. Bronchoscopic washings These chronic patients can be identified at presentation by certain risk can be sent for cultures for fungi and tuberculosis. For the pathologist, factors at presentation such as fibrosis on chest roentgenogram, pres- the more tissue that is provided, the more comfortable is the diagnosis ence of lupus pernio, bone cysts, cardiac or neurologic disease (except of sarcoidosis. A needle aspirate may be adequate in an otherwise clas-isolated seventh nerve paralysis), and presence of renal calculi due to sic case of sarcoidosis, but may be insufficient in a patient in whom hypercalciuria. Recent studies also indicate that patients who require lymphoma or fungal infection is a likely alternative diagnosis. Because glucocorticoids for any manifestation of their disease in the first 6 granulomas can be seen on the edge of a lymphoma, the presence months of presentation have a >50% chance of having chronic disease.

1	of a few granulomas from a needle aspirate may not be sufficient to In contrast, <10% of patients who require no systemic therapy in the clarify the diagnosis. Mediastinoscopy provides a larger sample to first 6 months will require chronic therapy. confirm the presence or absence of lymphoma in the mediastinum. Alternatively, for most patients, evidence of extrathoracic disease (e.g., eye involvement) may further support the diagnosis of sarcoidosis.

1	For patients with negative pathology, positive supportive tests may Indications for therapy should be based on symptoms or presence increase the likelihood of the diagnosis of sarcoidosis. These tests of organor life-threatening disease, including disease involving include an elevated ACE level, which can also be elevated in other the eye, heart, or nervous system. The patient with asymptomatic granulomatous diseases but not in malignancy. A positive PET scan elevated liver function tests or an abnormal chest roentgenogram can support the diagnosis if multiple organs are affected. A BAL is probably does not benefit from treatment. However, these patients often performed during the bronchoscopy. An increase in the per-should be monitored for evidence of progressive, symptomatic centage of lymphocytes supports the diagnosis of sarcoidosis. The use disease.

1	of the lymphocyte markers CD4 and CD8 can be used to determine One approach to therapy is summarized in Figs. 390-9 and the CD4/CD8 ratio of these increased lymphocytes in the BAL fluid. 390-10. We have divided the approach into treating acute versus A ratio of >3.5 is strongly supportive of sarcoidosis but is less sensi-chronic disease. For acute disease, no therapy remains a viable tive than an increase in lymphocytes alone. Although in general, an option for patients with no or mild symptoms. For symptoms con- increase in BAL lymphocytes is supportive of the diagnosis, other fined to only one organ, topical therapy is preferable. For multi- conditions must be considered. organ disease or disease too extensive for topical therapy, an

1	Supportivefindings,whencombinedwithcommonlyassociatedbut approach to systemic therapy is outlined. Glucocorticoids remain nondiagnostic clinical features of the disease, improve the diagnostic the drugs of choice for this disease. However, the decision to con-probability of sarcoidosis. These clinical features include uveitis, renal tinue to treat with glucocorticoids or to add steroid-sparing agents stones, hypercalcemia, seventh cranial nerve paralysis, or erythema depends on the tolerability, duration, and dosage of glucocorticoids.

1	Abnormalities of neurologic, cardiac, ocular, calcium Yes: consider systemic therapy No: no therapy and observe Acute disease Minimal to no symptoms Single organ disease Symptomatic multiple organs Affecting only: anterior eye, localized skin, cough Systemic therapy: glucocorticoids (e.g., prednisone) Yes: try topical steroids No: systemic therapy Taper to <10 mg in less than 6 months: continue prednisone Cannot taper to <10 mg in 6 months or glucocorticoid toxicity Consider methotrexate, hydroxy-chloroquine, azathioprine FIGUrE 390-9 The management of acute sarcoidosis is based on level of symptoms and extent of organ involvement. In patients with mild symptoms, no therapy may be needed unless specified manifestations are noted.

1	Table 390-2 summarizes the dosage and monitoring of several commonly used drugs. According to the available trials, evidence-based recommendations are made. Most of these recommendations are for pulmonary disease because most of the trials were performed only in pulmonary disease. Treatment recommendations for extrapulmonary disease are usually similar with a few modifications. For example, the dosage of glucocorticoids is usually higher for neurosarcoidosis and lower for cutaneous disease. There was some suggestion that higher doses would be beneficial for cardiac sarcoidosis, but one study found that initial prednisone doses >40 mg/d were associated with a worse outcome because of toxicity.

1	Systemic therapies for sarcoidosis are usually immunosuppressive including glucocorticoids, cytotoxics, or biologics. Although most patients receive glucocorticoids as their initial systemic therapy, toxicity associated with prolonged therapy often leads to steroid-sparing alternatives. The antimalarial drugs such as hydroxychloroquine are more effective for skin than pulmonary disease. Minocycline may also be useful for cutaneous sarcoidosis.

1	For pulmonary and other extrapulmonary disease, cytotoxic agents are often used. These include methotrexate, azathioprine, leflunomide, mycophenolate, and cyclophosphamide. The most widely studied cytotoxic agent has been methotrexate. This agent works in approximately two-thirds of sarcoidosis patients, regardless of the disease manifestation. In one retrospective study comparing methotrexate to azathioprine, both drugs were equally effective. However, methotrexate was associated with significantly less toxicity. As noted in Table 390-2, specific guidelines for monitoring therapy have been recommended. Cytokine modulators such as thalidomide and pentoxifylline have also been used in a limited number of cases.

1	The biologic anti-TNF agents have recently been studied in sarcoidosis, with prospective randomized trials completed for both etanercept and infliximab. Etanercept has a limited role as a steroid-sparing agent. Conversely, infliximab significantly improved lung function when administered to glucocorticoid and cytotoxic pretreated patients with chronic disease The difference in response between these two agents is similar to that observed in Glucocorticoids tolerated Glucocorticoids not tolerated Glucocorticoids not effective Dose <10 mg/d Seek alternative agents Alternative agents Methotrexate Hydroxychloroquine Azathioprine Leflunomide Mycophenolate Minocycline Try alternative agents If effective, taper off glucocorticoids If not effective, consider: Multiple agents Infliximab Cyclophosphamide Thalidomide No Yes FIGUrE 390-10 Approach to chronic disease is based on whether glucocorticoid therapy is tolerated or not.

1	FIGUrE 390-10 Approach to chronic disease is based on whether glucocorticoid therapy is tolerated or not. Hydroxychloroquine 200–400 mg qd 400 mg qd Eye exam q6–12 mo Ocular B: Some forms of D: Routine eye exam disease Azathioprine 50–150 mg qd 50–200 mg qd CBC, renal q2mo Hematologic, nausea C: Some forms D: Routine hematologic chronic disease monitoring Abbreviations: CBC, complete blood count; PPD, purified protein derivative test for tuberculosis. Source: Adapted from RP Baughman, O Selroos: Evidence-based approach to treatment of sarcoidosis, in PG Gibson et al (eds): Evidence-Based Respiratory Medicine. Oxford, BMJ Books Blackwell, 2005, pp 491–508.

1	Crohns disease, where infliximab is effective and etanercept is not. However, there is a higher risk for reactivation of tuberculosis with infliximab compared to etanercept. The differential response rate could be explained by differences in mechanism of action because etanercept is a TF receptor antagonist and infliximab is a monoclonal antibody against TF. In contrast to etanercept, infliximab also binds to TF on the surface of some cells that release TF, which leads to cell lysis. This effect has been documented in Crohn disease. Adalimumab is a humanized monoclonal anti-TF antibody that also appears effective for sarcoidosis when dosed at higher strengths, as recommended for the treatment of Crohn disease. The role of the newer therapeutic agents for sarcoidosis is still evolving. However, these targeted therapies confirm that TF may be an important target, especially in the treatment of chronic disease. However, these agents are not a panacea, because sarcoidosis-like disease has

1	these targeted therapies confirm that TF may be an important target, especially in the treatment of chronic disease. However, these agents are not a panacea, because sarcoidosis-like disease has occurred in patients treated with anti-TF agents for nonsarcoidosis indications.

1	IgG4-related Disease John H. Stone IgG4-related disease (IgG4-RD) is a fibroinflammatory condition characterized by a tendency to form tumefactive lesions. The clinical manifestations of this disease, however, are protean, and continue to be defined. IgG4-RD has now been described in virtually every organ 391e system. Commonly affected organs are the biliary tree, salivary glands, periorbital tissues, kidneys, lungs, lymph nodes, and retroperitoneum. In addition, IgG4-RD involvement of the meninges, aorta, prostate, thyroid, pericardium, skin, and other organs is well described. The disease is believed to affect the brain parenchyma, the joints, the bone marrow, and the bowel mucosa only rarely (if ever).

1	The clinical features of IgG4-RD are numerous, but the pathologic findings are consistent across all affected organs. These findings include a lymphoplasmacytic infiltrate with a high percentage of IgG4-positive plasma cells; a characteristic pattern of fibrosis termed “storiform”; a tendency to target blood vessels, particularly veins, through an obliterative process (“obliterative phlebitis”); and a mild to moderate tissue eosinophilia. IgG4-RD encompasses a number of conditions previously regarded as separate, organ-specific entities. A condition once known as “lymphoplasmacytic sclerosing pancreatitis” (among many other terms) became the paradigm of IgG4-RD in 2000, when Japanese investigators recognized that these patients had elevated serum concentrations of IgG4. This form of sclerosing pancreatitis is now termed type 1 (IgG4related) autoimmune pancreatitis (AIP). By 2003, extrapancreatic

1	Orbits and periorbital Painless eyelid or periocular tissue swelling; orbital pseudotumor; dacryoadenitis; dacryocystitis; orbital myositis; and mass lesions tissues extending into the pterygopalatine fossa and infiltrating along the trigeminal nerve Ears, nose, and sinuses Allergic phenomena (nasal polyps, asthma, allergic rhinitis, peripheral eosinophilia); nasal obstruction, rhinorrhea, anosmia, chronic sinusitis; occasional bone-destructive lesions Meninges Headache, radiculopathy, cranial nerve palsies, or other symptoms resulting from spinal cord compression; tendency to form mass lesions; magnetic resonance imaging shows marked thickening and enhancement of dura

1	Hypothalamus and Clinical syndromes resulting from involvement of the hypothalamus and pituitary, e.g., anterior pituitary hormone deficiency, central pituitary diabetes insipidus, or both; imaging reveals thickened pituitary stalk or mass formation on the stalk, swelling of the pituitary gland, or mass formation within the pituitary Lymph nodes Generalized lymphadenopathy or localized disease adjacent to a specific affected organ; the lymph nodes involved are generally 1–2 cm in diameter and nontender Thyroid gland Riedel’s thyroiditis; fibrosing variant of Hashimoto’s thyroiditis

1	Thyroid gland Riedel’s thyroiditis; fibrosing variant of Hashimoto’s thyroiditis Lungs Asymptomatic finding on lung imaging; cough, hemoptysis, dyspnea, pleural effusion, or chest discomfort; associated with parenchymal lung involvement, pleural disease, or both; four main clinical syndromes: inflammatory pseudotumor, central airway disease, localized or diffuse interstitial pneumonia, and pleuritis; pleural lesions have severe, nodular thickening of the visceral or parietal pleura with diffuse sclerosing inflammation, sometimes associated with pleural effusion Aorta Asymptomatic finding on radiologic studies; surprise finding at elective aortic surgery; aortic dissection; clinicopathologic syndromes described include lymphoplasmacytic aortitis of thoracic or abdominal aorta, aortic dissection, periaortitis and periarteritis, and inflammatory abdominal aneurysm

1	Retroperitoneum Backache, lower abdominal pain, lower extremity edema, hydronephrosis from ureteral involvement, asymptomatic finding on radiologic studies Kidneys Tubulointerstitial nephritis; membranous glomerulonephritis in a small minority; asymptomatic tumoral lesions, typically multiple and bilateral, are sometimes detected on radiologic studies; renal involvement strongly associated with hypocomplementemia Pancreas Type 1 autoimmune pancreatitis, presenting as mild abdominal pain; weight loss; acute, obstructive jaundice, mimicking adenocarcinoma of the pancreas (including a pancreatic mass); between 20 and 50% of patients present with acute glucose intolerance; imaging shows diffuse (termed “sausage-shaped pancreas”) or segmental pancreatic enlargement, with loss of normal lobularity; a mass often raises the suspicion of malignancy

1	Biliary tree Obstructive jaundice associated with autoimmunity in most cases; weight loss; steatorrhea; abdominal pain; and new-onset diabetes mellitus; mimicker of primary sclerosing cholangitis Liver Painless jaundice associated with mild to moderate abdominal discomfort, weight loss, steatorrhea; new-onset diabetes mellitus; mimicker of primary sclerosing cholangitis and cholangiocarcinoma

1	Other organs involved Gallbladder, breast (pseudotumor), prostate (prostatism), pericardium (constrictive pericarditis), mesentery (sclerosing mesenteritis), mediastinum (fibrosing mediastinitis), skin (erythematous or flesh-colored papules), peripheral nerve (perineural inflammation) disease manifestations had been identified in patients with type 1 AIP, 391e-1 and since then, the manifestations of IgG4-RD in many organs have been catalogued. Mikulicz’s disease, once considered to be a subset of Sjren’s syndrome that affected the lacrimal, parotid, and sub-mandibular glands, is now considered part of the IgG4-RD spectrum. Similarly, a subset of patients previously diagnosed as having primary sclerosing cholangitis was known to respond well to glucocorticoids, in contrast to the majority of patients with that diagnosis. This steroid-responsive subset is now explained by the fact that such patients actually have a separate disease, i.e., IgG4-related sclerosing cholangitis. In this

1	of patients with that diagnosis. This steroid-responsive subset is now explained by the fact that such patients actually have a separate disease, i.e., IgG4-related sclerosing cholangitis. In this manner, the understanding of IgG4-RD has extended to include nearly every specialty of medicine.

1	The major organ lesions are summarized in Table 391e-1. IgG4-RD usually presents subacutely, and most patients do not have severe constitutional symptoms. Fevers and dramatic elevations of C-reactive protein are unusual; however, some patients report substantial weight loss occurring over periods of months. Clinically apparent disease can evolve over months, years, or even decades before the manifestations within a given organ becomes sufficiently severe to bring the patient to medical attention. Some patients have disease that is marked by the appearance and then resolution or temporary improvement in symptoms within a particular organ. Other patients accumulate new organ involvement as their disease persists in previously affected organs. Many patients with IgG4-RD are misdiagnosed as having other conditions, particularly malignancies, or their findings are attributed initially to nonspecific inflammation. The disorder is often identified incidentally through radiologic findings or

1	other conditions, particularly malignancies, or their findings are attributed initially to nonspecific inflammation. The disorder is often identified incidentally through radiologic findings or unexpectedly in pathology specimens.

1	Multiorgan disease may be evident at diagnosis but can also evolve over months to years. Some patients have disease confined to a single organ for many years. Others have either known or subclinical organ involvement at the same time as the major clinical feature. Patients with type 1 AIP may have their major disease focus in the pancreas; however, thorough evaluations by history, physical examination, blood tests, urinalysis, and cross-sectional imaging may demonstrate lacrimal gland enlargement, sialoadenitis, lymphadenopathy, a variety of pulmonary findings, tubulointerstitial nephritis, hepatobiliary disease, aortitis, retroperitoneal fibrosis, or other organ involvement. Spontaneous improvement, sometimes leading to clinical resolution of certain organ system manifestations, is reported in a small percentage of patients.

1	Two common characteristics of IgG4-RD are allergic disease and the tendency to form tumefactive lesions that mimic malignancies (Fig. 391e-1). Many IgG4-RD patients have allergic features such as atopy, eczema, asthma, nasal polyps, sinusitis, and modest peripheral eosinophilia. IgG4-RD also appears to account for a significant proportion of tumorous swellings—pseudotumors—in many organ systems. Some patients undergo major surgeries (e.g., Whipple procedures or thyroidectomy) for the purpose of resecting malignancies before the correct diagnosis is identified. Frequent sites of pseudotumors are the major salivary glands, lacrimal glands, lungs, and kidneys; however, nearly all organs have been affected with this manifestation. FIgURE 391e-1 A major clinical feature of IgG4-related disease is its tendency to form tumefactive lesions. Shown here are mass lesions of the lacrimal glands (A) and the submandibular glands (B).

1	IgG4-RD often causes major morbidity and can lead to organ failure; however, its general pattern is to cause damage in a subacute manner. Destructive bone lesions in the sinuses, head, and middle ear spaces that mimic granulomatous polyangiitis (formerly Wegener’s granulomatosis) also occur in IgG4-RD; less aggressive lesions are the rule in most organs. In regions such as the retroperitoneum, substantial fibrosis often occurs before the diagnosis is established, leading to ureteral entrapment, hydronephrosis, postobstructive uropathy, renal atrophy, and chronic pain, possibly resulting from the encasement of peripheral nerves by the inflammatory process. Undiagnosed or undertreated IgG4-related cholangitis can lead to hepatic failure within months. Similarly, IgG4-related aortitis, believed to be associated with between 10 and 50% of cases of inflammatory aortitis, can cause aneurysms and dissections. Substantial renal dysfunction and even renal failure can ensue from IgG4-related

1	to be associated with between 10 and 50% of cases of inflammatory aortitis, can cause aneurysms and dissections. Substantial renal dysfunction and even renal failure can ensue from IgG4-related tubulointerstitial nephritis, and renal atrophy is a frequent sequel to this disease complication.

1	The majority of patients with IgG4-RD have elevated serum IgG4 concentrations; however, the range of elevation varies widely. Serum concentrations of IgG4 as high as 30 or 40 times the upper limit of normal sometimes occur, usually in patients with disease that affects multiple organ systems simultaneously. Approximately 30% of patients have normal serum IgG4 concentrations despite classic histopathologic and immunohistochemical findings. Such patients tend to have disease that affects fewer organs. Patients with IgG4-related retroperitoneal fibrosis have a high likelihood of normal serum IgG4 concentrations, perhaps because the process has advanced to a fibrotic stage by the time the diagnosis is considered.

1	The correlation between serum IgG4 concentrations and disease activity and the need for treatment is imperfect. Serum IgG4 concentrations typically decline swiftly with the institution of therapy but often do not normalize completely. Patients can achieve clinical remissions yet have persistently elevated serum IgG4 concentrations. Rapidly rising serum IgG4 concentrations may identify patients at greatest risk for clinical flares and monitoring of serial IgG4 concentrations identifies early relapse in some patients; however, the temporal relationship between modest IgG4 elevations and the need for clinical treatment is poor. Clinical relapses occur in some patients despite persistently normal IgG4 concentrations.

1	IgG4 concentrations in serum are usually measured by nephelometry assays. These assays can lead to reports of spuriously low IgG4 values because of the prozone effect. This effect can be corrected by dilution of the serum sample in the laboratory. The prozone effect should be considered when the results of serologic testing for IgG4 concentrations appear to be at odds with the patient’s clinical features. The typical patient with IgG4-RD is a middle-aged to elderly man. This epidemiology stands in stark contrast to that of many classic autoimmune conditions, which tend to affect young women. Studies of AIP patients in Japan indicate that the male-to-female ratio in that disease subset is on the order of 3:1. Even more striking, male predominance has been reported in IgG4-related tubulointerstitial nephritis and IgG4related retroperitoneal fibrosis. Among IgG4-RD manifestations that involve organs of the head and neck, the sex ratio may be closer to 1:1.

1	The key histopathology characteristics of IgG4-RD are a dense lymphoplasmacytic infiltrate (Fig. 391e-2) that is organized in a storiform pattern (resembling a basket-weave), obliterative phlebitis, and a mild to moderate eosinophilic infiltrate. Lymphoid follicles and germinal centers are frequently observed. The infiltrate tends to aggregate around ductal structures when it affects glands such as the lacrimal, submandibular, and parotid glands or the pancreas. The inflammatory lesion often aggregates into tumefactive masses that destroy the involved tissue. Obliterative arteritis is observed in some organs, particularly the lung; however, venous involvement is more common (and is indeed

1	Obliterative arteritis is observed in some organs, particularly the lung; however, venous involvement is more common (and is indeed FIgURE 391e-2 Hallmark histopathology characteristics of IgG4related disease (IgG4-RD) are a dense lymphoplasmacytic infiltrate and a mild to moderate eosinophilic infiltrate. The cellular inflammation is often encased in a distinctive type of fibrosis termed “storiform,” which often has a basket weave pattern. Abundant fibroblasts and strands of fibrosis accompany the lymphoplasmacytic infiltrate and eosinophils in this figure. This biopsy was taken from a nodular lesion on the cheek; however, the findings are identical to the pathology found in the pancreas, kidneys, lungs, salivary glands, and other organs affected by IgG4-RD.

1	a hallmark of IgG4-RD). Several histopathology features are uncommon in IgG4-RD and, when detected, mitigate against the diagnosis of IgG4-RD. These include intense neutrophilic infiltration, leukocytoclasis, granulomatous inflammation, multinucleated giant cells, and fibrinoid necrosis. The inflammatory infiltrate is composed of an admixture of B and T lymphocytes. B cells are typically organized in germinal centers. Plasma cells staining for CD19, CD138, and IgG4 appear to radiate out from the germinal centers. In contrast, the T cells, usually CD4+, are distributed more diffusely throughout the lesion and generally represent the most abundant cell type. Fibroblasts, histiocytes, and eosinophils can all be observed in moderate numbers. Some biopsy samples are particularly enriched with eosinophils. In other samples, particularly from long-standing cases, fibrosis predominates.

1	The histologic appearance of IgG4-RD, although highly characteristic, requires immunohistochemical confirmation of the diagnosis with IgG4 immunostaining. IgG4-positive plasma cells predominate within the lesion, but plasma cells containing immunoglobulins from each subclass can be found. The number of IgG4-positive plasma cells can be quantified by either counting the number of cells per high-power field (HPF) or by calculating the ratio of IgG4to IgG-bearing plasma cells. Tissue fibrosis predominates in the latter phases of organ involvement, and in this relatively acellular phase of inflammation, both the IgG4:total IgG ratio and the pattern of tissue fibrosis are more important than the number of IgG4-positive cells per HPF in establishing the diagnosis. In situ hybridization techniques are also now used to circumvent problems posed by increased background staining in conventional immunostaining techniques.

1	The IgG4 molecule is believed to play an indirect role in the pathophysiology of disease in most organs. However, the molecule has properties that are unique among the immunoglobulin subclasses and that may contribute to tissue injury in some circumstances. As an example, IgG4 molecules have the ability to undergo Fab exchange, a phenomenon in which the two halves of the molecule dissociate from each other and reassociate with dissimilar hemi-molecules from other IgG4 molecules. This property is unique among the immunoglobulin subclasses. Partly as a result of Fab exchange, however, IgG4 antibodies bind antigen loosely. The molecules have low affinities for Fc receptors and C1q and are regarded generally as noninflammatory immunoglobulins. The low affinities for Fc receptors and C1q impair the ability of IgG4 antibodies to induce phagocyte activation, antibody-dependent cellular cytotoxicity, and complement-mediated damage. It is possible that the increased concentrations of IgG4 in

1	the ability of IgG4 antibodies to induce phagocyte activation, antibody-dependent cellular cytotoxicity, and complement-mediated damage. It is possible that the increased concentrations of IgG4 in serum and IgG4-bearing plasma cells in tissue are merely the result of other effector pathways, such as TH2/Treg cytokines, that are more central to the inflammation and tissue damage.

1	Not every disease manifestation of IgG4-RD requires immediate treatment because the disease takes an indolent form in many patients. IgG4-related lymphadenopathy, for example, can be asymptomatic for years, without evolution to other disease manifestations. Thus, watchful waiting is prudent in some cases. Vital organ involvement must be treated aggressively, however, because IgG4-RD can lead to serious organ dysfunction and failure. Aggressive disease can lead quickly to end-stage liver disease, permanent impairment of pancreatic function, renal atrophy, aortic dissection or aneurysms, and destructive lesions in the sinuses and nasopharynx.

1	Glucocorticoids are the first line of therapy. Treatment regimens, extrapolated from experience with the management of type 1 AIP, generally begin with 40 mg/d of prednisone, with tapering to discontinuation or maintenance doses of 5 mg/d within 2 or 3 months. The clinical response to glucocorticoids is usually swift and striking; however, longitudinal data indicate that disease flares occur in more than 90% of patients within 3 years. Conventional steroid-sparing agents such as azathioprine and mycophenolate mofetil have been used in some patients; however, evidence for their efficacy is lacking.

1	For patients with relapsing or glucocorticoid-resistant disease, B cell depletion with rituximab is an excellent second-line therapy. Rituximab treatment (two doses of 1 g IV, separated by approximately 15 days) leads to a targeted, precipitous decline in serum IgG4 concentrations, suggesting that rituximab achieves its effects in part by preventing the repletion of short-lived plasma cells that produce IgG4. More important than its effects on IgG4 concentrations, however, may be the effect of B cell depletion on T cell function. Specific effects of rituximab on CD4+ effector T cells have been documented in IgG4-RD. Rituximab may be an appropriate first-line therapy for some patients, particularly those at high risk for glucocorticoid toxicity and patients with immediately organ-threatening disease. The optimal approaches to remission maintenance, by either re-treatment with rituximab or continuous low-dose glucocorticoid therapy, require further study.

1	Immune-Mediated, Inflammatory, and Rheumatologic Disorders familial Mediterranean fever and Other hereditary autoinflammatory Diseases Daniel L. Kastner FamilialMediterraneanfever(FMF)istheprototypeofagroupof392 inherited diseases (Table 392-1) that are characterized by recurrent episodes of fever with serosal, synovial, or cutaneous inflammation and, in some individuals, the eventual development of systemic AA amyloidosis (Chap. 137). Because of the relative infrequency of high-titer autoantibodies or antigen-specific T cells, the term autoinflammatory has been proposed to describe these disorders, rather than autoimmune. The innate immune system, with its myeloid effector cells and germline receptors for pathogen-associated molecular patterns and endogenous danger signals, plays a predominant role in the pathogenesis of the autoinflammatory diseases. Although the hereditary recurrent fevers comprise a major category of the autoinflammatory diseases, other inherited disorders of

1	role in the pathogenesis of the autoinflammatory diseases. Although the hereditary recurrent fevers comprise a major category of the autoinflammatory diseases, other inherited disorders of inflammation in which recurrent fever plays a less prominent role are now also considered to be autoinflammatory.

1	FMF was first recognized among Armenians, Arabs, Turks, and non-Ashkenazi (primarily North African and Iraqi) Jews. With the advent of genetic testing, FMF has been documented with increasing frequency among Ashkenazi Jews, Italians, and other Mediterranean populations, and occasional cases have been confirmed even in the absence of known Mediterranean ancestry. FMF is generally regarded as recessively inherited, but there is an increasing awareness of clearcut clinical cases with only a single demonstrable genetic mutation, and, for certain relatively rare FMF mutations, there is strong evidence for dominant inheritance. Particularly in countries where families are small, a positive family history can only be elicited in ~50% of cases. DNA testing demonstrates carrier frequencies as high as 1:3 among affected populations, suggesting a heterozygote advantage.

1	The FMF gene encodes a 781-amino acid, ~95 kDa protein denoted pyrin (or marenostrin) that is expressed in granulocytes, eosinophils, monocytes, dendritic cells, and synovial and peritoneal fibroblasts. The N-terminal 92 amino acids of pyrin define a motif, the PYRIN domain, that is similar in structure to death domains, death effector aA substantial percentage of patients with clinical FMF have only a single demonstrable MEFV mutation on DNA sequencing. Abbreviations: FCAS, familial cold autoinflammatory syndrome; FMF, familial Mediterranean fever; HIDS, hyperimmunoglobulinemia D with periodic fever syndrome; IL, interleukin; MWS, Muckle-Wells syndrome; NOMID, neonatal-onset multisystem inflammatory disease; NSAIDs, nonsteroidal anti-inflammatories; TNF, tumor necrosis factor; TRAPS, TNF receptor-associated periodic syndrome.

1	domains, and caspase recruitment domains. PYRIN domains medi-Over 90% of FMF patients experience abdominal attacks at some ate homotypic protein-protein interactions and have been found in time. Episodes range in severity from dull, aching pain and distention severalotherproteins,includingcryopyrin(NLRP3),whichismutated withmildtenderness ondirectpalpation toseveregeneralizedpain in three other recurrent fever syndromes. Through a number of with absent bowel sounds, rigidity, rebound tenderness, and air-fluid mechanisms, including the interaction of the PYRIN domain with an levels on upright radiographs. Computed tomography (CT) scanning intermediary adaptor protein, pyrin regulates caspase-1 (interleukin may demonstrate a small amount of fluid in the abdominal cavity. If [IL]1β-convertingenzyme),andtherebyIL-1βsecretion.Micebearing such patients undergo exploratory laparotomy, a sterile, neutrophilFMF-associated pyrin mutations exhibit inflammation and excessive rich peritoneal

1	such patients undergo exploratory laparotomy, a sterile, neutrophilFMF-associated pyrin mutations exhibit inflammation and excessive rich peritoneal exudate is present, sometimes with adhesions from IL-1 production. previous episodes. Ascites is rare.

1	Pleural attacks are usually manifested by unilateral, sharp, stabbing chest pain. Radiographs may show atelectasis and sometimes an effusion. If performed, thoracentesis demonstrates an exudative Febrile episodes in FMF may begin even in early infancy; 90% of fluid rich in neutrophils. After repeated attacks, pleural thickening patients have had their first attack by age 20. Typical FMF episodes may develop. generally last 24–72 h, with arthritic attacks tending to last somewhat FMF arthritis is most frequent among individuals homozygous for longer. In some patients, the episodes occur with great regularity, but the M694V mutation, which is especially common in the non-Ashkemore often, the frequency of attacks varies over time, ranging from as nazi Jewish population. Acute arthritis in FMF is usually monoarticuoftenasonceeveryfewdaystoremissionslastingseveralyears.Attacks lar, affecting the knee, ankle, or hip, although other patterns can be are often unpredictable, although some

1	FMF is usually monoarticuoftenasonceeveryfewdaystoremissionslastingseveralyears.Attacks lar, affecting the knee, ankle, or hip, although other patterns can be are often unpredictable, although some patients relate them to physi-seen, particularly inchildren. Largesterileeffusionsrichin neutrophils cal exertion, emotional stress, or menses; pregnancy may be associated are frequent, without commensurate erythema or warmth. Even after with remission. repeated arthritic attacks, radiographic changes are rare. Before the

1	If measured, fever is nearly always present throughout FMF attacks. advent of colchicine prophylaxis, chronic arthritis of the knee or hip Severehyperpyrexiaand evenfebrileseizuresmaybe seenininfants, wasseenin~5%ofFMFpatientswitharthritis.Chronicsacroiliitiscan andfeverissometimestheonlymanifestationofFMFinyoungchildren. occurinFMFirrespectiveoftheHLA-B27antigen,eveninthefaceof 2214 colchicine therapy. In the United States, FMF patients are much more likely to have arthralgia than arthritis. The most characteristic cutaneous manifestation of FMF is erysipelas-like erythema, a raised erythematous rash that most commonly occurs on the dorsum of the foot, ankle, or lower leg alone or in combination with abdominal pain, pleurisy, or arthritis. Biopsy demonstrates perivascular infiltrates of granulocytes and monocytes. This rash is seen most often in M694V homozygotes and is relatively rare in the United States. Exercise-induced (nonfebrile) myalgia is common in FMF, and a small

1	of granulocytes and monocytes. This rash is seen most often in M694V homozygotes and is relatively rare in the United States. Exercise-induced (nonfebrile) myalgia is common in FMF, and a small percentage of patients develop a protracted febrile myalgia that can last several weeks. Symptomatic pericardial disease is rare, although some patients have small pericardial effusions as an incidental echocardiographic finding. Unilateral acute scrotal inflammation may occur in prepubertal boys. Aseptic meningitis has been reported in FMF, but the causal connection is controversial. Vasculitis, including Henoch-Schönlein purpura and polyarteritis nodosa (Chap. 385), may be seen at increased frequency in FMF. The M694V FMF mutation has recently been shown to be a risk factor for Behçet’s disease. Laboratory features of FMF attacks are consistent with acute inflammation and include an elevated erythrocyte sedimentation rate, leukocytosis, thrombocytosis (in children), and elevations in

1	Laboratory features of FMF attacks are consistent with acute inflammation and include an elevated erythrocyte sedimentation rate, leukocytosis, thrombocytosis (in children), and elevations in C-reactiveprotein,fibrinogen,haptoglobin,andserumimmunoglobulins. Transient albuminuria and hematuria may also be seen.

1	Before the advent of colchicine prophylaxis, systemic amyloidosis was a common complication of FMF. It is caused by deposition of a fragment of serum amyloid A, an acute-phase reactant, in the kidneys, adrenals, intestine, spleen, lung, and testes (Chap. 137). Amyloidosis should be suspected in patients who have proteinuria between attacks; renal or rectal biopsy is used most often to establish the diagnosis. Risk factors include the M694V homozygous genotype, positive family history (independent of FMF mutational status), the SAA 1 genotype, male gender, noncompliance with colchicine therapy, and having grown up in the Middle East.

1	For typical cases, physicians experienced with FMF can often make the diagnosis on clinical grounds alone. Clinical criteria sets for FMF have been shown to have high sensitivity and specificity in parts of the world where the pretest probability of FMF is high. Genetic testing can provide a useful adjunct in ambiguous cases or for physicians not experienced in FMF. Most of the more severe disease-associated FMF mutations are in exon 10 of the gene, with a smaller group of milder variants in exon 2. An updated list of mutations for FMF and other hereditary recurrent fevers can be found online at http://fmf.igh.cnrs .fr/infevers/.

1	Genetic testing has permitted a broadening of the clinical spectrum and geographic distribution of FMF and may be of prognostic value. Most studies indicate that M694V homozygotes have an earlier age of onset and a higher frequency of arthritis, rash, and amyloidosis. In contrast, the E148Q variant is quite common in certain populations and is more likely to affect overall levels of inflammation than to cause clinical FMF. E148Q is sometimes found in cis with exon 10 mutations, which may complicate the interpretation of genetic test results. Only~70%ofpatientswithclinicallytypicalFMFhavetwoidentifiable mutations in trans. The inability to identify a second mutation even after intensive molecular analysis suggests that one FMF mutation may be sufficient to cause disease under some circumstances. In these cases clinical judgment is very important, and sometimes a therapeutic trial ofcolchicinemay help to confirm thediagnosis. Genetic testing of unaffected individuals is usually

1	circumstances. In these cases clinical judgment is very important, and sometimes a therapeutic trial ofcolchicinemay help to confirm thediagnosis. Genetic testing of unaffected individuals is usually inadvisable, because of the possibility of nonpenetrance and the potential impact of a positive test on future insurability.

1	Ifa patientisseenduringhisor herfirstattack,the differential diagnosis may be broad, although delimited by the specific organ involvement. After several attacks the differential diagnosis may include the other hereditary recurrent fever syndromes (Table 392-1); the syndrome of periodic fever with aphthous ulcers, pharyngitis, and cervical adenopathy (PFAPA); systemic-onset juvenile rheumatoid arthritis or adult Still’s disease; porphyria; hereditary angioedema; inflammatory bowel disease; and, in women, gynecologic disorders.

1	The treatment of choice for FMF is daily oral colchicine, which decreases the frequency and intensity of attacks and prevents the development of amyloidosis in compliant patients. Intermittent dosing at the onset of attacks is not as effective as daily prophylaxis and is of unproven value in preventing amyloidosis. The usual adult dose of colchicine is 1.2–1.8 mg/d, which causes substantial reduction in symptoms in two-thirds of patients and some improvement in >90%. Children may require lower doses, although not proportionately to body weight.

1	Common side effects of colchicine include bloating, abdominal cramps, lactose intolerance, and diarrhea. They can be minimized by starting at a low dose and gradually advancing as tolerated, splitting the dose, use of simethicone for flatulence, and avoidance of dairy products. If taken by either parent at the time of conception, colchicine may cause a small increase in the risk of trisomy 21 (Down’s syndrome). In elderly patients with renal insufficiency, colchicine can cause a myoneuropathy characterized by proximal muscle weakness and elevation of the creatine kinase. Cyclosporine inhibits hepatic excretion of colchicine by its effects on the MDR-1 transport system, sometimes leading to colchicine toxicity in patients who have undergone renal transplantation for amyloidosis. Intravenous colchicine should generally not be administered to patients already taking oral colchicine, because severe, sometimes fatal, toxicity can occur in this setting.

1	For FMF patients who do not respond to colchicine or cannot tolerate therapeutic doses, injectable IL-1 inhibitors appear to be effective in preventing the acute attacks. In a small randomized placebo-controlled trial, weekly subcutaneous rilonacept, a recombinant IL-1 receptor fusion protein, significantly reduced the frequency of attacks. There is also substantial anecdotal experience with daily subcutaneous anakinra, a recombinant IL-1 receptor antagonist, in preventing the acute attacks of FMF, and in some cases reducing established amyloid deposits. Canakinumab, a monoclonal antibody to IL-1β, and tumor necrosis factor (TNF) inhibitors may also have a role in the treatment of colchicine-unresponsive or intolerant patients. Bone marrow transplantation has been suggested for refractory FMF, but the risk-benefit ratio is currently regarded as unacceptable.

1	Within 5 years of the discovery of the FMF gene, three additional genes causing five other hereditary recurrent fever syndromes were identified, catalyzing a paradigm shift in diagnosis and treatment of these disorders.

1	TRAPS is caused by dominantly inherited mutations in the extracellular domains of the 55-kDa TNF receptor (TNFR1, p55). Although originallydescribedinalargeIrishfamily(andhencethenamefamilial Hibernian fever), TRAPS has a broad ethnic distribution. TRAPS episodes often begin in childhood. The duration of attacks ranges from 1–2 days to as long as several weeks, and in severe cases symptoms maybe nearly continuous. Inadditionto peritoneal, pleural,and synovial attacks similar to FMF, TRAPS patients frequently have ocular inflammation (most often conjunctivitis and/or periorbital edema), and a distinctive migratory myalgia with overlying painful erythema may be present. TRAPS patients generally respond better to glucocorticoids than to prophylactic colchicine. Untreated, about 15% develop amyloidosis. The diagnosis of TRAPS is based on the demonstration of TNFRSF1A mutations in the presence of characteristic symptoms. Two particular variants, R92Q and P46L, are common in certain

1	amyloidosis. The diagnosis of TRAPS is based on the demonstration of TNFRSF1A mutations in the presence of characteristic symptoms. Two particular variants, R92Q and P46L, are common in certain populations and may act more as functional polymorphisms than as disease-causing mutations. In contrast, pathogenic TNFRSF1A mutations, including a number of substitutions at highly conserved cysteine residues, are associated with intracellular TNFR1 misfolding, aggregation, and retention, with consequent ligand-independent kinase activation,mitochondrialreactiveoxygenspeciesproduction,andproinflammatorycytokinerelease.Etanercept,aTNFinhibitor,ameliorates TRAPS attacks, but the long-term experience with this agent has been less favorable. Perhaps because of the ligand-independent signaling abnormalities in TRAPS, IL-1 inhibition has been beneficial in a large percentage of the patients in whom it has been used. Monoclonal antiTNFantibodiesshouldbeavoided,becausetheymayexacerbateTRAPS attacks.

1	HIDS is a recessively inherited recurrent fever syndrome found primarily in individuals of northern European ancestry. It is caused by mutations in mevalonate kinase (MVK), encoding an enzyme involved in the synthesis of cholesterol and nonsterol isoprenoids. Attacks usually begin in infancy and last 3–5 days. Clinically distinctive features include painful cervical adenopathy, a diffuse maculopapular rash sometimes affecting the palms and soles, and aphthous ulcers; pleurisy is rare, as is amyloidosis. Although originally defined by the persistent elevation of serum IgD, disease activity is not related to IgD levels, and some patients with FMF or TRAPS may have modestly increased serum IgD. Moreover, occasional patients with MVK mutations and recurrent fever have normal IgD levels. For these reasons, some have proposed renaming this disorder mevalonate kinase deficiency (MKD). All patients with mutations have markedly elevated urinary mevalonatelevels duringtheir febrileattacks,

1	these reasons, some have proposed renaming this disorder mevalonate kinase deficiency (MKD). All patients with mutations have markedly elevated urinary mevalonatelevels duringtheir febrileattacks, althoughtheinflammatorymanifestations are likely to be due to a deficiency of isoprenoids rather than an excess of mevalonate. There is currently no established treatment for HIDS/MKD, although intermittent or continuous IL-1 inhibition and TNF inhibitors have been effective in small series.

1	THE CrYOPYrINOPaTHIES, Or CrYOPYrIN-aSSOCIaTED PErIODIC SYNDrOMES (CaPS)

1	Three hereditary febrile syndromes, familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID), are all caused by mutations in NLRP3 (formerly known as CIAS1), the gene encoding cryopyrin (or NLRP3), and represent a clinical spectrum of disease. FCASpatientsdevelopchills,fever,headache,arthralgia,conjunctivitis, and an urticaria-like rash in response to generalized cold exposure. In MWS, an urticarial rash is noted, but it is not usually induced by cold; MWSpatientsalsodevelopfevers,abdominalpain,limbpain,arthritis, conjunctivitis, and, over time, sensorineural hearing loss. NOMID is the most severe of the three disorders, with chronic aseptic meningitis, a characteristic arthropathy, and rash. Like the FMF protein, pyrin, cryopyrin has an N-terminal PYRIN domain. Cryopyrin regulates IL-1βproductionthroughtheformationofamacromolecularcomplex 2215 termed the inflammasome. Peripheral blood leukocytes from

1	protein, pyrin, cryopyrin has an N-terminal PYRIN domain. Cryopyrin regulates IL-1βproductionthroughtheformationofamacromolecularcomplex 2215 termed the inflammasome. Peripheral blood leukocytes from patients with FCAS, MWS, and NOMID release increased amounts of IL-1β upon in vitro stimulation, relative to healthy controls. Macrophages from cryopyrin-deficient mice exhibit decreased IL-1β production in response to certain gram-positive bacteria, bacterial RNA, and mono-sodium urate crystals. Patients with all three cryopyrinopathies show a dramatic response to injections of IL-1 inhibitors. Approximately one-third of patients with clinical manifestations of NOMID do not have germline mutations in NLRP3, but have been found to be mosaic for somatic NLRP3 mutations. Such patients also respond dramatically to IL-1 inhibition. Similarly, somatic mosaicism in NLRP3 has been found in Schnitzler’s syndrome, which presents in middle age with recurrent fever, urticarial rash, elevated acute

1	dramatically to IL-1 inhibition. Similarly, somatic mosaicism in NLRP3 has been found in Schnitzler’s syndrome, which presents in middle age with recurrent fever, urticarial rash, elevated acute phase reactants, monoclonal IgM gammopathy, and abnormal bone remodeling. IL-1 inhibition is the treatment of choice for Schnitzler’s syndrome.

1	There are a number of other Mendelian autoinflammatory diseases in which recurrent fevers are not a prominent clinical sign but that involve abnormalities of innate immunity. The syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA) is a dominantly inherited disorder that presents with episodes of sterile pyogenic monoarthritis often induced by trauma, severe pyoderma gangrenosum, and severe cystic acne, usually beginning in puberty. It is caused by mutations in PSTPIP1, which encodes a pyrin-binding protein, and the arthritic manifestations often respond to IL-1 inhibition. Patients with the recessively inherited deficiency of the IL-1 receptor antagonist (DIRA) present with a generalized pustular rash and multifocal sterile osteomyelitis, and show dramatic clinical responses to anakinra, the recombinant form of the protein they lack. IL-36 is another member of the IL-1 family of cytokines that is regulated by an endogenous receptor antagonist. The recessively

1	responses to anakinra, the recombinant form of the protein they lack. IL-36 is another member of the IL-1 family of cytokines that is regulated by an endogenous receptor antagonist. The recessively inherited deficiency of the IL-36 receptor antagonist (DITRA) presents with episodes of generalized pustular psoriasis and dramatic systemic inflammation.

1	Whereas PAPA, DIRA, and DITRA all involve mutations in IL-1related molecules, other autoinflammatory diseases are caused by mutations in other components of innate immunity. Blau’s syndrome is caused by mutations in CARD15 (also known as NOD2), which regulates nuclear factor-κB activation. Blau’s syndrome is characterized by granulomatous dermatitis, uveitis, and arthritis; distinct CARD15 variants predispose to Crohn’s disease. Recessive mutations in one or more components of the proteasome lead to excessive interferon signaling and the syndrome of chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), a severe form of generalized panniculitis. De novo gain-of-function mutations in TMEM173, encoding the stimulator of interferon genes (STING), cause severe vasculopathy and pulmonary fibrosis. Recessive loss-of-function mutations in CERCR1, encoding adenosine deaminase 2 (ADA2),causeavasculopathythatcanmanifestas livedoidrash,earlyonset lacunar

1	severe vasculopathy and pulmonary fibrosis. Recessive loss-of-function mutations in CERCR1, encoding adenosine deaminase 2 (ADA2),causeavasculopathythatcanmanifestas livedoidrash,earlyonset lacunar strokes, or polyarteritis nodosa.

1	Finally, it should be noted that a number of common, genetically complex disorders are now sometimes considered autoinflammatory, because of evidence that components of the innate immune system, such as the inflammasome, may play a role in the pathogenesis. Two prominent examples are gout and atherosclerosis. Large clinical trials of IL-1 inhibitors have been initiated in both conditions. 2216 approach to articular and Musculoskeletal Disorders John J. Cush Musculoskeletalcomplaintsaccountfor>315millionoutpatientvisitsperyearandover20%ofalloutpatientvisitsintheUnited393 SeC tIOn 3

1	States. The Centers for Disease Control and Prevention estimate that 22.7% (52.5 million) of the U.S. population has physician-diagnosed arthritis and 22 million have significant functional limitation. While many patients will have self-limited conditions requiring minimal evaluation and only symptomatic therapy and reassurance, specific musculoskeletal presentations or their persistence may herald a more serious condition that requires further evaluation or laboratory testing to establish a diagnosis. The goal of the musculoskeletal evaluation is to formulate a differential diagnosis that leads to an accurate diagnosis and timely therapy, while avoiding excessive diagnostic testing and unnecessary treatment (Table 393-1). There are several urgent conditions that must be diagnosed promptly to avoid significant morbid or mortal sequelae. These “red flag” diagnoses include septic arthritis, acute crystal-induced arthritis (e.g., gout), and fracture. Each may be suspected by its acute

1	to avoid significant morbid or mortal sequelae. These “red flag” diagnoses include septic arthritis, acute crystal-induced arthritis (e.g., gout), and fracture. Each may be suspected by its acute onset and monarticular or focal musculoskeletal pain (see below).

1	Despite well-known links between certain disorders and laboratory testing, the majority of individuals with musculoskeletal complaints can be diagnosed with a thorough history and a comprehensive physical and musculoskeletal examination. The initial encounter should determine whether the musculoskeletal complaint signals a red flag condition (septic arthritis, gout, or fracture) or not. The evaluation should proceed to ascertain if the complaint is (1) articular or non-articular in origin, (2) inflammatory or noninflammatory in nature, (3) acute or chronic in duration, and (4) localized (monarticular) or widespread (polyarticular) in distribution.

1	With such an approach and an understanding of the pathophysiologic processes, the musculoskeletal complaint or presentation can be characterized (e.g., acute inflammatory monarthritis or a chronic noninflammatory, nonarticular widespread pain) to narrow the diagnostic possibilities. A diagnosis can be made in the vast majority of individuals. However, some patients will not fit immediately into an established diagnostic category. Many musculoskeletal disorders resemble each other at the outset, andsome may take weeks or months (but not years) to evolve into a recognizable diagnostic entity. This consideration should temper the desire to establish a definitive diagnosis at the first encounter. Timely provision of therapy Avoidance of unnecessary diagnostic testing Identification of acute, focal/monarticular “red flag” conditions Determination of chronology (acute vs chronic) Determination of the nature of the pathologic process (inflammatory vs noninflammatory)

1	Determination of chronology (acute vs chronic) Determination of the nature of the pathologic process (inflammatory vs noninflammatory) Determination of the extent of involvement (monarticular, polyarticular, focal, widespread) Anatomic localization of complaint (articular vs nonarticular) Consider the most common disorders first

1	The musculoskeletal evaluation must discriminate the anatomic origin(s) of the patient’s complaint. For example, ankle pain can result from a variety of pathologic conditions involving disparate anatomic structures, including gonococcal arthritis, calcaneal fracture, Achilles tendinitis, plantar fasciitis, cellulitis, and peripheral or entrapment neuropathy. Distinguishing between articular and nonarticular conditions requires a careful and detailed examination. Articular structures include the synovium, synovial fluid, articular cartilage, intraarticular ligaments, joint capsule, and juxtaarticular bone. Nonarticular (or periarticular) structures, such as supportive extraarticular ligaments, tendons, bursae, muscle, fascia, bone, nerve, and overlying skin, may be involved in the pathologic process. Although musculoskeletal complaints are often ascribed to the joints, nonarticular disorders more frequently underlie such complaints. Distinguishing between these potential sources of

1	process. Although musculoskeletal complaints are often ascribed to the joints, nonarticular disorders more frequently underlie such complaints. Distinguishing between these potential sources of pain may be challenging to the unskilled examiner. Articular disorders may be characterized by deep or diffuse pain, pain or limited range of motion on active and passive movement, and swelling (caused by synovial proliferation, effusion, or bony enlargement), crepitation, instability, “locking,” or deformity. By contrast, nonarticular disorders tend to be painful on active, but not passive (or assisted), range of motion. Periarticular conditions often demonstrate point orfocaltenderness in regionsadjacentto articular structures,are elicited with a specific movement or position, and have physical findings remote from the joint capsule. Moreover, nonarticular disorders seldom demonstrate swelling, crepitus, instability, or deformity of the joint itself.

1	In the course of a musculoskeletal evaluation, the examiner should determinethenatureoftheunderlyingpathologicprocessandwhether inflammatory or noninflammatory findings exist. Inflammatory disorders may be infectious (Neisseria gonorrhoeae or Mycobacterium tuberculosis), crystal-induced (gout, pseudogout), immune-related (rheumatoidarthritis[RA],systemiclupuserythematosus[SLE]),reactive (rheumatic fever, reactive arthritis), or idiopathic. Inflammatory disorders may be identified by any of the four cardinal signs of inflammation (erythema, warmth, pain, or swelling), systemic symptoms (fatigue, fever, rash, weight loss), or laboratory evidence of inflammation (elevated erythrocyte sedimentation rate [ESR] or C-reactive protein [CRP], thrombocytosis, anemia of chronic disease, or hypoalbuminemia). Articular stiffness commonly accompanies chronic musculoskeletaldisorders. Thedurationofstiffnessmaybeprolonged (hours) with inflammatory disorders (such as RA or polymyalgia rheumatica) and

1	Articular stiffness commonly accompanies chronic musculoskeletaldisorders. Thedurationofstiffnessmaybeprolonged (hours) with inflammatory disorders (such as RA or polymyalgia rheumatica) and may improve with activity. By contrast, intermittent stiffness (also known as gel phenomenon) is typical of noninflammatory conditions (such as osteoarthritis [OA]), shorter in duration (<60 min), and exacerbated by activity. Fatigue may accompany inflammation (as seen in RA and polymyalgia rheumatica) but may also be a consequence of fibromyalgia (a noninflammatory disorder), chronic pain, poor sleep, depression, anemia, cardiac failure, endocrinopathy, or malnutrition. Noninflammatory disorders may be related to trauma (rotator cuff tear), repetitive use (bursitis, tendinitis), degeneration or ineffective repair (OA), neoplasm (pigmented villonodular synovitis), or pain amplification (fibromyalgia). Noninflammatory disorders are often characterized by pain without synovial swelling or warmth,

1	repair (OA), neoplasm (pigmented villonodular synovitis), or pain amplification (fibromyalgia). Noninflammatory disorders are often characterized by pain without synovial swelling or warmth, absence of inflammatory or systemic features, daytime gel phenomena rather than morning stiffness, and normal (for age) or negative laboratory investigations.

1	Identification of the nature of the underlying process and the site of the complaint will enable the examiner to characterize the musculoskeletal presentation (e.g., acute inflammatory monarthritis, chronic noninflammatory, nonarticular widespread pain), narrow the diagnostic considerations, and assess the need for immediate diagnostic or Initial rheumatic history and physical exam to determine 1. Is it articular? 2. Is it acute or chronic? 3. Is inflammation present? 4. How many/which joints are involved?

1	Is it articular?Nonarticular condition Consider • Trauma/fracture • Fibromyalgia • Polymyalgia rheumatica • Bursitis • Tendinitis Is complaint > 6 wk? Acute Chronic Consider • Acute arthritis• Infectious arthritis • Gout • Pseudogout • Reactive arthritis • Initial presentation of chronic arthritis Chronic inflammatory arthritis How many joints involved? Are DIP, CMC1, hip or knee joints involved? Chronic inflammatory mono/oligoarthritis Consider • Indolent infection • Psoriatic arthritis • Reactive arthritis • Pauciarticular JIA Chronic inflammatory polyarthritis Is involvement symmetric? Are PIP, MCP, or MTP joints involved? Consider • Psoriatic arthritis • Reactive arthritis Rheumatoid arthritis Osteoarthritis No Yes Yes No No Yes No Yes No Yes No Yes >31– 3 Unlikely to be osteoarthritis Consider • Osteonecrosis • Charcot arthritis Chronic noninflammatory arthritis Unlikely to be rheumatoid arthritis Consider • SLE • Scleroderma • Polymyositis FIGUrE 393-1 Algorithm for the

1	Consider • Osteonecrosis • Charcot arthritis Chronic noninflammatory arthritis Unlikely to be rheumatoid arthritis Consider • SLE • Scleroderma • Polymyositis FIGUrE 393-1 Algorithm for the diagnosis of musculoskeletal complaints. Anapproachtoformulatingadifferentialdiagnosis(showninitalics).CMC,carpometacarpal;CRP,C-reactiveprotein;DIP,distalinterphalangeal;ESR,erythrocytesedimentationrate;JIA,juvenileidiopathicarthritis;MCP,metacarpophalangeal;MTP,metatarsophalangeal;PIP,proximalinterphalangeal;PMR,polymyalgiarheumatica;SLE,systemiclupuserythematosus.

1	Is inflammation present? 1. Is there prolonged morning stiffness? 2. Is there soft tissue swelling? 3. Are there systemic symptoms? 4. Is the ESR or CRP elevated? Approach to Articular and Musculoskeletal Disorders therapeutic intervention or for continued observation. Figure 393-1 presents an algorithmic approach to the evaluation of patients with musculoskeletal complaints. This approach relies on clinical and historic features, rather than laboratory testing, to diagnose many common rheumatic disorders.

1	Asimpler,alternativeapproachwouldconsiderthemostcommonly encountered complaints first, based on frequency in younger versus older populations. The most prevalent causes of musculoskeletal complaints are shown in Fig. 393-2. Because trauma, fracture, overuse syndromes, and fibromyalgia are among the most common causes of joint pain, these should be considered during the initial encounter. If these possibilities are excluded, other frequently occurring disorders should be considered according to the patient’s age. Hence, those younger than 60 years are commonly affected by repetitive use/ strain disorders, gout (men only), RA, spondyloarthritis, and uncommonly, infectious arthritis. Patients over age 60 years are frequently affected by OA, crystal (gout and pseudogout) arthritis, polymyalgia rheumatica, osteoporotic fracture, and uncommonly, septic arthritis.

1	Trauma/fracture Low back pain Age <60 years Age >60 years Repetitive strain injury (Tendinitis, Bursitis) Osteoarthritis Gout (males only) Gout Pseudogout Rheumatoid arthritis Polymyalgia rheumatica Osteoporotic fracture Infectious arthritis (GC, viral, bacterial, Lyme) Septic arthritis (bacterial) Fibromyalgia Orthopedic evaluation FREQUENCY More Less Psoriatic, Reactive arthritis, IBD arthritis FIGUrE 393-2 Algorithm for consideration of the most common musculoskeletal conditions. GC, gonococcal; IBD, inflammatory bowel disease. These conditions are between 10 and 100 times more prevalent than other serious autoimmune conditions, such as SLE, scleroderma, polymyositis, and vasculitis.

1	Additional historic features may reveal important clues to the diagnosis. Aspects of the patient profile, complaint chronology, extent of joint involvement, and precipitating factors can provide important information. Certain diagnoses are more frequent in different age groups. SLE and reactive arthritis occur more frequently in the young, whereas fibromyalgia and RA are frequent in middle age, and OA and polymyalgia rheumatica are more prevalent among the elderly. Diagnostic clustering is also evident when sex and race are considered. Gout, spondyloarthritis, and ankylosing spondylitis are more common in men, whereas RA, fibromyalgia, and lupus are more frequent in women. Racial predilections may be evident. Thus, polymyalgia rheumatica, giant cell arteritis, and granulomatosis with polyangiitis (GPA; formerly called Wegener’s granulomatosis) commonly affect whites, whereas sarcoidosis and SLE more commonly affect African Americans. Familial aggregation is most common with ankylosing

1	(GPA; formerly called Wegener’s granulomatosis) commonly affect whites, whereas sarcoidosis and SLE more commonly affect African Americans. Familial aggregation is most common with ankylosing spondylitis, gout, and Heberden’s nodes of OA.

1	The chronology of the complaint is an important diagnostic feature and can be divided into the onset, evolution, and duration. The onset of disorders such as septic arthritis or gout tends to be abrupt, whereas OA, RA, and fibromyalgia may have more indolent presentations. The patients’ complaints may evolve differently and be classified as chronic (OA), intermittent (crystal or Lyme arthritis), migratory (rheumatic fever, gonococcal or viral arthritis), or additive (RA, psoriatic arthritis). Musculoskeletal disorders are typically classified as acute or chronic based on a symptom duration that is either less than or greater than 6 weeks, respectively. Acute arthropathies tend to be infectious, crystal-induced, or reactive. Chronic conditions include noninflammatory or immunologic arthritides (e.g., OA, RA) and nonarticular disorders (e.g., fibromyalgia).

1	The extent or distribution of articular involvement is often informative. Articular disorders are classified based on the number of joints involved, as either monarticular (one joint), oligoarticular or pauciarticular (two or three joints), or polyarticular (four or more joints). Although crystal and infectious arthritis are often mono-or oligoarticular, OA and RA are polyarticular disorders. Nonarticular disorders may be classified as either focal or widespread. Complaints secondary to tendinitis or carpal tunnel syndrome are typically focal, whereas weakness and myalgia, caused by polymyositis or fibromyalgia, are more widespread in their presentation. Joint involvement in RA tends to be symmetric and polyarticular. By contrast, spondyloarthritis, reactive arthritis, gout, and sarcoid are often asymmetric and oligoarticular. OA and psoriatic arthritis may be either symmetric or asymmetric and oligo-or polyarticular. The upper extremities are frequently involved inRAandOA, whereas

1	are often asymmetric and oligoarticular. OA and psoriatic arthritis may be either symmetric or asymmetric and oligo-or polyarticular. The upper extremities are frequently involved inRAandOA, whereas lower extremityarthritis is characteristic of reactive arthritis and gout at their onset. Involvement of the axial skeleton is common in OA and ankylosing spondylitis but is infrequent in RA, with the notable exception of the cervical spine.

1	The clinical history should also identify precipitating events, such as trauma (osteonecrosis, meniscal tear), drug administration (Table 393-2), antecedent or intercurrent infection (rheumatic fever, reactive arthritis, hepatitis), or illnesses that may have contributed to the patient’s complaint. Certain comorbidities may have musculoskeletal consequences. This is especially so for diabetes mellitus (carpal tunnel syndrome), renal insufficiency (gout), depression or insomnia (fibromyalgia), myeloma (low back pain), cancer (myositis), and osteoporosis (fracture) or when using certain drugs such as glucocorticoids (osteonecrosis, septic arthritis) and diuretics or chemotherapy (gout) (Table 393-2).

1	Lastly, a thorough rheumatic review of systems may disclose useful diagnostic information. A variety of musculoskeletal disorders may be associated with systemic features such as fever (SLE, infection), rash (SLE, psoriatic arthritis), nail abnormalities (psoriatic or reactive arthritis), myalgias (fibromyalgia, statin-or drug-induced myopathy), Quinidine, cimetidine, beta blockers, quinolones, chronic acyclovir, interferons, IL-2, nicardipine, vaccines, rifabutin, aromatase and HIV protease inhibitors Glucocorticoids, penicillamine, hydroxychloroquine, AZT, lovastatin, simvastatin, atorvastatin, pravastatin, clofibrate, amiodarone, interferon, IL-2, alcohol, cocaine, paclitaxel, docetaxel, imatinib mesylate, colchicine, quinolones, cyclosporine, tacrolimus, protease inhibitors Quinolones, glucocorticoids, isotretinoin, statins, collagenase injections Diuretics, aspirin, cytotoxics, cyclosporine, alcohol, moonshine, ethambutol, fructose-containing soft drinks

1	Quinolones, glucocorticoids, isotretinoin, statins, collagenase injections Diuretics, aspirin, cytotoxics, cyclosporine, alcohol, moonshine, ethambutol, fructose-containing soft drinks Hydralazine, procainamide, quinidine, phenytoin, carbamazepine, methyl-dopa, isoniazid, chlorpromazine, lithium, penicillamine, tetracyclines, TNF inhibitors, ACE inhibitors, ticlopidine Proton pump inhibitors, calcium channel blockers (diltiazem), ACE inhibitors, TNF inhibitors, terbinafine, interferons (α and β-1a), paclitaxel, docetaxel, HCTZ Glucocorticoids, alcohol, radiation, bisphosphonates Glucocorticoids, chronic heparin, phenytoin Vinyl chloride, bleomycin, baricitinib, pentazocine, organic solvents, carbidopa, tryptophan, rapeseed oil Allopurinol, amphetamines, cocaine (often levamisole adulterated), thiazides, penicillamine, propylthiouracil, montelukast, TNF inhibitors, hepatitis B vaccine, trimethoprim/sulfamethoxazole, minocycline, hydralazine

1	Abbreviations: ACE, angiotensin-converting enzyme; AZT, zidovudine; HCTZ, hydrochlorothiazide; IL-2, interleukin 2; TNF, tumor necrosis factor. or weakness (polymyositis, neuropathy). In addition, some conditions are associated with involvement of other organ systems including the eyes (Behçet’s disease, sarcoidosis, spondyloarthritis), gastrointestinal tract (scleroderma, inflammatory bowel disease), genitourinary tract (reactive arthritis, gonococcemia), or nervous system (Lyme disease, vasculitis).

1	The incidence of rheumatic diseases rises with age, such that 58% of those >65 years will have joint complaints. Musculoskeletal disorders inelderlypatientsareoftennotdiagnosed becausethesigns and symptoms may be insidious, overlooked, or overshadowed by comorbidities. These difficulties are compounded by the diminished reliability of laboratory testing in the elderly, who often manifest nonpathologic abnormal results. For example, the ESR may be misleadingly elevated, and low-titer positive tests for rheumatoid factor and antinuclear antibodies (ANAs) may be seen in up to 15% of elderly patients. Although nearly all rheumatic disorders afflict the elderly, geriatric patients are particularly prone to OA, osteoporosis, osteoporotic fractures, gout, pseudogout, polymyalgia rheumatica, vasculitis, and drug-induced disorders (Table 393-2). The elderly should be approached in the same manner as other patients with musculoskeletal complaints, but with an emphasis on identifying the

1	vasculitis, and drug-induced disorders (Table 393-2). The elderly should be approached in the same manner as other patients with musculoskeletal complaints, but with an emphasis on identifying the potential rheumatic consequences of medicalcomorbiditiesandtherapies.The physicalexaminationshould identify the nature of the musculoskeletal complaint as well as coexisting diseases that may influence diagnosis and choice of treatment.

1	Evaluation of a hospitalized patient with rheumatic complaints differs from that of an outpatient, owing to greater symptom severity, more acute presentations, and greater interplay of comorbidities. Patients with rheumatic disorders tend to be admitted for one of several reasons: (1) acute onset of inflammatory arthritis; (2) undiagnosed systemic or febrile illness; (3) musculoskeletal trauma; (4) exacerbation or deterioration of an existing autoimmune disorder (e.g., SLE); or (5) new medical comorbidities (e.g., thrombotic event, lymphoma, infection) arising in patients with an established rheumatic disorder. Notably, rheumatic patients are seldom if ever admitted because of widespread pain or serologic abnormalities or for the initiation of new therapies.

1	Acute monarticular inflammatory arthritis may be a “red flag” condition (e.g., septic arthritis, gout, pseudogout) that will require arthrocentesis and, on occasion, hospitalization if infection is suspected. However, new-onset inflammatory polyarthritis will have a wider differential diagnosis (e.g., RA, hepatitis-related arthritis, serum sickness, drug-induced lupus, polyarticular septic arthritis) and may require targeted laboratory investigations rather than synovial fluid analyses. Patients with febrile, multisystem disorders will require exclusion of crystal, infectious, or neoplastic etiologies and an evaluation driven by the dominant symptom/finding with the greatest specificity. Conditions worthy of consideration may include gout or pseudogout, vasculitis (giant cell arteritis in the elderly or polyarteritis nodosa in younger patients), adult-onset Still’s disease, SLE, antiphospholipid antibody syndrome, and sarcoidosis. Because the misdiagnosis of connective tissue

1	in the elderly or polyarteritis nodosa in younger patients), adult-onset Still’s disease, SLE, antiphospholipid antibody syndrome, and sarcoidosis. Because the misdiagnosis of connective tissue disorders is common, patients who present with a reported preexisting rheumatic condition (e.g., SLE, RA, ankylosing spondylitis) should have their diagnosis confirmed by careful history, physical and musculoskeletal examination, and review of their medical records. It is important to note that when established rheumatic disease patients are admitted to the hospital, it is usually not for a medical problem related to their autoimmune disease, but ratherbecauseofeitheracomorbid conditionorcomplicationofdrug therapy. Patients with chronic inflammatory disorders (e.g., RA, SLE, psoriasis) have an augmented risk of infection, cardiovascular events, and neoplasia.

1	Certain conditions, such as acute gout, can be precipitated in hospitalized patients by surgery, dehydration, or other events and should be consideredwhenhospitalizedpatientsareevaluatedfortheacuteonset ofamusculoskeletalcondition.Lastly,overlyaggressiveandunfocused 2219 laboratory testing will often yield abnormal findings that are better explained by the patient’s preexisting condition(s) rather than a new inflammatory or autoimmune disorder.

1	The goal of the physical examination is to ascertain the structures involved, the nature of the underlying pathology, the functional consequencesoftheprocess,andthepresence ofsystemicor extraarticular manifestations. A knowledge of topographic anatomy is necessary to identify the primary site(s) of involvement and differentiate articular from nonarticular disorders. The musculoskeletal examination depends largely on careful inspection, palpation, and a variety of specific physical maneuvers to elicit diagnostic signs (Table 393-3). Although most articulations of the appendicular skeleton can be examined in this manner, adequate inspection and palpation are not possible for many axial (e.g., zygapophyseal) and inaccessible (e.g., sacroiliac or hip) joints. For such joints, there is a greater reliance on specific maneuvers and imaging for assessment.

1	Examination of involved and uninvolved joints will determine whether pain, warmth, erythema, or swelling is present. The locale and level of pain elicited by palpation or movement should be quantified. One standard would be to count the number of tender joints on palpation of 28 easily examined joints (proximal interphalangeals, metacarpophalangeals, wrists, elbows, shoulders, and knees). Similarly, the number of swollen joints (0–28) can be counted and recorded. Careful examination should distinguish between true articular swelling (caused by bony hypertrophy, synovial effusion or proliferation) and nonarticular (or periarticular) involvement, which usually extends beyond the normal joint margins. Synovial effusion can be distinguished from synovial hypertrophy or bony hypertrophy by palpation or specific maneuvers. For example, small to moderate knee effusions may be identified by the “bulge sign” or “ballottement of the patellae.” Bursal effusions (e.g., effusions of the olecranon

1	or specific maneuvers. For example, small to moderate knee effusions may be identified by the “bulge sign” or “ballottement of the patellae.” Bursal effusions (e.g., effusions of the olecranon or prepatellar bursa) are often focal, periarticular, overlie bony prominences, and are fluctuant with sharply defined borders. Joint stability can be assessed by stabilizing the proximal joint, by palpation, and by the application of manual stress to the distal appendage. Subluxation or dislocation, which may be secondary to traumatic, mechanical, or inflammatory

1	A palpable (less commonly audible) vibratory or crackling sensation elicited with joint motion; fine joint crepitus is common and often insignificant in large joints; coarse joint crepitus indicates advanced cartilaginous and degenerative changes (as in osteoarthritis) Alteration of joint alignment such that articulating surfaces incompletely approximate each other Abnormal displacement of articulating surfaces such that the surfaces are not in contact Range of motion For diarthrodial joints, the arc of measurable movement through which the joint moves in a single plane Loss of full movement resulting from a fixed resistance caused either by tonic spasm of muscle (reversible) or by fibrosis of periarticular structures (permanent) Abnormal shape or size resulting from bony hypertrophy, malalignment of articulating structures, or damage to periarticular supportive structures Inflammation of the entheses (tendinous or ligamentous insertions on

1	Approach to Articular and Musculoskeletal Disorders 2220 causes, can be assessed by inspection and palpation. Joint swelling or volume can be assessed by palpation. Distention of the articular capsule usually causes pain and evident enlargement or fluctuance. The patient will attempt to minimize the pain by maintaining the joint in the position of least intraarticular pressure and greatest volume, usually partial flexion. For this reason, inflammatory effusions may give rise to flexion contractures. Clinically, this may be detected as fluctuant or “squishy” swelling in larger joints and grape-like compressibility in smaller joints. Inflammation may result in fixed flexion deformities or diminished range of motion—especially on extension, when intraarticular pressure is increased. Active and passive range of motion should be assessed in all planes, with contralateral comparison. A goniometer may be used to quantify the arc of movement. Each joint should be passively manipulated through

1	passive range of motion should be assessed in all planes, with contralateral comparison. A goniometer may be used to quantify the arc of movement. Each joint should be passively manipulated through its full range of motion (including, as appropriate, flexion, extension, rotation, abduction, adduction, lateral bending, inversion, eversion, supination, pronation, medial/lateral deviation, and plantar-or dorsiflexion). Extreme range of motion may be seen with hypermobility syndrome, with joint pain and connective tissue laxity, often associated with Ehlers-Danlos or Marfan’s syndrome. Limitation of motion is frequently caused by inflammation, effusion, pain, deformity, contracture, or restriction from neuromyopathic causes. If passivemotion exceeds active motion, a periarticular process (e.g., tendinitis, tendon rupture, or myopathy) should be considered. Contractures may reflect antecedent synovial inflammation or trauma. Minor joint crepitus is common during joint palpation and

1	(e.g., tendinitis, tendon rupture, or myopathy) should be considered. Contractures may reflect antecedent synovial inflammation or trauma. Minor joint crepitus is common during joint palpation and maneuvers but only indicates significant cartilage degeneration as it becomes coarser (e.g., OA). Joint deformity usually indicatesalong-standingoraggressivepathologicprocess.Deformities may result from ligamentous destruction, soft tissue contracture, bony enlargement, ankylosis, erosive disease, subluxation, trauma, or loss of proprioception. Examination of the musculature will document strength, atrophy, pain, or spasm. Appendicular muscle weakness should be characterized as proximal or distal. Muscle strength should be assessed by observing the patient’s performance (e.g., walking, rising from a chair, grasping, writing). Strength may also be graded on a 5-point scale: 0 for no movement; 1 for trace movement or twitch; 2 for movement with gravity eliminated; 3 for movement against

1	from a chair, grasping, writing). Strength may also be graded on a 5-point scale: 0 for no movement; 1 for trace movement or twitch; 2 for movement with gravity eliminated; 3 for movement against gravity only; 4 for movement against gravity and resistance; and 5 for normal strength. The examiner should assess for often-overlooked nonarticular or periarticular involvement, especially when articular complaints are not supported by objective findings referable to the joint capsule. The identification of soft tissue/nonarticular pain will prevent unwarranted and often expensive additional evaluations. Specific maneuvers may reveal common nonarticular abnormalities, such as a carpal tunnel syndrome (which can be identified by Tinel’s or Phalen’s sign). Other examples of soft tissue abnormalities include olecranon bursitis, epicondylitis (e.g., tennis elbow), enthesitis (e.g., Achilles tendinitis), and tender trigger points associated with fibromyalgia.

1	Although all patients should be evaluated in a logical and thorough manner, many cases with focal musculoskeletal complaints are caused by commonly encountered disorders that exhibit a predictable pattern of onset, evolution, and localization; they can often be diagnosed immediately on the basis of limited historic information and selected maneuvers or tests. Although nearly every joint could be approached in this manner, the evaluation of four common involved anatomic regions—the hand, shoulder, hip, and knee—are reviewed here.

1	Focal or unilateral hand pain may result from trauma, overuse, infection, or a reactive or crystal-induced arthritis. By contrast, bilateral hand complaints commonly suggest a degenerative (e.g., OA), systemic, or inflammatory/immune (e.g., RA) etiology. The distribution orpattern of joint involvement is highly suggestive of certain disorders (Fig. 393-3). Thus, OA (or degenerative arthritis) may manifest as distal interphalangeal (DIP) and proximal interphalangeal (PIP) joint pain with bony hypertrophy sufficient to produce Heberden’s and Bouchard’s nodes, respectively. Pain, with or without bony swelling, 1st CMC: OA de Quervain's tenosynovitis Wrist: RA, pseudogout, gonococcal arthritis, juvenile arthritis, carpal tunnel syndrome FIGUrE 393-3 Sites of hand or wrist involvement and their poten- DIP: OA, psoriatic or reactive arthritis PIP: OA, SLE, RA, psoriatic arthritis

1	MCP: RA, pseudogout, hemochromatosis tial disease associations. CMC, carpometacarpal; DIP, distal interphalangeal; MCP, metacarpophalangeal; OA, osteoarthritis; PIP, proximal interphalangeal; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus. (From JJ Cush et al: Evaluation of musculoskeletal complaints, in Rheumatology: Diagnosis and Therapeutics, 2nd ed, JJ Cush et al [eds]. Philadelphia, Lippincott Williams & Wilkins, 2005, pp 3–20. Used with permission from Dr. John J. Cush.) involving the base of the thumb (first carpometacarpal joint) is also highly suggestive of OA. By contrast, RA tends to cause symmetric, polyarticular involvement of the PIP, metacarpophalangeal (MCP), intercarpal, and carpometacarpal joints (wrist) with pain and palpable synovial tissue hypertrophy. Psoriatic arthritis may mimic the pattern of joint involvement seen in OA (DIP and PIP joints), but can be distinguished by the presence of inflammatory signs (erythema, warmth, synovial swelling), with

1	arthritis may mimic the pattern of joint involvement seen in OA (DIP and PIP joints), but can be distinguished by the presence of inflammatory signs (erythema, warmth, synovial swelling), with or without carpal involvement, nail pitting, or onycholysis. Whereas lateral or medial subluxations at the PIP or DIP joints are most likely due to inflammatory OA or psoriatic arthritis, dorsal or ventral deformities (swan neck or boutonnière deformities) are typical of RA. Hemochromatosis should be considered when degenerative changes (bony hypertrophy) are seen at the second and third MCP joints with associated radiographic chondrocalcinosis or episodic, inflammatory wrist arthritis.

1	Dactylitis manifests as soft tissue swelling of the whole digit and may have a sausage-like appearance. Common causes of dactylitis include psoriatic arthritis, spondyloarthritis, juvenile spondylitis, mixed connective tissue disease, scleroderma, sarcoidosis, and sickle cell disease. Soft tissue swelling over the dorsum of the hand and wrist may suggest an inflammatory extensor tendon tenosynovitis possibly caused by gonococcal infection, gout, or inflammatory arthritis (e.g., RA). Tenosynovitis is suggested by localized warmth, swelling, or pitting edema and may be confirmed when the soft tissue swelling tracks with tendon movement, such as flexion and extension of fingers, or when pain is induced while stretching the extensor tendon sheaths (flexing the digits distal to the MCP joints and maintaining the wrist in a fixed, neutral position).

1	Focal wrist pain localized to the radial aspect may be caused by de Quervain’s tenosynovitis resulting from inflammation of the tendon sheath(s) involving the abductor pollicis longus or extensor pollicis brevis (Fig. 393-3). This commonly results from overuse or follows pregnancy and may be diagnosed with Finkelstein’s test. A positive result is present when radial wrist pain is induced after the thumb is flexed and placed inside a clenched fist and the patient actively deviates the hand downward with ulnar deviation at the wrist. Carpal tunnel syndrome is another common disorder of the upper extremity and results from compression of the median nerve within the carpal tunnel. Manifestations include pain in the wrist that may radiate with paresthesia to the thumb, second and third fingers, and radial half of the fourth finger and, at times, atrophy of thenar musculature. Carpal tunnel syndrome is commonly associated with pregnancy, edema, trauma, OA, inflammatory arthritis, and

1	and radial half of the fourth finger and, at times, atrophy of thenar musculature. Carpal tunnel syndrome is commonly associated with pregnancy, edema, trauma, OA, inflammatory arthritis, and infiltrative disorders (e.g., amyloidosis). The diagnosis may be suggested by a positive Tinel’s or Phalen’s sign. With each test, paresthesia in a median nerve distribution is induced or increased by either “thumping” the volar aspect of the wrist (Tinel’s sign) or pressing the extensor surfaces of both flexed wrists against each other (Phalen’s sign). The low sensitivity and moderate specificity of these tests may require nerve conduction velocity testing to confirm a suspected diagnosis.

1	During the evaluation of shoulder disorders, the examiner should carefully note any history of trauma, fibromyalgia, infection, inflammatory disease, occupational hazards, or previous cervical disease. In addition, the patient should be questioned as to the activities or movement(s) that elicit shoulder pain. While arthritis is suggested by pain on movement in all planes, pain with specific active motion suggests a periarticular (nonarticular) process. Shoulder pain may originateintheglenohumeral oracromioclavicularjoints,subacromial (subdeltoid) bursa, periarticular soft tissues (e.g., fibromyalgia, rotator cuff tear/tendinitis), or cervical spine (Fig. 393-4). Shoulder pain is referred frequently from the cervical spine but may also be referred from intrathoracic lesions (e.g., a Pancoast tumor) or from gallbladder, hepatic, or diaphragmatic disease. These same visceral causes may also manifest as focal scapular pain. Fibromyalgia should be suspected

1	FIGUrE 393-4 Origins of shoulder pain. The schematic diagram of the shoulder indicates with arrows the anatomic origins of shoulder pain.

1	when glenohumeral pain is accompanied by diffuse periarticular (i.e., 2221 subacromial, bicipital) pain and tender points (i.e., trapezius or supraspinatus). The shoulder should be put through its full range of motion bothactivelyandpassively(withexaminerassistance):forwardflexion, extension, abduction, adduction, and internal and external rotation. Manual inspection of the periarticular structures will often provide important diagnostic information. Glenohumeral involvement is best detected by placing the thumb over the glenohumeral joint just medial and inferior to the coracoid process and applying pressure anteriorly while internally and externally rotating the humeral head. Pain localized to this region is indicative of glenohumeral pathology. Synovial effusion or tissue is seldom palpable but, if present, may suggest infection, RA, amyloidosis, or an acute tear of the rotator cuff. The examiner should apply direct manual pressure over the subacromial bursa that lies lateral to

1	but, if present, may suggest infection, RA, amyloidosis, or an acute tear of the rotator cuff. The examiner should apply direct manual pressure over the subacromial bursa that lies lateral to and immediately beneath the acromion (Fig. 393-4). Subacromial bursitis is a frequent cause of shoulder pain. Anterior to the subacromial bursa, the bicipital tendon traverses the bicipital groove. This tendon is best identified by palpating it in its groove as the patient rotates the humerus internally and externally. Direct pressure over the tendon may reveal pain indicative of bicipital tendinitis. Palpation of the acromioclavicular joint may disclose local pain, bony hypertrophy,or,uncommonly,synovialswelling.WhereasOAandRA commonly affect the acromioclavicular joint, OA seldom involves the glenohumeral joint, unless there is a traumatic or occupational cause.

1	Rotator cuff tendinitis or tear is a very common cause of shoulder pain. Nearly 30 percent of the elderly will have shoulder pain, with rotator cuff tendinitis or tear as the primary cause. The rotator cuff is formed by four tendons that attach the scapula to the proximal humerus (supraspinatus, infraspinatus, teres minor, and subscapularis tendons). Of these, the supraspinatus muscle is the most commonly damaged. Rotator cuff tendinitis is suggested by pain on active abduction (but not passive abduction), pain over the lateral deltoid muscle, nightpain,andevidenceoftheimpingementsigns(painwithoverhead arm activities). The Neer test for impingement is performed by the examiner raising the patient’s arm into forced flexion while stabilizing and preventing rotation of the scapula. A positive sign is present if pain develops before 180° of forward flexion. Tear of the rotator cuff is common in the elderly and often results from trauma; it may manifest in the same manner as tendinitis.

1	sign is present if pain develops before 180° of forward flexion. Tear of the rotator cuff is common in the elderly and often results from trauma; it may manifest in the same manner as tendinitis. The drop arm test is abnormal with supraspinatus pathology and is demonstrated by passive abduction of the arm to 90° by the examiner. If the patient is unable to hold the arm up actively or unable to lower the arm slowly without dropping, the test is positive. Tendinitis or tear of the rotator cuff is best confirmed by magnetic resonance imaging (MRI) or ultrasound.

1	Knee pain may result from intraarticular (OA, RA) or periarticular (anserine bursitis, collateral ligament strain) processes or be referred from hip pathology. A careful history should delineate the chronology of the knee complaint and whether there are predisposing conditions, trauma, or medications that might underlie the complaint. For example, patellofemoral disease (e.g., OA) may cause anterior knee pain that worsens with climbing stairs. Observation of the patient’s gait is also important. The knee should be carefully inspected in the upright (weight-bearing) and supine positions for swelling, erythema, malalignment, visible trauma, muscle wasting, and leg length discrepancy. The most common malalignment in the knee is genu varum (bowlegs) or genu valgum (knock-knees) resulting from asymmetric cartilage loss medially or laterally. Bony swelling of the knee joint commonly results from hypertrophic osseous changes seen with disorders such as OA and neuropathic arthropathy.

1	from asymmetric cartilage loss medially or laterally. Bony swelling of the knee joint commonly results from hypertrophic osseous changes seen with disorders such as OA and neuropathic arthropathy. Swelling caused by hypertrophy of the synovium or synovial effusion may manifest as a fluctuant, ballotable, or soft tissue enlargement in the suprapatellar pouch (suprapatellar reflection of the synovial cavity) or regions lateral and medial to the patella. Synovial effusions may also be detected by balloting the patella downward toward the femoral groove or by eliciting a “bulge sign.” With the knee extended, the examiner should manually compress, or “milk,” synovial fluid down from the suprapatellar pouch and lateral to the patellae. The

1	Approach to Articular and Musculoskeletal Disorders 2222 application of manual pressure lateral to the patella may cause an observable shift in synovial fluid (bulge) to the medial aspect. The examiner should note that this maneuver is only effective in detecting small to moderate effusions (<100 mL). Inflammatory disorders such as RA, gout, pseudogout, and psoriatic arthritis may involve the knee joint and produce significant pain, stiffness, swelling, or warmth. A popliteal or Baker’s cyst may be palpated with the knee partially flexed and is best viewed posteriorly with the patient standing and knees fully extended to visualize isolated or unilateral popliteal swelling or fullness. Anserine bursitis is an often missed periarticular cause of knee pain in adults. The pes anserine bursa underlies the insertion of the conjoined tendons (sartorius, gracilis, semitendinosus) on the anteromedial proximal tibia and may be painful following trauma, overuse, or inflammation. It is often

1	underlies the insertion of the conjoined tendons (sartorius, gracilis, semitendinosus) on the anteromedial proximal tibia and may be painful following trauma, overuse, or inflammation. It is often tender in patients with fibromyalgia, obesity, and knee OA. Other forms of bursitis may also present as knee pain. The prepatellar bursa is superficial and is located over the inferior portion of the patella. The infrapatellar bursa is deeper and lies beneath the patellar ligament before its insertion on the tibial tubercle. Internal derangement of the knee may result from trauma or degenerative processes. Damage to the meniscal cartilage (medial or lateral) frequently presents as chronic or intermittent knee pain. Such an injury should be suspected when there is a history of trauma, athletic activity, or chronic knee arthritis, and when the patient relates symptoms of “locking” or “giving way” of the knee. With the knee flexed 90° and the patient’s foot on the table, pain elicited during

1	activity, or chronic knee arthritis, and when the patient relates symptoms of “locking” or “giving way” of the knee. With the knee flexed 90° and the patient’s foot on the table, pain elicited during palpation over the joint line or when the knee is stressed laterally or medially may suggest a meniscal tear. A positive McMurray test may also indicate a meniscal tear.Toperformthistest,thekneeisfirstflexedat90°,andthelegisthen extended while the lower extremity is simultaneously torqued medially or laterally. A painful click during inward rotation may indicate a lateral meniscus tear, and pain during outward rotation may indicate a tear in the medial meniscus. Lastly, damage to the cruciate ligaments should be suspected with acute onset of pain, possibly with swelling, a history of trauma, or a synovial fluid aspirate that is grossly bloody. Examination of the cruciate ligaments is best accomplished by eliciting a drawer sign. With the patient recumbent, the knee should be partially

1	or a synovial fluid aspirate that is grossly bloody. Examination of the cruciate ligaments is best accomplished by eliciting a drawer sign. With the patient recumbent, the knee should be partially flexed and the footstabilizedon the examining surface. Theexaminer shouldmanually attempt to displace the tibia anteriorly or posteriorly with respect to the femur. If anterior movement is detected, then anterior cruciate ligament damage is likely. Conversely, significant posterior movement may indicate posterior cruciate damage. Contralateral comparison will assist the examiner in detecting significant anterior or posterior movement.

1	The hip is best evaluated by observing the patient’s gait and assessing range of motion. The vast majority of patients reporting “hip pain” localize their pain unilaterally to the posterior Enthesitis gluteal musculature (Fig. 393-5). Such pain tends to radiate down the posterolateral aspect of the thigh and may or may not be associated

1	True hip pain, with complaints of low back pain. This pre sentation frequently results from degenerative arthritis of the lumbosacral spine or disks and commonly follows a dermatomal distribution Meralgia with involvement of nerve roots between L4 and S1. Sciatica is caused by impingement of the L4, L5, or S1 nerve (i.e., from a herniated disk) and manifests as unilateral neuropathic pain extending from the gluteal region down the posterolateral leg to the foot. Some individuals instead localize their “hip pain” laterally to the area overlying the trochanteric bursa. Because of the depth of this bursa, swelling and warmth are FIGUrE 393-5 Origins of hip pain and dysesthesias. (From JJ Cush et al: Evaluation of mus-usually absent. Diagnosis of trochanteric bursitis culoskeletal complaints, in Rheumatology: Diagnosis and Therapeutics, 2nd ed, JJ Cush et al [eds]. or enthesitis can be confirmed by inducing point Philadelphia, Lippincott Williams & Wilkins, 2005, pp 3–20. Used with

1	in Rheumatology: Diagnosis and Therapeutics, 2nd ed, JJ Cush et al [eds]. or enthesitis can be confirmed by inducing point Philadelphia, Lippincott Williams & Wilkins, 2005, pp 3–20. Used with permission from Dr. John tenderness over the trochanteric bursa. Gluteal J. Cush.) and trochanteric pain are common findings in fibromyalgia. Range of movement may be limited by pain. Pain in the hip joint is less common and tends to be located anteriorly, over the inguinal ligament; it may radiate medially to the groin. Uncommonly, iliopsoas bursitis may mimic true hip joint pain. Diagnosis of iliopsoas bursitis may be suggestedbyahistoryoftraumaorinflammatoryarthritis.Painassociated with iliopsoas bursitis is localized to the groin or anterior thigh and tends to worsen with hyperextension of the hip; many patients prefer to flex and externally rotate the hip to reduce the pain from a distended bursa.

1	The vast majority of musculoskeletal disorders can be easily diagnosed by a complete history and physical examination. An additional objective of the initial encounter is to determine whether additional investigations or immediate therapy is required. Additional evaluation is indicated with: (1) monarticular conditions; (2) traumatic or inflammatory conditions; (3) the presence of neurologic findings; (4) systemic manifestations; or (5) chronic symptoms (>6 weeks) and a lack of response to symptomatic measures. The extent and nature of the additional investigation should be dictated by the clinical features and suspected pathologic process. Laboratory tests should be used to confirm a specific clinical diagnosis and not be used to screen or evaluate patients with vague rheumatic complaints. Indiscriminate use of broad batteries of diagnostic tests and radiographic procedures is rarely a useful or cost-effective means to establish a diagnosis.

1	Besidesacomplete bloodcount,includingawhitebloodcell(WBC) and differential count, the routine evaluation should include a determination of an acute-phase reactant such as the ESR or CRP, which can be useful in discriminating inflammatory from noninflammatory disorders. Both are inexpensive, easily obtained, and may be elevated with infection, inflammation, autoimmune disorders, neoplasia, pregnancy, renal insufficiency, advanced age, or hyperlipidemia. Extreme elevation of the acute-phase reactants (CRP, ESR) is seldom seen without evidence of serious illness (e.g., sepsis, pleuropericarditis, polymyalgia rheumatica, giant cell arteritis, adult Still’s disease).

1	Serum uric acid determinations are useful in the diagnosis of gout and in monitoring the response to urate-lowering therapy. Uric acid, the end product of purine metabolism, is primarily excreted in the urine. Serum values range from 238 to 516 μmol/L (4.0–8.6 mg/dL) in men; the lower values (178–351 μmol/L [3.0–5.9 mg/dL]) seen in women are caused by the uricosuric effects of estrogen. Urinary uric acid levels are normally <750 mg per 24 h. Although hyperuricemia (especiallylevels>535μmol/L[9mg/dL])isassociatedwithanincreased incidence of gout and nephrolithiasis, levels may not correlate with the severity of articular disease. Uric acid levels (and the risk of gout) may be increased by inborn errors of metabolism (Lesch-Nyhan syndrome), disease states (renal insufficiency, myeloproliferative disease, psoriasis), or drugs (alcohol, cytotoxic therapy, thiazides). Although nearly all patients with gout will demonstrate hyperuricemia at some time during their illness, up to 50% of

1	disease, psoriasis), or drugs (alcohol, cytotoxic therapy, thiazides). Although nearly all patients with gout will demonstrate hyperuricemia at some time during their illness, up to 50% of patients with an acute gouty attack will have normal serum uric acid levels. Monitoring serum uric acid may be useful in assessing the response to urate-lowering therapy or chemotherapy, with the target goal being a serum urate <6 mg/dL.

1	Serologic tests for rheumatoid factor (RF), cyclic anticitrullinated peptide (CCP or ACPA) antibodies, ANAs, complement levels, Lyme and antineutrophil cytoplasmic antibodies (ANCA), or antistreptolysin O (ASO) titer should be carried out only when there is clinical evidence to specifically suggest an associated diagnosis, because these have poor predictive value when used for screening, especially when the pretest probability is low. For most of these, there is no value to repeated or serial serologic testing. Although 4–5% of a healthy population will have positive tests for RF and ANAs, only 1% and <0.4% of the population will haveRA orSLE, respectively. IgM RF(autoantibodies against theFc portionofIgG)isfoundin80%ofpatientswithRAandmayalsobeseen in low titers in patients with chronic infections (tuberculosis, leprosy, hepatitis); other autoimmune diseases (SLE, Sjögren’s syndrome); and chronic pulmonary, hepatic, or renal diseases. When considering RA, both serum RF and anti-CCP

1	infections (tuberculosis, leprosy, hepatitis); other autoimmune diseases (SLE, Sjögren’s syndrome); and chronic pulmonary, hepatic, or renal diseases. When considering RA, both serum RF and anti-CCP antibodies should be obtained as these are complementary. Both are comparably sensitive, but CCP antibodies are more specific than RF. In RA, the presence of anti-CCP and RF antibodies may indicate a greater risk for more severe, erosive polyarthritis. ANAs are found in nearly all patients with SLE and may also be seen in patients with other autoimmune diseases (polymyositis, scleroderma, antiphospholipid syndrome, Sjögren’s syndrome), drug-induced lupus (Table 393-2), chronic liver or renal disorders, and advanced age. Positive ANAs are found in 5% of adults and in up to 14% of elderly or chronically ill individuals. The ANA test is very sensitive but poorly specific for lupus, as only 1–2% of all positive results will be caused by lupus alone. The interpretation of a positive ANA test

1	ill individuals. The ANA test is very sensitive but poorly specific for lupus, as only 1–2% of all positive results will be caused by lupus alone. The interpretation of a positive ANA test may depend on the magnitude of the titer and the pattern observed by immunofluorescence microscopy (Table 393-4). Diffuse and speckled patterns are least specific, whereas a peripheral, or rim, pattern (related to autoantibodies against double-strand [native] DNA) is highly specific and suggestive of lupus. Centromeric patterns are seen in patients with limited

1	Abbreviations: ANA, antinuclear antibody; CREST, calcinosis, Raynaud phenomenon, esophageal involvement, sclerodactyly, and telangiectasia; MCTD, mixed connective tissue disease; PSS, progressive systemic sclerosis; SCLE, subacute cutaneous lupus erythematosus; SLE, systemic lupus erythematosus. scleroderma (calcinosis, Raynaud’s phenomenon, esophageal involve-2223 ment, sclerodactyly, telangiectasia [CREST] syndrome) or primary biliary sclerosis, and nucleolar patterns may be seen in patients with diffuse systemic sclerosis or inflammatory myositis.

1	Aspiration and analysis of synovial fluid are always indicated in acute monarthritis or when an infectious or crystal-induced arthropathy is suspected. Synovial fluid may distinguish between noninflammatory and inflammatory processes by analysis of the appearance, viscosity,andcellcount.Testsforsynovialfluidglucose,protein,lactate dehydrogenase, lactic acid, or autoantibodies are not recommended because they have no diagnostic value. Normal synovial fluid is clear or a pale straw color and is viscous, primarily because of the high levels of hyaluronate. Noninflammatory synovial fluid is clear, viscous, and amber-colored, with a WBC count of <2000/μL and a predominance of mononuclear cells. The viscosity of synovial fluid is assessed by expressing fluid from the syringe one drop at a time. Normally, there is a stringing effect, with a long tail behind each synovial drop. Effusions caused by OAor traumawillhavenormalviscosity. Inflammatoryfluid is turbid and yellow, with an increased

1	Normally, there is a stringing effect, with a long tail behind each synovial drop. Effusions caused by OAor traumawillhavenormalviscosity. Inflammatoryfluid is turbid and yellow, with an increased WBC count (2000–50,000/μL) and a polymorphonuclear leukocyte predominance. Inflammatory fluid has reduced viscosity, diminished hyaluronate, and little or no tail following each drop of synovial fluid. Such effusions are found in RA, gout, andotherinflammatoryarthritides.Septicfluidisopaqueandpurulent, with a WBC count usually >50,000/μL, a predominance of polymorphonuclear leukocytes (>75%), and low viscosity. Such effusions are typical of septic arthritis but may also occur with RA or gout. In addition, hemorrhagic synovial fluid may be seen with trauma, hemarthro sis, or neuropathic arthritis. An algorithm for synovial fluid aspiration and analysis is shown in Fig. 393-6. Synovial fluid should be analyzed immediately for appearance, viscosity, and cell count. Monosodium urate crystals

1	An algorithm for synovial fluid aspiration and analysis is shown in Fig. 393-6. Synovial fluid should be analyzed immediately for appearance, viscosity, and cell count. Monosodium urate crystals (observed in gout) are seen by polarized microscopy and arelong,needle-shaped,negativelybirefringent,andusuallyintracellular. In chondrocalcinosis and pseudogout, calcium pyrophosphate dihydrate crystals are usually short, rhomboid-shaped, and positively birefringent. Whenever infection is suspected, synovial fluid should be Gram stained and cultured appropriately. If gonococcal arthritis is suspected, nucleic acid amplification tests should be used to detect either Chlamydia trachomatis or N. gonorrhoeae infection. Synovial fluid from patients withchronicmonarthritisshouldalsobeculturedfor M. tuberculosis and fungi. Last, it should be noted that crystal-induced arthritis and septic arthritis occasionally occur together in the same joint.

1	Conventional radiography has been a valuable tool in the diagnosis and staging of articular disorders. Plain x-rays are most appropriate and cost effective when there is a history of trauma, suspected chronic infection, progressive disability, or monarticular involvement; when therapeutic alterations are considered; or when a baseline assessment is desired for what appears to be a chronic process. However, in acute inflammatory arthritis, early radiography is rarely helpful in establishinga diagnosisand may only reveal soft tissueswelling or juxtaarticular demineralization.Asthediseaseprogresses,calcification(ofsofttissues, cartilage,orbone),jointspacenarrowing,erosions,bonyankylosis,new bone formation (sclerosis, osteophytes, or periostitis), or subchondral cysts may develop and suggest specific clinical entities. Consultation with a radiologist will help define the optimal imaging modality, technique, or positioning and prevent the need for further studies.

1	Additional imaging techniques may possess greater diagnostic sensitivity and facilitate early diagnosis in a limited number of articular disorders and in selected circumstances and are indicated when conventional radiography is inadequate or nondiagnostic (Table 393-5). Ultrasonography is useful in the detection of soft tissue abnormalities, such as tendinitis, tenosynovitis, enthesitis, bursitis, and entrapment neuropathies. Wider use, lower cost, better technology, and enhanced site-specific transducers now allow for routine use in outpatient care. Owing to low cost, portability, and wider use, ultrasound use has grown and is the preferred method for the evaluation of synovial (Baker’s) cysts, rotator cuff tears, tendinitis and tendon injury, and Approach to Articular and Musculoskeletal Disorders Strongly consider synovial fluid aspiration and analysis if there is Trauma with joint effusion Monarthritis in a patient with chronic polyarthritis

1	Strongly consider synovial fluid aspiration and analysis if there is Trauma with joint effusion Monarthritis in a patient with chronic polyarthritis Suspicion of joint infection, crystal-induced arthritis, or hemarthrosis

1	Analyze fluid for • Appearance, viscosity• WBC count, differential• Gram stain, culture, and sensitivity (if indicated)• Crystal identification by polarized microscopyInflammatory ornoninflammatoryarticular condition Consider• Trauma or mechanical derangement• Coagulopathy• Neuropathic arthropathy• OtherIs the WBC > 2000/˜L? Consider otherinflammatoryor septic arthritides • Gram stain, culture mandatoryIs the % PMNs > 75%? Are crystals present? Considernoninflammatoryarticular conditions • Osteoarthritis• Trauma• OtherIs the WBC > 50,000/˜L? Crystal identification for specific diagnosis • Gout• PseudogoutProbable inflammatory arthritisPossible septic arthritis Consider inflammatoryor septic arthritis Is the effusion hemorrhagic? NoYes NoYes NoYes Yes NoYes NoFIGUrE 393-6 Algorithmic approach to the use and interpretation of synovial fluid aspiration and analysis. PMNs,polymorphonucle-ar(leukocytes);WBC,whitebloodcell(count).

1	crystal deposition on cartilage. Use of power Doppler allows for early detection of synovitis and bony erosions. Radionuclide scintigraphy is a very sensitive, but poorly specific, means of detecting inflammatory or metabolic alterations in bone or periarticular soft tissue structures. Scintigraphy is best suited for total-body assessment (extent and distribution) of skeletal involvement (neoplasia, Paget’s disease) and the assessment of patients with undiagnosed polyarthralgias, looking for occult arthritis. The use of scintigraphy has declined with greater use and declining cost of ultrasound and MRI. The limited tissue contrast resolution of scintigraphy may obscure the distinction between a bony or periarticular process and may necessitate the additional use of MRI. Scintigraphy using 99mTc, 67Ga, or 111In-labeled WBCs has been applied to a variety of articular disorders with variable success (Table 393-5). Although [99mTc] diphosphate scintigraphy may be useful in identifying

1	99mTc, 67Ga, or 111In-labeled WBCs has been applied to a variety of articular disorders with variable success (Table 393-5). Although [99mTc] diphosphate scintigraphy may be useful in identifying osseous infection, neoplasia, inflammation, increasedbloodflow,boneremodeling,heterotopicboneformation,or avascular necrosis, MRI is preferred in most instances. Gallium scanning uses 67Ga, which binds serum and cellular transferrin and lactoferrin and is preferentially taken up by neutrophils, macrophages,

1	Imaging Method Time, h Costa Current Indications aRelative cost for imaging study. Abbreviations: NA, not commercially available; WBC, white blood cell. bacteria, and tumor tissue (e.g., lymphoma). As such, it is primarily used in the identification of occult infection or malignancy. Scanning with 111In-labeled WBCs has been used to detect osteomyelitis and infectious or inflammatory arthritis. Despite their utility, 111In-labeled WBC or 67Ga scanning has largely been replaced by MRI, except when there is a suspicion of septic joint or prosthetic joint infections.

1	Computed tomography (CT) provides detailed visualization of the axial skeleton. Articulationspreviously considered difficult to visualize by radiography (e.g., zygapophyseal, sacroiliac, sternoclavicular, hip joints) can be effectively evaluated using CT. CT has been demonstrated to be useful in the diagnosis of low back pain syndromes (e.g., spinal stenosis vs herniated disk), sacroiliitis, osteoid osteoma, and stress fractures. Helical or spiral CT (with or without contrast angiography) is a novel technique that is rapid, cost effective, and sensitive in diagnosing pulmonary embolism or obscure fractures, often in the setting of initially equivocalfindings. High-resolutionCTcan beadvocated in the evaluation of suspected or established infiltrative lung disease (e.g., scleroderma or rheumatoid lung). The recent use of hybrid (positron emission tomography [PET] or single-photon emission CT [SPECT]) CT scans in metastatic evaluations has incorporated CT to provide better anatomic

1	rheumatoid lung). The recent use of hybrid (positron emission tomography [PET] or single-photon emission CT [SPECT]) CT scans in metastatic evaluations has incorporated CT to provide better anatomic localization of scintigraphic abnormalities.

1	FIGUrE 393-7 Dual-energy computed tomography (DECT) scan from a 45-year-old woman with right ankle swelling around the lateral malleolus. Three-dimensionalvolume-renderedcoronalreformattedDECTimageshowsthatthemassiscomposedofmonosodiumurate(red)inkeepingwithtophus(arrow).(Used with permission from S Nicolaou et al: AJR 194:1072, 2010.) 18F-Fluorodeoxyglucose (FDG) is the most commonly used radio-pharmaceutical in PET scanning. FDG-PET/CT scans have been seldom used in the evaluation of septic or inflammatory arthritis. Dual-energy CT (DECT) scanning, developed in urology to identify urinary calculi, has been a highly sensitive and specific method used to identify and quantify uric acid deposition in tissues (Fig. 393-7).

1	MRI has significantly advanced the ability to image musculoskeletal structures. MRI has the advantages of providing multiplanar images with fine anatomic detail and contrast resolution (Fig. 393-8) that allows for the superior ability to visualize bone marrow and soft tissue periarticular structures. Although more costly with a longer proceduraltimethanCT,theMRIhasbecomethepreferredtechniquewhen evaluating complex musculoskeletal disorders.

1	MRI can image fascia, vessels, nerve, muscle, cartilage, ligaments, tendons, pannus, synovial effusions, and bone marrow. Visualization of particular structures can be enhanced by altering the pulse sequence to produce either T1-or T2-weighted spin echo, gradient echo, or inversion recovery (including short tau inversion recovery [STIR]) images. Because of its sensitivity to changes in marrow fat, MRI is a sensitivebutnonspecificmeansofdetectingosteonecrosis,osteomyelitis, and marrow inflammation indicating overlying synovitis or osteitis (Fig. 393-8). Because of its enhanced soft tissue resolution, MRI is more sensitive than arthrography or CT in the diagnosis of soft tissue injuries (e.g., meniscal and rotator cuff tears); intraarticular derangements; marrow abnormalities (osteonecrosis, myeloma); and spinal cord or nerve root damage, synovitis, or cartilage damage or loss. The author acknowledges the contributions of Dr. Peter E. Lipsky to this chapter in previous editions.

1	The author acknowledges the contributions of Dr. Peter E. Lipsky to this chapter in previous editions. Approach to Articular and Musculoskeletal Disorders FIGUrE 393-8 Superior sensitivity of magnetic resonance imaging (MRI) in the diagnosis of osteonecrosis of the femoral head. A 45-year-old woman receiving high-dose glucocorticoids developed right hip pain. Conventional x-rays (top) demonstrated only mild sclerosis of the right femoral head. T1-weighted MRI (bottom) demonstrated low-density signal in the right femoral head, diagnostic of osteonecrosis. 2226 Osteoarthritis David T. Felson Osteoarthritis(OA)isthemostcommontypeofarthritis.Itshighprevalence,especiallyintheelderly,andthehighrateofdisabilityrelatedtodiseasemakeitaleadingcauseofdisabilityintheelderly.BecauseoftheagingofWesternpopulationsandbecauseobesity,a394

1	FIGUrE 394-2 Severe osteoarthritis of the hands affectingthedis-talinterphalangealjoints(Heberden’snodes)andtheproximalinter-phalangealjoints(Bouchard’snodes).Thereisnoclearbonyenlarge-mentoftheothercommonsiteinthehands,thethumbbase. major risk factor, is increasing in prevalence, the occurrence of OA is on the rise. In the United States, OA prevalence will increase by 66–100% by 2020.

1	major risk factor, is increasing in prevalence, the occurrence of OA is on the rise. In the United States, OA prevalence will increase by 66–100% by 2020. OA affects certain joints, yet spares others (Fig. 394-1). Commonly affected joints include the cervical and lumbosacral spine, hip, knee, and first metatarsal phalangeal joint (MTP). In the hands, the distal and proximal interphalangeal joints and the base of the thumb are often affected. Usually spared are the wrist, elbow, and ankle. Our joints were designed, in an evolutionary sense, for brachiating apes, animals that still walked on four limbs. We thus develop OA in joints that were ill designed for human tasks such as pincer grip (OA in the thumb base) and walking upright (OA in knees and hips). Some joints, like the ankles, may be spared because their articular cartilage may be uniquely resistant to loading stresses.

1	OA can be diagnosed based on structural abnormalities or on the symptoms these abnormalities evoke. According to cadaveric studies, by elderly years, structural changes of OA are nearly universal. These include cartilage loss (seen as joint space loss on x-rays) and osteophytes. Many persons with x-ray evidence of OA have no joint symptoms, and although the prevalence of structural abnormalities is of interest in understanding disease pathogenesis, what matters more from a clinical perspective is the prevalence of symptomatic OA. Symptoms, usually joint pain, determine disability, visits to clinicians, and disease costs.

1	SymptomaticOAoftheknee(painonmostdaysofarecentmonthin akneeplusx-rayevidenceofOAinthatknee)occursin~12%ofpersons age ≥60 in the United States and 6% of all adults age ≥30. Symptomatic hipOAisroughlyone-thirdascommonasdiseaseintheknee.Although radiographically evident hand OA and the appearance of bony enlargementinaffectedhandjoints(Fig. 394-2) areextremelycommoninolder persons, most cases are often not symptomatic. Even so, symptomatic FIGUrE 394-1 Joints commonly affected by osteoarthritis. hand OA occurs in ~10% of elderly individuals and often produces measurable limitation in function. The prevalence of OA rises strikingly with age. Regardless of how it is defined, OA is uncommon in adults under age 40 and highly prevalent inthoseoverage 60.Itisalsoa disease that, atleast inmiddle-aged and elderly persons, is much more common in women than in men, and sex differences in prevalence increase with age.

1	X-ray evidence of OA is common in the lower back and neck, but backpain and neck pain have not been tied to findings of OA on x-ray. Thus, back pain and neck pain are treated separately (Chap. 22). OA is joint failure, a disease in which all structures of the joint have undergone pathologic change, often in concert. The pathologic sine qua non of disease is hyaline articular cartilage loss, present in a focal and, initially, nonuniform manner. This is accompanied by increasing thickness and sclerosis of the subchondral bony plate, by outgrowth of osteophytes at the joint margin, by stretching of the articular capsule, by mild synovitis in many affected joints, and by weakness of muscles bridging the joint. In knees, meniscal degeneration is part of the disease. There are numerous pathways that lead to joint failure, but the initial step is often joint injury in the setting of a failure of protective mechanisms.

1	Joint protectors include joint capsule and ligaments, muscle, sensory afferents, and underlying bone. Joint capsule and ligaments serve as joint protectors by providing a limit to excursion, thereby fixing the range of joint motion. Synovial fluid reduces friction between articulating cartilage surfaces, thereby serving as a protector against friction-induced cartilage wear. This lubrication function depends on hyaluronic acid and on lubricin, a mucinous glycoprotein secreted by synovial fibroblasts whose concentration diminishes after joint injury and in the face of synovial inflammation.

1	The ligaments, along with overlying skin and tendons, contain mechanoreceptor sensory afferent nerves. These mechanoreceptors fire at different frequencies throughout a joint’s range of motion, providing feedback by way of the spinal cord to muscles andtendons. As a consequence, these muscles and tendons can assume the right tension at appropriate points in joint excursion to act as optimal joint protectors, anticipating joint loading. Muscles and tendons that bridge the joint are key joint protectors. Their coordinated contractions at the appropriate time in joint movement provide the appropriate power and acceleration for the limb to accomplish its tasks. Focal stress across the joint is minimized by muscle contractionthatdeceleratesthejointbeforeimpactandassuresthatwhen joint impact arrives, it is distributed broadly across the joint surface.

1	Failure of these joint protectors increases the risk of joint injury and OA.Forexample,inanimals,OAdevelopsrapidlywhenasensorynerve to the joint is sectioned and joint injury induced. Similarly, in humans, Charcot’s arthropathy, a severe and rapidly progressive OA, develops when minor joint injury occurs in the presence of posterior column peripheral neuropathy. Another example of joint protector failure is rupture of ligaments, a well-known cause of the early development of OA. In addition to being a primary target tissue for disease, cartilage also functions as a joint protector. A thin rim of tissue at the ends of two opposing bones, cartilage is lubricated by synovial fluid to provide an almost frictionless surface across which these two bones move. The compressible stiffness of cartilage compared to bone provides the joint with impact-absorbing capacity.

1	The earliest changes of OA may occur in cartilage, and abnormalities there can accelerate disease development. The two major macromolecules in cartilage are type 2 collagen, which provides cartilage its tensile strength, and aggrecan, a proteoglycan macromolecule linked with hyaluronic acid, which consists of highly negatively charged glycosaminoglycans. In normal cartilage, type 2 collagen is woven tightly, constraining the aggrecan molecules in the interstices between collagen strands, forcing these highly negatively charged molecules into close proximity with one another. The aggrecan molecule, through electrostatic repulsion of its negative charges, gives cartilage its compressive stiffness. Chondrocytes, the cells within this avascular tissue, synthesize all elements of the matrix and produce enzymes that break down the matrix. Synovium and chondrocytes synthesize and release cytokines and growth factors, which provide feedback that modulates synthesis of matrix molecules (Fig.

1	produce enzymes that break down the matrix. Synovium and chondrocytes synthesize and release cytokines and growth factors, which provide feedback that modulates synthesis of matrix molecules (Fig. 394-3). Cartilage matrix synthesis and catabolism are in a dynamic equilibrium influenced by the cytokine and growth factor environment. Mechanical and osmotic stress on chondrocytes induces these cells to alter gene expression and increase production of inflammatory cytokines and matrix-degrading enzymes. While chondrocytes synthesize numerous enzymes, matrix metalloproteinases (MMP) (especially collagenases and ADAMTS-5) arecriticalenzymesinthebreakdownofcartilagematrix.Bothcollagenase and aggrecanases act primarily in the territorial matrix surrounding chondrocytes; however, as the osteoarthritic process develops, their activities and effects spread throughout the matrix, especially in the superficial layers of cartilage.

1	The synovium, cartilage, and bone all influence disease develop-2227 ment through cytokines, chemokines, and even complement activation (Fig. 394-3). These act on chondrocyte cell surface receptors and ultimately have transcriptional effects. Matrix fragments released from cartilage stimulate synovitis. Among the most important cytokines are interleukin (IL) 1β, which exerts transcriptional effects on chondrocytes, stimulating production of proteinases and suppressing cartilage matrix synthesis. Tumor necrosis factor (TNF) α may play a similar role to that of IL-1. These cytokines also induce chondrocytes to synthesize prostaglandin E2 and nitric oxide, which have complex effects on matrix synthesis and degradation. At early stages in the matrix response to injury and in the healthy response to loading, the net effect of cytokine stimulation may be matrix synthesis, but ultimately, the combination of effects on chondrocytes triggers matrix degradation. Enzymes in the matrix are held

1	to loading, the net effect of cytokine stimulation may be matrix synthesis, but ultimately, the combination of effects on chondrocytes triggers matrix degradation. Enzymes in the matrix are held in check by activation inhibitors, including tissue inhibitor of metalloproteinase (TIMP). Growth factors are also part of this complex network, with BMP-2 and transforming growth factor β playing prominent roles in stimulating the development of osteophytes. Whereas healthy articular cartilage is avascular in part due to angiogenesis inhibitors present in cartilage, disease is characterized by the invasion of blood vessels into cartilage from underlying bone and proliferation of vessels within synovium. This is influenced by vascular endothelial growth factor (VEGF) synthesis in the cartilage and bone. With these blood vessels come nerves that may bring nociceptive innervation.

1	Probably as a result of chronic oxidative damage, articular chondrocytes exhibit an age-related decline in synthetic capacity while maintaining the ability to produce proinflammatory mediators and matrix-degrading enzymes, findings characteristic of a senescent secretory phenotype. These chondrocytes are unable to maintain tissue homeostasis (such as after insults of a mechanical or inflammatory nature). Thus, with age, cartilage is easily damaged by minor sometimesunnoticedinjuries,includingthosethatarepartofdailyactivities. OA cartilage is characterized by gradual depletion of aggrecan, an unfurling of the tightly woven collagen matrix, and loss of type 2 collagen. With these changes comes increasing vulnerability of cartilage, which loses its compressive stiffness. Joint vulnerability and joint loading are the two major factors contributing to the development of OA. On the one hand, a vulnerable joint whose protectors are dysfunctional can develop OA with minimal

1	Hyalinecartilage(non-calcified)RANKL,OPG, uPAMMPs, IL-5,IL-8OsteocyteVEGFSclerostinVascularinvasionMatrix fragmentseophyt formation CalcifiedcartilageVEGFBONE REMODELINGSYNOVITISCell surface receptors on chondrocytesactivated by complement attack complex,DAMPS, cytokines and chemokines, WNTs(frizzled), fibronectin fragments and others SYNOVITIS mmentents BONBONBONBONBONBONERERERERERERE REMOEMOEMOEMOEMOEMOEMODELDELDELDELDELDELDELINGNGINGINGINGINGINGINGING titititit nnn ininvasiasionon˜BMPGFVascularGFGGGOstevOstOsteopeophhyteformatmationionHyaline cartilage (non-calcified) S100 proteins (alarmins), DAMPs, complements, IL-1˜, TNF°, IL-15, CCL 19, MCP-1, MIP-1˜RANKL, OPG, uPA MMPs, IL-5, IL-8 TGF--2 OsteocyteVESclerostin invasion Matrix fragments Osteophyte formationCalcified cartilage VEBONE REMODELING SYNOVITIS OOOOsOsststtOOOOOstt TGF-˜BMP-2 ooststststoostst BBBBBBBB tteeeeootteeo llclcl oeroerolcl roero oococcoco yytyteeyoococyte SSScScSSc EEEVEVEVEEEVEVE VascularVascular

1	VEBONE REMODELING SYNOVITIS OOOOsOsststtOOOOOstt TGF-˜BMP-2 ooststststoostst BBBBBBBB tteeeeootteeo llclcl oeroerolcl roero oococcoco yytyteeyoococyte SSScScSSc EEEVEVEVEEEVEVE VascularVascular inviasioninvasion FGGFGFGGGGGFFFFFGGG FFF EGEGEGGFG F EGEGEGEGEGEGVEVEVEVVEVEVE EGEG TGF-˜BMP-2 xfrmagmexfr magme agag T BB VascularVascular MatrixfMatrixf MM Cell surface receptorbyDAMPSreceptory t b pp yy ememe tnt att kack co lod hssbbanthethers Cell surface receptors on chondrocytes activated by complement attack complex, DAMPS, cytokines and chemokines, WNTs (frizzled), fibronectin fragments and others

1	FIGUrE 394-3 Selected factors involved in the osteoarthritic process including chondrocytes, bone, and synovium. Synovitis causes release of cytokines, alarmins, damage-associated molecular pattern (DAMP) molecules, and complement, which activate chondrocytes through cell surface receptors. Chondrocytes produce matrix molecules (collagen type 2, aggrecan) and the enzymes responsible for the degradation of the matrix (e.g., ADAMTS-5 and matrix metalloproteinases [MMPs]). Bone invasion occurs through the calcified cartilage, triggered by vascular endothelial growth factor (VEGF) and other molecules. IL, interleukin; TGF, transforming growth factor; TNF, tumor necrosis factor. (From RF Loeser et al: Arthritis Rheum 64:1697, 2012.) Previous damage (e.g., meniscectomy) Bridging muscle weakness Increasing bone density Malalignment Proprioceptive deficiences Susceptibility to OA

1	Previous damage (e.g., meniscectomy) Bridging muscle weakness Increasing bone density Malalignment Proprioceptive deficiences Susceptibility to OA FIGUrE 394-4 Risk factors for osteoarthritis (OA) either contribute to the susceptibility of the joint (systemic factors or factors in the local joint environment) or increase risk by the load they put on the joint. Usually a combination of loading and susceptibility factors is required to cause disease or its progression. levels of loading, perhaps even levels encountered during everyday activities. On the other hand, in a young joint with competent protectors, a major acute injury or long-term overloading is necessary to precipitate disease. Risk factors for OA can be understood in terms of their effect either on joint vulnerability or on loading (Fig. 394-4).

1	Age is the most potent risk factor for OA. Radiographic evidence of OA is rare in individuals under age 40; however, in some joints, such as the hands, OA occurs in >50% of persons over age 70. Aging increases joint vulnerability through several mechanisms. Whereas dynamic loading of joints stimulates cartilage matrix synthesis by chondrocytes in young cartilage, aged cartilage is less responsive to these stimuli. Partly because of this failure to synthesize matrix with loading, cartilage thins with age, and thinner cartilage experiences higher shear stress at basal layers and is at greater risk of cartilage damage. Also, joint protectors fail more often with age. Muscles that bridge the joint become weaker with age and also respond less quickly to oncoming impulses. Sensory nerve input slows with age, retarding the feedback loop of mechanoreceptors to muscles and tendons related to their tension and position. Ligaments stretch with age, making them less

1	loop of mechanoreceptors to muscles and tendons related to their tension and position. Ligaments stretch with age, making them less able to absorb impulses. These factors work in concert to increase the vulnerability of older joints to OA.

1	Older women are at high risk of OA in all joints, a risk that emerges as women reach their sixth decade. Although hormone loss with menopause may contribute to this risk, there is little understanding of the unique vulnerability of older women versus men to OA. OA is a highly heritable disease, but its heritability is joint specific. Fifty percent of the hand and hip OA in the community is attributable to inheritance, i.e., to disease present in other members of the family. However, the heritable proportion of knee OA is at most 30%, with some studies suggesting no heritability at all. Whereas many people with OA have disease in multiple joints, this “generalized OA” phenotype is rarely inherited and is more often a consequence of aging.

1	Emerging evidence has identified genetic mutations that confer a high risk of OA, the best replicated is a polymorphism within the growth differentiation factor 5 gene. This polymorphism diminishes the quantity of GDF5; GDF5 has its main influence on joint shape, and genes predisposing to OA are likely to increase risk of disease based on their effects on joint development and shape. United States. However, OA in the knees is at least as com mon, if not more so, in Chinese than in whites from the United States, and knee OA represents a major cause of disability in China, especially in rural areas. Anatomic differences between Chinese and white hips may account for much of the difference in hip OA prevalence, with white hips having a higher prevalence of anatomic predispositions to the development of OA. Persons from Africa, but not African Americans, may also have a very low rate of hip OA.

1	Some risk factors increase vulnerability of the joint through local effects on the joint environment. With changes in joint anatomy, for example, load across the joint is no longer distributed evenly across the joint surface, but rather shows an increase in focal stress. In the hip, three uncommon developmental abnormalities occurring in utero or in childhood, congenital dysplasia, Legg-Perthes disease, and slipped capital femoral epiphysis, leave a child with distortions of hip joint anatomy that often lead to OA later in life. Girls are predominantly affected by acetabular dysplasia, a mild form of congenital dislocation, whereas the other abnormalities more often affect boys. Depending on the severity of the anatomic abnormalities, hip OA occurs either in young adulthood (severe abnormalities) or middle age (mild abnormalities).

1	Major injuries to a joint also can produce anatomic abnormalities that leave the joint susceptible to OA. For example, a fracture through the joint surface often causes OA in joints in which the disease is otherwise rare such as the ankle and the wrist. Avascular necrosis can lead to collapse of dead bone at the articular surface, producing anatomic irregularities and subsequent OA. Tears of ligamentous and fibrocartilaginous structures that protect the joints, such as the anterior cruciate ligament and the meniscus in the knee and the labrum in the hip, can lead to premature OA. Meniscal tears increase with age and when chronic are often asymptomatic but lead to adjacent cartilage damage and accelerated OA. Even injuries in which the affected person never received a diagnosis may increase risk of OA. For example, in the Framingham Study subjects, men with a history of major knee injury, but no surgery, had a 3.5-fold increased risk for subsequent knee OA.

1	Another source of anatomic abnormality is malalignment across the joint (Fig. 394-5). This factor has been best studied in the knee, which is the fulcrum of the longest lever arm in the body. Varus (bowlegged) knees with OA are at exceedingly high risk of cartilage loss in the medial or inner compartment of the knee, whereas valgus (knockkneed) malalignment predisposes to rapid cartilage loss in the lateral compartment. Malalignment causes this effect by increasing stress on a focal area of cartilage, which then breaks down. There is evidence that malalignment in the knee not only causes cartilage loss but leads FIGUrE 394-5 The two types of limb malalignment in the frontal plane: varus, in which the stress is placed across the medial compartment of the knee joint, and valgus, which places excess stress across the lateral compartment of the knee.

1	to underlying bone damage, producing bone marrow lesions seen on magnetic resonance imaging (MRI). Malalignment in the knee often produces such a substantial increase in focal stress within the knee (as evidenced by its destructive effects on subchondral bone) that severely malaligned knees may be destined to progress regardless of the status of other risk factors. Weaknessin the quadriceps muscles bridging theknee increasesthe risk of the development of painful OA in the knee. Patients with knee OA have impaired proprioception across their knees, and this may predispose them to further disease progression. The role of bone in serving as a shock absorber for impact load is not well understood, but persons with increased bone density are at high risk of OA, suggesting that the resistance of bone to impact during joint use may play a role in disease development.

1	LOaDING FaCTOrS Obesity Three to six times body weight is transmitted across the knee during single-leg stance. Any increase in weight may be multiplied by this factor to reveal the excess force across the knee in overweight persons during walking. Obesity is a well-recognized and potent risk factorforthedevelopmentofkneeOAand,lessso,forhipOA.Obesity precedes the development of disease and is not just a consequence of the inactivity present in those with disease. It is a stronger risk factor for disease in women than in men, and in women, the relationship of weight to the risk of disease is linear, so that with each increase in weight, there is a commensurate increase in risk. Weight loss in women lowers the risk of developing symptomatic disease. Not only is obesity a risk factor for OA in weight-bearing joints, but obese persons have more severe symptoms from the disease.

1	Obesity’s effect on the development and progression of disease is mediated mostly through the increased loading in weight-bearing joints that occurs in overweight persons. However, a modest association of obesity with an increased risk of hand OA suggests that there may be a systemic metabolic factor circulating in obese persons that affects disease risk also.

1	repeated Use of Joint and Exercise There are two categories of repetitive joint use, occupational use and leisure time physical activities. Workers performing repetitive tasks as part of their occupations for many years are at high risk of developing OA in the joints they use repeatedly. For example, farmers are at high risk for hip OA, and miners have high rates of OA in knees and spine. Workers whose jobs require regular knee bending or lifting or carrying heavy loads have a high rate of knee OA. One reason why workers may get disease is that during long days at work, their muscles may gradually become exhausted, no longer serving as effective joint protectors.

1	It is widely recommended for people to adopt an exercise-filled lifestyle, and long-term studies of exercise suggest no consistent association of exercise with OA risk in the majority of persons. However, persons who already have injured joints may put themselves at greater risk by engaging in certain types of exercise. For example, persons who have already sustained major knee injuries are at increased risk of progressive knee OA as a consequence of running. In addition, compared to nonrunners, elite runners (professional runners and those on Olympic teams) have high risks of both knee and hip OA. Lastly, although recreational runners are not at increased risk of knee OA, studies suggest that they have a modest increased risk of disease in the hip.

1	The pathology of OA provides evidence of the involvement of many joint structures in disease. Cartilage initially shows surface fibrillation and irregularity. As disease progresses, focal erosions develop there, and these eventually extend down to the subjacent bone. With further progression,cartilageerosiondowntoboneexpandstoinvolvealarger proportion of the joint surface, even though OA remains a focal disease with nonuniform loss of cartilage (Fig. 394-6). After an injury to cartilage, chondrocytes undergo mitosis and clustering. Although the metabolic activity of these chondrocyte clusters is FIGUrE 394-6 Pathologic changes of osteoarthritis in a toe joint.

1	FIGUrE 394-6 Pathologic changes of osteoarthritis in a toe joint. Notethenonuniformlossofcartilage(arrowhead vssolid arrow),theincreasedthicknessofthesubchondralboneenvelope(solid arrow),andtheosteophyte(open arrow).(From the American College of Rheumatology slide collection.) high, theneteffect of this activityisto promoteproteoglycandepletion in the matrix surrounding the chondrocytes. This is because the cata bolicisgreaterthanthesyntheticactivity.Asdiseasedevelops,collagen matrix becomes damaged, the negative charges of proteoglycans get exposed, and cartilage swells from ionic attraction to water molecules. Because in damaged cartilage proteoglycans are no longer forced into close proximity, cartilage does not bounce back after loading as it did when healthy, and cartilage becomes vulnerable to further injury. Chondrocytes at the basal level of cartilage undergo apoptosis.

1	With loss of cartilage come alterations in subchondral bone. Stimulatedbygrowthfactorsandcytokines,osteoclastsandosteoblasts in the subchondral bony plate, just underneath cartilage, become activated. Bone formation produces a thickening and stiffness of the subchondral plate that occurs even before cartilage ulcerates. Trauma to bone during joint loading may be the primary factor driving this bone response, with healing from injury (including microcracks) producing stiffness. Small areas of osteonecrosis usually exist in joints with advanced disease. Bone death may also be caused by bone trauma with shearing of microvasculature, leading to a cutoff of vascular supply to some bone areas.

1	At the margin of the joint, near areas of cartilage loss, osteophytes form. These start as outgrowths of new cartilage, and with neurovascular invasion from the bone, this cartilage ossifies. Osteophytes are an important radiographic hallmark of OA. In malaligned joints, osteophytes grow larger on the side of the joint subject to most loading stress(e.g.,in varusknees,osteophytesgrowlargeron themedialside). The synovium produces lubricating fluids that minimize shear stress during motion. In healthy joints, the synovium consists of a single discontinuous layer filled with fat and containing two types of cells, macrophages and fibroblasts, but in OA, it can sometimes become edematousand inflamed.Thereisamigrationofmacrophages from the periphery into the tissue, and cells lining the synovium proliferate. Enzymes secreted by the synovium digest cartilage matrix that has been released from the surface of the cartilage.

1	Additionalpathologicchangesoccurinthecapsule,whichstretches, becomes edematous, and can become fibrotic. The pathology of OA is not identical across joints. In hand joints with severe OA, for example, there are often cartilage erosions in the center ofthejointprobablyproduced by bony pressure from the opposite side of the joint. Basic calcium phosphate and calcium pyrophosphate dihydrate crystals are present microscopically in most joints with end-stage OA. Their role in osteoarthritic cartilage is unclear, but their release from cartilage into the joint space and joint fluid likely triggers synovial 2230 inflammation, which can, in turn, produce release of enzymes and trigger nociceptive stimulation.

1	Because cartilage is aneural, cartilage loss in a joint is not accompanied by pain. Thus, pain in OA likely arises from structures outside the cartilage. Innervated structures in the joint include the synovium, ligaments, joint capsule, muscles, and subchondralbone.Mostof these are not visualized by the x-ray, and the severity of x-ray changes in OA correlates poorly with pain severity.

1	Based on MRI studies in osteoarthritic knees comparing those with and without pain and on studies mapping tenderness in unanesthetized joints, likely sources of pain include synovial inflammation, joint effusions, and bone marrow edema. Modest synovitis develops in many but not all osteoarthritic joints. Some diseased joints have no synovitis, whereas others have synovial inflammation that approaches the severity of joints with rheumatoid arthritis (Chap. 380). The presence of synovitis on MRI is correlated with the presence and severity of knee pain. Capsular stretching from fluid in the joint stimulates nociceptive fibers there, inducing pain. Increased focal loading as part of the disease not only damages cartilage but probably also injures the underlying bone. As a consequence, bone marrow edema appears on the MRI; histologically, this edema signals the presence of microcracks and scar, which are the consequences of trauma. These lesions may stimulate bone nociceptive fibers.

1	marrow edema appears on the MRI; histologically, this edema signals the presence of microcracks and scar, which are the consequences of trauma. These lesions may stimulate bone nociceptive fibers. Also, hemostatic pressure within bone rises in OA, and the increased pressure itself may stimulate nociceptive fibers, causing pain.

1	Painmayarisefromoutside the joint also, including bursaenearthe joints.Commonsourcesofpainnearthekneeareanserinebursitisand iliotibial band syndrome. Persons with chronic OA pain may develop nervous system alterations as a consequence of disease, changes which decrease inhibitory controls on nociception and its distribution. This may produce allodynia and hyperalgesia in some patients with OA.

1	Joint pain from OA is activity-related. Pain comes on either during or just after joint use and then gradually resolves. Examples include knee or hip pain with going up or down stairs, pain in weight-bearing joints when walking, and, for hand OA, pain when cooking. Early in disease, pain is episodic, triggered often by a day or two of overactive useofadiseasedjoint,suchasapersonwithkneeOAtakingalongrun and noticing a few days of pain thereafter. As disease progresses, the pain becomes continuous and even begins to be bothersome at night. Stiffness of the affected joint may be prominent, but morning stiffness is usually brief (<30 min).

1	In knees, buckling may occur, in part, due to weakness of muscles crossing the joint. Mechanical symptoms, such as buckling, catching, or locking, could also signify internal derangement, such as meniscal tears, and need to be evaluated. In the knee, pain with activities requiring knee flexion, such as stair climbing and arising from a chair, often emanates from the patellofemoral compartment of the knee, which does not actively articulate until the knee is bent ~35°.

1	OA is the most common cause of chronic knee pain in persons over age 45, but the differential diagnosis is long. Inflammatory arthritis is likely if there is prolonged morning stiffness and many other joints are affected. Bursitis occurs commonly around knees and hips. A physical examination should focus on whether tenderness is over the joint line (at the junction of the two bones around which the joint is articulating) or is outside of it. Anserine bursitis, medial and distal to the knee, is an extremely common cause of chronic knee pain that may respond to a glucocorticoid injection. Prominent nocturnal pain in the absence of end-stage OA merits a distinct workup. For hip pain, OA can be detected by loss of internal rotation on passive movement, and pain isolated to an area lateral to the hip joint usually reflects the presence of trochanteric bursitis. No blood tests are routinely indicated for workup of patients with OA unless symptoms and signs suggest inflammatory arthritis.

1	No blood tests are routinely indicated for workup of patients with OA unless symptoms and signs suggest inflammatory arthritis. FIGUrE 394-7 X-ray of knee with medial osteoarthritis. Notethenarrowedjointspaceonmedialsideofthejointonly(white arrow),thesclerosisoftheboneinthemedialcompartmentprovidingevi-denceofcorticalthickening(black arrow),andtheosteophytesinthemedialfemur(white wedge). Examination of the synovial fluid is often more helpful diagnostically than an x-ray. If the synovial fluid white count is >1000/μL, inflammatory arthritis or gout or pseudogout is likely, the latter two being also identified by the presence of crystals.

1	X-rays are indicated to evaluate chronic hand pain and hip pain thought to be due to OA, as the diagnosis is often unclear without confirming radiographs. For knee pain, x-rays should be obtained if symptoms or signs are not typical of OA or if knee pain persists after inauguration of effective treatment. In OA, radiographic findings (Fig. 394-7) correlate poorly with the presence and severity of pain. Further, radiographs may be normal in early disease as they are insensitive to cartilage loss and other early findings. Although MRI may reveal the extent of pathology in an osteoarthritic joint, it is not indicated as part of the diagnostic workup. Findings such as meniscal tears andcartilage and bone lesions occurin most patients with OA in the knee, but almost never warrant a change in therapy.

1	The goals of the treatment of OA are to alleviate pain and minimize loss of physical function. To the extent that pain and loss of function are consequences of inflammation, of weakness across the joint, and of laxity and instability, the treatment of OA involves addressing each of these impairments. Comprehensive therapy consists of a multimodality approach including nonpharmacologic and pharmacologic elements. Patients with mild and intermittent symptoms may need only reassurance or nonpharmacologic treatments. Patients with on-going, disabling pain are likely to need both nonpharmacotherapy and pharmacotherapy. Treatments for knee OA have been more completely evaluated than those for hip and hand OA or for disease in other joints. Thus, although the principles of treatment are identical for OA in all joints, we shall focus below on the treatment of knee OA, noting specific recommendations for disease in other joints, especially when they differ from those for the knee.

1	Because OA is a mechanically driven disease, the mainstay of treatment involves altering loading across the painful joint and improving the function of joint protectors, so they can better distribute load across the joint. Ways of lessening focal load across the joint include: 1. avoiding activities that overload the joint, as evidenced by their causing pain; 2. improving the strength and conditioning of muscles that bridge the joint, so as to optimize their function; and 3. unloading the joint, either by redistributing load within the joint with a brace or a splint or by unloading the joint during weight bearing with a cane or a crutch.

1	unloading the joint, either by redistributing load within the joint with a brace or a splint or by unloading the joint during weight bearing with a cane or a crutch. The simplest effective treatment for many patients is to avoid activities that precipitate pain. For example, for the middle-aged patient whose long-distance running brings on symptoms of knee OA, a less demanding form of weight-bearing activity may alleviate all symptoms. For an older person whose daily constitutionals up and down hills bring on knee pain, routing the constitutional away from hills might eliminate symptoms. Each pound of weight increases the loading across the knee threeto sixfold. Weight loss may have a commensurate multiplier effect, unloading both knees and hips and probably relieving pain in those joints.

1	In hand joints affected by OA, splinting, by limiting motion, often minimizes pain for patients with involvement especially in the base of the thumb. Weight-bearing joints such as knees and hips can be unloaded by using a cane in the hand opposite to the affected joint for partial weight bearing. A physical therapist can help teach the patient how to use the cane optimally, including ensuring that its height is optimal for unloading. Crutches or walkers can serve a similar beneficial function. Exercise Osteoarthritic pain in knees or hips during weight bearing results in lack of activity and poor mobility, and because OA is so common, the inactivity that results represents a public health concern, increasing the risk of cardiovascular disease and obesity. Aerobic capacity is poor in most elders with symptomatic knee OA, worse than others of the same age.

1	Weakness in muscles that bridge osteoarthritic joints is multi-factorial in etiology. First, there is a decline in strength with age. Second, with limited mobility comes disuse muscle atrophy. Third, patients with painful knee or hip OA alter their gait so as to lessen loading across the affected joint, and this further diminishes muscle use. Fourth, “arthrogenous inhibition” may occur, whereby contraction of muscles bridging the joint is inhibited by a nerve afferent feedback loop emanating in a swollen and stretched joint capsule; this prevents maximal attainment of voluntary maximal strength. Because adequate muscle strength and conditioning are critical to joint protection, weakness in a muscle that bridges a diseased joint makes the joint more susceptible to further damage and pain. The degree of weakness correlates strongly with the severity of joint pain and the degree of physical limitation. One of the cardinal elements of the treatment of OA is to improve the functioning of

1	The degree of weakness correlates strongly with the severity of joint pain and the degree of physical limitation. One of the cardinal elements of the treatment of OA is to improve the functioning of muscles surrounding the joint.

1	For knee and hip OA, trials have shown that exercise lessens pain and improves physical function. Most effective exercise regimens consist of aerobic and/or resistance training, the latter of which focuses on strengthening muscles across the joint. Exercises are likely to be effective, especially if they train muscles for the activities a person performs daily. Activities that increase pain in the joint should be avoided, and the exercise regimen needs to be individualized to optimize effectiveness. Range-of-motion exercises, which do not strengthen muscles, and isometric exercises that strengthen muscles, but not through range of motion, are unlikely to be effective by themselves. Low-impact exercises, including water aerobics and water resistance training, are often better tolerated by patients than exercises involving impact loading, such as running or treadmill exercises. A patient should be referred to an exercise class or to a therapist who can create an individualized regimen,

1	patients than exercises involving impact loading, such as running or treadmill exercises. A patient should be referred to an exercise class or to a therapist who can create an individualized regimen, and then an individualized home-based regimen can be crafted. In addition to conventional exercise regimens, tai chi may be effective for knee OA. However, there is no strong evidence that patients with hand OA benefit from therapeutic exercise.

1	Adherence over the long term is the major challenge to an exer-2231 cise prescription. In trials involving patients with knee OA, who are engaged in exercise treatment, from a third to over half of patients stopped exercising by 6 months. Less than 50% continued regular exercise at 1 year. The strongest predictor of a patient’s continued exercise is a previous personal history of successful exercise. Physicians should reinforce the exercise prescription at each clinic visit, help the patient recognize barriers to ongoing exercise, and identify convenient times for exercise to be done routinely. The combination of exercise with calorie restriction and weight loss is especially effective in lessening pain.

1	Correction of Malalignment Malalignment in the frontal plane (varusvalgus) markedly increases the stress across the joint, which can lead to progression of disease and to pain and disability (Fig. 394-5). Correcting malalignment, either surgically or with bracing, may relieve pain in persons whose knees are malaligned. Malalignment develops over years as a consequence of gradual anatomic alterations of the joint and bone, and correcting it is often very challeng ing. One way is with a fitted brace, which takes an often varus osteoarthritic knee and straightens it by putting valgus stress across the knee. Unfortunately, many patients are unwilling to wear a realigning knee brace; in addition, in patients with obese legs, braces may slip with usage and lose their realigning effect. They are indicated for willing patients who can learn to put them on correctly and on whom they do not slip.

1	Other ways of correcting malalignment across the knee include the use of orthotics in footwear. Unfortunately, although they may have modest effects on knee alignment, trials have heretofore not demonstrated efficacy of a lateral wedge orthotic versus placebo wedges. Pain from the patellofemoral compartment of the knee can be caused by tilting of the patella or patellar malalignment with the patella riding laterally or medially in the femoral trochlear groove. Using a brace to realign the patella, or tape to pull the patella back into the trochlear sulcus or reduce its tilt, has been shown, when compared to placebo taping in clinical trials, to lessen patellofemoral pain. However, patients may find it difficult to apply tape, and skin irritation from the tape is common. Commercial patellar braces may be a solution, but there is insufficient evidence on their efficacy to recommend them.

1	Although their effect on malalignment is questionable, neoprene sleeves pulled to cover the knee lessen pain and are easy to use and popular among patients. The explanation for their therapeutic effect on pain is unclear. In patients with knee OA, acupuncture produces modest pain relief compared to placebo needles and may be an adjunctive treatment. Although nonpharmacologic approaches to therapy constitute its mainstay, pharmacotherapy serves an important adjunctive role in OA treatment. Available drugs are administered using oral, topical, and intraarticular routes. acetaminophen, Nonsteroidal anti-Inflammatory Drugs (NSaIDs), and Cyclooxygenase-2 (COX-2) Inhibitors Acetaminophen (paracetamol) is the initial analgesic of choice for patients with OA in knees, hips, or hands. For some patients, it is adequate to control symptoms, in which case more toxic drugs such as NSAIDs can be avoided. Doses up to 1 g three times daily can be used (Table 394-1).

1	NSAIDs are the most popular drugs to treat osteoarthritic pain. They can be administered either topically or orally. In clinical trials, oral NSAIDs produce ~30% greater improvement in pain than high-dose acetaminophen. Occasional patients treated with NSAIDs experience dramatic pain relief, whereas others experience little improvement. Initially, NSAIDs should be administered topically or taken orally on an “as needed” basis because side effects are less frequent with low intermittent doses. If occasional medication use is insufficiently effective, then daily treatment may be indicated, with Acetaminophen Up to 1 g tid Prolongs half-life of warfarin. Make sure patient is not taking other treatments containing acetaminophen to avoid hepatic toxicity.

1	Acetaminophen Up to 1 g tid Prolongs half-life of warfarin. Make sure patient is not taking other treatments containing acetaminophen to avoid hepatic toxicity. aPatients at high risk include those with previous gastrointestinal events, persons ≥60 years, and persons taking glucocorticoids. Trials have shown the efficacy of proton pump inhibitors and misoprostol in the prevention of ulcers and bleeding. Misoprostol is associated with a high rate of diarrhea and cramping; therefore, proton pump inhibitors are more widely used to reduce NSAID-related gastrointestinal symptoms. Abbreviations: COX-2, cyclooxygenase-2; NSAIDs, nonsteroidal anti-inflammatory drugs. Source: Adapted from DT Felson: N Engl J Med 354:841, 2006. an anti-inflammatory dose selected (Table 394-1). Patients should be reminded to take low-dose aspirin and ibuprofen at different times to eliminate a drug interaction.

1	NSAIDs taken orally have substantial and frequent side effects, the most common of which is upper gastrointestinal toxicity, including dyspepsia, nausea, bloating, gastrointestinal bleeding, and ulcer disease. Some 30–40% of patients experience upper gastrointestinal (GI) side effects so severe as to require discontinuation of medication. To minimize the risk of nonsteroidal-related GI side effects, patients should not take two NSAIDs and should take medications after food; if risk is high, patients should take a gastroprotective agent, such as a proton pump inhibitor. Certain oral agents are safer to the stomach than others, including nonacetylated salicylates and nabumetone. Major NSAID-related GI side effects can occur in patients who do not complain of upper GI symptoms. In one study of patients hospitalized for GI bleeding, 81% had no premonitory symptoms.

1	Because of the increased rates of cardiovascular events associated with COX-2 inhibitors and with some conventional NSAIDs such as diclofenac, many of these drugs are not appropriate long-term treatment choices for older persons with OA, especially those at high risk of heart disease or stroke. The American Heart Association has identified rofecoxib and all other COX-2 inhibitors as putting patients at high risk, although low doses of celecoxib (≤200 mg/d) may not be associated with an elevation of risk. The only conventional NSAID that appears safe from a cardiovascular perspective is naproxen, but it does have GI toxicity.

1	There are other common side effects of NSAIDs, including the tendency to develop edema because of prostaglandin inhibition of afferent blood supply to glomeruli in the kidneys and, for similar reasons, a predilection toward reversible renal insufficiency. Blood pressure may increase modestly in some NSAID-treated patients. Oral NSAIDs should not be used in patients with stage IV or V renal disease and should be used with caution in those with stage III disease.

1	NSAIDs can be placed into a gel or topical solution with another chemical modality that enhances penetration of the skin barrier creating a topical NSAID. When absorbed through the skin, plasma concentrations are an order of magnitude lower than with the same amount of drug administered orally or parenterally. However, when these drugs are administered topically in proximity to a superficial joint (knees, hands, but not hips), the drug can be found in joint tissues such as the synovium and cartilage. Trial results have varied but generally have found that topical NSAIDs are slightly less efficacious than oral agents, but have far fewer GI and systemic side effects. Unfortunately, topical NSAIDs often cause local skin irritation where the medication is applied, inducing redness, burning, or itching in up to 40% of patients (see Table 394-1).

1	Intraarticular Injections: Glucocorticoids and Hyaluronic acid Because synovial inflammation is likely to be a major cause of pain in patients with OA, local anti-inflammatory treatments administered intraarticularly may be effective in ameliorating pain, at least temporarily. Glucocorticoid injections provide such efficacy, but response is variable, with some patients having little relief of pain whereas others experience pain relief lasting several months. Glucocorticoid injections are useful to get patients over acute flares of pain and may be especially indicated if the patient has coexistent OA and crystal deposition disease, especially from calcium pyrophosphate dihydrate crystals (Chap. 395). There is no evidence that repeated glucocorticoid injections into the joint are dangerous. Hyaluronic acid injections can be given for treatment of symptoms in knee and hip OA, but there is controversy as to whether they have efficacy versus placebo (Table 394-1).

1	Hyaluronic acid injections can be given for treatment of symptoms in knee and hip OA, but there is controversy as to whether they have efficacy versus placebo (Table 394-1). Other Classes of Drugs and Nutraceuticals For patients with symptomatic knee or hip OA who have not had an adequate response to the treatments above and are either unwilling to undergo or are not candidates for total joint arthroplasty, opioid analgesics have shown modest efficacy and can be tried. Opioid management plans and patient selection are critical. Another option is the use of duloxetine, which has demonstrated modest efficacy in OA. Recent guidelines recommend against the use of glucosamine or chondroitin for OA. Large publicly supported trials have failed to show that, compared with placebo, these compounds relieve pain in persons with disease.

1	Optimal nonsurgical therapy for OA is often achieved by trial and error, with each patient having idiosyncratic responses to specific treatments. When medical therapies have failed and the patient has an unacceptable reduction in their quality of life and ongoing pain gout and Other Crystal-associated arthropathies H. Ralph Schumacher, Lan X. Chen Theuseofpolarizinglightmicroscopyduringsynovialfluidanalysisin1961byMcCartyandHollanderandthesubsequentapplication395 and disability, then at least for knee and hip OA, total joint arthroplasty is indicated.

1	For knee OA, several operations are available. Arthroscopic debridement and lavage have diminished in popularity after randomized trials evaluating this operation have showed that its efficacy is no greater than that of sham surgery or no treatment for relief of pain or disability. Even mechanical symptoms such as buckling, which are extremely common in patients with knee OA, do not respond to arthroscopic debridement. Although arthroscopic meniscectomy is indicated for acute meniscal tears in which symptoms such as locking and acute pain are clearly related temporally to a knee injury that produced the tear, recent trials show that doing a partial meniscectomy in persons with OA and a symptomatic meniscal tear does not relieve knee pain or improve function.

1	For patients with knee OA isolated to the medial compartment, operations to realign the knee to lessen medial loading can relieve pain. These include a high tibial osteotomy, in which the tibia is broken just below the tibial plateau and realigned so as to load the lateral, nondiseased compartment, or a unicompartmental replacement with realignment. Each surgery may provide the patient with years of pain relief before a total knee replacement is required.

1	Ultimately, when the patient with knee or hip OA has failed medical treatment modalities and remains in pain, with limitations of physical function that compromise the quality of life, the patient should be referred for total knee or hip arthroplasty. These are highly efficacious operations that relieve pain and improve function in the vast majority of patients, although rates of success are higher for hip than knee replacement. Currently failure rates for both are ~1% per year, although these rates are higher in obese patients. The chance of surgical success is greater in centers where at least 25 such operations are performed yearly or with surgeons who perform multiple operations annually. The timing of knee or hip replacement is critical. If the patient suffers for many years until their functional status has declined substantially, with considerable muscle weakness, postoperative functional status may not improve to a level achieved by others who underwent operation earlier in

1	their functional status has declined substantially, with considerable muscle weakness, postoperative functional status may not improve to a level achieved by others who underwent operation earlier in their disease course.

1	Cartilage regeneration Chondrocyte transplantation has not been found to be efficacious in OA, perhaps because OA includes pathology of joint mechanics, which is not corrected by chondrocyte transplants. Similarly, abrasion arthroplasty (chondroplasty) has not been well studied for efficacy in OA, but it produces fibrocartilage in place of damaged hyaline cartilage. Both of these surgical attempts to regenerate and reconstitute articular cartilage may be more likely to be efficacious early in disease when joint malalignment and many of the other noncartilage abnormalities that characterize OA have not yet developed. of other crystallographic techniques, such as electron microscopy, energy-dispersive elemental analysis, and x-ray diffraction, have allowed investigators to identify the roles of different microcrystals, including monosodium urate (MSU), calcium pyrophosphate (CPP), calcium apatite (apatite), and calcium oxalate (CaOx), in inducing

1	Enthesitis Peculiar type of osteoarthritis acute or chronic arthritis or periarthritis. Theclinical events that result from deposition of MSU, CPP, apatite, and CaOx have many similarities but also have important differences. Because of often similar clinical presentations, the need to perform synovial fluid analysis to distinguish the type of crystal involved must be emphasized. Polarized light microscopy alone can identify most typical crystals; apatite, however, is an exception. Aspiration and analysis of effusions are also important to assess the possibility of infection. Apart from the identification of specific microcrystalline materials or organisms, synovial fluid characteristics in crystal-associated diseases are nonspecific, and synovial fluid can be inflammatory or noninflammatory. Without crystal identification, these diseases can be confused with rheumatoid or other types of arthritis. A list of possible musculoskeletal manifestations of crystal-associated arthritis is

1	Without crystal identification, these diseases can be confused with rheumatoid or other types of arthritis. A list of possible musculoskeletal manifestations of crystal-associated arthritis is shown in Table 395-1.

1	Goutisametabolicdiseasethatmostoftenaffectsmiddle-agedtoelderly men and postmenopausal women. It results from an increased body poolofuratewithhyperuricemia.Ittypicallyischaracterizedbyepisodic acute arthritis or chronic arthritis caused by deposition of MSU crystals in joints and connective tissue tophi and the risk for deposition in kidney interstitium or uric acid nephrolithiasis (Chap. 431e).

1	Acute arthritis is the most common early clinical manifestation of gout. Usually, only one joint is affected initially, but polyarticular acute gout can occur in subsequent episodes. The metatarsophalangeal joint of the first toe often is involved, but tarsal joints, ankles, and knees also are affected commonly. Especially in elderly patients or in advanced disease, finger joints may be involved. Inflamed Heberden’s or Bouchard’s nodes may be a first manifestation of gouty arthritis. Thefirstepisodeofacutegoutyarthritisfrequentlybeginsatnightwith dramaticjointpainandswelling.Jointsrapidlybecome warm,red,and tender, with a clinical appearance that often mimics that of cellulitis. Early attacks tend to subside spontaneously within 3–10 days, and most patients have intervals of varying length with no residual symptoms until the next episode. Several events may precipitate acute gouty arthritis: dietary excess, trauma, surgery, excessive ethanol ingestion, hypouricemic therapy, and

1	length with no residual symptoms until the next episode. Several events may precipitate acute gouty arthritis: dietary excess, trauma, surgery, excessive ethanol ingestion, hypouricemic therapy, and serious medical illnesses such as myocardial infarction and stroke.

1	After many acute mono-or oligoarticular attacks, a proportion of gouty patients may present with a chronic nonsymmetric synovitis, causing potential confusion with rheumatoid arthritis (Chap. 380). Less commonly, chronic gouty arthritis will be the only manifestation, and, more rarely, the disease will manifest only as periarticular tophaceous deposits in the absence of synovitis. Women represent only 5–20% of all patients with gout. Most women with gouty arthritis are postmenopausalandelderly,haveosteoarthritis and arterial hypertension that causes mild renal insufficiency, and usually are receiving diuretics. Premenopausal gout is rare. Kindreds of precocious gout in young females caused by decreased renal urate clearance and renal insufficiency have been described.

1	Laboratory Diagnosis Even if the clinical appearance strongly suggests gout, the presumptive diagnosis ideally should be confirmed by needle aspiration of acutely or chronically involved joints or tophaceous deposits. Acute septic arthritis, several of the other crystalline-associated arthropathies, palindromic rheumatism, and psoriatic arthritis may present with similar clinical features. During acute gouty FIGUrE 395-1 Extracellular and intracellular monosodium urate crystals, as seen in a fresh preparation of synovial fluid, illustrate needle-and rod-shaped crystals. These crystals are strongly negative birefringent crystals under compensated polarized light microscopy; 400×.

1	attacks, needle-shaped MSU crystals typically are seen both intracellularly and extracellularly (Fig. 395-1). With compensated polarized light, thesecrystalsarebrightlybirefringentwithnegativeelongation.Synovial fluid leukocyte counts are elevated from 2000 to 60,000/μL. Effusions appear cloudy due to the increased numbers of leukocytes. Large amounts of crystals occasionally produce a thick pasty or chalky joint fluid. Bacterial infection can coexist with urate crystals in synovial fluid; if there is any suspicion of septic arthritis, joint fluid must be cultured. MSU crystals also can often be demonstrated in the first metatarsophalangeal joint and in knees not acutely involved with gout. Arthrocentesis of these joints is a useful technique to establish the diagnosis of gout between attacks.

1	Serum uric acid levels can be normal or low at the time of an acute attack, as inflammatory cytokines can be uricosuric and effective initiation of hypouricemic therapy can precipitate attacks. This limits the value of serum uric acid determinations for the diagnosis of gout. Nevertheless, serum urate levels are almost always elevated at some time and are important to use to follow the course of hypouricemic therapy. A 24-h urine collection for uric acid can, in some cases, be useful in assessing the risk of stones, elucidating overproduction or underexcretion of uric acid, and deciding whether it may be appropriate to use a uricosuric therapy (Chap. 431e). Excretion of >800 mg of uric acid per 24 h on a regular diet suggests that causes of overproduction of purine should be considered. Urinalysis, serum creatinine, hemoglobin, white blood cell (WBC) count, liver function tests, and serum lipids should be obtained because of possible pathologic sequelae of gout and other associated

1	Urinalysis, serum creatinine, hemoglobin, white blood cell (WBC) count, liver function tests, and serum lipids should be obtained because of possible pathologic sequelae of gout and other associated diseases requiring treatment and as baselines because of possible adverse effects of gout treatment.

1	radiographic Features Cystic changes, well-defined erosions with sclerotic margins (often with overhanging bony edges), and soft tissue masses are characteristic radiographic features of advanced chronic tophaceous gout. Ultrasound may aid earlier diagnosis by showing a double contour sign overlying the articular cartilage. Dual-energy computed tomography (CT) can show specific features establishing the presence of urate crystals.

1	The mainstay of treatment during an acute attack is the administration of anti-inflammatory drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, or glucocorticoids. NSAIDs are used most often in individuals without complicating comorbid conditions. Both colchicine and NSAIDs may be poorly tolerated and dangerous in the elderly and in the presence of renal insufficiency and gastrointestinal disorders. Ice pack applications and rest of the involved joints can be helpful. Colchicine given orally is a traditional and effective treatment if used early in an attack. Useful regimens are one 0.6-mg tablet given every 8 h with subsequent tapering or 1.2 mg followed by 0.6 mg in 1 h with subsequent day dosing depending on response. This is generally better tolerated than the formerly advised higher dose regimens. The drug must be at least temporarily discontinued promptly at the first sign of loose stools, and symptomatic treatment must be given for the diarrhea. Intravenous

1	advised higher dose regimens. The drug must be at least temporarily discontinued promptly at the first sign of loose stools, and symptomatic treatment must be given for the diarrhea. Intravenous colchicine has been taken off the market. NSAIDs given in full anti-inflammatory doses are effective in ∼90% of patients, and the resolution of signs and symptoms usually occurs in 5–8 days. The most effective drugs are any of those with a short half-life and include indomethacin, 25–50 mg tid; naproxen, 500 mg bid; ibuprofen, 800 mg tid; diclofenac, 50 mg tid; and celecoxib 800 mg followed by 400 mg 12 h later, then 400 mg bid.

1	Glucocorticoids given IM or orally, for example, prednisone, 30–50 mg/d as the initial dose and gradually tapered with the resolution of the attack, can be effective in polyarticular gout. For a single joint or a few involved joints, intraarticular triamcinolone acetonide, 20–40 mg, or methylprednisolone, 25–50 mg, have been effective and well tolerated. Based on recent evidence on the essential role of the inflammasome and interleukin 1β (IL-1β) in acute gout, anakinra has been used, and other inhibitors of IL-1β, including canakinumab and rilonacept, are under investigation.

1	Ultimate control of gout requires correction of the basic underlying defect: the hyperuricemia. Attempts to normalize serum uric acid to <300–360 μmol/L (5.0–6.0 mg/dL) to prevent recurrent gouty attacks and eliminate tophaceous deposits are critical and entail a commitment to hypouricemic regimens and medications that generally are required for life. Hypouricemic drug therapy should be considered when, as in most patients, the hyperuricemia cannot be corrected by simple means (control of body weight, low-purine diet, increase in liquid intake, limitation of ethanol use, decreased use of fructose-containing foods and beverages, and avoidance of diuretics). The decision to initiate hypouricemic therapy usually is made taking into consideration the number of acute attacks (urate lowering may be cost-effective after two attacks), serum uric acid levels (progression is more rapid in patients with serum uric acid >535 μmol/L [>9.0 mg/dL]), the patient’s willingness to commit to lifelong

1	may be cost-effective after two attacks), serum uric acid levels (progression is more rapid in patients with serum uric acid >535 μmol/L [>9.0 mg/dL]), the patient’s willingness to commit to lifelong therapy, or the presence of uric acid stones. Urate-lowering therapy should be initiated in any patient who already has tophi or chronic gouty arthritis. Uricosuric agents such as probenecid can be used in patients with good renal function who underexcrete uric acid, with <600 mg in a 24-h urine sample. Urine volume should be maintained by ingestion of 1500 mL of water every day. Probenecid can be started at a dose of 250 mg twice daily and increased gradually as needed up to 3 g per day to achieve and maintain a serum uric acid level of less than 6 mg/dL. Probenecid is generally not effective in patients with serum creatinine levels >177 μmol/L (2 mg/dL). These patients may require allopurinol or benzbromarone (not available in the United States). Benzbromarone is another uricosuric drug

1	in patients with serum creatinine levels >177 μmol/L (2 mg/dL). These patients may require allopurinol or benzbromarone (not available in the United States). Benzbromarone is another uricosuric drug that is more effective in patients with chronic kidney disease. Some agents used to treat common comorbidities, including losartan, fenofibrate, and amlodipine, have some mild uricosuric effects.

1	The xanthine oxidase inhibitor allopurinol is by far the most commonly used hypouricemic agent and is the best drug to lower serum urate in overproducers, urate stone formers, and patients with renal disease. It can be given in a single morning dose, usually 100 mg initially and increasing up to 800 mg if needed. In patients with chronic renal disease, the initial allopurinol dose should be lower and adjusted depending on the serum creatinine concentration; for example, with a creatinine clearance of 10 mL/min, one generally would use 100 mg every other day. Doses can be increased gradually to reach the target urate level of less than 6 mg/dL. Toxicity of allopurinol has been recognized increasingly in patients who use thiazide diuretics, in patients allergic to penicillin and ampicillin, and in Asians expressing HLA-B*5801. The most serious side effects include life-threatening toxic epidermal necrolysis, systemic vasculitis, bone marrow suppression, granulomatous hepatitis, and

1	and in Asians expressing HLA-B*5801. The most serious side effects include life-threatening toxic epidermal necrolysis, systemic vasculitis, bone marrow suppression, granulomatous hepatitis, and renal failure. Patients with mild cutaneous reactions to allopurinol can reconsider the use of a uricosuric agent, undergo an attempt at desensitization to allopurinol, or take febuxostat, a new, chemically unrelated specific xanthine oxidase inhibitor. Febuxostat is approved in the United States at 40 or 80 mg once a day and does not require dose adjustment in mild to moderate renal disease. Pegloticase is a pegylated uricase, now available for patients who do not tolerate or fail full doses of other treatments. It is given intravenously usually at 8 mg every 2 weeks and can dramatically lower serum uric acid in up to 50% of such patients. New uricosurics are also undergoing investigation.

1	Urate-lowering drugs are generally not initiated during acute attacks but after the patient is stable and low-dose colchicine has been initiated to decrease the risk of the flares that often occur with urate lowering. Colchicine anti-inflammatory prophylaxis in doses of 0.6 mg one to two times daily should be given along with the hypouricemic therapy until the patient is normouricemic and without gouty attacks for 6 months or as long as tophi are present. Colchicine should not be used in dialysis patients and is given in lower doses in patients with renal disease or with P glycoprotein or CYP3A4 inhibitors such as clarithromycin that can increase toxicity of colchicine.

1	The deposition of CPP crystals in articular tissues is most common in the elderly, occurring in 10–15% of persons age 65–75 years and 30–50% of those >85years. In most cases,this processis asymptomatic and the cause of CPPD is uncertain. Because >80% of patients are >60 years and 70% have preexisting joint damage from other conditions, it is likely that biochemical changes in aging or diseased cartilage favor crystal nucleation. In patients with CPPD arthritis, there is increased production of inorganic pyrophosphate and decreased levels of pyrophosphatases in cartilage extracts. Mutations in the ANKH gene, as described in both familial and sporadic cases, can increase elaboration and extracellular transport of pyrophosphate. The increase in pyrophosphate production appears to be related to enhanced activity of ATP pyrophosphohydrolase and 5′-nucleotidase, which catalyze the reaction of ATP to adenosine and pyrophosphate. This pyrophosphate could combine with calcium to form CPP

1	to enhanced activity of ATP pyrophosphohydrolase and 5′-nucleotidase, which catalyze the reaction of ATP to adenosine and pyrophosphate. This pyrophosphate could combine with calcium to form CPP crystals in matrix vesicles or on collagen fibers. There are decreased levels of cartilage glycosaminoglycans that normally inhibit and regulate crystal nucleation. Highactivities of transglutaminaseenzymes also may contribute to the deposition of CPP crystals.

1	ReleaseofCPPcrystalsintothejointspaceisfollowedbythephagocytosis of those crystals by monocyte-macrophages and neutrophils, which respond by releasing chemotactic and inflammatory substances and, as with MSU crystals, activating the inflammasome.

1	A minority of patients with CPPD arthropathy have metabolic abnormalities or hereditary CPP disease (Table 395-2). These associations suggest that a variety of different metabolic products may enhance CPP crystal deposition either by directly altering cartilage or by inhibiting inorganic pyrophosphatases. Included among these conditions are hyperparathyroidism, hemochromatosis, hypophosphatasia, hypomagnesemia, and possibly myxedema. The presence of CPPD arthritis in individuals <50 years old should lead to consideration of these metabolic disorders (Table 395-2) and inherited forms of disease, including those identified in a variety of ethnic groups. Genomic DNA studies performed on different kindreds have shown a possible location of genetic defects on chromosome 8q or on chromosome 5p in a region that expresses the gene of the membrane pyrophosphate channel (ANKH gene). As noted above, mutations described in the ANKH gene in kindreds with CPPD arthritis can increase extracellular

1	in a region that expresses the gene of the membrane pyrophosphate channel (ANKH gene). As noted above, mutations described in the ANKH gene in kindreds with CPPD arthritis can increase extracellular pyrophosphate and induce CPP crystal formation. Investigation of younger patients with CPPD should include inquiry for evidence of familial aggregation and evaluation of serum calcium, phosphorus, alkaline phosphatase, magnesium, iron, and transferrin.

1	CPPD arthropathy may be asymptomatic, acute, subacute, or chronic or may cause acute synovitis superimposed on chronically involved joints. Acute CPPD arthritis originally was termed pseudogout by McCarty and co-workers because of its striking similarity to gout. Other clinical manifestations of CPPD include (1) association with or enhancement of peculiar forms of osteoarthritis; (2) induction of severe destructive disease that may radiographically mimic neuropathic arthritis; (3) production of chronic symmetric synovitis that is clinically similar to rheumatoid arthritis; (4) intervertebral disk and ligament calcification with restriction of spine mobility, the crowned dens syndrome, or spinal stenosis (most commonly seen in the elderly); and (5) rarely periarticular tophus-like nodules.

1	The knee is the joint most frequently affected in CPPD arthropathy. Other sites include the wrist, shoulder, ankle, elbow, and hands. The temporomandibular joint may be involved. Clinical and radiographic evidenceindicates that CPPDdeposition is polyarticularinat least two-thirds of patients. When the clinical picture resembles that of slowly progressive osteoarthritis, diagnosis may be difficult. Joint distribution may provide important clues suggesting CPPD disease. For example, primary osteoarthritis less often involves metacarpophalangeal, wrist, elbow, shoulder, or ankle joints. If radiographs or ultrasound reveal punctate and/or linear radiodense deposits within fibrocartilaginous joint menisci or articular hyaline cartilage (chondrocalcinosis), the diagnostic likelihood of CPPD disease is further increased. Definitive diagnosis requires demonstration of typical rhomboid or rodlike crystals (generally weakly positively birefringent or nonbirefringent with polarized light) in

1	disease is further increased. Definitive diagnosis requires demonstration of typical rhomboid or rodlike crystals (generally weakly positively birefringent or nonbirefringent with polarized light) in synovial fluid or articular tissue (Fig. 395-2). In the absence of joint effusion or indications to obtain a synovial biopsy, chondrocalcinosis is presumptive of CPPD. One exception is chondrocalcinosis due to CaOx in some patients with chronic renal failure.

1	Acute attacks of CPPD arthritis may be precipitated by trauma. Rapid diminution of serum calcium concentration, as may occur in severe medical illness or after surgery (especially parathyroidectomy), can also lead to attacks.

1	Inasmanyas50%ofcases,episodesofCPPD-inducedinflammation are associated with low-grade fever and, on occasion, temperatures as high as 40°C (104°F). In such cases, synovial fluid analysis with microbial cultures is essential to rule out the possibility of infection. In fact, infection in a joint with any microcrystalline deposition process can lead to crystal shedding and subsequent synovitis from both crystals and microorganisms. The leukocyte count in synovial fluid in acute CPPD can range from several thousand cells to 100,000 cells/μL, with the mean being about 24,000 cells/μL and the predominant cell being the neutrophil. CPP crystals may be seen inside tissue fragments and fibrin clots and in neutrophils (Fig. 395-2). CPP crystals may coexist with MSU and apatite in some cases.

1	FIGUrE 395-2 Intracellular and extracellular calcium pyrophos-phate (CPP) crystals, asseeninafreshpreparationofsynovialfluid,illustraterectangular,rod-shaped,andrhomboidcrystalsthatareweaklypositivelyornonbirefringentcrystals(compensatedpolarizedlightmicroscopy;400×).

1	Untreated acute attacks may last a few days to as long as a month. Treatment by rest, joint aspiration, and NSAIDs or by intraarticular glucocorticoid injection may result in more rapid return to prior status. For patients with frequent recurrent attacks, daily prophylactic treatment with low doses of colchicine may be helpful in decreasing the frequency of the attacks. Severe polyarticular attacks usually require short courses of glucocorticoids or, as recently reported, an IL-1β antagonist, anakinra. Unfortunately, there is no effective way to remove CPP deposits from cartilage and synovium. Uncontrolled studies suggest that the administration of NSAIDS (with a gastric protective agent if required), hydroxychloroquine, or even methotrexate may be helpful in controlling persistent synovitis. Patients with progressive destructive large-joint arthropathy may require joint replacement.

1	Apatite is the primary mineral of normal bone and teeth. Abnormal accumulationofbasiccalciumphosphates,largelycarbonatesubstituted apatite, can occur in areas of tissue damage (dystrophic calcification), hypercalcemicorhyperparathyroidstates(metastaticcalcification),and certain conditions of unknown cause (Table 395-3). In chronic renal failure, hyperphosphatemia can contribute to extensive apatite deposition both in and around joints. Familial aggregation is rarely seen; no association with ANKH mutations has been described thus far. Apatite crystals are deposited primarily on matrix vessels. Incompletely understood alterations in matrix proteoglycans, phosphatases, hormones, and cytokines probably can influence crystal formation.

1	Apatite aggregates are commonly present in synovial fluid in an extremely destructive chronic arthropathy of the elderly that occurs mostoftenintheshoulders(Milwaukeeshoulder)andinasimilarprocess in hips, knees, and erosive osteoarthritis of fingers. Joint destruction is associated with damage to cartilage and supporting structures, leading to instability and deformity. Progression tends to be indolent. Symptoms range from minimal to severe pain and disability that may lead to joint replacement surgery. Whether severely affected patients represent an extreme synovial tissue response to the apatite crystals that are so common in osteoarthritis is uncertain. Synovial lining cell or fibroblast cultures exposed to apatite (or CPP) crystals can undergo Hemorrhagic shoulder effusions in the elderly (Milwaukee shoulder) Tendinitis, bursitis Connective tissue diseases (e.g., systemic sclerosis, dermatomyositis, SLE)

1	Hemorrhagic shoulder effusions in the elderly (Milwaukee shoulder) Tendinitis, bursitis Connective tissue diseases (e.g., systemic sclerosis, dermatomyositis, SLE) Heterotopic calcification after neurologic catastrophes (e.g., stroke, spinal Bursitis, arthritis Abbreviation: SLE, systemic lupus erythematosus. mitosis and markedly increase the release of prostaglandin E2, various cytokines, and also collagenases and neutral proteases, underscoring the destructivepotential of abnormallystimulated synovial lining cells.

1	Periarticular or articular deposits may occur and may be associated with acute reversible inflammation and/or chronic damage to the joint capsule, tendons, bursa, or articular surfaces. The most common sites of apatite deposition include bursae and tendons in and/or around the knees, shoulders, hips, and fingers. Clinical manifestations include asymptomatic radiographic abnormalities, acute synovitis, bursitis, tendinitis, and chronic destructive arthropathy. Although the true incidence of apatite arthritis is not known, 30–50% of patients with osteoarthritis have apatite microcrystals in their synovial fluid. Such crystals frequently can be identified in clinically stable osteoarthritic joints, but they are more likely to come to attention in persons experiencing acute or subacute worsening of joint pain and swelling. The synovial fluid leukocyte count in apatite arthritis is usually low (<2000/μL) despite dramatic symptoms, with predominance of mononuclear cells.

1	Intra-and/or periarticular calcifications with or without erosive, destructive, or hypertrophic changes may be seen on radiographs (Fig. 395-3). They should be distinguished from the linear calcifications typical of CPPD.

1	Definitive diagnosis of apatite arthropathy, also called basic calcium phosphate disease, depends on identification of crystals from synovial fluid or tissue (Fig. 395-3). Individual crystals are very small and can be seen only by electron microscopy. Clumps of crystals may appear as 1-to 20-μm shiny intra-or extracellular nonbirefringent globules or aggregates that stain purplish with Wright’s stain and bright red with alizarin red S. Tetracycline binding and other investigative techniques are under consideration as labeling alternatives. Absolute identification depends on electron microscopy with energy-dispersive elemental analysis, x-ray diffraction, infrared spectroscopy, or Raman microspectroscopy, but these techniques usually are not required in clinical diagnosis.

1	Treatment of apatite arthritis or periarthritis is nonspecific. Acute attacks of bursitis or synovitis may be self-limiting, resolving in days to several weeks. Aspiration of effusions and the use of either NSAIDs or oral colchicine for 2 weeks or intraor periarticular injection of a depot glucocorticoid appear to shorten the duration and intensity of symptoms. Local injection of disodium ethylenediaminetetraacetic acid (EDTA) and SC anakinra have been suggested as effective in single studies of acute calcific tendinitis at the shoulder. Other reports have described that IV gamma globulin, rituximab, calcium channel blockers, or bisphosphonates may help diffuse calcinosis. Periarticular apatite deposits may be resorbed with resolution of attacks. Agents to lower serum phosphate levels may lead to resorption of deposits in renal failure patients receiving hemodialysis. In patients with underlying severe destructive articular changes, response to medical therapy is usually less

1	may lead to resorption of deposits in renal failure patients receiving hemodialysis. In patients with underlying severe destructive articular changes, response to medical therapy is usually less rewarding.

1	FIGUrE 395-3 A. Radiographshowingcalcificationduetoapatitecrystalssurroundinganerodedjoint.B. Anelectronmicrographdem-onstratesdarkneedle-shapedapatitecrystalswithinavacuoleofasynovialfluidmononuclearcell(30,000×). FIGUrE 395-4 Bipyramidal and small polymorphic calcium oxa-late crystals fromsynovialfluidareaclassicfindingincalciumoxalatearthropathy(ordinarylightmicroscopy;400×). different enzyme defects, leading to hyperoxalemia and deposition of CaOx crystals in tissues. Nephrocalcinosis and renal failure are typical results. Acute and/or chronic CaOx arthritis, periarthritis, and bone disease may complicate primary oxalosis during later years of illness.

1	Secondary oxalosis is more common than the primary disorder. In chronic renal disease, calcium oxalate deposits have long been recognized in visceral organs, blood vessels, bones, and cartilage and arenow known to be one of the causes of arthritis in chronic renal failure. Thus far, reported patients have been dependent on long-term hemodialysis or peritonealdialysis (Chap. 336), and many had receivedascorbic acid supplements. Ascorbic acid is metabolized to oxalate, which is inadequately cleared in uremia and by dialysis. Such supplements and foods highinoxalatecontentusuallyareavoidedindialysisprogramsbecause of the risk of enhancing hyperoxalosis and its sequelae.

1	CaOx aggregates can be found in bone, articular cartilage, synovium, and periarticular tissues. From these sites, crystals may be shed, causing acute synovitis. Persistent aggregates of CaOx can, like apatite and CPP, stimulate synovial cell proliferation and enzyme release, resulting in progressive articular destruction. Deposits have been documented in fingers, wrists, elbows, knees, ankles, and feet. Clinical features of acute CaOx arthritis may not be distinguishable from those due to urate, CPP, or apatite. Radiographs may reveal chondrocalcinosis or soft tissue calcifications. CaOx-induced synovial effusions are usually noninflammatory, with <2000 leukocytes/μL, or mildly inflammatory. Neutrophils or mononuclear cells can predominate. CaOx crystals have a variable shape and variable birefringence to polarized light. The most easily recognized forms are bipyramidal, have strong birefringence (Fig. 395-4), and stain with alizarin red S.

1	Treatment of CaOx arthropathy with NSAIDs, colchicine, intra-articular glucocorticoids, and/or an increased frequency of dialysis has produced only slight improvement. In primary oxalosis, liver transplantation has induced a significant reduction in crystal deposits (Chap. 434e). AcknowledgmentPaTHOGENESIS This chapter has been revised for this and the previous two editions from Primary oxalosis is a rare hereditary metabolic disorder (Chap. 434e). an original version written by Antonio Reginato, MD, in earlier editions Enhanced production of oxalic acid may result from at least two of Harrison’s Principles of Internal Medicine.

1	2238 fibromyalgia Leslie J. Crofford DEFINITION Fibromyalgia(FM)ischaracterizedbychronicwidespreadmuscu-loskeletalpainandtenderness.AlthoughFMisdefinedprimarilyasapainsyndrome,patientsalsocommonlyreportassociatedneuro-396 psychological symptoms of fatigue, unrefreshing sleep, cognitive dysfunction, anxiety, and depression. Patients with FM have an increased prevalence of other syndromes associated with pain and fatigue, including chronic fatigue syndrome (Chap. 464e), temporomandibular disorder, chronic headaches, irritable bowel syndrome, interstitial cystitis/painful bladder syndrome, and other pelvic pain syndromes. Available evidence implicates the central nervous system askeytomaintainingpainandothercoresymptomsofFMandrelated conditions. The presence of FM is associated with substantial negative consequences for physical and social functioning.

1	In clinical settings, a diagnosis of FM is made in ∼2% of the population and is far more common in women than in men, with a ratio of ∼9:1. However, in population-based survey studiesworldwide,theprevalencerateis ∼2–5%,withafemale-to-male ratio of only 2–3:1 and with some variability depending on the method of ascertainment. The prevalence data are similar across socioeconomic classes. Cultural factors may play a role in determining whether patients with FM symptoms seek medical attention; however, even in cultures in which secondary gain is not expected to play a significant role, the prevalence of FM remains in this range.

1	CLINICaL MaNIFESTaTIONS Pain and Tenderness At presentation, patients with FM most commonly report “pain all over.” These patients have pain that is typically both above and below the waist on both sides of the body and involves the axial skeleton (neck, back, or chest). The pain attributable to FM is poorly localized, difficult to ignore, severe in its intensity, and associated with a reduced functional capacity. For a diagnosis of FM, pain should have been present most of the day on most days for at least 3 months.

1	The clinical pain of FM is associated with increased evoked pain sensitivity. In clinical practice, this elevated sensitivity may be determined by a tender-point examination in which the examiner uses the thumbnail to exert pressure of ∼4 kg/m2 (or the amount of pressure leading to blanching of the tip of the thumbnail) on well-defined musculotendinous sites (Fig. 396-1). Previously, the classification criteria of the American College of Rheumatology required that 11 of 18 sites be perceived as painful for a diagnosis of FM. In practice, tenderness is a continuous variable, and strict application of a categorical threshold for diagnostic specifics is not necessary. Newer criteria eliminate the need for tender points and focus instead on clinical symptoms of widespread pain and neuropsychological symptoms. The newer criteria perform well in a clinical setting in comparison to the older, tender-point criteria. However, it appears that when the new criteria

1	pain and neuropsychological symptoms. The newer criteria perform well in a clinical setting in comparison to the older, tender-point criteria. However, it appears that when the new criteria areappliedtopopulations,theresultisanincreaseinprevalenceofFM and a change in the sex ratio (see “Epidemiology,” earlier).

1	Patients with FM often have peripheral pain generators that are thought to serve as triggers for the more widespread pain attributed to central nervous system factors. Potential pain generators such as arthritis, bursitis, tendinitis, neuropathies, and other inflammatory or degenerative conditions should be identified by history and physical examination. More subtle pain generators may include joint hypermobility and scoliosis. In addition, patients may have chronic myalgias triggered by infectious, metabolic, or psychiatric conditions that can also serve as triggers for the development of FM. These conditions are often identified in the differential diagnosis of patients with FM, and a major challenge is to distinguish the ongoing activity of a triggering condition from FM that is occurring as a consequence of a comorbid condition and that should itself be treated.

1	Neuropsychological Symptoms In addition to widespread pain, FM patients typically report fatigue, stiffness, sleep disturbance, cognitive dysfunction, anxiety, and depression. These symptoms are present to varyingdegreesinmostFMpatientsbutarenotpresentineverypatient or at all times in a given patient. Relative to pain, such symptoms may, however, have an equal or even greater impact on function and quality of life. Fatigue is highly prevalent in patients under primary care who ultimately are diagnosed with FM. Pain, stiffness, and fatigue often Occiput: Trapezius: midpoint of the upper border Supraspinatus: above the medial border of the scapular spine Gluteal: upper outer quadrants of buttocks Greater trochanter: posterior to the trochanteric prominence Low cervical: anterior aspects of the intertransverse spaces at C5-C7 Second rib: Lateral epicondyle: 2 cm distal to the epicondyles Knee: medial fat pad proximal to the joint line

1	FIGUrE 396-1 Tender-point assessment in patients with fibromyalgia. (Figure created using data from F Wolfe et al: Arthritis Care Res 62:600, 2010.) are worsened by exercise or unaccustomed activity (postexertional malaise). The sleep complaints include difficulty falling asleep, difficulty staying asleep, and early-morning awakening. Regardless of the specific complaint, patients awake feeling unrefreshed. Patients with FM may meet criteria for restless legs syndrome and sleep-disordered breathing; frank sleep apnea can also be documented. Cognitive issues are characterized as slowness in processing, difficulties with attention or concentration, problems with word retrieval, and short-term memory loss. Studies have demonstrated altered cognitive function in these domains in patients with FM, though speed of processing is age-appropriate. Symptoms of anxiety and depression are common, and the lifetime prevalence of mood disorders in patients with FM approaches 80%. Although depression

1	FM, though speed of processing is age-appropriate. Symptoms of anxiety and depression are common, and the lifetime prevalence of mood disorders in patients with FM approaches 80%. Although depression is neither necessary nor sufficient for the diagnosis of FM, it is important to screen for major depressive disorders by querying for depressed mood and anhedonia. Analysis of genetic factors that are likely to predispose to FM reveals shared neurobiologic pathways with mood disorders, providing the basis for comorbidity (see later in this chapter).

1	Overlapping Syndromes Because FM can overlap in presentation with other chronic pain conditions, review of systems often reveals headaches, facial/jaw pain, regional myofascial pain particularly involving the neck or back, and arthritis. Visceral pain involving the gastrointestinal tract, bladder, and pelvic or perineal region is often present as well. Patients may or may not meet defined criteria for specific syndromes.Itisimportantforpatients tounderstandthatsharedpathwaysmay mediate symptoms andthattreatmentstrategieseffectivefor one condition may help with global symptom management.

1	Comorbid Conditions FM is often comorbid with chronic musculoskeletal, infectious, metabolic, or psychiatric conditions. Whereas FM affects only 2–5% of the general population, it occurs in 20% or more of patients with degenerative or inflammatory rheumatic disorders, likely because these conditions serve as peripheral pain generators to alter central pain-processing pathways. Similarly, chronic infectious, metabolic, or psychiatric diseases associated with musculoskeletal pain can mimic FM and/or serve as a trigger for the development of FM. It is particularly important for clinicians to be sensitive to pain management of these comorbid conditions so that when FM emerges—characterized by pain outside the boundaries of what could reasonably be explained by the triggering condition, development of neuropsychological symptoms, or tenderness on physical examination—treatment of central pain processes will be undertaken as opposed to a continued focus on treatment of peripheral or

1	development of neuropsychological symptoms, or tenderness on physical examination—treatment of central pain processes will be undertaken as opposed to a continued focus on treatment of peripheral or inflammatory causes of pain.

1	Psychosocial Considerations Symptoms of FM often have their onset and are exacerbated during periods of high-level real or perceived stress. This pattern may reflect an interaction among central stress physiology, vigilance or anxiety, and central pain-processing pathways. An understanding of current psychosocial stressors will aid in patient management, as many factors that exacerbate symptoms cannot be addressed by pharmacologic approaches. Furthermore, there is a high prevalence of exposure to previous interpersonal and other forms of violence in patients with FM and related conditions. If post-traumatic stress disorder is an issue, the clinician should be aware of it and consider treatment options.

1	Functional Impairment It is crucial to evaluate the impact of FM symptoms on functionand role fulfillment. Indefining the successof a management strategy, improved function is a key measure. Functional assessment should include physical, mental, and social domains. A recognition of the ways in which role functioning falls short will be helpful in the establishment of treatment goals. Because musculoskeletal pain is such a common complaint, the differential diagnosis of FM is broad. Table 396-1 lists some of the more common conditions that should be considered. Patients with inflammatory causes for widespread pain should be identifiable on the basis of specific history, physical findings, and laboratory or radiographic tests. Polymyalgia rheumatica Inflammatory arthritis: rheumatoid arthritis, spondyloarthritides Connective tissue diseases: systemic lupus erythematosus, Sjögren’s

1	Polymyalgia rheumatica Inflammatory arthritis: rheumatoid arthritis, spondyloarthritides Connective tissue diseases: systemic lupus erythematosus, Sjögren’s Degenerative joint/spine/disk disease Myofascial pain syndromes Bursitis, tendinitis, repetitive strain injuries Routine laboratory and radiographic tests yield normal results in FM. Thus diagnostic testing is focused on exclusion of other diagnoses and evaluation for pain generators or comorbid conditions (Table 396-2). Mostpatientswithnewchronicwidespreadpainshouldbeassessedfor the most common entities in the differential diagnosis. Radiographic testing should be used sparingly and only for diagnosis of inflammatory arthritis. After the patient has been evaluated thoroughly, repeat testing is discouraged unless the symptom complex changes. Particularly to be discouraged is advanced imaging (MRI) of the spine unless there are features suggesting inflammatory spine disease or neurologic symptoms.

1	As in most complex diseases, it is likely that a number of genes contribute to vulnerability to the development of FM. To date, these genes appear to be in pathways controlling pain and stress Source: LM Arnold et al: J Women’s Health 21:231, 2012; MA Fitzcharles et al: J Rheumatol 40:1388, 2013.

1	2240 responses. Some of the genetic underpinnings of FM are shared across other chronic pain conditions. Genes associated with metabolism, transport, and receptors of serotonin and other monoamines have been implicated in FM and overlapping conditions. Genes associated with other pathways involved in pain transmission have also been describedasvulnerabilityfactorsforFM.Takentogether,thepathways in which polymorphisms have been identified in FM patients further implicate central factors in mediation of the physiology that leads to the clinical manifestations of FM. Psychophysical testing of patients with FM has demonstrated altered sensory afferent pain processing and impaired descending noxious inhibitory control leading to hyperalgesia and allodynia. Functional MRI and other research imaging procedures clearly demonstrate activation of the brain regions involved in the experience of pain in response to stimuli that are innocuous in study participants without FM. Pain perception in FM

1	procedures clearly demonstrate activation of the brain regions involved in the experience of pain in response to stimuli that are innocuous in study participants without FM. Pain perception in FM patients is influenced by the emotional and cognitive dimensions, such as catastrophizing and perceptions of control, providing a solid basis for recommendations for cognitive and behavioral treatment strategies.

1	APPROACH TO THE PATIENT: FM is common and has an extraordinary impact on the patient’s function and health-related quality of life. However, its symptoms and impact can be managed effectively by physicians and other health professionals. Developing a partnership with patients is essential for improving the outcome of FM, with a goal of understanding the factors involved, implementing a treatment strategy, and choosing appropriate nonpharmacologic and pharmacologic treatments.

1	Patients with chronic pain, fatigue, and other neuropsychological symptoms require a framework for understanding the symptoms that have such an important impact on their function and quality of life. Explaining the genetics, triggers, and physiology of FM can be an important adjunct in relieving associated anxiety and in reducing the overall cost of health care resources. In addition, patients must be educated regarding expectations for treatment. The physician should focus on improved function and quality of life rather than elimination of pain. Illness behaviors, such as frequent physician visits, should be discouraged and behaviors that focus on improved function strongly encouraged.

1	Treatment strategies should include physical conditioning, with encouragement to begin at low levels of aerobic exercise and to proceed with slow but consistent advancement. Patients who have been physically inactive or who report postexertional malaise may do best in supervised or water-based programs at the start. Activities that promote improved physical function with relaxation, such as yoga and Tai Chi, may also be helpful. Strength training may be recommended after patients reach their aerobic goals. Exercise programs are helpful in reducing tenderness and enhancing self-efficacy. Cognitive-behavioral strategies to improve sleep hygiene and reduce illness behaviors can also be helpful in management.

1	It is essential for the clinician to treat any comorbid triggering condition and to clearly delineate for the patient the treatment goals for each medication. For example, glucocorticoids or nonsteroidal anti-inflammatory drugs may be useful for management of inflammatory triggers but are not effective against FM-related symptoms. At present, the treatment approaches that have proved most successful in FM patients target afferent or descending pain pathways. Table 396-3 lists the drugs with demonstrated effectiveness. It should be emphasized Antidepressants: balanced serotonin–norepinephrine reuptake inhibitors Duloxetineb,c Milnacipranb,c Anticonvulsants: ligands of the alpha-2-delta subunit of voltage-gated calcium channels aRA Moore et al: Cochrane Database Syst Rev 12:CD008242, 2012. bApproved by the U.S. Food and Drug Administration. cW Hauser et al: Cochrane Database Syst Rev 1: CD010292, 2013. Source: LM Arnold: Arthritis Rheum 56:1336, 2007.

1	Source: LM Arnold: Arthritis Rheum 56:1336, 2007. strongly that opioid analgesics are to be avoided in patients with FM. These agents have no demonstrated efficacy in FM and are associated with opioid-induced hyperalgesia that can worsen both symptoms and function. Use of single agents to treat multiple symptom domains is strongly encouraged. For example, if a patient’s symptom complex is dominated by pain and sleep disturbance, use of an agent that exerts both analgesic and sleep-promoting effects is desirable. These agents include sedating antidepressants such as amitriptyline and alpha-2delta ligands such as gabapentin and pregabalin. For patients whose pain is associated with fatigue, anxiety, or depression, drugs that have both analgesic and antidepressant/anxiolytic effects, such as duloxetine or milnacipran, may be the best first choice. arthritis associated with Systemic Disease, and Other arthritides Carol A. Langford, Brian F. Mandell

1	arthritis associated with Systemic Disease, and Other arthritides Carol A. Langford, Brian F. Mandell Acromegaly is the result of excessive production of growth hormone by an adenoma in the anterior pituitary gland (Chap. 403). The excessive secretion of growth hormone along with insulin-like growth factor I stimulates proliferation of cartilage, periarticular connective tissue, and bone, resulting in several musculoskeletal problems, including osteoarthritis, back pain, muscle weakness, and carpal tunnel syndrome.

1	Osteoarthritis is a common feature, most often affecting the knees, shoulders, hips, and hands. Single or multiple joints may be affected. Hypertrophy of cartilage initially produces radiographic widening of the joint space. The newly synthesized cartilage is abnormally susceptible to fissuring, ulceration, and destruction. Ligamental laxity of joints further contributes to the development of osteoarthritis. Cartilage degrades, the joint space narrows, and subchondral sclerosis and osteophytes develop. Joint examination reveals crepitus and laxity. Joint fluid is noninflammatory. Calcium pyrophosphate dihydrate crystals are found in the cartilage in some cases of acromegaly arthropathy and, when shed into the joint, can elicit attacks of pseudogout. Chondrocalcinosis may be observed on radiographs. Back pain is extremely common, perhaps as a result of spine hyper-mobility. Spine radiographs show normal or widened intervertebral disk spaces, hypertrophic anterior osteophytes, and

1	on radiographs. Back pain is extremely common, perhaps as a result of spine hyper-mobility. Spine radiographs show normal or widened intervertebral disk spaces, hypertrophic anterior osteophytes, and ligamental calcification. The latter changes are similar to those observed in patients with diffuse idiopathic skeletal hyperostosis. Dorsal kyphosis in conjunction with elongation of the ribs contributes to the development of the barrel chest seen in acromegalic patients. The hands and feet become enlarged as a result of soft tissue proliferation. The fingers are thickened and have spadelike distal tufts. One-third of patients have a thickened heel pad. Approximately 25% of patients exhibit Raynaud’s phenomenon.Carpaltunnelsyndromeoccursinabouthalfofpatients. The median nerve is compressed by excess connective tissue in the carpal tunnel. Patients with acromegaly may develop proximal muscle weakness, which is thought to be caused by the effect of growth hormone on muscle. Serum muscle

1	by excess connective tissue in the carpal tunnel. Patients with acromegaly may develop proximal muscle weakness, which is thought to be caused by the effect of growth hormone on muscle. Serum muscle enzyme levels and electromyographic findings arenormal. Muscle biopsy specimens containmuscle fibers of varying size without inflammation.

1	Hemochromatosis is a disorder of iron storage. Absorption of excessive amounts of iron from the intestine leads to iron deposition in parenchymal cells, which results in impairment of organ function (Chap. 428). Symptoms of hemochromatosis usually begin between the ages of 40 and 60 but can appear earlier. Arthropathy, which occurs in 20–40% of patients, usually begins after the age of 50 and may be the first clinical feature of hemochromatosis. The arthropathy is an osteoarthritis-like disorder affecting the small joints of the hands and later the larger joints, such as knees, ankles, shoulders, and hips. The second and third metacarpophalangeal joints of both hands are often the first and most prominent joints affected; this clinical picture may provide an important clue to the possibility of hemochromatosis becausethese jointsarenotpredominantlyaffectedby “routine”osteoarthritis.Patients experience some morningstiffness andpainwith use of involved joints. The affected joints are

1	of hemochromatosis becausethese jointsarenotpredominantlyaffectedby “routine”osteoarthritis.Patients experience some morningstiffness andpainwith use of involved joints. The affected joints are enlarged and mildly tender. Radiographs show narrowing of the joint space, subchondral sclerosis, subchondral cysts, and juxtaarticular proliferation of bone. Hooklike osteophytes are seen in up to 20% of patients; although they are regarded as a characteristic feature of hemochromatosis, they can also occur in osteoarthritis and are not disease specific. The synovial fluid is noninflammatory. The synovium shows mild to moderate proliferation of iron-containing lining cells, fibrosis, and some mononuclear cell infiltration. In approximately half of patients, there is evidence of calcium pyrophosphate deposition disease, and some patients late in the course of disease experience episodes of acute pseudogout (Chap. 395). An early diagnosis is suggested by high serum transferrin saturation, which

1	deposition disease, and some patients late in the course of disease experience episodes of acute pseudogout (Chap. 395). An early diagnosis is suggested by high serum transferrin saturation, which is more sensitive than ferritin elevation.

1	Iron may damage the articular cartilage in several ways. Iron catalyzes superoxide-dependent lipid peroxidation, which may play a role in joint damage. In animal models, ferric iron has been shown to interfere with collagen formation and increase the release of lysosomal enzymes from cells in the synovial membrane. Iron inhibits synovial tissue pyrophosphatase in vitro and therefore may inhibit pyrophosphatase in vivo, resulting in chondrocalcinosis. The treatment of hemochromatosis is repeated phlebotomy. Unfortunately, this treatment has little effect on established arthritis, which, along with chondrocalcinosis, may progress. Symptom-based treatment of the arthritis consists of administration of acetaminophen and nonsteroidal anti-inflammatory drugs (NSAIDs), as tolerated. Acute pseudogout attacks are treated with high doses of an NSAID or a short course of glucocorticoids. Hip or knee total joint replacement has been successful in advanced disease.

1	Hemophilia is a sex-linked recessive genetic disorder characterized by the absence or deficiency of factor VIII (hemophilia A, orclassic hemophilia) or factor IX (hemophilia B, or Christmas disease) (Chap. 141). Hemophilia A constitutes 85% of cases. Spontaneous hemarthrosis is a common problem with both types of hemophilia and can lead to a deforming arthritis. The frequency and severity of hemarthrosis are related to the degree of clotting factor deficiency. Hemarthrosis is not common in other disorders of coagulation such as von Willebrand disease, factor V deficiency, warfarin therapy, or thrombocytopenia. Hemarthrosis occurs after 1 year of age, when a child begins to walk 2241 and run. In order of frequency, the joints most commonly affected are theknees,ankles,elbows,shoulders,andhips.Smalljointsofthehands and feet are occasionally involved.

1	In the initial stage of arthropathy, hemarthrosis produces a warm, tensely swollen, and painful joint. The patient holds the affected joint in flexion and guards against any movement. Blood in the joint remains liquid because of the absence of intrinsic clotting factors and the absence of tissue thromboplastin in the synovium. The synovial blood is resorbed over a period of ≥1 week, with the precise interval depending on the size of the hemarthrosis. Joint function usually returns to normal or baseline in~2 weeks. Low-grade temperature elevation may accompany hemarthrosis, but a fever >101°F (38.3°C) warrants concern about infection. Recurrent hemarthrosis may result in chronic arthritis. The involved joints remain swollen, and flexion deformities develop. Joint motion may be restricted and function severely limited. Restricted jointmotionorlaxitywithsubluxationisafeatureofend-stagedisease.

1	Bleeding into muscle and soft tissue also causes musculoskeletal dysfunction. When bleeding into the iliopsoas muscle occurs, the hip is held in flexion because of the pain, resulting in a hip flexion contracture. Rotation of the hip is preserved, which distinguishes this problem from hemarthrosis or other causes of hip synovitis. Expansion of the hematoma may place pressure on the femoral nerve, resulting in femoral neuropathy. Hemorrhage into a closed compartment space, such as the calf or the volar compartment in the forearm, can result in muscle necrosis, neuropathy, and flexion deformities of the ankles, wrists, and fingers. When bleeding involves periosteum or bone, a painful pseudotumor forms. These pseudotumors occur distal to the elbows or knees in children and improve with treatment of hemophilia. Surgical removal is indicated if the pseudotumor continues to enlarge. In adults, pseudotumors develop in the femur and pelvis and are usually refractory to treatment. When

1	treatment of hemophilia. Surgical removal is indicated if the pseudotumor continues to enlarge. In adults, pseudotumors develop in the femur and pelvis and are usually refractory to treatment. When bleeding occurs in muscle, cysts may develop within the muscle. Needle aspiration of a cyst is contraindicated because this procedure can induce further bleeding; however, if the cyst becomes secondarily infected, drainage may be necessary (after factor repletion).

1	Septic arthritis is rare in hemophilia and is difficult to distinguish from acute hemarthrosis on physical examination. If there is serious suspicion of an infected joint, the joint should be aspirated immediately, the fluid cultured, and treatment with broad-spectrum antibiotics administered, with coverage for microorganisms including Staphylococcus, until culture results become available. Clotting-factor deficiency should be corrected before arthrocentesis to minimize the risk of traumatic bleeding. Radiographs of joints reflect the stage of disease. In early stages, there is only capsule distention; later, juxtaarticular osteopenia, marginal erosions, and subchondral cysts develop. Late in the disease, the joint space is narrowed and there is bony overgrowth similar to that in osteoarthritis.

1	The treatment of musculoskeletal bleeding is initiated with the immediate infusion of factor VIII or IX at the first sign of joint or muscle hemorrhage. Patients who have developed factor inhibitors are at elevated risk for joint damage and may benefit from receiving recombinant activated factor VII or activated prothrombin complex concentrate. The joint should be rested in a position of forced extension, as tolerated, to avoid contracture. Analgesia should be provided; nonselective NSAIDs, which can diminish platelet function, should be avoided if possible. Selective cyclooxygenase-2 inhibitors do not interfere with platelet function, although cardiovascular and gastrointestinal risks must still be weighed. Synovectomy—open or arthroscopic—may be attempted in patients with chronic symptomatic synovial proliferation and recurrent hemarthrosis, although hypertrophied synovium is highly vascular and subject to bleeding. Both types of synovectomy reduce the number of hemarthroses. Open

1	synovial proliferation and recurrent hemarthrosis, although hypertrophied synovium is highly vascular and subject to bleeding. Both types of synovectomy reduce the number of hemarthroses. Open surgical synovectomy, however, is associated with some loss

1	Arthritis Associated with Systemic Disease, and Other Arthritides 2242 of range of motion. Both require aggressive prophylaxis against bleeding. Radiosynovectomy with either yttrium 90 silicate or phosphorus 31 colloid has been effective and may be attempted when surgical synovectomy is not practical. Total joint replacement is indicated for severe joint destruction and incapacitating pain.

1	arTHrOPaTHIES aSSOCIaTED WITH HEMOGLOBINOPaTHIES Sickle Cell Disease Sickle cell disease (Chap. 127) is associated with several musculoskeletal abnormalities (Table 397-1). Children under the ageof 5 yearsmay develop diffuse swelling, tenderness, and warmth of the hands and feet lasting 1–3 weeks. This condition, referred to as sickle cell dactylitis or hand-foot syndrome, has also been observed in sickle cell thalassemia. Dactylitis is believed to result from infarction of the bone marrow and cortical bone leading to periostitis and soft tissue swelling. Radiographs show periosteal elevation, subperiosteal new-bone formation, and areas of radiolucency and increased density involving the metacarpals, metatarsals, and proximal phalanges. These bonechangesdisappearafterseveralmonths.Thesyndromeleaveslittle ornoresidual damage.Becausehematopoiesisceases inthe small bones of the hands and feet with age, the syndrome is rarely seen after age 5.

1	Sickle cell crisis is associated with periarticular pain and occasionally with joint effusions. The joint and periarticular area are warm and tender. Knees and elbows are most often affected, but other joints can be involved. Joint effusions are usually noninflammatory. Acute synovial infarction can cause a sterile effusion with high neutrophil counts in synovial fluid. Synovial biopsies have shown mild lining-cell proliferation and microvascular thrombosis with infarctions. Scintigraphic studies have shown decreased marrow uptake adjacent to the involved joint. The treatment for sickle cell crisis is detailed in Chap. 127.

1	Patients with sickle cell disease seem predisposed to osteomyelitis, which commonly involves the long tubular bones (Chap. 158); Salmonella is a particularly common cause (Chap. 190). Radiographs of the involved site initially show periosteal elevation, with subsequent disruption of the cortex. Treatment of the infection results in healing of the bone lesion. In addition, sickle cell disease is associated with bone infarction resulting from vaso-occlusion secondary to the sickling of red cells. Bone infarction also occurs in hemoglobin sickle cell disease and sickle cell thalassemia (Chap. 127). The bone pain in sickle cell crisis is due to infarction of bone and bone marrow. In children, infarction of the epiphyseal growth plate interferes with normal growth of the affected extremity. Radiographically, infarction of the bone cortex results in periosteal elevation and irregular thickening of the bone cortex. Infarction in the bone marrow leads to lysis, fibrosis, and new bone

1	Radiographically, infarction of the bone cortex results in periosteal elevation and irregular thickening of the bone cortex. Infarction in the bone marrow leads to lysis, fibrosis, and new bone formation. Clinical distinction between osteomyelitis and bone infarctions can be difficult; imaging can be helpful.

1	Avascular necrosis of the head of the femur occurs in ~5% of patients. It also occurs in the humeral head and less commonly in the distal femur, tibial condyles, distal radius, vertebral bodies, and other juxtaarticular sites. Irregularity of the femoral head and other articular surfaces often results in degenerative joint disease. Radiography of the affected joint may show patchy radiolucency and density followed by flattening of the bone. MRI is a sensitive technique for detecting early avascular necrosis as well as bone infarction elsewhere. Total hip replacement and placement of prostheses in other joints may improve function and relieve joint pain in these patients. Septic arthritis is occasionally encountered in sickle cell disease (Chap. 157). Multiple joints may be infected. Joint infection may result from bacteremia due to splenic dysfunction or from contiguous Joint effusions in sickle cell crises Bone changes secondary to marrow hyperplasia

1	Joint effusions in sickle cell crises Bone changes secondary to marrow hyperplasia Infarction of bone Gouty arthritis Infarction of bone marrow osteomyelitis.ThemorecommonmicroorganismsincludeStaphylococcus aureus,Streptococcus,and Salmonella.Salmonella doesnotcauseseptic arthritis as frequently as it causes osteomyelitis. Acute gouty arthritis is uncommon in sickle cell disease, even though 40% of patients are hyperuricemic. However, it may occur in patients generally not expected to get gout (young patients, female patients). Hyperuricemia is due to overproduction of uric acid secondary to increased red cell turnover as well as suboptimal renal excretion. Attacks may be polyarticular, and diagnostic arthrocentesis should be performed to distinguish infection from gout or synovial infarction.

1	The bone marrow hyperplasia in sickle cell disease results in widening of the medullary cavities, thinning of the cortices, and coarse trabeculations and central cupping of the vertebral bodies. These changes are also seen to a lesser degree in hemoglobin sickle cell disease and sickle cell thalassemia. In normal individuals red marrow is located mostly in the axial skeleton, but in sickle cell disease red marrow is found in the bones of the extremities and even in the tarsal and carpal bones. Vertebral compression may lead to dorsal kyphosis, and softening of the bone in the acetabulum may result in protrusio acetabuli.

1	Thalassemia A congenital disorder of hemoglobin synthesis, β thalassemia is characterized by impaired production of β chains (Chap. 127). Bone and joint abnormalities occur in β thalassemia, being most common in the major and intermedia groups. In one study, ~50% of patients with β thalassemia had evidence of symmetric ankle arthropathy characterized by a dull aching pain that was aggravated by weight bearing. The onset came most often in the second or third decade of life. The degree of ankle pain in these patients varied. Some patients experienced self-limited ankle pain that occurred only after strenuous physical activity and lasted several days or weeks. Other patients had chronic ankle pain that became worse with walking. Symptoms eventually abated in a few patients. Compression of the ankle, calcaneus, or forefoot was painful in some patients. Synovial fluid from two patients was noninflammatory. Radiographs of the ankle showed osteopenia, widened medullary spaces, thin

1	of the ankle, calcaneus, or forefoot was painful in some patients. Synovial fluid from two patients was noninflammatory. Radiographs of the ankle showed osteopenia, widened medullary spaces, thin cortices, and coarse trabeculations— findings that are largely the result of bone marrow expansion. The joint space was preserved. Specimens of bone from three patients revealed osteomalacia, osteopenia, and microfractures. Increased numbers of osteoblasts as well as increased foci of bone resorption were present on the bone surface. Iron staining was found in the bone trabeculae, in osteoid, and in the cement line. Synovium showed hyperplasia of lining cells, which contained deposits of hemosiderin. This arthropathy was considered to be related to the underlying bone pathology. The role of iron overload or abnormal bone metabolism in the pathogenesis of this arthropathy is not known. The arthropathy was treated with analgesics and splints. Patients also received transfusions to decrease

1	overload or abnormal bone metabolism in the pathogenesis of this arthropathy is not known. The arthropathy was treated with analgesics and splints. Patients also received transfusions to decrease hematopoiesis and bone marrow expansion.

1	In patients with β-thalassemia major and β-thalassemia intermedia, other joints are also involved, including the knees, hips, and shoulders. Acquired hemochromatosis with arthropathy has been described in a patient with thalassemia. Gouty arthritis and septic arthritis can occur. Avascular necrosis is not a feature of thalassemia because there is no sickling of red cells leading to thrombosis and infarction.

1	β-Thalassemia minor (also known as β-thalassemia trait) is likewise associated with joint manifestations. Chronic seronegative oligoarthritis affecting predominantly ankles, wrists, and elbows has been described; the affected patients had mild persistent synovitis without large effusions or joint erosions. Recurrent episodes of acute asymmetric arthritis have also been reported; episodes last <1 week and may affect the knees, ankles, shoulders, elbows, wrists, and metacarpal phalangeal joints. The mechanism underlying this arthropathy is unknown. Treatment with NSAIDs is not particularly effective.

1	(See also Chap. 421) Musculoskeletal or cutaneous manifestations may be the first clinical indication of a specific hereditary disorder of lipoprotein metabolism. Patients with familial hypercholesterolemia (previously referred to as type II hyperlipoproteinemia) may have recurrent migratory polyarthritis involving the knees and other large peripheral joints and, to a lesser degree, peripheral small joints. Pain ranges from moderate to incapacitating. The involved joints can be warm, erythematous, swollen, and tender. Arthritis usually has a sudden onset, lasts from a few days to 2 weeks, and does not cause joint damage. Episodes may suggest acute gout attacks. Several attacks occur per year. Synovial fluid from involved joints is not inflammatory and contains few white cells and no crystals. Joint involvement may actually represent inflammatory periarthritis or peritendinitis and not true arthritis. The recurrent, transient nature of the arthritis may suggest rheumatic fever,

1	crystals. Joint involvement may actually represent inflammatory periarthritis or peritendinitis and not true arthritis. The recurrent, transient nature of the arthritis may suggest rheumatic fever, especially because patients with hyperlipoproteinemia may have an elevated erythrocyte sedimentation rate and elevated antistreptolysin O titers (the latter being quite common). Attacks of tendinitis, including the large Achilles and patellar tendons, may come on gradually and last only a few days or may be acute as described above. Patients may be asymptomatic between attacks. Achilles tendinitis and other joint manifestations often precede the appearance of xanthomas and may be the first clinical indication of hyperlipoproteinemia. Attacks of tendinitis may follow treatment with a lipid-lowering drug. Over time, patients may develop tendinous xanthomas in the Achilles, patellar, and extensor tendons of the hands and feet. Xanthomas have also been reported in the peroneal tendon, the

1	drug. Over time, patients may develop tendinous xanthomas in the Achilles, patellar, and extensor tendons of the hands and feet. Xanthomas have also been reported in the peroneal tendon, the plantar aponeurosis, and the periosteum overlying the distal tibia. These xanthomas are located within tendon fibers. Tuberous xanthomas are soft subcutaneous masses located over the extensor surfaces of the elbows, knees, and hands as well as on the buttocks. They appear during childhood in homozygous patients and after the age of 30 in heterozygous patients. Patients with elevated plasma levels of verylow-density lipoprotein (VLDL) and triglycerides (previously referred to as type IV hyperlipoproteinemia)may also have a mild inflammatory arthritis affecting large and small peripheral joints, usually in an asymmetric pattern, with only a few joints involved at a time. The onset of arthritis usually comes in middle age. Arthritis may be persistent or recurrent, with episodes lasting a few days or

1	in an asymmetric pattern, with only a few joints involved at a time. The onset of arthritis usually comes in middle age. Arthritis may be persistent or recurrent, with episodes lasting a few days or weeks. Some patients may experience severejointpainor morning stiffness.Joint tenderness and periarticular hyperesthesia may also be present, as may synovial thickening.Jointfluidisusuallynoninflammatoryandwithoutcrystals but may have increased white blood cell counts with predominantly mononuclear cells. Radiographs may show juxtaarticular osteopenia and cystic lesions. Large bone cysts have been noted in a few patients. Xanthoma and bone cysts are also observed in other lipoprotein disorders. The pathogenesis of arthritis in patients with familial hypercholesterolemia or with elevated levels of VLDL and triglycerides is not well understood. NSAIDs or analgesics usually provide adequate relief of symptoms when used on an as-needed basis.

1	Patients may improve clinically as they are treated with lipid-lowering agents; however, patients treated with an HMG-CoA reductase inhibitor may experience myalgias, and a few patients develop myopathy, myositis, or even rhabdomyolysis. Patients who develop myositis during statin therapy may be susceptible to this adverse effect because of an underlying muscle disorder and should be reevaluated after discontinuation of the drug. Myositis has also been reported with the use of niacin (Chap. 388) but is less common than myalgias. Musculoskeletal syndromes have not clearly been associated with the more common mixed hyperlipidemias seen in general practice.

1	Musculoskeletal syndromes have not clearly been associated with the more common mixed hyperlipidemias seen in general practice. Neuropathic joint disease (Charcot joint) is a progressive destructive arthritisassociatedwithlossofpainsensation,proprioception,orboth. Normalmuscularreflexesthat modulatejointmovementareimpaired. Without these protective mechanisms, joints are subjected to repeated trauma, resulting in progressive cartilage and bone damage. Today, diabetes mellitus is the most frequent cause of neuropathic joint disease (Fig. 397-1). A variety of other disorders are associated with neuropathic arthritis, including tabes dorsalis, leprosy, yaws, syringomyelia, meningomyelocele, congenital indifference to pain, peroneal muscular atrophy (Charcot-Marie-Tooth disease), and amyloidosis. An arthritis resembling neuropathic joint disease has been reported in

1	FIGUrE 397-1 Charcot arthropathy associated with diabetes mel-litus. Lateralfootradiographdemonstratingcompletelossofthearchduetobonyfragmentationanddislocationinthemidfoot.(Courtesy of Andrew Neckers, MD, and Jean Schils, MD; with permission.) patients who have received intraarticular glucocorticoid injections, but thisisararecomplicationandwasnotobservedinoneseriesofpatients with knee osteoarthritis who received intraarticular glucocorticoid injections every 3 months for 2 years. The distribution of joint involvement depends on the underlying neurologic disorder (Table 397-2). In tabes dorsalis, the knees, hips, and ankles are most commonly affected; in syringomyelia, the glenohumeral joint, elbow, and wrist; and in diabetes mellitus, the tarsal and tarsometatarsal joints.

1	The pathologic changes in the neuropathic joint are similar to those found in the severe osteoarthritic joint. There is fragmentation and eventual loss of articular cartilage with eburnation of the underlying bone.Osteophytesarefoundatthejointmargins.Withmoreadvanced disease, erosions are present on the joint surface. Fractures, devitalized bone, intraarticular loose bodies, and microscopic fragments of cartilage and bone may be present.

1	At least two underlying mechanisms are believed to be involved in the pathogenesis of neuropathic arthritis. An abnormal autonomic nervous system is thought to be responsible for the dysregulated blood flowtothejoint withsubsequentresorptionofbone.Lossofbone,particularly in the diabetic foot, may be the initial finding. With the loss of deep pain, proprioception, and protective neuromuscular reflexes, the joint is subjected to repeated microtrauma, resulting in ligamental tearsandbonefractures.Theinjurythatfollowsfrequentintraarticular glucocorticoid injections is thought to be due to the analgesic effect of glucocorticoids, leading to overuse of an already damaged joint; the result is accelerated cartilage damage, although steroid-induced cartilage damage be more common in some other animal species than in humans. It is not understood why only a few patients with neuropathy develop clinically evident neuropathic arthritis.

1	Neuropathic joint disease usually begins in a single joint and then becomes apparent in other joints, depending on the underlying neurologic disorder. The involved joint becomes progressively enlarged as a result of bony overgrowth and synovial effusion. Loose bodies may be palpated in the joint cavity. Joint instability, subluxation, and crepitus occur as the disease progresses. Neuropathic joints may develop rapidly, and a totally disorganized joint with multiple bony fragments may evolve within weeks or months. The amount of pain experienced Meningomyelocele Congenital indifference to pain

1	Arthritis Associated with Systemic Disease, and Other Arthritides 2244 bythe patient is less than wouldbe anticipatedfrom the degree of joint damage.Patientsmay experiencesuddenjointpainfrom intraarticular fractures of osteophytes or condyles. Neuropathic arthritis is encountered most often in patients with diabetes mellitus, with an incidence of ~0.5%. The onset of disease usually comes at an age of ≥50 years in a patient who has had diabetes for several years, but exceptions occur. The tarsal and tarsometatarsal joints are most often affected, with the metatarsophalangeal and talotibial joints next most commonly involved. The knees and spine are occasionally involved. Patients often attribute the onset of foot pain to antecedent trauma such as twisting of the foot. Neuropathic changes may develop rapidly after a foot fracture or dislocation. The foot and ankle areoftenswollen. Downward collapseofthe tarsal bones leads to convexity of the sole, referred to as a “rocker foot.” Large

1	may develop rapidly after a foot fracture or dislocation. The foot and ankle areoftenswollen. Downward collapseofthe tarsal bones leads to convexity of the sole, referred to as a “rocker foot.” Large osteophytes may protrude from the top of the foot. Calluses frequently form over the metatarsal heads and may lead to infected ulcers and osteomyelitis. The value of protective inserts and orthotics, as well as regular foot examination, cannot be overstated. Radiographs may show resorption and tapering of the distal metatarsal bones. The term Lisfranc fracture-dislocation issometimes used to describe thedestructivechangesatthe tarsometatarsal joints.

1	The diagnosis of neuropathic arthritis is based on the clinical features and characteristic radiographic findings in a patient with underlying sensory neuropathy. The differential diagnosis of neuropathic arthritis depends upon the severity of the process and includes osteomyelitis, avascular necrosis, advanced osteoarthritis, stress fractures, and calcium pyrophosphate deposition disease. Radiographs in neuropathic arthritis initially show changes of osteoarthritis with joint space narrowing, subchondral bone sclerosis, osteophytes, and joint effusions; marked destructive and hypertrophic changes follow later. The radiographic findings of neuropathic arthritis may be difficult to differentiate from those of osteomyelitis, especially in the diabetic foot. The joint margins in a neuropathic joint tend to be distinct, while in osteomyelitis they are blurred. Imaging studies may be helpful, but cultures of tissue from the joint are often required to exclude osteomyelitis. MRI and bone

1	joint tend to be distinct, while in osteomyelitis they are blurred. Imaging studies may be helpful, but cultures of tissue from the joint are often required to exclude osteomyelitis. MRI and bone scans using indium 111–labeled white blood cells or indium 111–labeled immunoglobulin G, which will show increased uptake in osteomyelitis but not in a neuropathic joint, may be useful. A technetium bone scan will not distinguish osteomyelitis from neuropathic arthritis, as increased uptake is observed in both. The joint fluid in neuropathic arthritis is noninflammatory; may be xanthochromic or even bloody; and may contain fragments of synovium, cartilage, and bone. The finding of calcium pyrophosphate dihydrate crystals supports the diagnosis of crystal-associated arthropathy. In the absence of such crystals, an increased number of leukocytes may indicate osteomyelitis.

1	The primary focus of treatment is to stabilize the joint. Treatment of the underlying disorder, even if successful, does not usually affect established joint disease. Braces and splints are helpful. Their use requires close surveillance, because patients may be unable to appreciate pressure from a poorly adjusted brace. In the diabetic patient, early recognition of Charcot foot and its treatment—prohibition of weight bearing by the foot for at least 8 weeks—may possibly prevent severe disease from developing. Fusion of an unstable joint may improve function and reduce pain, but nonunion is frequent, especially when immobilization of the joint is inadequate.

1	Hypertrophic osteoarthropathy (HOA) is characterized by clubbing of digits and, in more advanced stages, by periosteal new-bone formation and synovial effusions. HOA may be primary or familial and may begin in childhood. Secondary HOA is associated with intrathoracic malignancies, suppurative and some hypoxemic lung diseases, congenital heart disease, and a variety of other disorders. Clubbing is almost always a feature of HOA but can occur as an isolated manifestation (Fig. 397-2). The presence of clubbing in isolation may be congenital or represent either an early stage or one element in the spectrum of HOA. Isolated acquired clubbing has the same clinical significance as clubbing associated with periostitis. FIGUrE 397-2 Clubbing of the fingers. (Reprinted from the Clinical Slide Collection on the Rheumatic Diseases, © 1991, 1995. Used by permis-sion of the American College of Rheumatology.)

1	Pathology and Pathophysiology of acquired HOa In HOA, bone changes in the distal extremities begin as periostitis followed by new bone formation. At this stage, a radiolucent area may be observed between the new periosteal bone and the subjacent cortex. As the process progresses, multiple layers of new bone are deposited and become contiguouswiththecortex,withconsequentcorticalthickening.Theouter portion of the bone is laminated in appearance, with an irregular surface. Initially, the process of periosteal new-bone formation involves the proximal and distal diaphyses of the tibia, fibula, radius, and ulna and, lessfrequently,thefemur, humerus,metacarpals,metatarsals, and phalanges. Occasionally, scapulae, clavicles, ribs, and pelvic bones are also affected. The adjacent interosseous membranes may become ossified. The distribution of bone manifestations is usually bilateral and symmetric. The soft tissue overlying the distal third of the arms and legs may be thickened. Proliferation of

1	may become ossified. The distribution of bone manifestations is usually bilateral and symmetric. The soft tissue overlying the distal third of the arms and legs may be thickened. Proliferation of connective tissue occurs in the nail bed and volar pad of digits, giving the distal phalanges a clubbed appearance. Small blood vessels in the clubbed digits are dilated and have thickened walls. In addition, the number of arteriovenous anastomoses is increased.

1	Several theories have been suggested for the pathogenesis of HOA, but many have been disproved or have not explained the condition’s development in all clinical disorders with which it is associated. Previously proposed neurogenic and humoral theories are no longer considered likely explanations for HOA. Studies have suggested a role for platelets in the development of HOA. It has been observed that megakaryocytes and large platelet particles present in the venous circulation are fragmented in their passage through normal lung. In patients with cyanotic congenital heart disease and in other disorders associated with right-to-left shunts, these large platelet particles bypass the lung and reach the distal extremities, where they can interact with endothelial cells. Platelet–endothelial cell activation in the distal portion of the extremities may result in the release of platelet-derived growth factor (PDGF) and other factors leading to the proliferation of connective tissue and

1	cell activation in the distal portion of the extremities may result in the release of platelet-derived growth factor (PDGF) and other factors leading to the proliferation of connective tissue and periosteum. Stimulation of fibroblasts by PDGF and transforming growth factor β results in cell growth and collagen synthesis. ElevatedplasmalevelsofvonWillebrandfactorantigenhavebeenfound in patients with both primary and secondary forms of HOA, indicating endothelial activation or damage. Abnormalities of collagen synthesis have been demonstrated in the involved skin of patients with primary HOA. Other factors are undoubtedly involved in the pathogenesis of HOA, and further studies are needed to elucidate this disorder.

1	Clinical Manifestations Primary or familial HOA, also referred to as pachydermoperiostitis or Touraine-Solente-Golé syndrome, usually begins insidiously at puberty. In a smaller proportion of patients, the onset comes in the first year of life. The disorder is inherited as an autosomal dominant trait with variable expression and is nine times more common among boys than among girls. Approximately one-third of patients have a family history of primary HOA.

1	Primary HOA is characterized by clubbing, periostitis, and unusual skin features. A small number of patients with this syndrome do not express clubbing. The skin changes and periostitis are prominent features of this syndrome. The skin becomes thickened and coarse. Deep nasolabial folds develop, and the forehead may become furrowed. Patients may have heavy-appearing eyelids and ptosis. The skin is often greasy, and there may be excessive sweating of the hands and feet.Patientsmay alsoexperience acne vulgaris,seborrhea,and folliculitis. In a few patients, the skin over the scalp becomes very thick and corrugated, a feature that has been descriptively termed cutis verticis gyrata. The distal extremities, particularly the legs, become thickened as aconsequence of the proliferation of new bone and soft tissue; when the process is extensive, the distal lower extremities resemble those of an elephant. The periostitis usually is not painful, which it can be in secondary HOA. Clubbing of the

1	and soft tissue; when the process is extensive, the distal lower extremities resemble those of an elephant. The periostitis usually is not painful, which it can be in secondary HOA. Clubbing of the fingers may be extensive, producing large, bulbous deformities and clumsiness. Clubbing also affects the toes. Patients may experience articular and periarticular pain, especially in the ankles and knees, and joint motion may be mildly restricted by periarticular bone overgrowth. Noninflammatory effusions occur in the wrists, knees, and ankles. Synovial hypertrophy is not found. Associated abnormalitiesobserved in patients with primary HOA include hypertrophic gastropathy, bone marrow failure, female escutcheon, gynecomastia, and cranial suture defects. In patients with primary HOA, the symptoms disappear when adulthood is reached.

1	HOA secondary to an underlying disease occurs more frequently than primary HOA. It accompanies a variety of disorders and may precede clinical features of the associated disorder by months. Clubbing is more frequent than the full syndrome of HOA in patients with associated illnesses. Because clubbing evolves over months and is usually asymptomatic, it is often recognized first by the physician and not the patient. Patients may experience a burning sensation in their fingertips. Clubbing is characterized by widening of the fingertips, enlargement of the distal volar pad, convexity of the nail contour, and the loss of the normal 15° angle between the proximal nail and cuticle. The thickness of the digit at the base of the nail is greater than the thicknessatthedistalinterphalangealjoint.Anobjectivemeasurement of finger clubbing can be made by determining the diameter at the base of the nail and at the distal interphalangeal joint of all 10 digits. Clubbing is present when the sum of the

1	of finger clubbing can be made by determining the diameter at the base of the nail and at the distal interphalangeal joint of all 10 digits. Clubbing is present when the sum of the individual digit ratios is >10. At thebedside, clubbingcanbe appreciated by having the patient place the dorsal surface of the distal phalanges of the fourth fingers together with the nails opposing each other. Normally, an open area is visible betweenthebasesoftheopposingfingernails;whenclubbingispresent, this open space is no longer visible. The base of the nail feels spongy when compressed, and the nail can be easily rocked on its bed. When clubbing is advanced, the finger may have a drumstick appearance, and the distal interphalangeal joint can be hyperextended. Periosteal involvement in the distal extremities may produce a burning or deep-seated aching pain. The pain, which can be quite incapacitating, is aggravated by dependency and relieved by elevation of the affected limbs. Pressure applied over

1	may produce a burning or deep-seated aching pain. The pain, which can be quite incapacitating, is aggravated by dependency and relieved by elevation of the affected limbs. Pressure applied over the distal forearms and legs or gentle percussion of distal long bones like the tibia may be quite painful.

1	Patients may experience joint pain, most often in the ankles, wrists, and knees. Joint effusions may be present; usually, they are small and noninflammatory. The small joints of the hands are rarely affected. Severe joint or long bone pain may be the presenting symptom of an underlying lung malignancy and may precede the appearance of clubbing. In addition, the progression of HOA tends to be more rapid when associated with malignancies, most notably bronchogenic carcinoma. Noninflammatory but variably painful knee effusions may occur prior to the appearance of clubbing and symptoms of distal periostitis. Unlike primary HOA, secondary HOA does not commonly include excessive sweating and oiliness of the skin or thickening of the facial skin. Lung abscesses, empyema, bronchiectasis Neoplasms: esophagus, liver, bowel Aneurysm of major extremity arterya Arteriovenous fistula of major extremity vessela

1	Lung abscesses, empyema, bronchiectasis Neoplasms: esophagus, liver, bowel Aneurysm of major extremity arterya Arteriovenous fistula of major extremity vessela Hyperthyroidism (Graves’ disease) aUnilateral involvement. bBilateral lower-extremity involvement.

1	Hyperthyroidism (Graves’ disease) aUnilateral involvement. bBilateral lower-extremity involvement. HOA occurs in 5–10% of patients with intrathoracic malignancies, the most common being bronchogenic carcinoma and pleural tumors (Table 397-3). Lung metastases infrequently cause HOA. HOA is also seen in patients with intrathoracic infections, including lung abscesses, empyema, and bronchiectasis, but is uncommon in pulmonary tuberculosis. HOA may accompany chronic interstitial pneumonitis, sarcoidosis, and cystic fibrosis. In cystic fibrosis, clubbing is more common than the full syndrome of HOA. Other causes of clubbing include congenital heart disease with right-to-left shunts, bacterial endocarditis, Crohn’s disease, ulcerative colitis, sprue, and neoplasms of the esophagus, liver, and small and large bowel. In patients who have congenital heart disease with right-to-left shunts, clubbing alone occurs more often than the full syndrome of HOA.

1	Unilateral clubbing has been found in association with aneurysms of major extremity arteries, with infected arterial grafts, and with arteriovenous fistulas of brachial vessels. Clubbing of the toes but not the fingers has been associated with an infected abdominal aortic aneurysm and patent ductus arteriosus. Clubbing of a single digit may follow trauma and has been reported in tophaceous gout and sarcoidosis. While clubbing occurs more commonly than the full syndrome in most diseases, periostitis in the absence of clubbing has been observed in the affected limb of patients with infected arterial grafts.

1	Hyperthyroidism (Graves’ disease), treated or untreated, is occasionally associated with clubbing and periostitis of the bones of the hands and feet. This condition is referred to as thyroid acropachy. Periostitis may be asymptomatic and occurs in the midshaft and diaphyseal portionofthe metacarpal andphalangeal bones.Significant hand-joint pain may occur; this pain may respond to successful therapy for thyroid dysfunction. The long bones of the extremities are seldom affected. Elevated levels of long-acting thyroid stimulator are found in the sera of these patients.

1	Laboratory Findings The laboratory abnormalities reflect the underlying disorder. The synovial fluid of involved joints has <500 white cells/μL, and the cells are predominantly mononuclear. Radiographs show a faint radiolucent line beneath the new periosteal bone along the shaft of long bones at their distal end. These changes are observed most frequently at the ankles, wrists, and knees. The ends of the distal phalanges may show osseous resorption. Radionuclide studies show pericortical linear uptake along the cortical margins of long bones that may precede any radiographic changes. Arthritis Associated with Systemic Disease, and Other Arthritides Myofascial pain most often involves the posterior neck, low back, shoulders, and chest. Chronic pain in the muscles of the posterior

1	Myofascial pain most often involves the posterior neck, low back, shoulders, and chest. Chronic pain in the muscles of the posterior The treatment of HOA aims to identify the associated disorder and treat it appropriately. The symptoms and signs of HOA may disappear completely with removal of or effective chemotherapy for a tumor or with antibiotic therapy for a chronic pulmonary infection and drainage of the infected site. Vagotomy or percutaneous block of the vagus nerve leads to symptomatic relief in some patients. NSAIDs or analgesics may help control symptoms of HOA.

1	The reflex sympathetic dystrophy syndrome is now referred to as complex regional pain syndrome, type 1, according to the new classification system of the International Association for the Study of Pain. This syndrome is characterized by pain and swelling, usually of a distal extremity, accompanied by vasomotor instability, trophic skin changes, and the rapid development of bony demineralization. Reflex sympathetic dystrophy syndrome, including its treatment, is covered in greater detail in Chap. 454.

1	Tietze syndrome is manifested by painful swelling of one or more costochondral articulations. The age of onset is usually before 40, and both sexes are affected equally. In most patients, only one joint is involved, usually the second or third costochondral joint. The onset of anterior chest pain may be sudden or gradual. The pain may radiate to the arms or shoulders and is aggravated by sneezing, coughing, deep inspirations, or twisting motions of the chest. The term costochondritis is often used interchangeably with Tietze syndrome, but some workers restrict the former term to pain of the costochondral articulations without swelling. Costochondritis is observed in patients over age 40; tends to affect the third, fourth, and fifth costochondral joints; and occurs more often in women. Both syndromes may mimic cardiac or upper abdominal causes of pain. Rheumatoid arthritis, ankylosing spondylitis, and reactive arthritis may involve costochondral joints but are distinguished easily by

1	syndromes may mimic cardiac or upper abdominal causes of pain. Rheumatoid arthritis, ankylosing spondylitis, and reactive arthritis may involve costochondral joints but are distinguished easily by their other clinical features. Other skeletal causes of anterior chest wall pain are xiphoidalgia and the slipping rib syndrome, which usually involves the tenth rib. Malignancies such as breast cancer, prostate cancer, plasma cell cytoma, and sarcoma can invade the ribs, thoracic spine, or chest wall and produce symptoms suggesting Tietze syndrome. Patients with osteomalacia may have significant rib pain, with or without documented microfractures. These conditions should be distinguishable by radiography, bone scanning, vitamin D measurement, or biopsy. Analgesics, anti-inflammatory drugs, and local glucocorticoid injections usually relieve symptoms of costochondritis/Tietze syndrome. Care should be taken to avoid overdiagnosing these syndromes in patients with acute chest pain syndromes;

1	local glucocorticoid injections usually relieve symptoms of costochondritis/Tietze syndrome. Care should be taken to avoid overdiagnosing these syndromes in patients with acute chest pain syndromes; many patients will be tender to overly vigorous palpation of the costochondral joints.

1	Myofascial pain syndrome is characterized by multiple areas of localized musculoskeletal pain and tenderness in association with tender points. The pain is deep and aching and may be accompanied by a burning sensation. Myofascial pain may be regional and follow trauma, overuse, or prolonged static contraction of a muscle or muscle group, which may occur when an individual is reading or writing at a desk or working at a computer. In addition, this syndrome may be associated with underlying osteoarthritis of the neck or low back. Pain may be referred from tender points to defined areas distant from the area of original tenderness. Palpation of the tender point reproduces or accentuates the pain. The tender points are usually located in the center of a muscle belly, but they can occur at other sites such as costosternal junctions, the xiphoid process, ligamentous and tendinous insertions, fascia, and fatty areas. Tender point sites in muscle have been described as feeling indurated and

1	sites such as costosternal junctions, the xiphoid process, ligamentous and tendinous insertions, fascia, and fatty areas. Tender point sites in muscle have been described as feeling indurated and taut, and palpation may cause the muscle to twitch. These findings, however, have been shown not to be unique to myofascial pain syndrome: in a controlled study, they were also present in some “normal” subjects.

1	neck may involve referral of pain from a tender point in the erector neck muscle or upper trapezius to the head, leading to persistent headaches that may last for days. Tender points in the paraspinal muscles of the low back may refer pain to the buttock. Pain may be referred down the leg from a tender point in the gluteus medius and can mimic sciatica. A tender point in the infraspinatus muscle may produce local and referred pain over the lateral deltoid and down the outside of the arm into the hand. Injection of a local anesthetic such as 1% lidocaine into the tender point site often results in at least transient pain relief. Another useful technique is first to spray an agent such as ethyl chloride from the tender point toward the area of referred pain and then to stretch the muscle. This maneuver may need to be repeated several times. Massage and application of ultrasound to the affected area also may be beneficial. Patients should be instructed in methods to prevent muscle

1	This maneuver may need to be repeated several times. Massage and application of ultrasound to the affected area also may be beneficial. Patients should be instructed in methods to prevent muscle stresses related to work and recreation. Posture and resting positions are important in preventing muscle tension. The prognosis in most patients is good. In some patients, regionally localized myofascial pain syndrome may seem to evolve into more generalized fibromyalgia (Chap. 396). Abnormal or nonrestorative sleep is a common accompaniment in these patients and may need to be specifically addressed.

1	Primarytumors and tumor-like disorders of synovium areuncommon but should be considered in the differential diagnosis of monarticular jointdisease.Inaddition,metastasestoboneandprimarybonetumors adjacent to a joint may produce joint symptoms. Pigmented villonodular synovitis (PVNS) is characterized by the slowly progressive, exuberant, benign proliferation of synovial tissue, usually involving a single joint. The most common age of onset is in the third decade, and women are affected slightly more often than men. The cause of this disorder is unknown. The synovium has a brownish color and numerous large, finger-like villi that fuse to form pedunculated nodules. There is marked hyperplasia of synovial cells in the stroma of the villi. Hemosiderin granules and lipids are found in the cytoplasm of macrophages and in the interstitial tissue. Multinucleated giant cells may be present. The proliferative synovium grows into the subsynovial tissue and invades adjacent cartilage and bone.

1	The clinical picture of PVNS is characterized by the insidious onset of persistent swelling and pain in affected joints, most commonly the knee. Other joints affected include the hips, ankles, calcaneocuboid joints, elbows, and small joints of the fingers or toes. The disease may also involve the common flexor sheath of the hands or fingers. Less often, tendon sheaths in the wrist, ankle, or foot may be involved. Symptoms of pain, a catching sensation, or stiffness may initially be mild and intermittent and may be present for years before the patient seeks medical attention. Radiographs may show joint space narrowing, erosions, and subchondral cysts. The diagnosis of PVNS is strongly suggested by gradient echo MRI, which reveals a synovial mass lesion of low signal intensity typical of tissue containing hemosiderin (Fig. 397-3). The joint fluid contains blood and is dark red or almost black in color. Lipid-containing macrophages may be present in the fluid. The joint fluid may be

1	tissue containing hemosiderin (Fig. 397-3). The joint fluid contains blood and is dark red or almost black in color. Lipid-containing macrophages may be present in the fluid. The joint fluid may be clear if hemorrhage has not occurred. Some patients have polyarticular involvement.

1	The treatment for PVNS is complete synovectomy. With incomplete synovectomy, the villonodular synovitis recurs, and the rate of tissue growth may be faster than it was originally. Irradiation of the involved joint has been successful in some patients. Synovial chondromatosis is a disorder characterized by multiple focal metaplastic growths of normal-appearing cartilage in the synovium or tendon sheath. Segments of cartilage break loose and continue to grow as loose bodies. When calcification and ossification of loose bodies occur, the disorder is referred to as synovial osteochondromatosis. The disorder is usually monarticular and affects young to middle-aged individuals. The knee is most often involved, followed by hip, elbow, and shoulder. Symptoms are pain, swelling, and decreased motion of the joint. Radiographs may show several rounded calcifications within the joint cavity. Treatment is synovectomy; however, as in PVNS, the tumor may recur.

1	FIGUrE 397-3 Pigmented villonodular synovitis. MRIgradientechosagittalimageshowingamassthatabutstheneckofthetaluswithmarkedlowsignaltypicaloftissuecontaininghemosiderin.(Courtesy of Donald Flemming, MD; with permission.)

1	Synovial sarcoma is a malignant neoplasm often found near a large joint of both upper and lower extremities, being more common in the lower extremity. It seldom arises within the joint itself. Synovial sarcomas constitute 10% of soft tissue sarcomas. The tumor is believed to arise from primitive mesenchymal tissue that differentiates into epithelial cells and/or spindle cells. Small foci of calcification may be present in the tumor mass. Synovial sarcoma occurs most often in young adults and is more common in men. The tumor presents as a slowly growing deep-seated mass near a joint, without much pain. The area of the knee is the most common site, followed by the foot, ankle, elbow, and shoulder.Other primary sitesincludethebuttocks,abdominal wall, retroperitoneum, and mediastinum. The tumor spreads along tissue planes. The most common site of visceral metastasis is the lung. The diagnosis is made by biopsy. Treatment consists of wide resection of the tumor, including adjacent muscle

1	spreads along tissue planes. The most common site of visceral metastasis is the lung. The diagnosis is made by biopsy. Treatment consists of wide resection of the tumor, including adjacent muscle and regional lymph nodes, followed by chemotherapy and radiation therapy. Amputation of the involved distal extremity may be required. Chemotherapy may be beneficial in some patients with metastatic disease. Isolated sites of pulmonary metastasis can be surgically removed. The 5-year survival rate with treatment is variable and depends on the staging of the tumor, ranging from ~25% to ≥60%. Synovial sarcomas tend to recur locally and metastasize to regional lymph nodes, lungs, and skeleton.

1	In addition to the rare direct metastases of solid cell tumors to the highly vascular synovium, neoplasia arising from nonarticular organ sites can affect joints in other ways. Acute leukemias in children can mimic juvenile inflammatory arthritis with severe joint pain and fever. In adults, chronic and acute myeloid leukemia can infiltrate the synovium in rare instances. The rarely occurring hairy cell leukemia has a peculiar tendency to cause episodic inflammatory oligoarthritis and tenosynovitis; these episodes are dramatic and mimic acute gout attacks. They respond to potent anti-inflammatory therapy with glucocorticoids; with remission of the leukemia, they may abate. Carcinomas can be associated with several paraneoplastic articular syndromes, including HOA (discussed above). Acute palmar fasciitis withpolyarthritisisawell-describedbutrareconditionassociatedwith certain cancers, mainly adenocarcinomas. Clinically, this syndrome is

1	including HOA (discussed above). Acute palmar fasciitis withpolyarthritisisawell-describedbutrareconditionassociatedwith certain cancers, mainly adenocarcinomas. Clinically, this syndrome is fairlyabruptinonset,withpaininthemetacarpophalangealandproximal interphalangeal joints of the hands and rapidly evolving contractures of the fingers due to thickening of the palmar (flexor) tendons. A similar syndrome can be seen in diabetics. Paraneoplastic arthritis has been described and may occur in several patterns: asymmetric disease predominantly affecting the lower extremity joints and sym-2247 metric polyarthritis with hand joint involvement. Tumors are often found after the onset of the arthritis, and many patients have a preceding period of malaise or weight loss. The onset is often acute, and patients tend to be older men. These features should raise the specter of an underlying malignancy (or a viral infection such as hepatitis C) as the cause of the arthritis. In one series, the

1	and patients tend to be older men. These features should raise the specter of an underlying malignancy (or a viral infection such as hepatitis C) as the cause of the arthritis. In one series, the symptoms resolved with successful therapy for the malignancy and did not recur with relapse of the malignancy. Dermatomyositis has a well-described association with neoplasms and may include joint pain and arthritis. Malignancy-associated arthritis may be responsive to NSAIDs and to treatment of the primary neoplasm.

1	This chapter represents a revised version of the chapter authored by Dr. Bruce C. Gilliland that appeared in previous editions of Harrison’s. Dr. Gilliland passed away on February 17, 2007. He had been a contributor to Harrison’s Principles of Internal Medicine since the 11th edition. periarticular Disorders of the Carol A. Langford A number of periarticular disorders have become increasingly common, due in part to greater participation in recreational sports by individuals of a wide range of ages. Periarticular disorders most commonly affect the knee or shoulder. With the exception of bursitis, hip pain is most often articular or is being referred from disease affecting anotherstructure (Chap 393).Thischapterdiscussessomeofthemore common periarticular disorders.

1	Bursitis is inflammation ofabursa,whichisa thin-walledsaclinedwith synovial tissue. The function of the bursa is to facilitate movement of tendonsandmusclesoverbonyprominences.Excessivefrictionalforces from overuse, trauma, systemic disease (e.g., rheumatoid arthritis, gout), or infection may cause bursitis. Subacromial bursitis (subdeltoid bursitis) is the most common form of bursitis. The subacromial bursa, which is contiguous with the subdeltoid bursa, is located between the undersurface of the acromion and the humeral head and is covered by thedeltoidmuscle.Bursitisiscausedby repetitiveoverheadmotion and often accompanies rotator cuff tendinitis. Another frequently encountered form is trochanteric bursitis, which involves the bursa around the insertion of the gluteus medius onto the greater trochanter of the femur. Patients experience pain over the lateral aspect of the hip and upperthighand havetendernessoverthe posterioraspect of the greater trochanter. External rotation and

1	greater trochanter of the femur. Patients experience pain over the lateral aspect of the hip and upperthighand havetendernessoverthe posterioraspect of the greater trochanter. External rotation and resisted abduction of the hip elicit pain. Olecranon bursitis occurs over the posterior elbow, and when the area is acutely inflamed, infection or gout should be excluded by aspirating the bursa and performing a Gram stain and culture on the fluid aswellasexaminingthefluidforuratecrystals. Achilles bursitis involves the bursa located abovethe insertion of thetendon to the calcaneus and results from overuse and wearing tight shoes. Retrocalcaneal bursitis involves the bursa that is located between the calcaneus and posterior surface of the Achilles tendon. The pain is experienced at the back of the heel, and swelling appears on the medial and/or lateral side of the tendon. It occurs in association with spondyloarthritides, rheumatoid arthritis, gout, ortrauma. Ischial bursitis affects the

1	the heel, and swelling appears on the medial and/or lateral side of the tendon. It occurs in association with spondyloarthritides, rheumatoid arthritis, gout, ortrauma. Ischial bursitis affects the bursa separating the gluteusmediusfromtheischialtuberosityanddevelopsfromprolonged sitting and pivoting on hard surfaces. Iliopsoas bursitis affects the bursa that lies between the iliopsoas muscle and hip joint and is lateral to the femoral vessels. Pain is experienced over this area and is made worse

1	Periarticular Disorders of the Extremities 2248 by hip extension and flexion. Anserine bursitis is an inflammation of the sartorius bursa located over the medial side of the tibia just below the knee and under the conjoint tendon and is manifested by pain on climbing stairs. Tenderness is present over the insertion of the conjoint tendon of the sartorius, gracilis, and semitendinosus. Prepatellar bursitis occurs in the bursa situated between the patella and overlying skin and is caused by kneeling on hard surfaces. Gout or infection may also occur at this site. Bursitis is typically diagnosed by history and physical examination, but visualization by ultrasound may play a useful role in selected instances for diagnosis and directed guidance of glucocorticoid injection. Treatment of bursitis consists of prevention of the aggravating situation, rest of the involved part, administration of a nonsteroidal anti-inflammatory drug (NSAID) where appropriate for an individual patient, or local

1	consists of prevention of the aggravating situation, rest of the involved part, administration of a nonsteroidal anti-inflammatory drug (NSAID) where appropriate for an individual patient, or local glucocorticoid injection.

1	Tendinitis of the rotator cuff is the major cause of a painful shoulder andiscurrentlythoughttobecausedbyinflammationofthetendon(s). The rotator cuff consists of the tendons of the supraspinatus, infraspinatus, subscapularis, and teres minor muscles, and inserts on the humeral tuberosities. Of the tendons forming the rotator cuff, the supraspinatustendonis the most often affected,probablybecauseofits repeated impingement (impingement syndrome) between the humeral head and the undersurface of the anterior third of the acromion and coracoacromial ligament above as well as the reduction in its blood supply that occurs with abduction of the arm (Fig. 398-1). The tendon oftheinfraspinatusandthatofthelongheadofthebicepsarelesscommonlyinvolved.Theprocessbeginswithedemaandhemorrhageofthe rotatorcuff,whichevolvestofibroticthickeningandeventuallytorotator cuff degeneration with tendon tears and bone spurs. Subacromial bursitis also accompanies this syndrome. Symptoms usually appear after injury

1	cuff degeneration with tendon tears and bone spurs. Subacromial bursitis also accompanies this syndrome. Symptoms usually appear after injury or overuse, especially with activities involving elevation of the arm with some degree of forward flexion. Impingement syndrome occurs in persons participating in baseball, tennis, swimming, or occupations that require repeated elevation of the arm. Those over age 40 are particularly susceptible. Patients complain of a dull aching in the shoulder, which may interfere with sleep. Severe pain is experienced when the arm is actively abducted into an overhead position. The arc between 60° and 120° is especially painful. Tenderness is present over the lateral aspect of the humeral head just below the acromion. NSAIDs, local glucocorticoid injection, and physical therapy may relieve symptoms. Surgical decompression of the subacromial space may be necessary in patients refractory to conservative treatment.

1	FIGUrE 398-1 Coronal section of the shoulder illustrating the relationships of the glenohumeral joint, the joint capsule, the subacromial bursa, and the rotator cuff (supraspinatus tendon). (From F Kozin, in Arthritis and Allied Conditions, 13th ed, WJ Koopman [ed]. Baltimore, Williams & Wilkins, 1997, with permission.) Patients may tear the supraspinatus tendon acutely by falling on an outstretched arm or lifting a heavy object. Symptoms are pain along with weakness of abduction and external rotation of the shoulder. Atrophy of the supraspinatus muscles develops. The diagnosis is establishedbyarthrogram,ultrasound,ormagneticresonanceimaging (MRI).Surgicalrepairmaybenecessaryin patientswhofail torespond to conservative measures. In patients with moderate-to-severe tears and functional loss, surgery is indicated.

1	Thisconditionischaracterizedbydepositionofcalciumsalts,primarily hydroxyapatite, within a tendon. The exact mechanism of calcification is not known but may be initiated by ischemia or degeneration of the tendon. The supraspinatus tendon is most often affected because it is frequently impinged on and has a reduced blood supply when the arm is abducted. The condition usually develops after age 40. Calcification within the tendon may evoke acute inflammation, producing sudden and severe pain in the shoulder. However, it may be asymptomatic or not related to the patient’s symptoms. Diagnosis of calcific tendonitis can be made by ultrasound or radiograph. Most cases are self-limited and respond to conservative therapy with physical therapy and/or NSAIDs. A subset of patients is refractory and requires ultrasound-guided percutaneous needle aspiration and lavage or surgery.

1	Bicipital tendinitis, or tenosynovitis, is produced by friction on the tendon of the long head of the biceps as it passes through the bicipital groove. When the inflammation is acute, patients experience anterior shoulder pain that radiates down the biceps into the forearm. Abduction and external rotation of the arm are painful and limited. The bicipital groove is very tender to palpation. Pain may be elicited along the course of the tendon by resisting supination of the forearm with the elbow at 90° (Yergason’s supination sign). Acute rupture of the tendon may occur with vigorous exercise of the arm and is often painful. In a young patient, it should be repaired surgically. Rupture of the tendon in an older person may be associated with little or no pain and is recognized by the presence of persistent swelling of the biceps produced by the retraction of the long head of the biceps. Surgery is usually not necessary in this setting.

1	In this condition, inflammation involves the abductor pollicis longus and the extensor pollicis brevis as these tendons pass through a fibroussheathattheradialstyloidprocess.Theusualcauseisrepetitive twisting of the wrist. It may occur in pregnancy, and it also occurs in mothers who hold their babies with the thumb outstretched. Patients experience pain on grasping with their thumb, such as with pinching. Swelling and tenderness are often present over the radial styloid process. The Finkelstein sign is positive, which is elicited by having the patient place the thumb in the palm and close the fingers over it. The wrist is then ulnarly deviated, resulting in pain over the involved tendon sheath in the area of the radial styloid. Treatment consists initially of splinting the wrist and an NSAID. When severe or refractory to conservative treatment, glucocorticoid injections can be very effective.

1	Tendinitis involves the patellar tendon at its attachment to the lower pole of the patella. Patients may experience pain when jumping during basketball or volleyball, going up stairs, or doing deep knee squats. Tenderness is noted on examination over the lower pole of the patella. Treatment consists of rest, icing, and NSAIDs,followed by strengthening and increasing flexibility.

1	The iliotibial band is a thick connective tissue that runs from the ilium to the fibula. Patients with iliotibial band syndrome most commonly present with aching or burning pain at the site where the band courses over the lateral femoral condyle of the knee; pain may also radiate up the thigh, toward the hip. Predisposing factors for iliotibial band syndrome include a varus alignment of the knee, excessive running distance,poorlyfittedshoes,orcontinuousrunningonuneventerrain. Treatment consists of rest, NSAIDs, physical therapy, and addressing risk factors such as shoes and running surface. Glucocorticoid injection into thearea of tenderness can providerelief, butrunning mustbe avoided for at least 2 weeks after the injection. Surgical release of the iliotibial band has been helpful in rare patients for whom conservative treatment has failed.

1	Often referred to as “frozen shoulder,” adhesive capsulitis is characterized by pain and restricted movement of the shoulder, usually in the absence of intrinsic shoulder disease. Adhesive capsulitis may follow bursitis or tendinitis of the shoulder or be associated with systemic disorders such as chronic pulmonary disease, myocardial infarction, and diabetes mellitus. Prolonged immobility of the arm contributes to the development of adhesive capsulitis. Pathologically, the capsule of the shoulder is thickened, and a mild chronic inflammatory infiltrate and fibrosis may be present.

1	Adhesive capsulitis occurs more commonly in women after age 50. Pain and stiffness usually develop gradually but progress rapidly in some patients. Night pain is often present in the affected shoulder, and pain may interfere with sleep. The shoulder is tender to palpation, and both active and passive movement are restricted. Radiographs of the shoulder show osteopenia. The diagnosis is typically made by physical examination but can be confirmed if necessary by arthrography, in that only a limited amount of contrast material, usually <15 mL, can be injected under pressure into the shoulder joint.

1	In most patients, the condition improves spontaneously 1–3 years after onset. While pain usually improves, many patients are left with some limitation of shoulder motion. Early mobilization of the arm following an injury to the shoulder may prevent the development of this disease. Physical therapy provides the foundation of treatment for adhesive capsulitis. Local injections of glucocorticoids and NSAIDs may also provide relief of symptoms. Slow but forceful injection of contrast material into the joint may lyse adhesions and stretch the capsule, resulting in improvement of shoulder motion. Manipulation under anesthesia may be helpful in some patients.

1	Lateral epicondylitis, or tennis elbow, is a painful condition involving the soft tissue over the lateral aspect of the elbow. The pain originates ator near the siteof attachment of the commonextensorsto the lateral epicondyle and may radiate into the forearm and dorsum of the wrist. The pain usually appears after work or recreational activities involving repeated motions of wrist extension and supination against resistance. Most patients with this disorder injure themselves in activities other than tennis, such as pulling weeds, carrying suitcases or briefcases, or using a screwdriver. The injury in tennis usually occurs when hitting a backhand with the elbow flexed. Shaking hands and opening doors can reproduce the pain. Striking the lateral elbow against a solid object may also induce pain.

1	The treatment is usually rest along with administration of an NSAID. Ultrasound, icing, and friction massage may also help relieve pain. When pain is severe, the elbow is placed in a sling or splinted at 90° of flexion. When the pain is acute and well localized, injection of a glucocorticoid using a small-gauge needle may be effective. Following injection, the patient should be advised to rest the arm for at least 1 month and avoid activities that would aggravate the elbow. Once symptoms have subsided, the patient should begin rehabilitation to strengthen and increase flexibility of the extensor muscles before resuming physical activity involving the arm. A forearm band placed 2.5–5.0 cm (1–2 in.) below the elbow may help to reduce tension on the extensor muscles at their attachment to the lateral epicondyle. The patient should be advised to restrict activities requiring forcible extension and supination of the wrist. Improvement may take several months. The patient may continue to

1	the lateral epicondyle. The patient should be advised to restrict activities requiring forcible extension and supination of the wrist. Improvement may take several months. The patient may continue to experience mild pain but, with care, can usually avoid the return of debilitating pain. Occasionally, 2249 surgical release of the extensor aponeurosis may be necessary.

1	Medial epicondylitis is an overuse syndrome resulting in pain over the medial side of the elbow with radiation into the forearm. The cause of this syndrome is considered to be repetitive resisted motions of wrist flexion and pronation, which lead to microtears and granulation tissue at the origin of the pronator teres and forearm flexors, particularly the flexor carpi radialis. This overuse syndrome is usually seen in patients >35 years and is much less common than lateral epicondylitis. It occurs most often in work-related repetitive activities but also occurs with recreational activities such as swinging a golf club or throwing a baseball. On physical examination, there is tenderness just distal to the medial epicondyle over the origin of the forearm flexors. Pain can be reproduced by resisting wrist flexion and pronation with the elbow extended. Radiographs are usually normal. The differential diagnosis of patients with medial elbow symptoms includes tears of the pronator teres,

1	resisting wrist flexion and pronation with the elbow extended. Radiographs are usually normal. The differential diagnosis of patients with medial elbow symptoms includes tears of the pronator teres, acute medial collateral ligament tear, and medial collateral ligament instability. Ulnar neuritis has been found in 25–50% of patients with medial epicondylitis and is associated with tenderness over the ulnar nerve at the elbow as well as hypesthesia and paresthesia on the ulnar side of the hand.

1	The initial treatment of medial epicondylitis is conservative, involving rest, NSAIDs, friction massage, ultrasound, and icing. Some patients may require splinting. Injections of glucocorticoids at the painful site may also be effective. Patients should be instructed to rest for at least 1 month. Also, patients should start physical therapy once the pain has subsided. In patients with chronic debilitating medial epicondylitis that remains unresponsive after at least a year of treatment, surgical release of the flexor muscle at its origin may be necessary and is often successful.

1	Plantar fasciitisis a common cause of footpain in adults,with thepeak incidence occurring in people betweenthe agesof 40and 60 years. The painoriginatesatornearthesiteoftheplantarfasciaattachmenttothe medialtuberosity of the calcaneus.Severalfactorsthatincrease therisk of developing plantar fasciitis include obesity, pes planus (flat foot or absence of the foot arch when standing), pes cavus (high-arched foot), limited dorsiflexion of the ankle, prolonged standing, walking on hard surfaces, and faulty shoes. In runners, excessive running and a change to a harder running surface may precipitate plantar fasciitis.

1	The diagnosis of plantar fasciitis can usually be made on the basis of history and physical examination alone. Patients experience severe pain with the first steps on arising in the morning or following inactivity during the day. The pain usually lessens with weight-bearing activityduringtheday,onlytoworsenwithcontinuedactivity.Painismade worse on walking barefoot or up stairs. On examination, maximal tendernessis elicitedonpalpation over the inferiorheel corresponding to the site of attachment of the plantar fascia. Imaging studies may be indicated when the diagnosis is not clear. Plain radiographs may show heel spurs, which are of little diagnostic significance. Ultrasonography in plantar fasciitis can demonstrate thickening of the fascia and diffuse hypoechogenicity, indicating edema at the attachment of the plantar fascia to the calcaneus. MRI is a sensitive method for detecting plantar fasciitis, but it is usually not required for establishing the diagnosis.

1	The differential diagnosis of inferior heel pain includes calcaneal stress fractures, the spondyloarthritides, rheumatoid arthritis, gout, neoplastic or infiltrative bone processes, and nerve compression/ entrapment syndromes. Resolutionofsymptomsoccurswithin12monthsinmorethan80% of patients with plantar fasciitis. The patient is advised to reduce or discontinue activities that can exacerbate plantar fasciitis. Initial treatment consists of ice, heat, massage, and stretching. Orthotics provide medial arch support and can be effective. Foot strapping or taping are commonly performed, and some patients may benefit by wearing a

1	Periarticular Disorders of the Extremities 2250 night splint designed to keep the ankle in a neutral position. A short Acknowledgmentcourse of NSAIDs can be given to patients when the benefits outweigh This chapter represents a revised version of the chapter authored by the risks. Local glucocorticoid injections have also been shown to be Dr. Bruce C. Gilliland that was in the previous editions of Harrison’s. efficacious but may carry an increased risk for plantar fascia rupture. Dr. Gilliland passed away on February 17, 2007, and had been a Plantar fasciotomy is reserved for those patients who have failed to contributor to Harrison’s Principles of Internal Medicine since the improve after at least 6–12 months of conservative treatment. 11th edition. Approach to the Patient with Endocrine Disorders J. Larry Jameson The management of endocrine disorders requires a broad under-standing of intermediary metabolism, reproductive physiology, bone 399 SEC Tion 1 EnDoCRinology

1	Approach to the Patient with Endocrine Disorders metabolism, and growth. Accordingly, the practice of endocrinology is intimately linked to a conceptual framework for understanding hormone secretion, hormone action, and principles of feedback control (Chap. 400e). The endocrine system is evaluated primarily by measuring hormone concentrations, arming the clinician with valuable diagnostic information. Most disorders of the endocrine system are amenable to effective treatment once the correct diagnosis is determined. Endocrine deficiency disorders are treated with physiologic hormone replacement; hormone excess conditions, which usually are caused by benign glandular adenomas, are managed by removing tumors surgically or reducing hormone levels medically.

1	The specialty of endocrinology encompasses the study of glands and the hormones they produce. The term endocrine was coined by Starling to contrast the actions of hormones secreted internally (endocrine) with those secreted externally (exocrine) or into a lumen, such as the gastrointestinal tract. The term hormone, derived from a Greek phrase meaning “to set in motion,” aptly describes the dynamic actions of hormones as they elicit cellular responses and regulate physiologic processes through feedback mechanisms.

1	Unlike many other specialties in medicine, it is not possible to define endocrinology strictly along anatomic lines. The classic endocrine glands—pituitary, thyroid, parathyroid, pancreatic islets, adrenals, and gonads—communicate broadly with other organs through the nervous system, hormones, cytokines, and growth factors. In addition to its traditional synaptic functions, the brain produces a vast array of peptide hormones, and this has led to the discipline of neuroendocrinology. Through the production of hypothalamic releasing factors, the central nervous system (CNS) exerts a major regulatory influence over pituitary hormone secretion (Chap. 401e). The peripheral nervous system stimulates the adrenal medulla. The immune and endocrine systems are also intimately intertwined. The adrenal hormone cortisol is a powerful immunosuppressant. Cytokines and interleukins (ILs) have profound effects on the functions of the pituitary, adrenal, thyroid, and gonads. Common endocrine diseases

1	hormone cortisol is a powerful immunosuppressant. Cytokines and interleukins (ILs) have profound effects on the functions of the pituitary, adrenal, thyroid, and gonads. Common endocrine diseases such as autoimmune thyroid disease and type 1 diabetes mellitus are caused by dysregulation of immune surveillance and tolerance. Less common diseases such as polyglandular failure, Addison’s disease, and lymphocytic hypophysitis also have an immunologic basis.

1	The interdigitation of endocrinology with physiologic processes in other specialties sometimes blurs the role of hormones. For example, hormones play an important role in maintenance of blood pressure, intravascular volume, and peripheral resistance in the cardiovascular system. Vasoactive substances such as catecholamines, angiotensin II, endothelin, and nitric oxide are involved in dynamic changes of vascular tone in addition to their multiple roles in other tissues. The heart is the principal source of atrial natriuretic peptide, which acts in classic endocrine fashion to induce natriuresis at a distant target organ (the kidney). Erythropoietin, a traditional circulating hormone, is made in the kidney and stimulates erythropoiesis in bone marrow (Chap. 77). The kidney is also integrally involved in the renin-angiotensin axis (Chap. 406) and is a primary target of several hormones, including parathyroid hormone (PTH), mineralocorticoids, and vasopressin. The gastrointestinal tract

1	involved in the renin-angiotensin axis (Chap. 406) and is a primary target of several hormones, including parathyroid hormone (PTH), mineralocorticoids, and vasopressin. The gastrointestinal tract produces a surprising number of peptide hormones, such as cholecystokinin, ghrelin, gastrin, secretin, and vasoactive intestinal peptide, among many others. Carcinoid and islet tumors can secrete excessive amounts of these hormones, leading to specific clinical syndromes (Chap. 113). Many of these gastrointestinal hormones are also produced in the CNS, where their functions are poorly understood. Adipose tissue produces leptin, which acts centrally to control appetite, along with adiponectin, resistin, and other hormones that regulate metabolism. As hormones such as inhibin, ghrelin, and leptin are discovered, they become integrated into the science and practice of medicine on the basis of their functional roles rather than their tissues of origin.

1	Characterization of hormone receptors frequently reveals unexpected relationships to factors in nonendocrine disciplines. The growth hormone (GH) and leptin receptors, for example, are members of the cytokine receptor family. The G protein–coupled receptors (GPCRs), which mediate the actions of many peptide hormones, are used in numerous physiologic processes, including vision, smell, and neurotransmission. Endocrine diseases can be divided into three major types of conditions: (1) hormone excess, (2) hormone deficiency, and (3) hormone resistance (Table 399-1).

1	Syndromes of hormone excess can be caused by neoplastic growth of endocrine cells, autoimmune disorders, and excess hormone administration. Benign endocrine tumors, including parathyroid, pituitary, and adrenal adenomas, often retain the capacity to produce hormones, perhaps reflecting the fact that these tumors are relatively well differentiated. Many endocrine tumors exhibit subtle defects in their “set points” for feedback regulation. For example, in Cushing’s disease, impaired feedback inhibition of adrenocorticotropic hormone (ACTH) secretion is associated with autonomous function. However, the tumor cells are not completely resistant to feedback, as evidenced by ACTH suppression by higher doses of dexamethasone (e.g., high-dose dexamethasone test) (Chap. 406). Similar set point defects are also typical of parathyroid adenomas and autonomously functioning thyroid nodules.

1	The molecular basis of some endocrine tumors, such as the multiple endocrine neoplasia (MEN) syndromes (MEN 1, 2A, 2B), have provided important insights into tumorigenesis (Chap. 408). MEN 1 is characterized primarily by the triad of parathyroid, pancreatic islet, and pituitary tumors. MEN 2 predisposes to medullary thyroid carcinoma, pheochromocytoma, and hyperparathyroidism. The MEN1 gene, located on chromosome 11q13, encodes a putative tumor-suppressor gene, menin. Analogous to the paradigm first described for retinoblastoma, the affected individual inherits a mutant copy of the MEN1 gene, and tumorigenesis ensues after a somatic “second hit” Type of Endocrine Disorder Examples

1	Type of Endocrine Disorder Examples Multiple endocrine neoplasia (MEN) Autoimmune Iatrogenic Infectious/inflammatory Activating receptor mutations Hypofunction Pituitary adenomas, hyperparathyroidism, autonomous thyroid or adrenal nodules, pheochromocytoma Adrenal cancer, medullary thyroid cancer, carcinoid Ectopic ACTH, SIADH secretion MEN 1, MEN 2 Graves’ disease Cushing’s syndrome, hypoglycemia Subacute thyroiditis LH, TSH, Ca2+, PTH receptors, Gsα Hashimoto’s thyroiditis, type 1 diabetes mellitus, Addison’s disease, polyglandular failure Radiation-induced hypopituitarism, hypothyroidism, surgical Adrenal insufficiency, hypothalamic sarcoidosis GH, LHβ, FSHβ, vasopressin 21-Hydroxylase deficiency Kallmann syndrome, Turner’s syndrome, transcription factors Vitamin D deficiency, iodine deficiency Sheehan’s syndrome, adrenal insufficiency

1	Abbreviations: ACTH, adrenocorticotropic hormone; AR, androgen receptor; ER, estrogen receptor; FSH, follicle-stimulating hormone; GHRH, growth hormone–releasing hormone; GnRH, gonadotropin-releasing hormone; GR, glucocorticoid receptor; LH, luteinizing hormone; PPAR, peroxisome proliferator activated receptor; PTH, parathyroid hormone; SIADH, syndrome of inappropriate antidiuretic hormone; TR, thyroid hormone receptor; TSH, thyroid-stimulating hormone; VDR, vitamin D receptor. leads to loss of function of the normal MEN1 gene (through deletion or point mutations).

1	leads to loss of function of the normal MEN1 gene (through deletion or point mutations). In contrast to inactivation of a tumor-suppressor gene, as occurs in MEN 1 and most other inherited cancer syndromes, MEN 2 is caused by activating mutations in a single allele. In this case, activating mutations of the RET protooncogene, which encodes a receptor tyrosine kinase, leads to thyroid C cell hyperplasia in childhood before the development of medullary thyroid carcinoma. Elucidation of this pathogenic mechanism has allowed early genetic screening for RET mutations in individuals at risk for MEN 2, permitting identification of those who may benefit from prophylactic thyroidectomy and biochemical screening for pheochromocytoma and hyperparathyroidism.

1	Mutations that activate hormone receptor signaling have been identified in several GPCRs. For example, activating mutations of the luteinizing hormone (LH) receptor cause a dominantly transmitted form of male-limited precocious puberty, reflecting premature stimulation of testosterone synthesis in Leydig cells (Chap. 411). Activating mutations in these GPCRs are located predominantly in the trans-membrane domains and induce receptor coupling to Gsα even in the absence of hormone. Consequently, adenylate cyclase is activated, and cyclic adenosine monophosphate (AMP) levels increase in a manner that mimics hormone action. A similar phenomenon results from activating mutations in Gsα. When these mutations occur early in development, they cause McCune-Albright syndrome. When they occur only in somatotropes, the activating Gsα mutations cause GH-secreting tumors and acromegaly (Chap. 403).

1	In autoimmune Graves’ disease, antibody interactions with the thyroid-stimulating hormone (TSH) receptor mimic TSH action, leading to hormone overproduction (Chap. 405). Analogous to the effects of activating mutations of the TSH receptor, these stimulating autoantibodies induce conformational changes that release the receptor from a constrained state, thereby triggering receptor coupling to G proteins. Most examples of hormone deficiency states can be attributed to glandular destruction caused by autoimmunity, surgery, infection, inflammation, infarction, hemorrhage, or tumor infiltration (Table 399-1). Autoimmune damage to the thyroid gland (Hashimoto’s thyroiditis) and pancreatic islet β cells (type 1 diabetes mellitus) is a prevalent cause of endocrine disease. Mutations in a number of hormones, hormone receptors, transcription factors, enzymes, and channels can also lead to hormone deficiencies.

1	Most severe hormone resistance syndromes are due to inherited defects in membrane receptors, nuclear receptors, or the pathways that transduce receptor signals. These disorders are characterized by defective hormone action despite the presence of increased hormone levels. In complete androgen resistance, for example, mutations in the androgen receptor result in a female phenotypic appearance in genetic (XY) males, even though LH and testosterone levels are increased (Chap. 408). In addition to these relatively rare genetic disorders, more common acquired forms of functional hormone resistance include insulin resistance in type 2 diabetes mellitus, leptin resistance in obesity, and GH resistance in catabolic states. The pathogenesis of functional resistance involves receptor downregulation and postreceptor desensitization of signaling pathways; functional forms of resistance are generally reversible.

1	Because most glands are relatively inaccessible, the physical examination usually focuses on the manifestations of hormone excess or deficiency as well as direct examination of palpable glands, such as the thyroid and gonads. For these reasons, it is important to evaluate patients in the context of their presenting symptoms, review of systems, family and social history, and exposure to medications that may affect the endocrine system. Astute clinical skills are required to detect subtle symptoms and signs suggestive of underlying endocrine disease. For example, a patient with Cushing’s syndrome may manifest specific findings, such as central fat redistribution, striae, and proximal muscle weakness, in addition to features seen commonly in the general population, such as obesity, plethora, hypertension, and glucose intolerance. Similarly, the insidious onset of hypothyroidism— with mental slowing, fatigue, dry skin, and other features—can be difficult to distinguish from similar,

1	hypertension, and glucose intolerance. Similarly, the insidious onset of hypothyroidism— with mental slowing, fatigue, dry skin, and other features—can be difficult to distinguish from similar, nonspecific findings in the general population. Clinical judgment that is based on knowledge of disease prevalence and pathophysiology is required to decide when to embark on more extensive evaluation of these disorders. Laboratory testing plays an essential role in endocrinology by allowing quantitative assessment of hormone levels and dynamics. Radiologic imaging tests such as computed tomography (CT) scan, magnetic resonance imaging (MRI), thyroid scan, and ultrasound are also used for the diagnosis of endocrine disorders. However, these tests generally are employed only after a hormonal abnormality has been established by biochemical testing.

1	Immunoassays are the most important diagnostic tool in endocrinology, as they allow sensitive, specific, and quantitative determination of steady-state and dynamic changes in hormone concentrations. Immunoassays use antibodies to detect specific hormones. For many peptide hormones, these measurements are now configured to use two different antibodies to increase binding affinity and specificity. There are many variations of these assays; a common format involves using one antibody to capture the antigen (hormone) onto an immobilized surface and a second antibody, coupled to a chemiluminescent (immunochemiluminescent assay [ICMA]) or radioactive (immunoradiometric assay [IRMA]) signal, to detect the antigen. These assays are sensitive enough to detect plasma hormone concentrations in the picomolar to nanomolar range, and they can readily distinguish structurally related proteins, such as PTH from PTH-related peptide (PTHrP). A variety of other techniques are used to measure specific

1	picomolar to nanomolar range, and they can readily distinguish structurally related proteins, such as PTH from PTH-related peptide (PTHrP). A variety of other techniques are used to measure specific hormones, including mass spectroscopy, various forms of chromatography, and enzymatic methods; bioassays are now rarely used.

1	Most hormone measurements are based on plasma or serum samples. However, urinary hormone determinations remain useful for the evaluation of some conditions. Urinary collections over 24 h provide an integrated assessment of the production of a hormone or metabolite, many of which vary during the day. It is important to assure complete collections of 24-h urine samples; simultaneous measurement of creatinine provides an internal control for the adequacy of collection and can be used to normalize some hormone measurements. A 24-h urine free cortisol measurement largely reflects the amount of unbound cortisol, thus providing a reasonable index of biologically available hormone. Other commonly used urine determinations include 17-hydroxycorticosteroids, 17-ketosteroids, vanillylmandelic acid, metanephrine, catecholamines, 5-hydroxyindoleacetic acid, and calcium.

1	The value of quantitative hormone measurements lies in their cor-2253 rect interpretation in a clinical context. The normal range for most hormones is relatively broad, often varying by a factor of twoto tenfold. The normal ranges for many hormones are sexand age-specific. Thus, using the correct normative database is an essential part of interpreting hormone tests. The pulsatile nature of hormones and factors that can affect their secretion, such as sleep, meals, and medications, must also be considered. Cortisol values increase fivefold between midnight and dawn; reproductive hormone levels vary dramatically during the female menstrual cycle.

1	For many endocrine systems, much information can be gained from basal hormone testing, particularly when different components of an endocrine axis are assessed simultaneously. For example, low testosterone and elevated LH levels suggest a primary gonadal problem, whereas a hypothalamic-pituitary disorder is likely if both LH and testosterone are low. Because TSH is a sensitive indicator of thyroid function, it is generally recommended as a first-line test for thyroid disorders. An elevated TSH level is almost always the result of primary hypothyroidism, whereas a low TSH is most often caused by thyrotoxicosis. These predictions can be confirmed by determining the free thyroxine level. In the less common circumstance when free thyroxine and TSH are both low, it is important to consider secondary hypopituitarism caused by hypothalamic-pituitary disease. Elevated calcium and PTH levels suggest hyperparathyroidism, whereas PTH is suppressed in hypercalcemia caused by malignancy or

1	secondary hypopituitarism caused by hypothalamic-pituitary disease. Elevated calcium and PTH levels suggest hyperparathyroidism, whereas PTH is suppressed in hypercalcemia caused by malignancy or granulomatous diseases. A suppressed ACTH in the setting of hypercortisolemia, or increased urine free cortisol, is seen with hyperfunctioning adrenal adenomas.

1	It is not uncommon, however, for baseline hormone levels associated with pathologic endocrine conditions to overlap with the normal range. In this circumstance, dynamic testing is useful to separate the two groups further. There are a multitude of dynamic endocrine tests, but all are based on principles of feedback regulation, and most responses can be rationalized based on principles that govern the regulation of endocrine axes. Suppression tests are used in the setting of suspected endocrine hyperfunction. An example is the dexamethasone suppression test used to evaluate Cushing’s syndrome (Chaps. 403 and 406). Stimulation tests generally are used to assess endocrine hypofunction. The ACTH stimulation test, for example, is used to assess the adrenal gland response in patients with suspected adrenal insufficiency. Other stimulation tests use hypothalamic-releasing factors such as corticotropin-releasing hormone (CRH) and growth hormone–releasing hormone (GHRH) to evaluate pituitary

1	adrenal insufficiency. Other stimulation tests use hypothalamic-releasing factors such as corticotropin-releasing hormone (CRH) and growth hormone–releasing hormone (GHRH) to evaluate pituitary hormone reserve (Chap. 403). Insulin-induced hypoglycemia also evokes pituitary ACTH and GH responses. Stimulation tests based on reduction or inhibition of endogenous hormones are now used infrequently. Examples include metyrapone inhibition of cortisol synthesis and clomiphene inhibition of estrogen feedback.

1	Many endocrine disorders are prevalent in the adult population (Table 399-2) and can be diagnosed and managed by general internists, family practitioners, or other primary health care providers. The high prevalence and clinical impact of certain endocrine diseases justifies vigilance for features of these disorders during routine physical examinations; laboratory screening is indicated in selected high-risk populations. Approach to the Patient with Endocrine Disorders Approx. Prevalence in Disorder Adultsa Screening/Testing Recommendationsb Chapter(s) Hypogonadism, male Gynecomastia 35% 5–10%, women 0.5–2%, men 1–3%, women 0.1%, men 2–5% palpable >25% by ultrasound 5–10%, women 2–5%, men 0.1–0.5%, women > men 10%, couples 5–10%, women

1	Hypogonadism, male Gynecomastia 35% 5–10%, women 0.5–2%, men 1–3%, women 0.1%, men 2–5% palpable >25% by ultrasound 5–10%, women 2–5%, men 0.1–0.5%, women > men 10%, couples 5–10%, women Median age, 51 15% in women with amenorrhea or galactorrhea 0.2%, men 0.03%, women Calculate BMI Measure waist circumference Exclude secondary causes Consider comorbid complications Beginning at age 45, screen every 3 years, or earlier in high-risk groups: Fasting plasma glucose (FPG) >126 mg/dL Random plasma glucose >200 mg/dL An elevated HbA1c Consider comorbid complications Cholesterol screening at least every 5 years; more often in high-risk groups Lipoprotein analysis (LDL, HDL) for increased cholesterol, CAD, diabetes Consider secondary causes Measure waist circumference, FPG, BP, lipids TSH; confirm with free T4 Screen women after age 35 and every 5 years thereafter TSH, free T4

1	Physical examination of thyroid Fine-needle aspiration biopsy Bone mineral density measurements in women >65 years or in post- menopausal women or men at risk Exclude secondary causes Serum calcium PTH, if calcium is elevated Assess comorbid conditions Investigate both members of couple Semen analysis in male Assess ovulatory cycles in female Specific tests as indicated Free testosterone, DHEAS Consider comorbid conditions Free testosterone, DHEAS Exclude secondary causes Additional tests as indicated FSH PRL level MRI, if not medication-related Careful history, PRL, testosterone Consider secondary causes (e.g., diabetes) Testosterone, LH Often, no tests are indicated Consider Klinefelter’s syndrome Consider medications, hypogonadism, liver disease Karyotype Testosterone Measure serum 25-OH vitamin D Consider secondary causes Karyotype Consider comorbid conditions 411, 412 aThe prevalence of most disorders varies among ethnic groups and with aging. Data based primarily on U.S.

1	25-OH vitamin D Consider secondary causes Karyotype Consider comorbid conditions 411, 412 aThe prevalence of most disorders varies among ethnic groups and with aging. Data based primarily on U.S. population. bSee individual chapters for additional information on evaluation and treatment. Early testing is indicated in patients with signs and symptoms of disease and in those at increased risk.

1	Abbreviations: BMI, body mass index; BP, blood pressure; CAD, coronary artery disease; DHEAS, dehydroepiandrosterone; FSH, follicle-stimulating hormone; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LH, luteinizing hormone; MRI, magnetic resonance imaging; PRL, prolactin; PTH, parathyroid hormone; TSH, thyroid-stimulating hormone. Mechanisms of Hormone Action J. Larry Jameson CLASSES OF HORMONES Hormones can be divided into five major types: (1) amino acid deriva-400e tives such as dopamine, catecholamine, and thyroid hormone; (2) small neuropeptides such as gonadotropin-releasing hormone (GnRH), thyrotropin-releasing hormone (TRH), somatostatin, and vasopressin; (3) large proteins such as insulin, luteinizing hormone (LH), and parathyroid hormone (PTH); (4) steroid hormones such as cortisol and estrogen that are synthesized from cholesterol-based precursors; and (5) vitamin derivatives such as retinoids (vitamin A) and vitamin

1	D. A variety of peptide growth factors, most of which act locally, share actions with hormones. As a rule, amino acid derivatives and peptide hormones interact with cell-surface membrane receptors. Steroids, thyroid hormones, vitamin D, and retinoids are lipid-soluble and interact with intracellular nuclear receptors, although many also interact with membrane receptors or intracellular signaling proteins as well. Hormones and receptors can be grouped into families, reflecting structural similarities and evolutionary origins (Table 400e-1). The evolution of these families generates diverse but highly selective pathways of hormone action. Recognition of these relationships has proven useful for extrapolating information gleaned from one hormone or receptor to other family members.

1	The glycoprotein hormone family, consisting of thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), LH, and human chorionic gonadotropin (hCG), illustrates many features of related hormones. The glycoprotein hormones are heterodimers that share the α subunit in common; the β subunits are distinct and confer β-Adrenergic, LH, FSH, TSH GSα, adenylate Stimulation of cyclic AMP procyclase duction, protein kinase A Glucagon, PTH, PTHrP, Ca2+ channels Calmodulin, Ca2+-dependent ACTH, MSH, GHRH, CRH α-Adrenergic, Giα Inhibition of cyclic AMP somatostatin Activation of K+, Ca2+ channels TRH, GnRH Gq, G11 Phospholipase C, diacylglycerol, IP3, protein kinase C, voltage-dependent Ca2+ channels Insulin, IGF-I Tyrosine MAP kinases, PI 3-kinase; AKT kinases, IRS EGF, NGF Tyrosine Raf, MAP kinases, RSK kinases, ras GH, PRL JAK, tyrosine STAT, MAP kinase, PI 3-kinase, kinases IRS-1 Activin, TGF-β, MIS Serine kinase Smads

1	EGF, NGF Tyrosine Raf, MAP kinases, RSK kinases, ras GH, PRL JAK, tyrosine STAT, MAP kinase, PI 3-kinase, kinases IRS-1 Activin, TGF-β, MIS Serine kinase Smads Abbreviations: IP3, inositol triphosphate; IRS, insulin receptor substrates; MAP, mitogenactivated protein; MSH, melanocyte-stimulating hormone; NGF, nerve growth factor; PI, phosphatidylinositol; RSK, ribosomal S6 kinase; TGF-β, transforming growth factor β. For all other abbreviations, see text. Note that most receptors interact with multiple effectors and activate networks of signaling pathways. specific biologic actions. The overall three-dimensional architecture 400e-1 of the β subunits is similar, reflecting the locations of conserved disulfide bonds that restrain protein conformation. The cloning of the β-subunit genes from multiple species suggests that this family arose from a common ancestral gene, probably by gene duplication and subsequent divergence to evolve new biologic functions.

1	As hormone families enlarge and diverge, their receptors must co-evolve to derive new biologic functions. Related G protein–coupled receptors (GPCRs), for example, have evolved for each of the glycoprotein hormones. These receptors are structurally similar, and each is coupled predominantly to the Gsα signaling pathway. However, there is minimal overlap of hormone binding. For example, TSH binds with high specificity to the TSH receptor but interacts minimally with the LH or FSH receptors. Nonetheless, there can be subtle physiologic consequences of hormone cross-reactivity with other receptors. Very high levels of hCG during pregnancy stimulate the TSH receptor and increase thyroid hormone levels, resulting in a compensatory decrease in TSH.

1	Insulin and insulin-like growth factor I (IGF-I) and IGF-II have structural similarities that are most apparent when precursor forms of the proteins are compared. In contrast to the high degree of specificity seen with the glycoprotein hormones, there is moderate cross-talk among the members of the insulin/IGF family. High concentrations of an IGF-II precursor produced by certain tumors (e.g., sarcomas) can cause hypoglycemia, partly because of binding to insulin and IGF-I receptors (Chap. 424). High concentrations of insulin also bind to the IGF-I receptor, perhaps accounting for some of the clinical manifestations seen in conditions with chronic hyperinsulinemia.

1	Another important example of receptor cross-talk is seen with PTH and parathyroid hormone–related peptide (PTHrP) (Chap. 424). PTH is produced by the parathyroid glands, whereas PTHrP is expressed at high levels during development and by a variety of tumors (Chap. 121). These hormones have amino acid sequence similarity, particularly in their amino-terminal regions. Both hormones bind to a single PTH receptor that is expressed in bone and kidney. Hypercalcemia and hypophosphatemia therefore may result from excessive production of either hormone, making it difficult to distinguish hyperparathyroidism from hypercalcemia of malignancy solely on the basis of serum chemistries. However, sensitive and specific assays for PTH and PTHrP now allow these disorders to be distinguished more readily.

1	Based on their specificities for DNA binding sites, the nuclear receptor family can be subdivided into type 1 receptors (glucocorticoid receptor, mineralocorticoid receptor, androgen receptor, estrogen receptor, progesterone receptor) that bind steroids and type 2 receptors (thyroid hormone receptor, vitamin D receptor, retinoic acid receptor, peroxisome proliferator activated receptor) that bind thyroid hormone, vitamin D, retinoic acid, or lipid derivatives. Certain functional domains in nuclear receptors, such as the zinc finger DNA-binding domains, are highly conserved. However, selective amino acid differences within this domain confer DNA sequence specificity. The hormone-binding domains are more variable, providing great diversity in the array of small molecules that bind to different nuclear receptors. With few exceptions, hormone binding is highly specific for a single type of nuclear receptor. One exception involves the glucocorticoid and mineralocorticoid receptors. Because

1	nuclear receptors. With few exceptions, hormone binding is highly specific for a single type of nuclear receptor. One exception involves the glucocorticoid and mineralocorticoid receptors. Because the mineralocorticoid receptor also binds glucocorticoids with high affinity, an enzyme (11β-hydroxysteroid dehydrogenase) in renal tubular cells inactivates glucocorticoids, allowing selective responses to mineralocorticoids such as aldosterone. However, when very high glucocorticoid concentrations occur, as in Cushing’s syndrome, the glucocorticoid degradation pathway becomes saturated, allowing excessive cortisol levels to exert mineralocorticoid effects (sodium retention, potassium wasting). This phenomenon is particularly pronounced in ectopic adrenocorticotropic hormone (ACTH) syndromes (Chap. 406). Another example of relaxed nuclear receptor specificity involves the estrogen receptor, which can bind an array of compounds, some of which have little apparent structural similarity to the

1	406). Another example of relaxed nuclear receptor specificity involves the estrogen receptor, which can bind an array of compounds, some of which have little apparent structural similarity to the high-affinity ligand estradiol. This feature of the estrogen receptor makes it susceptible to activation by “environmental estrogens” such as resveratrol, octylphenol, and many other aromatic hydrocarbons. However, this lack of specificity

1	CHAPTER 400e Mechanisms of Hormone Action provides an opportunity to synthesize a remarkable series of clinically useful antagonists (e.g., tamoxifen) and selective estrogen response modulators (SERMs) such as raloxifene. These compounds generate distinct conformations that alter receptor interactions with components of the transcription machinery (see below), thereby conferring their unique actions. The synthesis of peptide hormones and their receptors occurs through a classic pathway of gene expression: transcription → mRNA → protein → posttranslational protein processing → intracellular sorting, followed by membrane integration or secretion (Chap. 82).

1	Many hormones are embedded within larger precursor polypeptides that are proteolytically processed to yield the biologically active hormone. Examples include proopiomelanocortin (POMC) → ACTH; proglucagon → glucagon; proinsulin → insulin; and pro-PTH → PTH, among others. In many cases, such as POMC and proglucagon, these precursors generate multiple biologically active peptides. It is provocative that hormone precursors are typically inactive, presumably adding an additional level of regulatory control. Prohormone conversion occurs not only for peptide hormones but also for certain steroids (testosterone → dihydrotestosterone) and thyroid hormone (T4 → T3).

1	Peptide precursor processing is intimately linked to intracellular sorting pathways that transport proteins to appropriate vesicles and enzymes, resulting in specific cleavage steps, followed by protein folding and translocation to secretory vesicles. Hormones destined for secretion are translocated across the endoplasmic reticulum under the guidance of an amino-terminal signal sequence that subsequently is cleaved. Cell-surface receptors are inserted into the membrane via short segments of hydrophobic amino acids that remain embedded within the lipid bilayer. During translocation through the Golgi and endoplasmic reticulum, hormones and receptors are subject to a variety of posttranslational modifications, such as glycosylation and phosphorylation, which can alter protein conformation, modify circulating half-life, and alter biologic activity.

1	Synthesis of most steroid hormones is based on modifications of the precursor, cholesterol. Multiple regulated enzymatic steps are required for the synthesis of testosterone (Chap. 411), estradiol (Chap. 412), cortisol (Chap. 406), and vitamin D (Chap. 423). This large number of synthetic steps predisposes to multiple genetic and acquired disorders of steroidogenesis.

1	Endocrine genes contain regulatory DNA elements similar to those found in many other genes, but their exquisite control by hormones reflects the presence of specific hormone response elements. For example, the TSH genes are repressed directly by thyroid hormones acting through the thyroid hormone receptor (TR), a member of the nuclear receptor family. Steroidogenic enzyme gene expression requires specific transcription factors, such as steroidogenic factor-1 (SF-1), acting in conjunction with signals transmitted by trophic hormones (e.g., ACTH or LH). For some hormones, substantial regulation occurs at the level of translational efficiency. Insulin biosynthesis, although it requires ongoing gene transcription, is regulated primarily at the translational and secretory levels in response to elevated levels of glucose or amino acids. HORMONE SECRETION, TRANSPORT, AND DEGRADATION

1	The level of a hormone is determined by its rate of secretion and its circulating half-life. After protein processing, peptide hormones (e.g., GnRH, insulin, growth hormone [GH]) are stored in secretory granules. As these granules mature, they are poised beneath the plasma membrane for imminent release into the circulation. In most instances, the stimulus for hormone secretion is a releasing factor or neural signal that induces rapid changes in intracellular calcium concentrations, leading to secretory granule fusion with the plasma membrane and release of its contents into the extracellular environment and bloodstream. Steroid hormones, in contrast, diffuse into the circulation as they are synthesized. Thus, their secretory rates are closely aligned with rates of synthesis. For example, ACTH and LH induce steroidogenesis by stimulating the activity of the steroidogenic acute regulatory (StAR) protein (transports cholesterol into the mitochondrion) along with other rate-limiting steps

1	and LH induce steroidogenesis by stimulating the activity of the steroidogenic acute regulatory (StAR) protein (transports cholesterol into the mitochondrion) along with other rate-limiting steps (e.g., cholesterol side-chain cleavage enzyme, CYP11A1) in the steroidogenic pathway.

1	Hormone transport and degradation dictate the rapidity with which a hormonal signal decays. Some hormone signals are evanescent (e.g., somatostatin), whereas others are longer-lived (e.g., TSH). Because somatostatin exerts effects in virtually every tissue, a short half-life allows its concentrations and actions to be controlled locally. Structural modifications that impair somatostatin degradation have been useful for generating long-acting therapeutic analogues such as octreotide (Chap. 403). In contrast, the actions of TSH are highly specific for the thyroid gland. Its prolonged half-life accounts for relatively constant serum levels even though TSH is secreted in discrete pulses.

1	An understanding of circulating hormone half-life is important for achieving physiologic hormone replacement, as the frequency of dosing and the time required to reach steady state are intimately linked to rates of hormone decay. T4, for example, has a circulating half-life of 7 days. Consequently, >1 month is required to reach a new steady state, and single daily doses are sufficient to achieve constant hormone levels. T3, in contrast, has a half-life of 1 day. Its administration is associated with more dynamic serum levels, and it must be administered two to three times per day. Similarly, synthetic glucocorticoids vary widely in their half-lives; those with longer half-lives (e.g., dexamethasone) are associated with greater suppression of the hypothalamic-pituitaryadrenal (HPA) axis. Most protein hormones (e.g., ACTH, GH, prolactin [PRL], PTH, LH) have relatively short half-lives (<20 min), leading to sharp peaks of secretion and decay. The only accurate way to profile the pulse

1	Most protein hormones (e.g., ACTH, GH, prolactin [PRL], PTH, LH) have relatively short half-lives (<20 min), leading to sharp peaks of secretion and decay. The only accurate way to profile the pulse frequency and amplitude of these hormones is to measure levels in frequently sampled blood (every 10 min or less) over long durations (8–24 h). Because this is not practical in a clinical setting, an alternative strategy is to pool three to four samples drawn at about 30-min intervals, or interpret the results in the context of a relatively wide normal range. Rapid hormone decay is useful in certain clinical settings. For example, the short half-life of PTH allows the use of intraoperative PTH determinations to confirm successful removal of an adenoma. This is particularly valuable diagnostically when there is a possibility of multicentric disease or parathyroid hyperplasia, as occurs with multiple endocrine neoplasia (MEN) or renal insufficiency.

1	Many hormones circulate in association with serum-binding proteins. Examples include (1) T4 and T3 binding to thyroxine-binding globulin (TBG), albumin, and thyroxine-binding prealbumin (TBPA); cortisol binding to cortisol-binding globulin (CBG); (3) androgen and estrogen binding to sex hormone–binding globulin (SHBG); IGF-I and -II binding to multiple IGF-binding proteins (IGFBPs);

1	GH interactions with GH-binding protein (GHBP), a circulating fragment of the GH receptor extracellular domain; and (6) activin binding to follistatin. These interactions provide a hormonal reservoir, prevent otherwise rapid degradation of unbound hormones, restrict hormone access to certain sites (e.g., IGFBPs), and modulate the unbound, or “free,” hormone concentrations. Although a variety of binding protein abnormalities have been identified, most have little clinical consequence aside from creating diagnostic problems. For example, TBG deficiency can reduce total thyroid hormone levels greatly but the free concentrations of T4 and T3 remain normal. Liver disease and certain medications can also influence binding protein levels (e.g., estrogen increases TBG) or cause displacement of hormones from binding proteins (e.g., salsalate displaces T4 from TBG). In general, only unbound hormone is available to interact with receptors and thus elicit a biologic response. Short-term

1	of hormones from binding proteins (e.g., salsalate displaces T4 from TBG). In general, only unbound hormone is available to interact with receptors and thus elicit a biologic response. Short-term perturbations in binding proteins change the free hormone concentration, which in turn induces compensatory adaptations through feedback loops. SHBG changes in women are an exception to this self-correcting mechanism. When SHBG decreases because of insulin resistance or androgen excess, the unbound testosterone concentration is increased, potentially leading to hirsutism (Chap. 68). The increased unbound testosterone level does not result in an adequate compensatory feedback correction because estrogen, not testosterone, is the primary regulator of the reproductive axis.

1	An additional exception to the unbound hormone hypothesis involves megalin, a member of the low-density lipoprotein (LDL) receptor family that serves as an endocytotic receptor for carrier-bound vitamins A and D and SHBG-bound androgens and estrogens. After internalization, the carrier proteins are degraded in lysosomes and release their bound ligands within the cells. Membrane transporters have also been identified for thyroid hormones. Hormone degradation can be an important mechanism for regulating concentrations locally. As noted above, 11β-hydroxysteroid dehydrogenase inactivates glucocorticoids in renal tubular cells, preventing actions through the mineralocorticoid receptor. Thyroid hormone deiodinases convert T4 to T3 and can inactivate T3. During development, degradation of retinoic acid by Cyp26b1 prevents primordial germ cells in the male from entering meiosis, as occurs in the female ovary.

1	Receptors for hormones are divided into two major classes: membrane and nuclear. Membrane receptors primarily bind peptide hormones and catecholamines. Nuclear receptors bind small molecules that can diffuse across the cell membrane, such as steroids and vitamin D. Certain general principles apply to hormone-receptor interactions regardless of the class of receptor. Hormones bind to receptors with specificity and an affinity that generally coincides with the dynamic range of circulating hormone concentrations. Low concentrations of free hormone (usually 10−12 to 10−9M) rapidly associate and dissociate from receptors in a bimolecular reaction such that the occupancy of the receptor at any given moment is a function of hormone concentration and the receptor’s affinity for the hormone. Receptor numbers vary greatly in different target tissues, providing one of the major determinants of specific tissue responses to circulating hormones. For example, ACTH receptors are located almost

1	Receptor numbers vary greatly in different target tissues, providing one of the major determinants of specific tissue responses to circulating hormones. For example, ACTH receptors are located almost exclusively in the adrenal cortex, and FSH receptors are found predominantly in the gonads. In contrast, insulin and TRs are widely distributed, reflecting the need for metabolic responses in all tissues.

1	Membrane receptors for hormones can be divided into several major groups: (1) seven transmembrane GPCRs, (2) tyrosine kinase receptors, (3) cytokine receptors, and (4) serine kinase receptors (Fig. 400e-1). The seven transmembrane GPCR family binds a remarkable array of hormones, including large proteins (e.g., LH, PTH), small peptides (e.g., TRH, somatostatin), catecholamines (epinephrine, dopamine), and even minerals (e.g., calcium). The extracellular domains of GPCRs vary widely in size and are the major binding site for large hormones. 400e-3 The transmembrane-spanning regions are composed of hydrophobic α-helical domains that traverse the lipid bilayer. Like some channels, these domains are thought to circularize and form a hydrophobic pocket into which certain small ligands fit. Hormone binding induces conformational changes in these domains, transducing structural changes to the intracellular domain, which is a docking site for G proteins.

1	The large family of G proteins, so named because they bind guanine nucleotides (guanosine triphosphate [GTP], guanosine diphosphate [GDP]), provides great diversity for coupling receptors to different signaling pathways. G proteins form a heterotrimeric complex that is composed of various α and βγ subunits. The α subunit contains the guanine nucleotide–binding site and hydrolyzes GTP → GDP. The βγ subunits are tightly associated and modulate the activity of the α subunit as well as mediating their own effector signaling pathways. G protein activity is regulated by a cycle that involves GTP hydrolysis and dynamic interactions between the α and αβ subunits. Hormone binding to the receptor induces GDP dissociation, allowing Gα to bind GTP and dissociate from the αβ complex. Under these conditions, the Gα subunit is activated and mediates signal transduction through various enzymes, such as adenylate cyclase and phospholipase C. GTP hydrolysis to GDP allows reassociation with the βγ

1	conditions, the Gα subunit is activated and mediates signal transduction through various enzymes, such as adenylate cyclase and phospholipase C. GTP hydrolysis to GDP allows reassociation with the βγ subunits and restores the inactive state. As described below, a variety of endocrinopathies result from G protein mutations or from mutations in receptors that modify their interactions with G proteins. G proteins interact with other cellular proteins, including kinases, channels, G protein–coupled receptor kinases (GRKs), and arrestins, that mediate signaling as well as receptor desensitization and recycling.

1	The tyrosine kinase receptors transduce signals for insulin and a variety of growth factors, such as IGF-I, epidermal growth factor (EGF), nerve growth factor, platelet-derived growth factor, and fibroblast growth factor. The cysteine-rich extracellular ligand-binding domains contain growth factor binding sites. After ligand binding, this class of receptors undergoes autophosphorylation, inducing interactions with intracellular adaptor proteins such as Shc and insulin receptor substrates (IRS). In the case of the insulin receptor, multiple kinases are activated, including the Raf-Ras-MAPK and the Akt/protein kinase B pathways. The tyrosine kinase receptors play a prominent role in cell growth and differentiation as well as in intermediary metabolism. The GH and PRL receptors belong to the cytokine receptor family. Analogous to the tyrosine kinase receptors, ligand binding induces CHAPTER400e Mechanisms of Hormone Action PKA, PKC Ras/Raf

1	The GH and PRL receptors belong to the cytokine receptor family. Analogous to the tyrosine kinase receptors, ligand binding induces CHAPTER400e Mechanisms of Hormone Action PKA, PKC Ras/Raf FIGURE 400e-1 Membrane receptor signaling. MAPK, mitogen-activated protein kinase; PKA, C, protein kinase A, C; TGF, transforming growth factor. For other abbreviations, see text. receptor interaction with intracellular kinases—the Janus kinases (JAKs), which phosphorylate members of the signal transduction and activators of transcription (STAT) family—as well as with other signaling pathways (Ras, PI3-K, MAPK). The activated STAT proteins translocate to the nucleus and stimulate expression of target genes.

1	The serine kinase receptors mediate the actions of activins, transforming growth factor β, müllerian-inhibiting substance (MIS, also known as anti-müllerian hormone, AMH), and bone morphogenic proteins (BMPs). This family of receptors (consisting of type I and II subunits) signals through proteins termed smads (fusion of terms for Caenorhabditis elegans sma + mammalian mad). Like the STAT proteins, the smads serve a dual role of transducing the receptor signal and acting as transcription factors. The pleomorphic actions of these growth factors dictate that they act primarily in a local (paracrine or autocrine) manner. Binding proteins such as follistatin (which binds activin and other members of this family) function to inactivate the growth factors and restrict their distribution.

1	The family of nuclear receptors has grown to nearly 100 members, many of which are still classified as orphan receptors because their ligands, if they exist, have not been identified (Fig. 400e-2). Otherwise, most nuclear receptors are classified on the basis of their ligands. Although all nuclear receptors ultimately act to increase or decrease gene transcription, some (e.g., glucocorticoid receptor) reside primarily in the cytoplasm, whereas others (e.g., TR) are located in the nucleus. After ligand binding, the cytoplasmically localized receptors translocate to the nucleus. There is growing evidence that certain nuclear receptors (e.g., glucocorticoid, estrogen) can also act at the membrane or in the cytoplasm to activate or repress signal transduction pathways, providing a mechanism for cross-talk between membrane and nuclear receptors.

1	The structures of nuclear receptors have been studied extensively, including by x-ray crystallography. The DNA binding domain, consisting of two zinc fingers, contacts specific DNA recognition sequences in target genes. Most nuclear receptors bind to DNA as dimers. Consequently, each monomer recognizes an individual DNA motif, referred to as a “half-site.” The steroid receptors, including the glucocorticoid, estrogen, progesterone, and androgen receptors, bind to DNA as homodimers. Consistent with this twofold symmetry, their DNA recognition half-sites are palindromic. The thyroid, retinoid, peroxisome proliferator activated, and vitamin D receptors bind to DNA preferentially as heterodimers in combination with retinoid X receptors (RXRs). Their DNA half-sites are typically arranged as direct repeats.

1	The carboxy-terminal hormone-binding domain mediates transcriptional control. For type II receptors such as TR and retinoic acid receptor (RAR), co-repressor proteins bind to the receptor in the absence of ligand and silence gene transcription. Hormone binding induces conformational changes, triggering the release of corepressors and inducing the recruitment of coactivators that stimulate transcription. Thus, these receptors are capable of mediating dramatic changes in the level of gene activity. Certain disease states are associated with defective regulation of these events. For example, mutations in the TR prevent co-repressor dissociation, resulting in an autosomal dominant form of hormone resistance (Chap. 405). In promyelocytic leukemia, fusion of RARα to other nuclear proteins causes aberrant gene silencing that prevents normal cellular differentiation. Treatment with retinoic acid reverses this repression and allows cellular differentiation and apoptosis to occur. Most type 1

1	aberrant gene silencing that prevents normal cellular differentiation. Treatment with retinoic acid reverses this repression and allows cellular differentiation and apoptosis to occur. Most type 1 steroid receptors interact weakly with co-repressors, but ligand binding still induces interactions with an array of coactivators. X-ray crystallography shows that various SERMs induce distinct estrogen receptor conformations. The tissue-specific responses caused by these agents in breast, bone, and uterus appear to reflect distinct interactions with coactivators. The receptorcoactivator complex stimulates gene transcription by several pathways, including (1) recruitment of enzymes (histone acetyl transferases) that modify chromatin structure, (2) interactions with additional transcription factors on the target gene, and (3) direct interactions with components of the general transcription apparatus to enhance the rate of RNA polymerase II–mediated transcription. Studies of nuclear

1	factors on the target gene, and (3) direct interactions with components of the general transcription apparatus to enhance the rate of RNA polymerase II–mediated transcription. Studies of nuclear receptor-mediated transcription show that these are dynamic events that involve relatively rapid (e.g., 30–60 min) cycling of transcription complexes on any specific target gene.

1	The functions of individual hormones are described in detail in subsequent chapters. Nevertheless, it is useful to illustrate how most biologic responses require integration of several different hormone pathways. The physiologic functions of hormones can be divided into three general areas: (1) growth and differentiation, (2) maintenance of homeostasis, and (3) reproduction. FIGURE 400e-2 Nuclear receptor signaling. AR, androgen receptor; DAX, dosage-sensitive sex-reversal, adrenal hypoplasia congenita, X-chromosome; ER, estrogen receptor; GR, glucocorticoid receptor; HNF4α, hepatic nuclear factor 4α; PPAR, peroxisome proliferator activated receptor; PR, progesterone receptor; RAR, retinoic acid receptor; SF-1, steroidogenic factor-1; TR, thyroid hormone receptor; VDR, vitamin D receptor.

1	Multiple hormones and nutritional factors mediate the complex phenomenon of growth (Chap. 401e). Short stature may be caused by GH deficiency, hypothyroidism, Cushing’s syndrome, precocious puberty, malnutrition, chronic illness, or genetic abnormalities that affect the epiphyseal growth plates (e.g., FGFR3 and SHOX mutations). Many factors (GH, IGF-I, thyroid hormones) stimulate growth, whereas others (sex steroids) lead to epiphyseal closure. Understanding these hormonal interactions is important in the diagnosis and management of growth disorders. For example, delaying exposure to high levels of sex steroids may enhance the efficacy of GH treatment. Although virtually all hormones affect homeostasis, the most important among them are the following: 1. Thyroid hormone—controls about 25% of basal metabolism in most tissues 2. Cortisol—exerts a permissive action for many hormones in addition to its own direct effects 3. 4. 5.

1	Thyroid hormone—controls about 25% of basal metabolism in most tissues 2. Cortisol—exerts a permissive action for many hormones in addition to its own direct effects 3. 4. 5. Mineralocorticoids—control vascular volume and serum electrolyte (Na+, K+) concentrations 6. Insulin—maintains euglycemia in the fed and fasted states The defense against hypoglycemia is an impressive example of integrated hormone action (Chap. 420). In response to the fasting state and falling blood glucose, insulin secretion is suppressed, resulting in decreased glucose uptake and enhanced glycogenolysis, lipolysis, proteolysis, and gluconeogenesis to mobilize fuel sources. If hypoglycemia develops (usually from insulin administration or sulfonylureas), an orchestrated counterregulatory response occurs—glucagon and epinephrine rapidly stimulate glycogenolysis and gluconeogenesis, whereas GH and cortisol act over several hours to raise glucose levels and antagonize insulin action.

1	Although free-water clearance is controlled primarily by vasopressin, cortisol and thyroid hormone are also important for facilitating renal tubular responses to vasopressin (Chap. 404). PTH and vitamin D function in an interdependent manner to control calcium metabolism (Chap. 423). PTH stimulates renal synthesis of 1,25-dihydroxyvitamin D, which increases calcium absorption in the gastrointestinal tract and enhances PTH action in bone. Increased calcium, along with vitamin D, feeds back to suppress PTH, thus maintaining calcium balance.

1	Depending on the severity of a specific stress and whether it is acute or chronic, multiple endocrine and cytokine pathways are activated to mount an appropriate physiologic response. In severe acute stress such as trauma or shock, the sympathetic nervous system is activated and catecholamines are released, leading to increased cardiac output and a primed musculoskeletal system. Catecholamines also increase mean blood pressure and stimulate glucose production. Multiple stress-induced pathways converge on the hypothalamus, stimulating several hormones, including vasopressin and corticotropin-releasing hormone (CRH). These hormones, in addition to cytokines (tumor necrosis factor α, interleukin [IL] 2, IL-6) increase ACTH and GH production. ACTH stimulates the adrenal gland, increasing cortisol, which in turn helps sustain blood pressure and dampen the inflammatory response. Increased vasopressin acts to conserve free water.

1	The stages of reproduction include (1) sex determination during fetal development (Chap. 410); (2) sexual maturation during puberty (Chaps. 411 and 412); (3) conception, pregnancy, lactation, and child rearing (Chap. 412); and (4) cessation of reproductive capability at menopause (Chap. 413). Each of these stages involves an orchestrated interplay of multiple hormones, a phenomenon well illustrated by the dynamic hormonal changes that occur during each 28-day menstrual cycle. In the early follicular phase, pulsatile secretion of LH and FSH stimulates the progressive maturation of the ovarian follicle. This 400e-5 results in gradually increasing estrogen and progesterone levels, leading to enhanced pituitary sensitivity to GnRH, which, when combined with accelerated GnRH secretion, triggers the LH surge and rupture of the mature follicle. Inhibin, a protein produced by the granulosa cells, enhances follicular growth and feeds back to the pituitary to selectively suppress FSH without

1	the LH surge and rupture of the mature follicle. Inhibin, a protein produced by the granulosa cells, enhances follicular growth and feeds back to the pituitary to selectively suppress FSH without affecting LH. Growth factors such as EGF and IGF-I modulate follicular responsiveness to gonadotropins. Vascular endothelial growth factor and prostaglandins play a role in follicle vascularization and rupture.

1	During pregnancy, the increased production of prolactin, in combination with placentally derived steroids (e.g., estrogen and progesterone), prepares the breast for lactation. Estrogens induce the production of progesterone receptors, allowing for increased responsiveness to progesterone. In addition to these and other hormones involved in lactation, the nervous system and oxytocin mediate the suckling response and milk release.

1	Feedback control, both negative and positive, is a fundamental feature of endocrine systems. Each of the major hypothalamic-pituitaryhormone axes is governed by negative feedback, a process that maintains hormone levels within a relatively narrow range (Chap. 401e). Examples of hypothalamic-pituitary negative feedback include (1) thyroid hormones on the TRH-TSH axis, (2) cortisol on the CRHACTH axis, (3) gonadal steroids on the GnRH-LH/FSH axis, and (4) IGF-I on the growth hormone–releasing hormone (GHRH)-GH axis (Fig. 400e-3). These regulatory loops include both positive (e.g., TRH, TSH) and negative (e.g., T4, T3) components, allowing for exquisite control of hormone levels. As an example, a small reduction of thyroid hormone triggers a rapid increase of TRH and TSH secretion, resulting in thyroid gland stimulation and increased thyroid hormone production. When thyroid hormone reaches a normal level, it feeds back to suppress TRH and TSH, and a new steady state is attained. Feedback

1	in thyroid gland stimulation and increased thyroid hormone production. When thyroid hormone reaches a normal level, it feeds back to suppress TRH and TSH, and a new steady state is attained. Feedback regulation also occurs for endocrine systems that do not involve the pituitary gland, such as calcium feedback on PTH, glucose inhibition of insulin secretion, and leptin feedback on the hypothalamus. An

1	CHAPTER 400e Mechanisms of Hormone Action FIGURE 400e-3 Feedback regulation of endocrine axes. CNS, central nervous system. understanding of feedback regulation provides important insights into endocrine testing paradigms (see below). Positive feedback control also occurs but is not well understood. The primary example is estrogen-mediated stimulation of the midcycle LH surge. Although chronic low levels of estrogen are inhibitory, gradually rising estrogen levels stimulate LH secretion. This effect, which is illustrative of an endocrine rhythm (see below), involves activation of the hypothalamic GnRH pulse generator. In addition, estrogen-primed gonadotropes are extraordinarily sensitive to GnRH, leading to amplification of LH release.

1	The previously mentioned examples of feedback control involve classic endocrine pathways in which hormones are released by one gland and act on a distant target gland. However, local regulatory systems, often involving growth factors, are increasingly recognized. Paracrine regulation refers to factors released by one cell that act on an adjacent cell in the same tissue. For example, somatostatin secretion by pancreatic islet δ cells inhibits insulin secretion from nearby β cells. Autocrine regulation describes the action of a factor on the same cell from which it is produced. IGF-I acts on many cells that produce it, including chondrocytes, breast epithelium, and gonadal cells. Unlike endocrine actions, paracrine and autocrine control are difficult to document because local growth factor concentrations cannot be measured readily.

1	Anatomic relationships of glandular systems also greatly influence hormonal exposure: the physical organization of islet cells enhances their intercellular communication; the portal vasculature of the hypothalamic-pituitary system exposes the pituitary to high concentrations of hypothalamic releasing factors; testicular seminiferous tubules gain exposure to high testosterone levels produced by the interdigitated Leydig cells; the pancreas receives nutrient information and local exposure to peptide hormones (incretins) from the gastrointestinal tract; and the liver is the proximal target of insulin action because of portal drainage from the pancreas.

1	The feedback regulatory systems described above are superimposed on hormonal rhythms that are used for adaptation to the environment. Seasonal changes, the daily occurrence of the light-dark cycle, sleep, meals, and stress are examples of the many environmental events that affect hormonal rhythms. The menstrual cycle is repeated on average every 28 days, reflecting the time required to follicular maturation and ovulation (Chap. 412). Essentially all pituitary hormone rhythms are entrained to sleep and to the circadian cycle, generating reproducible patterns that are repeated approximately every 24 h. The HPA axis, for example, exhibits characteristic peaks of ACTH and cortisol production in the early morning, with a nadir during the night. Recognition of these rhythms is important for endocrine testing and treatment. Patients with Cushing’s syndrome characteristically exhibit increased midnight cortisol levels compared with normal individuals (Chap. 406). In contrast, morning cortisol

1	testing and treatment. Patients with Cushing’s syndrome characteristically exhibit increased midnight cortisol levels compared with normal individuals (Chap. 406). In contrast, morning cortisol levels are similar in these groups, as cortisol is normally high at this time of day in normal individuals. The HPA axis is more susceptible to suppression by glucocorticoids administered at night as they blunt the early-morning rise of ACTH. Understanding these rhythms allows glucocorticoid replacement that mimics diurnal production by administering larger doses in the morning than in the afternoon. Disrupted sleep rhythms can alter hormonal regulation. For example, sleep deprivation causes mild insulin resistance, food craving, and hypertension, which are reversible, at least in the short term. Emerging evidence indicates that circadian clock pathways not only regulate sleep-wake cycles but also play important roles in virtually every cell type. For example, tissue-specific deletion of clock

1	evidence indicates that circadian clock pathways not only regulate sleep-wake cycles but also play important roles in virtually every cell type. For example, tissue-specific deletion of clock genes alters rhythms and levels of gene expression, as well as metabolic responses in liver, adipose, and other tissues.

1	Other endocrine rhythms occur on a more rapid time scale. Many peptide hormones are secreted in discrete bursts every few hours. LH and FSH secretion are exquisitely sensitive to GnRH pulse frequency. Intermittent pulses of GnRH are required to maintain pituitary sensitivity, whereas continuous exposure to GnRH causes pituitary gonadotrope desensitization. This feature of the hypothalamic-pituitarygonadotrope axis forms the basis for using long-acting GnRH agonists to treat central precocious puberty or to decrease testosterone levels in the management of prostate cancer. It is important to be aware of the pulsatile nature of hormone secretion and the rhythmic patterns of hormone production in relating serum hormone measurements to normal values. For some hormones, integrated markers have been developed to circumvent hormonal fluctuations. Examples include 24-h urine collections for cortisol, IGF-I as a biologic marker of GH action, and HbA1c as an index of long-term (weeks to months)

1	been developed to circumvent hormonal fluctuations. Examples include 24-h urine collections for cortisol, IGF-I as a biologic marker of GH action, and HbA1c as an index of long-term (weeks to months) blood glucose control.

1	Often, one must interpret endocrine data only in the context of other hormones. For example, PTH levels typically are assessed in combination with serum calcium concentrations. A high serum calcium level in association with elevated PTH is suggestive of hyperparathyroidism, whereas a suppressed PTH in this situation is more likely to be caused by hypercalcemia of malignancy or other causes of hypercalcemia. Similarly, TSH should be elevated when T4 and T3 concentrations are low, reflecting reduced feedback inhibition. When this is not the case, it is important to consider secondary hypothyroidism, which is caused by a defect at the level of the pituitary. Anterior Pituitary: Physiology of Pituitary Hormones Shlomo Melmed, J. Larry Jameson The anterior pituitary often is referred to as the “master gland” because, together with the hypothalamus, it orchestrates the complex 401e

1	Tissue-specific T-Pit Prop-1, Pit-1 Prop-1, Pit-1 Prop-1, Pit-1, TEF SF-1, DAX-1 transcription factor Fetal appearance 6 weeks 8 weeks 12 weeks 12 weeks 12 weeks Hormone POMC GH PRL TSH FSH, LH Protein Polypeptide Polypeptide Polypeptide Glycoprotein α, β Glycoprotein α, β subunits subunits Amino acids 266 (ACTH 1–39) 191 199 211 210, 204 Stimulators CRH, AVP, gp-130 GHRH, ghrelin Estrogen, TRH, VIP TRH GnRH, activins, estrogen cytokines Inhibitors Glucocorticoids Somatostatin, IGF-I Dopamine T3, T4, dopamine, soma-Sex steroids, inhibin tostatin, glucocorticoids Target gland Adrenal Liver, bone, other tissues Breast, other tissues Thyroid Ovary, testis Trophic effect Steroid production IGF-I production, Milk production T4 synthesis and Sex steroid production, growth induction, secretion follicle growth, germ cell insulin antagonism maturation Normal range ACTH, 4–22 pg/L <0.5 μg/La M <15 μg/L; F <20 μg/L 0.1–5 mU/L M, 5–20 IU/L, F (basal), 5–20 IU/L aHormone secretion integrated over

1	growth, germ cell insulin antagonism maturation Normal range ACTH, 4–22 pg/L <0.5 μg/La M <15 μg/L; F <20 μg/L 0.1–5 mU/L M, 5–20 IU/L, F (basal), 5–20 IU/L aHormone secretion integrated over 24 h.

1	Abbreviations: M, male; F, female. For other abbreviations, see text.

1	regulatory functions of many other endocrine glands. The anterior pituitary gland produces six major hormones: (1) prolactin (PRL), (2) growth hormone (GH), (3) adrenocorticotropic hormone (ACTH), (4) luteinizing hormone (LH), (5) follicle-stimulating hormone (FSH), and (6) thyroid-stimulating hormone (TSH) (Table 401e-1). Pituitary hormones are secreted in a pulsatile manner, reflecting stimulation by an array of specific hypothalamic releasing factors. Each of these pituitary hormones elicits specific responses in peripheral target tissues. The hormonal products of those peripheral glands, in turn, exert feedback control at the level of the hypothalamus and pituitary to modulate pituitary function (Fig. 401e-1). Pituitary tumors cause characteristic hormone excess syndromes. Hormone deficiency may be inherited or acquired. Fortunately, there are efficacious treatments for many pituitary hormone excess and deficiency syndromes. Nonetheless, these diagnoses are often elusive; this

1	deficiency may be inherited or acquired. Fortunately, there are efficacious treatments for many pituitary hormone excess and deficiency syndromes. Nonetheless, these diagnoses are often elusive; this emphasizes the importance of recognizing subtle clinical manifestations and performing the correct laboratory diagnostic tests. For discussion of disorders of the posterior pituitary, or neurohypophysis, see Chap. 404.

1	The pituitary gland weighs ~600 mg and is located within the sella turcica ventral to the diaphragma sella; it consists of anatomically and functionally distinct anterior and posterior lobes. The bony sella is contiguous to vascular and neurologic structures, including the cavernous sinuses, cranial nerves, and optic chiasm. Thus, expanding intrasellar pathologic processes may have significant central mass effects in addition to their endocrinologic impact.

1	Hypothalamic neural cells synthesize specific releasing and inhibiting hormones that are secreted directly into the portal vessels of the pituitary stalk. Blood supply of the pituitary gland comes from the superior and inferior hypophyseal arteries (Fig. 401e-2). The hypothalamic-pituitary portal plexus provides the major blood source for the anterior pituitary, allowing reliable transmission of hypothalamic peptide pulses without significant systemic dilution; consequently, pituitary cells are 401e-1 exposed to releasing or inhibiting factors and in turn release their hormones as discrete pulses into the systemic circulation (Fig. 401e-3).

1	The posterior pituitary is supplied by the inferior hypophyseal arteries. In contrast to the anterior pituitary, the posterior lobe is directly innervated by hypothalamic neurons (supraopticohypophyseal and tuberohypophyseal nerve tracts) via the pituitary stalk (Chap. 404). Thus, posterior pituitary production of vasopressin (antidiuretic hormone [ADH]) and oxytocin is particularly sensitive to neuronal damage by lesions that affect the pituitary stalk or hypothalamus.

1	The embryonic differentiation and maturation of anterior pituitary cells have been elucidated in considerable detail. Pituitary development from Rathke’s pouch involves a complex interplay of lineage-specific transcription factors expressed in pluripotent precursor cells and gradients of locally produced growth factors (Table 401e-1). The transcription factor Prop-1 induces pituitary development of Pit-1specific lineages as well as gonadotropes. The transcription factor Pit-1 determines cell-specific expression of GH, PRL, and TSH in somatotropes, lactotropes, and thyrotropes. Expression of high levels of estrogen receptors in cells that contain Pit-1 favors PRL expression, whereas thyrotrope embryonic factor (TEF) induces TSH expression. Pit-1 binds to GH, PRL, and TSH gene regulatory elements as well as to recognition sites on its own promoter, providing a mechanism for maintaining specific pituitary hormone phenotypic stability. Gonadotrope cell development is further defined by

1	elements as well as to recognition sites on its own promoter, providing a mechanism for maintaining specific pituitary hormone phenotypic stability. Gonadotrope cell development is further defined by the cell-specific expression of the nuclear receptors steroidogenic factor (SF-1) and d osage-sensitive sex reversal, a drenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1). Development of corticotrope cells, which express the proopiomelanocortin (POMC) gene, requires the T-Pit transcription factor. Abnormalities of pituitary development caused by mutations of Pit-1, Prop-1, SF-1, DAX-1, and T-Pit result in a rare, selective or combined pituitary hormone deficit syndromes.

1	Each anterior pituitary hormone is under unique control, and each exhibits highly specific normal and dysregulated secretory characteristics. PROLACTIN Synthesis PRL consists of 198 amino acids and has a molecular mass of 21,500 kDa; it is weakly homologous to GH and human placental lactogen (hPL), reflecting the duplication and divergence of a common GH-PRL-hPL precursor gene. PRL is synthesized in lactotropes, CHAPTER 401e Anterior Pituitary: Physiology of Pituitary Hormones Source: Adapted from I Shimon, S Melmed, in S Melmed, P Conn (eds): Endocrinology: Basic and Clinical Principles. Totowa, NJ, Humana, 2005. FIgURE 401e-2 Diagram of hypothalamic-pituitary vasculature. The hypothalamic nuclei produce hormones that traverse the portal system and impinge on anterior pituitary cells to regulate pituitary hormone secretion. Posterior pituitary hormones are derived from direct neural extensions.

1	PRL synthesis and secretion. Targeted disruption (gene knockout) of the murine D2 receptor in mice results in hyperprolactinemia and lactotrope proliferation. As discussed below, dopamine agonists play a central role in the management of hyperprolactinemic disorders. Thyrotropin-releasing hormone (TRH) (pyro Glu-His-Pro-NH2) is a hypothalamic tripeptide that elicits PRL release within 15–30 min after intravenous injection. The physiologic relevance of TRH for PRL regulation is unclear, and it appears primarily to regulate TSH (Chap. 405). Vasoactive intestinal peptide (VIP) also induces PRL release, whereas FIgURE 401e-1 Diagram of pituitary axes. Hypothalamic hormones glucocorticoids and thyroid hormone weakly suppress PRL secretion. regulate anterior pituitary trophic hormones that in turn determine target gland secretion. Peripheral hormones feed back to regulate hypothalamic and pituitary hormones. For abbreviations, see text.

1	which constitute about 20% of anterior pituitary cells. Lactotropes and somatotropes are derived from a common precursor cell that may give rise to a tumor that secretes both PRL and GH. Marked lactotrope cell hyperplasia develops during pregnancy and the first few months of lactation. These transient functional changes in the lactotrope popula- Serum PRL levels rise transiently after exercise, meals, sexual intercourse, minor surgical procedures, general anesthesia, chest wall injury, acute myocardial infarction, and other forms of acute stress. PRL levels increase markedly (about tenfold) during pregnancy and decline rapidly within 2 weeks of parturition. If breast-feeding is initiated, basal PRL levels remain elevated; suckling stimulates transient reflex increases in PRL levels that last for about 30–45 min. Breast suckling activates neural afferent pathways in the hypothalamus that tion are induced by estrogen.

1	Secretion Normal adult serum PRL levels are about 10–25 μg/L in women and 10–20 μg/L in men. PRL secretion is pulsatile, with the highest secretory peaks occurring during rapid eye movement sleep. Peak serum PRL levels (up to 30 μg/L) occur between 4:00 and 6:00 a.m. The circulating half-life of PRL is about 50 min. PRL is unique among the pituitary hormones in that the predomi- nant central control mechanism is inhibitory, reflecting dopamine-mediated suppression of PRL release. This regulatory pathway accounts for the spontaneous PRL hypersecretion that occurs with pituitary stalk section, often a consequence of compressive mass lesions at the skull FIgURE 401e-3 Hypothalamic gonadotropin-releasing hormone base. Pituitary dopamine type 2 (D2) receptors mediate inhibition of (GnRH) pulses induce secretory pulses of luteinizing hormone (LH). induce PRL release. With time, suckling-induced responses diminish and interfeeding PRL levels return to normal.

1	induce PRL release. With time, suckling-induced responses diminish and interfeeding PRL levels return to normal. Action The PRL receptor is a member of the type I cytokine receptor family that also includes GH and interleukin (IL) 6 receptors. Ligand binding induces receptor dimerization and intracellular signaling by Janus kinase (JAK), which stimulates translocation of the signal transduction and activators of transcription (STAT) family to activate target genes. In the breast, the lobuloalveolar epithelium proliferates in response to PRL, placental lactogens, estrogen, progesterone, and local paracrine growth factors, including insulin-like growth factor I (IGF-I).

1	PRL acts to induce and maintain lactation, decrease reproductive function, and suppress sexual drive. These functions are geared toward ensuring that maternal lactation is sustained and not interrupted by pregnancy. PRL inhibits reproductive function by suppressing hypothalamic gonadotropin-releasing hormone (GnRH) and pituitary gonadotropin secretion and by impairing gonadal steroidogenesis in both women and men. In the ovary, PRL blocks folliculogenesis and inhibits granulosa cell aromatase activity, leading to hypoestrogenism and anovulation. PRL also has a luteolytic effect, generating a shortened, or inadequate, luteal phase of the menstrual cycle. In men, attenuated LH secretion leads to low testosterone levels and decreased spermatogenesis. These hormonal changes decrease libido and reduce fertility in patients with hyperprolactinemia.

1	gROWTH HORMONE Synthesis GH is the most abundant anterior pituitary hormone, and GH-secreting somatotrope cells constitute up to 50% of the total anterior pituitary cell population. Mammosomatotrope cells, which coexpress PRL with GH, can be identified by using double immunostaining techniques. Somatotrope development and GH transcription are determined by expression of the cell-specific Pit-1 nuclear transcription factor. Five distinct genes encode GH and related proteins. The pituitary GH gene (hGH-N) produces two alternatively spliced products that give rise to 22-kDa GH (191 amino acids) and a less abundant 20-kDa GH molecule with similar biologic activity. Placental syncytiotrophoblast cells express a GH variant (hGH-V) gene; the related hormone human chorionic somatotropin (HCS) is expressed by distinct members of the gene cluster.

1	Secretion GH secretion is controlled by complex hypothalamic and peripheral factors. GH-releasing hormone (GHRH) is a 44-aminoacid hypothalamic peptide that stimulates GH synthesis and release. Ghrelin, an octanoylated gastric-derived peptide, and synthetic agonists of the GHS-R induce GHRH and also directly stimulate GH release. Somatostatin (somatotropin-release inhibiting factor [SRIF]) is synthesized in the medial preoptic area of the hypothalamus and inhibits GH secretion. GHRH is secreted in discrete spikes that elicit GH pulses, whereas SRIF sets basal GH secretory tone. SRIF also is expressed in many extrahypothalamic tissues, including the central nervous system (CNS), gastrointestinal tract, and pancreas, where it also acts to inhibit islet hormone secretion. IGF-I, the peripheral target hormone for GH, feeds back to inhibit GH; estrogen induces GH, whereas chronic glucocorticoid excess suppresses GH release.

1	Surface receptors on the somatotrope regulate GH synthesis and secretion. The GHRH receptor is a G protein–coupled receptor (GPCR) that signals through the intracellular cyclic AMP pathway to stimulate somatotrope cell proliferation as well as GH production. Inactivating mutations of the GHRH receptor cause profound dwarfism. A distinct surface receptor for ghrelin, the gastric-derived GH secretagogue, is expressed in both the hypothalamus and pituitary. Somatostatin binds to five distinct receptor subtypes (SSTR1 to SSTR5); SSTR2 and SSTR5 subtypes preferentially suppress GH (and TSH) secretion.

1	GH secretion is pulsatile, with highest peak levels occurring at night, generally correlating with sleep onset. GH secretory rates decline markedly with age so that hormone levels in middle age are about 15% of pubertal levels. These changes are paralleled by an age-related decline in lean muscle mass. GH secretion is also reduced in obese individuals, although IGF-I levels may not be suppressed, suggesting a change in the setpoint for feedback control. Elevated GH levels occur within an 401e-3 hour of deep sleep onset as well as after exercise, physical stress, and trauma and during sepsis. Integrated 24-h GH secretion is higher in women and is also enhanced by estrogen replacement likely reflective of increased peripheral GH-resistance. Using standard assays, random GH measurements are undetectable in ~50% of daytime samples obtained from healthy subjects and are also undetectable in most obese and elderly subjects. Thus, single random GH measurements do not distinguish patients

1	undetectable in ~50% of daytime samples obtained from healthy subjects and are also undetectable in most obese and elderly subjects. Thus, single random GH measurements do not distinguish patients with adult GH deficiency from normal persons.

1	GH secretion is profoundly influenced by nutritional factors. Using newer ultrasensitive GH assays with a sensitivity of 0.002 μg/L, a glucose load suppresses GH to <0.7 μg/L in women and to <0.07 μg/L in men. Increased GH pulse frequency and peak amplitudes occur with chronic malnutrition or prolonged fasting. GH is stimulated by intravenous L-arginine, dopamine, and apomorphine (a dopamine receptor agonist), as well as by α-adrenergic pathways. β-Adrenergic blockade induces basal GH and enhances GHRHand insulin-evoked GH release. Action The pattern of GH secretion may affect tissue responses. The higher GH pulsatility observed in men compared with the relatively continuous basal GH secretion in women may be an important biologic determinant of linear growth patterns and liver enzyme induction.

1	The 70-kDa peripheral GH receptor protein has structural homology with the cytokine/hematopoietic superfamily. A fragment of the receptor extracellular domain generates a soluble GH binding protein (GHBP) that interacts with GH in the circulation. The liver and cartilage contain the greatest number of GH receptors. GH binding to preformed receptor dimers is followed by internal rotation and subsequent signaling through the JAK/STAT pathway. Activated STAT proteins translocate to the nucleus, where they modulate expression of GH-regulated target genes. GH analogues that bind to the receptor but are incapable of mediating receptor signaling are potent antagonists of GH action. A GH receptor antagonist (pegvisomant) is approved for treatment of acromegaly.

1	GH induces protein synthesis and nitrogen retention and impairs glucose tolerance by antagonizing insulin action. GH also stimulates lipolysis, leading to increased circulating fatty acid levels, reduced omental fat mass, and enhanced lean body mass. GH promotes sodium, potassium, and water retention and elevates serum levels of inorganic phosphate. Linear bone growth occurs as a result of complex hormonal and growth factor actions, including those of IGF-I. GH stimulates epiphyseal prechondrocyte differentiation. These precursor cells produce IGF-I locally, and their proliferation is also responsive to the growth factor.

1	Insulin-Like growth Factors Although GH exerts direct effects in target tissues, many of its physiologic effects are mediated indirectly through IGF-I, a potent growth and differentiation factor. The liver is the major source of circulating IGF-I. In peripheral tissues, IGF-I also exerts local paracrine actions that appear to be both dependent on and independent of GH. Thus, GH administration induces circulating IGF-I as well as stimulating local IGF-I production in multiple tissues. Both IGF-I and IGF-II are bound to high-affinity circulating IGF-binding proteins (IGFBPs) that regulate IGF bioactivity. Levels of IGFBP3 are GH-dependent, and it serves as the major carrier protein for circulating IGF-I. GH deficiency and malnutrition usually are associated with low IGFBP3 levels. IGFBP1 and IGFBP2 regulate local tissue IGF action but do not bind appreciable amounts of circulating IGF-I.

1	Serum IGF-I concentrations are profoundly affected by physiologic factors. Levels increase during puberty, peak at 16 years, and subsequently decline by >80% during the aging process. IGF-I concentrations are higher in women than in men. Because GH is the major determinant of hepatic IGF-I synthesis, abnormalities of GH synthesis or action (e.g., pituitary failure, GHRH receptor defect, GH receptor defect or pharmacologic GH receptor blockade) reduce IGF-I levels. Hypocaloric states are associated with GH resistance; IGF-I levels are therefore low with cachexia, malnutrition, and sepsis. In acromegaly, IGF-I levels are invariably high and reflect a log-linear relationship with circulating GH concentrations. CHAPTER 401e Anterior Pituitary: Physiology of Pituitary Hormones

1	CHAPTER 401e Anterior Pituitary: Physiology of Pituitary Hormones IGF-I physIoloGy Injected IGF-I (100 μg/kg) induces hypoglycemia, and lower doses improve insulin sensitivity in patients with severe insulin resistance and diabetes. In cachectic subjects, IGF-I infusion (12 μg/kg per hour) enhances nitrogen retention and lowers cholesterol levels. Longer-term subcutaneous IGF-I injections enhance protein synthesis and are anabolic. Although bone formation markers are induced, bone turnover also may be stimulated by IGF-I. IGF-I has only been approved for use in patients with GH-resistance syndromes. IGF-I side effects are dose-dependent, and overdose may result in hypoglycemia, hypotension, fluid retention, temporomandibular jaw pain, and increased intracranial pressure, all of which are reversible. Avascular femoral head necrosis has been reported. Chronic excess IGF-I administration presumably would result in features of acromegaly. (See also Chap. 406)

1	(See also Chap. 406) Synthesis ACTH-secreting corticotrope cells constitute about 20% of the pituitary cell population. ACTH (39 amino acids) is derived from the POMC precursor protein (266 amino acids) that also generates several other peptides, including β-lipotropin, β-endorphin, met-enkephalin, α-melanocyte-stimulating hormone (α-MSH), and corticotropin-like intermediate lobe protein (CLIP). The POMC gene is potently suppressed by glucocorticoids and induced by corticotropinreleasing hormone (CRH), arginine vasopressin (AVP), and proinflammatory cytokines, including IL-6, as well as leukemia inhibitory factor. CRH, a 41-amino-acid hypothalamic peptide synthesized in the paraventricular nucleus as well as in higher brain centers, is the predominant stimulator of ACTH synthesis and release. The CRH receptor is a GPCR that is expressed on the corticotrope and signals to induce POMC transcription.

1	Secretion ACTH secretion is pulsatile and exhibits a characteristic circadian rhythm, peaking at about 6 a.m. and reaching a nadir about midnight. Adrenal glucocorticoid secretion, which is driven by ACTH, follows a parallel diurnal pattern. ACTH circadian rhythmicity is determined by variations in secretory pulse amplitude rather than changes in pulse frequency. Superimposed on this endogenous rhythm, ACTH levels are increased by physical and psychological stress, exercise, acute illness, and insulin-induced hypoglycemia. Glucocorticoid-mediated negative regulation of the hypothalamic-pituitary-adrenal (HPA) axis occurs as a consequence of both hypothalamic CRH suppression and direct attenuation of pituitary POMC gene expression and ACTH release. In contrast, loss of cortisol feedback inhibition, as occurs in primary adrenal failure, results in extremely high ACTH levels.

1	Acute inflammatory or septic insults activate the HPA axis through the integrated actions of proinflammatory cytokines, bacterial toxins, and neural signals. The overlapping cascade of ACTH-inducing cytokines (tumor necrosis factor [TNF]; IL-1, -2, and -6; and leukemia inhibitory factor) activates hypothalamic CRH and AVP secretion, pituitary POMC gene expression, and local pituitary paracrine cytokine networks. The resulting cortisol elevation restrains the inflammatory response and enables host protection. Concomitantly, cytokine-mediated central glucocorticoid receptor resistance impairs glucocorticoid suppression of the HPA. Thus, the neuroendocrine stress response reflects the net result of highly integrated hypothalamic, intrapituitary, and peripheral hormone and cytokine signals acting to regulate cortisol secretion.

1	Action The major function of the HPA axis is to maintain metabolic homeostasis and mediate the neuroendocrine stress response. ACTH induces adrenocortical steroidogenesis by sustaining adrenal cell proliferation and function. The receptor for ACTH, designated melanocortin-2 receptor, is a GPCR that induces steroidogenesis by stimulating a cascade of steroidogenic enzymes (Chap. 406). gONADOTROPINS: FSH AND LH Synthesis and Secretion Gonadotrope cells constitute about 10% of anterior pituitary cells and produce two gonadotropin hormones— LH and FSH. Like TSH and hCG, LH and FSH are glycoprotein hormones that comprise α and β subunits. The α subunit is common to these glycoprotein hormones; specificity of hormone function is conferred by the β subunits, which are expressed by separate genes.

1	Gonadotropin synthesis and release are dynamically regulated. This is particularly true in women, in whom rapidly fluctuating gonadal steroid levels vary throughout the menstrual cycle. Hypothalamic GnRH, a 10-amino-acid peptide, regulates the synthesis and secretion of both LH and FSH. Brain kisspeptin, a product of the KISSI gene regulates hypothalamic GnRH release. GnRH is secreted in discrete pulses every 60–120 min, and the pulses in turn elicit LH and FSH pulses (Fig. 401e-3). The pulsatile mode of GnRH input is essential to its action; pulses prime gonadotrope responsiveness, whereas continuous GnRH exposure induces desensitization. Based on this phenomenon, long-acting GnRH agonists are used to suppress gonadotropin levels in children with precocious puberty and in men with prostate cancer (Chap. 115) and are used in some ovulation-induction protocols to reduce levels of endogenous gonadotropins (Chap. 412). Estrogens act at both the hypothalamus and the pituitary to modulate

1	cancer (Chap. 115) and are used in some ovulation-induction protocols to reduce levels of endogenous gonadotropins (Chap. 412). Estrogens act at both the hypothalamus and the pituitary to modulate gonadotropin secretion. Chronic estrogen exposure is inhibitory, whereas rising estrogen levels, as occur during the preovulatory surge, exert positive feedback to increase gonadotropin pulse frequency and amplitude. Progesterone slows GnRH pulse frequency but enhances gonadotropin responses to GnRH. Testosterone feedback in men also occurs at the hypothalamic and pituitary levels and is mediated in part by its conversion to estrogens.

1	Although GnRH is the main regulator of LH and FSH secretion, FSH synthesis is also under separate control by the gonadal peptides inhibin and activin, which are members of the transforming growth factor β (TGF-β) family. Inhibin selectively suppresses FSH, whereas activin stimulates FSH synthesis (Chap. 412). Action The gonadotropin hormones interact with their respective GPCRs expressed in the ovary and testis, evoking germ cell development and maturation and steroid hormone biosynthesis. In women, FSH regulates ovarian follicle development and stimulates ovarian estrogen production. LH mediates ovulation and maintenance of the corpus luteum. In men, LH induces Leydig cell testosterone synthesis and secretion, and FSH stimulates seminiferous tubule development and regulates spermatogenesis.

1	THYROID-STIMULATINg HORMONE Synthesis and Secretion TSH-secreting thyrotrope cells constitute 5% of the anterior pituitary cell population. TSH shares a common α subunit with LH and FSH but contains a specific TSH β subunit. TRH is a hypothalamic tripeptide (pyroglutamyl histidylprolinamide) that acts through a pituitary GPCR to stimulate TSH synthesis and secretion; it also stimulates the lactotrope cell to secrete PRL. TSH secretion is stimulated by TRH, whereas thyroid hormones, dopamine, somatostatin, and glucocorticoids suppress TSH by overriding TRH induction.

1	Thyrotrope cell proliferation and TSH secretion are both induced when negative feedback inhibition by thyroid hormones is removed. Thus, thyroid damage (including surgical thyroidectomy), radiation-induced hypothyroidism, chronic thyroiditis, and prolonged goitrogen exposure are associated with increased TSH levels. Long-standing untreated hypothyroidism can lead to elevated TSH levels as well as thyrotrope hyperplasia and pituitary enlargement, which may be evident on magnetic resonance imaging. Action TSH is secreted in pulses, although the excursions are modest in comparison to other pituitary hormones because of the low amplitude of the pulses and the relatively long half-life of TSH. Consequently, single determinations of TSH suffice to precisely assess its circulating levels. TSH binds to a GPCR on thyroid follicular cells to stimulate thyroid hormone synthesis and release (Chap. 405).

1	2255 Hypopituitarism Shlomo Melmed, J. Larry Jameson Inadequate production of anterior pituitary hormones leads to features of hypopituitarism. Impaired production of one or more of the ante-rior pituitary trophic hormones can result from inherited disorders; more commonly, adult hypopituitarism is acquired and reflects the 402 compressive mass effects of tumors or the consequences of local pituitary or hypothalamic traumatic, inflammatory, or vascular damage. These processes also may impair synthesis or secretion of hypothalamic hormones, with resultant pituitary failure (Table 402-1). DEVELOPMENTAL AND GENETIC CAUSES OF HYPOPITUITARISM Pituitary Dysplasia Pituitary dysplasia may result in aplastic, hypo-plastic, or ectopic pituitary gland development. Because pituitary development follows midline cell migration from the nasopharyngeal Rathke’s pouch, midline craniofacial disorders may be associated with ETiology of HyPoPiTuiTARiSma

1	ETiology of HyPoPiTuiTARiSma Development/structural Transcription factor defect Pituitary dysplasia/aplasia Congenital central nervous system mass, encephalocele Primary empty sella Congenital hypothalamic disorders (septo-optic dysplasia, Prader-Willi syndrome, Laurence-Moon-Biedl syndrome, Kallmann syndrome) Neoplastic Pituitary adenoma Parasellar mass (germinoma, ependymoma, glioma) Rathke’s cyst Craniopharyngioma Hypothalamic hamartoma, gangliocytoma Pituitary metastases (breast, lung, colon carcinoma) Lymphoma and leukemia Meningioma

1	Vascular Pituitary apoplexy Pregnancy-related (infarction with diabetes; postpartum necrosis) Sickle cell disease Arteritis aTrophic hormone failure associated with pituitary compression or destruction usually occurs sequentially: growth hormone > follicle-stimulating hormone > luteinizing hormone > thyroid-stimulating hormone > adrenocorticotropic hormone. During childhood, growth retardation is often the presenting feature, and in adults, hypogonadism is the earliest symptom. 2256 pituitary dysplasia. Acquired pituitary failure in the newborn also can be caused by birth trauma, including cranial hemorrhage, asphyxia, and breech delivery.

1	2256 pituitary dysplasia. Acquired pituitary failure in the newborn also can be caused by birth trauma, including cranial hemorrhage, asphyxia, and breech delivery. Septo-optic dySplaSia Hypothalamic dysfunction and hypopituitarism may result from dysgenesis of the septum pellucidum or corpus callosum. Affected children have mutations in the HESX1 gene, which is involved in early development of the ventral prosencephalon. These children exhibit variable combinations of cleft palate, syndactyly, ear deformities, hypertelorism, optic nerve hypoplasia, micropenis, and anosmia. Pituitary dysfunction leads to diabetes insipidus, growth hormone (GH) deficiency and short stature, and, occasionally, thyroid-stimulating hormone (TSH) deficiency.

1	Tissue-Specific Factor Mutations Several pituitary cell–specific transcription factors, such as Pit-1 and Prop-1, are critical for determining the development and committed function of differentiated anterior pituitary cell lineages. Autosomal dominant or recessive Pit-1 mutations cause combined GH, prolactin (PRL), and TSH deficiencies. These patients usually present with growth failure and varying degrees of hypothyroidism. The pituitary may appear hypoplastic on magnetic resonance imaging (MRI).

1	Prop-1 is expressed early in pituitary development and appears to be required for Pit-1 function. Familial and sporadic PROP1 mutations result in combined GH, PRL, TSH, and gonadotropin deficiency. Over 80% of these patients have growth retardation; by adulthood, all are deficient in TSH and gonadotropins, and a small minority later develop adrenocorticotropic hormone (ACTH) deficiency. Because of gonadotropin deficiency, these individuals do not enter puberty spontaneously. In some cases, the pituitary gland appears enlarged on MRI. TPIT mutations result in ACTH deficiency associated with hypocortisolism.

1	Kallmann syndrome results from defective hypothalamic gonadotropin-releasing hormone (GnRH) synthesis and is associated with anosmia or hyposmia due to olfactory bulb agenesis or hypoplasia (Chap. 411). Classically, the syndrome may also be associated with color blindness, optic atrophy, nerve deafness, cleft palate, renal abnormalities, cryptorchidism, and neurologic abnormalities such as mirror movements. The initial genetic cause was identified in the X-linked KAL gene, mutations of which impair embryonic migration of GnRH neurons from the hypothalamic olfactory placode to the hypothalamus. Based on further studies, at least a dozen other genetic abnormalities, in addition to KAL mutations, have been found to cause isolated GnRH deficiency. Autosomal recessive (i.e., GPR54, KISS1) and dominant (i.e., FGFR1) modes of transmission have been described, and there is a growing list of genes associated with GnRH deficiency (GNRH1, PROK2, PROKR2, CH7, PCSK1, FGF8, NELF, WDR11, TAC3,

1	and dominant (i.e., FGFR1) modes of transmission have been described, and there is a growing list of genes associated with GnRH deficiency (GNRH1, PROK2, PROKR2, CH7, PCSK1, FGF8, NELF, WDR11, TAC3, TACR3). A fraction of patients have digenic mutations. Associated clinical features, in addition to GnRH deficiency, vary depending on the genetic cause. GnRH deficiency prevents progression through puberty. Males present with delayed puberty and pronounced hypogonadal features, including micropenis, probably the result of low testosterone levels during infancy. Females present with primary amenorrhea and failure of secondary sexual development.

1	Kallmann syndrome and other causes of congenital GnRH deficiency are characterized by low luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels and low concentrations of sex steroids (testosterone or estradiol). In sporadic cases of isolated gonadotropin deficiency, the diagnosis is often one of exclusion after other known causes of hypothalamic-pituitary dysfunction have been eliminated. Repetitive GnRH administration restores normal pituitary gonadotropin responses, pointing to a hypothalamic defect in these patients. Long-term treatment of males with human chorionic gonadotropin (hCG) or testosterone restores pubertal development and secondary sex characteristics; women can be treated with cyclic estrogen and progestin. Fertility also may be restored by the administration of gonadotropins or by using a portable infusion pump to deliver subcutaneous, pulsatile GnRH.

1	Bardet-Biedl Syndrome This very rare genetically heterogeneous disorder is characterized by mental retardation, renal abnormalities, obesity, and hexadactyly, brachydactyly, or syndactyly. Central diabetes insipidus may or may not be associated. GnRH deficiency occurs in 75% of males and half of affected females. Retinal degeneration begins in early childhood, and most patients are blind by age 30. Numerous subtypes of Bardet-Biedl syndrome (BBS) have been identified, with genetic linkage to at least nine different loci. Several of the loci encode genes involved in basal body cilia function, and this may account for the diverse clinical manifestations. leptin and leptin receptor mutationS Deficiencies of leptin or its receptor cause a broad spectrum of hypothalamic abnormalities, including hyperphagia, obesity, and central hypogonadism (Chap. 415e). Decreased GnRH production in these patients results in attenuated pituitary FSH and LH synthesis and release.

1	prader-Willi Syndrome This is a contiguous gene syndrome that results from deletion of the paternal copies of the imprinted SNRPN gene, the NECDIN gene, and possibly other genes on chromosome 15q. Prader-Willi syndrome is associated with hypogonadotropic hypogonadism, hyperphagia-obesity, chronic muscle hypotonia, mental retardation, and adult-onset diabetes mellitus (Chap. 83e). Multiple somatic defects also involve the skull, eyes, ears, hands, and feet. Diminished hypothalamic oxytocinand vasopressin-producing nuclei have been reported. Deficient GnRH synthesis is suggested by the observation that chronic GnRH treatment restores pituitary LH and FSH release.

1	Hypopituitarism may be caused by accidental or neurosurgical trauma; vascular events such as apoplexy; pituitary or hypothalamic neoplasms, craniopharyngioma, lymphoma, or metastatic tumors; inflammatory disease such as lymphocytic hypophysitis; infiltrative disorders such as sarcoidosis, hemochromatosis (Chap. 428), and tuberculosis; or irradiation. Increasing evidence suggests that patients with brain injury, including contact sports trauma, subarachnoid hemorrhage, and irradiation, have transient hypopituitarism and require intermittent long-term endocrine follow-up, because permanent hypothalamic or pituitary dysfunction will develop in 25–40% of these patients.

1	Hypothalamic Infiltration Disorders These disorders—including sarcoidosis, histiocytosis X, amyloidosis, and hemochromatosis— frequently involve both hypothalamic and pituitary neuronal and neurochemical tracts. Consequently, diabetes insipidus occurs in half of patients with these disorders. Growth retardation is seen if attenuated GH secretion occurs before puberty. Hypogonadotropic hypogonadism and hyperprolactinemia are also common. Inflammatory Lesions Pituitary damage and subsequent secretory dysfunction can be seen with chronic site infections such as tuberculosis, with opportunistic fungal infections associated with AIDS, and in tertiary syphilis. Other inflammatory processes, such as granulomas and sarcoidosis, may mimic the features of a pituitary adenoma. These lesions may cause extensive hypothalamic and pituitary damage, leading to trophic hormone deficiencies.

1	Cranial Irradiation Cranial irradiation may result in long-term hypothalamic and pituitary dysfunction, especially in children and adolescents, as they are more susceptible to damage after whole-brain or head and neck therapeutic irradiation. The development of hormonal abnormalities correlates strongly with irradiation dosage and the time interval after completion of radiotherapy. Up to two-thirds of patients ultimately develop hormone insufficiency after a median dose of 50 Gy (5000 rad) directed at the skull base. The development of hypopituitarism occurs over 5–15 years and usually reflects hypothalamic damage rather than primary destruction of pituitary cells. Although the pattern of hormone loss is variable, GH deficiency is most common, followed by gonadotropin and ACTH deficiency. When deficiency of one or more hormones is documented, the possibility of diminished reserve of other hormones is likely. Accordingly, anterior pituitary function should be continually evaluated over

1	When deficiency of one or more hormones is documented, the possibility of diminished reserve of other hormones is likely. Accordingly, anterior pituitary function should be continually evaluated over the long term in previously irradiated patients, and replacement therapy instituted when appropriate (see below).

1	Lymphocytic Hypophysitis This occurs most often in postpartum women; it usually presents with hyperprolactinemia and MRI evidence of a prominent pituitary mass that often resembles an adenoma, with mildly elevated PRL levels. Pituitary failure caused by diffuse lymphocytic infiltration may be transient or permanent but requires immediate evaluation and treatment. Rarely, isolated pituitary hormone deficiencies have been described, suggesting a selective autoimmune process targeted to specific cell types. Most patients manifest symptoms of progressive mass effects with headache and visual disturbance. The erythrocyte sedimentation rate often is elevated. Because the MRI image may be indistinguishable from that of a pituitary adenoma, hypophysitis should be considered in a postpartum woman with a newly diagnosed pituitary mass before an unnecessary surgical intervention is undertaken. The inflammatory process often resolves after several months of glucocorticoid treatment, and pituitary

1	a newly diagnosed pituitary mass before an unnecessary surgical intervention is undertaken. The inflammatory process often resolves after several months of glucocorticoid treatment, and pituitary function may be restored, depending on the extent of damage.

1	Pituitary Apoplexy Acute intrapituitary hemorrhagic vascular events can cause substantial damage to the pituitary and surrounding sellar structures. Pituitary apoplexy may occur spontaneously in a preexisting adenoma; postpartum (Sheehan’s syndrome); or in association with diabetes, hypertension, sickle cell anemia, or acute shock. The hyperplastic enlargement of the pituitary, which occurs normally during pregnancy, increases the risk for hemorrhage and infarction. Apoplexy is an endocrine emergency that may result in severe hypoglycemia, hypotension and shock, central nervous system (CNS) hemorrhage, and death. Acute symptoms may include severe headache with signs of meningeal irritation, bilateral visual changes, ophthalmoplegia, and, in severe cases, cardiovascular collapse and loss of consciousness. Pituitary computed tomography (CT) or MRI may reveal signs of intratumoral or sellar hemorrhage, with pituitary stalk deviation and compression of pituitary tissue.

1	Patients with no evident visual loss or impaired consciousness can be observed and managed conservatively with high-dose glucocorticoids. Those with significant or progressive visual loss, cranial nerve palsy, or loss of consciousness require urgent surgical decompression. Visual recovery after sellar surgery is inversely correlated with the length of time after the acute event. Therefore, severe ophthalmoplegia or visual deficits are indications for early surgery. Hypopituitarism is common after apoplexy.

1	Empty Sella A partial or apparently totally empty sella is often an incidental MRI finding, and may be associated with intracranial hypertension. These patients usually have normal pituitary function, implying that the surrounding rim of pituitary tissue is fully functional. Hypopituitarism, however, may develop insidiously. Pituitary masses also may undergo clinically silent infarction and involution with development of a partial or totally empty sella by cerebrospinal fluid (CSF) filling the dural herniation. Rarely, small but functional pituitary adenomas may arise within the rim of normal pituitary tissue, and they are not always visible on MRI.

1	The clinical manifestations of hypopituitarism depend on which hormones are lost and the extent of the hormone deficiency. GH deficiency causes growth disorders in children and leads to abnormal body composition in adults (see below). Gonadotropin deficiency causes menstrual disorders and infertility in women and decreased sexual function, infertility, and loss of secondary sexual characteristics in men. TSH and ACTH deficiency usually develop later in the course of pituitary failure. TSH deficiency causes growth retardation in children and features of hypothyroidism in children and adults. The secondary form of adrenal insufficiency caused by ACTH deficiency leads to hypocortisolism with relative preservation of mineralocorticoid production. PRL deficiency causes failure of lactation. When lesions involve the posterior pituitary, polyuria and polydipsia reflect loss of vasopressin secretion. In patients with long-standing pituitary damage, epidemiologic studies document an increased

1	lesions involve the posterior pituitary, polyuria and polydipsia reflect loss of vasopressin secretion. In patients with long-standing pituitary damage, epidemiologic studies document an increased mortality rate, primarily from increased cardiovascular 2257 and cerebrovascular disease. Previous head or neck irradiation is also a determinant of increased mortality rates in patients with hypopituitarism, especially from cerebrovascular disease.

1	Biochemical diagnosis of pituitary insufficiency is made by demonstrating low levels of respective pituitary trophic hormones in the setting of low levels of target hormones. For example, low free thyroxine in the setting of a low or inappropriately normal TSH level suggests secondary hypothyroidism. Similarly, a low testosterone level without elevation of gonadotropins suggests hypogonadotropic hypogonadism. Provocative tests may be required to assess pituitary reserve (Table 402-2). GH responses to insulin-induced hypoglycemia, arginine, L-dopa, growth hormone–releasing hormone (GHRH), or growth hormone–releasing peptides (GHRPs) can be used to assess GH reserve. Corticotropinreleasing hormone (CRH) administration induces ACTH release, and administration of synthetic ACTH (cosyntropin) evokes adrenal cortisol release as an indirect indicator of pituitary ACTH reserve (Chap. 406). ACTH reserve is most reliably assessed by measuring ACTH and cortisol levels during insulin-induced

1	evokes adrenal cortisol release as an indirect indicator of pituitary ACTH reserve (Chap. 406). ACTH reserve is most reliably assessed by measuring ACTH and cortisol levels during insulin-induced hypoglycemia. However, this test should be performed cautiously in patients with suspected adrenal insufficiency because of enhanced susceptibility to hypoglycemia and hypotension. Administering insulin to induce hypoglycemia is contraindicated in patients with active coronary artery disease or known seizure disorders.

1	Hormone replacement therapy, including glucocorticoids, thyroid hormone, sex steroids, growth hormone, and vasopressin, is usually safe and free of complications. Treatment regimens that mimic physiologic hormone production allow for maintenance of satisfactory clinical homeostasis. Effective dosage schedules are outlined in Table 402-3. Patients in need of glucocorticoid replacement require careful dose adjustments during stressful events such as acute illness, dental procedures, trauma, and acute hospitalization.

1	DISORDERS OF GROWTH AND DEVELOPMENT Skeletal Maturation and Somatic Growth The growth plate is dependent on a variety of hormonal stimuli, including GH, insulin-like growth factor (IGF) I, sex steroids, thyroid hormones, paracrine growth factors, and cytokines. The growth-promoting process also requires caloric energy, amino acids, vitamins, and trace metals and consumes about 10% of normal energy production. Malnutrition impairs chondrocyte activity, increases GH resistance, and reduces circulating IGF-I and IGFBP3 levels.

1	Linear bone growth rates are very high in infancy and are pituitary-dependent. Mean growth velocity is ~6 cm/year in later childhood and usually is maintained within a given range on a standardized percentile chart. Peak growth rates occur during midpuberty when bone age is 12 (girls) or 13 (boys). Secondary sexual development is associated with elevated sex steroids that cause progressive epiphyseal growth plate closure. Bone age is delayed in patients with all forms of true GH deficiency or GH receptor defects that result in attenuated GH action. Short stature may occur as a result of constitutive intrinsic growth defects or because of acquired extrinsic factors that impair growth. In general, delayed bone age in a child with short stature is suggestive of a hormonal or systemic disorder, whereas normal bone age in a short child is more likely to be caused by a genetic cartilage dysplasia or growth plate disorder (Chap. 427).

1	GH Deficiency in Children • GH deficiency Isolated GH deficiency is characterized by short stature, micropenis, increased fat, high-pitched voice, and a propensity to hypoglycemia due to relatively unopposed insulin action. Familial modes of inheritance are seen in at least one-third of these individuals and may be autosomal dominant, recessive, Abbreviations: T3, triiodothyronine; T4, thyroxine; TRH, thyrotropin-releasing hormone. For other abbreviations, see text.

1	or X-linked. About 10% of children with GH deficiency have muta-Circulating GH receptor antibodies may rarely cause peripheral GH tions in the GH-N gene, including gene deletions and a wide range of insensitivity. point mutations. Mutations in transcription factors Pit-1 and Prop-1, nutritional SHort Stature Caloric deprivation and malnutrition,which control somatotrope development, result in GH deficiency in uncontrolled diabetes, and chronic renal failure represent second-combination with other pituitary hormone deficiencies, which may ary causes of abrogated GH receptor function. These conditions alsobecome manifest only in adulthood. The diagnosis of idiopathic GH stimulate production of proinflammatory cytokines, which act to exacdeficiency (IGHD) should be made only after known molecular defects erbate the block of GH-mediated signal transduction. Children withhave been rigorously excluded.

1	these conditions typically exhibit features of acquired short stature GHrH receptor mutationS Recessive mutations of the GHRH receptor with normal or elevated GH and low IGF-I levels. gene in subjects with severe proportionate dwarfism are associated pSycHoSocial SHort Stature Emotional and social deprivation lead towith low basal GH levels that cannot be stimulated by exogenous growth retardation accompanied by delayed speech, discordant hyper-GHRH, GHRP, or insulin-induced hypoglycemia, as well as anterior phagia, and an attenuated response to administered GH. A nurturingpituitary hypoplasia The syndrome exemplifies the importance of environment restores growth rates. the GHRH receptor for somatotrope cell proliferation and hormonal responsiveness.

1	GH inSenSitivity This is caused by defects of GH receptor structure or Short stature is commonly encountered in clinical practice, andsignaling. Homozygous or heterozygous mutations of the GH receptor the decision to evaluate these children requires clinical judgmentare associated with partial or complete GH insensitivity and growth in association with auxologic data and family history. Short staturefailure (Laron’s syndrome). The diagnosis is based on normal or high should be evaluated comprehensively if a patient’s height is >3 stan-GH levels, with decreased circulating GH-binding protein (GHBP), dard deviations (SD) below the mean for age or if the growth rateand low IGF-I levels. Very rarely, defective IGF-I, IGF-I receptor, or has decelerated. Skeletal maturation is best evaluated by measur-IGF-I signaling defects are also encountered. STAT5B mutations result ing a radiologic bone age, which is based mainly on the degree ofin both immunodeficiency as well as abrogated GH

1	by measur-IGF-I signaling defects are also encountered. STAT5B mutations result ing a radiologic bone age, which is based mainly on the degree ofin both immunodeficiency as well as abrogated GH signaling, leading wrist bone growth plate fusion. Final height can be predicted using to short stature with normal or elevated GH levels and low IGF-I levels. standardized scales (Bayley-Pinneau or Tanner-Whitehouse) or aAll doses shown should be individualized for specific patients and should be reassessed during stress, surgery, or pregnancy. Male and female fertility requirements should be managed as discussed in Chaps. 411 and 412.

1	Note: For abbreviations, see text. estimated by adding 6.5 cm (boys) or subtracting 6.5 cm (girls) from the midparental height. Because GH secretion is pulsatile, GH deficiency is best assessed by examining the response to provocative stimuli, including exercise, insulin-induced hypoglycemia, and other pharmacologic tests that normally increase GH to >7 μg/L in children. Random GH measurements do not distinguish normal children from those with true GH deficiency. Adequate adrenal and thyroid hormone replacement should be assured before testing. Ageand sex-matched IGF-I levels are not sufficiently sensitive or specific to make the diagnosis but can be useful to confirm GH deficiency. Pituitary MRI may reveal pituitary mass lesions or structural defects. Molecular analyses for known mutations should be undertaken when the cause of short stature remains cryptic, or when additional clinical features suggest a genetic cause.

1	Replacement therapy with recombinant GH (0.02–0.05 mg/kg per day SC) restores growth velocity in GH-deficient children to ~10 cm/year. If pituitary insufficiency is documented, other associated hormone deficits should be corrected, especially adrenal steroids. GH treatment is also moderately effective for accelerating growth rates in children with Turner’s syndrome and chronic renal failure. In patients with GH insensitivity and growth retardation due to mutations of the GH receptor, treatment with IGF-I bypasses the dysfunctional GH receptor. This disorder usually is caused by acquired hypothalamic or pituitary somatotrope damage. Acquired pituitary hormone deficiency follows a typical pattern in which loss of adequate GH reserve foreshadows fEATuRES of ADulT gRowTH HoRmonE DEfiCiEnCy Impaired quality of life Decreased energy and drive Poor concentration Low self-esteem Social isolation

1	Impaired quality of life Decreased energy and drive Poor concentration Low self-esteem Social isolation Pituitary: mass or structural damage Bone: reduced bone mineral density Abdomen: excess omental adiposity Evoked GH <3 ng/mL IGF-I and IGFBP3 low or normal Increased LDL cholesterol Concomitant gonadotropin, TSH, and/or ACTH reserve deficits may be present Abbreviation: LDL, low-density lipoprotein. For other abbreviations, see text. subsequent hormone deficits. The sequential order of hormone loss is usually GH → FSH/LH → TSH → ACTH. Patients previously diagnosed with childhood-onset GH deficiency should be retested as adults to affirm the diagnosis.

1	The clinical features of AGHD include changes in body composition, lipid metabolism, and quality of life and cardiovascular dysfunction (Table 402-4). Body composition changes are common and include reduced lean body mass, increased fat mass with selective deposition of intraabdominal visceral fat, and increased waist-to-hip ratio. Hyperlipidemia, left ventricular dysfunction, hypertension, and increased plasma fibrinogen levels also may be present. Bone mineral content is reduced, with resultant increased fracture rates. Patients may experience social isolation, depression, and difficulty maintaining gainful employment. Adult hypopituitarism is associated with a threefold increase in cardiovascular mortality rates in comparison to ageand sex-matched controls, and this may be due to GH deficiency, as patients in these studies were replaced with other deficient pituitary hormones.

1	AGHD is rare, and in light of the nonspecific nature of associated clinical symptoms, patients appropriate for testing should be selected carefully on the basis of well-defined criteria. With few exceptions, testing should be restricted to patients with the following predisposing factors: (1) pituitary surgery, (2) pituitary or hypothalamic tumor or granulomas, (3) history of cranial irradiation, (4) radiologic evidence of a pituitary lesion, (5) childhood requirement for GH replacement therapy, and rarely (6) unexplained low ageand sex-matched IGF-I levels. The transition of a GH-deficient adolescent to adulthood 2260 requires retesting to document subsequent adult GH deficiency. Up to 20% of patients previously treated for childhood-onset GH deficiency are found to be GH-sufficient on repeat testing as adults. A significant proportion (~25%) of truly GH-deficient adults have low-normal IGF-I levels. Thus, as in the evaluation of GH deficiency in children, valid ageand sex-matched

1	repeat testing as adults. A significant proportion (~25%) of truly GH-deficient adults have low-normal IGF-I levels. Thus, as in the evaluation of GH deficiency in children, valid ageand sex-matched IGF-I measurements provide a useful index of therapeutic responses but are not sufficiently sensitive for diagnostic purposes. The most validated test to distinguish pituitary-sufficient patients from those with AGHD is insulin-induced (0.05–0.1 U/kg) hypoglycemia. After glucose reduction to ~40 mg/dL, most individuals experience neuroglycopenic symptoms (Chap. 420), and peak GH release occurs at 60 min and remains elevated for up to 2 h. About 90% of healthy adults exhibit GH responses >5 μg/L; AGHD is defined by a peak GH response to hypoglycemia of <3 μg/L. Although insulin-induced hypoglycemia is safe when performed under appropriate supervision, it is contraindicated in patients with diabetes, ischemic heart disease, cerebrovascular disease, or epilepsy and in elderly patients.

1	is safe when performed under appropriate supervision, it is contraindicated in patients with diabetes, ischemic heart disease, cerebrovascular disease, or epilepsy and in elderly patients. Alternative stimulatory tests include intravenous arginine (30 g), GHRH (1 μg/kg), GHRP-6 (90 μg), and glucagon (1 mg). Combinations of these tests may evoke GH secretion in subjects who are not responsive to a single test.

1	Once the diagnosis of AGHD is unequivocally established, replacement of GH may be indicated. Contraindications to therapy include the presence of an active neoplasm, intracranial hypertension, and uncontrolled diabetes and retinopathy. The starting dose of 0.1–0.2 mg/d should be titrated (up to a maximum of 1.25 mg/d) to maintain IGF-I levels in the mid-normal range for ageand sex-matched controls (Fig. 402-1). Women require higher doses than men, and elderly patients require less GH. Long-term GH maintenance sustains normal IGF-I levels and is associated with persistent body composition changes (e.g., enhanced lean body mass and lower body fat). High-density lipoprotein cholesterol increases, but total cholesterol and insulin levels may not change significantly. Lumbar spine bone mineral density increases, but this response is gradual (>1 year). Many patients note significant improvement in quality of life when evaluated by standardized questionnaires. The effect of GH replacement on

1	density increases, but this response is gradual (>1 year). Many patients note significant improvement in quality of life when evaluated by standardized questionnaires. The effect of GH replacement on mortality rates in GH-deficient patients is currently the subject of long-term prospective investigation.

1	FIGURE 402-1 Management of adult growth hormone (GH) deficiency. IGF, insulin-like growth factor; Rx, Treatment. About 30% of patients exhibit reversible dose-related fluid retention, joint pain, and carpal tunnel syndrome, and up to 40% exhibit myalgias and paresthesia. Patients receiving insulin require careful monitoring for dosing adjustments, as GH is a potent counterregulatory hormone for insulin action. Patients with type 2 diabetes mellitus initially develop further insulin resistance. However, glycemic control usually improves with the sustained loss of abdominal fat associated with long-term GH replacement. Headache, increased intracranial pressure, hypertension, and tinnitus occur rarely. Pituitary tumor regrowth and progression of skin lesions or other tumors are being assessed in long-term surveillance programs. To date, development of these potential side effects does not appear significant.

1	Secondary adrenal insufficiency occurs as a result of pituitary ACTH deficiency. It is characterized by fatigue, weakness, anorexia, nausea, vomiting, and, occasionally, hypoglycemia. In contrast to primary adrenal failure, hypocortisolism associated with pituitary failure usually is not accompanied by hyperpigmentation or mineralocorticoid deficiency.

1	ACTH deficiency is commonly due to glucocorticoid withdrawal after treatment-associated suppression of the hypothalamic-pituitaryadrenal (HPA) axis. Isolated ACTH deficiency may occur after surgical resection of an ACTH-secreting pituitary adenoma that has suppressed the HPA axis; this phenomenon is in fact suggestive of a surgical cure. The mass effects of other pituitary adenomas or sellar lesions may lead to ACTH deficiency, but usually in combination with other pituitary hormone deficiencies. Partial ACTH deficiency may be unmasked in the presence of an acute medical or surgical illness, when clinically significant hypocortisolism reflects diminished ACTH reserve. Rarely, TPIT or POMC mutations result in primary ACTH deficiency.

1	Inappropriately low ACTH levels in the setting of low cortisol levels are characteristic of diminished ACTH reserve. Low basal serum cortisol levels are associated with blunted cortisol responses to ACTH stimulation and impaired cortisol response to insulin-induced hypoglycemia, or testing with metyrapone or CRH. For a description of provocative ACTH tests, see Chap. 406.

1	Glucocorticoid replacement therapy improves most features of ACTH deficiency. The total daily dose of hydrocortisone replace- regulate GnRH neuron migration, development, and function (see above). Mutations in GPR54, DAX1, kisspeptin, the GnRH receptor, and the LHβ or FSHβ subunit genes also cause pituitary gonadotropin ment preferably should not exceed 25 mg daily, divided into two or three doses. Prednisone (5 mg each morning) is longer acting and has fewer mineralocorticoid effects than hydrocortisone. Some authorities advocate lower maintenance doses in an effort to avoid cushingoid side effects. Doses should be increased severalfold dur-ing periods of acute illness or stress. GONADOTROPIN DEFICIENC Y Hypogonadism is the most common presenting feature of adult hypo-pituitarism even when other pituitary hormones are also deficient. It is often a harbinger of hypothalamic or pituitary lesions that impair GnRH production or delivery through the pituitary stalk. As noted below,

1	when other pituitary hormones are also deficient. It is often a harbinger of hypothalamic or pituitary lesions that impair GnRH production or delivery through the pituitary stalk. As noted below, hypogonadotropic hypogonadism is a common presenting feature of hyperprolactinemia. A variety of inherited and acquired disorders are associated with isolated hypogonadotropic hypogonadism (IHH) (Chap. 411). Hypothalamic defects associated with GnRH deficiency includeKallmann syndrome and mutations in more than a dozen genes that History of pituitary pathology Clinical features present Evoked GH < 3 °g/L Treat with GH 0.1–0.3 mg/d Exclude contraindications Titrate GH dose up to 1.25 mg/d Check IGF-I after 1 mo No response Response 6 mo Discontinue Rx Monitor IGF-I Levels 2261deficiency. Acquired forms of GnRH deficiency leading to hypogonado-tropism are seen in association with anorexia nervosa, stress, starvation, and extreme exercise but also may be idiopathic. Hypogonadotropic hypogonadism

1	of GnRH deficiency leading to hypogonado-tropism are seen in association with anorexia nervosa, stress, starvation, and extreme exercise but also may be idiopathic. Hypogonadotropic hypogonadism in these disorders is reversed by removal of the stressful stimulus or by caloric replenishment. PRESENTATION AND DIAGNOSIS In premenopausal women, hypogonadotropic hypogonadism presents as diminished ovarian function leading to oligomenorrhea or amenor-rhea, infertility, decreased vaginal secretions, decreased libido, and breast atrophy. In hypogonadal adult men, secondary testicular failure is associated with decreased libido and potency, infertility, decreased muscle mass with weakness, reduced beard and body hair growth, soft testes, and characteristic fine facial wrinkles. Osteoporosis occurs in both untreated hypogonadal women and men. LABORATORY INVESTIGATION Central hypogonadism is associated with low or inappropriately normal serum gonadotropin levels in the setting of low sex hormone

1	in both untreated hypogonadal women and men. LABORATORY INVESTIGATION Central hypogonadism is associated with low or inappropriately normal serum gonadotropin levels in the setting of low sex hormone concentrations (testosterone in men, estradiol in women). Because gonadotropin secretion is pulsatile, valid assessments may require repeated measurements or the use of pooled serum samples. Men have reduced sperm counts. Intravenous GnRH (100 μg) stimulates gonadotropes to secrete LH (which peaks within 30 min) and FSH (which plateaus during the ensuing 60 min). Normal responses vary according to menstrual cycle stage, age, and sex of the patient. Generally, LH levels increase about threefold, whereas FSH responses are less pronounced. In the setting of gonadotropin deficiency, a normal gonadotropin response to GnRH indicates intact pituitary gonadotrope function and sug-gests a hypothalamic abnormality. An absent response, however, does not reliably distinguish pituitary from

1	gonadotropin response to GnRH indicates intact pituitary gonadotrope function and sug-gests a hypothalamic abnormality. An absent response, however, does not reliably distinguish pituitary from hypothalamic causes of hypogonadism. For this reason, GnRH testing usually adds little to the information gained from baseline evaluation of the hypothalamic-pituitary-gonadotrope axis except in cases of isolated GnRH defi-ciency (e.g., Kallmann syndrome). MRI examination of the sellar region and assessment of other pituitary functions usually are indicated in patients with documented central hypogonadism. In males, testosterone replacement is necessary to achieve and maintain normal growth and development of the external genitalia, secondary sex characteristics, male sexual behavior, and andro-genic anabolic effects, including maintenance of muscle function and bone mass. Testosterone may be administered by intramus-cular injections every 1–4 weeks or by using skin patches that are replaced

1	anabolic effects, including maintenance of muscle function and bone mass. Testosterone may be administered by intramus-cular injections every 1–4 weeks or by using skin patches that are replaced daily (Chap. 411). Testosterone gels are also available. Gonadotropin injections (hCG or human menopausal gonadotropin [hMG]) over 12–18 months are used to restore fertility. Pulsatile GnRH therapy (25–150 ng/kg every 2 h), administered by a subcuta-neous infusion pump, is also effective for treatment of hypothalamic hypogonadism when fertility is desired. In premenopausal women, cyclical replacement of estrogen and progesterone maintains secondary sexual characteristics and integrity of genitourinary tract mucosa and prevents premature osteoporosis (Chap. 412). Gonadotropin therapy is used for ovula-tion induction. Follicular growth and maturation are initiated using hMG or recombinant FSH; hCG or human luteinizing hormone (hLH) is subsequently injected to induce ovulation. As in men,

1	for ovula-tion induction. Follicular growth and maturation are initiated using hMG or recombinant FSH; hCG or human luteinizing hormone (hLH) is subsequently injected to induce ovulation. As in men, pulsatile GnRH therapy can be used to treat hypothalamic causes of gonado-tropin deficiency. Anterior Pituitary Tumor Syndromes Shlomo Melmed, J. Larry Jameson HYPOTHALAMIC, PITUITARY, AND OTHER SELLAR MASSES EVALUATION OF SELLAR MASSES 403

1	See Chap. 404 for diagnosis and treatment of diabetes insipidus. Local Mass Effects Clinical manifestations of sellar lesions vary, depending on the anatomic location of the mass and the direction of its extension (Table 403-1). The dorsal sellar diaphragm presents the least resistance to soft tissue expansion from the sella; consequently, pituitary adenomas frequently extend in a suprasellar direction. Bony invasion may occur as well. Headaches are common features of small intrasellar tumors, even with no demonstrable suprasellar extension. Because of the confined nature of the pituitary, small changes in intrasellar pressure stretch the dural plate; however, headache severity correlates poorly with adenoma size or extension.

1	Suprasellar extension can lead to visual loss by several mechanisms, the most common being compression of the optic chiasm, but rarely, direct invasion of the optic nerves or obstruction of cerebrospinal fluid (CSF) flow leading to secondary visual disturbances can occur. Pituitary stalk compression by a hormonally active or inactive intrasellar mass may compress the portal vessels, disrupting pituitary access to hypothalamic hormones and dopamine; this results in early hyperprolactinemia and later concurrent loss of other pituitary hormones. This “stalk section” phenomenon may also be caused by trauma, whiplash injury with posterior clinoid stalk compression, or skull base fractures. Lateral mass invasion may impinge on the cavernous sinus and compress its neural contents, leading to cranial nerve III, aAs the intrasellar mass expands, it first compresses intrasellar pituitary tissue, then usually invades dorsally through the dura to lift the optic chiasm or laterally to the

1	to cranial nerve III, aAs the intrasellar mass expands, it first compresses intrasellar pituitary tissue, then usually invades dorsally through the dura to lift the optic chiasm or laterally to the cavernous sinuses. Bony erosion is rare, as is direct brain compression. Microadenomas may present with headache.

1	2262 IV, and VI palsies as well as effects on the ophthalmic and maxillary branches of the fifth cranial nerve (Chap. 455). Patients may present with diplopia, ptosis, ophthalmoplegia, and decreased facial sensation, depending on the extent of neural damage. Extension into the sphenoid sinus indicates that the pituitary mass has eroded through the sellar floor. Aggressive tumors rarely invade the palate roof and cause nasopharyngeal obstruction, infection, and CSF leakage. Temporal and frontal lobe involvement may rarely lead to uncinate seizures, personality disorders, and anosmia. Direct hypothalamic encroachment by an invasive pituitary mass may cause important metabolic sequelae, including precocious puberty or hypogonadism, diabetes insipidus, sleep disturbances, dysthermia, and appetite disorders.

1	Magnetic Resonance Imaging Sagittal and coronal T1-weighted magnetic resonance imaging (MRI) before and after administration of gadolinium allows precise visualization of the pituitary gland with clear delineation of the hypothalamus, pituitary stalk, pituitary tissue and surrounding suprasellar cisterns, cavernous sinuses, sphenoid sinus, and optic chiasm. Pituitary gland height ranges from 6 mm in children to 8 mm in adults; during pregnancy and puberty, the height may reach 10–12 mm. The upper aspect of the adult pituitary is flat or slightly concave, but in adolescent and pregnant individuals, this surface may be convex, reflecting physiologic pituitary enlargement. The stalk should be midline and vertical. Computed tomography (CT) scan is reserved to define the extent of bony erosion or the presence of calcification.

1	Anterior pituitary gland soft tissue consistency is slightly heterogeneous on MRI, and signal intensity resembles that of brain matter on T1-weighted imaging (Fig. 403-1). Adenoma density is usually lower than that of surrounding normal tissue on T1-weighted imaging, and the signal intensity increases with T2-weighted images. The high phospholipid content of the posterior pituitary results in a “pituitary bright spot.”

1	Sellar masses are encountered commonly as incidental findings on MRI, and most of them are pituitary adenomas (incidentalomas). In the absence of hormone hypersecretion, these small intrasellar lesions can be monitored safely with MRI, which is performed annually and then less often if there is no evidence of further growth. Resection should be considered for incidentally discovered larger macroadenomas, because about one-third become invasive or cause local pressure effects. If hormone hypersecretion is evident, specific therapies are indicated as described below. When larger masses (>1 cm) are encountered, they should also be distinguished from nonadenomatous lesions. Meningiomas often are associated with bony hyperostosis;

1	FIGURE 403-1 Pituitary adenoma. Coronal T1-weighted postcontrast magnetic resonance image shows a homogeneously enhancing mass (arrowheads) in the sella turcica and suprasellar region compatible with a pituitary adenoma; the small arrows outline the carotid arteries. Acromegaly Serum IGF-I Interpret IGF-I relative to ageand sex-matched controls Oral glucose tolerance Normal subjects should test with GH obtained suppress growth hormone at 0, 30, and 60 min to <1 g/L MRI of the sella should be ordered if PRL is elevated Cushing’s disease 24-h urinary free cortisol Ensure urine collection is total and accurate Dexamethasone (1 mg) Normal subjects suppress to at 11 P.M. and fasting <5 g/dL plasma cortisol measured at 8 A.M. Abbreviations: ACTH, adrenocorticotropin hormone; GH, growth hormone; IGF-I, insulin-like growth factor I; MRI, magnetic resonance imaging; PRL, prolactin.

1	Abbreviations: ACTH, adrenocorticotropin hormone; GH, growth hormone; IGF-I, insulin-like growth factor I; MRI, magnetic resonance imaging; PRL, prolactin. craniopharyngiomas may be calcified and are usually hypodense, whereas gliomas are hyperdense on T2-weighted images.

1	craniopharyngiomas may be calcified and are usually hypodense, whereas gliomas are hyperdense on T2-weighted images. Ophthalmologic Evaluation Because optic tracts may be contiguous to an expanding pituitary mass, reproducible visual field assessment using perimetry techniques should be performed on all patients with sellar mass lesions that impinge the optic chiasm (Chap. 39). Bitemporal hemianopia, often more pronounced superiorly, is observed classically. It occurs because nasal ganglion cell fibers, which cross in the optic chiasm, are especially vulnerable to compression of the ventral optic chiasm. Occasionally, homonymous hemianopia occurs from postchiasmal compression or monocular temporal field loss from prechiasmal compression. Invasion of the cavernous sinus can produce diplopia from ocular motor nerve palsy. Early diagnosis reduces the risk of optic atrophy, vision loss, or eye misalignment.

1	Laboratory Investigation The presenting clinical features of functional pituitary adenomas (e.g., acromegaly, prolactinomas, or Cushing’s syndrome) should guide the laboratory studies (Table 403-2). However, for a sellar mass with no obvious clinical features of hormone excess, laboratory studies are geared toward determining the nature of the tumor and assessing the possible presence of hypopituitarism. When a pituitary adenoma is suspected based on MRI, initial hormonal evaluation usually includes (1) basal prolactin (PRL); (2) insulin-like growth factor (IGF) I; (3) 24-h urinary free cortisol (UFC) and/or overnight oral dexamethasone (1 mg) suppression test; (4) α subunit, follicle-stimulating hormone (FSH), and luteinizing hormone (LH); and (5) thyroid function tests. Additional hormonal evaluation may be indicated based on the results of these tests. Pending more detailed assessment of hypopituitarism, a menstrual history, measurement of testosterone and 8 A.M. cortisol levels,

1	evaluation may be indicated based on the results of these tests. Pending more detailed assessment of hypopituitarism, a menstrual history, measurement of testosterone and 8 A.M. cortisol levels, and thyroid function tests usually identify patients with pituitary hormone deficiencies that require hormone replacement before further testing or surgery.

1	Histologic Evaluation Immunohistochemical staining of pituitary tumor specimens obtained at transsphenoidal surgery confirms clinical and laboratory studies and provides a histologic diagnosis when hormone studies are equivocal and in cases of clinically nonfunctioning tumors. Occasionally, ultrastructural assessment by electron microscopy is required for diagnosis. TREATmEnT HypotHalamic, pituitary, anD otHer sellar masses OVERVIEW Successful management of sellar masses requires accurate diagnosis as well as selection of optimal therapeutic modalities. Most pituitary tumors are benign and slow-growing. Clinical features result from local mass effects and hormonal hyperor hyposecretion syndromes caused directly by the adenoma or occurring as a consequence of treatment. Thus, lifelong management and follow-up are necessary for these patients.

1	MRI with gadolinium enhancement for pituitary visualization, new advances in transsphenoidal surgery and in stereotactic radiotherapy (including gamma-knife radiotherapy), and novel therapeutic agents have improved pituitary tumor management. The goals of pituitary tumor treatment include normalization of excess pituitary secretion, amelioration of symptoms and signs of hormonal hypersecretion syndromes, and shrinkage or ablation of large tumor masses with relief of adjacent structure compression. Residual anterior pituitary function should be preserved during treatment and sometimes can be restored by removing the tumor mass. Ideally, adenoma recurrence should be prevented.

1	TRANSSPHENOIDAL SURGERY Transsphenoidal rather than transfrontal resection is the desired surgical approach for pituitary tumors, except for the rare invasive suprasellar mass surrounding the frontal or middle fossa or the optic nerves or invading posteriorly behind the clivus. Intraoperative microscopy facilitates visual distinction between adenomatous and normal pituitary tissue as well as microdissection of small tumors that may not be visible by MRI (Fig. 403-2). Transsphenoidal surgery also avoids the cranial invasion and manipulation of brain tissue required by subfrontal surgical approaches. Endoscopic techniques with three-dimensional nerve of cavernous

1	FIGURE 403-2 Transsphenoidal resection of pituitary mass via the endonasal approach. (Adapted from R Fahlbusch: Endocrinol Metab Clin 21:669, 1992.) intraoperative localization have also improved visualization and access 2263 to tumor tissue. Individual surgical experience is a major determinant of outcome efficacy with these techniques.

1	In addition to correction of hormonal hypersecretion, pituitary surgery is indicated for mass lesions that impinge on surrounding structures. Surgical decompression and resection are required for an expanding pituitary mass accompanied by persistent headache, progressive visual field defects, cranial nerve palsies, hydrocephalus, and, occasionally, intrapituitary hemorrhage and apoplexy. Transsphenoidal surgery sometimes is used for pituitary tissue biopsy to establish a histologic diagnosis.Whenever possible, the pituitary mass lesion should be selectively excised; normal pituitary tissue should be manipulated or resected only when critical for effective mass dissection. Nonselective hemihypophysectomy or total hypophysectomy may be indicated if no hypersecreting mass lesion is clearly discernible, multifocal lesions are present, or the remaining nontumorous pituitary tissue is obviously necrotic. This strategy, however, increases the likelihood of hypopituitarism and the need for

1	discernible, multifocal lesions are present, or the remaining nontumorous pituitary tissue is obviously necrotic. This strategy, however, increases the likelihood of hypopituitarism and the need for lifelong hormone replacement.

1	Preoperative mass effects, including visual field defects and compromised pituitary function, may be reversed by surgery, particularly when the deficits are not long-standing. For large and invasive tumors, it is necessary to determine the optimal balance between maximal tumor resection and preservation of anterior pituitary function, especially for preserving growth and reproductive function in younger patients. Similarly, tumor invasion outside the sella is rarely amenable to surgical cure; the surgeon must judge the risk-versus-benefit ratio of extensive tumor resection.

1	Side Effects Tumor size, the degree of invasiveness, and experience of the surgeon largely determine the incidence of surgical complications. Operative mortality rate is about 1%. Transient diabetes insipidus and hypopituitarism occur in up to 20% of patients. Permanent diabetes insipidus, cranial nerve damage, nasal septal perforation, or visual disturbances may be encountered in up to 10% of patients. CSF leaks occur in 4% of patients. Less common complications include carotid artery injury, loss of vision, hypothalamic damage, and menin gitis. Permanent side effects are rare after surgery for microadenomas.

1	Radiation is used either as a primary therapy for pituitary or parasellar masses or, more commonly, as an adjunct to surgery or medical therapy. Focused megavoltage irradiation is achieved by precise MRI localization, using a high-voltage linear accelerator and accurate isocentric rotational arcing. A major determinant of accurate irradiation is reproduction of the patient’s head position during multiple visits and maintenance of absolute head immobility. A total of <50 Gy (5000 rad) is given as 180-cGy (180-rad) fractions divided over about 6 weeks. Stereotactic radiosurgery delivers a large single high-energy dose from a cobalt-60 source (gamma knife), linear accelerator, or cyclotron. Long-term effects of gamma-knife surgery are unclear but appear to be similar to those encountered with conventional radiation. Proton beam therapy is available in some centers and provides concentrated radiation doses within a localized region.

1	The role of radiation therapy in pituitary tumor management depends on multiple factors, including the nature of the tumor, the age of the patient, and the availability of surgical and radiation expertise. Because of its relatively slow onset of action, radiation therapy is usually reserved for postsurgical management. As an adjuvant to surgery, radiation is used to treat residual tumor and in an attempt to prevent regrowth. Irradiation offers the only means for potentially ablating significant postoperative residual nonfunctioning tumor tissue. In contrast, PRLand growth hormone (GH)secreting tumor tissues are amenable to medical therapy.

1	Side Effects In the short term, radiation may cause transient nausea and weakness. Alopecia and loss of taste and smell may be more long-lasting. Failure of pituitary hormone synthesis is common in patients who have undergone head and neck or pituitary-directed irradiation. More than 50% of patients develop loss of GH, adrenocorticotropin 2264 hormone (ACTH), thyroid-stimulating hormone (TSH), and/or gonadotropin secretion within 10 years, usually due to hypothalamic damage. Lifelong follow-up with testing of anterior pituitary hormone reserve is therefore required after radiation treatment. Optic nerve damage with impaired vision due to optic neuritis is reported in about 2% of patients who undergo pituitary irradiation. Cranial nerve damage is uncommon now that radiation doses are ≤2 Gy (200 rad) at any one treatment session and the maximum dose is <50 Gy (5000 rad). The use of stereotactic radiotherapy may reduce damage to adjacent structures. Radiotherapy for pituitary tumors has

1	(200 rad) at any one treatment session and the maximum dose is <50 Gy (5000 rad). The use of stereotactic radiotherapy may reduce damage to adjacent structures. Radiotherapy for pituitary tumors has been associated with adverse mortality rates, mainly from cerebrovascular disease. The cumulative risk of developing a secondary tumor after conventional radiation is 1.3% after 10 years and 1.9% after 20 years.

1	Medical therapy for pituitary tumors is highly specific and depends on tumor type. For prolactinomas, dopamine agonists are the treatment of choice. For acromegaly, somatostatin analogues and GH receptor antagonists are indicated. For TSH-secreting tumors, somatostatin analogues and occasionally dopamine agonists are indicated. ACTH-secreting tumors and nonfunctioning tumors are generally not responsive to medications and require surgery and/ or irradiation. Sellar masses other than pituitary adenomas may arise from brain, hypothalamic, or pituitary tissues. Each exhibit features related to the lesion location but also unique to the specific etiology.

1	Hypothalamic Lesions Lesions involving the anterior and preoptic hypothalamic regions cause paradoxical vasoconstriction, tachycardia, and hyperthermia. Acute hyperthermia usually is due to a hemorrhagic insult, but poikilothermia may also occur. Central disorders of thermoregulation result from posterior hypothalamic damage. The periodic hypothermia syndrome is characterized by episodic attacks of rectal temperatures <30°C (86°F), sweating, vasodilation, vomiting, and bradycardia (Chap. 478e). Damage to the ventromedial hypothalamic nuclei by craniopharyngiomas, hypothalamic trauma, or inflammatory disorders may be associated with hyperphagia and obesity. This region appears to contain an energy-satiety center where melanocortin receptors are influenced by leptin, insulin, pro-opiomelanocortin (POMC) products, and gastrointestinal peptides (Chap. 415e). Polydipsia and hypodipsia are associated with damage to central osmoreceptors located in preoptic nuclei (Chap. 404). Slow-growing

1	(POMC) products, and gastrointestinal peptides (Chap. 415e). Polydipsia and hypodipsia are associated with damage to central osmoreceptors located in preoptic nuclei (Chap. 404). Slow-growing hypothalamic lesions can cause increased somnolence and disturbed sleep cycles as well as obesity, hypothermia, and emotional outbursts. Lesions of the central hypothalamus may stimulate sympathetic neurons, leading to elevated serum catecholamine and cortisol levels. These patients are predisposed to cardiac arrhythmias, hypertension, and gastric erosions.

1	Craniopharyngiomas are benign, suprasellar cystic masses that present with headaches, visual field deficits, and variable degrees of hypopituitarism. They are derived from Rathke’s pouch and arise near the pituitary stalk, commonly extending into the suprasellar cistern. Craniopharyngiomas are often large, cystic, and locally invasive. Many are partially calcified, exhibiting a characteristic appearance on skull x-ray and CT images. More than half of all patients present before age 20, usually with signs of increased intracranial pressure, including headache, vomiting, papilledema, and hydrocephalus. Associated symptoms include visual field abnormalities, personality changes and cognitive deterioration, cranial nerve damage, sleep difficulties, and weight gain. Hypopituitarism can be documented in about 90%, and diabetes insipidus occurs in about 10% of patients. About half of affected children present with growth retardation. MRI is generally superior to CT for evaluating cystic

1	documented in about 90%, and diabetes insipidus occurs in about 10% of patients. About half of affected children present with growth retardation. MRI is generally superior to CT for evaluating cystic structure and tissue components of craniopharyngiomas. CT is useful to define calcifications and evaluate invasion into surrounding bony structures and sinuses.

1	Treatment usually involves transcranial or transsphenoidal surgical resection followed by postoperative radiation of residual tumor. Surgery alone is curative in less than half of patients because of recurrences due to adherence to vital structures or because of small tumor deposits in the hypothalamus or brain parenchyma. The goal of surgery is to remove as much tumor as possible without risking complications associated with efforts to remove firmly adherent or inaccessible tissue. In the absence of radiotherapy, about 75% of craniopharyngiomas recur, and 10-year survival is less than 50%. In patients with incomplete resection, radiotherapy improves 10-year survival to 70–90% but is associated with increased risk of secondary malignancies. Most patients require lifelong pituitary hormone replacement.

1	Developmental failure of Rathke’s pouch obliteration may lead to Rathke’s cysts, which are small (<5 mm) cysts entrapped by squamous epithelium and are found in about 20% of individuals at autopsy. Although Rathke’s cleft cysts do not usually grow and are often diagnosed incidentally, about a third present in adulthood with compressive symptoms, diabetes insipidus, and hyperprolactinemia due to stalk compression. Rarely, hydrocephalus develops. The diagnosis is suggested preoperatively by visualizing the cyst wall on MRI, which distinguishes these lesions from craniopharyngiomas. Cyst contents range from CSF-like fluid to mucoid material. Arachnoid cysts are rare and generate an MRI image that is isointense with CSF.

1	Sella chordomas usually present with bony clival erosion, local invasiveness, and, on occasion, calcification. Normal pituitary tissue may be visible on MRI, distinguishing chordomas from aggressive pituitary adenomas. Mucinous material may be obtained by fine-needle aspiration. Meningiomas arising in the sellar region may be difficult to distinguish from nonfunctioning pituitary adenomas. Meningiomas typically enhance on MRI and may show evidence of calcification or bony erosion. Meningiomas may cause compressive symptoms. Histiocytosis X includes a variety of syndromes associated with foci of eosinophilic granulomas. Diabetes insipidus, exophthalmos, and punched-out lytic bone lesions (Hand-Schüller-Christian disease) are associated with granulomatous lesions visible on MRI, as well as a characteristic axillary skin rash. Rarely, the pituitary stalk may be involved.

1	Pituitary metastases occur in ~3% of cancer patients. Bloodborne metastatic deposits are found almost exclusively in the posterior pituitary. Accordingly, diabetes insipidus can be a presenting feature of lung, gastrointestinal, breast, and other pituitary metastases. About half of pituitary metastases originate from breast cancer; about 25% of patients with metastatic breast cancer have such deposits. Rarely, pituitary stalk involvement results in anterior pituitary insufficiency. The MRI diagnosis of a metastatic lesion may be difficult to distinguish from an aggressive pituitary adenoma; the diagnosis may require histologic examination of excised tumor tissue. Primary or metastatic lymphoma, leukemias, and plasmacytomas also occur within the sella.

1	Hypothalamic hamartomas and gangliocytomas may arise from astrocytes, oligodendrocytes, and neurons with varying degrees of differentiation. These tumors may overexpress hypothalamic neuropeptides, including gonadotropin-releasing hormone (GnRH), growth hormone–releasing hormone (GHRH), and corticotropin-releasing hormone (CRH). With GnRH-producing tumors, children present with precocious puberty, psychomotor delay, and laughing-associated seizures. Medical treatment of GnRH-producing hamartomas with long-acting GnRH analogues effectively suppresses gonadotropin secretion and controls premature pubertal development. Rarely, hamartomas also are associated with craniofacial abnormalities; imperforate anus; cardiac, renal, and lung disorders; and pituitary failure as features of Pallister-Hall syndrome, which is caused by mutations in the carboxy terminus of the GLI3 gene. Hypothalamic hamartomas are often contiguous with the pituitary, and preoperative MRI diagnosis may not be possible.

1	syndrome, which is caused by mutations in the carboxy terminus of the GLI3 gene. Hypothalamic hamartomas are often contiguous with the pituitary, and preoperative MRI diagnosis may not be possible. Histologic evidence of hypothalamic neurons in tissue resected at transsphenoidal surgery may be the first indication of a primary hypothalamic lesion.

1	Hypothalamic gliomas and optic gliomas occur mainly in childhood and usually present with visual loss. Adults have more aggressive tumors; about a third are associated with neurofibromatosis. Brain germ cell tumors may arise within the sellar region. They include dysgerminomas, which frequently are associated with diabetes insipidus and visual loss. They rarely metastasize. Germinomas, embryonal carcinomas, teratomas, and choriocarcinomas may arise in the parasellar region and produce hCG. These germ cell tumors present with precocious puberty, diabetes insipidus, visual field defects, and thirst disorders. Many patients are GH-deficient with short stature.

1	Pituitary adenomas are the most common cause of pituitary hormone hypersecretion and hyposecretion syndromes in adults. They account for ~15% of all intracranial neoplasms and have been identified with a population prevalence of ~80/100,000. At autopsy, up to one-quarter of all pituitary glands harbor an unsuspected microadenoma (<10 mm diameter). Similarly, pituitary imaging detects small clinically inapparent pituitary lesions in at least 10% of individuals.

1	Pathogenesis Pituitary adenomas are benign neoplasms that arise from one of the five anterior pituitary cell types. The clinical and biochemical phenotypes of pituitary adenomas depend on the cell type from which they are derived. Thus, tumors arising from lactotrope (PRL), somatotrope (GH), corticotrope (ACTH), thyrotrope (TSH), or gonadotrope (LH, FSH) cells hypersecrete their respective hormones (Table 403-3). Plurihormonal tumors express various combinations of GH, PRL, TSH, ACTH, or the glycoprotein hormone α or β subunits. They may be diagnosed by careful immunocytochemistry or may manifest as clinical syndromes that combine features of these hormonal hypersecretory syndromes. Morphologically, these tumors may arise from a single polysecreting cell type or include cells with mixed function within the same tumor.

1	Hormonally active tumors are characterized by autonomous hormone secretion with diminished feedback responsiveness to physiologic inhibitory pathways. Hormone production does not always correlate with tumor size. Small hormone-secreting adenomas may cause significant clinical perturbations, whereas larger adenomas that produce less hormone may be clinically silent and remain undiagnosed (if no central compressive effects occur). About one-third of all adenomas are clinically nonfunctioning and produce no distinct clinical hyper-secretory syndrome. Most of them arise from gonadotrope cells and may secrete small amounts of αand β-glycoprotein hormone subunits or, very rarely, intact circulating gonadotropins. True pituitary carcinomas with documented extracranial metastases are exceedingly rare.

1	Almost all pituitary adenomas are monoclonal in origin, implying the acquisition of one or more somatic mutations that confer a selective growth advantage. Consistent with their clonal origin, complete surgical resection of small pituitary adenomas usually cures hormone aHormone-secreting tumors are listed in decreasing order of frequency. All tumors may cause local pressure effects, including visual disturbances, cranial nerve palsy, and headache. Note: For abbreviations, see text. Source: Adapted from S Melmed, in JL Jameson (ed): Principles of Molecular Medicine. Totowa, NJ, Humana Press, 1998. hypersecretion. Nevertheless, hypothalamic hormones such as GHRH 2265 and CRH also enhance mitotic activity of their respective pituitary target cells in addition to their role in pituitary hormone regulation. Thus, patients who harbor rare abdominal or chest tumors that elaborate ectopic GHRH or CRH may present with somatotrope or corticotrope hyperplasia with GH or ACTH hypersecretion.

1	Several etiologic genetic events have been implicated in the development of pituitary tumors. The pathogenesis of sporadic forms of acromegaly has been particularly informative as a model of tumorigenesis. GHRH, after binding to its G protein–coupled somatotrope receptor, uses cyclic adenosine monophosphate (AMP) as a second messenger to stimulate GH secretion and somatotrope proliferation. A subset (~35%) of GH-secreting pituitary tumors contains sporadic mutations in Gsα (Arg 201 → Cys or His; Gln 227 → Arg). These mutations attenuate intrinsic GTPase activity, resulting in constitutive elevation of cyclic AMP, Pit-1 induction, and activation of cyclic AMP response element binding protein (CREB), thereby promoting somatotrope cell proliferation and GH secretion.

1	Characteristic loss of heterozygosity (LOH) in various chromosomes has been documented in large or invasive macroadenomas, suggesting the presence of putative tumor suppressor genes at these loci in up to 20% of sporadic pituitary tumors, including GH-, PRL-, and ACTH-producing adenomas and some nonfunctioning tumors. Lineage-specific cell cycle disruptions with elevated levels of CDK inhibitors are present in most of these adenomas.

1	Compelling evidence also favors growth factor promotion of pituitary tumor proliferation. Basic fibroblast growth factor (bFGF) is abundant in the pituitary and stimulates pituitary cell mitogenesis, whereas epi thelial growth factor (EGF) receptor signaling induces both hormone synthesis and cell proliferation. Other factors involved in initiation and promotion of pituitary tumors include loss of negative-feedback inhibition (as seen with primary hypothyroidism or hypogonadism) and estrogen-mediated or paracrine angiogenesis. Growth characteristics and neoplastic behavior also may be influenced by several activated oncogenes, including RAS and pituitary tumor transforming gene (PTTG), or inactivation of growth suppressor genes, including MEG3. Genetic Syndromes Associated with Pituitary Tumors Several familial syndromes are associated with pituitary tumors, and the genetic mechanisms for some of them have been unraveled (Table 403-4).

1	Genetic Syndromes Associated with Pituitary Tumors Several familial syndromes are associated with pituitary tumors, and the genetic mechanisms for some of them have been unraveled (Table 403-4). Multiple endocrine neoplasia (MEN) 1 is an autosomal dominant syndrome characterized primarily by a genetic predisposition to parathyroid, pancreatic islet, and pituitary adenomas (Chap. 408). MEN1 is caused by inactivating germline mutations in MENIN, a constitutively expressed tumor-suppressor gene located on chromosome 11q13.

1	2266 Loss of heterozygosity or a somatic mutation of the remaining normal MENIN allele leads to tumorigenesis. About half of affected patients develop prolactinomas; acromegaly and Cushing’s syndrome are less commonly encountered. Carney’s syndrome is characterized by spotty skin pigmentation, myxomas, and endocrine tumors, including testicular, adrenal, and pituitary adenomas. Acromegaly occurs in about 20% of these patients. A subset of patients have mutations in the R1α regulatory subunit of protein kinase A (PRKAR1A). McCune-Albright syndrome consists of polyostotic fibrous dysplasia, pigmented skin patches, and a variety of endocrine disorders, including acromegaly, adrenal adenomas, and autonomous ovarian function (Chap. 426e). Hormonal hypersecretion results from constitutive cyclic AMP production caused by inactivation of the GTPase activity of Gsα. The Gsα mutations occur postzygotically, leading to a mosaic pattern of mutant expression. Familial acromegaly is a rare disorder

1	AMP production caused by inactivation of the GTPase activity of Gsα. The Gsα mutations occur postzygotically, leading to a mosaic pattern of mutant expression. Familial acromegaly is a rare disorder in which family members may manifest either acromegaly or gigantism. A subset of families with a predisposition for familial pituitary tumors, especially acromegaly, have been found to harbor germline mutations in the AIP gene, which encodes the aryl hydrocarbon receptor interacting protein.

1	HYPERPROLACTINEMIA Etiology Hyperprolactinemia is the most common pituitary hormone hypersecretion syndrome in both men and women. PRL-secreting pituitary adenomas (prolactinomas) are the most common cause of PRL levels >200 μg/L (see below). Less pronounced PRL elevation can also be seen with microprolactinomas but is more commonly caused by drugs, pituitary stalk compression, hypothyroidism, or renal failure (Table 403-5). Pregnancy and lactation are the important physiologic causes of hyperprolactinemia. Sleep-associated hyperprolactinemia reverts to normal within an hour of awakening. Nipple stimulation and sexual orgasm also may increase PRL. Chest wall stimulation or trauma (including chest surgery and herpes zoster) invoke the reflex suckling arc with resultant hyperprolactinemia. Chronic renal failure elevates PRL by decreasing peripheral clearance. Primary hypothyroidism is associated with mild hyperprolactinemia, probably because of compensatory TRH secretion.

1	Lesions of the hypothalamic-pituitary region that disrupt hypothalamic dopamine synthesis, portal vessel delivery, or lactotrope responses are associated with hyperprolactinemia. Thus, hypothalamic tumors, cysts, infiltrative disorders, and radiation-induced damage cause elevated PRL levels, usually in the range of 30–100 μg/L. Plurihormonal adenomas (including GH and ACTH tumors) may hypersecrete PRL directly. Pituitary masses, including clinically nonfunctioning pituitary tumors, may compress the pituitary stalk to cause hyperprolactinemia.

1	Drug-induced inhibition or disruption of dopaminergic receptor function is a common cause of hyperprolactinemia (Table 403-5). Thus, antipsychotics and antidepressants are a relatively common cause of mild hyperprolactinemia. Most patients receiving risperidone have elevated prolactin levels, sometimes exceeding 200 μg/L. Methyldopa inhibits dopamine synthesis, and verapamil blocks dopamine release, also leading to hyperprolactinemia. Hormonal agents that induce PRL include estrogens and thyrotropin-releasing hormone (TRH).

1	Presentation and Diagnosis Amenorrhea, galactorrhea, and infertility are the hallmarks of hyperprolactinemia in women. If hyperprolactinemia develops before menarche, primary amenorrhea results. More commonly, hyperprolactinemia develops later in life and leads to oligomenorrhea and ultimately to amenorrhea. If hyperprolactinemia is sustained, vertebral bone mineral density can be reduced compared with age-matched controls, particularly when it is associated with pronounced hypoestrogenemia. Galactorrhea is present in up to 80% of hyperprolactinemic women. Although usually bilateral and spontaneous, it may be unilateral or expressed only manually. Patients also may complain of decreased libido, weight gain, and mild hirsutism. In men with hyperprolactinemia, diminished libido, infertility, and visual loss (from optic nerve compression) are the usual presenting symptoms. Gonadotropin suppression leads to reduced testosterone, ETiology of HyPERPRolACTinEmiA

1	ETiology of HyPERPRolACTinEmiA I. Physiologic hypersecretion II. Hypothalamic–pituitary stalk damage Empty sella Lymphocytic hypophysitis Adenoma with stalk Compression Granulomas Rathke’s cyst Irradiation Trauma III. Pituitary hypersecretion IV. Systemic disorders V. Drug-induced hypersecretion Dopamine receptor blockers Atypical antipsychotics: risperidone Phenothiazines: chlorpromazine, perphenazine Butyrophenones: haloperidol Thioxanthenes Metoclopramide Cimetidine, ranitidine Imipramines Amitriptyline, amoxapine Serotonin reuptake inhibitors Fluoxetine Note: Hyperprolactinemia >200 μg/L almost invariably is indicative of a prolactin-secreting pituitary adenoma. Physiologic causes, hypothyroidism, and drug-induced hyperprolactinemia should be excluded before extensive evaluation.

1	impotence, and oligospermia. True galactorrhea is uncommon in men with hyperprolactinemia. If the disorder is long-standing, secondary effects of hypogonadism are evident, including osteopenia, reduced muscle mass, and decreased beard growth. The diagnosis of idiopathic hyperprolactinemia is made by exclusion of known causes of hyperprolactinemia in the setting of a normal pituitary MRI. Some of these patients may harbor small microadenomas below visible MRI sensitivity (~2 mm).

1	Galactorrhea, the inappropriate discharge of milk-containing fluid from the breast, is considered abnormal if it persists longer than 6 months after childbirth or discontinuation of breast-feeding. Postpartum galactorrhea associated with amenorrhea is a self-limiting disorder usually associated with moderately elevated PRL levels. Galactorrhea may occur spontaneously, or it may be elicited by nipple pressure. In both men and women, galactorrhea may vary in color and consistency (transparent, milky, or bloody) and arise either unilaterally or bilaterally. Mammography or ultrasound is indicated for bloody discharges (particularly from a single nipple), which may be caused by breast cancer. Galactorrhea is commonly associated with hyperprolactinemia caused by any of the conditions listed in Table 403-5. Acromegaly is associated with galactorrhea in about one-third of patients. Treatment of galactorrhea usually involves managing the underlying disorder (e.g., replacing T4 for

1	listed in Table 403-5. Acromegaly is associated with galactorrhea in about one-third of patients. Treatment of galactorrhea usually involves managing the underlying disorder (e.g., replacing T4 for hypothyroidism, discontinuing a medication, treating prolactinoma).

1	Laboratory Investigation Basal, fasting morning PRL levels (normally <20 μg/L) should be measured to assess hypersecretion. Both false-positive and false-negative results may be encountered. In patients with markedly elevated PRL levels (>1000 μg/L), reported results may be falsely lowered because of assay artifacts; sample dilution is required to measure these high values accurately. Falsely elevated values may be caused by aggregated forms of circulating PRL, which are usually biologically inactive (macroprolactinemia). Hypothyroidism should be excluded by measuring TSH and T4 levels.

1	Treatment of hyperprolactinemia depends on the cause of elevated PRL levels. Regardless of the etiology, however, treatment should be aimed at normalizing PRL levels to alleviate suppressive effects on gonadal function, halt galactorrhea, and preserve bone mineral density. Dopamine agonists are effective for most causes of hyperprolactinemia (see the treatment section for prolactinoma, below) regardless of the underlying cause.

1	If the patient is taking a medication known to cause hyperprolactinemia, the drug should be withdrawn, if possible. For psychiatric patients who require neuroleptic agents, supervised dose titration or the addition of a dopamine agonist can help restore normoprolactinemia and alleviate reproductive symptoms. However, dopamine agonists may worsen the underlying psychiatric condition, especially at high doses. Hyperprolactinemia usually resolves after adequate thyroid hormone replacement in hypothyroid patients or after renal transplantation in patients undergoing dialysis. Resection of hypothalamic or sellar mass lesions can reverse hyperprolactinemia caused by stalk compression and reduced dopamine tone. Granulomatous infiltrates occasionally respond to glucocorticoid administration. In patients with irreversible hypothalamic damage, no treatment may be warranted. In up to 30% of patients with hyperprolactinemia—usually without a visible pituitary microadenoma— the condition may

1	In patients with irreversible hypothalamic damage, no treatment may be warranted. In up to 30% of patients with hyperprolactinemia—usually without a visible pituitary microadenoma— the condition may resolve spontaneously.

1	PROLACTINOMA Etiology and Prevalence Tumors arising from lactotrope cells account for about half of all functioning pituitary tumors, with a population prevalence of ~10/100,000 in men and ~30/100,000 in women. Mixed tumors that secrete combinations of GH and PRL, ACTH and PRL, and rarely TSH and PRL are also seen. These plurihormonal tumors are usually recognized by immunohistochemistry, sometimes without apparent clinical manifestations from the production of additional 2267 hormones. Microadenomas are classified as <1 cm in diameter and usually do not invade the parasellar region. Macroadenomas are >1 cm in diameter and may be locally invasive and impinge on adjacent structures. The female-to-male ratio for microprolactinomas is 20:1, whereas the sex ratio is near 1:1 for macroadenomas. Tumor size generally correlates directly with PRL concentrations; values >250 μg/L usually are associated with macroadenomas. Men tend to present with larger tumors than women, possibly because the

1	Tumor size generally correlates directly with PRL concentrations; values >250 μg/L usually are associated with macroadenomas. Men tend to present with larger tumors than women, possibly because the features of male hypogonadism are less readily evident. PRL levels remain stable in most patients, reflecting the slow growth of these tumors. About 5% of microadenomas progress in the long term to macroadenomas.

1	Presentation and Diagnosis Women usually present with amenorrhea, infertility, and galactorrhea. If the tumor extends outside the sella, visual field defects or other mass effects may be seen. Men often present with impotence, loss of libido, infertility, or signs of central nervous system (CNS) compression, including headaches and visual defects. Assuming that physiologic and medication-induced causes of hyperprolactinemia are excluded (Table 403-5), the diagnosis of prolactinoma is likely with a PRL level >200 μg/L. PRL levels <100 μg/L may be caused by microadenomas, other sellar lesions that decrease dopamine inhibition, or nonneoplastic causes of hyperprolactinemia. For this reason, an MRI should be performed in all patients with hyperprolactinemia. It is important to remember that hyperprolactinemia caused secondarily by the mass effects of nonlactotrope lesions is also corrected by treatment with dopamine agonists despite failure to shrink the underlying mass. Consequently, PRL

1	caused secondarily by the mass effects of nonlactotrope lesions is also corrected by treatment with dopamine agonists despite failure to shrink the underlying mass. Consequently, PRL suppression by dopamine agonists does not necessarily indicate that the underlying lesion is a prolactinoma.

1	Because microadenomas rarely progress to become macroadenomas, no treatment may be needed if patients are asymptomatic and fertility is not desired; these patients should be monitored by regular serial PRL measurements and MRI scans.For symptomatic microadenomas, therapeutic goals include control of hyperprolactinemia, reduction of tumor size, restoration of menses and fertility, and resolution of galactorrhea. Dopamine agonist doses should be titrated to achieve maximal PRL suppression and restoration of reproductive function (Fig. 403-3). A normalized PRL level does not ensure reduced tumor size. However, tumor shrinkage usually is not seen in those who do not respond with lowered PRL levels. For macroadenomas, formal visual field testing should be performed before initiating dopamine agonists. MRI and visual fields should be assessed at 6to 12-month intervals until the mass shrinks and annually thereafter until maximum size reduction has occurred.

1	Oral dopamine agonists (cabergoline and bromocriptine) are the mainstay of therapy for patients with microor macroprolactinomas. Dopamine agonists suppress PRL secretion and synthesis as well as lactotrope cell proliferation. In patients with microadenomas who have achieved normoprolactinemia and significant reduction of tumor mass, the dopamine agonist may be withdrawn after 2 years. These patients should be monitored carefully for evidence of prolactinoma recurrence. About 20% of patients (especially males) are resistant to dopaminergic treatment; these adenomas may exhibit decreased D2 dopamine receptor numbers or a postreceptor defect. D2 receptor gene mutations in the pituitary have not been reported.

1	Cabergoline An ergoline derivative, cabergoline is a long-acting dopamine agonist with high D2 receptor affinity. The drug effectively suppresses PRL for >14 days after a single oral dose and induces prolactinoma shrinkage in most patients. Cabergoline (0.5–1.0 mg twice weekly) achieves normoprolactinemia and resumption of normal gonadal function in ~80% of patients with microadenomas; galactorrhea improves or resolves in 90% of patients. Cabergoline normalizes PRL and shrinks ~70% of macroprolactinomas. Mass FIGURE 403-3 Management of prolactinoma. MRI, magnetic resonance imaging; PRL, prolactin.

1	FIGURE 403-3 Management of prolactinoma. MRI, magnetic resonance imaging; PRL, prolactin. effect symptoms, including headaches and visual disorders, usually improve dramatically within days after cabergoline initiation; improvement of sexual function requires several weeks of treatment but may occur before complete normalization of prolactin levels. After initial control of PRL levels has been achieved, cabergoline should be reduced to the lowest effective maintenance dose. In ~5% of treated patients harboring a microadenoma, hyperprolactinemia may resolve and not recur when dopamine agonists are discontinued after long-term treatment. Cabergoline also may be effective in patients resistant to bromocriptine. Adverse effects and drug intolerance are encountered less commonly than with bromocriptine.

1	The ergot alkaloid bromocriptine mesylate is a dopamine receptor agonist that suppresses prolactin secretion. Because it is short-acting, the drug is preferred when pregnancy is desired. In microadenomas, bromocriptine rapidly lowers serum prolactin levels to normal in up to 70% of patients, decreases tumor size, and restores gonadal function. In patients with macroadenomas, prolactin levels are also normalized in 70% of patients, and tumor mass shrinkage (≥50%) is achieved in most patients. Therapy is initiated by administering a low bromocriptine dose (0.625–1.25 mg) at bedtime with a snack, followed by gradually increasing the dose. Most patients are controlled with a daily dose of ≤7.5 mg (2.5 mg tid).

1	Side effects of dopamine agonists include constipation, nasal stuffiness, dry mouth, nightmares, insomnia, and vertigo; decreasing the dose usually alleviates these problems. Nausea, vomiting, and postural hypotension with faintness may occur in ~25% of patients after the initial dose. These symptoms may persist in some patients. In general, fewer side effects are reported with cabergoline. For the approximately 15% of patients who are intolerant of oral bromocriptine, cabergoline may be better tolerated. Intravaginal administration of bromocriptine is often efficacious in patients with intractable gastrointestinal side effects. Auditory hallucinations, delusions, and mood swings have been reported in up to 5% of patients and may be due to the dopamine agonist properties or to the lysergic acid derivative of the compounds. Rare reports of leukopenia, thrombocytopenia, pleural fibrosis, cardiac arrhythmias, and hepatitis have been described. Patients with Parkinson’s disease who

1	acid derivative of the compounds. Rare reports of leukopenia, thrombocytopenia, pleural fibrosis, cardiac arrhythmias, and hepatitis have been described. Patients with Parkinson’s disease who receive at least 3 mg of cabergoline daily have been reported to be at risk for development of cardiac valve regurgitation. Studies analyzing over 500 prolactinoma patients receiving recommended doses of cabergoline (up to 2 mg weekly) have shown no evidence for an increased incidence of valvular disorders. Nevertheless, because no controlled prospective studies in pituitary tumor patients are available, it is prudent to perform echocardiograms before initiating standard-dose cabergoline therapy.

1	Surgery Indications for surgical adenoma debulking include dopamine resistance or intolerance and the presence of an invasive macroadenoma with compromised vision that fails to improve after drug treatment. Initial PRL normalization is achieved in about 70% of microprolactinomas after surgical resection, but only 30% of macroadenomas can be resected successfully. Follow-up studies have shown that hyperprolactinemia recurs in up to 20% of patients within the first year after surgery; long-term recurrence rates exceed 50% for macroadenomas. Radiotherapy for prolactinomas is reserved for patients with aggressive tumors that do not respond to maximally tolerated dopamine agonists and/or surgery.

1	The pituitary increases in size during pregnancy, reflecting the stimulatory effects of estrogen and perhaps other growth factors on pituitary vascularity and lactotrope cell hyperplasia. About 5% of microadenomas significantly increase in size, but 15–30% of macroadenomas grow during pregnancy. Bromocriptine has been used for more than 30 years to restore fertility in women with hyperprolactinemia, without evidence of teratogenic effects. Nonetheless, most authorities recommend strategies to minimize fetal exposure to the drug. For women taking bromocriptine who desire pregnancy, mechanical contraception should be used through three regular menstrual cycles to allow for conception timing. When pregnancy is confirmed, bromocriptine should be discontinued and PRL levels followed serially, especially if headaches or visual symptoms occur. For women harboring macroadenomas, regular visual field testing is recommended, and the drug should be reinstituted if tumor growth is apparent.

1	especially if headaches or visual symptoms occur. For women harboring macroadenomas, regular visual field testing is recommended, and the drug should be reinstituted if tumor growth is apparent. Although pituitary MRI may be safe during pregnancy, this procedure should be reserved for symptomatic patients with severe headache and/or visual field defects. Surgical decompression may be indicated if vision is threatened. Although comprehensive data support the efficacy and relative safety of bromocriptine-facilitated fertility, patients should be advised of potential unknown deleterious effects and the risk of tumor growth during pregnancy. Because cabergoline is long-acting with a high D2receptor affinity, it is not recommended for use in women when fertility is desired.

1	ACROMEGALY Etiology GH hypersecretion is usually the result of a somatotrope adenoma but may rarely be caused by extrapituitary lesions (Table 403-6). In addition to the more common GH-secreting somatotrope adenomas, mixed mammosomatotrope tumors and acidophilic stem-cell adenomas secrete both GH and PRL. In patients with acidophilic stem-cell adenomas, features of hyperprolactinemia (hypogonadism and galactorrhea) predominate over the less clinically evident signs of acromegaly. Occasionally, mixed plurihormonal tumors are encountered that also secrete ACTH, the glycoprotein hormone α subunit, or TSH in addition to GH. Patients with partially empty sellas may present with GH hypersecretion due to a small GH-secreting adenoma within the compressed rim of pituitary tissue; some of these may reflect the spontaneous necrosis of tumors that were previously larger. GH-secreting tumors rarely arise from ectopic pituitary tissue remnants in the nasopharynx or midline sinuses.

1	There are case reports of ectopic GH secretion by tumors of pancreatic, ovarian, lung, or hematopoietic origin. Rarely, excess GHRH production may cause acromegaly because of chronic stimulation of somatotropes. These patients present with classic features of acromegaly, elevated GH levels, pituitary enlargement on MRI, and pathologic characteristics of pituitary hyperplasia. The most common cause of GHRH-mediated acromegaly is a chest or abdominal carcinoid tumor. Prevalence, % Source: Adapted from S Melmed: N Engl J Med 355:2558–2573, 2006. Although these tumors usually express positive GHRH immunoreac-2269 tivity, clinical features of acromegaly are evident in only a minority of patients with carcinoid disease. Excessive GHRH also may be elaborated by hypothalamic tumors, usually choristomas or neuromas.

1	Presentation and Diagnosis Protean manifestations of GH and IGF-I hypersecretion are indolent and often are not clinically diagnosed for 10 years or more. Acral bony overgrowth results in frontal bossing, increased hand and foot size, mandibular enlargement with prognathism, and widened space between the lower incisor teeth. In children and adolescents, initiation of GH hypersecretion before epiphyseal long bone closure is associated with development of pituitary gigantism (Fig. 403-4). Soft tissue swelling results in increased heel pad thickness, increased shoe or glove size, ring tightening, characteristic coarse facial features, and a large fleshy nose. Other commonly encountered clinical features include hyperhidrosis, a deep and hollow-sounding voice, oily skin, arthropathy, kyphosis, carpal tunnel syndrome, proximal muscle weakness and fatigue, acanthosis nigricans, and skin tags. Generalized visceromegaly occurs, including cardiomegaly, macroglossia, and thyroid gland

1	kyphosis, carpal tunnel syndrome, proximal muscle weakness and fatigue, acanthosis nigricans, and skin tags. Generalized visceromegaly occurs, including cardiomegaly, macroglossia, and thyroid gland enlargement.

1	The most significant clinical impact of GH excess occurs with respect to the cardiovascular system. Coronary heart disease, cardiomyopathy with arrhythmias, left ventricular hypertrophy, decreased diastolic function, and hypertension ultimately occur in most patients if untreated. Upper airway obstruction with sleep apnea occurs in more than 60% of patients and is associated with both soft tissue laryngeal airway obstruction and central sleep dysfunction. Diabetes mellitus develops in 25% of patients with acromegaly, and most patients are intolerant of a glucose load (as GH counteracts the action of insulin). Acromegaly is associated with an increased risk of colon polyps and mortality from colonic malignancy; polyps are diagnosed in up to one-third of patients. Overall mortality is increased about threefold and is due primarily to cardiovascular and cerebrovascular disorders and respiratory disease. Unless GH levels are controlled, survival is reduced by an average of 10 years

1	increased about threefold and is due primarily to cardiovascular and cerebrovascular disorders and respiratory disease. Unless GH levels are controlled, survival is reduced by an average of 10 years compared with an age-matched control population.

1	Laboratory Investigation Age-matched serum IGF-I levels are elevated in acromegaly. Consequently, an IGF-I level provides a useful laboratory screening measure when clinical features raise the possibility of acromegaly. Due to the pulsatility of GH secretion, measurement of a single random GH level is not useful for the diagnosis or exclusion of acromegaly and does not correlate with disease severity. The diagnosis of acromegaly is confirmed by demonstrating the failure of GH suppression to <0.4 μg/L within 1–2 h of an oral glucose load (75 g). When newer ultrasensitive GH assays are used, normal nadir GH levels are even lower (<0.05 μg/L). About 20% of patients exhibit a paradoxical GH rise after glucose. PRL should be measured, as it is elevated in ~25% of patients with acromegaly. Thyroid function, gonadotropins, and sex steroids may be attenuated because of tumor mass effects. Because most patients will undergo surgery with glucocorticoid coverage, tests of ACTH reserve in

1	Thyroid function, gonadotropins, and sex steroids may be attenuated because of tumor mass effects. Because most patients will undergo surgery with glucocorticoid coverage, tests of ACTH reserve in asymptomatic patients are more efficiently deferred until after surgery.

1	The goal of treatment is to control GH and IGF-I hypersecretion, ablate or arrest tumor growth, ameliorate comorbidities, restore mortality rates to normal, and preserve pituitary function. Surgical resection of GH-secreting adenomas is the initial treatment for most patients (Fig. 403-5). Somatostatin analogues are used as adjuvant treatment for preoperative shrinkage of large invasive macroadenomas, immediate relief of debilitating symptoms, and reduction of GH hypersecretion; in frail patients experiencing morbidity; and in patients who decline surgery or, when surgery fails, to achieve biochemical control. Irradiation or repeat surgery may be required for patients who cannot tolerate or do not respond to adjunctive medical therapy. The high rate of late hypopituitarism and the slow rate (5–15 years) of biochemical response are the

1	FIGURE 403-4 Features of acromegaly/gigantism. A 22-year-old man with gigantism due to excess growth hormone is shown to the left of his identical twin. The increased height and prognathism (A) and enlarged hand (B) and foot (C) of the affected twin are apparent. Their clinical features began to diverge at the age of approximately 13 years. (Reproduced from R Gagel, IE McCutcheon: N Engl J Med 324:524, 1999; with permission.) main disadvantages of radiotherapy. Irradiation is also relatively SURGERY ineffective in normalizing IGF-I levels. Stereotactic ablation of Transsphenoidal surgical resection by an experienced surgeon is the GH-secreting adenomas by gamma-knife radiotherapy is promising, preferred primary treatment for both microadenomas (remission but initial reports suggest that long-term results and side effects are rate ~70%) and macroadenomas (<50% in remission). Soft tissue similar to those observed with conventional radiation. Somatostatin swelling improves immediately

1	long-term results and side effects are rate ~70%) and macroadenomas (<50% in remission). Soft tissue similar to those observed with conventional radiation. Somatostatin swelling improves immediately after tumor resection. GH levels analogues may be required while awaiting the full benefits of return to normal within an hour, and IGF-I levels are normalized radiotherapy. Systemic co-morbid sequelae of acromegaly, includ-within 3–4 days. In ~10% of patients, acromegaly may recur several ing cardiovascular disease, diabetes, and arthritis, should be man-years after apparently successful surgery; hypopituitarism develops aged aggressively. Mandibular surgical repair may be indicated. in up to 15% of patients after surgery.

1	GH-Secreting Adenoma Surgery Debulking required for CNS pressure effects Somatostatin analogue Somatostatin analogue Assess likelihood of surgical cure Measure GH/IGF-I Measure GH/IGF-I Increase dose/frequency of somatostatin analogue; add GH receptor antagonist; or add dopamine agonist Likely Unlikely Monitor controlled controlled Measure GH/IGF-I • GH receptor antagonist • Radiation therapy• ReoperationMonitor controlled uncontrolled Monitor controlled uncontrolled elevated Measure GH/IGF-I FIGURE 403-5 Management of acromegaly. GH, growth hormone; CNS, central nervous system; IGF, insulin-like growth factor. (Adapted from S Melmed et al: J Clin Endocrinol Metab 94:1509–1517, 2009; © The Endocrine Society.)

1	Somatostatin analogues exert their therapeutic effects through SSTR2 and SSTR5 receptors, both of which are expressed by GH-secreting tumors. Octreotide acetate is an eight-amino-acid synthetic somatostatin analogue. In contrast to native somatostatin, the analogue is relatively resistant to plasma degradation. It has a 2-h serum half-life and possesses fortyfold greater potency than native somatostatin to suppress GH. Octreotide is administered by subcutaneous injection, beginning with 50 μg tid; the dose can be increased gradually up to 1500 μg/d. Fewer than 10% of patients do not respond to the analogue. Octreotide suppresses integrated GH levels and normalizes IGF-I levels in ~60% of treated patients.

1	The long-acting somatostatin depot formulations, octreotide and lanreotide, are the preferred medical treatment for patients with acromegaly. Sandostatin-LAR is a sustained-release, long-acting formulation of octreotide incorporated into microspheres that sustain drug levels for several weeks after intramuscular injection. GH suppression occurs for as long as 6 weeks after a 30-mg intramuscular injection; long-term monthly treatment sustains GH and IGF-I suppression and also reduces pituitary tumor size in ~50% of patients. Lanreotide autogel, a slow-release depot somatostatin preparation, is a cyclic somatostatin octapeptide analogue that suppresses GH and IGF-I hypersecretion after a 60-mg subcutaneous injection. Longterm (4–6 weeks) administration controls GH hypersecretion in about two-thirds of treated patients and improves patient compliance because of the long interval required between drug injections. Rapid relief of headache and soft tissue swelling occurs in ~75% of patients

1	of treated patients and improves patient compliance because of the long interval required between drug injections. Rapid relief of headache and soft tissue swelling occurs in ~75% of patients within days to weeks of somatostatin analogue initiation. Most patients report symptomatic improvement, including amelioration of headache, perspiration, obstructive apnea, and cardiac failure.

1	Side Effects Somatostatin analogues are well tolerated in most patients. Adverse effects are short-lived and mostly relate to drug-induced suppression of gastrointestinal motility and secretion. Transient nausea, abdominal discomfort, fat malabsorption, diarrhea, and flatulence occur in one-third of patients, and these symptoms usually remit within 2 weeks. Octreotide suppresses postprandial gallbladder contractility and delays gallbladder emptying; up to 30% of patients develop long-term echogenic sludge or asymptomatic cholesterol gallstones. Other side effects include mild glucose intolerance due to transient insulin suppression, asymptomatic bradycardia, hypothyroxinemia, and local injection site discomfort.

1	Pegvisomant antagonizes endogenous GH action by blocking peripheral GH binding to its receptor. Consequently, serum IGF-I levels are suppressed, reducing the deleterious effects of excess endogenous GH. Pegvisomant is administered by daily subcutaneous injection (10–20 mg) and normalizes IGF-I in ~70% of patients. GH levels, however, remain elevated as the drug does not target the pituitary adenoma. Side effects include reversible liver enzyme elevation, lipodystrophy, and injection site pain. Tumor size should be monitored by MRI. Combined treatment with monthly somatostatin analogues and weekly or biweekly pegvisomant injections has been used effectively in resistant patients.

1	Combined treatment with monthly somatostatin analogues and weekly or biweekly pegvisomant injections has been used effectively in resistant patients. Bromocriptine and cabergoline may modestly suppress GH secretion in some patients. Very high doses of bromocriptine (≥20 mg/d) or cabergoline (0.5 mg/d) are usually required to achieve modest GH therapeutic efficacy. Combined treatment with octreotide and cabergoline may induce additive biochemical control compared with either drug alone. External radiation therapy or high-energy stereotactic techniques are used as adjuvant therapy for acromegaly. An advantage of radiation is that patient compliance with long-term treatment is not required. Tumor mass is reduced, and GH levels are attenuated over time.

1	However, 50% of patients require at least 8 years for GH levels to 2271 be suppressed to <5 μg/L; this level of GH reduction is achieved in about 90% of patients after 18 years but represents suboptimal GH suppression. Patients may require interim medical therapy for several years before attaining maximal radiation benefits. Most patients also experience hypothalamic-pituitary damage, leading to gonadotropin, ACTH, and/or TSH deficiency within 10 years of therapy. In summary, surgery is the preferred primary treatment for GH-secreting microadenomas (Fig. 403-5). The high frequency of GH hypersecretion after macroadenoma resection usually necessitates adjuvant or primary medical therapy for these larger tumors. Patients unable to receive or respond to unimodal medical treatment may benefit from combined treatments, or can be offered radiation. (See also Chap. 406)

1	(See also Chap. 406) Etiology and Prevalence Pituitary corticotrope adenomas account for 70% of patients with endogenous causes of Cushing’s syndrome. However, it should be emphasized that iatrogenic hypercortisolism is the most common cause of cushingoid features. Ectopic tumor ACTH production, cortisol-producing adrenal adenomas, adrenal carcinoma, and adrenal hyperplasia account for the other causes; rarely, ectopic tumor CRH production is encountered.

1	ACTH-producing adenomas account for about 10–15% of all pituitary tumors. Because the clinical features of Cushing’s syndrome often lead to early diagnosis, most ACTH-producing pituitary tumors are relatively small microadenomas. However, macroadenomas also are seen and some ACTH-expressing adenomas are clinically silent. Cushing’s disease is 5–10 times more common in women than in men. These pituitary adenomas exhibit unrestrained ACTH secretion, with resultant hypercortisolemia. However, they retain partial suppressibility in the presence of high doses of administered glucocorticoids, providing the basis for dynamic testing to distinguish pituitary from nonpituitary causes of Cushing’s syndrome.

1	Presentation and Diagnosis The diagnosis of Cushing’s syndrome presents two great challenges: (1) to distinguish patients with pathologic cortisol excess from those with physiologic or other disturbances of cortisol production and (2) to determine the etiology of pathologic cortisol excess.

1	Typical features of chronic cortisol excess include thin skin, central obesity, hypertension, plethoric moon facies, purple striae and easy bruisability, glucose intolerance or diabetes mellitus, gonadal dysfunction, osteoporosis, proximal muscle weakness, signs of hyperandrogenism (acne, hirsutism), and psychological disturbances (depression, mania, and psychoses) (Table 403-7). Hematopoietic features of hypercortisolism include leukocytosis, lymphopenia, and eosinopenia. Immune suppression includes delayed hypersensitivity and infection propensity. These protean yet commonly encountered manifestations of hypercortisolism make it challenging to decide which patients mandate formal laboratory evaluation. Certain features make pathologic causes of hypercortisolism more likely; they include characteristic central redistribution of fat, thin skin with striae and bruising, and proximal muscle weakness. In children and young females, early osteoporosis may be particularly prominent. The

1	characteristic central redistribution of fat, thin skin with striae and bruising, and proximal muscle weakness. In children and young females, early osteoporosis may be particularly prominent. The primary cause of death is cardiovascular disease, but life-threatening infections and risk of suicide are also increased.

1	Rapid development of features of hypercortisolism associated with skin hyperpigmentation and severe myopathy suggests an ectopic tumor source of ACTH. Hypertension, hypokalemic alkalosis, glucose intolerance, and edema are also more pronounced in these patients. Serum potassium levels <3.3 mmol/L are evident in ~70% of patients with ectopic ACTH secretion but are seen in <10% of patients with pituitary-dependent Cushing’s syndrome. Laboratory Investigation The diagnosis of Cushing’s syndrome is based on laboratory documentation of endogenous hypercortisolism. Measurement of 24-h urine free cortisol (UFC) is a precise and cost-effective screening test. Alternatively, the failure to suppress plasma cortisol after an overnight 1-mg dexamethasone suppression test can Symptoms/Signs Frequency, % Source: Adapted from MA Magiokou et al, in ME Wierman (ed): Diseases of the Pituitary. Totowa, NJ, Humana, 1997.

1	be used to identify patients with hypercortisolism. As nadir levels of cortisol occur at night, elevated midnight serum or salivary samples of cortisol are suggestive of Cushing’s syndrome. Basal plasma ACTH levels often distinguish patients with ACTH-independent (adrenal or exogenous glucocorticoid) from those with ACTH-dependent (pituitary, ectopic ACTH) Cushing’s syndrome. Mean basal ACTH levels are about eightfold higher in patients with ectopic ACTH secretion than in those with pituitary ACTH-secreting adenomas. However, extensive overlap of ACTH levels in these two disorders precludes using ACTH measurements to make the distinction. Preferably, dynamic testing based on differential sensitivity to glucocorticoid feedback or ACTH stimulation in response to CRH or cortisol reduction is used to distinguish ectopic from pituitary sources of excess ACTH (Table 403-8). Very rarely, circulating CRH levels are elevated, reflecting ectopic tumor-derived secretion of CRH and often ACTH.

1	is used to distinguish ectopic from pituitary sources of excess ACTH (Table 403-8). Very rarely, circulating CRH levels are elevated, reflecting ectopic tumor-derived secretion of CRH and often ACTH. For further discussion of dynamic testing for Cushing’s syndrome, see Chap. 406.

1	Most ACTH-secreting pituitary tumors are <5 mm in diameter, and about half are undetectable by sensitive MRI. The high prevalence of incidental pituitary microadenomas diminishes the ability to distinguish ACTH-secreting pituitary tumors accurately from nonsecreting incidentalomas.

1	Inferior Petrosal Venous Sampling Because pituitary MRI with gadolinium enhancement is insufficiently sensitive to detect small (<2 mm) pituitary ACTH-secreting adenomas, bilateral inferior petrosal sinus ACTH sampling before and after CRH administration may be required to distinguish these lesions from ectopic ACTH-secreting tumors that may have similar clinical and biochemical characteristics. Simultaneous assessment of ACTH in each inferior petrosal vein and in the diagnosis of peripheral circulation provides a strategy for confirming and localizing pituitary ACTH production. Sampling is performed at baseline and 2, 5, and 10 min after intravenous bovine CRH (1 μg/kg) injection. An increased ratio (>2) of inferior petrosal:peripheral vein ACTH confirms pituitary Cushing’s syndrome. After CRH injection, peak petrosal:peripheral ACTH ratios ≥3 confirm the presence of a pituitary ACTH-secreting tumor. The sensitivity of this test is >95%, with very rare false-positive results.

1	After CRH injection, peak petrosal:peripheral ACTH ratios ≥3 confirm the presence of a pituitary ACTH-secreting tumor. The sensitivity of this test is >95%, with very rare false-positive results. False-negative results may be encountered in patients with aberrant venous drainage. Petrosal sinus catheterizations are technically difficult, and about 0.05% of patients develop neurovascular complications. The procedure should not be performed in patients with hypertension, in patients with known cerebrovascular disease, or in the presence of a well-visualized pituitary adenoma on MRI.

1	aACTH-independent causes of Cushing’s syndrome are diagnosed by suppressed ACTH levels and an adrenal mass in the setting of hypercortisolism. Iatrogenic Cushing’s syndrome is excluded by history. Abbreviations: ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; F, female; M, male. Selective transsphenoidal resection is the treatment of choice for Cushing’s disease (Fig. 403-6). The remission rate for this procedure is ~80% for microadenomas but <50% for macroadenomas. However, surgery is rarely successful when the adenoma is not visible on MRI. After successful tumor resection, most patients experience a postoperative period of symptomatic ACTH deficiency that may last up to 12 months. This usually requires low-dose cortisol replacement, as patients experience both steroid withdrawal symptoms and have a suppressed hypothalamic-pituitary-adrenal axis. Biochemical recurrence occurs in approximately 5% of patients in whom surgery was initially successful.

1	When initial surgery is unsuccessful, repeat surgery is sometimes indicated, particularly when a pituitary source for ACTH is well documented. In older patients, in whom issues of growth and fertility are less important, hemior total hypophysectomy may be necessary if a discrete pituitary adenoma is not recognized. Pituitary irradiation may be used after unsuccessful surgery, but it cures only about 15% of patients. Because the effects of radiation are slow and only partially effective in adults, steroidogenic inhibitors are used in combination with pituitary irradiation to block adrenal effects of persistently high ACTH levels.

1	Pasireotide (600 or 900 ug/day subcutaneously), a somatostatin analog with high affinity for SST5 > SST2 receptors, has been approved for treating patients with ACTH-secreting pituitary tumors when surgery is not an option or has been unsuccessful. In clinical trials, the drug lowered plasma ACTH levels, normalized 24-h urinary free cortisol levels in about 25% of patients, and resulted in up to 40% mean pituitary tumor shrinkage. Side effects include development of enlargement and increased pigmentation secondary to high ACTH 2273 levels. Prophylactic radiation therapy may be indicated to prevent the development of Nelson’s syndrome after adrenalectomy. Glucocorticoid replacement, if needed ?Irradiation Follow-up: Risk of Nelson’s syndrome FIGURE 403-6 Management of Cushing’s syndrome. ACTH, adrenocorticotropin hormone; MRI, magnetic resonance imaging. *, Not usually required.

1	Follow-up: Risk of Nelson’s syndrome FIGURE 403-6 Management of Cushing’s syndrome. ACTH, adrenocorticotropin hormone; MRI, magnetic resonance imaging. *, Not usually required. hyperglycemia and diabetes in about 70% of patients, likely due to suppressed pancreatic secretion of insulin and incretins. Because patients with hypercortisolism are insulin-resistant, hyperglycemia should be rigorously managed. Other side effects are similar to those encountered for somatostatin analogs and include transient abdominal discomfort, diarrhea, nausea, and gallstones (20% of patients). The drug requires consistent long-term administration. Ketoconazole, an imidazole derivative antimycotic agent, inhibits several P450 enzymes and effectively lowers cortisol in most patients with Cushing’s disease when administered twice daily (600–1200 mg/d). Elevated hepatic transaminases, gynecomastia, impotence, gastrointestinal upset, and edema are common side effects.

1	Mifepristone (300–1200 mg/d), a glucocorticoid receptor antagonist, blocks peripheral cortisol action and is approved to treat hyperglycemia in Cushing’s disease. Because the drug does not target the pituitary tumor, both ACTH and cortisol levels remain elevated, thus obviating a reliable circulating biomarker. Side effects are largely due to general antagonism of other steroid hormones and include hypokalemia, endometrial hyperplasia, hypoadrenalism, and hypertension.

1	Metyrapone (2–4 g/d) inhibits 11β-hydroxylase activity and normalizes plasma cortisol in up to 75% of patients. Side effects include nausea and vomiting, rash, and exacerbation of acne or hirsutism. Mitotane (o,p ‘-DDD; 3–6 g/d orally in four divided doses) suppresses cortisol hypersecretion by inhibiting 11β-hydroxylase and cholesterol side-chain cleavage enzymes and by destroying adrenocortical cells. Side effects of mitotane include gastrointestinal symptoms, dizziness, gynecomastia, hyperlipidemia, skin rash, and hepatic enzyme elevation. It also may lead to hypoaldosteronism. Other agents include aminoglutethimide (250 mg tid), trilostane (200–1000 mg/d), cyproheptadine (24 mg/d), and IV etomidate (0.3 mg/kg per hour). Glucocorticoid insufficiency is a potential side effect of agents used to block steroidogenesis.

1	The use of steroidogenic inhibitors has decreased the need for bilateral adrenalectomy. Surgical removal of both adrenal glands corrects hypercortisolism but may be associated with significant morbidity rates and necessitates permanent glucocorticoid and mineralocorticoid replacement. Adrenalectomy in the setting of residual corticotrope adenoma tissue predisposes to the development of Nelson’s syndrome, a disorder characterized by rapid pituitary tumor

1	NONFUNCTIONING AND GONADOTROPIN-PRODUCING PITUITARY ADENOMAS Etiology and Prevalence Nonfunctioning pituitary adenomas include those that secrete little or no pituitary hormones as well as tumors that produce too little hormone to result in recognizable clinical features. They are the most common type of pituitary adenoma and are usually macroadenomas at the time of diagnosis because clinical features are not apparent until tumor mass effects occur. Based on immunohistochemistry, most clinically nonfunctioning adenomas can be shown to originate from gonadotrope cells. These tumors typically produce small amounts of intact gonadotropins (usually FSH) as well as uncombined α, LH β, and FSH β subunits. Tumor secretion may lead to elevated α and FSH β subunits and, rarely, to increased LH β subunit levels. Some adenomas express α subunits without FSH or LH. TRH administration often induces an atypical increase of tumor-derived gonadotropins or subunits.

1	Presentation and Diagnosis Clinically nonfunctioning tumors often present with optic chiasm pressure and other symptoms of local expansion or may be incidentally discovered on an MRI performed for another indication (incidentaloma). Rarely, menstruwal disturbances or ovarian hyperstimulation occur in women with large tumors that produce FSH and LH. More commonly, adenoma compression of the pituitary stalk or surrounding pituitary tissue leads to attenuated LH and features of hypogonadism. PRL levels are usually slightly increased, also because of stalk compression. It is important to distinguish this circumstance from true prolactinomas, as nonfunctioning tumors do not shrink in response to treatment with dopamine agonists.

1	Laboratory Investigation The goal of laboratory testing in clinically nonfunctioning tumors is to classify the type of the tumor, identify hormonal markers of tumor activity, and detect possible hypopituitarism. Free α subunit levels may be elevated in 10–15% of patients with nonfunctioning tumors. In female patients, perior postmenopausal basal FSH concentrations are difficult to distinguish from tumor-derived FSH elevation. Premenopausal women have cycling FSH levels, also preventing clear-cut diagnostic distinction from tumor-derived FSH. In men, gonadotropin-secreting tumors may be diagnosed because of slightly increased gonadotropins (FSH > LH) in the setting of a pituitary mass. Testosterone levels are usually low despite the normal or increased LH level, perhaps reflecting reduced LH bioactivity or the loss of normal LH pulsatility. Because this pattern of hormone test results is also seen in primary gonadal failure and, to some extent, with aging (Chap. 411), the finding of

1	LH bioactivity or the loss of normal LH pulsatility. Because this pattern of hormone test results is also seen in primary gonadal failure and, to some extent, with aging (Chap. 411), the finding of increased gonadotropins alone is insufficient for the diagnosis of a gonadotropin-secreting tumor. In the majority of patients with gonadotrope adenomas, TRH administration stimulates LH β subunit secretion; this response is not seen in normal individuals. GnRH testing, however, is not helpful for making the diagnosis. For nonfunctioning and gonadotropinsecreting tumors, the diagnosis usually rests on immunohistochemical analyses of surgically resected tumor tissue, as the mass effects of these tumors usually necessitate resection.

1	Although acromegaly or Cushing’s syndrome usually presents with unique clinical features, clinically inapparent (silent) somatotrope or corticotrope adenomas may only be diagnosed by immunostaining of resected tumor tissue. If PRL levels are <100 μg/L in a patient harboring a pituitary mass, a nonfunctioning adenoma causing pituitary stalk compression should be considered. Asymptomatic small nonfunctioning microadenomas adenomas with no threat to vision may be followed with regular MRI and visual field testing without immediate intervention. However, for macroadenomas, transsphenoidal surgery is indicated to reduce tumor size and relieve mass effects (Fig. 403-7). Although it is not usually FIGURE 403-7 Management of a nonfunctioning pituitary mass. MRI, magnetic resonance imaging.

1	FIGURE 403-7 Management of a nonfunctioning pituitary mass. MRI, magnetic resonance imaging. possible to remove all adenoma tissue surgically, vision improves in 70% of patients with preoperative visual field defects. Preexisting hypopituitarism that results from tumor mass effects may improve or resolve completely. Beginning about 6 months postoperatively, MRI scans should be performed yearly to detect tumor regrowth. Within 5–6 years after successful surgical resection, ~15% of non-functioning tumors recur. When substantial tumor remains after transsphenoidal surgery, adjuvant radiotherapy may be indicated to prevent tumor regrowth. Radiotherapy may be deferred if no postoperative residual mass is evident. Nonfunctioning pituitary tumors respond poorly to dopamine agonist treatment and somatostatin analogues are largely ineffective for shrinking these tumors. The selective GnRH antagonist Nal-Glu GnRH suppresses FSH hypersecretion but has no effect on adenoma size.

1	TSH-producing macroadenomas are very rare but are often large and locally invasive when they occur. Patients usually present with thyroid goiter and hyperthyroidism, reflecting overproduction of TSH. Diagnosis is based on demonstrating elevated serum free T4 levels, inappropriately normal or high TSH secretion, and MRI evidence of a pituitary adenoma. Elevated uncombined α subunits are seen in many patients.

1	It is important to exclude other causes of inappropriate TSH secretion, such as resistance to thyroid hormone, an autosomal dominant disorder caused by mutations in the thyroid hormone β receptor (Chap. 405). The presence of a pituitary mass and elevated β subunit levels are suggestive of a TSH-secreting tumor. Dysalbuminemic hyperthyroxinemia syndromes, caused by mutations in serum thyroid hormone binding proteins, are also characterized by elevated thyroid hormone levels, but with normal rather than suppressed TSH levels. Moreover, free thyroid hormone levels are normal in these disorders, most of which are familial.

1	The initial therapeutic approach is to remove or debulk the tumor mass surgically, usually using a transsphenoidal approach. Total resection is not often achieved as most of these adenomas are large and locally invasive. Normal circulating thyroid hormone levels are achieved in about two-thirds of patients after surgery. Thyroid ablation or antithyroid drugs (methimazole and propylthiouracil) can be used to reduce thyroid hormone levels. Somatostatin analogue treatment effectively normalizes TSH and α subunit hypersecretion, shrinks the tumor mass in 50% of patients, and improves visual fields in 75% of patients; euthyroidism is restored in most patients. Because somatostatin analogues markedly suppress TSH, biochemical hypothyroidism often requires concomitant thyroid hormone replacement, which may also further control tumor growth. Disorders of the neurohypophysis Gary L. Robertson

1	Disorders of the neurohypophysis Gary L. Robertson The neurohypophysis, or posterior pituitary, is formed by axons that originate in large cell bodies in the supraoptic and paraventricular nuclei of the hypothalamus. It produces two hormones: (1) arginine vasopressin (AVP), also known as antidiuretic hormone, and (2) oxytocin. AVP acts on the renal tubules to reduce water loss by concentrating the urine. Oxytocin stimulates postpartum milk letdown in response to suckling. A deficiency of AVP secretion or action causes diabetes insipidus (DI), a syndrome characterized by the production of large amounts of dilute urine. Excessive or inappropriate AVP production impairs urinary water excretion and predisposes to hyponatremia if water intake is not reduced in parallel with urine output.

1	AVP is a nonapeptide composed of a six-member disulfide ring and a tripeptide tail (Fig. 404-1). It is synthesized via a polypeptide precursor that includes AVP, neurophysin, and copeptin, all encoded by a single gene on chromosome 20. After preliminary processing and folding, the precursor is packaged in neurosecretory vesicles, where it is transported down the axon; further processed to AVP, neurophysin, and copeptin; and stored in neurosecretory vesicles until released by exocytosis into peripheral blood. AVP secretion is regulated primarily by the “effective” osmotic pressure of body fluids. This control is mediated by specialized hypothalamic cells known as osmoreceptors, which are extremely sensitive FIGURE 404-1 Primary structures of arginine vasopressin (AVP), oxytocin, and desmopressin (DDAVP).

1	to small changes in the plasma concentration of sodium and its anions but normally are insensitive to other solutes such as urea and glucose. The osmoreceptors appear to include inhibitory as well as stimulatory components that function in concert to form a threshold, or set point, control system. Below this threshold, plasma AVP is suppressed to levels that permit the development of a maximum water diuresis. Above it, plasma AVP rises steeply in direct proportion to plasma osmolarity, quickly reaching levels sufficient to effect a maximum antidiuresis. The absolute levels of plasma osmolarity/sodium at which minimally and maximally effective levels of plasma AVP occur, vary appreciably from person to person, apparently due to genetic influences on the set and sensitivity of the system. However, the average threshold, or set point, for AVP release corresponds to a plasma osmolarity or sodium of about 280 mosmol/L or 135 meq/L, respectively; levels only 2–4% higher normally result in

1	However, the average threshold, or set point, for AVP release corresponds to a plasma osmolarity or sodium of about 280 mosmol/L or 135 meq/L, respectively; levels only 2–4% higher normally result in maximum antidiuresis.

1	Although it is relatively stable in a healthy adult, the set point of the osmoregulatory system can be lowered by pregnancy, the menstrual cycle, estrogen, and relatively large, acute reductions in blood pressure or volume. Those reductions are mediated largely by neuronal afferents that originate in transmural pressure receptors of the heart and large arteries and project via the vagus and glossopharyngeal nerves to the brainstem, from which postsynaptic projections ascend to the hypothalamus. These pathways maintain a tonic inhibitory tone that decreases when blood volume or pressure falls by >10–20%. This baroregulatory system is probably of minor importance in the physiology of AVP secretion because the hemodynamic changes required to affect it usually do not occur during normal activities. However, the baroregulatory system undoubtedly plays an important role in AVP secretion in patients with disorders that produce large, acute disturbances of hemodynamic function. However, the

1	However, the baroregulatory system undoubtedly plays an important role in AVP secretion in patients with disorders that produce large, acute disturbances of hemodynamic function. However, the baroregulatory system undoubtedly plays an important role in AVP secretion in patients with disorders that produce large, acute disturbances of hemodynamic function.

1	AVP secretion also can be stimulated by nausea, acute hypoglycemia, glucocorticoid deficiency, smoking, and, possibly, hyperangiotensinemia. The emetic stimuli are extremely potent since they typically elicit immediate, 50to 100-fold increases in plasma AVP even when the nausea is transient and is not associated with vomiting or other symptoms. They appear to act via the emetic center in the medulla and can be blocked completely by treatment with antiemetics such as fluphenazine. There is no evidence that pain or other noxious stresses have any effect on AVP unless they elicit a vasovagal reaction with its associated nausea and hypotension.

1	The most important, if not the only, physiologic action of AVP is to reduce water excretion by promoting concentration of urine. This antidiuretic effect is achieved by increasing the hydroosmotic permeability of cells that line the distal tubule and medullary collecting ducts of the kidney (Fig. 404-2). In the absence of AVP, these cells are impermeable to water and reabsorb little, if any, of the relatively large volume of dilute filtrate that enters from the proximal nephron. The lack of reabsorption results in the excretion of very large volumes (as much as 0.2 mL/kg per min) of maximally dilute urine (specific gravity and osmolarity ~1.000 and 50 mosmol/L, respectively), a condition known as water diuresis. In the presence of AVP, these cells become selectively permeable to water, allowing the water to diffuse back down the osmotic gradient created by the hypertonic renal medulla. As a result, the dilute fluid passing through the tubules is concentrated and the rate of urine flow

1	the water to diffuse back down the osmotic gradient created by the hypertonic renal medulla. As a result, the dilute fluid passing through the tubules is concentrated and the rate of urine flow decreases.

1	The magnitude of this effect varies in direct proportion to the plasma 2275 AVP concentration and the rate of solute excretion. At maximum levels of AVP and normal rates of solute excretion, it approximates a urine flow rate as low as 0.35 mL/min and a urine osmolarity as high as 1200 mosmol/L. This effect is reduced by a solute diuresis such as glucosuria in diabetes mellitus. Antidiuresis is mediated via binding to G protein– coupled V2 receptors on the serosal surface of the cell, activation of adenyl cyclase, and insertion into the luminal surface of water channels composed of a protein known as aquaporin 2 (AQP2). The V2 receptors and aquaporin 2 are encoded by genes on chromosomes Xq28 and 12q13, respectively.

1	At high concentrations, AVP also causes contraction of smooth muscle in blood vessels in the skin and gastrointestinal tract, induces glycogenolysis in the liver, and potentiates adrenocorticotropic hormone (ACTH) release by corticotropin-releasing factor. These effects are mediated by V1a or V1b receptors that are coupled to phospholipase C. Their role, if any, in human physiology/pathophysiology is uncertain. AVP distributes rapidly into a space roughly equal to the extracellular fluid volume. It is cleared irreversibly with a half-life (t 1/2) of 10–30 min. Most AVP clearance is due to degradation in the liver and kidneys. During pregnancy, the metabolic clearance of AVP is increased three-to fourfold due to placental production of an N-terminal peptidase.

1	Because AVP cannot reduce water loss below a certain minimum level obligated by urinary solute load and evaporation from skin and lungs, a mechanism for ensuring adequate intake is essential for preventing dehydration. This vital function is performed by the thirst mechanism. Like AVP, thirst is regulated primarily by an osmostat that is situated in the anteromedial hypothalamus and is able to detect very small changes in the plasma concentration of sodium and its anions. The thirst osmostat appears to be “set” about 3% higher than the AVP osmostat. This arrangement ensures that thirst, polydipsia, and dilution of body fluids do not occur until plasma osmolarity/sodium starts to exceed the defensive capacity of the antidiuretic mechanism.

1	Oxytocin is also a nonapeptide that differs from AVP only at positions 3 and 8 (Fig. 404-1). However, it has relatively little antidiuretic effect and seems to act mainly on mammary ducts to facilitate milk letdown during nursing. It also may help initiate or facilitate labor by stimulating contraction of uterine smooth muscle, but it is not clear if this action is physiologic or necessary for normal delivery.

1	DIABETES INSIPIDUS Clinical Characteristics A decrease of 75% or more in the secretion or action of AVP usually results in DI, a syndrome characterized by the production of abnormally large volumes of dilute urine. The 24-h urine volume exceeds 50 mL/kg body weight, and the osmolarity is less than 300 mosmol/L. The polyuria produces symptoms of urinary frequency, enuresis, and/or nocturia, which may disturb sleep and cause mild daytime fatigue or somnolence. It also results in a slight rise in plasma osmolarity that stimulates thirst and a commensurate increase in fluid intake (polydipsia). Overt clinical signs of dehydration are uncommon unless thirst and/or the compensatory increase of fluid intake are also impaired.

1	Etiology A primary deficiency of AVP secretion usually results from agenesis or irreversible destruction of the neurohypophysis. It is referred to variously as neurohypophyseal DI, neurogenic DI, pituitary DI, cranial DI, or central DI. It can be caused by a variety of congenital, acquired, or genetic disorders, but in about one-half of all adult patients, it is idiopathic (Table 404-1). Pituitary DI caused by surgery in or around the neurohypophysis usually appears within 24 h. After a few days, it may transition to a 2to 3-week period of inappropriate antidiuresis, after which the DI may or may not recur permanently. Disorders of the Neurohypophysis

1	FIGURE 404-2 Antidiuretic effect of arginine vasopressin (AVP) in the regulation of urine volume. In a typical 70-kg adult, the kidney filters ~180 L/d of plasma. Of this, ~144 L (80%) is reabsorbed isosmotically in the proximal tubule and another 8 L (4–5%) is reabsorbed without solute in the descending limb of Henle’s loop. The remainder is diluted to an osmolarity of ~60 mmol/kg by selective reabsorption of sodium and chloride in the ascending limb. In the absence of AVP, the urine issuing from the loop passes largely unmodified through the distal tubules and collecting ducts, resulting in a maximum water diuresis. In the presence of AVP, solute-free water is reabsorbed osmotically through the principal cells of the collecting ducts, resulting in the excretion of a much smaller volume of concentrated urine. This antidiuretic effect is mediated via a G protein–coupled V2 receptor that increases intracellular cyclic AMP, thereby inducing translocation of aquaporin 2 (AQP 2) water

1	of concentrated urine. This antidiuretic effect is mediated via a G protein–coupled V2 receptor that increases intracellular cyclic AMP, thereby inducing translocation of aquaporin 2 (AQP 2) water channels into the apical membrane. The resultant increase in permeability permits an influx of water that diffuses out of the cell through AQP 3 and AQP 4 water channels on the basal-lateral surface. The net rate of flux across the cell is determined by the number of AQP 2 water channels in the apical membrane and the strength of the osmotic gradient between tubular fluid and the renal medulla. Tight junctions on the lateral surface of the cells serve to prevent unregulated water flow.

1	Five genetic forms of pituitary DI are now known. By far, the most common is transmitted in an autosomal dominant mode and is caused by diverse mutations in the coding region of one allele of the AVP– neurophysin II (or AVP-NPII) gene. All the mutations alter one or more amino acids known to be critical for correct processing and/or folding of the prohormone, thus interfering with its trafficking through the endoplasmic reticulum. The misfolded mutant precursor accumulates and interferes with production of AVP by the normal allele, eventually destroying the magnocellular neurons in which it is produced. The AVP deficiency and DI are usually not present at birth but develop gradually over a period of several months to years, progressing from partial to severe at different rates depending on the mutation. Once established, the deficiency of AVP is permanent, but for unknown reasons, the DI occasionally improves or remits spontaneously in late middle age. The parvocellular neurons that

1	on the mutation. Once established, the deficiency of AVP is permanent, but for unknown reasons, the DI occasionally improves or remits spontaneously in late middle age. The parvocellular neurons that make AVP and the magnocellular neurons that make oxytocin appear to be unaffected. There are also rare autosomal recessive forms of pituitary DI. One is due to an inactivating mutation in the AVP portion of the gene; another is due to a large deletion involving the majority of the AVP gene and regulatory sequences in the intergenic region. A third form is caused by mutations of the WFS 1 gene responsible for Wolfram’s syndrome (DI, diabetes mellitus, optic atrophy, and neural deafness [DIDMOAD]). An X-linked recessive form linked to a region on Xq28 has also been described.

1	A primary deficiency of plasma AVP also can result from increased metabolism by an N-terminal aminopeptidase produced by the placenta. It is referred to as gestational DI because the signs and symptoms manifest during pregnancy and usually remit several weeks after delivery.

1	Secondary deficiencies of AVP secretion result from inhibition by excessive intake of fluids. They are referred to as primary polydipsia and can be divided into three subcategories. One of them, dipsogenic DI, is characterized by inappropriate thirst caused by a reduction in the set of the osmoregulatory mechanism. It sometimes occurs in association with multifocal diseases of the brain such as neurosarcoid, tuberculous meningitis, and multiple sclerosis but is often idiopathic. The second subtype, psychogenic polydipsia, is not associated with thirst, and the polydipsia seems to be a feature of psychosis or obsessive compulsive disorder. The third subtype, iatrogenic polydipsia, results from recommendations to increase fluid intake for its presumed health benefits.

1	Primary deficiencies in the antidiuretic action of AVP result in nephrogenic DI. The causes can be genetic, acquired, or drug induced (Table 404-1). The most common genetic form is transmitted in a semirecessive X-linked manner. It is caused by mutations in the coding region of the V2 receptor gene that impair trafficking and/or ligand binding of the mutant receptor. There are also autosomal recessive or dominant forms of nephrogenic DI. They are caused by AQP2 gene mutations that result in complete or partial defects in trafficking and function of the water channels that mediate antidiuresis in the distal and collecting tubules of the kidney. Secondary deficiencies in the antidiuretic response to AVP result from polyuria per se. They are caused by washout of the medullary concentration gradient and/or suppression of aquaporin function. They usually resolve 24–48 h after the polyuria is corrected but can complicate interpretation of some acute tests used for differential diagnosis.

1	Pathophysiology In pituitary, gestational, or nephrogenic DI, the polyuria results in a small (1–2%) decrease in body water and a commensurate increase in plasma osmolarity and sodium that stimulates thirst and a compensatory increase in water intake. As a result, hypernatremia and other overt physical or laboratory signs of dehydration do not develop unless the patient also has a defect in thirst or fails to increase fluid intake for some other reason. CAuSES of DiAbETES inSiPiDuS Metastatic (lung, breast) Hematologic (lymphoma, leukemia) Inflammatory Lymphocytic infundibuloneurohypophysitis Granulomatosis with polyangiitis (Wegener’s) Lupus erythematosus Scleroderma Congenital malformations Septo-optic dysplasia Midline craniofacial defects Holoprosencephaly Hypogenesis, ectopia of pituitary

1	Metabolic Hypercalcemia, hypercalciuria Hypokalemia causes excessive intake of fluids and an increase in 2277 body water that reduces plasma osmolarity/sodium, AVP secretion, and urinary concentration. Dilution of the urine, in turn, results in a compensatory increase in urinary free-water excretion that usually offsets the increase in intake and stabilizes plasma osmolarity/sodium at a level only 1–2% below basal. Thus, hyponatremia or clinically appreciable overhydration is uncommon unless the polydipsia is very severe or the compensatory water diuresis is impaired by a drug or disease that stimulates or mimics the antiduretic effect of endogenous AVP. A rise in plasma osmolarity and sodium produced by fluid deprivation or hypertonic saline infusion increases plasma AVP normally. However, the resultant increase in urine concentration is often subnormal because polyuria per se temporarily reduces the capacity of the kidney to concentrate the urine. Thus, the maximum level of urine

1	the resultant increase in urine concentration is often subnormal because polyuria per se temporarily reduces the capacity of the kidney to concentrate the urine. Thus, the maximum level of urine osmolarity achieved during fluid deprivation is often indistinguishable from that in patients with partial pituitary or partial nephrogenic DI.

1	Differential Diagnosis When symptoms of urinary frequency, enuresis, nocturia, and/or persistent thirst are present in the absence of glucosuria, the possibility of DI should be evaluated by collecting a 24-h urine on ad libitum fluid intake. If the volume exceeds 50 mL/kg per day (3500 mL in a 70-kg male) and the osmolarity is below 300 mosmol/L, DI is confirmed and the patient should be evaluated further to determine the type in order to select the appropriate therapy.

1	The type of DI can sometimes be inferred from the clinical setting or medical history. Often, however, such information is lacking, ambiguous, or misleading, and other approaches to differential diagnosis are needed. If basal plasma osmolarity and sodium are within normal limits, the traditional approach is to determine the effect of fluid deprivation and injection of antidiuretic hormone on urine osmolarity. This approach suffices for differential diagnosis if fluid deprivation raises plasma osmolarity and sodium above the normal range without inducing concentration of the urine. In that event, primary polydipsia and partial defects in AVP secretion and action are excluded, and the effect on urine osmolarity of injecting 2 μg of the AVP analogue, desmopressin, indicates whether the patient has severe pituitary DI or severe nephrogenic DI. However, this approach is of little or no diagnostic value if fluid deprivation results in concentration of the urine because the increases in

1	has severe pituitary DI or severe nephrogenic DI. However, this approach is of little or no diagnostic value if fluid deprivation results in concentration of the urine because the increases in urine osmolarity achieved both before and after the injection of desmopressin are similar in patients with partial pituitary DI,

1	Disorders of the Neurohypophysis

1	In pituitary and nephrogenic DI, the severity of the defect in AVP partial nephrogenic DI, and primary polydipsia. These disorders can secretion or action varies significantly from patient to patient. In some, the be differentiated by measuring plasma AVP during fluid deprivation defect is so severe that it cannot be overcome by even an intense stimulus and relating it to the concurrent level of plasma and urine osmolarity such as nausea or severe dehydration. In others, the defect in AVP secre-(Fig. 404-3). However, this approach does not always differentiate tion or action is incomplete, and a modest stimulus such as a few hours clearly between partial pituitary DI and primary polydipsia unless of fluid deprivation, smoking, or a vasovagal reaction can raise urine the measurement is made when plasma osmolarity and sodium are at osmolarity as high as 800 mosmol/L. However, even when the defects are or above the normal range. This level is difficult to achieve by fluid partial, the

1	when plasma osmolarity and sodium are at osmolarity as high as 800 mosmol/L. However, even when the defects are or above the normal range. This level is difficult to achieve by fluid partial, the relation of urine osmolarity to plasma AVP in patients with deprivation alone once urinary concentration occurs. Therefore it nephrogenic DI (Fig. 404-3A) or of plasma AVP to plasma osmolarity is usually necessary to give a short infusion of 3% saline condition and sodium in patients with pituitary DI (Fig. 404-3B) is subnormal. (0.1 mL/kg body weight per minute for 60 to 90 minutes) and repeat

1	In primary polydipsia, the pathogenesis of the polydipsia and the measurement of plasma AVP. polyuria is the reverse of that in pituitary, nephrogenic, and gesta-A simpler but equally reliable way to differentiate between pituitary tional DI. In primary polydipsia, an abnormality in cognition or thirst DI, nephrogenic DI, and primary polydipsia is to measure basal plasma Urine osmolarity, mosmol/L 0.1 0.5 1 3 B60 40 20 15 10 5 0 Plasma vasopressin, pg/mL Plasma vasopressin, pg/mL Plasma osmolarity, mosmol/L FIGURE 404-3 Relationship of plasma AVP to urine osmolarity (A) and plasma osmolarity (B) before and during fluid deprivation–hypertonic saline infusion test in patients who are normal or have primary polydipsia (blue zones), pituitary diabetes insipidus (green zones), or nephrogenic diabetes insipidus (pink zones).

1	AVP to determine if a brain magnetic resonance imaging (MRI) is needed and sufficient for diagnosis (Fig. 404-4). If plasma AVP on ad libitum fluid intake is normal or elevated (>1 pg/mL) when measured by a sensitive and specific assay, both primary polydipsia and pituitary DI are excluded and the diagnosis of nephrogenic DI can be confirmed, if desired, by a 1to 2-day outpatient trial of desmopressin therapy. If, however, basal plasma AVP is low or undetectable (<1 pg/ mL), nephrogenic DI is very unlikely and MRI of the brain can be used to differentiate pituitary DI from primary polydipsia. In most healthy adults and children, the posterior pituitary emits a hyperintense signal visible in T1-weighted midsagittal images. This “bright spot” is almost always present in patients with primary polydipsia but is always absent or abnormally small in patients with pituitary DI, even if their AVP deficiency is partial. The MRI is also useful in searching for pathology responsible for

1	primary polydipsia but is always absent or abnormally small in patients with pituitary DI, even if their AVP deficiency is partial. The MRI is also useful in searching for pathology responsible for pituitary DI or the dipsogenic form of primary polydipsia (Fig. 404-2). The principal caveat is that MRI is not reliable for differential diagnosis of DI in patients with empty sella because they typically lack a bright spot even when their AVP secretion and action are normal. MRI also cannot be used to differentiate pituitary from nephrogenic DI because many patients with nephrogenic DI also lack a posterior pituitary bright spot, probably because they have an abnormally high rate of AVP secretion and turnover.

1	Brain MRI Urinary frequency, nocturia, enuresis 24-h urine volume and osmolarity on unrestrictedfluid intake Volume >40 mL/kg Osmolarity <300 mosm/L Basal plasma AVP >1 pg/mL <1 pg/mL Pituitary bright spot Present Absent Anatomy Pathology? GU evaluation Volume <40 mL/kg Osmolarity >300 mosm/L If MRI and/or AVP assays with the requisite sensitivity and specificity are unavailable and a fluid deprivation test is impractical or undesirable, a third way to differentiate between pituitary DI, nephrogenic DI, and primary polydipsia is a trial of desmopressin therapy. Such a trial should be conducted with very close monitoring of serum sodium as well as urine output, preferably in hospital, because desmopressin will produce hyponatremia in 8–24 h if the patient has primary polydipsia.

1	The signs and symptoms of uncomplicated pituitary DI can be eliminated by treatment with desmopressin (DDAVP), a synthetic analogue of AVP (Fig. 404-1). DDAVP acts selectively at V2 receptors to increase urine concentration and decrease urine flow in a dose-dependent manner. It is also more resistant to degradation than is AVP and has a threeto fourfold longer duration of action. DDAVP can be given by IV or SC injection, nasal inhalation, or orally by means of a tablet of melt. The doses required to control pituitary DI completely vary widely, depending on the patient and the route of administration. However, among adults, they usually range from 1–2 μg qd or bid by injection, 10–20 μg bid or tid by nasal spray, or 100–400 μg bid or tid orally. The onset of antidiuresis is rapid, ranging from as little as 15 min after injection to 60 min after oral administration. When given in a dose that normalizes 24-h urinary osmolarity (400–800 mosmol/L) and volume (15–30 mL/kg body weight),

1	from as little as 15 min after injection to 60 min after oral administration. When given in a dose that normalizes 24-h urinary osmolarity (400–800 mosmol/L) and volume (15–30 mL/kg body weight), DDAVP produces a slight (1–3%) increase in total body water and a decrease in plasma osmolarity/sodium that rapidly eliminates thirst and polydipsia (Fig. 404-5). Consequently, water balance is maintained within the normal range. Hyponatremia does not develop unless urine volume is reduced too far (to less than 10 mL/kg per day) or fluid intake is excessive due to an associated

1	FIGURE 404-4 Simplified approach to the differential diagnosis of diabetes insipidus. When symptoms suggest diabetes insipidus (DI), the syndrome should be differentiated from a genitourinary (GU) abnormality by measuring the 24-h urine volume and osmolarity on unrestricted fluid intake. If DI is confirmed, basal plasma arginine vasopressin (AVP) should be measured on unrestricted fluid intake. If AVP is normal or elevated (>1 pg/mL), the patient probably has nephrogenic DI. However, if plasma AVP is low or undetectable, the patient has either pituitary DI or primary polydipsia. In that case, magnetic resonance imaging (MRI) of the brain can be performed to differentiate between these two conditions by determining whether or not the normal posterior pituitary bright spot is visible on T1-weighted midsagittal images. In addition, the MRI anatomy of the pituitary hypothalamic area can be examined to look for evidence of pathology that sometimes causes pituitary DI or the dipsogenic form

1	midsagittal images. In addition, the MRI anatomy of the pituitary hypothalamic area can be examined to look for evidence of pathology that sometimes causes pituitary DI or the dipsogenic form of primary polydipsia. MRI is not reliable for differential diagnosis unless nephrogenic DI has been excluded because the bright spot is also absent, small, or faint in this condition.

1	of hypovolemia such as tachycardia, postural hypotension, azotemia, 2279 hyperuricemia, and hypokalemia due to secondary hyperaldosteronism. Muscle weakness, pain, rhabdomyolysis, hyperglycemia, hyperlipidemia, and acute renal failure may also occur. Obtundation or coma may be present but are often absent. Despite inappropriately low levels of plasma AVP, DI usually is not evident at presentation but may develop during rehydration as blood volume, blood pressure, and plasma osmolarity/ sodium return toward normal, further reducing plasma AVP. Etiology Hypodipsia is usually due to hypogenesis or destruction of the osmoreceptors in the anterior hypothalamus that regulate thirst. These defects can result from various congenital malformations of midline brain structures or may be acquired due to diseases such as occlusions of the anterior communicating artery, primary or Days of treatment

1	FIGURE 404-5 Effect of desmopressin therapy on fluid intake (blue bars), urine output (orange bars), and plasma osmolarity (red line) in a patient with uncomplicated pituitary diabetes insipidus. Note metastatic tumors in the hypothalamus, head trauma, surgery, granulomatous diseases such as sarcoidosis and histiocytosis, AIDS, and cytomegalovirus encephalitis. Because of their proximity, the osmoreceptors that regulate AVP secretion also are usually impaired. Thus, AVP secretion responds poorly or not at all to hyperosmotic stimulation (Fig. 404-6) but, in most cases, increases normally to nonosmotic that treatment rapidly reduces fluid intake and urine output to normal, with only a slight increase in body water as evidenced by the 20 slight decrease in plasma osmolarity. 18 abnormality in thirst or cognition. Fortunately, thirst abnormalities are rare, and if the patient is taught to drink only when truly thirsty, DDAVP can be given safely in doses sufficient to normalize urine

1	in thirst or cognition. Fortunately, thirst abnormalities are rare, and if the patient is taught to drink only when truly thirsty, DDAVP can be given safely in doses sufficient to normalize urine output (~15– 30 mL/kg per day) without the need for allowing intermittent escape to prevent water intoxication.

1	Primary polydipsia cannot be treated safely with DDAVP or any other antidiuretic drug because eliminating the polyuria does not eliminate the urge to drink. Therefore, it invariably produces hypona tremia and/or other signs of water intoxication, usually within 8–24 Plasma vasopressin, pg/mL h if urine output is normalized completely. There is no consistently 2 effective way to correct dipsogenic or psychogenic polydipsia, but the iatrogenic form may respond to patient education. To minimize the 0 risk of water intoxication, all patients should be warned about the use Disorders of the Neurohypophysis of other drugs such as thiazide diuretics or carbamazepine (Tegretol) that can impair urinary free-water excretion directly or indirectly.

1	Disorders of the Neurohypophysis of other drugs such as thiazide diuretics or carbamazepine (Tegretol) that can impair urinary free-water excretion directly or indirectly. The polyuria and polydipsia of nephrogenic DI are not affected by treatment with standard doses of DDAVP. If resistance is partial, it may be overcome by tenfold higher doses, but this treatment is too expensive and inconvenient for long-term use. However, treatment with conventional doses of a thiazide diuretic and/or amiloride in conjunction with a low-sodium diet and a prostaglandin synthesis inhibitor (e.g., indomethacin) usually reduces the polyuria and polydipsia by 30–70% and may eliminate them completely in some patients. Side effects such as hypokalemia and gastric irritation can be minimized by the use of amiloride or potassium supplements and by taking medications with meals.

1	An increase in plasma osmolarity/sodium above the normal range (hypertonic hypernatremia) can be caused by either a decrease in total body water or an increase in total body sodium. The former results from a failure to drink enough to replace normal or increased urinary and insensible water loss. The deficient intake can be due either to water deprivation or a lack of thirst (hypodipsia). The most common cause of an increase in total body sodium is primary hyperaldosteronism (Chap. 406). Rarely, it can also result from ingestion of hypertonic saline in the form of sea water or incorrectly prepared infant formula. However, even in these forms of hypernatremia, inadequate intake of water also contributes. This chapter focuses on hypodipsic hypernatremia, the form of hypernatremia due to a primary defect in the thirst mechanism.

1	Clinical Characteristics Hypodipsic hypernatremia is a syndrome characterized by chronic or recurrent hypertonic dehydration. The hypernatremia varies widely in severity and usually is associated with signs adipsic hypernatremia (AH) and the syndrome of inappropriate antidiuresis (SIAD). Each line depicts schematically the relationship of plasma arginine vasopressin (AVP) to plasma osmolarity during water loading and/or infusion of 3% saline in a patient with either AH (open symbols) or SIAD (closed symbols). The shaded area indicates the normal range of the relationship. The horizontal broken line indicates the plasma AVP level below which the hormone is undetectable and urinary concentration usually does not occur. Lines P and T represent patients with a selective deficiency in the osmoregulation of thirst and AVP that is either partial ( ) or total ( ). In the latter, plasma AVP does not change in response to increases or decreases in plasma osmolarity but remains within a range

1	of thirst and AVP that is either partial ( ) or total ( ). In the latter, plasma AVP does not change in response to increases or decreases in plasma osmolarity but remains within a range sufficient to concentrate the urine even if overhydration produces hypotonic hyponatremia. In contrast, if the osmoregulatory deficiency is partial ( ), rehydration of the patient suppresses plasma AVP to levels that result in urinary dilution and polyuria before plasma osmolarity and sodium are reduced to normal. Lines a –d represent different defects in the osmoregulation of plasma AVP observed in patients with SIADH or SIAD. In a ( ), plasma AVP is markedly elevated and fluctuates widely without relation to changes in plasma osmolarity, indicating complete loss of osmoregulation. In b ( ), plasma AVP remains fixed at a slightly elevated level until plasma osmolarity reaches the normal range, at which point it begins to rise appropriately, indicating a selective defect in the inhibitory component of

1	fixed at a slightly elevated level until plasma osmolarity reaches the normal range, at which point it begins to rise appropriately, indicating a selective defect in the inhibitory component of the osmoregulatory mechanism. In c ( ), plasma AVP rises in close correlation with plasma osmolarity before the latter reaches the normal range, indicating downward resetting of the osmostat. In d ( ), plasma AVP appears to be osmoregulated normally, suggesting that the inappropriate antidiuresis is caused by some other abnormality.

1	2280 stimuli such as nausea or large reductions in blood volume or blood pressure, indicating that the neurohypophysis is intact.

1	Pathophysiology Hypodipsia results in a failure to drink enough water to replenish obligatory renal and extrarenal losses. Consequently, plasma osmolarity and sodium rise often to extremely high levels before the disorder is recognized. In most cases, urinary loss of water contributes little, if any, to the dehydration because AVP continues to be secreted in the small amounts necessary to concentrate the urine. In some patients this appears to be due to hypovolemic stimulation and/or incomplete destruction of AVP osmoreceptors because plasma AVP declines and DI develops during rehydration (Fig. 404-6). In others, however, plasma AVP does not decline during rehydration even if they are overhydrated. Consequently, they develop a hyponatremic syndrome indistinguishable from inappropriate antidiuresis. This suggests that the AVP osmoreceptors normally provide inhibitory and stimulatory input to the neurohypophysis and the patients can no longer osmotically stimulate or suppress tonic

1	antidiuresis. This suggests that the AVP osmoreceptors normally provide inhibitory and stimulatory input to the neurohypophysis and the patients can no longer osmotically stimulate or suppress tonic secretion of the hormone because both inputs have been totally eliminated by the same pathology that destroyed the osmoregulation of thirst. In a few patients, the neurohypophysis is also destroyed, resulting in a combination of chronic pituitary DI and hypodipsia that is particularly difficult to manage.

1	Differential Diagnosis Hypodipsic hypernatremia usually can be distinguished from other causes of inadequate fluid intake (e.g., coma, paralysis, restraints, absence of fresh water) by the clinical history and setting. Previous episodes and/or denial of thirst and failure to drink spontaneously when the patient is conscious, unrestrained, and hypernatremic are virtually diagnostic. The hypernatremia caused by excessive retention or intake of sodium can be distinguished by the presence of thirst as well as the physical and laboratory signs of hypervolemia rather than hypovolemia.

1	Hypodipsic hypernatremia should be treated by administering water orally if the patient is alert and cooperative or by infusing hypotonic fluids (0.45% saline or 5% dextrose and water) if the patient is not. The amount of free water in liters required to correct the deficit (ΔFW) can be estimated from body weight in kg (BW) and the serum sodium concentration in mmol/L (SNa) by the formula ΔFW = 0.5BW × ([S – 140]/140). If serum glucose (S ) is elevated, the mea- sured S should be corrected (S *) by the formula S *= S + ([S – 90]/36).

1	This amount plus an allowance for continuing insensible and urinary losses should be given over a 24to 48-h period. Close monitoring of serum sodium as well as fluid intake and urinary output is essential because, depending on the extent of osmoreceptor deficiency, some patients will develop AVP-deficient DI, requiring DDAVP therapy to complete rehydration; others will develop hyponatremia and a syndrome of inappropriate antidiuresis (SIAD)-like picture if overhydrated. If hyperglycemia and/or hypokalemia are present, insulin and/or potassium supplements should be given with the expectation that both can be discontinued soon after rehydration is complete. Plasma urea/creatinine should be monitored closely for signs of acute renal failure caused by rhabdomyolysis, hypovolemia, and hypotension.

1	Once the patient has been rehydrated, an MRI of the brain and tests of anterior pituitary function should be performed to look for the cause and collateral defects in other hypothalamic functions. A long-term management plan to prevent or minimize recurrence of the fluid and electrolyte imbalance also should be developed. This should include a practical method to regulate fluid intake in accordance with variations in water balance as indicated by changes in body weight or serum sodium determined by home monitoring analyzers. Prescribing a constant fluid intake is ineffective and potentially dangerous because it does not take into account the large, uncontrolled variations in insensible loss that inevitably result from changes in ambient temperature and physical activity.

1	A decrease in plasma osmolarity/sodium below the normal range (hypotonic hyponatremia) can be due to any of three different types of salt and water imbalance: (1) an increase in total body water that exceeds the increase in total body sodium (hypervolemic hyponatremia); (2) a decrease in body sodium greater than the decrease in body water (hypovolemic hyponatremia); or (3) an increase in body water with little or no change in body sodium (euvolemic hyponatremia) (Chap. 63). All three forms are associated with a failure to fully dilute the urine and mount a water diuresis in the face of hypotonic hyponatremia. The hypervolemic form typically occurs in disorders like severe congestive heart failure or cirrhosis. The hypovolemic form typically occurs in disorders such as severe diarrhea, diuretic abuse, or mineralocorticoid deficiency. Euvolemic hyponatremia, however, is due mainly to expansion of total body water caused by excessive intake in the face of a defect in urinary dilution.

1	abuse, or mineralocorticoid deficiency. Euvolemic hyponatremia, however, is due mainly to expansion of total body water caused by excessive intake in the face of a defect in urinary dilution. The impaired dilution is usually caused by a defect in the osmotic suppression of AVP that can have either of two causes. One is a nonhemodynamic stimulus such as nausea or a cortisol deficiency, which can be corrected quickly by treatment with antiemetics or cortisol. The other is a primary defect in osmoregulation caused by another disorder such as malignancy, stroke, or pneumonia that cannot be easily or quickly corrected. The latter is commonly known as the syndrome of inappropriate antidiuretic hormone (SIADH). Much less often, euvolemic hyponatremia can also result from AVP-independent activation of renal V2 receptors, a variant known as nephrogenic inappropriate antidiuresis or NSIAD. Both of the latter will be discussed in this chapter.

1	Clinical Characteristics Antidiuresis of any cause decreases the volume and increases the concentration of urine. If not accompanied by a commensurate reduction in fluid intake or an increase in insensible loss, the reduction in urine output results in excess water retention which expands and dilutes body fluids. If the hyponatremia develops gradually or has been present for more than a few days, it may be largely asymptomatic. However, if it develops acutely, it is usually accompanied by symptoms and signs of water intoxication that may include mild headache, confusion, anorexia, nausea, vomiting, coma, and convulsions. Severe acute hyponatremia may be lethal. Other clinical signs and symptoms vary greatly, depending on the type of hyponatremia. The hypervolemic form is characterized by generalized edema and other signs of marked volume expansion. The opposite is evident in the hypovolemic form. However, overt signs of volume expansion or contraction are absent in SIADH, SIAD, and

1	generalized edema and other signs of marked volume expansion. The opposite is evident in the hypovolemic form. However, overt signs of volume expansion or contraction are absent in SIADH, SIAD, and other forms of euvolemic hyponatremia.

1	Etiology In SIADH, the inappropriate secretion of AVP can have many different causes. They include ectopic production of AVP by lung cancer or other neoplasms; eutopic release induced by various diseases or drugs; and exogenous administration of AVP, DDAVP, or large doses of oxytocin (Table 404-2). The ectopic forms result from abnormal expression of the AVP-NPII gene by primary or metastatic malignancies. The eutopic forms occur most often in patients with acute infections or strokes but have also been associated with many other neurologic diseases and injuries. The mechanisms by which these diseases interfere with osmotic suppression of AVP are not known. The defect in osmoregulation can take any of four distinct forms (Fig. 404-6). In one of the most common (reset osmostat), AVP secretion remains fully responsive to changes in plasma osmolarity/sodium, but the threshold, or set point, of the osmoregulatory system is abnormally low. These patients differ from those with the other

1	remains fully responsive to changes in plasma osmolarity/sodium, but the threshold, or set point, of the osmoregulatory system is abnormally low. These patients differ from those with the other types of SIADH in that they are able to maximally suppress plasma AVP and dilute their urine if their fluid intake is high enough to reduce their plasma osmolarity and/or sodium to the new set point. In most patients, SIADH is self-limited and remits spontaneously within 2–3 weeks, but about 10% of cases are chronic. Another, smaller subgroup (~10% of the total) has inappropriate antidiuresis without a demonstrable defect in the osmoregulation of plasma AVP (Fig. 404-6). In some of them, all young boys, the inappropriate antidiuresis has been traced to a constitutively activating mutation of the V2 receptor gene. This unusual variant may be referred to as familial nephrogenic SIAD (NSIAD) to distinguish it from

1	CAuSES of SynDRomE of inAPPRoPRiATE AnTiDiuRETiC HoRmonE (SiADH) Bladder, ureter Psychosis Pneumothorax Pneumonia, bacterial or viral Positive-pressure respiration Abscess, lung or brain Vasopressin or desmopressin Tuberculosis, lung or brain Serotonin reuptake inhibitors Meningitis, bacterial or viral Oxytocin, high dose Encephalitis Nicotine Cerebrovascular occlusions, other possible causes of the syndrome. The inappropriate antidiuresis in these patients appears to be permanent, although the hyponatremia is variable owing presumably to individual differences in fluid intake.

1	Pathophysiology Impaired osmotic suppression of antidiuresis results in excessive retention of water and dilution of body fluids only if water intake exceeds insensible and urinary losses. The excess intake is sometimes due to an associated defect in the osmoregulation of thirst (dipsogenic) but can also be psychogenic or iatrogenic, including excessive IV administration of hypotonic fluids. In SIADH and other forms of euvolemic hyponatremia, the decrease in plasma osmolarity/ sodium and the increase in extracellular and intracellular volume are proportional to the amount of water retained. Thus, an increase in body water of 10% (~4 L in a 70-kg adult) reduces plasma osmolarity and sodium by approximately 10% (~28 mosmol/L or 14 meq/L). An increase in body water of this magnitude is rarely detectable on physical examination but will be reflected in a weight gain of about 4 kg. It also increases glomerular filtration and atrial natriuretic hormone and suppresses plasma renin activity,

1	detectable on physical examination but will be reflected in a weight gain of about 4 kg. It also increases glomerular filtration and atrial natriuretic hormone and suppresses plasma renin activity, thereby increasing urinary sodium excretion. The resultant reduction in total body sodium decreases the expansion of extracellular volume but aggravates the hyponatremia and further expands intracellular volume. The latter further increases brain swelling and intracranial pressure, which probably produces most of the symptoms of acute water intoxication. Within a few days, this swelling may be counteracted by inactivation or elimination of intracellular solutes, resulting in the remission of symptoms even though the hyponatremia persists.

1	In type I (hypervolemic) or type II (hypovolemic) hyponatremia, osmotic suppression of AVP secretion appears to be counteracted by a hemodynamic stimulus resulting from a large reduction in cardiac output and/or effective blood volume. The resultant antidiuresis is enhanced by decreased distal delivery of glomerular filtrate that results from increased reabsorption of sodium in proximal nephron. 2281 If the reduction in urine output is not associated with a commensurate reduction in water intake or an increase in insensible loss, body fluids are expanded and diluted, resulting in hyponatremia despite an increase in body sodium. Unlike SIADH and other forms of euvolemic hyponatremia, however, glomerular filtration is reduced and plasma renin activity and aldosterone are elevated. Thus, the rate of urinary sodium excretion is low (unless sodium reabsorption is impaired by a diuretic), and the hyponatremia is usually accompanied by edema, hypokalemia, azotemia, and hyperuricemia. In type

1	rate of urinary sodium excretion is low (unless sodium reabsorption is impaired by a diuretic), and the hyponatremia is usually accompanied by edema, hypokalemia, azotemia, and hyperuricemia. In type II (hypovolemic) hyponatremia, sodium and water are also retained as an appropriate compensatory response to the severe depletion.

1	Differential Diagnosis SIADH is a diagnosis of exclusion that usually can be made from the history, physical examination, and basic laboratory data. If hyperglycemia is present, its contribution to the reduction in plasma sodium can be estimated either by measuring plasma osmolarity for a more accurate estimate of the true “effective” tonicity of body fluids or by correcting the measured plasma sodium for the reduction caused by the hyperglycemia using the simplified formula where P = plasma sodium in meq/L and P = plasma glucose in mg/dL.

1	If the plasma osmolarity and/or corrected plasma sodium are below normal limits, hypotonic hyponatremia is present and further evaluation to determine the type should be undertaken in order to administer safe and effective treatment. This differentiation is usually possible by evaluating standard clinical indicators of the extracellular fluid volume (Table 404-3). If these findings are ambiguous or contradictory, measuring plasma renin activity or the rate of urinary sodium excretion may be helpful provided that the hyponatremia is not in the recovery phase or is due to a primary defect in renal conservation of sodium, diuretic abuse, or hyporeninemic hypoaldosteronism. The latter may be suspected if serum potassium is elevated instead of low, as it usually is in types I and II hyponatremia. Measurements of plasma AVP are currently of no value in differentiating SIADH from the other types of hyponatremia since the plasma levels are elevated similarly in all. In patients who fulfill

1	Measurements of plasma AVP are currently of no value in differentiating SIADH from the other types of hyponatremia since the plasma levels are elevated similarly in all. In patients who fulfill the clinical criteria for type III (euvolemic) hyponatremia, morning plasma cortisol should also be measured to exclude secondary adrenal insufficiency. If it is normal and there is no history of nausea/vomiting, the diagnosis of SIADH is confirmed, and a careful search for occult lung cancer or other common causes of the syndrome (Table 404-2) should be undertaken.

1	SIAD due to an activating mutation of the V2 receptor gene should be suspected if the hyponatremia occurs in a child or several members of the family or is refractory to treatment with a vaptan (see below). In that case, plasma AVP should be measured to confirm that it is appropriately suppressed while the hyponatremia and antidiuresis are present, and the V2 receptor gene should be sequenced, if possible.

1	The management of hyponatremia differs depending on the type and the severity and duration of symptoms. In acute symptomatic SIADH, the aim should be to raise plasma osmolarity and/or plasma sodium at a rate approximating 1% an hour until they reach levels of about 270 mosmol/L or 130 meq/L, respectively. This can be accomplished in either of two ways. One is to infuse hypertonic (3%) saline at a rate of about 0.05 mL/kg body weight per minute. This treatment also has the advantage of correcting the sodium deficiency that is partly responsible for the hyponatremia and often produces a solute diuresis that serves to remove some of the excess water. The other treatment is to reduce body water by giving an AVP receptor-2 antagonist (vaptan) to block the antidiuretic effect of AVP and increase urine output (Fig. 404-7). One of the vaptans, a combined V2/V1a antagonist (Conivaptan), has been approved for short-term, in-hospital IV treatment of SIADH, and others are in various stages of

1	urine output (Fig. 404-7). One of the vaptans, a combined V2/V1a antagonist (Conivaptan), has been approved for short-term, in-hospital IV treatment of SIADH, and others are in various stages of development. With either approach, fluid intake should be restricted to less than urine

1	Disorders of the Neurohypophysis SIADH and SIAD Clinical Findings Type I, Hypervolemic Type II, Hypovolemic Type III, Euvolemic Euvolemic CHF, cirrhosis, or nephrosis Generalized edema, ascites BUN, creatinine

1	Urinary sodium (meq per unit of time)g aPostural hypotension may occur in secondary (ACTH-dependent) adrenal insufficiency even though extracellular fluid volume and aldosterone are usually normal. bSerum potassium may be high if hypovolemia is due to aldosterone deficiency. cSerum potassium may be low if vomiting causes alkalosis. dSerum cortisol is low if hypovolemia is due to primary adrenal insufficiency (Addison’s disease). eSerum cortisol will be normal or high if the cause is nausea and vomiting rather than secondary (ACTH-dependent) adrenal insufficiency. fPlasma renin activity may be high if the cause is secondary (ACTH) adrenal insufficiency. gUrinary sodium should be expressed as the rate of excretion rather than the concentration. In a hyponatremic adult, an excretion rate >25 meq/d (or 25 μeq/mg of creatinine) could be considered high. hThe rate of urinary sodium excretion may be high if the hypovolemia is due to diuretic abuse, primary adrenal insufficiency, or other

1	>25 meq/d (or 25 μeq/mg of creatinine) could be considered high. hThe rate of urinary sodium excretion may be high if the hypovolemia is due to diuretic abuse, primary adrenal insufficiency, or other causes of renal sodium wasting. iThe rate of urinary sodium excretion may be low if intake is curtailed by symptoms or treatment.

1	Abbreviations: ACTH, adrenocorticotropic hormone; BUN, blood urea nitrogen; CHF, congestive heart failure; SIAD, syndrome of inappropriate antidiuresis. output, and serum sodium should be checked at least once every 2h to ensure it is not raised too fast or too far. Doing so may result in central pontine myelinolysis, an acute, potentially fatal neurologic syndrome characterized by quadriparesis, ataxia, and abnormal extraocular movements.

1	In chronic and/or minimally symptomatic SIADH, the hyponatremia can and should be corrected more gradually. This can be achieved by restricting total fluid intake to less than the sum of urinary and insensible losses. Because the water derived from food (300–700 mL/d) usually approximates basal insensible losses in adults, the aim should be to reduce total discretionary intake (all liquids) to approximately 500 mL less than urinary output. Adherence to this regimen is often problematic and, even if achieved, usually reduces body water and increases serum sodium by only about 1–2% per day. Hence, additional approaches are usually desirable if not necessary. The best approach for treatment of chronic SIADH is the administration of an oral vaptan, tolvaptan, a selective V2 antagonist that also increases urinary water excretion by blocking the antidiuretic effect of AVP. Some restriction of fluid intake may also be necessary to achieve satisfactory control of the hyponatremia. It is

1	also increases urinary water excretion by blocking the antidiuretic effect of AVP. Some restriction of fluid intake may also be necessary to achieve satisfactory control of the hyponatremia. It is approved for treatment of nonemergent SIADH with initial in-hospital dosing. Other approaches include demeclocycline, 150–300 mg PO tid or qid, or fludrocortisone, 0.05–0.2 mg PO bid. The effect of the demeclocycline manifests in 7–14 days and is due to induc- tion of a reversible form of nephrogenic DI. Potential side effects include phototoxicity and azotemia. The effect of fludrocortisone also requires 1–2 weeks and is partly due to increased retention of sodium and possibly inhibition of thirst. It also increases urinary potassium excretion, which may require replacement through dietary adjustments or supplements and may induce hypertension, occasionally necessitating discontinuation of the treatment.

1	vomiting or isolated glucocorticoid deficiency (type III), all abnormalities can be corrected quickly and completely by giving an antiemetic or stress doses of hydrocortisone (for glucocorticoid deficiency). As with other treatments, care must be taken to ensure that serum sodium does not rise too quickly or too far. In SIAD due to an activating mutation of the V2 receptor, the V2 FIGURE 404-7 The effect of vaptan therapy on water balance in a patient with chronic syndrome of inappropriate antidiuretic hormone (SIADH). The periods of vaptan (V) therapy are indicated by the green shaded boxes at the top. Urine output is indicated by orange bars. Fluid intake is shown by the open bars. Intake was restricted to 1 L/d throughout. Serum sodium is indicated by the black line. Note that sodium increased progressively when vaptan increased urine output to levels that clearly exceeded fluid intake.

1	antagonists usually do not block the antidiuresis or raise plasma osmolarity/sodium. In that condition, use of an osmotic diuretic such as urea is reported to be effective in preventing or correcting hyponatremia. However, some vaptans may be effective in patients with a different type of activating mutation so the response to this therapy may be neither predictable nor diagnostic.

1	In hypervolemic hyponatremia, fluid restriction is also appropriate and somewhat effective if it can be maintained. However, infusion of hypertonic saline is contraindicated because it further increases total body sodium and edema and may precipitate cardiovascular decompensation. However, as in SIADH, the V2 receptor antagonists are also safe and effective in the treatment of hypervolemic hyponatremia caused by congestive heart failure. Tolvaptan is approved by the Food and Drug Administration for this indication with the caveat that treatment should be initiated or reinitiated in hospital. Its use should also be limited to 30 days at a time because of reports that longer periods may be associated with abnormal liver chemistries.

1	In hypovolemic hyponatremia, the defect in AVP secretion and water balance usually can be corrected easily and quickly by stopping the loss of sodium and water and/or replacing the deficits by mouth or IV infusion of normal or hypertonic saline. As with the treatment of other forms of hyponatremia, care must be taken to ensure that plasma sodium does not increase too rapidly or too far. Fluid restriction and administration of AVP antagonists are contraindicated in type II hyponatremia because they would only aggravate the underlying volume depletion and could result in hemodynamic collapse.

1	The incidence, clinical characteristics, etiology, pathophysiology, differential diagnosis, and treatments of fluid and electrolyte disorders in tropical and nonindustrialized countries differ in some respects from those in the United States and other industrialized parts of the world. Hyponatremia, for example, appears to be more common and is more likely to be due to infectious diseases such as cholera, shigellosis, and other diarrheal disorders. In these circumstances, hyponatremia is probably due to gastrointestinal losses of salt and water (hypovolemia type II), but other abnormalities, including undefined infectious toxins, also may contribute. The causes of DI are similar worldwide except that malaria and venoms from snake or insect bites are much more common.

1	Disorders of the Thyroid gland J. Larry Jameson, Susan J. Mandel, Anthony P. Weetman The thyroid gland produces two related hormones, thyroxine (T4) and triiodothyronine (T3) (Fig. 405-1). Acting through thyroid hormone receptors α and β, these hormones play a critical role in cell differentia-tion during development and help maintain thermogenic 405 and metabolic homeostasis in the adult. Autoimmune disorders of the thyroid gland can stimulate overproduction of thyroid hormones (thyrotoxicosis) or cause glandular destruction and hormone deficiency (hypothyroidism). In addition, benign nodules and various forms of thyroid cancer are relatively common and amenable to detection by physical examination. The thyroid (Greek thyreos, shield, plus eidos, form) consists of two lobes connected by an isthmus. It is located I anterior to the trachea between the cricoid cartilage and

1	The thyroid (Greek thyreos, shield, plus eidos, form) consists of two lobes connected by an isthmus. It is located I anterior to the trachea between the cricoid cartilage and The thyroid gland develops from the floor of the primitive pharynx 2283 during the third week of gestation. The developing gland migrates along the thyroglossal duct to reach its final location in the neck. This feature accounts for the rare ectopic location of thyroid tissue at the base of the tongue (lingual thyroid) as well as the occurrence of thyroglossal duct cysts along this developmental tract. Thyroid hormone synthesis normally begins at about 11 weeks’ gestation.

1	Neural crest derivatives from the ultimobranchial body give rise to thyroid medullary C cells that produce calcitonin, a calcium-lowering hormone. The C cells are interspersed throughout the thyroid gland, although their density is greatest in the juncture of the upper one-third and lower two-thirds of the gland. Calcitonin plays a minimal role in calcium homeostasis in humans but the C-cells are important because of their involvement in medullary thyroid cancer.

1	Thyroid gland development is orchestrated by the coordinated expression of several developmental transcription factors. Thyroid transcription factor (TTF)-1, TTF-2, and paired homeobox-8 (PAX-8) are expressed selectively, but not exclusively, in the thyroid gland. In combination, they dictate thyroid cell development and the induction of thyroid-specific genes such as thyroglobulin (Tg), thyroid peroxidase (TPO), the sodium iodide symporter (Na+/I–, NIS), and the thyroid-stimulating hormone receptor (TSH-R). Mutations in these developmental transcription factors or their downstream target genes are rare causes of thyroid agenesis or dyshormonogenesis, although the causes of most forms of congenital hypothyroidism remain unknown (Table 405-1). Because congenital hypothyroidism occurs in approximately 1 in 4000 newborns, neonatal screening is now performed in most industrialized countries (see below). Transplacental passage of maternal thyroid hormone occurs before the fetal thyroid

1	approximately 1 in 4000 newborns, neonatal screening is now performed in most industrialized countries (see below). Transplacental passage of maternal thyroid hormone occurs before the fetal thyroid gland begins to function and provides partial hormone support to a fetus with congenital hypothyroidism. Early thyroid hormone replacement in newborns with congenital hypothyroidism prevents potentially severe developmental abnormalities.

1	The thyroid gland consists of numerous spherical follicles composed of thyroid follicular cells that surround secreted colloid, a proteinaceous fluid containing large amounts of thyroglobulin, the protein precursor of thyroid hormones (Fig. 405-2). The thyroid follicular cells are polarized—the basolateral surface is apposed to the bloodstream and an apical surface faces the follicular lumen. Increased demand for thyroid hormone is regulated by thyroid-stimulating hormone (TSH), which binds to its receptor on the basolateral surface of the follicular cells. This binding leads to Tg reabsorption from the follicular lumen and proteolysis within the cytoplasm, yielding thyroid hormones for secretion into the bloodstream. TSH, secreted by the thyrotrope cells of the anterior pituitary, plays a pivotal role in control of the thyroid axis and serves as the most useful 3,5,3',5'-Tetraiodothyronine Disorders of the Thyroid Gland the suprasternal notch. The normal thyroid is 12–20 g HO

1	Disorders of the Thyroid Gland the suprasternal notch. The normal thyroid is 12–20 g HO CH2 CH COOH in size, highly vascular, and soft in consistency. Four parathyroid glands, which produce parathyroid hormone II (Chap. 424), are located posterior to each pole of the Triiodothyronine (T3) Reverse T3 (rT3) 3,5,3'-Triiodothyronine 3,3',5'-Triiodothyronine thyroid. The recurrent laryngeal nerves traverse the lateral borders of the thyroid gland and must be identified FIGURE 405-1 Structures of thyroid hormones. Thyroxine (T4) contains four during thyroid surgery to avoid injury and vocal cord iodine atoms. Deiodination leads to production of the potent hormone triiodothyparalysis. ronine (T ) or the inactive hormone reverse T . PROP-1 Autosomal recessive Combined pituitary hormone deficiencies with preservation of adrenocorticotropic hormone PIT-1 Autosomal recessive Combined deficiencies of growth hormone, prolactin, thyroid-

1	PIT-1 Autosomal recessive Combined deficiencies of growth hormone, prolactin, thyroid- TTF-1 (TITF-1) Autosomal dominant Variable thyroid hypoplasia, choreoathetosis, pulmonary problems hereditary osteodystrophy) Na+/Isymporter Autosomal recessive Inability to transport iodide DUOX2 (THOX2) Autosomal dominant Organification defect physiologic marker of thyroid hormone action. TSH is a 31-kDa hormone composed of α and β subunits; the α subunit is common to the other glycoprotein hormones (luteinizing hormone, follicle-stimulating hormone, human chorionic gonadotropin [hCG]), whereas the TSH β subunit is unique to TSH. The extent and nature of carbohydrate modification are modulated by thyrotropin-releasing hormone (TRH) stimulation and influence the biologic activity of the hormone.

1	The thyroid axis is a classic example of an endocrine feedback loop. Hypothalamic TRH stimulates pituitary production of TSH, which, in turn, stimulates thyroid hormone synthesis and secretion. Thyroid hormones act via negative feedback predominantly through thyroid hormone receptor β2 (TRβ2) to inhibit TRH and TSH production (Fig. 405-2). The “set-point” in this axis is established by TSH. TRH is the major positive regulator of TSH synthesis and secretion. Peak TSH secretion occurs ~15 min after administration of exogenous TRH. Dopamine, glucocorticoids, and somatostatin suppress TSH but are not of major physiologic importance except when these agents are administered in pharmacologic doses. Reduced levels of thyroid hormone increase basal TSH production and enhance TRH-mediated stimulation of TSH. High thyroid hormone levels rapidly and directly suppress TSH gene expression secretion and inhibit TRH stimulation of TSH, indicating that thyroid hormones are the dominant regulator of

1	of TSH. High thyroid hormone levels rapidly and directly suppress TSH gene expression secretion and inhibit TRH stimulation of TSH, indicating that thyroid hormones are the dominant regulator of TSH production. Like other pituitary hormones, TSH is released in a pulsatile manner and exhibits a diurnal rhythm; its highest levels occur at night. However, these TSH excursions are modest in comparison to those of other pituitary hormones, in part, because TSH has a relatively long plasma half-life (50 min). Consequently, single measurements of TSH are adequate for assessing its circulating level. TSH is measured using immunoradiometric assays that are highly sensitive and specific. These assays readily distinguish between normal and suppressed TSH values; thus, TSH can be used for the diagnosis of hyperthyroidism (low TSH) as well as hypothyroidism (high TSH).

1	THYROID HORMONE SYNTHESIS, METABOLISM, AND ACTION Thyroid hormones are derived from Tg, a large iodinated glycoprotein. After secretion into the thyroid follicle, Tg is iodinated on tyrosine FIGURE 405-2 Regulation of thyroid hormone synthesis. Left. Thyroid hormones T4 and T3 feed back to inhibit hypothalamic production of thyrotropin-releasing hormone (TRH) and pituitary production of thyroid-stimulating hormone (TSH). TSH stimulates thyroid gland production of T4 and T3. Right. Thyroid follicles are formed by thyroid epithelial cells surrounding proteinaceous colloid, which contains thyroglobulin. Follicular cells, which are polarized, synthesize thyroglobulin and carry out thyroid hormone biosynthesis (see text for details). DIT, diiodotyrosine; MIT, monoiodotyrosine; NIS, sodium iodide symporter; Tg, thyroglobulin; TPO, thyroid peroxidase; TSH-R, thyroid-stimulating hormone receptor.

1	residues that are subsequently coupled via an ether linkage. Reuptake of Tg into the thyroid follicular cell allows proteolysis and the release of newly synthesized T4 and T3.

1	Iodine Metabolism and Transport Iodide uptake is a critical first step in thyroid hormone synthesis. Ingested iodine is bound to serum proteins, particularly albumin. Unbound iodine is excreted in the urine. The thyroid gland extracts iodine from the circulation in a highly efficient manner. For example, 10–25% of radioactive tracer (e.g., 123I) is taken up by the normal thyroid gland over 24 h; this value can rise to 70–90% in Graves’ disease. Iodide uptake is mediated by NIS, which is expressed at the basolateral membrane of thyroid follicular cells. NIS is most highly expressed in the thyroid gland, but low levels are present in the salivary glands, lactating breast, and placenta. The iodide transport mechanism is highly regulated, allowing adaptation to variations in dietary supply. Low iodine levels increase the amount of NIS and stimulate uptake, whereas high iodine levels suppress NIS expression and uptake. The selective expression of NIS in the thyroid allows isotopic

1	Low iodine levels increase the amount of NIS and stimulate uptake, whereas high iodine levels suppress NIS expression and uptake. The selective expression of NIS in the thyroid allows isotopic scanning, treatment of hyperthyroidism, and ablation of thyroid cancer with radioisotopes of iodine, without significant effects on other organs. Mutation of the NIS gene is a rare cause of congenital hypothyroidism, underscoring its importance in thyroid hormone synthesis. Another iodine transporter, pendrin, is located on the apical surface of thyroid cells and mediates iodine efflux into the lumen. Mutation of the pendrin gene causes Pendred syndrome, a disorder characterized by defective organification of iodine, goiter, and sensorineural deafness.

1	Iodine deficiency is prevalent in many mountainous regions and in central Africa, central South America, and northern

1	Asia (Fig. 405-3). Europe remains mildly iodine-deficient, and health surveys indicate that iodine intake has been falling in the United States and Australia. The World Health Organization (WHO) estimates that about 2 billion people are iodine-deficient, based on urinary excretion data. In areas of relative iodine deficiency, there is an increased prevalence of goiter and, when deficiency is severe, hypothyroidism and cretinism. Cretinism is characterized by mental and growth retardation and occurs when children who live in iodine-deficient regions are not treated with iodine or thyroid hormone to restore normal thyroid hormone levels during early life. These children are often born to mothers with iodine deficiency, and it is likely that maternal thyroid hormone deficiency worsens the condition. Concomitant selenium deficiency may also contribute to the neurologic manifestations of cretinism. Iodine supplementation of salt, bread, and other food substances has markedly reduced the

1	condition. Concomitant selenium deficiency may also contribute to the neurologic manifestations of cretinism. Iodine supplementation of salt, bread, and other food substances has markedly reduced the prevalence of cretinism. Unfortunately, however, iodine deficiency remains the most common cause of preventable mental deficiency, often because of societal resistance to food additives or the cost of supplementation. In addition to overt cretinism, mild iodine deficiency can lead to subtle reduction of IQ. Oversupply of iodine, through supplements or foods enriched in iodine (e.g., shellfish, kelp), is associated with an increased incidence of autoimmune thyroid disease. The recommended average daily intake of iodine is 150–250 μg/d for adults, 90–120 μg/d for children, and 250 μg/d for pregnant and lactating women. Urinary iodine is >10 μg/dL in iodine-sufficient populations.

1	Organification, Coupling, Storage, and Release After iodide enters the thyroid, it is trapped and transported to the apical membrane of thyroid follicular cells, where it is oxidized in an organification reaction that involves TPO and hydrogen peroxide produced by dual oxidase (DUOX) and DUOX maturation factor (DUOXA). The reactive iodine atom is added to selected tyrosyl residues within Tg, a large (660 kDa) dimeric protein that consists of 2769 amino acids. The iodotyrosines in Tg are then coupled via an ether linkage in a reaction that is also catalyzed by TPO. Either T4 or T3 can be produced by this reaction, depending on the number of iodine atoms present in the iodotyrosines. After coupling, Tg is taken back into the thyroid cell, where it is processed in lysosomes to release T4 and T3. Uncoupled monoand diiodotyrosines (MIT, DIT) are deiodinated by the enzyme dehalogenase, thereby recycling any iodide that is not converted into thyroid hormones.

1	Disorders of thyroid hormone synthesis are rare causes of con-2285 genital hypothyroidism. The vast majority of these disorders are due to recessive mutations in TPO or Tg, but defects have also been identified in the TSH-R, NIS, pendrin, hydrogen peroxide generation, and dehalogenase. Because of the biosynthetic defect, the gland is incapable of synthesizing adequate amounts of hormone, leading to increased TSH and a large goiter.

1	TSH Action TSH regulates thyroid gland function through the TSHR, a seven-transmembrane G protein–coupled receptor (GPCR). The TSH-R is coupled to the α subunit of stimulatory G protein (GSα), which activates adenylyl cyclase, leading to increased production of cyclic adenosine monophosphate (AMP). TSH also stimulates phosphatidylinositol turnover by activating phospholipase C. The functional role of the TSH-R is exemplified by the consequences of naturally occurring mutations. Recessive loss-of-function mutations cause thyroid hypoplasia and congenital hypothyroidism. Dominant gain-of-function mutations cause sporadic or familial hyperthyroidism that is characterized by goiter, thyroid cell hyperplasia, and autonomous function. Most of these activating mutations occur in the transmembrane domain of the receptor. They mimic the conformational changes induced by TSH binding or the interactions of thyroid-stimulating immunoglobulins (TSI) in Graves’ disease. Activating TSH-R mutations

1	domain of the receptor. They mimic the conformational changes induced by TSH binding or the interactions of thyroid-stimulating immunoglobulins (TSI) in Graves’ disease. Activating TSH-R mutations also occur as somatic events, leading to clonal selection and expansion of the affected thyroid follicular cell and autonomously functioning thyroid nodules (see below).

1	Other Factors That Influence Hormone Synthesis and Release Although TSH is the dominant hormonal regulator of thyroid gland growth and function, a variety of growth factors, most produced locally in the thyroid gland, also influence thyroid hormone synthesis. These include insulin-like growth factor I (IGF-I), epidermal growth factor, transforming growth factor β (TGF-β), endothelins, and various cytokines. The quantitative roles of these factors are not well understood, but they are important in selected disease states. In acromegaly, for example, increased levels of growth hormone and IGF-I are associated with goiter and predisposition to multinodular goiter (MNG). Certain cytokines and interleukins (ILs) produced in association with autoimmune thyroid disease induce thyroid growth, whereas others lead to apoptosis. Iodine deficiency increases thyroid blood flow and upregulates the NIS, stimulating more efficient iodine uptake. Excess iodide transiently inhibits thyroid iodide

1	whereas others lead to apoptosis. Iodine deficiency increases thyroid blood flow and upregulates the NIS, stimulating more efficient iodine uptake. Excess iodide transiently inhibits thyroid iodide organification, a phenomenon known as the Wolff-Chaikoff effect. In individuals with a normal thyroid, the gland escapes from this inhibitory effect and iodide organification resumes; the suppressive action of high iodide may persist, however, in patients with underlying autoimmune thyroid disease.

1	THYROID HORMONE TRANSPORT AND METABOLISM Serum Binding Proteins T4 is secreted from the thyroid gland in about twentyfold excess over T3 (Table 405-2). Both hormones are bound to plasma proteins, including thyroxine-binding globulin (TBG), transthyretin (TTR, formerly known as thyroxinebinding prealbumin, or TBPA), and albumin. The plasma-binding proteins increase the pool of circulating hormone, delay hormone clearance, and may modulate hormone delivery to selected tissue sites. The concentration of TBG is relatively low (1–2 mg/dL), but because of its high affinity for thyroid hormones (T4 > T3), it carries about 80% of the bound hormones. Albumin has relatively low affinity for thyroid hormones CHAPTER 405 Disorders of the Thyroid Gland until a new steady state is reached, thereby restoring euthyroidism. Circulating factors associated with acute illness may also displace thyroid hormone from binding proteins (see “Sick Euthyroid Syndrome,” below).

1	Total hormone 8 μg/dL 0.14 μg/dL Deiodinases T4 may be thought of as a precursor for the more potent Fraction of total hormone in the unbound form 0.02% 0.3% T . T is converted to T by the deiodinase enzymes (Fig. 405-1). Type I deiodinase, which is located primarily in thyroid, liver, and kidneys, Serum half-life 7 d 2 d has a relatively low affinity for T4. Type II deiodinase has a higher affin-Fraction directly from the thyroid 100% 20% ity for T4 and is found primarily in the pituitary gland, brain, brown fat, Production rate, including peripheral conversion 90 μg/d 32 μg/d and thyroid gland. Expression of type II deiodinase allows it to regulate T concentrations locally, a property that may be important in the context of levothyroxine (T ) replacement. Type II deiodinase is also

1	T concentrations locally, a property that may be important in the context of levothyroxine (T ) replacement. Type II deiodinase is also Relative metabolic potency 0.3 1 4 regulated by thyroid hormone; hypothyroidism induces the enzyme, resulting in enhanced T4 → T3 conversion in tissues such as brain and pituitary. T4 → T3 conversion is impaired by fasting, systemic illness When the effects of the various binding proteins are combined, or acute trauma, oral contrast agents, and a variety of medications approximately 99.98% of T and 99.7% of T are protein-bound. (e.g., propylthiouracil, propranolol, amiodarone, glucocorticoids). Because T is less tightly bound than T , the fraction of unbound T Type III deiodinase inactivates T and T and is the most important is greater than unbound T , but there is less unbound T in the cir-source of reverse T3 (rT3), including in the sick euthyroid syndrome.

1	culation because it is produced in smaller amounts and cleared more This enzyme is expressed in the human placenta but is not active in rapidly than T . The unbound or “free” concentrations of the hor-healthy individuals. In the sick euthyroid syndrome, especially with mones are ~2 × 10−11 M for T and ~6 × 10−12 M for T , which roughly hypoperfusion, the type III deiodinase is activated in muscle and liver. correspond to the thyroid hormone receptor binding constants for Massive hemangiomas that express type III deiodinase are a rare cause these hormones (see below). The unbound hormone is thought to be of hypothyroidism in infants. biologically available to tissues. Nonetheless, the homeostatic mechanisms that regulate the thyroid axis are directed toward maintenance THYROID HORMONE ACTION of normal concentrations of unbound hormones. Thyroid Hormone Transport Circulating thyroid hormones enter cells by passive diffusion and via specific transporters such as the mono-

1	Abnormalities of Thyroid Hormone Binding Proteins A number of inher carboxylate 8 transporter (MCT8), MCT10, and organic anionited and acquired abnormalities affect thyroid hormone binding transporting polypeptide 1C1. Mutations in the MCT8 gene have been proteins. X-linked TBG deficiency is associated with very low levels identified in patients with X-linked psychomotor retardation andof total T4 and T3. However, because unbound hormone levels are thyroid function abnormalities (low T4, high T3, and high TSH). Afternormal, patients are euthyroid and TSH levels are normal. It is imporentering cells, thyroid hormones act primarily through nuclear receptant to recognize this disorder to avoid efforts to normalize total T4 tors, although they also have nongenomic actions through stimulatinglevels, because this leads to thyrotoxicosis and is futile because of rapid mitochondrial enzymatic responses and may act directly on blood veshormone clearance in the absence of TBG. TBG levels are

1	because this leads to thyrotoxicosis and is futile because of rapid mitochondrial enzymatic responses and may act directly on blood veshormone clearance in the absence of TBG. TBG levels are elevated sels and the heart through integrin receptors.

1	by estrogen, which increases sialylation and delays TBG clearance. Consequently, in women who are pregnant or taking estrogen-Nuclear Thyroid Hormone Receptors Thyroid hormones bind with high containing contraceptives, elevated TBG increases total T4 and T3 levels; affinity to nuclear thyroid hormone receptors (TRs) α and β. Both TRα and however, unbound T4 and T3 levels are normal. These features are TRβ are expressed in most tissues, but their relative expression levels vary part of the explanation for why women with hypothyroidism require increased amounts of L-thyroxine replacement as TBG levels are increased by pregnancy or estrogen treatment. Mutations in TBG, TTR, and albumin may increase the bind-ing affinity for T4 and/or T3 and cause TAblE 405-3 ConDiTionS ASSoCiATED wiTH EuTHyRoiD HyPERTHyRoxinEmiA Disorder Cause Transmission Characteristics Familial dysalbuminemic hyperthyroxinemia (FDH) Albumin mutations, usually R218H AD Increased T4 Normal unbound T4

1	Rarely increased T3 roxinemia or familial dysalbuminemic TBG hyperthyroxinemia (FDH) (Table 405-3). Familial excess Increased TBG production XL Increased total T4, T3 disorders known as euthyroid hyperthy- Normal unbound T , T and/or T , but unbound hormone lev T4 3 Acquired excess Medications (estrogen), Acquired Increased total T4, T43 3 els are normal. The familial nature of the pregnancy, cirrhosis, Normal unbound T4, T3 disorders, and the fact that TSH levels are normal rather than suppressed, should suggest this diagnosis. Unbound hor- Excess Islet tumors Acquired Usually normal T4, T3 Mutations Increased affinity for T4 AD Increased total T4, T3 are normal in FDH. The diagnosis can be

1	Excess Islet tumors Acquired Usually normal T4, T3 Mutations Increased affinity for T4 AD Increased total T4, T3 are normal in FDH. The diagnosis can be Normal unbound T4, T3 confirmed by using tests that measure the affinities of radiolabeled hormone bind-Medications: propranolol, ipo-Decreased T4 → T3 Acquired Increased T4 ing to specific transport proteins or by date, iopanoic acid, amiodarone conversion performing DNA sequence analyses of the abnormal transport protein genes. Resistance to thyroid hormone Thyroid hormone receptor AD Increased unbound T4, T3Certain medications, such as salicylates (RTH) β mutations and salsalate, can displace thyroid hor- mones from circulating binding proteins. aAlso known as thyroxine-binding prealbumin (TBPA). the thyroid axis by increasing free thyroid hormone levels, TSH is suppressed Abbreviations: AD, autosomal dominant; TBG, thyroxine-binding globulin; TSH, thyroid-stimulating hormone; XL, X-linked.

1	the thyroid axis by increasing free thyroid hormone levels, TSH is suppressed Abbreviations: AD, autosomal dominant; TBG, thyroxine-binding globulin; TSH, thyroid-stimulating hormone; XL, X-linked. among organs; TRα is particularly abundant in brain, kidneys, gonads, muscle, and heart, whereas TRβ expression is relatively high in the pituitary and liver. Both receptors are variably spliced to form unique isoforms. The TRβ2 isoform, which has a unique amino terminus, is selectively expressed in the hypothalamus and pituitary, where it plays a role in feedback control of the thyroid axis (see above). The TRα2 isoform contains a unique carboxy terminus that precludes thyroid hormone binding; it may function to block the action of other TR isoforms.

1	The TRs contain a central DNA-binding domain and a C-terminal ligand-binding domain. They bind to specific DNA sequences, termed thyroid response elements (TREs), in the promoter regions of target genes (Fig. 405-4). The receptors bind as homodimers or, more commonly, as heterodimers with retinoic acid X receptors (RXRs) (Chap. 400e). The activated receptor can either stimulate gene transcription (e.g., myosin heavy chain α) or inhibit transcription (e.g., TSH β-subunit gene), depending on the nature of the regulatory elements in the target gene.

1	Thyroid hormones (T3 and T4) bind with similar affinities to TRα and TRβ. However, structural differences in the ligand binding domains provide the potential for developing receptor-selective agonists or antagonists, and these are under investigation. T3 is bound with 10–15 times greater affinity than T4, which explains its increased hormonal potency. Although T4 is produced in excess of T3, receptors are occupied mainly by T3, reflecting T4 → T3 conversion by peripheral tissues, greater T3 bioavailability in the plasma, and the greater affinity of receptors for T3. After binding to TRs, thyroid hormone induces conformational changes in the receptors that modify its interactions with accessory transcription factors. Importantly, in the absence of thyroid hormone binding, the aporeceptors bind to co-repressor proteins that inhibit gene transcription. Hormone binding dissociates the co-repressors and allows the recruitment of co-activators that enhance transcription. The discovery of TR

1	to co-repressor proteins that inhibit gene transcription. Hormone binding dissociates the co-repressors and allows the recruitment of co-activators that enhance transcription. The discovery of TR interactions with co-repressors explains the fact that TR silences gene expression in the absence of hormone binding. Consequently, hormone deficiency has a profound effect on gene expression because it causes gene repression as well as loss of hormone-induced stimulation. This concept has been corroborated by the finding that targeted deletion of the TR genes in mice has a less pronounced phenotypic effect than hormone deficiency.

1	FIGURE 405-4 Mechanism of thyroid hormone receptor action. The thyroid hormone receptor (TR) and retinoid X receptor (RXR) form heterodimers that bind specifically to thyroid hormone response elements (TRE) in the promoter regions of target genes. In the absence of hormone, TR binds co-repressor (CoR) proteins that silence gene expression. The numbers refer to a series of ordered reactions that occur in response to thyroid hormone: (1) T4 or T3 enters the nucleus; (2) T3 binding dissociates CoR from TR; (3) co-activators (CoA) are recruited to the T3-bound receptor; and (4) gene expression is altered.

1	Thyroid Hormone Resistance Resistance to thyroid hormone (RTH) 2287 is an autosomal dominant disorder characterized by elevated thyroid hormone levels and inappropriately normal or elevated TSH. Individuals with RTH do not, in general, exhibit signs and symptoms that are typical of hypothyroidism because hormone resistance is partial and is compensated by increased levels of thyroid hormone. The clinical features of RTH can include goiter, attention deficit disorder, mild reduction in IQ, delayed skeletal maturation, tachycardia, and impaired metabolic responses to thyroid hormone.

1	Classical forms of RTH are caused by mutations in the TRβ gene. These mutations, located in restricted regions of the ligand-binding domain, cause loss of receptor function. However, because the mutant receptors retain the capacity to dimerize with RXRs, bind to DNA, and recruit co-repressor proteins, they function as antagonists of the remaining normal TRβ and TRα receptors. This property, referred to as “dominant negative” activity, explains the autosomal dominant mode of transmission. The diagnosis is suspected when unbound thyroid hormone levels are increased without suppression of TSH. Similar hormonal abnormalities are found in other affected family members, although the TRβ mutation arises de novo in about 20% of patients. DNA sequence analysis of the TRβ gene provides a definitive diagnosis. RTH must be distinguished from other causes of euthyroid hyperthyroxinemia (e.g., FDH) and inappropriate secretion of TSH by TSH-secreting pituitary adenomas (Chap. 403). In most patients,

1	diagnosis. RTH must be distinguished from other causes of euthyroid hyperthyroxinemia (e.g., FDH) and inappropriate secretion of TSH by TSH-secreting pituitary adenomas (Chap. 403). In most patients, no treatment is indicated; the importance of making the diagnosis is to avoid inappropriate treatment of mistaken hyperthyroidism and to provide genetic counseling.

1	A distinct form of RTH is caused by mutations in the TRα gene. Affected patients have many clinical features of congenital hypothyroidism including growth retardation, skeletal dysplasia, and severe constipation. In contrast to RTH caused by mutations in TRβ, thyroid function tests include normal TSH, low or normal T4, and normal or elevated T3 levels. These distinct clinical and laboratory features underscore the different tissue distribution and functional roles of TRβ and TRα. Optimal treatment of patients with RTH caused by TRα mutations has not been established.

1	In addition to the examination of the thyroid itself, the physical examination should include a search for signs of abnormal thyroid function and the extrathyroidal features of ophthalmopathy and dermopathy (see below). Examination of the neck begins by inspecting the seated patient from the front and side and noting any surgical scars, obvious masses, or distended veins. The thyroid can be palpated with both hands from behind or while facing the patient, using the thumbs to palpate each lobe. It is best to use a combination of these methods, especially when nodules are small. The patient’s neck should be slightly flexed to relax the neck muscles. After locating the cricoid cartilage, the isthmus, which is attached to the lower one-third of the thyroid lobes, can be identified and then followed laterally to locate either lobe (normally, the right lobe is slightly larger than the left). By asking the patient to swallow sips of water, thyroid consistency can be better appreciated as the

1	laterally to locate either lobe (normally, the right lobe is slightly larger than the left). By asking the patient to swallow sips of water, thyroid consistency can be better appreciated as the gland moves beneath the examiner’s fingers.

1	Features to be noted include thyroid size, consistency, nodularity, and any tenderness or fixation. An estimate of thyroid size (normally 12–20 g) should be made, and a drawing is often the best way to record findings. However, ultrasound is the method of choice when it is important to determine thyroid size accurately. The size, location, and consistency of any nodules should also be defined. A bruit or thrill over the gland, located over the insertion of the superior and inferior thyroid arteries (superoor inferolaterally), indicates increased vascularity, as occurs in hyperthyroidism. If the lower borders of the thyroid lobes are not clearly felt, a goiter may be retrosternal. Large retrosternal goiters can cause venous distention over the neck and difficulty breathing, especially when the arms are raised (Pemberton’s sign). With any central mass above the thyroid, the tongue should be extended, as thyroglossal cysts then move upward. The thyroid examination is not complete without

1	the arms are raised (Pemberton’s sign). With any central mass above the thyroid, the tongue should be extended, as thyroglossal cysts then move upward. The thyroid examination is not complete without assessment for lymphadenopathy in the supraclavicular and cervical regions of the neck.

1	CHAPTER 405 Disorders of the Thyroid Gland 2288 LABORATORY EVALUATION Measurement of Thyroid Hormones The enhanced sensitivity and specificity of TSH assays have greatly improved laboratory assessment of thyroid function. Because TSH levels change dynamically in response to alterations of T4 and T3, a logical approach to thyroid testing is to first determine whether TSH is suppressed, normal, or elevated. With rare exceptions (see below), a normal TSH level excludes a primary abnormality of thyroid function. This strategy depends on the use of immunochemiluminometric assays (ICMAs) for TSH that are sensitive enough to discriminate between the lower limit of the reference range and the suppressed values that occur with thyrotoxicosis. Extremely sensitive (fourth-generation) assays can detect TSH levels ≤0.004 mIU/L, but, for practical purposes, assays sensitive to ≤0.1 mIU/L are sufficient. The widespread availability of the TSH ICMA has rendered the TRH stimulation test obsolete,

1	detect TSH levels ≤0.004 mIU/L, but, for practical purposes, assays sensitive to ≤0.1 mIU/L are sufficient. The widespread availability of the TSH ICMA has rendered the TRH stimulation test obsolete, because the failure of TSH to rise after an intravenous bolus of 200–400 μg TRH has the same implications as a suppressed basal TSH measured by ICMA. The finding of an abnormal TSH level must be followed by measurements of circulating thyroid hormone levels to confirm the diagnosis of hyperthyroidism (suppressed TSH) or hypothyroidism (elevated TSH). Radioimmunoassays are widely available for serum total T4 and total T3. T4 and T3 are highly protein-bound, and numerous factors (illness, medications, genetic factors) can influence protein binding. It is useful, therefore, to measure the free, or unbound, hormone levels, which correspond to the biologically available hormone pool. Two direct methods are used to measure unbound thyroid hormones: (1) unbound thyroid hormone competition with

1	or unbound, hormone levels, which correspond to the biologically available hormone pool. Two direct methods are used to measure unbound thyroid hormones: (1) unbound thyroid hormone competition with radiolabeled T4 (or an analogue) for binding to a solid-phase antibody, and (2) physical separation of the unbound hormone fraction by ultracentrifugation or equilibrium dialysis. Although early unbound hormone immunoassays suffered from artifacts, newer assays correlate well with the results of the more technically demanding and expensive physical separation methods. An indirect method that is now less commonly used to estimate unbound thyroid hormone levels is to calculate the free T3 or free T4 index from the total T4 or T3 concentration and the thyroid hormone binding ratio (THBR). The latter is derived from the T3-resin uptake test, which determines the distribution of radiolabeled T3 between an absorbent resin and the unoccupied thyroid hormone binding proteins in the sample. The

1	latter is derived from the T3-resin uptake test, which determines the distribution of radiolabeled T3 between an absorbent resin and the unoccupied thyroid hormone binding proteins in the sample. The binding of the labeled T3 to the resin is increased when there is reduced unoccupied protein binding sites (e.g., TBG deficiency) or increased total thyroid hormone in the sample; it is decreased under the opposite circumstances. The product of THBR and total T3 or T4 provides the free T3 or T4 index. In effect, the index corrects for anomalous total hormone values caused by abnormalities in hormone-protein binding. Total thyroid hormone levels are elevated when TBG is increased due to estrogens (pregnancy, oral contraceptives, hormone therapy, tamoxifen, selective estrogen receptor modulators, inflammatory liver disease) and decreased when TBG binding is reduced (androgens, nephrotic syndrome). Genetic disorders and acute illness can also cause abnormalities in thyroid hormone binding

1	inflammatory liver disease) and decreased when TBG binding is reduced (androgens, nephrotic syndrome). Genetic disorders and acute illness can also cause abnormalities in thyroid hormone binding proteins, and various drugs (phenytoin, carbamazepine, salicylates, and nonsteroidal anti-inflammatory drugs [NSAIDs]) can interfere with thyroid hormone binding. Because unbound thyroid hormone levels are normal and the patient is euthyroid in all of these circumstances, assays that measure unbound hormone are preferable to those for total thyroid hormones. For most purposes, the unbound T4 level is sufficient to confirm thyrotoxicosis, but 2–5% of patients have only an elevated T3 level (T3 toxicosis). Thus, unbound T3 levels should be measured in patients with a suppressed TSH but normal unbound T4 levels. There are several clinical conditions in which the use of TSH as a screening test may be misleading, particularly without simultaneous unbound T4 determinations. Any severe nonthyroidal

1	T4 levels. There are several clinical conditions in which the use of TSH as a screening test may be misleading, particularly without simultaneous unbound T4 determinations. Any severe nonthyroidal illness can cause abnormal TSH levels (see below). Although hypothyroidism is the most common cause of an elevated TSH level, rare causes include a TSH-secreting pituitary tumor (Chap. 403), thyroid hormone resistance, and assay artifact. Conversely, a suppressed TSH level, particularly <0.01 mIU/L, usually indicates thyrotoxicosis. However, subnormal TSH levels between 0.01 and 0.1 mIU/L may be seen during the first trimester of pregnancy (due to hCG secretion), after treatment of hyperthyroidism (because TSH can remain suppressed for several months), and in response to certain medications (e.g., high doses of glucocorticoids or dopamine). Importantly, secondary hypothyroidism, caused by hypothalamic-pituitary disease, is associated with a variable (low to high-normal) TSH level, which is

1	high doses of glucocorticoids or dopamine). Importantly, secondary hypothyroidism, caused by hypothalamic-pituitary disease, is associated with a variable (low to high-normal) TSH level, which is inappropriate for the low T4 level. Thus, TSH should not be used as an isolated laboratory test to assess thyroid function in patients with suspected or known pituitary disease.

1	Tests for the end-organ effects of thyroid hormone excess or depletion, such as estimation of basal metabolic rate, tendon reflex relaxation rates, or serum cholesterol, are not useful as clinical determinants of thyroid function. Tests to Determine the Etiology of Thyroid Dysfunction Autoimmune thyroid disease is detected most easily by measuring circulating antibodies against TPO and Tg. Because antibodies to Tg alone are uncommon, it is reasonable to measure only TPO antibodies. About 5–15% of euthyroid women and up to 2% of euthyroid men have thyroid antibodies; such individuals are at increased risk of developing thyroid dysfunction. Almost all patients with autoimmune hypothyroidism, and up to 80% of those with Graves’ disease, have TPO antibodies, usually at high levels.

1	TSIs are antibodies that stimulate the TSH-R in Graves’ disease. They are most commonly measured by commercially available tracer displacement assays called TRAb (TSH receptor antibody) with the assumption that elevated levels in the setting of clinical hyperthyroidism reflect stimulatory effects on the TSH receptor. A bioassay is less commonly used. The main use of these assays is to predict neonatal thyrotoxicosis caused by high maternal levels of TRAb or TSI (>3× upper limit of normal) in the last trimester of pregnancy.

1	Serum Tg levels are increased in all types of thyrotoxicosis except thyrotoxicosis factitia caused by self-administration of thyroid hormone. Tg levels are particularly increased in thyroiditis, reflecting thyroid tissue destruction and release of Tg. The main role for Tg measurement, however, is in the follow-up of thyroid cancer patients. After total thyroidectomy and radioablation, Tg levels should be undetectable; in the absence of anti-Tg antibodies, measurable levels indicate incomplete ablation or recurrent cancer. Radioiodine Uptake and Thyroid Scanning The thyroid gland selectively transports radioisotopes of iodine (123I, 125I, 131I) and 99mTc pertechnetate, allowing thyroid imaging and quantitation of radioactive tracer fractional uptake.

1	Nuclear imaging of Graves’ disease is characterized by an enlarged gland and increased tracer uptake that is distributed homogeneously. Toxic adenomas appear as focal areas of increased uptake, with suppressed tracer uptake in the remainder of the gland. In toxic MNG, the gland is enlarged—often with distorted architecture—and there are multiple areas of relatively increased (functioning nodules) or decreased tracer uptake (suppressed thyroid parenchyma or nonfunctioning nodules). Subacute, viral, and postpartum thyroiditis are associated with very low uptake because of follicular cell damage and TSH suppression. Thyrotoxicosis factitia is also associated with low uptake. In addition, if there is excessive circulating exogenous iodine (e.g., from dietary sources of iodinated contrast dye), the radionuclide uptake is low even in the presence of increased thyroid hormone production.

1	Thyroid scintigraphy is not used in the routine evaluation of patients with thyroid nodules, but should be performed if the serum TSH level is subnormal to determine if functioning thyroid nodules are present. Functioning or “hot” nodules are almost never malignant, and fine-needle aspiration (FNA) biopsy is not indicated. The vast majority of thyroid nodules do not produce thyroid hormone (“cold” nodules), and these are more likely to be malignant (~5–10%). Whole-body and thyroid scanning is also used in the treatment and surveillance of thyroid cancer. After thyroidectomy for thyroid cancer, the TSH level is raised by either using a thyroid hormone withdrawal protocol or recombinant human TSH injection (see below). Administration of 131I allows whole-body scanning (WBS) to confirm remnant ablation and to detect any functioning metastases. In addition, WBS may be helpful in surveillance of patients at risk for recurrence.

1	Thyroid Ultrasound Ultrasonography is valuable for the diagnosis and evaluation of patients with nodular thyroid disease (Table 405-4). Evidence-based guidelines recommend thyroid ultrasonography for all patients suspected of having thyroid nodules by either physical examination or another imaging study. Using 10to 12-MHz linear transducers, resolution and image quality are excellent, allowing the characterization of nodules and cysts >3 mm. Certain sonographic patterns are highly suggestive of malignancy (e.g., hypoechoic solid nodules with infiltrative borders and microcalcifications), whereas other features correlate with benignity (e.g., spongiform nodules defined as those with multiple small internal cystic areas) (Fig. 405-5). In addition to evaluating thyroid nodules, ultrasound is useful for monitoring nodule size and for the aspiration of nodules or cystic lesions. Ultrasound-guided FNA biopsy of thyroid lesions lowers the rate of inadequate sampling and decreases sample

1	is useful for monitoring nodule size and for the aspiration of nodules or cystic lesions. Ultrasound-guided FNA biopsy of thyroid lesions lowers the rate of inadequate sampling and decreases sample error, thereby reducing the false-negative rate of FNA cytology. Ultrasonography of the central and lateral cervical lymph node compartments is indispensable in the evaluation thyroid cancer patients, preoperatively and during follow-up.

1	Iodine deficiency remains a common cause of hypothyroidism worldwide. In areas of iodine sufficiency, autoimmune disease (Hashimoto’s Autoimmune hypothyroidism: Hashimoto’s thyroiditis, atrophic thyroiditis Iatrogenic: 131I treatment, subtotal or total thyroidectomy, external irradiation of neck for lymphoma or cancer Drugs: iodine excess (including iodine-containing contrast media and amiodarone), lithium, antithyroid drugs, p-aminosalicylic acid, interferon α and other cytokines, aminoglutethimide, tyrosine kinase inhibitors (e.g., sunitinib) Congenital hypothyroidism: absent or ectopic thyroid gland, dyshormonogenesis, TSH-R mutation Infiltrative disorders: amyloidosis, sarcoidosis, hemochromatosis, scleroderma, cystinosis, Riedel’s thyroiditis Overexpression of type 3 deiodinase in infantile hemangioma and other tumors Silent thyroiditis, including postpartum thyroiditis Subacute thyroiditis Withdrawal of supraphysiologic thyroxine treatment in individuals with an

1	Silent thyroiditis, including postpartum thyroiditis Subacute thyroiditis Withdrawal of supraphysiologic thyroxine treatment in individuals with an Hypopituitarism: tumors, pituitary surgery or irradiation, infiltrative disorders, Sheehan’s syndrome, trauma, genetic forms of combined pituitary hormone deficiencies Hypothalamic disease: tumors, trauma, infiltrative disorders, idiopathic Abbreviations: TSH, thyroid-stimulating hormone; TSH-R, TSH receptor. thyroiditis) and iatrogenic causes (treatment of hyperthyroidism) are most common (Table 405-5).

1	Abbreviations: TSH, thyroid-stimulating hormone; TSH-R, TSH receptor. thyroiditis) and iatrogenic causes (treatment of hyperthyroidism) are most common (Table 405-5). CONGENITAL HYPOTHYROIDISM Prevalence Hypothyroidism occurs in about 1 in 4000 newborns. It may be transient, especially if the mother has TSH-R blocking antibodies or has received antithyroid drugs, but permanent hypothyroidism occurs in the majority. Neonatal hypothyroidism is due to thyroid gland dysgenesis in 80–85%, to inborn errors of thyroid hormone synthesis in 10–15%, and is TSH-R antibody-mediated in 5% of affected newborns. The developmental abnormalities are twice as common in girls. Mutations that cause congenital hypothyroidism are being increasingly identified, but most remain idiopathic (Table 405-1). Disorders of the Thyroid Gland

1	Disorders of the Thyroid Gland FIGURE 405-5 Sonographic patterns of thyroid nodules. A. High suspicion ultrasound pattern for thyroid malignancy (hypoechoic solid nodule with irregular borders and microcalcifications). B. Very low suspicion ultrasound pattern for thyroid malignancy (spongiform nodule with microcystic areas comprises over >50% of nodule volume). Tiredness, weakness Dry coarse skin; cool peripheral extremities Dry skin Puffy face, hands, and feet Weight gain with poor appetite Carpal tunnel syndrome

1	Tiredness, weakness Dry coarse skin; cool peripheral extremities Dry skin Puffy face, hands, and feet Weight gain with poor appetite Carpal tunnel syndrome Clinical Manifestations The majority of infants appear normal at birth, and <10% are diagnosed based on clinical features, which include prolonged jaundice, feeding problems, hypotonia, enlarged tongue, delayed bone maturation, and umbilical hernia. Importantly, permanent neurologic damage results if treatment is delayed. Typical features of adult hypothyroidism may also be present (Table 405-6). Other congenital malformations, especially cardiac, are four times more common in congenital hypothyroidism.

1	Diagnosis and Treatment Because of the severe neurologic consequences of untreated congenital hypothyroidism, neonatal screening programs have been established. These are generally based on measurement of TSH or T4 levels in heel-prick blood specimens. When the diagnosis is confirmed, T4 is instituted at a dose of 10–15 μg/kg per day, and the dose is adjusted by close monitoring of TSH levels. T4 requirements are relatively great during the first year of life, and a high circulating T4 level is usually needed to normalize TSH. Early treatment with T4 results in normal IQ levels, but subtle neurodevelopmental abnormalities may occur in those with the most severe hypothyroidism at diagnosis or when treatment is delayed or suboptimal.

1	AUTOIMMUNE HYPOTHYROIDISM Classification Autoimmune hypothyroidism may be associated with a goiter (Hashimoto’s, or goitrous thyroiditis) or, at the later stages of the disease, minimal residual thyroid tissue (atrophic thyroiditis). Because the autoimmune process gradually reduces thyroid function, there is a phase of compensation when normal thyroid hormone levels are maintained by a rise in TSH. Although some patients may have minor symptoms, this state is called subclinical hypothyroidism. Later, unbound T4 levels fall and TSH levels rise further; symptoms become more readily apparent at this stage (usually TSH >10 mIU/L), which is referred to as clinical hypothyroidism or overt hypothyroidism.

1	Prevalence The mean annual incidence rate of autoimmune hypothyroidism is up to 4 per 1000 women and 1 per 1000 men. It is more common in certain populations, such as the Japanese, probably because of genetic factors and chronic exposure to a high-iodine diet. The mean age at diagnosis is 60 years, and the prevalence of overt hypothyroidism increases with age. Subclinical hypothyroidism is found in 6–8% of women (10% over the age of 60) and 3% of men. The annual risk of developing clinical hypothyroidism is about 4% when subclinical hypothyroidism is associated with positive TPO antibodies.

1	Pathogenesis In Hashimoto’s thyroiditis, there is a marked lymphocytic infiltration of the thyroid with germinal center formation, atrophy of the thyroid follicles accompanied by oxyphil metaplasia, absence of colloid, and mild to moderate fibrosis. In atrophic thyroiditis, the fibrosis is much more extensive, lymphocyte infiltration is less pronounced, and thyroid follicles are almost completely absent. Atrophic thyroiditis likely represents the end stage of Hashimoto’s thyroiditis rather than a distinct disorder.

1	As with most autoimmune disorders, susceptibility to autoimmune hypothyroidism is determined by a combination of genetic and environmental factors, and the risk of either autoimmune hypothyroidism or Graves’ disease is increased among siblings. HLA-DR polymorphisms are the best documented genetic risk factors for autoimmune hypothyroidism, especially HLA-DR3, -DR4, and -DR5 in Caucasians. A weak association also exists between polymorphisms in CTLA-4, a T cell–regulatory gene, and autoimmune hypothyroidism. Both of these genetic associations are shared by other autoimmune diseases, which may explain the relationship between autoimmune hypothyroidism and other autoimmune diseases, especially type 1 diabetes mellitus, Addison’s disease, pernicious anemia, and vitiligo. HLA-DR and CTLA-4 polymorphisms account for approximately half of the genetic susceptibility to autoimmune hypothyroidism. Other contributory loci remain to be identified. A gene on chromosome 21 may be responsible for

1	account for approximately half of the genetic susceptibility to autoimmune hypothyroidism. Other contributory loci remain to be identified. A gene on chromosome 21 may be responsible for the association between autoimmune hypothyroidism and Down’s syndrome. The female preponderance of thyroid autoimmunity is most likely due to sex steroid effects on the immune response, but an X chromosome–related genetic factor is also possible and may account for the high frequency of autoimmune hypothyroidism in Turner’s syndrome. Environmental susceptibility factors are poorly defined at present. A high iodine intake and decreased exposure to microorganisms in childhood increase the risk of autoimmune hypothyroidism. These factors may account for the increase in prevalence over the last two to three decades.

1	The thyroid lymphocytic infiltrate in autoimmune hypothyroidism is composed of activated CD4+ and CD8+ T cells as well as B cells. Thyroid cell destruction is primarily mediated by the CD8+ cytotoxic T cells, which destroy their targets by either perforin-induced cell necrosis or granzyme B–induced apoptosis. In addition, local T cell production of cytokines, such as tumor necrosis factor (TNF), IL-1, and interferon γ (IFN-γ), may render thyroid cells more susceptible to apoptosis mediated by death receptors, such as Fas, which are activated by their respective ligands on T cells. These cytokines also impair thyroid cell function directly and induce the expression of other proinflammatory molecules by the thyroid cells themselves, such as cytokines, HLA class I and class II molecules, adhesion molecules, CD40, and nitric oxide. Administration of high concentrations of cytokines for therapeutic purposes (especially IFN-α) is associated with increased autoimmune thyroid disease,

1	adhesion molecules, CD40, and nitric oxide. Administration of high concentrations of cytokines for therapeutic purposes (especially IFN-α) is associated with increased autoimmune thyroid disease, possibly through mechanisms similar to those in sporadic disease.

1	Antibodies to TPO and Tg are clinically useful markers of thyroid autoimmunity, but any pathogenic effect is restricted to a secondary role in amplifying an ongoing autoimmune response. TPO antibodies fix complement, and complement membrane-attack complexes are present in the thyroid in autoimmune hypothyroidism. However, transplacental passage of Tg or TPO antibodies has no effect on the fetal thyroid, which suggests that T cell–mediated injury is required to initiate autoimmune damage to the thyroid.

1	Up to 20% of patients with autoimmune hypothyroidism have antibodies against the TSH-R, which, in contrast to TSI, do not stimulate the receptor but prevent the binding of TSH. These TSH-R-blocking antibodies, therefore, cause hypothyroidism and, especially in Asian patients, thyroid atrophy. Their transplacental passage may induce transient neonatal hypothyroidism. Rarely, patients have a mixture of TSI and TSH-R-blocking antibodies, and thyroid function can oscillate between hyperthyroidism and hypothyroidism as one or the other antibody becomes dominant. Predicting the course of disease in such individuals is difficult, and they require close monitoring of thyroid function. Bioassays can be used to document that TSH-R-blocking antibodies reduce the cyclic AMP–inducing effect of TSH on cultured TSH-R-expressing cells, but these assays are difficult to perform. Thyrotropin-binding inhibitory immunoglobulin (TBII) assays that measure the binding of antibodies to the receptor by

1	on cultured TSH-R-expressing cells, but these assays are difficult to perform. Thyrotropin-binding inhibitory immunoglobulin (TBII) assays that measure the binding of antibodies to the receptor by competition with radiolabeled TSH do not distinguish between TSIand TSH-Rblocking antibodies, but a positive result in a patient with spontaneous hypothyroidism is strong evidence for the presence of blocking antibodies. The use of these assays does not generally alter clinical management, although it may be useful to confirm the cause of transient neonatal hypothyroidism.

1	Clinical Manifestations The main clinical features of hypothyroidism are summarized in Table 405-6. The onset is usually insidious, and the patient may become aware of symptoms only when euthyroidism is restored. Patients with Hashimoto’s thyroiditis may present because of goiter rather than symptoms of hypothyroidism. The goiter may not be large, but it is usually irregular and firm in consistency. It is often possible to palpate a pyramidal lobe, normally a vestigial remnant of the thyroglossal duct. Rarely is uncomplicated Hashimoto’s thyroiditis associated with pain.

1	Patients with atrophic thyroiditis or the late stage of Hashimoto’s thyroiditis present with symptoms and signs of hypothyroidism. The skin is dry, and there is decreased sweating, thinning of the epidermis, and hyperkeratosis of the stratum corneum. Increased dermal glycosaminoglycan content traps water, giving rise to skin thickening without pitting (myxedema). Typical features include a puffy face with edematous eyelids and nonpitting pretibial edema (Fig. 405-6). There is pallor, often with a yellow tinge to the skin due to carotene accumulation. Nail growth is retarded, and hair is dry, brittle, difficult to manage, and falls out easily. In addition to diffuse alopecia, there is thinning of the outer third of the eyebrows, although this is not a specific sign of hypothyroidism.

1	Other common features include constipation and weight gain (despite a poor appetite). In contrast to popular perception, the weight gain is usually modest and due mainly to fluid retention in the myxedematous tissues. Libido is decreased in both sexes, and there may be oligomenorrhea or amenorrhea in long-standing disease, but menorrhagia is also common. Fertility is reduced, and the incidence of miscarriage is increased. Prolactin levels are often modestly increased (Chap. 403) and may contribute to alterations in libido and fertility and cause galactorrhea. Myocardial contractility and pulse rate are reduced, leading to a reduced stroke volume and bradycardia. Increased peripheral resistance may be accompanied by hypertension, particularly diastolic. Blood flow is diverted from the skin, producing cool extremities. Pericardial effusions occur in up to 30% of patients but rarely compromise cardiac

1	FIGURE 405-6 Facial appearance in hypothyroidism. Note puffy eyes and thickened skin. function. Although alterations in myosin heavy chain isoform expres-2291 sion have been documented, cardiomyopathy is unusual. Fluid may also accumulate in other serous cavities and in the middle ear, giving rise to conductive deafness. Pulmonary function is generally normal, but dyspnea may be caused by pleural effusion, impaired respiratory muscle function, diminished ventilatory drive, or sleep apnea.

1	Carpal tunnel and other entrapment syndromes are common, as is impairment of muscle function with stiffness, cramps, and pain. On examination, there may be slow relaxation of tendon reflexes and pseudomyotonia. Memory and concentration are impaired. Experimentally, positron emission tomography (PET) scans examining glucose metabolism in hypothyroid subjects show lower regional activity in the amygdala, hippocampus, and perigenual anterior cingulated cortex, among other regions, and this activity corrects after thyroxine replacement. Rare neurologic problems include reversible cerebellar ataxia, dementia, psychosis, and myxedema coma. Hashimoto’s encephalopathy has been defined as a steroid-responsive syndrome associated with TPO antibodies, myoclonus, and slow-wave activity on electroencephalography, but the relationship with thyroid autoimmunity or hypothyroidism is not established. The hoarse voice and occasionally clumsy speech of hypothyroidism reflect fluid accumulation in the

1	but the relationship with thyroid autoimmunity or hypothyroidism is not established. The hoarse voice and occasionally clumsy speech of hypothyroidism reflect fluid accumulation in the vocal cords and tongue.

1	The features described above are the consequence of thyroid hormone deficiency. However, autoimmune hypothyroidism may be associated with signs or symptoms of other autoimmune diseases, particularly vitiligo, pernicious anemia, Addison’s disease, alopecia areata, and type 1 diabetes mellitus. Less common associations include celiac disease, dermatitis herpetiformis, chronic active hepatitis, rheumatoid arthritis, systemic lupus erythematosus (SLE), myasthenia gravis, and Sjögren’s syndrome. Thyroid-associated ophthalmopathy, which usually occurs in Graves’ disease (see below), occurs in about 5% of patients with autoimmune hypothyroidism.

1	Autoimmune hypothyroidism is uncommon in children and usually presents with slow growth and delayed facial maturation. The appearance of permanent teeth is also delayed. Myopathy, with muscle swelling, is more common in children than in adults. In most cases, puberty is delayed, but precocious puberty sometimes occurs. There may be intellectual impairment if the onset is before 3 years and the hormone deficiency is severe.

1	Laboratory Evaluation A summary of the investigations used to determine the existence and cause of hypothyroidism is provided in Fig. 405-7. A normal TSH level excludes primary (but not secondary) hypothyroidism. If the TSH is elevated, an unbound T4 level is needed to confirm the presence of clinical hypothyroidism, but T4 is inferior to TSH when used as a screening test, because it will not detect subclinical hypothyroidism. Circulating unbound T3 levels are normal in about 25% of patients, reflecting adaptive deiodinase responses to hypothyroidism. T3 measurements are, therefore, not indicated.

1	Once clinical or subclinical hypothyroidism is confirmed, the etiology is usually easily established by demonstrating the presence of TPO antibodies, which are present in >90% of patients with autoimmune hypothyroidism. TBII can be found in 10–20% of patients, but measurement is not needed routinely. If there is any doubt about the cause of a goiter associated with hypothyroidism, FNA biopsy can be used to confirm the presence of autoimmune thyroiditis. Other abnormal laboratory findings in hypothyroidism may include increased creatine phosphokinase, elevated cholesterol and triglycerides, and anemia (usually normocytic or macrocytic). Except when accompanied by iron deficiency, the anemia and other abnormalities gradually resolve with thyroxine replacement.

1	Differential Diagnosis An asymmetric goiter in Hashimoto’s thyroiditis may be confused with a MNG or thyroid carcinoma, in which thyroid antibodies may also be present. Ultrasound can be used to show the presence of a solitary lesion or an MNG rather than the heterogeneous thyroid enlargement typical of Hashimoto’s thyroiditis. FNA biopsy is useful in the investigation of focal nodules. Other causes of hypothyroidism are discussed below and in Table 405-5 but rarely cause diagnostic confusion. Disorders of the Thyroid Gland however, lower doses suffice until

1	Disorders of the Thyroid Gland however, lower doses suffice until Measure unbound T4 Mild Primary Pituitary disease suspected? Measure unbound T4 hypothyroidism hypothyroidism tests Measure TSH Elevated Normal Low Normal Normal Low Autoimmune hypothyroidismRule out otherYesNo causes of hypothyroidism T4 treatment Annual follow-up T4 treatment TPOAb+ or TPOAb– , no TPOAb+ TPOAb– symptomatic symptoms No further Rule out drug effects, sick euthyroid syndrome, then evaluate anterior pituitary function No further tests residual thyroid tissue is destroyed. In patients who develop hypothyroidism after the treatment of Graves’ disease, there is often underlying autonomous function, necessitating lower replacement doses (typically 75–125 μg/d).

1	Adult patients under 60 years old without evidence of heart disease may be started on 50–100 μg levothyroxine (T4) daily. The dose is adjusted on the basis of TSH levels, with the goal of treatment being a normal TSH, ideally in the lower half of the reference range. TSH responses are gradual and should be measured about 2 months after instituting treatment or after any subsequent change in levothyroxine dosage. The clinical effects of levothyroxine replacement are slow to appear. Patients may not experience full relief from symptoms until 3–6 months after normal TSH levels are FIGURE 405-7 Evaluation of hypothyroidism. TPOAb+, thyroid peroxidase antibodies present; restored. Adjustment of levothyroxine TPOAb−, thyroid peroxidase antibodies not present; TSH, thyroid-stimulating hormone. dosage is made in 12.5or 25-μg increments if the TSH is high; decrements of the same magnitude should be made if the TSH is suppressed. Patients with a suppressed TSH of any

1	dosage is made in 12.5or 25-μg increments if the TSH is high; decrements of the same magnitude should be made if the TSH is suppressed. Patients with a suppressed TSH of any Iatrogenic hypothyroidism is a common cause of hypothyroidism and cause, including T4 overtreatment, have an increased risk of atrial can often be detected by screening before symptoms develop. In the fibrillation and reduced bone density. first 3–4 months after radioiodine treatment, transient hypothyroid- ism may occur due to reversible radiation damage. Low-dose thy- USP) are available, they are not recommended because the ratio of roxine treatment can be withdrawn if recovery occurs. Because TSH

1	USP) are available, they are not recommended because the ratio of roxine treatment can be withdrawn if recovery occurs. Because TSH T to T is nonphysiologic. The use of levothyroxine combined with levels are suppressed by hyperthyroidism, unbound T4 levels are a 34 liothyronine (triiodothyronine, T ) has been investigated, but benefit better measure of thyroid function than TSH in the months following 3 has not been confirmed in prospective studies. There is no place radioiodine treatment. Mild hypothyroidism after subtotal thyroidec for liothyronine alone as long-term replacement, because the short tomy may also resolve after several months, as the gland remnant is half-life necessitates three or four daily doses and is associated with stimulated by increased TSH levels. fluctuating T levels. Iodine deficiency is responsible for endemic goiter and cretinism 3

1	fluctuating T levels. Iodine deficiency is responsible for endemic goiter and cretinism 3 Once full replacement is achieved and TSH levels are stable, but is an uncommon cause of adult hypothyroidism unless the iodine follow-up measurement of TSH is recommended at annual intervals intake is very low or there are complicating factors, such as the con- and may be extended to every 2–3 years if a normal TSH is main sumption of thiocyanates in cassava or selenium deficiency. Although tained over several years. It is important to ensure ongoing adher hypothyroidism due to iodine deficiency can be treated with thyroxine, ence, however, as patients do not feel any symptomatic difference public health measures to improve iodine intake should be advocated after missing a few doses of levothyroxine, and this sometimes leads to eliminate this problem. Iodized salt or bread or a single bolus of oral to self-discontinuation. or intramuscular iodized oil have all been used successfully.

1	or intramuscular iodized oil have all been used successfully. In patients of normal body weight who are taking ≥200 μg of levo- Paradoxically, chronic iodine excess can also induce goiter and thyroxine per day, an elevated TSH level is often a sign of poor adher hypothyroidism. The intracellular events that account for this effect ence to treatment. This is also the likely explanation for fluctuating are unclear, but individuals with autoimmune thyroiditis are espe- TSH levels, despite a constant levothyroxine dosage. Such patients cially susceptible. Iodine excess is responsible for the hypothyroidism often have normal or high unbound T4 levels, despite an elevated that occurs in up to 13% of patients treated with amiodarone (see

1	TSH, because they remember to take medication for a few days below). Other drugs, particularly lithium, may also cause hypothy before testing; this is sufficient to normalize T , but not TSH levels. It roidism. Transient hypothyroidism caused by thyroiditis is discussed 4 is important to consider variable adherence, because this pattern of below. thyroid function tests is otherwise suggestive of disorders associated

1	Secondary hypothyroidism is usually diagnosed in the context of with inappropriate TSH secretion (Table 405-3). Because T has a long half-life (7 days), patients who miss a dose can be advised to take is very rare (Chap. 402). TSH levels may be low, normal, or even two doses of the skipped tablets at once. Other causes of increased slightly increased in secondary hypothyroidism; the latter is due to levothyroxine requirements must be excluded, particularly malab secretion of immunoactive but bioinactive forms of TSH. The diagno sorption (e.g., celiac disease, small-bowel surgery), estrogen or selec sis is confirmed by detecting a low unbound T level. The goal of treat 4 tive estrogen receptor modulator therapy, ingestion with a meal, ment is to maintain T levels in the upper half of the reference range, 4 and drugs that interfere with T absorption or metabolism such as because TSH levels cannot be used to monitor therapy. 4 cholestyramine, ferrous sulfate, calcium supplements, proton

1	range, 4 and drugs that interfere with T absorption or metabolism such as because TSH levels cannot be used to monitor therapy. 4 cholestyramine, ferrous sulfate, calcium supplements, proton pump inhibitors, lovastatin, aluminum hydroxide, rifampicin, amiodarone, carbamazepine, phenytoin, and tyrosine kinase inhibitors.

1	If there is no residual thyroid function, the daily replacement dose By definition, subclinical hypothyroidism refers to biochemical of levothyroxine is usually 1.6 μg/kg body weight (typically 100–150 evidence of thyroid hormone deficiency in patients who have few μg), ideally taken at least 30 min before breakfast. In many patients, or no apparent clinical features of hypothyroidism. There are no universally accepted recommendations for the management of subclinical hypothyroidism, but levothyroxine is recommended if the patient is a woman who wishes to conceive or is pregnant, or when TSH levels are above 10 mIU/L. When TSH levels are below 10 mIU/L, treatment should be considered when patients have suggestive symptoms of hypothyroidism, positive TPO antibodies, or any evidence of heart disease. It is important to confirm that any elevation of TSH is sustained over a 3-month period before treatment is given. As long as excessive treatment is avoided, there is no risk in correcting

1	disease. It is important to confirm that any elevation of TSH is sustained over a 3-month period before treatment is given. As long as excessive treatment is avoided, there is no risk in correcting a slightly increased TSH. Treatment is administered by starting with a low dose of levothyroxine (25–50 μg/d) with the goal of normalizing TSH. If levothyroxine is not given, thyroid function should be evaluated annually.

1	Rarely, levothyroxine replacement is associated with pseudotumor cerebri in children. Presentation appears to be idiosyncratic and occurs months after treatment has begun.

1	Women with a history or high risk of hypothyroidism should ensure that they are euthyroid prior to conception and during early pregnancy because maternal hypothyroidism may adversely affect fetal neural development and cause preterm delivery. The presence of thyroid autoantibodies alone, in a euthyroid patient, is also associated with miscarriage and preterm delivery; it is unclear if levothyroxine therapy improves outcomes. Thyroid function should be evaluated immediately after pregnancy is confirmed and every 4 weeks during the first half of the pregnancy, with less frequent testing after 20 weeks’ gestation (every 6–8 weeks depending on whether levothyroxine dose adjustment is ongoing). The levothyroxine dose may need to be increased by up to 50% during pregnancy, with a goal TSH of less than 2.5 mIU/L during the first trimester and less than 3.0 mIU/L during the second and third trimesters. After delivery, thyroxine doses typically return to prepregnancy levels. Pregnant women

1	less than 2.5 mIU/L during the first trimester and less than 3.0 mIU/L during the second and third trimesters. After delivery, thyroxine doses typically return to prepregnancy levels. Pregnant women should be counseled to separate ingestion of prenatal vitamins and iron supplements from levothyroxine by at least 4 h.

1	Elderly patients may require 20% less thyroxine than younger patients. In the elderly, especially patients with known coronary artery disease, the starting dose of levothyroxine is 12.5–25 μg/d with similar increments every 2–3 months until TSH is normalized. In some patients, it may be impossible to achieve full replacement despite optimal antianginal treatment. Emergency surgery is generally safe in patients with untreated hypothyroidism, although routine surgery in a hypothyroid patient should be deferred until euthyroidism is achieved.

1	Myxedema coma still has a 20–40% mortality rate, despite intensive treatment, and outcomes are independent of the T4 and TSH levels. Clinical manifestations include reduced level of consciousness, sometimes associated with seizures, as well as the other features of hypothyroidism (Table 405-6). Hypothermia can reach 23°C (74°F). There may be a history of treated hypothyroidism with poor compliance, or the patient may be previously undiagnosed. Myxedema coma almost always occurs in the elderly and is usually precipitated by factors that impair respiration, such as drugs (especially sedatives, anesthetics, and antidepressants), pneumonia, congestive heart failure, myocardial infarction, gastrointestinal bleeding, or cerebrovascular accidents. Sepsis should also be suspected. Exposure to cold may also be a risk factor. Hypoventilation, leading to hypoxia and hypercapnia, plays a major role in pathogenesis; hypoglycemia and dilutional hyponatremia also contribute to the development of

1	cold may also be a risk factor. Hypoventilation, leading to hypoxia and hypercapnia, plays a major role in pathogenesis; hypoglycemia and dilutional hyponatremia also contribute to the development of myxedema coma.

1	Levothyroxine can initially be administered as a single IV bolus of 500 μg, which serves as a loading dose. Although further levothyroxine is not strictly necessary for several days, it is usually continued at a dose of 50–100 μg/d. If suitable IV preparation is not available, the same initial dose of levothyroxine can be given by nasogastric tube (although absorption may be impaired in myxedema). An alternative is to give liothyronine (T3) intravenously or via nasogastric tube, in doses ranging from 10 to 25 μg every 8–12 h. This treatment has been advocated because T4 → T3 conversion is impaired in myxedema coma. However, excess liothyronine has the potential to provoke 2293 arrhythmias. Another option is to combine levothyroxine (200 μg) and liothyronine (25 μg) as a single, initial IV bolus followed by daily treatment with levothyroxine (50–100 μg/d) and liothyronine (10 μg every 8 h).

1	Supportive therapy should be provided to correct any associated metabolic disturbances. External warming is indicated only if the temperature is <30°C, as it can result in cardiovascular collapse (Chap. 478e). Space blankets should be used to prevent further heat loss. Parenteral hydrocortisone (50 mg every 6 h) should be administered, because there is impaired adrenal reserve in profound hypothyroidism. Any precipitating factors should be treated, including the early use of broad-spectrum antibiotics, pending the exclusion of infection. Ventilatory support with regular blood gas analysis is usually needed during the first 48 h. Hypertonic saline or IV glucose may be needed if there is severe hyponatremia or hypoglycemia; hypotonic IV fluids should be avoided because they may exacerbate water retention secondary to reduced renal perfusion and inappropriate vasopressin secretion. The metabolism of most medications is impaired, and sedatives should be avoided if possible or used in

1	water retention secondary to reduced renal perfusion and inappropriate vasopressin secretion. The metabolism of most medications is impaired, and sedatives should be avoided if possible or used in reduced doses. Medication blood levels should be monitored, when available, to guide dosage.

1	Thyrotoxicosis is defined as the state of thyroid hormone excess and is not synonymous with hyperthyroidism, which is the result of excessive thyroid function. However, the major etiologies of thyrotoxicosis are hyperthyroidism caused by Graves’ disease, toxic MNG, and toxic adenomas. Other causes are listed in Table 405-7. GRAVES’ DISEASE Epidemiology Graves’ disease accounts for 60–80% of thyrotoxicosis. The prevalence varies among populations, reflecting genetic factors and iodine intake (high iodine intake is associated with an increased prevalence of Graves’ disease). Graves’ disease occurs in up to 2% of women but is one-tenth as frequent in men. The disorder rarely begins before adolescence and typically occurs between 20 and 50 years of age; it also occurs in the elderly. Activating mutation of the TSH receptor Activating mutation of GSα (McCune-Albright syndrome) Drugs: iodine excess (Jod-Basedow phenomenon)

1	Activating mutation of the TSH receptor Activating mutation of GSα (McCune-Albright syndrome) Drugs: iodine excess (Jod-Basedow phenomenon) Subacute thyroiditis Silent thyroiditis Other causes of thyroid destruction: amiodarone, radiation, infarction of adenoma Ingestion of excess thyroid hormone (thyrotoxicosis factitia) or thyroid tissue Thyroid hormone resistance syndrome: occasional patients may have features of thyrotoxicosis Chorionic gonadotropin-secreting tumorsa Gestational thyrotoxicosisa aCirculating TSH levels are low in these forms of secondary hyperthyroidism. Abbreviations: TSH, thyroid-stimulating hormone.

1	Disorders of the Thyroid Gland 2294 Pathogenesis As in autoimmune hypothyroidism, a combination of environmental and genetic factors, including polymorphisms in HLA-DR, the immunoregulatory genes CTLA-4, CD25, PTPN22, FCRL3, and CD226, as well as the TSH-R, contribute to Graves’ disease susceptibility. The concordance for Graves’ disease in monozygotic twins is 20–30%, compared to <5% in dizygotic twins. Indirect evidence suggests that stress is an important environmental factor, presumably operating through neuroendocrine effects on the immune system. Smoking is a minor risk factor for Graves’ disease and a major risk factor for the development of ophthalmopathy. Sudden increases in iodine intake may precipitate Graves’ disease, and there is a threefold increase in the occurrence of Graves’ disease in the postpartum period. Graves’ disease may occur during the immune reconstitution phase after highly active antiretroviral therapy (HAART) or alemtuzumab treatment. The hyperthyroidism

1	disease in the postpartum period. Graves’ disease may occur during the immune reconstitution phase after highly active antiretroviral therapy (HAART) or alemtuzumab treatment. The hyperthyroidism of Graves’ disease is caused by TSI that are synthesized in the thyroid gland as well as in bone marrow and lymph nodes. Such antibodies can be detected by bioassays or by using the more widely available TBII assays. The presence of TBII in a patient with thyrotoxicosis implies the existence of TSI, and these assays are useful in monitoring pregnant Graves’ patients in whom high levels of TSI can cross the placenta and cause neonatal thyrotoxicosis. Other thyroid autoimmune responses, similar to those in autoimmune hypothyroidism (see above), occur concurrently in patients with Graves’ disease. In particular, TPO antibodies occur in up to 80% of cases and serve as a readily measurable marker of autoimmunity. Because the coexisting thyroiditis can also affect thyroid function, there is no

1	In particular, TPO antibodies occur in up to 80% of cases and serve as a readily measurable marker of autoimmunity. Because the coexisting thyroiditis can also affect thyroid function, there is no direct correlation between the level of TSI and thyroid hormone levels in Graves’ disease. In the long term, spontaneous autoimmune hypothyroidism may develop in up to 15% of patients with Graves’ disease. Cytokines appear to play a major role in thyroid-associated ophthalmopathy. There is infiltration of the extraocular muscles by activated T cells; the release of cytokines such as IFN-γ, TNF, and IL-1 results in fibroblast activation and increased synthesis of glycosaminoglycans that trap water, thereby leading to characteristic muscle swelling. Late in the disease, there is irreversible fibrosis of the muscles. Orbital fibroblasts may be particularly sensitive to cytokines, perhaps explaining the anatomic localization of the immune response. Though the pathogenesis of thyroid-associated

1	of the muscles. Orbital fibroblasts may be particularly sensitive to cytokines, perhaps explaining the anatomic localization of the immune response. Though the pathogenesis of thyroid-associated ophthalmopathy remains unclear, there is mounting evidence that the TSH-R may be a shared autoantigen that is expressed in the orbit; this would explain the close association with autoimmune thyroid disease. Increased fat is an additional cause of retrobulbar tissue expansion. The increase in intraorbital pressure can lead to proptosis, diplopia, and optic neuropathy.

1	Clinical Manifestations Signs and symptoms include features that are common to any cause of thyrotoxicosis (Table 405-8) as well as those specific for Graves’ disease. The clinical presentation depends on the severity of thyrotoxicosis, the duration of disease, individual susceptibility to excess thyroid hormone, and the patient’s age. In the elderly, features of thyrotoxicosis may be subtle or masked, and patients may present mainly with fatigue and weight loss, a condition known as apathetic thyrotoxicosis. Hyperactivity, irritability, dysphoria Tachycardia; atrial fibrillation in the elderly Fatigue and weakness Warm, moist skin Weight loss with increased appetite Muscle weakness, proximal myopathy Oligomenorrhea, loss of libido aExcludes the signs of ophthalmopathy and dermopathy specific for Graves’ disease.

1	Thyrotoxicosis may cause unexplained weight loss, despite an enhanced appetite, due to the increased metabolic rate. Weight gain occurs in 5% of patients, however, because of increased food intake. Other prominent features include hyperactivity, nervousness, and irritability, ultimately leading to a sense of easy fatigability in some patients. Insomnia and impaired concentration are common; apathetic thyrotoxicosis may be mistaken for depression in the elderly. Fine tremor is a frequent finding, best elicited by having patients stretch out their fingers while feeling the fingertips with the palm. Common neurologic manifestations include hyperreflexia, muscle wasting, and proximal myopathy without fasciculation. Chorea is rare. Thyrotoxicosis is sometimes associated with a form of hypokalemic periodic paralysis; this disorder is particularly common in Asian males with thyrotoxicosis, but it occurs in other ethnic groups as well.

1	The most common cardiovascular manifestation is sinus tachycardia, often associated with palpitations, occasionally caused by supra-ventricular tachycardia. The high cardiac output produces a bounding pulse, widened pulse pressure, and an aortic systolic murmur and can lead to worsening of angina or heart failure in the elderly or those with preexisting heart disease. Atrial fibrillation is more common in patients >50 years of age. Treatment of the thyrotoxic state alone converts atrial fibrillation to normal sinus rhythm in about half of patients, suggesting the existence of an underlying cardiac problem in the remainder.

1	The skin is usually warm and moist, and the patient may complain of sweating and heat intolerance, particularly during warm weather. Palmar erythema, onycholysis, and, less commonly, pruritus, urticaria, and diffuse hyperpigmentation may be evident. Hair texture may become fine, and a diffuse alopecia occurs in up to 40% of patients, persisting for months after restoration of euthyroidism. Gastrointestinal transit time is decreased, leading to increased stool frequency, often with diarrhea and occasionally mild steatorrhea. Women frequently experience oligomenorrhea or amenorrhea; in men, there may be impaired sexual function and, rarely, gynecomastia. The direct effect of thyroid hormones on bone resorption leads to osteopenia in longstanding thyrotoxicosis; mild hypercalcemia occurs in up to 20% of patients, but hypercalciuria is more common. There is a small increase in fracture rate in patients with a previous history of thyrotoxicosis.

1	In Graves’ disease, the thyroid is usually diffusely enlarged to two to three times its normal size. The consistency is firm, but not nodular. There may be a thrill or bruit, best detected at the inferolateral margins of the thyroid lobes, due to the increased vascularity of the gland and the hyperdynamic circulation.

1	Lid retraction, causing a staring appearance, can occur in any form of thyrotoxicosis and is the result of sympathetic overactivity. However, Graves’ disease is associated with specific eye signs that comprise Graves’ ophthalmopathy (Fig. 405-8A). This condition is also called thyroid-associated ophthalmopathy, because it occurs in the absence of hyperthyroidism in 10% of patients. Most of these individuals have autoimmune hypothyroidism or thyroid antibodies. The onset of Graves’ ophthalmopathy occurs within the year before or after the diagnosis of thyrotoxicosis in 75% of patients but can sometimes precede or follow thyrotoxicosis by several years, accounting for some cases of euthyroid ophthalmopathy.

1	Some patients with Graves’ disease have little clinical evidence of ophthalmopathy. However, the enlarged extraocular muscles typical of the disease, and other subtle features, can be detected in almost all patients when investigated by ultrasound or computed tomography (CT) imaging of the orbits. Unilateral signs are found in up to 10% of patients. The earliest manifestations of ophthalmopathy are usually a sensation of grittiness, eye discomfort, and excess tearing. About one-third of patients have proptosis, best detected by visualization of the sclera between the lower border of the iris and the lower eyelid, with the eyes in the primary position. Proptosis can be measured using an exophthalmometer. In severe cases, proptosis may cause corneal exposure and damage, especially if the lids fail to close during sleep. Periorbital edema, scleral injection, and chemosis are also frequent. In 5–10% of patients, the muscle swelling is so severe that diplopia results, typically, but not

1	lids fail to close during sleep. Periorbital edema, scleral injection, and chemosis are also frequent. In 5–10% of patients, the muscle swelling is so severe that diplopia results, typically, but not exclusively, when the patient looks up and laterally. The most serious manifestation is compression of the optic nerve at

1	Primary thyrotoxicosis Features of Graves’ diseasea? T3 toxicosis Subclinical hyperthyroidism TSH low, unbound T4 high Measure TSH, unbound T4 Normal Measure unbound T3 TSH normal or increased, high unbound T4 TSH-secreting pituitary adenoma or thyroid hormone resistance syndrome Follow up in 6-12 weeks High TSH low, unbound T4 normal TSH and unbound T4 normal No further tests Graves’ disease Multinodular goiter or toxic adenomab? Yes No Toxic nodular hyperthyroidism Low radionuclide uptake? Yes No Destructive thyroiditis, iodine excess or excess thyroid hormone Rule out other causes including stimulation by chorionic gonadotropin

1	Disorders of the Thyroid Gland that assess disease activity are preferable for monitoring purposes. When Graves’ eye disease is active and severe, referral to an ophthalmologist is indicated and objective measurements are needed, such as lid-fissure width; corneal staining with fluorescein; and evaluation of extraocular muscle function (e.g., Hess chart), intra-ocular pressure and visual fields, acuity, and color vision. Thyroid dermopathy occurs in <5% of patients with Graves’ disease (Fig. 405-8B), almost always in the presence of moderate or severe ophthalmopathy. Although most frequent over the anterior and lateral aspects of the lower leg FIGURE 405-9 Evaluation of thyrotoxicosis. aDiffuse goiter, positive TPO antibodies or TRAb, (hence the term pretibial myxedema), ophthalmopathy, dermopathy. bCan be confirmed by radionuclide scan. TSH, thyroid-stimulating skin changes can occur at other sites, hormone.

1	FIGURE 405-8 Features of Graves’ disease. A. Ophthalmopathy in Graves’ disease; lid retraction, periorbital edema, conjunctival injection, and proptosis are marked. B. Thyroid dermopathy over the lateral aspects of the shins. C. Thyroid acropachy. the apex of the orbit, leading to papilledema; peripheral field defects; and, if left untreated, permanent loss of vision. The “NO SPECS” scoring system to evaluate ophthalmopathy is an acronym derived from the following changes: 0 = No signs or symptoms 1 = Only signs (lid retraction or lag), no symptoms 2 = Soft tissue involvement (periorbital

1	Although useful as a mnemonic, the NO SPECS scheme is inadequate to describe the eye disease fully, and patients do not necessarily progress from one class to another; alternative scoring systems particularly after trauma. The typical lesion is a noninflamed, indu-2295 rated plaque with a deep pink or purple color and an “orange skin” appearance. Nodular involvement can occur, and the condition can rarely extend over the whole lower leg and foot, mimicking elephantiasis. Thyroid acropachy refers to a form of clubbing found in <1% of patients with Graves’ disease (Fig. 405-8C). It is so strongly associated with thyroid dermopathy that an alternative cause of clubbing should be sought in a Graves’ patient without coincident skin and orbital involvement.

1	Laboratory Evaluation Investigations used to determine the existence and cause of thyrotoxicosis are summarized in Fig. 405-9. In Graves’ disease, the TSH level is suppressed, and total and unbound thyroid hormone levels are increased. In 2–5% of patients (and more in areas of borderline iodine intake), only T3 is increased (T3 toxicosis). The converse state of T4 toxicosis, with elevated total and unbound T4 and normal T3 levels, is occasionally seen when hyperthyroidism is induced by excess iodine, providing surplus substrate for thyroid hormone synthesis. Measurement of TPO antibodies or TRAb may be useful if the diagnosis is unclear clinically but is not needed routinely. Associated abnormalities that may cause diagnostic confusion in thyrotoxicosis include elevation of bilirubin, liver enzymes, and ferritin. Microcytic anemia and thrombocytopenia may occur.

1	Differential Diagnosis Diagnosis of Graves’ disease is straightforward in a patient with biochemically confirmed thyrotoxicosis, diffuse goiter on palpation, ophthalmopathy, and often a personal or family history of autoimmune disorders. For patients with thyrotoxicosis who lack these features, the diagnosis is generally established by a radionuclide (99mTc, 123I, or 131I) scan and uptake of the thyroid, which will distinguish the diffuse, high uptake of Graves’ disease from destructive thyroiditis, ectopic thyroid tissue, and factitious thyrotoxicosis. Scintigraphy is the preferred diagnostic test; however, TRAb measurement can be used to assess autoimmune activity. In secondary hyperthyroidism due to a 2296 TSH-secreting pituitary tumor, there is also a diffuse goiter. The presence of a nonsuppressed TSH level and the finding of a pituitary tumor on CT or magnetic resonance scan (MRI) scan suggest this diagnosis. Clinical features of thyrotoxicosis can mimic certain aspects of other

1	nonsuppressed TSH level and the finding of a pituitary tumor on CT or magnetic resonance scan (MRI) scan suggest this diagnosis. Clinical features of thyrotoxicosis can mimic certain aspects of other disorders, including panic attacks, mania, pheochromocytoma, and weight loss associated with malignancy. The diagnosis of thyrotoxicosis can be easily excluded if the TSH and unbound T4 and T3 levels are normal. A normal TSH also excludes Graves’ disease as a cause of diffuse goiter.

1	Clinical Course Clinical features generally worsen without treatment; mortality was 10–30% before the introduction of satisfactory therapy. Some patients with mild Graves’ disease experience spontaneous relapses and remissions. Rarely, there may be fluctuation between hypoand hyperthyroidism due to changes in the functional activity of TSH-R antibodies. About 15% of patients who enter remission after treatment develop hypothyroidism 10–15 years later as a result of the destructive autoimmune process.

1	The clinical course of ophthalmopathy does not follow that of the thyroid disease. Ophthalmopathy typically worsens over the initial 3–6 months, followed by a plateau phase over the next 12–18 months, with spontaneous improvement, particularly in the soft tissue changes. However, the course is more fulminant in up to 5% of patients, requiring intervention in the acute phase if there is optic nerve compression or corneal ulceration. Diplopia may appear late in the disease due to fibrosis of the extraocular muscles. Radioiodine treatment for hyperthyroidism worsens the eye disease in a small proportion of patients (especially smokers). Antithyroid drugs or surgery have no adverse effects on the clinical course of ophthalmopathy. Thyroid dermopathy, when it occurs, usually appears 1–2 years after the development of Graves’ hyperthyroidism; it may improve spontaneously.

1	The hyperthyroidism of Graves’ disease is treated by reducing thyroid hormone synthesis, using antithyroid drugs, or reducing the amount of thyroid tissue with radioiodine (131I) treatment or by thyroidectomy. Antithyroid drugs are the predominant therapy in many centers in Europe and Japan, whereas radioiodine is more often the first line of treatment in North America. These differences reflect the fact that no single approach is optimal and that patients may require multiple treatments to achieve remission.

1	The main antithyroid drugs are the thionamides, such as propylthiouracil, carbimazole (not available in the United States), and the active metabolite of the latter, methimazole. All inhibit the function of TPO, reducing oxidation and organification of iodide. These drugs also reduce thyroid antibody levels by mechanisms that remain unclear, and they appear to enhance rates of remission. Propylthiouracil inhibits deiodination of T4 → T3. However, this effect is of minor benefit, except in the most severe thyrotoxicosis, and is offset by the much shorter half-life of this drug (90 min) compared to methimazole (6 h). Due to the hepatotoxicity of propylthiouracil, the U.S. Food and Drug Administration (FDA) has limited indications for its use to the first trimester of pregnancy, the treatment of thyroid storm, and patients with minor adverse reactions to methimazole. If propylthiouracil is used, monitoring of liver function tests is recommended.

1	There are many variations of antithyroid drug regimens. The initial dose of carbimazole or methimazole is usually 10–20 mg every 8 or 12 h, but once-daily dosing is possible after euthyroidism is restored. Propylthiouracil is given at a dose of 100–200 mg every 6–8 h, and divided doses are usually given throughout the course. Lower doses of each drug may suffice in areas of low iodine intake. The starting dose of antithyroid drugs can be gradually reduced (titration regimen) as thyrotoxicosis improves. Alternatively, high doses may be given combined with levothyroxine supplementation (block-replace regimen) to avoid drug-induced hypothyroidism. The titration regimen is preferred to minimize the dose of antithyroid drug and provide an index of treatment response.

1	Thyroid function tests and clinical manifestations are reviewed 4–6 weeks after starting treatment, and the dose is titrated based on unbound T4 levels. Most patients do not achieve euthyroidism until 6–8 weeks after treatment is initiated. TSH levels often remain suppressed for several months and therefore do not provide a sensitive index of treatment response. The usual daily maintenance doses of antithyroid drugs in the titration regimen are 2.5–10 mg of carbimazole or methimazole and 50–100 mg of propylthiouracil. In the block-replace regimen, the initial dose of antithyroid drug is held constant, and the dose of levothyroxine is adjusted to maintain normal unbound T4 levels. When TSH suppression is alleviated, TSH levels can also be used to monitor therapy.

1	Maximum remission rates (up to 30–60% in some populations) are achieved by 12–18 months for the titration regimen and by 6 months for the block-replace regimen. For unclear reasons, remission rates appear to vary in different geographic regions. Younger patients, males, smokers, and patients with severe hyperthyroidism and large goiters are most likely to relapse when treatment stops, but outcomes are difficult to predict. All patients should be followed closely for relapse during the first year after treatment and at least annually thereafter.

1	The common minor side effects of antithyroid drugs are rash, urticaria, fever, and arthralgia (1–5% of patients). These may resolve spontaneously or after substituting an alternative antithyroid drug. Rare but major side effects include hepatitis (propylthiouracil; avoid use in children) and cholestasis (methimazole and carbimazole); an SLE-like syndrome; and, most important, agranulocytosis (<1%). It is essential that antithyroid drugs are stopped and not restarted if a patient develops major side effects. Written instructions should be provided regarding the symptoms of possible agranulocytosis (e.g., sore throat, fever, mouth ulcers) and the need to stop treatment pending an urgent complete blood count to confirm that agranulocytosis is not present. Management of agranulocytosis is described in Chap. 130. It is not useful to monitor blood counts prospectively, because the onset of agranulocytosis is idiosyncratic and abrupt.

1	Propranolol (20–40 mg every 6 h) or longer-acting selective β1 receptor blockers such as atenolol may be helpful to control adrenergic symptoms, especially in the early stages before antithyroid drugs take effect. Beta blockers are also useful in patients with thyrotoxic periodic paralysis, pending correction of thyrotoxicosis. In consultation with a cardiologist, anticoagulation with warfarin should be considered in all patients with atrial fibrillation who often spontaneously revert to sinus rhythm with control of hyperthyroidism. Decreased warfarin doses are required when patients are thyrotoxic. If digoxin is used, increased doses are often needed in the thyrotoxic state.

1	Radioiodine causes progressive destruction of thyroid cells and can be used as initial treatment or for relapses after a trial of antithyroid drugs. There is a small risk of thyrotoxic crisis (see below) after radioiodine, which can be minimized by pretreatment with antithyroid drugs for at least a month before treatment. Antecedent treatment with antithyroid drugs should be considered for all elderly patients or for those with cardiac problems to deplete thyroid hormone stores before administration of radioiodine. Carbimazole or methimazole must be stopped 3–5 days before radioiodine administration to achieve optimum iodine uptake. Propylthiouracil appears to have a prolonged radioprotective effect and should be stopped for a longer period before radioiodine is given, or a larger dose of radioiodine will be necessary.

1	Efforts to calculate an optimal dose of radioiodine that achieves euthyroidism without a high incidence of relapse or progression to hypothyroidism have not been successful. Some patients inevitably relapse after a single dose because the biologic effects of radiation vary between individuals, and hypothyroidism cannot be uniformly avoided even using accurate dosimetry. A practical strategy is to give a fixed dose based on clinical features, such as the severity of thyrotoxicosis, the size of the goiter (increases the dose needed), and the level of radioiodine uptake (decreases the dose needed). 131I dosage generally ranges between 370 MBq (10 mCi) and 555 MBq (15 mCi). Most authorities favor an approach aimed at thyroid ablation (as opposed to euthyroidism), given that levothyroxine replacement is straightforward and most patients ultimately progress to hypothyroidism over 5–10 years, frequently with some delay in the diagnosis of hypothyroidism.

1	Certain radiation safety precautions are necessary in the first few days after radioiodine treatment, but the exact guidelines vary depending on local protocols. In general, patients need to avoid close, prolonged contact with children and pregnant women for 5–7 days because of possible transmission of residual isotope and exposure to radiation emanating from the gland. Rarely, there may be mild pain due to radiation thyroiditis 1–2 weeks after treatment. Hyperthyroidism can persist for 2–3 months before radioiodine takes full effect. For this reason, β-adrenergic blockers or antithyroid drugs can be used to control symptoms during this interval. Persistent hyperthyroidism can be treated with a second dose of radioiodine, usually 6 months after the first dose. The risk of hypothyroidism after radioiodine depends on the dosage but is at least 10–20% in the first year and 5% per year thereafter. Patients should be informed of this possibility before treatment and require close follow-up

1	radioiodine depends on the dosage but is at least 10–20% in the first year and 5% per year thereafter. Patients should be informed of this possibility before treatment and require close follow-up during the first year followed by annual thyroid function testing.

1	Pregnancy and breast-feeding are absolute contraindications to radioiodine treatment, but patients can conceive safely 6 months after treatment. The presence of severe ophthalmopathy requires caution, and some authorities advocate the use of prednisone, 40 mg/d, at the time of radioiodine treatment, tapered over 6–12 weeks to prevent exacerbation of ophthalmopathy. The overall risk of cancer after radioiodine treatment in adults is not increased. Although many physicians avoid radioiodine in children and adolescents because of the theoretical risks of malignancy, emerging evidence suggests that radioiodine can be used safely in older children.

1	Subtotal or near-total thyroidectomy is an option for patients who relapse after antithyroid drugs and prefer this treatment to radioiodine. Some experts recommend surgery in young individuals, particularly when the goiter is very large. Careful control of thyrotoxicosis with antithyroid drugs, followed by potassium iodide (3 drops SSKI orally tid), is needed prior to surgery to avoid thyrotoxic crisis and to reduce the vascularity of the gland. The major complications of surgery—bleeding, laryngeal edema, hypoparathyroidism, and damage to the recurrent laryngeal nerves—are unusual when the procedure is performed by highly experienced surgeons. Recurrence rates in the best series are <2%, but the rate of hypothyroidism is only slightly less than that following radioiodine treatment.

1	The titration regimen of antithyroid drugs should be used to manage Graves’ disease in pregnancy because transplacental passage of these drugs may produce fetal hypothyroidism and goiter if the maternal dose is excessive. If available, propylthiouracil should be used in early gestation because of the association of rare cases of fetal aplasia cutis and other defects, such as choanal atresia with carbimazole and methimazole. As noted above, because of its rare association with hepatotoxicity, propylthiouracil should be limited to the first trimester and then maternal therapy should be converted to methimazole (or carbimazole) at a ratio of 15–20 mg of propylthiouracil to 1 mg of methimazole The lowest effective antithyroid drug dose should be used throughout gestation to maintain the maternal serum free T4 level at the upper limit of the nonpregnant normal reference range. It is often possible to stop treatment in the last trimester because TSIs tend to decline in pregnancy.

1	the maternal serum free T4 level at the upper limit of the nonpregnant normal reference range. It is often possible to stop treatment in the last trimester because TSIs tend to decline in pregnancy. Nonetheless, the transplacental transfer of these antibodies rarely causes fetal or neonatal thyrotoxicosis. Poor intrauterine growth, a fetal heart rate of >160 beats/min, and high levels of maternal TSI in the last trimester may herald this complication. Antithyroid drugs given to the mother can be used to treat the fetus and may be needed for 1–3 months after delivery, until the maternal antibodies disappear from the baby’s circulation. The postpartum period is a time of major risk for relapse of Graves’ disease. Breast-feeding is safe with low doses of antithyroid drugs. Graves’ disease in children is usually managed with methimazole or carbimazole (avoid propylthiouracil), often given as a prolonged 2297 course of the titration regimen. Surgery or radioiodine may be indicated for

1	in children is usually managed with methimazole or carbimazole (avoid propylthiouracil), often given as a prolonged 2297 course of the titration regimen. Surgery or radioiodine may be indicated for severe disease.

1	Thyrotoxic crisis, or thyroid storm, is rare and presents as a life-threatening exacerbation of hyperthyroidism, accompanied by fever, delirium, seizures, coma, vomiting, diarrhea, and jaundice. The mortality rate due to cardiac failure, arrhythmia, or hyperthermia is as high as 30%, even with treatment. Thyrotoxic crisis is usually precipitated by acute illness (e.g., stroke, infection, trauma, diabetic ketoacidosis), surgery (especially on the thyroid), or radioiodine treatment of a patient with partially treated or untreated hyperthyroidism. Management requires intensive monitoring and supportive care, identification and treatment of the precipitating cause, and measures that reduce thyroid hormone synthesis. Large doses of propylthiouracil (500–1000 mg loading dose and 250 mg every 4 h) should be given orally or by nasogastric tube or per rectum; the drug’s inhibitory action on T4 → T3 conversion makes it the antithyroid drug of choice. If not available, methimazole can be used in

1	should be given orally or by nasogastric tube or per rectum; the drug’s inhibitory action on T4 → T3 conversion makes it the antithyroid drug of choice. If not available, methimazole can be used in doses up to 30 mg every 12 h. One hour after the first dose of propylthiouracil, stable iodide is given to block thyroid hormone synthesis via the Wolff-Chaikoff effect (the delay allows the antithyroid drug to prevent the excess iodine from being incorporated into new hormone). A saturated solution of potassium iodide (5 drops SSKI every 6 h) or, where available, ipodate or iopanoic acid (500 mg per 12 h) may be given orally. Sodium iodide, 0.25 g IV every 6 h, is an alternative but is not generally available. Propranolol should also be given to reduce tachycardia and other adrenergic manifestations (60–80 mg PO every 4 h; or 2 mg IV every 4 h). Although other β-adrenergic blockers can be used, high doses of propranolol decrease T4 → T3 conversion, and the doses can be easily adjusted.

1	(60–80 mg PO every 4 h; or 2 mg IV every 4 h). Although other β-adrenergic blockers can be used, high doses of propranolol decrease T4 → T3 conversion, and the doses can be easily adjusted. Caution is needed to avoid acute negative inotropic effects, but controlling the heart rate is important, as some patients develop a form of high-output heart failure. Short-acting IV esmolol can be used to decrease heart rate while monitoring for signs of heart failure. Additional therapeutic measures include glucocorticoids (e.g., hydrocortisone 300 mg IV bolus, then 100 mg every 8 h), antibiotics if infection is present, cooling, oxygen, and IV fluids.

1	Ophthalmopathy requires no active treatment when it is mild or moderate, because there is usually spontaneous improvement. General measures include meticulous control of thyroid hormone levels, cessation of smoking, and an explanation of the natural history of ophthalmopathy. Discomfort can be relieved with artificial tears (e.g., 1% methylcellulose), eye ointment, and the use of dark glasses with side frames. Periorbital edema may respond to a more upright sleeping position or a diuretic. Corneal exposure during sleep can be avoided by using patches or taping the eyelids shut. Minor degrees of diplopia improve with prisms fitted to spectacles. Severe ophthalmopathy, with optic nerve involvement or chemosis resulting in corneal damage, is an emergency requiring joint management with an ophthalmologist. Pulse therapy with IV methylprednisolone (e.g., 500 mg of methylprednisolone once weekly for 6 weeks, then 250 mg once weekly for 6 weeks) is preferable to oral glucocorticoids, which

1	Pulse therapy with IV methylprednisolone (e.g., 500 mg of methylprednisolone once weekly for 6 weeks, then 250 mg once weekly for 6 weeks) is preferable to oral glucocorticoids, which are used for moderately active disease. When glucocorticoids are ineffective, orbital decompression can be achieved by removing bone from any wall of the orbit, thereby allowing displacement of fat and swollen extraocular muscles. The transantral route is used most often because it requires no external incision. Proptosis recedes an average of 5 mm, but there may be residual or even worsened diplopia. Once the eye disease has stabilized, surgery may be indicated for relief of diplopia and correction of the appearance. External beam radiotherapy of the orbits has been used for many years, but the efficacy of this therapy remains unclear, and it is best reserved for those with moderately active disease who have failed or are not candidates for glucocorticoid therapy. Other immunosuppressive agents such as

1	this therapy remains unclear, and it is best reserved for those with moderately active disease who have failed or are not candidates for glucocorticoid therapy. Other immunosuppressive agents such as rituximab have shown some benefit, but their role is yet to be established.

1	Thyroid dermopathy does not usually require treatment, but it can cause cosmetic problems or interfere with the fit of shoes. Surgical Disorders of the Thyroid Gland 2298 removal is not indicated. If necessary, treatment consists of topical, high-potency glucocorticoid ointment under an occlusive dressing. Octreotide may be beneficial in some cases.

1	Destructive thyroiditis (subacute or silent thyroiditis) typically presents with a short thyrotoxic phase due to the release of preformed thyroid hormones and catabolism of Tg (see “Subacute Thyroiditis,” below). True hyperthyroidism is absent, as demonstrated by a low radionuclide uptake. Circulating Tg levels are usually increased. Other causes of thyrotoxicosis with low or absent thyroid radionuclide uptake include thyrotoxicosis factitia, iodine excess, and, rarely, ectopic thyroid tissue, particularly teratomas of the ovary (struma ovarii) and functional metastatic follicular carcinoma. Whole-body radionuclide studies can demonstrate ectopic thyroid tissue, and thyrotoxicosis factitia can be distinguished from destructive thyroiditis by the clinical features and low levels of Tg. Amiodarone treatment is associated with thyrotoxicosis in up to 10% of patients, particularly in areas of low iodine intake (see below).

1	TSH-secreting pituitary adenoma is a rare cause of thyrotoxicosis. It is characterized by the presence of an inappropriately normal or increased TSH level in a patient with hyperthyroidism, diffuse goiter, and elevated T4 and T3 levels (Chap. 403). Elevated levels of the α-subunit of TSH, released by the TSH-secreting adenoma, support this diagnosis, which can be confirmed by demonstrating the pituitary tumor on MRI or CT scan. A combination of transsphenoidal surgery, sella irradiation, and octreotide may be required to normalize TSH, because many of these tumors are large and locally invasive at the time of diagnosis. Radioiodine or antithyroid drugs can be used to control thyrotoxicosis. Thyrotoxicosis caused by toxic MNG and hyperfunctioning solitary nodules is discussed below. A clinically useful classification of thyroiditis is based on the onset and duration of disease (Table 405-9).

1	Acute thyroiditis is rare and due to suppurative infection of the thyroid. In children and young adults, the most common cause is the presence of a piriform sinus, a remnant of the fourth branchial pouch that connects the oropharynx with the thyroid. Such sinuses are predominantly left-sided. A long-standing goiter and degeneration in a thyroid malignancy are risk factors in the elderly. The patient presents with thyroid pain, often referred to the throat or ears, and a small, tender goiter that may be asym-metric. Fever, dysphagia, and erythema over the thyroid are common, as are systemic symptoms of a febrile illness and lymphadenopathy. TAblE 405-9 CAuSES of THyRoiDiTiS Acute Bacterial infection: especially Staphylococcus, Streptococcus, and Enterobacter Fungal infection: Aspergillus, Candida, Coccidioides, Histoplasma, and Pneumocystis Radiation thyroiditis after 131I treatment 50 ESR TSH (mU/L)UT4 (pmol/L)5 0.5 0.01 40 30 10 0 20ESR (mm/h) 100 0 50 UT4 TSH

1	Viral (or granulomatous) thyroiditis Silent thyroiditis (including postpartum thyroiditis) Mycobacterial infection Drug induced (interferon, amiodarone) Autoimmunity: focal thyroiditis, Hashimoto’s thyroiditis, atrophic thyroiditis Riedel’s thyroiditis Parasitic thyroiditis: echinococcosis, strongyloidiasis, cysticercosis Traumatic: after palpation

1	The differential diagnosis of thyroid pain includes subacute or, rarely, chronic thyroiditis; hemorrhage into a cyst; malignancy including lymphoma; and, rarely, amiodarone-induced thyroiditis or amyloidosis. However, the abrupt presentation and clinical features of acute thyroiditis rarely cause confusion. The erythrocyte sedimentation rate (ESR) and white cell count are usually increased, but thyroid function is normal. FNA biopsy shows infiltration by polymorphonuclear leukocytes; culture of the sample can identify the organism. Caution is needed in immunocompromised patients as fungal, mycobacterial, or Pneumocystis thyroiditis can occur in this setting. Antibiotic treatment is guided initially by Gram stain and, subsequently, by cultures of the FNA biopsy. Surgery may be needed to drain an abscess, which can be localized by CT scan or ultrasound. Tracheal obstruction, septicemia, retropharyngeal abscess, mediastinitis, and jugular venous thrombosis may complicate acute

1	to drain an abscess, which can be localized by CT scan or ultrasound. Tracheal obstruction, septicemia, retropharyngeal abscess, mediastinitis, and jugular venous thrombosis may complicate acute thyroiditis but are uncommon with prompt use of antibiotics.

1	This is also termed de Quervain’s thyroiditis, granulomatous thyroiditis, or viral thyroiditis. Many viruses have been implicated, including mumps, coxsackie, influenza, adenoviruses, and echoviruses, but attempts to identify the virus in an individual patient are often unsuccessful and do not influence management. The diagnosis of subacute thyroiditis is often overlooked because the symptoms can mimic pharyngitis. The peak incidence occurs at 30–50 years, and women are affected three times more frequently than men.

1	Pathophysiology The thyroid shows a characteristic patchy inflammatory infiltrate with disruption of the thyroid follicles and multinucleated giant cells within some follicles. The follicular changes progress to granulomas accompanied by fibrosis. Finally, the thyroid returns to normal, usually several months after onset. During the initial phase of follicular destruction, there is release of Tg and thyroid hormones, leading to increased circulating T4 and T3 and suppression of TSH (Fig. 405-10). During this destructive phase, radioactive iodine uptake is low or undetectable. After several weeks, the thyroid is depleted of stored thyroid hormone and a phase of hypothyroidism typically occurs, with low unbound T4 (and sometimes T3) and moderately increased TSH levels. Radioactive iodine uptake returns to normal or is even increased as a result of the rise in TSH. Finally, thyroid hormone and TSH levels return to normal as the disease subsides.

1	Clinical Manifestations The patient usually presents with a painful and enlarged thyroid, sometimes accompanied by fever. There may be FIGURE 405-10 Clinical course of subacute thyroiditis. The release of thyroid hormones is initially associated with a thyrotoxic phase and suppressed thyroid-stimulating hormone (TSH). A hypothyroid phase then ensues, with low T4 and TSH levels that are initially low but gradually increase. During the recovery phase, increased TSH levels combined with resolution of thyroid follicular injury lead to normalization of thyroid function, often several months after the beginning of the illness. ESR, erythrocyte sedimentation rate; UT4, free or unbound T4.

1	features of thyrotoxicosis or hypothyroidism, depending on the phase of the illness. Malaise and symptoms of an upper respiratory tract infection may precede the thyroid-related features by several weeks. In other patients, the onset is acute, severe, and without obvious antecedent. The patient typically complains of a sore throat, and examination reveals a small goiter that is exquisitely tender. Pain is often referred to the jaw or ear. Complete resolution is the usual outcome, but late-onset permanent hypothyroidism occurs in 15% of cases, particularly in those with coincidental thyroid autoimmunity. A prolonged course over many months, with one or more relapses, occurs in a small percentage of patients.

1	Laboratory Evaluation As depicted in Fig. 405-10, thyroid function tests characteristically evolve through three distinct phases over about 6 months: (1) thyrotoxic phase, (2) hypothyroid phase, and (3) recovery phase. In the thyrotoxic phase, T4 and T3 levels are increased, reflecting their discharge from the damaged thyroid cells, and TSH is suppressed. The T4/T3 ratio is greater than in Graves’ disease or thyroid autonomy, in which T3 is often disproportionately increased. The diagnosis is confirmed by a high ESR and low uptake of radioiodine (<5%) or 99mTc pertechnetate (as compared to salivary gland pertechnetate concentration). The white blood cell count may be increased, and thyroid antibodies are negative. If the diagnosis is in doubt, FNA biopsy may be useful, particularly to distinguish unilateral involvement from bleeding into a cyst or neoplasm.

1	Relatively large doses of aspirin (e.g., 600 mg every 4–6 h) or NSAIDs are sufficient to control symptoms in many cases. If this treatment is inadequate, or if the patient has marked local or systemic symptoms, glucocorticoids should be given. The usual starting dose is 40–60 mg of prednisone, depending on severity. The dose is gradually tapered over 6–8 weeks, in response to improvement in symptoms and the ESR. If a relapse occurs during glucocorticoid withdrawal, treatment should be started again and withdrawn more gradually. In these patients, it is useful to wait until the radioactive iodine uptake normalizes before stopping treatment. Thyroid function should be monitored every 2–4 weeks using TSH and unbound T4 levels. Symptoms of thyrotoxicosis improve spontaneously but may be ameliorated by β-adrenergic blockers; antithyroid drugs play no role in treatment of the thyrotoxic phase. Levothyroxine replacement may be needed if the hypothyroid phase is prolonged, but doses should be

1	by β-adrenergic blockers; antithyroid drugs play no role in treatment of the thyrotoxic phase. Levothyroxine replacement may be needed if the hypothyroid phase is prolonged, but doses should be low enough (50–100 μg daily) to allow TSH-mediated recovery.

1	Painless thyroiditis, or “silent” thyroiditis, occurs in patients with underlying autoimmune thyroid disease and has a clinical course similar to that of subacute thyroiditis. The condition occurs in up to 5% of women 3–6 months after pregnancy and is then termed postpartum thyroiditis. Typically, patients have a brief phase of thyrotoxicosis lasting 2–4 weeks, followed by hypothyroidism for 4–12 weeks, and then resolution; often, however, only one phase is apparent. The condition is associated with the presence of TPO antibodies antepartum, and it is three times more common in women with type 1 diabetes mellitus. As in subacute thyroiditis, the uptake of 99mTc pertechnetate or radioactive iodine is initially suppressed. In addition to the painless goiter, silent thyroiditis can be distinguished from subacute thyroiditis by a normal ESR and the presence of TPO antibodies. Glucocorticoid treatment is not indicated for silent thyroiditis. Severe thyrotoxic symptoms can be managed with a

1	from subacute thyroiditis by a normal ESR and the presence of TPO antibodies. Glucocorticoid treatment is not indicated for silent thyroiditis. Severe thyrotoxic symptoms can be managed with a brief course of propranolol, 20–40 mg three or four times daily. Thyroxine replacement may be needed for the hypothyroid phase but should be withdrawn after 6–9 months, as recovery is the rule. Annual follow-up thereafter is recommended, because a proportion of these individuals develop permanent hypothyroidism. The condition may recur in subsequent pregnancies.

1	Patients receiving cytokines such as IFN-α or IL-2 may develop painless thyroiditis. IFN-α, which is used to treat chronic hepatitis B or C and hematologic and skin malignancies, causes thyroid dysfunction in up to 5% of treated patients. It has been associated with painless thyroiditis, hypothyroidism, and Graves’ disease, and is most common in women with TPO antibodies prior to treatment. For discussion of amiodarone, see “Amiodarone Effects on Thyroid Function,” below.

1	Focal thyroiditis is present in 20–40% of euthyroid autopsy cases and is associated with serologic evidence of autoimmunity, particularly the presence of TPO antibodies. The most common clinically apparent cause of chronic thyroiditis is Hashimoto’s thyroiditis, an autoimmune disorder that often presents as a firm or hard goiter of variable size (see above). Riedel’s thyroiditis is a rare disorder that typically occurs in middle-aged women. It presents with an insidious, painless goiter with local symptoms due to compression of the esophagus, trachea, neck veins, or recurrent laryngeal nerves. Dense fibrosis disrupts normal gland architecture and can extend outside the thyroid capsule. Despite these extensive histologic changes, thyroid dysfunction is uncommon. The goiter is hard, nontender, often asymmetric, and fixed, leading to suspicion of a malignancy. Diagnosis requires open biopsy as FNA biopsy is usually inadequate. Treatment is directed to surgical relief of compressive

1	often asymmetric, and fixed, leading to suspicion of a malignancy. Diagnosis requires open biopsy as FNA biopsy is usually inadequate. Treatment is directed to surgical relief of compressive symptoms. Tamoxifen may also be beneficial. There is an association between Riedel’s thyroiditis and IgG4-related systemic disease causing idiopathic fibrosis at other sites (retroperitoneum, mediastinum, biliary tree, lung, and orbit).

1	Any acute, severe illness can cause abnormalities of circulating TSH or thyroid hormone levels in the absence of underlying thyroid disease, making these measurements potentially misleading. The major cause of these hormonal changes is the release of cytokines such as IL-6. Unless a thyroid disorder is strongly suspected, the routine testing of thyroid function should be avoided in acutely ill patients.

1	The most common hormone pattern in sick euthyroid syndrome (SES) is a decrease in total and unbound T3 levels (low T3 syndrome) with normal levels of T4 and TSH. The magnitude of the fall in T3 correlates with the severity of the illness. T4 conversion to T3 via peripheral 5′ (outer ring) deiodination is impaired, leading to increased reverse T3 (rT3). Since rT3 is metabolized by 5′ deiodination, its clearance is also reduced. Thus, decreased clearance rather than increased production is the major basis for increased rT3. Also, T4 is alternately metabolized to the hormonally inactive T3 sulfate. It is generally assumed that this low T3 state is adaptive, because it can be induced in normal individuals by fasting. Teleologically, the fall in T3 may limit catabolism in starved or ill patients.

1	Very sick patients may exhibit a dramatic fall in total T4 and T3 levels (low T4 syndrome). With decreased tissue perfusion, muscle and liver expression of the type 3 deiodinase leads to accelerated T4 and T3 metabolism. This state has a poor prognosis. Another key factor in the fall in T4 levels is altered binding to TBG. The commonly used free T4 assays are subject to artifact when serum binding proteins are low and underestimate the true free T4 level. Fluctuation in TSH levels also creates challenges in the interpretation of thyroid function in sick patients. TSH levels may range from <0.1 mIU/L in very ill patients, especially with dopamine or glucocorticoid therapy, to >20 mIU/L during the recovery phase of SES. The exact mechanisms underlying the subnormal TSH seen in 10% of sick patients and the increased TSH seen in 5% remain unclear but may be mediated by cytokines including IL-12 and IL-18.

1	Any severe illness can induce changes in thyroid hormone levels, but certain disorders exhibit a distinctive pattern of abnormalities. Acute liver disease is associated with an initial rise in total (but not unbound) T3 and T4 levels due to TBG release; these levels become subnormal with progression to liver failure. A transient increase in total and unbound T4 levels, usually with a normal T3 level, is seen in 5–30% of acutely ill psychiatric patients. TSH values may be transiently

1	Disorders of the Thyroid Gland 2300 low, normal, or high in these patients. In the early stage of HIV infection, T3 and T4 levels rise, even if there is weight loss. T3 levels fall with progression to AIDS, but TSH usually remains normal. Renal disease is often accompanied by low T3 concentrations, but with normal rather than increased rT3 levels, due to an unknown factor that increases uptake of rT3 into the liver. The diagnosis of SES is challenging. Historic information may be limited, and patients often have multiple metabolic derangements. Useful features to consider include previous history of thyroid disease and thyroid function tests, evaluation of the severity and time course of the patient’s acute illness, documentation of medications that may affect thyroid function or thyroid hormone levels, and measurements of rT3 together with unbound thyroid hormones and TSH. The diagnosis of SES is frequently presumptive, given the clinical context and pattern of laboratory values;

1	hormone levels, and measurements of rT3 together with unbound thyroid hormones and TSH. The diagnosis of SES is frequently presumptive, given the clinical context and pattern of laboratory values; only resolution of the test results with clinical recovery can clearly establish this disorder. Treatment of SES with thyroid hormone (T4 and/or T3) is controversial, but most authorities recommend monitoring the patient’s thyroid function tests during recovery, without administering thyroid hormone, unless there is historic or clinical evidence suggestive of hypothyroidism. Sufficiently large randomized controlled trials using thyroid hormone are unlikely to resolve this therapeutic controversy in the near future, because clinical presentations and outcomes are highly variable.

1	Amiodarone is a commonly used type III antiarrhythmic agent (Chap. 277). It is structurally related to thyroid hormone and contains 39% iodine by weight. Thus, typical doses of amiodarone (200 mg/d) are associated with very high iodine intake, leading to greater than forty-fold increases in plasma and urinary iodine levels. Moreover, because amiodarone is stored in adipose tissue, high iodine levels persist for >6 months after discontinuation of the drug. Amiodarone inhibits deiodinase activity, and its metabolites function as weak antagonists of thyroid hormone action. Amiodarone has the following effects on thyroid function: (1) acute, transient suppression of thyroid function; (2) hypothyroidism in patients susceptible to the inhibitory effects of a high iodine load; and (3) thyrotoxicosis that may be caused by either a Jod-Basedow effect from the iodine load, in the setting of MNG or incipient Graves’ disease, or a thyroiditis-like condition.

1	The initiation of amiodarone treatment is associated with a transient decrease of T4 levels, reflecting the inhibitory effect of iodine on T4 release. Soon thereafter, most individuals escape from iodide-dependent suppression of the thyroid (Wolff-Chaikoff effect), and the inhibitory effects on deiodinase activity and thyroid hormone receptor action become predominant. These events lead to the following pattern of thyroid function tests: increased T4, decreased T3, increased rT3, and a transient TSH increase (up to 20 mIU/L). TSH levels normalize or are slightly suppressed within 1–3 months.

1	The incidence of hypothyroidism from amiodarone varies geographically, apparently correlating with iodine intake. Hypothyroidism occurs in up to 13% of amiodarone-treated patients in iodine-replete countries, such as the United States, but is less common (<6% incidence) in areas of lower iodine intake, such as Italy or Spain. The pathogenesis appears to involve an inability of the thyroid gland to escape from the Wolff-Chaikoff effect in autoimmune thyroiditis. Consequently, amiodarone-associated hypothyroidism is more common in women and individuals with positive TPO antibodies. It is usually unnecessary to discontinue amiodarone for this side effect, because levothyroxine can be used to normalize thyroid function. TSH levels should be monitored, because T4 levels are often increased for the reasons described above.

1	The management of amiodarone-induced thyrotoxicosis (AIT) is complicated by the fact that there are different causes of thyrotoxicosis and because the increased thyroid hormone levels exacerbate underlying arrhythmias and coronary artery disease. Amiodarone treatment causes thyrotoxicosis in 10% of patients living in areas of low iodine intake and in 2% of patients in regions of high iodine intake. There are two major forms of AIT, although some patients have features of both. Type 1 AIT is associated with an underlying thyroid abnormality (preclinical Graves’ disease or nodular goiter). Thyroid hormone synthesis becomes excessive as a result of increased iodine exposure (Jod-Basedow phenomenon). Type 2 AIT occurs in individuals with no intrinsic thyroid abnormalities and is the result of drug-induced lysosomal activation leading to destructive thyroiditis with histiocyte accumulation in the thyroid; the incidence rises as cumulative amiodarone dosage increases. Mild forms of type 2

1	drug-induced lysosomal activation leading to destructive thyroiditis with histiocyte accumulation in the thyroid; the incidence rises as cumulative amiodarone dosage increases. Mild forms of type 2 AIT can resolve spontaneously or can occasionally lead to hypothyroidism. Color-flow Doppler thyroid scanning shows increased vascularity in type 1 AIT but decreased vascularity in type 2 AIT. Thyroid scintiscans are difficult to interpret in this setting because the high endogenous iodine levels diminish tracer uptake. However, the presence of normal or rarely increased uptake favors type 1 AIT.

1	In AIT, the drug should be stopped, if possible, although this is often impractical because of the underlying cardiac disorder. Discontinuation of amiodarone will not have an acute effect because of its storage and prolonged half-life. High doses of antithyroid drugs can be used in type 1 AIT but are often ineffective. In type 2 AIT, oral contrast agents, such as sodium ipodate (500 mg/d) or sodium tyropanoate (500 mg, 1–2 doses/d), rapidly reduce T4 and T3 levels, decrease T4 → T3 conversion, and may block tissue uptake of thyroid hormones. Potassium perchlorate, 200 mg every 6 h, has been used to reduce thyroidal iodide content. Perchlorate treatment has been associated with agranulocytosis, although the risk appears relatively low with short-term use. Glucocorticoids, as administered for subacute thyroiditis, have modest benefit in type 2 AIT. Lithium blocks thyroid hormone release and can also provide some benefit. Near-total thyroidectomy rapidly decreases thyroid hormone levels

1	subacute thyroiditis, have modest benefit in type 2 AIT. Lithium blocks thyroid hormone release and can also provide some benefit. Near-total thyroidectomy rapidly decreases thyroid hormone levels and may be the most effective long-term solution if the patient can undergo the procedure safely.

1	Five factors alter thyroid function in pregnancy: (1) the transient increase in hCG during the first trimester, which stimulates the TSHR; (2) the estrogen-induced rise in TBG during the first trimester, which is sustained during pregnancy; (3) alterations in the immune system, leading to the onset, exacerbation, or amelioration of an underlying autoimmune thyroid disease (see above); (4) increased thyroid hormone metabolism by the placenta; and (5) increased urinary iodide excretion, which can cause impaired thyroid hormone production in areas of marginal iodine sufficiency. Women with a precarious iodine intake (<50 μg/d) are most at risk of developing a goiter during pregnancy or giving birth to an infant with a goiter and hypothyroidism. The World Health Organization recommends a daily iodine intake of 250 μg during pregnancy and prenatal vitamins should contain 150 μg per tablet.

1	The rise in circulating hCG levels during the first trimester is accompanied by a reciprocal fall in TSH that persists into the middle of pregnancy. This reflects the weak binding of hCG, which is present at very high levels, to the TSH-R. Rare individuals have been described with variant TSH-R sequences that enhance hCG binding and TSH-R activation. hCG-induced changes in thyroid function can result in transient gestational hyperthyroidism that may be associated with hyperemesis gravidarum, a condition characterized by severe nausea and vomiting and risk of volume depletion. However, since the hyperthyroidism is not causal, antithyroid drugs are not indicated unless concomitant Graves’ disease is suspected. Parenteral fluid replacement usually suffices until the condition resolves.

1	During pregnancy, subclinical hypothyroidism occurs in 2% of women, but overt hypothyroidism is present in only 1 in 500. Prospective randomized controlled trials have not shown a benefit for universal thyroid disease screening in pregnancy. Targeted TSH testing for hypothyroidism is recommended for women planning a pregnancy if they have a strong family history of autoimmune thyroid disease, other autoimmune disorders (e.g., type 1 diabetes), prior preterm delivery or recurrent miscarriage, or signs or symptoms of thyroid disease. Thyroid hormone requirements are increased by up to 50% during pregnancy in levothyroxine-treated hypothyroid women (see above section on treatment of hypothyroidism).

1	Goiter refers to an enlarged thyroid gland. Biosynthetic defects, iodine deficiency, autoimmune disease, and nodular diseases can each lead to goiter, although by different mechanisms. Biosynthetic defects and iodine deficiency are associated with reduced efficiency of thyroid hormone synthesis, leading to increased TSH, which stimulates thyroid growth as a compensatory mechanism to overcome the block in hormone synthesis. Graves’ disease and Hashimoto’s thyroiditis are also associated with goiter. In Graves’ disease, the goiter results mainly from the TSH-R–mediated effects of TSI. The goitrous form of Hashimoto’s thyroiditis occurs because of acquired defects in hormone synthesis, leading to elevated levels of TSH and its consequent growth effects. Lymphocytic infiltration and immune system–induced growth factors also contribute to thyroid enlargement in Hashimoto’s thyroiditis. Nodular disease is characterized by the disordered growth of thyroid cells, often combined with the

1	system–induced growth factors also contribute to thyroid enlargement in Hashimoto’s thyroiditis. Nodular disease is characterized by the disordered growth of thyroid cells, often combined with the gradual development of fibrosis. Because the management of goiter depends on the etiology, the detection of thyroid enlargement on physical examination should prompt further evaluation to identify its cause.

1	Nodular thyroid disease is common, occurring in about 3–7% of adults when assessed by physical examination. Using ultrasound, nodules are present in up to 50% of adults, with the majority being <1 cm in diameter. Thyroid nodules may be solitary or multiple, and they may be functional or nonfunctional.

1	DIFFUSE NONTOXIC (SIMPLE) GOITER Etiology and Pathogenesis When diffuse enlargement of the thyroid occurs in the absence of nodules and hyperthyroidism, it is referred to as a diffuse nontoxic goiter. This is sometimes called simple goiter, because of the absence of nodules, or colloid goiter, because of the presence of uniform follicles that are filled with colloid. Worldwide, diffuse goiter is most commonly caused by iodine deficiency and is termed endemic goiter when it affects >5% of the population. In nonendemic regions, sporadic goiter occurs, and the cause is usually unknown. Thyroid enlargement in teenagers is sometimes referred to as juvenile goiter. In general, goiter is more common in women than men, probably because of the greater prevalence of underlying autoimmune disease and the increased iodine demands associated with pregnancy.

1	In iodine-deficient areas, thyroid enlargement reflects a compensatory effort to trap iodide and produce sufficient hormone under conditions in which hormone synthesis is relatively inefficient. Somewhat surprisingly, TSH levels are usually normal or only slightly increased, suggesting increased sensitivity to TSH or activation of other pathways that lead to thyroid growth. Iodide appears to have direct actions on thyroid vasculature and may indirectly affect growth through vasoactive substances such as endothelins and nitric oxide. Endemic goiter is also caused by exposure to environmental goitrogens such as cassava root, which contains a thiocyanate; vegetables of the Cruciferae family (known as cruciferous vegetables) (e.g., Brussels sprouts, cabbage, and cauliflower); and milk from regions where goitrogens are present in grass. Although relatively rare, inherited defects in thyroid hormone synthesis lead to a diffuse nontoxic goiter. Abnormalities at each step in hormone

1	from regions where goitrogens are present in grass. Although relatively rare, inherited defects in thyroid hormone synthesis lead to a diffuse nontoxic goiter. Abnormalities at each step in hormone synthesis, including iodide transport (NIS), Tg synthesis, organification and coupling (TPO), and the regeneration of iodide (dehalogenase), have been described.

1	If thyroid function is preserved, most goiters are asymptomatic. Examination of a diffuse goiter reveals a symmetrically enlarged, nontender, generally soft gland without palpable nodules. Goiter is defined, somewhat arbitrarily, as a lateral lobe with a volume greater than the thumb of the individual being examined. If the thyroid is markedly enlarged, it can cause tracheal or esophageal compression. These features are unusual, however, in the absence of nodular disease and fibrosis. Substernal goiter may obstruct the thoracic inlet. Pemberton’s sign refers to symptoms of faintness with evidence of facial congestion and external jugular venous obstruction when the arms are raised above the head, a maneuver that draws the thyroid into the thoracic inlet. Respiratory flow measurements and CT or MRI should be used to evaluate substernal goiter in patients with obstruc-2301 tive signs or symptoms.

1	Thyroid function tests should be performed in all patients with goiter to exclude thyrotoxicosis or hypothyroidism. It is not unusual, particularly in iodine deficiency, to find a low total T4, with normal T3 and TSH, reflecting enhanced T4 → T3 conversion. A low TSH with a normal free T3 and free T4, particularly in older patients, suggests the possibility of thyroid autonomy or undiagnosed Graves’ disease, and is termed subclinical thyrotoxicosis. The benefit of treatment (typically with radioiodine) in subclinical thyrotoxicosis, versus follow-up and implementing treatment if free T3 or free T4 levels become abnormal, is unclear, but treatment is increasingly recommended in the elderly to reduce the risk of atrial fibrillation and bone loss. TPO antibodies may be useful to identify patients at increased risk of autoimmune thyroid disease. Low urinary iodine levels (<50 μg/L) support a diagnosis of iodine deficiency. Thyroid scanning is not generally necessary but will reveal

1	patients at increased risk of autoimmune thyroid disease. Low urinary iodine levels (<50 μg/L) support a diagnosis of iodine deficiency. Thyroid scanning is not generally necessary but will reveal increased uptake in iodine deficiency and most cases of dyshormonogenesis. Ultrasound is not generally indicated in the evaluation of diffuse goiter unless a nodule is palpable on physical examination.

1	Iodine replacement induces variable regression of goiter in iodine deficiency, depending on how long it has been present and the degree of fibrosis that has developed. Surgery is rarely indicated for diffuse goiter. Exceptions include documented evidence of tracheal compression or obstruction of the thoracic inlet, which are more likely to be associated with substernal MNGs (see below). Subtotal or near-total thyroidectomy for these or cosmetic reasons should be performed by an experienced surgeon to minimize complication rates. Surgery should be followed by replacement with levothyroxine, with the aim of keeping the TSH level at the lower end of the reference range to prevent regrowth of the goiter.

1	NONTOXIC MULTINODULAR GOITER Etiology and Pathogenesis Depending on the population studied, MNG or nodular enlargement of the thyroid occurs in up to 12% of adults. MNG is more common in women than men and increases in prevalence with age. It is more common in iodine-deficient regions but also occurs in regions of iodine sufficiency, reflecting multiple genetic, autoimmune, and environmental influences on the pathogenesis.

1	There is typically wide variation in nodule size. Histology reveals a spectrum of morphologies ranging from hypercellular regions to cystic areas filled with colloid. Fibrosis is often extensive, and areas of hemorrhage or lymphocytic infiltration may be seen. Using molecular techniques, most nodules within an MNG are polyclonal in origin, suggesting a hyperplastic response to locally produced growth factors and cytokines. TSH, which is usually not elevated, may play a permissive or contributory role. Monoclonal lesions also occur within an MNG, reflecting mutations in genes that confer a selective growth advantage to the progenitor cell.

1	Clinical Manifestations Most patients with nontoxic MNG are asymptomatic and euthyroid. MNG typically develops over many years and is detected on routine physical examination, when an individual notices an enlargement in the neck, or as an incidental finding on imaging. If the goiter is large enough, it can ultimately lead to compressive symptoms including difficulty swallowing, respiratory distress (tracheal compression), or plethora (venous congestion), but these symptoms are uncommon. Symptomatic MNGs are usually extraordinarily large and/or develop fibrotic areas that cause compression. Sudden pain in an MNG is usually caused by hemorrhage into a nodule but should raise the possibility of invasive malignancy. Hoarseness, reflecting laryngeal nerve involvement, also suggests malignancy.

1	Diagnosis On examination, thyroid architecture is distorted, and multiple nodules of varying size can be appreciated. Because many nodules are deeply embedded in thyroid tissue or reside in posterior or

1	Disorders of the Thyroid Gland 2302 substernal locations, it is not possible to palpate all nodules. Pemberton’s sign, characterized by facial suffusion when the patient’s arms are elevated above the head, suggests that the goiter has increased pressure in the thoracic inlet. A TSH level should be measured to exclude subclinical hyperor hypothyroidism, but thyroid function is usually normal. Tracheal deviation is common, but compression must usually exceed 70% of the tracheal diameter before there is significant airway compromise. Pulmonary function testing can be used to assess the functional effects of compression, which characteristically causes inspiratory stridor. CT or MRI can be used to evaluate the anatomy of the goiter and the extent of substernal extension or tracheal narrowing. A barium swallow may reveal the extent of esophageal compression. The risk of malignancy in MNG is similar to that in solitary nodules. Ultrasonography can be used to identify which nodules should be

1	A barium swallow may reveal the extent of esophageal compression. The risk of malignancy in MNG is similar to that in solitary nodules. Ultrasonography can be used to identify which nodules should be biopsied based on sonographic features (see section above on ultrasound) and size. For nodules with more suspicious imaging characteristics (e.g., hypoechogenicity, microcalcifications, irregular margins), biopsy is recommended when ≥1 cm.

1	Most nontoxic MNGs can be managed conservatively. T4 suppression is rarely effective for reducing goiter size and introduces the risk of subclinical or overt thyrotoxicosis, particularly if there is underlying autonomy or if it develops during treatment. If levothyroxine is used, it should be started at low doses (50 μg daily) and advanced gradually while monitoring the TSH level to avoid excessive suppression. Contrast agents and other iodine-containing substances should be avoided because of the risk of inducing the Jod-Basedow effect, characterized by enhanced thyroid hormone production by autonomous nodules. Radioiodine is used with increasing frequency in areas where large goiters are more prevalent because it can decrease goiter size and may selectively ablate regions of autonomy. Dosage of 131I depends on the size of the goiter and radioiodine uptake but is usually about 3.7 MBq (0.1 mCi) per gram of tissue, corrected for uptake (typical dose 370–1070 MBq [10 to 29 mCi]).

1	Dosage of 131I depends on the size of the goiter and radioiodine uptake but is usually about 3.7 MBq (0.1 mCi) per gram of tissue, corrected for uptake (typical dose 370–1070 MBq [10 to 29 mCi]). Repeat treatment may be needed and effectiveness may be increased by concurrent administration of low-dose recombinant TSH (0.1 mg IM). It is possible to achieve a 40–50% reduction in goiter size in most patients. Earlier concerns about radiation-induced thyroid swelling and tracheal compression have diminished, as studies have shown this complication to be rare. When acute compression occurs, glucocorticoid treatment or surgery may be needed. Radiation-induced hypothyroidism is less common than after treatment for Graves’ disease. However, posttreatment autoimmune thyrotoxicosis may occur in up to 5% of patients treated for nontoxic MNG. Surgery remains highly effective but is not without risk, particularly in older patients with underlying cardiopulmonary disease.

1	The pathogenesis of toxic MNG appears to be similar to that of nontoxic MNG; the major difference is the presence of functional autonomy in toxic MNG. The molecular basis for autonomy in toxic MNG remains unknown. As in nontoxic goiters, many nodules are polyclonal, whereas others are monoclonal and vary in their clonal origins. Genetic abnormalities known to confer functional autonomy, such as activating TSH-R or Gsα mutations (see below), are not usually found in the autonomous regions of toxic MNG goiter.

1	In addition to features of goiter, the clinical presentation of toxic MNG includes subclinical hyperthyroidism or mild thyrotoxicosis. The patient is usually elderly and may present with atrial fibrillation or palpitations, tachycardia, nervousness, tremor, or weight loss. Recent exposure to iodine, from contrast dyes or other sources, may precipitate or exacerbate thyrotoxicosis. The TSH level is low. The uncombined T4 level may be normal or minimally increased; T3 is often elevated to a greater degree than T4. Thyroid scan shows heterogeneous uptake with multiple regions of increased and decreased uptake; 24-h uptake of radioiodine may not be increased but is usually in the upper normal range.

1	Prior to definitive treatment of the hyperthyroidism, ultrasound imaging should be performed to assess the presence of discrete nodules corresponding to areas of decreased uptake (“cold” nodules). If present, FNA may be indicated based on sonographic features and size cutoffs. The cytology results, if indeterminate or suspicious, may direct the therapy to surgery.

1	Antithyroid drugs normalize thyroid function and are particularly useful in the elderly or ill patients with limited lifespan. In contrast to Graves’ disease, spontaneous remission does not occur and so treatment is long-term. Radioiodine is generally the treatment of choice; it treats areas of autonomy as well as decreasing the mass of the goiter. Sometimes, however, a degree of autonomy remains, presumably because multiple autonomous regions emerge as soon as others are treated, and further radioiodine treatment may be necessary. Surgery provides definitive treatment of underlying thyrotoxicosis as well as goiter. Patients should be rendered euthyroid using an antithyroid drug before operation.

1	A solitary, autonomously functioning thyroid nodule is referred to as toxic adenoma. The pathogenesis of this disorder has been unraveled by demonstrating the functional effects of mutations that stimulate the TSH-R signaling pathway. Most patients with solitary hyperfunctioning nodules have acquired somatic, activating mutations in the TSH-R (Fig. 405-11). These mutations, located primarily in the receptor transmembrane domain, induce constitutive receptor coupling to GSα, increasing cyclic AMP levels and leading to enhanced thyroid follicular cell proliferation and function. Less commonly, somatic mutations are identified in GSα. These mutations, which are similar to those seen in Transmembrane domains Activating mutations 4 75 6 Cell growth, differentiation Hormone synthesis GS˜AC cyclic AMP TSH-R Extracellular domain

1	Transmembrane domains Activating mutations 4 75 6 Cell growth, differentiation Hormone synthesis GS˜AC cyclic AMP TSH-R Extracellular domain FIGURE 405-11 Activating mutations of the thyroid-stimulating hormone receptor (TSH-R). Mutations (*) that activate TSH-R reside mainly in transmembrane 5 and intracellular loop 3, although mutations have occurred in a variety of different locations. The effect of these mutations is to induce conformational changes that mimic TSH binding, thereby leading to coupling to stimulatory G protein (GSα) and activation of adenylate cyclase (AC), an enzyme that generates cyclic AMP. McCune-Albright syndrome (Chap. 412) or in a subset of somatotrope adenomas (Chap. 403), impair guanosine triphosphate (GTP) hydrolysis, causing constitutive activation of the cyclic AMP signaling pathway. In most series, activating mutations in either the TSH-R or the GSα subunit genes are identified in >90% of patients with solitary hyperfunctioning nodules.

1	Thyrotoxicosis is usually mild. The disorder is suggested by a subnormal TSH level; the presence of the thyroid nodule, which is generally large enough to be palpable; and the absence of clinical features suggestive of Graves’ disease or other causes of thyrotoxicosis. A thyroid scan provides a definitive diagnostic test, demonstrating focal uptake in the hyperfunctioning nodule and diminished uptake in the remainder of the gland, as activity of the normal thyroid is suppressed.

1	Radioiodine ablation is usually the treatment of choice. Because normal thyroid function is suppressed, 131I is concentrated in the hyperfunctioning nodule with minimal uptake and damage to normal thyroid tissue. Relatively large radioiodine doses (e.g., 370–1110 MBq [10–29.9 mCi] 131I) have been shown to correct thyrotoxicosis in about 75% of patients within 3 months. Hypothyroidism occurs in <10% of those patients over the next 5 years. Surgical resection is also effective and is usually limited to enucleation of the adenoma or lobectomy, thereby preserving thyroid function and minimizing risk of hypoparathyroidism or damage to the recurrent laryngeal nerves. Medical therapy using antithyroid drugs and beta blockers can normalize thyroid function but is not an optimal long-term treatment. Using ultrasound guidance, repeated ethanol injections and percutaneous radiofrequency thermal ablation have been used successfully in some centers to ablate hyperfunctioning nodules, and these

1	Using ultrasound guidance, repeated ethanol injections and percutaneous radiofrequency thermal ablation have been used successfully in some centers to ablate hyperfunctioning nodules, and these techniques have also been used to reduce the size of nonfunctioning thyroid nodules.

1	The various types of benign thyroid nodules are listed in Table 405-10. These lesions are common (5–10% adults), particularly when assessed by sensitive techniques such as ultrasound. The risk of malignancy is very low for macrofollicular adenomas and normofollicular adenomas. Microfollicular, trabecular, and Hürthle cell variants raise greater concern, and the histology is more difficult to interpret. Many are mixed cystic/solid lesions on ultrasound and may appear spongiform reflecting the pathology of macrofollicular structure. However, the majority of solid nodules (whether hypo-, iso-, or hyperechoic) are also benign. FNA, usually performed with ultrasound guidance, is the diagnostic procedure of choice to evaluate thyroid nodules (see the “Approach to the Patient” section on thyroid nodules). Pure thyroid cysts, <2% of all thyroid growths, consist of colloid and are benign as well. Cysts frequently recur, even after repeated aspiration, and may require surgical excision if they

1	nodules). Pure thyroid cysts, <2% of all thyroid growths, consist of colloid and are benign as well. Cysts frequently recur, even after repeated aspiration, and may require surgical excision if they are large. Ethanol ablation to sclerose the cyst has been used successfully for patients who are symptomatic.

1	TSH suppression with levothyroxine therapy does not decrease thyroid nodule size in iodine-sufficient populations. However, if there is relative iodine deficiency, both iodine and levothyroxine therapy may decrease nodule volume. If levothyroxine is administered in this situation and the nodule has not decreased in size after 6–12 months of suppressive therapy, treatment should be discontinued because little benefit is likely to accrue from long-term treatment; the risk of iatrogenic subclinical thyrotoxicosis should also be considered. Thyroid carcinoma is the most common malignancy of the endocrine system. Malignant tumors derived from the follicular epithelium are classified according to histologic features. Differentiated tumors, such as papillary thyroid cancer (PTC) or follicular thyroid cancer (FTC), are often curable, and the prognosis is good for patients identified with early-stage disease. In contrast, anaplastic thyroid cancer (ATC) Malignant Approximate Prevalence, %

1	Malignant Approximate Prevalence, % Tall cell, columnar cell variants Others Abbreviation: MEN, multiple endocrine neoplasia. is aggressive, responds poorly to treatment, and is associated with a bleak prognosis.

1	Tall cell, columnar cell variants Others Abbreviation: MEN, multiple endocrine neoplasia. is aggressive, responds poorly to treatment, and is associated with a bleak prognosis. The incidence of thyroid cancer is ~12/100,000 per year in the United States and increases with age. Prognosis is worse in older persons (>65 years). Thyroid cancer is twice as common in women as men, but male gender is associated with a worse prognosis. Additional important risk factors include a history of childhood head or neck irradiation, large nodule size (≥4 cm), evidence for local tumor fixation or invasion into lymph nodes, and the presence of metastases (Table 405-11). Several unique features of thyroid cancer facilitate its management: (1) thyroid nodules are amenable to biopsy by FNA; (2) iodine radioisotopes can be used to diagnose (123I) and treat (131I) differentiated thyroid cancer, reflecting the unique uptake of this anion by the thyroid gland; and (3) serum markers allow the detection of

1	History of head and neck irradiation, Family history of thyroid cancer, MEN including total-body irradiation for 2, or other genetic syndromes associ bone marrow transplant and brain ated with thyroid malignancy (e.g., radiation for childhood leukemia Cowden’s syndrome, familial polypo sis, Carney complex) Exposure to ionizing radiation from fallout in childhood or adolescence Vocal cord paralysis, hoarse voice Age <20 or >65 years Nodule fixed to adjacent structures Abbreviation: MEN, multiple endocrine neoplasia. Disorders of the Thyroid Gland 2304 residual or recurrent disease, including the use of Tg levels for PTC and FTC, and calcitonin for medullary thyroid cancer (MTC).

1	Disorders of the Thyroid Gland 2304 residual or recurrent disease, including the use of Tg levels for PTC and FTC, and calcitonin for medullary thyroid cancer (MTC). Thyroid neoplasms can arise in each of the cell types that populate the gland, including thyroid follicular cells, calcitonin-producing C cells, lymphocytes, and stromal and vascular elements, as well as metastases from other sites (Table 405-10). The American Joint Committee on Cancer (AJCC) has designated a staging system using the tumor, node, metastasis (TNM) classification (Table 405-12). Several other classification and staging systems are also widely used, some of which place greater emphasis on histologic features or risk factors such as age or gender.

1	PATHOGENESIS AND GENETIC BASIS Radiation Early studies of the pathogenesis of thyroid cancer focused on the role of external radiation, which predisposes to chromosomal breaks, leading to genetic rearrangements and loss of tumor-suppressor genes. External radiation of the mediastinum, face, head, and neck region was administered in the past to treat an array of conditions, including acne and enlargement of the thymus, tonsils, and adenoids. Radiation exposure increases the risk of benign and malignant thyroid nodules, is associated with multicentric cancers, and shifts the incidence of thyroid cancer to an earlier age group. Radiation from nuclear fallout also increases the risk of thyroid cancer. Children seem more predisposed to the effects of radiation than adults. Of note, radiation derived from 131I therapy appears to contribute minimal increased risk of thyroid cancer.

1	TSH and Growth Factors Many differentiated thyroid cancers express TSH receptors and, therefore, remain responsive to TSH. Higher serum TSH levels, even within normal range, are associated with increased thyroid cancer risk in patients with thyroid nodules. These <45 years >45 years Stage I Any T, any N, M0 T1, N0, M0 Stage II Any T, any N, M1 T2, N0, M0 Stage III — T3, N0, M0 T1–T3, N1a, M0 Stage IVA — T4a, any N, M0 T1–T3, N1b, M0 Stage IVB T4b, any N, M0 Stage IVC Any T, any N, M1

1	T1–T3, N1a, M0 Stage IVA — T4a, any N, M0 T1–T3, N1b, M0 Stage IVB T4b, any N, M0 Stage IVC Any T, any N, M1 Stage IV All cases are stage IV aCriteria include: T, the size and extent of the primary tumor (T1a ≤1 cm; T1b >1 cm but ≤2 cm; T2 >2 cm but ≤4 cm; T3 >4 cm or any tumor with extension into perithyroidal soft tissue or sternothyroid muscle; T4a invasion into subcutaneous soft tissues, larynx, trachea, esophagus, or recurrent laryngeal nerve; T4b invasion into prevertebral fascia or encasement of carotid artery or mediastinal vessels); N, the absence (N0) or presence (N1a level IV central compartment; N1b levels II–V lateral compartment, upper mediastinal or retro/ parapharyngeal) of regional node involvement; M, the absence (M0) or presence (M1) of distant metastases. Source: American Joint Committee on Cancer staging system for thyroid cancers using the TNM classification, 7th edition.

1	Source: American Joint Committee on Cancer staging system for thyroid cancers using the TNM classification, 7th edition. observations provide the rationale for T4 suppression of TSH in patients with thyroid cancer. Residual expression of TSH receptors also allows TSH-stimulated uptake of 131I therapy (see below).

1	observations provide the rationale for T4 suppression of TSH in patients with thyroid cancer. Residual expression of TSH receptors also allows TSH-stimulated uptake of 131I therapy (see below). Oncogenes and Tumor-Suppressor Genes Thyroid cancers are monoclonal in origin, consistent with the idea that they originate as a consequence of mutations that confer a growth advantage to a single cell. In addition to increased rates of proliferation, some thyroid cancers exhibit impaired apoptosis and features that enhance invasion, angiogenesis, and metastasis. Thyroid neoplasms have been analyzed for a variety of genetic alterations, but without clear evidence of an ordered acquisition of somatic mutations as they progress from the benign to the malignant state. On the other hand, certain mutations are relatively specific for thyroid neoplasia, some of which correlate with histologic classification (Table 405-13).

1	As described above, activating mutations of the TSH-R and the subunit are associated with autonomously functioning nodules. Although these mutations induce thyroid cell growth, this type of nodule is almost always benign.

1	Activation of the RET-RAS-BRAF signaling pathway is seen in up to 70% of PTCs, although the types of mutations are heterogeneous. A variety of rearrangements involving the RET gene on chromosome 10 bring this receptor tyrosine kinase under the control of other promoters, leading to receptor overexpression. RET rearrangements occur in 20–40% of PTCs in different series and were observed with increased frequency in tumors developing after the Chernobyl radiation accident. Rearrangements in PTC have also been observed for another tyrosine kinase gene, TRK1, which is located on chromosome 1. To date, the identification of PTC with RET or TRK1 rearrangements has not proven useful for predicting prognosis or treatment responses. BRAF V600E mutations appear to be the most common genetic alteration in PTC. These mutations activate the kinase, which stimulates the mitogen-activated protein MAP kinase (MAPK) cascade. RAS mutations, which also stimulate the MAPK cascade, are found in about

1	in PTC. These mutations activate the kinase, which stimulates the mitogen-activated protein MAP kinase (MAPK) cascade. RAS mutations, which also stimulate the MAPK cascade, are found in about 20–30% of thyroid neoplasms (NRAS > HRAS > KRAS), including both PTC and FTC. Of note, simultaneous RET, BRAF, and RAS mutations rarely occur in the same tumor, suggesting that activation of the MAPK cascade is critical for tumor development, independent of the step that initiates the cascade.

1	RAS mutations also occur in FTCs. In addition, a rearrangement of the thyroid developmental transcription factor PAX8 with the nuclear receptor PPARγ is identified in a significant fraction of FTCs. Overall, about 70% of follicular cancers have mutations or genetic rearrangements. Loss of heterozygosity of 3p or 11q, consistent with deletions of tumor-suppressor genes, is also common in FTCs. Most of the mutations seen in differentiated thyroid cancers have also been detected in ATCs. BRAF mutations are seen in up to 50% of ATCs. Mutations in CTNNB1, which encodes β-catenin, occur in about two-thirds of ATCs, but not in PTC or FTC. Mutations of the tumor-suppressor P53 also play an important role in the development of ATC. Because P53 plays a role in cell cycle surveillance, DNA repair, and apoptosis, its loss may contribute to the rapid acquisition of genetic instability as well as poor treatment responses (Chap. 102e) (Table 405-13).

1	The role of molecular diagnostics in the clinical management of thyroid cancer is under investigation. In principle, analyses of specific mutations might aid in classification, prognosis, or choice of treatment. Although BRAF V600E mutations are associated with loss of iodine uptake by tumor cells, there is no clear evidence to date that this information alters clinical decision making. Higher recurrence rates have been variably reported in patients with BRAF-positive PTC, but the impact on survival rates is unclear. Sequencing of thyroid cancers as part of the Cancer Genome Atlas (TCGA) is likely to lead to new classification schemes based on molecular abnormalities in tumors. MTC, when associated with multiple endocrine neoplasia (MEN) type 2, harbors an inherited mutation of the RET gene. Unlike the rearrangements of RET seen in PTC, the mutations in MEN 2 are

1	Abbreviations: APC, adenomatous polyposis coli; ATC, anaplastic thyroid cancer; BRAF, v-raf homologue, B1; CDKN2A, cyclin-dependent kinase inhibitor 2A; c-MYC, cellular homologue of myelocytomatosis virus protooncogene; ELE1/TK, RET-activating gene ele1/tyrosine kinase; GPCR, G protein–coupled receptor; GSα, G-protein stimulating α-subunit; MEK, mitogen extracellular signal-regulated kinase; MEN 2, multiple endocrine neoplasia-2; MET, met protooncogene (hepatocyte growth factor receptor); MTS, multiple tumor suppressor; p53, p53 tumor suppressor gene; PTC, papillary thyroid cancer; PTEN, phosphatase and tensin homologue; RAS, rat sarcoma protooncogene; RET, rearranged during transfection protooncogene; p21, p21 tumor suppressor; PAX8, paired domain transcription factor; PPARγ1, peroxisome-proliferator activated receptor γ1; TRK, tyrosine kinase receptor; TSH, thyroid-stimulating hormone; WAF, wild-type p53 activated fragment.

1	Source: Adapted with permission from P Kopp, JL Jameson, in JL Jameson (ed): Principles of Molecular Medicine. Totowa, NJ, Humana Press, 1998. point mutations that induce constitutive activity of the tyrosine kinase spread is debated. Lymph node involvement by thyroid cancer can be (Chap. 408). MTC is preceded by hyperplasia of the C cells, raising well tolerated but appears to increase the risk of recurrence and morthe likelihood that as-yet-unidentified “second hits” lead to cellular tality, particularly in older patients. The staging of PTC by the TNM transformation. A subset of sporadic MTC contains somatic mutations system is outlined in Table 405-12. Most papillary cancers are identithat activate RET. fied in the early stages (>80% stages I or II) and have an excellent prog nosis, with survival curves similar to expected survival (Fig. 405-12).

1	Mortality is markedly increased in stage IV disease, especially in thePapillary PTC is the most common type of thyroid cancer, accounting presence of distant metastases (stage IVC), but this group comprisesfor 70–90% of well-differentiated thyroid malignancies. Microscopic only about 1% of patients. The treatment of PTC is described below.

1	PTC is present in up to 25% of thyroid glands at autopsy, but most of these lesions are very small (several millimeters) and are not clinically Follicular The incidence of FTC varies widely in different parts of the significant. Characteristic cytologic features of PTC help make the world; it is more common in iodine-deficient regions. Currently, FTC diagnosis by FNA or after surgical resection; these include psammoma accounts for only about 5% of all thyroid cancers diagnosed in the bodies, cleaved nuclei with an “orphan-Annie” appearance caused by United States. FTC is difficult to diagnose by FNA because the distinclarge nucleoli, and the formation of papillary structures. tion between benign and malignant follicular neoplasms rests largely

1	PTC tends to be multifocal and to invade locally within the thyroid on evidence of invasion into vessels, nerves, or adjacent structures. gland as well as through the thyroid capsule and into adjacent struc-FTC tends to spread by hematogenous routes leading to bone, lung, tures in the neck. It has a propensity to spread via the lymphatic system and central nervous system metastases. Mortality rates associated with but can metastasize hematogenously as well, particularly to bone and FTC are less favorable than for PTC, in part because a larger proporlung. Because of the relatively slow growth of the tumor, a significant tion of patients present with stage IV disease. Poor prognostic features burden of pulmonary metastases may accumulate, sometimes with include distant metastases, age >50 years, primary tumor size >4 cm, remarkably few symptoms. The prognostic implication of lymph node Hürthle cell histology, and the presence of marked vascular invasion.

1	Disorders of the Thyroid Gland of papillary cancer. (Adapted with permission from Edge SB, Byrd DR: Thyroid, in Compton CC, Fritz AB, Greene FL, Trotti A [eds]: AJCC Cancer Staging Manual, 7th ed. New York, Springer, 2010, pp 87–92.)

1	All well-differentiated thyroid cancers should be surgically excised. In addition to removing the primary lesion, surgery allows accurate histologic diagnosis and staging, and multicentric disease is commonly found in the contralateral thyroid lobe. Preoperative sonography should be performed in all patients to assess the central and lateral cervical lymph node compartments for suspicious adenopathy, which if present, can undergo FNA and then be removed at surgery. Bilateral, near-total thyroidectomy has been shown to reduce recurrence rates in all patients except those with T1a tumors (≤1 cm). If cytology is diagnostic for thyroid cancer, bilateral surgery should be done. If malignancy is identified pathologically after lobectomy, completion surgery is recommended unless the tumor is T1a or is a minimally invasive follicular cancer. Bilateral surgery for patients at higher risk allows monitoring of serum Tg levels and administration of radioiodine for remnant ablation and potential

1	or is a minimally invasive follicular cancer. Bilateral surgery for patients at higher risk allows monitoring of serum Tg levels and administration of radioiodine for remnant ablation and potential treatment of iodine-avid metastases, if indicated. Therefore, near-total thyroidectomy is preferable in almost all patients; complication rates are acceptably low if the surgeon is highly experienced in the procedure.

1	Because most tumors are still TSH-responsive, levothyroxine suppression of TSH is a mainstay of thyroid cancer treatment. Although TSH suppression clearly provides therapeutic benefit, there are no prospective studies that define the optimal level of TSH suppression. The degree of TSH suppression should be individualized based on a patient’s risk of recurrence. It should be adjusted over time as surveillance blood tests and imaging confirm absence of disease or, alternatively, indicate possible residual/recurrent cancer. For patients at low risk of recurrence, TSH should be suppressed into the low but detectable range (0.1–0.5 mIU/L). If subsequent surveillance testing indicates no evidence of disease, the TSH target may rise to the lower half of the normal range. For patients at high risk of recurrence or with known metastatic disease, TSH levels should be kept to <0.1 mIU/L if there are no strong contraindications to mild thyrotoxicosis. In this instance, unbound T4 must also be

1	risk of recurrence or with known metastatic disease, TSH levels should be kept to <0.1 mIU/L if there are no strong contraindications to mild thyrotoxicosis. In this instance, unbound T4 must also be monitored to avoid excessive treatment.

1	After near-total thyroidectomy, substantial thyroid tissue often remains, particularly in the thyroid bed and surrounding the parathyroid glands. Postsurgical radioablation of the remnant thyroid eliminates residual normal thyroid, facilitating the use of Tg determinations and radioiodine scanning for long-term follow-up. In addition, well-differentiated thyroid cancer often incorporates radioiodine, although less efficiently than normal thyroid follicular cells. Radioiodine uptake is determined primarily by expression of the NIS and is stimulated by TSH, requiring expression of the TSH R. The retention time for radioactivity is influenced by the extent to which the tumor retains differentiated functions such as iodide trapping and organification. Consequently, for patients at risk of recurrence and for those with known distant metastatic disease, 131I ablation may also potentially treat residual tumor cells.

1	Indications Not all patients benefit from radioiodine therapy. Neither recurrence nor survival rates are improved in stage I patients with T1 tumors (≤2 cm) confined to the thyroid. However, in higher risk patients (larger tumors, more aggressive variants of papillary cancer, tumor vascular invasion, presence of large-volume lymph node metastases), radioiodine reduces recurrence and may increase survival.

1	131I Thyroid Ablation and Treatment As noted above, the decision to use 131I for thyroid ablation should be coordinated with the surgical approach, because radioablation is much more effective when there is minimal remaining normal thyroid tissue. Radioiodine is administered after iodine depletion (patient follows a low-iodine diet for 1≤2 weeks) and in the presence of elevated serum TSH levels to stimulate uptake of the isotope into both the remnant and potentially any residual tumor. To achieve high serum TSH levels, there are two approaches. A patient may be withdrawn from thyroid hormone so that endogenous TSH is secreted and, ideally, the serum TSH level is >25 mIU/L at the time of 131I therapy. A typical strategy is to treat the patient for several weeks postoperatively with liothyronine (25 μg qd or bid), followed by thyroid hormone withdrawal for 2 weeks. Alternatively, recombinant human TSH (rhTSH) is administered as two daily consecutive injections (0.9 mg) with

1	with liothyronine (25 μg qd or bid), followed by thyroid hormone withdrawal for 2 weeks. Alternatively, recombinant human TSH (rhTSH) is administered as two daily consecutive injections (0.9 mg) with administration of 131I 24 h after the second injection. The patient can continue to take levothyroxine and remains euthyroid. Both approaches have equal success in achieving remnant ablation.

1	A pretreatment scanning dose of 131I (usually 111–185 MBq [3–5 mCi]) or 123I (74 MBq [2 mCi]) can reveal the amount of residual tissue and provides guidance about the dose needed to accomplish ablation. However, because of concerns about radioactive “stunning” that impairs subsequent treatment, there is a trend to avoid pretreatment scanning with 131I and use either 123I or proceed directly to ablation, unless there is suspicion that the amount of residual tissue will alter therapy or that there is distant metastatic disease. In the United States, outpatient doses of up to 6475 MBq (175 mCi) can be given at most centers. The administered dose depends on the indication for therapy with lower doses of 1850–2775 MBq (50–75 mCi) given for remnant ablation but higher doses of 3700–5500 MBq (100–150 mCi) used as adjuvant therapy when residual disease may be present. A WBS following radioiodine treatment is used to confirm the 131I uptake in the remnant and to identify possible metastatic

1	mCi) used as adjuvant therapy when residual disease may be present. A WBS following radioiodine treatment is used to confirm the 131I uptake in the remnant and to identify possible metastatic disease.

1	Follow-Up Whole-Body Thyroid Scanning and Thyroglobulin Determinations Serum thyroglobulin is a sensitive marker of residual/ recurrent thyroid cancer after ablation of the residual postsurgical thyroid tissue. However, newer Tg assays have functional sensitivities as low as 0.1 ng/mL, as opposed to older assays with functional sensitivities of 1 ng/mL, reducing the number of patients with truly undetectable serum Tg levels. Because the vast majority of papillary thyroid cancer recurrences are in cervical lymph nodes, a neck ultrasound should be performed about 6 months after thyroid ablation; ultrasound has been shown to be more sensitive than WBS in this scenario.

1	In low-risk patients who have no clinical evidence of residual disease after ablation and a basal Tg <1 ng/mL on levothyroxine, an rhTSH-stimulated Tg level should be obtained 6–12 months after ablation, without WBS. If stimulated Tg levels are low (<1 ng/mL) and, ideally, undetectable, the risk of recurrence is <5% at 5 years. Newer data indicate that rhTSH stimulation may not be required for patients with undetectable basal Tg levels in sensitive assays, if there is documented absence of Tg antibodies. These patients can be followed with unstimulated Tg every 6–12 months and neck ultrasound as indicated. Levothyroxine dosing may then be titrated to a higher TSH level of 0.5–1.5 mIU/L. The use of WBS is reserved for patients with known iodine-avid metastases or those with elevated serum thyroglobulin levels and negative imaging with ultrasound, chest CT, and neck cross-sectional imaging who may require additional 131I therapy.

1	In addition, most authorities advocate radioiodine treatment for scan-negative, Tg-positive (Tg >5–10 ng/mL) patients, as many derive therapeutic benefit from a large dose of 131I. For such patients, rhTSH preparation is not FDA approved for the treatment of metastatic disease, and the traditional approach of thyroid hormone withdrawal should be followed. This involves switching patients from levothyroxine (T4) to the more rapidly cleared hormone liothyronine (T3), thereby allowing TSH to increase more quickly. Whenever 131I is administered, posttherapy WBS is the gold standard to assess iodine-avid metastases. In addition to radioiodine, external beam radiotherapy is also used to treat specific metastatic lesions, particularly when they cause bone pain or threaten neurologic injury (e.g., vertebral metastases).

1	New Potential Therapies Kinase inhibitors are being explored as a means to target pathways known to be active in thyroid cancer, including the RAS, BRAF, EGFR, VEGFR, and angiogenesis pathways. A multicenter randomized controlled trial of the multikinase inhibitor sorafenib in 417 patients with progressive metastatic thyroid cancer reported a doubling of progression-free survival to 10.8 months in the treatment group compared with the placebo group. Ongoing trials are exploring whether differentiation protocols with kinase inhibitors or other approaches might enhance radioiodine uptake and efficacy.

1	ANAPLASTIC AND OTHER FORMS OF THYROID CANCER Anaplastic Thyroid Cancer As noted above, ATC is a poorly differentiated and aggressive cancer. The prognosis is poor, and most patients die within 6 months of diagnosis. Because of the undifferentiated state of these tumors, the uptake of radioiodine is usually negligible, but it can be used therapeutically if there is residual uptake. Chemotherapy has been attempted with multiple agents, including anthracyclines and paclitaxel, but it is usually ineffective. External beam radiation therapy can be attempted and continued if tumors are responsive.

1	Thyroid Lymphoma Lymphoma in the thyroid gland often arises in the background of Hashimoto’s thyroiditis. A rapidly expanding thyroid mass suggests the possibility of this diagnosis. Diffuse large-cell lymphoma is the most common type in the thyroid. Biopsies reveal sheets of lymphoid cells that can be difficult to distinguish from small-cell lung cancer or ATC. These tumors are often highly sensitive to external radiation. Surgical resection should be avoided as initial therapy because it may spread disease that is otherwise localized to the thyroid. If staging indicates disease outside of the thyroid, treatment should follow guidelines used for other forms of lymphoma (Chap. 134).

1	MTC can be sporadic or familial and accounts for about 5% of thyroid cancers. There are three familial forms of MTC: MEN 2A, MEN 2B, and familial MTC without other features of MEN (Chap. 408). In general, MTC is more aggressive in MEN 2B than in MEN 2A, and familial MTC is more aggressive than sporadic MTC. Elevated serum calcitonin provides a marker of residual or recurrent disease. All patients with MTC should be tested for RET mutations, because genetic counseling and testing of family members can be offered to those individuals who test positive for mutations. The management of MTC is primarily surgical. Unlike tumors 2307 derived from thyroid follicular cells, these tumors do not take up radioiodine. External radiation treatment and chemotherapy may provide palliation in patients with advanced disease (Chap. 408). APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: Palpable thyroid nodules are found in about 5% of adults, but the prevalence varies considerably worldwide. Given this high prevalence rate, practitioners commonly identify thyroid nodules either on physical examination or as incidental findings on imaging performed for another indication (e.g., carotid ultrasound, cervical spine MRI). The main goal of this evaluation is to identify, in a cost-effective manner, the small subgroup of individuals with malignant lesions.

1	Nodules are more common in iodine-deficient areas, in women, and with aging. Most palpable nodules are >1 cm in diameter, but the ability to feel a nodule is influenced by its location within the gland (superficial versus deeply embedded), the anatomy of the patient’s neck, and the experience of the examiner. More sensitive methods of detection, such as CT, thyroid ultrasound, and pathologic studies, reveal thyroid nodules in up to 50% of glands in individuals over the age of 50. The presence of these thyroid incidentalomas has led to much debate about how to detect nodules and which nodules to investigate further.

1	An approach to the evaluation of a solitary nodule is outlined in Fig. 405-13. Most patients with thyroid nodules have normal thyroid function tests. Nonetheless, thyroid function should be assessed by measuring a TSH level, which may be suppressed by one or more autonomously functioning nodules. If the TSH is suppressed, a radionuclide scan is indicated to determine if the identified nodule is “hot,” as lesions with increased uptake are almost never malignant and FNA is unnecessary. Otherwise, the next step in evaluation is performance of a thyroid ultrasound for three reasons: (1) Ultrasound will confirm if the palpable nodule is indeed a nodule. About 15% of “palpable” nodules are not confirmed on imaging, and therefore, no further evaluation is required. (2) Ultrasound will assess if there are additional nonpalpable nodules for which FNA may be recommended based on imaging features and size. (3) Ultrasound will characterize the imaging features of the nodule, which, combined with

1	there are additional nonpalpable nodules for which FNA may be recommended based on imaging features and size. (3) Ultrasound will characterize the imaging features of the nodule, which, combined with the nodule’s size, facilitate decision making about FNA.

1	Evidence-based guidelines from both the American Thyroid Association and the American Association of Clinical Endocrinologists provide recommendations for nodule FNA based on sonographic imaging features and size cut offs, with lower size cut offs for nodules with more suspicious ultrasound characteristics. FNA biopsy, ideally performed with ultrasound guidance, has good sensitivity and specificity when performed by physicians familiar with the procedure and when the results are interpreted by experienced cytopathologists. The technique is particularly useful for detecting PTC. However, the distinction between benign and malignant follicular lesions is often not possible using cytology alone.

1	In several large studies, FNA biopsies yielded the following findings: 65% benign, 5% malignant or suspicious for malignancy, 10% nondiagnostic or yielding insufficient material for diagnosis, and 20% indeterminate. The Bethesda System is now widely used to provide more uniform terminology for reporting thyroid nodule FNA cytology results. This six-tiered classification system with the respective estimated malignancy rates is shown in Table 405-14. Specifically, the Bethesda System subcategorized cytology specimens previously labeled as indeterminate into three categories: atypia or follicular lesion of undetermined significance (AUS/ FLUS), follicular neoplasm, and suspicious for malignancy. Disorders of the Thyroid Gland

1	Evaluate and Rx for hyperthyroidism Surgery if indicated with pre-op US for LN assessment Atypia or follicular lesion of undetermined significance (AUS/FLUS) Repeat US-guided FNA or consider molecular testing Surgery if indicated with pre-op US for LN assessment Nondiagnostic NondiagnosticRepeat US-Guided FNA Malignant Suspicious for PTC Follicular neoplasm Consider molecular testing Surgery Benign Follow Pre-op US for LN assessment Close follow-up or surgery Diagnostic US Nodule not functioning Radionuclide scanning Hyperfunctioning nodule Results of FNA cytology History, physical examination, TSH Normal or high TSH Low TSH Bethesda System Cytology Reporting EVALUATION OF THYROID NODULES DETECTED BY PALPATION OR IMAGING Nodule(s) detected on US Do FNA based on US imaging features and size FIGURE 405-13 Approach to the patient with a thyroid nodule. See text and references for details. FNA, fine-needle aspiration; LN, lymph node; PTC, papillary thyroid cancer; TSH, thyroid-stimulating

1	FIGURE 405-13 Approach to the patient with a thyroid nodule. See text and references for details. FNA, fine-needle aspiration; LN, lymph node; PTC, papillary thyroid cancer; TSH, thyroid-stimulating hormone; US, ultrasound.

1	Cytology results indicative of malignancy mandate surgery, after performing preoperative sonography to evaluate the cervical lymph nodes. Nondiagnostic cytology specimens generally result from cystic lesions but may also occur in fibrous long-standing nodules. Ultrasound-guided FNA is indicated when a repeat FNA is necessary. Repeat FNA will yield a diagnostic cytology in about 50% of cases. Benign nodules should be monitored by ultrasound for growth, and repeat FNA should be considered if the nodule enlarges. The use of levothyroxine to suppress serum TSH is not effective in shrinking nodules in iodine-replete populations, and therefore, levothyroxine should not be used. The three new cytology classifications introduced by the Bethesda System are associated with different risks of malignancy (Table 405-14). Diagnostic Category Risk of Malignancy Nondiagnostic or unsatisfactory 1–5% Benign 2–4% Atypia or follicular lesion of unknown 15–20%

1	Diagnostic Category Risk of Malignancy Nondiagnostic or unsatisfactory 1–5% Benign 2–4% Atypia or follicular lesion of unknown 15–20% For nodules with suspicious for malignancy cytology, surgery is recommended after ultrasound assessment of cervical lymph nodes. Options to be discussed with the patient include: (1) lobectomy with intraoperative frozen section; (2) near-total thyroidectomy; and (3) mutational analysis mainly for BRAF V600E, which is virtually diagnostic of PTC, and bilateral rather than unilateral thyroid surgery is required.

1	On the other hand, the majority of nodules with AUS/FLUS and follicular neoplasm cytology results are benign; only 10–30% are malignant. The traditional approach for these patients is diagnostic lobectomy for histopathologic diagnosis. Therefore, up to 85% of patients undergo surgery for benign nodules. A high-sensitivity (~90%) novel molecular test using gene expression profiling technology may reduce the need for unnecessary surgery in these two groups. In a multicenter trial of over 265 such nodules, a negative gene expression classifier test reduced the risk of malignancy to about 6%, leading to clinical recommendations for follow-up rather than surgery.

1	The evaluation of a thyroid nodule is stressful for most patients. They are concerned about the possibility of thyroid cancer, whether verbalized or not. It is constructive, therefore, to review the diagnostic approach and to reassure patients when no malignancy is found. When a suspicious lesion or thyroid cancer is identified, the generally favorable prognosis and available treatment options can be reassuring. the result of neoplasia, leading to increased production of adreno-2309 corticotropic hormone (ACTH) by the pituitary or neuroendocrine Disorders of the Adrenal Cortex cells (ectopic ACTH) or increased production of glucocorticoids, mineralocorticoids, or adrenal androgen precursors by adrenal nod ules. Adrenal nodules are increasingly identified incidentally during abdominal imaging performed for other reasons.

1	The adrenal cortex produces three classes of corticosteroid hormones: glucocorticoids (e.g., cortisol), mineralocorticoids (e.g., aldosterone), and adrenal androgen precursors (e.g., dehydroepiandrosterone [DHEA]) (Fig. 406-1). Glucocorticoids and mineralocorticoids act through specific nuclear receptors, regulating aspects of the physi-ologic stress response as well as blood pressure and electrolyte homeo-stasis. Adrenal androgen precursors are converted in the gonads and peripheral target cells to sex steroids that act via nuclear androgen and estrogen receptors. Disorders of the adrenal cortex are characterized by deficiency or excess of one or several of the three major corticosteroid classes. Hormone deficiency can be caused by inherited glandular or enzy-matic disorders or by destruction of the pituitary or adrenal gland by autoimmune disorders, infection, infarction, or iatrogenic events such as surgery or hormonal suppression. Hormone excess is usually CH3HOH3CH3CH3CH3CHHHH

1	Disorders of the Adrenal CortexCHAPTER 406OHOHHOH3CH3CHHOH3CCH3H3COHHHHOH3CH3CHHOH3CH3COHHHOCH3OOH3CH3CHHHCH3OOH3CH3CHHHOOOH3CH3CHHHCH3OOHOH3CH3CHHHOHOOHOH3CH3CHHHOOHOHOH3CHOH3CHHHOOHOH3CHHHOOHHOOCHOH3CHHHOOHHOHOHOOOHOH3CH3CHHHOHOHHHHH3CH3CHOHHOH3CH3CHOHHOH3CH3CHOHHHHPregnenolone Cholesterol Mineralocorticoid precursors Glucocorticoid precursors Progesterone HSD17BHSD17B SRD5A HSD11B1 HSD11B217-hydroxy-pregnenolone 17-hydroxy-progesterone (17OHP) 11-Deoxycortisol Cortisol Andro-stenedione Testosterone 5-Dihydrotestosterone Deoxycortico-sterone Cortico-sterone 18OH-Cortico-sterone Aldosterone Cortisone DHEA DHEAS CYP11A1ADXHSD3B2HSD3B2HSD3B2CYP21A2PORCYP17A1PORCYP17A1PORCYP17A1PORSULT2A1PAPSS2CYP17A1PORCYP11B2ADXCYP11B1ADXCYP11B2ADXCYP11B1ADXH6PDHCYP21A2PORCYP11B2ADXFIGURE 406-1 Adrenal steroidogenesis. ADX, adrenodoxin; CYP11A1, side chain cleavage enzyme; CYP11B1, 11β-hydroxylase; CYP11B2, aldosterone synthase; CYP17A1, 17α-hydroxylase/17,20 lyase; CYP21A2, 21-hydroxylase; DHEA,

1	steroidogenesis. ADX, adrenodoxin; CYP11A1, side chain cleavage enzyme; CYP11B1, 11β-hydroxylase; CYP11B2, aldosterone synthase; CYP17A1, 17α-hydroxylase/17,20 lyase; CYP21A2, 21-hydroxylase; DHEA, dehydroepiandrosterone; DHEAS, dehydro-epiandrosterone sulfate; H6PDH, hexose-6-phosphate dehydrogenase; HSD11B1, 11β-hydroxysteroid dehydrogenase type 1; HSD11B2, 11β-hydroxysteroid dehydrogenase type 2; HSD17B, 17β-hydroxysteroid dehydrogenase; HSD3B2, 3β-hydroxysteroid dehydrogenase type 2; PAPSS2, PAPS synthase type 2; POR, P450 oxidoreductase; SRD5A, 5α-reductase; SULT2A1, DHEA sulfotransferase.

1	The normal adrenal glands weigh 6–11 g each. They are located above the kidneys and have their own blood supply. Arterial blood flows initially to the subcapsular region and then meanders from the outer cortical zona glomerulosa through the intermediate zona fasciculata to the inner zona reticularis and eventually to the adrenal medulla. The right suprarenal vein drains directly into the vena cava, while the left suprarenal vein drains into the left renal vein. During early embryonic development, the adrenals originate from the urogenital ridge and then separate from gonads and kidneys at about the sixth week of gestation. Concordant with the time of sexual differentiation (seventh to ninth week of gestation, Chap. 410), the adrenal cortex starts to produce cortisol and the adrenal sex steroid 2310 ++Hypothalamus CircadianrhythmStressors(physical, emotional, including fever, hypoglycemia, hypotension) Anterior pituitary Adrenal Cortex CRH Neurotransmitters Circulating Cortisol ACTH

1	FIGURE 406-2 Regulation of the hypothalamic-pituitary-adrenal the hypothalamus, specifically its suprachiasmatic nucleus (SCN), with additional regulation by a complex network of cell-specific clock genes. Reflecting the pattern of ACTH secretion, adrenal cortisol secretion exhibits a distinct circadian rhythm, starting to rise in the early morning hours prior to awakening, with peak levels in the morning and low levels in the evening (Fig. 406-3). Diagnostic tests assessing the HPA axis make use of the fact that it is regulated by negative feedback. Glucocorticoid excess is diagnosed by employing a dexamethasone suppression test. Dexamethasone, a potent synthetic glucocorticoid, suppresses CRH/ACTH by binding hypothalamic-pituitary glucocorticoid receptors and, therefore, results in downregulation of endogenous cortisol synthesis. Various versions of the dexamethasone suppression test are described in detail in Chap.

1	403. If cortisol production is autonomous (e.g., adrenal nodule), ACTH is already suppressed and dexamethasone has little additional effect. If cortisol production is driven by an ACTH-producing pituitary adenoma, dexamethasone suppression is ineffective at low doses but usually induces suppression at high doses. If cortisol production is driven by an ectopic source of ACTH, the tumors are usually resistant to dexamethasone suppression. Thus, the dexamethasone suppression test is useful to establish the diagnosis of Cushing’s syndrome and to assist with the differential diagnosis of cortisol excess.

1	Conversely, to assess glucocorticoid deficiency, ACTH stimulation of cortisol production is used. The ACTH peptide contains 39 amino acids but the first 24 are sufficient to elicit a physiologic response. The standard ACTH stimulation test involves administration of cosyntropin (ACTH 1-24), 0.25 mg IM or IV, and collection of blood samples at 0, 30, and 60 min for cortisol. A normal response is defined as a cortisol level >20 μg/dL (>550 nmol/L) 30–60 min after cosyntropin stimulation. A low-dose (1 μg cosyntropin IV) version of this test has been advocated; however, it has no superior diagnostic value and is more cumbersome to carry out. Alternatively, an insulin tolerance test (ITT) can be used to assess adrenal function. It involves injection of insulin to induce hypoglycemia, which represents a strong stress signal that triggers hypothalamic CRH release and activation of the entire HPA axis. The ITT involves administration of regular insulin 0.1 U/kg IV (dose (HPA) axis. ACTH,

1	represents a strong stress signal that triggers hypothalamic CRH release and activation of the entire HPA axis. The ITT involves administration of regular insulin 0.1 U/kg IV (dose (HPA) axis. ACTH, adrenocorticotropic hormone; CRH, corticotropin should be lower if hypopituitarism is likely) and collection of blood releasing hormone.

1	samples at 0, 30, 60, and 120 min for glucose, cortisol, and growth hormone (GH), if also assessing the GH axis. Oral or IV glucose is precursor DHEA. The orphan nuclear receptors SF1 (steroidogenic administered after the patient has achieved symptomatic hypoglycefactor 1; encoded by the gene NR5A1) and DAX1 (dosage-sensitive sex mia (usually glucose <40 mg/dL). A normal response is defined as a reversal gene 1; encoded by the gene NR0B1), among others, play a cru-cortisol >20 μg/dL and GH >5.1 μg/L. The ITT requires careful clinical cial role during this period of development, as they regulate a multitude of adrenal genes involved in steroidogenesis. 600 Nadir: 0015 h MESOR: 5.25 µg/dl (145 nmol/L) Acrophase: 0830 h Production of glucocorticoids and adrenal androgens is under the control of the hypothalamic-pituitary-adrenal (HPA) axis, whereas mineralocorticoids are regulated by the renin angiotensin-aldosterone (RAA) system.

1	Glucocorticoid synthesis is under inhibitory feedback control by the hypothalamus and the pituitary (Fig. 406-2). Hypothalamic release of corticotropin-releasing hormone (CRH) occurs in response to endogenous or exogenous stress. CRH stimulates the cleavage of the 241–amino acid polypeptide proopiomelanocortin (POMC) by pituitary-specific prohormone convertase 1 (PC1), yielding the 39–amino acid peptide ACTH. ACTH is released by the corticotrope cells of the anterior pituitary and acts as the pivotal regulator of adrenal cortisol synthesis, with additional short-term effects on mineralocorticoid and adrenal androgen synthesis. The release of CRH, and subsequently ACTH, occurs in a pulsatile fashion that follows a circadian rhythm under the control of

1	FIGURE 406-3 Physiologic cortisol circadian rhythm. Circulating cortisol concentrations (geometrical mean ± standard deviation values and fitted cosinor) drop under the rhythm-adjusted mean (MESOR) in the early evening hours, with nadir levels around midnight and a rise in the early morning hours; peak levels are observed ~8:30 AM (acrophase). (Modified after M Debono et al: Modified-release hydrocortisone to provide circadian cortisol profiles. J Clin Endocrinol Metab 94:1548, 2009.) Angiotensin II Adrenal Aldosterone release Activation of Angiotensin II receptor type 1 (AT1 receptor) Angiotensin converting enzyme (ACE) Vasoconstriction pressure Juxtaglomerular cells Angiotensin I Angiotensinogen Renin release FIGURE 406-4 Regulation of the renin-angiotensin-aldosterone (RAA) system.

1	FIGURE 406-4 Regulation of the renin-angiotensin-aldosterone (RAA) system. Disorders of the Adrenal Cortex monitoring and sequential measurements of glucose. It is contraindicated in patients with coronary disease, cerebrovascular disease, or seizure disorders, which has made the short cosyntropin test the commonly accepted first-line test.

1	Mineralocorticoid production is controlled by the RAA regulatory cycle, which is initiated by the release of renin from the juxtaglomerular cells in the kidney, resulting in cleavage of angiotensinogen to angiotensin I in the liver (Fig. 406-4). Angiotensin-converting enzyme (ACE) cleaves angiotensin I to angiotensin II, which binds and activates the angiotensin II receptor type 1 (AT1 receptor [AT1R]), resulting in increased adrenal aldosterone production and vasoconstriction. Aldosterone enhances sodium retention and potassium excretion, and increases the arterial perfusion pressure, which in turn regulates renin release. Because mineralocorticoid synthesis is primarily under the control of the RAA system, hypothalamic-pituitary damage does not significantly impact the capacity of the adrenal to synthesize aldosterone.

1	Similar to the HPA axis, the assessment of the RAA system can be used for diagnostic purposes. If mineralocorticoid excess is present, there is a counter-regulatory downregulation of plasma renin (see below for testing). Conversely, in mineralocorticoid deficiency, plasma renin is markedly increased. Physiologically, oral or IV sodium loading results in suppression of aldosterone, a response that is attenuated or absent in patients with autonomous mineralocorticoid excess. STEROID HORMONE SYNTHESIS, METABOLISM, AND ACTION

1	ACTH stimulation is required for the initiation of steroidogenesis. The ACTH receptor MC2R (melanocortin 2 receptor) interacts with the MC2R-accessory protein MRAP, and the complex is transported to the adrenocortical cell membrane, where it binds to ACTH (Fig. 406-5). ACTH stimulation generates cyclic AMP (cAMP), which upregulates the protein kinase A (PKA) signaling pathway. Inactive PKA is a tetramer of two regulatory and two catalytic subunits that is dissociated by cAMP into a dimer of two regulatory subunits bound to cAMP and two free and active catalytic subunits. PKA activation impacts steroidogenesis in three distinct ways: (1) increases the import of cholesterol esters; (2) increases the activity of hormone-sensitive lipase, which cleaves cholesterol esters to cholesterol for import into the mitochondrion; and (3) increases the availability and phosphorylation of CREB (cAMP response element binding), a transcription factor that enhances transcription of CYP11A1 and other

1	import into the mitochondrion; and (3) increases the availability and phosphorylation of CREB (cAMP response element binding), a transcription factor that enhances transcription of CYP11A1 and other enzymes required for glucocorticoid synthesis.

1	Adrenal steroidogenesis occurs in a zone-specific fashion, with mineralocorticoid synthesis occurring in the outer zona glomerulosa, glucocorticoid synthesis in the zona fasciculata, and adrenal androgen synthesis in the inner zona reticularis (Fig. 406-1). All steroidogenic pathways require cholesterol import into the mitochondrion, a process initiated by the action of the steroidogenic acute regulatory (StAR) protein, which shuttles cholesterol from the outer to the inner mitochondrial membrane. The majority of steroidogenic enzymes are cytochrome P450 (CYP) enzymes, which are either located in the mitochondrion (side chain cleavage enzyme, CYP11A1; 11β-hydroxylase, CYP11B1; aldosterone synthase, CYP11B2) or in the endoplasmic reticulum membrane (17α-hydroxylase, CYP17A1; 21-hydroxylase, CYP21A2; aromatase, CYP19A1). These enzymes require electron donation via specific redox cofactor enzymes, P450 oxidoreductase (POR), and adrenodoxin/adrenodoxin reductase (ADX/ADR) for the

1	CYP21A2; aromatase, CYP19A1). These enzymes require electron donation via specific redox cofactor enzymes, P450 oxidoreductase (POR), and adrenodoxin/adrenodoxin reductase (ADX/ADR) for the microsomal and mitochondrial CYP enzymes, respectively. In addition, the short-chain dehydrogenase 3β-hydroxysteroid dehydrogenase type 2 (3β-HSD2), also termed Δ4,Δ5 isomerase, plays a major role in adrenal steroidogenesis.

1	The cholesterol side chain cleavage enzyme CYP11A1 generates pregnenolone. Glucocorticoid synthesis requires conversion of pregnenolone to progesterone by 3β-HSD2, followed by conversion to 17-hydroxyprogesterone by CYP17A1, further hydroxylation at carbon 21 by CYP21A2, and eventually, 11β-hydroxylation by CYP11B1 to generate active cortisol (Fig. 406-1). Mineralocorticoid synthesis also requires progesterone, which is first converted to deoxycorticosterone by CYP21A2 and then converted via corticosterone and 18-hydroxycorticosterone to aldosterone in three steps catalyzed by CYP11B2. For adrenal androgen synthesis, pregnenolone undergoes conversion by CYP17A1, which uniquely catalyzes two enzymatic reactions. Via its 17α-hydroxylase activity, CYP17A1 converts pregnenolone to 17-hydroxypregnenolone, followed by generation of the universal sex steroid precursor DHEA via CYP17A1 17,20 lyase activity. The majority of DHEA is secreted by the adrenal in the form of its sulfate ester,

1	followed by generation of the universal sex steroid precursor DHEA via CYP17A1 17,20 lyase activity. The majority of DHEA is secreted by the adrenal in the form of its sulfate ester, DHEAS, generated by DHEA sulfotransferase (SULT2A1).

1	FIGURE 406-5 ACTH effects on adrenal steroidogenesis. ACTH, adrenocorticotropic hormone; binding protein; MRAP, MC2R-accessory protein; protein kinase A catalytic subunit (C; PRKACA), PKA regulatory subunit (R; PRKAR1A); StAR, steroidogenic acute regulatory (protein); TSPO, translocator protein.

1	Following its release from the adrenal, cortisol circulates in the bloodstream mainly bound to cortisol-binding globulin (CBG) and to a lesser extent to albumin, with only a minor fraction circulating as free, unbound hormone. Free cortisol is thought to enter cells directly, not requiring active transport. In addition, in a multitude of peripheral target tissues of glucocorticoid action, including adipose, liver, muscle, and brain, cortisol is generated from inactive cortisone within the cell by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) (Fig. 406-6). Thereby, 11β-HSD1 functions as a tissue-specific prereceptor regulator of glucocorticoid action. For the conversion of inactive cortisone to active cortisol, 11β-HSD1 requires nicotinamide adenine dinucleotide phosphate (NADPH [reduced form]), which is provided by the enzyme hexose-6-phosphate dehydrogenase (H6PDH). Like the catalytic domain of 11β-HSD1, H6PDH is located in the lumen of the endoplasmic reticulum, and

1	[reduced form]), which is provided by the enzyme hexose-6-phosphate dehydrogenase (H6PDH). Like the catalytic domain of 11β-HSD1, H6PDH is located in the lumen of the endoplasmic reticulum, and converts glucose-6-phosphate (G6P) to 6-phosphogluconate (6PGL), thereby regenerating NADP+ to NADPH, which drives the activation of cortisol from cortisone by 11β-HSD1.

1	In the cytosol of target cells, cortisol binds and activates the glucocorticoid receptor (GR), which results in dissociation of heat shock FIGURE 406-6 Prereceptor activation of cortisol and glucocorticoid receptor (GR) action. AP-1 activator protein-1; G6P, glucose-6phosphate; GRE, glucocorticoid response elements; HSP, heat shock proteins; NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); 6PGL, 6-phosphogluconate. FIGURE 406-7 Prereceptor inactivation of cortisol and mineralocorticoid receptor action. ENaC, epithelial sodium channel; HRE, hormone response element; NADH, nicotinamide adenine dinucleotide; SGK-1, serum glucocorticoid-inducible kinase-1.

1	proteins (HSP) from the receptor and subsequent dimerization (Fig. 406-6). Cortisol-bound GR dimers translocate to the nucleus and activate glucocorticoid response elements (GRE) in the DNA sequence, thereby enhancing transcription of glucocorticoid-regulated genes (GR transactivation). However, cortisol-bound GR can also form heterodimers with transcription factors such as AP-1 or NF-κB, resulting in transrepression of proinflammatory genes, a mechanism of major importance for the anti-inflammatory action of glucocorticoids. It is important to note that corticosterone also exerts glucocorticoid activity, albeit much weaker than cortisol itself. However, in rodents, corticosterone is the major glucocorticoid, and in patients with 17-hydroxylase deficiency, lack of cortisol can be compensated for by higher concentrations of corticosterone that accumulates as a consequence of the enzymatic block.

1	Cortisol is inactivated to cortisone by the microsomal enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) (Fig. 406-7), mainly in the kidney, but also in the colon, salivary glands, and other target tissues. Cortisol and aldosterone bind the mineralocorticoid receptor (MR) with equal affinity; however, cortisol circulates in the bloodstream at about a thousandfold higher concentration. Thus, only rapid inactivation of cortisol to cortisone by 11β-HSD2 prevents MR activation by excess cortisol, thereby acting as a tissue-specific modulator of the MR pathway. In addition to cortisol and aldosterone, deoxycorticosterone (DOC) (Fig. 406-1) also exerts mineralocorticoid activity. DOC accumulation due to 11β-hydroxylase deficiency or due to tumor-related excess production can result in mineralocorticoid excess.

1	Aldosterone synthesis in the adrenal zona glomerulosa cells is driven by the enzyme aldosterone synthase (CYP11B2). The binding of angiotensin II to the AT1 receptor causes glomerulosa cell membrane depolarization by increasing intracellular sodium through inhibition of sodium potassium (Na+/K+) ATPase enzymes as well as potassium channels. This drives an increase in intracellular calcium by opening of voltage-dependent calcium channels or inhibition of calcium (Ca2+) ATPase enzymes. Consequently, the calcium signaling pathway is triggered, resulting in upregulation of CYP11B2 transcription (Fig. 406-8).

1	Analogous to cortisol action via the GR, aldosterone (or cortisol) binding to the MR in the kidney tubule cell dissociates the HSP–receptor complex, allowing homodimerization of the MR, and translocation of the hormone-bound MR dimer to the nucleus (Fig. 406-7). The activated MR enhances transcription of the epithelial sodium channel (ENaC) and serum glucocorticoid-inducible kinase 1 (SGK-1). In the cytosol, interaction of ENaC with Nedd4 prevents cell surface expression of ENaC. However, SGK-1 phosphorylates serine residues within the Nedd4 protein, reduces the interaction between Nedd4 and ENaC, and consequently, enhances the trafficking of ENaC to the cell surface, where it mediates sodium retention.

1	(See also Chap. 403) Cushing’s syndrome reflects a constellation of clinical features that result from chronic exposure to excess glucocorticoids of any etiology. The disorder can be ACTH-dependent (e.g., pituitary corticotrope adenoma, ectopic secretion of ACTH by nonpituitary tumor) or ACTH-independent (e.g., adrenocortical adenoma, adrenocortical carcinoma, nodular adrenal hyperplasia), as well as iatrogenic (e.g., administration of exogenous glucocorticoids to treat various inflammatory conditions). The term Cushing’s disease refers specifically to Cushing’s syndrome caused by a pituitary corticotrope adenoma.

1	Epidemiology Cushing’s syndrome is generally considered a rare disease. It occurs with an incidence of 1–2 per 100,000 population per year. However, it is debated whether mild cortisol excess may be more prevalent among patients with several features of Cushing’s such as centripetal obesity, type 2 diabetes, and osteoporotic vertebral fractures, recognizing that these are relatively nonspecific and common in the population. In the overwhelming majority of patients, Cushing’s syndrome is caused by an ACTH-producing corticotrope adenoma of the pituitary (Table 406-1), as initially described by Harvey Cushing in 1912. Cushing’s disease more frequently affects women, with the exception of prepubertal cases, where it is more common in boys. By contrast, Disorders of the Adrenal Cortex Na+, K+– Na+, Ca2+ Ca2+ Ca2+–

1	Disorders of the Adrenal Cortex Na+, K+– Na+, Ca2+ Ca2+ Ca2+– ATPase AT1R Ang II K+ channel exchanger channel ATPase Na+ Depolarization Ca2+ Ca2+ Ca2+ Na+ K+ K+ Nucleus CYP11B2 Aldosterone FIGURE 406-8 Regulation of adrenal aldosterone synthesis. AngII, angiotensin II; AT1R, angiotensin II receptor type 1; CYP11B2, aldosterone synthase. (Modified after F Beuschlein: Regulation of aldosterone secretion: from physiology to disease. Eur J Endocrinol 168:R85, 2013.) ectopic ACTH syndrome is more frequently identified in men. Only 10% of patients with Cushing’s syndrome have a primary, adrenal cause of their disease (e.g., autonomous cortisol excess independent of ACTH), and most of these patients are women. Overall, the medical use of glucocorticoids for immunosuppression, or for the treatment of inflammatory disorders, is the most common cause of Cushing’s syndrome.

1	Etiology In at least 90% of patients with Cushing’s disease, ACTH excess is caused by a corticotrope pituitary microadenoma, often only a few millimeters in diameter. Pituitary macroadenomas (i.e., tumors >1 cm in size) are found in only 5–10% of patients. Pituitary corticotrope adenomas usually occur sporadically but very rarely can be found in the context of multiple endocrine neoplasia type 1 (MEN 1) (Chap. 408). Ectopic ACTH production is predominantly caused by occult carcinoid tumors, most frequently in the lung, but also in thymus or pancreas. Because of their small size, these tumors are often difficult to locate. Advanced small-cell lung cancer can cause ectopic ACTH production. In rare cases, ectopic CRH and/or ACTH production has been found to originate from medullary thyroid carcinoma or pheochromocytoma, the latter co-secreting catecholamines and ACTH.

1	The majority of patients with ACTH-independent cortisol excess harbor a cortisol-producing adrenal adenoma; intratumor mutations, i.e., somatic mutations in the PKA catalytic subunit PRKACA, have been identified as cause of disease in 40% of these tumors. Adrenocortical carcinomas may also cause ACTH-independent disease and are often large, with excess production of several corticosteroid classes.

1	A rare but notable cause of adrenal cortisol excess is macronodular adrenal hyperplasia with low circulating ACTH, but with evidence for autocrine stimulation of cortisol production via intraadrenal ACTH production. These hyperplastic nodules are often also characterized by ectopic expression of G protein–coupled receptors not usually found in the adrenal, including receptors for luteinizing hormone, vasopressin, serotonin, interleukin 1, catecholamines, or gastric inhibitory peptide (GIP), the cause of food-dependent Cushing’s. Activation of these receptors results in upregulation of PKA signaling, as physiologically occurs with ACTH, with a subsequent increase in cortisol production. A combination of germline and somatic mutations in the tumor-suppressor gene ARMC5 have been identified as a prevalent cause of Cushing’s due to macronodular adrenal hyperplasia. Germline mutations in the PKA catalytic subunit PRKACA can represent a rare cause of macronodular adrenal hyperplasia

1	as a prevalent cause of Cushing’s due to macronodular adrenal hyperplasia. Germline mutations in the PKA catalytic subunit PRKACA can represent a rare cause of macronodular adrenal hyperplasia associated with cortisol excess.

1	Mutations in one of the regulatory subunits of PKA, PRKAR1A, are found in patients with primary pigmented nodular adrenal disease (PPNAD) as part of Carney’s complex, an autosomal dominant multiple neoplasia condition associated with cardiac myxomas, hyperlentiginosis, Sertoli cell tumors, and PPNAD. PPNAD can present as micronodular or macronodular hyperplasia, or both. Phosphodiesterases can influence intracellular cAMP and can thereby impact PKA activation. Mutations in PDE11A and PDE8B have been identified in patients with bilateral adrenal hyperplasia and Cushing’s, with and without evidence of PPNAD. Another rare cause of ACTH-independent Cushing’s is McCune-Albright syndrome, also associated with polyostotic fibrous dysplasia, unilateral café-au-lait spots, and precocious puberty. McCune-Albright syndrome is caused by activating mutations in the stimulatory Body fat Weight gain, central obesity, rounded face, fat pad on back of neck (“buffalo hump”)

1	Body fat Weight gain, central obesity, rounded face, fat pad on back of neck (“buffalo hump”) Skin Facial plethora, thin and brittle skin, easy bruising, broad and purple stretch marks, acne, hirsutism Bone Osteopenia, osteoporosis (vertebral fractures), decreased linear growth in children Muscle Weakness, proximal myopathy (prominent atrophy of gluteal and upper leg muscles with difficulty climb Cardiovascular Hypertension, hypokalemia, edema, atherosclerosis system Metabolism Glucose intolerance/diabetes, dyslipidemia Reproductive system Decreased libido, in women amenorrhea (due to cortisol-mediated inhibition of gonadotropin release) Central nervous Irritability, emotional lability, depression, sometimes system cognitive defects; in severe cases, paranoid psychosis Blood and immune Increased susceptibility to infections, increased white system blood cell count, eosinopenia, hypercoagulation with increased risk of deep vein thrombosis and pulmo

1	Blood and immune Increased susceptibility to infections, increased white system blood cell count, eosinopenia, hypercoagulation with increased risk of deep vein thrombosis and pulmo G protein alpha subunit 1, GNAS-1 (guanine nucleotide binding protein alpha stimulating activity polypeptide 1), and such mutations have also been found in bilateral macronodular hyperplasia without other McCune-Albright features and, in rare instances, also in isolated cortisol-producing adrenal adenomas (Table 406-1; Chap. 426e).

1	Clinical Manifestations Glucocorticoids affect almost all cells of the body, and thus signs of cortisol excess impact multiple physiologic systems (Table 406-2), with upregulation of gluconeogenesis, lipolysis, and protein catabolism causing the most prominent features. In addition, 2315 excess glucocorticoid secretion overcomes the ability of 11β-HSD2 to rapidly inactivate cortisol to cortisone in the kidney, thereby exerting mineralocorticoid actions, manifest as diastolic hypertension, hypokalemia, and edema. Excess glucocorticoids also interfere with central regulatory systems, leading to suppression of gonadotropins with subsequent hypogonadism and amenorrhea, and suppression of the hypothalamic-pituitary-thyroid axis, resulting in decreased thyroid-stimulating hormone (TSH) secretion.

1	The majority of clinical signs and symptoms observed in Cushing’s syndrome are relatively nonspecific and include features such as obesity, diabetes, diastolic hypertension, hirsutism, and depression that are commonly found in patients who do not have Cushing’s. Therefore, careful clinical assessment is an important aspect of evaluating suspected cases. A diagnosis of Cushing’s should be considered when several clinical features are found in the same patient, in particular when more specific features are found. These include fragility of the skin, with easy bruising and broad (>1 cm), purplish striae (Fig. 406-9), and signs of proximal myopathy, which becomes most obvious when trying to stand up from a chair without the use of hands or when climbing stairs. Clinical manifestations of Cushing’s do not differ substantially among the different causes of Cushing’s. In ectopic ACTH syndrome, hyperpigmentation of the knuckles, scars, or skin areas exposed to increased friction can be

1	Cushing’s do not differ substantially among the different causes of Cushing’s. In ectopic ACTH syndrome, hyperpigmentation of the knuckles, scars, or skin areas exposed to increased friction can be observed (Fig. 406-9) and is caused by stimulatory effects of excess ACTH and other POMC cleavage products on melanocyte pigment production. Furthermore, patients with ectopic ACTH syndrome, and some with adrenocortical carcinoma as the cause of Cushing’s, may have a more brisk onset and rapid progression of clinical signs and symptoms.

1	Patients with Cushing’s syndrome can be acutely endangered by deep vein thrombosis, with subsequent pulmonary embolism due to a hypercoagulable state associated with Cushing’s. The majority of patients also experience psychiatric symptoms, mostly in the form of anxiety or depression, but acute paranoid or depressive psychosis FIGURE 406-9 Clinical features of Cushing’s syndrome. A. Note central obesity and broad, purple stretch marks (B. close-up). C. Note thin and brittle skin in an elderly patient with Cushing’s syndrome. D. Hyperpigmentation of the knuckles in a patient with ectopic adrenocorticotropic hormone (ACTH) excess.

1	Disorders of the Adrenal Cortex 2316 may also occur. Even after cure, long-term health may be affected by A diagnosis of Cushing’s can be considered as established if the persistently impaired health-related quality of life and increased risk results of several tests are consistently suggestive of Cushing’s. These of cardiovascular disease and osteoporosis with vertebral fractures, tests may include increased 24-h urinary free cortisol excretion in depending on the duration and degree of exposure to significant cor-three separate collections, failure to appropriately suppress morning tisol excess. cortisol after overnight exposure to dexamethasone, and evidence of loss of diurnal cortisol secretion with high levels at midnight, theDiagnosis The most important first step in the management of time of the physiologically lowest secretion (Fig. 406-10). Factorspatients with suspected Cushing’s syndrome is to establish the correct potentially affecting the outcome of these diagnostic tests

1	time of the physiologically lowest secretion (Fig. 406-10). Factorspatients with suspected Cushing’s syndrome is to establish the correct potentially affecting the outcome of these diagnostic tests have to bediagnosis. Most mistakes in clinical management, leading to unneces-excluded such as incomplete 24-h urine collection or rapid inactivasary imaging or surgery, are made because the diagnostic protocol is tion of dexamethasone due to concurrent intake of CYP3A4-inducingnot followed (Fig. 406-10). This protocol requires establishing the drugs (e.g., antiepileptics, rifampicin). Concurrent intake of oral con-diagnosis of Cushing’s beyond doubt prior to employing any tests traceptives that raise CBG and thus total cortisol can cause failure toused for the differential diagnosis of the condition. In principle, after suppress after dexamethasone. If in doubt, testing should be repeatedexcluding exogenous glucocorticoid use as the cause of clinical signs after 4–6 weeks off estrogens.

1	In principle, after suppress after dexamethasone. If in doubt, testing should be repeatedexcluding exogenous glucocorticoid use as the cause of clinical signs after 4–6 weeks off estrogens. Patients with pseudo-Cushing states, i.e.,and symptoms, suspected cases should be tested if there are multiple alcohol-related, and those with cyclic Cushing’s may require further and progressive features of Cushing’s, particularly features with a testing to safely confirm or exclude the diagnosis of Cushing’s. Inpotentially higher discriminatory value. Exclusion of Cushing’s is also addition, the biochemical assays employed can affect the test results,indicated in patients with incidentally discovered adrenal masses. with specificity representing a common problem with antibody-based

1	Clinical suspicion of Cushing’s (Central adiposity, proximal myopathy, striae, amenorrhea, hirsutism, impaired glucose tolerance, diastolic hypertension, and osteoporosis) Screening/confirmation of diagnosis a.m.after 1 mg dexamethasone at 11 p.m.) If further confirmation needed/desired: • Low dose DEX test (Plasma cortisol >50 nmol/L after 0.5 mg dexamethasone q6h for 2 days) Pos. Neg. Neg.

1	Pos. Neg. Neg. Adrenal tumor workup Differential diagnosis 1: Plasma ACTH ACTH normal or high>15 pg/ml CRH test and highdose DEX positive ACTH suppressed to <5 pg/ml ACTH-dependent Cushing’s ACTH-independent Cushing’s Bilateral micronodular or macronodular adrenal hyperplasia Bilateral adrenal-ectomy Unilateral Unilateral adrenal mass CRH test and highEquivocal adrenal-ectomy Differential diagnosis 2 • MRI pituitary Unenhanced CT• CRH test (ACTH increase >40% at 15-30 min + cortisol increase >20%at 45-60 min after CRH 100 µg IV)• High dose DEX test (Cortisol suppression >50% after q6h 2 mg DEX for 2 days) Trans-Inferior petrosal sinus sampling Locate and sphenoidal(petrosal/peripheralremove surgery ACTH ratio >2 atbaseline, >3 at 2–5 minectopic after CRH 100 µg i.v.) source ACTH Ectopic ACTH production Cushing’s disease pituitary dose DEX negative results adrenals

1	FIGURE 406-10 Management of the patient with suspected Cushing’s syndrome. ACTH, adrenocorticotropic hormone; CRH, corticotropinreleasing hormone; CT, computed tomography; DEX, dexamethasone; MRI, magnetic resonance imaging. assays for the measurement of urinary free cortisol. These assays have been greatly improved by the introduction of highly specific tandem mass spectrometry.

1	Differential Diagnosis The evaluation of patients with confirmed Cushing’s should be carried out by an endocrinologist and begins with the differential diagnosis of ACTH-dependent and ACTH-independent cortisol excess (Fig. 406-10). Generally, plasma ACTH levels are suppressed in cases of autonomous adrenal cortisol excess, as a consequence of enhanced negative feedback to the hypothalamus and pituitary. By contrast, patients with ACTH-dependent Cushing’s have normal or increased plasma ACTH, with very high levels being found in some patients with ectopic ACTH syndrome. Importantly, imaging should only be used after it is established whether the cortisol excess is ACTH-dependent or ACTH-independent, because nodules in the pituitary or the adrenal are a common finding in the general population. In patients with confirmed ACTH-independent excess, adrenal imaging is indicated (Fig. 406-11), preferably using an unenhanced computed tomography (CT) scan. This allows assessment of adrenal

1	In patients with confirmed ACTH-independent excess, adrenal imaging is indicated (Fig. 406-11), preferably using an unenhanced computed tomography (CT) scan. This allows assessment of adrenal morphology and determination of precontrast tumor density in Hounsfield units (HU), which helps to distinguish between benign and malignant adrenal lesions.

1	For ACTH-dependent cortisol excess (Chap. 403), a magnetic resonance image (MRI) of the pituitary is the investigation of choice, but it may not show an abnormality in up to 40% of cases because of small tumors below the sensitivity of detection. Characteristically, pituitary corticotrope adenomas fail to enhance following gadolinium administration on T1-weighted MRI images. In all cases of confirmed ACTH-dependent Cushing’s, further tests are required for the differential diagnosis of pituitary Cushing’s disease and ectopic ACTH syndrome.

1	These tests exploit the fact that most pituitary corticotrope adenomas 2317 still display regulatory features, including residual ACTH suppression by high-dose glucocorticoids and CRH responsiveness. In contrast, ectopic sources of ACTH are typically resistant to dexamethasone suppression and unresponsive to CRH (Fig. 406-10). However, it should be noted that a small minority of ectopic ACTH-producing tumors exhibit dynamic responses similar to pituitary corticotrope tumors. If the two tests show discordant results, or if there is any other reason for doubt, the differential diagnosis can be further clarified by performing bilateral inferior petrosal sinus sampling (IPSS) with concurrent blood sampling for ACTH in the right and left inferior petrosal sinus and a peripheral vein. An increased central/peripheral plasma ACTH ratio >2 at baseline and >3 at 2–5 min after CRH injection is indicative of Cushing’s disease (Fig. 406-10), with very high sensitivity and specificity. Of note, the

1	central/peripheral plasma ACTH ratio >2 at baseline and >3 at 2–5 min after CRH injection is indicative of Cushing’s disease (Fig. 406-10), with very high sensitivity and specificity. Of note, the results of the IPSS cannot be reliably used for lateralization (i.e., prediction of the location of the tumor within the pituitary), because there is broad interindividual variability in the venous drainage of the pituitary region. Importantly, no cortisollowering agents should be used prior to IPSS.

1	If the differential diagnostic testing indicates ectopic ACTH syndrome, then further imaging should include high-resolution, fine-cut CT scanning of the chest and abdomen for scrutiny of the lung, thymus, and pancreas. If no lesions are identified, an MRI of the chest can be considered because carcinoid tumors usually show high signal intensity on T2-weighted images. Furthermore, octreotide scintigraphy can be helpful in some cases because ectopic ACTH-producing tumors often express somatostatin receptors. Depending on the suspected cause, patients with ectopic ACTH syndrome should also undergo blood sampling for fasting gut hormones, chromogranin A, calcitonin, and biochemical exclusion of pheochromocytoma.

1	FIGURE 406-11 Adrenal imaging in Cushing’s syndrome. A. Adrenal computed tomography (CT) showing normal bilateral adrenal morphology (arrows). B. CT scan depicting a right adrenocortical adenoma (arrow) causing Cushing’s syndrome. C. Magnetic resonance imaging (MRI) showing bilateral adrenal hyperplasia due to excess adrenocorticotropic hormone stimulation in Cushing’s disease. D. MRI showing bilateral macronodular hyperplasia causing Cushing’s syndrome. Disorders of the Adrenal Cortex Overt Cushing’s is associated with a poor prognosis if left untreated. In ACTH-independent disease, treatment consists of surgical removal of the adrenal tumor. For smaller tumors, a minimally invasive approach can be used, whereas for larger tumors and those suspected of malignancy, an open approach is preferred.

1	In Cushing’s disease, the treatment of choice is selective removal of the pituitary corticotrope tumor, usually via an endoscopic transsphenoidal approach. This results in an initial cure rate of 70–80% when performed by a highly experienced surgeon. However, even after initial remission following surgery, long-term follow-up is important because late relapse occurs in a significant number of patients. If pituitary disease recurs, there are several options, including second surgery, radiotherapy, stereotactic radiosurgery, and bilateral adrenalectomy. These options need to be applied in a highly individualized fashion.

1	In some patients with very severe, overt Cushing’s (e.g., difficult to control hypokalemic hypertension or acute psychosis), it may be necessary to introduce medical therapy to rapidly control the cortisol excess during the period leading up to surgery. Similarly, patients with metastasized, glucocorticoid-producing carcinomas may require long-term antiglucocorticoid drug treatment. In case of ectopic ACTH syndrome, in which the tumor cannot be located, one must carefully weigh whether drug treatment or bilateral adrenalectomy is the most appropriate choice, with the latter facilitating immediate cure but requiring life-long corticosteroid replacement. In this instance, it is paramount to ensure regular imaging follow-up for identification of the ectopic ACTH source.

1	Oral agents with established efficacy in Cushing’s syndrome are metyrapone and ketoconazole. Metyrapone inhibits cortisol synthesis at the level of 11β-hydroxylase (Fig. 406-1), whereas the antimycotic drug ketoconazole inhibits the early steps of steroidogenesis. Typical starting doses are 500 mg tid for metyrapone (maximum dose, 6 g) and 200 mg tid for ketoconazole (maximum dose, 1200 mg). Mitotane, a derivative of the insecticide o,p’DDD, is an adrenolytic agent that is also effective for reducing cortisol. Because of its side effect profile, it is most commonly used in the context of adrenocortical carcinoma, but low-dose treatment (500–1000 mg/d) has also been used in benign Cushing’s. In severe cases of cortisol excess, etomidate can be used to lower cortisol. It is administered by continuous IV infusion in low, nonanesthetic doses.

1	After the successful removal of an ACTHor cortisol-producing tumor, the HPA axis will remain suppressed. Thus, hydrocortisone replacement needs to be initiated at the time of surgery and slowly tapered following recovery, to allow physiologic adaptation to normal cortisol levels. Depending on degree and duration of cortisol excess, the HPA axis may require many months or even years to resume normal function. MINERALOCORTICOID EXCESS Epidemiology Following the first description of a patient with an aldosterone-producing adrenal adenoma (Conn’s syndrome), mineralocorticoid excess was thought to represent a rare cause of hypertension. However, in studies systematically screening all patients with hypertension, a much higher prevalence is now recognized, ranging from 5 to 12%. The prevalence is higher when patients are preselected for hypokalemic hypertension.

1	Etiology The most common cause of mineralocorticoid excess is primary aldosteronism, reflecting excess production of aldosterone by the adrenal zona glomerulosa. Bilateral micronodular hyperplasia is somewhat more common than unilateral adrenal adenomas (Table 406-3). Somatic mutations in channels and enzymes responsible for increasing sodium and calcium influx in adrenal zona glomerulosa cells have been identified as prevalent causes of aldosterone-producing adrenal adenomas (Table 406-3) and, in the case of germline mutations, also of primary aldosteronism due to bilateral macronodular adrenal hyperplasia. However, bilateral adrenal hyperplasia as a cause of mineralocorticoid excess is usually micronodular but can also contain larger nodules that might be mistaken for a unilateral adenoma. In rare instances, primary aldosteronism is caused by an adrenocortical carcinoma. Carcinomas should be considered in younger patients and in those with larger tumors, because benign

1	adenoma. In rare instances, primary aldosteronism is caused by an adrenocortical carcinoma. Carcinomas should be considered in younger patients and in those with larger tumors, because benign aldosterone-producing adenomas usually measure <2 cm in diameter.

1	A rare cause of aldosterone excess is glucocorticoid-remediable aldosteronism (GRA), which is caused by a chimeric gene resulting Causes of Mineralocorticoid Excess Mechanism % brane channel in open conformation for longer, enhancing mineralocorticoid action Abbreviations: ACTH, adrenocorticotropic hormone; DOC, deoxycorticosterone; ENaC, epithelial sodium channel; GR, glucocorticoid receptor; HSD11B2, 11β-hydroxysteroid dehydrogenase type 2; MR, mineralocorticoid receptor.

1	from cross-over of promoter sequences between the CYP11B1 and CYP11B2 genes that are involved in glucocorticoid and mineralocorticoid synthesis, respectively (Fig. 406-1). This rearrangement brings CYP11B2 transcription under the control of ACTH receptor signaling; consequently, aldosterone production is regulated by ACTH rather than by renin. The family history can be helpful because there may be evidence for dominant transmission of hypertension. Recognition of the disorder is important because it can be associated with early-onset hypertension and strokes. In addition, glucocorticoid suppression can reduce aldosterone production.

1	Other rare causes of mineralocorticoid excess are listed in Table 406-3. An important cause is excess binding and activation of the mineralocorticoid receptor by a steroid other than aldosterone. Cortisol acts as a potent mineralocorticoid if it escapes efficient inactivation to cortisone by 11β-HSD2 in the kidney (Fig. 406-7). This can be caused by inactivating mutations in the HSD11B2 gene resulting in the syndrome of apparent mineralocorticoid excess (SAME) that characteristically manifests with severe hypokalemic hypertension in childhood. However, milder mutations may cause normokalemic hypertension manifesting in adulthood (type II SAME). Inhibition of 11β-HSD2 by excess licorice ingestion also results in hypokalemic hypertension, as does overwhelming of 11β-HSD2 conversion capacity by cortisol excess in Cushing’s syndrome. Deoxycorticosterone (DOC) also binds and activates the mineralocorticoid receptor and can cause hypertension if its circulating concentrations are increased.

1	by cortisol excess in Cushing’s syndrome. Deoxycorticosterone (DOC) also binds and activates the mineralocorticoid receptor and can cause hypertension if its circulating concentrations are increased. This can arise through autonomous DOC secretion by an adrenocortical carcinoma, but also when DOC accumulates as a consequence of an adrenal enzymatic block, as seen in congenital adrenal hyperplasia due to CYP11B1 (11β-hydroxylase) or CYP17A1 (17α-hydroxylase) deficiency (Fig. 406-1). Progesterone can cause hypokalemic hypertension in rare individuals who harbor a mineralocorticoid receptor mutation that enhances binding and activation by progesterone; physiologically, progesterone normally exerts antimineralocorticoid activity. Finally, excess mineralocorticoid activity can be caused by mutations in the β or γ subunits of the ENaC, disrupting its interaction with Nedd4 (Fig. 406-7), and thereby decreasing receptor internalization and degradation. The constitutively active ENAC drives

1	in the β or γ subunits of the ENaC, disrupting its interaction with Nedd4 (Fig. 406-7), and thereby decreasing receptor internalization and degradation. The constitutively active ENAC drives hypokalemic hypertension, resulting in an autosomal dominant disorder termed Liddle’s syndrome.

1	Clinical Manifestations Excess activation of the mineralocorticoid receptor leads to potassium depletion and increased sodium retention, with the latter causing an expansion of extracellular and plasma volume. Increased ENaC activity also results in hydrogen depletion that can cause metabolic alkalosis. Aldosterone also has direct effects on the vascular system, where it increases cardiac remodeling and decreases compliance. Aldosterone excess may cause direct damage to the myocardium and the kidney glomeruli, in addition to secondary damage due to systemic hypertension.

1	The clinical hallmark of mineralocorticoid excess is hypokalemic hypertension; serum sodium tends to be normal due to the concurrent fluid retention, which in some cases can lead to peripheral edema. Hypokalemia can be exacerbated by thiazide drug treatment, which leads to increased delivery of sodium to the distal renal tubule, thereby driving potassium excretion. Severe hypokalemia can be associated with muscle weakness, overt proximal myopathy, or even hypokalemic paralysis. Severe alkalosis contributes to muscle cramps and, in severe cases, can cause tetany.

1	Diagnosis Diagnostic screening for mineralocorticoid excess is not currently recommended for all patients with hypertension, but should be restricted to those who exhibit hypertension associated with drug resistance, hypokalemia, an adrenal mass, or onset of disease before the age of 40 years (Fig. 406-12). The accepted screening test is concurrent measurement of plasma renin and aldosterone with subsequent calculation of the aldosterone-renin ratio (ARR) (Fig. 406-12); serum potassium needs to be normalized prior to testing. Stopping antihypertensive medication can be cumbersome, particularly in patients with severe hypertension. Thus, for practical purposes, in the first instance the patient can remain on the usual antihypertensive medications, with the exception that mineralocorticoid receptor antagonists need to be ceased at least 4 weeks prior to ARR measurement. The remaining antihypertensive 2319 drugs usually do not affect the outcome of ARR testing, except that beta blocker

1	receptor antagonists need to be ceased at least 4 weeks prior to ARR measurement. The remaining antihypertensive 2319 drugs usually do not affect the outcome of ARR testing, except that beta blocker treatment can cause false-positive results and ACE/AT1R inhibitors can cause false-negative results in milder cases (Table 406-4).

1	ARR screening is positive if the ratio is >750 pmol/L per ng/mL per hour, with a concurrently high normal or increased aldosterone (Fig. 406-12). If one relies on the ARR only, the likelihood of a false-positive ARR becomes greater when renin levels are very low. The characteristics of the biochemical assays are also important. Some labs measure plasma renin activity, whereas others measure plasma renin concentrations. Antibody-based assays for the measurement of serum aldosterone lack the reliability of tandem mass spectrometry assays, but these are not yet ubiquitously available.

1	Diagnostic confirmation of mineralocorticoid excess in a patient with positive ARR screening result should be undertaken by an endocrinologist as the tests lack optimized validation. The most straightforward is the saline infusion test, which involves the IV administration of 2 L of physiologic saline over a 4-h period. Failure of aldosterone to suppress below 140 pmol/L (5 ng/dL) is indicative of autonomous mineralocorticoid excess. Alternative tests are the oral sodium loading test (300 mmol NaCl/d for 3 days) or the fludrocortisone suppression test (0.1 mg q6h with 30 mmol NaCl q8h for 4 days); the latter can be difficult because of the risk of profound hypokalemia and increased hypertension. In patients with overt hypokalemic hypertension, strongly positive ARR, and concurrently increased aldosterone levels, confirmatory testing is usually not necessary.

1	Differential Diagnosis and Treatment After the diagnosis of hyperaldosteronism is established, the next step is to use adrenal imaging to further assess the cause. Fine-cut CT scanning of the adrenal region is the method of choice because it provides excellent visualization of adrenal morphology. CT will readily identify larger tumors suspicious of malignancy but may miss lesions smaller than 5 mm. The differentiation between bilateral micronodular hyperplasia and a unilateral adenoma is only required if a surgical approach is feasible and desired. Consequently, selective adrenal vein sampling (AVS) should only be carried out in surgical candidates with either no obvious lesion on CT or evidence of a unilateral lesion in patients older than 40 years, because the latter patients have a high likelihood of harboring a coincidental, endocrine-inactive adrenal adenoma (Fig. 406-12). AVS is used to compare aldosterone levels in the inferior vena cava and between the right and left adrenal

1	likelihood of harboring a coincidental, endocrine-inactive adrenal adenoma (Fig. 406-12). AVS is used to compare aldosterone levels in the inferior vena cava and between the right and left adrenal veins. AVS requires concurrent measurement of cortisol to document correct placement of the catheter in the adrenal veins and should demonstrate a cortisol gradient >3 between the vena cava and each adrenal vein. Lateralization is confirmed by an aldosterone/cortisol ratio that is at least twofold higher on one side than the other. AVS is a complex procedure that requires a highly skilled interventional radiologist. Even then, the right adrenal vein can be difficult to cannulate correctly, which, if not achieved, invalidates the procedure. There is also no agreement as to whether the two adrenal veins should be cannulated simultaneously or successively and whether ACTH stimulation enhances the diagnostic value of AVS.

1	Patients younger than 40 years with confirmed mineralocorticoid excess and a unilateral lesion on CT can go straight to surgery, which is also indicated in patients with confirmed lateralization documented by a valid AVS procedure. Laparoscopic adrenalectomy is the preferred approach. Patients who are not surgical candidates, or with evidence of bilateral hyperplasia based on CT or AVS, should be treated medically (Fig. 406-12). Medical treatment, which can also be considered prior to surgery to avoid postsurgical hypoaldosteronism, consists primarily of the mineralocorticoid receptor antagonist spironolactone. It can be started at 12.5–50 mg bid and titrated up to a maximum of 400 mg/d to control blood pressure and normalize potassium. Side effects include menstrual irregularity, decreased libido, and gynecomastia. The more selective MR antagonist eplerenone can also be used. Doses start at 25 mg bid, and it can be titrated up to 200 mg/d. Another useful drug is the sodium channel

1	libido, and gynecomastia. The more selective MR antagonist eplerenone can also be used. Doses start at 25 mg bid, and it can be titrated up to 200 mg/d. Another useful drug is the sodium channel blocker amiloride (5–10 mg bid).

1	In patients with normal adrenal morphology and family history of early-onset, severe hypertension, a diagnosis of GRA should be Disorders of the Adrenal Cortex Clinical suspicion of mineralocorticoid excess Patients with hypertension and Severe hypertension (>3 BP drugs, drug-resistant) or Family history of early-onset hypertension or cerebrovascular events at < 40 years of age Measurement of aldosterone-renin ratio (ARR) on current blood pressure medication (stop spironolactone for 4 wks) and with hypokalemia corrected (ARR screen positive if ARR >750 pmol/L: ng/ml/h and aldosterone >450 pmol/L) (consider repeat off ˜-blockers for 2 wks if results are equivocal) Negative E.g., saline infusion test (2 liters physiologic saline over 4 h IV), oral sodium loading, fludrocortisone suppression Confirmation of diagnosis Rare: Both renin and Aldo suppressed

1	Unilateral adrenalectomy Drug treatment (MR antagonists, amiloride) Dexamethasone 0.125-0.5 mg/d Adrenal vein sampling Bilateral micronodular hyperplasia Normal adrenal morphology 24-h urinary steroid profile (GC/MS) Diagnostic for • Apparent mineralocorti-coid excess (HSD11B2 def.) • CAH (CYP11B1 or CYP17A1 def.) • Adrenal tumor-related desoxycorticosterone excess If negative, consider • Liddle’s syndrome (ENaC mutations) (responsive to amiloride trial) Family history of early onset hypertension? Screen for glucocorticoid-remediable aldosteronism Unenhanced CT adrenals Age <40 years Age >40 years (if surgery practical and desired) Pos. Pos. Neg. Neg. Unilateral adrenal mass* FIGURE 406-12 Management of patients with suspected mineralocorticoid excess. *Perform adrenal tumor workup (see Fig. 406-13). BP, blood pressure; CAH, congenital adrenal hyperplasia; CT, computed tomography; GC/MS, gas chromatography/mass spectrometry; PRA, plasma renin activity.

1	considered and can be evaluated using genetic testing. Treatment of GRA consists of administering dexamethasone, using the lowest dose possible to control blood pressure. Some patients also require additional MR antagonist treatment.

1	The diagnosis of nonaldosterone-related mineralocorticoid excess is based on documentation of suppressed renin and suppressed aldo-Effect on Effect on Net Effect sterone in the presence of hypokalemic hypertension. This testing is best carried out by employing urinary steroid metabolite profiling by β Blockers ↓↑ ↑ gas chromatography/mass spectrometry (GC/MS). An increased free α1 Blockers →→ → cortisol over free cortisone ratio is suggestive of SAME and can be treated with dexamethasone. Steroid profiling by GC/MS also detects the steroids associated with CYP11B1 and CYP17A1 deficiency or the carcinoma (Fig. 406-12). If the GC/MS profile is normal, then Liddle’s syndrome should be considered. It is very sensitive to amiloride treat-Diuretics (↑)(↑) →/(↓) ment but will not respond to MR antagonist treatment, because the Abbreviations: ACE, angiotensin-converting enzyme; AT1R, angiotensin II receptor type 1. defect is due to a constitutively active ENaC.

1	APPROACH TO THE PATIENT: INCIDENTALLY DISCOVERED ADRENAL MASS Epidemiology Incidentally discovered adrenal masses, commonly termed adrenal “incidentalomas,” are common, with a prevalence of at least 2% in the general population as documented in CT and autopsy series. The prevalence increases with age, with 1% of 40-year-olds and 7% of 70-year-olds harboring an adrenal mass. Etiology Most solitary adrenal tumors are monoclonal neoplasms. Several genetic syndromes, including MEN 1 (MEN1), MEN 2 (RET), Carney’s complex (PRKAR1A), and McCune-Albright (GNAS1), can have adrenal tumors as one of their features. Somatic mutations in MEN1, GNAS1, and PRKAR1A have been identified in a small proportion of sporadic adrenocortical adenomas. Aberrant expression of membrane receptors (gastric inhibitory peptide, αand β-adrenergic, luteinizing hormone, vasopressin V1, and interleukin 1 receptors) have been identified in some sporadic cases of macronodular adrenocortical hyperplasia.

1	The majority of adrenal nodules are endocrine-inactive adrenocortical adenomas. However, larger series suggest that up to 25% of adrenal nodules are hormonally active, due to a cortisolor aldosteroneproducing adrenocortical adenoma or a pheochromocytoma associated with catecholamine excess (Table 406-5). Adrenocortical carcinoma is rare but is the cause of an adrenal mass in 5% of patients. However, the most common cause of a malignant adrenal mass is metastasis originating from another solid tissue tumor (Table 406-5). Differential Diagnosis and Treatment Patients with an adrenal mass >1 cm require a diagnostic evaluation. Two key questions need to be addressed: (1) Does the tumor autonomously secrete hormones that could have a detrimental effect on health? (2) Is the adrenal mass benign or malignant?

1	Hormone secretion by an adrenal mass occurs along a continuum, with a gradual increase in clinical manifestations in parallel with hormone levels. Exclusion of catecholamine excess from a pheochromocytoma arising from the adrenal medulla is a mandatory part of the diagnostic workup (Fig. 406-13). Furthermore, autonomous cortisol and aldosterone secretion resulting in Cushing’s syndrome or primary Aldosterone-producing 2–5 Pheochromocytoma 5–10 Adrenal myelolipoma <1 Adrenal ganglioneuroma <0.1 Adrenal hemangioma <0.1 Adrenal cyst <1 Adrenal hematoma/hemorrhagic <1 Note: Bilateral adrenal enlargement/masses may be caused by congenital adrenal hyperplasia, bilateral macronodular hyperplasia, bilateral hemorrhage (due to antiphospholipid syndrome or sepsis-associated Waterhouse-Friderichsen syndrome), granuloma, amyloidosis, or infiltrative disease including tuberculosis.

1	aldosteronism, respectively, require exclusion. Adrenal incidentalomas 2321 can be associated with lower levels of autonomous cortisol secretion, and patients may lack overt clinical features of Cushing’s syndrome. Nonetheless, they may exhibit one or more components of the metabolic syndrome (e.g., obesity, type 2 diabetes, or hypertension). There is ongoing debate about the optimal treatment for these patients with mild or subclinical Cushing’s syndrome. Overproduction of adrenal androgen precursors, DHEA and its sulfate, is rare and most frequently seen in the context of adrenocortical carcinoma, as are increased levels of steroid precursors such as 17-hydroxyprogesterone.

1	For the differentiation of benign from malignant adrenal masses, imaging is relatively sensitive, although specificity is suboptimal. CT is the procedure of choice for imaging the adrenal glands (Fig. 406-11). The risk of adrenocortical carcinoma, pheochromocytoma, and benign adrenal myelolipoma increases with the diameter of the adrenal mass. However, size alone is of poor predictive value, with only 80% sensitivity and 60% specificity for the differentiation of benign from malignant masses when using a 4-cm cut-off. Metastases are found with similar frequency in adrenal masses of all sizes. Tumor density on unenhanced CT is of additional diagnostic value, with most adrenocortical adenomas being lipid rich and thus presenting with low attenuation values (i.e., densities of <10 HU). By contrast, adrenocortical carcinomas, but also pheochromocytomas, usually have high attenuation values (i.e., densities >20 HU on precontrast scans). Generally, benign lesions are rounded and homogenous,

1	adrenocortical carcinomas, but also pheochromocytomas, usually have high attenuation values (i.e., densities >20 HU on precontrast scans). Generally, benign lesions are rounded and homogenous, whereas most malignant lesions appear lobulated and inhomogeneous. Pheochromocytoma and adrenomyelolipoma may also exhibit lobulated and inhomogeneous features.

1	Additional information can be obtained from CT by assessment of contrast wash-out after 15 min, which is >50% in benign lesions but <40% in malignant lesions, which usually have a more extensive vascularization. MRI also allows for the visualization of the adrenal glands with somewhat lower resolution than CT. However, because it does not involve exposure to ionizing radiation, it is preferred in children, young adults, and during pregnancy. MRI has a valuable role in the characterization of indeterminate adrenal lesions using chemical shift analysis, with malignant tumors rarely showing loss of signal on opposed-phase MRI.

1	Fine-needle aspiration (FNA) or CT-guided biopsy of an adrenal mass is almost never indicated. FNA of a pheochromocytoma can cause a life-threatening hypertensive crisis. FNA of an adrenocortical carcinoma violates the tumor capsule and can cause needle track metastasis. FNA should only be considered in a patient with a history of nonadrenal malignancy and a newly detected adrenal mass, after careful exclusion of pheochromocytoma, and if the outcome will influence therapeutic management. It is important to recognize that in 25% of patients with a previous history of nonadrenal malignancy, a newly detected mass on CT is not a metastasis.

1	Adrenal masses associated with confirmed hormone excess or suspected malignancy are usually treated surgically (Fig. 406-13) or, if adrenalectomy is not feasible or desired, with medication. Preoperative exclusion of glucocorticoid excess is particularly important for the prediction of postoperative suppression of the contralateral adrenal gland, which requires glucocorticoid replacement periand postoperatively. If the initial decision is for observation, imaging and biochemical testing should be repeated about a year after the first assessment. However, this may be performed earlier in patients with borderline imaging or hormonal findings. There is no agreement with regard to the required long-term follow-up beyond 1 year in patients with normal biochemistry and no evidence of increased tumor size at follow-up.

1	Adrenocortical carcinoma (ACC) is a rare malignancy with an annual incidence of 1–2 per million population. ACC is generally considered a highly malignant tumor; however, it presents with broad interindividual variability with regard to biologic characteristics and clinical behavior. Somatic mutations in the tumor-suppressor gene TP53 are found in 25% of apparently sporadic ACC. Germline TP53 mutations are the cause of the Li-Fraumeni syndrome associated with multiple solid organ cancers including ACC and are found in 25% of pediatric Disorders of the Adrenal Cortex 24-h urine for free cortisol excretion, plasma ACTH, midnight plasma (or salivary) cortisol, dexamethasone 1 mg overnight test (perform at least two out of four tests) If tumor >2 cm: Serum 17-hydroxyprogesterone and DHEAS imaging suggestive of malignancy: • Size >4 cm Negative and imaging not suggestive of malignancy: Neg. Pos. Neg. Pos.

1	If tumor >2 cm: Serum 17-hydroxyprogesterone and DHEAS imaging suggestive of malignancy: • Size >4 cm Negative and imaging not suggestive of malignancy: Neg. Pos. Neg. Pos. FIGURE 406-13 Management of the patient with an incidentally discovered adrenal mass. CT, computed tomography; F/U, follow-up; MRI, Neg. magnetic resonance imaging. ACC cases; the TP53 mutation R337H is found in almost all pediatric ACC in Brazil. Other genetic changes identified in ACC include alterations in the Wnt/β-catenin pathway and in the insulin-like growth factor 2 (IGF2) cluster; IGF2 overexpression is found in 90% of ACC.

1	Patients with large adrenal tumors suspicious of malignancy should be managed by a multidisciplinary specialist team, including an endocrinologist, an oncologist, a surgeon, a radiologist, and a histopathologist. FNA is not indicated in suspected ACC: first, cytology and also histopathology of a core biopsy cannot differentiate between benign and malignant primary adrenal masses; second, FNA violates the tumor capsule and may even cause needle canal metastasis. Even when the entire tumor specimen is available, the histopathologic differentiation between benign and malignant lesions is a diagnostic challenge. The most common histopathologic classification is the Weiss score, taking into account high nuclear grade; mitotic rate (>5/HPF); atypical mitosis; <25% clear cells; diffuse architecture; and presence of necrosis, venous invasion, and invasion of sinusoidal structures and tumor capsule. The presence of three or more elements suggests ACC.

1	Although 60–70% of ACCs show biochemical evidence of steroid overproduction, in many patients, this is not clinically apparent due to the relatively inefficient steroid production by the adrenocortical cancer cells. Excess production of glucocorticoids and adrenal androgen precursors are most common. Mixed excess production of several corticosteroid classes by an adrenal tumor is generally indicative of malignancy.

1	Tumor staging at diagnosis (Table 406-6) has important prognostic implications and requires scanning of the chest and abdomen for local organ invasion, lymphadenopathy, and metastases. Intravenous contrast medium is necessary for maximum sensitivity for hepatic metastases. An adrenal origin may be difficult to determine on standard axial CT imaging if the tumors are large and invasive, but CT reconstructions and MRI are more informative (Fig. 406-14) using multiple planes and different sequences. Vascular and adjacent organ invasion is diagnostic of malignancy. 18-Fluoro-2-deoxy-D-glucose positron emission tomography (18-FDG PET) is highly sensitive for the detection of malignancy and can be used to detect small metastases or local recurrence that may not be obvious on CT (Fig. 406-14). However, FDG PET is not specific and therefore cannot be used for differentiating benign from malignant adrenal lesions. Metastasis in ACC most frequently occurs to liver and lung.

1	There is no established grading system for ACC, and the Weiss score carries no prognostic value; the most important prognostic histopathologic parameter is the Ki67 proliferation index, with Ki67 <10% indicative of slow to moderate growth velocity, whereas a Ki67 ≥10% is associated with poor prognosis including high risk of recurrence and rapid progression.

1	Cure of ACC can only be achieved by early detection and complete surgical removal. Capsule violation during primary surgery, metastasis at diagnosis, and primary treatment in a nonspecialist center are major determinants of poor survival. If the primary tumor invades adjacent organs, en bloc removal of kidney and spleen should be considered to reduce the risk of recurrence. Surgery can also be considered in a patient with metastases if there is severe tumor-related hormone excess. This indication needs to be carefully weighed against surgical risk, including thromboembolic complications, and the resulting delay in the introduction of other therapeutic options. Patients with confirmed ACC and successful removal of the primary tumor should receive adjuvant treatment with mitotane (o,p’DDD), particularly in patients with a high risk of recurrence as determined by tumor size >8 cm, histopathologic signs of vascular invasion, capsule invasion or violation, and a Ki67 proliferation index

1	particularly in patients with a high risk of recurrence as determined by tumor size >8 cm, histopathologic signs of vascular invasion, capsule invasion or violation, and a Ki67 proliferation index ≥10%. Adjuvant mitotane should be continued for at least 2 years, if the patient can tolerate side effects. Regular monitoring of plasma mitotane levels is mandatory (therapeutic range 14–20 mg/L; neurotoxic complications more frequent at >20 mg/L). Mitotane is usually started at 500 mg tid, with stepwise increases to a maximum dose of 2000 mg tid

1	Abbreviations: ENSAT, European Network for the Study of Adrenal Tumors; TNM, tumor, node, metastasis.

1	in days (high-dose saturation) or weeks (low-dose saturation) as tolerated. Once therapeutic range plasma mitotane levels are achieved, the dose can be tapered to maintenance doses mostly ranging from 1000 to 1500 mg tid. Mitotane treatment results in disruption of cortisol synthesis and thus requires glucocorticoid replacement; glucocorticoid replacement dose should be at least double of that usually used in adrenal insufficiency (i.e., 20 mg tid) because mitotane induces hepatic CYP3A4 activity resulting in rapid inactivation of glucocorticoids. Mitotane also increases circulating CBG, thereby decreasing the available free cortisol fraction. Single metastases can be addressed surgically or with radiofrequency ablation as appropriate. If the tumor recurs or progresses during mitotane treatment, chemotherapy should be considered; the established first-line chemotherapy regimen is the combination of cisplatin, etoposide, and doxorubicin plus continuing mitotane. Painful bone metastasis

1	chemotherapy should be considered; the established first-line chemotherapy regimen is the combination of cisplatin, etoposide, and doxorubicin plus continuing mitotane. Painful bone metastasis responds to irradiation. Overall survival in ACC is still poor, with 5-year survival rates of 30–40% and a median survival of 15 months in metastatic ACC.

1	ADRENAL INSUFFICIENCY Epidemiology The prevalence of well-documented, permanent adrenal insufficiency is 5 in 10,000 in the general population. Hypothalamic-pituitary origin of disease is most frequent, with a prevalence of 3 in 10,000, whereas primary adrenal insufficiency has a prevalence of 2 in 2323 10,000. Approximately one-half of the latter cases are acquired, mostly caused by autoimmune destruction of the adrenal glands; the other one-half are genetic, most commonly caused by distinct enzymatic blocks in adrenal steroidogenesis affecting glucocorticoid synthesis (i.e., congenital adrenal hyperplasia.) Adrenal insufficiency arising from suppression of the HPA axis as a consequence of exogenous glucocorticoid treatment is much more common, occurring in 0.5–2% of the population in developed countries.

1	Etiology Primary adrenal insufficiency is most commonly caused by autoimmune adrenalitis. Isolated autoimmune adrenalitis accounts for 30–40%, whereas 60–70% develop adrenal insufficiency as part of autoimmune polyglandular syndromes (APS) (Chap. 408) (Table 406-7). APS1, also termed APECED (autoimmune polyendocrinopathy-candidiasisectodermal dystrophy), is the underlying cause in 10% of patients affected by APS. APS1 is transmitted in an autosomal recessive manner and is caused by mutations in the autoimmune regulator gene AIRE. Associated autoimmune conditions overlap with those seen in APS2, but may also include total alopecia, primary hypoparathyroidism, and, in rare cases, lymphoma. APS1 patients invariably develop chronic mucocutaneous candidiasis, usually manifest in childhood, and preceding adrenal insufficiency by years or decades. The much more prevalent APS2 is of polygenic inheritance, with confirmed associations with the HLA-DR3 gene region in the major histocompatibility

1	preceding adrenal insufficiency by years or decades. The much more prevalent APS2 is of polygenic inheritance, with confirmed associations with the HLA-DR3 gene region in the major histocompatibility complex and distinct gene regions involved in immune regulation (CTLA-4, PTPN22, CLEC16A). Coincident autoimmune disease most frequently includes thyroid autoimmune disease, vitiligo, and premature ovarian failure. Less commonly, additional features may include type 1 diabetes and pernicious anemia caused by vitamin B12 deficiency.

1	X-linked adrenoleukodystrophy has an incidence of 1:20,000 males and is caused by mutations in the X-ALD gene encoding the peroxisomal membrane transporter protein ABCD1; its disruption results in accumulation of very long chain (>24 carbon atoms) fatty acids. Approximately 50% of cases manifest in early childhood with rapidly progressive white matter disease (cerebral ALD); 35% present during adolescence or in early adulthood with neurologic features indicative of myelin and peripheral nervous system involvement (adrenomyeloneuropathy [AMN]). In the remaining 15%, adrenal insufficiency is the sole manifestation of disease. Of note, distinct mutations manifest with variable penetrance and phenotypes within affected families. Rarer causes of adrenal insufficiency involve destruction of the adrenal glands as a consequence of infection, hemorrhage, or infiltration

1	Rarer causes of adrenal insufficiency involve destruction of the adrenal glands as a consequence of infection, hemorrhage, or infiltration FIGURE 406-14 Imaging in adrenocortical carcinoma. Magnetic resonance imaging scan with (A) frontal and (B) lateral views of a right adrenocortical carcinoma that was detected incidentally. Computed tomography (CT) scan with (C) coronal and (D) transverse views depicting a right-sided adrenocortical carcinoma. Note the irregular border and inhomogeneous structure. CT scan (E) and positron emission tomography/ CT (F) visualizing a peritoneal metastasis of an adrenocortical carcinoma in close proximity to the right kidney (arrow). Disorders of the Adrenal Cortex Abbreviations: AIRE, autoimmune regulator; CMV, cytomegalovirus; DSD, disordered sex development; MC2R, ACTH receptor; MCM4, mini chromosome maintenance-deficient 4 homologue; MRAP, MC2R-accessory protein; NNT, nicotinamide nucleotide transhydrogenase.

1	(Table 406-7); tuberculous adrenalitis is still a frequent cause of disease in developing countries. Adrenal metastases rarely cause adrenal insufficiency, and this occurs only with bilateral, bulky metastases. Inborn causes of primary adrenal insufficiency other than congenital adrenal hyperplasia are rare, causing less than 1% of cases. However, their elucidation provides important insights into adrenal gland development and physiology. Mutations causing primary adrenal insufficiency (Table 406-7) include factors regulating adrenal development and steroidogenesis (DAX-1, SF-1), cholesterol synthesis, import and cleavage (DHCR7, StAR, CYP11A1), and elements of the adrenal ACTH response pathway (MC2R, MRAP) (Fig. 406-5), and factors involved in redox regulation (NNT) and DNA repair (MCM4, CDKN1C).

1	Secondary adrenal insufficiency is the consequence of dysfunction of the hypothalamic-pituitary component of the HPA axis (Table 406-8). Excluding iatrogenic suppression, the overwhelming majority of cases are caused by pituitary or hypothalamic tumors or their treatment by surgery or irradiation (Chap. 403). Rarer causes include pituitary apoplexy, either as a consequence of an infarcted pituitary adenoma or transient reduction in the blood supply of the pituitary during surgery or after rapid blood loss associated with parturition, also termed Sheehan’s syndrome. Isolated ACTH deficiency is rarely caused by autoimmune disease or pituitary infiltration (Table 406-8). Mutations in the ACTH precursor POMC or in factors regulating pituitary development are genetic causes of ACTH deficiency (Table 406-8).

1	Clinical Manifestations In principle, the clinical features of primary adrenal insufficiency (Addison’s disease) are characterized by the loss of both glucocorticoid and mineralocorticoid secretion (Table 406-9). In secondary adrenal insufficiency, only glucocorticoid deficiency is present, as the adrenal itself is intact and thus still amenable to regulation by the RAA system. Adrenal androgen secretion is disrupted in both primary and secondary adrenal insufficiency (Table 406-9). Hypothalamic-pituitary disease can lead to additional clinical manifestations due to involvement of other endocrine axes (thyroid, gonads, growth hormone, prolactin) or visual impairment with bitemporal hemianopia caused by chiasmal compression. It is important to recognize that iatrogenic adrenal insufficiency caused by exogenous glucocorticoid suppression of the HPA axis may result in all symptoms associated with glucocorticoid deficiency (Table 406-9), if exogenous glucocorticoids are stopped abruptly.

1	caused by exogenous glucocorticoid suppression of the HPA axis may result in all symptoms associated with glucocorticoid deficiency (Table 406-9), if exogenous glucocorticoids are stopped abruptly. However, patients will appear clinically cushingoid as a result of the preceding overexposure to glucocorticoids.

1	Chronic adrenal insufficiency manifests with relatively nonspecific signs and symptoms such as fatigue and loss of energy, often resulting in delayed or missed diagnoses (e.g., as depression or anorexia). A distinguishing feature of primary adrenal insufficiency is hyperpigmentation, which is caused by excess ACTH stimulation of melanocytes. Hyperpigmentation is most pronounced in skin areas exposed to increased friction or shear stress and is increased by sunlight (Fig. 406-15). Conversely, in secondary adrenal insufficiency, the skin has an alabaster-like paleness due to lack of ACTH secretion.

1	Hyponatremia is a characteristic biochemical feature in primary adrenal insufficiency and is found in 80% of patients at presentation. Hyperkalemia is present in 40% of patients at initial diagnosis. Hyponatremia is primarily caused by mineralocorticoid deficiency but can also occur in secondary adrenal insufficiency due to diminished inhibition of antidiuretic hormone (ADH) release by cortisol, resulting in mild syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Glucocorticoid deficiency also results in slightly increased TSH concentrations that normalize within days to weeks after initiation of glucocorticoid replacement.

1	Acute adrenal insufficiency usually occurs after a prolonged period of nonspecific complaints and is more frequently observed in patients with primary adrenal insufficiency, due to the loss of both glucocorticoid and mineralocorticoid secretion. Postural hypotension may progress to hypovolemic shock. Adrenal insufficiency may mimic features of acute abdomen with abdominal tenderness, nausea, vomiting, and fever. In some cases, the primary presentation may resemble neurologic disease, with decreased responsiveness, progressing to stupor and coma. An adrenal crisis can be triggered by an intercurrent illness, surgical or other stress, or increased glucocorticoid inactivation (e.g., hyperthyroidism). Abbreviations: ACTH, adrenocorticotropic hormone; GH, growth hormone; LH/FSH, luteinizing hormone/follicle-stimulating hormone; PRL, prolactin; TSH, thyroid-stimulating hormone.

1	Diagnosis The diagnosis of adrenal insufficiency is established by the short cosyntropin test, a safe and reliable tool with excellent predictive diagnostic value (Fig. 406-16). The cut-off for failure is usually defined at cortisol levels of <500–550 nmol/L (18–20 μg/dL) sampled 30–60 min after ACTH stimulation; the exact cut-off is dependent on the locally available assay. During the early phase of HPA disruption (e.g., within 4 weeks of pituitary insufficiency), patients may still respond to exogenous ACTH stimulation. In this circumstance, the ITT is an alternative choice but is more invasive and should be carried out only under a specialist’s supervision (see above). Induction of hypoglycemia is contraindicated in individuals with diabetes mellitus, cardiovascular disease, or history of seizures. Random serum cortisol measurements are of limited diagnostic value, because baseline cortisol levels may be coincidentally low due to the physiologic diurnal rhythm of cortisol secretion

1	of seizures. Random serum cortisol measurements are of limited diagnostic value, because baseline cortisol levels may be coincidentally low due to the physiologic diurnal rhythm of cortisol secretion (Fig. 406-3). Similarly, many patients with secondary adrenal insufficiency have relatively normal baseline cortisol levels but fail to mount an appropriate cortisol response to ACTH, which can only be revealed by stimulation testing. Importantly, tests to establish the diagnosis of adrenal insufficiency should never delay treatment. Thus, in a patient with suspected adrenal crisis, it is reasonable to draw baseline cortisol levels, provide replacement therapy, and defer formal stimulation testing until a later time.

1	TB>Fatigue, lack of energy Weight loss, anorexia Myalgia, joint pain Fever Normochromic anemia, lymphocytosis, eosinophilia Slightly increased TSH (due to loss of feedback inhibition of TSH release) Hypoglycemia (more frequent in children) Low blood pressure, postural hypotension Hyponatremia (due to loss of feedback inhibition of AVP release) Abdominal pain, nausea, vomiting Dizziness, postural hypotension Salt craving Low blood pressure, postural hypotension Increased serum creatinine (due to volume depletion) Hyponatremia Hyperkalemia Lack of energy Dry and itchy skin (in women) Loss of libido (in women) Loss of axillary and pubic hair (in women) Hyperpigmentation (primary adrenal insufficiency only) (due to excess of proopiomelanocortin [POMC]-derived peptides) Alabaster-colored pale skin (secondary adrenal insufficiency only) (due to deficiency of POMC-derived peptides) Abbreviations: AVP, arginine vasopressin; TSH, thyroid-stimulating hormone.

1	Once adrenal insufficiency is confirmed, measurement of plasma ACTH is the next step, with increased or inappropriately low levels defining primary and secondary origin of disease, respectively (Fig. 406-16). In primary adrenal insufficiency, increased plasma renin will confirm the presence of mineralocorticoid deficiency. At initial presentation, patients with primary adrenal insufficiency should undergo screening for steroid autoantibodies as a marker of autoimmune adrenalitis. If these tests are negative, adrenal imaging by CT is indicated to investigate possible hemorrhage, infiltration, or masses. In male patients with negative autoantibodies in the plasma, verylong-chain fatty acids should be measured to exclude X-ALD. Patients with inappropriately low ACTH, in the presence of confirmed cortisol deficiency, should undergo hypothalamic-pituitary imaging by MRI. Features suggestive of preceding pituitary apoplexy, such as sudden-onset severe headache or history of previous head

1	cortisol deficiency, should undergo hypothalamic-pituitary imaging by MRI. Features suggestive of preceding pituitary apoplexy, such as sudden-onset severe headache or history of previous head trauma, should be carefully explored, particularly in patients with no obvious MRI lesion.

1	Acute adrenal insufficiency requires immediate initiation of rehydration, usually carried out by saline infusion at initial rates of 1 L/h with continuous cardiac monitoring. Glucocorticoid replacement should be initiated by bolus injection of 100 mg hydrocortisone, followed by the administration of 100–200 mg hydrocortisone over 24 h, either by continuous infusion or by bolus IV or IM injections. Mineralocorticoid replacement can be initiated once the daily hydrocortisone dose has been reduced to <50 mg because at higher doses hydrocortisone provides sufficient stimulation of mineralocorticoid receptors. Glucocorticoid replacement for the treatment of chronic adrenal insufficiency should be administered at a dose that replaces the physiologic daily cortisol production, which is usually achieved by Disorders of the Adrenal Cortex

1	Disorders of the Adrenal Cortex FIGURE 406-15 Clinical features of Addison’s disease. Note the hyperpigmentation in areas of increased friction including (A) palmar creases, (B) dorsal foot, (C) nipples and axillary region, and (D) patchy hyperpigmentation of the oral mucosa.

1	the oral administration of 15–25 mg hydrocortisone in two to three divided doses. Pregnancy may require an increase in hydrocortisone dose by 50% during the last trimester. In all patients, at least one-half of the daily dose should be administered in the morning. Currently available glucocorticoid preparations fail to mimic the physiologic cortisol secretion rhythm (Fig. 406-3). Long-acting glucocorticoids such as prednisolone or dexamethasone are not preferred because they result in increased glucocorticoid exposure due to extended glucocorticoid receptor activation at times of physiologically low cortisol secretion. There are no well-established dose equivalencies, but as a guide, equipotency can be assumed for 1 mg hydrocortisone, 1.6 mg cortisone acetate, 0.2 mg prednisolone, 0.25 mg prednisone, and 0.025 mg dexamethasone.

1	Monitoring of glucocorticoid replacement is mainly based on the history and examination for signs and symptoms suggestive of glucocorticoid overor underreplacement, including assessment of body weight and blood pressure. Plasma ACTH, 24-h urinary free cortisol, or serum cortisol day curves reflect whether hydrocortisone has been taken or not, but do not convey reliable information about replacement quality. In patients with isolated primary adrenal insufficiency, monitoring should include screening for autoimmune thyroid disease, and female patients should be made aware of the possibility of premature ovarian failure. Supraphysiologic glucocorticoid treatment with doses equivalent to 30 mg hydrocortisone or more will affect bone metabolism, and these patients should undergo regular bone mineral density evaluation. All patients with adrenal insufficiency need to be instructed about the requirement for stress-related glucocorticoid dose adjustments. These generally consist of doubling

1	mineral density evaluation. All patients with adrenal insufficiency need to be instructed about the requirement for stress-related glucocorticoid dose adjustments. These generally consist of doubling the routine oral glucocorticoid dose in the case of intercurrent illness with fever and bed rest and the need for IV hydrocortisone injection at a daily dose of 100 mg in cases of prolonged vomiting, surgery, or trauma. Patients living or traveling in regions with delayed access to acute health care should carry a hydrocortisone self-injection emergency kit, in addition to their usual steroid emergency cards and bracelets.

1	Mineralocorticoid replacement in primary adrenal insufficiency should be initiated at a dose of 100–150 μg fludrocortisone. The adequacy of treatment can be evaluated by measuring blood pressure, sitting and standing, to detect a postural drop indicative of hypovolemia. In addition, serum sodium, potassium, and plasma renin should be measured regularly. Renin levels should be kept in the upper normal reference range. Changes in glucocorticoid dose may also impact on mineralocorticoid replacement as cortisol also binds the mineralocorticoid receptor; 40 mg hydrocortisone is equivalent to 100 μg fludrocortisone. In patients living or traveling in areas with hot or tropical weather conditions, the fludrocortisone dose should be increased by 50–100 μg during the summer. Mineralocorticoid dose may also need to be adjusted during pregnancy, due to the antimineralocorticoid activity of progesterone, but this is less often required than hydrocortisone dose adjustment.

1	Clinical suspicion of adrenal insufficiency (weight loss, fatigue, postural hypotension, hyperpigmentation, hyponatremia) Screening/confirmation of diagnosis CBC, serum sodium, potassium, creatinine, urea, TSH Differential diagnosis Plasma ACTH, plasma renin, serum aldosterone

1	Negative Adrenal autoantibodies • Autoimmune adrenalitis; • Autoimmune polyglandular syndrome (APS) Hypothalamic-pituitary mass lesion • History of exogenous glucocorticoid treatment? • History of head trauma? • Consider isolated ACTHdeficiency MRI Pituitary Secondary adrenal insufficiency (Low-normal ACTH, normal renin, normal aldosterone ) Primary adrenal insufficiency (High ACTH, high renin, low aldosterone) Glucocorticoid + mineralocorticoid replacement Glucocorticoid replacement Positive Positive Negative Positive Negative • Adrenal infection (tuberculosis), • Infiltration (e.g., lymphoma) • Hemorrhage;• Congenital adrenal hyperplasia (17OHP˜) • Autoimmune adrenalitis most likely diagnosis; • In men, consideradrenoleukodystrophy (VLCFA˜) • Chest x-ray • Serum 17OHP • In men: plasma very long chain fatty acids (VLCFA) • Adrenal CTFIGURE 406-16 Management of the patient with suspected adrenal insufficiency. ACTH, adrenocorticotropic hormone; CBC, complete blood count; MRI, magnetic

1	long chain fatty acids (VLCFA) • Adrenal CTFIGURE 406-16 Management of the patient with suspected adrenal insufficiency. ACTH, adrenocorticotropic hormone; CBC, complete blood count; MRI, magnetic resonance imaging; PRA, plasma renin activity; TSH, thyroid-stimulating hormone.

1	Plasma renin cannot serve as a monitoring tool during pregnancy, because renin rises physiologically during gestation. Adrenal androgen replacement is an option in patients with lack of energy, despite optimized glucocorticoid and mineralocorticoid replacement. It may also be indicated in women with features of androgen deficiency, including loss of libido. Adrenal androgen replacement can be achieved by once-daily administration of 25–50 mg DHEA. Treatment is monitored by measurement of DHEAS, androstenedione, testosterone, and sex hormone–binding globulin (SHBG) 24 h after the last DHEA dose.

1	(See also Chap. 410) Congenital adrenal hyperplasia (CAH) is caused by mutations in genes encoding steroidogenic enzymes involved in glucocorticoid synthesis (CYP21A2, CYP17A1, HSD3B2, CYP11B1) or in the cofactor enzyme P450 oxidoreductase that serves as an electron donor to CYP21A2 and CYP17A1 (Fig. 406-1). Invariably, patients affected by CAH exhibit glucocorticoid deficiency. Depending on the Disorders of the Adrenal Cortex exact step of enzymatic block, they may also have excess production of mineralocorticoids or deficient production of sex steroids (Table 406-10). The diagnosis of CAH is readily established by measurement of the steroids accumulating before the distinct enzymatic block, either in serum or in urine, preferably by the use of mass spectrometry–based assays (Table 406-10).

1	Mutations in CYP21A2 are the most prevalent cause of CAH, responsible for 90–95% of cases. 21-Hydroxylase deficiency disrupts glucocorticoid and mineralocorticoid synthesis (Fig. 406-1), resulting in diminished negative feedback via the HPA axis. This leads to increased pituitary ACTH release, which drives increased synthesis of adrenal androgen precursors and subsequent androgen excess. The degree of impairment of glucocorticoid and mineralocorticoid secretion depends on the severity of mutations. Major loss-of-function mutations result in combined glucocorticoid and mineralocorticoid deficiency (classic CAH, neonatal presentation), whereas less severe mutations affect glucocorticoid synthesis only (simple virilizing CAH, neonatal or early childhood presentation). The mildest mutations result in the least severe clinical phenotype, nonclassic CAH, usually presenting during adolescence and early adulthood and with preserved glucocorticoid production.

1	Androgen excess is present in all patients and manifests with broad phenotypic variability, ranging from severe virilization of the external genitalia in neonatal girls (e.g., 46,XX disordered sex development [DSD]) to hirsutism and oligomenorrhea resembling a polycystic ovary syndrome phenotype in young women with nonclassic CAH. In countries without neonatal screening for CAH, boys with classic CAH usually present with life-threatening adrenal crisis in the first few weeks of life (salt-wasting crisis); a simple-virilizing genotype manifests with precocious pseudo-puberty and advanced bone age in early childhood, whereas men with nonclassic CAH are usually detected only through family screening.

1	Glucocorticoid treatment is more complex than for other causes of primary adrenal insufficiency as it not only needed to replace missing glucocorticoids but also to control the increased ACTH drive and subsequent androgen excess. Current treatment is hampered by the lack of glucocorticoid preparations that mimic the diurnal cortisol secretion profile, resulting in a prolonged period of ACTH stimulation and subsequent androgen production during the early morning hours. In childhood, optimization of growth and pubertal development are important goals of glucocorticoid treatment, in addition to prevention of adrenal crisis and treatment of 46,XX DSD. In adults, the focus shifts to preserving fertility and preventing side effects of glucocorticoid overtreatment, namely, the metabolic syndrome and osteoporosis. Fertility can be compromised in women due to oligomenorrhea/amenorrhea with chronic anovulation as a consequence of androgen excess. Men may develop so-called testicular adrenal

1	and osteoporosis. Fertility can be compromised in women due to oligomenorrhea/amenorrhea with chronic anovulation as a consequence of androgen excess. Men may develop so-called testicular adrenal rest tumors (Fig. 406-17). These consist of hyperplastic cells with adrenocortical characteristics located in the rete testis and should not be confused with testicular tumors. Testicular adrenal rest tissue can compromise sperm production and induce fibrosis that may be irreversible.

1	Hydrocortisone is a good treatment option for the prevention of adrenal crisis, but longer acting prednisolone may be needed to control androgen excess. In children, hydrocortisone is given in divided doses at 1–1.5 times the normal cortisol production rate (about 10–13 mg/m2 per day). In adults, if hydrocortisone does not suffice, intermediate-acting glucocorticoids (e.g., prednisone) may be

1	FIGURE 406-17 Imaging in congenital adrenal hyperplasia (CAH). Adrenal computed tomography scans showing homogenous bilateral hyperplasia in a young patient with classic CAH (A) and macronodular bilateral hyperplasia (B) in a middle-aged patient with classic CAH with longstanding poor disease control. Magnetic resonance imaging scan with T1-weighted (C) and T2-weighted (D) images showing bilateral testicular adrenal rest tumors (arrows) in a young patient with salt-wasting congenital adrenal hyperplasia. (Courtesy of N. Reisch.) given, using the lowest dose necessary to suppress excess androgen production. For achieving fertility, dexamethasone treatment may be required, but should be only given for the shortest possible time period to limit adverse metabolic side effects. Biochemical monitoring should include androstenedione and testosterone, aiming for the normal sex-specific reference range. 17-Hydroxyprogesterone (17OHP) is a useful marker of overtreatment, indicated by 17OHP

1	monitoring should include androstenedione and testosterone, aiming for the normal sex-specific reference range. 17-Hydroxyprogesterone (17OHP) is a useful marker of overtreatment, indicated by 17OHP levels within the normal range of healthy controls. Glucocorticoid overtreatment may suppress the hypothalamic-pituitary-gonadal axis. Thus, treatment needs to be carefully titrated against clinical features of disease control. Stress dose glucocorticoids should be given at double or triple the daily dose for surgery, acute illness, or severe trauma. Poorly controlled CAH can result in adrenocortical hyperplasia, which gave the disease its name, and may present as macronodular hyperplasia subsequent to long-standing ACTH excess (Fig. 406-17). The nodular areas can develop autonomous adrenal androgen production and may be unresponsive to glucocorticoid treatment.

1	Mineralocorticoid requirements change during life and are higher in children, explained by relative mineralocorticoid resistance that diminishes with ongoing maturation of the kidney. Children with CAH usually receive mineralocorticoid and salt replacement. However, young adults with CAH should undergo reassessment of their mineralocorticoid reserve. Plasma renin should be regularly monitored and kept within the upper half of the normal reference range. Hartmut P. H. Neumann

1	Hartmut P. H. Neumann Pheochromocytomas and paragangliomas are catecholamine-producing tumors derived from the sympathetic or parasympathetic nervous system. These tumors may arise sporadically or be inherited as features of multiple endocrine neoplasia type 2, von Hippel–Lindau disease, or several other pheochromocytoma-associated syndromes. The diagnosis of pheochromocytomas identifies a potentially correctable cause of hypertension, and their removal can prevent hypertensive crises that can be lethal. The clinical presentation is variable, ranging from an adrenal incidentaloma to a hypertensive crisis with associated cerebrovascular or cardiac complications.

1	Pheochromocytoma is estimated to occur in 2–8 of 1 million persons per year, and ∼0.1% of hypertensive patients harbor a pheochromocytoma. The mean age at diagnosis is ∼40 years, although the tumors can occur from early childhood until late in life. The classic “rule of tens” for pheochromocytomas states that ∼10% are bilateral, 10% are extra-adrenal, and 10% are malignant.

1	Pheochromocytomas and paragangliomas are well-vascularized tumors that arise from cells derived from the sympathetic (e.g., adrenal medulla) or parasympathetic (e.g., carotid body, glomus vagale) paraganglia (Fig. 407-1). The name pheochromocytoma reflects the black-colored staining caused by chromaffin oxidation of catecholamines; although a variety of terms have been used to describe these tumors, most clinicians use this designation to describe symptomatic catecholamine-producing tumors, including those in extra-adrenal retroperitoneal, pelvic, and thoracic sites. The term paraganglioma is used to describe catecholamine-producing tumors in the skull base and neck; these tumors may secrete little or no catecholamine. In contrast to common clinical parlance, the World Health Organization (WHO) restricts the term pheochromocytoma to adrenal tumors and applies the term paraganglioma to tumors at all other sites.

1	The etiology of sporadic pheochromocytomas and paragangliomas 2329 is unknown. However, 25–33% of patients have an inherited condition, including germ-line mutations in the classically recognized RET, VHL, NF1, SDHB, SDHC, and SDHD genes or in the more recently recognized SDHA, SDHAF2, TMEM127, and MAX genes. Biallelic gene inactivation has been demonstrated for the VHL, NF1, and SDH genes, whereas RET mutations activate receptor tyrosine kinase activity. SDH is an enzyme of the Krebs cycle and the mitochondrial respiratory chain. The VHL protein is a component of a ubiquitin E3 ligase. VHL mutations reduce protein degradation, resulting in upregulation of components involved in cell cycle progression, glucose metabolism, and oxygen sensing.

1	Its clinical presentation is so variable that pheochromocytoma has been termed “the great masquerader” (Table 407-1). Among the presenting manifestations, episodes of palpitation, headache, and profuse sweating are typical, and these manifestations constitute a classic triad. The presence of all three manifestations in association with hypertension makes pheochromocytoma a likely diagnosis. However, a pheochromocytoma can be asymptomatic for years, and some tumors grow to a considerable size before patients note symptoms.

1	The dominant sign is hypertension. Classically, patients have episodic hypertension, but sustained hypertension is also common. Catecholamine crises can lead to heart failure, pulmonary edema, arrhythmias, and intracranial hemorrhage. During episodes of hormone release, which can occur at widely divergent intervals, patients are anxious and pale, and they experience tachycardia and palpitations. These paroxysms generally last <1 h and may be precipitated by surgery, positional changes, exercise, pregnancy, urination (particularly with bladder pheochromocytomas), and various medications (e.g., tricyclic antidepressants, opiates, metoclopramide). The diagnosis is based on documentation of catecholamine excess by biochemical testing and localization of the tumor by imaging. These two criteria are of equal importance, although measurement of catecholamines or metanephrines (their methylated metabolites) is traditionally the first step in diagnosis.

1	These two criteria are of equal importance, although measurement of catecholamines or metanephrines (their methylated metabolites) is traditionally the first step in diagnosis. Biochemical testing Pheochromocytomas and paragangliomas synthesize and store catecholamines, which include norepinephrine (noradrenaline), epinephrine (adrenaline), and dopamine. Elevated plasma and urinary levels of catecholamines and metanephrines form the cornerstone of diagnosis. The characteristic fluctuations in the hormonal activity of tumors results in considerable variation in serial catecholamine measurements. However, most tumors continuously leak O-methylated metabolites, which are detected by measurement of metanephrines.

1	Catecholamines and metanephrines can be measured by different methods, including high-performance liquid chromatography, enzyme-linked immunosorbent assay, and liquid chromatography/ mass spectrometry. When pheochromocytoma is suspected on clinical grounds (i.e., when values are three times the upper limit of normal), this diagnosis is highly likely regardless of the assay used. However, as summarized in Table 407-2, the sensitivity and specificity of available biochemical tests vary greatly, and these differences are important in assessing patients with borderline elevations of different compounds. Urinary tests for metanephrines (total or fractionated) and catecholamines are widely available and are used commonly for initial evaluation. Among these tests, those for the fractionated metanephrines and catecholamines are the most sensitive. Plasma tests are more convenient and include measurements of catecholamines and metanephrines. Measurements of plasma metanephrine are the most

1	and catecholamines are the most sensitive. Plasma tests are more convenient and include measurements of catecholamines and metanephrines. Measurements of plasma metanephrine are the most sensitive and are less susceptible to false-positive elevations from stress, including venipuncture. Although the incidence of false-positive test results has been reduced by the introduction of newer assays, physiologic stress responses and medications that increase catecholamine levels still can confound testing. Because the tumors are relatively rare, borderline elevations are likely to represent false-positive results. In 2330 Jugular v. Vagus n. Tympanic n. Jugular p. Intravagal p. Intercarotid p. Sup. laryngeal p. Inf. laryngeal p. Subclavian p. Pulmonary p. Descending aorta Glossopharyngeal n. Jugular ganglion Nodose ganglion Sup. laryngeal a. Int. laryngeal a. Recurrent laryngeal n. Aortico-pulmonary p. Coronary p. A Adrenal B Extra-adrenal C Head and neck paraganglioma

1	FIGURE 407-1 The paraganglial system and topographic sites (in red) of pheochromocytomas and paragangliomas. (Parts A and B from WM Manger, RW Gifford: Clinical and experimental pheochromocytoma. Cambridge, Blackwell Science, 1996; Part C from GG Glenner, PM Grimley: Tumors of the Extra-adrenal Paraganglion System [Including Chemoreceptors], Atlas of Tumor Pathology, 2nd Series, Fascicle 9. Washington, DC, AFIP, 1974.)

1	CliniCAl fEATuRES ASSoCiATED wiTH PHEoCHRomoCyTomA, this circumstance, it is important to exclude dietary or drug-related liSTED by fR£EquEnCy of oCCuRREnCE factors (withdrawal of levodopa or use of sympathomimetics, diuret 1. Headaches 10. Weight loss ics, tricyclic antidepressants, alpha and beta blockers) that might cause false-positive results and then to repeat testing or perform a clonidine 2. Profuse sweating 11. Paradoxical response to antihypertensive drugs suppression test (i.e., the measurement of plasma normetanephrine 3 h 3. Palpitations and tachycardia after oral administration of 300 μg of clonidine). Other pharmacologic 12. Polyuria and polydipsia 4. Hypertension, sustained or tests, such as the phentolamine test and the glucagon provocation test, paroxysmal 13. Constipation are of relatively low sensitivity and are not recommended. 5. Anxiety and panic attacks 14. Orthostatic hypotension

1	5. Anxiety and panic attacks 14. Orthostatic hypotension Diagnostic Imaging A variety of methods have been used to local 6. Pallor 15. Dilated cardiomyopathy ize pheochromocytomas and paragangliomas (Table 407-2). CT and 7. Nausea 16. Erythrocytosis MRI are similar in sensitivity and should be performed with contrast. 8. Abdominal pain 17. Elevated blood sugar T2-weighted MRI with gadolinium contrast is optimal for detecting 9. Weakness 18. Hypercalcemia pheochromocytomas and is somewhat better than CT for imaging extraadrenal pheochromocytomas and paragangliomas. About 5% of adrenal incidentalomas, which usually are detected by CT or MRI, prove to be pheochromocytomas upon endocrinologic evaluation.

1	Tumors also can be localized by procedures using radioactive tracers, including 131Ior 123I-metaiodobenzylguanidine (MIBG) scintigraphy, 111In-somatostatin analogue scintigraphy, 18F-DOPA positron emission tomography (PET), or 18F-fluorodeoxyglucose (FDG) PET. Diagnostic Method Sensitivity Specificity Because these agents exhibit selective uptake in paragangliomas, nuclear imaging is particularly useful in the hereditary syndromes.

1	Differential Diagnosis When the possibility of a pheochromocytoma isFractionated ++++ ++ being entertained, other disorders to consider include essential hyper tension, anxiety attacks, use of cocaine or amphetamines, mastocytosisTotal metanephrines +++ ++++ or carcinoid syndrome (usually without hypertension), intracranial Plasma tests lesions, clonidine withdrawal, autonomic epilepsy, and factitious crises Catecholamines +++ ++ (usually from use of sympathomimetic amines). When an asymptomatic adrenal mass is identified, likely diagnoses other than pheochromocytoma include a nonfunctioning adrenal adenoma, an aldoste- ronoma, and a cortisol-producing adenoma (Cushing’s syndrome).

1	Somatostatin receptor ++ ++ Complete tumor removal, the ultimate therapeutic goal, can bescintigraphya achieved by partial or total adrenalectomy. It is important to pre18Fluoro-DOPA PET/CT +++ ++++ serve the normal adrenal cortex, particularly in hereditary disorders aValues are particularly high in head and neck paragangliomas. in which bilateral pheochromocytomas are most likely. Preoperative preparation of the patient is important. Before surgery, blood

1	Abbreviations: MIBG, metaiodobenzylguanidine; PET/CT, positron emission tomography plus CT. For the biochemical tests, the ratings correspond globally to sensitivity and speci-pressure should be consistently below 160/90 mmHg. Classically, ficity rates as follows: ++, <85%; +++, 85–95%; and ++++, >95%. blood pressure has been controlled by α-adrenergic blockers (oral phenoxybenzamine, 0.5–4 mg/kg of body weight). Because patients are volume-constricted, liberal salt intake and hydration are necessary to avoid severe orthostasis. Oral prazosin or intravenous phentolamine can be used to manage paroxysms while adequate alpha blockade is awaited. Beta blockers (e.g., 10 mg of propranolol three or four times per day) can then be added. Other antihypertensives, such as calcium channel blockers or angiotensin-converting enzyme inhibitors, have also been used effectively.

1	Surgery should be performed by teams of surgeons and anesthesiologists with experience in the management of pheochromocytomas. Blood pressure can be labile during surgery, particularly at the outset of intubation or when the tumor is manipulated. Nitroprusside infusion is useful for intraoperative hypertensive crises, and hypotension usually responds to volume infusion. Minimally invasive techniques (laparoscopy or retroperitoneoscopy) have become the standard approaches in pheochromocytoma surgery. They are associated with fewer complications, a faster recovery, and optimal cosmetic results. Extra-adrenal abdominal and most thoracic pheochromocytomas also can also be removed endoscopically. Postoperatively, catecholamine normalization should be documented. An adrenocorticotropic hormone test should be used to exclude cortisol deficiency when bilateral adrenal cortex–sparing surgery has been performed.

1	About 5–10% of pheochromocytomas and paragangliomas are malignant. The diagnosis of malignant pheochromocytoma is problematic. The typical histologic criteria of cellular atypia, presence of mitoses, and invasion of vessels or adjacent tissues are insufficient for the diagnosis of malignancy in pheochromocytoma. Thus, the term malignant pheochromocytoma is restricted to tumors with distant metastases, most commonly found by nuclear medicine imaging in lungs, bone, or liver—locations suggesting a vascular pathway of spread. Because hereditary syndromes are associated with multifocal tumor sites, these features should be anticipated in patients with germ-line mutations of 2331 RET, VHL, SDHD, or SDHB. However, distant metastases also occur in these syndromes, especially in carriers of SDHB mutations.

1	Treatment of malignant pheochromocytoma or paraganglioma is challenging. Options include tumor mass reduction, alpha blockers for symptoms, chemotherapy, and nuclear medicine radiotherapy. The first-line choice is nuclear medicine therapy for scintigraphically documented metastases, preferably with 131I-MIBG in 200-mCi doses at monthly intervals over three to six cycles. Averbuch’s chemotherapy protocol includes dacarbazine (600 mg/m2 on days 1 and 2), cyclophosphamide (750 mg/m2 on day 1), and vincristine (1.4 mg/m2 on day 1), all repeated every 21 days for three to six cycles. Palliation (stable disease to shrinkage) is achieved in about one-half of patients. Other chemotherapeutic options are sunitinib and temozolomide/ thalidomide. The prognosis of metastatic pheochromocytoma or paraganglioma is variable, with 5-year survival rates of 30–60%.

1	Pheochromocytomas occasionally are diagnosed in pregnancy. Endoscopic removal, preferably in the fourth to sixth month of gestation, is possible and can be followed by uneventful childbirth. Regular screening in families with inherited pheochromocytomas provides an opportunity to identify and remove asymptomatic tumors in women of reproductive age. About 25–33% of patients with a pheochromocytoma or paragan glioma have an inherited syndrome. At diagnosis, patients with inherited syndromes are a mean of ∼15 years younger than patients with sporadic tumors. Neurofibromatosis type 1 (NF1) was the first described pheochromocytoma-associated syndrome (Chap. 118). The NF1 gene functions as a tumor suppressor by regulating the Ras signaling cascade. Classic features of neurofibromatosis include multiple neurofibromas, café au lait spots, axillary freckling of the skin, and Lisch nodules of the iris (Fig. 407-2). Pheochromocytomas occur in only ∼1% of these patients

1	FIGURE 407-2 Neurofibromatosis. A. MRI of bilateral adrenal pheochromocytoma. B. Cutaneous neurofibromas. C. Lisch nodules of the iris.

1	D. Axillary freckling. (Part A from HPH Neumann et al: Keio J Med 54:15, 2005; with permission.) 2332 and are located predominantly in the adrenals. Malignant pheochromocytoma is not uncommon. The best-known pheochromocytoma-associated syndrome is the autosomal dominant disorder multiple endocrine neoplasia type 2 (MEN2) (Chap. 408). Both types of MEN2 (2A and 2B) are caused by mutations in RET (rearranged during transfection), which encodes a tyrosine kinase. The locations of RET mutations correlate with the severity of disease and the type of MEN2 (Chap. 408). MEN2A is characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism; MEN2B also includes MTC and pheochromocytoma as well as multiple mucosal neuromas, marfanoid habitus, and other developmental disorders, though it typically lacks hyperparathyroidism. MTC is found in virtually all patients with MEN2, but pheochromocytoma occurs in only ∼50% of these patients. Nearly all pheochromocytomas in

1	disorders, though it typically lacks hyperparathyroidism. MTC is found in virtually all patients with MEN2, but pheochromocytoma occurs in only ∼50% of these patients. Nearly all pheochromocytomas in MEN2 are benign and located in the adrenals, often bilaterally (Fig. 407-3). Pheochromocytoma may be symptomatic before MTC. Prophylactic thyroidectomy is being performed in many carriers of RET mutations; pheochromocytomas should be excluded before any surgery in these patients. Von Hippel–Lindau syndrome (VHL) is an autosomal dominant disorder that predisposes to retinal and cerebellar hemangioblastomas, which also occur in the brainstem and spinal cord (Fig. 407-4). Other important features of VHL are clear cell renal carcinomas, pancreatic neuroendocrine tumors, endolymphatic sac tumors of the inner ear, cystadenomas of the epididymis and broad ligament, and multiple pancreatic or renal cysts.

1	The VHL gene (among other genes) encodes an E3 ubiquitin ligase that regulates expression of hypoxia-inducible factor 1. Loss of VHL is associated with increased expression of vascular endothelial growth factor (VEGF), which induces angiogenesis. Although the VHL gene can be inactivated by all types of mutations, patients with pheochromocytoma predominantly have missense mutations. About 20–30% of patients with VHL have pheochromocytomas, but in some families the incidence can reach 90%. The recognition of pheochromocytoma as a VHL-associated feature provides an opportunity to diagnose retinal, central nervous system, renal, and pancreatic tumors at a stage when effective treatment may still be possible.

1	The paraganglioma syndromes (PGLs) have been classified by genetic analyses of families with head and neck paragangliomas. The susceptibility genes encode subunits of the enzyme succinate dehydrogenase (SDH), a component in the Krebs cycle and the mitochondrial electron transport chain. SDH is formed by four subunits (A–D). Mutations of SDHB (PGL4), SDHC (PGL3), SDHD (PGL1), and SDHAF2 (PGL2) predispose to the PGLs. The transmission of the disease in carriers of SDHB and SDHC germ-line mutations is autosomal dominant. In contrast, in SDHD and SDHAF2 families, only the progeny of affected fathers develop tumors if they inherit the mutation. PGL1 is most common, followed by PGL4; PGL2 and PGL3 are rare. Adrenal, extra-adrenal abdominal, and thoracic pheochromocytomas, which are components of PGL1 and PGL4, are rare in PGL3 and absent in PGL2 (Fig. 407-5). About one-third of patients with PGL4 develop metastases.

1	FIGURE 407-3 Multiple endocrine neoplasia type 2. A, B. Multifocal medullary thyroid carcinoma shown by MIBG scintigraphy (A) and operative specimen (B). Arrows demonstrate the tumors; arrowheads show the tissue bridge of the cut specimen. C–E. Bilateral adrenal pheochromocytoma shown by MIBG scintigraphy (C), CT imaging (D), and operative specimens (E). (From HPH Neumann et al: Keio J Med 54:15, 2005; with permission.)

1	FIGURE 407-4 Von Hippel–Lindau disease. A. Retinal angioma. All subsequent panels show findings on MRI: B–D. Hemangioblastomas of the cerebellum (B) in brainstem (C) and spinal cord (D). E. Bilateral pheochromocytomas and bilateral renal clear cell carcinomas F. Multiple pancreatic cysts. (Parts A and D from HPH Neumann et al: Adv Nephrol Necker Hosp 27:361, 1997. © Elsevier. Part B from SH Morgan, J-P Grunfeld [eds]: Inherited Disorders of the Kidney. Oxford, UK, Oxford University Press, 1998. Part F from HPH Neumann et al: Contrib Nephrol 136:193, 2001. © S. Karger AG, Basel.)

1	FIGURE 407-5 Paraganglioma syndrome. A patient with the SDHD W5X mutation and PGL1 underwent incomplete resection of a left carotid body tumor. A. 18F-DOPA positron emission tomography demonstrating tumor uptake in the right jugular glomus, the right carotid body, the left carotid body, the left coronary glomus, and the right adrenal gland. Note the physiologic accumulation of the radiopharmaceutical agent in the kidneys, liver, gallbladder, renal pelvis, and urinary bladder. B and C. CT angiography with three-dimensional reconstruction. Arrows point to the paraganglial tumors. (From S Hoegerle et al: Eur J Nucl Med Mol Imaging 30:689, 2003; with permission.) 2334 Familial pheochromocytoma (FP) has been attributed to hereditary, germ-line mutations even in patients without a known family history. mainly adrenal tumors in patients with germ-line mutations in the A first step is to search for clinical features of inherited syndromes genes TMEM127, MAX, and SDHA. Transmission is also

1	history. mainly adrenal tumors in patients with germ-line mutations in the A first step is to search for clinical features of inherited syndromes genes TMEM127, MAX, and SDHA. Transmission is also autosomal and to obtain an in-depth, multigenerational family history. Each dominant, and mutations of MAX, like those of SDHD, cause tumors of these syndromes exhibits autosomal dominant transmission with only if inherited from the father. variable penetrance, but a proband with a mother affected by paraganglial tumors is not predisposed to PLG1 (SDHD mutation carrier). GUIDELINES FOR GENETIC SCREENING OF PATIENTS WITH Cutaneous neurofibromas, café au lait spots, and axillary freckling PHEOCHROMOCYTOMA OR PARAGANGLIOMA suggest neurofibromatosis. Germ-line mutations in NF1 have not In addition to family history, general features suggesting an inher-been reported in patients with sporadic pheochromocytomas. Thus,ited syndrome include young age, multifocal tumors, extra-adrenal NF1 testing

1	to family history, general features suggesting an inher-been reported in patients with sporadic pheochromocytomas. Thus,ited syndrome include young age, multifocal tumors, extra-adrenal NF1 testing need not be performed in the absence of other clinicaltumors, and malignant tumors (Fig. 407-6). Because of the relatively features of neurofibromatosis. A personal or family history of MTC or high prevalence of familial syndromes among patients who present an elevation of serum calcitonin strongly suggests MEN 2 and shouldwith pheochromocytoma or paraganglioma, it is useful to identify prompt testing for RET mutations. A history of visual impairment or

1	Percentages of germ-line mutations in pheochromocytoma susceptibility genes

1	FIGURE 407-6 Mutation distribution in the VHL, RET, SDHB, SDHC, SDHD, and NF1 genes in 2021 patients with pheochromocytomas and paragangliomas from the European-American Pheochromocytoma-Paraganglioma Registry based in Freiburg, Germany, as updated on March 1, 2014. A. Correlation with age. The bars depict the frequency of sporadic (spor) or various inherited forms of pheochromocytoma in different age groups. The inherited disorders are much more common among younger individuals presenting with pheochromocytoma. Patients with mutations in the TMEM127, MAX, and SDHA genes are not included, since they contribute <1% in decades 4–7 only. B–F. Germ-line mutations according to multiple (B), extra-adrenal retroperitoneal (C), thoracic (D), and malignant (E) pheochromocytomas and head and neck paragangliomas (F). (Data from the Freiburg International Pheochromocytoma and Paraganglioma Registry, 2014.) tumors of the cerebellum, kidney, brainstem, or spinal cord suggests the possibility of

1	paragangliomas (F). (Data from the Freiburg International Pheochromocytoma and Paraganglioma Registry, 2014.) tumors of the cerebellum, kidney, brainstem, or spinal cord suggests the possibility of VHL. A personal and/or family history of head and neck paraganglioma suggests PGL1 or PGL4.

1	A single adrenal pheochromocytoma in a patient with an otherwise unremarkable history may still be associated with mutations of VHL, RET, SDHB, or SDHD (in decreasing order of frequency). Two-thirds of extra-adrenal tumors are associated with one of these syndromes, and multifocal tumors occur with decreasing frequency in carriers of RET, SDHD, VHL, and SDHB mutations. About 30% of head and neck paragangliomas are associated with germ-line mutations of one of the SDH subunit genes (most often SDHD) and are rare in carriers of VHL, RET, and TMEM127 mutations (Fig. 407-6F). Immunohistochemistry is helpful in the preselection of hereditary pheochromocytoma. Negative immunostaining with antibodies to SDHB, TMEM127, and MAX may predict mutations of the SDH, TMEM127, and MAX genes, respectively.

1	Once the underlying syndrome is diagnosed, the benefit of genetic testing can be extended to relatives. For this purpose, it is necessary to identify the germ-line mutation in the proband and, after genetic counseling, to perform DNA sequence analyses of the responsible gene in relatives to determine whether they are affected (Chap. 84). Other family members may benefit when individuals who carry a germ-line mutation are biochemically screened for paraganglial tumors.

1	Asymptomatic paraganglial tumors, now often detected in patients with hereditary tumors and their relatives, are challenging to manage. Watchful waiting strategies have been introduced. Head and neck paragangliomas—mainly carotid body, jugular, and vagal tumors—are increasingly treated by radiation, since surgery is frequently associated with permanent palsy of cranial nerves II, VII, IX, X, XI, and XII. Nevertheless, tympanic paragangliomas are symptomatic early, and most of these tumors can easily be resected, with subsequent improve- Rajesh V. Thakker

1	Multiple endocrine neoplasia (MEN) is characterized by a predilection for tumors involving two or more endocrine glands. Four major forms of MEN are recognized and referred to as MEN types 1–4 (MEN 1–4) (Table 408-1). Each type of MEN is inherited as an autosomal dominant syndrome or may occur sporadically; that is, without a family history. However, this distinction between familial and sporadic forms is often difficult because family members with the disease may have died before symptoms developed. In addition to MEN 1–4, at least six other syndromes are associated with multiple endocrine and other organ neoplasias (MEONs) (Table 408-2). These MEONs include the hyperparathyroidism-jaw tumor syndrome, Carney complex, von Hippel-Lindau disease (Chap. 407), neurofibromatosis type 1 (Chap. 118), Cowden’s syndrome, and McCune-Albright syndrome (Chap. 426e); all of these are inherited as autosomal dominant disorders, except for McCune-Albright syndrome, which is caused by mosaic

1	1 (Chap. 118), Cowden’s syndrome, and McCune-Albright syndrome (Chap. 426e); all of these are inherited as autosomal dominant disorders, except for McCune-Albright syndrome, which is caused by mosaic expression of a postzygotic somatic cell mutation (Table 408-2).

1	A diagnosis of a MEN or MEON syndrome may be established in an individual by one of three criteria: (1) clinical features (two or more of the associated tumors [or lesions] in an individual); (2) familial pattern (one of the associated tumors [or lesions] in a first-degree relative of a patient with a clinical diagnosis of the syndrome); and (3) genetic analysis (a germline mutation in the associated gene in an individual, who may be clinically affected or asymptomatic). Mutational analysis in MEN and MEON syndromes is helpful in clinical practice to: (1) confirm the clinical diagnosis; (2) identify family members who harbor the mutation and require screening for relevant tumor detection and early/appropriate treatment; and (3) identify the ~50% of family members who do not harbor the germline mutation and can, therefore, 2335 be alleviated of the anxiety of developing associated tumors. This latter aspect also helps to reduce health care costs by reducing the need for unnecessary

1	germline mutation and can, therefore, 2335 be alleviated of the anxiety of developing associated tumors. This latter aspect also helps to reduce health care costs by reducing the need for unnecessary biochemical and radiologic investigations.

1	MULTIPLE ENDOCRINE NEOPLASIA TYPE 1 Clinical Manifestations MEN type 1 (MEN 1), which is also referred to as Wermer’s syndrome, is characterized by the triad of tumors involving the parathyroids, pancreatic islets, and anterior pituitary. In addition, adrenal cortical tumors, carcinoid tumors usually of the foregut, meningiomas, facial angiofibromas, collagenomas, and lipomas may also occur in some patients with MEN 1. Combinations of the affected glands and their pathologic features (e.g., hyperplastic adenomas of the parathyroid glands) may differ in members of the same family and even between identical twins. In addition, a nonfamilial (e.g., sporadic) form occurs in 8–14% of patients with MEN 1, and molecular genetic studies have confirmed the occurrence of de novo mutations of the MEN1 gene in approximately 10% of patients with MEN 1. The prevalence of MEN 1 is approximately 0.25% based on randomly chosen postmortem studies but is 1–18% among patients with primary

1	of the MEN1 gene in approximately 10% of patients with MEN 1. The prevalence of MEN 1 is approximately 0.25% based on randomly chosen postmortem studies but is 1–18% among patients with primary hyperparathyroidism, 16–38% among patients with pancreatic islet tumors, and <3% among patients with pituitary tumors. The disorder affects all age groups, with a reported age range of 5 to 81 years, with clinical and biochemical manifestations developing in the vast majority by the fifth decade. The clinical manifestations of MEN 1 are related to the sites of tumors and their hormonal products. In the absence of treatment, endocrine tumors are associated with an earlier mortality in patients with MEN 1, with a 50% probability of death by the age of 50 years. The cause of death is usually a malignant tumor, often from a pancreatic neuroendocrine tumor (NET) or foregut carcinoid. In addition, the treatment outcomes of patients with MEN 1–associated tumors are not as successful as those in

1	tumor, often from a pancreatic neuroendocrine tumor (NET) or foregut carcinoid. In addition, the treatment outcomes of patients with MEN 1–associated tumors are not as successful as those in patients with non–MEN 1 tumors. This is because MEN 1–associated tumors, with the exception of pituitary NETs, are usually multiple, making it difficult to achieve a successful surgical cure. Occult metastatic disease is also more prevalent in MEN 1, and the tumors may be larger, more aggressive, and resistant to treatment.

1	Parathyroid Tumors (See also Chap. 424) Primary hyperparathyroidism occurs in approximately 90% of patients and is the most common feature of MEN 1. Patients may have asymptomatic hypercalcemia or vague symptoms associated with hypercalcemia (e.g., polyuria, polydipsia, constipation, malaise, or dyspepsia). Nephrolithiasis and osteitis fibrosa cystica (less commonly) may also occur. Biochemical investigations reveal hypercalcemia, usually in association with elevated circulating parathyroid hormone (PTH) (Table 408-3). The hypercalcemia is usually mild, and severe hypercalcemia or parathyroid cancer is a rare occurrence. Additional differences in the primary hyperparathyroidism of patients with MEN 1, as opposed to those without MEN 1, include an earlier age at onset (20–25 years vs 55 years) and an equal male-to-female ratio (1:1 vs 1:3). Preoperative imaging (e.g., neck ultrasound with 99mTc-sestamibi parathyroid scintigraphy) is of limited benefit because all parathyroid glands may

1	and an equal male-to-female ratio (1:1 vs 1:3). Preoperative imaging (e.g., neck ultrasound with 99mTc-sestamibi parathyroid scintigraphy) is of limited benefit because all parathyroid glands may be affected, and neck exploration may be required irrespective of preoperative localization studies.

1	Surgical removal of the abnormally overactive parathyroids in patients with MEN 1 is the definitive treatment. However, it is controversial whether to perform subtotal (e.g., removal of 3.5 glands) or total parathyroidectomy with or without autotransplantation of parathyroid tissue in the forearm, and whether surgery should be performed at an early or late stage. Minimally invasive parathyroidectomy is not recommended because all four parathyroid glands are usually affected with multiple adenomas or hyperplasia. Surgical experience should be taken into account given the variability in pathology in MEN 1. Calcimimetics (e.g., cinacalcet), which act via MEN 2 (10 cen-10q11.2) MEN 2A MTC only MEN 2B (also known as MEN 3)

1	MEN 2 (10 cen-10q11.2) MEN 2A MTC only MEN 2B (also known as MEN 3) Parathyroid adenomaa Pituitary adenomaa Reproductive organ tumorsa (e.g., testicular cancer, neuroendocrine cervical carcinoma) ?Adrenal + renal tumorsa 83/84, 4-bp del (≈4%) 119, 3-bp del (≈3%) 209-211, 4-bp del (≈8%) 418, 3-bp del (≈4%) 514-516, del or ins (≈7%) Intron 4 ss (≈10%) 634, e.g., Cys → Arg (~85%) RET 618, missense (>50%) RET 918, Met → Thr (>95%) CDKN1B; no common mutations identified to date aInsufficient numbers reported to provide prevalence information. Note: Autosomal dominant inheritance of the MEN syndromes has been established. Abbreviations: del, deletion; ins, insertion; MTC, medullary thyroid cancer; NET, neuroendocrine tumor; PPoma, pancreatic polypeptide–secreting tumor; VIPoma, vasoactive intestinal polypeptide–secreting tumor. Source: Reproduced from RV Thakker et al: J Clin Endocrinol Metab 97:2990, 2012.

1	Source: Reproduced from RV Thakker et al: J Clin Endocrinol Metab 97:2990, 2012. the calcium-sensing receptor, have been used to treat primary hyperparathyroidism in some patients when surgery is unsuccessful or contraindicated. Pancreatic Tumors (See also Chap. 113) The incidence of pancreatic islet cell tumors, which are NETs, in patients with MEN 1 ranges from 30 to 80% in different series. Most of these tumors (Table 408-1) produce excessive amounts of hormone (e.g., gastrin, insulin, glucagon, vasoactive intestinal polypeptide [VIP]) and are associated with distinct clinical syndromes, although some are nonfunctioning or non-secretory. These pancreatic islet cell tumors have an earlier age at onset in patients with MEN 1 than in patients without MEN 1.

1	Gastrinoma Gastrin-secreting tumors (gastrinomas) are associated with marked gastric acid production and recurrent peptic ulcerations, a combination referred to as the Zollinger-Ellison syndrome. Gastrinomas occur more often in patients with MEN 1 who are older than age 30 years. Recurrent severe multiple peptic ulcers, which may perforate, and cachexia are major contributors to the high mortality. Patients with Zollinger-Ellison syndrome may also suffer from diarrhea and steatorrhea. The diagnosis is established by demonstration of an elevated fasting serum gastrin concentration in association with increased basal gastric acid secretion (Table 408-3). However, the diagnosis of Zollinger-Ellison syndrome may be difficult in hypercalcemic MEN 1 patients, because hypercalcemia can also cause hypergastrinemia.

1	aThe inheritance for these disorders is autosomal dominant, except MAS, which is due to mosaicism that results from the postzygotic somatic cell mutation of the GNAS1 gene, encoding Gsα. b?, unknown. Ultrasonography, endoscopic ultrasonography, computed tomography (CT), nuclear magnetic resonance imaging (MRI), selective abdominal angiography, venous sampling, and somatostatin receptor scintigraphy are helpful in localizing the tumor prior to surgery. Gastrinomas represent more than 50% of all pancreatic NETs in patients with MEN 1, and approximately 20% of patients with gastrinomas will be found to have MEN 1. Gastrinomas, which may also occur in the duodenal mucosa, are the major cause of morbidity and mortality in patients with MEN 1. Most MEN 1 gastrinomas are malignant and metastasize before a diagnosis is established.

1	Medical treatment of patients with MEN 1 and Zollinger-Ellison syndrome is directed toward reducing basal acid output to <10 mmol/L. Parietal cell H+-K+-adenosine triphosphatase (ATPase) inhibitors (e.g., omeprazole or lansoprazole) reduce acid output and are the drugs of choice for gastrinomas. Some patients may also require additional treatment with the histamine H2 receptor antagonists, cimetidine or ranitidine. The role of surgery in the treatment of gastrinomas in patients with MEN 1 is controversial. The goal of surgery is to reduce the risk of distant metastatic disease and improve survival. For a nonmetastatic gastrinoma situated in the pancreas, surgical excision is often effective. However, the risk of hepatic metastases increases with tumor size, such that 25–40% of patients with pancreatic NETs >4 cm develop hepatic metastases, and 2337 50–70% of patients with tumors 2–3 cm in size have lymph node metastases. Survival in MEN 1 patients with gastrinomas <2.5 cm in size is

1	pancreatic NETs >4 cm develop hepatic metastases, and 2337 50–70% of patients with tumors 2–3 cm in size have lymph node metastases. Survival in MEN 1 patients with gastrinomas <2.5 cm in size is 100% at 15 years, but 52% at 15 years, if metastatic disease is present. The presence of lymph node metastases does not appear to adversely affect survival. Surgery for gastrinomas that are >2–2.5 cm has been recommended, because the disease-related survival in these patients is improved following surgery. In addition, duodenal gastrinomas, which occur more frequently in patients with MEN 1, have been treated successfully with surgery. However, in most patients with MEN 1, gastrinomas are multiple or extrapancreatic, and with the exception of duodenal gastrinomas, surgery is rarely successful. For example, the results of one study revealed that only ~15% of patients with MEN 1 were free of disease immediately after surgery, and at 5 years, this number had decreased to ~5%; the respective

1	For example, the results of one study revealed that only ~15% of patients with MEN 1 were free of disease immediately after surgery, and at 5 years, this number had decreased to ~5%; the respective outcomes in patients without MEN 1 were better, at 45% and 40%. Given these findings, most specialists recommend a nonsurgical management for gastrinomas in MEN 1, except as noted earlier for smaller, isolated lesions. Treatment of disseminated gastrinomas is difficult. Chemotherapy with streptozotocin and 5-fluorouracil; hormonal therapy with octreotide or lanreotide, which are human somatostatin analogues; hepatic artery embolization; administration of human leukocyte interferon; and removal of all resectable tumor have been successful in some patients.

1	Insulinoma These β islet cell insulin-secreting tumors represent 10–30% of all pancreatic tumors in patients with MEN 1. Patients with an insulinoma present with hypoglycemic symptoms (e.g., weakness, headaches, sweating, faintness, seizures, altered behavior, weight gain) that typically develop after fasting or exertion and improve after glucose intake. The most reliable test is a supervised 72-h fast. Biochemical investigations reveal increased plasma insulin concentrations in association with hypoglycemia (Table 408-3). Circulating concentrations of C peptide and proinsulin, which are also increased, are useful in establishing the diagnosis. It also is important to demonstrate the absence of sulfonylureas in plasma and urine samples obtained during the investigation of hypoglycemia (Table 408-3). Surgical success is greatly enhanced by preoperative localization by endoscopic ultrasonography, CT scanning, or celiac axis angiography. Additional localization methods may include

1	(Table 408-3). Surgical success is greatly enhanced by preoperative localization by endoscopic ultrasonography, CT scanning, or celiac axis angiography. Additional localization methods may include preoperative and perioperative percutaneous transhepatic portal venous sampling, selective intraarterial stimulation with hepatic venous sampling, and intraoperative direct pancreatic ultrasonography. Insulinomas occur in association with gastrinomas in 10% of patients with MEN 1, and the two tumors may arise at different times. Insulinomas occur more often in patients with MEN 1 who are younger than 40 years, and some arise in individuals younger than 20 years. In contrast, in patients without MEN 1, insulinomas generally occur in those older than 40 years. Insulinomas may be the first manifestation of MEN 1 in 10% of patients, and approximately 4% of patients with insulinomas will have MEN 1.

1	Abbreviations: CT, computed tomography; EUS, endoscopic ultrasound; IGF-I, insulin-like growth factor I; MRI, magnetic resonance imaging; PTH, parathyroid hormone. Source: Reproduced from RV Thakker et al: J Clin Endocrinol Metab 97:2990, 2012. Medical treatment, which consists of frequent carbohydrate meals and diazoxide or octreotide, is not always successful, and surgery is the optimal treatment. Surgical treatment, which ranges from enucleation of a single tumor to a distal pancreatectomy or partial pancreatectomy, has been curative in many patients. Chemotherapy may include streptozotocin, 5-fluorouracil, and doxorubicin. Hepatic artery embolization has been used for metastatic disease.

1	Glucagonoma These glucagon-secreting pancreatic NETs occur in <3% of patients with MEN 1. The characteristic clinical manifestations of a skin rash (necrolytic migratory erythema), weight loss, anemia, and stomatitis may be absent. The tumor may have been detected in an asymptomatic patient with MEN 1 undergoing pancreatic imaging or by the finding of glucose intolerence and hyperglucagonemia. Surgical removal of the glucagonoma is the treatment of choice. However, treatment may be difficult because approximately 50–80% of patients have metastases at the time of diagnosis. Medical treatment with somatostatin analogues (e.g., octreotide or lanreotide) or chemotherapy with streptozotocin and 5-fluorouracil has been successful in some patients, and hepatic artery embolization has been used to treat metastatic disease.

1	Vasoactive Intestinal Peptide (VIP) Tumors (VIPomas) VIPomas have been reported in only a few patients with MEN 1. This clinical syndrome is characterized by watery diarrhea, hypokalemia, and achlorhydria and is also referred to as the Verner-Morrison syndrome, the WDHA (watery diarrhea, hypokalemia, and achlorhydria) syndrome, or the VIPoma syndrome. The diagnosis is established by excluding laxative and diuretic abuse, by confirming a stool volume in excess of 0.5–1.0 L/d during a fast, and by documenting a markedly increased plasma VIP concentration. Surgical management of VIPomas, which are mostly located in the tail of the pancreas, can be curative. However, in patients with unresectable tumor, somatostatin analogues, such as octreotide and lanreotide, may be effective. Streptozotocin with 5-fluorouracil may be beneficial, along with hepatic artery embolization for the treatment of metastases.

1	Pancreatic Polypeptide-Secreting Tumors (Ppomas) and Nonfunctioning Pancreatic NETs PPomas are found in a large number of patients with MEN 1. No pathologic sequelae of excessive polypeptide (PP) secretion are apparent, and the clinical significance of PP is unknown. Many PPomas may have been unrecognized or classified as nonfunctioning pancreatic NETs, which likely represent the most common enteropancreatic NET associated with MEN 1 (Fig. 408-1). The absence of both a clinical syndrome and specific biochemical abnormalities may result in a delayed diagnosis of nonfunctioning pancreatic NETs, which are associated with a worse prognosis than other functioning tumors, including insulinoma and gastrinoma. The optimum screening method and its timing interval for nonfunctioning pancreatic NETs remain to be established. At present, endoscopic ultrasound likely represents the most sensitive method of detecting small pancreatic tumors, but somatostatin receptor scintography is the most

1	NETs remain to be established. At present, endoscopic ultrasound likely represents the most sensitive method of detecting small pancreatic tumors, but somatostatin receptor scintography is the most reliable method for detecting metastatic disease (Table 408-3).

1	The management of nonfunctioning pancreatic NETs in the asymptomatic patient is controversial. One recommendation is to undertake surgery irrespective of tumor size after biochemical

1	FIGURE 408-1 Pancreatic nonfunctioning neuroendocrine tumor (NET) in a 14-year-old patient with multiple endocrine neoplasia type 1 (MEN 1). A. An abdominal magnetic resonance imaging scan revealed a low-intensity >2.0 cm (anteroposterior maximal diameter) tumor within the neck of pancreas. There was no evidence of invasion of adjacent structures or metastases. The tumor is indicated by white dashed circle. B. The pancreatic NET was removed by surgery, and macroscopic examination confirmed the location of the tumor (white dashed circles) in the neck of the pancreas. Immunohistochemistry showed the tumor to immunostain for chromogranin A, but not gastrointestinal peptides or menin, thereby confirming that it was a non-secreting NET due to loss of menin expression. (Part A adapted with permission from PJ Newey et al: J Clin Endocrinol Metab 10:3640, 2009.) assessment is complete. Alternatively, other experts recommend surgery based on tumor size, using either >1 cm or >3 cm at different

1	from PJ Newey et al: J Clin Endocrinol Metab 10:3640, 2009.) assessment is complete. Alternatively, other experts recommend surgery based on tumor size, using either >1 cm or >3 cm at different centers. Pancreatoduodenal surgery is successful in removing the tumors in 80% of patients, but more than 40% of patients develop complications, including diabetes mellitus, frequent steatorrhea, early and late dumping syndromes, and other gastrointestinal symptoms. However, ~50–60% of patients treated surgically survive >5 years. When considering these recommendations, it is important to consider that occult metastatic disease (e.g., tumors not detected by imaging investigations) is likely to be present in a substantial proportion of these patients at the time of presentation. Inhibitors of tyrosine kinase receptors (TKRs) and of the mammalian target of rapamycin (mTOR) signaling pathway have been reported to be effective in treating pancreatic NETs and in doubling the progression-free

1	tyrosine kinase receptors (TKRs) and of the mammalian target of rapamycin (mTOR) signaling pathway have been reported to be effective in treating pancreatic NETs and in doubling the progression-free survival time.

1	Other Pancreatic NETs NETs secreting growth hormone–releasing hormone (GHRH), GHRHomas, have been reported rarely in patients with MEN 1. It is estimated that ~33% of patients with GHRHomas have other MEN 1–related tumors. GHRHomas may be diagnosed by demonstrating elevated serum concentrations of growth hormone and GHRH. More than 50% of GHRHomas occur in the lung, 30% occur in the pancreas, and 10% are found in the small intestine. Somatostatinomas secrete somatostatin, a peptide that inhibits the secretion of a variety of hormones, resulting in hyperglycemia, cholelithiasis, low acid output, steatorrhea, diarrhea, abdominal pain, anemia, and weight loss. Although 7% of pancreatic NETs secrete somatostatin, the clinical features of somatostatinoma syndrome are unusual in patients with MEN 1.

1	Pituitary Tumors (See also Chap. 403) Pituitary tumors occur in 15–50% of patients with MEN 1 (Table 408-1). These occur as early as 5 years of age or as late as the ninth decade. MEN 1 pituitary adenomas are more frequent in women than men and significantly are macroadenomas (i.e., diameter >1 cm). Moreover, about one-third of these pituitary tumors show invasive features such as infiltration of tumor cells into surrounding normal juxtatumoral pituitary tissue. However, no specific histologic parameters differentiate between MEN 1 and non–MEN 1 pituitary tumors. Approximately 60% of MEN 1–associated pituitary tumors secrete prolactin, <25% secrete growth hormone, 5% secrete adrenocorticotropic hormone (ACTH), and the remainder appear to be nonfunctioning, with some secreting glycoprotein subunits (Table 408-1). However, pituitary tumors derived from MEN 1 patients may exhibit immunoreactivity to several hormones. In particular, there is a greater frequency of somatolactotrope tumors.

1	subunits (Table 408-1). However, pituitary tumors derived from MEN 1 patients may exhibit immunoreactivity to several hormones. In particular, there is a greater frequency of somatolactotrope tumors. Prolactinomas are the first manifestation of MEN 1 in ~15% of patients, whereas somatotrope tumors occur more often in patients older than 40 years of age. Fewer than 3% of patients with anterior pituitary tumors will have MEN 1. Clinical manifestations are similar to those in patients with sporadic pituitary tumors without MEN 1 and depend on the hormone secreted and the size of the pituitary tumor. Thus, patients may have symptoms of hyperprolactinemia (e.g., amenorrhea, infertility, and galactorrhea in women, or impotence and infertility in men) or have features of acromegaly or Cushing’s disease. In addition, enlarging pituitary tumors may compress adjacent structures such as the optic chiasm or normal pituitary tissue, causing visual disturbances and/or hypopituitarism. In

1	Cushing’s disease. In addition, enlarging pituitary tumors may compress adjacent structures such as the optic chiasm or normal pituitary tissue, causing visual disturbances and/or hypopituitarism. In asymptomatic patients with MEN 1, periodic biochemical monitoring of serum prolactin and insulin-like growth factor I (IGF-I) levels, as well as MRI of the pituitary, can lead to early identification of pituitary tumors (Table 408-3). In patients with abnormal results, hypothalamic-pituitary testing should characterize the nature of the pituitary lesion and its effects on the secretion of other pituitary hormones.

1	Treatment of pituitary tumors in patients with MEN 1 consists of therapies similar to those used in patients without MEN 1 and includes appropriate medical therapy (e.g., bromocriptine or cabergoline for prolactinoma; or octreotide or lanreotide for somatotrope tumors) or selective transsphenoidal adenomectomy, if feasible, with radiotherapy reserved for residual unresectable tumor tissue. Pituitary tumors in MEN 1 patients may be more aggressive and less responsive to medical or surgical treatments. Associated Tumors Patients with MEN 1 may also develop carcinoid tumors, adrenal cortical tumors, facial angiofibromas, collagenomas, thyroid tumors, and lipomatous tumors.

1	Carcinoid Tumors (See also Chap. 113) Carcinoid tumors occur in more than 3% of patients with MEN 1 (Table 408-1). The carcinoid tumor may be located in the bronchi, gastrointestinal tract, pancreas, or 2339 thymus. At the time of diagnosis, most patients are asymptomatic and do not have clinical features of the carcinoid syndrome. Importantly, no hormonal or biochemical abnormality (e.g., plasma chromogranin A) is consistently observed in individuals with thymic or bronchial carcinoid tumors. Thus, screening for these tumors is dependent on radiologic imaging. The optimum method for screening has not been established. CT and MRI are sensitive for detecting thymic and bronchial tumors (Table 408-3), although repeated CT scanning raises concern about exposure to repeated doses of ionizing radiation. Octreotide scintigraphy may also reveal some thymic and bronchial carcinoids, although there is insufficient evidence to recommend its routine use. Gastric carcinoids, of which the type II

1	radiation. Octreotide scintigraphy may also reveal some thymic and bronchial carcinoids, although there is insufficient evidence to recommend its routine use. Gastric carcinoids, of which the type II gastric enterochromaffin-like (ECL) cell carcinoids (ECLomas) are associated with MEN 1 and Zollinger-Ellison syndrome, may be detected incidentally at the time of gastric endoscopy for dyspeptic symptoms in MEN 1 patients. These tumors, which may be found in >10% of MEN 1 patients, are usually multiple and smaller than 1.5 cm. Bronchial carcinoids in patients with MEN 1 occur predominantly in women (male-to-female ratio, 1:4). In contrast, thymic carcinoids in European patients with MEN 1 occur predominantly in men (male-to-female ratio, 20:1), with cigarette smokers having a higher risk for these tumors; thymic carcinoids in Japanese patients with MEN 1 have a less marked sex difference (male-to-female ratio 2:1). The course of thymic carcinoids in MEN 1 appears to be particularly

1	for these tumors; thymic carcinoids in Japanese patients with MEN 1 have a less marked sex difference (male-to-female ratio 2:1). The course of thymic carcinoids in MEN 1 appears to be particularly aggressive. The presence of thymic tumors in patients with MEN 1 is associated with a median survival after diagnosis of approximately 9.5 years, with 70% of patients dying as a direct result of the tumor.

1	If resectable, surgical removal of carcinoid tumors is the treatment of choice. For unresectable tumors and those with metastatic disease, treatment with radiotherapy or chemotherapeutic agents (e.g., cisplatin, etoposide) may be used. In addition, somatostatin analogues, such as octreotide or lanreotide, have resulted in symptom improvement and regression of some tumors. Little is known about the malignant potential of gastric type II ECLomas, but treatment with somatostatin analogues, such as octreotide or lanreotide, has resulted in regression of these ECLomas.

1	Adrenocortical Tumors (See also Chap. 406) Asymptomatic adrenocortical tumors occur in 20–70% of patients with MEN 1 depending on the radiologic screening methods used (Table 408-1). Most of these tumors, which include cortical adenomas, hyperplasia, multiple adenomas, nodular hyperplasia, cysts, and carcinomas, are nonfunctioning. Indeed, <10% of patients with enlarged adrenal glands have hormonal hypersecretion, with primary hyperaldosteronism and ACTH-independent Cushing’s syndrome being encountered most commonly. Occasionally, hyperandrogenemia may occur in association with adrenocortical carcinoma. Pheochromocytoma in association with MEN 1 is rare. Biochemical investigation (e.g., plasma renin and aldosterone concentrations, low-dose dexamethasone suppression test, urinary catecholamines, and/or metanephrines) should be undertaken in those with symptoms or signs suggestive of functioning adrenal tumors or in those with tumors >1 cm. Adrenocortical carcinoma occurs in

1	catecholamines, and/or metanephrines) should be undertaken in those with symptoms or signs suggestive of functioning adrenal tumors or in those with tumors >1 cm. Adrenocortical carcinoma occurs in approximately 1% of MEN 1 patients but increases to >10% for adrenal tumors larger than 1 cm.

1	Consensus has not been reached about the management of MEN 1–associated nonfunctioning adrenal tumors, because the majority are benign. However, the risk of malignancy increases with size, particularly for tumors with a diameter >4 cm. Indications for surgery for adrenal tumors include: size >4 cm in diameter; atypical or suspicious radiologic features (e.g., increased Hounsfield unit on unenhanced CT scan) and size of 1–4 cm in diameter; or significant 2340 measurable growth over a 6-month period. The treatment of functioning (e.g., hormone-secreting) adrenal tumors is similar to that for tumors occurring in non–MEN 1 patients.

1	Meningioma Central nervous system (CNS) tumors, including ependymomas, schwannomas, and meningiomas, have been reported in MEN 1 patients (Table 408-1). Meningiomas are found in <10% of patients with other clinical manifestations of MEN 1 (e.g., primary hyperparathyroidism) for >15 years. The majority of meningiomas are not associated with symptoms, and 60% do not enlarge. The treatment of MEN 1–associated meningiomas is similar to that in non–MEN 1 patients. Lipomas Subcutaneous lipomas occur in >33% of patients with MEN 1 (Table 408-1) and are frequently multiple. In addition, visceral, pleural, or retroperitoneal lipomas may occur in patients with MEN 1. Management is conservative. However, when surgically removed for cosmetic reasons, they typically do not recur.

1	Facial Angiofibromas and Collagenomas The occurrence of multiple facial angiofibromas in patients with MEN 1 may range from >20 to >90%, and occurrence of collagenomas may range from 0 to >70% (Table 408-1). These cutaneous findings may allow presymptomatic diagnosis of MEN 1 in the relatives of a patient with MEN 1. Treatment for these cutaneous lesions is usually not required. Thyroid Tumors Thyroid tumors, including adenomas, colloid goiters, and carcinomas, have been reported to occur in >25% of patients with MEN 1. However, the prevalence of thyroid disorders in the general population is high, and it has been suggested that the association of thyroid abnormalities in patients with MEN 1 may be incidental. The treatment of thyroid tumors in MEN 1 patients is similar to that for non–MEN 1 patients.

1	Genetics and Screening The MEN1 gene is located on chromo some 11q13 and consists of 10 exons, which encode a 610– amino acid protein, menin, that regulates transcription, genome stability, cell division, and proliferation. The pathophysiology of MEN 1 follows the Knudson two-hit hypothesis with a tumor-suppressor role for menin. Inheritance of a germline MEN1 mutation predisposes an individual to developing a tumor that arises following a somatic mutation, which may be a point mutation or more commonly a deletion, leading to loss of heterozygosity (LOH) in the tumor DNA. The germline mutations of the MEN1 gene are scattered throughout the entire 1830-bp coding region and splice sites, and there is no apparent correlation between the location of MEN1 mutations and clinical manifestations of the disorder, in contrast with the situation in patients with MEN 2 (Table 408-1). More than 10% of MEN1 germline mutations arise de novo and may be transmitted to subsequent generations. Some

1	of the disorder, in contrast with the situation in patients with MEN 2 (Table 408-1). More than 10% of MEN1 germline mutations arise de novo and may be transmitted to subsequent generations. Some families with MEN 1 mutations develop parathyroid tumors as the sole endocrinopathy, and this condition is referred to as familial isolated hyperparathyroidism (FIHP). However, between 5 and 25% of patients with MEN 1 do not harbor germline mutations or deletions of the MEN1 gene. Such patients with MEN 1–associated tumors but without MEN1 mutations may represent phenocopies or have mutations involving other genes. Other genes associated with MEN 1–like features include: CDC73, which encodes parafibromin, whose mutations result in the hyperparathyroid-jaw tumor syndrome; the calcium-sensing receptor gene (CaSR), whose mutations result in familial benign hypocalciuric hypercalcemia (FBHH); and the aryl hydrocarbon receptor interacting protein gene (AIP), a tumor suppressor located on

1	receptor gene (CaSR), whose mutations result in familial benign hypocalciuric hypercalcemia (FBHH); and the aryl hydrocarbon receptor interacting protein gene (AIP), a tumor suppressor located on chromosome 11q13 whose mutations are associated with familial isolated pituitary adenomas (FIPA). Genetic testing to determine the MEN1 mutation status in symptomatic family members within a MEN 1 kindred, as well as to all index cases (e.g., patients) with two or more endocrine tumors, is advisable. If an MEN1 mutation is not identified in the index case with two or more endocrine tumors, then clinical and genetic tests for other disorders such as hyperparathyroid-jaw tumor syndrome, FBHH, FIPA, MEN 2, or MEN 4 should be considered, because these patients may represent phenocopies for MEN 1.

1	The current guidelines recommend that MEN1 mutational analysis should be undertaken in: (1) an index case with two or more MEN 1– associated endocrine tumors (e.g., parathyroid, pancreatic, or pituitary tumors); (2) asymptomatic first-degree relatives of a known MEN1 mutation carrier; and (3) first-degree relatives of a MEN1 mutation carrier with symptoms, signs, or biochemical or radiologic evidence for one or more MEN 1–associated tumors. In addition, MEN1 mutational analysis should be considered in patients with suspicious or atypical MEN 1. This would include individuals with parathyroid adenomas before the age of 30 years or multigland parathyroid disease; individuals with gastrinoma or multiple pancreatic NETs at any age; or individuals who have two or more MEN 1–associated tumors that are not part of the classical triad of parathyroid, pancreatic islet, and anterior pituitary tumors (e.g., parathyroid tumor plus adrenal tumor). Family members, including asymptomatic individuals

1	are not part of the classical triad of parathyroid, pancreatic islet, and anterior pituitary tumors (e.g., parathyroid tumor plus adrenal tumor). Family members, including asymptomatic individuals who have been identified to harbor a MEN1 mutation, will require biochemical and radiologic screening (Table 408-3). In contrast, relatives who do not harbor the MEN1 mutation have a risk of developing MEN 1–associated endocrine tumors that is similar to that of the general population; thus, relatives without the MEN1 mutation do not require repeated screening.

1	Mutational analysis in asymptomatic individuals should be undertaken at the earliest opportunity and, if possible, in the first decade of life because tumors have developed in some children by the age of 5 years. Appropriate biochemical and radiologic investigations (Table 408-3) aimed at detecting the development of tumors should then be undertaken in affected individuals. Mutant gene carriers should undergo biochemical screening at least once per annum and also have baseline pituitary and abdominal imaging (e.g., MRI or CT), which should then be repeated at 1to 3-year intervals (Table 408-3). Screening should commence after 5 years of age and should continue for life because the disease may develop as late as the eighth decade. The screening history and physical examination elicit the symptoms and signs of hypercalcemia, nephrolithiasis, peptic ulcer disease, neuroglycopenia, hypopituitarism, galactorrhea and amenorrhea in women, acromegaly, Cushing’s disease, and visual field loss

1	and signs of hypercalcemia, nephrolithiasis, peptic ulcer disease, neuroglycopenia, hypopituitarism, galactorrhea and amenorrhea in women, acromegaly, Cushing’s disease, and visual field loss and the presence of subcutaneous lipomas, angiofibromas, and collagenomas. Biochemical screening should include measurements of serum calcium, PTH, gastrointestinal hormones (e.g., gastrin, insulin with a fasting glucose, glucagon, VIP, PP), chromogranin A, prolactin, and IGF-I in all individuals. More specific endocrine function tests should be undertaken in individuals who have symptoms or signs suggestive of a specific clinical syndrome. Biochemical screening for the development of MEN 1 tumors in asymptomatic members of families with MEN 1 is of great importance to reduce morbidity and mortality from the associated tumors.

1	MULTIPLE ENDOCRINE NEOPLASIA TYPE 2 AND TYPE 3 Clinical Manifestations MEN type 2 (MEN 2), which is also called Sipple’s syndrome, is characterized by the association of medullary thyroid carcinoma (MTC), pheochromocytomas, and parathyroid tumors (Table 408-1). Three clinical variants of MEN 2 are recognized: MEN 2A, MEN 2B, and MTC only. MEN 2A, which is often referred to as MEN 2, is the most common variant. In MEN 2A, MTC is associated with pheochromocytomas in 50% of patients (may be bilateral) and with parathyroid tumors in 20% of patients. MEN 2A may rarely occur in association with Hirschsprung’s disease, caused by the absence of autonomic ganglion cells in the terminal hindgut, resulting in colonic dilatation, severe constipation, and obstruction. MEN 2A may also be associated with cutaneous lichen amyloidosis, which is a pruritic lichenoid lesion that is usually located on the upper back. MEN 2B, which is also referred to as MEN 3, represents 5% of all cases of MEN 2 and is

1	cutaneous lichen amyloidosis, which is a pruritic lichenoid lesion that is usually located on the upper back. MEN 2B, which is also referred to as MEN 3, represents 5% of all cases of MEN 2 and is characterized by the occurrence of MTC and pheochromocytoma in association with a Marfanoid habitus; mucosal neuromas of the lips, tongue, and eyelids; medullated corneal fibers; and intestinal autonomic ganglion dysfunction leading to multiple diverticulae and megacolon. Parathyroid tumors do not usually occur in MEN 2B. MTC only (FMTC) is a variant in which MTC is the sole manifestation of the syndrome. However, the distinction between aAdapted from American Thyroid Association Guidelines, RT Kloos et al: Thyroid 6:565, 2009. bRisk for early development of metastasis and aggressive growth of medullary thyroid cancer: ++++, highest; +++, high; ++, intermediate; and +, lowest. cMutations associated with MEN 2A (or medullary thyroid carcinoma only). dConsider surgery at 5 years or later if

1	medullary thyroid cancer: ++++, highest; +++, high; ++, intermediate; and +, lowest. cMutations associated with MEN 2A (or medullary thyroid carcinoma only). dConsider surgery at 5 years or later if serum calcitonin is normal, neck ultrasound is normal, and there is a less aggressive family history and family preference. eConsider surgery before 5 years or later if serum calcitonin is normal, neck ultrasound is normal, and there is a less aggressive family history and family preference. fMutations associated with MEN 2B (MEN 3). gNot required because PHPT is not a feature of MEN 2B (MEN 3).

1	Abbreviations: ASAP, as soon as possible; MEN, multiple endocrine neoplasia; PHPT, primary hyperparathyroidism. FMTC and MEN 2A is difficult and should only be considered if there are at least four family members above the age of 50 years who are affected by MTC but not pheochromocytomas or primary hyperparathyroidism. All of the MEN 2 variants are due to mutations of the rearranged during transfection (RET) protooncogene, which encodes a TKR. Moreover, there is a correlation between the locations of RET mutations and MEN 2 variants. Thus, ~95% of MEN 2A patients have mutations involving the cysteine-rich extracellular domain, with mutations of codon 634 accounting for ~85% of MEN 2A mutations; FMTC patients also have mutations of the cysteine-rich extracellular domain, with most mutations occurring in codon 618. In contrast, ~95% of MEN 2B/MEN 3 patients have mutations of codon 918 of the intracellular tyrosine kinase domain (Table 408-1 and Table 408-4).

1	Medullary Thyroid Carcinoma MTC is the most common feature of MEN 2A and MEN 2B and occurs in almost all affected individuals. MTC represents 5–10% of all thyroid gland carcinomas, and 20% of MTC patients have a family history of the disorder. The use of RET mutational analysis to identify family members at risk for hereditary forms of MTC has altered the presentation of MTC from that of symptomatic tumors to a preclinical disease for which prophylactic thyroidectomy (Table 408-4) is undertaken to improve the prognosis and ideally result in cure. However, in patients who do not have a known family history of MEN 2A, FMTC, or MEN 2B, and therefore have not had RET mutational analysis, MTC may present as a palpable mass in the neck, which may be asymptomatic or associated with symptoms of pressure or dysphagia in >15% of patients. Diarrhea occurs in 30% of patients and is associated either with elevated circulating concentrations of calcitonin or tumor-related secretion of serotonin and

1	or dysphagia in >15% of patients. Diarrhea occurs in 30% of patients and is associated either with elevated circulating concentrations of calcitonin or tumor-related secretion of serotonin and prostaglandins. Some patients may also experience flushing. In addition, ectopic ACTH production by MTC may cause Cushing’s syndrome. The diagnosis of MTC relies on the demonstration of hypercalcitoninemia (>90 pg/mL in the basal state); stimulation tests using IV pentagastrin (0.5 mg/kg) and or calcium infusion (2 mg/kg) are rarely used now, reflecting improvements in the assay for calcitonin. Neck ultrasonography with fine-needle aspiration of the nodules can confirm the diagnosis. Radionucleotide thyroid scans may reveal MTC tumors as “cold” nodules. Radiography may reveal dense irregular calcification within the involved portions of the thyroid gland and in lymph nodes involved with metastases. Positron emission tomography (PET) may help to identify the MTC and metastases (Fig. 408-2).

1	within the involved portions of the thyroid gland and in lymph nodes involved with metastases. Positron emission tomography (PET) may help to identify the MTC and metastases (Fig. 408-2). Metastases of MTC usually occur to the cervical lymph nodes in the early stages and to the mediastinal nodes, lung, liver, trachea, adrenal, esophagus, and bone in later stages. Elevations in serum calcitonin concentrations are often the first sign of recurrence or persistent disease, and the serum calcitonin doubling time is useful for determining prognosis. MTC can have an aggressive clinical course, with early metastases and death in approximately 10% of patients. A family history of aggressive MTC or MEN 2B may be elicited.

1	Individuals with RET mutations who do not have clinical manifestations of MTC should be offered prophylactic surgery between the ages of <1 and 5 years. The timing of surgery will depend on the type of RET mutation and its associated risk for early development, metastasis, and aggressive growth of MTC (Table 408-4). Such patients should have a total thyroidectomy with a systematic central neck dissection to remove occult nodal metastasis, although FIGURE 408-2 Fluorodeoxyglucose (FDG) positron emission tomography scan in a patient with multiple endocrine neoplasia type 2A, showing medullary thyroid cancer (MTC) with hepatic and skeletal (left arm) metastasis and a left adrenal pheochromocytoma. Note the presence of excreted FDG compound in the bladder.

1	(Reproduced with permission from A Naziat et al: Clin Endocrinol [Oxf] 78:966, 2013.) 2342 the value of undertaking a central neck dissection has been subject to debate. Prophylactic thyroidectomy, with life-long thyroxine replacement, has dramatically improved outcomes in patients with MEN 2 and MEN 3, such that ~90% of young patients with RET mutations who had a prophylactic thyroidectomy have no evidence of persistent or recurrent MTC at 7 years after surgery. In patients with clinically evident MTC, a total thyroidectomy with bilateral central resection is recommended, and an ipsilateral lateral neck dissection should be undertaken if the primary tumor is >1 cm in size or there is evidence of nodal metastasis in the central neck. Surgery is the only curative therapy for MTC. The 10-year survival in patients with metastatic MTC is ~20%. For inoperable MTC or metastatic disease, the tyrosine kinase inhibitors, vandetanib and cabozantinib, have improved the progression-free survival

1	survival in patients with metastatic MTC is ~20%. For inoperable MTC or metastatic disease, the tyrosine kinase inhibitors, vandetanib and cabozantinib, have improved the progression-free survival times. Other types of chemotherapy are of limited efficacy, but radiotherapy may help to palliate local disease.

1	Pheochromocytoma (See also Chap. 407) These noradrenalineand adrenaline-secreting tumors occur in >50% of patients with MEN 2A and MEN 2B and are a major cause of morbidity and mortality. Patients may have symptoms and signs of catecholamine secretion (e.g., headaches, palpitations, sweating, poorly controlled hypertension), or they may be asymptomatic with detection through biochemical screening based on a history of familial MEN 2A, MEN 2B, or MTC. Pheochromocytomas in patients with MEN 2A and MEN 2B differ significantly in distribution when compared with patients without MEN 2A and MEN 2B. Extra-adrenal pheochromocytomas, which occur in 10% of patients without MEN 2A and MEN 2B, are observed rarely in patients with MEN 2A and MEN 2B. Malignant pheochromocytomas are much less common in patients with MEN 2A and MEN 2B. The biochemical and radiologic investigation of pheochromocytoma in patients with MEN 2A and MEN 2B is similar to that in non– MEN 2 patients and includes the

1	in patients with MEN 2A and MEN 2B. The biochemical and radiologic investigation of pheochromocytoma in patients with MEN 2A and MEN 2B is similar to that in non– MEN 2 patients and includes the measurement of plasma (obtained from supine patients) and urinary free fractionated metanephrines (e.g., normetanephrine and metanephrines measured separately), CT or MRI scanning, radionuclide scanning with meta-iodo-(123I or 131I)-benzyl guanidine (MIBG), and PET using (18F)-fluorodopamine or (18F)-fluoro-2-dexoxy-d-glucose (Fig. 408-2).

1	Surgical removal of pheochromocytoma, using α and β adrenoreceptor blockade before and during the operation, is the recommended treatment. Endoscopic adrenal-sparing surgery, which decreases postoperative morbidity, hospital stay, and expense, as opposed to open surgery, has become the method of choice. Parathyroid Tumors (See also Chap. 424) Parathyroid tumors occur in 10–25% of patients with MEN 2A. However, >50% of these patients do not have hypercalcemia. The presence of abnormally enlarged parathyroids, which are unusually hyperplastic, is often seen in the normocalcemic patient undergoing thyroidectomy for MTC. The biochemical investigation and treatment of hypercalcemic patients with MEN 2A is similar to that of patients with MEN 1.

1	Genetics and Screening To date, approximately 50 different RET mutations have been reported, and these are located in exons 5, 8, 10, 11, 13, 14, 15, and 16. RET germline mutations are detected in >95% of MEN 2A, FMTC, and MEN 2B families, with Cys634Arg being most common in MEN 2A, Cys618Arg being most common in FMTC, and Met918Thr being most common in MEN 2B (Tables 408-1 and 408-4). Between 5 and 10% of patients with MTC or MEN 2A– associated tumors have de novo RET germline mutations, and ~50% of patients with MEN 2B have de novo RET germline mutations. These de novo RET germline mutations always occur on the paternal allele. Approximately 5% of patients with sporadic pheochromocytoma have a germline RET mutation, but such germline RET mutations do not appear to be associated with sporadic primary hyperparathyroidism.

1	Thus, RET mutational analysis should be performed in: (1) all patients with MTC who have a family history of tumors associated with MEN 2, FMTC, or MEN 3, such that the diagnosis can be confirmed and genetic testing offered to asymptomatic relatives; (2) all patients with MTC and pheochromocytoma without a known family history of MEN 2 or MEN 3; (3) all patients with MTC, but without a family history of MEN 2, FMTC, or MEN 3, because these patients may have a de novo germline RET mutations; (4) all patients with bilateral pheochromocytoma; and (5) patients with unilateral pheochromocytoma, particularly if this occurs with increased calcitonin levels.

1	Screening for MEN 2/MEN 3–associated tumors in patients with RET germline mutations should be undertaken annually and include serum calcitonin measurements, a neck ultrasound for MTC, plasma and 24-h urinary fractionated metanephrines for pheochromocytoma, and albumin-corrected serum calcium or ionized calcium with PTH for primary hyperparathyroidism. In patients with MEN 2–associated RET mutations, screening for MTC should begin by 3 to 5 years; for pheochromocytoma by 20 years; and for primary hyperparathyroidism by 20 years of age (Table 408-4).

1	MULTIPLE ENDOCRINE NEOPLASIA TYPE 4 Clinical Manifestations Patients with MEN 1–associated tumors, such as parathyroid adenomas, pituitary adenomas, and pancreatic NETs, occurring in association with gonadal, adrenal, renal, and thyroid tumors have been reported to have mutations of the gene encoding the 196–amino acid cyclin-dependent kinase inhibitor (CK1) p27 kip1 (CDNKIB). Such families with MEN 1–associated tumors and CDNKIB mutations are designated to have MEN 4 (Table 408-1). The investigations and treatments for the MEN 4–associated tumors are similar to those for MEN 1 and non–MEN 1 tumors.

1	Genetics and Screening To date, eight different MEN 4–associ ated mutations of CDNKIB, which is located on chromosome 12p13, have been reported, and all of these are associated with a loss of function. These MEN 4 patients may represent ~3% of the 5–10% of patients with MEN 1 who do not have mutations of the MEN1 gene. Germline CDNKIB mutations may rarely be found in patients with sporadic (i.e., nonfamilial) forms of primary hyperparathyroidism.

1	HYPERPARATHYROIDISM-JAW TUMOR SYNDROME (SEE ALSO CHAP. 424) Clinical Manifestations Hyperparathyroidism-jaw tumor (HPT-JT) syndrome is an autosomal dominant disorder characterized by the development of parathyroid tumors (15% are carcinomas) and fibroosseous jaw tumors. In addition, some patients may also develop Wilms’ tumors, renal cysts, renal hematomas, renal cortical adenomas, papillary renal cell carcinomas, pancreatic adenocarcinomas, uterine tumors, testicular mixed germ cell tumors with a major seminoma component, and Hürthle cell thyroid adenomas. The parathyroid tumors may occur in isolation and without any evidence of jaw tumors, and this may cause confusion with other hereditary hypercalcemic disorders, such as MEN 1. However, genetic testing to identify the causative mutation will help to establish the correct diagnosis. The investigation and treatment for HPT-JT-associated tumors are similar to those in non-HPT-JT patients, except that early parathyroidectomy is

1	mutation will help to establish the correct diagnosis. The investigation and treatment for HPT-JT-associated tumors are similar to those in non-HPT-JT patients, except that early parathyroidectomy is advisable because of the increased frequency of parathyroid carcinoma.

1	Genetics and Screening The gene that causes HPT-JT is located on chromosome 1q31.2 and encodes a 531–amino acid protein, parafibromin (Table 408-2). Parafibromin is also referred to as cell division cycle protein 73 (CDC73) and has a role in transcription. Genetic testing in families helps to identify mutation carriers who should be periodically screened for the development of tumors (Table 408-5). VON HIPPEL-LINDAU DISEASE (SEE ALSO CHAP. 407) Clinical Manifestations von Hippel-Lindau (VHL) disease is an autosomal dominant disorder characterized by hemangioblastomas of the retina and CNS; cysts involving the kidneys, pancreas, and epididymis; renal cell carcinomas; pheochromocytomas; and pancreatic islet cell tumors. The retinal and CNS hemangioblastomas are benign vascular tumors that may be multiple; those in the CNS may cause symptoms Parathyroid Serum Ca, PTH 6–12 months Ossifying jaw fibroma Panoramic jaw x-ray with neck 5 years shieldingc

1	Parathyroid Serum Ca, PTH 6–12 months Ossifying jaw fibroma Panoramic jaw x-ray with neck 5 years shieldingc Renal Abdominal MRIc,d 5 years aScreening for most common HPT-JT–associated tumors is considered. Assessment for other reported tumor types may be indicated (e.g., pancreatic, thyroid, testicular tumors). bFrequency of repeating test after baseline tests performed. cX-rays and imaging involving ionizing radiation should ideally be avoided to minimize risk of generating subsequent mutations. dUltrasound scan recommended if MRI unavailable. eSuch selective pelvic imaging should be considered after obtaining a detailed menstrual history. Abbreviations: Ca, calcium; D&C, dilatation and curettage; HPT-JT, hyperparathyroidismjaw tumor syndrome; MRI, magnetic resonance imaging; PTH, parathyroid hormone. Source: Reproduced from PJ Newey et al: Hum Mutat 31:295, 2010.

1	by compressing adjacent structures and/or increasing intracranial pressure. In the CNS, the cerebellum and spinal cord are the most frequently involved sites. The renal abnormalities consist of cysts and carcinomas, and the lifetime risk of a renal cell carcinoma (RCC) in VHL is 70%. The endocrine tumors in VHL consist of pheochromocytomas and pancreatic islet cell tumors. The clinical presentation of pheochromocytoma in VHL disease is similar to that in sporadic cases, except there is a higher frequency of bilateral or multiple tumors, which may involve extra-adrenal sites in VHL disease. The most frequent pancreatic lesions in VHL are multiple cyst-adenomas, which rarely cause clinical disease. However, nonsecreting pancreatic islet cell tumors occur in <10% of VHL patients, who are usually asymptomatic. The pancreatic tumors in these patients are often detected by regular screening using abdominal imaging. Pheochromocytomas should be investigated and treated as described earlier

1	asymptomatic. The pancreatic tumors in these patients are often detected by regular screening using abdominal imaging. Pheochromocytomas should be investigated and treated as described earlier for MEN 2. The pancreatic islet cell tumors frequently become malignant, and early surgery is recommended.

1	Genetics and Screening The VHL gene, which is located on chro mosome 3p26-p25, is widely expressed in human tissues and encodes a 213–amino acid protein (pVHL) (Table 408-2). A wide variety of germline VHL mutations have been identified. VHL acts as a tumor-suppressor gene. A correlation appears to exist between the type of mutation and the clinical phenotype; large deletions and protein-truncating mutations are associated with a low incidence of pheochromocytomas, whereas some missense mutations in VHL patients are associated with pheochromocytoma (referred to as VHL type 2C). Other missense mutations may be associated with hemangioblastomas and RCC but not pheochromocytoma (referred to as VHL type 1), whereas distinct missense mutations are associated with hemangioblastomas, RCC, and pheochromocytoma (VHL type 2B). VHL type 2A, which refers to the occurrence of hemangioblastomas and pheochromocytoma without RCC, is associated with rare missense mutations. The basis for these complex

1	(VHL type 2B). VHL type 2A, which refers to the occurrence of hemangioblastomas and pheochromocytoma without RCC, is associated with rare missense mutations. The basis for these complex genotype-phenotype relationships remains to be elucidated. One major function of pVHL, which is also referred to as elongin, is to downregulate the expression of vascular endothelial growth factor (VEGF) and other hypoxia-inducible mRNAs. Thus, pVHL, in complex with other proteins, regulates the expression of hypoxia-inducible factors (HIF-1 and HIF-2) such that loss of functional pVHL leads to a stabilization of the HIF protein complexes, resulting in VEGF overexpression and tumor angiogenesis. Screening for the development of pheochromocytomas and pancreatic islet cell tumors is as described earlier for MEN 2 and MEN 1, respectively (Tables 408-3 and 408-4).

1	NEUROFIBROMATOSIS Clinical Manifestations Neurofibromatosis type 1 (NF1), which is also referred to as von Recklinghausen’s disease, is an autosomal dominant disorder characterized by the following manifestations: neurologic (e.g., peripheral and spinal neurofibromas); ophthalmologic (e.g., optic gliomas and iris hamartomas such as Lisch nodules); dermatologic 2343 (e.g., café au lait macules); skeletal (e.g., scoliosis, macrocephaly, short stature, and pseudoarthrosis); vascular (e.g., stenoses of renal and intra-cranial arteries); and endocrine (e.g., pheochromocytoma, carcinoid tumors, and precocious puberty). Neurofibromatosis type 2 (NF2) is also an autosomal dominant disorder but is characterized by the development of bilateral vestibular schwannomas (acoustic neuromas) that lead to deafness, tinnitus, or vertigo. Some patients with NF2 also develop meningiomas, spinal schwannomas, peripheral nerve neurofibromas, and café au lait macules. Endocrine abnormalities are not found in

1	deafness, tinnitus, or vertigo. Some patients with NF2 also develop meningiomas, spinal schwannomas, peripheral nerve neurofibromas, and café au lait macules. Endocrine abnormalities are not found in NF2 and are associated solely with NF1. Pheochromocytomas, carcinoid tumors, and precocious puberty occur in about 1% of patients with NF1, and growth hormone deficiency has been also reported. The features of pheochromocytomas in NF1 are similar to those in nonNF1 patients, with 90% of tumors being located within the adrenal medulla and the remaining 10% at an extra-adrenal location, which often involves the para-aortic region. Primary carcinoid tumors are often periampullary and may also occur in the ileum but rarely in the pancreas, thyroid, or lungs. Hepatic metastases are associated with symptoms of the carcinoid syndrome, which include flushing, diarrhea, bronchoconstriction, and tricuspid valve disease. Precocious puberty is usually associated with the extension of an optic glioma

1	symptoms of the carcinoid syndrome, which include flushing, diarrhea, bronchoconstriction, and tricuspid valve disease. Precocious puberty is usually associated with the extension of an optic glioma into the hypothalamus with resultant early activation of gonadotropin-releasing hormone secretion. Growth hormone deficiency has also been observed in some NF1 patients, who may or may not have optic chiasmal gliomas, but it is important to note that short stature is frequent in the absence of growth hormone deficiency in patients with NF1. The investigation and treatment for tumors are similar to those undertaken for each respective tumor type in non-NF1 patients. Genetics and Screening The NF1 gene, which is located on chro mosome 17q11.2 and acts as a tumor suppressor, consists of 60 exons that span more than 350 kb of genomic DNA (Table 408-2). Mutations in NF1 are of diverse types and are scattered throughout the exons. The NF1 gene product is the protein neurofibromin, which has

1	that span more than 350 kb of genomic DNA (Table 408-2). Mutations in NF1 are of diverse types and are scattered throughout the exons. The NF1 gene product is the protein neurofibromin, which has homologies to the p120GAP (GTPase activating protein) and acts on p21ras by converting the active GTP bound form to its inactive GDP form. Mutations of NF1 impair this downregulation of the p21ras signaling pathways, which in turn results in abnormal cell proliferation. Screening for the development of pheochromocytomas and carcinoid tumors is as described earlier for MEN 2 and MEN 1, respectively (Tables 408-3 and 408-4).

1	CARNEY COMPLEX Clinical Manifestations Carney complex (CNC) is an autosomal dominant disorder characterized by spotty skin pigmentation (usually of the face, labia, and conjunctiva), myxomas (usually of the eyelids and heart, but also the tongue, palate, breast, and skin), psammomatous melanotic schwannomas (usually of the sympathetic nerve chain and upper gastrointestinal tract), and endocrine tumors that involve the adrenals, Sertoli cells, somatotropes, thyroid, and ovary. Cushing’s syndrome, the result of primary pigmented nodular adrenal disease (PPNAD), is the most common endocrine manifestation of CNC and may occur in one-third of patients. Patients with CNC and Cushing’s syndrome often have an atypical appearance by being thin (as opposed to having truncal obesity). In addition, they may have short stature, muscle and skin wasting, and osteoporosis. These patients often have levels of urinary free cortisol that are normal or increased only marginally. Cortisol production may

1	they may have short stature, muscle and skin wasting, and osteoporosis. These patients often have levels of urinary free cortisol that are normal or increased only marginally. Cortisol production may fluctuate periodically with days or weeks of hypercortisolism; this pattern is referred to as “periodic Cushing’s syndrome.” Patients with Cushing’s syndrome usually have loss of the circadian rhythm of cortisol production. Acromegaly, the result of a somatotrope tumor, affects ~10% of patients with CNC. Testicular tumors may also occur in one-third of patients with CNC. These may either be large-cell calcifying Sertoli cell tumors, adrenocortical rests, or Leydig cell tumors. The Sertoli cell tumors occasionally may be estrogen-secreting and lead to precocious puberty or gynecomastia. Some patients with CNC have been reported to develop thyroid follicular tumors, ovarian cysts, or breast duct adenomas.

1	Genetics and Screening CNC type 1 (CNC1) is due to mutations of the protein kinase A (PKA) regulatory subunit 1 α (R1α) (PPKAR1A), a tumor suppressor, whose gene is located on chromosome 17q.24.2 (Table 408-2). The gene causing CNC type 2 (CNC2) is located on chromosome 2p16 and has not yet been identified. It is interesting to note, however, that some tumors do not show LOH of 2p16 but instead show genomic instability, suggesting that this CNC gene may not be a tumor suppressor. Screening and treatment of these endocrine tumors are similar to those described earlier for patients with MEN 1 and MEN 2 (Tables 408-3 and 408-4).

1	COWDEN’S SYNDROME Clinical Manifestations Multiple hamartomatous lesions, especially of the skin, mucous membranes (e.g., buccal, intestinal, and colonic), breast, and thyroid are characteristic of Cowden’s (CWD) syndrome, which is an autosomal dominant disorder. Thyroid abnormalities occur in two-thirds of patients with CWD syndrome, and these usually consist of multinodular goiters or benign adenomas, although <10% of patients may have a follicular thyroid carcinoma. Breast abnormalities occur in >75% of patients and consist of either fibrocystic disease or adenocarcinomas. The investigation and treatment for CWD tumors are similar to those undertaken for non-CWD patients.

1	Genetics and Screening CWD syndrome is genetically heterogenous, and six types (CWD1–6) are recognized (Table 408-2). CWD is due to mutations of the phosphate and tensin homologue deleted on chromosome 10 (PTEN) gene, located on chromosome 10q23.31. CWD2 is caused by mutations of the succinate dehydrogenase subunit B (SDHB) gene, located on chromosome 1p36.13; and CWD3 is caused by mutations of the SDHD gene, located on chromosome 11q13.1. SDHB and SDHD mutations are also associated with pheochromocytoma. CWD4 is caused by hypermethylation of the Killin (KLLN) gene, the promoter of which shares the same transcription site as PTEN on chromosome 10q23.31. CWD5 is caused by mutations of the phosphatidylinositol 3-kinase catalytic alpha (PIK3CA) gene on chromosome 3q26.32, and CWD6 is caused by mutations of the V-Akt murine thymoma viral oncogene homolog 1 (AKT1) gene on chromosome 14q32.33. Screening for thyroid abnormalities entails neck ultrasonography and fine-needle aspiration with

1	by mutations of the V-Akt murine thymoma viral oncogene homolog 1 (AKT1) gene on chromosome 14q32.33. Screening for thyroid abnormalities entails neck ultrasonography and fine-needle aspiration with analysis of cell cytology.

1	MCCUNE-ALBRIGHT SYNDROME (SEE ALSO CHAP. 426e) Clinical Manifestations McCune-Albright syndrome (MAS) is characterized by the triad of polyostotic fibrous dysplasia, which may be associated with hypophosphatemic rickets; café au lait skin pigmentation; and peripheral precocious puberty; other endocrine abnormalities include thyrotoxicosis, which may be associated with a multinodular goiter, somatotrope tumors, and Cushing’s syndrome (due to adrenal tumors). Investigation and treatment for each endocrinopathy are similar to those used in patients without MAS.

1	Genetics and Screening MAS is a disorder of mosaicism that results from postzygotic somatic cell mutations of the G protein α stimulating subunit (Gsα), encoded by the GNAS1 gene, located on chromosome 20q13.32 (Table 408-2). The Gsα mutations, which include Arg201Cys, Arg201His, Glu227Arg, or Glu227His, are activating and are found only in cells of the abnormal tissues. Screening for hyperfunction of relevant endocrine glands and development of hypophosphatemia, which may be associated with elevated serum fibroblast growth factor 23 (FGF23) concentrations, is undertaken in MAS patients. The author is grateful to the Medical Research Council (UK) for support and to Mrs. Tracey Walker for typing the manuscript. Peter A. Gottlieb

1	Polyglandular deficiency syndromes have been given many different names, reflecting the wide spectrum of disorders that have been associated with these syndromes and the heterogeneity of their clinical presentations. The name used in this chapter for this group of disorders is autoimmune polyendocrine syndrome (APS). In general, these disorders are divided into two major categories, APS type 1 (APS-1) and APS type 2 (APS-2). Some groups have further subdivided APS-2 into APS type 3 (APS-3) and APS type 4 (APS-4) depending on the type of autoimmunity involved. For the most part, this additional classification does not clarify our understanding of disease pathogenesis or prevention of complications in individual patients. Importantly, there are many nonendocrine disease associations included in these syndromes, suggesting that although the underlying autoimmune disorder predominantly involves endocrine targets, it does not exclude other tissues. The disease associations found in APS-1

1	in these syndromes, suggesting that although the underlying autoimmune disorder predominantly involves endocrine targets, it does not exclude other tissues. The disease associations found in APS-1 and APS-2 are summarized in Table 409-1. Understanding these syndromes and their disease manifestations can lead to early diagnosis and treatment of additional disorders in patients and their family members.

1	Abbreviations: DIDMOAD, diabetes insipidus, diabetes mellitus, progressive bilateral optic atrophy, and sensorineural deafness; POEMS, polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes. Note: Italics denote less common disorders.

1	APS-1 (Online Mendelian Inheritance in Man [OMIM] 240300) has also been called autoimmune polyendocrinopathy–candidiasis– ectodermal dystrophy (APECED). Mucocutaneous candidiasis, hypoparathyroidism, and Addison’s disease form the three major components of this disorder. However, as summarized in Table 409-1, many other organ systems can be involved over time. APS-1 is rare, with fewer than 500 cases reported in the literature. It is an autosomal recessive disorder caused by mutations in the AIRE gene (autoimmune regulator gene) found on chromosome 21. This gene is most highly expressed in thymic medullary epithelial cells (mTECs) where it appears to control the expression of tissue-specific self-antigens (e.g., insulin). Deletion of this regulator leads to decreased expression of tissue-specific self-antigens and is hypothesized to allow autoreactive T cells to avoid clonal deletion, which normally occurs during T cell maturation in the thymus. The AIRE gene is also expressed in

1	self-antigens and is hypothesized to allow autoreactive T cells to avoid clonal deletion, which normally occurs during T cell maturation in the thymus. The AIRE gene is also expressed in epithelial cells found in peripheral lymphoid organs, but its role in these extrathymic cells remains controversial. A number of mutations have been described in this gene, and there is a higher frequency within certain ethnic groups including Iranian Jews, Sardinians, Finns, Norwegians, and Irish.

1	Clinical Manifestations APS-1 develops very early in life, often in infancy (Table 409-2). Chronic mucocutaneous candidiasis without signs of systemic disease is often the first manifestation. It affects the mouth and nails more frequently than the skin and esophagus. Chronic oral candidiasis can result in atrophic disease with areas suggestive of leukoplakia, which can pose a risk for future carcinoma. The etiology is associated with anticytokine autoantibodies (anti-IL-17A, -IL-17F, and -IL-22) related to T helper (TH) 17 T cells and depressed production of these cytokines by peripheral blood mononuclear cells. Hypoparathyroidism usually develops next, followed by adrenal insufficiency. The time from development of one component of the disorder to the next can be many years, and the order of disease appearance is variable.

1	Chronic candidiasis is nearly always present and is not very responsive to treatment. Hypoparathyroidism is found in >85% of cases, and Addison’s disease is found in nearly 80%. Gonadal failure appears to affect women more than men (70% vs 25%, respectively), and hypoplasia of the dental enamel also occurs frequently (77% of patients). Other endocrine disorders that occur less frequently include type 1 diabetes (23%) and autoimmune thyroid disease (18%). Nonendocrine manifestations that present less frequently include alopecia (40%), vitiligo (26%), intestinal malabsorption (18%), pernicious anemia (31%), chronic active hepatitis (17%), and nail dystrophy. An unusual and debilitating manifestation of the disorder is the development of refractory diarrhea/obstipation that may be related to autoantibody-mediated destruction of enterochromaffin or enterochromaffin-like cells.

1	Early onset: infancy Later onset Siblings often affected and at risk Multigenerational Equivalent sex distribution Females > males affected Monogenic: AIRE gene, chromo-Polygenic: HLA, MICA, PTNP22, CTLA4 some 21, autosomal recessive Not HLA associated for entire syn-DR3/DR4 associated; other HLA class III drome, some specific component gene associations noted risk Autoantibodies to type 1 interfer-No autoantibodies to cytokines Autoantibodies to specific target Autoantibodies to specific target organs organs Asplenism No defined immunodeficiency Mucocutaneous candidiasis Association with other nonendocrine Abbreviations: APS, autoimmune polyendocrine syndrome; IL, interleukin. The incidence rates for many of these disorders peak in the first or 2345 second decade of life, but the individual disease components continue to emerge over time. Therefore, prevalence rates may be higher than originally reported.

1	Diagnosis The diagnosis of APS-1 is usually made clinically when two of the three major component disorders are found in an individual patient. Siblings of individuals with APS-1 should be considered affected even if only one component disorder has been detected due to the known inheritance of the syndrome. Genetic analysis of the AIRE gene should be undertaken to identify mutations. Initial sequencing may detect the common mutations, but rare mutations are continually being noted, and an initial negative genetic analysis should not dissuade one from the clinical diagnosis until more extensive DNA sequencing can be performed. Detection of anti–interferon α and anti–interferon ο antibodies can identify nearly 100% of cases with APS-1. The autoantibody arises independent of the type of AIRE gene mutation and is not found in other autoimmune disorders.

1	Diagnosis of each underlying disorder should be done based on their typical clinical presentations (Table 409-3). Mucocutaneous candidiasis may present throughout the gastrointestinal tract, and it may be detected in the oral mucosa or from stool samples. Evaluation by a gastroenterologist to examine the esophagus for candidiasis or secondary stricture may be merited based on symptoms. Other gastrointestinal manifestations of APS-1, including malabsorption and obstipation, may also bring these young patients to the attention of gastroenterologists for first evaluation. Specific physical examination findings of hyperpigmentation, vitiligo, alopecia, tetany, and signs of hyperor hypothyroidism should be considered as signs of development of component disorders.

1	The development of disease-specific autoantibody assays can help confirm disease and also detect risk for future disease. For example, where possible, detection of anticytokine antibodies to interleukin (IL) 17 and IL-22 would confirm the diagnosis of mucocutaneous candidiasis due to APS-1. The presence of anti-21-hydroxylase antibody or anti-17-hydroxylase antibody (which may be found more commonly in adrenal insufficiency associated with APS-1) would confirm the presence or risk for Addison’s disease. Other autoantibodies found in type 1 diabetes (e.g., anti-GAD65), pernicious anemia, and other component conditions should be screened for on a regular basis (6to 12-month intervals depending on the age of the subject).

1	Laboratory tests, including a complete metabolic panel, phosphorous and magnesium, thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH; morning), hemoglobin A1c, plasma vitamin B12 level, and complete blood count with peripheral smear looking for Howell-Jolly bodies (asplenism), should also be performed at these time points. Detection of abnormal physical findings or test results should prompt subsequent examinations of the relevant organ system (e.g., presence of Howell-Jolly bodies indicates need for ultrasound of spleen).

1	Therapy of individual disease components is carried out as outlined in other relevant chapters. Replacement of deficient hormones (e.g., adrenal, pancreas, ovaries/testes) will treat most of the endocrinopathies noted. Several unique issues merit special emphasis. Adrenal insufficiency can be masked by primary hypothyroidism by prolonging the half-life of cortisol. The caveat therefore is that replacement therapy with thyroid hormone can precipitate an adrenal crisis in an undiagnosed individual. Hence, all patients with hypothyroidism and the possibility of APS should be screened for adrenal insufficiency to allow treatment with glucocorticoids prior to the initiation of thyroid hormone replacement. Treatment of mucocutaneous candidiasis with ketoconazole in an individual with subclinical adrenal insufficiency may also precipitate adrenal crisis. Furthermore, mucocutaneous candidiasis may be difficult to eradicate entirely. Severe cases of disease involvement may require systemic

1	adrenal insufficiency may also precipitate adrenal crisis. Furthermore, mucocutaneous candidiasis may be difficult to eradicate entirely. Severe cases of disease involvement may require systemic immunomodulatory therapy, but this is not commonly needed.

1	Addison’s disease Sodium, potassium, ACTH, cortisol, 21and 17-hydroxylase autoantibodies Hypoparathyroidism Serum calcium, phosphate, PTH Male hypogonadism FSH/LH, testosterone Malabsorption Physical examination, anti-IL-17 and anti-IL-22 autoantibodies Mucocutaneous Physical examination, mucosal swab, stool samples Ovarian failure FSH/LH, estradiol Pernicious anemia CBC, vitamin B12 levels Type 1 diabetes Glucose, hemoglobin A1c, diabetes-associated autoantibodies (insulin, GAD65, IA-2, ZnT8) Addison’s disease 21-Hydroxylase autoantibodies, ACTH stimulation testing if positive or hypothyroidism autoantibodies, anti-TSH receptor Ab Cerebellar ataxia Dictated by signs and symptoms of disease Chronic inflamma-Dictated by signs and symptoms of disease tory demyelinating polyneuropathy Hypophysitis Dictated by signs and symptoms of disease, anti-Pit1 Idiopathic heart Dictated by signs and symptoms of disease Myasthenia gravis Dictated by signs and symptoms of disease,

1	Hypophysitis Dictated by signs and symptoms of disease, anti-Pit1 Idiopathic heart Dictated by signs and symptoms of disease Myasthenia gravis Dictated by signs and symptoms of disease, Myocarditis Dictated by signs and symptoms of disease CBC, vitamin B12 levels if positive Serositis Dictated by signs and symptoms of disease Stiff man syndrome Dictated by signs and symptoms of disease Vitiligo Physical examination, NALP-1 polymorphism Abbreviations: Ab, antibody; ACTH, adrenocorticotropic hormone; APS, autoimmune polyendocrine syndrome; CBC, complete blood count; FSH, follicle-stimulating hormone; IL, interleukin; LH, luteinizing hormone; PTH, parathyroid hormone; TSH, thyroid-stimulating hormone.

1	APS-2 (OMIM 269200) is more common than APS-1 with a prevalence of 1 in 100,000. It has a gender bias and occurs more often in female patients with a ratio of at least 3:1 compared to male patients. In contrast to APS-1, APS-2 often has its onset in adulthood with a peak incidence between 20 and 60 years of age. It shows a familial, multigenerational heritage (Table 409-2). The presence of two or more of the following endocrine deficiencies in the same patient defines the presence of APS-2: primary adrenal insufficiency (Addison’s disease; 50–70%), Graves’ disease or autoimmune thyroiditis (15–69%), type 1 diabetes mellitus (T1D; 40–50%), and primary hypogonadism. Frequently associated autoimmune conditions include celiac disease (3–15%), myasthenia gravis, vitiligo, alopecia, serositis, and pernicious anemia. These conditions occur with increased frequency in affected patients but are also are found in their family members (Table 409-3).

1	Genetic Considerations The overwhelming risk factor for APS-2 has been localized to the genes in the human lymphocyte antigen complex on chromosome 6. Primary adrenal insufficiency in APS-2, but not APS-1, is strongly associated with both HLA-DR3 and HLA-DR4. Other class I and class II genes and alleles, such as HLA-B8, HLA-DQ2 and HLA-DQ8, and HLA-DR subtype such as DRB1*0404, appear to contribute to organ-specific disease susceptibility (Table 409-4). HLA-B8and HLA-DR3-associated illnesses include selective IgA deficiency, juvenile dermatomyositis, dermatitis herpetiformis, alopecia, scleroderma, autoimmune thrombocytopenia purpura, hypophysitis, metaphyseal osteopenia, and serositis.

1	Several other immune genes have been proposed to be associated with Addison’s disease and therefore with APS-2 (Table 409-3). The “5.1” allele of a major histocompatibility complex (MHC) gene is an atypical class I HLA molecule MIC-A. The MIC-A5.1 allele has a very strong association with Addison’s disease that is not accounted for by linkage disequilibrium with DR3 or DR4. Its role is complicated because certain HLA class I genes can offset this effect. PTPN22 codes for a polymorphism in a protein tyrosine phosphatase, which acts on intracellular signaling pathways in both T and B lymphocytes. It has been implicated in T1D, Addison’s disease, and other autoimmune conditions. CTLA4 is a receptor on the T cell surface that modulates the activation state of the cell as part of the signal 2 pathway. Polymorphisms of this gene appear to cause downregulation of the cell surface expression of the receptor, leading to decreased T cell activation and proliferation. This appears to contribute

1	pathway. Polymorphisms of this gene appear to cause downregulation of the cell surface expression of the receptor, leading to decreased T cell activation and proliferation. This appears to contribute to disease in Addison’s disease and potentially other components of APS-2. Allelic variants of the IL-2Rα are linked to development of T1D and autoimmune thyroid disease and could contribute to the phenotype of APS-2 in certain individuals.

1	Diagnosis When one of the component disorders is present, a second associated disorder occurs more commonly than in the general population (Table 409-3). There is controversy as to which tests to use and how often to screen individuals for disease. A strong family history of autoimmunity should raise suspicion in an individual with an initial component diagnosis. The development of a rarer form of autoimmunity, such as Addison’s disease, should prompt more extensive screening for other linked disorders compared to the diagnosis of autoimmune thyroid disease, which is relatively common.

1	Circulating autoantibodies, as previously discussed, can precede the development of disease by many years but would allow the clinician to follow the patient and identify the disease onset at its earliest time point (Tables 409-3 and 409-4). For each of the endocrine components of the disorder, appropriate autoantibody assays are listed and, if positive, should prompt physiologic testing to diagnose clinical or subclinical disease. For Addison’s disease, antibodies to 21-hydroxylase antibodies are highly diagnostic for risk of adrenal insufficiency. However, individuals may take many years to develop overt hypoadrenalism. Screening of 21-hydroxylase antibody–positive patients can be performed measuring morning ACTH and cortisol on a yearly basis. Rising ACTH values over time or low morning cortisol in association with signs or symptoms of adrenal insufficiency should prompt testing via the cosyntropin stimulation test (Chap. 406). T1D can be screened for by measuring autoantibodies

1	cortisol in association with signs or symptoms of adrenal insufficiency should prompt testing via the cosyntropin stimulation test (Chap. 406). T1D can be screened for by measuring autoantibodies including anti-insulin, anti-GAD65, anti-IA-2, and anti-ZnT8. Risk for progression to disease can be based on the number of antibodies, and in some cases the titer (insulin autoantibody), as well as other metabolic factors (impaired oral glucose tolerance test). National Institutes of Health–sponsored trial groups such as Type 1 Diabetes TrialNet are screening firstand second-degree family members for these autoantibodies and identifying prediabetic individuals who may qualify for intervention trials to change the course of the disease prior to onset.

1	Abbreviations: APS, autoimmune polyendocrine syndrome; SLE, systemic lupus erythematosus; TSH, thyroid-stimulating hormone.

1	Screening tests for thyroid disease can include anti–thyroid peroxidase (TPO) or anti-thyroglobulin autoantibodies or anti-TSH receptor antibodies for Graves’ disease. Yearly measurements of TSH can then be used to follow these individuals. Celiac disease can be screened for using the anti–tissue transglutaminase (tTg) antibody test. For those <20 years of age, testing every 1–2 years should be performed, whereas less frequent testing is indicated after the age of 20 because the majority of individuals who develop celiac disease have the antibody earlier in life. Positive tTg antibody test results should be confirmed on repeat testing, followed by small-bowel biopsy to document pathologic changes of celiac disease. Many patients have asymptomatic celiac disease that is nevertheless associated with osteopenia and impaired growth. If left untreated, symptomatic celiac disease has been reported to be associated with an increased risk of gastrointestinal malignancy, especially lymphoma.

1	The knowledge of the particular disease associations should guide other autoantibody or laboratory testing. A complete history and physical examination should be performed every 1–3 years including CBC, metabolic panel, TSH, and vitamin B12 levels to screen for most of the possible abnormalities. More specific tests should be based on specific findings from the history and physical.

1	With the exception of Graves’ disease, the management of each of the endocrine components of APS-2 involves hormone replacement and is covered in detail in the chapters on adrenal (Chap. 406), thyroid (Chap. 405), gonadal (Chaps. 411 and 412), and parathyroid disease (Chap. 424). As noted for APS-1, adrenal insufficiency can be masked by primary hypothyroidism and should be considered and treated as discussed above. In patients with T1D, decreasing insulin requirements or hypoglycemia, without obvious secondary causes, may indicate the emergence of adrenal insufficiency. Hypocalcemia in APS-2 patients is more likely due to malabsorption than hypoparathyroidism.

1	Immunotherapy for autoimmune endocrine disease has been reserved for T1D, for the most part, reflecting the lifetime burden of the disease for the individual patient and society. Although several immunotherapies (e.g., modified anti-CD3, rituximab, abatacept) can prolong the honeymoon phase of T1D, none has achieved long-term success. Active research using new approaches and combination therapy may change the treatment of this disease or other autoimmune conditions that share similar pathways. Furthermore, treatment of subclinical disease diagnosed by the presence of autoantibodies may provide a mechanism to preempt the development of overt disease and is the subject of active basic and clinical research.

1	Immune dysregulation, polyendocrinopathy, enteropathy, and X-linked disease (IPEX; OMIM 304790) is a rare X-linked recessive disorder. The disease onset is in infancy and is characterized by severe enteropathy, T1D, and skin disease, as well as variable association with several other autoimmune disorders. Many infants die within the first days of life, but the course is variable, with some children surviving for 12–15 years. Early onset of T1D, often at birth, is highly suggestive of the diagnosis because nearly 80% of IPEX patients develop T1D. Although treatment of the individual disorders can temporarily improve the situation, treatment of the underlying immune deficiency is required and includes immunosuppressive therapy generally followed by hematopoietic stem cell transplantation. Transplantation is the only life-saving form of therapy and can be fully curative by normalizing the imbalanced immune system found in this disorder.

1	2348 IPEX is caused by mutations in the FOXP3 gene, which is also mutated in the Scurfy mouse, an animal model that shares much of the phenotype of IPEX patients. The FOXP3 transcription factor is expressed in regulatory T cells designated CD4+CD25+FOXP3+ (Treg). Lack of this factor causes a profound deficiency of this Treg population and results in rampant autoimmunity due to the lack of peripheral tolerance normally provided by these cells. Certain mutations may lead to varying forms of expression of the full syndrome, and there are rare cases where the FOXP3 gene is intact but other genes involved in this pathway (e.g., CD25, IL-2Rα) may be causative.

1	Thymomas and thymic hyperplasia are associated with several autoimmune diseases, with the most common being myasthenia gravis (44%) and red cell aplasia (20%). Graves’ disease, T1D, and Addison’s disease may also be associated with thymic tumors. Patients with myasthenia gravis and thymoma may have unique anti–acetylcholine receptor autoantibodies. Many thymomas lack AIRE expression within the thymoma, and this could be a potential factor in the development of autoimmunity. In support of this concept, thymoma is the one other disease with “frequent” development of anticytokine antibodies and mucocutaneous candidiasis in adults. The majority of tumors are malignant, and temporary remissions of the autoimmune condition can occur with resection of the tumor.

1	This is a very rare disorder where severe insulin resistance (type B) is caused by the presence of anti-insulin receptor antibodies. It is associated with acanthosis nigricans, which can also be associated with other forms of less severe insulin resistance. About one-third of patients have an associated autoimmune illness such as systemic lupus erythematosus or Sjögren’s syndrome. Therefore, the presence of antinuclear antibodies, elevated erythrocyte sedimentation rate, hyperglobulinemia, leukopenia, and hypocomplementemia may accompany the presentation. The presence of anti-insulin receptor autoantibodies leads to marked insulin resistance, requiring more than 100,000 units of insulin to be given daily with only partial control of hyperglycemia. Patients can also have severe hypoglycemia due to partial activation of the insulin receptor by the antibody. The course of the disease is variable, and several patients have had spontaneous remissions. Therapy targeting B lymphocytes

1	due to partial activation of the insulin receptor by the antibody. The course of the disease is variable, and several patients have had spontaneous remissions. Therapy targeting B lymphocytes including rituximab, cyclophosphamide, and pulse steroids can induce remission of the disease.

1	The insulin autoimmune syndrome, associated with Graves’ disease and methimazole therapy (or other sulfhydryl-containing medications), is of particular interest due to a remarkably strong association with a specific HLA haplotype. Such patients with elevated titers of anti-insulin autoantibodies frequently present with hypoglycemia. In Japan, the disease is restricted to HLA-DR4-positive individuals with DRB1*0406. Curiously, a recent report demonstrated that five out of six Caucasian patients taking lipoic acid (sulfhydryl group) who developed insulin autoimmune syndrome were primarily DRB1*0403 (which is related to DRB1*0406); the sixth was DRB1*0406. In Hirata’s syndrome the anti-insulin autoantibodies are often polyclonal. Discontinuation of the medication generally leads to resolution of the syndrome over time.

1	POEMS (polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes; also known as Crow-Fukase syndrome; OMIM 192240) patients usually present with a progressive sensorimotor polyneuropathy, diabetes mellitus (50%), primary gonadal failure (70%), and a plasma cell dyscrasia with sclerotic bony lesions. Associated findings can be hepatosplenomegaly, lymphadenopathy, and hyper-pigmentation. Patients often present in the fifth to sixth decade of life and have a median survival after diagnosis of less than 3 years. The syndrome is assumed to be secondary to circulating immunoglobulins, but patients have excess vascular endothelial growth factor as well as elevated levels of other inflammatory cytokines such as IL1-β, IL-6, and tumor necrosis factor α. A small series of patients have been treated with thalidomide, leading to a decrease in vascular endothelial growth factor. Hyperglycemia responds to small, subcutaneous doses of insulin. The hypogonadism is due to primary

1	have been treated with thalidomide, leading to a decrease in vascular endothelial growth factor. Hyperglycemia responds to small, subcutaneous doses of insulin. The hypogonadism is due to primary gonadal disease with elevated plasma levels of follicle-stimulating hormone and luteinizing hormone. Temporary resolution of the features of POEMS, including normalization of blood glucose, may occur after radiotherapy for localized plasma cell lesions of bone or after chemotherapy, thalidomide, plasmapheresis, autologous stem cell transplantation, or treatment with all-trans-retinoic acid.

1	Other diseases can exhibit polyendocrine deficiencies, including Kearns-Sayre syndrome, DIDMOAD syndrome (diabetes insipidus, diabetes mellitus, progressive bilateral optic atrophy, and sensorineural deafness; also termed Wolfram’s syndrome), Down’s syndrome or trisomy 21 (OMIM 190685), Turner’s syndrome (monosomy X, 45,X), and congenital rubella.

1	Kearns-Sayre syndrome (OMIM 530000) is a rare mitochondrial DNA disorder characterized by myopathic abnormalities leading to ophthalmoplegia and progressive weakness in association with several endocrine abnormalities, including hypoparathyroidism, primary gonadal failure, diabetes mellitus, and hypopituitarism. Crystalline mitochondrial inclusions are found in muscle biopsy specimens, and such inclusions have also been observed in the cerebellum. Antiparathyroid antibodies have not been described; however, antibodies to the anterior pituitary gland and striated muscle have been identified, and the disease may have autoimmune components. These mitochondrial DNA mutations occur sporadically and do not appear to be associated with a familial syndrome.

1	Wolfram’s syndrome (OMIM 222300, chromosome 4; OMIM 598500, mitochondrial) is a rare autosomal recessive disease that is also called DIDMOAD. Neurologic and psychiatric disturbances are prominent in most patients and can cause severe disability. The disease is caused by defects in wolframin, a 100-kDa transmembrane protein that has been localized to the endoplasmic reticulum and is found in neuronal and neuroendocrine tissue. Its expression induces ion channel activity with a resultant increase in intracellular calcium and may play an important role in intracellular calcium homeostasis. Wolfram’s syndrome appears to be a slowly progressive neurodegenerative process, and there is nonautoimmune selective destruction of the pancreatic beta cells. Diabetes mellitus with an onset in childhood is usually the first manifestation. Diabetes mellitus and optic atrophy are present in all reported cases, but expression of the other features is variable.

1	Down’s syndrome, or trisomy 21 (OMIM 190685), is associated with the development of T1D, thyroiditis, and celiac disease. Patients with Turner’s syndrome also appear to be at increased risk for the development of thyroid disease and celiac disease. It is recommended to screen patients with trisomy 21 and Turner’s syndrome for associated autoimmune diseases on a regular basis. (sex-determining region on the Y chromosome) that encodes an HMG Disorders of Sex Development box transcription factor. SRY is expressed transiently in cells destined to become Sertoli cells and serves as a pivotal switch to establish the testis John C. Achermann, J. Larry Jameson lineage. Mutation of SRY prevents testis development in 46,XY individu-

1	Sex development begins in utero but continues into young adulthood with the achievement of sexual maturity and reproductive capability. The major determinants of sex development can be divided into three components: chromosomal sex, gonadal sex (sex determination), and phenotypic sex (sex differentiation) (Fig. 410-1). Variations at each of these stages can result in disorders (or differences) of sex development (DSDs) (Table 410-1). In the newborn period, approximately 1 in 4000 babies require investigation because of ambiguous (atypical) genitalia. Urgent assessment is required, because some causes such as congenital adrenal hyperplasia (CAH) can be associated with life-threatening adrenal crises. Support for the parents and clear communication about the diagnosis and management options are essential. The involvement of an experienced multidisciplinary team is important for counseling, planning appropriate investigations, and discussing long-term wellbeing. DSDs can also present at

1	are essential. The involvement of an experienced multidisciplinary team is important for counseling, planning appropriate investigations, and discussing long-term wellbeing. DSDs can also present at other ages and to a range of health professionals. Subtler forms of gonadal dysfunction (e.g., Klinefelter’s syndrome [KS], Turner’s syndrome [TS]) often are diagnosed later in life by internists. Because these conditions are associated with a variety of psychological, reproductive, and potential medical consequences, an open dialogue must be established between the patient and health care providers to ensure continuity and attention to these issues.

1	Chromosomal sex, defined by a karyotype, describes the X and/or Y chromosome complement (46,XY; 46,XX) that is established at the time of fertilization. The presence of a normal Y chromosome determines that testis development will occur even in the presence of multiple X chromosomes (e.g., 47,XXY or 48,XXXY). The loss of an X chromosome impairs gonad development (45,X or 45,X/46,XY mosaicism). Fetuses with no X chromosome (45,Y) are not viable. Gonadal sex refers to the histologic and functional characteristics of gonadal tissue as testis or ovary. The embryonic gonad is bipotential and can develop (from ~42 days after conception) into either a testis or an ovary, depending on which genes are expressed (Fig. 410-2). Testis development is initiated by expression of the Y chromosome gene SRY (T, DHT, AMH/MIS)

1	FIGURE 410-1 Sex development can be divided into three major components: chromosomal sex, gonadal sex, and phenotypic sex. DHT, dihydrotestosterone; MIS, müllerian-inhibiting substance also known as anti-müllerian hormone, AMH; T, testosterone.

1	als, whereas translocation of SRY in 46,XX individuals is sufficient to induce testis development and a male phenotype. Other genes are necessary to continue testis development. SOX9 (SRY-related HMG-box gene 9) is upregulated by SRY in the developing testis but is suppressed in the ovary. WT1 (Wilms’ tumor–related gene 1) acts early in the genetic pathway and regulates the transcription of several genes, including SFI (NR5A1), DAX1 (NR0B1), and AMH (encoding müllerian-inhibiting substance [MIS]). SF1 encodes steroidogenic factor 1, a nuclear receptor that functions in cooperation with other transcription factors to regulate a large array of adrenal and gonadal genes, including SOX9 and many genes involved in steroidogenesis. SF1 mutations causing loss of function are found in ~10% of XY patients with gonadal dysgenesis and impaired androgenization. In contrast, duplication of a related gene DAX1 also impairs testis development, revealing the exquisite sensitivity of the

1	~10% of XY patients with gonadal dysgenesis and impaired androgenization. In contrast, duplication of a related gene DAX1 also impairs testis development, revealing the exquisite sensitivity of the testis-determining pathway to gene dosage effects. DAX1 loss-of-function mutations cause adrenal hypoplasia, hypogonadotropic hypogonadism, and testicular dysgenesis. In addition to the genes mentioned above, studies of humans and mice indicate that at least 30 other genes are also involved in gonad development (Fig. 410-2). These genes encode an array of signaling molecules and paracrine growth factors in addition to transcription factors.

1	Although ovarian development once was considered a “default” process, it is now clear that specific genes are expressed during the earliest stages of ovary development. Some of these factors may repress testis development (e.g., WNT4, R-spondin-1) (Fig. 410-2). Once the ovary has formed, additional factors are required for normal follicular development (e.g., follicle-stimulating hormone [FSH] receptor, GDF9). Steroidogenesis in the ovary requires the development of follicles that contain granulosa cells and theca cells surrounding the oocytes (Chap. 412). Thus, there is relatively limited ovarian steroidogenesis until puberty.

1	Germ cells also develop in a sex dimorphic manner. In the developing ovary, primordial germ cells (PGCs) proliferate and enter meiosis, whereas they proliferate and then undergo mitotic arrest in the developing testis. PGC entry into meiosis is initiated by retinoic acid that activates STRA8 (stimulated by retinoic acid 8) and other genes involved in meiosis. The developing testis produces high levels of CYP26B1, an enzyme that degrades retinoic acid, preventing PGC entry into meiosis. Approximately 7 million germ cells are present in the fetal ovary in the second trimester, and 1 million remain at birth. Only 400 are ovulated during a woman’s reproductive life span (Chap. 412).

1	Phenotypic sex refers to the structures of the external and internal genitalia and secondary sex characteristics. The developing testis releases anti-müllerian hormone (AMH; also known as müllerianinhibiting substance [MIS]) from Sertoli cells and testosterone from Leydig cells. AMH is a member of the transforming growth factor (TGF) β family and acts through specific receptors to cause regression of the müllerian structures from 60–80 days after conception. At ~60–140 days after conception, testosterone supports the development of wolffian structures, including the epididymides, vasa deferentia, and seminal vesicles. Testosterone is the precursor for dihydrotestosterone (DHT), a potent androgen that promotes development of the external genitalia, including the penis and scrotum (65–100 days, and thereafter) (Fig. 410-3). The urogenital sinus develops into the prostate and prostatic urethra in the male and into the urethra and lower portion of the vagina in the female. The genital

1	days, and thereafter) (Fig. 410-3). The urogenital sinus develops into the prostate and prostatic urethra in the male and into the urethra and lower portion of the vagina in the female. The genital tubercle becomes the glans penis in the male and the clitoris in the female. The urogenital swellings form the scrotum or the labia majora, and the urethral folds fuse to form the shaft of the penis and the male urethra or the labia minora. In the female, wolffian ducts regress and the müllerian ducts form the

1	Disorders of Sex Development Sex Chromosome DSD 46,XY DSD (see Table 410-3) 46,XX DSD (see Table 410-4) 47,XXY (Klinefelter’s syndrome and Disorders of gonadal (testis) development variants) Complete or partial gonadal dysgenesis (e.g., SRY, SOX9, SF1, 45,X (Turner’s syndrome and variants) WT1, DHH, MAP3K1) 45,X/46,XY mosaicism (mixed gonadal Impaired fetal Leydig cell function (e.g., SF1/NR5A1, CXorf6/ dysgenesis) MAMLD1) 46,XX/46,XY (chimerism/mosaicism) Ovotesticular DSD Testis regression Disorders of androgen biosynthesis LH receptor (LHCGR) Smith-Lemli-Opitz syndrome Steroidogenic acute regulatory (StAR) protein Cholesterol side-chain cleavage (CYP11A1) 3β-Hydroxysteroid dehydrogenase II (HSD3B2) 17α-Hydroxylase/17,20-lyase (CYP17A1) P450 oxidoreductase (POR) Cytochrome b5 (CYB5A) 17β-Hydroxysteroid dehydrogenase III (HSD17B3) 5α-Reductase II (SRD5A2) Aldo-keto reductase 1C2 (AKR1C2)

1	Disorders of androgen action Androgen insensitivity syndrome Drugs and environmental modulators Syndromic associations of male genital development Persistent müllerian duct syndrome Vanishing testis syndrome Isolated hypospadias Congenital hypogonadotropic hypogonadism Cryptorchidism Environmental influences Source: Modified from IA Hughes: Arch Dis Child 91:554, 2006. Disorders of gonadal (ovary) development Gonadal dysgenesis Ovotesticular DSD Testicular DSD (e.g., SRY+, dup SOX9, RSPO1) Maternal Maternal virilizing tumors (e.g., luteomas) Androgenic drugs

1	Maternal Maternal virilizing tumors (e.g., luteomas) Androgenic drugs Syndromic associations (e.g., cloacal anomalies) Müllerian agenesis/hypoplasia (e.g., MRKH) Uterine abnormalities (e.g., MODY5) Vaginal atresia (e.g., McKusick-Kaufman) Labial adhesions fallopian tubes, uterus, and upper segment of the vagina. A female phe-firm (median length 2.5 cm [4 mL volume]; almost always <3.5 cm notype will develop in the absence of the gonad, but estrogen is needed [12 mL]) and typically seem inappropriately small for the degree of for maturation of the uterus and breast at puberty. androgenization. Biopsies are not usually necessary but typically reveal seminiferous tubule hyalinization and azoospermia. Other clinical fea- tures of KS are listed in Table 410-2. Plasma concentrations of FSH and Variations in sex chromosome number and structure can present as luteinizing hormone (LH) are increased in most adults with 47,XXY,

1	DSDs (e.g., 45,X/46,XY). KS (47,XXY) and TS (45,X) do not usually and plasma testosterone is decreased (50–75%), reflecting primary present with genital ambiguity but are associated with gonadal dys-gonadal failure. Estradiol is often increased, likely because of chronic function (Table 410-2). Leydig cell stimulation by LH and aromatization of androstenedione by adipose tissue; the increased ratio of estradiol-to-testosterone KLINEFELTER’S SYNDROME (47,XXY) results in gynecomastia (Chap. 411). Patients with mosaic forms of Pathophysiology The classic form of KS (47,XXY) occurs after meiotic KS have less severe clinical features, have larger testes, and sometimes nondisjunction of the sex chromosomes during gametogenesis (40% achieve spontaneous fertility. during spermatogenesis, 60% during oogenesis) (Chap. 83e). Mosaic forms of KS (46,XY/47,XXY) are thought to result from chromosomal mitotic nondisjunction within the zygote and occur in at least 10% of individuals with this

1	60% during oogenesis) (Chap. 83e). Mosaic forms of KS (46,XY/47,XXY) are thought to result from chromosomal mitotic nondisjunction within the zygote and occur in at least 10% of individuals with this condition. Other chromosomal variants of KS

1	Growth, endocrine function, and bone mineralization should be (e.g., 48,XXYY, 48,XXXY) have been reported but are less common.

1	monitored, especially from adolescence. Educational and psycho-Clinical Features KS is characterized by small testes, infertility, gyne-logical support is important for many individuals with KS. Androgen comastia, tall stature/increased leg length, and hypogonadism in supplementation improves virilization, libido, energy, hypofibrinolyphenotypic males. It has an incidence of at least 1 in 1000 men, but sis, and bone mineralization in men with low testosterone levels but approximately 75% of cases are not diagnosed. Of those who are may occasionally worsen gynecomastia (Chap. 411). Gynecomastia diagnosed, only 10% are identified prepubertally, usually because of can be treated by surgical reduction if it causes concern (Chap. small genitalia or cryptorchidism. Others are diagnosed after puberty, 411). Fertility has been achieved by using in vitro fertilization in usually based on impaired androgenization and/or gynecomastia. men with oligospermia or with intracytoplasmic sperm

1	after puberty, 411). Fertility has been achieved by using in vitro fertilization in usually based on impaired androgenization and/or gynecomastia. men with oligospermia or with intracytoplasmic sperm injection Developmental delay, speech difficulties, and poor motor skills may (ICSI) after retrieval of spermatozoa by testicular sperm extraction be features but are variable, especially in adolescence. Later in life, techniques. In specialized centers, successful spermatozoa retrieval body habitus or infertility leads to the diagnosis. Testes are small and using this technique is possible in >50% of men with nonmosaic

1	WNT4 46,XX 46,XY DMRT 1,2 FIGURE 410-2 The genetic regulation of gonadal development. AMH, anti-müllerian hormone (müllerian-inhibiting substance); ATRX, α-thalassemia, mental retardation on the X; BMP2 and 15, bone morphogenic factors 2 and 15; CBX2, chromobox homologue 2; DAX1, dosage sensitive sex-reversal, adrenal hypoplasia congenita on the X chromosome, gene 1; DHH, desert hedgehog; DHT, dihydrotestosterone; DMRT 1,2, doublesex MAB3-related transcription factor 1,2; FOXL2, forkhead transcription factor L2; GATA4, GATA binding protein 4; GDF9, growth differentiation factor 9; MAMLD1, mastermind-like domain containing 1; MAP3K1, mitogen-activated protein kinase kinase kinase 1; RSPO1, R-spondin 1; SF1, steroidogenic factor 1 (also known as NR5A1); SOX9, SRY-related HMG-box gene 9; SRY, sex-determining region on the Y chromosome; WNT4, wingless-type MMTV integration site 4; WT1, Wilms’ tumor–related gene 1.

1	KS. Results may be better in younger men. After ICSI and embryo transfer, successful pregnancies can be achieved in ~50% of these cases. The risk of transmission of this chromosomal abnormality needs to be considered, and preimplantation screening may be desired, although this outcome is much less common than originally 2351 predicted. Long-term monitoring of men with KS is important given the increased risk of breast cancer, cardiovascular disease, metabolic syndrome, and autoimmune disorders. Because most men with KS are never diagnosed, it is important that all internists consider this diagnosis in men with these features who might be seeking medical advice for other conditions.

1	TURNER’S SYNDROME (GONADAL DYSGENESIS; 45,X) Pathophysiology Approximately one-half of women with TS have a 45,X karyotype, about 20% have 45,X/46,XX mosaicism, and the remainder have structural abnormalities of the X chromosome such as X fragments, isochromosomes, or rings. The clinical features of TS result from haploinsufficiency of multiple X chromosomal genes (e.g., short stature homeobox, SHOX). However, imprinted genes also may be affected when the inherited X has different parental origins.

1	Clinical Features TS is characterized by bilateral streak gonads, primary amenorrhea, short stature, and multiple congenital anomalies in phenotypic females. It affects ~1 in 2500 women and is diagnosed at different ages depending on the dominant clinical features (Table 410-2). Prenatally, a diagnosis of TS usually is made incidentally after chorionic villus sampling or amniocentesis for unrelated reasons such as advanced maternal age. Prenatal ultrasound findings include increased nuchal translucency. The postnatal diagnosis of TS should be considered in female neonates or infants with lymphedema, nuchal folds, low hairline, or left-sided cardiac defects and in girls with unexplained growth failure or pubertal delay. Although limited spontaneous pubertal development occurs in up to 30% of girls with TS (10%, 45,X; 30–40%, 45,X/46,XX) and ~2% reach menarche, the vast majority of women with TS develop complete ovarian insufficiency. Therefore, this diagnosis should be considered in

1	of girls with TS (10%, 45,X; 30–40%, 45,X/46,XX) and ~2% reach menarche, the vast majority of women with TS develop complete ovarian insufficiency. Therefore, this diagnosis should be considered in all women who present with primary or secondary amenorrhea and elevated gonadotropin levels.

1	The management of girls and women with TS requires a multidisciplinary approach because of the number of potentially involved organ systems. Detailed cardiac and renal evaluation should be performed at the time of diagnosis. Individuals with congenital heart Disorders of Sex Development Klinefelter’s syndrome 47,XXY or 46,XY/47,XXY Hyalinized testes Male Male Gynecomastia Small testes, azoospermia, decreased facial and axillary hair, decreased libido, tall stature and increased leg length, decreased penile length, increased risk of breast tumors, thromboembolic disease, learning difficulties, speech delay and decreased verbal IQ, obesity, diabetes mellitus, metabolic syndrome, varicose veins, hypothyroidism, systemic lupus erythematosus, epilepsy Turner’s syndrome 45,X or 45,X/46,XX Streak gonad or Female Hypoplastic female Immature female immature ovary

1	Turner’s syndrome 45,X or 45,X/46,XX Streak gonad or Female Hypoplastic female Immature female immature ovary Infancy: lymphedema, web neck, shield chest, low-set hairline, cardiac defects and coarctation of the aorta, urinary tract malformations, and horseshoe kidney Childhood: short stature, cubitus valgus, short neck, short fourth metacarpals, hypoplastic nails, micrognathia, scoliosis, otitis media and sensorineural hearing loss, ptosis and amblyopia, multiple nevi and keloid formation, autoimmune thyroid disease, visuospatial learning difficulties Adulthood: pubertal failure and primary amenorrhea, hypertension, obesity, dyslipidemia, impaired glucose tolerance and insulin resistance, autoimmune thyroid disease, cardiovascular disease, aortic root dilation, osteoporosis, inflammatory bowel disease, chronic hepatic dysfunction, increased risk of colon cancer, hearing loss 45,X/46,XY mosaicism 45,X/46,XY Testis or streak gonad Variable Variable Usually male

1	Short stature, increased risk of gonadal tumors, some Turner’s syndrome features Ovotesticular DSD (true 46,XX/46,XY Testis and ovary or ovo-Variable Variable Gynecomastia hermaphroditism) Possible increased risk of gonadal tumors Ovary Fallopian tube Uterus Vagina FemaleMaleGonad Mesonephros Mullerian duct.. Wolffian duct Urogenital sinus Epididymis Testis Vas deferens Seminal vesicle Prostate

1	FIGURE 410-3 Sex development. A. Internal urogenital tract. B. External genitalia. (After E Braunwald et al [eds]: Harrison’s Principles of Internal Medicine, 15th ed. New York, McGraw-Hill, 2001.) defects (CHDs) (30%) (bicuspid aortic valve, 30–50%; coarctation of to regimens may be beneficial. Girls with evidence of ovarian the aorta, 30%; aortic root dilation, 5%) require long-term follow-up insufficiency require estrogen replacement to induce breast and by an experienced cardiologist, antibiotic prophylaxis for dental or uterine development, support growth, and maintain bone miner-surgical procedures, and serial magnetic resonance imaging (MRI) alization. Most physicians now initiate low-dose estrogen therapy of aortic root dimensions, because progressive aortic root dilation is (one-tenth to one-eighth of the adult replacement dose) to induce associated with increased risk of aortic dissection. Individuals found puberty at an age-appropriate time (~12 years). Doses of estrogen to

1	to one-eighth of the adult replacement dose) to induce associated with increased risk of aortic dissection. Individuals found puberty at an age-appropriate time (~12 years). Doses of estrogen to have congenital renal and urinary tract malformations (30%) are are increased gradually to allow development over a 2to 4-year at risk for urinary tract infections, hypertension, and nephrocalci-period. Progestins are added later to regulate withdrawal bleeds. nosis. Hypertension can occur independently of cardiac and renal Some women with TS have achieved successful pregnancy after malformations and should be monitored and treated as in other ovum donation and in vitro fertilization but are high risk, and car-patients with essential hypertension. Clitoral enlargement or other diac assessment is required. Long-term follow-up of women with evidence of virilization suggests the presence of covert, translocated TS involves careful surveillance of sex hormone replacement and Y chromosomal material

1	Long-term follow-up of women with evidence of virilization suggests the presence of covert, translocated TS involves careful surveillance of sex hormone replacement and Y chromosomal material and is associated with increased risk of reproductive function, bone mineralization, cardiac function and gonadoblastoma. Regular assessment of thyroid function, weight, aortic root dimensions, blood pressure, weight and glucose tolerdentition, hearing, speech, vision, and educational issues should be ance, hepatic and lipid profiles, thyroid function, and hearing. This performed during childhood. Otitis media and middle-ear disease service is provided by a dedicated TS clinic in some centers. are prevalent in childhood (50–85%), and sensorineural hearing loss becomes progressively common with age (70–90%). Autoimmune hypothyroidism (15–30%) can occur in childhood but has a mean 45,X/46,XY MOSAICISM (MIXED GONADAL DYSGENESIS) age of onset in the third decade. Counseling about long-term The

1	age (70–90%). Autoimmune hypothyroidism (15–30%) can occur in childhood but has a mean 45,X/46,XY MOSAICISM (MIXED GONADAL DYSGENESIS) age of onset in the third decade. Counseling about long-term The phenotype of individuals with 45,X/46,XY mosaicism (sometimes growth and fertility issues should be provided. Patient support called mixed gonadal dysgenesis) can vary considerably. Some have a groups are active throughout the world and can play an invaluable predominantly female phenotype with somatic features of TS, streak role. gonads, and müllerian structures, and are managed as TS with a Y

1	Short stature can be an issue for some girls because untreated chromosome. Most 45,X/46,XY individuals have a male phenotype final height rarely exceeds 150 cm in nonmosaic 45,X TS. High-dose and testes, and the diagnosis is made incidentally after amniocenterecombinant growth hormone stimulates growth rate in children sis or during investigation of infertility. In practice, most newborns with TS and is occasionally combined with low doses of the nonaro-referred for assessment have atypical genitalia and variable somatic matizable anabolic steroid oxandrolone (up to 0.05 mg/kg per day) features. Management is complex and needs to be individualized. A in an older child (>9 years). However, final height increments are female sex-of-rearing is often assigned if uterine structures are present, often about 5–10 cm, and individualization of treatment response gonads are intraabdominal, and phallic development is incomplete. In such situations, gonadectomy usually is considered to prevent

1	often about 5–10 cm, and individualization of treatment response gonads are intraabdominal, and phallic development is incomplete. In such situations, gonadectomy usually is considered to prevent further androgen secretion at puberty and prevent risk of gonadoblastoma (up to 25%). Individuals raised as males usually require reconstructive surgery for hypospadias and removal of dysgenetic or streak gonads if the gonads cannot be brought down into the scrotum. Scrotal testes can be preserved but require regular examination for tumor development and sonography at the time of puberty. Biopsy for carcinoma in situ is recommended in adolescence, and testosterone supplementation may be required to support androgenization in puberty or if low testosterone is detected in adulthood. Height potential is usually attenuated; some children receive recombinant growth hormone using TS protocols. Screening for cardiac, renal, and other TS features should be considered, and psychological support

1	is usually attenuated; some children receive recombinant growth hormone using TS protocols. Screening for cardiac, renal, and other TS features should be considered, and psychological support offered for the family and young person.

1	Ovotesticular DSD (formerly called true hermaphroditism) occurs when both an ovary and a testis—or when an ovotestis—are found in one individual. Most individuals with this diagnosis have a 46,XX karyotype, especially in sub-Saharan Africa, and present with ambiguous genitalia at birth or with breast development and phallic development at puberty. A 46,XX/46,XY chimeric karyotype is less common and has a variable phenotype. Disorders of gonadal and phenotypic sex can result in underandrogenization of individuals with a 46,XY karyotype (46,XY DSD) and the excess androgenization of individuals with a 46,XX karyotype (46,XX DSD) (Table 410-1). These disorders cover a spectrum of phenotypes ranging from “46,XY phenotypic females” or “46,XX phenotypic males” to individuals with atypical genitalia. 46,XY DSD

1	46,XY DSD Underandrogenization of the 46,XY fetus (formerly called male pseudohermaphroditism) reflects defects in androgen production or action. It can result from disorders of testis development, defects of androgen synthesis, or resistance to testosterone and DHT (Table 410-1).

1	Disorders of Testis Development • TesTicular dysgenesis Pure (or complete) gonadal dysgenesis (Swyer’s syndrome) is associated with streak gonads, müllerian structures (due to insufficient AMH/MIS secretion), and a complete absence of androgenization. Phenotypic females with this condition often present because of absent pubertal development and are found to have a 46,XY karyotype. Serum sex steroids, AMH/ MIS, and inhibin B are low, and LH and FSH are elevated. Patients with partial gonadal dysgenesis (dysgenetic testes) may produce enough MIS to regress the uterus and sufficient testosterone for partial androgenization, and therefore usually present in the newborn period with atypical genitalia. Gonadal dysgenesis can result from mutations or deletions of testis-promoting genes (WT1, CBX2, SF1, SRY, SOX9, MAP3K1, DHH, GATA4, ATRX, ARX, DMRT) or duplication of chromosomal loci containing “antitestis” genes (e.g., WNT4/RSPO1, DAX1) (Table 410-3). Among these, deletions or mutations of

1	SF1, SRY, SOX9, MAP3K1, DHH, GATA4, ATRX, ARX, DMRT) or duplication of chromosomal loci containing “antitestis” genes (e.g., WNT4/RSPO1, DAX1) (Table 410-3). Among these, deletions or mutations of SRY and heterozygous mutations of SF1 (NR5A1) appear to be most common but still account collectively for <25% of cases. Associated clinical features may be present, reflecting additional functional roles for these genes. For example, renal dysfunction occurs in patients with specific WT1 mutations (Denys-Drash and Frasier’s syndromes), primary adrenal failure occurs in some patients with SF1 mutations, and severe cartilage abnormalities (campomelic dysplasia) are the predominant clinical feature of SOX9 mutations. A family history of DSD, infertility, or early menopause is important because mutations in SF1/NR5A1 can be inherited from a mother in a sex-limited dominant manner (which can mimic X-linked inheritance). In some cases, a woman may later develop primary ovarian insufficiency

1	mutations in SF1/NR5A1 can be inherited from a mother in a sex-limited dominant manner (which can mimic X-linked inheritance). In some cases, a woman may later develop primary ovarian insufficiency because of the effect of SF1 on the ovary. Intraabdominal dysgenetic testes should be removed to prevent malignancy, and estrogens can be used to induce secondary sex characteristics and uterine development in 46,XY individuals raised as females, if it is felt that a female gender identity is established. Absent (vanishing) testis syndrome (bilateral anorchia) reflects regression 2353 of the testis during development. The etiology is unknown, but the absence of müllerian structures indicates adequate secretion of AMH early in utero. In most cases, androgenization of the external genitalia is either normal or slightly impaired (e.g., small penis, hypospadias). These individuals can be offered testicular prostheses and should receive androgen replacement in adolescence.

1	Disorders of Androgen Synthesis Defects in the pathway that regulates androgen synthesis (Fig. 410-4) cause underandrogenization of the 46,XY fetus (Table 410-1). Müllerian regression is unaffected because Sertoli cell function is preserved. Most of these conditions can present with a spectrum of genital phenotypes, ranging from female-typical external genitalia or clitoromegaly in the more severe situations to penoscrotal hypospadias or a small phallus in others. lH receptor Mutations in the LH receptor (LHCGR) cause Leydig cell hypoplasia and androgen deficiency, due to impaired actions of human chorionic gonadotropin in utero and LH late in gestation and during the neonatal period. As a result, testosterone and DHT synthesis are insufficient for complete androgenization.

1	SteroidoGenic enzyme patHWayS Mutations in steroidogenic acute regulatory protein (StAR) and CYP11A1 affect both adrenal and gonadal steroidogenesis (Fig. 410-4) (Chap. 406). Affected individuals (46,XY) usually have severe early-onset salt-losing adrenal failure and a female phenotype, although later-onset milder variants have been reported. Defects in 3β-hydroxysteroid dehydrogenase type 2 (HSD3β2) also cause adrenal insufficiency in severe cases, but the accumulation of dehydroepiandrosterone (DHEA) has a mild androgenizing effect, resulting in ambiguous genitalia or hypospadias. Salt loss occurs in many but not all cases. Patients with CAH due to 17α-hydroxylase (CYP17) deficiency have variable underandrogenization and develop hypertension and hypokalemia due to the potent salt-retaining effects of corticosterone and 11-deoxycorticosterone. Patients with complete loss of 17α-hydroxylase function often present as phenotypic females who fail to enter puberty and are found to have

1	effects of corticosterone and 11-deoxycorticosterone. Patients with complete loss of 17α-hydroxylase function often present as phenotypic females who fail to enter puberty and are found to have inguinal testes and hypertension in adolescence. Some mutations in CYP17 selectively impair 17,20-lyase activity without altering 17α-hydroxylase activity, leading to underandrogenization without mineralocorticoid excess and hypertension. Disruption of the coenzyme, cytochrome b5 (CYB5A), can present similarly, and methemoglobinemia is usually present. Mutations in P450 oxidoreductase (POR) affect multiple steroidogenic enzymes, leading to impaired androgenization and a biochemical pattern of apparent combined 21-hydroxylase and 17α-hydroxylase deficiency, sometimes with skeletal abnormalities (Antley-Bixler craniosynostosis). Defects in 17β-hydroxysteroid dehydrogenase type 3 (HSD17β3) and 5α-reductase type 2 (SRD5A2) interfere with the synthesis of testosterone and DHT, respectively. These

1	craniosynostosis). Defects in 17β-hydroxysteroid dehydrogenase type 3 (HSD17β3) and 5α-reductase type 2 (SRD5A2) interfere with the synthesis of testosterone and DHT, respectively. These conditions are characterized by minimal or absent androgenization in utero, but some phallic development can occur during adolescence due to the action of other enzyme isoforms. Individuals with 5α-reductase type 2 deficiency have normal wolffian structures and usually do not develop breast tissue. At puberty, the increase in testosterone induces muscle mass and other virilizing features despite DHT deficiency. Some individuals change gender from female to male at puberty. Thus, the management of this disorder is challenging. DHT cream can improve prepubertal phallic growth in patients raised as male. Gonadectomy before adolescence and estrogen replacement at puberty can be considered in individuals raised as females who have a female gender identity. Disruption of alternative pathways to fetal DHT

1	before adolescence and estrogen replacement at puberty can be considered in individuals raised as females who have a female gender identity. Disruption of alternative pathways to fetal DHT production might also present with 46,XY DSD (AKR1C2/AKR1C4).

1	Disorders of Androgen Action • androgen insensiTiviTy syndrome Mutations in the androgen receptor cause resistance to androgen (testosterone, DHT) action or the androgen insensitivity syndrome (AIS). AIS is a spectrum of disorders that affects at least 1 in 100,000 46,XY individuals. Because the androgen receptor is X-linked, only 46,XY offspring are affected if the mother is a carrier of a mutation. XY individuals with complete AIS (formerly called testicular feminization syndrome) have a female phenotype, normal breast development Disorders of Sex Development 2354 TAblE 410-3 SElECTED gEnETiC CAuSES of 46,xy DiSoRDERS of SEx DEvEloPmEnT (DSDs)

1	Abbreviations: AD, autosomal dominant; AKR1C2, aldo-keto reductase family 1 member 2; AR, autosomal recessive; ARX, aristaless related homeobox, X-linked; ATRX, α-thalassemia, mental retardation on the X; CAH, congenital adrenal hyperplasia; CBX2, chromobox homologue 2; CYB5A, cytochrome b5 POR, P450 oxidoreductase; CYP11A1, P450 cholesterol side-chain cleavage; CYP17, 17α-hydroxylase and 17,20-lyase; DAX1, dosage sensitive sex-reversal, adrenal hypoplasia congenita on the X chromosome, gene 1; DHEA, dehydroepiandrosterone; DHCR7, sterol 7 δ reductase; DHH, desert hedgehog; GATA4, GATA binding protein 4; HSD17B3, 17β-hydroxysteroid dehydrogenase type 3; HSD3B2, 3β-hydroxysteroid dehydrogenase type 2; LHR, LH receptor; MAP3K1, mitogen-activated protein kinase kinase kinase 1; SF1, steroidogenic factor 1; SL, sex-limited; SOX9, SRY-related HMG-box gene 9; SRD5A2, 5α-reductase type 2; SRY, sex-related gene on the Y chromosome; StAR, steroidogenic acute regulatory protein; WAGR, Wilms’

1	factor 1; SL, sex-limited; SOX9, SRY-related HMG-box gene 9; SRD5A2, 5α-reductase type 2; SRY, sex-related gene on the Y chromosome; StAR, steroidogenic acute regulatory protein; WAGR, Wilms’ tumor, aniridia, genitourinary anomalies, and mental retardation; WNT4, wingless-type mouse mammary tumor virus integration site, 4; WT1, Wilms’ tumor–related gene 1.

1	(due to aromatization of testosterone), a short vagina but no uterus offered for girls diagnosed in childhood, because there is a low risk of (because MIS production is normal), scanty pubic and axillary hair, malignancy, and estrogen replacement is prescribed. Alternatively, the and a female gender identity and sex role behavior. Gonadotropins gonads can be left in situ until breast development is complete and and testosterone levels can be low, normal, or elevated, depending on removed because of tumor risk. Some adults with complete AIS decline the degree of androgen resistance and the contribution of estradiol gonadectomy, but should be counseled about the risk of malignancy, to feedback inhibition of the hypothalamic-pituitary-gonadal axis. especially because early detection of premalignant changes by imag-AMH/MIS levels in childhood are normal or high. Most patients ing or biomarkers is currently not possible. The use of graded dilators present with inguinal hernias (containing

1	changes by imag-AMH/MIS levels in childhood are normal or high. Most patients ing or biomarkers is currently not possible. The use of graded dilators present with inguinal hernias (containing testes) in childhood or with in adolescence is usually sufficient to dilate the vagina for sexual primary amenorrhea in late adolescence. Gonadectomy sometimes is intercourse.

1	Cholesterol StAR (Cholesterol side chain cleavage enzyme) CYP11A1 hyperplasiaCYP17, (17,20-Lyase), CYB5A (Cytochrome b5) HSD17B3 (17˜-Hydroxysteroid dehydrogenase 3) CYP21A2 (21-Hydroxylase) SRD5A2 (5°-Reductase) CYP11B1 (11-Hydroxylase) 17-Hydroxyprogesterone 11-Deoxycortisol Androstenedione Cortisol Testosterone Glucocorticoid Pathway Androgen Pathway LH (testis) ACTH (adrenal) Dihydrotestosterone and 46,XX androgenization SOX9, or defects in RSPO1 (Table 410-4). deficiency (conGenital adrenal HyperplaSia) The classic form of 21-hydroxylase deficiency (21OHD) is the most common cause of CAH (Chap. 406). It has an incidence between 1 in

1	deficiency (conGenital adrenal HyperplaSia) The classic form of 21-hydroxylase deficiency (21OHD) is the most common cause of CAH (Chap. 406). It has an incidence between 1 in FIGURE 410-4 Simplified overview of glucocorticoid and androgen synthesis 10,000 and 1 in 15,000 and is the most common pathways. Defects in CYP21A2 and CYP11B1 shunt steroid precursors into the androgen cause of androgenization in chromosomal 46,XX pathway and cause androgenization of the 46,XX fetus. Testosterone is synthesized in females (Table 410-4). Affected individuals are the testicular Leydig cells and converted to dihydrotestosterone peripherally. Defects in homozygous or compound heterozygous for enzymes involved in androgen synthesis result in underandrogenization of the 46,XY fetus. severe mutations in the enzyme 21-hydroxylase StAR, steroidogenic acute regulatory protein. (After E Braunwald et al [eds]: Harrison’s Principles (CYP21A2). This mutation causes a block in

1	Disorders of Sex Development of Internal Medicine, 15th ed. New York, McGraw-Hill, 2001.)

1	Partial AIS (Reifenstein’s syndrome) results from androgen receptor mutations that maintain residual function. Patients often present in infancy with penoscrotal hypospadias and small undescended testes and with gynecomastia at the time of puberty. Those individuals raised as males usually require hypospadias repair in childhood and may need breast reduction in adolescence. Some boys enter puberty spontaneously. High-dose testosterone has been given to support development if puberty does not progress, but long-term data are limited. More severely underandrogenized patients present with clitoral enlargement and labial fusion and may be raised as females. The surgical and psychosexual management of these patients is complex and requires active involvement of the parents and the patient during the appropriate stages of development. Azoospermia and male-factor infertility also have been described in association with mild loss-offunction mutations in the androgen receptor.

1	OTHER DISORDERS AFFECTING 46,XY MALES

1	Persistent müllerian duct syndrome is the presence of a uterus in an otherwise phenotypic male. This condition can result from mutations in AMH or its receptor (AMHR2). The uterus may be removed, but only if damage to the vasa deferentia and blood supply can be avoided. Isolated hypospadias occurs in ~1 in 250 males and is usually repaired surgically. Most cases are idiopathic, although evidence of penoscrotal hypospadias, poor phallic development, and/or bilateral cryptorchidism requires investigation for an underlying DSD (e.g., partial gonadal dysgenesis, mild defect in testosterone action, or even severe adrenal glucocorticoid and mineralocorticoid synthesis, increasing 17-hydroxyprogesterone and shunting steroid precursors into the androgen synthesis pathway (Fig. 410-4). Glucocorticoid insufficiency causes a compensatory elevation of adrenocorticotropin (ACTH), resulting in adrenal hyperplasia and additional synthesis of steroid precursors proximal to the enzymatic block.

1	insufficiency causes a compensatory elevation of adrenocorticotropin (ACTH), resulting in adrenal hyperplasia and additional synthesis of steroid precursors proximal to the enzymatic block. Increased androgen synthesis in utero causes androgenization of the 46,XX fetus in the first trimester. Ambiguous genitalia are seen at birth, with varying degrees of clitoral enlargement and labial fusion. Excess androgen production causes gonadotropin-independent precocious puberty in males with 21-OHD.

1	The salt-wasting form of 21-OHD results from severe combined glucocorticoid and mineralocorticoid deficiency. A salt-wasting crisis usually manifests between 5 and 21 days of life and is a potentially life-threatening event that requires urgent fluid resuscitation and steroid treatment. Thus, a diagnosis of 21-OHD should be considered in any baby with atypical genitalia with bilateral nonpalpable gonads. Males (46,XY) with 21-OHD have no genital abnormalities at birth but are equally susceptible to adrenal insufficiency and salt-losing crises.

1	Females with the classic simple virilizing form of 21-OHD also present with genital ambiguity. They have impaired cortisol biosynthesis but do not develop salt loss. Patients with nonclassic 21-OHD produce normal amounts of cortisol and aldosterone but at the expense of producing excess androgens. Hirsutism (60%), oligomenorrhea (50%), and acne (30%) are the most common presenting features. This is one of the most common recessive disorders in humans, with an incidence as high as 1 in 100 to 500 in many populations and 1 in 27 in Ashkenazi Jews of Eastern European origin. 2356 TAblE 410-4 SElECTED gEnETiC CAuSES of 46,xx DiSoRDERS of SEx DEvEloPmEnT (DSDs) SRY Translocation Testis or ovotestis − Male or ambiguous SOX9 dup17q24 Unknown − Male or ambiguous RSPO1 AR Testis or ovotestis ± Male or ambiguous Palmar plantar hyperkeratosis, squamous cell skin WNT4 AR Testis or ovotestis − Male or ambiguous SERKAL syndrome (renal dysgenesis, adrenal and lung hypoplasia)

1	WNT4 AR Testis or ovotestis − Male or ambiguous SERKAL syndrome (renal dysgenesis, adrenal and lung hypoplasia) CAH, primary adrenal failure, mild androgenization due to ↑ DHEA CAH, phenotypic spectrum from severe salt-losing forms associated with adrenal failure to simple virilizing forms with compensated adrenal function, ↑ 17-hydroxyprogesterone Mixed features of 21-hydroxylase deficiency and 17α-hydroxylase/17,20-lyase deficiency, sometimes associated with Antley-Bixler craniosynostosis CAH, hypertension due to ↑ 11-deoxycortisol and 11-deoxycorticosterone Maternal virilization during pregnancy, absent breast development at puberty ↑ ACTH, 17-hydroxyprogesterone and cortisol; failure of dexamethasone suppression

1	Maternal virilization during pregnancy, absent breast development at puberty ↑ ACTH, 17-hydroxyprogesterone and cortisol; failure of dexamethasone suppression Abbreviations: ACTH, adrenocorticotropin; AR, autosomal recessive; CAH, congenital adrenal hyperplasia; CYP11B1, 11β-hydroxylase; CYP19, aromatase; CYP21A2, 21-hydroxylase; DHEA, dehydroepiandrosterone; HSD3B2, 3β-hydroxysteroid dehydrogenase type 2; POR, P450 oxidoreductase; RSPO1, R-spondin 1; SOX9, SRY-related HMG-box gene 9; SRY, sex-related gene on the Y chromosome.

1	Biochemical features of acute salt-wasting 21-OHD are hyponatremia, hyperkalemia, hypoglycemia, inappropriately low cortisol and aldosterone, and elevated 17-hydroxyprogesterone, ACTH, and plasma renin activity. Presymptomatic diagnosis of classic 21-OHD is now made by neonatal screening tests for increased 17-hydroxyprogesterone in many centers. In most cases, 17-hydroxyprogesterone is markedly increased. In adults, ACTH stimulation (0.25 mg of cosyntropin IV) with assays for 17-hydroxyprogesterone at 0 and 30 min can be useful for detecting nonclassic 21-OHD and heterozygotes (Chap. 406).

1	Acute salt-wasting crises require fluid resuscitation, IV hydrocortisone, and correction of hypoglycemia. Once the patient is stabilized, glucocorticoids must be given to correct the cortisol insufficiency and suppress ACTH stimulation, thereby preventing further virilization, rapid skeletal maturation, and the development of polycystic ovaries. Typically, hydrocortisone (10–15 mg/m2 per day in three divided doses) is used in childhood with a goal of partially suppressing 17-hydroxyprogesterone (100 to <1000 ng/dL). The aim of treatment is to use the lowest glucocorticoid dose that adequately suppresses adrenal androgen production without causing signs of glucocorticoid excess such as impaired growth and obesity. Salt-wasting conditions are treated with mineralocorticoid replacement. Infants usually need salt supplements up to the first year of life. Plasma renin activity and electrolytes are used to monitor mineralocorticoid replacement. Some patients with simple virilizing 21-OHD

1	usually need salt supplements up to the first year of life. Plasma renin activity and electrolytes are used to monitor mineralocorticoid replacement. Some patients with simple virilizing 21-OHD also benefit from mineralocorticoid supplements. Parents and patients should be educated about the need for increased doses of steroids during sickness, and patients should carry medic alert systems.

1	Steroid treatment for older adolescents and adults varies depending on lifestyle, age, and factors such as a desire to optimize fertility. Hydrocortisone remains a useful approach, but treatment with prednisolone at night may provide more complete ACTH suppression. Steroid doses should be adjusted to individual requirements because overtreatment can result in iatrogenic Cushing’s-like features, including weight gain, insulin resistance, hypertension, and osteopenia. Because it is long acting, dexamethasone given at night is useful for ACTH suppression but is often associated with more side effects, making hydrocortisone or prednisolone preferable for most patients. Androstenedione and testosterone may be useful measurements of long-term control, with less fluctuation than 17-hydroxyprogesterone. Mineralocorticoid requirements often decrease in adulthood, and doses should be reassessed and reduced to avoid hypertension in adults. In very severe cases, adrenalectomy has been advocated

1	Mineralocorticoid requirements often decrease in adulthood, and doses should be reassessed and reduced to avoid hypertension in adults. In very severe cases, adrenalectomy has been advocated but incurs the risks of surgery and total adrenal insufficiency.

1	Girls with significant genital androgenization due to classic 21-OHD usually undergo vaginal reconstruction and sometimes clitoral reduction (maintaining the glans and nerve supply), but the optimal timing of these procedures is debated, as is the need for the individual to be able to consent. There is a higher threshold for undertaking clitoral surgery in some centers because longterm sensation and ability to achieve orgasm can be affected, but the long-term results of newer techniques are not yet known. Full information about all options should be provided. If surgery is performed in infancy, surgical revision or regular vaginal dilatation may be needed in adolescence or adulthood, and long-term psychological support and psychosexual counseling may be appropriate. Women with 21-OHD frequently develop polycystic ovaries and have reduced fertility, especially when control is poor. Fecundity is achieved in 60–90% of women with good metabolic control, but ovulation induction (or even

1	develop polycystic ovaries and have reduced fertility, especially when control is poor. Fecundity is achieved in 60–90% of women with good metabolic control, but ovulation induction (or even adrenalectomy) may be required. Dexamethasone should be avoided in pregnancy. Men with poorly controlled 21-OHD may develop testicular adrenal rests and are at risk for reduced fertility. Prenatal treatment of 21-OHD by the administration of dexamethasone to mothers is still under evaluation. However, pending methods to diagnose the disorder early in pregnancy, both affected and nonaffected fetuses will be exposed because treatment is started ideally before 6 to 7 weeks. The longterm effects of prenatal dexamethasone exposure on fetal development are still under evaluation, and current guidelines recommend full informed consent before treatment, ideally in a protocol that allows long-term follow-up of all children treated. Newer techniques such as cell-free fetal DNA testing may potentially reduce

1	full informed consent before treatment, ideally in a protocol that allows long-term follow-up of all children treated. Newer techniques such as cell-free fetal DNA testing may potentially reduce treatment of nonaffected fetuses.

1	of treatment and support may be limited. Disorders of the Testes and male Reproductive System Shalender Bhasin, J. Larry Jameson The male reproductive system regulates sex differentiation, viriliza-tion, and the hormonal changes that accompany puberty, ultimately 411 Disorders of the Testes and Male Reproductive System The treatment of other forms of CAH includes mineralocorticoid and glucocorticoid replacement for salt-losing conditions (e.g., StAR, CYP11A1, HSD3β2), suppression of ACTH drive with glucocorticoids in disorders associated with hypertension (e.g., CYP17, CYP11B1), and appropriate sex hormone replacement in adolescence and adulthood, when necessary.

1	otHer cauSeS Increased androgen synthesis can also occur in CAH due to defects in POR, 11β-hydroxylase (CYP11B1), and 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) and with mutations in the genes encoding aromatase (CYP19) and the glucocorticoid receptor. Increased androgen exposure in utero can occur with maternal virilizing tumors and with ingestion of androgenic compounds. OTHER DISORDERS AFFECTING 46,XX FEMALES Congenital absence of the vagina occurs in association with müllerian agenesis or hypoplasia as part of the Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome (rarely caused by WNT4 mutations). This diagnosis should be considered in otherwise phenotypically normal females with primary amenorrhea. Associated features include renal (agenesis) and cervical spinal abnormalities.

1	The approach to a child or adolescent with ambiguous genita lia or another DSD requires cultural sensitivity, as the con cepts of sex and gender vary widely. Rare genetic DSDs can occur more frequently in specific populations (e.g., 5α-reductase type 2 in the Dominican Republic). Different forms of CAH also show ethnic and geographic variability. In many countries, appropriate biochemical tests may not be readily available, and access to appropriate forms leading to spermatogenesis and fertility. Under the control of the pituitary hormones—luteinizing hormone (LH) and follicle-stimulating hormone (FSH)—the Leydig cells of the testes produce testosterone and germ cells are nurtured by Sertoli cells to divide, differentiate, and mature into sperm. During embryonic development, testosterone and dihydrotestosterone (DHT) induce the wolffian duct and virilization of the external genitalia. During puberty, testosterone promotes somatic growth and the development of secondary sex

1	and dihydrotestosterone (DHT) induce the wolffian duct and virilization of the external genitalia. During puberty, testosterone promotes somatic growth and the development of secondary sex characteristics. In the adult, testosterone is necessary for spermatogenesis, stimulation of libido and normal sexual function, and maintenance of muscle and bone mass. This chapter focuses on the physiology of the testes and disorders associated with decreased androgen production, which may be caused by gonadotropin deficiency or by primary testis dysfunction. A variety of testosterone formulations now allow more physiologic androgen replacement. Infertility occurs in ~5% of men and is increasingly amenable to treatment by hormone replacement or by using sperm transfer techniques. For further discussion of sexual dysfunction, disorders of the prostate, and testicular cancer, see Chaps. 67, 115, and 116, respectively.

1	The fetal testis develops from the undifferentiated gonad after expression of a genetic cascade that is initiated by the SRY (sex-related gene on the Y chromosome) (Chap. 410). SRY induces differentiation of Sertoli cells, which surround germ cells and, together with peritubular 2357 myoid cells, form testis cords that will later develop into seminiferous tubules. Fetal Leydig cells and endothelial cells migrate into the gonad from the adjacent mesonephros but may also arise from interstitial cells that reside between testis cords. Leydig cells produce testosterone, which supports the growth and differentiation of wolffian duct structures that develop into the epididymis, vas deferens, and seminal vesicles. Testosterone is also converted to DHT (see below), which induces formation of the prostate and the external male genitalia, including the penis, urethra, and scrotum. Testicular descent through the inguinal canal is controlled in part by Leydig cell production of insulin-like

1	the prostate and the external male genitalia, including the penis, urethra, and scrotum. Testicular descent through the inguinal canal is controlled in part by Leydig cell production of insulin-like factor 3 (INSL3), which acts via a receptor termed Great (G protein–coupled receptor affecting testis descent). Sertoli cells produce müllerian-inhibiting substance (MIS), which causes regression of the müllerian structures, including the fallopian tube, uterus, and upper segment of the vagina.

1	Although puberty commonly refers to the maturation of the reproductive axis and the development of secondary sex characteristics, it involves a coordinated response of multiple hormonal systems including the adrenal gland and the growth hormone (GH) axis (Fig. 411-1). The development of secondary sex characteristics is initiated by adrenarche, which usually occurs between 6 and 8 years of age when the adrenal gland begins to produce greater amounts of androgens from the zona reticularis, the principal site of dehydroepiandrosterone (DHEA) production. The sex maturation process is greatly accelerated by the activation of the hypothalamic-pituitary axis and the production of gonadotropin-releasing hormone (GnRH). The GnRH pulse generator in the hypothalamus is active during fetal life and early infancy but is restrained until the early stages of puberty by a neuroendocrine brake imposed by the inhibitory actions of glutamate, γ-aminobutyric acid (GABA), and neuropeptide Y. Although the

1	infancy but is restrained until the early stages of puberty by a neuroendocrine brake imposed by the inhibitory actions of glutamate, γ-aminobutyric acid (GABA), and neuropeptide Y. Although the pathways that initiate reactivation of the GnRH pulse generator at the onset of puberty have been elusive, mounting evidence supports involvement of GPR54, a G protein–coupled receptor that binds an endogenous ligand, kisspeptin. Individuals with mutations of GPR54 fail to enter puberty, and experiments in primates demonstrate that infusion of the ligand is sufficient to induce premature puberty. Kisspeptin signaling plays an important role in mediating the feedback action of sex steroids on gonadotropin secretion and in regulating the tempo of sexual maturation at puberty. Leptin, a hormone produced by adipose cells, plays a permissive role in the resurgence of GnRH secretion at the onset of puberty, as leptin-deficient individuals also fail to enter puberty (Chap. 415e). The adipocyte

1	produced by adipose cells, plays a permissive role in the resurgence of GnRH secretion at the onset of puberty, as leptin-deficient individuals also fail to enter puberty (Chap. 415e). The adipocyte hormone leptin, gut hormone ghrelin, neuropeptide Y, and kisspeptin integrate the signals

1	FIGURE 411-1 Pubertal events in males. Sexual maturity ratings for genitalia and pubic hair and divided into five stages. (From WA Marshall, JM Tanner: Variations in the pattern of pubertal changes in boys. Arch Dis Child 45:13, 1970.) 2358 originating in energy stores and metabolic tissues with mechanisms that control onset of puberty through regulation of GnRH secretion. Energy deficit and excess and metabolic stress are associated with disturbed reproductive maturation and timing of pubertal onset. The early stages of puberty are characterized by nocturnal surges of LH and FSH. Growth of the testes is usually the first sign of puberty, reflecting an increase in seminiferous tubule volume. Increasing levels of testosterone deepen the voice and increase muscle growth. Conversion of testosterone to DHT leads to growth of the external genitalia and pubic hair. DHT also stimulates prostate and facial hair growth and initiates recession of the temporal hairline. The growth spurt occurs

1	to DHT leads to growth of the external genitalia and pubic hair. DHT also stimulates prostate and facial hair growth and initiates recession of the temporal hairline. The growth spurt occurs at a testicular volume of about 10–12 mL. GH increases early in puberty and is stimulated in part by the rise in gonadal steroids. GH increases the level of insulin-like growth factor I (IGF-I), which enhances linear bone growth. The prolonged pubertal exposure to gonadal steroids (mainly estradiol) ultimately causes epiphyseal closure and limits further bone growth.

1	Hypothalamic GnRH regulates the production of the pituitary gonadotropins LH and FSH (Fig. 411-2). GnRH is released in discrete pulses approximately every 2 h, resulting in corresponding pulses of LH and FSH. These dynamic hormone pulses account in part for the wide variations in LH and testosterone, even within the same individual. LH acts primarily on the Leydig cell to stimulate testosterone synthesis. The regulatory control of androgen synthesis is mediated by testosterone and estrogen feedback on both the hypothalamus and the pituitary. FSH acts on the Sertoli cell to regulate spermatogenesis and the production of Sertoli products such as inhibin B, which acts to selectively suppress pituitary FSH. Despite these somewhat distinct Leydig and Sertoli cell–regulated pathways, testis function is integrated at several levels: GnRH regulates both gonadotropins; spermatogenesis requires high levels of testosterone; and numerous paracrine interactions between Leydig and Sertoli cells are

1	is integrated at several levels: GnRH regulates both gonadotropins; spermatogenesis requires high levels of testosterone; and numerous paracrine interactions between Leydig and Sertoli cells are necessary for normal testis function.

1	THE LEYDIG CELL: ANDROGEN SYNTHESIS

1	LH binds to its seven-transmembrane, G protein–coupled receptor to activate the cyclic AMP pathway. Stimulation of the LH receptor induces steroid acute regulatory (StAR) protein, along with several steroidogenic enzymes involved in androgen synthesis. LH receptor mutations cause Leydig cell hypoplasia or agenesis, underscoring the importance of this pathway for Leydig cell development and function. The rate-limiting process in testosterone synthesis is the delivery of cholesterol by the StAR protein to the inner mitochondrial membrane. Peripheral benzodiazepine receptor, a mitochondrial cholesterol-binding protein, is also an acute regulator of Leydig cell steroidogenesis. The five major enzymatic steps involved in testosterone synthesis are summarized in Fig. 411-3. After cholesterol transport into the mitochondrion, the formation of pregnenolone by CYP11A1 (side chain cleavage enzyme) is a limiting enzymatic step. The 17α-hydroxylase and the 17,20-lyase reactions are catalyzed by a

1	into the mitochondrion, the formation of pregnenolone by CYP11A1 (side chain cleavage enzyme) is a limiting enzymatic step. The 17α-hydroxylase and the 17,20-lyase reactions are catalyzed by a single enzyme, CYP17; posttranslational modification (phosphorylation) of this enzyme and the presence of specific enzyme cofactors confer 17,20-lyase activity selectively in the testis and zona reticularis of the adrenal gland. Testosterone can be converted to the more potent DHT by 5α-reductase, or it can be aromatized to estradiol by CYP19 (aromatase). Two isoforms of steroid 5α-reductase, SRD5A1 and SRD5A2, have been described; all known kindreds with 5α-reductase deficiency have had mutations in SRD5A2, the predominant form in the prostate and the skin.

1	Testosterone Transport and Metabolism In males, 95% of circulating testosterone is derived from testicular production (3–10 mg/d). Direct secretion of testosterone by the adrenal and the peripheral conversion of androstenedione to testosterone collectively account for another 0.5 mg/d of testosterone. Only a small amount of DHT (70 μg/d) is secreted directly by the testis; most circulating DHT is derived from peripheral conversion of testosterone. Most of the daily production of estradiol (~45 μg/d) in men is derived from aromatase-mediated peripheral conversion of testosterone and androstenedione.

1	+––++HypothalamusGonadotrope (LH, FSH) GnRH-producing neuron Tunica albuginea Seminiferous tubules Seminiferous tubules Interstitial Leydig cells (testosterone) AnteriorpituitaryVas deferens Testosterone Inhibin B E2 DHT LH FSH LH FSH Epididymis Sertoli cell (Inhibin B) Spermatid Spermatogonium FIGURE 411-2 Human pituitary gonadotropin axis, structure of testis, and seminiferous tubule. E2, 17β-estradiol; DHT, dihydrotes-tosterone; FSH, follicle-stimulating hormones; GnRH, gonadotropin-releasing; LH, luteinizing hormone.

1	Circulating testosterone is bound to two plasma proteins: sex hormone–binding globulin (SHBG) and albumin (Fig. 411-4). SHBG binds testosterone with much greater affinity than albumin. Only 0.5–3% of testosterone is unbound. According to the “free hormone” hypothesis, only the unbound fraction is biologically active; however, albumin-bound hormone dissociates readily in the capillaries and may be bioavailable. SHBG-bound testosterone also may be internalized through endocytic pits by binding to a protein called megalin. SHBG concentrations are decreased by androgens, obesity, diabetes mellitus, insulin, and nephrotic syndrome. Conversely, estrogen administration, hyperthyroidism, many chronic inflammatory illnesses, infections such as HIV or hepatitis B and C, and aging are associated with high SHBG concentrations. Testosterone is metabolized predominantly in the liver, although some degradation occurs in peripheral tissues, particularly the (17,20-Lyase)

1	Testosterone is metabolized predominantly in the liver, although some degradation occurs in peripheral tissues, particularly the (17,20-Lyase) FIGURE 411-3 The biochemical pathway in the conversion of 27-carbon sterol cholesterol to androgens and estrogens. • External genitalia• Prostate growth• Acne• Facial/body hair• Scalp hair loss• Hypothalamic/ pituitary feedback• Bone resorption• Epiphyseal closure• Gynecomastia• Some vascular and behavioral effectsAlbumin(50-70%)Estradiol• Wolffian duct• Bone formation• Muscle mass• SpermatogenesisSHBG(30-45%)Unbound(0.5-3.0%)TestosteroneExcretion(90%)Dihydrotestosterone(DHT)Aromatase(0.3%)5˜-Reductase(6-8%)Testosterone (5 mg/d) FIGURE 411-4 Androgen metabolism and actions. SHBG, sex hormone–binding globulin.

1	FIGURE 411-4 Androgen metabolism and actions. SHBG, sex hormone–binding globulin. prostate and the skin. In the liver, testosterone is converted by 2359 a series of enzymatic steps that involve 5αand 5β-reductases, 3αand 3β-hydroxysteroid dehydrogenases, and 17β-hydroxysteroid dehydrogenase into androsterone, etiocholanolone, DHT, and 3-α-androstanediol. These compounds undergo glucuronidation or sulfation before being excreted by the kidneys.

1	Mechanism of Androgen Action Testosterone exerts some of its biologic effects by binding to androgen receptor, either directly or after its conversion to DHT by the steroid 5-α reductase. Testosterone’s effects on the skeletal muscle, erythropoiesis, and bone in men do not require its obligatory conversion to DHT. However, the conversion of testosterone to DHT is necessary for the masculinization of the urogenital sinus and genital tubercle. Aromatization of testosterone to estradiol mediates additional effects of testosterone on the bone resorption, epiphyseal closure, sexual desire, vascular endothelium, and fat. DHT can also be converted in some tissues by 3-keto reductase/3β-hydroxysteroid dehydrogenase enzymes to 5α-androstane-3β,17β-diol, which is a high-affinity ligand and agonist of estrogen receptor β.

1	The androgen receptor (AR) is structurally related to the nuclear receptors for estrogen, glucocorticoids, and progesterone (Chap. 400e). The AR is encoded by a gene on the long arm of the X chromosome and has a molecular mass of about 110 kDa. A polymorphic region in the amino terminus of the receptor, which contains a variable number of glutamine repeats, modifies the transcriptional activity of the receptor. The AR protein is distributed in both the cytoplasm and the nucleus. The ligand binding to the AR induces con formational changes that allow the recruitment and assembly of tissue-specific cofactors and causes it to translocate into the nucleus, where it binds to DNA or other transcription factors already bound to DNA. Thus, the AR is a ligand-regulated transcription factor that regulates the expression of androgen-dependent genes in a tissue-specific manner. Some androgen effects may be mediated by nongenomic AR signal transduction pathways. Testosterone binds to AR with half

1	the expression of androgen-dependent genes in a tissue-specific manner. Some androgen effects may be mediated by nongenomic AR signal transduction pathways. Testosterone binds to AR with half the affinity of DHT. The DHT-AR complex also has greater thermostability and a slower dissociation rate than the testosterone-AR complex. However, the molecular basis for selective testosterone versus DHT actions remains incompletely explained.

1	THE SEMINIFEROUS TUBULES: SPERMATOGENESIS

1	The seminiferous tubules are convoluted, closed loops with both ends emptying into the rete testis, a network of progressively larger efferent ducts that ultimately form the epididymis (Fig. 411-2). The seminiferous tubules total about 600 m in length and comprise about two-thirds of testis volume. The walls of the tubules are formed by polarized Sertoli cells that are apposed to peritubular myoid cells. Tight junctions between Sertoli cells create a blood-testis barrier. Germ cells compose the majority of the seminiferous epithelium (~60%) and are intimately embedded within the cytoplasmic extensions of the Sertoli cells, which function as “nurse cells.” Germ cells progress through characteristic stages of mitotic and meiotic divisions. A pool of type A spermatogonia serve as stem cells capable of self-renewal. Primary spermatocytes are derived from type B spermatogonia and undergo meiosis before progressing to spermatids that undergo spermiogenesis (a differentiation process

1	cells capable of self-renewal. Primary spermatocytes are derived from type B spermatogonia and undergo meiosis before progressing to spermatids that undergo spermiogenesis (a differentiation process involving chromatin condensation, acquisition of an acrosome, elongation of cytoplasm, and formation of a tail) and are released from Sertoli cells as mature spermatozoa. The complete differentiation process into mature sperm requires 74 days. Peristaltic-type action by peritubular myoid cells transports sperm into the efferent ducts. The spermatozoa spend an additional 21 days in the epididymis, where they undergo further maturation and capacitation. The normal adult testes produce >100 million sperm per day.

1	Naturally occurring mutations in the FSHβ gene and in the FSH receptor confirm an important, but not essential, role for this pathway in spermatogenesis. Females with these mutations are hypogonadal and infertile because ovarian follicles do not mature; males exhibit variable degrees of reduced spermatogenesis, presumably because of impaired Sertoli cell function. Because Sertoli cells produce inhibin B, an inhibitor of FSH, seminiferous tubule damage (e.g., by radiation)

1	Disorders of the Testes and Male Reproductive System 2360 causes a selective increase of FSH. Testosterone reaches very high concentrations locally in the testis and is essential for spermatogenesis. The cooperative actions of FSH and testosterone are important in the progression of meiosis and spermiation. FSH and testosterone regulate germ cell survival via the intrinsic and the extrinsic apoptotic mechanisms. FSH may also play an important role in supporting spermatogonia. Gonadotropin-regulated testicular RNA helicase (GRTH/ DDX25), a testis-specific gonadotropin/androgen-regulated RNA helicase, is present in germ cells and Leydig cells and may be an important factor in the paracrine regulation of germ cell development. Several cytokines and growth factors are also involved in the regulation of spermatogenesis by paracrine and autocrine mechanisms. A number of knockout mouse models exhibit impaired germ cell development or spermatogenesis, presaging possible mutations associated

1	of spermatogenesis by paracrine and autocrine mechanisms. A number of knockout mouse models exhibit impaired germ cell development or spermatogenesis, presaging possible mutations associated with male infertility. The human Y chromosome contains a small pseudoautosomal region that can recombine with homologous regions of the X chromosome. Most of the Y chromosome does not recombine with the X chromosome and is referred to as the male-specific region of the Y (MSY). The MSY contains 156 transcription units that encode for 26 proteins, including nine families of Y-specific multicopy genes; many of these Y-specific genes are also testis-specific and necessary for spermatogenesis. Microdeletions of several Y chromosome azoospermia factor (AZF) genes (e.g., RNA-binding motif, RBM; deleted in azoospermia, DAZ) are associated with oligospermia or azoospermia.

1	Treatment options for male factor infertility have expanded greatly in recent years. Secondary hypogonadism is highly amenable to treatment with pulsatile GnRH or gonadotropins (see below). Assisted reproductive technologies such as the in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) have provided new opportunities for patients with primary testicular failure and disorders of sperm transport. Choice of initial treatment options depends on sperm concentration and motility. Expectant management should be attempted initially in men with mild male factor infertility (sperm count of 15–20 × 106/mL and normal motility). Moderate male factor infertility (10–15 × 106/mL and 20–40% motility) should begin with intrauterine insemination alone or in combination with treatment of the female partner with clomiphene or gonadotropins, but it may require IVF with or without ICSI. For men with a severe defect (sperm count of <10 × 106/mL, 10% motility), IVF with ICSI or donor

1	of the female partner with clomiphene or gonadotropins, but it may require IVF with or without ICSI. For men with a severe defect (sperm count of <10 × 106/mL, 10% motility), IVF with ICSI or donor sperm should be used.

1	The history should focus on developmental stages such as puberty and growth spurts, as well as androgen-dependent events such as early morning erections, frequency and intensity of sexual thoughts, and frequency of masturbation or intercourse. Although libido and the overall frequency of sexual acts are decreased in androgen-deficient men, young hypogonadal men may achieve erections in response to visual erotic stimuli. Men with acquired androgen deficiency often report decreased energy and increased irritability.

1	The physical examination should focus on secondary sex characteristics such as hair growth, gynecomastia, testicular volume, prostate, and height and body proportions. Eunuchoid proportions are defined as an arm span >2 cm greater than height and suggest that androgen deficiency occurred before epiphyseal fusion. Hair growth in the face, axilla, chest, and pubic regions is androgen-dependent; however, changes may not be noticeable unless androgen deficiency is severe and prolonged. Ethnicity also influences the intensity of hair growth (Chap. 68). Testicular volume is best assessed by using a Prader orchidometer. Testes range from 3.5 to 5.5 cm in length, which corresponds to a volume of 12–25 mL. Advanced age does not influence testicular size, although the consistency becomes less firm. Asian men generally have smaller testes than Western Europeans, independent of differences in body size. Because of its possible role in infertility, the presence of varicocele should be sought by

1	Asian men generally have smaller testes than Western Europeans, independent of differences in body size. Because of its possible role in infertility, the presence of varicocele should be sought by palpation while the patient is standing; it is more common on the left side. Patients with Klinefelter’s syndrome have markedly reduced testicular volumes (1–2 mL). In congenital hypogonadotropic hypogonadism, testicular volumes provide a good index for the degree of gonadotropin deficiency and the likelihood of response to therapy.

1	LH and FSH are measured using two-site immunoradiometric, immunofluorometric, or chemiluminescent assays, which have very low cross-reactivity with other pituitary glycoprotein hormones and human chorionic gonadotropin (hCG) and have sufficient sensitivity to measure the low levels present in patients with hypogonadotropic hypogonadism. In men with a low testosterone level, an LH level can distinguish primary (high LH) versus secondary (low or inappropriately normal LH) hypogonadism. An elevated LH level indicates a primary defect at the testicular level, whereas a low or inappropriately normal LH level suggests a defect at the hypothalamic-pituitary level. LH pulses occur about every 1–3 h in normal men. Thus, gonadotropin levels fluctuate, and samples should be pooled or repeated when results are equivocal. FSH is less pulsatile than LH because it has a longer half-life. Selective increase in FSH suggests damage to the seminiferous tubules. Inhibin B, a Sertoli cell product that

1	results are equivocal. FSH is less pulsatile than LH because it has a longer half-life. Selective increase in FSH suggests damage to the seminiferous tubules. Inhibin B, a Sertoli cell product that suppresses FSH, is reduced with seminiferous tubule damage. Inhibin B is a dimer with α-βB subunits and is measured by two-site immunoassays.

1	GnRH Stimulation Testing The GnRH test is performed by measuring LH and FSH concentrations at baseline and at 30 and 60 min after intravenous administration of 100 μg of GnRH. A minimally acceptable response is a twofold LH increase and a 50% FSH increase. In the prepubertal period or with severe GnRH deficiency, the gonadotrope may not respond to a single bolus of GnRH because it has not been primed by endogenous hypothalamic GnRH; in these patients, GnRH responsiveness may be restored by chronic, pulsatile GnRH administration. With the availability of sensitive and specific LH assays, GnRH stimulation testing is used rarely except to evaluate gonadotrope function in patients who have undergone pituitary surgery or have a space-occupying lesion in the hypothalamic-pituitary region.

1	TESTOSTERONE ASSAYS Total Testosterone Total testosterone includes both unbound and protein-bound testosterone and is measured by radioimmunoassays, immunometric assays, or liquid chromatography tandem mass spectrometry (LC-MS/MS). LC-MS/MS involves extraction of serum by organic solvents, separation of testosterone from other steroids by high-performance liquid chromatography and mass spectrometry, and quantitation of unique testosterone fragments by mass spectrometry. LC-MS/MS provides accurate and sensitive measurements of testosterone levels even in the low range and is emerging as the method of choice for testosterone measurement. Laboratories that have been certified by the Centers for Disease Control and Prevention (CDC) Hormone Standardization Program for Testosterone (HoST) can ensure that testosterone measurements are accurate and calibrated to an international standard. A single fasting morning sample provides a good approximation of the average testosterone concentration

1	ensure that testosterone measurements are accurate and calibrated to an international standard. A single fasting morning sample provides a good approximation of the average testosterone concentration with the realization that testosterone levels fluctuate in response to pulsatile LH. Testosterone is generally lower in the late afternoon and is reduced by acute illness. The testosterone concentration in healthy young men ranges from 300 to 1000 ng/dL in most laboratories, and efforts are under way to generate harmonized population-based reference ranges that can be applied to all CDC-certified laboratories. Alterations in SHBG levels due to aging, obesity, diabetes mellitus, hyperthyroidism, some types of medications, or chronic illness or on a congenital basis can affect total testosterone levels. Heritable factors contribute substantially to the population-level variation in testosterone levels, and genome-wide association studies have revealed polymorphisms in the SHBG gene as

1	levels. Heritable factors contribute substantially to the population-level variation in testosterone levels, and genome-wide association studies have revealed polymorphisms in the SHBG gene as important contributors to variation in testosterone levels.

1	Measurement of Unbound Testosterone Levels Most circulating testosterone is bound to SHBG and to albumin; only 0.5–3% of circulating testosterone is unbound, or “free.” The unbound testosterone concentration can be measured by equilibrium dialysis or calculated from total testosterone, SHBG, and albumin concentrations. Recent research has shown that testosterone binding to SHBG is a multistep process that involves complex homoallostery within the SHBG dimer; a novel allosteric model of testosterone binding to SHBG dimers provides good estimates of free testosterone concentrations. The previous law of mass action equations based on linear models of testosterone binding to SHBG have been shown to be erroneous. Tracer analogue methods are relatively inexpensive and convenient, but they are inaccurate. Bioavailable testosterone refers to unbound testosterone plus testosterone that is loosely bound to albumin; it can be determined by the ammonium sulfate precipitation method.

1	hCG Stimulation Test The hCG stimulation test is performed by administering a single injection of 1500–4000 IU of hCG intramuscularly and measuring testosterone levels at baseline and 24, 48, 72, and 120 h after hCG injection. An alternative regimen involves three injections of 1500 units of hCG on successive days and measuring testosterone levels 24 h after the last dose. An acceptable response to hCG is a doubling of the testosterone concentration in adult men. In prepubertal boys, an increase in testosterone to >150 ng/dL indicates the presence of testicular tissue. No response may indicate an absence of testicular tissue or marked impairment of Leydig cell function. Measurement of MIS, a Sertoli cell product, is also used to detect the presence of testes in prepubertal boys with cryptorchidism.

1	Semen analysis is the most important step in the evaluation of male infertility. Samples are collected by masturbation following a period of abstinence for 2–3 days. Semen volumes and sperm concentrations vary considerably among fertile men, and several samples may be needed before concluding that the results are abnormal. Analysis should be performed within an hour of collection. Using semen samples from over 4500 men in 14 countries, whose partners had a time-to-pregnancy of less than 12 months, the World Health Organization (WHO) has generated the following one-sided reference limits for semen parameters: semen volume, 1.5 mL; total sperm number, 39 million per ejaculate; sperm concentration, 15 million/mL; vitality, 58% live; progressive motility, 32%; total (progressive + nonprogressive) motility, 40%; morphologically normal forms, 4.0%. Some men with low sperm counts are nevertheless fertile. A variety of tests for sperm function can be performed in specialized laboratories, but

1	motility, 40%; morphologically normal forms, 4.0%. Some men with low sperm counts are nevertheless fertile. A variety of tests for sperm function can be performed in specialized laboratories, but these add relatively little to the treatment options.

1	Testicular biopsy is useful in some patients with oligospermia or azoospermia as an aid in diagnosis and indication for the feasibility of treatment. Using local anesthesia, fine-needle aspiration biopsy is performed to aspirate tissue for histology. Alternatively, open biopsies can be performed under local or general anesthesia when more tissue is required. A normal biopsy in an azoospermic man with a normal FSH level suggests obstruction of the vas deferens, which may be correctable surgically. Biopsies are also used to harvest sperm for ICSI and to classify disorders such as hypospermatogenesis (all stages present but in reduced numbers), germ cell arrest (usually at primary spermatocyte stage), and Sertoli cell–only syndrome (absent germ cells) or hyalinization (sclerosis with absent cellular elements). See Chap. 410.

1	See Chap. 410. The onset and tempo of puberty varies greatly in the general population and is affected by genetic and environmental factors. Although some of the variance in the timing of puberty is explained by heritable factors, the genes involved remain unknown. Puberty in boys before age 9 is considered precocious. Isosexual precocity refers to premature sexual development consistent with phenotypic sex and includes features such as the development of facial hair and phallic growth. Isosexual precocity is divided into gonadotropindependent and gonadotropin-independent causes of androgen excess (Table 411-1). Heterosexual precocity refers to the premature development of estrogenic features in boys, such as breast development.

1	Gonadotropin-Dependent Precocious Puberty This disorder, called central precocious puberty (CPP), is less common in boys than in girls. It is caused by premature activation of the GnRH pulse generator, sometimes because of central nervous system (CNS) lesions such as hypothalamic hamartomas, but it is often idiopathic. CPP is characterized by gonadotropin levels that are inappropriately elevated for age. Because pituitary priming has occurred, GnRH elicits LH and FSH responses typical of those seen in puberty or in adults. Magnetic resonance imaging (MRI) should be performed to exclude a mass, structural defect, infection, or inflammatory process. Mutations in MKRN3, an imprinted gene encoding makorin ring-finger protein 3, which is expressed only from the paternally inherited allele, have been associated with CPP.

1	Gonadotropin-Independent Precocious Puberty In gonadotropinindependent precocious puberty, androgens from the testis or the adrenal are increased, but gonadotropins are low. This group of disorders includes hCG-secreting tumors; congenital adrenal hyperplasia; sex steroid–producing tumors of the testis, adrenal, and ovary; accidental or deliberate exogenous sex steroid administration; hypothyroidism; and activating mutations of the LH receptor or Gsα subunit. CAuSES of PRECoCiouS oR DElAyED PubERTy in boyS I. Precocious puberty A. Gonadotropin-dependent 1. 2. 3. B. Gonadotropin-independent 1. 2. 3. 4. 5. II. A. Constitutional delay of growth and puberty B. 1. 2. 3. C. CNS tumors and their treatment (radiotherapy and surgery) D. Hypothalamic-pituitary causes of pubertal failure (low gonadotropins) 1. 2. a. b. E. Gonadal causes of pubertal failure (elevated gonadotropins) 1. 2. 3. 4. 5. F. Androgen insensitivity

1	D. Hypothalamic-pituitary causes of pubertal failure (low gonadotropins) 1. 2. a. b. E. Gonadal causes of pubertal failure (elevated gonadotropins) 1. 2. 3. 4. 5. F. Androgen insensitivity Abbreviations: CNS, central nervous system; GnRH, gonadotropin-releasing hormone; hCG, human chronic gonadotropin; LH, luteinizing hormone.

1	2. 3. 4. 5. F. Androgen insensitivity Abbreviations: CNS, central nervous system; GnRH, gonadotropin-releasing hormone; hCG, human chronic gonadotropin; LH, luteinizing hormone. Disorders of the Testes and Male Reproductive System 2362 familial male-limited precociouS puBerty Also called testotoxicosis, familial male-limited precocious puberty is an autosomal dominant disorder caused by activating mutations in the LH receptor, leading to constitutive stimulation of the cyclic AMP pathway and testosterone production. Clinical features include premature androgenization in boys, growth acceleration in early childhood, and advanced bone age followed by premature epiphyseal fusion. Testosterone is elevated, and LH is suppressed. Treatment options include inhibitors of testosterone synthesis (e.g., ketoconazole), AR antagonists (e.g., flutamide and bicalutamide), and aromatase inhibitors (e.g., anastrazole).

1	mccune-alBriGHt Syndrome This is a sporadic disorder caused by somatic (postzygotic) activating mutations in the Gsα subunit that links G protein–coupled receptors to intracellular signaling pathways (Chap. 426e). The mutations impair the guanosine triphosphatase activity of the Gsα protein, leading to constitutive activation of adenylyl cyclase. Like activating LH receptor mutations, this stimulates testosterone production and causes gonadotropin-independent precocious puberty. In addition to sexual precocity, affected individuals may have autonomy in the adrenals, pituitary, and thyroid glands. Café au lait spots are characteristic skin lesions that reflect the onset of the somatic mutations in melanocytes during embryonic development. Polyostotic fibrous dysplasia is caused by activation of the parathyroid hormone receptor pathway in bone. Treatment is similar to that in patients with activating LH receptor mutations. Bisphosphonates have been used to treat bone lesions.

1	conGenital adrenal HyperplaSia Boys with congenital adrenal hyperplasia (CAH) who are not well controlled with glucocorticoid suppression of adrenocorticotropic hormone (ACTH) can develop premature virilization because of excessive androgen production by the adrenal gland (Chaps. 406 and 410). LH is low, and the testes are small. Adrenal rests may develop within the testis of poorly controlled patients with CAH because of chronic ACTH stimulation; adrenal rests do not require surgical removal and regress with effective glucocorticoid therapy. Some children with CAH may develop gonadotropin-dependent precocious puberty with early maturation of the hypothalamic-pituitary-gonadal axis, elevated gonadotropins, and testicular growth.

1	Heterosexual Sexual Precocity Breast enlargement in prepubertal boys can result from familial aromatase excess, estrogen-producing tumors in the adrenal gland, Sertoli cell tumors in the testis, marijuana smoking, or exogenous estrogens or androgens. Occasionally, germ cell tumors that secrete hCG can be associated with breast enlargement due to excessive stimulation of estrogen production (see “Gynecomastia,” below). APPROACH TO THE PATIENT:

1	After verification of precocious development, serum LH and FSH levels should be measured to determine whether gonadotropins are increased in relation to chronologic age (gonadotropin-dependent) or whether sex steroid secretion is occurring independent of LH and FSH (gonadotropin-independent). In children with gonadotropindependent precocious puberty, CNS lesions should be excluded by history, neurologic examination, and MRI scan of the head. If organic causes are not found, one is left with the diagnosis of idiopathic central precocity. Patients with high testosterone but suppressed LH concentrations have gonadotropin-independent sexual precocity; in these patients, DHEA sulfate (DHEAS) and 17α-hydroxyprogesterone should be measured. High levels of testosterone and 17α-hydroxyprogesterone suggest the possibility of CAH due to 21α-hydroxylase or 11β-hydroxylase deficiency. If testosterone and DHEAS are elevated, adrenal tumors should be excluded by obtaining a computed tomography (CT)

1	suggest the possibility of CAH due to 21α-hydroxylase or 11β-hydroxylase deficiency. If testosterone and DHEAS are elevated, adrenal tumors should be excluded by obtaining a computed tomography (CT) scan of the adrenal glands. Patients with elevated testosterone but without increased 17α-hydroxyprogesterone or DHEAS should undergo careful evaluation of the testis by palpation and ultrasound to exclude a Leydig cell neoplasm. Activating mutations of the LH receptor should be considered in children with gonadotropinindependent precocious puberty in whom CAH, androgen abuse, and adrenal and testicular neoplasms have been excluded.

1	In patients with a known cause (e.g., a CNS lesion or a testicular tumor), therapy should be directed toward the underlying disorder. In patients with idiopathic CPP, long-acting GnRH analogues can be used to suppress gonadotropins and decrease testosterone, halt early pubertal development, delay accelerated bone maturation, prevent early epiphyseal closure, promote final height gain, and mitigate the psychosocial consequences of early pubertal development without causing osteoporosis. The treatment is most effective for increasing final adult height if it is initiated before age 6. Puberty resumes after discontinuation of the GnRH analogue. Counseling is an important aspect of the overall treatment strategy.

1	In children with gonadotropin-independent precocious puberty, inhibitors of steroidogenesis, such as ketoconazole, and AR antagonists have been used empirically. Long-term treatment with spironolactone (a weak androgen antagonist) and ketoconazole has been reported to normalize growth rate and bone maturation and to improve predicted height in small, nonrandomized trials in boys with familial male-limited precocious puberty. Aromatase inhibitors, such as testolactone and letrozole, have been used as an adjunct to antiandrogen and GnRH analogue therapy for children with familial male-limited precocious puberty, CAH, and McCune-Albright syndrome.

1	Puberty is delayed in boys if it has not ensued by age 14, an age that is 2–2.5 standard deviations above the mean for healthy children. Delayed puberty is more common in boys than in girls. There are four main categories of delayed puberty: (1) constitutional delay of growth and puberty (~60% of cases); (2) functional hypogonadotropic hypogonadism caused by systemic illness or malnutrition (~20% of cases); (3) hypogonadotropic hypogonadism caused by genetic or acquired defects in the hypothalamic-pituitary region (~10% of cases); and (4) hypergonadotropic hypogonadism secondary to primary gonadal failure (~15% of cases) (Table 411-1). Functional hypogonadotropic hypogonadism is more common in girls than in boys. Permanent causes of hypogonadotropic or hypergonadotropic hypogonadism are identified in >25% of boys with delayed puberty. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: Any history of systemic illness, eating disorders, excessive exercise, social and psychological problems, and abnormal patterns of linear growth during childhood should be verified. Boys with pubertal delay may have accompanying emotional and physical immaturity relative to their peers, which can be a source of anxiety. Physical examination should focus on height; arm span; weight; visual fields; and secondary sex characteristics, including hair growth, testicular volume, phallic size, and scrotal reddening and thinning. Testicular size >2.5 cm generally indicates that the child has entered puberty.

1	The main diagnostic challenge is to distinguish those with constitutional delay, who will progress through puberty at a later age, from those with an underlying pathologic process. Constitutional delay should be suspected when there is a family history and when there are delayed bone age and short stature. Pituitary priming by pulsatile GnRH is required before LH and FSH are synthesized and secreted normally. Thus, blunted responses to exogenous GnRH can be seen in patients with constitutional delay, GnRH deficiency, or pituitary disorders (see “GnRH Stimulation Testing,” above). On the other hand, low-normal basal gonadotropin levels or a normal response to exogenous GnRH is consistent with an early stage of puberty, which is often heralded by nocturnal GnRH secretion. Thus, constitutional delay is a diagnosis of exclusion that requires ongoing evaluation until the onset of puberty and the growth spurt.

1	If therapy is considered appropriate, it can begin with 25–50 mg testosterone enanthate or testosterone cypionate every 2 weeks, or by using a 2.5-mg testosterone patch or 25-mg testosterone gel. Because aromatization of testosterone to estrogen is obligatory for mediating androgen effects on epiphyseal fusion, concomitant treatment with aromatase inhibitors may allow attainment of greater final adult height. Testosterone treatment should be interrupted after 6 months to determine if endogenous LH and FSH secretion have ensued. Other causes of delayed puberty should be considered when there are associated clinical features or when boys do not enter puberty spontaneously after a year of observation or treatment.

1	Reassurance without hormonal treatment is appropriate for many individuals with presumed constitutional delay of puberty. However, the impact of delayed growth and pubertal progression on a child’s social relationships and school performance should be weighed. Also, boys with constitutional delay of puberty are less likely to achieve their full genetic height potential and have reduced total-body bone mass as adults, mainly due to narrow limb bones and vertebrae as a result of impaired periosteal expansion during puberty. Administration of androgen therapy to boys with constitutional delay does not affect final height, and when administered with an aromatase inhibitor, it may improve final height.

1	Because LH and FSH are trophic hormones for the testes, impaired secretion of these pituitary gonadotropins results in secondary hypogonadism, which is characterized by low testosterone in the setting of low LH and FSH. Those with the most severe deficiency have complete absence of pubertal development, sexual infantilism, and, in some cases, hypospadias and undescended testes. Patients with partial gonadotropin deficiency have delayed or arrested sex development. The 24-h LH secretory profiles are heterogeneous in patients with hypogonadotropic hypogonadism, reflecting variable abnormalities of LH pulse frequency or amplitude. In severe cases, basal LH is low and there are no LH pulses. A smaller subset of patients has low-amplitude LH pulses or markedly reduced pulse frequency. Occasionally, only sleep-entrained LH pulses occur, reminiscent of the pattern seen in the early stages of puberty. Hypogonadotropic hypogonadism can be classified into congenital and acquired disorders.

1	only sleep-entrained LH pulses occur, reminiscent of the pattern seen in the early stages of puberty. Hypogonadotropic hypogonadism can be classified into congenital and acquired disorders. Congenital disorders most commonly involve GnRH deficiency, which leads to gonadotropin deficiency. Acquired disorders are much more common than congenital disorders and may result from a variety of sellar mass lesions or infiltrative diseases of the hypothalamus or pituitary.

1	Congenital Disorders Associated with Gonadotropin Deficiency Congenital hypogonadotropic hypogonadism is a heterogeneous group of disorders characterized by decreased gonadotropin secretion and testicular dysfunction either due to impaired function of the GnRH pulse generator or the gonadotrope. The disorders characterized by GnRH deficiency represent a family of oligogenic disorders whose phenotype spans a wide spectrum. Some individuals with GnRH deficiency may suffer from complete absence of pubertal development, while others may manifest varying degrees of gonadotropin deficiency and pubertal delay, and a subset that carries the same mutations as their affected family members may even have normal reproductive function. In approximately 10% of men with idiopathic hypogonadotropic hypogonadism, reversal of gonadotropin deficiency may occur in adult life after sex 2363 steroid therapy. Also, a small fraction of men with idiopathic hypogonadotropic hypogonadism may present with

1	hypogonadism, reversal of gonadotropin deficiency may occur in adult life after sex 2363 steroid therapy. Also, a small fraction of men with idiopathic hypogonadotropic hypogonadism may present with androgen deficiency and infertility in adult life after having gone through apparently normal pubertal development. Nutritional, emotional, or metabolic stress may unmask gonadotropin deficiency and reproductive dysfunction (analogous to hypothalamic amenorrhea) in some patients who harbor mutations in the candidate genes but who previously had normal reproductive function. The clinical phenotype may include isolated anosmia or hyposmia. These striking variations in phenotypic presentation of GnRH deficiency have highlighted the important role of oligogenicity and gene-gene and gene-environment interactions in shaping the clinical phenotype.

1	Mutations in a number of genes involved in the development and migration of GnRH neurons or in the regulation of GnRH secretion have been linked to GnRH deficiency, although the genetic defect remains elusive in nearly two-thirds of cases. Familial hypogonadotropic hypogonadism can be transmitted as an X-linked (20%), autosomal recessive (30%), or autosomal dominant (50%) trait. Some individuals with idiopathic hypogonadotropic hypogonadism (IHH) have sporadic mutations in the same genes that cause inherited forms of the disorder. The genetic defects associated with GnRH deficiency can be conveniently classified as anosmic (Kallmann’s syndrome) or normosmic (Table 411-2), although the occurrence of both anosmic and normosmic forms of GnRH deficiency in the same families suggests commonality of pathophysiologic mechanisms. Kallmann’s syndrome, the anosmic form of GnRH deficiency, can result from mutations in one or more genes associated with olfactory bulb morphogenesis and the

1	of pathophysiologic mechanisms. Kallmann’s syndrome, the anosmic form of GnRH deficiency, can result from mutations in one or more genes associated with olfactory bulb morphogenesis and the migration of GnRH neurons from their origin in the region of the olfactory placode, along the scaffold established by the olfactory nerves, through the cribriform plate into their final location into the preoptic region of the hypothalamus. Thus, mutations in KAL1, FGF8, FGFR1, NELF, PROK2, PROK2R, and CHD7 have been described in patients with Kallmann’s syndrome. An X-linked form of IHH is caused by mutations in the KAL1 gene, which encodes anosmin, a protein that mediates the migration of neural progenitors of the olfactory bulb and GnRH-producing neurons. These individuals have GnRH deficiency and variable combinations of anosmia or hyposmia, renal defects, and neurologic abnormalities including mirror movements. Mutations in the FGFR1 gene cause an autosomal dominant form of hypogonadotropic

1	variable combinations of anosmia or hyposmia, renal defects, and neurologic abnormalities including mirror movements. Mutations in the FGFR1 gene cause an autosomal dominant form of hypogonadotropic hypogonadism that clinically resembles Kallmann’s syndrome; mutations in its putative ligand, FGF8 gene product, have also been associated with IHH. Prokineticin 2 (PROK2) also encodes a protein involved in migration and development of olfactory and GnRH neurons. Recessive mutations in PROK2 or in its receptor, PROKR2, have been associated with both anosmic and normosmic forms of hypogonadotropic hypogonadism.

1	Normosmic GnRH deficiency results from defects in pulsatile GnRH secretion, its regulation, or its action on the gonadotrope and has been associated with mutations in GnRHR, GNRH1, KISS1R, TAC3, TACR3, and NROB1 (DAX1). Some mutations, such as those in PROK2, PROKR2, and CHD7, have been associated with both the anosmic and normosmic forms of IHH. GnRHR mutations, the most frequent identifiable cause of normosmic IHH, account for ~40% of autosomal recessive and 10% of sporadic cases of hypogonadotropic hypogonadism. These patients have decreased LH response to exogenous GnRH. Some receptor mutations alter GnRH binding affinity, allowing apparently normal responses to pharmacologic doses of exogenous GnRH, whereas other mutations may alter signal transduction downstream of hormone binding. Mutations of the GnRH1 gene have also been reported in patients with hypogonadotropic hypogonadism, although they are rare. G protein–coupled receptor KISS1R (GPR54) and its cognate ligand, kisspeptin

1	of the GnRH1 gene have also been reported in patients with hypogonadotropic hypogonadism, although they are rare. G protein–coupled receptor KISS1R (GPR54) and its cognate ligand, kisspeptin (KISS1), are important regulators of sexual maturation in primates. Recessive mutations in GPR54 cause gonadotropin deficiency without anosmia. Patients retain responsiveness to exogenous GnRH, suggesting an abnormality in the neural pathways controlling GnRH release. The genes encoding neurokinin B (TAC3), which is involved in preferential activation of GnRH release in early development, and its receptor (TAC3R) have been implicated

1	Disorders of the Testes and Male Reproductive System A. Hypogonadotropic Hypogonadism due to GnRH Deficiency A1. GnRH Deficiency Associated with Hyposmia or Anosmia KAL1 Xp22 X-linked Anosmia, renal agenesis, synkinesia, cleft lip/palate, oculomotor/visuospatial defects, gut malformations NELF 9q34.3 AR Anosmia, hypogonadotropic hypogonadism FGFR1 8p11-p12 AD Anosmia, cleft lip/palate, synkinesia, syndactyly PROK2R 20p12.3 AR Variable CHD7 8q12.1 Anosmia, other features of CHARGE syndrome A2. GnRH Deficiency with Normal Sense of Smell GNRHR 4q21 AR None GnRH1 8p21 AR None KISS1R 19p13 AR None TAC3 12q13 AR Microphallus, cryptorchidism, reversal of GnRH deficiency TAC3R 4q25 AR Microphallus, cryptorchidism, reversal B. Hypogonadotropic Hypogonadism not due to GnRH Deficiency

1	Abbreviations: ACTH, adrenocorticotropic hormone; AD, autosomal dominant; AR, autosomal recessive; CHARGE, eye coloboma, choanal atresia, growth and developmental retardation, genitourinary anomalies, ear anomalies; CPHD, combined pituitary hormone deficiency; DAX1, dosage-sensitive sex-reversal, adrenal hypoplasia congenita, X-chromosome; FGFR1, fibroblast growth factor receptor 1; FSH, follicle-stimulating hormone; FSHβ, follicle-stimulating hormone β-subunit; GH, growth hormone; GnRH, gonadotropinreleasing hormone; GNRHR, gonadotropin-releasing hormone receptor; GPR54, G protein– coupled receptor 54; HESX1, homeo box gene expressed in embryonic stem cells 1; KAL1, interval-1 gene; LEP, leptin; LEPR, leptin receptor; LH, luteinizing hormone; LHβ, luteinizing hormone β-subunit; LHX3, LIM homeobox gene 3; NELF, nasal embryonic LHRH factor; PC1, prohormone convertase 1; PROK2, prokineticin 2; PROP1, Prophet of Pit 1; SF1, steroidogenic factor 1; TAC3, tachykinin 3; TAC3R, tachykinin 3

1	homeobox gene 3; NELF, nasal embryonic LHRH factor; PC1, prohormone convertase 1; PROK2, prokineticin 2; PROP1, Prophet of Pit 1; SF1, steroidogenic factor 1; TAC3, tachykinin 3; TAC3R, tachykinin 3 receptor.

1	in some families with normosmic IHH. Mutations in more than one gene (digenicity or oligogenicity) may contribute to clinical heterogeneity in IHH patients. X-linked hypogonadotropic hypogonadism also occurs in adrenal hypoplasia congenita, a disorder caused by mutations in the DAX1 gene, which encodes a nuclear receptor in the adrenal gland and reproductive axis. Adrenal hypoplasia congenita is characterized by absent development of the adult zone of the adrenal cortex, leading to neonatal adrenal insufficiency. Puberty usually does not occur or is arrested, reflecting variable degrees of gonadotropin deficiency. Although sexual differentiation is normal, most patients have testicular dysgenesis and impaired spermatogenesis despite gonadotropin replacement. Less commonly, adrenal hypoplasia congenita, sex reversal, and hypogonadotropic hypogonadism can be caused by mutations of steroidogenic factor 1 (SF1). Rarely, recessive mutations in the LHβ or FSHβ gene have been described in

1	congenita, sex reversal, and hypogonadotropic hypogonadism can be caused by mutations of steroidogenic factor 1 (SF1). Rarely, recessive mutations in the LHβ or FSHβ gene have been described in patients with selective deficiencies of these gonadotropins. In approximately 10% of men with IHH, reversal of gonadotropin deficiency may occur in adult life.

1	Also, a small fraction of men with IHH may present with androgen deficiency and infertility in adult life after having gone through apparently normal pubertal development. A number of homeodomain transcription factors are involved in the development and differentiation of the specialized hormone-producing cells within the pituitary gland (Table 411-2). Patients with mutations of PROP1 have combined pituitary hormone deficiency that includes GH, prolactin (PRL), thyroid-stimulating hormone (TSH), LH, and FSH, but not ACTH. LHX3 mutations cause combined pituitary hormone deficiency in association with cervical spine rigidity. HESX1 mutations cause septo-optic dysplasia and combined pituitary hormone deficiency.

1	Prader-Willi syndrome is characterized by obesity, hypotonic musculature, mental retardation, hypogonadism, short stature, and small hands and feet. Prader-Willi syndrome is a genomic imprinting disorder caused by deletions of the proximal portion of the paternally derived chromosome 15q11-15q13 region, which contains a bipartite imprinting center, uniparental disomy of the maternal alleles, or mutations of the genes/loci involved in imprinting (Chap. 83e). Laurence-Moon syndrome is an autosomal recessive disorder characterized by obesity, hypogonadism, mental retardation, polydactyly, and retinitis pigmentosa. Recessive mutations of leptin, or its receptor, cause severe obesity and pubertal arrest, apparently because of hypothalamic GnRH deficiency (Chap. 415e).

1	Acquired Hypogonadotropic Disorders • severe illness, sTress, malnuTri-Tion, and exercise These factors may cause reversible gonadotropin deficiency. Although gonadotropin deficiency and reproductive dysfunction are well documented in these conditions in women, men exhibit similar but less pronounced responses. Unlike women, most male runners and other endurance athletes have normal gonadotropin and sex steroid levels, despite low body fat and frequent intensive exercise. Testosterone levels fall at the onset of illness and recover during recuperation. The magnitude of gonadotropin suppression generally correlates with the severity of illness. Although hypogonadotropic hypogonadism is the most common cause of androgen deficiency in patients with acute illness, some have elevated levels of LH and FSH, which suggest primary gonadal dysfunction. The pathophysiology of reproductive dysfunction during acute illness is unknown but likely involves a combination of cytokine and/or

1	levels of LH and FSH, which suggest primary gonadal dysfunction. The pathophysiology of reproductive dysfunction during acute illness is unknown but likely involves a combination of cytokine and/or glucocorticoid effects. There is a high frequency of low testosterone levels in patients with chronic illnesses such as HIV infection, end-stage renal disease, chronic obstructive lung disease, and many types of cancer and in patients receiving glucocorticoids. About 20% of HIV-infected men with low testosterone levels have elevated LH and FSH levels; these patients presumably have primary testicular dysfunction. The remaining 80% have either normal or low LH and FSH levels; these men have a central hypothalamic-pituitary defect or a dual defect involving both the testis and the hypothalamic-pituitary centers. Muscle wasting is common in chronic diseases associated with hypogonadism, which also leads to debility, poor quality of life, and adverse outcome of disease. There is great interest

1	centers. Muscle wasting is common in chronic diseases associated with hypogonadism, which also leads to debility, poor quality of life, and adverse outcome of disease. There is great interest in exploring strategies that can reverse androgen deficiency or attenuate the sarcopenia associated with chronic illness.

1	Men using opioids for relief of cancer or noncancerous pain or because of addiction often have suppressed testosterone and LH levels and high prevalence of sexual dysfunction and osteoporosis; the degree of suppression is dose-related and particularly severe with long-acting opioids such as methadone. Opioids suppress GnRH secretion and alter the sensitivity to feedback inhibition by gonadal steroids. Men who are heavy users of marijuana have decreased testosterone secretion and sperm production. The mechanism of marijuana-induced hypogonadism is decreased GnRH secretion. Gynecomastia observed in marijuana users can also be caused by plant estrogens in crude preparations. Androgen deprivation therapy in men with prostate cancer has been associated with increased risk of bone fractures, diabetes mellitus, cardiovascular events, fatigue, sexual dysfunction, and poor quality of life.

1	oBeSity In men with mild to moderate obesity, SHBG levels decrease in proportion to the degree of obesity, resulting in lower total testosterone levels. However, free testosterone levels usually remain within the normal range. The decrease in SHBG levels is caused by increased circulating insulin, which inhibits SHBG production. Estradiol levels are higher in obese men compared to healthy, nonobese controls, because of aromatization of testosterone to estradiol in adipose tissue. Weight loss is associated with reversal of these abnormalities including an increase in total and free testosterone levels and a decrease in estradiol levels. A subset of obese men with moderate to severe obesity may have a defect in the hypothalamic-pituitary axis as suggested by low free testosterone in the absence of elevated gonadotropins. Weight gain in adult men can accelerate the rate of age-related decline in testosterone levels.

1	Hyperprolactinemia (See also Chap. 403) Elevated PRL levels are associated with hypogonadotropic hypogonadism. PRL inhibits hypothalamic GnRH secretion either directly or through modulation of tuberoinfundibular dopaminergic pathways. A PRL-secreting tumor may also destroy the surrounding gonadotropes by invasion or compression of the pituitary stalk. Treatment with dopamine agonists reverses gonadotropin deficiency, although there may be a delay relative to PRL suppression.

1	Sellar maSS leSionS Neoplastic and nonneoplastic lesions in the hypothalamus or pituitary can directly or indirectly affect gonadotrope function. In adults, pituitary adenomas constitute the largest category of space-occupying lesions affecting gonadotropin and other pituitary hormone production. Pituitary adenomas that extend into the suprasellar region can impair GnRH secretion and mildly increase PRL secretion (usually <50 μg/L) because of impaired tonic inhibition by dopaminergic pathways. These tumors should be distinguished from prolactinomas, which typically secrete higher PRL levels. The presence of diabetes insipidus suggests the possibility of a craniopharyngioma, infiltrative disorder, or other hypothalamic lesions (Chap. 404).

1	HemocHromatoSiS (See also Chap. 428) Both the pituitary and testis can be affected by excessive iron deposition. However, the pituitary defect is the predominant lesion in most patients with hemochromatosis and hypogonadism. The diagnosis of hemochromatosis is suggested by the association of characteristic skin discoloration, hepatic enlargement or dysfunction, diabetes mellitus, arthritis, cardiac conduction defects, and hypogonadism. Common causes of primary testicular dysfunction include Klinefelter’s syndrome, uncorrected cryptorchidism, cancer chemotherapy, radiation to the testes, trauma, torsion, infectious orchitis, HIV infection, anorchia syndrome, and myotonic dystrophy. Primary testicular disorders may be associated with impaired spermatogenesis, decreased androgen production, or both. See Chap. 410 for disorders of testis development, androgen synthesis, and androgen action.

1	Klinefelter’s Syndrome (See also Chap. 410) Klinefelter’s syndrome is the most common chromosomal disorder associated with testicular dysfunction and male infertility. It occurs in about 1 in 600 live-born males. Azoospermia is the rule in men with Klinefelter’s syndrome who have the 47,XXY karyotype; however, men with mosaicism may have germ cells, especially at a younger age. The clinical phenotype of Klinefelter’s syndrome can be heterogeneous possibly because of mosaicism, polymorphisms in AR gene, variable testosterone levels, or other genetic factors. Testicular histology shows hyalinization of seminiferous tubules and absence of spermatogenesis. Although their function is impaired, the number of Leydig cells appears to increase. Testosterone is decreased and estradiol is increased, leading to clinical features of undervirilization and gynecomastia. Men with Klinefelter’s syndrome are at increased risk of systemic lupus erythematosus, Sjögren’s syndrome, breast cancer, diabetes

1	leading to clinical features of undervirilization and gynecomastia. Men with Klinefelter’s syndrome are at increased risk of systemic lupus erythematosus, Sjögren’s syndrome, breast cancer, diabetes mellitus, osteoporosis, non-Hodgkin’s lymphoma, and lung cancer, and reduced risk of prostate cancer. Periodic mammography for breast cancer surveillance is recommended for men with Klinefelter’s syndrome. Fertility has been achieved by intracytoplasmic injection of sperm retrieved surgically from testicular biopsies of men with Klinefelter’s syndrome, including some men with the nonmosaic form of Klinefelter’s syndrome. The 2365 karyotypes 48,XXXY and 49,XXXXY are associated with a more severe phenotype, increased risk of congenital malformations, and lower intelligence than 47,XXY individuals.

1	Cryptorchidism Cryptorchidism occurs when there is incomplete descent of the testis from the abdominal cavity into the scrotum. About 3% of full-term and 30% of premature male infants have at least one undescended testis at birth, but descent is usually complete by the first few weeks of life. The incidence of cryptorchidism is <1% by 9 months of age. Androgens regulate predominantly the inguinoscrotal descent of the testes through degeneration of the craniosuspensory ligament and a shortening of the gubernaculums, respectively. Mutations in INSL3 and leucine-rich repeat family of G protein–coupled receptor 8 (LGR8), which regulate the transabdominal portion of testicular descent, have been found in some patients with cryptorchidism.

1	Cryptorchidism is associated with increased risk of malignancy, infertility, inguinal hernia, and torsion. Unilateral cryptorchidism, even when corrected before puberty, is associated with decreased sperm count, possibly reflecting unrecognized damage to the fully descended testis or other genetic factors. Epidemiologic, clinical, and molecular evidence supports the idea that cryptorchidism, hypospadias, impaired spermatogenesis, and testicular cancer may be causally related to common genetic and environment perturbations and are components of the testicular dysgenesis syndrome.

1	Acquired Testicular Defects Viral orchitis may be caused by the mumps virus, echovirus, lymphocytic choriomeningitis virus, and group B arboviruses. Orchitis occurs in as many as one-fourth of adult men with mumps; the orchitis is unilateral in about two-thirds and bilateral in the remainder. Orchitis usually develops a few days after the onset of parotitis but may precede it. The testis may return to normal size and function or undergo atrophy. Semen analysis returns to normal for three-fourths of men with unilateral involvement but for only one-third of men with bilateral orchitis. Trauma, including testicular torsion, can also cause secondary atrophy of the testes. The exposed position of the testes in the scrotum renders them susceptible to both thermal and physical trauma, particularly in men with hazardous occupations.

1	The testes are sensitive to radiation damage. Doses >200 mGy (20 rad) are associated with increased FSH and LH levels and damage to the spermatogonia. After ~800 mGy (80 rad), oligospermia or azoospermia develops, and higher doses may obliterate the germinal epithelium. Permanent androgen deficiency in adult men is uncommon after therapeutic radiation; however, most boys given direct testicular radiation therapy for acute lymphoblastic leukemia have permanently low testosterone levels. Sperm banking should be considered before patients undergo radiation treatment or chemotherapy. Drugs interfere with testicular function by several mechanisms, including inhibition of testosterone synthesis (e.g., ketoconazole), blockade of androgen action (e.g., spironolactone), increased estrogen (e.g., marijuana), or direct inhibition of spermatogenesis (e.g., chemotherapy).

1	Combination chemotherapy for acute leukemia, Hodgkin’s disease, and testicular and other cancers may impair Leydig cell function and cause infertility. The degree of gonadal dysfunction depends on the type of chemotherapeutic agent and the dose and duration of therapy. Because of high response rates and the young age of these men, infertility and androgen deficiency have emerged as important longterm complications of cancer chemotherapy. Cyclophosphamide and combination regimens containing procarbazine are particularly toxic to germ cells. Thus, 90% of men with Hodgkin’s lymphoma receiving MOPP (mechlorethamine, vincristine, procarbazine, prednisone) therapy develop azoospermia or extreme oligozoospermia; newer regimens that do not include procarbazine, such as ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine), are less toxic to germ cells.

1	Alcohol, when consumed in excess for prolonged periods, decreases testosterone, independent of liver disease or malnutrition. Elevated estradiol and decreased testosterone levels may occur in men taking digitalis.

1	Disorders of the Testes and Male Reproductive System 2366 The occupational and recreational history should be carefully evaluated in all men with infertility because of the toxic effects of many chemical agents on spermatogenesis. Known environmental hazards include pesticides (e.g., vinclozolin, dicofol, atrazine), sewage contaminants (e.g., ethinyl estradiol in birth control pills, surfactants such as octylphenol, nonyphenol), plasticizers (e.g., pthalates), flame retardants (e.g., polychlorinated biphenyls, polybrominated diphenol ethers), industrial pollutants (e.g., heavy metals cadmium and lead, dioxins, polycyclic aromatic hydrocarbons), microwaves, and ultrasound. In some populations, sperm density is said to have declined by as much as 40% in the past 50 years. Environmental estrogens or antiandrogens may be partly responsible. Testicular failure also occurs as a part of polyglandular autoimmune insufficiency (Chap. 408). Sperm antibodies can cause isolated male infertility.

1	or antiandrogens may be partly responsible. Testicular failure also occurs as a part of polyglandular autoimmune insufficiency (Chap. 408). Sperm antibodies can cause isolated male infertility. In some instances, these antibodies are secondary phenomena resulting from duct obstruction or vasectomy. Granulomatous diseases can affect the testes, and testicular atrophy occurs in 10–20% of men with lepromatous leprosy because of direct tissue invasion by the mycobacteria. The tubules are involved initially, followed by endarteritis and destruction of Leydig cells. Systemic disease can cause primary testis dysfunction in addition to suppressing gonadotropin production. In cirrhosis, a combined testicular and pituitary abnormality leads to decreased testosterone production independent of the direct toxic effects of ethanol. Impaired hepatic extraction of adrenal androstenedione leads to extraglandular conversion to estrone and estradiol, which partially suppresses LH. Testicular atrophy and

1	toxic effects of ethanol. Impaired hepatic extraction of adrenal androstenedione leads to extraglandular conversion to estrone and estradiol, which partially suppresses LH. Testicular atrophy and gynecomastia are present in approximately one-half of men with cirrhosis. In chronic renal failure, androgen synthesis and sperm production decrease despite elevated gonadotropins. The elevated LH level is due to reduced clearance, but it does not restore normal testosterone production. About one-fourth of men with renal failure have hyperprolactinemia. Improvement in testosterone production with hemodialysis is incomplete, but successful renal transplantation may return testicular function to normal. Testicular atrophy is present in one-third of men with sickle cell anemia. The defect may be at either the testicular or the hypothalamic-pituitary level. Sperm density can decrease temporarily after acute febrile illness in the absence of a change in testosterone production. Infertility in men

1	either the testicular or the hypothalamic-pituitary level. Sperm density can decrease temporarily after acute febrile illness in the absence of a change in testosterone production. Infertility in men with celiac disease is associated with a hormonal pattern typical of androgen resistance, namely elevated testosterone and LH levels. Neurologic diseases associated with altered testicular function include myotonic dystrophy, spinobulbar muscular atrophy, and paraplegia. In myotonic dystrophy, small testes may be associated with impairment of both spermatogenesis and Leydig cell function. Spinobulbar muscular atrophy is caused by an expansion of the glutamine repeat sequences in the amino-terminal region of the AR; this expansion impairs function of the AR, but it is unclear how the alteration is related to the neurologic manifestations. Men with spinobulbar muscular atrophy often have undervirilization and infertility as a late manifestation. Spinal cord lesions that cause paraplegia can

1	is related to the neurologic manifestations. Men with spinobulbar muscular atrophy often have undervirilization and infertility as a late manifestation. Spinal cord lesions that cause paraplegia can lead to a temporary decrease in testosterone levels and may cause persistent defects in spermatogenesis; some patients retain the capacity for penile erection and ejaculation.

1	Mutations in the AR cause resistance to the action of testosterone and DHT. These X-linked mutations are associated with variable degrees of defective male phenotypic development and undervirilization (Chap. 410). Although not technically hormone-insensitivity syndromes, two genetic disorders impair testosterone conversion to active sex steroids. Mutations in the SRD5A2 gene, which encodes 5α-reductase type 2, prevent the conversion of testosterone to DHT, which is necessary for the normal development of the male external genitalia. Mutations in the CYP19 gene, which encodes aromatase, prevent testosterone conversion to estradiol. Males with CYP19 mutations have delayed epiphyseal fusion, tall stature, eunuchoid proportions, and osteoporosis, consistent with evidence from an estrogen receptor– deficient individual that these testosterone actions are mediated indirectly via estrogen.

1	Gynecomastia refers to enlargement of the male breast. It is caused by excess estrogen action and is usually the result of an increased estrogen-to-androgen ratio. True gynecomastia is associated with glandular breast tissue that is >4 cm in diameter and often tender. Glandular tissue enlargement should be distinguished from excess adipose tissue: glandular tissue is firmer and contains fibrous-like cords. Gynecomastia occurs as a normal physiologic phenomenon in the newborn (due to transplacental transfer of maternal and placental estrogens), during puberty (high estrogen-to-androgen ratio in early stages of puberty), and with aging (increased fat tissue and increased aromatase activity), but it can also result from pathologic conditions associated with androgen deficiency or estrogen excess. The prevalence of gynecomastia increases with age and body mass index (BMI), likely because of increased aromatase activity in adipose tissue. Medications that alter androgen metabolism or

1	excess. The prevalence of gynecomastia increases with age and body mass index (BMI), likely because of increased aromatase activity in adipose tissue. Medications that alter androgen metabolism or action may also cause gynecomastia. The relative risk of breast cancer is increased in men with gynecomastia, although the absolute risk is relatively small.

1	Any cause of androgen deficiency can lead to gynecomastia, reflecting an increased estrogen-to-androgen ratio, because estrogen synthesis still occurs by aromatization of residual adrenal and gonadal androgens. Gynecomastia is a characteristic feature of Klinefelter’s syndrome (Chap. 410). Androgen insensitivity disorders also cause gynecomastia. Excess estrogen production may be caused by tumors, including Sertoli cell tumors in isolation or in association with Peutz-Jeghers syndrome or Carney complex. Tumors that produce hCG, including some testicular tumors, stimulate Leydig cell estrogen synthesis. Increased conversion of androgens to estrogens can be a result of increased availability of substrate (androstenedione) for extraglandular estrogen formation (CAH, hyperthyroidism, and most feminizing adrenal tumors) or of diminished catabolism of androstenedione (liver disease) so that estrogen precursors are shunted to aromatase in peripheral sites. Obesity is associated with

1	and most feminizing adrenal tumors) or of diminished catabolism of androstenedione (liver disease) so that estrogen precursors are shunted to aromatase in peripheral sites. Obesity is associated with increased aromatization of androgen precursors to estrogens. Extraglandular aromatase activity can also be increased in tumors of the liver or adrenal gland or rarely as an inherited disorder. Several families with increased peripheral aromatase activity inherited as an autosomal dominant or as an X-linked disorder have been described. In some families with this disorder, an inversion in chromosome 15q21.2-3 causes the CYP19 gene to be activated by the regulatory elements of contiguous genes, resulting in excessive estrogen production in the fat and other extragonadal tissues. Drugs can cause gynecomastia by acting directly as estrogenic substances (e.g., oral contraceptives, phytoestrogens, digitalis) or by inhibiting androgen synthesis (e.g., ketoconazole) or action (e.g.,

1	can cause gynecomastia by acting directly as estrogenic substances (e.g., oral contraceptives, phytoestrogens, digitalis) or by inhibiting androgen synthesis (e.g., ketoconazole) or action (e.g., spironolactone).

1	Because up to two-thirds of pubertal boys and half of hospitalized men have palpable glandular tissue that is benign, detailed investigation or intervention is not indicated in all men presenting with gynecomastia (Fig. 411-5). In addition to the extent of gynecomastia, recent onset, rapid growth, tender tissue, and occurrence in a lean subject should prompt more extensive evaluation. This should include a careful drug history, measurement and examination of the testes, assessment of virilization, evaluation of liver function, and hormonal measurements including testosterone, estradiol, and androstenedione, LH, and hCG. A karyotype should be obtained in men with very small testes to exclude Klinefelter’s syndrome. Despite extensive evaluation, the etiology is established in fewer than one-half of patients.

1	When the primary cause can be identified and corrected, breast enlargement usually subsides over several months. However, if gynecomastia is of long duration, surgery is the most effective therapy. Indications for surgery include severe psychological and/or cosmetic problems, continued growth or tenderness, or suspected malignancy. In patients who have painful gynecomastia and in whom surgery cannot be performed, treatment with antiestrogens such as tamoxifen (20 mg/d) can reduce pain and breast tissue size in over half the patients. Estrogen receptor antagonists, tamoxifen and raloxifene, have been reported in small trials to reduce breast size in men with pubertal gynecomastia, although complete regression of breast enlargement is unusual with the use of estrogen receptor antagonists. Aromatase inhibitors can be effective in the early proliferative phase of the disorder. However, in a randomized trial in men with established gynecomastia, anastrozole proved no more effective than

1	Aromatase inhibitors can be effective in the early proliferative phase of the disorder. However, in a randomized trial in men with established gynecomastia, anastrozole proved no more effective than placebo in reducing breast size. Tamoxifen is effective in the prevention and treatment of breast enlargement and breast pain in men with prostate cancer who are receiving antiandrogen therapy.

1	Increased aromatization of androgen to estrogen (obesity, feminizing adrenal tumors, Sertoli cell tumors, inherited dysregulation of aromatase) Breast enlargement True glandular enlargement Breast mass hard or fixed to the underlying tissue Recent onset and rapid growth Mammography and/or biopsy to exclude malignancy Follow-up with serial examinations Increased E2, normal T, altered E2/T ratio Increased hCG˜Exclude hCG secreting tumors Low T, high E2/T ratio Androgen deficiency syndrome Serum T, LH, FSH, estradiol, and hCG˜Onset in neonatal or peripubertal period Causative drugs Known liver disease Size <4 cm Clinical evidence of androgen deficiency Breast tenderness Very small testes Glandular tissue >4 cm in diameter Absence of causative drugs or liver disease Increased adipose tissue FIGURE 411-5 Evaluation of gynecomastia. E2, 17β-estradiol; hCGβ, human chorionic gonadotropin β; T, testosterone.

1	A number of cross-sectional and longitudinal studies (e.g., The Baltimore Longitudinal Study of Aging, the Framingham Heart Study, the Massachusetts Male Aging Study, and the European Male Aging Study) have established that testosterone concentrations decrease with advancing age. This age-related decline starts in the third decade of life and progresses slowly; the rate of decline in testosterone concentrations is greater in obese men, men with chronic illness, and those taking medications than in healthy older men. Because SHBG concentrations are higher in older men than in younger men, free or bioavailable testosterone concentrations decline with aging to a greater extent than total testosterone concentrations. The age-related decline in testosterone is due to defects at all levels of the hypothalamic-pituitarytesticular axis: pulsatile GnRH secretion is attenuated, LH response to GnRH is reduced, and testicular response to LH is impaired. However, the gradual rise of LH with aging

1	hypothalamic-pituitarytesticular axis: pulsatile GnRH secretion is attenuated, LH response to GnRH is reduced, and testicular response to LH is impaired. However, the gradual rise of LH with aging suggests that testis dysfunction is

1	FIGURE 411-6 Evaluation of hypogonadism. GnRH, gonadotropinreleasing hormone; LH, luteinizing hormone; T, testosterone. the main cause of declining androgen levels. The term andropause has been used to denote age-related decline in testosterone concentrations; this term is a misnomer because there is no discrete time when testosterone concentrations decline abruptly. The approach to evaluating hypogonadism is summarized in Fig. 411-6.

1	In epidemiologic surveys, low total and bioavailable testosterone concentrations have been associated with decreased appendicular skeletal muscle mass and strength, decreased self-reported physical function, higher visceral fat mass, insulin resistance, and increased risk of coronary artery disease and mortality, although the associations are weak. An analysis of signs and symptoms in older men in the European Male Aging Study revealed a syndromic association of sexual symptoms with total testosterone levels below 320 ng/dL and free testosterone levels below 64 pg/mL in community-dwelling older men. In systematic reviews of randomized controlled trials, testosterone therapy of healthy older men with low or low-normal testosterone levels was associated with greater increments in lean body mass, grip strength, and self-reported physical function compared with placebo. Testosterone therapy also induced greater improvement in vertebral but not femoral bone mineral density. Testosterone

1	mass, grip strength, and self-reported physical function compared with placebo. Testosterone therapy also induced greater improvement in vertebral but not femoral bone mineral density. Testosterone therapy of older men with sexual dysfunction and unequivocally low testosterone levels improves libido, but testosterone effects on erectile function and response to selective phosphodiesterase inhibitors have been inconsistent. Testosterone therapy has not been shown to improve depression scores, fracture risk, cognitive function, response to phosphodiesterase inhibitors, or clinical outcomes in older men. Furthermore, neither the long-term risks nor clinical benefits of testosterone therapy in older men have been demonstrated in adequately powered trials. Although there is no evidence that testosterone causes prostate cancer, there is concern that testosterone therapy might cause subclinical prostate cancers to grow. Testosterone therapy is associated with increased risk of detection of

1	causes prostate cancer, there is concern that testosterone therapy might cause subclinical prostate cancers to grow. Testosterone therapy is associated with increased risk of detection of prostate events (Fig. 411-7).

1	Disorders of the Testes and Male Reproductive System Spitzer 2012 4 70 2 70 2.06 [0.37; 11.63] 4.0% Hoyos 2012 1 33 0 34 3.18 [0.13; 81.01] 1.1% 0.01 0.1 1 10 100 + + + + + + + + + + + + + + + + + + + + + + + + + + 1.00 [0.06; 16.37] 1.5% Kaufman 2011 11 234 0 40 4.17 [0.24; 72.13] 1.5% Jones 2011 5 108 12 112 0.40 [0.14; 1.19] 10.3% Aversa 2010 0 40 1 10 0.08 [0.00; 2.07] 1.1% 6.05 [2.22; 16.51] 11.9% Kalinchenko 2010 0 113 2 71 0.12 [0.01; 2.59] 1.3% Srinivas-Shankar 2010 5 138 2 136 2.52 [0.48; 13.21] 4.4% Caminiti 2009 2 35 1 35 2.06 [0.18; 23.83] 2.0% Chapman 2009 1 11 1 12 1.10 [0.06; 20.01] 1.4% Legros 2009 1 237 0 79 1.01 [0.04; 25.01] 1.2% Emmelot-Vonk 2008 8 120 3 117 2.71 [0.70; 10.49] 6.6% Svartberg 2008 1 19 0 19 3.16 [0.12; 82.64] 1.1% 1.20 [0.34; 4.18] 7.7% Malkin 2006 4 37 4 39 1.06 [0.25; 4.59] 5.6% Merza 2006 0 20 1 19 0.30 [0.01; 7.85] 1.1% 1.32 [0.39; 4.50] 8.0% Amory 2004 1 24 0 24 3.13 [0.12; 80.68] 1.1% Kenny 2004 0 6 1 5 0.23 [0.01; 7.05] 1.0%

1	Merza 2006 0 20 1 19 0.30 [0.01; 7.85] 1.1% 1.32 [0.39; 4.50] 8.0% Amory 2004 1 24 0 24 3.13 [0.12; 80.68] 1.1% Kenny 2004 0 6 1 5 0.23 [0.01; 7.05] 1.0% Svartberg 2004 0 15 1 14 0.29 [0.01; 7.74] 1.1% Snyder 2001 9 54 5 54 1.96 [0.61; 6.29] 8.8% English 2000 2 25 0 25 5.43 [0.25; 118.96] 1.3% Sih 1997 1 17 1 15 0.88 [0.05; 15.33] 1.5% Hall 1996 0 17 2 18 0.19 [0.01; 4.23] 1.2% Marin 1993 1 11 0 10 3.00 [0.11; 82.40] 1.1% Copenhagen 1986 16 134 5 87 2.22 [0.78; 6.31] 11.0% Fixed effect model 115 1733 65 1261 1.54 [1.09; 2.18] 100% Heterogeneity: I-squared = 7.8%, tau-squared = 0.0742, p = 0.3484 A < Testosterone Favors Placebo >

1	Fixed effect model 115 1733 65 1261 1.54 [1.09; 2.18] 100% Heterogeneity: I-squared = 7.8%, tau-squared = 0.0742, p = 0.3484 A < Testosterone Favors Placebo > FIGURE 411-7 Meta-analyses of cardiovascular and prostate adverse events associated with testosterone therapy. A. A meta-analysis of cardiovascular-related events in randomized testosterone trials of 12 weeks or longer in duration. Randomization to testosterone was associated with a significantly increased risk of cardiovascular-related event (odds ratio [OR] 1.54). (Modified with permission from L Xu et al: Testosterone therapy and cardiovascular events among men: a systematic review and meta-analysis of placebo-controlled randomized trials BMC Med 11:108, 2013.)

1	B. The relative risk of prostate events and the associated 95% confidence intervals (CIs) in a meta-analysis of randomized testosterone trials. PSA, prostate-specific antigen. (Data were derived from a meta-analysis by MM Fernández-Balsells et al: J Clin Endocrinol Metab 95:2560, 2010, and the figure was reproduced with permission from M Spitzer et al: Nat Rev Endocrinol 9:414, 2013.)

1	One randomized testosterone trial in older men with mobility limi-Population screening of all older men for low testosterone levtation and high burden of chronic conditions, such as diabetes, heart els is not recommended, and testing should be restricted to men disease, hypertension, and hyperlipidemia, reported a greater number who have symptoms or physical features attributable to androof cardiovascular events in men randomized to the testosterone arm of gen deficiency. Testosterone therapy is not recommended for all the study than in those randomized to the placebo arm. Since then, two older men with low testosterone levels. In older men with siglarge retrospective analyses of patient databases have reported higher nificant symptoms of androgen deficiency who have testosterone frequency of cardiovascular events, including myocardial infarction, in levels below 200 ng/dL, testosterone therapy may be considered on older men with preexisting heart disease (Fig. 411-7). an

1	frequency of cardiovascular events, including myocardial infarction, in levels below 200 ng/dL, testosterone therapy may be considered on older men with preexisting heart disease (Fig. 411-7). an individualized basis and should be instituted after careful discussion of the risks and benefits (see “Testosterone Replacement,” below).

1	Testicular morphology, semen production, and fertility are maintained up to a very old age in men. Although concern has been expressed about age-related increases in germ cell mutations and impairment of DNA repair mechanisms, there is no clear evidence that the frequency of chromosomal aneuploidy is increased in the sperm of older men. However, the incidence of autosomal dominant diseases, such as achondroplasia, polyposis coli, Marfan’s syndrome, and Apert’s syndrome, increases in the offspring of men who are advanced in age, consistent with transmission of sporadic missense mutations. Advanced paternal age may be associated with increased rates of de novo mutations, which may contribute to an increased risk of neurodevelopmental diseases such as schizophrenia and autism. The somatic mutations in male germ cells that enhance the proliferation of germ cells could lead to within-testis expansion of mutant clonal lines, thus favoring the propagation of germ cells carrying these

1	mutations in male germ cells that enhance the proliferation of germ cells could lead to within-testis expansion of mutant clonal lines, thus favoring the propagation of germ cells carrying these pathogenic mutations and increasing the risk of mutations in the offspring of older fathers (the “selfish spermatogonial selection” hypothesis).

1	APPROACH TO THE PATIENT:

1	Hypogonadism is often characterized by decreased sex drive, reduced frequency of sexual activity, inability to maintain erections, reduced beard growth, loss of muscle mass, decreased testicular size, and gynecomastia. Erectile dysfunction and androgen deficiency are two distinct clinical disorders that can coexist in middle-aged and older men. Less than 10% of patients with erectile dysfunction have testosterone deficiency. Thus, it is useful to evaluate men presenting with erectile dysfunction for androgen deficiency. Except when extreme, these clinical features of androgen deficiency may be difficult to distinguish from changes that occur with normal aging. Moreover, androgen deficiency may develop gradually. Several epidemiologic studies, such as the Framingham Heart Study, the Massachusetts Male Aging Study, the Baltimore Longitudinal Study of Aging, and the Study of Osteoporotic Fractures in Men, have reported a high prevalence of low testosterone levels in middle-aged and older

1	Male Aging Study, the Baltimore Longitudinal Study of Aging, and the Study of Osteoporotic Fractures in Men, have reported a high prevalence of low testosterone levels in middle-aged and older men. The age-related decline in testosterone should be distinguished from classical hypogonadism due to diseases of the testes, the pituitary, and the hypothalamus.

1	When symptoms or clinical features suggest possible androgen deficiency, the laboratory evaluation is initiated by the measurement of total testosterone, preferably in the morning using a reliable assay, such as LC-MS/MS that has been calibrated to an international testosterone standard (Fig. 411-6). A consistently low total testosterone level <300 ng/dL measured by a reliable assay, in association with symptoms, is evidence of testosterone deficiency. An early-morning testosterone level >400 ng/dL makes the diagnosis of androgen deficiency unlikely. In men with testosterone levels between 200 and 400 ng/dL, the total testosterone level should be repeated and a free testosterone level should be measured. In older men and in patients with other clinical states that are associated with alterations in SHBG levels, a direct measurement of free testosterone level by equilibrium dialysis can be useful in unmasking testosterone deficiency.

1	When androgen deficiency has been confirmed by the consistently low testosterone concentrations, LH should be measured to classify the patient as having primary (high LH) or secondary (low or inappropriately normal LH) hypogonadism. An elevated LH level indicates that the defect is at the testicular level. Common causes of primary testicular failure include Klinefelter’s syndrome, HIV infection, uncorrected cryptorchidism, cancer chemotherapeutic agents, radiation, surgical orchiectomy, or prior infectious orchitis. Unless causes of primary testicular failure are known, a karyotype should be performed in men with low testosterone and elevated LH to exclude Klinefelter’s syndrome. Men who have low testosterone levels but “inappropriately normal” or low LH levels have secondary hypogonadism; their defect resides at the hypothalamic-pituitary level. Common causes of acquired secondary hypogonadism include space-occupying lesions of the sella, hyperprolactinemia, chronic illness,

1	their defect resides at the hypothalamic-pituitary level. Common causes of acquired secondary hypogonadism include space-occupying lesions of the sella, hyperprolactinemia, chronic illness, hemochromatosis, excessive exercise, and the use of anabolic-androgenic steroids, opiates, marijuana, glucocorticoids, and alcohol. Measurement of PRL and MRI scan of the hypothalamic-pituitary region can help exclude the presence of a space-occupying lesion. Patients in whom known causes of hypogonadotropic hypogonadism have been excluded are classified as having IHH. It is not unusual for congenital causes of hypogonadotropic hypogonadism, such as Kallmann’s syndrome, to be diagnosed in young adults.

1	Gonadotropin therapy is used to establish or restore fertility in patients with gonadotropin deficiency of any cause. Several gonadotropin preparations are available. Human menopausal gonadotropin (hMG; purified from the urine of postmenopausal women) contains 75 IU FSH and 75 IU LH per vial. hCG (purified from the urine of pregnant women) has little FSH activity and resembles LH in its ability to stimulate testosterone production by Leydig cells. Recombinant LH is now available. Because of the expense of hMG, treatment is usually begun with hCG alone, and hMG is added later to promote the FSH-dependent stages of spermatid development. Recombinant human FSH (hFSH) is now available and is indistinguishable from purified urinary hFSH in its biologic activity and pharmacokinetics in vitro and in vivo, although the mature β subunit of recombinant hFSH has seven fewer amino acids. Recombinant hFSH is available in ampoules containing 75 IU (~7.5 μg FSH), which accounts for >99% of protein

1	in vivo, although the mature β subunit of recombinant hFSH has seven fewer amino acids. Recombinant hFSH is available in ampoules containing 75 IU (~7.5 μg FSH), which accounts for >99% of protein content. Once spermatogenesis is restored using combined FSH and LH therapy, hCG alone is often sufficient to maintain spermatogenesis.

1	Although a variety of treatment regimens are used, 1000–2000 IU of hCG or recombinant human LH (rhLH) administered intramuscularly three times weekly is a reasonable starting dose. Testosterone levels should be measured 6–8 weeks later and 48–72 h after the hCG or rhLH injection; the hCG/rhLH dose should be adjusted to achieve testosterone levels in the mid-normal range. Sperm counts should be monitored on a monthly basis. It may take several months for spermatogenesis to be restored; therefore, it is important to forewarn patients about the potential length and expense of the treatment and to provide conservative estimates of success rates. If testosterone levels are in the mid-normal range but the sperm concentrations are low after 6 months of therapy with hCG alone, FSH should be added. This can be done by using hMG, highly purified urinary hFSH, or recombinant hFSH. The selection of FSH dose is empirical. A common practice is to start with the addition of 75 IU FSH three times

1	This can be done by using hMG, highly purified urinary hFSH, or recombinant hFSH. The selection of FSH dose is empirical. A common practice is to start with the addition of 75 IU FSH three times each week in conjunction with the hCG/rhLH injections. If sperm densities are still low after 3 months of combined treatment, the FSH dose should be increased to 150 IU. Occasionally, it may take ≥18–24 months for spermatogenesis to be restored.

1	The two best predictors of success using gonadotropin therapy in hypogonadotropic men are testicular volume at presentation and time of onset. In general, men with testicular volumes >8 mL have better response rates than those who have testicular volumes >4 mL. Patients who became hypogonadotropic after puberty experience higher success rates than those who have never undergone pubertal changes. Spermatogenesis can usually be reinitiated by hCG alone, with high rates of success for men with postpubertal onset of hypogonadotropism. The presence of a primary testicular abnormality, such as cryptorchidism, will attenuate testicular response to gonadotropin therapy. Prior androgen therapy does not preclude subsequent response to gonadotropin therapy, although some studies suggest that it may attenuate response to subsequent gonadotropin therapy.

1	Disorders of the Testes and Male Reproductive System 2370 TESTOSTERONE REPLACEMENT Androgen therapy is indicated to restore testosterone levels to normal to correct features of androgen deficiency. Testosterone replacement improves libido and overall sexual activity; increases energy, lean muscle mass, and bone density; and decreases fat mass. The benefits of testosterone replacement therapy have only been proven in men who have documented androgen deficiency, as demonstrated by testosterone levels that are well below the lower limit of normal.

1	Testosterone is available in a variety of formulations with distinct pharmacokinetics (Table 411-3). Testosterone serves as a prohormone and is converted to 17β-estradiol by aromatase and to 5α-dihydrotestosterone by steroid 5α-reductase. Therefore, when evaluating testosterone formulations, it is important to consider whether the formulation being used can achieve physiologic estradiol and DHT concentrations, in addition to normal testosterone concentrations. Although testosterone concentrations at the lower end of the normal male range can restore sexual function, it is not aThese formulations are not approved for clinical use in the United States, but are available outside the United States in many countries. Physicians in those countries where these formulations are available should follow the approved drug regimens. Abbreviations: DHT, dihydrotestosterone; E2, estradiol; T, testosterone.

1	Abbreviations: DHT, dihydrotestosterone; E2, estradiol; T, testosterone. clear whether low-normal testosterone levels can maintain bone mineral density and muscle mass. The current recommendation is to restore testosterone levels to the mid-normal range.

1	Oral Derivatives of Testosterone Testosterone is well-absorbed after oral administration but is quickly degraded during the first pass through the liver. Therefore, it is difficult to achieve sustained blood levels of testosterone after oral administration of crystalline testosterone. 17α-Alkylated derivatives of testosterone (e.g., 17α-methyl testosterone, oxandrolone, fluoxymesterone) are relatively resistant to hepatic degradation and can be administered orally; however, because of the potential for hepatotoxicity, including cholestatic jaundice, peliosis, and hepatoma, these formulations should not be used for testosterone replacement. Hereditary angioedema due to C1 esterase deficiency is the only exception to this general recommendation; in this condition, oral 17α-alkylated androgens are useful because they stimulate hepatic synthesis of the C1 esterase inhibitor.

1	Injectable Forms of Testosterone The esterification of testosterone at the 17β-hydroxy position makes the molecule hydrophobic and extends its duration of action. The slow release of testosterone ester from an oily depot in the muscle accounts for its extended duration of action. The longer the side chain, the greater is the hydrophobicity of the ester and the longer is the duration of action. Thus, testosterone enanthate, cypionate, and undecanoate with longer side chains have longer duration of action than testosterone propionate. Within 24 h after intramuscular administration of 200 mg testosterone enanthate or cypionate, testosterone levels rise into the high-normal or supraphysiologic range and then gradually decline into the hypogonadal range over the next 2 weeks. A bimonthly regimen of testosterone enanthate or cypionate therefore results in peaks and troughs in testosterone levels that are accompanied by changes in a patient’s mood, sexual desire, and energy level. The

1	regimen of testosterone enanthate or cypionate therefore results in peaks and troughs in testosterone levels that are accompanied by changes in a patient’s mood, sexual desire, and energy level. The kinetics of testosterone enanthate and cypionate are similar. Estradiol and DHT levels are normal if testosterone replacement is physiologic.

1	Transdermal Testosterone Patch The nongenital testosterone patch, when applied in an appropriate dose, can normalize testosterone, DHT, and estradiol levels 4–12 h after application. Sexual function and well-being are restored in androgen-deficient men treated with the nongenital patch. One 5-mg patch may not be sufficient to increase testosterone into the mid-normal male range in all hypogonadal men; some patients may need two 5-mg patches daily to achieve the targeted testosterone concentrations. The use of testosterone patches may be associated with skin irritation in some individuals.

1	Testosterone Gel Several transdermal testosterone gels (e.g., Androgel, Testim, Fortesta, and Axiron), when applied topically to the skin in appropriate doses (Table 411-3), can maintain total and free testosterone concentrations in the normal range in hypogonadal men. The current recommendations are to begin with an initial U.S. Food and Drug Administration–approved dose and adjust the dose based on testosterone levels. The advantages of the testosterone gel include the ease of application and its flexibility of dosing. A major concern is the potential for inadvertent transfer of the gel to a sexual partner or to children who may come in close contact with the patient. The ratio of DHT to testosterone concentrations is higher in men treated with the testosterone gel than in healthy men. Also, there is considerable intraand interindividual variation in serum testosterone levels in men treated with the transdermal gel due to variations in transdermal absorption and plasma clearance of

1	Also, there is considerable intraand interindividual variation in serum testosterone levels in men treated with the transdermal gel due to variations in transdermal absorption and plasma clearance of testosterone. Therefore, monitoring of serum testosterone levels and multiple dose adjustments may be required to achieve and maintain testosterone levels in the target range.

1	Buccal Adhesive Testosterone A buccal testosterone tablet, which adheres to the buccal mucosa and releases testosterone as it is slowly dissolved, has been approved. After twice-daily application of 30-mg tablets, serum testosterone levels are maintained within the normal male range in a majority of treated hypogonadal men. 2371 The adverse effects include buccal ulceration and gum problems in a few subjects. The effects of food and brushing on absorption have not been studied in detail. Implants of crystalline testosterone can be inserted in the subcutaneous tissue by means of a trocar through a small skin incision. Testosterone is released by surface erosion of the implant and absorbed into the systemic circulation. Two to six 200-mg implants can maintain testosterone in the midto high-normal range for up to 6 months. Potential drawbacks include incising the skin for insertion and removal and spontaneous extrusions and fibrosis at the site of the implant.

1	Testosterone Formulations Not Available in the United States Testosterone undecanoate, when administered orally in oleic acid, is absorbed preferentially through the lymphatics into the systemic circulation and is spared the first-pass degradation in the liver. Doses of 40–80 mg orally, two or three times daily, are typically used. However, the clinical responses are variable and suboptimal. DHTto-testosterone ratios are higher in hypogonadal men treated with oral testosterone undecanoate, as compared to eugonadal men. After initial priming, long-acting testosterone undecanoate in oil, when administered intramuscularly every 12 weeks, maintains serum testosterone, estradiol, and DHT in the normal male range and corrects symptoms of androgen deficiency in a majority of treated men. However, large injection volume (4 mL) is its relative drawback.

1	Novel Androgen Formulations A number of androgen formulations with better pharmacokinetics or more selective activity profiles are under development. A long-acting ester, testosterone undecanoate, when injected intramuscularly, can maintain circulating testosterone concentrations in the male range for 7–12 weeks. Initial clinical trials have demonstrated the feasibility of administering testosterone by the sublingual or buccal routes. 7α-Methyl-19-nortestosterone is an androgen that cannot be 5α-reduced; therefore, compared to testosterone, it has relatively greater agonist activity in muscle and gonadotropin suppression but lesser activity on the prostate.

1	Selective AR modulators (SARMs) are a class of AR ligands that bind the AR and display tissue-selective actions. A number of non-steroidal SARMs that act as full agonists on the muscle and bone and that spare the prostate to varying degrees have advanced to phase 3 human trials. Nonsteroidal SARMs do not serve as substrates for either the steroid 5α-reductase or the CYP19 aromatase. SARM binding to AR induces specific conformational changes in the AR protein, which then modulates protein-protein interactions between AR and its coregulators, resulting in tissue-specific regulation of gene expression.

1	Pharmacologic Uses of Androgens Androgens and SARMs are being evaluated as anabolic therapies for functional limitations associated with aging and chronic illness. Testosterone supplementation increases skeletal muscle mass, maximal voluntary strength, and muscle power in healthy men, hypogonadal men, older men with low testosterone levels, HIV-infected men with weight loss, and men receiving glucocorticoids. These anabolic effects of testosterone are related to testosterone dose and circulating concentrations. Systematic reviews have confirmed that testosterone therapy of HIV-infected men with weight loss promotes improvements in body weight, lean body mass, muscle strength, and depression indices, leading to the recommendation that testosterone be considered as an adjunctive therapy in HIV-infected men who are experiencing unexplained weight loss and who have low testosterone levels. Similarly, in glucocorticoid-treated men, testosterone therapy should be considered to maintain

1	in HIV-infected men who are experiencing unexplained weight loss and who have low testosterone levels. Similarly, in glucocorticoid-treated men, testosterone therapy should be considered to maintain muscle mass and strength and vertebral bone mineral density. It is unknown whether testosterone therapy of older men with functional limitations is safe and effective in improving physical function, vitality, and health-related quality of life and reducing disability. Concerns about potential adverse effects of testosterone on prostate and cardiovascular event rates

1	Disorders of the Testes and Male Reproductive System 2372 have encouraged the development of SARMs that are preferentially anabolic and spare the prostate. Testosterone administration induces hypertrophy of both type 1 and 2 fibers and increases satellite cell (muscle progenitor cells) and myonuclear number. Androgens promote the differentiation of mesenchymal, multipotent progenitor cells into the myogenic lineage and inhibit their differentiation into the adipogenic lineage. Testosterone may have additional effects on satellite cell replication and muscle protein synthesis, which may contribute to an increase in skeletal muscle mass. Other indications for androgen therapy are in selected patients with anemia due to bone marrow failure (an indication largely supplanted by erythropoietin) or for hereditary angioedema.

1	Male Hormonal Contraception Based on Combined Administration of Testosterone and Gonadotropin Inhibitors Supraphysiologic doses of testosterone (200 mg testosterone enanthate weekly) suppress LH and FSH secretion and induce azoospermia in 50% of Caucasian men and >95% of Chinese men. The WHO-supported multicenter efficacy trials have demonstrated that suppression of spermatogenesis to azoospermia or severe oligozoospermia (<3 million/mL) by administration of testosterone enanthate to men results in highly effective contraception. Because of concern about long-term adverse effects of supraphysiologic testosterone doses, regimens that combine other gonadotropin inhibitors, such as GnRH antagonists and progestins with replacement doses of testosterone, are being investigated. Oral etonogestrel daily in combination with intramuscular testosterone decanoate every 4–6 weeks induced azoospermia or severe oligozoospermia (sperm density <1 million/mL) in 99% of treated men over a 1-year

1	daily in combination with intramuscular testosterone decanoate every 4–6 weeks induced azoospermia or severe oligozoospermia (sperm density <1 million/mL) in 99% of treated men over a 1-year period. This regimen was associated with weight gain, deceased testicular volume, and decreased plasma high-density lipoprotein (HDL) cholesterol, and its long-term safety has not been demonstrated. SARMs that are more potent inhibitors of gonadotropins than testosterone and spare the prostate hold promise for their contraceptive potential.

1	Recommended Regimens for Androgen Replacement Testosterone esters are administered typically at doses of 75–100 mg intramuscularly every week, or 150–200 mg every 2 weeks. One or two 5-mg nongenital testosterone patches can be applied daily over the skin of the back, thigh, or upper arm away from pressure areas. Testosterone gels are typically applied over a covered area of skin at initial doses that vary with the formulation; patients should wash their hands after gel application. Bioadhesive buccal testosterone tablets at a dose of 30 mg are typically applied twice daily on the buccal mucosa.

1	Establishing Efficacy of Testosterone Replacement Therapy Because a clinically useful marker of androgen action is not available, restoration of testosterone levels to the mid-normal range remains the goal of therapy. Measurements of LH and FSH are not useful in assessing the adequacy of testosterone replacement. Testosterone should be measured 3 months after initiating therapy to assess adequacy of therapy. There is substantial interindividual variability in serum testosterone levels, especially with transdermal gels, presumably due to genetic differences in testosterone clearance and transdermal absorption. In patients who are treated with testosterone enanthate or cypionate, testosterone levels should be 350–600 ng/dL 1 week after the injection. If testosterone levels are outside this range, adjustments should be made either in the dose or in the interval between injections. In men on transdermal patch, gel, or buccal testosterone therapy, testosterone levels should be in the

1	this range, adjustments should be made either in the dose or in the interval between injections. In men on transdermal patch, gel, or buccal testosterone therapy, testosterone levels should be in the mid-normal range (500–700 ng/dL) 4–12 h after application. If testosterone levels are outside this range, the dose should be adjusted. Multiple dose adjustments are often necessary to achieve testosterone levels in the desired therapeutic range.

1	Restoration of sexual function, secondary sex characteristics, energy, and well-being and maintenance of muscle and bone health are important objectives of testosterone replacement therapy. The patient should be asked about sexual desire and activity, the presence of early morning erections, and the ability to achieve and maintain erections adequate for sexual intercourse. Some hypogonadal men continue to complain about sexual dysfunction even after testosterone replacement has been instituted; these patients may benefit from counseling. The hair growth in response to androgen replacement is variable and depends on ethnicity. Hypogonadal men with prepubertal onset of androgen deficiency who begin testosterone therapy in their late twenties or thirties may find it difficult to adjust to their newly found sexuality and may benefit from counseling. If the patient has a sexual partner, the partner should be included in counseling because of the dramatic physical and sexual changes that

1	their newly found sexuality and may benefit from counseling. If the patient has a sexual partner, the partner should be included in counseling because of the dramatic physical and sexual changes that occur with androgen treatment.

1	Contraindications for Androgen Administration Testosterone administration is contraindicated in men with a history of prostate or breast cancer (Table 411-4). Testosterone therapy should not be administered without further urologic evaluation to men with a palpable prostate nodule or induration; to men with prostate-specific antigen levels >4 ng/mL or >3 ng/mL in men at high risk for prostate cancer such as African Americans or men with first-degree relatives with prostate cancer; or to men with severe lower urinary tract symptoms (American Urological Association lower urinary tract symptom score >19). Testosterone replacement should not be administered to men with baseline hematocrit ≥50%, severe untreated obstructive sleep apnea, uncontrolled or poorly controlled congestive heart failure, or myocardial infarction, stroke, or acute coronary syndrome in the preceding 6 months.

1	Monitoring Potential Adverse Experiences The clinical effectiveness and safety of testosterone replacement therapy should be assessed 3 to 6 months after initiating testosterone therapy and annually thereafter (Table 411-5). Potential adverse effects include acne, oiliness of skin, erythrocytosis, breast tenderness and enlargement, leg edema, induction and exacerbation of obstructive sleep apnea, and increased risk of detection of prostate events. In addition, there may be formulation-specific adverse effects such as skin irritation with transdermal patch, risk of gel transfer to a sexual partner with testosterone gels, buccal ulceration and gum problems with buccal testosterone, and pain and mood fluctuation with injectable testosterone esters. Older men with preexisting heart disease may be at increased risk of cardiovascular events after initiation of testosterone therapy.

1	HemoGloBin levelS Administration of testosterone to androgen-deficient men is typically associated with a ~3% increase in hemoglobin ConDiTionS in wHiCH TESToSTERonE ADminiSTRATion iS ASSoCiATED wiTH A RiSk of ADvERSE ouTComE Conditions in which testosterone administration is associated with very high risk of serious adverse outcomes: Conditions in which testosterone administration is associated with moderate to high risk of adverse outcomes: Undiagnosed prostate nodule or induration PSA >4 ng/mL (>3 ng/mL in individuals at high risk for prostate cancer, such as African Americans or men with first-degree relatives who have prostate cancer) Erythrocytosis (hematocrit >50%) Severe lower urinary tract symptoms associated with benign prostatic hypertrophy as indicated by American Urological Association/International Prostate Symptom Score >19 Uncontrolled or poorly controlled congestive heart failure Myocardial infarction, stroke, or acute coronary syndrome in the preceding 6 months

1	Uncontrolled or poorly controlled congestive heart failure Myocardial infarction, stroke, or acute coronary syndrome in the preceding 6 months Abbreviation: PSA, prostate-specific antigen. Source: Reproduced from the Endocrine Society Guideline for Testosterone Therapy of Androgen Deficiency Syndromes in Men (S Bhasin et al: J Clin Endocrinol Metab 95:2536, 2010). 1. Evaluate the patient 3–6 months after treatment initiation and then annually to assess whether symptoms have responded to treatment and whether the patient is suffering from any adverse effects. 2. Monitor testosterone level 3–6 months after initiation of testosterone therapy: Therapy should aim to raise serum testosterone level into the mid-normal range. Injectable testosterone enanthate or cypionate: Measure serum testosterone level midway between injections. If testosterone is >700 ng/dL (24.5 nmol/L) or >400 ng/dL (14.1 nmol/L), adjust dose or frequency.

1	Transdermal patches: Assess testosterone level 3–12 h after application of the patch; adjust dose to achieve testosterone level in the mid-normal range. Buccal testosterone bioadhesive tablet: Assess level immediately before or after application of fresh system. Transdermal gels and solution: Assess testosterone level 2–8 h after patient has been on treatment for at least 2 weeks; adjust dose to achieve serum testosterone level in the mid-normal range. Testosterone pellets: Measure testosterone levels at the end of the dosing interval. Adjust the number of pellets and/or the dosing interval to achieve serum testosterone levels in the normal range. Oral testosterone undecanoatea: Monitor serum testosterone level 3–5 h after ingestion. Injectable testosterone undecanoate: Measure serum testosterone level just prior to each subsequent injection and adjust the dosing interval to maintain serum testosterone in mid-normal range. 3.

1	Injectable testosterone undecanoate: Measure serum testosterone level just prior to each subsequent injection and adjust the dosing interval to maintain serum testosterone in mid-normal range. 3. Check hematocrit at baseline, at 3–6 months, and then annually. If hematocrit is >54%, stop therapy until hematocrit decreases to a safe level; evaluate the patient for hypoxia and sleep apnea; reinitiate therapy with a reduced dose. 4. Measure bone mineral density of lumbar spine and/or femoral neck after 1–2 years of testosterone therapy in hypogonadal men with osteoporosis or low trauma fracture, consistent with regional standard of care. 5. In men 40 years of age or older with baseline PSA >0.6 ng/mL, perform digital rectal examination and check PSA level before initiating treatment, at 3–6 months, and then in accordance with guidelines for prostate cancer screening depending on the age and race of the patient. 6. Obtain urologic consultation if there is:

1	6. Obtain urologic consultation if there is: An increase in serum PSA concentration >1.4 ng/mL within any 12-month period of testosterone treatment. A PSA velocity of >0.4 ng/mL per year using the PSA level after 6 months of testosterone administration as the reference (only applicable if PSA data are available for a period exceeding 2 years). Detection of a prostatic abnormality on digital rectal examination. An AUA/IPSS prostate symptom score of >19. 7. Evaluate formulation-specific adverse effects at each visit: Buccal testosterone tablets: Inquire about alterations in taste and examine the gums and oral mucosa for irritation. Injectable testosterone esters (enanthate, cypionate, and undecanoate): Ask about fluctuations in mood or libido and, rarely, cough after injections. Testosterone patches: Look for skin reaction at the application site.

1	Testosterone patches: Look for skin reaction at the application site. Testosterone gels: Advise patients to cover the application sites with a shirt and to wash the skin with soap and water before having skin-to-skin contact, because testosterone gels leave a testosterone residue on the skin that can be transferred to a woman or child who might come in close contact. Serum testosterone levels are maintained when the application site is washed 4–6 h after application of the testosterone gel. • Testosterone pellets: Look for signs of infection, fibrosis, or pellet extrusion. aNot approved for clinical use in the United States. Abbreviations: AUA/IPSS, American Urological Association/International Prostate Symptom Score; PSA, prostate-specific antigen. Source: Reproduced with permission from the Endocrine Society Guideline for Testosterone Therapy of Androgen Deficiency Syndromes in Adult Men (S Bhasin et al: J Clin Endocrinol Metab 95:2536, 2010).

1	Disorders of the Testes and Male Reproductive System levels, due to increased erythropoiesis, suppression of hepcidin, and increased iron availability for erythropoiesis. The magnitude of hemoglobin increase during testosterone therapy is greater in older men than younger men and in men who have sleep apnea, a significant smoking history, or chronic obstructive lung disease. The frequency of erythrocytosis is higher in hypogonadal men treated with injectable testosterone esters than in those treated with transdermal formulations, presumably due to the higher testosterone dose delivered by the typical regimens of testosterone esters. Erythrocytosis is the most frequent adverse event reported in testosterone trials in middle-aged and older men and is also the most frequent cause of treatment discontinuation in these trials. If hematocrit rises above 54%, testosterone therapy should be stopped until hematocrit has fallen to <50%. After evaluation of the patient for hypoxia and sleep

1	discontinuation in these trials. If hematocrit rises above 54%, testosterone therapy should be stopped until hematocrit has fallen to <50%. After evaluation of the patient for hypoxia and sleep apnea, testosterone therapy may be reinitiated at a lower dose.

1	proState and Serum proState-Specific antiGen levelS Testosterone replacement therapy increases prostate volume to the size seen in age-matched controls but does not increase prostate volume beyond that expected for age. There is no evidence that testosterone therapy causes prostate cancer. However, androgen administration can exacerbate preexisting metastatic prostate cancer. Many older men harbor microscopic foci of cancer in their prostates. It is not known whether long-term testosterone administration will induce these microscopic foci to grow into clinically significant cancers.

1	Prostate-specific antigen (PSA) levels are lower in testosterone-deficient men and are restored to normal after testosterone replacement. There is considerable test-retest variability in PSA measurements. Increments in PSA levels after testosterone supplementation in androgen-deficient men are generally <0.5 ng/mL, and increments >1.0 ng/mL over a 3to 6-month period are unusual. The 90% confidence interval for the change in PSA values in men with benign prostatic hypertrophy, measured 3–6 months apart, is 1.4 ng/mL. Therefore, the Endocrine Society expert panel suggested that an increase in PSA >1.4 ng/mL in any 1 year after starting testosterone therapy, if confirmed, should lead to urologic evaluation. PSA velocity criterion can be used for patients who have sequential PSA measurements for >2 years; a change of >0.40 ng/mL per year merits closer urologic follow-up.

1	cardiovaScular riSK In epidemiologic studies, testosterone concentrations are negatively related to the risk of diabetes mellitus, heart disease, and all-cause and cardiovascular mortality. A recent testosterone trial in older men with mobility limitation was stopped early because of the higher rates of cardiovascular events in the testosterone arm than in the placebo arm of this trial. Meta-analyses 2374 of testosterone trials have found a significant increase in cardiovascular event rates in older men receiving testosterone therapy. Inferences about adverse events from previous trials included in these meta-analyses are limited by poor ascertainment, small numbers of events, heterogeneity of study populations, and small numbers of participants. Two retrospective analyses also found a higher frequency of cardiovascular events in association with testosterone therapy in older men with preexisting heart disease. Retrospective database analyses are limited by their inherent inability to

1	frequency of cardiovascular events in association with testosterone therapy in older men with preexisting heart disease. Retrospective database analyses are limited by their inherent inability to verify the indication for treatment, diagnoses, or other relevant quantitative information and are susceptible to confounding by many other factors. Adequately powered prospective studies are needed to determine the effect on testosterone replacement on cardiovascular risk.

1	Androgen Abuse by Athletes and Recreational Bodybuilders The illicit use of androgenic-anabolic steroids (AAS) to enhance athletic performance first surfaced in the 1950s among power lifters and spread rapidly to other sports, professional as well as high school athletes, and recreational bodybuilders. In the early 1980s, the use of AAS spread beyond the athletic community into the general population, and now as many as 3 million Americans, most of them men, have likely used these compounds. Most AAS users are not athletes, but rather recreational weightlifters, who use these drugs to look lean and more muscular. The most commonly used AAS include testosterone esters, nandrolone, stanozolol, methandienone, and methenolol. AAS users generally use increasing doses of multiple steroids in a practice known as stacking.

1	The adverse effects of long-term AAS abuse remain poorly understood. Most of the information about the adverse effects of AAS has emerged from case reports, uncontrolled studies, or clinical trials that used replacement doses of testosterone. The adverse event data from clinical trials using physiologic replacement doses of testosterone have been extrapolated unjustifiably to AAS users who may administer 10–100 times the replacement doses of testosterone over many years and to support the claim that AAS use is safe. A substantial fraction of androgenic steroid users also use other drugs that are perceived to be muscle building or performance enhancing, such as GH; erythropoiesis-stimulating agents; insulin; and stimulants such as amphetamine, clenbuterol, cocaine, ephedrine, and thyroxine; and drugs perceived to reduce adverse effects such as hCG, aromatase inhibitors, or estrogen antagonists. The men who abuse androgenic steroids are more likely to engage in other high-risk behaviors

1	and drugs perceived to reduce adverse effects such as hCG, aromatase inhibitors, or estrogen antagonists. The men who abuse androgenic steroids are more likely to engage in other high-risk behaviors than nonusers. The adverse events associated with AAS use may be due to AAS themselves, concomitant use of other drugs, high-risk behaviors, and host characteristics that may render these individuals more susceptible to AAS use or to other high-risk behaviors.

1	The high rates of mortality and morbidities observed in AAS users are alarming. One Finnish study reported 4.6 times the risk of death among elite power lifters than in age-matched men from the general population. The causes of death among power lifters included suicides, myocardial infarction, hepatic coma, and non-Hodgkin’s lymphoma. A retrospective review of patient records in Sweden also reported higher standardized mortality ratios for AAS users than for nonusers. Thiblin and colleagues found that 32% of deaths among AAS users were suicidal, 26% homicidal, and 35% accidental. The median age of death among AAS users (24 years) is even lower than that for heroin or amphetamine users.

1	Numerous reports of cardiac death among young AAS users raise concerns about the adverse cardiovascular effects of AAS. High doses of AAS may induce proatherogenic dyslipidemia, increase thrombosis risk via effects on clotting factors and platelets, and induce vasospasm through their effects on vascular nitric oxide. Replacement doses of testosterone, when administered parenterally, are associated with only a small decrease in HDL cholesterol and little or no effect on total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglyceride levels. In contrast, supraphysiologic doses of testosterone and orally administered, 17α-alkylated, nonaromatizable AAS are associated with marked reductions in HDL cholesterol and increases in LDL cholesterol.

1	Recent studies of AAS users using tissue Doppler and strain imaging and MRI have reported diastolic and systolic dysfunction, including significantly lower early and late diastolic tissue velocities, reduced E/A ratio, and reduced peak systolic strain in AAS users than in nonusers. Power athletes using AAS often have short QT intervals but increased QT dispersion, which may predispose them to ventricular arrhythmias. Long-term AAS use may be associated with myocardial hypertrophy and fibrosis. Myocardial tissue of power lifters using AAS has been shown to be infiltrated with fibrous tissue and fat droplets. The finding of ARs on myocardial cells suggests that AAS might be directly toxic to myocardial cells.

1	Long-term AAS use suppresses LH and FSH secretion and inhibits endogenous testosterone production and spermatogenesis. Men who have used AAS for more than a few months experience marked suppression of the hypothalamic-pituitary-testicular (HPT) axis after stopping AAS that may be associated with sexual dysfunction, fatigue, infertility, and depression; in some AAS users, HPT suppression may last more than a year, and in a few individuals, complete recovery may never occur. The symptoms of androgen deficiency caused by androgen withdrawal may cause some men to revert back to using AAS, leading to continued use and AAS dependence. As many as 30% of AAS users develop a syndrome of AAS dependence, characterized by long-term AAS use despite adverse medical and psychiatric effects. Supraphysiologic doses of testosterone may also impair insulin sensitivity. Orally administered androgens also have been associated with insulin resistance and diabetes.

1	Supraphysiologic doses of testosterone may also impair insulin sensitivity. Orally administered androgens also have been associated with insulin resistance and diabetes. Unsafe injection practices, high-risk behaviors, and increased rates of incarceration render AAS users at increased risk of HIV and hepatitis B and C. In one survey, nearly 1 in 10 gay men had injected AAS or other substances, and AAS users were more likely to report high-risk unprotected anal sex than other men. Some AAS users develop hypomanic or manic symptoms during AAS exposure (irritability, aggressiveness, reckless behavior, and occasional psychotic symptoms, sometimes associated with violence) and major depression (sometimes associated with suicidality) during AAS withdrawal. Users may also develop other forms of illicit drug use, which may be potentiated or exacerbated by AAS.

1	Elevated liver enzymes, cholestatic jaundice, hepatic neoplasms, and peliosis hepatis have been reported with oral, 17α-alkylated AAS. AAS use may cause muscle hypertrophy without compensatory adaptations in tendons, ligaments, and joints, thus increasing the risk of tendon and joint injuries. AAS use is associated with acne, baldness, and increased body hair.

1	The suspicion of AAS use may be raised by the increased hemoglobin and hematocrit, suppressed LH and FSH and testosterone levels, low high-density lipoproteins cholesterol, and low testicular volume and sperm density in a person who looks highly muscular. Accredited laboratories use gas chromatography–mass spectrometry or liquid chromatography–mass spectrometry to detect anabolic steroid abuse. In recent years, the availability of high-resolution mass spectrometry and tandem mass spectrometry has further improved the sensitivity of detecting androgen abuse. Illicit testosterone use is detected generally by the application of the measurement of the urinary testosterone-to-epitestosterone ratio and further confirmed by the use of the 13C:12C ratio in testosterone by the use of isotope ratio combustion mass spectrometry. Exogenous testosterone administration increases urinary testosterone glucuronide excretion and consequently the testosterone-to-epitestosterone ratio. Ratios >4 suggest

1	combustion mass spectrometry. Exogenous testosterone administration increases urinary testosterone glucuronide excretion and consequently the testosterone-to-epitestosterone ratio. Ratios >4 suggest exogenous testosterone use but can also reflect genetic variation. Genetic variations in the uridine diphosphoglucuronyl transferase 2B17 (UGT2B17), the major enzyme for testosterone glucuronidation, affect the testosterone-to-epitestosterone ratio. Synthetic testosterone has a lower 13C:12C ratio than endogenously produced testosterone, and these differences in 13C:12C ratio can be detected by isotope ratio combustion mass spectrometry, which is used to confirm exogenous testosterone use in individuals with a high testosterone-to-epitestosterone ratio.

1	Disorders of the female Reproductive System Janet E. Hall The female reproductive system regulates the hormonal changes respon-sible for puberty and adult reproductive function. Normal reproductive function in women requires the dynamic integration of hormonal sig-412 nals from the hypothalamus, pituitary, and ovary, resulting in repetitive cycles of follicle development, ovulation, and preparation of the endometrial lining of the uterus for implantation should conception occur. It is critical to understand pubertal development in normal girls (and boys) as a yardstick for identifying precocious and delayed puberty. For further discussion of related topics, see the following chapters: amenorrhea and pelvic pain (Chap. 69), infertility and contraception (Chap. 414), menopause (Chap. 413), disorders of sex development (Chap. 410), and disorders of the male reproductive system (Chap. 411).

1	The ovary orchestrates the development and release of a mature oocyte and also elaborates hormones (e.g., estrogen, progesterone, inhibin, relaxin) that are critical for pubertal development and preparation of the uterus for conception, implantation, and the early stages of pregnancy. To achieve these functions in repeated monthly cycles, the ovary undergoes some of the most dynamic changes of any organ in the body. Primordial germ cells can be identified by the third week of gestation, and their migration to the genital ridge is complete by 6 weeks of gestation. Germ cells persist within the genital ridge, are then referred to as oogonia, and are essential for induction of ovarian development. Although one X chromosome undergoes X inactivation in somatic cells, it is reactivated in oogonia and genes on both X chromosomes are required for normal ovarian development. A 2375 streak ovary containing only stromal cells is found in patients with 45,X Turner’s syndrome (Chap. 410).

1	The germ cell population expands, and starting at ~8 weeks of gestation, oogonia begin to enter prophase of the first meiotic division and become primary oocytes. This allows the oocyte to be surrounded by a single layer of flattened granulosa cells to form a primordial follicle (Fig. 412-1). Granulosa cells are derived from mesonephric cells that invade the ovary early in its development, pushing the germ cells to the periphery. The weight of evidence supports the concept that for the most part, the ovary contains a nonrenewable pool of germ cells. Through the combined processes of mitosis, meiosis, and atresia, the population of oogonia reaches its maximum of 6–7 million by 20 weeks of gestation, after which there is a progressive loss of both oogonia and primordial follicles through the process of atresia. At birth, oogonia are no longer present in the ovary, and only 1–2 million germ cells remain in the form of primordial follicles (Fig. 412-2). The oocyte persists in prophase of

1	process of atresia. At birth, oogonia are no longer present in the ovary, and only 1–2 million germ cells remain in the form of primordial follicles (Fig. 412-2). The oocyte persists in prophase of the first meiotic division until just before ovulation, when meiosis resumes.

1	The quiescent primordial follicles are recruited to further growth and differentiation through a highly regulated process that limits the size of the developing cohort to ensure that folliculogenesis can continue throughout the reproductive life span. This initial recruitment of primordial follicles to form primary follicles (Fig. 412-1) is characterized by growth of the oocyte and the transition from squamous to cuboidal granulosa cells. The theca interna cells that surround the developing follicle begin to form as the primary follicle grows. Acquisition of a zona pellucida by the oocyte and the presence of several layers of surrounding cuboidal granulosa cells mark the development of secondary follicles. It is at this stage that granulosa cells develop follicle-stimulating hormone (FSH), estradiol, and androgen receptors and communicate with one another through the development of gap junctions.

1	Bidirectional signaling between the germ cells and the somatic cells in the ovary is a necessary component underlying the maturation Prophase of first Disorders of the Female Reproductive System Resumption of meiosis FIGURE 412-1 Stages of ovarian development from the arrival of the migratory germ cells at the genital ridge through gonadotropinindependent and gonadotropin-dependent phases that ultimately result in ovulation of a mature oocyte. FSH, follicle-stimulating hormone; LH, luteinizing hormone. FIGURE 412-2 Ovarian germ cell number is maximal at mid-gestation and then decreases precipitously.

1	FIGURE 412-2 Ovarian germ cell number is maximal at mid-gestation and then decreases precipitously. of the oocyte and the capacity for hormone secretion. For example, oocyte-derived growth differentiation factor 9 (GDF-9) and bone morphogenic protein-15 (BMP-15), also known as GDF-9b, are required for migration of pregranulosa and pretheca cells to the outer surface of the developing follicle and, hence, initial follicle formation. GDF-9 is also required for formation of secondary follicles, as are granulosa cell–derived KIT ligand (KITL) and the forkhead transcription factor (FOXL2). All of these genes are potential candidates for premature ovarian failure in women, and mutations in the human FOXL2 gene have been shown to cause the syndrome of blepharophimosis/ptosis/ epicanthus inversus, which is associated with ovarian failure.

1	The early stages of follicle growth are primarily driven by intraovarian factors and may take up to a year from development of the primary follicle to the dominant follicle stage. Further maturation to the preovulatory state, including the resumption of meiosis in the oocyte, requires the combined stimulus of FSH and luteinizing hormone (LH) (Fig. 412-1) and can be accomplished within weeks. Recruitment of secondary follicles from the resting follicle pool requires the direct action of FSH, whereas anti-müllerian hormone (AMH) produced from small growing follicles, restrains this effect of FSH. Accumulation of follicular fluid between the layers of granulosa cells creates an antrum that divides the granulosa cells into two functionally distinct groups: mural cells that line the follicle wall and cumulus cells that surround the oocyte (Fig. 412-3). Recent evidence suggests that, in addition to its role in normal development of the müllerian system, the WNT signaling pathway is required

1	and cumulus cells that surround the oocyte (Fig. 412-3). Recent evidence suggests that, in addition to its role in normal development of the müllerian system, the WNT signaling pathway is required for normal antral follicle development and may also play a role in ovarian steroidogenesis. A single dominant follicle emerges from the growing follicle pool within the first 5–7 days after the onset of menses, and the majority of follicles fall off their growth trajectory and become atretic. Autocrine actions of activin and BMP-6, derived from the granulosa cells, and paracrine actions of GDF-9, BMP-15, BMP-6, and Gpr149, derived from the oocyte, are involved in granulosa cell proliferation and modulation of FSH responsiveness. Differential exposure to these factors may explain the mechanism whereby a given follicle is selected for continued growth to the preovulatory stage. The dominant follicle can be distinguished by its size, evidence of granulosa cell proliferation, large number of FSH

1	a given follicle is selected for continued growth to the preovulatory stage. The dominant follicle can be distinguished by its size, evidence of granulosa cell proliferation, large number of FSH receptors, high aromatase activity, and elevated concentrations of estradiol and inhibin A in follicular fluid.

1	FIGURE 412-3 Development of ovarian follicles. The Graafian follicle is also known as a tertiary or preovulatory follicle. (Courtesy of JH Eichhorn and D. Roberts, Massachusetts General Hospital; with permission.)

1	The dominant follicle undergoes rapid expansion during the 5–6 days prior to ovulation, reflecting granulosa cell proliferation and accumulation of follicular fluid. FSH induces LH receptors on the granulosa cells, and the preovulatory, or Graafian, follicle moves to the outer ovarian surface in preparation for ovulation. The LH surge triggers the resumption of meiosis, the suppression of granulosa cell proliferation, and the induction of cyclooxygenase 2 (COX-2), prostaglandins, the progesterone receptor, and the epidermal growth factor (EGF)-like growth factors amphiregulin, epiregulin, betacellulin, and neuroregulin 1, all of which are required for ovulation. EGF-like factors are thought to mediate these follicular responses to LH. Ovulation also involves production of extracellular matrix leading to expansion of the cumulus cell population that surrounds the oocyte and the controlled expulsion of the egg and follicular fluid. Both progesterone and prostaglandins (induced by the

1	matrix leading to expansion of the cumulus cell population that surrounds the oocyte and the controlled expulsion of the egg and follicular fluid. Both progesterone and prostaglandins (induced by the ovulatory stimulus) are essential for this process. After ovulation, luteinization is induced by LH in conjunction with the acquisition of a rich vascular network in response to vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF). Traditional regulators of central reproductive control, gonadotropin-releasing hormone (GnRH) and its receptor (GnRHR), are also produced in the ovary and may be involved in corpus luteum function.

1	GnRH neurons develop from epithelial cells outside the central nervous system and migrate, initially alongside the olfactory neurons, to the medial basal hypothalamus. Studies in GnRH-deficient patients who fail to undergo puberty have provided insights into genes that control the ontogeny and function of GnRH neurons (Fig. 412-4). KAL1, FGF8/FGFR1, PROK2/PROKR2, NSMF, and CDH7, among others (Chap. 411), have been implicated in the migration of GnRH neurons to the hypothalamus. Approximately 7000 GnRH neurons, scattered throughout the medial basal hypothalamus, establish contacts with capillaries of the pituitary portal system in the median eminence. GnRH is secreted into the pituitary portal system in discrete pulses to stimulate synthesis and secretion of LH and FSH from pituitary gonadotropes, which comprise ~10% of cells in the pituitary (Chap. 401e). Functional connections of GnRH neurons with the portal system are established by the end of the first trimester, coinciding with

1	which comprise ~10% of cells in the pituitary (Chap. 401e). Functional connections of GnRH neurons with the portal system are established by the end of the first trimester, coinciding with the production of pituitary gonadotropins. Thus, like the ovary, the hypothalamic and pituitary components of the reproductive system are present before birth. However, the high levels of estradiol and progesterone produced by the placenta suppress hypothalamic-pituitary stimulation of ovarian hormonal secretion in the fetus.

1	After birth and the loss of placenta-derived steroids, gonadotropin levels rise. FSH levels are much higher in girls than in boys. This rise in FSH results in ovarian activation (evident on ultrasound) and increased inhibin B and estradiol levels. Studies that have identified mutations in TAC3, which encodes neurokinin B, and its receptor, TAC3R, in patients with GnRH deficiency indicate that both are involved in control of GnRH secretion and may be particularly important at this early stage of development. By 12–20 months of age, the reproductive axis is again suppressed, and a period of relative quiescence persists until puberty (Fig. 412-5). At the onset of puberty, pulsatile GnRH secretion induces pituitary gonadotropin production. In the early stages of puberty, LH and FSH secretion are apparent only during sleep, but as puberty develops, pulsatile gonadotropin secretion occurs throughout the day and night.

1	FIGURE 412-4 Establishment of a functional gonadotropin-releasing hormone (GnRH) system requires the participation of a number of genes that are essential for development and migration of GnRH neurons from the olfactory placode to the hypothalamus in addition to genes involved in the functional control of GnRH secretion and action. KAL 1 FGR8/FGFR1 NSMF PROK2/PROKR2 OlfactoryplacodePituitaryHypothalamusGnRH1 GnRHR TAC3R KISS1R KISS1 TAC3 MigrationFunction

1	The mechanisms responsible for the childhood quiescence and pubertal reactivation of the reproductive axis remain incompletely understood. GnRH neurons in the hypothalamus respond to both excitatory and inhibitory factors. Increased sensitivity to the inhibitory influence of gonadal steroids has long been implicated in the inhibition of GnRH secretion during childhood but has not been definitively established in the human. Metabolic signals, such as adipocyte-derived leptin, play a permissive role in reproductive function (Chap. 415e). Studies of patients with isolated GnRH deficiency reveal that mutations in the G protein–coupled receptor 54 (GPR54) gene (now known as KISS1R) preclude the onset of puberty. The ligand for this receptor, metastin, is derived from the parent peptide, kisspeptin-1 (KISS1), and is a powerful stimulant for GnRH release. A potential role for kisspeptin in the onset of puberty has been suggested by upregulation of KISS1 and KISS1R transcripts in the

1	kisspeptin-1 (KISS1), and is a powerful stimulant for GnRH release. A potential role for kisspeptin in the onset of puberty has been suggested by upregulation of KISS1 and KISS1R transcripts in the hypothalamus at the time of puberty. TAC3 and dynorphin (Dyn), which appear to play an inhibitory rather than stimulatory role in GnRH control, are co-expressed

1	FIGURE 412-5 Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are increased during the neonatal years but go through a period of childhood quiescence before increasing again during puberty. Gonadotropin levels are cyclic during the reproductive years and increase dramatically with the loss of negative feedback that accompanies menopause. Granulosa cell Androstenedione Testosterone EstroneEstradiolaromatase Cholesterol LH pregnenolone progesterone 17-OHP 3˜HSD 17 hydroxylase 17,20 lyase AndrostenedioneTestosterone17 ˜HSD FIGURE 412-6 Estrogen production in the ovary requires the cooperative function of the theca and granulosa cells under the control of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). HSD, hydroxysteroid dehydrogenase; OHP, hydroxyprogesterone. with KISS1 in KNDy neurons that project to GnRH neurons. This system is intimately involved with estrogen negative feedback regulation of GnRH secretion.

1	Ovarian steroid-producing cells do not store hormones but produce them in response to LH and FSH during the normal menstrual cycle. The sequence of steps and the enzymes involved in the synthesis of steroid hormones are similar in the ovary, adrenal, and testis. However, the enzymes required to catalyze specific steps are compartmentalized and may not be abundant or even present in all cell types. Within the developing ovarian follicle, estrogen synthesis from cholesterol requires close integration between theca and granulosa cells—sometimes called the two-cell model for steroidogenesis (Fig. 412-6). FSH receptors are confined to the granulosa cells, whereas LH receptors are restricted to the theca cells until the late stages of follicular development, when they are also found on granulosa cells. The theca cells surrounding the follicle are highly vascularized and use cholesterol, derived primarily from circulating lipoproteins, as the starting point for the synthesis of

1	on granulosa cells. The theca cells surrounding the follicle are highly vascularized and use cholesterol, derived primarily from circulating lipoproteins, as the starting point for the synthesis of androstenedione and testosterone under the control of LH. Androstenedione and testosterone are transferred across the basal lamina to the granulosa cells, which receive no direct blood supply. The mural granulosa cells are particularly rich in aromatase and, under the control of FSH, produce estradiol, the primary steroid secreted from the follicular phase ovary and the most potent estrogen. Theca cell–produced androstenedione and, to a lesser extent, testosterone are also secreted into peripheral blood, where they can be converted to dihydrotestosterone in skin and to estrogens in adipose tissue. The hilar interstitial cells of the ovary are functionally similar to Leydig cells and are also capable of secreting androgens. Although stromal cells proliferate in response to androgens (as in

1	The hilar interstitial cells of the ovary are functionally similar to Leydig cells and are also capable of secreting androgens. Although stromal cells proliferate in response to androgens (as in polycystic ovarian syndrome [PCOS]), they do not secrete androgens.

1	Development of the rich capillary network following rupture of the follicle at the time of ovulation makes it possible for large molecules such as low-density lipoprotein (LDL) to reach the luteinized granulosa and theca lutein cells. As in the follicle, both cell types are required for steroidogenesis in the corpus luteum. The large luteinized granulosa cells are the main source of progesterone production, whereas the smaller theca lutein cells produce 17-hydroxyprogesterone, a substrate for aromatization to estradiol by the luteinized granulosa cells. LH is critical for normal structure and function of the corpus luteum. Because LH and human chorionic gonadotropin (hCG) bind to a common receptor, the role of LH in support of the corpus luteum can be replaced by hCG in the first 10 weeks after conception, and hCG is commonly used for luteal phase support in the treatment of infertility.

1	Disorders of the Female Reproductive System 2378 Steroid Hormone Actions Both estrogen and progesterone play critical roles in the expression of secondary sexual characteristics in women (Chap. 400e). Estrogen promotes development of the ductule system in the breast, whereas progesterone is responsible for glandular development. In the reproductive tract, estrogens create a receptive environment for fertilization and support pregnancy and parturition through carefully coordinated changes in the endometrium, thickening of the vaginal mucosa, thinning of the cervical mucus, and uterine growth and contractions. Progesterone induces secretory activity in the estrogen-primed endometrium, increases the viscosity of cervical mucus, and inhibits uterine contractions. Both gonadal steroids play critical roles in the negative and positive feedback controls of gonadotropin secretion. Progesterone also increases basal body temperature and has therefore been used clinically as a marker of

1	play critical roles in the negative and positive feedback controls of gonadotropin secretion. Progesterone also increases basal body temperature and has therefore been used clinically as a marker of ovulation. The vast majority of circulating estrogens and androgens are carried in the blood bound to carrier proteins, which restrain their free diffusion into cells and prolong their clearance, serving as a reservoir. High-affinity binding proteins include sex hormone–binding globulin (SHBG), which binds androgens with somewhat greater affinity than estrogens, and corticosteroid-binding globulin (CBG), which also binds progesterone. Modulations in binding protein levels by insulin, androgens, and estrogens contribute to high bioavailable testosterone levels in PCOS and to high circulating estrogen and progesterone levels during pregnancy. Estrogens act primarily through binding to the nuclear receptors, estrogen receptor (ER) α and β. Transcriptional coactivators and corepressors

1	estrogen and progesterone levels during pregnancy. Estrogens act primarily through binding to the nuclear receptors, estrogen receptor (ER) α and β. Transcriptional coactivators and corepressors modulate ER action (Chap. 400e). Both ER subtypes are present in the hypothalamus, pituitary, ovary, and reproductive tract. Although ERα and -β exhibit some functional redundancy, there is also a high degree of specificity, particularly in expression within cell types. For example, ERα functions in the ovarian theca cells, whereas ERβ is critical for granulosa cell function. There is also evidence for membrane-initiated signaling by estrogen. Similar signaling mechanisms pertain for progesterone with evidence of transcriptional regulation through progesterone receptor (PR) A and B protein isoforms, as well as rapid membrane signaling.

1	Inhibin was initially isolated from gonadal fluids based on its ability to selectively inhibit FSH secretion from pituitary cells. Inhibin is a heterodimer composed of an α subunit and a βA or βB subunit to form inhibin A or inhibin B, both of which are secreted from the ovary. Activin is a homodimer of inhibin β subunits with the capacity to stimulate the synthesis and secretion of FSH. Inhibins and activins are members of the transforming growth factor β (TGF-β) superfamily of growth and differentiation factors. During the purification of inhibin, follistatin, an unrelated monomeric protein that inhibits FSH secretion, was discovered. Within the pituitary, follistatin inhibits FSH secretion indirectly through binding and neutralizing activin.

1	Inhibin B is secreted from the granulosa cells of small antral follicles, whereas inhibin A is present in both granulosa and theca cells and is secreted by dominant follicles. Inhibin A is also present in luteinized granulosa cells and is a major secretory product of the corpus luteum. Inhibin B is constitutively secreted by granulosa cells and increases in serum in conjunction with recruitment of secondary follicles to the pool of actively growing follicles under the control of FSH. Inhibin B has been used clinically as a marker of ovarian reserve. Inhibin B is an important inhibitor of FSH, independent of estradiol, during the menstrual cycle. Although activin is also secreted from the ovary, the excess of follistatin in serum, combined with its nearly irreversible binding of activin, make it unlikely that ovarian activin plays an endocrine role in FSH regulation. However, there is evidence that activin plays an autocrine/paracrine role in the ovary, in addition to its

1	of activin, make it unlikely that ovarian activin plays an endocrine role in FSH regulation. However, there is evidence that activin plays an autocrine/paracrine role in the ovary, in addition to its intrapituitary role in modulation of FSH production.

1	AMH (also known as müllerian-inhibiting substance) is important in ovarian biology in addition to the function from which it derived its name (i.e., promotion of the degeneration of the müllerian system during embryogenesis in the male). AMH is produced by granulosa cells from small follicles and, like inhibin B, is a marker of ovarian reserve. AMH inhibits the recruitment of primordial follicles into the follicle pool and counters FSH stimulation of aromatase expression. Relaxin, which is produced by the theca lutein cells of the corpus luteum, is thought to play a role in decidualization of the endometrium and suppression of myometrial contractile activity, both of which are essential for the early establishment of pregnancy.

1	The sequence of changes responsible for mature reproductive function is coordinated through a series of negative and positive feedback loops that alter pulsatile GnRH secretion, the pituitary response to GnRH, and the relative secretion of LH and FSH from the gonadotrope. The frequency and amplitude of pulsatile GnRH secretion differentially modulate the synthesis and secretion of LH and FSH, with slow frequencies favoring FSH synthesis and increased amplitudes favoring LH synthesis. Activin is produced in both pituitary gonadotropes and folliculostellate cells and stimulates the synthesis and secretion of FSH. Inhibins function as potent antagonists of activins through sequestration of the activin receptors. Although inhibin is expressed in the pituitary, gonadal inhibin is the principal source of feedback inhibition of FSH.

1	For the majority of the cycle, the reproductive system functions in a classic endocrine negative feedback mode. Estradiol and progesterone inhibit GnRH secretion, and the inhibins act at the pituitary to selectively inhibit FSH synthesis and secretion (Fig. 412-7). This negative feedback control of FSH is critical for development of the single mature oocyte that characterizes normal reproductive function in women. In addition to these negative feedback controls, the menstrual cycle is uniquely dependent on estrogen-induced positive feedback to produce an LH surge that is essential for ovulation of a mature follicle. Estrogen negative feedback in women occurs primarily at the hypothalamus with a small pituitary contribution, whereas estrogen positive feedback occurs at the pituitary with hypothalamic GnRH secretion playing a permissive role.

1	The follicular phase is characterized by recruitment of a cohort of secondary follicles and the ultimate selection of a dominant preovulatory follicle (Fig. 412-8). The follicular phase begins, by convention, on the first day of menses. However, follicle recruitment is initiated by the rise in FSH that begins in the late luteal phase of the previous cycle in conjunction with the loss of negative feedback of gonadal steroids and likely inhibin A. The fact that a 20–30% increase in FSH is adequate for follicular recruitment speaks to the marked sensitivity of the resting follicle pool to FSH. The resultant granulosa cell proliferation is responsible for increasing early follicular phase levels of inhibin B. Inhibin B in conjunction with rising levels of estradiol, and

1	FIGURE 412-7 The reproductive system in women is critically dependent on both negative feedback of gonadal steroids and inhibin to modulate follicle-stimulating hormone (FSH) secretion and on estrogen positive feedback to generate the preovulatory luteinizing hormone (LH) surge. GnRH, gonadotropin-releasing hormone. FIGURE 412-8 Relationship between gonadotropins, follicle development, gonadal secretion, and endometrial changes during the normal menstrual cycle. E2, estradiol; Endo, endometrium; FSH, follicle-stimulating hormone; LH, luteinizing hormone; Prog, progesterone.

1	probably inhibin A, restrain FSH secretion during this critical period such that only a single follicle matures in the vast majority of cycles. The increased risk of multiple gestation associated with the increased levels of FSH characteristic of advanced maternal age, or with exogenous gonadotropin administration in the treatment of infertility, attests to the importance of negative feedback regulation of FSH. With further growth of the dominant follicle, estradiol and inhibin A increase exponentially and the follicle acquires LH receptors. Increasing levels of estradiol are responsible for proliferative changes in the endometrium. The exponential rise in estradiol results in positive feedback on the pituitary, leading to the generation of an LH surge (and a smaller FSH surge), thereby triggering ovulation and luteinization of the granulosa cells.

1	The luteal phase begins with the formation of the corpus luteum from the ruptured follicle (Fig. 412-8). Progesterone and inhibin A are produced from the luteinized granulosa cells, which continue to aromatize theca-derived androgen precursors, producing estradiol. The combined actions of estrogen and progesterone are responsible for the secretory changes in the endometrium that are necessary for implantation. The corpus luteum is supported by LH but has a finite life span because of diminished sensitivity to LH. The demise of the corpus luteum results in a progressive decline in hormonal support of the endometrium. Inflammation or local hypoxia and ischemia result in vascular changes in the endometrium, leading to the release of cytokines, cell death, and shedding of the endometrium.

1	If conception occurs, hCG produced by the trophoblast binds to LH receptors on the corpus luteum, maintaining steroid hormone production and preventing involution of the corpus luteum. The corpus luteum is essential for the hormonal maintenance of the endometrium during the first 6–10 weeks of pregnancy until this function is taken over by the placenta. Menstrual bleeding should become regular within 2–4 years of menarche, although anovulatory and irregular cycles are common before that. For the remainder of adult reproductive life, the cycle shortening of cycle length with age such that women over the age of 2379 35 have cycles that are shorter than during their younger reproductive years. Anovulatory cycles increase as women approach menopause, and bleeding patterns may be erratic.

1	Women who report regular monthly bleeding with cycles that do not vary by >4 days generally have ovulatory cycles, but several other clinical signs can be used to assess the likelihood of ovulation. Some women experience mittelschmerz, described as midcycle pelvic discomfort that is thought to be caused by the rapid expansion of the dominant follicle at the time of ovulation. A constellation of premenstrual moliminal symptoms such as bloating, breast tenderness, and food cravings often occur several days before menses in ovulatory cycles, but their absence cannot be used as evidence of anovulation. Methods that can be used to determine whether ovulation is likely include a serum progesterone level >5 ng/mL ~7 days before expected menses, an increase in basal body temperature of 0.24°C (>0.5°F) in the second half of the cycle due to the thermoregulatory effect of progesterone, or the detection of the urinary LH surge using ovulation predictor kits. Because ovulation occurs ~36 h after

1	in the second half of the cycle due to the thermoregulatory effect of progesterone, or the detection of the urinary LH surge using ovulation predictor kits. Because ovulation occurs ~36 h after the LH surge, urinary LH can be helpful in timing intercourse to coincide with ovulation.

1	Ultrasound can be used to detect the growth of the fluid-filled antrum of the developing follicle and to assess endometrial proliferation in response to increasing estradiol levels in the follicular phase, as well as the characteristic echogenicity of the secretory endometrium of the luteal phase.

1	The first menstrual period (menarche) occurs relatively late in the series of developmental milestones that characterize normal pubertal development (Table 412-1). Menarche is preceded by the appearance of pubic and then axillary hair (adrenarche) as a result of maturation of the zona reticularis in the adrenal gland and increased adrenal androgen secretion, particularly dehydroepiandrosterone (DHEA). The triggers for adrenarche remain unknown but may involve increases in body mass index, as well as in utero and neonatal factors. Menarche is also preceded by breast development (thelarche). The breast is exquisitely sensitive to the very low levels of estrogen that result from peripheral conversion of adrenal androgens and the low levels of estrogen secreted from the ovary early in pubertal maturation. Breast development precedes the appearance of pubic and axillary hair in ~60% of girls. The interval between the onset of breast development and menarche is ~2 years. There has been a

1	maturation. Breast development precedes the appearance of pubic and axillary hair in ~60% of girls. The interval between the onset of breast development and menarche is ~2 years. There has been a gradual decline in the age of menarche over the past century, attributed in large part to improvement in nutrition, and there is a relationship between adiposity and earlier sexual maturation in girls. In the United States, menarche occurs at an average age of 12.5 years (Table 412-1). Much of the variation in the timing of puberty is due to genetic factors, with heritability estimates of 50–80%. Both adrenarche and thelarche occur ~1 year earlier in black compared with white girls, although the timing of menarche differs by only 6 months between these ethnic groups.

1	Other important hormonal changes also occur in conjunction with puberty. Growth hormone (GH) levels increase early in puberty, stimulated in part by the pubertal increases in estrogen secretion. GH increases insulin-like growth factor-I (IGF-I), which enhances linear growth. The growth spurt is generally less pronounced in girls than in boys, with a peak growth velocity of ~7 cm/year. Linear growth is ultimately limited by closure of epiphyses in the long bones as a result of prolonged exposure to estrogen. Puberty is also associated with mild insulin resistance. Disorders of the Female Reproductive System length counted from the first day of menses to the first day of subsequent menses is ~28 days, with a range of 25–35 days. However, cycle-to-cycle variability for an individual

1	Onset of Breast/ Age of Peak woman is ±2 days. Luteal phase length is relatively constant between 12 and 14 days in normal cycles; thus, the major variability in cycle length is due to variations in the fol-White 10.2 11.9 12.6 14.3 17.1 licular phase. The duration of menstrual bleeding in ovula-Black 9.6 11.5 12 13.6 16.5 tory cycles varies between 4 and 6 days. There is a gradual Source: From FM Biro et al: J Pediatr 148:234, 2006. Abbreviations: CNS, central nervous system; GnRH, gonadotropin-releasing hormone; hCG, human chorionic gonadotropin. The differential diagnosis of precocious and delayed puberty is similar in boys (Chap. 411) and girls. However, there are differences in the timing of normal puberty and differences in the relative frequency of specific disorders in girls compared with boys.

1	Precocious Puberty Traditionally, precocious puberty has been defined as the development of secondary sexual characteristics before the age of 8 in girls based on data from Marshall and Tanner in British girls studied in the 1960s. More recent studies led to recommendations that girls be evaluated for precocious puberty if breast development or pubic hair is present at <7 years of age for white girls or <6 years for black girls.

1	Precocious puberty in girls is most often centrally mediated (Table 412-2), resulting from early activation of the hypothalamic-pituitary-ovarian axis. It is characterized by pulsatile LH secretion (which is initially associated with deep sleep) and an enhanced LH and FSH response to exogenous GnRH (twoto threefold stimulation) (Table 412-3). True precocity is marked by advancement in bone age of >2 standard deviations, a recent history of growth acceleration, and progression of secondary sexual characteristics. In girls, centrally mediated precocious puberty is idiopathic in ~85% of cases; however, neurogenic causes must be considered. Mutations in genes associated with GnRH deficiency have been reported in small numbers of patients with idiopathic precocious puberty (KISS, KISS1R, TAC3, TAC3R, and DAX-1), but their frequency is insufficient to warrant their use in clinical testing. GnRH agonists that induce pituitary desensitization are the mainstay of treatment to prevent premature

1	TAC3R, and DAX-1), but their frequency is insufficient to warrant their use in clinical testing. GnRH agonists that induce pituitary desensitization are the mainstay of treatment to prevent premature epiphyseal closure and preserve adult height, as well as to manage psychosocial repercussions of precocious puberty.

1	Peripherally mediated precocious puberty does not involve activation of the hypothalamic-pituitary-ovarian axis and is characterized by suppressed gonadotropins in the presence of elevated estradiol. Management of peripheral precocious puberty involves treating the underlying disorder (Table 412-2) and limiting the effects of gonadal steroids using aromatase inhibitors, inhibitors of steroidogenesis, and ER blockers. It is important to be aware that central precocious puberty can also develop in girls whose precocity was initially peripherally mediated, as in McCune-Albright syndrome and congenital adrenal hyperplasia. Incomplete and intermittent forms of precocious puberty may also occur. For example, premature breast development may occur in girls before the age of 2 years, with no further progression and without significant advancement in bone age, estrogen production, or compromised

1	History and physical × × Assessment of growth velocity × × Bone age × × LH, FSH × × Estradiol, testosterone × × DHEAS × × 17-Hydroxyprogesterone × TSH, T4 × × Complete blood count × Sedimentation rate, C-reactive protein Electrolytes, renal function × IGF-I, IGFBP-3 Abbreviations: ACTH, adrenocorticotropic hormone; DHEAS, dehydroepiandrosterone sulfate; FSH, follicle-stimulating hormone; hCG, human chorionic gonadotropin; IGF-I, insulin-like growth factor-I; IGFBP-3, IGF-binding protein 3; LH, luteinizing hormone; MRI, magnetic resonance imaging; TSH, thyroid-stimulating hormone; T4, thyroxine. height. Premature adrenarche can also occur in the absence of progressive pubertal development, but it must be distinguished from late-onset congenital adrenal hyperplasia and androgen-secreting tumors, in which case it may be termed heterosexual precocity. Premature adrenarche may be associated with obesity, hyperinsulinemia, and the subsequent predisposition to PCOS.

1	Delayed Puberty Delayed puberty (Table 412-4) is defined as the absence of secondary sexual characteristics by age 13 in girls. The diagnostic considerations are very similar to those for primary amenorrhea (Chap. 69). Between 25 and 40% of delayed puberty in girls is of ovarian origin, with Turner’s syndrome accounting for the majority of such patients. Functional hypogonadotropic hypogonadism encompasses diverse etiologies such as systemic illnesses, including celiac disease and chronic renal disease, and endocrinopathies such as diabetes and hypothyroidism. In addition, girls appear to be particularly susceptible to the adverse effects of decreased energy balance resulting from exercise, dieting, and/or eating disorders. Together these reversible conditions account for ~25% of delayed puberty in girls. Congenital hypogonadotropic hypogonadism in girls or boys can be caused by mutations in several different genes or combinations of genes (Fig. 412-4, Chap. 411, Table 411-2).

1	puberty in girls. Congenital hypogonadotropic hypogonadism in girls or boys can be caused by mutations in several different genes or combinations of genes (Fig. 412-4, Chap. 411, Table 411-2). Approximately 50% of girls with congenital hypogonadotropic hypogonadism, with or without anosmia, have a history of some degree of breast development, and 10% report one to two episodes of vaginal bleeding. Family studies suggest that genes identified in association with absent puberty may also cause delayed puberty, and recent reports have further suggested that a genetic susceptibility to environmental stresses such as diet and exercise may account for at least some cases of functional hypothalamic amenorrhea. Although neuroanatomic causes of delayed puberty are considerably less common in girls than in boys, it is always important to rule these out in the setting of hypogonadotropic hypogonadism.

1	menopause and Postmenopausal Hormone Therapy JoAnn E. Manson, Shari S. Bassuk Menopause is the permanent cessation of menstruation due to loss of ovarian follicular function. It is diagnosed retrospectively after 12 413 LH or FSH, IU/L perimenopause precedes the final menses by 2–8 years, with a mean 2381 duration of 4 years. Smoking accelerates the menopausal transition by 2 years. Although the periand postmenopausal transitions share many symptoms, the physiology and clinical management of the two differ. Estradiol or estrone, pg/mL

1	Although the periand postmenopausal transitions share many symptoms, the physiology and clinical management of the two differ. Estradiol or estrone, pg/mL FIGURE 413-1 Mean serum levels of ovarian and pituitary hormones during the menopausal transition. FSH, follicle-stimulating hormone; LH, luteinizing hormone. (From JL Shifren, I Schiff: J Womens Health Gend Based Med 9 Suppl 1:S3, 2000.) months of amenorrhea. The average age at menopause is 51 years among U.S. women. Perimenopause refers to the time period preceding menopause, when fertility wanes and menstrual cycle irregularity increases, until the first year after cessation of menses. The onset of FSHβ, LHR, FSHR KAL1, FGF8, FGFR1, NSMF, PROK2, PROKR2, KISS1, KISS1R, TAC3, TAC3R, GnRH1, GnRHR, SEM3A, HS6ST1, WDR11, CHD7 Abnormalities of pituitary development/function CNS tumors/infiltrative disorders Craniopharyngioma Astrocytoma, germinoma, glioma Prolactinomas, other pituitary tumors Histiocytosis X

1	Abnormalities of pituitary development/function CNS tumors/infiltrative disorders Craniopharyngioma Astrocytoma, germinoma, glioma Prolactinomas, other pituitary tumors Histiocytosis X Abbreviations: CHD7, chromodomain-helicase-DNA-binding protein 7; CNS, central nervous system; FGF8, fibroblast growth factor 8; FGFR1, fibroblast growth factor 1 receptor; FSHβ, follicle-stimulating hormone β chain; FSHR, FSH receptor; GNRHR, gonadotropinreleasing hormone receptor; HESX1, homeobox, embryonic stem cell expressed 1; HS6ST1, heparin sulfate 6-O sulfotransferase 1; IHH, idiopathic hypogonadotropic hypogonadism; KAL, Kallmann; KISS1, kisspeptin 1; KISSR1, KISS1 receptor; LHR, luteinizing hormone receptor; NSMF, NMDA receptor synaptonuclear signaling and neuronal migration factor; PROK2, prokineticin 2; PROKR2 prokineticin receptor 2; PROP1, prophet of Pit1, paired-like homeodomain transcription factor SEMA3A, semaphorin-3A; WDR11, WD repeat-containing protein 11.

1	Low-dose oral contraceptives have become a therapeutic mainstay in perimenopause, whereas postmenopausal hormone therapy (HT) has been a common method of symptom alleviation after menstruation ceases.

1	Ovarian mass and fertility decline sharply after age 35 and even more precipitously during perimenopause; depletion of primary follicles, a process that begins before birth, occurs steadily until menopause (Chap. 412). In perimenopause, intermenstrual intervals shorten significantly (typically by 3 days) as a result of an accelerated follicular phase. Follicle-stimulating hormone (FSH) levels rise because of altered folliculogenesis and reduced inhibin secretion. In contrast to the consistently high FSH and low estradiol levels seen in menopause, perimenopause is characterized by “irregularly irregular” hormone levels. The propensity for anovulatory cycles can produce a hyperestrogenic, hypoprogestagenic environment that may account for the increased incidence of endometrial hyperplasia or carcinoma, uterine polyps, and leiomyoma observed among women of perimenopausal age. Mean serum levels of selected ovarian and pituitary hormones during the menopausal transition are shown in Fig.

1	or carcinoma, uterine polyps, and leiomyoma observed among women of perimenopausal age. Mean serum levels of selected ovarian and pituitary hormones during the menopausal transition are shown in Fig. 413-1. With transition into menopause, estradiol levels fall markedly, whereas estrone levels are relatively preserved, a pattern reflecting peripheral aromatization of adrenal and ovarian androgens. Levels of FSH increase more than those of luteinizing hormone, presumably because of the loss of inhibin as well as estrogen feedback.

1	The Stages of Reproductive Aging Workshop +10 (STRAW+10) classification provides a comprehensive framework for the clinical assessment of ovarian aging. As shown in Fig. 413-2, menstrual cycle characteristics are the principal criteria for characterizing the menopausal transition, with biomarker measures as supportive criteria. Because of their extreme intraindividual variability, FSH and estradiol levels are imperfect diagnostic indicators of perimenopause in menstruating women. However, a consistently low FSH level in the early follicular phase (days 2–5) of the menstrual cycle does not support a diagnosis of perimenopause, while levels >25 IU/L in a random blood sample are characteristic of the late menopause transition. FSH measurement can also aid in assessing fertility; levels of <20 IU/L, 20 to <30 IU/L, and ≥30 IU/L measured on day 3 of the cycle indicate a good, fair, and poor likelihood of achieving pregnancy, respectively. Antimüllerian hormone and inhibin B may also be

1	<20 IU/L, 20 to <30 IU/L, and ≥30 IU/L measured on day 3 of the cycle indicate a good, fair, and poor likelihood of achieving pregnancy, respectively. Antimüllerian hormone and inhibin B may also be useful for assessing reproductive aging.

1	Menopause, years = elevated. **Approximate expected level based on assays using current international pituitary standard. FIGURE 413-2 The Stages of Reproductive Aging Workshop +10 (STRAW +10) staging system for reproductive aging in women. AMH, antimüllerian hormone; FSH, follicle-stimulating hormone. (From SD Harlow et al: Menopause 14:387, 2012. Reproduced with permission.)

1	Determining whether symptoms that develop in midlife are due to ovarian senescence or to other age-related changes is difficult. There is strong evidence that the menopausal transition can cause hot flashes, night sweats, irregular bleeding, and vaginal dryness, and there is moderate evidence that it can cause sleep disturbances in some women. There is inconclusive or insufficient evidence that ovarian aging is a major cause of mood swings, depression, impaired memory or concentration, somatic symptoms, urinary incontinence, or sexual dysfunction. In one U.S. study, nearly 60% of women reported hot flashes in the 2 years before their final menses. Symptom intensity, duration, frequency, and effects on quality of life are highly variable.

1	For women with irregular or heavy menses or hormone-related symptoms that impair quality of life, low-dose combined oral contraceptives are a staple of therapy. Static doses of estrogen and progestin (e.g., 20 μg of ethinyl estradiol and 1 mg of norethindrone acetate daily for 21 days each month) can eliminate vasomotor symptoms and restore regular cyclicity. Oral contraceptives provide other benefits, including protection against ovarian and endometrial cancers and increased bone density, although it is not clear whether use during perimenopause decreases fracture risk later in life. Moreover, the contraceptive benefit is important, given that the unintentional pregnancy rate among women in their forties rivals that of adolescents. Contraindications to oral contraceptive use include cigarette smoking, liver disease, a history of thromboembolism or cardiovascular disease, breast cancer, or unexplained vaginal bleeding. Progestin-only formulations (e.g., 0.35 mg of norethindrone daily)

1	smoking, liver disease, a history of thromboembolism or cardiovascular disease, breast cancer, or unexplained vaginal bleeding. Progestin-only formulations (e.g., 0.35 mg of norethindrone daily) or medroxyprogesterone (Depo-Provera) injections (e.g., 150 mg IM every 3 months) may provide an alternative for the treatment of perimenopausal menorrhagia in women who smoke or have cardiovascular risk factors. Although progestins neither regularize cycles nor reduce the number of bleeding days, they reduce the volume of menstrual flow.

1	Nonhormonal strategies to reduce menstrual flow include the use of nonsteroidal anti-inflammatory agents such as mefenamic acid (an initial dose of 500 mg at the start of menses, then 250 mg qid for 2–3 days) or, when medical approaches fail, endometrial ablation. It should be noted that menorrhagia requires an evaluation to rule out uterine disorders. Transvaginal ultrasound with saline enhancement is useful for detecting leiomyomata or polyps, and endometrial aspiration can identify hyperplastic changes.

1	For sexually active women using contraceptive hormones to alleviate perimenopausal symptoms, the question of when and if to switch to HT must be individualized. Doses of estrogen and progestogen (either synthetic progestins or natural forms of progesterone) in HT are lower than those in oral contraceptives and have not been documented to prevent pregnancy. Although a 1-year absence of spontaneous menses reliably indicates ovulation cessation, it is not possible to assess the natural menstrual pattern while a woman is taking an oral contraceptive. Women willing to switch to a barrier method of contraception should do so; if menses occur spontaneously, oral contraceptive use can be resumed. The average age of final menses among relatives can serve as a guide for when to initiate this process, which can be repeated yearly until menopause has occurred.

1	One of the most complex health care decisions facing women is whether to use postmenopausal HT. Once prescribed primarily to relieve vasomotor symptoms, HT has been promoted as a strategy to forestall various disorders that accelerate after menopause, including osteoporosis and cardiovascular disease. In 2000, nearly 40% of post-menopausal women age 50–74 in the United States had used HT. This widespread use occurred despite the paucity of conclusive data, until recently, on the health consequences of such therapy. Although many women rely on their health care providers for a definitive answer to the question of whether to use postmenopausal hormones, balancing the benefits and risks for an individual patient is challenging.

1	Although observational studies suggest that HT prevents cardiovascular and other chronic diseases, the apparent benefits may result at least in part from differences between women who opt to take postmenopausal hormones and women who do not. Those choosing HT tend to be healthier, have greater access to medical care, are more compliant with prescribed treatments, and maintain a more health-promoting lifestyle. Randomized trials, which eliminate these confounding factors, have not consistently confirmed the benefits found in observational studies. Indeed, the largest HT trial to date, the Women’s Health Initiative (WHI), which examined more than 27, 000 postmenopausal women age 50–79 (mean age, 63) for an average of 5–7 years, was stopped early because of an overall unfavorable benefit-risk ratio in the estrogen-progestin arm and an excess risk of stroke that was not offset by a reduced risk of coronary heart disease (CHD) in the estrogen-only arm.

1	The following summary offers a decision-making guide based on a synthesis of currently available evidence. Prevention of cardiovascular disease is eliminated from the equation due to lack of evidence for such benefits in recent randomized clinical trials. See Table 413-1.

1	Definite Benefits • SymptomS of menopauSe Compelling evidence, including data from randomized clinical trials, indicates that estrogen therapy is highly effective for controlling vasomotor and genitourinary symptoms. Alternative approaches, including the use of antidepressants (such as paroxetine, 7.5 mg/d; or venlafaxine, 75–150 mg/d), gabapentin (300–900 mg/d), clonidine (0.1–0.2 mg/d), or vitamin E (400–800 IU/d), or the consumption of soy-based products or other phytoestrogens, may also alleviate vasomotor symptoms, although they are less effective than HT. Paroxetine is the only nonhormonal drug approved by the U.S. Food and Drug Administration for treatment of vasomotor symptoms. Bazedoxifene, an estrogen agonist/antagonist, in combination with conjugated estrogens has also received approval for vasomotor symptom management. For genitourinary symptoms, the efficacy of vaginal estrogen is similar to that of oral or transdermal estrogen; oral ospemifene is an additional option.

1	oSteoporoSiS (See also Chap. 425) Bone density By reducing bone turnover and resorption rates, estrogen slows the aging-related bone loss experienced by most postmenopausal women. More than 50 randomized trials have demonstrated that postmenopausal estrogen therapy, with or without a progestogen, rapidly increases bone mineral density at the spine by 4–6% and at the hip by 2–3% and that those increases are maintained during treatment.

1	Fractures Data from observational studies indicate a 50–80% lower risk of vertebral fracture and a 25–30% lower risk of hip, wrist, and other peripheral fractures among current estrogen users; addition of a progestogen does not appear to modify this benefit. In the WHI, 5–7 years of either combined estrogen-progestin or estrogen-only therapy was associated with a 33% reduction in hip fractures and 25–30% fewer total fractures among a population unselected for osteoporosis. Bisphosphonates (such as alendronate, 10 mg/d or 70 mg once per week; risedronate, 5 mg/d or 35 mg once per week; or ibandronate, 2.5 mg/d or 150 mg once per month or 3 mg every 3 months IV) and raloxifene (60 mg/d), a selective estrogen receptor modulator (SERM), have been shown in randomized trials to increase bone mass density and decrease fracture rates. Other options for treatment of osteoporosis are bazedoxifene in combination with conjugated estrogens and parathyroid hormone (teriparatide, 20 μg/d SC). These

1	density and decrease fracture rates. Other options for treatment of osteoporosis are bazedoxifene in combination with conjugated estrogens and parathyroid hormone (teriparatide, 20 μg/d SC). These agents, unlike 2383 estrogen, do not appear to have adverse effects on the endometrium or breast. Increased physical activity, adequate calcium intake (1000–1200 mg/d through diet or supplements in two or three divided doses), and adequate vitamin D intake (600–1000 IU/d) may also reduce the risk of osteoporosis-related fractures. According to the Institute of Medicine’s 2011 report, 25-hydroxyvitamin D blood levels of ≥50 nmol/L are sufficient for bone-density maintenance and fracture prevention. The Fracture Risk Assessment (FRAX®) score, an algorithm that combines an individual’s bone-density score with age and other risk factors to predict her 10-year risk of hip and major osteoporotic fracture, may be of use in guiding decisions about pharmacologic treatment (see www .shef.ac.uk/FRAX/).

1	Definite Risks • endometrial cancer (WitH eStroGen alone) A combined analysis of 30 observational studies found a tripling of endometrial cancer risk among short-term users (1–5 years) of unopposed estrogen and a nearly tenfold increased risk among long-term users (≥10 years). These findings are supported by results from the randomized Postmenopausal Estrogen/Progestin Interventions (PEPI) trial, in which 24% of women assigned to unopposed estrogen for 3 years developed atypical endometrial hyperplasia—a premalignant lesion— as opposed to only 1% of women assigned to placebo. Use of a progestogen, which opposes the effects of estrogen on the endometrium, eliminates these risks and may even reduce risk (see later).

1	venouS tHromBoemBoliSm A meta-analysis of observational studies found that current oral estrogen use was associated with a 2.5-fold increase in risk of venous thromboembolism in postmenopausal women. A meta-analysis of randomized trials, including the WHI, found a 2.1-fold increase in risk. Results from the WHI indicate a nearly twofold increase in risk of pulmonary embolism and deep vein thrombosis with estrogen-progestin and a 35–50% increase in these risks with estrogen-only therapy. Transdermal estrogen, taken alone or with certain progestogens (micronized progesterone or pregnane derivatives), appears to be a safer alternative with respect to throm botic risk.

1	BreaSt cancer (WitH eStroGen-proGeStin) An increased risk of breast cancer has been found among current or recent estrogen users in observational studies; this risk is directly related to duration of use. In a meta-analysis of 51 case-control and cohort studies, short-term use (<5 years) of postmenopausal HT did not appreciably elevate breast cancer incidence, whereas long-term use (≥5 years) was associated with a 35% increase in risk. In contrast to findings for endometrial cancer, combined estrogen-progestin regimens appear to increase breast cancer risk more than estrogen alone. Data from randomized trials also indicate that estrogen-progestin raises breast cancer risk. In the WHI, women assigned to receive combination hormones for an average of 5.6 years were 24% more likely to develop breast cancer than women assigned to placebo, but 7.1 years of estrogen-only therapy did not increase risk. Indeed, the WHI showed a trend toward a reduction in breast cancer risk with estrogen

1	breast cancer than women assigned to placebo, but 7.1 years of estrogen-only therapy did not increase risk. Indeed, the WHI showed a trend toward a reduction in breast cancer risk with estrogen alone, although it is unclear whether this finding would pertain to formulations of estrogen other than conjugated equine estrogens or to treatment durations of >7 years. In the Heart and Estrogen/Progestin Replacement Study (HERS), 4 years of combination therapy was associated with a 27% increase in breast cancer risk. Although the latter finding was not statistically significant, the totality of evidence strongly implicates estrogen-progestin therapy in breast carcinogenesis.

1	Some observational data suggest that the length of the interval between menopause onset and HT initiation may influence the association between such therapy and breast cancer risk, with a “gap time” of <3–5 years conferring a higher HT-associated breast cancer risk. (This pattern of findings contrasts with that for CHD, as discussed later in this Chapter.) However, this association remains inconclusive and may be a spurious finding attributable to higher rates of screening mammography and thus earlier cancer detection in HT users than in nonusers, especially in early menopause. Indeed, in the WHI trial, hazard ratios for HT and breast cancer risk did not differ among women bEnEfiTS AnD RiSkS of PoSTmEnoPAuSAl HoRmonE THERAPy in THE ovERAll STuDy PoPulATion of womEn 50–79 yEARS of AgE in THE inTERvEnTion PHASE of THE womEn’S HEAlTH iniTiATivE (wHi) ESTRogEn-PRogESTin AnD ESTRogEn-AlonE TRiAlSa diseased women and women many years past (n.s.) (41 vs. 35) infarction for estrogen alone,

1	THE inTERvEnTion PHASE of THE womEn’S HEAlTH iniTiATivE (wHi) ESTRogEn-PRogESTin AnD ESTRogEn-AlonE TRiAlSa diseased women and women many years past (n.s.) (41 vs. 35) infarction for estrogen alone, with reduced risk in (n.s.) (35 vs. 29) trend by age = .02)

1	Stroke Probable increase in risk ↑37% increased risk 9 excess cases ↑35% increased risk 11 excess cases (33 vs. 24) (45 vs. 34) Ovarian cancer Probable increase in risk with long-term ↑41% increased risk 1 excess cases Not available Not available use (≥5 years) (n.s.) (5 vs. 4) Endometrial cancer Probable decrease in risk with estrogen-↓33% decreased riskf 3 fewer cases See above See above progestin during long-term follow-up (7 vs. 10) (see above for estrogen alone) (1661 vs. 1112) (2255 vs. 1403) Colorectal cancer Probable decrease in risk with estrogen-↓38% decreased risk 6.5 fewer cases No increase or No difference in riske progestin; possible increase in risk in (10 vs. 17) decrease in riske older women with estrogen alone (p for trend by age = .02 for estrogen alone)

1	Type 2 diabetes Probable decrease in risk ↓19% decreased risk 16 fewer cases ↓14% decreased risk 21 fewer cases (72 vs. 88) (134 vs. 155) (46 vs. 23) (n.s.) (44 vs. 29) studies and randomized trials) pausal women (p for trend by age <.05

1	Global indexg Probable increase in risk or no effect ↑12% increased risk 20.5 excess cases No increase in riske No difference in riske among older women and women many (189 vs. 168) years past menopause; possible decrease in risk or no effect in younger or recently menopausal women (p for trend by age = 0.02 for estrogen alone) aThe estrogen-progestin arm of the WHI assessed 5.6 years of conjugated equine estrogen (0.625 mg/d) plus medroxyprogesterone acetate (2.5 mg/d) versus placebo. The estrogen-alone arm of the WHI assessed 7.1 years of conjugated equine estrogen (0.625 mg/d) versus placebo. bNumber of cases per 10, 000 women per year. cThe WHI was not designed to assess the effect of HT on menopausal symptoms. Data from other randomized trials suggest that HT reduces risk for menopausal symptoms by 65–90%. dCoronary heart disease is defined as nonfatal myocardial infarction or coronary death. eThere was a significant interaction by age; that is, the association between HT and the

1	symptoms by 65–90%. dCoronary heart disease is defined as nonfatal myocardial infarction or coronary death. eThere was a significant interaction by age; that is, the association between HT and the specified outcome was different in younger women and older women. f This is the risk reduction that was observed during a cumulative 12-year follow-up period (5.6 years of treatment plus 6.8 years of postintervention observation). gThe global index is a composite outcome representing the first event for each participant from among the following: coronary heart disease, stroke, pulmonary embolism, breast cancer, colorectal cancer, endometrial cancer (estrogen-progestin arm only), hip fracture, and death. Because participants can experience more than one type of event, the global index cannot be derived by a simple summing of the component events. hIncludes some outcomes where results were divergent between the estrogen-progestin arm and the estrogen-alone arm.

1	Abbreviation: n.s., not statistically significant. Source: Data from JE Manson et al: JAMA 310:1353, 2013. 50–59, those 60–69, and those 70–79 years of age at trial entry. (There was insufficient power to examine finer age categories.) Additional research is needed to clarify the issue. GallBladder diSeaSe Large observational studies report a twoto threefold increased risk of gallstones or cholecystectomy among postmenopausal women taking oral estrogen. In the WHI, women randomized to estrogen-progestin or estrogen alone were ∼55% more likely to develop gallbladder disease than those assigned to placebo. Risks were also increased in HERS. Transdermal HT might be a safer alternative, but further research is needed.

1	Probable or Uncertain Risks and Benefits • coronary Heart diSeaSe/ StroKe Until recently, HT had been enthusiastically recommended as a possible cardioprotective agent. In the past three decades, multiple observational studies suggested, in the aggregate, that estrogen use leads to a 35–50% reduction in CHD incidence among postmenopausal women. The biologic plausibility of such an association is supported by data from randomized trials demonstrating that exogenous estrogen lowers plasma low-density lipoprotein (LDL) cholesterol levels and raises high-density lipoprotein (HDL) cholesterol levels by 10–15%. Administration of estrogen also favorably affects lipoprotein(a) levels, LDL oxidation, endothelial vascular function, fibrinogen, and plasminogen activator inhibitor 1. However, estrogen therapy has unfavorable effects on other biomarkers of cardiovascular risk: it boosts triglyceride levels; promotes coagulation via factor VII, prothrombin fragments 1 and 2, and fibrinopeptide A

1	therapy has unfavorable effects on other biomarkers of cardiovascular risk: it boosts triglyceride levels; promotes coagulation via factor VII, prothrombin fragments 1 and 2, and fibrinopeptide A elevations; and raises levels of the inflammatory marker C-reactive protein.

1	Randomized trials of estrogen or combined estrogen-progestin in women with preexisting cardiovascular disease have not confirmed the benefits reported in observational studies. In HERS (a secondary-prevention trial designed to test the efficacy and safety of estrogen-progestin therapy with regard to clinical cardiovascular outcomes), the 4-year incidence of coronary death and nonfatal myocardial infarction was similar in the active-treatment and placebo groups, and a 50% increase in risk of coronary events was noted during the first year among participants assigned to the active-treatment group. Although it is possible that progestin may mitigate estrogen’s benefits, the Estrogen Replacement and Atherosclerosis (ERA) trial indicated that angiographically determined progression of coronary atherosclerosis was unaffected by either opposed or unopposed estrogen treatment. Moreover, no cardiovascular benefit was found in the Papworth Hormone Replacement Therapy Atherosclerosis Study, a

1	atherosclerosis was unaffected by either opposed or unopposed estrogen treatment. Moreover, no cardiovascular benefit was found in the Papworth Hormone Replacement Therapy Atherosclerosis Study, a trial of transdermal estradiol with and without norethindrone; the Women’s Estrogen for Stroke Trial (WEST), a trial of oral 17β-estradiol; or the Estrogen in the Prevention of Reinfarction Trial (ESPRIT), a trial of oral estradiol valerate. Thus, in clinical trials, HT has not proved effective for the secondary prevention of cardiovascular disease in postmenopausal women.

1	Primary-prevention trials also suggest an early increase in cardiovascular risk and an absence of cardioprotection with postmenopausal HT. In the WHI, women assigned to 5.6 years of estrogen-progestin therapy were 18% more likely to develop CHD (defined in primary analyses as nonfatal myocardial infarction or coronary death) than those assigned to placebo, although this risk elevation was not statistically significant. However, during the trial’s first year, there was a significant 80% increase in risk, which diminished in subsequent years (p for trend by time = .03). In the estrogen-only arm of the WHI, no overall effect on CHD was observed during the 7.1 years of the trial or in any specific year of follow-up. This pattern of results was similar to that for the outcome of total myocardial infarction.

1	However, a closer look at available data suggests that timing of initiation of HT may critically influence the association between such therapy and CHD. Estrogen may slow early stages of atherosclerosis but have adverse effects on advanced atherosclerotic lesions. It has been hypothesized that the prothrombotic and proinflammatory effects of estrogen manifest themselves predominantly among women with subclinical lesions who initiate HT well after the menopausal transition, whereas women with less arterial damage who start HT early in menopause may derive cardiovascular benefit because they have not yet developed advanced lesions. Nonhuman primate data support this concept. Conjugated estrogens had no effect on the extent 2385 of coronary artery plaque in cynomolgus monkeys assigned to receive estrogen alone or combined with progestin starting 2 years (∼6 years in human terms) after oophorectomy and well after the establishment of atherosclerosis. However, administration of exogenous

1	estrogen alone or combined with progestin starting 2 years (∼6 years in human terms) after oophorectomy and well after the establishment of atherosclerosis. However, administration of exogenous hormones immediately after oophorectomy, during the early stages of atherosclerosis, reduced the extent of plaque by 70%.

1	Lending further credence to this hypothesis are results of subgroup analyses of observational and clinical trial data. For example, among women who entered the WHI trial with a relatively favorable cholesterol profile, estrogen with or without progestin led to a 40% lower risk of incident CHD. Among women who entered with a worse cholesterol profile, therapy resulted in a 73% higher risk (p for interaction = .02). The presence or absence of the metabolic syndrome (Chap. 422) also strongly influenced the relation between HT and incident CHD. Among women with the metabolic syndrome, HT more than doubled CHD risk, whereas no association was observed among women without the syndrome. Moreover, although there was no association between estrogen-only therapy and CHD in the WHI trial cohort as a whole, such therapy was associated with a CHD risk reduction of 40% among participants age 50–59; in contrast, a risk reduction of only 5% was observed among those age 60–69, and a risk increase of

1	a whole, such therapy was associated with a CHD risk reduction of 40% among participants age 50–59; in contrast, a risk reduction of only 5% was observed among those age 60–69, and a risk increase of 9% was found among those age 70–79 (p for trend by age = .08). For the outcome of total myocardial infarction, estrogen alone was associated with a borderline-significant 45% reduction and a nonsignificant 24% increase in risk among the youngest and oldest women, respectively (p for trend by age = .02). Estrogen was also associated with lower levels of coronary artery calcified plaque in the younger age group. Although age did not have a similar effect in the estrogen-progestin arm of the WHI, CHD risks increased with years since menopause (p for trend = .08), with a significantly elevated risk among women who were ≥20 years past menopause. For the outcome of total myocardial infarction, estrogen-progestin was associated with a 9% risk reduction among women <10 years past menopause as

1	risk among women who were ≥20 years past menopause. For the outcome of total myocardial infarction, estrogen-progestin was associated with a 9% risk reduction among women <10 years past menopause as opposed to a 16% increase in risk among women 10–19 years past menopause and a twofold increase in risk among women >20 years past menopause (p for trend = .01). In the large observational Nurses’ Health Study, women who chose to start HT within 4 years of menopause experienced a lower risk of CHD than did nonusers, whereas those who began therapy ≥10 years after menopause appeared to receive little coronary benefit. Observational studies include a high proportion of women who begin HT within 3–4 years of menopause, whereas clinical trials include a high proportion of women ≥12 years past menopause; this difference helps to reconcile some of the apparent discrepancies between the two types of studies.

1	For the outcome of stroke, WHI participants assigned to estrogenprogestin or estrogen alone were ∼35% more likely to suffer a stroke than those assigned to placebo. Whether or not age at initiation of HT influences stroke risk is not well understood. In the WHI and the Nurses’ Health Study, HT was associated with an excess risk of stroke in all age groups. Further research is needed on age, time since menopause, and other individual characteristics (including biomarkers) that predict increases or decreases in cardiovascular risk associated with exogenous HT. Furthermore, it remains uncertain whether different doses, formulations, or routes of administration of HT will produce different cardiovascular effects.

1	colorectal cancer Observational studies have suggested that HT reduces risks of colon and rectal cancer, although the estimated magnitudes of the relative benefits have ranged from 8% to 34% in various meta-analyses. In the WHI (the sole trial to examine the issue), estrogen-progestin was associated with a significant 38% reduction in colorectal cancer over a 5.6-year period, although no benefit was seen with 7 years of estrogen-only therapy. However, a modifying effect of age was observed, with a doubling of risk with HT in women age 70–79 but no risk elevation in younger women (p for trend by age = .02).

1	coGnitive decline and dementia A meta-analysis of 10 case-control and two cohort studies suggested that postmenopausal HT is associated with a 34% decreased risk of dementia. Subsequent randomized trials (including the WHI), however, have failed to demonstrate any benefit 2386 of estrogen or estrogen-progestin therapy on the progression of mild to moderate Alzheimer’s disease and/or have indicated a potential adverse effect of HT on the incidence of dementia, at least in women ≥65 years of age. Among women randomized to HT (as opposed to placebo) at age 50–55 in the WHI, no effect on cognition was observed during the postintervention phase. Determining whether timing of initiation of HT influences cognitive outcomes will require further study.

1	ovarian cancer and otHer diSorderS On the basis of limited observational and randomized data, it has been hypothesized that HT increases the risk of ovarian cancer and reduces the risk of type 2 diabetes mellitus. Results from the WHI support these hypotheses. The WHI also found that HT use was associated with an increased risk of urinary incontinence and that estrogen-progestin was associated with increased rates of lung cancer mortality. endometrial cancer (WitH eStroGen-proGeStin) In the WHI, use of estrogen-progestin was associated with a nonsignificant 17% reduction in risk of endometrial cancer. A significant reduction in risk emerged during the postintervention period (see later).

1	all-cauSe mortality In the overall WHI cohort, estrogen with or without progestin was not associated with all-cause mortality. However, there was a trend toward reduced mortality in younger women, particularly with estrogen alone. For women 50–59, 60–69, and 70–79 years of age, relative risks (RRs) associated with estrogen-only therapy were 0.70, 1.01, and 1.21, respectively (p for trend = .04).

1	overall Benefit-riSK profile Estrogen-progestin was associated with an unfavorable benefit-risk profile (excluding relief from menopausal symptoms) as measured by a “global index”—a composite outcome including CHD, stroke, pulmonary embolism, breast cancer, colorectal cancer, endometrial cancer, hip fracture, and death (Table 413-1)— in the WHI cohort as a whole, and this association did not vary by 10-year age group. Estrogen-only therapy was associated with a neutral benefit-risk profile in the WHI cohort as a whole. However, there was a significant trend toward a more favorable benefit-risk profile among younger women and a less favorable profile among older women, with RRs of 0.84, 0.99, and 1.17 for women 50–59, 60–69, and 70–79 years of age, respectively (p for trend by age = .02).

1	cHanGeS in HealtH StatuS after diScontinuation of Hormone tHerapy In the WHI, many but not all risks and benefits associated with active use of HT dissipated within 5–7 years after discontinuation of therapy. For estrogen-progestin, an elevated risk of breast cancer persisted (RR = 1.28 [95% confidence interval, 1.11–1.48]) during a cumulative 12-year follow-up period (5.6 years of treatment plus 6.8 years of postintervention observation), but most cardiovascular disease risks became neutral. A reduction in hip fracture risk persisted (RR = 0.81 [0.68–0.97]), and a significant reduction in endometrial cancer risk emerged (RR = 0.67 [0.49–0.91]). For estrogen alone, the reduction in breast cancer risk became statistically significant (RR = 0.79 [0.65–0.97]) during a cumulative 12-year follow-up period (6.8 years of treatment plus 5.1 years of postintervention observation), and significant differences by age group persisted for total myocardial infarction and the global index, with more

1	period (6.8 years of treatment plus 5.1 years of postintervention observation), and significant differences by age group persisted for total myocardial infarction and the global index, with more favorable results for younger women.

1	APPROACH TO THE PATIENT:

1	The rational use of postmenopausal HT requires balancing the potential benefits and risks. Figure 413-3 provides one approach to decision making. The clinician should first determine whether the patient has moderate to severe menopausal symptoms—the primary indication for initiation of systemic HT. Systemic HT may also be used to prevent osteoporosis in women at high risk of fracture who cannot tolerate alternative osteoporosis therapies. (Vaginal estrogen or other medications may be used to treat urogenital symptoms in the absence of vasomotor symptoms.) The benefits and risks of such therapy should be reviewed with the patient, giving more emphasis to absolute than to relative measures of effect and pointing out uncertainties in clinical knowledge where relevant. Because chronic disease rates generally increase with age, absolute risks tend to be greater in older women, even when relative risks remain similar. Potential side effects—especially vaginal bleeding that may result from

1	rates generally increase with age, absolute risks tend to be greater in older women, even when relative risks remain similar. Potential side effects—especially vaginal bleeding that may result from use of the combined estrogen-progestogen formulations recommended for women with an intact uterus—should be noted. The patient’s own preference regarding therapy should be elicited and factored into the decision. Contraindications to HT should be assessed routinely and include unexplained vaginal bleeding, active liver disease, venous thromboembolism, history of endometrial cancer (except stage 1 without deep invasion) or breast cancer, and history of CHD, stroke, transient ischemic attack, or diabetes. Relative contraindications include hypertriglyceridemia (>400 mg/dL) and active gallbladder disease; in such cases, transdermal estrogen may be an option. Primary prevention of heart disease should not be viewed as an expected benefit of HT, and an increase in the risk of stroke as well as a

1	in such cases, transdermal estrogen may be an option. Primary prevention of heart disease should not be viewed as an expected benefit of HT, and an increase in the risk of stroke as well as a small early increase in the risk of coronary artery disease should be considered. Nevertheless, such therapy may be appropriate if the noncoronary benefits of treatment clearly outweigh the risks. A woman who suffers an acute coronary event or stroke while taking HT should discontinue therapy immediately.

1	Short-term use (<5 years for estrogen-progestogen and <7 years for estrogen alone) is appropriate for relief of menopausal symptoms among women without contraindications to such use. However, such therapy should be avoided by women with an elevated baseline risk of future cardiovascular events. Women who have contraindications for or are opposed to HT may derive benefit from the use of certain antidepressants (including venlafaxine, fluoxetine, or paroxetine), gabapentin, clonidine, soy, or black cohosh and, for genitourinary symptoms, intravaginal estrogen creams or devices, or ospemifene.

1	Long-term use (≥5 years for estrogen-progestogen and ≥7 years for estrogen alone) is more problematic because a heightened risk of breast cancer must be factored into the decision, especially for estrogen-progestogen. Reasonable candidates for such use include the small percentage of postmenopausal women who have persistent severe vasomotor symptoms along with an increased risk of osteoporosis (e.g., those with osteopenia, a personal or family history of nontraumatic fracture, or a weight below 125 lbs), who also have no personal or family history of breast cancer in a first-degree relative or other contraindications, and who have a strong personal preference for therapy. Poor candidates are women with elevated cardiovascular risk, those at increased risk of breast cancer (e.g., women who have a first-degree relative with breast cancer, susceptibility genes such as BRCA1 or BRCA2, or a personal history of cellular atypia detected by breast biopsy), and those at low risk of

1	women who have a first-degree relative with breast cancer, susceptibility genes such as BRCA1 or BRCA2, or a personal history of cellular atypia detected by breast biopsy), and those at low risk of osteoporosis. Even for reasonable candidates, strategies to minimize dose and duration of use should be employed. For example, women using HT to relieve intense vasomotor symptoms in early postmenopause should consider discontinuing therapy within 5 years, resuming it only if such symptoms persist. Because of the role of progestogens in increasing breast cancer risk, regimens that employ cyclic rather than continuous progestogen exposure as well as formulations other than medroxyprogesterone acetate should be considered if treatment is extended. For prevention of osteoporosis, alternative therapies such as bisphosphonates or SERMs should be considered. Research on alternative progestogens and androgen-containing preparations has been limited, particularly with respect to long-term safety.

1	such as bisphosphonates or SERMs should be considered. Research on alternative progestogens and androgen-containing preparations has been limited, particularly with respect to long-term safety. Additional research on the effects of these agents on cardiovascular disease, glucose tolerance, and breast cancer will be of particular interest.

1	In addition to HT, lifestyle choices such as smoking abstention, adequate physical activity, and a healthy diet can play a role in controlling symptoms and preventing chronic disease. An expanding array of pharmacologic options (e.g., bisphosphonates, SERMs, and other agents for osteoporosis; cholesterol-lowering or antihypertensive agents for cardiovascular disease) should also reduce the widespread reliance on hormone use. However, short-term HT may still benefit some women. Avoid HThYes Yes No No Free of contraindicationscto HT and no h/o CHD, stroke, or TIA? AND No increased risk of stroke (<10% by Framingham Stroke Score)?dSignificant symptoms of menopause (moderate-to-severe hot flashes, night sweats)?bAssess CHD risk and years since last menstrual period Years since last menstrual periodfAvoid HT Low (5% to <10%) Moderate (10% to 20%) 6 to 10

1	Decision about duration of use: continued moderate-to-severe symptoms; patient preference; weigh baseline risks of breast cancer vs osteoporosis FIGURE 413-3 Chart for identifying appropriate candidates for postmenopausal hormone therapy (HT).a aReassess each step at least once every 6–12 months (assuming the patient’s continued preference for HT). bWomen who are at high risk of osteoporotic fracture but are unable to tolerate alternative preventive medications may also be reasonable candidates for systemic HT even if they do not have moderate to severe vasomotor symptoms. Women who have vaginal dryness without moderate to severe vasomotor symp-toms may be candidates for vaginal estrogen. cTraditional contraindications are unexplained vaginal bleeding; active liver disease; history of venous thromboembolism due to pregnancy, oral contraceptive use, or an unknown etiology; blood-clotting disorder; history of breast or endo-metrial cancer; and diabetes. Oral HT should be avoided but

1	venous thromboembolism due to pregnancy, oral contraceptive use, or an unknown etiology; blood-clotting disorder; history of breast or endo-metrial cancer; and diabetes. Oral HT should be avoided but transdermal HT may be an option (see g below) for other contraindications, includ-ing high triglyceride levels (>400 mg/dL); active gallbladder disease; and history of venous thromboembolism due to past immobility, surgery, or bone fracture. dTen-year risk of stroke, based on Framingham Stroke Risk Score (RB D’Agostino et al: Stroke risk profile: Adjustment for antihyper-tensive medication. The Framingham Study. Stroke 25:40, 1994), as modified by JE Manson, SS Bassuk: Hot Flashes, Hormones & Your Health. New York, McGraw-Hill, 2007. eTen-year risk of CHD, based on Framingham Coronary Heart Disease Risk Score (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults: JAMA 285:2486, 2001), as modified by JE Manson, SS Bassuk: Hot Flashes, Hormones & Your

1	Disease Risk Score (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults: JAMA 285:2486, 2001), as modified by JE Manson, SS Bassuk: Hot Flashes, Hormones & Your Health. New York, McGraw-Hill, 2007. f Women >10 years past menopause are not good candidates for initiation (first use) of HT. gAvoid oral HT. Transdermal HT may be an option because it has a less adverse effect on clotting factors, triglyceride levels, and inflammation factors than oral HT. hConsider selective serotonin or serotonin–norepinephrine reuptake inhibitor, gabapentin, clonidine, soy, or another alternative. Abbreviations: CHD, coronary heart disease; h/o, history of; TIA, transient ischemic attack. (Adapted from JE Manson, SS Bassuk: Hot Flashes, Hormones & Your Health. New York, McGraw-Hill, 2007. Copyright © 2007 by the President and Fellows of Harvard College. All rights reserved.) infertility and Contraception Janet E. Hall INFERTILITY DEFINITION AND PREVALENCE Infertility

1	2007. Copyright © 2007 by the President and Fellows of Harvard College. All rights reserved.) infertility and Contraception Janet E. Hall INFERTILITY DEFINITION AND PREVALENCE Infertility has traditionally been defined as the inability to conceive after 12 months of unprotected sexual intercourse. In women who ultimately conceived, pregnancy occurred in ~50% within 3 months, 75–82% within 6 months, and 85–92% within 12 months. The World Health Organization (WHO) considers infertility as a disability (an impairment of function) and thus access to health care falls under the Convention on the Rights of Persons with Disability. Thirty-four mil-lion women, predominantly from developing countries, have infertility resulting from maternal sepsis and unsafe abortion. In populations <60 years old, infertility is ranked the fifth highest serious global dis-ability. In the United States, the rate of infertility in married women age 15–44 is 6% based on the National Survey of Family Growth, 414

1	infertility is ranked the fifth highest serious global dis-ability. In the United States, the rate of infertility in married women age 15–44 is 6% based on the National Survey of Family Growth, 414 although prospective studies suggest that it may be as high as 12–15%. The infertility rate has remained relatively stable over the past 30 years in most countries. However, the proportion of couples without children has risen, reflecting both higher numbers of couples in childbearing years and a trend to delay childbearing. This trend has important implications because of an age-related decrease in fecundability: the incidence of primary infertility increases from ~8% between the ages of 18 and 38 to 25% and 30% between the ages of 35 and 39 and 40 and 44, respectively. It is estimated that 14% of couples in the United States have received medical assistance for infertility; of these, two-thirds received counseling, ~12% underwent infertility testing of the female and/or male partner, and

1	of couples in the United States have received medical assistance for infertility; of these, two-thirds received counseling, ~12% underwent infertility testing of the female and/or male partner, and 17% received drugs to induce ovulation.

1	The spectrum of infertility ranges from reduced conception rates or the need for medical intervention to irreversible causes of infertility. Infertility can be attributed primarily to male factors in 25% of couples and female factors in 58% of couples and is unexplained in about 17% of couples (Fig. 414-1). Not uncommonly, both male and female factors contribute to infertility. Decreases in the ability to conceive as a Infertility14% of reproductive aged women 5 million couples in the U.S. Female causes 58% Male causes 25% Unexplained 17% Primary hypogonadism ( FSH) 30–40% Secondary hypogonadism ( FSH, LH) 2% Disordered sperm transport 10–20% Unknown 40–50% FIGURE 414-1 Causes of infertility. FSH, follicle-stimulating hormone; LH, luteinizing hormone.

1	FIGURE 414-1 Causes of infertility. FSH, follicle-stimulating hormone; LH, luteinizing hormone. function of age in women has led to recommendations that women >34 years old who are not at increased risk of infertility seek attention after 6 months, rather than 12 months as suggested for younger women, and receive an expedited work-up and approach to treatment. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: In all couples presenting with infertility, the initial evaluation includes discussion of the appropriate timing of intercourse and discussion of modifiable risk factors such as smoking, alcohol, caffeine, and obesity. The range of required investigations should be reviewed as well as a brief description of infertility treatment options, including adoption. Initial investigations are focused on determining whether the primary cause of the infertility is male, female, or both. These investigations include a semen analysis in the male, confirmation of ovulation in the female, and, in the majority of situations, documentation of tubal patency in the female. In some cases, after an extensive workup excluding all male and female factors, a specific cause cannot be identified, and infertility may ultimately be classified as unexplained.

1	Infertility is invariably associated with psychological stress related not only to the diagnostic and therapeutic procedures themselves but also to repeated cycles of hope and loss associated with each new procedure or cycle of treatment that does not result in the birth of a child. These feelings are often combined with a sense of isolation from friends and family. Counseling and stress-management techniques should be introduced early in the evaluation of infertility. Importantly, infertility and its treatment do not appear to be associated with long-term psychological sequelae.

1	Abnormalities in menstrual function constitute the most common cause of female infertility. These disorders, which include ovulatory dysfunction and abnormalities of the uterus or outflow tract, may present as amenorrhea or as irregular or short menstrual cycles. A careful history and physical examination and a limited number of laboratory tests will help to determine whether the abnormality is (1) hypothalamic or pituitary (low follicle-stimulating hormone [FSH], luteinizing hormone [LH], and estradiol with or without an increase in prolactin), (2) polycystic ovary syndrome (PCOS; irregular cycles and hyperandrogenism in the absence of other causes of androgen excess), (3) ovarian (low estradiol with increased FSH), or (4) a uterine or outflow tract abnormality. The frequency of these diagnoses depends on whether the amenorrhea is primary or occurs after normal puberty and menarche (see Fig. 69-2).

1	The approach to further evaluation of these disorders is described in detail in Chap. 69. Ovulatory Dysfunction In women with a history of regular menstrual cycles, evidence of ovulation should be sought (Chap. 412). Even in the presence of ovulatory cycles, evaluation of ovarian reserve is recommended for women age >35 years if they are interested in fertility. Measurement of FSH on day 3 of the cycle (an FSH level <10 IU/ mL on cycle day 3 predicts adequate ovarian oocyte reserve) is the most cost-effective test. Other tests include measurement of FSH in response to clomiphene citrate (blocks estrogen negative feedback on FSH), antral follicle count on ultrasound, and anti-müllerian hormone (AMH; <0.5 ng/mL predicts reduced ovarian reserve although there is variability between labs).

1	Tubal Disease Tubal dysfunction may result from pelvic inflammatory disease (PID), appendicitis, endometriosis, pelvic adhesions, tubal surgery, previous use of an intrauterine device (IUD), and a previous ectopic pregnancy. However, a cause is not identified in up to 50% of patients with documented tubal factor infertility. Because of the high prevalence of tubal disease, evaluation of tubal patency by hysterosalpingogram (HSG) or laparoscopy should occur early in the majority of couples with infertility. Subclinical infections with Chlamydia trachomatis may be an underdiagnosed cause of tubal infertility and requires the treatment of both partners.

1	Endometriosis Endometriosis is defined as the presence of endometrial glands or stroma outside the endometrial cavity and uterine musculature and accounts for 40% of infertility not due to ovulatory disorders, tubal obstruction, or male factor. Its presence is suggested by a history of dyspareunia (painful intercourse), worsening dysmenorrhea that often begins before menses, or a thickened rectovaginal septum or deviation of the cervix on pelvic examination. Mild endometriosis does not appear to impair fertility; the pathogenesis of the infertility associated with moderate and severe endometriosis may be multifactorial with impairments of folliculogenesis, fertilization, and implantation, as well as adhesions. Endometriosis is often clinically silent, however, and can only be excluded definitively by laparoscopy. MALE CAUSES (SEE ALSO CHAP. 411)

1	MALE CAUSES (SEE ALSO CHAP. 411) Known causes of male infertility include primary testicular disease, genetic disorders (particularly Y chromosome microdeletions), disorders of sperm transport, and hypothalamic-pituitary disease resulting in secondary hypogonadism. However, the etiology is not ascertained in up to one-half of men with suspected male factor infertility. The key initial diagnostic test is a semen analysis. Testosterone levels should be measured if the sperm count is low on repeated examination or if there is clinical evidence of hypogonadism. Gonadotropin levels will help to determine a gonadal versus a central cause of hypogonadism.

1	In addition to addressing the negative impact of smoking on fertility and pregnancy outcome, counseling about nutrition and weight is a fundamental component of infertility and pregnancy management. Both low and increased body mass index (BMI) are associated with infertility in women and with increased morbidity during pregnancy. Obesity has also been associated with infertility in men. The treatment of infertility should be tailored to the problems unique to each couple. In many situations, including unexplained infertility, mild-to-moderate endometriosis, and/or borderline semen parameters, a stepwise approach to infertility is optimal, beginning with low-risk interventions and moving to more invasive, higher risk interventions only if necessary. After determination of all infertility factors and their correction, if possible, this approach might include, in increasing order of complexity: (1) expectant management, (2) clomiphene citrate or an aromatase inhibitor (see below) with or

1	and their correction, if possible, this approach might include, in increasing order of complexity: (1) expectant management, (2) clomiphene citrate or an aromatase inhibitor (see below) with or without intrauterine insemination (IUI), (3) gonadotropins with or without IUI, and (4) in vitro fertilization (IVF). The time used for evaluation, correction of problems identified, and expectant management can be longer in women age <30 years, but this process should be advanced rapidly in women age >35 years. In some situations, expectant management will not be appropriate.

1	Treatment of ovulatory dysfunction should first be directed at identification of the etiology of the disorder to allow specific management when possible. Dopamine agonists, for example, may be indicated in patients with hyperprolactinemia (Chap. 403); lifestyle modification may be successful in women with obesity, low body weight, or a history of intensive exercise.

1	Medications used for ovulation induction include agents that increase FSH through alteration of negative feedback, gonadotropins, and pulsatile GnRH. Clomiphene citrate is a nonsteroidal estrogen antagonist that increases FSH and LH levels by blocking estrogen negative feedback at the hypothalamus. The efficacy of clomiphene for ovulation induction is highly dependent on patient selection. In appropriate patients, it induces ovulation in ~60% of women with PCOS and has traditionally been the initial treatment of choice. Combination with agents that modify insulin levels such as metformin does not appear to improve outcome. Clomiphene citrate is less successful in patients with hypogonadotropic hypogonadism. Aromatase inhibitors have also been investigated for the treatment of infertility. Studies suggest they may have advantages over clomiphene, but these medications have not been approved for this indication.

1	Gonadotropins are highly effective for ovulation induction in women with hypogonadotropic hypogonadism and PCOS and are used to induce the development of multiple follicles in unexplained infertility and in older reproductive-age women. Disadvantages include a significant risk of multiple gestation and the risk of ovarian hyperstimulation, particularly in women with polycystic ovaries, with or without other features of PCOS. Careful monitoring and a conservative approach to ovarian stimulation reduce these risks. Currently available gonadotropins include urinary preparations of LH and FSH, highly purified FSH, and recombinant FSH. Although FSH is the key component, LH is essential for steroidogenesis in hypogonadotropic patients, and LH or human chorionic gonadotropin (hCG) may improve results through effects on terminal differentiation of the oocyte. These methods are commonly combined with IUI.

1	None of these methods are effective in women with premature ovarian failure, in whom donor oocyte or adoption is the method of choice. If hysterosalpingography suggests a tubal or uterine cavity abnormality or if a patient is age ≥35 at the time of initial evaluation, laparoscopy with tubal lavage is recommended, often with a hysteroscopy. Although tubal reconstruction may be attempted if tubal disease is identified, it is generally being replaced by the use of IVF. These patients are at increased risk of developing an ectopic pregnancy.

1	Although 60% of women with minimal or mild endometriosis may conceive within 1 year without treatment, laparoscopic resection or ablation appears to improve conception rates. Medical management of advanced stages of endometriosis is widely used for symptom control but has not been shown to enhance fertility. In moderate and severe endometriosis, conservative surgery is associated with pregnancy rates of 50 and 39%, respectively, compared with rates of 2389 25 and 5% with expectant management alone. In some patients, IVF may be the treatment of choice.

1	The treatment options for male factor infertility have expanded greatly in recent years (Chap. 411). Secondary hypogonadism is highly amenable to treatment with gonadotropins or pulsatile gonadotropin-releasing hormone (GnRH) where available. In vitro techniques have provided new opportunities for patients with primary testicular failure and disorders of sperm transport. Choice of initial treatment options depends on sperm concentration and motility. Expectant management should be attempted initially in men with mild male factor infertility (sperm count of 15 to 20 × 106/ mL and normal motility). Moderate male factor infertility (10 to 15 × 106/mL and 20–40% motility) should begin with IUI alone or in combination with treatment of the female partner with ovulation induction, but it may require IVF with or without intracytoplasmic sperm injection (ICSI). For men with a severe defect (sperm count of <10 × 106/mL, 10% motility), IVF with ICSI or donor sperm should be used. If ICSI is

1	require IVF with or without intracytoplasmic sperm injection (ICSI). For men with a severe defect (sperm count of <10 × 106/mL, 10% motility), IVF with ICSI or donor sperm should be used. If ICSI is performed because of azoospermia due to congenital bilateral absence of the vas deferens, genetic testing and counseling should be provided because of the risk of cystic fibrosis.

1	The development of assisted reproductive technologies (ARTs) has dramatically altered the treatment of male and female infertility. IVF is indicated for patients with many causes of infertility that have not been successfully managed with more conservative approaches. IVF or ICSI is often the treatment of choice in couples with a significant male factor or tubal disease, whereas IVF using donor oocytes is used in patients with premature ovarian failure and in women of advanced reproductive age. Success rates are influenced by cause of infertility and age, varying between 15 and 40%. Success rates are highest in anovulatory women and lowest in women with decreased ovarian reserve. In the United States, success rates are higher in white than in black, Asian, or Hispanic women. Although often effective, IVF is expensive and requires careful monitoring of ovulation induction and invasive techniques, including the aspiration of multiple follicles. IVF is associated with a significant risk

1	effective, IVF is expensive and requires careful monitoring of ovulation induction and invasive techniques, including the aspiration of multiple follicles. IVF is associated with a significant risk of multiple gestation, particularly in women age <35, in whom the rate can be as high as 30%, which has led to specific recommendations for numbers of embryos or blastocysts to transfer based on age and specific prognostic factors.

1	Although use of contraception worldwide has increased in the last two decades, as of 2010, 146 million women worldwide age 15–49 years who were married or in a union had an unmet need for family planning. The absolute number of married women who use contraception or have an unmet need for family planning is projected to grow from 900 million (876–922 million) in 2010 to 962 million (927–992 million) in 2015. Only 15% of couples in the United States report having unprotected sexual intercourse in the past 3 months. However, despite the wide availability and widespread use of a variety of effective methods of contraception, approximately one-half of all births in the United States are the result of unintended pregnancy. Teenage pregnancies continue to represent a serious public health problem in the United States, with >1 million unintended pregnancies each year—a significantly greater incidence than in other industrialized nations.

1	Of the contraceptive methods available (Table 414-1), a reversible form of contraception is used by >50% of couples, whereas sterilization (male or female) has been used as a permanent form of contraception by over one-third of couples. Pregnancy termination is relatively safe when directed by health care professionals but is rarely the option of choice. 2390 surgical interruption of the fallopian tubes in women or the vas deferens in men. TAblE 414-1 EffECTivEnESS of DiffEREnT foRmS of ConTRACEPTion Method of Theoreticala Actuala Percent Continuing Methods Used by are potentially reversible, these proce- Contraception Effectiveness, % Effectiveness, % Use at 1 Yearb U.S. Womenc patient counseling. Several methods of tubal ligation Cervical cap 94 82 50 <1 have been developed, all of which are Spermicides 97 79 43 1 highly effective with a 10-year cumulative pregnancy rate of 1.85 per 100 women. However, when pregnancy does occur, the

1	Male 99.9 99.9 100 9 risk of ectopic pregnancy may be as high Female 99.8 99.6 100 27 as 30%. The success rate of tubal reanasto- 1 mosis depends on the method of ligationCopper T380 99 97 78 used, but even after successful reversal, the risk of ectopic pregnancy remains high. In Mirena 99.9 99.8 addition to prevention of pregnancy, tubal Hormonal 99.7 92 72 31 ligation reduces the risk of ovarian cancer, contraceptives possibly by limiting the upward migration of potential carcinogens. Vasectomy is a highly effective outpa- tient surgical procedure that has little risk. The development of azoospermia may be delayed for 2–6 months, and other forms sterility. Reanastomosis may restore fertilaAdapted from J Trussel et al: Obstet Gynecol 76:558, 1990. bAdapted from Contraceptive Technology Update. Contraceptive ity in 30–50% of men, but the success rateTechnology, Feb. 1996, Vol 17, No 1, pp 13–24. cAdapted from LJ Piccinino, WD Mosher: Fam Plan Perspective 30:4, 1998.

1	declines with time after vasectomy and may be influenced by factors such as the development of antisperm antibodies.

1	No single contraceptive method is ideal, although all are safer than INTRAUTERINE DEVICES carrying a pregnancy to term. The effectiveness of a given method of IUDs inhibit pregnancy through several mechanisms, primarily via a contraception does not just depend on the efficacy of the method itself. spermicidal effect caused by a sterile inflammatory reaction induced Discrepancies between theoretical and actual effectiveness emphasize by the presence of a foreign body in the uterine cavity (copper IUDs) the importance of patient education and compliance when consider-or by the release of progestins (Progestasert, Mirena). IUDs provide aing various forms of contraception (Table 414-1). Knowledge of the high level of efficacy in the absence of systemic metabolic effects, and advantages and disadvantages of each contraceptive is essential for ongoing motivation is not required to ensure efficacy once the devicecounseling an individual about the methods that are safest and most has been

1	disadvantages of each contraceptive is essential for ongoing motivation is not required to ensure efficacy once the devicecounseling an individual about the methods that are safest and most has been placed. However, only 1% of women in the United States consistent with his or her lifestyle. The WHO has extensive family use this method compared to a utilization rate of 15–30% in much ofplanning resources for the physician and patient that can be accessed Europe and Canada, despite evidence that the newer devices are notonline. Similar resources for determining medical eligibility are avail-associated with increased rates of pelvic infection and infertility, asable through the Centers for Disease Control and Prevention (CDC). occurred with earlier devices. An IUD should not be used in women at Considerations for contraceptive use in obese patients and after bariat-high risk for development of STI or in women at high risk for bacterialric surgery are discussed below. endocarditis. The

1	at Considerations for contraceptive use in obese patients and after bariat-high risk for development of STI or in women at high risk for bacterialric surgery are discussed below. endocarditis. The IUD may not be effective in women with uterine leiomyomas because they alter the size or shape of the uterine cavity.

1	IUD use is associated with increased menstrual blood flow, althoughBarrier contraceptives (such as condoms, diaphragms, and cervical this is less pronounced with the progestin-releasing IUD, which iscaps) and spermicides are easily available, reversible, and have fewer associated with a more frequent occurrence of spotting or amenorrhea. side effects than hormonal methods. However, their effectiveness is highly dependent on adherence and proper use (Table 414-1). A major advantage of barrier contraceptives is the protection pro-Oral Contraceptive Pills Because of their ease of use and efficacy,vided against sexually transmitted infections (STIs) (Chap. 163).

1	oral contraceptive pills are the most widely used form of hormonalConsistent use is associated with a decreased risk of HIV, gonorcontraception. They act by suppressing ovulation, changing cervical rhea, nongonococcal urethritis, and genital herpes, probably due mucus, and altering the endometrium. The current formulations arein part to the concomitant use of spermicides. Natural membrane made from synthetic estrogens and progestins. The estrogen com-condoms may be less effective than latex condoms, and petroleumponent of the pill consists of ethinyl estradiol or mestranol, whichbased lubricants can degrade condoms and decrease their efficacy for is metabolized to ethinyl estradiol. Multiple synthetic progestins arepreventing HIV infection. Barrier methods used by women include used. Norethindrone and its derivatives are used in many formuthe diaphragm, cervical cap, and contraceptive sponge. The cervical lations. Low-dose norgestimate and the more recently developedcap and sponge are

1	and its derivatives are used in many formuthe diaphragm, cervical cap, and contraceptive sponge. The cervical lations. Low-dose norgestimate and the more recently developedcap and sponge are less effective than the diaphragm, and there have (third-generation) progestins (desogestrel, gestodene, drospirenone)been rare reports of toxic shock syndrome with the diaphragm and have a less androgenic profile; levonorgestrel appears to be the mostcontraceptive sponge.

1	androgenic of the progestins and should be avoided in patients with STERILIzATION hyperandrogenism. The three major formulations of oral contracep-Sterilization is the method of birth control most frequently chosen tives are (1) fixed-dose estrogen-progestin combination, (2) phasic by fertile men and multiparous women >30 years old (Table 414-1). estrogen-progestin combination, and (3) progestin only. Each of these Sterilization refers to a procedure that prevents fertilization by formulations is administered daily for 3 weeks followed by a week of Absolute Previous thromboembolic event or stroke History of an estrogen-dependent tumor Active liver disease Pregnancy Undiagnosed abnormal uterine bleeding Hypertriglyceridemia Women age >35 years who smoke heavily Coronary heart disease—increased in smokers >35; no relation to proges Hypertension—relative risk 1.8 (current users) and 1.2 (previous users)

1	Coronary heart disease—increased in smokers >35; no relation to proges Hypertension—relative risk 1.8 (current users) and 1.2 (previous users) Venous thrombosis—relative risk ~4; may be higher with third-generation progestin, drospirenone, and patch; compounded by obesity (tenfold increased risk compared with nonobese, no OCP); markedly increased with factor V Leiden or prothrombin gene mutations Stroke—slight increase; unclear relation to migraine headache Cerebral vein thrombosis—relative risk ~13–15; synergistic with prothrombin gene mutation Breast cancer—may increase risk in carriers of BRCA1 and possibly BRCA2 Abbreviation: OCP, oral contraceptive pill.

1	no medication during which menstrual bleeding generally occurs. Two extended oral contraceptives are approved for use in the United States; Seasonale is a 3-month preparation with 84 days of active drug and 7 days of placebo, whereas Lybrel is a continuous preparation. Current doses of ethinyl estradiol range from 10 to 50 μg. However, indications for the 50-μg dose are rare, and the majority of formulations contain 30–35 μg of ethinyl estradiol. The reduced estrogen and progestin content in the secondand third-generation pills has decreased both side effects and risks associated with oral contraceptive use (Table 414-2). At the currently used doses, patients must be cautioned not to miss pills due to the potential for ovulation. Side effects, including breakthrough bleeding, amenorrhea, breast tenderness, and weight gain, often respond to a change in formulation. Even the lower dose oral contraceptives have been associated with an increased risk of cardiovascular disease (myocardial

1	breast tenderness, and weight gain, often respond to a change in formulation. Even the lower dose oral contraceptives have been associated with an increased risk of cardiovascular disease (myocardial infarction, stroke, venous thromboembolism [VTE]), but the absolute excess risk is extremely low. VTE risk is higher with the third-generation than the second-generation progestins, and the risk of stroke and VTE is also higher with drospirenone (although not cyproterone), but the absolute excess risk is small and may be outweighed by contraceptive benefits and reduction in ovarian and endometrial cancer risk.

1	The microdose progestin-only minipill is less effective as a contraceptive, having a pregnancy rate of 2–7 per 100 women-years. However, it may be appropriate for women at increased risk for cardiovascular disease or for women who cannot tolerate synthetic estrogens.

1	Alternative Methods A weekly contraceptive patch (Ortho Evra) is available and has similar efficacy to oral contraceptives. Approximately 2% of patches fail to adhere, and a similar percentage of women have skin reactions. Efficacy is lower in women weighing >90 kg. The amount of estrogen delivered may be comparable to that of a 40-μg ethinyl estradiol oral contraceptive, raising the possibility of increased risk of 2391 VTE, which must be balanced against potential benefits for women not able to successfully use other methods. A monthly contraceptive estrogen/progestin injection (Lunelle) is highly effective, with a first-year failure rate of <0.2%, but it may be less effective in obese women. Its use is associated with bleeding irregularities that diminish over time. Fertility returns rapidly after discontinuation. A monthly vaginal ring (NuvaRing) that is intended to be left in place during intercourse is also available for contraceptive use. It is highly effective, with a 12-month

1	after discontinuation. A monthly vaginal ring (NuvaRing) that is intended to be left in place during intercourse is also available for contraceptive use. It is highly effective, with a 12-month failure rate of 0.7%. Ovulation returns within the first recovery cycle after discontinuation.

1	Long-Term Contraceptives Long-term progestin administration acts primarily by inhibiting ovulation and causing changes in the endometrium and cervical mucus that result in decreased implantation and sperm transport. Depot medroxyprogesterone acetate (Depo-Provera, DMPA), the only injectable form available in the United States, is effective for 3 months, but return of fertility after discontinuation may be delayed for up to 12–18 months. DMPA is now available for both SC and IM injection. Irregular bleeding, amenorrhea, and weight gain are the most common side effects. This form of contraception may be particularly good for women in whom an estrogen-containing contraceptive is contraindicated (e.g., migraine exacerbation, sickle cell anemia, fibroids).

1	The probability of pregnancy without relation to time of the month is 8%, but the probability varies significantly in relation to proximity to ovulation and may be as high has 30%. In order of efficacy, methods of postcoital contraception include the following: 1. Copper IUD insertion within a maximum of 5 days has a reported efficacy of 99–100% and prevents pregnancy by its spermicidal effect; insertion is frequently available through family planning clinics. 2. Oral antiprogestins (ulipristal acetate, 30 mg single dose, available worldwide, or mifepristone, 600 mg single dose, not available for this indication in the United States) prevent pregnancy by delaying or preventing ovulation; when administered, ideally within 72 h but up to 120 h after intercourse, they have an efficacy of 98–99%; require a prescription. 3.

1	3. Levonorgestrel (1.5 mg as a single dose) delays or prevents ovulation and is not effective after ovulation; should be taken within 72 h of unprotected intercourse, and has an efficacy that varies between 60 and 94%; it is available over the counter. Combined estrogen and progestin regimens have lower efficacy and are no longer recommended. A pregnancy test is not necessary before the use of oral methods, but pregnancy should be excluded before IUD insertion. Risk factors for failure of oral regimens include close proximity to ovulation and unprotected intercourse after use. In addition, there is an increased risk of pregnancy in obese and overweight women using levonorgestrel for postcoital contraception and an increased risk in obese women using an antiprogestin.

1	Approximately one-third of adults in the United States are obese. Although obesity is associated with some reduction in fertility, the vast majority of obese women can conceive. The risk of pregnancy-associated complications is higher in obese women. Intrauterine contraception may be more effective than oral or transdermal methods for obese women. The WHO guidelines provide no restrictions (class 1) for the use of intrauterine contraception, DMPA, and progestin-only pills for obese women (BMI ≥30) in the absence of coexistent medical problems, whereas methods that include estrogen (pill, patch, ring) are considered class 2 (advantages generally outweigh theoretical or proven risks) due to the increased risk of thromboembolic disease. There are no restrictions to the use of any contraceptive methods following restrictive bariatric surgery procedures, but both combined and progestin-only pills are relatively less effective following procedures associated with malabsorption.

1	415e-1 SECTiOn 3 OBESiTy, DiABETES MElliTuS, AnD METABOliC SynDROME intraabdominal and abdominal subcutaneous fat have more significanceBiology of Obesity than subcutaneous fat present in the buttocks and lower extremities. This distinction is most easily made clinically by determining

1	Jeffrey S. Flier, Eleftheria Maratos-Flier the waist-to-hip ratio, with a ratio >0.9 in women and >1.0 in men being abnormal. Many of the most important complications of obesity, such as insulin resistance, diabetes, hypertension, hyperlipid-In a world where food supplies are intermittent, the ability to store emia, and hyperandrogenism in women, are linked more stronglyenergy in excess of what is required for immediate use is essential for to intraabdominal and/or upper body fat than to overall adipositysurvival. Fat cells, residing within widely distributed adipose tissue (Chap. 422). The mechanism underlying this association is unknowndepots, are adapted to store excess energy efficiently as triglyceride but may relate to the fact that intraabdominal adipocytes are more lipoand, when needed, to release stored energy as free fatty acids for use at lytically active than those from other depots. Release of free fatty acidsother sites. This physiologic system, orchestrated through

1	needed, to release stored energy as free fatty acids for use at lytically active than those from other depots. Release of free fatty acidsother sites. This physiologic system, orchestrated through endocrine into the portal circulation has adverse metabolic actions, especially onand neural pathways, permits humans to survive starvation for as the liver. Adipokines and cytokines that are differentially secreted by adilong as several months. However, in the presence of nutritional abunpocyte depots may play a role in the systemic complications of obesity.

1	dance and a sedentary lifestyle, and influenced importantly by genetic endowment, this system increases adipose energy stores and produces PREVALENCE adverse health consequences. Data from the National Health and Nutrition Examination Surveys (NHANES) show that the percentage of the American adult population Obesity is a state of excess adipose tissue Weight Height kg lb cm in. mass. Although often viewed as equivalent to increased body weight, this need not be 340 the case—lean but very muscular individu-150 dards without having increased adiposity. 130 Body weights are distributed continuously 130 in populations, so that choice of a medi-120 and obese is somewhat arbitrary. Obesity is therefore defined by assessing its linkage to morbidity or mortality.

1	Although not a direct measure of adi posity, the most widely used method to gauge obesity is the body mass index (BMI), which is equal to weight/height2 (in kg/m2) (Fig. 415e-1). Other approaches to quanti-75 fold thickness), densitometry (underwater weighing), computed tomography (CT) or magnetic resonance imaging (MRI), and electrical impedance. Using data from the

1	Metropolitan Life Tables, BMIs for the mid-55 point of all heights and frames among both men and women range from 19 to 26 kg/m2; at a similar BMI, women have more body fat than men. Based on data of substantial morbidity, a BMI of 30 is most commonly used as a threshold for obesity in both men 40 85 and women. Most but not all large-scale epidemiologic studies suggest that all-cause, metabolic, cancer, and cardiovascular mor-35 bidity begin to rise (albeit at a slow rate) when BMIs are ≥25. Most authorities use the term overweight (rather than obese) to describe individuals with BMIs between 25 and 30. A BMI between 25 and 30 should be viewed as medically significant and wor-25 thy of therapeutic intervention in the pres ence of risk factors that are influenced by adiposity, such as hypertension and glucose FIgURE 415e-1 Nomogram for determining body mass index. To use this nomogram, intolerance. place a ruler or other straight edge between the body weight (without clothes) in

1	hypertension and glucose FIgURE 415e-1 Nomogram for determining body mass index. To use this nomogram, intolerance. place a ruler or other straight edge between the body weight (without clothes) in kilograms or

1	The distribution of adipose tissue in dif-pounds located on the left-hand line and the height (without shoes) in centimeters or inches ferent anatomic depots also has substan-located on the right-hand line. The body mass index is read from the middle of the scale and is tial implications for morbidity. Specifically, in metric units. (Copyright 1979, George A. Bray, MD; used with permission.)

1	CHAPTER 415e Biology of Obesity with obesity (BMI >30) has increased from 14.5% (between 1976 and 1980) to 35.7% (between 2009 and 2010). As many as 68% of U.S. adults aged ≥20 years were overweight (defined as BMI >25) between the years of 2007 and 2008. Extreme obesity (BMI ≥40) has also increased and affects 5.7% of the population. The increasing prevalence of medically significant obesity raises great concern. Overall, the prevalence of obesity is comparable in men and women. In women, poverty is associated with increased prevalence. Obesity is more common among blacks and Hispanics. The prevalence in children and adolescents has been rising at a worrisome rate, reaching 15.9% in 2009/2010, but may be leveling off.

1	Substantial evidence suggests that body weight is regulated by both endocrine and neural components that ultimately influence the effector arms of energy intake and expenditure. This complex regulatory system is necessary because even small imbalances between energy intake and expenditure will ultimately have large effects on body weight. For example, a 0.3% positive imbalance over 30 years would result in a 9-kg (20-lb) weight gain. This exquisite regulation of energy balance cannot be monitored easily by calorie-counting in relation to physical activity. Rather, body weight regulation or dysregulation depends on a complex interplay of hormonal and neural signals. Alterations in stable weight by forced overfeeding or food deprivation induce physiologic changes that resist these perturbations: with weight loss, appetite increases and energy expenditure falls; with overfeeding, appetite falls and energy expenditure increases. This latter compensatory mechanism frequently fails,

1	with weight loss, appetite increases and energy expenditure falls; with overfeeding, appetite falls and energy expenditure increases. This latter compensatory mechanism frequently fails, however, permitting obesity to develop when food is abundant and physical activity is limited. A major regulator of these adaptive responses is the adipocyte-derived hormone leptin, which acts through brain circuits (predominantly in the hypothalamus) to influence appetite, energy expenditure, and neuroendocrine function (see below).

1	Appetite is influenced by many factors that are integrated by the brain, most importantly within the hypothalamus (Fig. 415e-2). Signals that impinge on the hypothalamic center include neural afferents, hormones, and metabolites. Vagal inputs are particularly important, bringing information from viscera, such as gut distention. Hormonal signals include leptin, insulin, cortisol, and gut peptides. Among the latter is ghrelin, which is made in the stomach and stimulates feeding, and peptide YY (PYY) and cholecystokinin, which is made in the small intestine and signals to the brain through direct action on hypothalamic control centers and/or via the vagus nerve. Metabolites, including glucose, can influence appetite, as seen by the effect of hypoglycemia to induce hunger; however, glucose is not

1	FIgURE 415e-2 The factors that regulate appetite through effects on central neural circuits. Some factors that increase or decrease appetite are listed. AgRP, Agouti-related peptide; CART, cocaineand amphetamine-related transcript; CCK, cholecystokinin; GLP-1, glucagonrelated peptide-1; MCH, melanin-concentrating hormone; α-MSH, α-melanocyte-stimulating hormone; NPY, neuropeptide Y.

1	normally a major regulator of appetite. These diverse hormonal, metabolic, and neural signals act by influencing the expression and release of various hypothalamic peptides (e.g., neuropeptide Y [NPY], Agoutirelated peptide [AgRP], α-melanocyte-stimulating hormone [α-MSH], and melanin-concentrating hormone [MCH]) that are integrated with serotonergic, catecholaminergic, endocannabinoid, and opioid signaling pathways (see below). Psychological and cultural factors also play a role in the final expression of appetite. Apart from rare genetic syndromes involving leptin, its receptor, and the melanocortin system, specific defects in this complex appetite control network that influence common cases of obesity are not well defined.

1	Energy expenditure includes the following components: (1) resting or basal metabolic rate; (2) the energy cost of metabolizing and storing food; (3) the thermic effect of exercise; and (4) adaptive thermo-genesis, which varies in response to long-term caloric intake (rising with increased intake). Basal metabolic rate accounts for ~70% of daily energy expenditure, whereas active physical activity contributes 5–10%. Thus, a significant component of daily energy consumption is fixed.

1	Genetic models in mice indicate that mutations in certain genes (e.g., targeted deletion of the insulin receptor in adipose tissue) protect against obesity, apparently by increasing energy expenditure. Adaptive thermogenesis occurs in brown adipose tissue (BAT), which plays an important role in energy metabolism in many mammals. In contrast to white adipose tissue, which is used to store energy in the form of lipids, BAT expends stored energy as heat. A mitochondrial uncoupling protein (UCP-1) in BAT dissipates the hydrogen ion gradient in the oxidative respiration chain and releases energy as heat. The metabolic activity of BAT is increased by a central action of leptin, acting through the sympathetic nervous system that heavily innervates this tissue. In rodents, BAT deficiency causes obesity and diabetes; stimulation of BAT with a specific adrenergic agonist (β3 agonist) protects against diabetes and obesity. BAT exists in humans (especially neonates), and although its physiologic

1	and diabetes; stimulation of BAT with a specific adrenergic agonist (β3 agonist) protects against diabetes and obesity. BAT exists in humans (especially neonates), and although its physiologic role is not yet established, identification of functional BAT in many adults using positron emission tomography (PET) imaging has increased interest in the implications of the tissue for pathogenesis and therapy of obesity. Beige fat cells, recently described, resemble BAT cells in expressing UCP-1. They are scattered through white adipose tissue, and their thermogenic potential is uncertain.

1	Adipose tissue is composed of the lipid-storing adipose cell and a stromal/vascular compartment in which cells including preadipocytes and macrophages reside. Adipose mass increases by enlargement of adipose cells through lipid deposition, as well as by an increase in the number of adipocytes. Obese adipose tissue is also characterized by increased numbers of infiltrating macrophages. The process by which adipose cells are derived from a mesenchymal preadipocyte involves an orchestrated series of differentiation steps mediated by a cascade of specific transcription factors. One of the key transcription factors is peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor that binds the thiazolidinedione class of insulin-sensitizing drugs used in the treatment of type 2 diabetes (Chap. 418).

1	Although the adipocyte has generally been regarded as a storage depot for fat, it is also an endocrine cell that releases numerous molecules in a regulated fashion (Fig. 415e-3). These include the energy balance–regulating hormone leptin, cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6, complement factors such as factor D (also known as adipsin), prothrombotic agents such as plasminogen activator inhibitor I, and a component of the blood pressure–regulating system, angiotensinogen. Adiponectin, an abundant adipose-derived protein whose levels are reduced in obesity, enhances insulin sensitivity and lipid oxidation and has vascular-protective effects, whereas resistin and RBP4, whose levels are increased in obesity, may induce insulin resistance. These factors, and others not yet identified, play a role in the physiology of lipid homeostasis, insulin sensitivity, blood pressure control, coagulation, and vascular health, and are likely to contribute to

1	and others not yet identified, play a role in the physiology of lipid homeostasis, insulin sensitivity, blood pressure control, coagulation, and vascular health, and are likely to contribute to obesity-related pathologies.

1	FIgURE 415e-3 Factors released by the adipocyte that can affect peripheral tissues. IL-6, interleukin 6; PAI, plasminogen activator inhibitor; RBP4, retinal binding protein 4; TNF, tumor necrosis factor. Although the molecular pathways regulating energy balance are beginning to be illuminated, the causes of obesity remain elusive. In part, this reflects the fact that obesity is a heterogeneous group of disorders. At one level, the pathophysiology of obesity seems simple: a chronic excess of nutrient intake relative to the level of energy expenditure. However, due to the complexity of the neuroendocrine and metabolic systems that regulate energy intake, storage, and expenditure, it has been difficult to quantitate all the relevant parameters (e.g., food intake and energy expenditure) over time in human subjects.

1	Role of genes Versus Environment Obesity is commonly seen in families, and the heritability of body weight is similar to that for height. Inheritance is usually not Mendelian, however, and it is difficult to distinguish the role of genes and environmental factors. Adoptees more closely resemble their biologic than adoptive parents with respect to obesity, providing strong support for genetic influences. Likewise, identical twins have very similar BMIs whether reared together or apart, and their BMIs are much more strongly correlated than those of dizygotic twins. These genetic effects appear to relate to both energy intake and expenditure. Currently, identified genetic variants, both common and rare, account for less than 5% of the variance of body weight.

1	Whatever the role of genes, it is clear that the environment plays a key role in obesity, as evidenced by the fact that famine prevents obesity in even the most obesity-prone individual. In addition, the recent increase in the prevalence of obesity in the United States is far too rapid to be due to changes in the gene pool. Undoubtedly, genes influence the susceptibility to obesity in response to specific diets and availability of nutrition. Cultural factors are also important—these relate to both availability and composition of the diet and to changes in the level of physical activity. In industrial societies, obesity is more common among poor women, whereas in underdeveloped countries, wealthier women are more often obese. In children, obesity correlates to some degree with time spent watching television. Although the role of diet composition in obesity continues to generate controversy, it appears that high-fat diets may, when combined with simple, rapidly absorbed carbohydrates,

1	watching television. Although the role of diet composition in obesity continues to generate controversy, it appears that high-fat diets may, when combined with simple, rapidly absorbed carbohydrates, promote obesity. Specific genes are likely to influence the response to specific diets, but these genes are largely unidentified.

1	Additional environmental factors may contribute to the increasing obesity prevalence. Both epidemiologic correlations and experimental data suggest that sleep deprivation leads to increased obesity. Changes in gut microbiome with capacity to alter energy balance are receiving experimental support from animal studies, and a possible role for obesigenic viral infections continues to receive sporadic attention. Specific genetic Syndromes For many years, obesity in rodents has been known to be caused by a number of distinct mutations distributed through the genome. Most of these single-gene mutations cause both hyperphagia and diminished energy expenditure, suggesting a physiologic link between these two parameters of energy homeostasis. Identification of the ob gene mutation in genetically obese (ob/ob) mice represented a major breakthrough in the field. The ob/ob mouse

1	FIgURE 415e-4 The physiologic system regulated by leptin. Rising or falling leptin levels act through the hypothalamus to influence appetite, energy expenditure, and neuroendocrine function and through peripheral sites to influence systems such as the immune system.

1	develops severe obesity, insulin resistance, and hyperphagia, as well as efficient metabolism (e.g., it gets fat even when ingesting the same number of calories as lean litter mates). The product of the ob gene is the peptide leptin, a name derived from the Greek root leptos, meaning thin. Leptin is secreted by adipose cells and acts primarily through the hypothalamus. Its level of production provides an index of adipose energy stores (Fig. 415e-4). High leptin levels decrease food intake and increase energy expenditure. Another mouse mutant, db/db, which is resistant to leptin, has a mutation in the leptin receptor and develops a similar syndrome. The ob gene is present in humans where it is also expressed in fat. Several families with morbid, early-onset obesity caused by inactivating mutations in either leptin or the leptin receptor have been described, thus demonstrating the biologic relevance of the leptin pathway in humans. Obesity in these individuals begins shortly after

1	mutations in either leptin or the leptin receptor have been described, thus demonstrating the biologic relevance of the leptin pathway in humans. Obesity in these individuals begins shortly after birth, is severe, and is accompanied by neuroendocrine abnormalities. The most prominent of these is hypogonadotropic hypogonadism, which is reversed by leptin replacement in the leptin-deficient subset. Central hypothyroidism and growth retardation are seen in the mouse model, but their occurrence in leptin-deficient humans is less clear. Mutations in the leptin or leptin receptor genes do not play a prominent role in common forms of obesity.

1	Mutations in several other genes cause severe obesity in humans (Table 415e-1); each of these syndromes is rare. Mutations in the gene encoding proopiomelanocortin (POMC) cause severe obesity through failure to synthesize α-MSH, a key neuropeptide that inhibits appetite in the hypothalamus. The absence of POMC also causes secondary adrenal insufficiency due to absence of adrenocorticotropic hormone (ACTH), as well as pale skin and red hair due to absence of α-MSH. Proenzyme convertase 1 (PC-1) mutations are thought to cause obesity by preventing synthesis of α-MSH from its precursor peptide, POMC. α-MSH binds to the type 4 melanocortin receptor (MC4R), a key hypothalamic receptor that inhibits eating. Heterozygous loss-of-function mutations of this receptor account for as much as 5% of severe obesity. Loss of function of MRAP2, a protein required for normal MC4R signaling, has been found in rare cases of severe obesity. These six genetic defects define a pathway through which leptin

1	obesity. Loss of function of MRAP2, a protein required for normal MC4R signaling, has been found in rare cases of severe obesity. These six genetic defects define a pathway through which leptin (by stimulating POMC and increasing α-MSH) restricts food intake and limits weight

1	CHAPTER 415e Biology of Obesity mouse deficient in the peptide MCH, whose administration causes feeding, is lean. Gene Gene Product Mechanism of Obesity In Human In Rodent A number of complex human syndromes Leptin, a fat-derived hormone Proopiomelanocortin, a precursor of several hormones and neuropeptides Agouti-related peptide, a neuropeptide expressed in the hypothalamus Prohormone convertase 1, a processing enzyme Carboxypeptidase E, a processing enzyme Tub, a hypothalamic protein of unknown function TrkB, a neurotrophin receptor Mutation prevents leptin from delivering satiety signal; brain perceives starvation Same as above Mutation prevents synthesis of melanocyte-stimulating hormone (MSH), a satiety signal Mutation prevents reception of satiety signal from MSH Mutation prevents synthesis of neuropeptide, probably MSH Same as above

1	Mutation prevents reception of satiety signal from MSH Mutation prevents synthesis of neuropeptide, probably MSH Same as above Hyperphagia due to uncharacterized hypothalamic defect (Fig. 415e-5). The results of genomewide association studies to identify genetic loci responsible for obesity in the general population have so far been disappointing. More than 40 replicated loci linked to obesity have been identified, but together they account for less than 3% of interindividual variation in BMI. The most replicated of these is a gene named FTO, which is of unknown function, but like many of the other recently described candidates, is expressed in the brain. Because the heritability of obesity is estimated to be 40–70%, it is likely that many more loci remain to be identified. It is possible that epistatic interactions between causative loci or unknown gene-environment interactions explain the poor success at identifying causal loci.

1	In addition to these human obesity genes, studies in rodents reveal several other molecular candidates for hypothalamic mediators of human obesity or leanness. The tub gene encodes a hypothalamic peptide of unknown function; mutation of this gene causes late-onset obesity. The fat gene encodes carboxypeptidase E, a peptide-processing enzyme; mutation of this gene is thought to cause obesity by disrupting production of one or more neuropeptides. AgRP is coexpressed with NPY in arcuate nucleus neurons. AgRP antagonizes α-MSH action at MC4 receptors, and its overexpression induces obesity. In contrast, a

1	FIgURE 415e-5 A central pathway through which leptin acts to regulate appetite and body weight. Leptin signals through proopiomelanocortin (POMC) neurons in the hypothalamus to induce increased production of α-melanocyte-stimulating hormone (α-MSH), requiring the processing enzyme PC-1 (proenzyme convertase 1). α-MSH acts as an agonist on melanocortin-4 receptors to inhibit appetite, and the neuropeptide AgRp (Agouti-related peptide) acts as an antagonist of this receptor. Mutations that cause obesity in humans are indicated by the solid green arrows. At least 12 genetic loci have been identified, and most of the encoded proteins form two multiprotein complexes that are involved in ciliary function and microtubule-based intracellular transport. Some evidence suggests that mutations might disrupt leptin receptor trafficking in key hypothalamic neurons, causing leptin resistance. Other Specific Syndromes Associated with Obesity • CUSHING’S SYNDROME

1	Other Specific Syndromes Associated with Obesity • CUSHING’S SYNDROME Although obese patients commonly have central obesity, hypertension, and glucose intolerance, they lack other specific stigmata of Cushing’s syndrome (Chap. 406). Nonetheless, a potential diagnosis of Cushing’s syndrome is often entertained. Cortisol production and urinary metabolites (17OH steroids) may be increased in simple obesity. Unlike in Cushing’s syndrome, however, cortisol levels in blood and urine in the basal state and in response to corticotropinreleasing hormone (CRH) or ACTH are normal; the overnight 1-mg dexamethasone suppression test is normal in 90%, with the remainder being normal on a standard 2-day low-dose dexamethasone suppression test. Obesity may be associated with excessive local reactivation of cortisol in fat by 11β-hydroxysteroid dehydrogenase 1, an enzyme that converts inactive cortisone to cortisol.

1	HYPOTHYROIDISM The possibility of hypothyroidism should be considered, but it is an uncommon cause of obesity; hypothyroidism is easily ruled out by measuring thyroid-stimulating hormone (TSH). Much of the weight gain that occurs in hypothyroidism is due to myxedema (Chap. 405). INSULINOMA Patients with insulinoma often gain weight as a result of overeating to avoid hypoglycemic symptoms (Chap. 420). The increased substrate plus high insulin levels promote energy storage in fat. This can be marked in some individuals but is modest in most.

1	Whether through tumors, trauma, or inflammation, hypothalamic dysfunction of systems controlling satiety, hunger, and energy expenditure can cause varying degrees of obesity (Chap. 402). It is uncommon to identify a discrete anatomic basis for these disorders. Subtle hypothalamic dysfunction is probably a more common cause of obesity than can be documented using currently available imaging techniques. Growth hormone (GH), which exerts lipolytic activity, is diminished in obesity and is increased with weight loss. Despite low GH levels, insulin-like growth factor (IGF) I (somatomedin) production is normal, suggesting that GH suppression may be a compensatory response to increased nutritional supply.

1	Pathogenesis of Common Obesity Obesity can result from increased energy intake, decreased energy expenditure, or a combination of the two. Thus, identifying the etiology of obesity should involve measurements of both parameters. However, it is difficult to perform direct and accurate measurements of energy intake in free-living individuals; and the obese, in particular, often underreport intake. Measurements of chronic energy expenditure are possible using doubly labeled water or metabolic chamber/rooms. In subjects at stable weight and body composition, energy intake equals expenditure. Consequently, these techniques allow assessment of energy intake in free-living individuals. The level of energy expenditure differs in established obesity, during periods of weight gain or loss, and in the preor postobese state. Studies that fail to take note of this phenomenon are not easily interpreted.

1	There is continued interest in the concept of a body weight “set point.” This idea is supported by physiologic mechanisms centered around a sensing system in adipose tissue that reflects fat stores and a receptor, or “adipostat,” that is in the hypothalamic centers. When fat stores are depleted, the adipostat signal is low, and the hypothalamus responds by stimulating hunger and decreasing energy expenditure to conserve energy. Conversely, when fat stores are abundant, the signal is increased, and the hypothalamus responds by decreasing hunger and increasing energy expenditure. The recent discovery of the ob gene, and its product leptin, and the db gene, whose product is the leptin receptor, provides important elements of a molecular basis for this physiologic concept (see above).

1	What Is the Status of Food Intake in Obesity? (Do the Obese Eat More Than the Lean?) This question has stimulated much debate, due in part to the methodologic difficulties inherent in determining food intake. Many obese individuals believe that they eat small quantities of food, and this claim has often been supported by the results of food intake questionnaires. However, it is now established that average energy expenditure increases as individuals get more obese, due primarily to the fact that metabolically active lean tissue mass increases with obesity. Given the laws of thermodynamics, the obese person must therefore eat more than the average lean person to maintain their increased weight. It may be the case, however, that a subset of individuals who are predisposed to obesity have the capacity to become obese initially without an absolute increase in caloric consumption.

1	What Is the State of Energy Expenditure in Obesity? The average total daily energy expenditure is higher in obese than lean individuals when measured at stable weight. However, energy expenditure falls as weight is lost, due in part to loss of lean body mass and to decreased sympathetic nerve activity. When reduced to near-normal weight and maintained there for a while, (some) obese individuals have lower energy expenditure than (some) lean individuals. There is also a tendency for those who will develop obesity as infants or children to have lower resting energy expenditure rates than those who remain lean. The physiologic basis for variable rates of energy expenditure (at a given body weight and level of energy intake) is essentially unknown.

1	Another component of thermogenesis, called nonexercise activity thermogenesis (NEAT), has been linked to obesity. It is the thermogenesis that accompanies physical activities other than volitional exercise such as the activities of daily living, fidgeting, spontaneous muscle contraction, and maintaining posture. NEAT accounts for about two-thirds of the increased daily energy expenditure induced by overfeeding. The wide variation in fat storage seen in overfed individuals is predicted by the degree to which NEAT is induced. The molecular basis for NEAT and its regulation is unknown.

1	Leptin in Typical Obesity The vast majority of obese persons have increased leptin levels but do not have mutations of either leptin or its receptor. They appear, therefore, to have a form of functional “leptin resistance.” Data suggesting that some individuals produce less leptin per unit fat mass than others or have a form of relative leptin deficiency that predisposes to obesity are at present contradictory and unsettled. The mechanism for leptin resistance, and whether it can be overcome by raising leptin levels or combining leptin with other treatments in a subset of obese individuals, is not yet established. Some data suggest that leptin may not effectively cross the blood-brain barrier as levels rise. It is also apparent from animal studies that leptin-signaling inhibitors, such as SOCS3 and PTP1b, are involved in the leptinresistant state.

1	(See also Chap. 416) Obesity has major adverse effects on health. Obesity is associated with an increase in mortality, with a 50–100% increased risk of death from all causes compared to normal-weight CHAPTER 415e Biology of Obesity individuals, mostly due to cardiovascular causes. Obesity and overweight together are the second leading cause of preventable death in the United States, accounting for 300,000 deaths per year. Mortality rates rise as obesity increases, particularly when obesity is associated with increased intraabdominal fat (see above). Life expectancy of a moderately obese individual could be shortened by 2–5 years, and a 20to 30-year-old male with a BMI >45 may lose 13 years of life. It is likely that the degree to which obesity affects particular organ systems is influenced by susceptibility genes that vary in the population.

1	Insulin Resistance and Type 2 Diabetes Mellitus Hyperinsulinemia and insulin resistance are pervasive features of obesity, increasing with weight gain and diminishing with weight loss (Chap. 422). Insulin resistance is more strongly linked to intraabdominal fat than to fat in other depots. Molecular links between obesity and insulin resistance in fat, muscle, and liver have been sought for many years. Major factors include: (1) insulin itself, by inducing receptor downregulation; (2) free fatty acids that are increased and capable of impairing insulin action; (3) intracellular lipid accumulation; and (4) several circulating peptides produced by adipocytes, including the cytokines TNF-α and IL-6, RBP4, and the “adipokines” adiponectin and resistin, which have altered expression in obese adipocytes and can modify insulin action. Additional mechanisms are obesity-linked inflammation, including infiltration of macrophages into tissues including fat, and induction of the endoplasmic

1	adipocytes and can modify insulin action. Additional mechanisms are obesity-linked inflammation, including infiltration of macrophages into tissues including fat, and induction of the endoplasmic reticulum stress response, which can bring about resistance to insulin action in cells. Despite the prevalence of insulin resistance, most obese individuals do not develop diabetes, suggesting that diabetes requires an interaction between obesity-induced insulin resistance and other factors such as impaired insulin secretion (Chap. 417). Obesity, however, is a major risk factor for diabetes, and as many as 80% of patients with type 2 diabetes mellitus are obese. Weight loss and exercise, even of modest degree, increase insulin sensitivity and often improve glucose control in diabetes.

1	Reproductive Disorders Disorders that affect the reproductive axis are associated with obesity in both men and women. Male hypogonadism is associated with increased adipose tissue, often distributed in a pattern more typical of females. In men whose weight is >160% ideal body weight (IBW), plasma testosterone and sex hormone– binding globulin (SHBG) are often reduced, and estrogen levels (derived from conversion of adrenal androgens in adipose tissue) are increased (Chap. 411). Gynecomastia may be seen. However, masculinization, libido, potency, and spermatogenesis are preserved in most of these individuals. Free testosterone may be decreased in morbidly obese men whose weight is >200% IBW.

1	Obesity has long been associated with menstrual abnormalities in women, particularly in women with upper body obesity (Chap. 412). Common findings are increased androgen production, decreased SHBG, and increased peripheral conversion of androgen to estrogen. Most obese women with oligomenorrhea have polycystic ovarian syndrome (PCOS), with its associated anovulation and ovarian hyperandrogenism; 40% of women with PCOS are obese. Most nonobese women with PCOS are also insulin-resistant, suggesting that insulin resistance, hyperinsulinemia, or the combination of the two are causative or contribute to the ovarian pathophysiology in PCOS in both obese and lean individuals. Increasing evidence supports a role for adipokines in mediating a link between obesity and the reproductive dysfunction of PCOS. In obese women with PCOS, weight loss or treatment with insulin-sensitizing drugs often restores normal menses. The increased conversion of androstenedione to estrogen, which occurs to a

1	of PCOS. In obese women with PCOS, weight loss or treatment with insulin-sensitizing drugs often restores normal menses. The increased conversion of androstenedione to estrogen, which occurs to a greater degree in women with lower body obesity, may contribute to the increased incidence of uterine cancer in postmenopausal women with obesity.

1	Cardiovascular Disease The Framingham Study revealed that obesity was an independent risk factor for the 26-year incidence of cardiovascular disease in men and women (including coronary disease, stroke, and congestive heart failure). The waist-to-hip ratio may be the best predictor of these risks. When the additional effects of hypertension and glucose intolerance associated with obesity are included, the adverse impact of obesity is even more evident. The effect of obesity on cardiovascular mortality in women may be seen at BMIs as low as 25. Obesity, especially abdominal obesity, is associated with an atherogenic lipid profile; with increased low-density lipoprotein cholesterol, very-low-density lipoprotein, and triglyceride; and with decreased high-density lipoprotein cholesterol and decreased levels of the vascular protective adipokine adiponectin (Chap. 421). Obesity is also associated with hypertension. Measurement of blood pressure in the obese requires use of a larger cuff

1	decreased levels of the vascular protective adipokine adiponectin (Chap. 421). Obesity is also associated with hypertension. Measurement of blood pressure in the obese requires use of a larger cuff size to avoid artifactual increases. Obesity-induced hypertension is associated with increased peripheral resistance and cardiac output, increased sympathetic nervous system tone, increased salt sensitivity, and insulin-mediated salt retention; it is often responsive to modest weight loss.

1	Pulmonary Disease Obesity may be associated with a number of pulmonary abnormalities. These include reduced chest wall compliance, increased work of breathing, increased minute ventilation due to increased metabolic rate, and decreased functional residual capacity and expiratory reserve volume. Severe obesity may be associated with obstructive sleep apnea and the “obesity hypoventilation syndrome” with attenuated hypoxic and hypercapnic ventilatory responses. Sleep apnea can be obstructive (most common), central, or mixed and is associated with hypertension. Weight loss (10–20 kg) can bring substantial improvement, as can major weight loss following gastric bypass or restrictive surgery. Continuous positive airway pressure has been used with some success.

1	Hepatobiliary Disease Obesity is frequently associated with nonalcoholic fatty liver disease (NAFLD), and this association represents one of the most common causes of liver disease in industrialized countries. The hepatic fatty infiltration of NAFLD progresses in a subset to inflammatory nonalcoholic steatohepatitis (NASH) and more rarely to cirrhosis and hepatocellular carcinoma. Steatosis typically improves following weight loss, secondary to diet or bariatric surgery. The mechanism for the association remains unclear. Obesity is associated with enhanced biliary secretion of cholesterol, supersaturation of bile, and a higher incidence of gallstones, particularly cholesterol gallstones (Chap. 369). A person 50% above IBW has about a sixfold increased incidence of symptomatic gallstones. Paradoxically, fasting increases supersaturation of bile by decreasing the phospholipid component. Fasting-induced cholecystitis is a complication of extreme diets.

1	Cancer Obesity is associated with increased risk of several cancer types, and in addition can lead to poorer treatment outcomes and increased cancer mortality. Obesity in males is associated with higher mortality from cancer of the esophagus, colon, rectum, pancreas, liver, and prostate; obesity in females is associated with higher mortality from cancer of the gallbladder, bile ducts, breasts, endometrium, cervix, and ovaries. Some of the latter may be due to increased rates of conversion of androstenedione to estrone in adipose tissue of obese individuals. Other possible mechanistic links may involve hormones, growth factors, and cytokines whose levels are linked to nutritional state, including insulin, leptin, adiponectin, and IGF-I, as well as activation of signaling pathways linked to both obesity and cancer. It has been estimated that obesity accounts for 14% of cancer deaths in men and 20% in women in the United States.

1	Bone, Joint, and Cutaneous Disease Obesity is associated with an increased risk of osteoarthritis, no doubt partly due to the trauma of added weight bearing, but potentially linked as well to activation of inflammatory pathways that could promote synovial pathology. The prevalence of gout may also be increased (Chap. 395). One of the skin problems associated with obesity is acanthosis nigricans, manifested by darkening and thickening of the skinfolds on the neck, elbows, and dorsal interphalangeal spaces. Acanthosis reflects the severity of underlying insulin resistance and diminishes with weight loss. Friability of skin may be increased, especially in skinfolds, enhancing the risk of fungal and yeast infections. Finally, venous stasis is increased in the obese. 2392 SEC Tion 3 obESiT y, DiAbETES mElliTuS, AnD mETAboliC SynDRomE Evaluation and management of Robert F. Kushner

1	2392 SEC Tion 3 obESiT y, DiAbETES mElliTuS, AnD mETAboliC SynDRomE Evaluation and management of Robert F. Kushner More than 66% of U.S. adults are categorized as overweight or obese, and the prevalence of obesity is increasing rapidly in most of the industrialized world. Children and adolescents also are becoming more obese, indicating that the current trends will accelerate over time. Obesity is associated with an increased risk of multiple health problems, including hypertension, type 2 diabetes, dyslipidemia, obstructive sleep apnea, nonalcoholic fatty liver disease, degenerative joint disease, and some malignancies. Thus, it is important for physicians to identify, evaluate, and treat patients for obesity and associated comorbid conditions.

1	Physicians should screen all adult patients for obesity and offer intensive counseling and behavioral interventions to promote sustained weight loss. The five main steps in the evaluation of obesity, as described below, are (1) a focused obesity-related history, (2) a physical examination to determine the degree and type of obesity, (3) assessment of comorbid conditions, (4) determination of fitness level, and (5) assessment of the patient’s readiness to adopt lifestyle changes. The Obesity-Focused History Information from the history should address the following seven questions: What factors contribute to the patient’s obesity? How is the obesity affecting the patient’s health? What is the patient’s level of risk from obesity? What does the patient find difficult about managing weight? What are the patient’s goals and expectations? Is the patient motivated to begin a weight management program? What kind of help does the patient need?

1	What are the patient’s goals and expectations? Is the patient motivated to begin a weight management program? What kind of help does the patient need? Although the vast majority of cases of obesity can be attributed to behavioral factors that affect diet and physical activity patterns, the history may suggest secondary causes that merit further evaluation. Disorders to consider include polycystic ovarian syndrome, hypothyroidism, Cushing’s syndrome, and hypothalamic disease. Drug-induced weight gain also should be considered. Common causes include medications for diabetes (insulin, sulfonylureas, thiazolidinediones); steroid hormones; psychotropic agents; mood stabilizers (lithium); antidepressants (tricyclics, monoamine oxidase inhibitors, paroxetine, mirtazapine); and antiepileptic drugs (valproate, gabapentin, carbamazepine). Other medications, such as nonsteroidal anti-inflammatory drugs and calcium channel blockers, may cause peripheral edema but do not increase body fat.

1	The patient’s current diet and physical activity patterns may reveal factors that contribute to the development of obesity and may identify behaviors to target for treatment. This type of historic information is best obtained by the combination of a questionnaire and an interview. Body Mass Index (BMI) and Waist Circumference Three key anthropometric measurements are important in evaluating the degree of obesity: weight, height, and waist circumference. The BMI, calculated as weight (kg)/height (m)2 or as weight (lbs)/height (inches)2 × 703, is used to classify weight status and risk of disease (Tables 416-1 and 416-2). BMI provides an estimate of body fat and is related to disease risk. Lower BMI thresholds for overweight and obesity have been proposed for the Asia-Pacific region since this population appears to be at risk for glucose and lipid abnormalities at lower body weights.

1	Excess abdominal fat, assessed by measurement of waist circumference or waist-to-hip ratio, is independently associated with a higher risk for diabetes mellitus and cardiovascular disease. Measurement of the waist circumference is a surrogate for visceral adipose tissue and should be performed in the horizontal plane above the iliac crest (Table 416-3). Physical Fitness Several prospective studies have demonstrated that physical fitness, reported by questionnaire or measured by a maximal treadmill exercise test, is an important predictor of all-cause mortality rate independent of BMI and body composition. These observations highlight the importance of taking a physical activity and exercise history during examination as well as emphasizing physical activity as a treatment approach.

1	Obesity-Associated Comorbid Conditions The evaluation of comorbid conditions should be based on presentation of symptoms, risk factors, and index of suspicion. For all patients, a fasting lipid panel should be performed (total, low-density lipoprotein, and high-density lipoprotein cholesterol and triglyceride levels) and a fasting blood glucose level and blood pressure determined. Symptoms and diseases that are directly or indirectly related to obesity are listed in Table 416-4. Although individuals vary, the number and severity of organ-specific comorbid conditions usually rise with increasing levels of obesity. Patients at very high absolute risk include those with the following: established coronary heart disease; presence of other atherosclerotic diseases, such as peripheral arterial disease, abdominal aortic aneurysm, and symptomatic carotid artery disease; type 2 diabetes; and sleep apnea.

1	Assessing the Patient’s Readiness to Change An attempt to initiate lifestyle changes when the patient is not ready usually leads to frustration and may hamper future weight-loss efforts. Assessment includes patient motivation and support, stressful life events, psychiatric status, A helpful method to begin a readiness assessment is to use the motitime availability and constraints, and appropriateness of goals and vational interviewing technique of “anchoring” the patient’s interest expectations. Readiness can be viewed as the balance of two opposing and confidence to change on a numerical scale. With this technique, forces: (1) motivation, or the patient’s desire to change; and (2) resis-the patient is asked to rate—on a scale from 0 to 10, with 0 being not so tance, or the patient’s resistance to change. important (or confident) and 10 being very important (or confident)— his or her level of interest in and confidence about losing weight at this time. This exercise helps establish

1	to change. important (or confident) and 10 being very important (or confident)— his or her level of interest in and confidence about losing weight at this time. This exercise helps establish readiness to change and also serves as a basis for further dialogue.

1	Underweight <18.5 — — TREATmEnT obesity Healthy weight 18.5–24.9 — — Overweight 25.0–29.9 — Increased The primary goals of treatment are to improve obesity-related Obesity 30.0–34.9 I High comorbid conditions and to reduce the risk of developing future Obesity 35.0–39.9 II Very high comorbidities. Information obtained from the history, physical examination, and diagnostic tests is used to determine risk and Source: Adapted from the National Institutes of Health, National Heart, Lung, and Blood develop a treatment plan (Fig. 416-1). The decision of how aggres-Institute: Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight sively to treat the patient and which modalities to use is determined and Obesity in Adults. U.S. Department of Health and Human Services, U.S. Public Health Service, 1998. by the patient’s risk status, expectations, and available resources. Evaluation and Management of Obesity

1	Service, 1998. by the patient’s risk status, expectations, and available resources. Evaluation and Management of Obesity Eastern (Arab) populations Source: From KGMM Alberti et al for the IDF Epidemiology Task Force Consensus Group: Lancet 366:1059, 2005. Not all patients who are deemed obese by BMI alone need to be treated, as exemplified by the concepts of obesity paradox or the metabolically healthy obese. However, patients who present with obesity-related comorbidities and who would benefit from weight loss intervention should be managed proactively. Therapy for obesity always begins with lifestyle management and may include pharmacotherapy or surgery, depending on BMI risk category (Table 416-5). Setting an initial weight-loss goal of 8–10% over 6 months is a realistic target. Integument Dementia Striae distensae Stasis pigmentation of legs Lymphedema Cellulitis Intertrigo, carbuncles Acanthosis nigricans Acrochordons (skin tags) Hidradenitis suppurativa

1	Integument Dementia Striae distensae Stasis pigmentation of legs Lymphedema Cellulitis Intertrigo, carbuncles Acanthosis nigricans Acrochordons (skin tags) Hidradenitis suppurativa Obesity care involves attention to three essential elements of lifestyle: dietary habits, physical activity, and behavior modification. Because obesity is fundamentally a disease of energy imbalance, all patients must learn how and when energy is consumed (diet), how and when energy is expended (physical activity), and how to incorporate this information into their daily lives (behavioral therapy). Lifestyle management has been shown to result in a modest (typically 3–5 kg) weight loss when compared with no treatment or usual care.

1	Diet Therapy The primary focus of diet therapy is to reduce overall calorie consumption. Guidelines from the National Heart, Lung, and Blood Institute recommend initiating treatment with a calorie deficit of 500–1000 kcal/d compared with the patient’s habitual diet. This reduction is consistent with a goal of losing ~1–2 lbs per week. The calorie deficit can be instituted through dietary substitutions or alternatives. Examples include choosing smaller portion sizes, eating more fruits and vegetables, consuming more whole-grain cereals, selecting leaner cuts of meat and skimmed dairy products, reducing consumption of fried foods and other foods with added fats and oils, and drinking water instead of sugar-sweetened beverages. It is important that dietary counseling remain patient centered and that the goals set be practical, realistic, and achievable.

1	The macronutrient composition of the diet will vary with the patient’s preference and medical condition. The 2010 U.S. Department of Agriculture Dietary Guidelines for Americans (Chap. 95e), which focus on health promotion and risk reduction, can be applied to treatment of overweight or obese patients. The recommendations include maintaining a diet rich in whole grains, fruits, vegetables, and dietary fiber; consuming two servings (8 oz) of fish high in omega 3 fatty acids per week; decreasing sodium intake to <2300 mg/d; consuming 3 cups of milk (or equivalent low-fat or fat-free dairy products) per day; limiting cholesterol intake to <300 mg/d; and keeping total fat intake at 20–35% of daily calories and saturated fat intake at <10% of daily calories. Application of these guidelines to specific calorie goals can be found on the website www.choosemyplate.gov. The revised Dietary Reference Intakes for Macronutrients released by the Institute of Medicine recommends that 45–65% of

1	to specific calorie goals can be found on the website www.choosemyplate.gov. The revised Dietary Reference Intakes for Macronutrients released by the Institute of Medicine recommends that 45–65% of calories come from carbohydrates, 20–35% from fat, and 10–35% from protein. The guidelines also recommend daily fiber intake of 38 g (men) and 25 g (women) for persons over 50 years of age and 30 g (men) and 21 g (women) for those under age 50.

1	Since portion control is one of the most difficult strategies for patients to manage, the use of pre-prepared products such as meal replacements is a simple and convenient suggestion. Examples include frozen entrees, canned beverages, and bars. Use of meal replacements in the diet has been shown to result in a 7–8% weight loss.

1	Patient encounter Hx of ˜ 25 BMI? BMI measured in past 2 years? No • Measure weight, height and waist circumference • Calculate BMI BMI ˜ 25 OR waist circumference > 88 cm (F) >102 cm (M) Hx BMI ˜ 25? Brief reinforcement/ educate on weight management Advise to maintain weight, address other risk factors Assess risk factors BMI ˜ 30 OR {[BMI 26 to 29.9 OR waist circumference > 88 cm (F) >102 cm (M)] AND ˜ 2 risk factors} Does patient want to lose weight? Progress being made/goal achieved? Maintenance counseling: • Dietary therapy • Behavior therapy • Physical therapy Assess reasons for failure to lose weight Periodic weight check 4 5 6 Yes 7 Yes Yes No 8 9 12 11 10 No Yes 1315 14 No Yes 16 No No Clinician and patient devise goals and treatment strategy for weight loss and risk factor control

1	FIGURE 416-1 Algorithm for the treatment of obesity. This algorithm applies only to assessment for overweight and obesity and subsequent decisions based on that assessment. It does not reflect initial overall assessment for other conditions that the physician may wish to perform. BMI, body mass index; Hx, history. (From the National, Heart, Lung, and Blood Institute: Clinical guidelines on the identification, evaluation, and treatment of overweight and obesity in adults: The evidence report. Washington, DC, US Department of Health and Human Services, 1998.) Evaluation and Management of Obesity

1	Evaluation and Management of Obesity Numerous randomized trials comparing diets of different macro-patient’s taste preferences, cooking style, and culture. However, the nutrient composition (e.g., low-carbohydrate, low-fat, Mediterranean) patient’s underlying medical problems are also important in guiding have shown that weight loss depends primarily on reduction of total the recommended dietary composition. The dietary prescription will caloric intake and adherence to the prescribed diet, not the spe-vary according to the patient’s metabolic profile and risk factors. A cific proportions of carbohydrate, fat, and protein in the diet. The consultation with a registered dietitian for medical nutrition therapy macronutrient composition will ultimately be determined by the is particularly useful in considering patient preference and treatment of comorbid diseases.

1	Another dietary approach to consider is based on the concept of energy density, which refers to the BMI Category (kg/m2) number of calories (i.e., amount of energy) a food Treatment 25–26.9 27–29.9 30–34.9 35–39.9 ≥40 contains per unit of weight. People tend to ingest a constant volume of food regardless of caloric or Diet, exercise, With With + + + behavioral therapy comorbidities comorbidities macronutrient content. Adding water or fiber to a food decreases its energy density by increasing weight without affecting caloric content. Examples of foods with low-energy density include soups, fruits, vegetables, oatmeal, and lean meats. Dry foods and high-fat foods such as pretzels, cheese, egg

1	Source: From the National Heart, Lung, and Blood Institute, North American Association for the Study of Obesity (2000). yolks, potato chips, and red meat have a high-energy 2396 density. Diets containing low-energy-dense foods have been shown to control hunger and thus to result in decreased caloric intake and weight loss. Occasionally, very low-calorie diets (VLCDs) are prescribed as a form of aggressive dietary therapy. The primary purpose of a VLCD is to promote a rapid and significant (13to 23-kg) short-term weight loss over a 3to 6-month period. The proprietary formulas designed for this purpose typically supply ≤800 kcal, 50–80 g of protein, and 100% of the recommended daily intake for vitamins and minerals. According to a review by the National Task Force on the Prevention and Treatment of Obesity, indications for initiating a VLCD include the involvement of well-motivated individuals who are moderately to severely obese (BMI, >30 kg/m2), have failed at more conservative

1	Treatment of Obesity, indications for initiating a VLCD include the involvement of well-motivated individuals who are moderately to severely obese (BMI, >30 kg/m2), have failed at more conservative approaches to weight loss, and have a medical condition that would be immediately improved with rapid weight loss. These conditions include poorly controlled type 2 diabetes, hypertriglyceridemia, obstructive sleep apnea, and symptomatic peripheral edema. The risk for gallstone formation increases exponentially at rates of weight loss >1.5 kg/week (3.3 lb/week). Prophylaxis against gallstone formation with ursodeoxycholic acid (600 mg/d) is effective in reducing this risk. Because of the need for close metabolic monitoring, VLCDs usually are prescribed by physicians specializing in obesity care.

1	Physical Activity Therapy Although exercise alone is only moderately effective for weight loss, the combination of dietary modification and exercise is the most effective behavioral approach for the treatment of obesity. The most important role of exercise appears to be in the maintenance of the weight loss. The 2008 Physical Activity Guidelines for Americans (www.health.gov/paguidelines) recommend that adults should engage in 150 min of moderate-intensity or 75 min a week of vigorous-intensity aerobic physical activity per week, performed in episodes of at least 10 min and preferably spread throughout the week. Focusing on simple ways to add physical activity into the normal daily routine through leisure activities, travel, and domestic work should be suggested. Examples include walking, using the stairs, doing housework and yard work, and engaging in sports. Asking the patient to wear a pedometer or accelerometer to monitor total accumulation of steps or kcal expended as part of the

1	using the stairs, doing housework and yard work, and engaging in sports. Asking the patient to wear a pedometer or accelerometer to monitor total accumulation of steps or kcal expended as part of the activities of daily living is a useful strategy. Step counts are highly correlated with activity level. Studies have demonstrated that lifestyle activities are as effective as structured exercise programs for improving cardiorespiratory fitness and weight loss. A high level of physical activity (>300 min of moderate-intensity activity per week) is often needed to lose weight and sustain weight loss. These exercise recommendations are daunting to most patients and need to be implemented gradually. Consultation with an exercise physiologist or personal trainer may be helpful.

1	Behavioral Therapy Cognitive behavioral therapy is used to help change and reinforce new dietary and physical activity behaviors. Strategies include self-monitoring techniques (e.g., journaling, weighing, and measuring food and activity); stress management; stimulus control (e.g., using smaller plates, not eating in front of the television or in the car); social support; problem solving; and cognitive restructuring to help patients develop more positive and realistic thoughts about themselves. When recommending any behavioral lifestyle change, the patient should be asked to identify what, when, where, and how the behavioral change will be performed. The patient should keep a record of the anticipated behavioral change so that progress can be reviewed at the next office visit. Because these techniques are time-consuming to implement, their supervision is often undertaken by ancillary office staff, such as a nurse-clinician or registered dietitian.

1	Adjuvant pharmacologic treatments should be considered for patients with a BMI ≥30 kg/m2 or—for patients who have concomitant obesity-related diseases and for whom dietary and physical activity therapy has not been successful—a BMI ≥27 kg/m2. When an antiobesity medication is prescribed, patients should be actively engaged in a lifestyle program that provides the strategies and skills needed to use the drug effectively, since such support increases total weight loss.

1	Medications for obesity have traditionally fallen into two major categories: appetite suppressants (anorexiants) and gastrointestinal fat blockers. Appetite-suppressing medications have primarily targeted three monoamine receptor systems in the hypothalamus: noradrenergic, dopaminergic, and serotonergic receptors. Two new appetite suppressants were approved by the U.S. Food and Drug Administration (FDA) in 2012: lorcaserin and phentermine/topiramate (PHEN/TPM) extended release. Gastrointestinal fat blockers reduce the absorption of selective macronutrients, such as fat, from the gastrointestinal tract.

1	Centrally Acting Anorexiant Medications Anorexiants affect satiety (the absence of hunger after eating) and hunger (the biologic sensation that prompts eating). By increasing satiety and decreasing hunger, these agents help patients reduce caloric intake without a sense of deprivation. The target site for the actions of anorexiants is the ventromedial and lateral hypothalamic regions in the central nervous system (Chap. 415e). The biologic effect of these agents on appetite regulation is produced by augmentation of the neurotransmission of three monoamines: norepinephrine; serotonin (5-hydroxytryptamine, or 5-HT); and, to a lesser degree, dopamine. The classic sympathomimetic adrenergic agents (benzphetamine, phendimetrazine, diethylpropion, mazindol, and phentermine) function by stimulating norepinephrine release or by blocking its reuptake. Among the anorexiants, phentermine has been the most commonly prescribed; there is limited long-term data on its effectiveness. A 2002 review of

1	norepinephrine release or by blocking its reuptake. Among the anorexiants, phentermine has been the most commonly prescribed; there is limited long-term data on its effectiveness. A 2002 review of six randomized, placebo-controlled trials of phentermine for weight control found that patients lost 0.6–6.0 additional kilograms of weight over 2–24 weeks of treatment. The most common side effects of the amphetamine-derived anorexiants are restlessness, insomnia, dry mouth, constipation, and increased blood pressure and heart rate.

1	PHEN/TPM is a combination drug that contains a catecholamine releaser (phentermine) and an anticonvulsant (topiramate). Topiramate is approved by the FDA as an anticonvulsant for the treatment of epilepsy and for the prophylaxis of migraine headaches. Weight loss was identified as an unintended side effect of topiramate during clinical trials for epilepsy. The mechanism responsible for weight loss is uncertain but is thought to be mediated through the drug’s modulation of γ-aminobutyric acid receptors, inhibition of carbonic anhydrase, and antagonism of glutamate. PHEN/TPM has undergone two 1-year pivotal randomized, placebo-controlled, double-blind trials of efficacy and safety: EQUIP and CONQUER. In a third study, SEQUEL, 78% of CONQUER participants continued to receive their blinded treatment for an additional year. All participants received diet and exercise counseling. Participant numbers, eligibility, characteristics, and weight loss outcomes are displayed in Table 416-6.

1	treatment for an additional year. All participants received diet and exercise counseling. Participant numbers, eligibility, characteristics, and weight loss outcomes are displayed in Table 416-6. Intention-to-treat 1-year placebo-subtracted weight loss for the PHEN/TPM 15-mg/92-mg dose was 9.3% and 8.6%, respectively, in the EQUIP and CONQUER trials. Clinical and statistical dose-dependent improvements were seen in selected cardiovascular and metabolic outcome measurements that were related to the weight loss. The most common adverse events experienced by the drug-randomized group were paresthesias, dry mouth, constipation, dysgeusia, and insomnia. Because of an increased risk of congenital fetal oral-cleft formation from topiramate, the FDA approval of PHEN/TPM stipulated a Risk Evaluation and Mitigation Strategies requirement to educate prescribers about the need for active birth control among women of childbearing age and a contraindication for use during pregnancy.

1	Lorcaserin is a selective 5-HT2C receptor agonist with a functional selectivity ~15 times that of 5-HT2A receptors and 100 times that of 5-HT2B receptors. This selectivity is important, since the drug-induced valvulopathy documented with two other serotonergic agents that were removed from the market—fenfluramine No. of participants (ITT-LOCF) 3182 4008 1230 2448 Age (years) 18–65 18–65 ≥35 27–45 BMI (kg/m2) 27–45 27–45 18–70 18–70 Comorbid conditions (cardio-≥1 ≥1 ≥1 ≥2

1	No. of participants (ITT-LOCF) 3182 4008 1230 2448 Age (years) 18–65 18–65 ≥35 27–45 BMI (kg/m2) 27–45 27–45 18–70 18–70 Comorbid conditions (cardio-≥1 ≥1 ≥1 ≥2 Mean weight loss (%) with 5.8 vs. 2.2 4.8 vs. 2.8 11 vs. 1.6 10.4 vs. 1.8 treatment vs. placebo Placebo-subtracted weight 3.6 3.0 9.3 8.6 loss (%) Categorical change in 5% 47.5 vs. 20.3 47.2 vs. 25 67 vs. 17 70 vs. 21 weight loss with treatment vs. placebo Completion rate (%) Lorcaserin, 55.4; placebo, 45.1 55.5 59.9 62 aTable shows a comparison of two 1-year prospective, randomized, double-blind trials of lorcaserin (BLOOM and BLOSSOM) and phentermine-topiramate extended release (EQUIP and CONQUER). bLorcaserin dose: 10 mg bid. cLorcaserin dose: 10 mg bid or qd. dPhentermine-topiramate extended release dose: 15 mg/92 mg. Abbreviations: BMI, body mass index (see Table 416-1); ITT-LOCF, intention to treat, last observation carried forward; PHEN/TPM, phentermine-topiramate extended release.

1	Abbreviations: BMI, body mass index (see Table 416-1); ITT-LOCF, intention to treat, last observation carried forward; PHEN/TPM, phentermine-topiramate extended release. and dexfenfluramine—was due to activation of the 5-HT2B receptors expressed on cardiac valvular interstitial cells. By activating the 5-HT2C receptor, lorcaserin is thought to decrease food intake through the pro-opiomelanocortin system of neurons.

1	Lorcaserin has undergone two randomized, placebo-controlled, double-blind trials for efficacy and safety. Participants were randomized to receive lorcaserin (10 mg bid) or placebo in the BLOOM study and to receive lorcaserin (10 mg bid or qd) or placebo in the BLOSSOM study. All participants received diet and exercise counseling. Participant numbers, eligibility, characteristics, and weight loss outcomes are displayed in Table 416-6. Overweight or obese subjects had at least one coexisting condition (hypertension, dyslipidemia, cardiovascular disease, impaired glucose tolerance, or sleep apnea)—medical conditions that are commonly seen in the office setting. Intention-to-treat 1-year placebo-subtracted weight loss was 3.6% and 3.0%, respectively, in the BLOOM and BLOSSOM trials. Echocardiography was performed at the screening visit and at scheduled time points over the course of the studies. There was no difference in the development of FDA-defined valvulopathy between drug-treated

1	was performed at the screening visit and at scheduled time points over the course of the studies. There was no difference in the development of FDA-defined valvulopathy between drug-treated and placebo-treated participants at 1 year or 2 years. Modest statistical improvements consistent with the weight loss were seen in selected cardiovascular and metabolic outcome measurements. The most common adverse events experienced by the drug group were headache, dizziness, and nausea.

1	In approving both PHEN/TPM and lorcaserin, the FDA introduced a new provision with important clinical relevance: a prescription trial period to assess effectiveness. Response to both medications should be assessed after 3 months of treatment. For lorcaserin, the medication should be discontinued if the patient has not lost at least 5% of body weight by that point. For PHEN/TPM, if the patient has not lost at least 3% of body weight at 3 months, the clinician can either escalate the dose and reassess progress at 6 months or discontinue treatment entirely.

1	Peripherally Acting Medications Orlistat (XenicalTM) is a synthetic hydrogenated derivative of a naturally occurring lipase inhibitor, lipostatin, that is produced by the mold Streptomyces toxytricini. This drug is a potent, slowly reversible inhibitor of pancreatic, gastric, and carboxylester lipases and phospholipase A2, which are required for the hydrolysis of dietary fat into fatty acids and monoacylglycerols. Orlistat acts in the lumen of the stomach and small intestine by forming a covalent bond with the active site of these lipases. Taken at a therapeutic dose of 120 mg tid, orlistat blocks the digestion and absorption of ~30% of dietary fat. After discontinuation of the drug, fecal fat content usually returns to normal within 48–72 h.

1	Multiple randomized, double-blind, placebo-controlled studies have shown that, after 1 year, orlistat produces a weight loss of ~9–10%, whereas placebo recipients have a 4–6% weight loss. Because orlistat is minimally (<1%) absorbed from the gastrointestinal tract, it has no systemic side effects. The drug’s tolerability is related to the malabsorption of dietary fat and the subsequent passage of fat in the feces. Adverse gastrointestinal effects, including flatus with discharge, fecal urgency, fatty/oily stool, and increased defecation, are reported in at least 10% of orlistat-treated patients. These side effects generally are experienced early, diminish as patients control their dietary fat intake, and only infrequently cause patients to withdraw from clinical trials. When taken concomitantly, psyllium mucilloid is helpful in controlling orlistat-induced gastrointestinal side effects. Because serum concentrations of the fat-soluble vitamins D and E and β-carotene may be reduced by

1	psyllium mucilloid is helpful in controlling orlistat-induced gastrointestinal side effects. Because serum concentrations of the fat-soluble vitamins D and E and β-carotene may be reduced by orlistat treatment, vitamin supplements are recommended to prevent potential deficiencies. Orlistat was approved for over-the-counter use in 2007.

1	Antiobesity Drugs in Development Two additional medications are currently in development. Bupropion and naltrexone (ContraveTM)—a dopamine and norepinephrine reuptake inhibitor and an opioid receptor antagonist, respectively—are theoretically combined to dampen the motivation/reinforcement that food brings (dopamine effect) and the pleasure/palatability of eating (opioid effect). In the COR-1 randomized, double-blind, placebo-controlled trial, 1742 enrolled participants, who were 18–65 years of age and had BMIs of 30–45 kg/m2, were randomized to receive naltrexone (16 mg/d) plus bupropion (360 mg/d), naltrexone (32 mg/d) plus bupropion (360 mg/d), or placebo. Mean change in body weight for the three groups was 5.0%, 6.1%, and 1.3%, respectively. The most common adverse events were nausea, headache, constipation, dizziness, vomiting, and dry mouth. However, the FDA rejected the drug in 2011 because of cardiovascular concerns and concluded that a large-scale study of the long-term

1	headache, constipation, dizziness, vomiting, and dry mouth. However, the FDA rejected the drug in 2011 because of cardiovascular concerns and concluded that a large-scale study of the long-term cardiovascular effects of naltrexone would be needed before approval could be considered.

1	Liraglutide, a glucagon-like peptide 1 receptor agonist currently approved for the treatment of type 2 diabetes, has independent weight loss effects via hypothalamic neural activation causing appetite suppression. In a double-blind, placebo-controlled trial, 564 adults with BMIs of 30–40 kg/m2 were randomized to receive once-daily SC liraglutide (1.2, 1.8, 2.4, or 3.0 mg), placebo, or open-label orlistat (120 mg tid) for 1 year. The liraglutide and placebo recipients were switched to 2.4 mg of liraglutide during the second year and then to 3.0 mg for an additional year. One-year placebo-subtracted mean weight loss was 5.8 kg for liraglutide and 3.8 kg more than those on orlistat. The most common side effects were nausea, vomiting, and change in bowel habits.

1	Evaluation and Management of Obesity 2398 SURGERY Bariatric surgery (Fig. 416-2) can be considered for patients with severe obesity (BMI, ≥40 kg/m2) or for those with moderate obesity (BMI, ≥35 kg/m2) associated with a serious medical condition. Weight loss surgeries have traditionally been classified into three categories on the basis of anatomic changes: restrictive, restrictive malabsorptive, and malabsorptive. More recently, however, the clinical benefits of bariatric surgery in achieving weight loss and alleviating metabolic comorbidities have been attributed largely to changes in the physiologic responses of gut hormones and in adipose tissue metabolism. Metabolic effects resulting from bypassing the foregut include altered responses of ghrelin, glucagon-like peptide 1, peptide YY3-36, and oxyntonodulin. Additional effects on food intake and body weight control may be attributed to changes in vagal signaling. The loss of fat mass, particularly visceral fat, is associated with

1	and oxyntonodulin. Additional effects on food intake and body weight control may be attributed to changes in vagal signaling. The loss of fat mass, particularly visceral fat, is associated with multiple metabolic, adipokine, and inflammatory changes that include improved insulin sensitivity and glucose disposal; reduced free fatty acid flux; increased adiponectin levels; and decreased interleukin 6, tumor necrosis factor α, and high-sensitivity C-reactive protein levels. Restrictive surgeries limit the amount of food the stomach can hold and slow the rate of gastric emptying. Laparoscopic adjustable gastric banding is the prototype of this category. The first banding device, the LAP-BAND, was approved for use in the United States in 2001 and the second, the REALIZE band, in 2007. In contrast to previous devices, these bands have diameters that are adjustable by way of their connection to a reservoir that is implanted under the skin. Injection of saline into the reservoir and removal

1	to previous devices, these bands have diameters that are adjustable by way of their connection to a reservoir that is implanted under the skin. Injection of saline into the reservoir and removal of saline from the reservoir tighten and loosen the band’s internal diameter, respectively, thus changing the size of the gastric opening. The mean percentage of total body weight lost at 5 years is estimated at 20–25%. In laparoscopic sleeve gastrectomy, the stomach is restricted by stapling and dividing it vertically, removing ~80% of the greater curvature, and leaving a slim banana-shaped remnant stomach along the lesser curvature. Weight loss after this procedure is superior to that after laparoscopic adjustable gastric banding.

1	The three restrictive-malabsorptive bypass procedures combine the elements of gastric restriction and selective malabsorption. These procedures are Roux-en-Y gastric bypass, biliopancreatic diversion, and biliopancreatic diversion with duodenal switch (Fig. 416-2). Roux-en-Y is the most commonly undertaken and most accepted bypass procedure. It may be performed with an open incision or by laparoscopy.

1	These procedures generally produce a 30–35% average total body weight loss that is maintained in nearly 60% of patients at 5 years. In general, mean weight loss is greater after the combined restrictive-malabsorptive procedures than after the restrictive procedures. Significant improvement in multiple obesity-related comorbid conditions, including type 2 diabetes, hypertension, dyslipidemia, obstructive sleep apnea, quality of life, and long-term cardiovascular events, has been reported. A meta-analysis of controlled clinical trials comparing bariatric surgery versus no surgery showed that surgery was associated with a reduced odds ratio (OR) risk of global mortality (OR = 0.55), cardiovascular death (OR = 0.58), and all-cause mortality (OR = 0.70).

1	Among the observed improvements in comorbidities, the prevention and treatment of type 2 diabetes resulting from bariatric surgery has garnered the most attention. Fifteen-year data from the Swedish Obese Subjects study demonstrated a marked reduction (i.e., by 78%) in the incidence of type 2 diabetes development among obese patients who underwent bariatric surgery. Several randomized controlled studies have shown greater weight loss and more improved glycemic control at 1 and 2 years among surgical patients than among patients receiving conventional medical therapy. A retrospective cohort study of more than 4000 adults with diabetes found that, overall, 68.2% of patients experienced an initial complete type 2 diabetes remission within 5 years after surgery. However, among these patients, one-third redeveloped type 2 diabetes within 5 years. The rapid improvement seen in diabetes after restrictive-malabsorptive procedures is thought to be due to surgery-specific, weight-independent

1	one-third redeveloped type 2 diabetes within 5 years. The rapid improvement seen in diabetes after restrictive-malabsorptive procedures is thought to be due to surgery-specific, weight-independent effects on glucose homeostasis brought about by alteration of gut hormones.

1	The mortality rate from bariatric surgery is generally <1% but varies with the procedure, the patient’s age and comorbid conditions, and the experience of the surgical team. The most common surgical complications include stomal stenosis or marginal ulcers (occurring in 5–15% of patients) that present as prolonged nausea and vomiting after eating or inability to advance the diet to solid foods. These complications typically are treated by endoscopic balloon dilation and acid suppression therapy, respectively. For patients who undergo laparoscopic adjustable gastric banding, there are no intestinal absorptive abnormalities other than mechanical reduction in gastric size and outflow. Therefore, selective deficiencies are uncommon unless eating habits become unbalanced. In contrast, the restrictive-malabsorptive procedures carry an increased risk for micronutrient deficiencies

1	FIGURE 416-2 Bariatric surgical procedures. Examples of operative interventions of vitamin B12, iron, folate, calcium, and vitamin used for surgical manipulation of the gastrointestinal tract. A. Laparoscopic adjustable D. Patients with restrictive-malabsorptive proce gastric banding. B. Laparoscopic sleeve gastrectomy. C. The Roux-en-Y gastric bypass. dures require lifelong supplementation with these D. Biliopancreatic diversion with duodenal switch. E. Biliopancreatic diversion. (From micronutrients. ML Kendrick, GF Dakin: Mayo Clin Proc 815:518, 2006; with permission.)

1	Diabetes mellitus: Diagnosis, Classification, and Pathophysiology Alvin C. Powers Diabetes mellitus (DM) refers to a group of common metabolic disor-417 ders that share the phenotype of hyperglycemia. Several distinct types of DM are caused by a complex interaction of genetics and environmental factors. Depending on the etiology of the DM, factors contributing to hyperglycemia include reduced insulin secretion, decreased glucose utilization, and increased glucose production. The metabolic dysregulation associated with DM causes secondary pathophysiologic changes in multiple organ systems that impose a tremendous burden on the individual with diabetes and on the health care system. In the United States, DM is the leading cause of end-stage renal disease (ESRD), nontraumatic lower extremity amputations, and adult blindness. It also predisposes to cardiovascular diseases. With an increasing incidence worldwide, DM will be likely a leading cause of morbidity and mortality in the future.

1	DM is classified on the basis of the pathogenic process that leads to hyperglycemia, as opposed to earlier criteria such as age of onset or type of therapy (Fig. 417-1). There are two broad categories of DM, designated type 1 and type 2 (Table 417-1). However, there is increasing recognition of other forms of diabetes in which the pathogenesis is better understood. These other forms of diabetes may share features of Type of <5.6 mmol/L 5.6–6.9 mmol/L ˜7.0 mmol/L (100 mg/dL) 2-h PG <7.8 mmol/L 7.8–11.0 mmol/L ˜11.1 mmol/L (140 mg/dL) HbA1C <5.6% 5.7–6.4% ˜6.5%

1	FIGURE 417-1 Spectrum of glucose homeostasis and diabetes mellitus (DM). The spectrum from normal glucose tolerance to diabetes in type 1 DM, type 2 DM, other specific types of diabetes, and gestational DM is shown from left to right. In most types of DM, the individual traverses from normal glucose tolerance to impaired glucose tolerance to overt diabetes (these should be viewed not as abrupt categories but as a spectrum). Arrows indicate that changes in glucose tolerance may be bidirectional in some types of diabetes. For example, individuals with type 2 DM may return to the impaired glucose tolerance category with weight loss; in gestational DM, diabetes may revert to impaired glucose tolerance or even normal glucose tolerance after delivery. The fasting plasma glucose (FPG), the 2-h plasma glucose (PG) after a glucose challenge, and the hemoglobin A1c (HbA1c) for the different categories of glucose tolerance are shown at the lower part of the figure. These values do not apply to

1	plasma glucose (PG) after a glucose challenge, and the hemoglobin A1c (HbA1c) for the different categories of glucose tolerance are shown at the lower part of the figure. These values do not apply to the diagnosis of gestational DM. Some types of DM may or may not require insulin for survival. *Some use the term increased risk for diabetes or intermediate hyperglycemia (World Health Organization) rather than prediabetes. (Adapted from the American Diabetes Association, 2014.)

1	ETiologiC ClASSifiCATion of DiAbETES mElliTuS 1. Type 1 diabetes (beta cell destruction, usually leading to absolute insulin deficiency) A. Immune-mediated B. Idiopathic II. Type 2 diabetes (may range from predominantly insulin resistance with relative insulin deficiency to a predominantly insulin secretory defect with insulin resistance) III. Other specific types of diabetes A. Genetic defects of beta cell development or function characterized by mutations in: 1. 2. Glucokinase (MODY 2) 3. HNF-1α (MODY 3) 4. Insulin promoter factor-1 (IPF-1; MODY 4) 5. HNF-1β (MODY 5) 6. NeuroD1 (MODY 6) 7. 8. Subunits of ATP-sensitive potassium channel 9. 10. Other pancreatic islet regulators/proteins such as KLF11, PAX4, BLK, GATA4, GATA6, SLC2A2 (GLUT2), RFX6, GLIS3 B. Genetic defects in insulin action 1. 2. 3. 4.

1	10. Other pancreatic islet regulators/proteins such as KLF11, PAX4, BLK, GATA4, GATA6, SLC2A2 (GLUT2), RFX6, GLIS3 B. Genetic defects in insulin action 1. 2. 3. 4. C. Diseases of the exocrine pancreas—pancreatitis, pancreatectomy, neoplasia, cystic fibrosis, hemochromatosis, fibrocalculous pancreatopathy, mutations in carboxyl ester lipase D. Endocrinopathies—acromegaly, Cushing’s syndrome, glucagonoma, pheochromocytoma, hyperthyroidism, somatostatinoma, aldosteronoma E. Drugor chemical-induced—glucocorticoids, vacor (a rodenticide), pentamidine, nicotinic acid, diazoxide, β-adrenergic agonists, thiazides, calcineurin and mTOR inhibitors, hydantoins, asparaginase, α-interferon, protease inhibitors, antipsychotics (atypicals and others), epinephrine F. Infections—congenital rubella, cytomegalovirus, coxsackievirus G. Uncommon forms of immune-mediated diabetes—”stiff-person” syndrome, anti-insulin receptor antibodies

1	F. Infections—congenital rubella, cytomegalovirus, coxsackievirus G. Uncommon forms of immune-mediated diabetes—”stiff-person” syndrome, anti-insulin receptor antibodies H. Other genetic syndromes sometimes associated with diabetes— Wolfram’s syndrome, Down’s syndrome, Klinefelter’s syndrome, Turner’s syndrome, Friedreich’s ataxia, Huntington’s chorea, Laurence-Moon-Biedl syndrome, myotonic dystrophy, porphyria, Prader-Willi syndrome IV. Gestational diabetes mellitus (GDM) Abbreviation: MODY, maturity-onset diabetes of the young. Source: Adapted from American Diabetes Association: Diabetes Care 37(Suppl 1):S14, 2014.

1	IV. Gestational diabetes mellitus (GDM) Abbreviation: MODY, maturity-onset diabetes of the young. Source: Adapted from American Diabetes Association: Diabetes Care 37(Suppl 1):S14, 2014. type 1 and/or type 2 DM. Both type 1 and type 2 DM are preceded by a phase of abnormal glucose homeostasis as the pathogenic processes progress. Type 1 DM is the result of complete or near-total insulin deficiency. Type 2 DM is a heterogeneous group of disorders characterized by variable degrees of insulin resistance, impaired insulin secretion, and increased glucose production. Distinct genetic and metabolic defects in insulin action and/or secretion give rise to the common phenotype of hyperglycemia in type 2 DM and have important potential therapeutic implications now that pharmacologic agents are available to target specific metabolic derangements. Type 2 DM is preceded by a period of abnormal glucose homeostasis classified as impaired fasting glucose (IFG) or impaired glucose tolerance (IGT).

1	Two features of the current classification of DM merit emphasis from previous classifications. First, the terms insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) are obsolete. Because many individuals with type 2 DM eventually require insulin treatment for control of Diabetes Mellitus: Diagnosis, Classification, and Pathophysiology 2400 glycemia, the use of the term NIDDM generated considerable confusion. A second difference is that age or treatment modality is not a criterion. Although type 1 DM most commonly develops before the age of 30, an autoimmune beta cell destructive process can develop at any age. It is estimated that between 5 and 10% of individuals who develop DM after age 30 years have type 1 DM. Although type 2 DM more typically develops with increasing age, it is now being diagnosed more frequently in children and young adults, particularly in obese adolescents.

1	Other etiologies for DM include specific genetic defects in insulin secretion or action, metabolic abnormalities that impair insulin secretion, mitochondrial abnormalities, and a host of conditions that impair glucose tolerance (Table 417-1). Maturity-onset diabetes of the young (MODY) and monogenic diabetes are subtypes of DM characterized by autosomal dominant inheritance, early onset of hyperglycemia (usually <25 years; sometimes in neonatal period), and impaired insulin secretion (discussed below). Mutations in the insulin receptor cause a group of rare disorders characterized by severe insulin resistance.

1	DM can result from pancreatic exocrine disease when the majority of pancreatic islets are destroyed. Cystic fibrosis–related DM is an important consideration in that patient population. Hormones that antagonize insulin action can also lead to DM. Thus, DM is often a feature of endocrinopathies such as acromegaly and Cushing’s disease. Viral infections have been implicated in pancreatic islet destruction but are an extremely rare cause of DM. A form of acute onset of type 1 diabetes, termed fulminant diabetes, has been noted in Japan and may be related to viral infection of islets.

1	Glucose intolerance developing during pregnancy is classified as gestational diabetes mellitus (GDM). Insulin resistance is related to the metabolic changes of late pregnancy, and the increased insulin requirements may lead to IGT or diabetes. GDM occurs in ~7% (range 1–14%) of pregnancies in the United States; most women revert to normal glucose tolerance postpartum but have a substantial risk (35–60%) of developing DM in the next 10–20 years. The International Association of the Diabetes and Pregnancy Study Groups and the American Diabetes Association (ADA) recommend that diabetes diagnosed at the initial prenatal visit should be classified as “overt” diabetes rather than GDM. With the rising rates of obesity, the number of women being diagnosed with GDM or overt diabetes is rising worldwide.

1	The worldwide prevalence of DM has risen dramatically over the past two decades, from an estimated 30 million cases in 1985 to 382 million in 2013 (Fig. 417-2). Based on current trends, the International Diabetes Federation projects that 592 million individuals will have diabetes by the year 2035 (see http://www.idf .org/). Although the prevalence of both type 1 and type 2 DM is increasing worldwide, the prevalence of type 2 DM is rising much more rapidly, presumably because of increasing obesity, reduced activity levels as countries become more industrialized, and the aging of the population. In 2013, the prevalence of diabetes in individuals from age 20–79 ranged from 23 to 37% in the 10 countries with the highest prevalence (Tuvalu, Federated States of Micronesia, Marshall Islands, Kiribati, Vanuatu, Cook Islands, Saudi Arabia, Nauru, Kuwait, and Qatar, in descending order of prevalence). The countries with the greatest number of individuals with diabetes in 2013 are China (98.4

1	FIGURE 417-2 Worldwide prevalence of diabetes mellitus. Global estimate is 382 million indi-viduals with diabetes. Regional estimates of the number of individuals with diabetes (20–79 years of age) are shown (2013). (Used with permission from the IDF Diabetes Atlas, the International Diabetes Federation, 2013.) million), India (65.1 million), United States (24.4 million), Brazil (11.9 million), and the Russian Federation (10.9 million). Up to 80% of individuals with diabetes live in low-income or medium-income countries. In the most recent estimate for the United States (2012), the Centers for Disease Control and Prevention (CDC) estimated that 9.3% of the population had diabetes (~28% of the individuals with diabetes were undiagnosed; globally, it is estimated that 50% of individuals may be undiagnosed). The CDC estimated that the incidence and prevalence of diabetes doubled from 1990–2008, but appears to have plateaued from 2008–2012. DM increases with age. In 2012, the prevalence

1	be undiagnosed). The CDC estimated that the incidence and prevalence of diabetes doubled from 1990–2008, but appears to have plateaued from 2008–2012. DM increases with age. In 2012, the prevalence of DM in the United Sates was estimated to be 0.2% in individuals age <20 years and 12% in individuals age >20 years. In individuals age >65 years, the prevalence of DM was 26.9%. The prevalence is similar in men and women throughout most age ranges (14% and 11%, respectively, in individuals age >20 years). Worldwide, most individuals with diabetes are between the ages of 40 and 59 years.

1	There is considerable geographic variation in the incidence of both type 1 and type 2 DM. Scandinavia has the highest incidence of type 1 DM; the lowest incidence is in the Pacific Rim where it is 20to 30-fold lower. Northern Europe and the United States have an intermediate rate. Much of the increased risk of type 1 DM is believed to reflect the frequency of high-risk human leukocyte antigen (HLA) alleles among ethnic groups in different geographic locations. The prevalence of type 2 DM and its harbinger, IGT, is highest in certain Pacific islands and the Middle East and intermediate in countries such as India and the United States. This variability is likely due to genetic, behavioral, and environmental factors. DM prevalence also varies among different ethnic populations within a given country, with indigenous populations usually having a greater incidence of diabetes than the general population of the country. For example, the CDC estimated that the age-adjusted prevalence of DM

1	country, with indigenous populations usually having a greater incidence of diabetes than the general population of the country. For example, the CDC estimated that the age-adjusted prevalence of DM in the United States (age >20 years; 2010–2012) was 8% in non-Hispanic whites, 9% in Asian Americans, 13% in Hispanics, 13% in non-Hispanic blacks, and 16% in American-Indian and Alaskan native populations. The onset of type 2 DM occurs, on average, at an earlier age in ethnic groups other than non-Hispanic whites. In Asia, the prevalence of diabetes is increasing rapidly, and the diabetes phenotype appears to be somewhat different from that in the United States and Europe, with an onset at a lower body mass index (BMI) and younger age, greater visceral adiposity, and reduced insulin secretory capacity.

1	Diabetes is a major cause of mortality, but several studies indicate that diabetes is likely underreported as a cause of death. In the United States, diabetes was listed as the seventh leading cause of death in 2010. A recent estimate suggested that diabetes was responsible for almost 5.1 million deaths or 8% of deaths worldwide in 2013. In 2013, it was estimated that $548 billion or 11% of health care expenditures worldwide were spent on individuals with diabetes. CRiTERiA foR THE DiAgnoSiS of DiAbETES mElliTuS • Symptoms of diabetes plus random blood glucose concentration ≥11.1 mmol/L (200 mg/dL)a or Fasting plasma glucose ≥7.0 mmol/L (126 mg/dL)b or

1	CRiTERiA foR THE DiAgnoSiS of DiAbETES mElliTuS • Symptoms of diabetes plus random blood glucose concentration ≥11.1 mmol/L (200 mg/dL)a or Fasting plasma glucose ≥7.0 mmol/L (126 mg/dL)b or Hemoglobin A1c ≥ 6.5%c or 2-h plasma glucose ≥11.1 mmol/L (200 mg/dL) during an oral glucose aRandom is defined as without regard to time since the last meal. bFasting is defined as no caloric intake for at least 8 h. cHemoglobin A1c test should be performed in a laboratory using a method approved by the National Glycohemoglobin Standardization Program and correlated to the reference assay of the Diabetes Control and Complications Trial. Point-of-care hemoglobin A1c should not be used for diagnostic purposes. dThe test should be performed using a glucose load containing the equivalent of 75 g anhydrous glucose dissolved in water, not recommended for routine clinical use.

1	Note: In the absence of unequivocal hyperglycemia and acute metabolic decompensation, these criteria should be confirmed by repeat testing on a different day. Source: Adapted from American Diabetes Association: Diabetes Care 37(Suppl 1):S14, 2014.

1	Glucose tolerance is classified into three broad categories: normal glucose homeostasis, DM, or impaired glucose homeostasis. Glucose tolerance can be assessed using the fasting plasma glucose (FPG), the response to oral glucose challenge, or the hemoglobin A1c (HbA1c). An FPG <5.6 mmol/L (100 mg/dL), a plasma glucose <140 mg/dL (11.1 mmol/L) following an oral glucose challenge, and an HbA1c <5.7% are considered to define normal glucose tolerance. The International Expert Committee with members appointed by the ADA, the European Association for the Study of Diabetes, and the International Diabetes Federation have issued diagnostic criteria for DM (Table 417-2) based on the following premises: (1) the FPG, the response to an oral glucose challenge (oral glucose tolerance test [OGTT]), and HbA1c differ among individuals, and (2) DM is defined as the level of glycemia at which diabetes-specific complications occur rather than on deviations from a population-based mean. For example, the

1	HbA1c differ among individuals, and (2) DM is defined as the level of glycemia at which diabetes-specific complications occur rather than on deviations from a population-based mean. For example, the prevalence of retinopathy in Native Americans (Pima Indian population) begins to increase at an FPG >6.4 mmol/L (116 mg/dL) (Fig. 417-3).

1	An FPG ≥7.0 mmol/L (126 mg/dL), a glucose ≥11.1 mmol/L (200 mg/dL) 2 h after an oral glucose challenge, or an HbA1c ≥6.5% warrants the diagnosis of DM (Table 417-2). A random plasma glucose

1	FIGURE 417-3 Relationship of diabetes-specific complication and glucose tolerance. This figure shows the incidence of retinopathy in Pima Indians as a function of the fasting plasma glucose (FPG), the 2-h plasma glucose after a 75-g oral glucose challenge (2-h PG), or the hemoglobin A1c (HbA1c). Note that the incidence of retinopathy greatly increases at a fasting plasma glucose >116 mg/dL, a 2-h plasma glucose of 185 mg/dL, or an HbA1c >6.5%. (Blood glucose values are shown in mg/dL; to convert to mmol/L, divide value by 18.) (Copyright 2002, American Diabetes Association. From Diabetes Care 25[Suppl 1]: S5–S20, 2002.) 70 89 93 97 100 105 109 116 136 226 38 94 106 116 126 138 156 185 244 364 3.4 4.8 5.0 5.2 5.3 5.5 5.7 6.0 6.7 9.5 concentration ≥11.1 mmol/L (200 mg/dL) accompanied by classic 2401 symptoms of DM (polyuria, polydipsia, weight loss) is also sufficient for the diagnosis of DM (Table 417-2).

1	Abnormal glucose homeostasis (Fig. 417-1) is defined as (1) FPG = 5.6–6.9 mmol/L (100–125 mg/dL), which is defined as impaired fasting glucose (IFG); (2) plasma glucose levels between 7.8 and 11 mmol/L (140 and 199 mg/dL) following an oral glucose challenge, which is termed impaired glucose tolerance (IGT); or (3) HbA1c of 5.7–6.4%. An HbA1c of 5.7–6.4%, IFG, and IGT do not identify the same individuals, but individuals in all three groups are at greater risk of progressing to type 2 DM, have an increased risk of cardiovascular disease, and should be counseled about ways to decrease these risks (see below). Some use the terms prediabetes, increased risk of diabetes, or intermediate hyperglycemia (World Health Organization) for this category. These values for the fasting plasma glucose, the glucose following an oral glucose challenge, and HbA1c are continuous variables and not discrete categories. The current criteria for the diagnosis of DM emphasize the HbA1c or the FPG as the most

1	glucose following an oral glucose challenge, and HbA1c are continuous variables and not discrete categories. The current criteria for the diagnosis of DM emphasize the HbA1c or the FPG as the most reliable and convenient tests for identifying DM in asymptomatic individuals (however, some individuals may meet criteria for one test but not the other). OGTT, although still a valid means for diagnosing DM, is not often used in routine clinical care.

1	The diagnosis of DM has profound implications for an individual from both a medical and a financial standpoint. Thus, abnormalities on screening tests for diabetes should be repeated before making a definitive diagnosis of DM, unless acute metabolic derangements or a markedly elevated plasma glucose are present (Table 417-2). These criteria also allow for the diagnosis of DM to be withdrawn in situations when the glucose intolerance reverts to normal.

1	Widespread use of the FPG or the HbA1c as a screening test for type 2 DM is recommended because (1) a large number of individuals who meet the current criteria for DM are asymptomatic and unaware that they have the disorder, (2) epidemiologic studies suggest that type 2 DM may be present for up to a decade before diagnosis, (3) some individuals with type 2 DM have one or more diabetes-specific complications at the time of their diagnosis, (4) treatment of type 2 DM may favorably alter the natural history of DM, diagnosis of prediabetes should spur efforts for diabetes prevention. The ADA recommends screening all individuals >45 years every 3 years and screening individuals at an earlier age if they are overweight (BMI >25 kg/m2 or ethnically relevant definition for overweight) and have one additional risk factor for diabetes (Table 417-3). In contrast to type 2 DM, a long asymptomatic period of hyperglycemia is rare prior to the diagnosis of type 1 DM. A number of immunologic markers

1	additional risk factor for diabetes (Table 417-3). In contrast to type 2 DM, a long asymptomatic period of hyperglycemia is rare prior to the diagnosis of type 1 DM. A number of immunologic markers for type 1 DM are becoming available (discussed below), but their routine use outside a clinical trial is discouraged, pending the identification of clinically beneficial interventions for individuals at high risk for developing type 1 DM.

1	Family history of diabetes (i.e., parent or sibling with type 2 diabetes) Race/ethnicity (e.g., African American, Latino, Native American, Asian American, Pacific Islander) Previously identified with IFG, IGT, or an hemoglobin A1c of 5.7–6.4% History of GDM or delivery of baby >4 kg (9 lb) HDL cholesterol level <35 mg/dL (0.90 mmol/L) and/or a triglyceride level >250 mg/dL (2.82 mmol/L) History of cardiovascular disease Abbreviations: BMI, body mass index; GDM, gestational diabetes mellitus; HDL, high-density lipoprotein; IFG, impaired fasting glucose; IGT, impaired glucose tolerance. Source: Adapted from American Diabetes Association: Diabetes Care 37(Suppl 1):S14, 2014. Diabetes Mellitus: Diagnosis, Classification, and Pathophysiology

1	Glucose homeostasis reflects a balance between hepatic glucose production and peripheral glucose uptake and utilization. Insulin is the most important regulator of this metabolic equilibrium, but neural input, metabolic signals, and other hormones (e.g., glucagon) result in integrated control of glucose supply and utilization (Fig. 417-4). The organs that regulate glucose and lipids communicate by neural and humoral mechanisms with fat and muscle producing adipokines, myokines, and metabolites that influence liver function. In the fasting state, low insulin levels increase glucose production by promoting hepatic gluconeogenesis and glycogenolysis and reduce glucose uptake in insulin-sensitive tissues (skeletal muscle and fat), thereby promoting mobilization of stored precursors such as amino acids and free fatty acids (lipolysis). Glucagon, secreted by pancreatic alpha cells when blood glucose or insulin levels are low, stimulates glycogenolysis and gluconeogenesis by the liver and

1	amino acids and free fatty acids (lipolysis). Glucagon, secreted by pancreatic alpha cells when blood glucose or insulin levels are low, stimulates glycogenolysis and gluconeogenesis by the liver and renal medulla (Chap. 420). Postprandially, the glucose load elicits a rise in insulin and fall in glucagon, leading to a reversal of these processes. Insulin, an anabolic hormone, promotes the storage of carbohydrate and fat and protein synthesis. The major portion of postprandial glucose is used by skeletal muscle, an effect of insulin-stimulated glucose uptake. Other tissues, most notably the brain, use glucose in an insulin-independent fashion. Factors secreted by skeletal myocytes (irisin), adipocytes (leptin, resistin, adiponectin, etc.), and bone also influence glucose homeostasis.

1	Insulin is produced in the beta cells of the pancreatic islets. It is initially synthesized as a single-chain 86-amino-acid precursor polypeptide, preproinsulin. Subsequent proteolytic processing removes the amino-terminal signal peptide, giving rise to proinsulin. Proinsulin is structurally related to insulin-like growth factors I and II, which bind weakly to the insulin receptor. Cleavage of an internal 31-residue fragment from proinsulin generates the C peptide and the A (21 amino acids) and B (30 amino acids) chains of insulin, which are connected by disulfide bonds. The mature insulin molecule and C peptide are stored together and co-secreted from secretory granules in the beta cells. Because C peptide is cleared more slowly than insulin, it is a useful marker of insulin secretion and allows discrimination of endogenous and exogenous sources of insulin in the evaluation of hypoglycemia (Chaps. 420 and 113). Pancreatic beta cells co-secrete islet amyloid polypeptide (IAPP) or

1	and allows discrimination of endogenous and exogenous sources of insulin in the evaluation of hypoglycemia (Chaps. 420 and 113). Pancreatic beta cells co-secrete islet amyloid polypeptide (IAPP) or amylin, a 37-amino-acid peptide, along with insulin. The role of IAPP in normal physiology is incompletely defined, but it is the major component of the amyloid fibrils found in the islets of patients with type 2 diabetes, and an analogue

1	FIGURE 417-4 Regulation of glucose homeostasis. The organs shown contribute to glucose utilization, production, or storage. See text for a description of the communications (arrows), which can be neural or humoral. is sometimes used in treating type 1 and type 2 DM. Human insulin is produced by recombinant DNA technology; structural alterations at one or more amino acid residues modify its physical and pharmacologic characteristics (Chap. 418).

1	Glucose is the key regulator of insulin secretion by the pancreatic beta cell, although amino acids, ketones, various nutrients, gastrointestinal peptides, and neurotransmitters also influence insulin secretion. Glucose levels >3.9 mmol/L (70 mg/dL) stimulate insulin synthesis, primarily by enhancing protein translation and processing. Glucose stimulation of insulin secretion begins with its transport into the beta cell by a facilitative glucose transporter (Fig. 417-5). Glucose phosphorylation by glucokinase is the rate-limiting step that controls glucose-regulated insulin secretion. Further metabolism of glucose-6-phosphate via glycolysis generates ATP, which inhibits the activity of an ATP-sensitive K+ channel. This channel consists of two separate proteins: one is the binding site for certain oral hypoglycemics (e.g., sulfonylureas, meglitinides); the other is an inwardly rectifying K+ channel protein (Kir6.2). Inhibition of this K+ channel induces beta cell membrane

1	site for certain oral hypoglycemics (e.g., sulfonylureas, meglitinides); the other is an inwardly rectifying K+ channel protein (Kir6.2). Inhibition of this K+ channel induces beta cell membrane depolarization, which opens voltage-dependent calcium channels (leading to an influx of calcium) and stimulates insulin secretion. Insulin secretory profiles reveal a pulsatile pattern of hormone release, with small secretory bursts occurring about every 10 min, superimposed upon greater amplitude oscillations of about 80–150 min. Incretins are released from neuroendocrine cells of the gastrointestinal tract following food ingestion and amplify glucose-stimulated insulin secretion and suppress glucagon secretion. Glucagon-like peptide 1 (GLP-1), the most potent incretin, is released from L cells in the small intestine and stimulates insulin secretion only when the blood glucose is above the fasting level. Incretin analogues or pharmacologic agents that prolong the activity of endogenous GLP-1

1	the small intestine and stimulates insulin secretion only when the blood glucose is above the fasting level. Incretin analogues or pharmacologic agents that prolong the activity of endogenous GLP-1 enhance insulin secretion.

1	Once insulin is secreted into the portal venous system, ~50% is removed and degraded by the liver. Unextracted insulin enters the FIGURE 417-5 Mechanisms of glucose-stimulated insulin secretion and abnormalities in diabetes. Glucose and other nutrients regulate insulin secretion by the pancreatic beta cell. Glucose is transported by a glucose transporter (GLUT1 and/or GLUT2 in humans, GLUT2 in rodents); subsequent glucose metabolism by the beta cell alters ion channel activity, leading to insulin secretion. The SUR receptor is the binding site for some drugs that act as insulin secretagogues. Mutations in the events or proteins underlined are a cause of monogenic forms of diabetes. ADP, adenosine diphosphate; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; IAPP, islet amyloid polypeptide or amylin; SUR, sulfonylurea receptor.

1	systemic circulation where it binds to receptors in target sites. Insulin binding to its receptor stimulates intrinsic tyrosine kinase activity, leading to receptor autophosphorylation and the recruitment of intracellular signaling molecules, such as insulin receptor substrates (IRS). IRS and other adaptor proteins initiate a complex cascade of phosphorylation and dephosphorylation reactions, resulting in the widespread metabolic and mitogenic effects of insulin. As an example, activation of the phosphatidylinositol-3′-kinase (PI-3-kinase) pathway stimulates translocation of a facilitative glucose transporter (e.g., GLUT4) to the cell surface, an event that is crucial for glucose uptake by skeletal muscle and fat. Activation of other insulin receptor signaling pathways induces glycogen synthesis, protein synthesis, lipogenesis, and regulation of various genes in insulin-responsive cells.

1	Type 1 DM is the result of interactions of genetic, environmental, and immunologic factors that ultimately lead to the destruction of the pancreatic beta cells and insulin deficiency. Type 1 DM, which can develop at any age, develops most commonly before 20 years of age. Worldwide, the incidence of type 1 DM is increasing at the rate of 3–4% per year for uncertain reasons. Type 1 DM results from autoimmune beta cell destruction, and most, but not all, individuals have evidence of islet-directed autoimmunity. Some individuals who have the clinical phenotype of type 1 DM lack immunologic markers indicative of an autoimmune process involving the beta cells and the genetic markers of type 1 DM. These individuals are thought to develop insulin deficiency by unknown, nonimmune mechanisms and may be ketosis prone; many are African American or Asian in heritage. The temporal development of type 1 DM is shown schematically as a function of beta cell mass in Fig. 417-6. Individuals with a

1	may be ketosis prone; many are African American or Asian in heritage. The temporal development of type 1 DM is shown schematically as a function of beta cell mass in Fig. 417-6. Individuals with a genetic susceptibility are thought to have normal beta cell mass at birth but begin to lose beta cells secondary to autoimmune destruction that occurs over months to years. This autoimmune process is thought to be triggered by an infectious or environmental stimulus and to be sustained by a beta cell–specific molecule. In the majority of patients, immunologic

1	FIGURE 417-6 Temporal model for development of type 1 diabetes. Individuals with a genetic predisposition are exposed to a trigger that initiates an autoimmune process, resulting in a gradual decline in beta cell mass. The downward slope of the beta cell mass varies among individuals and may not be continuous. This progressive impairment in insulin release results in diabetes when ~80% of the beta cell mass is destroyed. A “honeymoon” phase may be seen in the first 1 or 2 years after the onset of diabetes and is associated with reduced insulin requirements. (Adapted from ER Kaufman: Medical Management of Type 1 Diabetes, 6th ed. American Diabetes Association, Alexandria, VA, 2012.) markers appear after the triggering event but before diabetes becomes 2403 clinically overt. Beta cell mass then begins to decrease, and insulin secretion progressively declines, although normal glucose tolerance is maintained. The rate of decline in beta cell mass varies widely among individuals, with some

1	begins to decrease, and insulin secretion progressively declines, although normal glucose tolerance is maintained. The rate of decline in beta cell mass varies widely among individuals, with some patients progressing rapidly to clinical diabetes and others evolving more slowly. Features of diabetes do not become evident until a majority of beta cells are destroyed (70–80%). At this point, residual functional beta cells exist but are insufficient in number to maintain glucose tolerance. The events that trigger the transition from glucose intolerance to frank diabetes are often associated with increased insulin requirements, as might occur during infections or puberty. After the initial clinical presentation of type 1 DM, a “honeymoon” phase may ensue during which time glycemic control is achieved with modest doses of insulin or, rarely, insulin is not needed. However, this fleeting phase of endogenous insulin production from residual beta cells disappears and the individual becomes

1	achieved with modest doses of insulin or, rarely, insulin is not needed. However, this fleeting phase of endogenous insulin production from residual beta cells disappears and the individual becomes insulin deficient. Many individuals with long-standing type 1 DM produce a small amount of insulin (as reflected by C-peptide production), and some individuals with more than 50 years of type 1 DM have insulin-positive cells in the pancreas at autopsy.

1	Susceptibility to type 1 DM involves multiple genes. The concor dance of type 1 DM in identical twins ranges between 40 and 60%, indicating that additional modifying factors are likely involved in determining whether diabetes develops. The major susceptibility gene for type 1 DM is located in the HLA region on chromosome 6. Polymorphisms in the HLA complex account for 40–50% of the genetic risk of developing type 1 DM. This region contains genes that encode the class II major histocompatibility complex (MHC) molecules, which present antigen to helper T cells and thus are involved in initiating the immune response (Chap. 373e). The ability of class II MHC molecules to present antigen is dependent on the amino acid composition of their antigen-binding sites. Amino acid substitutions may influence the specificity of the immune response by altering the binding affinity of different antigens for class II molecules.

1	Most individuals with type 1 DM have the HLA DR3 and/or DR4 haplotype. Refinements in genotyping of HLA loci have shown that the haplotypes DQA1*0301, DQB1*0302, and DQB1*0201 are most strongly associated with type 1 DM. These haplotypes are present in 40% of children with type 1 DM as compared to 2% of the normal U.S. population. However, most individuals with predisposing haplotypes do not develop diabetes. In addition to MHC class II associations, genome association studies have identified at least 20 different genetic loci that contribute susceptibility to type 1 DM (polymorphisms in the promoter region of the insulin gene, the CTLA-4 gene, interleukin 2 receptor, CTLA4, and PTPN22, etc.). Genes that confer protection against the development of the disease also exist. The haplotype DQA1*0102, DQB1*0602 is extremely rare in individuals with type 1 DM (<1%) and appears to provide protection from type 1 DM.

1	Although the risk of developing type 1 DM is increased tenfold in relatives of individuals with the disease, the risk is relatively low: 3–4% if the parent has type 1 DM and 5–15% in a sibling (depending on which HLA haplotypes are shared). Hence, most individuals with type 1 DM do not have a first-degree relative with this disorder.

1	Pathophysiology Although other islet cell types (alpha cells [glucagonproducing], delta cells [somatostatin-producing], or PP cells [pancreatic polypeptide-producing]) are functionally and embryologically similar to beta cells and express most of the same proteins as beta cells, they are spared from the autoimmune destruction. Pathologically, the pancreatic islets have a modest infiltration of lymphocytes (a process termed insulitis). After beta cells are destroyed, it is thought that the inflammatory process abates and the islets become atrophic. Studies of the autoimmune process in humans and in animal models of type 1 DM (NOD mouse and BB rat) have identified the following abnormalities in the humoral and cellular arms of the immune system: (1) islet cell autoantibodies; (2) activated lymphocytes in the islets, Beta cell mass (% of max)

1	Beta cell mass (% of max) Diabetes Mellitus: Diagnosis, Classification, and Pathophysiology 2404 peripancreatic lymph nodes, and systemic circulation; (3) T lymphocytes that proliferate when stimulated with islet proteins; and (4) release of cytokines within the insulitis. Beta cells seem to be particularly susceptible to the toxic effect of some cytokines (tumor necrosis factor α [TNF-α], interferon γ, and interleukin 1 [IL-1]). The precise mechanisms of beta cell death are not known but may involve formation of nitric oxide metabolites, apoptosis, and direct CD8+ T cell cytotoxicity. The islet destruction is mediated by T lymphocytes rather than islet autoantibodies, as these antibodies do not generally react with the cell surface of islet cells and are not capable of transferring DM to animals. Efforts to suppress the autoimmune process at the time of diagnosis of diabetes have largely been ineffective or only temporarily effective in slowing beta cell destruction.

1	Pancreatic islet molecules targeted by the autoimmune process include insulin, glutamic acid decarboxylase (GAD; the biosynthetic enzyme for the neurotransmitter GABA), ICA-512/IA-2 (homology with tyrosine phosphatases), and a beta cell–specific zinc transporter (ZnT-8). Most of the autoantigens are not beta cell–specific, which raises the question of how the beta cells are selectively destroyed. Current theories favor initiation of an autoimmune process directed at one beta cell molecule, which then spreads to other islet molecules as the immune process destroys beta cells and creates a series of secondary autoantigens. The beta cells of individuals who develop type 1 DM do not differ from beta cells of normal individuals because islets transplanted from a genetically identical twin are destroyed by a recurrence of the autoimmune process of type 1 DM.

1	Immunologic Markers Islet cell autoantibodies (ICAs) are a composite of several different antibodies directed at pancreatic islet molecules such as GAD, insulin, IA-2/ICA-512, and ZnT-8, and serve as a marker of the autoimmune process of type 1 DM. Assays for autoantibodies to GAD-65 are commercially available. Testing for ICAs can be useful in classifying the type of DM as type 1 and in identifying nondiabetic individuals at risk for developing type 1 DM. ICAs are present in the majority of individuals (>85%) diagnosed with new-onset type 1 DM, in a significant minority of individuals with newly diagnosed type 2 DM (5–10%), and occasionally in individuals with GDM (<5%). ICAs are present in 3–4% of first-degree relatives of individuals with type 1 DM. In combination with impaired insulin secretion after IV glucose tolerance testing, they predict a >50% risk of developing type 1 DM within 5 years. At present, the measurement of ICAs in nondiabetic individuals is a research tool

1	secretion after IV glucose tolerance testing, they predict a >50% risk of developing type 1 DM within 5 years. At present, the measurement of ICAs in nondiabetic individuals is a research tool because no treatments have been demonstrated to prevent the occurrence or progression to type 1 DM.

1	Environmental Factors Numerous environmental events have been proposed to trigger the autoimmune process in genetically susceptible individuals; however, none have been conclusively linked to diabetes. Identification of an environmental trigger has been difficult because the event may precede the onset of DM by several years (Fig. 417-6). Putative environmental triggers include viruses (coxsackie, rubella, enteroviruses most prominently), bovine milk proteins, and nitrosourea compounds. There is increasing interest in the microbiome and type 1 diabetes (Chap. 86e). Prevention of Type 1 DM A number of interventions have prevented diabetes in animal models. None of these interventions have been successful in preventing type 1 DM in humans. For example, the Diabetes Prevention Trial–Type 1 concluded that administering insulin (IV or PO) to individuals at high risk for developing type 1 DM did not prevent type 1 DM. This is an area of active clinical investigation.

1	Insulin resistance and abnormal insulin secretion are central to the development of type 2 DM. Although the primary defect is controversial, most studies support the view that insulin resistance precedes an insulin secretory defect but that diabetes develops only when insulin secretion becomes inadequate. Type 2 DM likely encompasses a range of disorders with common phenotype of hyperglycemia. Most of our current understanding (and the discussion below) of the pathophysiology and genetics is based on studies of individuals of European descent. It is becoming increasing apparent that DM in other ethnic groups (Asian, African, and Latin American) has a somewhat different, but yet undefined, pathophysiology. In general, Latinos have greater insulin resistance and East Asians and South Asians have more beta cell dysfunction, but both defects are present in both populations. East and South Asians appear to develop type 2 DM at a younger age and a lower BMI. In some groups, DM that is

1	have more beta cell dysfunction, but both defects are present in both populations. East and South Asians appear to develop type 2 DM at a younger age and a lower BMI. In some groups, DM that is ketosis prone (often obese) or ketosis-resistant (often lean) is seen.

1	Type 2 DM has a strong genetic component. The concordance of type 2 DM in identical twins is between 70 and 90%. Individuals with a parent with type 2 DM have an increased risk of diabetes; if both parents have type 2 DM, the risk approaches 40%. Insulin resistance, as demonstrated by reduced glucose utilization in skeletal muscle, is present in many nondiabetic, first-degree relatives of individuals with type 2 DM. The disease is polygenic and multifactorial, because in addition to genetic susceptibility, environmental factors (such as obesity, nutrition, and physical activity) modulate the phenotype. The in utero environment also contributes, and either increased or reduced birth weight increases the risk of type 2 DM in adult life. The genes that predispose to type 2 DM are incompletely identified, but recent genome-wide association studies have identified a large number of genes that convey a relatively small risk for type 2 DM (>70 genes, each with a relative risk of 1.06–1.5).

1	identified, but recent genome-wide association studies have identified a large number of genes that convey a relatively small risk for type 2 DM (>70 genes, each with a relative risk of 1.06–1.5). Most prominent is a variant of the transcription factor 7–like 2 gene that has been associated with type 2 DM in several populations and with IGT in one population at high risk for diabetes. Genetic polymorphisms associated with type 2 DM have also been found in the genes encoding the peroxisome proliferator– activated receptor γ, inward rectifying potassium channel, zinc transporter, IRS, and calpain 10. The mechanisms by which these genetic loci increase the susceptibility to type 2 DM are not clear, but most are predicted to alter islet function or development or insulin secretion. Although the genetic susceptibility to type 2 DM is under active investigation (it is estimated that <10% of genetic risk is determined by loci identified thus far), it is currently not possible to use a

1	the genetic susceptibility to type 2 DM is under active investigation (it is estimated that <10% of genetic risk is determined by loci identified thus far), it is currently not possible to use a combination of known genetic loci to predict type 2 DM.

1	Pathophysiology Type 2 DM is characterized by impaired insulin secretion, insulin resistance, excessive hepatic glucose production, and abnormal fat metabolism. Obesity, particularly visceral or central (as evidenced by the hip-waist ratio), is very common in type 2 DM (≥80% of patients are obese). In the early stages of the disorder, glucose tolerance remains near-normal, despite insulin resistance, because the pancreatic beta cells compensate by increasing insulin output (Fig. 417-7). As insulin resistance and compensatory hyperinsulinemia progress, the pancreatic islets in certain individuals are unable to sustain the hyperinsulinemic state. IGT, characterized by elevations in postprandial glucose, then develops. A further decline in insulin secretion and an increase in hepatic glucose production lead to overt diabetes with fasting hyperglycemia. Ultimately, beta cell failure ensues. Although both insulin resistance and impaired insulin secretion contribute to the pathogenesis of

1	production lead to overt diabetes with fasting hyperglycemia. Ultimately, beta cell failure ensues. Although both insulin resistance and impaired insulin secretion contribute to the pathogenesis of type 2 DM, the relative contribution of each varies from individual to individual.

1	Metabolic Abnormalities • abnormal muscle and faT meTabolism Insulin resistance, the decreased ability of insulin to act effectively on target tissues (especially muscle, liver, and fat), is a prominent feature of type 2 DM and results from a combination of genetic susceptibility and obesity. Insulin resistance is relative, however, because supranormal levels of circulating insulin will normalize the plasma glucose. Insulin dose-response curves exhibit a rightward shift, indicating reduced sensitivity, and a reduced maximal response, indicating an overall decrease in maximum glucose utilization (30–60% lower than in normal individuals). Insulin resistance impairs glucose utilization by insulin-sensitive tissues and increases hepatic glucose output; both effects contribute to the hyperglycemia. Increased hepatic glucose output predominantly accounts for increased FPG levels, whereas decreased peripheral glucose usage results in postprandial hyperglycemia. In skeletal muscle, there is a

1	Increased hepatic glucose output predominantly accounts for increased FPG levels, whereas decreased peripheral glucose usage results in postprandial hyperglycemia. In skeletal muscle, there is a greater impairment in nonoxidative

1	FIGURE 417-7 Metabolic changes during the development of type 2 diabetes mellitus (DM). Insulin secretion and insulin sensitivity are related, and as an individual becomes more insulin resistant (by moving from point A to point B), insulin secretion increases. A failure to compensate by increasing the insulin secretion results initially in impaired glucose tolerance (IGT; point C) and ultimately in type 2 DM (point D). NGT, normal glucose tolerance. (Adapted from SE Kahn: J Clin Endocrinol Metab 86:4047, 2001; RN Bergman, M Ader: Trends Endocrinol Metab 11:351, 2000.) glucose usage (glycogen formation) than in oxidative glucose metabolism through glycolysis. Glucose metabolism in insulin-independent tissues is not altered in type 2 DM.

1	The precise molecular mechanism leading to insulin resistance in type 2 DM has not been elucidated. Insulin receptor levels and tyrosine kinase activity in skeletal muscle are reduced, but these alterations are most likely secondary to hyperinsulinemia and are not a primary defect. Therefore, “postreceptor” defects in insulin-regulated phosphorylation/dephosphorylation appear to play the predominant role in insulin resistance. Abnormalities include the accumulation of lipid within skeletal myocytes, which may impair mitochondrial oxidative phosphorylation and reduce insulin-stimulated mitochondrial ATP production. Impaired fatty acid oxidation and lipid accumulation within skeletal myocytes also may generate reactive oxygen species such as lipid peroxides. Of note, not all insulin signal transduction pathways are resistant to the effects of insulin (e.g., those controlling cell growth and differentiation using the mitogenic-activated protein kinase pathway). Consequently,

1	signal transduction pathways are resistant to the effects of insulin (e.g., those controlling cell growth and differentiation using the mitogenic-activated protein kinase pathway). Consequently, hyperinsulinemia may increase the insulin action through these pathways, potentially accelerating diabetes-related conditions such as atherosclerosis.

1	The obesity accompanying type 2 DM, particularly in a central or visceral location, is thought to be part of the pathogenic process (Chap. 415e). In addition to these white fat depots, humans now are recognized to have brown fat, which has much greater thermogenic capacity. Efforts are under way to increase the activity or quantity of brown fat (e.g., a myokine, irisin, may convert white to brown fat). The increased adipocyte mass leads to increased levels of circulating free fatty acids and other fat cell products. For example, adipocytes secrete a number of biologic products (nonesterified free fatty acids, retinolbinding protein 4, leptin, TNF-α, resistin, IL-6, and adiponectin). In addition to regulating body weight, appetite, and energy expenditure, adipokines also modulate insulin sensitivity. The increased production of free fatty acids and some adipokines may cause insulin resistance in skeletal muscle and liver. For example, free fatty acids impair glucose utilization in

1	sensitivity. The increased production of free fatty acids and some adipokines may cause insulin resistance in skeletal muscle and liver. For example, free fatty acids impair glucose utilization in skeletal muscle, promote glucose production by the liver, and impair beta cell function. In contrast, the production by adipocytes of adiponectin, an insulin-sensitizing peptide, is reduced in obesity, and this may contribute to hepatic insulin resistance. Adipocyte products and adipokines also produce an inflammatory state and may explain why markers of inflammation such as IL-6 and C-reactive protein are often elevated in type 2 DM. In addition, inflammatory cells have been found infiltrating adipose tissue. Inhibition of inflammatory 2405 signaling pathways such as the nuclear factor-κB (NF-κB) pathway appears to reduce insulin resistance and improve hyperglycemia in animal models and is being tested in humans.

1	impaired inSulin Secretion Insulin secretion and sensitivity are interrelated (Fig. 417-7). In type 2 DM, insulin secretion initially increases in response to insulin resistance to maintain normal glucose tolerance. Initially, the insulin secretory defect is mild and selectively involves glucose-stimulated insulin secretion, including a greatly reduced first secretory phase. The response to other nonglucose secretagogues, such as arginine, is preserved, but overall beta function is reduced by as much as 50% at the onset of type 2 DM. Abnormalities in proinsulin processing are reflected by increased secretion of proinsulin in type 2 DM. Eventually, the insulin secretory defect is progressive.

1	The reason(s) for the decline in insulin secretory capacity in type 2 DM is unclear. The assumption is that a second genetic defect— superimposed upon insulin resistance—leads to beta cell failure. Beta cell mass is decreased by approximately 50% in individuals with longstanding type 2 DM. Islet amyloid polypeptide or amylin, co-secreted by the beta cell, forms the amyloid fibrillar deposit found in the islets of individuals with long-standing type 2 DM. Whether such islet amyloid deposits are a primary or secondary event is not known. The metabolic environment of diabetes may also negatively impact islet function. For example, chronic hyperglycemia paradoxically impairs islet function (“glucose toxicity”) and leads to a worsening of hyperglycemia. Improvement in glycemic control is often associated with improved islet function. In addition, elevation of free fatty acid levels (“lipotoxicity”) and dietary fat may also worsen islet function. Reduced GLP-1 action may contribute to the

1	associated with improved islet function. In addition, elevation of free fatty acid levels (“lipotoxicity”) and dietary fat may also worsen islet function. Reduced GLP-1 action may contribute to the reduced insulin secretion.

1	increaSed Hepatic GlucoSe and lipid production In type 2 DM, insulin resistance in the liver reflects the failure of hyperinsulinemia to suppress gluconeogenesis, which results in fasting hyperglycemia and decreased glycogen storage by the liver in the postprandial state. Increased hepatic glucose production occurs early in the course of diabetes, although likely after the onset of insulin secretory abnormalities and insulin resistance in skeletal muscle. As a result of insulin resistance in adipose tissue, lipolysis and free fatty acid flux from adipocytes are increased, leading to increased lipid (very-low-density lipoprotein [VLDL] and triglyceride) synthesis in hepatocytes. This lipid storage or steatosis in the liver may lead to nonalcoholic fatty liver disease (Chap. 367e) and abnormal liver function tests. This is also responsible for the dyslipidemia found in type 2 DM (elevated triglycerides, reduced high-density lipoprotein [HDL], and increased small dense low-density

1	abnormal liver function tests. This is also responsible for the dyslipidemia found in type 2 DM (elevated triglycerides, reduced high-density lipoprotein [HDL], and increased small dense low-density lipoprotein [LDL] particles).

1	Insulin Resistance Syndromes The insulin resistance condition comprises a spectrum of disorders, with hyperglycemia representing one of the most readily diagnosed features. The metabolic syndrome, the insulin resistance syndrome, and syndrome X are terms used to describe a constellation of metabolic derangements that includes insulin resistance, hypertension, dyslipidemia (decreased HDL and elevated triglycerides), central or visceral obesity, type 2 DM or IGT/IFG, and accelerated cardiovascular disease. This syndrome is discussed in Chap. 422.

1	A number of relatively rare forms of severe insulin resistance include features of type 2 DM or IGT (Table 417-1). Mutations in the insulin receptor that interfere with binding or signal transduction are a rare cause of insulin resistance. Acanthosis nigricans and signs of hyperandrogenism (hirsutism, acne, and oligomenorrhea in women) are also common physical features. Two distinct syndromes of severe insulin resistance have been described in adults: (1) type A, which affects young women and is characterized by severe hyperinsulinemia, obesity, and features of hyperandrogenism; and (2) type B, which affects middle-aged women and is characterized by severe hyperinsulinemia, features of hyperandrogenism, and autoimmune disorders. Individuals with the type A insulin resistance syndrome have an undefined defect in the insulin-signaling pathway; individuals with the type B insulin resistance syndrome have autoantibodies directed at

1	Diabetes Mellitus: Diagnosis, Classification, and Pathophysiology 2406 the insulin receptor. These receptor autoantibodies may block insulin binding or may stimulate the insulin receptor, leading to intermittent hypoglycemia. Polycystic ovary syndrome (PCOS) is a common disorder that affects premenopausal women and is characterized by chronic anovulation and hyperandrogenism (Chap. 412). Insulin resistance is seen in a significant subset of women with PCOS, and the disorder substantially increases the risk for type 2 DM, independent of the effects of obesity.

1	Prevention Type 2 DM is preceded by a period of IGT or IFG, and a number of lifestyle modifications and pharmacologic agents prevent or delay the onset of DM. Individuals with prediabetes or increased risk of diabetes should be referred to a structured program to reduce body weight and increase physical activity as well as being screened for cardiovascular disease. The Diabetes Prevention Program (DPP) demonstrated that intensive changes in lifestyle (diet and exercise for 30 min/d five times/week) in individuals with IGT prevented or delayed the development of type 2 DM by 58% compared to placebo. This effect was seen in individuals regardless of age, sex, or ethnic group. In the same study, metformin prevented or delayed diabetes by 31% compared to placebo. The lifestyle intervention group lost 5–7% of their body weight during the 3 years of the study. Studies in Finnish and Chinese populations noted similar efficacy of diet and exercise in preventing or delaying type 2 DM. A number

1	lost 5–7% of their body weight during the 3 years of the study. Studies in Finnish and Chinese populations noted similar efficacy of diet and exercise in preventing or delaying type 2 DM. A number of agents, including α-glucosidase inhibitors, metformin, thiazolidinediones, GLP-1 receptor pathway modifiers, and orlistat, prevent or delay type 2 DM but are not approved for this purpose. Individuals with a strong family history of type 2 DM and individuals with IFG or IGT should be strongly encouraged to maintain a normal BMI and engage in regular physical activity. Pharmacologic therapy for individuals with prediabetes is currently controversial because its cost-effectiveness and safety profile are not known. The ADA has suggested that metformin be considered in individuals with both IFG and IGT who are at very high risk for progression to diabetes (age <60 years, BMI ≥35 kg/m2, family history of diabetes in first-degree relative, and women with a history of GDM). Individuals with IFG,

1	IGT who are at very high risk for progression to diabetes (age <60 years, BMI ≥35 kg/m2, family history of diabetes in first-degree relative, and women with a history of GDM). Individuals with IFG, IGT, or an HbA1c of 5.7–6.4% should be monitored annually to determine if diagnostic criteria for diabetes are present.

1	GENETICALLY DEFINED, MONOGENIC FORMS OF DIABETES MELLITUS RELATED TO REDUCED INSULIN SECRETION

1	Several monogenic forms of DM have been identified. More than 10 different variants of MODY, caused by mutations in genes encoding islet-enriched transcription factors or glucokinase (Fig. 417-5; Table 417-1), are transmitted as autosomal dominant disorders. MODY 1, MODY 3, and MODY 5 are caused by mutations in hepatocyte nuclear transcription factor (HNF) 4α, HNF-1α, and HNF-1β, respectively. As their names imply, these transcription factors are expressed in the liver but also in other tissues, including the pancreatic islets and kidney. These factors most likely affect islet development or the expression of genes important in glucose-stimulated insulin secretion or the maintenance of beta cell mass. For example, individuals with an HNF-1α mutation (MODY 3) have a progressive decline in glycemic control but may respond to sulfonylureas. In fact, some of these patients were initially thought to have type 1 DM but were later shown to respond to a sulfonylurea, and insulin was

1	in glycemic control but may respond to sulfonylureas. In fact, some of these patients were initially thought to have type 1 DM but were later shown to respond to a sulfonylurea, and insulin was discontinued. Individuals with a HNF-1β mutation have progressive impairment of insulin secretion and hepatic insulin resistance, and require insulin treatment (minimal response to sulfonylureas). These individuals often have other abnormalities such as renal cysts, mild pancreatic exocrine insufficiency, and abnormal liver function tests. Individuals with MODY 2, the result of mutations in the glucokinase gene, have mild-to-moderate, stable hyperglycemia that does not respond to oral hypoglycemic agents. Glucokinase catalyzes the formation of glucose-6-phosphate from glucose, a reaction that is important for glucose sensing by the beta cells (Fig. 417-5) and for glucose utilization by the liver. As a result of glucokinase mutations, higher glucose levels are required to elicit insulin

1	is important for glucose sensing by the beta cells (Fig. 417-5) and for glucose utilization by the liver. As a result of glucokinase mutations, higher glucose levels are required to elicit insulin secretory responses, thus altering the set point for insulin secretion. Studies of populations with type 2 DM suggest that mutations in MODY-associated genes are an uncommon (<5%) cause of type 2 DM. Mutations in mitochondrial DNA are associated with diabetes and deafness.

1	Transient or permanent neonatal diabetes (onset <12 months of age) occurs. Permanent neonatal diabetes may be caused by several genetic mutations, usually requires treatment with insulin, and phenotypically is similar to type 1 DM. Mutations in the ATP-sensitive potassium channel subunits (Kir6.2 and ABCC8) and the insulin gene (interfere with proinsulin folding and processing) (Fig. 417-5) are the major causes of permanent neonatal diabetes. Although these activating mutations in the ATP-sensitive potassium channel subunits impair glucose-stimulated insulin secretion, these individuals may respond to sulfonylureas and can be treated with these agents. These mutations are often associated with a spectrum of neurologic dysfunction. MODY 4 is a rare variant caused by mutations in the insulin promoter factor (IPF) 1, a transcription factor that regulates pancreatic development and insulin gene transcription. Homozygous inactivating mutations cause pancreatic agenesis, whereas

1	the insulin promoter factor (IPF) 1, a transcription factor that regulates pancreatic development and insulin gene transcription. Homozygous inactivating mutations cause pancreatic agenesis, whereas heterozygous mutations may result in DM. Mutations in the transcription factor of GATA6 are the most common cause of pancreatic agenesis. Homozygous glucokinase mutations cause a severe form of neonatal diabetes.

1	APPROACH TO THE PATIENT: Once the diagnosis of DM is made, attention should be directed to symptoms related to diabetes (acute and chronic) and classifying the type of diabetes. DM and its complications produce a wide range of symptoms and signs; those secondary to acute hyperglycemia may occur at any stage of the disease, whereas those related to chronic hyperglycemia begin to appear during the second decade of hyperglycemia (Chap. 419). Individuals with previously undetected type 2 DM may present with chronic complications of DM at the time of diagnosis. The history and physical examination should assess for symptoms or signs of acute hyperglycemia and should screen for the chronic complications and conditions associated with DM.

1	A complete medical history should be obtained with special emphasis on DM-relevant aspects such as weight, family history of DM and its complications, risk factors for cardiovascular disease, exercise, smoking, and ethanol use. Symptoms of hyperglycemia include polyuria, polydipsia, weight loss, fatigue, weakness, blurry vision, frequent superficial infections (vaginitis, fungal skin infections), and slow healing of skin lesions after minor trauma. Metabolic derangements relate mostly to hyperglycemia (osmotic diuresis) and to the catabolic state of the patient (urinary loss of glucose and calories, muscle breakdown due to protein degradation and decreased protein synthesis). Blurred vision results from changes in the water content of the lens and resolves as the hyperglycemia is controlled.

1	In a patient with established DM, the initial assessment should also include special emphasis on prior diabetes care, including the type of therapy, prior HbA1c levels, self-monitoring blood glucose results, frequency of hypoglycemia, presence of DM-specific complications, and assessment of the patient’s knowledge about diabetes, exercise, and nutrition. Diabetes-related complications may afflict several organ systems, and an individual patient may exhibit some, all, or none of the symptoms related to the complications of DM (Chap. 419). In addition, the presence of DM-related comorbidities should be sought (cardiovascular disease, hypertension, dyslipidemia). Pregnancy plans should be ascertained in women of childbearing age.

1	In addition to a complete physical examination, special attention should be given to DM-relevant aspects such as weight or BMI, retinal examination, orthostatic blood pressure, foot examination, peripheral pulses, and insulin injection sites. Blood pressure >140/80 mmHg is considered hypertension in individuals with diabetes. Because periodontal disease is more frequent in DM, the teeth and gums should also be examined.

1	An annual foot examination should (1) assess blood flow, sensation (vibratory sensation [128-MHz tuning fork at the base of the great toe], the ability to sense touch with a monofilament [5.07, 10-g monofilament], pinprick sensation, testing for ankle reflexes, and vibration perception threshold using a biothesiometer), ankle reflexes, and nail care; (2) look for the presence of foot deformities such as hammer or claw toes and Charcot foot; and (3) identify sites of potential ulceration. The ADA recommends annual screening for distal symmetric neuropathy beginning with the initial diagnosis of diabetes and annual screening for autonomic neuropathy 5 years after diagnosis of type 1 DM and at the time of diagnosis of type 2 DM. This includes testing for loss of protective sensation (LOPS) using monofilament testing plus one of the following tests: vibration, pinprick, ankle reflexes, or vibration perception threshold (using a biothesiometer). If the monofilament test or one of the other

1	monofilament testing plus one of the following tests: vibration, pinprick, ankle reflexes, or vibration perception threshold (using a biothesiometer). If the monofilament test or one of the other tests is abnormal, the patient is diagnosed with LOPS and counseled accordingly (Chap. 419).

1	The etiology of diabetes in an individual with new-onset disease can usually be assigned on the basis of clinical criteria. Individuals with type 1 DM tend to have the following characteristics: (1) onset of disease prior to age 30 years; (2) lean body habitus; (3) requirement of insulin as the initial therapy; (4) propensity to develop ketoacidosis; and (5) an increased risk of other autoimmune disorders such as autoimmune thyroid disease, adrenal insufficiency, pernicious anemia, celiac disease, and vitiligo. In contrast, individuals with type 2 DM often exhibit the following features: (1) develop diabetes after the age of 30 years; (2) are usually obese (80% are obese, but elderly individuals may be lean); (3) may not require insulin therapy initially; and (4) may have associated conditions such as insulin resistance, hypertension, cardiovascular disease, dyslipidemia, or PCOS. In type 2 DM, insulin resistance is often associated with abdominal obesity (as opposed to hip and thigh

1	such as insulin resistance, hypertension, cardiovascular disease, dyslipidemia, or PCOS. In type 2 DM, insulin resistance is often associated with abdominal obesity (as opposed to hip and thigh obesity) and hypertriglyceridemia. Although most individuals diagnosed with type 2 DM are older, the age of diagnosis is declining, and there is a marked increase among overweight children and adolescents. Some individuals with phenotypic type 2 DM present with diabetic ketoacidosis but lack autoimmune markers and may be later treated with oral glucose-lowering agents rather than insulin (this clinical picture is sometimes referred to as ketosis-prone type 2 DM). On the other hand, some individuals (5–10%) with the phenotypic appearance of type 2 DM do not have absolute insulin deficiency but have autoimmune markers (GAD and other ICA autoantibodies) suggestive of type 1 DM (termed latent autoimmune diabetes of the adult). Such individuals are more likely to be <50 years of age, thinner, and

1	autoimmune markers (GAD and other ICA autoantibodies) suggestive of type 1 DM (termed latent autoimmune diabetes of the adult). Such individuals are more likely to be <50 years of age, thinner, and have a personal or family history of other autoimmune disease than individuals with type 2 DM. They are much more likely to require insulin treatment within 5 years. Monogenic forms of diabetes (discussed above) should be considered in those with diabetes onset at <30 years of age, an autosomal pattern of diabetes inheritance, and the lack of nearly complete insulin deficiency. Despite recent advances in the understanding of the pathogenesis of diabetes, it remains difficult to categorize some patients unequivocally. Individuals who deviate from the clinical profile of type 1 and type 2 DM, or who have other associated defects such as deafness, pancreatic exocrine disease, and other endocrine disorders, should be classified accordingly (Table 417-1).

1	The laboratory assessment should first determine whether the patient meets the diagnostic criteria for DM (Table 417-2) and then assess the degree of glycemic control (Chap. 418). In addition to the standard laboratory evaluation, the patient should be screened for DM-associated conditions (e.g., albuminuria, dyslipidemia, thyroid dysfunction). The classification of the type of DM may be facilitated by laboratory assessments. Serum insulin or C-peptide measurements often do not distinguish type 1 from type 2 DM, but a low C-peptide level confirms a patient’s need for insulin. Many individuals with new-onset type 1 DM retain some C-peptide production. Measurement of islet cell antibodies at the time of diabetes onset may be useful if the type of DM is not clear based on the characteristics described above. Diabetes mellitus: management Alvin C. Powers

1	Diabetes mellitus: management Alvin C. Powers The goals of therapy for type 1 or type 2 diabetes mellitus (DM) are to (1) eliminate symptoms related to hyperglycemia, (2) reduce or eliminate the long-term microvascular and macrovascular complications of DM (Chap. 419), and (3) allow the patient to achieve as normal a lifestyle as possible. To reach these goals, the physician should identify a target level of glycemic control for each patient, provide the patient with the educational and pharmacologic resources necessary to reach this level, and monitor/treat DM-related complications. Symptoms of diabetes usually resolve when the plasma glucose is <11.1 mmol/L (200 mg/dL), and thus most DM treatment focuses on achieving the second and third goals. This chapter first reviews the ongoing treatment of diabetes in the outpatient setting and then discusses the treatment of severe hyperglycemia, as well as the treatment of diabetes in hospitalized patients.

1	The care of an individual with either type 1 or type 2 DM requires a multidisciplinary team. Central to the success of this team are the patient’s participation, input, and enthusiasm, all of which are essential for optimal diabetes management. Members of the health care team include the primary care provider and/or the endocrinologist or diabetologist, a certified diabetes educator, a nutritionist, and a psychologist. In addition, when the complications of DM arise, sub-specialists (including neurologists, nephrologists, vascular surgeons, cardiologists, ophthalmologists, and podiatrists) with experience in DM-related complications are essential.

1	A number of names are sometimes applied to different approaches to diabetes care, such as intensive insulin therapy, intensive glycemic control, and “tight control.” The current chapter, and other sources, uses the term comprehensive diabetes care to emphasize the fact that optimal diabetes therapy involves more than plasma glucose management and medications. Although glycemic control is central to optimal diabetes therapy, comprehensive diabetes care of both type 1 and type 2 DM should also detect and manage DM-specific complications (Chap. 419) and modify risk factors for DM-associated diseases. The key elements of comprehensive diabetes care are summarized in Table 418-1. In addition to the physical aspects of DM, social, family, financial, cultural, and employment-related issues may impact diabetes care. The International Diabetes Federation (IDF), recognizing that resources available for diabetes care vary widely throughout the world, has issued guidelines for “recommended care”

1	diabetes care. The International Diabetes Federation (IDF), recognizing that resources available for diabetes care vary widely throughout the world, has issued guidelines for “recommended care” (a well-developed service base and with health care funding systems consuming a significant part of their national wealth), “limited care” (health care settings with very limited resources), and “comprehensive care” (health care settings with considerable resources). This chapter provides guidance

1	Diabetes Mellitus: Management and Therapies guiDElinES foR ongoing, ComPREHEnSivE mEDiCAl CARE foR PATiEnTS wiTH DiAbETES Self-monitoring of blood glucose (individualized frequency) • HbA1c testing (2–4 times/year) Eye examination (annual or biannual; Chap. 419) Chap. 419) Screening for diabetic nephropathy (annual; Chap. 419) Lipid profile and serum creatinine (estimate GFR) (annual; Chap. 419) Consider antiplatelet therapy (Chap. 419) Abbreviations: GFR, glomerular filtration rate; HbA1c, hemoglobin A1c. for this comprehensive level of diabetes care. The treatment goals for patients with diabetes are summarized in Table 418-2 and should be individualized.

1	The morbidity and mortality rates of DM-related complications (Chap. 419) can be greatly reduced by timely and consistent surveillance procedures (Table 418-1). These screening procedures are indicated for all individuals with DM, but many individuals with diabetes do not receive comprehensive diabetes care. A comprehensive eye examination should be performed by a qualified optometrist or ophthalmologist. Because many individuals with type 2 DM have had asymptomatic diabetes for several years before diagnosis, the American Diabetes Association (ADA) recommends the following ophthalmologic examination schedule: (1) individuals with type 1 DM should have an initial eye examination within 5 years of diagnosis, (2) individuals with type 2 DM should have an initial eye examination at the time of diabetes diagnosis, (3) women with DM who are pregnant or contemplating pregnancy should have an eye examination prior to conception and during the first trimester, and (4) if eye exam is normal,

1	of diabetes diagnosis, (3) women with DM who are pregnant or contemplating pregnancy should have an eye examination prior to conception and during the first trimester, and (4) if eye exam is normal, repeat examination in 2–3 years is appropriate.

1	<7.0%c 4.4–7.2 mmol/L (80–130 mg/dL) <10.0 mmol/L (<180 mg/dL) <2.6 mmol/L (100 mg/dL)g >1 mmol/L (40 mg/dL) in men >1.3 mmol/L (50 mg/dL) in women <1.7 mmol/L (150 mg/dL) aAs recommended by the American Diabetes Association; goals should be individualized for each patient (see text). Goals may be different for certain patient populations. bHbA1c is primary goal. cDiabetes Control and Complications Trial–based assay. d1–2 h after beginning of a meal. eGoal of <130/80 mmHg may be appropriate for younger individuals fIn decreasing order of priority. Recent guidelines from the American College of Cardiology and American Heart Association no longer advocate specific LDL and HDL goals (see Chaps. 291e and 419). gGoal of <1.8 mmol/L (70 mg/dL) may be appropriate for individuals with cardiovascular disease. Abbreviation: HbA1c, hemoglobin A1c. Source: Adapted from American Diabetes Association: Diabetes Care 38(Suppl 1):S1, 2015. PATIENT EDUCATION ABOUT DM, NUTRITION, AND EXERCISE

1	The patient with type 1 or type 2 DM should receive education about nutrition, exercise, care of diabetes during illness, and medications to lower the plasma glucose. Along with improved compliance, patient education allows individuals with DM to assume greater responsibility for their care. Patient education should be viewed as a continuing process with regular visits for reinforcement; it should not be a process that is completed after one or two visits to a nurse educator or nutritionist. The ADA refers to education about the individualized management plan for the patient as diabetes self-management education (DSME) and diabetes self-management support (DSMS). DSME and DSMS are ways to improve the patient’s knowledge, skills, and abilities necessary for diabetes self-care and should also emphasize psychosocial issues and emotional well-being. More frequent contact between the patient and the diabetes management team (e.g., electronic, telephone) improves glycemic control.

1	Diabetes Education The diabetes educator is a health care professional (nurse, dietician, or pharmacist) with specialized patient education skills who is certified in diabetes education (e.g., American Association of Diabetes Educators). Education topics important for optimal diabetes care include self-monitoring of blood glucose; urine ketone monitoring (type 1 DM); insulin administration; guidelines for diabetes management during illnesses; prevention and management of hypoglycemia (Chap. 420); foot and skin care; diabetes management before, during, and after exercise; and risk factor–modifying activities.

1	Psychosocial Aspects Because the individual with DM can face challenges that affect many aspects of daily life, psychosocial assessment and treatment are a critical part of providing comprehensive diabetes care. The individual with DM must accept that he or she may develop complications related to DM. Even with considerable effort, normoglycemia can be an elusive goal, and solutions to worsening glycemic control may not be easily identifiable. The patient should view himor herself as an essential member of the diabetes care team and not as someone who is cared for by the diabetes management team. Emotional stress may provoke a change in behavior so that individuals no longer adhere to a dietary, exercise, or therapeutic regimen. This can lead to the appearance of either hyperor hypoglycemia. Eating disorders, including binge eating disorders, bulimia, and anorexia nervosa, appear to occur more frequently in individuals with type 1 or type 2 DM.

1	Nutrition Medical nutrition therapy (MNT) is a term used by the ADA to describe the optimal coordination of caloric intake with other aspects of diabetes therapy (insulin, exercise, weight loss). Primary prevention measures of MNT are directed at preventing or delaying the onset of type 2 DM in high-risk individuals (obese or with prediabetes) by promoting weight reduction. Medical treatment of obesity is a rapidly evolving area and is discussed in Chap. 416. Secondary prevention measures of MNT are directed at preventing or delaying diabetes-related complications in diabetic individuals by improving glycemic control. Tertiary prevention measures of MNT are directed at managing diabetes-related complications (cardiovascular disease, nephropathy) in diabetic individuals. MNT in patients with diabetes and cardiovascular disease should incorporate dietary principles used in nondiabetic patients with cardiovascular disease. Although the recommendations for all three types of MNT overlap,

1	diabetes and cardiovascular disease should incorporate dietary principles used in nondiabetic patients with cardiovascular disease. Although the recommendations for all three types of MNT overlap, this chapter emphasizes secondary prevention measures of MNT. Pharmacologic approaches that facilitate weight loss and bariatric surgery should be considered in selected patients (Chaps. 415e and 416).

1	In general, the components of optimal MNT are similar for individuals with type 1 or type 2 DM and similar to those for the general population (fruits, vegetables, fiber-containing foods, and low fat; Table 418-3). MNT education is an important component of comprehensive diabetes care and should be reinforced by regular patient education. Historically, nutrition education imposed restrictive, complicated regimens on the patient. Current practices have greatly changed, although many patients and health care providers still view the diabetic diet as monolithic and static. For example, MNT now includes foods • Hypocaloric diet that is low-carbohydrate Fat in diet (optimal % of diet is not known; should be individualized) Carbohydrate in diet (optimal % of diet is not known; should be individualized) Monitor carbohydrate intake in regard to calories

1	Carbohydrate in diet (optimal % of diet is not known; should be individualized) Monitor carbohydrate intake in regard to calories Sucrose-containing foods may be consumed with adjustments in insulin dose, but minimize intake • Amount of carbohydrate determined by estimating grams of carbohydrate • Use glycemic index to predict how consumption of a particular food may Protein in diet (optimal % of diet is not known; should be individualized) • Dietary fiber, vegetable, fruits, whole grains, dairy products, and sodium intake as advised for general population Routine supplements of vitamins, antioxidants, or trace elements not aSee text for differences for patients with type 1 or type 2 diabetes. Source: Adapted from American Diabetes Association: Diabetes Care 37(Suppl 1):S14, 2014.

1	Source: Adapted from American Diabetes Association: Diabetes Care 37(Suppl 1):S14, 2014. with sucrose and seeks to modify other risk factors such as hyperlipidemia and hypertension rather than focusing exclusively on weight loss in individuals with type 2 DM. The glycemic index is an estimate of the postprandial rise in the blood glucose when a certain amount of that food is consumed. Consumption of foods with a low glycemic index appears to reduce postprandial glucose excursions and improve glycemic control. Reduced-calorie and nonnutritive sweeteners are useful. Currently, evidence does not support supplementation of the diet with vitamins, antioxidants (vitamin C and E), or micronutrients (chromium) in patients with diabetes.

1	The goal of MNT in the individual with type 1 DM is to coordinate and match the caloric intake, both temporally and quantitatively, with the appropriate amount of insulin. MNT in type 1 DM and self-monitoring of blood glucose must be integrated to define the optimal insulin regimen. The ADA encourages patients and providers to use carbohydrate counting or exchange systems to estimate the nutrient content of a meal or snack. Based on the patient’s estimate of the carbohydrate content of a meal, an insulin-to-carbohydrate ratio determines the bolus insulin dose for a meal or snack. MNT must be flexible enough to allow for exercise, and the insulin regimen must allow for deviations in caloric intake. An important component of MNT in type 1 DM is to minimize the weight gain often associated with intensive diabetes management.

1	The goals of MNT in type 2 DM should focus on weight loss and address the greatly increased prevalence of cardiovascular risk factors (hypertension, dyslipidemia, obesity) and disease in this population. The majority of these individuals are obese, and weight loss is strongly encouraged and should remain an important goal. Hypocaloric diets and modest weight loss (5–7%) often result in rapid and dramatic glucose lowering in individuals with new-onset type 2 DM. Nevertheless, numerous studies document that long-term weight loss is uncommon. MNT for type 2 DM should emphasize modest caloric reduction (low-carbohydrate) and increased physical activity. Increased consumption of soluble, dietary fiber may improve glycemic control in individuals with type 2 DM. Weight loss and exercise improve insulin resistance.

1	Exercise Exercise has multiple positive benefits including cardiovascular risk reduction, reduced blood pressure, maintenance of muscle mass, reduction in body fat, and weight loss. For individuals with type 2409 1 or type 2 DM, exercise is also useful for lowering plasma glucose (during and following exercise) and increasing insulin sensitivity. In patients with diabetes, the ADA recommends 150 min/week (distributed over at least 3 days) of moderate aerobic physical activity with no gaps longer than 2 days. The exercise regimen should also include resistance training.

1	Despite its benefits, exercise presents challenges for individuals with DM because they lack the normal glucoregulatory mechanisms (normally, insulin falls and glucagon rises during exercise). Skeletal muscle is a major site for metabolic fuel consumption in the resting state, and the increased muscle activity during vigorous, aerobic exercise greatly increases fuel requirements. Individuals with type 1 DM are prone to either hyperglycemia or hypoglycemia during exercise, depending on the preexercise plasma glucose, the circulating insulin level, and the level of exercise-induced catecholamines. If the insulin level is too low, the rise in catecholamines may increase the plasma glucose excessively, promote ketone body formation, and possibly lead to ketoacidosis. Conversely, if the circulating insulin level is excessive, this relative hyperinsulinemia may reduce hepatic glucose production (decreased glycogenolysis, decreased gluconeogenesis) and increase glucose entry into muscle,

1	insulin level is excessive, this relative hyperinsulinemia may reduce hepatic glucose production (decreased glycogenolysis, decreased gluconeogenesis) and increase glucose entry into muscle, leading to hypoglycemia.

1	To avoid exercise-related hyperor hypoglycemia, individuals with type 1 DM should (1) monitor blood glucose before, during, and after exercise; (2) delay exercise if blood glucose is >14 mmol/L (250 mg/ dL) and ketones are present; (3) if the blood glucose is <5.6 mmol/L (100 mg/dL), ingest carbohydrate before exercising; (4) monitor glu cose during exercise and ingest carbohydrate to prevent hypoglycemia; (5) decrease insulin doses (based on previous experience) before exercise and inject insulin into a nonexercising area; and (6) learn individual glucose responses to different types of exercise and increase food intake for up to 24 h after exercise, depending on intensity and duration of exercise. In individuals with type 2 DM, exercise-related hypoglycemia is less common but can occur in individuals taking either insulin or insulin secretagogues.

1	younger age in both type 1 and type 2 DM, routine screening for coronary artery disease has not been shown to be effective and is not recommended (Chap. 419). Untreated proliferative retinopathy is a relative contraindication to vigorous exercise, because this may lead to vitreous hemorrhage or retinal detachment. Optimal monitoring of glycemic control involves plasma glucose measurements by the patient and an assessment of long-term control by the physician (measurement of hemoglobin A1c [HbA1c] and review of the patient’s self-measurements of plasma glucose). These measurements are complementary: the patient’s measurements provide a picture of short-term glycemic control, whereas the HbA1c reflects average glycemic control over the previous 2–3 months.

1	Self-Monitoring of Blood Glucose Self-monitoring of blood glucose (SMBG) is the standard of care in diabetes management and allows the patient to monitor his or her blood glucose at any time. In SMBG, a small drop of blood and an easily detectable enzymatic reaction allow measurement of the capillary plasma glucose. Many glucose monitors can rapidly and accurately measure glucose (calibrated to provide plasma glucose value even though blood glucose is measured) in small amounts of blood (3–10 μL) obtained from the fingertip; alternative testing sites (e.g., forearm) are less reliable, especially when the blood glucose is changing rapidly (postprandially). A large number of blood glucose monitors are available, and the certified diabetes educator is critical in helping the patient select the optimal device and learn to use it properly. By combining glucose measurements with diet history, medication changes, and exercise history, the diabetes management team and patient can improve the

1	optimal device and learn to use it properly. By combining glucose measurements with diet history, medication changes, and exercise history, the diabetes management team and patient can improve the treatment program.

1	The frequency of SMBG measurements must be individualized and adapted to address the goals of diabetes care. Individuals with type 1

1	Diabetes Mellitus: Management and Therapies 2410 DM or individuals with type 2 DM taking multiple insulin injections each day should routinely measure their plasma glucose three or more times per day to estimate and select mealtime boluses of short-acting insulin and to modify long-acting insulin doses. Most individuals with type 2 DM require less frequent monitoring, although the optimal frequency of SMBG has not been clearly defined. Individuals with type 2 DM who are taking insulin should use SMBG more frequently than those on oral agents. Individuals with type 2 DM who are on oral medications should use SMBG as a means of assessing the efficacy of their medication and the impact of diet. Because plasma glucose levels fluctuate less in these individuals, one to two SMBG measurements per day (or fewer in patients who are on oral agents or are diet-controlled) may be sufficient. Most measurements in individuals with type 1 or type 2 DM should be performed prior to a meal and

1	per day (or fewer in patients who are on oral agents or are diet-controlled) may be sufficient. Most measurements in individuals with type 1 or type 2 DM should be performed prior to a meal and supplemented with postprandial measurements to assist in reaching postprandial glucose targets (Table 418-2). Devices for continuous glucose monitoring (CGM) have been approved by the U.S. Food and Drug Administration (FDA), and others are in various stages of development. These devices do not replace the need for traditional glucose measurements and require calibration with SMBG. This rapidly evolving technology requires substantial expertise on the part of the diabetes management team and the patient. Current CGM systems measure the glucose in interstitial fluid, which is in equilibrium with the blood glucose. These devices provide useful short-term information about the patterns of glucose changes as well as an enhanced ability to detect hypoglycemic episodes. Alarms notify the patient if

1	blood glucose. These devices provide useful short-term information about the patterns of glucose changes as well as an enhanced ability to detect hypoglycemic episodes. Alarms notify the patient if the blood glucose falls into the hypoglycemic range. Clinical experience with these devices is rapidly growing, and they are most useful in individuals with hypoglycemia unawareness, individuals with frequent hypoglycemia, or those who have not achieved glycemic targets despite major efforts. The utility of CGM in the intensive care unit (ICU) setting remains to be determined.

1	Assessment of Long-Term Glycemic Control Measurement of glycated hemoglobin (HbA1c) is the standard method for assessing long-term glycemic control. When plasma glucose is consistently elevated, there is an increase in nonenzymatic glycation of hemoglobin; this alteration reflects the glycemic history over the previous 2–3 months, because erythrocytes have an average life span of 120 days (glycemic level in the preceding month contributes about 50% to the HbA1c value). Measurement of HbA1c at the “point of care” allows for more rapid feedback and may therefore assist in adjustment of therapy.

1	HbA1c should be measured in all individuals with DM during their initial evaluation and as part of their comprehensive diabetes care. As the primary predictor of long-term complications of DM, the HbA1c should mirror, to a certain extent, the short-term measurements of SMBG. These two measurements are complementary in that recent intercurrent illnesses may impact the SMBG measurements but not the HbA1c. Likewise, postprandial and nocturnal hyperglycemia may not be detected by the SMBG of fasting and preprandial capillary plasma glucose but will be reflected in the HbA1c. In standardized assays, the HbA1c approximates the following mean plasma glucose values: an HbA1c of 6% = 7.0 mmol/L (126 mg/dL), 7% = 8.6 mmol/L (154 mg/dL), 8% = 10.2 mmol/L (183 mg/dL), 9% = 11.8 mmol/L (212 mg/dL), 10% = 13.4 mmol/L (240 mg/dL), 11% = 14.9 mmol/L (269 mg/dL), and 12% = 16.5 mmol/L (298 mg/dL). In patients achieving their glycemic goal, the ADA recommends measurement of the HbA1c at least twice per

1	= 13.4 mmol/L (240 mg/dL), 11% = 14.9 mmol/L (269 mg/dL), and 12% = 16.5 mmol/L (298 mg/dL). In patients achieving their glycemic goal, the ADA recommends measurement of the HbA1c at least twice per year. More frequent testing (every 3 months) is warranted when glycemic control is inadequate or when therapy has changed. Laboratory standards for the HbA1c test have been established and should be correlated to the reference assay of the Diabetes Control and Complications Trial (DCCT). Clinical conditions such hemoglobinopathies, anemias, reticulocytosis, transfusions, and uremia may interfere with the HbA1c result. The degree of glycation of other proteins, such as albumin, can be used as an alternative indicator of glycemic control when the HbA1c is inaccurate. The fructosamine assay (measuring glycated albumin) reflects the glycemic status over the prior 2 weeks.

1	Comprehensive care of type 1 and type 2 DM requires an emphasis on nutrition, exercise, and monitoring of glycemic control but also usually involves glucose-lowering medication(s). This chapter discusses classes of such medications but does not describe every glucose-lowering agent available worldwide. The initial step is to select an individualized, glycemic goal for the patient. Because the complications of DM are related to glycemic control, normoglycemia or near-normoglycemia is the desired, but often elusive, goal for most patients. Normalization or near-normalization of the plasma glucose for long periods of time is extremely difficult, as demonstrated by the DCCT and United Kingdom Prospective Diabetes Study (UKPDS). Regardless of the level of hyperglycemia, improvement in glycemic control will lower the risk of diabetes-specific complications (Chap. 419).

1	The target for glycemic control (as reflected by the HbA1c) must be individualized, and the goals of therapy should be developed in consultation with the patient after considering a number of medical, social, and lifestyle issues. The ADA calls this a patient-centered approach, and other organizations such as the IDF and American Association of Clinical Endocrinologists (AACE) also suggest an individualized glycemic goal. Important factors to consider include the patient’s age and ability to understand and implement a complex treatment regimen, presence and severity of complications of diabetes, known cardiovascular disease (CVD), ability to recognize hypoglycemic symptoms, presence of other medical conditions or treatments that might affect survival or the response to therapy, lifestyle and occupation (e.g., possible consequences of experiencing hypoglycemia on the job), and level of support available from family and friends.

1	In general, the ADA suggests that the goal is to achieve an HbA1c as close to normal as possible without significant hypoglycemia. In most individuals, the target HbA1c should be <7% (Table 418-2) with a more stringent target for some patients. For instance, the HbA1c goal in a young adult with type 1 DM may be 6.5%. A higher HbA1c goal may be appropriate for the very young or old or in individuals with limited life span or comorbid conditions. For example, an appropriate HbA1c goal in elderly individuals with multiple, chronic illnesses and impaired activities of daily living might be 8.0 or 8.5%. A major consideration is the frequency and severity of hypoglycemia, because this becomes more common with a more stringent HbA1c goal.

1	More stringent glycemic control (HbA1c of ≤6%) is not beneficial, and may be detrimental, in patients with type 2 DM and a high risk of CVD. Large clinical trials (UKPDS, Action to Control Cardiovascular Risk in Diabetes [ACCORD], Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation [ADVANCE], Veterans Affairs Diabetes Trial [VADT]; Chap. 419) have examined glycemic control in type 2 DM in individuals with low risk of CVD, with high risk of CVD, or with established CVD and have found that more intense glycemic control is not beneficial and, in some patient populations, may have a negative impact on some outcomes. These divergent outcomes stress the need for individualized glycemic goals based on the following general guidelines: (1) early in the course of type 2 diabetes when the CVD risk is lower, improved glycemic control likely leads to improved cardiovascular outcome, but this benefit occurs more than a decade after the period of improved

1	of type 2 diabetes when the CVD risk is lower, improved glycemic control likely leads to improved cardiovascular outcome, but this benefit occurs more than a decade after the period of improved glycemic control; (2) intense glycemic control in individuals with established CVD or at high risk for CVD is not advantageous, and may be deleterious, over a follow-up of 3–5 years; an HbA1c goal <7.0% is not appropriate in this population; (3) hypoglycemia in such high-risk populations (elderly, CVD) should be avoided; and (4) improved glycemic control reduces microvascular complications of diabetes (Chap. 419) even if it does not improve macrovascular complications like CVD.

1	TYPE 1 DIABETES MELLITUS General Aspects The ADA recommendations for fasting and bedtime glycemic goals and HbA1c targets are summarized in Table 418-2. The goal is to design and implement insulin regimens that mimic physiologic insulin secretion. Because individuals with type 1 DM partially or completely lack endogenous insulin production, administration of basal insulin is essential for regulating glycogen breakdown, gluconeogenesis, lipolysis, and ketogenesis. Likewise, insulin replacement for meals should be appropriate for the carbohydrate intake and promote normal glucose utilization and storage.

1	Intensive Management Intensive diabetes management has the goal of achieving euglycemia or near-normal glycemia. This approach requires multiple resources, including thorough and continuing patient education, comprehensive recording of plasma glucose measurements and nutrition intake by the patient, and a variable insulin regimen that matches glucose intake and insulin dose. Insulin regimens usually include multiple-component insulin regimens, multiple daily injections (MDIs), or insulin infusion devices (each discussed below).

1	The benefits of intensive diabetes management and improved glycemic control include a reduction in the microvascular complications of DM and a reduction in diabetes-related complications. From a psychological standpoint, the patient experiences greater control over his or her diabetes and often notes an improved sense of well-being, greater flexibility in the timing and content of meals, and the capability to alter insulin dosing with exercise. In addition, intensive diabetes management prior to and during pregnancy reduces the risk of fetal malformations and morbidity. Intensive diabetes management is encouraged in newly diagnosed patients with type 1 DM because it may prolong the period of C-peptide production, which may result in better glycemic control and a reduced risk of serious hypoglycemia. Although intensive management confers impressive benefits, it is also accompanied by significant personal and financial costs and is therefore not appropriate for all individuals.

1	Insulin Preparations Current insulin preparations are generated by recombinant DNA technology and consist of the amino acid sequence of human insulin or variations thereof. In the United States, most insulin is formulated as U-100 (100 units/mL). Regular insulin formulated as U-500 (500 units/mL) is available and sometimes useful in patients with severe insulin resistance. Human insulin has been formulated with distinctive pharmacokinetics or genetically modified to more closely mimic physiologic insulin secretion. Insulins can be classified as short-acting or long-acting (Table 418-4). For example, one short-acting insulin formulation, insulin lispro, is an insulin analogue in which the 28th and 29th amino acids (lysine and proline) on the insulin B chain have been reversed by recombinant DNA technology. Insulin aspart and insulin glulisine are genetically modified insulin analogues with properties similar to lispro. All three of the insulin analogues have full biologic activity but

1	DNA technology. Insulin aspart and insulin glulisine are genetically modified insulin analogues with properties similar to lispro. All three of the insulin analogues have full biologic activity but less tendency for self-aggregation, resulting in more rapid absorption and onset of action and a shorter duration of action. These characteristics are particularly advantageous for allowing entrainment of insulin injection and action to rising plasma glucose levels following meals. The shorter duration of action also appears to be associated with a decreased number of hypoglycemic episodes, primarily because the decay of insulin action corresponds to the decline in plasma glucose after a meal. Thus, insulin aspart, lispro, or glulisine is preferred over regular insulin for prandial coverage. Insulin glargine is a long-acting biosynthetic human insulin that differs from normal insulin in that asparagine is replaced by glycine at amino acid 21, and two arginine residues are added to the C

1	Insulin glargine is a long-acting biosynthetic human insulin that differs from normal insulin in that asparagine is replaced by glycine at amino acid 21, and two arginine residues are added to the C terminus of the B chain. Compared to neutral protamine Hagedorn (NPH) insulin, the onset of insulin glargine action is later, the duration of action is longer (~24 h), and there is a less pronounced peak. A lower incidence of hypoglycemia, especially at night, has been reported with insulin glargine when compared to NPH insulin. The most recent evidence does not support an association between glargine and increased cancer risk. Insulin detemir has a fatty acid side chain that prolongs its action by slowing absorption and catabolism. Twice-daily injections of glargine or detemir are sometimes required to provide 24-h coverage. Regular and NPH insulin have the native insulin amino acid sequence.

1	Basal insulin requirements are provided by long-acting (NPH insulin, insulin glargine, or insulin detemir) insulin formulations. These TAblE 418-4 PRoPERTiES of inSulin PREPARATionSa Time of Action aInsulin preparations available in the United States; others are available in the United Kingdom and Europe. bGlargine and detemir have minimal peak activity. cDuration is dose-dependent (shorter at lower doses). dOther insulin combinations are available eDual: two peaks—one at 2–3 h and the second one several hours later. Source: Adapted from FR Kaufman: Medical Management of Type 1 Diabetes, 6th edition. Alexandria, VA: American Diabetes Association, 2012.

1	are usually prescribed with short-acting insulin in an attempt to mimic physiologic insulin release with meals. Although mixing of NPH and short-acting insulin formulations is common practice, this mixing may alter the insulin absorption profile (especially the short-acting insulins). For example, lispro absorption is delayed by mixing with NPH. The alteration in insulin absorption when the patient mixes different insulin formulations should not prevent mixing insulins. However, the following guidelines should be followed: (1) mix the different insulin formulations in the syringe immediately before injection (inject within 2 min after mixing); (2) do not store insulin as a mixture; (3) follow the same routine in terms of insulin mixing and administration to standardize the physiologic response to injected insulin; and (4) do not mix insulin glargine or detemir with other insulins. The miscibility of some insulins allows for the production of combination insulins that contain 70% NPH

1	to injected insulin; and (4) do not mix insulin glargine or detemir with other insulins. The miscibility of some insulins allows for the production of combination insulins that contain 70% NPH and 30% regular (70/30), or equal mixtures of NPH and regular (50/50). By including the insulin analogue mixed with protamine, several combinations have a short-acting and long-acting profile (Table 418-4). Although more convenient for the patient (only two injections/day), combination insulin formulations do not allow independent adjustment of short-acting and long-acting activity. Several insulin formulations are available as insulin “pens,” which may be more convenient for some patients. Insulin delivery by inhalation has recently been approved but is not yet available. Other insulins, such as one with a duration of action of several days, are under development but are not currently available in the United States.

1	Insulin Regimens Representations of the various insulin regimens that may be used in type 1 DM are illustrated in Fig. 418-1. Although the insulin profiles are depicted as “smooth,” symmetric curves, there is considerable patient-to-patient variation in the peak and duration. In all regimens, long-acting insulins (NPH, glargine, or detemir) supply basal insulin, whereas regular, insulin aspart, glulisine, or lispro insulin provides prandial insulin. Short-acting insulin analogues should be injected just before (<10 min) or just after a meal; regular insulin is given 30–45 min prior to a meal. Sometimes short-acting insulin analogues are injected just after a meal (gastroparesis, unpredictable food intake). A shortcoming of current insulin regimens is that injected insulin immediately enters the systemic circulation, whereas endogenous Diabetes Mellitus: Management and Therapies

1	FIGURE 418-1 Representative insulin regimens for the treatment of diabetes. For each panel, the y-axis shows the amount of insulin effect and the x-axis shows the time of day. B, breakfast; HS, bedtime; L, lunch; S, supper. *Lispro, glulisine, or insulin aspart can be used. The time of insulin injection is shown with a vertical arrow. The type of insulin is noted above each insulin curve. A. Multiple-component insulin regimen consisting of long-acting insulin (∧glargine or detemir) to provide basal insulin coverage and three shots of glulisine, lispro, or insulin aspart to provide glycemic coverage for each meal. B. Injection of two shots of long-acting insulin (NPH) and short-acting insulin analogue (glulisine, lispro, insulin aspart [solid red line], or regular insulin [green dashed line]). Only one formulation of short-acting insulin is used. C. Insulin administration by insulin infusion device is shown with the basal insulin and a bolus injection at each meal. The basal insulin

1	Only one formulation of short-acting insulin is used. C. Insulin administration by insulin infusion device is shown with the basal insulin and a bolus injection at each meal. The basal insulin rate is decreased during the evening and increased slightly prior to the patient awakening in the morning. Glulisine, lispro, or insulin aspart is used in the insulin pump.

1	(Adapted from H Lebovitz [ed]: Therapy for Diabetes Mellitus. American Diabetes Association, Alexandria, VA, 2004.) insulin is secreted into the portal venous system. Thus, exogenous insulin administration exposes the liver to subphysiologic insulin levels. No insulin regimen reproduces the precise insulin secretory pattern of the pancreatic islet. However, the most physiologic regimens entail more frequent insulin injections, greater reliance on short-acting insulin, and more frequent capillary plasma glucose measurements. In general, individuals with type 1 DM require 0.5–1 U/kg per day of insulin divided into multiple doses, with ~50% of the insulin given as basal insulin.

1	Multiple-component insulin regimens refer to the combination of basal insulin and bolus insulin (preprandial short-acting insulin). The timing and dose of short-acting, preprandial insulin are altered to accommodate the SMBG results, anticipated food intake, and physical activity. Such regimens offer the patient with type 1 diabetes more flexibility in terms of lifestyle and the best chance for achieving near normoglycemia. One such regimen, shown in Fig. 418-1B, consists of basal insulin with glargine or detemir and preprandial lispro, glulisine, or insulin aspart. The insulin aspart, glulisine, or lispro dose is based on individualized algorithms that integrate the preprandial glucose and the anticipated carbohydrate intake. To determine the meal component of the preprandial insulin dose, the patient uses an insulin-tocarbohydrate ratio (a common ratio for type 1 DM is 1–1.5 units/10 g of carbohydrate, but this must be determined for each individual). To this insulin dose is added

1	the patient uses an insulin-tocarbohydrate ratio (a common ratio for type 1 DM is 1–1.5 units/10 g of carbohydrate, but this must be determined for each individual). To this insulin dose is added the supplemental or correcting insulin based on the preprandial blood glucose (one formula uses 1 unit of insulin for every 2.7 mmol/L [50 mg/dL] over the preprandial glucose target; another formula uses [body weight in kg] × [blood glucose – desired glucose in mg/dL]/1500). An alternative multiple-component insulin regimen consists of bedtime NPH insulin, a small dose of NPH insulin at breakfast (20–30% of bedtime dose), and preprandial short-acting insulin. Other variations of this regimen are in use but have the disadvantage that NPH has a significant peak, making hypoglycemia more common. Frequent SMBG (more than three times per day) is absolutely essential for these types of insulin regimens.

1	In the past, one commonly used regimen consisted of twice-daily injections of NPH mixed with a short-acting insulin before the morning and evening meals (Fig. 418-1B). Such regimens usually prescribe two-thirds of the total daily insulin dose in the morning (with about two-thirds given as long-acting insulin and one-third as short-acting) and one-third before the evening meal (with approximately one-half given as long-acting insulin and one-half as short-acting). The drawback to such a regimen is that it forces a rigid schedule on the patient, in terms of daily activity and the content and timing of meals. Although it is simple and effective at avoiding severe hyperglycemia, it does not generate near-normal glycemic control in individuals with type 1 DM. Moreover, if the patient’s meal pattern or content varies or if physical activity is increased, hyperglycemia or hypoglycemia may result. Moving the long-acting insulin from before the evening meal to bedtime may avoid nocturnal

1	pattern or content varies or if physical activity is increased, hyperglycemia or hypoglycemia may result. Moving the long-acting insulin from before the evening meal to bedtime may avoid nocturnal hypoglycemia and provide more insulin as glucose levels rise in the early morning (so-called dawn phenomenon). The insulin dose in such regimens should be adjusted based on SMBG results with the following general assumptions: (1) the fasting glucose is primarily determined by the prior evening long-acting insulin; (2) the pre-lunch glucose is a function of the morning short-acting insulin; (3) the pre-supper glucose is a function of the morning long-acting insulin; and (4) the bedtime glucose is a function of the pre-supper, short-acting insulin. This is not an optimal regimen for the patient with type 1 DM, but is sometimes used for patients with type 2 DM.

1	Continuous SC insulin infusion (CSII) is a very effective insulin regimen for the patient with type 1 DM (Fig. 418-1C). To the basal insulin infusion, a preprandial insulin (“bolus”) is delivered by the insulin infusion device based on instructions from the patient, who uses an individualized algorithm incorporating the preprandial plasma glucose and anticipated carbohydrate intake. These sophisticated insulin infusion devices can accurately deliver small doses of insulin (microliters per hour) and have several advantages: (1) multiple basal infusion rates can be programmed to accommodate nocturnal versus daytime basal insulin requirement; (2) basal infusion rates can be altered during periods of exercise; (3) different waveforms of insulin infusion with meal-related bolus allow better matching of insulin depending on meal composition; and (4) programmed algorithms consider prior insulin administration and blood glucose values in calculating the insulin dose. These devices require

1	matching of insulin depending on meal composition; and (4) programmed algorithms consider prior insulin administration and blood glucose values in calculating the insulin dose. These devices require instruction by a health professional with considerable experience with insulin-infusion devices and very frequent patient interactions with the diabetes management team. Insulin-infusion devices present unique challenges, such as infection at the infusion site, unexplained hyperglycemia because the infusion set becomes obstructed, or diabetic ketoacidosis if the pump becomes disconnected. Because most physicians use lispro, glulisine, or insulin aspart in CSII, the extremely short half-life of these insulins quickly leads to insulin deficiency if the delivery system is interrupted. Essential to the safe use of infusion devices is thorough patient education about pump function and frequent SMBG. Efforts to create a closed-loop system in which data from continuous glucose measurement

1	to the safe use of infusion devices is thorough patient education about pump function and frequent SMBG. Efforts to create a closed-loop system in which data from continuous glucose measurement regulate the insulin infusion rate are under way.

1	Other Agents That Improve Glucose Control The role of amylin, a 37-amino-acid peptide co-secreted with insulin from pancreatic beta cells, in normal glucose homeostasis is uncertain. However, based on the rationale that patients who are insulin deficient are also amylin deficient, an analogue of amylin (pramlintide) was created and found to reduce postprandial glycemic excursions in type 1 and type 2 diabetic patients taking insulin. Pramlintide injected just before a meal slows gastric emptying and suppresses glucagon but does not alter insulin levels. Pramlintide is approved for insulin-treated patients with type 1 and type 2 DM. Addition of pramlintide produces a modest reduction in the HbA1c and seems to dampen meal-related glucose excursions. In type 1 DM, pramlintide is started as a 15-μg SC injection before each meal and titrated up to a maximum of 30–60 μg as tolerated. In type 2 DM, pramlintide is started as a 60-μg SC injection before each meal and may be titrated up to a

1	a 15-μg SC injection before each meal and titrated up to a maximum of 30–60 μg as tolerated. In type 2 DM, pramlintide is started as a 60-μg SC injection before each meal and may be titrated up to a maximum of 120 μg. The major side effects are nausea and vomiting, and dose escalations should be slow to limit these side effects. Because pramlintide slows gastric emptying, it may influence absorption of other medications and should not be used in combination with other drugs that slow GI motility. The short-acting insulin given before the meal should initially be reduced to avoid hypoglycemia and then titrated as the effects of the pramlintide become evident. α-Glucosidase inhibitors are sometimes used with insulin in type 1 DM.

1	TYPE 2 DIABETES MELLITUS General Aspects The goals of glycemia-controlling therapy for type 2 DM are similar to those in type 1 DM. Whereas glycemic control tends to dominate the management of type 1 DM, the care of individuals with type 2 DM must also include attention to the treatment of conditions associated with type 2 DM (e.g., obesity, hypertension, dyslipidemia, CVD) and detection/management of DM-related complications (Fig. 418-2). Reduction in cardiovascular risk is of paramount importance because this is the leading cause of mortality in these individuals.

1	Type 2 DM management should begin with MNT (discussed above). An exercise regimen to increase insulin sensitivity and promote weight loss should also be instituted. Pharmacologic approaches to the management of type 2 DM include oral glucose-lowering agents, insulin, and other agents that improve glucose control; most physicians and patients prefer oral glucose-lowering agents as the initial choice. Any therapy that improves glycemic control reduces “glucose toxicity” to beta cells and improves endogenous insulin secretion. However, type 2 DM is a progressive disorder and ultimately requires multiple therapeutic agents and often insulin in most patients.

1	Glucose-Lowering Agents Advances in the therapy of type 2 DM have generated oral glucose-lowering agents that target different pathophysiologic processes in type 2 DM. Based on their mechanisms of action, glucose-lowering agents are subdivided into agents that increase insulin secretion, reduce glucose production, increase insulin sensitivity, enhance GLP-1 action, or promote urinary excretion of glucose (Table 418-5). Glucose-lowering agents other than insulin (with the exception of amylin analogue and α-glucosidase inhibitors) are ineffective in type 1 DM and should not be used for glucose management of severely ill individuals with type 2 DM. Insulin is sometimes the initial glucose-lowering agent in type 2 DM. BiGuanideS Metformin, representative of this class of agents, reduces hepatic glucose production and improves peripheral glucose utilization slightly (Table 418-5). Metformin activates AMP-dependent protein kinase and enters cells through organic cation transporters

1	FIGURE 418-2 Essential elements in comprehensive care of type 2 diabetes.

1	(polymorphisms of these may influence the response to metformin). 2413 Recent evidence indicates that metformin’s mechanism for reducing hepatic glucose production is to antagonize glucagon’s ability to generate cAMP in hepatocytes. Metformin reduces fasting plasma glucose (FPG) and insulin levels, improves the lipid profile, and promotes modest weight loss. An extended-release form is available and may have fewer gastrointestinal side effects (diarrhea, anorexia, nausea, metallic taste). Because of its relatively slow onset of action and gastrointestinal symptoms with higher doses, the initial dose should be low and then escalated every 2–3 weeks based on SMBG measurements. Metformin is effective as monotherapy and can be used in combination with other oral agents or with insulin. The major toxicity of metformin, lactic acidosis, is very rare and can be prevented by careful patient selection. Vitamin B12 levels are ~30% lower during metformin treatment. Metformin should not be used

1	toxicity of metformin, lactic acidosis, is very rare and can be prevented by careful patient selection. Vitamin B12 levels are ~30% lower during metformin treatment. Metformin should not be used in patients with renal insufficiency (glomerular filtration rate [GFR] <60 mL/min), any form of acidosis, unstable congestive heart failure (CHF), liver disease, or severe hypoxemia. Some feel that that these guidelines are too restrictive and prevent individuals with mild to moderate renal impairment from being safely treated with metformin. The National Institute for Health and Clinical Excellence in the United Kingdom suggests that metformin be used at a GFR >30 mL/min, with a reduced dose when the GFR is <45 mL/min. Metformin should be discontinued in hospitalized patients, in patients who can take nothing orally, and in those receiving radiographic contrast material. Insulin should be used until metformin can be restarted.

1	inSulin SecretaGoGueS—aGentS tHat affect tHe atp-SenSitive K+ cHannel Insulin secretagogues stimulate insulin secretion by interacting with the ATP-sensitive potassium channel on the beta cell (Chap. 417). These drugs are most effective in individuals with type 2 DM of relatively recent onset (<5 years) who have residual endogenous insulin production. First-generation sulfonylureas (chlorpropamide, tolazamide, tolbutamide) have a longer half-life, a greater incidence of hypoglycemia, and more frequent drug interactions, and are no longer used. Second-generation sulfonylureas have a more rapid onset of action and better coverage of the postprandial glucose rise, but the shorter half-life of some agents may require more than once-a-day dosing. Sulfonylureas reduce both fasting and postprandial glucose and should be initiated at low doses and increased at 1to 2-week intervals based on SMBG. In general, sulfonylureas increase insulin acutely and thus should be taken shortly before a meal;

1	glucose and should be initiated at low doses and increased at 1to 2-week intervals based on SMBG. In general, sulfonylureas increase insulin acutely and thus should be taken shortly before a meal; with chronic therapy, though, the insulin release is more sustained. Glimepiride and glipizide can be given in a single daily dose and are preferred over glyburide, especially in the elderly. Repaglinide, nateglinide, and mitiglinide are not sulfonylureas but also interact with the ATP-sensitive potassium channel. Because of their short half-life, these agents are given with each meal or immediately before to reduce meal-related glucose excursions.

1	Insulin secretagogues, especially the longer acting ones, have the potential to cause hypoglycemia, especially in elderly individuals. Hypoglycemia is usually related to delayed meals, increased physical activity, alcohol intake, or renal insufficiency. Individuals who ingest an overdose of some agents develop prolonged and serious hypoglycemia and should be monitored closely in the hospital (Chap. 420). Most sulfonylureas are metabolized in the liver to compounds (some of which are active) that are cleared by the kidney. Thus, their use in individuals with significant hepatic or renal dysfunction is not advisable. Weight gain, a common side effect of sulfonylurea therapy, results from the increased insulin levels and improvement in glycemic control. Some sulfonylureas have significant drug interactions with alcohol and some medications including warfarin, aspirin, ketoconazole, α-glucosidase inhibitors, and fluconazole. A related isoform of ATP-sensitive potassium channels is present

1	interactions with alcohol and some medications including warfarin, aspirin, ketoconazole, α-glucosidase inhibitors, and fluconazole. A related isoform of ATP-sensitive potassium channels is present in the myocardium and the brain. All of these agents except glyburide have a low affinity for this isoform. Despite concerns that this agent might affect the myocardial response to ischemia and observational studies suggesting that sulfonylureas increase cardiovascular risk, studies have not shown an increased cardiac mortality with glyburide or other agents in this class.

1	Diabetes Mellitus: Management and Therapies aExamples are approved for use in at least one country, but may not be available in the United States or all countries. Examples may not include all agents in the class. bHbA1c reduction (absolute) depends partly on starting HbA1c. cUsed for treatment of type 2 diabetes. dUsed in conjunction with insulin for treatment of type 1 diabetes. Cost of agent: *low, **moderate, ***high, ****variable. Note: Some agents used to treat type 2 DM are not included in table (see text). Abbreviations: ACE, angiotensin-converting enzyme; CHF, congestive heart failure; CV, cardiovascular; GI, gastrointestinal; HbA1c, hemoglobin A1c.

1	insulin secreTagogues—agenTs THaT enHance glP-1 recePTor signaling peptidase IV [DPP-IV]). Thus, exenatide has prolonged GLP-1-like “Incretins” amplify glucose-stimulated insulin secretion (Chap. 417). action and binds to GLP-1 receptors found in islets, the gastrointesti-Agents that either act as a GLP-1 receptor agonist or enhance endog-nal tract, and the brain. Liraglutide, another GLP-1 receptor agonist, enous GLP-1 activity are approved for the treatment of type 2 DM is almost identical to native GLP-1 except for an amino acid substitu(Table 418-5). Agents in this class do not cause hypoglycemia because tion and addition of a fatty acyl group (coupled with a γ-glutamic acid of the glucose-dependent nature of incretin-stimulated insulin secre-spacer) that promote binding to albumin and plasma proteins and protion (unless there is concomitant use of an agent that can lead to long its half-life. GLP-1 receptor agonists increase glucose-stimulated hypoglycemia—sulfonylureas, etc.).

1	plasma proteins and protion (unless there is concomitant use of an agent that can lead to long its half-life. GLP-1 receptor agonists increase glucose-stimulated hypoglycemia—sulfonylureas, etc.). Exenatide, a synthetic version of insulin secretion, suppress glucagon, and slow gastric emptying. These a peptide initially identified in the saliva of the Gila monster (exen-agents do not promote weight gain; in fact, most patients experience din-4), is an analogue of GLP-1. Unlike native GLP-1, which has a modest weight loss and appetite suppression. Treatment with these half-life of >5 min, differences in the exenatide amino acid sequence agents should start at a low dose to minimize initial side effects (nausea render it resistant to the enzyme that degrades GLP-1 (dipeptidyl being the limiting one). GLP-1 receptor agonists, available in twice daily, daily, and weekly injectable formulations, can be used as combination therapy with metformin, sulfonylureas, and thiazolidinediones. Some

1	one). GLP-1 receptor agonists, available in twice daily, daily, and weekly injectable formulations, can be used as combination therapy with metformin, sulfonylureas, and thiazolidinediones. Some patients taking insulin secretagogues may require a reduction in those agents to prevent hypoglycemia. The major side effects are nausea, vomiting, and diarrhea. Some formulations carry a black box warning from the FDA because of an increased risk of thyroid C-cell tumors in rodents and are contraindicated in individuals with medullary carcinoma of the thyroid or multiple endocrine neoplasia. Because GLP-1 receptor agonists slow gastric emptying, they may influence the absorption of other drugs. Whether GLP-1 receptor agonists enhance beta cell survival, promote beta cell proliferation, or alter the natural history of type 2 DM is not known. Other GLP-1 receptor agonists and formulations are under development.

1	DPP-IV inhibitors inhibit degradation of native GLP-1 and thus enhance the incretin effect. DPP-IV, which is widely expressed on the cell surface of endothelial cells and some lymphocytes, degrades a wide range of peptides (not GLP-1 specific). DPP-IV inhibitors promote insulin secretion in the absence of hypoglycemia or weight gain and appear to have a preferential effect on postprandial blood glucose. The levels of GLP-1 action in the patient are greater with the GLP-1 receptor agonists than with DPP-IV inhibitors. DPP-IV inhibitors are used either alone or in combination with other oral agents in type 2 DM. Reduced doses should be given to patients with renal insufficiency. Initial concerns about the pancreatic side effects of GLP-1 receptor agonists and DPP-IV inhibitors (pancreatitis, possible premalignant lesions) appear to be unfounded.

1	α-GlucoSidaSe inHiBitorS α-Glucosidase inhibitors reduce postprandial hyperglycemia by delaying glucose absorption; they do not affect glucose utilization or insulin secretion (Table 418-5). Postprandial hyperglycemia, secondary to impaired hepatic and peripheral glucose disposal, contributes significantly to the hyperglycemic state in type 2 DM. These drugs, taken just before each meal, reduce glucose absorption by inhibiting the enzyme that cleaves oligosaccharides into simple sugars in the intestinal lumen. Therapy should be initiated at a low dose with the evening meal and increased to a maximal dose over weeks to months. The major side effects (diarrhea, flatulence, abdominal distention) are related to increased delivery of oligosaccharides to the large bowel and can be reduced somewhat by gradual upward dose titration. α-Glucosidase inhibitors may increase levels of sulfonylureas and increase the incidence of hypoglycemia. Simultaneous treatment with bile acid resins and

1	somewhat by gradual upward dose titration. α-Glucosidase inhibitors may increase levels of sulfonylureas and increase the incidence of hypoglycemia. Simultaneous treatment with bile acid resins and antacids should be avoided. These agents should not be used in individuals with inflammatory bowel disease, gastroparesis, or a serum creatinine >177 μmol/L (2 mg/dL). This class of agents is not as potent as other oral agents in lowering the HbA1c but is unique because it reduces the postprandial glucose rise even in individuals with type 1 DM. If hypoglycemia from other diabetes treatments occurs while taking these agents, the patient should consume glucose because the degradation and absorption of complex carbohydrates will be retarded.

1	tHiazolidinedioneS Thiazolidinediones (Table 418-5) reduce insulin resistance by binding to the PPAR-γ (peroxisome proliferator–activated receptor γ) nuclear receptor (which forms a heterodimer with the retinoid X receptor). The PPAR-γ receptor is found at highest levels in adipocytes but is expressed at lower levels in many other tissues. Agonists of this receptor regulate a large number of genes, promote adipocyte differentiation, reduce hepatic fat accumulation, and promote fatty acid storage. Thiazolidinediones promote a redistribution of fat from central to peripheral locations. Circulating insulin levels decrease with use of the thiazolidinediones, indicating a reduction in insulin resistance. Although direct comparisons are not available, the two currently available thiazolidinediones appear to have similar efficacy. The prototype of this class of drugs, troglitazone, was withdrawn from the U.S. market after reports of hepatotoxicity and an association with an idiosyncratic

1	appear to have similar efficacy. The prototype of this class of drugs, troglitazone, was withdrawn from the U.S. market after reports of hepatotoxicity and an association with an idiosyncratic liver reaction that sometimes led to hepatic failure. Although rosiglitazone and pioglitazone do not appear to induce the liver abnormalities seen with troglitazone, the FDA recommends measurement of liver function tests prior to initiating therapy.

1	Rosiglitazone raises low-density lipoprotein (LDL), high-density 2415 lipoprotein (HDL), and triglycerides slightly. Pioglitazone raises HDL to a greater degree and LDL a lesser degree but lowers triglycerides. The clinical significance of the lipid changes with these agents is not known and may be difficult to ascertain because most patients with type 2 DM are also treated with a statin.

1	Thiazolidinediones are associated with weight gain (2–3 kg), a small reduction in the hematocrit, and a mild increase in plasma volume. Peripheral edema and CHF are more common in individuals treated with these agents. These agents are contraindicated in patients with liver disease or CHF (class III or IV). The FDA has issued an alert that rare patients taking these agents may experience a worsening of diabetic macular edema. An increased risk of fractures has been noted in women taking these agents. Thiazolidinediones have been shown to induce ovulation in premenopausal women with polycystic ovary syndrome. Women should be warned about the risk of pregnancy because the safety of thiazolidinediones in pregnancy is not established.

1	Concerns about increased cardiovascular risk associated with rosiglitazone led to considerable restrictions on its use and to the FDA issuing a “black box” warning in 2007. However, based on new information, the FDA has revised its guidelines and categorizes rosiglitazone similar to other drugs for type 2 DM. Because of a possible increased risk of bladder cancer, pioglitazone is part of an ongoing FDA safety review.

1	(Table 418-5) lower the blood glucose by selectively inhibiting this co-transporter, which is expressed almost exclusively in the proximal, convoluted tubule in the kidney. This inhibits glucose reabsorption, lowers the renal threshold for glucose, and leads to increased urinary glucose excretion. Thus, the glucose-lowering effect is insulin independent and not related to changes in insulin sensitivity or secretion. Because these agents are the newest class to treat type 2 DM (Table 418-5), clinical experience is limited. Due to the increased urinary glucose, urinary or vaginal infections are more common, and the diuretic effect can lead to reduced intravascular volume. As part of the FDA approval of canagliflozin in 2013, postmarketing studies for cardiovascular outcomes and for monitoring bladder and urinary cancer risk are under way.

1	Bile acid–binding resins Evidence indicates that bile acids, by signaling through nuclear receptors, may have a role in metabolism. Bile acid metabolism is abnormal in type 2 DM. The bile acid–binding resin colesevelam has been approved for the treatment of type 2 DM (already approved for treatment of hypercholesterolemia). Because bile acid–binding resins are minimally absorbed into the systemic circulation, how bile acid–binding resins lower blood glucose is not known. The most common side effects are gastrointestinal (constipation, abdominal pain, and nausea). Bile acid–binding resins can increase plasma triglycerides and should be used cautiously in patients with a tendency for hypertriglyceridemia. The role of this class of drugs in the treatment of type 2 DM is not yet defined.

1	Bromocriptine A formulation of the dopamine receptor agonist bromocriptine (Cycloset) has been approved by the FDA for the treatment of type 2 DM. However, its role in the treatment of type 2 DM is uncertain. insulin THeraPy in TyPe 2 dm Insulin should be considered as the initial therapy in type 2 DM, particularly in lean individuals or those with severe weight loss, in individuals with underlying renal or hepatic disease that precludes oral glucose-lowering agents, or in individuals who are hospitalized or acutely ill. Insulin therapy is ultimately required by a substantial number of individuals with type 2 DM because of the progressive nature of the disorder and the relative insulin deficiency that develops in patients with long-standing diabetes. Both physician and patient reluctance often delay the initiation of insulin therapy, but glucose control and patient well-being are improved by insulin therapy in patients who have not reached the glycemic target.

1	Diabetes Mellitus: Management and Therapies 2416 Because endogenous insulin secretion continues and is capable of providing some coverage of mealtime caloric intake, insulin is usually initiated in a single dose of long-acting insulin (0.3–0.4 U/kg per day), given in the evening (NPH) or just before bedtime (NPH, glargine, detemir). Because fasting hyperglycemia and increased hepatic glucose production are prominent features of type 2 DM, bedtime insulin is more effective in clinical trials than a single dose of morning insulin. Glargine given at bedtime has less nocturnal hypoglycemia than NPH insulin. Some physicians prefer a relatively low, fixed starting dose of long-acting insulin (5–15 units) or a weight-based dose (0.2 units/kg). The insulin dose may then be adjusted in 10% increments as dictated by SMBG results. Both morning and bedtime long-acting insulin may be used in combination with oral glucose-lowering agents. Initially, basal insulin may be sufficient, but often

1	as dictated by SMBG results. Both morning and bedtime long-acting insulin may be used in combination with oral glucose-lowering agents. Initially, basal insulin may be sufficient, but often prandial insulin coverage with multiple insulin injections is needed as diabetes progresses (see insulin regimens used for type 1 DM). Other insulin formulations that have a combination of short-acting and long-acting insulin (Table 418-4) are sometimes used in patients with type 2 DM because of convenience but do not allow independent adjustment of short-acting and long-acting insulin dose and often do not achieve the same degree of glycemic control as basal/bolus regimens. In selected patients with type 2 DM, insulin-infusion devices may be considered.

1	cHoice of initial GlucoSe-loWerinG aGent The level of hyperglycemia and the patient’s individualized goal (see “Establishment of Target Level of Glycemic Control”) should influence the initial choice of therapy. Assuming that maximal benefit of MNT and increased physical activity has been realized, patients with mild to moderate hyperglycemia (FPG <11.1–13.9 mmol/L [200–250 mg/dL]) often respond well to a single, oral glucose-lowering agent. Patients with more severe hyperglycemia (FPG >13.9 mmol/L [250 mg/dL]) may respond partially but are unlikely to achieve normoglycemia with oral monotherapy. A stepwise approach that starts with a single agent and adds a second agent to achieve the glycemic target can be used (see “Combination therapy with glucose-lowering agents,” below). Insulin can be used as initial therapy in individuals with severe hyperglycemia (FPG <13.9–16.7 mmol/L [250–300 mg/dL]) or in those who are symptomatic from the hyperglycemia. This approach is based on the

1	can be used as initial therapy in individuals with severe hyperglycemia (FPG <13.9–16.7 mmol/L [250–300 mg/dL]) or in those who are symptomatic from the hyperglycemia. This approach is based on the rationale that more rapid glycemic control will reduce “glucose toxicity” to the islet cells, improve endogenous insulin secretion, and possibly allow oral glucose-lowering agents to be more effective. If this occurs, the insulin may be discontinued.

1	Insulin secretagogues, biguanides, α-glucosidase inhibitors, thiazolidinediones, GLP-1 receptor agonists, DPP-IV inhibitors, SLGT2 inhibitors, and insulin are approved for monotherapy of type 2 DM. Although each class of oral glucose-lowering agents has advantages and disadvantages (Table 418-5), certain generalizations apply: (1) insulin secretagogues, biguanides, GLP-1 receptor agonists, and thiazolidinediones improve glycemic control to a similar degree (1–2% reduction in HbA1c) and are more effective than α-glucosidase inhibitors, DPP-IV inhibitors, and SLGT2 inhibitors; (2) assuming a similar degree of glycemic improvement, no clinical advantage to one class of drugs has been demonstrated; any therapy that improves glycemic control is likely beneficial; (3) insulin secretagogues, GLP-1 receptor agonists, DPP-IV inhibitors, α-glucosidase inhibitors, and SLGT2 inhibitors begin to lower the plasma glucose immediately, whereas the glucose-lowering effects of the biguanides and

1	GLP-1 receptor agonists, DPP-IV inhibitors, α-glucosidase inhibitors, and SLGT2 inhibitors begin to lower the plasma glucose immediately, whereas the glucose-lowering effects of the biguanides and thiazolidinediones are delayed by weeks; (4) not all agents are effective in all individuals with type 2 DM; (5) biguanides, α-glucosidase inhibitors, GLP-1 receptor agonists, DPP-IV inhibitors, thiazolidinediones, and SLGT2 inhibitors do not directly cause hypoglycemia; (6) most individuals will eventually require treatment with more than one class of oral glucose-lowering agents or insulin, reflecting the progressive nature of type 2 DM; and (7) durability of glycemic control is slightly less for glyburide compared to metformin or rosiglitazone.

1	Considerable clinical experience exists with metformin and sulfonylureas because they have been available for several decades. It is assumed that the α-glucosidase inhibitors, GLP-1 agonists, DPP-IV inhibitors, thiazolidinediones, and SLGT2 inhibitors will reduce DM-related complications by improving glycemic control, but longterm data are not yet available. The thiazolidinediones are theoretically attractive because they target a fundamental abnormality in type 2 DM, namely insulin resistance. However, all of these agents are currently more costly than metformin and sulfonylureas. Patient with type 2 diabetes Individualized glycemic goal Medical nutrition therapy, increased physical activity, weight loss + metformin

1	Patient with type 2 diabetes Individualized glycemic goal Medical nutrition therapy, increased physical activity, weight loss + metformin Insulin + metformin Reassess HbA1c Reassess HbA1c Reassess HbA1c Combination therapy -metformin + second agent Combination therapy -metformin + two other agents FIGURE 418-3 Glycemic management of type 2 diabetes. See text for discussion of treatment of severe hyperglycemia or symptomatic hyperglycemia. Agents that can be combined with metformin include insulin secretagogues, thiazolidinediones, α-glucosidase inhibitors, DPP-IV inhibitors, GLP-1 receptor agonists, SLGT2 inhibitors, and insu-lin. HbA1c, hemoglobin HbA1c.

1	Treatment algorithms by several professional societies (ADA/ European Association for the Study of Diabetes [EASD], IDF, AACE) suggest metformin as initial therapy because of its efficacy, known side effect profile, and low cost (Fig. 418-3). Metformin’s advantages are that it promotes mild weight loss, lowers insulin levels, and improves the lipid profile slightly. Based on SMBG results and the HbA1c, the dose of metformin should be increased until the glycemic target is achieved or maximum dose is reached. If metformin is not tolerated, then initial therapy with an insulin secretagogue or DPP-IV inhibitor is reasonable.

1	comBination tHerapy WitH GlucoSe-loWerinG aGentS A number of combinations of therapeutic agents are successful in type 2 DM (metformin + second oral agent, metformin + GLP-1 receptor agonist, or metformin + insulin), and the dosing of agents in combination is the same as when the agents are used alone. Because mechanisms of action of the first and second agents should be different, the effect on glycemic control is usually additive. There are little data to support the choice of one combination over another combination. Medication costs vary considerably (Table 418-5), and this often factors into medication choice. Several fixed-dose combinations of oral agents are available, but evidence that they are superior to titration of single agent to a maximum dose and then addition of a second agent is lacking. If adequate control is not achieved with the combination of two agents (based on reassessment of the HbA1c every 3 months), a third oral agent or basal insulin should be added (Fig.

1	agent is lacking. If adequate control is not achieved with the combination of two agents (based on reassessment of the HbA1c every 3 months), a third oral agent or basal insulin should be added (Fig. 418-3). Treatment approaches vary considerably from country to country. For example, α-glucosidase inhibitors are used commonly in South Asian patients (Indian), but infrequently in the United States or Europe. Whether this reflects an underlying difference in the disease or physician preference is not clear.

1	Treatment with insulin becomes necessary as type 2 DM enters the phase of relative insulin deficiency (as seen in long-standing DM) and is signaled by inadequate glycemic control with one or two oral glucose-lowering agents. Insulin alone or in combination should be used in patients who fail to reach the glycemic target. For example, a single dose of long-acting insulin at bedtime is often effective in combination with metformin. In contrast, insulin secretagogues have little utility once insulin therapy is started. Experience using incretin therapies and insulin is limited. As endogenous insulin production falls further, multiple injections of long-acting and short-acting insulin regimens are necessary to control postprandial glucose excursions. These insulin regimens are identical to the long-acting and short-acting combination regimens discussed above for type 1 DM. Because the hyperglycemia of type 2 DM tends to be more “stable,” these regimens can be increased in 10% increments

1	long-acting and short-acting combination regimens discussed above for type 1 DM. Because the hyperglycemia of type 2 DM tends to be more “stable,” these regimens can be increased in 10% increments every 2–3 days using the fasting blood glucose results. Weight gain and hypoglycemia are the major adverse effects of insulin therapy. The daily insulin dose required can become quite large (1–2 units/kg per day) as endogenous insulin production falls and insulin resistance persists. Individuals who require >1 unit/kg per day of long-acting insulin should be considered for combination therapy with metformin or a thiazolidinedione. The addition of metformin or a thiazolidinedione can reduce insulin requirements in some individuals with type 2 DM, while maintaining or even improving glycemic control. Insulin plus a thiazolidinedione promotes weight gain and is associated with peripheral edema. Addition of a thiazolidinedione to a patient’s insulin regimen may necessitate a reduction in the

1	Insulin plus a thiazolidinedione promotes weight gain and is associated with peripheral edema. Addition of a thiazolidinedione to a patient’s insulin regimen may necessitate a reduction in the insulin dose to avoid hypoglycemia. Patients requiring large doses of insulin (>200 units/day) can be treated with a more concentrated form of insulin, U-500.

1	Whole pancreas transplantation (performed concomitantly with a renal transplant) may normalize glucose tolerance and is an important therapeutic option in type 1 DM with end-stage renal disease, although it requires substantial expertise and is associated with the side effects of immunosuppression. Pancreatic islet transplantation has been plagued by limitations in pancreatic islet supply and graft survival and remains an area of clinical investigation. Many individuals with longstanding type 1 DM still produce very small amounts of insulin or have insulin-positive cells within the pancreas. This suggests that beta cells may slowly regenerate but are quickly destroyed by the autoimmune process. Thus, efforts to suppress the autoimmune process and to stimulate beta cell regeneration are being tested both at the time of diagnosis and in years after the diagnosis of type 1 DM. Closed-loop pumps that infuse the appropriate amount of insulin in response to changing glucose levels are

1	being tested both at the time of diagnosis and in years after the diagnosis of type 1 DM. Closed-loop pumps that infuse the appropriate amount of insulin in response to changing glucose levels are potentially feasible now that CGM technology has been developed. Bi-hormonal pumps that deliver both insulin and glucagon are under development. New therapies under development for type 2 DM include activators of glucokinase, inhibitors of 11 β-hydroxysteroid dehydrogenase-1, GPR40 agonists, monoclonal antibodies to reduce inflammation, and salsalate.

1	Bariatric surgery for obese individuals with type 2 DM has shown considerable promise, sometimes with dramatic resolution of the diabetes or major reductions in the needed dose of glucose-lowering therapies (Chap. 416). Several large, unblinded clinical trials have demonstrated a much greater efficacy of bariatric surgery compared to medical management in the treatment of type 2 DM; the durability of the diabetes reversal or improvement is uncertain. The ADA clinical guidelines state that bariatric surgery should be considered in individuals with DM and a body mass index >35 kg/m2.

1	As with any therapy, the benefits of efforts directed toward glycemic control must be balanced against the risks of treatment (Table 418-5). Side effects of intensive treatment include an increased frequency of serious hypoglycemia, weight gain, increased economic costs, and greater demands on the patient. In the DCCT, quality of life was very similar in 2417 the intensive and standard therapy groups. The most serious complication of therapy for DM is hypoglycemia, and its treatment with oral glucose or glucagon injection is discussed in Chap. 420. Severe, recurrent hypoglycemia warrants examination of treatment regimen and glycemic goal for the individual patient. Weight gain occurs with most (insulin, insulin secretagogues, thiazolidinediones) but not all (metformin, α-glucosidase inhibitors, GLP-1 receptor agonists, DPP-IV inhibitors) therapies. The weight gain is partially due to the anabolic effects of insulin and the reduction in glucosuria. As a result of recent controversies

1	GLP-1 receptor agonists, DPP-IV inhibitors) therapies. The weight gain is partially due to the anabolic effects of insulin and the reduction in glucosuria. As a result of recent controversies about the optimal glycemic goal and concerns about safety, the FDA now requires information about the cardiovascular safety profile as part of its evaluation of new treatments for type 2 DM.

1	Individuals with type 1 or type 2 DM and severe hyperglycemia (>16.7 mmol/L [300 mg/dL]) should be assessed for clinical stability, including mentation and hydration. Depending on the patient and the rapidity and duration of the severe hyperglycemia, an individual may require more intense and rapid therapy to lower the blood glucose. However, many patients with poorly controlled diabetes and hyperglycemia have few symptoms. The physician should assess if the patient is stable or if diabetic ketoacidosis or a hyperglycemic hyperosmolar state should be considered. Ketones, an indicator of diabetic ketoacidosis, should be measured in individuals with type 1 DM when the plasma glucose is >16.7 mmol/L (300 mg/dL), during a concurrent illness, or with symptoms such as nausea, vomiting, or abdominal pain. Blood measurement of β-hydroxybutyrate is preferred over urine testing with nitroprusside-based assays that measure only acetoacetate and acetone.

1	Diabetic ketoacidosis (DKA) and hyperglycemic hyperosmolar state (HHS) are acute, severe disorders directly related to diabetes. DKA was formerly considered a hallmark of type 1 DM, but also occurs in individuals who lack immunologic features of type 1 DM and who can sometimes subsequently be treated with oral glucose-lowering agents (these obese individuals with type 2 DM are often of Hispanic or African-American descent). HHS is primarily seen in individuals with type 2 DM. Both disorders are associated with absolute or relative insulin deficiency, volume depletion, and acid-base abnormalities. DKA and HHS exist along a continuum of hyperglycemia, with or without ketosis. The metabolic similarities and differences in DKA and HHS are highlighted in Table 418-6. Both Diabetes Mellitus: Management and Therapies aLarge changes occur during treatment of DKA. bAlthough plasma levels may be normal or high at presentation, total-body stores are usually depleted.

1	Diabetes Mellitus: Management and Therapies aLarge changes occur during treatment of DKA. bAlthough plasma levels may be normal or high at presentation, total-body stores are usually depleted. 2418 disorders are associated with potentially serious complications if not promptly diagnosed and treated.

1	DIABETIC KETOACIDOSIS Clinical Features The symptoms and physical signs of DKA are listed in Table 418-7 and usually develop over 24 h. DKA may be the initial symptom complex that leads to a diagnosis of type 1 DM, but more frequently, it occurs in individuals with established diabetes. Nausea and vomiting are often prominent, and their presence in an individual with diabetes warrants laboratory evaluation for DKA. Abdominal pain may be severe and can resemble acute pancreatitis or ruptured viscus. Hyperglycemia leads to glucosuria, volume depletion, and tachycardia. Hypotension can occur because of volume depletion in combination with peripheral vasodilatation. Kussmaul respirations and a fruity odor on the patient’s breath (secondary to metabolic acidosis and increased acetone) are classic signs of the disorder. Lethargy and central nervous system depression may evolve into coma with severe DKA but should also prompt evaluation for other reasons for altered mental status (e.g.,

1	classic signs of the disorder. Lethargy and central nervous system depression may evolve into coma with severe DKA but should also prompt evaluation for other reasons for altered mental status (e.g., infection, hypoxemia). Cerebral edema, an extremely serious complication of DKA, is seen most frequently in children. Signs of infection, which may precipitate DKA, should be sought on physical examination, even in the absence of fever. Tissue ischemia (heart, brain) can also be a precipitating factor. Omission of insulin because of an eating disorder, mental health disorders, or an unstable psychosocial environment may sometimes be a factor precipitating DKA.

1	Pathophysiology DKA results from relative or absolute insulin deficiency combined with counterregulatory hormone excess (glucagon, catecholamines, cortisol, and growth hormone). Both insulin deficiency and glucagon excess, in particular, are necessary for DKA to develop. The decreased ratio of insulin to glucagon promotes gluconeogenesis, glycogenolysis, and ketone body formation in the liver, as well as increases in substrate delivery from fat and muscle (free fatty acids, amino acids) to the liver. Markers of inflammation (cytokines, C-reactive protein) are elevated in both DKA and HHS

1	The combination of insulin deficiency and hyperglycemia reduces the hepatic level of fructose-2,6-bisphosphate, which alters the activity of phosphofructokinase and fructose-1,6-bisphosphatase. Glucagon excess decreases the activity of pyruvate kinase, whereas insulin deficiency increases the activity of phosphoenolpyruvate carboxykinase. These changes shift the handling of pyruvate toward glucose synthesis and away from glycolysis. The increased levels of glucagon and catecholamines in the face of low insulin levels promote glycogenolysis. Insulin deficiency also reduces levels of the GLUT4 glucose transporter, which impairs glucose uptake into skeletal muscle and fat and reduces intracellular glucose metabolism.

1	Ketosis results from a marked increase in free fatty acid release from adipocytes, with a resulting shift toward ketone body synthesis in the liver. Reduced insulin levels, in combination with elevations in catecholamines and growth hormone, increase lipolysis and the mAnifESTATionS of DiAbETiC kEToACiDoSiS Shortness of breath Abdominal tenderness (may edema/possibly coma Infarction (cerebral, coronary, mesenteric, peripheral) Abbreviation: UTI, urinary tract infection.

1	Abbreviation: UTI, urinary tract infection. release of free fatty acids. Normally, these free fatty acids are converted to triglycerides or very-low-density lipoprotein (VLDL) in the liver. However, in DKA, hyperglucagonemia alters hepatic metabolism to favor ketone body formation, through activation of the enzyme carnitine palmitoyltransferase I. This enzyme is crucial for regulating fatty acid transport into the mitochondria, where beta oxidation and conversion to ketone bodies occur. At physiologic pH, ketone bodies exist as ketoacids, which are neutralized by bicarbonate. As bicarbonate stores are depleted, metabolic acidosis ensues. Increased lactic acid production also contributes to the acidosis. The increased free fatty acids increase triglyceride and VLDL production. VLDL clearance is also reduced because the activity of insulin-sensitive lipoprotein lipase in muscle and fat is decreased. Hypertriglyceridemia may be severe enough to cause pancreatitis.

1	DKA is often precipitated by increased insulin requirements, as occurs during a concurrent illness (Table 418-7). Failure to augment insulin therapy often compounds the problem. Complete omission or inadequate administration of insulin by the patient or health care team (in a hospitalized patient with type 1 DM) may precipitate DKA. Patients using insulin-infusion devices with short-acting insulin may develop DKA, because even a brief interruption in insulin delivery (e.g., mechanical malfunction) quickly leads to insulin deficiency.

1	Laboratory Abnormalities and Diagnosis The timely diagnosis of DKA is crucial and allows for prompt initiation of therapy. DKA is characterized by hyperglycemia, ketosis, and metabolic acidosis (increased anion gap) along with a number of secondary metabolic derangements (Table 418-6). Occasionally, the serum glucose is only minimally elevated. Serum bicarbonate is frequently <10 mmol/L, and arterial pH ranges between 6.8 and 7.3, depending on the severity of the acidosis. Despite a total-body potassium deficit, the serum potassium at presentation may be mildly elevated, secondary to the acidosis. Total-body stores of sodium, chloride, phosphorus, and magnesium are reduced in DKA but are not accurately reflected by their levels in the serum because of hypovolemia and hyperglycemia. Elevated blood urea nitrogen (BUN) and serum creatinine levels reflect intravascular volume depletion. Interference from acetoacetate may falsely elevate the serum creatinine measurement. Leukocytosis,

1	blood urea nitrogen (BUN) and serum creatinine levels reflect intravascular volume depletion. Interference from acetoacetate may falsely elevate the serum creatinine measurement. Leukocytosis, hypertriglyceridemia, and hyperlipoproteinemia are commonly found as well. Hyperamylasemia may suggest a diagnosis of pancreatitis, especially when accompanied by abdominal pain. However, in DKA the amylase is usually of salivary origin and thus is not diagnostic of pancreatitis. Serum lipase should be obtained if pancreatitis is suspected.

1	The measured serum sodium is reduced as a consequence of the hyperglycemia (1.6-mmol/L [1.6-meq] reduction in serum sodium for each 5.6-mmol/L [100-mg/dL] rise in the serum glucose). A normal serum sodium in the setting of DKA indicates a more profound water deficit. In “conventional” units, the calculated serum osmolality (2 × [serum sodium + serum potassium] + plasma glucose [mg/dL]/ 18 + BUN/2.8) is mildly to moderately elevated, although to a lesser degree than that found in HHS (see below).

1	In DKA, the ketone body, β-hydroxybutyrate, is synthesized at a threefold greater rate than acetoacetate; however, acetoacetate is preferentially detected by a commonly used ketosis detection reagent (nitroprusside). Serum ketones are present at significant levels (usually positive at serum dilution of ≥1:8). The nitroprusside tablet, or stick, is often used to detect urine ketones; certain medications such as captopril or penicillamine may cause false-positive reactions. Serum or plasma assays for β-hydroxybutyrate are preferred because they more accurately reflect the true ketone body level.

1	The metabolic derangements of DKA exist along a spectrum, beginning with mild acidosis with moderate hyperglycemia evolving into more severe findings. The degree of acidosis and hyperglycemia do not necessarily correlate closely because a variety of factors determine the level of hyperglycemia (oral intake, urinary glucose loss). Ketonemia is a consistent finding in DKA and distinguishes it from simple hyperglycemia. The differential diagnosis of DKA includes starvation ketosis, alcoholic ketoacidosis (bicarbonate usually >15 meq/L), and other forms of increased anion-gap acidosis (Chap. 66).

1	The management of DKA is outlined in Table 418-8. After initiating IV fluid replacement and insulin therapy, the agent or event that precipitated the episode of DKA should be sought and aggressively treated. If the patient is vomiting or has altered mental status, a nasogastric tube should be inserted to prevent aspiration of gastric contents. Central to successful treatment of DKA is careful monitoring and frequent reassessment to ensure that the patient and the metabolic derangements are improving. A comprehensive flow sheet should record chronologic changes in vital signs, fluid intake and output, and laboratory values as a function of insulin administered.

1	After the initial bolus of normal saline, replacement of the sodium and free water deficit is carried out over the next 24 h (fluid deficit is often 3–5 L). When hemodynamic stability and adequate urine output are achieved, IV fluids should be switched to 0.45% saline depending on the calculated volume deficit. The change to 0.45% saline helps to reduce the trend toward hyperchloremia later in the course of DKA. Alternatively, initial use of lactated Ringer’s IV solution may reduce the hyperchloremia that commonly occurs with normal saline.

1	A bolus of IV (0.1 units/kg) short-acting insulin should be administered immediately (Table 418-8), and subsequent treatment should provide continuous and adequate levels of circulating insulin. IV administration is preferred (0.1 units/kg of regular insulin per hour) because it ensures rapid distribution and allows adjustment of the infusion rate as the patient responds to therapy. In mild episodes of DKA, short-acting insulin can be used SC. IV insulin mAnAgEmEnT of DiAbETiC kEToACiDoSiS 1. Confirm diagnosis (↑ plasma glucose, positive serum ketones, metabolic acidosis). 2. Admit to hospital; intensive care setting may be necessary for frequent monitoring or if pH <7.00 or unconscious. 3. Assess: Serum electrolytes (K+, Na+, Mg2+, Cl−, bicarbonate, phosphate) Acid-base status—pH, HCO3−, PCO2, β-hydroxybutyrate Renal function (creatinine, urine output) 4.

1	3. Assess: Serum electrolytes (K+, Na+, Mg2+, Cl−, bicarbonate, phosphate) Acid-base status—pH, HCO3−, PCO2, β-hydroxybutyrate Renal function (creatinine, urine output) 4. Replace fluids: 2–3 L of 0.9% saline over first 1–3 h (10–20 mL/kg per hour); subsequently, 0.45% saline at 250–500 mL/h; change to 5% glucose and 0.45% saline at 150–250 mL/h when plasma glucose reaches 250 mg/dL (13.9 mmol/L). 5. Administer short-acting insulin: IV (0.1 units/kg), then 0.1 units/kg per hour by continuous IV infusion; increase twoto threefold if no response by 2–4 h. If the initial serum potassium is <3.3 mmol/L (3.3 meq/L), do not administer insulin until the potassium is corrected. 6. Assess patient: What precipitated the episode (noncompliance, infection, trauma, pregnancy, infarction, cocaine)? Initiate appropriate workup for precipitating event (cultures, CXR, ECG). 7.

1	6. Assess patient: What precipitated the episode (noncompliance, infection, trauma, pregnancy, infarction, cocaine)? Initiate appropriate workup for precipitating event (cultures, CXR, ECG). 7. Measure capillary glucose every 1–2 h; measure electrolytes (especially K+, bicarbonate, phosphate) and anion gap every 4 h for first 24 h. 8. Monitor blood pressure, pulse, respirations, mental status, fluid intake and output every 1–4 h. 9. Replace K+: 10 meq/h when plasma K+ <5.0–5.2 meq/L (or 20–30 meq/L of infusion fluid), ECG normal, urine flow and normal creatinine documented; administer 40–80 meq/h when plasma K+ <3.5 meq/L or if bicarbonate is given. If initial serum potassium is >5.2 mmol/L (5.2 meq/L), do not supplement K+ until the potassium is corrected. 10. See text about bicarbonate or phosphate supplementation. 11.

1	10. See text about bicarbonate or phosphate supplementation. 11. Continue above until patient is stable, glucose goal is 8.3–13.9 mmol/L (150–250 mg/dL), and acidosis is resolved. Insulin infusion may be decreased to 0.05–0.1 units/kg per hour. 12. Administer long-acting insulin as soon as patient is eating. Allow for a 2–4 hour overlap in insulin infusion and SC insulin injection. Abbreviations: CXR, chest x-ray; ECG, electrocardiogram. Source: Adapted from M Sperling, in Therapy for Diabetes Mellitus and Related Disorders, American Diabetes Association, Alexandria, VA, 1998; and AE Kitabchi et al: Diabetes Care 32:1335, 2009.

1	Source: Adapted from M Sperling, in Therapy for Diabetes Mellitus and Related Disorders, American Diabetes Association, Alexandria, VA, 1998; and AE Kitabchi et al: Diabetes Care 32:1335, 2009. should be continued until the acidosis resolves and the patient is 2419 metabolically stable. As the acidosis and insulin resistance associated with DKA resolve, the insulin infusion rate can be decreased (to 0.05–0.1 units/kg per hour). Long-acting insulin, in combination with SC short-acting insulin, should be administered as soon as the patient resumes eating, because this facilitates transition to an outpatient insulin regimen and reduces length of hospital stay. It is crucial to continue the insulin infusion until adequate insulin levels are achieved by administering long-acting insulin by the SC route. Even relatively brief periods of inadequate insulin administration in this transition phase may result in DKA relapse.

1	Hyperglycemia usually improves at a rate of 4.2–5.6 mmol/L (75–100 mg/dL) per hour as a result of insulin-mediated glucose disposal, reduced hepatic glucose release, and rehydration. The latter reduces catecholamines, increases urinary glucose loss, and expands the intravascular volume. The decline in the plasma glucose within the first 1–2 h may be more rapid and is mostly related to volume expansion. When the plasma glucose reaches 13.9 mmol/L (250 mg/dL), glucose should be added to the 0.45% saline infusion to maintain the plasma glucose in the 8.3–13.9 mmol/L (150–250 mg/dL) range, and the insulin infusion should be continued. Ketoacidosis begins to resolve as insulin reduces lipolysis, increases peripheral ketone body use, suppresses hepatic ketone body formation, and promotes bicarbonate regeneration. However, the acidosis and ketosis resolve more slowly than hyperglycemia. As ketoacidosis improves, β-hydroxybutyrate is converted to ace toacetate. Ketone body levels may appear

1	regeneration. However, the acidosis and ketosis resolve more slowly than hyperglycemia. As ketoacidosis improves, β-hydroxybutyrate is converted to ace toacetate. Ketone body levels may appear to increase if measured by laboratory assays that use the nitroprusside reaction, which only detects acetoacetate and acetone. The improvement in acidosis and anion gap, a result of bicarbonate regeneration and decline in ketone bodies, is reflected by a rise in the serum bicarbonate level and the arterial pH. Depending on the rise of serum chloride, the anion gap (but not bicarbonate) will normalize. A hyperchloremic acidosis (serum bicarbonate of 15–18 mmol/L [15–18 meq/L]) often follows successful treatment and gradually resolves as the kidneys regenerate bicarbonate and excrete chloride.

1	Potassium stores are depleted in DKA (estimated deficit 3–5 mmol/kg [3–5 meq/kg]). During treatment with insulin and fluids, various factors contribute to the development of hypokalemia. These include insulin-mediated potassium transport into cells, resolution of the acidosis (which also promotes potassium entry into cells), and urinary loss of potassium salts of organic acids. Thus, potassium repletion should commence as soon as adequate urine output and a normal serum potassium are documented. If the initial serum potassium level is elevated, then potassium repletion should be delayed until the potassium falls into the normal range. Inclusion of 20–40 meq of potassium in each liter of IV fluid is reasonable, but additional potassium supplements may also be required. To reduce the amount of chloride administered, potassium phosphate or acetate can be substituted for the chloride salt. The goal is to maintain the serum potassium at >3.5 mmol/L (3.5 meq/L).

1	Despite a bicarbonate deficit, bicarbonate replacement is not usually necessary. In fact, theoretical arguments suggest that bicarbonate administration and rapid reversal of acidosis may impair cardiac function, reduce tissue oxygenation, and promote hypokalemia. The results of most clinical trials do not support the routine use of bicarbonate replacement, and one study in children found that bicarbonate use was associated with an increased risk of cerebral edema. However, in the presence of severe acidosis (arterial pH <7.0), the ADA advises bicarbonate (50 mmol/L [meq/L] of sodium bicarbonate in 200 mL of sterile water with 10 meq/L KCl per hour for 2 h until the pH is >7.0). Hypophosphatemia may result from increased glucose usage, but randomized clinical trials have not demonstrated that phosphate replacement is beneficial in DKA. If the serum phosphate is <0.32 mmol/L (1 mg/dL), then phosphate supplement should be considered and the serum calcium monitored. Hypomagnesemia may

1	that phosphate replacement is beneficial in DKA. If the serum phosphate is <0.32 mmol/L (1 mg/dL), then phosphate supplement should be considered and the serum calcium monitored. Hypomagnesemia may develop during DKA therapy and may also require supplementation.

1	Diabetes Mellitus: Management and Therapies 2420 With appropriate therapy, the mortality rate of DKA is low (<1%) and is related more to the underlying or precipitating event, such as infection or myocardial infarction. Venous thrombosis, upper gastrointestinal bleeding, and acute respiratory distress syndrome occasionally complicate DKA. The major nonmetabolic complication of DKA therapy is cerebral edema, which most often develops in children as DKA is resolving. The etiology of and optimal therapy for cerebral edema are not well established, but overreplacement of free water should be avoided. Following treatment, the physician and patient should review the sequence of events that led to DKA to prevent future recurrences. Foremost is patient education about the symptoms of DKA, its precipitating factors, and the management of diabetes during a concurrent illness. During illness or when oral intake is compromised, patients should (1) frequently measure the capillary blood glucose;

1	factors, and the management of diabetes during a concurrent illness. During illness or when oral intake is compromised, patients should (1) frequently measure the capillary blood glucose; (2) measure urinary ketones when the serum glucose is >16.5 mmol/L (300 mg/dL); (3) drink fluids to maintain hydration; (4) continue or increase insulin; and (5) seek medical attention if dehydration, persistent vomiting, or uncontrolled hyperglycemia develop. Using these strategies, early DKA can be prevented or detected and treated appropriately on an outpatient basis.

1	HYPERGLYCEMIC HYPEROSMOLAR STATE Clinical Features The prototypical patient with HHS is an elderly individual with type 2 DM, with a several-week history of polyuria, weight loss, and diminished oral intake that culminates in mental confusion, lethargy, or coma. The physical examination reflects profound dehydration and hyperosmolality and reveals hypotension, tachycardia, and altered mental status. Notably absent are symptoms of nausea, vomiting, and abdominal pain and the Kussmaul respirations characteristic of DKA. HHS is often precipitated by a serious, concurrent illness such as myocardial infarction or stroke. Sepsis, pneumonia, and other serious infections are frequent precipitants and should be sought. In addition, a debilitating condition (prior stroke or dementia) or social situation that compromises water intake usually contributes to the development of the disorder.

1	Pathophysiology Relative insulin deficiency and inadequate fluid intake are the underlying causes of HHS. Insulin deficiency increases hepatic glucose production (through glycogenolysis and gluconeogenesis) and impairs glucose utilization in skeletal muscle (see above discussion of DKA). Hyperglycemia induces an osmotic diuresis that leads to intravascular volume depletion, which is exacerbated by inadequate fluid replacement. The absence of ketosis in HHS is not understood. Presumably, the insulin deficiency is only relative and less severe than in DKA. Lower levels of counterregulatory hormones and free fatty acids have been found in HHS than in DKA in some studies. It is also possible that the liver is less capable of ketone body synthesis or that the insulin/glucagon ratio does not favor ketogenesis.

1	Laboratory Abnormalities and Diagnosis The laboratory features in HHS are summarized in Table 418-6. Most notable are the marked hyperglycemia (plasma glucose may be >55.5 mmol/L [1000 mg/dL]), hyperosmolality (>350 mosmol/L), and prerenal azotemia. The measured serum sodium may be normal or slightly low despite the marked hyperglycemia. The corrected serum sodium is usually increased (add 1.6 meq to measured sodium for each 5.6-mmol/L [100-mg/dL] rise in the serum glucose). In contrast to DKA, acidosis and ketonemia are absent or mild. A small anion-gap metabolic acidosis may be present secondary to increased lactic acid. Moderate ketonuria, if present, is secondary to starvation.

1	Volume depletion and hyperglycemia are prominent features of both HHS and DKA. Consequently, therapy of these disorders shares several elements (Table 418-8). In both disorders, careful monitoring of the patient’s fluid status, laboratory values, and insulin infusion rate is crucial. Underlying or precipitating problems should be aggressively sought and treated. In HHS, fluid losses and dehydration are usually more pronounced than in DKA due to the longer duration of the illness. The patient with HHS is usually older, more likely to have mental status changes, and more likely to have a life-threatening precipitating event with accompanying comorbidities. Even with proper treatment, HHS has a substantially higher mortality rate than DKA (up to 15% in some clinical series).

1	Fluid replacement should initially stabilize the hemodynamic status of the patient (1–3 L of 0.9% normal saline over the first 2–3 h). Because the fluid deficit in HHS is accumulated over a period of days to weeks, the rapidity of reversal of the hyperosmolar state must balance the need for free water repletion with the risk that too rapid a reversal may worsen neurologic function. If the serum sodium is >150 mmol/L (150 meq/L), 0.45% saline should be used. After hemodynamic stability is achieved, the IV fluid administration is directed at reversing the free water deficit using hypotonic fluids (0.45% saline initially, then 5% dextrose in water [D5W]). The calculated free water deficit (which averages 9–10 L) should be reversed over the next 1–2 days (infusion rates of 200–300 mL/h of hypotonic solution). Potassium repletion is usually necessary and should be dictated by repeated measurements of the serum potassium. In patients taking diuretics, the potassium deficit can be quite

1	hypotonic solution). Potassium repletion is usually necessary and should be dictated by repeated measurements of the serum potassium. In patients taking diuretics, the potassium deficit can be quite large and may be accompanied by magnesium deficiency. Hypophosphatemia may occur during therapy and can be improved by using KPO4 and beginning nutrition.

1	As in DKA, rehydration and volume expansion lower the plasma glucose initially, but insulin is also required. A reasonable regimen for HHS begins with an IV insulin bolus of 0.1 unit/kg followed by IV insulin at a constant infusion rate of 0.1 unit/kg per hour. If the serum glucose does not fall, increase the insulin infusion rate by twofold. As in DKA, glucose should be added to IV fluid when the plasma glucose falls to 13.9 mmol/L (250 mg/dL), and the insulin infusion rate should be decreased to 0.05–0.1 unit/kg per hour. The insulin infusion should be continued until the patient has resumed eating and can be transferred to a SC insulin regimen. The patient should be discharged from the hospital on insulin, although some patients can later switch to oral glucose-lowering agents.

1	Virtually all medical and surgical subspecialties are involved in the care of hospitalized patients with diabetes. Hyperglycemia, whether in a patient with known diabetes or in someone without known diabetes, appears to be a predictor of poor outcome in hospitalized patients. General anesthesia, surgery, infection, or concurrent illness raises the levels of counterregulatory hormones (cortisol, growth hormone, catecholamines, and glucagon) and cytokines that may lead to transient insulin resistance and hyperglycemia. These factors increase insulin requirements by increasing glucose production and impairing glucose utilization and thus may worsen glycemic control. The concurrent illness or surgical procedure may lead to variable insulin absorption and also prevent the patient with DM from eating normally and, thus, may promote hypoglycemia. Glycemic control should be assessed on admission using the HbA1c. Electrolytes, renal function, and intravascular volume status should be assessed

1	eating normally and, thus, may promote hypoglycemia. Glycemic control should be assessed on admission using the HbA1c. Electrolytes, renal function, and intravascular volume status should be assessed as well. The high prevalence of CVD in individuals with DM (especially in type 2 DM) may necessitate preoperative cardiovascular evaluation (Chap. 419).

1	The goals of diabetes management during hospitalization are nearnormoglycemia, avoidance of hypoglycemia, and transition back to the outpatient diabetes treatment regimen. Upon hospital admission, frequent glycemic monitoring should begin, as should planning for diabetes management after discharge. Glycemic control appears to improve the clinical outcomes in a variety of settings, but optimal glycemic goals for the hospitalized patient are incompletely defined.

1	In a number of cross-sectional studies of patients with diabetes, a greater degree of hyperglycemia was associated with worse cardiac, neurologic, and infectious outcomes. In some studies, patients who do not have preexisting diabetes but who develop modest blood glucose elevations during their hospitalization appear to benefit from achieving near-normoglycemia using insulin treatment. However, a large randomized clinical trial (Normoglycemia in Intensive Care Evaluation Survival Using Glucose Algorithm Regulation [NICESUGAR]) of individuals in the ICU (most of whom were receiving mechanical ventilation) found an increased mortality rate and a greater number of episodes of severe hypoglycemia with very strict glycemic control (target blood glucose of 4.5–6 mmol/L or 81–108 mg/dL) compared to individuals with a more moderate glycemic goal (mean blood glucose of 8 mmol/L or 144 mg/dL). Currently, most data suggest that very strict blood glucose control in acutely ill patients likely

1	to individuals with a more moderate glycemic goal (mean blood glucose of 8 mmol/L or 144 mg/dL). Currently, most data suggest that very strict blood glucose control in acutely ill patients likely worsens outcomes and increases the frequency of hypoglycemia. The ADA suggests the following glycemic goals for hospitalized patients: (1) in critically ill patients: glucose of 7.8–10.0 mmol/L or 140–180 mg/dL; (2) in non–critically ill patients: premeal glucose <7.8 mmol/L (140 mg/dL) and at other times blood glucose <10 mmol/L (180 mg/dL).

1	Critical aspects for optimal diabetes care in the hospital include the following. (1) A hospital system approach to treatment of hyperglycemia and prevention of hypoglycemia is needed. Inpatient diabetes management teams consisting of nurse practitioners and physicians are increasingly common. (2) Diabetes treatment plans should focus on the transition from the ICU and the transition from the inpatient to outpatient setting. (3) Adjustment of the discharge treatment regimen of patients whose diabetes was poorly controlled on admission (as reflected by the HbA1c) is necessary.

1	The physician caring for an individual with diabetes in the perioperative period, during times of infection or serious physical illness, or simply when the patient is fasting for a diagnostic procedure must monitor the plasma glucose vigilantly, adjust the diabetes treatment regimen, and provide glucose infusion as needed. Hypoglycemia is frequent in hospitalized patients, and many of these episodes are avoidable. Hospital systems should have a diabetes management protocol to avoid inpatient hypoglycemia. Measures to reduce or prevent hypoglycemia include frequent glucose monitoring and anticipating potential modifications of insulin/glucose administration because of changes in the clinical situation or treatment (e.g., tapering of glucocorticoids) or interruption of enteral or parenteral infusions or PO intake.

1	Depending on the severity of the patient’s illness and the hospital setting, the physician can use either an insulin infusion or SC insulin. Insulin infusions are preferred in the ICU or in a clinically unstable setting. The absorption of SC insulin may be variable in such situations. Insulin infusions can also effectively control plasma glucose in the perioperative period and when the patient is unable to take anything by mouth. Regular insulin is used rather than insulin analogues for IV insulin infusion because it is less expensive and equally effective. The physician must consider carefully the clinical setting in which an insulin infusion will be used, including whether adequate ancillary personnel are available to monitor the plasma glucose frequently and whether they can adjust the insulin infusion rate to maintain the plasma glucose within the optimal range. Insulin-infusion algorithms should integrate the insulin sensitivity of the patient, frequent blood glucose monitoring,

1	insulin infusion rate to maintain the plasma glucose within the optimal range. Insulin-infusion algorithms should integrate the insulin sensitivity of the patient, frequent blood glucose monitoring, and the trend of changes in the blood glucose to determine the insulin-infusion rate. Insulin-infusion algorithms jointly developed and implemented by nursing and physician staff are advised. Because of the short half-life of IV regular insulin, it is necessary to administer long-acting insulin prior to discontinuation of the insulin infusion (2–4 h before the infusion is stopped) to avoid a period of insulin deficiency.

1	In patients who are not critically ill or not in the ICU, basal or “scheduled” insulin is provided by SC, long-acting insulin supplemented by prandial and/or “corrective” insulin using a short-acting insulin (insulin analogues preferred). The use of “sliding scale,” short-acting insulin alone, where no insulin is given unless the blood glucose is elevated, is inadequate for inpatient glucose management 2421 and should not be used. The short-acting, preprandial insulin dose should include coverage for food consumption (based on anticipated carbohydrate intake) plus a corrective or supplemental insulin based on the patient’s insulin sensitivity and the blood glucose. For example, if the patient is thin (and likely insulin-sensitive), a corrective insulin supplement might be 1 unit for each 2.7 mmol/L (50 mg/dL) over the glucose target. If the patient is obese and insulin-resistant, then the insulin supplement might be 2 units for each 2.7 mmol/L (50 mg/dL) over the glucose target. It is

1	mmol/L (50 mg/dL) over the glucose target. If the patient is obese and insulin-resistant, then the insulin supplement might be 2 units for each 2.7 mmol/L (50 mg/dL) over the glucose target. It is critical to individualize the regimen and adjust the basal or “scheduled” insulin dose frequently, based on the corrective insulin required. A consistent carbohydrate diabetes meal plan for hospitalized patients provides a predictable amount of carbohydrate for a particular meal each day (but not necessarily the same amount for breakfast, lunch, and supper). The hospital diet should be determined by a nutritionist; terms such as ADA diet or low-sugar diet are no longer used.

1	Individuals with type 1 DM who are undergoing general anesthesia and surgery or who are seriously ill should receive continuous insulin, either through an IV insulin infusion or by SC administration of a reduced dose of long-acting insulin. Short-acting insulin alone is insufficient. Prolongation of a surgical procedure or delay in the recovery room is not uncommon and may result in periods of insulin deficiency leading to DKA. Insulin infusion is the preferred method for managing patients with type 1 DM in the perioperative period or when serious concurrent illness is present (0.5–1.0 units/h of regular insulin). If the diagnostic or surgical procedure is brief and performed under local or regional anesthesia, a reduced dose of SC, long-acting insulin may suffice (30–50% reduction, with short-acting insulin withheld or reduced). This approach facilitates the transition back to long-acting insulin after the procedure. Glucose may be infused to prevent hypoglycemia. The blood glucose

1	short-acting insulin withheld or reduced). This approach facilitates the transition back to long-acting insulin after the procedure. Glucose may be infused to prevent hypoglycemia. The blood glucose should be monitored frequently during the illness or in the perioperative period.

1	Individuals with type 2 DM can be managed with either an insulin infusion or SC long-acting insulin (25–50% reduction depending on clinical setting) plus preprandial, short-acting insulin. Oral glucose- lowering agents should be discontinued upon admission and are not useful in regulating the plasma glucose in clinical situations where the insulin requirements and glucose intake are changing rapidly. Moreover, these oral agents may be dangerous if the patient is fasting (e.g., hypoglycemia with sulfonylureas). Metformin should be withheld when radiographic contrast media will be given or if unstable CHF, acidosis, or declining renal function is present.

1	(See also Chap. 98e) Total parenteral nutrition (TPN) greatly increases insulin requirements. In addition, individuals not previously known to have DM may become hyperglycemic during TPN and require insulin treatment. IV insulin infusion is the preferred treatment for hyperglycemia, and rapid titration to the required insulin dose is done most efficiently using a separate insulin infusion. After the total insulin dose has been determined, insulin may be added directly to the TPN solution or, preferably, given as a separate infusion or subcutaneously. Often, individuals receiving either TPN or enteral nutrition receive their caloric loads continuously and not at “meal times”; consequently, SC insulin regimens must be adjusted.

1	Glucocorticoids increase insulin resistance, decrease glucose utilization, increase hepatic glucose production, and impair insulin secretion. These changes lead to a worsening of glycemic control in individuals with DM and may precipitate diabetes in other individuals (“steroidinduced diabetes”). The effects of glucocorticoids on glucose homeostasis are dose-related, usually reversible, and most pronounced in the postprandial period. If the FPG is near the normal range, oral diabetes agents (e.g., sulfonylureas, metformin) may be sufficient to reduce Diabetes Mellitus: Management and Therapies 2422 hyperglycemia. If the FPG is >11.1 mmol/L (200 mg/dL), oral agents are usually not efficacious and insulin therapy is required. Short-acting Diabetes mellitus: Complications insulin may be required to supplement long-acting insulin in order to Alvin C. Powers control postprandial glucose excursions.

1	Reproductive capacity in either men or women with DM appears to be normal. Menstrual cycles may be associated with alterations in glycemic control in women with DM. Pregnancy is associated with marked insulin resistance; the increased insulin requirements often precipitate DM and lead to the diagnosis of gestational diabetes mellitus (GDM). Glucose, which at high levels is a teratogen to the developing fetus, readily crosses the placenta, but insulin does not. Thus, hyperglycemia from the maternal circulation may stimulate insulin secretion in the fetus. The anabolic and growth effects of insulin may result in macrosomia. GDM complicates ~7% (range 1–14%) of pregnancies. The incidence of GDM is greatly increased in certain ethnic groups, including African Americans and Latinas, consistent with a similar increased risk of type 2 DM. Current recommendations advise screening for glucose intolerance between weeks 24 and 28 of pregnancy in women with increased risk for GDM (≥25 years;

1	with a similar increased risk of type 2 DM. Current recommendations advise screening for glucose intolerance between weeks 24 and 28 of pregnancy in women with increased risk for GDM (≥25 years; obesity; family history of DM; member of an ethnic group such as Latina, Native American, Asian American, African American, or Pacific Islander). Therapy for GDM is similar to that for individuals with pregnancy-associated diabetes and involves MNT and insulin, if hyperglycemia persists. Oral glucose-lowering agents are not approved for use during pregnancy, but studies using metformin or glyburide have shown efficacy and have not found toxicity. However, many physicians use insulin to treat GDM. With current practices, the morbidity and mortality rates of the mother with GDM and the fetus are not different from those in the nondiabetic population. Individuals who develop GDM are at marked increased risk for developing type 2 DM in the future and should be screened periodically for DM. Most

1	different from those in the nondiabetic population. Individuals who develop GDM are at marked increased risk for developing type 2 DM in the future and should be screened periodically for DM. Most individuals with GDM revert to normal glucose tolerance after delivery, but some will continue to have overt diabetes or impairment of glucose tolerance after delivery. In addition, children of women with GDM appear to be at risk for obesity and glucose intolerance and have an increased risk of diabetes beginning in the later stages of adolescence.

1	Pregnancy in individuals with known DM requires meticulous planning and adherence to strict treatment regimens. Intensive diabetes management and normalization of the HbA1c are essential for individuals with existing DM who are planning pregnancy. The most crucial period of glycemic control is soon after fertilization. The risk of fetal malformations is increased 4–10 times in individuals with uncontrolled DM at the time of conception, and normal plasma glucose during the preconception period and throughout the periods of organ development in the fetus should be the goal. Lipodystrophy, or the loss of subcutaneous fat tissue, may be generalized in certain genetic conditions such as leprechaunism. Generalized lipodystrophy is associated with severe insulin resistance and is often accompanied by acanthosis nigricans and dyslipidemia. Localized lipodystrophy associated with insulin injections has been reduced considerably by the use of human insulin.

1	Protease Inhibitors and Lipodystrophy Protease inhibitors used in the treatment of HIV disease (Chap. 226) have been associated with a centripetal accumulation of fat (visceral and abdominal area), accumulation of fat in the dorsocervical region, loss of extremity fat, decreased insulin sensitivity (elevations of the fasting insulin level and reduced glucose tolerance on IV glucose tolerance testing), and dyslipidemia. Although many aspects of the physical appearance of these individuals resemble Cushing’s syndrome, increased cortisol levels do not account for this appearance. The possibility remains that this is related to HIV infection by some undefined mechanism, because some features of the syndrome were observed before the introduction of protease inhibitors. Therapy for HIV-related lipodystrophy is not well established.

1	Diabetes-related complications affect many organ systems and are responsible for the majority of morbidity and mortality associated with the disease. Strikingly, in the United States, diabetes is the leading cause of new blindness in adults, renal failure, and nontraumatic lower extremity amputation. Diabetes-related complications usually do not appear until the second decade of hyperglycemia. Because type 2 diabetes mellitus (DM) often has a long asymptomatic period of hyperglycemia before diagnosis, many individuals with type 2 DM have complications at the time of diagnosis. Fortunately, many of the diabetes-related complications can be prevented or delayed with early detection, aggressive glycemic control, and efforts to minimize the risks of complications.

1	Diabetes-related complications can be divided into vascular and nonvascular complications and are similar for type 1 and type 2 DM (Table 419-1). The vascular complications of DM are further subdivided into microvascular (retinopathy, neuropathy, nephropathy) and macrovascular complications (coronary heart disease [CHD], peripheral arterial disease [PAD], cerebrovascular disease). Microvascular complications are diabetes-specific, whereas macrovascular complications are similar to those in nondiabetics but occur at greater frequency in individuals with diabetes. Nonvascular complications include gastroparesis, infections, skin changes, and hearing loss. Whether type 2 DM increases the risk of dementia or impaired cognitive function is not clear.

1	The microvascular complications of both type 1 and type 2 DM result from chronic hyperglycemia (Fig. 419-1). Evidence implicating a causative role for chronic hyperglycemia in the development of macrovascular complications is less conclusive. CHD events and mortality rate are two to four times greater in patients with type 2 DM and correlate with fasting and postprandial plasma glucose levels as well the Gastrointestinal (gastroparesis, diarrhea) Other comorbid conditions associated with diabetes (relationship to hyperglycemia is uncertain): depression, obstructive sleep apnea, fatty liver disease, hip fracture, osteoporosis (in type 1 diabetes), cognitive impairment or dementia, low testosterone in men aThickened skin and reduced joint mobility. Length of follow-up, years

1	Length of follow-up, years FIGURE 419-1 Relationship of glycemic control and diabetes duration to diabetic retinopathy. The progression of retinopathy in individuals in the Diabetes Control and Complications Trial is graphed as a function of the length of follow-up with different curves for different hemoglobin A1c (HbA1c) values. (Adapted from The Diabetes Control and Complications Trial Research Group: Diabetes 44:968, 1995.) hemoglobin A1c (HbA1c). Other factors such as dyslipidemia and hypertension also play important roles in macrovascular complications.

1	The Diabetes Control and Complications Trial (DCCT) provided definitive proof that reduction in chronic hyperglycemia can prevent many complications of type 1 DM (Fig. 419-1). This large multicenter clinical trial randomized more than 1400 individuals with type 1 DM to either intensive or conventional diabetes management and prospectively evaluated the development of diabetes-related complications during a mean follow-up of 6.5 years. Individuals in the intensive diabetes management group received multiple administrations of insulin each day (injection or pump) along with extensive educational, psychological, and medical support. Individuals in the conventional diabetes management group received twice-daily insulin injections and quarterly nutritional, educational, and clinical evaluation. The goal in the former group was normoglycemia; the goal in the latter group was prevention of symptoms of diabetes. Individuals in the intensive diabetes management group achieved a substantially

1	The goal in the former group was normoglycemia; the goal in the latter group was prevention of symptoms of diabetes. Individuals in the intensive diabetes management group achieved a substantially lower HbA1c (7.3%) than individuals in the conventional diabetes management group (9.1%). After the DCCT results were reported in 1993, study participants continue to be followed in the Epidemiology of Diabetes Intervention and Complications (EDIC) trial, which recently completed 30 years of follow-up (DCCT + EDIC). At the end of the DCCT phase, study participants in both intensive and conventional arms were offered intensive therapy. However, during the subsequent follow-up of more than 18 years, the initial separation in glycemic control disappeared with both arms maintaining a mean HbA1c of 8.0%.

1	The DCCT phase demonstrated that improvement of glycemic control reduced nonproliferative and proliferative retinopathy (47% reduction), microalbuminuria (39% reduction), clinical nephropathy (54% reduction), and neuropathy (60% reduction). Improved glycemic control also slowed the progression of early diabetic complications. During the DCCT phase, weight gain (4.6 kg) and severe hypoglycemia (requiring assistance of another person to treat) were more common in the intensive therapy group. The benefits of an improvement in glycemic control occurred over the entire range of HbA1c values (Fig. 419-1), indicating that at any HbA1c level, an improvement in glycemic control is beneficial. The results of the DCCT predicted that individuals in the intensive diabetes management group would gain 7.7 additional years of vision, 5.8 additional years free from end-stage renal disease (ESRD), and 5.6 years free from lower extremity amputations. If all complications of DM were combined, individuals

1	additional years of vision, 5.8 additional years free from end-stage renal disease (ESRD), and 5.6 years free from lower extremity amputations. If all complications of DM were combined, individuals in the intensive diabetes management group would experience 15.3 more years of life without significant microvascular or neurologic complications of DM, compared to individuals who received standard therapy. This translates into an additional 5.1 years of life expectancy for individuals in the intensive diabetes management group. The 30-year follow-up data in the intensively treated group show a continued reduction in retinopathy, nephropathy, and cardiovascular disease. For example, individuals in the intensive therapy group had a 42–57% reduction in

1	Retinopathy progression, rate cardiovascular events (nonfatal myocardial infarction [MI], stroke, or 2423 death from a cardiovascular event) at a mean follow-up of 17 years, even though their subsequent glycemic control was the same as those in the conventional diabetes management group from years 6.5–17. During the EDIC phase, less than 1% of the cohort had become blind, lost a limb to amputation, or required dialysis.

1	The United Kingdom Prospective Diabetes Study (UKPDS) studied the course of >5000 individuals with type 2 DM for >10 years. This study used multiple treatment regimens and monitored the effect of intensive glycemic control and risk factor treatment on the development of diabetic complications. Newly diagnosed individuals with type 2 DM were randomized to (1) intensive management using various combinations of insulin, a sulfonylurea, or metformin or (2) conventional therapy using dietary modification and pharmacotherapy with the goal of symptom prevention. In addition, individuals were randomly assigned to different antihypertensive regimens. Individuals in the intensive treatment arm achieved an HbA1c of 7%, compared to a 7.9% HbA1c in the standard treatment group. The UKPDS demonstrated that each percentage point reduction in HbA1c was associated with a 35% reduction in microvascular complications. As in the DCCT, there was a continuous relationship between glycemic control and

1	that each percentage point reduction in HbA1c was associated with a 35% reduction in microvascular complications. As in the DCCT, there was a continuous relationship between glycemic control and development of complications. Improved glycemic control also reduced the cardiovascular event rate in the follow-up period of >10 years.

1	One of the major findings of the UKPDS was that strict blood pressure control significantly reduced both macroand microvascular complications. In fact, the beneficial effects of blood pressure control were greater than the beneficial effects of glycemic control. Lowering blood pressure to moderate goals (144/82 mmHg) reduced the risk of DM-related death, stroke, microvascular endpoints, retinopathy, and heart failure (risk reductions between 32 and 56%).

1	Similar reductions in the risks of retinopathy and nephropathy were also seen in a small trial of lean Japanese individuals with type 2 DM randomized to either intensive glycemic control or standard therapy with insulin (Kumamoto study). These results demonstrate the effectiveness of improved glycemic control in individuals of different ethnicity and, presumably, a different etiology of DM (i.e., phenotypically different from those in the DCCT and UKPDS). The Action to Control Cardiovascular Risk in Diabetes (ACCORD) and Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation (ADVANCE) trials also found that improved glycemic control reduced microvascular complications.

1	Thus, these large clinical trials in type 1 and type 2 DM indicate that chronic hyperglycemia plays a causative role in the pathogenesis of diabetic microvascular complications. In both the DCCT and the UKPDS, cardiovascular events were reduced at follow-up of >10 years, even though the improved glycemic control was not maintained. The positive impact of a period of improved glycemic control on later disease has been termed a legacy effect or metabolic memory.

1	A summary of the features of diabetes-related complications includes the following. (1) Duration and degree of hyperglycemia correlate with complications. (2) Intensive glycemic control is beneficial in all forms of DM. (3) Blood pressure control is critical, especially in type 2 DM. (4) Survival in patients with type 1 DM is improving, and diabetes-related complications are declining. (5) Not all individuals with diabetes develop diabetes-related complications. Other incompletely defined factors appear to modulate the development of complications. For example, despite long-standing DM, some individuals never develop nephropathy or retinopathy. Many of these patients have glycemic control that is indistinguishable from those who develop microvascular complications, suggesting a genetic susceptibility for developing particular complications.

1	Although chronic hyperglycemia is an important etiologic factor leading to complications of DM, the mechanism(s) by which it leads to such diverse cellular and organ dysfunction is unknown. An emerging hypothesis is that hyperglycemia leads to epigenetic changes (Chap. 82) that influence gene expression in affected cells. For example, this may explain the legacy effect or metabolic memory mentioned above.

1	Diabetes Mellitus: Complications 2424 Four theories, which are not mutually exclusive, on how hyperglycemia might lead to the chronic complications of DM include the following pathways. (1) Increased intracellular glucose leads to the formation of advanced glycosylation end products, which bind to a cell surface receptor, via the nonenzymatic glycosylation of intraand extracellular proteins, leading to cross-linking of proteins, accelerated atherosclerosis, glomerular dysfunction, endothelial dysfunction, and altered extracellular matrix composition. (2) Hyperglycemia increases glucose metabolism via the sorbitol pathway related to the enzyme aldose reductase. However, testing of this theory in humans, using aldose reductase inhibitors, has not demonstrated beneficial effects. (3) Hyperglycemia increases the formation of diacylglycerol, leading to activation of protein kinase C, which alters the transcription of genes for fibronectin, type IV collagen, contractile proteins, and

1	increases the formation of diacylglycerol, leading to activation of protein kinase C, which alters the transcription of genes for fibronectin, type IV collagen, contractile proteins, and extracellular matrix proteins in endothelial cells and neurons. (4) Hyperglycemia increases the flux through the hexosamine pathway, which generates fructose-6-phosphate, a substrate for O-linked glycosylation and proteoglycan production, leading to altered function by glycosylation of proteins such as endothelial nitric oxide synthase or by changes in gene expression of transforming growth factor β (TGF-β) or plasminogen activator inhibitor-1. Growth factors may play an important role in some diabetes-related complications, and their production is increased by most of these proposed pathways. Vascular endothelial growth factor A (VEGF-A) is increased locally in diabetic proliferative retinopathy and decreases after laser photocoagulation. TGF-β is increased in diabetic nephropathy and stimulates

1	endothelial growth factor A (VEGF-A) is increased locally in diabetic proliferative retinopathy and decreases after laser photocoagulation. TGF-β is increased in diabetic nephropathy and stimulates basement membrane production of collagen and fibronectin by mesangial cells. A possible unifying mechanism is that hyperglycemia leads to increased production of reactive oxygen species or superoxide in the mitochondria; these compounds may activate all four of the pathways described above. Although hyperglycemia serves as the initial trigger for complications of diabetes, it is still unknown whether the same pathophysiologic processes are operative in all complications or whether some pathways predominate in certain organs.

1	DM is the leading cause of blindness between the ages of 20 and 74 in the United States. The gravity of this problem is highlighted by the finding that individuals with DM are 25 times more likely to become legally blind than individuals without DM. Severe vision loss is primarily the result of progressive diabetic retinopathy and clinically significant macular edema. Diabetic retinopathy is classified into two stages: nonproliferative and proliferative. Nonproliferative diabetic retinopathy usually appears late in the first decade or early in the second decade of the disease and is marked by retinal vascular microaneurysms, blot hemorrhages, and cotton-wool spots (Fig. 419-2). Mild nonproliferative retinopathy may progress to more extensive disease, characterized by changes in venous vessel caliber, intraretinal microvascular

1	FIGURE 419-2 Diabetic retinopathy results in scattered hemorrhages, yellow exudates, and neovascularization. This patient has neovascular vessels proliferating from the optic disc, requiring urgent panretinal laser photocoagulation. abnormalities, and more numerous microaneurysms and hemorrhages. The pathophysiologic mechanisms invoked in nonproliferative retinopathy include loss of retinal pericytes, increased retinal vascular permeability, alterations in retinal blood flow, and abnormal retinal microvasculature, all of which can lead to retinal ischemia. A new concept is that the pathology involves inflammatory processes in the retinal neurovascular unit, which consists of neurons, glia, astrocytes, Muüller cells, and specialized vasculature.

1	The appearance of neovascularization in response to retinal hypoxemia is the hallmark of proliferative diabetic retinopathy (Fig. 419-2). These newly formed vessels appear near the optic nerve and/or macula and rupture easily, leading to vitreous hemorrhage, fibrosis, and ultimately retinal detachment. Not all individuals with nonproliferative retinopathy go on to develop proliferative retinopathy, but the more severe the nonproliferative disease, the greater the chance of evolution to proliferative retinopathy within 5 years. This creates an important opportunity for early detection and treatment of diabetic retinopathy. Clinically significant macular edema can occur in the context of non-proliferative or proliferative retinopathy. Fluorescein angiography and optical coherence tomography are useful to detect macular edema, which is associated with a 25% chance of moderate visual loss over the next 3 years. Duration of DM and degree of glycemic control are the best predictors of the

1	are useful to detect macular edema, which is associated with a 25% chance of moderate visual loss over the next 3 years. Duration of DM and degree of glycemic control are the best predictors of the development of retinopathy; hypertension and nephropathy are also risk factors. Nonproliferative retinopathy is found in many individuals who have had DM for >20 years. Although there is genetic susceptibility for retinopathy, it confers less influence than either the duration of DM or the degree of glycemic control.

1	The most effective therapy for diabetic retinopathy is prevention. Intensive glycemic and blood pressure control will delay the development or slow the progression of retinopathy in individuals with either type 1 or type 2 DM. Paradoxically, during the first 6–12 months of improved glycemic control, established diabetic retinopathy may transiently worsen. Fortunately, this progression is temporary, and in the long term, improved glycemic control is associated with less diabetic retinopathy. Individuals with known retinopathy may be candidates for prophylactic laser photocoagulation when initiating intensive therapy. Once advanced retinopathy is present, improved glycemic control imparts less benefit, although adequate ophthalmologic care can prevent most blindness.

1	Regular, comprehensive eye examinations are essential for all individuals with DM (see Table 418-1). Most diabetic eye disease can be successfully treated if detected early. Routine, nondilated eye examinations by the primary care provider or diabetes specialist are inadequate to detect diabetic eye disease, which requires an ophthalmologist for optimal care of these disorders. Laser photo-coagulation is very successful in preserving vision. Proliferative retinopathy is usually treated with panretinal laser photocoagulation, whereas macular edema is treated with focal laser photocoagulation and anti–vascular endothelial growth factor therapy (ocular injection). Aspirin therapy (650 mg/d) does not appear to influence the natural history of diabetic retinopathy.

1	Diabetic nephropathy is the leading cause of chronic kidney disease (CKD), ESRD, and CKD requiring renal replacement therapy. Furthermore, the prognosis of diabetic patients on dialysis is poor, with survival comparable to many forms of cancer. Albuminuria in individuals with DM is associated with an increased risk of cardiovascular disease. Individuals with diabetic nephropathy commonly have diabetic retinopathy.

1	Like other microvascular complications, the pathogenesis of diabetic nephropathy is related to chronic hyperglycemia. The mechanisms by which chronic hyperglycemia leads to diabetic nephropathy, although incompletely defined, involve the effects of soluble factors (growth factors, angiotensin II, endothelin, advanced glycation end products [AGEs]), hemodynamic alterations in the renal microcirculation (glomerular hyperfiltration or hyperperfusion, increased glomerular capillary pressure), and structural changes in the glomerulus (increased extracellular matrix, basement membrane thickening, mesangial expansion, fibrosis). Some of these effects may be mediated through angiotensin II receptors. Smoking accelerates the decline in renal function. Because only 20–40% of patients with diabetes develop diabetic nephropathy, additional genetic or environmental susceptibility factors remain unidentified. Known risk factors include race and a family history of diabetic nephropathy. Diabetic

1	develop diabetic nephropathy, additional genetic or environmental susceptibility factors remain unidentified. Known risk factors include race and a family history of diabetic nephropathy. Diabetic nephropathy and ESRD secondary to DM develop more commonly in African Americans, Native Americans, and Hispanic individuals with diabetes.

1	The natural history of diabetic nephropathy is characterized by a fairly predictable sequence of events that was initially defined for individuals with type 1 DM but appears to be similar in type 2 DM (Fig. 419-3). Glomerular hyperperfusion and renal hypertrophy occur in the first years after the onset of DM and are associated with an increase of the glomerular filtration rate (GFR). During the first 5 years of DM, thickening of the glomerular basement membrane, glomerular hypertrophy, and mesangial volume expansion occur as the GFR returns to normal. After 5–10 years of type 1 DM, many individuals begin to excrete small amounts of albumin in the urine. The American Diabetes Association (ADA) recently suggested that the terms previously used to refer to increased urinary protein (microalbuminuria as defined as 30–299 mg/d in a 24-h collection or 30–299 μg/mg creatinine in a spot collection or macroalbuminuria as defined as >300 mg/24 h) be replaced by the phrases “persistent

1	(microalbuminuria as defined as 30–299 mg/d in a 24-h collection or 30–299 μg/mg creatinine in a spot collection or macroalbuminuria as defined as >300 mg/24 h) be replaced by the phrases “persistent albuminuria (30–299 mg/24 h)” and “persistent albuminuria (≥300 mg/24 h)” to better reflect the continuous nature of albumin excretion in the urine as risk factor for nephropathy and cardiovascular disease (CVD). This chapter uses the terms microalbuminuria and macroalbuminuria. Although the appearance of microalbuminuria in type 1 DM is an important risk factor for progression to macroalbuminuria, only ~50% of individuals progress to macroalbuminuria over the next 10 years. In some individuals with type 1 diabetes and microalbuminuria of short duration, the microalbuminuria regresses. Microalbuminuria is also a risk factor for CVD. Once macroalbuminuria is present, there is a steady decline in GFR, and ~50% of individuals reach ESRD in 7–10 years. Once macroalbuminuria develops, blood

1	is also a risk factor for CVD. Once macroalbuminuria is present, there is a steady decline in GFR, and ~50% of individuals reach ESRD in 7–10 years. Once macroalbuminuria develops, blood pressure rises slightly and the pathologic changes are likely irreversible.

1	The nephropathy that develops in type 2 DM differs from that of type 1 DM in the following respects: (1) microalbuminuria or macroalbuminuria may be present when type 2 DM is diagnosed, reflecting its long asymptomatic period; (2) hypertension more commonly accompanies microalbuminuria or macroalbuminuria in type 2 DM; and (3) microalbuminuria may be less predictive of diabetic nephropathy and likelihood of progression to macroalbuminuria in type 2 DM, in large part due to increased CV mortality in this population. Finally, it should be noted that albuminuria in type 2 DM may be secondary to factors unrelated to DM, such as hypertension, congestive heart failure (CHF), prostate disease, or infection.

1	As part of comprehensive diabetes care (Chap. 418), albuminuria should be detected at an early stage when effective therapies can be instituted. Because some individuals with type 1 or type 2 DM have a decline in GFR in the absence of albuminuria, annual measurement of the serum creatinine to estimate GFR should also be performed. FIGURE 419-4 Screening for microalbuminuria should be performed in patients with type 1 diabetes for ≥5 years, in patients with type 2 diabetes, and during pregnancy. Non-diabetes-related conditions that might increase microalbuminuria are urinary tract infection, hematuria, heart failure, febrile illness, severe hyperglycemia, severe hypertension, and vigorous exercise. (Adapted from RA DeFranzo, in Therapy for Diabetes Mellitus and Related Disorders, 3rd ed. American Diabetes Association, Alexandria, VA, 1998.)

1	An annual microalbuminuria measurement (albumin-to-creatinine ratio in spot urine) is advised in individuals with type 1 or type 2 DM (Fig. 419-4). The urine protein measurement in a routine urinalysis does not detect these low levels of albumin excretion. Screening for albuminuria should commence 5 years after the onset of type 1 DM and at the time of diagnosis of type 2 DM.

1	Type IV renal tubular acidosis (hyporeninemic hypoaldosteronism) may occur in type 1 or 2 DM. These individuals develop a propensity to hyperkalemia and acidemia, which may be exacerbated by medications (especially angiotensin-converting enzyme [ACE] inhibitors, angiotensin receptor blockers [ARBs], and spironolactone). Patients with DM are predisposed to radiocontrast-induced nephrotoxicity. Risk factors for radiocontrast-induced nephrotoxicity are preexisting nephropathy and volume depletion. Individuals with DM undergoing radiographic procedures with contrast dye should be well hydrated before and after dye exposure, and the serum creatinine should be monitored for 24–48 h following the procedure. Metformin should be held if indicated.

1	The optimal therapy for diabetic nephropathy is prevention by control of glycemia (Chap. 418 outlines glycemic goals and approaches). Interventions effective in slowing progression of albuminuria include (1) improved glycemic control, (2) strict blood pressure control, and (3) administration of an ACE inhibitor or ARB. Dyslipidemia should also be treated. Diabetes Mellitus: Complications Time from onset of diabetes, years 0351015 20 25 GFR, mL/min 120 150 150 120 60 <10 Serum creatinine, mg/dL 1.0 0.8 0.8 1.0 >2.0 >5

1	FIGURE 419-3 Time course of development of diabetic nephropathy. The relationship of time from onset of diabetes, the glomerular filtration rate (GFR), and the serum creatinine are shown. (Adapted from RA DeFranzo, in Therapy for Diabetes Mellitus and Related Disorders, 3rd ed. American Diabetes Association, Alexandria, VA, 1998.) 2426 Improved glycemic control reduces the rate at which microalbuminuria appears and progresses in type 1 and type 2 DM. However, once macroalbuminuria is present, it is unclear whether improved glycemic control will slow progression of renal disease. During the later phase of declining renal function, insulin requirements may fall as the kidney is a site of insulin degradation. As the GFR decreases with progressive nephropathy, the use and dose of glucose-lowering agents should be reevaluated (see Table 418-5). Some glucose-lowering medications (sulfonylureas and metformin) are contraindicated in advanced renal insufficiency. Many individuals with type 1

1	agents should be reevaluated (see Table 418-5). Some glucose-lowering medications (sulfonylureas and metformin) are contraindicated in advanced renal insufficiency. Many individuals with type 1 or type 2 DM develop hypertension. Numerous studies in both type 1 and type 2 DM demonstrate the effectiveness of strict blood pressure control in reducing albumin excretion and slowing the decline in renal function. Blood pressure should be maintained at <140/90 mmHg in diabetic individuals. Either ACE inhibitors or ARBs should be used to reduce the albuminuria and the associated decline in GFR that accompanies it in individuals with type 1 or type 2 DM (see “Hypertension,” below). Although direct comparisons of ACE inhibitors and ARBs are lacking, most experts believe that the two classes of drugs are equivalent in patient with diabetes. ARBs can be used as an alternative in patients who develop ACE inhibitor–associated cough or angioedema. After 2–3 months of therapy in patients with

1	of drugs are equivalent in patient with diabetes. ARBs can be used as an alternative in patients who develop ACE inhibitor–associated cough or angioedema. After 2–3 months of therapy in patients with microalbuminuria, the drug dose is increased until the maximum tolerated dose is reached. Recent studies do not show benefit of intervention prior to onset of microalbuminuria. The combination of an ACE inhibitor and an ARB is not recommended and appears to be detrimental. If use of either ACE inhibitors or ARBs is not possible or the blood pressure is not controlled, then, diuretics, calcium channel blockers (nondihydropyridine class), or beta blockers should be used. These salutary effects are mediated by reducing intraglomerular pressure and inhibition of angiotensin-driven sclerosing pathways, in part through inhibition of TGF-β-mediated pathways. The ADA does not suggest restriction of protein intake in diabetic individuals with albuminuria because studies have failed to show

1	pathways, in part through inhibition of TGF-β-mediated pathways. The ADA does not suggest restriction of protein intake in diabetic individuals with albuminuria because studies have failed to show benefit. Nephrology consultation should be considered when albuminuria appears and again when the estimated GFR is <60 mL/min per 1.743 m2. As compared with nondiabetic individuals, hemodialysis in patients with DM is associated with more frequent complications, such as hypotension (due to autonomic neuropathy or loss of reflex tachycardia), more difficult vascular access, and accelerated progression of retinopathy. Complications of atherosclerosis are the leading cause of death in diabetic individuals with nephropathy and hyperlipidemia should be treated aggressively. Renal transplantation from a living related donor is the preferred therapy but requires chronic immunosuppression. Combined pancreas-kidney transplant offers the promise of normoglycemia and freedom from dialysis.

1	Diabetic neuropathy occurs in ~50% of individuals with long-standing type 1 and type 2 DM. It may manifest as polyneuropathy, mononeuropathy, and/or autonomic neuropathy. As with other complications of DM, the development of neuropathy correlates with the duration of diabetes and glycemic control. Additional risk factors are body mass index (BMI) (the greater the BMI, the greater the risk of neuropathy) and smoking. The presence of CVD, elevated triglycerides, and hypertension is also associated with diabetic peripheral neuropathy. Both myelinated and unmyelinated nerve fibers are lost. Because the clinical features of diabetic neuropathy are similar to those of other neuropathies, the diagnosis of diabetic neuropathy should be made only after other possible etiologies are excluded (Chap. 459).

1	Polyneuropathy/Mononeuropathy The most common form of diabetic neuropathy is distal symmetric polyneuropathy. It most frequently presents with distal sensory loss and pain, but up to 50% of patients do not have symptoms of neuropathy. Hyperesthesia, paresthesia, and dysesthesia also may occur. Any combination of these symptoms may develop as neuropathy progresses. Symptoms may include a sensation of numbness, tingling, sharpness, or burning that begins in the feet and spreads proximally. Neuropathic pain develops in some of these individuals, occasionally preceded by improvement in their glycemic control. Pain typically involves the lower extremities, is usually present at rest, and worsens at night. Both an acute (lasting <12 months) and a chronic form of painful diabetic neuropathy have been described. The acute form is sometimes treatment-related, occurring in the context of improved glycemic control. As diabetic neuropathy progresses, the pain subsides and eventually disappears,

1	been described. The acute form is sometimes treatment-related, occurring in the context of improved glycemic control. As diabetic neuropathy progresses, the pain subsides and eventually disappears, but a sensory deficit in the lower extremities persists. Physical examination reveals sensory loss, loss of ankle deep-tendon reflexes, and abnormal position sense.

1	Diabetic polyradiculopathy is a syndrome characterized by severe disabling pain in the distribution of one or more nerve roots. It may be accompanied by motor weakness. Intercostal or truncal radiculopathy causes pain over the thorax or abdomen. Involvement of the lumbar plexus or femoral nerve may cause severe pain in the thigh or hip and may be associated with muscle weakness in the hip flexors or extensors (diabetic amyotrophy). Fortunately, diabetic polyradiculopathies are usually self-limited and resolve over 6–12 months.

1	Mononeuropathy (dysfunction of isolated cranial or peripheral nerves) is less common than polyneuropathy in DM and presents with pain and motor weakness in the distribution of a single nerve. Mononeuropathies can occur at entrapment sites such as carpal tunnel or be noncompressive. A vascular etiology for noncompressive mononeuropathies has been suggested, but the pathogenesis is unknown. Involvement of the third cranial nerve is most common and is heralded by diplopia. Physical examination reveals ptosis and ophthalmoplegia with normal pupillary constriction to light. Sometimes other cranial nerves, such as IV, VI, or VII (Bell’s palsy), are affected. Peripheral mononeuropathies or simultaneous involvement of more than one nerve (mononeuropathy multiplex) may also occur.

1	Autonomic Neuropathy Individuals with long-standing type 1 or 2 DM may develop signs of autonomic dysfunction involving the cholinergic, noradrenergic, and peptidergic (peptides such as pancreatic polypeptide, substance P, etc.) systems. DM-related autonomic neuropathy can involve multiple systems, including the cardiovascular, gastrointestinal, genitourinary, sudomotor, and metabolic systems. Autonomic neuropathies affecting the cardiovascular system cause a resting tachycardia and orthostatic hypotension. Reports of sudden death have also been attributed to autonomic neuropathy. Gastroparesis and bladder-emptying abnormalities are often caused by the autonomic neuropathy seen in DM (discussed below). Hyperhidrosis of the upper extremities and anhidrosis of the lower extremities result from sympathetic nervous system dysfunction. Anhidrosis of the feet can promote dry skin with cracking, which increases the risk of foot ulcers. Autonomic neuropathy may reduce counterregulatory

1	from sympathetic nervous system dysfunction. Anhidrosis of the feet can promote dry skin with cracking, which increases the risk of foot ulcers. Autonomic neuropathy may reduce counterregulatory hormone release (especially catecholamines), leading to an inability to sense hypoglycemia appropriately (hypoglycemia unawareness; Chap. 420), thereby subjecting the patient to the risk of severe hypoglycemia and complicating efforts to improve glycemic control.

1	Treatment of diabetic neuropathy is less than satisfactory. Improved glycemic control should be aggressively pursued and will improve nerve conduction velocity, but symptoms of diabetic neuropathy may not necessarily improve. Efforts to improve glycemic control in long-standing diabetes may be confounded by autonomic neuropathy and hypoglycemia unawareness. Risk factors for neuropathy such as hypertension and hypertriglyceridemia should be treated. Avoidance of neurotoxins (alcohol) and smoking, supplementation with vitamins for possible deficiencies (B12, folate; Chap. 96e), and symptomatic treatment are the mainstays of therapy. Loss of sensation in the foot places the patient at risk for ulceration and its sequelae; consequently, prevention of such problems is of paramount importance. Patients with symptoms or signs of neuropathy should check their feet daily and take precautions (footwear) aimed at preventing calluses or ulcerations. If foot deformities are present, a podiatrist

1	Patients with symptoms or signs of neuropathy should check their feet daily and take precautions (footwear) aimed at preventing calluses or ulcerations. If foot deformities are present, a podiatrist should be involved.

1	Chronic, painful diabetic neuropathy is difficult to treat but may respond to duloxetine, amitriptyline, gabapentin, valproate, pregabalin, or opioids. Two agents, duloxetine and pregabalin, have been approved by the U.S. Food and Drug Administration (FDA) for pain associated with diabetic neuropathy, but no treatments are satisfactory. No direct comparisons of agents are available, and it is reasonable to switch agents if there is no response or if side effects develop. Referral to a pain management center may be necessary. Because the pain of acute diabetic neuropathy may resolve over time, medications may be discontinued as progressive neuronal damage from DM occurs.

1	Therapy of orthostatic hypotension secondary to autonomic neuropathy is also challenging. A variety of agents have limited success (fludrocortisone, midodrine, clonidine, octreotide, and yohimbine), but each has significant side effects. Nonpharmacologic maneuvers (adequate salt intake, avoidance of dehydration and diuretics, and lower extremity support hose) may offer some benefit.

1	Long-standing type 1 and 2 DM may affect the motility and function of the gastrointestinal (GI) and genitourinary systems. The most prominent GI symptoms are delayed gastric emptying (gastroparesis) and altered smalland large-bowel motility (constipation or diarrhea). Gastroparesis may present with symptoms of anorexia, nausea, vomiting, early satiety, and abdominal bloating. Microvascular complications (retinopathy and neuropathy) are usually present. Nuclear medicine scintigraphy after ingestion of a radiolabeled meal may document delayed gastric emptying, but may not correlate well with the patient’s symptoms. Noninvasive “breath tests” following ingestion of a radio-labeled meal have been developed, but are not yet validated. Although parasympathetic dysfunction secondary to chronic hyperglycemia is important in the development of gastroparesis, hyperglycemia itself also impairs gastric emptying. Nocturnal diarrhea, alternating with constipation, is a feature of DM-related GI

1	hyperglycemia is important in the development of gastroparesis, hyperglycemia itself also impairs gastric emptying. Nocturnal diarrhea, alternating with constipation, is a feature of DM-related GI autonomic neuropathy. In type 1 DM, these symptoms should also prompt evaluation for celiac sprue because of its increased frequency. Esophageal dysfunction in long-standing DM may occur but is usually asymptomatic.

1	Diabetic autonomic neuropathy may lead to genitourinary dysfunction including cystopathy and female sexual dysfunction (reduced sexual desire, dyspareunia, reduced vaginal lubrication). Symptoms of diabetic cystopathy begin with an inability to sense a full bladder and a failure to void completely. As bladder contractility worsens, bladder capacity and the postvoid residual increase, leading to symptoms of urinary hesitancy, decreased voiding frequency, incontinence, and recurrent urinary tract infections. Diagnostic evaluation includes cystometry and urodynamic studies. Erectile dysfunction and retrograde ejaculation are very common in DM and may be one of the earliest signs of diabetic neuropathy (Chap. 67). Erectile dysfunction, which increases in frequency with the age of the patient and the duration of diabetes, may occur in the absence of other signs of diabetic autonomic neuropathy.

1	Current treatments for these complications of DM are inadequate. Improved glycemic control should be a primary goal, because some aspects (neuropathy, gastric function) may improve. Smaller, more frequent meals that are easier to digest (liquid) and low in fat and fiber may minimize symptoms of gastroparesis. Metoclopramide has been used but is now restricted in both the United States and Europe and not advised for long-term use. Gastric electrical stimulatory devices are available but not approved. Diabetic diarrhea in the absence of bacterial overgrowth is treated symptomatically (Chap. 349).

1	Diabetic cystopathy should be treated with scheduled voiding or 2427 self-catheterization. Drugs that inhibit type 5 phosphodiesterase are effective for erectile dysfunction, but their efficacy in individuals with DM is slightly lower than in the nondiabetic population (Chap. 67). Sexual dysfunction in women may be improved with use of vaginal lubricants, treatment of vaginal infections, and systemic or local estrogen replacement.

1	CVD is increased in individuals with type 1 or type 2 DM. The Framingham Heart Study revealed a marked increase in PAD, coronary artery disease, MI, and CHF (risk increase from oneto fivefold) in DM. In addition, the prognosis for individuals with diabetes who have coronary artery disease or MI is worse than for nondiabetics. CHD is more likely to involve multiple vessels in individuals with DM. In addition to CHD, cerebrovascular disease is increased in individuals with DM (threefold increase in stroke). Thus, after controlling for all known cardiovascular risk factors, type 2 DM increases the cardiovascular death rate twofold in men and fourfold in women.

1	The American Heart Association has designated DM as a “CHD risk equivalent,” and type 2 DM patients without a prior MI have a similar risk for coronary artery–related events as nondiabetic individuals who have had a prior MI. However, the cardiovascular risk assessment in type 2 DM should encompass a more nuanced approach. Cardiovascular risk is lower and not equivalent in a younger individual with a brief duration of type 2 DM compared to an older individual with long-standing type 2DM. Because of the extremely high prevalence of underlying CVD in individuals with diabetes (especially in type 2 DM), evidence of atherosclerotic vascular disease (e.g., cardiac stress test) should be sought in an individual with diabetes who has symptoms suggestive of cardiac ischemia or peripheral or carotid arterial disease. The screening of asymptomatic individuals with diabetes for CHD, even with a risk-factor scale, is not recommended because recent studies have not shown a clinical benefit. The

1	arterial disease. The screening of asymptomatic individuals with diabetes for CHD, even with a risk-factor scale, is not recommended because recent studies have not shown a clinical benefit. The absence of chest pain (“silent ischemia”) is common in individuals with diabetes, and a thorough cardiac evaluation should be considered prior to major surgical procedures.

1	The increase in cardiovascular morbidity and mortality rates in diabetes appears to relate to the synergism of hyperglycemia with other cardiovascular risk factors. Risk factors for macrovascular disease in diabetic individuals include dyslipidemia, hypertension, obesity, reduced physical activity, and cigarette smoking. Additional risk factors more prevalent in the diabetic population include microalbuminuria, macroalbuminuria, an elevation of serum creatinine, abnormal platelet function and endothelial dysfunction The possibility of atherogenic potential of insulin is suggested by the data in nondiabetic individuals showing higher serum insulin levels (indicative of insulin resistance) in association with greater risk of cardiovascular morbidity and mortality. However, treatment with insulin and the sulfonylureas did not increase the risk of CVD in individuals with type 2 DM.

1	In general, the treatment of coronary disease is not different in the diabetic individual (Chap. 293). Revascularization procedures for CHD, including percutaneous coronary interventions (PCI) and coronary artery bypass grafting (CABG), may be less efficacious in the diabetic individual. Initial success rates of PCI in diabetic individuals are similar to those in the nondiabetic population, but diabetic patients have higher rates of restenosis and lower long-term patency and survival rates in older studies. Aggressive cardiovascular risk modification in all individuals with DM and glycemic control should be individualized, as discussed in Chap. 418. In patients with known CHD and type 2 DM, an ACE inhibitor (or ARB), a statin, and acetylsalicylic acid (ASA; aspirin) should be considered. Past trepidation about using beta blockers in individuals who have diabetes should not prevent use of these agents because they clearly benefit diabetic patients after MI. In

1	Diabetes Mellitus: Complications 2428 patients with CHF, thiazolidinediones should not be used (Chap. 418). However, metformin can be used in patients with stable CHF if the renal function is normal. Antiplatelet therapy reduces cardiovascular events in individuals with DM who have CHD and is recommended. Current recommendations by the ADA include the use of aspirin for primary prevention of coronary events in diabetic individuals with an increased 10-year cardiovascular risk >10% (at least one risk factor such as hypertension, smoking, family history, albuminuria, or dyslipidemia in men >50 years or women >60 years of age). ASA is not recommended for primary prevention in those with a 10-year cardiovascular risk <10%. The aspirin dose is the same as in nondiabetic individuals.

1	Cardiovascular Risk Factors • dysliPidemia Individuals with DM may have several forms of dyslipidemia (Chap. 421). Because of the additive cardiovascular risk of hyperglycemia and hyperlipidemia, lipid abnormalities should be assessed aggressively and treated as part of comprehensive diabetes care (Chap. 418). The most common pattern of dyslipidemia is hypertriglyceridemia and reduced high-density lipoprotein (HDL) cholesterol levels. DM itself does not increase levels of low-density lipoprotein (LDL), but the small dense LDL particles found in type 2 DM are more atherogenic because they are more easily glycated and susceptible to oxidation.

1	Almost all treatment studies of diabetic dyslipidemia have been performed in individuals with type 2 DM because of the greater frequency of dyslipidemia in this form of diabetes. Interventional studies have shown that the beneficial effects of LDL reduction with statins are similar in the diabetic and nondiabetic populations. Large prospective trials of primary and secondary intervention for CHD have included some individuals with type 2 DM, and subset analyses have consistently found that reductions in LDL reduce cardiovascular events and morbidity in individuals with DM. No prospective studies have addressed similar questions in individuals with type 1 DM. Because the frequency of CVD is low in children and young adults with diabetes, assessment of cardiovascular risk should be incorporated into the guidelines discussed below.

1	Based on the guidelines provided by the ADA, priorities in the treatment of dyslipidemia are as follows: (1) lower the LDL cholesterol, (2) raise the HDL cholesterol, and (3) decrease the triglycerides. A treatment strategy depends on the pattern of lipoprotein abnormalities. Initial therapy for all forms of dyslipidemia should include dietary changes, as well as the same lifestyle modifications recommended in the nondiabetic population (smoking cessation, blood pressure control, weight loss, increased physical activity). The dietary recommendations for individuals with DM include increased monounsaturated fat and carbohydrates and reduced saturated fats and cholesterol (Chap. 421). According to guidelines of the ADA, the target lipid values in diabetic individuals (age >40 years) without CVD should be as follows: LDL <2.6 mmol/L (100 mg/dL); HDL >1 mmol/L (40 mg/ dL) in men and >13 mmol/L (50 mg/dL) in women; and triglycerides <1.7 mmol/L (150 mg/dL). In patients >40 years, the ADA

1	CVD should be as follows: LDL <2.6 mmol/L (100 mg/dL); HDL >1 mmol/L (40 mg/ dL) in men and >13 mmol/L (50 mg/dL) in women; and triglycerides <1.7 mmol/L (150 mg/dL). In patients >40 years, the ADA recommends addition of a statin, regardless of the LDL level, in patients with CHD and those without CHD who have CHD risk factors. Recently released guidelines by the American College of Cardiology (ACC) and American Heart Association (AHA) differ slightly and recommend that diabetic individuals aged 40–75 without CHD and a LDL of 70–189 mg/dl receive “moderate” intensity statin therapy (Chap. 291e). Improvement in glycemic control will lower triglycerides and have a modest beneficial effect by raising HDL.

1	If the patient is known to have CHD, the ADA recommends an LDL goal of <18 mmol/L (70 mg/dL) as an “option” (in keeping with evidence that such a goal is beneficial in nondiabetic individuals with CHD [Chap. 421]). The ACC/AHA guidelines do not advocate a specific LDL for statin therapy. HMG-CoA reductase inhibitors are the agents of choice for lowering LDL. Combination therapy with an HMG-CoA reductase inhibitor and a fibrate or another lipid-lowering agent (ezetimibe, niacin) may be considered but increases the possibility of side effects such as myositis and has not been shown to be beneficial. Nicotinic acid effectively raises HDL and can be used in patients with diabetes, but may worsen glycemic control and increase insulin resistance and has not been shown to provide additional benefit beyond statin therapy alone. Bile acid–binding resins should not be used if hypertriglyceridemia is present. In large clinical trials, statin usage is associated with a mild increase in the risk

1	beyond statin therapy alone. Bile acid–binding resins should not be used if hypertriglyceridemia is present. In large clinical trials, statin usage is associated with a mild increase in the risk of developing type 2 DM. This risk is greatest in individuals with other risk factors for type 2 DM (Chap. 417). However, the cardiovascular benefits of statin use outweigh the mildly increased risk of diabetes.

1	HypertenSion Hypertension can accelerate other complications of DM, particularly CVD, nephropathy, and retinopathy. In targeting a goal of blood pressure of <140/80 mmHg, therapy should first emphasize lifestyle modifications such as weight loss, exercise, stress management, and sodium restriction. The BP goal should be individualized. In some younger individuals, the provider may target a blood pressure of <130/80 mmHg. Realizing that more than one agent is usually required to reach the blood pressure goal, the ADA recommends that all patients with diabetes and hypertension be treated with an ACE inhibitor or an ARB. Subsequently, agents that reduce cardiovascular risk (beta blockers, thiazide diuretics, and calcium channel blockers) should be incorporated into the regimen. ACE inhibitors and ARBs are likely equivalent in most patients with diabetes and renal disease. Serum potassium and renal function should be monitored.

1	Because of the high prevalence of atherosclerotic disease in individuals with type 2 DM, the possibility of renovascular hypertension should be considered when the blood pressure is not readily controlled.

1	DM is the leading cause of nontraumatic lower extremity amputation in the United States. Foot ulcers and infections are also a major source of morbidity in individuals with DM. The reasons for the increased incidence of these disorders in DM involve the interaction of several pathogenic factors: neuropathy, abnormal foot biomechanics, PAD, and poor wound healing. The peripheral sensory neuropathy interferes with normal protective mechanisms and allows the patient to sustain major or repeated minor trauma to the foot, often without knowledge of the injury. Disordered proprioception causes abnormal weight bearing while walking and subsequent formation of callus or ulceration. Motor and sensory neuropathy lead to abnormal foot muscle mechanics and to structural changes in the foot (hammer toe, claw toe deformity, prominent metatarsal heads, Charcot joint). Autonomic neuropathy results in anhidrosis and altered superficial blood flow in the foot, which promote drying of the skin and

1	toe, claw toe deformity, prominent metatarsal heads, Charcot joint). Autonomic neuropathy results in anhidrosis and altered superficial blood flow in the foot, which promote drying of the skin and fissure formation. PAD and poor wound healing impede resolution of minor breaks in the skin, allowing them to enlarge and to become infected.

1	Many individuals with type 2 DM develop a foot ulcer (great toe or metatarsophalangeal areas are most common), and a significant subset who develop an ulceration will ultimately undergo amputation (14–24% risk with that ulcer or subsequent ulceration). Risk factors for foot ulcers or amputation include male sex, diabetes for >10 years, peripheral neuropathy, abnormal structure of foot (bony abnormalities, callus, thickened nails), PAD, smoking, history of previous ulcer or amputation, visual impairment, and poor glycemic control. Large calluses are often precursors to or overlie ulcerations.

1	The optimal therapy for foot ulcers and amputations is prevention through identification of high-risk patients, education of the patient, and institution of measures to prevent ulceration. High-risk patients should be identified during the routine, annual foot examination performed on all patients with DM (see “Ongoing Aspects of Comprehensive Diabetes Care” in Chap. 418). If the monofilament test or one of the other tests is abnormal, the patient is diagnosed with loss of protective sensation (LOPS; Chap. 417). Providers should consider screening for asymptomatic PAD in individuals >50 years of age who have diabetes and other risk factors using anklebrachial index testing in high-risk individuals (Chap. 302). Patient education should emphasize (1) careful selection of footwear, (2) daily inspection of the feet to detect early signs of poor-fitting footwear or minor trauma, (3) daily foot hygiene to keep the skin clean and moist, (4) avoidance of self-treatment of foot abnormalities

1	inspection of the feet to detect early signs of poor-fitting footwear or minor trauma, (3) daily foot hygiene to keep the skin clean and moist, (4) avoidance of self-treatment of foot abnormalities and high-risk behavior (e.g., walking barefoot), and (5) prompt consultation with a health care provider if an abnormality arises. Patients at high risk for ulceration or amputation may benefit from evaluation by a foot care specialist. Calluses and nail deformities should be treated by a podiatrist. Interventions directed at risk factor modification include orthotic shoes and devices, callus management, nail care, and prophylactic measures to reduce increased skin pressure from abnormal bony architecture. Attention to other risk factors for vascular disease (smoking, dyslipidemia, hypertension) and improved glycemic control are also important.

1	Despite preventive measures, foot ulceration and infection are common and represent a serious problem. Due to the multifactorial pathogenesis of lower extremity ulcers, management of these lesions is multidisciplinary and often demands expertise in orthopedics, vascular surgery, endocrinology, podiatry, and infectious diseases. The plantar surface of the foot is the most common site of ulceration. Ulcers may be primarily neuropathic (no accompanying infection) or may have surrounding cellulitis or osteomyelitis. Cellulitis without ulceration is also frequent and should be treated with antibiotics that provide broad-spectrum coverage, including anaerobes (see below).

1	An infected ulcer is a clinical diagnosis, because superficial culture of any ulceration will likely find multiple possible bacterial species. The infection surrounding the foot ulcer is often the result of multiple organisms, with aerobic gram-positive cocci (staphylococci including MRSA, Group A and B streptococci) being most common and with aerobic gram-negative bacilli and/or obligate anaerobes as co-pathogens.

1	Gas gangrene may develop in the absence of clostridial infection. Cultures taken from the surface of the ulcer are not helpful; a culture from the debrided ulcer base or from purulent drainage or aspiration of the wound is the most helpful. Wound depth should be determined by inspection and probing with a blunt-tipped sterile instrument. Plain radiographs of the foot should be performed to assess the possibility of osteomyelitis in chronic ulcers that have not responded to therapy. Magnetic resonance imaging (MRI) is the most specific modality, with nuclear medicine scans and labeled white cell studies as alternatives. Surgical debridement is often necessary.

1	Osteomyelitis is best treated by a combination of prolonged antibiotics (IV, then oral) and/or possibly debridement of infected bone. The possible contribution of vascular insufficiency should be considered in all patients. Peripheral arterial bypass procedures are often effective in promoting wound healing and in decreasing the need for amputation of the ischemic limb (Chap. 302).

1	A consensus statement from the ADA identified six interventions with demonstrated efficacy in diabetic foot wounds: (1) off-loading, (2) debridement, (3) wound dressings, (4) appropriate use of antibiotics, (5) revascularization, and (6) limited amputation. Off-loading is the complete avoidance of weight bearing on the ulcer, which removes the mechanical trauma that retards wound healing. Bed rest and a variety of orthotic devices or contact casting limit weight bearing on wounds or pressure points. Surgical debridement is important and effective, but clear efficacy of other modalities for wound cleaning (enzymes, soaking, whirlpools) is lacking. Dressings such as hydrocolloid dressings promote wound healing by creating a moist environment and protecting the wound. Antiseptic agents should be avoided. Topical antibiotics are of limited value. Referral for physical therapy, orthotic evaluation, and rehabilitation should occur once the infection is controlled.

1	Mild or non-limb-threatening infections can be treated with oral antibiotics directed predominantly at methicillin-susceptible staphylococci and streptococci (e.g., dicloxacillin, cephalosporin, amoxicillin/clavulanate). However the increasing prevalence of MRSA often requires the use of clindamycin, doxycycline, or trimethoprim-sulfamethoxazole. Trimethoprim-sulfamethoxazole exhibits less reliable coverage of streptococci than the β-lactams, and dia-2429 betic patients may develop adverse effects including acute kidney injury and hyperkalemia. Surgical debridement of necrotic tissue, local wound care (avoidance of weight bearing over the ulcer), and close surveillance for progression of infection are crucial. More severe infections require IV antibiotics as well as bed rest and local wound care. Urgent surgical debridement may be required. Optimization of glycemic control should be a goal. IV antibiotics should provide broad-spectrum coverage directed toward Staphylococcus aureus,

1	care. Urgent surgical debridement may be required. Optimization of glycemic control should be a goal. IV antibiotics should provide broad-spectrum coverage directed toward Staphylococcus aureus, including MRSA, streptococci, gram-negative aerobes, and anaerobic bacteria. Initial antimicrobial regimens include vancomycin plus a β-lactam/β-lactamase inhibitor or carbapenem or vancomycin plus a combination of quinolone plus metronidazole. Daptomycin, ceftaroline, or linezolid may be substituted for vancomycin. If the infection surrounding the ulcer is not improving with IV antibiotics, reassessment of antibiotic coverage and reconsideration of the need for surgical debridement or revascularization are indicated. With clinical improvement, oral antibiotics and local wound care can be continued on an outpatient basis with close follow-up.

1	Individuals with DM have a greater frequency and severity of infection. The reasons for this include incompletely defined abnormalities in cell-mediated immunity and phagocyte function associated with hyperglycemia, as well as diminished vascularization. Hyperglycemia aids the colonization and growth of a variety of organisms (Candida and other fungal species). Many common infections are more frequent and severe in the diabetic population, whereas several rare infections are seen almost exclusively in the diabetic population. Examples of this latter category include rhinocerebral mucormycosis, emphysematous infections of the gallbladder and urinary tract, and “malignant” or invasive otitis externa. Invasive otitis externa is usually secondary to

1	P. aeruginosa infection in the soft tissue surrounding the external auditory canal, usually begins with pain and discharge, and may rapidly progress to osteomyelitis and meningitis. These infections should be sought, in particular, in patients presenting with severe hyperglycemia (Chap. 418).

1	Pneumonia, urinary tract infections, and skin and soft tissue infections are all more common in the diabetic population. In general, the organisms that cause pulmonary infections are similar to those found in the nondiabetic population; however, gram-negative organisms, S. aureus, and Mycobacterium tuberculosis are more frequent pathogens. Urinary tract infections (either lower tract or pyelonephritis) are the result of common bacterial agents such as Escherichia coli, although several yeast species (Candida and Torulopsis glabrata) are commonly observed. Complications of urinary tract infections include emphysematous pyelonephritis and emphysematous cystitis. Bacteriuria occurs frequently in individuals with diabetic cystopathy. Susceptibility to furunculosis, superficial candidal infections, and vulvovaginitis are increased. Poor glycemic control is a common denominator in individuals with these infections. Diabetic individuals have an increased rate of colonization of S. aureus in

1	and vulvovaginitis are increased. Poor glycemic control is a common denominator in individuals with these infections. Diabetic individuals have an increased rate of colonization of S. aureus in the skinfolds and nares. Diabetic patients also have a greater risk of postoperative wound infections.

1	The most common skin manifestations of DM are xerosis and pruritus and are usually relieved by skin moisturizers. Protracted wound healing and skin ulcerations are also frequent complications. Diabetic dermopathy, sometimes termed pigmented pretibial papules, or “diabetic skin spots,” begins as an erythematous macule or papule that evolves into an area of circular hyperpigmentation. These lesions result from minor mechanical trauma in the pretibial region and are more common in elderly men with DM. Bullous diseases, such as bullosa diabeticorum (shallow ulcerations or erosions in the pretibial region), are also seen. Necrobiosis lipoidica diabeticorum is an uncommon disorder, accompanying diabetes in predominantly young women. This usually begins in the pretibial region as an erythematous plaque or papules that gradually enlarge, darken, and develop irregular margins,

1	Diabetes Mellitus: Complications 2430 with atrophic centers and central ulceration. They are often painful. Vitiligo occurs at increased frequency in individuals with type 1 DM. Acanthosis nigricans (hyperpigmented velvety plaques seen on the neck, axilla, or extensor surfaces) is sometimes a feature of severe insulin resistance and accompanying diabetes. Generalized or localized granuloma annulare (erythematous plaques on the extremities or trunk) and scleredema (areas of skin thickening on the back or neck at the site of previous superficial infections) are more common in the diabetic population. Lipoatrophy and lipohypertrophy can occur at insulin injection sites but are now unusual with the use of human insulin.

1	Hypoglycemia Philip E. Cryer, Stephen N. Davis Hypoglycemia is most commonly caused by drugs used to treat diabe-tes mellitus or by exposure to other drugs, including alcohol. However, a number of other disorders, including critical organ failure, sepsis and inanition, hormone deficiencies, non-β-cell tumors, insulinoma, 420 and prior gastric surgery, can cause hypoglycemia (Table 420-1). Hypoglycemia is most convincingly documented by Whipple’s triad: (1) symptoms consistent with hypoglycemia, (2) a low plasma glucose concentration measured with a precise method (not a glucose monitor), and (3) relief of symptoms after the plasma glucose level is raised. The lower limit of the fasting plasma glucose concentration is normally ∼70 mg/dL (∼3.9 mmol/L), but lower venous glucose levels occur normally, late after a meal, during pregnancy, and during prolonged fasting (>24 h). Hypoglycemia can cause serious morbidity; if severe and prolonged, it can be fatal. It should be considered in any

1	normally, late after a meal, during pregnancy, and during prolonged fasting (>24 h). Hypoglycemia can cause serious morbidity; if severe and prolonged, it can be fatal. It should be considered in any patient with episodes of confusion, an altered level of consciousness, or a seizure.

1	Glucose is an obligate metabolic fuel for the brain under physiologic conditions. The brain cannot synthesize glucose or store more than a few minutes’ supply as glycogen and therefore requires a continuous supply of glucose from the arterial circulation. As the arterial plasma glucose concentration falls below the physiologic range, blood-tobrain glucose transport becomes insufficient to support brain energy metabolism and function. However, redundant glucose counterregulatory mechanisms normally prevent or rapidly correct hypoglycemia.

1	Plasma glucose concentrations are normally maintained within a relatively narrow range—roughly 70–110 mg/dL (3.9–6.1 mmol/L) in the fasting state, with transient higher excursions after a meal— despite wide variations in exogenous glucose delivery from meals and in endogenous glucose utilization by, for example, exercising muscle. Between meals and during fasting, plasma glucose levels are maintained by endogenous glucose production, hepatic glycogenolysis, and hepatic (and renal) gluconeogenesis (Fig. 420-1). Although hepatic glycogen stores are usually sufficient to maintain plasma glucose levels for ∼8 h, this period can be shorter if glucose demand is increased by exercise or if glycogen stores are depleted by illness or starvation.

1	Gluconeogenesis normally requires low insulin levels and the presence of anti-insulin (counterregulatory) hormones together with a coordinated supply of precursors from muscle and adipose tissue to the liver (and kidneys). Muscle provides lactate, pyruvate, alanine, glutamine, and other amino acids. Triglycerides in adipose tissue are broken down into fatty acids and glycerol, which is a gluconeogenic precursor. Fatty acids provide an alternative oxidative fuel to tissues other than the brain (which requires glucose). Systemic glucose balance—maintenance of the normal plasma glucose concentration—is accomplished by a network of hormones, neural signals, and substrate effects that regulate endogenous glucose production and glucose utilization by tissues other than the brain (Chap. 417). Among the regulatory factors, insulin plays a dominant role (Table 420-2; Fig. 420-1). As plasma glucose levels decline within 1. 2.

1	2. Critical illness Hepatic, renal or cardiac failure Sepsis Inanition 3. 4. 5. Endogenous hyperinsulinism Insulinoma Functional β-cell disorders (nesidioblastosis) Antibody to insulin Antibody to insulin receptor 6. Accidental, surreptitious, or malicious hypoglycemia Source: From PE Cryer et al: J Clin Endocrinol Metab 94:709, 2009. ©The Endocrine Society, 2009. the physiologic range in the fasting state, pancreatic β-cell insulin secretion decreases, thereby increasing hepatic glycogenolysis and hepatic (and renal) gluconeogenesis. Low insulin levels also reduce glucose utilization in peripheral tissues, inducing lipolysis and proteolysis and consequently releasing gluconeogenic precursors. Thus, a decrease in insulin secretion is the first defense against hypoglycemia.

1	As plasma glucose levels decline just below the physiologic range, glucose counterregulatory (plasma glucose–raising) hormones are released (Table 420-2; Fig. 420-1). Among these, pancreatic α-cell glucagon, which stimulates hepatic glycogenolysis, plays a primary role. Glucagon is the second defense against hypoglycemia. Adrenomedullary epinephrine, which stimulates hepatic glycogenolysis and gluconeogenesis (and renal gluconeogenesis), is not normally critical. However, it becomes critical when glucagon is deficient. Epinephrine is the third defense against hypoglycemia. When hypoglycemia is prolonged beyond ∼4 h, cortisol and growth hormone also support glucose production and restrict glucose utilization to a limited amount (∼20% compared to epinephrine). Thus cortisol and growth hormone play no role in defense against acute hypoglycemia.

1	As plasma glucose levels fall further, symptoms prompt behavioral defense against hypoglycemia, including the ingestion of food (Table 420-2; Fig. 420-1). The normal glycemic thresholds for these responses to decreasing plasma glucose concentrations are shown in Table 420-2. However, these thresholds are dynamic. They shift to higher-thannormal glucose levels in people with poorly controlled diabetes, who can experience symptoms of hypoglycemia when their glucose levels decline toward the normal range (pseudohypoglycemia). On the other hand, thresholds shift to lower-than-normal glucose levels in people with recurrent hypoglycemia; e.g., patients with aggressively treated diabetes or an insulinoma have symptoms at glucose levels lower than those that cause symptoms in healthy individuals.

1	Clinical Manifestations Neuroglycopenic manifestations of hypoglycemia are the direct result of central nervous system glucose deprivation. These features include behavioral changes, confusion, fatigue, seizure, loss of consciousness, and, if hypoglycemia is severe and prolonged, Brain Pituitary Growth hormone (ACTH) Adrenal cortex Cortisol Pancreas Insulin Liver Kidneys Glucose production Arterial glucoseFat Muscle Gluconeogenic precursor (lactate, amino acids, glycerol) Glucagon Sympathoadrenal outflow Sympathetic postganglionic neurons Adrenal medullae Epinephrine Norepinephrine Acetylcholine Glucose clearance Symptoms (Ingestion) FIGURE 420-1 Physiology of glucose counterregulation: mechanisms that normally prevent or rapidly correct hypoglycemia. In insulin-deficient diabetes, the key counterregulatory responses—suppression of insulin and increases in glucagon—are lost, and stimulation of sympa-thoadrenal outflow is attenuated. ACTH, adrenocorticotropic hormone.

1	death. Neurogenic (or autonomic) manifestations of hypoglycemia result from the perception of physiologic changes caused by the central nervous system–mediated sympathoadrenal discharge that is triggered by hypoglycemia. They include adrenergic symptoms (mediated largely by norepinephrine released from sympathetic postganglionic neurons but perhaps also by epinephrine released from the adrenal medullae), such as palpitations, tremor, and anxiety, as well as cholinergic symptoms (mediated by acetylcholine released from sympathetic postganglionic neurons), such as sweating, hunger, and paresthesias. Clearly, these are nonspecific symptoms. Their attribution to hypoglycemia requires that the corresponding plasma glucose concentration be low and that the symptoms resolve after the glucose level is raised (as delineated by Whipple’s triad).

1	Common signs of hypoglycemia include diaphoresis and pallor. Heart rate and systolic blood pressure are typically increased but may not be raised in an individual who has experienced repeated, recent episodes of hypoglycemia. Neuroglycopenic manifestations are often observable. Transient focal neurologic deficits occur occasionally. Permanent neurologic deficits are rare. Etiology and Pathophysiology Hypoglycemia is most commonly a result of the treatment of diabetes. This topic is therefore addressed before other causes of hypoglycemia are considered.

1	Etiology and Pathophysiology Hypoglycemia is most commonly a result of the treatment of diabetes. This topic is therefore addressed before other causes of hypoglycemia are considered. HYPOGLYCEMIA IN DIABETES Impact and Frequency Hypoglycemia is the limiting factor in the glycemic management of diabetes mellitus. First, it causes recurrent morbidity in most people with type 1 diabetes (T1DM) and in many with advanced type 2 diabetes (T2DM), and it is sometimes fatal. Second, it precludes maintenance of euglycemia over a lifetime of diabetes and thus full realization of the well-established microvascular benefits of glycemic control. Third, it causes a vicious cycle of recurrent hypoglycemia by producing hypoglycemia-associated autonomic failure—i.e., the clinical syndromes of defective glucose counterregulation and of hypoglycemia unawareness (see later).

1	Hypoglycemia is a fact of life for people with T1DM. They suffer an average of two episodes of symptomatic hypoglycemia per week and at least one episode of severe, at least temporarily disabling hypoglycemia each year. An estimated 6–10% of people with T1DM die as a result Glycemic Threshold, Physiologic Role in Prevention or Correction of Hypoglycemia Response mmol/L (mg/dL) Effects (Glucose Counterregulation) Note: Ra, rate of glucose appearance, glucose production by the liver and kidneys; Rc, rate of glucose clearance, glucose utilization relative to the ambient plasma glucose by insulin-sensitive tissues; Rd, rate of glucose disappearance, glucose utilization by insulin-sensitive tissues such as skeletal muscle. Rd by the brain is not altered by insulin, glucagon, epinephrine, cortisol, or growth hormone. Source: From PE Cryer, in S Melmed et al (eds): Williams Textbook of Endocrinology, 12th ed. New York, Elsevier, 2012.

1	Source: From PE Cryer, in S Melmed et al (eds): Williams Textbook of Endocrinology, 12th ed. New York, Elsevier, 2012. decrements in insulin and increments in glucagon and epinephrine), and hypoglycemia unaware- ness compromises behavioral defense (ingestion of carbohydrate). defective GlucoSe counterreGulation In the setting of absolute endogenous insulin deficiency, insu- lin levels do not decrease as plasma glucose levels of hypoglycemia fall; the first defense against hypoglycemia is lost. Furthermore, probably because the decrement in intraislet insulin is normally a signal to stimulate glucagon secretion, glucagon levels do not increase against hypoglycemia is lost. Finally, the increase in epinephrine levels, a third defense against hypogly cemia, in response to a given level of hypoglycemia is typically attenuated. The glycemic threshold for the sympathoadrenal (adrenomedullary epinephrine is shifted to lower plasma glucose concentrations.

1	That shift is typically the result of recent antecedent iatrogenic hypoglycemia. In the setting of absent decrements in insulin and of absent increments in glucagon, the attenuated increment in epinephrine causes the clinical syndrome of defective glucose counterregulation. Affected patients are at ≥25-fold greater risk of severe iatrogenic hypoglycemia during are patients with normal epinephrine responses. FIGURE 420-2 Hypoglycemia-associated autonomic failure (HAAF) in insulin- This functional—and potentially reversible— deficient diabetes. T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus.

1	disorder is distinct from classic diabetic autonomic (Modified from PE Cryer: Hypoglycemia in Diabetes. Pathophysiology, Prevalence, and neuropathy—a structural and irreversible disorder. Prevention, 2nd ed. © American Diabetes Association, 2012.) glucose control in either inpatient or outpatient settings have reported a high prevalence of severe hypoglycemia. In the NICE-SUGAR study, attempts to control in-hospital plasma glucose values towards physiologic levels resulted in increased mortality risk. The ADVANCE and ACCORD studies and the Veterans Affairs Diabetes Trial (VADT) also found a significant incidence of severe hypoglycemia among T2DM patients. Severe hypoglycemia with accompanying serious cardiovascular morbidity and mortality also occurred in the standard (e.g., not receiving intensified treatment) control group in both the ACCORD study and the VADT. Thus, severe hypoglycemia can and does occur at HbA1c values of 8–9% in both T1DM and T2DM. Somewhat surprisingly, all

1	intensified treatment) control group in both the ACCORD study and the VADT. Thus, severe hypoglycemia can and does occur at HbA1c values of 8–9% in both T1DM and T2DM. Somewhat surprisingly, all three studies found little or no benefit of intensive glucose control to reduce macrovascular events in T2DM. In fact, the ACCORD study was ended early because of the increased mortality rate in the intensive glucose control arm. Whether iatrogenic hypoglycemia was the cause of the increased mortality risk is not known. In light of these findings, some new recommendations and paradigms have been formulated. Whereas there is little debate regarding the need to reduce hyperglycemia in the hospital, the glycemic maintenance goals have been modified to lie between 140 and 180 mg/dL. Accordingly, the benefits of insulin therapy and reduced hyperglycemia can be obtained while the prevalence of hypoglycemia is reduced.

1	Similarly, evidence exists that intensive glucose control can reduce the prevalence of microvascular disease in both T1DM and T2DM. These benefits need to be weighed against the increased prevalence of hypoglycemia. Certainly, the level of glucose control (i.e., the HbA1c level) should be evaluated for each patient. Multicenter trials have demonstrated that individuals with recently diagnosed T1DM or T2DM can have better glycemic control with less hypoglycemia. In addition, there is still long-term benefit in reducing HbA1c values from higher to lower, albeit still above recommended levels. Perhaps a reasonable therapeutic goal is the lowest HbA1c level that does not cause severe hypoglycemia and that preserves awareness of hypoglycemia.

1	Pancreatic transplantation (both whole-organ and islet-cell) has been used in part as a treatment for severe hypoglycemia. Generally, rates of hypoglycemia are reduced after transplantation. This decrease appears to be due to increased physiologic insulin and glucagon responses during hypoglycemia. The use of continuous glucose monitors offers some promise as a method of reducing hypoglycemia while improving HbA1c. Other interventions to stimulate counterregulatory responses, such as selective serotonin-reuptake inhibitors, β-adrenergic receptor antagonists, opiate receptor antagonists, and fructose, remain experimental and have not been assessed in large-scale clinical trials.

1	Thus, intensive glycemic therapy (Chap. 418) needs to be applied along with the patient’s education and empowerment, frequent self-monitoring of blood glucose, flexible insulin (and other drug) regimens (including the use of insulin analogues, both shortand longer-acting), individualized glycemic goals, and ongoing professional guidance, support, and consideration of both the conventional risk factors and those indicative of compromised glucose counterregulation. Given a history of hypoglycemia unawareness, a 2to 3-week period of scrupulous avoidance of hypoglycemia is indicated.

1	There are many causes of hypoglycemia (Table 420-1). Because hypoglycemia is common in insulinor insulin secretagogue–treated diabetes, it is often reasonable to assume that a clinically suspicious episode is the result of hypoglycemia. On the other hand, because hypoglycemia is rare in the absence of relevant drug-treated diabetes, it is reasonable to conclude that a hypoglycemic disorder is present only in patients in whom Whipple’s triad can be demonstrated. Particularly when patients are ill or medicated, the initial diagnostic focus should be on the possibility of drug involvement and then on critical illnesses, hormone deficiency, or non–islet cell tumor hypoglycemia. In the absence of any of these etiologic factors and in a seemingly well individual, the focus should shift to possible endogenous hyperinsulinism or accidental, surreptitious, or even malicious hypoglycemia.

1	Drugs Insulin and insulin secretagogues suppress glucose produc-2433 tion and stimulate glucose utilization. Ethanol blocks gluconeogenesis but not glycogenolysis. Thus, alcohol-induced hypoglycemia typically occurs after a several-day ethanol binge during which the person eats little food, with consequent glycogen depletion. Ethanol is usually measurable in blood at the time of presentation, but its levels correlate poorly with plasma glucose concentrations. Because gluconeogenesis becomes the predominant route of glucose production during prolonged hypoglycemia, alcohol can contribute to the progression of hypoglycemia in patients with insulin-treated diabetes. Many other drugs have been associated with hypoglycemia. These include commonly used drugs such as angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists, β-adrenergic receptor antagonists, quinolone antibiotics, indomethacin, quinine, and sulfonamides.

1	Critical Illness Among hospitalized patients, serious illnesses such as renal, hepatic, or cardiac failure; sepsis; and inanition are second only to drugs as causes of hypoglycemia. Rapid and extensive hepatic destruction (e.g., toxic hepatitis) causes fasting hypoglycemia because the liver is the major site of endogenous glucose production. The mechanism of hypoglycemia in patients with cardiac failure is unknown. Hepatic congestion and hypoxia may be involved. Although the kidneys are a source of glucose production, hypoglycemia in patients with renal failure is also caused by the reduced clearance of insulin and the reduced mobilization of gluconeogenic precursors in renal failure.

1	Sepsis is a relatively common cause of hypoglycemia. Increased glucose utilization is induced by cytokine production in macrophage-rich tissues such as the liver, spleen, and lung. Hypoglycemia develops if glucose production fails to keep pace. Cytokine-induced inhibition of gluconeogenesis in the setting of nutritional glycogen depletion, in combination with hepatic and renal hypoperfusion, may also contribute to hypoglycemia. Hypoglycemia can be seen with starvation, perhaps because of loss of whole-body fat stores and subsequent depletion of gluconeogenic pre cursors (e.g., amino acids), necessitating increased glucose utilization.

1	Hormone Deficiencies Neither cortisol nor growth hormone is critical to the prevention of hypoglycemia, at least in adults. Nonetheless, hypoglycemia can occur with prolonged fasting in patients with primary adrenocortical failure (Addison’s disease) or hypopituitarism. Anorexia and weight loss are typical features of chronic cortisol deficiency and likely result in glycogen depletion. Cortisol deficiency is associated with impaired gluconeogenesis and low levels of gluconeogenic precursors; these associations suggest that substrate-limited gluconeogenesis, in the setting of glycogen depletion, is the cause of hypoglycemia. Growth hormone deficiency can cause hypoglycemia in young children. In addition to extended fasting, high rates of glucose utilization (e.g., during exercise or in pregnancy) or low rates of glucose production (e.g., after alcohol ingestion) can precipitate hypoglycemia in adults with previously unrecognized hypopituitarism.

1	Hypoglycemia is not a feature of the epinephrine-deficient state that results from bilateral adrenalectomy when glucocorticoid replacement is adequate, nor does it occur during pharmacologic adrenergic blockade when other glucoregulatory systems are intact. Combined deficiencies of glucagon and epinephrine play a key role in the pathogenesis of iatrogenic hypoglycemia in people with insulin-deficient diabetes, as discussed earlier. Otherwise, deficiencies of these hormones are not usually considered in the differential diagnosis of a hypoglycemic disorder.

1	Non-β-Cell Tumors Fasting hypoglycemia, often termed non–islet cell tumor hypoglycemia, occurs occasionally in patients with large mesenchymal or epithelial tumors (e.g., hepatomas, adrenocortical carcinomas, carcinoids). The glucose kinetic patterns resemble those of hyperinsulinism (see next), but insulin secretion is suppressed appropriately during hypoglycemia. In most instances, hypoglycemia is due to overproduction of an incompletely processed form of insulin-like growth factor II (“big IGF-II”) that does not complex normally with circulating binding proteins and thus more readily gains access to 2434 target tissues. The tumors are usually apparent clinically, plasma ratios of IGF-II to IGF-I are high, and free IGF-II levels (and levels of proIGF-II [1–21]) are elevated. Curative surgery is seldom possible, but reduction of tumor bulk may ameliorate hypoglycemia. Therapy with a glucocorticoid, a growth hormone, or both has also been reported to alleviate hypoglycemia.

1	surgery is seldom possible, but reduction of tumor bulk may ameliorate hypoglycemia. Therapy with a glucocorticoid, a growth hormone, or both has also been reported to alleviate hypoglycemia. Hypoglycemia attributed to ectopic IGF-I production has been reported but is rare.

1	Endogenous Hyperinsulinism Hypoglycemia due to endogenous hyperinsulinism can be caused by (1) a primary β-cell disorder—typically a β-cell tumor (insulinoma), sometimes multiple insulinomas, or a functional β-cell disorder with β-cell hypertrophy or hyperplasia; (2) an antibody to insulin or to the insulin receptor; (3) a β-cell secretagogue such as a sulfonylurea; or perhaps (4) ectopic insulin secretion, among other very rare mechanisms. None of these causes is common.

1	The fundamental pathophysiologic feature of endogenous hyperinsulinism caused by a primary β-cell disorder or an insulin secretagogue is the failure of insulin secretion to fall to very low levels during hypoglycemia. This feature is assessed by measurement of plasma insulin, C-peptide (the connecting peptide that is cleaved from proinsulin to produce insulin), proinsulin, and glucose concentrations during hypoglycemia. Insulin, C-peptide, and proinsulin levels need not be high relative to normal, euglycemic values; rather, they are inappropriately high in the setting of a low plasma glucose concentration. Critical diagnostic findings are a plasma insulin concentration ≥3 μU/mL (≥18 pmol/L), a plasma C-peptide concentration ≥0.6 ng/mL (≥0.2 nmol/L), and a plasma proinsulin concentration ≥5.0 pmol/L when the plasma glucose concentration is <55 mg/dL (<3.0 mmol/L) with symptoms of hypoglycemia. A low plasma β-hydroxybutyrate concentration (≤2.7 mmol/L) and an increment in plasma glucose

1	pmol/L when the plasma glucose concentration is <55 mg/dL (<3.0 mmol/L) with symptoms of hypoglycemia. A low plasma β-hydroxybutyrate concentration (≤2.7 mmol/L) and an increment in plasma glucose level of >25 mg/dL (>1.4 mmol/L) after IV administration of glucagon (1.0 mg) indicate increased insulin (or IGF) actions.

1	The diagnostic strategy is (1) to measure plasma glucose, insulin, C-peptide, proinsulin, and β-hydroxybutyrate concentrations and to screen for circulating oral hypoglycemic agents during an episode of hypoglycemia and (2) to assess symptoms during the episode and seek their resolution following correction of hypoglycemia by IV injection of glucagon (i.e., to document Whipple’s triad). This is straightforward if the patient is hypoglycemic when seen. Since endogenous hyperinsulinemic disorders usually, but not invariably, cause fasting hypoglycemia, a diagnostic episode may develop after a relatively short outpatient fast. Serial sampling during an inpatient diagnostic fast of up to 72 h or after a mixed meal is more problematic. An alternative is to give patients a detailed list of the required measurements and ask them to present to an emergency room, with the list, during a symptomatic episode. Obviously, a normal plasma glucose concentration during a symptomatic episode indicates

1	measurements and ask them to present to an emergency room, with the list, during a symptomatic episode. Obviously, a normal plasma glucose concentration during a symptomatic episode indicates that the symptoms are not the result of hypoglycemia.

1	An insulinoma—an insulin-secreting pancreatic islet β-cell tumor— is the prototypical cause of endogenous hyperinsulinism and therefore should be sought in patients with a compatible clinical syndrome. However, insulinoma is not the only cause of endogenous hyperinsulinism. Some patients with fasting endogenous hyperinsulinemic hypoglycemia have diffuse islet involvement with β-cell hypertrophy and sometimes hyperplasia. This pattern is commonly referred to as nesidioblastosis, although β-cells budding from ducts are not invariably found. Other patients have a similar islet pattern but with postprandial hypoglycemia, a disorder termed noninsulinoma pancreatogenous hypoglycemia. Postgastric bypass postprandial hypoglycemia, which most often follows Roux-en-Y gastric bypass, is also characterized by diffuse islet involvement and endogenous hyperinsulinism. Some have suggested that exaggerated GLP-1 responses to meals cause hyperinsulinemia and hypoglycemia, but the relevant pathogenesis

1	by diffuse islet involvement and endogenous hyperinsulinism. Some have suggested that exaggerated GLP-1 responses to meals cause hyperinsulinemia and hypoglycemia, but the relevant pathogenesis has not been clearly established. If medical treatments with agents such as an α-glucosidase inhibitor, diazoxide, or octreotide fail, partial pancreatectomy may be required. Autoimmune hypoglycemias include those caused by an antibody to insulin that binds post-meal insulin and then gradually disassociates, with consequent late postprandial hypoglycemia. Alternatively, an insulin receptor antibody can function as an agonist. The presence of an insulin secretagogue, such as a sulfonylurea or a glinide, results in a clinical and biochemical pattern similar to that of an insulinoma but can be distinguished by the presence of the circulating secretagogue. Finally, there are reports of very rare phenomena such as ectopic insulin secretion, a gain-of-function insulin receptor mutation, and

1	distinguished by the presence of the circulating secretagogue. Finally, there are reports of very rare phenomena such as ectopic insulin secretion, a gain-of-function insulin receptor mutation, and exercise-induced hyperinsulinemia.

1	Insulinomas are uncommon, with an estimated yearly incidence of 1 in 250, 000. Because more than 90% of insulinomas are benign, they are a treatable cause of potentially fatal hypoglycemia. The median age at presentation is 50 years in sporadic cases, but the tumor usually presents in the third decade when it is a component of multiple endocrine neoplasia type 1 (Chap. 408). More than 99% of insulinomas are within the substance of the pancreas, and the tumors are usually small (<2.0 cm in diameter in 90% of cases). Therefore, they come to clinical attention because of hypoglycemia rather than mass effects. CT or MRI detects ∼70–80% of insulinomas. These methods detect metastases in the roughly 10% of patients with a malignant insulinoma. Transabdominal ultrasound often identifies insulinomas, and endoscopic ultrasound has a sensitivity of ∼90%. Somatostatin receptor scintigraphy is thought to detect insulinomas in about half of patients. Selective pancreatic arterial calcium

1	insulinomas, and endoscopic ultrasound has a sensitivity of ∼90%. Somatostatin receptor scintigraphy is thought to detect insulinomas in about half of patients. Selective pancreatic arterial calcium injections, with the endpoint of a sharp increase in hepatic venous insulin levels, regionalize insulinomas with high sensitivity, but this invasive procedure is seldom necessary except to confirm endogenous hyperinsulinism in the diffuse islet disorders. Intraoperative pancreatic ultrasonography almost invariably localizes insulinomas that are not readily palpable by the surgeon. Surgical resection of a solitary insulinoma is generally curative. Diazoxide, which inhibits insulin secretion, or the somatostatin analogue octreotide can be used to treat hypoglycemia in patients with unresectable tumors; everolimus, an mTOR (mammalian target of rapamycin) inhibitor, is promising.

1	ACCIDENTAL, SURREPTITIOUS, OR MALICIOUS HYPOGLYCEMIA Accidental ingestion of an insulin secretagogue (e.g., as the result of a pharmacy or other medical error) or even accidental administration of insulin can occur. Factitious hypoglycemia, caused by surreptitious or even malicious administration of insulin or an insulin secretagogue, shares many clinical and laboratory features with insulinoma. It is most common among health care workers, patients with diabetes or their relatives, and people with a history of other factitious illnesses. However, it should be considered in all patients being evaluated for hypoglycemia of obscure cause. Ingestion of an insulin secretagogue causes hypoglycemia with increased C-peptide levels, whereas exogenous insulin causes hypoglycemia with low C-peptide levels reflecting suppression of insulin secretion.

1	Analytical error in the measurement of plasma glucose concentrations is rare. On the other hand, glucose monitors used to guide treatment of diabetes are not quantitative instruments, particularly at low glucose levels, and should not be used for the definitive diagnosis of hypoglycemia. Even with a quantitative method, low measured glucose concentrations can be artifactual—e.g., the result of continued glucose metabolism by the formed elements of the blood ex vivo, particularly in the presence of leukocytosis, erythrocytosis, or thrombocytosis or with delayed separation of the serum from the formed elements (pseudohypoglycemia). Nondiabetic hypoglycemia also results from inborn errors of metabolism. Such hypoglycemia most commonly occurs in infancy but can also occur in adulthood. Cases in adults can be classified into those resulting in fasting hypoglycemia, postprandial hypoglycemia, and exercise-induced hypoglycemia.

1	Fasting Hypoglycemia Although rare, disorders of glycogenolysis can result in fasting hypoglycemia. These disorders include glycogen storage disease (GSD) of types 0, I, III, and IV and Fanconi-Bickel syndrome (Chap. 433e). Patients with GSD types I and III characteristically have high blood lactate levels before and after meals, respectively. Both groups have hypertriglyceridemia, but ketones are high in GSD type III. Defects in fatty acid oxidation also result in fasting hypoglycemia. These defects can include (1) defects in the carnitine cycle; (2) fatty-acid β-oxidation disorders; (3) electron transfer disturbances; and (4) ketogenesis disorders. Finally, defects in gluconeogenesis (fructose-1, 6-biphosphatase) have been reported to result in recurrent hypoglycemia and lactic acidosis.

1	Postprandial Hypoglycemia Inborn errors of metabolism resulting in postprandial hypoglycemia are also rare. These errors include (1) glucokinase, SUR1, and Kir6.2 potassium channel mutations; (2) congenital disorders of glycosylation; and (3) inherited fructose intolerance. Exercise-Induced Hypoglycemia Exercise-induced hypoglycemia, by definition, follows exercise. It results in hyperinsulinemia caused by increased activity of monocarboxylate transporter 1 in β cells. APPROACH TO THE PATIENT: In addition to the recognition and documentation of hypoglycemia as well as its treatment (often on an urgent basis), diagnosis of the hypoglycemic mechanism is critical for the selection of therapy that prevents, or at least minimizes, recurrent hypoglycemia.

1	Hypoglycemia is suspected in patients with typical symptoms; in the presence of confusion, an altered level of consciousness, or a seizure; or in a clinical setting in which hypoglycemia is known to occur. Blood should be drawn, whenever possible, before the administration of glucose to allow documentation of a low plasma glucose concentration. Convincing documentation of hypoglycemia requires the fulfillment of Whipple’s triad. Thus, the ideal time to measure the plasma glucose level is during a symptomatic episode. A normal glucose level excludes hypoglycemia as the cause of the symptoms. A low glucose level confirms that hypoglycemia is the cause of the symptoms, provided the latter resolve after the glucose level is raised. When the cause of the hypoglycemic episode is obscure, additional measurements—made while the glucose level is low and before treatment—should include plasma insulin, C-peptide, proinsulin, and β-hydroxybutyrate levels; also critical are screening for

1	additional measurements—made while the glucose level is low and before treatment—should include plasma insulin, C-peptide, proinsulin, and β-hydroxybutyrate levels; also critical are screening for circulating oral hypoglycemic agents and assessment of symptoms before and after the plasma glucose concentration is raised.

1	When the history suggests prior hypoglycemia and no potential mechanism is apparent, the diagnostic strategy is to evaluate the patient as just described and assess for Whipple’s triad during and after an episode of hypoglycemia. On the other hand, while it cannot be ignored, a distinctly low plasma glucose concentration measured in a patient without corresponding symptoms raises the possibility of an artifact (pseudohypoglycemia).

1	In a patient with documented hypoglycemia, a plausible hypoglycemic mechanism can often be deduced from the history, physical examination, and available laboratory data (Table 420-1). Drugs, particularly alcohol or agents used to treat diabetes, should be the first consideration—even in the absence of known use of a relevant drug—given the possibility of surreptitious, accidental, or malicious drug administration. Other considerations include evidence of a relevant critical illness, hormone deficiencies (less commonly), and a non-β-cell tumor that can be pursued diagnostically (rarely). Absent one of these mechanisms in an otherwise seemingly well individual, the physician should consider endogenous hyperinsulinism and proceed with measurements and assessment of symptoms during spontaneous hypoglycemia or under conditions that might elicit hypoglycemia.

1	If the patient is able and willing, oral treatment with glucose tablets or glucose-containing fluids, candy, or food is appropriate. A reasonable initial dose is 20 g of glucose. If the patient is unable or unwilling (because of neuroglycopenia) to take carbohydrates orally, parenteral therapy is necessary. IV administration of glucose (25 g) should be followed by a glucose infusion guided by serial plasma glucose measurements. If IV therapy is not practical, SC or IM glucagon (1.0 mg in adults) can be used, particularly in patients with T1DM. Because it acts by stimulating glycogenolysis, glucagon is ineffective in glycogen-depleted individuals (e.g., those with alcohol-induced hypoglycemia). Glucagon also stimulates insulin secretion and is therefore less useful in T2DM. The somatostatin analogue octreotide can be used to suppress insulin secretion in sulfonylurea-induced hypoglycemia. These treatments raise plasma glucose concentrations only transiently, and patients should

1	analogue octreotide can be used to suppress insulin secretion in sulfonylurea-induced hypoglycemia. These treatments raise plasma glucose concentrations only transiently, and patients should therefore be urged to eat as soon as is practical to replete glycogen stores.

1	Prevention of recurrent hypoglycemia requires an understanding of the hypoglycemic mechanism. Offending drugs can be discontinued or their doses reduced. Hypoglycemia caused by a sulfonylurea can persist for hours or even days. Underlying critical illnesses can often be treated. Cortisol and growth hormone can be replaced if levels are deficient. Surgical, radiotherapeutic, or chemotherapeutic reduction of a non–islet cell tumor can alleviate hypoglycemia even if the tumor cannot be cured; glucocorticoid or growth hormone administration also may reduce hypoglycemic episodes in such patients. Surgical resection of an insulinoma is curative; medical therapy with diazoxide or octreotide can be used if resection is not possible and in patients with a nontumor β-cell disorder. Partial pancreatectomy may be necessary in the latter patients. The treatment of autoimmune hypoglycemia (e.g., with glucocorticoid or immunosuppressive drugs) is problematic, but these disorders are sometimes

1	may be necessary in the latter patients. The treatment of autoimmune hypoglycemia (e.g., with glucocorticoid or immunosuppressive drugs) is problematic, but these disorders are sometimes self-limited. Failing these treatments, frequent feedings and avoidance of fasting may be required. Administration of uncooked cornstarch at bedtime or even an overnight intragastric infusion of glucose may be necessary for some patients.

1	Disorders of lipoprotein Daniel J. Rader, Helen H. Hobbs

1	Lipoproteins are complexes of lipids and proteins that are essential for transport of cholesterol, triglycerides, and fat-soluble vitamins. Previously, lipoprotein disorders were the purview of specialized lipidologists, but the demonstration that lipid-lowering therapy significantly reduces the clinical complications of atherosclerotic cardiovascular disease (ASCVD) has brought the diagnosis and treatment of these disorders into the domain of the internist. The number of individuals who are candidates for lipid-lowering therapy continues to increase. Therefore, the appropriate diagnosis and management of lipoprotein disorders is of critical importance in the practice of medicine. This chapter reviews normal lipoprotein physiology, the pathophysiology of disorders of lipoprotein metabolism, the effects of diet and other environmental factors that influence lipoprotein metabolism, and the practical approaches to the diagnosis and management of lipoprotein disorders.

1	Lipoproteins are large macromolecular complexes composed of lipids and proteins that transport poorly soluble lipids (primarily triglycerides, cholesterol, and fat-soluble vitamins) through body fluids Disorders of Lipoprotein Metabolism Density, g/mL 0.95 1.006 1.02 FIGURE 421-1 The density and size distribution of the major classes of lipoprotein particles. Lipoproteins are classified by density and size, which are inversely related. HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; VLDL, very-low-density lipoprotein. (plasma, interstitial fluid, and lymph) to and from tissues. Lipoproteins play an essential role in the absorption of dietary cholesterol, long-chain fatty acids, and fat-soluble vitamins; the transport of triglycerides, cholesterol, and fat-soluble vitamins from the liver to peripheral tissues; and the transport of cholesterol from peripheral tissues to the liver and intestine.

1	Lipoproteins contain a core of hydrophobic lipids (triglycerides and cholesteryl esters) surrounded by a shell of hydrophilic lipids (phospholipids, unesterified cholesterol) and proteins (called apolipoproteins) that interact with body fluids. The plasma lipoproteins are divided into five major classes based on their relative density (Fig. 421-1 and Table 421-1): chylomicrons, very-low-density lipoproteins (VLDLs), intermediate-density lipoproteins (IDLs), low-density lipoproteins (LDLs), and high-density lipoproteins (HDLs). Each lipoprotein class comprises a family of particles that vary in density, size, and protein composition. Because lipid is less dense than water, the density of a lipoprotein particle is primarily determined by the amount of lipid per particle. Chylomicrons are the most lipid-rich and therefore least dense lipoprotein particles, whereas HDLs have the least lipid and are therefore the most dense lipoproteins. In addition to their density, lipoprotein particles

1	most lipid-rich and therefore least dense lipoprotein particles, whereas HDLs have the least lipid and are therefore the most dense lipoproteins. In addition to their density, lipoprotein particles can be classified according to their size, determined either by nondenaturing gel electrophoresis

1	ApoA-I Intestine, liver ApoA-IV Intestine, ApoC-I Liver ApoC-II Liver ApoC-III Liver, HDL, chylomicrons HDL, chylomicrons HDL, chylomicrons VLDL, chylomicrons Chylomicrons, chylomicron remnants VLDL, IDL, LDL, Lp(a) Chylomicrons, VLDL, HDL Chylomicrons, VLDL, HDL Chylomicrons, VLDL, HDL Chylomicron remnants, IDL, HDL Structural protein for HDL Structural protein for VLDL, LDL, IDL, Lp(a) Ligand for binding to LDL receptor Inhibits LPL activity and lipoprotein binding to receptors Ligand for binding to LDL receptor and other receptors Abbreviations: HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; LCAT, lecithin-cholesterol acyltransferase; LDL, low-density lipoprotein; Lp(a), lipoprotein A; LPL, lipoprotein lipase; VLDL, very-low-density lipoprotein.

1	or by nuclear magnetic resonance profiling. There is a strong inverse relationship between density and size, with the largest particles being the most buoyant (chylomicrons) and the smallest particles being the most dense (HDL). The proteins associated with lipoproteins, called apolipoproteins (Table 421-2), are required for the assembly, structure, function, and metabolism of lipoproteins. Apolipoproteins activate enzymes important in lipoprotein metabolism and act as ligands for cell surface receptors. ApoB is a very large protein and is the major structural protein of chylomicrons, VLDLs, IDLs, and LDLs; one molecule Electrophoretic Lipoprotein Density, g/mLa Size, nmb Mobilityc Major Other Other Constituents Chylomicrons 0.930 75–1200 Origin ApoB-48 A-I, A-V, C-I, C-II, Retinyl esters C-III, E Chylomicron 0.930–1.006 30–80 Slow pre-β ApoB-48 A-I, A-V, C-I, C-II, Retinyl esters remnants C-III, E VLDL 0.930–1.006 30–80 Pre-β ApoB-100 A-I, A-II, A-V, C-I, C-II, Vitamin E

1	Chylomicron 0.930–1.006 30–80 Slow pre-β ApoB-48 A-I, A-V, C-I, C-II, Retinyl esters remnants C-III, E VLDL 0.930–1.006 30–80 Pre-β ApoB-100 A-I, A-II, A-V, C-I, C-II, Vitamin E C-III, E IDL 1.006–1.019 25–35 Slow pre-β ApoB-100 C-I, C-II, C-III, E Vitamin E LDL 1.019–1.063 18–25 β ApoB-100 HDL 1.063–1.210 5–12 α ApoA-I A-II, A-IV, A-V, C-III, E LCAT, CETP, paroxonase Lp(a) 1.050–1.120 25 Pre-β ApoB-100 Apo(a) Oxidized phospholipids aThe density of the particle is determined by ultracentrifugation. bThe size of the particle is measured using gel electrophoresis. cThe electrophoretic mobility of the particle on agarose gel electrophores reflects the size and surface charge of the particle, with β being the position of LDL and α being the position of HDL. Note: All of the lipoprotein classes contain phospholipids, esterified and unesterified cholesterol, and triglycerides to varying degrees.

1	Note: All of the lipoprotein classes contain phospholipids, esterified and unesterified cholesterol, and triglycerides to varying degrees. Abbreviations: CETP, cholesteryl ester transfer protein; HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; LCAT, lecithin-cholesterol acyltransferase; LDL, low-density lipoprotein; Lp(a), lipoprotein A; VLDL, very-low-density lipoprotein.

1	of apoB, either apoB-48 (chylomicron) or apoB-100 (VLDL, IDL, or LDL), is present on each lipoprotein particle. The human liver synthesizes apoB-100, and the intestine makes apoB-48, which is derived from the same gene by mRNA editing. HDLs have different apolipoproteins that define this lipoprotein class, most importantly apoA-I, which is synthesized in the liver and intestine and is found on virtually all HDL particles. ApoA-II is the second most abundant HDL apolipoprotein and is on approximately two-thirds of the HDL particles. ApoC-I, apoC-II, and apoC-III participate in the metabolism of triglyceride-rich lipoproteins. ApoE also plays a critical role in the metabolism and clearance of triglyceride-rich particles. Most apolipoproteins, other than apoB, exchange actively among lipoprotein particles in the blood. Apolipoprotein(a) [apo(a)] is a distinctive apolipoprotein and is discussed more below.

1	One critical role of lipoproteins is the efficient transport of dietary lipids from the intestine to tissues that require fatty acids for energy or store and metabolize lipids (Fig. 421-2). Dietary triglycerides are hydrolyzed by lipases within the intestinal lumen and emulsified with bile acids to form micelles. Dietary cholesterol, fatty acids, and fat-soluble vitamins are absorbed in the proximal small intestine. Cholesterol and retinol are esterified (by the addition of a fatty acid) in the enterocyte to form cholesteryl esters and retinyl esters, respectively. Longer-chain fatty acids (>12 carbons) are incorporated into triglycerides and packaged with apoB-48, cholesteryl esters, retinyl esters, phospholipids, and cholesterol to form chylomicrons. Nascent chylomicrons are secreted into the intestinal lymph and delivered via the thoracic duct directly to the systemic circulation, where they are extensively processed by peripheral tissues before reaching the liver. The particles

1	into the intestinal lymph and delivered via the thoracic duct directly to the systemic circulation, where they are extensively processed by peripheral tissues before reaching the liver. The particles encounter lipoprotein lipase (LPL), which is anchored to a glycosylphosphatidylinositol-anchored protein, GPIHBP1, that 2437 is attached to the endothelial surfaces of capillaries in adipose tissue, heart, and skeletal muscle (Fig. 421-2). The triglycerides of chylomicrons are hydrolyzed by LPL, and free fatty acids are released. ApoC-II, which is transferred to circulating chylomicrons from HDL, acts as a required cofactor for LPL in this reaction. The released free fatty acids are taken up by adjacent myocytes or adipocytes and either oxidized to generate energy or reesterified and stored as triglyceride. Some of the released free fatty acids bind albumin before entering cells and are transported to other tissues, especially the liver. The chylomicron particle progressively shrinks in

1	as triglyceride. Some of the released free fatty acids bind albumin before entering cells and are transported to other tissues, especially the liver. The chylomicron particle progressively shrinks in size as the hydrophobic core is hydrolyzed and the hydrophilic lipids (cholesterol and phospholipids) and apolipoproteins on the particle surface are transferred to HDL, creating chylomicron remnants.

1	Chylomicron remnants are rapidly removed from the circulation by the liver through a process that requires apoE as a ligand for receptors in the liver. Consequently, few, if any, chylomicrons or chylomicron remnants are generally present in the blood after a 12-h fast, except in patients with certain disorders of lipoprotein metabolism.

1	Another key role of lipoproteins is the transport of hepatic lipids from the liver to the periphery (Fig. 421-2). VLDL particles resemble chylomicrons in protein composition but contain apoB-100 rather than apoB-48 and have a higher ratio of cholesterol to triglyceride (~1 mg of cholesterol for every 5 mg of triglyceride). The triglycerides of VLDL are derived predominantly from the esterification of long-chain fatty acids in the liver. The packaging of hepatic triglycerides with the other major components of the nascent VLDL particle (apoB-100, cholesteryl esters, phospholipids, and vitamin E) requires the action of the enzyme microsomal triglyceride transfer protein (MTP). After secretion into the plasma, VLDL acquires multiple copies of apoE and apolipoproteins of the C series by transfer from HDL. As with chylomicrons, the triglycerides of VLDL are hydrolyzed by LPL, especially in muscle, heart, and adipose tissue. After the VLDL remnants dissociate from LPL, they are referred to

1	from HDL. As with chylomicrons, the triglycerides of VLDL are hydrolyzed by LPL, especially in muscle, heart, and adipose tissue. After the VLDL remnants dissociate from LPL, they are referred to as IDLs, which contain roughly similar amounts of cholesterol and triglyceride. The liver removes approximately 40–60% of IDL by LDL receptor–mediated endocytosis via binding to apoE. The remainder of IDL is remodeled by hepatic lipase (HL) to form LDL. During this process, phospholipids and triglyceride in the particle are hydrolyzed, and all apolipoproteins except apoB-100 are transferred to other lipoproteins. Approximately 70% of LDL is removed from the circulation by the liver in a similar manner as IDL; however, in this case, apoB, rather than apoE, binds the LDL receptor.

1	Lp(a) is a lipoprotein similar to LDL in lipid and pro tein composition, but it contains an additional protein called apolipoprotein(a) [apo(a)]. Apo(a) is synthesized IDL in the liver and attached to apoB-100 by a disulfide link- Disorders of Lipoprotein Metabolism age. The major site of clearance of Lp(a) is the liver, but the uptake pathway is not known.

1	All nucleated cells synthesize cholesterol, but only hepatocytes and enterocytes can effectively excrete cholesterol from the body, into either the bile or the gut lumen. In the liver, cholesterol is secreted into the bile, either directly or after conversion to bile acids. Cholesterol in peripheral cells is transported from the plasma mem-FIGURE 421-2 The exogenous and endogenous lipoprotein metabolic path-branes of peripheral cells to the liver and intestine by ways. The exogenous pathway transports dietary lipids to the periphery and the a process termed “reverse cholesterol transport” that is liver. The endogenous pathway transports hepatic lipids to the periphery. FFA, free facilitated by HDL (Fig. 421-3). fatty acid; HL, hepatic lipase; IDL, intermediate-density lipoprotein; LDL, low-density Nascent HDL particles are synthesized by the intestine lipoprotein; LDLR, low-density lipoprotein receptor; LPL, lipoprotein lipase; VLDL, and the liver. Newly secreted apoA-I rapidly

1	low-density Nascent HDL particles are synthesized by the intestine lipoprotein; LDLR, low-density lipoprotein receptor; LPL, lipoprotein lipase; VLDL, and the liver. Newly secreted apoA-I rapidly acquires very-low-density lipoprotein. phospholipids and unesterified cholesterol from its site or drug). Many, but not all, patients with dyslipidemia are at increased risk for ASCVD, the primary reason for making the diagnosis, as intervention may reduce this risk. In addition, patients with substantially elevated levels of triglycerides may be at risk for acute pancreatitis and require intervention to reduce this risk.

1	Although literally hundreds of proteins influence lipoprotein metabolism and may interact to produce dyslipidemia in an individual patient, there are a limited number of discrete “nodes” that regulate lipoprotein metabolism. These include: (1) assembly and secretion of triglyceride-rich VLDLs by the liver; (2) lipolysis of triglyceride-rich lipoproteins by LPL; (3) receptor-mediated uptake of apoB-containing lipoproteins by the liver; (4) cellular cholesterol metabolism in the hepatocyte and the enterocyte; and (5) neutral lipid transfer and phospholipid hydrolysis in the plasma. The following discussion

1	FIGURE 421-3 High-density lipoprotein (HDL) metabolism and reverse choles-will focus on these regulatory nodes, recognizing thatterol transport. This pathway transports excess cholesterol from the periphery back in many cases these nodes interact with and influenceto the liver for excretion in the bile. The liver and the intestine produce nascent each other. HDLs. Free cholesterol is acquired from macrophages and other peripheral cells and esterified by lecithin-cholesterol acyltransferase (LCAT), forming mature HDLs. HDL DYSLIPIDEMIA CAUSED BY EXCESSIVE HEPATIC SECRETION cholesterol can be selectively taken up by the liver via SR-BI (scavenger receptor OF VLDL class BI). Alternatively, HDL cholesteryl ester can be transferred by cholesteryl ester Excessive production of VLDL by the liver is one of transfer protein (CETP) from HDLs to very-low-density lipoproteins (VLDLs) and chy-the most common causes of dyslipidemia. Individuals lomicrons, which can then be taken up by the liver.

1	is one of transfer protein (CETP) from HDLs to very-low-density lipoproteins (VLDLs) and chy-the most common causes of dyslipidemia. Individuals lomicrons, which can then be taken up by the liver. IDL, intermediate-density lipo-with excessive hepatic VLDL production usually have protein; LDL, low-density lipoprotein; LDLR, low-density lipoprotein receptor. elevated fasting triglycerides and low levels of HDL cholesterol (HDL-C), with variable elevations in LDL cholesterol (LDL-C) but usually elevated plasma levels of synthesis (intestine or liver) via efflux promoted by the membrane of apoB. A cluster of other metabolic risk factors are often found protein ATP-binding cassette protein A1 (ABCA1). This process in association with VLDL overproduction, including obesity, gluresults in the formation of discoidal HDL particles, which then recruit cose intolerance, insulin resistance, and hypertension (the so-called additional unesterified cholesterol from cells or circulating

1	in the formation of discoidal HDL particles, which then recruit cose intolerance, insulin resistance, and hypertension (the so-called additional unesterified cholesterol from cells or circulating lipopro-metabolic syndrome, Chap. 422). Some of the major factors that teins. Within the HDL particle, the cholesterol is esterified by lecithin-drive hepatic VLDL secretion include obesity, insulin resistance, a cholesterol acyltransferase (LCAT), a plasma enzyme associated with high-carbohydrate diet, alcohol use, exogenous estrogens, and genetic HDL, and the more hydrophobic cholesteryl ester moves to the core of predisposition. the HDL particle. As HDL acquires more cholesteryl ester, it becomes

1	Secondary Causes of VLDL Overproduction • HigH-carboHydraTe dieT spherical, and additional apolipoproteins and lipids are transferred Dietary carbohydrates are converted to fatty acids in the liver. Some to the particles from the surfaces of chylomicrons and VLDLs during of the newly synthesized fatty acids are esterified forming triglycerideslipolysis. (TGs) and secreted as constituents of VLDL. Thus, excessive intake ofHDL cholesterol is transported to hepatocytes by both an indirect calories as carbohydrates, which is frequent in Western societies, leads and a direct pathway. HDL cholesteryl esters can be transferred to to increased hepatic VLDL-TG secretion.

1	apoB-containing lipoproteins in exchange for triglyceride by the cholesteryl ester transfer protein (CETP). The cholesteryl esters are then alcoHol Regular alcohol consumption inhibits hepatic oxidation removed from the circulation by LDL receptor–mediated endocytosis. of free fatty acids, thus promoting hepatic TG synthesis and VLDL HDL cholesterol can also be taken up directly by hepatocytes via the secretion. Regular alcohol use also raises plasma levels of HDL-C and scavenger receptor class B1 (SR-B1), a cell surface receptor that medi-should be considered in patients with the unusual combination of ates the selective transfer of lipids to cells. elevated TGs and elevated HDL-C.

1	HDL particles undergo extensive remodeling within the plasma oBeSity and inSulin reSiStance (See also Chaps. 416 and 417) Obesity compartment by a variety of lipid transfer proteins and lipases. The and insulin resistance are frequently accompanied by dyslipidemia phospholipid transfer protein (PLTP) transfers phospholipids from characterized by elevated plasma levels of TG, low HDL-C, variable other lipoproteins to HDL or among different classes of HDL particles. levels of LDL-C, and increased levels of small dense LDL. The increase After CETPand PLTP-mediated lipid exchange, the triglyceride-enriched HDL becomes a much better substrate for HL, which hydro-in adipocyte mass and accompanying decreased insulin sensitivity associated with obesity have multiple effects on lipid metabolism, with lyzes the triglycerides and phospholipids to generate smaller HDL one of the major effects being excessive hepatic VLDL production.

1	particles. A related enzyme called endothelial lipase hydrolyzes HDL More free fatty acids are delivered from the expanded and insulin phospholipids, generating smaller HDL particles that are catabolized resistant adipose tissue to the liver, where they are reesterified in faster. Remodeling of HDL influences the metabolism, function, and hepatocytes to form TGs, which are packaged into VLDLs for secretion plasma concentrations of HDL. into the circulation. In addition, the increased insulin levels promote DISORDERS OF ELEVATED CHOLESTEROL AND TRIGLYCERIDES increased fatty acid synthesis in the liver. In insulin-resistant patients who progress to type 2 diabetes mellitus, dyslipidemia remains com- Disorders of lipoprotein metabolism are collectively referred to as mon, even when the patient is under relatively good glycemic control.

1	Disorders of lipoprotein metabolism are collectively referred to as mon, even when the patient is under relatively good glycemic control. “dyslipidemias.” Dyslipidemias are generally characterized clinically In addition to increased VLDL production, insulin resistance can alsoby increased plasma levels of cholesterol, triglycerides, or both, variresult in decreased LPL activity, resulting in reduced catabolism ofably accompanied by reduced levels of HDL cholesterol. Because chylomicrons and VLDLs and more severe hypertriglyceridemia (seeplasma lipids are commonly screened (see below), dyslipidemia is below).

1	frequently seen in clinical practice. The majority of patients with dyslipidemia have some combination of genetic predisposition (often poly-nepHrotic Syndrome (See also Chap. 335) Nephrotic syndrome genic) and environmental contribution (lifestyle, medical condition, is a classic cause of excessive VLDL production. The molecular mechanism of VLDL overproduction remains poorly understood but has been attributed to the effects of hypoalbuminemia leading to increased hepatic protein synthesis. Effective treatment of the underlying renal disease often normalizes the lipid profile, but most patients with chronic nephrotic syndrome require lipid-lowering drug therapy.

1	cuSHinG’S Syndrome (See also Chap. 406) Endogenous or exogenous glucocorticoid excess is associated with increased VLDL synthesis and secretion and hypertriglyceridemia. Patients with Cushing’s syndrome frequently have dyslipidemia especially characterized by hypertriglyceridemia and low HDL-C, although elevations in plasma levels of LDL-C can also be seen. Primary (Genetic) Causes of VLDL Overproduction Genetic variation influences hepatic VLDL production. A number of genes have been identified in which common and low-frequency variants likely contribute to increased VLDL production, likely involving interactions with diet and other environmental factors. The best recognized inherited condition associated with VLDL overproduction is familial combined hyperlipidemia.

1	familial comBined Hyperlipidemia (fcHl) FCHL is generally characterized by elevations in plasma levels of TGs (VLDL) and LDL-C (including small dense LDL) and reduced plasma levels of HDL-C. It is estimated to occur in approximately 1 in 100–200 individuals and is an important cause of premature coronary heart disease (CHD); approximately 20% of patients who develop CHD under age 60 have FCHL. FCHL can manifest in childhood but is usually not fully expressed until adulthood. The disease clusters in families, with affected family members typically have one of three possible phenotypes: (1) elevated plasma levels of LDL-C, (2) elevated plasma levels of TGs due to elevation in VLDL, or (3) elevated plasma levels of both LDL-C and TG. The lipoprotein profile can switch among these three phenotypes in the same individual over time and may depend on factors such as diet, exercise, weight, and insulin sensitivity. Patients with FCHL almost always have significantly elevated plasma levels of

1	in the same individual over time and may depend on factors such as diet, exercise, weight, and insulin sensitivity. Patients with FCHL almost always have significantly elevated plasma levels of apoB. The levels of apoB are disproportionately high relative to the plasma LDL-C concentration, indicating the presence of small, dense LDL particles, which are characteristic of this syndrome.

1	Individuals with this phenotype generally share the same metabolic defect, namely overproduction of VLDL by the liver. The molecular etiology of this condition remains poorly understood, and no single gene has been identified in which mutations cause this disorder. It is likely that defects in a combination of genes can cause the condition, suggesting that a more appropriate term for the disorder might be polygenic combined hyperlipidemia.

1	The presence of a mixed dyslipidemia (plasma TG levels between 200 and 600 mg/dL and total cholesterol levels between 200 and 400 mg/dL, usually with HDL-C levels <40 mg/dL in men and <50 mg/dL in women) and a family history of mixed dyslipidemia and/or premature CHD strongly suggests the diagnosis. Individuals with this phenotype should be treated aggressively due to significantly increased risk of premature CHD. Decreased dietary intake of simple carbohydrates, aerobic exercise, and weight loss can all have beneficial effects on the lipid profile. Patients with diabetes should be aggressively treated to maintain good glucose control. Most patients with FCHL require lipid-lowering drug therapy, starting with statins, to reduce lipoprotein levels and lower the risk of cardiovascular disease.

1	lipodyStropHy Lipodystrophy is a condition in which the generation of adipose tissue generally or in certain fat depots is impaired. Lipodystrophies are often associated with insulin resistance and elevated plasma levels of VLDL and chylomicrons due to increased fatty acid synthesis and VLDL production, as well as reduced clearance of TG-rich particles. This disorder can be especially difficult to control. Patients with congenital generalized lipodystrophy are very rare and have nearly complete absence of subcutaneous fat, accompanied by profound insulin resistance and leptin deficiency, and accumulation of TGs in multiple tissues including the liver. Some patients with generalized lipodystrophy have been treated successfully with leptin administration. Partial lipodystrophy is somewhat more common and 2439 can be caused by mutations in several different genes, most notably lamin A. Partial lipodystrophy is usually characterized by increased truncal fat accompanied by markedly reduced

1	common and 2439 can be caused by mutations in several different genes, most notably lamin A. Partial lipodystrophy is usually characterized by increased truncal fat accompanied by markedly reduced or absent subcutaneous fat in the extremities and buttocks. These patients generally have insulin resistance, often quite severe, accompanied by type 2 diabetes, hepatosteatosis, and dyslipidemia. The dyslipidemia is usually characterized by elevated TGs and cholesterol and can be difficult to manage clinically. Patients with partial lipodystrophy are at substantially increased risk of atherosclerotic vascular disease and should therefore be treated aggressively for their dyslipidemia with statins and, if necessary, additional lipid-lowering therapies.

1	Impaired lipolysis of the TGs in TG-rich lipoproteins (TRLs) also commonly contributes to dyslipidemia. As noted above, LPL is the key enzyme responsible for hydrolyzing the TGs in chylomicrons and VLDL. LPL is synthesized and secreted into the extracellular space from adipocytes, myocytes, and cardiomyocytes. It is then transported from the subendothelial to the vascular endothelial surfaces by GPIHPB1. LPL is also synthesized in macrophages. Individuals with impaired LPL activity, whether secondary or due to a primary genetic disorder, have elevated fasting TGs and low levels of HDL-C, usually without elevation in LDL-C or apoB. Insulin resistance, in addition to causing excessive VLDL production, can also cause impaired LPL activity and lipolysis. A number of common and low-frequency genetic variants have been described that influence LPL activity, and single-gene Mendelian disorders that reduce LPL activity have also been described (Table 421-3).

1	Secondary Causes of Impaired Lipolysis of TRLs • obesiTy and insulin reSiStance (See also Chaps. 415e, 416, and 417) In addition to hepatic overproduction of VLDL, as discussed above, obesity, insulin resistance, and type 2 diabetes have been reported to be associated with variably reduced LPL activity. This may be due in part to the effects of tissue insulin resistance leading to reduced transcription of LPL in skeletal muscle and adipose, as well as to increased production of the LPL inhibitor apoC-III by the liver. This reduction in LPL activity often contributes to the dyslipidemia seen in these patients.

1	Primary (Genetic) Causes and Genetic Predisposition to Impaired Lipolysis of TRLs • familial cHylomicronemia syndrome As noted above, LPL is required for the hydrolysis of TGs in chylomicrons and VLDLs, and apoC-II is a cofactor for LPL. Genetic deficiency or inactivity of either protein results in impaired lipolysis and profound elevations in plasma chylomicrons. These patients can also have elevated plasma levels of VLDL, but chylomicronemia predominates. The fasting plasma is turbid, and if left at 4°C (39.2°F) for a few hours, the chylomicrons float to the top and form a creamy supernatant. In these disorders, collectively called the familial chylomicronemia syndrome, fasting TG levels are almost invariably >1000 mg/dL. Fasting cholesterol levels are also elevated but to a lesser degree.

1	LPL deficiency has autosomal recessive inheritance and has a frequency of approximately 1 in 1 million in the population. ApoC-II deficiency is also recessive in inheritance pattern and is even less common than LPL deficiency. Multiple different mutations in the LPL and APOC2 genes cause these diseases. Obligate LPL heterozygotes often have mild-to-moderate elevations in plasma TG levels, whereas individuals heterozygous for mutation in apoC-II do not have hypertriglyceridemia.

1	Both LPL and apoC-II deficiency usually present in childhood with recurrent episodes of severe abdominal pain due to acute pancreatitis. On funduscopic examination, the retinal blood vessels are opalescent (lipemia retinalis). Eruptive xanthomas, which are small, yellowish-white papules, often appear in clusters on the back, buttocks, and extensor surfaces of the arms and legs. These typically painless skin lesions may become pruritic. Hepatosplenomegaly results from the uptake of circulating chylomicrons by reticuloendothelial cells in the liver and spleen. For unknown reasons, some patients with persistent Disorders of Lipoprotein Metabolism Lipoprotein lipase deficiency LPL (LPL) Chylomicrons, VLDL Eruptive xanthomas, hepatosplenomegaly, AR ~1/1,000,000 pancreatitis Familial apoC-II deficiency ApoC-II (APOC2) Chylomicrons, VLDL Eruptive xanthomas, hepatosplenomegaly, AR <1/1,000,000 pancreatitis

1	Familial apoC-II deficiency ApoC-II (APOC2) Chylomicrons, VLDL Eruptive xanthomas, hepatosplenomegaly, AR <1/1,000,000 pancreatitis ApoA-V deficiency ApoA-V (APOA5) Chylomicrons, VLDL Eruptive xanthomas, hepatosplenomegaly, AR <1/1,000,000 pancreatitis GPIHBP1 deficiency GPIHBP1 Chylomicrons Eruptive xanthomas, pancreatitis AR <1/1,000,000 Familial hepatic lipase Hepatic lipase (LIPC) VLDL remnants, HDL Pancreatitis, CHD AR <1/1,000,000 deficiency Familial ApoE (APOE) Chylomicron remnants, Palmar and tuberoeruptive xanthomas, AR ~1/10,000 dysbetalipoproteinemia VLDL remnants CHD, PVD cholesterolemia, type 3 Tendon xanthomas, CHD Tendon xanthomas, CHD Tendon xanthomas, CHD Tendon xanthomas, CHD Tendon xanthomas, CHD AD ~1/250 to 1/500 AD <~1/1500 AD <1/1,000,000 AR <1/1,000,000 AR <1/1,000,000

1	Tendon xanthomas, CHD Tendon xanthomas, CHD Tendon xanthomas, CHD Tendon xanthomas, CHD Tendon xanthomas, CHD AD ~1/250 to 1/500 AD <~1/1500 AD <1/1,000,000 AR <1/1,000,000 AR <1/1,000,000 Abbreviations: AD, autosomal dominant; apo, apolipoprotein; AR, autosomal recessive; ARH, autosomal recessive hypercholesterolemia; CHD, coronary heart disease; LDL, low-density lipoprotein; LPL, lipoprotein lipase; PVD, peripheral vascular disease; VLDL, very-low density lipoprotein. and pronounced chylomicronemia never develop pancreatitis, eruptive xanthomas, or hepatosplenomegaly. Premature CHD is not generally a feature of familial chylomicronemia syndromes.

1	and pronounced chylomicronemia never develop pancreatitis, eruptive xanthomas, or hepatosplenomegaly. Premature CHD is not generally a feature of familial chylomicronemia syndromes. The diagnoses of LPL and apoC-II deficiency are established enzymatically in specialized laboratories by assaying TG lipolytic activity in postheparin plasma. Blood is sampled after an IV heparin injection to release the endothelial-bound LPL. LPL activity is profoundly reduced in both LPL and apoC-II deficiency; in patients with apoC-II deficiency, it normalizes after the addition of normal plasma (providing a source of apoC-II). Molecular sequencing of the genes can be used to confirm the diagnosis.

1	The major therapeutic intervention in familial chylomicronemia syndrome is dietary fat restriction (to as little as 15 g/d) with fat-soluble vitamin supplementation. Consultation with a registered dietician familiar with this disorder is essential. Caloric supplementation with medium-chain TGs, which are absorbed directly into the portal circulation, can be useful, but there is uncertainty about their hepatic safety with prolonged use. If dietary fat restriction alone is not successful in resolving the chylomicronemia, fish oils have been effective in some patients. In patients with apoC-II deficiency, apoC-II can be provided by infusing fresh-frozen plasma to resolve the chylomicronemia in the acute setting. Management of patients with familial chylomicronemia syndrome is particularly challenging during pregnancy when VLDL production is increased. A gene therapy approach, called alipogene tiparvovec, is approved for LPL deficiency in Europe; it involves multiple intramuscular

1	challenging during pregnancy when VLDL production is increased. A gene therapy approach, called alipogene tiparvovec, is approved for LPL deficiency in Europe; it involves multiple intramuscular injections of an adeno-associated viral vector encoding a gain-of-function LPL variant, leading to skeletal myocyte expression of LPL.

1	apoa-v deficiency Another apolipoprotein, ApoA-V, facilitates the association of VLDL and chylomicrons with LPL and promotes their hydrolysis. Individuals harboring loss-of-function mutations in both APOA5 alleles develop hyperchylomicronemia. Heterozygosity for variants in APOA5 that reduce its function contributes to the polygenic basis of hypertriglyceridemia. gPiHbP1 deficiency Homozygosity for mutations that interfere with GPIHBP1 synthesis or folding cause severe hypertriglyceridemia by compromising the transport of LPL to the vascular endothelium. The frequency of chylomicronemia due to mutations in GHIHBP1 has not been established but appears to be very rare.

1	familial HypertriGlyceridemia (fHtG) FHTG is characterized by elevated fasting TGs without a clear secondary cause, average to below average LDL-C levels, low HDL-C levels, and a family history of hypertriglyceridemia. Plasma LDL-C levels are often reduced due to defective conversion of TG-rich particles to LDL. In contrast to FCHL, apoB levels are not elevated. The identification of other first-degree relatives with hypertriglyceridemia is useful in making the diagnosis. Unlike in FCHL, this condition is not generally associated with a significantly increased risk of CHD. However, if the hypertriglyceridemia is exacerbated by environmental factors, medical conditions, or drugs, the TGs can rise to a level at which acute pancreatitis is a risk. Indeed, management of patients with this condition is mostly geared toward reduction of TGs to prevent pancreatitis.

1	Individuals with this phenotype generally have reduced lipolysis of TRLs, although overproduction of VLDL by the liver can also contribute. No single gene has been identified in which mutations cause this disorder, whereas combinations of gene variants have been shown to cause this phenotype. A more appropriate term for this condition might be polygenic hypertriglyceridemia.

1	It is important to consider and rule out secondary causes of the hypertriglyceridemia as discussed above. Increased intake of simple carbohydrates, obesity, insulin resistance, alcohol use, estrogen treatment, and certain medications can exacerbate this phenotype. Patients who are at high risk for CHD due to other risk factors should be treated with statin therapy. In patients who are otherwise not at high risk for CHD, lipid-lowering drug therapy can frequently be avoided with appropriate dietary and lifestyle changes. Patients with plasma TG levels >500 mg/ dL after a trial of diet and exercise should be considered for drug therapy with a fibrate or fish oil to reduce TGs in order to prevent pancreatitis.

1	Impaired uptake of LDL and remnant lipoproteins by the liver is another common cause of dyslipidemia. As discussed above, the LDL receptor is the major receptor responsible for uptake of LDL and remnant particles by the liver. Downregulation of LDL receptor activity or genetic variation that reduces the activity of the LDL receptor pathway leads to elevations in LDL-C. One major factor that reduces LDL receptor activity is a diet high in saturated and trans fats. Other medical conditions that reduce LDL receptor activity include hypothyroidism and estrogen deficiency. In addition, genetic variation in a number of genes influences LDL clearance, and mutations in some of these genes cause several discrete Mendelian disorders of elevated LDL-C (Table 421-3).

1	Secondary Causes of Impaired Hepatic Uptake of Lipoproteins • HyPo tHyroidiSm (See also Chap. 405) Hypothyroidism is associated with elevated plasma LDL-C levels due primarily to a reduction in hepatic LDL receptor function and delayed clearance of LDL. Thyroid hormone increases hepatic expression of the LDL receptor. Hypothyroid patients also frequently have increased levels of circulating IDL, and some patients with hypothyroidism also have mild hypertriglyceridemia. Because hypothyroidism is often subtle and therefore easily overlooked, all patients presenting with elevated plasma levels of LDL-C, especially if there has been an unexplained increase in LDL-C, should be screened for hypothyroidism. Thyroid replacement therapy usually ameliorates the hypercholesterolemia; if not, the patient probably has a primary lipoprotein disorder and may require lipid-lowering drug therapy with a statin.

1	cHronic Kidney diSeaSe (See also Chap. 335) Chronic kidney disease (CKD) is often associated with mild hypertriglyceridemia (<300 mg/ dL) due to the accumulation of VLDLs and remnant lipoproteins in the circulation. TG lipolysis and remnant clearance are both reduced in patients with renal failure. Because the risk of ASCVD is increased in end-stage renal disease, subjects with hyperlipidemia, they should usually be aggressively treated with lipid-lowering agents, even though there is inadequate data at present to indicate that this population benefits from LDL-lowering therapy. Patients with solid organ transplants often have increased lipid levels due to the effect of the drugs required for immunosuppression. These patients can present a difficult clinical management problem, since statins should be used cautiously in these patients due to untoward muscle-related side effects. Primary (Genetic) Causes of Impaired Hepatic Uptake of Lipoproteins

1	Genetic variation contributes substantially to elevated LDL-C levels in the general population. It has been estimated that at least 50% of variation in LDL-C is genetically determined. Many patients with elevated LDL-C have polygenic hypercholesterolemia characterized by hypercholesterolemia in the absence of secondary causes of hypercholesterolemia (other than dietary factors) or a primary Mendelian disorder. In patients who are genetically predisposed to higher LDL-C levels, diet plays a key role; indeed increased saturated and trans fats in the diet shifts the entire distribution of LDL levels in the population to the right. Inheritance of several variants that together elevate LDLC, coupled with diet, is generally the cause of this condition; <10% of first-degree relatives themselves have hypercholesterolemia. However, single-gene (Mendelian) causes of elevated LDL-C are relatively common and should be considered in the differential diagnosis of elevated LDL-C.

1	familial HypercHoleSterolemia (fH) FH, also known as autosomal dominant hypercholesterolemia (ADH) type 1, is an autosomal co-dominant disorder characterized by elevated plasma levels of LDL-C in the absence of hypertriglyceridemia. FH is caused by loss-of-function mutations in the gene encoding the LDL receptor. The reduction in LDL receptor activity in the liver results in a reduced rate of clearance of LDL from the circulation. The plasma level of LDL increases to a level such that the rate of LDL production equals the rate of LDL clearance by residual LDL receptor as well as non-LDL receptor mechanisms. More than 1600 different mutations have been reported in association with FH. The elevated levels of LDL-C in FH are primarily due to delayed removal of LDL from the blood; in addition, because the removal of IDL is also delayed, the production of LDL from IDL is also increased. Individuals with two mutated LDL receptor alleles (FH homozygotes, or compound heterozygotes) have much

1	because the removal of IDL is also delayed, the production of LDL from IDL is also increased. Individuals with two mutated LDL receptor alleles (FH homozygotes, or compound heterozygotes) have much higher LDL-C levels than those with one mutant allele (FH heterozygotes).

1	Heterozygous FH is caused by the inheritance of one mutant

1	LDL receptor allele. The population frequency of heterozy gous FH due to LDL receptor mutations was originally estimated to be 1 in 500 individuals, but recent data suggest it may be 2441 as high as approximately 1 in 250 individuals, making it one of the most common single-gene disorders in humans. FH has a higher prevalence in certain founder populations, such as South African Afrikaners, Christian Lebanese, and French Canadians. Heterozygous FH is characterized by elevated plasma levels of LDL-C (usually 200– 400 mg/dL) and normal levels of TGs. Patients with heterozygous FH have hypercholesterolemia from birth, and disease recognition is usually based on detection of hypercholesterolemia on routine screening, the appearance of tendon xanthomas, or the development of symptomatic cardiovascular disease. Inheritance is dominant, meaning that the condition was inherited from one parent and ~50% of the patient’s siblings can be expected to have hypercholesterolemia. The family history

1	disease. Inheritance is dominant, meaning that the condition was inherited from one parent and ~50% of the patient’s siblings can be expected to have hypercholesterolemia. The family history is frequently positive for premature CHD on the side of the family from which the mutation was inherited. Physical findings in many, but not all, patients with heterozygous FH include corneal arcus and tendon xanthomas particularly involving the dorsum of the hands and the Achilles tendons. Untreated heterozygous FH is associated with a markedly increased risk of cardiovascular disease. Untreated men with heterozygous FH have an ~50% chance of having a myocardial infarction before age 60 years, and women with heterozygous FH are at substantially increased risk as well. The age of onset of cardiovascular disease is highly variable and depends on the specific molecular defect, the level of LDL-C, and coexisting cardiovascular risk factors. FH heterozygotes with elevated plasma levels of Lp(a) (see

1	disease is highly variable and depends on the specific molecular defect, the level of LDL-C, and coexisting cardiovascular risk factors. FH heterozygotes with elevated plasma levels of Lp(a) (see below) appear to be at greater risk for cardiovascular disease.

1	No definitive diagnostic test for heterozygous FH is available, except in certain founder populations where selected mutations predominate. Most LDL receptor mutations are private and require sequencing of the LDL receptor gene for identification. Sequencing for clinical diagnosis is available but not standard of care and is rarely performed in the United States, because the clinical utility of identifying the specific mutation has not been demonstrated. A family history of hypercholesterolemia and/or premature coronary disease is supportive of the diagnosis. Secondary causes of significant hypercholesterolemia such as hypothyroidism, nephrotic syndrome, and obstructive liver disease should be excluded.

1	Heterozygous FH patients should be aggressively treated to lower plasma levels of LDL-C, starting in childhood. Initiation of a diet low in saturated and trans fats is recommended, but heterozygous FH patients virtually always require lipid-lowering drug therapy for effective control of their LDL-C levels. Statins are effective in heterozygous FH and are clearly the drug class of choice, and usually a more potent member of the class. However, some heterozygous FH patients cannot achieve adequate control of their LDL-C levels even with high-dose statin therapy and require additional drugs; a cholesterol absorption inhibitor and/or a bile acid sequestrant are the next-line classes of drugs. Currently, heterozygous FH patients whose LDL-C levels remain markedly elevated (>200 mg/dL with cardiovascular disease [CVD] or >300 mg/dL without CVD) on maximally tolerated drug therapy are candidates for LDL apheresis, a physical method of purging the blood of LDL in which the LDL particles are

1	disease [CVD] or >300 mg/dL without CVD) on maximally tolerated drug therapy are candidates for LDL apheresis, a physical method of purging the blood of LDL in which the LDL particles are selectively removed from the circulation; LDL apheresis is usually performed every 2 weeks. A new class of drugs known as PCSK9 inhibitors is under clinical development and has the potential to effectively control LDL-C levels in the vast majority of patients with heterozygous FH who are inadequately controlled on a statin alone or who are statin intolerant.

1	Homozygous FH is caused by mutations in both alleles of the LDL receptor and therefore much rarer than heterozygous FH. Patients with homozygous FH have been classified into those patients with virtually no detectable LDL receptor activity (receptor negative) and those patients with markedly reduced but detectable LDL receptor activity (receptor defective). LDL-C levels in patients with homozygous FH range from about 400 to >1000 mg/dL, with receptor-defective patients at the lower end and receptor-negative patients at the higher end of the range. TGs are usually normal. Many patients with homozygous FH, particularly receptor-negative patients, present in childhood with cutaneous xanthomas on the hands, wrists, elbows, knees, heels, or

1	Disorders of Lipoprotein Metabolism 2442 buttocks. The devastating consequence of homozygous FH is accelerated ASCVD, which often presents in childhood or early adulthood. Atherosclerosis often develops first in the aortic root, where it can cause aortic valvular or supravalvular stenosis, and typically extends into the coronary ostia, which become stenotic. Symptoms can be atypical, and sudden death is not uncommon. Untreated, receptor-negative patients with homozygous FH rarely survive beyond the second decade; patients with receptor-defective LDL receptor defects have a better prognosis but almost invariably develop clinically apparent atherosclerotic vascular disease by age 30, and often much sooner. Carotid and femoral disease develops later in life and is usually not clinically significant. Homozygous FH should be suspected in a child or young adult with LDL >400 mg/dL without secondary cause. Cutaneous xanthomas, evidence of CVD, and hypercholesterolemia in both parents all are

1	Homozygous FH should be suspected in a child or young adult with LDL >400 mg/dL without secondary cause. Cutaneous xanthomas, evidence of CVD, and hypercholesterolemia in both parents all are supportive of the diagnosis. Although the specific mutations in the LDL receptor can usually be identified by DNA sequencing, this is not generally performed, and the diagnosis is usually made on clinical grounds. Patients with homozygous FH must be treated aggressively to delay the onset and progression of CVD. Receptor defective patients sometimes respond to statins and other LDL-lowering drug classes such as a cholesterol absorption inhibitor or a bile acid sequestrant, which upregulate the LDL receptor activity. Two drugs that reduce the hepatic production of VLDL and thus LDL, a small-molecule inhibitor of the microsomal TG transfer protein (MTP) and an antisense oligonucleotide to apoB, are approved in the United States for the treatment of adults with homozygous FH and can be considered.

1	inhibitor of the microsomal TG transfer protein (MTP) and an antisense oligonucleotide to apoB, are approved in the United States for the treatment of adults with homozygous FH and can be considered. PCSK9 inhibitors, which work through increasing LDL receptor availability, appear to have some benefit in receptor-defective patients and are under clinical development. LDL apheresis is used to lower plasma LDL levels in these patients and can promote regression of xanthomas as well as slow the progression of atherosclerosis. Because the liver is quantitatively the most important tissue for removing circulating LDLs via the LDL receptor, liver transplantation is effective in decreasing plasma LDL-C levels in this disorder but is infrequently used because of the associated problems with immunosuppression.

1	familial defecTive aPob-100 (fdb) FDB, also known as autosomal dominant hypercholesterolemia (ADH) type 2, is a dominantly inherited disorder that clinically resembles heterozygous FH with elevated LDL-C levels and normal TGs. FDB is caused by mutations in the gene encoding apoB-100, specifically in LDL receptor–binding domain of apoB-100. Several different mutations have been identified, but a single mutation predominates: substitution of glutamine for arginine at position 3500. The mutation results in a reduction in the affinity of LDL binding to the LDL receptor, so LDL is removed from the circulation at a reduced rate. FDB is less common than FH but is more prevalent in individuals of central European descent; the Lancaster County (United States) Amish are a founder population in which the prevalence of FDB is as high as 1 in 10 individuals. FDB is characterized by elevated plasma LDL-C levels with normal TGs; tendon xanthomas can be seen, although not as frequently as in FH, and

1	the prevalence of FDB is as high as 1 in 10 individuals. FDB is characterized by elevated plasma LDL-C levels with normal TGs; tendon xanthomas can be seen, although not as frequently as in FH, and there is an associated increase in risk of CHD. Patients with FDB cannot be clinically distinguished from patients with heterozygous FH, although patients with FDB tend to have somewhat lower plasma levels of LDL-C than FH heterozygotes, presumably due to the fact that IDL clearance is not impaired in this disorder. Homozygotes for FDB mutations have higher LDL-C levels than FDB heterozygotes but are not as severely affected as homozygous FH patients. The apoB-100 gene mutations can be detected directly through sequencing of the receptor-binding region of the apoB gene or genotyping for the most common mutation, but genetic diagnosis is not generally performed because there is no direct implication for clinical management. As with FH, patients are treated with statins first and, if

1	the most common mutation, but genetic diagnosis is not generally performed because there is no direct implication for clinical management. As with FH, patients are treated with statins first and, if necessary, with additional classes of LDL-lowering drugs.

1	autoSomal dominant HypercHoleSterolemia due to mutationS in pcSK9 (adH-pcSK9 or adH3) ADH-PCSK9, also known as autosomal dominant hypercholesterolemia (ADH) type 3, is a very rare autosomal dominant disorder caused by gain-of-function mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 is a secreted protein that binds to the LDL receptor, targeting it for degradation. Normally, after LDL binds to the LDL receptor, it is internalized along with the receptor, and in the low pH of the endosome, the LDL receptor dissociates from the LDL and recycles to the cell surface. When PCSK9 binds the receptor, the complex is internalized and the receptor is directed to the lysosome, rather than to the cell surface. The missense mutations in PCSK9 that cause hypercholesterolemia enhance the activity of PCSK9. As a consequence, the number of hepatic LDL receptors is reduced. Patients with ADH-PCSK9 are similar clinically to patients with FH. They may be particularly responsive

1	the activity of PCSK9. As a consequence, the number of hepatic LDL receptors is reduced. Patients with ADH-PCSK9 are similar clinically to patients with FH. They may be particularly responsive to PCSK9 inhibitors in clinical development. Loss-of-function mutations in PCSK9 cause low LDL-C levels (see below).

1	autoSomal receSSive HypercHoleSterolemia (arH) ARH is a very rare disorder that is mostly seen in individuals of Sardinian descent. The disease is caused by mutations in a protein, ARH (also called LDLR adaptor protein, LDLRAP), which is required for LDL receptor–mediated endocytosis in the liver. ARH binds to the cytoplasmic domain of the LDL receptor and links the receptor to the endocytic machinery. In the absence of LDLRAP, LDL binds to the extracellular domain of the LDL receptor, but the lipoprotein-receptor complex fails to be internalized. ARH, like homozygous FH, is characterized by hypercholesterolemia, tendon xanthomas, and premature coronary artery disease (CAD). The levels of plasma LDL-C tend to be intermediate between the levels present in FH homozygotes and FH heterozygotes, and CAD is not usually symptomatic until the third decade. LDL receptor function in cultured fibroblasts is normal or only modestly reduced in ARH, whereas LDL receptor function in lymphocytes and

1	and CAD is not usually symptomatic until the third decade. LDL receptor function in cultured fibroblasts is normal or only modestly reduced in ARH, whereas LDL receptor function in lymphocytes and the liver is negligible. Unlike FH homozygotes, the hyperlipidemia responds to treatment with statins, but these patients usually require additional therapy to lower plasma LDL-C to acceptable levels.

1	SitoSterolemia Sitosterolemia is a rare autosomal recessive disease that can result in severe hypercholesterolemia, tendon xanthomas, and premature ASCVD. Sitosterolemia is caused by loss-of-function mutations in either of two members of the ATP-binding cassette (ABC) half transporter family, ABCG5 and ABCG8. These genes are expressed in enterocytes and hepatocytes. The proteins heterodimerize to form a functional complex that transports plant sterols such as sitosterol and campesterol, and animal sterols, predominantly cholesterol, across the biliary membrane of hepatocytes into the bile and across the intestinal luminal surface of enterocytes into the gut lumen. In normal individuals, <5% of dietary plant sterols are absorbed by the proximal small intestine. The small amounts of plant sterols that enter the circulation are preferentially excreted into the bile. Thus, levels of plant sterols are kept very low in tissues. In sitosterolemia, the intestinal absorption of sterols is

1	sterols that enter the circulation are preferentially excreted into the bile. Thus, levels of plant sterols are kept very low in tissues. In sitosterolemia, the intestinal absorption of sterols is increased and biliary and fecal excretion of the sterols is reduced, resulting in increased plasma and tissue levels of both plant sterols and cholesterol. The increase in hepatic sterol levels results in transcriptional suppression of the expression of the LDL receptor, resulting in reduced uptake of LDL and substantially increased LDL-C levels. In addition to the usual clinical picture of hypercholesterolemia (i.e., tendon xanthomas and premature ASCVD), these patients also have anisocytosis and poikilocytosis of erythrocytes and megathrombocytes due to the incorporation of plant sterols into cell membranes. Episodes of hemolysis and splenomegaly are a distinctive clinical feature of this disease compared to other genetic forms of hypercholesterolemia and can be a clue to the diagnosis.

1	Sitosterolemia should be suspected in a patient with severe hypercholesterolemia without a family history of such or who responds dramatically to dietary therapy and/or ezetimibe but not statins. Sitosterolemia can be diagnosed by a laboratory finding of a substantial increase in the plasma level of sitosterol and/or other plant sterols. It is important to make the diagnosis, because bile acid sequestrants and cholesterol-absorption inhibitors are the most effective agents to reduce LDL-C and plasma plant sterol levels in these patients.

1	cHoleSteryl eSter StoraGe diSeaSe (ceSd) CESD, also known as lysosomal acid lipase deficiency, is an autosomal recessive disorder characterized by elevated LDL-C, usually in association with low HDLC, together with progressive fatty liver ultimately leading to hepatic fibrosis. Plasma TG levels can also be mild to moderately increased in this disorder. The most severe form of this disorder, Wolman’s disease, presents in infancy and is rapidly fatal. Both Wolman’s disease and CESD are caused by loss-of-function variants in both alleles of the gene encoding lysosomal acid lipase (LAL; gene name LIPA). LAL is responsible for hydrolyzing neutral lipids, particularly TGs and cholesteryl esters, after delivery to the lysosome by cell-surface receptors such as the LDL receptor. It is particularly important in the liver, which clears large amounts of lipoproteins from the circulation. Genetic deficiency of LAL results in accumulation of neutral lipid in the hepatocytes, leading to

1	particularly important in the liver, which clears large amounts of lipoproteins from the circulation. Genetic deficiency of LAL results in accumulation of neutral lipid in the hepatocytes, leading to hepatosplenomegaly, microvesicular steatosis, and ultimately fibrosis and end-stage liver disease. The etiology of the elevated LDL-C levels is uncertain; one study suggested that VLDL production is increased, but impaired LDL receptor–mediated clearance of LDL is also likely.

1	CESD should be particularly suspected in nonobese patients with elevated LDL-C, low HDL-C, and evidence of fatty liver in the absence of overt insulin resistance. The diagnosis can be made with a dried blood spot assay of LAL activity and confirmed by DNA genotyping for the most common mutation, followed if necessary by sequencing of the gene to find the second mutation. Liver biopsy is required to assess the degree of inflammation and fibrosis. It is important to make the diagnosis because it has implications for liver monitoring and potentially for therapeutic approaches under development.

1	familial dySBetalipoproteinemia (fdBl) FDBL (also known as type III hyperlipoproteinemia) is usually a recessive disorder characterized by a mixed hyperlipidemia (elevated cholesterol and TGs) due to the accumulation of remnant lipoprotein particles (chylomicron remnants and VLDL remnants, or IDL). ApoE is present in multiple copies on chylomicron remnants and IDL, and mediates their removal via hepatic lipoprotein receptors (Fig. 421-2). FDBL is due to genetic variants of apoE, most commonly apoE2, that result in an apoE protein with reduced ability to bind lipoprotein receptors. The APOE gene is polymorphic in sequence, resulting in the expression of three common isoforms: apoE3, which is the most common; and apoE2 and apoE4, which both differ from apoE3 by a single amino acid. Although associated with slightly higher LDL-C levels and increased CHD risk, the apoE4 allele is not associated with FDBL. Individuals who carry one or two apoE4 alleles have an increased risk of Alzheimer’s

1	associated with slightly higher LDL-C levels and increased CHD risk, the apoE4 allele is not associated with FDBL. Individuals who carry one or two apoE4 alleles have an increased risk of Alzheimer’s disease. ApoE2 has a lower affinity for the LDL receptor; therefore, chylomicron remnants and IDL containing apoE2 are removed from plasma at a slower rate. Individuals who are homozygous for the E2 allele (the E2/E2 genotype) comprise the most common subset of patients with FDBL.

1	Approximately 0.5% of the general population are apoE2/E2 homo-zygotes, but only a small minority of these individuals actually develop hyperlipidemia characteristic of FDBL. In most cases, an additional, sometimes identifiable, factor precipitates the development of hyperlipoproteinemia. The most common precipitating factors are a high-fat diet, diabetes mellitus, obesity, hypothyroidism, renal disease, HIV infection, estrogen deficiency, alcohol use, or certain drugs. The disease seldom presents in women before menopause. Other mutations in apoE can cause a dominant form of FDBL where the hyperlipidemia is fully manifest in the heterozygous state, but these mutations are very rare.

1	Patients with FDBL usually present in adulthood with hyperlipidemia, xanthomas, or premature coronary or peripheral vascular disease. In FDBL, in contrast to other disorders of elevated TGs, the plasma levels of cholesterol and TG are often elevated to a similar degree, and the level of HDL-C is usually normal or reduced. Two distinctive types of xanthomas, tuberoeruptive and palmar, are seen in FDBL patients. Tuberoeruptive xanthomas begin as clusters of small papules on the elbows, knees, or buttocks and can grow to the size of small grapes. Palmar xanthomas (alternatively called xanthomata striata palmaris) are orange-yellow discolorations of the creases in the palms and wrists. Both of these xanthoma types are virtually pathognomonic for FDBL. 2443 Subjects with FDBL have premature ASCVD and tend to have more peripheral vascular disease than is typically seen in FH.

1	The definitive diagnosis of FDBL can be made either by the documentation of very high levels of remnant lipoproteins or by identification of the apoE2/E2 genotype. A variety of methods are used to identify remnant lipoproteins in the plasma, including “β-quantification” by ultracentrifugation (ratio of directly measured VLDL-C to total plasma TG >0.30), lipoprotein electrophoresis (broad β band), or nuclear magnetic resonance lipoprotein profiling. The Friedewald formula for calculation of LDL-C is not valid in FDBL because the VLDL particles are depleted in TG and enriched in cholesterol. The plasma levels of LDL-C are actually low in this disorder due to defective metabolism of VLDL to LDL. DNA-based methods (apoE genotyping) can be performed to confirm homozygosity for apoE2. However, absence of the apoE2/E2 genotype does not strictly rule out the diagnosis of FDBL, because other mutations in apoE can (rarely) cause this condition.

1	Because FDBL is associated with increased risk of premature ASCVD, it should be treated aggressively. Other metabolic conditions that can worsen the hyperlipidemia (see above) should be managed. Patients with FDBL are typically diet-responsive and can respond favorably to weight reduction and to low-cholesterol, low-fat diets. Alcohol intake should be curtailed. Pharmacologic therapy is often required, and statins are the first line in management. In the event of statin intolerance or insufficient control of hyperlipidemia, cholesterol absorption inhibitors, fibrates, and niacin are also effective in the treatment of FDBL.

1	Hepatic lipaSe deficiency Hepatic lipase (HL; gene name LIPC) is a member of the same gene family as LPL and hydrolyzes TGs and phospholipids in remnant lipoproteins and HDL. Hydrolysis of lipids in remnant particles by HL contributes to their hepatic uptake via an apoE-mediated process. HL deficiency is a very rare autosomal recessive disorder characterized by elevated plasma levels of cholesterol and TGs (mixed hyperlipidemia) due to the accumulation of lipoprotein remnants, accompanied by elevated plasma level of HDL-C. The diagnosis is confirmed by measuring HL activity in postheparin plasma and/or confirmation of loss-of-function mutations in both alleles of HL/LIPC. Due to the small number of patients with HL deficiency, the association of this genetic defect with ASCVD is not entirely clear, although anecdotally patients with HL deficiency who have premature CVD have been described. As with FDBL, statin therapy is recommended to reduce remnant lipoproteins and cardiovascular

1	clear, although anecdotally patients with HL deficiency who have premature CVD have been described. As with FDBL, statin therapy is recommended to reduce remnant lipoproteins and cardiovascular risk.

1	Additional Secondary Causes of Dyslipidemia Many of the secondary causes of dyslipidemia (Table 421-4) have been described above. Additional considerations are discussed here. liver diSorderS (See also Chap. 357) Because the liver is the principal site of formation and clearance of lipoproteins, liver disorders can affect plasma lipid levels in a variety of ways. Hepatitis due to infection, drugs, or alcohol is often associated with increased VLDL synthesis and mild to moderate hypertriglyceridemia. Severe hepatitis and liver failure are associated with dramatic reductions in plasma cholesterol and TGs due to reduced lipoprotein biosynthetic capacity.

1	Cholestasis is associated with hypercholesterolemia, which can be very severe. A major pathway by which cholesterol is excreted from the body is via secretion into bile, either directly or after conversion to bile acids, and cholestasis blocks this critical excretory pathway. In cholestasis, free cholesterol, coupled with phospholipids, is secreted into the plasma as a constituent of a lamellar particle called LP-X. The particles can deposit in skinfolds, producing lesions resembling those seen in patients with FDBL (xanthomata strata palmaris). Planar and eruptive xanthomas can also be seen in patients with cholestasis.

1	druGS Many drugs have an impact on lipid metabolism and can result in significant alterations in the lipoprotein profile (Table 421-4). Estrogen administration is associated with increased VLDL and HDL synthesis, resulting in elevated plasma levels of both TGs and HDL-C. This lipoprotein pattern is distinctive because the levels of plasma TG and HDL-C are typically inversely related. Plasma TG levels should be monitored when birth control pills or postmenopausal estrogen Disorders of Lipoprotein Metabolism Renal failure Sepsis Stress Cushing’s syndrome Pregnancy Acromegaly Lipodystrophy Drugs: estrogen, beta blockers, glucocorticoids, bile acid binding resins, retinoic acid Hypothyroidism Hypothyroidism Acromegaly Drugs: growth hor mone, isotretinoin Abbreviations: DM, diabetes mellitus; HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; Lp(a), lipoprotein A; VLDL, very-low-density lipoprotein.

1	Abbreviations: DM, diabetes mellitus; HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; Lp(a), lipoprotein A; VLDL, very-low-density lipoprotein. therapy is initiated to ensure that the increase in VLDL production does not lead to severe hypertriglyceridemia. Use of low-dose preparations of estrogen or the estrogen patch can minimize the effect of exogenous estrogen on lipids. Plasma concentrations of LDL-C <60 mg/dL are unusual. Although in some cases LDL-C levels in this range may be reflective of malnutrition or serious chronic illness, LDL-C <60 mg/dL in an otherwise healthy individual suggests an inherited condition. The major inherited causes of low LDL-C are reviewed here.

1	Abetalipoproteinemia The synthesis and secretion of apoB-containing lipoproteins in the enterocytes of the proximal small bowel and in the hepatocytes of the liver involve a complex series of events that coordinate the coupling of various lipids with apoB-48 and apoB100, respectively. Abetalipoproteinemia is a rare autosomal recessive disease caused by loss-of-function mutations in the gene encoding microsomal TG transfer protein (MTP; gene name MTTP), a protein that transfers lipids to nascent chylomicrons and VLDLs in the intestine and liver, respectively. Plasma levels of cholesterol and TG are extremely low in this disorder, and chylomicrons, VLDLs, LDLs, and apoB are undetectable in plasma. The parents of patients with abetalipoproteinemia (obligate heterozygotes) have normal plasma lipid and apoB levels. Abetalipoproteinemia usually presents in early childhood with diarrhea and failure to thrive due to fat malabsorption. The initial neurologic manifestations are loss of deep

1	lipid and apoB levels. Abetalipoproteinemia usually presents in early childhood with diarrhea and failure to thrive due to fat malabsorption. The initial neurologic manifestations are loss of deep tendon reflexes, followed by decreased distal lower extremity vibratory and proprioceptive sense, dysmetria, ataxia, and the development of a spastic gait, often by the third or fourth decade. Patients with abetalipoproteinemia also develop a progressive pigmented retinopathy presenting with decreased night and color vision, followed by reductions in daytime visual acuity and ultimately progressing to near-blindness. The presence of spinocerebellar degeneration and pigmented retinopathy in this disease has resulted in some patients with abetalipoproteinemia being misdiagnosed as having Friedreich’s ataxia.

1	Most of the clinical manifestations of abetalipoproteinemia result from defects in the absorption and transport of fat-soluble vitamins.

1	Vitamin E and retinyl esters are normally transported from enterocytes to the liver by chylomicrons, and vitamin E is dependent on VLDL for transport out of the liver and into the circulation. As a consequence of the inability of these patients to secrete apoB-containing particles, patients with abetalipoproteinemia are markedly deficient in vitamin E and are also mildly to moderately deficient in vitamins A and K. Patients with abetalipoproteinemia should be referred to specialized centers for confirmation of the diagnosis and appropriate therapy. Treatment consists of a low-fat, high-caloric, vitamin-enriched diet accompanied by large supplemental doses of vitamin E. It is imperative that treatment be initiated as soon as possible to prevent development of neurologic sequelae, which can progress even with appropriate therapy. New therapies for this serious disease are needed.

1	Familial Hypobetalipoproteinemia (FHBL) FHBL generally refers to a condition of low total cholesterol, LDL-C, and apoB due to mutations in apoB. Most of the mutations causing FHBL result in a truncated apoB protein, resulting in impaired assembly and secretion of chylomicrons from enterocytes and VLDL from the liver. Mutations that result in VLDL particles containing a truncated apoB protein are cleared from the circulation at an accelerated rate, which also contributes to patients with this disorder having low levels of LDL-C and apoB. Individuals heterozygous for these mutations usually have LDL-C levels <60–80 mg/dL and also tend to have lower levels of plasma TG. Many FHBL patients have elevated levels of hepatic fat (due to reduced VLDL export) and sometimes have increased levels of liver transaminases, although it appears that these patients infrequently develop associated inflammation and fibrosis.

1	Mutations in both apoB alleles cause homozygous FHBL, an extremely rare disorder resembling abetalipoproteinemia with nearly undetectable LDL-C and apoB. The neurologic defects in this form of hypobetalipoproteinemia tend to be less severe than is typically seen in abetalipoproteinemia. Homozygous hypobetalipoproteinemia can be distinguished from abetalipoproteinemia by examining the inheritance pattern of the plasma LDL-C level. The levels of LDL-C and apoB are normal in the parents of patients with abetalipoproteinemia and low in those of patients with homozygous hypobetalipoproteinemia.

1	PCSK9 Deficiency Another inherited cause of low LDL-C results from loss-of-function mutations in PCSK9. PCSK9 is a secreted protein that binds to the extracellular domain of the LDL receptor in the liver and promotes the degradation of the receptor. Heterozygosity for nonsense mutations in PCSK9 that interfere with the synthesis of the protein are associated with increased hepatic LDL receptor activity and reduced plasma levels of LDL-C. Such mutations are particularly frequent in individuals of African descent. Individuals who are heterozygous for a loss-of-function mutation in PCSK9 have an ~30–40% reduction in plasma levels of LDL-C and have a substantial protection from CHD relative to those without a PCSK9 mutation, presumably due to having lower plasma cholesterol levels since birth. This observation led to the development of PCSK9 inhibitors as a new strategy for reducing LDL-C levels and cardiovascular risk. Homozygotes for these nonsense mutations have been reported and have

1	This observation led to the development of PCSK9 inhibitors as a new strategy for reducing LDL-C levels and cardiovascular risk. Homozygotes for these nonsense mutations have been reported and have extremely low LDL-C levels (<20 mg/dL) but appear otherwise healthy. A sequence variation of somewhat higher frequency (R46L) is found predominantly in individuals of European descent. This mutation impairs, but does not completely destroy, PCSK9 function. As a consequence, the plasma levels of LDL-C in individuals carrying this mutation are more modestly reduced (~15–20%); individuals with these mutations have a 45% reduction in ASCVD risk.

1	Low levels of HDL-C are very commonly encountered in clinical practice. Low HDL-C is an important independent predictor of increased cardiovascular risk and has been used regularly in standardized risk calculators, including the most recent one from the American Heart Association (AHA)/American College of Cardiology (ACC). However, it remains very uncertain whether low HDL-C is directly causal for the development of ASCVD. HDL metabolism is strongly influenced by TRLs, insulin resistance, and inflammation, among other environmental and medical factors. Thus the HDL-C measurement integrates a number of cardiovascular risk factors, potentially explaining its strong inverse association with ASCVD.

1	The majority of patients with low HDL-C have some combination of genetic predisposition and secondary factors. Variants in dozens of genes have been shown to influence HDL-C levels. Even more important quantitatively, obesity and insulin resistance have strong suppressive effects on HDL-C, and low HDL-C in these conditions is widely observed. Furthermore, the vast majority of patients with elevated TGs have reduced levels of HDL-C. Most patients with low HDL-C who have been studied in detail have accelerated catabolism of HDL and its associated apoA-I as the physiologic basis for the low HDL C. Importantly, although HDL-C remains an important biomarker for assessing cardiovascular risk, it is not currently a direct target of intervention for raising the level in order to reduce cardiovascular risk. Certain therapeutic approaches in clinical development, such as inhibitors of CETP (see below), have the potential to change this paradigm.

1	Mutations in genes encoding proteins that play critical roles in HDL synthesis and catabolism can result in reductions in plasma levels of HDL-C. Unlike the genetic forms of hypercholesterolemia, which are invariably associated with premature coronary atherosclerosis, genetic forms of hypoalphalipoproteinemia (low HDL-C) are often not associated with clearly increased risk of ASCVD.

1	Gene Deletions in the APOA5-A1-C3-A4 Locus and Coding Mutations in APOA1 Complete genetic deficiency of apoA-I due to a complete deletion of the APOA1 gene results in the virtual absence of circulating HDL and appears to increase the risk of premature ASCVD. The genes encoding APOA5, APOA1, APOC3, and APOA4 are clustered together on chromosome 11. Some patients with no apoA-I have genomic deletions that include other genes in the cluster. ApoA-I is required for LCAT activity. In the absence of LCAT, free cholesterol levels increase in both plasma (not HDL) and in tissues. The free cholesterol can form deposits in the cornea and in the skin, resulting in corneal 2445 opacities and planar xanthomas. Premature CHD is associated with apoA-I deficiency.

1	Missense and nonsense mutations in the apoA-I gene are present in some patients with low plasma levels of HDL-C (usually 15–30 mg/ dL), but are a rare cause of low plasma HDL-C levels. Most individuals with low plasma HDL-C levels due to missense mutations in apoA-I do not appear to have premature CHD. Patients who are heterozygous for an Arg173Cys substitution in apoA-I (so-called apoA-IMilano) have very low plasma levels of HDL-C due to impaired LCAT activation and accelerated clearance of the HDL particles containing the abnormal apoA-I. Despite having very low plasma levels of HDL-C, these individuals do not have an increased risk of premature CHD. A few selected missense mutations in apoA-I and apoA-II promote the formation of amyloid fibrils, which can cause systemic amyloidosis.

1	Tangier Disease (ABCA1 Deficiency) Tangier disease is a rare autosomal co-dominant form of extremely low plasma HDL-C levels that is caused by mutations in the gene encoding ABCA1, a cellular transporter that facilitates efflux of unesterified cholesterol and phospholipids from cells to apoA-I (Fig. 421-3). ABCA1 in the liver and intestine rapidly lipidates the apoA-I secreted from the basolateral membranes of these tissues. In the absence of ABCA1, the nascent, poorly lipidated apoA-I is immediately cleared from the circulation. Thus, patients with Tangier disease have extremely low circulating plasma levels of HDL-C (<5 mg/dL) and apoA-I (<5 mg/dL). Cholesterol accumulates in the reticuloendothelial system of these patients, resulting in hepatosplenomegaly and pathognomonic enlarged, grayish yellow or orange tonsils. An intermittent peripheral neuropathy (mononeuritis multiplex) or a sphingomyelia-like neurologic disorder can also be seen in this disorder. Tangier disease is

1	grayish yellow or orange tonsils. An intermittent peripheral neuropathy (mononeuritis multiplex) or a sphingomyelia-like neurologic disorder can also be seen in this disorder. Tangier disease is probably associated with some increased risk of premature atherosclerotic disease, although the association is not as robust as might be anticipated, given the very low levels of HDL-C and apoA-I in these patients. Patients with Tangier disease also have low plasma levels of LDL-C, which may attenuate the atherosclerotic risk. Obligate heterozygotes for ABCA1 mutations have moderately reduced plasma HDL-C levels (15–30 mg/dL), and their risk of premature CHD remains uncertain.

1	Familial LCAT Deficiency This rare autosomal recessive disorder is caused by mutations in LCAT, an enzyme synthesized in the liver and secreted into the plasma, where it circulates associated with lipoproteins (Fig. 421-3). As reviewed above, the enzyme is activated by apoA-I and mediates the esterification of cholesterol to form cholesteryl esters. Consequently, in familial LCAT deficiency, the proportion of free cholesterol in circulating lipoproteins is greatly increased (from ~25% to >70% of total plasma cholesterol). Deficiency in this enzyme interferes with the maturation of HDL particles and results in rapid catabolism of circulating apoA-I.

1	Two genetic forms of familial LCAT deficiency have been described in humans: complete deficiency (also called classic LCAT deficiency) and partial deficiency (also called fish-eye disease). Progressive corneal opacification due to the deposition of free cholesterol in the cornea, very low plasma levels of HDL-C (usually <10 mg/dL), and variable hypertriglyceridemia are characteristic of both disorders. In partial LCAT deficiency, there are no other known clinical sequelae. In contrast, patients with complete LCAT deficiency have hemolytic anemia and progressive renal insufficiency that eventually leads to end-stage renal disease. Remarkably, despite the extremely low plasma levels of HDL-C and apoA-I, premature ASCVD is not a consistent feature of either LCAT deficiency or fish eye disease. The diagnosis can be confirmed in a specialized laboratory by assaying plasma LCAT activity or by sequencing the LCAT gene.

1	Primary Hypoalphalipoproteinemia The condition of low plasma levels of HDL-C (the “alpha lipoprotein”) is referred to as hypoalphalipoproteinemia. Primary hypoalphalipoproteinemia is defined as a plasma HDL-C level below the tenth percentile in the setting of relatively normal cholesterol and TG levels, no apparent secondary causes of low plasma HDL-C, and no clinical signs of LCAT deficiency or Tangier

1	Disorders of Lipoprotein Metabolism 2446 disease. This syndrome is often referred to as isolated low HDL. A family history of low HDL-C facilitates the diagnosis of an inherited condition, which may follow an autosomal dominant pattern. The metabolic etiology of this disease appears to be primarily accelerated catabolism of HDL and its apolipoproteins. Some of these patients may have ABCA1 mutations and therefore technically have heterozygous Tangier disease. Several kindreds with primary hypoalphalipoproteinemia and an increased incidence of premature CHD have been described, although it is not clear if the low HDL-C level is the cause of the accelerated atherosclerosis in these families. Association of hypoalphalipoproteinemia with premature CHD may depend on the specific nature of the gene defect or the underlying metabolic defect that either directly or indirectly causes the low plasma HDL-C level.

1	INHERITED CAUSES OF VERY HIGH LEVELS OF HDL-C CETP Deficiency Loss-of-function mutations in both alleles of the gene encoding CETP cause substantially elevated HDL-C levels (usually >150 mg/dL). As noted above, CETP transfers cholesteryl esters from HDL to apoB-containing lipoproteins (Fig. 421-3). Absence of this transfer activity results in an increase in the cholesteryl ester content of HDL and a reduction in plasma levels of LDL-C. The large, cholesterol-rich HDL particles circulating in these patients are cleared at a reduced rate. CETP deficiency was first diagnosed in Japanese persons and is rare outside of Japan. The relationship of CETP deficiency to ASCVD remains unresolved. Heterozygotes for CETP deficiency have only modestly elevated HDL-C levels. Based on the phenotype of high HDL-C in CETP deficiency, pharmacologic inhibition of CETP is under development as a new therapeutic approach to both raise HDL-C levels and lower LDL-C levels, but whether it will reduce risk of

1	HDL-C in CETP deficiency, pharmacologic inhibition of CETP is under development as a new therapeutic approach to both raise HDL-C levels and lower LDL-C levels, but whether it will reduce risk of ASCVD remains to be determined.

1	SCREENING, DIAGNOSIS, AND MANAGEMENT OF DISORDERS OF LIPOPROTEIN METABOLISM

1	Plasma lipid and lipoprotein levels should be measured in all adults, preferably after a 12-h overnight fast. In most clinical laboratories, the total cholesterol and TGs in the plasma are measured enzymatically, and then the cholesterol in the supernatant is measured after precipitation of apoB-containing lipoproteins to determine the HDL-C. The LDL-C is then estimated using the following equation: (The VLDL cholesterol content is estimated by dividing the plasma TG by 5, reflecting the ratio of TG to cholesterol in VLDL particles.) This formula (the Friedewald formula) is reasonably accurate if test results are obtained on fasting plasma and if the TG level does not exceed ~200 mg/dL; by convention it cannot be used if the TG level is >400 mg/dL. LDL-C can be directly measured by a number of methods. Further evaluation and treatment are based primarily on the clinical assessment of absolute cardiovascular risk using risk calculators such as the AHA/ACC risk calculator based on a

1	of methods. Further evaluation and treatment are based primarily on the clinical assessment of absolute cardiovascular risk using risk calculators such as the AHA/ACC risk calculator based on a large amount of observational data.

1	A critical first step in managing a lipoprotein disorder is to attempt to determine the class or classes of lipoproteins that are increased or decreased in the patient. Once the hyperlipidemia is accurately classified, efforts should be directed to rule out any possible secondary causes of the hyperlipidemia (Table 421-4). Although many patients with hyperlipidemia have a primary (i.e., genetic) cause of their lipid disorder, secondary factors frequently contribute to the hyperlipidemia. A careful social, medical, and family history should be obtained. A fasting glucose should be obtained in the initial workup of all subjects with an elevated TG level. Nephrotic syndrome and chronic renal insufficiency should be excluded by obtaining urine protein and serum creatinine. Liver function tests should be performed to rule out hepatitis and cholestasis. Hypothyroidism should be ruled out by measuring serum thyroid-stimulating hormone.

1	Once secondary causes have been ruled out, attempts should be made to diagnose the primary lipid disorder because the underlying genetic defect can provide important prognostic information regarding the risk of developing CHD, the response to drug therapy, and the management of other family members. Obtaining the correct diagnosis often requires a detailed family medical history, lipid analyses in family members, and sometimes specialized testing.

1	Severe Hypertriglyceridemia If the fasting plasma TG level is >1000 mg/dL, the patient has chylomicronemia. If the cholesterol-to-TG ratio is >10, familial chylomicronemia syndrome must be considered, and LPL activity measured in postheparin plasma can help with making that diagnosis. Most adults with chylomicronemia also have elevated VLDL levels. These individuals usually do not have a Mendelian disorder but instead are genetically predisposed and have secondary factors (diet, obesity, glucose intolerance, alcohol ingestion, estrogen therapy) that contribute to the hyperlipidemia. Such patients are a risk of acute pancreatitis and should be treated to reduce their TG levels and thus their risk of pancreatitis.

1	Severe Hypercholesterolemia If the levels of LDL-C are very high (greater than a ninety-fifth percentile for age and sex), it is likely that the patient has a genetic cause of hypercholesterolemia. At present, there is no compelling reason to perform molecular studies to further refine the molecular diagnosis because the clinical management is not affected. Recessive forms of severe hypercholesterolemia are rare, but if a patient with severe hypercholesterolemia has parents with normal cholesterol levels, ARH, sitosterolemia, and CESD should be considered. Patients with more moderate hypercholesterolemia that does not segregate in families as a monogenic trait are likely to have polygenic hypercholesterolemia.

1	Combined Hyperlipidemia The most common errors in the diagnosis of lipid disorders involve patients with combined hyperlipidemia. Elevations in the plasma levels of both cholesterol and TGs are seen in patients with increased plasma levels of VLDL and LDL or of remnant lipoproteins. A β-quantification to determine the VLDL cholesterol/TG ratio in plasma (see discussion of FDBL) or a direct measurement of the plasma LDL-C should be performed at least once prior to initiation of lipid-lowering therapy to determine if the hyperlipidemia is due to the accumulation of remnants or to an increase in both LDL and VLDL. Measurement of plasma apoB levels can help identify patients with FCHL who may require more aggressive treatment. APPROACH TO THE PATIENT: The major goals in the clinical management of lipoprotein disorders are: (1) prevention of acute pancreatitis in patients with severe hypertriglyceridemia; and (2) prevention of CVD and related cardiovascular events.

1	Although the observational relationship between severe hypertriglyceridemia, particularly chylomicronemia, and acute pancreatitis is well-established, there has never been a clinical trial designed or powered to prove that intervention to reduce TGs reduces the risk of pancreatitis. Nevertheless, it is generally considered appropriate medical practice to intervene in patients with TGs >500 mg/dL in order to reduce the risk of pancreatitis. It remains controversial whether individuals with severe hypertriglyceridemia are at increased risk for ASCVD.

1	Lifestyle Modifying the lifestyle of the patient with severe hypertriglyceridemia often is associated with a significant reduction in plasma TG level. Patients who drink alcohol should be encouraged to decrease or preferably eliminate their intake. Patients with severe hypertriglyceridemia often benefit from a formal dietary consultation with a dietician intimately familiar with counseling patients on the dietary management of high TGs. Dietary fat intake should be restricted to reduce the formation of chylomicrons in the intestine. The excessive intake of simple carbohydrates should be discouraged because insulin drives TG production in the liver. Aerobic exercise and even increase in regular physical activity can have a positive effect in reducing TG levels and should be strongly encouraged. For patients who are overweight, weight loss can help to reduce TG levels. In extreme cases, bariatric surgery has been shown to not only produce effective weight loss but also substantially

1	For patients who are overweight, weight loss can help to reduce TG levels. In extreme cases, bariatric surgery has been shown to not only produce effective weight loss but also substantially reduce plasma TG levels.

1	Pharmacologic Therapy for Severe Hypertriglyceridemia Despite the above interventions, however, many patients with severe hypertriglyceridemia require pharmacologic therapy (Table 421-5). Patients who persist in having fasting TG >500 mg/dL despite active lifestyle management are candidates for pharmacologic therapy. There are three classes of drugs that are used for management of these patients: fibrates, omega-3 fatty acids (fish oils), and niacin. In addition, statins can reduce plasma TG levels and also reduce ASCVD risk.

1	fiBrateS Fibric acid derivatives, or fibrates, are agonists of PPARα, a nuclear receptor involved in the regulation of lipid metabolism. Fibrates stimulate LPL activity (enhancing TG hydrolysis), reduce apoC-III synthesis (enhancing lipoprotein remnant clearance), promote β-oxidation of fatty acids, and may reduce VLDL TG production. Fibrates are a first-line therapy for severe hypertriglyceridemia (>500 mg/dL). This class of therapeutic agents sometimes lowers but more often raises the plasma level of LDL-C in individuals with severe hypertriglyceridemia. Fibrates are generally well tolerated, but are associated with an increase in the incidence of gallstones. Fibrates can cause myopathy, especially when combined with other lipid-lowering therapy (statins, niacin), and can raise creatinine. Fibrates should be used with caution in patients with CKD. Importantly, fibrates can potentiate the effect of warfarin and certain oral hypoglycemic agents, so the anticoagulation status and

1	Fibrates should be used with caution in patients with CKD. Importantly, fibrates can potentiate the effect of warfarin and certain oral hypoglycemic agents, so the anticoagulation status and plasma glucose levels should be closely monitored in patients on these agents.

1	omeGa 3 fatty acidS (fiSH oilS) Omega-3 fatty acids, or omega-3 polyunsaturated fatty acids (n-3 PUFAs), commonly known as fish oils, are present in high concentration in fish and in flaxseed. The most widely used n-3 PUFAs for the treatment of hyperlipidemias are the two active molecules in fish oil: eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). n-3 PUFAs have been concentrated into tablets and in doses of 3–4 g/d are effective at lowering fasting TG levels. Fish oils are a reasonable consideration for first-line therapy in patients with severe hypertriglyceridemia (>500 mg/ dL) to prevent pancreatitis. Fish oils can cause an increase in plasma LDL-C levels in some patients. In general, fish oils are well tolerated, with the major side effect being dyspepsia. They appear to be safe, at least at doses up to 3–4 g, but can be associated with a prolongation in the bleeding time.

1	nicotinic acid Nicotinic acid, or niacin, is a B-complex vitamin that has been used as a lipid-modifying agent for more than five decades. Niacin suppresses lipolysis in the adipocyte through its effect on the niacin receptor GPR109A and has other effects on hepatic lipid metabolism that are poorly understood. Niacin reduces plasma TG and LDL-C levels and also raises the plasma concentration of HDL-C. Because it has a number of side effects and can be difficult to use, it is at best a third-line agent for the management of severe hypertriglyceridemia. Niacin therapy is generally started at lower doses and gradually titrated up to higher doses. The most frequent side effect of niacin is cutaneous flushing, which is mediated by activating GPR109A in the skin. Niacin can cause dyspepsia and can exacerbate esophageal reflux and peptic ulcer disease. Mild elevations in transaminases occur in up to 15% of patients treated with any form of niacin. Niacin can raise plasma levels of uric acid

1	can exacerbate esophageal reflux and peptic ulcer disease. Mild elevations in transaminases occur in up to 15% of patients treated with any form of niacin. Niacin can raise plasma levels of uric acid and precipitate gouty attacks in susceptible patients. Acanthosis nigricans, a dark-colored coarse skin lesion, and maculopathy are infrequent side effects of niacin.

1	In contrast to hypertriglyceridemia and pancreatitis, there are abundant and compelling data that intervention to reduce LDL-C substantially reduces the risk of CVD, including myocardial infarction and stroke, as well as total mortality. Thus, it is imperative that patients with hypercholesterolemia be assessed for cardiovascular risk and for the need for intervention. It is also worth noting that patients at high risk for CVD who have plasma LDL-C levels in the “normal” or average range also benefit from intervention to reduce LDL-C levels.

1	Lifestyle The first approach to a patient with hypercholesterolemia and high cardiovascular risk is to make any necessary lifestyle changes. In obese patients, efforts should be made to reduce body weight to the ideal level. Patients should receive dietary counseling to reduce the content of saturated fats, trans fats, and cholesterol in the diet. Regular aerobic exercise has relatively little impact on reducing plasma LDL-C levels, although it has cardiovascular benefits independent of LDL lowering. Pharmacologic Therapy for Hypercholesterolemia The decision to use LDL-lowering drug therapy (Table 421-5)—with a statin being first-line therapy—depends on the level of LDL-C as well as the level of cardiovascular risk. In general, patients with a Mendelian disorder of elevated LDL-C such as FH must be treated to reduce the very high lifetime risk of CVD, and treatment should be initiated as early as possible in adulthood or, in some cases, during childhood.

1	Otherwise, the decision to initiate LDL-lowering drug therapy is generally determined by the level of cardiovascular risk. In patients with established CVD, statin therapy is well supported by clinical trial data and should be used regardless of the LDL-C level. For patients >40 years old without clinical CVD, the AHA/ACC risk calculator (http://my.americanheart .org/professional/StatementsGuidelines/PreventionGuidelines/ Prevention-Guidelines_UCM_457698_SubHomePage.jsp) can be used to determine the 10-year absolute risk for CVD, and current guidelines suggest that a 10-year risk >7.5% merits consideration of statin therapy regardless of plasma LDL-C level. For younger patients, the assessment of lifetime risk of CVD may help inform the decision to start a statin.

1	HmG-coa reductaSe inHiBitorS (StatinS) Statins inhibit HMG-CoA reductase, a key enzyme in cholesterol biosynthesis. By inhibiting cholesterol biosynthesis, statins lead to increased hepatic LDL receptor activity and accelerated clearance of circulating LDL, resulting in a dose-dependent reduction in plasma levels of LDL-C. The magnitude of LDL lowering associated with statin treatment varies widely among individuals, but once a patient is on a statin, the doubling of the statin dose produces an ~6% further reduction in the level of plasma LDL-C. The statins currently available differ in their LDL-C–reducing potency (Table 421-5). Currently, there is no convincing evidence that any of the different statins confer an advantage that is independent of the effect on LDL-C. Statins also reduce plasma TGs in a dose-dependent fashion, which is roughly proportional to their LDL-C–lowering effects (if the TGs are <400 mg/dL). Statins have a modest HDL-raising effect (5–10%) that is not

1	reduce plasma TGs in a dose-dependent fashion, which is roughly proportional to their LDL-C–lowering effects (if the TGs are <400 mg/dL). Statins have a modest HDL-raising effect (5–10%) that is not generally dose-dependent.

1	Statins are well tolerated and can be taken in tablet form once a day. Potential side effects include dyspepsia, headaches, fatigue, and muscle or joint pains. Severe myopathy and even rhabdomyolysis occur rarely with statin treatment. The risk of statin-associated myopathy is increased by the presence of older age, frailty, renal insufficiency, and coadministration of drugs that interfere with the metabolism of statins, such as erythromycin and related antibiotics, antifungal agents, immunosuppressive drugs, and fibric acid deriva- Disorders of Lipoprotein Metabolism Abbreviations: GI, gastrointestinal; HDL-C, high-density lipoprotein cholesterol; HoFH, homozygous familial hypercholesterolemia; LDL, low-density lipoprotein; LDL-C, LDL-cholesterol; LPL, lipoprotein lipase; TG, triglyceride; VLDL, very-low-density lipoprotein.

1	tives (particularly gemfibrozil). Severe myopathy can usually be less frequent monitoring of transaminases in patients taking statins. avoided by careful patient selection, avoidance of interacting drugs, The statin-associated elevation in liver enzymes resolves upon dis-and instructing the patient to contact the physician immediately in continuation of the medication. the event of unexplained muscle pain. In the event of muscle symp-Statins appear to be remarkably safe. Meta-analyses of large toms, the plasma creatine kinase (CK) level should be obtained to randomized controlled clinical trials with statins do not suggest an differentiate myopathy from myalgia. Serum CK levels need not be increase in any major noncardiac diseases except type 2 diabetes. A monitored on a routine basis in patients taking statins, because an small excess percentage of those taking statins will develop diabetes elevated CK in the absence of symptoms does not predict the devel-but the benefits associated

1	patients taking statins, because an small excess percentage of those taking statins will develop diabetes elevated CK in the absence of symptoms does not predict the devel-but the benefits associated with the reduction in cardiovascular opment of myopathy and does not necessarily suggest the need for events outweigh the increase in incidence of diabetes. Statins are the discontinuing the drug. drug class of choice for LDL-C reduction and are by far the most

1	Another consequence of statin therapy can be elevation in widely used class of lipid-lowering drugs. liver transaminases (alanine aminotransferase [ALT] and aspartate aminotransferase [AST]). They should be checked before starting cHoleSterol aBSorption inHiBitorS Cholesterol within the lumen therapy, at 2–3 months, and then annually. Substantial (greater than of the small intestine is derived from the diet (about one-third) three times the upper limit of normal) elevation in transaminases is and the bile (about two-thirds) and is actively absorbed by the relatively rare, and mild-to-moderate (one to three times normal) enterocyte through a process that involves the protein NPC1L1. elevation in transaminases in the absence of symptoms need not Ezetimibe (Table 421-5) is a cholesterol absorption inhibitor that mandate discontinuing the medication. Severe clinical hepatitis binds directly to and inhibits NPC1L1 and blocks the intestinal associated with statins is exceedingly rare, and

1	inhibitor that mandate discontinuing the medication. Severe clinical hepatitis binds directly to and inhibits NPC1L1 and blocks the intestinal associated with statins is exceedingly rare, and the trend is toward absorption of cholesterol. Ezetimibe (10 mg) inhibits cholesterol absorption by almost 60%, resulting in a reduction in delivery of dietary sterols in the liver and an increase in hepatic LDL receptor expression. The mean reduction in plasma LDL-C on ezetimibe (10 mg) is 18%, and the effect is additive when used in combination with a statin. Effects on TG and HDL-C levels are negligible. When used in combination with a statin, monitoring of liver transaminases is recommended. The only roles for ezetimibe in monotherapy are in patients who do not tolerate statins and in sitosterolemia.

1	Bile acid SeQueStrantS (reSinS) Bile acid sequestrants bind bile acids in the intestine and promote their excretion rather than reabsorption in the ileum. To maintain the bile acid pool size, the liver diverts cholesterol to bile acid synthesis. The decreased hepatic intracellular cholesterol content results in upregulation of the LDL receptor and enhanced LDL clearance from the plasma. Bile acid sequestrants, including cholestyramine, colestipol, and colesevelam (Table 421-5), primarily reduce plasma LDL-C levels but can cause an increase in plasma TGs. Therefore, patients with hypertriglyceridemia generally should not be treated with bile acid–binding resins. Cholestyramine and colestipol are insoluble resins that must be suspended in liquids. Colesevelam is available as tablets but generally requires up to six to seven tablets per day for effective LDL-C lowering. Most side effects of resins are limited to the gastrointestinal tract and include bloating and constipation. Because

1	requires up to six to seven tablets per day for effective LDL-C lowering. Most side effects of resins are limited to the gastrointestinal tract and include bloating and constipation. Because bile acid sequestrants are not systemically absorbed, they are very safe and the cholesterol-lowering drug of choice in children and in women of childbearing age who are lactating, pregnant, or could become pregnant. They are effective in combination with statins and in combination with ezetimibe and are particularly useful with one or both of these drugs for patients with severe hypercholesterolemia or those with statin intolerance.

1	Specialized druGS for HomozyGouS fH Two “orphan” drugs are approved specifically for the management of homozygous FH. They include a small-molecule inhibitor of MTP, called lomitapide, and an antisense oligonucleotide against apoB, called mipomersen. These drugs reduce VLDL production and LDL-C levels in homozygous FH patients. Due to their mechanism of action, each drug causes an increase in hepatic fat, the long-term consequences of which are unknown. In addition, lomitapide is associated with gastrointestinal-related side effects, and mipomersen is associated with skin reactions and flu-like symptoms.

1	ldl apHereSiS Patients who remain severely hypercholesterolemic despite optimally tolerated drug therapy are candidates for LDL apheresis. In this process, the patient’s plasma is passed over a column that selectively removes the LDL, and the LDL-depleted plasma is returned to the patient. Patients on maximally tolerated combination drug therapy who have CHD and a plasma LDL-C level >200 mg/dL or no CHD and a plasma LDL-C level >300 mg/ dL are candidates for every-other-week LDL apheresis and should The metabolic Syndrome Robert H. Eckel

1	The metabolic Syndrome Robert H. Eckel The metabolic syndrome (syndrome X, insulin resistance syndrome) consists of a constellation of metabolic abnormalities that confer increased risk of cardiovascular disease (CVD) and diabetes mellitus. Evolution of the criteria for the metabolic syndrome since the original definition by the World Health Organization in 1998 reflects growing clinical evidence and analysis by a variety of consensus conferences and professional organizations. The major features of the metabolic syndrome include central obesity, hypertriglyceridemia, low levels of high-density lipoprotein (HDL) cholesterol, hyperglycemia, and 2449 hypertension (Table 422-1).

1	The most challenging feature of the metabolic syndrome to define is waist circumference. Intraabdominal circumference (visceral adipose tissue) is considered most strongly related to insulin resistance and risk of diabetes and CVD, and for any given waist circumference the distribution of adipose tissue between SC and visceral depots varies substantially. Thus, within and between populations, there is a lesser vs. greater risk at the same waist circumference. These differences in populations are reflected in the range of waist circumferences considered to confer risk in different geographic locations (Table 422-1).

1	The prevalence of the metabolic syndrome varies around the world, in part reflecting the age and ethnicity of the populations studied and the diagnostic criteria applied. In general, the prevalence of the metabolic syndrome increases with age. The highest recorded prevalence worldwide is among Native Americans, with nearly 60% of women ages 45–49 and 45% of men ages 45–49 meeting the criteria of the National Cholesterol Education Program and Adult Treatment Panel III (NCEP:ATPIII). In the United States, the metabolic syndrome is less common among African-American men and more common among Mexican-American women. Based on data from the National Health and Nutrition Examination Survey (NHANES) 2003–2006, the age-adjusted prevalence of the metabolic syndrome in U.S. adults without diabetes is 28% for men and 30% for women. In France, studies of a cohort of 30to 60-year-olds have shown a <10% prevalence for each sex, although 17.5% of people 60–64 years of age are affected. Greater global

1	28% for men and 30% for women. In France, studies of a cohort of 30to 60-year-olds have shown a <10% prevalence for each sex, although 17.5% of people 60–64 years of age are affected. Greater global industrialization is associated with rising rates of obesity, which are expected to increase the prevalence of the metabolic syndrome dramatically, especially as the population ages. Moreover, the rising prevalence and severity of obesity among children is reflected in features of the metabolic syndrome in a younger population.

1	The frequency distribution of the five components of the syndrome for the U.S. population (NHANES III) is summarized in Fig. 422-1. Increases in waist circumference predominate among women, whereas increases in fasting plasma triglyceride levels (i.e., to >150 mg/dL), reductions in HDL cholesterol levels, and hyperglycemia are more likely in men. RISK FACTORS Overweight/Obesity Although the metabolic syndrome was first described in the early twentieth century, the worldwide overweight/ obesity epidemic has recently been the force driving its increasing recognition. Central adiposity is a key feature of the syndrome, and the syndrome’s prevalence reflects the strong relationship between waist circumference and increasing adiposity. However, despite the importance of obesity, patients who are of normal weight may also be insulin resistant and may have the metabolic syndrome.

1	Sedentary Lifestyle Physical inactivity is a predictor of CVD events and the related risk of death. Many components of the metabolic syndrome are associated with a sedentary lifestyle, including increased adipose tissue (predominantly central), reduced HDL cholesterol, and increased triglycerides, blood pressure, and glucose in genetically susceptible persons. Compared with individuals who watch television or videos or use the computer <1 h daily, those who do so for >4 h daily have a twofold increased risk of the metabolic syndrome. Aging The metabolic syndrome affects nearly 50% of the U.S. population older than age 50, and at >60 years of age women are more often affected than men. The age dependency of the syndrome’s prevalence is seen in most populations around the world.

1	Diabetes Mellitus Diabetes mellitus is included in both the NCEP and the harmonizing definitions of the metabolic syndrome. It is estimated that the great majority (~75%) of patients with type 2 diabetes or impaired glucose tolerance have the metabolic syndrome. The presence of the metabolic syndrome in these populations relates to a The Metabolic Syndrome NCEP:ATPIII 2001 Harmonizing Definitionb Low HDLc cholesterol: <40 mg/dL and <50 mg/dL for men and women, respectively, or specific medication Hypertension: blood pressure ≥130 mmHg systolic or

1	Hypertension: blood pressure ≥130 mmHg systolic or HDL cholesterol level <40 mg/dL and <50 mg/dL for men and women, respectively, or • Fasting plasma glucose level ≥100 mg/dL (alternative indication: drug treatment of aNational Cholesterol Education Program and Adult Treatment Panel III. bIn this analysis, the following thresholds for waist circumference were used: white men, ≥94 cm; African-American men, ≥94 cm; Mexican-American men, ≥90 cm; white women, ≥80 cm; African-American women, ≥80 cm; Mexican-American women, ≥80 cm. For participants whose designation was “other race—including multiracial,” thresholds that were once based on Europid cutoffs (≥94 cm for men and ≥80 cm for women) and on South Asian cutoffs (≥90 cm for men and ≥80 cm for women) were used. For participants who were considered “other Hispanic,” the International Diabetes Federation thresholds for ethnic South and Central Americans were used. cHigh-density lipoprotein.

1	higher prevalence of CVD than in patients who have type 2 diabetes or impaired glucose tolerance but do not have this syndrome. Cardiovascular Disease Individuals with the metabolic syndrome are twice as likely to die of cardiovascular disease as those who do not, and their risk of an acute myocardial infarction or stroke is threefold higher. The approximate prevalence of the metabolic syndrome among patients with coronary heart disease (CHD) is 50%, with a prevalence of ~35% among patients with premature coronary artery disease (before or at age 45) and a particularly high prevalence among women. With appropriate cardiac rehabilitation and changes in lifestyle (e.g., nutrition, physical activity, weight reduction, and—in some cases—pharmacologic therapy), the prevalence of the syndrome can be reduced.

1	Lipodystrophy Lipodystrophic disorders in general are associated with the metabolic syndrome. Both genetic lipodystrophy (e.g., Berardinelli-Seip congenital lipodystrophy, Dunnigan familial partial lipodystrophy) and acquired lipodystrophy (e.g., HIV-related lipodystrophy in % of subjects FIGURE 422-1 Prevalence of the metabolic syndrome components, from NHANES 2003–2006. NHANES, National Health and Nutrition Examination Survey; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; BP, blood pressure. The prevalence of elevated glucose includes individuals with known diabetes mellitus. (Created from data in ES Ford et al: J Diabetes 2:1753, 2010.) patients receiving antiretroviral therapy) may give rise to severe insulin resistance and many of the components of the metabolic syndrome.

1	ETIOLOGY Insulin Resistance The most accepted and unifying hypothesis to describe the pathophysiology of the metabolic syndrome is insulin resistance, which is caused by an incompletely understood defect in insulin action (Chap. 417). The onset of insulin resistance is heralded by postprandial hyperinsulinemia, which is followed by fasting hyperinsulinemia and ultimately by hyperglycemia.

1	An early major contributor to the development of insulin resistance is an overabundance of circulating fatty acids (Fig. 422-2). Plasma albumin-bound free fatty acids are derived predominantly from adipose-tissue triglyceride stores released by intracellular lipolytic enzymes. Fatty acids are also derived from the lipolysis of triglyceride-rich lipoproteins in tissues by lipoprotein lipase. Insulin mediates both antilipolysis and the stimulation of lipoprotein lipase in adipose tissue. Of note, the inhibition of lipolysis in adipose tissue is the most sensitive pathway of insulin action. Thus, when insulin resistance develops, increased lipolysis produces more fatty acids, which further decrease the antilipolytic effect of insulin. Excessive fatty acids enhance substrate availability and create insulin resistance by modifying downstream signaling. Fatty acids impair insulin-mediated glucose uptake and accumulate as triglycerides in both skeletal and cardiac muscle, whereas increased

1	create insulin resistance by modifying downstream signaling. Fatty acids impair insulin-mediated glucose uptake and accumulate as triglycerides in both skeletal and cardiac muscle, whereas increased glucose production and triglyceride accumulation take place in the liver.

1	Leptin resistance has also been raised as a possible pathophysiologic mechanism to explain the metabolic syndrome. Physiologically, leptin reduces appetite, promotes energy expenditure, and enhances insulin sensitivity. In addition, leptin may regulate cardiac and vascular function through a nitric oxide–dependent mechanism. However, when obesity develops, hyperleptinemia ensues, with evidence of leptin resistance in the brain and other tissues resulting in inflammation, insulin resistance, hyperlipidemia, and a plethora of cardiovascular disorders, such as hypertension, atherosclerosis, CHD, and heart failure.

1	The oxidative stress hypothesis provides a unifying theory for aging and the predisposition to the metabolic syndrome. In studies of insulin-resistant individuals with obesity or type 2 diabetes, the offspring of patients with type 2 diabetes, and the elderly, a defect in mitochondrial oxidative phosphorylation that leads to the accumulation of triglycerides and related lipid molecules in muscle has been identified.

1	Recently, the gut microbiome has emerged as an important contributor to the development of obesity and related metabolic disorders, including the metabolic syndrome. Although the mechanism remains uncertain, interaction among genetic predisposition, diet, and the compensate for defects in insulin action, insulin secretion and/or clear-intestinal flora is important. ance must be modified so that euglycemia is sustained. Ultimately, this Increased Waist Circumference Waist circumference is an important compensatory mechanism fails, usually because of defects in insulin component of the most recent and frequently applied diagnostic secretion, resulting in progression from impaired fasting glucose and/ criteria for the metabolic syndrome. However, measuring waist cir-or impaired glucose tolerance to diabetes mellitus. cumference does not reliably distinguish increases in SC adipose tissue

1	Hypertension The relationship between insulin resistancefrom those in visceral fat; this distinction requires CT or MRI. With and hypertension is well established. Paradoxically, under increases in visceral adipose tissue, adipose tissue–derived free fatty normal physiologic conditions, insulin is a vasodilator withacids are directed to the liver. In contrast, increases in abdominal SC secondary effects on sodium reabsorption in the kidney. However, infat release lipolysis products into the systemic circulation and avert the setting of insulin resistance, the vasodilatory effect of insulin is lostmore direct effects on hepatic metabolism. Relative increases in vis- but the renal effect on sodium reabsorption is preserved. Sodium reabceral versus SC adipose tissue with increasing waist circumference sorption is increased in whites with the metabolic syndrome but not inin Asians and Asian Indians may explain the greater prevalence of

1	Africans or Asians. Insulin also increases the activity of the sympathe syndrome in those populations than in African-American men, thetic nervous system, an effect that may be preserved in the setting ofin whom SC fat predominates. It is also possible that visceral fat is a insulin resistance. Insulin resistance is characterized by pathway-marker for—but not the source of—excess postprandial free fatty acids specific impairment in phosphatidylinositol-3-kinase signaling. In thein obesity.

1	endothelium, this impairment may cause an imbalance between the Dyslipidemia (See also Chap. 421) In general, free fatty acid flux to production of nitric oxide and the secretion of endothelin 1, with a the liver is associated with increased production of ApoB-containing, consequent decrease in blood flow. Although these mechanisms are triglyceride-rich, very low-density lipoproteins (VLDLs). The effect provocative, evaluation of insulin action by measurement of fasting of insulin on this process is complex, but hypertriglyceridemia is an insulin levels or by homeostasis model assessment shows that insulin 2452 resistance contributes only partially to the increased prevalence of hypertension in the metabolic syndrome. Another possible mechanism underlying hypertension in the metabolic syndrome is the vasoactive role of perivascular adipose tissue. Reactive oxygen species released by NADPH oxidase impair endothelial function and result in local vasoconstriction. Other paracrine effects

1	is the vasoactive role of perivascular adipose tissue. Reactive oxygen species released by NADPH oxidase impair endothelial function and result in local vasoconstriction. Other paracrine effects could be mediated by leptin or other proinflammatory cytokines released from adipose tissue, such as tumor necrosis factor α. Hyperuricemia is another consequence of insulin resistance and is commonly observed in the metabolic syndrome. There is growing evidence not only that uric acid is associated with hypertension but also that reduction of uric acid normalizes blood pressure in hyperuricemic adolescents with hypertension. The mechanism appears to be related to an adverse effect of uric acid on nitric acid synthase in the macula densa of the kidney and stimulation of the renin-angiotensin aldosterone system.

1	Proinflammatory Cytokines The increases in proinflammatory cytokines—including interleukins 1, 6, and 18; resistin; tumor necrosis factor α; and the systemic biomarker C-reactive protein—reflect overproduction by the expanded adipose tissue mass (Fig. 422-2). Adipose tissue–derived macrophages may be the primary source of proinflammatory cytokines locally and in the systemic circulation. It remains unclear, however, how much of the insulin resistance is caused by the paracrine effects of these cytokines and how much by the endocrine effects.

1	Adiponectin Adiponectin is an anti-inflammatory cytokine produced exclusively by adipocytes. Adiponectin enhances insulin sensitivity and inhibits many steps in the inflammatory process. In the liver, adiponectin inhibits the expression of gluconeogenic enzymes and the rate of glucose production. In muscle, adiponectin increases glucose transport and enhances fatty acid oxidation, partially through the activation of AMP kinase. Adiponectin levels are reduced in the metabolic syndrome. The relative contributions of adiponectin deficiency and overabundance of the proinflammatory cytokines are unclear.

1	CLINICAL FEATURES Symptoms and Signs The metabolic syndrome typically is not associated with symptoms. On physical examination, waist circumference may be expanded and blood pressure elevated. The presence of either or both of these signs should prompt the clinician to search for other biochemical abnormalities that may be associated with the metabolic syndrome. Less frequently, lipoatrophy or acanthosis nigricans is found on examination. Because these physical findings characteristically are associated with severe insulin resistance, other components of the metabolic syndrome should be expected.

1	Associated Diseases • cardiovaScular diSeaSe The relative risk for new-onset CVD in patients with the metabolic syndrome who do not have diabetes averages 1.5–3 fold. However, an 8-year follow-up of middle-aged participants in the Framingham Offspring Study documented that the population-attributable CVD risk in the metabolic syndrome was 34% among men and only 16% among women. In the same study, both the metabolic syndrome and diabetes predicted ischemic stroke, with greater risk among patients with the metabolic syndrome than among those with diabetes alone (19% vs. 7%) and a particularly large difference among women (27% vs. 5%). Patients with the metabolic syndrome are also at increased risk for peripheral vascular disease.

1	TyPe 2 diabeTes Overall, the risk for type 2 diabetes among patients with the metabolic syndrome is increased threeto fivefold. In the Framingham Offspring Study’s 8-year follow-up of middle-aged participants, the population-attributable risk for developing type 2 diabetes was 62% among men and 47% among women. Other Associated Conditions In addition to the features specifically associated with the metabolic syndrome, other metabolic alterations accompany insulin resistance. Those alterations include increases in ApoB and ApoCIII, uric acid, prothrombotic factors (fibrinogen, plasminogen activator inhibitor 1), serum viscosity, asymmetric dimethylarginine, homocysteine, white blood cell count, proinflammatory cytokines, C-reactive protein, microalbuminuria, nonalcoholic fatty liver disease and/or nonalcoholic steatohepatitis, polycystic ovary syndrome, and obstructive sleep apnea.

1	nonalcoHolic fatty liver diSeaSe (See alSo cHap. 367e) Fatty liver is a relatively common condition, affecting 25–45% of the U.S. population. However, in nonalcoholic steatohepatitis, triglyceride accumulation and inflammation coexist. Nonalcoholic steatohepatitis is now present in 3–12% of the population of the United States and other Western countries. Of patients with the metabolic syndrome, ~25–60% have nonalcoholic fatty liver disease and up to 35% have nonalcoholic steatohepatitis. As the prevalence of overweight/obesity and the metabolic syndrome increases, nonalcoholic steatohepatitis may become one of the more common causes of end-stage liver disease and hepatocellular carcinoma.

1	Hyperuricemia (see also cHaP. 431e) Hyperuricemia reflects defects in insulin action on the renal tubular reabsorption of uric acid and may contribute to hypertension through its effect on the endothelium. An increase in asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, also relates to endothelial dysfunction. In addition, microalbuminuria may be caused by altered endothelial pathophysiology in the insulin-resistant state. polycyStic ovary Syndrome (see also cHaP. 412) Polycystic ovary syndrome is highly associated with insulin resistance (50–80%) and the metabolic syndrome, with a prevalence of the syndrome between 40% and 50%. Women with polycystic ovary syndrome are two to four times more likely to have the metabolic syndrome than are women without polycystic ovary syndrome.

1	oBStructive Sleep apnea (See alSo cHap. 38) Obstructive sleep apnea is commonly associated with obesity, hypertension, increased circulating cytokines, impaired glucose tolerance, and insulin resistance. With these associations, it is not surprising that individuals with obstructive sleep apnea frequently have the metabolic syndrome. Moreover, when biomarkers of insulin resistance are compared between patients with obstructive sleep apnea and weight-matched controls, insulin resistance is found to be more severe in those with apnea. Continuous positive airway pressure treatment improves insulin sensitivity in patients with obstructive sleep apnea.

1	The diagnosis of the metabolic syndrome relies on fulfillment of the criteria listed in Table 422-1, as assessed using tools at the bedside and in the laboratory. The medical history should include evaluation of symptoms for obstructive sleep apnea in all patients and polycystic ovary syndrome in premenopausal women. Family history will help determine risk for CVD and diabetes mellitus. Blood pressure and waist circumference measurements provide information necessary for the diagnosis.

1	Laboratory Tests Measurement of fasting lipids and glucose is needed in determining whether the metabolic syndrome is present. The measurement of additional biomarkers associated with insulin resistance can be individualized. Such tests might include those for ApoB, high-sensitivity C-reactive protein, fibrinogen, uric acid, urinary microalbumin, and liver function. A sleep study should be performed if symptoms of obstructive sleep apnea are present. If polycystic ovary syndrome is suspected on the basis of clinical features and anovulation, testosterone, luteinizing hormone, and follicle-stimulating hormone should be measured. LIFESTYLE (SEE ALSO CHAP. 416)

1	LIFESTYLE (SEE ALSO CHAP. 416) Obesity is the driving force behind the metabolic syndrome. Thus, weight reduction is the primary approach to the disorder. With weight reduction, improvement in insulin sensitivity is often accompanied by favorable modifications in many components of the metabolic syndrome. In general, recommendations for weight loss include a combination of caloric restriction, increased physical activity, and behavior modification. Caloric restriction is the most important component, whereas increases in physical activity are important for maintenance of weight loss. Some but not all evidence suggests that the addition of exercise to caloric restriction may promote greater weight loss from the visceral depot. The tendency for weight regain after successful weight reduction underscores the need for long-lasting behavioral changes.

1	Diet Before prescribing a weight-loss diet, it is important to emphasize that it has taken the patient a long time to develop an expanded fat mass; thus, the correction need not occur quickly. Given that ~3500 kcal = 1 lb of fat, ~500-kcal restriction daily equates to weight reduction of 1 lb per week. Diets restricted in carbohydrate typically provide a rapid initial weight loss. However, after 1 year, the amount of weight reduction is minimally reduced or no different from that with caloric restriction alone. Thus, adherence to the diet is more important than which diet is chosen. Moreover, there is concern about low-carbohydrate diets enriched in saturated fat, particularly for patients at risk for CVD. Therefore, a high-quality dietary pattern—i.e., a diet enriched in fruits, vegetables, whole grains, lean poultry, and fish—should be encouraged to maximize overall health benefit.

1	Physical Activity Before a physical activity recommendation is provided to patients with the metabolic syndrome, it is important to ensure that the increased activity does not incur risk. Some high-risk patients should undergo formal cardiovascular evaluation before initiating an exercise program. For an inactive participant, gradual increases in physical activity should be encouraged to enhance adherence and avoid injury. Although increases in physical activity can lead to modest weight reduction, 60–90 min of daily activity is required to achieve this goal. Even if an overweight or obese adult is unable to undertake this level of activity, a significant health benefit will follow from at least 30 min of moderate-intensity activity daily. The caloric value of 30 min of a variety of activities can be found at www.heart.org/HEARTORG/GettingHealthy/WeightManagement/ LosingWeight/Losing-Weight_UCM_307904_Article.jsp. Of note, a variety of routine activities, such as gardening, walking,

1	can be found at www.heart.org/HEARTORG/GettingHealthy/WeightManagement/ LosingWeight/Losing-Weight_UCM_307904_Article.jsp. Of note, a variety of routine activities, such as gardening, walking, and housecleaning, require moderate caloric expenditure. Thus, physical activity need not be defined solely in terms of formal exercise such as jogging, swimming, or tennis.

1	Behavior Modification Behavioral treatment typically includes recommendations for dietary restriction and more physical activity, resulting in weight loss that benefits metabolic health. The subsequent challenge is the duration of the program because weight regain so often follows successful weight reduction. Long-term outcomes may be enhanced by a variety of methods, such as the Internet, social media, and telephone follow-up to maintain contact between providers and patients.

1	Obesity (See also Chap. 416) In some patients with the metabolic syndrome, treatment options need to extend beyond lifestyle intervention. Weight-loss drugs come in two major classes: appetite suppressants and absorption inhibitors. Appetite suppressants approved by the U.S. Food and Drug Administration include phentermine (for short-term use [3 months] only) as well as the more recent additions phentermine/topiramate and lorcaserin, which are approved without restrictions on the duration of therapy. In clinical trials, the phentermine/topiramate combination has resulted in ~10% weight loss in 50% of patients. Side effects include palpitations, headache, paresthesias, constipation, and insomnia. Lorcaserin results in less weight loss—typically ~5% beyond placebo—but can cause headache and nasopharyngitis. Orlistat inhibits fat absorption by ~30% and is moderately effective compared with placebo (~5% more weight loss). Orlistat has been shown to reduce the incidence of type 2 diabetes,

1	nasopharyngitis. Orlistat inhibits fat absorption by ~30% and is moderately effective compared with placebo (~5% more weight loss). Orlistat has been shown to reduce the incidence of type 2 diabetes, an effect that was especially evident among patients with impaired glucose tolerance at baseline. This drug is often difficult of take because of oily leakage per rectum.

1	Metabolic or bariatric surgery is an option for patients with the 2453 metabolic syndrome who have a body mass index >40 kg/m2, or >35 kg/m2 with comorbidities. An evolving application for metabolic surgery includes patients with a body mass index as low as 30 kg/m2 and type 2 diabetes. Gastric bypass or vertical sleeve gastrectomy results in dramatic weight reduction and improvement in the features of the metabolic syndrome. A survival benefit with gastric bypass has also been realized. LDL CHOLESTEROL (SEE ALSO CHAP. 421)

1	LDL CHOLESTEROL (SEE ALSO CHAP. 421) The rationale for the NCEP:ATPIII’s development of criteria for the metabolic syndrome was to go beyond LDL cholesterol in identifying and reducing the risk of CVD. The working assumption by the panel was that LDL cholesterol goals had already been achieved and that increasing evidence supports a linear reduction in CVD events as a result of progressive lowering of LDL cholesterol with statins. For patients with the metabolic syndrome and diabetes, a statin should be prescribed. For those patients with diabetes and known CVD, the current evidence supports a maximum of penultimate dose of a potent statin (e.g., atorvastatin or rosuvastatin). For those patients with the metabolic syndrome but without diabetes, a score that predicts a 10-year CVD risk exceeding 7.5% should also take a statin. With a 10-year risk of <7.5%, use of statin therapy is not evidence based.

1	Diets restricted in saturated fats (<7% of calories) and trans-fats (as few as possible) should be applied aggressively. Although less evidence exists, dietary cholesterol should also be restricted. If LDL cholesterol remains elevated, pharmacologic intervention is needed. Treatment with statins, which lower LDL cholesterol by 15–60%, is evidence based and is the first-choice medication intervention. Of note, for each doubling of the statin dose, LDL cholesterol is further lowered by only ~6%. Hepatotoxicity (more than a threefold increase in hepatic aminotransferases) is rare, and myopathy is seen in ~10% of patients. The cholesterol absorption inhibitor ezetimibe is well tolerated and should be the second- choice medication intervention. Ezetimibe typically reduces LDL cholesterol by 15–20%. The bile acid sequestrants cholestyramine, colestipol, and colesevalam may be more effective than ezetimibe but, because they can increase triglyceride levels, must be used with caution in

1	by 15–20%. The bile acid sequestrants cholestyramine, colestipol, and colesevalam may be more effective than ezetimibe but, because they can increase triglyceride levels, must be used with caution in patients with the metabolic syndrome. In general, bile sequestrants should not be administered when fasting triglyceride levels are >250 mg/dL. Side effects include gastrointestinal symptoms (palatability, bloating, belching, constipation, anal irritation). Nicotinic acid has modest LDL cholesterol–lowering capabilities (<20%). Fibrates are best employed to lower LDL cholesterol when both LDL cholesterol and triglycerides are elevated. Fenofibrate may be more effective than gemfibrozil in this setting.

1	TRIGLYCERIDES (SEE ALSO CHAP. 421) The NCEP:ATPIII has focused on non-HDL cholesterol rather than on triglycerides. However, a fasting triglyceride value of <150 mg/ dL is recommended. In general, the response of fasting triglycerides relates to the amount of weight reduction achieved: a weight reduction of >10% is necessary to lower fasting triglyceride levels.

1	A fibrate (gemfibrozil or fenofibrate) is the drug of choice to lower fasting triglyceride levels, which are typically reduced by 30–45%. Concomitant administration with drugs metabolized by the 3A4 cytochrome P450 system (including some statins) increases the risk of myopathy. In these cases, fenofibrate may be preferable to gemfibrozil. In the Veterans Affairs HDL Intervention Trial, gemfibrozil was administered to men with known CHD and levels of HDL cholesterol <40 mg/dL. A coronary disease event and mortality rate benefit was experienced predominantly among men with hyperinsulinemia and/or diabetes, many of whom were identified retrospectively as having the metabolic syndrome. Of note, the degree of triglyceride lowering in this trial did not predict benefit. Although levels of LDL cholesterol did not change, a decrease in LDL particle number correlated with benefit. Several additional clinical trials have not shown The Metabolic Syndrome

1	Endocrinology and Metabolism Health and Disease F. Richard Bringhurst, Marie B. Demay, Stephen M. Krane, Henry M. Kronenberg BONE STRUCTURE AND METABOLISM 423 SEC Tion 4 2454 clear evidence that fibrates reduce CVD risk; however, post hoc analyses of several studies demonstrated that patients with baseline triglyceride levels >200 mg/dL and HDL cholesterol levels <35 mg/ dL did benefit. Other drugs that lower triglyceride levels include statins, nicotinic acid, and—in high doses—omega-3 fatty acids. For this purpose, an intermediate or high dose of the “more potent” statins (atorvastatin, rosuvastatin) is needed. The effect of nicotinic acid on fasting triglycerides is dose related and ~20–35%, an effect that is less pronounced than that of fibrates. In patients with the metabolic syndrome and diabetes, nicotinic acid may increase fasting glucose levels. Omega-3 fatty acid preparations that include high doses of docosahexaenoic acid plus eicosapentaenoic acid (~1.5–4.5 g/d) or

1	syndrome and diabetes, nicotinic acid may increase fasting glucose levels. Omega-3 fatty acid preparations that include high doses of docosahexaenoic acid plus eicosapentaenoic acid (~1.5–4.5 g/d) or eicosapentaenoic acid alone lower fasting triglyceride levels by ~30–40%. No drug interactions with fibrates or statins occur, and the main side effect of their use is eructation with a fishy taste. This taste can be partially blocked by ingestion of the nutraceutical after freezing. Clinical trials of nicotinic acid or high-dose omega-3 fatty acids in patients with the metabolic syndrome have not been reported.

1	HDL CHOLESTEROL (SEE ALSO CHAP. 421) Very few lipid-modifying compounds increase HDL cholesterol levels. Statins, fibrates, and bile acid sequestrants have modest effects (5–10%), whereas ezetimibe and omega-3 fatty acids have no effect. Nicotinic acid is the only currently available drug with predictable HDL cholesterol-raising properties. The response is dose related, and nicotinic acid can increase HDL cholesterol by ~30% above baseline. After several trials of nicotinic acid versus placebo in statin-treated patients, there is still no evidence that raising HDL with nicotinic acid beneficially affects CVD events in patients with or without the metabolic syndrome. BLOOD PRESSURE (SEE ALSO CHAP. 298) The direct relationship between blood pressure and all-cause mortality rate has been well established in studies comparing patients

1	BLOOD PRESSURE (SEE ALSO CHAP. 298) The direct relationship between blood pressure and all-cause mortality rate has been well established in studies comparing patients Bone is a dynamic tissue that is remodeled constantly throughout life. The arrangement of compact and cancellous bone provides strength and density suitable for both mobility and protection. In addition, bone provides a reservoir for calcium, magnesium, phosphorus, sodium, and other ions necessary for homeostatic functions. Bone also hosts and regulates hematopoiesis by providing niches for hematopoietic cell proliferation and differentiation. The skeleton is highly vascular and receives about 10% of the cardiac output. Remodeling of bone is accomplished by two distinct cell types: osteoblasts produce bone matrix, and osteoclasts resorb the matrix.

1	The extracellular components of bone consist of a solid mineral phase in close association with an organic matrix, of which 90–95% is type I collagen (Chap. 427). The noncollagenous portion of the organic matrix is heterogeneous and contains serum proteins such as albumin as well as many locally produced proteins, whose functions are incompletely understood. Those proteins include cell attachment/ with hypertension (>140/90 mmHg), patients with pre-hypertension (>120/80 mmHg but <140/90 mmHg), and individuals with normal blood pressure (<120/80 mmHg). In patients who have the metabolic syndrome without diabetes, the best choice for the initial antihypertensive medication is an angiotensin-converting enzyme (ACE) inhibitor or an angiotensin II receptor blocker, as these two classes of drugs appear to reduce the incidence of new-onset type 2 diabetes. In all patients with hypertension, a sodium-restricted dietary pattern enriched in fruits and vegetables, whole grains, and low-fat dairy

1	appear to reduce the incidence of new-onset type 2 diabetes. In all patients with hypertension, a sodium-restricted dietary pattern enriched in fruits and vegetables, whole grains, and low-fat dairy products should be advocated. Home monitoring of blood pressure may assist in maintaining good blood-pressure control.

1	IMPAIRED FASTING GLUCOSE (SEE ALSO CHAP. 417) In patients with the metabolic syndrome and type 2 diabetes, aggressive glycemic control may favorably modify fasting levels of triglycerides and/or HDL cholesterol. In patients with impaired fasting glucose who do not have diabetes, a lifestyle intervention that includes weight reduction, dietary fat restriction, and increased physical activity has been shown to reduce the incidence of type 2 diabetes. Metformin also reduces the incidence of diabetes, although the effect is less pronounced than that of lifestyle intervention. INSULIN RESISTANCE (SEE ALSO CHAP. 418)

1	INSULIN RESISTANCE (SEE ALSO CHAP. 418) Several drug classes (biguanides, thiazolidinediones [TZDs]) increase insulin sensitivity. Because insulin resistance is the primary pathophysiologic mechanism for the metabolic syndrome, representative drugs in these classes reduce its prevalence. Both metformin and TZDs enhance insulin action in the liver and suppress endogenous glucose production. TZDs, but not metformin, also improve insulin-mediated glucose uptake in muscle and adipose tissue. Benefits of both drugs have been seen in patients with nonalcoholic fatty liver disease and polycystic ovary syndrome, and the drugs have been shown to reduce markers of inflammation. signaling proteins such as thrombospondin, osteopontin, and fibronectin; calcium-binding proteins such as matrix gla protein and osteocalcin; and proteoglycans such as biglycan and decorin. Some of the proteins organize collagen fibrils; others influence mineralization and binding of the mineral phase to the matrix.

1	The mineral phase is made up of calcium and phosphate and is best characterized as a poorly crystalline hydroxyapatite. The mineral phase of bone is deposited initially in intimate relation to the collagen fibrils and is found in specific locations in the “holes” between the collagen fibrils. This architectural arrangement of mineral and matrix results in a two-phase material well suited to withstand mechanical stresses. The organization of collagen influences the amount and type of mineral phase formed in bone. Although the primary structures of type I collagen in skin and bone tissues are similar, there are differences in posttranslational modifications and distribution of intermolecular cross-links. The holes in the packing structure of the collagen are larger in mineralized collagen of bone and dentin than in unmineralized collagens such as those in tendon. Single amino acid substitutions in the helical portion of either the α1 (COL1A1) or α2 (COL1A2) chains of type I collagen

1	of bone and dentin than in unmineralized collagens such as those in tendon. Single amino acid substitutions in the helical portion of either the α1 (COL1A1) or α2 (COL1A2) chains of type I collagen disrupt the organization of bone in osteogenesis imperfecta. The severe skeletal fragility associated with this group of disorders highlights the importance of the fibrillar matrix in the structure of bone (Chap. 427).

1	Osteoblasts synthesize and secrete the organic matrix and regulate its mineralization. They are derived from cells of mesenchymal origin (Fig. 423-1A). Active osteoblasts are found on the surface of newly BMPs PTH, Vit D, IGFs, 2455 BMPs, Wnts Mesenchymal osteoblast progenitor Osteoblast precursor Collagen (I) Alkaline phosphatase Osteocalcin, osteopontin Bone sialoprotein c-src+ ˜3 integrin+ PYK2 kinase+ Cathepsin K+ TRAF+ Carbonic anhydrase II+ Runx 2 Hematopoietic osteoclast progenitor M-CSF Osteoclast precursor Active osteoblast Mononuclear osteoclast Quiescent osteoclast Active osteoclast PU-1+ c-fos+ NK°B+ TRAF+ CommitmentRANK Ligand DifferentiationM-CSF RANK Ligand IL-1,IL-6 FusionRANK Ligand IL-1 A B

1	FIGURE 423-1 Pathways regulating development of (A) osteoblasts and (B) osteoclasts. Hormones, cytokines, and growth factors that control cell proliferation and differentiation are shown above the arrows. Transcription factors and other markers specific for various stages of development are depicted below the arrows. BMPs, bone morphogenic proteins; IGFs, insulin-like growth factors; IL-1, interleukin 1; IL-6, interleukin 6; M-CSF, macrophage colony-stimulating factor; NFκB, nuclear factor κB; PTH, parathyroid hormone; PU-1, a monocyteand B lymphocyte–specific ets family transcription factor; RANK ligand, receptor activator of NFκB ligand; Runx2, Runt-related transcription factor 2; TRAF, tumor necrosis factor receptor–associated factors; Vit D, vitamin D; wnts, wingless-type mouse mammary tumor virus integration site.

1	(Modified from T Suda et al: Endocr Rev 20:345, 1999, with permission.) forming bone. As an osteoblast secretes matrix, which then is mineralized, the cell becomes an osteocyte, still connected with its blood supply through a series of canaliculi. Osteocytes account for the vast majority of the cells in bone. They are thought to be the mechanosensors in bone that communicate signals to surface osteoblasts and their progenitors through the canalicular network and thereby serve as master regulators of bone formation and resorption. Remarkably, osteocytes also secrete fibroblast growth factor 23 (FGF23), a major regulator of phosphate metabolism (see below). Mineralization of the matrix, both in trabecular bone and in osteones of compact cortical bone (Haversian systems), begins soon after the matrix is secreted (primary mineralization) but is not completed for several weeks or even longer (secondary mineralization). Although this mineralization takes advantage of the high concentrations

1	matrix is secreted (primary mineralization) but is not completed for several weeks or even longer (secondary mineralization). Although this mineralization takes advantage of the high concentrations of calcium and phosphate, already near saturation in serum, mineralization is a carefully regulated process that is dependent on the activity of osteoblast-derived alkaline phosphatase, which probably works by hydrolyzing inhibitors of mineralization.

1	Genetic studies in humans and mice have identified several key genes that control osteoblast development. Runx2 is a transcription factor expressed specifically in chondrocyte (cartilage cells) and osteoblast progenitors as well as in hypertrophic chondrocytes and mature osteoblasts. Runx2 regulates the expression of several important osteoblast proteins, including osterix (another transcription factor needed for osteoblast maturation), osteopontin, bone sialoprotein, type I collagen, osteocalcin, and receptor-activator of NFκB (RANK) ligand. Runx2 expression is regulated in part by bone morphogenic proteins (BMPs). Runx2-deficient mice are devoid of osteoblasts, whereas mice with a deletion of only one allele (Runx2 +/−) exhibit a delay in formation of the clavicles and some cranial bones. The latter abnormalities are similar to those in the human disorder cleidocranial dysplasia, which is also caused by heterozygous inactivating mutations in Runx2.

1	The paracrine signaling molecule, Indian hedgehog (Ihh), also plays a critical role in osteoblast development, as evidenced by Ihh-deficient mice that lack osteoblasts in the type of bone formed on a cartilage mold (endochondral ossification). Signals originating from members of the wnt (wingless-type mouse mammary tumor virus integration site) family of paracrine factors are also important for osteoblast proliferation and differentiation. Numerous other growth-regulatory factors affect osteoblast function, including the three closely related transforming growth factor βs, fibroblast growth factors (FGFs) 2 and 18, platelet-derived growth factor, and insulin-like growth factors (IGFs) I and II. Hormones such as parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,25[OH]2D) activate receptors expressed by osteoblasts to assure mineral homeostasis and influence a variety of bone cell functions.

1	Resorption of bone is carried out mainly by osteoclasts, multinucleated cells that are formed by fusion of cells derived from the common precursor of macrophages and osteoclasts. Thus, these cells derive from the hematopoietic lineage, quite different from the mesenchymal cells that become osteoblasts. Multiple factors that regulate osteoclast development have been identified (Fig. 423-1B). Factors produced by osteoblasts or marrow stromal cells allow osteoblasts to control osteoclast development and activity. Macrophage colony-stimulating factor (M-CSF) plays a critical role during several steps in the pathway and ultimately leads to fusion of osteoclast progenitor cells to form multinucleated, active osteoclasts. RANK ligand, a member of the tumor necrosis factor (TNF) family, is expressed on the surface of osteoblast progenitors and stromal fibroblasts. In a process involving cell-cell interactions, RANK ligand binds to the RANK receptor on osteoclast progenitors, stimulating

1	on the surface of osteoblast progenitors and stromal fibroblasts. In a process involving cell-cell interactions, RANK ligand binds to the RANK receptor on osteoclast progenitors, stimulating osteoclast differentiation and activation. Alternatively, a soluble decoy receptor, referred to as osteoprotegerin, can bind RANK ligand and inhibit osteoclast ~3 weeks ~3 months Osteoid Osteocyte Osteoclast Active osteoclast precursors Bone remodeling unit Osteoblast Lining cells Activation Resorption Bone formation Mineralization Cement line Reversal Resting bone surface FIGURE 423-2 Schematic representation of bone remodeling. The cycle of bone remodeling 2456 differentiation. Several growth factors and Osteoclast cytokines (including interleukins 1, 6, precursor Osteoblast and 11; TNF; and interferon γ) modulate osteoclast differentiation and function. Most hormones that influence osteoclast function do not target these cells directly but instead act on cells of the osteoblast lineage to

1	γ) modulate osteoclast differentiation and function. Most hormones that influence osteoclast function do not target these cells directly but instead act on cells of the osteoblast lineage to increase production of M-CSF and RANK. Both PTH and 1,25(OH)2D increase osteoclast number and activity by this indirect mechanism. Calcitonin, in contrast, binds to its receptor on the basal surface of osteoclasts and directly inhibits osteoclast function. Estradiol has is carried out by the basic multicellular unit (BMU), which consists of a group of osteoclasts and multiple cellular targets in bone, includ-osteoblasts. In cortical bone, the BMUs tunnel through the tissue, whereas in cancellous bone, ing osteoclasts, immune cells, and osteo-they move across the trabecular surface. The process of bone remodeling is initiated by contracblasts; actions on all these cells serve to tion of the lining cells and the recruitment of osteoclast precursors. These precursors fuse to form decrease osteoclast

1	remodeling is initiated by contracblasts; actions on all these cells serve to tion of the lining cells and the recruitment of osteoclast precursors. These precursors fuse to form decrease osteoclast number and decrease multinucleated, active osteoclasts that mediate bone resorption. Osteoclasts adhere to bone and bone resorption. subsequently remove it by acidification and proteolytic digestion. As the BMU advances, osteo

1	Osteoclast-mediated resorption of clasts leave the resorption site and osteoblasts move in to cover the excavated area and begin bone takes place in scalloped spaces the process of new bone formation by secreting osteoid, which eventually is mineralized into new (Howship’s lacunae) where the osteoclasts bone. After osteoid mineralization, osteoblasts flatten and form a layer of lining cells over new are attached through a specific αvβ3 inte-bone. grin to components of the bone matrix such as osteopontin. The osteoclast forms a tight seal to the underlying matrix and secretes protons, chloride, and The response of bone to fractures, infection, and interruption of proteinases into a confined space that has been likened to an extracel-blood supply and to expanding lesions is relatively limited. Dead bone lular lysosome. The active osteoclast surface forms a ruffled border must be resorbed, and new bone must be formed, a process carried that contains a specialized proton pump ATPase that

1	Dead bone lular lysosome. The active osteoclast surface forms a ruffled border must be resorbed, and new bone must be formed, a process carried that contains a specialized proton pump ATPase that secretes acid and out in association with growth of new blood vessels into the involved solubilizes the mineral phase. Carbonic anhydrase (type II isoenzyme) area. In injuries that disrupt the organization of the tissue such as a within the osteoclast generates the needed protons. The bone matrix fracture in which apposition of fragments is poor or when motion is resorbed in the acid environment adjacent to the ruffled border by exists at the fracture site, progenitor stromal cells recapitulate the proteases, such as cathepsin K, that act at low pH. endochondral bone formation of early development and form carti-

1	In the embryo and the growing child, bone develops mostly by lage that is replaced by bone and, variably, fibrous tissue. When there remodeling and replacing previously calcified cartilage (endochon-is good apposition with fixation and little motion at the fracture site, dral bone formation) or, in a few bones, is formed without a cartilage repair occurs predominantly by formation of new bone without other matrix (intramembranous bone formation). During endochondral mediating tissue. bone formation, chondrocytes proliferate, secrete and mineralize a Remodeling of bone occurs along lines of force generated by matrix, enlarge (hypertrophy), and then die, enlarging bone and pro-mechanical stress. The signals from these mechanical stresses are viding the matrix and factors that stimulate endochondral bone for-sensed by osteocytes, which transmit signals to osteoclasts and osteomation. This program is regulated by both local factors, such as IGF-I blasts or their precursors. One such

1	endochondral bone for-sensed by osteocytes, which transmit signals to osteoclasts and osteomation. This program is regulated by both local factors, such as IGF-I blasts or their precursors. One such signal made by osteocytes is and -II, Ihh, PTH-related peptide (PTHrP), and FGFs, and by systemic sclerostin, an inhibitor of wnt signaling. Mechanical forces suppress hormones, such as growth hormone, glucocorticoids, and estrogen. sclerostin production and thus increase bone formation by osteo-

1	New bone, whether formed in infants or in adults during repair, blasts. Expanding lesions in bone such as tumors induce resorption has a relatively high ratio of cells to matrix and is characterized by at the surface in contact with the tumor by producing ligands such as coarse fiber bundles of collagen that are interlaced and randomly PTHrP that stimulate osteoclast differentiation and function. Even in dispersed (woven bone). In adults, the more mature bone is orga-a disorder as architecturally disruptive as Paget’s disease, remodeling is nized with fiber bundles regularly arranged in parallel or concentric dictated by mechanical forces. Thus, bone plasticity reflects the inter-sheets (lamellar bone). In long bones, deposition of lamellar bone in action of cells with each other and with the environment. a concentric arrangement around blood vessels forms the Haversian Measurement of the products of osteoblast and osteoclast activsystems. Growth in length of bones is dependent on

1	the environment. a concentric arrangement around blood vessels forms the Haversian Measurement of the products of osteoblast and osteoclast activsystems. Growth in length of bones is dependent on proliferation of ity can assist in the diagnosis and management of bone diseases.

1	cartilage cells and the endochondral sequence at the growth plate. Osteoblast activity can be assessed by measuring serum bone-specific Growth in width and thickness is accomplished by formation of alkaline phosphatase. Similarly, osteocalcin, a protein secreted from bone at the periosteal surface and by resorption at the endosteal osteoblasts, is made virtually only by osteoblasts. Osteoclast activity surface, with the rate of formation exceeding that of resorption. In can be assessed by measurement of products of collagen degradation. adults, after the growth plates of cartilage close, growth in length and Collagen molecules are covalently linked to each other in the extracelendochondral bone formation cease except for some activity in the lular matrix through the formation of hydroxypyridinium cross-links cartilage cells beneath the articular surface. Even in adults, however, (Chap. 427). After digestion by osteoclasts, these cross-linked peptides remodeling of bone (within

1	cross-links cartilage cells beneath the articular surface. Even in adults, however, (Chap. 427). After digestion by osteoclasts, these cross-linked peptides remodeling of bone (within Haversian systems as well as along the can be measured both in urine and in blood. surfaces of trabecular bone) continues throughout life. In adults, ~4% of the surface of trabecular bone (such as iliac crest) is involved in active resorption, whereas 10–15% of trabecular surfaces are covered with osteoid, unmineralized new bone formed by osteoblasts. Over 99% of the 1–2 kg of calcium present normally in the adult Radioisotope studies indicate that as much as 18% of the total skeletal human body resides in the skeleton, where it provides mechanical stacalcium is deposited and removed each year. Thus, bone is an active bility and serves as a reservoir sometimes needed to maintain extracelmetabolizing tissue that requires an intact blood supply. The cycle of lular fluid (ECF) calcium concentration (Fig.

1	is an active bility and serves as a reservoir sometimes needed to maintain extracelmetabolizing tissue that requires an intact blood supply. The cycle of lular fluid (ECF) calcium concentration (Fig. 423-3). Skeletal calcium bone resorption and formation is a highly orchestrated process carried accretion first becomes significant during the third trimester of fetalout by the basic multicellular unit, which is composed of a group of life, accelerates throughout childhood and adolescence, reaches a peak osteoclasts and osteoblasts (Fig. 423-2). in early adulthood, and gradually declines thereafter at rates that rarely 0.4–1.5 g 0.25–0.5 g 0.25–0.5 g 0.1–0.2 g 0.25–0.5 g 7.9–9.7 g 0.3–1 g 0.15–.3 g

1	FIGURE 423-3 Calcium homeostasis. Schematic illustration of calcium content of extracellular fluid (ECF) and bone as well as of diet and feces; magnitude of calcium flux per day as calculated by various methods is shown at sites of transport in intestine, kidney, and bone. Ranges of values shown are approximate and were chosen to illustrate certain points discussed in the text. In conditions of calcium balance, rates of calcium release from and uptake into bone are equal. exceed 1–2% per year. These slow changes in total skeletal calcium content contrast with relatively high daily rates of closely matched fluxes of calcium into and out of bone (~250–500 mg each), a process mediated by coupled osteoblastic and osteoclastic activity. Another 0.5–1% of skeletal calcium is freely exchangeable (e.g., in chemical equilibrium) with that in the ECF.

1	The concentration of ionized calcium in the ECF must be maintained within a narrow range because of the critical role calcium plays in a wide array of cellular functions, especially those involved in neuromuscular activity, secretion, and signal transduction. Intracellular cytosolic free calcium levels are ~100 nmol/L and are 10,000-fold lower than ionized calcium concentrations in the blood and ECF (1.1–1.3 mmol/L). Cytosolic calcium does not play the structural role played by extracellular calcium; instead, it serves a signaling function. The steep chemical gradient of calcium from outside to inside the cell promotes rapid calcium influx through various membrane calcium channels that can be activated by hormones, metabolites, or neurotransmitters, swiftly changing cellular function. In blood, total calcium concentration is normally 2.2–2.6 mM (8.5–10.5 mg/dL), of which ~50% is ionized. The remainder is bound ionically to negatively charged proteins (predominantly albumin and

1	In blood, total calcium concentration is normally 2.2–2.6 mM (8.5–10.5 mg/dL), of which ~50% is ionized. The remainder is bound ionically to negatively charged proteins (predominantly albumin and immunoglobulins) or loosely complexed with phosphate, citrate, sulfate, or other anions. Alterations in serum protein concentrations directly affect the total blood calcium concentration even if the ionized calcium concentration remains normal. An algorithm to correct for protein changes adjusts the total serum calcium (in mg/dL) upward by 0.8 times the deficit in serum albumin (g/dL) or by 0.5 times the deficit in serum immunoglobulin (in g/dL). Such corrections provide only rough approximations of actual free calcium concentrations, however, and may be misleading, particularly during acute illness. Acidosis also alters ionized calcium by reducing its association with proteins. The best practice is to measure blood ionized calcium directly by a method that employs calcium-selective

1	illness. Acidosis also alters ionized calcium by reducing its association with proteins. The best practice is to measure blood ionized calcium directly by a method that employs calcium-selective electrodes in acute settings during which calcium abnormalities might occur.

1	Control of the ionized calcium concentration in the ECF ordinarily is accomplished by adjusting the rates of calcium movement across intestinal and renal epithelia. These adjustments are mediated mainly via changes in blood levels of the hormones, PTH and 1,25(OH)2D. Blood ionized calcium directly suppresses PTH secretion by activating calcium-sensing receptors (CaSRs) in parathyroid cells. Also, ionized 2457 calcium indirectly affects PTH secretion by lowering 1,25(OH)2D production. This active vitamin D metabolite inhibits PTH production by an incompletely understood mechanism of negative feedback (Chap. 424).

1	Normal dietary calcium intake in the United States varies widely, ranging from 10–37 mmol/d (400–1500 mg/d). An Institute of Medicine report recommends a daily allowance of 25–30 mmol (1000–1200 mg) for most adults. Intestinal absorption of ingested calcium involves both active (transcellular) and passive (paracellular) mechanisms. Passive calcium absorption is nonsaturable and approximates 5% of daily calcium intake, whereas active absorption involves apical calcium entry via specific ion channels (TRPV5 and TRPV6), whose expression is controlled principally by 1,25(OH)2D, and normally ranges from 20 to 70%. Active calcium transport occurs mainly in the proximal small bowel (duodenum and proximal jejunum), although some active calcium absorption occurs in most segments of the small intestine. Optimal rates of calcium absorption require gastric acid. This is especially true for weakly dissociable calcium supplements such as calcium carbonate. In fact, large boluses of calcium

1	intestine. Optimal rates of calcium absorption require gastric acid. This is especially true for weakly dissociable calcium supplements such as calcium carbonate. In fact, large boluses of calcium carbonate are poorly absorbed because of their neutralizing effect on gastric acid. In achlorhydric subjects and for those taking drugs that inhibit gastric acid secretion, supplements should be taken with meals to optimize their absorption. Use of calcium citrate may be preferable in these circumstances. Calcium absorption may also be blunted in disease states such as pancreatic or biliary insufficiency, in which ingested calcium remains bound to unabsorbed fatty acids or other food constituents. At high levels of calcium intake, synthesis of 1,25(OH)2D is reduced; this decreases the rate of active intestinal calcium absorption. The opposite occurs with dietary calcium restriction. Some calcium, ~2.5–5 mmol/d (100–200 mg/d), is excreted as an obligate component of intestinal secretions and

1	intestinal calcium absorption. The opposite occurs with dietary calcium restriction. Some calcium, ~2.5–5 mmol/d (100–200 mg/d), is excreted as an obligate component of intestinal secretions and is not regulated by calciotropic hormones.

1	The feedback-controlled hormonal regulation of intestinal absorptive efficiency results in a relatively constant daily net calcium absorption of ~5–7.5 mmol/d (200–400 mg/d) despite large changes in daily dietary calcium intake. This daily load of absorbed calcium is excreted by the kidneys in a manner that is also tightly regulated by the concentration of ionized calcium in the blood. Approximately 8–10 g/d of calcium is filtered by the glomeruli, of which only 2–3% appears in the urine. Most filtered calcium (65%) is reabsorbed in the proximal tubules via a passive, paracellular route that is coupled to concomitant NaCl reabsorption and not specifically regulated. The cortical thick ascending limb of Henle’s loop (cTAL) reabsorbs roughly another 20% of filtered calcium, also via a paracellular mechanism. Calcium reabsorption in the cTAL requires a tight-junctional protein called paracellin-1 and is inhibited by increased blood concentrations of calcium or magnesium, acting via the

1	mechanism. Calcium reabsorption in the cTAL requires a tight-junctional protein called paracellin-1 and is inhibited by increased blood concentrations of calcium or magnesium, acting via the CaSR, which is highly expressed on basolateral membranes in this nephron segment. Operation of the renal CaSR provides a mechanism, independent of those engaged directly by PTH or 1,25(OH)2D, by which serum ionized calcium can control renal calcium reabsorption. Finally, ~10% of filtered calcium is reabsorbed in the distal convoluted tubules (DCTs) by a transcellular mechanism. Calcium enters the luminal surface of the cell through specific apical calcium channels (TRPV5), whose number is regulated. It then moves across the cell in association with a specific calcium-binding protein (calbindin-D28k) that buffers cytosolic calcium concentrations from the large mass of transported calcium. Ca2+-ATPases and Na+/Ca2+ exchangers actively extrude calcium across the basolateral surface and thereby

1	that buffers cytosolic calcium concentrations from the large mass of transported calcium. Ca2+-ATPases and Na+/Ca2+ exchangers actively extrude calcium across the basolateral surface and thereby maintain the transcellular calcium gradient. All these processes are stimulated directly or indirectly by PTH. The DCT is also the site of action of thiazide diuretics, which lower urinary calcium excretion by inducing sodium depletion and thereby augmenting proximal calcium reabsorption. Conversely, dietary sodium loads, or increased distal sodium delivery caused by loop diuretics or saline infusion, induce calciuresis.

1	The homeostatic mechanisms that normally maintain a constant serum ionized calcium concentration may fail at extremes of calcium intake or when the hormonal systems or organs involved are compromised. Thus, even with maximal activity of the vitamin D–dependent 2458 intestinal active transport system, sustained calcium intakes <5 mmol/d (<200 mg/d) cannot provide enough net calcium absorption to replace obligate losses via the intestine, the kidney, sweat, and other secretions. In this case, increased blood levels of PTH and 1,25(OH)2D activate osteoclastic bone resorption to obtain needed calcium from bone, which leads to progressive bone loss and negative calcium balance. Increased PTH and 1,25(OH)2D also enhance renal calcium reabsorption, and 1,25(OH)2D enhances calcium absorption in the gut. At very high calcium intakes (>100 mmol/d [>4 g/d]), passive intestinal absorption continues to deliver calcium into the ECF despite maximally downregulated intestinal active transport and

1	the gut. At very high calcium intakes (>100 mmol/d [>4 g/d]), passive intestinal absorption continues to deliver calcium into the ECF despite maximally downregulated intestinal active transport and renal tubular calcium reabsorption. This can cause severe hypercalciuria, nephrocalcinosis, progressive renal failure, and hypercalcemia (e.g., “milk-alkali syndrome”). Deficiency or excess of PTH or vitamin D, intestinal disease, and renal failure represent other commonly encountered challenges to normal calcium homeostasis (Chap. 424).

1	Although 85% of the ~600 g of body phosphorus is present in bone mineral, phosphorus is also a major intracellular constituent both as the free anion(s) and as a component of numerous organophosphate compounds, including structural proteins, enzymes, transcription factors, carbohydrate and lipid intermediates, high-energy stores (adenosine triphosphate [ATP], creatine phosphate), and nucleic acids. Unlike calcium, phosphorus exists intracellularly at concentrations close to those present in ECF (e.g., 1–2 mmol/L). In cells and in the ECF, phosphorus exists in several forms, predominantly as H2PO4− or NaHPO4−, with perhaps 10% as HPO42−. This mixture of anions will be referred to here as “phosphate.” In serum, about 12% of phosphorus is bound to proteins. Concentrations of phosphates in blood and ECF generally are expressed in terms of elemental phosphorus, with the normal range in adults being 0.75–1.45 mmol/L (2.5–4.5 mg/dL). Because the volume of the intracellular fluid compartment

1	and ECF generally are expressed in terms of elemental phosphorus, with the normal range in adults being 0.75–1.45 mmol/L (2.5–4.5 mg/dL). Because the volume of the intracellular fluid compartment is twice that of the ECF, measurements of ECF phosphate may not accurately reflect phosphate availability within cells that follows even modest shifts of phosphate from one compartment to the other.

1	Phosphate is widely available in foods and is absorbed efficiently (65%) by the small intestine even in the absence of vitamin D. However, phosphate absorptive efficiency may be enhanced (to 85–90%) via active transport mechanisms that are stimulated by 1,25(OH)2D. These mechanisms involve activation of Na+/PO42− co-transporters that move phosphate into intestinal cells against an unfavorable electrochemical gradient. Daily net intestinal phosphate absorption varies widely with the composition of the diet but is generally in the range of 500–1000 mg/d. Phosphate absorption can be inhibited by large doses of calcium salts or by sevelamer hydrochloride (Renagel), strategies commonly used to control levels of serum phosphate in renal failure. Aluminum hydroxide antacids also reduce phosphate absorption but are used less commonly because of the potential for aluminum toxicity. Low serum phosphate stimulates renal proximal tubular synthesis of 1,25(OH)2D, perhaps by suppressing blood

1	absorption but are used less commonly because of the potential for aluminum toxicity. Low serum phosphate stimulates renal proximal tubular synthesis of 1,25(OH)2D, perhaps by suppressing blood levels of FGF23 (see below).

1	Serum phosphate levels vary by as much as 50% on a normal day. This reflects the effect of food intake but also an underlying circadian rhythm that produces a nadir between 7:00 and 10:00 a.m. Carbohydrate administration, especially as IV dextrose solutions in fasting subjects, can decrease serum phosphate by >0.7 mmol/L (2 mg/ dL) due to rapid uptake into and utilization by cells. A similar response is observed in the treatment of diabetic ketoacidosis and during metabolic or respiratory alkalosis. Because of this wide variation in serum phosphate, it is best to perform measurements in the basal, fasting state.

1	Control of serum phosphate is determined mainly by the rate of renal tubular reabsorption of the filtered load, which is ~4–6 g/d. Because intestinal phosphate absorption is highly efficient, urinary excretion is not constant but varies directly with dietary intake. The fractional excretion of phosphate (ratio of phosphate to creatinine clearance) is generally in the range of 10–15%. The proximal tubule is the principal site at which renal phosphate reabsorption is regulated. This is accomplished by changes in the levels of apical expression and activity of specific Na+/PO42− co-transporters (NaPi-2a and NaPi-2c) in the proximal tubule. Levels of these transporters at the apical surface of these cells are reduced rapidly by PTH, a major hormonal regulator of renal phosphate excretion. FGF23 can impair phosphate reabsorption dramatically by a similar mechanism. Activating FGF23 mutations cause the rare disorder autosomal dominant hypophosphatemic rickets. In contrast to PTH, FGF23 also

1	can impair phosphate reabsorption dramatically by a similar mechanism. Activating FGF23 mutations cause the rare disorder autosomal dominant hypophosphatemic rickets. In contrast to PTH, FGF23 also leads to reduced synthesis of 1,25(OH)2D, which may worsen the resulting hypophosphatemia by lowering intestinal phosphate absorption. Renal reabsorption of phosphate is responsive to changes in dietary intake such that experimental dietary phosphate restriction leads to a dramatic lowering of urinary phosphate within hours, preceding any decline in serum phosphate (e.g., filtered load). This physiologic renal adaptation to changes in dietary phosphate availability occurs independently of PTH and may be mediated in part by changes in levels of serum FGF23. Findings in FGF23-knockout mice suggest that FGF23 normally acts to lower blood phosphate and 1,25(OH)2D levels. In turn, elevation of blood phosphate increases blood levels of FGF23.

1	Renal phosphate reabsorption is impaired by hypocalcemia, hypomagnesemia, and severe hypophosphatemia. Phosphate clearance is enhanced by ECF volume expansion and impaired by dehydration. Phosphate retention is an important pathophysiologic feature of renal insufficiency (Chap. 335). HYPOPHOSPHATEMIA Causes Hypophosphatemia can occur by one or more of three primary mechanisms: (1) inadequate intestinal phosphate absorption, (2) excessive renal phosphate excretion, and (3) rapid redistribution of phosphate from the ECF into bone or soft tissue (Table 423-1). Because phosphate is so abundant in foods, inadequate intestinal absorption is almost never observed now that aluminum hydroxide antacids, which bind phosphate in the gut, are no longer widely used. Fasting or starvation, however, may result in depletion of body phosphate and predispose to subsequent hypophosphatemia during refeeding, especially if this is accomplished with IV glucose alone.

1	Chronic hypophosphatemia usually signifies a persistent renal tubular phosphate-wasting disorder. Excessive activation of PTH/ PTHrP receptors in the proximal tubule as a result of primary or secondary hyperparathyroidism or because of the PTHrP-mediated hypercalcemia syndrome in malignancy (Chap. 424) is among the more common causes of renal hypophosphatemia, especially because of the high prevalence of vitamin D deficiency in older Americans. Familial hypocalciuric hypercalcemia and Jansen’s chondrodystrophy are rare examples of genetic disorders in this category (Chap. 424).

1	Several genetic and acquired diseases cause PTH/PTHrPindependent tubular phosphate wasting with associated rickets and osteomalacia. All these diseases manifest severe hypophosphatemia; renal phosphate wasting, sometimes accompanied by aminoaciduria; inappropriately low blood levels of 1,25(OH)2D; low-normal serum levels of calcium; and evidence of impaired cartilage or bone mineralization. Analysis of these diseases led to the discovery of the hormone FGF23, which is an important physiologic regulator of phosphate metabolism. FGF23 decreases phosphate reabsorption in the proximal tubule and also suppresses the 1α-hydroxylase responsible for synthesis of 1,25(OH)2D. FGF23 is synthesized by cells of the osteoblast lineage, primarily osteocytes. High-phosphate diets increase FGF23 levels, and low-phosphate diets decrease them. Autosomal dominant hypophosphatemic rickets (ADHR) was the first disease linked to abnormalities in FGF23. ADHR results from activating mutations in the gene that

1	low-phosphate diets decrease them. Autosomal dominant hypophosphatemic rickets (ADHR) was the first disease linked to abnormalities in FGF23. ADHR results from activating mutations in the gene that encodes FGF23. These mutations alter a cleavage site that ordinarily allows for inactivation of intact FGF23. Several other genetic disorders exhibit elevated FGF23 and hypophosphatemia. The most common of these is X-linked hypophosphatemic rickets (XLH), which results from inactivating mutations in an endopeptidase termed PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) that is expressed most abundantly on the surface of osteocytes and mature osteoblasts. Patients with XLH usually have high FGF23 levels, and ablation of the FGF23 gene reverses the hypophosphatemia found in the mouse version of XLH. How inactivation

1	CAuSES of HyPoPHoSPHATEmiA I. Reduced renal tubular phosphate reabsorption A. PTH/PTHrP-dependent 1. 2. a. b. c. d. Autosomal recessive renal hypercalciuria with hypomagnesemia 3. PTHrP-dependent hypercalcemia of malignancy 4. B. PTH/PTHrP-independent 1. Excess FGF23 or other “phosphatonins” a. b. c. Autosomal dominant hypophosphatemic rickets (ADHR) (DMP1, ENPP1 deficiency) d. e. f. 2. Intrinsic renal disease a. b. c. Wilson’s disease d. NaPi-2a or NaPi-2c mutations 3. Other systemic disorders a. b. c. d. e. f. g. h. 4. Drugs or toxins a. b. Acetazolamide, other diuretics c. d. Heavy metals (lead, cadmium, saccharated ferric oxide) e. Toluene, N-methyl formamide f. Cisplatin, ifosfamide, foscarnet, rapamycin II. A. B. III. Shifts of extracellular phosphate into cells A. B. C. Catecholamines (epinephrine, dopamine, albuterol) D. E. Gram-negative sepsis, toxic shock syndrome F. G. 1. 2.

1	II. A. B. III. Shifts of extracellular phosphate into cells A. B. C. Catecholamines (epinephrine, dopamine, albuterol) D. E. Gram-negative sepsis, toxic shock syndrome F. G. 1. 2. Intensive erythropoietin, other growth factor therapy IV. A. B. Treatment of vitamin D deficiency, Paget’s disease C. Osteoblastic metastases Abbreviations: PTH, parathyroid hormone; PTHrP, parathyroid hormone–related peptide.

1	Osteoblastic metastases Abbreviations: PTH, parathyroid hormone; PTHrP, parathyroid hormone–related peptide. of PHEX leads to increased levels of FGF23 has not been determined. 2459 Two rare autosomal recessive hypophosphatemic syndromes associated with elevated FGF23 are due to inactivating mutations of dentin matrix protein-1 (DMP1) and ectonucleotide pyrophosphatase/ phosphodiesterase 1 (ENPP1), both of which normally are highly expressed in bone and regulate FGF23 production. An unusual hypophosphatemic disorder, tumor-induced osteomalacia (TIO), is an acquired disorder in which tumors, usually of mesenchymal origin and generally histologically benign, secrete FGF23 and/or other molecules that induce renal phosphate wasting. The hypophosphatemic syndrome resolves completely within hours to days after successful resection of the responsible tumor. Such tumors typically express large amounts of FGF23 mRNA, and patients with TIO usually exhibit elevations of FGF23 in their blood.

1	Dent’s disease is an X-linked recessive disorder caused by inactivating mutations in CLCN5, a chloride transporter expressed in endosomes of the proximal tubule; features include hypercalciuria, hypophosphatemia, and recurrent kidney stones. Renal phosphate wasting is common among poorly controlled diabetic patients and alcoholics, who therefore are at risk for iatrogenic hypophosphatemia when treated with insulin or IV glucose, respectively. Diuretics and certain other drugs and toxins can cause defective renal tubular phosphate reabsorption (Table 423-1).

1	In hospitalized patients, hypophosphatemia is often attributable to massive redistribution of phosphate from the ECF into cells. Insulin therapy for diabetic ketoacidosis is a paradigm for this phenomenon, in which the severity of the hypophosphatemia is related to the extent of antecedent depletion of phosphate and other electrolytes (Chap. 417). The hypophosphatemia is usually greatest at a point many hours after initiation of insulin therapy and is difficult to predict from baseline measurements of serum phosphate at the time of presentation, when prerenal azotemia can obscure significant phosphate depletion. Other factors that may contribute to such acute redistributive hypophosphatemia include antecedent starvation or malnutrition, administration of IV glucose without other nutrients, elevated blood catecholamines (endogenous or exogenous), respiratory alkalosis, and recovery from metabolic acidosis.

1	Hypophosphatemia also can occur transiently (over weeks to months) during the phase of accelerated net bone formation that follows parathyroidectomy for severe primary hyperparathyroidism or during treatment of vitamin D deficiency or lytic Paget’s disease. This is usually most prominent in patients who preoperatively have evidence of high bone turnover (e.g., high serum levels of alkaline phosphatase). Osteoblastic metastases can also lead to this syndrome.

1	Clinical and Laboratory Findings The clinical manifestations of severe hypophosphatemia reflect a generalized defect in cellular energy metabolism because of ATP depletion, a shift from oxidative phosphorylation toward glycolysis, and associated tissue or organ dysfunction. Acute, severe hypophosphatemia occurs mainly or exclusively in hospitalized patients with underlying serious medical or surgical illness and preexisting phosphate depletion due to excessive urinary losses, severe malabsorption, or malnutrition. Chronic hypophosphatemia tends to be less severe, with a clinical presentation dominated by musculoskeletal complaints such as bone pain, osteomalacia, pseudo-fractures, and proximal muscle weakness or, in children, rickets and short stature.

1	Neuromuscular manifestations of severe hypophosphatemia are variable but may include muscle weakness, lethargy, confusion, disorientation, hallucinations, dysarthria, dysphagia, oculomotor palsies, anisocoria, nystagmus, ataxia, cerebellar tremor, ballismus, hyporeflexia, impaired sphincter control, distal sensory deficits, paresthesia, hyperesthesia, generalized or Guillain-Barré–like ascending paralysis, seizures, coma, and even death. Serious sequelae such as paralysis, confusion, and seizures are likely only at phosphate concentrations <0.25 mmol/L (<0.8 mg/dL). Rhabdomyolysis may develop during rapidly progressive hypophosphatemia. The diagnosis of hypophosphatemiainduced rhabdomyolysis may be overlooked, as up to 30% of patients with acute hypophosphatemia (<0.7 mM) have creatine phosphokinase 2460 elevations that peak 1–2 days after the nadir in serum phosphate, when the release of phosphate from injured myocytes may have led to a near normalization of circulating levels of

1	phosphokinase 2460 elevations that peak 1–2 days after the nadir in serum phosphate, when the release of phosphate from injured myocytes may have led to a near normalization of circulating levels of phosphate. Respiratory failure and cardiac dysfunction, which are reversible with phosphate treatment, may occur at serum phosphate levels of 0.5–0.8 mmol/L (1.5–2.5 mg/dL). Renal tubular defects, including tubular acidosis, glycosuria, and impaired reabsorption of sodium and calcium, may occur. Hematologic abnormalities correlate with reductions in intracellular ATP and 2,3-diphosphoglycerate and may include erythrocyte microspherocytosis and hemolysis; impaired oxyhemoglobin dissociation; defective leukocyte chemotaxis, phagocytosis, and bacterial killing; and platelet dysfunction with spontaneous gastrointestinal hemorrhage.

1	Severe hypophosphatemia (<0.75 mmol/L [<2 mg/dL]), particularly in the setting of underlying phosphate depletion, constitutes a dangerous electrolyte abnormality that should be corrected promptly. Unfortunately, the cumulative deficit in body phosphate cannot be predicted easily from knowledge of the circulating level of phosphate, and therapy must be approached empirically. The threshold for IV phosphate therapy and the dose administered should reflect consideration of renal function, the likely severity and duration of the underlying phosphate depletion, and the presence and severity of symptoms consistent with those of hypophosphatemia. In adults, phosphate may be safely administered IV as neutral mixtures of sodium or potassium phosphate salts at initial doses of 0.2–0.8 mmol/kg of elemental phosphorus over 6 h (e.g., 10–50 mmol over 6 h), with doses >20 mmol/6 h reserved for those who have serum levels <0.5 mmol/L (1.5 mg/dL) and normal renal function. A suggested approach is

1	elemental phosphorus over 6 h (e.g., 10–50 mmol over 6 h), with doses >20 mmol/6 h reserved for those who have serum levels <0.5 mmol/L (1.5 mg/dL) and normal renal function. A suggested approach is presented in Table 423-2. Serum levels of phosphate and calcium must be monitored closely (every 6–12 h) throughout treatment. It is necessary to avoid a serum calcium-phosphorus product >50 to reduce the risk of heterotopic calcification. Hypocalcemia, if present, should be corrected before administering IV phosphate. Less severe hypophosphatemia, in the range of 0.5–0.8 mmol/L (1.5–2.5 mg/dL), usually can be treated with oral phosphate in divided doses of 750–2000 mg/d as elemental phosphorus; higher doses can cause bloating and diarrhea.

1	Management of chronic hypophosphatemia requires knowledge of the cause(s) of the disorder. Hypophosphatemia related to the secondary hyperparathyroidism of vitamin D deficiency usually responds to treatment with vitamin D and calcium alone. XLH, ADHR, TIO, and related renal tubular disorders usually are managed with divided oral doses of phosphate, often with calcium and 1,25(OH)2D supplements to bypass the block in renal 1,25(OH)2D synthesis and prevent secondary hyperparathyroidism caused by suppression of ECF calcium levels. Thiazide diuretics may be used to prevent nephrocalcinosis in patients who are managed this way. Complete normalization of hypophosphatemia is generally not possible in these conditions. Optimal therapy for TIO is extirpation of the responsible tumor, which may be localized by radiographic skeletal survey or bone scan (many are located in bone) or by radionuclide scanning using sestamibi or labeled octreotide. Successful treatment of TIO-induced

1	which may be localized by radiographic skeletal survey or bone scan (many are located in bone) or by radionuclide scanning using sestamibi or labeled octreotide. Successful treatment of TIO-induced hypophosphatemia with octreotide has been reported in a small number of patients.

1	HYPERPHOSPHATEMIA Causes When the filtered load of phosphate and glomerular filtration rate (GFR) are normal, control of serum phosphate levels is achieved by adjusting the rate at which phosphate is reabsorbed by the proximal tubular NaPi-2 co-transporters. The principal hormonal regulators of NaPi-2 activity are PTH and FGF23. Hyperphosphatemia, defined in adults as a fasting serum phosphate concentration >1.8 mmol/L (5.5 mg/dL), usually results from impaired glomerular filtration, hypoparathyroidism, excessive delivery of phosphate into the ECF (from bone, gut, or parenteral phosphate therapy), or a combination of these factors (Table 423-3). The upper limit of normal serum phosphate concentrations is higher in children and neonates (2.4 mmol/L [7 mg/ dL]). It is useful to distinguish hyperphosphatemia caused by impaired renal phosphate excretion from that which results from excessive delivery of phosphate into the ECF (Table 423-3).

1	In chronic renal insufficiency, reduced GFR leads to phosphate retention. Hyperphosphatemia in turn further impairs renal synthesis of 1,25(OH)2D, increases FGF23 levels, and stimulates PTH secretion and hypertrophy both directly and indirectly (by lowering blood ionized calcium levels). Thus, hyperphosphatemia is a major cause of the secondary hyperparathyroidism of renal failure and must be addressed early in the course of the disease (Chaps. 335 and 424).

1	Hypoparathyroidism leads to hyperphosphatemia via increased expression of NaPi-2 co-transporters in the proximal tubule. Hypoparathyroidism, or parathyroid suppression, has multiple potential causes, including autoimmune disease; developmental, surgical, or radiation-induced absence of functional parathyroid tissue; vitamin D intoxication or other causes of PTH-independent hypercalcemia; cellular PTH resistance (pseudohypoparathyroidism or hypomagnesemia); infiltrative disorders such as Wilson’s disease and hemochromatosis; and impaired PTH secretion caused by hypermagnesemia, severe hypomagnesemia, or activating mutations in the CaSR. Hypocalcemia may also contribute directly to impaired phosphate clearance, as calcium infusion can induce phosphaturia in hypoparathyroid subjects. Increased tubular phosphate reabsorption also occurs in acromegaly, during heparin administration, and in tumoral calcinosis. Tumoral calcinosis is caused by a rare group of genetic disorders in which FGF23

1	tubular phosphate reabsorption also occurs in acromegaly, during heparin administration, and in tumoral calcinosis. Tumoral calcinosis is caused by a rare group of genetic disorders in which FGF23 is processed in a way that leads to low levels of active FGF23 in the bloodstream. This may result from mutations in the FGF23 sequence or via inactivating mutations in the GALNT3 gene, which encodes a galactosaminyl transferase that normally adds sugar residues to FGF23 that slow its proteolysis. A similar syndrome results from FGF23 resistance due to inactivating mutations of the FGF23 co-receptor Klotho. These abnormalities cause elevated serum 1,25(OH)2D, parathyroid

1	Likely severity of underlying phosphate depletion Concurrent parenteral glucose administration Presence of neuromuscular, cardiopulmonary, or hematologic complications of hypophosphatemia Renal function (reduce dose by 50% if serum creatinine >220 μmol/L [>2.5 mg/dL]) Serum calcium level (correct hypocalcemia first; reduce dose by 50% in hypercalcemia) Guidelines Serum Phosphorus, mM (mg/dL) Rate of Infusion, mmol/h Duration, h Total Administered, mmol <0.8 (<2.5) 2 6 12 <0.5 (<1.5) 4 6 24 <0.3 (<1) 8 6 48 Note: Rates shown are calculated for a 70-kg person; levels of serum calcium and phosphorus must be measured every 6–12 h during therapy; infusions can be repeated to achieve stable serum phosphorus levels >0.8 mmol/L (>2.5 mg/dL); most formulations available in the United States provide 3 mmol/mL of sodium or potassium phosphate. CAuSES of HyPERPHoSPHATEmiA I. Impaired renal phosphate excretion A. Renal insufficiency B. Hypoparathyroidism 1. 2. 3. 4.

1	CAuSES of HyPERPHoSPHATEmiA I. Impaired renal phosphate excretion A. Renal insufficiency B. Hypoparathyroidism 1. 2. 3. 4. Activating mutations of the calcium-sensing receptor C. Parathyroid suppression 1. Parathyroid-independent hypercalcemia a. b. Sarcoidosis, other granulomatous diseases c. Immobilization, osteolytic metastases d. 2. Severe hypermagnesemia or hypomagnesemia D. Pseudohypoparathyroidism E. Acromegaly F. Tumoral calcinosis G. Heparin therapy II. A. Rapid administration of exogenous phosphate (intravenous, oral, rectal) B. 1. 2. 3. 4. 5. 6. C. Transcellular phosphate shifts 1. 2. suppression, increased intestinal calcium absorption, and focal hyperostosis with large, lobulated periarticular heterotopic ossifications (especially at shoulders or hips) and are accompanied by hyperphosphatemia. In some forms of tumoral calcinosis, serum phosphorus levels are normal.

1	When large amounts of phosphate are delivered rapidly into the ECF, hyperphosphatemia can occur despite normal renal function. Examples include overzealous IV phosphate therapy, oral or rectal administration of large amounts of phosphate-containing laxatives or enemas (especially in children), extensive soft tissue injury or necrosis (crush injuries, rhabdomyolysis, hyperthermia, fulminant hepatitis, cytotoxic chemotherapy), extensive hemolytic anemia, and transcellular phosphate shifts induced by severe metabolic or respiratory acidosis.

1	Clinical Findings The clinical consequences of acute, severe hyperphosphatemia are due mainly to the formation of widespread calcium phosphate precipitates and resulting hypocalcemia. Thus, tetany, seizures, accelerated nephrocalcinosis (with renal failure, hyperkalemia, hyperuricemia, and metabolic acidosis), and pulmonary or cardiac calcifications (including development of acute heart block) may occur. The severity of these complications relates to the elevation of serum phosphate levels, which can reach concentrations as high as 7 mmol/L (20 mg/dL) in instances of massive soft tissue injury or tumor lysis syndrome.

1	Therapeutic options for management of severe hyperphosphatemia are limited. Volume expansion may enhance renal phosphate clearance. Aluminum hydroxide antacids or sevelamer may be helpful in chelating and limiting absorption of offending phosphate salts present in the intestine. Hemodialysis is the most effective thera-2461 peutic strategy and should be considered early in the course of severe hyperphosphatemia, especially in the setting of renal failure and symptomatic hypocalcemia.

1	Magnesium is the major intracellular divalent cation. Normal concentrations of extracellular magnesium and calcium are crucial for normal neuromuscular activity. Intracellular magnesium forms a key complex with ATP and is an important cofactor for a wide range of enzymes, transporters, and nucleic acids required for normal cellular function, replication, and energy metabolism. The concentration of magnesium in serum is closely regulated within the range of 0.7–1 mmol/L (1.5–2 meq/L; 1.7–2.4 mg/dL), of which 30% is protein-bound and another 15% is loosely complexed to phosphate and other anions. One-half of the 25 g (1000 mmol) of total body magnesium is located in bone, only one-half of which is insoluble in the mineral phase. Almost all extraskeletal magnesium is present within cells, where the total concentration is 5 mM, 95% of which is bound to proteins and other macromolecules. Because only 1% of body magnesium resides in the ECF, measurements of serum magnesium levels may not

1	where the total concentration is 5 mM, 95% of which is bound to proteins and other macromolecules. Because only 1% of body magnesium resides in the ECF, measurements of serum magnesium levels may not accurately reflect the level of total body magnesium stores.

1	Dietary magnesium content normally ranges from 6 to 15 mmol/d (140–360 mg/d), of which 30–40% is absorbed, mainly in the jejunum and ileum. Intestinal magnesium absorptive efficiency is stimulated by 1,25(OH)2D and can reach 70% during magnesium deprivation. Urinary magnesium excretion normally matches net intestinal absorption and is ~4 mmol/d (100 mg/d). Regulation of serum magnesium concentrations is achieved mainly by control of renal magnesium reabsorption. Only 20% of filtered magnesium is reabsorbed in the proximal tubule, whereas 60% is reclaimed in the cTAL and another 5–10% in the DCT. Magnesium reabsorption in the cTAL occurs via a paracellular route that requires both a lumen-positive potential, created by NaCl reabsorption, and tight-junction proteins encoded by members of the Claudin gene family. Magnesium reabsorption in the cTAL is increased by PTH but inhibited by hypercalcemia or hypermagnesemia, both of which activate the CaSR in this nephron segment.

1	HYPOMAGNESEMIA Causes Hypomagnesemia usually signifies substantial depletion of body magnesium stores (0.5–1 mmol/kg). Hypomagnesemia can result from intestinal malabsorption; protracted vomiting, diarrhea, or intestinal drainage; defective renal tubular magnesium reabsorption; or rapid shifts of magnesium from the ECF into cells, bone, or third spaces (Table 423-4). Dietary magnesium deficiency is unlikely except possibly in the setting of alcoholism. A rare genetic disorder that causes selective intestinal magnesium malabsorption has been described (primary infantile hypomagnesemia). Another rare inherited disorder (hypomagnesemia with secondary hypocalcemia) is caused by mutations in the gene encoding TRPM6, a protein that, along with TRPM7, forms a channel important for both intestinal and distal-tubular renal transcellular magnesium transport. Malabsorptive states, often compounded by vitamin D deficiency, can critically limit magnesium absorption and produce hypomagnesemia

1	and distal-tubular renal transcellular magnesium transport. Malabsorptive states, often compounded by vitamin D deficiency, can critically limit magnesium absorption and produce hypomagnesemia despite the compensatory effects of secondary hyperparathyroidism and of hypocalcemia and hypomagnesemia to enhance cTAL magnesium reabsorption. Diarrhea or surgical drainage fluid may contain ≥5 mmol/L of magnesium. Proton pump inhibitors (omeprazole and others) may produce hypomagnesemia by an unknown mechanism that does not involve renal wasting of magnesium.

1	Several genetic magnesium-wasting syndromes have been described, including inactivating mutations of genes encoding the DCT NaCl co-transporter (Gitelman’s syndrome), proteins required for cTAL Na-K-2Cl transport (Bartter’s syndrome), claudin 16 or claudin 19 (autosomal recessive renal hypomagnesemia with hypercalciuria), a DCT Na+,K+-ATPase γ-subunit (autosomal dominant renal hypomagnesemia with hypocalciuria), DCT K+ channels (Kv1.1, Kir4.1), and a mitochondrial gene encoding a tRNA. Activating mutations CAuSES of HyPomAgnESEmiA I. Impaired intestinal absorption A. Hypomagnesemia with secondary hypocalcemia (TRPM6 mutations) B. Malabsorption syndromes C. Vitamin D deficiency D. Proton pump inhibitors II. A. B. Intestinal drainage, fistulas III. A. 1. 2. 3. 4. Potassium channel mutations (Kv1.1, Kir4.1) 5. Na+,K+-ATPase γ-subunit mutations (FXYD2) B. Acquired renal disease 1. 2. Postobstruction, ATN (diuretic phase) 3. C. Drugs and toxins 1. 2.

1	4. Potassium channel mutations (Kv1.1, Kir4.1) 5. Na+,K+-ATPase γ-subunit mutations (FXYD2) B. Acquired renal disease 1. 2. Postobstruction, ATN (diuretic phase) 3. C. Drugs and toxins 1. 2. Diuretics (loop, thiazide, osmotic) 3. 4. Pentamidine, foscarnet 5. 6. Aminoglycosides, amphotericin B 7. D. Other 1. 2. 3. 4. 5. 6. 7. 8. IV. A. 1. 2. 3. Correction of respiratory acidosis 4. B. Accelerated bone formation 1. 2. Treatment of vitamin D deficiency 3. C. Other 1. Pancreatitis, burns, excessive sweating 2. Abbreviations: ATN, acute tubular necrosis; SIADH, syndrome of inappropriate antidiuretic hormone.

1	C. Other 1. Pancreatitis, burns, excessive sweating 2. Abbreviations: ATN, acute tubular necrosis; SIADH, syndrome of inappropriate antidiuretic hormone. of the CaSR can cause hypomagnesemia as well as hypocalcemia. ECF expansion, hypercalcemia, and severe phosphate depletion may impair magnesium reabsorption, as can various forms of renal injury, including those caused by drugs such as cisplatin, cyclosporine, aminoglycosides, and pentamidine as well as the epidermal growth factor (EGF) receptor inhibitory antibody, cetuximab (EGF action is required for normal DCT apical expression of TRPM6) (Table 423-4). A rising blood concentration of ethanol directly impairs tubular magnesium reabsorption, and persistent glycosuria with osmotic diuresis leads to magnesium wasting and probably contributes to the high frequency of hypomagnesemia in poorly controlled diabetic patients. Magnesium depletion is aggravated by metabolic acidosis, which causes intracellular losses as well.

1	Hypomagnesemia due to rapid shifts of magnesium from ECF into the intracellular compartment can occur during recovery from diabetic ketoacidosis, starvation, or respiratory acidosis. Less acute shifts may be seen during rapid bone formation after parathyroidectomy, with treatment of vitamin D deficiency, or with osteoblastic metastases. Large amounts of magnesium may be lost with acute pancreatitis, extensive burns, or protracted and severe sweating and during pregnancy and lactation.

1	Clinical and Laboratory Findings Hypomagnesemia may cause generalized alterations in neuromuscular function, including tetany, tremor, seizures, muscle weakness, ataxia, nystagmus, vertigo, apathy, depression, irritability, delirium, and psychosis. Patients are usually asymptomatic when serum magnesium concentrations are >0.5 mmol/L (1 meq/L; 1.2 mg/dL), although the severity of symptoms may not correlate with serum magnesium levels. Cardiac arrhythmias may occur, including sinus tachycardia, other supraventricular tachycardias, and ventricular arrhythmias. Electrocardiographic abnormalities may include prolonged PR or QT intervals, T-wave flattening or inversion, and ST straightening. Sensitivity to digitalis toxicity may be enhanced.

1	Other electrolyte abnormalities often seen with hypomagnesemia, including hypocalcemia (with hypocalciuria) and hypokalemia, may not be easily corrected unless magnesium is administered as well. The hypocalcemia may be a result of concurrent vitamin D deficiency, although hypomagnesemia can cause impaired synthesis of 1,25(OH)2D, cellular resistance to PTH, and, at very low serum magnesium (<0.4 mmol/L [0.8 meq/L; <1 mg/dL]), a defect in PTH secretion; these abnormalities are reversible with therapy.

1	Mild, asymptomatic hypomagnesemia may be treated with oral magnesium salts (MgCl2, MgO, Mg[OH]2) in divided doses totaling 20–30 mmol/d (40–60 meq/d). Diarrhea may occur with larger doses. More severe hypomagnesemia should be treated parenterally, preferably with IV MgCl2, which can be administered safely as a continuous infusion of 50 mmol/d (100 meq Mg2+/d) if renal function is normal. If GFR is reduced, the infusion rate should be lowered by 50–75%. Use of IM MgSO4 is discouraged; the injections are painful and provide relatively little magnesium (2 mL of 50% MgSO4 supplies only 4 mmol). MgSO4 may be given IV instead of MgCl2, although the sulfate anions may bind calcium in serum and urine and aggravate hypocalcemia. Serum magnesium should be monitored at intervals of 12–24 h during therapy, which may continue for several days because of impaired renal conservation of magnesium (only 50–70% of the daily IV magnesium dose is retained) and delayed repletion of intracellular deficits,

1	which may continue for several days because of impaired renal conservation of magnesium (only 50–70% of the daily IV magnesium dose is retained) and delayed repletion of intracellular deficits, which may be as high as 1–1.5 mmol/kg (2–3 meq/kg).

1	It is important to consider the need for calcium, potassium, and phosphate supplementation in patients with hypomagnesemia. Vitamin D deficiency frequently coexists and should be treated with oral or parenteral vitamin D or 25(OH)D (but not with 1,25[OH]2D, which may impair tubular magnesium reabsorption, possibly via PTH suppression). In severely hypomagnesemic patients with concomitant hypocalcemia and hypophosphatemia, administration of IV magnesium alone may worsen hypophosphatemia, provoking neuromuscular symptoms or rhabdomyolysis, due to rapid stimulation of PTH secretion. This is avoided by administering both calcium and magnesium. HYPERMAGNESEMIA Causes Hypermagnesemia is rarely seen in the absence of renal insufficiency, as normal kidneys can excrete large amounts (250 mmol/d) of CAuSES of HyPERmAgnESEmiA I. Excessive magnesium intake A. Cathartics, urologic irrigants B. Parenteral magnesium administration II. A. Trauma, shock, sepsis B. C. III. A. B. IV. A.

1	CAuSES of HyPERmAgnESEmiA I. Excessive magnesium intake A. Cathartics, urologic irrigants B. Parenteral magnesium administration II. A. Trauma, shock, sepsis B. C. III. A. B. IV. A. B. C. magnesium. Mild hypermagnesemia due to excessive reabsorption in the cTAL occurs with CaSR mutations in familial hypocalciuric hypercalcemia and has been described in some patients with adrenal insufficiency, hypothyroidism, or hypothermia. Massive exogenous magnesium exposures, usually via the gastrointestinal tract, can overwhelm renal excretory capacity and cause life-threatening hypermagnesemia (Table 423-5). A notable example of this is prolonged retention of even normal amounts of magnesium-containing cathartics in patients with intestinal ileus, obstruction, or perforation. Extensive soft tissue injury or necrosis can also deliver large amounts of magnesium into the ECF in patients who have suffered trauma, shock, sepsis, cardiac arrest, or severe burns.

1	Clinical and Laboratory Findings The most prominent clinical manifestations of hypermagnesemia are vasodilation and neuromuscular blockade, which may appear at serum magnesium concentrations >2 mmol/L (>4 meq/L; >4.8 mg/dL). Hypotension that is refractory to vasopressors or volume expansion may be an early sign. Nausea, lethargy, and weakness may progress to respiratory failure, paralysis, and coma, with hypoactive tendon reflexes, at serum magnesium levels >4 mmol/L. Other findings may include gastrointestinal hypomotility or ileus; facial flushing; pupillary dilation; paradoxical bradycardia; prolongation of PR, QRS, and QT intervals; heart block; and, at serum magnesium levels approaching 10 mmol/L, asystole. Hypermagnesemia, acting via the CaSR, causes hypocalcemia and hypercalciuria due to both parathyroid suppression and impaired cTAL calcium reabsorption.

1	Hypermagnesemia, acting via the CaSR, causes hypocalcemia and hypercalciuria due to both parathyroid suppression and impaired cTAL calcium reabsorption. Successful treatment of hypermagnesemia generally involves identifying and interrupting the source of magnesium and employing measures to increase magnesium clearance from the ECF. Use of magnesium-free cathartics or enemas may be helpful in clearing ingested magnesium from the gastrointestinal tract. Vigorous IV hydration should be attempted, if appropriate. Hemodialysis is effective and may be required in patients with significant renal insufficiency. Calcium, administered IV in doses of 100–200 mg over 1–2 h, has been reported to provide temporary improvement in signs and symptoms of hypermagnesemia.

1	1,25-Dihydroxyvitamin D (1,25[OH]2D) is the major steroid hormone involved in mineral ion homeostasis regulation. Vitamin D and its metabolites are hormones and hormone precursors rather than vitamins, since in the proper biologic setting, they can be synthesized endogenously (Fig. 423-4). In response to ultraviolet radiation of the 1,25(OH)2D FIGURE 423-4 Vitamin D synthesis and activation. Vitamin D is synthesized in the skin in response to ultraviolet radiation and also is absorbed from the diet. It is then transported to the liver, where it undergoes 25-hydroxylation. This metabolite is the major circulating form of vitamin D. The final step in hormone activation, 1α-hydroxylation, occurs in the kidney.

1	skin, a photochemical cleavage results in the formation of vitamin D from 7-dehydrocholesterol. Cutaneous production of vitamin D is decreased by melanin and high solar protection factor sunblocks, which effectively impair skin penetration by ultraviolet light. The increased use of sunblocks in North America and Western Europe and a reduction in the magnitude of solar exposure of the general population over the last several decades has led to an increased reliance on dietary sources of vitamin D. In the United States and Canada, these sources largely consist of fortified cereals and dairy products, in addition to fish oils and egg yolks. Vitamin D from plant sources is in the form of vitamin D2, whereas that from animal sources is vitamin D3. These two forms have equivalent biologic activity and are activated equally well by the vitamin D hydroxylases in humans. Vitamin D enters the circulation, whether absorbed from the intestine or synthesized cutaneously, bound to vitamin D–binding

1	and are activated equally well by the vitamin D hydroxylases in humans. Vitamin D enters the circulation, whether absorbed from the intestine or synthesized cutaneously, bound to vitamin D–binding protein, an α-globulin synthesized in the liver. Vitamin D is subsequently 25-hydroxylated in the liver by cytochrome P450–like enzymes in the mitochondria and microsomes. The activity of this hydroxylase is not tightly regulated, and the resultant metabolite, 25-hydroxyvitamin D (25[OH]D), is the major circulating and storage form of vitamin D. Approximately 88% of 25(OH)D circulates bound to the vitamin D–binding protein, 0.03% is free, and the rest circulates bound to albumin. The half-life of 25(OH)D is approximately 2–3 weeks; however, it is shortened dramatically when vitamin D–binding protein levels are reduced, as can occur with increased urinary losses in the nephrotic syndrome.

1	1,25(OH)2D3 Ca2+HPO42– Calcification Ca2+HPO42– KidneyParathyroidglandsBone25(OH)D-1˜-hydroxylaseand other factors PTH PTH Blood calcium IntestinePi /+1,25(OH)2D3 / FIGURE 423-5 Schematic representation of the hormonal control loop for vitamin D metabolism and function. A reduction in the serum calcium below ~2.2 mmol/L (8.8 mg/dL) prompts a proportional increase in the secretion of parathyroid hormone (PTH) and so mobilizes additional calcium from the bone. PTH promotes the synthesis of 1,25(OH)2D in the kidney, which in turn stimulates the mobilization of calcium from bone and intestine and regulates the synthesis of PTH by negative feedback.

1	The second hydroxylation, required for the formation of the mature hormone, occurs in the kidney (Fig. 423-5). The 25-hydroxyvitamin D-1α-hydroxylase is a tightly regulated cytochrome P450–like mixed-function oxidase expressed in the proximal convoluted tubule cells of the kidney. PTH and hypophosphatemia are the major inducers of this microsomal enzyme, whereas calcium, FGF23, and the enzyme’s product, 1,25(OH)2D, repress it. The 25-hydroxyvitamin D-1α-hydroxylase is also present in epidermal keratinocytes, but keratinocyte production of 1,25(OH)2D is not thought to contribute to circulating levels of this hormone. In addition to being present in the trophoblastic layer of the placenta, the 1α-hydroxylase is produced by macrophages associated with granulomas and lymphomas. In these latter pathologic states, the activity of the enzyme is induced by interferon γ and TNF-α but is not regulated by calcium or 1,25(OH)2D; therefore, hypercalcemia, associated with elevated levels of

1	latter pathologic states, the activity of the enzyme is induced by interferon γ and TNF-α but is not regulated by calcium or 1,25(OH)2D; therefore, hypercalcemia, associated with elevated levels of 1,25(OH)2D, may be observed. Treatment of sarcoidosis-associated hypercalcemia with glucocorticoids, ketoconazole, or chloroquine reduces 1,25(OH)2D production and effectively lowers serum calcium. In contrast, chloroquine has not been shown to lower the elevated serum 1,25(OH)2D levels in patients with lymphoma.

1	The major pathway for inactivation of vitamin D metabolites is an additional hydroxylation step by the vitamin D 24-hydroxylase, an enzyme that is expressed in most tissues. 1,25(OH)2D is the major inducer of this enzyme; therefore, this hormone promotes its own inactivation, thereby limiting its biologic effects. Mutations of the gene encoding this enzyme (CYP24A1) can lead to infantile hypercalcemia and, in those less severely affected, long-standing hypercalciuria, nephrocalcinosis, and nephrolithiasis. Polar metabolites of 1,25(OH)2D are secreted into the bile and reabsorbed via the enterohepatic circulation. Impairment of this recirculation, which is seen with diseases of the terminal ileum, leads to accelerated losses of vitamin D metabolites.

1	ACTIONS OF 1,25(OH)2D 1,25(OH)2D mediates its biologic effects by binding to a member of the nuclear receptor superfamily, the vitamin D receptor (VDR). This receptor belongs to the subfamily that includes the thyroid hormone receptors, the retinoid receptors, and the peroxisome proliferator– activated receptors; however, in contrast to the other members of this subfamily, only one VDR isoform has been isolated. The VDR binds to target DNA sequences as a heterodimer with the retinoid X receptor, recruiting a series of coactivators that modify chromatin and approximate the VDR to the basal transcriptional apparatus, resulting in the induction of target gene expression. The mechanism of transcriptional repression by the VDR varies with different target genes but has been shown to involve either interference with the action of activating transcription factors or the recruitment of novel proteins to the VDR complex, resulting in transcriptional repression.

1	The affinity of the VDR for 1,25(OH)2D is approximately three orders of magnitude higher than that for other vitamin D metabolites. In normal physiologic circumstances, these other metabolites are not thought to stimulate receptor-dependent actions. However, in states of vitamin D toxicity, the markedly elevated levels of 25(OH)D may lead to hypercalcemia by interacting directly with the VDR and by displacing 1,25(OH)2D from vitamin D–binding protein, resulting in increased bioavailability of the active hormone.

1	The VDR is expressed in a wide range of cells and tissues. The molecular actions of 1,25(OH)2D have been studied most extensively in tissues involved in the regulation of mineral ion homeostasis. This hormone is a major inducer of calbindin 9K, a calcium-binding protein expressed in the intestine, which is thought to play an important role in the active transport of calcium across the enterocyte. The two major calcium transporters expressed by intestinal epithelia, TRPV5 and TRPV6 (transient receptor potential vanilloid), are also vitamin D responsive. By inducing the expression of these and other genes in the small intestine, 1,25(OH)2D increases the efficiency of intestinal calcium absorption, and it also has been shown to have several important actions in the skeleton. The VDR is expressed in osteoblasts and regulates the expression of several genes in this cell. These genes include the bone matrix proteins osteocalcin and osteopontin, which are upregulated by 1,25(OH)2D, in

1	expressed in osteoblasts and regulates the expression of several genes in this cell. These genes include the bone matrix proteins osteocalcin and osteopontin, which are upregulated by 1,25(OH)2D, in addition to type I collagen, which is transcriptionally repressed by 1,25(OH)2D. Both 1,25(OH)2D and PTH induce the expression of RANK ligand, which promotes osteoclast differentiation and increases osteoclast activity, by binding to RANK on osteoclast progenitors and mature osteoclasts. This is the mechanism by which 1,25(OH)2D induces bone resorption. However, the skeletal features associated with VDR-knockout mice (rickets, osteomalacia) are largely corrected by increasing calcium and phosphorus intake, underscoring the importance of vitamin D action in the gut.

1	The VDR is expressed in the parathyroid gland, and 1,25(OH)2D has been shown to have antiproliferative effects on parathyroid cells and to suppress the transcription of the PTH gene. These effects of 1,25(OH)2D on the parathyroid gland are an important part of the rationale for current therapies directed at preventing and treating hyperparathyroidism associated with renal insufficiency. The VDR is also expressed in tissues and organs that do not play a role in mineral ion homeostasis. Notable in this respect is the observation that 1,25(OH)2D has an antiproliferative effect on several cell types, including keratinocytes, breast cancer cells, and prostate cancer cells. The effects of 1,25(OH)2D and the VDR on keratinocytes are particularly intriguing. Alopecia is seen in humans and mice with mutant VDRs but is not a feature of vitamin D deficiency; thus, the effects of the VDR on the hair follicle are ligand-independent.

1	The mounting concern about the relationship between solar exposure and the development of skin cancer has led to increased reliance on dietary sources of vitamin D. Although the prevalence of vitamin D deficiency varies, the third National Health and Nutrition Examination Survey (NHANES III) revealed that vitamin D deficiency is prevalent throughout the United States. The clinical syndrome of vitamin D deficiency can be a result of deficient production of vitamin D in the skin, lack of dietary intake, accelerated losses of vitamin D, impaired vitamin D activation, or resistance to the biologic effects of 1,25(OH)2D (Table 423-6). The elderly and nursing home residents are particularly at risk for vitamin D deficiency, since both the efficiency of vitamin D synthesis in the skin and the absorption of vitamin D from the intestine decline with age. Similarly, intestinal malabsorption of dietary fats and short bowel syndrome, including that associated with intestinal bypass surgery, can

1	of vitamin D from the intestine decline with age. Similarly, intestinal malabsorption of dietary fats and short bowel syndrome, including that associated with intestinal bypass surgery, can lead to vitamin D deficiency. This is further exacerbated in the presence of terminal ileal disease, which results in impaired enterohepatic circulation of vitamin D metabolites. In addition to intestinal diseases, accelerated inactivation of vitamin D metabolites can be seen with drugs that induce hepatic cytochrome P450 mixed-function oxidases such as barbiturates, phenytoin, and rifampin. Impaired 25-hydroxylation, associated with severe liver disease or isoniazid, is an uncommon cause of vitamin D deficiency. A mutation in the gene responsible for 25-hydroxylation has been identified in one kindred. Impaired 1α-hydroxylation is prevalent in the population with profound renal dysfunction due to an increase in circulating FGF23 levels and a decrease in functional renal mass. Thus, therapeutic

1	Impaired 1α-hydroxylation is prevalent in the population with profound renal dysfunction due to an increase in circulating FGF23 levels and a decrease in functional renal mass. Thus, therapeutic interventions should be considered in patients whose creatinine clearance is <0.5 mL/s (30 mL/min). Mutations in the renal 1α-hydroxylase are the basis for the genetic disorder, pseudovitamin D–deficiency rickets. This autosomal recessive disorder presents with the syndrome of vitamin D deficiency in the first year of life. Patients present with growth retardation, rickets, and hypocalcemic seizures. Serum 1,25(OH)2D levels are low despite normal 25(OH)D levels and elevated PTH levels. Treatment with vitamin D metabolites that do not require 1α-hydroxylation results in disease remission, although lifelong therapy is required. A second autosomal recessive disorder, hereditary vitamin D–resistant rickets, a consequence of vitamin D receptor mutations, is a greater therapeutic challenge. These

1	lifelong therapy is required. A second autosomal recessive disorder, hereditary vitamin D–resistant rickets, a consequence of vitamin D receptor mutations, is a greater therapeutic challenge. These patients present in a similar fashion during the first year of life, but alopecia often accompanies the disorder, demonstrating a functional role of the VDR in postnatal hair regeneration. Serum levels of 1,25(OH)2D are dramatically elevated in these individuals both because of increased production due to stimulation of 1α-hydroxylase activity as a consequence of secondary hyperparathyroidism and because of impaired inactivation, since induction of the 24-hydroxylase by 1,25(OH)2D requires an intact VDR. Because the receptor mutation results in hormone resistance, daily calcium and phosphorus infusions may be required to bypass the defect in intestinal mineral ion absorption.

1	CAuSES of imPAiRED viTAmin D ACTion Accelerated loss of vitamin D 1α-hydroxylase mutation Increased metabolism (barbitu-Oncogenic osteomalacia rates, phenytoin, rifampin) Liver disease, isoniazid

1	Regardless of the cause, the clinical manifestations of vitamin D 2465 deficiency are largely a consequence of impaired intestinal calcium absorption. Mild to moderate vitamin D deficiency is asymptomatic, whereas long-standing vitamin D deficiency results in hypocalcemia accompanied by secondary hyperparathyroidism, impaired mineralization of the skeleton (osteopenia on x-ray or decreased bone mineral density), and proximal myopathy. Vitamin D deficiency also has been shown to be associated with an increase in overall mortality, including cardiovascular causes. In the absence of an intercurrent illness, the hypocalcemia associated with long-standing vitamin D deficiency rarely presents with acute symptoms of hypocalcemia such as numbness, tingling, and seizures. However, the concurrent development of hypomagnesemia, which impairs parathyroid function, or the administration of potent bisphosphonates, which impair bone resorption, can lead to acute symptomatic hypocalcemia in vitamin

1	development of hypomagnesemia, which impairs parathyroid function, or the administration of potent bisphosphonates, which impair bone resorption, can lead to acute symptomatic hypocalcemia in vitamin D–deficient individuals.

1	Rickets and Osteomalacia In children, before epiphyseal fusion, vitamin D deficiency results in growth retardation associated with an expansion of the growth plate known as rickets. Three layers of chondrocytes are present in the normal growth plate: the reserve zone, the proliferating zone, and the hypertrophic zone. Rickets associated with impaired vitamin D action is characterized by expansion of the hyper-trophic chondrocyte layer. The proliferation and differentiation of the chondrocytes in the rachitic growth plate are normal, and the expansion of the growth plate is a consequence of impaired apoptosis of the late hypertrophic chondrocytes, an event that precedes replacement of these cells by osteoblasts during endochondral bone formation. Investigations in murine models demonstrate that hypophosphatemia, which in vitamin D deficiency is a consequence of secondary hyperparathyroidism, is a key etiologic factor in the development of the rachitic growth plate.

1	The hypocalcemia and hypophosphatemia that accompany vitamin D deficiency result in impaired mineralization of bone matrix proteins, a condition known as osteomalacia. Osteomalacia is also a feature of long-standing hypophosphatemia, which may be a consequence of renal phosphate wasting or chronic use of etidronate or phosphate-binding antacids. This hypomineralized matrix is biomechanically inferior to normal bone; as a result, patients with vitamin D deficiency are prone to bowing of weight-bearing extremities and skeletal fractures. Vitamin D and calcium supplementation have been shown to decrease the incidence of hip fracture among ambulatory nursing home residents in France, suggesting that undermineralization of bone contributes significantly to morbidity in the elderly. Proximal myopathy is a striking feature of severe vitamin D deficiency both in children and in adults. Rapid resolution of the myopathy is observed upon vitamin D treatment.

1	Although vitamin D deficiency is the most common cause of rickets and osteomalacia, many disorders lead to inadequate mineralization of the growth plate and bone. Calcium deficiency without vitamin D deficiency, the disorders of vitamin D metabolism previously discussed, and hypophosphatemia can all lead to inefficient mineralization. Even in the presence of normal calcium and phosphate levels, chronic acidosis and drugs such as bisphosphonates can lead to osteomalacia. The inorganic calcium/phosphate mineral phase of bone cannot form at low pH, and bisphosphonates bind to and prevent mineral crystal growth. Because alkaline phosphatase is necessary for normal mineral deposition, probably because the enzyme can hydrolyze inhibitors of mineralization such as inorganic pyrophosphate, genetic inactivation of the alkaline phosphatase gene (hereditary hypophosphatasia) also can lead to osteomalacia in the setting of normal calcium and phosphate levels.

1	Diagnosis of Vitamin D Deficiency, Rickets, and Osteomalacia The most specific screening test for vitamin D deficiency in otherwise healthy individuals is a serum 25(OH)D level. Although the normal ranges vary, levels of 25(OH)D <37 nmol/L (<15 ng/mL) are associated with increasing PTH levels and lower bone density. The Institute of Medicine has defined vitamin D sufficiency as a vitamin D level >50 nmol/L (>20 ng/mL), although higher levels may be required to 2466 optimize intestinal calcium absorption in the elderly and those with underlying disease states. Vitamin D deficiency leads to impaired intestinal absorption of calcium, resulting in decreased serum total and ionized calcium values. This hypocalcemia results in secondary hyperparathyroidism, a homeostatic response that initially maintains serum calcium levels at the expense of the skeleton. Due to the PTH-induced increase in bone turnover, alkaline phosphatase levels are often increased. In addition to increasing bone

1	maintains serum calcium levels at the expense of the skeleton. Due to the PTH-induced increase in bone turnover, alkaline phosphatase levels are often increased. In addition to increasing bone resorption, PTH decreases urinary calcium excretion while promoting phosphaturia. This results in hypophosphatemia, which exacerbates the mineralization defect in the skeleton. With prolonged vitamin D deficiency resulting in osteomalacia, calcium stores in the skeleton become relatively inaccessible, since osteoclasts cannot resorb unmineralized osteoid, and frank hypocalcemia ensues. Because PTH is a major stimulus for the renal 25(OH)D 1α-hydroxylase, there is increased synthesis of the active hormone, 1,25(OH)2D. Paradoxically, levels of this hormone are often normal in severe vitamin D deficiency. Therefore, measurements of 1,25(OH)2D are not accurate reflections of vitamin D stores and should not be used to diagnose vitamin D deficiency in patients with normal renal function. Radiologic

1	Therefore, measurements of 1,25(OH)2D are not accurate reflections of vitamin D stores and should not be used to diagnose vitamin D deficiency in patients with normal renal function. Radiologic features of vitamin D deficiency in children include a widened, expanded growth plate that is characteristic of rickets. These findings not only are apparent in the long bones but also are present at the costochondral junction, where the expansion of the growth plate leads to swellings known as the “rachitic rosary.” Impairment of intramembranous bone mineralization leads to delayed fusion of the calvarial sutures and a decrease in the radiopacity of cortical bone in the long bones. If vitamin D deficiency occurs after epiphyseal fusion, the main radiologic finding is a decrease in cortical thickness and relative radiolucency of the skeleton. A specific radiologic feature of osteomalacia, whether associated with phosphate wasting or vitamin D deficiency, is pseudofractures, or Looser’s zones.

1	and relative radiolucency of the skeleton. A specific radiologic feature of osteomalacia, whether associated with phosphate wasting or vitamin D deficiency, is pseudofractures, or Looser’s zones. These are radiolucent lines that occur where large arteries are in contact with the underlying skeletal elements; it is thought that the arterial pulsations lead to the radiolucencies. As a result, these pseudofractures are usually a few millimeters wide, are several centimeters long, and are seen particularly in the scapula, the pelvis, and the femoral neck.

1	Based on the Institute of Medicine 2010 report, the recommended daily intake of vitamin D is 600 IU from 1 to 70 years of age, and 800 IU for those over 70. Based on the observation that 800 IU of vitamin D, with calcium supplementation, decreases the risk of hip fractures in elderly women, this higher dose is thought to be an appropriate daily intake for prevention of vitamin D deficiency in adults. The safety margin for vitamin D is large, and vitamin D toxicity usually is observed only in patients taking doses in the range of 40,000 IU daily. Treatment of vitamin D deficiency should be directed at the underlying disorder, if possible, and also should be tailored to the severity of the condition. Vitamin D should always be repleted in conjunction with calcium supplementation because most of the consequences of vitamin D deficiency are a result of impaired mineral ion homeostasis. In patients in whom 1α-hydroxylation is impaired, metabolites that do not require this activation step

1	of the consequences of vitamin D deficiency are a result of impaired mineral ion homeostasis. In patients in whom 1α-hydroxylation is impaired, metabolites that do not require this activation step are the treatment of choice. They include 1,25(OH)2D3 (calcitriol [Rocaltrol], 0.25–0.5 μg/d) and 1α-hydroxyvitamin D2 (Hectorol, 2.5–5 μg/d). If the pathway required for activation of vitamin D is intact, severe vitamin D deficiency can be treated with pharmacologic repletion initially (50,000 IU weekly for 3–12 weeks), followed by maintenance therapy (800 IU daily). Pharmacologic doses may be required for maintenance therapy in patients who are taking medications, such as barbiturates or phenytoin, that accelerate metabolism of or cause resistance to 1,25(OH)2D. Calcium supplementation should include 1.5–2 g/d of elemental calcium. Normocalcemia is usually observed within 1 week of the institution of therapy, although increases in PTH and alkaline phosphatase levels may persist for 3–6

1	include 1.5–2 g/d of elemental calcium. Normocalcemia is usually observed within 1 week of the institution of therapy, although increases in PTH and alkaline phosphatase levels may persist for 3–6 months. The most efficacious methods to monitor treatment and resolution of vitamin D deficiency are serum and urinary calcium measurements. In patients who are vitamin D replete and are taking adequate calcium supplementation, the 24-h urinary calcium excretion should be in the range of 100–250 mg/24 h. Lower levels suggest problems with adherence to the treatment regimen or with absorption of calcium or vitamin D supplements. Levels >250 mg/24 h predispose to nephrolithiasis and should lead to a reduction in vitamin D dosage and/or calcium supplementation.

1	Disorders of the Parathyroid 424 gland and Calcium Homeostasis John T. Potts, Jr., Harald Jpner

1	The four parathyroid glands are located posterior to the thyroid gland. They produce parathyroid hormone (PTH), which is the primary regulator of calcium physiology. PTH acts directly on bone, where it induces calcium release; on the kidney, where it enhances calcium reabsorption in the distal tubules; and in the proximal renal tubules, where it synthesizes 1,25-dihydroxyvitamin D (1,25[OH]2D), a hormone that increases gastrointestinal calcium absorption. Serum PTH levels are tightly regulated by a negative feedback loop. Calcium, acting through the calcium-sensing receptor, and vitamin D, acting through its nuclear receptor, reduce PTH release and synthesis. Additional evidence indicates that fibroblast growth factor 23 (FGF23), a phosphaturic hormone, can suppress PTH secretion. Understanding the hormonal pathways that regulate calcium levels and bone metabolism is essential for effective diagnosis and management of a wide array of hyperand hypocalcemic disorders.

1	Hyperparathyroidism, characterized by excess production of PTH, is a common cause of hypercalcemia and is usually the result of autonomously functioning adenomas or hyperplasia. Surgery for this disorder is highly effective and has been shown to reverse some of the deleterious effects of long-standing PTH excess on bone density. Humoral hypercalcemia of malignancy is also common and is usually due to the overproduction of parathyroid hormone–related peptide (PTHrP) by cancer cells. The similarities in the biochemical characteristics of hyperparathyroidism and humoral hypercalcemia of malignancy, first noted by Albright in 1941, are now known to reflect the actions of PTH and PTHrP through the same G protein–coupled PTH/PTHrP receptor.

1	The genetic basis of multiple endocrine neoplasia (MEN) types 1 and 2, familial hypocalciuric hypercalcemia (FHH), different forms of pseudohypoparathyroidism, Jansen’s syndrome, disorders of vitamin D synthesis and action, and the molecular events associated with parathyroid gland neoplasia have provided new insights into the regulation of calcium homeostasis. PTH and possibly some of its analogues are promising therapeutic agents for the treatment of postmenopausal or senile osteoporosis, and calcimimetic agents, which activate the calcium-sensing receptor, have provided new approaches for PTH suppression.

1	The primary function of PTH is to maintain the extracellular fluid (ECF) calcium concentration within a narrow normal range. The hormone acts directly on bone and kidney and indirectly on the intestine through its effects on synthesis of 1,25(OH)2D to increase serum calcium concentrations; in turn, PTH production is closely regulated by the concentration of serum ionized calcium. This feedback system is the critical homeostatic mechanism for maintenance of ECF calcium. Any tendency toward hypocalcemia, as might be induced by calciumor vitamin D–deficient diets, is counteracted by an increased secretion of PTH. This in turn (1) increases the rate of dissolution of bone mineral, thereby increasing the flow of calcium from bone into blood; (2) reduces the renal clearance of calcium, returning more of the calcium and phosphate filtered at the glomerulus into ECF; and (3) increases the efficiency of calcium absorption in the intestine by stimulating the production of 1,25(OH)2D. Immediate

1	more of the calcium and phosphate filtered at the glomerulus into ECF; and (3) increases the efficiency of calcium absorption in the intestine by stimulating the production of 1,25(OH)2D. Immediate control of blood calcium is due to PTH effects on bone and, to a lesser extent, on renal calcium clearance. Maintenance of steady-state calcium balance, on the other hand, probably results from the effects of 1,25(OH)2D on calcium absorption (Chap. 423). The renal actions of the hormone are exerted at multiple sites and include inhibition of phosphate transport (proximal tubule), augmentation of calcium reabsorption (distal tubule), and stimulation of the renal 25(OH)D-1α-hydroxylase. As much as 12 mmol (500 mg) of calcium is transferred between the ECF and bone each day (a large amount in relation to the total ECF calcium pool), and PTH has a major effect on this transfer. The homeostatic role of the hormone can preserve calcium concentration in blood at the cost of bone demineralization.

1	PTH has multiple actions on bone, some direct and some indirect. PTH-mediated changes in bone calcium release can be seen within minutes. The chronic effects of PTH are to increase the number of bone cells, both osteoblasts and osteoclasts, and to increase the remodeling of bone; these effects are apparent within hours after the hormone is given and persist for hours after PTH is withdrawn. Continuous exposure to elevated PTH (as in hyperparathyroidism or long-term infusions in animals) leads to increased osteoclast-mediated bone resorption. However, the intermittent administration of PTH, elevating hormone levels for 1–2 h each day, leads to a net stimulation of bone formation rather than bone breakdown. Striking increases, especially in trabecular bone in the spine and hip, have been reported with the use of PTH in combination with estrogen. PTH(1–34) as monotherapy caused a highly significant reduction in fracture incidence in a worldwide placebo-controlled trial.

1	Osteoblasts (or stromal cell precursors), which have PTH/PTHrP receptors, are crucial to this bone-forming effect of PTH; osteoclasts, which mediate bone breakdown, lack such receptors. PTH-mediated stimulation of osteoclasts is indirect, acting in part through cytokines released from osteoblasts to activate osteoclasts; in experimental studies of bone resorption in vitro, osteoblasts must be present for PTH to activate osteoclasts to resorb bone (Chap. 423).

1	PTH is an 84-amino-acid single-chain peptide. The amino-terminal portion, PTH(1–34), is highly conserved and is critical for the biologic actions of the molecule. Modified synthetic fragments of the amino-terminal sequence as small as PTH(1–11) are sufficient to activate the PTH/PTHrP receptor (see below). The carboxyl-terminal region of the full-length PTH(1–84) molecule also can bind to a separate binding protein/receptor (cPTH-R), but this receptor has been incompletely characterized. Fragments shortened at the amino-terminus possibly by binding to cPTH-R can reduce, directly or indirectly, some of the biologic actions of full-length PTH(1–84) and of PTH(1–34).

1	BIOSYNTHESIS, SECRETION, AND METABOLISM Synthesis Parathyroid cells have multiple methods of adapting to increased needs for PTH production. Most rapid (within minutes) is secretion of preformed hormone in response to hypocalcemia. Second, within hours, PTH mRNA expression is induced by sustained hypocalcemia. Finally, protracted challenge leads within days to cellular replication to increase parathyroid gland mass. PTH is initially synthesized as a larger molecule (preproparathyroid hormone, consisting of 115 amino acids). After a first cleavage step to remove the “pre” sequence of 25 amino acid residues, a second cleavage step removes the “pro” sequence of 6 amino acid residues before secretion of the mature peptide comprising 84 residues. Mutations in the preprotein region of the gene can cause hypoparathyroidism by interfering with hormone synthesis, transport, or secretion.

1	Transcriptional suppression of the PTH gene by calcium is nearly maximal at physiologic calcium concentrations. Hypocalcemia increases transcriptional activity within hours. 1,25(OH)2D strongly suppresses 2467 PTH gene transcription. In patients with renal failure, IV administration of supraphysiologic levels of 1,25(OH)2D or analogues of this active metabolite can dramatically suppress PTH overproduction, which is sometimes difficult to control due to severe secondary hyperparathyroidism. Regulation of proteolytic destruction of preformed hormone (posttranslational regulation of hormone production) is an important mechanism for mediating rapid (within minutes) changes in hormone availability. High calcium increases and low calcium inhibit the proteolytic destruction of stored hormone.

1	Regulation of PTH Secretion PTH secretion increases steeply to a maximum value of about five times the basal rate of secretion as the calcium concentration falls from normal to the range of 1.9–2.0 mmol/L (7.6–8.0 mg/dL; measured as total calcium). However, the ionized fraction of blood calcium is the important determinant of hormone secretion. Severe intracellular magnesium deficiency impairs PTH secretion (see below).

1	ECF calcium controls PTH secretion by interaction with a calcium-sensing receptor (CaSR), a G protein–coupled receptor (GPCR) for which Ca2+ ions act as the primary ligand (see below). This receptor is a member of a distinctive subgroup of the GPCR superfamily that mediates its actions through the alpha-subunits of two related signaling G proteins, namely Gq and G11, and is characterized by a large extracellular domain suitable for “clamping” the small-molecule ligand. Stimulation of the CaSR by high calcium levels suppresses PTH secretion. The CaSR is present in parathyroid glands and the calcitonin-secreting cells of the thyroid (C cells), as well as in multiple other sites, including brain and kidney. Genetic evidence has revealed a key biologic role for the CaSR in parathyroid gland responsiveness to calcium and in renal calcium clearance. Heterozygous loss-of-function mutations in CaSR cause the syndrome of FHH, in which the blood calcium abnormality resembles that observed in

1	responsiveness to calcium and in renal calcium clearance. Heterozygous loss-of-function mutations in CaSR cause the syndrome of FHH, in which the blood calcium abnormality resembles that observed in hyperparathyroidism but with hypocalciuria; two more recently defined variants of FHH, FHH2 and FHH3, are caused either by heterozygous mutations in G11, one of the signaling proteins downstream of the CaSR, or by hetero zygous mutations in AP2S1. Homozygous loss-of-function mutations in the CaSR are the cause of severe neonatal hyperparathyroidism, a disorder that can be lethal if not treated within the first days of life. On the other hand, heterozygous gain-of-function mutations cause a form of hypocalcemia resembling hypoparathyroidism (see below).

1	Metabolism The secreted form of PTH is indistinguishable by immunologic criteria and by molecular size from the 84-amino-acid peptide (PTH[1–84]) extracted from glands. However, much of the immunoreactive material found in the circulation is smaller than the extracted or secreted hormone. The principal circulating fragments of immunoreactive hormone lack a portion of the critical amino-terminal sequence required for biologic activity and, hence, are biologically inactive fragments (so-called middle and carboxyl-terminal fragments). Much of the proteolysis of the hormone occurs in the liver and kidney. Peripheral metabolism of PTH does not appear to be regulated by physiologic states (high versus low calcium, etc.); hence, peripheral metabolism of hormone, although responsible for rapid clearance of secreted hormone, appears to be a high-capacity, metabolically invariant catabolic process.

1	The rate of clearance of the secreted 84-amino-acid peptide from blood is more rapid than the rate of clearance of the biologically inactive fragment(s) corresponding to the middle and carboxyl-terminal regions of PTH. Consequently, the interpretation of results obtained with earlier PTH radioimmunoassays was influenced by the nature of the peptide fragments detected by the antibodies. Although the problems inherent in PTH measurements have been largely circumvented by use of double-antibody immunometric assays, it is now known that some of these assays detect, besides the intact molecule, large amino-terminally truncated forms of PTH, which are present in normal and uremic individuals in addition to PTH(1–84). The concentration of these fragments relative to that of intact PTH(1–84) is higher with induced hypercalcemia than in eucalcemic

1	Disorders of the Parathyroid Gland and Calcium Homeostasis 2468 or hypocalcemic conditions and is higher in patients with impaired especially of the squamous cell type as well as renal cell carcinomas, renal function. PTH(7–84) has been identified as a major component lead to massive overproduction of the hormone and hypercalcemia. of these amino-terminally truncated fragments. Growing evidence suggests that the PTH(7–84) (and probably related amino-terminally PTH AND PTHrP HORMONE ACTION truncated fragments) can act, through yet undefined mechanisms, Both PTH and PTHrP bind to and activate the PTH/PTHrP receptor.as an inhibitor of PTH action and may be of clinical significance, The PTH/PTHrP receptor (also known as the PTH-1 receptor, PTH1R) particularly in patients with chronic kidney disease. In this group of belongs to a subfamily of GPCRs that includes the receptors for calcitopatients, efforts to prevent secondary hyperparathyroidism by a variety nin, glucagon, secretin,

1	disease. In this group of belongs to a subfamily of GPCRs that includes the receptors for calcitopatients, efforts to prevent secondary hyperparathyroidism by a variety nin, glucagon, secretin, vasoactive intestinal peptide, and other peptides.of measures (vitamin D analogues, higher calcium intake, higher dialy-Although both ligands activate the PTH1R, the two peptides induce sate calcium, phosphate-lowering strategies, and calcimetic drugs) distinct responses in the receptor, which explains how a single receptorcan lead to oversuppression of the parathyroid glands since some without isoforms can serve two biologic roles. The extracellular regions amino-terminally truncated PTH fragments, such as PTH(7–84), react of the receptor are involved in hormone binding, and the intracellularin many immunometric PTH assays (now termed second-generation domains, after hormone activation, bind G protein subunits to trans-assays; see below under “Diagnosis”), thus overestimating the levels duce

1	immunometric PTH assays (now termed second-generation domains, after hormone activation, bind G protein subunits to trans-assays; see below under “Diagnosis”), thus overestimating the levels duce hormone signaling into cellular responses through the stimulationof biologically active, intact PTH. Such excessive parathyroid gland of second messenger formation. A second receptor that binds PTH,suppression in chronic kidney disease can lead to adynamic bone originally termed the PTH-2 receptor (PTH2R), is primarily expresseddisease (see below), which has been associated with further impaired in brain, pancreas, and testis. Different mammalian PTH1Rs respondgrowth in children and increased bone fracture rates in adults, and can equivalently to PTH and PTHrP, at least when tested with traditionalfurthermore lead to significant hypercalcemia. The measurement of assays, whereas only the human PTH2R responds efficiently to PTHPTH with newer third-generation immunoassays, which use detection

1	lead to significant hypercalcemia. The measurement of assays, whereas only the human PTH2R responds efficiently to PTHPTH with newer third-generation immunoassays, which use detection (but not to PTHrP). PTH2Rs from other species show little or no stimuantibodies directed against extreme amino-terminal PTH epitopes and lation of second-messenger formation in response to PTH or PTHrP.thus detect only full-length PTH(1–84), may provide some advantage The endogenous ligand of the PTH2R was shown to be a hypothalamicto prevent bone disease in chronic kidney disease. peptide referred to as tubular infundibular peptide of 39 residues, TIP39, that is distantly related to PTH and PTHrP. The PTH1R and the PTH2R can be traced backward in evolutionary time to fish; in fact, the zebrafish genome contains, in addition to the PTH1R and the PTH2R orthologs, a PTHrP is responsible for most instances of humoral hypercalcemia third receptor, the PTH3R, that is more closely related to the fish PTH1R of

1	in addition to the PTH1R and the PTH2R orthologs, a PTHrP is responsible for most instances of humoral hypercalcemia third receptor, the PTH3R, that is more closely related to the fish PTH1R of malignancy (Chap. 121), a syndrome that resembles primary than to the fish PTH2R. The evolutionary conservation of structure and hyperparathyroidism but without elevated PTH levels. Most cell types function suggests important biologic roles for these receptors, even in normally produce PTHrP, including brain, pancreas, heart, lung, fish, which lack discrete parathyroid glands but produce two molecules mammary tissue, placenta, endothelial cells, and smooth muscle. In that are closely related to mammalian PTH. fetal animals, PTHrP directs transplacental calcium transfer, and Studies using the cloned PTH1R confirm that it can be coupled high concentrations of PTHrP are produced in mammary tissue and to more than one G protein and second-messenger pathway, apparsecreted into milk, but the biologic

1	PTH1R confirm that it can be coupled high concentrations of PTHrP are produced in mammary tissue and to more than one G protein and second-messenger pathway, apparsecreted into milk, but the biologic significance of the very high con-ently explaining the multiplicity of pathways stimulated by PTH. centrations of this hormone in breast milk is unknown. PTHrP also Activation of protein kinases (A and C) and calcium transport chanplays an essential role in endochondral bone formation and in branch-nels is associated with a variety of hormone-specific tissue responses. ing morphogenesis of the breast, and possibly in uterine contraction These responses include inhibition of phosphate and bicarbonate and other biologic functions. transport, stimulation of calcium transport, and activation of renal

1	PTH and PTHrP, although products of different genes, exhibit con-1α-hydroxylase in the kidney. The responses in bone include effects siderable functional and structural homology (Fig. 424-1) and have on collagen synthesis, alkaline phosphatase, ornithine decarboxylase, evolved from a shared ancestral gene. The structure of the gene encod-citrate decarboxylase, and glucose-6-phosphate dehydrogenase activiing human PTHrP, however, is more complex than that of PTH, con-ties; phospholipid synthesis; and calcium and phosphate transport. taining multiple additional exons, which can undergo alternate splic-Ultimately, these biochemical events lead to an integrated hormonal ing patterns during formation of the mature mRNA. Protein products response in bone turnover and calcium homeostasis. PTH also actiof 139, 141, and 173 amino acids are produced, and other molecular vates Na+/Ca2+ exchangers at renal distal tubular sites and stimulates forms may result from tissue-specific degradation at

1	also actiof 139, 141, and 173 amino acids are produced, and other molecular vates Na+/Ca2+ exchangers at renal distal tubular sites and stimulates forms may result from tissue-specific degradation at accessible internal translocation of preformed calcium transport channels, moving them cleavage sites. The biologic roles of these various molecular species and from the interior to the apical surface to increase tubular uptake of the nature of the circulating forms of PTHrP are unclear. In fact, it is calcium. PTH-dependent stimulation of phosphate excretion (reducuncertain whether PTHrP circulates at any significant level in adults. ing reabsorption—the opposite effect from actions on calcium in As a paracrine factor, PTHrP may be produced, act, and be destroyed the kidney) involves the downregulation of two sodium-dependent locally within tissues. In adults, PTHrP appears to have little influence phosphate co-transporters, NPT2a and NPT2c, and their expression on calcium homeostasis,

1	of two sodium-dependent locally within tissues. In adults, PTHrP appears to have little influence phosphate co-transporters, NPT2a and NPT2c, and their expression on calcium homeostasis, except in disease states, when large tumors, at the apical membrane, thereby reducing phosphate reabsorption in

1	FIGURE 424-1 Schematic diagram to illustrate similarities and differences in structure of human parathyroid hormone (PTH) and human PTH-related peptide (PTHrP). Close structural (and functional) homology exists between the first 30 amino acids of hPTH and hPTHrP. The PTHrP sequence may be ≥144 amino acid residues in length. PTH is only 84 residues long; after residue 30, there is little structural homology between the two. Dashed lines in the PTHrP sequence indicate identity; underlined residues, although different from those of PTH, still represent conservative changes (charge or polarity preserved). Ten amino acids are identical, and a total of 20 of 30 are homologues.

1	FIGURE 424-2 Dual role for the actions of the PTH/PTHrP receptor (PTH1R). Parathyroid hormone (PTH; endocrine-calcium homeostasis) and PTH-related peptide (PTHrP; paracrine–multiple tissue actions including growth plate cartilage in developing bone) use the single receptor for their disparate functions mediated by the amino-terminal 34 residues of either peptide. Other regions of both ligands interact with other receptors (not shown). the proximal renal tubules. Similar mechanisms may be involved in other renal tubular transporters that are influenced by PTH. Recent studies reaffirm the critical linkage of blood phosphate lowering to net calcium entry into blood by PTH action and emphasize the participation of bone cells other than osteoclasts in the rapid calcium-elevating actions of PTH.

1	PTHrP exerts important developmental influences on fetal bone development and in adult physiology. A homozygous ablation of the gene encoding PTHrP (or disruption of the PTH1R gene) in mice causes a lethal phenotype in which animals are born with pronounced acceleration of chondrocyte maturation that resembles a lethal form of chondrodysplasia in humans that is caused by homozygous or compound heterozygous, inactivating PTH1R mutations (Fig. 424-2). Heterozygous PTH1R mutations in humans furthermore can be a cause of delayed tooth eruption, and mice that are heterozygous for ablation of the PTHrP gene display reduced mineral density consistent with osteoporosis. Experiments with these mouse models point to a hitherto unappreciated role of PTHrP as a paracrine/autocrine factor that modulates bone metabolism in adults as well as during bone development.

1	(See also Chap. 408) Calcitonin is a hypocalcemic peptide hormone that in several mammalian species acts as an indirect antagonist to the calcemic actions of PTH. Calcitonin seems to be of limited physiologic significance in humans, at least with regard to calcium homeostasis. It is of medical significance because of its role as a tumor marker in sporadic and hereditary cases of medullary carcinoma and its medical use as an adjunctive treatment in severe hypercalcemia and in Paget’s disease of bone.

1	The hypocalcemic activity of calcitonin is accounted for primarily by inhibition of osteoclast-mediated bone resorption and secondarily by stimulation of renal calcium clearance. These effects are mediated by receptors on osteoclasts and renal tubular cells. Calcitonin exerts additional effects through receptors present in the brain, the gastrointestinal tract, and the immune system. The hormone, for example, exerts analgesic effects directly on cells in the hypothalamus and related structures, possibly by interacting with receptors for related peptide hormones such as calcitonin gene–related peptide (CGRP) or amylin. Both of these ligands have specific high-affinity receptors that share considerable structural similarity with the PTH1R and can also bind to and activate calcitonin receptors. The calcitonin receptor shares considerable structural similarity with the PTH1R.

1	The thyroid is the major source of the hormone, and the cells 2469 involved in calcitonin synthesis arise from neural crest tissue. During embryogenesis, these cells migrate into the ultimobranchial body, derived from the last branchial pouch. In submammalian vertebrates, the ultimobranchial body constitutes a discrete organ, anatomically separate from the thyroid gland; in mammals, the ultimobranchial gland fuses with and is incorporated into the thyroid gland. The naturally occurring calcitonins consist of a peptide chain of 32 amino acids. There is considerable sequence variability among species. Calcitonin from salmon, which is used therapeutically, is 10–100 times more potent than mammalian forms in lowering serum calcium.

1	There are two calcitonin genes, α and β; the transcriptional control of these genes is complex. Two different mRNA molecules are transcribed from the α gene; one is translated into the precursor for calcitonin, and the other message is translated into an alternative product, CGRP. CGRP is synthesized wherever the calcitonin mRNA is expressed (e.g., in medullary carcinoma of the thyroid). The β, or CGRP-2, gene is transcribed into the mRNA for CGRP in the central nervous system (CNS); this gene does not produce calcitonin, however. CGRP has cardiovascular actions and may serve as a neurotransmitter or play a developmental role in the CNS.

1	The circulating level of calcitonin in humans is lower than that in many other species. In humans, even extreme variations in calcitonin production do not change calcium and phosphate metabolism; no definite effects are attributable to calcitonin deficiency (totally thyroidectomized patients receiving only replacement thyroxine) or excess (patients with medullary carcinoma of the thyroid, a calcitonin secreting tumor) (Chap. 408). Calcitonin has been a useful pharmacologic agent to suppress bone resorption in Paget’s disease (Chap. 426e) and osteoporosis (Chap. 425) and in the treatment of hypercalcemia of malignancy (see below). However, bisphosphonates are usually more effective, and the physiologic role, if any, of calcitonin in humans is uncertain. On the other hand, ablation of the calcitonin gene (combined because of the close proximity with ablation of the CGRP gene) in mice leads to reduced bone mineral density, suggesting that its biologic role in mammals is still not fully

1	calcitonin gene (combined because of the close proximity with ablation of the CGRP gene) in mice leads to reduced bone mineral density, suggesting that its biologic role in mammals is still not fully understood.

1	(See also Chap. 65) Hypercalcemia can be a manifestation of a serious illness such as malignancy or can be detected coincidentally by laboratory testing in a patient with no obvious illness. The number of patients recognized with asymptomatic hypercalcemia, usually hyperparathyroidism, increased in the late twentieth century. Whenever hypercalcemia is confirmed, a definitive diagnosis must be established. Although hyperparathyroidism, a frequent cause of asymptomatic hypercalcemia, is a chronic disorder in which manifestations, if any, may be expressed only after months or years, hypercalcemia can also be the earliest manifestation of malignancy, the second most common cause of hypercalcemia in the adult. The causes of hypercalcemia are numerous (Table 424-1), but hyperparathyroidism and cancer account for 90% of all cases.

1	Before undertaking a diagnostic workup, it is essential to be sure that true hypercalcemia, not a false-positive laboratory test, is present. A false-positive diagnosis of hypercalcemia is usually the result of inadvertent hemoconcentration during blood collection or elevation in serum proteins such as albumin. Hypercalcemia is a chronic problem, and it is cost-effective to obtain several serum calcium measurements; these tests need not be in the fasting state.

1	Clinical features are helpful in differential diagnosis. Hypercalcemia in an adult who is asymptomatic is usually due to primary hyperparathyroidism. In malignancy-associated hypercalcemia, the disease is usually not occult; rather, symptoms of malignancy bring the patient to the physician, and hypercalcemia is discovered during the evaluation. In such patients, the interval between detection of hypercalcemia and death, especially without vigorous treatment, is often <6 months. Accordingly, if an asymptomatic individual has had hypercalcemia or some manifestation of hypercalcemia such as kidney stones for more than 1 or 2 years, it is unlikely that malignancy Disorders of the Parathyroid Gland and Calcium Homeostasis ClASSifiCATion of CAuSES of HyPERCAlCEmiA I. Parathyroid-Related A. Primary hyperparathyroidism 1. 2. 3. B. Lithium therapy C. Familial hypocalciuric hypercalcemia II. Malignancy-Related A. Solid tumor with metastases (breast)

1	I. Parathyroid-Related A. Primary hyperparathyroidism 1. 2. 3. B. Lithium therapy C. Familial hypocalciuric hypercalcemia II. Malignancy-Related A. Solid tumor with metastases (breast) B. Solid tumor with humoral mediation of hypercalcemia (lung, kidney) C. Hematologic malignancies (multiple myeloma, lymphoma, leukemia) III. Vitamin D–Related A. Vitamin D intoxication B. ↑ 1,25(OH)2D; sarcoidosis and other granulomatous diseases C. ↑ 1,25(OH)2D; impaired 1,25(OH)2D metabolism due to 24-hydroxylase deficiency IV. Associated with High Bone Turnover A. Hyperthyroidism B. Immobilization C. Thiazides D. Vitamin A intoxication E. Fat necrosis V. Associated with Renal Failure A. Severe secondary hyperparathyroidism B. Aluminum intoxication

1	A. Hyperthyroidism B. Immobilization C. Thiazides D. Vitamin A intoxication E. Fat necrosis V. Associated with Renal Failure A. Severe secondary hyperparathyroidism B. Aluminum intoxication C. Milk-alkali syndrome is the cause. Nevertheless, differentiating primary hyperparathyroidism from occult malignancy can occasionally be difficult, and careful evaluation is required, particularly when the duration of the hypercalcemia is unknown. Hypercalcemia not due to hyperparathyroidism or malignancy can result from excessive vitamin D action, impaired metabolism of 1,25(OH)2D, high bone turnover from any of several causes, or renal failure (Table 424-1). Dietary history and a history of ingestion of vitamins or drugs are often helpful in diagnosing some of the less frequent causes. Immunometric PTH assays serve as the principal laboratory test in establishing the diagnosis.

1	Hypercalcemia from any cause can result in fatigue, depression, mental confusion, anorexia, nausea, vomiting, constipation, reversible renal tubular defects, increased urine output, a short QT interval in the electrocardiogram, and, in some patients, cardiac arrhythmias. There is a variable relation from one patient to the next between the severity of hypercalcemia and the symptoms. Generally, symptoms are more common at calcium levels >2.9–3.0 mmol/L (11.6–12.0 mg/dL), but some patients, even at this level, are asymptomatic. When the calcium level is >3.2 mmol/L (12.8 mg/dL), calcification in kidneys, skin, vessels, lungs, heart, and stomach occurs and renal insufficiency may develop, particularly if blood phosphate levels are normal or elevated due to impaired renal excretion. Severe hypercalcemia, usually defined as ≥3.7–4.5 mmol/L (14.8–18.0 mg/dL), can be a medical emergency; coma and cardiac arrest can occur.

1	Acute management of the hypercalcemia is usually successful. The type of treatment is based on the severity of the hypercalcemia and the nature of associated symptoms, as outlined below.

1	PRIMARY HYPERPARATHYROIDISM Natural History and Incidence Primary hyperparathyroidism is a generalized disorder of calcium, phosphate, and bone metabolism due to an increased secretion of PTH. The elevation of circulating hormone usually leads to hypercalcemia and hypophosphatemia. There is great variation in the manifestations. Patients may present with multiple signs and symptoms, including recurrent nephrolithiasis, peptic ulcers, mental changes, and, less frequently, extensive bone resorption. However, with greater awareness of the disease and wider use of multiphasic screening tests, including measurements of blood calcium, the diagnosis is frequently made in patients who have no symptoms and minimal, if any, signs of the disease other than hypercalcemia and elevated levels of PTH. The manifestations may be subtle, and the disease may have a benign course for many years or a lifetime. This milder form of the disease is usually termed asymptomatic hyperparathyroidism. Rarely,

1	The manifestations may be subtle, and the disease may have a benign course for many years or a lifetime. This milder form of the disease is usually termed asymptomatic hyperparathyroidism. Rarely, hyperparathyroidism develops or worsens abruptly and causes severe complications such as marked dehydration and coma, so-called hypercalcemic parathyroid crisis.

1	The annual incidence of the disease is calculated to be as high as 0.2% in patients >60, with an estimated prevalence, including undiscovered asymptomatic patients, of ≥1%; some reports suggest the incidence may be declining. If confirmed, these changing estimates may reflect less frequent routine testing of serum calcium in recent years, earlier overestimates in incidence, or unknown factors. The disease has a peak incidence between the third and fifth decades but occurs in young children and in the elderly. Etiology Parathyroid tumors are most often encountered as isolated adenomas without other endocrinopathy. They may also arise in hereditary syndromes such as MEN syndromes. Parathyroid tumors may also arise as secondary to underlying disease (excessive stimulation in secondary hyperparathyroidism, especially chronic renal failure) or after other forms of excessive stimulation such as lithium therapy. These etiologies are discussed below.

1	Solitary adenomaS A single abnormal gland is the cause in ~80% of patients; the abnormality in the gland is usually a benign neoplasm or adenoma and rarely a parathyroid carcinoma. Some surgeons and pathologists report that the enlargement of multiple glands is common; double adenomas are reported. In ~15% of patients, all glands are hyperfunctioning; chief cell parathyroid hyperplasia is usually hereditary and frequently associated with other endocrine abnormalities.

1	Hereditary SyndromeS and multiple paratHyroid tumorS Hereditary hyperparathyroidism can occur without other endocrine abnormalities but is usually part of a multiple endocrine neoplasia (MEN) syndrome (Chap. 408). MEN 1 (Wermer’s syndrome) consists of hyperparathyroidism and tumors of the pituitary and pancreas, often associated with gastric hypersecretion and peptic ulcer disease (Zollinger-Ellison syndrome). MEN 2A is characterized by pheochromocytoma and medullary carcinoma of the thyroid, as well as hyperparathyroidism; MEN 2B has additional associated features such as multiple neuromas but usually lacks hyperparathyroidism. Each of these MEN syndromes is transmitted in an apparent autosomal dominant manner, although, as noted below, the genetic basis of MEN 1 involves biallelic loss of a tumor suppressor.

1	The hyperparathyroidism jaw tumor (HPT-JT) syndrome occurs in families with parathyroid tumors (sometimes carcinomas) in association with benign jaw tumors. This disorder is caused by mutations in CDC73 (HRPT2), and mutations in this gene are also observed in parathyroid cancers. Some kindreds exhibit hereditary hyperparathyroidism without other endocrinopathies. This disorder is often termed nonsyndromic familial isolated hyperparathyroidism (FIHP). There is speculation that these families may be examples of variable expression of the other syndromes such as MEN 1, MEN 2, or the HPT-JT syndrome, but they may also have distinctive, still unidentified genetic causes.

1	Pathology Adenomas are most often located in the inferior parathyroid glands, but in 6–10% of patients, parathyroid adenomas may be located in the thymus, the thyroid, the pericardium, or behind the esophagus. Adenomas are usually 0.5–5 g in size but may be as large as 10–20 g (normal glands weigh 25 mg on average). Chief cells are predominant in both hyperplasia and adenoma. With chief cell hyperplasia, the enlargement may be so asymmetric that some involved glands appear grossly normal. If generalized hyperplasia is present, however, histologic examination reveals a uniform pattern of chief cells and disappearance of fat even in the absence of an increase in gland weight. Thus, microscopic examination of biopsy specimens of several glands is essential to interpret findings at surgery.

1	Parathyroid carcinoma is often not aggressive. Long-term survival without recurrence is common if at initial surgery the entire gland is removed without rupture of the capsule. Recurrent parathyroid carcinoma is usually slow-growing with local spread in the neck, and surgical correction of recurrent disease may be feasible. Occasionally, however, parathyroid carcinoma is more aggressive, with distant metastases (lung, liver, and bone) found at the time of initial operation. It may be difficult to appreciate initially that a primary tumor is carcinoma; increased numbers of mitotic figures and increased fibrosis of the gland stroma may precede invasion. The diagnosis of carcinoma is often made in retrospect. Hyperparathyroidism from a parathyroid carcinoma may be indistinguishable from other forms of primary hyperparathyroidism but is usually more severe clinically. A potential clue to the diagnosis is offered by the degree of calcium elevation. Calcium values of 3.5–3.7 mmol/L (14–15

1	forms of primary hyperparathyroidism but is usually more severe clinically. A potential clue to the diagnosis is offered by the degree of calcium elevation. Calcium values of 3.5–3.7 mmol/L (14–15 mg/dL) are frequent with carcinoma and may alert the surgeon to remove the abnormal gland with care to avoid capsular rupture. Recent findings concerning the genetic basis of parathyroid carcinoma (distinct from that of benign adenomas) indicate the need, in these kindreds, for family screening (see below).

1	As in many other types of neoplasia, two fundamental types of genetic defects have been identified in parathyroid gland tumors: (1) overactivity of protooncogenes and (2) loss of function of tumor-suppressor genes. The former, by definition, can lead to uncontrolled cellular growth and function by activation (gain-of-function mutation) of a single allele of the responsible gene, whereas the latter requires loss of function of both allelic copies. Biallelic loss of function of a tumor-suppressor gene is usually characterized by a germline defect (all cells) and an additional somatic deletion/mutation in the tumor (Fig. 424-3).

1	Mutations in the MEN1 gene locus, encoding the protein MENIN, 2471 on chromosome 11q13 are responsible for causing MEN 1; the normal allele of this gene fits the definition of a tumor-suppressor gene. Inheritance of one mutated allele in this hereditary syndrome, followed by loss of the other allele via somatic cell mutation, leads to monoclonal expansion and tumor development. Also, in ~15–20% of sporadic parathyroid adenomas, both alleles of the MEN1 locus on chromosome 11 are somatically deleted, implying that the same defect responsible for MEN 1 can also cause the sporadic disease (Fig. 424-3A). Consistent with the Knudson hypothesis for two-step neoplasia in certain inherited cancer syndromes (Chap. 101e), the earlier onset of hyperparathyroidism in the hereditary syndromes reflects the need for only one mutational event to trigger the monoclonal outgrowth. In sporadic adenomas, typically occurring later in life, two different somatic events must occur before the MEN1 gene is

1	the need for only one mutational event to trigger the monoclonal outgrowth. In sporadic adenomas, typically occurring later in life, two different somatic events must occur before the MEN1 gene is silenced.

1	Other presumptive anti-oncogenes involved in hyperparathyroidism include a still unidentified gene mapped to chromosome 1p seen in 40% of sporadic parathyroid adenomas and a gene mapped to chromosome Xp11 in patients with secondary hyperparathyroidism and renal failure, who progressed to “tertiary” hyperparathyroidism, now known to reflect monoclonal outgrowths within previously hyperplastic glands. A more complex pattern, still incompletely resolved, arises with genetic defects and carcinoma of the parathyroids. This appears to be due to biallelic loss of a functioning copy of a gene, HRPT2 (or CDC73), originally identified as the cause of the HPT-JT syndrome. Several inactivating mutations have been identified in HRPT2 (located on chromosome 1q21-31), which encodes a 531-amino-acid protein called parafibromin. The responsible genetic mutations in HRPT2 appear to be necessary, but not sufficient, for parathyroid cancer.

1	In general, the detection of additional genetic defects in these parathyroid tumor–related syndromes and the variations seen in Somatic mutation of one copy of the Clonal progenitor cell lacks HRPT2 tumor suppressor gene on functional HRPT2 gene product 1q21–31 no adverse consequences A to parathyroid cell

1	FIGURE 424-3 A. Schematic diagram indicating molecular events in tumor susceptibility. The patient with the hereditary abnormality (multiple endocrine neoplasia [MEN]) is envisioned as having one defective gene inherited from the affected parent on chromosome 11, but one copy of the normal gene is present from the other parent. In the monoclonal tumor (benign tumor), a somatic event, here partial chromosomal deletion, removes the remaining normal gene from a cell. In nonhereditary tumors, two successive somatic mutations must occur, a process that takes a longer time. By either pathway, the cell, deprived of growth-regulating influence from this gene, has unregulated growth and becomes a tumor. A different genetic locus also involving loss of a tumor-suppressor gene termed HRPT2 is involved in the pathogenesis of parathyroid carcinoma. (From A Arnold: J Clin Endocrine Metab 77:1108, 1993. Copyright 1993, The Endocrine Society.) B. Schematic illustration of the mechanism and

1	is involved in the pathogenesis of parathyroid carcinoma. (From A Arnold: J Clin Endocrine Metab 77:1108, 1993. Copyright 1993, The Endocrine Society.) B. Schematic illustration of the mechanism and consequences of gene rearrangement and overexpression of the PRAD1 protooncogene (pericentromeric inversion of chromosome 11) in parathyroid adenomas. The excessive expression of PRAD1 (a cell cycle control protein, cyclin D1) by the highly active parathyroid hormone (PTH) gene promoter in the parathyroid cell contributes to excess cellular proliferation. (From J Habener et al, in L DeGroot, JL Jameson [eds]: Endocrinology, 4th ed. Philadelphia, Saunders, 2001; with permission.)

1	Disorders of the Parathyroid Gland and Calcium Homeostasis Mutant copy of putative tumor Clonal progenitor cell lacks suppressor gene on 11q13 is functional gene product inherited in MEN-1 and present in all parathyroid cells

1	Mutation of one allele of same gene may occur somatically in other patients, present in specific parathyroid cell(s) 2472 phenotypic expression/penetrance indicate the multiplicity of the genetic factors responsible. Nonetheless, the ability to detect the presence of the major genetic contributors has greatly aided a more informed management of family members of patients identified in the hereditary syndromes such as MEN 1, MEN 2, and HPT-JT. An important contribution from studies on the genetic origin of parathyroid carcinoma has been the realization that the mutations involve a different pathway than that involved with the benign gland enlargements. Unlike the pathogenesis of genetic alterations seen in colon cancer, where lesions evolve from benign adenomas to malignant disease by progressive genetic changes, the alterations commonly seen in most parathyroid cancers (HRPT2 mutations) are infrequently seen in sporadic parathyroid adenomas. Abnormalities at the Rb gene were the first

1	genetic changes, the alterations commonly seen in most parathyroid cancers (HRPT2 mutations) are infrequently seen in sporadic parathyroid adenomas. Abnormalities at the Rb gene were the first to be noted in parathyroid cancer. The Rb gene, a tumor-suppressor gene located on chromosome 13q14, was initially associated with retinoblastoma but has since been implicated in other neoplasias, including parathyroid carcinoma. Early studies implicated allelic deletions of the Rb gene in many parathyroid carcinomas and decreased or absent expression of the Rb protein. However, because there are often large deletions in chromosome 13 that include many genes in addition to the Rb locus (with similar findings in some pituitary carcinomas), it remains possible that other tumor-suppressor genes on chromosome 13 may be playing a role in parathyroid carcinoma. Study of the parathyroid cancers found in some patients with the HPT-JT syndrome has led to identification of a much larger role for mutations

1	13 may be playing a role in parathyroid carcinoma. Study of the parathyroid cancers found in some patients with the HPT-JT syndrome has led to identification of a much larger role for mutations in the HRPT2 gene in most parathyroid carcinomas, including those that arise sporadically, without apparent association with the HPT-JT syndrome. Mutations in the coding region have been identified in 75–80% of all parathyroid cancers analyzed, leading to the conclusion that, with addition of presumed mutations in the noncoding regions, this genetic defect may be seen in essentially all parathyroid carcinomas. Of special importance was the discovery that, in some sporadic parathyroid cancers, germline mutations have been found; this, in turn, has led to careful investigation of the families of these patients and a new clinical indication for genetic testing in this setting. Hypercalcemia occurring in family members (who are also found to have the germline mutations) can lead to the finding, at

1	patients and a new clinical indication for genetic testing in this setting. Hypercalcemia occurring in family members (who are also found to have the germline mutations) can lead to the finding, at parathyroid surgery, of premalignant parathyroid tumors. Overall, it seems there are multiple factors in parathyroid cancer, in addition to the HRPT2 and Rb gene, although the HRPT2 gene mutation is the most invariant abnormality. RET encodes a tyrosine kinase type receptor; specific inherited germline mutations lead to a constitutive activation of the receptor, thereby explaining the autosomal dominant mode of transmission and the relatively early onset of neoplasia. In the MEN 2 syndrome, the RET protooncogene may be responsible for the earliest disorder detected, the polyclonal disorder (C cell hyperplasia, which then is transformed into a clonal outgrowth—a medullary carcinoma with the participation of other, still uncharacterized genetic defects). In some parathyroid adenomas,

1	(C cell hyperplasia, which then is transformed into a clonal outgrowth—a medullary carcinoma with the participation of other, still uncharacterized genetic defects). In some parathyroid adenomas, activation of a protooncogene has been identified (Fig. 424-3B). A reciprocal translocation involving chromosome 11 has been identified that juxtaposes the PTH gene promoter upstream of a gene product termed PRAD-1, encoding a cyclin D protein that plays a key role in normal cell division. This translocation plus other mechanisms that cause an equivalent overexpression of cyclin D1 are found in 20–40% of parathyroid adenomas. Mouse models have confirmed the role of several of the major identified genetic defects in parathyroid disease and the MEN syndromes. Loss of the MEN1 gene locus or overexpression of the PRAD-1 protooncogene or the mutated RET protooncogene have been analyzed by genetic manipulation in mice, with the expected onset of parathyroid tumors or medullary carcinoma,

1	overexpression of the PRAD-1 protooncogene or the mutated RET protooncogene have been analyzed by genetic manipulation in mice, with the expected onset of parathyroid tumors or medullary carcinoma, respectively.

1	Signs and Symptoms Many patients with hyperparathyroidism are asymptomatic. Manifestations of hyperparathyroidism involve primarily the kidneys and the skeletal system. Kidney involvement, due either to deposition of calcium in the renal parenchyma or to recurrent nephrolithiasis, was present in 60–70% of patients prior to 1970. With earlier detection, renal complications occur in <20% of patients in many large series. Renal stones are usually composed of either calcium oxalate or calcium phosphate. In occasional patients, repeated episodes of nephrolithiasis or the formation of large calculi may lead to urinary tract obstruction, infection, and loss of renal function. Nephrocalcinosis may also cause decreased renal function and phosphate retention.

1	The distinctive bone manifestation of hyperparathyroidism is osteitis fibrosa cystica, which occurred in 10–25% of patients in series reported 50 years ago. Histologically, the pathognomonic features are an increase in the giant multinucleated osteoclasts in scalloped areas on the surface of the bone (Howship’s lacunae) and a replacement of the normal cellular and marrow elements by fibrous tissue. X-ray changes include resorption of the phalangeal tufts and replacement of the usually sharp cortical outline of the bone in the digits by an irregular outline (subperiosteal resorption). In recent years, osteitis fibrosa cystica is very rare in primary hyperparathyroidism, probably due to the earlier detection of the disease.

1	Dual-energy x-ray absorptiometry (DEXA) of the spine provides reproducible quantitative estimates (within a few percent) of spinal bone density. Similarly, bone density in the extremities can be quantified by densitometry of the hip or of the distal radius at a site chosen to be primarily cortical. Computed tomography (CT) is a very sensitive technique for estimating spinal bone density, but reproducibility of standard CT is no better than 5%. Newer CT techniques (spiral, “extreme” CT) are more reproducible but are currently available in a limited number of medical centers. Cortical bone density is reduced while cancellous bone density, especially in the spine, is relatively preserved. In symptomatic patients, dysfunctions of the CNS, peripheral nerve and muscle, gastrointestinal tract, and joints also occur. It has been reported that severe neuropsychiatric manifestations may be reversed by parathyroidectomy. When present in symptomatic patients, neuromuscular manifestations may

1	and joints also occur. It has been reported that severe neuropsychiatric manifestations may be reversed by parathyroidectomy. When present in symptomatic patients, neuromuscular manifestations may include proximal muscle weakness, easy fatigability, and atrophy of muscles and may be so striking as to suggest a primary neuromuscular disorder. The distinguishing feature is the complete regression of neuromuscular disease after surgical correction of the hyperparathyroidism.

1	Gastrointestinal manifestations are sometimes subtle and include vague abdominal complaints and disorders of the stomach and pancreas. Again, cause and effect are unclear. In MEN 1 patients with hyperparathyroidism, duodenal ulcer may be the result of associated pancreatic tumors that secrete excessive quantities of gastrin (Zollinger-Ellison syndrome). Pancreatitis has been reported in association with hyperparathyroidism, but the incidence and the mechanism are not established. Much attention has been paid in recent years to the manifestations of and optimum management strategies for asymptomatic hyperparathyroidism. This is now the most prevalent form of the disease. Asymptomatic primary hyperparathyroidism is defined as biochemically confirmed hyperparathyroidism (elevated or inappropriately normal PTH levels despite hypercalcemia) with the absence of signs and symptoms typically associated with more severe hyperparathyroidism such as features of renal or bone disease.

1	Three conferences on the topic have been held in the United States over the past two decades, with the most recent in 2008. The published proceedings include discussion of more subtle manifestations of disease, its natural history (without parathyroidectomy), and guidelines both for indications for surgery and medical monitoring in nonoperated patients.

1	Issues of concern include the potential for cardiovascular deterioration, the presence of subtle neuropsychiatric symptoms, and the longer-term status of skeletal integrity in patients not treated surgically. The current consensus is that medical monitoring rather than surgical correction of hyperparathyroidism may be justified in certain patients. The current recommendation is that patients who show mild disease, as defined by specific criteria (Table 424-2), can be safely followed under management guidelines (Table 424-3). There is, however, growing uncertainty about subtle disease manifestations and whether surgery is therefore indicated in most patients. Among the issues is the evidence of eventual (>8 years) deterioration in bone mineral density after a decade circulating biologically inactive fragments, detected by the original 2473 first-generation assays. Double-antibody assays are now referred to as second-generation. Such PTH assays have in some centers and testing covered

1	inactive fragments, detected by the original 2473 first-generation assays. Double-antibody assays are now referred to as second-generation. Such PTH assays have in some centers and testing covered that large PTH fragments, devoid of only the extreme amino- terminal portion of the PTH molecule, are also present in blood and are detected, incorrectly, as intact PTH. These amino-terminally trun-

1	Bone density T score <−2.5 at any of 3 sitesc aJP Bilezikian et al: Guidelines for the management of asymptomatic primary hyperparathyroidism: Summary statement from the third international workshop. J Clin Endocrinol Metab 94:335, 2009. bCreatinine clearance calculated by Cockcroft-Gault equation or Modification of Diet in Renal Disease (MDRD) equation. cSpine, distal radius, hip. of relative stability. There is concern that this late-onset deterioration in bone density in nonoperated patients could contribute significantly to the well-known age-dependent fracture risk (osteoporosis). One study reported significant and sustained improvements in bone mineral density after successful parathyroidectomy, again raising the issue regarding benefits of surgery. Other randomized studies, however, did not report major gains after surgery.

1	Cardiovascular disease including left ventricular hypertrophy, carcated PTH fragments were prevented from registering in the newer third-generation assays by use of a detection antibody directed against the extreme amino-terminal epitope. These assays may be useful for clinical research studies as in management of chronic renal disease, but the consensus is that either secondor third-generation assays are useful in the diagnosis of primary hyperparathyroidism and for the diagnosis of high-turnover bone disease in chronic kidney disease. Many tests based on renal responses to excess PTH (renal calcium and phosphate clearance; blood phosphate, chloride, magnesium; urinary or nephrogenous cyclic AMP [cAMP]) were used in earlier decades. These tests have low specificity for hyperparathyroidism and are therefore not cost-effective; they have been replaced by PTH immunometric assays combined with simultaneous blood calcium measurements (Fig. 424-4).

1	diac functional defects, and endothelial dysfunction have been reported as reversible in European patients with more severe symptomatic disease after surgery, leading to numerous studies of these cardiovascular features in those with milder disease. There are reports of endothelial Surgical excision of the abnormal parathyroid tissue is the defini tive therapy for this disease. As noted above, medical surveillance dysfunction in patients with mild asymptomatic hyperparathyroidism, without operation for patients with mild, asymptomatic disease is, but the expert panels concluded that more observation is needed, espe cially regarding whether there is reversibility with surgery.

1	A topic of considerable interest and some debate is assessment of neuropsychiatric status and health-related quality of life (QOL) status in hyperparathyroid patients both before surgery and in response to parathyroidectomy. Several observational studies suggest considerable however, still preferred by some physicians and patients, particu larly when the patients are more elderly. Evidence favoring surgery, if medically feasible, is growing because of concerns about skeletal, improvements in symptom score after surgery. Randomized studies 1000 of surgery versus observation, however, have yielded inconclusive results, especially regarding benefits of surgery. Most studies report 800 that hyperparathyroidism is associated with increased neuropsychi-600 atric symptoms, so the issue remains a significant factor in decisions regarding the impact of surgery in this disease. Disorders of the Parathyroid Gland and Calcium Homeostasis

1	Disorders of the Parathyroid Gland and Calcium Homeostasis The diagnosis is typically made by detecting an elevated immunoreactive PTH level in a patient with asymptomatic hypercalcemia (see “Differential Diagnosis: Special Tests,” below). Serum phosphate is usually low but may be normal, especially if renal failure has developed.

1	Several modifications in PTH assays have been introduced in efforts to improve their utility in light of information about metabolism of PTH (as discussed above). First-generation assays were based on displacement of radiolabeled PTH from antibodies that reacted with PTH (often also PTH fragments). Double-antibody or immunometric assays (one antibody that is usually directed against the carboxylterminal portion of intact PTH to capture the hormone and a second radioor enzyme-labeled antibody that is usually directed against the amino-terminal portion of intact PTH) greatly improved the diagnostic discrimination of the tests by eliminating interference from aUpdates guidelines (JP Bilezikian et al: J Clin Endocrinol Metab 2014; epub ahead of print). bCreatinine clearance calculated by Cockcroft-Gault equation or Modification of Diet in Renal Disease (MDRD) equation.

1	FIGURE 424-4 Levels of immunoreactive parathyroid hormone (PTH) detected in patients with primary hyperparathyroidism, hypercalcemia of malignancy, and hypoparathyroidism. Boxed area represents the upper and normal limits of blood calcium and/or immunoreactive PTH. (From SR Nussbaum, JT Potts, Jr, in L DeGroot, JL Jameson [eds]: Endocrinology, 4th ed. Philadelphia, Saunders, 2001; with permission.) 2474 cardiovascular, and neuropsychiatric disease, even in mild hyperparathyroidism. Two surgical approaches are generally practiced. The conventional parathyroidectomy procedure was neck exploration with general anesthesia; this procedure is being replaced in many centers, whenever feasible, by an outpatient procedure with local anesthesia, termed minimally invasive parathyroidectomy. Parathyroid exploration is challenging and should be undertaken by an experienced surgeon. Certain features help in predicting the pathology (e.g., multiple abnormal glands in familial cases). However, some

1	exploration is challenging and should be undertaken by an experienced surgeon. Certain features help in predicting the pathology (e.g., multiple abnormal glands in familial cases). However, some critical decisions regarding management can be made only during the operation. With conventional surgery, one approach is still based on the view that typically only one gland (the adenoma) is abnormal. If an enlarged gland is found, a normal gland should be sought. In this view, if a biopsy of a normal-sized second gland confirms its histologic (and presumed functional) normality, no further exploration, biopsy, or excision is needed. At the other extreme is the minority viewpoint that all four glands be sought and that most of the total parathyroid tissue mass be removed. The concern with the former approach is that the recurrence rate of hyperparathyroidism may be high if a second abnormal gland is missed; the latter approach could involve unnecessary surgery and an unacceptable rate of

1	former approach is that the recurrence rate of hyperparathyroidism may be high if a second abnormal gland is missed; the latter approach could involve unnecessary surgery and an unacceptable rate of hypoparathyroidism. When normal glands are found in association with one enlarged gland, excision of the single adenoma usually leads to cure or at least years free of symptoms. Long-term follow-up studies to establish true rates of recurrence are limited. Recently, there has been growing experience with new surgical strategies that feature a minimally invasive approach guided by improved preoperative localization and intraoperative monitoring by PTH assays. Preoperative 99mTc sestamibi scans with single-photon emission CT (SPECT) are used to predict the location of an abnormal gland and intraoperative sampling of PTH before and at 5-min intervals after removal of a suspected adenoma to confirm a rapid fall (>50%) to normal levels of PTH. In several centers, a combination of preoperative

1	sampling of PTH before and at 5-min intervals after removal of a suspected adenoma to confirm a rapid fall (>50%) to normal levels of PTH. In several centers, a combination of preoperative sestamibi imaging, cervical block anesthesia, minimal surgical incision, and intraoperative PTH measurements has allowed successful outpatient surgical management with a clear-cut cost benefit compared to general anesthesia and more extensive neck surgery. The use of these minimally invasive approaches requires clinical judgment to select patients unlikely to have multiple gland disease (e.g., MEN or secondary hyperparathyroidism). The growing acceptance of the technique and its relative ease for the patient has lowered the threshold for surgery. Severe hypercalcemia may provide a preoperative clue to the presence of parathyroid carcinoma. In such cases, when neck exploration is undertaken, the tissue should be widely excised; care is taken to avoid rupture of the capsule to prevent local seeding of

1	presence of parathyroid carcinoma. In such cases, when neck exploration is undertaken, the tissue should be widely excised; care is taken to avoid rupture of the capsule to prevent local seeding of tumor cells. Multiple-gland hyperplasia, as predicted in familial cases, poses more difficult questions of surgical management. Once a diagnosis of hyperplasia is established, all the glands must be identified. Two schemes have been proposed for surgical management. One is to totally remove three glands with partial excision of the fourth gland; care is taken to leave a good blood supply for the remaining gland. Other surgeons advocate total parathyroidectomy with immediate transplantation of a portion of a removed, minced parathyroid gland into the muscles of the forearm, with the view that surgical excision is easier from the ectopic site in the arm if there is recurrent hyperfunction. In a minority of cases, if no abnormal parathyroid glands are found in the neck, the issue of further

1	surgical excision is easier from the ectopic site in the arm if there is recurrent hyperfunction. In a minority of cases, if no abnormal parathyroid glands are found in the neck, the issue of further exploration must be decided. There are documented cases of five or six parathyroid glands and of unusual locations for adenomas such as in the mediastinum. When a second parathyroid exploration is indicated, the minimally invasive techniques for preoperative localization such as ultrasound, CT scan, and isotope scanning are combined with venous sampling and/or selective digital arteriography in one of the centers specializing in these procedures. Intraoperative monitoring of PTH levels by rapid PTH immunoassays may be useful in guiding the surgery. At one center, long-term cures have been achieved with selective embolization or injection of large amounts of contrast material into the end-arterial circulation feeding the parathyroid tumor.

1	A decline in serum calcium occurs within 24 h after successful surgery; usually blood calcium falls to low-normal values for 3–5 days until the remaining parathyroid tissue resumes full hormone secretion. Acute postoperative hypocalcemia is likely only if severe bone mineral deficits are present or if injury to all the normal parathyroid glands occurs during surgery. In general, there are few problems encountered in patients with uncomplicated disease such as a single adenoma (the clear majority), who do not have symptomatic bone disease or a large deficit in bone mineral, who are vitamin D and magnesium sufficient, and who have good renal and gastrointestinal function. The extent of postoperative hypocalcemia varies with the surgical approach. If all glands are biopsied, hypocalcemia may be transiently symptomatic and more prolonged. Hypocalcemia is more likely to be symptomatic after second parathyroid explorations, particularly when normal parathyroid tissue was removed at the

1	may be transiently symptomatic and more prolonged. Hypocalcemia is more likely to be symptomatic after second parathyroid explorations, particularly when normal parathyroid tissue was removed at the initial operation and when the manipulation and/or biopsy of the remaining normal glands are more extensive in the search for the missing adenoma.

1	Patients with hyperparathyroidism have efficient intestinal calcium absorption due to the increased levels of 1,25(OH)2D stimulated by PTH excess. Once hypocalcemia signifies successful surgery, patients can be put on a high-calcium intake or be given oral calcium supplements. Despite mild hypocalcemia, most patients do not require parenteral therapy. If the serum calcium falls to <2 mmol/L (8 mg/dL), and if the phosphate level rises simultaneously, the possibility that surgery has caused hypoparathyroidism must be considered. With unexpected hypocalcemia, coexistent hypomagnesemia should be considered, because it interferes with PTH secretion and causes functional hypoparathyroidism (Chap. 423).

1	Signs of hypocalcemia include symptoms such as muscle twitching, a general sense of anxiety, and positive Chvostek’s and Trousseau’s signs coupled with serum calcium consistently <2 mmol/L (8 mg/dL). Parenteral calcium replacement at a low level should be instituted when hypocalcemia is symptomatic. The rate and duration of IV therapy are determined by the severity of the symptoms and the response of the serum calcium to treatment. An infusion of 0.5–2 mg/kg per hour or 30–100 mL/h of a 1-mg/mL solution usually suffices to relieve symptoms. Usually, parenteral therapy is required for only a few days. If symptoms worsen or if parenteral calcium is needed for >2–3 days, therapy with a vitamin D analogue and/or oral calcium (2–4 g/d) should be started (see below). It is cost-effective to use calcitriol (doses of 0.5–1 μg/d) because of the rapidity of onset of effect and prompt cessation of action when stopped, in comparison to other forms of vitamin D. A rise in blood calcium after

1	use calcitriol (doses of 0.5–1 μg/d) because of the rapidity of onset of effect and prompt cessation of action when stopped, in comparison to other forms of vitamin D. A rise in blood calcium after several months of vitamin D replacement may indicate restoration of parathyroid function to normal. It is also appropriate to monitor serum PTH serially to estimate gland function in such patients.

1	If magnesium deficiency was present, it can complicate the postoperative course since magnesium deficiency impairs the secretion of PTH. Hypomagnesemia should be corrected whenever detected. Magnesium replacement can be effective orally (e.g., MgCl2, MgOH2), but parenteral repletion is usual to ensure postoperative recovery, if magnesium deficiency is suspected due to low blood magnesium levels. Because the depressant effect of magnesium on central and peripheral nerve functions does not occur at levels <2 mmol/L (normal range 0.8–1.2 mmol/L), parenteral replacement can be given rapidly. A cumulative dose as great as 0.5–1 mmol/kg of body weight can be administered if severe hypomagnesemia is present; often, however, total doses of 20–40 mmol are sufficient.

1	The guidelines for recommending surgical intervention, if feasible (Table 424-2), as well as for monitoring patients with asymptomatic hyperparathyroidism who elect not to undergo parathyroidectomy (Table 424-3), reflect the changes over time since the first conference on the topic in 1990. Medical monitoring rather than corrective surgery is still acceptable, but it is clear that surgical intervention is the more frequently recommended option for the reasons noted above. Tightened guidelines favoring surgery include lowering the recommended level of serum calcium elevation, more careful attention to skeletal integrity through reference to peak skeletal mass at baseline (T scores) rather than age-adjusted bone density (Z scores), as well as the presence of any fragility fracture. The other changes noted in the two guidelines (Tables 424-2 and 424-3) reflect accumulated experience and practical consideration, such as a difficulty in quantity of urine collections. Despite the usefulness

1	changes noted in the two guidelines (Tables 424-2 and 424-3) reflect accumulated experience and practical consideration, such as a difficulty in quantity of urine collections. Despite the usefulness of the guidelines, the importance of individual patient and physician judgment and preference is clear in all recommendations.

1	When surgery is not selected, or not medically feasible, there is interest in the potential value of specific medical therapies. There is no long-term experience regarding specific clinical outcomes such as fracture prevention, but it has been established that bisphosphonates increase bone mineral density significantly without changing serum calcium (as does estrogen, but the latter is not favored because of reported adverse effects in other organ systems). Calcimimetics that lower PTH secretion lower calcium but do not affect bone mineral density.

1	OTHER PARATHYROID-RELATED CAUSES OF HYPERCALCEMIA Lithium Therapy Lithium, used in the management of bipolar depression and other psychiatric disorders, causes hypercalcemia in ~10% of treated patients. The hypercalcemia is dependent on continued lithium treatment, remitting and recurring when lithium is stopped and restarted. The parathyroid adenomas reported in some hypercalcemic patients with lithium therapy may reflect the presence of an independently occurring parathyroid tumor; a permanent effect of lithium on parathyroid gland growth need not be implicated as most patients have complete reversal of hypercalcemia when lithium is stopped. However, long-standing stimulation of parathyroid cell replication by lithium may predispose to development of adenomas (as is documented in secondary hyperparathyroidism and renal failure).

1	At the levels achieved in blood in treated patients, lithium can be shown in vitro to shift the PTH secretion curve to the right in response to calcium; i.e., higher calcium levels are required to lower PTH secretion, probably acting at the calcium sensor (see below). This effect can cause elevated PTH levels and consequent hypercalcemia in otherwise normal individuals. Fortunately, there are usually alternative medications for the underlying psychiatric illness. Parathyroid surgery should not be recommended unless hypercalcemia and elevated PTH levels persist after lithium is discontinued.

1	GENETIC DISORDERS CAUSING HYPERPARATHYROID-LIKE SYNDROMES Familial Hypocalciuric Hypercalcemia FHH (also called familial benign hypercalcemia) is inherited as an autosomal dominant trait. Affected individuals are discovered because of asymptomatic hypercalcemia. Most cases of FHH (FHH1) are caused by an inactivating mutation in a single allele of the CaSR (see below), leading to inappropriately normal or even increased secretion of PTH, whereas another hypercalcemic disorder, namely the exceedingly rare Jansen’s disease, is caused by a constitutively active PTH/PTHrP receptor in target tissues. Neither FHH1 nor Jansen’s disease, however, is a growth disorder of the parathyroids. Other forms of FHH are caused either by heterozygous mutations in GNA11 (encoding G11), one of the signaling proteins downstream of the CaSR (FHH2), or by mutations in AP2S1 (FHH3).

1	The pathophysiology of FHH1 is now understood. The primary defect is abnormal sensing of the blood calcium by the parathyroid gland and renal tubule, causing inappropriate secretion of PTH and excessive reabsorption of calcium in the distal renal tubules. The CaSR is a member of the third family of GPCRs (type C or type III). The receptor responds to increased ECF calcium concentration by suppressing PTH secretion through second-messenger signaling involving the G protein alpha-subunits G11 and Gq, thereby providing negative-feedback regulation of PTH secretion. Many different inac-2475 tivating CaSR mutations have been identified in patients with FHH1. These mutations lower the capacity of the sensor to bind calcium, and the mutant receptors function as though blood calcium levels were low; excessive secretion of PTH occurs from an otherwise normal gland. Approximately two-thirds of patients with FHH have mutations within the protein-coding region of the CaSR gene. The remaining

1	low; excessive secretion of PTH occurs from an otherwise normal gland. Approximately two-thirds of patients with FHH have mutations within the protein-coding region of the CaSR gene. The remaining one-third of kindreds may have mutations in the promoter of the CaSR gene or are caused by mutations in other genes.

1	Even before elucidation of the pathophysiology of FHH, abundant clinical evidence served to separate the disorder from primary hyperparathyroidism; these clinical features are still useful in differential diagnosis. Patients with primary hyperparathyroidism have <99% renal calcium reabsorption, whereas most patients with FHH have >99% reabsorption. The hypercalcemia in FHH is often detectable in affected members of the kindreds in the first decade of life, whereas hypercalcemia rarely occurs in patients with primary hyperparathyroidism or the MEN syndromes who are age <10 years. PTH may be elevated in the different forms of FHH, but the values are usually normal or lower for the same degree of calcium elevation than is observed in patients with primary hyperparathyroidism. Parathyroid surgery performed in a few patients with FHH before the nature of the syndrome was understood led to permanent hypoparathyroidism; nevertheless, hypocalciuria persisted, establishing that hypocalciuria

1	performed in a few patients with FHH before the nature of the syndrome was understood led to permanent hypoparathyroidism; nevertheless, hypocalciuria persisted, establishing that hypocalciuria is not PTH-dependent (now known to be due to the abnormal CaSR in the kidney).

1	Few clinical signs or symptoms are present in patients with FHH, whereas other endocrine abnormalities are not. Most patients are detected as a result of family screening after hypercalcemia is detected in a proband. In those patients inadvertently operated upon for primary hyperparathyroidism, the parathyroids appeared normal or moderately hyperplastic. Parathyroid surgery is not appropriate, nor, in view of the lack of symptoms, does medical treatment seem needed to lower the calcium. One striking exception to the rule against parathyroid surgery in this syndrome is the occurrence, usually in consanguineous marriages (due to the rarity of the gene mutation), of a homozygous or compound heterozygote state, resulting in severe impairment of CaSR function. In this condition, neonatal severe hypercalcemia, total parathyroidectomy is mandatory, but calcimetics have been used as a temporary measure. Rare but well-documented cases of acquired hypocalciuric hypercalcemia are reported due to

1	hypercalcemia, total parathyroidectomy is mandatory, but calcimetics have been used as a temporary measure. Rare but well-documented cases of acquired hypocalciuric hypercalcemia are reported due to antibodies against the CaSR. They appear to be a complication of an underlying autoimmune disorder and respond to therapies directed against the underlying disorder.

1	Jansen’s Disease Activating mutations in the PTH/PTHrP receptor (PTH1R) have been identified as the cause of this rare autosomal dominant syndrome. Because the mutations lead to constitutive activation of receptor function, one abnormal copy of the mutant receptor is sufficient to cause the disease, thereby accounting for its dominant mode of transmission. The disorder leads to short-limbed dwarfism due to abnormal regulation of chondrocyte maturation in the growth plates of the bone that are formed through the endochondral process. In adult life, there are numerous abnormalities in bone, including multiple cystic resorptive areas resembling those seen in severe hyperparathyroidism. Hypercalcemia and hypophosphatemia with undetectable or low PTH levels are typically observed. The pathogenesis of the growth plate abnormalities in Jansen’s disease has been confirmed by transgenic experiments in which targeted expression of the mutant PTH/PTHrP receptor to the proliferating chondrocyte

1	of the growth plate abnormalities in Jansen’s disease has been confirmed by transgenic experiments in which targeted expression of the mutant PTH/PTHrP receptor to the proliferating chondrocyte layer of growth plate emulated several features of the human disorder. Some of these genetic mutations in the parathyroid gland or PTH target cells that affect Ca2+ metabolism are illustrated in Fig. 424-5.

1	MALIGNANCY-RELATED HYPERCALCEMIA Clinical Syndromes and Mechanisms of Hypercalcemia Hypercalcemia due to malignancy is common (occurring in as many as 20% of cancer patients, especially with certain types of tumor such as lung carcinoma), often severe and difficult to manage, and, on rare occasions, Disorders of the Parathyroid Gland and Calcium Homeostasis Loss-of-function FHH1, Blomstrand’s lethal NSHPT chondrodysplasia C Cellular events, RC R including HDAC4 Transcription factors, e.g. GATA3, GCM2, AIRE, FAM111A Gq/11 IP3 + DAG Acrodysostosis with (e.g. kidney, bone, or cartilage)

1	FIGURE 424-5 Illustration of some genetic mutations that alter calcium metabolism by effects on the parathyroid cell or target cells of parathyroid hormone (PTH) action. Alterations in PTH production by the parathyroid cell can be caused by changes in the response to extracellular fluid calcium (Ca2+) that are detected by the calcium-sensing receptor (CaSR). Furthermore, PTH (or PTH-related peptide [PTHrP]) can show altered efficacy in target cells such as in proximal tubular cells, by altered function of its receptor (PTH/PTHrP receptor) or the signal transduction proteins, G proteins such as Gsα, which is linked to adenylate cyclase (AC), the enzyme responsible for producing cyclic AMP (cAMP) (also illustrated are Gq/11, which activate an alternate pathway of receptor signal transmission involving the generation of inositol triphosphate [IP3] or diacylglycerol [DAG]). Heterozygous loss-of-function mutations in the CaSR cause familial benign hypocalciuric hypercalcemia (FBHH),

1	involving the generation of inositol triphosphate [IP3] or diacylglycerol [DAG]). Heterozygous loss-of-function mutations in the CaSR cause familial benign hypocalciuric hypercalcemia (FBHH), homozygous mutations (both alleles mutated), and severe neonatal hyperparathyroidism (NSHPT); heterozygous gain-of-function causes autosomal dominant hypercalciuric hypocalcemia (ADHH). Other defects in parathyroid cell function that occur at the level of gene regulation (oncogenes or tumor-suppressor genes) or transcription factors are discussed in the text. Blomstrand’s lethal chondrodysplasia is due to homozygous or compound heterozygous loss-of-function mutations in the PTH/PTHrP receptor, a neonatally lethal disorder, while pseudohypoparathyroidism involves inactivation at the level of the G proteins, specifically mutations that eliminate or reduce Gsα activity in the kidney (see text for details). Acrodysostosis can occur with (acrodysostosis with hormonal resistance [ADOHR]; mutant

1	G proteins, specifically mutations that eliminate or reduce Gsα activity in the kidney (see text for details). Acrodysostosis can occur with (acrodysostosis with hormonal resistance [ADOHR]; mutant regulatory subunit of PKA) or without hormonal resistance (ADOP4; mutant PDE4D). Jansen’s metaphyseal chondrodysplasia and McCune-Albright syndrome represent gain-of-function mutations in the PTH/PTHrP receptor and Gsα protein, respectively.

1	difficult to distinguish from primary hyperparathyroidism. Although produced by activated normal lymphocytes and by myeloma and malignancy is often clinically obvious or readily detectable by medical lymphoma cells, originally termed osteoclast activation factor, now history, hypercalcemia can occasionally be due to an occult tumor. appears to represent the biologic action of several different cytokines, Previously, hypercalcemia associated with malignancy was thought to probably interleukin 1 and lymphotoxin or tumor necrosis factor be due to local invasion and destruction of bone by tumor cells; many (TNF). In some lymphomas, there is a third mechanism, caused by cases are now known to result from the elaboration by the malignant an increased blood level of 1,25(OH)2D, produced by the abnormal cells of humoral mediators of hypercalcemia. PTHrP is the responsible lymphocytes. humoral agent in most solid tumors that cause hypercalcemia. In the more common mechanism, usually termed

1	abnormal cells of humoral mediators of hypercalcemia. PTHrP is the responsible lymphocytes. humoral agent in most solid tumors that cause hypercalcemia. In the more common mechanism, usually termed humoral hyper-

1	The histologic character of the tumor is more important than the calcemia of malignancy, solid tumors (cancers of the lung and kidney, extent of skeletal metastases in predicting hypercalcemia. Small-cell in particular), in which bone metastases are absent, minimal, or not carcinoma (oat cell) and adenocarcinoma of the lung, although the detectable clinically, secrete PTHrP measurable by immunoassay. most common lung tumors associated with skeletal metastases, rarely Secretion by the tumors of the PTH-like factor, PTHrP, activates the cause hypercalcemia. By contrast, many patients with squamous cell PTH1R, resulting in a pathophysiology closely resembling hyperparacarcinoma of the lung develop hypercalcemia. Histologic studies of thyroidism, but with normal or suppressed PTH levels. The clinical bone in patients with squamous cell or epidermoid carcinoma of the picture resembles primary hyperparathyroidism (hypophosphatemia lung, in sites invaded by tumor as well as areas remote from

1	bone in patients with squamous cell or epidermoid carcinoma of the picture resembles primary hyperparathyroidism (hypophosphatemia lung, in sites invaded by tumor as well as areas remote from tumor accompanies hypercalcemia), and elimination or regression of the invasion, reveal increased bone resorption. primary tumor leads to disappearance of the hypercalcemia.

1	Two main mechanisms of hypercalcemia are operative in cancer As in hyperparathyroidism, patients with the humoral hypercalcehypercalcemia. Many solid tumors associated with hypercalcemia, mia of malignancy have elevated urinary nephrogenous cAMP excreparticularly squamous cell and renal tumors, produce and secrete tion, hypophosphatemia, and increased urinary phosphate clearance. PTHrP that causes increased bone resorption and mediate the hyper-However, in humoral hypercalcemia of malignancy, immunoreactive calcemia through systemic actions on the skeleton. Alternatively, PTH is undetectable or suppressed, making the differential diagnosis direct bone marrow invasion occurs with hematologic malignancies easier. Other features of the disorder differ from those of true hyper-such as leukemia, lymphoma, and multiple myeloma. Lymphokines parathyroidism. Although the biologic actions of PTH and PTHrP and cytokines (including PTHrP) produced by cells involved in are exerted through the same

1	lymphoma, and multiple myeloma. Lymphokines parathyroidism. Although the biologic actions of PTH and PTHrP and cytokines (including PTHrP) produced by cells involved in are exerted through the same receptor, subtle differences in receptor the marrow response to the tumors promote resorption of bone activation by the two ligands must account for some of the discordance through local destruction. Several hormones, hormone analogues, in pathophysiology, when an excess of one or the other peptide occurs. cytokines, and growth factors have been implicated as the result of Other cytokines elaborated by the malignancy may contribute to the clinical assays, in vitro tests, or chemical isolation. The etiologic factor variations from hyperparathyroidism in these patients as well. Patients with humoral hypercalcemia of malignancy may have low to normal levels of 1,25(OH)2D instead of elevated levels as in true hyperparathyroidism. In some patients with the humoral hypercalcemia of malignancy,

1	hypercalcemia of malignancy may have low to normal levels of 1,25(OH)2D instead of elevated levels as in true hyperparathyroidism. In some patients with the humoral hypercalcemia of malignancy, osteoclastic resorption is unaccompanied by an osteoblastic or bone-forming response, implying inhibition of the normal coupling of bone formation and resorption.

1	Several different assays (singleor double-antibody, different epitopes) have been developed to detect PTHrP. Most data indicate that circulating PTHrP levels are undetectable (or low) in normal individuals except perhaps in pregnancy (high in human milk) and elevated in most cancer patients with the humoral syndrome. The etiologic mechanisms in cancer hypercalcemia may be multiple in the same patient. For example, in breast carcinoma (metastatic to bone) and in a distinctive type of T cell lymphoma/leukemia initiated by human T cell lymphotropic virus I, hypercalcemia is caused by direct local lysis of bone as well as by a humoral mechanism involving excess production of PTHrP. Hyperparathyroidism has been reported to coexist with the humoral cancer syndrome, and rarely, ectopic hyperparathyroidism due to tumor elaboration of true PTH is reported.

1	Diagnostic Issues Levels of PTH measured by the double-antibody technique are undetectable or extremely low in tumor hypercalcemia, as would be expected with the mediation of the hypercalcemia by a factor other than PTH (the hypercalcemia suppresses the normal parathyroid glands). In a patient with minimal symptoms referred for hypercalcemia, low or undetectable PTH levels would focus attention on a possible occult malignancy (except for very rare cases of ectopic hyperparathyroidism).

1	Ordinarily, the diagnosis of cancer hypercalcemia is not difficult because tumor symptoms are prominent when hypercalcemia is detected. Indeed, hypercalcemia may be noted incidentally during the workup of a patient with known or suspected malignancy. Clinical suspicion that malignancy is the cause of the hypercalcemia is heightened when there are other signs or symptoms of a paraneoplastic process such as weight loss, fatigue, muscle weakness, or unexplained skin rash, or when symptoms specific for a particular tumor are present. Squamous cell tumors are most frequently associated with hypercalcemia, particularly tumors of the lung, kidney, head and neck, and urogenital tract. Radiologic examinations can focus on these areas when clinical evidence is unclear. Bone scans with technetium-labeled bisphosphonate are useful for detection of osteolytic metastases; the sensitivity is high, but specificity is low; results must be confirmed by conventional x-rays to be certain that areas of

1	bisphosphonate are useful for detection of osteolytic metastases; the sensitivity is high, but specificity is low; results must be confirmed by conventional x-rays to be certain that areas of increased uptake are due to osteolytic metastases per se. Bone marrow biopsies are helpful in patients with anemia or abnormal peripheral blood smears.

1	Treatment of the hypercalcemia of malignancy is first directed to control of tumor; reduction of tumor mass usually corrects hypercalcemia. If a patient has severe hypercalcemia yet has a good chance for effective tumor therapy, treatment of the hypercalcemia should be vigorous while awaiting the results of definitive therapy. If hypercalcemia occurs in the late stages of a tumor that is resistant to antitumor therapy, the treatment of the hypercalcemia should be judicious as high calcium levels can have a mild sedating effect. Standard therapies for hypercalcemia (discussed below) are applicable to patients with malignancy.

1	Hypercalcemia caused by vitamin D can be due to excessive ingestion or abnormal metabolism of the vitamin. Abnormal metabolism of the vitamin is usually acquired in association with a widespread granulomatous disorder. Vitamin D metabolism is carefully regulated, particularly the activity of renal 1α-hydroxylase, the enzyme responsible for the production of 1,25(OH)2D (Chap. 423). The regulation of 1α-hydroxylase and the normal feedback suppression by 1,25(OH)2D 2477 seem to work less well in infants than in adults and to operate poorly, if at all, in sites other than the renal tubule; these phenomena may explain the occurrence of hypercalcemia secondary to excessive 1,25(OH)2D production in infants with Williams’ syndrome (see below) and in adults with sarcoidosis or lymphoma.

1	Vitamin D Intoxication Chronic ingestion of 40–100 times the normal physiologic requirement of vitamin D (amounts >40,000–100,000 U/d) is usually required to produce significant hypercalcemia in otherwise healthy individuals. The stated upper limit of safe dietary intake is 2000 U/d (50 μg/d) in adults because of concerns about potential toxic effects of cumulative supraphysiologic doses. These recommendations are now regarded as too restrictive, because some estimates are that in elderly individuals in northern latitudes, 2000 U/d or more may be necessary to avoid vitamin D insufficiency.

1	Hypercalcemia in vitamin D intoxication is due to an excessive biologic action of the vitamin, perhaps the consequence of increased levels of 25(OH)D rather than merely increased levels of the active metabolite 1,25(OH)2D (the latter may not be elevated in vitamin D intoxication). 25(OH)D has definite, if low, biologic activity in the intestine and bone. The production of 25(OH)D is less tightly regulated than is the production of 1,25(OH)2D. Hence concentrations of 25(OH)D are elevated several-fold in patients with excess vitamin D intake. The diagnosis is substantiated by documenting elevated levels of 25(OH)D >100 mg/mL. Hypercalcemia is usually controlled by restriction of dietary calcium intake and appropriate attention to hydration.

1	These measures, plus discontinuation of vitamin D, usually lead to resolution of hypercalcemia. However, vitamin D stores in fat may be substantial, and vitamin D intoxication may persist for weeks after vitamin D ingestion is terminated. Such patients are responsive to glucocorticoids, which in doses of 100 mg/d of hydrocortisone or its equivalent usually return serum calcium levels to normal over several days; severe intoxication may require intensive therapy.

1	Sarcoidosis and Other Granulomatous Diseases In patients with sarcoid osis and other granulomatous diseases, such as tuberculosis and fungal infections, excess 1,25(OH)2D is synthesized in macrophages or other cells in the granulomas. Indeed, increased 1,25(OH)2D levels have been reported in anephric patients with sarcoidosis and hypercalcemia. Macrophages obtained from granulomatous tissue convert 25(OH)D to 1,25(OH)2D at an increased rate. There is a positive correlation in patients with sarcoidosis between 25(OH)D levels (reflecting vitamin D intake) and the circulating concentrations of 1,25(OH)2D, whereas normally there is no increase in 1,25(OH)2D with increasing 25(OH) D levels due to multiple feedback controls on renal 1α-hydroxylase (Chap. 423). The usual regulation of active metabolite production by calcium and phosphate or by PTH does not operate in these patients. Clearance of 1,25(OH)2D from blood may be decreased in sarcoidosis as well. PTH levels are usually low and

1	metabolite production by calcium and phosphate or by PTH does not operate in these patients. Clearance of 1,25(OH)2D from blood may be decreased in sarcoidosis as well. PTH levels are usually low and 1,25(OH)2D levels are elevated, but primary hyperparathyroidism and sarcoidosis may coexist in some patients.

1	Management of the hypercalcemia can often be accomplished by avoiding excessive sunlight exposure and limiting vitamin D and calcium intake. Presumably, however, the abnormal sensitivity to vitamin D and abnormal regulation of 1,25(OH)2D synthesis will persist as long as the disease is active. Alternatively, glucocorticoids in the equivalent of 100 mg/d of hydrocortisone or equivalent doses of glucocorticoids may help control hypercalcemia. Glucocorticoids appear to act by blocking excessive production of 1,25(OH)2D, as well as the response to it in target organs. Idiopathic Hypercalcemia of Infancy This rare disorder, usually referred to as Williams’ syndrome, is an autosomal dominant disorder characterized by multiple congenital development defects, including supravalvular aortic stenosis, mental retardation, and an elfin facies, in association with hypercalcemia due to abnormal sensitivity to vitamin

1	D. The hypercalcemia associated with the syndrome was first recognized in England after fortification of milk with vitamin D. The cardiac and developmental abnormalities were independently described, Disorders of the Parathyroid Gland and Calcium Homeostasis 2478 but the connection between these defects and hypercalcemia were not described until later. Levels of 1,25(OH)2D can be elevated, ranging from 46 to 120 nmol/L (150–500 pg/mL). The mechanism of the abnormal sensitivity to vitamin D and of the increased circulating levels of 1,25(OH)2D is still unclear. Studies suggest that genetic mutations involving microdeletions at the elastin locus and perhaps other genes on chromosome 7 may play a role in the pathogenesis. Another cause of hypercalcemia in infants and young children is a 24-hydroxylase deficiency that impairs metabolism of 1,25(OH)2D.

1	HYPERCALCEMIA ASSOCIATED WITH HIGH BONE TURNOVER Hyperthyroidism As many as 20% of hyperthyroid patients have high-normal or mildly elevated serum calcium concentrations; hypercalciuria is even more common. The hypercalcemia is due to increased bone turnover, with bone resorption exceeding bone formation. Severe calcium elevations are not typical, and the presence of such suggests a concomitant disease such as hyperparathyroidism. Usually, the diagnosis is obvious, but signs of hyperthyroidism may occasionally be occult, particularly in the elderly (Chap. 405). Hypercalcemia is managed by treatment of the hyperthyroidism. Reports that thyroid-stimulating hormone (TSH) itself normally has a bone-protective effect suggest that suppressed TSH levels also play a role in hypercalcemia.

1	Immobilization Immobilization is a rare cause of hypercalcemia in adults in the absence of an associated disease but may cause hypercalcemia in children and adolescents, particularly after spinal cord injury and paraplegia or quadriplegia. With resumption of ambulation, the hypercalcemia in children usually returns to normal. The mechanism appears to involve a disproportion between bone formation and bone resorption; the former decreased and the latter increased. Hypercalciuria and increased mobilization of skeletal calcium can develop in normal volunteers subjected to extensive bed rest, although hypercalcemia is unusual. Immobilization of an adult with a disease associated with high bone turnover, however, such as Paget’s disease, may cause hypercalcemia.

1	Thiazides Administration of benzothiadiazines (thiazides) can cause hypercalcemia in patients with high rates of bone turnover. Traditionally, thiazides are associated with aggravation of hypercalcemia in primary hyperparathyroidism, but this effect can be seen in other high-boneturnover states as well. The mechanism of thiazide action is complex. Chronic thiazide administration leads to reduction in urinary calcium; the hypocalciuric effect appears to reflect the enhancement of proximal tubular resorption of sodium and calcium in response to sodium depletion. Some of this renal effect is due to augmentation of PTH action and is more pronounced in individuals with intact PTH secretion. However, thiazides cause hypocalciuria in hypoparathyroid patients on high-dose vitamin D and oral calcium replacement if sodium intake is restricted. This finding is the rationale for the use of thiazides as an adjunct to therapy in hypoparathyroid patients, as discussed below. Thiazide administration

1	replacement if sodium intake is restricted. This finding is the rationale for the use of thiazides as an adjunct to therapy in hypoparathyroid patients, as discussed below. Thiazide administration to normal individuals causes a transient increase in blood calcium (usually within the high-normal range) that reverts to preexisting levels after a week or more of continued administration. If hormonal function and calcium and bone metabolism are normal, homeostatic controls are reset to counteract the calcium-elevating effect of the thiazides. In the presence of hyperparathyroidism or increased bone turnover from another cause, homeostatic mechanisms are ineffective. The abnormal effects of the thiazide on calcium metabolism disappear within days of cessation of the drug.

1	Vitamin A Intoxication Vitamin A intoxication is a rare cause of hypercalcemia and is most commonly a side effect of dietary faddism (Chap. 96e). Calcium levels can be elevated into the 3–3.5-mmol/L (12–14 mg/dL) range after the ingestion of 50,000–100,000 units of vitamin A daily (10–20 times the minimum daily requirement). Typical features of severe hypercalcemia include fatigue, anorexia, and, in some, severe muscle and bone pain. Excess vitamin A intake is presumed to increase bone resorption. The diagnosis can be established by history and by measurement of vitamin A levels in serum. Occasionally, skeletal x-rays reveal periosteal calcifications, particularly in the hands. Withdrawal of the vitamin is usually associated with prompt disappearance of the hypercalcemia and reversal of the skeletal changes. As in vitamin D intoxication, administration of 100 mg/d of hydrocortisone or its equivalent leads to a rapid return of the serum calcium to normal.

1	HYPERCALCEMIA ASSOCIATED WITH RENAL FAILURE Severe Secondary Hyperparathyroidism The pathogenesis of secondary hyperparathyroidism in chronic kidney disease is incompletely understood. Resistance to the normal level of PTH is a major factor contributing to the development of hypocalcemia, which, in turn, is a stimulus to parathyroid gland enlargement. However, recent findings have indicated that an increase of FGF23 production by osteocytes (and possibly osteoblasts) in bone occurs well before an elevation in PTH is detected. FGF23 is a potent inhibitor of the renal 1-alpha hydroxylase, and the FGF23-dependent reduction in 1,25(OH)2 vitamin D seems to be an important stimulus for the development of secondary hyperparathyroidism.

1	Secondary hyperparathyroidism occurs not only in patients with renal failure but also in those with osteomalacia due to multiple causes (Chap. 423), including deficiency of vitamin D action and pseudohypoparathyroidism (deficient response to PTH downstream of PTHR1). For both disorders, hypocalcemia seems to be the common denominator in initiating the development of secondary hyperparathyroidism. Primary (1°) and secondary (2°) hyperparathyroidism can be distinguished conceptually by the autonomous growth of the parathyroid glands in primary hyperparathyroidism (presumably irreversible) and the adaptive response of the parathyroids in secondary hyperparathyroidism (typically reversible). In fact, reversal over weeks from an abnormal pattern of secretion, presumably accompanied by involution of parathyroid gland mass to normal, occurs in patients with osteomalacia who have been treated effectively with calcium and vitamin D. However, it is now recognized that a true clonal outgrowth

1	of parathyroid gland mass to normal, occurs in patients with osteomalacia who have been treated effectively with calcium and vitamin D. However, it is now recognized that a true clonal outgrowth (irreversible) can arise in long-standing, inadequately treated chronic kidney disease (e.g., tertiary [3°] hyperparathyroidism; see below).

1	Patients with secondary hyperparathyroidism may develop bone pain, ectopic calcification, and pruritus. The bone disease seen in patients with secondary hyperparathyroidism and chronic kidney disease is termed renal osteodystrophy and affects primarily bone turnover. However, osteomalacia is frequently encountered as well and may be related to the circulating levels of FGF23.

1	Two other skeletal disorders have been frequently associated in the past with chronic kidney disease (CKD) patients treated by longterm dialysis, who received aluminum-containing phosphate binders. Aluminum deposition in bone (see below) leads to an osteomalacialike picture. The other entity is a low-turnover bone disease termed “aplastic” or “adynamic” bone disease; PTH levels are lower than typically observed in CKD patients with secondary hyperparathyroidism. It is believed that the condition is caused, at least in part, by excessive PTH suppression, which may be even greater than previously appreciated in light of evidence that some of the immunoreactive PTH detected by most commercially available PTH assays is not the full-length biologically active molecule (as discussed above) but may consist of amino-terminally truncated fragments that do not activate the PTH1R.

1	Medical therapy to reverse secondary hyperparathyroidism in CKD includes reduction of excessive blood phosphate by restriction of dietary phosphate, the use of nonabsorbable phosphate binders, and careful, selective addition of calcitriol (0.25–2 μg/d) or related analogues. Calcium carbonate became preferred over aluminum-containing antacids to prevent aluminum-induced bone disease. However, synthetic gels that also bind phosphate (such as sevelamer; Chap. 335) are now widely used, with the advantage of avoiding not only aluminum retention, but also excess calcium loading, which may contribute to cardiovascular calcifications. Intravenous calcitriol (or related analogues), administered as several pulses each week, helps control secondary hyperparathyroidism. Aggressive but carefully administered medical therapy can often, but not always, reverse hyperparathyroidism and its symptoms and manifestations.

1	Occasional patients develop severe manifestations of secondary hyperparathyroidism, including hypercalcemia, pruritus, extraskeletal calcifications, and painful bones, despite aggressive medical efforts to suppress the hyperparathyroidism. PTH hypersecretion no longer responsive to medical therapy, a state of severe hyperparathyroidism in patients with CKD that requires surgery, has been referred to as tertiary hyperparathyroidism. Parathyroid surgery is necessary to control this condition. Based on genetic evidence from examination of tumor samples in these patients, the emergence of autonomous parathyroid function is due to a monoclonal outgrowth of one or more previously hyperplastic parathyroid glands. The adaptive response has become an independent contributor to disease; this finding seems to emphasize the importance of optimal medical management to reduce the proliferative response of the parathyroid cells that enables the irreversible genetic change.

1	Aluminum Intoxication Aluminum intoxication (and often hypercalcemia as a complication of medical treatment) in the past occurred in patients on chronic dialysis; manifestations included acute dementia and unresponsive and severe osteomalacia. Bone pain, multiple non-healing fractures, particularly of the ribs and pelvis, and a proximal myopathy occur. Hypercalcemia develops when these patients are treated with vitamin D or calcitriol because of impaired skeletal responsiveness. Aluminum is present at the site of osteoid mineralization, osteoblastic activity is minimal, and calcium incorporation into the skeleton is impaired. The disorder is now rare because of the avoidance of aluminum-containing antacids or aluminum excess in the dialysis regimen (Chap. 429).

1	Milk-Alkali Syndrome The milk-alkali syndrome is due to excessive ingestion of calcium and absorbable antacids such as milk or calcium carbonate. It is much less frequent since proton pump inhibitors and other treatments became available for peptic ulcer disease. For a time, the increased use of calcium carbonate in the management of secondary hyperparathyroidism led to reappearance of the syndrome. Several clinical presentations—acute, subacute, and chronic—have 2479 been described, all of which feature hypercalcemia, alkalosis, and renal failure. The chronic form of the disease, termed Burnett’s syndrome, is associated with irreversible renal damage. The acute syndromes reverse if the excess calcium and absorbable alkali are stopped.

1	Individual susceptibility is important in the pathogenesis, because some patients are treated with calcium carbonate and alkali regimens without developing the syndrome. One variable is the fractional calcium absorption as a function of calcium intake. Some individuals absorb a high fraction of calcium, even with intakes ≥2 g of elemental calcium per day, instead of reducing calcium absorption with high intake, as occurs in most normal individuals. Resultant mild hypercalcemia after meals in such patients is postulated to contribute to the generation of alkalosis. Development of hypercalcemia causes increased sodium excretion and some depletion of total-body water. These phenomena and perhaps some suppression of endogenous PTH secretion due to mild hypercalcemia lead to increased bicarbonate resorption and to alkalosis in the face of continued calcium carbonate ingestion. Alkalosis per se selectively enhances calcium resorption in the distal nephron, thus aggravating the

1	bicarbonate resorption and to alkalosis in the face of continued calcium carbonate ingestion. Alkalosis per se selectively enhances calcium resorption in the distal nephron, thus aggravating the hypercalcemia. The cycle of mild hypercalcemia → bicarbonate retention → alkalosis → renal calcium retention → severe hypercalcemia perpetuates and aggravates hypercalcemia and alkalosis as long as calcium and absorbable alkali are ingested.

1	DIFFERENTIAL DIAGNOSIS: SPECIAL TESTS Differential diagnosis of hypercalcemia is best achieved by using clini cal criteria, but immunometric assays to measure PTH are especially useful in distinguishing among major causes (Fig. 424-6). The clinical features that deserve emphasis are the presence or absence of symptoms or signs of disease and evidence of chronicity. If one discounts fatigue or depression, >90% of patients with primary hyperparathyroidism have asymptomatic hypercalcemia; symptoms of malignancy are usually present in cancer-associated hypercalcemia. Disorders other than hyperparathyroidism and malignancy cause <10% of cases of hypercalcemia, and some of the nonparathyroid causes are associated with clear-cut manifestations such as renal failure.

1	Hyperparathyroidism is the likely diagnosis in patients with chronic hypercalcemia. If hypercalcemia has been manifest for >1 year, malignancy can usually be excluded as the cause. A striking feature of malignancy-associated hypercalcemia is the rapidity of the course, (lithium, thiazides) Immobilization FIGURE 424-6 Algorithm for the evaluation of patients with hypercalcemia. See text for details. FHH, familial hypocalciuric hypercalcemia; MEN, multiple endocrine neoplasia; PTH, parathyroid hormone; PTHrP, parathyroid hormone–related peptide.

1	2480 whereby signs and symptoms of the underlying malignancy are evident within months of the detection of hypercalcemia. Although clinical considerations are helpful in arriving at the correct diagnosis of the cause of hypercalcemia, appropriate laboratory testing is essential for definitive diagnosis. The immunoassay for PTH usually separates hyperparathyroidism from all other causes of hypercalcemia (exceptions are very rare reports of ectopic production of excess PTH by nonparathyroid tumors). Patients with hyperparathyroidism have elevated PTH levels despite hypercalcemia, whereas patients with malignancy and the other causes of hypercalcemia (except for disorders mediated by PTH such as lithium-induced hypercalcemia) have levels of hormone below normal or undetectable levels. Assays based on the double-antibody method for PTH exhibit very high sensitivity (especially if serum calcium is simultaneously evaluated) and specificity for the diagnosis of primary hyperparathyroidism

1	based on the double-antibody method for PTH exhibit very high sensitivity (especially if serum calcium is simultaneously evaluated) and specificity for the diagnosis of primary hyperparathyroidism (Fig. 424-4). In summary, PTH values are elevated in >90% of parathyroid-related causes of hypercalcemia, undetectable or low in malignancy-related hypercalcemia, and undetectable or normal in vitamin D– related and high-bone-turnover causes of hypercalcemia. In view of the specificity of the PTH immunoassay and the high frequency of hyperparathyroidism in hypercalcemic patients, it is cost-effective to measure the PTH level in all hypercalcemic patients unless malignancy or a specific nonparathyroid disease is obvious. False-positive PTH assay results are rare. Immunoassays for PTHrP are helpful in diagnosing certain types of malignancy-associated hypercalcemia. Although FHH is parathyroid-related, the disease should be managed distinctively from hyperparathyroidism. Clinical features and

1	in diagnosing certain types of malignancy-associated hypercalcemia. Although FHH is parathyroid-related, the disease should be managed distinctively from hyperparathyroidism. Clinical features and the low urinary calcium excretion can help make the distinction. Because the incidence of malignancy and hyperparathyroidism both increase with age, they can coexist as two independent causes of hypercalcemia. 1,25(OH)2D levels are elevated in many (but not all) patients with primary hyperparathyroidism. In other disorders associated with hypercalcemia, concentrations of 1,25(OH)2D are low or, at the most, normal. However, this test is of low specificity and is not cost-effective, as not all patients with hyperparathyroidism have elevated 1,25(OH)2D levels and not all nonparathyroid hypercalcemic patients have suppressed 1,25(OH)2D. Measurement of 1,25(OH)2D is, however, critically valuable in establishing the cause of hypercalcemia in sarcoidosis and certain lymphomas. A useful general

1	patients have suppressed 1,25(OH)2D. Measurement of 1,25(OH)2D is, however, critically valuable in establishing the cause of hypercalcemia in sarcoidosis and certain lymphomas. A useful general approach is outlined in Fig. 424-6. If the patient is asymptomatic and there is evidence of chronicity to the hypercalcemia, hyperparathyroidism is almost certainly the cause. If PTH levels (usually measured at least twice) are elevated, the clinical impression is confirmed and little additional evaluation is necessary. If there is only a short history or no data as to the duration of the hypercalcemia, occult malignancy must be considered; if the PTH levels are not elevated, then a thorough workup must be undertaken for malignancy, including chest x-ray, CT of chest and abdomen, and bone scan. Immunoassays for PTHrP may be especially useful in such situations. Attention should also be paid to clues for underlying hematologic disorders such as anemia, increased plasma globulin, and abnormal

1	Immunoassays for PTHrP may be especially useful in such situations. Attention should also be paid to clues for underlying hematologic disorders such as anemia, increased plasma globulin, and abnormal serum immunoelectrophoresis; bone scans can be negative in some patients with metastases such as in multiple myeloma. Finally, if a patient with chronic hypercalcemia is asymptomatic and malignancy therefore seems unlikely on clinical grounds, but PTH values are not elevated, it is useful to search for other chronic causes of hypercalcemia such as occult sarcoidosis. A careful history of dietary supplements and drug use may suggest intoxication with vitamin D or vitamin A or the use of thiazides.

1	The approach to medical treatment of hypercalcemia varies with its severity (Table 424-4). Mild hypercalcemia, <3 mmol/L (12 mg/ dL), can be managed by hydration. More severe hypercalcemia (levels of 3.2–3.7 mmol/L [13–15 mg/dL]) must be managed aggressively; above that level, hypercalcemia can be life-threatening and requires emergency measures. By using a combination of approaches in severe hypercalcemia, the serum calcium concentration can be decreased by 0.7–2.2 mmol/L (3–9 mg/dL) within 24–48 h in most patients, enough to relieve acute symptoms, prevent death from hypercalcemic crisis, and permit diagnostic evaluation. Therapy can then be directed at the underlying disorder—the second priority.

1	Hypercalcemia develops because of excessive skeletal calcium release, increased intestinal calcium absorption, or inadequate renal calcium excretion. Understanding the particular pathogenesis helps guide therapy. For example, hypercalcemia in patients with malignancy is primarily due to excessive skeletal calcium release and is, therefore, minimally improved by restriction of dietary calcium. On the other hand, patients with vitamin D hypersensitivity or vitamin D intoxication have excessive intestinal calcium absorption, and restriction of dietary calcium is beneficial. Decreased renal function or ECF depletion decreases urinary calcium excretion. In such situations, rehydration may rapidly reduce or reverse the hypercalcemia, even though increased bone resorption persists. As outlined below, the more severe the hypercalcemia, the greater the number of combined therapies that should be used. Rapid-acting (hours) approaches—rehydration, forced diuresis, and calcitonin—can be used with

1	the more severe the hypercalcemia, the greater the number of combined therapies that should be used. Rapid-acting (hours) approaches—rehydration, forced diuresis, and calcitonin—can be used with the most effective antiresorptive agents such as bisphosphonates (since severe hypercalcemia usually involves excessive bone resorption).

1	HYDRATION, INCREASED SALT INTAKE, MILD AND FORCED DIURESIS

1	The first principle of treatment is to restore normal hydration. Many hypercalcemic patients are dehydrated because of vomiting, inanition, and/or hypercalcemia-induced defects in urinary concentrating ability. The resultant drop in glomerular filtration rate is accompanied by an additional decrease in renal tubular sodium and calcium clearance. Restoring a normal ECF volume corrects these abnormalities and increases urine calcium excretion by 2.5–7.5 mmol/d (100–300 mg/d). Increasing urinary sodium excretion to 400–500 mmol/d increases urinary calcium excretion even further than simple rehydration. After rehydration has been achieved, saline can be administered, or furosemide or ethacrynic acid can be given twice daily to depress the tubular reabsorptive mechanism for calcium (care must be taken to prevent dehydration). The combined use of these therapies can increase urinary calcium excretion to ≥12.5 mmol/d (500 mg/d) in most hypercalcemic patients. Because this is a substantial

1	be taken to prevent dehydration). The combined use of these therapies can increase urinary calcium excretion to ≥12.5 mmol/d (500 mg/d) in most hypercalcemic patients. Because this is a substantial percentage of the exchangeable calcium pool, the serum calcium concentration usually falls 0.25–0.75 mmol/L (1–3 mg/dL) within 24 h. Precautions should be taken to prevent potassium and magnesium depletion; calcium-containing renal calculi are a potential complication.

1	Under life-threatening circumstances, the preceding approach can be pursued more aggressively, but the availability of effective agents to block bone resorption (such as bisphosphonates) has reduced the need for extreme diuresis regimens (Table 424-4). Depletion of potassium and magnesium is inevitable unless replacements are given; pulmonary edema can be precipitated. The potential complications can be reduced by careful monitoring of central venous pressure and plasma or urine electrolytes; catheterization of the bladder may be necessary. Dialysis treatment may be needed when renal function is compromised.

1	The bisphosphonates are analogues of pyrophosphate, with high affinity for bone, especially in areas of increased bone turnover, where they are powerful inhibitors of bone resorption. These bone-seeking compounds are stable in vivo because phosphatase enzymes cannot hydrolyze the central carbon-phosphorus-carbon bond. The bisphosphonates are concentrated in areas of high bone turnover and are taken up by and inhibit osteoclast action; the mechanism of action is complex. The bisphosphonate molecules that contain amino groups in the side chain structure (see below) interfere with prenylation of proteins and can lead to cellular apoptosis. The highly active nonamino group–containing bisphosphonates are also metabolized to cytotoxic products.

1	The initial bisphosphonate widely used in clinical practice, etidronate, was effective but had several disadvantages, including the capacity to inhibit bone formation as well as blocking resorption. Subsequently, a number of secondor third-generation compounds have become the mainstays of antiresorptive therapy for treatment of hypercalcemia and osteoporosis. The newer bisphosphonates have a highly favorable ratio of blocking resorption versus inhibiting bone formation; they inhibit osteoclast-mediated skeletal resorption yet do not cause mineralization defects at ordinary doses. Although the bisphosphonates have similar structures, the routes of administration, efficacy, toxicity, and side effects vary. The potency of the compounds for inhibition of bone resorption varies more than 10,000-fold, increasing in the order of etidronate, tiludronate, pamidronate, alen-2481 dronate, risedronate, and zoledronate. The IV use of pamidronate and zoledronate is approved for the treatment of

1	increasing in the order of etidronate, tiludronate, pamidronate, alen-2481 dronate, risedronate, and zoledronate. The IV use of pamidronate and zoledronate is approved for the treatment of hypercalcemia; between 30 and 90 mg pamidronate, given as a single IV dose over a few hours, returns serum calcium to normal within 24–48 h with an effect that lasts for weeks in 80–100% of patients. Zoledronate given in doses of 4 or 8 mg/5-min infusion has a more rapid and more sustained effect than pamidronate in direct comparison.

1	These drugs are used extensively in cancer patients. Absolute survival improvements are noted with pamidronate and zoledronate in multiple myeloma, for example. However, although still rare, there are increasing reports of jaw necrosis, especially after dental surgery, mainly in cancer patients treated with multiple doses of the more potent bisphosphonates.

1	Calcitonin acts within a few hours of its administration, principally through receptors on osteoclasts, to block bone resorption. Calcitonin, after 24 h of use, is no longer effective in lowering calcium. Tachyphylaxis, a known phenomenon with this drug, seems to explain the results, since the drug is often effective in the first 24 h of use. Therefore, in life-threatening hypercalcemia, calcitonin can be used effectively within the first 24 h in combination with rehydration and saline diuresis while waiting for more sustained effects from a simultaneously administered bisphosphonate such as pamidronate. Usual doses of calcitonin are 2–8 U/kg of body weight IV, SC, or IM every 6–12 h.

1	IV, SC, or IM every 6–12 h. Denosumab, an antibody that blocks the RANK ligand (RANKL) and dramatically reduces osteoclast number and function, is approved for therapy of osteoporosis. It also appears to be an effective treatment to reverse hypercalcemia of malignancy, but is not yet approved for this indication. Plicamycin (formerly mithramycin), which inhibits bone resorption, and gallium nitrate, which exerts a hypocalcemic action also by inhibiting bone resorption, are no longer used because of superior alternatives such as bisphosphonates.

1	Glucocorticoids have utility, especially in hypercalcemia complicating certain malignancies. They increase urinary calcium excretion and decrease intestinal calcium absorption when given in pharmacologic doses, but they also cause negative skeletal calcium balance. In normal individuals and in patients with primary hyperparathyroidism, glucocorticoids neither increase nor decrease the serum calcium concentration. In patients with hypercalcemia due to certain osteolytic malignancies, however, glucocorticoids may be effective as a result of antitumor effects. The malignancies in which hypercalcemia responds to glucocorticoids include multiple myeloma, leukemia, Hodgkin’s disease, other lymphomas, and carcinoma of the breast, at least early in the course of the disease. Glucocorticoids are also effective in treating hypercalcemia due to vitamin D intoxication and sarcoidosis. Glucocorticoids are also useful in the rare form of hypercalcemia, now recognized in certain autoimmune disorders

1	effective in treating hypercalcemia due to vitamin D intoxication and sarcoidosis. Glucocorticoids are also useful in the rare form of hypercalcemia, now recognized in certain autoimmune disorders in which inactivating antibodies against the receptor imitate FHH. Elevated PTH and calcium levels are effectively lowered by the glucocorticoids. In all the preceding situations, the hypocalcemic effect develops over several days, and the usual glucocorticoid dosage is 40–100 mg prednisone (or its equivalent) daily in four divided doses. The side effects of chronic glucocorticoid therapy may be acceptable in some circumstances.

1	Dialysis is often the treatment of choice for severe hypercalcemia complicated by renal failure, which is difficult to manage medically. Peritoneal dialysis with calcium-free dialysis fluid can remove 5–12.5 mmol (200–500 mg) of calcium in 24–48 h and lower the serum calcium concentration by 0.7–3 mmol/L (3–12 mg/dL). Large quantities of phosphate are lost during dialysis, and serum inorganic phosphate concentration usually falls, potentially aggravating hypercalcemia. Therefore, the serum inorganic phosphate concentration

1	Disorders of the Parathyroid Gland and Calcium Homeostasis 2482 should be measured after dialysis, and phosphate supplements should be added to the diet or to dialysis fluids if necessary. Phosphate therapy, PO or IV, has a limited role in certain circumstances (Chap. 423). Correcting hypophosphatemia lowers the serum calcium concentration by several mechanisms, including bone/calcium exchange. The usual oral treatment is 1–1.5 g of phosphorus per day for several days, given in divided doses. It is generally believed, but not established, that toxicity does not occur if therapy is limited to restoring serum inorganic phosphate concentrations to normal. Raising the serum inorganic phosphate concentration above normal decreases serum calcium levels, sometimes strikingly. Intravenous phosphate is one of the most dramatically effective treatments available for severe hypercalcemia but is toxic and even dangerous (fatal hypocalcemia). For these reasons, it is used rarely and only in

1	is one of the most dramatically effective treatments available for severe hypercalcemia but is toxic and even dangerous (fatal hypocalcemia). For these reasons, it is used rarely and only in severely hypercalcemic patients with cardiac or renal failure where dialysis, the preferable alternative, is not feasible or is unavailable.

1	The various therapies for hypercalcemia are listed in Table 424-4. The choice depends on the underlying disease, the severity of the hypercalcemia, the serum inorganic phosphate level, and the renal, hepatic, and bone marrow function. Mild hypercalcemia (≤3 mmol/L [12 mg/dL]) can usually be managed by hydration. Severe hypercalcemia (≥3.7 mmol/L [15 mg/dL]) requires rapid correction. Calcitonin should be given for its rapid, albeit short-lived, blockade of bone resorption, and IV pamidronate or zoledronate should be administered, although its onset of action is delayed for 1–2 days. In addition, for the first 24–48 h, aggressive sodium-calcium diuresis with IV saline should be given and, following rehydration, large doses of furosemide or ethacrynic acid, but only if appropriate monitoring is available and cardiac and renal function are adequate. Intermediate degrees of hypercalcemia between 3 and 3.7 mmol/L (12 and 15 mg/dL) should be approached with vigorous hydration and then the

1	is available and cardiac and renal function are adequate. Intermediate degrees of hypercalcemia between 3 and 3.7 mmol/L (12 and 15 mg/dL) should be approached with vigorous hydration and then the most appropriate selection for the patient of the combinations used with severe hypercalcemia.

1	(See also Chap. 65) PATHOPHYSIOLOGY OF HYPOCALCEMIA: CLASSIFICATION BASED ON MECHANISM Chronic hypocalcemia is less common than hypercalcemia; causes include chronic renal failure, hereditary and acquired hypoparathyroidism, vitamin D deficiency, pseudohypoparathyroidism, and hypomagnesemia (Table 424-5).

1	Acute rather than chronic hypocalcemia is seen in critically ill patients or as a consequence of certain medications and often does not require specific treatment. Transient hypocalcemia is seen with severe sepsis, burns, acute kidney injury, and extensive transfusions with citrated blood. Although as many as one-half of patients in an intensive care setting are reported to have calcium concentrations of <2.1 mmol/L (8.5 mg/dL), most do not have a reduction in ionized calcium. Patients with severe sepsis may have a decrease in ionized calcium (true hypocalcemia), but in other severely ill individuals, hypoalbuminemia is the primary cause of the reduced total calcium concentration. Alkalosis increases calcium binding to proteins, and in this setting, direct measurements of ionized calcium should be made.

1	Medications such as protamine, heparin, and glucagon may cause transient hypocalcemia. These forms of hypocalcemia are usually not associated with tetany and resolve with improvement in the overall medical condition. The hypocalcemia after repeated transfusions of citrated blood usually resolves quickly. Patients with acute pancreatitis have hypocalcemia that persists during the acute inflammation and varies in degree with disease severity. The cause of hypocalcemia remains unclear. PTH values are ↓ Dietary intake or sunlight Vitamin D–dependent rickets type II Defective metabolism: Pseudohypoparathyroidism Anticonvulsant therapy Vitamin D–dependent rickets reported to be low, normal, or elevated, and both resistance to PTH and impaired PTH secretion have been postulated. Occasionally, a chronic low total calcium and low ionized calcium concentration are detected in an elderly patient without obvious cause and with a paucity of symptoms; the pathogenesis is unclear.

1	Chronic hypocalcemia, however, is usually symptomatic and requires treatment. Neuromuscular and neurologic manifestations of chronic hypocalcemia include muscle spasms, carpopedal spasm, facial grimacing, and, in extreme cases, laryngeal spasm and convulsions. Respiratory arrest may occur. Increased intracranial pressure occurs in some patients with long-standing hypocalcemia, often in association with papilledema. Mental changes include irritability, depression, and psychosis. The QT interval on the electrocardiogram is prolonged, in contrast to its shortening with hypercalcemia. Arrhythmias occur, and digitalis effectiveness may be reduced. Intestinal cramps and chronic malabsorption may occur. Chvostek’s or Trousseau’s sign can be used to confirm latent tetany.

1	The classification of hypocalcemia shown in Table 424-5 is based on an organizationally useful premise that PTH is responsible for minute-to-minute regulation of plasma calcium concentration and, therefore, that the occurrence of hypocalcemia must mean a failure of the homeostatic action of PTH. Failure of the PTH response can occur if there is hereditary or acquired parathyroid gland failure, if PTH is ineffective in target organs, or if the action of the hormone is overwhelmed by the loss of calcium from the ECF at a rate faster than it can be replaced.

1	Whether hereditary or acquired, hypoparathyroidism has a number of common components. Symptoms of untreated hypocalcemia are shared by both types of hypoparathyroidism, although the onset of hereditary hypoparathyroidism can be more gradual and associated with other developmental defects. Basal ganglia calcification and extrapyramidal syndromes are more common and earlier in onset in hereditary hypoparathyroidism. In previous decades, acquired hypoparathyroidism secondary to surgery in the neck was more common than hereditary hypoparathyroidism, but the frequency of surgically induced parathyroid failure has diminished as a result of improved surgical techniques that spare the parathyroid glands and increased use of nonsurgical therapy for hyperthyroidism. Pseudohypoparathyroidism, an example of ineffective PTH action rather than a failure of parathyroid gland production, may share several features with hypoparathyroidism, including extraosseous calcification and extrapyramidal

1	an example of ineffective PTH action rather than a failure of parathyroid gland production, may share several features with hypoparathyroidism, including extraosseous calcification and extrapyramidal manifestations such as choreoathetotic movements and dystonia.

1	Papilledema and raised intracranial pressure may occur in both hereditary and acquired hypoparathyroidism, as do chronic changes in fingernails and hair and lenticular cataracts, the latter usually reversible with treatment of hypocalcemia. Certain skin manifestations, including alopecia and candidiasis, are characteristic of hereditary hypoparathyroidism associated with autoimmune polyglandular failure (Chap. 408).

1	Hypocalcemia associated with hypomagnesemia is associated with both deficient PTH release and impaired responsiveness to the hormone. Patients with hypocalcemia secondary to hypomagnesemia have absent or low levels of circulating PTH, indicative of diminished hormone release despite a maximum physiologic stimulus by hypocalcemia. Plasma PTH levels return to normal with correction of the hypomagnesemia. Thus hypoparathyroidism with low levels of PTH in blood can be due to hereditary gland failure, acquired gland failure, or acute but reversible gland dysfunction (hypomagnesemia).

1	Genetic Abnormalities and Hereditary Hypoparathyroidism Hereditary hypoparathyroidism can occur as an isolated entity without other endocrine or dermatologic manifestations. More typically, it occurs in association with other abnormalities such as defective development of the thymus or failure of other endocrine organs such as the adrenal, thyroid, or ovary (Chap. 408). Hereditary hypoparathyroidism is often manifest within the first decade but may appear later. Genetic defects associated with hypoparathyroidism serve to illuminate the complexity of organ development, hormonal biosynthesis and secretion, and tissue-specific patterns of endocrine effector function (Fig. 424-5). Often, hypoparathyroidism is isolated, signifying a highly specific functional disturbance. When hypoparathyroidism is associated with other developmental or organ defects, treatment of the hypocalcemia can still be effective.

1	A form of hypoparathyroidism associated with defective development of both the thymus and the parathyroid glands is termed the DiGeorge syndrome, or the velocardiofacial syndrome. Congenital cardiovascular, facial, and other developmental defects are present, and patients may die in early childhood with severe infections, hypocalcemia and seizures, or cardiovascular complications. Patients can survive into adulthood, and milder, incomplete forms occur. Most cases are sporadic, but an autosomal dominant form involving micro-deletions of chromosome 22q11.2 has been described. Smaller deletions in chromosome 22 are seen in incomplete forms of the DiGeorge syndrome, appearing in childhood or adolescence, that are manifest primarily by parathyroid gland failure. The chromosome 22 defect is now termed DSG1; more recently, a defect in chromosome 10p is also recognized—now called DSG2. The phenotypes seem similar. Studies on the chromosome 22 defect have pinpointed a transcription factor,

1	now termed DSG1; more recently, a defect in chromosome 10p is also recognized—now called DSG2. The phenotypes seem similar. Studies on the chromosome 22 defect have pinpointed a transcription factor, TBX1. Deletions of the orthologous mouse gene show a phenotype similar to the human syndrome.

1	Another autosomal dominant developmental defect, featuring hypoparathyroidism, deafness, and renal dysplasia (HDR), has been studied at the genetic level. Cytogenetic abnormalities in some, but not all kindreds, point to translocation defects on chromosome 10, as in DiGeorge syndrome. However, the lack of immunodeficiency and heart defects distinguishes the two syndromes. Mouse models, as well as deletional analysis in some HDR patients, has identified the transcription factor GATA3, which is important in embryonic development and is expressed in developing kidney, ear structures, and the parathyroids.

1	Another pair of linked developmental disorders involving the parathyroids is recognized. Kenney-Caffey syndrome type I features hypoparathyroidism, short stature, osteosclerosis, and thick cortical bones. A defect seen in Middle Eastern patients, particularly in Saudi Arabia, termed Sanjad-Sakati syndrome, also exhibits growth failure and other dysmorphic features. This syndrome, which is clearly autosomal recessive, involves a gene on chromosome 1q42-q43. Both syndromes apparently involve a chaperone protein, called TBCE, relevant to tubulin function. Recently, a defect in FAM111A was identified as the cause of Kenney-Caffey syndrome type 2.

1	Hypoparathyroidism can occur in association with a complex hereditary autoimmune syndrome involving failure of the adrenals, the ovaries, the immune system, and the parathyroids in association 2483 with recurrent mucocutaneous candidiasis, alopecia, vitiligo, and pernicious anemia (Chap. 408). The responsible gene on chromosome 21q22.3 has been identified. The protein product, which resembles a transcription factor, has been termed the autoimmune regulator, or AIRE. A stop codon mutation occurs in many Finnish families with the disorder, commonly referred to as polyglandular autoimmune type 1 deficiency, whereas another AIRE mutation (Y85C) is typically observed in Jews of Iraqi and Iranian descent.

1	Hypoparathyroidism is seen in two disorders associated with mitochondrial dysfunction and myopathy, one termed the Kearns-Sayre syndrome (KSS), with ophthalmoplegia and pigmentary retinopathy, and the other termed the MELAS syndrome (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes). Mutations or deletions in mitochondrial genes have been identified.

1	Several forms of hypoparathyroidism, each rare in frequency, are seen as isolated defects; the genetic mechanisms are varied. The inheritance includes autosomal dominant, autosomal recessive, and X-linked modes. Three separate autosomal defects involving the parathyroid gene have been recognized: one is dominant and the other two are recessive. The dominant form has a point mutation in the signal sequence, a critical region involved in intracellular transport of the hormone precursor. An Arg for Cys mutation interferes with processing of the precursor and is believed to trigger an apoptotic cellular response, hence acting as a dominant negative. The other two forms are recessive. One point mutation also blocks cleavage of the PTH precursor but requires both alleles to cause hypoparathyroidism. The third involves a single-nucleotide base change that results in an exon splicing defect; the lost exon contains the promoter—hence, the gene is silenced. An X-linked recessive form of

1	The third involves a single-nucleotide base change that results in an exon splicing defect; the lost exon contains the promoter—hence, the gene is silenced. An X-linked recessive form of hypoparathyroidism has been described in males, and the defect has been localized to chromosome Xq26-q27, perhaps involving the SOX3 gene.

1	Abnormalities in the CaSR are detected in three distinctive hypocalcemic disorders. All are rare, but more than 10 different gain-offunction mutations have been found in one form of hypocalcemia termed autosomal dominant hypocalcemic hypercalciuria (ADHH). The receptor senses the ambient calcium level as excessive and suppresses PTH secretion, leading to hypocalcemia. The hypocalcemia is aggravated by constitutive receptor activity in the renal tubule causing excretion of inappropriate amounts of calcium. Recognition of the syndrome is important because efforts to treat the hypocalcemia with vitamin D analogues and increased oral calcium exacerbate the already excessive urinary calcium excretion (several grams or more per 24 h), leading to irreversible renal damage from stones and ectopic calcification.

1	Other causes of isolated hypoparathyroidism include homozygous, inactivating mutations in the parathyroid-specific transcription factor GCM2, which lead to an autosomal recessive form of the disease, or heterozygous point mutations in GCM2, which have a dominant negative effect on the wild-type protein and thus lead to an autosomal dominant form of hypoparathyroidism. Furthermore, heterozygous mutations in G11, one of the two signaling proteins downstream of the CaSR, have been identified as a cause of autosomal dominant hypoparathyroidism.

1	Bartter’s syndrome is a group of disorders associated with disturbances in electrolyte and acid/base balance, sometimes with nephrocalcinosis and other features. Several types of ion channels or transporters are involved. Curiously, Bartter’s syndrome type V has the electrolyte and pH disturbances seen in the other syndromes but appears to be due to a gain of function in the CaSR. The defect may be more severe than in ADHH and explains the additional features seen beyond hypocalcemia and hypercalciuria. As with autoimmune disorders that block the CaSR (discussed above under hypercalcemic conditions), there are autoantibodies that at least transiently activate the CaSR, leading to suppressed PTH secretion and hypocalcemia. This disorder may wax and wane. Acquired Hypoparathyroidism Acquired chronic hypoparathyroidism is usually the result of inadvertent surgical removal of all the parathyroid

1	Disorders of the Parathyroid Gland and Calcium Homeostasis 2484 glands; in some instances, not all the tissue is removed, but the remainder undergoes vascular supply compromise secondary to fibrotic changes in the neck after surgery. In the past, the most frequent cause of acquired hypoparathyroidism was surgery for hyperthyroidism. Hypoparathyroidism now usually occurs after surgery for hyperparathyroidism when the surgeon, facing the dilemma of removing too little tissue and thus not curing the hyperparathyroidism, removes too much. Parathyroid function may not be totally absent in all patients with postoperative hypoparathyroidism. Rare causes of acquired chronic hypoparathyroidism include radiation-induced damage subsequent to radioiodine therapy of hyperthyroidism and glandular damage in patients with hemochromatosis or hemosiderosis after repeated blood transfusions. Infection may involve one or more of the parathyroids but usually does not cause hypoparathyroidism because all

1	in patients with hemochromatosis or hemosiderosis after repeated blood transfusions. Infection may involve one or more of the parathyroids but usually does not cause hypoparathyroidism because all four glands are rarely involved. Transient hypoparathyroidism is frequent following surgery for hyperparathyroidism. After a variable period of hypoparathyroidism, normal parathyroid function may return due to hyperplasia or recovery of remaining tissue. Occasionally, recovery occurs months after surgery.

1	Treatment involves replacement with vitamin D or 1,25(OH)2D (calcitriol) combined with a high oral calcium intake. In most patients, blood calcium and phosphate levels are satisfactorily regulated, but some patients show resistance and a brittleness, with a tendency to alternate between hypocalcemia and hypercalcemia. For many patients, vitamin D in doses of 40,000–120,000 U/d (1–3 mg/d) combined with ≥1 g elemental calcium is satisfactory. The wide dosage range reflects the variation encountered from patient to patient; precise regulation of each patient is required. Compared to typical daily requirements in euparathyroid patients of 200 U/d (or in older patients as high as 800 U/d), the high dose of vitamin D (as much as 100-fold higher) reflects the reduced conversion of vitamin D to 1,25(OH)2D. Many physicians now use 0.5–1 μg of calcitriol in management of such patients, especially if they are difficult to control. Because of its storage in fat, when vitamin D is withdrawn, weeks

1	Many physicians now use 0.5–1 μg of calcitriol in management of such patients, especially if they are difficult to control. Because of its storage in fat, when vitamin D is withdrawn, weeks are required for the disappearance of the biologic effects, compared with a few days for calcitriol, which has a rapid turnover.

1	Oral calcium and vitamin D restore the overall calcium-phosphate balance but do not reverse the lowered urinary calcium reabsorption typical of hypoparathyroidism. Therefore, care must be taken to avoid excessive urinary calcium excretion after vitamin D and calcium replacement therapy; otherwise, nephrocalcinosis and kidney stones can develop, and the risk of CKD is increased. Thiazide diuretics lower urine calcium by as much as 100 mg/d in hypoparathyroid patients on vitamin D, provided they are maintained on a low-sodium diet. Use of thiazides seems to be of benefit in mitigating hypercalciuria and easing the daily management of these patients. There are now trials of parenterally administered PTH (either PTH[1–34] or PTH[1–84]) in patients with hypoparathyroidism providing greater ease of maintaining serum calcium and reducing urinary calcium excretion (desirable to protect any renal damage). However, PTH therapy for the treatment of hypoparathyroidism is not approved as of yet.

1	Hypomagnesemia Severe hypomagnesemia (<0.4 mmol/L; <0.8 meq/L) is associated with hypocalcemia (Chap. 423). Restoration of the total-body magnesium deficit leads to rapid reversal of hypocalcemia. There are at least two causes of the hypocalcemia—impaired PTH secretion and reduced responsiveness to PTH. For further discussion of causes and treatment of hypomagnesemia, see Chap. 423.

1	The effects of magnesium on PTH secretion are similar to those of calcium; hypermagnesemia suppresses and hypomagnesemia stimulates PTH secretion. The effects of magnesium on PTH secretion are normally of little significance, however, because the calcium effects dominate. Greater change in magnesium than in calcium is needed to influence hormone secretion. Nonetheless, hypomagnesemia might be expected to increase hormone secretion. It is therefore surprising to find that severe hypomagnesemia is associated with blunted secretion of PTH. The explanation for the paradox is that severe, chronic hypomagnesemia leads to intracellular magnesium deficiency, which interferes with secretion and peripheral responses to PTH. The mechanism of the cellular abnormalities caused by hypomagnesemia is unknown, although effects on adenylate cyclase (for which magnesium is a cofactor) have been proposed.

1	PTH levels are undetectable or inappropriately low in severe hypomagnesemia despite the stimulus of severe hypocalcemia, and acute repletion of magnesium leads to a rapid increase in PTH level. Serum phosphate levels are often not elevated, in contrast to the situation with acquired or idiopathic hypoparathyroidism, probably because phosphate deficiency is often seen in hypomagnesmia (Chap. 393). Diminished peripheral responsiveness to PTH also occurs in some patients, as documented by subnormal response in urinary phosphorus and urinary cAMP excretion after administration of exogenous PTH to patients who are hypocalcemic and hypomagnesemic. Both blunted PTH secretion and lack of renal response to administered PTH can occur in the same patient. When acute magnesium repletion is undertaken, the restoration of PTH levels to normal or supra-normal may precede restoration of normal serum calcium by several days.

1	Repletion of magnesium cures the condition. Repletion should be parenteral. Attention must be given to restoring the intracellular deficit, which may be considerable. After IV magnesium administration, serum magnesium may return transiently to the normal range, but unless replacement therapy is adequate, serum magnesium will again fall. If the cause of the hypomagnesemia is renal magnesium wasting, magnesium may have to be given long-term to prevent recurrence (Chap. 423).

1	PTH is not sufficiently active to fully prevent hypocalcemia (although retaining phosphaturic activity, for example). This problem occurs when the PTH1R–signaling protein complex is defective (as in the different forms of pseudohypoparathyroidism [PHP], discussed below); when PTH action to promote calcium absorption from the diet via the synthesis of 1,25(OH)2D is insufficient because of vitamin D deficiency or because vitamin D is ineffective (defects in vitamin D receptor or vitamin D synthesis); or in CKD in which the calcium-elevating action of PTH is impaired. Typically, hypophosphatemia is more severe than hypocalcemia in vitamin D deficiency states because of the increased secretion of PTH, which, although only partly effective in elevating blood calcium, is readily capable of promoting urinary phosphate excretion.

1	PHP, on the other hand, has a pathophysiology that is different from the other disorders of ineffective PTH action. PHP resembles hypoparathyroidism (in which PTH synthesis is deficient) and is manifested by hypocalcemia and hyperphosphatemia, yet elevated PTH levels. The cause of the disorder is defective PTH-dependent activation of the stimulatory G protein complex or the downstream effector protein kinase A, resulting in failure of PTH to increase intracellular cAMP or to respond to elevated cAMP levels (see below).

1	Chronic Kidney Disease Improved medical management of CKD now allows many patients to survive for decades and hence allows time enough to develop features of renal osteodystrophy, which must be controlled to avoid additional morbidity. Impaired production of 1,25(OH)2D is now thought to be the principal factor that causes calcium deficiency, secondary hyperparathyroidism, and bone disease; hyperphosphatemia typically occurs only in the later stages of the disease. Low levels of 1,25(OH)2D due to increased FGF23 production in bone are critical in the development of hypocalcemia. The uremic state also causes impairment of intestinal absorption by mechanisms other than defects in vitamin D metabolism. Nonetheless, treatment with supraphysiologic amounts of vitamin D or calcitriol can correct the impaired calcium absorption. Because increased FGF23 levels are seen even in early stages of CKD and have been reported to correlate with increased mortality and left ventricular hypertrophy,

1	the impaired calcium absorption. Because increased FGF23 levels are seen even in early stages of CKD and have been reported to correlate with increased mortality and left ventricular hypertrophy, there is current interest in approaches to lower intestinal phosphate absorption early during the course of kidney disease and to thereby lower FGF23 levels. However, there is concern as to whether vitamin D supplementation increases the circulating FGF23 levels in CKD patients. Although vitamin D analogs improve survival in this patient population, it is notable that there are often dramatic elevations of FGF23.

1	Hyperphosphatemia in CKD lowers blood calcium levels by several mechanisms, including extraosseous deposition of calcium and phosphate, impairment of the bone-resorbing action of PTH, and reduction in 1,25(OH)2D production by remaining renal tissue.

1	Therapy of CKD (Chap. 335) involves appropriate management of patients prior to dialysis and adjustment of regimens once dialysis is initiated. Attention should be paid to restriction of phosphate in the diet; avoidance of aluminum-containing phosphate-binding antacids to prevent the problem of aluminum intoxication; provision of an adequate calcium intake by mouth, usually 1–2 g/d; and supplementation with 0.25–1 μg/d calcitriol or other activated forms of vitamin D. Each patient must be monitored closely. The aim of therapy is to restore normal calcium balance to prevent osteomalacia and severe secondary hyperparathyroidism (it is usually recommended to maintain PTH levels between 100 and 300 pg/ mL) and, in light of evidence of genetic changes and monoclonal outgrowths of parathyroid glands in CKD patients, to prevent secondary hyperparathyroidism from becoming autonomous hyperparathyroidism. Reduction of hyperphosphatemia and restoration of normal intestinal calcium absorption by

1	in CKD patients, to prevent secondary hyperparathyroidism from becoming autonomous hyperparathyroidism. Reduction of hyperphosphatemia and restoration of normal intestinal calcium absorption by calcitriol can improve blood calcium levels and reduce the manifestations of secondary hyperparathyroidism. Because adynamic bone disease can occur in association with low PTH levels, it is important to avoid excessive suppression of the parathyroid glands while recognizing the beneficial effects of controlling the secondary hyperparathyroidism. These patients should probably be closely monitored with PTH assays that detect only the full-length PTH(1–84) to ensure that biologically active PTH and not inactive, inhibitory PTH fragments are measured. Use of phosphate-binding agents such as sevelamer is approved only in end-stage renal disease, but it may be necessary to initiate such treatment much earlier during the course of kidney disease to prevent the increase in FGF23 and its “off-target”

1	is approved only in end-stage renal disease, but it may be necessary to initiate such treatment much earlier during the course of kidney disease to prevent the increase in FGF23 and its “off-target” effects.

1	Vitamin D Deficiency due to Inadequate Diet and/or Sunlight Vitamin D deficiency due to inadequate intake of dairy products enriched with vitamin D, lack of vitamin supplementation, and reduced sunlight exposure in the elderly, particularly during winter in northern latitudes, is more common in the United States than previously recognized. Biopsies of bone in elderly patients with hip fracture (documenting osteomalacia) and abnormal levels of vitamin D metabolites, PTH, calcium, and phosphate indicate that vitamin D deficiency may occur in as many as 25% of elderly patients, particularly in northern latitudes in the United States. Concentrations of 25(OH)D are low or low-normal in these patients. Quantitative histomorphometric analysis of bone biopsy specimens from such individuals reveals widened osteoid seams consistent with osteomalacia (Chap. 423). PTH hypersecretion compensates for the tendency for the blood calcium to fall but also increases renal phosphate excretion and thus

1	widened osteoid seams consistent with osteomalacia (Chap. 423). PTH hypersecretion compensates for the tendency for the blood calcium to fall but also increases renal phosphate excretion and thus causes osteomalacia.

1	Treatment involves adequate replacement with vitamin D and cal-2485 cium until the deficiencies are corrected. Severe hypocalcemia rarely occurs in moderately severe vitamin D deficiency of the elderly, but vitamin D deficiency must be considered in the differential diagnosis of mild hypocalcemia. Mild hypocalcemia, secondary hyperparathyroidism, severe hypophosphatemia, and a variety of nutritional deficiencies occur with gastrointestinal diseases. Hepatocellular dysfunction can lead to reduction in 25(OH)D levels, as in portal or biliary cirrhosis of the liver, and malabsorption of vitamin D and its metabolites, including 1,25(OH)2D, may occur in a variety of bowel diseases, hereditary or acquired. Hypocalcemia itself can lead to steatorrhea, due to deficient production of pancreatic enzymes and bile salts. Depending on the disorder, vitamin D or its metabolites can be given parenterally, guaranteeing adequate blood levels of active metabolites.

1	Defective Vitamin D Metabolism • anticonvulSant tHerapy Anticonvulsant therapy with any of several agents induces acquired vitamin D deficiency by increasing the conversion of vitamin D to inactive compounds and/or causing resistance to its action. The more marginal the vitamin D intake in the diet, the more likely that anticonvulsant therapy will lead to abnormal mineral and bone metabolism.

1	vitamin d–dependent ricKetS type i Vitamin D–dependent rickets type I, previously termed pseudo-vitamin D–resistant rickets, differs from true vitamin D–resistant rickets (vitamin D–dependent rickets type II, see below) in that it is typically less severe and the biochemical and radiographic abnormalities can be reversed with appropriate doses of the vitamin’s active metabolite, 1,25(OH)2D. Physiologic amounts of calcitriol cure the disease (Chap. 423). This finding fits with the pathophysiology of the disorder, which is autosomal recessive, and is now known to be caused by mutations in the gene encoding 25(OH) D-1α-hydroxylase. Both alleles are inactivated in affected patients, and compound heterozygotes, harboring distinct mutations, are common.

1	Clinical features include hypocalcemia, often with tetany or con vulsions, hypophosphatemia, secondary hyperparathyroidism, and osteomalacia, often associated with skeletal deformities and increased alkaline phosphatase. Treatment involves physiologic replacement doses of 1,25(OH)2D (Chap. 423). vitamin d–dependent ricKetS type ii Vitamin D–dependent rickets type II results from end-organ resistance to the active metabolite 1,25(OH)2D. The clinical features resemble those of the type I disorder and include hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and rickets but also partial or total alopecia. Plasma levels of 1,25(OH)2D are elevated, in keeping with the refractoriness of the end organs. This disorder is caused by mutations in the gene encoding the vitamin D receptor; treatment is difficult and requires regular, usually nocturnal calcium infusions, which dramatically improve growth but do not restore hair growth (Chap. 423).

1	Pseudohypoparathyroidism PHP refers to a group of distinct inherited disorders. Patients affected by PHP type Ia (PHP-Ia) are characterized by symptoms and signs of hypocalcemia in association with distinctive skeletal and developmental defects. The hypocalcemia is due to a deficient response to PTH, which is probably restricted to the proximal renal tubules. Hyperplasia of the parathyroids, a response to hormone-resistant hypocalcemia, causes elevation of PTH levels. Studies, both clinical and basic, have clarified some aspects of these disorders, including the variable clinical spectrum, the pathophysiology, the genetic defects, and their mode of inheritance.

1	A working classification of the various forms of PHP is given in Table 424-6. The classification scheme is based on the signs of ineffective PTH action (low calcium and high phosphate), low or normal urinary cAMP response to exogenous PTH, the presence or absence of Albright’s hereditary osteodystrophy (AHO), and assays to measure the concentration of the Gsα subunit of the adenylate cyclase enzyme. Using these criteria, there are four types: PHP types Ia and Ib; pseudopseudohypoparathyroidism (PPHP), and PHP-II. Disorders of the Parathyroid Gland and Calcium Homeostasis PHP-Ia Yes ↓↑ Yes Yes PPHP No Normal Normal Yes Yes PHP-Ib Yes ↓↑ No No PHP-II Yes Normal ↑ No No Acrodysostosis with Yes Normal (but ↓↑ No Yes phosphaturic response) Abbreviations: ↓, decreased; ↑, increased; AHO, Albright’s hereditary osteodystrophy; PTH, parathyroid hormone.

1	pHp-ia and pHp-iB Individuals with PHP-I, the most common of the disorders, show a deficient urinary cAMP response to administration of exogenous PTH. Patients with PHP-I are divided into type Ia and type Ib. Patients with PHP-Ia show evidence for AHO and reduced amounts of Gsα protein/activity, as determined in readily accessible tissues such as erythrocytes, lymphocytes, and fibroblasts. Patients with PHP-Ib typically lack evidence for AHO and they have normal G α activity. PHP-Ic, sometimes listed as a third form of PHP-I, is really sa variant of PHP-Ia, although the mutant G α shows normal activity in certain in vitro assays. s

1	Most patients who have PHP-Ia reveal characteristic features of AHO, which consist of short stature, round face, obesity, skeletal anomalies (brachydactyly), intellectual impairment, and/or heterotopic calcifications. Patients have low calcium and high phosphate levels, as with true hypoparathyroidism. PTH levels, however, are elevated, reflecting resistance to hormone action. Amorphous deposits of calcium and phosphate are found in the basal ganglia in about one-half of patients. The defects in metacarpal and metatarsal bones are sometimes accompanied by short phalanges as well, possibly reflecting premature closing of the epiphyses. The typical findings are short fourth and fifth metacarpals and metatarsals. The defects are usually bilateral. Exostoses and radius curvus are frequent.

1	Inheritance and Genetic Defects Multiple defects at the GNAS locus have now been identified in PHP-Ia, PHP-Ib, and PPHP patients. This gene, which is located on chromosome 20q13.3, encodes the α-subunit of the stimulatory G protein (Gsα), among other products (see below). Mutations include abnormalities in splice junctions associated with deficient mRNA production, point mutations, insertions, and/or deletion that all result in a protein with defective function resulting in a 50% reduction of Gsα activity in erythrocytes or other cells.

1	Detailed analyses of disease transmission in affected kindreds have clarified many features of PHP-Ia, PPHP, and PHP-Ib (Fig. 424-7). The former two entities, often traced through multiple generations, have an inheritance pattern consistent with genetic imprinting. The phenomenon of gene imprinting, involving methylation of genetic loci, independent of any mutation, impairs transcription from either the maternal or the paternal allele (Chap. 82). The Gsα transcript is biallelically expressed in most tissues; expression from paternal allele is silenced through as-of-yet unknown mechanisms in some tissues including the proximal renal tubules and the thyroid; consequently, inheritance of a defective paternal allele has no implications with regard to hormonal function. Thus, females affected by either PHP-Ia or PPHP will have offspring with PHP-Ia, if these children inherit the allele carrying the GNAS mutation; in contrast, if the mutant allele is inherited from a male affected by either

1	either PHP-Ia or PPHP will have offspring with PHP-Ia, if these children inherit the allele carrying the GNAS mutation; in contrast, if the mutant allele is inherited from a male affected by either disorder, the offspring will exhibit PPHP. Consistent with these data in humans, gene-ablation studies in mice have shown that inheritance of the mutant G α allele from the female causes much reduced Gsα protein in renal cortex, hypocalcemia, and resistance to PTH. Offspring inheriting the mutant allele from the male showed no evidence of PTH resistance or hypocalcemia.

1	Imprinting is tissue selective. Paternal Gsα expression is not silenced in most tissues. It seems likely, therefore, that the AHO phenotype recognized in PPHP as well as PHP-Ia reflects G α haploinsufficiency during embryonic or postnatal development. s The complex mechanisms that control the GNAS gene contribute to challenges involved in unraveling the pathogenesis of these disorders, especially that of PHP-Ib. Much intensive work with families in which multiple members are affected by PHP-Ib, as well as studies of the complex regulation of the GNAS gene locus, have now shown that PHP-Ib is caused by microdeletions within or upstream of the GNAS locus, which are associated with a loss of DNA methylation at one or several loci of the maternal allele (Table 424-6). These abnormalities in methylation silence the expression of the gene. This leads in the proximal renal tubules—where Gsα appears to be expressed exclusively from the maternal allele—to PTH resistance.

1	PHP-Ib, lacking the AHO phenotype in most instances, shares with PHP-Ia the hypocalcemia and hyperphosphatemia caused by PTH resistance, and thus the blunted urinary cAMP response to administered PTH, a standard test to assess the presence or absence of hormone resistance (Table 424-6). Furthermore, these endocrine abnormalities become apparent only if the disease-causing mutation is inherited maternally. Bone responsiveness may be excessive rather than blunted in PHP-Ib (and in PHP-Ia) patients, based on case reports that have emphasized an osteitis fibrosa–like pattern in several PHP-Ib patients. PHP-II refers to patients with hypocalcemia and hyperphosphatemia, who have a normal urinary cAMP but an impaired urinary phosphaturic

1	PHP-II refers to patients with hypocalcemia and hyperphosphatemia, who have a normal urinary cAMP but an impaired urinary phosphaturic FIGURE 424-7 Paternal imprinting of renal parathyroid hormone (PTH) resistance. An impaired excretion of urinary cyclic AMP and phosphate is observed in patients with pseudohypoparathyroidism type Ia (PHP-Ia). In the renal cortex, there is selective silencing of paternal Gsα expression. The disease becomes manifest only in patients who inherit the defective gene from an obligate female carrier (left). If the genetic defect is inherited from an obligate male gene carrier, there is no biochemical abnormality; administration of PTH causes an appropriate increase in the urinary cyclic AMP and phosphate concentration (pseudo-PHP [PPHP]; right). Both patterns of inheritance lead to Albright’s hereditary osteodystrophy (AHO), perhaps because of haplotype insufficiency—i.e., both copies of G α must be active for normal bone development.

1	response to PTH. In a PHP-II variant, referred to as acrodysostosis with hormonal resistance (ADOHR), patients have a defect in the regulatory subunit of PKA (PRKAR1A) that mediates the response to PTH distal to cAMP production. Acrodysostosis without hormonal resistance is caused by mutations in the cAMP-selective phosphodiesterase 4 (ADOP4). It remains unclear why the PTH-resistance in some patients, labeled as PHP-II without bony abnormalities, resolves upon treatment with vitamin D supplements.

1	The diagnosis of these hormone-resistant states can usually be made without difficulty when there is a positive family history for features of AHO, in association with the signs and symptoms of hypocalcemia. In both categories—PHP-Ia and PHP-Ib—serum PTH levels are elevated, particularly when patients are hypocalcemic. However, patients with PHP-Ib or PHP-II without acrodysostosis present only with hypocalcemia and high PTH levels, as evidence for hormone resistance. In PHP-Ia and PHP-Ib, the response of urinary cAMP to the administration of exogenous PTH is blunted. The diagnosis of PHP-II, in the absence of acrodysostosis, is more complex, and vitamin D deficiency must be excluded before such a diagnosis can be entertained.

1	Treatment of PHP is similar to that of hypoparathyroidism, except that calcium and vitamin D doses are usually higher. Patients with PHP show no PTH-resistance in the distal tubules—hence, urinary calcium clearance is typically reduced, and they are not at risk of developing nephrocalcinosis as are patients with true hypoparathyroidism, unless overtreatment occurs, for example, after the completion of pubertal development and skeletal mutation, when calcium and 1,25(OH)2D treatment should be reduced. Variability in response makes it necessary to establish the optimal regimen for each patient, based on maintaining appropriate blood calcium level and urinary calcium excretion and keeping the PTH level within or slightly above the normal range.

1	Occasionally, loss of calcium from the ECF is so severe that PTH cannot compensate. Such situations include acute pancreatitis and severe, acute hyperphosphatemia, often in association with renal failure, conditions in which there is rapid efflux of calcium from the ECF. Severe hypocalcemia can occur quickly; PTH rises in response to hypocalcemia but does not return blood calcium to normal.

1	Severe, Acute Hyperphosphatemia Severe hyperphosphatemia is associated with extensive tissue damage or cell destruction (Chap. 423). The combination of increased release of phosphate from muscle and impaired ability to excrete phosphorus because of renal failure causes moderate to severe hyperphosphatemia, the latter causing calcium loss from the blood and mild to moderate hypocalcemia. Hypocalcemia is usually reversed with tissue repair and restoration of renal function as phosphorus and creatinine values return to normal. There may even be a mild hypercalcemic period in the oliguric phase of renal function recovery. This sequence, severe hypocalcemia followed by mild hypercalcemia, reflects widespread deposition of calcium in muscle and subsequent redistribution of some of the calcium to the ECF after phosphate levels return to normal.

1	Other causes of hyperphosphatemia include hypothermia, massive hepatic failure, and hematologic malignancies, either because of high cell turnover of malignancy or because of cell destruction by chemotherapy. TREATmEnT severe, acute HyperpHospHatemia Treatment is directed toward lowering of blood phosphate by the administration of phosphate-binding antacids or dialysis, often needed for the management of CKD. Although calcium replacement may be necessary if hypocalcemia is severe and symptomatic, calcium administration during the hyperphosphatemic period tends to increase extraosseous calcium deposition and aggravate tissue 2487 damage. The levels of 1,25(OH)2D may be low during the hyperphosphatemic phase and return to normal during the oliguric phase of recovery.

1	Osteitis Fibrosa after Parathyroidectomy Severe hypocalcemia after parathyroid surgery is rare now that osteitis fibrosa cystica is an infrequent manifestation of hyperparathyroidism. When osteitis fibrosa cystica is severe, however, bone mineral deficits can be large. After parathyroidectomy, hypocalcemia can persist for days if calcium replacement is inadequate. Treatment may require parenteral administration of calcium; addition of calcitriol and oral calcium supplementation is sometimes needed for weeks to a month or two until bone defects are filled (which, of course, is of therapeutic benefit in the skeleton), making it possible to discontinue parenteral calcium and/or reduce the amount.

1	Care must be taken to ensure that true hypocalcemia is present; in addition, acute transient hypocalcemia can be a manifestation of a variety of severe, acute illnesses, as discussed above. Chronic hypocalcemia, however, can usually be ascribed to a few disorders associated with absent or ineffective PTH. Important clinical criteria include the duration of the illness, signs or symptoms of associated disorders, and the presence of features that suggest a hereditary abnormality. A nutritional history can be helpful in recognizing a low intake of vitamin D and calcium in the elderly, and a history of excessive alcohol intake may suggest magnesium deficiency.

1	Hypoparathyroidism and PHP are typically lifelong illnesses, usually (but not always) appearing by adolescence; hence, a recent onset of hypocalcemia in an adult is more likely due to nutritional deficiencies, renal failure, or intestinal disorders that result in deficient or ineffective vitamin D. Neck surgery, even long past, however, can be associated with a delayed onset of postoperative hypoparathyroidism. A history of seizure disorder raises the issue of anticonvulsive medication. Developmental defects may point to the diagnosis of PHP. Rickets and a variety of neuromuscular syndromes and deformities may indicate ineffective vitamin D action, either due to defects in vitamin D metabolism or to vitamin D deficiency.

1	A pattern of low calcium with high phosphorus in the absence of renal failure or massive tissue destruction almost invariably means hypoparathyroidism or PHP. A low calcium and low phosphorus pattern points to absent or ineffective vitamin D, thereby impairing the action of PTH on calcium metabolism (but not phosphate clearance). The relative ineffectiveness of PTH in calcium homeostasis in vitamin D deficiency, anticonvulsant therapy, gastrointestinal disorders, and hereditary defects in vitamin D metabolism leads to secondary hyperparathyroidism as a compensation. The excess PTH on renal tubule phosphate transport accounts for renal phosphate wasting and hypophosphatemia.

1	Exceptions to these patterns may occur. Most forms of hypomagnesemia are due to long-standing nutritional deficiency as seen in chronic alcoholics. Despite the fact that the hypocalcemia is principally due to an acute absence of PTH, phosphate levels are usually low, rather than elevated, as in hypoparathyroidism. Chronic renal failure is often associated with hypocalcemia and hyperphosphatemia, despite secondary hyperparathyroidism.

1	Diagnosis is usually established by application of the PTH immunoassay, tests for vitamin D metabolites, and measurements of the urinary cAMP response to exogenous PTH. In hereditary and acquired hypoparathyroidism and in severe hypomagnesemia, PTH is either undetectable or inappropriately in the normal range (Fig. 424-4). This finding in a hypocalcemic patient is supportive of hypoparathyroidism, as distinct from ineffective PTH action, in which even mild hypocalcemia is associated with elevated PTH levels. Hence a failure to detect elevated PTH levels establishes the diagnosis of hypoparathyroidism; elevated levels suggest the presence of secondary hyperparathyroidism, as found in many of the situations in which the hormone is

1	Disorders of the Parathyroid Gland and Calcium Homeostasis 2488 ineffective due to associated abnormalities in vitamin D action. Assays –2.5. Postmenopausal women who fall at the lower end of the young for 25(OH)D can be helpful. Low or low-normal 25(OH)D indicates normal range (a T-score <–1.0) are defined as having low bone density vitamin D deficiency due to lack of sunlight, inadequate vitamin D and are also at increased risk of osteoporosis. Although risk is lower intake, or intestinal malabsorption. Recognition that mild hypocalce-in this group, more than 50% of fractures among postmenopausal mia, rickets, and hypophosphatemia are due to anticonvulsant therapy women, including hip fractures, occur in this group with low bone is made by history. density, because the number of individuals in this category is so much larger than that in the osteoporosis range. As a result, there are ongo- ing attempts to identify individuals within the low bone density range who are at high risk of

1	in this category is so much larger than that in the osteoporosis range. As a result, there are ongo- ing attempts to identify individuals within the low bone density range who are at high risk of fracture and might benefit from pharmacologic intervention. Furthermore, some have advocated using fracture risk as

1	The management of hypoparathyroidism, PHP, chronic renal failure, and hereditary defects in vitamin D metabolism involves the use of vitamin D or vitamin D metabolites and calcium supplementation. Vitamin D itself is the least expensive form of vitamin D replacement and is frequently used in the management of uncomplicated hypoparathyroidism and some disorders associated with ineffective vitamin D action. When vitamin D is used prophylactically, as in the elderly or in those with chronic anticonvulsant therapy, there is a wider margin of safety than with the more potent metabolites. However, most of the conditions in which vitamin D is administered chronically for hypocalcemia require amounts 50–100 times the daily replacement dose because the formation of 1,25(OH)2D is deficient. In such situations, vitamin D is no safer than the active metabolite because intoxication can occur with high-dose therapy (because of storage in fat). Calcitriol is more rapid in onset of action and also

1	situations, vitamin D is no safer than the active metabolite because intoxication can occur with high-dose therapy (because of storage in fat). Calcitriol is more rapid in onset of action and also has a short biologic half-life.

1	Vitamin D (at least 1000 U/d [2–3 μg/d] [higher levels required in older persons]) or calcitriol (0.25–1 μg/d) is required to prevent rickets in normal individuals. In contrast, 40,000–120,000 U (1–3 mg) of vitamin D2 or D3 is typically required in hypoparathyroidism. The dose of calcitriol is unchanged in hypoparathyroidism, because the defect is in hydroxylation by the 25(OH)D-1α-hydroxylase. Calcitriol is also used in disorders of 25(OH)D-1α-hydroxylase; vitamin D receptor defects are much more difficult to treat.

1	Patients with hypoparathyroidism should be given 2–3 g of elemental calcium PO each day. The two agents, vitamin D or calcitriol and oral calcium, can be varied independently. Urinary calcium excretion needs to be monitored carefully. If hypocalcemia alternates with episodes of hypercalcemia in high-brittleness patients with hypoparathyroidism, administration of calcitriol and use of thiazides, as discussed above, may make management easier. Clinical trials with PTH(1–34) or PTH(1–84) are promising, but these alternative treatments have not yet been approved. the “diagnostic” criterion for osteoporosis.

1	the “diagnostic” criterion for osteoporosis. In the United States, as many as 9 million adults have osteoporosis (T-score <–2.5 in either spine or hip), and an additional 48 million individuals have bone mass levels that put them at increased risk of developing osteoporosis (e.g., bone mass T-score <–1.0). Osteoporosis occurs more frequently with increasing age as bone tissue is lost progressively. In women, the loss of ovarian function at menopause (typically about age 50) precipitates rapid bone loss so that most women meet the diagnostic criterion for osteoporosis by age 70–80. As the population continues to age, the number of individuals with osteoporosis and fractures will also continue to increase, despite a recognized reduction in age-specific risk. It is estimated that about 2 million fractures occur each year in the United States as a consequence of osteoporosis, and that number is expected to increase as the population continues to age.

1	The epidemiology of fractures follows the trend for loss of bone density, with exponential increases in both hip and vertebral fractures with age. Fractures of the distal radius have a somewhat different epidemiology, increasing in frequency before age 50 and plateauing by age 60, with only a modest age-related increase thereafter. In contrast, incidence rates for hip fractures double every 5 years after age 70 (Fig. 425-1). This distinct epidemiology may be related to the way the elderly fall as they age, with fewer falls on an outstretched hand and more falls directly on the hip. About 300,000 hip fractures occur each year in the United States, most of which require hospital admission and surgical intervention. The probability that a 50-year-old white individual will have a hip fracture during his or her lifetime is 14% for women and 5% for men; the risk for African Americans is lower (about one-half those rates), and the risk for Asians is roughly equal to that for whites. Hip

1	during his or her lifetime is 14% for women and 5% for men; the risk for African Americans is lower (about one-half those rates), and the risk for Asians is roughly equal to that for whites. Hip fractures are associated with a high incidence of deep vein thrombosis and pulmonary embolism (20–50%) and a mortality rate between 5 and 20% during the year after surgery.

1	osteoporosis Robert Lindsay, Felicia Cosman Osteoporosis, a condition characterized by decreased bone strength, is prevalent among postmenopausal women but also occurs in men and women with underlying conditions or major risk factors associ-ated with bone demineralization. Its chief clinical manifestations are 425 Incidence/100,000 person-year 3,000 2,000 1,000 vertebral and hip fractures, although fractures can occur at almost any skeletal site. Osteoporosis affects almost 10 million individuals in the United States, but only a small proportion are diagnosed and treated. Osteoporosis is defined as a reduction in the strength of bone that leads to an increased risk of fractures. Loss of bone tissue is associated

1	Osteoporosis is defined as a reduction in the strength of bone that leads to an increased risk of fractures. Loss of bone tissue is associated Age group, year with deterioration in skeletal microarchitecture. The World Health Organization (WHO) operationally defines osteoporosis as a bone FIGURE 425-1 Epidemiology of vertebral, hip, and Colles’ fracdensity that falls 2.5 standard deviations (SD) below the mean for tures with age. (Adapted from C Cooper, LJ Melton III: Trends Endocrinol young healthy adults of the same sex—also referred to as a T-score of Metab 3:224, 1992; with permission.) FIGURE 425-2 Lateral spine x-ray showing severe osteopenia and a severe wedge-type deformity (severe anterior compression). There is also significant morbidity, with about 20–40% of survivors requiring long-term care, and many who are unable to function as they did before the fracture.

1	There is also significant morbidity, with about 20–40% of survivors requiring long-term care, and many who are unable to function as they did before the fracture. There are about 550,000 vertebral crush fractures per year in the United States. Only a fraction (estimated to be one-third) of them are recognized clinically, because many are relatively asymptomatic and are identified incidentally during radiography for other purposes (Fig. 425-2). Vertebral fractures rarely require hospitalization but are associated with long-term morbidity and a slight increase in mortality rates, primarily related to pulmonary disease. Multiple vertebral fractures lead to height loss (often of several inches), kyphosis, and secondary pain and discomfort related to altered biomechanics of the back. Thoracic fractures can be associated with restrictive lung disease, whereas lumbar fractures are associated with abdominal symptoms that include distention, early satiety, and constipation.

1	Approximately 400,000 wrist fractures and 135,000 pelvic fractures occur in the United States each year. Fractures of the humerus and other bones (estimated to be about 675,000 per year) also occur with osteoporosis; this is not surprising in light of the fact that bone loss is a systemic phenomenon. Although some fractures result from major trauma, the threshold for fracture is reduced for an osteoporotic bone (Fig. 425-3). In addition to bone density, there are a number of risk factors for fracture; the common ones are summarized in Table 425-1 Age, prior fractures (especially recent fractures), a family history of osteoporosis-related fractures, low body weight, smoking, and excessive alcohol use are all independent predictors of fracture. Chronic diseases with inflammatory components that increase skeletal remodeling such as rheumatoid arthritis, increase the risk of osteoporosis, as do FIGURE 425-3 Factors leading to osteoporotic fractures.

1	FIGURE 425-3 Factors leading to osteoporotic fractures. diseases associated with malabsorption. Chronic diseases that increase 2489 the risk of falling or frailty, including dementia, Parkinson’s disease, and multiple sclerosis, also increase fracture risk. In the United States and Europe, osteoporosis-related fractures are more common among women than men, presumably due to a lower peak bone mass as well as postmenopausal bone loss in women. However, this sex difference in bone density and age-related increase in hip fractures is not as apparent in some other cultures, possibly due to genetics, physical activity level, or diet.

1	Fractures are themselves risk factors for future fractures (Table 425-1). Vertebral fractures increase the risk of other vertebral fractures as well as fractures of the peripheral skeleton such as the hip and wrist. Wrist fractures also increase the risk of vertebral and hip fractures. The risk for subsequent fractures is particularly high in the first several years after the first fracture, and the risk wanes considerably thereafter. Consequently, among individuals over age 50, any fracture should be considered as potentially related to osteoporosis regardless of the circumstances of the fracture. Osteoporotic bone is more likely to fracture than normal bone at any level of trauma, and a fracture in a person over 50 should trigger evaluation for osteoporosis. This often does not occur because postfracture care is not always well coordinated.

1	Osteoporosis results from bone loss due to age-related changes in bone remodeling as well as extrinsic and intrinsic factors that exaggerate this process. These changes may be superimposed on a low peak bone mass. Consequently, understanding the bone remodeling process is fundamental to understanding the pathophysiology of osteoporosis (Chap. 423). During growth, the skeleton increases in size by linear growth and by apposition of new bone tissue on the outer surfaces of the cortex (Fig. 425-4). The latter process is called modeling, a process that also allows the long bones to adapt in shape to the stresses placed on them. Increased sex hormone production at puberty is required for skeletal maturation, which reaches maximum mass and density in early adulthood. It is around puberty that the sexual dimorphism in skeletal size becomes obvious, although true bone density remains similar between the sexes. Nutrition and lifestyle also play an important role in growth, although genetic

1	the sexual dimorphism in skeletal size becomes obvious, although true bone density remains similar between the sexes. Nutrition and lifestyle also play an important role in growth, although genetic factors primarily determine peak skeletal mass and density. Numerous genes control skeletal growth, peak bone mass, and body size, as well as skeletal structure and density. Heritability estimates of 50–80% for bone density and size have been derived on the basis of twin studies. Although peak bone mass is often lower among individuals with a family history of osteoporosis, association studies of candidate genes (vitamin D receptor; type I collagen, the estrogen receptor [ER], and interleukin 6 [IL-6]; and insulin-like growth factor I [IGF-I]) and bone mass, bone turnover, and fracture prevalence have been inconsistent. Linkage studies suggest that a genetic locus on chromosome 11 is associated with high bone mass. Families with high bone mass and without much apparent age-related bone loss

1	have been inconsistent. Linkage studies suggest that a genetic locus on chromosome 11 is associated with high bone mass. Families with high bone mass and without much apparent age-related bone loss have been shown to have a point mutation in LRP5, a low-density lipoprotein receptor–related protein. The role of this gene in the general population is not clear, although a nonfunctional mutation results in osteoporosispseudoglioma syndrome, and LRP5 signaling appears to be important in controlling bone formation. LRP5 acts through the Wnt signaling pathway. With LRP5 and Wnt activation, beta-catenin is translocated to the nucleus, allowing stimulation of osteoblast formation, activation, and life span as well as suppression of osteoclast activity, thereby increasing bone formation. The osteocyte product, sclerostin, is a negative inhibitor of Wnt signaling.

1	Genome-wide scans for low bone mass suggest multiple genes are involved, many of which are also implicated in control of body size. In adults, bone remodeling, not modeling, is the principal metabolic skeletal process. Bone remodeling has two primary functions: (1) to repair microdamage within the skeleton to maintain skeletal strength and ensure the relative youth of the skeleton and (2) to supply calcium from the skeleton to maintain serum calcium. Remodeling may be activated by microdamage to bone as a result of excessive or accumulated stress. Acute demands for calcium involve 2490 TAblE 425-1 ConDiTionS, DiSEASES, AnD mEDiCATionS THAT ConTRibuTE To oSTEoPoRoSiS AnD fRACTuRES Parental history of hip fracture Porphyria Riley-Day syndrome

1	Parental history of hip fracture Porphyria Riley-Day syndrome Thiazolidinediones (such as pioglitazone and rosiglitazone) osteoclast-mediated resorption as well as calcium transport by osteocytes. Chronic demands for calcium result in secondary hyperparathyroidism, increased bone remodeling, and overall loss of bone tissue.

1	Bone remodeling also is regulated by several circulating hormones, including estrogens, androgens, vitamin D, and parathyroid hormone (PTH), as well as locally produced growth factors such as IGF-I and immunoreactive growth hormone II (IGH-II), transforming growth factor β (TGF-β), parathyroid hormone–related peptide (PTHrP), interleukins (ILs), prostaglandins, and members of the tumor necrosis factor (TNF) superfamily. These factors primarily modulate the rate at which new remodeling sites are activated, a process that results initially in bone resorption by osteoclasts, followed by a period of repair during which new bone tissue is synthesized by osteoblasts. The cytokine responsible for communication between the osteoblasts, other marrow cells, and osteoclasts is RANK ligand (RANKL; receptor activator of nuclear factor-κB [NF-κB]). RANKL, a member of the TNF family, is secreted by osteoblasts and certain cells of the immune system (Chap. 423). The osteoclast receptor for this

1	receptor activator of nuclear factor-κB [NF-κB]). RANKL, a member of the TNF family, is secreted by osteoblasts and certain cells of the immune system (Chap. 423). The osteoclast receptor for this protein is referred to as RANK. Activation of RANK by RANKL is a final common path in osteoclast development, activation, and life span. A humoral decoy for RANKL, also secreted by osteoblasts, is osteoprotegerin (Fig. 425-5). Modulation of osteoclast recruitment and activity appears to be related to the interplay among these three factors. It appears that estrogens are pivotal in modulating secretion of osteoprotegerin (OPG) and perhaps also RANKL. Additional influences include nutrition (particularly calcium intake) and physical activity level.

1	In young adults, resorbed bone is replaced by an equal amount of new bone tissue. Thus, the mass of the skeleton remains constant after peak bone mass is achieved in adulthood. After age 30–45, however, the resorption and formation processes become imbalanced, and resorption exceeds formation. This imbalance may begin at different ages and varies at different skeletal sites; it becomes exaggerated in women after menopause. Excessive bone loss can be due to an increase in osteoclastic activity and/or a decrease in osteoblastic activity. In addition, an increase in remodeling activation frequency, and thus the number of remodeling sites, can magnify the small imbalance seen at each remodeling unit. Increased recruitment of bone remodeling sites produces a reversible reduction in bone tissue but also can result in permanent loss of tissue and disrupted skeletal architecture. In trabecular bone, if the osteoclasts penetrate trabeculae, they leave no template for new bone formation to

1	but also can result in permanent loss of tissue and disrupted skeletal architecture. In trabecular bone, if the osteoclasts penetrate trabeculae, they leave no template for new bone formation to occur, and, consequently, rapid bone loss ensues and cancellous connectivity becomes impaired. A higher number of remodeling sites increases the likelihood of this event. In cortical bone, increased activation of remodeling creates more porous bone. The effect of this increased porosity on cortical bone strength may be modest if the overall diameter of the bone is not changed. However, decreased apposition of new bone on the periosteal surface coupled with increased endocortical resorption of bone decreases the biomechanical strength of long bones. Even a slight exaggeration in normal bone loss increases the risk of osteoporosis-related fractures because of the architectural changes that occur, and osteoporosis is primarily a disease of disordered skeletal architecture. The main clinically

1	increases the risk of osteoporosis-related fractures because of the architectural changes that occur, and osteoporosis is primarily a disease of disordered skeletal architecture. The main clinically available tool (dual-energy x-ray absorptiometry) measures mass not architecture. Emerging data from high-resolution peripheral quantitative computed tomography (CT) scans suggest that aging is associated with

1	Source: From the 2014 National Osteoporosis Foundation Clinician’s Guide to the Prevention and changes in microstructure of bone tissue, including increased Treatment of Osteoporosis. © National Osteoporosis Foundation. cortical porosity and reduced cortical thickness.

1	cortical porosity and reduced cortical thickness. FIGURE 425-4 Mechanism of bone remodeling. The basic molecular unit (BMU) moves along the trabecular surface at a rate of about 10 μm/d. The figure depicts remodeling over ~120 days. A. Origination of BMU-lining cells contracts to expose collagen and attract preosteoclasts. B. Osteoclasts fuse into multinucleated cells that resorb a cavity. Mononuclear cells continue resorption, and preosteoblasts are stimulated to proliferate. C. Osteoblasts align at bottom of cavity and start forming osteoid (black). D. Osteoblasts continue formation and mineralization. Previous osteoid starts to mineralize (horizontal lines). E. Osteoblasts begin to flatten. F. Osteoblasts turn into lining cells; bone remodeling at initial surface (left of drawing) is now complete, but BMU is still advancing (to the right). (Adapted from SM Ott, in JP Bilezikian et al [eds]: Principles of Bone Biology, vol. 18. San Diego, Academic Press, 1996, pp 231–241.)

1	Peak bone mass may be impaired by inadequate calcium intake during growth among other nutritional factors (calories, protein, and other minerals), leading to increased risk of osteoporosis later in life. During the adult phase of life, insufficient calcium intake contributes to relative secondary hyperparathyroidism and an increase in the rate of bone remodeling to maintain normal serum calcium levels. PTH stimulates the hydroxylation of vitamin D in the kidney, leading to increased levels of 1,25-dihydroxyvitamin D [1,25(OH)2D] and enhanced gastrointestinal calcium absorption. PTH also reduces renal calcium loss. Although these are all appropriate compensatory homeostatic responses for adjusting calcium economy, the long-term effects are detrimental to the skeleton because the increased remodeling rates and the ongoing imbalance between resorption and formation at remodel-2491 ing sites combine to accelerate loss of bone tissue.

1	Total daily calcium intakes <400 mg are detrimental to the skeleton, and intakes in the range of 600–800 mg, which is about the average intake among adults in the United States, are also probably suboptimal. The recommended daily required intake of 1000–1200 mg for adults accommodates population heterogeneity in controlling calcium balance (Chap. 95e). Such intakes should preferentially come from dietary sources, and supplements should be used only when dietary intakes fall short. The supplement should contain enough calcium to bring total intake to about 1200 mg/d.

1	(See also Chap. 423) Severe vitamin D deficiency causes rickets in children and osteomalacia in adults. However, there is accumulating evidence that vitamin D insufficiency may be more prevalent than previously thought, particularly among individuals at increased risk such as the elderly; those living in northern latitudes; and individuals with poor nutrition, malabsorption, or chronic liver or renal disease. Dark-skinned individuals are also at high risk of vitamin D deficiency. There is controversy regarding optimal levels of serum 25-hydroxyvitamin D [25(OH)D], with some advocating levels >20 ng/mL and others advocating optimal targets >75 nmol/L (30 ng/mL). To achieve this level for most adults requires an intake of 800–1000 units/d, particularly in individuals who avoid sunlight or routinely use ultraviolet-blocking lotions. Vitamin D insufficiency leads to compensatory secondary hyperparathyroidism and is an important risk factor for osteoporosis and fractures. Some studies have

1	use ultraviolet-blocking lotions. Vitamin D insufficiency leads to compensatory secondary hyperparathyroidism and is an important risk factor for osteoporosis and fractures. Some studies have shown that >50% of inpatients on a general medical service exhibit biochemical features of vitamin D deficiency, including increased levels of PTH and alkaline phosphatase and lower levels of ionized calcium. In women living in northern latitudes, vitamin D levels decline during the winter months. This is associated with seasonal bone loss, reflecting increased bone turnover. Even among healthy ambulatory individuals, mild vitamin D deficiency is increasing in prevalence, in part due to decreased exposure to sunlight coupled with increased use of potent sunscreens.

1	Treatment with vitamin D can return levels to normal and prevent the associated increase in bone remodeling, bone loss, and fractures. Improved muscle function and gait associated with reduced falls and fracture rates also have been documented among individuals in northern latitudes who have greater vitamin D intake and higher 25(OH)D levels (see below). Vitamin D adequacy also may affect risk and/or severity of other diseases, including cancers (colorectal, prostate, and breast), autoimmune diseases, and diabetes; however, many observational studies suggesting these potential extraskeletal benefits have not been confirmed with randomized controlled trials.

1	Estrogen deficiency probably causes bone loss by two distinct but interrelated mechanisms: (1) activation of new bone remodeling sites and (2) exaggeration of the imbalance between bone formation and resorption. The change in activation frequency causes a transient bone loss until a new steady state between resorption and formation is achieved. The remodeling imbalance, however, results in a permanent decrement in mass. In addition, the very presence of more remodeling sites in the skeleton increases the probability that trabeculae will be penetrated, eliminating the template on which new bone can be formed and accelerating the loss of bony tissue.

1	The most common estrogen-deficient state is the cessation of ovarian function at the time of menopause, which occurs on average at age 51 (Chap. 413). Thus, with current life expectancy, an average woman will spend about 30 years without an ovarian supply of estrogen. The mechanism by which estrogen deficiency causes bone loss is summarized in Fig. 425-5. Marrow cells (macrophages, monocytes, osteoclast precursors, mast cells) as well as bone cells (osteoblasts, osteocytes, osteoclasts) express ERs α and β. Loss of estrogen increases production of RANKL and may reduce production of OPG, increasing osteoclast recruitment. Estrogen also may play an important role in determining the life span of bone cells by controlling the rate of apoptosis. Thus,

1	CFU-GM Activated T lymphocytes Activated synovial fibroblasts Activated dendritic cells RANKL RANK OPG Preosteoclast Multinucleated osteoclast Activated osteoclast Bone T Osteoblasts or bone marrow stromal cells Proresorptive and calciotropic factors 1,25(OH)2 vitamin D3. PTH, PTHrP, PGE2, IL-1, IL-6, TNF, prolactin, corticosteroids, oncostatin M, LIF M-CSF AApoptotic osteoclast T Anabolic or anti-resorptive factors Estrogens, calcitonin, BMP 2/4, TGF-˜, TPO, IL-17, PDGF, calcium M-CSF B

1	FIGURE 425-5 Hormonal control of bone resorption. A. Proresorptive and calciotropic factors. B. Anabolic and antiosteoclastic factors. RANK ligand (RANKL) expression is induced in osteoblasts, activated T cells, synovial fibroblasts, and bone marrow stromal cells. It binds to membrane-bound receptor RANK to promote osteoclast differentiation, activation, and survival. Conversely, osteoprotegerin (OPG) expression is induced by factors that block bone catabolism and promote anabolic effects. OPG binds and neutralizes RANKL, leading to a block in osteoclastogenesis and decreased survival of preexisting osteoclasts. CFU-GM, colony-forming units, granulocyte macrophage; IL, interleukin; LIF, leukemia inhibitory factor; M-CSF, macrophage colony-stimulating factor; OPG-L, osteoprotegerin-ligand; PDGF, platelet-derived growth factor; PGE2, prostaglandin E2; PTH, parathyroid hormone; RANKL, receptor activator of nuclear factor nuclear factor-κB; TGF-β, transforming growth factor β; TNF, tumor

1	PDGF, platelet-derived growth factor; PGE2, prostaglandin E2; PTH, parathyroid hormone; RANKL, receptor activator of nuclear factor nuclear factor-κB; TGF-β, transforming growth factor β; TNF, tumor necrosis factor; TPO, thrombospondin. (From WJ Boyle et al: Nature 423: 337, 2003.) in situations of estrogen deprivation, the life span of osteoblasts may be decreased, whereas the longevity and activity of osteoclasts are increased. The rate and duration of bone loss after menopause are heterogeneous and unpredictable. Once surfaces are lost in cancellous bone, the rate of bone loss must decline. In cortical bone, loss is slower but continues for a longer time period.

1	Because remodeling is initiated at the surface of bone, it follows that trabecular bone—which has a considerably larger surface area (80% of the total) than cortical bone—will be affected preferentially by estrogen deficiency. Fractures occur earliest at sites where trabecular bone contributes most to bone strength; consequently, vertebral fractures are the most common early consequence of estrogen deficiency.

1	Inactivity, such as prolonged bed rest or paralysis, results in significant bone loss. Concordantly, athletes have higher bone mass than does the general population. These changes in skeletal mass are most marked when the stimulus begins during growth and before the age of puberty. Adults are less capable than children of increasing bone mass after restoration of physical activity. Epidemiologic data support the beneficial effects on the skeleton of chronic high levels of physical activity. Fracture risk is lower in rural communities and in countries where physical activity is maintained into old age. However, when exercise is initiated during adult life, the effects of moderate exercise on the skeleton are modest, with a bone mass increase of 1–2% in short-term studies of <2 years in duration. It is argued that more active individuals are less likely to fall and are more capable of protecting themselves upon falling, thereby reducing fracture risk.

1	Various genetic and acquired diseases are associated with an increase in the risk of osteoporosis (Table 425-1). Mechanisms that contribute to bone loss are unique for each disease and typically result from multiple factors, including nutrition, reduced physical activity levels, and factors that affect rates of bone remodeling. In most, but not all, circumstances the primary diagnosis is made before osteoporosis presents clinically.

1	A large number of medications used in clinical practice have potentially detrimental effects on the skeleton (Table 425-1). Glucocorticoids are the most common cause of medication-induced osteoporosis. It is often not possible to determine the extent to which osteoporosis is related to glucocorticoids or to other factors, because treatment is superimposed on the effects of the primary disease, which in itself may be associated with bone loss (e.g., rheumatoid arthritis). Excessive doses of thyroid hormone can accelerate bone remodeling and result in bone loss.

1	Other medications have less detrimental effects on the skeleton than pharmacologic doses of glucocorticoids. Anticonvulsants are thought to increase the risk of osteoporosis, although many affected individuals have concomitant insufficiency of 1,25(OH)2D, as some anticonvulsants induce the cytochrome P450 system and vitamin D metabolism. Patients undergoing transplantation are at high risk for rapid bone loss and fracture not only from glucocorticoids but also from treatment with other immunosuppressants such as cyclosporine and tacrolimus (FK506). In addition, these patients often have underlying metabolic abnormalities, such as hepatic or renal failure, that predispose to bone loss.

1	Aromatase inhibitors, which potently block the aromatase enzyme that converts androgens and other adrenal precursors to estrogen, reduce circulating postmenopausal estrogen levels dramatically. These agents, which are used in various stages for breast cancer treatment, also have been shown to have a detrimental effect on bone density and risk of fracture. More recently a variety of agents have been implicated in increased bone loss and fractures. These include selective serotonin reuptake inhibitors, proton pump inhibitors, and thiazolidinediones. It is difficult in some cases to separate the risk accrued by the underlying disease from that attributable to the medication. For example, both depression and diabetes are risk factors for fracture by themselves.

1	The use of cigarettes over a long period has detrimental effects on bone mass. These effects may be mediated directly by toxic effects on osteoblasts or indirectly by modifying estrogen metabolism. On average, cigarette smokers reach menopause 1–2 years earlier than the general population. Cigarette smoking also produces secondary effects that can modulate skeletal status, including intercurrent respiratory and other illnesses, frailty, decreased exercise, poor nutrition, and the need for additional medications (e.g., glucocorticoids for lung disease).

1	Several noninvasive techniques are available for estimating skeletal mass or density. They include dual-energy x-ray absorptiometry (DXA), single-energy x-ray absorptiometry (SXA), quantitative CT, and ultrasound (US). DXA is a highly accurate x-ray technique that has become the standard for measuring bone density. Although it can be used for measurement in any skeletal site, clinical determinations usually are made of the lumbar spine and hip. DXA also can be used to measure body composition. In the DXA technique, two x-ray energies are used to estimate the area of mineralized tissue, and the mineral content is divided by the area, which partially corrects for body size. However, this correction is only partial because DXA is a two-dimensional scanning technique and cannot estimate the depth or posteroanterior length of the bone. Thus, small slim people tend to have lower than average bone mineral density (BMD), a feature that is important in interpreting BMD measurements when

1	the depth or posteroanterior length of the bone. Thus, small slim people tend to have lower than average bone mineral density (BMD), a feature that is important in interpreting BMD measurements when performed in young adults, and something that must be taken into account at any age. Bone spurs, which are common in osteoarthritis, tend to falsely increase bone density of the spine and are a particular problem in measuring the spine in older individuals. Because DXA instrumentation is provided by several different manufacturers, the output varies in absolute terms. Consequently, it has become standard practice to relate the results to “normal” values by using T-scores (a T-score of 1 equals 1 SD), which compare individual results to those in a young population that is matched for race and sex. Z-scores (also measured in SD) compare individual results to those of an age-matched population that also is matched for race and sex. Thus, a 60-year-old woman with a Z-score of –1 (1 SD below

1	sex. Z-scores (also measured in SD) compare individual results to those of an age-matched population that also is matched for race and sex. Thus, a 60-year-old woman with a Z-score of –1 (1 SD below mean for age) has a T-score of –2.5 (2.5 SD below mean for a young control group) (Fig. 425-6). A T-score below –2.5 in the lumbar spine, femoral neck, or total hip has been defined as a diagnosis of osteoporosis. As noted above, because more than 50% of fractures occur in individuals with low bone mass rather than BMD osteoporosis, attempts are ongoing to redefine the disease as a fracture risk rather than a specific BMD. Consistent with this concept, fractures of the spine and hip that occur in the absence of major trauma would be considered to be sufficient to diagnose osteoporosis, regardless of BMD. Fractures of other sites, such as pelvis, proximal humerus, and wrist, would be tantamount to an osteoporosis diagnosis in the presence of low BMD. CT can also be used to measure the spine

1	of BMD. Fractures of other sites, such as pelvis, proximal humerus, and wrist, would be tantamount to an osteoporosis diagnosis in the presence of low BMD. CT can also be used to measure the spine and the

1	T-Score = –2.5 Z-Score=-1 FIGURE 425-6 Relationship between Z-scores and T-scores in a 60-year-old woman. BMD, bone mineral density; SD, standard deviation. hip, but is rarely used clinically, in part because of higher radiation 2493 exposure and cost, in addition to a lesser body of data confirming its ability to predict fracture risk, compared with BMD by DXA. High-resolution peripheral CT is used to measure bone in the forearm or tibia as a research tool to noninvasively provide some measure of skeletal architecture. Magnetic resonance imaging (MRI) can also be used in research settings to obtain some architectural information on the forearm and perhaps the hip.

1	DXA equipment can also be used to obtain lateral images of the spine, from T4 through L4, a technique called vertebral fracture assessment (VFA). Although not as definitive as radiography, it is a useful screening tool when height loss, back pain, or postural change suggests the presence of an undiagnosed vertebral fracture. Furthermore, because vertebral fractures are so prevalent with advancing age, screening vertebral imaging is recommended in women and men with low bone mass (T-score <1) by age 70 and 80, respectively. US is used to measure bone mass by calculating the attenuation of the signal as it passes through bone or the speed with which it traverses the bone. It is unclear whether US assesses properties of bone other than mass (e.g., quality), but this is a potential advantage of the technique. Because of its relatively low cost and mobility, US is amenable for use as a screening procedure in stores or at health fairs.

1	All of these techniques for measuring BMD have been approved by the U.S. Food and Drug Administration (FDA) on the basis of their capacity to predict fracture risk. The hip is the preferred site of measurement in most individuals, because it predicts the risk of hip fracture, the most important consequence of osteoporosis, better than any other bone density measurement site. When hip measurements are performed by DXA, the spine can be measured at the same time. In younger individuals such as perimenopausal or early postmenopausal women, spine measurements may be the most sensitive indicator of bone loss. A risk assessment tool (FRAX) incorporates femoral neck BMD to assess 10-year fracture risk (see below).

1	Clinical guidelines have been developed for the use of bone densitometry in clinical practice. The original National Osteoporosis Foundation guidelines recommend bone mass measurements in postmenopausal women, assuming they have one or more risk factors for osteoporosis in addition to age, sex, and estrogen deficiency. The guidelines further recommend that bone mass measurement be considered in all women by age 65, a position ratified by the U.S. Preventive Health Services Task Force. Criteria approved for Medicare reimbursement of BMD are summarized in Table 425-2.

1	Most guidelines suggest that patients be considered for treatment when BMD is >2.5 SD below the mean value for young adults (T-score ≤–2.5), in either spine, total hip, or femoral neck. Treatment also should also be considered in postmenopausal women with fracture risk factors even if BMD is not in the osteoporosis range. Risk factors (age, prior fracture, family history of hip fracture, low body weight, cigarette consumption, excessive alcohol use, steroid use, and rheumatoid arthritis) can be combined with BMD to assess the likelihood of a Consider BMD testing in the following individuals: • Women age 65 and older and men age 70 and older, regardless of clinical • Younger postmenopausal women, women in the menopausal transition and men age 50–69 with clinical risk factors for fracture

1	Adults with a condition (e.g., rheumatoid arthritis) or taking a medication (e.g., glucocorticoids in a daily dose ≥5 mg prednisone or equivalent for ≥3 months) associated with low bone mass or bone loss Source: From the 2014 National Osteoporosis Foundation Clinician’s Guide to the Prevention and Treatment of Osteoporosis. © National Osteoporosis Foundation. 2494 fracture over a 5or 10-year period. Treatment threshold depends on cost-effectiveness analyses but probably is ~1% per year of risk in the United States. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: The perimenopausal transition is a good opportunity to initiate a discussion about risk factors for osteoporosis and consideration of indications for a BMD test. A careful history and physical examination should be performed to identify risk factors for osteoporosis. A low Z-score increases the suspicion of a secondary disease. Height loss >2.5–3.8 cm (>1–1.5 in.) is an indication for VFA by DXA or radiography to rule out asymptomatic vertebral fractures, as is the presence of significant kyphosis or back pain, particularly if it began after menopause. In appropriate individuals, screening BMD and screening vertebral imaging should be recommended as above, even in the absence of any specific risk factors (Table 425-3). For patients who present with fractures, it is important to ensure that the fractures are not caused by an underlying malignancy. Usually this is clear on routine radiography, but on occasion, CT, MRI, or radionuclide scans may be necessary.

1	There is no established algorithm for the evaluation of women who present with osteoporosis. A general evaluation that includes complete blood count, serum and 24-h urine calcium, renal and hepatic function tests, and a 25(OH)D level is useful for identifying selected secondary causes of low bone mass, particularly for women with fractures or very low Z-scores. An elevated serum calcium level suggests hyperparathyroidism or malignancy, whereas a reduced serum calcium level may reflect malnutrition and osteomalacia. In the presence of hypercalcemia, a serum PTH level differentiates between hyperparathyroidism (PTH↑) and malignancy (PTH↓), and a high PTHrP level can help document the presence of humoral hypercalcemia of malignancy (Chap. 424). A low urine calcium (<50 mg/24 h) suggests osteomalacia, malnutrition, or malabsorption; a high urine calcium (>300 mg/24 h) is indicative of hypercalciuria and must be investigated further. Hypercalciuria occurs primarily in three situations: (1)

1	malnutrition, or malabsorption; a high urine calcium (>300 mg/24 h) is indicative of hypercalciuria and must be investigated further. Hypercalciuria occurs primarily in three situations: (1) a renal calcium leak, which is more common in males with osteoporosis; (2) absorptive hypercalciuria, which can be idiopathic or associated with increased 1,25(OH)2D in granulomatous disease; or (3) hematologic malignancies or conditions associated with excessive bone turnover such as Paget’s disease, hyperparathyroidism, and hyperthyroidism. Renal hypercalciuria is treated with thiazide diuretics, which lower urine calcium and help improve calcium economy.

1	Individuals who have osteoporosis-related fractures or bone density in the osteoporotic range should have a measurement of serum 25(OH)D level, because the intake of vitamin D required to achieve a target level >20–30 ng/mL is highly variable. Vitamin D levels should be optimized in all individuals being treated for Consider vertebral imaging tests in the following individuals: mineral density (BMD) T-score is –1.0 or below In women age 65–69 and men age 75–79 if BMD T-score is –1.5 or below In postmenopausal women age 50–64 and men age 50–69 with specific risk factors: º Historical height loss of 1.5 in. or more (4 cm) º Prospective height loss of 0.8 in. or more (2 cm) Source: From the 2014 National Osteoporosis Foundation Clinician’s Guide to the Prevention and Treatment of Osteoporosis. © National Osteoporosis Foundation. osteoporosis. Hyperthyroidism should be evaluated by measuring thyroid-stimulating hormone (TSH).

1	When there is clinical suspicion of Cushing’s syndrome, urinary free cortisol levels or a fasting serum cortisol should be measured after overnight dexamethasone. When bowel disease, malabsorption, or malnutrition is suspected, serum albumin, cholesterol, and a complete blood count should be checked. Asymptomatic malabsorption may be heralded by anemia (macrocytic—vitamin B12 or folate deficiency; microcytic—iron deficiency) or low serum cholesterol or urinary calcium levels. If these or other features suggest malabsorption, further evaluation is required. Asymptomatic celiac disease with selective malabsorption is being found with increasing frequency; the diagnosis can be made by testing for antigliadin, antiendomysial, or transglutaminase antibodies but may require endoscopic biopsy. A trial of a gluten-free diet can be confirmatory (Chap. 349). When osteoporosis is found associated with symptoms of rash, multiple allergies, diarrhea, or flushing, mastocytosis should be excluded by

1	trial of a gluten-free diet can be confirmatory (Chap. 349). When osteoporosis is found associated with symptoms of rash, multiple allergies, diarrhea, or flushing, mastocytosis should be excluded by using 24-h urine histamine collection or serum tryptase.

1	Myeloma can masquerade as generalized osteoporosis, although it more commonly presents with bone pain and characteristic “punched-out” lesions on radiography. Serum and urine electrophoresis and or serum free light chains are required to exclude this diagnosis. More commonly, a monoclonal gammopathy of unclear significance (MGUS) is found, and the patient is subsequently monitored to ensure that this is not an incipient myeloma. Approximately 1% of patients with MGUS progress to myeloma each year. A bone marrow biopsy may be required to rule out myeloma (in patients with equivocal electrophoretic results) and also can be used to exclude mastocytosis, leukemia, and other marrow infiltrative disorders such as Gaucher’s disease. MGUS syndromes, although benign, may also be associated with reduced bone mass and elevated bone turnover.

1	Tetracycline labeling of the skeleton allows determination of the rate of remodeling as well as evaluation for other metabolic bone diseases. The current use of BMD tests, in combination with hormonal evaluation and biochemical markers of bone remodeling, has largely replaced the clinical use of bone biopsy, although it remains an important tool in clinical research and assessment of mechanism of action of medication for osteoporosis. Several biochemical tests are available that provide an index of the overall rate of bone remodeling (Table 425-4). Biochemical markers usually are characterized as those related primarily to bone formation or bone resorption. These tests measure the overall state of bone remodeling at a single point in time. Clinical use of these tests has been hampered by biologic variability (in part related to circadian rhythm) as well as analytic variability, although the latter is improving. Biochemical markers of bone turnover may:

1	Biochemical markers of bone turnover may: Predict risk of fracture independently of bone density. Predict extent of fracture risk reduction when repeated after 3–6 months of treatment with FDA-approved therapies. Predict magnitude of BMD increases with FDA-approved therapies. Predict rapidity of bone loss. Help determine adequacy of patient compliance and persistence with osteoporosis therapy. • Help determine duration of “drug holiday” (data are quite limited to support this use, but studies are under way). Abbreviations: BMD, bone mineral density; FDA, U.S. Food and Drug Administration. Source: Adapted from the 2014 National Osteoporosis Foundation Clinician’s Guide to the Prevention and Treatment of Osteoporosis. © National Osteoporosis Foundation.

1	Biochemical markers of bone resorption may help in the prediction of fracture risk, independently of bone density, particularly in older individuals. In women ≥65 years, when bone density results are greater than the usual treatment thresholds noted above, a high level of bone resorption should prompt consideration of treatment. The primary use of biochemical markers is for monitoring the response to treatment. With the introduction of antiresorptive therapeutic agents, bone remodeling declines rapidly, with the fall in resorption occurring earlier than the fall in formation. Inhibition of bone resorption is maximal within 3 months or so. Thus, measurement of bone resorption (C-telopeptide [CTX] is the preferred marker) before initiating therapy and 3–6 months after starting therapy provides an earlier estimate of patient response than does bone densitometry. A decline in resorptive markers can be ascertained after treatment with potent antiresorptive agents such as bisphosphonates,

1	an earlier estimate of patient response than does bone densitometry. A decline in resorptive markers can be ascertained after treatment with potent antiresorptive agents such as bisphosphonates, denosumab, or standard-dose estrogen; this effect is less marked after treatment with weaker agents such as raloxifene or intranasal calcitonin. A biochemical marker response to therapy is particularly useful for asymptomatic patients and may help ensure long-term adherence to treatment. Bone turnover markers are also useful in monitoring the effects of osteoanabolic agents such as 1-34hPTH, or teriparatide, which rapidly increases bone formation (P1NP is preferred, but osteocalcin is a reasonable alternative) and later bone resorption. The recent suggestion of “drug holidays” (see below) has created another use for biochemical markers, allowing evaluation of the off effect of drugs such as bisphosphonates.

1	Treatment of a patient with osteoporosis frequently involves management of acute fractures as well as treatment of the underlying disease. Hip fractures almost always require surgical repair if the patient is to become ambulatory again. Depending on the location and severity of the fracture, condition of the neighboring joint, and general status of the patient, procedures may include open reduction and internal fixation with pins and plates, hemiarthroplasties, and total arthroplasties. These surgical procedures are followed by intense rehabilitation in an attempt to return patients to their prefracture functional level. Long bone fractures (e.g., wrist) often require either external or internal fixation. Other fractures (e.g., vertebral, rib, and pelvic fractures) usually are managed with supportive care, requiring no specific orthopedic treatment.

1	Only ~25–30% of vertebral compression fractures present with sudden-onset back pain. For acutely symptomatic fractures, treatment with analgesics is required, including nonsteroidal anti-inflammatory agents and/or acetaminophen, sometimes with the addition of a narcotic agent (codeine or oxycodone). A few small, randomized clinical trials suggest that calcitonin may reduce pain related to acute vertebral compression fracture. Percutaneous injection of artificial cement (polymethylmethacrylate) into the vertebral body (vertebroplasty or kyphoplasty) may offer significant immediate pain relief in patients with severe pain from acute or subacute vertebral fractures. Safety concerns include extravasation of cement with neurologic sequelae and increased risk of fracture in neighboring vertebrae due to mechanical rigidity of the treated bone. Exactly which patients are the optimal candidates for this procedure remains unknown. Short periods of bed rest may be helpful for pain management,

1	due to mechanical rigidity of the treated bone. Exactly which patients are the optimal candidates for this procedure remains unknown. Short periods of bed rest may be helpful for pain management, but in general, early mobilization is recommended because it helps prevent further bone loss associated with immobilization. Occasionally, use of a soft elastic-style brace may facilitate earlier mobilization. Muscle spasms often occur with acute compression fractures and can be treated with muscle relaxants and heat treatments.

1	Severe pain usually resolves within 6–10 weeks. More chronic severe pain might suggest the possibility of multiple myeloma or underlying metastatic disease. Chronic pain following vertebral fracture is probably not bony in origin; instead, it is related to abnormal strain on muscles, ligaments, and tendons and to secondary facet-joint arthritis associated with alterations in thoracic and/or abdominal shape. Chronic pain is difficult to treat effectively and may require analgesics, sometimes including narcotic analgesics. Frequent intermittent rest in a supine or semireclining position is often required to allow the soft tissues, which are under tension, to relax. Back-strengthening exercises (paraspinal) may be beneficial. Heat treatments help relax muscles and reduce the muscular component of discomfort. Various physical modalities, such as US and trans-cutaneous nerve stimulation, may be beneficial in some patients. Pain also occurs in the neck region, not as a result of compression

1	of discomfort. Various physical modalities, such as US and trans-cutaneous nerve stimulation, may be beneficial in some patients. Pain also occurs in the neck region, not as a result of compression fractures (which almost never occur in the cervical spine as a result of osteoporosis) but because of chronic strain associated with trying to elevate the head in a person with a significant thoracic kyphosis.

1	Multiple vertebral fractures often are associated with psychological symptoms; this is not always appreciated. The changes in body configuration and back pain can lead to marked loss of self-image and a secondary depression. Altered balance, precipitated by the kyphosis and the anterior movement of the body’s center of gravity, leads to a fear of falling, a consequent tendency to remain indoors, and the onset of social isolation. These symptoms sometimes can be alleviated by family support and/or psychotherapy. Medication may be necessary when depressive features are present. Multiple thoracic vertebral fractures may be associated with restrictive lung disease symptoms and increased pulmonary infections. Multiple lumbar vertebral fractures are often associated with abdominal pain, constipation, protuberance, and early satiety. Multiple vertebral fractures are associated with greater age-specific mortality.

1	Multiple studies show that the majority of patients presenting in adulthood with fractures are not evaluated or treated for osteoporosis. Estimates suggest only about 20% of fracture patients receive follow-up care. Patients who sustain acute fractures are at dramatically elevated risk for more fractures, particularly within the first several years, and pharmacologic intervention can reduce that risk substantially. Recently, several studies have demonstrated the effectiveness of a relatively simple and inexpensive program that reduces the risk of subsequent fractures. In the Kaiser system, it is estimated that a 20% decline in hip fracture occurrence was seen with the introduction of what is called a fracture liaison service. This typically involves a health care professional (usually a nurse) whose job is to coordinate follow-up care and education of fracture patients. If the Kaiser experience can be repeated, there would be significant savings of health care dollars, as well as a

1	nurse) whose job is to coordinate follow-up care and education of fracture patients. If the Kaiser experience can be repeated, there would be significant savings of health care dollars, as well as a dramatic drop in hip fracture incidence and a marked improvement in morbidity and mortality among the aging population.

1	Patients presenting with typical osteoporosis-related fractures (certainly hip and spine) can be assumed to have osteoporosis and can be treated appropriately. Patients with osteoporosis by BMD are handled in a similar fashion. Other fracture patients and those with reduced bone mass can be classified according to their future risk of fracture and treated if that risk is sufficiently high. It must be emphasized, however, that risk assessment is an inexact science when applied to individual patients. Fractures are chance occurrences that can happen to anyone. Patients often do not understand the relative benefits of medications, compared to the perceived risks of the medications themselves.

1	Risk Factor Reduction Several tools exist for risk assessment. The most commonly available is the FRAX tool, developed by a working party for the WHO, and available as part of the report from many DXA machines. It is also available online (http://www.shef.ac.uk/ FRAX/tool.jsp?locationValue=9) (Fig. 425-7). In the United States, it has been estimated that it is cost-effective to treat a patient if the 10-year major fracture risk (including hip, clinical spine, proximal humerus, and tibia) from FRAX is ≥20% and/or the 10-year risk of hip fracture is ≥3%. FRAX is an imperfect tool because it does not include FIGURE 425-7 FRAX calculation tool. When the answers to the indicated questions are filled in, the calculator can be used to assess the 10-year probability of fracture. The calculator (available online at http://www.shef.ac.uk/FRAX/tool.jsp?locationValue=9) also can risk adjust for various ethnic groups.

1	any assessment of fall risk and secondary causes are excluded when BMD is entered. Moreover, it does not include any term for multiple fractures or recent versus remote fracture. Nonetheless, it is useful as an educational tool for patients.

1	After risk assessment, patients should be thoroughly educated to reduce the impact of modifiable risk factors associated with bone loss and falling. All medications that increase risk of falls, bone loss, or fractures should be reviewed to ensure that they are necessary and being used at the lowest required dose. For those on thyroid hormone replacement, TSH testing should be performed to confirm that an excessive dose is not being used, because biochemical and symptomatic thyrotoxicosis can be associated with increased bone loss. In patients who smoke, efforts should be made to facilitate smoking cessation. Reducing risk factors for falling also include alcohol abuse treatment and a review of the medical regimen for any drugs that might be associated with orthostatic hypotension and/ or sedation, including hypnotics and anxiolytics. If nocturia occurs, the frequency should be reduced, if possible (e.g., by decreasing or modifying diuretic use), because arising in the middle of sleep

1	sedation, including hypnotics and anxiolytics. If nocturia occurs, the frequency should be reduced, if possible (e.g., by decreasing or modifying diuretic use), because arising in the middle of sleep is a common precipitant of a fall. Patients should be instructed about environmental safety with regard to eliminating exposed wires, curtain strings, slippery rugs, and mobile tables. Avoiding stocking feet on wood floors, checking carpet condition (particularly on stairs), and providing good light in paths to bathrooms and outside the home are important preventive measures. Treatment for impaired vision is recommended, particularly a problem with depth perception, which is specifically associated with increased falling risk. Elderly patients with neurologic impairment (e.g., stroke, Parkinson’s disease, Alzheimer’s disease) are particularly at risk of falling and require specialized supervision and care.

1	Nutritional Recommendations • calcium A large body of data indicates that optimal calcium intake reduces bone loss and suppresses bone turnover. Recommended intakes from an Institute of Medicine report are shown in Table 425-5. The National Health and Nutrition Examination Surveys (NHANES) have consistently documented that average calcium intakes fall considerably short of these recommendations. Food sources of calcium are dairy products (milk, yogurt, and cheese) and fortified Estimated Adequate Daily Life Stage Group Calcium Intake, mg/d Note: Pregnancy and lactation needs are the same as for nonpregnant women (e.g., 1300 mg/d for adolescents/young adults and 1000 mg/d for ≥19 years). Source: Adapted from the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes. Food and Nutrition Board. Institute of Medicine. Washington, DC, 1997, National Academy Press. STEP 1: Estimate calcium intake from calcium-rich foods

1	STEP 1: Estimate calcium intake from calcium-rich foods Estimated # of calcium/ Product Servings/d serving, in mg Calcium in mg Milk (8 oz.) __________ × 300 = __________ Yogurt (6 oz.) __________ × 300 = __________ Cheese (1 oz. or 1 cubic in.) __________ × 200 = __________ Fortified foods or juices __________ × 80 to 1000 = __________ STEP 2: Total from above + 250 mg for nondairy sources = total dietary calcium TOTAL Calcium, in mg = __________ Source: Adapted from SM Krane, MF Holick, Chap. 355, in Harrison’s Principles of Internal Medicine, 14th ed. New York, McGraw-Hill, 1998.

1	Source: Adapted from SM Krane, MF Holick, Chap. 355, in Harrison’s Principles of Internal Medicine, 14th ed. New York, McGraw-Hill, 1998. foods such as certain cereals, waffles, snacks, juices, and crackers. Some of these fortified foods contain as much calcium per serving as milk. Green leafy vegetables and nuts, particularly almonds, are also sources of calcium, although their bioavailability may be lower than with dairy products. Calcium intake from the diet can also be assessed (Table 425-6) and calculators are available at NOF.org or NYSOPEP.org.

1	If a calcium supplement is required, it should be taken in doses sufficient to supplement dietary intake to bring total intake to the required level (1000–1200 mg/d). Doses of supplements should be ≤600 mg at a time, because the calcium absorption fraction decreases at higher doses. Calcium supplements should be calculated on the basis of the elemental calcium content of the supplement, not the weight of the calcium salt. Calcium supplements containing carbonate are best taken with food because they require acid for solubility. Calcium citrate supplements can be taken at any time. To confirm bioavailability, calcium supplements can be placed in distilled vinegar. They should dissolve within 30 min.

1	Several controlled clinical trials of calcium, mostly plus vitamin D, have confirmed reductions in clinical fractures, including fractures of the hip (~20–30% risk reduction). All recent studies of pharmacologic agents have been conducted in the context of calcium replacement (± vitamin D). Thus, it is standard practice to ensure an adequate calcium and vitamin D intake in patients with osteoporosis whether they are receiving additional pharmacologic therapy or not. A systematic review confirmed a greater BMD response to antiresorptive therapy when calcium intake was adequate.

1	Although side effects from supplemental calcium are minimal (eructation and constipation mostly with carbonate salts), individuals with a history of kidney stones should have a 24-h urine calcium determination before starting increased calcium to avoid significant hypercalciuria. Many studies confirm a small but significant increase in the risk of renal stones with calcium supplements, but not dietary calcium. A recent analysis of published data has suggested that high intakes of calcium from supplements are associated with an increase in the risk of heart disease. This is an evolving story with additional studies that confirm or refute this finding. Because high calcium supplement intakes increase the risk of renal stones and confer no extra benefit to the skeleton, the recommendation that total intakes should be between 1000 and 1200 mg/d is reasonable.

1	vitamin d Vitamin D is synthesized in skin under the influence of heat and ultraviolet light (Chap. 423). However, large segments of the population do not obtain sufficient vitamin D to maintain what is now considered an adequate supply [serum 25(OH)D consistently >75 μmol/L (30 ng/mL)]. Because vitamin D supplementation at doses that would achieve these serum levels is safe and inexpensive, the Institute of Medicine (based on obtaining a serum level of 20 ng/mL) recommends daily intakes of 200 IU for adults <50 years of age, 400 IU for those 50–70 years, and 600 IU for those >70 years. Multivitamin tablets usually contain 400 IU, and many calcium supplements also contain vitamin D. Some data suggest 2497 that higher doses (≥1000 IU) may be required in the elderly and chronically ill. The Institute of Medicine report suggests that it is safe to take up to 4000 IU/d. For those with osteoporosis or those at risk of osteoporosis, 1000–2000 IU/d can usually maintain serum 25(OH)D above 30

1	Institute of Medicine report suggests that it is safe to take up to 4000 IU/d. For those with osteoporosis or those at risk of osteoporosis, 1000–2000 IU/d can usually maintain serum 25(OH)D above 30 ng/mL.

1	otHer nutrientS Other nutrients such as salt, high animal protein intakes, and caffeine may have modest effects on calcium excretion or absorption. Adequate vitamin K status is required for optimal carboxylation of osteocalcin. States in which vitamin K nutrition or metabolism is impaired, such as with long-term warfarin therapy, have been associated with reduced bone mass. Research concerning cola intake is controversial but suggests a possible link to reduced bone mass through factors that are independent of caffeine. Although dark green leafy vegetables such as spinach and kale contain a fair amount of calcium, the high oxalate content reduces absorption of this calcium (but does not inhibit absorption of calcium from other food eaten simultaneously).

1	Magnesium is abundant in foods, and magnesium deficiency is quite rare in the absence of a serious chronic disease. Magnesium supplementation may be warranted in patients with inflammatory bowel disease, celiac disease, chemotherapy, severe diarrhea, malnutrition, or alcoholism. Dietary phytoestrogens, which are derived primarily from soy products and legumes (e.g., garbanzo beans [chickpeas] and lentils), exert some estrogenic activity but are insufficiently potent to justify their use in place of a pharmacologic agent in the treatment of osteoporosis. Patients with hip fractures are often frail and relatively malnourished. Some data suggest an improved outcome in such patients when they are provided calorie and protein supplementation. Excessive protein intake can increase renal calcium excretion, but this can be corrected by an adequate calcium intake.

1	Exercise Exercise in young individuals increases the likelihood that they will attain the maximal genetically determined peak bone mass. Meta-analyses of studies performed in postmenopausal women indicate that weight-bearing exercise helps prevent bone loss but does not appear to result in substantial gain of bone mass. This beneficial effect wanes if exercise is discontinued. Most of the studies are short term, and a more substantial effect on bone mass is likely if exercise is continued over a long period. Exercise also has beneficial effects on neuromuscular function, and it improves coordination, balance, and strength, thereby reducing the risk of falling. A walking program is a practical way to start. Other activities, such as dancing, racquet sports, cross-country skiing, and use of gym equipment, are also recommended, depending on the patient’s personal preference and general condition. Even women who cannot walk benefit from swimming or water exercises, not so much for the

1	of gym equipment, are also recommended, depending on the patient’s personal preference and general condition. Even women who cannot walk benefit from swimming or water exercises, not so much for the effects on bone, which are quite minimal, but because of effects on muscle. Exercise habits should be consistent, optimally at least three times a week.

1	Before the mid-1990s, estrogen treatment, either by itself or in concert with a progestin, was the primary therapeutic agent for prevention or treatment of osteoporosis. There are now a number of new medications approved for osteoporosis and more under development. Some are agents that specifically treat osteoporosis (bisphosphonates, calcitonin, denosumab, and teriparatide [1-34hPTH]); others, such as selective estrogen response modulators (SERMs) and, most recently, an estrogen/SERM combination medication, have broader effects. The availability of these drugs allows therapy to be tailored to the needs of an individual patient.

1	Estrogens A large body of clinical trial data indicates that various types of estrogens (conjugated equine estrogens, estradiol, estrone, esterified estrogens, ethinyl estradiol, and mestranol) reduce bone turnover, prevent bone loss, and induce small increases in bone mass of the spine, hip, and total body. The effects of estrogen are 2498 seen in women with natural or surgical menopause and in late postmenopausal women with or without established osteoporosis. Estrogens are efficacious when administered orally or transdermally. For both oral and transdermal routes of administration, combined estrogen/progestin preparations are now available in many countries, obviating the problem of taking two tablets or using a patch and oral progestin.

1	Dose of Estrogen For oral estrogens, the standard recommended doses have been 0.3 mg/d for esterified estrogens, 0.625 mg/d for conjugated equine estrogens, and 5 μg/d for ethinyl estradiol. For transdermal estrogen, the commonly used dose supplies 50 μg estradiol per day, but a lower dose may be appropriate for some individuals. Dose-response data for conjugated equine estrogens indicate that lower doses (0.3 and 0.45 mg/d) are effective. Doses even lower have been associated with bone mass protection.

1	fracture data Epidemiologic databases indicate that women who take estrogen replacement have a 50% reduction, on average, of osteoporotic fractures, including hip fractures. The beneficial effect of estrogen is greatest among those who start replacement early and continue the treatment; the benefit declines after discontinuation to the extent that there is no residual protective effect against fracture by 10 years after discontinuation. The first clinical trial evaluating fractures as secondary outcomes, the Heart and Estrogen-Progestin Replacement Study (HERS) trial, showed no effect of hormone therapy on hip or other clinical fractures in women with established coronary artery disease. These data made the results of the Women’s Health Initiative (WHI) exceedingly important (Chap. 413). The estrogen-progestin arm of the WHI in >16,000 postmenopausal healthy women indicated that hormone therapy reduces the risk of hip and clinical spine fracture by 34% and reduces the risk of all

1	The estrogen-progestin arm of the WHI in >16,000 postmenopausal healthy women indicated that hormone therapy reduces the risk of hip and clinical spine fracture by 34% and reduces the risk of all clinical fractures by 24%. There was similar antifracture efficacy seen with estrogen alone in women who had had a hysterectomy.

1	A few smaller clinical trials have evaluated spine fracture occurrence as an outcome with estrogen therapy. They have consistently shown that estrogen treatment reduces the incidence of vertebral compression fracture.

1	The WHI has provided a vast amount of data on the multisystemic effects of hormone therapy. Although earlier observational studies suggested that estrogen replacement might reduce heart disease, the WHI showed that combined estrogen-progestin treatment increased risk of fatal and nonfatal myocardial infarction by ~29%, confirming data from the HERS study. Other important relative risks included a 40% increase in stroke, a 100% increase in venous thromboembolic disease, and a 26% increase in risk of breast cancer. Subsequent analyses have confirmed the increased risk of stroke and, in a substudy, showed a twofold increase in dementia. Benefits other than the fracture reductions noted above included a 37% reduction in the risk of colon cancer. These relative risks have to be interpreted in light of absolute risk (Fig. 425-8). For example, out of 10,000 women treated with estrogen-progestin for 1 year, there will be 8 excess heart attacks, 8 excess breast cancers, 18 excess venous

1	in light of absolute risk (Fig. 425-8). For example, out of 10,000 women treated with estrogen-progestin for 1 year, there will be 8 excess heart attacks, 8 excess breast cancers, 18 excess venous thromboembolic events, 5 fewer hip fractures, 44 fewer clinical fractures, and 6 fewer colorectal cancers. These numbers must be multiplied by years of hormone treatment. There was no effect of hormone treatment on the risk of uterine cancer or total mortality.

1	It is important to note that these WHI findings apply specifically to hormone treatment in the form of conjugated equine estrogen plus medroxyprogesterone acetate. The relative benefits and risks of unopposed estrogen in women who had hysterectomies vary somewhat. They still show benefits against fracture occurrence and increased risk of venous thrombosis and stroke, similar in magnitude to the risks for combined hormone therapy. In contrast, though, the estrogen-only arm of WHI indicated no increased risk of heart attack or breast cancer. The data suggest that at least some of the detrimental effects of combined therapy are related to the progestin component. In addition, there is the possibility, suggested by primate data, that the risk accrues mainly to women who have Number of casesin 10,000 women/year

1	Number of casesin 10,000 women/year FIGURE 425-8 Effects of hormone therapy on event rates: green, placebo; purple, estrogen and progestin. CHD, coronary heart disease; VTE, venous thromboembolic events. (Adapted from Women’s Health Initiative. WHI HRT Update. Available at http://www.nhlbi.nih.gov/health/ women/upd2002.htm.) some years of estrogen deficiency before initiating treatment. (The average woman in the WHI was more than 10 years from the last menstrual period). Nonetheless, there is reluctance among women to use estrogen/hormone therapy, and the U.S. Preventive Services Task Force has specifically suggested that estrogen/hormone therapy not be used for disease prevention.

1	mode of action Two subtypes of ERs, α and β, have been identified in bone and other tissues. Cells of monocyte lineage express both ERα and ERβ, as do osteoblasts. Estrogen-mediated effects vary with the receptor type. Using ER knockout mouse models, elimination of ERα produces a modest reduction in bone mass, whereas mutation of ERβ has less of an effect on bone. A male patient with a homozygous mutation of ERα had markedly decreased bone density as well as abnormalities in epiphyseal closure, confirming the important role of ERα in bone biology. The mechanism of estrogen action in bone is an area of active investigation (Fig. 425-5). Although data are conflicting, estrogens may inhibit osteoclasts directly. However, the majority of estrogen (and androgen) effects on bone resorption are mediated through paracrine factors produced by osteoblasts and osteocytes. These actions include decreasing RANKL production and increasing OPG production by osteoblasts.

1	Progestins In women with a uterus, daily progestin or cyclical progestins at least 12 days per month are prescribed in combination with estrogens to reduce the risk of uterine cancer. Medroxyprogesterone acetate and norethindrone acetate blunt the high-density lipoprotein response to estrogen, but micronized progesterone does not. Neither medroxyprogesterone acetate nor micronized progesterone appears to have an independent effect on bone; at lower doses of estrogen, norethindrone acetate may have an additive benefit. On breast tissue, progestins may increase the risk of breast cancer.

1	Two SERMs are used currently in postmenopausal women: raloxifene, which is approved for the prevention and treatment of osteoporosis as well as the prevention of breast cancer, and tamoxifen, which is approved for the prevention and treatment of breast cancer. A third SERM, bazedoxifene, has been complexed with conjugated estrogen, creating a tissue selective estrogen complex (TSEC). This agent has been approved for prevention of osteoporosis.

1	Tamoxifen reduces bone turnover and bone loss in postmenopausal women compared with placebo groups. These findings support the concept that tamoxifen acts as an estrogenic agent in bone. There are limited data on the effect of tamoxifen on fracture risk, but the Breast Cancer Prevention Study indicated a possible reduction in clinical vertebral, hip, and Colles’ fractures. The major benefit of tamoxifen is on breast cancer occurrence. The breast cancer prevention trial indicated that tamoxifen administration over 4–5 years reduced the incidence of new invasive and noninvasive breast cancer by ~45% in women at increased risk of breast cancer. The incidence of ER-positive breast cancers was reduced by 65%. Tamoxifen increases the risk of uterine cancer and increases risk of venous thrombosis, cataracts, and possibly stroke in post-menopausal women, limiting its use for breast cancer prevention in women at low or moderate risk.

1	Raloxifene (60 mg/d) has effects on bone turnover and bone mass that are very similar to those of tamoxifen, indicating that this agent is also estrogenic on the skeleton. The effect of raloxifene on bone density (+1.4–2.8% vs placebo in the spine, hip, and total body) is somewhat less than that seen with standard doses of estrogens. Raloxifene reduces the occurrence of vertebral fracture by 30–50%, depending on the population; however, there are no data confirming that raloxifene can reduce the risk of nonvertebral fractures over 8 years of observation.

1	Raloxifene, like tamoxifen and estrogen, has effects in other organ systems. The most beneficial effect appears to be a reduction in invasive breast cancer (mainly decreased ER-positive) occurrence of ~65% in women who take raloxifene compared to placebo. In a head-to-head study, raloxifene was as effective as tamoxifen in preventing breast cancer in high-risk women, and raloxifene is now FDA approved for this indication. In a further study, raloxifene had no effect on heart disease in women with increased risk for this outcome. In contrast to tamoxifen, raloxifene is not associated with an increase in the risk of uterine cancer or benign uterine disease. Raloxifene increases the occurrence of hot flashes but reduces serum total and low-density lipoprotein cholesterol, lipoprotein(a), and fibrinogen. Raloxifene, with positive effects on breast cancer and vertebral fractures, has become a useful agent for the treatment of the younger asymptomatic postmenopausal woman. In some women, a

1	fibrinogen. Raloxifene, with positive effects on breast cancer and vertebral fractures, has become a useful agent for the treatment of the younger asymptomatic postmenopausal woman. In some women, a recurrence of menopausal hot flashes may occur. Usually this is evanescent, but occasionally, it is sufficiently impactful on daily life and sleep that the drug must be withdrawn. Raloxifene increases the risk of deep vein thrombosis and may increase the risk of death from stroke among older women. Consequently, it is not usually recommended for women over 70 years of age.

1	The main advantage of the bazedoxifene/conjugated estrogen compound is that the bazedoxifene protects uterine tissue from the effects of estrogen and makes it possible to avoid taking a progestin, while using an estrogen primarily for control of menopausal symptoms. The TSEC prevents bone loss somewhat more potently than raloxifene alone and appears safe for the breast.

1	mode of action of SermS All SERMs bind to the ER, but each agent produces a unique receptor-drug conformation. As a result, specific co-activator or co-repressor proteins are bound to the receptor (Chap. 400e), resulting in differential effects on gene transcription that vary depending on other transcription factors present in the cell. Another aspect of selectivity is the affinity of each SERM for the different ERα and ERβ subtypes, which are expressed differentially in various tissues. These tissue-selective effects of SERMs offer the possibility of tailoring estrogen therapy to best meet the needs and risk factor profile of an individual patient.

1	Bisphosphonates Alendronate, risedronate, ibandronate, and zoledronic acid are approved for the prevention and treatment of post-menopausal osteoporosis. Alendronate, risedronate, and zoledronic acid are also approved for the treatment of steroid-induced osteoporosis, and risedronate and zoledronic acid are approved for prevention of steroid-induced osteoporosis. Alendronate, risedronate, and zoledronic acid are approved for treatment of osteoporosis in men.

1	Alendronate has been shown to decrease bone turnover and increase bone mass in the spine by up to 8% versus placebo and by 6% versus placebo in the hip. Multiple trials have evaluated its effect on fracture occurrence. The Fracture Intervention Trial provided evidence in >2000 women with prevalent vertebral fractures that daily alendronate treatment (5 mg/d for 2 years and 10 mg/d for 9 months afterward) reduces vertebral fracture risk by about 50%, multiple vertebral fractures by up to 90%, and hip fractures 2499 by up to 50%. Several subsequent trials have confirmed these findings (Fig. 425-9). For example, in a study of >1900 women with low bone mass treated with alendronate (10 mg/d) versus placebo, the incidence of all nonvertebral fractures was reduced by ~47% after only 1 year. In the United States, the 10-mg dose is approved for treatment of osteoporosis and 5 mg/d is used for prevention.

1	Trials comparing once-weekly alendronate, 70 mg, with daily 10-mg dosing have shown equivalence with regard to bone mass and bone turnover responses. Consequently, once-weekly therapy generally is preferred because of the low incidence of gastrointestinal side effects and ease of administration. Alendronate should be given with a full glass of water before breakfast, because bisphosphonates are poorly absorbed. Because of the potential for esophageal irritation, alendronate is contraindicated in patients who have stricture or inadequate emptying of the esophagus. It is recommended that patients remain upright for at least 30 min after taking the medication to avoid esophageal irritation. Cases of esophagitis, esophageal ulcer, and esophageal stricture have been described, but the incidence appears to be low. In clinical trials, overall gastrointestinal symptomatology was no different with alendronate than with placebo. Alendronate is also available in a preparation that contains

1	appears to be low. In clinical trials, overall gastrointestinal symptomatology was no different with alendronate than with placebo. Alendronate is also available in a preparation that contains vitamin D.

1	Risedronate also reduces bone turnover and increases bone mass. Controlled clinical trials have demonstrated 40–50% reduction in vertebral fracture risk over 3 years, accompanied by a 40% reduction in clinical nonspine fractures. The only clinical trial specifically designed to evaluate hip fracture outcome (HIP) indicated that risedronate reduced hip fracture risk in women in their seventies with confirmed osteoporosis by 40%. In contrast, risedronate was not effective at reducing hip fracture occurrence in older women (80+ years) without proven osteoporosis. Studies have shown that 35 mg of risedronate administered once weekly is therapeutically equivalent to 5 mg/d and that 150 mg once monthly is therapeutically equivalent to 35 mg once weekly. Patients should take risedronate with a full glass of plain water to facilitate delivery to the stomach and should not lie down for 30 min after taking the drug. The incidence of gastrointestinal side effects in trials with risedronate was

1	full glass of plain water to facilitate delivery to the stomach and should not lie down for 30 min after taking the drug. The incidence of gastrointestinal side effects in trials with risedronate was similar to that of placebo. A new preparation, which allows risedronate to be taken with food, was recently approved.

1	Etidronate was the first bisphosphonate to be approved, initially for use in Paget’s disease and hypercalcemia. This agent has also been used in osteoporosis trials of smaller magnitude than those performed for alendronate and risedronate but is not approved by the FDA for treatment of osteoporosis. Etidronate probably has some efficacy against vertebral fracture when given as an intermittent cyclical regimen (2 weeks on, 2.5 months off). Its effectiveness against nonvertebral fractures has not been studied.

1	Ibandronate is the third amino-bisphosphonate approved in the United States. Ibandronate (2.5 mg/d) has been shown in clinical trials to reduce vertebral fracture risk by ~40% but with no overall effect on nonvertebral fractures. In a post hoc analysis of subjects with a femoral neck T-score of –3 or below, ibandronate reduced the risk of nonvertebral fractures by ~60%. In clinical trials, ibandronate doses of 150 mg/month PO or 3 mg every 3 months IV had greater effects on turnover and bone mass than did 2.5 mg/d. Patients should take oral ibandronate in the same way as other bisphosphonates, but with 1 h elapsing before other food or drink (other than plain water).

1	Zoledronic acid is a potent bisphosphonate with a unique administration regimen (5 mg by slow IV infusion annually). The data confirm that it is highly effective in fracture risk reduction. In a study of >7000 women followed for 3 years, zoledronic acid (three annual infusions) reduced the risk of vertebral fractures by 70%, nonvertebral fractures by 25%, and hip fractures by 40%. These results were associated with less height loss and disability. In the treated population, there was an increased risk of transient postdose symptoms (acute-phase reaction) manifested by fever, arthralgia, myalgias, and headache. The symptoms usually last less than 48 h. An increased risk of atrial fibrillation and transient but Alendronate Risedronate Ibandronate Zoledronate pooled, post hoc pooled, post hoc preplanned preplanned Percent of patients

1	Alendronate Risedronate Ibandronate Zoledronate pooled, post hoc pooled, post hoc preplanned preplanned Percent of patients Alendronate pooled, post hoc Risedronate pooled, post hoc Zoledronate preplanned * * 0 12 24 36 0 5 10 15 Percent of patients PLB ALN 27%˜* * * ** * ** * * * PLB RIS 0 0 12 24 36 5 10 15 59%˜* 0 0 12 24 36 5 10 15 PLB ZOL 25%˜* ? Cumulative incidence of hip fractures over 3 years Time to first hip fracture (months)

1	FIGURE 425-9 Effects of various bisphosphonates on clinical vertebral fractures (A), nonvertebral fractures (B), and hip fractures (C). PLB, placebo; RRR, relative risk reduction. (After DM Black et al: J Clin Endocrinol Metab 85:4118, 2000; C Roux et al: Curr Med Res Opin 4:433, 2004; CH Chesnut et al: J Bone Miner Res 19:1241, 2004; DM Black et al: N Engl J Med 356:1809, 2007; JT Harrington et al: Calcif Tissue Int 74:129, 2003.) not permanent reduction in renal function was seen in comparison fractures was reduced significantly by about 35%, and there was to placebo. Detailed evaluation of all bisphosphonates failed to a trend toward reduced risk of a second hip fracture (effect size confirm that these agents increased the risk of atrial fibrillation. similar to that seen above). There was also a reduction in mortality Zoledronic acid is the only osteoporosis agent that has been stud-of about 30% that was not completely accounted for the reduced ied in the elderly with a prior hip

1	was also a reduction in mortality Zoledronic acid is the only osteoporosis agent that has been stud-of about 30% that was not completely accounted for the reduced ied in the elderly with a prior hip fracture. The risk of all clinical hip fracture risk.

1	Recently there has been concern about two potential side effects associated with bisphosphonate use. The first is osteonecrosis of the jaw (ONJ). ONJ usually follows a dental procedure in which bone is exposed (extractions or dental implants). It is presumed that the exposed bone becomes infected and dies. It is not uncommon among cancer victims with multiple myeloma or patients receiving high doses of bisphosphonates for skeletal metastases, but is rare among persons with osteoporosis on usual doses of bisphosphonates. The second side effect is called atypical femur fracture. These are unusual fractures that occur distal to the lesser trochanter and anywhere along the femoral shaft. They are often preceded by pain in the lateral thigh or groin that can be present for weeks or months before the fracture. The fractures occur with trivial trauma, sometimes completely spontaneously, and are primarily transverse, with a medial break when complete and minimally comminuted. A localized

1	before the fracture. The fractures occur with trivial trauma, sometimes completely spontaneously, and are primarily transverse, with a medial break when complete and minimally comminuted. A localized periosteal reaction, consistent with a stress fracture, is often seen in the lateral cortex (Fig. 425-10). The overall risk is low (suggested to be about one-one hundredth to one-tenth that of hip fracture) but appears to increase in incidence with long-term use of bisphosphonates. Although the fractures may be bisphosphonate related in many individuals, they clearly occur in patients with no prior bisphosphonate exposure. When complete, they require surgical fixation and may be difficult to heal. Anabolic medication may accelerate healing of these fractures in some patients, and surgery can sometimes be avoided. Patients initiating bisphosphonates need to be warned that if they develop thigh or groin pain they must notify their physician. Routine x-rays will sometimes pick up cortical

1	sometimes be avoided. Patients initiating bisphosphonates need to be warned that if they develop thigh or groin pain they must notify their physician. Routine x-rays will sometimes pick up cortical thickening or even a stress fracture, but more commonly MRI or technetium bone scan is required. The presence of an abnormality requires at minimum a period of modified weight bearing and may need prophylactic rodding of the femur. It is important to realize that these fractures may be bilateral, and when an abnormality is found, the other femur should be investigated.

1	mode of action Bisphosphonates are structurally related to pyrophosphates, compounds that are incorporated into bone matrix. Bisphosphonates specifically impair osteoclast function and reduce osteoclast number, in part by inducing apoptosis. Recent evidence suggests that the nitrogen-containing bisphosphonates also inhibit protein prenylation, one of the end products in the mevalonic

1	FIGURE 425-10 An atypical femur fracture (AFF) of the femoral diaphysis. A. Note the transverse fracture line in the lateral cortex that becomes oblique as it progresses medially across the femur (white arrow). B. On radiograph obtained immediately after intramedullary rod placement, a small area of periosteal thickening of the lateral cortex is visible (white arrow). C. On radiograph obtained at 6 weeks, note callus formation of the fracture site (white arrow). D. On radiograph obtained at 3 months, there is a mature callus that has failed to bridge the cortical gap (white arrow). Note the localized periosteal and/or endosteal thickening of the lateral cortex at the fracture site (white arrow). (From E Shane et al: J Bone Min Res 29:1-23, 2014. Courtesy of Fergus McKiernan.) acid pathway, by inhibiting the enzyme farnesyl pyrophosphate 2501 synthase. This effect disrupts intracellular protein trafficking and ultimately may lead to apoptosis. Some bisphosphonates have very long

1	by inhibiting the enzyme farnesyl pyrophosphate 2501 synthase. This effect disrupts intracellular protein trafficking and ultimately may lead to apoptosis. Some bisphosphonates have very long retention in the skeleton and may exert long-term effects. The consequences of this, if any, are unknown.

1	Calcitonin Calcitonin is a polypeptide hormone produced by the thyroid gland (Chap. 424). Its physiologic role is unclear because no skeletal disease has been described in association with calcitonin deficiency or excess. Calcitonin preparations are approved by the FDA for Paget’s disease, hypercalcemia, and osteoporosis in women >5 years past menopause. Concerns have been raised about an increase in the incidence of cancer associated with calcitonin use. Initially, the cancer noted was of the prostate, but an analysis of all data suggested a more general increase in cancer risk. In Europe, the European Medicines Agency (EMA) has removed the osteoporosis indication, and an FDA Advisory Committee has voted for a similar change in the United States.

1	Injectable calcitonin produces small increments in bone mass of the lumbar spine. However, difficulty of administration and frequent reactions, including nausea and facial flushing, make general use limited. A nasal spray containing calcitonin (200 IU/d) is available for treatment of osteoporosis in postmenopausal women. One study suggests that nasal calcitonin produces small increments in bone mass and a small reduction in new vertebral fractures in calcitonintreated patients versus those on calcium alone. There has been no proven effectiveness against nonvertebral fractures. Calcitonin is not indicated for prevention of osteoporosis and is not sufficiently potent to prevent bone loss in early postmenopausal women. Calcitonin might have an analgesic effect on bone pain, both in the subcutaneous and possibly the nasal form.

1	mode of action Calcitonin suppresses osteoclast activity by direct action on the osteoclast calcitonin receptor. Osteoclasts exposed to calcitonin cannot maintain their active ruffled border, which normally maintains close contact with underlying bone.

1	Denosumab A novel agent that was given twice yearly by SC administration in a randomized controlled trial in postmenopausal women with osteoporosis has been shown to increase BMD in the spine, hip, and forearm and reduce vertebral, hip, and nonvertebral fractures over a 3-year period by 70, 40, and 20%, respectively (Fig. 425-11). Other clinical trials indicate ability to increase bone mass in postmenopausal women with low bone mass (above osteoporosis range) and in postmenopausal women with breast cancer treated with hormonal agents. Furthermore, a study of men with prostate cancer treated with gonadotropin-releasing hormone (GnRH) agonist therapy indicated the ability of denosumab to improve bone mass and reduce vertebral fracture occurrence. Denosumab was approved by the FDA in 2010 for the treatment of postmenopausal women who have a high risk for osteoporotic fractures, including those with a history of fracture or multiple risk factors for fracture, and those who have failed or

1	the treatment of postmenopausal women who have a high risk for osteoporotic fractures, including those with a history of fracture or multiple risk factors for fracture, and those who have failed or are intolerant to other osteoporosis therapy. Denosumab is also approved for the treatment of osteoporosis in men at high risk, men with prostate cancer on GnRH agonist therapy, and women with breast cancer on aromatase inhibitor therapy.

1	mode of action Denosumab is a fully human monoclonal antibody to RANKL, the final common effector of osteoclast formation, activity, and survival. Denosumab binds to RANKL, inhibiting its ability to initiate formation of mature osteoclasts from osteoclast precursors and to bring mature osteoclasts to the bone surface and initiate bone resorption. Denosumab also plays a role in reducing the survival of the osteoclast. Through these actions on the osteoclast, denosumab induces potent antiresorptive action, as assessed biochemically and histomorphometrically, and may contribute to the occurrence of ONJ. Atypical femur fractures have also been noted. Serious adverse reactions include hypocalcemia, skin infections (usually cellulitis of the lower extremity), and dermatologic reactions such as dermatitis, rashes, and eczema. The effects of denosumab are rapidly reversible. Effect of teriparatide on the risk of new vertebral fractures

1	Effect of teriparatide on the risk of new vertebral fractures Relative: Relative: 10 65% 69% Absolute: Absolute: 40 9.3% 9.9% Number of women with 1 or % of women % of women Effect of teriparatide on the risk of nonvertebral fragility fractures Relative: 53% Absolute: 2.9% Relative: 54% Absolute: 3.0% Risk reduction Placebo TPTD20 TPTD40 30 14 14 Number of women with

1	Relative: 53% Absolute: 2.9% Relative: 54% Absolute: 3.0% Risk reduction Placebo TPTD20 TPTD40 30 14 14 Number of women with Effect of teriparatide on the risk of nonvertebral fragility fractures (time to first fracture) *P <0.05 vs. placebo PlaceboTPTD20 TPTD40 * * % of women** tures (A) and nonvertebral fragility fractures (B and C). (After RM Neer et al: N Engl J Med 344:1434, 2001.) a single daily injection given for a maximum of 2 years. Teriparatide produces increases in bone mass and mediates architectural improvements in skeletal structure. These effects are lower when patients have been exposed previously to bisphosphonates, possibly in proportion to the potency of the antiresorptive effect. When teriparatide is being considered for treatment-naive patients, it is best administered as monotherapy and followed by an antiresorptive agent such as a bisphosphonate. If teriparatide treatment is not followed by an antiresorptive agent, the bone gained is rapidly lost.

1	Side effects of teriparatide are generally mild and can include leg cramps, muscle pain, weakness, dizziness, headache, and nausea. Rodents given prolonged treatment with PTH in relatively high doses developed osteogenic sarcomas. Long-term surveillance studies suggest no association between 2 years of teriparatide administration and osteosarcoma risk in humans. PTH use may be limited by its mode of administration; alternative modes of delivery are being investigated. The optimal frequency of administration also remains to be established, and it is possible that FIGURE 425-11 Effects of denosumab on new vertebral fractures (A) and times to nonvertebral and hip fracture (B and C). RR, relative risk. (After SR Cummings et al: N Engl J Med 361:756, 2009.) If denosumab is stopped, bone will be lost rapidly if another agent is not used.

1	Parathyroid Hormone Endogenous PTH is an 84-amino-acid peptide that is largely responsible for calcium homeostasis (Chap. 424). Although chronic elevation of PTH, as occurs in hyperparathyroidism, is associated with bone loss (particularly cortical bone), PTH when given exogenously as a daily injection exerts anabolic effects on bone. Teriparatide (1-34hPTH) is approved for the treatment of osteoporosis in both men and women at high risk for fracture. In a pivotal study (median time of treatment, 19 months’ duration), 20 μg of teriparatide daily by SC injection reduced vertebral fractures by 65% and nonvertebral fractures by 45% (Fig. 425-12). Treatment is administered as PTH might be effective when used intermittently. Cost also may be a limiting factor. In some settings, the effect of PTH might be enhanced by combination with an antiresorptive agent. This might be particularly important in patients who have been treated previously with bisphosphonate medications.

1	FIGURE 425-13 Effect of parathyroid hormone (PTH) treatment on bone microarchitecture. Paired biopsy specimens from a 64-year-old woman before (A) and after (B) treatment with PTH. (From DW Dempster et al: J Bone Miner Res 16:1846, 2001.) mode of action Exogenously administered PTH appears to have direct actions on osteoblast activity, with biochemical and histomorphometric evidence of de novo bone formation early in response to PTH, before activation of bone resorption. Subsequently, PTH activates bone remodeling but still appears to favor bone formation over bone resorption. PTH stimulates Wnt signaling, IGF-I, and collagen production and appears to increase osteoblast number by stimulating replication, enhancing osteoblast recruitment, and inhibiting apoptosis. Unlike all other treatments, PTH produces a true increase in bone tissue and an apparent restoration of bone microarchitecture (Fig. 425-13).

1	Fluoride Fluoride has been available for many years and is a potent stimulator of osteoprogenitor cells when studied in vitro. It has been used in multiple osteoporosis studies with conflicting results, in part because of the use of varying doses and preparations. Despite increments in bone mass of up to 10%, there are no consistent effects of fluoride on vertebral or nonvertebral fracture; the latter may actually increase when high doses of fluoride are used. Fluoride remains an experimental agent despite its long history and multiple studies.

1	Strontium Ranelate Strontium ranelate is approved in several European countries for the treatment of osteoporosis. It increases bone mass throughout the skeleton; in clinical trials, the drug reduced the risk of vertebral fractures by 37% and that of nonvertebral fractures by 14%. It appears to be modestly antiresorptive while at the same time not causing as much of a decrease in bone formation (measured biochemically). Strontium is incorporated into hydroxyapatite, replacing calcium, a feature that might explain some of its fracture benefits. Small increased risks of venous thrombosis, sometimes severe dermatologic reactions, seizures, and abnormal cognition have been seen and require further study. An increase in risk of cardiovascular disease has also been associated with use of strontium, such that the EMA has restricted its use at present.

1	Other Potential Anabolic Agents Several small studies of growth hormone (GH), alone or in combination with other agents, have not shown consistent or substantial positive effects on skeletal mass. Many of these studies have been relatively short term, and the effects of GH, growth hormone–releasing hormone, and the IGFs are still under investigation. Anabolic steroids, mostly derivatives of testosterone, act primarily as antiresorptive agents to reduce bone turnover but also may stimulate osteoblastic activity. Effects on bone mass remain unclear but appear weak in general, and use is limited by masculinizing side effects. Several observational studies suggested that the statin drugs, used to treat hypercholesterolemia, 2503 may be associated with increased bone mass and reduced fractures, but conclusions from clinical trials have been largely negative. Early studies with sclerostin antibodies, which inhibit sclerostin, activate Wnt, and might be highly anabolic to bone, are under

1	but conclusions from clinical trials have been largely negative. Early studies with sclerostin antibodies, which inhibit sclerostin, activate Wnt, and might be highly anabolic to bone, are under development. Odanacatib is a mixed antiresorptive, partial bone formation stimulator that is currently in the late stages of development.

1	In some early studies, protective pads worn around the outer thigh, which cover the trochanteric region of the hip, were able to prevent hip fractures in elderly residents in nursing homes. Randomized controlled trials of hip protectors have been unable to confirm these early findings. Therefore, the efficacy of hip protectors remains controversial at this time. Kyphoplasty and vertebroplasty are also useful nonpharmacologic approaches for the treatment of painful vertebral fractures. However, no long-term data are available.

1	There are currently no well-accepted guidelines for monitoring treatment of osteoporosis. Because most osteoporosis treatments produce small or moderate bone mass increments on average, it is reasonable to consider BMD as a monitoring tool. Changes must exceed ~4% in the spine and 6% in the hip to be considered significant in any individual. The hip is the preferred site due to larger surface area and greater reproducibility. Medication-induced increments may require several years to produce changes of this magnitude (if they do at all). Consequently, it can be argued that BMD should be repeated at intervals >2 years. Only significant BMD reductions should prompt a change in medical regimen, because it is expected that many individuals will not show responses greater than the detection limits of the current measurement techniques.

1	Biochemical markers of bone turnover may prove useful for treatment monitoring, but little hard evidence currently supports this concept; it remains unclear which endpoint is most useful. If bone turnover markers are used, a determination should be made before therapy is started and repeated ≥4 months after therapy is initiated. In general, a change in bone turnover markers must be 30–40% lower than the baseline to be significant because of the biologic and technical variability in these tests. A positive change in biochemical markers and/or bone density can be useful to help patients adhere to treatment regimens.

1	Osteoporotic fractures are a well-characterized consequence of the hypercortisolism associated with Cushing’s syndrome. However, the therapeutic use of glucocorticoids is by far the most common form of glucocorticoid-induced osteoporosis. Glucocorticoids are used widely in the treatment of a variety of disorders, including chronic lung disorders, rheumatoid arthritis and other connective tissue diseases, inflammatory bowel disease, and after transplantation. Osteoporosis and related fractures are serious side effects of chronic glucocorticoid therapy. Because the effects of glucocorticoids on the skeleton are often superimposed on the consequences of aging and menopause, it is not surprising that women and the elderly are most frequently affected. The skeletal response to steroids is remarkably heterogeneous, however, and even young, growing individuals treated with glucocorticoids can present with fractures.

1	The risk of fractures depends on the dose and duration of glucocorticoid therapy, although recent data suggest that there may be no completely safe dose. Bone loss is more rapid during the early months of treatment, and trabecular bone is affected more severely than cortical bone. As a result, fractures have been shown to increase within 3 months of steroid treatment. There is an increase in fracture risk in both the axial skeleton and the appendicular skeleton, including risk of hip fracture. Bone loss can occur with any route of steroid administration, including high-dose inhaled glucocorticoids and intraarticular 2504 injections. Alternate-day delivery does not appear to ameliorate the skeletal effects of glucocorticoids.

1	Glucocorticoids increase bone loss by multiple mechanisms, including (1) inhibition of osteoblast function and an increase in osteoblast apoptosis, resulting in impaired synthesis of new bone; (2) stimulation of bone resorption, probably as a secondary effect; (3) impairment of the absorption of calcium across the intestine, probably by a vitamin D–independent effect; (4) increase of urinary calcium loss and perhaps induction of some degree of secondary hyperparathyroidism; (5) reduction of adrenal androgens and suppression of ovarian and testicular secretion of estrogens and androgens; and (6) induction of glucocorticoid myopathy, which may exacerbate effects on skeletal and calcium homeostasis as well as increase the risk of falls.

1	Because of the prevalence of glucocorticoid-induced bone loss, it is important to evaluate the status of the skeleton in all patients starting or already receiving long-term glucocorticoid therapy. Modifiable risk factors should be identified, including those for falls. Examination should include testing of height and muscle strength. Laboratory evaluation should include an assessment of 24-h urinary calcium. All patients on long-term (>3 months) glucocorticoids should have measurement of bone mass at both the spine and the hip using DXA. If only one skeletal site can be measured, it is best to assess the spine in individuals <60 years and the hip in those >60 years.

1	Bone loss caused by glucocorticoids can be prevented and the risk of fractures significantly reduced. Strategies must include using the lowest dose of glucocorticoid for disease management. Topical and inhaled routes of administration are preferred, where appropriate. Risk factor reduction is important, including smoking cessation, limitation of alcohol consumption, and participation in weight-bearing exercise, when appropriate. All patients should receive an adequate calcium and vitamin D intake from the diet or from supplements. Several bisphosphonates (alendronate, risedronate, and zoledronic acid) have been demonstrated in large clinical trials to reduce the risk of vertebral fractures in patients being treated with glucocorticoids, as well as improve bone mass in spine and hip. Teriparatide also improves bone mass and reduces fracture risk in glucocorticoid-treated osteoporosis compared to an active comparator (alendronate).

1	Paget’s Disease and Other Dysplasias of Bone Murray J. Favus, Tamara J. Vokes PAGET’S DISEASE OF BONE Paget’s disease is a localized bone-remodeling disorder that affects 426e widespread, noncontiguous areas of the skeleton. The pathologic process is initiated by overactive osteoclastic bone resorption followed by a compensatory increase in osteoblastic new bone formation, resulting in a structurally disorganized mosaic of woven and lamellar bone. Pagetic bone is expanded, less compact, and more vascular; thus, it is more susceptible to deformities and fractures. Although most patients are asymptomatic, symptoms resulting directly from bony involvement (bone pain, secondary arthritis, fractures) or secondarily from the expansion of bone causing compression of surrounding neural tissue are not uncommon. Epidemiology There is a marked geographic variation in the frequency of Paget’s disease, with high prevalence in Western

1	Epidemiology There is a marked geographic variation in the frequency of Paget’s disease, with high prevalence in Western Europe (Great Britain, France, and Germany, but not Switzerland or Scandinavia) and among those who have immigrated to Australia, New Zealand, South Africa, and North and South America. The disease is rare in native populations of the Americas, Africa, Asia, and the Middle East; when it does occur, the affected subjects usually have evidence of European ancestry, supporting the migration theory. For unclear reasons, the prevalence and severity of Paget’s disease are decreasing, and the age of diagnosis is increasing.

1	The prevalence is greater in males and increases with age. Autopsy series reveal Paget’s disease in about 3% of those over age 40. Prevalence of positive skeletal radiographs in patients over age 55 is 2.5% for men and 1.6% for women. Elevated alkaline phosphatase (ALP) levels in asymptomatic patients have an age-adjusted incidence of 12.7 and 7 per 100,000 person-years in men and women, respectively. Etiology The etiology of Paget’s disease of bone remains unknown, but evidence supports both genetic and viral etiologies. A positive family history is found in 15–25% of patients and, when present, raises the prevalence of the disease sevento tenfold among first-degree relatives.

1	A clear genetic basis has been established for several rare familial bone disorders that clinically and radiographically resemble Paget’s disease but have more severe presentation and earlier onset. A homozygous deletion of the TNFRSF11B gene, which encodes osteoprotegrin (Fig. 426e-1), causes juvenile Paget’s disease, also known as familial idiopathic hyperphosphatasia, a disorder characterized by uncontrolled osteoclastic differentiation and resorption. Familial patterns of disease in several large kindred are consistent with an autosomal dominant pattern of inheritance with variable penetrance. Familial expansile osteolysis, expansile skeletal hyperphosphatasia, and early-onset Paget’s disease are associated with mutations in TNFRSF11A gene, which encodes RANK (receptor activator of nuclear factor-κB), a member of the tumor necrosis factor superfamily critical for osteoclast differentiation (Fig. 426e-1). Finally, mutations in the gene for valosin-containing protein cause a rare

1	nuclear factor-κB), a member of the tumor necrosis factor superfamily critical for osteoclast differentiation (Fig. 426e-1). Finally, mutations in the gene for valosin-containing protein cause a rare syndrome with autosomal dominant inheritance and variable penetrance known as inclusion body myopathy with Paget’s disease and frontotemporal dementia (IBMPFD). The role of genetic factors is less clear in the more common form of late-onset Paget’s disease. Although a few families with mutations in the gene encoding RANK have been reported, the most common mutations identified in familial and sporadic cases of Paget’s disease have been in the SQSTM1 gene (sequestasome-1 or p62 protein) in the C-terminal ubiquitin-binding domain. The p62 protein is involved in nuclear factor κB (NF-κB) signaling and regulates osteoclastic differentiation. The phenotypic variability in patients with SQSTM1 mutations suggests that additional factors, such as other genetic influences or viral infection, may

1	and regulates osteoclastic differentiation. The phenotypic variability in patients with SQSTM1 mutations suggests that additional factors, such as other genetic influences or viral infection, may influence clinical expression of the disease.

1	Mesenchymal cell Collagen osteocalcin Osteoclast Osteoclast precursor Osteoblasts Osteoblasts IGF-1 IGF-2 OPG M-CSF IL-1, IL-6 c-fms + RANK L RANK

1	FIGurE 426e-1 Diagram illustrating factors that promote differentiation and function of osteoclasts and osteoblasts and the role of the RANK pathway. Stromal bone marrow (mesenchymal) cells and differentiated osteoblasts produce multiple growth factors and cytokines, including macrophage colony-stimulating factor (M-CSF), to modulate osteoclastogenesis. RANKL (receptor activator of nuclear factor-κB ligand) is produced by osteoblast progenitors and mature osteoblasts and can bind to a soluble decoy receptor known as OPG (osteoprotegerin) to inhibit RANKL action. Alternatively, a cell-cell interaction between osteoblast and osteoclast progenitors allows RANKL to bind to its membrane-bound receptor, RANK, thereby stimulating osteoclast differentiation and function. RANK binds intracellular proteins called TRAFs (tumor necrosis factor receptor–associated factors) that mediate receptor signaling through transcription factors such as NF-κB. M-CSF binds to its receptor, c-fms, which is the

1	proteins called TRAFs (tumor necrosis factor receptor–associated factors) that mediate receptor signaling through transcription factors such as NF-κB. M-CSF binds to its receptor, c-fms, which is the cellular homologue of the fms oncogene. See text for the potential role of these pathways in disorders of osteoclast function such as Paget’s disease and osteopetrosis. IL, interleukin; IGF, insulin-like growth factor.

1	Several lines of evidence suggest that a viral infection may contribute to the clinical manifestations of Paget’s disease, including (1) the presence of cytoplasmic and nuclear inclusions resembling paramyxoviruses (measles and respiratory syncytial virus) in pagetic osteoclasts and (2) viral mRNA in precursor and mature osteoclasts. The viral etiology is further supported by conversion of osteoclast precursors to pagetic-like osteoclasts by vectors containing the measles virus nucleocapsid or matrix genes. However, the viral etiology has been questioned by the inability to culture a live virus from pagetic bone and by failure to clone the full-length viral genes from material obtained from patients with Paget’s disease.

1	Pathophysiology The principal abnormality in Paget’s disease is the increased number and activity of osteoclasts. Pagetic osteoclasts are large, increased 10to 100-fold in number, and have a greater number of nuclei (as many as 100 compared to 3–5 nuclei in the normal osteoclast). The overactive osteoclasts may create a sevenfold increase in resorptive surfaces and an erosion rate of 9 μg/d (normal is 1 μg/d). Several causes for the increased number and activity of pagetic osteoclasts have been identified: (1) osteoclastic precursors are hypersensitive to 1,25(OH)2D3; (2) osteoclasts are hyperresponsive to RANK ligand (RANKL), the osteoclast stimulatory factor that mediates the effects of most osteotropic factors on osteoclast formation; (3) marrow stromal cells from pagetic lesions have increased RANKL expression; (4) osteoclast precursor recruitment is increased by interleukin (IL) 6, which is increased in the blood of patients with active Paget’s disease and is overexpressed in

1	increased RANKL expression; (4) osteoclast precursor recruitment is increased by interleukin (IL) 6, which is increased in the blood of patients with active Paget’s disease and is overexpressed in pagetic osteoclasts; (5) expression of the protooncogene c-fos, which increases osteoclastic activity, is increased; and

1	CHAPTER 426e Paget’s Disease and Other Dysplasias of Bone (6) the antiapoptotic oncogene Bcl-2 in pagetic bone is overexpressed. Numerous osteoblasts are recruited to active resorption sites and produce large amounts of new bone matrix. As a result, bone turnover is high, and bone mass is normal or increased, not reduced, unless there is concomitant deficiency of calcium and/or vitamin D.

1	The characteristic feature of Paget’s disease is increased bone resorption accompanied by accelerated bone formation. An initial osteolytic phase involves prominent bone resorption and marked hypervascularization. Radiographically, this manifests as an advancing lytic wedge, or “blade of grass” lesion. The second phase is a period of very active bone formation and resorption that replaces normal lamellar bone with haphazard (woven) bone. Fibrous connective tissue may replace normal bone marrow. In the final sclerotic phase, bone resorption declines progressively and leads to a hard, dense, less vascular pagetic or mosaic bone, which represents the so-called burned-out phase of Paget’s disease. All three phases may be present at the same time at different skeletal sites.

1	Clinical Manifestations Diagnosis is often made in asymptomatic patients because they have elevated ALP levels on routine blood chemistry testing or an abnormality on a skeletal radiograph obtained for another indication. The skeletal sites most commonly involved are the pelvis, vertebral bodies, skull, femur, and tibia. Familial cases with an early presentation often have numerous active sites of skeletal involvement.

1	The most common presenting symptom is pain, which may result from increased bony vascularity, expanding lytic lesions, fractures, bowing, or other deformities. Bowing of the femur or tibia causes gait abnormalities and abnormal mechanical stresses with secondary osteoarthritis of the hip or knee joints. Long bone bowing also causes extremity pain by stretching the muscles attached to the bone softened by the pagetic process. Back pain results from enlarged pagetic vertebrae, vertebral compression fractures, spinal stenosis, degenerative changes of the joints, and altered body mechanics with kyphosis and forward tilt of the upper back. Rarely, spinal cord compression may result from bone enlargement or from the vascular steal syndrome. Skull involvement may cause headaches, symmetric or asymmetric enlargement of the parietal or frontal bones (frontal bossing), and increased head size. Cranial expansion may narrow cranial foramens and cause neurologic complications including hearing

1	asymmetric enlargement of the parietal or frontal bones (frontal bossing), and increased head size. Cranial expansion may narrow cranial foramens and cause neurologic complications including hearing loss from cochlear nerve damage from temporal bone involvement, cranial nerve palsies, and softening of the base of the skull (platybasia) with the risk of brainstem compression. Pagetic involvement of the facial bones may cause facial deformity; loss of teeth and other dental conditions; and, rarely, airway compression.

1	Fractures are serious complications of Paget’s disease and usually occur in long bones at areas of active or advancing lytic lesions. Common fracture sites are the femoral shaft and subtrochanteric regions. Neoplasms arising from pagetic bone are rare (<0.5%). The incidence of sarcoma appears to be decreasing, possibly because of earlier, more effective treatment with potent antiresorptive agents. The majority of tumors are osteosarcomas, which usually present with new pain in a long-standing pagetic lesion. Osteoclast-rich benign giant cell tumors may arise in areas adjacent to pagetic bone, and they respond to glucocorticoid therapy.

1	Cardiovascular complications may occur in patients with involvement of large (15–35%) portions of the skeleton and a high degree of disease activity (ALP four times above normal). The extensive arteriovenous shunting and marked increases in blood flow through the vascular pagetic bone lead to a high-output state and cardiac enlargement. However, high-output heart failure is relatively rare and usually develops in patients with concomitant cardiac pathology. In addition, calcific aortic stenosis and diffuse vascular calcifications have been associated with Paget’s disease.

1	Diagnosis The diagnosis may be suggested on clinical examination by the presence of an enlarged skull with frontal bossing, bowing of an extremity, or short stature with simian posturing. An extremity with an area of warmth and tenderness to palpation may suggest an underlying pagetic lesion. Other findings include bony deformity of the pelvis, skull, spine, and extremities; arthritic involvement of the joints adjacent to lesions; and leg-length discrepancy resulting from deformities of the long bones.

1	Paget’s disease is usually diagnosed from radiologic and biochemical abnormalities. Radiographic findings typical of Paget’s disease include enlargement or expansion of an entire bone or area of a long bone, cortical thickening, coarsening of trabecular markings, and typical lytic and sclerotic changes. Skull radiographs (Fig. 426e-2) reveal regions of “cotton wool,” or osteoporosis circumscripta, thickening of diploic areas, and enlargement and sclerosis of a portion or all of one or more skull bones. Vertebral cortical thickening of the superior and inferior end plates creates a “picture frame” vertebra. Diffuse radiodense enlargement of a vertebra is referred to as “ivory vertebra.” Pelvic radiographs may demonstrate disruption or fusion of the sacroiliac joints; porotic and radiodense lesions of the ilium with whorls of coarse trabeculation; thickened and sclerotic iliopectineal line (brim sign); and softening with protrusio acetabuli, with axial migration of the hips and

1	lesions of the ilium with whorls of coarse trabeculation; thickened and sclerotic iliopectineal line (brim sign); and softening with protrusio acetabuli, with axial migration of the hips and functional flexion contracture. Radiographs of long bones reveal bowing deformity and typical pagetic changes of cortical thickening and expansion and areas of lucency and sclerosis (Fig. 426e-3). Radionuclide 99mTc bone scans are less specific but are more sensitive than standard radiographs for identifying sites of active skeletal lesions. Although computed tomography (CT) and magnetic resonance imaging (MRI) studies are not necessary in most cases,

1	FIGurE 426e-2 A 48-year-old woman with Paget’s disease of the skull. Left. Lateral radiograph showing areas of both bone resorption and sclerosis. Right. 99mTc HDP bone scan with anterior, posterior, and lateral views of the skull showing diffuse isotope uptake by the frontal, parietal, occipital, and petrous bones. FIGurE 426e-3 Radiograph of a 73-year-old man with Paget’s disease of the right proximal femur. Note the coarsening of the trabecular pattern with marked cortical thickening and narrowing of the joint space consistent with osteoarthritis secondary to pagetic deformity of the right femur. CT may be useful for the assessment of possible fracture, and MRI is necessary to assess the possibility of sarcoma, giant cell tumor, or metastatic disease in pagetic bone. Definitive diagnosis of malignancy often requires bone biopsy.

1	Biochemical evaluation is useful in the diagnosis and management of Paget’s disease. The marked increase in bone turnover can be monitored using biochemical markers of bone formation and resorption. The parallel rise in markers of bone formation and resorption confirms the coupling of bone formation and resorption in Paget’s disease. The degree of bone marker elevation reflects the extent and severity of the disease. Patients with the highest elevation of ALP (10 × the upper limit of normal) typically have involvement of the skull and at least one other skeletal site. Lower values suggest less extensive involvement or a quiescent phase of the disease. For most patients, serum total ALP remains the test of choice both for diagnosis and assessing response to therapy. Occasionally, a symptomatic patient with evidence of progression at a single site may have a normal total ALP level but increased bone-specific ALP. For unclear reasons, serum osteocalcin, another marker of bone formation,

1	patient with evidence of progression at a single site may have a normal total ALP level but increased bone-specific ALP. For unclear reasons, serum osteocalcin, another marker of bone formation, is not always elevated and is not recommended for use in diagnosis or management of Paget’s disease. Bone resorption markers (serum or urine N-telopeptide or C-telopeptide measured in the blood or urine) are also elevated in active Paget’s disease and decrease more rapidly in response to therapy than does ALP.

1	Serum calcium and phosphate levels are normal in Paget’s disease. Immobilization of a patient with active Paget’s disease may rarely cause hypercalcemia and hypercalciuria and increase the risk for nephrolithiasis. However, the discovery of hypercalcemia, even in the presence of immobilization, should prompt a search for another cause of hypercalcemia. In contrast, hypocalcemia or mild secondary hyperparathyroidism may develop in Paget’s patients with very active bone formation and insufficient calcium and vitamin D intake, particularly during bisphosphonate therapy when bone resorption is rapidly suppressed and active bone formation continues. Therefore, adequate calcium and vitamin D intake should be instituted prior to administration of bisphosphonates.

1	The development of effective and potent pharmacologic agents (Table 426e-1) has changed the treatment philosophy from treating only symptomatic patients to treating asymptomatic patients who are at risk for complications. Pharmacologic therapy is indicated in

1	Dose and Mode Name of Delivery Normalization of ALP the following circumstances: to control symptoms caused by metabolically active Paget’s disease such as bone pain, fracture, headache, pain from pagetic radiculopathy or arthropathy, or neurologic complications; to decrease local blood flow and minimize operative blood loss in patients who need surgery at an active pagetic site; to reduce hypercalciuria that may occur during immobilization; and to decrease the risk of complications when disease activity is high (elevated ALP) and when the site of involvement involves weight-bearing bones, areas adjacent to major joints, vertebral bodies, and the skull. Whether or not early therapy prevents late complications remains to be determined. A randomized study of over 1200 patients from the United Kingdom showed no difference in bone pain, fracture rates, quality of life, and hearing loss between patients who received pharmacologic therapy to control symptoms (bone pain) and those receiving

1	Kingdom showed no difference in bone pain, fracture rates, quality of life, and hearing loss between patients who received pharmacologic therapy to control symptoms (bone pain) and those receiving bisphosphonates to normalize serum ALP. However, the most potent agent (zoledronic acid) was not used, and the duration of observation (mean of 3 years with a range of 2 to 5 years) may not be long enough to assess the impact of treatment on long-term outcomes. It seems likely that the restoration of normal bone architecture following suppression of pagetic activity will prevent further deformities and complications.

1	Agents approved for treatment of Paget’s disease suppress the very high rates of bone resorption and secondarily decrease the high rates of bone formation (Table 426e-1). As a result of decreasing bone turnover, pagetic structural patterns, including areas of poorly mineralized woven bone, are replaced by more normal cancellous or lamellar bone. Reduced bone turnover can be documented by a decline in serum ALP and urine or serum resorption markers (N-telopeptide, C-telopeptide).

1	The first clinically useful agent, etidronate, is now rarely used because the doses required to suppress bone resorption may impair mineralization, necessitating that the drug be given for a maximum of 6 months followed by a 6-month drug-free period. The second-generation oral bisphosphonates—tiludronate, alendronate, and risedronate—are more potent than etidronate in controlling bone turnover and, thus, induce a longer remission at a lower dose. The lower doses reduce the risks of impaired mineralization and osteomalacia. Oral bisphosphonates should be taken first thing in the morning on an empty stomach, followed by maintenance of upright posture with no food, drink, or other medications for 30–60 min. The efficacy of different agents, based on their ability to normalize or decrease ALP levels, is summarized in Table 426e-1, although the response rates are not comparable because they are obtained from different studies.

1	Intravenous bisphosphonates approved for Paget’s disease include pamidronate and zoledronic acid. Although the recommended dose for pamidronate is 30 mg dissolved in 500 mL of normal saline or dextrose IV over 4 h on 3 consecutive days, a more commonly used simpler regimen is a single infusion of 60–90 mg in patients with mild elevation of serum ALP and multiple 90-mg

1	CHAPTER 426e Paget’s Disease and Other Dysplasias of Bone infusions in those with higher levels of ALP. In many patients, particularly those who have severe disease or need rapid normalization of bone turnover (neurologic symptoms, severe bone pain due to a lytic lesion, risk of an impending fracture, or pretreatment prior to elective surgery in an area of active disease), treatment with zoledronic acid is the first choice. It normalizes ALP in about 90% of patients by 6 months, and the therapeutic effect persists for at least 6 more months in most patients. About 10–20% of patients experience a flulike syndrome after the first infusion, which can be partly ameliorated by pretreatment with acetaminophen or nonsteroidal anti-inflammatory drugs (NSAIDs). In patients with high bone turnover, vitamin D and calcium should be provided to prevent hypocalcemia and secondary hyperparathyroidism. Remission following treatment with IV bisphosphonates, particularly zoledronic acid, may persist

1	vitamin D and calcium should be provided to prevent hypocalcemia and secondary hyperparathyroidism. Remission following treatment with IV bisphosphonates, particularly zoledronic acid, may persist for well over 1 year. Bisphosphonates should not be used in patients with renal insufficiency (glomerular filtration rate <35 mL/min).

1	The subcutaneous injectable form of salmon calcitonin is approved for the treatment of Paget’s disease. The common side effects of calcitonin therapy are nausea and facial flushing. Secondary resistance after prolonged use may be due to either the formation of anticalcitonin antibodies or downregulation of osteoclastic cell–surface calcitonin receptors. The lower potency and injectable mode of delivery make this agent a less attractive treatment option that should be reserved for patients who either do not tolerate bisphosphonates or have a contraindication to their use. In early reports, denosumab, an antibody to RANKL, has shown promise but has not been approved for this indication.

1	Osteopetrosis refers to a group of disorders caused by severe impairment of osteoclast-mediated bone resorption. Other terms that are often used include marble bone disease, which captures the solid x-ray appearance of the involved skeleton, and Albers-Schonberg disease, which refers to the milder, adult form of osteopetrosis also known as autosomal dominant osteopetrosis type II. The major types of osteopetrosis include malignant (severe, infantile, autosomal recessive) osteopetrosis and benign (adult, autosomal dominant) osteopetrosis types I and II. A rare autosomal recessive intermediate form has a more benign prognosis. Autosomal recessive carbonic anhydrase (CA) II deficiency produces osteopetrosis of intermediate severity associated with renal tubular acidosis and cerebral calcification.

1	Etiology and Genetics Naturally occurring and gene-knockout animal models with phenotypes similar to those of the human disorders have been used to explore the genetic basis of osteopetrosis. The primary defect in osteopetrosis is the loss of osteoclastic bone resorption and preservation of normal osteoblastic bone formation. Osteoprotegerin (OPG) is a soluble decoy receptor that binds osteoblast-derived RANK ligand, which mediates osteoclast differentiation and activation (Fig. 426e-1). Transgenic mice that overexpress OPG develop osteopetrosis, presumably by blocking RANK ligand. Mice deficient in RANK lack osteoclasts and develop severe osteopetrosis.

1	Recessive mutations of CA II prevent osteoclasts from generating an acid environment in the clear zone between its ruffled border and the adjacent mineral surface. Absence of CA II, therefore, impairs osteoclastic bone resorption. Other forms of human disease have less clear genetic defects. About one-half of the patients with malignant infantile osteopetrosis have a mutation in the TCIRG1 gene encoding the osteoclast-specific subunit of the vacuolar proton pump, which mediates the acidification of the interface between bone mineral and the osteoclast ruffled border. Mutations in the CICN7 chloride channel gene cause autosomal dominant osteopetrosis type II.

1	Clinical Presentation The incidence of autosomal recessive severe (malignant) osteopetrosis ranges from 1 in 200,000 to 1 in 500,000 live births. As bone and cartilage fail to undergo modeling, paralysis of one or more cranial nerves may occur due to narrowing of the cranial foramens. Failure of skeletal modeling also results in inadequate marrow space, leading to extramedullary hematopoiesis with hypersplenism and pancytopenia. Hypocalcemia due to lack of osteoclastic bone resorption may occur in infants and young children. The untreated infantile disease is fatal, often before age 5.

1	Adult (benign) osteopetrosis is an autosomal dominant disease that is usually diagnosed by the discovery of typical skeletal changes in young adults who undergo radiologic evaluation of a fracture. The prevalence is 1 in 100,000 to 1 in 500,000 adults. The course is not always benign, because fractures may be accompanied by loss of vision, deafness, psychomotor delay, mandibular osteomyelitis, and other complications usually associated with the juvenile form. In some kindred, nonpenetrance results in skip generations, while in other families, severely affected children are born into families with benign disease. The milder form of the disease does not usually require treatment.

1	radiography Typically, there are generalized symmetric increases in bone mass with thickening of both cortical and trabecular bone. Diaphyses and metaphyses are broadened, and alternating sclerotic and lucent bands may be seen in the iliac crests, at the ends of long bones, and in vertebral bodies. The cranium is usually thickened, particularly at the base of the skull, and the paranasal and mastoid sinuses are underpneumatized. Laboratory Findings The only significant laboratory findings are elevated serum levels of osteoclast-derived tartrate-resistant acid phosphatase (TRAP) and the brain isoenzyme of creatine kinase. Serum calcium may be low in severe disease, and parathyroid hormone and 1,25-dihydroxyvitamin D levels may be elevated in response to hypocalcemia.

1	Allogeneic HLA-identical bone marrow transplantation has been successful in some children. Following transplantation, the marrow contains progenitor cells and normally functioning osteoclasts. A cure is most likely when children are transplanted before age 4. Marrow transplantation from nonidentical HLA-matched donors has a much higher failure rate. Limited studies in small numbers of patients have suggested variable benefits following treatment with interferon γ-1β, 1,25-dihydroxyvitamin D (which stimulates osteoclasts directly), methylprednisolone, and a low-calcium/highphosphate diet. Surgical intervention is indicated to decompress optic or auditory nerve compression. Orthopedic management is required for the surgical treatment of fractures and their complications including malunion and postfracture deformity.

1	This is an autosomal recessive form of osteosclerosis that is believed to have affected the French impressionist painter Henri de Toulouse-Lautrec. The molecular basis involves mutations in the gene that encodes cathepsin K, a lysosomal metalloproteinase highly expressed in osteoclasts and important for bone-matrix degradation. Osteoclasts are present but do not function normally. Pyknodysostosis is a form of short-limb dwarfism that presents with frequent fractures but usually a normal life span. Clinical features include short stature; kyphoscoliosis and deformities of the chest; high arched palate; proptosis; blue sclerae; dysmorphic features including small face and chin, frontooccipital prominence, pointed beaked nose, large cranium, and obtuse mandibular angle; and small, square hands with hypoplastic nails. Radiographs demonstrate a generalized increase in bone density, but in contrast to osteopetrosis, the long bones are normally shaped. Separated cranial sutures, including

1	with hypoplastic nails. Radiographs demonstrate a generalized increase in bone density, but in contrast to osteopetrosis, the long bones are normally shaped. Separated cranial sutures, including the persistent patency of the anterior fontanel, are characteristic of the disorder. There may also be hypoplasia of the sinuses, mandible, distal clavicles, and terminal phalanges. Persistence of deciduous teeth and sclerosis of the calvarium and base of the skull are also common. Histologic evaluation shows normal cortical bone architecture with decreased osteoblastic and osteoclastic activities. Serum chemistries are normal, and unlike osteopetrosis, there is no anemia. There is no known treatment for this condition, and there are no reports of attempted bone marrow transplant.

1	Also known as Camurati-Engelmann disease, progressive diaphyseal dysplasia is an autosomal dominant disorder that is characterized radiographically by diaphyseal hyperostosis and a symmetric thickening and increased diameter of the endosteal and periosteal surfaces of the diaphyses of the long bones, particularly the femur and tibia, and, less often, the fibula, radius, and ulna. The genetic defect responsible for the disease has been localized to the area of chromosome 19q13.2 encoding tumor growth factor (TGF) β1. The mutation promotes activation of TGF-β1. The clinical severity is variable. The most common presenting symptoms are pain and tenderness of the involved areas, fatigue, muscle wasting, and gait disturbance. The weakness may be mistaken for muscular dystrophy. Characteristic body habitus includes thin limbs with little muscle mass yet prominent and palpable bones and, when the skull is involved, large head with prominent forehead and proptosis. Patients may also display

1	body habitus includes thin limbs with little muscle mass yet prominent and palpable bones and, when the skull is involved, large head with prominent forehead and proptosis. Patients may also display signs of cranial nerve palsies, hydrocephalus, central hypogonadism, and Raynaud’s phenomenon. Radiographically, patchy progressive endosteal and periosteal new bone formation is observed along the diaphyses of the long bones. Bone scintigraphy shows increased radiotracer uptake in involved areas.

1	Treatment with low-dose glucocorticoids relieves bone pain and may reverse the abnormal bone formation. Intermittent bisphosphonate therapy has produced clinical improvement in a limited number of patients.

1	This is also known as van Buchem’s disease; it is an autosomal recessive disorder characterized by endosteal hyperostosis in which osteosclerosis involves the skull, mandible, clavicles, and ribs. The major manifestations are due to narrowed cranial foramens with neural compressions that may result in optic atrophy, facial paralysis, and deafness. Adults may have an enlarged mandible. Serum ALP levels may be elevated, which reflect the uncoupled bone remodeling with high osteoblastic formation rates and low osteoclastic resorption. As a result, there is increased accumulation of normal bone. Endosteal hyperostosis with syndactyly, known as sclerosteosis, is a more severe form. The genetic defects for both sclerosteosis and van Buchem’s disease have been assigned to the same region of the chromosome 17q12-q21. It is possible that both conditions may have deactivating mutations in the BEER (bone-expressed equilibrium regulator) gene.

1	Melorheostosis (Greek, “flowing hyperostosis”) may occur sporadically or follow a pattern consistent with an autosomal recessive disorder. The major manifestation is progressive linear hyperostosis in one or more bones of one limb, usually a lower extremity. The name comes from the radiographic appearance of the involved bone, which resembles melted wax that has dripped down a candle. Symptoms appear during childhood as pain or stiffness in the area of sclerotic bone. There may be associated ectopic soft tissue masses, composed of cartilage or osseous tissue, and skin changes overlying the involved bone, consisting of scleroderma-like areas and hypertrichosis. The disease does not progress in adults, but pain and stiffness may persist. Laboratory tests are unremarkable. No specific etiology is known. There is no specific treatment. Surgical interventions to correct contractures are often unsuccessful.

1	The literal translation of osteopoikilosis is “spotted bones”; it is a benign autosomal dominant condition in which numerous small, variably shaped (usually round or oval) foci of bony sclerosis are seen in the epiphyses and adjacent metaphyses. The lesions may involve any bone except the skull, ribs, and vertebrae. They may be misidentified 426e-5 as metastatic lesions. The main differentiating points are that bony lesions of osteopoikilosis are stable over time and do not accumulate radionucleotide on bone scanning. In some kindred, osteopoikilosis is associated with connective tissue nevi known as dermatofibrosis lenticularis disseminata, also known as Buschke-Ollendorff syndrome. Histologic inspection reveals thickened but otherwise normal trabeculae and islands of normal cortical bone. No treatment is indicated.

1	Hepatitis C–associated osteosclerosis (HCAO) is a rare acquired diffuse osteosclerosis in adults with prior hepatitis C infection. After a latent period of several years, patients develop diffuse appendicular bone pain and a generalized increase in bone mass with elevated serum ALP. Bone biopsy and histomorphometry reveal increased rates of bone formation, decreased bone resorption with a marked decrease in osteoclasts, and dense lamellar bone. One patient had increased serum OPG levels, and bone biopsy showed large numbers of osteoblasts positive for OPG and reduced osteoclast number. Empirical therapy includes pain control, and there may be beneficial response to bisphosphonate. Long-term antiviral therapy may reverse the bone disease.

1	This is a rare inherited disorder that presents as rickets in infants and children or osteomalacia in adults with paradoxically low serum levels of ALP. The frequency of the severe neonatal and infantile forms is about 1 in 100,000 live births in Canada, where the disease is most common because of its high prevalence among Mennonites and Hutterites. It is rare in African Americans. The severity of the disease is remarkably variable, ranging from intrauterine death associated with profound skeletal hypomineralization at one extreme to premature tooth loss as the only manifestation in some adults. Severe cases are inherited in an autosomal recessive manner, but the genetic patterns are less clear for the milder forms. The disease is caused by a deficiency of tissue nonspecific (bone/liver/kidney) ALP (TNSALP), which, although ubiquitous, results only in bone abnormalities. Protein levels and functions of the other ALP isozymes (germ cell, intestinal, placental) are normal. Defective ALP

1	ALP (TNSALP), which, although ubiquitous, results only in bone abnormalities. Protein levels and functions of the other ALP isozymes (germ cell, intestinal, placental) are normal. Defective ALP permits accumulation of its major naturally occurring substrates including phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and pyridoxal 5′-phosphate (PLP). The accumulation of PPi interferes with mineralization through its action as a potent inhibitor of hydroxyapatite crystal growth.

1	Perinatal hypophosphatasia becomes manifest during pregnancy and is often complicated by polyhydramnios and intrauterine death. The infantile form becomes clinically apparent before the age of 6 months with failure to thrive, rachitic deformities, functional craniosynostosis despite widely open fontanels (which are actually hypomineralized areas of the calvarium), raised intracranial pressure, and flail chest with predisposition to pneumonia. Hypercalcemia and hypercalciuria are common. This form has a mortality rate of about 50%. Prognosis seems to improve for the children who survive infancy. Childhood hypophosphatasia has variable clinical presentation. Premature loss of deciduous teeth (before age 5) is the hallmark of the disease. Rickets causes delayed walking with waddling gait, short stature, and dolichocephalic skull with frontal bossing. The disease often improves during puberty but may recur in adult life. Adult hypophosphatasia presents during middle age with painful,

1	short stature, and dolichocephalic skull with frontal bossing. The disease often improves during puberty but may recur in adult life. Adult hypophosphatasia presents during middle age with painful, poorly healing metatarsal stress fractures or thigh pain due to femoral pseudofractures.

1	Laboratory investigation reveals low ALP levels and normal or elevated levels of serum calcium and phosphorus despite clinical and radiologic evidence of rickets or osteomalacia. Serum parathyroid hormone, 25-hydroxyvitamin D, and 1,25-dihydroxyvitamin D levels are normal. The elevation of PLP is specific for the disease and may even be present in asymptomatic parents of severely affected children. Because vitamin B6 increases PLP levels, vitamin B6 supplements should be discontinued 1 week before testing. Clinical testing is available to CHAPTER 426e Paget’s Disease and Other Dysplasias of Bone detect loss-of-function mutation(s) within the ALPL gene that encodes TNSALP.

1	CHAPTER 426e Paget’s Disease and Other Dysplasias of Bone detect loss-of-function mutation(s) within the ALPL gene that encodes TNSALP. There is no established medical therapy. In contrast to other forms of rickets and osteomalacia, calcium and vitamin D supplementation should be avoided because they may aggravate hypercalcemia and hypercalciuria. A low-calcium diet, glucocorticoids, and calcitonin have been used in a small number of patients with variable responses. Because fracture healing is poor, placement of intramedullary rods is best for acute fracture repair and for prophylactic prevention of fractures.

1	This is a rare disorder characterized by defective skeletal mineralization despite normal serum calcium and phosphate levels. Clinically, the disorder presents in middle-aged or elderly men with chronic axial skeletal discomfort. Cervical spine pain may also be present. Radiographic findings are mainly osteosclerosis due to coarsened trabecular patterns typical of osteomalacia. Spine, pelvis, and ribs are most commonly affected. Histologic changes show defective mineralization and flat, inactive osteoblasts. The primary defect appears to be an acquired defect in osteoblast function. The course is benign, and there is no established treatment. Calcium and vitamin D therapies are not effective.

1	This is a rare condition of unknown etiology. It presents in both sexes; in middle age or later; and with progressive, intractable skeletal pain and fractures; worsening immobilization; and a debilitating course. Radiographic evaluation reveals generalized osteomalacia, osteopenia, and occasional pseudofractures. Histologic features include a tangled pattern of collagen fibrils with abundant osteoblasts and osteoclasts. There is no effective treatment. Spontaneous remission has been reported in a small number of patients. Calcium and vitamin D have not been beneficial.

1	Fibrous dysplasia is a sporadic disorder characterized by the presence of one (monostotic) or more (polyostotic) expanding fibrous skeletal lesions composed of bone-forming mesenchyme. The association of the polyostotic form with café au lait spots and hyperfunction of an endocrine system such as pseudoprecocious puberty of ovarian origin is known as McCune-Albright syndrome (MAS). A spectrum of the phenotypes is caused by activating mutations in the GNAS1 gene, which encodes the α subunit of the stimulatory G protein (Gsα). As the postzygotic mutations occur at different stages of early development, the extent and type of tissue affected are variable and explain the mosaic pattern of skin and bone changes. GTP binding activates the Gsα regulatory protein and mutations in regions of Gsα that selectively inhibit GTPase activity, which results in constitutive stimulation of the cyclic AMP–protein kinase A signal transduction pathway. Such mutations of the Gsα protein–coupled receptor

1	that selectively inhibit GTPase activity, which results in constitutive stimulation of the cyclic AMP–protein kinase A signal transduction pathway. Such mutations of the Gsα protein–coupled receptor may cause autonomous function in bone (parathyroid hormone receptor); skin (melanocyte-stimulating hormone receptor); and various endocrine glands including ovary (follicle-stimulating hormone receptor), thyroid (thyroid-stimulating hormone receptor), adrenal (adrenocorticotropic hormone receptor), and pituitary (growth hormone–releasing hormone receptor). The skeletal lesions are composed largely of mesenchymal cells that do not differentiate into osteoblasts, resulting in the formation of imperfect bone. In some areas of bone, fibroblast-like cells develop features of osteoblasts in that they produce extracellular matrix that organizes into woven bone. Calcification may occur in some areas. In other areas, cells have features of chondrocytes and produce cartilage-like extracellular

1	they produce extracellular matrix that organizes into woven bone. Calcification may occur in some areas. In other areas, cells have features of chondrocytes and produce cartilage-like extracellular matrix.

1	Clinical Presentation Fibrous dysplasia occurs with equal frequency in both sexes, whereas MAS with precocious puberty is more common (10:1) in girls. The monostotic form is the most common and is usually diagnosed in patients between 20 and 30 years of age without associated skin lesions. The polyostotic form typically manifests in children <10 years old and may progress with age. Early-onset disease is generally more severe. Lesions may become quiescent in puberty and progress during pregnancy or with estrogen therapy. In polyostotic fibrous dysplasia, the lesions most commonly involve the maxilla and other craniofacial bones, ribs, and metaphyseal or diaphyseal portions of the proximal femur or tibia. Expanding bone lesions may cause pain, deformity, fractures, and nerve entrapment. Sarcomatous degeneration involving the facial bones or femur is infrequent (<1%). The risk of malignant transformation is increased by radiation, which has proven to be ineffective treatment. In rare

1	Sarcomatous degeneration involving the facial bones or femur is infrequent (<1%). The risk of malignant transformation is increased by radiation, which has proven to be ineffective treatment. In rare patients with widespread lesions, renal phosphate wasting and hypophosphatemia may cause rickets or osteomalacia. Hypophosphatemia may be due to production of a phosphaturic factor by the abnormal fibrous tissue.

1	MAS patients may have café au lait spots, which are flat, hyperpigmented skin lesions that have rough borders (“coast of Maine”) in contrast to the café au lait lesions of neurofibromatosis that have smooth borders (“coast of California”). The most common endocrinopathy is isosexual pseudoprecocious puberty in girls. Other less common endocrine disorders include thyrotoxicosis, Cushing’s syndrome, acromegaly, hyperparathyroidism, hyperprolactinemia, and pseudoprecocious puberty in boys.

1	radiographic Findings In long bones, the fibrous dysplastic lesions are typically well-defined, radiolucent areas with thin cortices and a ground-glass appearance. Lesions may be lobulated with trabeculated areas of radiolucency (Fig. 426e-4). Involvement of facial bones usually presents as radiodense lesions, which may create a leonine appearance (leontiasis osea). Expansile cranial lesions may narrow foramens and cause optic lesions, reduce hearing, and create other manifestations of cranial nerve compression. Laboratory results Serum ALP is occasionally elevated but calcium, parathyroid hormone, 25-hydroxyvitamin D, and 1,25-dihydroxyvitamin D levels are normal. Patients with extensive polyostotic lesions may have hypophosphatemia, hyperphosphaturia, and osteomalacia. The hypophosphatemia and phosphaturia are directly related to the levels of fibroblast growth factor 23 (FGF23). Biochemical markers of bone turnover may be elevated.

1	FIGurE 426e-4 Radiograph of a 16-year-old male with fibrous dysplasia of the right proximal femur. Note the multiple cystic lesions, including the large lucent lesion in the proximal midshaft with scalloping of the interior surface. The femoral neck contains two lucent cystic lesions. Spontaneous healing of the lesions does not occur, and there is no established effective treatment. Improvement in bone pain and partial or complete resolution of radiographic lesions have been reported after IV bisphosphonate therapy. Surgical stabilization is used to prevent pathologic fracture or destruction of a major joint space and to relieve nerve root or cranial nerve compression or sinus obstruction.

1	Pachydermoperiostosis, or hypertrophic osteoarthropathy (primary or idiopathic), is an autosomal dominant disorder characterized by periosteal new bone formation that involves the distal extremities. The lesions present as clubbing of the digits and hyperhidrosis and thickening of the skin, primarily of the face and forehead. The changes usually appear during adolescence, progress over the next decade, and then become quiescent. During the active phase, progressive enlargement of the hands and feet produces a paw-like appearance, which may be mistaken for acromegaly. Arthralgias, pseudogout, and limited mobility may also occur. The disorder must be differentiated from secondary hypertrophic osteopathy that develops during the course of serious pulmonary disorders. The two conditions can be differentiated by standard radiography of the digits in which secondary pachydermoperiostosis has exuberant periosteal new bone formation and a smooth and undulating surface. In contrast, primary

1	be differentiated by standard radiography of the digits in which secondary pachydermoperiostosis has exuberant periosteal new bone formation and a smooth and undulating surface. In contrast, primary hypertrophic osteopathy has an irregular periosteal surface.

1	There are no diagnostic blood or urine tests. Synovial fluid does not have an inflammatory profile. There is no specific therapy, although a limited experience with colchicine suggests some benefit in controlling the arthralgias. These include several hundred heritable disorders of connective tissue. These primary abnormalities of cartilage manifest as disturbances in cartilage and bone growth. Selected growth-plate chondrodysplasias are described here. For discussion of chondrodysplasias, see Chap. 427.

1	Achondrodysplasia This is a relatively common form of short-limb dwarfism that occurs in 1 in 15,000 to 1 in 40,000 live births. The disease is caused by a mutation of the fibroblast growth factor receptor 3 (FGFR3) gene that results in a gain-of-function state. Most cases are sporadic mutations. However, when the disorder appears in families, the inheritance pattern is consistent with an autosomal dominant disorder. The primary defect is abnormal chondrocyte proliferation at the growth plate that causes development of short, but proportionately thick, long bones. Other regions of the long bones may be relatively unaffected. The disorder is manifest by the presence of short limbs (particularly the proximal portions), normal trunk, large head, saddle nose, and an exaggerated lumbar lordosis. Severe spinal deformity may lead to cord compression. The homozygous disorder is more serious than the sporadic form and may cause neonatal death. Pseudoachondroplasia clinically resembles

1	lordosis. Severe spinal deformity may lead to cord compression. The homozygous disorder is more serious than the sporadic form and may cause neonatal death. Pseudoachondroplasia clinically resembles achondrodysplasia but has no skull abnormalities.

1	Enchondromatosis This is also called dyschondroplasia or Ollier’s disease; it is also a disorder of the growth plate in which the primary cartilage is not resorbed. Cartilage ossification proceeds normally, but it is not resorbed normally, leading to cartilage accumulation. The changes are most marked at the ends of long bones, where the highest growth rates occur. Chondrosarcoma develops infrequently. The association of enchondromatosis and cavernous hemangiomas of the skin and soft tissues is known as Maffucci’s syndrome. Both Ollier’s disease and Maffucci’s syndrome are associated with various malignancies, including granulosa cell tumor of the ovary and cerebral glioma.

1	Multiple Exostoses This is also called diaphyseal aclasis or osteochon-426e-7 dromatosis; it is a genetic disorder that follows an autosomal dominant pattern of inheritance. In this condition, areas of growth plates become displaced, presumably by growing through a defect in the perichondrium. The lesion begins with vascular invasion of the growth-plate cartilage, resulting in a characteristic radiographic finding of a mass that is in direct communication with the marrow cavity of the parent bone. The underlying cortex is resorbed. The disease is caused by inactivating mutations of the EXT1 and EXT2 genes, whose products normally regulate processing of chondrocyte cytoskeletal proteins. The products of the EXT gene likely function as tumor suppressors, with the loss-of-function mutation resulting in abnormal proliferation of growth-plate cartilage. Solitary or multiple lesions are located in the metaphyses of long bones. Although usually asymptomatic, the lesions may interfere with

1	resulting in abnormal proliferation of growth-plate cartilage. Solitary or multiple lesions are located in the metaphyses of long bones. Although usually asymptomatic, the lesions may interfere with joint or tendon function or compress peripheral nerves. The lesions stop growing when growth ceases but may recur during pregnancy. There is a small risk for malignant transformation into chondrosarcoma.

1	Deposition of calcium phosphate crystals (calcification) or formation of true bone (ossification) in nonosseous soft tissue may occur by one of three mechanisms: (1) metastatic calcification due to a supranormal calcium × phosphate concentration product in extracellular fluid; (2) dystrophic calcification due to mineral deposition into metabolically impaired or dead tissue despite normal serum levels of calcium and phosphate; and (3) ectopic ossification, or true bone formation. Disorders that may cause extraskeletal calcification or ossification are listed in Table 426e-2.

1	Soft tissue calcification may complicate diseases associated with significant hypercalcemia, hyperphosphatemia, or both. In addition, vitamin D and phosphate treatments or calcium administration in the presence of mild hyperphosphatemia, such as during hemodialysis, may induce ectopic calcification. Calcium phosphate precipitation may complicate any disorder when the serum calcium × phosphate concentration product is >75. The initial calcium phosphate deposition is in the form of small, poorly organized crystals, which subsequently organize into hydroxyapatite crystals. Calcifications that occur in hypercalcemic states with normal or low phosphate have a predilection for kidney, lungs, and gastric mucosa. Hyperphosphatemia with normal or low serum calcium may promote soft tissue calcification with predilection for the kidney and arteries. The disturbances of calcium and phosphate in renal failure and hemodialysis are common causes of soft tissue (metastatic) calcification.

1	Therapy with vitamin D and phosphate CHAPTER 426e Paget’s Disease and Other Dysplasias of Bone

1	This is a rare genetic disorder characterized by masses of metastatic calcifications in soft tissues around major joints, most often shoulders, hips, and ankles. Tumoral calcinosis differs from other disorders in that the periarticular masses contain hydroxyapatite crystals or amorphous calcium phosphate complexes, while in fibrodysplasia ossificans progressiva (below), true bone is formed in soft tissues. About one-third of tumoral calcinosis cases are familial, with both autosomal recessive and autosomal dominant modes of inheritance reported. The disease is also associated with a variably expressed abnormality of dentition marked by short bulbous roots, pulp calcification, and radicular dentin deposited in swirls. The primary defect responsible for the metastatic calcification appears to be hyperphosphatemia resulting from the increased capacity of the renal tubule to reabsorb filtered phosphate. Spontaneous soft tissue calcification is related to the elevated serum phosphate,

1	to be hyperphosphatemia resulting from the increased capacity of the renal tubule to reabsorb filtered phosphate. Spontaneous soft tissue calcification is related to the elevated serum phosphate, which, along with normal serum calcium, exceeds the concentration product of 75.

1	All of the North American patients reported have been African American. The disease usually presents in childhood and continues throughout the patient’s life. The calcific masses are typically painless and grow at variable rates, sometimes becoming large and bulky. The masses are often located near major joints but remain extracapsular. Joint range of motion is not usually restricted unless the tumors are very large. Complications include compression of neural structures and ulceration of the overlying skin with drainage of chalky fluid and risk of secondary infection. Small deposits not detected by standard radiographs may be detected by 99mTc bone scanning. The most common laboratory findings are hyperphosphatemia and elevated serum 1,25-dihydroxyvitamin D levels. Serum calcium, parathyroid hormone, and ALP levels are usually normal. Renal function is also usually normal. Urine calcium and phosphate excretions are low, and calcium and phosphate balances are positive.

1	An acquired form of the disease may occur with other causes of hyperphosphatemia, such as secondary hyperparathyroidism associated with hemodialysis, hypoparathyroidism, pseudohypoparathyroidism, and massive cell lysis following chemotherapy for leukemia. Tissue trauma from joint movement may contribute to the periarticular calcifications. Metastatic calcifications are also seen in conditions associated with hypercalcemia, such as in sarcoidosis, vitamin D intoxication, milk-alkali syndrome, and primary hyperparathyroidism. In these conditions, however, mineral deposits are more likely to occur in proton-transporting organs such as kidney, lungs, and gastric mucosa in which an alkaline milieu is generated by the proton pumps.

1	Therapeutic successes have been achieved with surgical removal of subcutaneous calcified masses, which tend not to recur if all calcification is removed from the site. Reduction of serum phosphate by chronic phosphorus restriction may be accomplished using low dietary phosphorus intake alone or in combination with oral phosphate binders. The addition of the phosphaturic agent acetazolamide may be useful. Limited experience using the phosphaturic action of calcitonin deserves further testing.

1	Posttraumatic calcification may occur with normal serum calcium and phosphate levels and normal ion-solubility product. The deposited mineral is either in the form of amorphous calcium phosphate or hydroxyapatite crystals. Soft tissue calcification complicating connective tissue disorders such as scleroderma, dermatomyositis, and systemic lupus erythematosus may involve localized areas of the skin or deeper subcutaneous tissue and is referred to as calcinosis circumscripta. Mineral deposition at sites of deeper tissue injury including periarticular sites is called calcinosis universalis.

1	True extraskeletal bone formation that begins in areas of fasciitis following surgery, trauma, burns, or neurologic injury is referred to as myositis ossificans. The bone formed is organized as lamellar or trabecular, with normal osteoblasts and osteoclasts conducting active remodeling. Well-developed haversian systems and marrow elements may be present. A second cause of ectopic bone formation occurs in an inherited disorder, fibrodysplasia ossificans progressiva.

1	This is also called myositis ossificans progressiva; it is a rare autosomal dominant disorder characterized by congenital deformities of the hands and feet and episodic soft tissue swellings that ossify. Ectopic bone formation occurs in fascia, tendons, ligaments, and connective tissue within voluntary muscles. Tender, rubbery induration, sometimes precipitated by trauma, develops in the soft tissue and gradually calcifies. Eventually, heterotopic bone forms at these sites of soft tissue trauma. Morbidity results from heterotopic bone interfering with normal movement and function of muscle and other soft tissues. Mortality is usually related to restrictive lung disease caused by an inability of the chest to expand. Laboratory tests are unremarkable.

1	There is no effective medical therapy. Bisphosphonates, glucocorticoids, and a low-calcium diet have largely been ineffective in halting progression of the ossification. Surgical removal of ectopic bone is not recommended, because the trauma of surgery may precipitate formation of new areas of heterotopic bone. Dental complications including frozen jaw may occur following injection of local anesthetics. Thus, CT imaging of the mandible should be undertaken to detect early sites of soft tissue ossification before they are appreciated by standard radiography.

1	Heritable Disorders of Connective Tissue Darwin J. Prockop, John F. Bateman CLASSIFICATION OF CONNECTIVE TISSUE DISORDERS Some of the most common conditions that are transmitted genetically 427 SEC Tion 5 Endocrinology and Metabolism in families are disorders that produce clinically obvious changes in the skeleton, skin, or other relatively acellular tissues that have been loosely defined as connective tissues. Because of their heritability, the disorders were recognized as potentially traceable to mutated genes soon after the principles of genetics were introduced into medicine. In the last several decades, many of these disorders have been linked to mutations in several hundred different genes. However, classifying the disorders on the basis of either their clinical presentations or the mutations causing them presents a challenge for both the clinician and the geneticist.

1	A major development in the field was made by McKusick, who suggested that a group of disorders that included brittle bones in children (osteogenesis imperfecta), hyperextensible skin (Ehlers-Danlos syndrome), and characteristic distortions of skeleton (Marfan’s syndrome) be considered as “heritable disorders of connective tissue” and that mutations causing the disorders would be found in the genes coding for proteins of the tissues.

1	The information on the disorders has continued to develop on two levels. The initial clinical classifications suggested by McKusick, and others, had to be refined as additional patients were examined. For example, some patients had skin changes similar to those commonly seen in Ehlers-Danlos syndrome, but this feature was overshadowed by other features such as extreme hypotonia or sudden rupture of large blood vessels. To account for the full spectrum of presentations in patients and families, many of the disorders have been reclassified several times, and each has been divided into a series of subtypes. For example, a recent effort to classify all the heritable disorders that alter the skeleton defined 456 distinctive conditions that were divided into 40 major groups.

1	The identification of mutations causing the diseases has developed on a parallel track. The first genes cloned for connective tissues were the two genes coding for type I collagen, the most abundant protein in bones, skin, tendons, and several other tissues. Some of the first assays in patients with osteogenesis imperfecta (OI) revealed mutations in type I collagen genes. Biochemical data developed using cultures of skin fibroblasts from affected patients demonstrated that the mutations dramatically altered the synthesis or structure of collagen fibers. The results stimulated efforts to identify additional mutations in genes coding for structural proteins. Genes for collagens provided an attractive paradigm to search for mutations, since a series of different types of collagens were found in different connective tissues and the collagen genes were readily isolated by their unique signature sequences. Also, the collagen genes are particularly vulnerable to a large number different

1	in different connective tissues and the collagen genes were readily isolated by their unique signature sequences. Also, the collagen genes are particularly vulnerable to a large number different mutations because of unusual structural requirements of the protein. The search for mutations in collagen genes proved fruitful in that mutations were found in most patients with OI, in many patients with hyperextensible skin, in some patients with dwarfism, and in patients with other disorders, including Alport’s syndrome, that were not initially classified as disorders of connective tissue. Also, mutations in collagen genes were found in subset of patients with a diagnosis of osteoarthritis and a subset of patients with the diagnosis of osteoporosis. However, the search for mutations quickly expanded to hundreds of other genes that included those for other structural proteins, for the posttranslational processing of structural proteins, and for growth factors and their receptors, and other

1	to hundreds of other genes that included those for other structural proteins, for the posttranslational processing of structural proteins, and for growth factors and their receptors, and other genes whose functions are still not fully understood.

1	In many instances, the mutations helped to define the clinical subtype of the disorder. In others, however, they did not. Some patients with the same clinical presentations were found to have mutations in different genes. Also, some patients with different manifestations were found to have mutations in the same genes. In addition, it was difficult to establish whether a change in the structure of a gene caused the phenotypic changes in patients and was not simply a neutral polymorphism. Therefore, there has been a continuing debate as to whether the disorders should be classified by their clinical presentations or by the genetic abnormalities. As an illustration of the problem, mutations in 226 genes have been found to be associated with the 456 defined disorders of the skeleton, but the latest nosology remains a “hybrid” between a list of clinically defined disorders, waiting for molecular clarification, and an annotated database documenting the phenotypic spectrum pro-2505 duced by

1	latest nosology remains a “hybrid” between a list of clinically defined disorders, waiting for molecular clarification, and an annotated database documenting the phenotypic spectrum pro-2505 duced by mutations in a given gene. A simpler system of classification proved feasible for one rare heritable disorder of skin, epidermolysis bullosa. The disorder was first defined by the presence of friction-induced blister. It was then divided into subtypes that were defined by the ultrastructural layers of the skin that cleaved and blistered. Most patients in each subtype were subsequently shown to have mutations in genes expressed in the corresponding layer of skin. Even with these patients, however, the strength of the genotype-phenotype correlation varies, and mutations have not yet been found in every patient.

1	In the end, consensus reports by experts in the field and sources such as the Online Mendelian Inheritance in Man database provide valuable resources for physicians searching for diagnoses of patients with unusual clinical features. However, patients with the most common forms of the disorders have mutations in a limited number of genes. This chapter will focus primarily on these more common disorders.

1	Connective tissues such as skin, bone, cartilage, ligaments, and tendons are the critical structural frameworks of the body important for development and function. They consist of a complex interacting extracellular matrix network of collagens, proteoglycans, and a large number of noncollagenous glycoproteins and proteins. Although these precise combinations of up to ~500 potential extracellular matrix building blocks provide tissue-specific function, there are many overarching similarities in composition, such as the role of composite collagen fibrils in providing strength and form, elastin fibrils and proteoglycans and other interacting proteins, and glycoproteins that fine-tune function (Table 427-1). The most abundant components are three similar fibrillar collagens (types I, II, and III). They have a similar tensile

1	Heritable Disorders of Connective Tissue aOver 30 proteoglycans have been identified. They differ in the structures of their core proteins and their contents of glycosaminoglycan side chains of chondroitin-4-sulfate, chondroitin6-sulfate, dermatan sulfate, and keratin sulfate. Basal lamina contains a proteoglycan with a side chain of heparan sulfate that resembles heparin. 2506 strength as steel wires. The three fibrillar collagens are distributed in a tissue-specific manner: type I collagen accounts for most of the protein of dermis, ligaments, tendons, and demineralized bone; type I and type III are the most abundant proteins of large blood vessels; and type II is the most abundant protein of cartilage.

1	Connective tissues are among the most stable components in living organisms, but they are not inert. During embryonic development, connective tissue membranes appear as early as the four-cell blastocyst to provide strength and a structural scaffold for the developing embryo. With the development of blood vessels and skeleton, there is a rapid increase in the synthesis, degradation, and resynthesis of connective tissues. The turnover continues at a slower, but still rapid pace throughout postnatal development and then spikes during the growth spurt of puberty. During adulthood, the metabolic turnover of most connective tissues is slow, but it continues at a moderate pace in bone. With age, malnutrition, physical inactivity, and low gravitational stress, the rate of degradation of most connective tissues, especially in bone and skin, begins to exceed the rate of synthesis and the tissues shrink. In starvation, a large fraction of the collagen in skin and other connective tissues is

1	connective tissues, especially in bone and skin, begins to exceed the rate of synthesis and the tissues shrink. In starvation, a large fraction of the collagen in skin and other connective tissues is degraded and provides amino acids for gluconeogenesis (Chap. 97). In both osteoarthritis and rheumatoid arthritis, there is extensive degradation of articular cartilage collagen. Glucocorticoids weaken most tissues by decreasing collagen synthesis. In many pathologic states, however, collagen is deposited in excess. With most injuries to tissues, inflammatory and immune responses stimulate the deposition of collagen fibrils in the form of fibrotic scars. The deposition of the fibrils is largely irreversible and prevents regeneration of normal tissues in hepatic cirrhosis, pulmonary fibrosis, atherosclerosis, and nephrosclerosis.

1	Structure and Biosynthesis of Fibrillar Collagens The tensile strength of collagen fibers derives primarily from the self-assembly of protein monomers into large fibril structures in a process that resembles crystallization. The self-assembly requires monomers of highly uniform and relatively rigid structure. It also requires a complex series of post-translational processing steps that maintain the solubility of the monomers until they are transported to the appropriate extracellular sites for fibril assembly. Because of the stringent requirements for correct self-assembly, it is not surprising that mutations in genes for fibrillar collagens cause many of the diseases of connective tissues.

1	The monomers of the three fibrillar collagens are formed from three polypeptide chains, called α chains, that are wrapped around each other into a rope-like triple-helical conformation. The triple helix is a unique structure among proteins, and it provides rigidity to the molecule. It also orients the side chains of amino acids in an “inside out” manner relative to most other proteins so that the charged and hydrophobic residues on the surface can direct self-assembly of the monomers into fibrils. The triple-helical conformation of the monomer is generated because each of the α chains has a repetitive amino acid sequence in which glycine (Gly) appears as every third amino acid. Each α chain contains about 1000 amino acids. Therefore, the sequence of each α chain can be designated as (-Gly-X-Y-)n, where X and Y represent amino acids other than glycine and n is >338. The presence of glycine, the smallest amino acid, in every third position in the sequence is critical because this

1	(-Gly-X-Y-)n, where X and Y represent amino acids other than glycine and n is >338. The presence of glycine, the smallest amino acid, in every third position in the sequence is critical because this residue must fit in a sterically restricted space in the middle of the helix where the three chains come together. The requirement for a glycine residue at every third position explains the severe effects of mutations that convert any of the glycine residues to an amino acid with a bulkier side chain (see below). Many of the Xand Y-position amino acids are proline and hydroxyproline, which, because of their ring structures, provide additional rigidity to the triple helix. Other Xand Y-positions are occupied by charged or hydrophobic amino acids that precisely direct lateral and longitudinal assembly of the monomers into highly ordered fibrils. Mutations that substitute amino acids in some Xand Y-positions can, in rare instances, also produce genetic diseases.

1	The fibers formed by the three fibrillar collagens differ in thickness and length, but they have a similar fine structure. As viewed by electron microscopy, they all have a characteristic pattern of cross-striations that are about one-quarter the length of the monomers and reflect the precise packing into fibrils. The three fibrillar collagens, however, differ in sequences found in the Xand Y-positions of the α chains and therefore in some of their physical properties. Type I collagen is composed of two identical α1(I) chains and a third α2(I) chain that differs slightly in its amino acid sequence. Type II collagen is composed of three identical α(II) chains. Type III collagen is composed of three identical α1(III) chains.

1	To deliver a monomer of the correct structure to the appropriate site of fibril assembly, the biosynthesis of fibrillar collagens involves a large number of unique processing steps (Fig. 427-1). The monomer, first synthesized as a soluble precursor called procollagen, contains an additional globular domain at each end. As the proα chains of procollagen are synthesized on ribosomes, the free N-terminal ends move into the cisternae of the rough endoplasmic reticulum. Signal peptides at the N-termini are cleaved, and additional posttranslational reactions begin. Proline residues in the Y-position of the repeating -Gly-X-Ysequences are converted to hydroxyproline by the enzyme prolyl hydroxylase. The hydroxylation of prolyl residues is essential for the three α chains of the monomer to fold into a triple helix at body temperature. The enzyme requires ascorbic acid as one of its essential cofactors, an observation that explains why wounds fail to heal in scurvy (Chap. 96e). In scurvy, some

1	a triple helix at body temperature. The enzyme requires ascorbic acid as one of its essential cofactors, an observation that explains why wounds fail to heal in scurvy (Chap. 96e). In scurvy, some of the underhydroxylated and unfolded protein accumulates in the cisternae of the rough endoplasmic reticulum and is degraded. Lysine residues in the Y-position are also hydroxylated to hydroxylysine by a separate lysyl hydroxylase. Many of the hydroxylysine residues are glycosylated with galactose or with galactose and glucose. A large mannose-rich oligosaccharide is assembled on the C-terminal propeptide of each chain. The proα chains are assembled by interactions among these C-terminal propeptides that control the selection of the appropriate partner chains to form heteroor homotrimers and provide the correct chain registration required for subsequent formation of the collagen triple helix. After the C-terminal propeptides assemble the three proα chains, a nucleus of triple helix is

1	provide the correct chain registration required for subsequent formation of the collagen triple helix. After the C-terminal propeptides assemble the three proα chains, a nucleus of triple helix is formed near the C terminus, and the helical conformation is propagated toward the N terminus in a zipper-like manner that resembles crystallization. The folding into the triple helix is spontaneous in solution, but as discussed below, identification of rare mutations causing OI demonstrated that the folding in cellulo is assisted by ancillary proteins. The fully folded protein is then secreted. After secretion, procollagen is processed to collagen by cleavage of the N-propeptides and C-propeptides by two specific proteinases. The release of the propeptides decreases the solubility of the protein about 1000-fold. The entropic energy that is released drives the self-assembly of the collagen into fibrils. Self-assembled collagen fibers have considerable tensile strength, but their strength is

1	about 1000-fold. The entropic energy that is released drives the self-assembly of the collagen into fibrils. Self-assembled collagen fibers have considerable tensile strength, but their strength is increased further by cross-linking reactions that form covalent bonds between α chains in one molecule and α chains in adjacent molecules.

1	Although the assembly of collagen monomers into fibers is a spontaneous reaction, the process in tissues is modulated by the presence of less abundant collagens (type V with type I, and type XI with type II) and by other components such as a series of small leucine-rich proteins (SLRPs). Some of the less abundant components alter the rate of fibril assembly, whereas others change the morphology of the fibers or their interactions with cells and other molecules. Collagen fibers are resistant to most proteases, but during degradation of connective tissues, they are cleaved by specific matrix metalloproteinases (collagenases) that cause partial unfolding of the triple helices into gelatin-like structures that are further degraded by less specific proteinases. The unique properties of the triple helix are used to define a family of at least 28 collagens that contain repetitive -Gly-X-Ysequences and form triple helices of varying length and complexity. The proteins are

1	O-Gal-Glc-Gal-GlcOHOHOHOHOHOHOHOHOHOH OH OH OH OH OH OH OHOH Glc Gal O OH OH OHOH OH OH OH OH OH O Gal (Man)n GlcNAc S S S S Glc Gal O OH OH OHOH OH OH OH OH OH O Gal (Man)n GlcNAc SH SH SH SH O-Gal Assembly of three procollagen chains Polypeptide synthesis Collagen prolyl 4-hydroxylase Lysyl hydroxylase Prolyl 3-hydroxylase Collagen gal-transferase and glc-transferase N glycosylated residue Protein disulfide isomerase Assembly of triple helix Secretion of procollagen in transport vesicles Endoplasmic reticulum Late transport vesicles and extracellular matrix FIGURE 427-1 Schematic summary of biosynthesis of fibrillar collagens. (Modified and reproduced with permission from J Myllyharju, KI Kivirikko: Trends in Genetics 20:33, 2004.) Cleavage of propeptides

1	Cleavage of propeptides Formation of covalent cross-links heterogeneous both in structure and function, and many are the sites of mutations causing genetic diseases. For example, the type IV collagen found in basement membranes is composed of three α chains synthesized from any of six different genes. Mutations in any of the six genes can cause Alport’s syndrome.

1	Fibrillin Aggregates and Elastin In addition to tensile strength, many tissues such as the lung, large blood vessels, and ligaments require elasticity. The elasticity was originally ascribed to an amorphous rubber-like protein named elastin. Subsequent analyses, largely sparked by discoveries of mutations causing the Marfan’s syndrome (MFS), demonstrated that the elasticity resided in thin fibrils composed primarily of large glycoproteins named fibrillins. The fibrillins contain large numbers of epidermal growth factor–like domains interspersed with characteristic cysteine-rich domains that are also found in latent transforming growth factor β (TGF-β) binding proteins. The fibrillins assemble into long beadlike strands that also contain numerous other components including small and variable amounts of elastin, bone morphogenic proteins (BMPs), and microfibril-associated glycoproteins (MAGPs). The principles whereby the fibrils provide elasticity to tissue and their biosynthetic

1	variable amounts of elastin, bone morphogenic proteins (BMPs), and microfibril-associated glycoproteins (MAGPs). The principles whereby the fibrils provide elasticity to tissue and their biosynthetic assembly are still under investigation. As well as contributing to extracellular matrix structure, the fibrillins play a major role in TGF-β signaling.

1	Proteoglycans The resiliency to compression of connective tissues such as cartilage or the aorta is largely explained by the presence of proteoglycans. Proteoglycans are composed of a core protein to which are attached a large series of negatively charged polymers of disaccharides (largely chondroitin sulfates). At least 30 proteoglycans have been identified. They vary in their binding to collagens and other components of matrix, but specific functions have not been assigned to most. The major proteoglycan of cartilage, called aggrecan, has a core protein of 2000 amino acids that is decorated with about 100 side chains of chondroitin sulfate and keratin sulfate. The core protein, in turn, binds to long chains of the polymeric disaccharide hyaluronan to form proteoglycan aggregates, one of the largest soluble macromolecular structures in nature. Because of its highly negative charge and extended structure, the proteoglycan aggregate binds large amounts of water and small ions to

1	one of the largest soluble macromolecular structures in nature. Because of its highly negative charge and extended structure, the proteoglycan aggregate binds large amounts of water and small ions to distend the three-dimensional arcade of collagen fibers found in the same tissues. It thereby makes the cartilage resilient to pressure.

1	The central feature of OI is a severe decrease in bone mass that makes bones brittle. The disorder is frequently associated with blue sclerae, dental abnormalities (dentinogenesis imperfecta), progressive hearing loss, and a positive family history. Most patients have mutations in one of the two genes coding for type I collagen. Classification OI was originally classified into two subtypes of congenita and tarda depending on the age of onset of the symptoms. Sillence suggested a series of subtypes based on clinical and radiologic findings and mode of inheritance. As with the other disorders discussed here, the description of rare recessive forms of OI and discovery of mutations in new genes have opened a debate as to whether the disorders should be classified by the clinical phenotypes or by the genes at fault. For the near term, the classification based on the clinical presentations seems the most useful (Table 427-2).

1	Type I is the mildest subtype and can produce either mild or no apparent deformities of the skeleton. Most patients have distinctly blue sclerae. Type II produces bone so brittle that it is lethal in utero or shortly after birth; it can be subclassified into types IIA, IIB, and IIC, depending on radiologic findings. Of the nonlethal forms, type III is progressively deforming with moderate to severe bone deformity, and type IV (common variable OI with normal sclerae) has mild to moderate bone fragility. The classifications of patients by types of OI do not consistently predict the clinical course of the disease. Some patients appear normal at birth and become progressively worse; others have multiple fractures in infancy and childhood, improve after puberty, and fracture more frequently later in life. Women are particularly prone to fracture during Heritable Disorders of Connective Tissue Nondeforming OI with I blue sclerae Common variable OI IV with normal sclerae

1	Heritable Disorders of Connective Tissue Nondeforming OI with I blue sclerae Common variable OI IV with normal sclerae OI with calcification of V the interosseous membranes Bruck syndrome type 2 Mild to moderate bone fragility, AD normal or near-normal stature, blue sclerae, normal dentition in most, hearing loss in ~50% Extreme bone fragility, short stature, AD long bone bowing, blue sclerae Normal/pale blue sclerae, normal AR Moderate to severe bone deformity, AD blue sclerae at birth, hearing loss and abnormal dentition common Mild to moderate, bone fragility, AD normal sclerae, variable dentition, hearing loss in <10% Calcification of the interosseous AD membranes in forearm and legs and/or hypertrophic callus; variable bone deformity, normal sclerae and dentition Contractures with pterygia, fractures AR in infancy or early childhood, postnatal short stature, severe limb deformity, and progressive scoliosis

1	Contractures with pterygia, fractures AR in infancy or early childhood, postnatal short stature, severe limb deformity, and progressive scoliosis Abbreviations: AD, autosomal dominant; AR, autosomal recessive. Note: Predominant OI gene mutations (>90%) are in COL1A1 and COL1A2 (in bold typeface). pregnancy and after menopause. A few women from families with mild variants of OI do not develop fractures until after menopause, and their disease may be difficult to distinguish from postmenopausal osteoporosis. Incidence Type I OI has a frequency of about 1 in 15,000–20,000 births. Type II OI has a reported incidence of about 1 in 60,000. Only a limited number of patients with the severe forms of OI have been reported, and the combined incidence of the severe forms that are recognizable at birth (types II, III, and IV) may be higher than 1 in 60,000.

1	Skeletal Effects In type I OI, the fragility of bones may be severe enough to limit physical activity or be so mild that individuals are unaware of any disability. Radiographs of the skull in patients with mild disease may show a mottled appearance because of small islands of irregular ossification. In type II OI, ossification of many bones is frequently incomplete. Continuously beaded or broken ribs and crumpled long bones (accordina femora) may be present. For reasons that are not apparent, the long bones may be either thick or thin. In types III and IV, multiple fractures from minor physical stress can produce severe deformities. Kyphoscoliosis can impair respiration, cause cor pulmonale, and predispose to pulmonary infections. The appearance of “popcorn-like” deposits of mineral in x-rays of the ends of long bones is an ominous sign. Progressive neurologic symptoms may Proteolytic removal of procollagen N-propeptide PEDF growth factor signaling? Cation channel, Ca2+ release

1	Proteolytic removal of procollagen N-propeptide PEDF growth factor signaling? Cation channel, Ca2+ release Transcription factor, bone formation defect Transcription factor, bone formation defect Collagen posttranslational modification of lysine result from basilar compression and communicating hydrocephalus. Type V OI is recognized by the presence of dislocated radial heads and hyperplastic callus formation. In all forms of OI, bone mineral density is decreased. However, the degree of osteopenia may be difficult to evaluate because recurrent fractures limit exercise and thereby diminish bone mass. Surprisingly, fractures appear to heal normally. Ocular Features The sclerae can be normal, gray, slightly bluish, or bright blue. The color is probably caused by a thinness of the collagen layers of the sclerae that allows the choroid layers to be seen. Blue sclerae, however, are an inherited trait in some families who do not have increased bone fragility.

1	Dentinogenesis The teeth may be normal, moderately discolored, or grossly abnormal. The enamel generally appears normal, but the teeth may have a characteristic amber, yellowish brown, or translucent bluish gray color because of a deficiency of dentin that is rich in type I collagen. The deciduous teeth are usually smaller than normal, whereas permanent teeth are frequently bell-shaped and restricted at the base. In some patients, the teeth readily fracture and need to be extracted. Similar tooth defects, however, can be inherited without any evidence of OI. Hearing Loss Hearing loss usually begins during the second decade of life and occurs in more than 50% of individuals over age 30. The loss can be conductive, sensorineural, or mixed, and it varies in severity. The middle ear usually exhibits maldevelopment, deficient ossification, persistence of cartilage in areas that are normally ossified, and abnormal calcium deposits.

1	Other Features Changes in other connective tissues can include thin skin that scars extensively, joint laxity with permanent dislocations indistinguishable from those of Ehlers-Danlos syndrome (EDS), and occasionally, cardiovascular manifestations such as aortic regurgitation, floppy mitral valves, mitral incompetence, and fragility of large blood vessels. For unknown reasons, some patients develop bouts of a hypermetabolic state with elevated serum thyroxine levels, hyperthermia, and excessive sweating. Molecular Defects Of the ~1360 unique gene mutations now described in OI, more than 90% are heterozygous mutations in either COL1A1 or COL1A2, the genes coding for the proα1 or proα2 chain of type I procollagen (Table 427-2). Most patients with type I OI and blue sclerae have mutations that reduce the synthesis of proα1 chains to about one-half. Mutations that reduce the synthesis of proα2 chains produce slightly more severe phenotypes and skin defects similar to EDS.

1	In contrast to the null mutations found in type I OI, most of the severe variants (types II, III, and IV) are caused by mutations that produce structurally abnormal proα chains that have compromised assembly or abnormal folding of the triple helix. As with collagen mutations in other connective tissue diseases, these structural mutations generally fall into two functional categories. First, the relatively rare mutations in the C-propeptide domain can prevent or seriously impair initial assembly of the procollagen trimers. These misfolded chains are retained in the endoplasmic reticulum (ER) and targeted for degradation by the ER-associated proteasomal pathway. Because these mutations induce an ER stress response, the unfolded protein response (UPR) may have many downstream effects on cells. ER stress is a new concept in the pathophysiology of connective tissue disease and has been best characterized for chondrodysplasias (see below).

1	The most common type I collagen mutations, however, are single base substitutions that introduce an amino acid with a bulky side chain for one of the glycine residues that appear as every third amino acid in the triple helix. In effect, any of the 338 glycine residues in the helical domain of either the proα1 or proα2 chain of type I procollagen is a potential site for a disease-producing mutation. These mutations compromise the structural integrity of the triple helix, causing disruption to helix folding, retention of the mutant trimers in the ER, and increased posttranslational hydroxylation and glycosylation of lysines. Collagen-containing helix mutations can form insoluble aggregates in the ER that are degraded by the autophagosome-endosome system, rather than the proteasomes.

1	A similar sequence of events occurs with less common mutations that produce partial gene deletions, partial gene duplications, and splicing mutations. In addition to their intracellular effects, the structurally abnormal mutant-containing collagen that is secreted by the cell can also have important extracellular affects. For example, the presence of one abnormal proα chain in a procollagen molecule can interfere with cleavage of the N-propeptide from the protein. The persistence of the N-propeptide on a fraction of the molecules interferes with the self-assembly of normal collagen so that thin and irregular collagen fibrils are formed. Furthermore, if structurally abnormal collagens are incorporated into fibrils, they may have a destabilizing effect and be selectively degraded, or they may alter the interactions of collagen with other connective tissue components, disturbing architecture and stability.

1	Several generalizations can be made about mutations in type I collagen genes. One is that unrelated patients rarely have the same mutation in the same gene. Glycine substitutions in the N-terminal region of the triple helix tend to produce milder phenotypes, apparently because they have less effect on the zipper-like propagation of the triple-helical conformation from the C terminus. Rare substitutions 2509 of charged amino acids (Asp, Arg) or a branched amino acid (Val) in X-or Y-positions produce lethal phenotypes, apparently because they are located at sites for lateral assembly of the monomers or binding of other components of the matrix.

1	The search for mutations causing the less common and autosomal recessive forms of OI identified mutations in genes for a series of proteins that are essential for the timely folding of the procollagen monomer: cartilage-associated protein (CRTAP), prolyl-3-hydroxylase (LEPRE1/P3H1), cyclophilin B (PPIB), collagen chaperone-like protein HSP47 (SERPINH1), and the procollagen chaperone protein FKBP65 (FKBP10). Recently, mutations have been characterized in additional downstream components of the collagen fibrillogenesis pathway: BMP1, the gene coding for a metalloproteinase that cleaves the C-propeptide of type I procollagen, and PLOD2 (LH2, lysyl oxidase 2), which is involved in establishing collagen cross-links. In addition to these mutations that affect the collagen assembly pathway, mutations have been characterized in genes involved in the regulation of bone formation and mineralization such as SP7 (osterix), IFITM5, WNT1, and TMEM38B (Table 427-2).

1	Inheritance and Mosaicism in Germline Cells and in Somatic Cells Type I OI is inherited as an autosomal dominant trait. However, some patients with type I OI appear to represent sporadic new mutations or a diagnosis that was missed in earlier generations. Most lethal OI is the result of sporadic mutations that occur in the germ line in one of the parents. Because of the possibility for germline mosaicism for newly generated mutations, there is about a 7% probability that a second child could inherit a severe variant of OI.

1	Diagnosis OI is usually diagnosed on the basis of clinical criteria. The presence of fractures together with blue sclerae, dentinogenesis imperfecta, or family history of the disease is usually sufficient to make the diagnosis. Other causes of pathologic fractures must be excluded, including battered child syndrome, nutritional deficiencies, malignancies, and other inherited disorders such as chondrodysplasias and hypophosphatasia that can have overlapping presentations. The absence of superficial bruises can be helpful in distinguishing OI from battered child syndrome. X-rays usually reveal a decrease in bone density that can be verified by photon or x-ray absorptiometry. Bone microscopy can be helpful in the diagnosis. The diagnosis, as in other genetic disorders, is now routinely determined using targeted candidate gene sequencing and exome sequencing, but whole-genome sequencing may be used in the future.

1	Many patients with OI lead productive and successful lives despite severe deformities. Those with mild forms of the disease may need little treatment when fractures decrease after puberty, but women require special attention during pregnancy and after menopause, when fractures again increase. More severely affected children require a comprehensive program of physical therapy and surgical management of fractures and skeletal deformities.

1	Many fractures are only slightly displaced and have little soft tissue swelling and, therefore, can be treated with minimal support or traction for a week or two followed by a light cast. If fractures are relatively painless, physical therapy can be initiated early. A judicious amount of exercise prevents loss of bone mass secondary to physical inactivity. Some physicians advocate insertion of steel rods into long bones to correct limb deformities; the risk/benefits and cost/benefits of such procedures are difficult to evaluate. Aggressive conventional intervention is usually warranted for pneumonia and cor pulmonale. For severe hearing loss, stapedectomy or replacement of the stapes with a prosthesis may be successful. Moderately to severely affected patients should be evaluated periodically to anticipate possible neurologic problems. About half of children have a substantial increase in growth when given growth hormone. Treatment with bisphosphonates to decrease bone loss has been

1	to anticipate possible neurologic problems. About half of children have a substantial increase in growth when given growth hormone. Treatment with bisphosphonates to decrease bone loss has been introduced for moderate to severe forms of OI. Improvements in bone mineral

1	Heritable Disorders of Connective Tissue 2510 density are consistently seen in patients. Some clinical trials observed improvements in bone pain and fracture incidence; however, there are still unresolved questions about the best delivery protocols and the risks associated with long-term use in OI patients. For these reasons, the current consensus is that bisphosphonate therapy should be restricted to moderate to severe OI, where the possible benefits outweigh risks. Also, a clinical trial was performed in which patients were treated by intravenous infusion of cells from bone marrow referred to as mesenchymal stem cells, or multipotential stromal cells (MSCs; see Chap. 90e). Promising results were obtained, but the trial required a prior bone marrow transplant with marrow from a normal donor who subsequently was used as a source of normal MSCs. As a result, the procedure has not been widely adopted. However, the results raise the possibility that it may be possible in the future to

1	donor who subsequently was used as a source of normal MSCs. As a result, the procedure has not been widely adopted. However, the results raise the possibility that it may be possible in the future to develop effective stem cell therapies for OI. Counseling and emotional support are important for patients and their parents; lay organizations in some countries provide help in these areas. Prenatal ultrasonography will detect severely affected fetuses at about 16 weeks of pregnancy. Diagnosis is routinely performed on DNA from blood.

1	EDS is characterized by hyperextensible skin and hypermobile joints, but the category includes rare patients with other distinctive features. Mutations in different types of collagen are found in many patients, but other genes are at fault in rare forms. Contrary to initial expectations, no patients have been found with mutations in the gene for elastin in EDS.

1	Classification Several types of EDS have been defined, based on the extent to which the skin, joints, and other tissues are involved, mode of inheritance, and molecular and biochemical analysis (Table 427-3). Classical EDS includes a severe form of the disease (type I) and a milder form (type II), both characterized by joint hypermobility and skin that is velvety in texture, hyperextensible, and easily scarred. In hypermobile EDS (type III), joint hypermobility is more prominent than skin changes. In vascular-type EDS (type IV), the skin changes are more prominent than joint changes, and the patients are predisposed to sudden death from rupture of large blood vessels or other hollow organs. EDS type V is similar to EDS type II but is inherited as an X-linked trait. The ocular-scoliotic type of EDS (type VI) is characterized by scoliosis, ocular fragility, and a cone-shaped deformity of the cornea (keratoconus). The arthrochalasic type of EDS (type VIIA and VIIB) is characterized by

1	type of EDS (type VI) is characterized by scoliosis, ocular fragility, and a cone-shaped deformity of the cornea (keratoconus). The arthrochalasic type of EDS (type VIIA and VIIB) is characterized by marked joint hypermobility that is difficult to distinguish from EDS III except by the specific molecular defects in the processing of type I procollagen to collagen. The periodontotic-type EDS (type VIII) is distinguished by prominent periodontal changes. EDS types IX, X, and XI were defined on the basis of preliminary biochemical and clinical data. EDS due to tenascin X deficiency has not been assigned a type; it is an autosomal recessive form of the syndrome similar to EDS II. The cardiac valvular form of EDS has similar features to EDS II, but also involves severe changes to the aorta. The progeroid form of EDS displays features of both EDS and progeria. Because of overlapping signs and symptoms, many patients and families with some of the features of EDS cannot be assigned to any of

1	progeroid form of EDS displays features of both EDS and progeria. Because of overlapping signs and symptoms, many patients and families with some of the features of EDS cannot be assigned to any of the defined types.

1	Incidence The overall incidence of EDS is about 1 in 5000 births, with a higher rate for blacks. Classical and hypermobile types of EDS are the most common. Patients with milder forms frequently do not seek medical attention. Skin Skin changes vary from thin and velvety to skin that is either dramatically hyperextensible (“rubber person” syndrome) or easily Classic (EDS I—severe and Skin hyperextensibility and fragility, joint hypermobil-EDS II—mild) ity, tissue fragility manifested by widened atrophic scarring Hypermobile (EDS III) Joint hypermobility, moderate skin involvement, absence of tissue fragility Vascular (EDS IV) Markedly reduced life span due to spontaneous rupture of internal organs such as arteries and intestines; skin is thin, translucent, and fragile, with extensive bruising; hypermobile minor joints; characteristic facial appearance X-linked EDS (EDS V) Similar to classic type Ocular-scoliotic EDS VI (EDS Features of classic EDS as well as severe muscular

1	X-linked EDS (EDS V) Similar to classic type Ocular-scoliotic EDS VI (EDS Features of classic EDS as well as severe muscular VIA and EDS VIB) hypotonia after birth, progressive kyphoscoliosis, a Marfanoid habitus, osteopenia, occasionally rupture of the eye globe and great arteries Arthrochalasic EDS VII (EDS Congenital bilateral hip dislocation, hypermobile VIIA and EDS VIIB) joints, moderate skin involvement, osteopenia Dermatosparactic EDS VII C Redundant and fragile skin, prominent hernias, joint laxity, dysmorphic features Periodontotic EDS VIII Absorptive periodontosis with premature loss of permanent teeth, fragility of the skin, skin lesions EDS due to tenascin X Similar to EDS II deficiency EDS, progeroid form Abbreviations: AD, autosomal dominant; AR, autosomal recessive. AD, AR AD AD AR AD AR AR? COL1A1 Proα1 (I) and proα2 (I) chains of procollagen I PLOD1 Deficiency of procollagenlysine 5-dioxygenase activity (EDS VIA)

1	AD, AR AD AD AR AD AR AR? COL1A1 Proα1 (I) and proα2 (I) chains of procollagen I PLOD1 Deficiency of procollagenlysine 5-dioxygenase activity (EDS VIA) COL1A1 Mutations that prevent cleavage of the N propeptides ADAMTS2 Deficiency of procollagen I N-terminal proteinase B4GALT7 Deficiency of galactosyltransferase 7 (defective synthesis of dermatan sulfate proteoglycans) torn or scarred. Patients with classical EDS develop characteristic “cigarette-paper” scars. In vascular-type EDS, extensive scars and hyperpigmentation develop over bony prominences, and the skin may be so thin that subcutaneous blood vessels are visible. In the periodontotic type of EDS, the skin is more fragile than hyperextensible, and it heals with atrophic, pigmented scars. Easy bruisability occurs in several types of EDS.

1	Ligament and Joint Changes Laxity and hypermobility of joints vary from mild to unreducible dislocations of hips and other large joints. In mild forms, patients learn to avoid dislocations by limiting physical activity. In more severe forms, surgical repair may be required. Some patients have progressive difficulty with age. Other Features Mitral valve prolapse and hernias occur, particularly with type I. Pes planus and mild to moderate scoliosis are common. Extreme joint laxity and repeated dislocations may lead to degenerative arthritis. In the ocular-scoliotic type of EDS, the eye may rupture with minimal trauma, and kyphoscoliosis can cause respiratory impairment. Also, sclerae may be blue.

1	Molecular Defects Subsets of patients with different types of EDS have mutations in the structural genes for collagens (Table 4273). These include mutations in the COL1A1 gene in a few patients with moderately severe classical EDS (type I); mutations in COL1A2 in rare patients with an aortic valvular form of EDS; mutations in two of the three genes (COL5A1 and COL5A2) for type V collagen, a minor collagen found in association with type I collagen, in about half the patients with classical EDS (types I and II); and mutations in the COL3A1 gene for type III collagen, which is abundant in the aorta in patients with the frequently lethal vascular EDS (type IV). Some of the type I collagen-related mutations alter processing of the protein or genes for the processing enzymes. Arthrochalasic EDS (type

1	VII) is caused by mutations in the amino acid sequence that make type I procollagen resistant to cleavage by procollagen N-proteinase or by mutations that decrease the activity of the enzyme. The persistence of the N propeptide causes the formation of collagen fibrils that are thin and irregular. Some of the patients have fragile bones and therefore a phenotype that overlaps with OI. The ocular-scoliotic type of EDS (type VI) is caused by homozygous or compound heterozygous mutations in the PLOD1 gene, which encodes procollagen-lysine 5-dioxygenase (lysyl hydroxylase 1), an enzyme required for formation of stable cross-links in collagen fibers.

1	Some patients with the hypermobile EDS (type III) and a few with mild EDS (type II) have mutations in the TNXB gene, which encodes tenascin X, another minor component of connective tissue that appears to regulate the assembly of collagen fibers. Mutations in proteoglycans have been found in a few patients. The progeroid form of EDS results from autosomal recessive mutations in B4GALT7, the gene for β-1,4-galactosyltransferase 7, a key enzyme in the addition of glycosaminoglycan chains to proteoglycans. Diagnosis The diagnosis is based on clinical criteria and increasingly on DNA sequencing. Correlations between genotype and phenotype can be challenging, but gene or biochemical tests are particularly useful for the diagnosis of vascular type IV EDS with its dire prognosis.

1	As with other heritable diseases of connective tissue, there is a large degree of variability among members of the same family carrying the same mutation. Some patients have increased fractures and are difficult to distinguish from OI. A few families with heritable aortic aneurysms have mutations in the gene for type III collagen without any evidence of EDS or OI. Surgical repair and tightening of joint ligaments require careful evaluation of individual patients, as the ligaments frequently do not hold sutures. Patients with easy bruisability should be evaluated for bleeding disorders. Patients with type IV EDS and members of their families should be evaluated at regular intervals for early detection of aneurysms, but surgical repair may be difficult because of friable 2511 tissues. Also, women with type IV EDS should be counseled about the increased risk of uterine rupture, bleeding, and other complications of pregnancy.

1	(See also Chap. 426e) Chondrodysplasias (CDs), also referred to as skeletal dysplasias, are heritable skeletal disorders that are characterized by dwarfism and abnormal body proportions. The category also includes some individuals with normal stature and body proportions who have features such as ocular changes or cleft palate, which are common in more severe CDs. Many patients develop degenerative joint changes, and mild CD in adults may be difficult to differentiate from primary generalized osteoarthritis. An undefined number of patients have mutations in either the most abundant collagen in cartilage (type II) or the less abundant collagens (types X or XI). Other patients have mutations in genes that code for other components of cartilage or for proteins required for the embryonic development of cartilage, including a common mutation in a gene for a fibroblast growth factor receptor.

1	Classification Over 200 distinct types and subtypes have been defined based on criteria such as “bringing death” (thanatophoric), causing “twisted” bones (diastrophic), affecting metaphyses (metaphyseal), affecting epiphyses (epiphyseal), affecting spine (spondylo-), and producing histologic changes such as an apparent increase in the fibrous material in the epiphyses (fibrochondrogenesis). Also, a number of eponyms are based on the first or most comprehensive case reports. Severe forms of the diseases produce dwarfism with gross distortions of most cartilaginous structures and of other structures including the eye. Mild forms are more difficult to classify. Among the features are cataracts, degeneration of the vitreous, retinal detachment, high forehead hypoplastic facies, cleft palate, short extremities, and gross distortions of the epiphyses, metaphyses, and joint surfaces. Patients with Stickler’s syndrome (hereditary arthro-ophthalmopathy) have been classified into three types

1	short extremities, and gross distortions of the epiphyses, metaphyses, and joint surfaces. Patients with Stickler’s syndrome (hereditary arthro-ophthalmopathy) have been classified into three types based on a combination of the ocular phenotype and mutated genes.

1	The overall incidence of all forms of CD ranges from 1 per 2500 to 1 per 4000 births. Data on the frequency of individual CDs are incomplete, but the incidence of Stickler’s syndrome is 1 in 10,000. Therefore, the disease is probably among the more common heritable disorders of connective tissue.

1	Molecular Defects Mutations in the COL2A1 gene for the type II collagen of cartilage are found in a fraction of patients with both mild and severe CDs. For example, a mutation in the gene substituting a cysteine residue for an arginine was found in three unrelated families with spondyloepiphyseal dysplasia (SED) and precocious generalized osteoarthritis (OA). Mutations in the gene, often glycine substitution mutations with the collagen II triple helix, were also found in some lethal CDs characterized by gross deformities of bones and cartilage, such as those found in spondyloepiphyseal dysplasia congenita, spondyloepimetaphyseal dysplasia congenita, hypochondrogenesis/achondrogenesis type II, and Kniest’s syndrome. The highest incidence of COL2A1 mutations, however, occurs in patients with the distinctive features of Stickler’s syndrome, which is characterized by skeletal changes, orofacial abnormalities, and auditory abnormalities. Most of the mutations in COL2A1 are premature stop

1	the distinctive features of Stickler’s syndrome, which is characterized by skeletal changes, orofacial abnormalities, and auditory abnormalities. Most of the mutations in COL2A1 are premature stop codons that produce haploinsufficiency. In addition, some patients with Stickler’s syndrome or a closely related syndrome have mutations in two genes specific for type XI collagen, which is an unusual heterotrimer formed from α chains encoded by the gene for type II collagen (COL2A1) and two distinctive genes for type XI collagen (COL11A1 and COL11A2). Mutations in the COL11A1 gene are also found in patients with Marshall’s syndrome, which is similar to classic Stickler’s syndrome, but with more severe hearing loss and dysmorphic features, such as a flat or retracted midface with a flat nasal bridge, short nose, anteverted nostrils, long philtrum, and large-appearing eyes.

1	Heritable Disorders of Connective Tissue 2512 CDs are also caused by mutations in the less abundant collagens found in cartilage. For example, patients with Schmid metaphyseal CD have mutations in the gene for type X collagen, a short, network-forming collagen found in the hypertrophic zone of endochondral cartilage. The syndrome is characterized by short stature, coxa vara, flaring metaphyses, and waddling gait. As with other collagen genes, the most common mutations are of two types: nonsense mutations that lead to haploinsufficiency and structural mutations that compromise collagen assembly. In type X collagen, all the structural mutations detected occur in the C-terminal NC1 domain that coordinates the formation of the trimers. This NC1 domain is functionally equivalent to the C-propeptide of the fibrillar collagens. These mutations disturb the structure of the NC1 domain, leading to misfolding and initiation of cellular ER stress via the unfolded protein response (UPR). While the

1	of the fibrillar collagens. These mutations disturb the structure of the NC1 domain, leading to misfolding and initiation of cellular ER stress via the unfolded protein response (UPR). While the UPR evolved to allow cells to adjust their ER folding capacity to differing protein folding loads, it is deployed by cells when mutant misfolded proteins accumulate in the ER. Activation of the UPR attenuates protein translation and activates mutant protein degradation pathways such as ER-associated degradation. If these strategies do not sufficiently reduce the stress response, cell death may occur. In Schmid metaphyseal CD, mutant misfolded type X collagen induces the UPR, resulting in downstream consequences that contribute to the pathophysiology. This general mechanism may also contribute to pathology in other CDs (and in other connective tissues disorders) where gene mutations lead to protein structural abnormalities. Some patients have mutations in genes for proteins that interact with

1	pathology in other CDs (and in other connective tissues disorders) where gene mutations lead to protein structural abnormalities. Some patients have mutations in genes for proteins that interact with collagens. Patients with pseudoachondroplasia or autosomal dominant multiple epiphyseal dysplasia have mutations in the gene for the cartilage oligomeric matrix protein (COMP), a protein that interacts with both collagens and proteoglycans in cartilage. However, some families with multiple epiphyseal dysplasia have a defect in one of the three genes for type IX collagen (COL9A1, COL9A2, and COL9A3) or in matrilin-3, another extracellular protein found in cartilage. With misfolding mutations in COMP and matrilin-3, the activation of the UPR has been described, providing further evidence that the UPR is a component of pathology of these conditions. Some CDs are caused by mutations in genes that affect early development of cartilage and related structures. The most common form of

1	that the UPR is a component of pathology of these conditions. Some CDs are caused by mutations in genes that affect early development of cartilage and related structures. The most common form of short-limbed dwarfism, achondroplasia, is caused by mutations in the gene for a receptor for a fibroblastic growth factor (FGFR3). The mutations in the FGFR3 gene causing achondroplasias are unusual in several respects. The same single-base mutation in the gene that converts glycine to arginine at position 380 in the FGFR3 gene is present in over 90% of patients. Most patients harbor sporadic new mutations, and therefore, this nucleotide change must be one of the most common recurring mutations in the human genome. The mutation causes unregulated signal transduction through the receptor and inappropriate development of cartilage. Mutations that alter other domains of FGFR3 have been found in patients with the more severe disorders of hypochondroplasia and thanatophoric dysplasia and in a few

1	development of cartilage. Mutations that alter other domains of FGFR3 have been found in patients with the more severe disorders of hypochondroplasia and thanatophoric dysplasia and in a few families with a variant of craniosynostosis. However, most patients with craniosynostosis appear to have mutations in the related FGFR2 gene. The similarities between the phenotypes produced by mutations in genes for FGF receptors and mutations in structural proteins of cartilage are probably explained by the observation that the activity of FGFs is regulated in part by binding of FGFs to proteins sequestered in the extracellular matrix. Therefore, the situation parallels the interactions between transforming growth factors (TGFs) and fibrillin in MFS (see below). Other mutations involve the proteoglycans of cartilage, aggrecan (AGC1), and perlecan (HSPG2) and the proteoglycan posttranslational sulphation pathway (DTDST, PAPSS2, and CHST3). Mutations in more than 45 other genes have been defined

1	of cartilage, aggrecan (AGC1), and perlecan (HSPG2) and the proteoglycan posttranslational sulphation pathway (DTDST, PAPSS2, and CHST3). Mutations in more than 45 other genes have been defined in chondrodysplasias.

1	Diagnosis The diagnosis of CDs is made on the basis of the physical appearance, slit-lamp eye examinations, x-ray findings, histologic changes, and clinical course. Evaluation of patients by specialists in the field is usually required for a diagnosis. Targeted gene and exome sequencing or more global sequencing strategies are used for molecular diagnosis. Given the wide spectrum of CD phenotypes, these gene tests are becoming critical diagnostic tools. For Stickler’s syndrome, more precise diagnostic criteria have made it possible to identify type I variants with mutations in the COL2A1 gene with a high degree of accuracy. It has been suggested that the type II variant with mutations in the COL11A1 gene can be identified on the basis of a “beaded” vitreous phenotype, and the type III variant with mutations in the COL11A2 gene can be identified on the basis of the characteristic systemic features without the ocular involvement. Prenatal diagnosis based on analysis of DNA obtained from

1	with mutations in the COL11A2 gene can be identified on the basis of the characteristic systemic features without the ocular involvement. Prenatal diagnosis based on analysis of DNA obtained from chorionic villus or amniotic fluid is possible.

1	The treatment is symptomatic and is directed to secondary features such as degenerative arthritis. Many patients require joint replacement surgery and corrective surgery for cleft palate. The eyes should be monitored carefully for the development of cataracts and the need for laser therapy to prevent retinal detachment. In general, patients should be advised to avoid obesity and contact sports. Counseling for the psychological problems of short stature is critical, and support groups have formed in many countries. MFS includes features that primarily affect the skeleton, the cardiovascular system, and the eyes. Most patients have mutations in the gene for fibrillin-1.

1	MFS includes features that primarily affect the skeleton, the cardiovascular system, and the eyes. Most patients have mutations in the gene for fibrillin-1. Classification MFS was initially characterized by a triad of features: (1) skeletal changes that include long, thin extremities, frequently associated with loose joints; (2) reduced vision as the result of dislocations of the lenses (ectopia lentis); and (3) aortic aneurysms. An international panel has developed a series of revised “Ghent criteria” that are useful in classifying patients. Incidence and Inheritance The incidence of MFS is among the highest of any heritable disorder: about 1 in 3000/5000 births in most racial and ethnic groups. The related syndromes are less common. Mutations are generally inherited as autosomal dominant traits, but about one-fourth of patients have sporadic new mutations.

1	Skeletal Effects Patients have long limbs and are usually tall compared to other members of the same family. The ratio of the upper segment (top of the head to the top of the pubic ramus) to the lower segment (top of the pubic ramus to the floor) is usually two standard deviations below mean for age, race, and sex. The fingers and hands are long and slender and have a spider-like appearance (arachnodactyly). Many patients have severe chest deformities, including depression (pectus excavatum), protrusion (pectus carinatum), or asymmetry. Scoliosis is frequent and usually accompanied by kyphosis. High-arched palate and high pedal arches or pes planus are common. A few patients have severe joint hypermobility similar to EDS. Computed tomography or magnetic resonance imaging examinations of the lumbar sacral region frequently reveal enlargement of the neural canal, thinning of the pedicles and laminae, widening of the foramina, or anterior meningocele (dural ectasia).

1	Cardiovascular Features Cardiovascular abnormalities are the major source of morbidity and mortality (Chap. 301). Mitral valve prolapse develops early in life and progresses to mitral valve regurgitation of increasing severity in about one-quarter of patients. Dilation of the root of the aorta and the sinuses of Valsalva are characteristic and ominous features of the disease that can develop at any age. The rate of dilation is unpredictable, but it can lead to aortic regurgitation, dissection of the aorta, and rupture. Dilation is probably accelerated by physical and emotional stress, as well as by pregnancy. Patients usually differ from those with familial aortic aneurysms who tend to develop aneurysms in the abdominal aorta. The location of the aneurysms, however, is somewhat variable, and the high incidence of aortic aneurysms in the general population (1 in 100) makes the differential diagnosis difficult unless other features of MFS are clearly present.

1	Ocular Features Upward displacement of the lens is common. It is usually not progressive but may contribute to the formation of cataracts. The ocular globe is frequently elongated, and most patients are myopic, but with adequate vision. Retinal detachment can occur. Other Features Striae may occur over the shoulders and buttocks. A number of patients develop spontaneous pneumothorax. Inguinal and incisional hernias are common. Patients are typically thin with little subcutaneous fat, but adults may develop centripetal obesity.

1	Molecular Defects More than 90% of patients clinically classified as having MFS by the “Ghent criteria” have a mutation in the gene for fibrillin-1 (FBN1). Mutations in the same gene are found in a few patients who do not meet the Ghent criteria. Also, a few MFS patients without mutations in the FBN1 gene have mutations in the gene for TGF-β receptor 2 (TGFBR2). In addition, mutations in either TGFBR2 or TGFBR1 are found in the related Loeys-Dietz syndrome, which is characterized by aortic aneurysms, cleft palate, and hypertelorism. Mutations in the FBN2 gene, which is structurally similar to the FBN1 gene, are found in patients with MFS-like syndrome of congenital contractual arachnodactyly.

1	FBN1 gene mutations are scattered throughout its 65 coding exons. Most are private mutations, but about 10% are recurrent new mutations that are largely located in CpG sequences known to be “hot spots.” Most severe mutations are located in the central codons (24–32). About one-third of the mutations introduce premature termination codons, and about two-thirds are missense mutations that alter calcium-binding domains in the repetitive epidermal growth factor– like domains of the protein. Rarer mutations alter the processing of the protein. As in many genetic diseases, the severity of the phenotype cannot be predicted from the nature of the mutation.

1	The discovery that syndromes similar to MFS are caused by mutations in TGFBR1 and TGFBR2 refocused attention on structural similarity between fibrillin-1 and TGF-β binding proteins that sequester TGF-β in the extracellular matrix. As a result, some of the manifestations of MFS have been shown to arise from alterations in binding sites that modulate TGF-β bioavailability during development of the skeleton and other tissues. Likewise, TGFBR1 and TGFBR2 mutations in Loeys-Dietz syndrome alter TGF-β signaling. In both MFS and Loeys-Dietz syndrome, the pathogenic mechanisms involve increased TGF-β signaling, which contributes to aneurysm formation.

1	Diagnosis All patients with a suspected diagnosis of MFS should have a slit-lamp examination and an echocardiogram. Also, homocystinuria should be ruled out by amino acid analysis of plasma (Chap. 434e). The diagnosis of MFS according to the international Ghent standards places emphasis on major criteria that include presence of at least four skeletal abnormalities: ectopia lentis; dilation of the ascending aorta with or without dissection; dural ectasia; and a blood relative who meets the same criteria, with or without a DNA diagnosis. A final diagnosis is based on a balanced assessment of the major criteria together with several minor criteria. The absence of ocular changes suggests the Loeys-Dietz syndrome, and the presence of contractures with some of the signs of OI suggests congenital contractual arachnodactyly.

1	Diagnostic tests based on gene sequencing or detection of protein defects are available. These results are unlikely to alter the treatment or prognosis but are helpful to inform the patients and families and to rapidly exclude the diagnosis in unaffected family members. Propranolol or other β-adrenergic blocking agents are used to lower blood pressure and thereby delay or prevent aortic dilation. Surgical correction of the aorta, aortic valve, and mitral valve has been successful in many patients, but tissues are frequently friable. Patients should be advised that the risks are increased by severe physical exertion, emotional stress, and pregnancy.

1	The scoliosis tends to be progressive and should be treated by mechanical bracing and physical therapy if >20° or by surgery if it progresses to >45°. Dislocated lenses rarely require surgical removal, but patients should be followed closely for retinal detach-2513 ment. The finding that MFS pathophysiology involves alterations in TGF-β signaling has raised the possibility of new therapeutic strategies. Attenuation of TGF-β signaling with agents such as angiotensin II receptor blockers (e.g., losartan) was effective in animal studies and has been very promising in small observational studies on MFS patients, significantly reducing progressive aortic enlargement. Based on these results, large randomized clinical trials of angiotensin receptor blockers in MFS are under way.

1	Mutations in the elastin gene (ELN) have been found in patients with supravalvular aortic stenosis and skin that hangs in loose and redundant folds (cutis laxa). As indicated in Table 427-3, patients with several forms of EDS have similar changes in skin that were initially thought to reflect changes in elastin. EB has been defined as the category of heritable disorders involving skin that is specifically characterized by blistering as a result of friction. Using this criterion, it was possible to define subtypes by the ultra-structural layer of skin in which the cleavage and blistering occurred. These functional and anatomical criteria made it possible to establish that most patients with a specific subtype have mutations in genes coding for a structural protein, or a cell adherence protein, expressed in the corresponding layer of skin.

1	Classification and Incidence The four major types of EB are: (1) EB simplex in which cleavage occurs within the epidermis, (2) junctional EB in which cleavage occurs within the lamina lucida, (3) dystrophic EB in which cleavage occurs within the sublamina densa, and (4) Kindler’s syndrome with a mixed level of cleavage in different layers. Patients are then separated into major and minor subtypes based on clinical features and analysis of mutations. The incidence of EB in the United States is about 1 in 50,000.

1	The incidence of EB in the United States is about 1 in 50,000. Molecular Defects The distinctive anatomic locations in skin have made it possible to relate the clinical subtypes of EB to mutations for specific components. In EB simplex, mutations are found primarily in the genes for the major keratins of basal epithelial cells (keratins 5 and 14) and the cell adhesion proteins plectin, α6β4 integrin, plakophilin-1, and desmoplakin. Patients with the related syndrome, epidermolytic ichthyosis, have mutations in keratin 1 and keratin 10. In junctional EB, mutations occur in type XVII collagen, a laminin (laminin-332), and α6β4 integrin. In the severe syndrome of dystrophic EB, mutations are found in the gene that codes for type VII collagen, which forms long loops anchoring the epidermis to the dermis. Patients with more complex features of what is classified as Kindler’s syndrome have mutations in kindlin-1, a focal adhesion protein involved in integrin activation.

1	Diagnosis and Treatment The diagnosis is based on skin that readily breaks and forms blisters from minor trauma. EB simplex is generally milder than junctional EB or dystrophic EB. Dystrophic EB variants usually have large and prominent scars. Precise classification within subtypes usually requires immunofluorescent mapping. DNA diagnostic tests have been developed as research tools but are not widely available. The treatment is symptomatic. Novel therapeutic approaches such as gene therapy, protein replacement therapy, and cell therapy are being explored.

1	AS is an inherited disorder characterized by hematuria and several associated features. It was not initially considered as a disorder of connective tissue. However, the search for mutations in the genes coding for the collagen found that most patients had mutations in collagen found in basement membranes (type IV). Four forms of AS are now recognized: (1) classic AS, which is inherited as an X-linked disorder with hematuria, sensorineural deafness, and conical deformation of Heritable Disorders of Connective Tissue 2514 the anterior surface of the lens (lenticonus); (2) an X-linked form associated with diffuse leiomyomatosis; (3) an autosomal recessive form; and (4) an autosomal dominant form. Both autosomal recessive and dominant forms can cause renal disease without deafness or lenticonus. Incidence The incidence of AS is about 1 in 10,000 births in the general population and as high as 1 in 5000 in some ethnic groups. About 80% of AS patients have the classical X-linked variant.

1	Molecular Defects Most patients have mutations in four of the six genes for the chains of type IV collagen (COL4A3, COL4A4, COL4A5, and COL4A6). The genes for the proteins are arranged in tandem pairs on different chromosomes in an unusual head-to-head orientation and with overlapping promoters; i.e., the COL4A1 and COL4A2 genes are head-to-head on chromosome 13q34, the COL4A3 and COL4A4 genes are on chromosome 2q35–37, and the COL4A5 and COL4A6 genes are on chromosome Xq22. The X-linked variants are caused by either mutations in the COL4A5 gene or by partial deletions of both of the adjacent COL4A4 and COL4A5 genes. The autosomal recessive variants are caused by mutations in either the COL4A3 or COL4A4 gene. The mutations responsible for the autosomal dominant variants are still unknown, but they have been mapped to the same locus as the COL4A3 and COL4A4 genes.

1	Diagnosis The diagnosis of classic AS is based on X-linked inheritance of hematuria, sensorineural deafness, and lenticonus. The lenticonus together with hematuria is pathognomonic of classic AS. The sensorineural deafness is primarily in the high-tone range. It can frequently be detected only by an audiogram and is usually not progressive. Because of the X-linked transmission, women are generally underdiagnosed and are usually less severely affected than men. The hematuria usually progresses to nephritis and may cause renal failure in late adolescence in affected males and at older ages in some women. Renal transplantation is usually successful. The authors acknowledge the contributions of Helena Kuivaniemi, Gerard Tromp, Leena Ala-Kokko, and Malwina Czarny-Ratajcak to this chapter in previous editions of Harrison’s. The authors also wish to thank David Sillence for his expert advice on the classifications of types of OI.

1	Hemochromatosis Lawrie W. Powell DEFINITION Hemochromatosis is a common inherited disorder of iron metabolism in which dysregulation of intestinal iron absorption results in deposi-tion of excessive amounts of iron in parenchymal cells with eventual 428Endocrinology and Metabolism tissue damage and impaired function in a wide range of organs. The iron-storage pigment in tissues is called hemosiderin because it was believed to be derived from the blood. The term hemosiderosis is used to describe the presence of stainable iron in tissues, but tissue iron must be quantified to assess body-iron status accurately (see below and Chap. 126). Hemochromatosis refers to a group of genetic diseases that predispose to iron overload, potentially leading to fibrosis and organ failure. Cirrhosis of the liver, diabetes mellitus, arthritis, cardiomyopathy, and hypogonadotropic hypogonadism are the major clinical manifestations.

1	Although there is debate about definitions, the following terminology is widely accepted. 1. Hereditary hemochromatosis is most often caused by a mutant gene, termed HFE, which is tightly linked to the HLA-A locus on Hemochromatosis, HFE-related (type 1) C282Y homozygosity C282Y/H63D compound heterozygosity Hemochromatosis, non-HFE-related Juvenile hemochromatosis (type 2A) (hemojuvelin mutations) Juvenile hemochromatosis (type 2B) (hepcidin mutation) Mutated transferrin receptor 2, TFR2 (type 3) Mutated ferroportin 1 gene, SLC11A3 (type 4)

1	Sideroblastic anemia Alcoholic cirrhosis, especially when advanced chromosome 6p (see “Genetic Basis,” below). Persons who are homozygous for the mutation are at increased risk of iron overload and account for 80–90% of clinical hereditary hemochromatosis in persons of northern European descent. In such subjects, the presence of hepatic fibrosis, cirrhosis, arthropathy, or hepatocellular carcinoma constitutes iron overload–related disease. Rarer forms of non-HFE hemochromatosis are caused by mutations in other genes involved in iron metabolism (Table 428-1). The disease can be recognized during its early stages when iron overload and organ damage are minimal. At this stage, the disease is best referred to as early hemochromatosis or precirrhotic hemochromatosis.

1	2. Secondary iron overload occurs as a result of an iron-loading anemia, such as thalassemia or sideroblastic anemia, in which erythropoiesis is increased but ineffective. In the acquired iron-loading disorders, massive iron deposits in parenchymal tissues can lead to the same clinical and pathologic features as in hemochromatosis.

1	HFE-associated hemochromatosis mutations are among the most common inherited disease alleles, although the prevalence varies in different ethnic groups. It is most common in populations of northern European extraction in whom approximately 1 in 10 persons are heterozygous carriers and 0.3–0.5% are homozygotes. However, expression of the disease is variable and modified by several factors, especially alcohol consumption and dietary iron intake, blood loss associated with menstruation and pregnancy, and blood donation. Recent population studies indicate that approximately 30% of homozygous men develop iron overload–related disease and about 6% develop hepatic cirrhosis; for women, the figure is closer to 1%. Presumably there are as yet unidentified modifying genes responsible for expression and there is some early evidence to support this. Nearly 70% of untreated patients develop the first symptoms between ages 40 and 60. The disease is rarely evident before age 20, although with family

1	and there is some early evidence to support this. Nearly 70% of untreated patients develop the first symptoms between ages 40 and 60. The disease is rarely evident before age 20, although with family screening (see “Screening for Hemochromatosis,” below) and periodic health examinations, asymptomatic subjects with iron overload can be identified, including young menstruating women.

1	In contrast to HFE-associated hemochromatosis, the non-HFEassociated forms of hemochromatosis (Table 428-1) are rare, but they affect all races and young people (juvenile hemochromatosis).

1	A homozygous G to A mutation in the HFE gene resulting in a cysteine to tyrosine substitution at position 282 (C282Y) is the most common mutation. It is identified in 85–90% of patients with hereditary hemochromatosis in populations of northern European descent but is found in only 60% of cases from Mediterranean populations (e.g., southern Italy). A second, relatively common HFE mutation (H63D) results in a substitution of histidine to aspartic acid at codon 63. Homozygosity for H63D is not associated with clinically significant iron overload. Some compound heterozygotes (e.g., one copy each of C282Y and H63D) have mild to moderately increased body-iron stores but develop clinical disease only in association with cofactors such as heavy alcohol intake or hepatic steatosis. Thus, HFE-associated hemochromatosis is inherited as an autosomal recessive trait; heterozygotes have no, or minimal, increase in iron stores. However, this slight increase in hepatic iron can act as a cofactor

1	hemochromatosis is inherited as an autosomal recessive trait; heterozygotes have no, or minimal, increase in iron stores. However, this slight increase in hepatic iron can act as a cofactor that may modify the expression of other diseases such as porphyria cutanea tarda (PCT) or nonalcoholic steatohepatitis.

1	Mutations in other genes involved in iron metabolism are responsible for non-HFE-associated hemochromatosis, including juvenile hemochromatosis, which affects persons in the second and third decades of life (Table 428-1). Mutations in the genes encoding hepcidin, transferrin receptor 2 (TfR2), and hemojuvelin (Fig. 428-1) result in clinicopathologic features that are indistinguishable from HFE-associated hemochromatosis. However, mutations in ferroportin, responsible for the efflux of iron from enterocytes and most other cell types, result in iron loading of reticuloendothelial cells and macrophages as well as parenchymal cells.

1	Normally, the body-iron content of 3–4 g is maintained such that intestinal mucosal absorption of iron is equal to iron loss. This amount is approximately 1 mg/d in men and 1.5 mg/d in menstruating women. In hemochromatosis, mucosal absorption is greater than body requirements and amounts to 4 mg/d or more. The progressive accumulation of iron increases plasma iron and saturation of transferrin and results in a progressive increase of plasma ferritin (Fig. 428-2). A liver-derived peptide, hepcidin, represses basolateral iron transport in the intestine and iron release from macrophages and other cells by binding to ferroportin. Hepcidin, in turn, responds to signals in the liver mediated by HFE, TfR2, and hemojuvelin (Fig. 428-1). Thus, hepcidin is a crucial molecule in iron metabolism, linking body stores with intestinal iron absorption.

1	The HFE gene encodes a 343-amino-acid protein that is structurally related to MHC class I proteins (HFE). The basic defect in HFE-associated hemochromatosis is a lack of cell surface expression of HFE (due to the C282Y mutation). The normal (wild-type) HFE protein forms a complex with β2-microglobulin and transferrin receptor 1 (TfR1). The C282Y mutation completely abrogates this interaction. As a result, the mutant HFE protein remains trapped intracellularly, reducing TfR1-mediated iron uptake by the intestinal crypt cell. This impaired TfR1-mediated iron uptake leads to upregulation of the divalent metal transporter (DMT1) on the brush border of the villus cells, causing inappropriately increased intestinal iron absorption (Fig. 428-1). In advanced disease, the body may contain 20 g or more of iron that is deposited mainly in parenchymal cells of the liver, pancreas, and heart. Iron may be increased 50to 100-fold in the liver and pancreas

1	TransferrinBoneMarrowFPNHepcidinHepcidinHepcidinHepcidinHFE/TfR1TfR2HJVTMPRSS6BMP6BMPRSMADLiver?PPPDcytbDMT1VillusCryptDuodenumHephFPN

1	FIGURE 428-1 Pathways of normal iron homeostasis. Dietary inorganic iron traverses the brush border membrane of duodenal enterocytes via the divalent metal-ion transporter 1 (DMT1) after reduction of ferric (Fe3+) iron to the ferrous (Fe2+) state by duodenal cytochrome B (DcytB). Iron then moves from the enterocyte to the circulation via a process requiring the basolateral iron exporter ferroportin (FPN) and the iron oxidase hephaestin (Heph). In the circulation, iron binds to plasma transferrin and is thereby distributed to sites of iron utilization and storage. Much of the diferric transferrin supplies iron to immature erythrocyte cells in the bone marrow for hemoglobin synthesis. At the end of their life, senescent red blood cells (RBCs) are phagocytosed by macrophages, and iron is returned to the circulation after export through ferroportin. The liver-derived peptide hepcidin represses basolateral iron transport in the gut as well as iron released from macrophages and other cells

1	to the circulation after export through ferroportin. The liver-derived peptide hepcidin represses basolateral iron transport in the gut as well as iron released from macrophages and other cells and serves as a central regulator of body-iron traffic. Hepcidin responds to changes in body-iron requirements by signals mediated by diferric transferrin through two mechanisms. One involves HFE and TfR2, whereas the other involves hemojuvelin (HJV) and the bone morphogenetic protein (BMP)/SMAD pathway. TMPRSS6 is a protease that modulates HJV activity. Heme is metabolized by heme oxygenase within the enterocytes, and the released iron then follows the same pathway. Mutations in the genes encoding HFE, TfR2, hemojuvelin, and hepcidin all lead to decreased hepcidin release and increased iron absorption, resulting in hemochromatosis (Table 428-1).

1	(yrs.) Cirrhosis, organ failure Progressive tissue injury Increased total body iron Increased hepatic iron Increased serum iron Increased iron absorption FIGURE 428-2 Sequence of events in genetic hemochromatosis and their correlation with the serum ferritin concentration. Increased iron absorption is present throughout life. Overt, symptomatic disease usually develops between ages 40 and 60, but latent disease can be detected long before this. and 5to 25-fold in the heart. Iron deposition in the pituitary causes hypogonadotropic hypogonadism in both men and women. Tissue injury may result from disruption of iron-laden lysosomes, from lipid peroxidation of subcellular organelles by excess iron, or from stimulation of collagen synthesis by activated stellate cells.

1	Secondary iron overload with deposition in parenchymal cells occurs in chronic disorders of erythropoiesis, particularly in those due to defects in hemoglobin synthesis or ineffective erythropoiesis such as sideroblastic anemia and thalassemia (Chap. 127). In these disorders, iron absorption is increased. Moreover, these patients require blood transfusions and are frequently treated inappropriately with iron. PCT, a disorder characterized by a defect in porphyrin biosynthesis (Chap. 430), can also be associated with excessive parenchymal iron deposits. The magnitude of the iron load in PCT is usually insufficient to produce tissue damage. However, some patients with PCT also have mutations in the HFE gene, and some have associated hepatitis C virus (HCV) infection. Although the relationship between these disorders remains to be clarified, iron overload accentuates the inherited enzyme deficiency in PCT and should be avoided along with other agents (alcohol, estrogens, haloaromatic

1	between these disorders remains to be clarified, iron overload accentuates the inherited enzyme deficiency in PCT and should be avoided along with other agents (alcohol, estrogens, haloaromatic compounds) that may exacerbate PCT. Another cause of hepatic parenchymal iron overload is hereditary aceruloplasminemia. In this disorder, impairment of iron mobilization due to deficiency of ceruloplasmin (a ferroxidase) causes iron overload in hepatocytes.

1	Excessive iron ingestion over many years rarely results in hemochromatosis. An important exception has been reported in South Africa among groups who brew fermented beverages in vessels made of iron. Hemochromatosis has been described in apparently normal persons who have taken medicinal iron over many years, but such individuals probably had genetic disorders. The common denominator in all patients with hemochromatosis is excessive amounts of iron in parenchymal tissues. Parenteral administration of iron in the form of blood transfusions or iron preparations results predominantly in reticuloendothelial cell iron overload. This appears to lead to less tissue damage than iron loading of parenchymal cells.

1	In the liver, parenchymal iron is in the form of ferritin and hemosiderin. In the early stages, these deposits are seen in the periportal parenchymal cells, especially within lysosomes in the pericanalicular cytoplasm of the hepatocytes. This stage progresses to perilobular fibrosis and eventually to deposition of iron in bile-duct epithelium, Kupffer cells, and fibrous septa due to activation of stellate cells. In the advanced stage, a macronodular or mixed macroand micronodular cirrhosis develops. Hepatic fibrosis and cirrhosis correlate significantly with hepatic iron concentration. At autopsy, the enlarged nodular liver and pancreas are rusty in color. Histologically, iron is increased in many organs, particularly in the liver, heart, and pancreas, and, to a lesser extent, in the endocrine glands. The epidermis of the skin is thin, and melanin is increased in the cells of the basal layer and dermis. Deposits of iron are present around the synovial lining cells of the joints.

1	C282Y homozygotes can be characterized by the stage of progression as follows: (1) a genetic predisposition without abnormalities; (2) iron overload without symptoms; (3) iron overload with symptoms (e.g., arthritis and fatigue); and (4) iron overload with organ damage—in particular, cirrhosis. Thus, many subjects with significant iron overload are asymptomatic. For example, in a study of 672 asymptomatic C282Y homozygous subjects—identified by either family screening or routine health examinations—there was hepatic iron overload (grades 2–4) in 56% and 34.5% of male and female subjects, respectively; hepatic fibrosis (stages 2–4) in 18.4% and 5.4%, respectively; and cirrhosis in 5.6% and 1.9%, respectively.

1	Initial symptoms are often nonspecific and include lethargy, arthralgia, change in skin color, loss of libido, and features of diabetes mellitus. Hepatomegaly, increased pigmentation, spider angiomas, splenomegaly, arthropathy, ascites, cardiac arrhythmias, congestive heart failure, loss of body hair, testicular atrophy, and jaundice are prominent in advanced disease.

1	The liver is usually the first organ to be affected, and hepatomegaly is present in more than 95% of symptomatic patients. Hepatic enlargement may exist in the absence of symptoms or of abnormal liver-function tests. Manifestations of portal hypertension and esophageal varices occur less commonly than in cirrhosis from other causes. Hepatocellular carcinoma develops in about 30% of patients with cirrhosis, and it is the most common cause of death in treated patients— hence the importance of early diagnosis and therapy. The incidence increases with age, it is more common in men, and it occurs almost exclusively in cirrhotic patients.

1	Excessive skin pigmentation is present in patients with advanced disease. The characteristic metallic or slate-gray hue is sometimes referred to as bronzing and results from increased melanin and iron in the dermis. Pigmentation usually is diffuse and generalized, but it may be more pronounced on the face, neck, extensor aspects of the lower forearms, dorsa of the hands, lower legs, and genital regions, as well as in scars. Diabetes mellitus occurs in about 65% of patients with advanced disease and is more likely to develop in those with a family history of diabetes, suggesting that direct damage to the pancreatic islets by iron deposition occurs in combination with other risk factors. The management is similar to that of other forms of diabetes, although insulin resistance is more common in association with hemochromatosis. Late complications are the same as seen in other causes of diabetes mellitus.

1	Arthropathy develops in 25–50% of symptomatic patients. It usually occurs after age 50 but may occur as a first manifestation or long after therapy. The joints of the hands, especially the second and third metacarpophalangeal joints, are usually the first joints involved, a feature that helps to distinguish the chondrocalcinosis associated with hemochromatosis from the idiopathic form (Chap. 395). A progressive polyarthritis involving wrists, hips, ankles, and knees may also ensue. Acute brief attacks of synovitis may be associated with deposition of calcium pyrophosphate (chondrocalcinosis or pseudogout), mainly in the knees. Radiologic manifestations include cystic changes of the subchondral bones, loss of articular cartilage with narrowing of the joint space, diffuse demineralization, hypertrophic bone proliferation, and calcification of the synovium. The arthropathy tends to progress despite removal of iron by phlebotomy. Although the relation of these abnormalities to iron

1	hypertrophic bone proliferation, and calcification of the synovium. The arthropathy tends to progress despite removal of iron by phlebotomy. Although the relation of these abnormalities to iron metabolism is not known, the fact that similar changes occur in other forms of iron overload suggests that iron is directly involved.

1	Cardiac involvement is the presenting manifestation in about 15% of symptomatic patients. The most common manifestation is congestive heart failure, which occurs in about 10% of young adults with the disease, especially those with juvenile hemochromatosis. Symptoms of congestive heart failure may develop suddenly, with rapid progression to death if untreated. The heart is diffusely enlarged; this may be misdiagnosed as idiopathic cardiomyopathy if other overt manifestations are absent. Cardiac arrhythmias include premature supraventricular beats, paroxysmal tachyarrhythmias, atrial flutter, atrial fibrillation, and varying degrees of atrioventricular block.

1	Hypogonadism occurs in both sexes and may antedate other clinical features. Manifestations include loss of libido, impotence, amenorrhea, testicular atrophy, gynecomastia, and sparse body hair. These changes are primarily the result of decreased production of gonadotropins due to impairment of hypothalamic-pituitary function by iron deposition. Adrenal insufficiency, hypothyroidism, and hypoparathyroidism are rare manifestations. The association of (1) hepatomegaly, (2) skin pigmentation, (3) diabetes mellitus, (4) heart disease, (5) arthritis, and (6) hypogonadism should suggest the diagnosis. However, as stated above, significant iron overload may exist with none or only some of these manifestations. Therefore, a high index of suspicion is needed to make the diagnosis early. Treatment before permanent organ damage occurs can reverse the iron toxicity and restore life expectancy to normal.

1	The history should be particularly detailed in regard to disease in other family members; alcohol ingestion; iron intake; and ingestion of large doses of ascorbic acid, which promotes iron absorption (Chap. 96e). Appropriate tests should be performed to exclude iron deposition due to hematologic disease. The presence of liver, pancreatic, cardiac, and joint disease should be confirmed by physical examination, radiography, and standard function tests of these organs.

1	The degree of increase in total body iron stores can be assessed by (1) measurement of serum iron and the percent saturation of transferrin (or the unsaturated iron-binding capacity), (2) measurement of serum ferritin concentration, (3) liver biopsy with measurement of the iron concentration and calculation of the hepatic iron index (Table 428-2), and (4) magnetic resonance imaging (MRI) of the liver. In addition, a retrospective assessment of body-iron storage is also provided by performing weekly phlebotomy and calculating the amount of iron removed before iron stores are exhausted (1 mL blood = approximately 0.5 mg iron).

1	Each of these methods for assessing iron stores has advantages and limitations. The serum iron level and percent saturation of transferrin are elevated early in the course, but their specificity is reduced by significant false-positive and false-negative rates. For example, serum iron concentration may be increased in patients with alcoholic liver disease without iron overload; in this situation, however, the hepatic 2517 iron index is usually not increased as in hemochromatosis (Table 428-1). In otherwise healthy persons, a fasting serum transferrin saturation greater than 45% is abnormal and suggests homozygosity for hemochromatosis.

1	The serum ferritin concentration is usually a good index of body-iron stores, whether decreased or increased. In fact, an increase of 1 μg/L in serum ferritin level reflects an increase of about 5 mg in body stores. In most untreated patients with hemochromatosis, the serum ferritin level is significantly increased (Fig. 428-2 and Table 428-1), and a serum ferritin level >1000 μg/L is the strongest predictor of disease expression among individuals homozygous for the C282Y mutation. However, in patients with inflammation and hepatocellular necrosis, serum ferritin levels may be elevated out of proportion to body-iron stores due to increased release from tissues. Therefore, a repeat determination of serum ferritin should be carried out after acute hepatocellular damage has subsided (e.g., in alcoholic liver disease). Ordinarily, the combined measurements of the percent transferrin saturation and serum ferritin level provide a simple and reliable screening test for hemochromatosis,

1	in alcoholic liver disease). Ordinarily, the combined measurements of the percent transferrin saturation and serum ferritin level provide a simple and reliable screening test for hemochromatosis, including the precirrhotic phase of the disease. If either of these tests is abnormal, genetic testing for hemochromatosis should be performed (Fig. 428-3).

1	The role of liver biopsy in the diagnosis and management of hemochromatosis has been reassessed as a result of the widespread availability of genetic testing for the C282Y mutation. The absence of severe fibrosis can be accurately predicted in most patients using clinical and biochemical variables. Thus, there is virtually no risk of severe fibrosis in a C282Y homozygous subject with (1) serum ferritin level less than 1000 μg/L, (2) normal serum alanine aminotransferase values, (3) no hepatomegaly, and (4) no excess alcohol intake. However, it should be emphasized that liver biopsy is the only reliable method for establishing or excluding the presence of hepatic cirrhosis, which is the critical factor determining prognosis and the risk of developing hepatocellular carcinoma. Biopsy also permits histochemical estimation of tissue iron and measurement of hepatic iron concentration. Increased density of the liver due to iron deposition can be demonstrated by computed tomography (CT) or

1	permits histochemical estimation of tissue iron and measurement of hepatic iron concentration. Increased density of the liver due to iron deposition can be demonstrated by computed tomography (CT) or MRI, and with improved technology, MRI has become more accurate in determining hepatic iron concentration.

1	When the diagnosis of hemochromatosis is established, it is important to counsel and screen other family members (Chap. 84). Asymptomatic and symptomatic family members with the disease usually have an increased saturation of transferrin and an increased serum ferritin concentration. These changes occur even before the iron stores are greatly increased (Fig. 428-2). All adult first-degree relatives of patients with hemochromatosis should be tested for the C282Y and H63D mutations and counseled appropriately (Fig. 428-3). In affected individuals, it is important to confirm or exclude the presence of cirrhosis and begin therapy as early as possible. For children of an identified proband, testing for HFE of the other parent is helpful because if TAblE 428-2 REPRESEnTATivE iRon vAluES in noRmAl SubJECTS, PATiEnTS wiTH HEmoCHRomAToSiS, AnD PATiEnTS wiTH AlCoHoliC livER DiSEASE

1	TAblE 428-2 REPRESEnTATivE iRon vAluES in noRmAl SubJECTS, PATiEnTS wiTH HEmoCHRomAToSiS, AnD PATiEnTS wiTH AlCoHoliC livER DiSEASE Adult first-degree relative of patient with HH Subjects with unexplained liver disease Individual with suggestive symptoms (see text) Transferrin saturation and serum ferritin* TS <45% SF <300 TS ˜45% and/or SF >300 °gL Reassure, possibly retest later HFE Genotype PhlebotomyNormal Counsel and consider non-HFE hemochromatosis Serum ferritin – 300–1000 °g/L LFT normal Serum ferritin > 1000 °g/L and/or LFT abnormal Serum ferritin <300 °g/L LFT normal Observe retest in 1–2 years C282Y Homozygote C282Y/H63D (Compound Heterozygote) Confirmed iron overload *For convenience both genotype and phenotype (iron tests) can be performed together at a single visit in first-degree relatives. Liver biopsy No iron overload Investigate and treat as appropriate

1	FIGURE 428-3 Algorithm for screening for HFE-associated hemochromatosis. HH, hereditary hemochromatosis, homozygous subject (C282Y +/+); LFT, liver function tests; SF, serum ferritin concentration; TS, transferrin saturation. normal, the child is merely an obligate heterozygote and at no risk. Otherwise, for practical purposes, children need not be checked before they are 18 years old. The role of population screening for hemochromatosis is controversial. Recent studies indicate that it is highly effective for primary care physicians to screen subjects using transferrin saturation and serum ferritin levels. Such screening also detects iron deficiency. Genetic screening of the normal population is feasible but is probably not cost effective.

1	The therapy of hemochromatosis involves removal of the excess body iron and supportive treatment of damaged organs. Iron removal is best accomplished by weekly or twice-weekly phlebotomy of 500 mL. Although there is an initial modest decline in the volume of packed red blood cells to about 35 mL/dL, the level stabilizes after several weeks. The plasma transferrin saturation remains increased until the available iron stores are depleted. In contrast, the plasma ferritin concentration falls progressively, reflecting the gradual decrease in body-iron stores. One 500-mL unit of blood contains 200–250 mg of iron, and up to 25 g of iron or more may have to be removed. Therefore, in patients with advanced disease, weekly phlebotomy may be required for 1–2 years, and it should be continued until the serum ferritin level is <50 μg/L. Thereafter, phlebotomies are performed at appropriate intervals to maintain ferritin levels between 50 and 100 μg/L. Usually one phlebotomy every 3 months will

1	the serum ferritin level is <50 μg/L. Thereafter, phlebotomies are performed at appropriate intervals to maintain ferritin levels between 50 and 100 μg/L. Usually one phlebotomy every 3 months will suffice.

1	Chelating agents such as deferoxamine, when given parenterally, remove 10–20 mg of iron per day, which is much less than that mobilized by once-weekly phlebotomy. Phlebotomy is also less expensive, more convenient, and safer for most patients. However, chelating agents are indicated when anemia or hypoproteinemia is severe enough to preclude phlebotomy. Subcutaneous infusion of deferoxamine using a portable pump is the most effective means of its administration. An effective oral iron chelating agent, deferasirox (Exjade), has recently become available but is still in clinical trials. This agent is effective in thalassemia and secondary iron overload, but its role in primary iron overload has yet to be established.

1	Alcohol consumption should be severely curtailed or eliminated because it increases the risk of cirrhosis in hereditary hemochromatosis nearly tenfold. Dietary adjustments are unnecessary, although vitamin C and iron supplements should be avoided. The management of hepatic failure, cardiac failure, and diabetes mellitus is similar to conventional therapy for these conditions. Loss of libido and change in secondary sex characteristics are managed with testosterone replacement or gonadotropin therapy (Chap. 411). End-stage liver disease may be an indication for liver transplantation, although results are improved if the excess iron can be removed beforehand. The available evidence indicates that the fundamental metabolic abnormality in hemochromatosis is reversed by successful liver transplantation. The principal causes of death are cardiac failure, hepatocellular failure or portal hypertension, and hepatocellular carcinoma.

1	Life expectancy is improved by removal of the excessive stores of iron and maintenance of these stores at near-normal levels. The 5-year survival rate with therapy increases from 33 to 89%. With repeated phlebotomy, the liver decreases in size, liver function improves, pigmentation of skin decreases, and cardiac failure may be reversed. Diabetes improves in about 40% of patients, but removal of excess iron has little effect on hypogonadism or arthropathy. Hepatic fibrosis may decrease, but established cirrhosis is irreversible. Hepatocellular carcinoma occurs as a late sequela in patients who are cirrhotic at presentation. The apparent increase in its incidence in treated patients is probably related to their increased life span. Hepatocellular carcinoma rarely develops if the disease is treated in the precirrhotic stage. Indeed, the life expectancy of homozygotes treated before the development of cirrhosis is normal.

1	The importance of family screening and early diagnosis and treatment cannot be overemphasized. Asymptomatic individuals detected by family studies should have phlebotomy therapy if iron stores are moderately to severely increased. Assessment of iron stores at appropriate intervals is also important. With this management approach, most manifestations of the disease can be prevented.

1	There is considerable interest in the role of HFE mutations and hepatic iron in several other liver diseases. Several studies have shown an increased prevalence of HFE mutations in PCT patients. Iron accentuates the inherited enzyme deficiency in PCT and clinical manifestations of PCT. The situation in nonalcoholic steatohepatitis (NASH) is less clear, but some studies have shown an increased prevalence of HFE mutations in NASH patients. The role of phlebotomy therapy, however, is unproven. In chronic HCV infection, HFE mutations are not more common, but some subjects have increased hepatic iron. Before initiating antiviral therapy in these patients, it is reasonable to perform phlebotomy therapy to remove excess iron stores, because this reduces liver enzyme levels. HFE mutations are not increased in frequency in alcoholic liver disease. Hemochromatosis in a heavy drinker can be distinguished from alcoholic liver disease by the presence of the C282Y mutation.

1	HFE mutations are not increased in frequency in alcoholic liver disease. Hemochromatosis in a heavy drinker can be distinguished from alcoholic liver disease by the presence of the C282Y mutation. End-stage liver disease may also be associated with iron overload of the degree seen in hemochromatosis. The mechanism is uncertain, although studies have shown that alcohol suppresses hepatic hepcidin secretion. Hemolysis also plays a role. HFE mutations are uncommon. A recent large population study has suggested that subjects homozygous for C282Y are at increased risk of breast and colorectal cancer.

1	A recent large population study has suggested that subjects homozygous for C282Y are at increased risk of breast and colorectal cancer. The HFE mutation is of northern European origin (Celtic or Nordic) with a heterozygous carrier rate of approximately 1 in 10 (1 in 8 in Ireland). Thus, HFE-associated hemochromatosis is quite rare in non-European populations, e.g., Asia. However, nonHFE-associated hemochromatosis resulting from mutations in other genes involved in iron metabolism (Fig. 428-1) is ubiquitous and should be considered when one encounters iron overload. wilson’s Disease George J. Brewer Wilson’s disease is an autosomal recessive disorder caused by muta-tions in the ATP7B gene, which encodes a membrane-bound, copper-transporting ATPase. Clinical manifestations are caused by copper toxicity and primarily involve the liver and the brain. Because effective 429 treatment is available, it is important to make this diagnosis early.

1	The frequency of Wilson’s disease in most populations is about 1 in 30,000–40,000, and the frequency of carriers of ATP7B mutations is ∼1%. Siblings of a diagnosed patient have a 1 in 4 risk of Wilson’s disease, whereas children of an affected patient have about a 1 in 200 risk. Because a large number of inactivating mutations have been reported in the ATP7B gene, mutation screening for diagnosis is not routine, although this approach may be practical in the future. DNA haplotype analysis can be used to genotype siblings of an affected patient. A rare multisystem disorder of copper metabolism with features of both Menkes and Wilson’s diseases has been reported. It is termed the MEDNIK syndrome (mental retardation, enteropathy, deafness, neuropathy, ichthyosis, keratodermia) and is caused by mutations in the AP1S1 gene, which encodes an adaptor protein necessary for intracellular trafficking of copper pump proteins ATP7A (Menkes disease) and ATP7B (Wilson’s disease).

1	ATP7B protein deficiency impairs biliary copper excretion, resulting in positive copper balance, hepatic copper accumulation, and copper toxicity from oxidant damage. Excess hepatic copper is initially bound to metallothionein; liver damage begins as this storage capacity is exceeded, sometimes by 3 years of age. Defective copper incorporation into apoceruloplasmin leads to excess catabolism and low blood levels of ceruloplasmin. Serum copper levels are usually lower than normal because of low blood levels of ceruloplasmin, which normally binds >90% of serum copper. As the disease progresses, nonceruloplasmin serum copper (“free” copper) levels increase, resulting in copper buildup in other parts of the body (e.g., in the brain, with consequent neurologic and psychiatric disease).

1	CLINICAL PRESENTATION Hepatic Features Wilson’s disease may present as hepatitis, cirrhosis, or hepatic decompensation. Patients typically present in the midto late teenage years in Western countries, although the age of presentation is quite broad and extends into the fifth decade of life. An episode of hepatitis may occur—with elevated serum aminotransferase levels, with or without jaundice—and then spontaneously regress. Hepatitis often recurs, and most of these patients eventually develop cirrhosis. Hepatic decompensation is associated with elevated serum bilirubin, reduced serum albumin and coagulation factors, ascites, peripheral edema, and hepatic encephalopathy. In severe hepatic failure, hemolytic anemia may develop because large amounts of copper derived from hepatocellular necrosis are released into the bloodstream. The association of hemolysis and liver disease makes Wilson’s disease a likely diagnosis.

1	Neurologic Features The neurologic manifestations of Wilson’s dis-2519 ease typically occur in patients in their early twenties, although the age of onset extends into the sixth decade of life. MRI and CT scans reveal damage in the basal ganglia and occasionally in the pons, medulla, thalamus, cerebellum, and subcortical areas. The three main movement disorders include dystonia, incoordination, and tremor. Dysarthria and dysphagia are common. In some patients, the clinical picture closely resembles that of Parkinson’s disease. Dystonia can involve any part of the body and eventually leads to grotesque positions of the limbs, neck, and trunk. Autonomic disturbances may include orthostatic hypotension and sweating abnormalities as well as bowel, bladder, and sexual dysfunction. Memory loss, migraine-type headaches, and seizures may occur. Patients have difficulty focusing on tasks, but cognition usually is not grossly impaired. Sensory abnormalities and muscular weakness are not

1	loss, migraine-type headaches, and seizures may occur. Patients have difficulty focusing on tasks, but cognition usually is not grossly impaired. Sensory abnormalities and muscular weakness are not features of the disease.

1	Psychiatric Features Half of patients with neurologic disease have a history of behavioral disturbances with onset in the 5 years before diagnosis. The features are diverse and may include loss of emotional control (temper tantrums, crying bouts), depression, hyperactivity, or loss of sexual inhibition. Other Manifestations Some female patients have repeated spontaneous abortions, and most become amenorrheic prior to diagnosis. Cholelithiasis and nephrolithiasis occur with increased frequency. Some patients have osteoarthritis, particularly of the knee. Microscopic hematuria is common, and levels of urinary excretion of phosphates, amino acids, glucose, or urates may increase; however, a full-blown Fanconi syndrome is rare. Sunflower cataracts and Kayser-Fleischer rings (copper deposits in the outer rim of the cornea) may be seen. Electrocardiographic and other cardiac abnormalities have been reported but are not common.

1	Diagnostic tests for Wilson’s disease are listed in Table 429-1. Serum ceruloplasmin levels should not be used for definitive diagnosis, because they are normal in up to 10% of affected patients and are reduced in 20% of carriers. Kayser-Fleischer rings (Fig. 429-1) can be definitively diagnosed only by an ophthalmologist using a slit lamp. They are present in >99% of patients with neurologic/psychiatric forms of the disease and have been described very rarely in the absence of Wilson’s disease. Kayser-Fleischer rings are present in only ∼30–50% of patients diagnosed in the hepatic or presymptomatic state; thus, the absence of rings does not exclude the diagnosis.

1	Urine copper measurement is an important diagnostic tool, but urine must be collected carefully to avoid contamination. Symptomatic patients invariably have urine copper levels >1.6 μmol (>100 μg) per 24 h. Heterozygotes have values <1.3 μmol (<80 μg) per 24 h. About half of presymptomatic patients who are ultimately affected have diagnostically elevated urine copper values, but the other half have levels that are in an intermediate range between 0.9 and 1.6 μmol (60–100 μg) per 24 h. Because heterozygotes may have values up to 1.3 μmol (80 μg) per 24 h, patients in this range may require a liver biopsy for definitive diagnosis.

1	The gold standard for diagnosis remains liver biopsy with quantitative copper assays. Affected patients have values >3.1 μmol/g (>200 μg/g [dry weight] of liver). Copper stains are not reliable. False-positive results can occur with long-standing obstructive liver disease, which can elevate hepatic and urine copper concentrations and rarely causes Kayser-Fleischer rings. Recommended anticopper treatments are listed in Table 429-2. Penicillamine was previously the primary anticopper treatment but now plays only a minor role because of its toxicity and because it often worsens existing neurologic disease if used as initial therapy. If penicillamine is given, it should always be accompanied by pyridoxine (25 mg/d). Trientine is a less toxic chelator and is supplanting penicillamine when a chelator is indicated.

1	Absent Absent Present in >99% if neurologic or psychiatric symptoms are present 0.3–0.8 μmol (20–50 μg) Normal to 1.3 μmol (80 μg) >1.6 μmol (>100 μg) in symptomatic patients; 0.9 to >1.6 μmol (60 to >100 μg) in presymptomatic patients 0.3–0.8 μmol/g (20–50 Normal to 2.0 μmol (125 μg) >3.1 μmol (>200 μg) (Obstructive liver disease can μg/g of tissue) cause false-positive results.) aUsefulness range: + (somewhat useful) to ++++ (very useful).

1	For patients with hepatitis or cirrhosis but without evidence of hepatic decompensation or neurologic/psychiatric symptoms, zinc is the therapy of choice although some experts advocate therapy with trientine. Zinc has proven efficacy in Wilson’s disease and is essentially nontoxic. It produces a negative copper balance by blocking intestinal absorption of copper, and it induces hepatic metallothionein synthesis, thereby sequestering additional toxic copper. All presymptomatic patients should be treated prophylactically because the disease is close to 100% penetrant.

1	The first step in evaluating patients presenting with hepatic decompensation is to establish disease severity, which can be estimated with the Nazer prognostic index (Table 429-3). Patients with scores <7 can usually be managed with medical therapy. Patients with scores >9 should be considered immediately for liver transplantation. For patients with scores between 7 and 9, clinical judgment is required in deciding whether to recommend transplantation or medical therapy. A combination of trientine and zinc has been used to treat patients with Nazer scores as high as 9, but such patients should be watched carefully for indications of hepatic deterioration, which mandates transplantation.

1	For initial medical treatment of patients with hepatic decompensation, the recommended regimen is a chelator (preferably trientine) plus zinc (Table 429-2). Zinc should not, however, be ingested simultaneously with trientine, which chelates zinc and forms therapeutically ineffective complexes. Administration of the two drugs should be separated by at least 1 h.

1	For initial neurologic therapy, tetrathiomolybdate is emerging as the drug of choice because of its rapid control of free copper, preservation of neurologic function, and low toxicity. Penicillamine and trientine should be avoided because both have a high risk of worsening the neurologic condition. Until tetrathiomolybdate is commercially available, zinc therapy is recommended. Although it is relatively slow-acting, zinc itself does not exacerbate neurologic abnormalities. Although hepatic transplantation may alleviate neurologic symptoms, it does so only by copper removal, which can be done more safely and inexpensively with anticopper drugs. Pregnant patients should be treated with zinc or trientine throughout pregnancy but without tight copper control because copper deficiency can be teratogenic.

1	FIGURE 429-1 A Kayser-Fleischer ring. Although in this case, the brownish ring rimming the cornea is clearly visible to the naked eye, confirmation is usually made by slit-lamp examination. Anticopper therapy must be lifelong. With treatment, liver function usually recovers after about a year although residual liver damage is usually present. Neurologic and psychiatric symptoms usually improve after 6–24 months of treatment. When trientine or penicillamine is first used, it is necessary to monitor for drug toxicity, particularly bone marrow suppression and proteinuria. Complete blood counts, standard biochemical profiles, and a urinalysis should be performed at weekly intervals for 1 month, then at twice-weekly intervals for 2 or 3 months, then at monthly intervals for 3 or 4 months, and at 4to 6-month intervals thereafter.

1	The anticopper effects of trientine and penicillamine can be monitored by following 24-h “free” serum copper levels. Changes in urine copper levels are more difficult to interpret because excretion reflects the effect of the drug as well as body loading with copper. Free serum copper is calculated by subtracting the ceruloplasmin copper from aZinc acetate is supplied as Galzin, manufactured by Gate Pharmaceutical. The recommended adult dose for all the above indications is 50 mg of elemental zinc three times daily, with each dose separated by at least 1 h from consumption of food and beverages other than water as well as from trientine or penicillamine doses. bTrientine is supplied as Syprine and penicillamine as Cuprimine, both manufactured by Merck. The recommended adult dosage for both drugs is 500 mg twice daily, with each dose at least 0.5 h before or 2 h after meals and separated by at least 1 h from zinc administration. cTetrathiomolybdate is being studied in clinical trials.

1	the total serum copper. Each 10 mg/L (1 mg/dL) of ceruloplasmin contributes 0.5 μmol/L (3 μg/dL) of serum copper. The normal serum free copper value is 1.6–2.4 μmol/L (10–15 μg/dL); the level is often as high as 7.9 μmol/L (50 μg/dL) in untreated Wilson’s disease. With treatment, the serum free copper should be <3.9 μmol/L (<25 μg/dL).

1	Zinc treatment does not require monitoring of blood or urine for toxicity. Its only significant side effect is gastric burning or nausea in ∼10% of patients, usually with the first morning dose. This effect can be mitigated if the first dose is taken an hour after breakfast or if zinc is taken with a small amount of protein. Because zinc mainly affects stool copper, 24-h urine copper can be used to reflect body loading. The typical value in untreated symptomatic patients is >3.1 μmol (>200 μg) per 24 h. This level should decrease during the first 1–2 years of therapy to <2.0 μmol (<125 μg) per 24 h. A normal value (0.3–0.8 μmol [20–50 μg]) is rarely reached during the first decade of therapy and should raise concern about overtreatment (copper deficiency), the first sign of which is anemia and/or leukopenia.

1	The age of onset of clinical disease may be considerably younger in India and the Far East; in these regions, onset often occurs in children at only 5 or 6 years of age. The incidence of the disease may be increased in certain populations as a result of founder effects. For example, in Sardinia, the incidence may be 1 in 3000. In countries where penicillamine, trientine, and zinc acetate (as Galzin) are not available or are unaffordable, zinc salts such as gluco nate or sulfate provide an alternative treatment option. The Porphyrias Robert J. Desnick, Manisha Balwani The porphyrias are metabolic disorders, each resulting from the deficiency of a specific enzyme in the heme biosynthetic pathway (Fig. 430-1 and Table 430-1).These enzyme deficiencies are inherited as autosomal dominant, autosomal recessive, or X-linked traits, with 430 the exception of porphyria cutanea tarda (PCT), which usually is sporadic (Table 430-1). The porphyrias are classified as either hepatic or

1	dominant, autosomal recessive, or X-linked traits, with 430 the exception of porphyria cutanea tarda (PCT), which usually is sporadic (Table 430-1). The porphyrias are classified as either hepatic or erythropoietic, depending on the primary site of overproduction and accumulation of their respective porphyrin precursors or porphyrins (Tables 430-1 and 430-2), although some have overlapping features. For example, PCT, the most common porphyria, is hepatic and presents with blistering cutaneous photosensitivity, which is typically characteristic of the erythropoietic porphyrias. The major manifestations of the acute hepatic porphyrias are neurologic, including neuropathic abdominal pain, peripheral motor neuropathy, and mental disturbances, with attacks often precipitated by dieting, certain drugs, and hormonal changes. While hepatic porphyrias are symptomatic primarily in adults, rare homozygous variants of the autosomal dominant hepatic porphyrias usually manifest clinically prior to

1	drugs, and hormonal changes. While hepatic porphyrias are symptomatic primarily in adults, rare homozygous variants of the autosomal dominant hepatic porphyrias usually manifest clinically prior to puberty.

1	In contrast, the erythropoietic porphyrias usually present at birth or in early childhood with cutaneous photosensitivity, or in the case of congenital erythropoietic porphyria (CEP), even in utero as nonimmune hydrops fetalis. Cutaneous sensitivity to sunlight results from excitation of excess porphyrins in the skin by long-wave ultraviolet light, leading to cell damage, scarring, and disfigurement. Thus, the porphyrias are metabolic disorders in which environmental, physiologic, and genetic factors interact to cause disease.

1	Because many symptoms of the porphyrias are nonspecific, diagnosis is often delayed. Laboratory measurement of porphyrin precursors (5′-aminolevulinic acid [ALA] and porphobilinogen [PBG]) or porphyrins in urine, plasma, erythrocytes, or feces is required to confirm or exclude the various types of porphyria (see below). However, a definite diagnosis requires demonstration of the specific gene defect (Table 430-3). The genes encoding all the heme biosynthetic enzymes have been characterized, permitting identification of the mutations causing each porphyria (Table 430-2). Molecular genetic analyses now make it possible to provide precise heterozygote or homozygote identification and prenatal diagnoses in families with known mutations.

1	In addition to recent reviews of the porphyrias, informative and upto-date websites are sponsored by the American Porphyria Foundation (www.porphyriafoundation.com) and the European Porphyria Initiative (www.porphyria-europe.org). An extensive list of unsafe and safe drugs for individuals with acute porphyrias is provided at the Drug Database for Acute Porphyrias (www.drugs-porphyria.com).

1	The porphyrias are panethnic metabolic diseases that affect individuals around the globe. The acute hepatic porphyrias— acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), and variegate porphyria (VP)—are autosomal dominant disorders. The frequency of AIP, the most common acute hepatic porphyria, is ~1 in 20,000 among Caucasian individuals of Western European ancestry, and it is particularly frequent in Scandinavians, with a frequency of ~1 in 10,000 in Sweden. VP is particularly frequent in South Africa, where its high prevalence (>10,000 affected patients) is in part due to a genetic “founder effect.” The autosomal recessive acute hepatic porphyria, ALA dehydratase-deficient porphyria (ADP), is very rare, and less than 20 patients have been identified worldwide.

1	The erythropoietic protoporphyrias—CEP, erythropoietic protoporphyria (EPP), and X-linked protoporphyria (XLP)—also are pan-ethnic. EPP is the most common porphyria in children, whereas CEP is very rare, with about 200 reported cases worldwide. The frequency of EPP varies globally because most patients have the common low expression FECH mutation that varies in frequency in different populations. It rarely occurs in Africans, is present in about 10% of whites, and is frequent (~30%) in the Japanese. The autosomal recessive porphyrias—ADP, CEP, EPP, and hepatoerythropoietic porphyria (HEP)—are more frequent in regions with high rates of consanguineous unions. PCT, which is typically sporadic, occurs more frequently in countries in which its predisposing risk factors such as hepatitis C and HIV are more prevalent. Heme biosynthesis involves eight enzymatic steps in the conversion of glycine and succinyl-CoA to heme (Fig. 430-2 and Table 430-2). The Porphyrias

1	Heme biosynthesis involves eight enzymatic steps in the conversion of glycine and succinyl-CoA to heme (Fig. 430-2 and Table 430-2). The Porphyrias FIGURE 430-1 The human heme biosynthetic pathway indicating in linked boxes the enzyme that, when deficient, causes the respective porphyria. Hepatic porphyrias are shown in yellow boxes and erythropoietic porphyrias in pink boxes.

1	These eight enzymes are encoded by nine genes, as the first enzyme in the cytosol. Heme is required for a variety of hemoproteins such in the pathway, 5′-aminolevulinate synthase (ALA synthase), has as hemoglobin, myoglobin, respiratory cytochromes, and the cytochrome two genes that encode unique housekeeping (ALAS1) and erythroid-P450 enzymes (CYPs). Hemoglobin synthesis in erythroid precurspecific (ALAS2) isozymes. The first and last three enzymes in the sor cells accounts for approximately 85% of daily heme synthesis pathway are located in the mitochondrion, whereas the other four are in humans. Hepatocytes account for most of the rest, primarily for X-linked protoporphyria (XLP) ALA synthase 2 XL CP >100c Protoporphyrin — Protoporphyrin aPolymorphism in intron 3 of wild-type allele affects level of enzyme activity and clinical expression. bType I isomers. cIncreased activity due to “gain-of-function” mutations in ALAS2 exon 11.

1	Abbreviations: AD, autosomal dominant; ALA, 5-aminolevulinic acid; AR, autosomal recessive; COPRO I, coproporphyrin I; COPRO III, coproporphyrin III; CP, cutaneous photosensitivity; ISOCOPRO, isocoproporphyrin; + Nv, neurovisceral; PBG, porphobilinogen; PROTO, protoporphyrin IX; URO I, uroporphyrin I; URO III, uroporphyrin III; XL, X-linked. aNumber of exons and those encoding separate housekeeping and erythroid-specific forms indicated in parentheses. bNumber of known mutations from the Human Gene Mutation Database (www.hgmd.org). cCrystallized from human (H), murine (M), Escherichia coli (E), Bacillus subtilis (B), or yeast (Y) purified enzyme; references in Protein Data Bank (www.rcsb.org). Abbreviations: C, cytoplasm; M, mitochondria. Source: From KE Anderson et al: Disorders of heme biosynthesis: X-linked sideroblastic anemia and the porphyrias, in The Metabolic and Molecular Bases of Inherited Diseases, CR Scriver et al (eds). New York, McGraw-Hill, 2001, pp 2991–3062.

1	aNonspecific increases in zinc protoporphyrins are common in other porphyrias. Rule out other causes of elevated ALA; ↓↓RBC ALA dehydratase activity (<10%); ALA dehydratase mutation analysis Measure RBC HMB synthase: normal activity Measure RBC HMB synthase: normal activity RBC URO decarboxylase activity: half-normal in familial PCT (~20% of all PCT cases); substantially deficient in HEP URO decarboxylase mutation analysis: mutation(s) present in familial PCT (heterozygous) and HEP (homozygous) The Porphyrias

1	The Porphyrias Abbreviations: ADP, 5-ALA dehydratase-deficient porphyria; AIP, acute intermittent porphyria; ALA, 5-aminolevulinic acid; CEP, congenital erythropoietic porphyria; COPRO I, coproporphyrin I; COPRO III, coproporphyrin III; EPP, erythropoietic protoporphyria; F, fecal; HCP, hereditary coporphyria; HEP, ; ISOCOPRO, isocoproporphyrin; P, plasma; PBG, porphobilinogen; PCT, porphyria cutanea tarda; PROTO, protoporphyrin IX; RBC, erythrocytes; U, urine; URO I, uroporphyrin I; URO III, uroporphyrin III; VP, variegate porphyria; XLP, X-linked protoporphyria. Source: Based on KE Anderson et al: Ann Intern Med 142:439, 2005. FIGURE 430-2 The heme biosynthetic pathway showing the eight enzymes and their substrates and products. Four of the enzymes are localized in the mitochondria and four in the cytosol.

1	the synthesis of CYPs, which are especially abundant in the liver (e.g., housekeeping) and erythroid-specific forms of ALA synthase are endoplasmic reticulum, and turn over more rapidly than many other encoded by separate genes located on chromosome 3p21.1 (ALAS1) hemoproteins, such as the mitochondrial respiratory cytochromes. As and Xp11.2 (ALAS2), respectively. Defects in the erythroid gene shown in Fig. 430-2, pathway intermediates are the porphyrin precur-ALAS2 that decrease its activity cause X-linked sideroblastic anemia sors, ALA and PBG, and porphyrins (mostly in their reduced forms, (XLSA). Recently, gain-of-function mutations in the last exon (11) of known as porphyrinogens). At least in humans, these intermediates do ALAS2 that increase its activity have been shown to cause an X-linked not accumulate in significant amounts under normal conditions or form of EPP, known as X-linked protoporphyria (XLP). have important physiologic functions. The second enzyme, ALA

1	to cause an X-linked not accumulate in significant amounts under normal conditions or form of EPP, known as X-linked protoporphyria (XLP). have important physiologic functions. The second enzyme, ALA dehydratase, catalyzes the condensation

1	The first enzyme, ALA synthase, catalyzes the condensation of gly-of two molecules of ALA to form PBG. Hydroxymethylbilane synthase cine, activated by pyridoxal phosphate and succinyl coenzyme A, to (HMB synthase; also known as PBG deaminase) catalyzes the head-toform ALA. In the liver, this rate-limiting enzyme can be induced by a tail condensation of four PBG molecules by a series of deaminations variety of drugs, steroids, and other chemicals. Distinct nonerythroid to form the linear tetrapyrrole, HMB. Uroporphyrinogen III synthase (URO synthase) catalyzes the rearrangement and rapid cyclization of HMB to form the asymmetric, physiologic, octacarboxylate porphyrinogen, uroporphyrinogen (URO’gen) III.

1	The fifth enzyme in the pathway, uroporphyrinogen decarboxylase (URO decarboxylase), catalyzes the sequential removal of the four carboxyl groups from the acetic acid side chains of URO’gen III to form coproporphyrinogen (COPRO’gen) III, a tetracarboxylate porphyrinogen. This compound then enters the mitochondrion via a specific transporter, ABCB6, where COPRO oxidase, the sixth enzyme, catalyzes the decarboxylation of two of the four propionic acid groups to form the two vinyl groups of protoporphyrinogen (PROTO’gen) IX, a decarboxylate porphyrinogen. Next, PROTO oxidase oxidizes PROTO gen to protoporphyrin IX by the removal of six hydrogen atoms. The product of the reaction is a porphyrin (oxidized form), in contrast to the preceding tetrapyrrole intermediates, which are porphyrinogens (reduced forms). Finally, ferrous iron is inserted into protoporphyrin to form heme, a reaction catalyzed by the eighth enzyme in the pathway, ferrochelatase (also known as heme synthetase or

1	(reduced forms). Finally, ferrous iron is inserted into protoporphyrin to form heme, a reaction catalyzed by the eighth enzyme in the pathway, ferrochelatase (also known as heme synthetase or protoheme ferrolyase).

1	Regulation of heme synthesis differs in the two major heme-forming tissues, the liver and erythron. In the liver, the concentration of “free” heme regulates the synthesis and mitochondrial translocation of the housekeeping form of ALA synthase 1. Heme represses the synthesis of the ALA synthase 1 mRNA and interferes with the transport of the enzyme from the cytosol into mitochondria. Hepatic ALA synthase 1 is increased by many of the same chemicals that induce the cytochrome P450 enzymes in the endoplasmic reticulum of the liver. Because most of the heme in the liver is used for the synthesis of cytochrome P450 enzymes, hepatic ALA synthase 1 and the cytochrome P450s are regulated in a coordinated fashion, and many drugs that induce hepatic ALA synthase 1 also induce the CYP genes. The other hepatic heme biosynthetic enzymes are presumably expressed at constant levels, although their relative activities and kinetic properties differ. For example, normal individuals have high

1	The other hepatic heme biosynthetic enzymes are presumably expressed at constant levels, although their relative activities and kinetic properties differ. For example, normal individuals have high activities of ALA dehydratase, but low activities of HMB synthase, the latter being the second rate-limiting step in the pathway.

1	In the erythron, novel regulatory mechanisms allow for the production of the very large amounts of heme needed for hemoglobin synthesis. The response to stimuli for hemoglobin synthesis occurs during cell differentiation, leading to an increase in cell number. In contrast, the erythroid-specific ALA synthase 2 is expressed at higher levels than the housekeeping enzyme, and erythroid-specific control mechanisms regulate other pathway enzymes as well as iron transport into erythroid cells. Separate erythroid-specific and nonerythroid or “housekeeping” transcripts are known for the first four enzymes in the pathway. As noted above, housekeeping and erythroid-specific ALA synthases are encoded by genes on different chromosomes, but for each of the next three genes in the pathway, both erythroid and nonerythroid transcripts are transcribed by alternative promoters from their single respective genes (Table 430-2).

1	As mentioned above, the porphyrias can be classified as either hepatic or erythropoietic, depending on whether the heme biosynthetic intermediates that accumulate arise initially from the liver or developing erythrocytes, or as acute or cutaneous, based on their clinical manifestations. Table 430-1 lists the porphyrias, their principal symptoms, and major biochemical abnormalities. Four of the five hepatic porphyr-ias—AIP, HCP, VP, and ADP—present during adult life with acute attacks of neurologic manifestations and elevated levels of one or both of the porphyrin precursors, ALA and PBG, and are thus classified as acute porphyrias. Patients with ADP have presented in infancy and adolescence. The fifth hepatic disorder, PCT, presents with blistering skin lesions. HCP and VP also may have cutaneous manifestations similar to PCT.

1	The erythropoietic porphyrias—CEP, EPP, and the recently described XLP—are characterized by elevations of porphyrins in bone marrow and erythrocytes and present with cutaneous photosensitiv-2525 ity. The skin lesions in CEP resemble PCT but are usually much more severe, whereas EPP and XLP cause a more immediate, painful, and nonblistering type of photosensitivity. EPP is the most common porphyria to cause symptoms before puberty. Around 20% of EPP patients develop minor abnormalities of liver function, with up to about 5% developing hepatic complications that can become life-threatening. XLP has a clinical presentation similar to EPP causing photosensitivity and liver disease.

1	A few specific and sensitive first-line laboratory tests should be used whenever symptoms or signs suggest the diagnosis of porphyria (Table 430-3). If a first-line test is significantly abnormal, more comprehensive testing should follow to establish the type of porphyria, including the specific causative gene mutation.

1	Acute Porphyrias An acute porphyria should be suspected in patients with neurovisceral symptoms after puberty, such as abdominal pain, and when the initial clinical evaluation does not suggest another cause. The urinary porphyrin precursors (ALA and PBG) should be measured (Fig. 430-2). Urinary PBG is virtually always increased during acute attacks of AIP, HCP, and VP and is not substantially increased in any other medical condition. Therefore, this measurement is both sensitive and specific. A method for rapid, in-house testing for urinary PBG, such as the Trace PBG kit (Thermo Scientific), can be used. Results from spot (single-void) urine specimens are highly informative because very substantial increases in PBG are expected during acute attacks of porphyria. A 24-h collection can unnecessarily delay diagnosis. The same spot urine specimen should be saved for quantitative determination of ALA, PBG, and creatinine, in order to confirm the qualitative PBG result and also to detect

1	delay diagnosis. The same spot urine specimen should be saved for quantitative determination of ALA, PBG, and creatinine, in order to confirm the qualitative PBG result and also to detect patients with ADP. Urinary porphyrins may remain increased longer than porphyrin precursors in HCP and VP. Therefore, it is useful to measure total urinary porphyrins in the same sample, keeping in mind that urinary porphyrin increases are often nonspecific. Measurement of urinary porphyrins alone should be avoided for screening, because these may be increased in disorders other than porphyrias, such as chronic liver disease, and misdiagnoses of porphyria can result from minimal increases in urinary porphyrins that have no diagnostic significance. Measurement of erythrocyte HMB synthase is not useful as a first-line test. Moreover, the enzyme activity is not decreased in all AIP patients, a borderline low normal value is not diagnostic, and the enzyme is not deficient in other acute porphyrias.

1	Cutaneous Porphyrias Blistering skin lesions due to porphyria are virtually always accompanied by increases in total plasma porphyrins. A fluorometric method is preferred, because the porphyrins in plasma in VP are mostly covalently linked to plasma proteins and may be less readily detected by high-performance liquid chromatography (HPLC). The normal range for plasma porphyrins is somewhat increased in patients with end-stage renal disease.

1	Although a total plasma porphyrin determination will usually detect EPP and XLP, an erythrocyte protoporphyrin determination is more sensitive. Increases in erythrocyte protoporphyrin occur in many other conditions. Therefore, the diagnosis of EPP must be confirmed by showing a predominant increase in free protoporphyrin rather than zinc protoporphyrin. In XLP, both free and zinc protoporphyrin are markedly increased in approximately equal proportions. Interpretation of laboratory reports can be difficult, because the term free erythrocyte protoporphyrin sometimes actually represents zinc protoporphyrin.

1	More extensive testing is justified when an initial test is positive. A substantial increase in PBG may be due to AIP, HCP, or VP. These acute porphyrias can be distinguished by measuring urinary porphyrins (using the same spot urine sample), fecal porphyrins, and plasma porphyrins. Assays for COPRO oxidase or PROTO oxidase are not widely available. More specifically, mutation analysis by sequencing the genes encoding HMB synthase, COPRO oxidase, and PROTO oxidase will detect almost all disease-causing mutations, and will be

1	The Porphyrias 2526 diagnostic even when the levels of urinary ALA and PBG have returned to normal or near normal. The various porphyrias that cause blistering skin lesions are differentiated by measuring porphyrins in urine, feces, and plasma. These porphyrias also should be confirmed at the DNA level by the demonstration of the causative gene mutation(s). It is often difficult to diagnose or “rule out” porphyria in patients who have had suggestive symptoms months or years in the past, and in relatives of patients with acute porphyrias, because porphyrin precursors and porphyrins may be normal. In those situations, detection of the specific gene mutation in the index case can make the diagnosis and facilitate the diagnosis and genetic counseling of at-risk relatives. Consultation with a specialist laboratory and physician will assist in selecting the heme biosynthetic gene or genes to be sequenced.

1	Markedly elevated plasma and urinary concentrations of the porphyrin precursors, ALA and/or PBG, which originate from the liver, are especially evident during attacks of neurologic manifestations of the four acute porphyrias—ADP, AIP, HCP, and VP. In PCT, excess porphyrins also accumulate initially in the liver and cause chronic blistering of sun-exposed areas of the skin.

1	ADP is a rare autosomal recessive acute hepatic porphyria caused by a severe deficiency of ALA dehydratase activity. To date, there are only a few documented cases, some in children or young adults, in which specific gene mutations have been identified. These affected homozygotes had <10% of normal ALA dehydratase activity in erythrocytes, but their clinically asymptomatic parents and heterozygous relatives had about half-normal levels of activity and did not excrete increased levels of ALA. The frequency of ADP is unknown, but the frequency of heterozygous individuals with <50% normal ALA dehydratase activity was ~2% in a screening study in Sweden. Because there are multiple causes for deficient ALA dehydratase activity, it is important to confirm the diagnosis of ADP by mutation analysis.

1	Clinical Features The clinical presentation depends on the amount of residual ALA dehydratase activity. Four of the documented patients were male adolescents with symptoms resembling those of AIP, including abdominal pain and neuropathy. One patient was an infant with more severe disease, including failure to thrive beginning at birth. The earlier age of onset and more severe manifestations in this patient reflect a more significant deficiency of ALA dehydratase activity. Another patient developed an acute motor polyneuropathy at age 63 that was associated with a myeloproliferative disorder. He was heterozygous for an ALAD mutation that presumably was present in erythroblasts that underwent clonal expansion due to the bone marrow malignancy.

1	Diagnosis All patients had significantly elevated levels of plasma and urinary ALA and urinary coproporphyrin (COPRO) III; ALAD activities in erythrocytes were <10% of normal. Hereditary tyrosinemia type 1 (fumarylacetoacetase deficiency) and lead intoxication should be considered in the differential diagnosis because either succinylacetone (which accumulates in hereditary tyrosinemia and is structurally similar to ALA) or lead can inhibit ALA dehydratase, increase urinary excretion of ALA and COPRO III, and cause manifestations that resemble those of the acute porphyrias. Heterozygotes are clinically asymptomatic and do not excrete increased levels of ALA but can be detected by demonstration of intermediate levels of erythrocyte ALA dehydratase activity or a specific mutation in the ALAD gene. To date, molecular studies of ADP patients have identified nine point mutations, two splice-site mutations, and a two-base deletion in the ALAD gene (Human Gene Mutation Database;

1	in the ALAD gene. To date, molecular studies of ADP patients have identified nine point mutations, two splice-site mutations, and a two-base deletion in the ALAD gene (Human Gene Mutation Database; www.hgmd.org). The parents in each case were not consanguineous, and the index cases had inherited a different ALAD mutation from each parent. Prenatal diagnosis of this disorder is possible by determination of ALA dehydratase activity and/or gene mutations in cultured chorionic villi or amniocytes.

1	The treatment of ADP acute attacks is similar to that of AIP (see below). The severely affected infant referred to above was supported by hyperalimentation and periodic blood transfusions but did not respond to intravenous hemin and died after liver transplantation. This hepatic porphyria is an autosomal dominant condition resulting from the half-normal level of HMB synthase activity. The disease is widespread but is especially common in Scandinavia and Great Britain. Clinical expression is highly variable, and activation of the disease is often related to environmental or hormonal factors, such as drugs, diet, and steroid hormones. Attacks can be prevented by avoiding known precipitating factors. Rare homozygous dominant AIP also has been described in children (see below).

1	Clinical Features Induction of the rate-limiting hepatic enzyme ALA synthase in heterozygotes who have half-normal HMB synthase activity is thought to underlie the acute attacks in AIP. The disorder remains latent (or asymptomatic) in the great majority of those who are heterozygous for HMBS mutations, and this is almost always the case prior to puberty. In patients with no history of acute symptoms, porphyrin precursor excretion is usually normal, suggesting that half-normal hepatic HMB synthase activity is sufficient and that hepatic ALA synthase activity is not increased. However, under conditions where heme synthesis is increased in the liver, half-normal HMB synthase activity may become limiting, and ALA, PBG, and other heme pathway intermediates may accumulate and be excreted in the urine. Common precipitating factors include endogenous and exogenous steroids, porphyrinogenic drugs, alcohol ingestion, and low-calorie diets, usually instituted for weight loss.

1	The fact that AIP is almost always latent before puberty suggests that adult levels of steroid hormones are important for clinical expression. Symptoms are more common in women, suggesting a role for estrogens or progestins. Premenstrual attacks are probably due to endogenous progesterone. Acute porphyrias are sometimes exacerbated by exogenous steroids, including oral contraceptive preparations containing progestins. Surprisingly, pregnancy is usually well tolerated, suggesting that beneficial metabolic changes may ameliorate the effects of high levels of progesterone. Table 430-4 provides a partial list of the major drugs that are harmful in AIP (and also in HCP and VP). Extensive lists of unsafe and safe drugs are available on websites sponsored by the American Porphyria Foundation (www .porphyriafoundation.com) and the European Porphyria Initiative (www.porphyria-europe.org), and at the Drug Database for Acute Porphyrias website (www.drugs-porphyria.com). Reduced intake of

1	(www .porphyriafoundation.com) and the European Porphyria Initiative (www.porphyria-europe.org), and at the Drug Database for Acute Porphyrias website (www.drugs-porphyria.com). Reduced intake of calories and carbohydrate, as may occur with illness or attempts to lose weight, can also increase porphyrin precursor excretion and induce attacks of porphyria. Increased carbohydrate intake may ameliorate attacks. Studies in a knockout AIP mouse model indicate that the hepatic ALAS1 gene is regulated by the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). Hepatic PGC-1α is induced by fasting, which in turn activates ALAS1 transcription, resulting in increased heme biosynthesis. This finding suggests an important link between nutritional status and the attacks in acute porphyrias. Attacks also can be provoked by infections, surgery, and ethanol.

1	Because the neurovisceral symptoms rarely occur before puberty and are often nonspecific, a high index of suspicion is required to make the diagnosis. The disease can be disabling but is rarely fatal. Abdominal pain, the most common symptom, is usually steady and poorly localized but may be cramping. Ileus, abdominal distention, and decreased bowel sounds are common. However, increased bowel sounds and diarrhea may occur. Abdominal tenderness, fever, and leukocytosis are usually absent or mild because the symptoms are neurologic rather than inflammatory. Nausea; vomiting; constipation; tachycardia; hypertension; mental symptoms; pain in the limbs, head, neck, or chest; muscle weakness; sensory loss; dysuria; and urinary The Porphyrias

1	The Porphyrias Note: Based on list in “Patient’s and Doctor’s Guide to Medication in Acute Porphyria,” Swedish Porphyria Association and Porphyria Centre Sweden. Also see the website Drug Database for Acute Porphyrias (www.drugs-porphyria.com) for a searchable list of safe and unsafe drugs. retention are characteristic. Tachycardia, hypertension, restlessness, tremors, and excess sweating are due to sympathetic overactivity.

1	retention are characteristic. Tachycardia, hypertension, restlessness, tremors, and excess sweating are due to sympathetic overactivity. The peripheral neuropathy is due to axonal degeneration (rather than demyelinization) and primarily affects motor neurons. Significant neuropathy does not occur with all acute attacks; abdominal symptoms are usually more prominent. Motor neuropathy affects the proximal muscles initially, more often in the shoulders and arms. The course and degree of involvement are variable and sometimes may be focal and involve cranial nerves. Deep tendon reflexes initially may be normal or hyperactive but become decreased or absent as the neuropathy advances. Sensory changes such as paresthesia and loss of sensation are less prominent. Progression to respiratory and bulbar paralysis and death occurs especially when the diagnosis and treatment are delayed. Sudden death may result from sympathetic overactivity and cardiac arrhythmia.

1	Mental symptoms such as anxiety, insomnia, depression, disorientation, hallucinations, and paranoia can occur in acute attacks. Seizures can be due to neurologic effects or to hyponatremia. Treatment of seizures is difficult because most antiseizure drugs can exacerbate AIP (clonazepam may be safer than phenytoin or barbiturates). Hyponatremia results from hypothalamic involvement and inappropriate vasopressin secretion or from electrolyte depletion due to vomiting, diarrhea, poor intake, or excess renal sodium loss. Persistent hypertension and impaired renal function may occur. When an attack resolves, abdominal pain may disappear within hours, and paresis begins to improve within days and may continue to improve over several years.

1	Homozygous dominant AIP is a rare form of AIP in which patients inherit HMBS mutations from each of their heterozygous parents and, therefore, have very low (<2%) enzyme activity. The disease has been described in a Dutch girl, two young British siblings, and a Spanish boy. In these homozygous affected patients, the disease presented in infancy with failure to thrive, developmental delay, bilateral cataracts, and/or hepatosplenomegaly. Urinary ALA and PBG concentrations were markedly elevated. All of these patients’ HMBS mutations (R167W, R167Q, and R172Q) were in exon 10 within five bases of each other. Studies of the brain magnetic resonance images (MRIs) of children with homozygous AIP have suggested damage primarily in white matter that was myelinated postnatally, while tracks that myelinated prenatally were normal. Most children with homozygous AIP die at an early age.

1	Diagnosis ALA and PBG levels are substantially increased in plasma and urine, especially during acute attacks, and become normal only after prolonged latency. For example, urinary PBG excretion during an attack is usually 50–200 mg/24 h (220–880 μmol/24 h) (normal, 0–4 mg/24 h, [0–18 μmol/24 h]), and urinary ALA excretion is 20–100 mg/24 h (150–760 μmol/24 h) (normal, 1–7 mg/24 h [8–53 μmol/24 h]). Because levels often remain high after symptoms resolve, the diagnosis of an acute attack in a patient with biochemically proven AIP is based primarily on clinical features. Excretion of ALA and PBG decreases over a few days after intravenous hemin administration. A normal urinary PBG level before hemin effectively excludes AIP as a cause for current symptoms. Fecal porphyrins are usually normal or minimally increased in AIP, in contrast to HCP and VP. Most AIP heterozygotes with no history of symptoms have normal urinary excretion of ALA and PBG. Therefore, the detection of the family’s

1	or minimally increased in AIP, in contrast to HCP and VP. Most AIP heterozygotes with no history of symptoms have normal urinary excretion of ALA and PBG. Therefore, the detection of the family’s HMBS mutation will diagnose asymptomatic family members.

1	Patients with HMBS mutations in the initiation of translation codon in exon 1 and in the intron 15′-splice donor site have normal enzyme levels in erythrocytes and deficient activity only in nonerythroid tissues. This occurs because the erythroid and housekeeping forms of HMB synthase are encoded by a single gene, which has two promoters. Thus, the enzyme assay may not be diagnostic, and genetic testing should be used to confirm the diagnosis. More than 390 HMBS mutations have been identified in AIP, including missense, nonsense, and splicing mutations and insertions and deletions, with most mutations found in only one or a few families (Human Gene Mutation Database, www.hgmd.org). The prenatal diagnosis of a fetus at risk can be made with cultured amniotic cells or chorionic villi. However, this is seldom done, because the prognosis of individuals with HMBS mutations is generally favorable.

1	During acute attacks, narcotic analgesics may be required for abdominal pain, and phenothiazines are useful for nausea, vomiting, anxiety, and restlessness. Chloral hydrate can be given for insomnia, and benzodiazepines are probably safe in low doses if a minor tranquilizer is required. Carbohydrate loading, usually with intravenous glucose (at least 300 g daily), may be effective in milder acute attacks of porphyria (without paresis, hyponatremia, etc.) if hemin is not available. Intravenous hemin is more effective and should be used as first-line therapy for all acute attacks. The standard regimen is 3–4 mg/kg of heme, in the form of lyophilized hematin (Recordati Pharmaceuticals), heme albumin (hematin reconstituted with human albumin), or heme arginate (Orphan Europe), infused daily for 4 days. Heme arginate and heme albumin are chemically stable and are less likely than hematin to produce phlebitis or an anticoagulant effect. Recovery depends on the degree of neuronal damage and

1	4 days. Heme arginate and heme albumin are chemically stable and are less likely than hematin to produce phlebitis or an anticoagulant effect. Recovery depends on the degree of neuronal damage and usually is rapid if therapy is started early. Recovery from severe motor neuropathy may require months or years. Identification and avoidance of inciting factors can hasten recovery from an attack and prevent future attacks. Inciting factors are usually multiple, and removal of one or more hastens recovery and helps prevent future attacks. Frequent attacks that occur during the luteal phase of the menstrual cycle may be prevented with a gonadotropin-releasing hormone analogue, which prevents ovulation and progesterone production, or by prophylactic hematin administration.

1	The long-term risk of hypertension and chronic renal disease is increased in AIP; a number of patients have undergone successful renal transplantation. Chronic, low-grade abnormalities in liver function tests are common, and the risk of hepatocellular carcinoma is increased. Hepatic imaging is recommended at least yearly for early detection of these tumors. An allogeneic liver transplant was performed on a 19-year-old female AIP heterozygote who had 37 acute attacks in the 29 months

1	The Porphyrias 2530 prior to transplantation. After transplantation, her elevated urinary ALA and BPG levels returned to normal in 24 h, and she did not experience acute neurologic attacks for more than 3 years after transplant. Two AIP patients had combined liver and kidney transplants secondary to uncontrolled acute porphyria attacks, chronic peripheral neuropathy, and renal failure requiring dialysis. Both patients had a marked improvement with no attacks and normal urinary PBG levels after transplantation, as well as improvement of their neuropathic manifestations. More recently, a group from the United Kingdom reported their experience with liver transplantation in 10 AIP patients with recurrent attacks that were refractory to medical management and impaired quality of life. Patients had a complete biochemical and symptomatic resolution after transplant. The investigators reported a high rate of hepatic artery thrombosis in their series. Clearly, liver transplantation is a

1	had a complete biochemical and symptomatic resolution after transplant. The investigators reported a high rate of hepatic artery thrombosis in their series. Clearly, liver transplantation is a high-risk procedure and should be considered as a last resort in patients with severe recurrent attacks. Recently, liver-directed gene therapy has proven successful in the prevention of drug-induced biochemical attacks in a murine model of human AIP, and clinical trials of AAV-HMBS gene transfer have been initiated. In addition, preclinical studies of a hepatic-targeted RNA interference (RNAi) therapy directed to inhibit the markedly elevated hepatic ALAS1 mRNA in the AIP mouse model prevented induced biochemical attacks and rapidly reduced the ALAS1 mRNA during an ongoing attack.

1	PCT, the most common of the porphyrias, can be either sporadic (type 1) or familial (type 2) and can also develop after exposure to halogenated aromatic hydrocarbons. Hepatic URO decarboxylase is deficient in all types of PCT, and for clinical symptoms to manifest, this enzyme deficiency must be substantial (~20% of normal activity or less); it is currently attributed to generation of an URO decarboxylase inhibitor in the liver, which forms uroporphomethene in the presence of iron and under conditions of oxidative stress. The majority of PCT patients (~80%) have no UROD mutations and are said to have sporadic (type 1) disease. PCT patients heterozygous for UROD mutations have familial (type 2) PCT. In these patients, inheritance of a UROD mutation from one parent results in half-normal enzyme activity in liver and all other tissues, which is a significant predisposing factor, but is insufficient by itself to cause symptomatic PCT. As discussed below, other genetic and environmental

1	enzyme activity in liver and all other tissues, which is a significant predisposing factor, but is insufficient by itself to cause symptomatic PCT. As discussed below, other genetic and environmental factors contribute to susceptibility for both types of PCT. Because penetrance of the genetic trait is low, many patients with familial (type 2) PCT have no family history of the disease. HEP is an autosomal recessive form of porphyria that results from the marked systemic deficiency of URO decarboxylase activity with clinical symptoms in childhood.

1	Clinical Features Blistering skin lesions that appear most commonly on the backs of the hands are the major clinical feature (Fig. 430-3). These rupture and crust over, leaving areas of atrophy and scarring. Lesions may also occur on the forearms, face, legs, and feet. Skin friability and small white papules termed milia are common, especially on the backs of the hands and fingers. Hypertrichosis and hyperpigmentation, especially of the face, are especially troublesome in women. Occasionally, the skin over sun-exposed areas becomes severely thickened, with scarring and calcification that resembles systemic sclerosis. Neurologic features are absent.

1	A number of susceptibility factors, in addition to inherited UROD mutations in type 2 PCT, can be recognized clinically and can affect management. These include hepatitis C, HIV, excess alcohol, elevated iron levels, and estrogens. The importance of excess hepatic iron as a precipitating factor is underscored by the finding that the incidence of the common hemochromatosis-causing mutations, hemochromatosis gene (HFE) mutations C282Y and H63D, are increased in patients with types 1 and 2 PCT (Chap. 428). Excess alcohol is a long-recognized contributor, as is estrogen use in women. HIV is probably an independent but less common risk factor that, like hepatitis C, does not cause PCT in isolation. Multiple susceptibility factors that appear to act synergistically can be identified in the individual PCT patient. Patients with PCT characteristically have chronic liver disease and sometimes cirrhosis and are at risk for hepatocellular carcinoma. Various chemicals can also induce PCT; an

1	individual PCT patient. Patients with PCT characteristically have chronic liver disease and sometimes cirrhosis and are at risk for hepatocellular carcinoma. Various chemicals can also induce PCT; an epidemic of PCT occurred in eastern Turkey in the 1950s as a consequence of wheat contaminated with the fungicide hexachlorobenzene. PCT also occurs after exposure to other chemicals, including diand trichlorophenols and 2,3,7,8-tetrachlorodibenzo-(p)-dioxin (TCDD, dioxin).

1	FIGURE 430-3 Typical cutaneous lesions in a patient with porphyria cutanea tarda. Chronic, crusted lesions resulting from blistering due to photosensitivity on the dorsum of the hand of a patient with porphyria cutanea tarda. (Courtesy of Dr. Karl E. Anderson; with permission.) Diagnosis Porphyrins are increased in the liver, plasma, urine, and stool. The urinary ALA level may be slightly increased, but the PBG level is normal. Urinary porphyrins consist mostly of uroporphyins and heptacarboxylate porphyrin, with lesser amounts of coproporphyrin and hexaand pentacarboxylate porphyrins. Plasma porphyrins are also increased, and fluorometric scanning of diluted plasma at neutral pH can rapidly distinguish VP and PCT (Table 430-3). Isocoproporphyrins, which are increased in feces and sometimes in plasma and urine, are diagnostic for hepatic URO decarboxylase deficiency.

1	Type 2 PCT and HEP can be distinguished from type 1 by finding decreased URO decarboxylase in erythrocytes. URO decarboxylase activity in liver, erythrocytes, and cultured skin fibroblasts in type 2 PCT is approximately 50% of normal in affected individuals and in family members with latent disease. In HEP, the URO decarboxylase activity is markedly deficient, with typical levels of 3–10% of normal. Over 121 mutations have been identified in the UROD gene (Human Gene Mutation Database; www.hgmd.org). Of the mutations listed in the database, ~65% are missense or nonsense and ~10% are splice-site mutations. Most UROD mutations have been identified in only one or two families.

1	Alcohol, estrogens, iron supplements, and, if possible, any drugs that may exacerbate the disease should be discontinued, but this step does not always lead to improvement. A complete response can almost always be achieved by the standard therapy, repeated phlebotomy, to reduce hepatic iron. A unit (450 mL) of blood can be removed every 1–2 weeks. The aim is to gradually reduce excess hepatic iron until the serum ferritin level reaches the lower limits of normal. Because iron overload is not marked in most cases, remission may occur after only five or six phlebotomies; however, PCT patients with hemochromatosis may require more treatments to bring their iron levels down to the normal range. To document improvement in PCT, it is most convenient to follow the total plasma porphyrin concentration, which becomes normal some time after the target ferritin level is reached. Hemoglobin levels or hematocrits and serum ferritin should be followed closely to prevent development of iron

1	concentration, which becomes normal some time after the target ferritin level is reached. Hemoglobin levels or hematocrits and serum ferritin should be followed closely to prevent development of iron deficiency and anemia. After remission, continued phlebotomy may not be needed. Plasma porphyrin levels are followed at 6to 12-month intervals for early detection of recurrences, which are treated by additional phlebotomy.

1	An alternative when phlebotomy is contraindicated or poorly tolerated is a low-dose regimen of chloroquine or hydroxychloroquine, both of which complex with the excess porphyrins and promote their excretion. Small doses (e.g., 125 mg chloroquine phosphate twice weekly) should be given, because standard doses can induce transient, sometimes marked increases in photosensitivity and hepatocellular damage. Recent studies indicate that low-dose hydroxychloroquine is as safe and effective as phlebotomy in PCT. Hepatic imaging can diagnose or exclude complicating hepatocellular carcinoma. Treatment of PCT in patients with end-stage renal disease is facilitated by administration of erythropoietin.

1	HCP is an autosomal dominant hepatic porphyria that results from the half-normal activity of COPRO oxidase. The disease presents with acute attacks, as in AIP. Cutaneous photosensitivity also may occur, but much less commonly than in VP. HCP patients may have acute attacks and cutaneous photosensitivity together or separately. HCP is less common than AIP and VP. Homozygous dominant HCP and harderoporphyria, a biochemically distinguishable variant of HCP, present with clinical symptoms in children (see below). Clinical Features HCP is influenced by the same factors that cause attacks in AIP. The disease is latent before puberty, and symptoms, which are virtually identical to those of AIP, are more common in women. HCP is generally less severe than AIP. Blistering skin lesions are identical to PCT and VP and begin in childhood in rare homozygous cases.

1	Diagnosis COPRO III is markedly increased in the urine and feces in symptomatic patients, and often persists, especially in feces, when there are no symptoms. Urinary ALA and PBG levels are increased (but less than in AIP) during acute attacks, but may revert to normal more quickly than in AIP when symptoms resolve. Plasma porphyrins are usually normal or only slightly increased, but they may be higher in cases with skin lesions. The diagnosis of HCP is readily confirmed by increased fecal porphyrins consisting almost entirely of COPRO III, which distinguishes it from other porphyrias.

1	Although the diagnosis can be confirmed by measuring COPRO oxidase activity, the assays for this mitochondrial enzyme are not widely available and require cells other than erythrocytes. To date, over 64 mutations have been identified in the CPOX gene, 67% of which are missense or nonsense (Human Gene Mutation Database; www.hgmd .org). Detection of a CPOX mutation in a symptomatic individual permits the identification of asymptomatic family members. Neurologic symptoms are treated as in AIP (see above). Phlebotomy and chloroquine are not effective for the cutaneous lesions.

1	Neurologic symptoms are treated as in AIP (see above). Phlebotomy and chloroquine are not effective for the cutaneous lesions. VP is an autosomal dominant hepatic porphyria that results from the deficient activity of PROTO oxidase, the seventh enzyme in the heme biosynthetic pathway, and can present with neurologic symptoms, photosensitivity, or both. VP is particularly common in South Africa, where 3 of every 1000 whites have the disorder. Most are descendants of a couple who emigrated from Holland to South Africa in 1688. In other countries, VP is less common than AIP. Rare cases of homozygous dominant VP, presenting in childhood with cutaneous symptoms, also have been reported.

1	Clinical Features VP can present with skin photosensitivity, acute neurovisceral crises, or both. In two large studies of VP patients, 59% had only skin lesions, 20% had only acute attacks, and 22% had both. Acute attacks are identical to those in AIP and are precipitated by the same factors as AIP (see above). Blistering skin manifestations are similar to those in PCT, but are more difficult to treat and usually are 2531 of longer duration. Homozygous VP is associated with photosensitivity, neurologic symptoms, and developmental disturbances, including growth retardation, in infancy or childhood; all cases had increased erythrocyte levels of zinc protoporphyrin, a characteristic finding in all homozygous porphyrias so far described.

1	Diagnosis Urinary ALA and PBG levels are increased during acute attacks, but may return to normal more quickly than in AIP. Increases in fecal protoporphyrin and COPRO III and in urinary COPRO III are more persistent. Plasma porphyrin levels also are increased, particularly when there are cutaneous lesions. VP can be distinguished rapidly from all other porphyrias by examining the fluorescence emission spectrum of porphyrins in plasma since VP has a unique fluorescence peak at neutral pH. Assays of PROTO oxidase activity in cultured fibroblasts or lymphocytes are not widely available. Over 174 mutations have been identified in the PPOX gene from unrelated VP patients (Human Gene Mutation Database; www.hgmd.org). The missense mutation R59W is the common mutation in most South Africans with VP of Dutch descent. Five missense mutations were common in English and French VP patients; however, most mutations have been found in only one or two families.

1	Acute attacks are treated as in AIP, and hemin should be started early in most cases. Other than avoiding sun exposure, there are few effective measures for treating the skin lesions. β-Carotene, phlebotomy, and chloroquine are not helpful. In the erythropoietic porphyrias, excess porphyrins from bone marrow erythrocyte precursors are transported via the plasma to the skin and lead to cutaneous photosensitivity. XLSA results from the deficient activity of the erythroid form of ALA synthase (ALA synthase 2) and is associated with ineffective erythropoiesis, weakness, and pallor.

1	XLSA results from the deficient activity of the erythroid form of ALA synthase (ALA synthase 2) and is associated with ineffective erythropoiesis, weakness, and pallor. Clinical Features Typically, males with XLSA develop refractory hemolytic anemia, pallor, and weakness during infancy. They have secondary hypersplenism, become iron overloaded, and can develop hemosiderosis. The severity depends on the level of residual erythroid ALA synthase activity and on the responsiveness of the specific mutation to pyridoxal 5′-phosphate supplementation (see below). Peripheral blood smears reveal a hypochromic, microcytic anemia with striking anisocytosis, poikilocytosis, and polychromasia; the leukocytes and platelets appear normal. Hemoglobin content is reduced, and the mean corpuscular volume and mean corpuscular hemoglobin concentration are decreased. Patients with milder, late-onset disease have been reported recently.

1	Diagnosis Bone marrow examination reveals hypercellularity with a left shift and megaloblastic erythropoiesis with an abnormal maturation. A variety of Prussian blue-staining sideroblasts are observed. Levels of urinary porphyrin precursors and of both urinary and fecal porphyrins are normal. The activity of erythroid ALA synthase 2 is decreased in bone marrow, but this enzyme is difficult to measure in the presence of the normal ALA synthase 1 housekeeping enzyme. Definitive diagnosis requires the demonstration of mutations in the erythroid ALAS2 gene. The severe anemia may respond to pyridoxine supplementation. This cofactor is essential for ALA synthase activity, and mutations in the pyridoxine binding site of the enzyme have been found in The Porphyrias 2532 several responsive patients. Cofactor supplementation may make it possible to eliminate or reduce the frequency of transfusion. Unresponsive patients may be transfusion-dependent and require chelation therapy.

1	CEP, also known as Günther’s disease, is an autosomal recessive disorder. It is due to the markedly deficient, but not absent, activity of URO synthase and the resultant accumulation of URO I and COPRO I isomers. CEP is associated with hemolytic anemia and cutaneous lesions.

1	Clinical Features Severe cutaneous photosensitivity typically begins in early infancy. The skin over light-exposed areas is friable, and bullae and vesicles are prone to rupture and infection. Skin thickening, focal hypoand hyperpigmentation, and hypertrichosis of the face and extremities are characteristic. Secondary infection of the cutaneous lesions can lead to disfigurement of the face and hands. Porphyrins are deposited in teeth and in bones. As a result, the teeth are brownish and fluoresce on exposure to long-wave ultraviolet light. Hemolysis is probably due to the marked increase in erythrocyte porphyrins and leads to splenomegaly. Adults with a milder later-onset form of the disease also have been described.

1	Diagnosis URO and COPRO (mostly type I isomers) accumulate in the bone marrow, erythrocytes, plasma, urine, and feces. The predominant porphyrin in feces is COPRO I. The diagnosis of CEP can be confirmed by demonstration of markedly deficient URO synthase activity and/or by the identification of specific mutations in the UROS gene. The disease can be detected in utero by measuring porphyrins in amniotic fluid and URO synthase activity in cultured amniotic cells or chorionic villi, or by the detection of the family’s specific gene mutations. Molecular analyses of the mutant alleles from unrelated patients have revealed the presence of over 48 mutations in the UROS gene, including four in the erythroid-specific promoter of the UROS gene. Genotype/phenotype correlations can predict the severity of the disease. The CEP phenotype may be modulated by sequence variations in the erythroid specific ALA synthase 2, mutation of which typically causes XLP. One mutation (p.ArgR216WTrp) in GATA1,

1	of the disease. The CEP phenotype may be modulated by sequence variations in the erythroid specific ALA synthase 2, mutation of which typically causes XLP. One mutation (p.ArgR216WTrp) in GATA1, encoding the X-linked erythroid-specific transcription factor GATA binding protein 1 (GATA1), has been identified in an individual with CEP, thrombocytopenia, and β thalassemia.

1	Severe cases often require transfusions for anemia. Chronic transfusions of sufficient blood to suppress erythropoiesis are effective in reducing porphyrin production but result in iron overload. Splenectomy may reduce hemolysis and decrease transfusion requirements. Protection from sunlight and from minor skin trauma is important. β-Carotene may be of some value. Complicating bacterial infections should be treated promptly. Recently, bone marrow and cord blood transplantation has proven curative in several transfusion-dependent children, providing the rationale for stem cell gene therapy.

1	EPP is an inherited disorder resulting from the deficient activity of ferrochelatase (FECH), the last enzyme in the heme biosynthetic pathway. EPP is the most common erythropoietic porphyria in children and, after PCT, the second most common porphyria in adults. EPP patients have FECH activities as low as 15–25% of normal in lymphocytes and cultured fibroblasts. Protoporphyrin accumulates in bone marrow reticulocytes and then appears in plasma, is taken up in the liver, and is excreted in bile and feces. Protoporphyrin transported to the vessels in the skin causes the nonblistering photosensitivity. In most symptomatic patients (~90%) with this autosomal recessive disorder, a mutation in one FECH allele is inherited with a relatively common (~10% of normal whites) intronic 3 (IVS3) alteration (IVS3– 48T>C) that results in the low expression of the normal enzyme. In about 10% of EPP families, two FECH mutations have been found. Recently, deletion mutations in exon 11 of the ALAS2 gene

1	(IVS3– 48T>C) that results in the low expression of the normal enzyme. In about 10% of EPP families, two FECH mutations have been found. Recently, deletion mutations in exon 11 of the ALAS2 gene have been described, causing XLP that is clinically indistinguishable from EPP. The deletion of the C-terminal amino acids of ALAS2 results in its increased activity and the accumulation of protoporphyrin. XLP accounts for approximately 2–10% of cases with the EPP phenotype in Europe and North America.

1	Clinical Features Skin photosensitivity, which differs from that in other porphyrias, usually begins in childhood and consists of pain, redness, and itching occurring within minutes of sunlight exposure (Fig. 430-4). Photosensitivity is associated with substantial elevations in erythrocyte protoporphyrin and occurs only in patients with genotypes that result in ferrochelatase activities below ~35% of normal. Vesicular lesions are uncommon. Redness, swelling, burning, and itching can develop shortly after sun exposure and resemble angioedema. Pain symptoms may seem out of proportion to the visible skin involvement. Sparse vesicles and bullae occur in ~10% of cases. Chronic skin changes may include lichenification, leathery pseudovesicles, labial grooving, and nail changes. Severe scarring is rare, as are pigment changes, friability, and hirsutism. Unless hepatic or other complications develop, protoporphyrin levels and symptoms of photosensitivity remain remarkably stable over many

1	is rare, as are pigment changes, friability, and hirsutism. Unless hepatic or other complications develop, protoporphyrin levels and symptoms of photosensitivity remain remarkably stable over many years in most patients. Factors that exacerbate the hepatic porphyrias play little or no role in EPP.

1	The primary source of excess protoporphyrin is the bone marrow reticulocytes. Erythrocyte protoporphyrin is free (not complexed with zinc) and is mostly bound to hemoglobin. In plasma, protoporphyrin is bound to albumin. Hemolysis and anemia are usually absent or mild.

1	Although EPP is an erythropoietic porphyria, up to 20% of EPP patients may have minor abnormalities of liver function, and in about 5% of these patients the accumulation of protoporphyrins causes chronic liver disease that can progress to liver failure and death. Protoporphyrin is insoluble, and excess amounts form crystalline structures in liver cells (Fig. 430-4) and can decrease hepatic bile flow. Studies in the mouse model of EPP have shown that the bile duct epithelium may be damaged by toxic bile, leading to biliary fibrosis. Thus, rapidly progressive liver disease appears to be related to the cholestatic effects of protoporphyrins and is associated with increasing hepatic protoporphyrin levels due to impaired hepatobiliary excretion and increased photosensitivity. The hepatic complications also are often characterized by increasing levels of protoporphyrins in erythrocytes and plasma as well as severe abdominal and back pains, especially in the right upper quadrant. Gallstones

1	FIGURE 430-4 Erythema and edema of the hands due to acute photosensitivity in a 10-year-old boy with erythropoietic protoporphyria. (From P Poblette-Gutierrez et al: Eur J Dermatol 16:230, 2006.) composed at least in part of protoporphyrin occur in some patients. Hepatic complications appear to be higher in autosomal recessive EPP due to two FECH mutations and in XLP.

1	diagnosis A substantial increase in erythrocyte protoporphyrin, which is predominantly free and not complexed with zinc, is the hallmark of EPP. Protoporphyrin levels are also variably increased in bone marrow, plasma, bile, and feces. Erythrocyte protoporphyrin concentrations are increased in other conditions such as lead poisoning, iron deficiency, various hemolytic disorders, all homozygous forms of other porphyrias, and sometimes even in acute porphyrias. In all these conditions, however, in contrast to EPP, protoporphyrin is complexed with zinc. Therefore, after an increase in erythrocyte protoporphyrin is found in a suspected EPP patient, it is important to confirm the diagnosis by an assay that distinguishes free and zinc-complexed protoporphyrin. Erythrocytes in EPP also exhibit red fluorescence under a fluorescence microscopy at 620 nm. Urinary levels of porphyrins and porphyrin precursors are normal. Ferrochelatase activity in cultured lymphocytes or fibroblasts is

1	exhibit red fluorescence under a fluorescence microscopy at 620 nm. Urinary levels of porphyrins and porphyrin precursors are normal. Ferrochelatase activity in cultured lymphocytes or fibroblasts is decreased. DNA diagnosis by mutation analysis is recommended to detect the causative FECH mutation(s) and/or the presence of the IVS3–48T>C low expression allele. To date, over 190 mutations have been identified in the FECH gene, many of which result in an unstable or absent enzyme protein (null alleles) (Human Gene Mutation Database; www.hgmd.org). Studies suggest that EPP patients with a null allele (and the IVS3–48T>C low expression allele) have a greater risk for developing severe liver complications.

1	In XLP, the erythrocyte protoporphyrin levels appear to be higher than other forms of EPP and the proportions of free and zinc protopophyrins may reach 50%. To date, four ALAS2 mutations, three deletions of one to four bases, and one novel nonsense mutation have been described, which markedly increase ALA synthase 2 activity and cause XLP. XLP accounts for about 2% of patients with the EPP phenotype in Western Europe. Recent studies show that about 10% of North American patients with the EPP phenotype have XLP.

1	Avoiding sunlight exposure and wearing clothing designed to provide protection for conditions with chronic photosensitivity are essential. Oral β-carotene (120–180 mg/dL) may improve tolerance to sunlight in some patients. The beneficial effects of β-carotene may involve quenching of singlet oxygen or free radicals. The dosage may need to be adjusted to maintain serum carotene levels in the recommended range of 10–15 mmol/L (600–800 mg/dL). Mild skin discoloration due to carotenemia is the only significant side effect. Afamelanotide, an α-melanocyte-stimulating hormone (MSH) analogue has completed phase III clinical trials in the United States for patients with EPP and XLP.

1	Treatment of hepatic complications, which may be accompanied by motor neuropathy, is difficult. Cholestyramine and other porphyrin absorbents such as activated charcoal may interrupt the enterohepatic circulation of protoporphyrin and promote its fecal excretion, leading to some improvement. Splenectomy may be helpful when the disease is accompanied by hemolysis and significant splenomegaly. Plasmapheresis and intravenous hemin are sometimes beneficial.

1	Liver transplantation has been carried out in some EPP and XLP patients with severe liver complications and is often successful in the short term. However, the disease often recurs in the transplanted liver due to continued bone marrow production of excess protoporphyrin. In a retrospective study of 17 liver-transplanted EPP patients, 11 (65%) had recurrent EPP liver disease. Posttransplantation treatment with hematin and plasmapheresis should be considered to prevent the recurrence of liver disease. However, bone marrow transplantation, which has been successful in human EPP and which prevented liver disease in a mouse model, should be considered after liver transplantation, if a suitable donor can be found.

1	The authors thank Dr. Karl E. Anderson for his review of the manuscript and helpful comments and suggestions. This work is supported in part by the Porphyrias Consortium (U54 DK083909), a part of the National Institutes of Health (NIH) Rare Disease Clinical Research Network (RDCRN), supported through collaboration between the NIH Office of Rare Diseases Research (ORDR) at the National Center for Advancing Translational Science (NCATS), and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. MB is supported by a career development award K23-DK-095946 The Porphyrias

1	The Porphyrias Disorders of Purine and Pyrimidine Metabolism Christopher M. Burns, Robert L. Wortmann Purines (adenine and guanine) and pyrimidines (cytosine, thymine, uracil) serve fundamental roles in the replication of genetic mate-431e rial, gene transcription, protein synthesis, and cellular metabolism. Disorders that involve abnormalities of nucleotide metabolism range from relatively common diseases such as hyperuricemia and gout, in which there is increased production or impaired excretion of a metabolic end product of purine metabolism (uric acid), to rare enzyme deficiencies that affect purine and pyrimidine synthesis or degradation. Understanding these biochemical pathways has led, in some instances, to the development of specific forms of treatment, such as the use of allopurinol and febuxostat to reduce uric acid production.

1	Uric acid is the final breakdown product of purine degradation in humans. It is a weak diprotic acid with pK a values of 5.75 and 10.3. Urates, the ionized forms of uric acid, predominate in plasma, extracellular fluid, and synovial fluid, with ~98% existing as monosodium urate at pH 7.4. Plasma is saturated with monosodium urate at a concentration of 405 μmol/L (6.8 mg/dL) at 37°C. At higher concentrations, plasma is therefore supersaturated—a situation that creates the potential for urate crystal precipitation. However, plasma urate concentrations can reach 4800 μmol/L (80 mg/dL) without precipitation, perhaps because of the presence of solubilizing substances.

1	The pH of urine greatly influences the solubility of uric acid. At pH 5.0, urine is saturated with uric acid at concentrations ranging from 360 to 900 μmol/L (6–15 mg/dL). At pH 7, saturation is reached at concentrations from 9840 to 12,000 μmol/L (158–200 mg/dL). Ionized forms of uric acid in urine include monosodium, disodium, potassium, ammonium, and calcium urates. Although purine nucleotides are synthesized and degraded in all tissues, urate is produced only in tissues that contain xanthine oxidase, primarily the liver and small intestine. Urate production varies with the purine content of the diet and with rates of purine biosynthesis, degradation, and salvage (Fig. 431e-1). Normally, two-thirds to three-fourths of

1	FIgURE 431e-1 The total-body urate pool is the net result between urate production and excretion. Urate production is influenced by dietary intake of purines and the rates of de novo biosynthesis of purines from nonpurine precursors, nucleic acid turnover, and salvage by phosphoribosyltransferase activities. The formed urate is normally excreted by urinary and intestinal routes. Hyperuricemia can result from increased production, decreased excretion, or a combination of both mechanisms. When hyperuricemia exists, urate can precipitate and deposit in tissues as tophi. urate is excreted by the kidneys, and most of the remainder is eliminated 431e-1 through the intestines.

1	The kidneys clear urate from the plasma and maintain physiologic balance by utilizing specific organic anion transporters (OATs), including urate transporter 1 (URAT1, SLC22A12) (Fig. 431e-2). In humans, OAT1 (SLC22A6), OAT2 (SLC22A7), and OAT3 (SLC22A8) are located on the basolateral membrane of renal proximal tubule cells. OAT4 (SLC22A11), OAT10 (SLC22A13), and URAT1 are located on the apical brush-border membrane of these cells. The latter transporters carry urate and other organic anions into the tubular cells from the lumen in exchange for intracellular organic anions. Once inside the cell, urate must pass to the basolateral side of the lumen in a process controlled by voltage-dependent carriers, including glucose transporter 9 (GLUT9, SLC2A9). Uricosuric compounds (Table 431e-1) directly inhibit URAT1 on the apical side of the tubular cell (so-called cisinhibition). In contrast, antiuricosuric compounds (those that promote hyperuricemia), such as nicotinate, pyrazinoate,

1	directly inhibit URAT1 on the apical side of the tubular cell (so-called cisinhibition). In contrast, antiuricosuric compounds (those that promote hyperuricemia), such as nicotinate, pyrazinoate, lactate, and other aromatic organic acids, serve as the exchange anion inside the cell, thereby stimulating anion exchange and urate reabsorption (trans-stimulation). The activities of URAT1, other OATs, and sodium anion transporters result in excretion of 8–12% of the filtered urate as uric acid.

1	Most children have serum urate concentrations of 180–240 μmol/L (3–4 mg/dL). Levels begin to rise in males during puberty but remain low in females until menopause. The most recent mean serum urate values for men and premenopausal women in the United States are 415 and 360 μmol/L (6.14 and 4.87 mg/dL), respectively, according to National Health and Nutrition Evaluation Survey (NHANES) data for 2007–2008. After menopause, values for women increase to approximately those for men. In adulthood, concentrations rise steadily over time and vary with height, body weight, blood pressure, renal function, and alcohol intake. Hyperuricemia can result from increased production or decreased excretion of uric acid or from a combination of the two processes. Sustained hyperuricemia predisposes some individuals to develop clinical manifestations including gouty arthritis (Chap. 395), urolithiasis, and renal dysfunction (see below).

1	In general, hyperuricemia is defined as a plasma (or serum) urate concentration >405 μmol/L (>6.8 mg/dL). The risk of developing gouty arthritis or urolithiasis increases with higher urate levels and escalates in proportion to the degree of elevation. The prevalence of hyperuricemia is increasing among ambulatory adults and even more markedly among hospitalized patients. The prevalence of gout in the United States more than doubled between the 1960s and the 1990s. Based on NHANES data from 2007–2008, these trends continue, with an approximate prevalence of gout among men of 5.9% (6.1 million) and among women of 2.0% (2.2 million). Mean serum urate levels rose to 6.14 mg/dL among men and 4.87 mg/dL among women, with consequent hyperuricemia prevalences of 21.2% and 21.6%, respectively (with hyperuricemia defined as a serum urate level of >7.0 mg/ dL [415 μmol/L] for men and >5.7 mg/dL [340 μmol/L] for women). These numbers represent a 1.2% increase in the prevalence of gout, a

1	(with hyperuricemia defined as a serum urate level of >7.0 mg/ dL [415 μmol/L] for men and >5.7 mg/dL [340 μmol/L] for women). These numbers represent a 1.2% increase in the prevalence of gout, a 0.15-mg/dL increase in the serum urate level, and a 3.2% increase in the prevalence of hyperuricemia over figures reported in NHANESIII (1988–1994). These rises are thought to be driven by increased obesity and hypertension and perhaps also by better medical care and increased longevity.

1	Hyperuricemia may be classified as primary or secondary, depending on whether the cause is innate or an acquired disorder. However, it is more useful to classify hyperuricemia in relation to the underlying pathophysiology—i.e., whether it results from increased production, decreased excretion, or a combination of the two (Fig. 431e-1, Table 431e-2). Increased Urate Production Diet contributes to the serum urate concentration in proportion to its purine content. Strict restriction of purine intake reduces the mean serum urate level by ~60 μmol/L CHAPTER 431e Disorders of Purine and Pyrimidine Metabolism FIgURE 431e-2 Schematic for handling of uric acid by the kidney. A complex interplay of transporters on both the apical and basolateral aspects of the renal tubule epithelial cell is involved in the reabsorption of uric acid. See text for details. Most uricosuric compounds inhibit URAT1 on the apical side, as well as OAT1, OAT3, and GLUT9 on the basolateral side.

1	(~1 mg/dL) and urinary uric acid excretion by ~1.2 mmol/d (~200 mg/d). Foods high in nucleic acid content include liver, “sweetbreads” (i.e., thymus and pancreas), kidney, and anchovy. Endogenous sources of purine production also influence the serum urate level (Fig. 431e-3). De novo purine biosynthesis is a multistep process that forms inosine monophosphate (IMP). The rates of purine biosynthesis and urate production are predominantly determined by amidophosphoribosyltransferase (amidoPRT), which combines phosphoribosylpyrophosphate (PRPP) and glutamine. A secondary regulatory pathway is the salvage of purine bases by hypoxanthine phosphoribosyltransferase (HPRT). HPRT catalyzes the combination of the purine bases hypoxanthine and guanine with PRPP to form the respective ribonucleotides IMP and guanosine monophosphate (GMP).

1	Acetohexamide Glyceryl guaiacolate Adrenocorticotropic hormone Glycopyrrolate Ascorbic acid Halofenate Azauridine Losartan Benzbromarone Meclofenamate Calcitonin Phenolsulfonphthalein Chlorprothixene Phenylbutazone Citrate Probenecid Dicumarol Radiographic contrast agents Diflunisal Salicylates (>2 g/d) Estrogens Sulfinpyrazone Fenofibrate Tetracycline that is outdated Glucocorticoids Zoxazolamine Serum urate levels are closely coupled to the rates of de novo purine biosynthesis, which is driven in part by the level of PRPP, as evidenced by two X-linked inborn errors of purine metabolism (Table 431e-3). Both increased PRPP synthetase activity and HPRT deficiency are associated with overproduction of purines, hyperuricemia, and hyperuricaciduria (see below for clinical descriptions).

1	Accelerated purine nucleotide degradation can also cause hyperuricemia—i.e., with conditions of rapid cell turnover, proliferation, or cell death, as in leukemic blast crises, cytotoxic therapy for malignancy, hemolysis, or rhabdomyolysis. Hyperuricemia can result from excessive degradation of skeletal muscle ATP after strenuous physical exercise or status epilepticus and in glycogen storage disease types III, V, and VII (Chap. 433e). The hyperuricemia of myocardial infarction, smoke inhalation, and acute respiratory failure may also be related to accelerated breakdown of ATP.

1	Decreased Uric Acid Excretion More than 90% of individuals with sustained hyperuricemia have a defect in the renal handling of uric acid. For any given plasma urate concentration, patients who have gout excrete ~40% less uric acid than those who do not. When plasma urate levels are raised by purine ingestion or infusion, uric acid excretion increases in patients with and without gout; however, in those with gout, plasma urate concentrations must be 60–120 μmol/L (1–2 mg/ dL) higher than normal to achieve equivalent uric acid excretion rates. Uric acid ? retically result from decreased glomerular filtration, decreased tubular secretion, or enhanced tubular reabsorption. Decreased urate filtration does not appear to cause primary hyperuricemia but does contribute to the hyperuricemia of renal insufficiency. Although hyperuricemia is invariably present in chronic renal disease, the correlation among Glycogenosis III, V, and VII Purine-rich diet Lymphoproliferative diseases

1	Glycogenosis III, V, and VII Purine-rich diet Lymphoproliferative diseases Lactic acidosis Diabetic ketoacidosis Starvation ketosis Drug ingestion Berylliosis Salicylates (<2 g/d) Sarcoidosis Diuretics Lead intoxication Alcohol Hyperparathyroidism Levodopa Hypothyroidism Ethambutol Toxemia of pregnancy Pyrazinamide Bartter’s syndrome Nicotinic acid Down syndrome Cyclosporine Abbreviations: HPRT, hypoxanthine phosphoribosyltransferase; PRPP, phosphoribosylpyrophosphate. PRA SAICAR Guanosine Xanthine Urate IMP PRPP AICAR Adenine Inosine 2,8 Dihydroxyadenine NH3 Purine nucleotide cycle De novo biosynthesis PRPP Glutamine Ribose-5-P ATP Feedback inhibition Feedback inhibition 1 2 3 3 4 6 55 77 8 9 5 1010 10 GMP AMP Adenosine HypoxanthineGuanine PRPP FIgURE 431e-3 Abbreviated scheme of purine metabolism.

1	FIgURE 431e-3 Abbreviated scheme of purine metabolism. (1) Phosphoribosylpyrophosphate (PRPP) synthetase, (2) amidophosphoribosyltransferase (amidoPRT), (3) adenylosuccinate lyase, (4) (myo-)adenylate (AMP) deaminase, (5) 5′-nucleotidase, (6) adenosine deaminase, (7) purine nucleoside phosphorylase, (8) hypoxanthine phosphoribosyltransferase (HPRT), (9) adenine phosphoribosyltransferase (APRT), and (10) xanthine oxidase. PRA, phosphoribosylamine; SAICAR, succinylaminoimidazole carboxamide ribotide; AICAR, aminoimidazole carboxamide ribotide; GMP, guanylate; IMP, inosine mono-phosphate; ATP, adenosine triphosphate. serum creatinine, urea nitrogen, and urate concentrations is poor. Extrarenal clearance of uric acid increases as renal damage becomes more severe.

1	Many agents that cause hyperuricemia exert their effects by stimulating reabsorption rather than inhibiting secretion. This stimulation appears to occur through a process of “priming” renal urate reabsorption through the sodium-dependent loading of proximal tubular epithelial cells with anions capable of trans-stimulating urate reabsorption. The sodium-coupled monocarboxyl transporters SMCT1 and 2 (SLC5A8, SLC5A12) in the brush border of the proximal tubular cells mediate sodium-dependent loading of these cells with 431e-3 monocarboxylates. A similar transporter, SLC13A3, mediates sodium-dependent influx of dicarboxylates into the epithelial cell from the basolateral membrane. Some of these carboxylates are well known to cause hyperuricemia, including pyrazinoate (from pyrazinamide treatment), nicotinate (from niacin therapy), and the organic acids lactate, β-hydroxybutyrate, and acetoacetate. The monoand divalent anions then become substrates for URAT1 and OAT4, respectively, and are

1	nicotinate (from niacin therapy), and the organic acids lactate, β-hydroxybutyrate, and acetoacetate. The monoand divalent anions then become substrates for URAT1 and OAT4, respectively, and are exchanged for uric acid from the proximal tubule. Increased blood levels of these anions result in their increased glomerular filtration and greater reabsorption by proximal tubular cells. The increased intraepithelial cell concentrations lead to increased uric acid reabsorption by promoting URAT1-, OAT4-, and OAT10-dependent anion exchange. Low doses of salicylates also promote hyperuricemia by this mechanism. Sodium loading of proximal tubular cells also provokes urate retention by reducing extracellular fluid volume and increasing angiotensin II, insulin, and parathyroid hormone release. Additional organic anion transporters OAT1, OAT2, and OAT3 are involved in the movement of uric acid through the basolateral membrane, although the detailed mechanisms are still being elucidated.

1	GLUT9 (SLC2A9) is an electrogenic hexose transporter with splicing variants that mediate co-reabsorption of uric acid along with glucose and fructose at the apical membrane (GLUT9ΔN/SLC2A9v2) as well as through the basolateral membrane (SLC2A9v1) and thus into the circulation. GLUT9 has recently been identified as a high-capacity urate transporter, with rates 45–60 times faster than its glucose/fructose transport activity. GLUT9 may be responsible for the observed association of the consumption of fructose-sweetened soft drinks with an increased risk of hyperuricemia and gout. Genome-wide association scanning suggests that polymorphisms in SLC2A9 may play an important role in susceptibility to gout in the Caucasian population. The presence of one predisposing variant allele increases the relative risk of developing gout by 30–70%, most likely by increasing expression of the shorter isoform, SLC2A9v2 (GLUT9ΔN). Notably, though, genetic polymorphisms explain only ~6% of the differences

1	relative risk of developing gout by 30–70%, most likely by increasing expression of the shorter isoform, SLC2A9v2 (GLUT9ΔN). Notably, though, genetic polymorphisms explain only ~6% of the differences in serum uric acid levels in Caucasians. Clearly, gout is polygenic and complex, and at this time the utility of genetic testing for relevant polymorphisms remains investigational and of no clinical utility.

1	Alcohol promotes hyperuricemia because of increased urate production and decreased uric acid excretion. Excessive alcohol consumption accelerates hepatic breakdown of ATP to increase urate production. Alcohol consumption can also induce hyperlacticacidemia, which blocks uric acid secretion. The higher purine content in some alcoholic beverages may also be a factor. Consumption of beer confers a greater risk of gout than liquor, and moderate wine intake does not increase gout risk. Intake of red meat and fructose increases the risk of gout, whereas intake of low-fat dairy products, purine-rich vegetables, whole grains, nuts and legumes, less sugary fruits, coffee, and vitamin C reduces the risk. CHAPTER 431e Disorders of Purine and Pyrimidine Metabolism Hyperuricemia does not necessarily represent a disease, nor is it a specific indication for therapy. The decision to treat depends on the cause and the potential consequences of hyperuricemia in each individual.

1	Quantification of uric acid excretion can be used to determine whether hyperuricemia is caused by overproduction or decreased excretion. On a purine-free diet, men with normal renal function excrete <3.6 mmol/d (600 mg/d). Thus, the hyperuricemia of individuals who excrete uric acid above this level while on a purine-free diet is due to purine overproduction; for those who excrete lower amounts on the purine-free diet, it is due to decreased excretion. If the assessment is performed while the patient is on a regular diet, the level of 4.2 mmol/d (800 mg/d) can be used as the discriminating value.

1	The most recognized complication of hyperuricemia is gouty arthritis. NHANES 2007–2008 found a prevalence of gout among U.S. adults of 3.9%, with figures of ~6% for men and ~2% for women. The higher the serum urate level, the more likely an individual is to develop gout. In one study, the incidence of gout was 4.9% among individuals with serum urate concentrations >540 μmol/L (>9.0 mg/dL) as opposed to only 0.5% among those with values between 415 and 535 μmol/L (7.0 and 8.9 mg/dL). The complications of gout correlate with both the duration and the severity of hyperuricemia. For further discussion of gout, see Chap. 395.

1	Hyperuricemia also causes several renal problems: (1) nephrolithiasis; (2) urate nephropathy, a rare cause of renal insufficiency attributed to monosodium urate crystal deposition in the renal interstitium; and (3) uric acid nephropathy, a reversible cause of acute renal failure resulting from deposition of large amounts of uric acid crystals in the renal collecting ducts, pelvis, and ureters. Nephrolithiasis Uric acid nephrolithiasis occurs most commonly, but not exclusively, in individuals with gout. In gout, the prevalence of nephrolithiasis correlates with the serum and urinary uric acid levels, reaching ~50% with serum urate levels of 770 μmol/L (13 mg/dL) or urinary uric acid excretion >6.5 mmol/d (1100 mg/d).

1	Uric acid stones can develop in individuals with no evidence of arthritis, only 20% of whom are hyperuricemic. Uric acid can also play a role in other types of kidney stones. Some individuals who do not have gout but have calcium oxalate or calcium phosphate stones have hyperuricemia or hyperuricaciduria. Uric acid may act as a nidus on which calcium oxalate can precipitate or lower the formation product for calcium oxalate crystallization. Urate Nephropathy Urate nephropathy, sometimes referred to as urate nephrosis, is a late manifestation of severe gout and is characterized histologically by deposits of monosodium urate crystals surrounded by a giant-cell inflammatory reaction in the medullary interstitium and pyramids. The disorder is now rare and cannot be diagnosed in the absence of gouty arthritis. The lesions may be clinically silent or cause proteinuria, hypertension, and renal insufficiency.

1	Uric Acid Nephropathy This reversible cause of acute renal failure is due to precipitation of uric acid in renal tubules and collecting ducts that obstructs urine flow. Uric acid nephropathy develops following sudden urate overproduction and marked hyperuricaciduria. Factors that favor uric acid crystal formation include dehydration and acidosis. This form of acute renal failure occurs most often during an aggressive “blastic” phase of leukemia or lymphoma prior to or coincident with cytolytic therapy but has also been observed in individuals with other neoplasms, following epileptic seizures, and after vigorous exercise with heat stress. Autopsy studies have demonstrated intraluminal precipitates of uric acid, dilated proximal tubules, and normal glomeruli. The initial pathogenic events are believed to include obstruction of collecting ducts with uric acid and obstruction of the distal renal vasculature.

1	If recognized, uric acid nephropathy is potentially reversible. Appropriate therapy has reduced the mortality rate from ~50% to practically nil. Serum levels cannot be relied on for diagnosis because this condition has developed in the presence of urate concentrations varying from 720 to 4800 μmol/L (12–80 mg/dL). The distinctive feature is the urinary uric acid concentration. In most forms of acute renal failure with decreased urine output, urinary uric acid content is either normal or reduced, and the ratio of uric acid to creatinine is <1. In acute uric acid nephropathy, the ratio of uric acid to creatinine in a random urine sample or a 24-h specimen is >1, and a value that high is essentially diagnostic.

1	Metabolic syndrome (Chap. 422) is characterized by abdominal obesity with visceral adiposity, impaired glucose tolerance due to insulin resistance with hyperinsulinemia, hypertriglyceridemia, increased low-density lipoprotein cholesterol, decreased high-density lipoprotein cholesterol, and hyperuricemia. Hyperinsulinemia reduces the renal excretion of uric acid and sodium. Not surprisingly, hyperuricemia resulting from euglycemic hyperinsulinemia may precede the onset of type 2 diabetes, hypertension, coronary artery disease, and gout in individuals with metabolic syndrome.

1	Hyperuricemia is present in ~21% of the population and in at least 25% of hospitalized individuals. The vast majority of hyperuricemic persons are at no clinical risk. In the past, the association of hyperuricemia with cardiovascular disease and renal failure led to the use of urate-lowering agents for patients with asymptomatic hyperuricemia. This practice is no longer recommended except for individuals receiving cytolytic therapy for neoplastic disease, who are treated with urate-lowering agents in an effort to prevent uric acid nephropathy. Because hyperuricemia can be a component of the metabolic syndrome, its presence is an indication to screen for and aggressively treat any accompanying obesity, hyperlipidemia, diabetes mellitus, or hypertension.

1	Hyperuricemic individuals, especially those with higher serum urate levels, are at risk for the development of gouty arthritis. However, most hyperuricemic persons never develop gout, and prophylactic treatment is not indicated. Furthermore, neither structural kidney damage nor tophi are identifiable before the first attack. Reduced renal function cannot be attributed to asymptomatic hyperuricemia, and treatment of asymptomatic hyperuricemia does not alter the progression of renal dysfunction in patients with renal disease. An increased risk of stone formation in those with asymptomatic hyperuricemia has not been established.

1	Thus, because treatment with specific antihyperuricemic agents entails inconvenience, cost, and potential toxicity, routine treatment of asymptomatic hyperuricemia cannot be justified other than for prevention of acute uric acid nephropathy. In addition, routine screening for asymptomatic hyperuricemia is not recommended. If hyperuricemia is diagnosed, however, the cause should be determined. Causal factors should be corrected if the condition is secondary, and associated problems such as hypertension, hypercholesterolemia, diabetes mellitus, and obesity should be treated. See Chap. 395 for treatment of gout, including urate nephrosis.

1	Nephrolithiasis Antihyperuricemic therapy is recommended for the individual who has both gouty arthritis and either uric acid– or calcium-containing stones, both of which may occur in association with hyperuricaciduria. Regardless of the nature of the calculi, fluid ingestion should be sufficient to produce a daily urine volume >2 L. Alkalinization of the urine with sodium bicarbonate or acetazolamide may be justified to increase the solubility of uric acid. Specific treatment of uric acid calculi requires reducing the urine uric acid concentration with a xanthine oxidase inhibitor, such as allopurinol or febuxostat. These agents decrease the serum urate concentration and the urinary excretion of uric acid in the first 24 h, with a maximal reduction within 2 weeks. Allopurinol can be given once a day because of the long half-life (18 h) of its active metabolite, oxypurinol. In the febuxostat trials, the generally recommended dose of allopurinol (300 mg/d) was effective at achieving a

1	once a day because of the long half-life (18 h) of its active metabolite, oxypurinol. In the febuxostat trials, the generally recommended dose of allopurinol (300 mg/d) was effective at achieving a target serum urate concentration below 6.0 mg/dL (357 μmol/L) in <50% of patients; this result suggested that higher doses should be considered. The drug is effective in patients with renal insufficiency, but the dose should be reduced. Allopurinol is also useful in reducing the recurrence of calcium oxalate stones in patients with gout and in individuals with hyperuricemia or hyperuricaciduria who do not have gout. Febuxostat (40–80 mg/d) is also taken once daily, and doses do not need to be adjusted in the presence of mild to moderate renal dysfunction. Potassium citrate (30–80 mmol/d orally in divided doses) is an alternative therapy for patients with uric acid stones alone or mixed calcium/uric acid stones. A xanthine oxidase inhibitor is also indicated for the treatment of

1	orally in divided doses) is an alternative therapy for patients with uric acid stones alone or mixed calcium/uric acid stones. A xanthine oxidase inhibitor is also indicated for the treatment of 2,8-dihydroxyadenine kidney stones.

1	Uric Acid Nephropathy Uric acid nephropathy is often preventable, and immediate appropriate therapy has greatly reduced the mortality rate. Vigorous IV hydration and diuresis with furosemide dilute the uric acid in the tubules and promote urine flow to ≥100 mL/h. The administration of acetazolamide (240–500 mg every 6–8 h) and sodium bicarbonate (89 mmol/L) IV enhances urine alkalinity and thereby solubilizes more uric acid. It is important to ensure that the urine pH remains >7.0 and to watch for circulatory overload. In addition, antihyperuricemic therapy in the form of allopurinol in a single dose of 8 mg/kg is administered to reduce the amount of urate that reaches the kidney. If renal insufficiency persists, subsequent daily doses should be reduced to 100–200 mg because oxypurinol, the active metabolite of allopurinol, accumulates in renal failure. Despite these measures, hemodialysis may be required. Urate oxidase (rasburicase) can also be administered IV to prevent or to treat

1	active metabolite of allopurinol, accumulates in renal failure. Despite these measures, hemodialysis may be required. Urate oxidase (rasburicase) can also be administered IV to prevent or to treat tumor lysis syndrome.

1	Hypouricemia, defined as a serum urate concentration <120 μmol/L (<2.0 mg/dL), can result from decreased production of urate, increased excretion of uric acid, or a combination of both mechanisms. This condition occurs in <0.2% of the general population and <0.8% of hospitalized individuals. Hypouricemia causes no symptoms or pathology and therefore requires no therapy.

1	Most hypouricemia results from increased renal uric acid excretion. The finding of normal amounts of uric acid in a 24-h urine collection from an individual with hypouricemia is evidence for a renal cause. Medications with uricosuric properties (Table 431e-1) include aspirin (at doses >2.0 g/d), losartan, fenofibrate, x-ray contrast materials, and 431e-5 glyceryl guaiacolate. Total parenteral hyperalimentation can also cause hypouricemia, possibly a result of the high glycine content of the infusion formula. Other causes of increased urate clearance include conditions such as neoplastic disease, hepatic cirrhosis, diabetes mellitus, and inappropriate secretion of vasopressin; defects in renal tubular transport such as primary Fanconi syndrome and Fanconi syndromes caused by Wilson’s disease, cystinosis, multiple myeloma, and heavy metal toxicity; and isolated congenital defects in the bidirectional transport of uric acid. Hypouricemia can be a familial disorder that is generally

1	disease, cystinosis, multiple myeloma, and heavy metal toxicity; and isolated congenital defects in the bidirectional transport of uric acid. Hypouricemia can be a familial disorder that is generally inherited in an autosomal recessive manner. Most cases are caused by a loss of function mutation in SLC22A12, the gene that encodes URAT-1, resulting in increased renal urate clearance. Individuals with normal SLC22A12 most likely have a defect in other urate transporters. Although hypouricemia is usually asymptomatic, some patients suffer from urate nephrolithiasis or exercise-induced renal failure.

1	(See also Table 431e-3, Table 431e-4, Fig. 431e-3, and Fig. 431e-4) More than 30 defects in human purine and pyrimidine metabolic pathways have been identified thus far. Many are benign, but about half are associated with clinical manifestations, some causing major morbidity and mortality. Advances in genetics, along with high-performance liquid chromatography and tandem mass spectrometry, have facilitated diagnosis.

1	PURINE DISORDERS HPRT Deficiency The HPRT gene is located on the X chromosome. Affected males are hemizygous for the mutant gene; carrier females are asymptomatic. A complete deficiency of HPRT, the Lesch-Nyhan syndrome, is characterized by hyperuricemia, self-mutilative behavior, choreoathetosis, spasticity, and mental retardation. A partial deficiency of HPRT, the Kelley-Seegmiller syndrome, is associated with hyperuricemia but no central nervous system manifestations. In both disorders, the hyperuricemia results from urate overproduction and can cause uric acid crystalluria, nephrolithiasis, obstructive uropathy, and gouty arthritis. Early diagnosis and appropriate therapy with allopurinol can prevent or eliminate all the problems attributable to hyperuricemia without affecting behavioral or neurologic abnormalities.

1	Increased PRPP Synthetase Activity Like the HPRT deficiency states, PRPP synthetase overactivity is X-linked and results in gouty arthritis and uric acid nephrolithiasis. Nerve deafness occurs in some families. Adenine Phosphoribosyltransferase (APRT) Deficiency APRT deficiency is inherited as an autosomal recessive trait. Affected individuals develop kidney stones composed of 2,8-dihydroxyadenine. Caucasians with the disorder have a complete deficiency (type I), whereas Japanese CHAPTER 431e Disorders of Purine and Pyrimidine Metabolism

1	CHAPTER 431e Disorders of Purine and Pyrimidine Metabolism Carbamylphosphate +Aspartic acid Orotic AcidUTP RNA CTP or dCTP RNA or DNA UMPUDPdUMP CMP Cytidine Uracil °-Alanine °-Aminoisobutyric acid TTP 1 2 3 4 5 5 DNA dTMP Thymidine Thymine Uridine 2 FIgURE 431e-4 Abbreviated scheme of pyrimidine metabolism. (1) Thymidine kinase, (2) dihydropyrimidine dehydrogenase, (3) thymidylate synthase, (4) UMP synthase, (5) 5′-nucleotidase. CMP, cytidine-5′-monophosphate; UMP, uridine-5′-monophosphate; UDP, uridine-5′-diphosphate; dUMP, deoxyuridine-5′-monophosphate; dTMP, deoxythymidine-5′-monophosphate; TTP, thymidine triphosphate; UTP, uridine triphosphate. individuals have some measurable enzyme activity (type II). Expression of the defect is similar in the two populations, as is the frequency of the heterozygous state (0.4–1.1 per 100). Allopurinol treatment prevents stone formation.

1	Hereditary Xanthinuria A deficiency of xanthine oxidase causes all purine in the urine to occur in the form of hypoxanthine and xanthine. About two-thirds of deficient individuals are asymptomatic. The remainder develop kidney stones composed of xanthine. Myoadenylate Deaminase Deficiency Primary (inherited) and secondary (acquired) forms of myoadenylate deaminase deficiency have been described. The primary form is inherited as an autosomal recessive trait. Clinically, some persons may have relatively mild myopathic symptoms with exercise or other triggers, but most individuals with this defect are asymptomatic. Therefore, another explanation for the myopathy should be sought in symptomatic patients with this deficiency. The acquired deficiency occurs in association with a wide variety of neuromuscular diseases, including muscular dystrophies, neuropathies, inflammatory myopathies, and collagen vascular diseases.

1	Adenylosuccinate Lyase Deficiency Deficiency of this enzyme is due to an autosomal recessive trait and causes profound psychomotor retardation, seizures, and other movement disorders. All individuals with this deficiency are mentally retarded, and most are autistic. Adenosine Deaminase Deficiency and Purine Nucleoside Phosphorylase Deficiency See Chap. 374. The pyrimidine cytidine is found in both DNA and RNA; it is a complementary base pair for guanine. Thymidine is found only in DNA, where it is paired with adenine. Uridine is found only in RNA and can pair with either adenine or guanine in RNA secondary structures. Pyrimidines can be synthesized by a de novo pathway (Fig. 431e-4) or reused in a salvage pathway. Although more than 25 different enzymes are involved in pyrimidine metabolism, disorders of these pathways are rare. Seven disorders of pyrimidine metabolism have been discovered (Table 431e-4), three of which are discussed below.

1	Orotic Aciduria Hereditary orotic aciduria is caused by mutations in a bifunctional enzyme, uridine-5′monophosphate (UMP) synthase, which converts orotic acid to UMP in the de novo synthesis pathway (Fig. 431e-4). The disorder is characterized by hypochromic megaloblastic anemia that is unresponsive to vitamin B12 and folic acid, growth retardation, and neurologic abnormalities. Increased excretion of orotic acid causes crystalluria and obstructive uropathy. Replacement of uridine (100–200 mg/kg per day) corrects anemia, reduces orotic acid excretion, and improves the other sequelae of the disorder.

1	Pyrimidine 5′-nucleotidase Deficiency Pyrimidine 5′-nucleotidase catalyzes the removal of the phosphate group from pyrimidine ribonucleoside monophosphates (cytidine5′-monophosphate or UMP) (Fig. 431e-4). An inherited deficiency of this enzyme causes hemolytic anemia with prominent basophilic stippling of erythrocytes. The accumulation of pyrimidines or cytidine diphosphate choline is thought to induce hemolysis. There is no specific treatment. Acquired pyrimidine 5′-nucleotidase deficiency has been reported in lead poisoning and in thalassemia. Dihydropyrimidine Dehydrogenase Deficiency Dihydropyrimidine dehydrogenase is the rate-limiting enzyme in the pathway of uracil and thymine degradation (Fig. 431e-4). Deficiency of this enzyme causes excessive urinary excretion of uracil and thymine. In addition, this deficiency causes nonspecific cerebral dysfunction with convulsive disorders, motor retardation, and mental retardation. No specific treatment is available.

1	Medication Effect on Pyrimidine Metabolism A variety of medications can influence pyrimidine metabolism. The anticancer agents fluorodeoxyuridine and 5-fluorouracil and the antimicrobial agent fluorocytosine cause cytotoxicity when converted to fluorodeoxyuridylate, a specific suicide inhibitor of thymidylate synthase. Fluorocytosine must be converted to 5-fluorouracil to be effective. This conversion is catalyzed by cytosine deaminase activity. Fluorocytosine’s action is selective because cytosine deaminase is present in bacteria and fungi but not in human cells. Dihydropyrimidine dehydrogenase is involved in the degradation of 5-fluorouracil. Consequently, deficiency of this enzyme is associated with 5-fluorouracil neurotoxicity.

1	Leflunomide, which is used to treat rheumatoid arthritis, inhibits de novo pyrimidine synthesis by inhibiting dihydroorotate dehydrogenase, resulting in an antiproliferative effect on T cells. Allopurinol, which inhibits xanthine oxidase in the purine metabolic pathway, also inhibits the activity of orotidine-5′-phosphate decarboxylase, a step in UMP synthesis. Consequently, allopurinol use is associated with increased excretion of orotidine and orotic acid. There are no known clinical effects of this inhibition.

1	Lysosomal Storage Diseases Robert J. Hopkin, Gregory A. Grabowski Lysosomes are heterogeneous subcellular organelles containing specific hydrolyses that allow selective processing or degradation of proteins, nucleic acids, carbohydrates, and lipids. There are more than 40 dif-ferent lysosomal storage diseases (LSDs), classified according to the 432e nature of the stored material (Table 432e-1). Several of the most prevalent disorders are reviewed here: Tay-Sachs disease, Fabry disease, Gaucher disease, Niemann-Pick disease, lysosomal acid lipase deficiencies, the mucopolysaccharidoses, and Pompe disease. LSDs should be considered in the differential diagnosis of patients with neurologic, renal, or muscular degeneration and/or unexplained hepatomegaly, splenomegaly, cardiomyopathy, or skeletal dysplasias and deformations. Physical findings are disease specific, and enzyme assays or genetic testing can be used to make a definitive diagnosis. Although the nosology of LSDs segregates the

1	dysplasias and deformations. Physical findings are disease specific, and enzyme assays or genetic testing can be used to make a definitive diagnosis. Although the nosology of LSDs segregates the variants into distinct phenotypes, these are heuristic; in the clinic, each disease exhibits—to some degree—a continuous spectrum of manifestations, from severe to attenuated variants.

1	Lysosomal biogenesis involves ongoing synthesis of lysosomal hydrolases, membrane constitutive proteins, and new membranes. Lysosomes originate from the fusion of trans-Golgi network vesicles with late endosomes. Progressive vesicular acidification accompanies the maturation of these vesicles; this gradient facilitates the pH-dependent dissociation of receptors and ligands and also activates lysosomal hydrolases. Lysosomes are components of the lysosome/autophagy/ mitophagy system, which can be disrupted in the LSDs.

1	Abnormalities at any biosynthetic step can impair enzyme activation and lead to a lysosomal storage disorder. After leader sequence clipping, remodeling of complex oligosaccharides (including the lysosomal targeting ligand mannose-6-phosphate as well as high-mannose oligosaccharide chains of many soluble lysosomal hydrolases) occurs during transit through the Golgi. Lysosomal integral or associated membrane proteins are sorted to the membrane or interior of the lysosome by several different peptide signals. Phosphorylation, sulfation, additional proteolytic processing, and macromolecular assembly of heteromers occur concurrently. Such posttranslational modifications are critical to enzyme function, and defects can result in multiple enzyme/protein deficiencies.

1	The final common pathway for LSDs is the accumulation of specific macromolecules within tissues and cells that normally have a high flux of these substrates. The majority of lysosomal enzyme deficiencies result from point mutations or genetic rearrangements at a locus that encodes a single lysosomal hydrolase. However, some mutations cause deficiencies of several different lysosomal hydrolases by alteration of the enzymes/proteins involved in targeting, active site modifications, or macromolecular association or trafficking. All LSDs are inherited as autosomal recessive disorders except for Hunter (mucopolysaccharidosis type II), Danon, and Fabry diseases, which are X-linked. Substrate accumulation leads to lysosomal distortion, which has significant pathologic consequences. In addition, abnormal amounts of metabolites may also have pharmacologic effects important to disease pathophysiology and propagation.

1	For many LSDs, the accumulated substrates are endogenously synthesized within particular tissue sites of pathology. Other diseases have greater exogenous substrate supplies. For example, they are delivered by low-density lipoprotein receptor–mediated uptake in Fabry and cholesteryl ester storage diseases (CESDs) or by phagocytosis in Gaucher disease type 1. The threshold hypothesis refers to a level of enzyme activity below which disease develops; small changes in enzyme activity near the threshold can lead to or prevent disease. A critical element of this model is that enzymatic activity can be challenged by changes in substrate flux based on genetic background, cell turnover, recycling, or metabolic demands. Thus, a set level of residual 432e-1 enzyme may be adequate for substrate in some tissues or cells but not in others. In addition, several variants of each LSD exist at a clinical level. These disorders therefore represent a continuum of manifestations that are not easily

1	in some tissues or cells but not in others. In addition, several variants of each LSD exist at a clinical level. These disorders therefore represent a continuum of manifestations that are not easily dissociated into discrete entities. The bases for such variations have not been elucidated in any detail.

1	About 1 in 30 Ashkenazi Jews is a carrier for Tay-Sachs disease, which is caused by total hexosaminidase A (Hex A) deficiency resulting from defective α-chains. The infantile form is a fatal neurodegenerative disease with macrocephaly, loss of motor skills, increased startle reaction, and a macular cherry red spot. The juvenile-onset form presents as ataxia and dementia, with death by age 10–15 years. The adult-onset disorder is characterized by clumsiness in childhood; progressive motor weakness in adolescence; and additional spinocerebellar and lower-motor-neuron symptoms and dysarthria in adulthood. Intelligence declines slowly, and psychosis is common. Screening for Tay-Sachs disease carriers is recommended in the Ashkenazi Jewish population. Sandhoff disease, due to a deficiency in both Hex A and Hex B resulting from defective β-chains, is phenotypically similar to Tay-Sachs disease but also includes hepatosplenomegaly and bony dysplasias.

1	Fabry disease is an X-linked disorder that results from mutations in the α-galactosidase A gene. The estimated prevalence of hemizygous males ranges from 1 in 40,000 to 1 in 3500 in selected populations. Clinically, the disease manifests with angiokeratomas (telangiectatic skin lesions), hypohidrosis, corneal and lenticular opacities, acroparesthesia; and progressive small-vessel disease of the kidney, heart, and brain.

1	The angiokeratomas and acroparesthesias may appear in childhood. Angiokeratomas are punctate, dark red to blue-black, flat or slightly raised, and usually symmetric; they do not blanch with pressure. They range from barely visible to several millimeters in diameter and have a tendency to increase in size and number with age. They usually are most dense between the umbilicus and the knees—the “bathing suit area”—but may occur anywhere, including the mucosal surfaces. Angiokeratomas also occur in several other very rare LSDs. Corneal and lenticular lesions, detectable on slit-lamp examination, may help in establishing a diagnosis of Fabry disease. Debilitating episodic burning pain of the hands, feet, and proximal extremities (acroparesthesia) can last from minutes to days and can be precipitated by changes in temperature, exercise, fatigue, or fever. Abdominal pain can resemble that from appendicitis or renal colic. Proteinuria, isosthenuria, and progressive renal dysfunction occur in

1	by changes in temperature, exercise, fatigue, or fever. Abdominal pain can resemble that from appendicitis or renal colic. Proteinuria, isosthenuria, and progressive renal dysfunction occur in the second to fourth decades; ~5% of male patients with idiopathic renal failure have α-galactosidase A mutations. Hypertension, left ventricular hypertrophy, anginal chest pain, and congestive heart failure can occur in the third to fourth decades. About 1–3% of patients with idiopathic hypertrophic myocardiopathy have Fabry disease. Similarly, ~3–5% of male patients with idiopathic stroke at 35–50 years of age have α-galactosidase A mutations. Leg lymphedema without hypoproteinemia and episodic diarrhea also occur. Death is due to renal failure or cardiovascular or cerebrovascular disease in untreated male patients. Variants with residual α-galactosidase A activity may have late-onset manifestations that are limited to the cardiovascular system and resemble hypertrophic cardiomyopathy.

1	untreated male patients. Variants with residual α-galactosidase A activity may have late-onset manifestations that are limited to the cardiovascular system and resemble hypertrophic cardiomyopathy. Variants with predominant cardiac, renal, or central nervous system (CNS) manifestations are becoming better defined. Up to 70% of heterozygous females may exhibit clinical manifestations. However, in females, heart disease is the most common life-threatening manifestation, followed in frequency by stroke and then renal disease.

1	Phenytoin and carbamazepine diminish chronic and episodic acroparesthesia. Chronic hemodialysis or kidney transplantation can be lifesaving in patients with renal failure. Enzyme therapy clears stored lipids from a variety of cells, particularly those of the renal, cardiac, and skin vascular endothelium. Renal insufficiency appears to be irreversible. Early institution of enzyme therapy may prevent or slow the progression of life-threatening complications. Gaucher disease is an autosomal recessive disorder that results from defective activity of acid β-glucosidase; ~400 mutations have been described at the GBA1 locus of such patients. Disease variants are classified by the absence or presence and progression of neuronopathic involvement.

1	Gaucher disease type 1 is a nonneuronopathic disease that can present in childhood to adulthood as slowly to rapidly progressive visceral disease. About 55–60% of patients are diagnosed at <20 years of age in white populations and at even younger ages in other groups. This pattern of presentation is distinctly bimodal, with peaks at <10–15 years and at ~25 years. Younger patients tend to have a greater degree of hepatosplenomegaly and accompanying blood cytopenias. In contrast, the older group has a greater tendency for chronic bone disease. Hepatosplenomegaly occurs in virtually all symptomatic patients and can be minor or massive. Accompanying anemia and thrombocytopenia are variable and are not directly related to liver or spleen volumes. Severe liver dysfunction is unusual. Splenic infarctions can resemble an acute abdomen. Pulmonary hypertension and alveolar Gaucher cell accumulation are uncommon but life-threatening and can occur at any age. GBA1 mutations in the heteroor

1	infarctions can resemble an acute abdomen. Pulmonary hypertension and alveolar Gaucher cell accumulation are uncommon but life-threatening and can occur at any age. GBA1 mutations in the heteroor homozygous state are a significant risk factor for early-onset or more rapidly progressive Parkinson disease.

1	All patients with Gaucher disease have nonuniform infiltration of bone marrow by lipid-laden macrophages termed Gaucher cells. This phenomenon can lead to marrow packing with subsequent infarction, ischemia, necrosis, and cortical bone destruction. Bone marrow involvement spreads from proximal to distal in the limbs and can involve the axial skeleton extensively, causing vertebral collapse. In addition to bone marrow involvement, bone remodeling is defective, with loss of total bone calcium leading to osteopenia, osteonecrosis, avascular infarction, and vertebral compression fractures and spinal cord involvement. Aseptic necrosis of the femoral head is common, as is fracture of the femoral neck. The mechanism by which diseased bone marrow macrophages interact with osteoclasts and/or osteoblasts to cause bone disease is not well understood. Chronic, ill-defined bone pain can be debilitating and poorly correlated with radiographic findings. “Bone crises” are associated with localized

1	to cause bone disease is not well understood. Chronic, ill-defined bone pain can be debilitating and poorly correlated with radiographic findings. “Bone crises” are associated with localized excruciating pain and, on occasion, local erythema, fever, and leukocytosis. These crises represent acute infarctions of bone, as evidenced in nuclear scans by localized absent uptake of pyrophosphate agents. Decreased acid β-glucosidase activity (0–20% of normal) in nucleated cells establishes the diagnosis. The enzyme is not present in bodily fluids. The sensitivity of enzyme testing is poor for heterozygote detection; molecular testing by GBA1 sequencing is preferred. The disease frequency varies from about 1 in 1000 among Ashkenazi Jews to <1 in 100,000 in other populations; ~1 in 12–15 Ashkenazi Jews carries a Gaucher disease allele. Four common mutations account for ~85% of the mutations in that population of affected patients: N370S (1226G), 84GG (a G insertion at cDNA position 84), L444P

1	Jews carries a Gaucher disease allele. Four common mutations account for ~85% of the mutations in that population of affected patients: N370S (1226G), 84GG (a G insertion at cDNA position 84), L444P (1448C), and IVS-2 (an intron 2 splice junction mutation).

1	Genotype/phenotype studies indicate a significant, though not absolute, correlation between disease type and severity and the GBA1 genotype. The most common mutation in the Ashkenazi Jewish population (N370S) shares a 100% association with nonneuronopathic or type 1 Gaucher disease. The N370S/N370S and N370S/other mutant allele genotypes are associated with later-onset/less severe disease and with earlier-onset/severe disease, respectively. As many as 50–60% of individuals with the N370S/N370S genotype are asymptomatic. Other alleles include L444P (very low activity), 84GG (null), or IVS-2 (null) and rare/private or uncharacterized alleles. The L444P/L444P patients almost always have life-threatening to very severe/early-onset disease, and many, though not all, develop CNS involvement in the first two decades of life. Symptom-based treatment of blood cytopenias and joint replacement surgeries continue to have important roles in management.

1	Symptom-based treatment of blood cytopenias and joint replacement surgeries continue to have important roles in management. However, regular intravenous enzyme therapy is currently the treat-432e-5 ment of choice in significantly affected patients and is highly efficacious and safe in diminishing hepatosplenomegaly and improving hematologic values. Bone disease is decreased but not eliminated by enzyme therapy. Adult patients may benefit from adjunctive treatment with bisphosphonates to improve bone density. Patients who cannot be treated with enzyme, either because it is not effective or because they have an allergy or other hypersensitivities, may be receive substrate reduction therapy with medications that decrease the production of the complex lipid molecules that are broken down by acid β-glucosidase.

1	Gaucher disease type 2 is a rare, severe, progressive CNS disease that leads to death by 2 years of age. Gaucher disease type 3 has highly variable manifestations in the CNS and viscera. It can present in early childhood with rapidly progressive, massive visceral disease and slowly progress to static CNS involvement; in adolescence with dementia; or in early adulthood with rapidly progressive, uncontrollable myoclonic seizures and mild visceral disease. Visceral disease in type 3 is nearly identical to that in type 1 but is generally more severe. Early CNS findings may be limited to defects in lateral gaze tracking, which may remain static for decades. Mental retardation can be slowly progressive or static. This variant is most frequent among individuals of Swedish descent. Visceral—but not CNS—involvement responds to enzyme therapy.

1	Niemann-Pick diseases are autosomal recessive disorders that result from defects in acid sphingomyelinase. Types A and B are distinguished by the early age of onset and progressive CNS disease in type A. Type A typically has its onset in the first 6 months of life, with rapidly progressive CNS deterioration, spasticity, failure to thrive, and massive hepatosplenomegaly. Type B has a later, more variable onset and is characterized by a progression of hepatosplenomegaly, with eventual development of cirrhosis and hepatic replacement by foam cells. Affected patients develop progressive pulmonary disease with dyspnea, hypoxemia, and a reticular infiltrative pattern on chest x-ray. Foam cells are present in alveoli, lymphatic vessels, and pulmonary arteries. Progressive hepatic or lung disease leads to death in adolescence or early adulthood.

1	The diagnosis is established by markedly decreased (1–10% of normal) sphingomyelinase activity in nucleated cells. There is no specific treatment for Niemann-Pick disease. The efficacy of hepatic or bone marrow transplantation has not been clearly established. Clinical trials of enzyme therapy are in phases 2 and 3. Niemann-Pick C diseases are progressive CNS diseases due to mutations in either NPC1 or NPC2. They present with liver or splenic disease, but their major manifestations are progressive CNS disease over one to two decades. Treatment with substrate inhibition agents (e.g., Miglustat) and substrate depletion with cyclodextrin have shown promise.

1	Lysosomal acid lipase deficiency may result in Wolman disease (severe deficiency) or CESD, which presents later and has some (3–10%) residual enzyme activity. Wolman disease presents in early infancy with hepatosplenomegaly, diarrhea, vomiting, and abdominal distention, sometimes accompanied by adrenal calcification, anemia, and mixed hyperlipidemia. Death occurs before the age of 1 year and is often due to severe intestinal malabsorption. CESD is heterogeneous and presents with hepatomegaly and hepatosteatosis at any age in childhood or adulthood. CESD should be included in the differential diagnosis for all patients with isolated hypercholesterolemia. The disease may progress to hepatic fibrosis, cirrhosis, and liver failure. In addition, patients often develop very early-onset atherosclerotic vascular disease, which may be life-threatening in childhood. Preliminary results of treatment with enzyme replacement therapy are promising for Wolman disease and CESD.

1	Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disorder caused by deficiency of α-L-iduronidase. The continuum of involvement traditionally has been divided into three categories: (1) Hurler disease (MPS I H) for severe deficiency with neurodegeneration, (2) Scheie disease (MPS I S) for later-onset disease without neurologic involvement and with relatively less severe disease in other organ systems, and (3) Hurler-Scheie syndrome (MPS I H/S) for patients intermediate between these extremes. MPS I H/S is characterized by severe somatic disease, usually without overt neurologic deterioration. MPS I often presents in infancy or early childhood as chronic rhinitis, clouding of the corneas, and hepatosplenomegaly. As the disease progresses, nearly every organ system can be affected. In the more severe forms, cardiac and respiratory diseases become life-threatening in childhood. Skeletal disease can be quite severe, resulting in very limited mobility.

1	There are two current treatments for the MPS I diseases. Hematopoietic stem cell transplantation (HSCT) is the standard treatment for patients presenting at <2 years of age who appear to have or are at risk for neurologic degeneration. HSCT results in stabilization of CNS disease and reverses hepatosplenomegaly. It also beneficially affects cardiac and respiratory disease. HSCT does not eliminate corneal disease or result in the resolution of progressive skeletal disease. Enzyme therapy effectively addresses hepatosplenomegaly and alleviates cardiac and respiratory disease. The enzyme does not effectively penetrate the CNS and does not directly affect CNS disease. Enzyme therapy and HSCT appear to have similar effects on visceral signs and symptoms. Enzyme therapy poses a lower risk of life-threatening complications and may therefore be advantageous for patients who have attenuated manifestations without CNS disease. A combination of enzyme therapy and HSCT has been used, with enzyme

1	complications and may therefore be advantageous for patients who have attenuated manifestations without CNS disease. A combination of enzyme therapy and HSCT has been used, with enzyme therapy initiated prior to transplantation in an attempt to reduce the disease burden. The experience with this approach is not well documented, but it appears to have advantages over HSCT alone.

1	U.S. Food and Drug Administration (FDA) approval. This very rare autosomal recessive disorder is characterized by hepatosplenomegaly, bone disease, heart disease, and respiratory compromise similar to those seen in MPS I; however, MPS VI is due to deficiency of arylsulfatase B and is not associated with neurologic degeneration. Hunter disease (MPS II) is an X-linked disorder due to deficiency in iduronate sulfate sulfatase and has manifestations similar to those of MPS I, including neurologic degeneration. There is no corneal clouding or other eye disease. Like MPS I, MPS II is clinically variable, with CNS and non-CNS variants. HSCT has not been successful in treating CNS disease associated with MPS II. The FDA and the European Medicines Agency (EMA) have approved enzyme therapy for the visceral manifestations of MPS II.

1	Acid maltase (acid α-glucosidase, GAA) deficiency, also called Pompe disease, is the only glycogen LSD. The classic severe infantile form presents with hypotonia, myocardiopathy, and hepatosplenomegaly. This variant is rapidly progressive and generally results in death in the first year of life. However, as with other LSDs, there are earlyand late-onset forms of this disorder. The late-onset variants may be as common as 1 in 40,000; patients typically present with a slowly progressive myopathy that may resemble limb-girdle muscular dystrophy. Respiratory insufficiency may be the presenting sign or may develop with advancing disease. In late stages of the disease, patients may require mechanical ventilation, report swallowing difficulties, and experience loss of bowel and bladder control. Myocardiopathy is not usually seen in late-onset variants of Pompe disease.

1	The FDA and EMA have approved enzyme therapy for Pompe disease. This treatment clearly prolongs life in the infantile form, consistently resulting in improved cardiac function. Respiratory function is also improved in most treated infants. Some infants demonstrate marked improvement in motor functions, while others have minor changes in muscle tone or strength. Prevention of deterioration has been shown with GAA enzyme therapy in the late-onset forms. Early intervention with GAA enzyme therapy in such patients may limit or prevent deterioration, but very advanced disease will have significant irreversible components.

1	Glycogen Storage Diseases and Other Inherited Disorders of Carbohydrate Metabolism Priya S. Kishnani, Yuan-Tsong Chen Carbohydrate metabolism plays a vital role in cellular function by pro-433e viding the energy required for most metabolic processes. The relevant biochemical pathways involved in the metabolism of these carbohydrates are shown in Fig. 433e-1. Glucose is the principal substrate of energy metabolism in humans. Metabolism of glucose generates ATP through glycolysis and mitochondrial oxidative phosphorylation. The body obtains glucose through the ingestion of polysaccharides (primarily starch) and disaccharides (e.g., lactose, maltose, and sucrose). Galactose and fructose are two other monosaccharides that serve as sources of fuel for cellular metabolism; however, their role as fuel sources is much less significant than that of glucose. Galactose is derived from lactose (galactose + glucose), which is found in milk products, and is an important component of certain

1	role as fuel sources is much less significant than that of glucose. Galactose is derived from lactose (galactose + glucose), which is found in milk products, and is an important component of certain glycolipids, 433e-1 glycoproteins, and glycosaminoglycans. Fructose is found in fruits, vegetables, and honey. Sucrose (fructose + glucose) is another dietary source of fructose and is a commonly used sweetener.

1	Debrancher PaP PbKa PbKb Pb Pa ˜-Glucosidase Glucose + Pi ENDOPLASMIC RETICULUM Glucose LYSOSOME Brancher PaP Pa Pb GSa GSa UTP UDP-Gal Gal-1-P Uridyl transferase GSb Phosphoglucomutase Glucokinase, hexokinase Glc-1-P Gal-1-P UDP-Glc Pyrophosphorylase UDP-Gal-4-epimerase Galactose Galacto-kinase Fructose UDP-Glc Glc-6-PGlc-6-P F-6-P F-1, 6-P2 Glyceraldehyde-3-P 1, 3-Bisphosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate Dihydroxyacetone-P Phosphotriose isomerase Glyceraldehyde Aldolase Phosphohexose isomerase Phosphofructokinase Fructose 1,6-diphosphatase Aldolase Glyceraldehyde-3-P dehydrogenase Phosphoglycerate kinase Phosphoglycerate mutase Enolase Pyruvate kinase Lactate dehydrogenase Pyruvate Lactate NADHTCA Cycle CYTOSOL NAD GSb PbKa PbKb G CYTOSOL G G G G G Galactose GLUT2 Glucose Trans-locase GLUT2 GLUT2 Glucose Glucose Fructose F-1-P GLUT2 MITOCHONDRIA Glc-6-Pase FIGuRE 433e-1 Metabolic pathways related to glycogen storage diseases and galactose and

1	G Galactose GLUT2 Glucose Trans-locase GLUT2 GLUT2 Glucose Glucose Fructose F-1-P GLUT2 MITOCHONDRIA Glc-6-Pase FIGuRE 433e-1 Metabolic pathways related to glycogen storage diseases and galactose and fructose disorders. Nonstandard abbreviations are as follows: GS ainactive glycogen synthase; Pa, active phosphorylase; Pb, inactive phosphorylase; P

1	Glycogen, the storage form of glucose in animal cells, is composed of glucose residues joined in straight chains by α1-4 linkages and branched at intervals of 4–10 residues by α1-6 linkages. Glycogen forms a treelike molecule and can have a molecular weight of many millions. Glycogen may aggregate to form structures recognizable by electron microscopy. With the exception of type 0 disease, defects in glycogen metabolism typically cause an accumulation of glycogen in the tissues—hence the designation glycogen storage diseases (GSDs). The structure of stored glycogen can be normal or abnormal in the various disorders. Defects in gluconeogenesis or glycolytic pathways, including galactose and fructose metabolism, usually do not result in glycogen accumulation.

1	Clinical manifestations of the various disorders of carbohydrate metabolism differ markedly. The symptoms range from harmless to lethal. Unlike disorders of lipid metabolism, mucopolysaccharidoses, or other storage diseases, many carbohydrate disorders have been effectively managed with dietary therapy. All of the genes responsible for inherited defects of carbohydrate metabolism have been cloned, and mutations have been identified. Advances in our understanding of the molecular basis of these diseases are being used to improve diagnosis and management. Some of these disorders are candidates for enzyme replacement therapy, substrate reduction therapy, and early trials of gene therapy.

1	Historically, the GSDs were categorized numerically in the order in which the enzymatic defects were identified. They are also classified by the organs involved (liver, muscle, and/or heart) and clinical manifestations. The latter is the system followed in this chapter (Table 433e-1). The overall frequency of all forms of GSD is ~1 in 20,000 live births. Most are inherited as autosomal recessive traits; however, phosphoglycerate kinase deficiency—one form of liver phosphorylase kinase (PhK) deficiency—and lysosomal-associated membrane protein 2 (LAMP2) deficiency are X-linked disorders. The most common childhood disorders are glucose-6-phosphatase deficiency (type I), lysosomal acid α-glucosidase deficiency (type II), debrancher deficiency (type III), and liver PhK deficiency (type IX). The most common adult disorder is myophosphorylase deficiency (type V, or McArdle disease).

1	DISORDERS WITH HEPATOMEGALY AND HYPOGLYCEMIA Type I GSD (Glucose-6-Phosphatase or Translocase Deficiency, Von Gierke’s Disease) Type I GSD is an autosomal recessive , active glycogen synthase; GS , disorder caused by glucose-6-phospha aP, phosphorylase tase deficiency in liver, kidney, and intes- CHAPTER 433e Glycogen Storage Diseases and Other Inherited Disorders of Carbohydrate Metabolism α phosphatase; PbKa, active phosphorylase β kinase; PbKb, inactive phosphorylase β kinase; G, gly-tinal mucosa. There are two subtypes of cogenin, the primer protein for glycogen synthesis. (Modified from AR Beaudet, in KJ Isselbacher et al GSD I: type Ia, in which the glucose[eds]: Harrison’s Principles of Internal Medicine, 13th ed., New York, McGraw-Hill, 1994, p 1855.) 6-phosphatase enzyme is defective, and Disorders with hepatomegaly and hypoglycemia Disorders with liver cirrhosis

1	Disorders with hepatomegaly and hypoglycemia Disorders with liver cirrhosis Growth retardation, enlarged liver and kidney, hypoglycemia, elevated blood lactate, cholesterol, triglycerides, and uric acid As for Ia, with additional findings of neutropenia and neutrophil dysfunction Childhood: hepatomegaly, growth retardation, muscle weakness, hypoglycemia, hyperlipidemia, elevated liver aminotransferases. Liver symptoms improve with age. Adulthood: muscle atrophy and weakness; onset in third or fourth decades; variable cardiomyopathy, liver cirrhosis, progressive liver failure Liver symptoms same as in type IIIa; no muscle symptoms Hepatomegaly, variable hypoglycemia, hyperlipidemia, and ketosis. Symptoms may improve with age. Fasting hypoglycemia and ketosis, elevated lactic acid and hyperglycemia after glucose load, no hepatomegaly

1	Fasting hypoglycemia and ketosis, elevated lactic acid and hyperglycemia after glucose load, no hepatomegaly Failure to thrive, short stature, hypophosphatemic rickets, metabolic acidosis, hepatomegaly, proximal renal tubular dysfunction, impaired glucose and galactose utilization Common, severe hypoglycemia. Complications in adulthood include hepatic adenomas, hepatic carcinoma, and renal failure. ~10% of type I Common, intermediate severity of hypoglycemia. Hepatic adenomas, liver cirrhosis, and hepatic carcinoma can occur. ~15% of type III Rare, often a “benign” glycogenosis, severe cases being recognized Common, X-linked, typically less severe than autosomal forms; clinical variability within and between subtypes; severe cases being recognized Rare, consanguinity in 70% Fructose 1,6-bisphosphate aldolase A deficiency Fructose 1,6-bisphosphate aldolase A

1	Rare, consanguinity in 70% Fructose 1,6-bisphosphate aldolase A deficiency Fructose 1,6-bisphosphate aldolase A Muscle β-enolase Exercise intolerance, muscle cramps, myoglobinuria on strenuous exercise, increased CK, “second-wind” phenomenon As for type V, with additional findings of compensated hemolysis, myalgia As for type V, with additional findings of hemolytic anemia and CNS dysfunction As for type V, with additional findings of erythematous skin eruption and uterine stiffness resulting in childbirth difficulty in females As for type V, with additional finding of hemolytic anemia As for type V. Some patients may have muscle weakness and atrophy. Disorders with progressive skeletal muscle myopathy and/or cardiomyopathy Common, male predominance Prevalent in Ashkenazi Jews and Japanese Rare, X-linked Rare, most patients African American Rare Rare Rare Rare, autosomal recessive Rare

1	Common, male predominance Prevalent in Ashkenazi Jews and Japanese Rare, X-linked Rare, most patients African American Rare Rare Rare Rare, autosomal recessive Rare Danon’s disease Lysosomal-associated membrane protein 2 (LAMP2) Infantile: hypotonia, muscle weakness, cardiac enlargement and failure, fatal early late onset (juvenile and adult): progressive skeletal muscle weakness and atrophy, proximal muscle and respiratory muscle seriously affected Severe cardiomyopathy and early heart failure (9–55 years). Congenital fetal form is rapidly fatal with hypertrophic cardiomyopathy and Wolff-Parkinson-White syndrome. Severe cardiomyopathy and heart failure (8–15 years) Common, undetectable or very low level of enzyme activity in infantile form; variable residual enzyme activity in late-onset form Very rare, autosomal dominant Very rare, X-linked Abbreviations: CK, creatine kinase; CNS, central nervous system; M, muscle.

1	Very rare, autosomal dominant Very rare, X-linked Abbreviations: CK, creatine kinase; CNS, central nervous system; M, muscle. type Ib, in which the translocase that transports glucose-6-phosphate across the microsomal membrane is defective. The defects in both subtypes lead to inadequate conversion of glucose-6-phosphate to glucose in the liver and thus make affected individuals susceptible to fasting hypoglycemia.

1	CliniCal and laboratory findings Persons with type I GSD may develop hypoglycemia and lactic acidosis during the neonatal period; however, more commonly, they exhibit hepatomegaly at 3–4 months of age. Hypoglycemia, hypoglycemic seizures, and lactic acidosis can develop after a short fast. These children usually have doll-like faces with fat cheeks, relatively thin extremities, short stature, and a protuberant abdomen that is due to massive hepatomegaly. The kidneys are enlarged, but the spleen and heart are of normal size. The hepatocytes are distended by glycogen and fat, with large and prominent lipid vacuoles. Despite hepatomegaly, liver enzyme levels are usually normal or near normal. Easy bruising and epistaxis are associated with a prolonged bleeding time as a result of impaired platelet aggregation/adhesion. Hyperuricemia is present. Hyperlipidemia includes elevation of triglycerides, low-density lipoproteins, and phospholipids. Type Ib patients have additional findings of

1	platelet aggregation/adhesion. Hyperuricemia is present. Hyperlipidemia includes elevation of triglycerides, low-density lipoproteins, and phospholipids. Type Ib patients have additional findings of neutropenia and impaired neutrophil function, which result in recurrent bacterial infections and oral and intestinal mucosal ulceration. GSD I patients may experience intermittent diarrhea, which can worsen with age. In GSD Ib, diarrhea is largely due to loss of mucosal barrier function caused by inflammation.

1	long-term CompliCations Gout usually becomes symptomatic at puberty as a result of long-term hyperuricemia. Puberty is often delayed. Nearly all female patients have ultrasound findings consistent with polycystic ovaries; however, the other clinical features of polycystic ovary syndrome, such as acne and hirsutism, are not seen. Several reports of successful pregnancy in women with GSD I suggest that fertility is not affected. Increased bleeding during menstrual cycles, including life-threatening menorrhagia, has been reported. Secondary to lipid abnormalities, there is an increased risk of pancreatitis. Patients with GSD I may be at increased risk for cardiovascular disease. In adult patients, frequent fractures can occur and radiographic evidence of osteopenia/ osteoporosis can be found; in prepubertal patients, radial bone mineral content is significantly reduced. Pulmonary hypertension—although rare—has been reported. By the second or third decade of life, many patients with type

1	in prepubertal patients, radial bone mineral content is significantly reduced. Pulmonary hypertension—although rare—has been reported. By the second or third decade of life, many patients with type I GSD develop hepatic adenomas that can hemorrhage and, in some cases, become malignant. Renal disease is a serious late complication. Almost all patients older than 20 years have proteinuria, and many have hypertension, kidney stones, nephrocalcinosis, and altered creatinine clearance. Laboratory findings include abnormally high levels of blood lactate, triglycerides, cholesterol, and uric acid. In some patients, renal function deteriorates and progresses to complete failure, requiring dialysis or transplantation.

1	diagnosis Clinical presentation and abnormal plasma lactate and lipid values suggest that a patient may have GSD I, but gene-based mutation analysis provides a noninvasive means of reaching a definitive diagnosis for most patients with types Ia and Ib disease. Before the glucose-6-phosphatase and glucose-6-phosphate translocase genes were cloned, a definitive diagnosis required a liver biopsy to demonstrate a deficiency. Type III GSD (Debrancher Deficiency, Limit Dextrinosis) Type III GSD is an autosomal recessive disorder caused by a deficiency of glycogen debranching enzyme. Debranching and phosphorylase enzymes are responsible for complete degradation of glycogen. When the debranching enzyme is defective, glycogen breakdown is incomplete. Abnormal glycogen accumulates with short outer chains and resembles dextrin.

1	CliniCal and laboratory findings Deficiency of glycogen debranching enzyme causes hepatomegaly, hypoglycemia, short stature, variable skeletal myopathy, and cardiomyopathy. The disorder usually involves both liver and muscle and, in such cases, is termed type IIIa GSD. However, in ~15% of patients, the disease appears to involve only the liver and is classified as type IIIb. Hypoglycemia and hyperlipidemia occur in children. In type III disease (as opposed to type I disease), fasting ketosis can be prominent, aminotransferase levels are elevated, and blood lactate and uric acid concentrations are usually normal. Serum creatine kinase (CK) levels can sometimes be used to identify patients with muscle involvement, but normal levels do not rule out muscle enzyme deficiency. In most patients with type III disease, hepatomegaly improves with age; however, liver cirrhosis and hepatocellular carcinoma may occur in late adulthood. Hepatic adenomas may occur, although less commonly than in GSD

1	type III disease, hepatomegaly improves with age; however, liver cirrhosis and hepatocellular carcinoma may occur in late adulthood. Hepatic adenomas may occur, although less commonly than in GSD I. Left ventricular hypertrophy and life-threatening arrhythmias have been reported. Patients with type IIIa disease may experience muscle weakness in childhood that can become severe after the third or fourth decade of life. Polycystic ovaries are common in GSD III, and some patients develop features of polycystic ovarian syndrome, such as hirsutism and irregular menstrual cycles. Reports of successful pregnancy in women with GSD III suggest that fertility is normal.

1	diagnosis In type IIIa GSD, deficient debranching enzyme activity can be demonstrated in liver, skeletal muscle, and heart. In contrast, patients with type IIIb have debranching enzyme deficiency in the liver but not in muscle. The liver has distended hepatocytes due to glycogen buildup; areas of fibrosis are also noted very early in the disease course. In the past, definitive assignment of subtype required enzyme assays in both liver and muscle. DNA-based analyses now provide a noninvasive way of subtyping these disorders in most patients. However, the large size of the gene and the distribution of private mutations across it pose challenges in DNA-based analysis. CHAPTER 433e Glycogen Storage Diseases and Other Inherited Disorders of Carbohydrate Metabolism

1	Type IX GSD (Liver Phosphorylase Kinase Deficiency) Defects of PhK cause a heterogeneous group of glycogenoses. The PhK enzyme complex consists of four subunits (α, β, γ, and δ). Each subunit is encoded by different genes (X chromosome as well as autosomes) that are differentially expressed in various tissues. PhK deficiency can be divided into several subtypes on the basis of the gene/subunit involved, the tissues primarily affected, and the mode of inheritance. The most common subtype is X-linked liver PhK deficiency, which is also one of the most common liver glycogenoses. PhK activity may also be deficient in erythrocytes and leukocytes but is normal in muscle. Typically, a child between the ages of 1 year and 5 years presents with growth retardation and hepatomegaly. Children tend eventually to exhibit normal growth patterns initiated by a delayed growth spurt during puberty. Liver fibrosis has been identified in some patients, including children. Levels of cholesterol,

1	tend eventually to exhibit normal growth patterns initiated by a delayed growth spurt during puberty. Liver fibrosis has been identified in some patients, including children. Levels of cholesterol, triglycerides, and liver enzymes are mildly elevated. Fasting ketosis is another feature of the disease. Lactic and uric acid levels are usually normal. Hypoglycemia is typically mild; however, phenotypic variability is being increasingly recognized, with significant involvement in some cases of the X-linked form. The accumulated glycogen in liver (β particles, rosette form) has a frayed or burst appearance and is less compact than the glycogen seen in type I or type III GSD. Hepatomegaly and abnormal blood chemistries gradually return to normal with age. Most adults reach a normal final height and are practically asymptomatic, despite persistent PhK deficiency. The prognosis is usually good, and adult patients have minimal hepatomegaly. Some patients have significant ketosis and

1	height and are practically asymptomatic, despite persistent PhK deficiency. The prognosis is usually good, and adult patients have minimal hepatomegaly. Some patients have significant ketosis and progressive liver disease that can advance to fibrosis and liver failure. It is recommended that adult patients be monitored for hepatic complications with regular CT or MRI scans.

1	Treatment is symptom based. A high-carbohydrate diet and frequent feedings are effective in preventing hypoglycemia. Some patients require no specific treatment. Recent studies have shown that instituting a treatment regimen of cornstarch and protein feedings early, even in seemingly stable patients, may prevent long-term complications. Blood ketones and glucose should be evaluated during times of stress. Other subtypes of type IX GSD include an autosomal recessive form of liver and muscle PhK deficiency, an autosomal recessive form of liver PhK deficiency that often develops into liver cirrhosis, a muscle-specific PhK deficiency that causes cramps and myoglobinuria with exercise, and a cardiac-specific PhK deficiency that is lethal during infancy because of massive glycogen deposition in the myocardium. The finding of PhK deficiency in the cardiac-specific PhK deficiency may be a secondary phenomenon, as a subset of these patients have mutations in PRKAG2.

1	Other Liver Glycogenoses with Hepatomegaly and Hypoglycemia These disorders include hepatic phosphorylase deficiency (Hers disease, type VI) and hepatic glycogenosis with renal Fanconi syndrome (type XI). Patients with GSD type VI can have growth retardation, hyperlipidemia, and hyperketosis in addition to hepatomegaly and hypoglycemia. Some patients have a benign clinical course. GSD XI is caused by defects in the facilitative glucose transporter 2 (GLUT-2), which transports glucose and galactose in and out of hepatocytes, pancreatic cells, and the basolateral membranes of intestinal and renal epithelial cells. The disease is characterized by proximal renal tubular dysfunction, impaired glucose and galactose utilization, and accumulation of glycogen in liver and kidney.

1	Type V GSD (Muscle Phosphorylase Deficiency, McArdle Disease) Type V GSD is an autosomal recessive disorder caused by deficiency of muscle phosphorylase. McArdle disease is a prototypical muscle-energy disorder as the enzyme deficiency limits ATP generation by glycogenolysis and results in glycogen accumulation.

1	CliniCal and laboratory findings Usually, symptoms first develop in adulthood and involve exercise intolerance with muscle cramps. Two types of activity tend to cause symptoms: (1) brief exercise of great intensity, such as sprinting or carrying heavy loads; and (2) less intense but sustained activity, such as climbing stairs or walking uphill. Most patients can engage in moderate exercise, such as walking on level ground, for long periods. Patients often exhibit the “second-wind” phenomenon, in which, after a short break from the initiation of strenuous physical effort, they are able to continue the activity without pain. Although most patients experience episodic muscle pain and cramping as a result of exercise, 35% report permanent pain that seriously affects sleep and other activities. About half of patients report burgundy-colored urine after exercise; this coloration results from myoglobinuria secondary to rhabdomyolysis. Intense myoglobinuria after vigorous exercise can lead to

1	About half of patients report burgundy-colored urine after exercise; this coloration results from myoglobinuria secondary to rhabdomyolysis. Intense myoglobinuria after vigorous exercise can lead to renal failure. Clinical heterogeneity is uncommon; however, there are cases with symptom onset as late as the eighth decade and cases that present early with hypotonia, generalized muscle weakness, and progressive respiratory insufficiency, which is often fatal.

1	Although cardiac involvement is not usually associated with muscle phosphorylase deficiency, hypertrophic cardiomyopathy has been observed in an adult patient with GSD V. In rare cases, electromyographic findings may suggest inflammatory myopathy, a diagnosis that may be confused with polymyositis. These patients may be at risk for statin-induced myopathy and rhabdomyolysis. During rest, the serum CK level is usually elevated; after exercise, the CK level increases even more. Exercise also increases levels of blood ammonia, inosine, hypoxanthine, and uric acid; these abnormalities reflect residues of accelerated muscle purine nucleotide recycling as a result of insufficient ATP production. NADH is underproduced during physical exertion.

1	diagnosis Lack of an increase in blood lactate and exaggerated blood ammonia elevations after an ischemic exercise test are indicative of a muscle glycogenosis and suggest a defect in the conversion of glycogen or glucose to lactate. This abnormal exercise response, however, can also occur with other defects in glycogenolysis or glycolysis, such as deficiencies of muscle phosphofructokinase or debranching enzyme (when the test is done after fasting). The cycle test detects the hallmark heart rate observed during the second-wind phenomenon. A definitive diagnosis is made by enzymatic assay in muscle tissue or by mutation analysis of the myophosphorylase gene.

1	DISORDERS WITH PROGRESSIVE SKELETAL MuSCLE MYOPATHY AND/OR CARDIOMYOPATHY Pompe Disease, Type II GSD (Acid α-1,4 Glucosidase Deficiency) Pompe disease is an autosomal recessive disorder caused by a deficiency of lysosomal acid α-1,4 glucosidase, an enzyme responsible for the degradation of glycogen in the lysosomes. This disease is characterized by the accumulation of glycogen in the lysosomes as opposed to accumulation in cytoplasm (as in the other glycogenoses). CliniCal and laboratory findings The disorder encompasses a range of phenotypes. Each includes myopathy but differs in the age of onset, extent of organ involvement, and clinical severity. The most severe is the infantile form, with cardiomegaly, hypotonia, and death before the age of 1 year. Infants may appear normal at birth but soon develop generalized muscle weakness with feeding difficulties, macroglossia, hepatomegaly, and congestive heart failure due to hypertrophic cardiomyopathy.

1	The late-onset form (juvenile/late-childhood or adult form) is characterized by skeletal muscle manifestations, usually with minimal or no cardiac involvement, and a more slowly progressive course. The juvenile form typically presents as delayed motor milestones (if age of onset is early enough) and difficulty in walking. With disease progression, patients often develop swallowing difficulties, proximal muscle weakness, and respiratory muscle involvement. Death may occur before the end of the second decade.

1	Adults typically present between the second and seventh decades with slowly progressive myopathy without overt cardiac involvement. The clinical picture is dominated by slowly progressive proximal limb girdle muscle weakness. The pelvic girdle, paraspinal muscles, and diaphragm are most seriously affected. Respiratory symptoms include somnolence, morning headache, orthopnea, and exertional dyspnea. In rare instances, patients present with respiratory insufficiency as the initial symptom. Basilar artery aneurysms and dilation of the ascending aorta have been observed in patients with Pompe disease. Ptosis, lingual weakness, gastrointestinal dysmotility, and incontinence due to poor sphincter tone are now being recognized as part of the clinical spectrum. Individuals with advanced disease often require some form of ventilation and are dependent on a walking aid or wheelchair.

1	Laboratory findings include elevated levels of serum CK, aspartate aminotransferase, and lactate dehydrogenase. Levels of urine glucose tetrasaccharide (Hex4), a breakdown product of glycogen, are elevated, especially on the severe end of the disease spectrum. In infants, chest x-ray shows massive cardiomegaly, and electrocardiographic findings include a high-voltage QRS complex and a shortened PR interval. Muscle biopsy shows vacuoles that stain positive for glycogen; the muscle acid phosphatase level is increased, presumably from a compensatory increase of lysosomal enzymes. Electromyography reveals myopathic features, with irritability of muscle fibers and pseudomyotonic discharges. Serum CK is not always elevated in adults, and, depending on the muscle biopsied or tested, muscle histology or electromyography may not be abnormal. The affected muscle should be examined.

1	diagnosis The confirmatory step for a diagnosis of Pompe disease is enzyme assay demonstrating deficient acid α-glucosidase or a gene sequence with two pathogenic mutations in the GAA gene. Enzyme activity can be measured in muscle, cultured skin fibroblasts, or blood. Deficiency is usually more severe in the infantile form. Early diagnosis is the key to treatment efficacy.

1	GSD Mimicking Hypertrophic Cardiomyopathy Deficiency of LAMP2— also called Danon’s disease—or of the protein kinase, AMP-activated gamma 2 noncatalytic subunit (PRKAG2), results in the accumulation of glycogen in the heart and skeletal muscle. LAMP2 deficiency is X-linked, whereas PRKAG2 deficiency is autosomal dominant. Clinically, both subsets of patients present primarily with hypertrophic cardiomyopathy. Their electrophysiologic abnormalities, particularly ventricular preexcitation and conduction defects, can distinguish them from patients with hypertrophic cardiomyopathy resulting from defects in sarcomere-protein genes. In patients with LAMP2 deficiency, mental delays are common and the onset of cardiac symptoms, including chest pain, palpitation, syncope, and cardiac arrest, can occur between the ages of 8 and 15 years—i.e., earlier than the average age of 33 years for patients with PRKAG2 deficiency. Patients as young as 9 years old have presented with PRKAG2 deficiency. A

1	occur between the ages of 8 and 15 years—i.e., earlier than the average age of 33 years for patients with PRKAG2 deficiency. Patients as young as 9 years old have presented with PRKAG2 deficiency. A rapidly fatal congenital form of PRKAG2 presents in early infancy with severe hypertrophic cardiomyopathy and Wolff-Parkinson-White syndrome. In these patients, levels of PhK have been found to be low. The prognosis for LAMP2 deficiency is poor, with progressive end-stage heart failure early in adulthood. By contrast, except in the fatal congenital form, long-term survival is possible for patients with cardiomyopathy due to PRKAG2 mutations. Some patients may require the implantation of a pacemaker and aggressive control of arrhythmias. Congestive heart failure has been documented in patients with PRKAG2 deficiency.

1	Heart transplantation has been suggested as a preventive measure for LAMP2 deficiency and noncongenital PRKAG2 deficiency. “Classic” galactosemia is caused by galactose 1-phosphate uridyltransferase (GALT) deficiency. It is a serious disease with an incidence of 1 in 60,000 and an early onset of symptoms. The newborn infant normally receives up to 40% of caloric intake as lactose (glucose + galactose). Without the transferase, the infant is unable to metabolize galactose 1-phosphate (Fig. 433e-1), which consequently accumulates, resulting in injury to parenchymal cells of the kidney, liver, and brain. After the first feeding, infants can present with vomiting, diarrhea, hypotonia, jaundice, and hepatomegaly. Patients with galactosemia are at increased risk for Escherichia coli neonatal sepsis; the onset of sepsis often precedes the diagnosis of galactosemia.

1	Widespread newborn screening for galactosemia has identified 433e-5 these infants early and allowed them to be placed on dietary restriction. Elimination of galactose from the diet reverses growth failure as well as renal and hepatic dysfunction, improving the prognosis. However, on long-term follow-up, some patients still have ovarian failure manifesting as primary or secondary amenorrhea as well as developmental delays and learning disabilities that increase in severity with age. Of women with classic galactosemia, 80–90% or more report hypergonadotropic hypogonadism. While most female patients are infertile when they reach childbearing age, a few have given birth. Several mutations appear to be protective, particularly the p.Ser135Leu mutation, which is more common in the African-American population. Methods for fertility preservation, such as cryopreservation, are still in the experimental stages. In addition, most patients have speech disorders, and a smaller proportion

1	population. Methods for fertility preservation, such as cryopreservation, are still in the experimental stages. In addition, most patients have speech disorders, and a smaller proportion demonstrate poor growth and impaired motor function and balance (with or without overt ataxia). Adults on dairy-free diets have developed cataracts, tremors, and low bone density. The treatment of galactosemia to prevent long-term complications remains a challenge.

1	Deficiency of galactokinase (Fig. 433e-1) causes cataracts. Deficiency of uridine diphosphate galactose 4-epimerase can be benign when the enzyme deficiency is limited to blood cells but can be as severe as classic galactosemia when the enzyme deficiency is generalized. Fructokinase deficiency, or essential fructosemia (Fig. 433e-1), causes a benign condition that is usually an incidental finding made through the detection of fructose as a reducing substance in the urine.

1	Deficiency of fructose 1,6-bisphosphate aldolase (aldolase B; hereditary fructose intolerance) is a serious disease in infants. These patients are healthy and asymptomatic until fructose or sucrose (table sugar) is ingested (usually from fruit, sweetened cereal, or sucrose-containing formula). Clinical manifestations may include jaundice, hepatomegaly, vomiting, lethargy, irritability, and convulsions. The incidence of celiac disease is higher among patients with hereditary fructose intolerance (>10%) than in the general population (1–3%). Laboratory findings show prolonged clotting time, hypoalbuminemia, elevation of bilirubin and aminotransferase levels, and proximal renal tubular dysfunction. If the disease is not diagnosed and intake of the noxious sugar continues, hypoglycemic episodes recur, and liver and kidney failure progresses, eventually leading to death. Treatment requires the elimination of all sources of sucrose, fructose, and sorbitol from the diet. Through this

1	episodes recur, and liver and kidney failure progresses, eventually leading to death. Treatment requires the elimination of all sources of sucrose, fructose, and sorbitol from the diet. Through this treatment, liver and kidney dysfunction improve, and catch-up growth is common; intellectual development is usually unimpaired. Over time, the patient’s symptoms become milder, even after fructose ingestion, and the long-term prognosis is good.

1	Fructose 1,6-diphosphatase deficiency is characterized by childhood life-threatening episodes of acidosis, hypoglycemia, hyperventilation, convulsions, and coma. These episodes are triggered by febrile infections and gastroenteritis when oral food intake decreases. Laboratory findings show low blood glucose levels, high lactate and uric acid levels, and metabolic acidosis. Unlike hereditary fructose intolerance, this deficiency usually is not associated with an aversion to sweets, and renal tubular and liver functions are normal. Treatment of acute attacks requires the correction of hypoglycemia and acidosis by IV infusion. Later, avoidance of fasting and elimination of fructose and sucrose from the diet prevent further episodes. A slowly released carbohydrate such as cornstarch is useful for the long-term prevention of hypoglycemia. The prognosis is good, as patients who survive childhood develop normally.

1	CHAPTER 433e Glycogen Storage Diseases and Other Inherited Disorders of Carbohydrate Metabolism The GSDs and other inherited disorders of carbohydrate metabolism, although rare, have been reported in most ethnic populations. The prevalent genetic mutations for each disease may vary in different ethnic populations, but clinical symptoms are remarkably similar and treatment guidelines apply to all populations. The practice of newborn screening should be considered worldwide to intercept the rapid progression of many of these disorders.

1	Inherited Disorders of Amino Acid Metabolism in Adults Nicola Longo Amino acids are not only the building blocks of proteins but also serve as neurotransmitters (glycine, glutamate, γ-aminobutyric acid) or as 434e precursors of hormones, coenzymes, pigments, purines, or pyrimidines. Eight amino acids, referred to as essential, cannot be synthesized by humans and must be obtained from dietary sources. The others are formed endogenously. Each amino acid has a unique degradative pathway by which its nitrogen and carbon components are used for the synthesis of other amino acids, carbohydrates, and lipids. Disorders of amino acid metabolism and transport (Chap. 435e) are individually rare—the incidences range from 1 in 10,000 for cystinuria or phenylketonuria to 1 in 200,000 for homocystinuria or alkaptonuria—but collectively, they affect perhaps 1 in 1000 newborns. Almost all are transmitted as autosomal recessive traits.

1	The features of inherited disorders of amino acid catabolism are summarized in Table 434e-1. In general, these disorders are named for the compound that accumulates to highest concentration in blood (-emias) or urine (-urias). In the aminoacidopathies, the parent amino acid is found in excess, whereas products in the catabolic pathway accumulate in organic acidemias. Which compound(s) accumulates depends on the site of the enzymatic block, the reversibility of the reactions proximal to the lesion, and the availability of alternative pathways of metabolic “runoff.” Biochemical and genetic heterogeneity are common. Five distinct forms of hyperphenylalaninemia, nine forms of homocystinuria, and methylmalonic acidemia are recognized. Such heterogeneity reflects the presence of a large array of molecular defects.

1	The manifestations of these conditions differ widely (Table 434e-1). Some, such as sarcosinemia, produce no clinical consequences. At the other extreme, complete deficiency of ornithine transcarbamylase is lethal in the untreated neonate. Central nervous system (CNS) dysfunction, in the form of developmental retardation, seizures, alterations in sensorium, or behavioral disturbances, is present in more than half the disorders. Protein-induced vomiting, neurologic dysfunction, and hyperammonemia occur in many disorders of urea cycle intermediates. Metabolic ketoacidosis, often accompanied by hyperammonemia, is a frequent presenting finding in disorders of branched-chain amino acid metabolism. Some disorders produce focal tissue or organ involvement such as liver disease, renal failure, cutaneous abnormalities, or ocular lesions.

1	The analysis of plasma amino acids (by ion-exchange chromatography), urine organic acids (by gas chromatography/mass spectrometry), and plasma acylcarnitine profile (by tandem mass spectrometry) is commonly used to diagnose and monitor most of these disorders. The diagnosis is confirmed by enzyme assay on cells or tissues from the patients or by DNA testing. The clinical manifestations in many of these conditions can be prevented or mitigated if a diagnosis is achieved early and appropriate treatment (e.g., dietary protein or amino acid restriction or vitamin supplementation) is instituted promptly. For this reason, newborn screening programs seek to identify several of these disorders. Infants with a positive screening test need additional metabolic testing (usually suggested by the newborn screening program) to confirm or exclude the diagnosis. Confirmed cases should be referred to a metabolic center for initiation of therapy. The parents need to be counseled about the recurrence

1	screening program) to confirm or exclude the diagnosis. Confirmed cases should be referred to a metabolic center for initiation of therapy. The parents need to be counseled about the recurrence risk of the disease in future pregnancies. In some cases, parents need testing to exclude metabolic alterations seen in carriers for some of these disorders (such as some forms of homocystinuria) or because they might have a disorder themselves (such as glutaric acidemia type 1, methylcrotonyl coenzyme A carboxylase deficiency, or fatty acid oxidation defects). Some metabolic disorders can remain asymptomatic until adult age, presenting only when fasting or severe stress require full activity of affected metabolic pathways to provide energy.

1	Selected disorders that illustrate the principles, properties, and 434e-1 problems presented by the disorders of amino acid metabolism are discussed in this chapter.

1	The hyperphenylalaninemias (Table 434e-1) result from impaired conversion of phenylalanine to tyrosine. The most common and clinically important is phenylketonuria (frequency 1:10,000), which is an autosomal recessive disorder characterized by an increased concentration of phenylalanine and its by-products in body fluids and by severe mental retardation if untreated in infancy. It results from reduced activity of phenylalanine hydroxylase. The accumulation of phenylalanine inhibits the transport of other amino acids required for protein or neurotransmitter synthesis, reduces synthesis and increases degradation of myelin, and leads to inadequate formation of norepinephrine and serotonin. Phenylalanine is a competitive inhibitor of tyrosinase, a key enzyme in the pathway of melanin synthesis, and accounts for the hypopigmentation of hair and skin. Untreated children with classic phenylketonuria are normal at birth but fail to attain early developmental milestones, develop microcephaly,

1	and accounts for the hypopigmentation of hair and skin. Untreated children with classic phenylketonuria are normal at birth but fail to attain early developmental milestones, develop microcephaly, and demonstrate progressive impairment of cerebral function. Hyperactivity, seizures, and severe intellectual disability are major clinical problems later in life. Electroencephalographic abnormalities; “mousy” odor of skin, hair, and urine (due to phenylacetate accumulation); and a tendency to develop hypopigmentation and eczema complete the devastating clinical picture. In contrast, affected children who are detected and treated at birth show none of these abnormalities.

1	To prevent intellectual disability, diagnosis and initiation of dietary treatment of classic phenylketonuria must occur before the child is 2 weeks of age. For this reason, most newborns in North America, Australia, and Europe are screened by determinations of blood phenylalanine levels. Abnormal values are confirmed using quantitative analysis of plasma amino acids. Dietary phenylalanine restriction is usually instituted if blood phenylalanine levels are >360 μmol/L (6 mg/dL). Treatment consists of a special diet low in phenylalanine and supplemented with tyrosine, since tyrosine becomes an essential amino acid in phenylalanine hydroxylase deficiency. With therapy, plasma phenylalanine concentrations should be maintained between 120 and 360 μmol/L (2 and 6 mg/dL). Dietary restriction should be continued and monitored indefinitely. Some patients with milder forms of phenylketonuria (phenylalanine <1200 μm at presentation) show increased tolerance to dietary proteins and improved

1	should be continued and monitored indefinitely. Some patients with milder forms of phenylketonuria (phenylalanine <1200 μm at presentation) show increased tolerance to dietary proteins and improved metabolic control when treated with tetrahydrobiopterin (5–20 mg/ kg per day), an essential cofactor of phenylalanine hydroxylase.

1	A number of women with phenylketonuria who have been treated since infancy will reach adulthood and become pregnant. If maternal phenylalanine levels are not strictly controlled before and during pregnancy, their offspring are at increased risk for congenital defects and microcephaly (maternal phenylketonuria). After birth, these children have severe intellectual disability and growth retardation. Pregnancy risks can be minimized by continuing lifelong phenylalanine-restricted diets and assuring strict phenylalanine restriction 2 months prior to conception and throughout gestation. CHAPTER 434e Inherited Disorders of Amino Acid Metabolism in Adults The homocystinurias are nine biochemically and clinically distinct disorders (Table 434e-1) characterized by increased concentration of the sulfur-containing amino acid homocystine in blood and urine.

1	Classic homocystinuria, the most common (frequency 1:200,000), results from reduced activity of cystathionine β-synthase (Fig. 434e-1), the pyridoxal phosphate–dependent enzyme that condenses homocysteine with serine to form cystathionine. Most patients present between CHAPTER 434e Inherited Disorders of Amino Acid Metabolism in Adults Abbreviations: AD, autosomal dominant; AR, autosomal recessive; Cbl, cobalamin; DOPA, dihydroxyphenylalanine; GABA, γ-aminobutyric acid; GTP, guanosine 5′-triphosphate; XL, X-linked.

1	Abbreviations: AD, autosomal dominant; AR, autosomal recessive; Cbl, cobalamin; DOPA, dihydroxyphenylalanine; GABA, γ-aminobutyric acid; GTP, guanosine 5′-triphosphate; XL, X-linked. 5,10-Methylene THF Methylene Tetrahydro Folate Reductase (MTHFR) N5-methyl THF Methyl-cobalamin Cobalamin (B12) cbl C, D, F, J, X Glycine Serine TetraHydro Folate (THF) Remethylation Methionine Synthase Reductase (cblE) Methionine Synthase (cblG) Methionine Betaine Dimethylglycine Betaine homocysteine methyltransferase Homocysteine Serine Glycine Creatine AMPCystathionine Homoserine Cysteine Cystathionine ˜synthase (B6) Cystathionase (B6) Adenosine Trans-sulfuration Methyl transfer ATP Methionine adenosyl transferase (MAT) S-adenosyl methionine Methyltransferases N-methylglycine (Sarcosine) Glycine N-methyltransferase CH3 S-adenosyl homocysteine S-adenosyl homocysteine hydrolase CH3-S-(CH2)2-CH-COOH NH2 Guanidinoacetate Guanidinoacetate methyltransferase Adenosine kinase

1	FIgURE 434e-1 Pathways, enzymes, and coenzymes involved in the homocystinurias. Methionine transfers a methyl group during its conversion to homocysteine. Defects in methyl transfer or in the subsequent metabolism of homocysteine by the pyridoxal phosphate (vitamin B6)dependent cystathionine β-synthase increase plasma methionine levels. Homocysteine is transformed into methionine via remethylation. This occurs through methionine synthase, a reaction requiring methylcobalamin and folic acid. Deficiencies in these enzymes or lack of cofactors is associated with decreased or normal methionine levels. In an alternative pathway, homocysteine can be remethylated by betaine:homocysteine methyl transferase. 3 and 5 years of age with dislocated optic lenses and intellectual disability (in about half of cases). Some patients develop a marfanoid habitus and radiologic evidence of osteoporosis.

1	Life-threatening vascular complications (affecting coronary, renal, and cerebral arteries) can occur during the first decade of life and are the major cause of morbidity and mortality. Classic homocystinuria can be diagnosed with analysis of plasma amino acids, showing elevated methionine and presence of free homocystine. Total plasma homocysteine is also extremely elevated (usually >100 μM). Treatment consists of a special diet restricted in protein and methionine and supplemented with cystine. In approximately half of patients, oral pyridoxine (25–500 mg/d) produces a fall in plasma methionine and homocystine concentration in body fluids. Folate and vitamin B12 deficiency should be prevented by adequate supplementation. Betaine is also effective in reducing homocystine levels in pyridoxineunresponsive patients.

1	The other forms of homocystinuria are the result of impaired remethylation of homocysteine to methionine. This can be caused by defective methionine synthase or reduced availability of two essential cofactors, 5-methyltetrahydrofolate and methylcobalamin (methylvitamin B12).

1	Hyperhomocysteinemia refers to increased total plasma concentration of homocysteine with or without an increase in free homocystine (disulfide form). Hyperhomocysteinemia, in the absence of significant homocystinuria, is found in some heterozygotes for the genetic defects noted above or in homozygotes for milder variants. Changes of homocysteine levels are also observed with increasing age; with smoking; in postmenopausal women; in patients with renal failure, hypothyroidism, leukemias, inflammatory bowel disease, or psoriasis; and during therapy with drugs such as methotrexate, nitrous oxide, isoniazid, and some antiepileptic agents. Homocysteine acts as an atherogenic and thrombophilic agent. An increase in total plasma homocysteine is an independent risk factor for coronary, cerebrovascular, and peripheral arterial disease as well as for deep vein thrombosis (Chap. 291e). Homocysteine is synergistic with hypertension and smoking, and it is additive with other risk factors that

1	and peripheral arterial disease as well as for deep vein thrombosis (Chap. 291e). Homocysteine is synergistic with hypertension and smoking, and it is additive with other risk factors that predispose to peripheral arterial disease. In addition, hyperhomocysteinemia and folate and vitamin deficiency have been associated with an increased risk of neural tube defects in pregnant women. Vitamin supplements are effective in reducing plasma homocysteine levels in these cases, although there are limited effects on cardiovascular disease.

1	Alkaptonuria is a rare (frequency 1:200,000) disorder of tyrosine catabolism in which deficiency of homogentisate 1,2-dioxygenase (also known as homogentisic acid oxidase) leads to excretion of large amounts of homogentisic acid in urine and accumulation of oxidized homogentisic acid pigment in connective tissues (ochronosis). Alkaptonuria may go unrecognized until middle life, when degenerative joint disease develops. Prior to this time, about half of patients might be diagnosed for the presence of dark urine. Foci of gray-brown scleral pigment and generalized darkening of the concha, anthelix, and, finally, helix of the ear usually develop after age 30. Low back pain usually starts between 30 and 40 years of age. Ochronotic arthritis is heralded by pain, stiffness, and some limitation of motion of the hips, knees, and shoulders. Acute arthritis may resemble rheumatoid arthritis, but small joints are usually spared. Pigmentation of heart valves, larynx, tympanic membranes, and skin

1	of motion of the hips, knees, and shoulders. Acute arthritis may resemble rheumatoid arthritis, but small joints are usually spared. Pigmentation of heart valves, larynx, tympanic membranes, and skin occurs, and occasional patients develop pigmented renal or prostatic calculi. Pigment deposition in the heart and blood vessels leads to aortic stenosis necessitating valve replacement, especially after 60 years of age. The diagnosis should be suspected in a patient whose urine darkens to blackness. Homogentisic acid in urine is identified by urine organic acid analysis. Ochronotic arthritis is treated symptomatically with pain medications, spinal surgery, and arthroplasty (Chap. 394). Ascorbic acid and protein restriction are not effective in reducing homogentisic acid production. By contrast, nitisone (2-[2-nitro-4-trifluoromethylbenzoyl]-1,3-cyclohexanedione), a drug used in tyrosinemia type I, reduces urinary excretion of homogentisic acid and, in conjunction with a low-protein diet,

1	nitisone (2-[2-nitro-4-trifluoromethylbenzoyl]-1,3-cyclohexanedione), a drug used in tyrosinemia type I, reduces urinary excretion of homogentisic acid and, in conjunction with a low-protein diet, might prevent the long-term complications of alkaptonuria.

1	Excess ammonia generated from protein nitrogen is removed by the urea cycle, a process mediated by several enzymes and transporters (Table 434e-1). Complete absence of any of these enzymes usually causes severe hyperammonemia in newborns, while milder variants can be seen in adults. The accumulation of ammonia and glutamine leads to brain edema and direct neuronal toxicity. Deficiencies in urea cycle enzymes are individually rare, but as a group, they affect about 1:25,000 individuals. They are all transmitted as autosomal recessive traits, with the exception of ornithine transcarbamylase deficiency, which is X-linked. Hepatocytes of females with ornithine transcarbamylase deficiency express either the normal or the mutant allele due to random X-inactivation and may be unable to remove excess ammonia if mutant cells are predominant.

1	Infants with classic urea cycle defects present at 1–4 days of life with refusal to eat and lethargy progressing to coma and death. Milder enzyme deficiencies present with protein avoidance, recurrent vomiting, migraine, mood swings, chronic fatigue, irritability, and disorientation that can progress to coma. Females with ornithine transcarbamylase deficiency can present at time of childbirth due to the combination of involuntary fasting and stress that favors catabolism. The diagnosis requires measurement of plasma ammonia, plasma amino acids, and urine orotic acid, useful for differentiating ornithine transcarbamylase deficiency from carbamyl phosphate synthase-1 and N-acetylglutamate synthase deficiency. Hyperammonemia can also be 434e-5 caused by liver disease from any cause and several organic acidemias and fatty acid oxidation defects (the latter two excluded by the analysis of urine organic acids and plasma acylcarnitine profile).

1	Therapy is aimed at stopping catabolism and ammonia production by providing adequate calories (as IV glucose and lipids in the comatose patient) and, if needed, insulin. Excess nitrogen is removed by IV phenylacetate and benzoate (0.25 g/kg for the priming dose and subsequently as an infusion over 24 h) that conjugate with glutamine and glycine, respectively, to form phenylacetylglutamine and hippuric acid, water-soluble molecules efficiently excreted in urine. Arginine (200 mg/kg per day) becomes an essential amino acid (except in arginase deficiency) and should be provided intravenously to resume protein synthesis. If these measures fail to reduce ammonia, hemodialysis should be initiated promptly. Chronic therapy consists of a protein-restricted diet, phenylbutyrate, glycerol phenylbutyrate (a liquid drug better tolerated by most patients), arginine, or citrulline supplements, depending on the specific diagnosis. Liver transplantation should be considered in patients with severe

1	(a liquid drug better tolerated by most patients), arginine, or citrulline supplements, depending on the specific diagnosis. Liver transplantation should be considered in patients with severe urea cycle defects that are difficult to control medically.

1	Hyperammonemia due to a functional deficiency of glutamine synthase can occur in patients receiving chemotherapy for different malignancies or undergoing solid organ transplants. It can also be seen with hepatic cirrhosis. Several of these patients have been successfully rescued from hyperammonemia using the protocol described above for urea cycle defects. CHAPTER 434e Inherited Disorders of Amino Acid Metabolism in Adults

1	Inherited Defects of Membrane Transport Nicola Longo Specific membrane transporters mediate the passage of a wide variety of substances across cellular membranes. Classes of substrates include amino acids, sugars, cations, anions, vitamins, and water. The number 435e of inherited disorders of membrane transport continues to increase with the identification of new transporters on the plasma membrane or intracellular organelles and the clarification of the molecular basis of diseases with previously unknown pathophysiology. The first transport disorders identified affected the gut or the kidney, but transport processes are now proving essential for the normal function of every organ. Mutations in transporter molecules cause disorders of the heart, muscle, brain, and endocrine and sensory organs (Table 435e-1). Inherited defects impairing the transport of selected amino acids that can present in adults are discussed here as examples of the abnormalities encountered; others are considered

1	(Table 435e-1). Inherited defects impairing the transport of selected amino acids that can present in adults are discussed here as examples of the abnormalities encountered; others are considered elsewhere in this text.

1	Cystinuria (frequency of 1 in 10,000 to 1 in 15,000) is an autosomal recessive disorder caused by defective transporters in the apical brush border of proximal renal tubule and small intestinal cells. It is characterized by impaired reabsorption and excessive urinary excretion of the dibasic amino acids lysine, arginine, ornithine, and cystine. Because cystine is poorly soluble, its excess excretion predisposes to the formation of renal, ureteral, and bladder stones. Such stones are responsible for the signs and symptoms of the disorder. There are two variants of cystinuria. Homozygotes for both variants have high urinary excretion of cystine, lysine, arginine, and ornithine. Type I heterozygotes usually have normal urinary amino acid excretion, whereas most non–type I (formerly type II and type

1	III) heterozygotes have moderately increased urinary excretion of each of the four amino acids. The gene for type I cystinuria (SLC3A1, chromosome 2p16.3) encodes a membrane glycoprotein. Non–type I cystinuria is caused by mutations in SLC7A9 (chromosome 19q13) that encodes the b0,+ amino acid transporter. The glycoprotein encoded by SLC3A1 favors the correct processing of the b0,+ membrane transporter and explains why mutations in two different genes cause a similar disease. Class of Substance Individual Tissues Manifesting and Disorder Substrates Transport Defect Molecular Defect Major Clinical Manifestations Inheritance CHAPTER 435e Inherited Defects of Membrane Transport

1	CHAPTER 435e Inherited Defects of Membrane Transport Cystine stones account for 1–2% of all urinary tract calculi but are the most common cause of stones in children. Cystinuria homozygotes regularly excrete 2400–7200 μmol (600–1800 mg) of cystine daily. Since the maximum solubility of cystine in the physiologic urinary pH range of 4.5–7.0 is about 1200 μmol/L (300 mg/L), cystine needs to be diluted to 2.5–7 L of water to prevent crystalluria. Stone formation usually manifests in the second or third decade but may occur in the first year of life. Symptoms and signs are those typical of urolithiasis: hematuria, flank pain, renal colic, obstructive uropathy, and infection (Chap. 342). Recurrent urolithiasis may lead to progressive renal insufficiency.

1	Cystinuria is suspected after observing typical hexagonal crystals in the sediment of acidified, concentrated, chilled urine or after performing a urinary nitroprusside test. Quantitative urine amino acid analysis confirms the diagnosis of cystinuria by showing selective overexcretion of cystine, lysine, arginine, and ornithine. Quantitative measurements are important for differentiating heterozygotes from homozygotes and for following free cystine excretion during therapy.

1	Management is aimed at preventing cystine crystal formation by increasing urinary volume and by maintaining an alkaline urine pH. Fluid ingestion in excess of 4 L/d is essential, and 5–7 L/d is optimal. Urinary cystine concentration should be <1000 μmol/L (250 mg/L). The daily fluid ingestion necessary to maintain this dilution of excreted cystine should be spaced over 24 h, with one-third of the total volume ingested between bedtime and 3 A.M. Cystine solubility rises sharply above pH 7.5, and urinary alkalinization (with bicarbonate or potassium citrate) can be therapeutic. Penicillamine (1–3 g/d) and tiopronin (α-mercaptopropionylglycine, 800–1200 mg/d in four divided doses) undergo sulfhydryl-disulfide exchange with cystine to form mixed disulfides. Because these disulfides are much more soluble than cystine, pharmacologic therapy can prevent and promote dissolution of calculi. Penicillamine can have significant side effects and should be reserved for patients who fail to respond

1	soluble than cystine, pharmacologic therapy can prevent and promote dissolution of calculi. Penicillamine can have significant side effects and should be reserved for patients who fail to respond to hydration alone or who are in a high-risk category (e.g., one remaining kidney, renal insufficiency). When medical management fails, shock wave lithotripsy, ureteroscopy, and percutaneous nephrolithotomy are effective for most stones. Open urologic surgery is considered for complex staghorn stones or when the patient has concomitant renal or ureteral abnormalities. Occasional patients progress to renal failure and require kidney transplantation.

1	This disorder is characterized by a defect in renal tubular reabsorption of the three dibasic amino acids lysine, arginine, and ornithine but not cystine (lysinuric protein intolerance). Homozygotes show defective intestinal transport of dibasic amino acids as well as exaggerated renal losses. Lysinuric protein intolerance is most common in Finland (1 in 60,000), southern Italy, and Japan, but is rare elsewhere. The transport defect affects basolateral rather than luminal membrane transport and is associated with impairment of the urea cycle. The defective gene (SLC7A7, chromosome 14q11.2) encodes the y+LAT membrane transporter, which associates with the cell-surface glycoprotein 4F2 heavy chain to form the complete sodium-independent transporter y+L.

1	Manifestations are related to impairment of the urea cycle and to immune dysfunction potentially attributable to nitric oxide overproduction secondary to arginine intracellular trapping. Affected patients present in childhood with hepatosplenomegaly, protein intolerance, and episodic ammonia intoxication. Older patients may present with severe osteoporosis, impairment of kidney function, pulmonary alveolar proteinosis, various autoimmune disorders, and an incompletely characterized immune deficiency. Plasma concentrations of lysine, arginine, and ornithine are reduced, whereas urinary excretion of lysine and orotic acid are increased. Hyperammonemia may develop after the ingestion of protein loads or with infections, probably because of insufficient amounts of arginine and ornithine to maintain proper function of the urea cycle. Therapy consists of dietary protein restriction and supplementation of citrulline (2–8 g/d), a neutral amino acid that fuels the urea cycle when metabolized

1	proper function of the urea cycle. Therapy consists of dietary protein restriction and supplementation of citrulline (2–8 g/d), a neutral amino acid that fuels the urea cycle when metabolized to arginine and ornithine. Pulmonary disease responds to glucocorticoids or bronchoalveolar lavage in some patients. Women with lysinuric protein intolerance who become pregnant have an increased risk of anemia, toxemia, and bleeding complications during delivery. These can be minimized by aggressive nutritional therapy and control of blood pressure. Their infants can have intrauterine growth restriction but have normal neurologic function

1	Citrullinemia type 2 is a recessive condition caused by deficiency of the mitochondrial aspartate-glutamate carrier AGC2 (citrin). A defect in this transporter reduces the availability of cytoplasmic aspartate to combine with citrulline, impairing the urea cycle and decreasing the transfer of reducing equivalents from the cytosol to the mitochondria through the malate–aspartate NADH shuttle. Mutations in the SLC25A13 gene on chromosome 7q21.3 that encodes for this transporter are rare in Caucasians, but affect about 1:20,000 people with ancestry from Japan, China, and Southeast Asia with variable penetrance.

1	The disease usually presents with sudden onset between 20 and 50 years of age with recurring episodes of hyperammonemia with associated neuropsychiatric symptoms such as altered mental status, irritability, seizures, or coma resembling hepatic encephalopathy. Some patients might come to medical attention for hypertriglyceridemia, pancreatitis, hepatoma, or fatty liver histologically similar to nonalcoholic steatohepatitis. Without therapy, most patients die with cerebral edema within a few years of onset. Episodes are usually triggered by medications (such as acetaminophen), surgery, alcohol consumption or high sugar intake, the latter conditions causing excess NADH production. NADH is not generated by the metabolism of proteins or fats, and many individuals with citrullinemia type 2 spontaneously prefer foods such as meat, eggs, and fish, and avoid carbohydrates.

1	Laboratory studies during an acute attack include elevated ammonia, citrulline, and arginine with low or normal levels of glutamine (the latter is usually increased in classic urea cycle defects). The diagnosis is confirmed by demonstrating mutations in the SLC25A13 gene. Liver transplantation prevents progression of the disease and normalizes biochemical parameters. A diet high in fats and proteins and low in carbohydrates with supplements of arginine and pyruvate is also effective in preventing further episodes, at least in the short term.

1	Hartnup disease (frequency 1 in 24,000) is an autosomal recessive disorder characterized by pellagra-like skin lesions, variable neurologic manifestations, and neutral and aromatic aminoaciduria. Alanine, serine, threonine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, glutamine, asparagine, and histidine are excreted in urine in quantities 5–10 times greater than normal, and intestinal transport of these same amino acids is defective. The defective neutral amino acid transporter, B°AT1 encoded by the SLC6A19 gene on chromosome 5p15, requires either collectrin or angiotensin-converting enzyme 2 for surface expression in the kidney and intestine, respectively.

1	The clinical manifestations result from nutritional deficiency of the essential amino acid tryptophan, caused by its intestinal and renal malabsorption, and of niacin, which derives in part from tryptophan metabolism. Only a small fraction of patients with the chemical findings of this disorder develop a pellagra-like syndrome, implying that manifestations depend on other factors in addition to the transport defect. The diagnosis of Hartnup disease should be suspected in any patient with clinical features of pellagra who does not have a history of dietary niacin deficiency (Chap. 96e). The neurologic and psychiatric manifestations range from attacks of cerebellar ataxia to mild emotional lability to frank delirium, and they are usually accompanied by exacerbations of the erythematous, eczematoid skin rash. Fever, sunlight, stress, and sulfonamide therapy provoke clinical relapses. Diagnosis is made by detection of the neutral aminoaciduria, which does not occur in dietary niacin

1	eczematoid skin rash. Fever, sunlight, stress, and sulfonamide therapy provoke clinical relapses. Diagnosis is made by detection of the neutral aminoaciduria, which does not occur in dietary niacin deficiency. Treatment is directed at niacin repletion and includes a high-protein diet and daily nicotinamide supplementation (50–250 mg).

1	Atlas of Clinical Manifestations of Metabolic Diseases J. Larry Jameson The term metabolism is derived from the Greek metabol, meaning “to 436e change.” This term encompasses the broad array of chemical pathways that are necessary for normal development and homeostasis. In practice, clinicians generally use the term metabolism in reference to energy utilization for anabolism or catabolism. Alternatively, intermediary metabolism describes the myriad cellular pathways that convert energy sources from one form to another (e.g., the citric acid cycle). The emerging field of metabolomics is based on the premise that the identification and measurement of metabolic products will enhance our understanding of physiology and disease.

1	Over the years, the classification of metabolic diseases has extended beyond traditional pathways involved in fuel metabolism to include disorders such as lysosomal storage diseases and connective tissue diseases. Thus, metabolic diseases really reflect disorders of cell biology, and many have a well-defined genetic basis. For example, lysosomal storage diseases (Chap. 432e) result from a variety of genetic defects, usually in a lysosomal enzyme, causing accumulation of a substrate within the lysosome. Certain lipodystrophies and cardiomyopathies can be caused by mutations in lamin A, a structural protein in the nuclear envelope. Membrane defects (Chap. 435e), usually involving transporters of amino acids, sugars, or ions, cause disorders such as cystinuria, Hartnup’s disease, or Wilson’s disease (Chap. 429). Connective tissue diseases (Chap. 427) frequently involve defects in collagen synthesis or structure (osteogenesis imperfecta, Ehlers-Danlos syndrome, Alport’s syndrome) or in

1	disease (Chap. 429). Connective tissue diseases (Chap. 427) frequently involve defects in collagen synthesis or structure (osteogenesis imperfecta, Ehlers-Danlos syndrome, Alport’s syndrome) or in other extracellular matrix structural proteins such as fibrillin (Marfan syndrome). Many metabolic disorders originate from defects in enzymes involved in the synthesis or degradation of amino acids, carbohydrates, lipids, purines, or pyrimidines (Chaps. 431e, 433e, and 434e). Lipoprotein disorders (Chap. 421) are caused by defects in a wide array of cellular pathways including membrane receptors (the low-density lipoprotein receptor), enzyme defects (lipoprotein lipase), carrier proteins (apolipoprotein B100), or transporters (ATPbinding cassette transporter ABCA1). In some instances, metabolic abnormalities induce compensatory physiologic responses that reflect the interactions of multiple metabolic pathways. For example, the metabolic syndrome (Chap. 422), which includes a constellation

1	abnormalities induce compensatory physiologic responses that reflect the interactions of multiple metabolic pathways. For example, the metabolic syndrome (Chap. 422), which includes a constellation of clinical features (central obesity, hypertriglyceridemia, low high-density lipoprotein cholesterol, hyperglycemia, and hypertension), likely has multiple genetic and environmental origins. Cushing’s syndrome reflects the metabolic effects of excess cortisol on multiple tissues (Chap. 406).

1	This broader definition results in a plethora of metabolic diseases, numbering in the thousands. Fortunately, comprehensive reference sources exist, such as the Online Metabolic and Molecular Bases of Inherited Disease (OMMBID) (http://www.ommbid.com/) and the Online Mendelian Inheritance in Man (OMIM) (http://www.ncbi .nlm.nih.gov/entrez/query.fcgi?db=OMIM). The study of metabolic diseases has been invaluable for advancing our understanding of human genetics by providing insight into principles such as patterns of inheritance, variable expressivity, phenotypic variation, and novel approaches to therapy, including screening programs, blood and organ transplantation, gene therapy, and enzyme replacement (Chap. 82).

1	This atlas provides a visual survey of selected metabolic disorders 436e-1 with references to the topics elsewhere in the text. The author encourages submission of additional illustrations that might facilitate learning among our peers and thereby enhance the recognition and care of patients with these disorders. Figure 436e-1 “Gauntlet” of pellagra (niacin deficiency). Note indurated, lichenified, pigmented, and scaly skin on the dorsa of the hands. (Source: K Wolff et al: Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005.) See Chap. 96e. CHAPTER 436e Atlas of Clinical Manifestations of Metabolic Diseases

1	CHAPTER 436e Atlas of Clinical Manifestations of Metabolic Diseases Figure 436e-2 Scurvy (vitamin C deficiency). Note perifollicular hemorrhage on the leg. The follicles are often plugged by keratin (perifollicular hyperkeratosis). This eruption occurred in a 46-year-old alcoholic, homeless man who also had bleeding gums and loose teeth. (Source: K Wolff et al: Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005.) See Chap. 96e. Figure 436e-3 Podagra with gouty inflammation of the left first metatarsophalangeal joint. Note swelling and erythema. (From KJ Knoop et al: The Atlas of Emergency Medicine, 2nd ed. New York, McGraw-Hill, 2002. Courtesy of Kevin J. Knoop, MD, MS; with permission.) See Chaps. 395 and 431e. Figure 436e-4 Large tophi of gout located in and around the right knee. (Courtesy of Daniel L. Savitt, MD; with permission.) See Chaps. 395 and 431e.

1	Figure 436e-4 Large tophi of gout located in and around the right knee. (Courtesy of Daniel L. Savitt, MD; with permission.) See Chaps. 395 and 431e. Figure 436e-5 Gouty arthritis of the finger. The finger is an unusual site for gouty arthritis. Examination of the synovial fluid confirmed the diagnosis. (Courtesy of Alan B. Storrow, MD; with permission.) See Chaps. 395 and 431e. Figure 436e-6 Cushing’s syndrome. Note plethoric moon facies with erythema and telangiectases of cheek and forehead. The face and neck show increased deposition of fat, which was also seen in the supraclavicular areas (not depicted here). (Source: K Wolff et al: Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005.) See Chap. 406.

1	(Source: K Wolff et al: Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005.) See Chap. 406. Figure 436e-7 Necrobiosis lipoidica diabeticorum. A large symmetric plaque with active tan-pink, well-demarcated, raised, firm borders and a yellow center in the pretibial regions of a 28-year-old diabetic woman is shown. The central parts of the lesion are depressed with atrophic changes of epidermal thinning and telangiectasis against a yellow background. (Source: K Wolff et al: Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005.) See Chap. 417. Figure 436e-8 Patient with multiple endocrine neoplasia 2B syn-drome. Note the multiple neuromas on the lips and tongue and the marfanoid facies. (Source: DG Gardner, D Shoback, eds: Greenspan’s Basic & Clinical Endocrinology, 8th ed. New York, McGraw-Hill, 2006, www .accessmedicine.com.) See Chap. 408.

1	Figure 436e-10 Bone scan of a patient with severe Paget’s disease of the skull, ribs, spine, pelvis, right femur, and acetabulum. Note localization of bone-seeking isotope (99mTc-labeled bisphosphonate) in these areas. (Source: DG Gardner, D Shoback, eds: Greenspan’s Basic & Clinical Endocrinology, 8th ed. New York, McGraw-Hill, 2006, www.access medicine.com.) See Chap. 426e. CHAPTER 436e Atlas of Clinical Manifestations of Metabolic Diseases Figure 436e-9 Early and late radiographs of Paget’s disease of the tibia of a male patient, taken at 45 (A) and 65 years of age (B). (Source: HB Skinner: Current Diagnosis & Treatment in Orthopedics, 4th ed. New York, McGraw-Hill, 2007, www.accessmedicine.com.) See Chap. 426e. Figure 436e-11 Tendinous xanthomas. Large subcutaneous tumors adherent to the Achilles tendons. (Source: K Wolff et al: Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005.) See Chap. 421.

1	AB Figure 436e-12 Papular eruptive xanthomas. A. Multiple, discrete, red-to-yellow papules becoming confluent on the elbow of a white individual with uncontrolled diabetes mellitus; lesions were present on both elbows and buttocks. B. Papular eruptive xanthomas on the elbows and lower arms of an African-American patient. (Source: K Wolff et al: Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005.) See Chap. 421. Figure 436e-13 Forms of xanthomas and other lipid deposits frequently seen in familial hypercholesterolemia homozygotes. A. Arcus corneae. B, E, and F. Cutaneous planar xanthomas, which usually have a bright orange hue. C, D, and G. Tuberous xanthomas on the elbows.

1	(Source for panels C and D: CR Scriver et al [eds]: The Metabolic and Molecular Bases of Inherited Disease online, 8th ed. New York, McGraw-Hill, www.ommbid.com.) H. Tendon and tuberous xanthomas. (Panel H reproduced through the courtesy of Dr. A. Khachadurian; with permission.) See Chap. 421. Figure 436e-14 Examples of xanthomas in type III hyperlipo-proteinemic patients. A. Tuberoeruptive xanthomas of the elbows. B. Tuberous xanthomas of the digits and xanthomas of the palmar creases (xanthoma striata palmaris) (arrows). (Courtesy of Dr. Thomas P. Bersot; with permission.) See Chap. 421. Figure 436e-15 A 17-year-old patient with abetalipoproteinemia, with generalized weakness, kyphoscoliosis, and lordosis. (Courtesy of Drs. Peter Herbert, Gerd Assmann, Antonio M. Gotto, Jr., and Donald Fredrickson; with permission.) See Chap. 421.

1	Figure 436e-16 Porphyria cutanea tarda. Periorbital and malar violaceous coloration, hyperpigmentation, and hypertrichosis on the face; bullae, crusts, and scars on the dorsa of the hands. (Source: K Wolff et al: Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology, 5th ed. New York, McGraw-Hill, 2005.) See Chap. 430. CHAPTER 436e Atlas of Clinical Manifestations of Metabolic Diseases

1	CHAPTER 436e Atlas of Clinical Manifestations of Metabolic Diseases Figure 436e-17 Mucopolysaccharidosis type IH (Hurler’s syndrome) in a 4-year-old boy. The diagnosis was made at the age of 15 months, at which time he had developmental delay, hepatomegaly, and skeletal involvement. At the time of the picture, the patient had short stature, an enlarged tongue, persistent nasal discharge, stiff joints, and hydrocephalus. Verbal language skills consisted of four or five words. The patient had a severe hearing loss and wore hearing aids. (Source: CR Scriver et al [eds]: The Metabolic and Molecular Bases of Inherited Disease online, 8th ed. New York, McGraw-Hill, www.ommbid .com.) See Chap. 432e.

1	Figure 436e-18 Growth and development of two patients with type Ia glycogen storage disease. A. Patient at age 7 years and at age 39 years. B. Another patient at age 10 years and at age 33 years. Both patients survive despite inadequate treatment of their disease. Note that the abdomen is less protuberant with age. Hypoglycemia also improves with age. In adulthood, however, both patients continue to be short, and both have gout, multiple liver adenomas, and progressive renal disease. (Source: CR Scriver et al [eds]: The Metabolic and Molecular Bases of Inherited Disease online, 8th ed. New York, McGraw-Hill, www.ommbid.com.) See Chap. 433e.

1	Figure 436e-19 Progressive myopathy in a patient with type IIIa glycogen storage disease. The patient has a debrancher deficiency in both liver and muscle (subtype IIIa). As a child, he had hepatomegaly, hypoglycemia, and growth retardation. After puberty, he no longer had hepatomegaly, and his final height is normal. Note the muscle wasting in the lower legs and both hands at age 44 years of age (left panel); this condition progressed to pronounced muscle atrophy at age 53 years (two right panels). (Source: CR Scriver et al [eds]: The Metabolic and Molecular Bases of Inherited Disease online, 8th ed. New York, McGraw-Hill, www.ommbid.com.) See Chap. 433e.

1	Figure 436e-20 Skeletal features of Marfan’s syndrome in a 16-year-old girl. Note the long limbs (associated with disproportion-ately tall stature), long fingers, scoliosis, and genu valgum. (Source: CR Scriver et al [eds]: The Metabolic and Molecular Bases of Inherited Disease online, 8th ed. New York, McGraw-Hill, www.ommbid.com.) See Chap. 427. Figure 436e-21 Marfan’s syndrome. A. Long, narrow face. B. Arachnodactyly and positive wrist sign. C. High-arched palate. D. Ectopia lentis associated with aortic aneurysm and severe aortic regurgitation in a teenage girl. (Source: V Fuster et al [eds]: Hurst’s The Heart, 11th ed. New York, McGraw-Hill, 2004, www.accessmedicine.com.) See Chap. 427. Figure 436e-22 Ochronotic pigmentation of the femur of a 56-year-old alkaptonuric patient. (Courtesy of Dr. H. W. Edmonds of the Washington Hospital Center, Washington, DC; with permission.) See Chap. 434e.

1	Figure 436e-24 Two patients with type B Niemann-Pick disease (NPD). A. A 4.7-year-old patient. (From DS Fredrickson, HR Sloan, in JB Stanbury et al: The Metabolic Basis of Inherited Disease, 3rd ed. New York, McGraw-Hill, 1972. Used by permission.) B. A 44-year-old patient. See Chap. 432e. Figure 436e-23 Clusters of angiokeratomas (telangiectases) on the buttocks (A) and in the umbilical area (B) of a hemizygote with Fabry disease. (Source: CR Scriver et al [eds]: The Metabolic and Molecular Bases of Inherited Disease online, 8th ed. New York, McGraw-Hill, www .ommbid.com.) See Chap. 432e. Figure 436e-25 “Cherry red” spot in the eye of a Tay-Sachs patient. (From http://www.nei.nih.gov/resources/eyegene.asp.) See Chap. 432e. CHAPTER 436e Atlas of Clinical Manifestations of Metabolic Diseases

1	CHAPTER 436e Atlas of Clinical Manifestations of Metabolic Diseases Figure 436e-26 Kayser-Fleischer ring. This manifestation develops in Wilson’s disease from copper deposition in Descemet’s membrane, which produces brownish discoloration of the peripheral cornea. It should not be confused with the yellow-white lipid ring of arcus senilis, which is common in the elderly and occasionally signifies hyperlipidemia, especially when it appears at a young age. (Courtesy of Jonathan C. Horton, MD, PhD; with permission.) See Chap. 429.

1	Figure 436e-27 Anterior view of patients with different forms of lipodystrophy. A. Congenital generalized lipodystrophy: a 16-year-old girl with generalized loss of fat, acromegaloid features, severe acanthosis nigricans affecting the axillae and abdomen, and umbilical hernia. (From A Garg et al: J Clin Endocrinol Metab 84:3390, 1999; with permission.) B. Familial partial lipodystrophy, Dunnigan variety: a 43-year-old woman with marked loss of subcutaneous fat from both the limbs and the trunk and excess fat deposition in the face, chin, supraclavicular area, and labia majora. (From JM Peters et al: Nat Genet 18;292, 1998; with permission.) C. Acquired generalized lipodystrophy: a 10-year-old boy who developed generalized loss of fat that also affected the palms and soles after panniculitis at the age of 3 months. D. Acquired partial lipodystrophy: a 30-year-old woman with onset of lipodystrophy at age 14 years. Note loss of fat from the face, neck, upper limbs, trunk, and anterior

1	at the age of 3 months. D. Acquired partial lipodystrophy: a 30-year-old woman with onset of lipodystrophy at age 14 years. Note loss of fat from the face, neck, upper limbs, trunk, and anterior thighs. There is accumulation of excess fat in the hips and other regions of the lower limbs.

1	2535 approach to the patient with Neurologic Disease Daniel H. Lowenstein, Joseph B. Martin, Stephen L. Hauser Neurologic diseases are common and costly. According to estimates by the World Health Organization, neurologic disorders affect over 1 bil-lion people worldwide, constitute 12% of the global burden of disease, and cause 14% of global deaths (Table 437-1). These numbers are only expected to increase as the world’s population ages. Most patients with neurologic symptoms seek care from internists and other generalists rather than from neurologists. Because therapies now exist for many neurologic disorders, a skillful approach to diagnosis is essential. Errors commonly result from an overreliance on costly neuroimaging procedures and laboratory tests, which, while useful, do not substitute for an adequate history and examination. The proper approach to the patient with a neurologic illness begins with the patient and focuses the clinical problem first in anatomic and then in

1	substitute for an adequate history and examination. The proper approach to the patient with a neurologic illness begins with the patient and focuses the clinical problem first in anatomic and then in pathophysiologic terms; only then should a specific diagnosis be entertained. This method ensures that technology is judiciously applied, a correct diagnosis is established in an efficient manner, and treatment is promptly initiated. THE NEUROLOGIC METHOD 437 part 17: Neurologic Disorders seCtioN 1 DiaGNosis of NeuroLoGiC DisorDers

1	Approach to the Patient with Neurologic Disease The first priority is to identify the region of the nervous system that is likely to be responsible for the symptoms. Can the disorder be mapped to one specific location, is it multifocal, or is a diffuse process present? Are the symptoms restricted to the nervous system, or do they arise in the context of a systemic illness? Is the problem in the central nervous system (CNS), the peripheral nervous system (PNS), or both? If in the CNS, is the cerebral cortex, basal ganglia, brainstem, cerebellum, or spinal cord responsible? Are the pain-sensitive meninges involved? If in the PNS, could the disorder be located in peripheral nerves and, if so, are motor or sensory nerves primarily affected, or is a lesion in the neuromuscular junction or muscle more likely?

1	The first clues to defining the anatomic area of involvement appear in the history, and the examination is then directed to confirm or rule out these impressions and to clarify uncertainties. A more detailed examination of a particular region of the CNS or PNS is often indicated. For example, the examination of a patient who presents with a history of ascending paresthesias and weakness should be directed toward deciding, among other things, if the location of the lesion is in the spinal cord or peripheral nerves. Focal back pain, a spinal cord sensory level, and incontinence suggest a spinal cord origin, whereas a stocking-glove pattern of sensory loss suggests peripheral nerve disease; areflexia usually indicates peripheral neuropathy but may also be present with spinal shock in acute spinal cord disorders.

1	Deciding “where the lesion is” accomplishes the task of limiting the possible etiologies to a manageable, finite number. In addition, this strategy safeguards against making serious errors. Symptoms of recurrent vertigo, diplopia, and nystagmus should not trigger “multiple sclerosis” as an answer (etiology) but “brainstem” or “pons” (location); then a diagnosis of brainstem arteriovenous malformation will not be missed for lack of consideration. Similarly, the combination of optic neuritis and spastic ataxic paraparesis suggests optic nerve and spinal cord disease; multiple sclerosis (MS), CNS syphilis, and vitamin B12 deficiency are treatable disorders that can produce this syndrome. Once the question, “Where is the lesion?” is answered, then the question, “What is the lesion?” can be addressed.

1	Clues to the pathophysiology of the disease process may also be present in the history. Primary neuronal (gray matter) disorders may present as early cognitive disturbances, movement disorders, or seizures, whereas white matter involvement produces predominantly “long tract” disorders of motor, sensory, visual, and cerebellar pathways. Progressive and symmetric symptoms often have a metabolic or degenerative origin; in such cases lesions are usually not sharply circumscribed. Thus, a patient with paraparesis and a clear spinal cord sensory level is unlikely to have vitamin B12 deficiency as the explanation. A Lhermitte symptom (electric shock–like sensations evoked by neck flexion) is due to ectopic impulse generation in white matter pathways and occurs with demyelination in the cervical spinal cord; among many possible causes, this symptom may indicate MS in a young adult or compressive cervical spondylosis in an older person. Symptoms that worsen after exposure to heat or exercise

1	spinal cord; among many possible causes, this symptom may indicate MS in a young adult or compressive cervical spondylosis in an older person. Symptoms that worsen after exposure to heat or exercise may indicate conduction block in demyelinated axons, as occurs in MS. A patient with recurrent episodes of diplopia and dysarthria associated with exercise or fatigue may have a disorder of neuromuscular transmission such as myasthenia gravis. Slowly advancing visual scotoma with luminous edges, termed fortification spectra, indicates spreading cortical depression, typically with migraine.

1	Attention to the description of the symptoms experienced by the patient and substantiated by family members and others often permits an accurate localization and determination of the probable cause of the complaints, even before the neurologic examination is performed. The history also helps to bring a focus to the neurologic examination that follows. Each complaint should be pursued as far as possible to elucidate the location of the lesion, the likely underlying pathophysiology, and potential etiologies. For example, a patient complains of weakness of the right arm. What are the associated features? Does the patient have difficulty with brushing hair or reaching upward (proximal) or buttoning buttons or opening a twist-top bottle (distal)? Negative associations may also be crucial. A patient with a right hemiparesis without a language deficit likely has a lesion (internal capsule, brainstem, or spinal cord) different from that of a patient with a right hemiparesis and aphasia (left

1	with a right hemiparesis without a language deficit likely has a lesion (internal capsule, brainstem, or spinal cord) different from that of a patient with a right hemiparesis and aphasia (left hemisphere). Other pertinent features of the history include the following: 2536 1. Temporal course of the illness. It is important to determine the precise time of appearance and rate of progression of the symptoms experienced by the patient. The rapid onset of a neurologic complaint, occurring within seconds or minutes, usually indicates a vascular event, a seizure, or migraine. The onset of sensory symptoms located in one extremity that spread over a few seconds to adjacent portions of that extremity and then to the other regions of the body suggests a seizure. A more gradual onset and less well-localized symptoms point to the possibility of a transient ischemic attack (TIA). A similar but slower temporal march of symptoms accompanied by headache, nausea, or visual disturbance suggests

1	well-localized symptoms point to the possibility of a transient ischemic attack (TIA). A similar but slower temporal march of symptoms accompanied by headache, nausea, or visual disturbance suggests migraine. The presence of “positive” sensory symptoms (e.g., tingling or sensations that are difficult to describe) or involuntary motor movements suggests a seizure; in contrast, transient loss of function (negative symptoms) suggests a TIA. A stuttering onset where symptoms appear, stabilize, and then progress over hours or days also suggests cerebrovascular disease; an additional history of transient remission or regression indicates that the process is more likely due to ischemia rather than hemorrhage. A gradual evolution of symptoms over hours or days suggests a toxic, metabolic, infectious, or inflammatory process. Progressing symptoms associated with the systemic manifestations of fever, stiff neck, and altered level of consciousness imply an infectious process. Relapsing and

1	or inflammatory process. Progressing symptoms associated with the systemic manifestations of fever, stiff neck, and altered level of consciousness imply an infectious process. Relapsing and remitting symptoms involving different levels of the nervous system suggest MS or other inflammatory processes. Slowly progressive symptoms without remissions are characteristic of neurodegenerative disorders, chronic infections, gradual intoxications, and neoplasms.

1	2. Patients’ descriptions of the complaint. The same words often mean different things to different patients. “Dizziness” may imply impending syncope, a sense of disequilibrium, or true spinning vertigo. “Numbness” may mean a complete loss of feeling, a positive sensation such as tingling, or even weakness. “Blurred vision” may be used to describe unilateral visual loss, as in transient monocular blindness, or diplopia. The interpretation of the true meaning of the words used by patients to describe symptoms obviously becomes even more complex when there are differences in primary languages and cultures. 3.

1	3. Corroboration of the history by others. It is almost always helpful to obtain additional information from family, friends, or other observers to corroborate or expand the patient’s description. Memory loss, aphasia, loss of insight, intoxication, and other factors may impair the patient’s capacity to communicate normally with the examiner or prevent openness about factors that have contributed to the illness. Episodes of loss of consciousness necessitate that details be sought from observers to ascertain precisely what has happened during the event. 4.

1	4. Family history. Many neurologic disorders have an underlying genetic component. The presence of a Mendelian disorder, such as Huntington’s disease or Charcot-Marie-Tooth neuropathy, is often obvious if family data are available. More detailed questions about family history are often necessary in polygenic disorders such as MS, migraine, and many types of epilepsy. It is important to elicit family history about all illnesses, in addition to neurologic and psychiatric disorders. A familial propensity to hypertension or heart disease is relevant in a patient who presents with a stroke. There are numerous inherited neurologic diseases that are associated with multisystem manifestations that may provide clues to the correct diagnosis (e.g., neurofibromatosis, Wilson’s disease, mitochondrial disorders). 5.

1	Medical illnesses. Many neurologic diseases occur in the context of systemic disorders. Diabetes mellitus, hypertension, and abnormalities of blood lipids predispose to cerebrovascular disease. A solitary mass lesion in the brain may be an abscess in a patient with valvular heart disease, a primary hemorrhage in a patient with a coagulopathy, a lymphoma or toxoplasmosis in a patient with AIDS, or a metastasis in a patient with underlying cancer. Patients with malignancy may also present with a neurologic paraneoplastic syndrome (Chap. 122) or complications from chemotherapy or radiotherapy. Marfan’s syndrome and related collagen disorders predispose to dissection of the cranial arteries and aneurysmal subarachnoid hemorrhage; the latter may also occur with polycystic kidney disease. Various neurologic disorders occur with dysthyroid states or other endocrinopathies. It is especially important to look for the presence of systemic diseases in patients with peripheral neuropathy. Most

1	neurologic disorders occur with dysthyroid states or other endocrinopathies. It is especially important to look for the presence of systemic diseases in patients with peripheral neuropathy. Most patients with coma in a hospital setting have a metabolic, toxic, or infectious cause.

1	6.

1	Drug use and abuse and toxin exposure. It is essential to inquire about the history of drug use, both prescribed and illicit. Sedatives, antidepressants, and other psychoactive medications are frequently associated with acute confusional states, especially in the elderly. Aminoglycoside antibiotics may exacerbate symptoms of weakness in patients with disorders of neuromuscular transmission, such as myasthenia gravis, and may cause dizziness secondary to ototoxicity. Vincristine and other antineoplastic drugs can cause peripheral neuropathy, and immunosuppressive agents such as cyclosporine can produce encephalopathy. Excessive vitamin ingestion can lead to disease; examples include vitamin A and pseudotumor cerebri or pyridoxine and peripheral neuropathy. Many patients are unaware that over-the-counter sleeping pills, cold preparations, and diet pills are actually drugs. Alcohol, the most prevalent neurotoxin, is often not recognized as such by patients, and other drugs of abuse such

1	sleeping pills, cold preparations, and diet pills are actually drugs. Alcohol, the most prevalent neurotoxin, is often not recognized as such by patients, and other drugs of abuse such as cocaine and heroin can cause a wide range of neurologic abnormalities. A history of environmental or industrial exposure to neurotoxins may provide an essential clue; consultation with the patient’s coworkers or employer may be required.

1	7. Formulating an impression of the patient. Use the opportunity while taking the history to form an impression of the patient. Is the information forthcoming, or does it take a circuitous course? Is there evidence of anxiety, depression, or hypochondriasis? Are there any clues to problems with language, memory, insight, comportment, or behavior? The neurologic assessment begins as soon as the patient comes into the room and the first introduction is made.

1	The neurologic examination is challenging and complex; it has many components and includes a number of skills that can be mastered only through repeated use of the same techniques on a large number of individuals with and without neurologic disease. Mastery of the complete neurologic examination is usually important only for physicians in neurology and associated specialties. However, knowledge of the basics of the examination, especially those components that are effective in screening for neurologic dysfunction, is essential for all clinicians, especially generalists.

1	There is no single, universally accepted sequence of the examination that must be followed, but most clinicians begin with assessment of mental status followed by the cranial nerves, motor system, reflexes, sensory system, coordination, and gait. Whether the examination is basic or comprehensive, it is essential that it be performed in an orderly and systematic fashion to avoid errors and serious omissions. Thus, the best way to learn and gain expertise in the examination is to choose one’s own approach and practice it frequently and do it in the same exact sequence each time.

1	The detailed description that follows describes the more commonly used parts of the neurologic examination, with a particular emphasis on the components that are considered most helpful for the assessment of common neurologic problems. Each section also includes a brief description of the minimal examination necessary to adequately screen for abnormalities in a patient who has no symptoms suggesting neurologic dysfunction. A screening examination done in this way can be completed in 3–5 min.

1	Several additional points about the examination are worth noting. First, in recording observations, it is important to describe what is found rather than to apply a poorly defined medical term (e.g., “patient groans to sternal rub” rather than “obtunded”). Second, subtle CNS abnormalities are best detected by carefully comparing a patient’s performance on tasks that require simultaneous activation of both cerebral hemispheres (e.g., eliciting a pronator drift of an outstretched arm with the eyes closed; extinction on one side of bilaterally applied light touch, also with eyes closed; or decreased arm swing or a slight asymmetry when walking). Third, if the patient’s complaint is brought on by some activity, reproduce the activity in the office. If the complaint is of dizziness when the head is turned in one direction, have the patient do this and also look for associated signs on examination (e.g., nystagmus or dysmetria). If pain occurs after walking two blocks, have the patient

1	head is turned in one direction, have the patient do this and also look for associated signs on examination (e.g., nystagmus or dysmetria). If pain occurs after walking two blocks, have the patient leave the office and walk this distance and immediately return, and repeat the relevant parts of the examination. Finally, the use of tests that are individually tailored to the patient’s problem can be of value in assessing changes over time. Tests of walking a 7.5-m (25ft) distance (normal, 5–6 s; note assistance, if any), repetitive finger or toe tapping (normal, 20–25 taps in 5 s), or handwriting are examples.

1	• The bare minimum: During the interview, look for difficulties with communication and determine whether the patient has recall and insight into recent and past events. The mental status examination is under way as soon as the physician begins observing and speaking with the patient. If the history raises any concern for abnormalities of higher cortical function or if cognitive problems are observed during the interview, then detailed testing of the mental status is indicated. The patient’s ability to understand the language used for the examination, cultural background, educational experience, sensory or motor problems, or comorbid conditions need to be factored into the applicability of the tests and interpretation of results.

1	The Folstein mini-mental status examination (MMSE) is a standardized screening examination of cognitive function that is extremely easy to administer and takes <10 min to complete. Using age-adjusted values for defining normal performance, the test is ~85% sensitive and 85% specific for making the diagnosis of dementia that is moderate or severe, especially in educated patients. When there is sufficient time available, the MMSE is one of the best methods for documenting the current mental status of the patient, and this is especially useful as a baseline assessment to which future scores of the MMSE can be compared. Individual elements of the mental status examination can be subdivided into level of consciousness, orientation, speech and language, memory, fund of information, insight and judgment, abstract thought, and calculations.

1	Level of consciousness is the patient’s relative state of awareness of the self and the environment, and ranges from fully awake to comatose. When the patient is not fully awake, the examiner should describe the responses to the minimum stimulus necessary to elicit a reaction, ranging from verbal commands to a brief, painful stimulus such as a squeeze of the trapezius muscle. Responses that are directed toward the stimulus and signify some degree of intact cerebral function (e.g., opening the eyes and looking at the examiner or reaching to push away a painful stimulus) must be distinguished from reflex responses of a spinal origin (e.g., triple flexion response—flexion at the ankle, knee, and hip in response to a painful stimulus to the foot). Orientation is tested by asking the person to state his or her name, location, and time (day of the week and date); time is usually the first to be affected in a variety of conditions.

1	Orientation is tested by asking the person to state his or her name, location, and time (day of the week and date); time is usually the first to be affected in a variety of conditions. Speech is assessed by observing articulation, rate, rhythm, and prosody (i.e., the changes in pitch and accentuation of syllables and words). Language is assessed by observing the content of the patient’s verbal and written output, response to spoken commands, and ability to read. A typical testing sequence is to ask the patient to name successively more detailed components of clothing, a watch, or a pen; repeat the phrase “No ifs, ands, or buts”; follow a three-step, verbal command; write a sentence; and read and respond to a written command.

1	Memory should be analyzed according to three main time scales: (1) immediate memory is assessed by saying a list of three items and having the patient repeat the list immediately; (2) short-term memory is tested by asking the patient to recall the same three items 5 and 15 min later; and (3) long-term memory is evaluated by determining how well the patient is able to provide a coherent chronologic history of his or her illness or personal events. Fund of information is assessed by asking questions about major historic or current events, with special attention to educational level and life experiences. Abnormalities of insight and judgment are usually detected during 2537 the patient interview; a more detailed assessment can be elicited by asking the patient to describe how he or she would respond to situations having a variety of potential outcomes (e.g., “What would you do if you found a wallet on the sidewalk?”).

1	Abstract thought can be tested by asking the patient to describe similarities between various objects or concepts (e.g., apple and orange, desk and chair, poetry and sculpture) or to list items having the same attributes (e.g., a list of four-legged animals). Calculation ability is assessed by having the patient carry out a computation that is appropriate to the patient’s age and education (e.g., serial subtraction of 7 from 100 or 3 from 20; or word problems involving simple arithmetic). • The bare minimum: Check the fundi, visual fields, pupil size and reactivity, extraocular movements, and facial movements. The cranial nerves (CN) are best examined in numerical order, except for grouping together CN III, IV, and VI because of their similar function.

1	The cranial nerves (CN) are best examined in numerical order, except for grouping together CN III, IV, and VI because of their similar function. CN I (Olfactory) Testing is often omitted unless there is suspicion for inferior frontal lobe disease (e.g., meningioma). With eyes closed, ask the patient to sniff a mild stimulus such as toothpaste or coffee and identify the odorant.

1	CN II (Optic) Check visual acuity (with eyeglasses or contact lens correction) using a Snellen chart or similar tool. Test the visual fields by confrontation, i.e., by comparing the patient’s visual fields to your own. As a screening test, it is usually sufficient to examine the visual fields of both eyes simultaneously; individual eye fields should be tested if there is any reason to suspect a problem of vision by the history or other elements of the examination, or if the screening test reveals an abnormality. Face the patient at a distance of approximately 0.6–1.0 m (2–3 ft) and place your hands at the periphery of your visual fields in the plane that is equidistant between you and the patient. Instruct the patient to look directly at the center of your face and to indicate when and where he or she sees one of your fingers moving. Beginning with the two inferior quadrants and then the two superior quadrants, move your index finger of the right hand, left hand, or both hands

1	and where he or she sees one of your fingers moving. Beginning with the two inferior quadrants and then the two superior quadrants, move your index finger of the right hand, left hand, or both hands simultaneously and observe whether the patient detects the movements. A single small-amplitude movement of the finger is sufficient for a normal response. Focal perimetry and tangent screen examinations should be used to map out visual field defects fully or to search for subtle abnormalities. Optic fundi should be examined with an ophthalmoscope, and the color, size, and degree of swelling or elevation of the optic disc noted, as well as the color and texture of the retina. The retinal vessels should be checked for size, regularity, arteriovenous nicking at crossing points, hemorrhage, exudates, etc.

1	CN III, IV, VI (Oculomotor, Trochlear, Abducens) Describe the size and shape of pupils and reaction to light and accommodation (i.e., as the eyes converge while following your finger as it moves toward the bridge of the nose). To check extraocular movements, ask the patient to keep his or her head still while tracking the movement of the tip of your finger. Move the target slowly in the horizontal and vertical planes; observe any paresis, nystagmus, or abnormalities of smooth pursuit (saccades, oculomotor ataxia, etc.). If necessary, the relative position of the two eyes, both in primary and multidirectional gaze, can be assessed by comparing the reflections of a bright light off both pupils. However, in practice it is typically more useful to determine whether the patient describes diplopia in any direction of gaze; true diplopia should almost always resolve with one eye closed. Horizontal nystagmus is best assessed at 45° and not at extreme lateral gaze (which is uncomfortable for

1	in any direction of gaze; true diplopia should almost always resolve with one eye closed. Horizontal nystagmus is best assessed at 45° and not at extreme lateral gaze (which is uncomfortable for the patient); the target must often be held at the lateral position for at least a few seconds to detect an abnormality.

1	CN V (Trigeminal) Examine sensation within the three territories of the branches of the trigeminal nerve (ophthalmic, maxillary, and mandibular) on each side of the face. As with other parts of the sensory examination, testing of two sensory modalities derived from different anatomic pathways (e.g., light touch and temperature) is sufficient Approach to the Patient with Neurologic Disease 2538 for a screening examination. Testing of other modalities, the corneal reflex, and the motor component of CN V (jaw clench—masseter muscle) is indicated when suggested by the history.

1	CN VII (Facial) Look for facial asymmetry at rest and with spontaneous movements. Test eyebrow elevation, forehead wrinkling, eye closure, smiling, and cheek puff. Look in particular for differences in the lower versus upper facial muscles; weakness of the lower two-thirds of the face with preservation of the upper third suggests an upper motor neuron lesion, whereas weakness of an entire side suggests a lower motor neuron lesion. CN VIII (Vestibulocochlear) Check the patient’s ability to hear a finger rub or whispered voice with each ear. Further testing for air versus mastoid bone conduction (Rinne) and lateralization of a 512-Hz tuning fork placed at the center of the forehead (Weber) should be done if an abnormality is detected by history or examination. Any suspected problem should be followed up with formal audiometry. For further discussion of assessing vestibular nerve function in the setting of dizziness, hearing loss, or coma, see Chaps. 28, 43, and 328, respectively.

1	CN IX, X (Glossopharyngeal, Vagus) Observe the position and symmetry of the palate and uvula at rest and with phonation (“aah”). The pharyngeal (“gag”) reflex is evaluated by stimulating the posterior pharyngeal wall on each side with a sterile, blunt object (e.g., tongue blade), but the reflex is often absent in normal individuals. CN XI (Spinal Accessory) Check shoulder shrug (trapezius muscle) and head rotation to each side (sternocleidomastoid) against resistance. CN XII (Hypoglossal) Inspect the tongue for atrophy or fasciculations, position with protrusion, and strength when extended against the inner surface of the cheeks on each side. • The bare minimum: Look for muscle atrophy and check extremity tone. Assess upper extremity strength by checking for pronator drift and strength of wrist or finger extensors. Assess lower extremity strength by checking strength of the toe extensors and having the patient walk normally and on heels and toes.

1	The motor examination includes observations of muscle appearance, tone, and strength. Although gait is in part a test of motor function, it is usually evaluated separately at the end of the examination. Appearance Inspect and palpate muscle groups under good light and with the patient in a comfortable and symmetric position. Check for muscle fasciculations, tenderness, and atrophy or hypertrophy. Involuntary movements may be present at rest (e.g., tics, myoclonus, choreoathetosis), during maintained posture (pill-rolling tremor of Parkinson’s disease), or with voluntary movements (intention tremor of cerebellar disease or familial tremor).

1	Tone Muscle tone is tested by measuring the resistance to passive movement of a relaxed limb. Patients often have difficulty relaxing during this procedure, so it is useful to distract the patient to minimize active movements. In the upper limbs, tone is assessed by rapid pronation and supination of the forearm and flexion and extension at the wrist. In the lower limbs, while the patient is supine the examiner’s hands are placed behind the knees and rapidly raised; with normal tone, the ankles drag along the table surface for a variable distance before rising, whereas increased tone results in an immediate lift of the heel off the surface. Decreased tone is most commonly due to lower motor neuron or peripheral nerve disorders. Increased tone may be evident as spasticity (resistance determined by the angle and velocity of motion; corticospinal tract disease), rigidity (similar resistance in all angles of motion; extrapyramidal disease), or paratonia (fluctuating changes in resistance;

1	by the angle and velocity of motion; corticospinal tract disease), rigidity (similar resistance in all angles of motion; extrapyramidal disease), or paratonia (fluctuating changes in resistance; frontal lobe pathways or normal difficulty in relaxing). Cogwheel rigidity, in which passive motion elicits jerky interruptions in resistance, is seen in parkinsonism.

1	Strength Testing for pronator drift is an extremely useful method for screening upper limb weakness. The patient is asked to hold both arms fully extended and parallel to the ground with eyes closed. This position should be maintained for ~10 s; any flexion at the elbow or fingers or pronation of the forearm, especially if asymmetric, is a sign of potential weakness. Muscle strength is further assessed by having the patient exert maximal effort for the particular muscle or muscle group being tested. It is important to isolate the muscles as much as possible, i.e., hold the limb so that only the muscles of interest are active. It is also helpful to palpate accessible muscles as they contract. Grading muscle strength and evaluating the patient’s effort is an art that takes time and practice. Muscle strength is traditionally graded using the following scale: 1 = flicker or trace of contraction but no associated movement at 2 = movement with gravity eliminated 4− = movement against a mild

1	Muscle strength is traditionally graded using the following scale: 1 = flicker or trace of contraction but no associated movement at 2 = movement with gravity eliminated 4− = movement against a mild degree of resistance

1	However, in many cases, it is more practical to use the following terms: Paralysis = no movement Severe weakness = movement with gravity eliminated Moderate weakness = movement against gravity but not against mild resistance Mild weakness = movement against moderate resistance Full strength Noting the pattern of weakness is as important as assessing the magnitude of weakness. Unilateral or bilateral weakness of the upper limb extensors and lower limb flexors (“pyramidal weakness”) suggests a lesion of the pyramidal tract, bilateral proximal weakness suggests myopathy, and bilateral distal weakness suggests peripheral neuropathy. • The bare minimum: Check the biceps, patellar, and Achilles reflexes.

1	• The bare minimum: Check the biceps, patellar, and Achilles reflexes. Muscle Stretch Reflexes Those that are typically assessed include the biceps (C5, C6), brachioradialis (C5, C6), and triceps (C7, C8) reflexes in the upper limbs and the patellar or quadriceps (L3, L4) and Achilles (S1, S2) reflexes in the lower limbs. The patient should be relaxed and the muscle positioned midway between full contraction and extension. Reflexes may be enhanced by asking the patient to voluntarily contract other, distant muscle groups (Jendrassik maneuver). For example, upper limb reflexes may be reinforced by voluntary teeth-clenching, and the Achilles reflex by hooking the flexed fingers of the two hands together and attempting to pull them apart. For each reflex tested, the two sides should be tested sequentially, and it is important to determine the smallest stimulus required to elicit a reflex rather than the maximum response. Reflexes are graded according to the following scale:

1	Cutaneous Reflexes The plantar reflex is elicited by stroking, with a noxious stimulus such as a tongue blade, the lateral surface of the sole of the foot beginning near the heel and moving across the ball of the foot to the great toe. The normal reflex consists of plantar flexion of the toes. With upper motor neuron lesions above the S1 level of the spinal cord, a paradoxical extension of the toe is observed, associated with fanning and extension of the other toes (termed an extensor plantar response, or Babinski sign). However, despite its popularity, the reliability and validity of the Babinski sign for identifying upper motor neuron weakness is limited—it is far more useful to rely on tests of tone, strength, stretch reflexes, and coordination. Superficial abdominal reflexes are elicited by gently stroking the abdominal surface near the umbilicus in a diagonal fashion with a sharp object (e.g., the wooden end of a cotton-tipped swab) and observing the movement of the umbilicus.

1	by gently stroking the abdominal surface near the umbilicus in a diagonal fashion with a sharp object (e.g., the wooden end of a cotton-tipped swab) and observing the movement of the umbilicus. Normally, the umbilicus will pull toward the stimulated quadrant. With upper motor neuron lesions, these reflexes are absent. They are most helpful when there is preservation of the upper (spinal cord level T9) but not lower (T12) abdominal reflexes, indicating a spinal lesion between T9 and T12, or when the response is asymmetric. Other useful cutaneous reflexes include the cremasteric (ipsilateral elevation of the testicle following stroking of the medial thigh; mediated by L1 and L2) and anal (contraction of the anal sphincter when the perianal skin is scratched; mediated by S2, S3, S4) reflexes. It is particularly important to test for these reflexes in any patient with suspected injury to the spinal cord or lumbosacral roots.

1	Primitive Reflexes With disease of the frontal lobe pathways, several primitive reflexes not normally present in the adult may appear. The suck response is elicited by lightly touching with a tongue blade the center of the lips, and the root response the corner of the lips; the patient will move the lips to suck or root in the direction of the stimulus. The grasp reflex is elicited by touching the palm between the thumb and index finger with the examiner’s fingers; a positive response is a forced grasp of the examiner’s hand. In many instances, stroking the back of the hand will lead to its release. The palmomental response is contraction of the mentalis muscle (chin) ipsilateral to a scratch stimulus diagonally applied to the palm. • The bare minimum: Ask whether the patient can feel light touch and the temperature of a cool object in each distal extremity. Check double simultaneous stimulation using light touch on the hands. Perform the Romberg maneuver.

1	Evaluating sensation is usually the most unreliable part of the examination because it is subjective and is difficult to quantify. In the compliant and discerning patient, the sensory examination can be extremely helpful for the precise localization of a lesion. With patients who are uncooperative or lack an understanding of the tests, it may be useless. The examination should be focused on the suspected lesion. For example, in spinal cord, spinal root, or peripheral nerve abnormalities, all major sensory modalities should be tested while looking for a pattern consistent with a spinal level and dermatomal or nerve distribution. In patients with lesions at or above the brainstem, screening the primary sensory modalities in the distal extremities along with tests of “cortical” sensation is usually sufficient.

1	The five primary sensory modalities—light touch, pain, temperature, vibration, and joint position—are tested in each limb. Light touch is assessed by stimulating the skin with single, very gentle touches of the examiner’s finger or a wisp of cotton. Pain is tested using a new pin, and temperature is assessed using a metal object (e.g., tuning fork) that has been immersed in cold and warm water. Vibration is tested using a 128-Hz tuning fork applied to the distal phalanx of the great toe or index finger just below the nail bed. By placing a finger on the opposite side of the joint being tested, the examiner compares the patient’s threshold of vibration perception with his or her own. For joint position testing, the examiner grasps the digit or limb laterally and distal to the joint being assessed; small 1to 2-mm excursions can usually be sensed. The Romberg maneuver is primarily a test of proprioception. The patient is asked to stand with the feet as close together as necessary to

1	assessed; small 1to 2-mm excursions can usually be sensed. The Romberg maneuver is primarily a test of proprioception. The patient is asked to stand with the feet as close together as necessary to maintain balance while the eyes are open, and the eyes are then closed. A loss of balance with the eyes closed is an abnormal response.

1	“Cortical” sensation is mediated by the parietal lobes and represents an integration of the primary sensory modalities; testing cortical sensation is only meaningful when primary sensation is intact. Double simultaneous stimulation is especially useful as a screening test for cortical function; with the patient’s eyes closed, the examiner lightly touches one or both hands and asks the patient to identify the stimuli. With a parietal lobe lesion, the patient may be unable to identify the stimulus on the contralateral side when both hands are touched. Other modalities relying on the parietal cortex include the discrimination of two closely placed stimuli as separate (two-point discrimination), identification of an object by touch and manipulation alone (stereognosis), and the identification of numbers or letters written on the skin surface (graphesthesia).

1	• The bare minimum: Observe the patient at rest and during spontaneous movements. Test rapid alternating movements of the hands and feet and finger to nose. Coordination refers to the orchestration and fluidity of movements. Even simple acts require cooperation of agonist and antagonist muscles, maintenance of posture, and complex servomechanisms to control the rate and range of movements. Part of this integration relies on normal function of the cerebellar and basal ganglia systems. However, coordination also requires intact muscle strength and kinesthetic and proprioceptive information. Thus, if the examination has disclosed abnormalities of the motor or sensory systems, the patient’s coordination should be assessed with these limitations in mind.

1	Rapid alternating movements in the upper limbs are tested separately on each side by having the patient make a fist, partially extend the index finger, and then tap the index finger on the distal thumb as quickly as possible. In the lower limb, the patient rapidly taps the foot against the floor or the examiner’s hand. Finger-to-nose testing is primarily a test of cerebellar function; the patient is asked to touch his or her index finger repetitively to the nose and then to the examiner’s outstretched finger, which moves with each repetition. A similar test in the lower extremity is to have the patient raise the leg and touch the examiner’s finger with the great toe. Another cerebellar test in the lower limbs is the heel-knee-shin maneuver; in the supine position the patient is asked to slide the heel of each foot from the knee down the shin of the other leg. For all these movements, the accuracy, speed, and rhythm are noted.

1	• The bare minimum: Observe the patient while walking normally, on the heels and toes, and along a straight line. Watching the patient walk is the most important part of the neurologic examination. Normal gait requires that multiple systems—including strength, sensation, and coordination—function in a highly integrated fashion. Unexpected abnormalities may be detected that prompt the examiner to return in more detail to other aspects of the examination. The patient should be observed while walking and turning normally, walking on the heels, walking on the toes, and walking heel-to-toe along a straight line. The examination may reveal decreased arm swing on one side (corticospinal tract disease), a stooped posture and short-stepped gait (parkinsonism), a broad-based unstable gait (ataxia), scissoring (spasticity), or a high-stepped, slapping gait (posterior column or peripheral nerve disease), or the patient may appear to be stuck in place (apraxia with frontal lobe disease).

1	The clinical data obtained from the history and examination are interpreted to arrive at an anatomic localization that best explains the clinical findings (Table 437-2), to narrow the list of diagnostic possibilities, and to select the laboratory tests most likely to be informative. The laboratory assessment may include (1) serum electrolytes; complete blood count; and renal, hepatic, endocrine, and immune studies; (2) cerebrospinal fluid examination; (3) focused neuroimaging studies (Chap. 440e); or (4) electrophysiologic studies (Chap. 442e). The anatomic localization, mode of onset and course of illness, other medical data, and laboratory findings are then integrated to establish an etiologic diagnosis.

1	The neurologic examination may be normal even in patients with a serious neurologic disease, such as seizures, chronic meningitis, or a TIA. A comatose patient may arrive with no available history, and in such cases, the approach is as described in Chap. 328. In other patients, an inadequate history may be overcome by a succession of examinations from which the course of the illness can be inferred. In perplexing cases it is useful to remember that uncommon presentations of common diseases are more likely than rare etiologies. Thus, even in tertiary care settings, multiple strokes are usually due to emboli and not vasculitis, and dementia with myoclonus is usually Alzheimer’s disease and not a prion disorder or a paraneoplastic illness. Finally, the most important task of a primary care physician

1	Approach to the Patient with Neurologic Disease ing head and limbs Visual field abnormalities Movement abnormalities (e.g., diffuse incoordination, tremor, chorea) Brainstem Isolated cranial nerve abnormalities (single or multiple) “Crossed” weaknessa and sensory abnormalities of head and limbs, e.g., weakness of right face and left arm and leg Spinal cord Back pain or tenderness Weaknessa and sensory abnormalities sparing the head Mixed upper and lower motor neuron findings Sensory level Sphincter dysfunction Weaknessb or sensory abnormalities following root distribution (see Figs. 31-2 and 31-3) Loss of reflexes Weaknessb or sensory abnormalities following nerve distribution (see Figs. 31-2 and 31-3) “Stocking or glove” distribution of sensory loss Loss of reflexes

1	Weaknessb or sensory abnormalities following nerve distribution (see Figs. 31-2 and 31-3) “Stocking or glove” distribution of sensory loss Loss of reflexes Neuromuscular Bilateral weakness including face (ptosis, diplopia, dysjunction phagia) and proximal limbs Increasing weakness with exertion Sparing of sensation Muscle Bilateral proximal or distal weakness Sparing of sensation aWeakness along with other abnormalities having an “upper motor neuron” pattern, i.e., spasticity, weakness of extensors > flexors in the upper extremity and flexors > extensors in the lower extremity, and hyperreflexia. bWeakness along with other abnormalities having a “lower motor neuron” pattern, i.e., flaccidity and hyporeflexia.

1	faced with a patient who has a new neurologic complaint is to assess the urgency of referral to a specialist. Here, the imperative is to rapidly identify patients likely to have nervous system infections, acute strokes, and spinal cord compression or other treatable mass lesions and arrange for immediate care. The Neurologic Screening Exam Daniel H. Lowenstein Knowledge of the basic neurologic examination is an essential clinical skill. A simple neurologic screening examination—assessment of mental status, cranial nerves, motor system, sensory system, coordination, and gait—can be reliably performed in 3–5 min. Although the components of the examination may appear daunting at first, skills usually improve rapidly with repetition and practice. In this video, the technique of performing a simple and efficient screening examination is presented. CHAPTER 438e The Neurologic Screening Exam

1	CHAPTER 438e The Neurologic Screening Exam Video Atlas of the Detailed Neurologic Examination Martin A. Samuels The comprehensive neurologic examination is an irreplaceable tool for the efficient diagnosis of neurologic disorders. Mastery of its details 439e requires knowledge of normal nervous system anatomy and physiology combined with personal experience performing orderly and systematic examinations on large numbers of patients and healthy individuals. In the hands of a great clinician, the neurologic examination also becomes a thing of beauty—the pinnacle of the art of medicine. In this video, the most commonly used components of the examination are presented in detail, with a particular emphasis on those elements that are most helpful for assessment of common neurologic problems. CHAPTER 439e Video Atlas of the Detailed Neurologic Examination

1	CHAPTER 439e Video Atlas of the Detailed Neurologic Examination Neuroimaging in Neurologic Disorders William P. Dillon The clinician caring for patients with neurologic symptoms is faced with myriad imaging options, including computed tomography (CT), 440e

1	CT angiography (CTA), perfusion CT (pCT), magnetic resonance (MR) imaging (MRI), MR angiography (MRA), functional MRI (fMRI), MR spectroscopy (MRS), MR neurography (MRN), diffusion and diffusion tensor imaging, susceptibility-weighted MR imaging (SWI), arterial spin label MRI (ASL) and perfusion MRI (pMRI). In addition, an increasing number of interventional neuroradiologic techniques are available, including angiography catheter embolization, coiling, and stenting of vascular structures, and spine diagnostic and interventional techniques, such as diskography, transforaminal and translaminar epidural and nerve root injections, and blood patches. Multidetector CTA (MDCTA) and gadolinium-enhanced MRA have narrowed the indications for conventional angiography, which is now reserved for patients in whom small-vessel detail is essential for diagnosis or for whom concurrent interventional therapy is planned (Table 440e-1).

1	In general, MRI is more sensitive than CT for the detection of lesions affecting the central nervous system (CNS), particularly those of the spinal cord, cranial nerves, and posterior fossa structures. Diffusion MR, a sequence sensitive to the microscopic motion of water, is the most sensitive technique for detecting acute ischemic stroke of the brain or spinal cord, and it is also useful in the detection of encephalitis, abscesses, and prion diseases. CT, however, is quickly acquired and is widely available, making it a pragmatic choice for the initial evaluation of patients with acute changes in mental status, suspected acute stroke, hemorrhage, and intracranial or spinal trauma. CT is also more sensitive than MRI for visualizing fine osseous detail and is indicated in the initial imaging evaluation of conductive hearing loss as well as lesions affecting the skull base and calvarium. MR may, however, add important diagnostic information regarding bone marrow infiltrative processes

1	evaluation of conductive hearing loss as well as lesions affecting the skull base and calvarium. MR may, however, add important diagnostic information regarding bone marrow infiltrative processes that are difficult to detect on CT.

1	The CT image is a cross-sectional representation of anatomy created by a computer-generated analysis of the attenuation of x-ray beams passed through a section of the body. As the x-ray beam, collimated to the desired slice width, rotates around the patient, it passes through selected regions in the body. X-rays that are not attenuated by body structures are detected by sensitive x-ray detectors aligned 180° from the x-ray tube. A computer calculates a “back projection” image from the 360° x-ray attenuation profile. Greater x-ray attenuation (e.g., as caused by bone), results in areas of high “density” (whiter) on the scan, whereas soft tissue structures that have poor attenuation of x-rays, such as organs and air-filled cavities, are lower (blacker) in density. The resolution of an image depends on the radiation dose, the detector size, collimation (slice thickness), the field of view, and the matrix size of the display. A modern CT scanner is capable of obtaining sections as thin as

1	depends on the radiation dose, the detector size, collimation (slice thickness), the field of view, and the matrix size of the display. A modern CT scanner is capable of obtaining sections as thin as 0.5–1 mm with 0.4-mm in-plane resolution at a speed of 0.3 s per rotation; complete studies of the brain can be completed in 1–10 s.

1	Multidetector CT (MDCT) is now standard in most radiology departments. Single or multiple (from 4 to 320) solid-state detectors positioned opposite to the x-ray source result in multiple slices per revolution of the beam around the patient. The table moves continuously through the rotating x-ray beam, generating a continuous “helix” of information that can be reformatted into various slice thicknesses and planes. Advantages of MDCT include shorter scan times, reduced patient and organ motion, and the ability to acquire images dynamically during the infusion of intravenous contrast, which can be used to taBLe 440e-1 GuiDeLiNes for the use of Ct, uLtrasouND, aND Mri Hemorrhage Acute parenchymal CT, MR Subacute/chronic MRI Subarachnoid hemorrhage CT, CTA, lumbar puncture → angiography Aneurysm Angiography > CTA, MRA

1	Hemorrhage Acute parenchymal CT, MR Subacute/chronic MRI Subarachnoid hemorrhage CT, CTA, lumbar puncture → angiography Aneurysm Angiography > CTA, MRA Ischemic infarction Hemorrhagic infarction CT or MRI Bland infarction MRI with diffusion > CT, CTA, angiography Carotid or vertebral dissection MRI/MRA Vertebral basilar insufficiency CTA, MRI/MRA Carotid stenosis CTA, MRA > US Neoplasm, primary or MRI + contrast metastatic Infection/abscess MRI + contrast Immunosuppressed with focal MRI + contrast First time, no focal neurologic MRI > CT deficits Partial complex/refractory MRI Cranial neuropathy MRI with contrast Meningeal disease MRI with contrast Cervical spondylosis MRI, CT, CT myelography Infection MRI + contrast, CT Arteriovenous malformation MRI + contrast, angiography Abbreviations: CT, computed tomography; CTA, CT angiography; MRA, magnetic resonance angiography; MRI, magnetic resonance imaging.

1	Arteriovenous malformation MRI + contrast, angiography Abbreviations: CT, computed tomography; CTA, CT angiography; MRA, magnetic resonance angiography; MRI, magnetic resonance imaging. construct CT angiograms of vascular structures and perfusion images (Figs. 440e-1B and C). CTA can be displayed in three dimensions to yield angiogram-like images (Figs. 440e-1C, 440e-2E and F, and see Fig. 446-4). CTA has proved useful in assessing the cervical and intra-cranial arterial and venous anatomy.

1	Intravenous iodinated contrast is often administered to identify both vascular structures and to detect defects in the blood-brain barrier (BBB) that are caused by tumors, infarcts, and infections. In the normal CNS, only vessels and structures lacking a BBB (e.g., the pituitary gland, choroid plexus, and dura) enhance after contrast administration. The use of iodinated contrast agents carries a small risk of allergic reaction and adds additional expense. While helpful in characterizing mass lesions as well as essential for the acquisition of CTA studies, the decision to use contrast material should always be considered carefully.

1	Figure 440e-1 Computed tomography (CT) angiography (CTA) of ruptured anterior cerebral artery aneurysm in a patient presenting with acute headache. A. Noncontrast CT demonstrates sub-arachnoid hemorrhage and mild obstructive hydrocephalus. B. Axial maximum-intensity projection from CTA demonstrates enlargement of the anterior cerebral artery (arrow). C. Three-dimensional surface reconstruction using a workstation confirms the anterior cerebral aneurysm and demonstrates its orientation and relationship to nearby vessels (arrow). CTA image is produced by 0.5to 1-mm helical CT scans performed during a rapid bolus infusion of intravenous contrast medium.

1	CT is the primary study of choice in the evaluation of an acute change in mental status, focal neurologic findings, acute trauma to the brain and spine, suspected subarachnoid hemorrhage, and conductive hearing loss (Table 440e-1). CT is complementary to MR in the evaluation of the skull base, orbit, and osseous structures of the spine. In the spine, CT is useful in evaluating patients with osseous spinal stenosis and spondylosis, but MRI is often preferred in those with neurologic deficits. CT can also be obtained following intrathecal contrast injection to evaluate the intracranial cisterns (CT cisternography) for cerebrospinal fluid (CSF) fistula, as well as the spinal subarachnoid space (CT myelography), although intrathecal administration of gadolinium combined with MR may also be complementary.

1	CT is safe, fast, and reliable. Radiation exposure depends on the dose used but is normally between 2 and 5 mSv (millisievert) for a routine brain CT study. Care must be taken to reduce exposure when imaging children. With the advent of MDCT, CTA, and CT perfusion, the benefit must be weighed against the increased radiation doses associated with these techniques. Advanced noise reduction software now permits acceptable diagnostic CT scans at 30–40% lower radiation doses. The most frequent complications are those associated with use of intravenous contrast agents. While two broad categories of contrast media, ionic and nonionic, are in use, ionic agents have been largely replaced by safer nonionic compounds.

1	Contrast nephropathy may result from hemodynamic changes, renal tubular obstruction and cell damage, or immunologic reactions to contrast agents. A rise in serum creatinine of at least 85 μmol/L (1 mg/dL) within 48 h of contrast administration is often used as a definition of contrast nephropathy, although other causes of acute renal failure must be excluded. The prognosis is usually favorable, with serum creatinine levels returning to baseline within 1–2 weeks. Risk factors for contrast nephropathy include advanced age (>80 years), preexisting renal disease (serum creatinine exceeding 2 mg/dL), solitary kidney, diabetes mellitus, dehydration, paraproteinemia, concurrent use of nephrotoxic medication or chemotherapeutic agents, and high contrast dose. Patients with diabetes and those with mild renal failure should be well hydrated prior to the administration of contrast agents, although careful consideration should be given to alternative imaging techniques such as MRI, noncontrast

1	mild renal failure should be well hydrated prior to the administration of contrast agents, although careful consideration should be given to alternative imaging techniques such as MRI, noncontrast CT, or ultrasound (US). Nonionic, low-osmolar media produce fewer abnormalities in renal blood flow and less endothelial cell damage but should still be used carefully in patients at risk for allergic reaction. Estimated glomerular filtration rate (eGFR) is a more reliable indicator of renal function compared to creatinine alone because it takes into account age, race, and sex. In one study, 15% of outpatients with a normal serum creatinine had an estimated creatinine clearance of 50 mL/min/1.73 m2 or less (normal is ≥90 mL/min/1.73 m2). The exact eGFR threshold, below which withholding intravenous contrast should be considered, is controversial. The risk of contrast nephropathy increases in patients with an eGFR <60 mL/min/1.73 m2; however, the majority of these patients will only have a

1	contrast should be considered, is controversial. The risk of contrast nephropathy increases in patients with an eGFR <60 mL/min/1.73 m2; however, the majority of these patients will only have a temporary rise in creatinine. The risk of dialysis after receiving contrast significantly increases in patients with eGFR <30 mL/min/1.73 m2. Thus, an eGFR threshold between 60 and 30 mL/ min/1.73 m2 is appropriate; however, the exact number is somewhat arbitrary. A creatinine of 1.6 in a 70-year-old, non-African-American male corresponds to an eGFR of approximately 45 mL/min/1.73 m2. The American College of Radiology suggests using an eGFR of 45 mL/ min/1.73 m2 as a threshold below which iodinated contrast should not be given without serious consideration of the potential for contrast nephropathy. If contrast must be administered to a patient with an eGFR below 45 mL/min/1.73 m2, the patient should be well hydrated, and a reduction in the dose of contrast should be considered. Use of other

1	If contrast must be administered to a patient with an eGFR below 45 mL/min/1.73 m2, the patient should be well hydrated, and a reduction in the dose of contrast should be considered. Use of other agents such as bicarbonate and acetylcysteine may reduce the incidence of contrast nephropathy.

1	allergy Immediate reactions following intravenous contrast media can occur through several mechanisms. The most severe reactions

1	Figure 440e-2 Acute left hemiparesis due to middle cerebral artery occlusion. A. Axial noncontrast computed tomography (CT) scan demonstrates high density within the right middle cerebral artery (arrow) associated with subtle low density involving the right putamen (arrowheads). B. Mean transit time CT perfusion parametric map indicating prolonged mean transit time involving the right middle cerebral territory (arrows). C. Cerebral blood volume (CBV) map shows reduced CBV involving an area within the defect shown in B, indicating a high likelihood of infarction (arrows). D. Axial maximum-intensity projection from a CT angiography (CTA) study through the circle of Willis demonstrates an abrupt occlusion of the proximal right middle cerebral artery (arrow). E. Sagittal reformation through the right internal carotid artery demonstrates a low-density lipid-laden plaque (arrowheads) narrowing the lumen (black arrow). F. Three-dimensional surface-rendered CTA image demonstrates

1	through the right internal carotid artery demonstrates a low-density lipid-laden plaque (arrowheads) narrowing the lumen (black arrow). F. Three-dimensional surface-rendered CTA image demonstrates calcification and narrowing of the right internal carotid artery (arrow), consistent with atherosclerotic disease. G. Coronal maximum-intensity projection from magnetic resonance angiography shows right middle cerebral artery (MCA) occlusion (arrow). H. and I. Axial diffusion-weighted image (H) and apparent diffusion coefficient image (I) documents the presence of a right middle cerebral artery infarction.

1	GuiDeLiNes for preMeDiCatioN of patieNts with prior CoNtrast aLLerGy 12 h prior to examination: Prednisone, 50 mg PO or methylprednisolone, 32 mg PO 2 h prior to examination: Prednisone, 50 mg PO or methylprednisolone, 32 mg PO and cimetidine, 300 mg PO or ranitidine, 150 mg PO Immediately prior to examination:

1	Benadryl, 50 mg IV (alternatively, can be given PO 2 h prior to exam) are related to allergic hypersensitivity (anaphylaxis) and range from mild hives to bronchospasm and death. The pathogenesis of allergic hypersensitivity reactions is thought to include the release of mediators such as histamine, antibody-antigen reactions, and complement activation. Severe allergic reactions occur in ~0.04% of patients receiving nonionic media, sixfold lower than with ionic media. Risk factors include a history of prior contrast reaction (fivefold increased likelihood), food and or drug allergies, and atopy (asthma and hay fever). The predictive value of specific allergies, such as those to shellfish, once thought important, actually is now recognized to be unreliable. Nonetheless, in patients with a history worrisome for potential allergic reaction, a noncontrast CT or MRI procedure should be considered as an alternative to contrast administration. If iodinated contrast is absolutely required, a

1	history worrisome for potential allergic reaction, a noncontrast CT or MRI procedure should be considered as an alternative to contrast administration. If iodinated contrast is absolutely required, a nonionic agent should be used in conjunction with pretreatment with glucocorticoids and antihistamines (Table 440e-2); however, pretreatment does not guarantee safety. Patients with allergic reactions to iodinated contrast material do not usually react to gadolinium-based MR contrast material, although such reactions can occur. It would be wise to pretreat patients with a prior allergic history to MR contrast administration in a similar fashion. Nonimmediate (>1 h after injection) reactions are frequent and probably related to T cell–mediated immune reactions. These are typically urticarial but can occasionally be more severe. Drug provocation and skin testing may be required to determine the culprit agent involved as well as determine a safe alternative.

1	Other side effects of CT scanning are rare but include a sensation of warmth throughout the body and a metallic taste during intravenous administration of iodinated contrast media. Extravasation of contrast media, although rare, can be painful and lead to compartment syndrome. When this occurs, consultation with plastic surgery is indicated. Patients with significant cardiac disease may be at increased risk for contrast reactions, and in these patients, limits to the volume and osmolality of the contrast media should be considered. Patients who may undergo systemic radioactive iodine therapy for thyroid disease or cancer should not receive iodinated contrast media if possible, because this will decrease the uptake of the radioisotope into the tumor or thyroid (see the American College of Radiology Manual on Contrast Media, Version 9, 2013; http://www.acr.org/~/media/ACR/ Documents/PDF/QualitySafety/Resources/Contrast%20Manual/2013_ Contrast_Media.pdf).

1	MRI is a complex interaction between hydrogen protons in biologic tissues, a static magnetic field (the magnet), and energy in the form of radiofrequency (Rf) waves of a specific frequency introduced by coils placed next to the body part of interest. Images are made by computerized processing of resonance information received from protons in the body. Field strength of the magnet is directly related to signal-to-noise ratio. While 1.5-T magnets have become the standard high-field MRI units, 3-T magnets are now widely available and have distinct advantages in the brain and musculoskeletal systems. Even higher field magnets (7-T) and positron emission tomography (PET) MR machines promise increased resolution and anatomic-functional information on a variety of disorders. Spatial localization is achieved by magnetic gradients surrounding the main magnet, which impart slight changes in magnetic field throughout the imaging volume. Rf pulses transiently excite the energy state of the

1	is achieved by magnetic gradients surrounding the main magnet, which impart slight changes in magnetic field throughout the imaging volume. Rf pulses transiently excite the energy state of the hydrogen protons in the body. Rf is administered at a frequency specific for the field strength of the magnet. The subsequent return to equilibrium energy state (relaxation) of the hydrogen protons results in a release of Rf energy (the echo), which is detected by the coils that delivered the Rf pulses. Fourier analysis is used to transform the echo into the information used to form an MR image. The MR image thus consists of a map of the distribution of hydrogen protons, with signal intensity imparted by both density of hydrogen protons and differences in the relaxation times (see below) of hydrogen protons on different molecules. Although clinical MRI currently makes use of the ubiquitous hydrogen proton, research into sodium and carbon imaging and spectroscopy appears promising.

1	t1 and t2 relaxation times The rate of return to equilibrium of perturbed protons is called the relaxation rate. The relaxation rate varies among normal and pathologic tissues. The relaxation rate of a hydrogen proton in a tissue is influenced by local interactions with surrounding molecules and atomic neighbors. Two relaxation rates, T1 and T2, influence the signal intensity of the image. The T1 relaxation time is the time, measured in milliseconds, for 63% of the hydrogen protons to return to their normal equilibrium state, whereas the T2 relaxation is the time for 63% of the protons to become dephased owing to interactions among nearby protons. The intensity and image contrast of the signal within various tissues can be modulated by altering acquisition parameters such as the interval between Rf pulses (TR) and the time between the Rf pulse and the signal reception (TE). T1-weighted (T1W) images are produced by keeping the TR and TE relatively short, whereas using longer TR and TE

1	between Rf pulses (TR) and the time between the Rf pulse and the signal reception (TE). T1-weighted (T1W) images are produced by keeping the TR and TE relatively short, whereas using longer TR and TE times produces T2-weighted (T2W) images. Fat and subacute hemorrhage have relatively shorter T1 relaxation rates and thus higher signal intensity than brain on T1W images. Structures containing more water, such as CSF and edema, have long T1 and T2 relaxation rates, resulting in relatively lower signal intensity on T1W images and higher signal intensity on T2W images (Table 440e-3). Gray matter contains 10–15% more water than white matter, which accounts for much of the intrinsic contrast between the two on MRI (Fig. 440e-6B). T2W images are more sensitive than T1W images to edema, demyelination, infarction, and chronic hemorrhage, whereas T1W imaging is more sensitive to subacute hemorrhage and fat-containing structures.

1	Many different MR pulse sequences exist, and each can be obtained in various planes (Figs. 440e-2, 440e-3, and 440e-4). The selection of a proper protocol that will best answer a clinical question depends on an accurate clinical history and indication for the examination. Fluid-attenuated inversion recovery (FLAIR) is a useful pulse sequence that produces T2W images in which the normally high signal intensity of CSF is suppressed (Fig. 440e-6B). FLAIR images are more sensitive than standard spin echo images for any water-containing lesions or edema. Susceptibility-weighted imaging, such as gradient echo imaging, is very sensitive to magnetic susceptibility generated by blood, calcium, and air and routinely obtained in patients suspected of pathology that might result in microhemorrhages, such as amyloid, hemorrhagic metastases, and thrombotic states (Fig. 440e-5C). MR images can be generated in any plane without changing the patient’s position. Each sequence, however, must be obtained

1	as amyloid, hemorrhagic metastases, and thrombotic states (Fig. 440e-5C). MR images can be generated in any plane without changing the patient’s position. Each sequence, however, must be obtained separately and takes 1–10 min on average to complete. Three-dimensional volumetric imaging is also possible with MRI, resulting in a three-dimensional

1	FLAIR (T2) Long Long Low Medium High High Abbreviations: CSF, cerebrospinal fluid; FLAIR, fluid-attenuated inversion recovery; TE, interval between radiofrequency pulse and signal reception; TR, interval between radiofrequency pulses; T1W and T2W, T1and T2-weighted. Figure 440e-3 Cerebral abscess in a patient with fever and a right hemiparesis. A. Coronal postcontrast T1-weighted image demonstrates a ring-enhancing mass in the left frontal lobe. B. Axial diffusion-weighted image demonstrates restricted diffusion (high signal intensity) within the lesion, which in this setting is highly suggestive of cerebral abscess. volume of data that can be reformatted in any orientation to highlight certain disease processes.

1	mr Contrast material The heavy-metal element gadolinium forms the basis of all currently approved intravenous MR contrast agents. Gadolinium is a paramagnetic substance, which means that it reduces the T1 and T2 relaxation times of nearby water protons, resulting in a high signal on T1W images and a low signal on T2W images (the latter requires a sufficient local concentration, usually in the form of an intravenous bolus). Unlike iodinated contrast agents, the effect of MR contrast agents depends on the presence of local hydrogen protons on which it must act to achieve the desired effect. There are nine different gadolinium agents approved in the United States for use with MRI. These differ according the attached chelated moiety, which also affects the strength of chelation of the otherwise toxic gadolinium element. The chelating carrier molecule for gadolinium can be classified by whether it is macrocyclic or has linear geometry and whether it is ionic or nonionic. Most of these are

1	toxic gadolinium element. The chelating carrier molecule for gadolinium can be classified by whether it is macrocyclic or has linear geometry and whether it is ionic or nonionic. Most of these are excreted by the renal system. Cyclical agents are less likely to release the gadolinium element, and thus are considered the safest category.

1	ALLERGIC HYPERSENSITIVITY Gadolinium-DTPA (diethylenetriaminepentaacetic acid) does not normally cross the intact BBB immediately but will enhance lesions lacking a BBB (Fig. 440e-3A) as well as areas of the brain that normally are devoid of the BBB (pituitary, dura, choroid plexus). However, gadolinium contrast has been noted to slowly cross an intact BBB over time and especially in the setting of reduced renal clearance or inflamed meninges. The agents are generally well tolerated; overall adverse events after injection range from 0.07–2.4%. True allergic reactions are rare (0.004–0.7%) but have been reported. Severe life-threatening reactions are exceedingly rare; in one report, only 55 reactions out of 20 million doses occurred. However, the adverse reaction rate in patients with a prior history of reaction to gadolinium is eight times higher than normal. Other risk factors include atopy or asthma (3.7%); although there is no cross-reactivity to iodinated contrast material, those

1	history of reaction to gadolinium is eight times higher than normal. Other risk factors include atopy or asthma (3.7%); although there is no cross-reactivity to iodinated contrast material, those with a prior allergic response to iodine should be considered at higher risk. Gadolinium contrast material can be administered safely to children as well as adults, although these agents are generally avoided in those under 6 months of age.

1	NEPHROTOXICITY Contrast-induced renal failure does not occur with gadolinium agents. A rare complication, nephrogenic systemic fibrosis (NSF), has occurred in patients with severe renal insufficiency who have been exposed to gadolinium contrast agents. The onset of NSF has been reported between 5 and 75 days following exposure; histologic features include thickened collagen bundles with surrounding clefts, mucin deposition, and increased numbers of fibrocytes and elastic fibers in skin. In addition to dermatologic symptoms, other manifestations include widespread fibrosis of the skeletal muscle, bone, lungs, pleura, pericardium, myocardium, kidney, muscle, bone, testes, and dura. The American College of Radiology recommends that a glomerular filtration rate (GFR) assessment be obtained within 6 weeks prior to elective gadolinium-based MR contrast agent administration in patients with: 1. A history of renal disease (including solitary kidney, renal transplant, renal tumor) 2. 3.

1	A history of renal disease (including solitary kidney, renal transplant, renal tumor) 2. 3. History of hypertension 4. History of diabetes 5. History of severe hepatic disease, liver transplant, or pending liver transplant; for these patients, it is recommended that the patient’s GFR assessment be nearly contemporaneous with the MR examination The incidence of NSF in patients with severe renal dysfunction (GFR <30) varies from 0.19 to 4%. Other risk factors for NSF include acute kidney injury, the use of nonmacrocyclic agents, and repeated or high-dose exposure to gadolinium. The American College of Radiology Committee on Drugs and Contrast Media states that patients receiving any gadolinium-containing agent should be considered at risk of NSF if they are on dialysis (of any form); have severe or end-stage chronic renal disease (eGFR <30 mL/min/1.73 m2) without dialysis; eGFR of 30–40 mL/min/1.73 m2 without dialysis (as the GFR may fluctuate); or have acute renal insufficiency.

1	From the patient’s perspective, an MRI examination can be intimidating, and a higher level of cooperation is required than with CT. The patient lies on a table that is moved into a long, narrow gap within the magnet. Approximately 5% of the population experiences severe claustrophobia in the MR environment. This can be reduced by mild sedation but remains a problem for some. Because it takes between 3 and

1	Figure 440e-4 Herpes simplex encephalitis in a patient presenting with altered mental status and fever. A. and B. Coronal (A) and axial (B) T2-weighted fluid-attenuated inversion recovery images demonstrate expansion and high signal intensity involving the right medial temporal lobe and insular cortex (arrows). C. Coronal diffusion-weighted image demonstrates high signal intensity indicating restricted diffusion involving the right medial temporal lobe and hippocampus (arrows) as well as subtle involvement of the left inferior temporal lobe (arrowhead). This is most consistent with neuronal death and can be seen in acute infarction as well as encephalitis and other inflammatory conditions. The suspected diagnosis of herpes simplex encephalitis was confirmed by cerebrospinal fluid polymerase chain reaction analysis.

1	10 min per sequence, movement of the patient during an MR exam distorts all of the images; therefore, uncooperative patients should either be sedated for the MR study or scanned with CT. Generally, children under the age of 8 years usually require conscious sedation in order to complete the MR examination without motion degradation.

1	MRI is considered safe for patients, even at very high field strengths. Serious injuries have been caused, however, by attraction of ferromagnetic objects into the magnet, which act as missiles if brought too close to the magnet. Likewise, ferromagnetic implants, such as aneurysm clips, may torque within the magnet, causing damage to vessels and even death. Metallic foreign bodies in the eye have moved and caused intraocular hemorrhage; screening for ocular metallic fragments is indicated in those with a history of metal work or ocular metallic foreign bodies. Implanted cardiac pacemakers are generally a contraindication to MRI owing to the risk of induced arrhythmias; however, some newer pacemakers have been shown to be safe. All health care personnel and patients must be screened and educated thoroughly to prevent such disasters because the magnet is always “on.” Table 440e-4 lists common contraindications for MRI.

1	MR angiography is a general term describing several MR techniques that result in vascular-weighted images. These provide a vascular flow map rather than the anatomic map shown by conventional angiography. On routine spin echo MR sequences, moving protons (e.g., flowing blood, CSF) exhibit complex MR signals that range from highto low-signal intensity relative to background stationary tissue. Figure 440e-5 Susceptibility-weighted imaging in a patient with familial cavernous malformations. A. Noncontrast computed tomography scan shows one hyperdense lesion in the right hemisphere (arrow). B. T2-weighted fast spin echo image shows subtle low-intensity lesions (arrows). C. Susceptibility-weighted image shows numerous low-intensity lesions consistent with hemosiderin-laden cavernous malformations (arrow).

1	Fast-flowing blood returns no signal (flow void) on routine T1W or T2W spin echo MR images. Slower-flowing blood, as occurs in veins or distal to arterial stenosis, may appear high in signal. However, using special pulse sequences called gradient echo sequences, it is possible to increase the signal intensity of moving protons in contrast to the low signal background intensity of stationary tissue. This creates angiography-like images, which can be manipulated in three dimensions to highlight vascular anatomy and relationships.

1	So called time-of-flight (TOF) MRA relies on the suppression of nonmoving tissue to provide a low-intensity background for the high signal intensity of flowing blood entering the section; arterial or venous structures may be highlighted. A typical TOF MRA sequence results in a series of contiguous, thin MR sections (0.6–0.9 mm thick), which can be viewed as a stack and manipulated to create an angiographic image data set that can be reformatted and viewed in various planes and angles, much like that seen with conventional angiography (Fig. 440e-2G).

1	Phase-contrast MRA has a longer acquisition time than TOF MRA, but in addition to providing anatomic information similar to that of TOF imaging, it can be used to reveal the velocity and direction of blood flow in a given vessel. Through the selection of different imaging parameters, differing blood velocities can be highlighted; selective venous and arterial MRA images can thus be obtained. One advantage of phase-contrast MRA is the excellent suppression of high-signalintensity background structures. MRA can also be acquired during infusion of contrast material. Advantages include faster imaging times (1–2 min vs 10 min), fewer flow-related artifacts, and higher resolution images. Recently, CoMMoN CoNtraiNDiCatioNs to MaGNetiC resoNaNCe iMaGiNG

1	CoMMoN CoNtraiNDiCatioNs to MaGNetiC resoNaNCe iMaGiNG Cardiac pacemaker or permanent pacemaker leads Internal defibrillatory device Cochlear prostheses Bone growth stimulators Spinal cord stimulators Electronic infusion devices Intracranial aneurysm clips (some but not all) Ocular implants (some) or ocular metallic foreign body McGee stapedectomy piston prosthesis Duraphase penile implant Swan-Ganz catheter Magnetic stoma plugs Magnetic dental implants Magnetic sphincters Ferromagnetic inferior vena cava filters, coils, stents—safe 6 weeks after implantation Tattooed eyeliner (contains ferromagnetic material and may irritate eyes) Note: See also http://www.mrisafety.com. contrast-enhanced MRA has become the standard for extracranial vascular MRA. This technique entails rapid imaging using coronal three-dimensional TOF sequences during a bolus infusion of gadolinium contrast agent. Proper technique and timing of acquisition relative to bolus arrival are critical for success.

1	MRA has lower spatial resolution compared with conventional film-based angiography, and therefore the detection of small-vessel abnormalities, such as vasculitis and distal vasospasm, is problematic. MRA is also less sensitive to slowly flowing blood and thus may not reliably differentiate complete from near-complete occlusions. Motion, either by the patient or by anatomic structures, may distort the MRA images, creating artifacts. These limitations notwithstanding, MRA has proved useful in evaluation of the extracranial carotid and vertebral circulation as well as of larger-caliber intracranial arteries and dural sinuses. It has also proved useful in the noninvasive detection of intracranial aneurysms and vascular malformations.

1	Recent improvements in gradients, software, and high-speed computer processors now permit extremely rapid MRI of the brain. With echo-planar MRI (EPI), fast gradients are switched on and off at high speeds to create the information used to form an image. In routine spin echo imaging, images of the brain can be obtained in 5–10 min. With EPI, all of the information required for processing an image is accumulated in milliseconds, and the information for the entire brain can be obtained in less than 1–2 min, depending on the degree of resolution required or desired. Fast MRI reduces patient and organ motion and is the basis of perfusion imaging during contrast infusion and kinematic motion studies. EPI is also the sequence used to obtain diffusion imaging and tractography, as well as fMRI and arterial spin-labeled studies (Figs. 440e-2H, 440e-3, 440e-4C, and 440e-6; and see Fig. 446-16).

1	Perfusion and diffusion imaging are EPI techniques that are useful in early detection of ischemic injury of the brain and may be useful together to demonstrate infarcted tissue as well as ischemic but potentially viable tissue at risk of infarction (e.g., the ischemic penumbra). Diffusion-weighted imaging (DWI) assesses microscopic motion of water; abnormal restriction of motion appears as relative high-signal intensity on diffusion-weighted images. Infarcted tissue reduces the water motion within cells and in the interstitial tissues, resulting in high signal on DWI. DWI is the most sensitive technique for detection of acute cerebral infarction of <7 days in duration (Fig. 440e-2H). It is also quite sensitive for detecting dying or dead brain tissue secondary to encephalitis, as well as abscess formation (Fig. 440e-3B).

1	Perfusion MRI involves the acquisition of fast echo planar gradient images during a rapid intravenous bolus of gadolinium contrast material. Relative cerebral blood volume, mean transit time, and cerebral blood flow maps are then derived. Delay in mean transit time and reduction in cerebral blood volume and cerebral blood flow are typical of infarction. In the setting of reduced blood flow, a prolonged mean transit time of contrast but normal or elevated cerebral blood volume may indicate tissue supplied by collateral flow that is at risk of infarction. Perfusion MRI imaging can also be used in the assessment of brain tumors to differentiate intraaxial primary tumors, whose BBB is relatively intact, from extraaxial tumors or metastases, which demonstrate a relatively more permeable BBB.

1	Diffusion tensor imaging is derived from diffusion MRI imaging sequences, which assesses the direction of microscopic motion of water along white matter tracts. This technique has great potential in the assessment of brain maturation as well as disease entities that undermine the integrity of the white matter architecture. It has proven valuable in preoperative assessment of subcortical white matter tract anatomy prior to brain tumor surgery (Fig. 440e-6).

1	fMRI of the brain is an EPI technique that localizes regions of activity in the brain following task activation. Neuronal activity elicits a slight increase in the delivery of oxygenated blood flow to a specific region of activated brain. This results in an alteration in the balance of oxyhemoglobin and deoxyhemoglobin, which yields a 2–3% increase in signal intensity within veins and local capillaries. Further studies will determine whether these techniques are cost effective or clinically useful, but currently, preoperative somatosensory and auditory cortex localization is possible. This technique has proved useful to neuroscientists interested in interrogating the localization of certain brain functions.

1	ASL is a quantitative noninvasive MR technique that measures cerebral blood flow. Blood traversing in the neck is labeled by an MR pulse and then imaged in the brain after a short delay. The signal in the brain is reflective of blood flow. ASL is an especially important technique for patients with kidney failure and for pediatric patients in whom the use of radioactive tracers or exogenous contrast agents is contraindicated. Increased cerebral flow is more easily identified than slow flow, which can be sometimes difficult to quantify. This technique has also been shown useful in detecting arterial venous shunting in arteriovenous malformations and arteriovenous fistulas.

1	MRN is a T2W MR technique that shows promise in detecting increased signal in irritated, inflamed, or infiltrated peripheral nerves. Images are obtained with fat-suppressed fast spin echo imaging or short inversion recovery sequences. Irritated or infiltrated nerves will demonstrate high signal on T2W imaging. This is indicated in patients with radiculopathy whose conventional MR studies of the spine are normal, or in those suspected of peripheral nerve entrapment or trauma.

1	PET relies on the detection of positrons emitted during the decay of a radionuclide that has been injected into a patient. The most frequently used moiety is 2-[18F]fluoro-2-deoxy-D-glucose (FDG), which is an analogue of glucose and is taken up by cells competitively with 2-deoxyglucose. Multiple images of glucose uptake activity are formed after 45–60 min. Images reveal differences in regional glucose activity among normal and pathologic brain structures. FDG-PET is used primarily for the detection of extracranial metastatic disease; however, a lower activity of FDG in the parietal lobes is associated with Alzheimer’s disease, a finding that may simply reflect atrophy that occurs in the later stages of the disease. Combination PET-CT scanners, in which both CT and PET are obtained at one sitting, have largely replaced PET scans alone for most clinical indications. MR-PET scanners have also

1	Figure 440e-6 Diffusion tractography in cerebral glioma. Associative and descending pathways in a healthy subject (A) and in a patient with parietal lobe glioblastoma (B) presenting with a language deficit: the mass causes a disruption of the arcuate-SLF complex, in particular of its anterior portion (SLF III). Also shown are bilateral optic tract and left optic radiation pathways in a healthy subject (C) and in a patient with left occipital grade II oligoastrocytoma (D): the mass causes a disruption of the left optic radiation. Shown in neurologic orientation, i.e., the left brain appears on the left side of the image. AF, long segment of the arcuate fascicle; CST, corticospinal tract; IFOF: inferior fronto-occipital fascicle; ILF, inferior longitudinal fascicle; SLF III, superior longitudinal fascicle III or anterior segment of the arcuate fascicle; SLF-tp, temporo-parietal portion of the superior longitudinal fascicle or posterior segment of the arcuate fascicle; T, tumor; UF,

1	fascicle III or anterior segment of the arcuate fascicle; SLF-tp, temporo-parietal portion of the superior longitudinal fascicle or posterior segment of the arcuate fascicle; T, tumor; UF, uncinated fascicle. (Part D courtesy of Eduardo Caverzasi and Roland Henry.) been developed and may prove useful for imaging the brain and other myelography organs without the radiation exposure of CT. More recent PET ligand developments include amyloid tracers, such as Pittsburgh compound B Myelography involves the intrathecal instillation of specially formulated (PIB) and 18-F AV-45 (florbetapir), and tau PET tracers, such as water-soluble iodinated contrast medium into the lumbar or cervical18F-T807 and T808. Studies have shown an increased percentage of subarachnoid space. CT scanning is typically performed after myelograamyloid deposition in patients with Alzheimer’s disease compared phy (CT myelography) to better demonstrate the spinal cord and roots,with mild cognitive impairment and healthy

1	after myelograamyloid deposition in patients with Alzheimer’s disease compared phy (CT myelography) to better demonstrate the spinal cord and roots,with mild cognitive impairment and healthy controls; however, up to which appear as filling defects in the opacified subarachnoid space. Low25% of cognitively “normal” patients show abnormalities on amyloid dose CT myelography, in which CT is performed after the subarachnoidPET imaging. This may either reflect subclinical disease processes or injection of a small amount of relatively dilute contrast material, hasvariation of normal. Tau imaging may be more specific for Alzheimer’s replaced conventional myelography for many indications, thereby disease, and clinical studies are under way.

1	reducing exposure to radiation and contrast media. Newer multidetector scanners now obtain CT studies quickly so that reformations in sagittal and coronal planes, equivalent to traditional myelography projections, are now routine. Myelography has been largely replaced by CT myelography and MRI for diagnosis of diseases of the spinal canal and cord (Table 440e-1). Remaining indications for conventional plain-film myelography include the evaluation of suspected meningeal or arachnoid cysts and the localization of CSF fistulas. Conventional myelography and CT myelography provide the most precise information in patients with prior spinal fusion and spinal fixation hardware.

1	Myelography is relatively safe; however, it should be performed with caution in any patient with elevated intracranial pressure, evidence of a spinal block, or a history of allergic reaction to intrathecal contrast media. In patients with a suspected spinal block, MR is the preferred technique. If myelography is necessary, only a small amount of contrast medium should be instilled below the lesion in order to minimize the risk of neurologic deterioration. Lumbar puncture is to be avoided in patients with bleeding disorders, including patients receiving anticoagulant therapy, as well as in those with infections of the overlying soft tissues (Chap. 443e).

1	Headache is the most frequent complication of myelography and is reported to occur in 5–30% of patients. Nausea and vomiting may also occur rarely. Postural headache (post–lumbar puncture headache) is generally due to leakage of CSF from the puncture site, resulting in CSF hypotension. A higher incidence is noted among younger women and with the use of larger gauge cutting-type spinal needles. If significant headache persists for longer than 48 h, placement of an epidural blood patch should be considered. Management of lumbar puncture headache is discussed in Chap. 21. Vasovagal syncope may occur during lumbar puncture; it is accentuated by the upright position used during lumbar myelography. Adequate hydration before and after myelography will reduce the incidence of this complication.

1	Hearing loss is a rare complication of myelography. It may result from a direct toxic effect of the contrast medium or from an alteration of the pressure equilibrium between CSF and perilymph in the inner ear. Puncture of the spinal cord is a rare but serious complication of cervical (C1–2) or high lumbar puncture. The risk of cord puncture is greatest in patients with spinal stenosis, Chiari malformations, or conditions that reduce CSF volume. In these settings, a low-dose lumbar injection followed by thin-section CT or MRI is a safer alternative to cervical puncture. Intrathecal contrast reactions are rare, but aseptic meningitis and encephalopathy are reported complications. The latter is usually dose related and associated with contrast entering the intra-cranial subarachnoid space. Seizures occur following myelography in 0.1–0.3% of patients. Risk factors include a preexisting seizure disorder and the use of a total iodine dose of >4500 mg. Other reported complications include

1	Seizures occur following myelography in 0.1–0.3% of patients. Risk factors include a preexisting seizure disorder and the use of a total iodine dose of >4500 mg. Other reported complications include hyperthermia, hallucinations, depression, and anxiety states. These side effects have been reduced by the development of nonionic, water-soluble contrast agents as well as by head elevation and generous hydration following myelography.

1	The evaluation of back pain and radiculopathy may require diagnostic procedures that attempt either to reproduce the patient’s pain or relieve it, indicating its correct source prior to lumbar fusion. Diskography is performed by fluoroscopic placement of a 22to 25-gauge needle into the intervertebral disk and subsequent injection of 1–3 mL of contrast media. The intradiskal pressure is recorded, as is an assessment of the patient’s response to the injection of contrast material. Typically little or no pain is felt during injection of a normal disk, which does not accept much more than 1 mL of contrast material, even at pressures as high as 415–690 kPa (60–100 lb/in2). CT and plain films are obtained following the procedure. Concerns have been raised that diskography may contribute to an accelerated rate of disk degeneration.

1	Percutaneous selective nerve root and epidural blocks with glucocorticoid and anesthetic mixtures may be both therapeutic and diagnostic, especially if a patient’s pain is relieved. Typically, 1–2 mL of an equal mixture of a long-acting glucocorticoid such as betamethasone and a long-acting anesthetic such as bupivacaine 0.75% is instilled under CT or fluoroscopic guidance in the intraspinal epidural space or adjacent to an existing nerve root. Catheter angiography is indicated for evaluating intracranial small-vessel pathology (such as vasculitis), for assessing vascular malformations and aneurysms, and in endovascular therapeutic procedures (Table 440e-1). Angiography has been replaced for many indications by CT/CTA or MRI/MRA.

1	Angiography carries the greatest risk of morbidity of all diagnostic imaging procedures, owing to the necessity of inserting a catheter into a blood vessel, directing the catheter to the required location, injecting contrast material to visualize the vessel, and removing the catheter while maintaining hemostasis. Therapeutic transcatheter procedures (see below) have become important options for the treatment of some cerebrovascular diseases. The decision to undertake a diagnostic or therapeutic angiographic procedure requires careful assessment of the goals of the investigation and its attendant risks.

1	To improve tolerance to contrast agents, patients undergoing angiography should be well hydrated before and after the procedure. Because the femoral route is used most commonly, the femoral artery must be compressed after the procedure to prevent a hematoma from developing. The puncture site and distal pulses should be evaluated carefully after the procedure; complications can include thigh hematoma or lower extremity emboli.

1	A common femoral arterial puncture provides retrograde access via the aorta to the aortic arch and great vessels. The most feared complication of cerebral angiography is stroke. Thrombus can form on or inside the tip of the catheter, and atherosclerotic thrombus or plaque can be dislodged by the catheter or guide wire or by the force of injection and can embolize distally in the cerebral circulation. Risk factors for ischemic complications include limited experience on the part of the angiographer, atherosclerosis, vasospasm, low cardiac output, decreased oxygen-carrying capacity, advanced age, and prior history of migraine. The risk of a neurologic complication varies but is ~4% for transient ischemic attack and stroke, 1% for permanent deficit, and <0.1% for death.

1	Ionic contrast material injected into the cerebral vasculature can be neurotoxic if the BBB is breached, either by an underlying disease or by the injection of hyperosmolar contrast agent. Ionic contrast media are less well tolerated than nonionic media, probably because they can induce changes in cell membrane electrical potentials. Patients with dolichoectasia of the basilar artery can suffer reversible brainstem dysfunction and acute short-term memory loss during angiography, owing to the slow percolation of the contrast material and the consequent prolonged exposure of the brain. Rarely, an intracranial aneurysm ruptures during an angiographic contrast injection, causing subarachnoid hemorrhage, perhaps as a result of injection under high pressure. Spinal angiography may be indicated to evaluate vascular malformations and tumors and to identify the artery of Adamkiewicz (Chap.

1	Spinal angiography may be indicated to evaluate vascular malformations and tumors and to identify the artery of Adamkiewicz (Chap. 456) prior to aortic aneurysm repair. The procedure is lengthy and requires the use of relatively large volumes of contrast; the incidence of serious complications, including paraparesis, subjective visual blurring, and altered speech, is ~2%. Gadolinium-enhanced MRA has been used successfully in this setting, as has iodinated contrast CTA, which has promise for replacing diagnostic spinal angiography for some indications.

1	This rapidly developing field is providing new therapeutic options for patients with challenging neurovascular problems. Available procedures include detachable coil therapy for aneurysms, particulate or liquid adhesive embolization of arteriovenous malformations, stent retrieval systems for embolectomy, balloon angioplasty and stenting of arterial stenosis or vasospasm, transarterial or transvenous embolization of dural arteriovenous fistulas, balloon occlusion of carotid-cavernous and vertebral fistulas, endovascular treatment of vein-of-Galen malforma-440e-11 tions, preoperative embolization of tumors, and thrombolysis of acute arterial or venous thrombosis. Many of these disorders place the patient at high risk of cerebral hemorrhage, stroke, or death.

1	The highest complication rates are found with the therapies designed to treat the highest risk diseases. The advent of electrolytically detachable coils has ushered in a new era in the treatment of cerebral aneurysms. Two randomized trials found reductions of morbidity and mortality at 1 year among those treated for aneurysm with detachable coils compared with neurosurgical clipping. It remains to be determined what the role of coils will be relative to surgical options, but in many centers, coiling has become standard therapy for many aneurysms.

1	Atlas of Neuroimaging Andre D. Furtado, William P. Dillon This atlas comprises 48 cases to assist the clinician caring for patients with neurologic symptoms. The majority of the images shown are magnetic resonance imaging (MRI) scans; other techniques illustrated include magnetic resonance (MR) and conventional angiography 441e and computed tomography (CT) scans. Many different categories of neurologic disease are illustrated, including numerous examples of ischemic, inflammatory, inherited, vascular, and neoplastic etiologies. CHAPTER 441e Atlas of Neuroimaging FIGURE 441e-1 Limbic encephalitis (Chap. 122). Coronal (A, B), axial fluid-attenuated inversion recovery (FLAIR) (C, D), and axial T2-weighted (E) MR images demonstrate abnormal high signal involving the bilateral mesial temporal lobes (arrowheads) including the hippocampi (left greater than right) without significant mass effect (arrows). There was no enhancement on postgadolinium images (not shown).

1	FIGURE 441e-2 Central nervous system tuberculosis (Chap. 202). Axial T2-weighted MRI (A) demonstrates multiple lesions (arrows) with peripheral high signal and central low signal, located predominantly in the cortex and subcortical white matter, as well as in the basal ganglia. Axial T1-weighted MR images postgadolinium (B, C) demonstrate ring enhancement of the lesions (arrows) and additional lesions in the subarachnoid space (arrowheads). Sagittal T2-weighted MR image of the cervical spine (D) demonstrates a hypointense lesion in the subarachnoid space at the level of T5 (arrow). Sagittal T1-weighted postgadolinium MRI of the cervical spine (E) demonstrates enhancement of the lesion in the subarachnoid space at the level of T5 (arrow).

1	FIGURE 441e-3 Neurosyphilis (Chap. 206): Case I. Axial T2-weighted MRIs (A, B) demonstrate well-defined areas of abnormal high signal in the basal ganglia bilaterally and in a wedge-shaped distribution in the right parietal lobe (arrows). Axial (C, D) T1-weighted images postgadolinium. Coronal (E, F) T1-weighted images postgadolinium demonstrate irregular ring enhancement of the lesions (arrows). FIGURE 441e-4 Neurosyphilis (Chap. 206): Case II. Axial T2-weighted MRI (A) demonstrates a dural-based, peripherally hyperintense and centrally hypointense lesion located lateral to the left frontal lobe (arrow). Axial (B) and coronal (C) T1-weighted MRIs postgadolinium demonstrate peripheral enhancement of the lesion (arrows).

1	FIGURE 441e-5 Histoplasmosis of the pons (Chap. 236). Axial FLAIR (A) and T2-weighted (B) MRIs demonstrate a low signal mass in the right pons (arrows) with surrounding vasogenic edema. Axial T1-weighted MRI postgadolinium (C) demonstrates ring enhancement of the lesion in the right pons (arrow). Of note, there was no evidence of restricted diffusion (not shown). FIGURE 441e-6 Coccidiomycosis meningitis (Chap. 237). Axial postcontrast CT (A) and axial (B) and coronal (C) T1-weighted MRIs postgadolinium demonstrate enhancement of the perimesencephalic cisterns (arrows), as well as the sylvian and interhemispheric fissures.

1	FIGURE 441e-7 Candidiasis in a newborn (Chap. 240). Axial T2-weighted MRI (A) demonstrates multiple punctate foci of low signal diffusely distributed in the brain parenchyma (arrowhead). Axial T1-weighted MRIs postgadolinium (B, C) demonstrate marked enhancement of the lesions (arrowheads). Apparent diffusion coefficient (ADC) map (D, E) demonstrates restricted diffusion of water molecules in the lesions (arrowheads).

1	FIGURE 441e-8 Central nervous system (CNS) aspergillosis (Chap. 241). Axial FLAIR MRIs (A, B) demonstrate multiple areas of abnormal high signal in the basal ganglia as well as cortex and subcortical white matter (arrows). There is also abnormal high signal in the subarachnoid space adjacent to the lesions (arrowheads) that can correspond to blood or high protein content. Axial T2-weighted MRIs (C, D) demonstrate intrinsic low signal in the lesions (arrows), suggesting the presence of blood products. Some of the lesions also show vasogenic edema. Coronal (E) and axial (F) T1-weighted MRIs postgadolinium demonstrate peripheral enhancement of the lesions (arrows). FIGURE 441e-9 Invasive sinonasal aspergillosis (Chap. 241). Axial T2-weighted MRI (A) demonstrates an irregularly shaped low signal lesion involving the left orbital apex (arrow). B. T1-weighted image pregadolinium demonstrates low signal in left anterior clinoid process (arrow).

1	C. T1-weighted image postgadolinium demonstrates enhancement of lesion (arrow). FIGURE 441e-10 Behçet’s disease (Chap. 387). Axial FLAIR MRI demonstrates abnormal high signal involving the anterior pons (arrow); following gadolinium administration, the lesion was nonenhancing (not shown). Brainstem lesions are typical of Behçet’s disease, caused primarily by vasculitis and in some cases demyelinating lesions. FIGURE 441e-11 Neurosarcoid (Chap. 390): Case I. Coronal (A) and axial (B) T1-weighted images postgadolinium with fat suppression demonstrate a homogeneously enhancing well-circumscribed mass centered in the left Meckel’s cave (arrows). FIGURE 441e-12 Neurosarcoid (Chap. 390): Case II. Axial (A, B) and sagittal (C) T1-weighted images postgadolinium with fat suppression demonstrate a homogeneously enhancing mass involving the hypothalamus and the pituitary stalk (arrows).

1	FIGURE 441e-13 Neurosarcoid (Chap. 390): Case III. Axial FLAIR images (A–E) demonstrate abnormal high signal and slight expansion in the midbrain, dorsal pons, and pineal region (arrows) without significant mass effect. Sagittal T1-weighted images postgadolinium (F) with fat suppression demonstrate abnormal enhancement in the midbrain, dorsal pons, and pineal region (arrows). FIGURE 441e-14 Neurosarcoid (Chap. 390): Case IV. Axial T2-weighted images (A–D) demonstrate numerous areas of abnormal hyperintensity involving the corpus callosum, left internal capsule and globus pallidus, bilateral cerebral peduncles, bilateral gyrus rectus, right frontal lobe periventricular white matter, and patchy areas in bilateral temporal lobes. T1-weighted images postgadolinium (E–H) demonstrate abnormal enhancement of those areas with high T2 signal.

1	FIGURE 441e-15 Histiocytosis (Chap. 404). Sagittal T1-weighted image (A) demonstrates enlargement of the pituitary stalk (arrow) and absence of the posterior pituitary intrinsic T1 hyperintensity (arrowhead). Sagittal and coronal T1-weighted images postgadolinium (B, C) demonstrate enhancement of the pituitary stalk and infundibulum (arrows). FIGURE 441e-16 Middle cerebral artery stenosis (Chap. 446). Time-of-flight (TOF) MR angiography (MRA) (A, B) reveals narrowing within the left M1 segment that is likely secondary to atherosclerosis (arrows).

1	FIGURE 441e-17 Lacunar infarction (Chap. 446). Axial noncontrast CT (A) demonstrates abnormal hypodensity involving the left anterior putamen and anterior limb of internal capsule with ex-vacuo dilatation of the adjacent frontal horn of the left lateral ventricle, suggestive of an old infarction (arrow). A small area of slight hypodensity is also seen in the posterior limb of the right internal capsule that can correspond to an acute infarct (arrowhead). Axial FLAIR MRI (B) demonstrates abnormal high signal involving the left anterior putamen and anterior limb of internal capsule with ex-vacuo dilatation of the adjacent frontal horn of the left lateral ventricle, suggestive of an old infarction (arrow). A small area of slight hyperintensity is also seen in the posterior limb of the right internal capsule that can correspond to an acute lacunar infarct (arrowhead). Diffusion-weighted image (C) and apparent diffusion coefficient (ADC) map (D) demonstrate restricted water motion in the

1	internal capsule that can correspond to an acute lacunar infarct (arrowhead). Diffusion-weighted image (C) and apparent diffusion coefficient (ADC) map (D) demonstrate restricted water motion in the lesion of the posterior limb of the right internal capsule, strongly suggestive for an acute lacunar infarct (arrowhead). There is no evidence of restricted diffusion in the old infarct (arrow).

1	FIGURE 441e-18 Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) (Chap. 446). Axial T2-weighted MRIs (A, B) demonstrate multiple patchy areas of abnormal high signal in the periventricular white matter (arrows). Coronal FLAIR MRI (C, D) demonstrates multiple patchy areas of abnormal high signal in the periventricular white matter bilaterally, including the temporal lobes (arrows). In some of these areas, there are small areas of tissue loss (encephalomalacia) (arrowheads).

1	FIGURE 441e-19 CNS vasculitis (Chap. 446). Axial noncontrast CT (A) demonstrates a large hyperdense intraparenchymal hematoma surrounded by hypodense vasogenic edema in the right parietal lobe. Axial T2-weighted MRI (B) demonstrates a large hypointense intraparenchymal hematoma surrounded by hyperintense vasogenic edema in the right parietal lobe. Conventional angiography (C) demonstrates multiple segments of intracranial arterial narrowing, some of which have associated adjacent areas of focal arterial dilatation. These abnormalities are suggestive of vasculitis.

1	FIGURE 441e-20 Superior sagittal sinus thrombosis (Chap. 446). Noncontrast CT of the head (A) demonstrates increased density in the superior sagittal sinus, suggestive of thrombosis (arrow), and small linear hyperdensities in some temporal lobe sulci, suggestive of subarachnoid hemorrhage (arrowheads). Axial T1-weighted MRI (B) demonstrates absence of flow void in the superior sagittal sinus, suggestive of thrombosis. Coronal FLAIR images (C, D) demonstrate areas of abnormal high signal involving the gray and the subcortical white matter of the right frontal and left parietal lobes, as well as the adjacent sulci. These findings are suggestive of vasogenic edema with subarachnoid hemorrhage (arrowheads). Diffusion-weighted images (E, F) and ADC maps (G, H) demonstrate restricted diffusion of the abnormal areas on FLAIR, suggestive of infarct. Phase-contrast venography of the brain (I) demonstrates absence of signal in the superior sagittal sinus down to the torcular herophili, and left

1	the abnormal areas on FLAIR, suggestive of infarct. Phase-contrast venography of the brain (I) demonstrates absence of signal in the superior sagittal sinus down to the torcular herophili, and left transverse sinus and jugular vein. Axial (J) and coronal (K) T1-weighted images postgadolinium demonstrate a filling defect in the superior sagittal sinus, suggestive of thrombosis.

1	CHAPTER 441e Atlas of Neuroimaging FIGURE 441e-21 Multiple system atrophy (Chap. 449). Axial T2-weighted MRI (A) reveals symmetric poorly circumscribed abnormal high signal in the middle cerebellar peduncles bilaterally (arrowheads). Sagittal T1-weighted MRI (B) demonstrates pontine atrophy and enlarged cerebellar fissures as a result of cerebellar atrophy (arrows).

1	FIGURE 441e-22 Huntington’s disease (Chap. 449). Axial noncontrast CT (A) demonstrates symmetric bilateral severe atrophy involving the caudate nuclei, putamen, and globus pallidi bilaterally with consequent enlargement of the frontal horns of the lateral ventricles (arrows). There is also diffuse prominence of the sulci indicating generalized cortical atrophy. Axial (B) and coronal (C) FLAIR images demonstrate bilateral symmetric abnormal high signal in the caudate and putamen. Coronal T1-weighted image (D) demonstrates enlarged frontal horns with abnormal configuration. Also note diffusely decreased marrow signal, which could represent anemia or myeloproliferative disease.

1	FIGURE 441e-23 Bell’s palsy (Chap. 455). Axial T1-weighted images postgadolinium with fat suppression (A–C) demonstrate diffuse smooth linear enhancement along the left facial nerve, involving the second and third segments (genu, tympanic, and mastoid) within the temporal bone (arrows). Note that there is no evidence of a mass lesion. A potential pitfall for facial nerve enhancement in the stylomastoid foramen is the enhancement of the stylomastoid artery that enters the foramen and supplies the tympanic cavity, the tympanic antrum, mastoid cells, and the semicircular canals. Coronal T1-weighted images postgadolinium with fat suppression (D, E) demonstrate the course of the enhancing facial nerve (arrows). Although these findings are highly suggestive of Bell’s palsy, the diagnosis is established on clinical grounds.

1	FIGURE 441e-24 Spinal cord infarction (Chap. 456). Sagittal T2-weighted MRI of the lumbar spine (A) demonstrates poorly defined areas of abnormal high signal in the conus medullaris and mild cord expansion (arrow). T1-weighted MRI of the lumbar spine postgadolinium (B) demonstrates mild enhancement (arrow). Sagittal diffusion-weighted MRI of the lumbar spine (C) demonstrates restricted diffusion (arrow) in the areas of abnormal high signal on the T2-weighted image (A). FIGURE 441e-25 Acute transverse myelitis (Chap. 456). Sagittal T2-weighted MRI (A) demonstrates abnormal high signal in the cervical cord extending from C1 to T1 with associated cord expansion (arrows). Sagittal T1-weighted MRI postgadolinium (B) demonstrates abnormal enhancement in the posterior half of the cord from C2 to T1 (arrows).

1	FIGURE 441e-26 Acute disseminated encephalomyelitis (ADEM) (Chap. 458). Axial T2-weighted (A) and coronal FLAIR (B) images demonstrate abnormal areas of high signal involving predominantly the subcortical white matter of the frontal lobe bilaterally and left caudate head. Following administration of gadolinium, corresponding axial (C) and coronal (D) T1-weighted images demonstrate irregular enhancement consistent with blood-brain barrier breakdown and inflammation; some lesions show incomplete rim enhancement, typical for demyelination.

1	FIGURE 441e-27 Bals concentric sclerosis (a variant of multiple sclerosis) (Chap. 458). Coronal FLAIR MRI (A) demonstrates multiple areas of abnormal high signal in the supratentorial white matter bilaterally. The lesions are ovoid in shape, perpendicular to the orientation of the lateral ventricles, and with little mass effect. Axial (B) and sagittal (C–E) T2-weighted MRIs demonstrate multiple areas of abnormal high signal in the supratentorial white matter bilaterally, as well as the involvement of the body and splenium of the corpus callosum and the callosal-septal interface (arrowhead). Some of the lesions reveal concentric layers, typical of Bals concentric sclerosis (arrows). Sagittal (F) and axial (G, H) T1-weighted MRIs postgadolinium demonstrate abnormal enhancement of all lesions with some of the lesions demonstrating concentric ring enhancement (arrows).

1	FIGURE 441e-28 Hashimoto’s encephalopathy (Chap. 164). Axial FLAIR (A) demonstrates focal area of abnormal high signal involving the gray and white matter in the left frontal lobe. There is also a small area of abnormal high signal in the precentral gyrus. Axial T1-weighted images (B, C) pre and postgadolinium demonstrate cortical/pial enhancement in the region of high signal on FLAIR. FIGURE 441e-29 Brachial plexopathy (Chap. 459). Axial (A), sagittal (B), and coronal (C, D) short tau inversion recovery (STIR) MRIs demonstrate abnormal enlargement and abnormal high signal involving the right C6, C7, and C8 nerve roots, and the trunks and divisions that originate from these roots (arrows). Diffusion-weighted MRI (E) demonstrates abnormal reduced diffusion within the right C6, C7, and C8 nerve roots and their corresponding trunks and divisions (arrow). These findings are compatible with radiation-induced brachial plexopathy. CHAPTER 441e Atlas of Neuroimaging

1	CHAPTER 441e Atlas of Neuroimaging FIGURE 441e-31 CT facet fracture. Axial CT demonstrates fracture line along the C2 facet (arrow). FIGURE 441e-32 Compression fracture. Sagittal T2-weighted MRI demonstrates compression fracture of C7 (∗) and high signal within the spinous processes of C6-C7 (arrows) and to lesser degree C5-C6. This is suggestive of interspinous ligament injury. Note the pad under the patient’s neck to maintain neck alignment during the scanning time. FIGURE 441e-30 Anterior dens dislocation. Sagittal CT demonstrates the tip of the dens below the anterior arch of C2 (arrow), indicating anterior dislocation.

1	FIGURE 441e-30 Anterior dens dislocation. Sagittal CT demonstrates the tip of the dens below the anterior arch of C2 (arrow), indicating anterior dislocation. FIGURE 441e-33 Epidural hematoma. Axial noncontrast CT (A) demonstrates a high-density epidural collection in the cervical spine (∗), which is consistent with acute hemorrhage. Also noted is mass effect on the spinal cord (arrowheads). Sagittal reformatted CT image (B) demonstrates the extension of the acute epidural hematoma (∗) and a disk bulge (arrowhead), which further contributes to spinal canal narrowing. CT is the imaging procedure of choice to detect acute hematoma.

1	FIGURE 441e-34 Retropharyngeal soft tissue mass. Sagittal T1-weighted MRI demonstrates a hyperflexion fracture with retropulsion of the posterior wall in the canal at C5 and C6 (arrow). There is also a large retropharyngeal hematoma (∗). The distance from the posterior wall of the airway to the anterior wall of the vertebral body should not measure more than 6 mm at C2 or more than 20 mm at C6 (mnemonic “6 at 2 and 20 at 6”). FIGURE 441e-35 Jefferson fracture. Axial CT demonstrates four fracture lines (arrows) separating C1 in four parts. Jefferson fracture is usually caused by axial impact to the head such as diving in shallow water. FIGURE 441e-36 Ligament injury after trauma. Coronal CT reconstruction demonstrates abnormal asymmetry between the dens and the lateral masses of C1 indicating transverse ligament rupture.

1	FIGURE 441e-36 Ligament injury after trauma. Coronal CT reconstruction demonstrates abnormal asymmetry between the dens and the lateral masses of C1 indicating transverse ligament rupture. FIGURE 441e-38 Pathologic fracture. Sagittal T1-weighted MRI (A) demonstrates wedge-shaped T6 vertebral body (arrow). Sagittal postcontrast T1-weighted MRI (B) depicts tumor extension into the epidural space and the involvement of the posterior arch (∗), which are highly suggestive of metastatic or primary bone tumor. CHAPTER 441e Atlas of Neuroimaging FIGURE 441e-39 Sacral insufficiency fracture. Axial T2-weighted MRI (A) and T1-weighted MRI (B) demonstrate symmetric high T2 and low T1 signal involving the sacral alae longitudinally (arrows). FIGURE 441e-37 Odontoid fracture. Sagittal CT demonstrates disruption of the main reference cervical lines. 1: Anterior vertebral body line; 2: Posterior vertebral body line; 3: Spinolaminar line.

1	FIGURE 441e-37 Odontoid fracture. Sagittal CT demonstrates disruption of the main reference cervical lines. 1: Anterior vertebral body line; 2: Posterior vertebral body line; 3: Spinolaminar line. FIGURE 441e-40 Subdural hematoma. Sagittal T2-weighted MRI (A) and axial noncontrast T1-weighted MRI (B) demonstrate subdural collection in the lumbosacral region (∗∗). Note that the epidural fat is compressed but not involved (arrow). FIGURE 441e-41 Teardrop fracture. Sagittal CT (A) demonstrates fracture line separating the anteroinferior corner of C6 (arrow). Sagittal T2-weighted MRI (B) displays cord injury (arrow).

1	FIGURE 441e-41 Teardrop fracture. Sagittal CT (A) demonstrates fracture line separating the anteroinferior corner of C6 (arrow). Sagittal T2-weighted MRI (B) displays cord injury (arrow). FIGURE 441e-42 Demyelinating disease (multiple sclerosis, Chap. 458). Axial T2-weighted MRI (A, D) and axial T2 FLAIR MRI (B, E) demonstrate multiple hyperintense lesions involving the periventricular and subcortical white matter (arrows). Although not always present, the appearance of a “lesion within and lesion” (arrowheads) is typical of demyelinating disease. Axial T1-weighted postcontrast MRI (C, F) shows partial enhancement of the lesion (arrows), which is often peripheral, incomplete, and “C-shaped” (curved arrow). CHAPTER 441e Atlas of Neuroimaging

1	CHAPTER 441e Atlas of Neuroimaging FIGURE 441e-43 Neurofibromatosis type 1 (Chap. 118). Axial T2 FLAIR MRI (A, B) demonstrates multiple hyperintense lesions involving the brainstem and basal ganglia (arrows) as well as deep cerebellar hemispheres (arrowheads). Sagittal and corona T1-weighted postcontrast MRI (C, D) shows enlargement of the optic chiasm with an area of enhancement on the left, representing an optic pathway glioma (arrows). Coronal STIR MRI (E) shows thoracolumbar scoliosis and a large paravertebral plexiform neurofibroma (arrows).

1	FIGURE 441e-44 Neurofibromatosis type 2 (Chap. 118). Axial T1-weighted postcontrast MRI (A, B) shows enhancing expansible lesions in the bilateral cerebellopontine cisterns extending in the internal auditory canals, consistent with vestibular schwannomas (arrows), as well as in the bilateral prepontine cistern, consistent with trigeminal schwannomas (arrowheads). Coronal axial T1-weighted image postgadolinium (C ) demonstrates an intensely enhancing dural-based lesion typical for a small meningioma (arrows). Sagittal (D, E) T1-weighted images postgadolinium show intradural, extramedullary lesions, suggestive of multiple spinal schwannomas (small arrows). The flat dural-based lesion may represent a spinal meningioma (arrowhead). Axial T1-weighted image postgadolinium (F ) shows an enhancing intramedullary lesion, most consistent with an ependymoma. (curved arrow).

1	FIGURE 441e-45 Tuberous sclerosis (Chap. 118). Coronal T2-weighted MRI (A) shows multiple T2 hyperintense lesions in a cortical and subcortical distribution (arrows). Coronal and axial postcontrast T1-weighted image (B, C) demonstrates an expanding nodule with intense enhancement in the proximity of the right foramen of Monro, consistent with a subependymal giant-cell astrocytoma (SEGA) (arrowheads). Surveillance T1-weighted image (D) and postcontrast T1-weighted image with fat saturation (E) show multiple bilateral renal lesions with signal intensity of fat, consistent with angiomyolipomas (small arrows).

1	FIGURE 441e-46 Von Hippel–Lindau (VHL) (Chap. 408). Axial postcontrast T1 weighted images (A–C) demonstrates multiple enhancing nodules in the posterior fossa (arrows). Sagittal postcontrast T1-weighted image (D) shows vascular flow voids within the enhancing nodule in the region of the foramen of Magendie (arrow), indicating increased vascularity. Surveillance axial T2-weighted MRI of the abdomen (E) shows multiple small pancreatic cysts (arrowheads). This patient did not have an endolymphatic sac tumor, renal cell carcinoma, neuroendocrine pancreatic tumor, or pheochromocytoma, all of which may also occur in von Hippel–Lindau disease. CHAPTER 441e Atlas of Neuroimaging

1	CHAPTER 441e Atlas of Neuroimaging FIGURE 441e-47 Neurocutaneous melanosis. Coronal T1 weighted MRI (A–D) shows multiple lesions with intrinsic increased T1 signal in the bilateral amygdalae, right superior temporal gyrus, right cerebellar hemisphere, and right medial occipital cortex (arrows). Sagittal and axial T1-weighted images of the spine (E, F) show intradural, extramedullary lesions, also with intrinsic increased T1 signal, due to malignant melanoma (arrows). FIGURE 441e-48 Sturge-Weber syndrome. Coronal T1-weighted MRI (A) shows enlargement of the sulci in the left parietal lobe, consistent with brain parenchymal volume loss (arrows). Axial susceptibility weighted imaging shows susceptibility effect in this region, consistent with calcifications (arrows). Coronal and axial T1-weighted images postgadolinium (C, D) show increased leptomeningeal enhancement (arrows) and enlargement of the left choroid plexus (curved arrow).

1	FIGURE 441e-49 Multiple cavernomas (Chap. 446). Axial susceptibility weighted images (A–D) show multiple foci of susceptibility involving the bilateral cerebral hemispheres, pons, and left cerebellum, which in a young patient most likely represents multiple cavernomas (arrows). These lesions have variable signal intensity on T2-weighted (E) and T1-weighted MRI (F), related to the different stages of hemoglobin degradation (arrows). Cavernomas are not seen on the time-of-flight MR angiography (G); thus these are designated as angiographically occult vascular malformations. Of note, in elderly patients, amyloid angiopathy may have a similar appearance. CHAPTER 441e Atlas of Neuroimaging

1	CHAPTER 441e Atlas of Neuroimaging FIGURE 441e-50 Brainstem glioma (Chap. 118). Axial T2 FLAIR MRI (A) demonstrates increased T2 signal and marked enlargement of the pons (large arrows), which engulfs the basilar artery (small arrow). These findings are characteristic of brainstem glioma. At the time of diagnosis, these lesions are typically low grade without abnormal enhancement, as shown by axial postcontrast T1-weighted image (B). FIGURE 441e-51 Pilocytic astrocytoma (Chap. 118). Axial T2-weighted and T1-weighted postgadolinium images (A, B) show a cystic lesion with peripheral enhancement and an enhancing solid component located in the posterior fossa (arrows). These findings are suggestive of a pilocytic astrocytoma. Note that the lesion exerts mass effect on the fourth ventricle, which is compressed (curved arrows).

1	FIGURE 441e-52 Fourth ventricle ependymoma causing hydrocephalus (Chap. 118). Unenhanced axial CT images (A, B) demonstrate an expansile mass lesion, isodense with brain parenchyma, filling the fourth ventricle (arrows) and causing obstruction of cerebrospinal fluid flow, dilation of the lateral ventricles, and hydrocephalus (curved arrows). The hypoattenuation within periventricular white matter bilaterally represents transependymal flow (arrowheads). Axial T1-weighted postgadolinium MRI confirms the presence of a heterogeneously enhancing mass filling the fourth ventricle (arrow), which is suggestive of ependymoma.

1	FIGURE 441e-53 Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) (Chap. 462e). Axial T2 FLAIR MRI (A, B) shows areas of increased T2 signal involving the cortex and subcortical white matter in the posterior right frontal lobe and anterior left temporal lobe, consistent with edema (arrows). Axial diffusion-weighted images (C, D) show reduced diffusion from cytotoxic edema, consistent with infarcts (arrows). MR spectroscopy of the right frontal lesion (E) demonstrates markedly increased lactate (arrow), an expected finding in infarction regardless of etiology. However, MR spectroscopy of the normal-appearing contralateral brain parenchyma (F) shows mildly elevated lactate (arrow), which is suggestive of a mitochondrial disorder.

1	FIGURE 441e-54 Leigh’s disease (subacute necrotizing encephalomyelopathy) (Chap. 85e). Axial T2-weighted MRI (A, B) demonstrates increased T2 signal involving the substantia nigra (white arrows) and dorsal midbrain (black arrows), as well as the putamin bilaterally (curved arrows). This is a common pattern for the mitochondrial disorder Leigh’s disease secondary to cytochrome oxidase (CO IV) deficiency. CHAPTER 441e Atlas of Neuroimaging FIGURE 441e-55 Krabbe’s disease (Chap. 432e). Axial and coronal T2-weighted MRIs (A, B) show increased T2 signal involving predominantly the posterior white matter bilaterally (arrows) with sparing of the subcortical U-fibers (arrowheads). MR spectroscopy of the left parietal white matter (C) shows markedly decreased N-acetylaspartate (large arrow) and increased lactate/lipids (small arrow), consistent with severe neuronal injury.

1	FIGURE 441e-56 X-linked adrenoleukodystrophy (Chap. 459). Axial unenhanced CT (A) demonstrates areas of attenuation involving the posterior white matter bilaterally (arrows). Axial T2 FLAIR MRI (B) displays increased T2 signal consistent with edema (arrows). Axial T1-weighted image postgadolinium (C) shows peripheral enhancement of the parietal lesions bilaterally (arrows). These findings are typical of adrenoleukodystrophy.

1	FIGURE 441e-57 Sickle cell disease and moyamoya disease (Chap. 446). Axial T2-weighted MRI (A, B) shows multiple small areas of encephalomalacia from prior infarcts in the watershed zones between the anterior and middle cerebral artery territories (small arrows). There is also an area of edema involving the left basal ganglia from an evolving subacute infarct (arrow). An axial diffusion-weighted image (C) with corresponding ADC map (D) shows an area of restricted diffusion in the right frontoparietal region, consistent with an acute infarct (arrows). Time-of-flight MR angiography (E) shows absence of flow in the distal internal carotid arteries and proximal middle cerebral arteries (arrows) due to moyamoya disease. Also note that this patient is status post–bilateral encephalo-duro-arterio-synangiosis (EDAS) (arrowheads), a surgical procedure to create indirect anastomosis between branches of the external carotid artery with distal branches of the middle cerebral artery.

1	FIGURE 441e-58 Hepatic encephalopathy (Chap. 330). Axial T1-weighted MRI (A, B) shows increased intrinsic T1 signal involving the basal ganglia bilaterally, particularly the globus pallidus (arrows). FIGURE 441e-59 Guillain-Barré syndrome (Chap. 460). Axial precontrast T1-weighted MRI (A) and axial and sagittal T1-weighted postgadolinium (B, C) images show thickening and increased enhancement of the anterior roots of the cauda equina (arrows). FIGURE 441e-60 Hemiplegic migraine (Chap. 447). Axial noncontrast perfusion MRI using an arterial spin labeling technique (A) demonstrates decreased cerebral blood flow to the left hemisphere (arrows) in a patient with right hemiparesis associated with migraine symptoms. No abnormality was seen on T2-weighted images, diffusion-weighted images, or time-of-flight MR angiography (B–D) to suggest stroke. CHAPTER 441e Atlas of Neuroimaging

1	CHAPTER 441e Atlas of Neuroimaging Electrodiagnostic Studies of Nervous System Disorders: EEG, Evoked Potentials, and EMG Michael J. Aminoff ELECTROENCEPHALOGRAPHY 442e

1	The electrical activity of the brain (the electroencephalogram [EEG]) is easily recorded from electrodes placed on the scalp. The potential difference between pairs of electrodes on the scalp (bipolar derivation) or between individual scalp electrodes and a relatively inactive common reference point (referential derivation) is amplified and displayed on a computer monitor, oscilloscope, or paper. Digital systems allow the EEG to be reconstructed and displayed with any desired format and to be manipulated for more detailed analysis and also permit computerized techniques to be used to detect certain abnormalities. The characteristics of the normal EEG depend on the patient’s age and level of arousal. The rhythmic activity normally recorded represents the postsynaptic potentials of vertically oriented pyramidal cells of the cerebral cortex and is characterized by its frequency. In normal awake adults lying quietly with the eyes closed, an 8to 13-Hz alpha rhythm is seen posteriorly in

1	oriented pyramidal cells of the cerebral cortex and is characterized by its frequency. In normal awake adults lying quietly with the eyes closed, an 8to 13-Hz alpha rhythm is seen posteriorly in the EEG, intermixed with a variable amount of generalized faster (beta) activity (>13 Hz); the alpha rhythm is attenuated when the eyes are opened (Fig. 442e-1). During drowsiness, the alpha rhythm is also attenuated; with light sleep, slower activity in the theta (4–7 Hz) and delta (<4 Hz) ranges becomes more conspicuous.

1	Activating procedures are generally undertaken while the EEG is 442e-1 recorded in an attempt to provoke abnormalities. Such procedures commonly include hyperventilation (for 3 or 4 min), photic stimulation, sleep, and sleep deprivation on the night prior to the recording. Electroencephalography is relatively inexpensive and may aid clinical management in several different contexts.

1	The EEG is most useful in evaluating patients with suspected epilepsy. The presence of electrographic seizure activity—i.e., of abnormal, repetitive, rhythmic activity having an abrupt onset and termination and a characteristic evolution—clearly establishes the diagnosis. The absence of such electrocerebral accompaniment to an episodic behavioral disturbance does not exclude a seizure disorder, however, because there may be no changes in the scalp-recorded EEG during certain focal seizures. With generalized tonic-clonic seizures, the EEG is always abnormal during the episode. It is often not possible to obtain an EEG during clinical events that may represent seizures, especially when such events occur unpredictably or infrequently. Continuous monitoring for prolonged periods in video-EEG telemetry units has made it easier to capture the electrocerebral accompaniments of such clinical episodes. Monitoring by these means is sometimes helpful in confirming that seizures are occurring,

1	telemetry units has made it easier to capture the electrocerebral accompaniments of such clinical episodes. Monitoring by these means is sometimes helpful in confirming that seizures are occurring, characterizing the nature of clinically equivocal episodes, and determining the frequency of epileptic events.

1	The EEG findings in the interictal period may show certain abnormalities that are strongly supportive of a diagnosis of epilepsy. Such epileptiform activity consists of bursts of abnormal discharges containing spikes or sharp waves. The presence of epileptiform activity is not specific for epilepsy, but it has a much greater prevalence in epileptic patients than in normal individuals. However, even in individuals with epilepsy, the initial routine interictal EEG may be normal up to 60% of CHAPTER 442e Electrodiagnostic Studies of Nervous System Disorders: EEG, Evoked Potentials, and EMG

1	FIGuRE 442e-1 A. Normal electroencephalogram (EEG) showing a posteriorly situated 9-Hz alpha rhythm that attenuates with eye opening. B. Abnormal EEG showing irregular diffuse slow activity in an obtunded patient with encephalitis. C. Irregular slow activity in the right central region, on a diffusely slowed background, in a patient with a right parietal glioma. D. Periodic complexes occurring once every second in a patient with Creutzfeldt-Jakob disease. Horizontal calibration: 1 s; vertical calibration: 200 μV in A, 300 μV in other panels. In this and the following figure, electrode placements are indicated at the left of each panel and accord with the international 10:20 system. A, earlobe; C, central; F, frontal; Fp, frontal polar; P, parietal; T, temporal; O, occipital. Right-sided placements are indicated by even numbers, left-sided placements by odd numbers, and midline placements by Z. (From MJ Aminoff [ed]: Aminoff’s Electrodiagnosis in Clinical Neurology, 6th ed. Oxford,

1	placements are indicated by even numbers, left-sided placements by odd numbers, and midline placements by Z. (From MJ Aminoff [ed]: Aminoff’s Electrodiagnosis in Clinical Neurology, 6th ed. Oxford, Elsevier Saunders, 2012.) the time. Thus, the EEG cannot establish the diagnosis of epilepsy in many cases.

1	The EEG findings have been used in classifying seizure disorders and selecting appropriate anticonvulsant medication for individual patients (Fig. 442e-2). The episodic generalized spike-wave activity that occurs during and between seizures in patients with typical absence epilepsy contrasts with focal interictal epileptiform discharges or ictal patterns found in patients with focal seizures. These latter seizures may have no correlates in the scalp-recorded EEG or may be associated with abnormal rhythmic activity of variable frequency, a localized or generalized distribution, and a stereotyped pattern that varies with the patient. Focal or lateralized epileptogenic lesions are important to recognize, especially if surgical treatment is contemplated. Intensive long-term monitoring of clinical behavior and the EEG is required for operative candidates, however, and this generally also involves recording from intracranial (subdural, extradural, or intracerebral) electrodes.

1	The EEG findings may indicate the prognosis of seizure disorders: In general, a normal EEG implies a better prognosis than otherwise, whereas an abnormal background or profuse epileptiform activity suggests a poor outlook. The EEG findings are not helpful in determining which patients with head injuries, stroke, or brain tumors will go on to develop seizures, because in such circumstances epileptiform activity is FIGuRE 442e-2 Electrographic seizures. A. Onset of a tonic seizure showing generalized repetitive sharp activity with synchronous onset over both hemispheres. B. Burst of repetitive spikes occurring with sudden onset in the right temporal region during a clinical spell characterized by transient impairment of external awareness.

1	C. Generalized 3-Hz spike-wave activity occurring synchronously over both hemispheres during an absence (petit mal) attack. Horizontal calibration: 1 s; vertical calibration: 400 µV in A, 200 µV in B, and 750 µV in C. (From MJ Aminoff [ed]: Aminoff’s Electrodiagnosis in Clinical Neurology, 6th ed. Oxford, Elsevier Saunders, 2012.) commonly encountered regardless of whether seizures occur. The EEG findings are of limited utility in determining whether anticonvulsant medication can be discontinued after several seizure-free years. Further seizures may occur after withdrawal of anticonvulsant medication despite a normal EEG or, conversely, may not occur despite a continuing EEG abnormality. The decision to discontinue anticonvulsant medication is made on clinical grounds, and the EEG is helpful only for providing guidance when there is clinical ambiguity or the patient requires reassurance about a particular course of action.

1	The EEG has no role in the management of tonic-clonic status epilepticus except when there is clinical uncertainty about whether seizures are continuing in a comatose patient. In patients treated by drug-induced coma for refractory status epilepticus, the EEG findings indicate the level of anesthesia and whether seizures are occurring. During status epilepticus, the EEG shows repeated electrographic seizures or continuous spike-wave discharges. In nonconvulsive status epilepticus, a disorder that may not be recognized unless an EEG is performed, the EEG may also show continuous spike-wave activity (“spike-wave stupor”) or, less commonly, repetitive electrographic seizures (focal status epilepticus).

1	The EEG tends to become slower as consciousness is depressed, regardless of the underlying cause (Fig. 442e-1). Other findings may suggest diagnostic possibilities, as when electrographic seizures are found or a focal abnormality indicates a structural lesion. The EEG generally slows in metabolic encephalopathies, and triphasic waves may be present. The findings do not permit differentiation of the underlying metabolic disturbance but help to exclude other encephalopathic processes by indicating the diffuse extent of cerebral dysfunction. An EEG responsive to external stimulation is helpful prognostically because electrocerebral responsiveness implies a lighter level of coma than a nonreactive EEG, and thus a better prognosis. Serial records provide a better guide to prognosis than a single record and supplement the clinical examination in following the course of events. As the depth of coma increases, the EEG becomes nonreactive and may show a burst-suppression pattern, with bursts

1	record and supplement the clinical examination in following the course of events. As the depth of coma increases, the EEG becomes nonreactive and may show a burst-suppression pattern, with bursts of mixed-frequency activity separated by intervals of relative cerebral inactivity. In other instances there is a reduction in amplitude of the EEG until eventually activity cannot be detected. Such electrocerebral silence does not necessarily reflect irreversible brain damage, because it may occur reversibly in hypothermic patients or with drug overdose. The prognosis of electrocerebral silence, when recorded using an adequate technique, therefore depends on the clinical context in which it is found. In patients with severe cerebral anoxia, for example, electrocerebral silence in a technically satisfactory record implies that useful cognitive recovery will not occur.

1	In patients with clinically suspected brain death, an EEG recorded using appropriate technical standards may be confirmatory by showing electrocerebral silence, but disorders that may produce a similar but reversible EEG appearance must be excluded. The presence of residual EEG activity in suspected brain death fails to confirm the diagnosis but does not exclude it. The EEG is usually normal in patients with locked-in syndrome (Chap. 446), and helps in distinguishing this disorder from the comatose state with which it is sometimes confused clinically.

1	In developed countries, computed tomography (CT) scanning and magnetic resonance imaging (MRI) are used as a noninvasive means of screening for focal structural abnormalities of the brain, such as tumors, infarcts, or hematomas (Fig. 442e-1). The EEG is still used for this purpose in many parts of the world, however, although infratentorial or slowly expanding lesions may not be recognized. Focal slow-wave disturbances, a localized loss of electrocerebral activity, or more generalized electrocerebral disturbances are common findings but do not indicate the nature of the underlying pathology.

1	In patients with an acute encephalopathy, focal or lateralized periodic slow-wave complexes, sometimes with a sharpened outline, suggest a diagnosis of herpes simplex encephalitis, and periodic lateralizing epileptiform discharges (PLEDs) are commonly found with acute hemispheric pathology such as a hematoma, abscess, or rapidly expanding tumor. The EEG findings in dementia are usually nonspecific and do not distinguish reliably between different underlying causes except in rare instances when the presence of complexes occurring with a regular repetition rate (“periodic complexes”) supports a diagnosis of Creutzfeldt-Jakob disease (Fig. 442e-1) or subacute sclerosing panencephalitis. In most patients with dementia, the EEG is normal or diffusely slowed, and the findings alone cannot indicate whether a patient is demented or distinguish between dementia and pseudodementia.

1	The brief EEG obtained routinely in the laboratory often fails to reveal abnormalities that are transient and infrequent. Continuous monitoring over 12 or 24 hours or longer may detect abnormalities or capture clinical events that otherwise would be missed. The EEG is often recorded continuously in critically ill patients to detect early changes in neurologic status. Continuous EEG recording in this context has been used to detect acute events such as from nonconvulsive seizures or developing cerebral ischemia, to monitor cerebral function in patients with metabolic disorders such as liver failure, and to manage the level of anesthesia in pharmacologically induced coma.

1	Recording the magnetic field of the electrical activity of the brain (magnetoencephalography [MEG]) provides a means of examining cerebral activity that is less subject to distortion by other biologic tissues than the EEG. MEG is used in only a few specialized centers because of the complexity and expense of the necessary equipment. It permits the source of activity to be localized and coregistered with the MRI in a technique that is known as magnetic source imaging. In patients with focal epilepsy, MEG is useful in localizing epileptogenic foci for surgery and for guiding the placement of intracranial electrodes for electrophysiologic monitoring. MEG has also been used for mapping brain tumors, identifying the central fissure preoperatively, and localizing functionally eloquent cortical areas such as those concerned with language.

1	The noninvasive recording of spinal or cerebral potentials elicited by stimulation of specific afferent pathways allows the functional integrity of these pathways to be monitored but does not indicate the pathologic basis of lesions involving them. Such evoked potentials (EPs) are small compared to the background EEG activity, and the responses to a number of stimuli are therefore recorded and averaged with a computer to permit their recognition and definition. The background EEG activity, which has no fixed temporal relationship to the stimulus, is averaged out by this procedure.

1	Visual evoked potentials (VEPs) are elicited by monocular stimulation with a reversing checkerboard pattern and are recorded from the occipital region in the midline and on either side of the scalp. The component of major clinical importance is the so-called P100 response, a positive peak having a latency of approximately 100 ms. Its presence, latency, and symmetry over the two sides of the scalp are noted. Amplitude changes are less helpful for the recognition of pathology. VEPs are most useful in detecting dysfunction of the visual pathways anterior to the optic chiasm. In acute severe optic neuritis, the P100 is frequently lost or grossly attenuated; as clinical recovery occurs, it is restored but with an increased latency that generally remains abnormal indefinitely. The VEP findings are therefore helpful in indicating previous or subclinical optic neuritis. They may also be abnormal with ocular abnormalities and with other causes of optic nerve disease, such as ischemia or

1	are therefore helpful in indicating previous or subclinical optic neuritis. They may also be abnormal with ocular abnormalities and with other causes of optic nerve disease, such as ischemia or compression by a tumor. Flash-elicited VEPs may be normal in patients with cortical blindness.

1	Routine VEPs record a mass response over a relatively large cortical area and thus may be insensitive to localized waveform abnormalities. A newer technique, multifocal VEP, measures responses from 120 individual sectors within each affected eye and is more sensitive than routine VEP. Brainstem auditory evoked potentials (BAEPs) are elicited by monaural stimulation with repetitive clicks and are recorded between the vertex of the scalp and the mastoid process or earlobe. A series of potentials, designated by roman numerals, occurs in the first 10 ms after the stimulus and represents in part the sequential activation of different structures in the pathway between the auditory nerve (wave

1	I) and the inferior colliculus (wave V) in the midbrain. The presence, latency, and interpeak latency of the first five positive potentials recorded at the vertex are evaluated. The findings are helpful in screening for acoustic neuromas, detecting brainstem pathology, and evaluating comatose patients. The BAEPs are often normal in coma due to metabolic/toxic disorders or bihemispheric disease but are typically abnormal in the presence of brainstem pathology. Somatosensory evoked potentials (SSEPs) are recorded over the scalp 442e-3 and spine in response to electrical stimulation of a peripheral (mixed or cutaneous) nerve. The configuration, polarity, and latency of the responses depend on the nerve that is stimulated and on the recording arrangements. SSEPs are used to evaluate proximal (otherwise inaccessible) portions of the peripheral nervous system and the integrity of the central somatosensory pathways, especially in patients who are comatose or suspected to be brain dead.

1	Clinical utility of EPs EP studies may detect and localize lesions in afferent pathways in the central nervous system (CNS). They have been used particularly to investigate patients with suspected multiple sclerosis (MS), the diagnosis of which requires the recognition of multifocal white-matter lesions. In patients with clinical evidence of a single lesion, the electrophysiologic recognition of abnormalities in other sites helps to support the diagnosis but does not establish it unequivocally. Multimodality EP abnormalities are not specific for MS; they may occur in AIDS, Lyme disease, systemic lupus erythematosus, neurosyphilis, spinocerebellar degenerations, familial spastic paraplegia, and deficiency of vitamin E or B12, among other disorders. The diagnostic utility of the EP findings therefore depends on the circumstances in which they are found. Abnormalities may aid in the localization of lesions to broad areas of the CNS, but attempts at precise localization may be misleading

1	therefore depends on the circumstances in which they are found. Abnormalities may aid in the localization of lesions to broad areas of the CNS, but attempts at precise localization may be misleading because the generators of many components are unknown.

1	The EP findings are sometimes of prognostic relevance. Bilateral loss of cortically generated SSEP components implies that cognition may not be regained in posttraumatic or postanoxic coma, and EP studies may also be useful in evaluating patients with suspected brain death. In patients who are comatose for uncertain reasons, preserved BAEPs suggest either a metabolic-toxic etiology or bihemispheric disease. In patients with spinal cord injuries, SSEPs have been used to indicate the completeness of the lesion. The presence or early return of a cortically generated response to stimulation of a nerve below the injured segment of the cord indicates an incomplete lesion and thus a better prognosis for functional recovery than otherwise. In surgery, intraoperative EP monitoring of neural structures placed at risk by the procedure may permit the early recognition of dysfunction and thereby permit a neurologic complication to be averted or minimized.

1	Visual and auditory acuity may be determined using EP techniques in patients whose age or mental state precludes traditional ophthalmologic or audiologic examinations.

1	Certain EP components depend on the mental attention of the subject and the setting in which the stimulus occurs, rather than simply on the physical characteristics of the stimulus. Such “event-related” potentials (ERPs) or “endogenous” potentials are related in some manner to the cognitive aspects of distinguishing an infrequently occurring target stimulus from other stimuli occurring more frequently. For clinical purposes, attention has been directed particularly at the so-called P3 component of the ERP, which is also designated the P300 component because of its positive polarity and latency of approximately 300–400 ms after onset of an auditory target stimulus. The P3 component is prolonged in latency in many patients with dementia, whereas it is generally normal in patients with depression or other disorders simulating dementia. ERPs are, therefore, sometimes helpful in making this distinction when there is clinical uncertainty, although a response of normal latency does not

1	depression or other disorders simulating dementia. ERPs are, therefore, sometimes helpful in making this distinction when there is clinical uncertainty, although a response of normal latency does not exclude dementia.

1	The electrical potentials recorded from muscle or the spinal cord following stimulation of the motor cortex or central motor pathways are referred to as motor evoked potentials. For clinical purposes, such responses are recorded most often as the compound muscle action potentials elicited by transcutaneous magnetic stimulation of the motor cortex. A strong but brief magnetic field is produced by passing a current through a coil, and this induces stimulating currents in the subjacent neural tissue. The procedure is painless and apparently safe. CHAPTER 442e Electrodiagnostic Studies of Nervous System Disorders: EEG, Evoked Potentials, and EMG

1	CHAPTER 442e Electrodiagnostic Studies of Nervous System Disorders: EEG, Evoked Potentials, and EMG Abnormalities have been described in several neurologic disorders with clinical or subclinical involvement of central motor pathways, including MS and motor neuron disease. In addition to a possible role in diagnosis or evaluating the extent of pathologic involvement, the technique provides information of prognostic relevance (e.g., in suggesting the likelihood of recovery of motor function after stroke) and provides a means of monitoring intraoperatively the functional integrity of central motor tracts. Nevertheless, it is not used widely for clinical purposes.

1	The motor unit is the basic element subserving motor function. It is defined as an anterior horn cell, its axon and neuromuscular junctions, and all the muscle fibers innervated by the axon. The number of motor units in a muscle ranges from approximately 10 in the extraocular muscles to several thousand in the large muscles of the legs. There is considerable variation in the average number of muscle fibers within the motor units of an individual muscle, i.e., in the innervation ratio of different muscles. Thus the innervation ratio is <25 in the human external rectus or platysma muscle and between 1600 and 1700 in the medial head of the gastrocnemius muscle. The muscle fibers of individual motor units are divided into two general types by distinctive contractile properties, histochemical stains, and characteristic responses to fatigue. Within each motor unit, all of the muscle fibers are of the same type.

1	The pattern of electrical activity in muscle, i.e., the electromyogram (EMG), may be recorded both at rest and during activity from a needle electrode inserted into the muscle. The nature and pattern of abnormalities relate to disorders at different levels of the motor unit.

1	Relaxed muscle normally is electrically silent except in the end-plate region, but abnormal spontaneous activity (Fig. 442e-3) occurs in various neuromuscular disorders, especially those associated with denervation or inflammatory changes in affected muscle. Fibrillation potentials and positive sharp waves (which reflect muscle fiber irritability) and complex repetitive discharges are most often—but not always—found in denervated muscle and may also occur after muscle injury and in certain myopathic disorders, especially inflammatory disorders such as polymyositis. After an acute neuropathic lesion, they occur earlier in proximal than distal muscles and sometimes do not develop distally in the extremities for 4–6 weeks; once present, they may persist indefinitely unless reinnervation occurs or the muscle degenerates so completely that no viable tissue remains. Fasciculation potentials

1	FIGuRE 442e-3 Activity recorded during electromyography (EMG). A. Spontaneous fibrillation potentials and positive sharp waves. B. Complex repetitive discharges recorded in partially denervated muscle at rest. C. Normal triphasic motor unit action potential. D. Small, short-duration, polyphasic motor unit action potential such as is commonly encountered in myopathic disorders. E. Long-duration polyphasic motor unit action potential such as may be seen in chronic neuropathic disorders.

1	(which reflect the spontaneous activity of individual motor units) are characteristic of slowly progressive neuropathic disorders, especially those with degeneration of anterior horn cells (such as amyotrophic lateral sclerosis). Myotonic discharges—high-frequency discharges of potentials derived from single muscle fibers that wax and wane in amplitude and frequency—are the signature of myotonic disorders such as myotonic dystrophy or myotonia congenita but occur occasionally in polymyositis or other, rarer, disorders.

1	Slight voluntary contraction of a muscle leads to activation of a small number of motor units. The potentials generated by muscle fibers of these units that are within the pickup range of the needle electrode will be recorded (Fig. 442e-3). The parameters of normal motor unit action potentials depend on the muscle under study and age of the patient, but their duration is normally between 5 and 15 ms, amplitude is between 200 μV and 2 mV, and most are bior triphasic. The number of units activated depends on the degree of voluntary activity. An increase in muscle contraction is associated with an increase in the number of motor units that are activated (recruited) and in the frequency of discharge. With a full contraction, so many motor units are normally activated that individual motor unit action potentials can no longer be distinguished, and a complete interference pattern is said to have been produced.

1	The incidence of small, short-duration, polyphasic motor unit action potentials (i.e., having more than four phases) is usually increased in myopathic muscle, and an excessive number of units is activated for a specified degree of voluntary activity. By contrast, the loss of motor units that occurs in neuropathic disorders leads to a reduction in number of units activated during a maximal contraction and an increase in their firing rate, i.e., there is an incomplete or reduced interference pattern. The configuration and dimensions of the potentials may also be abnormal, depending on the duration of the neuropathic process. The surviving motor units are initially normal in configuration but, as reinnervation occurs, they increase in amplitude and duration and become polyphasic (Fig. 442e-3).

1	Action potentials from the same motor unit sometimes fire with a consistent temporal relationship to each other, so that double, triple, or multiple discharges are recorded, especially in tetany, hemifacial spasm, or myokymia. Electrical silence characterizes the involuntary, sustained muscle contraction that occurs in phosphorylase deficiency, which is designated a contracture. EMG enables disorders of the motor units to be detected and characterized as either neurogenic or myopathic. In neurogenic disorders, the pattern of affected muscles may localize the lesion to the anterior horn cells or to a specific site as the axons traverse a nerve root, limb plexus, and peripheral nerve to their terminal arborizations. The findings do not enable a specific etiologic diagnosis to be made, however, except in conjunction with the clinical findings and results of other laboratory studies.

1	The findings may provide a guide to the severity of an acute disorder of a peripheral or cranial nerve (by indicating whether denervation has occurred and the completeness of the lesion) and whether the pathologic process is active or progressive in chronic or degenerative disorders such as amyotrophic lateral sclerosis. Such information is important for prognostic purposes.

1	Various quantitative EMG approaches have been developed. The most common is to determine the mean duration and amplitude of 20 motor unit action potentials using a standardized technique. The technique of macro-EMG provides information about the number and size of muscle fibers in a larger volume of the motor unit territory and has also been used to estimate the number of motor units in a muscle. Scanning EMG is a computer-based technique that has been used to study the topography of motor unit action potentials and, in particular, the spatial and temporal distribution of activity in individual units. The technique of single-fiber EMG is discussed separately below. Recording of the electrical response of a muscle to stimulation of its motor nerve at two or more points along its course (Fig. 442e-4)

1	FIGuRE 442e-4 Arrangement for motor conduction studies of the ulnar nerve. Responses are recorded with a surface electrode from the abductor digiti minimi muscle to supramaximal stimulation of the nerve at different sites, and are shown in the lower panel. (From MJ Aminoff: Electromyography in Clinical Practice: Electrodiagnostic Aspects of Neuromuscular Disease, 3rd ed. New York, Churchill Livingstone, 1998.) permits conduction velocity to be determined in the fastest conducting motor fibers between the points of stimulation. The latency and amplitude of the electrical response of muscle (i.e., of the compound muscle action potential) to stimulation of its motor nerve at a distal site are also compared with values defined in normal subjects. Sensory nerve conduction studies are performed by determining the conduction velocity and amplitude of action potentials in sensory fibers when these fibers are stimulated at one point and the responses are recorded at another point along the

1	by determining the conduction velocity and amplitude of action potentials in sensory fibers when these fibers are stimulated at one point and the responses are recorded at another point along the course of the nerve. In adults, conduction velocity in the arms is normally between 50 and 70 m/s, and in the legs is between 40 and 60 m/s.

1	Nerve conduction studies complement the EMG examination, enabling the presence and extent of peripheral nerve pathology to be determined. They are particularly helpful in determining whether sensory symptoms are arising from pathology proximal or distal to the dorsal root ganglia (in the former instance, peripheral sensory conduction studies is normal) and whether neuromuscular dysfunction relates to peripheral nerve disease. In patients with a mononeuropathy, they are invaluable as a means of localizing a focal nerve lesion, determining the extent and severity of the underlying pathology, providing a guide to prognosis, and detecting subclinical involvement of other nerves. They enable a polyneuropathy to be distinguished from a mononeuropathy multiplex, which has important etiologic implications. Nerve conduction studies provide a means of following the progression and therapeutic response of peripheral nerve disorders and are used widely for this purpose in clinical trials. They

1	Nerve conduction studies provide a means of following the progression and therapeutic response of peripheral nerve disorders and are used widely for this purpose in clinical trials. They may suggest the underlying pathologic basis in individual cases. Conduction velocity is often markedly slowed, terminal motor latencies are prolonged, and compound motor and sensory nerve action potentials may be dispersed in the demyelinative neuropathies (such as in Guillain-Barré syndrome, chronic inflammatory polyneuropathy, metachromatic leukodystrophy, or certain hereditary neuropathies); conduction block is frequent in acquired varieties of 442e-5 these neuropathies. By contrast, conduction velocity is normal or slowed only mildly, sensory nerve action potentials are small or absent, and there is EMG evidence of denervation in axonal neuropathies such as occur in association with metabolic or toxic disorders.

1	The utility and complementary role of EMG and nerve conduction studies are best illustrated by reference to a common clinical problem. Numbness and paresthesias of the little finger and associated wasting of the intrinsic muscles of the hand may result from a spinal cord lesion, C8/T1 radiculopathy, brachial plexopathy (lower trunk or medial cord), or a lesion of the ulnar nerve. If sensory nerve action potentials can be recorded normally at the wrist following stimulation of the digital fibers in the affected finger, the pathology is probably proximal to the dorsal root ganglia (i.e., there is a radiculopathy or more central lesion); absence of the sensory potentials, by contrast, suggests distal pathology. EMG examination will indicate whether the pattern of affected muscles conforms to radicular or ulnar nerve territory or is more extensive (thereby favoring a plexopathy). Ulnar motor conduction studies will generally also distinguish between a radiculopathy (normal findings) and

1	radicular or ulnar nerve territory or is more extensive (thereby favoring a plexopathy). Ulnar motor conduction studies will generally also distinguish between a radiculopathy (normal findings) and ulnar neuropathy (abnormal findings) and will often identify the site of an ulnar nerve lesion. The nerve is stimulated at several points along its course to determine whether the compound action potential recorded from a distal muscle that it supplies shows a marked alteration in size or area or a disproportionate change in latency, with stimulation at a particular site. The electrophysiologic findings thus permit a definitive diagnosis to be made and specific treatment instituted in circumstances where there is clinical ambiguity.

1	Stimulation of a motor nerve causes impulses to travel antidromically (i.e., toward the spinal cord) as well as orthodromically (to the nerve terminals). Such antidromic impulses cause a few of the anterior horn cells to discharge, producing a small motor response that occurs considerably later than the direct response elicited by nerve stimulation. The F wave so elicited is sometimes abnormal (absent or delayed) with proximal pathology of the peripheral nervous system, such as a radiculopathy, and may therefore be helpful in detecting abnormalities when conventional nerve conduction studies are normal. In general, however, the clinical utility of F-wave studies has been disappointing, except perhaps in Guillain-Barré syndrome, where they are often absent or delayed.

1	The H reflex is easily recorded only from the soleus muscle (S1) in normal adults. It is elicited by low-intensity stimulation of the tibial nerve and represents a monosynaptic reflex in which spindle (Ia) afferent fibers constitute the afferent arc and alpha motor axons the efferent pathway. The H reflexes are often absent bilaterally in elderly patients or with polyneuropathies and may be lost unilaterally in S1 radiculopathies. The size of the electrical response of a muscle to supramaximal electrical stimulation of its motor nerve relates to the number of muscle fibers that are activated. Neuromuscular transmission can be tested by several different protocols, but the most helpful is to record with surface electrodes the electrical response of a muscle to supramaximal stimulation of its motor nerve by repetitive (2–3 Hz) shocks delivered before and at selected intervals after a maximal voluntary contraction.

1	There is normally little or no change in size of the muscle response to repetitive stimulation of a motor nerve at 2–3 Hz with stimuli delivered at intervals after voluntary contraction of the muscle for about 20–30 s, even though preceding activity in the junctional region influences the release of acetylcholine and thus the size of the end-plate potentials elicited by a test stimulus. This is because more acetylcholine is normally released than is required to bring the motor end-plate potentials to the threshold for generating muscle fiber action potentials. In disorders of neuromuscular transmission this safety

1	CHAPTER 442e Electrodiagnostic Studies of Nervous System Disorders: EEG, Evoked Potentials, and EMG factor is reduced. Thus in myasthenia gravis, repetitive stimulation, particularly at a rate of between 2 and 5 Hz, may lead to a depression of neuromuscular transmission, with a decrement in size of the response recorded from affected muscles. Similarly, immediately after a period of maximal voluntary activity, single or repetitive stimuli of the motor nerve may elicit larger muscle responses than before, indicating that more muscle fibers are responding. This postactivation facilitation of neuromuscular transmission is followed by a longer-lasting period of depression, maximal between 2 and 4 min after the conditioning period and lasting for as long as 10 min or so, during which responses are reduced in size.

1	Decrementing responses to repetitive stimulation at 2–5 Hz are common in myasthenia gravis but may also occur in the congenital myasthenic syndromes (Chap. 461). In Lambert-Eaton myasthenic syndrome, in which there is defective release of acetylcholine at the neuromuscular junction, the compound muscle action potential elicited by a single stimulus is generally very small. With repetitive stimulation at rates of up to 10 Hz, the first few responses may decline in size, but subsequent responses increase. If faster rates of stimulation are used (20–50 Hz), the increment may be dramatic so that the amplitude of compound muscle action potentials eventually reaches a size that is several times larger than the initial response. In patients with botulism, the response to repetitive stimulation is similar to that in Lambert-Eaton myasthenic syndrome, although the findings are somewhat more variable and not all muscles are affected.

1	This technique is particularly helpful in detecting disorders of neuromuscular transmission. A special needle electrode is placed within a muscle and positioned to record action potentials from two muscle fibers belonging to the same motor unit. The time interval between the two potentials will vary in consecutive discharges; this is called the neuromuscular jitter. The jitter can be quantified as the mean difference between consecutive interpotential intervals and is normally between 10 and 50 μs. This value is increased when neuromuscular transmission is disturbed for any reason, and in some instances impulses in individual muscle fibers may fail to occur because of impulse blocking at the neuromuscular junction. Single-fiber EMG is more sensitive than repetitive nerve stimulation or determination of acetylcholine receptor antibody levels in diagnosing myasthenia gravis.

1	Single-fiber EMG can also be used to determine the mean fiber density of motor units (i.e., mean number of muscle fibers per motor unit within the recording area) and to estimate the number of motor units in a muscle, but this is of less immediate clinical relevance. Electrical or mechanical stimulation of the supraorbital nerve on one side leads to two separate reflex responses of the orbicularis oculi—an ipsilateral R1 response having a latency of approximately 10 ms and a bilateral R2 response with a latency in the order of 30 ms. The trigeminal and facial nerves constitute the afferent and efferent arcs of the reflex, respectively. Abnormalities of either nerve or intrinsic lesions of the medulla or pons may lead to unior bilateral loss of the response, and the findings may therefore be helpful in identifying or localizing such pathology.

1	technique of Lumbar puncture Elizabeth Robbins, Stephen L. Hauser In experienced hands, lumbar puncture (LP) is usually a safe proce-dure. Major complications are extremely uncommon but can include cerebral herniation, injury to the spinal cord or nerve roots, hemor-rhage (spinal hematoma), or infection. Minor complications occur 443e with greater frequency and can include backache, post-LP headache, and radicular pain or numbness.

1	Patients with an altered level of consciousness, a focal neurologic deficit, new-onset seizure, papilledema, or an immunocompromised state are at increased risk for potentially fatal cerebellar or tentorial herniation following LP. Neuroimaging should be obtained in these patients prior to LP to exclude a focal mass lesion or diffuse swelling. Imaging studies should include the spine in patients with symptoms suggesting spinal cord compression, such as back pain, leg weakness, urinary retention, or incontinence. In patients with suspected meningitis who require neuroimaging prior to diagnostic LP, administration of antibiotics, preferably following blood culture, should precede the neuroimaging study. LP should not be performed through infected skin, as organisms can be introduced into the subarachnoid space (SAS).

1	LP should not be performed through infected skin, as organisms can be introduced into the subarachnoid space (SAS). Patients with coagulation defects including thrombocytopenia are at increased risk of post-LP spinal subdural or epidural hematomas, either of which can produce permanent nerve injury and/or paralysis. If a bleeding disorder is suspected, the platelet count, international normalized ratio (INR), and partial thromboplastin time should be checked prior to LP. There are no data available to assess the safety of LP in patients with low platelet counts; a count of <20,000/μL is considered to be a contraindication to LP. Bleeding complications rarely occur in patients with platelet counts ≥50,000/μL and an INR ≤1.5. Some institutions recommend that the platelet count be >40,000 prior to LP.

1	There is an increased risk of bleeding complications if an LP is performed in a patient receiving antiplatelet or anticoagulant medications. The risk is further increased when multiple anticoagulant medications are used or when the level of anticoagulation is high. The most common site of bleeding is the epidural space. Symptoms of bleeding following an LP can include a sensory or motor deficit and/ or bowel/bladder dysfunction; back pain occurs less commonly. For serious deficits such as paraparesis, immediate surgical intervention, ideally within 8 h of onset of weakness, is important to minimize permanent disability; surgical intervention after 24 h is associated with a poor outcome.

1	Only limited data are available to guide decisions about performing LPs in patients receiving anticoagulant drugs. Information about managing antiplatelet and anticoagulation drugs during invasive surgical procedures is often available from the prescribing information provided by the drug manufacturer. Evidence-based guidelines for management of regional anesthetic procedures including spinal and epidural blocks in patients receiving anticoagulation have been developed by the American Society of Regional Anesthesia and Pain (ASRA); these guidelines can help guide decisions by physicians considering LP in patients receiving anticoagulation. Management of these patients can be complex and needs to consider both the risk of LP-related hemorrhage as well as the risk of reversing therapeutic anticoagulation prior to LP. Guidelines for some commonly used anticoagulants are summarized below.

1	Unfractionated Heparin, Therapeutic Dosing The ASRA 2010 Practice Advisory recommends discontinuing unfractionated heparin (UFH) 2–4 h prior to removal of spinal or epidural catheters to minimize risk 443e-1 of hematoma. Similar guidelines are reasonable for patients undergoing LP: discontinue UFH 2–4 h prior to LP; document normal partial thromboplastin time (PTT) prior to the procedure; and document a normal platelet count in patients who have received heparin for 4 days or longer because of the risk of heparin-induced thrombocytopenia (HIT). The half-life of heparin is 60–90 min.

1	Unfractionated Heparin, Prophylactic Dosing There are only a few case reports of spinal hematoma resulting from spinal or epidural anesthetic procedures in patients receiving low-dose subcutaneous UFH; ASRA guidelines state that there is no contraindication to the use of these techniques for anesthesia in patients receiving prophylactic UFH at a dose of 5000 U subcutaneously twice daily. Similarly, LP in patients receiving 5000 U of UFH subcutaneously twice daily is unlikely to cause spinal hematoma. Precautions to minimize risk include the following: document a normal PTT prior to the LP; document a normal platelet count in patients who have received heparin for 4 days or longer; and perform the LP 1–2 h prior to the next heparin dose, when the heparin effect should be minimal.

1	Low-Molecular-Weight Heparin, Therapeutic Dose (e.g., Enoxaparin 1 mg/kg Subcutaneously q12h) Patients receiving low-molecular-weight heparin (LMWH) are at increased risk of post-LP spinal or epidural hematoma. LMWH dose should be held for at least 24 h before the procedure. Low-Molecular-Weight Heparin, Prophylactic Dose (e.g., Enoxaparin 0.5 mg/kg Subcutaneously q12h) Patients receiving prophylactic-dose LMWH have altered coagulation. ASRA guidelines recommend waiting at least 10–12 h after a prophylactic dose of LMWH before inserting a spinal or epidural catheter to minimize the risk of spinal or epidural hematoma. Similar guidelines are reasonable for patients undergoing LP. Warfarin Spinal puncture is contraindicated during warfarin therapy.

1	Warfarin Spinal puncture is contraindicated during warfarin therapy. Aspirin and Nonsteroidal Anti-inflammatory Drugs (NSAIDs) ASRA guidelines conclude that use of these drugs does not appear to be associated with an added significant risk of spinal bleeding in patients having spinal or epidural anesthesia. Similarly, LP in patients receiving one of these drugs is unlikely to cause bleeding. Reversal of drug effect on platelet function requires stopping the drug for approximately 10 days for aspirin and for 48 h for NSAIDs. Ticlopidine/Clopidogrel The actual risk of spinal hematoma with these drugs is unknown. Based on drug prescribing information and surgical reviews, ASRA guidelines suggest discontinuing ticlopidine 14 days prior to a spinal or epidural procedure and discontinuing clopidogrel 7 days prior to the procedure. Similar guidelines are reasonable for performing LP. Abciximab, Eptifibatide, and Other Platelet Glycoprotein IIb/IIIa Inhibitors

1	Abciximab, Eptifibatide, and Other Platelet Glycoprotein IIb/IIIa Inhibitors The actual risk of spinal hematoma with these drugs is unknown. Platelet aggregation remains abnormal for 24–48 h following discontinuation of abciximab and 4–8 h following discontinuation of eptifibatide. ASRA guidelines recommend avoiding spinal or epidural procedures until platelet function is normal. Similar guidelines are reasonable for performing LP. Direct Thrombin Inhibitors (e.g., Argatroban, Bivalirudin) ASRA guidelines recommend against performing spinal or epidural anesthesia in patients receiving thrombin inhibitors. Oral Factor Xa Inhibitor (e.g., Rivaroxaban) Rivaroxaban prescribing information includes a black box warning that epidural or spinal hematomas have occurred in patients treated with rivaroxaban who are receiving spinal or epidural anesthesia or undergoing LP. LP should be avoided in patients receiving this drug.

1	Anxiety and pain can be minimized prior to beginning the procedure. Anxiety can be allayed by the use of lorazepam, 1–2 mg given PO 30 min prior to the procedure or IV 5 min prior to the procedure. Chapter 443e Technique of Lumbar Puncture Level of the posterior superior iliac crest L3-L4 interspace L5L4L3L5L4L3 FIGURE 443e-1 Proper positioning of a patient in the lateral decubitus position. Note that the shoulders and hips are in a vertical plane; the torso is perpendicular to the bed. (From RP Simon et al [eds]: Clinical Neurology, 7th ed. New York, McGraw-Hill, 2009.) Topical anesthesia can be achieved by the application of a lidocainebased cream. Lidocaine 4% is effective when applied 30 min prior to the procedure; lidocaine/prilocaine requires 60–120 min. The cream should be applied in a thick layer so that it completely covers the skin; an occlusive dressing is used to keep the cream in place.

1	Proper positioning of the patient is essential. The procedure should be performed on a firm surface; if the procedure is to be performed at the bedside, the patient should be positioned at the edge of the bed and not in the middle. The patient is asked to lie on his or her side, facing away from the examiner, and to “roll up into a ball.” The neck is gently ante-flexed and the thighs pulled up toward the abdomen; the shoulders and pelvis should be vertically aligned without forward or backward tilt (Fig. 443e-1). The spinal cord terminates at approximately the L1 vertebral level in 94% of individuals. In the remaining 6%, the conus extends to the L2–L3 interspace. LP is therefore performed at or below the L3–L4 interspace. A useful anatomic guide is a line drawn between the posterior superior iliac crests, which corresponds closely to the level of the L3–L4 interspace. The interspace is chosen following gentle palpation to identify the spinous processes at each lumbar level.

1	An alternative to the lateral recumbent position is the seated position. The patient sits at the side of the bed, with feet supported on a chair. The patient is instructed to curl forward, trying to touch the nose to the umbilicus. It is important that the patient not simply lean forward onto a bedside tabletop, as this is not an optimal position for opening up the spinous processes. LP is sometimes more easily performed in obese patients if they are sitting. A disadvantage of the seated position is that measurement of opening pressure is not accurate. In situations in which LP is difficult using palpable spinal landmarks, bedside ultrasound to guide needle placement may be used. In some particularly difficult situations, a computed tomography (CT)-guided needle placement may be necessary.

1	Once the desired site for needle insertion has been identified, the examiner should put on sterile gloves. A mask is worn if the clinician will be injecting material into the spinal or epidural space to prevent droplet spread of oral flora during the procedure. After cleansing the skin with povidone-iodine or similar disinfectant, the area is draped with a sterile cloth; the needle insertion site is blotted dry using a sterile gauze pad. Proper local disinfection reduces the risk of introducing skin bacteria into the SAS or other sites. Local anesthetic, typically 1% lidocaine, 3–5 mL total, is injected into the subcutaneous tissue; in nonemergency situations, a topical anesthetic cream can be applied (see above). When time permits, pain associated with the injection of lidocaine can be minimized by slow, serial injections, each one progressively deeper than the last, over a period of ~5 min. Approximately 0.5–1 mL of lidocaine is injected at a time; the needle is not usually

1	can be minimized by slow, serial injections, each one progressively deeper than the last, over a period of ~5 min. Approximately 0.5–1 mL of lidocaine is injected at a time; the needle is not usually withdrawn between injections. A pause of ~15 s between injections helps to minimize the pain of the subsequent injection. The goal is to inject each mini-bolus of anesthetic into an area of skin that has become numb from the preceding injection. Approximately 5–10 mini-boluses are injected, using a total of ~5 mL of lidocaine.

1	If possible, the LP should be delayed for 10–15 min following the completion of the injection of anesthetic; this significantly decreases and can even eliminate pain from the procedure. Even a delay of 5 min will help to reduce pain.

1	The LP needle (typically 20to 22-gauge) is inserted in the midline, midway between two spinous processes, and slowly advanced. The bevel of the needle should be maintained in a horizontal position, parallel to the direction of the dural fibers and with the flat portion of the bevel pointed upward; this minimizes injury to the fibers as the dura is penetrated. When LP is performed in patients who are sitting, the bevel should be maintained in the vertical position. In most adults, the needle is advanced 4–5 cm (1–2 in.) before the SAS is reached; the examiner usually recognizes entry as a sudden release of resistance, a “pop.” If no fluid appears despite apparently correct needle placement, then the needle may be rotated 90°–180°. If there is still no fluid, the stylet is reinserted and the needle is advanced slightly. Some examiners halt needle advancement periodically to remove the stylet and check for flow of cerebrospinal fluid (CSF). If the needle cannot be advanced because it

1	the needle is advanced slightly. Some examiners halt needle advancement periodically to remove the stylet and check for flow of cerebrospinal fluid (CSF). If the needle cannot be advanced because it hits bone, if the patient experiences sharp radiating pain down one leg, or if no fluid appears (“dry tap”), the needle is partially withdrawn and reinserted at a different angle. If on the second attempt the needle still hits bone (indicating lack of success in introducing it between the spinous processes), then the needle should be completely withdrawn and the patient should be repositioned. The second attempt is sometimes more successful if the patient straightens the spine completely prior to repositioning. The needle can then be reinserted at the same level or at an adjacent one.

1	Once the SAS is reached, a manometer is attached to the needle and the opening pressure measured. The examiner should look for normal oscillations in CSF pressure associated with pulse and respirations. The upper limit of normal opening pressure with the patient supine is 180 mmH2O in adults but may be as high as 200–250 mmH2O in obese adults.

1	CSF is allowed to drip into collection tubes; it should not be withdrawn with a syringe. Depending on the clinical indication, fluid is obtained for studies including: (1) cell count with differential; (2) protein and glucose concentrations; (3) culture (bacterial, fungal, mycobacterial, viral); (4) smears (e.g., Gram’s and acid-fast stained smears); (5) antigen tests (e.g., latex agglutination); (6) polymerase chain reaction (PCR) amplification of DNA or RNA of microorganisms (e.g., herpes simplex virus, enteroviruses); (7) antibody levels against microorganisms; (8) immunoelectrophoresis for determination of γ-globulin level and oligoclonal banding; and (9) cytology. Although 15 mL of CSF is sufficient to obtain all of the listed studies, the yield of fungal and mycobacterial cultures and cytology increases when larger volumes are sampled. In general 20–30 mL may be safely removed from adults.

1	A bloody tap due to penetration of a meningeal vessel (a “traumatic tap”) may result in confusion with subarachnoid hemorrhage (SAH). In these situations a specimen of CSF should be centrifuged immediately after it is obtained; clear supernatant following CSF centrifugation supports the diagnosis of a bloody tap, whereas xanthochromic supernatant suggests SAH. In general, bloody CSF due to the penetration of a meningeal vessel clears gradually in successive tubes, whereas blood due to SAH does not. In addition to SAH, xanthochromic CSF may also be present in patients with liver disease and when the CSF protein concentration is markedly elevated (>1.5–2 g/L [150–200 mg/dL]).

1	Prior to removing the LP needle, the stylet is reinserted to avoid the possibility of entrapment of a nerve root in the dura as the needle is being withdrawn; entrapment could result in a dural CSF leak, causing headache. Some practitioners question the safety of this maneuver, with its potential risk of causing a needle-stick injury to the examiner. Injury is unlikely, however, given the flexibility of the small-diameter stylet, which tends to bend, rather than penetrate, on contact. Following LP, the patient is customarily positioned in a comfortable, recumbent position for 30–60 min before rising, although this may be unnecessary because it does not appear to affect the development of headache (see below).

1	The principal complication of LP is headache, occurring in 10–30% of patients. Younger age and female gender are associated with an increased risk of post-LP headache. Headache usually begins within 48 h but may be delayed for up to 12 days. Head pain is dramatically positional; it begins when the patient sits or stands upright; there is relief upon reclining or with abdominal compression. The longer the patient is upright, the longer the latency before head pain subsides. The pain is usually a dull ache but may be throbbing; its location is occipitofrontal. Nausea and stiff neck often accompany headache, and occasionally, patients report blurred vision, photophobia, tinnitus, and vertigo. In more than three-quarters of patients, symptoms completely resolve within a week, but in a minority they can persist for weeks or even months.

1	Post-LP headache is caused by a drop in CSF pressure related to persistent leakage of CSF at the site where the needle entered the SAS. Loss of CSF volume decreases the brain’s supportive cushion, so that when a patient is upright there is probably dilation and tension placed on the brain’s anchoring structures, the pain-sensitive dural sinuses, resulting in pain. Although intracranial hypotension is the usual explanation for severe LP headache, the syndrome can occur in patients with normal CSF pressure. Because post-LP headache usually resolves without specific treatment, care is largely supportive with oral analgesics (acetaminophen, NSAIDs, opioids [Chap. 18]) and antiemetics. Patients may obtain relief by lying in a comfortable (especially a recumbent or head-down Trendelenburg) position. For some patients, beverages with caffeine can provide temporary pain relief.

1	For patients with persistent pain, treatment with IV caffeine (500 mg in 500 mL saline administered over 2 h) may be effective; atrial fibrillation is a rare side effect. Alternatively, an epidural blood patch accomplished by injection of 15 mL of autologous whole blood is usually effective; the injection is directed at the epidural space at the level of the initial LP. This procedure is most often performed by a pain specialist or anesthesiologist. The blood patch has an immediate effect, making it unlikely that sealing off a dural hole with blood clot is its sole mechanism of action. The acute benefit may be due to compression of the CSF space by the clot, increasing CSF pressure. Some clinicians reserve epidural blood patch for patients who do not respond to caffeine, while others prefer to use blood patch as initial management for unremitting post-LP symptoms.

1	Strategies to decrease the incidence of post-LP headache are listed in Table 443e-1. Use of a smaller caliber needle is associated with a lower risk: in one study, the risk of headache following use of a 24to 27-gauge standard (Quincke) needle was 5–12%, compared to 20–40% when a 20-or 22-gauge needle was used. The smallest gauge needles usually require the use of an introducer needle and are associated with a slower CSF flow rate. Use of an “atraumatic” (Sprotte, “pencil Use of small-diameter needle (22-gauge or smaller) Use of atraumatic needle (Sprotte and others) Replacement of stylet prior to removal of needle Insertion of needle with bevel oriented in a cephalad to caudad direction Bed rest (up to 4 h) following LP Supplemental fluids Minimizing the volume of spinal fluid removed Immediate mobilization following LP Abbreviation: LP, lumbar puncture.

1	Bed rest (up to 4 h) following LP Supplemental fluids Minimizing the volume of spinal fluid removed Immediate mobilization following LP Abbreviation: LP, lumbar puncture. FIGURE 443e-2 Comparison of the standard (“cutting” or Quincke) lumbar puncture (LP) needle with the “atraumatic” (Sprotte). The “atraumatic” needle has its opening on the top surface of the needle, a design intended to reduce the chance of cutting dural fibers that, by protruding through the dura, could be responsible for subsequent cerebrospinal fluid leak and post-LP headache.

1	(From SR Thomas et al: BMJ 321:986, 2000.) point,” or “noncutting”) needle also reduces the incidence of moderate to severe headache compared with standard LP (Quincke, or “traumatic”) needles (Fig. 443e-2). However, because atraumatic needles are more difficult to use, more attempts may be required to perform the LP, particularly in overweight patients. It may also be necessary to use an introducer with the atraumatic needle, which does not have the customary cutting, beveled tip. There is a low risk of needle damage, e.g., breakage, with the Sprotte atraumatic needle. Another strategy to decrease the incidence of headache is to replace the stylet before removing the LP needle.

1	Patients are often advised to remain in a recumbent position for up to an hour following LP. However, studies comparing mobilization immediately following LP with bed rest for periods up to 4 h show no significant differences in the incidence of headache, suggesting that the customary practice of remaining in a recumbent position post-LP may be unnecessary. Chapter 443e Technique of Lumbar Puncture Glucose 2.22–3.89 mmol/L 40–70 mg/dL Lactate 1–2 mmol/L 10–20 mg/dL Total protein Lumbar 0.15–0.5 g/L 15–50 mg/dL Cisternal 0.15–0.25 g/L 15–25 mg/dL Ventricular 0.06–0.15 g/L 6–15 mg/dL Albumin 0.066–0.442 g/L 6.6–44.2 mg/dL IgG 0.009–0.057 g/L 0.9–5.7 mg/dL IgG indexb 0.29–0.59 Oligoclonal bands (OGB) <2 bands not present in

1	Albumin 0.066–0.442 g/L 6.6–44.2 mg/dL IgG 0.009–0.057 g/L 0.9–5.7 mg/dL IgG indexb 0.29–0.59 Oligoclonal bands (OGB) <2 bands not present in Total 0–5 mononuclear cells per mm3 Differential Lymphocytes 60–70% Monocytes 30–50% Neutrophils None aBecause CSF concentrations are equilibrium values, measurements of the same parameters in blood plasma obtained at the same time are recommended. However, there is a time lag in attainment of equilibrium, and cerebrospinal levels of plasma constituents that can fluctuate rapidly (such as plasma glucose) may not achieve stable values until after a significant lag phase. bIgG index = CSF IgG (mg/dL) × Serum albumin (g/dL)/Serum IgG (g/dL) × CSF albumin (mg/dL).

1	NORMAL VALUES in centrifuged or concentrated CSF specimens such as those used for cytologic examination. Red blood cells (RBCs) are not normally pres (See Table 443e-2) In uninfected CSF, the normal white blood cell ent in CSF; if RBCs are present from a traumatic tap, their numbercount is fewer than five mononuclear cells (lymphocytes and mono-decreases as additional CSF is collected. CSF glucose concentrationscytes) per μL. Polymorphonuclear leukocytes (PMNs) are not found <2.2 mmol/L (<40 mg/dL) are abnormal. in normal unconcentrated CSF; however, rare PMNs can be found noncoding sequences of DNA. For example, large intronic GGGGCC 444e-1 Biology of Neurologic Diseases repeat expansions in a gene of unknown function C9orf 72 (chromo some 9 open reading frame 72) were recently identified as a common Stephen L. Hauser, Stanley B. Prusiner, M. Flint Beal cause of both frontotemporal dementia and amyotrophic lateral scle-

1	Stephen L. Hauser, Stanley B. Prusiner, M. Flint Beal cause of both frontotemporal dementia and amyotrophic lateral scle- The human nervous system is the organ of consciousness, cognition, ethics, and behavior; as such, it is the most intricate structure known to exist. More than one-third of the 23,000 genes encoded in the human genome are expressed in the nervous system. Each mature brain is composed of 100 billion neurons, several million miles of axons and dendrites, and >1015 synapses. Neurons exist within a dense parenchyma of multifunctional glial cells that synthesize myelin, preserve homeostasis, and regulate immune responses. Measured against this background of complexity, the achievements of molecular neuroscience have been extraordinary. This chapter reviews selected themes in neuroscience that provide a context for understanding fundamental mechanisms underlying neurologic disorders.

1	The landscape of neurology has been transformed by modern molecular genetics. Several hundred neurologic and psychiatric disorders can now be diagnosed through genetic testing (http://www.ncbi.nlm.nih .gov/sites/GeneTests/?db=GeneTests). The vast majority of these represent highly penetrant mutations that cause rare neurologic disorders; alternatively, they represent rare monogenic causes of common phenotypes. Examples of the latter include mutations of the amyloid precursor protein in familial Alzheimer’s disease, the microtubule-associated protein tau (MAPT) in frontotemporal dementia, and α-synuclein in Parkinson’s disease. These discoveries have been profoundly important because the mutated gene in the familial disorder often encodes a protein that is also pathogenetically involved (although not mutated) in the typical, sporadic form. The common mechanism involves disordered processing and, ultimately, aggregation of the protein leading to cell death (see “Protein Aggregation and

1	not mutated) in the typical, sporadic form. The common mechanism involves disordered processing and, ultimately, aggregation of the protein leading to cell death (see “Protein Aggregation and Neurodegeneration,” below).

1	There is optimism that complex genetic disorders, caused by combinations of both genetic and environmental factors, have now become tractable problems. Genome-wide association studies (GWAS) have been carried out in many complex neurologic disorders, with many hundreds of variants identified, nearly all of which confer only a small increment in disease risk (1.15to 1.5-fold). GWAS studies are rooted in the “common disease, common variant” hypothesis, as they examine potential risk alleles that are relatively frequent (e.g. >5%) in the general population. More than 1500 GWAS studies have been carried out, with notable successes such as the identification of 110 risk alleles for multiple sclerosis (Chap. 458). Furthermore, using bioinformatics tools, risk variants can be aligned in functional biologic pathways to identify novel pathogenic mechanisms as well as to reveal heterogeneity (e.g., different pathways in different individuals). Despite these successes, many experienced

1	functional biologic pathways to identify novel pathogenic mechanisms as well as to reveal heterogeneity (e.g., different pathways in different individuals). Despite these successes, many experienced geneticists question the real value of common disease-associated variants, particularly whether they are actually causative or merely mark the approximate locations of more important—truly causative—rare mutations.

1	This debate has set the stage for the next revolution in human genetics, made possible by the development of increasingly efficient and cost-effective high-throughput sequencing methodologies. It is already possible to sequence an entire human genome in approximately an hour, at a cost of only $1300 for the entire coding sequence (“wholeexome”) or $3000 for the entire genome; it is certain that these costs will continue to decline. This makes it feasible to look for disease-causing sequence variations in individual patients with the possibility of identifying rare variants that cause disease. The utility of this approach was demonstrated by whole-genome sequencing in a patient with Charcot-Marie-Tooth neuropathy, in which compound heterozygous mutations were identified in the SH3TC2 gene, which then were shown to co-segregate with the disease in other members of the family.

1	It is increasingly recognized that not all genetic diseases or predispositions are caused by simple changes in the linear nucleotide sequence of genes. Disease-causative mutations also occur commonly in rosis (ALS). This mutation is the most common cause of both familial and sporadic ALS identified thus far. It was shown to be associated with TDP-43 (tar DNA binding protein-43) inclusions in both hippocampal and cerebral neurons. Interestingly, despite the absence of a start codon, the three alternate dipeptide sequences consisting of two amino acids are translated and found in postmortem brain tissue of affected patients. Three potential pathogenic mechanisms have been proposed, including (1) haploinsufficiency, (2) repeat RNA-mediated toxicity, and (3) dipeptide protein toxicity. The possibility of RNA toxicity is supported by the finding of intranuclear RNA foci containing C9orf 72 hexanucleotide repeats and specific RNA-binding proteins. The C9orf 72 mRNA forms quadruplexes of DNA

1	of RNA toxicity is supported by the finding of intranuclear RNA foci containing C9orf 72 hexanucleotide repeats and specific RNA-binding proteins. The C9orf 72 mRNA forms quadruplexes of DNA and RNA, which then can halt transcription and also bind transcription factors. Mutations in both TARDP (transactive region DNA binding protein) and FUS (fused in sarcoma) also encode DNA/RNA-processing polypeptides and are a cause of familial and sporadic ALS.

1	As the complex architecture of the human genome becomes better defined, many disorders that result from alterations in copy numbers of genes (“gene-dosage” effects) resulting from unequal crossing-over are also likely to be identified. As much as 5–10% of the human genome consists of nonhomologous duplications and deletions, and these appear to occur with a much higher mutational rate than is the case for single base pair mutations. The first copy-number disorders to be recognized were Charcot-Marie-Tooth disease type 1A (CMT1A), caused by a duplication in the gene encoding the myelin protein PMP22, and the reciprocal deletion of the gene causing hereditary liability to pressure palsies (Chap. 459). Gene-dosage effects are causative in some cases of Parkinson’s disease (α-synuclein), Alzheimer’s disease (amyloid precursor protein), spinal muscular atrophy (survival motor neuron 2), the dysmyelinating disorder Pelizaeus-Merzbacher syndrome (proteolipid protein 1), late-onset

1	Alzheimer’s disease (amyloid precursor protein), spinal muscular atrophy (survival motor neuron 2), the dysmyelinating disorder Pelizaeus-Merzbacher syndrome (proteolipid protein 1), late-onset leukodystrophy (lamin B1), and a variety of developmental neurologic disorders. It is likely that copy-number variations contribute substantially to normal human genomic variation for numerous genes involved in neurologic function, regulation of cell growth, and regulation of metabolism. It is also already clear that gene-dosage effects will influence many behavioral phenotypes, learning disorders, and autism spectrum disorders. Deletions at ch444eq and ch15q have been associated with schizophrenia, and deletions at 15q and 16p with autism. Interestingly, the 16p deletion is also associated with epilepsy. Duplications of the X-linked MeCP2 gene cause autism in males and psychiatric disorders with anxiety in females, whereas point mutations in this gene produce the neurodevelopmental disorder

1	epilepsy. Duplications of the X-linked MeCP2 gene cause autism in males and psychiatric disorders with anxiety in females, whereas point mutations in this gene produce the neurodevelopmental disorder Rett’s syndrome. The understanding of the role of copy number variation in human disease is still in its infancy.

1	The role of splicing variation as a contributor to neurologic disease is another area of active investigation. Alternative splicing refers to the inclusion of different combinations of exons in mature mRNA, resulting in the potential for many different protein products encoded by a single gene. Alternative splicing represents a powerful mechanism for generation of complexity and variation, and this mechanism appears to be highly prevalent in the nervous system, affecting key processes such as neurotransmitter receptors and ion channels. Numerous diseases are already known to result from abnormalities in alternative splicing. Increased inclusion of exon 10–containing transcripts of MAPT can cause frontotemporal dementia. Aberrant splicing also contributes to the pathogenesis of Duchenne’s, myotonic, and facioscapulohumeral muscular dystrophies; ataxia telangiectasia; neurofibromatosis; some inherited ataxias; and fragile X syndrome, among other disorders. It is also likely that subtle

1	myotonic, and facioscapulohumeral muscular dystrophies; ataxia telangiectasia; neurofibromatosis; some inherited ataxias; and fragile X syndrome, among other disorders. It is also likely that subtle variations of splicing will influence many genetically complex disorders. A splicing variant of the interleukin 7 receptor α chain, resulting in production of more soluble and less membrane-bound receptor, is associated with susceptibility to multiple sclerosis (MS) in multiple different populations.

1	Epigenetics refers to the mechanisms by which levels of gene expression can be exquisitely modulated, not by variations in the primary Chapter 444e Biology of Neurologic Diseases genetic sequence of DNA but rather by postgenomic alterations in DNA and chromatin structure, which influence how, when, and where genes are expressed. Category Disorder Type Mutated Gene Chap. Ref. DNA methylation and methylation and acetylation of histone proteins that interact with nuclear DNA to form chromatin are key mediators of these events. Epigenetic Ataxias Episodic ataxia-1 K KCNA1 processes appear to be dynamically active even in post- mitotic neurons. Imprinting refers to an epigenetic fea- Spinocerebellar ataxia-6 Ca CACNL1A ture, present for a subset of genes, in which the predomi-Migraine Familial hemiplegic migraine 1 Ca CACNL1A 447 nant expression of one allele is determined by its parent of origin. The distinctive neurodevelopmental disorders

1	Epilepsy Benign neonatal familial K KCNQ2, KCNQ3 445 Generalized epilepsy with (cortical atrophy, cerebellar dysmyelination, Purkinje cell loss) are classic examples of imprinting disorders whose distinctive features are determined by whether the paternal or maternal copy of the chromosome of the critical genetic region 15q11-13 is responsible. In a study of discordant monozygotic twins for MS in which the entire DNA sequence, transcriptome (e.g., mRNA levels), and methylome were assessed genome-wide, tantalizing allelic differences in the use of the paternal, Deafness Jervell and Lange-Nielsen syn-K KCNQ1, KCNE1 43 drome (deafness, prolonged compared to maternal, copy for a group of genes were

1	QT interval, and arrhythmia) identified. Preferential allelic expression, whether due to imprinting, resistance to X-inactivation, or other mechanisms, is likely to play a major role in determining complex behaviors and susceptibility to many neurologic and psychiatric disorders. Paraneoplastic Limbic encephalitis Kv1 — 122 Another advance is the development of transgenic mouse models of neurologic diseases, which has been particularly fruitful in producing models relevant to

1	Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and ALS. These models are useful in both studying disease pathogenesis and developing and testing new therapies. New transgenic mouse models with conditional expression have fostered investigations in which late gene epilepsy syndromes, and recently such mutations have been identified. expression avoids developmental compensation or in which the revers-These mutations appear to alter the normal gating function of these ibility of a disease phenotype can be examined by turning a gene off channels, increasing the inherent excitability of neuronal membranes after the disease phenotype has manifested. One can also examine in regions where the abnormal channels are expressed. the effects of gene expression in specific subsets of neurons, such as Whereas the specific clinical manifestations of channelopathies entorhinal cortex, or selectively in neurons, astrocytes, or microglia. are quite variable, one common feature is that

1	neurons, such as Whereas the specific clinical manifestations of channelopathies entorhinal cortex, or selectively in neurons, astrocytes, or microglia. are quite variable, one common feature is that manifestations tend to Models in both Caenorhabditis elegans and Drosophila have also been be intermittent or paroxysmal, such as occurs in epilepsy, migraine, extremely useful, particularly in studying genetic modifiers and thera-ataxia, myotonia, or periodic paralysis. Exceptions are clinically peutic interventions. progressive channel disorders such as autosomal dominant hearing impairment. The genetic channelopathies identified to date are all uncommon disorders caused by obvious mutations in channel genes.

1	As the full repertoire of human ion channels and related proteins is The resting potential of neurons and the action potentials responsible identified, it is likely that additional channelopathies will be discovfor impulse conduction are generated by ion currents and ion channels. ered. In addition to rare disorders that result from obvious mutations, Most ion channels are gated, meaning that they can transition between it is also likely that less penetrant allelic variations in channel genes conformations that are open or closed to ion conductance. Individual or in their pattern of expression might underlie susceptibility to some ion channels are distinguished by the specific ions they conduct; by apparently sporadic forms of epilepsy, migraine, or other disorders. their kinetics; and by whether they directly sense voltage, are linked to For example, mutations in the potassium channel gene Kir2.6 have receptors for neurotransmitters or other ligands such as neurotroph-been found in

1	whether they directly sense voltage, are linked to For example, mutations in the potassium channel gene Kir2.6 have receptors for neurotransmitters or other ligands such as neurotroph-been found in many individuals with thyrotoxic hypokalemic periodic ins, or are activated by second messengers. The diverse characteristics paralysis, a disorder similar to hypokalemic periodic paralysis but preof different ion channels provide a means by which neuronal excitabil-cipitated by stress from thyrotoxicosis or carbohydrate loading. ity can be exquisitely modulated at both the cellular and the subcellular levels. Disorders of ion channels—channelopathies—are responsible for a growing list of human neurologic diseases (Table 444e-1). Most Synaptic neurotransmission is the predominant means by which neuare caused by mutations in ion channel genes or by autoantibodies rons communicate with each other. Classic neurotransmitters are syn-against ion channel proteins. One example is epilepsy, a

1	which neuare caused by mutations in ion channel genes or by autoantibodies rons communicate with each other. Classic neurotransmitters are syn-against ion channel proteins. One example is epilepsy, a syndrome of thesized in the presynaptic region of the nerve terminal; stored in vesdiverse causes characterized by repetitive, synchronous firing of neu-icles; and released into the synaptic cleft, where they bind to receptors ronal action potentials. Action potentials are normally generated by the on the postsynaptic cell. Secreted neurotransmitters are eliminated by opening of sodium channels and the inward movement of sodium ions reuptake into the presynaptic neuron (or glia), by diffusion away from down the intracellular concentration gradient. Depolarization of the the synaptic cleft, and/or by specific inactivation. In addition to the neuronal membrane opens potassium channels, resulting in outward classic neurotransmitters, many neuropeptides have been identified movement of

1	and/or by specific inactivation. In addition to the neuronal membrane opens potassium channels, resulting in outward classic neurotransmitters, many neuropeptides have been identified movement of potassium ions, repolarization, closure of the sodium as definite or probable neurotransmitters; these include substance P, channel, and hyperpolarization. Sodium or potassium channel subunit neurotensin, enkephalins, β-endorphin, histamine, vasoactive intesgenes have long been considered candidate disease genes in inherited tinal polypeptide, cholecystokinin, neuropeptide Y, and somatostatin.

1	Peptide neurotransmitters are synthesized in the cell body rather than the nerve terminal and may colocalize with classic neurotransmitters in single neurons. A number of neuropeptides are important in pain modulation including substance P and calcitonin gene-related peptide (CGRP), which causes migraine-like headaches in patients. As a consequence, CGRP receptor antagonists have been developed and shown to be effective in treating migraine headaches. Nitric oxide and carbon monoxide are gases that appear also to function as neurotransmitters, in part by signaling in a retrograde fashion from the postsynaptic to the presynaptic cell.

1	Neurotransmitters modulate the function of postsynaptic cells by binding to specific neurotransmitter receptors, of which there are two major types. Ionotropic receptors are direct ion channels that open after engagement by the neurotransmitter. Metabotropic receptors interact with G proteins, stimulating production of second messengers and activating protein kinases, which modulate a variety of cellular events. Ionotropic receptors are multiple subunit structures, whereas metabotropic receptors are composed of single subunits only. One important difference between ionotropic and metabotropic receptors is that the kinetics of ionotropic receptor effects are fast (generally <1 ms) because 444e-3 neurotransmitter binding directly alters the electrical properties of the postsynaptic cell, whereas metabotropic receptors function over longer time periods. These different properties contribute to the potential for selective and finely modulated signaling by neurotransmitters.

1	Neurotransmitter systems are perturbed in a large number of clinical disorders, several of which are highlighted in Table 444e-2. One example is the involvement of dopaminergic neurons originating in the substantia nigra of the midbrain and projecting to the striatum (nigrostriatal pathway) in Parkinson’s disease and in heroin addicts after the ingestion of the toxin MPTP (1-methyl-4-phenyl-1,2,5,6tetrahydropyridine). A second important dopaminergic system arising in the midbrain is the mediocorticolimbic pathway, which is implicated in the pathogenesis of addictive behaviors including drug reward. Its key components include the midbrain ventral tegmental area (VTA), median forebrain bundle, and nucleus accumbens (see Fig. 465e-2). The cholinergic pathway originating in the nucleus basalis of Meynert plays a role in memory function in Alzheimer’s disease. Chapter 444e Biology of Neurologic Diseases Arcuate nucleus of hypothalamus → anterior pituitary (via portal veins)

1	Chapter 444e Biology of Neurologic Diseases Arcuate nucleus of hypothalamus → anterior pituitary (via portal veins) Locus coeruleus (pons) → limbic system, hypothalamus, cortex Medulla → locus coeruleus, spinal cord Postganglionic neurons of sympathetic nervous system Medulla/pons → dorsal horn of spinal cord Major excitatory neurotransmitter; located throughout CNS, including cortical pyramidal cells Acetylcholinesterases (nerve gases) Myasthenia gravis (antibodies to ACh receptor) Congenital myasthenic syndromes (mutations in ACh Lambert-Eaton syndrome (antibodies to Ca channels impair ACh release) Botulism (toxin disrupts ACh release by exocytosis) Alzheimer disease (selective cell death) Autosomal dominant frontal lobe epilepsy (mutations in CNS Parkinson’s disease (selective cell death) MPTP parkinsonism (toxin transported into neurons) Addiction, behavioral disorders Inhibits prolactin secretion

1	Parkinson’s disease (selective cell death) MPTP parkinsonism (toxin transported into neurons) Addiction, behavioral disorders Inhibits prolactin secretion Stiff person syndrome (antibodies to glutamic acid decarboxylase, the biosynthetic enzyme for GABA) Epilepsy (gabapentin and valproic acid increase GABA) Spasticity Hyperekplexia (myoclonic startle syndrome) due to mutations in glycine receptor Seizures due to ingestion of domoic acid (a glutamate Abbreviations: CNS, central nervous system; MAOA, monoamine oxidase A; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; SSRI, selective serotonin reuptake inhibitor.

1	Addictive drugs share the property of increasing dopamine release in the nucleus accumbens (Chap. 465e). Amphetamine increases intracellular release of dopamine from vesicles and reverses transport of dopamine through the dopamine transporters. Patients prone to addiction show increased activation of the nucleus accumbens following administration of amphetamine. Cocaine binds to dopamine transporters and inhibits dopamine reuptake. Ethanol inhibits inhibitory neurons in the VTA, leading to increased dopamine release in the nucleus accumbens. Opioids also disinhibit these dopaminergic neurons by binding to μ receptors expressed by γ-aminobutyric acid (GABA)–containing interneurons in the VTA. Nicotine increases dopamine release by activating nicotinic acetylcholine receptors on cell bodies and nerve terminals of dopaminergic VTA neurons. Tetrahydrocannabinol, the active ingredient of cannabis, also increases dopamine levels in the nucleus accumbens. Blockade of dopamine in the nucleus

1	and nerve terminals of dopaminergic VTA neurons. Tetrahydrocannabinol, the active ingredient of cannabis, also increases dopamine levels in the nucleus accumbens. Blockade of dopamine in the nucleus accumbens can terminate the rewarding effects of addictive drugs.

1	Not all cell-to-cell communication in the nervous system occurs via neurotransmission. Gap junctions provide for direct neuron-neuron electrical conduction and also create openings for the diffusion of ions and metabolites between cells. In addition to neurons, gap junctions are also widespread in glia, creating a syncytium that protects neurons by removing glutamate and potassium from the extracellular environment. Gap junctions consist of membrane-spanning proteins, termed connexins, that pair across adjacent cells. Mechanisms that involve gap junctions have been related to a variety of neurologic disorders. Mutations in connexin 32, a gap junction protein expressed by Schwann cells, are responsible for the X-linked form of Charcot-Marie-Tooth (CMT) disease (Chap. 459). Mutations in either of two gap junction proteins expressed in the inner ear—connexin 26 and connexin 31—result in autosomal dominant progressive hearing loss (Chap. 43). Glial calcium waves mediated through gap

1	either of two gap junction proteins expressed in the inner ear—connexin 26 and connexin 31—result in autosomal dominant progressive hearing loss (Chap. 43). Glial calcium waves mediated through gap junctions also appear to explain the phenomenon of spreading depression associated with migraine auras and the march of epileptic discharges. Spreading depression is a neural response that follows a variety of different stimuli and is characterized by a circumferentially expanding negative potential that propagates at a characteristic speed of 20 m/s and is associated with an increase in extracellular potassium.

1	The fundamental issue of how memory, learning, and thinking are encoded in the nervous system is likely to be clarified by identifying the signaling pathways involved in neuronal differentiation, axon guidance, and synapse formation, and by understanding how these pathways are modulated by experience. Many families of transcription factors, each comprising multiple individual components, are expressed in the nervous system. Elucidation of these signaling pathways has already begun to provide insights into the cause of a variety of neurologic disorders, including inherited disorders of cognition such as X-linked mental retardation. This problem affects ~1 in 500 males, and linkage studies in different families suggest that as many as 60 different X-chromosome-encoded genes may be responsible. The formation of RNA-DNA duplexes that block transcription has also been observed with the CGG repeat expansions that occur in fragile X gene-associated mental retardation. Rett’s syndrome, a

1	The formation of RNA-DNA duplexes that block transcription has also been observed with the CGG repeat expansions that occur in fragile X gene-associated mental retardation. Rett’s syndrome, a common cause of (dominant) X-linked progressive mental retardation in females, is due to a mutation in a gene (MECP2) encoding a DNA-binding protein involved in transcriptional repression. Because the X chromosome comprises only ~3% of germline DNA, then by extrapolation, the number of genes that potentially contribute to clinical disorders affecting intelligence in humans must be potentially very large. As discussed below, there is increasing evidence that abnormal gene transcription may play a role in neurodegenerative diseases, such as Huntington’s disease, in which proteins with polyglutamine expansions bind to and sequester transcription factors. A critical transcription factor for neuronal survival is CREB (cyclic adenosine monophosphate responsive element-binding) protein, which also plays

1	bind to and sequester transcription factors. A critical transcription factor for neuronal survival is CREB (cyclic adenosine monophosphate responsive element-binding) protein, which also plays an important role in memory in the hippocampus. The regulatory gene repressor element 1-silencing transcription factor (REST) coordinates the expression of neuroprotective stress genes during normal aging. It turns off genes involved in cell death and pathology and boosts protective factors. High levels of REST are associated with normal cognition even in the presence of both amyloid plaques and neurofibrillary tangles. Although REST increases with normal aging, it fails to increase in the nucleus in patients with Alzheimer’s disease and is found clumped with amyloid in autophagosomes.

1	Myelin is the multilayered insulating substance that surrounds axons and speeds impulse conduction by permitting action potentials to jump between naked regions of axons (nodes of Ranvier) and across myelinated segments. Molecular interactions between the myelin membrane and axon are required to maintain the stability, function, and normal life span of both structures. A single oligodendrocyte usually ensheaths multiple axons in the central nervous system (CNS), whereas in the peripheral nervous system (PNS), each Schwann cell typically myelinates a single axon. Myelin is a lipid-rich material formed by a spiraling process of the membrane of the myelinating cell around the axon, creating multiple membrane bilayers that are tightly apposed (compact myelin) by charged protein interactions. Several inhibitors of axon growth are expressed on the innermost (periaxonal) lamellae of the myelin membrane (see “Stem Cells and Transplantation,” below). A number of clinically important neurologic

1	inhibitors of axon growth are expressed on the innermost (periaxonal) lamellae of the myelin membrane (see “Stem Cells and Transplantation,” below). A number of clinically important neurologic disorders are caused by inherited mutations in myelin proteins of the CNS or PNS (Fig. 444e-1). Constituents of myelin also have a

1	FIGURE 444e-1 The molecular architecture of the myelin sheath illustrating the most important disease-related proteins. The illustration represents a composite of central nervous system (CNS) and peripheral nervous system (PNS) myelin. Proteins restricted to CNS myelin are shown in green, proteins of PNS myelin are lavender, and proteins present in both CNS and PNS are red. In the CNS, the X-linked allelic disorders, Pelizaeus-Merzbacher disease and one variant of familial spastic paraplegia, are caused by mutations in the gene for proteolipid protein (PLP) that normally promotes extracellular compaction between adjacent myelin lamellae. The homologue of PLP in the PNS is the P0 protein, mutations in which cause the neuropathy Charcot-Marie-Tooth disease (CMT) type 1B. The most common form of CMT is the 1A subtype caused by a duplication of the PMP22 gene; deletions in PMP22 are responsible for another inherited neuropathy termed hereditary liability to pressure palsies (Chap. 459).

1	In multiple sclerosis (MS), myelin basic protein (MBP) and the quantitatively minor CNS protein, myelin oligodendrocyte glycoprotein (MOG), are likely T cell and B cell antigens, respectively (Chap. 458). The location of MOG at the outermost lamella of the CNS myelin membrane may facilitate its targeting by autoantibodies. In the PNS, autoantibodies against myelin gangliosides are implicated in a variety of disorders, including GQ1b in the Fisher variant of Guillain-Barré syndrome, GM1 in multifocal motor neuropathy, and sulfatide constituents of myelin-associated glycoprotein (MAG) in peripheral neuropathies associated with monoclonal gammopathies (Chap. 460). Chapter 444e Biology of Neurologic Diseases Mitochondrial swelling, rupture of outer membrane NOS Activation of caspase cascadeDNA/RNA oxidation

1	FIGURE 444e-2 Involvement of mitochondria in cell death. A severe excitotoxic insult (A) results in cell death by necrosis, whereas a mild excitotoxic insult (B) results in apoptosis. After a severe insult (such as ischemia), there is a large increase in glutamate activation of N-methyl-D-aspartate (NMDA) receptors, an increase in intracellular Ca2+ concentrations, activation of nitric oxide synthase (NOS), and increased mitochondrial Ca2+ and superoxide generation followed by the formation of ONOO–. This sequence results in damage to cellular macromolecules including DNA, leading to activation of poly-ADP-ribose polymerase (PARS). Both mitochondrial accumulation of Ca2+ and oxidative damage lead to activation of the permeability transition pore (PTP) that is linked to excitotoxic cell death. A mild excitotoxic insult can occur due either to an abnormality in an excitotoxicity amino acid receptor, allowing more Ca2+ flux, or to impaired functioning of other ionic channels or of energy

1	A mild excitotoxic insult can occur due either to an abnormality in an excitotoxicity amino acid receptor, allowing more Ca2+ flux, or to impaired functioning of other ionic channels or of energy production, which may allow the voltage-dependent NMDA receptor to be activated by ambient concentrations of glutamate. This event can then lead to increased mitochondrial Ca2+ and free radical production, yet relatively preserved ATP generation. The mitochondria may then release cytochrome c (Cytc), caspase 9, apoptosis-inducing factor (Aif), and perhaps other mediators that lead to apoptosis. The precise role of the PTP in this mode of cell death is still being clarified, but there does appear to be involvement of the adenine nucleotide transporter that is a key component of the PTP.

1	propensity to be targeted as autoantigens in autoimmune demyelinating disorders (Fig. 444e-2).

1	Specification to oligodendrocyte precursor cells (OPCs) is transcriptionally regulated by the Olig 2 and Yin Yang 1 genes, whereas myelination mediated by postmitotic oligodendrocytes depends on a different transcription factor, myelin gene regulatory factor (MRF). It is noteworthy that in the normal adult brain, large numbers of OPCs (expressing PDGFR-α and NG2) are widely distributed but do not myelinate axons, even in demyelinating environments such as in lesions of MS. Several families of molecules have now been identified that regulate oligodendrocyte differentiation and myelination, including LINGO-1, PSA-NCAM, hyaluronan, Nogo-A, the Wnt pathway, notch signaling (and its receptor Jagged), and the retinoic acid receptor RXRγ; all are inhibitory, with the exception of RXRγ, which is excitatory. All are also potential targets for myelin repair therapies, and a monoclonal antibody against LINGO-1 is in clinical testing for remyelination in MS. Very recently, a series of

1	which is excitatory. All are also potential targets for myelin repair therapies, and a monoclonal antibody against LINGO-1 is in clinical testing for remyelination in MS. Very recently, a series of observations has called into question the traditional concept that axon-derived cues are always required for myelination to occur. Fixed (i.e., dead) axons could be efficiently myelinated by oligodendrocytes in vitro, as could artificial polystyrene nanowires of a similar diameter. This led to development of new high-throughput screening assays based on myelination of polystyrene nanowires to identify compounds that could promote myelination.

1	Macrophages and microglia represent the major cell types in the nervous system responsible for antigen presentation and innate immunity. Brain macrophages are derived from either hematopoietic stem cell–derived bone marrow monocytes or from brain microglia that migrate from the yolk sac early in embryogenesis before the blood-brain barrier is formed. In a murine model of autoimmune demyelination, experimental allergic encephalomyelitis (EAE) (Fig. 444e-3), macrophages derived from bone marrow monocytes, but not microglia, were found to represent the critical population that initiated inflammatory demyelination at paraxonal regions near nodes of Ranvier. An additional, unexpected role for brain microglia was also identified in the regulation of neural circuits through pruning of excitatory synapses and control of dendritic spine densities; mice depleted of microglia during development exhibited a variety of cognitive learning and behavioral deficits, including abnormal social

1	synapses and control of dendritic spine densities; mice depleted of microglia during development exhibited a variety of cognitive learning and behavioral deficits, including abnormal social behaviors. Remarkably, depletion of microglia in adult mice by administration of a selective inhibitor of colony-stimulating factor receptor 1 (CSFR1) was followed by their rapid repopulation, suggesting that a pool of resident microglial precursor cells may exist throughout the CNS.

1	proteins? activation macrophages Chemokines IL-1, IL-12 Brain tissue TNF, IFN, free radicals, vasoactive amines, complement, proteases, cytokines, eicosanoids FIGURE 444e-3 A model for experimental allergic encephalomyelitis (EAE). Crucial steps for disease initiation and progression include peripheral activation of preexisting autoreactive T cells; homing to the central nervous system (CNS) and extravasation across the blood-brain barrier; reactivation of T cells by exposed autoantigens; secretion of cytokines; activation of microglia and astrocytes and recruitment of a secondary inflammatory wave; and immune-mediated myelin destruction. ICAM, intercellular adhesion molecule; IFN, interferon; IL, interleukin; LFA-1, leukocyte function-associated antigen-1; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule.

1	The human microbiome (Chap. 86e) represents the collective set of genes from the 1014 organisms living in our gut, skin, mucosa, and other sites. Different microbial communities are associated with different ethnicities, diets, and environments. In any individual, the predominant gut microbiota can be remarkably stable over decades, but also can be altered by exposure to certain microbial species, for example by ingestion of probiotics.

1	There is compelling evidence that gut microbes can shape immune responses through the interaction of their metabolism with that of humans. These gut-brain interactions are likely to be important in understanding the pathogenesis of many autoimmune neurologic diseases. For example, mice treated with broad-spectrum antibiotics are resistant to EAE, an effect associated with decreases in production of proinflammatory cytokines, and conversely more production of the immunosuppressive cytokines interleukin (IL) 10 and IL-13 and an increase in regulatory T and B lymphocytes. Oral administration of polysaccharide A (PSA) from Bacillus fragilis also protects mice from EAE, via increases in IL-10.

1	In addition to nonspecific effects on immune homeostasis mediated by cytokines and regulatory cells, some microbial proteins can trigger, in susceptible individuals, a cross-reactive immune response against a homologous protein in the nervous system, a mechanism termed molecular mimicry. Examples include cross-reactivity between the astrocyte water channel aquaporin-4 and an ABC transporter permease from Clostridia perfringens in neuromyelitis optica (Chap. 458); the neural ganglioside Gm1 and similar sialic acid–containing structures from Campylobacter jejuni in Guillain-Barré syndrome (Chap. 460); and the sleep-promoting protein hypocretin and hemagglutinin from H1N1 influenza virus in narcolepsy (Chap. 38), among others.

1	Recently, a number of tantalizing observations have incriminated the microbial environment in the pathogenesis of a much wider spectrum of neurologic conditions and behaviors, extending well beyond the traditional boundaries of immune-mediated pathologies. This is perhaps not surprising, as it has long been known in neurology that gut bacteria can influence brain function, based mostly on classic studies demonstrating that products of gut microbes can worsen hepatic encephalopathy, forming the basis of treatment with antibiotics for this condition.

1	Mice that developed in a completely germ-free environment displayed less anxiety, lower responses to stressful situations, more exploratory locomotive behaviors, and impaired memory formation compared with non-germ-free counterparts. These behaviors were related to changes in gene expression in pathways related to neural signaling, synaptic function, and modulation of neurotransmitters. Moreover, this behavior could be reversed when the germ-free mice were co-housed with non-germ-free mice.

1	The enteric autonomic nervous system in humans provides a bidirectional neural connection between the brain and gut. The vagus nerve, which innervates the upper gut and proximal colon, has been implicated in anxietyand depression-like behaviors in mice. Ingestion of Lactobacillus rhamnosus induced changes in expression of the inhibitory neurotransmitter GABA1b in neurons of the limbic cortex, hippocampus, and amygdala, associated with reduced levels of corticosteroids and reduced anxietyand depression-like behaviors. Remarkably, these changes could be blocked by vagotomy.

1	Another area of emerging interest is in a possible contribution of the gut microbiome to autism and related disorders. Children with autistic spectrum disorders have long been known to have gastrointestinal disturbances, and it has been claimed that the severity of dysbiosis correlates with the severity of autism. A murine model of autism was recently induced in offspring after injecting the pregnant mother with the viral RNA mimic polyinosinic:polycytidylic acid (poly I:C). Remarkably, oral treatment of offspring with B. fragilis corrected a range of autistic behaviors in these mice and also improved gut permeability.

1	Neurotrophic factors (Table 444e-3) are secreted proteins that modulate neuronal growth, differentiation, repair, and survival; some have additional functions, including roles in neurotransmission and in the synaptic reorganization involved in learning and memory. The neurotrophin (NT) family contains nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT3, and NT4/5. The neurotrophins act at TrK and p75 receptors to promote survival of neurons. BDNF is linked to synaptogenesis. Certain polymorphisms are linked to increased risk for Alzheimer’s disease (AD), and BDNF is depleted in Huntington’s disease. Because of their survival-promoting and antiapoptotic effects, neurotrophic factors are in theory outstanding candidates for therapy of disorders characterized by premature death of neurons such as occurs in ALS and other degenerative motor neuron disorders. Knockout mice lacking receptors for ciliary neurotrophic factor (CNTF) or BDNF show loss of motor neurons, and

1	death of neurons such as occurs in ALS and other degenerative motor neuron disorders. Knockout mice lacking receptors for ciliary neurotrophic factor (CNTF) or BDNF show loss of motor neurons, and experimental motor neuron death can be rescued by treatment with various neurotrophic factors including CNTF, BDNF, and vascular endothelial growth factor (VEGF). However, in phase 3 clinical trials, growth factors were ineffective in human ALS. The growth factor glial-derived neurotrophic factor (GDNF) is important for survival of dopaminergic neurons. Direct infusions of GDNF showed initial promise in Parkinson’s disease, but the benefits were not replicated in a larger clinical trial.

1	The nervous system is traditionally considered to be a nonmitotic organ, in particular with respect to neurons. These concepts have been challenged by the finding that neural progenitor or stem cells exist in the adult CNS that are capable of differentiation, migration over long distances, and extensive axonal arborization and synapse formation with appropriate targets. These capabilities also indicate that the repertoire of factors required for growth, survival, differentiation, and migration of these cells exists in the mature nervous system. In rodents, neural stem cells, defined as progenitor cells capable of differentiating into mature cells of neural or glial lineage, have been experimentally propagated from fetal CNS and neuroectodermal tissues and also from adult germinal matrix and ependyma regions. Human fetal CNS tissue is also capable of differentiation into cells with neuronal, astrocyte, and oligodendrocyte morphology when cultured in the presence of growth factors.

1	Once the repertoire of signals required for cell type specification is better understood, differentiation into specific neural or glial subpopulations can be directed in vitro; such cells could also be engineered to express therapeutic molecules. Another promising approach is to use growth factors, such as BDNF, to stimulate endogenous stem cells to 444e-7 proliferate and migrate to areas of neuronal damage.

1	A major advance has been the development of induced pluripotent stem cells. Using this technique, adult somatic cells such as skin fibroblasts are treated with four pluripotency factors (SOX2, KLF4, cMYC, and Oct4), and this generates induced pluripotent stem cells (iPSCs). These adult-derived stem cells sidestep the ethical issues of using stem cells derived from human embryos. The development of these cells has tremendous promise for both studying disease mechanisms and testing therapeutics. As yet there is no consensus on the best way to generate and differentiate the iPSCs; however, techniques to avoid using viral vectors and use of Cre-lox systems to remove reprogramming factors result in a better match of gene expression profiles with those of embryonic stem cells. Over the years, the field of directed differentiation has used three main strategies to specify neural lineages from human pluripotent stem cells. These strategies are embryoid body formation, coculture on

1	years, the field of directed differentiation has used three main strategies to specify neural lineages from human pluripotent stem cells. These strategies are embryoid body formation, coculture on neural-inducing feeders, and direct neural induction. Thus far, iPSCs have been made from patients with all of the major human neurodegenerative diseases, and studies using them are under way.

1	Although stem cells hold tremendous promise for the treatment of debilitating neurologic diseases, such as Parkinson’s disease and spinal cord injury, it should be emphasized that medical application is in its infancy. Major obstacles are the generation of positionand neurotransmitter-defined subtypes of neurons and their isolation as pure populations of the desired cells. This is crucial to avoid persistence of undifferentiated embryonic stem (ES) cells, which can generate tumors. The establishment of appropriate neural connections and afferent control is also critical. For instance, human ES motor neurons will need to be introduced at multiple segments in the neuraxis, and then their axons will need to regenerate from the spinal cord to distal musculature.

1	Experimental transplantation of human fetal dopaminergic neurons in patients with Parkinson’s disease has shown that these transplanted cells can survive within the host striatum; however, some patients developed disabling dyskinesias, and this approach is no longer in clinical development. The possibility that iPSCs will be used in Parkinson’s disease was strengthened by studies showing that they can be differentiated into dopaminergic neurons. The dopaminergic neurons were then shown to rescue the parkinsonian phenotype in a MPTP-induced primate model with excellent dopaminergic neuron survival function and lack of neural overgrowth. The correction of tau mutations in iPSC-derived neurons has been shown to reverse the toxic phenotype in dendrite retraction and cell death.

1	Another new use for iPSCs is to screen drugs as potential treatments for neurodegenerative and other diseases. The feasibility of this has been shown using iPSC-induced macrophages from patients with Gaucher’s disease, and verifying the efficacy of protein chaperones in these cells as a means of stabilizing the mutant glucocerebrosidase and increasing its activity and the duration of its effects. Other approaches are to attempt to reduce expression of proteins, such as amyloid, tau, and α-synuclein, implicated in the pathogenesis of neurodegenerative diseases. One difficulty has been that reprogramming cells to iPSCs resets their identity back to an embryonic age, which is a hurdle in modeling of late-onset diseases. One approach to this has been to express a fragment of the mutated gene, such as a portion of lamin A, which causes premature aging in progeria. This approach showed that dendrite degeneration and progressive loss of tyrosine hydroxylase expression, as well as enlarged

1	such as a portion of lamin A, which causes premature aging in progeria. This approach showed that dendrite degeneration and progressive loss of tyrosine hydroxylase expression, as well as enlarged mitochondria and Lewy body precursor inclusions, were induced in iPSC-derived dopaminergic neurons with progerin-induced aging.

1	Studies of transplantation for patients with Huntington’s disease have also reported encouraging, although very preliminary, results. OPCs transplanted into mice with a dysmyelinating disorder effectively migrated in the new environment, interacted with axons, and mediated myelination; such experiments raise hope that similar transplantation strategies may be feasible in human disorders of myelin such as MS. The promise of stem cells for treatment of both neurodegenerative diseases and neural injury is great, but development has Chapter 444e Biology of Neurologic Diseases been slowed by unresolved concerns over safety (including the theoretical risk of malignant transformation of transplanted cells), ethics (particularly with respect to use of fetal tissue), and efficacy.

1	In developing brain, the extracellular matrix provides stimulatory and inhibitory signals that promote neuronal migration, neurite outgrowth, and axonal extension. After neuronal damage, reexpression of inhibitory molecules such as chondroitin sulfate proteoglycans may prevent tissue regeneration. Chondroitinase degraded these inhibitory molecules and enhanced axonal regeneration and motor recovery in a rat model of spinal cord injury. Several myelin proteins, specifically Nogo, oligodendrocyte myelin glycoprotein (OMGP), and myelin-associated glycoprotein (MAG), may also interfere with axon regeneration. Sialidase, which cleaves one class of receptors for MAG, enhances axonal outgrowth. Antibodies against Nogo promote regeneration after experimental focal ischemia or spinal cord injury. Nogo, OMGP, and MAG all bind to the same neural receptor, the Nogo receptor, which mediates its inhibitory function via the p75 neurotrophin receptor signaling.

1	CELL DEATH: EXCITOTOXICITY AND APOPTOSIS

1	Excitotoxicity refers to neuronal cell death caused by activation of excitatory amino acid receptors (Fig. 444e-4). Compelling evidence for a role of excitotoxicity, especially in ischemic neuronal injury, is derived from experiments in animal models. Experimental models of stroke are associated with increased extracellular concentrations of the excitatory amino acid neurotransmitter glutamate, and neuronal damage is attenuated by denervation of glutamate-containing neurons or the administration of glutamate receptor antagonists. The distribution of cells sensitive to ischemia corresponds closely with that of N-methyld-aspartate (NMDA) receptors (except for cerebellar Purkinje cells, which are vulnerable to hypoxia-ischemia but lack NMDA receptors); and competitive and noncompetitive NMDA antagonists are effective in preventing focal ischemia. In global cerebral ischemia, non-NMDA receptors (kainic acid and α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate [AMPA]) are activated, and

1	antagonists are effective in preventing focal ischemia. In global cerebral ischemia, non-NMDA receptors (kainic acid and α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate [AMPA]) are activated, and antagonists to these receptors are protective. Experimental brain damage induced by hypoglycemia is also attenuated by NMDA antagonists.

1	Excitotoxicity is not a single event but rather a cascade of cell injury. Excitotoxicity causes influx of calcium into cells, and much of the calcium is sequestered in mitochondria rather than in the cytoplasm. Increased cytoplasmic calcium causes metabolic dysfunction and free radical generation; activates protein kinases, phospholipases, nitric oxide synthase, proteases, and endonucleases; and inhibits protein synthesis. Activation of nitric oxide synthase generates nitric oxide (NO•), which can react with superoxide (O•2) to generate peroxynitrite (ONOO−), which may play a direct role in neuronal injury. Another critical pathway is activation of poly-ADP-ribose polymerase, which occurs in response to free radical–mediated DNA damage. Experimentally, mice with knockout mutations of neuronal nitric oxide synthase or poly-ADP-ribose polymerase, or those that overexpress superoxide dismutase, are resistant to focal ischemia.

1	Another aspect of excitotoxicity is that it has been demonstrated that stimulation of extrasynaptic NMDA receptors mediates cell death, where as stimulation of synaptic receptors is protective. This has been shown to play a role in excitotoxicity in transgenic mouse models of

1	FIGURE 444e-4 Neurodegeneration caused by prions. A. In sporadic neurodegenerative diseases (NDs), wild-type (Wt) prions multiply through self-propagating cycles of posttranslational modification, during which the precursor protein (green circle) is converted into the prion form (red square), which generally is high in β-sheet content. Pathogenic prions are most toxic as oligomers and less toxic after polymerization into amyloid fibrils. The small polygons (blue) represent proteolytic cleavage products of the prion. Depending on the protein, the fibrils coalesce into Aβ amyloid plaques in Alzheimer’s disease (AD), neurofibrillary tangles in AD and Pick’s disease, or Lewy bodies in Parkinson’s disease and Lewy body dementia. Drug targets for the development of therapeutics include: (1) lowering the precursor protein, (2) inhibiting prion formation, and (3) enhancing prion clearance. B. Late-onset heritable neurodegeneration argues for two discrete events: The (i) first event is the

1	lowering the precursor protein, (2) inhibiting prion formation, and (3) enhancing prion clearance. B. Late-onset heritable neurodegeneration argues for two discrete events: The (i) first event is the synthesis of mutant precursor protein (green circle), and the (ii) second event is the age-dependent formation of mutant prions (red square). The highlighted yellow bar in the DNA structure represents mutation of a base pair within an exon, and the small yellow circles signify the corresponding mutant amino acid substitution. Green arrows represent a normal process; red arrows, a pathogenic process; and blue arrows, a process that is known to occur but unknown whether it is normal or pathogenic. (Micrographs prepared by Stephen J. DeArmond. Reprinted with permission from SB Prusiner: Biology and genetics of prions causing neurodegeneration. Annu Rev Genet 47:601, 2013.)

1	Huntington’s disease, in which using low-dose memantine to selectively block the extrasynaptic receptors is beneficial.

1	Although excitotoxicity is clearly implicated in the pathogenesis of cell death in stroke, to date treatment with NMDA antagonists has not proven to be clinically useful. One approach has been to use an inhibitor of the postsynaptic density-95 protein that uncouples NMDA receptors from neurotoxic pathways, including the generation of nitric oxide. This approach was effective in a primate stroke model and in a phase 2 clinical trial of stroke associated with endovascular repair of cerebral aneurysms. Transient receptor potentials (TRPs) are calcium channels that are activated by oxidative stress in parallel with excitotoxic signal pathways. In addition, glutamate-independent pathways of calcium influx via acid-sensing ion channels have been identified. These channels transport calcium in the setting of acidosis and substrate depletion, and pharmacologic blockade of these channels markedly attenuates stroke injury. These channels offer a potential new therapeutic target for stroke.

1	Apoptosis, or programmed cell death, plays an important role in both physiologic and pathologic conditions. During embryogenesis, apoptotic pathways operate to destroy neurons that fail to differentiate appropriately or reach their intended targets. There is mounting evidence for an increased rate of apoptotic cell death in a variety of acute and chronic neurologic diseases. Apoptosis is characterized by neuronal shrinkage, chromatin condensation, and DNA fragmentation, whereas necrotic cell death is associated with cytoplasmic and mitochondrial swelling followed by dissolution of the cell membrane. Apoptotic and necrotic cell death can coexist or be sequential events, depending on the severity of the initiating insult. Cellular energy reserves appear to have an important role in these two forms of cell death, with apoptosis favored under conditions in which ATP levels are preserved. Evidence of DNA fragmentation has been found in a number of degenerative neurologic disorders,

1	two forms of cell death, with apoptosis favored under conditions in which ATP levels are preserved. Evidence of DNA fragmentation has been found in a number of degenerative neurologic disorders, including AD, Huntington’s disease, and ALS. The best characterized genetic neurologic disorder related to apoptosis is infantile spinal muscular atrophy (Werdnig-Hoffmann disease), in which two genes thought to be involved in the apoptosis pathways are causative.

1	Mitochondria are essential in controlling specific apoptosis pathways. The redistribution of cytochrome c, as well as apoptosis-inducing factor (AIF), from mitochondria during apoptosis leads to the activation of a cascade of intracellular proteases known as caspases. Caspase-independent apoptosis occurs after DNA damage, activation of poly-ADP-ribose polymerase, and translocation of AIF into the nucleus. Redistribution of cytochrome c is prevented by overproduction of the apoptotic protein BCL2 and is promoted by the proapoptotic protein BAX. These pathways may be triggered by activation of a large pore in the mitochondrial inner membrane known as the permeability transition pore, although in other circumstances, they occur independently. The permeability transition pore is made up of dimers of ATP synthase and is activated by cyclophilin D, leading to large calcium fluxes across the inner mitochondrial membrane. Certain forms of congenital muscular dystrophies are caused by

1	up of dimers of ATP synthase and is activated by cyclophilin D, leading to large calcium fluxes across the inner mitochondrial membrane. Certain forms of congenital muscular dystrophies are caused by mutations in collagen VI, which leads to increased activation of the permeability transition pore. Recent studies suggest that blocking the mitochondrial pore reduces both hypoglycemic and ischemic cell death. Mice deficient in cyclophilin D, a key protein involved in opening the permeability transition pore, are resistant to necrosis produced by focal cerebral ischemia.

1	The possibility that protein aggregation plays a role in the pathogenesis of neurodegenerative diseases is a major focus of current research. Protein aggregation is a major histopathologic hallmark of neurodegenerative diseases. Deposition of β-amyloid is strongly implicated in the pathogenesis of AD. Genetic mutations in familial AD cause increased production of β-amyloid with 42 amino acids, which has an increased propensity to aggregate, as compared to β-amyloid with 40 amino acids. Furthermore, mutations in the amyloid precursor protein (APP), which reduce the production of β-amyloid, protect against the development of AD and are associated with preserved cognition in the elderly. Mutations in genes encoding the MAPT lead 444e-9 to altered splicing of tau and the production of neurofibrillary tangles in frontotemporal dementia and progressive supranuclear palsy. Familial Parkinson’s disease is associated with mutations in leucinerich repeat kinase 2 (LRRK2), α-synuclein, parkin,

1	tangles in frontotemporal dementia and progressive supranuclear palsy. Familial Parkinson’s disease is associated with mutations in leucinerich repeat kinase 2 (LRRK2), α-synuclein, parkin, PINK1, and DJ-1. PINK1 is a mitochondrial kinase (see below), and DJ-1 is a protein involved in protection from oxidative stress. Parkin, which causes autosomal recessive early-onset Parkinson’s disease, is a ubiquitin ligase. The characteristic histopathologic feature of Parkinson’s disease is the Lewy body, an eosinophilic cytoplasmic inclusion that contains both neurofilaments and α-synuclein. Huntington’s disease and cerebellar degenerations are associated with expansions of polyglutamine repeats in proteins, which aggregate to produce neuronal intranuclear inclusions. Familial ALS is associated with superoxide dismutase mutations and cytoplasmic inclusions containing superoxide dismutase. An important finding was the discovery that ubiquinated inclusions observed in most cases of ALS and the

1	superoxide dismutase mutations and cytoplasmic inclusions containing superoxide dismutase. An important finding was the discovery that ubiquinated inclusions observed in most cases of ALS and the most common form of frontotemporal dementia are composed of TAR DNA binding protein 43 (TDP-43). Subsequently, mutations in the TDP-43 gene, and in the fused in sarcoma gene (FUS), were found in familial ALS. Both of these proteins are involved in transcription regulation as well as RNA metabolism. In autosomal dominant neurohypophyseal diabetes insipidus, mutations in vasopressin result in abnormal protein processing, accumulation in the endoplasmic reticulum, and cell death.

1	Another key mechanism linked to cell death is mitochondrial dynamics, which refers to the processes involved in movement of mitochondria, as well as in mitochondrial fission and fusion, which play a critical role mitochondrial turnover and in replenishment of damaged mitochondria. Mitochondrial dysfunction is strongly linked to the pathogenesis of a number of neurodegenerative diseases such as Friedreich’s ataxia, which is caused by mutations in an iron-binding protein that plays an important role in transferring iron to iron-sulfur clusters in aconitase and complex I and II of the electron transport chain. Mitochondrial fission is dependent on the dynamin-related proteins (Drp1), which bind to its receptor Fis, whereas mitofuscins 1 and 2 (MF 1/2) and optic atrophy protein 1 (OPA1) are responsible for fusion of the outer and inner mitochondrial membrane, respectively. Mutations in MFN2 cause Charcot-Marie-Tooth neuropathy type 2A, and mutations in OPA1 cause autosomal dominant optic

1	for fusion of the outer and inner mitochondrial membrane, respectively. Mutations in MFN2 cause Charcot-Marie-Tooth neuropathy type 2A, and mutations in OPA1 cause autosomal dominant optic atrophy. Both β-amyloid and mutant huntingtin protein induce mitochondrial fragmentation and neuronal cell death associated with increased activity of Drp1. In addition, mutations in genes causing autosomal recessive Parkinson’s disease, parkin and PINK1, cause abnormal mitochondrial morphology and result in impairment of the ability of the cell to remove damaged mitochondria by autophagy.

1	One major scientific question is whether protein aggregates directly contribute to neuronal death or whether they are merely secondary bystanders. A current focus in all the neurodegenerative diseases is on small protein aggregates termed oligomers. These may be the toxic species of β-amyloid, α-synuclein, and proteins with expanded polyglutamines such as are associated with Huntington’s disease. Protein aggregates are usually ubiquinated, which targets them for degradation by the 26S component of the proteasome. An inability to degrade protein aggregates could lead to cellular dysfunction, impaired axonal transport, and cell death by apoptotic mechanisms.

1	Autophagy is the degradation of cystolic components in lysosomes. There is increasing evidence that autophagy plays an important role in degradation of protein aggregates in the neurodegenerative diseases, and it is impaired in AD, Parkinson’s disease, and Huntington’s disease. Autophagy is particularly important to the health of neurons, and failure of autophagy contributes to cell death. In Huntington’s disease, a failure of cargo recognition occurs, contributing to protein aggregates and cell death. Rapamycin, which induces autophagy, exerts beneficial therapeutic effects in transgenic mouse models of AD, Parkinson’s disease, and Huntington’s disease. There is other evidence for lysosomal dysfunction and impaired autophagy in Parkinson’s disease. Mutations in glucocerebrosidase are associated with 5% of all Parkinson’s disease cases as well as 8–9% of patients with dementia with Lewy bodies. Therefore, this is the most

1	Chapter 444e Biology of Neurologic Diseases important genetic cause of both disorders thus far identified. There appear to be reciprocal interactions between glucocerebrosidase and α-synuclein. It has been shown that glucocerebrosidase concentrations and enzymatic activity are reduced in the substantia nigra of sporadic Parkinson’s disease patients. Furthermore, α-synuclein is degraded by chaperone-mediated and macro autophagy. The degradation of α-synuclein has been shown to be impaired in transgenic mice deficient in glucocerebrosidase as well as in mice in which the enzyme has been inhibited. Furthermore, it is known that α-synuclein inhibits the activity of glucocerebrosidase. Therefore, there is bidirectional feedback between α-synuclein and glucocerebrosidase. An attractive therapeutic intervention could be to use protein chaperones to increase the activity and duration of action of glucocerebrosidase. This would also reduce α-synuclein levels and block the degeneration of

1	intervention could be to use protein chaperones to increase the activity and duration of action of glucocerebrosidase. This would also reduce α-synuclein levels and block the degeneration of dopaminergic neurons.

1	The retromer complex is a conserved membrane-associated protein complex that functions in the endosome-to-Golgi complex. The retromer complex contains a cargo selective complex consisting of VPS35, VPS26, and VPS29, along with a sorting nexin dimer. Recently, mutations in VPS35 were shown to be a cause of late-onset autosomal dominant Parkinson’s disease. The retromer also traffics APP away from endosomes, where it is cleaved to generate β-amyloid. Deficiencies of VPS35 and VPS26 were also identified in hippocampal brain tissue from AD. A new therapeutic approach to these diseases might therefore be to use chaperones to stabilize the retromer and reduce the generation of β-amyloid and α-synuclein.

1	The LRRK2 mutations were shown to have effects on clearance of Golgi-derived vesicles through the autophagy-lysosome system both in vitro and in vivo. LRRK2 mutations also are linked to elevated protein synthesis mediated by ribosomal protein s15 phosphorylation. Blocking this phosphorylation reduces LRRK2-mediated neurite loss and cell death in human dopamine and cortical neurons.

1	Interestingly, in experimental models of Huntington’s disease and cerebellar degeneration, protein aggregates are not well correlated with neuronal death and may be protective. A substantial body of evidence suggests that the mutant proteins with polyglutamine expansions in these diseases bind to transcription factors and that this contributes to disease pathogenesis. In Huntington’s disease, there is dysfunction of the transcriptional co-regulator, PGC-1α, a key regulator of mitochondrial biogenesis. There is evidence that impaired function of PGC-1α is also important in both Parkinson’s disease and AD, making it an attractive target for treatments. Agents that upregulate gene transcription are neuroprotective in animal models of these diseases. A number of compounds have been developed to block β-amyloid production and/or aggregation, and these agents are being studied in early clinical trials in humans. Another approach under investigation is immunotherapy with antibodies that bind

1	block β-amyloid production and/or aggregation, and these agents are being studied in early clinical trials in humans. Another approach under investigation is immunotherapy with antibodies that bind β-amyloid, tau, or α-synuclein. These studies have shown efficacy in preventing the spread of amyloid, tau, and α-synuclein in animal studies, raising hopes that this could lead to effective therapies by blocking neuron-to-neuron propagation. Two large clinical trials of β-amyloid immunotherapy, however, did not show efficacy, although this therapeutic strategy is still being studied.

1	As we have learned more about the etiology and pathogenesis of the neurodegenerative diseases, it has become clear that the histologic abnormalities that were once curiosities, in fact, are likely to reflect the etiologies. For example, the amyloid plaques in kuru and Creutzfeldt-Jakob disease (CJD) are filled with the PrPSc prions that have assembled into fibrils. The past three decades have witnessed an explosion of new knowledge about prions. For many years, kuru, CJD, and scrapie of sheep were thought to be caused by slow-acting viruses, but a large body of experimental evidence argues that the infectious pathogens causing these diseases are devoid of nucleic acid. Such pathogens are called prions, which are composed of host-encoded proteins that adopt alternative conformations (Chap. 453e). Prions are self-propagating by imposing their conformations on the normal, precursor protein; most prions are enriched for β-sheet and can assemble into amyloid fibrils.

1	Similar to the plaques in kuru and CJD that are composed of PrP prions, the amyloid plaques in AD are filled with Aβ prions that have polymerized into fibrils. This relationship between the neuropathologic findings and the etiologic prion was strengthened by the genetic linkage between familial CJD and mutations in the PrP gene, as well as (as noted above) between familial AD and mutations in the APP gene. Moreover, a mutation in the APP gene that prevents Aβ peptide formation was correlated with a decreased incidence of AD in Iceland.

1	The heritable neurodegenerative diseases offer an important insight into the pathogenesis of the more common, sporadic ones. Although the mutant proteins that cause these disorders are expressed in the brains of people early in life, the diseases do not occur for many decades. Many explanations for the late onset of familial neurodegenerative diseases have been offered, but none are supported by substantial experimental evidence. The late onset might be due to a second event in which a mutant protein, after its conversion into a prion, begins to accumulate at some rather advanced age (Fig. 444e-5). Such a formulation is also consistent with data showing that the protein quality-control mechanisms diminish in efficiency with age. Thus, the prion forms of both wild-type and mutant proteins are likely to be efficiently degraded in younger people but are less well handled in older individuals. This explanation is consistent with the view that neurodegenerative diseases are disorders of

1	are likely to be efficiently degraded in younger people but are less well handled in older individuals. This explanation is consistent with the view that neurodegenerative diseases are disorders of the aging nervous system.

1	A new classification for neurodegenerative diseases can be proposed based on not only the traditional phenotypic presentation and neuropathology, but also the prion etiology (Table 444e-4). Over the past decade, an expanding body of experimental data has accumulated implicating prions in each of these illnesses. In addition to kuru and CJD, Gerstmann-Sträussler-Scheinker disease (GSS) and fatal insomnia in humans are caused by PrPSc prions. In animals, PrPSc prions cause scrapie of sheep and goats, bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD) of deer and elk, feline spongiform encephalopathy, and transmissible mink encephalopathy (TME). Similar to PrP, Aβ, tau, α-synuclein, superoxide dismutase 1 (SOD1), and possibly huntingtin all adopt alternative conformations that become self-propagating, and thus, each protein can become a prion and be transferred to synaptically connected neurons. Moreover, each of these prions causes a distinct constellation of

1	that become self-propagating, and thus, each protein can become a prion and be transferred to synaptically connected neurons. Moreover, each of these prions causes a distinct constellation of neurodegenerative diseases.

1	Evidence for a prion etiology of AD comes from a series of transmission experiments initially performed in marmosets and more recently in transgenic (Tg) mice inoculated with a synthetic Aβ peptide folded into a prion. Studies with the tau protein have shown that it not only features in the pathogenesis of AD, but also causes such illnesses as the frontotemporal dementias including chronic traumatic encephalopathy, which has been reported in both contact sport athletes and military personnel who have suffered traumatic brain injuries. A series of incisive studies using cultured cells and Tg mice has demonstrated that tau can become a prion and multiply in the brain. In contrast to the Aβ and tau prions, a strain of α-synuclein prions found in the brains of patients who died of multiple system atrophy (MSA) killed the Tg mouse host ~90 days after intracerebral inoculation, whereas α-synuclein prions formed spontaneously in Tg mouse brains killed recipient mice in ~200 days.

1	For many years, the most frequently cited argument against prions was the existence of strains that produced distinct clinical presentations and different patterns of neuropathologic lesions. Some investigators argued that the biologic information carried in different prion strains could only be encoded within a nucleic acid. Subsequently, many studies demonstrated that strain-specified variation is enciphered in the conformation of PrPSc, but the molecular mechanisms responsible for the storage of biologic information remains enigmatic. The neuroanatomical patterns of prion deposition have been shown to be dependent on the particular strain of prion. Convincing evidence in support of this proposition has been accumulated for PrP, Aβ, tau, and α-synuclein prions.

1	i) Experiment 1: Lifespan ii) Experiment 2: Training * Midline Frontal Theta Power (dB) Initial older adults Single-task training Multitask training No contact control Older adult post-training * = p < .05 –1.50 –1.10 –0.80 –0.45 –0.10 0.25 0.60 0.95 1.30 1.65 2.00 2.35 2.70 3.05 3.40

1	FIGURE 444e-5 Video game training can enhance cognitive performance. A. An older participant engaging in NeuroRacer training (driving while responding to target signs), with (B) a screen shot of the experimental training session. C. NeuroRacer multitasking costs for target discrimination increased (i.e., a larger percentage decrease from single task performance when multitasking) in (i) a linear fashion across the lifespan and with (ii) costs before training 1 month after training and 6 months after training, showing a differential benefit of multitasking training compared to a no-contact control group and a single-task training group. D. Midline frontal theta activity obtained with electroencephalogram showed significantly enhanced activity only following multitasking training, mimicking the pattern of change in the behavioral data as well as performance improvements on untrained tests of working memory and sustained attention (not presented). For details, see JA Anguera et al:

1	Nature 501:97, 2013. Although the number of prions identified in mammals and in fungi continues to expand, the existence of prions in other phylogeny remains undetermined. Some mammalian prions perform vital functions and do not cause disease; such nonpathogenic prions include the cytoplasmic polyadenylation element binding (CPEB) protein, the mitochondrial antiviral-signaling (MAVS) protein, and T cell– restricted intracellular antigen 1 (TIA-1).

1	All mammalian prion proteins adopt a β-sheet-rich conformation and appear to readily oligomerize as this process becomes self-propagating. Control of the self-propagating state of benign mammalian prions is not well understood but is critical for the well-being of the host. In contrast, pathogenic mammalian prions appear to multiply exponentially, but the mechanisms by which they cause disease are poorly defined. We do not know if prions multiply as monomers or as oligomers; notably, the ionizing radiation target size of PrPSc prions seems to suggest it is a trimer. The oligomeric states of pathogenic mammalian prions are thought to be the toxic forms, and assembly into larger polymers, such as amyloid fibrils, seems to be a mechanism for minimizing toxicity.

1	To date, there is no medication that halts or even slows a human neurodegenerative disease. The development of drugs designed to inhibit the conversion of the normal precursor proteins into prions or to enhance the degradation of prions focuses on the initial step in prion accumulation. Although several drugs that cross the blood-brain barrier have been identified that prolong the lives of mice infected with scrapie prions, none have been identified that extend the lives (BSE) Scrapie Chronic wasting disease (CWD) Feline spongiform encephalopathy Transmissible mink encephalopathy Alzheimer’s disease (AD) Aβ → tau Parkinson’s disease α-Synuclein Frontotemporal dementias (FTDs) Tau, TDP43, FUS (C9orf72,

1	Posttraumatic FTD, called chronic traumatic encephalopathy Amyotrophic lateral sclerosis SOD1, TDP43, FUS (C9orf72) Huntington’s disease Huntingtin of Tg mice that replicate human CJD prions. Despite doubling the length of incubation times in mice inoculated with scrapie prions, all of the mice eventually succumb to illness. Because all of the treated mice develop neurologic dysfunction at the same time, the mutation rate as judged by drug resistance is likely to approach 100%, which is much higher than mutation rates recorded for bacteria and viruses. Mutations in prions seem likely to represent conformational variants that are selected for in mammals where survival becomes limited by the fastest-replicating prions. The results of these studies make it likely that cocktails of drugs that attack a variety of prion conformers will be required for the development of effective therapeutics.

1	Systems neuroscience refers to study of the functions of neural circuits and how they relate to brain function, behavior, motor activity, and cognition. Brain imaging techniques, primarily functional magnetic resonance imaging (fMRI) and position emission tomography (PET), have made it possible to investigate, noninvasively and in awake individuals, cognitive processes such as perception, making judgments, paying attention, and thinking. This has allowed insights into how networks of neurons operate to produce behavior. Many of these studies at present are based on determining the connectivity of neural circuits and how they operate, and how this can be then modeled to produce improved understanding of physiologic processes. fMRI uses contrast mechanisms related to physiologic changes in tissue, and brain perfusion can be studied by observing the time course of changes in brain water signal as a bolus of injected paramagnetic gadolinium contrast moves through the brain. More recently,

1	and brain perfusion can be studied by observing the time course of changes in brain water signal as a bolus of injected paramagnetic gadolinium contrast moves through the brain. More recently, to study intrinsic contrast-related local changes in blood oxygenation with brain activity, blood-oxygen-level-dependent (BOLD) contrast has been used to provide a rapid noninvasive approach for functional assessment. These techniques have been reliably used in the field of both behavior and cognitive sciences. One example is the use of fMRI to demonstrate mirror neuron systems, imitative pathways activated when observing actions of others. Mirror neurons are thought to be important for social conditioning and for many forms of learning, and abnormalities in mirror neurons may underlie some autism disorders.

1	Both structural and functional connectivity methods show large-scale network dysfunction in AD and frontotemporal dementia. The networks targeted have been defined as the default network in AD and the salience network in frontotemporal dementia. The default network is characterized by an area of reduced glucose metabolism in the temporoparietal cortex, which precedes the onset of dementia and which is an area preferentially affected by amyloid deposition. These networks are now thought to be pathways accounting for the spread of abnormally templated proteins (prions; see above), including β-amyloid, tau, and α-synuclein.

1	Other examples of the use of fMRI include the study of memory, revealing that not only is hippocampal activity correlated with declarative memory consolidation, but it also involves activation in the ventral medial prefrontal cortex. Consolidation of memory over time results in decreased activity of the hippocampus and progressively stronger activation in the ventral medial prefrontal region associated with retrieval of consolidated memories. fMRI has also been used to identify sequences of brain activation involved in normal movements and alterations in their activation associated with both injury and recovery, to plan neurosurgical operations, and remarkably, to reconstruct actual visual images from the occipital cortex. Noninvasive brain-computer interfaces have extraordinary potential to advance the development of robotics and exoskeleton devices guided by brain activity for patients with a variety of nervous system afflictions. Diffusion tensor imaging is a recently developed MRI

1	to advance the development of robotics and exoskeleton devices guided by brain activity for patients with a variety of nervous system afflictions. Diffusion tensor imaging is a recently developed MRI technique that can measure macroscopic axonal organization in nervous system tissues; it appears to be useful in assessing myelin and axonal injuries as well as brain development. Advances in understanding neural processing have led to the development of the ability to demonstrate that humans have online voluntary control of human temporal lobe neurons.

1	Multitasking capabilities, including attention to tasks when faced with distractions, decline as we age due to a decline in medial prefrontal cognitive control system. When faced with a multitasking challenge, video game training can improve cognitive control capabilities by augmenting prefrontal suppression of the default network and, as measured by electroencephalography and fMRI, result in improved performance that is sustained and, importantly, transfers to other cognitive tasks not associated with the training paradigm. A significant recent advance in neuropathology has discovered that sodium dodecyl sulfate (SDS) detergent treatment can render the brain transparent (CLARITY), removing lipids while preserving most protein and structural elements and providing opportunities to identify brain structures and neural networks with unprecedented detail.

1	A therapeutic technology that has long-reaching implications for the development of novel interventions for neurologic, including behavioral, conditions has been the development of deep-brain stimulation as a highly effective therapeutic intervention for treating excessively firing neurons in the subthalamic nucleus of patients with Parkinson’s disease and the precingulate cortex in patients with depression.

1	The BRAIN initiative, grand in scope, was launched in 2013 to speed development of advances to understand, treat, repair, and prevent common neurologic disorders that, in aggregate, affect more than 1 billion people worldwide. The initial goal of BRAIN is to bring together experts in neurobiology (including optogenetics), engineering, information technology, and other fields to develop novel visualization and electrophysiologic methods to better define and understand neural circuits and all the connections among individual neurons. The announcement of the BRAIN initiative followed just weeks after a similarly ambitious program, the Human Brain Project (HBP), was unveiled by the European Union. The HBP seeks to model individual neurons, neural circuits, and ultimately the entire brain using computer technologies. Its architects also envision layering clinical and biomarker data from large health care databases to identify biosignatures associated with human phenotypes, possibly leading

1	computer technologies. Its architects also envision layering clinical and biomarker data from large health care databases to identify biosignatures associated with human phenotypes, possibly leading to a fundamental reclassification of disease, a concept also proposed by others, including in a 2011 report of the National Academy of Sciences (Toward Precision Medicine: Building a Knowledge Network for Biomedical Research and a New Taxonomy of Disease). These two ambitious projects are expected to be complementary and over time will hopefully become increasingly integrated. Emerging discoveries are also certain to stimulate a range of their potential military use. Both the BRAIN and HBP initiatives have 444e-13 ethical questions, many of which are not unique to the neurosciences put into place strong ethical components to ensure that these programs but do come into sharpest focus in this area. These include possible risks are carried out, from the outset and to the fullest extent

1	place strong ethical components to ensure that these programs but do come into sharpest focus in this area. These include possible risks are carried out, from the outset and to the fullest extent possible, consisto the privacy of personal information about our health, cognitive capa-tent with guiding ethical principles that include respect for individuals, bilities, or behavioral attributes; the sanctity of our private thoughts; as public beneficence, justice and fairness, democratic deliberation, and well as concerns regarding neuroenhancement technologies, including transparency.

1	Chapter 444e Biology of Neurologic Diseases Daniel H. Lowenstein A seizure (from the Latin sacire, “to take possession of”) is a paroxysmal event due to abnormal excessive or synchronous neuronal activity in the brain. Depending on the distribution of discharges, this abnormal brain activity can have various manifestations, ranging from dramatic convulsive activity to experiential phenomena not readily discernible by an observer. Although a variety of factors influence the incidence and prevalence of seizures, ~5–10% of the population will have at least one seizure, with the highest incidence occurring in early childhood and late adulthood.

1	The meaning of the term seizure needs to be carefully distinguished from that of epilepsy. Epilepsy describes a condition in which a person has recurrent seizures due to a chronic, underlying process. This definition implies that a person with a single seizure, or recurrent seizures due to correctable or avoidable circumstances, does not necessarily have epilepsy. Epilepsy refers to a clinical phenomenon rather than a single disease entity, because there are many forms and causes of epilepsy. However, among the many causes of epilepsy there are various epilepsy syndromes in which the clinical and pathologic characteristics are distinctive and suggest a specific underlying etiology. Using the definition of epilepsy as two or more unprovoked seizures, the incidence of epilepsy is ~0.3–0.5% in different populations throughout the world, and the prevalence of epilepsy has been estimated at 5–30 persons per 1000.

1	Determining the type of seizure that has occurred is essential for focusing the diagnostic approach on particular etiologies, selecting the appropriate therapy, and providing potentially vital information regarding prognosis. The International League against Epilepsy (ILAE) Commission on Classification and Terminology, 2005–2009 has provided an updated approach to classification of seizures (Table 445-1). This system is based on the clinical features of seizures and associated electroencephalographic findings. Other potentially distinctive features such as etiology or cellular substrate are not considered in this classification system, although this will undoubtedly change in the future as more is learned about the pathophysiologic mechanisms that underlie specific seizure types. A fundamental principle is that seizures may be either focal or generalized. Focal seizures originate within networks limited to one

1	A fundamental principle is that seizures may be either focal or generalized. Focal seizures originate within networks limited to one CLassifiCatioN of seiZures 1. Focal seizures (Can be further described as having motor, sensory, autonomic, cognitive, or other features) 2. Generalized seizures a. b. c. d. e. f. 3. May be focal, generalized, or unclear cerebral hemisphere (note that the term partial seizures is no longer used). Generalized seizures arise within and rapidly engage networks distributed across both cerebral hemispheres. Focal seizures are usually associated with structural abnormalities of the brain. In contrast, generalized seizures may result from cellular, biochemical, or structural abnormalities that have a more widespread distribution. There are clear exceptions in both cases, however.

1	Focal seizures arise from a neuronal network either discretely localized within one cerebral hemisphere or more broadly distributed but still within the hemisphere. With the new classification system, the subcategories of “simple focal seizures” and “complex focal seizures” have been eliminated. Instead, depending on the presence of cognitive impairment, they can be described as focal seizures with or without dyscognitive features. Focal seizures can also evolve into generalized seizures. In the past this was referred to as focal seizures with secondary generalization, but the new system relies on specific descriptions of the type of generalized seizures that evolve from the focal seizure.

1	The routine interictal (i.e., between seizures) electroencephalogram (EEG) in patients with focal seizures is often normal or may show brief discharges termed epileptiform spikes, or sharp waves. Because focal seizures can arise from the medial temporal lobe or inferior frontal lobe (i.e., regions distant from the scalp), the EEG recorded during the seizure may be nonlocalizing. However, the seizure focus is often detected using sphenoidal or surgically placed intracranial electrodes.

1	Focal Seizures Without Dyscognitive Features Focal seizures can cause motor, sensory, autonomic, or psychic symptoms without impairment of cognition. For example, a patient having a focal motor seizure arising from the right primary motor cortex near the area controlling hand movement will note the onset of involuntary movements of the contralateral, left hand. These movements are typically clonic (i.e., repetitive, flexion/extension movements) at a frequency of ~2–3 Hz; pure tonic posturing may be seen as well. Since the cortical region controlling hand movement is immediately adjacent to the region for facial expression, the seizure may also cause abnormal movements of the face synchronous with the movements of the hand. The EEG recorded with scalp electrodes during the seizure (i.e., an ictal EEG) may show abnormal discharges in a very limited region over the appropriate area of cerebral cortex if the seizure focus involves the cerebral convexity. Seizure activity occurring within

1	ictal EEG) may show abnormal discharges in a very limited region over the appropriate area of cerebral cortex if the seizure focus involves the cerebral convexity. Seizure activity occurring within deeper brain structures is sometimes not detected by the standard EEG, however, and may require intracranial electrodes for its detection.

1	Three additional features of focal motor seizures are worth noting. First, in some patients, the abnormal motor movements may begin in a very restricted region such as the fingers and gradually progress (over seconds to minutes) to include a larger portion of the extremity. This phenomenon, described by Hughlings Jackson and known as a “Jacksonian march,” represents the spread of seizure activity over a progressively larger region of motor cortex. Second, patients may experience a localized paresis (Todd’s paralysis) for minutes to many hours in the involved region following the seizure. Third, in rare instances, the seizure may continue for hours or days. This condition, termed epilepsia partialis continua, is often refractory to medical therapy.

1	Focal seizures may also manifest as changes in somatic sensation (e.g., paresthesias), vision (flashing lights or formed hallucinations), equilibrium (sensation of falling or vertigo), or autonomic function (flushing, sweating, piloerection). Focal seizures arising from the temporal or frontal cortex may also cause alterations in hearing, olfaction, or higher cortical function (psychic symptoms). This includes the sensation of unusual, intense odors (e.g., burning rubber or kerosene) or sounds (crude or highly complex sounds), or an epigastric sensation that rises from the stomach or chest to the head. Some patients describe odd, internal feelings such as fear, a sense of impending change, detachment, depersonalization, déjá vu, or illusions that objects are growing smaller (micropsia) or larger (macropsia). These subjective, “internal” events that are not directly observable by someone else are referred to as auras.

1	Focal Seizures with Dyscognitive Features Focal seizures may also be accompanied by a transient impairment of the patient’s ability to maintain normal contact with the environment. The patient is unable to respond appropriately to visual or verbal commands during the seizure and has impaired recollection or awareness of the ictal phase. The seizures frequently begin with an aura (i.e., a focal seizure without cognitive disturbance) that is stereotypic for the patient. The start of the ictal phase is often a sudden behavioral arrest or motionless stare, which marks the onset of the period of impaired awareness. The behavioral arrest is usually accompanied by automatisms, which are involuntary, automatic behaviors that have a wide range of manifestations. Automatisms may consist of very basic behaviors such as chewing, lip smacking, swallowing, or “picking” movements of the hands, or more elaborate behaviors such as a display of emotion or running. The patient is typically confused

1	basic behaviors such as chewing, lip smacking, swallowing, or “picking” movements of the hands, or more elaborate behaviors such as a display of emotion or running. The patient is typically confused following the seizure, and the transition to full recovery of consciousness may range from seconds up to an hour. Examination immediately following the seizure may show an anterograde amnesia or, in cases involving the dominant hemisphere, a postictal aphasia.

1	The range of potential clinical behaviors linked to focal seizures is so broad that extreme caution is advised before concluding that stereotypic episodes of bizarre or atypical behavior are not due to seizure activity. In such cases additional, detailed EEG studies may be helpful.

1	Focal seizures can spread to involve both cerebral hemispheres and produce a generalized seizure, usually of the tonic-clonic variety (discussed below). This evolution is observed frequently following focal seizures arising from a focus in the frontal lobe, but may also be associated with focal seizures occurring elsewhere in the brain. A focal seizure that evolves into a generalized seizure is often difficult to distinguish from a primary generalized-onset tonic-clonic seizure, because bystanders tend to emphasize the more dramatic, generalized convulsive phase of the seizure and overlook the more subtle, focal symptoms present at onset. In some cases, the focal onset of the seizure becomes apparent only when a careful history identifies a preceding aura. Often, however, the focal onset is not clinically evident and may be established only through careful EEG analysis. Nonetheless, distinguishing between these two entities is extremely important, because there may be substantial

1	is not clinically evident and may be established only through careful EEG analysis. Nonetheless, distinguishing between these two entities is extremely important, because there may be substantial differences in the evaluation and treatment of epilepsies associated with focal versus generalized seizures.

1	Generalized seizures are thought to arise at some point in the brain but immediately and rapidly engage neuronal networks in both cerebral hemispheres. Several types of generalized seizures have features that place them in distinctive categories and facilitate clinical diagnosis. Typical Absence Seizures Typical absence seizures are characterized by sudden, brief lapses of consciousness without loss of postural control. The seizure typically lasts for only seconds, consciousness returns as suddenly as it was lost, and there is no postictal confusion. Although the brief loss of consciousness may be clinically inapparent or the sole manifestation of the seizure discharge, absence seizures are usually accompanied by subtle, bilateral motor signs such as rapid blinking of the eyelids, chewing movements, or small-amplitude, clonic movements of the hands.

1	Typical absence seizures are associated with a group of genetically determined epilepsies with onset usually in childhood (ages 4–8 years) or early adolescence and are the main seizure type in 15–20% of children with epilepsy. The seizures can occur hundreds of times per day, but the child may be unaware of or unable to convey their existence. Because the clinical signs of the seizures are subtle, especially to parents who may not have had previous experience with seizures, it is not sur-2543 prising that the first clue to absence epilepsy is often unexplained “daydreaming” and a decline in school performance recognized by a teacher.

1	The electrophysiologic hallmark of typical absence seizures is a generalized, symmetric, 3-Hz spike-and-wave discharge that begins and ends suddenly, superimposed on a normal EEG background. Periods of spike-and-wave discharges lasting more than a few seconds usually correlate with clinical signs, but the EEG often shows many more brief bursts of abnormal cortical activity than were suspected clinically. Hyperventilation tends to provoke these electrographic discharges and even the seizures themselves and is routinely used when recording the EEG.

1	Atypical Absence Seizures Atypical absence seizures have features that deviate both clinically and electrophysiologically from typical absence seizures. For example, the lapse of consciousness is usually of longer duration and less abrupt in onset and cessation, and the seizure is accompanied by more obvious motor signs that may include focal or lateralizing features. The EEG shows a generalized, slow spike-andwave pattern with a frequency of ≤2.5 per second, as well as other abnormal activity. Atypical absence seizures are usually associated with diffuse or multifocal structural abnormalities of the brain and therefore may accompany other signs of neurologic dysfunction such as mental retardation. Furthermore, the seizures are less responsive to anticonvulsants compared to typical absence seizures.

1	Generalized, Tonic-Clonic Seizures Generalized-onset tonic-clonic seizures are the main seizure type in ~10% of all persons with epilepsy. They are also the most common seizure type resulting from metabolic derangements and are therefore frequently encountered in many different clinical settings. The seizure usually begins abruptly without warning, although some patients describe vague premonitory symptoms in the hours leading up to the seizure. This prodrome is distinct from the stereotypic auras associated with focal seizures that generalize. The initial phase of the seizure is usually tonic contraction of muscles throughout the body, accounting for a number of the classic features of the event. Tonic contraction of the muscles of expiration and the larynx at the onset will produce a loud moan or “ictal cry.” Respirations are impaired, secretions pool in the oropharynx, and cyanosis develops. Contraction of the jaw muscles may cause biting of the tongue. A marked enhancement of

1	a loud moan or “ictal cry.” Respirations are impaired, secretions pool in the oropharynx, and cyanosis develops. Contraction of the jaw muscles may cause biting of the tongue. A marked enhancement of sympathetic tone leads to increases in heart rate, blood pressure, and pupillary size. After 10–20 s, the tonic phase of the seizure typically evolves into the clonic phase, produced by the superimposition of periods of muscle relaxation on the tonic muscle contraction. The periods of relaxation progressively increase until the end of the ictal phase, which usually lasts no more than 1 min. The postictal phase is characterized by unresponsiveness, muscular flaccidity, and excessive salivation that can cause stridorous breathing and partial airway obstruction. Bladder or bowel incontinence may occur at this point. Patients gradually regain consciousness over minutes to hours, and during this transition, there is typically a period of postictal confusion. Patients subsequently complain of

1	occur at this point. Patients gradually regain consciousness over minutes to hours, and during this transition, there is typically a period of postictal confusion. Patients subsequently complain of headache, fatigue, and muscle ache that can last for many hours. The duration of impaired consciousness in the postictal phase can be extremely long (i.e., many hours) in patients with prolonged seizures or underlying central nervous system (CNS) diseases such as alcoholic cerebral atrophy.

1	The EEG during the tonic phase of the seizure shows a progressive increase in generalized low-voltage fast activity, followed by generalized high-amplitude, polyspike discharges. In the clonic phase, the high-amplitude activity is typically interrupted by slow waves to create a spike-and-wave pattern. The postictal EEG shows diffuse slowing that gradually recovers as the patient awakens. There are a number of variants of the generalized tonic-clonic seizure, including pure tonic and pure clonic seizures. Brief tonic seizures lasting only a few seconds are especially noteworthy since they are usually associated with specific epileptic syndromes having mixed seizure phenotypes, such as the Lennox-Gastaut syndrome (discussed below).

1	Atonic Seizures Atonic seizures are characterized by sudden loss of postural muscle tone lasting 1–2 s. Consciousness is briefly impaired, 2544 but there is usually no postictal confusion. A very brief seizure may cause only a quick head drop or nodding movement, whereas a longer seizure will cause the patient to collapse. This can be extremely dangerous, because there is a substantial risk of direct head injury with the fall. The EEG shows brief, generalized spike-and-wave discharges followed immediately by diffuse slow waves that correlate with the loss of muscle tone. Similar to pure tonic seizures, atonic seizures are usually seen in association with known epilepsy syndromes.

1	Myoclonic Seizures Myoclonus is a sudden and brief muscle contraction that may involve one part of the body or the entire body. A normal, common physiologic form of myoclonus is the sudden jerking movement observed while falling asleep. Pathologic myoclonus is most commonly seen in association with metabolic disorders, degenerative CNS diseases, or anoxic brain injury (Chap. 330). Although the distinction from other forms of myoclonus is imprecise, myoclonic seizures are considered to be true epileptic events because they are caused by cortical (versus subcortical or spinal) dysfunction. The EEG may show bilaterally synchronous spike-and-wave discharges synchronized with the myoclonus, although these can be obscured by movement artifact. Myoclonic seizures usually coexist with other forms of generalized seizures but are the predominant feature of juvenile myoclonic epilepsy (discussed below).

1	Not all seizure types can be designated as focal or generalized, and they should therefore be labeled as “unclassifiable” until additional evidence allows a valid classification. Epileptic spasms are such an example. These are characterized by a briefly sustained flexion or extension of predominantly proximal muscles, including truncal muscles. The EEG in these patients usually shows hypsarrhythmias, which consist of diffuse, giant slow waves with a chaotic background of irregular, multifocal spikes and sharp waves. During the clinical spasm, there is a marked suppression of the EEG background (the “electrodecremental response”). The electromyogram (EMG) also reveals a characteristic rhomboid pattern that may help distinguish spasms from brief tonic and myoclonic seizures. Epileptic spasms occur predominantly in infants and likely result from differences in neuronal function and connectivity in the immature versus mature CNS.

1	Epilepsy syndromes are disorders in which epilepsy is a predominant feature, and there is sufficient evidence (e.g., through clinical, EEG, radiologic, or genetic observations) to suggest a common underlying mechanism. Three important epilepsy syndromes are listed below; additional examples with a known genetic basis are shown in Table 445-2.

1	Juvenile myoclonic epilepsy (JME) is a generalized seizure disorder of unknown cause that appears in early adolescence and is usually characterized by bilateral myoclonic jerks that may be single or repetitive. The myoclonic seizures are most frequent in the morning after awakening and can be provoked by sleep deprivation. Consciousness is preserved unless the myoclonus is especially severe. Many patients also experience generalized tonic-clonic seizures, and up to one-third have absence seizures. Although complete remission is relatively uncommon, the seizures usually respond well to appropriate anticonvulsant medication. There is often a family history of epilepsy, and genetic linkage studies suggest a polygenic cause.

1	Lennox-Gastaut syndrome occurs in children and is defined by the following triad: (1) multiple seizure types (usually including generalized tonic-clonic, atonic, and atypical absence seizures); (2) an EEG showing slow (<3 Hz) spike-and-wave discharges and a variety of other abnormalities; and (3) impaired cognitive function in most but not all cases. Lennox-Gastaut syndrome is associated with CNS disease or dysfunction from a variety of causes, including de novo mutations, developmental abnormalities, perinatal hypoxia/ischemia, trauma, infection, and other acquired lesions. The multifactorial nature of this syndrome suggests that it is a nonspecific response of the brain to diffuse neural injury. Unfortunately, many patients have a poor prognosis due to the underlying CNS disease and the physical and psychosocial consequences of severe, poorly controlled epilepsy.

1	Mesial temporal lobe epilepsy (MTLE) is the most common syndrome associated with focal seizures with dyscognitive features and is an example of an epilepsy syndrome with distinctive clinical, electroencephalographic, and pathologic features (Table 445-3). High-resolution magnetic resonance imaging (MRI) can detect the characteristic hippocampal sclerosis that appears to be essential in the pathophysiology of MTLE for many patients (Fig. 445-1). Recognition of this syndrome is especially important because it tends to be refractory to treatment with anticonvulsants but responds well to surgical intervention. Advances in the understanding of basic mechanisms of epilepsy have come through studies of experimental models of MTLE, discussed below.

1	Seizures are a result of a shift in the normal balance of excitation and inhibition within the CNS. Given the numerous properties that control neuronal excitability, it is not surprising that there are many different ways to perturb this normal balance, and therefore many different causes of both seizures and epilepsy. Three clinical observations emphasize how a variety of factors determine why certain conditions may cause seizures or epilepsy in a given patient. 1.

1	1. The normal brain is capable of having a seizure under the appropriate circumstances, and there are differences between individuals in the susceptibility or threshold for seizures. For example, seizures may be induced by high fevers in children who are otherwise normal and who never develop other neurologic problems, including epilepsy. However, febrile seizures occur only in a relatively small proportion of children. This implies there are various underlying endogenous factors that influence the threshold for having a seizure. Some of these factors are genetic, as a family history of epilepsy has a clear influence on the likelihood of seizures occurring in otherwise normal individuals. Normal development also plays an important role, because the brain appears to have different seizure thresholds at different maturational stages. 2.

1	2. There are a variety of conditions that have an extremely high likelihood of resulting in a chronic seizure disorder. One of the best examples of this is severe, penetrating head trauma, which is associated with up to a 45% risk of subsequent epilepsy. The high propensity for severe traumatic brain injury to lead to epilepsy suggests that the injury results in a long-lasting pathologic change in the CNS that transforms a presumably normal neural network into one that is abnormally hyperexcitable. This process is known as epileptogenesis, and the specific changes that result in a lowered seizure threshold can be considered epileptogenic factors. Other processes associated with epileptogenesis include stroke, infections, and abnormalities of CNS development. Likewise, the genetic abnormalities associated with epilepsy likely involve processes that trigger the appearance of specific sets of epileptogenic factors. 3.

1	3. Seizures are episodic. Patients with epilepsy have seizures intermittently and, depending on the underlying cause, many patients are completely normal for months or even years between seizures. This implies there are important provocative or precipitating factors that induce seizures in patients with epilepsy. Similarly, precipitating factors are responsible for causing the single seizure in someone without epilepsy. Precipitants include those due to intrinsic physiologic processes such as psychological or physical stress, sleep deprivation, or hormonal changes associated with the menstrual cycle. They also include exogenous factors such as exposure to toxic substances and certain medications. Gene (Locus) Function of Gene Clinical Syndrome Comments CHRNA4 (20q13.2) Nicotinic acetylcholine receptor subunit; mutations cause alterations in Ca2+ flux through the receptor; this may reduce amount of GABA release in presynaptic terminals

1	CHRNA4 (20q13.2) Nicotinic acetylcholine receptor subunit; mutations cause alterations in Ca2+ flux through the receptor; this may reduce amount of GABA release in presynaptic terminals KCNQ2 (20q13.3) Voltage-gated potassium channel subunits; mutation in pore regions may cause a 20–40% reduction of potassium currents, which will lead to impaired repolarization SCN1A (2q24.3) α-Subunit of a voltage-gated sodium channel; numerous mutations affecting sodium currents that cause either gain or loss of function; network effects appear related to expression in excitatory or inhibitory cells LGI1 (10q24) Leucine-rich glioma-inactivated 1 gene; previous evidence for role in glial tumor progression; recent studies suggest an influence in the postnatal development of glutamatergic circuits in the hippocampus

1	DEPDC5 (22q12.2) Disheveled, Egl-10 and pleckstrin domain containing protein 5; exerts an inhibitory effect on mammalian target of rapamycin (mTOR)-mediated processes, such as cell growth and proliferation CSTB (21q22.3) Cystatin B, a noncaspase cysteine protease inhibitor; normal protein may block neuronal apoptosis by inhibiting caspases directly or indirectly (via cathepsins), or controlling proteolysis EPM2A (6q24) Laforin, a protein tyrosine phosphatase (PTP); involved in glycogen metabolism and may have antiapoptotic activity Doublecortin (Xq21-24) Doublecortin, expressed primarily in frontal lobes; directly regulates micro-tubule polymerization and bundling Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE); childhood onset; brief, nighttime seizures with prominent motor movements; often misdiagnosed as primary sleep disorder

1	Benign familial neonatal seizures (BFNS); autosomal dominant inheritance; onset in 1st week of life in infants who are otherwise normal; remission usually within weeks to months; long-term epilepsy in 10–15% Generalized epilepsy with febrile seizures plus (GEFS+); autosomal dominant inheritance; presents with febrile seizures at median 1 year, which may persist >6 years, then variable seizure types not associated with fever; numerous other syndromes, including almost 80% of patients with Dravet’s syndrome (severe myoclonic epilepsy of infancy) and some cases of Lennox-Gastaut syndrome Autosomal dominant partial epilepsy with auditory features (ADPEAF); a form of idiopathic lateral temporal lobe epilepsy with auditory symptoms or aphasia as a major focal seizure manifestation; age of onset usually between 10 and 25 years

1	Autosomal dominant familial focal epilepsy with variable foci (FFEVF); family members have seizures originating from different cortical regions; neuroimaging usually normal but may harbor subtle malformations; recent studies also suggest association with benign epilepsy with centrotemporal spikes Progressive myoclonus epilepsy (PME) (Unverricht-Lundborg disease); autosomal recessive inheritance; age of onset between 6 and 15 years, myoclonic seizures, ataxia, and progressive cognitive decline; brain shows neuronal degeneration Progressive myoclonus epilepsy (Lafora’s disease); autosomal recessive inheritance; age of onset 6–19 years, death within 10 years; brain degeneration associated with polyglucosan intracellular inclusion bodies in numerous organs

1	Classic lissencephaly associated with severe mental retardation and seizures in males; sub-cortical band heterotopia with more subtle findings in females (presumably due to random X-inactivation); X-linked dominant Rare; first identified in a large Australian family; other families found to have mutations in CHRNA2 or CHRNB2, and some families appear to have mutations at other loci Rare; other families found to have mutations in KCNQ3 or an inversion in chromosomal 5; sequence and functional homology to KCNQ1, mutations of which cause long QT syndrome and a cardiac-auditory syndrome Incidence uncertain; GEFS+ identified in other families with mutations in other sodium channel subunits (SCN2B and SCN2A) and GABAA receptor subunit (GABRG2 and GABRA1); significant phenotypic heterogeneity within same family, including members with febrile seizures only

1	Mutations found in up to 50% of families containing two or more subjects with idiopathic localization-related epilepsy with ictal auditory symptoms, suggesting that at least one other gene may underlie this syndrome. Study of families with limited number of affected members revealed mutations in approximately 12% of families; thus may be a relatively common cause of lesion-negative focal epilepsies with suspected genetic basis Overall rare, but relatively common in Finland and Western Mediterranean (>1 in 20,000); precise role of cystatin B in human disease unknown, although mice with null mutations of cystatin B have similar syndrome Most common PME in Southern Europe, Middle East, Northern Africa, and Indian subcontinent; genetic heterogeneity; unknown whether seizure phenotype due to degeneration or direct effects of abnormal laforin expression

1	Relatively rare but of uncertain incidence; recent increased ascertainment due to improved imaging techniques; relationship between migration defect and seizure phenotype unknown aThe first five syndromes listed in the table (ADNFLE, BFNC, GEFS+, ADPEAF, and FFEVF) are examples of idiopathic epilepsies associated with identified gene mutations. The last three syndromes are examples of the numerous Mendelian disorders in which seizures are one part of the phenotype. Abbreviations: GABA, γ-aminobutyric acid; PME, progressive myoclonus epilepsy.

1	Abbreviations: GABA, γ-aminobutyric acid; PME, progressive myoclonus epilepsy. These observations emphasize the concept that the many causes of seizures and epilepsy result from a dynamic interplay between endogenous factors, epileptogenic factors, and precipitating factors. The potential role of each needs to be carefully considered when determining the appropriate management of a patient with seizures. For example, the identification of predisposing factors (e.g., family history of epilepsy) in a patient with febrile seizures may increase the necessity for closer follow-up and a more aggressive diagnostic evaluation. Finding an epileptogenic lesion may help in the estimation of seizure recurrence and duration of therapy. Finally, removal or modification of a precipitating factor may be an effective and safer method for preventing further seizures than the prophylactic use of anticonvulsant drugs.

1	In practice, it is useful to consider the etiologies of seizures based on the age of the patient, because age is one of the most important factors determining both the incidence and the likely causes of seizures or epilepsy (Table 445-4). During the neonatal period and early infancy, potential causes include hypoxic-ischemic encephalopathy, trauma, CNS infection, congenital CNS abnormalities, and metabolic disorders. Babies born to mothers using neurotoxic drugs such as cocaine, heroin, or ethanol are susceptible to drug-withdrawal seizures in the first few days after delivery. Hypoglycemia and hypocalcemia, which can occur as secondary complications of perinatal injury, are also causes of seizures early after delivery. Seizures due to inborn errors of metabolism usually present once regular feeding begins, typically 2–3 days after birth. Pyridoxine (vitamin B6) deficiency, an important cause of neonatal seizures, can be effectively treated with pyridoxine replacement. The idiopathic

1	feeding begins, typically 2–3 days after birth. Pyridoxine (vitamin B6) deficiency, an important cause of neonatal seizures, can be effectively treated with pyridoxine replacement. The idiopathic or inherited forms of benign neonatal convulsions are also seen during this time period.

1	The most common seizures arising in late infancy and early childhood are febrile seizures, which are seizures associated with fevers but without evidence of CNS infection or other defined causes. The overall prevalence Small hippocampus with increased signal on T2-weighted sequences Small temporal lobe Enlarged temporal horn Highly selective loss of specific cell populations within hippocampus in most cases Abbreviations: EEG, electroencephalogram; MRI, magnetic resonance imaging; PET, positron emission tomography; SPECT, single-photon emission computed tomography.

1	Abbreviations: EEG, electroencephalogram; MRI, magnetic resonance imaging; PET, positron emission tomography; SPECT, single-photon emission computed tomography. is 3–5% and even higher in some parts of the world such as Asia. Patients often have a family history of febrile seizures or epilepsy. Febrile seizures usually occur between 3 months and 5 years of age and have a peak incidence between 18 and 24 months. The typical scenario is a child who has a generalized, tonic-clonic seizure during a febrile illness in the setting of a common childhood infection such as otitis media, respiratory

1	FIGURE 445-1 Mesial temporal lobe epilepsy. The electroencephalogram and seizure semiology were consistent with a left temporal lobe focus. This coronal high-resolution T2-weighted fast spin echo magnetic resonance image obtained at 3 tesla is at the level of the hippocampal bodies, and shows abnormal high signal intensity, blurring of internal laminar architecture, and reduced size of the left hippocampus (arrow) relative to the right. This triad of imaging findings is consistent with hippocampal sclerosis. taBLe 445-4 Causes of seiZures Abbreviation: CNS, central nervous system.

1	taBLe 445-4 Causes of seiZures Abbreviation: CNS, central nervous system. infection, or gastroenteritis. The seizure is likely to occur during the rising phase of the temperature curve (i.e., during the first day) rather than well into the course of the illness. A simple febrile seizure is a single, isolated event, brief, and symmetric in appearance. Complex febrile seizures are characterized by repeated seizure activity, duration >15 minutes, or by focal features. Approximately one-third of patients with febrile seizures will have a recurrence, but <10% have three or more episodes. Recurrences are much more likely when the febrile seizure occurs in the first year of life. Simple febrile seizures are not associated with an increase in the risk of developing epilepsy, while complex febrile seizures have a risk of 2–5%; other risk factors include the presence of preexisting neurologic deficits and a family history of nonfebrile seizures.

1	Childhood marks the age at which many of the well-defined epilepsy syndromes present. Some children who are otherwise normal develop idiopathic, generalized tonic-clonic seizures without other features that fit into specific syndromes. Temporal lobe epilepsy usually presents in childhood and may be related to mesial temporal lobe sclerosis (as part of the MTLE syndrome) or other focal abnormalities such as cortical dysgenesis. Other types of focal seizures, including those that evolve into generalized seizures, may be the relatively late manifestation of a developmental disorder, an acquired lesion such as head trauma, CNS infection (especially viral encephalitis), or very rarely a CNS tumor.

1	The period of adolescence and early adulthood is one of transition during which the idiopathic or genetically based epilepsy syndromes, including JME and juvenile absence epilepsy, become less common, while epilepsies secondary to acquired CNS lesions begin to predominate. Seizures that arise in patients in this age range may be associated with head trauma, CNS infections (including parasitic infections such as cysticercosis), brain tumors, congenital CNS abnormalities, illicit drug use, or alcohol withdrawal. Autoantibodies directed against CNS antigens such as potassium channels or glutamate receptors are a newly recognized cause of epilepsy that also begins to appear in this age group (although cases of autoimmunity are being increasingly described in the pediatric population), including patients without an identifiable cancer. This etiology should be suspected when a previously normal individual presents with a particularly aggressive seizure pattern developing over weeks to

1	patients without an identifiable cancer. This etiology should be suspected when a previously normal individual presents with a particularly aggressive seizure pattern developing over weeks to months and characterized by increasingly frequent and prolonged seizures combined with cognitive decline (Chap. 122).

1	Head trauma is a common cause of epilepsy in adolescents and adults. The head injury can be caused by a variety of mechanisms, and the likelihood of developing epilepsy is strongly correlated with the severity of the injury. A patient with a penetrating head wound, depressed skull fracture, intracranial hemorrhage, or prolonged post-traumatic coma or amnesia has a 30–50% risk of developing epilepsy, whereas a patient with a closed head injury and cerebral contusion has a 5–25% risk. Recurrent seizures usually develop within 1 year after head trauma, although intervals of >10 years are well known. In controlled studies, mild head injury, defined as a concussion with amnesia or loss of consciousness of <30 min, was found to be associated with only a slightly increased likelihood of epilepsy. Nonetheless, most epileptologists know of patients who have focal seizures within hours or days of a mild head injury and subsequently develop chronic seizures of the same type; such cases may

1	Nonetheless, most epileptologists know of patients who have focal seizures within hours or days of a mild head injury and subsequently develop chronic seizures of the same type; such cases may represent rare examples of chronic epilepsy resulting from mild head injury.

1	The causes of seizures in older adults include cerebrovascular disease, trauma (including subdural hematoma), CNS tumors, and degenerative diseases. Cerebrovascular disease may account for ~50% of new cases of epilepsy in patients older than age 65. Acute seizures (i.e., occurring at the time of the stroke) are seen more often with embolic rather than hemorrhagic or thrombotic stroke. Chronic seizures typically appear months to years after the initial event and are associated with all forms of stroke. Metabolic disturbances such as electrolyte imbalance, hypoor hyperglycemia, renal failure, and hepatic failure may cause seizures at any age. Similarly, endocrine disorders, hematologic disorders, vasculitides, and many other systemic diseases may cause seizures over a broad age range. A wide variety of medications and abused substances are known to precipitate seizures as well (Table 445-5).

1	Focal seizure activity can begin in a very discrete region of cortex and then slowly invade the surrounding regions. The hallmark of an established seizure is typically an electrographic “spike” due to intense near-simultaneous firing of a large number of local excitatory neurons, resulting in an apparent hypersynchronization of the excitatory bursts across a relatively large cortical region. The bursting activity in individual neurons (the “paroxysmal depolarization shift”) is caused by a relatively long-lasting depolarization of the neuronal membrane due to influx of extracellular calcium (Ca2+), which leads to the opening of voltage-dependent sodium (Na+) channels, influx of Na+, and generation of repetitive action potentials. This is followed by a hyper-polarizing afterpotential mediated by γ-aminobutyric acid (GABA) receptors or potassium (K+) channels, depending on the cell type. The synchronized bursts from a sufficient number of neurons result in a so-called spike discharge on

1	by γ-aminobutyric acid (GABA) receptors or potassium (K+) channels, depending on the cell type. The synchronized bursts from a sufficient number of neurons result in a so-called spike discharge on the EEG.

1	The spreading seizure wavefront is slowed and ultimately halted by intact hyperpolarization and a “surround” inhibition created by feed-forward activation of inhibitory neurons. With sufficient activation, there is a recruitment of surrounding neurons via a number of synaptic and nonsynaptic mechanisms, including: (1) an increase in extracellular K+, which blunts hyperpolarization and depolarizes neighboring neurons; (2) accumulation of Ca2+ in presynaptic terminals, leading to enhanced neurotransmitter release; (3) depolarization-induced Alkylating agents (e.g., busulfan, chlorambucil) Antimalarials (chloroquine, mefloquine) Cyclosporine OKT3 (monoclonal antibodies to T cells) Tacrolimus Interferons Antidepressants (e.g., bupropion) Antipsychotics (e.g., clozapine) Lithium Drugs of abuse aIn benzodiazepine-dependent patients.

1	Antidepressants (e.g., bupropion) Antipsychotics (e.g., clozapine) Lithium Drugs of abuse aIn benzodiazepine-dependent patients. activation of the N-methyl-d-aspartate (NMDA) subtype of the excitatory amino acid receptor, which causes additional Ca2+ influx and neuronal activation; and (4) ephaptic interactions related to changes in tissue osmolarity and cell swelling. The recruitment of a sufficient number of neurons leads to the propagation of excitatory currents into contiguous areas via local cortical connections and to more distant areas via long commissural pathways such as the corpus callosum.

1	Many factors control neuronal excitability, and thus there are many potential mechanisms for altering a neuron’s propensity to have bursting activity. Mechanisms intrinsic to the neuron include changes in the conductance of ion channels, response characteristics of membrane receptors, cytoplasmic buffering, second-messenger systems, and protein expression as determined by gene transcription, translation, and posttranslational modification. Mechanisms extrinsic to the neuron include changes in the amount or type of neurotransmitters present at the synapse, modulation of receptors by extracellular ions and other molecules, and temporal and spatial properties of synaptic and nonsynaptic input. Nonneural cells, such as astrocytes and oligodendrocytes, have an important role in many of these mechanisms as well.

1	Certain recognized causes of seizures are explained by these mechanisms. For example, accidental ingestion of domoic acid, which is an 2548 analogue of glutamate (the principal excitatory neurotransmitter in the brain), causes profound seizures via direct activation of excitatory amino acid receptors throughout the CNS. Penicillin, which can lower the seizure threshold in humans and is a potent convulsant in experimental models, reduces inhibition by antagonizing the effects of GABA at its receptor. The basic mechanisms of other precipitating factors of seizures such as sleep deprivation, fever, alcohol withdrawal, hypoxia, and infection, are not as well understood but presumably involve analogous perturbations in neuronal excitability. Similarly, the endogenous factors that determine an individual’s seizure threshold may relate to these properties as well. Knowledge of the mechanisms responsible for initiation and propagation of most generalized seizures (including tonic-clonic,

1	an individual’s seizure threshold may relate to these properties as well. Knowledge of the mechanisms responsible for initiation and propagation of most generalized seizures (including tonic-clonic, myoclonic, and atonic types) remains rudimentary and reflects the limited understanding of the connectivity of the brain at a systems level. Much more is understood about the origin of generalized spike-and-wave discharges in absence seizures. These appear to be related to oscillatory rhythms normally generated during sleep by circuits connecting the thalamus and cortex. This oscillatory behavior involves an interaction between GABAB receptors, T-type Ca2+ channels, and K+ channels located within the thalamus. Pharmacologic studies indicate that modulation of these receptors and channels can induce absence seizures, and there is good evidence that the genetic forms of absence epilepsy may be associated with mutations of components of this system.

1	Epileptogenesis refers to the transformation of a normal neuronal network into one that is chronically hyperexcitable. There is often a delay of months to years between an initial CNS injury such as trauma, stroke, or infection and the first seizure. The injury appears to initiate a process that gradually lowers the seizure threshold in the affected region until a spontaneous seizure occurs. In many genetic and idiopathic forms of epilepsy, epileptogenesis is presumably determined by developmentally regulated events.

1	Pathologic studies of the hippocampus from patients with temporal lobe epilepsy have led to the suggestion that some forms of epileptogenesis are related to structural changes in neuronal networks. For example, many patients with MTLE have a highly selective loss of neurons that may contribute to inhibition of the main excitatory neurons within the dentate gyrus. There is also evidence that, in response to the loss of neurons, there is reorganization or “sprouting” of surviving neurons in a way that affects the excitability of the network. Some of these changes can be seen in experimental models of prolonged electrical seizures or traumatic brain injury. Thus, an initial injury such as head injury may lead to a very focal, confined region of structural change that causes local hyperexcitability. The local hyperexcitability leads to further structural changes that evolve over time until the focal lesion produces clinically evident seizures. Similar models have provided strong evidence

1	The local hyperexcitability leads to further structural changes that evolve over time until the focal lesion produces clinically evident seizures. Similar models have provided strong evidence for long-term alterations in intrinsic, biochemical properties of cells within the network such as chronic changes in glutamate or GABA receptor function. Recent work has suggested that induction of inflammatory cascades may be a critical factor in these processes as well.

1	The most important recent progress in epilepsy research has been the identification of genetic mutations associated with a variety of epilepsy syndromes (Table 445-2). Although most of the mutations identified to date cause rare forms of epilepsy, their discovery has led to extremely important conceptual advances. For example, it appears that many of the inherited, idiopathic epilepsies (i.e., the relatively “pure” forms of epilepsy in which seizures are the phenotypic abnormality and brain structure and function are otherwise normal) are due to mutations affecting ion channel function. These syndromes are therefore part of the larger group of channelopathies causing paroxysmal disorders such as cardiac arrhythmias, episodic ataxia, periodic weakness, and familial hemiplegic migraine. In contrast, gene mutations observed in symptomatic epilepsies (i.e., disorders in which other neurologic abnormalities such as cognitive impairment coexist with seizures) are proving to be associated

1	contrast, gene mutations observed in symptomatic epilepsies (i.e., disorders in which other neurologic abnormalities such as cognitive impairment coexist with seizures) are proving to be associated with pathways influencing CNS development or neuronal homeostasis. De novo mutations may explain a significant proportion of these syndromes, especially those with onset in early childhood. A current challenge is to identify the multiple susceptibility genes that underlie the more common forms of idiopathic epilepsies. Recent studies suggest that ion channel mutations and copy number variants may contribute to causation in a subset of these patients.

1	Antiepileptic drugs appear to act primarily by blocking the initiation or spread of seizures. This occurs through a variety of mechanisms that modify the activity of ion channels or neurotransmitters, and in most cases, the drugs have pleiotropic effects. The mechanisms include inhibition of Na+-dependent action potentials in a frequency-dependent manner (e.g., phenytoin, carbamazepine, lamotrigine, topiramate, zonisamide, lacosamide, rufinamide), inhibition of voltage-gated Ca2+ channels (phenytoin, gabapentin, pregabalin), facilitating the opening of potassium channels (ezogabine), attenuation of glutamate activity (lamotrigine, topiramate, felbamate), potentiation of GABA receptor function (benzodiazepines and barbiturates), increase in the availability of GABA (valproic acid, gabapentin, tiagabine), and modulation of release of synaptic vesicles (levetiracetam). The two most effective drugs for absence seizures, ethosuximide and valproic acid, probably act by inhibiting T-type

1	tiagabine), and modulation of release of synaptic vesicles (levetiracetam). The two most effective drugs for absence seizures, ethosuximide and valproic acid, probably act by inhibiting T-type Ca2+ channels in thalamic neurons.

1	In contrast to the relatively large number of antiepileptic drugs that can attenuate seizure activity, there are currently no drugs known to prevent the formation of a seizure focus following CNS injury. The eventual development of such “antiepileptogenic” drugs will provide an important means of preventing the emergence of epilepsy following injuries such as head trauma, stroke, and CNS infection. APPROACH TO THE PATIENT: When a patient presents shortly after a seizure, the first priorities are attention to vital signs, respiratory and cardiovascular support, and treatment of seizures if they resume (see “Treatment: Seizures and Epilepsy”). Life-threatening conditions such as CNS infection, metabolic derangement, or drug toxicity must be recognized and managed appropriately.

1	When the patient is not acutely ill, the evaluation will initially focus on whether there is a history of earlier seizures (Fig. 445-2). If this is the first seizure, then the emphasis will be to: (1) establish whether the reported episode was a seizure rather than another paroxysmal event, (2) determine the cause of the seizure by identifying risk factors and precipitating events, and (3) decide whether anticonvulsant therapy is required in addition to treatment for any underlying illness. In the patient with prior seizures or a known history of epilepsy, the evaluation is directed toward: (1) identification of the underlying cause and precipitating factors, and (2) determination of the adequacy of the patient’s current therapy.

1	The first goal is to determine whether the event was truly a seizure. An in-depth history is essential, because in many cases the diagnosis of a seizure is based solely on clinical grounds—the examination and laboratory studies are often normal. Questions should focus on the symptoms before, during, and after the episode in order to differentiate a seizure from other paroxysmal events (see “Differential Diagnosis of Seizures” below). Seizures frequently occur out-of-hospital, and the patient may be unaware of the ictal and immediate postictal phases; thus, witnesses to the event should be interviewed carefully. The history should also focus on risk factors and predisposing events. Clues for a predisposition to seizures include a history of febrile seizures, earlier auras or brief seizures not recognized as such, and a family history of seizures. Epileptogenic factors such as prior head trauma, stroke, tumor, or CNS infection should be identified.

1	Normal Adult Patient with a Seizure History of epilepsy; currently treated with antiepileptics Assess: adequacy of antiepileptic therapy Side effects Serum levels No history of epilepsy Laboratory studies CBC Electrolytes, calcium, magnesium Serum glucose Liver and renal function tests Urinalysis Toxicology screen Negative metabolic screen Positive metabolic screen or symptoms/signs suggesting a metabolic or infectious disorder Abnormal or change in neurologic exam Treat identifiable metabolic abnormalities Assess cause of neurologic change Lumbar puncture Cultures Endocrine studies CT MRI if focal features present MRI scan and EEG Subtherapeutic antiepileptic levels Appropriate increase or resumption of dose Increase antiepileptic therapy to maximum tolerated dose; consider alternative antiepileptic drugs Therapeutic antiepileptic levels Focal features of seizures Focal abnormalities on clinical or lab examination Other evidence of neurologic dysfunction Treat underlying

1	antiepileptic drugs Therapeutic antiepileptic levels Focal features of seizures Focal abnormalities on clinical or lab examination Other evidence of neurologic dysfunction Treat underlying metabolic abnormality Idiopathic seizures Treat underlying disorder Yes No Consider: Mass lesion; stroke; CNS infection; trauma; degenerative disease Consider: Antiepileptic therapy Further workup Other causes of episodic cerebral dysfunction Syncope Transient ischemic attack Migraine Acute psychosis History Physical examination Exclude Consider: Antiepileptic therapy Consider: Antiepileptic therapy Consider CBC Electrolytes, calcium, magnesium Serum glucose Liver and renal function tests Urinalysis Toxicology screen FIGURE 445-2 Evaluation of the adult patient with a seizure. CBC, complete blood count; CNS, central nervous system; CT, computed tomography; EEG, electroencephalogram; MRI, magnetic resonance imaging.

1	In children, a careful assessment of developmental milestones may The general physical examination includes a search for signs of provide evidence for underlying CNS disease. Precipitating factors infection or systemic illness. Careful examination of the skin may such as sleep deprivation, systemic diseases, electrolyte or metabolic reveal signs of neurocutaneous disorders such as tuberous sclerosis derangements, acute infection, drugs that lower the seizure threshold or neurofibromatosis, or chronic liver or renal disease. A finding of (Table 445-5), or alcohol or illicit drug use should also be identified. organomegaly may indicate a metabolic storage disease, and limb 2550 asymmetry may provide a clue to brain injury early in development. Signs of head trauma and use of alcohol or illicit drugs should be sought. Auscultation of the heart and carotid arteries may identify an abnormality that predisposes to cerebrovascular disease. All patients require a complete neurologic

1	or illicit drugs should be sought. Auscultation of the heart and carotid arteries may identify an abnormality that predisposes to cerebrovascular disease. All patients require a complete neurologic examination, with particular emphasis on eliciting signs of cerebral hemispheric disease (Chap. 437). Careful assessment of mental status (including memory, language function, and abstract thinking) may suggest lesions in the anterior frontal, parietal, or temporal lobes. Testing of visual fields will help screen for lesions in the optic pathways and occipital lobes. Screening tests of motor function such as pronator drift, deep tendon reflexes, gait, and coordination may suggest lesions in motor (frontal) cortex, and cortical sensory testing (e.g., double simultaneous stimulation) may detect lesions in the parietal cortex.

1	Routine blood studies are indicated to identify the more common metabolic causes of seizures such as abnormalities in electrolytes, glucose, calcium, or magnesium, and hepatic or renal disease. A screen for toxins in blood and urine should also be obtained from all patients in appropriate risk groups, especially when no clear precipitating factor has been identified. A lumbar puncture is indicated if there is any suspicion of meningitis or encephalitis, and it is mandatory in all patients infected with HIV, even in the absence of symptoms or signs suggesting infection. Testing for autoantibodies in the serum and cerebrospinal fluid (CSF) should be considered in patients presenting with a seemingly aggressive form of epilepsy associated with other abnormalities such as cognitive disturbances. All patients who have a possible seizure disorder should be evaluated with an EEG as soon as possible. Details about the EEG are covered in Chap. 442e.

1	In the evaluation of a patient with suspected epilepsy, the presence of electrographic seizure activity during the clinically evident event (i.e., abnormal, repetitive, rhythmic activity having a discrete onset and termination) clearly establishes the diagnosis. The absence of electrographic seizure activity does not exclude a seizure disorder, however, because focal seizures may originate from a region of the cortex that cannot be detected by standard scalp electrodes. The EEG is always abnormal during generalized tonic-clonic seizures. Because seizures are typically infrequent and unpredictable, it is often not possible to obtain the EEG during a clinical event. Continuous monitoring for prolonged periods in video-EEG telemetry units for hospitalized patients or the use of portable equipment to record the EEG continuously for ≥24 h in ambulatory patients has made it easier to capture the electrophysiologic accompaniments of clinical events. In particular, video-EEG telemetry is now

1	to record the EEG continuously for ≥24 h in ambulatory patients has made it easier to capture the electrophysiologic accompaniments of clinical events. In particular, video-EEG telemetry is now a routine approach for the accurate diagnosis of epilepsy in patients with poorly characterized events or seizures that are difficult to control.

1	The EEG may also be helpful in the interictal period by showing certain abnormalities that are highly supportive of the diagnosis of epilepsy. Such epileptiform activity consists of bursts of abnormal discharges containing spikes or sharp waves. The presence of epileptiform activity is not specific for epilepsy, but it has a much greater prevalence in patients with epilepsy than in normal individuals. However, even in an individual who is known to have epilepsy, the initial routine interictal EEG may be normal up to 60% of the time. Thus, the EEG cannot establish the diagnosis of epilepsy in many cases.

1	The EEG is also used for classifying seizure disorders and aiding in the selection of anticonvulsant medications. For example, episodic generalized spike-wave activity is usually seen in patients with typical absence epilepsy and may be seen with other generalized epilepsy syndromes. Focal interictal epileptiform discharges would support the diagnosis of a focal seizure disorder such as temporal lobe epilepsy or frontal lobe seizures, depending on the location of the discharges.

1	The routine scalp-recorded EEG may also be used to assess the prognosis of seizure disorders; in general, a normal EEG implies a better prognosis, whereas an abnormal background or profuse epileptiform activity suggests a poor outcome. Unfortunately, the EEG has not proved to be useful in predicting which patients with predisposing conditions such as head injury or brain tumor will go on to develop epilepsy, because in such circumstances epileptiform activity is commonly encountered regardless of whether seizures occur.

1	Magnetoencephalography (MEG) provides another way of looking noninvasively at cortical activity. Instead of measuring electrical activity of the brain, it measures the small magnetic fields that are generated by this activity. The source of epileptiform activity seen on MEG can be analyzed, and its source in the brain can be estimated using a variety of mathematical techniques. These source estimates can then be plotted on an anatomic image of the brain such as an MRI (discussed below), to generate a magnetic source image (MSI). MSI can be useful to localize potential seizure foci.

1	Almost all patients with new-onset seizures should have a brain imaging study to determine whether there is an underlying structural abnormality that is responsible. The only potential exception to this rule is children who have an unambiguous history and examination suggestive of a benign, generalized seizure disorder such as absence epilepsy. MRI has been shown to be superior to computed tomography (CT) for the detection of cerebral lesions associated with epilepsy. In some cases, MRI will identify lesions such as tumors, vascular malformations, or other pathologies that need urgent therapy. The availability of newer MRI methods such as 3-tesla scanners, parallel imaging with multichannel head coils, three-dimensional structural imaging at submillimeter resolution, and widespread use of pulse sequences such as fluid-attenuated inversion recovery (FLAIR), has increased the sensitivity for detection of abnormalities of cortical architecture, including hippocampal atrophy associated

1	of pulse sequences such as fluid-attenuated inversion recovery (FLAIR), has increased the sensitivity for detection of abnormalities of cortical architecture, including hippocampal atrophy associated with mesial temporal sclerosis, as well as abnormalities of cortical neuronal migration. In such cases, the findings may not lead to immediate therapy, but they do provide an explanation for the patient’s seizures and point to the need for chronic antiepileptic drug therapy or possible surgical resection.

1	In the patient with a suspected CNS infection or mass lesion, CT scanning should be performed emergently when MRI is not immediately available. Otherwise, it is usually appropriate to obtain an MRI study within a few days of the initial evaluation. Functional imaging procedures such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are also used to evaluate certain patients with medically refractory seizures (discussed below). Disorders that may mimic seizures are listed in Table 445-6. In most cases, seizures can be distinguished from other conditions by meticulous attention to the history and relevant laboratory studies. On occasion, additional studies such as video-EEG monitoring, sleep studies, tilt-table analysis, or cardiac electrophysiology, may be required to reach a correct diagnosis. Two of the more common nonepileptic syndromes in the differential diagnosis are detailed below.

1	(See also Chap. 27) The diagnostic dilemma encountered most frequently is the distinction between a generalized seizure and syncope. Observations by the patient and bystanders that can help differentiate between the two are listed in Table 445-7. Characteristics of a seizure include the presence of an aura, cyanosis, unconsciousness, motor manifestations lasting >15 s, postictal disorientation, muscle soreness, and sleepiness. In contrast, a syncopal episode is more likely if the event was provoked by acute pain or anxiety or occurred immediately after arising from the lying or sitting position. Patients with syncope often describe a stereotyped transition from consciousness to unconsciousness that includes tiredness, sweating, nausea, and tunneling of vision, and they experience a relatively brief loss of consciousness. Headache or incontinence usually suggests a seizure but may on occasion also occur with syncope. A brief period (i.e., 1–10 s) of convulsive motor activity is

1	relatively brief loss of consciousness. Headache or incontinence usually suggests a seizure but may on occasion also occur with syncope. A brief period (i.e., 1–10 s) of convulsive motor activity is frequently seen immediately at the onset of a syncopal episode, especially if the patient remains in an upright posture

1	DiffereNtiaL DiaGNosis of seiZures Vasovagal syncope Basilar artery TIA Cardiac arrhythmia Sleep disorders Valvular heart disease Narcolepsy/cataplexy Cardiac failure Benign sleep myoclonus Orthostatic hypotension Movement disorders Psychological disorders Tics Psychogenic seizure Nonepileptic myoclonus Hyperventilation Paroxysmal choreoathetosis Panic attack Special considerations in children Metabolic disturbances Breath-holding spells Alcoholic blackouts Migraine with recurrent abdominal (e.g., hallucinogens) Sleepwalking after fainting (e.g., in a dentist’s chair) and therefore has a sustained decrease in cerebral perfusion. Rarely, a syncopal episode can induce a full tonic-clonic seizure. In such cases, the evaluation must focus on both the cause of the syncopal event as well as the possibility that the patient has a propensity for recurrent seizures.

1	Psychogenic seizures are nonepileptic behaviors that resemble seizures. They are often part of a conversion reaction precipitated by underlying psychological distress. Certain behaviors such as sideto-side turning of the head, asymmetric and large-amplitude shaking movements of the limbs, twitching of all four extremities without loss of consciousness, and pelvic thrusting are more commonly associated with psychogenic rather than epileptic seizures. Psychogenic seizures minutes to hours. However, the distinction is sometimes difficult on clinical grounds alone, and there are many examples of diagnostic errors made by experienced epileptologists. This is especially true for psychogenic seizures that resemble focal seizures with dyscognitive features, because the behavioral manifestations of focal seizures (especially of frontal lobe origin) can be extremely unusual, and in both cases, the routine surface EEG may be normal. Video-EEG monitoring is very useful when historic features are

1	focal seizures (especially of frontal lobe origin) can be extremely unusual, and in both cases, the routine surface EEG may be normal. Video-EEG monitoring is very useful when historic features are nondiagnostic. Generalized tonic-clonic seizures always produce marked EEG abnormalities during and after the seizure. For suspected focal seizures of temporal lobe origin, the use of additional electrodes beyond the standard scalp locations (e.g., sphenoidal electrodes) may be required to localize a seizure focus. Measurement of serum prolactin levels may also help to distinguish between organic and psychogenic seizures, because most generalized seizures and some focal seizures are accompanied by rises in serum prolactin (during the immediate 30-min postictal period), whereas psychogenic seizures are not. The diagnosis of psychogenic seizures does not exclude a concurrent diagnosis of epilepsy, because the two often coexist.

1	Therapy for a patient with a seizure disorder is almost always multi-modal and includes treatment of underlying conditions that cause or contribute to the seizures, avoidance of precipitating factors, suppression of recurrent seizures by prophylactic therapy with antiepileptic medications or surgery, and addressing a variety of psychological and social issues. Treatment plans must be individualized, given the many different types and causes of seizures as well as the differences in efficacy and toxicity of antiepileptic medications for each patient. In almost all cases, a neurologist with experience in the treatment of epilepsy should design and oversee implementation of the treatment strategy. Furthermore, patients with refractory epilepsy or those who require polypharmacy with antiepileptic drugs should remain under the regular care of a neurologist.

1	If the sole cause of a seizure is a metabolic disturbance such as an abnormality of serum electrolytes or glucose, then treatment is aimed at reversing the metabolic problem and preventing its recurrence. Therapy with antiepileptic drugs is usually unnecessary unless the metabolic disorder cannot be corrected promptly and the patient is at risk of having further seizures. If the apparent cause of a seizure was a medication (e.g., theophylline) or illicit drug use (e.g., cocaine), then appropriate therapy is avoidance of the drug; there is usually no need for antiepileptic medications unless subsequent seizures occur in the absence of these precipitants.

1	Seizures caused by a structural CNS lesion such as a brain tumor, vascular malformation, or brain abscess may not recur after appropriate treatment of the underlying lesion. However, despite removal of the structural lesion, there is a risk that the seizure focus will remain in the surrounding tissue or develop de novo as a result of gliosis and other processes induced by surgery, radiation, or other therapies. Most patients are therefore maintained on an antiepileptic medication for at least 1 year, and an attempt is made to withdraw medications only if the patient has been completely seizure free. If seizures are refractory to medication, the patient may benefit from surgical removal of the epileptic brain region (see below).

1	Unfortunately, little is known about the specific factors that determine precisely when a seizure will occur in a patient with epilepsy. Some patients can identify particular situations that appear to lower their seizure threshold; these situations should be avoided. For example, a patient who has seizures in the setting of sleep deprivation should obviously be advised to maintain a normal sleep schedule. Many patients note an association between alcohol 2552 intake and seizures, and they should be encouraged to modify their drinking habits accordingly. There are also relatively rare cases of patients with seizures that are induced by highly specific stimuli such as a video game monitor, music, or an individual’s voice (“reflex epilepsy”). Because there is often an association between stress and seizures, stress reduction techniques such as physical exercise, meditation, or counseling may be helpful.

1	Antiepileptic drug therapy is the mainstay of treatment for most patients with epilepsy. The overall goal is to completely prevent seizures without causing any untoward side effects, preferably with a single medication and a dosing schedule that is easy for the patient to follow. Seizure classification is an important element in designing the treatment plan, because some antiepileptic drugs have different activities against various seizure types. However, there is considerable overlap between many antiepileptic drugs such that the choice of therapy is often determined more by the patient’s specific needs, especially his or her assessment of side effects.

1	When to Initiate Antiepileptic Drug Therapy Antiepileptic drug therapy should be started in any patient with recurrent seizures of unknown etiology or a known cause that cannot be reversed. Whether to initiate therapy in a patient with a single seizure is controversial. Patients with a single seizure due to an identified lesion such as a CNS tumor, infection, or trauma, in which there is strong evidence that the lesion is epileptogenic, should be treated. The risk of seizure recurrence in a patient with an apparently unprovoked or idiopathic seizure is uncertain, with estimates ranging from 31 to 71% in the first 12 months after the initial seizure. This uncertainty arises from differences in the underlying seizure types and etiologies in various published epidemiologic studies. Generally accepted risk factors associated with recurrent seizures include the following: (1) an abnormal neurologic examination, (2) seizures presenting as status epilepticus, (3) postictal Todd’s paralysis,

1	accepted risk factors associated with recurrent seizures include the following: (1) an abnormal neurologic examination, (2) seizures presenting as status epilepticus, (3) postictal Todd’s paralysis, (4) a strong family history of seizures, or (5) an abnormal EEG. Most patients with one or more of these risk factors should be treated. Issues such as employment or driving may influence the decision whether to start medications as well. For example, a patient with a single, idiopathic seizure whose job depends on driving may prefer taking antiepileptic drugs rather than risk a seizure recurrence and the potential loss of driving privileges.

1	Selection of Antiepileptic Drugs Antiepileptic drugs available in the United States are shown in Table 445-8, and the main pharmacologic characteristics of commonly used drugs are listed in Table 445-9. Worldwide, older medications such as phenytoin, valproic acid, carbamazepine, phenobarbital, and ethosuximide are generally used as first-line therapy for most seizure disorders because, overall, they are as effective as recently marketed drugs and significantly less expensive overall. Most of the new drugs that have become available in the past decade are used as add-on or alternative therapy, although many are now being used as first-line monotherapy.

1	In addition to efficacy, factors influencing the choice of an initial medication include the convenience of dosing (e.g., once daily versus three or four times daily) and potential side effects. In this regard, a number of the newer drugs have the advantage of reduced drug-drug interactions and easier dosing. Almost all of the commonly used antiepileptic drugs can cause similar, dose-related side effects such as sedation, ataxia, and diplopia. Long-term use of some agents in adults, especially the elderly, can lead to osteoporosis. Close follow-up is required to ensure these side effects are promptly recognized and reversed. Most of the older drugs and some of the newer ones can also cause idiosyncratic toxicity such as rash, bone marrow suppression, or hepatotoxicity. Although rare, these side effects should be considered during drug selection, and patients must be instructed about symptoms or signs that should signal the need to alert their health care provider. For some drugs,

1	these side effects should be considered during drug selection, and patients must be instructed about symptoms or signs that should signal the need to alert their health care provider. For some drugs, laboratory tests (e.g., complete blood count and liver function tests) are recommended prior to the institution of therapy (to establish baseline values) and during initial dosing and titration of the agent. Importantly, studies have shown that Asian individuals carrying the human leukocyte antigen allele, HLA-B*1502, are at particularly high risk of developing serious skin reactions from carbamazepine and phenytoin. As a result, racial background and genotype are additional factors to consider in drug selection.

1	Antiepileptic Drug Selection for focAl SeizureS Carbamazepine (or a related drug, oxcarbazepine), lamotrigine, phenytoin, and levetiracetam are currently the drugs of choice approved for the initial treatment of focal seizures, including those that evolve into generalized seizures. Overall they have very similar efficacy, but differences in pharmacokinetics and toxicity are the main determinants for use in a given patient. For example, an advantage of carbamazepine (which is also available in an extended-release form) is that its metabolism follows first-order pharmacokinetics, which allows for a linear relationship between drug dose, serum levels, and toxicity. Carbamazepine can cause leukopenia, aplastic anemia, or hepatotoxicity and would therefore be contraindicated in patients with predispositions to these problems. Oxcarbazepine has the advantage of being metabolized in a way that avoids an intermediate metabolite associated with some of the side effects of carbamazepine.

1	with predispositions to these problems. Oxcarbazepine has the advantage of being metabolized in a way that avoids an intermediate metabolite associated with some of the side effects of carbamazepine. Oxcarbazepine also has fewer drug interactions than carbamazepine. Lamotrigine tends to be well tolerated in terms of side effects. However, patients need to be particularly vigilant about the possibility of a skin rash during the initiation of therapy. This can be extremely severe and lead to Stevens-Johnson syndrome if unrecognized and if the medication is not discontinued immediately. This risk can be reduced by the use of low initial doses and slow titration. Lamotrigine must be started at lower initial doses when used as add-on therapy with valproic acid, because valproic acid inhibits lamotrigine metabolism and results in a substantially prolonged half-life. Phenytoin has a relatively long half-life and offers the advantage of once or twice daily dosing compared to two or three

1	lamotrigine metabolism and results in a substantially prolonged half-life. Phenytoin has a relatively long half-life and offers the advantage of once or twice daily dosing compared to two or three times daily dosing for many of the other drugs. However, phenytoin shows properties of nonlinear kinetics, such that small increases in phenytoin doses above a standard maintenance dose can precipitate marked side effects. This is one of the main causes of acute phenytoin toxicity. Long-term use of phenytoin is associated with untoward cosmetic effects (e.g., hirsutism, coarsening of facial features, gingival hypertrophy) and effects on bone metabolism. Due to these side effects, phenytoin is often avoided in young patients who are likely to require the drug for many years. Levetiracetam has aExamples only; please refer to other sources for comprehensive listings of all potential drug-drug interactions. bPhenytoin, carbamazepine, phenobarbital. cExtended-release product available.

1	2556 the advantage of having no known drug-drug interactions, making it especially useful in the elderly and patients on other medications. However, a significant number of patients taking levetiracetam complain of irritability, anxiety, and other psychiatric symptoms. Topiramate can be used for both focal and generalized seizures. Similar to some of the other antiepileptic drugs, topiramate can cause significant psychomotor slowing and other cognitive problems. Additionally, it should not be used in patients at risk for the development of glaucoma or renal stones. Valproic acid is an effective alternative for some patients with focal seizures, especially when the seizures generalize. Gastrointestinal side effects are fewer when using the delayed-release formulation (Depakote). Laboratory testing is required to monitor toxicity because valproic acid can rarely cause reversible bone marrow suppression and hepatotoxicity. This drug should generally be avoided in patients with

1	Laboratory testing is required to monitor toxicity because valproic acid can rarely cause reversible bone marrow suppression and hepatotoxicity. This drug should generally be avoided in patients with preexisting bone marrow or liver disease. Irreversible, fatal hepatic failure appearing as an idiosyncratic rather than dose-related side effect is a relatively rare complication; its risk is highest in children <2 years old, especially those taking other anti-epileptic drugs or with inborn errors of metabolism. Zonisamide, tiagabine, gabapentin, lacosamide, and ezogabine are additional drugs currently used for the treatment of focal seizures with or without evolution into generalized seizures. Phenobarbital and other barbiturate compounds were commonly used in the past as first-line therapy for many forms of epilepsy. However, the barbiturates frequently cause sedation in adults, hyperactivity in children, and other more subtle cognitive changes; thus, their use should be limited to

1	for many forms of epilepsy. However, the barbiturates frequently cause sedation in adults, hyperactivity in children, and other more subtle cognitive changes; thus, their use should be limited to situations in which no other suitable treatment alternatives exist.

1	Antiepileptic Drug Selection for generAlizeD SeizureS Lamotrigine and valproic acid are currently considered the best initial choice for the treatment of primary generalized, tonic-clonic seizures. Topiramate, zonisamide, phenytoin, carbamazepine, and oxcarbazepine are suitable alternatives. Valproic acid is also particularly effective in absence, myoclonic, and atonic seizures. It is therefore the drug of choice in patients with generalized epilepsy syndromes having mixed seizure types. Importantly, carbamazepine, oxcarbazepine, and phenytoin can worsen certain types of generalized seizures, including absence, myoclonic, tonic, and atonic seizures. Ethosuximide is a particularly effective drug for the treatment of uncomplicated absence seizures, but it is not useful for tonic-clonic or focal seizures. Periodic monitoring of blood cell counts is required since ethosuximide rarely causes bone marrow suppression. Lamotrigine appears to be particularly effective in epilepsy syndromes

1	focal seizures. Periodic monitoring of blood cell counts is required since ethosuximide rarely causes bone marrow suppression. Lamotrigine appears to be particularly effective in epilepsy syndromes with mixed, generalized seizure types such as JME and Lennox-Gastaut syndrome. Topiramate, zonisamide, and felbamate may have similar broad efficacy.

1	Because the response to any antiepileptic drug is unpredictable, patients should be carefully educated about the approach to therapy. The goal is to prevent seizures and minimize the side effects of treatment; determination of the optimal dose is often a matter of trial and error. This process may take months or longer if the baseline seizure frequency is low. Most antiepileptic drugs need to be introduced relatively slowly to minimize side effects. Patients should expect that minor side effects such as mild sedation, slight changes in cognition, or imbalance will typically resolve within a few days. Starting doses are usually the lowest value listed under the dosage column in Table 445-9. Subsequent increases should be made only after achieving a steady state with the previous dose (i.e., after an interval of five or more half-lives).

1	Monitoring of serum antiepileptic drug levels can be very useful for establishing the initial dosing schedule. However, the published therapeutic ranges of serum drug concentrations are only an approximate guide for determining the proper dose for a given patient. The key determinants are the clinical measures of seizure frequency and presence of side effects, not the laboratory values. Conventional assays of serum drug levels measure the total drug (i.e., both free and protein bound). However, it is the concentration of free drug that reflects extracellular levels in the brain and correlates best with efficacy. Thus, patients with decreased levels of serum proteins (e.g., decreased serum albumin due to impaired liver or renal function) may have an increased ratio of free to bound drug, yet the concentration of free drug may be adequate for seizure control. These patients may have a “subtherapeutic” drug level, but the dose should be changed only if seizures remain uncontrolled, not

1	yet the concentration of free drug may be adequate for seizure control. These patients may have a “subtherapeutic” drug level, but the dose should be changed only if seizures remain uncontrolled, not just to achieve a “therapeutic” level. It is also useful to monitor free drug levels in such patients. In practice, other than during the initiation or modification of therapy, monitoring of antiepileptic drug levels is most useful for documenting adherence.

1	If seizures continue despite gradual increases to the maximum tolerated dose and documented compliance, then it becomes necessary to switch to another antiepileptic drug. This is usually done by maintaining the patient on the first drug while a second drug is added. The dose of the second drug should be adjusted to decrease seizure frequency without causing toxicity. Once this is achieved, the first drug can be gradually withdrawn (usually over weeks unless there is significant toxicity). The dose of the second drug is then further optimized based on seizure response and side effects. Monotherapy should be the goal whenever possible.

1	Overall, about 70% of children and 60% of adults who have their seizures completely controlled with antiepileptic drugs can eventually discontinue therapy. The following patient profile yields the greatest chance of remaining seizure free after drug withdrawal: (1) complete medical control of seizures for 1–5 years; (2) single seizure type, either focal or generalized; (3) normal neurologic examination, including intelligence; and (4) normal EEG. The appropriate seizure-free interval is unknown and undoubtedly varies for different forms of epilepsy. However, it seems reasonable to attempt withdrawal of therapy after 2 years in a patient who meets all of the above criteria, is motivated to discontinue the medication, and clearly understands the potential risks and benefits. In most cases, it is preferable to reduce the dose of the drug gradually over 2–3 months. Most recurrences occur in the first 3 months after discontinuing therapy, and patients should be advised to avoid potentially

1	is preferable to reduce the dose of the drug gradually over 2–3 months. Most recurrences occur in the first 3 months after discontinuing therapy, and patients should be advised to avoid potentially dangerous situations such as driving or swimming during this period.

1	Approximately one-third of patients with epilepsy do not respond to treatment with a single antiepileptic drug, and it becomes necessary to try a combination of drugs to control seizures. Patients who have focal epilepsy related to an underlying structural lesion or those with multiple seizure types and developmental delay are particularly likely to require multiple drugs. There are currently no clear guidelines for rational polypharmacy, although in theory a combination of drugs with different mechanisms of action may be most useful. In most cases, the initial combination therapy combines first-line drugs (i.e., carbamazepine, oxcarbazepine, lamotrigine, valproic acid, levetiracetam, and phenytoin). If these drugs are unsuccessful, then the addition of other drugs such as topiramate, zonisamide, lacosamide, or tiagabine is indicated. Patients with myoclonic seizures resistant to valproic acid may benefit from the addition of clonazepam or clobazam, and those with absence seizures may

1	lacosamide, or tiagabine is indicated. Patients with myoclonic seizures resistant to valproic acid may benefit from the addition of clonazepam or clobazam, and those with absence seizures may respond to a combination of valproic acid and ethosuximide. The same principles concerning the monitoring of therapeutic response, toxicity, and serum levels for monotherapy apply to polypharmacy, and potential drug interactions need to be recognized. If there is no improvement, a third drug can be added while the first two are maintained. If there is a response, the less effective or less well tolerated of the first two drugs should be gradually withdrawn.

1	Approximately 20–30% of patients with epilepsy continue to have seizures despite efforts to find an effective combination of anti-epileptic drugs. For some, surgery can be extremely effective in substantially reducing seizure frequency and even providing complete seizure control. Understanding the potential value of surgery is especially important when a patient’s seizures are not controlled with initial treatment, as such patients often do not respond to subsequent medication trials. Rather than submitting the patient to years of unsuccessful medical therapy and the psychosocial trauma and increased mortality associated with ongoing seizures, the patient should have an efficient but relatively brief attempt at medical therapy and then be referred for surgical evaluation.

1	The most common surgical procedure for patients with temporal lobe epilepsy involves resection of the anteromedial temporal lobe (temporal lobectomy) or a more limited removal of the underlying hippocampus and amygdala (amygdalohippocampectomy). Focal seizures arising from extratemporal regions may be abolished by a focal neocortical resection with precise removal of an identified lesion (lesionectomy). Localized neocortical resection without a clear lesion identified on MRI is also possible when other tests (e.g. MEG, PET, SPECT) implicate a focal cortical region as a seizure onset zone. When the cortical region cannot be removed, multiple subpial transection, which disrupts intracortical connections, is sometimes used to prevent seizure spread. Hemispherectomy or multilobar resection is useful for some patients with severe seizures due to hemispheric abnormalities such as hemimegalencephaly or other dysplastic abnormalities, and corpus callosotomy has been shown to be effective for

1	useful for some patients with severe seizures due to hemispheric abnormalities such as hemimegalencephaly or other dysplastic abnormalities, and corpus callosotomy has been shown to be effective for disabling tonic or atonic seizures, usually when they are part of a mixed-seizure syndrome (e.g., Lennox-Gastaut syndrome).

1	Presurgical evaluation is designed to identify the functional and structural basis of the patient’s seizure disorder. Inpatient video-EEG monitoring is used to define the anatomic location of the seizure focus and to correlate the abnormal electrophysiologic activity with behavioral manifestations of the seizure. Routine scalp or scalpsphenoidal recordings and a high-resolution MRI scan are usually sufficient for localization of the epileptogenic focus, especially when the findings are concordant. Functional imaging studies such as SPECT, PET, and MEG are adjunctive tests that may help to reveal or verify the localization of an apparent epileptogenic region. Once the presumed location of the seizure onset is identified, additional studies, including neuropsychological testing, the intracarotid amobarbital test (Wada test), and functional MRI may be used to assess language and memory localization and to determine the possible functional consequences of surgical removal of the

1	intracarotid amobarbital test (Wada test), and functional MRI may be used to assess language and memory localization and to determine the possible functional consequences of surgical removal of the epileptogenic region. In some cases, standard noninvasive evaluation is not sufficient to localize the seizure onset zone, and invasive electrophysiologic monitoring, such as implanted depth or subdural electrodes, is required for more definitive localization. The exact extent of the resection to be undertaken can also be determined by performing cortical mapping at the time of the surgical procedure, allowing for a tailored resection. This involves electrocorticographic recordings made with electrodes on the surface of the brain to identify the extent of epileptiform disturbances. If the region to be resected is within or near brain regions suspected of having sensorimotor or language function, electrical cortical stimulation mapping is performed on the awake patient to determine the

1	region to be resected is within or near brain regions suspected of having sensorimotor or language function, electrical cortical stimulation mapping is performed on the awake patient to determine the function of cortical regions in question in order to avoid resection of so-called eloquent cortex and thereby minimize postsurgical deficits.

1	Advances in presurgical evaluation and microsurgical techniques have led to a steady increase in the success of epilepsy surgery. Clinically significant complications of surgery are <5%, and the use of functional mapping procedures has markedly reduced the neurologic sequelae due to removal or sectioning of brain tissue. For example, about 70% of patients treated with temporal lobectomy will become seizure free, and another 15–25% will have at least a 90% reduction in seizure frequency. Marked improvement is also usually seen in patients treated with hemispherectomy for catastrophic seizure disorders due to large hemispheric abnormalities. Postoperatively, patients generally need to remain on antiepileptic drug therapy, but the marked reduction of seizures following resective surgery can have a very beneficial effect on quality of life.

1	Not all medically refractory patients are suitable candidates for resective surgery. For example, some patients have seizures arising from more than one location, making the risk of ongoing seizures 2557 or potential harm from the surgery unacceptably high. Vagus nerve stimulation (VNS) has been used in some of these cases, although the results are limited and it is difficult to predict who will benefit. A new implantable device that can detect the onset of a seizure (in some instances before the seizure becomes clinically apparent) and deliver an electrical stimulation (Responsive NeuroStimulation) has recently been approved and may be of benefit in selected patients. Studies are currently evaluating the efficacy of stereotactic radio-surgery, laser thermoablation, and deep brain stimulation (DBS) as other options for surgical treatment of refractory epilepsy.

1	Status epilepticus refers to continuous seizures or repetitive, discrete seizures with impaired consciousness in the interictal period. Status epilepticus has numerous subtypes, including generalized convulsive status epilepticus (GCSE) (e.g., persistent, generalized electrographic seizures, coma, and tonic-clonic movements) and nonconvulsive status epilepticus (e.g., persistent absence seizures or focal seizures with confusion or partially impaired consciousness, and minimal motor abnormalities). The duration of seizure activity sufficient to meet the definition of status epilepticus has traditionally been specified as 15–30 min. However, a more practical definition is to consider status epilepticus as a situation in which the duration of seizures prompts the acute use of anticonvulsant therapy. For GCSE, this is typically when seizures last beyond 5 min.

1	GCSE is an emergency and must be treated immediately, because cardiorespiratory dysfunction, hyperthermia, and metabolic derangements can develop as a consequence of prolonged seizures, and these can lead to irreversible neuronal injury. Furthermore, CNS injury can occur even when the patient is paralyzed with neuromuscular blockade but continues to have electrographic seizures. The most common causes of GCSE are anticonvulsant withdrawal or noncompliance, metabolic disturbances, drug toxicity, CNS infection, CNS tumors, refractory epilepsy, and head trauma.

1	GCSE is obvious when the patient is having overt convulsions. However, after 30–45 min of uninterrupted seizures, the signs may become increasingly subtle. Patients may have mild clonic movements of only the fingers or fine, rapid movements of the eyes. There may be paroxysmal episodes of tachycardia, hypertension, and pupillary dilation. In such cases, the EEG may be the only method of establishing the diagnosis. Thus, if the patient stops having overt seizures, yet remains comatose, an EEG should be performed to rule out ongoing status epilepticus. This is obviously also essential when a patient with GCSE has been paralyzed with neuromuscular blockade in the process of protecting the airway.

1	The first steps in the management of a patient in GCSE are to attend to any acute cardiorespiratory problems or hyperthermia, perform a brief medical and neurologic examination, establish venous access, and send samples for laboratory studies to identify metabolic abnormalities. Anticonvulsant therapy should then begin without delay; a treatment approach is shown in Fig. 445-3. The treatment of nonconvulsive status epilepticus is thought to be less urgent than GCSE, because the ongoing seizures are not accompanied by the severe metabolic disturbances seen with GCSE. However, evidence suggests that nonconvulsive status epilepticus, especially that caused by ongoing, focal seizure activity, is associated with cellular injury in the region of the seizure focus; therefore this condition should be treated as promptly as possible using the general approach described for GCSE. BEYOND SEIZURES: OTHER MANAGEMENT ISSUES

1	BEYOND SEIZURES: OTHER MANAGEMENT ISSUES The adverse effects of epilepsy often go beyond clinical seizures, and the extent of these effects largely depends on the etiology of epilepsy, seizure frequency and severity, and side effects from antiepileptic therapy. Many epilepsy patients are completely normal between seizures and live highly successful and productive lives. In contrast, patients with seizures secondary to developmental abnormalities or acquired brain injury may have impaired cognitive function and other neurologic deficits. Frequent interictal EEG abnormalities are associated with subtle dysfunction of memory and attention. Patients with many seizures, especially those emanating from the temporal lobe, often note an impairment of short-term memory that may progress over time.

1	Patients with epilepsy are at risk of developing a variety of psychiatric problems, including depression, anxiety, and psychosis. This risk varies considerably depending on many factors, including the etiology, frequency, and severity of seizures and the patient’s age and previous personal or family history of psychiatric disorder. Depression occurs in ~20% of patients, and the incidence of suicide is higher in patients with epilepsy than in the general population. Depression should be treated through counseling or medication. The selective serotonin reuptake inhibitors (SSRIs) typically have minimal effect on seizures, whereas tricyclic antidepressants may lower the seizure threshold. Anxiety can be a seizure symptom, and anxious or psychotic behavior can occur during a postictal delirium. Postictal psychosis is a rare phenomenon that typically occurs after a period of increased seizure frequency. There is usually a brief lucid interval lasting up to a week, followed by days to weeks

1	Postictal psychosis is a rare phenomenon that typically occurs after a period of increased seizure frequency. There is usually a brief lucid interval lasting up to a week, followed by days to weeks of agitated, psychotic behavior. The psychosis usually resolves spontaneously but frequently will require short-term treatment with antipsychotic or anxiolytic medications.

1	There is ongoing controversy as to whether some patients with epilepsy (especially temporal lobe epilepsy) have a stereotypical “interictal personality.” The predominant view is that atypical personality traits occur in diverse epilepsies (e.g., generalized and frontal lobe epilepsy) and may result from an underlying structural brain lesion, antiepileptic drug effects, and psychosocial factors related to suffering from a chronic disease, as well as the epilepsy itself.

1	Other approaches Surgery, VNS, rTMS, ECT, hypothermia Other anesthetics Isoflurane, desflurane, ketamine IV MDZ 0.2 mg/kg ˜ 0.2–0.6 mg/kg/h and/or IV PRO 2 mg/kg ˜ 2–10 mg/kg/h Focal-complex, myoclonic or absence SE Generalized convulsive or “subtle” SE Impending and early SE (5–30 minutes) Established and early refractory SE (30 minutes–48 hours) Late refractory SE (>48 hours) Further IV/PO antiepileptic drug VPA, LEV, LCM, TPM, PGB, or other Other medications Lidocaine, verapamil, magnesium, ketogenic diet, immunomodulation IV antiepileptic drug PHT 20 mg/kg, or VPA 20–30 mg/kg, or LEV 20–30 mg/kg IV benzodiazepine LZP 0.1 mg/kg, or MDZ 0.2 mg/kg, or CLZ 0.015 mg/kg PTB (THP) 5 mg/kg (1 mg/kg) ˜ 1–5 mg/kg/h FIGURE 445-3 Pharmacologic treatment of generalized tonic-clonic status epilepticus (SE) in adults. CLZ, clonazepam; ECT, electrocon-vulsive therapy; LCM, lacosamide; LEV, levetiracetam; LZP, lorazepam; MDZ, midazolam; PGB, pregabalin; PHT, phenytoin or fos-phenytoin; PRO,

1	(SE) in adults. CLZ, clonazepam; ECT, electrocon-vulsive therapy; LCM, lacosamide; LEV, levetiracetam; LZP, lorazepam; MDZ, midazolam; PGB, pregabalin; PHT, phenytoin or fos-phenytoin; PRO, propofol; PTB, pentobarbital; rTMS, repetitive transcranial magnetic stimulation; THP, thiopental; TPM, topiramate; VNS, vagus nerve stimulation; VPA, valproic acid. (From AO Rossetti, DH Lowenstein: Lancet Neurol 10:922, 2011.)

1	Patients with epilepsy have a risk of death that is roughly two to three times greater than expected in a matched population without epilepsy. Most of the increased mortality is due to the underlying etiology of epilepsy (e.g., tumors or strokes in older adults). However, a significant number of patients die from accidents, status epilepticus, and a syndrome known as sudden unexpected death in epilepsy (SUDEP), which usually affects young people with convulsive seizures and tends to occur at night. The cause of SUDEP is unknown; it may result from brainstem-mediated effects of seizures on pulmonary, cardiac, and arousal functions. Recent studies suggest that, in some cases, a genetic mutation may be the cause of both epilepsy and a cardiac conduction defect that gives rise to sudden death.

1	There continues to be a cultural stigma about epilepsy, although it is slowly declining in societies with effective health education programs. Many patients with epilepsy harbor fears such as the fear of becoming mentally retarded or dying during a seizure. These issues need to be carefully addressed by educating the patient about epilepsy and by ensuring that family members, teachers, fellow employees, and other associates are equally well informed. A useful source of educational material is the Web site www.epilepsy.com. EMPLOYMENT, DRIVING, AND OTHER ACTIVITIES

1	EMPLOYMENT, DRIVING, AND OTHER ACTIVITIES Many patients with epilepsy face difficulty in obtaining or maintaining employment, even when their seizures are well controlled. Federal and state legislation is designed to prevent employers from discriminating against patients with epilepsy, and patients should be encouraged to understand and claim their legal rights. Patients in these circumstances also benefit greatly from the assistance of health providers who act as strong patient advocates.

1	Loss of driving privileges is one of the most disruptive social consequences of epilepsy. Physicians should be very clear about local regulations concerning driving and epilepsy, because the laws vary considerably among states and countries. In all cases, it is the physician’s responsibility to warn patients of the danger imposed on themselves and others while driving if their seizures are uncontrolled (unless the seizures are not associated with impairment of consciousness or motor control). In general, most states allow patients to drive after a seizure-free interval (on or off medications) of between 3 months and 2 years.

1	Patients with incompletely controlled seizures must also contend with the risk of being in other situations where an impairment of consciousness or loss of motor control could lead to major injury or death. Thus, depending on the type and frequency of seizures, many patients need to be instructed to avoid working at heights or with machinery or to have someone close by for activities such as bathing and swimming. Some women experience a marked increase in seizure frequency around the time of menses. This is believed to be mediated by either the effects of estrogen and progesterone on neuronal excitability or changes in antiepileptic drug levels due to altered protein binding or metabolism. Some patients may benefit from increases in antiepileptic drug dosages during menses. Natural progestins or intramuscular medroxyprogesterone may be of benefit to a subset of women.

1	Most women with epilepsy who become pregnant will have an uncomplicated gestation and deliver a normal baby. However, epilepsy poses some important risks to a pregnancy. Seizure frequency during pregnancy will remain unchanged in ~50% of women, increase in 30%, and decrease in 20%. Changes in seizure frequency are attributed to endocrine effects on the CNS, variations in antiepileptic drug pharmacokinetics (such as acceleration of hepatic drug metabolism or effects on plasma protein binding), and changes in medication compliance. It is useful to see patients at frequent intervals during pregnancy and monitor serum antiepileptic drug levels. Measurement of the unbound drug concentrations may be useful if there is an increase in seizure frequency or worsening of side effects of antiepileptic drugs.

1	The overall incidence of fetal abnormalities in children born to mothers with epilepsy is 5–6%, compared to 2–3% in healthy women. Part of the higher incidence is due to teratogenic effects of antiepileptic drugs, and the risk increases with the number of medications used (e.g., 10–20% risk of malformations with three drugs) and possibly with higher doses. A meta-analysis of published pregnancy registries and cohorts found that the most common malformations were defects in the cardiovascular and musculoskeletal system (1.4–1.8%). Valproic acid is strongly associated with an increased risk of adverse fetal outcomes (7–20%). Recent findings from a large pregnancy registry suggest that, other than topiramate, the newer antiepileptic drugs are far safer than valproic acid.

1	Because the potential harm of uncontrolled convulsive seizures on the mother and fetus is considered greater than the teratogenic effects of antiepileptic drugs, it is currently recommended that pregnant women be maintained on effective drug therapy. When possible, it seems prudent to have the patient on monotherapy at the lowest effective dose, especially during the first trimester. For some women, however, the type and frequency of their seizures may allow for them to safely wean off antiepileptic drugs prior to conception. Patients should also take folate (1–4 mg/d), because the antifolate effects of anticonvulsants are thought to play a role in the development of neural tube defects, although the benefits of this treatment remain unproved in this setting.

1	Enzyme-inducing drugs such as phenytoin, carbamazepine, oxcarbazepine, topiramate, phenobarbital, and primidone cause a transient and reversible deficiency of vitamin K–dependent clotting factors in ~50% of newborn infants. Although neonatal hemorrhage is uncommon, the mother should be treated with oral vitamin K (20 2559 mg/d, phylloquinone) in the last 2 weeks of pregnancy, and the infant should receive intramuscular vitamin K (1 mg) at birth. Special care should be taken when prescribing antiepileptic medications for women who are taking oral contraceptive agents. Drugs such as carbamazepine, phenytoin, phenobarbital, and topiramate can significantly decrease the efficacy of oral contraceptives via enzyme induction and other mechanisms. Patients should be advised to consider alternative forms of contraception, or their contraceptive medications should be modified to offset the effects of the antiepileptic medications.

1	Antiepileptic medications are excreted into breast milk to a variable degree. The ratio of drug concentration in breast milk relative to serum ranges from ~5% (valproic acid) to 300% (levetiracetam). Given the overall benefits of breast-feeding and the lack of evidence for long-term harm to the infant by being exposed to antiepileptic drugs, mothers with epilepsy can be encouraged to breast-feed. This should be reconsidered, however, if there is any evidence of drug effects on the infant such as lethargy or poor feeding. Wade S. Smith, S. Claiborne Johnston, J. Claude Hemphill, III

1	Cerebrovascular diseases include some of the most common and devastating disorders: ischemic stroke and hemorrhagic stroke. Stroke is the second leading cause of death worldwide, causing 6.2 million deaths in 2011, and is double the rate of heart disease in China. Strokes cause ~200,000 deaths each year in the United States and are a major cause of disability. The incidence of cerebrovascular diseases increases with age, and the number of strokes is projected to increase as the elderly population grows, with a doubling in stroke deaths in the United States by 2030. A stroke, or cerebrovascular accident, is defined as an abrupt onset of a neurologic deficit that is attributable to a focal vascular cause. Thus, the definition of stroke is clinical, and laboratory studies including brain imaging are used to support the diagnosis. The clinical manifestations of stroke are highly variable because of the complex anatomy of the brain and its vasculature. Cerebral ischemia is caused by a

1	imaging are used to support the diagnosis. The clinical manifestations of stroke are highly variable because of the complex anatomy of the brain and its vasculature. Cerebral ischemia is caused by a reduction in blood flow that lasts longer than several seconds. Neurologic symptoms are manifest within seconds because neurons lack glycogen, so energy failure is rapid. If the cessation of flow lasts for more than a few minutes, infarction or death of brain tissue results. When blood flow is quickly restored, brain tissue can recover fully and the patient’s symptoms are only transient: this is called a transient ischemic attack (TIA). The definition of TIA requires that all neurologic signs and symptoms resolve within 24 h without evidence of brain infarction on brain imaging. Stroke has occurred if the neurologic signs and symptoms last for >24 h or brain infarction is demonstrated. A generalized reduction in cerebral blood flow due to systemic hypotension (e.g., cardiac arrhythmia,

1	occurred if the neurologic signs and symptoms last for >24 h or brain infarction is demonstrated. A generalized reduction in cerebral blood flow due to systemic hypotension (e.g., cardiac arrhythmia, myocardial infarction, or hemorrhagic shock) usually produces syncope (Chap. 27). If low cerebral blood flow persists for a longer duration, then infarction in the border zones between the major cerebral artery distributions may develop. In more severe instances, global hypoxia-ischemia causes widespread brain injury; the constellation of cognitive sequelae that ensues is called hypoxic-ischemic encephalopathy (Chap. 330). Focal ischemia or infarction, conversely, is usually caused by thrombosis of the cerebral vessels themselves or by emboli from a proximal arterial source or the heart. Intracranial hemorrhage is caused by bleeding directly into or around the brain; it produces neurologic symptoms by producing a 2560 mass effect on neural structures, from the toxic effects of blood

1	Intracranial hemorrhage is caused by bleeding directly into or around the brain; it produces neurologic symptoms by producing a 2560 mass effect on neural structures, from the toxic effects of blood itself, or by increasing intracranial pressure.

1	with acute neurologic symptoms due to hemorrhage, seizure, or hydrocephalus. Surprisingly, migraine (Chap. 447) can mimic stroke, even in patients without a significant migraine history. When migraine develops without head pain (acephalgic migraine), the diagnosis can be especially difficult. Patients without any prior history of migraine may develop acephalgic migraine even after age 65. A sensory disturbance is often prominent, and the sensory deficit, as well as any motor deficits, tends to migrate slowly across a limb, over minutes rather than seconds as with stroke. The diagnosis of migraine becomes more secure as the cortical disturbance begins to cross vascular boundaries or if typical visual symptoms are present such as scintillating scotomata. At times it may be impossible to make the diagnosis of migraine until there have been multiple episodes with no residual symptoms or signs and no changes on brain magnetic resonance imaging (MRI). Metabolic encephalopathies typically

1	the diagnosis of migraine until there have been multiple episodes with no residual symptoms or signs and no changes on brain magnetic resonance imaging (MRI). Metabolic encephalopathies typically produce fluctuating mental status changes without focal neurologic findings. However, in the setting of prior stroke or brain injury, a patient with fever or sepsis may manifest a recurrent hemiparesis, which clears rapidly when the infection is treated. The metabolic process serves to “unmask” a prior deficit.

1	Once the diagnosis of stroke is made, a brain imaging study is necessary to determine if the cause of stroke is ischemia or hemorrhage (Fig. 446-1). Computed tomography (CT) imaging of the brain is the standard imaging modality to detect the presence or absence of intracranial hemorrhage (see “Imaging Studies,” below). If the stroke is ischemic, administration of recombinant tissue plasminogen activator (rtPA) or endovascular mechanical thrombectomy may be beneficial in restoring cerebral perfusion (see “Treatment: Acute Ischemic Stroke”). Medical management to reduce the risk of complications becomes the next priority, followed by plans for secondary prevention. For ischemic stroke, several strategies can reduce the risk of subsequent stroke in all patients, while other strategies are effective for patients with specific causes of stroke such as cardiac embolus and carotid atherosclerosis. For hemorrhagic stroke, aneurysmal subarachnoid hemorrhage (SAH) and hypertensive intracerebral

1	for patients with specific causes of stroke such as cardiac embolus and carotid atherosclerosis. For hemorrhagic stroke, aneurysmal subarachnoid hemorrhage (SAH) and hypertensive intracerebral hemorrhage are two important causes. The treatment and prevention of hypertensive intracerebral hemorrhage are discussed later in this chapter. SAH is discussed in Chap. 330.

1	Acute occlusion of an intracranial vessel causes reduction in blood flow to the brain region it supplies. The magnitude of flow reduction is a function of collateral blood flow, and this depends on individual

1	Stroke or TIA ABCs, glucose Ischemic stroke/ TIA, 85% Hemorrhage 15% Consider thrombolysis/ thrombectomy Consider BP lowering Obtain brain imaging Establish cause Establish cause Atrial fibrillation, 17% Carotid disease, 4% Aneurysmal SAH, 4% Hyperten-sive ICH, 7% Other, 64% Other, 4% Consider warfarin Consider CEA or stent Clip or coil (Chap. 330) Consider surgery Treat specific cause Treat specific cause APPROACH TO THE PATIENT: Cerebrovascular Disease Rapid evaluation is essential for use of time-sensitive treatments such as thrombolysis. However, patients with acute stroke often do not seek medical assistance on their own because they are rarely in pain and also may lose the appreciation that something is wrong (anosognosia); it is often a family member or a bystander who calls for help. Therefore, patients and their family members should be counseled to call emergency medical services immediately if they experience or witness the sudden onset of any of the following: loss of

1	help. Therefore, patients and their family members should be counseled to call emergency medical services immediately if they experience or witness the sudden onset of any of the following: loss of sensory and/or motor function on one side of the body (nearly 85% of ischemic stroke patients have hemiparesis); change in vision, gait, or ability to speak or understand; or a sudden, severe headache. Other causes of sudden-onset neurologic symptoms that may mimic stroke include seizure, intracranial tumor, migraine, and metabolic encephalopathy. An adequate history from an observer that no convulsive activity occurred at the onset usually excludes seizure, although ongoing complex partial seizures without tonic-clonic activity can on occasion mimic stroke. Tumors may present

1	Deep venous thrombosis prophylaxis Physical, occupational, speech therapy Evaluate for rehab, discharge planning Secondary prevention based on disease FIGURE 446-1 Medical management of stroke and TIA. Rounded boxes are diagnoses; rectangles are interventions. Numbers are percentages of stroke overall. ABCs, airway, breathing, circulation; BP, blood pressure; CEA, carotid endarterectomy; ICH, intracerebral hemorrhage; SAH, subarachnoid hemorrhage; TIA, transient ischemic attack.

1	vascular anatomy (which may be altered by disease), the site of occlusion, and systemic blood pressure. A decrease in cerebral blood flow to zero causes death of brain tissue within 4–10 min; values <16–18 mL/100 g tissue per minute cause infarction within an hour; and values <20 mL/100 g tissue per minute cause ischemia without infarction unless prolonged for several hours or days. If blood flow is restored to ischemic tissue before significant infarction develops, the patient may experience only transient symptoms, and the clinical syndrome is called a TIA. Another important concept is the ischemic penumbra, defined as the ischemic but reversibly dysfunctional tissue surrounding a core area of infarction. The penumbra can be imaged by perfusion-diffusion imaging using MRI or CT (see below and Figs. 446-15 and 446-16). The ischemic penumbra will eventually progress to infarction if no change in flow occurs, and hence saving the ischemic penumbra is the goal of revascularization

1	below and Figs. 446-15 and 446-16). The ischemic penumbra will eventually progress to infarction if no change in flow occurs, and hence saving the ischemic penumbra is the goal of revascularization therapies.

1	Focal cerebral infarction occurs via two distinct pathways (Fig. 446-2): (1) a necrotic pathway in which cellular cytoskeletal breakdown is rapid, due principally to energy failure of the cell; and (2) an apoptotic pathway in which cells become programmed to die. Ischemia produces necrosis by starving neurons of glucose and oxygen, which in turn results in failure of mitochondria to produce ATP. Without ATP, membrane ion pumps stop functioning and neurons depolarize, allowing intracellular calcium to rise. Cellular depolarization also causes glutamate release from synaptic terminals; excess extracellular glutamate produces neurotoxicity by activating postsynaptic glutamate receptors that increase neuronal calcium influx. Free radicals are produced by degradation of membrane lipids and mitochondrial dysfunction. Free radicals cause catalytic destruction of membranes and likely damage other vital functions of cells. Lesser degrees of ischemia, as are seen within the ischemic penumbra,

1	dysfunction. Free radicals cause catalytic destruction of membranes and likely damage other vital functions of cells. Lesser degrees of ischemia, as are seen within the ischemic penumbra, favor apoptotic cellular death causing cells to die days to weeks later. Fever dramatically worsens brain injury during ischemia, as does hyperglycemia (glucose >11.1 mmol/L [200 mg/dL]), so it is reasonable to suppress fever and prevent

1	FIGURE 446-2 Major steps in the cascade of cerebral ischemia. See text for details. iNOS, inducible nitric oxide synthase; PARP, poly-A ribose polymerase. hyperglycemia as much as possible. The value of induced mild hypothermia to improve stroke outcomes is the subject of continuing clinical research.

1	After the clinical diagnosis of stroke is made, an orderly process of evaluation and treatment should follow (Fig. 446-1). The first goal is to prevent or reverse brain injury. Attend to the patient’s airway, breathing, and circulation (ABCs), and treat hypoglycemia or hyperglycemia if identified. Perform an emergency noncontrast head CT scan to differentiate between ischemic stroke and hemorrhagic stroke; there are no reliable clinical findings that conclusively separate ischemia from hemorrhage, although a more depressed level of consciousness, higher initial blood pressure, or worsening of symptoms after onset favor hemorrhage, and a deficit that is maximal at onset, or remits, suggests ischemia. Treatments designed to reverse or lessen the amount of tissue infarction and improve clinical outcome fall within six categories: (1) medical support, (2) IV thrombolysis, (3) endovascular revascularization, (4) antithrombotic treatment, (5) neuroprotection, and (6) stroke centers and

1	outcome fall within six categories: (1) medical support, (2) IV thrombolysis, (3) endovascular revascularization, (4) antithrombotic treatment, (5) neuroprotection, and (6) stroke centers and rehabilitation.

1	When ischemic stroke occurs, the immediate goal is to optimize cerebral perfusion in the surrounding ischemic penumbra. Attention is also directed toward preventing the common complications of bedridden patients—infections (pneumonia, urinary, and skin) and deep venous thrombosis (DVT) with pulmonary embolism. Subcutaneous heparin (unfractionated and low-molecular-weight) is safe and can be used concomitantly. Use of pneumatic compression stockings is of proven benefit in reducing risk of DVT and is a safe alternative to heparin.

1	Because collateral blood flow within the ischemic brain may be blood pressure dependent, there is controversy about whether blood pressure should be lowered acutely. Blood pressure should be lowered if there is malignant hypertension (Chap. 298) or concomitant myocardial ischemia, or if blood pressure is >185/110 mmHg and thrombolytic therapy is anticipated. When faced with the competing demands of myocardium and brain, lowering the heart rate with a β1-adrenergic blocker (such as esmolol) can be a first step to decrease cardiac work and maintain blood pressure. Routine lowering of blood pressure has been found to worsen outcomes. Fever is detrimental and should be treated with antipyretics and surface cooling. Serum glucose should be monitored and kept at <10.0 mmol/L (180 mg/dL) using an insulin infusion if necessary.

1	Between 5 and 10% of patients develop enough cerebral edema to cause obtundation or brain herniation. Edema peaks on the second or third day but can cause mass effect for ~10 days. The larger the infarct, the greater the likelihood that clinically significant edema will develop. Water restriction and IV mannitol may be used to raise the serum osmolarity, but hypovolemia should be avoided because this may contribute to hypotension and worsening infarction. Combined analysis of three randomized European trials of hemicraniectomy (craniotomy and temporary removal of part of the skull) shows that hemicraniectomy markedly reduces mortality, and the clinical outcomes of survivors are acceptable. The size of the diffusion-weighted imaging volume of brain infarction during the acute stroke is a predictor of deterioration requiring hemicraniectomy.

1	Special vigilance is warranted for patients with cerebellar infarction. These strokes may mimic labyrinthitis because of prominent vertigo and vomiting; the presence of head or neck pain should alert the physician to consider cerebellar stroke from vertebral artery dissection. Even small amounts of cerebellar edema can acutely increase intracranial pressure (ICP) by obstructing cerebrospinal fluid (CSF) flow leading to hydrocephalus or by directly compressing the brainstem. The resulting brainstem compression can manifest as coma and respiratory arrest and require emergency surgical decompression. Prophylactic suboccipital decompression of large cerebellar infarcts before brainstem compression, although not tested rigorously in a clinical trial, is practiced at most stroke centers.

1	The National Institute of Neurological Disorders and Stroke (NINDS) rtPA Stroke Study showed a clear benefit for IV rtPA in selected patients with acute stroke. The NINDS study used IV rtPA (0.9 mg/kg to a 90-mg maximum; 10% as a bolus, then the remainder over 60 min) versus placebo in ischemic stroke within 3 h of onset. One-half of the patients were treated within 90 min. Symptomatic intra-cranial hemorrhage occurred in 6.4% of patients on rtPA and 0.6% 2562 on placebo. In the rTPA group, there was a significant 12% absolute increase in the number of patients with only minimal disability (32% on placebo and 44% on rtPA) and a nonsignificant 4% reduction in mortality (21% on placebo and 17% on rtPA). Thus, despite an increased incidence of symptomatic intracranial hemorrhage, treatment with IV rtPA within 3 h of the onset of ischemic stroke improved clinical outcome. Three subsequent trials of IV rtPA did not confirm this benefit, perhaps because of the dose of rtPA used, the timing

1	IV rtPA within 3 h of the onset of ischemic stroke improved clinical outcome. Three subsequent trials of IV rtPA did not confirm this benefit, perhaps because of the dose of rtPA used, the timing of its delivery, and small sample size. When data from all randomized IV rtPA trails were combined, however, efficacy was confirmed in the <3-h time window, and efficacy likely extended to 4.5 h and possibly to 6 h. Based on these combined results, the European Cooperative Acute Stroke Study (ECASS) III explored the safety and efficacy of rtPA in the 3to 4.5-h time window. Unlike the NINDS study, patients older than 80 years of age and diabetic patients with a previous stroke were excluded. In this 821-patient randomized study, efficacy was again confirmed, although the treatment effect was less robust than in the 0to 3-h time window. In the rtPA group, 52.4% of patients achieved a good outcome at 90 days, compared to 45.2% of the placebo group (odds ratio [OR] 1.34, p = .04). The symptomatic

1	than in the 0to 3-h time window. In the rtPA group, 52.4% of patients achieved a good outcome at 90 days, compared to 45.2% of the placebo group (odds ratio [OR] 1.34, p = .04). The symptomatic intra-cranial hemorrhage rate was 2.4% in the rtPA group and 0.2% in the placebo group (p = .008). Based on these data, rtPA is approved in the 3to 4.5-h window in Europe and Canada, but is still only approved for 0–3 h in the United States and Canada. Use of IV tPA is now considered a central component of primary stroke centers (see below). It represents the first treatment proven to improve clinical outcomes in ischemic stroke and is cost-effective and cost-saving. Advanced neuroimaging techniques (see neuroimaging section below) may help to select patients beyond the 4.5-h window who will benefit from thrombolysis, but this is currently investigational. The time of stroke onset is defined as the time the patient’s symptoms were witnessed to begin or the time the patient was last seen as

1	from thrombolysis, but this is currently investigational. The time of stroke onset is defined as the time the patient’s symptoms were witnessed to begin or the time the patient was last seen as normal. Patients who awaken with stroke have the onset defined as when they went to bed. Table 446-1 summarizes eligibility criteria and instructions for administration of IV rtPA.

1	Ischemic stroke from large-vessel intracranial occlusion results in high rates of mortality and morbidity. Occlusions in such large vessels (middle cerebral artery [MCA], intracranial internal carotid artery, and the basilar artery) generally involve a large clot volume and often fail to open with IV rtPA alone. Therefore, there is growing interest in using thrombolytics via an intraarterial route to increase the concentration of drug at the clot and minimize systemic bleeding complications. The Prolyse in Acute Cerebral Thromboembolism (PROACT) II trial found benefit for intraarterial prourokinase in acute MCA occlusions up to the sixth hour following onset of stroke. Intraarterial treatment of basilar artery occlusions may also be beneficial for selected patients. Intraarterial administration of a thrombolytic agent for acute ischemic stroke (AIS) is not approved by the U.S. Food and Drug Administration (FDA); however, many stroke centers offer this treatment based on these data.

1	Endovascular mechanical thrombectomy has been studied as an alternative or adjunctive treatment of acute stroke in patients who are ineligible for, or have contraindications to, thrombolytics or in those who failed to achieve vascular recanalization with IV thrombolytics (see Fig. 446-15). The Mechanical Embolus Removal in Cerebral Ischemia (MERCI) and multi-MERCI single-arm trials found that an endovascular thrombectomy device restored patency of occluded intracranial vessels within 8 h of ischemic stroke symptoms compared with a historical control group. Recanalization of the target vessel occurred in 48–58% of treated patients and in 60–69% of patients after use of adjuvant endovascular methods, and successful recanalization at 90 days correlated well with favorable outcomes. Based on these nonrandomized data, the FDA approved this device as the first device for revascularization of occluded vessels in AIS even if the patient has been given rtPA and that therapy has failed.

1	Clinical diagnosis of stroke Sustained BP >185/110 mmHg despite treatment Onset of symptoms to time of drug administration ≤4.5 hb Platelets <100,000; HCT <25%; glucose <50 or >400 mg/dL edema of >1/3 of the MCA territory Use of heparin within 48 h and prolonged PTT, or elevated INR Administration of rtPA IV access with two peripheral IV lines (avoid arterial or central line placement) Administer 0.9 mg/kg IV (maximum 90 mg) IV as 10% of total dose by bolus, followed by remainder of total dose over 1 h For decline in neurologic status or uncontrolled blood pressure, stop infusion, give cryoprecipitate, and reimage brain emergently aSee Activase (tissue plasminogen activator) package insert for complete list of contraindications and dosing. bDepending on the country, IV rtPA may be approved for up to 4.5 h with additional restrictions.

1	Abbreviations: BP, blood pressure; CT, computed tomography; HCT, hematocrit; INR, international normalized ratio; MCA, middle cerebral artery; PTT, partial thromboplastin time. The Penumbra Pivotal Stroke trial tested another mechanical device that showed even higher rates of recanalization and led to FDA clearance of the tested device as well. More recently, two Stentriever devices (nondetachable stents) were shown to significantly improve vascular recanalization compared to the first approved MERCI device, approaching recanalization rates of 90% in most large intra-cranial vessels.

1	In 2013, three randomized endovascular trials with nonendovascular controls found no benefits to endovascular therapy. The largest was the Interventional Management of Stroke III trial that randomized 656 AIS patients within 3 h of onset to IV rtPA (0.9 mg/kg) alone versus IV rtPA (0.6 mg/kg) followed by endovascular adjuvant treatment with IA rtPA, or endovascular thrombectomy as soon as possible. Outcomes between these groups were not significantly different, and there were more complications (groin bleeding chiefly) in the endovascular group. The SYNTHESIS trial based in Italy randomized 363 patients to IV rtPA versus intraarterial rtPA for patients within 3 h of stroke onset. No differences were found between the groups at 90 days. These two relatively large trials indicate that endovascular therapy using principally intraarterial rtPA is not better than IV therapy, but many questions remain. Relatively few patients received mechanical clot retraction therapies, and those who did

1	therapy using principally intraarterial rtPA is not better than IV therapy, but many questions remain. Relatively few patients received mechanical clot retraction therapies, and those who did received what we now know were inferior devices. Trials assessing more efficacious thrombectomy devices are currently ongoing.

1	Because use of endovascular devices in combination with rtPA appears relatively safe, some centers continue to offer endovascular therapy. This applies to patients who are not eligible for IV rtPA (recent surgery, stroke following cardiac catheterization, etc.), and some continue to use thrombectomy because of perceived better outcomes in patients with more effective devices. Comprehensive stroke centers are now obtaining credentialing to offer this therapy in distinction to primary stroke centers that offer only IV rtPA.

1	ANTITHROMBOTIC TREATMENT Platelet Inhibition Aspirin is the only antiplatelet agent that has been proven effective for the acute treatment of ischemic stroke; there are several antiplatelet agents proven for the secondary prevention of stroke (see below). Two large trials, the International Stroke Trial (IST) and the Chinese Acute Stroke Trial (CAST), found that the use of aspirin within 48 h of stroke onset reduced both stroke recurrence risk and mortality minimally. Among 19,435 patients in IST, those allocated to aspirin, 300 mg/d, had slightly fewer deaths within 14 days (9.0 vs 9.4%), significantly fewer recurrent ischemic strokes (2.8 vs 3.9%), no excess of hemorrhagic strokes (0.9 vs 0.8%), and a trend toward a reduction in death or dependence at 6 months (61.2 vs 63.5%). In CAST, 21,106 patients with ischemic stroke received 160 mg/d of aspirin or a placebo for up to 4 weeks. There were very small reductions in the aspirin group in early mortality (3.3 vs 3.9%), recurrent

1	21,106 patients with ischemic stroke received 160 mg/d of aspirin or a placebo for up to 4 weeks. There were very small reductions in the aspirin group in early mortality (3.3 vs 3.9%), recurrent ischemic strokes (1.6 vs 2.1%), and dependency at discharge or death (30.5 vs 31.6%). These trials demonstrate that the use of aspirin in the treatment of AIS is safe and produces a small net benefit. For every 1000 acute strokes treated with aspirin, about 9 deaths or nonfatal stroke recurrences will be prevented in the first few weeks and ~13 fewer patients will be dead or dependent at 6 months.

1	Clopidogrel is being tested as a way to prevent stroke following TIA and minor ischemic stroke (see below).

1	Anticoagulation Numerous clinical trials have failed to demonstrate any benefit of anticoagulation in the primary treatment of atherothrombotic cerebral ischemia. Several trials have investigated antiplatelet versus anticoagulant medications given within 12–24 h of the initial event. The U.S. Trial of Organon 10172 in Acute Stroke Treatment (TOAST), an investigational low-molecular-weight heparin (LMWH), failed to show any benefit over aspirin. Use of SC unfractionated heparin versus aspirin was tested in IST. Heparin given SC afforded no additional benefit over aspirin and increased bleeding rates. Several trials of LMWHs have also shown no consistent benefit in AIS. Furthermore, trials generally have shown an excess risk of brain and systemic hemorrhage with acute anticoagulation. A recent meta-analysis of all forms of heparin found no benefit for acute stroke patients at high or low risk of thrombotic events. Therefore, trials do not support the use of heparin or other

1	A recent meta-analysis of all forms of heparin found no benefit for acute stroke patients at high or low risk of thrombotic events. Therefore, trials do not support the use of heparin or other anticoagulants for patients with atherothrombotic stroke.

1	Neuroprotection is the concept of providing a treatment that prolongs the brain’s tolerance to ischemia. Drugs that block the excitatory amino acid pathways have been shown to protect neurons and glia in animals, but despite multiple human trials, they have not yet been proven to be beneficial. Hypothermia is a powerful neuroprotective treatment in patients with cardiac arrest (Chap. 330) and is neuroprotective in animal models of stroke, but it has not been adequately studied in patients with ischemic stroke and is associated with an increase in pneumonia rates that could adversely impact stroke outcomes.

1	Patient care in stroke units followed by rehabilitation services improves neurologic outcomes and reduces mortality. Use of clinical pathways and staff dedicated to the stroke patient can improve care. This includes use of standardized stroke order sets. Stroke teams that provide emergency 24-h evaluation of acute stroke patients for acute medical management and consideration of thrombolysis or endovascular treatments are essential components of primary and comprehensive stroke centers, respectively.

1	Proper rehabilitation of the stroke patient includes early physical, occupational, and speech therapy. It is directed toward educating the patient and family about the patient’s neurologic deficit, preventing the complications of immobility (e.g., pneumonia, DVT 2563 and pulmonary embolism, pressure sores of the skin, and muscle contractures), and providing encouragement and instruction in overcoming the deficit. Use of pneumatic compression stockings is of proven benefit in reducing risk of DVT and is a safe alternative to heparin. The goal of rehabilitation is to return the patient to home and to maximize recovery by providing a safe, progressive regimen suited to the individual patient. Additionally, the use of constrained movement therapy (immobilizing the unaffected side) has been shown to improve hemiparesis following stroke, even years after the stroke, suggesting that physical therapy can recruit unused neural pathways. Newer robotic therapies appear promising as well. The

1	shown to improve hemiparesis following stroke, even years after the stroke, suggesting that physical therapy can recruit unused neural pathways. Newer robotic therapies appear promising as well. The human nervous system is more adaptable than previously thought, and developing physical and pharmacologic strategies to enhance long-term neural recovery is an active area of research.

1	(Figs. 446-1 and 446-3 and Table 446-2) Although the initial management of AIS often does not depend on the etiology, establishing a cause is essential to reduce the risk of recurrence. Particular focus should be on atrial fibrillation and carotid atherosclerosis, because these etiologies have proven secondary prevention strategies. The clinical presentation and examination findings often establish the cause of stroke or narrow the possibilities to a few. Judicious use of laboratory testing and imaging studies completes the initial evaluation. Nevertheless, nearly 30% of strokes remain unexplained despite extensive evaluation.

1	Clinical examination should focus on the peripheral and cervical vascular system (carotid auscultation for bruits and blood pressure), the heart (dysrhythmia, murmurs), extremities (peripheral emboli), and retina (effects of hypertension and cholesterol emboli [Hollenhorst plaques]). A complete neurologic examination is performed to localize the anatomic site of stroke. An imaging study of the brain is nearly always indicated and is required for patients being considered for thrombolysis; it may be combined with CTor MRI-based angiography to visualize the vasculature of the neck and intracranial vessels (see “Imaging Studies,” below). A chest x-ray, electrocardiogram (ECG), urinalysis, complete blood count, erythrocyte sedimentation rate (ESR), serum electrolytes, blood urea nitrogen (BUN), creatinine, blood glucose, serum lipid profile, prothrombin time (PT), and partial thromboplastin time (PTT) are often useful and should be considered in all patients. An ECG may demonstrate

1	(BUN), creatinine, blood glucose, serum lipid profile, prothrombin time (PT), and partial thromboplastin time (PTT) are often useful and should be considered in all patients. An ECG may demonstrate arrhythmias or reveal evidence of recent myocardial infarction (MI). Of all these studies, only brain imaging, blood glucose, and perhaps PTT/international normalized ratio (INR) are necessary prior to IV rtPA; the results of other studies should not delay the rapid administration of IV rtPA if the patient is eligible.

1	Cardioembolic Stroke Cardioembolism is responsible for ~20% of all ischemic strokes. Stroke caused by heart disease is primarily due to embolism of thrombotic material forming on the atrial or ventricular wall or the left heart valves. These thrombi then detach and embolize into the arterial circulation. The thrombus may fragment or lyse quickly, producing only a TIA. Alternatively, the arterial occlusion may last longer, producing stroke. Embolic strokes tend to occur suddenly with maximum neurologic deficit present at onset. With reperfusion following more prolonged ischemia, petechial hemorrhages can occur within the ischemic territory. These are usually of no clinical significance and should be distinguished from frank intracranial hemorrhage into a region of ischemic stroke where the mass effect from the hemorrhage can cause a significant decline in neurologic function.

1	Emboli from the heart most often lodge in the intracranial internal carotid artery, the MCA, the posterior cerebral artery (PCA), or one of their branches; infrequently, the anterior cerebral artery (ACA) is involved. Emboli large enough to occlude the stem of the MCA (3–4 mm) lead to large infarcts that involve both deep gray and white matter and some portions of the cortical surface and its underlying white matter. A smaller embolus may occlude a small cortical or penetrating arterial branch. The location and size of an infarct within a vascular territory depend on the extent of the collateral circulation. artery disease Carotid plaque with arteriogenic emboli

1	FIGURE 446-3 Pathophysiology of ischemic stroke. A. Diagram illustrating the three major mechanisms that underlie ischemic stroke: (1) occlusion of an intracranial vessel by an embolus that arises at a distant site (e.g., cardiogenic sources such as atrial fibrillation or artery-to-artery emboli from carotid atherosclerotic plaque), often affecting the large intracranial vessels; (2) in situ thrombosis of an intracranial vessel, typically affecting the small penetrating arteries that arise from the major intracranial arteries; (3) hypoperfusion caused by flow-limiting stenosis of a major extracranial (e.g., internal carotid) or intracranial vessel, often producing “watershed” ischemia. B. and C. Diagram and reformatted computed tomography angiogram of the common, internal, and external carotid arteries. High-grade stenosis of the internal carotid artery, which may be associated with either cerebral emboli or flow-limiting ischemia, was identified in this patient.

1	The most significant causes of cardioembolic stroke in most of the world are nonrheumatic (often called nonvalvular) atrial fibrillation, MI, prosthetic valves, rheumatic heart disease, and ischemic cardiomyopathy (Table 446-2). Cardiac disorders causing brain embolism are discussed in the chapters on heart diseases. A few pertinent aspects are highlighted here.

1	Nonrheumatic atrial fibrillation is the most common cause of cerebral embolism overall. The presumed stroke mechanism is thrombus formation in the fibrillating atrium or atrial appendage, with subsequent embolization. Patients with atrial fibrillation have an average annual risk of stroke of ~5%. The risk of stroke can be estimated by calculating the CHADS2 score (Table 446-3). Left atrial enlargement is an additional risk factor for formation of atrial thrombi. Rheumatic heart disease usually causes ischemic stroke when there is prominent mitral stenosis or atrial fibrillation. Recent MI may be a source of emboli, especially when transmural and involving the anteroapical ventricular wall, and prophylactic anticoagulation following MI has been shown to reduce stroke risk. Mitral valve prolapse is not usually a source of emboli unless the prolapse is severe.

1	Paradoxical embolization occurs when venous thrombi migrate to the arterial circulation, usually via a patent foramen ovale or atrial septal defect. Bubble-contrast echocardiography (IV injection of agitated saline coupled with either transthoracic or transesophageal echocardiography) can demonstrate a right-to-left cardiac shunt, revealing the conduit for paradoxical embolization. Alternatively, a right-to-left shunt is implied if immediately following IV injection of agitated saline, the ultrasound signature of bubbles is observed during transcranial Doppler insonation of the MCA; pulmonary arteriovenous malformations should be considered if this test is positive yet an echocardiogram fails to reveal an intracardiac shunt. Both techniques are highly sensitive for detection of right-to-left shunts. Besides venous clot, fat and tumor emboli, bacterial endocarditis, IV air, and amniotic fluid emboli at childbirth may occasionally be responsible for paradoxical embolization. The

1	shunts. Besides venous clot, fat and tumor emboli, bacterial endocarditis, IV air, and amniotic fluid emboli at childbirth may occasionally be responsible for paradoxical embolization. The importance of a patent foramen ovale (PFO) as a cause of stroke is debated, particularly because they are present in ~15% of the general population. Some studies have suggested that the risk is only elevated in the presence of a coexisting atrial septal aneurysm. The presence of a venous source of embolus, most commonly a deep venous thrombus, may provide confirmation of the importance of a PFO with an accompanying right-to-left shunt in a particular case. Three randomized trials of PFO occlusion for secondary prevention of ischemic stroke were negative, although each lacked sufficient power to be conclusive. At present, there is no supportive evidence to offer percutaneous PFO closure for stroke prevention.

1	Bacterial endocarditis can be a source of valvular vegetations that give rise to septic emboli. The appearance of multifocal symptoms and signs in a patient with stroke makes bacterial endocarditis more likely. Infarcts of microscopic size occur, and large septic infarcts may evolve into brain abscesses or cause hemorrhage into the infarct, which generally precludes use of anticoagulation or thrombolytics. Mycotic aneurysms caused by septic emboli may also present as SAH or intra-cerebral hemorrhage.

1	Artery-to-Artery Embolic Stroke Thrombus formation on atherosclerotic plaques may embolize to intracranial arteries producing an artery-to-artery embolic stroke. Less commonly, a diseased vessel may acutely thrombose. Unlike the myocardial vessels, artery-to-artery embolism, rather than local thrombosis, appears to be the dominant vascular mechanism causing large-vessel brain ischemia. Any diseased vessel may be an embolic source, including the aortic arch, common carotid, internal carotid, vertebral, and basilar arteries.

1	cArotiD AtheroScleroSiS Atherosclerosis within the carotid artery occurs most frequently within the common carotid bifurcation and proximal internal carotid artery; the carotid siphon (portion within the cavernous sinus) is also vulnerable to atherosclerosis. Male gender, older age, smoking, hypertension, diabetes, and hypercholesterolemia are risk factors for carotid disease, as they are for stroke in general (Table 446-4). Carotid atherosclerosis produces an estimated 10% of Spontaneous echo contrast Systemic vasculitis (PAN, granuloma- Stimulant drugs: cocaine, tosis with polyangiitis [Wegener’s], amphetamine Takayasu’s, giant cell arteritis) Primary CNS vasculitis Meningitis (syphilis, tuberculosis, fungal, bacterial, zoster) aChiefly cause venous sinus thrombosis. bMay be associated with any hypercoagulable disorder. Abbreviations: CNS, central nervous system; PAN, polyarteritis nodosa.

1	Abbreviations: CNS, central nervous system; PAN, polyarteritis nodosa. ischemic stroke. For further discussion of the pathogenesis of atherosclerosis, see Chap. 291e. Carotid disease can be classified by whether the stenosis is symptomatic or asymptomatic and by the degree of stenosis (percent narrowing of the narrowest segment compared to a nondiseased segment). Symptomatic carotid disease implies that the patient has experienced a stroke or TIA within the vascular distribution of the artery, and it is associated with a greater risk of subsequent stroke than asymptomatic stenosis, in which the patient is symptom free and the stenosis is detected through screening. Greater degrees of arterial narrowing are generally associated with a higher risk of stroke, except that those with near occlusions are at lower risk of stroke.

1	other cAuSeS of Artery-to-Artery embolic Stroke Intracranial atherosclerosis produces stroke either by an embolic mechanism or by in situ thrombosis of a diseased vessel. It is more common in patients of Asian 2565 and African-American descent. Recurrent stroke risk is ~15% per year, similar to symptomatic untreated carotid atherosclerosis.

1	Dissection of the internal carotid or vertebral arteries or even vessels beyond the circle of Willis is a common source of embolic stroke in young (age <60 years) patients. The dissection is usually painful and precedes the stroke by several hours or days. Extracranial dissections do not cause hemorrhage, presumably because of the tough adventitia of these vessels. Intracranial dissections, conversely, may produce SAH because the adventitia of intracranial vessels is thin and pseudoaneu rysms may form, requiring urgent treatment to prevent rerupture. Treating asymptomatic pseudoaneurysms following dissection is likely not necessary. The cause of dissection is usually unknown, and recurrence is rare. Ehlers-Danlos type IV, Marfan’s disease, cystic medial necrosis, and fibromuscular dysplasia are associated with dissections. Trauma (usually a motor vehicle accident or a sports injury) can cause carotid and vertebral artery dissections. Spinal manipulative therapy is associated with

1	are associated with dissections. Trauma (usually a motor vehicle accident or a sports injury) can cause carotid and vertebral artery dissections. Spinal manipulative therapy is associated with vertebral artery dissection and stroke. Most dissections heal spontaneously, and stroke or TIA is uncommon beyond 2 weeks. Although there are no trials comparing anticoagulation to antiplatelet agents, many physicians treat acutely with anticoagulants and then convert to antiplatelet therapy after demonstration of satisfactory vascular recanalization.

1	The term lacunar infarction refers to infarction following atherothrombotic or lipohyalinotic occlusion of a small artery in the brain. The term small-vessel stroke denotes occlusion of such a small penetrating artery and is now the preferred term. Small-vessel strokes account for ~20% of all strokes.

1	Pathophysiology The MCA stem, the arteries comprising the circle of Willis (A1 segment, anterior and posterior communicating arteries, and P1 segment), and the basilar and vertebral arteries all give rise to 30to 300-μm branches that penetrate the deep gray and white matter of the cerebrum or brainstem (Fig. 446-4). Each of these small branches can occlude either by atherothrombotic disease at its origin or by the development of lipohyalinotic thickening. Thrombosis of these vessels causes small infarcts that are referred to as lacunes (Latin for “lake” of fluid noted at autopsy). These infarcts range in size from 3 mm to 2 cm in diameter. Hypertension and age are the principal risk factors.

1	Clinical Manifestations The most common small-vessel stroke syndromes are the following: (1) Pure motor hemiparesis from an infarct in the posterior limb of the internal capsule or the pons; the face, arm, and leg are almost always involved; (2) pure sensory stroke from an infarct in the ventral thalamus; (3) ataxic hemiparesis from an infarct in the ventral pons or internal capsule; (4) and dysarthria and a clumsy hand or arm due to infarction in the ventral pons or in the genu of the internal capsule. Transient symptoms (small-vessel TIAs) may herald a small-vessel infarct; they may occur several times a day and last only a few minutes. Recovery from small-vessel strokes tends to be more rapid and complete than recovery from large-vessel strokes; in some cases, however, there is severe permanent disability.

1	A large-vessel source (either thrombosis or embolism) may manifest initially as a small-vessel infarction. Therefore, the search for embolic sources (carotid and heart) should not be completely abandoned in the evaluation of these patients. Secondary prevention of small-vessel stroke involves risk factor modification, specifically reduction in blood pressure (see “Treatment: Primary and Secondary Prevention of Stroke and TIA,” below). (Table 446-2) Hypercoagulable disorders (Chap. 78) primarily increase the risk of venous, including venous sinus, thrombosis. Systemic lupus erythematosus with Libman-Sacks endocarditis can be a cause of embolic stroke. These conditions overlap with the antiphospholipid syndrome, which probably requires long-term anticoagulation to With atrial fibrillation, previous embolization, or atrial appendage thrombus, or left atrial diameter >55 mm Without atrial fibrillation but systemic embolization, or otherwise cryptogenic stroke or TIA

1	Without atrial fibrillation but systemic embolization, or otherwise cryptogenic stroke or TIA Aortic position, bileaflet or Medtronic Hall tilting disk with normal left atrial size and sinus rhythm Mitral or aortic position, anterior-apical myocardial infarct or left atrial enlargement Mitral or aortic position, with atrial fibrillation, or hypercoagulable state, or low ejection fraction, or atherosclerotic vascular disease VKA INR 2.5, range 2–3 VKA INR 3.0, range 2.5–3.5 VKA INR 3.0, range 2.5–3.5 Aspirin plus VKA INR 3.0, range 2.5–3.5 Add aspirin and/or increase INR: prior target was 2.5 increase to 3.0, range 2.5–3.5; prior target was 3.0 increase to 3.5, range 3–4 aCHADS2 score calculated as follows: 1 point for age >75 years, 1 point for hypertension, 1 point for congestive heart failure, 1 point for diabetes, and 2 points for stroke or TIA; sum of points is the total CHADS2 score.

1	Note: Dose of aspirin is 50–325 mg/d; target INR for OAC is between 2 and 3 unless otherwise specified. Abbreviations: INR, international normalized ratio; LMWH, low-molecular-weight heparin; OAC, oral anticoagulant (VKA, thrombin inhibitor, oral factor Xa inhibitors); TIA, transient ischemic attack; VKA, vitamin K antagonist. Sources: Modified from DE Singer et al: Chest 133:546S, 2008; DN Salem et al: Chest 133:593S, 2008. prevent further stroke. Homocysteinemia may cause arterial thromboses as well; this disorder is caused by various mutations in the homocysteine pathways and responds to different forms of cobalamin depending on the mutation.

1	Venous sinus thrombosis of the lateral or sagittal sinus or of small cortical veins (cortical vein thrombosis) occurs as a complication of oral contraceptive use, pregnancy and the postpartum period, inflammatory bowel disease, intracranial infections (meningitis), and dehydration. It is also seen in patients with laboratory-confirmed thrombophilia including polycythemia, sickle cell anemia, deficiencies of proteins C and S, factor V Leiden mutation (resistance to activated protein C), antithrombin III deficiency, homocysteinemia, and the prothrombin G20210 mutation. Women who take oral contraceptives and have the prothrombin G20210 mutation may be at particularly high risk for sinus thrombosis. Patients present with headache and may also have focal neurologic signs (especially paraparesis) and seizures. Often, CT imaging is normal unless an intracranial venous hemorrhage has occurred, but the venous sinus occlusion is readily visualized using magnetic resonance (MR) or CT venography

1	and seizures. Often, CT imaging is normal unless an intracranial venous hemorrhage has occurred, but the venous sinus occlusion is readily visualized using magnetic resonance (MR) or CT venography or conventional x-ray angiography. With greater degrees of sinus thrombosis, the

1	Basilar a. Basilar a. Vertebral a. Deep branches Hypertension 2–5 38% 100–300 50–100 Atrial fibrillation 1.8–2.9 68% warfarin, 21% aspirin 20–83 13 Diabetes 1.8–6 No proven effect Smoking 1.8 50% at 1 year, baseline risk at 5 years postcessation Hyperlipidemia 1.8–2.6 16–30% 560 230 Asymptomatic 2.0 53% 85 N/A carotid stenosis aNumber needed to treat to prevent one stroke annually. Prevention of other cardiovascular outcomes is not considered here. Abbreviation: N/A, not applicable. Deep branches of the Anterior cerebral a. middle cerebral a. Anterior cerebral a. Internal carotid a. Middle cerebral a. Internal carotid a. Middle cerebral a. Vertebral a. of the basilar a.

1	Deep branches of the Anterior cerebral a. middle cerebral a. Anterior cerebral a. Internal carotid a. Middle cerebral a. Internal carotid a. Middle cerebral a. Vertebral a. of the basilar a. FIGURE 446-4 Diagrams and reformatted computed tomography (CT) angiograms in the coronal section illustrating the deep penetrating arteries involved in small-vessel strokes. In the anterior circulation, small penetrating arteries called lenticulostriates arise from the proximal portion of the anterior and middle cerebral arteries and supply deep subcortical structures (upper panels). In the posterior circulation, similar arteries arise directly from the vertebral and basilar arteries to supply the brainstem (lower panels). Occlusion of a single penetrating artery gives rise to a discrete area of infarct (pathologically termed a “lacune,” or lake). Note that these vessels are too small to be visualized on CT angiography. patient may develop signs of increased 2567

1	patient may develop signs of increased 2567 ICP and coma. Intravenous heparin, regardless of the presence of intracranial hemorrhage, reduces morbidity and mortality, and the long-term outcome is generally good. Heparin prevents further thrombosis and reduces venous hypertension and ischemia. If an underlying hypercoagulable state is not found, many physicians treat with vitamin K antagonists (VKAs) for 3–6 months and then convert to aspirin, depending on the degree of resolution of the venous sinus thrombus. Anticoagulation is often continued indefinitely if thrombophilia is diagnosed.

1	Sickle cell anemia (SS disease) is a common cause of stroke in children. A subset of homozygous carriers of this hemoglobin mutation develop stroke in childhood, and this may be predicted by documenting high-velocity blood flow within the MCAs using transcranial Doppler ultrasonography. In children who are identified to have high velocities, treatment with aggressive exchange transfusion dramatically reduces risk of stroke, and if exchange transfusion is ceased, their stroke rate increases again along with MCA velocities.

1	Fibromuscular dysplasia affects the cervical arteries and occurs mainly in women. The carotid or vertebral arteries show multiple rings of segmental narrowing alternating with dilatation. Vascular occlusion is usually incomplete. The process is often asymptomatic but occasionally is associated with an audible bruit, TIAs, or stroke. Involvement of the renal arteries is common and may cause hypertension. The cause and natural history of fibromuscular dysplasia are unknown (Chap. 302). TIA or stroke generally occurs only when the artery is severely narrowed or dissects. Anticoagulation or antiplatelet therapy may be helpful. Temporal (giant cell) arteritis (Chap.

1	Temporal (giant cell) arteritis (Chap. 385) is a relatively common affliction of elderly individuals in which the external carotid system, particularly the temporal arteries, undergo subacute granulomatous inflammation with giant cells. Occlusion of posterior ciliary arteries derived from the ophthalmic artery results in blindness in one or both eyes and can be prevented with glucocorticoids. It rarely causes stroke because the internal carotid artery is usually not inflamed. Idiopathic giant cell arteritis involving the great vessels arising from the aortic arch (Takayasu’s arteritis) may cause carotid or vertebral thrombosis; it is rare in the Western Hemisphere. Necrotizing (or granulomatous) arteritis, occurring alone or in association with generalized polyarteritis nodosa or granulomatosis with polyangiitis (Wegener’s), involves the distal small

1	Necrotizing (or granulomatous) arteritis, occurring alone or in association with generalized polyarteritis nodosa or granulomatosis with polyangiitis (Wegener’s), involves the distal small FIGURE 446-5 Cerebral angiogram from a 32-year-old male with central nervous system vasculopathy. Dramatic beading (arrows) typical of vasculopathy is seen.

1	FIGURE 446-5 Cerebral angiogram from a 32-year-old male with central nervous system vasculopathy. Dramatic beading (arrows) typical of vasculopathy is seen. branches (<2 mm diameter) of the main intracranial arteries and produces small ischemic infarcts in the brain, optic nerve, and spinal cord. The CSF often shows pleocytosis, and the protein level is elevated. Primary central nervous system vasculitis is rare; small or medium-sized vessels are usually affected, without apparent systemic vasculitis. The differential diagnosis includes other inflammatory vasculopathies including infection (tuberculous, fungal), sarcoidosis, angiocentric lymphoma, carcinomatous meningitis, and noninflammatory causes such as atherosclerosis, emboli, connective tissue disease, vasospasm, migraine-associated vasculopathy, and drug-associated causes. Some cases develop in the postpartum period and are self-limited.

1	Patients with any form of vasculopathy may present with insidious progression of combined white and gray matter infarctions, prominent headache, and cognitive decline. Brain biopsy or high-resolution conventional x-ray angiography is usually required to make the diagnosis (Fig. 446-5). An inflammatory profile found on lumbar puncture favors an inflammatory cause. In cases where inflammation is confirmed, aggressive immunosuppression with glucocorticoids, and often cyclophosphamide, is usually necessary to prevent progression; a diligent investigation for infectious causes such as tuberculosis is essential prior to immunosuppression. With prompt recognition and treatment, many patients can make an excellent recovery.

1	Drugs, in particular amphetamines and perhaps cocaine, may cause stroke on the basis of acute hypertension or drug-induced vasculopathy. No data exist on the value of any treatment. Phenylpropanolamine has been linked with intracranial hemorrhage, as has cocaine and methamphetamine, perhaps related to a drug-induced vasculopathy. Moyamoya disease is a poorly understood occlusive disease involving large intracranial arteries, especially the distal internal carotid artery and the stem of the MCA and ACA. Vascular inflammation is absent. The lenticulostriate arteries develop a rich collateral circulation around the occlusive lesion, which gives the impression of a “puff of smoke” (moyamoya in Japanese) on conventional x-ray angiography. Other collaterals include transdural anastomoses between the cortical surface branches of the meningeal and scalp arteries. The disease occurs mainly in Asian children or young adults, but the appearance may be identical in adults who have

1	between the cortical surface branches of the meningeal and scalp arteries. The disease occurs mainly in Asian children or young adults, but the appearance may be identical in adults who have atherosclerosis, particularly in association with diabetes. Intracranial hemorrhage may result from rupture of the transdural and pial anastomotic channels; thus, anticoagulation is risky. Breakdown of dilated lenticulostriate arteries may produce intraparenchymal hemorrhage, and progressive occlusion of large surface arteries can occur, producing large-artery distribution strokes. Surgical bypass of extracranial carotid arteries to the dura or MCAs may prevent stroke and hemorrhage.

1	Posterior reversible encephalopathy syndrome (PRES) can occur with head injury, seizure, migraine, sympathomimetic drug use, eclampsia, and in the postpartum period (Chap. 463e). The pathophysiology is uncertain but likely involves a hyperperfusion state with widespread segmental vasoconstriction and cerebral edema. Patients complain of headache and manifest fluctuating neurologic symptoms and signs, especially visual symptoms. Sometimes cerebral infarction ensues, but typically the clinical and imaging findings suggest that ischemia reverses completely. MRI findings are characteristic with the edema present within the occipital lobes but can be generalized and do not respect any single vascular territory. A closely related reversible cerebral vasoconstriction syndrome (RCVS) typically presents with sudden, severe headache closely mimicking SAH. Patients may experience ischemic infarction and intracerebral hemorrhage and typically have new-onset, severe hypertension. Conventional

1	presents with sudden, severe headache closely mimicking SAH. Patients may experience ischemic infarction and intracerebral hemorrhage and typically have new-onset, severe hypertension. Conventional x-ray angiography reveals changes in the vascular caliber throughout the hemispheres resembling vasculitis, but the process is noninflammatory. Oral calcium channel blockers may be effective in producing remission, and recurrence is rare.

1	Leukoaraiosis, or periventricular white matter disease, is the result of multiple small-vessel infarcts within the subcortical white matter. It is readily seen on CT or MRI scans as areas of white matter injury surrounding the ventricles and within the corona radiata. The pathophysiologic basis of the disease is lipohyalinosis of small penetrating arteries within the white matter, likely produced by chronic hypertension. Patients with periventricular white matter disease may develop a subcortical dementia syndrome, and it is likely that this common form of dementia may be delayed or prevented with antihypertensive medications (Chap. 448).

1	CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is an inherited disorder that presents as small-vessel strokes, progressive dementia, and extensive symmetric white matter changes often including the anterior temporal lobes visualized by MRI. Approximately 40% of patients have migraine with aura, often manifest as transient motor or sensory deficits. Onset is usually in the fourth or fifth decade of life. This autosomal dominant condition is caused by one of several mutations in Notch-3, a member of a highly conserved gene family characterized by epidermal growth factor repeats in its extracellular domain. Other monogenic ischemic stroke syndromes include cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) and hereditary endotheliopathy, retinopathy, nephropathy, and stroke (HERNS). Fabry’s disease also produces both a large-vessel arteriopathy and small-vessel infarctions.

1	TIAs are episodes of stroke symptoms that last only briefly; the standard definition of duration is <24 h, but most TIAs last <1 h. If a relevant brain infarction is identified on brain imaging, the clinical entity is now classified as stroke regardless of the duration of symptoms. The causes of TIA are similar to the causes of ischemic stroke, but because TIAs may herald stroke, they are an important risk factor that should be considered separately and urgently. TIAs may arise from emboli to the brain or from in situ thrombosis of an intracranial vessel. With a TIA, the occluded blood vessel reopens and neurologic function is restored.

1	The risk of stroke after a TIA is ~10–15% in the first 3 months, with most events occurring in the first 2 days. This risk can be directly estimated using the well-validated ABCD2 score (Table 446-5). Therefore, urgent evaluation and treatment are justified. Because etiologies for stroke and TIA are identical, evaluation for TIA should parallel that of stroke (Figs. 446-1 and 446-3). The improvement characteristic of TIA is a contraindication to thrombolysis. However, because the risk of subsequent stroke in the first few days after a TIA is high, the opportunity to give rtPA rapidly if a stroke occurs may justify hospital admission for most patients. The combination of aspirin and clopidogrel has been recently reported to prevent stroke following TIA better than aspirin alone in a large Chinese randomized trial and is undergoing similar evaluation in an ongoing National Institutes of Health (NIH)sponsored trial (POINT study). A: Age ≥60 years 1 B: SBP >140 mmHg or DBP >90 mmHg 1

1	A: Age ≥60 years 1 B: SBP >140 mmHg or DBP >90 mmHg 1 C: Clinical symptoms Unilateral weakness 2 Speech disturbance without weakness 1 D: D: ABCD2 Score Total 3-Month Rate of Stroke (%)a 00 12 23 33 48 5 12 6 17 7 22 aData ranges are from five cohorts. Abbreviations: DBP, diastolic blood pressure; SBP, systolic blood pressure. Source: SC Johnston et al: Validation and refinement of score to predict very early stroke risk after transient ischaemic attack. Lancet 369:283, 2007.

1	Source: SC Johnston et al: Validation and refinement of score to predict very early stroke risk after transient ischaemic attack. Lancet 369:283, 2007. A number of medical and surgical interventions, as well as lifestyle modifications, are available for preventing stroke. Some of these can be widely applied because of their low cost and minimal risk; others are expensive and carry substantial risk but may be valuable for selected high-risk patients. Identification and control of modifiable risk factors, and especially hypertension, is the best strategy to reduce the burden of stroke, and the total number of strokes could be reduced substantially by these means (Table 446-4).

1	The relationship of various factors to the risk of atherosclerosis is described in Chap. 291e. Older age, diabetes mellitus, hypertension, tobacco smoking, abnormal blood cholesterol (particularly, low high-density lipoprotein [HDL] and/or elevated low-density lipoprotein [LDL]), and other factors are either proven or probable risk factors for ischemic stroke, largely by their link to atherosclerosis. Risk of stroke is much greater in those with prior stroke or TIA. Many cardiac conditions predispose to stroke, including atrial fibrillation and recent MI. Oral contraceptives and hormone replacement therapy increase stroke risk, and although rare, certain inherited and acquired hypercoagulable states predispose to stroke.

1	Hypertension is the most significant of the risk factors; in general, all hypertension should be treated to a target of less than 140– 150/90 mmHg. However, many vascular neurologists recommend that guidelines for secondary prevention of stroke should aim for blood pressure reduction to 130/80 mmHg or lower. The presence of known cerebrovascular disease is not a contraindication to treatment aimed at achieving normotension. Also, the value of treating systolic hypertension in older patients has been clearly established. Lowering blood pressure to levels below those traditionally defining hypertension appears to reduce the risk of stroke even further. Data are particularly strong in support of thiazide diuretics and angiotensin-converting enzyme inhibitors.

1	Several trials have confirmed that statin drugs reduce the risk of 2569 stroke even in patients without elevated LDL or low HDL. The Stroke Prevention by Aggressive Reduction in Cholesterol Levels (SPARCL) trial showed benefit in secondary stroke reduction for patients with recent stroke or TIA who were prescribed atorvastatin, 80 mg/d. The primary prevention trial, Justification for the Use of Statins in Prevention: An Intervention Trial Evaluating Rosuvastatin (JUPITER), found that patients with low LDL (<130 mg/dL) caused by elevated C-reactive protein benefitted by daily use of this statin. Primary stroke occurrence was reduced by 51% (hazard ratio 0.49, p = .004), and there was no increase in the rates of intracranial hemorrhage. Meta-analysis has also supported a primary treatment effect for statins given acutely for ischemic stroke. Therefore, a statin should be considered in all patients with prior ischemic stroke. Tobacco smoking should be discouraged in all patients (Chap.

1	effect for statins given acutely for ischemic stroke. Therefore, a statin should be considered in all patients with prior ischemic stroke. Tobacco smoking should be discouraged in all patients (Chap. 470). The use of pioglitazone (an agonist of peroxisome proliferator-activated receptor gamma) in patients with type 2 diabetes and previous stroke may lower risk of recurrent stroke, MI, or vascular death, but no trial sufficiently powered to definitively detect a significant reduction in stroke in the general diabetic population has yet been performed.

1	Platelet antiaggregation agents can prevent atherothrombotic events, including TIA and stroke, by inhibiting the formation of intraarterial platelet aggregates. These can form on diseased arteries, induce thrombus formation, and occlude or embolize into the distal circulation. Aspirin, clopidogrel, and the combination of aspirin plus extended-release dipyridamole are the antiplatelet agents most commonly used for this purpose. Ticlopidine has been largely abandoned because of its adverse effects but may be used as an alternative to clopidogrel.

1	Aspirin is the most widely studied antiplatelet agent. Aspirin acetylates platelet cyclooxygenase, which irreversibly inhibits the formation in platelets of thromboxane A2, a platelet aggregating and vasoconstricting prostaglandin. This effect is permanent and lasts for the usual 8-day life of the platelet. Paradoxically, aspirin also inhibits the formation in endothelial cells of prostacyclin, an antiaggregating and vasodilating prostaglandin. This effect is transient. As soon as aspirin is cleared from the blood, the nucleated endothelial cells again produce prostacyclin. Aspirin in low doses given once daily inhibits the production of thromboxane A2 in platelets without substantially inhibiting prostacyclin formation. Higher doses of aspirin have not been proven to be more effective than lower doses.

1	Ticlopidine and clopidogrel block the adenosine diphosphate (ADP) receptor on platelets and thus prevent the cascade resulting in activation of the glycoprotein IIb/IIIa receptor that leads to fibrinogen binding to the platelet and consequent platelet aggregation. Ticlopidine is more effective than aspirin; however, it has the disadvantage of causing diarrhea, skin rash, and, in rare instances, neutropenia and thrombotic thrombocytopenic purpura (TTP). Clopidogrel rarely causes TTP but does not cause neutropenia. The Clopidogrel versus Aspirin in Patients at Risk of Ischemic Events (CAPRIE) trial, which led to FDA approval, found that it was only marginally more effective than aspirin in reducing risk of stroke. The Management of Atherothrombosis with Clopidogrel in High-Risk Patients (MATCH) trial was a large multicenter, randomized, double-blind study that compared clopidogrel in combination with aspirin to clopidogrel alone in the secondary prevention of TIA or stroke. The MATCH

1	trial was a large multicenter, randomized, double-blind study that compared clopidogrel in combination with aspirin to clopidogrel alone in the secondary prevention of TIA or stroke. The MATCH trial found no difference in TIA or stroke prevention with this combination, but did show a small but significant increase in major bleeding complications (3 vs 1%). In the Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management, and Avoidance (CHARISMA) trial, which included a subgroup of patients with prior stroke or TIA along with other groups at high risk of cardiovascular events, there was no benefit of clopidogrel combined with aspirin compared to aspirin alone. Lastly, the SPS3 trial looked at the long-term combination of clopidogrel and aspirin versus clopidogrel alone in small-vessel stroke and found no 2570 improvement in stroke prevention and a significant increase in both hemorrhage and death. Thus, the long-term use of clopidogrel in combination with

1	alone in small-vessel stroke and found no 2570 improvement in stroke prevention and a significant increase in both hemorrhage and death. Thus, the long-term use of clopidogrel in combination with aspirin is not recommended for stroke prevention. The short-term combination of clopidogrel with aspirin may be effective in preventing second stroke, however. A trial of 5170 Chinese patients enrolled within 24 h of TIA or minor ischemic stroke found that a clopidogrel-aspirin regimen (clopidogrel 300 mg load then 75 mg/d with aspirin 75 mg for the first 21 days) was superior to aspirin (75 mg/d) alone, with 90-day stroke risk decreased from 11.7 to 8.2% (p < .001) and no increase in major hemorrhage. An international NIH-sponsored trial of similar design is ongoing. Dipyridamole is an antiplatelet agent that inhibits the uptake of adenosine by a variety of cells, including those of the vascular endothelium. The accumulated adenosine is an inhibitor of aggregation. At least in part through

1	agent that inhibits the uptake of adenosine by a variety of cells, including those of the vascular endothelium. The accumulated adenosine is an inhibitor of aggregation. At least in part through its effects on platelet and vessel wall phosphodiesterases, dipyridamole also potentiates the antiaggregatory effects of prostacyclin and nitric oxide produced by the endothelium and acts by inhibiting platelet phosphodiesterase, which is responsible for the breakdown of cyclic AMP. The resulting elevation in cyclic AMP inhibits aggregation of platelets. Dipyridamole is erratically absorbed depending on stomach pH, but a newer formulation combines timed-release dipyridamole, 200 mg, with aspirin, 25 mg, and has better oral bioavailability. This combination drug was studied in three trials. The European Stroke Prevention Study (ESPS) II showed efficacy of both 50 mg/d of aspirin and extended-release dipyridamole in preventing stroke, and a significantly better risk reduction when the two agents

1	Stroke Prevention Study (ESPS) II showed efficacy of both 50 mg/d of aspirin and extended-release dipyridamole in preventing stroke, and a significantly better risk reduction when the two agents were combined. The open-label ESPRIT (European/Australasian Stroke Prevention in Reversible Ischaemia Trial) trial confirmed the ESPS-II results. After 3.5 years of follow-up, 13% of patients on aspirin and dipyridamole and 16% on aspirin alone (hazard ratio 0.80, 95% confidence index [CI] 0.66– 0.98) met the primary outcome of death from all vascular causes. In the Prevention Regimen for Effectively Avoiding Second Strokes (PRoFESS) trial, the combination of extended-release dipyridamole and aspirin was compared directly with clopidogrel with and without the angiotensin receptor blocker telmisartan; there were no differences in the rates of second stroke (9% each) or degree of disability in patients with median follow-up of 2.4 years. Telmisartan also had no effect on these outcomes. This

1	there were no differences in the rates of second stroke (9% each) or degree of disability in patients with median follow-up of 2.4 years. Telmisartan also had no effect on these outcomes. This suggests that these anti-platelet regimens are similar and also raises questions about default prescription of agents to block the angiotensin pathway in all stroke patients. The principal side effect of dipyridamole is headache. The combination capsule of extended-release dipyridamole and aspirin is approved for prevention of stroke.

1	Many large clinical trials have demonstrated clearly that most antiplatelet agents reduce the risk of all important vascular atherothrombotic events (i.e., ischemic stroke, MI, and death due to all vascular causes) in patients at risk for these events. The overall relative reduction in risk of nonfatal stroke is about 25–30% and of all vascular events is about 25%. The absolute reduction varies considerably, depending on the particular patient’s risk. Individuals at very low risk for stroke seem to experience the same relative reduction, but their risks may be so low that the “benefit” is meaningless. Conversely, individuals with a 10–15% risk of vascular events per year experience a reduction to about 7.5–11%.

1	Aspirin is inexpensive, can be given in low doses, and could be recommended for all adults to prevent both stroke and MI. However, it causes epigastric discomfort, gastric ulceration, and gastrointestinal hemorrhage, which may be asymptomatic or life threatening. Consequently, not every 40or 50-year-old should be advised to take aspirin regularly because the risk of atherothrombotic stroke is extremely low and is outweighed by the risk of adverse side effects. Conversely, every patient who has experienced an atherothrombotic stroke or TIA and has no contraindication should be taking an antiplatelet agent regularly because the average annual risk of another stroke is 8–10%; another few percent will experience an MI or vascular death. Clearly, the likelihood of benefit far outweighs the risks of treatment.

1	The choice of antiplatelet agent and dose must balance the risk of stroke, the expected benefit, and the risk and cost of treatment. However, there are no definitive data, and opinions vary. Many authorities believe low-dose (30–75 mg/d) and high-dose (650–1300 mg/d) aspirin are about equally effective. Some advocate very low doses to avoid adverse effects, and still others advocate very high doses to be sure the benefit is maximal. Most physicians in North America recommend 81–325 mg/d, whereas most Europeans recommend 50–100 mg. Clopidogrel and extended-release dipyridamole plus aspirin are being increasingly recommended as first-line drugs for secondary prevention. Similarly, the choice of aspirin, clopidogrel, or dipyridamole plus aspirin must balance the fact that the latter are more effective than aspirin but the cost is higher, and this is likely to affect long-term patient adherence. The use of platelet aggregation studies in individual patients taking aspirin is controversial

1	effective than aspirin but the cost is higher, and this is likely to affect long-term patient adherence. The use of platelet aggregation studies in individual patients taking aspirin is controversial because of limited data.

1	Several trials have shown that anticoagulation (INR range, 2–3) in patients with chronic nonvalvular (nonrheumatic) atrial fibrillation (NVAF) prevents cerebral embolism and stroke and is safe. For primary prevention and for patients who have experienced stroke or TIA, anticoagulation with a VKA reduces the risk by about 67%, which clearly outweighs the 1–3% risk per year of a major bleeding complication. VKAs are difficult to dose, their effects vary with dietary intake of vitamin K, and they require frequent blood monitoring of the PTT/INR. Several newer oral anticoagulants (OACs) have recently been shown to be more convenient and efficacious for stroke prevention in NVAF. A randomized trial compared the oral thrombin inhibitor dabigatran to VKAs in a noninferiority trial to prevent stroke or systemic embolization in NVAF. Two doses of dabigatran were used: 110 mg/d and 150 mg/d. Both dose tiers of dabigatran were noninferior to VKAs in preventing second stroke and systemic

1	stroke or systemic embolization in NVAF. Two doses of dabigatran were used: 110 mg/d and 150 mg/d. Both dose tiers of dabigatran were noninferior to VKAs in preventing second stroke and systemic embolization, and the higher dose tier was superior (relative risk 0.66; 95% CI 0.53–0.82; p < .001) and the rate of major bleeding was lower in the lower dose tier of dabigatran compared to VKAs. Dabigatran requires no blood monitoring to titrate the dose, and its effect is independent of oral intake of vitamin K. Newer oral factor Xa inhibitors have also been found to be equivalent or safer and more effective than VKAs in NVAF stroke prevention. In the Apixaban for Reduction in Stroke and Other Thromboembolic Events in Atrial Fibrillation (ARISTOTLE) trial, patients were randomized between apixaban, 5 mg twice daily, and dose-adjusted warfarin (INR 2–3). The combined endpoint of ischemic or hemorrhagic stroke or system embolism occurred in 1.27% of patients in the apixaban group and in 1.6%

1	5 mg twice daily, and dose-adjusted warfarin (INR 2–3). The combined endpoint of ischemic or hemorrhagic stroke or system embolism occurred in 1.27% of patients in the apixaban group and in 1.6% in the warfarin group (p < .001 for non-inferiority and p < .01 for superiority). Major bleeding was 1% less, favoring apixaban (p < .001). Similar results were obtained in the Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET-AF). Here, patients with NVAF were randomized to rivaroxaban versus warfarin: 1.7% of the factor Xa group and 2.2% of the warfarin group reached the endpoint of stroke and systemic embolism (p < .001 for noninferiority); intracranial hemorrhage was also lower with rivaroxaban. Finally, the factor Xa inhibitor edoxaban was also found to be noninferior to warfarin. Thus, oral factor Xa inhibitors are at least a suitable alternative to VKAs, and likely are

1	with rivaroxaban. Finally, the factor Xa inhibitor edoxaban was also found to be noninferior to warfarin. Thus, oral factor Xa inhibitors are at least a suitable alternative to VKAs, and likely are superior both in efficacy and perhaps compliance.

1	For patients who cannot take anticoagulant medications, clopidogrel plus aspirin was compared to aspirin alone in the Atrial Fibrillation Clopidogrel Trial with Irbesartan for Prevention of Vascular Events (ACTIVE-A). Clopidogrel combined with aspirin was more effective than aspirin alone in preventing vascular events, principally stroke, but increased the risk of major bleeding (relative risk 1.57, p < .001).

1	The decision to use anticoagulation for primary prevention is based primarily on risk factors (Table 446-3). The history of a TIA or stroke tips the balance in favor of anticoagulation regardless of other risk factors. Intermittent atrial fibrillation carries the same risk of stroke as chronic atrial fibrillation, and several ambulatory studies of seemingly “cryptogenic” stroke have found evidence of intermittent atrial fibrillation in nearly 20% of patients monitored for a few weeks. Interrogation of implanted pacemakers also confirms an association between subclinical atrial fibrillation and stroke risk. Therefore, for patients with otherwise cryptogenic embolic stroke (no evidence of any other cause for stroke), ambulatory monitoring for 3–4 weeks is a reasonable strategy to determine the best prophylactic therapy.

1	Because of the high annual stroke risk in untreated rheumatic heart disease with atrial fibrillation, primary prophylaxis against stroke has not been studied in a double-blind fashion. These patients generally should receive long-term anticoagulation. Dabigatran and the oral Xa inhibitors have not been studied in this population. Anticoagulation also reduces the risk of embolism in acute MI. Most clinicians recommend a 3-month course of anticoagulation when there is anterior Q-wave infarction, substantial left ventricular dysfunction, congestive heart failure, mural thrombosis, or atrial fibrillation. OACs are recommended long-term if atrial fibrillation persists.

1	Stroke secondary to thromboembolism is one of the most serious complications of prosthetic heart valve implantation. The intensity of anticoagulation and/or antiplatelet therapy is dictated by the type of prosthetic valve and its location. Dabigatran may be less effective than warfarin, and the oral Xa inhibitors have not been studied in this population. If the embolic source cannot be eliminated, anticoagulation should in most cases be continued indefinitely. Many neurologists recommend combining antiplatelet agents with anticoagulants for patients who “fail” anticoagulation (i.e., have another stroke or TIA), but the evidence basis for this is lacking.

1	Data do not support the use of long-term VKAs for preventing atherothrombotic stroke for either intracranial or extracranial cerebrovascular disease. The Warfarin-Aspirin Recurrent Stroke Study (WARSS) found no benefit of warfarin sodium (INR 1.4–2.8) over aspirin, 325 mg, for secondary prevention of stroke but did find a slightly higher bleeding rate in the warfarin group; a European study confirmed this finding. The Warfarin and Aspirin for Symptomatic Intracranial Disease (WASID) study (see below) demonstrated no benefit of warfarin (INR 2–3) over aspirin in patients with symptomatic intracranial atherosclerosis and also found a higher rate of bleeding complications. Carotid atherosclerosis can be removed surgically (endarterectomy) or mitigated with endovascular stenting with or without balloon angioplasty. Anticoagulation has not been directly compared with antiplatelet therapy for carotid disease.

1	Symptomatic carotid stenosis was studied in the North American Symptomatic Carotid Endarterectomy Trial (NASCET) and the European Carotid Surgery Trial (ECST). Both showed a substantial benefit for surgery in patients with stenosis of ≥70%. In NASCET, the average cumulative ipsilateral stroke risk at 2 years was 26% for patients treated medically and 9% for those receiving the same medical treatment plus a carotid endarterectomy. This 17% absolute reduction in the surgical group is a 65% relative risk reduction favoring surgery (Table 446-4). NASCET also showed a significant, although less robust, benefit for patients with 50–70% stenosis. ECST found harm for patients with stenosis <30% treated surgically.

1	A patient’s risk of stroke and possible benefit from surgery are related to the presence of retinal versus hemispheric symptoms, degree of arterial stenosis, extent of associated medical conditions (of note, NASCET and ECST excluded “high-risk” patients with sig-2571 nificant cardiac, pulmonary, or renal disease), institutional surgical morbidity and mortality, timing of surgery relative to symptoms, and other factors. A recent meta-analysis of the NASCET and ECST trials demonstrated that endarterectomy is most beneficial when performed within 2 weeks of symptom onset. In addition, benefit is more pronounced in patients >75 years, and men appear to benefit more than women.

1	In summary, a patient with recent symptomatic hemispheric ischemia, high-grade stenosis in the appropriate internal carotid artery, and an institutional perioperative morbidity and mortality rate of ≤6% generally should undergo carotid endarterectomy. If the perioperative stroke rate is >6% for any particular surgeon, however, the benefits of carotid endarterectomy are questionable.

1	The indications for surgical treatment of asymptomatic carotid disease have been clarified by the results of the Asymptomatic Carotid Atherosclerosis Study (ACAS) and the Asymptomatic Carotid Surgery Trial (ACST). ACAS randomized asymptomatic patients with ≥60% stenosis to medical treatment with aspirin or the same medical treatment plus carotid endarterectomy. The surgical group had a risk over 5 years for ipsilateral stroke (and any perioperative stroke or death) of 5.1%, compared to a risk in the medical group of 11%. Although this demonstrates a 53% relative risk reduction, the absolute risk reduction is only 5.9% over 5 years, or 1.2% annually (Table 446-4). Nearly one-half of the strokes in the surgery group were caused by preoperative angiograms. ACST randomized asymptomatic patients with >60% carotid stenosis to endarterectomy or medical therapy. The 5-year risk of stroke in the surgical group (including perioperative stroke or death) was 6.4%, compared to 11.8% in the

1	patients with >60% carotid stenosis to endarterectomy or medical therapy. The 5-year risk of stroke in the surgical group (including perioperative stroke or death) was 6.4%, compared to 11.8% in the medically treated group (46% relative risk reduction and 5.4% absolute risk reduction).

1	In both ACAS and ACST, the perioperative complication rate was higher in women, perhaps negating any benefit in the reduction of stroke risk within 5 years. It is possible that with longer follow-up, a clear benefit in women will emerge. At present, carotid endarterectomy in asymptomatic women remains particularly controversial.

1	In summary, the natural history of asymptomatic stenosis is an ~2% per year stroke rate, whereas symptomatic patients experience a 13% per year risk of stroke. Whether to recommend carotid revascularization for an asymptomatic patient is somewhat controversial and depends on many factors, including patient preference, degree of stenosis, age, gender, and comorbidities. Medical therapy for reduction of atherosclerosis risk factors, including cholesterol-lowering agents and antiplatelet medications, is generally recommended for patients with asymptomatic carotid stenosis. As with atrial fibrillation, it is imperative to counsel the patient about TIAs so that therapy can be revised if symptoms develop.

1	Balloon angioplasty coupled with stenting is being used with increasing frequency to open stenotic carotid arteries and maintain their patency. These techniques can treat carotid stenosis not only at the bifurcation but also near the skull base and in the intracranial segments. The Stenting and Angioplasty with Protection in Patients at High Risk for Endarterectomy (SAPPHIRE) trial randomized high-risk patients (defined as patients with clinically significant coronary or pulmonary disease, contralateral carotid occlusion, restenosis after endarterectomy, contralateral laryngeal-nerve palsy, prior radical neck surgery or radiation, or age >80) with symptomatic carotid stenosis >50% or asymptomatic stenosis >80% to either stenting combined with a distal emboli-protection device or endarterectomy. The risk of death, stroke, or MI within 30 days and ipsilateral stroke or death within 1 year was 12.2% in the stenting group and 20.1% in the endarterectomy group (p = .055), suggesting that

1	The risk of death, stroke, or MI within 30 days and ipsilateral stroke or death within 1 year was 12.2% in the stenting group and 20.1% in the endarterectomy group (p = .055), suggesting that stenting is at the very least comparable to endarterectomy as a treatment option for this patient group at high risk of surgery. However, the outcomes with both interventions may not have been better than leaving the carotid stenoses untreated, particularly for the asymptomatic patients, and much of the benefit seen in the 2572 stenting group was due to a reduction in periprocedure MI. Two randomized trials comparing stents to endarterectomy in lower-risk patients have been published. The Carotid Revascularization Endarterectomy versus Stenting Trial (CREST) enrolled patients with either asymptomatic or symptomatic stenosis. The 30-day risk of stroke was 4.1% in the stent group and 2.3% in the surgical group, but the 30-day risk of MI was 1.1% in the stent group and 2.3% in the surgery group,

1	or symptomatic stenosis. The 30-day risk of stroke was 4.1% in the stent group and 2.3% in the surgical group, but the 30-day risk of MI was 1.1% in the stent group and 2.3% in the surgery group, suggesting relative equivalence of risk between the procedures. At median follow-up of 2.5 years, the combined endpoint of stroke, MI, and death was the same (7.2% stent vs 6.8% surgery). The rate of restenosis at 2 years was also similar in both groups. The International Carotid Stenting (ICSS) trial randomized symptomatic patients to stents versus endarterectomy and found a different result: At 120 days, the incidence of stroke, MI, or death was 8.5% in the stenting group versus 5.2% in the endarterectomy group (p = .006); longer-term follow-up is currently under way. Differences between trial designs, selection of stent, and operator experience may explain these important differences. Until more data are available, there remains controversy as to who should receive a stent or have

1	trial designs, selection of stent, and operator experience may explain these important differences. Until more data are available, there remains controversy as to who should receive a stent or have endarterectomy; it is likely that the procedures carry similar risks if performed by experienced physicians.

1	Extracranial-to-intracranial (EC-IC) bypass surgery has been proven ineffective for atherosclerotic stenoses that are inaccessible to conventional carotid endarterectomy. In patients with recent stroke, an associated carotid occlusion, and evidence of inadequate perfusion of the brain as measured with positron emission tomography, no benefit from EC-IC bypass was found in a trial stopped for futility.

1	The WASID trial randomized patients with symptomatic stenosis (50–99%) of a major intracranial vessel to either high-dose aspirin (1300 mg/d) or warfarin (target INR, 2.0–3.0), with a combined primary endpoint of ischemic stroke, brain hemorrhage, or death from vascular cause other than stroke. The trial was terminated early because of an increased risk of adverse events related to warfarin anticoagulation. With a mean follow-up of 1.8 years, the primary endpoint was seen in 22.1% of patients in the aspirin group and 21.8% of the warfarin group. Death from any cause was seen in 4.3% of the aspirin group and 9.7% of the warfarin group; 3.2% of patients on aspirin experienced major hemorrhage, compared to 8.3% of patients taking warfarin.

1	Intracranial stenting of intracranial atherosclerosis was found to be dramatically harmful compared to aspirin in the Stenting and Aggressive Medical Management for Preventing Recurrent Stroke in Intracranial Stenosis (SAMMPRIS) trial. This trial enrolled newly symptomatic TIA or minor stroke patients with associated 70–99% intracranial stenosis to primary stenting with a self-expanding stent or to medical management. Both groups received clopidogrel, aspirin, statin, and aggressive control of blood pressure. The endpoint of stroke or death occurred in 14.7% of the stented group and 5.8% of the medically treated groups (p = .002). This low rate of second stroke was significantly lower than in the WASID trial and suggests that aggressive medical management had a marked influence on secondary stroke risk.

1	Dural Sinus Thrombosis Limited evidence exists to support short-term use of anticoagulants, regardless of the presence of intracranial hemorrhage, for venous infarction following sinus thrombosis. The long-term outcome for most patients, even those with intracerebral hemorrhage, is excellent. A careful history and neurologic examination can often localize the region of brain dysfunction; if this region corresponds to a particular arterial distribution, the possible causes responsible for the syndrome can be narrowed. This is of particular importance when the patient Uncus Caudate Anterior cerebral a. Internal carotid a. Middle cerebral a. Anterior cerebral a. Middle cerebral a. Deep branches of middle cerebral a. Postcerebral a. Deep branches of ant. cerebral a. FIGURE 446-6 Diagram of a cerebral hemisphere in coronal section showing the territories of the major cerebral vessels that branch from the internal carotid arteries.

1	FIGURE 446-6 Diagram of a cerebral hemisphere in coronal section showing the territories of the major cerebral vessels that branch from the internal carotid arteries. presents with a TIA and a normal examination. For example, if a patient develops language loss and a right homonymous hemianopia, a search for causes of left middle cerebral emboli should be performed. A finding of an isolated stenosis of the right internal carotid artery in that patient, for example, suggests an asymptomatic carotid stenosis, and the search for other causes of stroke should continue. The following sections describe the clinical findings of cerebral ischemia associated with cerebral vascular territories depicted in Figs. 446-4 and 446-6 through 446-14. Stroke syndromes are divided into: (1) large-vessel stroke within the anterior circulation, (2) large-vessel stroke within the posterior circulation, and (3) small-vessel disease of either vascular bed.

1	Stroke within the Anterior Circulation The internal carotid artery and its branches comprise the anterior circulation of the brain. These vessels can be occluded by intrinsic disease of the vessel (e.g., atherosclerosis or dissection) or by embolic occlusion from a proximal source as discussed above. Occlusion of each major intracranial vessel has distinct clinical manifestations. miDDle cerebrAl Artery Occlusion of the proximal MCA or one of its major branches is most often due to an embolus (artery-to-artery, cardiac, or of unknown source) rather than intracranial atherothrombosis. Atherosclerosis of the proximal MCA may cause distal emboli to the middle cerebral territory or, less commonly, may produce low-flow TIAs. Collateral formation via leptomeningeal vessels often prevents MCA stenosis from becoming symptomatic.

1	The cortical branches of the MCA supply the lateral surface of the hemisphere except for (1) the frontal pole and a strip along the superomedial border of the frontal and parietal lobes supplied by the ACA, and (2) the lower temporal and occipital pole convolutions supplied by the PCA (Figs. 446-6, 446-7, 446-8, and 446-9). The proximal MCA (M1 segment) gives rise to penetrating branches (termed lenticulostriate arteries) that supply the putamen, outer globus pallidus, posterior limb of the internal capsule, adjacent corona radiata, and most of the caudate nucleus (Fig. 446-6). In the sylvian fissure, the MCA in most patients divides into superior and inferior divisions Ant. parietal a. Rolandic a. Temporopolar a. Inf. division middle cerebral a. Ant. temporal a. Post. parietal a. Visual radiation Prerolandic a. Angular a. Lateral orbitofrontal a. Sup. division middle cerebral a. Post. temporal a.

1	Prerolandic a. Angular a. Lateral orbitofrontal a. Sup. division middle cerebral a. Post. temporal a. FIGURE 446-7 Diagram of a cerebral hemisphere, lateral aspect, showing the branches and distribution of the middle cerebral artery and the principal regions of cerebral localization. Note the bifurcation of the middle cerebral artery into a superior and inferior division. Signs and symptoms: Structures involved Paralysis of the contralateral face, arm, and leg; sensory impairment over the same area (pinprick, cotton touch, vibration, position, two-point discrimination, stereognosis, tactile localization, barognosis, cutaneographia): Somatic motor area for face and arm and the fibers descending from the leg area to enter the corona radiata and corresponding somatic sensory system Motor aphasia: Motor speech area of the dominant hemisphere

1	Motor aphasia: Motor speech area of the dominant hemisphere Central aphasia, word deafness, anomia, jargon speech, sensory agraphia, acalculia, alexia, finger agnosia, right-left confusion (the last four comprise the Gerstmann syndrome): Central, suprasylvian speech area and parietooccipital cortex of the dominant hemisphere Conduction aphasia: Central speech area (parietal operculum) Apractagnosia of the nondominant hemisphere, anosognosia, hemiasomatognosia, unilateral neglect, agnosia for the left half of external space, dressing “apraxia,” constructional “apraxia,” distortion of visual coordinates, inaccurate localization in the half field, impaired ability to judge distance, upside-down reading, visual illusions (e.g., it may appear that another person walks through a table): Nondominant parietal lobe (area corresponding to speech area in dominant hemisphere); loss of topographic memory is usually due to a nondominant lesion, occasionally to a dominant one

1	Homonymous hemianopia (often homonymous inferior quadrantanopia): Optic radiation deep to second temporal convolution Paralysis of conjugate gaze to the opposite side: Frontal contraversive eye field or projecting fibers (M2 branches). Branches of the inferior division supply the inferior parietal and temporal cortex, and those from the superior division supply the frontal and superior parietal cortex (Fig. 446-7). If the entire MCA is occluded at its origin (blocking both its penetrating and cortical branches) and the distal collaterals are limited, the clinical findings are contralateral hemiplegia, hemianesthesia, homonymous hemianopia, and a day or two of gaze preference to the ipsilateral side. Dysarthria is common because of facial weakness. When the dominant hemisphere is involved, global aphasia is present also, and when the nondominant hemisphere is affected, anosognosia, constructional apraxia, and neglect are found (Chap. 36).

1	Complete MCA syndromes occur most often when an embolus occludes the stem of the artery. Cortical collateral blood flow and differing arterial configurations are probably responsible for the development of many partial syndromes. Partial syndromes may also be due to emboli that enter the proximal MCA without complete occlusion, occlude distal MCA branches, or frag-2573 ment and move distally.

1	Partial syndromes due to embolic occlusion of a single branch include hand, or arm and hand, weakness alone (brachial syndrome) or facial weakness with nonfluent (Broca) aphasia (Chap. 36), with or without arm weakness (frontal opercular syndrome). A combination of sensory disturbance, motor weakness, and nonfluent aphasia suggests that an embolus has occluded the proximal superior division and infarcted large portions of the frontal and parietal cortices (Fig. 446-7). If a fluent (Wernicke’s) aphasia occurs without weakness, the inferior division of the MCA supplying the posterior part (temporal cortex) of the dominant hemisphere is probably involved. Jargon speech and an inability to comprehend written and spoken language are prominent features, often accompanied by a contralateral, homonymous superior quadrantanopia. Hemineglect or spatial agnosia without weakness indicates that the inferior division of the MCA in the nondominant hemisphere is involved.

1	Occlusion of a lenticulostriate vessel produces small-vessel (lacunar) stroke within the internal capsule (Fig. 4466). This produces pure motor stroke or sensory-motor stroke contralateral to the lesion. Ischemia within the genu of the internal capsule causes primarily facial weakness followed by arm and then leg weakness as the ischemia moves posterior within the capsule. Alternatively, the contralateral hand may become ataxic, and dysarthria will be prominent (clumsy hand, dysarthria lacunar syndrome). Lacunar infarction affecting the globus pallidus and putamen often has few clinical signs, but parkinsonism and hemiballismus have been reported.

1	Anterior cerebrAl Artery The ACA is divided into two segments: the precommunal (A1) circle of Willis, or stem, which connects the internal carotid artery to the anterior communicating artery, and the postcommunal (A2) segment distal to the anterior communicating artery (Figs. 446-4, 446-6, and 446-8). The A1 seg ment gives rise to several deep penetrating branches that supply the anterior limb of the internal capsule, the anterior perforate substance, amygdala, anterior hypothalamus, and the inferior part of the head of the caudate nucleus (Fig. 446-6).

1	Occlusion of the proximal ACA is usually well tolerated because of collateral flow through the anterior communicating artery and collaterals through the MCA and PCA. Occlusion of a single A2 segment results in the contralateral symptoms noted in Fig. 446-8. If both A2 segments arise from a single anterior cerebral stem (contralateral A1 segment atresia), the occlusion may affect both hemispheres. Profound abulia (a delay in verbal and motor response) and bilateral pyramidal signs with paraparesis or quadriparesis and urinary incontinence result. Anterior choroiDAl Artery This artery arises from the internal carotid artery and supplies the posterior limb of the internal capsule and the white matter posterolateral to it, through which pass some of rolandic a. Post. prerolandic a. Secondary Pericallosal a. Sensory parietal a. motor area cortex Medial Splenial a. Lateral posterior Callosomarginal a. choroidal a. Post. thalamic a. Parietooccipital a. Frontopolar a.

1	Secondary Pericallosal a. Sensory parietal a. motor area cortex Medial Splenial a. Lateral posterior Callosomarginal a. choroidal a. Post. thalamic a. Parietooccipital a. Frontopolar a. Visual cortex Ant. cerebral a. Medial orbitofrontal a. Calcarine a. Post. communicating a. Post. temporal a. Medial posterior choroidal a. Penetrating thalamosubthalamic Post. Hippocampal As. Ant. paramedian As. cerebral temporal a. FIGURE 446-8 Diagram of a cerebral hemisphere, medial aspect, showing the branches and distribution of the anterior cerebral artery and the principal regions of cerebral localization.

1	FIGURE 446-8 Diagram of a cerebral hemisphere, medial aspect, showing the branches and distribution of the anterior cerebral artery and the principal regions of cerebral localization. Signs and symptoms: Structures involved Paralysis of opposite foot and leg: Motor leg area A lesser degree of paresis of opposite arm: Arm area of cortex or fibers descending to corona radiata Cortical sensory loss over toes, foot, and leg: Sensory area for foot and leg Urinary incontinence: Sensorimotor area in paracentral lobule Contralateral grasp reflex, sucking reflex, gegenhalten (paratonic rigidity): Medial surface of the posterior frontal lobe; likely supplemental motor area Abulia (akinetic mutism), slowness, delay, intermittent interruption, lack of spontaneity, whispering, reflex distraction to sights and sounds:

1	Uncertain localization—probably cingulate gyrus and medial inferior portion of frontal, parietal, and temporal lobes Impairment of gait and stance (gait apraxia): Frontal cortex near leg motor area Dyspraxia of left limbs, tactile aphasia in left limbs: Corpus callosum the geniculocalcarine fibers (Fig. 446-9). The complete syndrome of in that eye or that the upper or lower half of vision disappeared. In anterior choroidal artery occlusion consists of contralateral hemiple-most cases, these symptoms last only a few minutes. Rarely, ischemia gia, hemianesthesia (hypesthesia), and homonymous hemianopia. or infarction of the ophthalmic artery or central retinal arteries occurs However, because this territory is also supplied by penetrating vessels at the time of cerebral TIA or infarction. of the proximal MCA and the posterior communicating and posterior A high-pitched prolonged carotid bruit fading into diastole is often choroidal arteries, minimal deficits may occur, and patients

1	of the proximal MCA and the posterior communicating and posterior A high-pitched prolonged carotid bruit fading into diastole is often choroidal arteries, minimal deficits may occur, and patients frequently associated with tightly stenotic lesions. As the stenosis grows tighter recover substantially. Anterior choroidal strokes are usually the result and flow distal to the stenosis becomes reduced, the bruit becomes of in situ thrombosis of the vessel, and the vessel is particularly vul-fainter and may disappear when occlusion is imminent. nerable to iatrogenic occlusion during surgical clipping of aneurysms common cArotiD Artery All symptoms and signs of internal carotidarising from the internal carotid artery.

1	occlusion may also be present with occlusion of the common carotid internAl cArotiD Artery The clinical picture of internal carotid occlu-artery. Jaw claudication may result from low flow in the external sion varies depending on whether the cause of ischemia is propagated carotid branches. Bilateral common carotid artery occlusions at their thrombus, embolism, or low flow. The cortex supplied by the MCA origin may occur in Takayasu’s arteritis (Chap. 385). territory is affected most often. With a competent circle of Willis,

1	Stroke Within the Posterior Circulation The posterior circulation is occlusion may go unnoticed. If the thrombus propagates up the composed of the paired vertebral arteries, the basilar artery, and theinternal carotid artery into the MCA or embolizes it, symptoms are paired posterior cerebral arteries. The vertebral arteries join to formidentical to proximal MCA occlusion (see above). Sometimes there is the basilar artery at the pontomedullary junction. The basilar artery massive infarction of the entire deep white matter and cortical surface.

1	divides into two posterior cerebral arteries in the interpeduncular fossaWhen the origins of both the ACA and MCA are occluded at the top (Figs. 446-4, 446-8, and 446-9). These major arteries give rise to long of the carotid artery, abulia or stupor occurs with hemiplegia, hemianand short circumferential branches and to smaller deep penetratingesthesia, and aphasia or anosognosia. When the PCA arises from the branches that supply the cerebellum, medulla, pons, midbrain, sub-internal carotid artery (a configuration called a fetal posterior cerebral thalamus, thalamus, hippocampus, and medial temporal and occipitalartery), it may also become occluded and give rise to symptoms refer-lobes. Occlusion of each vessel produces its own distinctive syndrome. able to its peripheral territory (Figs. 446-8 and 446-9).

1	able to its peripheral territory (Figs. 446-8 and 446-9). In addition to supplying the ipsilateral brain, the internal carotid poSterior cerebrAl Artery In 75% of cases, both PCAs arise from the artery perfuses the optic nerve and retina via the ophthalmic artery. bifurcation of the basilar artery; in 20%, one has its origin from the In ~25% of symptomatic internal carotid disease, recurrent transient ipsilateral internal carotid artery via the posterior communicating monocular blindness (amaurosis fugax) warns of the lesion. Patients artery; in 5%, both originate from the respective ipsilateral internal typically describe a horizontal shade that sweeps down or up across the carotid arteries (Figs. 446-8 and 446-9). The precommunal, or P1, segfield of vision. They may also complain that their vision was blurred ment of the true posterior cerebral artery is atretic in such cases. Ant. cerebral a. Post. communicating a. Visual Parietooccipital a. Internal carotid a.

1	Ant. cerebral a. Post. communicating a. Visual Parietooccipital a. Internal carotid a. Post. cerebral a. Ant. choroidal a. Medial posterior choroidal a. Mesencephalic paramedian As. Ant. temporal a. Splenial a. Hippocampal a. Calcarine a. Post. temporal a. Post. thalamic a. Lateral posterior choroidal a. FIGURE 446-9 Inferior aspect of the brain with the branches and distribution of the posterior cerebral artery and the principal anatomic structures shown. Signs and symptoms: Structures involved

1	Peripheral territory (see also Fig. 446-12). Homonymous hemianopia (often upper quadrantic): Calcarine cortex or optic radiation nearby. Bilateral homonymous hemianopia, cortical blindness, awareness or denial of blindness; tactile naming, achromatopia (color blindness), failure to see to-and-fro movements, inability to perceive objects not centrally located, apraxia of ocular movements, inability to count or enumerate objects, tendency to run into things that the patient sees and tries to avoid: Bilateral occipital lobe with possibly the parietal lobe involved. Verbal dyslexia without agraphia, color anomia: Dominant calcarine lesion and posterior part of corpus callosum. Memory defect: Hippocampal lesion bilaterally or on the dominant side only. Topographic disorientation and prosopagnosia: Usually with lesions of nondominant, calcarine, and lingual gyrus. Simultanagnosia, hemivisual neglect: Dominant visual cortex, contralateral hemisphere. Unformed visual hallucinations,

1	Usually with lesions of nondominant, calcarine, and lingual gyrus. Simultanagnosia, hemivisual neglect: Dominant visual cortex, contralateral hemisphere. Unformed visual hallucinations, peduncular hallucinosis, metamorphopsia, teleopsia, illusory visual spread, palinopsia, distortion of outlines, central photophobia: Calcarine cortex. Complex hallucinations: Usually nondominant hemisphere.

1	Central territory. Thalamic syndrome: sensory loss (all modalities), spontaneous pain and dysesthesias, choreoathetosis, intention tremor, spasms of hand, mild hemiparesis: Posteroventral nucleus of thalamus; involvement of the adjacent subthalamus body or its afferent tracts. Thalamoperforate syndrome: crossed cerebellar ataxia with ipsilateral third nerve palsy (Claude’s syndrome): Dentatothalamic tract and issuing third nerve. Weber’s syndrome: third nerve palsy and contralateral hemiplegia: Third nerve and cerebral peduncle. Contralateral hemiplegia: Cerebral peduncle. Paralysis or paresis of vertical eye movement, skew deviation, sluggish pupillary responses to light, slight miosis and ptosis (retraction nystagmus and “tucking” of the eyelids may be associated): Supranuclear fibers to third nerve, interstitial nucleus of Cajal, nucleus of Darkschewitsch, and posterior commissure. Contralateral rhythmic, ataxic action tremor; rhythmic postural or “holding” tremor (rubral tremor):

1	third nerve, interstitial nucleus of Cajal, nucleus of Darkschewitsch, and posterior commissure. Contralateral rhythmic, ataxic action tremor; rhythmic postural or “holding” tremor (rubral tremor): Dentatothalamic tract.

1	PCA syndromes usually result from atheroma formation or emboli that lodge at the top of the basilar artery; posterior circulation disease may also be caused by dissection of either vertebral artery or fibromuscular dysplasia. Two clinical syndromes are commonly observed with occlusion of 2575 the PCA: (1) P1 syndrome: midbrain, subthalamic, and thalamic signs, which are due to disease of the proximal P1 segment of the PCA or its penetrating branches (thalamogeniculate, Percheron, and posterior choroidal arteries); and (2) P2 syndrome: cortical temporal and occipital lobe signs, due to occlusion of the P2 segment distal to the junction of the PCA with the posterior communicating artery.

1	p1 SynDromeS Infarction usually occurs in the ipsilateral subthalamus and medial thalamus and in the ipsilateral cerebral peduncle and mid- brain (Figs. 446-9 and 446-14). A third nerve palsy with contralateral ataxia (Claude’s syndrome) or with contralateral hemiplegia (Weber’s syndrome) may result. The ataxia indicates involvement of the red nucleus or dentatorubrothalamic tract; the hemiplegia is localized to the cerebral peduncle (Fig. 446-14). If the subthalamic nucleus is involved, contralateral hemiballismus may occur. Occlusion of the artery of Percheron produces paresis of upward gaze and drowsiness and often abulia. Extensive infarction in the midbrain and sub-thalamus occurring with bilateral proximal PCA occlusion presents as coma, unreactive pupils, bilateral pyramidal signs, and decerebrate rigidity.

1	Occlusion of the penetrating branches of thalamic and thalamogeniculate arteries produces less extensive thalamic and thalamocapsular lacunar syndromes. The thalamic Déjérine-Roussy syndrome consists of contralateral hemisensory loss followed later by an agonizing, searing or burning pain in the affected areas. It is persistent and responds poorly to analgesics. Anticonvulsants (carbamazepine or gabapentin) or tricyclic antidepressants may be beneficial.

1	p2 SynDromeS (Figs. 446-8 and 446-9) Occlusion of the distal PCA causes infarction of the medial temporal and occipital lobes. Contralateral homonymous hemianopia with macula sparing is the usual manifestation. Occasionally, only the upper quadrant of visual field is involved. If the visual association areas are spared and only the calcarine cortex is involved, the patient may be aware of visual defects. Medial temporal lobe and hippocampal involvement may cause an acute disturbance in memory, particularly if it occurs in the dominant hemisphere. The defect usually clears because memory has bilateral representation. If the dominant hemisphere is affected and the infarct extends to involve the splenium of the corpus callosum, the patient may demonstrate alexia without agraphia. Visual agnosia for faces, objects, mathematical symbols, and colors and anomia with paraphasic errors (amnestic aphasia) may also occur, even without callosal involvement. Occlusion of the posterior cerebral

1	for faces, objects, mathematical symbols, and colors and anomia with paraphasic errors (amnestic aphasia) may also occur, even without callosal involvement. Occlusion of the posterior cerebral artery can produce peduncular hallucinosis (visual hallucinations of brightly colored scenes and objects).

1	Bilateral infarction in the distal PCAs produces cortical blindness (blindness with preserved pupillary light reaction). The patient is often unaware of the blindness or may even deny it (Anton’s syndrome). Tiny islands of vision may persist, and the patient may report that vision fluctuates as images are captured in the preserved portions. Rarely, only peripheral vision is lost and central vision is spared, resulting in “gun-barrel” vision. Bilateral visual association area lesions may result in Balint’s syndrome, a disorder of the orderly visual scanning of the environment (Chap. 36), usually resulting from infarctions secondary to low flow in the “watershed” between the distal PCA and MCA territories, as occurs after cardiac arrest. Patients may experience persistence of a visual image for several minutes despite gazing at another scene (palinopsia) or an inability to synthesize the whole of an image (asimultanagnosia). Embolic occlusion of the top of the basilar artery can produce

1	for several minutes despite gazing at another scene (palinopsia) or an inability to synthesize the whole of an image (asimultanagnosia). Embolic occlusion of the top of the basilar artery can produce any or all of the central or peripheral territory symptoms. The hallmark is the sudden onset of bilateral signs, including ptosis, pupillary asymmetry or lack of reaction to light, and somnolence.

1	VertebrAl AnD poSterior inferior cerebellAr ArterieS The vertebral artery, which arises from the innominate artery on the right and the subclavian artery on the left, consists of four segments. The first (V1) extends from its origin to its entrance into the sixth or fifth transverse vertebral foramen. The second segment (V2) traverses the vertebral Descending nucleus and tract 5th n. nucleus 12th n. Medullary syndrome: 12th n. 10th n. FIGURE 446-10 Axial section at the level of the medulla, depicted schematically on the left, with a corresponding magnetic resonance image on the right. Note that in Figs. 446-10 through 446-14, all drawings are oriented with the dorsal surface at the bottom, matching the orientation of the brainstem that is commonly seen in all modern neuroimaging studies. Approximate regions involved in medial and lateral medullary stroke syndromes are shown. Signs and symptoms: Structures involved 1.

1	Signs and symptoms: Structures involved 1. Medial medullary syndrome (occlusion of vertebral artery or of branch of vertebral or lower basilar artery) On side of lesion Paralysis with atrophy of one-half half the tongue: Ipsilateral twelfth nerve On side opposite lesion Paralysis of arm and leg, sparing face; impaired tactile and proprioceptive sense over one-half the body: Contralateral pyramidal tract and medial lemniscus 2. Lateral medullary syndrome (occlusion of any of five vessels may be responsible—vertebral, posterior inferior cerebellar, superior, middle, or inferior lateral medullary arteries) On side of lesion

1	Lateral medullary syndrome (occlusion of any of five vessels may be responsible—vertebral, posterior inferior cerebellar, superior, middle, or inferior lateral medullary arteries) On side of lesion Pain, numbness, impaired sensation over one-half the face: Descending tract and nucleus fifth nerve Ataxia of limbs, falling to side of lesion: Uncertain—restiform body, cerebellar hemisphere, cerebellar fibers, spinocerebellar tract (?) Nystagmus, diplopia, oscillopsia, vertigo, nausea, vomiting: Vestibular nucleus Horner’s syndrome (miosis, ptosis, decreased sweating): Descending sympathetic tract Dysphagia, hoarseness, paralysis of palate, paralysis of vocal cord, diminished gag reflex: Issuing fibers ninth and tenth nerves Loss of taste: Nucleus and tractus solitarius Numbness of ipsilateral arm, trunk, or leg: Cuneate and gracile nuclei Weakness of lower face: Genuflected upper motor neuron fibers to ipsilateral facial nucleus

1	On side opposite lesion Impaired pain and thermal sense over half the body, sometimes face: Spinothalamic tract 3. Total unilateral medullary syndrome (occlusion of vertebral artery): Combination of medial and lateral syndromes 4. Lateral pontomedullary syndrome (occlusion of vertebral artery): Combination of lateral medullary and lateral inferior pontine syndrome 5. Basilar artery syndrome (the syndrome of the lone vertebral artery is equivalent): A combination of the various brainstem syndromes plus those arising in the posterior cerebral artery distribution. Bilateral long tract signs (sensory and motor; cerebellar and peripheral cranial nerve abnormalities): Bilateral long tract; cerebellar and peripheral cranial nerves

1	Paralysis or weakness of all extremities, plus all bulbar musculature: Corticobulbar and corticospinal tracts bilaterally foramina from C6 to C2. The third (V3) passes through the transverse foramen and circles around the arch of the atlas to pierce the dura at the foramen magnum. The fourth (V4) segment courses upward to join the other vertebral artery to form the basilar artery; only the fourth segment gives rise to branches that supply the brainstem and cerebellum. The posterior inferior cerebellar artery (PICA) in its proximal segment supplies the lateral medulla and, in its distal branches, the inferior surface of the cerebellum.

1	Atherothrombotic lesions have a predilection for V1 and V4 segments of the vertebral artery. The first segment may become diseased at the origin of the vessel and may produce posterior circulation emboli; collateral flow from the contralateral vertebral artery or the ascending cervical, thyrocervical, or occipital arteries is usually sufficient to prevent low-flow TIAs or stroke. When one vertebral artery is atretic and an atherothrombotic lesion threatens the origin of the other, the collateral circulation, which may also include retrograde flow down the basilar artery, is often insufficient (Figs. 446-4 and 446-9). In this setting, low-flow TIAs may occur, consisting of syncope, vertigo, and alternating hemiplegia; this state also sets the stage for thrombosis. Disease of the distal fourth segment of the vertebral artery can promote thrombus formation manifest as embolism or with propagation as basilar artery thrombosis. Stenosis proximal to the origin of the PICA can threaten the

1	segment of the vertebral artery can promote thrombus formation manifest as embolism or with propagation as basilar artery thrombosis. Stenosis proximal to the origin of the PICA can threaten the lateral medulla and posterior inferior surface of the cerebellum.

1	If the subclavian artery is occluded proximal to the origin of the vertebral artery, there is a reversal in the direction of blood flow in the ipsilateral vertebral artery. Exercise of the ipsilateral arm may increase demand on vertebral flow, producing posterior circulation TIAs, or “subclavian steal.” 7th n. 6th n. peduncle 6th n. nucleus complex 7th n. nucleus cochlear Descending tract and nucleus of 5th n. 8th n. Inferior pontine syndrome: FIGURE 446-11 Axial section at the level of the inferior pons, depicted schematically on the left, with a corresponding magnetic resonance image on the right. Approximate regions involved in medial and lateral inferior pontine stroke syndromes are shown. Signs and symptoms: Structures involved 1. Medial inferior pontine syndrome (occlusion of paramedian branch of basilar artery)

1	Signs and symptoms: Structures involved 1. Medial inferior pontine syndrome (occlusion of paramedian branch of basilar artery) On side of lesion Paralysis of conjugate gaze to side of lesion (preservation of convergence): Center for conjugate lateral gaze Nystagmus: Vestibular nucleus Ataxia of limbs and gait: Likely middle cerebellar peduncle Diplopia on lateral gaze: Abducens nerve On side opposite lesion Paralysis of face, arm, and leg: Corticobulbar and corticospinal tract in lower pons Impaired tactile and proprioceptive sense over one-half of the body: Medial lemniscus 2. Lateral inferior pontine syndrome (occlusion of anterior inferior cerebellar artery)

1	On side of lesion Horizontal and vertical nystagmus, vertigo, nausea, vomiting, oscillopsia: Vestibular nerve or nucleus Facial paralysis: Seventh nerve Paralysis of conjugate gaze to side of lesion: Center for conjugate lateral gaze Deafness, tinnitus: Auditory nerve or cochlear nucleus Ataxia: Middle cerebellar peduncle and cerebellar hemisphere Impaired sensation over face: Descending tract and nucleus fifth nerve On side opposite lesion Impaired pain and thermal sense over one-half the body (may include face): Spinothalamic tract Although atheromatous disease rarely narrows the second and third segments of the vertebral artery, this region is subject to dissection, fibromuscular dysplasia, and, rarely, encroachment by osteophytic spurs within the vertebral foramina.

1	Embolic occlusion or thrombosis of a V4 segment causes ischemia of the lateral medulla. The constellation of vertigo, numbness of the ipsilateral face and contralateral limbs, diplopia, hoarseness, dysarthria, dysphagia, and ipsilateral Horner’s syndrome is called the lateral medullary (or Wallenberg’s) syndrome (Fig. 446-10). Most cases result from ipsilateral vertebral artery occlusion; in the remainder, PICA occlusion is responsible. Occlusion of the medullary penetrating branches of the vertebral artery or PICA results in partial syndromes. Hemiparesis is not a feature of vertebral artery occlusion; however, quadriparesis may result from occlusion of the anterior spinal artery. Rarely, a medial medullary syndrome occurs with infarction of the pyramid and contralateral hemiparesis of the arm and leg, sparing the face. If the medial lemniscus and emerging hypoglossal nerve fibers are involved, contralateral loss of joint position sense and ipsilateral tongue weakness occur.

1	Cerebellar infarction with edema can lead to sudden respiratory arrest due to raised ICP in the posterior fossa. Drowsiness, Babinski signs, dysarthria, and bifacial weakness may be absent, or present only briefly, before respiratory arrest ensues. Gait unsteadiness, headache, dizziness, nausea, and vomiting may be the only early symptoms and signs and should arouse suspicion of this impending complication, which may require neurosurgical decompression, often with an excellent outcome. Separating these symptoms from those of viral labyrinthitis can be a challenge, but headache, neck stiffness, and unilateral dysmetria favor stroke. bASilAr Artery Branches of the basilar artery supply the base of the pons and superior cerebellum and fall into three groups: (1) paramedian, 7–10 in number, which supply a wedge of pons on either side of the midline; (2) short circumferential, 5–7 in number, that supply 5th n. Lateral Superior cerebellar peduncle Midpontine syndrome:

1	Superior cerebellar peduncle Midpontine syndrome: Medial longitudinal fasciculus 5th n. sensory nucleus 5th n. motor nucleus Spinothalamic Medial lemniscus FIGURE 446-12 Axial section at the level of the midpons, depicted schematically on the left, with a corresponding magnetic resonance image on the right. Approximate regions involved in medial and lateral midpontine stroke syndromes are shown. Signs and symptoms: Structures involved 1. Medial midpontine syndrome (paramedian branch of midbasilar artery) On side of lesion Ataxia of limbs and gait (more prominent in bilateral involvement): Pontine nuclei On side opposite lesion Paralysis of face, arm, and leg: Corticobulbar and corticospinal tract Variable impaired touch and proprioception when lesion extends posteriorly: Medial lemniscus 2.

1	On side opposite lesion Paralysis of face, arm, and leg: Corticobulbar and corticospinal tract Variable impaired touch and proprioception when lesion extends posteriorly: Medial lemniscus 2. On side of lesion Ataxia of limbs: Middle cerebellar peduncle Paralysis of muscles of mastication: Motor fibers or nucleus of fifth nerve Impaired sensation over side of face: Sensory fibers or nucleus of fifth nerve On side opposite lesion Impaired pain and thermal sense on limbs and trunk: Spinothalamic tract the lateral two-thirds of the pons and middle and superior cerebellar peduncles; and (3) bilateral long circumferential (superior cerebellar and anterior inferior cerebellar arteries), which course around the pons to supply the cerebellar hemispheres.

1	Atheromatous lesions can occur anywhere along the basilar trunk but are most frequent in the proximal basilar and distal vertebral segments. Typically, lesions occlude either the proximal basilar and one or both vertebral arteries. The clinical picture varies depending on the availability of retrograde collateral flow from the posterior communicating arteries. Rarely, dissection of a vertebral artery may involve the basilar artery and, depending on the location of true and false lumen, may produce multiple penetrating artery strokes. Although atherothrombosis occasionally occludes the distal portion of the basilar artery, emboli from the heart or proximal vertebral or basilar segments are more commonly responsible for “top of the basilar” syndromes.

1	Because the brainstem contains many structures in close apposition, a diversity of clinical syndromes may emerge with ischemia, reflecting involvement of the corticospinal and corticobulbar tracts, ascending sensory tracts, and cranial nerve nuclei (Figs. 446-11, 446-12, 446-13, and 446-14).

1	The symptoms of transient ischemia or infarction in the territory of the basilar artery often do not indicate whether the basilar artery itself or one of its branches is diseased, yet this distinction has important implications for therapy. The picture of complete basilar occlusion, however, is easy to recognize as a constellation of bilateral long tract signs (sensory and motor) with signs of cranial nerve and cerebellar dysfunction. A “locked-in” state of preserved consciousness with quadriplegia and cranial nerve signs suggests complete pontine and lower midbrain infarction. The therapeutic goal is to identify impending basilar occlusion before devastating infarction occurs. A series of TIAs and a slowly progressive, fluctuating stroke are extremely significant, because they often herald an atherothrombotic occlusion of the distal vertebral or proximal basilar artery.

1	TIAs in the proximal basilar distribution may produce vertigo (often described by patients as “swimming,” “swaying,” “moving,” “unsteadiness,” or “light-headedness”). Other symptoms that warn of basilar thrombosis include diplopia, dysarthria, facial or circumoral numbness, and hemisensory symptoms. In general, symptoms of basilar branch TIAs affect one side of the brainstem, whereas symptoms of basilar artery TIAs usually affect both sides, although a “herald” hemiparesis has been emphasized as an initial symptom of basilar occlusion. Most often, TIAs, whether due to impending occlusion of the basilar artery or a basilar branch, are short lived (5–30 min) and repetitive, occurring several times a day. The pattern suggests intermittent reduction of flow. Many neurologists treat with heparin to prevent clot propagation. Atherothrombotic occlusion of the basilar artery with infarction usually causes bilateral brainstem signs. A gaze paresis or internuclear Superior pontine syndrome:

1	Atherothrombotic occlusion of the basilar artery with infarction usually causes bilateral brainstem signs. A gaze paresis or internuclear Superior pontine syndrome: FIGURE 446-13 Axial section at the level of the superior pons, depicted schematically on the left, with a corresponding magnetic resonance image on the right. Approximate regions involved in medial and lateral superior pontine stroke syndromes are shown. Signs and symptoms: Structures involved 1. Medial superior pontine syndrome (paramedian branches of upper basilar artery) On side of lesion Cerebellar ataxia (probably): Superior and/or middle cerebellar peduncle Internuclear ophthalmoplegia: Medial longitudinal fasciculus Myoclonic syndrome, palate, pharynx, vocal cords, respiratory apparatus, face, oculomotor apparatus, etc.: Localization uncertain—central tegmental bundle, dentate projection, inferior olivary nucleus

1	On side opposite lesion Paralysis of face, arm, and leg: Corticobulbar and corticospinal tract Rarely touch, vibration, and position are affected: Medial lemniscus 2. Lateral superior pontine syndrome (syndrome of superior cerebellar artery) On side of lesion Ataxia of limbs and gait, falling to side of lesion: Middle and superior cerebellar peduncles, superior surface of cerebellum, dentate nucleus Dizziness, nausea, vomiting; horizontal nystagmus: Vestibular nucleus Paresis of conjugate gaze (ipsilateral): Pontine contralateral gaze Skew deviation: Uncertain Miosis, ptosis, decreased sweating over face (Horner’s syndrome): Descending sympathetic fibers Tremor: Localization unclear—Dentate nucleus, superior cerebellar peduncle

1	On side opposite lesion Impaired pain and thermal sense on face, limbs, and trunk: Spinothalamic tract Impaired touch, vibration, and position sense, more in leg than arm (there is a tendency to incongruity of pain and touch deficits): Medial lemniscus (lateral portion) ophthalmoplegia associated with ipsilateral hemiparesis may be the only manifestation of bilateral brainstem ischemia. More often, unequivocal signs of bilateral pontine disease are present. Complete basilar thrombosis carries a high mortality. Occlusion of a branch of the basilar artery usually causes unilateral symptoms and signs involving motor, sensory, and cranial nerves. As long as symptoms remain unilateral, concern over pending basilar occlusion should be reduced.

1	Occlusion of the superior cerebellar artery results in severe ipsilateral cerebellar ataxia, nausea and vomiting, dysarthria, and contra-lateral loss of pain and temperature sensation over the extremities, body, and face (spinoand trigeminothalamic tract). Partial deafness, ataxic tremor of the ipsilateral upper extremity, Horner’s syndrome, and palatal myoclonus may occur rarely. Partial syndromes occur frequently (Fig. 446-13). With large strokes, swelling and mass effects may compress the midbrain or produce hydrocephalus; these symptoms may evolve rapidly. Neurosurgical intervention may be lifesaving in such cases.

1	Occlusion of the anterior inferior cerebellar artery produces variable degrees of infarction because the size of this artery and the territory it supplies vary inversely with those of the PICA. The principal symptoms include: (1) ipsilateral deafness, facial weakness, vertigo, nausea and vomiting, nystagmus, tinnitus, cerebellar ataxia, Horner’s syndrome, and paresis of conjugate lateral gaze; and (2) contralateral loss of pain and temperature sensation. An occlusion close to the origin of the artery may cause corticospinal tract signs (Fig. 446-11). Occlusion of one of the short circumferential branches of the basilar artery affects the lateral two-thirds of the pons and middle or superior cerebellar peduncle, whereas occlusion of one of the paramedian branches affects a wedge-shaped area on either side of the medial pons (Figs. 446-11 through 446-13). See also Chap. 440e. 2580 3rd n. Midbrain syndrome:

1	See also Chap. 440e. 2580 3rd n. Midbrain syndrome: FIGURE 446-14 Axial section at the level of the midbrain, depicted schematically on the left, with a corresponding magnetic resonance image on the right. Approximate regions involved in medial and lateral midbrain stroke syndromes are shown. Signs and symptoms: Structures involved 1. Medial midbrain syndrome (paramedian branches of upper basilar and proximal posterior cerebral arteries) On side of lesion Eye “down and out” secondary to unopposed action of fourth and sixth cranial nerves, with dilated and unresponsive pupil: Third nerve fibers On side opposite lesion Paralysis of face, arm, and leg: Corticobulbar and corticospinal tract descending in crus cerebri 2. Lateral midbrain syndrome (syndrome of small penetrating arteries arising from posterior cerebral artery)

1	On side of lesion Eye “down and out” secondary to unopposed action of fourth and sixth cranial nerves, with dilated and unresponsive pupil: Third nerve fibers and/or third nerve nucleus On side opposite lesion Hemiataxia, hyperkinesias, tremor: Red nucleus, dentatorubrothalamic pathway CT Scans CT radiographic images identify or exclude hemorrhage as the cause of stroke, and they identify extraparenchymal hemorrhages, neoplasms, abscesses, and other conditions masquerading as stroke. Brain CT scans obtained in the first several hours after an infarction generally show no abnormality, and the infarct may not be seen reliably for 24–48 h. CT may fail to show small ischemic strokes in the posterior fossa because of bone artifact; small infarcts on the cortical surface may also be missed.

1	Contrast-enhanced CT scans add specificity by showing contrast enhancement of subacute infarcts and allow visualization of venous structures. Coupled with multidetector scanners, CT angiography (CTA) can be performed with administration of IV iodinated contrast allowing visualization of the cervical and intracranial arteries, intracranial veins, aortic arch, and even the coronary arteries in one imaging session. Carotid disease and intracranial vascular occlusions are readily identified with this method (Fig. 446-3). After an IV bolus of contrast, deficits in brain perfusion produced by vascular occlusion can also be demonstrated (Fig. 446-15) and used to predict the region of infarcted brain and the brain at risk of further infarction (i.e., the ischemic penumbra, see “Pathophysiology of Ischemic Stroke” above). CT imaging is also sensitive for detecting SAH (although by itself does not rule it out), and CTA can readily identify intracranial aneurysms (Chap. 330). Because of its

1	of Ischemic Stroke” above). CT imaging is also sensitive for detecting SAH (although by itself does not rule it out), and CTA can readily identify intracranial aneurysms (Chap. 330). Because of its speed and wide availability, noncontrast head CT is the imaging modality of choice in patients with acute stroke (Fig. 446-1), and CTA and CT perfusion imaging may also be useful and convenient adjuncts.

1	MRI reliably documents the extent and location of infarction in all areas of the brain, including the posterior fossa and cortical surface.

1	It also identifies intracranial hemorrhage and other abnormalities and, using special sequences, can be as sensitive as CT for detecting acute intracerebral hemorrhage. MRI scanners with magnets of higher field strength produce more reliable and precise images. Diffusion-weighted imaging is more sensitive for early brain infarction than standard MR sequences or CT (Fig. 446-16), as is fluid-attenuated inversion recovery (FLAIR) imaging (Chap. 440e). Using IV administration of gadolinium contrast, MR perfusion studies can be performed. Brain regions showing poor perfusion but no abnormality on diffusion provide, compared to CT, an equivalent measure of the ischemic penumbra. MR angiography is highly sensitive for steno-sis of extracranial internal carotid arteries and of large intracranial vessels. With higher degrees of stenosis, MR angiography tends to overestimate the degree of stenosis when compared to conventional x-ray angiography. MRI with fat saturation is an imaging sequence

1	vessels. With higher degrees of stenosis, MR angiography tends to overestimate the degree of stenosis when compared to conventional x-ray angiography. MRI with fat saturation is an imaging sequence used to visualize extra or intracranial arterial dissection. This sensitive technique images clotted blood within the dissected vessel wall. Iron-sensitive imaging (ISI) is helpful to detect cerebral microbleeds that may be present in cerebral amyloid angiopathy and other hemorrhagic disorders.

1	MRI is more expensive and time consuming than CT and less readily available. Claustrophobia and the logistics of imaging acutely critically ill patients also limit its application. Most acute stroke protocols use CT because of these limitations. However, MRI is useful outside the acute period by more clearly defining the extent of tissue injury and discriminating new from old regions of brain infarction. MRI may have particular utility in patients with TIA, because it is also more likely to identify new infarction, which is a strong predictor of subsequent stroke.

1	FIGURE 446-15 Acute left middle cerebral artery (MCA) stroke with right hemiplegia but preserved language. A. Computed tomography (CT) perfusion mean-transit time map showing delayed perfusion of the left MCA distribution (blue). B. Predicted region of infarct (red) and penumbra (green) based on CT perfusion data. C. Conventional angiogram showing occlusion of the left internal carotid–MCA bifurcation (left panel), and revascularization of the vessels following successful thrombectomy 8 h after stroke symptom onset (right panel). D. The clot removed with a thrombectomy device (L5, Concentric Medical, Inc.). E. CT scan of the brain 2 days later; note infarction in the region predicted in B but preservation of the penumbral region by successful revascularization.

1	Cerebral Angiography Conventional x-ray cerebral angiography is the gold standard for identifying and quantifying atherosclerotic stenoses of the cerebral arteries and for identifying and characterizing other pathologies, including aneurysms, vasospasm, intraluminal thrombi, fibromuscular dysplasia, arteriovenous fistulae, vasculitis, and collateral channels of blood flow. Conventional angiography carries risks of arterial damage, groin hemorrhage, embolic stroke, and renal failure from contrast nephropathy, so it should be reserved for situations where less invasive means are inadequate. As reviewed earlier in this chapter, endovascular stroke therapy has not been proven effective in three randomized trials, and this remains an area of ongoing investigation.

1	Ultrasound Techniques Stenosis at the origin of the internal carotid artery can be identified and quantified reliably by ultrasonography that combines a B-mode ultrasound image with a Doppler ultrasound assessment of flow velocity (“duplex” ultrasound). Transcranial Doppler (TCD) assessment of MCA, ACA, and PCA flow and of vertebrobasilar flow is also useful. This latter technique can detect stenotic lesions in the large intracranial arteries because such lesions increase systolic flow velocity. TCD can assist thrombolysis and improve large artery recanalization following rtPA administration; the potential clinical benefit of this treatment is the subject of ongoing study. TCD can also detect microemboli from otherwise asymptomatic carotid plaques. In many cases, MR angiography combined with carotid and transcranial ultrasound studies eliminates the need for conventional x-ray angiography in evaluating vascular stenosis. Alternatively, CT angiography of the entire head and neck can be

1	carotid and transcranial ultrasound studies eliminates the need for conventional x-ray angiography in evaluating vascular stenosis. Alternatively, CT angiography of the entire head and neck can be performed during the initial imaging of acute stroke. Because this images the entire arterial system relevant to stroke, with the exception of the heart, much of the clinician’s stroke workup can be completed with this single imaging study.

1	Perfusion Techniques Both xenon techniques (principally xenon-CT) and positron emission tomography (PET) can quantify cerebral blood flow. These tools are generally used for research (Chap. 440e) but can be useful for determining the significance of arterial stenosis and planning for revascularization surgery. Single-photon emission computed tomography (SPECT) and MR perfusion techniques report relative cerebral blood flow. As noted above, CT imaging is used as the initial imaging modality for acute stroke, and some centers combine both CT angiography and CT perfusion imaging together with the noncontrast CT scan. CT perfusion imaging increases the sensitivity for detecting ischemia and can measure the ischemic penumbra (Fig. 446-15). Alternatively, MR perfusion can be combined with MR diffusion imaging to identify the ischemic penumbra as the mismatch between these two imaging sequences (Fig. 446-16).

1	Hemorrhages are classified by their location and the underlying vascular pathology. Bleeding into subdural and epidural spaces is

1	FIGURE 446-16 Magnetic resonance imaging (MRI) of acute stroke. A. MRI diffusion-weighted image (DWI) of an 82-year-old woman 2.5 h after onset of right-sided weakness and aphasia reveals restricted diffusion within the left basal ganglia and internal capsule (colored regions). B. Perfusion defect within the left hemisphere (colored signal) imaged after administration of an IV bolus of gadolinium contrast. The discrepancy between the region of poor perfusion shown in B and the diffusion deficit shown in A is called diffusion-perfusion mismatch and provides an estimate of the ischemic penumbra. Without specific therapy, the region of infarction will expand into much or all of the perfusion deficit. C. Cerebral angiogram of the left internal carotid artery in this patient before (left) and after (right) successful endovascular embolectomy. The occlusion is within the carotid terminus. D. Fluid-attenuated inversion recovery image obtained 3 days later showing a region of infarction

1	and after (right) successful endovascular embolectomy. The occlusion is within the carotid terminus. D. Fluid-attenuated inversion recovery image obtained 3 days later showing a region of infarction (coded as white) that corresponds to the initial DWI image in A, but not the entire area at risk shown in B, suggesting that successful embolectomy saved a large region of brain tissue from infarction. (Courtesy of Gregory Albers, MD, Stanford University; with permission.) principally produced by trauma. SAH results from trauma or the rupture of an intracranial aneurysm or arteriovenous malformation (AVM) (Chap. 330). Intracerebral and intraventricular hemorrhage will be considered here.

1	Intracranial hemorrhage is often discovered on noncontrast CT imaging of the brain during the acute evaluation of stroke. Because CT is more widely available and may be logistically easier, CT imaging is the preferred method for acute stroke evaluation (Fig. 446-1). The location of the hemorrhage narrows the differential diagnosis to a few entities. Table 446-6 lists the causes and anatomic spaces involved in hemorrhages.

1	Close attention should be paid to airway management because a reduction in the level of consciousness is common and often progressive. The initial blood pressure should be maintained until the results of the CT scan are reviewed and demonstrate an intracerebral hemorrhage (ICH). In theory, a higher blood pressure should promote hematoma expansion, but it remains unclear if lowering of blood pressure reduces hematoma growth. Recent clinical trials have shown that systolic blood pressure (SBP) can be safely lowered acutely and rapidly to <140 mmHg in patients with spontaneous ICH whose initial SBP was 150–220 mmHg.

1	The INTERACT2 trial is the only large phase 3 clinical trial to address the effect of acute blood pressure lowering on ICH functional outcome. INTERACT2 randomized patients with spontaneous ICH within 6 h of onset and a baseline SBP of 150–220 mmHg to two different SBP targets (<140 mmHg and <180 mmHg). In those with the target SBP <140 mmHg, 52% had an outcome of death or major disability at 90 days compared with 55.6% of those with a target SBP <180 mmHg (p = .06). There was a significant shift to improved outcomes in the lower blood pressure arm, whereas both groups had a similar mortality. This study shows that it is not harmful, and may be modestly beneficial, to lower blood pressure in acute ICH. Thus, it is reasonable to target an SBP <140 mmHg initially in this group of patients. In patients who have higher SBP on presentation or who are deeply comatose with possible elevated ICP, it is unclear whether the INTERACT2 results apply. In patients who have ICP monitors in place,

1	In patients who have higher SBP on presentation or who are deeply comatose with possible elevated ICP, it is unclear whether the INTERACT2 results apply. In patients who have ICP monitors in place, current recommendations are to maintain the cerebral perfusion pressure (mean arterial pressure [MAP] minus ICP) above 60 mmHg. Blood pressure should be lowered with nonvasodilating IV drugs such as nicardipine, labetalol, or esmolol. Patients with cerebellar hemorrhages or with depressed mental status and radiographic evidence of hydrocephalus should undergo urgent neurosurgical evaluation; these patients require close monitoring because they can deteriorate rapidly. Based on the clinical examination and CT findings, further imaging studies may be necessary, including MRI or conventional x-ray angiography. Stuporous or comatose patients with clinical and imaging signs of herniation are generally treated presumptively for elevated ICP, with tracheal intubation, administration of osmotic

1	angiography. Stuporous or comatose patients with clinical and imaging signs of herniation are generally treated presumptively for elevated ICP, with tracheal intubation, administration of osmotic diuretics such as mannitol or hypertonic saline, and elevation of the head of the bed while surgical consultation is obtained (Chap. 330). Reversal of coagulopathy and consideration of surgical evacuation of the hematoma (detailed below) are two other principal aspects of initial emergency management.

1	ICH accounts for ~10% of all strokes, and about 35–45% of patients die within the first month. Incidence rates are particularly high in Asians and blacks. Hypertension, coagulopathy, sympathomimetic drugs (cocaine, methamphetamine), and cerebral amyloid angiopathy cause the majority of these hemorrhages. Advanced age and heavy alcohol consumption increase the risk, and cocaine and methamphetamine use is one of the most important causes in the young.

1	Hypertensive Intracerebral Hemorrhage • pAthophySiology Hypertensive ICH usually results from spontaneous rupture of a small penetrating artery deep in the brain. The most common sites are the basal ganglia (especially the putamen), thalamus, cerebellum, and pons. The small arteries in these areas seem most prone to hypertension-induced vascular injury. When hemorrhages occur in other brain areas or in nonhypertensive patients, greater consideration should be given to other causes such as hemorrhagic disorders, neoplasms, vascular malformations, and cerebral amyloid angiopathy. The hemorrhage may be small, or a large clot may form and compress adjacent tissue, causing herniation and death. Blood may also dissect into the ventricular space, which substantially increases morbidity and may cause hydrocephalus.

1	Most hypertensive ICHs initially develop over 30–90 min, whereas those associated with anticoagulant therapy may evolve for as long as 24–48 h. However, it is now recognized that about a third of patients even with no coagulopathy may have significant hematoma expansion with the first day. Within 48 h, macrophages begin to phagocytize the hemorrhage at its outer surface. After 1–6 months, the hemorrhage is generally resolved to a slitlike orange cavity lined with glial scar and hemosiderin-laden macrophages. clinicAl mAnifeStAtionS ICH generally presents as the abrupt onset of a focal neurologic deficit. Seizures are uncommon. Although clinical symptoms may be maximal at onset, commonly the focal deficit worsens over 30–90 min and is associated with a diminishing level of consciousness and signs of increased ICP such as headache and vomiting.

1	The putamen is the most common site for hypertensive hemorrhage, and the adjacent internal capsule is usually damaged (Fig. 446-17). Contralateral hemiparesis is therefore the sentinel sign. When mild, the face sags on one side over 5–30 min, speech becomes slurred, the arm and leg gradually weaken, and the eyes deviate away from the side of the hemiparesis. The paralysis may worsen until the affected limbs become flaccid or extend rigidly. When hemorrhages are large, drowsiness gives way to stupor as signs of upper brainstem compression appear. Coma ensues, accompanied by deep, irregular, or intermittent respiration, a dilated and fixed ipsilateral pupil, and decerebrate rigidity. In milder cases, edema in adjacent brain tissue may cause progressive deterioration over 12–72 h.

1	Thalamic hemorrhages also produce a contralateral hemiplegia or hemiparesis from pressure on, or dissection into, the adjacent internal capsule. A prominent sensory deficit involving all modalities is usually present. Aphasia, often with preserved verbal repetition, may occur after hemorrhage into the dominant thalamus, and constructional apraxia or mutism occurs in some cases of nondominant hemorrhage. There may also be a homonymous visual field defect. Thalamic hemorrhages cause several typical ocular disturbances by virtue of extension FIGURE 446-17 Hypertensive hemorrhage. Transaxial noncontrast computed tomography scan through the region of the basal ganglia reveals a hematoma involving the left putamen in a patient with rapidly progressive onset of right hemiparesis. Occurs in 1–6% of ischemic strokes with predilection for large hemispheric infarctions Lung, choriocarcinoma, melanoma, renal cell carcinoma, thyroid, atrial myxoma Cocaine, amphetamine

1	Occurs in 1–6% of ischemic strokes with predilection for large hemispheric infarctions Lung, choriocarcinoma, melanoma, renal cell carcinoma, thyroid, atrial myxoma Cocaine, amphetamine Risk is ~2–3% per year for bleeding if previously unruptured Mycotic and nonmycotic forms of aneurysms Degenerative disease of intracranial vessels; associated with dementia, rare in patients <60 years Multiple cavernous angiomas linked to mutations in KRIT1, CCM2, and PDCD10 genes Rare cause of hemorrhage inferiorly into the upper midbrain. These include deviation of the eyes downward and inward so that they appear to be looking at the nose, unequal pupils with absence of light reaction, skew deviation with the eye opposite the hemorrhage displaced downward and medially, ipsilateral Horner’s syndrome, absence of convergence, paralysis of vertical gaze, and retraction nystagmus. Patients may later develop a chronic, contralateral pain syndrome (Déjérine-Roussy syndrome).

1	In pontine hemorrhages, deep coma with quadriplegia often occurs over a few minutes. Typically, there is prominent decerebrate rigidity and “pinpoint” (1 mm) pupils that react to light. There is impairment of reflex horizontal eye movements evoked by head turning (doll’s-head or oculocephalic maneuver) or by irrigation of the ears with ice water (Chap. 328). Hyperpnea, severe hypertension, and hyperhidrosis are common. Most patients with deep coma from pontine hemorrhage ultimately die, but small hemorrhages are compatible with survival.

1	Cerebellar hemorrhages usually develop over several hours and are characterized by occipital headache, repeated vomiting, and ataxia of gait. In mild cases, there may be no other neurologic signs except for gait ataxia. Dizziness or vertigo may be prominent. There is often paresis of conjugate lateral gaze toward the side of the hemorrhage, forced deviation of the eyes to the opposite side, or an ipsilateral sixth nerve palsy. Less frequent ocular signs include blepharospasm, involuntary closure of one eye, ocular bobbing, and skew deviation. Dysarthria and dysphagia may occur. As the hours pass, the patient often becomes stuporous and then comatose from brainstem compression or obstructive hydrocephalus; immediate surgical evacuation before brainstem compression occurs may be lifesaving. Hydrocephalus from fourth ventricle compression can be relieved by external ventricular drainage, but definitive hematoma evacuation is recommended. If the deep cerebellar nuclei are spared, full

1	Hydrocephalus from fourth ventricle compression can be relieved by external ventricular drainage, but definitive hematoma evacuation is recommended. If the deep cerebellar nuclei are spared, full recovery is common.

1	Lobar Hemorrhage The major neurologic deficit with an occipital hemorrhage is hemianopia; with a left temporal hemorrhage, aphasia and delirium; with a parietal hemorrhage, hemisensory loss; and with frontal hemorrhage, arm weakness. Large hemorrhages may be associated with stupor or coma if they compress the thalamus or midbrain. Most patients with lobar hemorrhages have focal headaches, and more than one-half vomit or are drowsy. Stiff neck and seizures are uncommon. Other Causes of Intracerebral Hemorrhage Cerebral amyloid angiopathy is a disease of the elderly in which arteriolar degeneration occurs and amyloid is deposited in the walls of the cerebral arteries. Amyloid angiopathy causes both single and recurrent lobar hemorrhages and is probably the most common cause of lobar hemorrhage in the elderly.

1	2584 It accounts for some intracranial hemorrhages associated with IV thrombolysis given for MI. This disorder can be suspected in patients who present with multiple hemorrhages (and infarcts) over several months or years or in patients with “microbleeds” seen on brain MRI sequences sensitive for hemosiderin (iron-sensitive imaging), but it is definitively diagnosed by pathologic demonstration of Congo red staining of amyloid in cerebral vessels. The ε2 and ε4 allelic variations of the apolipoprotein E gene are associated with increased risk of recurrent lobar hemorrhage and may therefore be markers of amyloid angiopathy. Currently, there is no specific therapy. OACs are typically avoided. Cocaine and methamphetamine are frequent causes of stroke in young (age <45 years) patients. ICH, ischemic stroke, and SAH are all associated with stimulant use. Angiographic findings vary from completely normal arteries to large-vessel occlusion or stenosis, vasospasm, or changes consistent with

1	ischemic stroke, and SAH are all associated with stimulant use. Angiographic findings vary from completely normal arteries to large-vessel occlusion or stenosis, vasospasm, or changes consistent with vasculopathy. The mechanism of sympathomimetic-related stroke is not known, but cocaine enhances sympathetic activity causing acute, sometimes severe, hypertension, and this may lead to hemorrhage. Slightly more than one-half of stimulant-related intracranial hemorrhages are intracerebral, and the rest are subarachnoid. In cases of SAH, a saccular aneurysm is usually identified. Presumably, acute hypertension causes aneurysmal rupture. Head injury often causes intracranial bleeding. The common sites are intraparenchymal (especially temporal and inferior frontal lobes) and into the subarachnoid, subdural, and epidural spaces. Trauma must be considered in any patient with an unexplained acute neurologic deficit (hemiparesis, stupor, or confusion), particularly if the deficit occurred in the

1	subdural, and epidural spaces. Trauma must be considered in any patient with an unexplained acute neurologic deficit (hemiparesis, stupor, or confusion), particularly if the deficit occurred in the context of a fall (Chap. 457e). Intracranial hemorrhages associated with anticoagulant therapy can occur at any location; they are often lobar or subdural. Anticoagulant-related ICHs may continue to evolve over 24–48 h, especially if coagulopathy is insufficiently reversed. Coagulopathy and thrombocytopenia should be reversed rapidly, as discussed below. ICH associated with hematologic disorders (leukemia, aplastic anemia, thrombocytopenic purpura) can occur at any site and may present as multiple ICHs. Skin and mucous membrane bleeding may be evident and offers a diagnostic clue. Hemorrhage into a brain tumor may be the first manifestation of neoplasm. Choriocarcinoma, malignant melanoma, renal cell carcinoma, and bronchogenic carcinoma are among the most common metastatic tumors

1	into a brain tumor may be the first manifestation of neoplasm. Choriocarcinoma, malignant melanoma, renal cell carcinoma, and bronchogenic carcinoma are among the most common metastatic tumors associated with ICH. Glioblastoma multiforme in adults and medulloblastoma in children may also have areas of ICH. Hypertensive encephalopathy is a complication of malignant hypertension. In this acute syndrome, severe hypertension is associated with headache, nausea, vomiting, convulsions, confusion, stupor, and coma. Focal or lateralizing neurologic signs, either transitory or permanent, may occur but are infrequent and therefore suggest some other vascular disease (hemorrhage, embolism, or atherosclerotic thrombosis). There are retinal hemorrhages, exudates, papilledema (hypertensive retinopathy), and evidence of renal and cardiac disease. In most cases, ICP and CSF protein levels are elevated. MRI brain imaging shows a pattern of typically posterior (occipital > frontal) brain edema that is

1	and evidence of renal and cardiac disease. In most cases, ICP and CSF protein levels are elevated. MRI brain imaging shows a pattern of typically posterior (occipital > frontal) brain edema that is reversible and termed reversible posterior leukoencephalopathy. The hypertension may be essential or due to chronic renal disease, acute glomerulonephritis, acute toxemia of pregnancy, pheochromocytoma, or other causes. Lowering the blood pressure reverses the process, but stroke can occur, especially if blood pressure is lowered too rapidly. Neuropathologic examination reveals multifocal to diffuse cerebral edema and hemorrhages of various sizes from petechial to massive. Microscopically, there are necrosis of arterioles, minute cerebral infarcts, and hemorrhages. The term hypertensive encephalopathy should be reserved for this syndrome and not for chronic recurrent headaches, dizziness, recurrent TIAs, or small strokes that often occur in association with high blood pressure. Primary

1	should be reserved for this syndrome and not for chronic recurrent headaches, dizziness, recurrent TIAs, or small strokes that often occur in association with high blood pressure. Primary intraventricular hemorrhage is rare and should prompt investigation for an underlying vascular anomaly. Sometimes bleeding begins within the periventricular substance of the brain and dissects into the ventricular system without leaving signs of intraparenchymal hemorrhage. Alternatively, bleeding can arise from periependymal veins. Vasculitis, usually polyarteritis nodosa or lupus erythematosus, can produce hemorrhage in any region of the central nervous system; most hemorrhages are associated with hypertension, but the arteritis itself may cause bleeding by disrupting the vessel wall. Nearly one-half of patients with primary intraventricular hemorrhage have identifiable bleeding sources seen using conventional angiography.

1	Sepsis can cause small petechial hemorrhages throughout the cerebral white matter. Moyamoya disease, mainly an occlusive arterial disease that causes ischemic symptoms, may on occasion produce ICH, particularly in the young. Hemorrhages into the spinal cord are usually the result of an AVM, cavernous malformation, or metastatic tumor. Epidural spinal hemorrhage produces a rapidly evolving syndrome of spinal cord or nerve root compression (Chap. 456). Spinal hemorrhages usually present with sudden back pain and some manifestation of myelopathy.

1	Laboratory and Imaging Evaluation Patients should have routine blood chemistries and hematologic studies. Specific attention to the platelet count and PT/PTT/INR is important to identify coagulopathy. CT imaging reliably detects acute focal hemorrhages in the supratentorial space. Rarely very small pontine or medullary hemorrhages may not be well delineated because of motion and bone-induced artifact that obscure structures in the posterior fossa. After the first 2 weeks, x-ray attenuation values of clotted blood diminish until they become isodense with surrounding brain. Mass effect and edema may remain. In some cases, a surrounding rim of contrast enhancement appears after 2–4 weeks and may persist for months. MRI, although more sensitive for delineating posterior fossa lesions, is generally not necessary for primary diagnosis in most instances. Images of flowing blood on MRI scan may identify AVMs as the cause of the hemorrhage. MRI, CT angiography (CTA), and conventional x-ray

1	not necessary for primary diagnosis in most instances. Images of flowing blood on MRI scan may identify AVMs as the cause of the hemorrhage. MRI, CT angiography (CTA), and conventional x-ray angiography are used when the cause of intracranial hemorrhage is uncertain, particularly if the patient is young or not hypertensive and the hematoma is not in one of the usual sites for hypertensive hemorrhage. CTA or postcontrast CT imaging may reveal one or more small areas of enhancement within a hematoma; this “spot sign” is thought to represent ongoing bleeding. The presence of a spot sign is associated with an increased risk of hematoma expansion, increased mortality, and lower likelihood of favorable functional outcome. Some centers routinely perform CT with CTA and postcontrast CT at the time of initial imaging to rapidly identify any macrovascular etiology of the hemorrhage and provide prognostic information at the same time. Because patients typically have focal neurologic signs and

1	of initial imaging to rapidly identify any macrovascular etiology of the hemorrhage and provide prognostic information at the same time. Because patients typically have focal neurologic signs and obtundation and often show signs of increased ICP, a lumbar puncture is generally unnecessary and should usually be avoided because it may induce cerebral herniation.

1	Although about 40% of patients with a hypertensive ICH die, others have a good to complete recovery if they survive the initial hemorrhage. The ICH Score (Table 446-7) is a validated clinical grading scale that is useful for stratification of mortality risk and clinical outcome. Any identified coagulopathy should be corrected as soon as possible. For patients taking VKAs, rapid correction of coagulopathy can be achieved by infusing prothrombin complex concentrates (PCC), which can be administered quickly, with vitamin K administered concurrently. Fresh frozen plasma is an alternative but generally requires larger fluid volumes and longer time to achieve adequate reversal than PCC. There is no effective antidote to ICH associated with oral thrombin inhibitor dabigatran, although FEIBA (factor VIII inhibitor bypassing activity) and recombinant factor VIIa have been tried in individual cases. PCC may partially reverse the effects of oral factor Xa inhibitors and are reasonable to

1	(factor VIII inhibitor bypassing activity) and recombinant factor VIIa have been tried in individual cases. PCC may partially reverse the effects of oral factor Xa inhibitors and are reasonable to administer if available. When ICH is associated with thrombocytopenia (platelet count <50,000/μL), transfusion of fresh platelets is indicated. The role of platelet transfusions either empirically or based on urgent platelet inhibition assays remains unclear.

1	Total Score Sum of each category above Note: Although an ICH Score of 6 is possible with the scale, this is rarely observed and is considered highly likely to be fatal. Abbreviations: CI, confidence interval; ICH, intracerebral hemorrhage. Sources: JC Hemphill et al: Stroke 32:891, 2001; JC Hemphill et al: Neurology 73:1088, 2009. Hematomas may expand for several hours following the initial hemorrhage, even in patients without coagulopathy. However, the precise mechanism is unclear. A phase 3 trial of treatment with recombinant factor VIIa reduced hematoma expansion; however, clinical outcomes were not improved, so use of this drug cannot be advocated at present. The theoretical risk of acutely elevated blood pressure on hematoma expansion forms the basis of the consideration for recently completed and ongoing clinical trials of acute blood pressure lowering.

1	Evacuation of supratentorial hematomas does not appear to improve outcome for most patients. The International Surgical Trial in Intracerebral Haemorrhage (STICH) randomized patients with supratentorial ICH to either early surgical evacuation or initial medical management. No benefit was found in the early surgery arm, although analysis was complicated by the fact that 26% of patients in the initial medical management group ultimately had surgery for neurologic deterioration. The follow-up study STICH-II found that surgery within 24 h of lobar, supratentorial hemorrhage did not improve overall outcome, but might have a role in select severely affected patients. Therefore, existing data do not support routine surgical evacuation of supratentorial hemorrhages in stable patients. However, many centers still consider surgery for patients deemed salvageable and who are having progressive neurologic deterioration due to herniation. Surgical techniques continue to evolve, and minimally

1	many centers still consider surgery for patients deemed salvageable and who are having progressive neurologic deterioration due to herniation. Surgical techniques continue to evolve, and minimally invasive endoscopic hematoma evacuation is currently being investigated in clinical trials.

1	For cerebellar hemorrhages, a neurosurgeon should be consulted immediately to assist with the evaluation; most cerebellar hematomas >3 cm in diameter will require surgical evacuation. If the patient is alert without focal brainstem signs and if the hematoma is <1 cm in diameter, surgical removal is usually unnecessary. Patients with signs of impaired consciousness, progressive hydrocephalus, and precipitous respiratory failure. Hydrocephalus due to cerebellar hematoma should not be treated solely with ventricular drainage. Tissue surrounding hematomas is displaced and compressed but not necessarily infarcted. Hence, in survivors, major improvement commonly occurs as the hematoma is reabsorbed and the adjacent tissue regains its function. Careful management of the patient during the acute phase of the hemorrhage can lead to considerable recovery.

1	Surprisingly, ICP is often normal even with large ICHs. However, if the hematoma causes marked midline shift of structures with consequent obtundation, coma, or hydrocephalus, osmotic agents can be instituted in preparation for placement of a ventriculostomy or parenchymal ICP monitor (Chap. 330). Once ICP is recorded, CSF drainage (if available), osmotic therapy, and blood pressure management can be tailored to the individual patient to keep cerebral perfusion pressure (MAP minus ICP) above 60 mmHg. For example, if ICP is found to be high, CSF can be drained from the ventricular space and osmotic therapy continued; persistent or progressive elevation in ICP may prompt surgical evacuation of the clot. Alternately, if ICP is normal or only mildly elevated, interventions such as osmotic therapy may be tapered. Because hyperventilation may actually produce ischemia by cerebral vasoconstriction, induced hyperventilation should be limited to acute resuscitation of the patient with

1	therapy may be tapered. Because hyperventilation may actually produce ischemia by cerebral vasoconstriction, induced hyperventilation should be limited to acute resuscitation of the patient with presumptive high ICP and eliminated once other treatments (osmotic therapy or surgical treatments) have been instituted. Glucocorticoids are not helpful for the edema from intra-cerebral hematoma.

1	Hypertension is the leading cause of primary ICH. Prevention is aimed at reducing chronic hypertension, eliminating excessive alcohol use, and discontinuing use of illicit drugs such as cocaine and amphetamines. Patients with amyloid angiopathy should generally avoid OACs, but antiplatelet agents may be administered if there is an indication based on atherothrombotic vascular disease. Vascular anomalies can be divided into congenital vascular malformations and acquired vascular lesions. True arteriovenous malformations (AVMs), venous anomalies, and capillary telangiectasias are lesions that usually remain clinically silent through life. AVMs are probably congenital, but cases of acquired lesions have been reported.

1	True AVMs are congenital shunts between the arterial and venous systems that may present with headache, seizures, and intracranial hemorrhage. AVMs consist of a tangle of abnormal vessels across the cortical surface or deep within the brain substance. AVMs vary in size from a small blemish a few millimeters in diameter to a large mass of tortuous channels composing an arteriovenous shunt of sufficient magnitude to raise cardiac output and precipitate heart failure. Blood vessels forming the tangle interposed between arteries and veins are usually abnormally thin and histologically resemble both arteries and veins. AVMs occur in all parts of the cerebral hemispheres, brainstem, and spinal cord, but the largest ones are most frequently in the posterior half of the hemispheres, commonly forming a wedge-shaped lesion extending from the cortex to the ventricle.

1	Bleeding, headache, and seizures are most common between the ages of 10 and 30, occasionally as late as the fifties. AVMs are more frequent in men, and rare familial cases have been described. Familial AVM may be a part of the autosomal dominant syndrome of hereditary hemorrhagic telangiectasia (Osler-Rendu-Weber) syndrome due to mutations in either endoglin or activin receptor-like kinase 1, both involved in transforming growth factor (TGF) signaling and angiogenesis.

1	2586 Headache (without bleeding) may be hemicranial and throbbing, like migraine, or diffuse. Focal seizures, with or without generalization, occur in ~30% of cases. One-half of AVMs become evident as ICHs. In most, the hemorrhage is mainly intraparenchymal with extension into the subarachnoid space in some cases. Blood is usually not deposited in the basal cisterns, and symptomatic cerebral vasospasm is rare. The risk of AVM rupture is strongly influenced by a history of prior rupture. Although unruptured AVMs have a hemorrhage rate of ~2–4% per year, previously ruptured AVMs may have a rate as high as 17% a year, at least for the first year. Hemorrhages may be massive, leading to death, or may be as small as 1 cm in diameter, leading to minor focal symptoms or no deficit. The AVM may be large enough to steal blood away from adjacent normal brain tissue or to increase venous pressure significantly to produce venous ischemia locally and in remote areas of the brain. This is seen most

1	large enough to steal blood away from adjacent normal brain tissue or to increase venous pressure significantly to produce venous ischemia locally and in remote areas of the brain. This is seen most often with large AVMs in the territory of the MCA. Large AVMs of the anterior circulation may be associated with a systolic and diastolic bruit (sometimes self-audible) over the eye, forehead, or neck and a bounding carotid pulse. Headache at the onset of AVM rupture is generally not as explosive as with aneurysmal rupture. MRI is better than CT for diagnosis, although noncontrast CT scanning sometimes detects calcification of the AVM and contrast may demonstrate the abnormal blood vessels. Once identified, conventional x-ray angiography is the gold standard for evaluating the precise anatomy of the AVM. Surgical treatment of AVMs presenting with hemorrhage often done in conjunction with preoperative embolization to reduce operative bleeding is usually indicated for accessible lesions.

1	of the AVM. Surgical treatment of AVMs presenting with hemorrhage often done in conjunction with preoperative embolization to reduce operative bleeding is usually indicated for accessible lesions. Stereotaxic radiation, an alternative to surgery, can produce a slow sclerosis of the AVM over 2–3 years. Several angiographic features can be used to help predict future bleeding risk. Paradoxically, smaller lesions seem to have a higher hemorrhage rate. The presence of deep venous drainage, venous outflow stenosis, and intranidal aneurysms may increase rupture risk. Because of the relatively low annual rate of hemorrhage and the risk of complications due to surgical or endovascular treatment, the indication for surgery in asymptomatic AVMs is debated. The ARUBA (A Randomized Trial of Unruptured Brain Arteriovenous Malformations) trial randomized patients to medical management versus intervention (surgery, endovascular embolization, combination embolization and surgery, or gamma-knife). The

1	Brain Arteriovenous Malformations) trial randomized patients to medical management versus intervention (surgery, endovascular embolization, combination embolization and surgery, or gamma-knife). The trial was stopped prematurely for harm, with the medical arm achieving the combined endpoint of death or symptomatic stroke in 10.1% of patients compared to 30.7% in the intervention group at an average follow-up time of 33 months. This highly significant finding argues against routine intervention for patients presenting without hemorrhage, although debate ensues regarding the generalizability of these results. Venous anomalies are the result of development of anomalous cerebral, cerebellar, or brainstem venous drainage. These structures, unlike AVMs, are functional venous channels. They are of little clinical significance and should be ignored if found incidentally on brain imaging studies. Surgical resection of these anomalies may result in venous infarction and hemorrhage. Venous

1	are of little clinical significance and should be ignored if found incidentally on brain imaging studies. Surgical resection of these anomalies may result in venous infarction and hemorrhage. Venous anomalies may be associated with cavernous malformations (see below), which do carry some bleeding risk. Capillary telangiectasias are true capillary malformations that often form extensive vascular networks through an otherwise normal brain structure. The pons and deep cerebral white matter are typical locations, and these capillary malformations can be seen in patients with hereditary hemorrhagic telangiectasia (Osler-Rendu-Weber) syndrome. If bleeding does occur, it rarely produces mass effect or significant symptoms. No treatment options exist.

1	Cavernous angiomas are tufts of capillary sinusoids that form within the deep hemispheric white matter and brainstem with no normal intervening neural structures. The pathogenesis is unclear. Familial cavernous angiomas have been mapped to several different genes: KRIT1, CCM2, and PDCD10. Both KRIT1 and CCM2 have roles in blood vessel formation, whereas PDCD10 is an apoptotic gene. Cavernous angiomas are typically <1 cm in diameter and are often associated with a venous anomaly. Bleeding is usually of small volume, causing slight mass effect only. The bleeding risk for single cavernous malformations is 0.7–1.5% per year and may be higher for patients with prior clinical hemorrhage or multiple malformations. Seizures may occur if the malformation is located near the cerebral cortex. Surgical resection eliminates bleeding risk and may reduce seizure risk, but it is usually reserved for those malformations that form near the brain surface. Radiation treatment has not been shown to be of

1	resection eliminates bleeding risk and may reduce seizure risk, but it is usually reserved for those malformations that form near the brain surface. Radiation treatment has not been shown to be of benefit.

1	Dural arteriovenous fistulas are acquired connections usually from a dural artery to a dural sinus. Patients may complain of a pulse-synchronous cephalic bruit (“pulsatile tinnitus”) and headache. Depending on the magnitude of the shunt, venous pressures may rise high enough to cause cortical ischemia or venous hypertension and hemorrhage, particularly SAH. Surgical and endovascular techniques are usually curative. These fistulas may form because of trauma, but most are idiopathic. There is an association between fistulas and dural sinus thrombosis. Fistulas have been observed to appear months to years following venous sinus thrombosis, suggesting that angiogenesis factors elaborated from the thrombotic process may cause these anomalous connections to form. Alternatively, dural arteriovenous fistulas can produce venous sinus occlusion over time, perhaps from the high pressure and high flow through a venous structure. Peter J. Goadsby, Neil H. Raskin

1	Peter J. Goadsby, Neil H. Raskin The general principles around headache as a cardinal symptom are covered elsewhere (Chap. 21); here we discuss disorders in which headache and associated features occur in the absence of any exogenous cause. The most common are migraine, tension-type headache, and the trigeminal autonomic cephalalgias, notably cluster headache; the complete list is summarized in Table 447-1.

1	Migraine, the second most common cause of headache, and the most common headache-related, and indeed neurologic, cause of disability in the world, afflicts approximately 15% of women and 6% of men over a 1-year period. It is usually an episodic headache associated with certain features such as sensitivity to light, sound, or movement; nausea and vomiting often accompany the headache. A useful description of migraine is a recurring syndrome of headache associated with other symptoms of neurologic dysfunction in varying admixtures (Table 447-2). Migraine can often be recognized by its activators, referred to as triggers.

1	The brain of the migraineur is particularly sensitive to environmental and sensory stimuli; migraine-prone patients do not habituate easily to sensory stimuli. This sensitivity is amplified in females during the menstrual cycle. Headache can be initiated or amplified by various triggers, including glare, bright lights, sounds, or other afferent stimulation; hunger; let-down from stress; physical exertion; stormy weather or barometric pressure changes; hormonal fluctuations during menses; lack of or excess sleep; and alcohol or other chemical stimulation, such as with nitrates. Knowledge of a patient’s susceptibility to specific triggers can be useful in management strategies involving lifestyle adjustments. Pathogenesis The sensory sensitivity that is characteristic of migraine is probably due to dysfunction of monoaminergic sensory control systems located in the brainstem and hypothalamus (Fig. 447-1).

1	Activation of cells in the trigeminal nucleus results in the release of vasoactive neuropeptides, particularly calcitonin gene–related peptide primary heaDaChe DisorDers, moDifieD from iNterNatioNaL CLassifiCatioN of heaDaChe DisorDers-iii-Beta (heaDaChe CLassifiCatioN Committee of the iNterNatioNaL heaDaChe soCiety, 2013) 1. 2. 3. 4.

1	1.1 Migraine without aura 1.2 Migraine with aura 1.2.1 Migraine with typical aura 1.2.1.1 Typical aura with headache 1.2.1.2 Typical aura without headache 1.2.2 Migraine with brainstem aura 1.2.3 Hemiplegic migraine 1.2.3.1 Familial hemiplegic migraine (FHM) 1.2.3.1.1 Familial hemiplegic migraine type 1 1.2.3.1.2 Familial hemiplegic migraine type 2 1.2.3.1.3 Familial hemiplegic migraine type 3 1.2.3.2 Sporadic hemiplegic migraine 1.2.4 Retinal migraine 1.3 Chronic migraine 1.4 Complications of migraine 1.4.1 Status migrainosus 1.4.2 Persistent aura without infarction 1.4.3 Migrainous infarction 1.4.4 Migraine aura-triggered seizure 1.5 Probable migraine 1.5.1 Probable migraine without aura 1.5.2 Probable migraine with aura 1.6 Episodic syndromes that may be associated with migraine 1.6.1 Recurrent gastrointestinal disturbance 1.6.1.1 Cyclical vomiting syndrome 1.6.1.2 Abdominal migraine 1.6.2 Benign paroxysmal vertigo 1.6.3 2.1 2.2 Frequent episodic tension-type headache 2.3 3.1 3.1.1

1	gastrointestinal disturbance 1.6.1.1 Cyclical vomiting syndrome 1.6.1.2 Abdominal migraine 1.6.2 Benign paroxysmal vertigo 1.6.3 2.1 2.2 Frequent episodic tension-type headache 2.3 3.1 3.1.1 Episodic cluster headache 3.1.2 Chronic cluster headache 3.2 Paroxysmal hemicrania 3.2.1 Episodic paroxysmal hemicrania 3.2.2 Chronic paroxysmal hemicrania 3.3 Short-lasting unilateral neuralgiform headache attacks 3.3.1 Short-lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing (SUNCT) 3.3.2 Short-lasting unilateral neuralgiform headache attacks with cranial autonomic symptoms (SUNA) 3.4 4.1 4.2 Primary exercise headache 4.3 Primary headache associated with sexual activity 4.4 Primary thunderclap headache 4.5 Cold-stimulus headache 4.5.1 Headache attributed to external application of a cold stimulus 4.5.2 Headache attributed to ingestion or inhalation of a cold stimulus 4.6 External-pressure headache 4.6.1 External-compression headache 4.6.2 External-traction

1	application of a cold stimulus 4.5.2 Headache attributed to ingestion or inhalation of a cold stimulus 4.6 External-pressure headache 4.6.1 External-compression headache 4.6.2 External-traction headache 4.7 Primary stabbing headache 4.8 Nummular headache 4.9 Hypnic headache 4.10 New daily persistent headache (NDPH)

1	Symptom Patients Affected, % Source: From NH Raskin: Headache, 2nd ed. New York, Churchill Livingston, 1988; with permission. (CGRP), at vascular terminations of the trigeminal nerve and within the trigeminal nucleus. CGRP receptor antagonists, gepants, have now been shown to be effective in the acute treatment of migraine, and monoclonal antibodies to CGRP have been shown effective in two early phase clinical trials. Centrally, the second-order trigeminal neurons cross the midline and project to ventrobasal and posterior nuclei of the thalamus for further processing. Additionally, there are projections to the periaqueductal gray and hypothalamus, from which reciprocal descending systems have established antinociceptive effects. Other brainstem regions likely to be involved in descending modulation of trigeminal pain include the nucleus locus coeruleus in the pons and the rostroventromedial medulla.

1	Pharmacologic and other data point to the involvement of the neurotransmitter 5-hydroxytryptamine (5-HT; also known as serotonin) in migraines. Approximately 60 years ago, methysergide was found to antagonize certain peripheral actions of 5-HT and was introduced as the first drug capable of preventing migraine attacks. The triptans were designed to stimulate selectively subpopulations of 5-HT receptors; at least 14 different 5-HT receptors exist in humans. The triptans are potent agonists of 5-HT1B and 5-HT1D receptors, and some are active at the 5-HT1F receptors; the latter’s exclusive agonists are called ditans. Triptans arrest nerve signaling in the nociceptive pathways of the trigeminovascular system, at least in the trigeminal nucleus caudalis and trigeminal sensory thalamus, in addition to cranial vasoconstriction, while ditans, now shown conclusively to be effective in acute migraine, act only at neural targets. An interesting range of neural targets is now being actively

1	addition to cranial vasoconstriction, while ditans, now shown conclusively to be effective in acute migraine, act only at neural targets. An interesting range of neural targets is now being actively pursed for the acute and preventive management of migraine.

1	Data also support a role for dopamine in the pathophysiology of migraine. Most migraine symptoms can be induced by dopaminergic stimulation. Moreover, there is dopamine receptor hypersensitivity in migraineurs, as demonstrated by the induction of yawning, nausea, vomiting, hypotension, and other symptoms of a migraine attack by dopaminergic agonists at doses that do not affect nonmigraineurs. Dopamine receptor antagonists are effective therapeutic agents in migraine, especially when given parenterally or concurrently with other antimigraine agents. Moreover, hypothalamic activation, anterior to that seen in cluster headache, has now been shown in the premonitory phase of migraine using functional imaging, and this may hold a key to understanding some part of the role of dopamine in the disorder.

1	Dura Dorsal raphe nucleusLocuscoeruleusSuperior salivatory nucleus Magnus raphenucleusThalamusHypothalamusDorsal raphe nucleus Locus coeruleus Superior salivatory nucleus Magnus raphe nucleus Sphenopalatine ganglion Trigeminal ganglion TCC Thalamus Hypothalamus QuintothalamictractQuintothalamic tract FIGURE 447-1 Brainstem pathways that modulate sensory input. The key pathway for pain in migraine is the trigeminovascular input from the meningeal vessels, which passes through the trigeminal ganglion and synapses on second-order neurons in the trigeminocervical complex (TCC). These neurons in turn project in the quintothalamic tract and, after decussating in the brainstem, synapse on neurons in the thalamus. Important modulation of the trigeminovascular nociceptive input comes from the dorsal raphe nucleus, locus coeruleus, and nucleus raphe magnus.

1	Migraine genes identified by studying families with familial hemiplegic migraine (FHM) reveal involvement of ion channels, suggesting that alterations in membrane excitability can predispose to migraine. Mutations involving the Cav2.1 (P/Q)–type voltage-gated calcium channel CACNA1A gene are now known to cause FHM 1; this mutation is responsible for about 50% of FHMs. Mutations in the Na+-K+ATPase ATP1A2 gene, designated FHM 2, are responsible for about 20% of FHMs. Mutations in the neuronal voltage-gated sodium channel SCN1A cause FHM 3. Functional neuroimaging has suggested that brainstem regions in migraine (Fig. 447-2) and the posterior hypothalamic gray matter region close to the human circadian pacemaker cells of the suprachiasmatic nucleus in cluster headache (Fig. 447-3) are good candidates for specific involvement in primary headache.

1	Diagnosis and Clinical Features Diagnostic criteria for migraine headache are listed in Table 447-3. A high index of suspicion is required to diagnose migraine: the migraine aura, consisting of visual disturbances with flashing lights or zigzag lines moving across the visual field or of other neurologic symptoms, is reported in only 20–25% of patients. 2589

1	A headache diary can often be helpful in making the diagnosis; this is also helpful in assessing disability and the frequency of treatment for acute attacks. Patients with episodes of migraine that occur daily or near-daily are considered to have chronic migraine (see “Chronic Daily Headache” in Chap. 21). Migraine must be differentiated from tension-type headache (discussed below), the most common primary headache syndrome seen in the population. Migraine has several forms that have been defined (Table 447-1): migraine with and without aura and chronic migraine, the latter occurring 15 days or more a month, as the most important. Migraine at its most basic level is headache with associated features, and tension-type headache is headache that is featureless. Most patients with disabling headache probably have migraine.

1	Patients with acephalgic migraine (typical aura without headache, 1.2.1.2 in Table 447-1) experience recurrent neurologic symptoms, often with nausea or vomiting, but with little or no headache. Vertigo can be prominent; it has been estimated that one-third of patients referred for vertigo or dizziness have a primary diagnosis of migraine. Migraine aura can have prominent brainstem symptoms, and the terms basilar artery and basilar-type migraine have now been replaced by migraine with brainstem aura (Table 447-1).

1	FIGURE 447-2 Positron emission tomography (PET) activation in migraine. Hypothalamic, dorsal midbrain, and dorsolateral pontine activation is seen in triggered attacks in the premonitory phase before pain, whereas in migraine attacks, dorsolateral pontine activation persists, as it does in chronic migraine (not shown). The dorsolateral pontine area, which includes the noradrenergic locus coeruleus, is fundamental to the expression of migraine. Moreover, lateralization of changes in this region of the brainstem correlates with lateralization of the head pain in hemicranial migraine; the scans shown in panels C and D are of patients with acute migraine headache on the right and left side, respectively. (Panel A from FH Maniyar et al: Brain 137:232, 2014; panel B from SK Afridi et al: Arch Neurol 2005;62:1270; Panels C and D from SK Afridi et al: Brain 128:932, 2005.)

1	(Panel A from FH Maniyar et al: Brain 137:232, 2014; panel B from SK Afridi et al: Arch Neurol 2005;62:1270; Panels C and D from SK Afridi et al: Brain 128:932, 2005.) FIGURE 447-3 A. Posterior hypothalamic gray matter activation by positron emission tomography in a patient with acute cluster headache. (From A May et al: Lancet 352:275, 1998.) B. High-resolution T1-weighted magnetic resonance image obtained using voxel-based morphometry demonstrates increased gray matter activity, lateralized to the side of pain in a patient with cluster headache. (From A May et al: Nat Med 5:836, 1999.) Once a diagnosis of migraine has been established, it is important to assess the extent of a patient’s disease and disability. The Migraine Disability Assessment Score (MIDAS) is a well-validated, easy-to-use tool (Fig. 447-4).

1	Patient education is an important aspect of migraine management. Information for patients is available at sites such as www.achenet.org, the website of the American Council for Headache Education (ACHE). It is helpful for patients to understand that migraine is an inherited tendency to headache; that migraine can be modified and controlled by lifestyle adjustments and medications, but it cannot be eradicated; and that, except in some occasions in women on oral estrogens or contraceptives, migraine is not associated with serious or life-threatening illnesses. Migraine can often be managed to some degree by a variety of nonpharmacologic approaches. Most patients benefit by the identification and avoidance of specific headache triggers. A regulated lifestyle is helpful, including a healthy diet, regular exercise, regular sleep patterns, avoidance of excess caffeine and alcohol, and avoidance of acute changes in stress levels, being particularly wary of the let-down effect.

1	The measures that benefit a given individual should be used routinely because they provide a simple, cost-effective approach to migraine management. Patients with migraine do not encounter more stress than headache-free individuals; over-responsiveness to changes in stress appears to be the issue. Because the stresses of everyday living cannot be eliminated, lessening one’s response to stress by various techniques is helpful for many patients. These may Repeated attacks of headache lasting 4–72 h in patients with a normal physical examination, no other reasonable cause for the headache, and: At Least 2 of the Plus at Least 1 of the Following Features: Following Features: Source: Adapted from the International Headache Society Classification (Headache Classification Committee of the International Headache Society, 2013).

1	Source: Adapted from the International Headache Society Classification (Headache Classification Committee of the International Headache Society, 2013). include yoga, transcendental meditation, hypnosis, and conditioning techniques such as biofeedback. For most patients, this approach is, at best, an adjunct to pharmacotherapy. Nonpharmacologic measures are unlikely to prevent all migraine attacks. If these measures fail to prevent an attack, pharmacologic approaches are then needed to abort an attack.

1	The mainstay of pharmacologic therapy is the judicious use of one or more of the many medicines that are effective in migraine (Table 447-4). The selection of the optimal regimen for a given patient depends on a number of factors, the most important of which is the severity of the attack. Mild migraine attacks can usually be managed by oral agents; the average efficacy rate is 50–70%. Severe migraine attacks may require parenteral therapy. Most drugs effective in the treatment of migraine are members of one of three major pharmacologic classes: nonsteroidal anti-inflammatory drugs, 5-HT receptor agonists, and dopamine receptor antagonists.

1	In general, an adequate dose of whichever agent is chosen should be used as soon as possible after the onset of an attack. If additional medication is required within 60 min because symptoms return or have not abated, the initial dose should be increased for subsequent attacks or a different class of drug tried as first-line treatment. Migraine therapy must be individualized; a standard approach for all patients is not possible. A therapeutic regimen may need to be constantly refined until one is identified that provides the patient with rapid, complete, and consistent relief with minimal side effects (Table 447-5).

1	Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) Both the severity and duration of a migraine attack can be reduced significantly by NSAIDs (Table 447-4). Indeed, many undiagnosed migraineurs self-treat with nonprescription NSAIDs. A general consensus is that NSAIDs are most effective when taken early in the migraine attack. However, the effectiveness of these agents in migraine is usually less than optimal in moderate or severe migraine attacks. The combination of acetaminophen, aspirin, and caffeine has been approved for use by the U.S. Food and Drug Administration (FDA) for the treatment of mild to moderate migraine. The combination of aspirin and metoclopramide has been shown to be comparable to a single dose of oral sumatriptan. Important side effects of NSAIDs include dyspepsia and gastrointestinal irritation. Oral Stimulation of 5-HT receptors can stop an acute migraine attack. Ergotamine and dihydroergotamine are nonselective receptor

1	Oral Stimulation of 5-HT receptors can stop an acute migraine attack. Ergotamine and dihydroergotamine are nonselective receptor INSTRUCTIONS: Please answer the following questions about ALL headaches you have had FIGURE 447-4 The Migraine Disability Assessment Score (MIDAS) Questionnaire. agonists, whereas the triptans are selective 5-HT receptor ago nists. A variety of triptans, 5-HT receptor agonists—sumatriptan, almotriptan, eletriptan, frovatriptan, naratriptan, rizatriptan, and zolmitriptan—are now available for the treatment of migraine.

1	Each drug in the triptan class has similar pharmacologic properties but varies slightly in terms of clinical efficacy. Rizatriptan and eletriptan are the most efficacious of the triptans currently available in the United States. Sumatriptan and zolmitriptan have similar rates of efficacy as well as time to onset, with an advantage of having multiple formulations, whereas almotriptan has a similar rate of efficacy to sumatriptan and is better tolerated, and frovatriptan and naratriptan are somewhat slower in onset and are better tolerated. Clinical efficacy appears to be related more to the t max (time to peak plasma level) than to the potency, half-life, or bioavailability. This observation is consistent with a large body of data indicating that faster-acting analgesics are more effective than slower-acting agents.

1	Unfortunately, monotherapy with a selective oral 5-HT recep tor agonist does not result in rapid, consistent, and complete relief of migraine in all patients. Triptans are generally not effective in migraine with aura unless given after the aura is completed and the headache initiated. Side effects are common, although often mild and transient. Moreover, 5-HT receptor agonists are contraindi cated in individuals with a history of cardiovascular and cerebrovascular disease. Recurrence of headache, within usual time course of an attack, is another important limitation of triptan use and occurs at least occasionally in most patients. Evidence from randomized controlled trials show that coadministration of a longer-acting NSAID, naproxen 500 mg, with sumatriptan will augment the initial effect of sumatriptan and, importantly, reduce rates of headache recurrence.

1	Ergotamine preparations offer a nonselective means of stimulating 5-HT1 receptors. A nonnauseating dose of ergotamine should be sought because a dose that provokes nausea is too high and may intensify head pain. Except for a sublingual formulation of ergotamine, oral formulations of ergotamine also contain 100 mg caffeine (theoretically to enhance ergotamine absorption and possibly to add additional analgesic activity). The average oral ergotamine dose for a migraine attack is 2 mg. Because the clinical studies demonstrating the efficacy of ergotamine in migraine predated the clinical trial methodologies used with the triptans, it is difficult to assess the clinical efficacy of ergotamine versus the triptans. In general, ergotamine appears to have a much higher incidence of nausea than triptans but less headache recurrence.

1	Nasal Nasal formulations of dihydroergotamine (Migranal), zolmitriptan (Zomig nasal), or sumatriptan can be useful in patients requiring a nonoral route of administration. The nasal sprays result in substantial blood levels within 30–60 min. Although in theory nasal sprays might provide faster and more effective relief of a migraine attack than oral formulations, their reported efficacy is only approximately 50–60%. Studies with a new inhalational formulation of dihydroergotamine indicate that its absorption problems can be overcome to produce rapid onset of action with good tolerability.

1	Parenteral Administration of drugs by injection, such as dihydroergotamine and sumatriptan, is approved by the FDA for the rapid relief of a migraine attack. Peak plasma levels of dihydroergotamine are achieved 3 min after IV dosing, 30 min after IM dosing, and 45 min after SC dosing. If an attack has not already peaked, SC or IM administration of 1 mg of dihydroergotamine suffices for about 80–90% of patients. Sumatriptan, 4–6 mg SC, is effective in ~50–80% of patients, and can now be administered by a needle-free device.

1	DOPAMINE RECEPTOR ANTAGONISTS Oral Oral dopamine receptor antagonists can be considered as adjunctive therapy in migraine. Drug absorption is impaired during migraine because of reduced gastrointestinal motility. Delayed absorption occurs even in the absence of nausea and is related to the severity of the attack and not its duration. Therefore, when oral NSAIDs and/or triptan agents fail, the addition of a dopamine receptor antagonist, such as metoclopramide 10 mg or domperidone 10 mg (not available in the United States), should be considered to aNot all drugs are specifically indicated by the FDA for migraine. Local regulations and guidelines should be consulted. Note: Antiemetics (e.g., domperidone 10 mg or ondansetron 4 or 8 mg) or prokinetics (e.g., metoclopramide 10 mg) are sometimes useful adjuncts. Abbreviations: 5-HT, 5-hydroxytryptamine; NSAIDs, nonsteroidal anti-inflammatory drugs.

1	enhance gastric absorption. In addition, dopamine receptor antago-the treatment of severe migraine is the administration over 2 min of a nists decrease nausea/vomiting and restore normal gastric motility. mixture of 5 mg of prochlorperazine and 0.5 mg of dihydroergotamine. Parenteral Dopamine receptor antagonists (e.g., chlorpromazine, prochlorperazine, metoclopramide) by injection can also provide sig-OTHER MEDICATIONS FOR ACUTE MIGRAINE nificant acute relief of migraine; they can be used in combination with Oral The combination of acetaminophen, dichloralphenazone, and parenteral 5-HT receptor agonists. A common IV protocol used for isometheptene, one to two capsules, has been classified by the FDA Almotriptan 12.5 mg PO Zolmitriptan 2.5 mg PO Naratriptan 2.5 mg PO Frovatriptan 2.5 mg PO usually with caffeine) Naratriptan 2.5 mg PO Almotriptan 12.5 mg PO Tolerating acute treatments poorly Naratriptan 2.5 mg Almotriptan 12.5 mg

1	Naratriptan 2.5 mg PO Frovatriptan 2.5 mg PO usually with caffeine) Naratriptan 2.5 mg PO Almotriptan 12.5 mg PO Tolerating acute treatments poorly Naratriptan 2.5 mg Almotriptan 12.5 mg Dihydroergotamine 1 mg IM Abbreviation: NSAIDs, nonsteroidal anti-inflammatory drugs. as “possibly” effective in the treatment of migraine. Because the clinical studies demonstrating the efficacy of this combination analgesic in migraine predated the clinical trial methodologies used with the triptans, it is difficult to compare the efficacy of this sympathomimetic compound to other agents. Nasal A nasal preparation of butorphanol is available for the treatment of acute pain. As with all opioids, the use of nasal butorphanol has little role in migraine treatment.

1	Parenteral Opioids are modestly effective in the acute treatment of migraine. For example, IV meperidine (50–100 mg) is given frequently in the emergency room. This regimen “works” in the sense that the pain of migraine is eliminated. However, this regimen is clearly suboptimal for patients with recurrent headache. Opioids do not treat the underlying headache mechanism; rather, they act to alter the pain sensation, and there is evidence their use may decrease the likelihood of a response to triptans in the future. Moreover, in patients taking oral opioids, such as oxycodone or hydrocodone, habituation or addiction can greatly confuse the treatment of migraine. Opioid craving and/or withdrawal can aggravate and accentuate migraine. Therefore, it is recommended that opioid use in migraine be limited to patients with severe, but infrequent, headaches that are unresponsive to to other therapies.

1	Acute attack medications, particularly opioid or barbiturate-containing compound analgesics, have a propensity to aggravate headache frequency and induce a state of refractory daily or near-daily headache called medication-overuse headache. This condition is likely not a separate headache entity but a reaction of the migraine patient to a particular medicine. Migraine patients who have two or more headache days a week should be cautioned about frequent analgesic use (see “Chronic Daily Headache” in Chap. 21).

1	Patients with an increasing frequency of migraine attacks or with attacks that are either unresponsive or poorly responsive to abortive treatments are good candidates for preventive agents. In general, a preventive medication should be considered in the subset of patients with four or more attacks a month. Significant side effects are associated with the use of many of these agents; furthermore, determination of dose can be difficult because the recommended doses have been derived for conditions other than migraine. The mechanism of action of these drugs is unclear; it seems likely that the brain sensitivity that underlies migraine is modified. Patients are usually started on a low dose of a chosen treatment; the dose is then gradually increased, up to a reasonable maximum, to achieve clinical benefit.

1	Drugs that have the capacity to stabilize migraine are listed in Table 447-6. Drugs must be taken daily, and there is usually a lag of between 2 to 12 weeks before an effect is seen. The drugs that have been approved by the FDA for the prophylactic treatment of migraine include propranolol, timolol, sodium valproate, topiramate, and methysergide (not available). In addition, a number of other drugs appear to display prophylactic efficacy. This group includes amitriptyline, nortriptyline, flunarizine, phenelzine, gabapentin, and cyproheptadine. Placebo-controlled trials of onabotulinum toxin type A in episodic migraine were negative, whereas, overall, placebo-controlled trials in chronic migraine were positive. Phenelzine and methysergide are usually reserved for recalcitrant cases because of their serious potential side effects. Phenelzine is a monoamine oxidase inhibitor (MAOI); therefore, tyramine-containing foods, decongestants, and meperidine are contraindicated. Methysergide may

1	of their serious potential side effects. Phenelzine is a monoamine oxidase inhibitor (MAOI); therefore, tyramine-containing foods, decongestants, and meperidine are contraindicated. Methysergide may cause retroperitoneal or cardiac valvular fibrosis when it is used for >6 months, and thus monitoring is required for patients using this drug; the risk of fibrosis is about 1:1500 and is likely to reverse after the drug is stopped.

1	The probability of success with any one of the antimigraine drugs is 50–75%. Many patients are managed adequately with low-dose amitriptyline, propranolol, candesartan, topiramate, or valproate. If these agents fail or lead to unacceptable side effects, second-line agents such as methysergide or phenelzine can be used. Once effective stabilization is achieved, the drug is continued for ~6 months and then slowly tapered to assess the continued need. Many patients are able to discontinue medication and experience fewer and milder attacks for long periods, suggesting that these drugs may alter the natural history of migraine.

1	TENSION-TYPE HEADACHE Clinical Features The term tension-type headache (TTH) is commonly used to describe a chronic head-pain syndrome characterized by bilateral tight, band-like discomfort. The pain typically builds slowly, fluctuates in severity, and may persist more or less continuously for many days. The headache may be episodic or chronic (present >15 days per month). A useful clinical approach is to diagnose TTH in patients whose headaches are completely without accompanying features such as nausea, vomiting, photophobia, phonophobia, osmophobia, throbbing, and aggravation with movement. Such an approach neatly separates reuptake inhibitors: fluoxetine aCommonly used preventives are listed with typical doses and common side effects. Not all listed medicines are approved by the U.S. Food and Drug Administration; local regulations and guidelines should be consulted. bNot available in the United States. cNot currently available worldwide.

1	migraine, which has one or more of these features and is the main differential diagnosis, from TTH. The International Headache Society’s main definition of TTH allows an admixture of nausea, photophobia, or phonophobia in various combinations, although the appendix definition does not; this illustrates the difficulty in distinguishing these two clinical entities. In clinical practice, dichotomizing patients on the basis of the presence of associated features (migraine) and the absence of associated features (TTH) is highly recommended. Indeed patients whose headaches fit the TTH phenotype and who have migraine at other times, along with a family history of migraine, migrainous illnesses of childhood, or typical migraine triggers to their migraine attacks, may be biologically different from those who have TTH headache with none of the features. TTH may be infrequent (episodic) or occur on 15 days or more a month (chronic).

1	Pathophysiology The pathophysiology of TTH is incompletely understood. It seems likely that TTH is due to a primary disorder of central nervous system pain modulation alone, unlike migraine, which involves a more generalized disturbance of sensory modulation. Data suggest a genetic contribution to TTH, but this may not be a valid finding: given the current diagnostic criteria, the studies undoubtedly included many migraine patients. The name tension-type headache implies that pain is a product of nervous tension, but there is no clear evidence for tension as an etiology. Muscle contraction has been considered to be a feature that distinguishes TTH from migraine, but there appear to be no differences in contraction between the two headache types.

1	The pain of TTH can generally be managed with simple analgesics such as acetaminophen, aspirin, or NSAIDs. Behavioral approaches including relaxation can also be effective. Clinical studies have demonstrated that triptans in pure TTH are not helpful, although triptans are effective in TTH when the patient also has migraine. For chronic TTH, amitriptyline is the only proven treatment (Table 447-6); other tricyclics, selective serotonin reuptake inhibitors, and the benzodiazepines have not been shown to be effective. There is no evidence for the efficacy of acupuncture. Placebo-controlled trials of onabotulinum toxin type A in chronic TTH were negative. TRIGEMINAL AUTONOMIC CEPHALALGIAS, INCLUDING CLUSTER HEADACHE

1	TRIGEMINAL AUTONOMIC CEPHALALGIAS, INCLUDING CLUSTER HEADACHE The trigeminal autonomic cephalalgias (TACs) describe a grouping of primary headaches including cluster headache, paroxysmal hemicrania, SUNCT (short-lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing)/SUNA (short-lasting unilateral neuralgiform headache attacks with cranial autonomic symptoms), and hemicrania continua (Table 447-1). TACs are characterized by relatively short-lasting attacks of head pain associated with cranial autonomic symptoms, such as lacrimation, conjunctival injection, or nasal congestion (Table 447-7). Pain is usually severe and may occur more than once a day. Because of the associated nasal congestion or rhinorrhea, patients are often misdiagnosed with “sinus headache” and treated with decongestants, which are ineffective.

1	TACs must be differentiated from short-lasting headaches that do not have prominent cranial autonomic syndromes, notably trigeminal neuralgia, primary stabbing headache, and hypnic headache. The cycling pattern and length, frequency, and timing of attacks are useful in classifying patients. Patients with TACs should undergo pituitary imaging and pituitary function tests because there is an excess of TAC presentations in patients with pituitary tumor–related headache.

1	Cluster Headache Cluster headache is a relatively rare form of primary headache with a population frequency of approximately 0.1%. The pain is deep, usually retroorbital, often excruciating in intensity, nonfluctuating, and explosive in quality. A core feature of cluster headache is periodicity. At least one of the daily attacks of pain recurs at about the same hour each day for the duration of a cluster bout. The typical cluster headache patient has daily bouts of one to two attacks of aIf conjunctival injection and tearing are not present, consider SUNA. bNausea, photophobia, or phonophobia; photophobia and phonophobia are typically unilateral on the side of the pain. cIndicates complete response to indomethacin. Abbreviations: SUNA, short-lasting unilateral neuralgiform headache attacks with cranial autonomic features; SUNCT, short-lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing.

1	relatively short-duration unilateral pain for 8 to 10 weeks a year; this is usually followed by a pain-free interval that averages a little less than 1 year. Cluster headache is characterized as chronic when there is less than 1 month of sustained remission without treatment. Patients are generally perfectly well between episodes. Onset is nocturnal in about 50% of patients, and men are affected three times more often than women. Patients with cluster headache tend to move about during attacks, pacing, rocking, or rubbing their head for relief; some may even become aggressive during attacks. This is in sharp contrast to patients with migraine, who prefer to remain motionless during attacks.

1	Cluster headache is associated with ipsilateral symptoms of cranial parasympathetic autonomic activation: conjunctival injection or lacrimation, rhinorrhea or nasal congestion, or cranial sympathetic dysfunction such as ptosis. The sympathetic deficit is peripheral and likely to be due to parasympathetic activation with injury to ascending sympathetic fibers surrounding a dilated carotid artery as it passes into the cranial cavity. When present, photophobia and phonophobia are far more likely to be unilateral and on the same side of the pain, rather than bilateral, as is seen in migraine. This phenomenon of unilateral photophobia/phonophobia is characteristic of TACs. Cluster headache is likely to be a disorder involving central pacemaker neurons in the posterior hypothalamic region (Fig. 447-3).

1	The most satisfactory treatment is the administration of drugs to prevent cluster attacks until the bout is over. However, treatment of acute attacks is required for all cluster headache patients at some time. Cluster headache attacks peak rapidly, and thus a treatment with quick onset is required. Many patients with acute cluster headache respond very well to oxygen inhalation. This should be given as 100% oxygen at 10–12 L/min for 15–20 min. It appears that high flow and high oxygen content are important. Sumatriptan 6 mg SC is rapid in onset and will usually shorten an attack to 10–15 min; there is no evidence of tachyphylaxis. Sumatriptan (20 mg) and zolmitriptan (5 mg) nasal sprays are both effective in acute cluster headache, offering a useful option for patients who may not wish to self-inject daily. Oral sumatriptan is not effective for prevention or for acute treatment of cluster headache.

1	The choice of a preventive treatment in cluster headache depends in part on the length of the bout. Patients with long bouts or those with chronic cluster headache require medicines that are safe when taken for long periods. For patients with relatively short bouts, limited courses of oral glucocorticoids or methysergide (not available in the United States) can be very useful. A 10-day course of prednisone, beginning at 60 mg daily for 7 days and followed by a rapid taper, may interrupt the pain bout for many patients. Lithium (400–800 mg/d) appears to be particularly useful for the chronic form of the disorder.

1	Many experts favor verapamil as the first-line preventive treatment for patients with chronic cluster headache or prolonged bouts. While verapamil compares favorably with lithium in practice, some patients require verapamil doses far in excess of those administered for cardiac disorders. The initial dose range is 40–80 mg twice daily; effective doses may be as high as 960 mg/d. Side effects such as constipation and leg swelling can be problematic. Of paramount concern, however, is the cardiovascular safety of verapamil, particularly Prednisone 1 mg/kg up to Verapamil 160–960 mg/d 60 mg qd, tapering over 21 days Gabapentinb 1200–3600 mg/d Melatoninb 9–12 mg/d aNot available worldwide. bUnproven but of potential benefit.

1	Prednisone 1 mg/kg up to Verapamil 160–960 mg/d 60 mg qd, tapering over 21 days Gabapentinb 1200–3600 mg/d Melatoninb 9–12 mg/d aNot available worldwide. bUnproven but of potential benefit. 2596 at high doses. Verapamil can cause heart block by slowing conduction in the atrioventricular node, a condition that can be monitored by following the PR interval on a standard electrocardiogram (ECG). Approximately 20% of patients treated with verapamil develop ECG abnormalities, which can be observed with doses as low as 240 mg/d; these abnormalities can worsen over time in patients on stable doses. A baseline ECG is recommended for all patients. The ECG is repeated 10 days after a dose change in patients whose dose is being increased above 240 mg daily. Dose increases are usually made in 80-mg increments. For patients on long-term verapamil, ECG monitoring every 6 months is advised.

1	When medical therapies fail in chronic cluster headache, neurostimulation strategies can be used. Deep-brain stimulation of the region of the posterior hypothalamic gray matter has proven successful in a substantial proportion of patients, although its risk-benefit ratio makes it inappropriate with so many other options now available. Favorable results have also been reported with the less-invasive approach of occipital nerve stimulation, with sphenopalatine ganglion stimulation and with a noninvasive vagal nerve stimulator.

1	Paroxysmal hemicrania (PH) is characterized by frequent unilateral, severe, short-lasting episodes of headache. Like cluster headache, the pain tends to be retroorbital but may be experienced all over the head and is associated with autonomic phenomena such as lacrimation and nasal congestion. Patients with remissions are said to have episodic PH, whereas those with the nonremitting form are said to have chronic PH. The essential features of PH are unilateral, very severe pain; short-lasting attacks (2–45 min); very frequent attacks (usually more than five a day); marked autonomic features ipsilateral to the pain; rapid course (<72 h); and excellent response to indomethacin. In contrast to cluster headache, which predominantly affects males, the male-tofemale ratio in PH is close to 1:1.

1	Indomethacin (25–75 mg tid), which can completely suppress attacks of PH, is the treatment of choice. Although therapy may be complicated by indomethacin-induced gastrointestinal side effects, currently there are no consistently effective alternatives. Topiramate is helpful in some cases. Piroxicam has been used, although it is not as effective as indomethacin. Verapamil, an effective treatment for cluster headache, does not appear to be useful for PH. In occasional patients, PH can coexist with trigeminal neuralgia (PH-tic syndrome); similar to cluster-tic syndrome, each component may require separate treatment.

1	Secondary PH has been reported with lesions in the region of the sella turcica, including arteriovenous malformation, cavernous sinus meningioma, pituitary pathology and epidermoid tumors. Secondary PH is more likely if the patient requires high doses (>200 mg/d) of indomethacin. In patients with apparent bilateral PH, raised cerebrospinal fluid (CSF) pressure should be suspected. It is important to note that indomethacin reduces CSF pressure. When a diagnosis of PH is considered, magnetic resonance imaging (MRI) is indicated to exclude a pituitary lesion.

1	SUNCT (short-lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing) is a rare primary headache syndrome characterized by severe, unilateral orbital or temporal pain that is stabbing or throbbing in quality. Diagnosis requires at least 20 attacks, lasting for 5–240 s; ipsilateral conjunctival injection and lacrimation should be present. In some patients, conjunctival injection or lacrimation is missing, and the diagnosis of SUNA (short-lasting unilateral neuralgiform headache attacks with cranial autonomic symptoms) can be made.

1	DiAgnoSiS The pain of SUNCT/SUNA is unilateral and may be located anywhere in the head. Three basic patterns can be seen: single stabs, which are usually short-lived; groups of stabs; or a longer attack comprising many stabs between which the pain does not completely resolve, thus giving a “saw-tooth” phenomenon with attacks lasting many minutes. Each pattern may be seen in the context of an underlying continuous head pain. Characteristics that lead to a suspected diagnosis of SUNCT are the cutaneous (or other) triggers of attacks, a lack of refractory period to triggering between attacks, and the lack of a response to indomethacin. Apart from trigeminal sensory disturbance, the neurologic examination is normal in primary SUNCT. The diagnosis of SUNCT/SUNA is often confused with trigeminal neuralgia (TN) particularly in first-division TN (Chap. 455). Minimal or no cranial autonomic symptoms and a clear refractory period to triggering indicate a diagnosis of TN.

1	SeconDAry (SymptomAtic) Sunct SUNCT can be seen with posterior fossa or pituitary lesions. All patients with SUNCT/SUNA should be evaluated with pituitary function tests and a brain MRI with pituitary views. Therapy of acute attacks is not a useful concept in SUNCT/SUNA because the attacks are of such short duration. However, IV lidocaine, which arrests the symptoms, can be used in hospitalized patients. Long-term prevention to minimize disability and hospitalization is the goal of treatment. The most effective treatment for prevention is lamotrigine, 200–400 mg/d. Topiramate and gabapentin may also be effective. Carbamazepine, 400–500 mg/d, has been reported by patients to offer modest benefit.

1	Surgical approaches such as microvascular decompression or destructive trigeminal procedures are seldom useful and often produce long-term complications. Greater occipital nerve injection has produced limited benefit in some patients. Occipital nerve stimulation is probably helpful in a subgroup of these patients. Complete control with deep-brain stimulation of the posterior hypothalamic region was reported in a single patient. For intractable cases, short-term prevention with IV lidocaine can be effective, as can occipital nerve stimulation.

1	Hemicrania Continua The essential features of hemicrania continua are moderate and continuous unilateral pain associated with fluctuations of severe pain; complete resolution of pain with indomethacin; and exacerbations that may be associated with autonomic features, including conjunctival injection, lacrimation, and photophobia on the affected side. The age of onset ranges from 11 to 58 years; women are affected twice as often as men. The cause is unknown.

1	Treatment consists of indomethacin; other NSAIDs appear to be of little or no benefit. The IM injection of 100 mg of indomethacin has been proposed as a diagnostic tool, and administration with a placebo injection in a blinded fashion can be very useful diagnostically. Alternatively, a trial of oral indomethacin, starting with 25 mg tid, then 50 mg tid, and then 75 mg tid, can be given. Up to 2 weeks at the maximal dose may be necessary to assess whether a dose has a useful effect. Topiramate can be helpful in some patients. Occipital nerve stimulation probably has a role in patients with hemicrania continua who are unable to tolerate indomethacin.

1	OTHER PRIMARY HEADACHES Primary Cough Headache Primary cough headache is a generalized headache that begins suddenly, lasts for several minutes, sometimes up to a few hours, and is precipitated by coughing; it is preventable by avoiding coughing or other precipitating events, which can include sneezing, straining, laughing, or stooping. In all patients with this syndrome, serious etiologies must be excluded before a diagnosis of “benign” primary cough headache can be established. A Chiari malformation or any lesion causing obstruction of CSF pathways or displacing cerebral structures can be the cause of the head pain. Other conditions that can present with cough or exertional headache as the initial symptom include cerebral aneurysm, carotid stenosis, and vertebrobasilar disease. Benign cough headache can resemble benign exertional headache (below), but patients with the former condition are typically older.

1	Indomethacin 25–50 mg two to three times daily is the treatment of choice. Some patients with cough headache obtain complete cessation of their attacks with lumbar puncture; this is a simple option when compared to prolonged use of indomethacin, and it is effective in about one-third of patients. The mechanism of this response is unclear. Primary Exercise Headache Primary exertional headache has features resembling both cough headache and migraine. It may be precipitated by any form of exercise; it often has the pulsatile quality of migraine. The pain, which can last from 5 min to 24 h, is bilateral and throbbing at onset; migrainous features may develop in patients susceptible to migraine. The duration tends to be shorter in adolescents than in older adults. Primary exertional headache can be prevented by avoiding excessive exertion, particularly in hot weather or at high altitude.

1	The mechanism of primary exertional headache is unclear. Acute venous distension likely explains one syndrome—the acute onset of headache with straining and breath holding, as in weightlifter’s headache. Because exertion can result in headache in a number of serious underlying conditions, these must be considered in patients with exertional headache. Pain from angina may be referred to the head, probably by central connections of vagal afferents, and may present as exertional headache (cardiac cephalgia). The link to exercise is the main clinical clue that headache is of cardiac origin. Pheochromocytoma may occasionally cause exertional headache. Intracranial lesions and stenosis of the carotid arteries are other possible etiologies.

1	Exercise regimens should begin modestly and progress gradually to higher levels of intensity. Indomethacin at daily doses from 25 to 150 mg is generally effective in benign exertional headache. Indomethacin (50 mg), ergotamine (1 mg orally), dihydroergotamine (2 mg by nasal spray), and methysergide (1–2 mg orally given 30–45 min before exercise) are useful prophylactic measures.

1	Primary Headache Associated with Sexual Activity Three types of sex headache are reported: a dull bilateral ache in the head and neck that intensifies as sexual excitement increases; a sudden, severe, explosive headache occurring at orgasm; and a postural headache developing after coitus that resembles the headache of low CSF pressure. The last arises from vigorous sexual activity and is a form of low CSF pressure headache (Chap. 21). Headaches developing at the time of orgasm are not always benign; 5–12% of cases of subarachnoid hemorrhage are precipitated by sexual intercourse. Sex headache is reported by men more often than women and may occur at any time during the years of sexual activity. It may develop on several occasions in succession and then not trouble the patient again, even without an obvious change in sexual activity. In patients who stop sexual activity when headache is first noticed, the pain may subside within a period of 5 min to 2 h. In about half of patients, sex

1	without an obvious change in sexual activity. In patients who stop sexual activity when headache is first noticed, the pain may subside within a period of 5 min to 2 h. In about half of patients, sex headache will subside within 6 months.

1	About half of patients with sex headache have a history of exertional 2597 headaches, but there is no excess of cough headache. Migraine is probably more common in patients with sex headache. Benign sex headaches recur irregularly and infrequently. Management can often be limited to reassurance and advice about ceasing sexual activity if a mild, warning headache develops. Propranolol can be used to prevent headache that recurs regularly or frequently, but the dosage required varies from 40 to 200 mg/d. An alternative is the calcium channel–blocking agent diltiazem, 60 mg tid. Ergotamine (1 mg) or indomethacin (25–50 mg) taken 30–45 min prior to sexual activity can also be helpful.

1	Primary Thunderclap Headache Sudden onset of severe headache may occur in the absence of any known provocation. The differential diagnosis includes the sentinel bleed of an intracranial aneurysm, cervicocephalic arterial dissection, and cerebral venous thrombosis. Headaches of explosive onset may also be caused by the ingestion of sympathomimetic drugs or of tyramine-containing foods in a patient who is taking MAOIs, or they may be a symptom of pheochromocytoma. Whether thunderclap headache can be the presentation of an unruptured cerebral aneurysm is uncertain. When neuroimaging studies and lumbar puncture exclude subarachnoid hemorrhage, patients with thunderclap headache usually do very well over the long term. In one study of patients whose computed tomography (CT) scans and CSF findings were negative, ~15% had recurrent episodes of thunderclap headache, and nearly half subsequently developed migraine or TTH.

1	The first presentation of any sudden-onset severe headache should be diligently investigated with neuroimaging (CT or, when possible, MRI with MR angiography) and CSF examination. Formal cerebral angiography should be reserved for those cases in which no primary diagnosis is forthcoming and for clinical situations that are particularly suggestive of intracranial aneurysm. Reversible segmental cerebral vasoconstriction may be seen in primary thunderclap headache without an intracranial aneurysm. In the presence of posterior leukoencephalopathy, the differential diagnosis includes cerebral angiitis, drug toxicity (cyclosporine, intrathecal methotrexate/cytarabine, pseudoephedrine, or cocaine), posttransfusion effects, and postpartum angiopathy. Treatment with nimodipine may be helpful, although by definition, the vasoconstriction of primary thunderclap headache resolves spontaneously.

1	Cold-Stimulus Headache This refers to head pain triggered by application or ingestion/inhalation of something cold. It is bought on quickly and typically resolves within 10–30 min of the stimulus being removed. It is best recognized as “brain-freeze” headache or ice-cream headache when due to ingestion. Although cold may be uncomfortable at some level for many people, it is the reliable, severe, and somewhat prolonged nature of these pains that set them apart. The transient receptor potential cation subfamily M member 8 (TRPM8) channel, a known cold temperature sensor, may be a mediator of this syndrome.

1	External Pressure Headache External pressure from compression or traction on the head can produce a pain that may have some generalized component, although the pain is largely focused around the site of the pressure. It typically resolves within an hour of the stimulus being removed. Examples of stimuli include helmets, swimming goggles, or very long ponytails. Treatment is to recognize the problem and remove the stimulus.

1	Primary Stabbing Headache The essential features of primary stabbing headache are stabbing pain confined to the head or, rarely, the face, lasting from 1 to many seconds or minutes and occurring as a alzheimer’s Disease and other Dementias William W. Seeley, Bruce L. Miller ALZHEIMER’S DISEASE Approximately 10% of all persons over the age of 70 years have sig-448 2598 single stab or a series of stabs; absence of associated cranial autonomic features; absence of cutaneous triggering of attacks; and a pattern of recurrence at irregular intervals (hours to days). The pains have been variously described as “ice-pick pains” or “jabs and jolts.” They are more common in patients with other primary headaches, such as migraine, the TACs, and hemicrania continua.

1	The response of primary stabbing headache to indomethacin (25–50 mg two to three times daily) is usually excellent. As a general rule, the symptoms wax and wane, and after a period of control on indomethacin, it is appropriate to withdraw treatment and observe the outcome. Nummular Headache Nummular headache is felt as a round or elliptical discomfort that is fixed in place, ranges in size from 1–6 cm, and may be continuous or intermittent. Uncommonly it may be multifocal. It may be episodic but is more often continuous during exacerbations. Accompanying the pain there may be a local sensory disturbance, such as allodynia or hypesthesia. Local dermatologic or bony lesions need to be excluded by examination and investigation. This condition can be difficult to treat; tricyclics, such as amitriptyline, or anticonvulsants, such as topiramate or valproate, are most often tried.

1	Hypnic Headache This headache syndrome typically begins a few hours after sleep onset. The headaches last from 15 to 30 min and are typically moderately severe and generalized, although they may be unilateral and can be throbbing. Patients may report falling back to sleep only to be awakened by a further attack a few hours later; up to three repetitions of this pattern occur through the night. Daytime naps can also precipitate head pain. Most patients are female, and the onset is usually after age 60 years. Headaches are bilateral in most, but may be unilateral. Photophobia, phonophobia, and nausea are usually absent. The major secondary consideration in this headache type is poorly controlled hypertension; 24-h blood pressure monitoring is recommended to detect this treatable condition.

1	Patients with hypnic headache generally respond to a bedtime dose of lithium carbonate (200–600 mg). For those intolerant of lithium, verapamil (160 mg) or methysergide (1–4 mg at bedtime) may be alternative strategies. One to two cups of coffee or caffeine, 60 mg orally, at bedtime may be effective in approximately one-third of patients. Case reports also suggest that flunarizine, 5 mg nightly, can be effective.

1	New Daily Persistent Headache Primary new daily persistent headache (NDPH) occurs in both males and females. It can be of the migrainous type, with features of migraine, or it can be featureless, appearing as new-onset TTH. Migrainous features are common and include unilateral headache and throbbing pain; each feature is present in about one-third of patients. Nausea, photophobia, and/or phonophobia occur in about half of patients. Some patients have a previous history of migraine; however, the proportion of NDPH sufferers with preexisting migraine is no greater than the frequency of migraine in the general population. At 24 months, ~86% of patients are headache-free. Treatment of migrainous-type primary NDPH consists of using the preventive therapies effective in migraine (see above). Featureless NDPH is one of the primary headache forms most refractory to treatment. Standard preventive therapies can be offered but are often ineffective. The secondary NDPHs are discussed elsewhere

1	Featureless NDPH is one of the primary headache forms most refractory to treatment. Standard preventive therapies can be offered but are often ineffective. The secondary NDPHs are discussed elsewhere (Chap. 21).

1	nificant memory loss, and in more than half, the cause is Alzheimer’s disease (AD). It is estimated that the median annual total cost of caring for a single patient with advanced AD is >$50,000, while the emotional toll for family members and caregivers is immeasurable. AD can manifest as young as the third decade, but it is the most common cause of dementia in the elderly. Patients most often present with an insidious loss of episodic memory followed by a slowly progressive dementia that evolves over years. In typical amnestic AD, brain imaging reveals atrophy that begins in the medial temporal lobes before spreading to lateral and medial parietal and temporal lobes and lateral frontal cortex. Microscopically, there are neuritic plaques containing amyloid beta (Aβ), neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau filaments, and Aβ accumulation of in blood vessel walls in cortex and leptomeninges (see “Pathology,” below). The identification of causative mutations

1	(NFTs) composed of hyperphosphorylated tau filaments, and Aβ accumulation of in blood vessel walls in cortex and leptomeninges (see “Pathology,” below). The identification of causative mutations and susceptibility genes for AD has provided a foundation for rapid progress in understanding the biological basis of the disorder. The major genetic risk for AD is apolipoprotein ε4 (Apo ε4). Carrying one Ε4 allele increases the risk for AD by 2to 3-fold, whereas two alleles increase the risk 16-fold.

1	The cognitive changes of AD tend to follow a characteristic pattern, beginning with memory impairment and progressing to language and visuospatial deficits. Yet, approximately 20% of patients with AD present with nonmemory complaints such as word-finding, organizational, or navigational difficulty. In other patients, upstream visual processing dysfunction (referred to as posterior cortical atrophy syndrome) or a progressive “logopenic” aphasia are the primary manifestations of AD for years before progressing to involve memory and other cognitive domains. Still other patients may present with an asymmetric akineticrigid-dystonic (“corticobasal”) syndrome or a dysexecutive “frontal variant” of AD.

1	In the early stages of typical amnestic AD, the memory loss may go unrecognized or be ascribed to benign forgetfulness of aging. Once the memory loss becomes noticeable to the patient and spouse and falls 1.5 standard deviations below normal on standardized memory tests, the term mild cognitive impairment (MCI) is applied. This construct provides useful prognostic information, because approximately 50% of patients with MCI (roughly 12% per year) will progress to AD over 4 years. Increasingly, the MCI construct is being replaced by the notion of “early symptomatic AD” to signify that AD is considered the underlying disease (based on clinical or biomarker evidence) in a patient who remains functionally compensated. Even earlier in the course, “prodromal AD” refers to a person with biomarker evidence of AD (amyloid imaging positive with positron emission tomography or low cerebrospinal Aβ42 and mildly elevated tau) in the absence of symptoms. These refinements have been developed in

1	evidence of AD (amyloid imaging positive with positron emission tomography or low cerebrospinal Aβ42 and mildly elevated tau) in the absence of symptoms. These refinements have been developed in anticipation of early-stage treatment and prevention trials that have already begun in humans. New evidence suggests that partial and sometimes generalized seizures herald AD and can occur even prior to dementia onset.

1	Eventually, with AD, the cognitive problems begin to interfere with daily activities, such as keeping track of finances, following instructions on the job, driving, shopping, and housekeeping. Some patients are unaware of these difficulties (anosognosia), but most remain acutely attuned to their deficits. Changes in environment (travel, relocation, hospitalization) tend to destabilize the patient. Over time patients become lost on walks or while driving. Social graces, routine behavior, and superficial conversation may be surprisingly intact, even into the later stages of the illness.

1	In the middle stages of AD, the patient is unable to work, is easily lost and confused, and requires daily supervision. Language becomes impaired—first naming, then comprehension, and finally fluency. Word-finding difficulties and circumlocution can be evident in the early stages, even when formal testing demonstrates intact naming and fluency. Apraxia emerges, and patients have trouble performing learned sequential motor tasks. Visuospatial deficits begin to interfere with dressing, eating, or even walking, and patients fail to solve simple puzzles or copy geometric figures. Simple calculations and clock reading become difficult in parallel.

1	In the late stages, some persons remain ambulatory, wandering aimlessly. Loss of judgment and reasoning is inevitable. Delusions are common, usually simple, with common themes of theft, infidelity, or misidentification. Approximately 10% of AD patients develop Capgras’ syndrome, believing that a caregiver has been replaced by an impostor. In contrast to dementia with Lewy bodies (DLB), where Capgras’ syndrome is an early feature, in AD this syndrome emerges late. Disinhibition and uncharacteristic belligerence may occur and alternate with passivity and withdrawal. Sleep-wake patterns are disrupted, and nighttime wandering becomes disturbing to the household. Some patients develop a shuffling gait with generalized muscle rigidity associated with slowness and awkwardness of movement. Patients often look parkinsonian (Chap. 449) but rarely have a high-amplitude, low-frequency tremor at rest. There is a strong overlap between Parkinson’s disease (PD) and AD, and some AD patients develop

1	often look parkinsonian (Chap. 449) but rarely have a high-amplitude, low-frequency tremor at rest. There is a strong overlap between Parkinson’s disease (PD) and AD, and some AD patients develop more classical PD features.

1	In the end stages, AD patients become rigid, mute, incontinent, and bedridden, and help is needed with eating, dressing, and toileting. Hyperactive tendon reflexes and myoclonic jerks (sudden brief contractions of various muscles or the whole body) may occur spontaneously or in response to physical or auditory stimulation. Often death results from malnutrition, secondary infections, pulmonary emboli, heart disease, or, most commonly, aspiration. The typical duration of AD is 8–10 years, but the course ranges from 1 to 25 years. For unknown reasons, some patients with AD show a steady decline in function while others have prolonged plateaus without major deterioration.

1	Early in the disease course, other etiologies of dementia should be excluded (see Tables 35-1, 35-3, and 35-4). Neuroimaging studies (computed tomography [CT] and magnetic resonance imaging [MRI]) do not show a single specific pattern with AD and may be normal early in the disease. As AD progresses, more distributed but usually posterior-predominant cortical atrophy becomes apparent, along with atrophy of the medial temporal memory structures (see Chap. 35, Fig. 35-1). The main purpose of imaging is to exclude other disorders, such as primary and secondary neoplasms, vascular dementia, diffuse white matter disease, and normal-pressure hydrocephalus (NPH). Imaging also helps to distinguish AD from other degenerative disorders, such as frontotemporal dementia (FTD) or Creutzfeldt-Jacob disease (CJD), which feature distinctive imaging patterns. Functional imaging studies, such as positron emission tomography (PET), reveal hypometabolism in the posterior temporal-parietal cortex in AD

1	(CJD), which feature distinctive imaging patterns. Functional imaging studies, such as positron emission tomography (PET), reveal hypometabolism in the posterior temporal-parietal cortex in AD (see Fig. 35-1). PET can also be used to detect the presence of fibrillar amyloid in the brain (see Fig. 35-4), and amyloid PET positivity is becoming required for entry into treatment trials for AD. Barriers to interpretation continue, however, to limit the use of amyloid PET in routine clinical evaluation. Although amyloid binding with PET is typical for AD, many asymptomatic healthy older individuals show amyloid uptake, and the likelihood that these individuals will convert to clinical AD is still under study. Similarly, dementia due to a non-AD disorder can be the underlying etiology in a patient who is amyloid positive on imaging. Electroencephalogram (EEG) is normal or shows nonspecific slowing; prolonged EEG can be used to seek out intermittent non-convulsive seizures. Routine spinal

1	who is amyloid positive on imaging. Electroencephalogram (EEG) is normal or shows nonspecific slowing; prolonged EEG can be used to seek out intermittent non-convulsive seizures. Routine spinal fluid examination is also normal. Cerebrospinal fluid (CSF) Aβ42 level is reduced, whereas the tau protein is elevated, but the test characteristics of these assays still make interpretation challenging in individual patients. Slowly progressive decline in memory and orientation, normal results on laboratory tests, and an MRI 2599 or CT scan showing only distributed or posteriorly predominant cortical and hippocampal atrophy are highly suggestive of AD. A clinical diagnosis of AD reached after careful evaluation is confirmed at autopsy about 90% of the time, with misdiagnosed cases usually representing one of the other dementing disorders described later in this chapter, a mixture of AD with vascular pathology, or DLB.

1	Simple clinical clues are useful in the differential diagnosis. Early prominent gait disturbance with only mild memory loss suggests vascular dementia or, rarely, NPH (see below). Resting tremor with stooped posture, bradykinesia, and masked facies suggest PD (Chap. 449). When dementia occurs after a well-established diagnosis of PD, PD dementia (PDD) is usually the correct diagnosis, but many patients with this diagnosis will show a mixture of AD and Lewy body disease at autopsy. The early appearance of parkinsonian features in association with fluctuating alertness, visual hallucinations, or delusional misidentification suggests DLB. Chronic alcoholism should prompt the search for vitamin deficiency. Loss of joint position and vibration sensibility accompanied by Babinski signs suggests vitamin B12 deficiency (Chap. 456). Early onset of a focal seizure suggests a metastatic or primary brain neoplasm (Chap. 118). Previous or ongoing depression raises suspicion for depression-related

1	vitamin B12 deficiency (Chap. 456). Early onset of a focal seizure suggests a metastatic or primary brain neoplasm (Chap. 118). Previous or ongoing depression raises suspicion for depression-related cognitive impairment, although AD can feature a depressive prodrome. A history of treatment for insomnia, anxiety, psychiatric disturbance, or epilepsy suggests chronic drug intoxication. Rapid progression over a few weeks or months associated with rigidity and myoclonus suggests CJD (Chap. 453e). Prominent behavioral changes with intact navigation and focal anterior-predominant atrophy on brain imaging are typical of FTD. A positive family history of dementia suggests either one of the familial forms of AD or one of the other genetic disorders associated with dementia, such as FTD (see below), HD (see below), prion disease (Chap. 453e), or rare hereditary ataxias (Chap. 450).

1	The most important risk factors for AD are old age and a positive family history. The prevalence of AD increases with each decade of adult life, reaching 20–40% of the population over the age of 85. A positive family history of dementia suggests a genetic contribution to AD, although autosomal dominant inheritance occurs in only 2% of patients. Female sex is a risk factor independent of the greater longevity of women, and women who carry an Apo ε4 allele are more susceptible than are male ε4 carriers. A history of head trauma with concussion increases the risk for AD. AD is more common in groups with low educational attainment, but education influences test-taking ability, and it is clear that AD can affect persons of all intellectual levels. One study found that the capacity to express complex written language in early adulthood correlated with a decreased risk for AD. Numerous environmental factors, including aluminum, mercury, and viruses, have been proposed as causes of AD, but

1	complex written language in early adulthood correlated with a decreased risk for AD. Numerous environmental factors, including aluminum, mercury, and viruses, have been proposed as causes of AD, but rigorous studies have failed to demonstrate to a significant role for any of these exposures. Similarly, several studies suggest that the use of nonsteroidal anti-inflammatory agents is associated with a decreased risk of AD, but this risk has not been confirmed in large prospective studies. Vascular disease, and stroke in particular, seems to lower the threshold for the clinical expression of AD. Also, in many patients with AD, amyloid angiopathy can lead to microhemorrhages, large lobar hemorrhages, ischemic infarctions most often in the subcortical white matter, or in rare cases an inflammatory leukoencephalopathy. Diabetes increases the risk of AD threefold. Elevated homocysteine and cholesterol levels; hypertension; diminished serum levels of folic acid; low dietary intake of fruits,

1	leukoencephalopathy. Diabetes increases the risk of AD threefold. Elevated homocysteine and cholesterol levels; hypertension; diminished serum levels of folic acid; low dietary intake of fruits, vegetables, and red wine; and low levels of exercise are all being explored as potential risk factors for AD.

1	At autopsy, the earliest and most severe degeneration is usually found in the medial temporal lobe (entorhinal/perirhinal cortex and hippocampus), lateral temporal cortex, and nucleus basalis of Meynert. The characteristic microscopic findings are neuritic plaques and NFTs (Fig. 448-1). These lesions may accumulate in small numbers during FIGURE 448-1 Neuropathology of Alzheimer’s disease. A. Early neurofibrillary degeneration, consisting of neurofibrillary tangles and neuropil threads, preferentially affects the medial temporal lobes, especially the stellate pyramidal neurons that compose the layer 2 islands of entorhinal cortex, as shown. B. Higher magnification view reveals the fibrillary nature of tangles (arrows) and the complex structure of neuritic plaques (arrowheads), whose major component is Aβ (inset shows immunohistochemistry for Aβ). Scale bars are 500 μM in A, 50 μM in B, and 20 μM in B inset.

1	normal brain aging but dominate the picture in AD. Increasing evidence suggests that soluble amyloid species called oligomers may cause cellular dysfunction and represent the early toxic molecule in AD. Eventually, further amyloid polymerization and fibril formation lead to neuritic plaques, which contain a central core of amyloid, proteoglycans, Apo ε4, α-antichymotrypsin, and other proteins. Aβ is a protein of 39–42 amino acids that is derived proteolytically from a larger transmembrane protein, amyloid precursor protein (APP), when APP is cleaved by β and γ secretases (Fig. 448-2). The normal Step 1: Cleavage by either ˜ or ˛ secretase Step 2: Cleavage by ˝ secretase

1	Step 1: Cleavage by either ˜ or ˛ secretase Step 2: Cleavage by ˝ secretase FIGURE 448-2 Amyloid precursor protein (APP) is catabolized by α, β, and γ secretases. A key initial step is the digestion by either β secretase (BASE) or α secretase (ADAM10 or ADAM17 [TACE]), producing smaller nontoxic products. Cleavage of the β secretase product by γ secretase (Step 2) results in either the toxic Aβ42 or the nontoxic Aβ40 peptide; cleavage of the α secretase product by γ secretase produces the nontoxic P3 peptide. Excess production of Aβ42 is a key initiator of cellular damage in Alzheimer’s disease (AD). Therapeutics for AD have focused on attempts to reduce accumulation of Aβ42 by antagonizing β or γ secretases, promoting α secretase, or clearing Aβ42 that has already formed by use of specific antibodies.

1	function of the Aβ peptides remains uncertain. APP has neurotrophic and neuroprotective properties. The plaque core is surrounded by a halo, which contains dystrophic, tau-immunoreactive neurites and activated microglia. The accumulation of Aβ in cerebral arterioles is termed amyloid angiopathy. NFTs are composed of silver-staining neuronal cytoplasmic fibrils composed of abnormally phosphorylated tau protein; they appear as paired helical filaments by electron microscopy. Tau binds to and stabilizes microtubules, supporting axonal transport of organelles, glycoproteins, neurotransmitters, and other important cargoes throughout the neuron. Once hyperphosphorylated, tau can no longer bind properly to microtubules and redistributes from the axon to throughout the neuronal cytoplasm and distal dendrites, compromising function. Finally, patients with AD often show comorbid DLB or vascular pathology. In animal models of AD, diminishing neuronal tau ameliorates the cognitive deficits and

1	dendrites, compromising function. Finally, patients with AD often show comorbid DLB or vascular pathology. In animal models of AD, diminishing neuronal tau ameliorates the cognitive deficits and seizures, even though Aβ42 continues to accumulate, raising hope for tau-lowering therapies in humans. Biochemically, AD is associated with a decrease in the cortical levels of several proteins and neurotransmitters, especially acetylcholine, its synthetic enzyme choline acetyltransferase, and nicotinic cholinergic receptors. Reduction of acetylcholine reflects degeneration of cholinergic neurons in the nucleus basalis of Meynert that project throughout the cortex. There is also noradrenergic and serotonergic depletion due to degeneration of brainstem nuclei such as the locus coeruleus and dorsal raphe, where tau-immunoreactive neuronal cytoplasmic inclusions can be identified even in individuals lacking entorhinal cortex NFTs.

1	Several genes play an important role in the pathogenesis of AD. One is the APP gene on chromosome 21. Adults with trisomy 21 (Down’s syndrome) consistently develop the typical neuropathologic hallmarks of AD if they survive beyond age 40 years, and many develop a progressive dementia superimposed on their baseline mental retardation. The extra dose of the APP gene on chromosome 21 is the initiating cause of AD in adult Down’s syndrome and results in excess cerebral amyloid production. Supporting this hypothesis, some families with early age-of-onset familial AD (FAD) have point mutations in APP. Although very rare, these families were the first examples of single-gene autosomal dominant transmission of AD.

1	Investigation of large families with multigenerational FAD led to the discovery of two additional AD-causing genes, the presenilins. Presenilin-1 (PS-1) is on chromosome 14 and encodes a protein called S182. Mutations in this gene cause an early-age-of-onset AD, with onset before the age of 60 and often before age 50, transmitted in an autosomal dominant, highly penetrant fashion. More than 100 different mutations have been found in the PS-1 gene in families from a wide range of ethnic backgrounds. Presenilin-2 (PS-2) is on chromosome 1 and encodes a protein called STM2. A mutation in the PS-2 gene was first found in a group of American families with Volga German ethnic background. Mutations in PS-1 are much more common than those in PS-2. The presenilins are highly homologous and encode similar proteins that at first appeared to have seven transmembrane domains (hence the designation STM), but subsequent studies have suggested eight such domains, with a ninth submembrane region. Both

1	proteins that at first appeared to have seven transmembrane domains (hence the designation STM), but subsequent studies have suggested eight such domains, with a ninth submembrane region. Both S182 and STM2 are cytoplasmic neuronal proteins that are widely expressed throughout the nervous system. They are homologous to a cell-trafficking protein, sel 12, found in the nematode Caenorhabditis elegans. Patients with mutations in the presenilin genes have elevated plasma levels of Aβ42, and PS-1 mutations produce increased Aβ42 in the media in cell culture. There is evidence that PS-1 is involved in the cleavage of APP at the γ secretase site and mutations in either gene (PS-1 or APP) may disturb γ secretase cleavage. Mutations in PS-1 are the most common cause of early-age-of-onset FAD, representing perhaps 40–70% of all cases. Mutations in PS-1 tend to produce AD with an earlier age of onset (mean onset 45 years) and a shorter, more rapidly progressive course (mean duration 6–7 years)

1	perhaps 40–70% of all cases. Mutations in PS-1 tend to produce AD with an earlier age of onset (mean onset 45 years) and a shorter, more rapidly progressive course (mean duration 6–7 years) than the disease caused by mutations in PS-2 (mean onset 53 years; duration 11 years). Although some carriers of PS-2 mutations have had onset of dementia after the age of 70, mutations in the presenilins rarely lead to late-ageof-onset AD. Clinical genetic testing for these uncommon mutations is available but likely to be revealing only in early-age-of-onset FAD and should be performed in association with formal genetic counseling.

1	The Apo ε gene on chromosome 19 is involved in the pathogenesis of AD. The protein, apolipoprotein E, participates in cholesterol transport (Chap. 421), and the gene has three alleles: ε2, ε3, and ε4. The Apo ε4 allele confers increased risk of AD in the general population, including sporadic and late-age-of-onset familial forms. Approximately 24–30% of the nondemented white population has at least one ε4 allele (12–15% allele frequency), and about 2% are ε4/ε4 homozygotes. Among patients with AD, 40–65% have at least one ε4 allele, a highly significant elevation compared with controls. Conversely, many AD patients have no ε4 allele, and ε4 carriers may never develop AD. Therefore, ε4 is neither necessary nor sufficient to cause AD. Nevertheless, the Apo ε4 allele represents the most important genetic risk factor for sporadic AD and acts as a dose-dependent disease modifier, with the earliest age of onset associated with the ε4 homozygosity. Precise mechanisms through which Apo ε4

1	genetic risk factor for sporadic AD and acts as a dose-dependent disease modifier, with the earliest age of onset associated with the ε4 homozygosity. Precise mechanisms through which Apo ε4 confers AD risk or hastens onset remain unclear, but ε4 leads to less efficient amyloid clearance and to the production of toxic fragments from cleavage of the molecule. Apo ε can be identified in neuritic plaques and may also be involved in neurofibrillary tangle formation, because it binds to tau protein. Apo ε4 decreases neurite outgrowth in dorsal root ganglion neuronal cultures, perhaps indicating a deleterious role in the brain’s response to injury. Some evidence suggests that the ε2 allele may reduce AD risk. Use of Apo ε testing in AD diagnosis remains controversial. It is not indicated as a predictive test in normal persons because its precise predictive value is unclear, and many individuals with the ε4 allele never develop dementia. Many cognitively normal ε4 heterozygotes and

1	as a predictive test in normal persons because its precise predictive value is unclear, and many individuals with the ε4 allele never develop dementia. Many cognitively normal ε4 heterozygotes and homozygotes show decreased cerebral cortical metabolic function with PET, suggesting presymptomatic abnormalities due to AD or an inherited vulnerability of the AD-targeted network. In demented persons who meet clinical criteria for AD, finding an ε4 allele increases the reliability of diagnosis; however, the absence of an ε4 allele cannot be considered evidence against AD. Furthermore, all patients with dementia, including those with an ε4 allele, require a search for reversible causes of their cognitive impairment. Nevertheless, Apo ε4 remains the single most important biologic marker associated with 2601 AD risk, and studies of ε4’s functional role and diagnostic utility are progressing rapidly. The ε4 allele is not associated with risk for FTD, DLB, or CJD, although some evidence

1	with 2601 AD risk, and studies of ε4’s functional role and diagnostic utility are progressing rapidly. The ε4 allele is not associated with risk for FTD, DLB, or CJD, although some evidence suggests that ε4 may exacerbate the phenotype of non-AD degenerative disorders, head trauma, and other brain injuries. Additional genes are also likely to be involved in AD, especially as minor risk alleles for sporadic forms of the disease. Genome-wide association studies have implicated the clusterin (CLU), phosphatidylinositol-binding clathrin assembly protein (PICALM), and complement component (3b/4b) receptor 1 (CR1) genes. CLU may play a role in synapse turnover, PICALM participates in clathrinmediated endocytosis, and CR1 may be involved in amyloid clearance through the complement pathway. TREM2 is a gene involved with inflammation that increases the likelihood of dementia. Homozygous mutation carriers develop a frontal dementia with bone cysts (Nasu-Hakola disease), whereas heterozygotes

1	is a gene involved with inflammation that increases the likelihood of dementia. Homozygous mutation carriers develop a frontal dementia with bone cysts (Nasu-Hakola disease), whereas heterozygotes are predisposed to the development of AD.

1	The management of AD is challenging and gratifying despite the absence of a cure or a robust pharmacologic treatment. The primary focus is on long-term amelioration of associated behavioral and neurologic problems, as well as providing caregiver support.

1	Building rapport with the patient, family members, and other caregivers is essential to successful management. In the early stages of AD, memory aids such as notebooks and posted daily reminders can be helpful. Family members should emphasize activities that are pleasant while curtailing those that increase stress on the patient. Kitchens, bathrooms, stairways, and bedrooms need to be made safe, and eventually patients will need to stop driving. Loss of independence and change of environment may worsen confusion, agitation, and anger. Communication and repeated calm reassurance are necessary. Caregiver “burnout” is common, often resulting in nursing home placement of the patient or new health problems for the caregiver. Respite breaks for the caregiver help to maintain a successful long-term therapeutic milieu. Use of adult day care centers can be helpful. Local and national support groups, such as the Alzheimer’s Association and the Family Caregiver Alliance, are valuable resources.

1	therapeutic milieu. Use of adult day care centers can be helpful. Local and national support groups, such as the Alzheimer’s Association and the Family Caregiver Alliance, are valuable resources. Internet access to these resources has become available to clinicians and families in recent years.

1	Donepezil (target dose, 10 mg daily), rivastigmine (target dose, 6 mg twice daily or 9.5-mg patch daily), galantamine (target dose 24 mg daily, extended-release), and memantine (target dose, 10 mg twice daily) are approved by the Food and Drug Administration (FDA) for the treatment of AD. Due to hepatotoxicity, tacrine is no longer used. Dose escalations for each of these medications must be carried out over 4–6 weeks to minimize side effects. The pharmacologic action of donepezil, rivastigmine, and galantamine is inhibition of the cholinesterases, primarily acetylcholinesterase, with a resulting increase in cerebral acetylcholine levels. Memantine appears to act by blocking overexcited N-methyl-D-aspartate (NMDA) glutamate receptors. Double-blind, placebo-controlled, crossover studies with cholinesterase inhibitors and memantine in moderate to severe AD have shown them to be associated with improved caregiver ratings of patients’ functioning and with an apparent decreased rate of

1	with cholinesterase inhibitors and memantine in moderate to severe AD have shown them to be associated with improved caregiver ratings of patients’ functioning and with an apparent decreased rate of decline in cognitive test scores over periods of up to 3 years. The average patient on an anticholinesterase inhibitor maintains his or her mini-mental state examination (MMSE) score for close to a year, whereas a placebo-treated patient declines 2–3 points over the same time period. Memantine, used in conjunction with cholinesterase inhibitors or by itself, slows cognitive deterioration and decreases caregiver burden for patients with moderate to severe AD but is not approved for mild AD. Each of these compounds has only modest efficacy for AD. Cholinesterase inhibitors are relatively easy to administer, and their major side effects are gastrointestinal symptoms (nausea, diarrhea, cramps), altered sleep with unpleasant or vivid dreams, bradycardia (usually benign), and muscle cramps.

1	2602 In a prospective observational study, the use of estrogen replacement therapy appeared to protect—by about 50%—against development of AD in women. This study seemed to confirm the results of two earlier case-controlled studies. Sadly, a prospective placebo-controlled study of a combined estrogen-progesterone therapy for asymptomatic postmenopausal women increased, rather than decreased, the prevalence of dementia. This study markedly dampened enthusiasm for hormonal treatments to prevent dementia. Additionally, no benefit has been found in the treatment of AD with estrogen alone. A controlled trial of an extract of Ginkgo biloba found modest improvement in cognitive function in subjects with AD and vascular dementia. Unfortunately, a comprehensive 6-year multicenter prevention study using ginkgo found no slowing of progression to dementia in the treated group. Vaccination against Aβ42 has proved highly efficacious in mouse models of AD, helping clear brain amyloid and preventing

1	using ginkgo found no slowing of progression to dementia in the treated group. Vaccination against Aβ42 has proved highly efficacious in mouse models of AD, helping clear brain amyloid and preventing further amyloid accumulation. In human trials, this approach led to life-threatening complications, including meningoencephalitis, in a minority of patients. Another experimental approach to AD treatment has been the use of β and γ secretase inhibitors that diminish the production of Aβ42, but the first two placebo-controlled trials of γ secretase inhibitors, tarenflurbil and semagacestat, were negative, and semagacestat may have accelerated cognitive decline compared to placebo. Passive immunization with monoclonal antibodies against Aβ42 has been tried in mild to moderate AD. These studies were negative, leading some to suggest that the patients treated were too advanced to respond to amyloid-lowering therapies. Therefore, new trials have started in asymptomatic individuals with mild

1	were negative, leading some to suggest that the patients treated were too advanced to respond to amyloid-lowering therapies. Therefore, new trials have started in asymptomatic individuals with mild AD, in asymptomatic autosomal dominant forms of AD, and in cognitively normal elderly who are amyloid positive with PET. Medications that modify tau phosphorylation and aggregation, including tau antibodies, are beginning to be studied as possible treatments for both AD and non-AD tau-related disorders including FTD and progressive supranuclear palsy. Several retrospective studies suggest that nonsteroidal anti-inflammatory agents and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) may have a protective effect on dementia if used prior to the onset of disease but do not influence clinically symptomatic AD. Finally, there is now a strong interest in the relationship between diabetes and AD, and insulin-regulating studies are being conducted. Mild to moderate

1	do not influence clinically symptomatic AD. Finally, there is now a strong interest in the relationship between diabetes and AD, and insulin-regulating studies are being conducted. Mild to moderate depression is common in the early stages of AD and may respond to antidepressants or cholinesterase inhibitors. Selective serotonin reuptake inhibitors (SSRIs) are commonly used due to their low anticholinergic side effects (for example, escitalopram, target dose 5–10 mg daily). Seizures can be treated with levetiracetam unless the patient had a different regimen that was effective prior to the onset of AD. Agitation, insomnia, hallucinations, and belligerence are especially troublesome characteristics of some AD patients, and these behaviors can lead to nursing home placement. The newer generation of atypical antipsychotics, such as risperidone, quetiapine, and olanzapine, are being used in low doses to treat these neuropsychiatric symptoms. The few controlled studies comparing drugs

1	of atypical antipsychotics, such as risperidone, quetiapine, and olanzapine, are being used in low doses to treat these neuropsychiatric symptoms. The few controlled studies comparing drugs against behavioral intervention in the treatment of agitation suggest mild efficacy with significant side effects related to sleep, gait, and cardiovascular complications, including an increased risk of death. All antipsychotics carry a black box FDA warning and should be used with caution in the demented elderly; however, careful, daily, nonpharmacologic behavior management is often not available, rendering medications necessary for some patients. Finally, medications with strong anticholinergic effects should be vigilantly avoided, including prescription and over-the-counter sleep aids (e.g., diphenhydramine) or incontinence therapies (e.g., oxybutynin).

1	Dementia associated with cerebrovascular disease can be divided into two general categories: multi-infarct dementia and diffuse white matter disease (also called leukoaraiosis, subcortical arteriosclerotic leukoencephalopathy, or Binswanger’s disease). Cerebrovascular disease appears to be a more common cause of dementia in Asia than in Europe and North America, perhaps due to the increased prevalence of intra-cranial atherosclerosis. Individuals who have had strokes may develop chronic cognitive deficits, commonly called multi-infarct dementia. The strokes may be large or small (sometimes lacunar) and usually involve several different brain regions. The occurrence of dementia depends partly on the total volume of damaged cortex. Patients typically report previous discrete episodes of sudden neurologic deterioration. Many patients with multi-infarct dementia have a history of hypertension, diabetes, coronary artery disease, or other manifestations of widespread atherosclerosis.

1	of sudden neurologic deterioration. Many patients with multi-infarct dementia have a history of hypertension, diabetes, coronary artery disease, or other manifestations of widespread atherosclerosis. Physical examination may show focal neurologic deficits such as hemiparesis, a unilateral Babinski sign, a visual field defect, or pseudobulbar palsy. Recurrent strokes result in a stepwise disease progression. Neuroimaging reveals multiple areas of infarction. Thus, the history and neuroimaging findings differentiate this condition from AD; however, both AD and multiple infarctions are common and sometimes co-occur. With normal aging, there is also an accumulation of amyloid in cerebral blood vessels, leading to a condition called cerebral amyloid angiopathy (without dementia), which predisposes older persons to lobar hemorrhage and brain microhemorrhages. AD patients appear to be at increased risk for amyloid angiopathy, and this association may explain some of the observed links

1	older persons to lobar hemorrhage and brain microhemorrhages. AD patients appear to be at increased risk for amyloid angiopathy, and this association may explain some of the observed links between AD and stroke.

1	Some individuals with dementia are discovered on MRI to have bilateral T2 signal hyperintensities in the subcortical white matter, termed diffuse white matter disease, often occurring in association with lacunar infarctions (see Fig. 35-2). The dementia may be insidious in onset and progress slowly, features that distinguish it from multi-infarct dementia, but other patients show a stepwise deterioration more typical of multi-infarct dementia. Early symptoms include mild confusion, apathy, anxiety, psychosis, and memory, spatial, or executive deficits. Marked difficulties in judgment and orientation and dependence on others for daily activities develop later. Euphoria, elation, depression, or aggressive behaviors are common as the disease progresses. Pyramidal and cerebellar signs may be present, and a gait disorder is seen in at least half of these patients. With advanced disease, urinary incontinence and dysarthria with or without other pseudobulbar features (e.g., dysphagia,

1	be present, and a gait disorder is seen in at least half of these patients. With advanced disease, urinary incontinence and dysarthria with or without other pseudobulbar features (e.g., dysphagia, emotional lability) are frequent. Seizures and myoclonic jerks appear in a minority of patients. Often, this disorder results from chronic ischemia due to occlusive disease of small, penetrating cerebral arteries and arterioles (microangiopathy). Any disease-causing stenosis of small cerebral vessels may be the critical underlying factor, although hypertension is the major cause. The term Binswanger’s disease should be used with caution, because it does not clearly identify a single entity.

1	Other rare causes of white matter disease also present with dementia, such as adult metachromatic leukodystrophy (arylsulfatase A deficiency) and progressive multifocal leukoencephalopathy (Chap. 164). A dominantly inherited form of white matter disease is known as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), discussed later in “Other Causes of Dementia.” Mitochondrial disorders can present with stroke-like episodes and can selectively injure basal ganglia or cortex. Many such patients show other findings suggestive of a neurologic or systemic disorder such as ophthalmoplegia, retinal degeneration, deafness, myopathy, neuropathy, or diabetes. Diagnosis is difficult, but serum or (especially) CSF levels of lactate and pyruvate may be abnormal, and biopsy of affected tissue, preferably muscle, may be diagnostic.

1	Treatment of vascular dementia must be focused on preventing new ischemic injury by stabilizing or removing the underlying causes, such as hypertension, diabetes, smoking, or lack of exercise. Recovery of lost cognitive function is not likely, although fluctuations with periods of improvement are common. Frontotemporal dementia (FTD) refers to a group of clinical syndromes united by underlying frontotemporal lobar degeneration (FTLD) pathology. FTD most often begins in the fifth to seventh decades and

1	FIGURE 448-3 Three major frontotemporal dementia (FTD) clinical syndromes. Coronal magnetic resonance imaging sections from representative patients with behavioral variant FTD (left), semantic dementia (center), and progressive nonfluent aphasia (right). Areas of early and severe atrophy in each syndrome are highlighted (white arrowheads). The behavioral variant features anterior cingulate and frontoinsular atrophy, spreading to orbital and dorsolateral prefrontal cortex. Semantic variant primary progressive aphasia (PPA) shows prominent temporopolar atrophy, more often on the left. Nonfluent/agrammatic variant PPA is associated with dominant frontal opercular and dorsal insula degeneration. is nearly as prevalent as AD in this age group. Early studies suggested that FTD may be more common in men than women, although more recent reports cast doubt on this finding. Although a family history of dementia is common, autosomal dominant inheritance is seen in only 10–20% of all FTD cases.

1	The clinical heterogeneity seen in familial and sporadic FTD is remarkable. Three core clinical syndromes have been described (Fig. 448-3). In the behavioral variant (bvFTD), the most common FTD syndrome, social and emotional systems dysfunction manifests as apathy, disinhibition, compulsivity, loss of empathy, and overeating, often but not always accompanied by deficits in executive control. Two forms of primary progressive aphasia (PPA), the semantic and nonfluent/agrammatic variants, are commonly due to FTLD and included under the FTD umbrella. In the semantic variant, patients slowly lose the ability to decode word, object, person-specific, and emotion meaning, whereas patients with the nonfluent/agrammatic variant develop profound inability to produce words, often with prominent motor speech impairment. Any of these three clinical syndromes, but most often bvFTD, may be accompanied by motor neuron disease (MND), in which case the term FTD-MND is applied. In addition, the

1	motor speech impairment. Any of these three clinical syndromes, but most often bvFTD, may be accompanied by motor neuron disease (MND), in which case the term FTD-MND is applied. In addition, the corticobasal syndrome (CBS) and progressive supranuclear palsy syndrome (PSP-S) can be considered part of the FTLD clinical spectrum. Furthermore, patients may evolve from any of the major syndromes described above to have prominent features of another syndrome.

1	Findings at the bedside are dictated by the anatomic localization of the disorder. Right hemisphere-predominant or symmetric anterior cingulate/medial prefrontal, orbital, and anterior insular degeneration predicts bvFTD. Patients with nonfluent/agrammatic PPA show left (dominant) frontal opercular and precentral gyrus degeneration, whereas left anterior temporal atrophy presents with semantic variant PPA. Visuoconstructive ability, arithmetic calculations, and navigation may remain normal late into any FTD syndrome. Many patients with nonfluent aphasia or bvFTD later develop PSP-S, as disease spreads into diencephalic and brainstem structures, or CBS-like features, as disease moves into dorsal and lateral perirolandic cortices.

1	The most common autosomal dominantly inherited mutations causing FTD involve the C9ORF72 (chromosome 9), GRN (chromosome 17), and MAPT (chromosome 17) genes. Hexanucleotide (GGGGCC) expansions in the noncoding portion of C9ORF72 are the most recently identified and represent the most common genetic cause of familial or sporadic FTD (usually presenting as bvFTD with or without MND) and amyotrophic lateral sclerosis (ALS). The expansion is associated with reduced C9ORF72 mRNA expression, nuclear mRNA foci containing transcribed portions of the expansion and other mRNAs, neuronal cytoplasmic inclusions containing dipeptide repeat proteins translated from the repeat mRNA, and transactive response DNA-binding protein of 43 kDa (TDP-43) neuronal cytoplasmic and glial inclusions. The pathogenic significance of these various features is a topic of vigorous investigation. MAPT mutations lead to a change in the alternate splicing of tau or cause loss of function in the tau molecule, thereby

1	significance of these various features is a topic of vigorous investigation. MAPT mutations lead to a change in the alternate splicing of tau or cause loss of function in the tau molecule, thereby altering microtubule binding. With GRN, mutations in the coding sequence of the gene encoding progranulin protein result in mRNA degradation due to nonsense-mediated decay, providing a rare example of an autosomal dominant mutation that leads to haploinsufficiency and leads to a ~50% reduction in circulating progranulin protein levels. Intriguingly, a patient with GRN mutations on both chromosomes was recently reported to develop neuronal ceroid lipofuscinosis, focusing investigators on the lysosome as a site of molecular dysfunction in GRN-related FTD. Progranulin is a growth factor that binds to tumor necrosis factor (TNF) receptors and participates in tissue repair and tumor growth. How progranulin mutations lead to FTD remains unknown, but the most likely mechanisms include lysosomal

1	to tumor necrosis factor (TNF) receptors and participates in tissue repair and tumor growth. How progranulin mutations lead to FTD remains unknown, but the most likely mechanisms include lysosomal dysfunction and enhanced neuroinflammation. Both MAPT and GRN mutations are associated with parkinsonian features, whereas ALS is rare. Infrequently, mutations in the valosin-containing protein (VCP, chromosome 9) and charged multivesicular body protein 2b (CHMP2b, chromosome 3) genes also lead to autosomal dominant familial FTD. Mutations in the TARDBP (encoding TDP-43) and FUS (encoding fused in sarcoma [FUS]) genes (see below) cause familial ALS, sometimes in association with an FTD syndrome, although a few patients presenting with FTD alone have been reported.

1	The gross pathologic hallmark of FTLD is a focal atrophy of frontal, insular, and/or temporal cortex, which can be visualized with neuroimaging studies (Fig. 448-3) and is often profound at autopsy. Despite the appearance of advanced disease, however, imaging studies suggest that atrophy often begins focally in one hemisphere before spreading to anatomically interconnected regions, including basal ganglia. Loss of cortical serotonergic innervation is seen in many patients. In contrast to AD, the cholinergic system is relatively spared in FTD, which accounts for the poor efficacy of acetylcholinesterase inhibitors in this group.

1	Although early studies suggested that 15–30% of patients with FTD showed underlying AD at autopsy, progressive refinement in clinical diagnosis has improved pathologic prediction accuracy, and most patients diagnosed with FTD at a dementia clinic with expertise in FTD will show underlying FTLD pathology. Microscopic findings seen across all patients with FTLD include gliosis, microvacuolation, and neuronal loss, but the disease is subtyped according to the protein composition of neuronal and glial inclusions, which contain either tau or TDP-43 in ~90% of patients, with the remaining ~10% showing inclusions containing FUS (Fig. 448-4).

1	The toxicity and spreading capacity of tau aggregates underlies the pathogenesis of many familial cases and is emerging as a key factor in sporadic tauopathies, although loss of tau microtubule stabilizing function may also play a role. TDP-43 and FUS, in contrast, are RNA/ DNA binding proteins whose roles in neuronal function are still being actively investigated, but one key role may be the chaperoning of Pick’s 3R tau FTDP-17 MAPTOther: CTE, AGD, MST, GGT CBD 4R tau PSP 4R tau aFTLD-U BIBD NIFID/ NIBD FUS NOS FUSType U (C9ORF72)(TARDBP)Type D VCPType A (PGRN)(C9ORF72)Type B (C9ORF72)Type C Alzheimer’s disease bvFTD svPPA nfvPPA FTD-MND CBS PSPS FTLD-tau FTLD-TDP* FTLD-FUS FTLD-3 CHMP2BFrontotemporal lobar degeneration (FTLD)

1	FIGURE 448-4 Frontotemporal dementia syndromes are united by underlying frontotemporal lobar degeneration pathology, which can be divided according to the presence of tau, TPD-43, or FUS-containing inclusions in neurons and glia. Correlations between clinical syndromes and major molecular classes are shown with colored shading. Despite improvements in clinical syndromic diagnosis, a small percentage of patients with some frontotemporal dementia syndromes will show Alzheimer’s disease neuropathology at autopsy (gray shading). aFTLD-U, atypical frontotemporal lobar degeneration with ubiquitin-positive inclusions; AGD, argyrophilic grain disease; BIBD, basophilic inclusion body disease; bvFTD, behavioral variant frontotemporal dementia; CBD, corticobasal degeneration; CBS, corticobasal syndrome; CTE, chronic traumatic encephalopathy; FTD-MND, frontotemporal dementia with motor neuron disease; FTDP-17, frontotemporal dementia with parkinsonism linked to chromosome 17; FUS, fused in

1	syndrome; CTE, chronic traumatic encephalopathy; FTD-MND, frontotemporal dementia with motor neuron disease; FTDP-17, frontotemporal dementia with parkinsonism linked to chromosome 17; FUS, fused in sarcoma; GGT, globular glial tauopathy; MST, multisystem tauopathy; nfvPPA, nonfluent/agrammatic variant primary progressive aphasia; NIBD, neurofilament inclusion body disease; NIFID, neuronal intermediate filament inclusion disease; PSP, progressive supranuclear palsy; PSPS, progressive supranuclear palsy syndrome; svPPA, semantic variant primary progressive aphasia; Type U, unclassifiable type.

1	mRNAs to the distal neuron for activity-dependent translation within dendritic spines. Because these proteins also form intracellular aggregates and produce similar anatomic progression, protein toxicity and spreading may also factor heavily in the pathogenesis of these FTLDTDP and FTLD-FUS. Increasingly, misfolded proteins in neurodegenerative disease are being recognized as having “prion-like” properties in that they can template the misfolding of their natively folded protein counterparts, a process that creates exponential amplification of protein misfolding within a cell and may promote transcellular and even transsynaptic protein propagation between cells. This hypothesis could provide a unifying explanation for the stereotypical patterns of disease spread observed in each syndrome (Chap. 444e).

1	Although the term Pick’s disease was once used to describe a progressive degenerative disorder characterized by selective involvement of the anterior frontal and temporal neocortex and pathologically by intraneuronal cytoplasmic inclusions (Pick bodies), it is now used only in reference to a specific FTLD-tau histopathologic entity. Classical Pick bodies are argyrophilic, staining positively with the Bielschowsky silver method (but not with the Gallyas method) and also with immunostaining for hyperphosphorylated tau. Recognition of the three FTLD major molecular classes has allowed delineation of distinct FTLD subtypes within each class. These subtypes, based on the morphology and distribution of the neuronal and glial inclusions (Fig. 448-5), account for the vast majority of patients, and some subtypes show strong clinical or genetic associations (Fig. 448-4). Despite this progress, available data do not allow reliable prediction of the underlying FTLD subtype, or even the major

1	and some subtypes show strong clinical or genetic associations (Fig. 448-4). Despite this progress, available data do not allow reliable prediction of the underlying FTLD subtype, or even the major molecular class, based on clinical features alone. Molecular PET imaging with ligands chosen to bind misfolded tau protein shows great promise and is already being applied to the study of patients with AD and FTD. Because FTLD-tau and FTLD-TDP account for 90% of FTLD patients, the ability to detect pathologic tau protein deposition in vivo will greatly improve prediction accuracy, especially when amyloid PET imaging is negative.

1	The burden on caregivers of patients with FTD is extremely high, especially when the illness disrupts core emotional and personality functions of the loved one. Treatment is symptomatic, and there are currently no therapies known to slow progression or improve symptoms. Many of the behaviors that may accompany FTD, such as depression, hyperorality, compulsions, and irritability, can be ameliorated with antidepressants, especially SSRIs. The co-association with motor disorders such as parkinsonism necessitates the careful use of antipsychotics, which can exacerbate this problem.

1	Progressive supranuclear palsy syndrome (PSP-S; also known as Steele-Richardson-Olszewski syndrome) is a degenerative disorder that involves the brainstem, basal ganglia, limbic structures, and selected areas of cortex. Clinically, PSP-S begins with falls and executive or subtle personality changes (such as mental rigidity, impulsivity, or apathy). Shortly thereafter, a progressive oculomotor syndrome ensues that begins with square wave jerks, followed by slowed saccades (vertical worse than horizontal) before resulting in progressive supranuclear ophthalmoparesis. Dysarthria, dysphagia, and symmetric axial rigidity can be prominent features that emerge at any point in the illness. A stiff, unstable posture with hyperextension of the neck and a slow, jerky, toppling gait are characteristic. Frequent unexplained and sometimes spectacular falls are common secondary to a combination of axial rigidity, inability to look down, and poor judgment. Even once patients have severely limited

1	Frequent unexplained and sometimes spectacular falls are common secondary to a combination of axial rigidity, inability to look down, and poor judgment. Even once patients have severely limited voluntary eye movements, they retain oculocephalic reflexes (demonstrated using a vertical doll’s head maneuver); thus, the oculomotor disorder is supranuclear. The dementia overlaps with bvFTD, featuring apathy, frontal-executive dysfunction, poor judgment, slowed thought processes, impaired verbal fluency, and difficulty with sequential actions and with shifting from one task to another. These features are common at presentation and often precede the motor syndrome. Some patients with a pathologic diagnosis of PSP begin with a nonfluent aphasia or motor speech disorder and progress to classical PSP-S. Response to l-dopa is limited or absent; no other treatments exist. Death occurs within 5–10 years of onset. Like Pick’s disease, increasingly the term PSP is used to refer to a specific

1	PSP-S. Response to l-dopa is limited or absent; no other treatments exist. Death occurs within 5–10 years of onset. Like Pick’s disease, increasingly the term PSP is used to refer to a specific histopathologic entity within the FTLD-tau class. In PSP, accumulation of hyperphosphorylated 4-repeat tau is seen within neurons and glia. Neuronal inclusions often take the form of NFTs, which may

1	FIGURE 448-5 Neuropathology in frontotemporal lobar degeneration (FTLD). FTLD-tau (A–C) and FTLD-TDP (D–F) account for over 90% of patients with FTLD, and immunohistochemistry reveals characteristic lesions in each of the major histopathologic subtypes within each class: small compact or crescentic neuronal cytoplasmic inclusions and short, then neuropil threads in FTLD-TDP, type A; (E) diffuse/granular neuronal cytoplasmic inclusions (with a relative paucity of neuropil threads) in FTLD-TDP, type B; and (F) long, tortuous dystrophic neurites in FTLD-TDP, type C. TDP can be seen within the nucleus in neurons lacking inclusions but mislocalizes to the cytoplasm and forms inclusions in FTLD-TDP. Immunostains are 3-repeat tau (A), phospho-tau (B and C), and TDP-43 (D–F). Sections are counterstained with hematoxylin. Scale bar applies to all panels and represents 50 μm in A, B, C, and E and 100 μm in D and F.

1	be large, spherical (“globose”), and coarse in brainstem, cerebellar dentate, and diencephalic neurons. Tau deposition is most prominent in subcortical structures (including the subthalamic nucleus, globus pallidus, substantia nigra, locus coeruleus, periaqueductal gray, tectum, oculomotor nuclei, and dentate nucleus of cerebellum). Neocortical NFTs, like those in AD, often take on a more flame-shaped morphology, but on electron microscopy, PSP tangles can be shown to consist of straight tubules rather than the paired helical filaments found in AD. Furthermore, PSP is associated with prominent tau-positive glial pathologies, such as tufted astrocytes (Fig. 448-5), thorny astrocytes, and coiled oligodendroglial inclusions (“coiled bodies”). Most patients with PSP-S show PSP at autopsy, although small numbers will show another tauopathy (corticobasal degeneration [CBD] or Pick’s disease; Fig. 448-4).

1	In addition to its overlap with FTD and CBS (see below), PSP is often confused with idiopathic Parkinson’s disease (PD). Although elderly patients with PD may have restricted upgaze, they do not develop downgaze paresis or other abnormalities of voluntary eye movements typical of PSP. Dementia occurs in ~20% of patients with PD, often due to the emergence of a full-blown DLB-like syndrome. Furthermore, the behavioral syndromes seen with DLB differ from PSP (see below). Dementia in PD becomes more likely with increasing age, increasing severity of extrapyramidal signs, long disease duration, and the presence of depression. Patients with PD who develop dementia also show cortical atrophy on brain imaging. Neuropathologically, there may be AD-related changes in the cortex, LBD-related α-synuclein inclusions in both the limbic system and cortex, or no specific microscopic changes other than gliosis and neuronal loss. PD is discussed in detail in Chap. 449.

1	Corticobasal syndrome (CBS) is a slowly progressive dementia-movement disorder associated with severe atrophy in perirolandic cortex and basal ganglia (substantia nigra and striatopallidum). Patients typically present with asymmetric onset of rigidity, dystonia, myoclonus, and apraxia of one limb, at times associated with alien limb phenomena in which the limb exhibits unintended motor actions such as grasping, groping, drifting, or undoing. Eventually CBS becomes bilateral and leads to dysarthria, slow gait, action tremor, and typically a frontal-predominant dementia. Whereas CBS refers to the clinical syndrome, CBD refers to a specific histopathologic FTLD-tau entity (Fig. 448-4). Although CBS was once thought to be pathognomonic for CBD, increasingly it has been recognize that CBS can be due to CBD, PSP, FTLD-TDP, or even AD. In CBD, the microscopic features include ballooned, achromatic, tau-positive neurons; astrocytic plaques (Fig. 448-5); and other dystrophic glial tau

1	CBS can be due to CBD, PSP, FTLD-TDP, or even AD. In CBD, the microscopic features include ballooned, achromatic, tau-positive neurons; astrocytic plaques (Fig. 448-5); and other dystrophic glial tau pathomorphologies that overlap with those seen in PSP. Most specifically, CBD features a severe tauopathy burden in the subcortical white matter, consisting of threads and oligodendroglial coiled bodies. As shown in Fig. 448-4, patients with bvFTD, nonfluent/agrammatic PPA, and PSP-S may also show CBD at autopsy, emphasizing the importance of distinguishing clinical and pathologic constructs and terminology. Treatment of CBS remains symptomatic; no disease-modifying therapies are available.

1	The parkinsonian dementia syndromes are under increasing study, with many cases unified by Lewy body and Lewy neurite pathology that ascends from the low brainstem up through the substantia nigra, 2606 limbic system, and cortex. The DLB clinical syndrome is characterized by visual hallucinations, parkinsonism, fluctuating alertness, falls, and often rapid eye movement (REM) sleep behavior disorder (RBD). Dementia can precede or follow the appearance of parkinsonism. Hence, one pathway occurs in patients with long-standing PD without cognitive impairment, who slowly develop a dementia that is associated with visual hallucinations and fluctuating alertness. When this occurs after an established diagnosis of PD, many use the term Parkinson’s disease dementia (PDD). In others, the dementia and neuropsychiatric syndrome precede or co-emerge with the parkinsonism, and this constellation is referred to as DLB. Both PDD and DLB may be accompanied or preceded by symptoms referable to brainstem

1	neuropsychiatric syndrome precede or co-emerge with the parkinsonism, and this constellation is referred to as DLB. Both PDD and DLB may be accompanied or preceded by symptoms referable to brainstem pathology below the substantia nigra including constipation, orthostatic lightheadedness, or RBD, and many researchers conceptualize these disorders as points on a spectrum of α-synuclein pathology. Patients with PDD and DLB are highly sensitive to metabolic perturbations, and in some patients, the first manifestation of illness is a delirium, often precipitated by an infection, new medicine, or other systemic disturbance. A hallucinatory delirium induced by l-dopa, prescribed for parkinsonian symptoms attributed to PD, may likewise provide the initial clue to a PDD or DLB diagnosis. Conversely, patients with mild cognitive deficits and hallucinations may receive typical or atypical antipsychotic medications, which induce profound parkinsonism at low doses due to a subclinical DLB-related

1	patients with mild cognitive deficits and hallucinations may receive typical or atypical antipsychotic medications, which induce profound parkinsonism at low doses due to a subclinical DLB-related nigral dopaminergic neuron loss. Even without an underlying precipitant, fluctuations can be marked in DLB, with episodic confusion or even stupor admixed with lucid intervals. Despite the fluctuating pattern, however, the core clinical features persist, unlike delirium, which resolves following correction of the inciting factor. Cognitively, DLB features relative preservation of memory but more severe visuospatial and executive deficits than seen in patients with early AD. The key neuropathologic feature in DLB is the presence of Lewy bodies and Lewy neurites throughout specific brainstem nuclei, substantia nigra, amygdala, cingulate gyrus, and, ultimately, the neocortex. Lewy bodies are intraneuronal cytoplasmic inclusions that stain with periodic acid–Schiff (PAS) and ubiquitin but are

1	substantia nigra, amygdala, cingulate gyrus, and, ultimately, the neocortex. Lewy bodies are intraneuronal cytoplasmic inclusions that stain with periodic acid–Schiff (PAS) and ubiquitin but are now identified with antibodies to the presynaptic protein, α-synuclein. Lewy bodies are composed of straight neurofilaments 7–20 nm long with surrounding amorphous material and contain epitopes recognized by antibodies against phosphorylated and nonphosphorylated neurofilament proteins, ubiquitin, and α-synuclein. Lewy bodies are typically found in the substantia nigra of patients with idiopathic PD, where they can be readily seen with hematoxylin-and-eosin staining. A profound cholinergic deficit, owing to basal forebrain and pedunculopontine nucleus involvement, is present in many patients with DLB and may be a factor responsible for the fluctuations, inattention, and visual hallucinations. Due to the frequent comorbidity with AD and the cholinergic deficit in DLB, cholinesterase inhibitors

1	DLB and may be a factor responsible for the fluctuations, inattention, and visual hallucinations. Due to the frequent comorbidity with AD and the cholinergic deficit in DLB, cholinesterase inhibitors often provide significant benefit, reducing hallucinosis, stabilizing delusional symptoms, and even helping with RBD in some patients. Exercise programs maximize motor function and protect against fall-related injury. Antidepressants are often necessary. Atypical antipsychotics may be required for psychosis but can worsen extrapyramidal syndromes, even at low doses, and increase risk of death. Patients with DLB are extremely sensitive to dopaminergic medications, which must be carefully titrated; tolerability may be improved by concomitant use of a cholinesterase inhibitor.

1	Prion diseases such as Creutzfeldt-Jakob disease (CJD) are rare neurodegenerative conditions (prevalence ~1 per million) that produce dementia. CJD is a rapidly progressive disorder associated with dementia, focal cortical signs, rigidity, and myoclonus, causing death <1 year after first symptoms appear. The rapidity of progression seen with CJD is uncommon in AD so that the distinction between the two disorders is usually straightforward. CBD and DLB, more rapid degenerative dementias with prominent movement abnormalities, are more likely to be mistaken for CJD. The differential diagnosis for CJD includes other rapidly progressive dementing conditions such as viral or bacterial encephalitides, Hashimoto’s encephalopathy, central nervous system (CNS) vasculitis, lymphoma, or paraneoplastic/ autoimmune syndromes. The markedly abnormal periodic complexes on EEG and cortical ribboning and basal ganglia hyperintensities on fluid-attenuate inversion recovery MRI are diagnostic features of

1	autoimmune syndromes. The markedly abnormal periodic complexes on EEG and cortical ribboning and basal ganglia hyperintensities on fluid-attenuate inversion recovery MRI are diagnostic features of CJD, although rarely, prolonged focal or generalized seizures can produce a similar imaging appearance. Prion diseases are discussed in detail in Chap. 453e.

1	Huntington’s disease (HD) (Chap. 449) is an autosomal dominant degenerative brain disorder. HD clinical hallmarks include chorea, behavioral disturbance, and executive impairment. Symptoms typically begin in the fourth or fifth decade, but there is a wide range, from childhood to >70 years. Memory is frequently not impaired until late in the disease, but attention, judgment, self-awareness, and executive functions are often deficient at an early stage. Depression, apathy, social withdrawal, irritability, and intermittent disinhibition are common. Delusions and obsessive-compulsive behavior may occur. Disease duration is variable but typically lasts approximately 15 years.

1	Normal-pressure hydrocephalus (NPH) is a relatively uncommon but treatable syndrome. The clinical, physiologic, and neuroimaging characteristics of NPH must be carefully distinguished from those of other dementias associated with gait impairment. Historically, many patients treated for NPH have suffered from other dementias, particularly AD, vascular dementia, DLB, and PSP. For NPH, the clinical triad includes an abnormal gait (ataxic or apractic), dementia (usually mild to moderate, with an emphasis on executive impairment), and urinary urgency or incontinence. Neuroimaging reveals enlarged lateral ventricles (hydrocephalus) with little or no cortical atrophy, although the sylvian fissures may appear propped open (so-called “boxcarring”), which can be mistaken for perisylvian atrophy. This syndrome is a communicating hydrocephalus with a patent aqueduct of Sylvius (see Fig. 35-3), in contrast to aqueductal stenosis, in which the aqueduct is small. Lumbar puncture opening pressure

1	This syndrome is a communicating hydrocephalus with a patent aqueduct of Sylvius (see Fig. 35-3), in contrast to aqueductal stenosis, in which the aqueduct is small. Lumbar puncture opening pressure falls in the high-normal range, and the CSF protein, glucose, and cell counts are normal. NPH may be caused by obstruction to normal CSF flow over the cerebral convexities and delayed resorption into the venous system. The indolent nature of the process results in enlarged lateral ventricles with relatively little increase in CSF pressure. Presumed edema, stretching, and distortion of subfrontal white matter tracts may lead to clinical symptoms, but the precise underlying pathophysiology remains unclear. Some patients provide a history of conditions that produce meningeal scarring (blocking CSF resorption) such as previous meningitis, sub-arachnoid hemorrhage, or head trauma. Others with long-standing but asymptomatic congenital hydrocephalus may have adult-onset deterioration in gait or

1	resorption) such as previous meningitis, sub-arachnoid hemorrhage, or head trauma. Others with long-standing but asymptomatic congenital hydrocephalus may have adult-onset deterioration in gait or memory that is confused with NPH. In contrast to AD, the patient with NPH complains of an early and prominent gait disturbance without cortical atrophy on CT or MRI.

1	Numerous attempts to improve NPH diagnosis with various special studies and predict the success of ventricular shunting have been undertaken. These tests include radionuclide cisternography (showing a delay in CSF absorption over the convexity) and various efforts to monitor and alter CSF flow dynamics, including a constant-pressure infusion test. None has proven to be specific or consistently useful. A transient improvement in gait or cognition may follow lumbar puncture (or serial punctures) with removal of 30–50 mL of CSF, but this finding has also not proved to be consistently predictive of postshunt improvement. Perhaps the most reliable strategy is a period of close inpatient evaluation before, during, and after lumbar CSF drainage. Occasionally, when a patient with AD presents with gait impairment (at times due to comorbid subfrontal vascular injury) and absent or only mild cortical atrophy on CT or MRI, distinguishing NPH from AD can be challenging. Hippocampal atrophy on MRI

1	gait impairment (at times due to comorbid subfrontal vascular injury) and absent or only mild cortical atrophy on CT or MRI, distinguishing NPH from AD can be challenging. Hippocampal atrophy on MRI favors AD, whereas a characteristic “magnetic” gait with external hip rotation, low foot clearance, and short strides, along with prominent truncal sway or instability, favors NPH. The diagnosis of NPH should be avoided when hydrocephalus is not detected on imaging studies, even if the symptoms otherwise fit. Thirty to fifty percent of patients identified by careful diagnosis as having NPH will improve with ventricular shunting. Gait may improve more than cognition, but many reported failures to improve cognitively may have resulted from comorbid AD. Short-lasting improvement is common. Patients should be carefully selected for shunting, because subdural hematoma, infection, and shunt failure are known complications and can be a cause for early nursing home placement in an elderly patient

1	should be carefully selected for shunting, because subdural hematoma, infection, and shunt failure are known complications and can be a cause for early nursing home placement in an elderly patient with previously mild dementia.

1	Intracranial hypotension, sometimes called sagging brain syndrome, is a disorder caused by low CSF pressure, leading to downward pressure on the subcortical structures and disruption of cerebral function. It presents in a variable manner with headache, often exacerbated by coughing or a Valsalva maneuver or by moving from lying to standing. Other common symptoms include dizziness, vomiting, disruption of sleep-wake cycles, and sometimes a progressive bvFTD-like syndrome. Although sometimes idiopathic, this syndrome can be caused by CSF leaks secondary to lumbar puncture, head trauma, or spinal cord arachnoid cysts. Treatment consists of finding and patching CSF leaks.

1	Dementia can accompany chronic alcoholism (Chap. 467) and may result from associated malnutrition, especially of B vitamins, particularly thiamine. Other poorly defined aspects of chronic alcoholism may, however, also produce cerebral damage. A rare idiopathic syndrome of dementia and seizures with degeneration of the corpus callosum has been reported primarily in male Italian red wine drinkers (Marchiafava-Bignami disease).

1	Thiamine (vitamin B1) deficiency causes Wernicke’s encephalopathy (Chap. 330). The clinical presentation features a malnourished patient (frequently but not necessarily alcoholic) with confusion, ataxia, and diplopia resulting from inflammation and necrosis of periventricular midline structures, including dorsomedial thalamus, mammillary bodies, midline cerebellum, periaqueductal gray matter, and trochlear and abducens nuclei. Damage to the dorsomedial thalamus correlates most closely with the memory loss. Prompt administration of parenteral thiamine (100 mg intravenously for 3 days followed by daily oral dosage) may reverse the disease if given in the first days of symptom onset. Prolonged untreated thiamine deficiency can result in an irreversible and profound amnestic syndrome (Korsakoff’s syndrome) or even death.

1	In Korsakoff’s syndrome, the patient is unable to recall new information despite normal immediate memory, attention span, and level of consciousness. Memory for new events is seriously impaired, whereas knowledge acquired prior to the illness remains relatively intact. Patients are easily confused, disoriented, and cannot store information for more than a few minutes. Superficially, they may be conversant, engaging, and able to perform simple tasks and follow immediate commands. Confabulation is common, although not always present. There is no specific treatment because the previous thiamine deficiency has produced irreversible damage to the medial thalamic nuclei and mammillary bodies. Mammillary body atrophy may be visible on MRI in the chronic phase (see Fig. 330-6).

1	Vitamin B12 deficiency, as can occur in pernicious anemia, causes a megaloblastic anemia and may also damage the nervous system (Chaps. 128 and 456). Neurologically, it most commonly produces a spinal cord syndrome (myelopathy) affecting the posterior columns (loss of vibration and position sense) and corticospinal tracts (hyperactive tendon reflexes with Babinski signs); it also damages peripheral nerves (neuropathy), resulting in sensory loss with depressed tendon reflexes. Damage to myelinated axons may also cause dementia. The mechanism of neurologic damage is unclear but may be related to a deficiency of S-adenosyl methionine (required for methylation of myelin phospholipids) due to reduced methionine synthase activity or accumulation of methylmalonate, homocysteine, and propionate, providing abnormal substrates for fatty acid synthesis in myelin. Use of histamine blockers or metformin, vegan diets, autoimmunity against gastric parietal cells, and various causes of malabsorption

1	providing abnormal substrates for fatty acid synthesis in myelin. Use of histamine blockers or metformin, vegan diets, autoimmunity against gastric parietal cells, and various causes of malabsorption are the typical causes for vitamin B12 deficiency. The neurologic sequelae of vitamin B12 deficiency may occur in the absence of hematologic manifestations, making it critical to avoid using the complete blood count (CBC) and blood smear as a substitute for measuring B12 blood levels. Treatment with parenteral vitamin B12 (1000 μg intramuscularly daily for a week, weekly for a month, and monthly for life for pernicious anemia) stops progression of the disease if instituted promptly, but complete reversal of advanced nervous system damage will not occur.

1	Deficiency of nicotinic acid (pellagra) is associated with skin rash 2607 over sun-exposed areas, glossitis, and angular stomatitis (Chap. 96e). Severe dietary deficiency of nicotinic acid along with other B vitamins such as pyridoxine may result in spastic paraparesis, peripheral neuropathy, fatigue, irritability, and dementia. This syndrome has been seen in prisoners of war and in concentration camps but should be considered in any malnourished individual. Low serum folate levels appear to be a rough index of malnutrition, but isolated folate deficiency has not been proved as a specific cause of dementia.

1	dromes. However, some chronic CNS infections, particularly those associated with chronic meningitis (Chap. 165), may produce a dementing illness. The possibility of chronic infectious meningitis should be suspected in patients presenting with a dementia or behavioral syndrome, who also have headache, meningismus, cranial neuropathy, and/or radiculopathy. Between 20 and 30% of patients in the advanced stages of HIV infection become demented (Chap. 226). Cardinal features include psychomotor retardation, apathy, and impaired memory. This syndrome may result from secondary opportunistic infections but can also be caused by direct infection of CNS neurons with HIV. Neurosyphilis (Chap. 206) was a common cause of dementia in the preantibiotic era; it is now uncommon but can still be encountered in patients with multiple sex partners, particularly among patients with HIV. Characteristic CSF changes consist of pleocytosis, increased protein, and a positive Venereal Disease Research

1	in patients with multiple sex partners, particularly among patients with HIV. Characteristic CSF changes consist of pleocytosis, increased protein, and a positive Venereal Disease Research Laboratory (VDRL) test.

1	Primary and metastatic neoplasms of the CNS (Chap. 118) usually produce focal neurologic findings and seizures rather than dementia, but if tumor growth begins in the frontal or temporal lobes, the initial manifestations may be memory loss or behavioral changes. A paraneoplastic syndrome of dementia associated with occult carcinoma (often small-cell lung cancer) is termed limbic encephalitis. In this syndrome, confusion, agitation, seizures, poor memory, emotional changes, and frank dementia may occur. Paraneoplastic encephalitis associated with NMDA receptor antibodies presents as a progressive psychiatric disorder with memory loss and seizures; affected patients are often young women with ovarian teratoma (Chap. 122).

1	A nonconvulsive seizure disorder (Chap. 445) may underlie a syndrome of confusion, clouding of consciousness, and garbled speech. Often, psychiatric disease is suspected, but an EEG demonstrates the epileptic nature of the illness. If recurrent or persistent, the condition may be termed complex partial status epilepticus. The cognitive disturbance often responds to anticonvulsant therapy. The etiology may be previous small strokes or head trauma; some cases are idiopathic. It is important to recognize systemic diseases that indirectly affect the brain and produce chronic confusion or dementia. Such conditions include hypothyroidism; vasculitis; and hepatic, renal, or pulmonary disease. Hepatic encephalopathy may begin with irritability and confusion and slowly progress to agitation, lethargy, and coma.

1	Isolated vasculitis of the CNS (CNS granulomatous angiitis) (Chaps. 385 and 446) occasionally causes a chronic encephalopathy associated with confusion, disorientation, and clouding of consciousness. Headache is common, and strokes and cranial neuropathies may occur. Brain imaging studies may be normal or nonspecifically abnormal. CSF analysis reveals a mild pleocytosis or protein elevation. Cerebral angiography can show multifocal stenoses involving medium-caliber vessels, but some patients have only small-vessel disease that is not revealed on angiography. The angiographic appearance is not specific and may be mimicked by atherosclerosis, infection, or other causes of vascular disease. Brain or meningeal biopsy demonstrates endothelial cell proliferation and mononuclear infiltrates within blood vessel walls. The prognosis is often poor, although the disorder may remit spontaneously. Some patients respond to glucocorticoids or chemotherapy.

1	Chronic metal exposure represents a rare cause of dementia. The key to diagnosis is to elicit a history of exposure at work or home. Chronic lead poisoning from inadequately fire-glazed pottery has been reported. Fatigue, depression, and confusion may be associated with episodic abdominal pain and peripheral neuropathy. Gray lead lines appear in the gums, usually accompanied by an anemia with basophilic stippling of red blood cells. The clinical presentation can 2608 resemble that of acute intermittent porphyria, including elevated levels of urine porphyrins as a result of the inhibition of δ-aminolevulinic acid dehydrase. The treatment is chelation therapy with agents such as ethylenediamine tetraacetic acid (EDTA). Chronic mercury poisoning produces dementia, peripheral neuropathy, ataxia, and tremulousness that may progress to a cerebellar intention tremor or choreoathetosis. The confusion and memory loss of chronic arsenic intoxication is also associated with nausea, weight loss,

1	and tremulousness that may progress to a cerebellar intention tremor or choreoathetosis. The confusion and memory loss of chronic arsenic intoxication is also associated with nausea, weight loss, peripheral neuropathy, pigmentation and scaling of the skin, and transverse white lines of the fingernails (Mees’ lines). Treatment is chelation therapy with dimercaprol (BAL). Aluminum poisoning is rare but was documented with the dialysis dementia syndrome, in which water used during renal dialysis was contaminated with excessive amounts of aluminum. This poisoning resulted in a progressive encephalopathy associated with confusion, nonfluent aphasia, memory loss, agitation, and, later, lethargy and stupor. Speech arrest and myoclonic jerks were common and associated with severe and generalized EEG changes. The condition has been eliminated by the use of deionized water for dialysis. Recurrent head trauma in professional athletes may lead to a dementia previously referred to as “punch-drunk”

1	changes. The condition has been eliminated by the use of deionized water for dialysis. Recurrent head trauma in professional athletes may lead to a dementia previously referred to as “punch-drunk” syndrome or dementia pugilistica but now known as chronic traumatic encephalopathy (CTE) to signify its relevance to contact sport athletes other than boxers. The symptoms can be progressive, beginning late in an athlete’s career or, more often, after retirement. Early in the course, a personality change associated with social instability and sometimes paranoia and delusions occurs. Later, memory loss progresses to full-blown dementia, often associated with parkinsonian signs and ataxia or intention tremor. At autopsy, the cerebral cortex shows changes tau-immunoreactive NFTs that are more prominent than amyloid plaques (which are usually diffuse or absent rather than neuritic). NFTs and tau-positive reactive astrocytes are often clustered in the depths of cortical sulci, and TDP-43

1	prominent than amyloid plaques (which are usually diffuse or absent rather than neuritic). NFTs and tau-positive reactive astrocytes are often clustered in the depths of cortical sulci, and TDP-43 inclusions have also been reported, highlighting the overlap with the FTLD spectrum. Loss of neurons in the substantia nigra is a variable feature. Chronic subdural hematoma (Chap. 457e) is also occasionally associated with dementia, often in the context of underlying cortical atrophy from conditions such as AD or HD. Transient global amnesia (TGA) is characterized by the sudden onset of a severe episodic memory deficit, usually occurring in persons over the age of 50 years. Often the amnesia occurs in the setting of an emotional stimulus or physical exertion. During the attack, the individual is alert and communicative, general cognition seems intact, and there are no other neurologic signs or symptoms. The patient may seem confused and repeatedly ask about his or her location in place and

1	alert and communicative, general cognition seems intact, and there are no other neurologic signs or symptoms. The patient may seem confused and repeatedly ask about his or her location in place and time. The ability to form new memories returns after a period of hours, and the individual returns to normal with no recall for the period of the attack. Frequently no cause is determined, but cerebrovascular disease, epilepsy (7% in one study), migraine, or cardiac arrhythmias have all been implicated. Approximately one-quarter of patients experience recurrent attacks. Rare instances of permanent memory loss have been reported in patients with TGA-like spells, usually representing ischemic infarction of the hippocampus or dorsomedial thalamic nucleus bilaterally. Seizure activity due to AD should always be suspected in this syndrome. The ALS/parkinsonian/dementia complex of Guam is a rare degenerative disease that has occurred in the Chamorro natives on the island of Guam. Individuals may

1	always be suspected in this syndrome. The ALS/parkinsonian/dementia complex of Guam is a rare degenerative disease that has occurred in the Chamorro natives on the island of Guam. Individuals may have any combination of parkinsonian features, dementia, and MND. The most characteristic pathologic features are the presence of NFTs in degenerating neurons of the cortex and substantia nigra and loss of motor neurons in the spinal cord, although recent reanalysis has shown that some patients with this illness also show coexisting TDP-43 pathology. Epidemiologic evidence supports a possible environmental cause, such as exposure to a neurotoxin or an infectious agent with a long latency period. One interesting but unproven candidate neurotoxin occurs in the seed of the false palm tree, which Guamanians traditionally used to make flour. The ALS syndrome is no longer present in Guam, but a dementing illness with rigidity continues to be seen. Rarely, adult-onset leukodystrophies, lysosomal

1	Guamanians traditionally used to make flour. The ALS syndrome is no longer present in Guam, but a dementing illness with rigidity continues to be seen. Rarely, adult-onset leukodystrophies, lysosomal storage diseases, and other genetic disorders can present as a dementia in middle to late life. Metachromatic leukodystrophy (MLD) causes a progressive psychiatric or dementia syndrome associated with extensive, confluent frontal white matter abnormality. MLD is diagnosed by measuring arylsulfatase A enzyme activity in white blood cells. Adult-onset presentations of adrenoleukodystrophy have been reported in female carriers, and these patients often feature spinal cord and posterior white matter involvement. Adrenoleukodystrophy is diagnosed with measurement of plasma very-long-chain fatty acids. CADASIL is another genetic syndrome associated with white matter disease, often frontally and temporally predominant. Diagnosis is made with skin biopsy, which shows osmophilic granules in

1	acids. CADASIL is another genetic syndrome associated with white matter disease, often frontally and temporally predominant. Diagnosis is made with skin biopsy, which shows osmophilic granules in arterioles, or, increasingly, through genetic testing for mutations in Notch 3. The neuronal ceroid lipofuscinoses are a genetically heterogeneous group of disorders associated with myoclonus, seizures, vision loss, and progressive dementia. Diagnosis is made by finding eosinophilic curvilinear inclusions within white blood cells or neuronal tissue.

1	Psychogenic amnesia for personally important memories can be seen. Whether this results from deliberate avoidance of unpleasant memories, outright malingering, or unconscious repression remains unknown and probably depends on the patient. Event-specific amnesia is more likely to occur after violent crimes such as homicide of a close relative or friend or sexual abuse. It may develop in association with severe drug or alcohol intoxication and sometimes with schizophrenia. More prolonged psychogenic amnesia occurs in fugue states that also commonly follow severe emotional stress. The patient with a fugue state suffers from a sudden loss of personal identity and may be found wandering far from home. In contrast to neurologic amnesia, fugue states are associated with amnesia for personal identity and events closely associated with the personal past. At the same time, memory for other recent events and the ability to learn and use new information are preserved. The episodes usually last

1	and events closely associated with the personal past. At the same time, memory for other recent events and the ability to learn and use new information are preserved. The episodes usually last hours or days and occasionally weeks or months while the patient takes on a new identity. On recovery, there is a residual amnesia gap for the period of the fugue. Very rarely does selective loss of autobiographic information reflect a focal injury to the brain areas involved with these functions.

1	Psychiatric diseases may mimic dementia. Severely depressed or anxious individuals may appear demented, a phenomenon sometimes called pseudodementia. Memory and language are usually intact when carefully tested, and a significant memory disturbance usually suggests an underlying dementia, even if the patient is depressed. Patients in this condition may feel confused and unable to accomplish routine tasks. Vegetative symptoms, such as insomnia, lack of energy, poor appetite, and concern with bowel function, are common. Onset is often more abrupt, and the psychosocial milieu may suggest prominent reasons for depression. Such patients respond to treatment of the underlying psychiatric illness. Schizophrenia is usually not difficult to distinguish from dementia, but occasionally the distinction can be problematic. Schizophrenia generally has a much earlier age of onset (second and third decades) than most dementing illnesses and is associated with intact memory. The delusions and

1	can be problematic. Schizophrenia generally has a much earlier age of onset (second and third decades) than most dementing illnesses and is associated with intact memory. The delusions and hallucinations of schizophrenia are usually more complex, bizarre, and threatening than those of dementia. Some chronic schizophrenics develop an unexplained progressive dementia late in life that is not related to AD. Conversely, FTD, HD, vascular dementia, DLB, AD, or leukoencephalopathy can begin with schizophrenia-like features, leading to the misdiagnosis of a psychiatric condition. Later age of onset, significant deficits on cognitive testing, or the presence of abnormal neuroimaging suggest a degenerative condition. Memory loss may also be part of a conversion disorder. In this situation, patients commonly complain bitterly of memory loss, but careful cognitive testing either does not confirm the deficits or demonstrates inconsistent or unusual patterns of cognitive problems. The patient’s

1	commonly complain bitterly of memory loss, but careful cognitive testing either does not confirm the deficits or demonstrates inconsistent or unusual patterns of cognitive problems. The patient’s behavior and “wrong” answers to questions often indicate that he or she understands the question and knows the correct answer.

1	Clouding of cognition by chronic drug or medication use, often prescribed by physicians, is an important cause of dementia. Sedatives, tranquilizers, and analgesics used to treat insomnia, pain, anxiety, or agitation may cause confusion, memory loss, and lethargy, especially in the elderly. Discontinuation of the offending medication often improves mentation.

1	parkinson’s Disease and other movement Disorders C. Warren Olanow, Anthony H.V. Schapira, Jose A. Obeso PARKINSON’S DISEASE AND RELATED DISORDERS Parkinson’s disease (PD) is the second commonest neurodegenerative 449 disease, exceeded only by Alzheimer’s disease (AD). Its cardinal clinical features were first described by the English physician James Parkinson in 1817. It is noteworthy that James Parkinson was a general physician who captured the essence of this condition based on a visual inspection of a mere handful of patients. It is estimated that approximately 1 million persons in the United States, 1 million in Western Europe, and 5 million worldwide suffer from this disorder. PD affects men and women of all races, all occupations, and all countries. The mean age of onset is about 60 years. The frequency of PD increases with aging, but cases can be seen in patients in their 20s and even younger. Based on the aging of the population and projected demographics, it is estimated that

1	years. The frequency of PD increases with aging, but cases can be seen in patients in their 20s and even younger. Based on the aging of the population and projected demographics, it is estimated that the prevalence of the disease will dramatically increase in the next several decades.

1	Clinically, PD is characterized by rest tremor, rigidity, bradykinesia (slowing), and gait impairment, known as the “cardinal features” of the disease. Additional features can include freezing of gait, postural instability, speech difficulty, autonomic disturbances, sensory alterations, mood disorders, sleep dysfunction, cognitive impairment, and dementia (Table 449-1).

1	Pathologically, the hallmark features of PD are degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc), reduced striatal dopamine, and intracytoplasmic proteinaceous inclusions known as Lewy bodies that primarily contain the protein alpha synuclein (Fig. 449-1). While interest has primarily focused on the dopamine system, neuronal degeneration with inclusion body formation can also affect cholinergic neurons of the nucleus basalis of Meynert (NBM), norepinephrine neurons of the locus coeruleus (LC), serotonin neurons in the raphe nuclei of the brainstem, and neurons of the olfactory system, cerebral hemispheres, spinal cord, and peripheral autonomic nervous system. This “nondopaminergic” pathology is likely responsible for the development of nondopaminergic clinical features listed in Table 449-1 characterized by their lack of satisfactory response to dopaminergic replacement therapy. There is evidence that Lewy body pathology first begins in the peripheral

1	features listed in Table 449-1 characterized by their lack of satisfactory response to dopaminergic replacement therapy. There is evidence that Lewy body pathology first begins in the peripheral autonomic nervous system, olfactory system, and dorsal motor nucleus of the vagus nerve in the lower brainstem, and then spreads in a predictable and sequential manner to affect the upper brainstem and cerebral hemispheres

1	Abbreviations: RBD, rapid eye movement behavior disorder; MCI, mild cognitive impairment. (Braak staging). These studies suggest that degeneration of dopamine 2609 neurons develops in a mid-stage of the disease. Indeed, epidemiologic studies suggest that clinical symptoms reflecting this nondopaminergic degeneration, such as constipation, anosmia, rapid eye movement (REM) behavior sleep disorder, and cardiac denervation, can precede the onset of the classic motor features of PD.

1	Parkinsonism is a generic term that is used to define a syndrome manifest as bradykinesia with rigidity and/or tremor. It has a differential diagnosis (Table 449-2) that reflects damage to different components of the basal ganglia. The basal ganglia are comprised of a group of subcortical nuclei that include the striatum (putamen and caudate nucleus), subthalamic nucleus (STN), globus pallidus pars externa (GPe), globus pallidus pars interna (GPi), and the SNc (Fig. 449-2). Among the different forms of parkinsonism, PD is the most common (approximately 75% of cases). Historically, PD was diagnosed based on the presence of two of three parkinsonian features (tremor, rigidity, bradykinesia). However, postmortem studies found a 24% error rate when diagnosis was based on these criteria. Clinicopathologic correlation studies subsequently determined that parkinsonism associated with rest tremor, asymmetry, and a good response to levodopa was more likely to predict the correct pathologic

1	correlation studies subsequently determined that parkinsonism associated with rest tremor, asymmetry, and a good response to levodopa was more likely to predict the correct pathologic diagnosis. With these revised criteria (known as the U.K. Brain Bank Criteria), a clinical diagnosis of PD is confirmed pathologically in as many as 99% of cases. A more complete definition of PD is now needed to incorporate the fact that there is widespread pathology beyond the dopaminergic system, nondopamine and nonmotor clinical features, and a premotor stage of the disease.

1	Imaging of the brain dopamine system in PD with positron emission tomography (PET) or single-photon emission computed tomography (SPECT) shows reduced uptake of striatal dopaminergic markers, particularly in the posterior putamen with relative sparing of the caudate nucleus (Fig. 449-3), reflecting the degeneration of nigrostriatal dopamine neurons. Imaging can be useful in patients where there is diagnostic uncertainty (e.g., dystonic tremor, essential tremor) or in research studies, but is rarely necessary in routine practice because the diagnosis can usually be established on clinical criteria alone. This may change in the future when there is a disease-modifying therapy and it is important to make the diagnosis as early as possible. Genetic testing is not routinely used at present, but can be helpful for identifying at-risk individuals in a research setting. Mutations of the LRRK2 gene (see below) have attracted particular interest because they are the commonest cause of familial

1	can be helpful for identifying at-risk individuals in a research setting. Mutations of the LRRK2 gene (see below) have attracted particular interest because they are the commonest cause of familial PD and are responsible for approximately 1% of typical sporadic cases of the disease. Mutations in LRRK2 are a particularly common cause of PD in Ashkenazi Jews and North African Berber Arabs. The penetrance of the most common LRRK2 mutation ranges from 28 to 74% and is strongly correlated to the age of the carrier, with 50% affected by age 60 years. Mutations in the parkin gene should be considered in patients with onset prior to 40 years of age.

1	Atypical and Secondary Parkinsonism Atypical parkinsonism refers to a group of neurodegenerative conditions that usually are associated with more widespread neurodegeneration than is found in PD (often involvement of striatum and/or globus pallidus as well as the SNc). As a group, they present with parkinsonism (rigidity and bradykinesia) but have a slightly different clinical picture than PD, reflecting differences in the underlying pathology. In these conditions, parkinsonism is typically characterized by early speech and gait impairment, absence of rest tremor, no motor asymmetry, poor or no response to levodopa, and an aggressive clinical course. In the early stages, they may show some modest benefit from levodopa and be difficult to distinguish from PD. Pathologically, neurodegeneration typically involves degeneration of the SNc but occurs without Lewy bodies (see below for individual conditions). Neuroimaging of the dopamine system is usually not helpful, because dopamine

1	typically involves degeneration of the SNc but occurs without Lewy bodies (see below for individual conditions). Neuroimaging of the dopamine system is usually not helpful, because dopamine depletion can be seen in both PD and atypical parkinsonism. By contrast, metabolic imaging of the basal ganglia/thalamus network (using 2-F-deoxiglucose PET) may be helpful, showing a pattern of decreased activity in the GPi with increased activity in the thalamus, the reverse of what is seen in PD.

1	FIGURE 449-1 Pathologic specimens from a patient with Parkinson’s disease (PD) compared to a normal control demonstrating (A) reduction of pigment in SNc in PD (right) versus control (left), (B) reduced numbers of cells in SNc in PD (right) compared to control (left), and (C) Lewy bodies (arrows) within melanized dopamine neurons in PD. SNc, substantia nigra pars compacta.

1	Multiple-system atrophy (MSA) manifests as a combination of parkinsonian, cerebellar, and autonomic features and can be divided into a predominant parkinsonian (MSA-p) or cerebellar (MSA-c) form. Clinically, MSA is suspected when a patient presents with atypical parkinsonism in conjunction with cerebellar signs and/or early and prominent autonomic dysfunction, usually orthostatic hypotension (Chap. 454). Pathologically, MSA is characterized by degeneration of the SNc, striatum, cerebellum, and inferior olivary nuclei coupled with characteristic glial cytoplasmic inclusions (GCIs) that stain for α-synuclein. Magnetic resonance imaging (MRI) can show pathologic iron accumulation in the striatum on T2-weighted scans, high signal change in the region of the external surface of the putamen (putaminal rim) in MSA-p, or cerebellar and brainstem atrophy (the pontine “hot

1	DiffereNtiaL DiaGNosis of parkiNsoNism cross buns” sign [Fig. 454-2]) in MSA-c. Mutations in the CoQ2 gene encoding parahydroxybenzoate-polyprenyl transferase, an enzyme involved in the biosynthesis of coenzyme Q10 (CoQ10), a cofactor of the mitochondrial respiratory chain, have been identified in familial and sporadic forms of MSA. Progressive supranuclear palsy (PSP) is a form of atypical parkinsonism that is characterized by slow ocular saccades, eyelid apraxia, and restricted eye movements with particular impairment of downward gaze. Patients frequently experience hyperextension of the neck with early gait disturbance and falls. In later stages, speech and swallowing difficulty and cognitive impairment become evident. MRI may reveal a characteristic atrophy of the midbrain with relative preservation of the pons, the “hummingbird sign” on midsagittal images. Pathologically, Dementia with Lewy bodies Atypical Parkinsonism

1	Dementia with Lewy bodies Atypical Parkinsonism Secondary Parkinsonism Drug-induced Tumor Infection Vascular Normal-pressure hydrocephalus Trauma Liver failure Toxins (e.g., carbon monoxide, manganese, MPTP, cyanide, hexane, methanol, carbon disulfide) Other Neurodegenerative Disorders Wilson’s disease Huntington’s disease Neurodegeneration with brain iron accumulation SCA 3 (spinocerebellar ataxia) Fragile X–associated ataxia-tremor-parkinsonism Prion disease Dystonia-parkinsonism (DYT3) Alzheimer’s disease with parkinsonism Abbreviations: MPTP, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine. FIGURE 449-2 Basal ganglia nuclei. Schematic (A) and postmortem (B) coronal sections illustrating the various components of the basal ganglia. SNc, substantia nigra pars compacta; STN, subthalamic nucleus.

1	PSP is characterized by degeneration of the SNc, striatum, subthalamic nucleus, midline thalamic nuclei, and pallidum along with neurofibrillary tangles and inclusions that stain for the tau protein. Corticobasal ganglionic degeneration is less common and is usually manifest by asymmetric dystonic contractions and clumsiness of one hand coupled with cortical sensory disturbances manifest as apraxia, agnosia, focal limb myoclonus, or alien limb phenomenon (where

1	FIGURE 449-3 [11C]Dihydrotetrabenazine positron emission tomography (a marker of VMAT2) in healthy control (A) and Parkinson’s disease (B) patient. Note the reduced striatal uptake of tracer, which is most pronounced in the posterior putamen and tends to be asymmetric. (Courtesy of Dr. Jon Stoessl.) the limb assumes a position in space without the patient being aware of it). Dementia may occur at any stage of the disease. Both cortical and basal ganglia features are required to make this diagnosis. MRI frequently shows asymmetric cortical atrophy. Pathologic findings include achromatic neuronal degeneration with tau deposits. Because other disorders such as PSP can present with a similar clinical picture, the term corticobasal ganglia syndrome should be used until a precise diagnosis can be confirmed pathologically.

1	Secondary parkinsonism can occur as a result of drugs, stroke, tumor, infection, or exposure to toxins such as carbon monoxide or manganese. Dopamine-blocking agents such as the neuroleptics are the commonest cause of secondary parkinsonism. These drugs are most widely used in psychiatry, but physicians should be aware that drugs such as metoclopramide and chlorpromazine, which are primarily used to treat gastrointestinal problems, are also neuroleptic agents and common causes of secondary parkinsonism (as well as acute and tardive dyskinesias; see below). Other drugs that can cause secondary parkinsonism include tetrabenazine, calcium channel blockers (flunarizine, cinnarizine), amiodarone, and lithium.

1	Finally, parkinsonism can be seen as a feature of other degenerative disorders such as Wilson’s disease, Huntington’s disease (especially the juvenile form known as the Westphal variant), dopa-responsive dystonia, and neurodegenerative disorders with brain iron accumulation such as pantothenate kinase (PANK)–associated neurodegeneration (formerly known as Hallervorden-Spatz disease). Some features that suggest parkinsonism might be due to a condition other than PD are shown in Table 449-3.

1	Most PD cases occur sporadically (~85–90%) and are of unknown cause. Twin studies suggest that environmental factors likely play an important role in patients older than 50 years, with genetic factors being more important in younger patients. Epidemiologic studies also suggest increased risk with exposure to pesticides, rural living, and drinking well water and reduced risk with cigarette smoking and caffeine. However, no environmental factor has yet been proven to cause typical PD. The environmental hypothesis received support with the demonstration in the 1980s that MPTP (1-methyl-4-phenyl1,2,5,6-tetrahydropyridine), a byproduct of the illicit manufacture of a heroin-like drug, caused a PD-like syndrome in addicts in northern California. MPTP is transported to the central nervous system, where it is oxidized to form MPP+, a mitochondrial toxin that is selectively taken up by, and damages, dopamine neurons. However, MPTP or MPTP-like compounds have not been linked to sporadic PD.

1	About 10–15% of cases are familial in origin, and multiple specific mutations and gene associations have been identified (Table 449-4). Genetic factors have also been linked to sporadic cases, with several typical PD cases found to carry the LRRK2 mutation, and genome-wide association studies (GWAS) implicating alpha synuclein, tau, and Abbreviations: MSA-c, multiple-system atrophy–cerebellar type; MSA-p, multiple-system atrophy–Parkinson’s type; PD, Parkinson’s disease; PSP, progressive supranuclear palsy. HLA as risk factors. It has been proposed that most cases of PD may be due to a “double hit” involving an interaction between a gene mutation that induces susceptibility coupled with exposure to a toxic environmental factor that may induce epigenetic or somatic DNA alterations. In this scenario, both factors are required for PD to ensue, while the presence of either one alone is not sufficient to cause the disease.

1	Several factors have been implicated in the pathogenesis of cell death in PD, including oxidative stress, inflammation, mitochondrial dysfunction, and proteolytic stress. Recent studies have demonstrated that with aging dopamine neurons switch from sodium to calcium pacing through calcium channels, potentially making these high-energy neurons vulnerable to calcium-mediated neurotoxicity. Whatever the pathogenic mechanism, cell death appears to occur, at least in part, by way of a signal-mediated apoptotic or “suicidal” process. Abbreviations: AD, autosomal dominant; AR, autosomal recessive; Chr, chromosome; Sp, sporadic. FIGURE 449-4 Schematic representation of how pathogenetic factors implicated in Parkinson’s disease interact in a network manner, ultimately leading to cell death. This figure illustrates how interference with any one of these factors may not necessarily stop the cell death cascade. (Adapted from CW Olanow: Movement Disorders 22:S-335, 2007.)

1	Each of these mechanisms offers a potential target for neuroprotective drugs. However, it is not clear which of these factors is primary, if the mechanism is the same in each individual case, if they act by way of a network such that a cocktail of agents might be required to provide neuroprotection, or if the findings to date merely represent epiphenomena unrelated to the true cause of cell death that remains undiscovered (Fig. 449-4). Gene mutations may not cause all cases of PD, but may be helpful in pointing to specific pathogenic pathways and mechanisms that are central to a neurodegenerative process that might be relevant to all forms of the disease. To date, most interest has focused on pathways implicated by mutations in α-synuclein, LRRK2, and PINK1/Parkin.

1	Most interest has focused on α-synuclein. Mutations in α-synuclein cause rare familial forms of PD, and α-synuclein constitutes the major component of Lewy bodies in patients with sporadic PD (Fig. 449-1). Furthermore, duplication or triplication of the wild-type α-synuclein can also cause a form of PD, indicating that increased production of the normal protein alone can cause the disease. More recently, Lewy pathology was discovered to have developed in healthy embryonic dopamine neurons that had been implanted into the striatum of PD patients, suggesting that the abnormal protein had transferred from affected cells to healthy unaffected dopamine neurons. Based on these findings, it has been proposed that α-synuclein is a prion and PD is a prion disorder. Here it is proposed that, like the prion protein PrPC, α-synuclein can misfold to form β-rich sheets, generate toxic oligomers and aggregates, polymerize to form amyloid plaques (i.e., Lewy bodies), cause neurodegeneration, and

1	the prion protein PrPC, α-synuclein can misfold to form β-rich sheets, generate toxic oligomers and aggregates, polymerize to form amyloid plaques (i.e., Lewy bodies), cause neurodegeneration, and spread to involve unaffected neurons. Indeed, injection of α-synuclein fibrils into the striatum promotes the development of Lewy pathology in host neurons, neurodegeneration, behavioral abnormalities, and the spread of α-synuclein pathology to anatomically connected sites. Further support for this hypothesis comes from the demonstration that inoculation of α-synuclein derived from human Lewy bodies induces widespread Lewy pathology in mice and primates. Collectively, this evidence supports the possibility that neuroprotective therapies for PD might be developed based on inhibiting accumulation or accelerating removal of α-synuclein aggregates.

1	Mutations in the glucocerebrosidase (GBA) gene associated with Gaucher’s disease numerically represent the most important risk factor for the development of PD. While the responsible mechanism is not precisely known, it is noteworthy that GBA mutations are associated with altered autophagy and lysosomal function and could impair the clearance of α-synuclein.

1	Six different LRRK2 mutations have been linked to PD, with Gly2019Ser being the most common. The mechanism responsible for cell death with this mutation is not known but is thought to involve changes in kinase activity with altered phosphorylation of target proteins (including autophosphorylation) and possibly lysosomal dysfunction. Kinase inhibitors can block toxicity associated with LRRK2 mutations in laboratory models, and there has been much interest in developing drugs directed at this target. However, kinase inhibitors are likely to be toxic, the physiologic role of LRRK2 is not known, and the large majority of PD patients do not carry a LRRK2 mutation.

1	Mutations in PINK1 and parkin have implicated mitochondrial dysfunction as a possible cause of PD. Recent studies suggest a role for parkin and PINK1 proteins in the turnover and clearance of damaged mitochondria (mitophagy), and mutations in parkin and PINK1 cause mitochondrial dysfunction in transgenic animals that can be corrected with overexpression of parkin. This is a particularly attractive target because postmortem studies in PD patients show a defect in complex I of the respiratory chain in SNc neurons. Thus, evidence is accumulating that genetics plays an important role in both familial and “sporadic” forms of PD. It is anticipated that better understanding of the pathways responsible for cell death caused by these mutations will permit the development of more relevant animal models of PD and targets for the development of neuroprotective drugs.

1	The classic model of the organization of the basal ganglia in the normal and PD states is provided in Fig. 449-5. With respect to motor function, a series of neuronal circuits or loops link the basal ganglia nuclei with corresponding cortical motor regions in a somatotopic manner. The striatum is the major input region of the basal ganglia, while the GPi and SNr are the major output regions. The input and output regions are connected via direct and indirect pathways that have reciprocal effects on the activity of the output pathway. The output of the basal ganglia provides inhibitory (GABAergic) tone to thalamic and brainstem neurons that in turn connect to motor systems in the cerebral cortex and spinal cord that control motor function. 2613 Physiologically, decreased neuronal activity in the GPi/SNr is associated with movement facilitation and vice versa. Dopaminergic projections from SNc neurons serve to modulate neuronal firing and to stabilize the basal ganglia network. The basal

1	the GPi/SNr is associated with movement facilitation and vice versa. Dopaminergic projections from SNc neurons serve to modulate neuronal firing and to stabilize the basal ganglia network. The basal ganglia and similar cortical loops are now thought to also play an important role in regulating normal behavioral, emotional, and cognitive functions. In PD, dopamine denervation with loss of dopaminergic tone leads to increased firing of neurons in the STN and GPi, excessive inhibition of the thalamus, reduced activation of cortical motor systems, and the development of parkinsonian features (Fig. 449-5). The current role of surgery in the treatment of PD is based on this model, which predicted that lesions or high-frequency stimulation of the STN or GPi might reduce this neuronal overactivity and improve PD features.

1	Since its introduction in the late 1960s, levodopa has been the mainstay of therapy for PD. Experiments in the late 1950s by Carlsson demonstrated that blocking dopamine uptake with reserpine caused rabbits to become parkinsonian; this could be reversed with the dopamine precursor, levodopa. Subsequently, Hornykiewicz demonstrated a dopamine deficiency in the striatum of PD patients and suggested the potential benefit of dopaminergic replacement therapy. Dopamine does not cross the blood-brain barrier (BBB), so clinical trials were initiated with levodopa, a precursor of dopamine. Studies over the course of the next decade confirmed the value of levodopa and revolutionized the treatment of PD.

1	Levodopa is routinely administered in combination with a peripheral decarboxylase inhibitor to prevent its peripheral metabolism to dopamine and the development of nausea and vomiting due to activation of dopamine receptors in the area postrema that are not protected by the BBB. In the United States, levodopa is combined with the decarboxylase inhibitor carbidopa (Sinemet), whereas in

1	FIGURE 449-5 Basal ganglia organization. Classic model of the organization of the basal ganglia in the normal (A), Parkinson’s disease (PD) (B), and levodopa-induced dyskinesia (C) state. Inhibitory connections are shown as blue arrows and excitatory connections as red arrows. The striatum is the major input region and receives its major input from the cortex. The GPi and SNr are the major output regions, and they project to the thalamocortical and brainstem motor regions. The striatum and GPi/SNr are connected by direct and indirect pathways. This model predicts that parkinsonism results from increased neuronal firing in the STN and GPi and that lesions or DBS of these targets might provide benefit. This concept led to the rationale for surgical therapies for PD. The model also predicts that dyskinesia results from decreased firing of the output regions, resulting in excessive cortical activation by the thalamus. This component of the model is not completely correct because lesions

1	that dyskinesia results from decreased firing of the output regions, resulting in excessive cortical activation by the thalamus. This component of the model is not completely correct because lesions of the GPi ameliorate rather than increase dyskinesia in PD, suggesting that firing frequency is just one of the components that lead to the development of dyskinesia. DBS, deep brain stimulation; GPe, external segment of the globus pallidus; GPi, internal segment of the globus pallidus; PPN, pedunculopontine nucleus; SNc, substantia nigra, pars compacta; SNr, substantia nigra, pars reticulata; STN, subthalamic nucleus; VL, ventrolateral thalamus. (Derived from JA Obeso et al: Trends Neurosci 23:S8, 2000.) 2614 many other countries, it is combined with benserazide (Madopar). Levodopa is also available in controlled-release formulations as well as in combination with a catechol-O-methyltransferase (COMT) inhibitor (see below). Levodopa remains the most effective symptomatic treatment for PD

1	in controlled-release formulations as well as in combination with a catechol-O-methyltransferase (COMT) inhibitor (see below). Levodopa remains the most effective symptomatic treatment for PD and the gold standard against which new therapies are compared. No current medical or surgical treatment provides antiparkinsonian benefits superior to what can be achieved with levodopa. Levodopa benefits the classic motor features of PD, prolongs independence and employability, improves quality of life, and increases life span. Almost all PD patients experience improvement, and failure to respond to an adequate trial should cause the diagnosis to be questioned. There are, however, important limitations of levodopa therapy. Acute dopaminergic side effects include nausea, vomiting, and orthostatic hypotension. These are usually transient and can generally be avoided by gradual titration. If they persist, they can be treated with additional doses of a peripheral decarboxylase inhibitor (e.g.,

1	hypotension. These are usually transient and can generally be avoided by gradual titration. If they persist, they can be treated with additional doses of a peripheral decarboxylase inhibitor (e.g., carbidopa) or a peripheral dopamine-blocking agent such as domperidone (not available in the United States). More important are motor complications (see below) that develop in the majority of patients treated long-term with levodopa. In addition, the disease continues to progress, and features such as falling, freezing, autonomic dysfunction, sleep disorders, and dementia may emerge with disease progression that are not adequately controlled by levodopa. Indeed, these nondopaminergic features (especially falling and dementia) are the primary source of disability and the main reason for nursing home placement for patients with advanced PD Levodopa-induced motor complications consist of fluctuations in motor response (“on” episodes when the drug is working and “off” episodes when parkinsonian

1	placement for patients with advanced PD Levodopa-induced motor complications consist of fluctuations in motor response (“on” episodes when the drug is working and “off” episodes when parkinsonian features return) and involuntary movements known as dyskinesias (Fig. 449-6). When patients initially take levodopa, benefits are long-lasting (many hours) even though the drug has a relatively short half-life (60–90 min). With continued treatment, however, the duration of benefit following an individual dose becomes progressively shorter until it approaches the half-life of the drug. This loss of benefit is known as the wearing-off effect. In more severe cases, patients may experience a delay in turning on (delayed-on) or no response at all to a given dose (noon). Dyskinesias tend to occur at the time of levodopa peak plasma concentration and maximal clinical benefit (peak-dose dyskinesia). They are usually choreiform in nature but can manifest as dystonic movements, myoclonus, or other

1	the time of levodopa peak plasma concentration and maximal clinical benefit (peak-dose dyskinesia). They are usually choreiform in nature but can manifest as dystonic movements, myoclonus, or other movement disorders. They are not troublesome when mild, but can be disabling when severe, and can limit the ability to fully use levodopa to control PD features. In more advanced states, patients may cycle between “on” periods complicated by disabling dyskinesias and “off” periods in which they suffer from severe parkinsonism and painful dystonic postures. Patients may also experience “diphasic dyskinesias,” which occur as the levodopa dose begins to take effect and again as it wears off. These dyskinesias typically consist of transient, stereotypic, rhythmic movements that predominantly involve the lower extremities and are frequently associated with parkinsonism in other body regions. They can be relieved by increasing the dose of levodopa, although higher doses may induce more severe

1	the lower extremities and are frequently associated with parkinsonism in other body regions. They can be relieved by increasing the dose of levodopa, although higher doses may induce more severe peak-dose dyskinesia.

1	The cause of levodopa-induced motor complications is not precisely known. They are more likely to occur in females, younger individuals with more severe disease, and with the use of higher doses (mg/kg) of levodopa. The classic model of the basal ganglia has been useful for understanding the origin of motor features in PD, but has proved less valuable for understanding levodopa-induced dyskinesias (Fig. 449-5). The model predicts that dopamine replacement might excessively inhibit the pallidal output system, thereby leading to increased thalamocortical activity, enhanced stimulation of cortical motor regions, and the development of dyskinesia. However, lesions of the pallidum that completely destroy its output are associated with amelioration rather than induction of dyskinesia as suggested by the classic model. It is now thought that dyskinesia results from levodopa-induced alterations in the GPi neuronal firing pattern (pauses, bursts, synchrony, etc.) and oscillatory activity, and

1	by the classic model. It is now thought that dyskinesia results from levodopa-induced alterations in the GPi neuronal firing pattern (pauses, bursts, synchrony, etc.) and oscillatory activity, and not simply the firing frequency alone. This in turn leads to the transmission of misinformation from pallidum to thalamus/cortex, resulting in dyskinesia. Surgical lesions or high-frequency stimulation might ameliorate dyskinesia by interfering with (blocking or masking) this abnormal neuronal activity and preventing the transfer of misinformation to motor systems.

1	Current information suggests that altered neuronal firing patterns and motor complications relate to nonphysiologic levodopa replacement. Striatal dopamine levels are normally maintained at a relatively constant level. In PD, dopamine neurons degenerate and striatal dopamine is dependent on the peripheral availability of levodopa. Intermittent doses of short-acting levodopa result in fluctuating plasma levels because of variability in transit of the drug from the stomach to the duodenum where it is absorbed and the short half-life of the drug. This variability results in exposure of dopamine receptors to pathologically high and low concentrations of dopamine. It has been hypothesized that more continuous delivery of levodopa might prevent the development of motor complications. Indeed, a recent controlled study demonstrated that continuous intraintestinal infusion of levodopa/carbidopa intestinal gel (Duodopa) is associated with significant improvement in “off” time and in “on” time

1	recent controlled study demonstrated that continuous intraintestinal infusion of levodopa/carbidopa intestinal gel (Duodopa) is associated with significant improvement in “off” time and in “on” time without dyskinesia in advanced PD patients compared with optimized standard oral levodopa.

1	Behavioral alterations can also be encountered in levodopatreated patients. A dopamine dysregulation syndrome has been described where patients have a craving for levodopa and take frequent and unnecessary doses of the drug in an addictive manner. PD patients taking high doses of levodopa can develop purposeless, stereotyped behaviors such as the meaningless assembly and disassembly or collection and sorting of objects. This is known as punding, a term taken from the Swedish description of the meaningless behaviors seen in chronic amphetamine users. Hypersexuality and other impulse-control disorders are occasionally encountered with levodopa, although these are more commonly seen with dopamine agonists.

1	Dopamine agonists are a diverse group of drugs that act directly on dopamine receptors. Unlike levodopa, they do not require metabolism to an active product and do not undergo oxidative metabolism. Initial dopamine agonists were ergot derivatives (e.g., bromocriptine, pergolide, cabergoline) and were associated with ergot-related side effects, including cardiac valvular damage. They have largely been replaced by a second generation of nonergot dopamine agonists (e.g., pramipexole, ropinirole, rotigotine). In general, dopamine agonists do not have comparable efficacy to levodopa. They were initially introduced as adjuncts to levodopa to enhance motor function and reduce “off” time in fluctuating patients. Subsequently, it was shown that dopamine agonists, possibly because they are relatively long-acting, are less prone than levodopa to induce dyskinesia. For this reason, many physicians initiate therapy with a dopamine agonist, although supplemental levodopa is eventually required in

1	long-acting, are less prone than levodopa to induce dyskinesia. For this reason, many physicians initiate therapy with a dopamine agonist, although supplemental levodopa is eventually required in virtually all patients. Both ropinirole and pramipexole are available as orally administered immediate (tid) and extended-release (qd) formulations. Rotigotine is administered as a once-daily transdermal patch. Apomorphine is a dopamine agonist with efficacy comparable to levodopa, but it must be administered parenterally and has a very short half-life and duration of activity (45 min). It is generally administered by injection as a rescue agent for the treatment of severe “off” episodes. Apomorphine can also be administered by continuous subcutaneous infusion and has been demonstrated to reduce both “off” time and dyskinesia in advanced patients. However, this approach has not been approved in the United States.

1	Dopamine agonist use is associated with a variety of side effects. Acute side effects are primarily dopaminergic and include nausea, vomiting, and orthostatic hypotension. As with levodopa, these can usually be avoided by slow titration. Side effects associated with chronic use include hallucinations and cognitive impairment. Sedation with sudden unintended episodes of falling asleep while driving a motor vehicle have been reported. Patients should be informed about this potential problem and should not drive when tired. Dopamine agonists can also be associated with impulse-control disorders, including pathologic gambling, hypersexuality, and compulsive eating and shopping. The precise cause of these problems, and why they appear to occur more frequently with dopamine agonists than levodopa, remains to be resolved, but reward systems associated with dopamine and alterations in the ventral striatum and orbitofrontal regions have been implicated. In general, chronic side effects are

1	remains to be resolved, but reward systems associated with dopamine and alterations in the ventral striatum and orbitofrontal regions have been implicated. In general, chronic side effects are dose-related and can be avoided or minimized with lower doses. Injections of apomorphine and patch delivery of rotigotine can be complicated by development of skin lesions at sites of administration.

1	Inhibitors of monoamine oxidase type B (MAO-B) block central dopamine metabolism and increase synaptic concentrations of the neurotransmitter. Selegiline and rasagiline are relatively selective suicide inhibitors of the MAO-B enzyme. Clinically, MAO-B inhibitors provide antiparkinsonian benefits when used as monotherapy in early disease and reduced “off” time when used as an adjunct to levodopa in patients with motor fluctuations. MAO-B inhibitors are generally safe and well tolerated. They may increase dyskinesia in levodopa-treated patients, but this can usually be controlled by down-titrating the dose of levodopa. Inhibition of the MAO-A isoform prevents metabolism of tyramine in the gut, leading to a potentially fatal hypertensive reaction known as a “cheese effect” because it can be precipitated by foods rich in tyramine such as some cheeses, aged meats, and red wine. Selegiline and rasagiline do not functionally inhibit MAO-A and are not associated with a 2615 cheese effect with

1	by foods rich in tyramine such as some cheeses, aged meats, and red wine. Selegiline and rasagiline do not functionally inhibit MAO-A and are not associated with a 2615 cheese effect with doses typically used in clinical practice. There are theoretical risks of a serotonin reaction in patients receiving concomitant selective serotonin reuptake inhibitor (SSRI) antidepressants, but these are rarely encountered.

1	Interest in MAO-B inhibitors has also focused on their potential to have disease-modifying effects. MPTP toxicity can be prevented experimentally by coadministration of an MAO-B inhibitor that blocks its conversion to the toxic pyridinium ion MPP+. MAO-B inhibitors also have the potential to block the oxidative metabolism of dopamine and prevent oxidative stress. In addition, both selegiline and rasagiline incorporate a propargyl ring within their molecular structure that provides antiapoptotic effects in laboratory models. The DATATOP study showed that selegiline significantly delayed the time until the emergence of disability, necessitating the introduction of levodopa, in untreated PD patients. However, it could not be determined whether this was due to a neuroprotective effect that slowed disease progression or a symptomatic effect that merely masked ongoing neurodegeneration. More recently, the ADAGIO study demonstrated that early treatment with rasagiline 1 mg/d, but not 2 mg/d,

1	disease progression or a symptomatic effect that merely masked ongoing neurodegeneration. More recently, the ADAGIO study demonstrated that early treatment with rasagiline 1 mg/d, but not 2 mg/d, provided benefits that could not be achieved when treatment with the same drug was initiated at a later time point, consistent with a disease-modifying effect; however, the long-term significance of these findings is uncertain.

1	When levodopa is administered with a decarboxylase inhibitor, it is primarily metabolized in the periphery by COMT. Inhibitors of COMT increase the elimination half-life of levodopa and enhance its brain availability. Combining levodopa with a COMT inhibitor reduces “off” time and prolongs “on” time in fluctuating patients while enhancing motor scores. Two COMT inhibitors have been approved, tolcapone and entacapone. There is also a combination tablet of levodopa, carbidopa, and entacapone (Stalevo).

1	Side effects of COMT inhibitors are primarily dopaminergic (nausea, vomiting, increased dyskinesia) and can usually be controlled by down-titrating the dose of levodopa by 20–30%. Severe diarrhea has been described with tolcapone, and to a lesser degree with entacapone, and necessitates stopping the medication in 5–10% of individuals. Cases of fatal hepatic toxicity have been reported with tolcapone, and periodic monitoring of liver function is required. This problem has not been encountered with entacapone. Discoloration of urine can be seen with both COMT inhibitors due to accumulation of a metabolite, but it is of no clinical concern.

1	It has been proposed that initiating levodopa in combination with a COMT inhibitor to enhance its elimination half-life could provide more continuous levodopa delivery if administered at frequent intervals and reduce the risk of motor complications. While this result has been demonstrated in a preclinical MPTP model, and continuous infusion reduces both “off” time and dyskinesia in advanced PD patients, no benefit of initiating levodopa with a COMT inhibitor compared to levodopa alone was detected in early PD patients in the STRIDE-PD study. This may have been because the combination was not administered at frequent enough intervals to provide continuous levodopa availability. For now, the main value of COMT inhibitors continues to be in patients who experience motor fluctuations.

1	Centrally acting anticholinergic drugs such as trihexyphenidyl and benztropine were used historically for the treatment of PD, but they lost favor with the introduction of dopaminergic agents. Their major clinical effect is on tremor, although it is not certain that this benefit is superior to what can be obtained with agents such as levodopa and dopamine agonists. Still, they can be helpful in individual patients with severe tremor. Their use is limited particularly in the elderly, due to their propensity to induce a variety of side effects including urinary dysfunction, glaucoma, and particularly cognitive impairment. Carbidopa/levodopa 10/100, 25/100, 25/ 200–1000 mg 250 mg levodopa/d 2–4 times/d Benserazide/levodopa 25/100, 50/200 mg Carbidopa/levodopa CR 25/100, 50/200 mg Benserazide/levodopa 25/200, 25/250 mg MDS Parcopa 10/100, 25/100, 25/250 Carbidopa/levodopa/ 12.5/50/200, entacapone 18.75/75/200, 25/100/200, 31.25/125/200, 37.5/150/200, 50/200/200 mg

1	Benserazide/levodopa 25/200, 25/250 mg MDS Parcopa 10/100, 25/100, 25/250 Carbidopa/levodopa/ 12.5/50/200, entacapone 18.75/75/200, 25/100/200, 31.25/125/200, 37.5/150/200, 50/200/200 mg Pramipexole 0.125, 0.25, 0.5, 1.0, 1.5 0.25–1.0 mg tid mg Pramipexole ER 0.375, 0.75, 1.5. 3.0, 4.5 1–3 mg/d mg Ropinirole 0.25, 0.5, 1.0, 3.0 mg 6–24 mg/d Ropinirole XL 2, 4, 6, 8 mg 6–24 mg/d Rotigotine patch 2-, 4-, 6-, 8-mg patches 4–24 mg/d Entacapone 200 mg 200 mg with each levodopa dose Tolcapone 100, 200 mg 100–200 mg tid Rasagiline 0.5, 1.0 mg 1.0 mg QAM aTreatment should be individualized. Generally, drugs should be started in low doses and titrated to optimal dose. Note: Drugs should not be withdrawn abruptly but should be gradually lowered or removed as appropriate. Abbreviations: COMT, catechol-O-methyltransferase; MAO-B, monoamine oxidase type B; QAM, every morning.

1	Abbreviations: COMT, catechol-O-methyltransferase; MAO-B, monoamine oxidase type B; QAM, every morning. Amantadine also has historical importance. Originally introduced as an antiviral agent, it was appreciated to also have antiparkinsonian effects that are now thought to be due to N-methyl-D-aspartate (NMDA) receptor antagonism. While some physicians use amantadine in patients with early disease for its mild symptomatic effects, it is most widely used as an antidyskinesia agent in patients with advanced PD. Indeed, it is the only oral agent that has been demonstrated in controlled studies to reduce dyskinesia without worsening parkinsonian features, although benefits may be relatively transient. Cognitive impairment is a major concern. Other side effects include livido reticularis and weight gain. Amantadine should always be discontinued gradually because patients can experience withdrawal-like symptoms.

1	Several new classes of drug are currently being investigated in an attempt to enhance antiparkinsonian effects, reduce off time, and treat or prevent dyskinesia. These include adenosine A2A antagonists, nicotinic agonists, glutamate antagonists, and 5-HT1A agonists. A list of the major drugs and available dosage strengths is provided in Table 449-5.

1	A list of the major drugs and available dosage strengths is provided in Table 449-5. Despite the many therapeutic agents available for the treatment of PD, patients continue to experience disease progression with intolerable disability. A neuroprotective therapy that slows or stops disease progression remains the major unmet therapeutic need in PD. As noted above, trials of certain drugs (e.g., selegiline and rasagiline) have provided positive results consistent with a disease-modifying effect. However, it is not possible to determine if the positive results were due to neuroprotection with slowing of disease progression or confounding symptomatic effects that mask ongoing progression. CoQ10, a mitochondrial bioenhancer and antioxidant, attracted attention with a positive preliminary trial, but this was not replicated in larger double-blind studies.

1	Surgical treatments for PD have been used for more than a century. Lesions placed in the motor cortex improved tremor but were associated with motor deficits, and this approach was abandoned. Subsequently, it was appreciated that lesions placed into the ventral intermediate (VIM) nucleus of the thalamus reduced contralateral tremor without inducing hemiparesis, but these lesions did not meaningfully help other more disabling features of PD. In the 1990s, it was shown that lesions placed in the posteroventral portion of the GPi (motor territory) improved rigidity and bradykinesia as well as tremor. Importantly, pallidotomy was also associated with marked improvement in contralateral dyskinesia. This procedure gained favor with greater understanding of the pathophysiology of PD (see above). However, this procedure is not optimal for patients with bilateral disease, because bilateral lesions are associated with side effects such as dysphagia, dysarthria, and impaired cognition, and has

1	However, this procedure is not optimal for patients with bilateral disease, because bilateral lesions are associated with side effects such as dysphagia, dysarthria, and impaired cognition, and has largely been replaced by deep brain stimulation (DBS). Unilateral lesions of the STN are associated with a larger antiparkinsonian benefit and reduced levodopa requirement, but there is a concern about the risk of hemiballismus, and this procedure is not commonly performed.

1	Most surgical procedures for PD performed today use DBS. Here, an electrode is placed into the target area and connected to a stimulator inserted SC over the chest wall. DBS simulates the effects of a lesion without necessitating making a brain lesion. The precise mechanism whereby DBS works is not fully resolved but may act by disrupting the abnormal signal associated with PD and motor complications. The stimulation variables can be adjusted with respect to electrode configuration, voltage, frequency, and pulse duration in order to maximize benefit and minimize adverse side effects. In cases with intolerable side effects, stimulation can be stopped and the system removed. The procedure does not require making a lesion in the brain and is thus suitable for performing bilateral procedures with relative safety.

1	DBS for PD primarily targets the STN or the GPi. It provides dramatic results, particularly with respect to reducing “off” time and dyskinesias, but does not improve or prevent the development of features that fail to respond to levodopa such as freezing, falling, and dementia. The procedure is thus primarily indicated for patients who suffer disability resulting from severe tremor, or levodopa-induced motor complications that cannot be satisfactorily controlled with drug manipulation. In such patients, DBS has been shown to improve quality of life in comparison to best medical therapy. Side effects can be seen with respect to the surgical procedure (hemorrhage, infarction, infection), the DBS system (infection, lead break, lead displacement, skin ulceration), or the stimulation itself (ocular and speech abnormalities, muscle twitches, paresthesias, depression, and rarely suicide). Recent studies indicate that benefits following DBS of the STN and GPi are comparable, but that GPi

1	(ocular and speech abnormalities, muscle twitches, paresthesias, depression, and rarely suicide). Recent studies indicate that benefits following DBS of the STN and GPi are comparable, but that GPi stimulation may be associated with a reduced frequency of depression. Although not all PD patients are candidates, the procedure is profoundly beneficial for many. Studies of DBS in early PD patients show benefits in comparison to medical therapy, but this must be weighed against the cost of the procedure and the risk of side effects. Long-term studies demonstrate continued benefits with respect to the classical motor features of PD, but DBS does not prevent the development of nondopaminergic features, which continue to be a source of disability. Studies continue to evaluate the optimal way to use DBS (lowvs high-frequency stimulation, closed systems, etc.). Comparison of DBS to other therapies aimed at improving motor function without causing dyskinesia, such as Duodopa and apomorphine

1	to use DBS (lowvs high-frequency stimulation, closed systems, etc.). Comparison of DBS to other therapies aimed at improving motor function without causing dyskinesia, such as Duodopa and apomorphine infusions, remains to be performed. Studies are examining additional DBS targets that might benefit gait dysfunction, depression, and cognitive impairment in PD patients.

1	There has been considerable scientific and public interest in a number of novel interventions that are being investigated as possible treatments for PD. These include cell-based therapies (such as transplantation of fetal nigral dopamine cells or dopamine neurons derived from stem cells), gene therapies, and trophic factors. Transplant strategies are based on the concept of implanting dopaminergic cells into the striatum to replace degenerating SNc dopamine neurons. Fetal nigral mesencephalic cells have been demonstrated to survive implantation, re-innervate the striatum in an organotypic manner, and restore motor function in PD models. However, two double-blind studies failed to show significant benefit of fetal nigral transplantation in comparison to a sham operation with respect to their primary endpoints. Additionally, grafting of fetal nigral cells is associated with a previously unrecognized form of dyskinesia that persists after lowering or even stopping levodopa. This has been

1	primary endpoints. Additionally, grafting of fetal nigral cells is associated with a previously unrecognized form of dyskinesia that persists after lowering or even stopping levodopa. This has been postulated to be related to unregulated release of dopamine from serotonin neurons. In addition, there is evidence that after many years, transplanted healthy embryonic dopamine neurons from unrelated donors develop PD pathology and become dysfunctional, suggesting transfer of α-synuclein from affected to unaffected neurons in a prion-like manner (see discussion above). Perhaps most importantly, it is not clear how replacing dopamine cells alone will improve nondopaminergic features such as falling and dementia, which are the major sources of disability for patients with advanced disease. These same concerns apply to dopamine neurons derived from stem cells, which have not yet been properly tested in PD patients and bear the additional concern of tumors and unanticipated side effects. The

1	same concerns apply to dopamine neurons derived from stem cells, which have not yet been properly tested in PD patients and bear the additional concern of tumors and unanticipated side effects. The short-term future for this technology as a treatment for PD, at least in its current state, is therefore not promising, and there is no scientific basis to warrant routine treatment with stem cells as is being marketed in some countries.

1	Trophic factors are a series of proteins that enhance neuronal growth and restore function to damaged neurons. There are several different trophic factors that have been demonstrated to have beneficial effects on dopamine neurons in laboratory studies. Glialderived neurotrophic factor (GDNF) and neurturin have attracted particular attention as possible therapies for PD. However, double-blind trials of intraventricular and intraputaminal infusions of GDNF failed to show benefits compared to placebo in PD patients, possibly because of inadequate delivery of the trophic molecule throughout the target region.

1	Gene delivery offers the potential of providing widespread delivery throughout a target region and long-term expression of a therapeutic protein with a single procedure. Gene therapy involves placing the DNA of a therapeutic protein into a viral vector that can then be delivered to specific target regions. The DNA of the therapeutic protein is then incorporated into the genome of the host cells and released on a continual basis. The AAV2 virus has been most often used as the viral vector because it does not promote an inflammatory response, is not incorporated into the host genome, and is associated with long-lasting transgene expression. Clinical trials of AAV2 delivery of the trophic factor neurturin showed promising results in open label trials but failed in double-blind trials, possibly because axonal damage in PD prevented retrograde transport of the protein to dopamine neurons in the SNc where it is required to induce upregulation of repair genes required for the trophic

1	possibly because axonal damage in PD prevented retrograde transport of the protein to dopamine neurons in the SNc where it is required to induce upregulation of repair genes required for the trophic response. However, a subsequent double-blind trial of AAV2-neurturin delivered into both the putamen and SNc also failed.

1	Gene delivery is also being explored as a means of delivering aromatic amino acid decarboxylase with or without tyrosine hydroxy-lase to the striatum to facilitate dopamine production and glutamic acid decarboxylase to the STN to inhibit overactive neuronal firing. None of these procedures has been established to be effective in PD patients. Furthermore, although gene delivery technology has great potential, this approach also carries the risk of unanticipated side 2617 effects, and current approaches directed at the nigrostriatal system do not address the nondopaminergic features of the illness.

1	Although PD management has primarily focused on the dopaminergic features of the disease, management of the nondopaminergic features should not be ignored. Some nonmotor features, although not thought to reflect dopaminergic pathology, nonetheless benefit from dopaminergic drugs. For example, problems such as anxiety, panic attacks, depression, sweating, sensory problems, freezing, and constipation all tend to be worse during “off” periods and may improve with better dopaminergic control. Approximately 50% of PD patients suffer depression during the course of the disease, and depression is frequently underdiagnosed and undertreated. Antidepressants should not be withheld, particularly for patients with major depression. Serotonin syndromes have been a theoretical concern with the combined use of SSRIs and MAO-B inhibitors but are rarely encountered. Anxiety can be treated with short-acting benzodiazepines.

1	Psychosis can be a problem for some PD patients. In contrast to AD, hallucinations are typically visual, formed, and nonthreatening. Importantly, they can limit the use of dopaminergic agents to obtain satisfactory motor control. Psychosis in PD often responds to low doses of atypical neuroleptics and permits higher doses of levodopa to be tolerated. Clozapine is the most effective drug, but it can be associated with agranulocytosis, and regular monitoring is required. For this reason, many physicians start with quetiapine even though it has not been established to be effective in placebocontrolled trials. Hallucinations in PD patients are often a harbinger of a developing dementia.

1	Dementia in PD (PDD) is common, ultimately affecting as many as 80% of patients. Its frequency increases with aging and, in contrast to AD, primarily affects executive functions and attention, with relative sparing of language, memory, and calculations. When dementia precedes, or develops within 1 year after, the onset of motor dysfunction, it is by convention referred to as dementia with Lewy bodies (DLB; Chap. 448). These patients are particularly prone to have hallucinations and diurnal fluctuations. Pathologically, DLB is characterized by Lewy bodies distributed throughout the cerebral cortex (especially the hippocampus and amygdala) and is often also associated with AD pathology. It is likely that DLB and PDD represent a PD spectrum rather than separate disease entities. Mild cognitive impairment (MCI) frequently precedes the onset of dementia and is a more reliable index of impending dementia in PD than in the general population. Dopaminergic drugs can worsen cognitive function

1	impairment (MCI) frequently precedes the onset of dementia and is a more reliable index of impending dementia in PD than in the general population. Dopaminergic drugs can worsen cognitive function in demented patients and should be stopped or reduced to try and provide a compromise between antiparkinsonian benefit and preserved cognitive function. Drugs are usually discontinued in the following sequence: anticholinergics, amantadine, dopamine agonists, COMT inhibitors, and MAO-B inhibitors. Eventually, patients with cognitive impairment should be managed with the lowest dose of standard levodopa that provides meaningful antiparkinsonian effects and does not worsen mental function. Anticholinesterase agents such as rivastigmine and donepezil reduce the rate of deterioration of measures of cognitive function and can improve attention, but do not typically improve cognitive function in any meaningful way.

1	Autonomic disturbances are common and frequently require attention. Orthostatic hypotension can be problematic and contribute to falling. Initial treatment should include adding salt to the diet and elevating the head of the bed to prevent overnight sodium natriuresis. Low doses of fludrocortisone (Florinef) or midodrine provide control for most cases. Vasopressin, erythropoietin, and the norepinephrine precursor 3-0-methylDOPS can be used in more severe or refractory cases. If orthostatic hypotension is prominent in early disease, MSA should be considered. Sexual dysfunction can be helped with sildenafil or tadalafil. Urinary problems, especially in males, should be treated in consultation with a urologist to exclude 2618 prostate problems. Anticholinergic agents, such as oxybutynin (Ditropan), may be helpful. Constipation can be a very important problem for PD patients. Mild laxatives or enemas can be useful, but physicians should first ensure that patients are drinking adequate

1	may be helpful. Constipation can be a very important problem for PD patients. Mild laxatives or enemas can be useful, but physicians should first ensure that patients are drinking adequate amounts of fluid and consuming a diet rich in bulk with green leafy vegetables and bran. Agents that promote gastrointestinal (GI) motility can also be helpful. Sleep disturbances are common in PD patients, with many experiencing fragmented sleep with excess daytime sleepiness. Restless leg syndrome, sleep apnea, and other sleep disorders should be treated as appropriate. REM behavior disorder (RBD) is a syndrome comprised of violent movements and vocalizations during REM sleep, possibly representing acting out of dreams due to a failure of the normal inhibition of motor movements that typically accompanies REM sleep. Many PD patients have a history of antecedent RBD preceding the onset of the classic motor features of PD, and most cases of RBD go on to develop an α-synucleinopathy (PD or MSA). Low

1	REM sleep. Many PD patients have a history of antecedent RBD preceding the onset of the classic motor features of PD, and most cases of RBD go on to develop an α-synucleinopathy (PD or MSA). Low doses of clonazepam (0.5–1 mg at bedtime) are usually effective in controlling this problem. Consultation with a sleep specialist and polysomnography may be necessary to identify and optimally treat sleep problems.

1	Gait dysfunction with falling is an important cause of disability in PD. Dopaminergic therapies can help patients whose gait is worse in “off” time, but there are currently no specific therapies available. Canes and walkers may become necessary to increase stability and reduce the risk of falling. Freezing, where patients suddenly become stuck in place for seconds to minutes as if their feet were glued to the ground, is a major cause of falling. Freezing may occur during “on” or “off” periods. Freezing during “off” periods may respond to dopaminergic therapies, but there are no specific treatments for “on” period freezing. Some patients will respond to sensory cues such as marching in place, singing a song, or stepping over an imaginary line.

1	Exercise, with a full range of active and passive movements, has been shown to maintain and even improve function for PD patients, and active and passive exercises with full range of motion reduce the risk of arthritis and frozen joints. Some laboratory studies suggest the possibility that exercise might also have neuroprotective effects, but this has not been confirmed in PD. Exercise is generally recommended for all PD patients. It is less clear that physical therapy or specific exercises such as tai chi are required. It is important for patients to maintain social and intellectual activities to the extent possible. Education, assistance with financial planning, social services, and attention to home safety are important elements of the overall care plan. Information is available through numerous PD foundations and on the web, but should be reviewed with physicians to ensure accuracy. The needs of the caregiver should not be neglected. Caring for a person with PD involves a

1	through numerous PD foundations and on the web, but should be reviewed with physicians to ensure accuracy. The needs of the caregiver should not be neglected. Caring for a person with PD involves a substantial work effort and there is an increased incidence of depression among caregivers. Support groups for patients and caregivers may be useful.

1	The management of PD should be tailored to the needs of the individual patient, and there is no single treatment approach that is universally accepted and applicable to all individuals. Clearly, if an agent could be demonstrated to have disease-modifying effects, it should be initiated at the time of diagnosis. Indeed, constipation, RBD, and anosmia may represent premotor features of PD and could permit the initiation of a disease-modifying therapy prior to the onset of the classical motor features of the disease. However, no therapy has yet been proved to be disease modifying. For now, physicians must use their judgment in deciding whether or not to introduce rasagiline (see above) or other drugs for their possible disease-modifying effects.

1	The next important issue to address is when to initiate symptomatic therapy. Several studies now suggest that it may be best to start therapy at the time of diagnosis (or soon after) in order to preserve beneficial compensatory mechanisms and possibly provide functional benefits even in the early stage of the disease. Levodopa remains the most effective symptomatic therapy for PD, and some recommend starting it immediately using low doses (≤400 mg/d), but others prefer to delay levodopa treatment, particularly in younger patients, in order to reduce the risk of inducing motor complications. An alternate approach is to begin with an MAO-B inhibitor and/or a dopamine agonist, and reserve levodopa for later stages when these drugs can no longer provide satisfactory control. In making this decision, the age, degree of disability, and side effect profile of the drug must all be considered. In patients with more severe disability, the elderly, those with cognitive impairment, or those in

1	decision, the age, degree of disability, and side effect profile of the drug must all be considered. In patients with more severe disability, the elderly, those with cognitive impairment, or those in whom the diagnosis is uncertain, most physicians would initiate therapy with levodopa. Regardless of initial choice, it is important not to deny patients levodopa when they cannot be adequately controlled with alternative medications.

1	If motor complications develop, patients can initially be treated by manipulating the frequency and dose of levodopa or by combining lower doses of levodopa with a dopamine agonist, a COMT inhibitor, or an MAO-B inhibitor. Amantadine is the only drug that has been demonstrated to treat dyskinesia without worsening parkinsonism, but benefits may be short-lasting, and there are important side effects related to cognitive function. In advanced cases, it may be necessary to consider a surgical therapy such as DBS if the patient is a suitable candidate, but as described above, these procedures have their own set of complications. Continuous intraintestinal infusion of levodopa/carbidopa intestinal gel (Duodopa) appears to offer similar benefits to DBS, but also requires a surgical intervention with potentially serious complications. Continuous infusion of apomorphine is another treatment option and does not require surgery but is associated with potentially troublesome skin nodules.

1	with potentially serious complications. Continuous infusion of apomorphine is another treatment option and does not require surgery but is associated with potentially troublesome skin nodules. Comparative studies of these approaches in more advanced patients are awaited. There are ongoing efforts aimed at developing a long-acting oral or transdermal formulation of levodopa that mirrors the pharmacokinetic properties of a levodopa infusion. Such a formulation might provide all of the benefits of levodopa without motor complications and avoid the need for polypharmacy and surgical intervention.

1	A decision tree that considers the various treatment options and decision points for the management of PD is provided in Fig. 449-7. Hyperkinetic movement disorders are characterized by involuntary movements unaccompanied by weakness and occurring in isolation or in combination (Table 449-6). The major hyperkinetic movement disorders and the diseases with which they are associated are considered in this section. Tremor consists of alternating contractions of agonist and antagonist muscles in an oscillating, rhythmic manner. It can be most prominent at rest (rest tremor), on assuming a posture (postural tremor), or on actively reaching for a target (kinetic tremor). Tremor is also assessed based on distribution, frequency, and related neurologic dysfunction.

1	PD is characterized by a resting tremor, essential tremor (ET) by a postural tremor (trying to sustain a posture), and cerebellar disease by an intention or kinetic tremor (on reaching to touch a target). Normal individuals can have a physiologic tremor that typically manifests as a mild, high-frequency (10–12 Hz), postural or action tremor that is usually of no clinical consequence and often is only appreciated with an accelerometer. An enhanced physiologic tremor (EPT) can be seen in up to 10% of the population, often in association with anxiety, fatigue, a metabolic disturbance (e.g., hyperthyroidism, electrolyte abnormalities), drugs (e.g., valproate, lithium), or toxins (e.g., alcohol). Treatment is initially directed to the control of any underlying disorder and, if necessary, can often be improved with a beta blocker.

1	Functional disability Parkinson’s disease Surgery/CDS Combination therapy Levodopa/dopamine agonist/COMT Inhibitor/MAO-B Inhibitor Nonpharmacologic intervention Pharmacologic intervention Neuroprotection —? Rasagiline Yes Levodopa No Dopamine agonists

1	FIGURE 449-7 Treatment options for the management of Parkinson’s disease (PD). Decision points include: (1) Introduction of a neuroprotective therapy: No drug has been established to have or is currently approved for neuroprotection or disease modification, but there are several agents that have this potential based on laboratory and preliminary clinical studies (e.g., rasagiline 1 mg/d, coenzyme Q10 1200 mg/d, the dopamine agonists ropinirole, and pramipexole). (2) When to initiate symptomatic therapy: There is a trend toward initiating therapy at the time of diagnosis or early in the course of the disease because patients may have some disability even at an early stage, and there is the possibility that early treatment may preserve beneficial compensatory mechanisms; however, some experts recommend waiting until there is functional disability before initiating therapy. (3) What therapy to initiate: Many experts favor starting with a monoamine oxidase type B (MAO-B) inhibitor in

1	recommend waiting until there is functional disability before initiating therapy. (3) What therapy to initiate: Many experts favor starting with a monoamine oxidase type B (MAO-B) inhibitor in mildly affected patients because of the good safety profile of the drug and the potential for a disease-modifying effect; dopamine agonists for younger patients with functionally significant disability to reduce the risk of motor complications; and levodopa for patients with more advanced disease, the elderly, or those with cognitive impairment. Recent studies suggest the early employment of polypharmacy using low doses of multiple drugs to avoid side effects associated with high doses of any one agent. (4) Management of motor complications: Motor complications are typically approached with combination therapy to try and reduce dyskinesia and enhance the “on” time. When medical therapies cannot provide satisfactory control, surgical therapies such as DBS or continuous infusion of

1	with combination therapy to try and reduce dyskinesia and enhance the “on” time. When medical therapies cannot provide satisfactory control, surgical therapies such as DBS or continuous infusion of levodopa/carbidopa intestinal gel can be considered. (5) Nonpharmacologic approaches: Interventions such as exercise, education, and support should be considered throughout the course of the disease. CDS, continuous dopaminergic stimulation; COMT, catechol-O-methyltransferase. (Adapted from CW Olanow et al: Neurology 72:S1, 2009.)

1	ET is the commonest movement disorder, affecting approximately 5–10 million persons in the United States. It can present in childhood but dramatically increases in prevalence over the age of 70 years. ET is characterized by a high-frequency tremor (6–10 Hz) that predominantly affects the upper extremities. The tremor is most often manifest as a postural or action (kinetic) tremor and, in severe cases, can interfere with functions such as eating and drinking. It is typically bilateral and symmetric but may begin on one side and remain asymmetric. Patients with severe ET can have an intention tremor with overshoot and slowness of movement. Tremor involves the head in ~30% of cases, voice in ~20%, tongue in ~20%, face/jaw in ~10%, and lower limbs in ~10%. The tremor is characteristically improved by alcohol and worsened by stress. Subtle impairment of coordination or tandem walking may be present, and disturbances of hearing, cognition, personality, mood, and olfaction have also been

1	improved by alcohol and worsened by stress. Subtle impairment of coordination or tandem walking may be present, and disturbances of hearing, cognition, personality, mood, and olfaction have also been described, but usually the neurologic examination is normal aside from tremor. The major differential is a dystonic tremor (see below) or PD. PD can usually be differentiated from ET based on the presence of bradykinesia, rigidity, micrographia, and other parkinsonian features. However, the examiner should be aware that PD patients may have a postural tremor and ET patients may develop a rest tremor. These typically begin after a latency of a few seconds (emergent tremor). The examiner must take care to differentiate the effect of tremor on measurement of tone in ET from the cogwheel rigidity found in PD.

1	The etiology and pathophysiology of ET are not known. Approximately 50% of cases have a positive family history with an autosomal dominant pattern of inheritance. Linkage studies have detected loci at chromosomes 3q13 (ETM-1), 2p22-25 (ETM-2), and 6p23 (ETM-3), but no causative genes have been identified to date. GWAS demonstrated an association with the LINGO1 gene, which is involved in oligodendrocyte differentiation and myelination, particularly in patients with young-onset ET. Recently, a nonsense mutation in the fused in sarcoma (FUS) gene was implicated as a cause of ET in a multigenerational family from Canada; this finding is of particular interest because different mutations in FUS are a known cause of familial amyotrophic lateral sclerosis (Chap. 452). It is likely that there are many other undiscovered genes for ET. The cerebellum and inferior olives have been implicated as possible sites of a “tremor pacemaker” based on the presence of cerebellar signs and increased

1	are many other undiscovered genes for ET. The cerebellum and inferior olives have been implicated as possible sites of a “tremor pacemaker” based on the presence of cerebellar signs and increased metabolic activity and blood flow in these regions in some patients. Some pathologic studies have described cerebellar pathology with a loss of Purkinje cells and axonal torpedoes, but these findings are controversial and the precise pathologic correlate of ET remains to be defined.

1	Many cases are mild and require no treatment other than reassurance. Occasionally, tremor can be severe and interfere with eating, writing, and activities of daily living. This is more likely to occur as the patient ages and is often associated with a reduction in tremor frequency. Beta blockers and primidone are the standard drug therapies for ET and help in about 50% of cases. Propranolol (20–120 mg daily, given in 2620 divided doses) is usually effective at relatively low doses, but higher doses may be effective in some patients. The drug is contraindicated in patients with bradycardia or asthma. Hand tremor tends to be most improved, while head tremor is often refractory. Primidone can be helpful but should be started at low doses (12.5 mg) and gradually increased (125–250 mg tid) to avoid sedation. Benefits have also been reported with gabapentin and topiramate. Botulinum toxin injections may be helpful for limb or voice tremor, but treatment can be associated with secondary

1	to avoid sedation. Benefits have also been reported with gabapentin and topiramate. Botulinum toxin injections may be helpful for limb or voice tremor, but treatment can be associated with secondary muscle weakness. Surgical therapies targeting the VIM nucleus of the thalamus can be very effective for severe and drug-resistant cases.

1	Dystonia is a disorder characterized by sustained (>100 ms) or repetitive involuntary muscle contractions frequently associated with twisting and abnormal postures. Dystonia can range from minor contractions in an individual muscle group to severe and disabling involvement of multiple muscle groups. The frequency is estimated to be 300,000 cases in the United States but is likely to be much higher because many cases are not recognized. Dystonia is often brought out by voluntary movements (action dystonia) and can extend to involve muscle groups and body regions not required for a given action (overflow). It can be aggravated by stress and fatigue and attenuated by relaxation and sensory tricks such as touching the affected body part (geste antagoniste). Dystonia can be classified according to age of onset (childhood vs adult), distribution (focal, multifocal, segmental, or generalized), or etiology (primary or secondary).

1	At least 16 gene mutations are associated with dystonia and classified as DYT1–DYT16. Idiopathic torsion dystonia (DYT1) or Oppenheim’s dystonia is predominantly a childhood-onset form of dystonia with an autosomal dominant pattern of inheritance that primarily affects Ashkenazi Jewish families. The majority of patients have an age of onset younger than 26 years (mean 14 years). In young-onset patients, dystonia typically begins in the foot or the arm and in 60–70% progresses to involve other limbs as well as the head and neck. In severe cases, patients can suffer disabling postural deformities that compromise mobility. Severity can vary within family members, with some affected relatives having severe disability and others a mild dystonia that may not even be appreciated. Most childhood-onset cases are linked to a mutation in the DYT1 gene located on chromosome 9q34, resulting in a trinucleotide GAG deletion with loss of one of a pair of glutamic acid residues in the protein torsin

1	cases are linked to a mutation in the DYT1 gene located on chromosome 9q34, resulting in a trinucleotide GAG deletion with loss of one of a pair of glutamic acid residues in the protein torsin A. DYT1 mutations are found in 90% of Ashkenazi Jewish patients with DYT1 dystonia and probably relate to a founder effect that occurred about 350 years ago. There is variable penetrance, with only about 30% of gene carriers expressing a clinical phenotype. Why some gene carriers express dystonia and others do not is not known. The function of torsin A is unknown, but it is a member of the AAA+ (ATPase) family that resembles heat-shock proteins and may be related to protein processing and transport. The precise pathology responsible for DYT1 dystonia is not known.

1	Dopa-responsive dystonia (DRD) or the Segawa variant (DYT5) is a dominantly inherited form of childhood-onset dystonia caused by a mutation in the gene that encodes GTP cyclohydrolase-I, the rate-limiting enzyme for the synthesis of tetrahydrobiopterin. This mutation leads to a defect in the biochemical synthesis of tyrosine hydroxylase, the rate-limiting enzyme in the formation of dopamine. DRD typically presents in early childhood (1–12 years) and is characterized by foot dystonia that interferes with walking. Patients often experience diurnal fluctuations, with worsening of gait as the day progresses and improvement with sleep. DRD is typified by an excellent and sustained response to small doses of levodopa. Some patients may present with parkinsonian features, but can be differentiated from juvenile PD by normal striatal dopamine imaging and the absence of levodopainduced dyskinesias. DRD may occasionally be confused with cerebral palsy because patients appear to have spasticity,

1	from juvenile PD by normal striatal dopamine imaging and the absence of levodopainduced dyskinesias. DRD may occasionally be confused with cerebral palsy because patients appear to have spasticity, increased reflexes, and Babinski responses (which likely reflect a dystonic contraction rather than an upper motor neuron lesion). Any patient suspected of having a childhood-onset dystonia should receive a trial of levodopa to exclude this treatable condition.

1	Mutations in the THAP1 gene (DYT6) on chromosome 8p21q22 have been identified in Amish families and are the cause of as many as 25% of cases of non-DYT1 young-onset primary torsion dystonia. These patients are more likely to have dystonia beginning in the brachial and cervical muscles, which later can become generalized and associated with speech impairment. Myoclonic dystonia (DYT11) results from a mutation in the epsilon-sarcoglycan gene on chromosome 7q21. It typically manifests as a combination of dystonia and myoclonic jerks, frequently accompanied by psychiatric disturbances.

1	These are the most common forms of dystonia. They typically present in the fourth to sixth decades and affect women more than men. The major types are as follows: (1) blepharospasm—dystonic contractions of the eyelids with increased blinking that can interfere with reading, watching television, and driving. This can sometimes be so severe as to cause functional blindness. (2) Oromandibular dystonia (OMD)—contractions of muscles of the lower face, lips, tongue, and jaw (opening or closing). Meige’s syndrome is a combination of OMD and blepharospasm that predominantly affects women older than age 60 years. (3) Spasmodic dysphonia—dystonic contractions of the vocal cords during phonation, causing impaired speech. Most cases affect the adductor muscles and cause speech to have a choking or strained quality. Less commonly, the abductors are affected, leading to speech with a breathy or whispering quality. (4) Cervical dystonia—dystonic contractions of neck muscles causing the head to

1	or strained quality. Less commonly, the abductors are affected, leading to speech with a breathy or whispering quality. (4) Cervical dystonia—dystonic contractions of neck muscles causing the head to deviate to one side (torticollis), in a forward direction (anterocollis), or in a backward direction (retrocollis). Muscle contractions can be painful and associated with a secondary cervical radiculopathy. (5) Limb dystonias—these can be present in either arms or legs and are often brought out by task-specific activities such as handwriting (writer’s cramp), playing a musical instrument (musician’s cramp), or putting (the yips). Focal dystonias can extend to involve other body regions (about 30% of cases) and are frequently misdiagnosed as psychiatric or orthopedic in origin. Their cause is not known, but genetic factors, autoimmunity, and trauma have been suggested. Focal dystonias are often associated with a high-frequency tremor that resembles ET. Dystonic tremor can usually be

1	is not known, but genetic factors, autoimmunity, and trauma have been suggested. Focal dystonias are often associated with a high-frequency tremor that resembles ET. Dystonic tremor can usually be distinguished from ET because it tends to occur in conjunction with the dystonic contraction and disappears when the dystonia is relieved.

1	These develop as a consequence of drugs or other neurologic disorders. Drug-induced dystonia is most commonly seen with neuroleptic drugs or after chronic levodopa treatment in PD patients and may be acute or chronic (see below). Secondary dystonia can also be observed following discrete lesions in the striatum and occasionally in the pallidum, thalamus, cortex, and brainstem due to infarction, anoxia, metabolic disorders, trauma, tumor, infection, or toxins such as manganese or carbon monoxide. In these cases, dystonia often assumes a segmental distribution, but it can be generalized when lesions are bilateral or widespread. More rarely, dystonia can develop following peripheral nerve injury and be associated with features of complex regional pain syndrome (Chap. 454). A psychogenic origin is responsible for some cases of dystonia presenting with fixed, immobile dystonic postures (see below).

1	Dystonia may occur as a part of another neurodegenerative conditions such as Huntington’s disease, PD, Wilson’s disease, corticobasilar ganglionic degeneration, PSP, the Lubag form of dystonia-parkinsonism (DYT3), and mitochondrial encephalopathies. In contrast to the primary dystonias, dystonia is usually not the dominant neurologic feature in these conditions.

1	The pathophysiologic basis of dystonia is not completely known. The phenomenon is characterized by co-contracting synchronous bursts of agonist and antagonist muscle groups with recruitment of muscle groups that are not required for a given movement (overflow). Dystonia is characterized by derangement of the basic physiological principle of action-selection, leading to abnormal recruitment of inappropriate muscles for a given action with inadequate inhibition of this undesired motor activity. Physiologically, loss of inhibition is observed at multiple levels of the motor system (e.g., cortex, brainstem, spinal cord) accompanied by increased cortical excitability and reorganization. Attention has focused on the basal ganglia as the site of origin of at least some types of dystonia because there are alterations in blood flow and metabolism in these structures. Further, lesions of the GPi can induce dystonia, and surgical ablation or DBS of the globus pallidus can ameliorate dystonia.

1	there are alterations in blood flow and metabolism in these structures. Further, lesions of the GPi can induce dystonia, and surgical ablation or DBS of the globus pallidus can ameliorate dystonia. The dopamine system has also been implicated, because dopaminergic therapies can both induce and treat some forms of dystonia. Interestingly, no specific pathology has been consistently identified in primary dystonia.

1	Treatment of dystonia is for the most part symptomatic except in rare cases where correction of a primary underlying condition is possible. Wilson’s disease should be ruled out in young patients with dystonia. Levodopa should be tried in all cases of childhood-onset dystonia to rule out DRD. High-dose anticholinergics (e.g., trihexyphenidyl 20–120 mg/d) may be beneficial in children, but adults can rarely tolerate high doses because of side effects related to cognitive impairment with hallucinations. Oral baclofen (20–120 mg) may also be helpful, but benefits, if present, are usually modest, and side effects of sedation, weakness, and memory loss can be problematic. Intrathecal infusion of baclofen is more likely to be useful, particularly for leg and trunk dystonia, but benefits are frequently not sustained, and complications can be serious and include infection, seizures, and coma. Tetrabenazine (the usual starting dose is 12.5 mg/d and the average treating dose is 25–75 mg/d) is

1	not sustained, and complications can be serious and include infection, seizures, and coma. Tetrabenazine (the usual starting dose is 12.5 mg/d and the average treating dose is 25–75 mg/d) is another consideration, but use may be limited by sedation and the development of parkinsonism. Neuroleptics can improve as well as induce dystonia, but they are typically not recommended because of their potential to induce parkinsonism and other movement disorders, including tardive dystonia. Clonazepam and diazepam are rarely effective.

1	Botulinum toxin has become the preferred treatment for patients with focal dystonia, particularly where involvement is limited to small muscle groups such as in blepharospasm, torticollis, and spasmodic dysphonia. Botulinum toxin acts by blocking the release of acetylcholine at the neuromuscular junction, leading to reduced dystonic muscle contractions, but excessive weakness may ensue and can be troublesome particularly if it involves neck and swallowing muscles. Two serotypes of botulinum toxin are available (A and B). Both are effective, and it is not clear that there are advantages of one over the other. No systemic side effects are encountered with the doses typically used, but benefits are transient, and repeat injections are required at 2to 5-month intervals. Some patients fail to respond after having experienced an initial benefit. This has been attributed to antibody formation, but improper muscle selection, injection technique, and inadequate dose should be excluded.

1	Surgical therapy is an alternative for patients with severe dystonia who are not responsive to other treatments. Peripheral procedures such as rhizotomy and myotomy were used in the past to treat cervical dystonia, but are now rarely used. DBS of the pallidum can provide dramatic benefits for patients with primary DYT1 dystonia. This represents a major therapeutic advance because previously there was no consistently effective therapy, especially for these patients who had severe disability. Benefits tend to be obtained with a lower frequency of stimulation and often occur after a relatively long latency (weeks) in comparison to PD. Better results are typically obtained in younger patients with shorter disease duration. Recent studies suggest that DBS may also be valuable for patients with focal and secondary dystonias, although results are less consistent. Supportive treatments such as physical therapy and education are important and should be a part of the treatment regimen.

1	Physicians should be aware of dystonic storm, a rare but poten-2621 tially fatal condition that can occur in response to a stress situation such as surgery in patients with preexisting dystonia. It consists of the acute onset of generalized and persistent dystonic contractions that can involve the vocal cords or laryngeal muscles, leading to airway obstruction. Patients may experience rhabdomyolysis with renal failure and should be managed in an intensive care unit with airway protection if required. Treatment can be instituted with one or a combination of anticholinergics, diphenhydramine, baclofen, benzodiazepines, and dopaminergic agents. Spasms may be difficult to control, and anesthesia with muscle paralysis may be required. Most, if not all, cases of dystonic storm are due to a secondary cause.

1	HD is a progressive, fatal, highly penetrant autosomal dominant disorder characterized by motor, behavioral, oculomotor, and cognitive dysfunction. The disease is named for George Huntington, a family physician who described cases on Long Island, New York, in the nineteenth century. Onset is typically between the ages of 25 and 45 years (range, 3–70 years) with a prevalence of 2–8 cases per 100,000 and an average age at death of 60 years. It is prevalent in Europe, North and South America, and Australia but is rare in African blacks and Asians. HD is characterized by rapid, nonpatterned, semipurposeful, involuntary choreiform movements, and for this reason was formerly referred to as Huntington’s chorea. In the early stages, the chorea tends to be focal or segmental, but progresses over time to involve multiple body regions. Dysarthria, gait disturbance, oculomotor abnormalities, behavioral disturbance, and cognitive impairment with dementia are also common features. With advancing

1	time to involve multiple body regions. Dysarthria, gait disturbance, oculomotor abnormalities, behavioral disturbance, and cognitive impairment with dementia are also common features. With advancing disease, there tends to be a reduction in chorea and the emergence of dystonia, rigidity, bradykinesia, and myoclonus. Functional decline is often predicted by progressive weight loss despite adequate calorie intake. In younger patients (~10% of cases), HD can present as an akinetic-rigid or parkinsonian syndrome (Westphal variant). HD patients eventually develop behavioral and cognitive disturbances, and the majority progress to dementia. Depression with suicidal tendencies, aggressive behavior, and psychosis can be prominent features. HD patients may also develop non-insulin-dependent diabetes mellitus and neuroendocrine abnormalities (e.g., hypothalamic dysfunction). A clinical diagnosis of HD can be strongly suspected in cases of chorea with a positive family history, but genetic

1	mellitus and neuroendocrine abnormalities (e.g., hypothalamic dysfunction). A clinical diagnosis of HD can be strongly suspected in cases of chorea with a positive family history, but genetic testing provides the ultimate confirmation of the diagnosis. The disease predominantly affects the striatum. Progressive atrophy of the heads of the caudate nuclei, which form the lateral margins of the lateral ventricles, can be visualized by MRI (Fig. 449-8), but the putamen can be equally or even more severely affected. More diffuse cortical atrophy is seen in the middle and late stages of the disease. Supportive studies include reduced metabolic activity in the caudate nucleus and putamen. Genetic testing can be used to confirm the diagnosis and to detect at-risk individuals in the family, but must be performed with caution and in conjunction with trained counselors, because positive results can worsen depression and generate suicidal reactions. The neuropathology of HD consists of prominent

1	be performed with caution and in conjunction with trained counselors, because positive results can worsen depression and generate suicidal reactions. The neuropathology of HD consists of prominent neuronal loss and gliosis in the caudate nucleus and putamen; similar changes are also widespread in the cerebral cortex. Intraneuronal inclusions containing aggregates of ubiquitin and the mutant protein huntingtin are found in the nuclei of affected neurons.

1	In anticipation of developing neuroprotective therapies, there has been an intensive effort to define the premanifest stage of HD. Subtle motor impairment, cognitive alterations, and imaging changes can be detected in at-risk individuals who later go on to develop the manifest form of the disease. Defining the rate of progression of these features is paramount for future studies of putative disease-modifying therapies. HD is caused by an increase in the number of polyglutamine (CAG) repeats (>40) in the coding sequence of the huntingtin gene located on FIGURE 449-8 Huntington’s disease. A. Coronal fluid attenuated inversion recovery (FLAIR) magnetic resonance imaging shows enlargement of the lateral ventricles reflecting typical atrophy (arrows). B. Axial FLAIR image demonstrates abnormal high signal in the caudate and putamen (arrows).

1	the short arm of chromosome 4. The larger the number of repeats, the earlier the disease is manifest. Intermediate forms of the disease with 36–39 repeats are described in some patients, typically with less severe clinical involvement. Acceleration of the process tends to occur, particularly in males, with subsequent generations having larger numbers of repeats and earlier age of disease onset, a phenomenon referred to as anticipation. The gene encodes the highly conserved cytoplasmic protein huntingtin, which is widely distributed in neurons throughout the central nervous system (CNS) but whose function is not known. Models of HD with striatal pathology can be induced by excitotoxic agents such as kainic acid and 3-nitropoprionic acid, which promote calcium entry into the cell and cytotoxicity. Mitochondrial dysfunction has been demonstrated in the striatum and skeletal muscle of symptomatic and presymptomatic individuals. Fragments of the mutant huntingtin protein can be toxic,

1	Mitochondrial dysfunction has been demonstrated in the striatum and skeletal muscle of symptomatic and presymptomatic individuals. Fragments of the mutant huntingtin protein can be toxic, possibly by translocating into the nucleus and interfering with transcriptional regulation of proteins. Neuronal inclusions found in affected regions in HD may represent a protective mechanism aimed at segregating and facilitating the clearance of these toxic proteins.

1	Although the gene for HD was identified more than two decades ago, there is still no disease-modifying therapy for this disorder. Current treatment involves a multidisciplinary approach, with medical, neuropsychiatric, social, and genetic counseling for patients and their families. Dopamine-blocking agents may control the choreatic movements. Tetrabenazine (a presynaptic dopamine depleting agent) has been approved for the treatment of chorea in the United States, but can cause secondary parkinsonism. Neuroleptics are generally not recommended because of their potential to induce other more troubling movement disorders and because HD chorea tends to be self-limited and is usually not disabling. Depression and anxiety can be greater problems, and patients should be treated with appropriate antidepressant and antianxiety drugs and monitored for mania and suicidal ideations. Psychosis can be treated with atypical anti-psychotics such as clozapine (50–600 mg/d), quetiapine (50–600 mg/d),

1	antidepressant and antianxiety drugs and monitored for mania and suicidal ideations. Psychosis can be treated with atypical anti-psychotics such as clozapine (50–600 mg/d), quetiapine (50–600 mg/d), and risperidone (2–8 mg/d). There is no adequate treatment for the cognitive or motor decline. A neuroprotective therapy that slows or stops disease progression is the major unmet medical need in HD. Drugs that enhance mitochondrial function and increase the clearance of defective mitochondria are being tested as possible disease-modifying therapies. Antiglutamate agents, dopamine stabilizers, caspase inhibitors, neurotrophic factors, and transplantation of fetal striatal cells are areas of active research, but none has as yet been demonstrated to have a beneficial effect in HD. The potential to use transcriptional blockade of the mutant huntingtin gene with small interfering RNAs (siRNAs) is an exciting area currently being explored.

1	A group of rare inherited conditions that can mimic HD, designated HD-like (HDL) disorders, have also been identified. HDL-1, -2, and -4 are autosomal dominant conditions that typically present in adulthood. HDL-1 is due to expansion of an octapeptide repeat in PRNP, the gene encoding the prion protein (Chap. 453e). Thus HDL-1 is properly considered a prion disease. Patients exhibit onset of personality change in the third or fourth decade, followed by chorea, rigidity, myoclonus, ataxia, and epilepsy. HDL-2 manifests in the third or fourth decade with a variety of movement disorders, including chorea, dystonia, or parkinsonism and dementia. Most patients are of African descent. Acanthocytosis can sometimes be seen in these patients, and this condition must be distinguished from neuroacanthocytosis. HDL-2 is caused by an abnormally expanded CTG/ CAG trinucleotide repeat expansion in the junctophilin-3 (JPH3) gene. The pathology of HDL-2 consists of intranuclear inclusions

1	neuroacanthocytosis. HDL-2 is caused by an abnormally expanded CTG/ CAG trinucleotide repeat expansion in the junctophilin-3 (JPH3) gene. The pathology of HDL-2 consists of intranuclear inclusions immunoreactive for ubiquitin and expanded polyglutamine repeats. HDL-4, the most common condition in this group, is caused by expansion of trinucleotide repeats in TBP, the gene that encodes the TATA box binding protein involved in regulating transcription; this condition is identical to spinocerebellar ataxia (SCA) 17 (Chap. 451e), and most patients present primarily with ataxia rather than chorea. Mutations of the C9Orf gene associated with amyotrophic lateral sclerosis have also been reported in some individuals with an HDL phenotype.

1	Chorea can be seen in a number of additional disorders. Sydenham’s chorea (originally called St. Vitus’s dance) is more common in females and is typically seen in childhood (5–15 years). It often develops in association with prior exposure to group A streptococcal infection and is thought to be autoimmune in nature. It is characterized by the acute onset of choreiform movements and behavioral disturbances. With the reduction in the incidence of rheumatic fever, the incidence of Sydenham’s chorea has fallen, but it can still be seen in developing countries. The chorea generally responds to dopamine-blocking agents, valproic acid, and carbamazepine, but is self-limited, and treatment is generally restricted to those with severe chorea. Chorea may recur in later life, particularly in association with pregnancy (chorea gravidarum) or treatment with sex hormones. Several reports have documented cases of chorea associated with NMDA receptor antibody–positive encephalitis following herpes

1	with pregnancy (chorea gravidarum) or treatment with sex hormones. Several reports have documented cases of chorea associated with NMDA receptor antibody–positive encephalitis following herpes simplex virus encephalitis.

1	Chorea-acanthocytosis (neuroacanthocytosis) is a progressive and typically fatal autosomal recessive disorder that is characterized by chorea coupled with red cell abnormalities on peripheral blood smear (acanthocytes). The chorea can be severe and associated with self-mutilating behavior, dystonia, tics, seizures, and a polyneuropathy. Mutations in the VPS13A gene encoding chorein have been described. A phenotypically similar X-linked form of the disorder has been described in older individuals who have reactivity with Kell blood group antigens (McLeod syndrome). A benign hereditary chorea of childhood (BHC1) due to mutations in the gene for thyroid transcription factor 1 and a late-onset benign senile chorea (BHC2) have also been described. It is important to ensure that patients with these types of choreas do not have HD.

1	Chorea may also occur in association with vascular diseases, hypoand hyperglycemia, and a variety of infections and degenerative disorders. Systemic lupus erythematosus is the most common systemic disorder that causes chorea, which can last for days to years. Chorea can also be seen with hyperthyroidism, autoimmune disorders including Sjögren’s syndrome, infectious disorders including HIV disease, metabolic alterations, and polycythemia rubra vera; following open-heart surgery in the pediatric population; and in association with many medications (especially anticonvulsants, cocaine, CNS stimulants, estrogens, and lithium). Chorea is commonly seen in association with chronic levodopa treatment (discussed in the section on PD above). Chorea can also be seen in paraneoplastic syndromes associated with anti-CRMP-5 or anti-Hu antibodies (Chap. 122).

1	Hemiballismus is a violent form of chorea comprised of wild, flinging, large-amplitude movements on one side of the body. Proximal limb muscles tend to be predominantly affected. These movements may affect just one limb (monoballism) or, more exceptionally, both upper or lower limbs (paraballism). The movements may be so severe as to cause exhaustion, dehydration, local injury, and, in extreme cases, death. Fortunately, dopamine-blocking drugs can be very helpful, and importantly, hemiballismus is usually self-limiting and tends to resolve spontaneously after weeks or months. The most common cause is a partial lesion (infarct or hemorrhage) in the STN, but cases can also be seen with lesions in the putamen, thalamus, and parietal cortex. In extreme cases, pallidotomy can be very effective. Interestingly, surgically induced lesions and DBS of the STN in PD patients are usually not associated with hemiballismus.

1	A tic is a brief, rapid, recurrent, and seemingly purposeless stereotyped motor contraction. Motor tics can be simple, with movement only affecting an individual muscle group (e.g., blinking, twitching of the nose, jerking of the neck), or complex, with coordinated involvement of multiple muscle groups (e.g., jumping, sniffing, head banging, and echopraxia [mimicking movements]). Phonic (or vocal) tics can also be simple (e.g., grunting) or complex (e.g., echolalia [repeating other people’s words], palilalia [repeating one’s own words], and coprolalia [expression of obscene words]). Patients may also experience sensory tics, composed of unpleasant focal sensations in the face, head, or neck. These can be mild and of little clinical consequence or severe and disabling to the patient.

1	TS is a neurobehavioral disorder named after the French neurologist Georges Gilles de la Tourette. It predominantly affects males, and the prevalence is estimated to be 0.03–1.6%, but it is likely that many mild cases do not come to medical attention. TS is characterized by multiple motor tics often accompanied by vocalizations (phonic tics). Patients characteristically can voluntarily suppress tics for short periods of time, but then experience an irresistible urge to express them. Tics vary in intensity and may be absent for days or weeks only to recur, occasionally in a different pattern. Tics tend to present between ages 2 and 15 years (mean 7 years) and often lessen or even disappear in adulthood. Associated behavioral disturbances include anxiety, depression, attention deficit hyperactivity disorder, and obsessive-compulsive disorder. Patients may experience personality disorders, self-destructive behaviors, difficulties in school, and impaired interpersonal relationships. Tics

1	disorder, and obsessive-compulsive disorder. Patients may experience personality disorders, self-destructive behaviors, difficulties in school, and impaired interpersonal relationships. Tics may present in adulthood and can also be seen in association with a variety of other disorders, including PD, HD, trauma, dystonia, drugs (e.g., levodopa, neuroleptics), and toxins.

1	Etiology and Pathophysiology TS is thought to be a genetic disorder, but no specific gene mutation has been identified. Current evidence supports a complex inheritance pattern, with one or more major genes, multiple loci, low penetrance, and environmental influences. The risk of a family with one affected child having a second is about 25%. The pathophysiology of TS is not known, but alterations in dopamine neurotransmission, opioids, and second-messenger systems have been 2623 proposed. Some cases of TS may be the consequence of an autoimmune response to β-hemolytic streptococcal infection (pediatric autoimmune neuropsychiatric disorder associated with streptococcal infection [PANDAS]); however, this entity remains controversial.

1	Patients with mild disease often only require education and counseling (for themselves and family members). Drug treatment is indicated when the tics are disabling and interfere with quality of life. Therapy is individualized, and there is no singular treatment regimen that has been properly evaluated in double-blind trials. Some physicians use the α-agonist clonidine, starting at low doses and gradually increasing the dose and frequency until satisfactory control is achieved. Guanfacine (0.5–2 mg/d) is an α-agonist that is preferred by some because it only requires once-a-day dosing. Other physicians prefer to use neuroleptics. Atypical neuroleptics are usually used initially (risperidone, olanzapine, ziprasidone) because they are thought to be associated with a reduced risk of tar-dive dyskinesia. If they are not effective, low doses of classical neuroleptics such as haloperidol, fluphenazine, pimozide, or tiapride can be tried because the risk of tardive dyskinesia in young people

1	dyskinesia. If they are not effective, low doses of classical neuroleptics such as haloperidol, fluphenazine, pimozide, or tiapride can be tried because the risk of tardive dyskinesia in young people is relatively low. Botulinum toxin injections can be effective in controlling focal tics that involve small muscle groups. Behavioral features, and particularly anxiety and compulsions, can be a disabling feature of TS and should be treated. The potential value of DBS targeting the anterior portion of the internal capsule, the GPi, or the thalamus is currently being explored.

1	Myoclonus is a brief, rapid (<100 ms), shock-like, jerky movement consisting of single or repetitive muscle discharges. Myoclonic jerks can be focal, multifocal, segmental, or generalized and can occur spontaneously, in association with voluntary movement (action myoclonus) or in response to an external stimulus (reflex or startle myoclonus). Negative myoclonus consists of a brief loss of muscle activity (e.g., asterixis in hepatic failure). Myoclonic jerks can be severe and interfere with normal movement or benign and of no clinical consequence as is commonly observed in normal people when waking up or falling asleep (hypnogogic jerks).

1	Myoclonic jerks differ from tics in that they are not typically repetitive, can interfere with normal voluntary movement, and are not suppressible. They can arise in association with abnormal neuronal discharges in cortical, subcortical, brainstem, or spinal cord regions and can be associated with lesions in each of these regions, particularly in association with hypoxemia (especially following cardiac arrest), encephalopathy, and neurodegeneration. Reversible myoclonus can be seen with metabolic disturbances (renal failure, electrolyte imbalance, hypocalcemia), toxins, and many medications. Essential myoclonus is a relatively benign familial condition characterized by multifocal, very brief, lightning-like movements that are frequently alcohol sensitive. A mutation in the epsilon-sarcoglycan gene has been associated with a variety of myoclonus seen in association with dystonia (myoclonic dystonia).

1	Treatment primarily consists of managing the underlying condition or removing an offending agent. Pharmacologic therapy involves one or a combination of GABAergic agents such as valproic acid (800–3000 mg/d), piracetam (8–20 g/d), clonazepam (2–15 mg/d), levetiracetam (1000–3000 mg/d), or primidone (500–1000 mg/d) and may be associated with striking clinical improvement in chronic cases (e.g., postanoxic myoclonus, progressive myoclonic epilepsy). The serotonin precursor 5-hydroxitriptophan (plus carbidopa) may be useful in some cases of postanoxic myoclonus.

1	This important group of movement disorders is primarily associated with drugs that block dopamine receptors (neuroleptics) or central dopaminergic transmission. These drugs are widely used in psychiatry, but it is important to appreciate that drugs used in the treatment of nausea or vomiting (e.g., prochlorperazine [Compazine]) or gastroesophageal disorders (e.g., metoclopramide) are neuroleptic agents. Hyperkinetic movement disorders secondary to neuroleptic drugs can be divided into those that present acutely, subacutely, or after prolonged exposure (tardive syndromes). Dopamine-blocking drugs can also be associated with a reversible parkinsonian syndrome for which anticholinergics are often concomitantly prescribed, but there is concern that this may increase the risk of developing a tardive syndrome.

1	Dystonia is the most common acute hyperkinetic drug reaction. It is typically generalized in children and focal in adults (e.g., blepharospasm, torticollis, or oromandibular dystonia). The reaction can develop within minutes of exposure and can be successfully treated in most cases with parenteral administration of anticholinergics (benztropine or diphenhydramine), benzodiazepines (lorazepam, clonazepam, or diazepam), or dopamine agonists. The abrupt onset of severe spasms may occasionally be confused with a seizure; however, there is no loss of consciousness, automatisms, or postictal features typical of epilepsy. The acute onset of chorea, stereotypic behavior, and tics may also be seen, particularly following exposure to CNS stimulants such as methylphenidate, cocaine, or amphetamines.

1	Akathisia is the commonest reaction in this category. It consists of motor restlessness with a need to move that is alleviated by movement. Therapy consists of removing the offending agent. When this is not possible, symptoms may be ameliorated with benzodiazepines, anticholinergics, beta blockers, or dopamine agonists.

1	These disorders develop months to years after initiation of neuroleptic treatment. Tardive dyskinesia (TD) is most common and typically presents with choreiform movements involving the mouth, lips, and tongue. In severe cases, the trunk, limbs, and respiratory muscles may also be affected. In approximately one-third of patients, TD remits within 3 months of stopping the drug, and most patients gradually improve over the course of several years. Abnormal movements may also develop or worsen after stopping the offending agent. The movements are often mild and more upsetting to the family than to the patient, but they can be severe and disabling, particularly in the context of an underlying psychiatric disorder. Atypical antipsychotics (e.g., clozapine, risperidone, olanzapine, quetiapine, ziprasidone, and aripiprazole) are thought to be associated with a lower risk of TD in comparison to traditional antipsychotics, although this remains to be established in controlled studies. Younger

1	ziprasidone, and aripiprazole) are thought to be associated with a lower risk of TD in comparison to traditional antipsychotics, although this remains to be established in controlled studies. Younger patients have a lower risk of developing neuroleptic-induced TD, whereas the elderly, females, and those with underlying organic cerebral dysfunction have been reported to be at greater risk. Chronic use is associated with increased risk, and specifically, the U.S. Food and Drug Administration has warned that use of metoclopramide for more than 12 weeks increases the risk of TD. Because TD can be permanent and resistant to treatment, antipsychotics should be used judiciously, atypical neuroleptics should be the preferred agent when possible, and the need for continued use should be regularly monitored.

1	Treatment primarily consists of stopping the offending agent. If the patient is receiving a traditional antipsychotic and withdrawal is not possible, replacement with an atypical antipsychotic should be tried. Abrupt cessation of a neuroleptic should be avoided because acute withdrawal can induce worsening. TD can persist after withdrawal of antipsychotics and can be difficult to treat. Benefits may occasionally be achieved with valproic acid, anticholinergics, or botulinum toxin injections. In refractory cases, catecholamine depleters such as tetrabenazine may be helpful, but this drug can be associated with dose-dependent sedation and orthostatic hypotension and may induce parkinsonism as a side effect. Other approaches include baclofen (40–80 mg/d), clonazepam (1–8 mg/d), or valproic acid (750–3000 mg/d). In some cases, the abnormal movement is refractory to therapy.

1	Chronic neuroleptic exposure can also be associated with tardive dystonia, with preferential involvement of axial muscles and characteristic rocking movements of the trunk and pelvis. Tardive dystonia can be more troublesome than tardive dyskinesia and frequently persists despite stopping medication. Valproic acid, anticholinergics, and botulinum toxin may occasionally be beneficial, but patients are frequently refractory to medical therapy. Tardive akathisia, tardive Tourette’s, and tardive tremor syndromes are rare but may also occur after chronic neuroleptic exposure.

1	Neuroleptic medications can also be associated with a neuroleptic malignant syndrome (NMS). NMS is characterized by the acute or subacute onset of muscle rigidity, elevated temperature, altered mental status, hyperthermia, tachycardia, labile blood pressure, renal failure, and markedly elevated creatine kinase levels. Symptoms typically evolve within days or weeks after initiating the drug. NMS can also be precipitated by the abrupt withdrawal of dopaminergic medications in PD patients. Treatment involves immediate cessation of the offending antipsychotic drug and the introduction of a dopaminergic agent (e.g., a dopamine agonist or levodopa), dantrolene, or a benzodiazepine. Treatment may need to be undertaken in an intensive care setting and include supportive measures such as control of body temperature (antipyretics and cooling blankets), hydration, electrolyte replacement, and control of renal function and blood pressure.

1	Drugs that have serotonin-like activity (tryptophan, MDMA or “ecstasy,” meperidine) or that block serotonin reuptake can induce a rare, but potentially fatal, serotonin syndrome that is characterized by confusion, hyperthermia, tachycardia, and coma as well as rigidity, ataxia, and tremor. Myoclonus is often a prominent feature, in contrast to NMS, which it resembles. Patients can be managed with propranolol, diazepam, diphenhydramine, chlorpromazine, or cyproheptadine as well as supportive measures.

1	A variety of drugs can also be associated with parkinsonism (see above) and hyperkinetic movement disorders. Some examples include phenytoin (chorea, dystonia, tremor, myoclonus), carbamazepine (tics and dystonia), tricyclic antidepressants (dyskinesias, tremor, myoclonus), fluoxetine (myoclonus, chorea, dystonia), oral contraceptives (dyskinesia), β-adrenergics (tremor), buspirone (akathisia, dyskinesias, myoclonus), and digoxin, cimetidine, diazoxide, lithium, methadone, and fentanyl (dyskinesias).

1	Paroxysmal dyskinesias are a group of rare disorders characterized by episodic, brief involuntary movements that can manifest as various types of hyperkinetic movements, including chorea, dystonia, tremor, and myoclonus. There are two main categories: (1) paroxysmal kinesigenic dyskinesia, where the involuntary movements are triggered by sudden movement, and (2) paroxysmal nonkinesigenic dyskinesias, where the attacks are not induced by movement. There are rare cases of exercise-induced dyskinesia, where attacks are induced by prolonged exercise.

1	Paroxysmal kinesigenic dyskinesia (PKD) is characterized by brief, self-limited attacks induced by movement onset such as running but also occasionally by unexpected sound or photic stimulation. Attacks may affect one side of the body, last seconds to minutes at a time, and recur several times a day. They usually manifest as dystonic posturing of a limb but may also become generalized. PKD is most commonly familial with an autosomal dominant pattern of inheritance but may also occur secondary to various brain disorders such as multiple sclerosis or hyperglycemia. PKD is more frequent in males (4:1), and the onset is typically in the first or second decade of life. About 70% report sensory symptoms such as tingling or numbness of the affected limb preceding the attack by a few milliseconds. The evolution is relatively benign, and there is a trend toward resolution of the attacks over time. The cause is not known, but a mutation in the prolinerich transmembrane protein 2 (PRRT2) gene

1	The evolution is relatively benign, and there is a trend toward resolution of the attacks over time. The cause is not known, but a mutation in the prolinerich transmembrane protein 2 (PRRT2) gene that may be involved in neurotransmitter release has now been identified. Treatment with low-dose anticonvulsant therapy such as carbamazepine or phenytoin is advised when the attacks are frequent and interfere with daily life activities and is effective in about 80% of patients. Some clinical features of PKD (abrupt and short-lasting attacks preceded by an “aura”) and its favorable response to anticonvulsant drugs have led to speculation that it is epileptic in origin, but this has not been established.

1	Paroxysmal nonkinesigenic dyskinesia (PNKD) involves attacks of generalized dyskinesias precipitated by alcohol, caffeine, stress, or fatigue. In comparison to PKD, the episodes have a relatively longer duration (minutes to hours) and are less frequent (one to three per day). PNKD is inherited as autosomal dominant with incomplete penetrance pattern in some 80% of cases. A missense mutation in the myofibrillogenesis regulator (MR-1) gene has been identified in several families. Recognition of the condition and elimination of the underlying precipitating factors, where possible, are the first priority. Tetrabenazine, neuroleptics, dopamine-blocking agents, propranolol, clonazepam, and baclofen may be helpful. Treatment may not be required if the condition is mild and self-limited. Most patients with PNKD do not benefit from anticonvulsant drugs, but some may respond to clonazepam or other benzodiazepines.

1	Restless legs syndrome (RLS) is a neurologic disorder that affects approximately 10% of the adult population (it is rare in Asians) and can cause significant morbidity in some. It was first described in the seventeenth century by an English physician (Thomas Willis), but has only recently been recognized as being a bona fide movement disorder. The four core symptoms required for diagnosis are as follows: an urge to move the legs, usually caused or accompanied by an unpleasant sensation in the legs; symptoms that begin or worsen with rest; partial or complete relief by movement; and worsening during the evening or night.

1	Symptoms most commonly begin in the legs, but can spread to or even begin in the upper limbs. The unpleasant sensation is often described as a creepy-crawly feeling, paresthesia, or burning. In about 80% of patients, RLS is associated with periodic leg movements (PLMs) during sleep and occasionally while awake. These involuntary movements are usually brief, lasting no more than a few seconds, and recur every 5–90 s. The restlessness and PLMs are a major cause of sleep disturbance in patients, leading to poor-quality sleep and daytime sleepiness.

1	RLS is a heterogeneous condition. Primary RLS is genetic, and several loci have been found with an autosomal dominant pattern of inheritance, although penetrance may be variable. The mean age of onset in genetic forms is 27 years, although pediatric cases are recognized. The severity of symptoms is variable. Secondary RLS may be associated with pregnancy or a range of underlying disorders, including anemia, ferritin deficiency, renal failure, and peripheral neuropathy. The pathogenesis probably involves disordered dopamine function, which may be peripheral or central, in association with an abnormality of iron metabolism. Diagnosis is made on clinical grounds but can be supported by polysomnography and the demonstration of PLMs. The neurologic examination is normal. Secondary RLS should be excluded, and ferritin levels, glucose, and renal function should be measured.

1	Most RLS sufferers have mild symptoms that do not require specific treatment. General measures to improve sleep hygiene and quality should be attempted first. If symptoms remain intrusive, low doses of dopamine agonists, e.g., pramipexole (0.25–0.5 mg) or ropinirole (1–2 mg), are given 1–2 h before bedtime. Levodopa can be effective but is frequently associated with augmentation (spread and worsening of restlessness and its appearance earlier in the day) or rebound (reappearance sometimes with worsening of symptoms at a time compatible with the drug’s short half-life). Other drugs that can be effective include anticonvulsants, analgesics, and opiates. Management of secondary RLS should be directed to correcting the underlying disorder; for example, iron replacement for anemia. Iron infusion may also be helpful for severe primary RLS but requires expert supervision.

1	Wilson’s disease (WD) is an autosomal recessive inherited disorder of copper metabolism that may manifest with neurologic, psychiatric, and liver disorders, alone or in combination. It is caused by mutations in the gene encoding a P-type ATPase. The disease was first comprehensively described by the English neurologist Kinnier Wilson at the beginning of the twentieth century, although at around the same time the German physicians Kayser and Fleischer separately noted the characteristic association of corneal pigmentation with hepatic and neurologic features. WD has a worldwide prevalence of approximately 1 in 30,000, with a gene carrier frequency of 1 in 90. About half of WD patients (especially younger patients) manifest with liver abnormalities. The remainder present with neurologic disease (with or without underlying liver abnormalities), and a small proportion have hematologic or psychiatric problems at disease onset.

1	Neurologic onset usually manifests in the second decade with tremor and rigidity. The tremor is usually in the upper limbs, bilateral, and asymmetric. Tremor can be on intention or occasionally resting and, in advanced disease, can take on a wing-beating characteristic. Other features include parkinsonism with bradykinesia, dystonia (particularly facial grimacing), dysarthria, and dysphagia. More than half of those with neurologic features have a history of psychiatric disturbances, including depression, mood swings, and overt psychosis. Kayser-Fleischer (KF) rings are seen in 80% of those with hepatic presentations and virtually all with neurologic features. KF rings represent the deposition of copper in Descemet’s membrane around the cornea. They consist of a characteristic grayish rim or circle at the limbus of the cornea and are best detected by slit-lamp examination. Neuropathologic examination is characterized by neurodegeneration and astrogliosis in the basal ganglia,

1	rim or circle at the limbus of the cornea and are best detected by slit-lamp examination. Neuropathologic examination is characterized by neurodegeneration and astrogliosis in the basal ganglia, particularly in the striatum.

1	WD should always be considered in the differential diagnosis of a movement disorder in the first decades of life. Low levels of blood copper and ceruloplasmin and high levels of urinary copper may be present, but normal levels do not exclude the diagnosis. A computed tomography (CT) scan usually reveals generalized brain atrophy in established cases, and ~50% have signal hypointensity in the caudate head, putamen, globus pallidum, substantia nigra, and red nucleus on T2-weighted MRI. However, correlation of imaging changes with clinical features is not good. It is very rare for WD patients with neurologic features not to have KF rings, and therefore when the diagnosis is considered, examination by slit-lamp is essential. Liver biopsy with demonstration of high copper levels remains the gold standard for the diagnosis.

1	In the absence of treatment, the course is progressive and leads to severe neurologic dysfunction and early death. Treatment is directed at reducing tissue copper levels and maintenance therapy to prevent reaccumulation. There is no clear consensus on treatment, and all patients should be managed in a unit with expertise in WD. Penicillamine is frequently used to increase copper excretion, but it may lead to a worsening of symptoms in the initial stages of therapy. Side effects are common and can to some degree be attenuated by coadministration of pyridoxine. Tetrathiomolybdate blocks the absorption of copper and can be used instead of penicillamine. Trientine and zinc are useful drugs for maintenance therapy. Effective treatment can reverse the neurologic features in most patients, particularly when started early. Some patients stabilize, and a few may still progress, especially those with hepatocerebral disease. KF rings tend to decrease after 3–6 months and disappear by 2 years.

1	when started early. Some patients stabilize, and a few may still progress, especially those with hepatocerebral disease. KF rings tend to decrease after 3–6 months and disappear by 2 years. Adherence to maintenance therapy is a major challenge in long-term care.

1	Neurodegeneration with brain iron accumulation (NBIA) represents a group of inherited disorders characterized by iron accumulation in the basal ganglia. Clinically, they can manifest as a progressive neurologic disorder manifesting a variety of features including parkinsonism, 2626 dystonia, neuropsychiatric abnormalities, and retinal degeneration. Cognitive disorders and cerebellar dysfunction may also be seen. Presentation is usually in childhood, but adult cases have been described. Multiple genes have been identified to date. Pantothenate kinase–associated neurodegeneration (PKAN) formerly known as Hallervorden-Spatz disease and caused by a mutation in the PANK2 gene is the most common form of NBIA, accounting for about 50% of cases. Onset is usually in early childhood and is manifest as a combination of dystonia, parkinsonism, and spasticity. MRI shows a characteristic low signal abnormality in the center of the globus pallidus on T2-weighted scans known as the “eye of the tiger”

1	as a combination of dystonia, parkinsonism, and spasticity. MRI shows a characteristic low signal abnormality in the center of the globus pallidus on T2-weighted scans known as the “eye of the tiger” sign caused by iron accumulation. Numerous other gene mutations have been described associated with iron accumulation including mutations in PLA2G6, C19orf12, FA2H, ATP13A2, WDR45, FTL, CP, and DCAF17. One must be cautious, however, not to assume that all cases with iron accumulation in the basal ganglia represent an NBIA, because iron accumulation in specific basal ganglia regions is normal, and excess iron accumulation may occur in the basal ganglia region as a consequence of neurodegeneration of multiple causes unrelated to a defect in iron metabolism.

1	Acanthocytosis, some hereditary spinocerebellar atrophies and spastic parapareses, and HD can also present with parkinsonian features associated with involuntary movements. Diagnosis in these cases is best established with genetic testing.

1	Virtually all movement disorders including tremor, tics, dystonia, myoclonus, chorea, ballism, and parkinsonism can be psychogenic in origin. Tremor affecting the upper limbs is the most common psychogenic movement disorder. Psychogenic movements can result from a somatoform or conversion disorder, malingering (e.g., seeking financial gain), or a factitious disorder (e.g., seeking psychological gain). Psychogenic movement disorders are common (estimated to be 2–3% of patients seen in a movement disorder clinic), more frequent in women, disabling for the patient and family, and expensive for society (estimated $20 billion annually). Clinical features suggesting a psychogenic movement disorder include an acute onset and a pattern of abnormal movement that is inconsistent with a known movement disorder. Diagnosis is based on the nonorganic quality of the movement, the absence of findings of an organic disease process, and positive features that specifically point to a psychogenic illness

1	disorder. Diagnosis is based on the nonorganic quality of the movement, the absence of findings of an organic disease process, and positive features that specifically point to a psychogenic illness such as variability and distractibility. For example, the magnitude of a psychogenic tremor is increased with attention and diminishes or even disappears when the patient is distracted by being asked to perform a different task or is unaware that he or she is being observed. Other positive features suggesting a psychogenic problem include a tremor frequency that is variable or that entrains with the frequency of a designated movement in the contralateral limb, and a positive response to placebo medication. Associated features can include nonanatomic sensory findings, give-way weakness, astasia-abasia (an odd, gyrating gait; Chap. 32), and multiple somatic complaints with no underlying pathology (somatoform disorder). Comorbid psychiatric problems such as anxiety, depression, and emotional

1	(an odd, gyrating gait; Chap. 32), and multiple somatic complaints with no underlying pathology (somatoform disorder). Comorbid psychiatric problems such as anxiety, depression, and emotional trauma may be present but are not necessary for the diagnosis of a psychogenic movement disorder to be made. Psychogenic movement disorders can occur as an isolated entity or in association with an underlying organic problem. The diagnosis can often be made based on clinical features alone, and unnecessary tests or medications can be avoided. Underlying psychiatric problems may be present and should be identified and treated, but many patients with psychogenic movement disorders have no obvious psychiatric pathology. Psychotherapy and hypnosis may be of value for patients with conversion reaction, and cognitive behavioral therapy may be helpful for patients with somatoform disorders. Patients with hypochondriasis, factitious disorders, and malingering have a poor prognosis.

1	Roger N. Rosenberg APPROACH TO THE PATIENT:

1	Symptoms and signs of ataxia consist of gait impairment, unclear (“scanning”) speech, visual blurring due to nystagmus, hand incoordination, and tremor with movement. These result from the involvement of the cerebellum and its afferent and efferent pathways, including the spinocerebellar pathways, and the frontopontocerebellar pathway originating in the rostral frontal lobe. True cerebellar ataxia must be distinguished from ataxia associated with vestibular nerve or labyrinthine disease, as the latter results in a disorder of gait associated with a significant degree of dizziness, light-headedness, or the perception of movement (Chap. 28). True cerebellar ataxia is devoid of these vertiginous complaints and is clearly an unsteady gait due to imbalance. Sensory disturbances can also on occasion simulate the imbalance of cerebellar disease; with sensory ataxia, imbalance dramatically worsens when visual input is removed (Romberg sign). Rarely, weakness of proximal leg muscles mimics

1	occasion simulate the imbalance of cerebellar disease; with sensory ataxia, imbalance dramatically worsens when visual input is removed (Romberg sign). Rarely, weakness of proximal leg muscles mimics cerebellar disease. In the patient who presents with ataxia, the rate and pattern of the development of cerebellar symptoms help to narrow the diagnostic possibilities (Table 450-1). A gradual and progressive increase in symptoms with bilateral and symmetric involvement suggests a genetic, metabolic, immune, or toxic etiology. Conversely, focal, unilateral symptoms with headache and impaired level of consciousness accompanied by ipsilateral cranial nerve palsies and contralateral weakness imply a space-occupying cerebellar lesion.

1	Progressive and symmetric ataxia can be classified with respect to onset as acute (over hours or days), subacute (weeks or months), or chronic (months to years). Acute and reversible ataxias include those caused by intoxication with alcohol, phenytoin, lithium, barbiturates, and other drugs. Intoxication caused by toluene exposure, gasoline sniffing, glue sniffing, spray painting, or exposure to methyl mercury or bismuth are additional causes of acute or subacute ataxia, as is treatment with cytotoxic chemotherapeutic drugs such as fluorouracil and paclitaxel. Patients with a postinfectious syndrome (especially after varicella) may develop gait ataxia and mild dysarthria, both of which are reversible (Chap. 458). Rare infectious causes of acquired ataxia include poliovirus, coxsackievirus, echovirus, Epstein-Barr virus, toxoplasmosis, Legionella, and Lyme disease.

1	The subacute development of ataxia of gait over weeks to months (degeneration of the cerebellar vermis) may be due to the combined effects of alcoholism and malnutrition, particularly with deficiencies of vitamins B1 and B12. Hyponatremia has also been associated with ataxia. Paraneoplastic cerebellar ataxia is associated with a number of different tumors (and autoantibodies) such as breast and ovarian cancers (anti-Yo), small-cell lung cancer (anti-PQ-type voltage-gated calcium channel), and Hodgkin’s disease (anti-Tr) (Chap. 122). Another paraneoplastic syndrome associated with myoclonus and opsoclonus occurs with breast (anti-Ri) and lung cancers and neuroblastoma. Elevated serum anti-glutamic acid decarboxylase (GAD) antibodies have been associated with a progressive ataxic syndrome affecting speech and gait. For all of these paraneoplastic ataxias, the neurologic syndrome may be the presenting symptom of the cancer. Another immune-mediated progressive ataxia is associated with

1	affecting speech and gait. For all of these paraneoplastic ataxias, the neurologic syndrome may be the presenting symptom of the cancer. Another immune-mediated progressive ataxia is associated with antigliadin (and antiendomysium) antibodies and the human leukocyte antigen (HLA) DQB1*0201 haplotype; in some affected patients, biopsy of the small intestine reveals villus atrophy consistent with gluten-sensitive enteropathy (Chap. 349). Finally, subacute progressive ataxia may be caused by a prion disorder,

1	Intoxication: alcohol, lithium, phenytoin, barbiturates (positive history and toxicology screen) Acute viral cerebellitis (CSF supportive of acute viral infection) Postinfection syndrome Intoxication: mercury, solvents, gasoline, glue; cytotoxic chemotherapeutic, hemotherapeutic drugs Abbreviations: CSF, cerebrospinal fluid; CT, computed tomography; MRI, magnetic resonance imaging. especially when an infectious etiology, such as transmission from contaminated human growth hormone, is responsible (Chap. 453e). Chronic symmetric gait ataxia suggests an inherited ataxia (discussed below), a metabolic disorder, or a chronic infection. Hypothyroidism must always be considered as a readily treatable and reversible form of gait ataxia. Infectious diseases that can present with ataxia are meningovascular syphilis and tabes dorsalis due to degeneration of the posterior columns and spinocerebellar pathways in the spinal cord.

1	Acute focal ataxia commonly results from cerebrovascular disease, usually ischemic infarction or cerebellar hemorrhage. These lesions typically produce cerebellar symptoms ipsilateral to the injured cerebellum and may be associated with an impaired level of consciousness due to brainstem compression and increased intracranial pressure; ipsilateral pontine signs, including sixth and seventh nerve palsies, may be present. Focal and worsening signs of acute ataxia should also prompt consideration of a posterior fossa subdural hematoma, bacterial abscess, or primary or metastatic cerebellar tumor. Computed tomography (CT) or magnetic resonance imaging (MRI) studies will reveal clinically significant processes of this type. Many of these lesions represent true neurologic emergencies, as sudden herniation, either rostrally through the tentorium or caudal herniation of cerebellar tonsils through the foramen magnum, can occur and is usually devastating. Acute surgical decompression may be

1	herniation, either rostrally through the tentorium or caudal herniation of cerebellar tonsils through the foramen magnum, can occur and is usually devastating. Acute surgical decompression may be required (Chap. 330). Lymphoma or progressive multifocal leukoencephalopathy (PML) in a patient with AIDS may present with an acute or subacute focal cerebellar syndrome. Chronic etiologies of progressive ataxia include multiple sclerosis (Chap. 458) and congenital lesions such as a Chiari malformation (Chap. 456) or a congenital cyst of the posterior fossa (Dandy-Walker syndrome).

1	These may show autosomal dominant, autosomal recessive, or maternal (mitochondrial) modes of inheritance. A genomic classification (Chap. 451e) has now largely superseded previous ones based on clinical expression alone. Although the clinical manifestations and neuropathologic findings of cerebellar disease dominate the clinical picture, there may also be characteristic changes in the basal ganglia, brainstem, spinal cord, optic nerves, retina, and peripheral nerves. In large families with dominantly inherited ataxias, many gradations are observed from purely cerebellar manifestations to mixed cerebellar and brainstem disorders, cerebellar and basal ganglia syndromes, and spinal cord or peripheral nerve disease. Rarely, dementia is present as well. The clinical picture may be homogeneous within a family with dominantly inherited ataxia, but sometimes most affected family members show one characteristic syndrome, while one or several members have an entirely different phenotype.

1	The autosomal spinocerebellar ataxias (SCAs) include SCA types 1 through 36, dentatorubropallidoluysian atrophy (DRPLA), and episodic ataxia (EA) types 1 to 7 (Chap. 451e). SCA1, SCA2, SCA3 (Machado-Joseph disease [MJD]), SCA6, SCA7, and SCA17 are caused by CAG triplet repeat expansions in different genes. SCA8 is due to an untranslated CTG repeat expansion, SCA12 is linked to an untranslated CAG repeat, and SCA10 is caused by an untranslated pentanucleotide repeat. The clinical phenotypes of these SCAs overlap. The genotype has become the gold standard for diagnosis and classification. CAG encodes glutamine, and these expanded CAG triplet repeat expansions result in expanded polyglutamine proteins, termed ataxins, that produce a toxic gain of function with autosomal dominant inheritance. Although the phenotype is variable for any given disease gene, a pattern of neuronal loss with gliosis is produced that is relatively unique for each ataxia. Immunohistochemical and biochemical

1	Although the phenotype is variable for any given disease gene, a pattern of neuronal loss with gliosis is produced that is relatively unique for each ataxia. Immunohistochemical and biochemical studies have shown cytoplasmic (SCA2), neuronal (SCA1, MJD, SCA7), and nucleolar (SCA7) accumulation of the specific mutant polyglutamine-containing ataxin proteins. Expanded polyglutamine ataxins with more than ~40 glutamines are potentially toxic to neurons for a variety of reasons including: high levels of gene expression for the mutant polyglutamine ataxin in affected neurons; conformational change of the aggregated protein to a β-pleated structure; abnormal transport of the ataxin into the nucleus (SCA1, MJD, SCA7); binding to other polyglutamine proteins, including the TATA-binding transcription protein and the CREB-binding protein, impairing their functions; altering the efficiency of the ubiquitin-proteasome system of protein turnover; and inducing neuronal apoptosis. An earlier age of

1	protein and the CREB-binding protein, impairing their functions; altering the efficiency of the ubiquitin-proteasome system of protein turnover; and inducing neuronal apoptosis. An earlier age of onset (anticipation) and more aggressive disease in subsequent generations are due to further expansion of the CAG triplet repeat and increased polyglutamine number in the mutant ataxin. The most common disorders are discussed below.

1	SCA1 was previously referred to as olivopontocerebellar atrophy, but genomic data have shown that that entity represents several different genotypes with overlapping clinical features. Symptoms and Signs SCA1 is characterized by the development in early or middle adult life of progressive cerebellar ataxia of the trunk and limbs, impairment of equilibrium and gait, slowness of voluntary movements, scanning speech, nystagmoid eye movements, and oscillatory tremor of the head and trunk. Dysarthria, dysphagia, and

1	FIGURE 450-1 Sagittal magnetic resonance imaging (MRI) of the brain of a 60-year-old man with gait ataxia and dysarthria due to spinocerebellar ataxia type 1 (SCA1), illustrating cerebellar atrophy (arrows). (Reproduced with permission from RN Rosenberg, P Khemani, in RN Rosenberg, JM Pascual [eds]: Rosenberg’s Molecular and Genetic Basis of Neurological and Psychiatric Disease, 5th ed. London, Elsevier, 2015.) oculomotor and facial palsies may also occur. Extrapyramidal symptoms include rigidity, an immobile face, and parkinsonian tremor. The reflexes are usually normal, but knee and ankle jerks may be lost, and extensor plantar responses may occur. Dementia may be noted but is usually mild. Impairment of sphincter function is common, with urinary and sometimes fecal incontinence. Cerebellar and brainstem atrophy are evident on MRI (Fig. 450-1).

1	Marked shrinkage of the ventral half of the pons, disappearance of the olivary eminence on the ventral surface of the medulla, and atrophy of the cerebellum are evident on gross postmortem inspection of the brain. Variable loss of Purkinje cells, reduced numbers of cells in the molecular and granular layer, demyelination of the middle cerebellar peduncle and the cerebellar hemispheres, and severe loss of cells in the pontine nuclei and olives are found on histologic examination. Degenerative changes in the striatum, especially the putamen, and loss of the pigmented cells of the substantia nigra may be found in cases with extrapyramidal features. More widespread degeneration in the central nervous system (CNS), including involvement of the posterior columns and the spinocerebellar fibers, is often present.

1	SCA1 encodes a gene product, called ataxin-1, which is a novel protein of unknown function. The mutant allele has 40 CAG repeats located within the coding region, whereas alleles from unaffected individuals have ≤36 repeats. A few patients with 38–40 CAG repeats have been described. There is a direct correlation between a larger number of repeats and a younger age of onset for SCA1. Juvenile patients have higher numbers of repeats, and anticipation is present in subsequent generations. Transgenic mice carrying SCA1 developed ataxia and Purkinje cell pathology. Nuclear localization, but not aggregation, of ataxin-1 appears to be required for cell death initiated by the mutant protein.

1	SCA2 Symptoms and Signs Another clinical phenotype, SCA2, has been described in patients from Cuba and India. Cuban patients probably are descendants of a common ancestor, and the population may be the largest homogeneous group of patients with ataxia yet described. The age of onset ranges from 2–65 years, and there is considerable clinical variability within families. Although neuropathologic and clinical findings are compatible with a diagnosis of SCA1, including slow saccadic eye movements, ataxia, dysarthria, parkinsonian rigidity, optic disc pallor, mild spasticity, and retinal degeneration, SCA2 is a unique form of cerebellar degenerative disease. The gene in SCA2 families also contains CAG repeat expansions coding for a polyglutamine-containing protein, ataxin-2. Normal alleles contain 15–32 repeats; mutant alleles have 35–77 repeats.

1	The gene in SCA2 families also contains CAG repeat expansions coding for a polyglutamine-containing protein, ataxin-2. Normal alleles contain 15–32 repeats; mutant alleles have 35–77 repeats. MJD was first described among the Portuguese and their descendants in New England and California. Subsequently, MJD has been found in families from Portugal, Australia, Brazil, Canada, China, England, France, India, Israel, Italy, Japan, Spain, Taiwan, and the United States. In most populations, it is the most common autosomal dominant ataxia.

1	Symptoms and Signs MJD has been classified into three clinical types. In type I MJD (amyotrophic lateral sclerosis-parkinsonism-dystonia type), neurologic deficits appear in the first two decades and involve weakness and spasticity of extremities, especially the legs, often with dystonia of the face, neck, trunk, and extremities. Patellar and ankle clonus are common, as are extensor plantar responses. The gait is slow and stiff, with a slightly broadened base and lurching from side to side; this gait results from spasticity, not true ataxia. There is no truncal titubation. Pharyngeal weakness and spasticity cause difficulty with speech and swallowing. Of note is the prominence of horizontal and vertical nystagmus, loss of fast saccadic eye movements, hypermetric and hypometric saccades, and impairment of upward vertical gaze. Facial fasciculations, facial myokymia, lingual fasciculations without atrophy, ophthalmoparesis, and ocular prominence are common early manifestations.

1	In type II MJD (ataxic type), true cerebellar deficits of dysarthria and gait and extremity ataxia begin in the second to fourth decades along with corticospinal and extrapyramidal deficits of spasticity, rigidity, and dystonia. Type II is the most common form of MJD. Ophthalmoparesis, upward vertical gaze deficits, and facial and lingual fasciculations are also present. Type II MJD can be distinguished from the clinically similar disorders SCA1 and SCA2. Type III MJD (ataxic-amyotrophic type) presents in the fifth to the seventh decades with a pancerebellar disorder that includes dysarthria and gait and extremity ataxia. Distal sensory loss involving pain, touch, vibration, and position senses and distal atrophy are prominent, indicating the presence of peripheral neuropathy. The deep tendon reflexes are depressed to absent, and there are no corticospinal or extrapyramidal findings.

1	The mean age of onset of symptoms in MJD is 25 years. Neurologic deficits invariably progress and lead to death from debilitation within 15 years of onset, especially in patients with types I and II disease. Usually, patients retain full intellectual function. The major pathologic findings are variable loss of neurons and glial replacement in the corpus striatum and severe loss of neurons in the pars compacta of the substantia nigra. A moderate loss of neurons occurs in the dentate nucleus of the cerebellum and in the red nucleus. Purkinje cell loss and granule cell loss occur in the cerebellar cortex. Cell loss also occurs in the dentate nucleus and in the cranial nerve motor nuclei. Sparing of the inferior olives distinguishes MJD from other dominantly inherited ataxias.

1	The gene for MJD maps to 14q24.3-q32. Unstable CAG repeat expansions are present in the MJD gene coding for a polyglutamine-containing protein named ataxin-3, or MJD-ataxin. An earlier age of onset is associated with longer repeats. Alleles from normal individuals have between 12 and 37 CAG repeats, whereas MJD alleles have 60–84 CAG repeats. Polyglutamine-containing aggregates of ataxin-3 (MJD-ataxin) have been described in neuronal nuclei undergoing degeneration. MJD-ataxin codes for a ubiquitin protease, which is inactive due to expanded polyglutamines. Proteosome function is impaired, resulting in altered clearance of proteins and cerebellar neuronal loss.

1	Genomic screening for CAG repeats in other families with autosomal dominant ataxia and vibratory and proprioceptive sensory loss have yielded another locus. Of interest is that different mutations in the same gene for the α1A voltage-dependent calcium channel subunit (CACNLIA4; also referred to as the CACNA1A gene) at 19p13 result in different clinical disorders. CAG repeat expansions (21–27 in patients; 4–16 triplets in normal individuals) result in late-onset progressive ataxia with cerebellar degeneration. Missense mutations in this gene result in familial hemiplegic migraine. Nonsense mutations resulting in termination of protein synthesis of the gene product yield hereditary paroxysmal cerebellar ataxia or EA. Some patients with familial hemiplegic migraine develop progressive ataxia and also have cerebellar atrophy.

1	This disorder is distinguished from all other SCAs by the presence of retinal pigmentary degeneration. The visual abnormalities first appear as blue-yellow color blindness and proceed to frank visual loss with macular degeneration. In almost all other respects, SCA7 resembles several other SCAs in which ataxia is accompanied by various noncerebellar findings, including ophthalmoparesis and extensor plantar responses. The genetic defect is an expanded CAG repeat in the SCA7 gene at 3p14-p21.1. The expanded repeat size in SCA7 is highly variable. Consistent with this, the severity of clinical findings varies from essentially asymptomatic to mild late-onset symptoms to severe, aggressive disease in childhood with rapid progression. Marked anticipation has been recorded, especially with paternal transmission. The disease protein, ataxin-7, forms aggregates in nuclei of affected neurons, as has also been described for SCA1 and SCA3/MJD.

1	This form of ataxia is caused by a CTG repeat expansion in an untranslated region of a gene on chromosome 13q21. There is marked maternal bias in transmission, perhaps reflecting contractions of the repeat during spermatogenesis. The mutation is not fully penetrant. Symptoms include slowly progressive dysarthria and gait ataxia beginning at ~40 years of age with a range between 20 and 65 years. Other features include nystagmus, leg spasticity, and reduced vibratory sensation. Severely affected individuals are nonambulatory by the fourth to sixth decades. MRI shows cerebellar atrophy. The mechanism of disease may involve a dominant “toxic” effect occurring at the RNA level, as occurs in myotonic dystrophy.

1	DRPLA has a variable presentation that may include progressive ataxia, choreoathetosis, dystonia, seizures, myoclonus, and dementia. DRPLA is due to unstable CAG triplet repeats in the open reading frame of a gene named atrophin located on chromosome 12p12-ter. Larger expansions are found in patients with earlier onset. The number of repeats is 49 in patients with DRPLA and ≤26 in normal individuals. Anticipation occurs in successive generations, with earlier onset of disease in association with an increasing CAG repeat number in children who inherit the disease from their father. One well-characterized family in North Carolina has a phenotypic variant known as the Haw River syndrome, now recognized to be due to the DRPLA mutation.

1	EA types 1 and 2 are two rare dominantly inherited disorders that have been mapped to chromosomes 12p (a potassium channel gene) for type 1 and 19p for type 2. Patients with EA-1 have brief episodes of ataxia with myokymia and nystagmus that last only minutes. Startle, sudden change in posture, and exercise can induce episodes. Acetazolamide or anticonvulsants may be therapeutic. Patients with EA-2 have episodes 2629 of ataxia with nystagmus that can last for hours or days. Stress, exercise, or excessive fatigue may be precipitants. Acetazolamide may be therapeutic and can reverse the relative intracellular alkalosis detected by magnetic resonance spectroscopy. Stop codon, nonsense mutations causing EA-2 have been found in the CACNA1A gene, encoding the voltage-dependent calcium channel subunit (see “SCA6,” above).

1	AUTOSOMAL RECESSIVE ATAXIAS Friedreich’s Ataxia This is the most common form of inherited ataxia, comprising one-half of all hereditary ataxias. It can occur in a classic form or in association with a genetically determined vitamin E deficiency syndrome; the two forms are clinically indistinguishable. SymptomS AnD SignS Friedreich’s ataxia presents before 25 years of age with progressive staggering gait, frequent falling, and titubation. The lower extremities are more severely involved than the upper ones. Dysarthria occasionally is the presenting symptom; rarely, progressive scoliosis, foot deformity, nystagmus, or cardiopathy is the initial sign.

1	The neurologic examination reveals nystagmus, loss of fast saccadic eye movements, truncal titubation, dysarthria, dysmetria, and ataxia of trunk and limb movements. Extensor plantar responses (with normal tone in trunk and extremities), absence of deep tendon reflexes, and weakness (greater distally than proximally) are usually found. Loss of vibratory and proprioceptive sensation occurs. The median age of death is 35 years. Women have a significantly better prognosis than men. Cardiac involvement occurs in 90% of patients. Cardiomegaly, symmetric hypertrophy, murmurs, and conduction defects are reported. Moderate mental retardation or psychiatric syndromes are present in a small percentage of patients. A high incidence of diabetes mellitus (20%) is found and is associated with insulin resistance and pancreatic β-cell dysfunction. Musculoskeletal deformities are common and include pes cavus, pes equinovarus, and scoliosis. MRI of the spinal cord shows atrophy (Fig. 450-2).

1	The primary sites of pathology are the spinal cord, dorsal root ganglion cells, and the peripheral nerves. Slight atrophy of the cerebellum and cerebral gyri may occur. Sclerosis and degeneration occur

1	FIGURE 450-2 Sagittal magnetic resonance imaging (MRI) of the brain and spinal cord of a patient with Friedreich’s ataxia, demonstrating spinal cord atrophy. (Reproduced with permission from RN Rosenberg, P Khemani, in RN Rosenberg, JM Pascual [eds]: Rosenberg’s Molecular and Genetic Basis of Neurological and Psychiatric Disease, 5th ed. London, Elsevier, 2015.) 2630 predominantly in the spinocerebellar tracts, lateral corticospinal tracts, and posterior columns. Degeneration of the glossopharyngeal, vagus, hypoglossal, and deep cerebellar nuclei is described. The cerebral cortex is histologically normal except for loss of Betz cells in the precentral gyri. The peripheral nerves are extensively involved, with a loss of large myelinated fibers. Cardiac pathology consists of myocytic hypertrophy and fibrosis, focal vascular fibromuscular dysplasia with subintimal or medial deposition of periodic acid-Schiff (PAS)-positive material, myocytopathy with unusual pleomorphic nuclei, and focal

1	and fibrosis, focal vascular fibromuscular dysplasia with subintimal or medial deposition of periodic acid-Schiff (PAS)-positive material, myocytopathy with unusual pleomorphic nuclei, and focal degeneration of nerves and cardiac ganglia.

1	The classic form of Friedreich’s ataxia has been mapped to 9q13q21.1, and the mutant gene, frataxin, contains expanded GAA triplet repeats in the first intron. There is homozygosity for expanded GAA repeats in >95% of patients. Normal persons have 7–22 GAA repeats, and patients have 200–900 GAA repeats. A more varied clinical syndrome has been described in compound heterozygotes who have one copy of the GAA expansion and the other copy a point mutation in the frataxin gene. When the point mutation is located in the region of the gene that encodes the amino-terminal half of frataxin, the phenotype is milder, often consisting of a spastic gait, retained or exaggerated reflexes, no dysarthria, and mild or absent ataxia.

1	Patients with Friedreich’s ataxia have undetectable or extremely low levels of frataxin mRNA, as compared with carriers and unrelated individuals; thus, disease appears to be caused by a loss of expression of the frataxin protein. Frataxin is a mitochondrial protein involved in iron homeostasis. Mitochondrial iron accumulation due to loss of the iron transporter coded by the mutant frataxin gene results in oxidized intramitochondrial iron. Excess oxidized iron results in turn in the oxidation of cellular components and irreversible cell injury.

1	Two forms of hereditary ataxia associated with abnormalities in the interactions of vitamin E (α-tocopherol) with very-low-density lipoprotein (VLDL) have been delineated. These are abetalipoproteinemia (Bassen-Kornzweig syndrome) and ataxia with vitamin E deficiency (AVED). Abetalipoproteinemia is caused by mutations in the gene coding for the larger subunit of the microsomal triglyceride transfer protein (MTP). Defects in MTP result in impairment of formation and secretion of VLDL in liver. This defect results in a deficiency of delivery of vitamin E to tissues, including the central and peripheral nervous system, as VLDL is the transport molecule for vitamin E and other fat-soluble substitutes. AVED is due to mutations in the gene for α-tocopherol transfer protein (α-TTP). These patients have an impaired ability to bind vitamin E into the VLDL produced and secreted by the liver, resulting in a deficiency of vitamin E in peripheral tissues. Hence, either absence of VLDL

1	These patients have an impaired ability to bind vitamin E into the VLDL produced and secreted by the liver, resulting in a deficiency of vitamin E in peripheral tissues. Hence, either absence of VLDL (abetalipoproteinemia) or impaired binding of vitamin E to VLDL (AVED) causes an ataxic syndrome. Once again, a genotype classification has proved to be essential in sorting out the various forms of the Friedreich’s disease syndrome, which may be clinically indistinguishable.

1	Ataxia Telangiectasia • SymptomS AnD SignS Patients with ataxia telangiectasia (AT) present in the first decade of life with progressive telangiectatic lesions associated with deficits in cerebellar function and nystagmus. The neurologic manifestations correspond to those in Friedreich’s disease, which should be included in the differential diagnosis. Truncal and limb ataxia, dysarthria, extensor plantar responses, myoclonic jerks, areflexia, and distal sensory deficits may develop. There is a high incidence of recurrent pulmonary infections and neoplasms of the lymphatic and reticuloendothelial system in patients with AT. Thymic hypoplasia with cellular and humoral (IgA and IgG2) immunodeficiencies, premature aging, and endocrine disorders such as type 1 diabetes mellitus are described. There is an increased incidence of lymphomas, Hodgkin’s disease, acute T cell leukemias, and breast cancer.

1	The most striking neuropathologic changes include loss of Purkinje, granule, and basket cells in the cerebellar cortex as well as of neurons in the deep cerebellar nuclei. The inferior olives of the medulla may also have neuronal loss. There is a loss of anterior horn neurons in the spinal cord and of dorsal root ganglion cells associated with posterior column spinal cord demyelination. A poorly developed or absent thymus gland is the most consistent defect of the lymphoid system. The gene for AT (the ATM gene) encodes a protein that is simi lar to several yeast and mammalian phosphatidylinositol-3´ kinases involved in mitogenic signal transduction, meiotic recombination, and cell cycle control. Defective DNA repair in AT fibroblasts exposed to ultraviolet light has been demonstrated. The discovery of ATM permits early diagnosis and identification of heterozygotes who are at risk for cancer (e.g., breast cancer).

1	Spinocerebellar syndromes have been identified with mutations in mitochondrial DNA (mtDNA). Thirty pathogenic mtDNA point mutations and 60 different types of mtDNA deletions are known, several of which cause or are associated with ataxia (Chap. 462e).

1	The most important goal in management of patients with ataxia is to identify treatable disease entities. Mass lesions must be recognized promptly and treated appropriately. Paraneoplastic disorders can often be identified by the clinical patterns of disease that they produce, measurement of specific autoantibodies, and uncovering the primary cancer; these disorders are often refractory to therapy, but some patients improve following removal of the tumor or immunotherapy (Chap. 122). Ataxia with antigliadin antibodies and gluten-sensitive enteropathy may improve with a gluten-free diet. Malabsorption syndromes leading to vitamin E deficiency may lead to ataxia. The vitamin E deficiency form of Friedreich’s ataxia must be considered, and serum vitamin E levels measured. Vitamin E therapy is indicated for these rare patients. Vitamin B1 and B12 levels in serum should be measured, and the vitamins administered to patients having deficient levels. Hypothyroidism is easily treated. The

1	is indicated for these rare patients. Vitamin B1 and B12 levels in serum should be measured, and the vitamins administered to patients having deficient levels. Hypothyroidism is easily treated. The cerebrospinal fluid should be tested for a syphilitic infection in patients with progressive ataxia and other features of tabes dorsalis. Similarly, antibody titers for Lyme disease and Legionella should be measured and appropriate antibiotic therapy should be instituted in antibody-positive patients. Aminoacidopathies, leukodystrophies, urea-cycle abnormalities, and mitochondrial encephalomyopathies may produce ataxia, and some dietary or metabolic therapies are available for these disorders. The deleterious effects of phenytoin and alcohol on the cerebellum are well known, and these exposures should be avoided in patients with ataxia of any cause.

1	There is no proven therapy for any of the autosomal dominant ataxias (SCA1 to SCA36). There is preliminary evidence that idebenone, a free-radical scavenger, can improve myocardial hypertrophy in patients with classic Friedreich’s ataxia; there is no current evidence, however, that it improves neurologic function. A small preliminary study in a mixed population of patients with different inherited ataxias raised the possibility that the glutamate antagonist riluzole may offer modest benefit. Iron chelators and antioxidant drugs are potentially harmful in Friedreich’s patients because they may increase heart muscle injury. Acetazolamide can reduce the duration of symptoms of episodic ataxia. At present, identification of an at-risk person’s genotype, together with appropriate family and genetic counseling, can reduce the incidence of these cerebellar syndromes in future generations (Chap. 84).

1	1. Baylor College of Medicine; Houston, Texas, 1-713-798-6522 http://www.bcm.edu/genetics/index.cfm?pmid=21387 2. GeneDx http://www.genedx.com 3. Transgenomic, 1-877-274-9432 http://www.transgenomic.com/labs/neurology Ataxias with autosomal dominant, autosomal recessive, X-linked, or mitochondrial forms of inheritance are present on a worldwide basis. Machado-Joseph disease (SCA3) (auto somal dominant) and Friedreich’s ataxia (autosomal recessive) are the most common types in most populations. Genetic markers are now commercially available to precisely identify the genetic mutation for correct diagnosis and also for family planning. Early detection of asymptomatic preclinical disease can reduce or eliminate the inherited form of ataxia in some families on a global, worldwide basis.

1	Classification of the Spinocerebellar Ataxias Roger N. Rosenberg Ataxias with autosomal dominant, autosomal recessive, X-linked, or mitochondrial forms of inheritance are present on a worldwide 451e basis. Machado-Joseph disease (SCA3) (autosomal dominant) and Friedreich’s ataxia (autosomal recessive) are the most common types in most populations. Mutation markers are now commercially available to identify carriers at risk in their families, which allows for precise identification of the genetic mutation for correct diagnosis and also for family planning. Identification of positive mutation carriers with family planning has allowed for early detection of asymptomatic preclinical disease to reduce or eliminate the inherited form of ataxia in specific families on a global, worldwide basis.

1	6p22-p23 with CAG repeats (exonic); leucine-rich acidic nuclear protein (LANP), region-specific interaction protein 12q23-q24.1 with CAG repeats (exonic) 14q24.3-q32 with CAG repeats (exonic); codes for ubiquitin protease (inactive with polyglutamine expansion); altered turnover of cellular proteins due to proteosome dysfunction 16q22.1-ter; pleckstrin homology domain-containing protein, family G, member 4 (PLEKHG4; puratrophin-1: Purkinje cell atrophy associated protein-1, including spectrin repeat and the guanine-nucleotide exchange factor, GEF for Rho GTPases) 11p12-q12; β-III spectrin mutations; (SPTBN2); stabilizes glutamate transporter EAAT4; descendants of President Abraham Lincoln 19p13.2 with CAG repeats in α1A-voltage–dependent calcium channel gene (exonic); CACNA1A protein, P/Q type calcium channel subunit 3p14.1-p21.1 with CAG repeats (exonic); ataxin-7; subunit of GCN5, histone acetyltransferase-containing complexes; ataxin-7 binding protein; Cbl-associated protein (CAP;

1	type calcium channel subunit 3p14.1-p21.1 with CAG repeats (exonic); ataxin-7; subunit of GCN5, histone acetyltransferase-containing complexes; ataxin-7 binding protein; Cbl-associated protein (CAP; SH3D5) 13q21 with CTG repeats; noncoding; 3‘ untranslated region of transcribed RNA; KLHL1AS 22q13; pentanucleotide repeat ATTCT repeat; noncoding, intron 9 15q14-q21.3 by linkage 5q31-q33 by linkage; CAG repeat; protein phosphatase 2A, regulatory subunit B, (PPP2R2B); protein PP2A, serine/ threonine phosphatase 19q13.3-q14.4; potassium channel voltage-gated; KCNC3 19q-13.4; protein kinase Cγ (PRKCG), missense mutations including in-frame deletion and a splice site mutation among others; serine/threonine kinase 3p24.2-3pter; inositol 1,4,5triphosphate receptor type 1 (ITPRI) 8q22.1-24.1 6q27; CAG expansion in the TATA-binding protein (TBP) gene 1p21-q21; KCND3; missense mutations; T352P; M373I; S390N; allelic with SCA22; overlaps with the locus of SCA22 11p13-q11; 260 kb duplication Ataxia

1	expansion in the TATA-binding protein (TBP) gene 1p21-q21; KCND3; missense mutations; T352P; M373I; S390N; allelic with SCA22; overlaps with the locus of SCA22 11p13-q11; 260 kb duplication Ataxia with ophthalmoparesis, pyramidal and extrapyramidal findings; genetic testing is available; 6% of all autosomal dominant (AD) cerebellar ataxia

1	Ataxia with slow saccades and minimal pyramidal and extrapyramidal findings; genetic testing available; 13% of all AD cerebellar ataxia Ataxia with ophthalmoparesis and variable pyramidal, extrapyramidal, and amyotrophic signs; dementia (mild); 23% of all AD cerebellar ataxia; genetic testing available Ataxia with normal eye movements, sensory axonal neuropathy, and pyramidal signs; genetic testing available Ataxia and dysarthria, nystagmus, mild proprioceptive sensory loss; genetic testing available Ophthalmoparesis, visual loss, ataxia, dysarthria, extensor plantar response, pigmentary retinal degeneration; genetic testing available Gait ataxia, dysarthria, nystagmus, leg spasticity, and reduced vibratory sensation; genetic testing available Gait ataxia, dysarthria, nystagmus; partial complex and generalized motor seizures; polyneuropathy; genetic testing available Slowly progressive gait and extremity ataxia, dysarthria, vertical nystagmus, hyperreflexia

1	Slowly progressive gait and extremity ataxia, dysarthria, vertical nystagmus, hyperreflexia Tremor, decreased movement, increased reflexes, dystonia, ataxia, dysautonomia, dementia, dysarthria; genetic testing available Ataxia, legs > arms; dysarthria, horizontal nystagmus; delayed motor development; mental developmental delay; tendon reflexes increased; MRI: cerebellar and pontine atrophy; genetic testing available Gait ataxia; leg > arm ataxia; dysarthria; pure ataxia with later onset; myoclonus; tremor of head and extremities; increased deep tendon reflexes at ankles; occasional dystonia and sensory neuropathy; genetic testing available Gait and extremity ataxia, dysarthria; nystagmus; MRI: superior vermis atrophy; sparing of hemispheres and tonsils Pure cerebellar ataxia and head tremor, gait ataxia, and dysarthria; horizontal gaze–evoked nystagmus; MRI: cerebellar atrophy; no brainstem changes

1	Pure cerebellar ataxia and head tremor, gait ataxia, and dysarthria; horizontal gaze–evoked nystagmus; MRI: cerebellar atrophy; no brainstem changes Gait ataxia, dementia, parkinsonism, dystonia, chorea, seizures; hyperreflexia; dysarthria and dysphagia; MRI shows cerebral and cerebellar atrophy; genetic testing available Ataxia; motor/sensory neuropathy; head tremor; dysarthria; extensor plantar responses in some patients; sensory axonal neuropathy; EMG denervation; MRI: cerebellar atrophy Ataxia, tremor, cognitive impairment, myoclonus; MRI: atrophy of cerebellum Dysarthria; gait ataxia; ocular gaze–evoked saccades; palatal tremor; dentate calcification on CT; MRI: cerebral atrophy Classification of the Spinocerebellar Ataxias Cerebellar ataxia, deafness, and narcolepsy (autosomal dominant) Familial dementia with amyloid angiopathy and spastic ataxia (autosomal dominant)

1	Sensory ataxic neuropathy and ophthalmoparesis (SANDO) with dysarthria (autosomal recessive) 7p21.3-p15.1 1p21-q23; deletion (in frame); V338E; G345V; T377M; allelic with SCA19; KCND3; Kv4.3 channels 20p13-12.3; prodynorphin (PDYN protein); missense R138S; L211S; R212W; R215C 19p13.3 18p11.22-q11.2; ATPase family gene 3like 2 (AFG3L2 protein) mutations: N432T; S674L; E691K; A694E; R702Q 4q34.3-q35.1; candidate gene ODZ3 16q22.1; associated with NEDD4 (BEAN) 20p13; large intronic expansion of GGCCTG (1500–2500); also phe265leu mutation; RNA gain of function; microRNA; MIR 1292 suppression 20p13;pro102leu; ala 117 val mutations; proteinase k resistant form PrP27-30 accumulates in brain; eponym: GerstmannStraüssler-Scheinker disease Glu200Lys mutation; increased octapeptide repeats; eponym: Creutzfeldt-Jakob disease 10q23.31; phosphatase and tensin homolog (PTEN); Cowden’s; Lhermitte-Duclos syndrome 19p13.2; exon 21; missense ala570val; val606phe mutations 1p36.31-p36.23 13q14.2;

1	Creutzfeldt-Jakob disease 10q23.31; phosphatase and tensin homolog (PTEN); Cowden’s; Lhermitte-Duclos syndrome 19p13.2; exon 21; missense ala570val; val606phe mutations 1p36.31-p36.23 13q14.2; integral membrane protein 2B (ITM2B) 12p13.31 with CAG repeats (exonic) 9q13-q21.1 with intronic GAA repeats, in intron at end of exon 1

1	Frataxin defective; abnormal regulation of mitochondrial iron metabolism; iron accumulates in mitochondria in yeast mutants 8q13.1-q13.3 (α-TTP deficiency) 15q25; mutations in DNA polymerase-gamma (POLG) gene that leads to mtDNA deletions 3p26-p25 Ataxia, dysarthria, extrapyramidal features of akinesia, rigidity, tremor, cognitive defect; reduced deep tendon reflexes; MRI: cerebellar atrophy, normal basal ganglia and brainstem Pure cerebellar ataxia; dysarthria; dysphagia; nystagmus; MRI: cerebellar atrophy Gait ataxia; dysarthria; extremity ataxia; ocular nystagmus, dysmetria; leg vibration loss; extensor plantar responses; MRI: cerebellar atrophy Ataxia, nystagmus; vibratory loss in the feet; pain loss in some; abdominal pain; nausea and vomiting may be prominent; absent ankle reflexes; sensory nerve action potentials are absent; MRI: cerebellar atrophy, normal brainstem Gait ataxia; extremity ataxia; dysarthria; nystagmus; MRI: cerebellar atrophy

1	Gait ataxia; extremity ataxia; dysarthria; nystagmus; MRI: cerebellar atrophy Tremor in extremities and head and orofacial dyskinesia; ataxia of arms > legs, gait ataxia; dysarthria; nystagmus; psychiatric symptoms; cognitive defect; MRI: cerebellar atrophy; genetic testing available Extremity and gait ataxia; dysarthria; nystagmus; ophthalmoparesis; leg hyperreflexia and extensor plantar responses; MRI: cerebellar atrophy Candidate gene ODZ3; gait ataxia, dysarthria, saccades; nystagmus, brisk tendon reflexes in legs; MRI: cerebellar atrophy Pentanucleotide (TGGAA)n repeat insertions; previously called SCA4; gait ataxia; limb dysmetria; MRI: cerebellar atrophy Ataxia, azoospermia, mental retardation; absent germ cells on testicular biopsy Ataxia; onset fifth to sixth decades; motor neuron disorder; grouped atrophy (muscle biopsy) fasciculations; increased reflexes; flexor plantars Ataxia; dementia third to seventh decades Ataxia; dementia; rigidity Ataxia, mental retardation

1	Ataxia; dementia third to seventh decades Ataxia; dementia; rigidity Ataxia, mental retardation Ataxia, choreoathetosis, dystonia, seizures, myoclonus, dementia; genetic testing available Ataxia, areflexia, extensor plantar responses, position sense deficits, cardiomyopathy, diabetes mellitus, scoliosis, foot deformities; optic atrophy; late-onset form, as late as 50 years with preserved deep tendon reflexes, slower progression, reduced skeletal deformities, associated with an intermediate number of GAA repeats and missense mutations in one allele of frataxin; genetic testing available Same as phenotype that maps to 9q but associated with vitamin E deficiency; genetic testing available Young adult–onset ataxia, sensory neuropathy, ophthalmoparesis, hearing loss, gastric symptoms; a variant of progressive external ophthalmoplegia; MRI: cerebellar and thalamic abnormalities; mildly increased lactate and creatine kinase

1	Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) Mitochondrial encephalopathy, lactic acidosis, and stroke syndrome (MELAS) (maternal inheritance) Episodic ataxia, type 1 (EA-1) (autosomal dominant) Episodic ataxia, type 2 (EA-2) (autosomal dominant) Episodic ataxia, type 3 (autosomal dominant) Episodic ataxia, type 4 (autosomal dominant) Episodic ataxia, type 5 (autosomal dominant) Episodic ataxia type 6 with seizures, migraine, and alternating hemiplegia (autosomal dominant) Episodic ataxia, type 7 (autosomal dominant) Episodic ataxia with paroxysmal choreoathetosis and spasticity (dystonia-9) (DYT9; CSE) (autosomal dominant) Early-onset cerebellar ataxia with retained deep tendon reflexes (autosomal recessive) Ataxia with oculomotor apraxia (AOA1) (autosomal recessive)

1	Early-onset cerebellar ataxia with retained deep tendon reflexes (autosomal recessive) Ataxia with oculomotor apraxia (AOA1) (autosomal recessive) Ataxia with oculomotor apraxia 2 (AOA2) (autosomal recessive) 21q22.3; cystatin B; extra repeats of 12–base pair tandem repeats 5q31; SIL 1 protein, nucleotide exchange factor for the heat-shock protein 70 (HSP70); chaperone HSPA5; homozygous 4-nucleotide duplication in exon 6; also compound heterozygote Chromosome 13q12; SACS gene; loss of sacsin peptide activity Mutation in mtDNA of the tRNAlys at 8344; also mutation at 8356 12p13; potassium voltage-gated channel gene, KCNA1; Phe249Leu mutation; variable syndrome 19p-13 (CACNA1A) (allelic with SCA6) (α1A-voltage–dependent calcium channel subunit); point mutations or small deletions; allelic with SCA6 and familial hemiplegic migraine SLC1A3; 5p13; EAAT1 protein; missense mutations; glial glutamate transporter (GLAST); 1047 C to G; proline to arginine

1	SLC1A3; 5p13; EAAT1 protein; missense mutations; glial glutamate transporter (GLAST); 1047 C to G; proline to arginine Xq27.3; CGG premutation expansion in FMR1 gene; expansions of 55–200 repeats in 5’ UTR of the FMR-1 mRNA; presumed dominant toxic RNA effect 11q22-23; ATM gene for regulation of cell cycle; mitogenic signal transduction and meiotic recombination; elevated serum alpha-fetoprotein level; immunoglobulin deficiency 9p21; protein is member of histidine triad superfamily, role in DNA repair; elevation of serum LDL cholesterol and low serum albumin level; APTX, aprataxin 9q34; senataxin protein, involved in RNA maturation and termination; helicase superfamily 1; elevated serum alphafetoprotein level; SETX, senataxin Myoclonus epilepsy; late-onset ataxia; responds to valproic acid, clonazepam; phenobarbital

1	Ataxia, dysarthria; nystagmus; retarded motor and mental maturation; rhabdomyolysis after viral illness; weakness; hypotonia; areflexia; cataracts in childhood; short stature; kyphoscoliosis; contractures; hypogonadism Childhood onset of ataxia, spasticity, dysarthria, distal muscle wasting, foot deformity, retinal striations, mitral valve prolapse Ptosis, ophthalmoplegia, pigmentary retinal degeneration, cardiomyopathy, diabetes mellitus, deafness, heart block, increased CSF protein, ataxia Myoclonic epilepsy, ragged red fiber myopathy, ataxia Headache, stroke, lactic acidosis, ataxia Episodic ataxia for minutes; provoked by startle or exercise; with facial and hand myokymia; cerebellar signs are not progressive; choreoathetotic movements; responds to phenytoin; genetic testing available

1	Episodic ataxia for days; provoked by stress, fatigue; with down-gaze nystagmus; vertigo; vomiting; headache; cerebellar atrophy results; progressive cerebellar signs; responds to acetazolamide; genetic testing available Episodic ataxia; 1 min to over 6 h; induced by movement; vertigo and tinnitus; headache; responds to acetazolamide Episodic ataxia; vertigo; diplopia; ocular slow pursuit defect; no response to acetazolamide Episodic ataxia; hours to weeks; seizures Ataxia, duration 2–4 days; episodic hypotonia; delayed motor milestones; seizures; migraine; alternating hemiplegia; mild truncal ataxia; coma; febrile illness as a trigger; MRI: cerebellar atrophy Episodic ataxia; vertigo, weakness; less than 24 h Ataxia; involuntary movements; dystonia; headache; spastic paraplegia; responds on occasion to acetazolamide

1	Episodic ataxia; vertigo, weakness; less than 24 h Ataxia; involuntary movements; dystonia; headache; spastic paraplegia; responds on occasion to acetazolamide Late-onset ataxia with tremor, cognitive impairment, occasional parkinsonism; males typically affected, although affected females also reported; syndrome is of high concern if affected male has grandson with mental retardation (fragile X syndrome); MRI shows increased T2 signal in middle cerebellar peduncles, cerebellar atrophy, and occasional widespread brain atrophy; genetic testing available Telangiectasia, ataxia, dysarthria, pulmonary infections, neoplasms of lymphatic system; IgA and IgG deficiencies; diabetes mellitus, breast cancer; genetic testing available; chorea; dystonia Ataxia; neuropathy; preserved deep tendon reflexes; impaired cognitive and visuospatial functions; MRI, cerebellar atrophy

1	Ataxia; neuropathy; preserved deep tendon reflexes; impaired cognitive and visuospatial functions; MRI, cerebellar atrophy Gait ataxia; choreoathetosis; dystonia; oculomotor apraxia; neuropathy, vibration loss, position sense loss, and mild light touch loss; absent leg deep tendon reflexes; extensor plantar response; genetic testing available Classification of the Spinocerebellar Ataxias Cerebellar ataxia with muscle coenzyme Q10 deficiency (autosomal recessive) Infantile-onset spinocerebellar ataxia of Nikali et al (autosomal recessive) Hypoceruloplasminemia with ataxia and dysarthria (autosomal recessive) Spinocerebellar ataxia with neuropathy (SCAN1) (autosomal recessive) 9q34.3

1	Hypoceruloplasminemia with ataxia and dysarthria (autosomal recessive) Spinocerebellar ataxia with neuropathy (SCAN1) (autosomal recessive) 9q34.3 Xq13; ATP-binding cassette 7 (ABCB7; ABC7) transporter; mitochondrial inner membrane; iron homeostasis; export from matrix to the intermembrane space 10q23.3-q24.1; twinkle protein (gene); homozygous for Tyr508Cys missense mutations 1q42;ADCK3 (CABC1); aarf-domain containing kinase 3; elevation of serum lactate and decreased coenzyme Q10 level 18q11; NPCI; NPCH1 and 2; skin biopsy (filipin staining) Ataxia; hypotonia; seizures; mental retardation; increased deep tendon reflexes; extensor plantar responses; coenzyme Q10 levels reduced with about 25% of patients with a block in transfer of electrons to complex 3; may respond to coenzyme 10 Infantile ataxia, sensory neuropathy; athetosis, hearing deficit, reduced deep tendon reflexes; ophthalmoplegia, optic atrophy; seizures; primary hypogonadism in females

1	Infantile ataxia, sensory neuropathy; athetosis, hearing deficit, reduced deep tendon reflexes; ophthalmoplegia, optic atrophy; seizures; primary hypogonadism in females Gait ataxia and dysarthria; hyperreflexia; cerebellar atrophy by MRI; iron deposition in cerebellum, basal ganglia, thalamus, and liver; onset in the fourth decade Onset in second decade; gait ataxia, dysarthria, seizures, cerebellar vermis atrophy on MRI, dysmetria Abbreviations: CSF, cerebrospinal fluid; CT, computed tomography; EMG, electromyogram; LDL, low-density lipoprotein; MRI, magnetic resonance imaging; REM, rapid eye movement; UTR, untranslated region. amyotrophic Lateral sclerosis and other motor Neuron Diseases Robert H. Brown, Jr. AMYOTROPHIC LATERAL SCLEROSIS Amyotrophic lateral sclerosis (ALS) is the most common form of 452 progressive motor neuron disease. It is a prime example of a neurodegenerative disease and is arguably the most devastating of the neurodegenerative disorders.

1	The pathologic hallmark of motor neuron degenerative disorders is death of lower motor neurons (consisting of anterior horn cells in the spinal cord and their brainstem homologues innervating bulbar muscles) and upper, or corticospinal, motor neurons (originating in layer five of the motor cortex and descending via the pyramidal tract to synapse with lower motor neurons, either directly or indirectly via interneurons) (Chap. 30). Although at its onset ALS may involve selective loss of function of only upper or lower motor neurons, it ultimately causes progressive loss of both categories of motor neurons. Indeed, in the absence of clear involvement of both motor neuron types, the diagnosis of ALS is questionable. In a subset of cases, ALS arises concurrently with frontotemporal dementia (Chap. 448); in these instances, there is degeneration of frontotemporal cortical neurons and corresponding cortical atrophy.

1	Other motor neuron diseases involve only particular subsets of motor neurons (Tables 452-1 and 452-2). Thus, in bulbar palsy and spinal muscular atrophy (SMA; also called progressive muscular atrophy), Abbreviations: CSF, cerebrospinal fluid; FUS/TLS, fused in sarcoma/translocated in liposarcoma; HTLV-1, human T-cell lymphotropic virus; MRI, magnetic resonance imaging; PTH, parathyroid; WBC, white blood cell. the lower motor neurons of brainstem and spinal cord, respectively, are most severely involved. By contrast, pseudobulbar palsy, primary lateral sclerosis (PLS), and familial spastic paraplegia (FSP) affect only upper motor neurons innervating the brainstem and spinal cord.

1	In each of these diseases, the affected motor neurons undergo shrinkage, often with accumulation of the pigmented lipid (lipofuscin) that normally develops in these cells with advancing age. In ALS, the motor neuron cytoskeleton is typically affected early in the illness. Focal enlargements are frequent in proximal motor axons; ultrastructurally, these “spheroids” are composed of accumulations of neurofilaments and other proteins. Commonly in both sporadic and familial Upper and lower motor neuron Amyotrophic lateral sclerosis Predominantly upper motor neuron Primary lateral sclerosis Predominantly lower motor neuron Multifocal motor neuropathy with Motor neuropathy with paraprotein Associated with other neurodegenerative disorders

1	Motor neuropathy with paraprotein Associated with other neurodegenerative disorders ALS, the affected neurons demonstrate ubiquitin-positive aggregates, typically associated with the protein TDP43 (see below). Also seen is proliferation of astroglia and microglia, the inevitable accompaniment of all degenerative processes in the central nervous system (CNS).

1	The death of the peripheral motor neurons in the brainstem and spinal cord leads to denervation and consequent atrophy of the corresponding muscle fibers. Histochemical and electrophysiologic evidence indicates that in the early phases of the illness denervated muscle can be reinnervated by sprouting of nearby distal motor nerve terminals, although reinnervation in this disease is considerably less extensive than in most other disorders affecting motor neurons (e.g., poliomyelitis, peripheral neuropathy). As denervation progresses, muscle atrophy is readily recognized in muscle biopsies and on clinical examination. This is the basis for the term amyotrophy. The loss of cortical motor neurons results in thinning of the corticospinal tracts that travel via the internal capsule (Fig. 452-1) and brainstem to the lateral and anterior white matter columns of the spinal cord. The loss of fibers in the lateral columns and resulting fibrillary gliosis impart a particular firmness (lateral

1	and brainstem to the lateral and anterior white matter columns of the spinal cord. The loss of fibers in the lateral columns and resulting fibrillary gliosis impart a particular firmness (lateral sclerosis). A remarkable feature of the disease is the selectivity of neuronal cell death. By light microscopy, the entire sensory apparatus, the regulatory mechanisms for the control and coordination of movement, remains intact. Except in cases of frontotemporal dementia, the components of the brain required for cognitive processing are also preserved. However, immunostaining indicates that neurons bearing ubiquitin, a marker for degeneration, are also detected in nonmotor systems. Moreover, studies of glucose metabolism in the illness also indicate that there is neuronal dysfunction outside of the motor system. Within the motor system, there is some selectivity of involvement. Thus, motor neurons required for ocular motility remain unaffected, as do the parasympathetic neurons in the sacral

1	motor system. Within the motor system, there is some selectivity of involvement. Thus, motor neurons required for ocular motility remain unaffected, as do the parasympathetic neurons in the sacral spinal cord (the nucleus of Onufrowicz, or Onuf ) that innervate the sphincters of the bowel and bladder.

1	The manifestations of ALS are somewhat variable depending on whether corticospinal neurons or lower motor neurons in the brain-stem and spinal cord are more prominently involved. With lower motor neuron dysfunction and early denervation, typically the first evidence of the disease is insidiously developing asymmetric weakness, usually first evident distally in one of the limbs. A detailed history often discloses recent development of cramping with volitional movements, typically in the early hours of the morning (e.g., while stretching in bed). Weakness caused by denervation is associated with progressive wasting and atrophy of muscles and, particularly early in the illness, spontaneous twitching of motor units, or fasciculations. In the hands, a

1	FIGURE 452-1 Amyotrophic lateral sclerosis. Axial T2-weighted magnetic resonance imaging (MRI) scan through the lateral ventricles of the brain reveals abnormal high signal intensity within the corticospinal tracts (arrows). This MRI feature represents an increase in water content in myelin tracts undergoing Wallerian degeneration secondary to cortical motor neuronal loss. This finding is commonly present in ALS, but can also be seen in AIDS-related encephalopathy, infarction, or other disease processes that produce corticospinal neuronal loss in a symmetric fashion.

1	preponderance of extensor over flexor weakness is common. When the initial denervation involves bulbar rather than limb muscles, the problem at onset is difficulty with chewing, swallowing, and movements of the face and tongue. Early involvement of the muscles of respiration may lead to death before the disease is far advanced elsewhere. With prominent corticospinal involvement, there is hyperactivity of the muscle-stretch reflexes (tendon jerks) and, often, spastic resistance to passive movements of the affected limbs. Patients with significant reflex hyperactivity complain of muscle stiffness often out of proportion to weakness. Degeneration of the corticobulbar projections innervating the brainstem results in dysarthria and exaggeration of the motor expressions of emotion. The latter leads to involuntary excess in weeping or laughing (pseudobulbar affect).

1	Virtually any muscle group may be the first to show signs of disease, but, as time passes, more and more muscles become involved until ultimately the disorder takes on a symmetric distribution in all regions. It is characteristic of ALS that, regardless of whether the initial disease involves upper or lower motor neurons, both will eventually be implicated. Even in the late stages of the illness, sensory, bowel and bladder, and cognitive functions are preserved. Even when there is severe brainstem disease, ocular motility is spared until the very late stages of the illness. As noted, in some cases (particularly those that are familial), ALS develops concurrently with frontotemporal dementia, characterized by early behavioral abnormalities with prominent behavioral features indicative of frontal lobe dysfunction.

1	A committee of the World Federation of Neurology has established diagnostic guidelines for ALS. Essential for the diagnosis is simultaneous upper and lower motor neuron involvement with progressive weakness and the exclusion of all alternative diagnoses. The disorder is ranked as “definite” ALS when three or four of the following are involved: bulbar, cervical, thoracic, and lumbosacral motor neurons. When two sites are involved, the diagnosis is “probable,” and when only one site is implicated, the diagnosis is “possible.” An exception is made for those who have progressive upper and lower motor neuron signs at only one site and a mutation in the gene encoding superoxide dismutase (SOD1; see below).

1	The illness is relentlessly progressive, leading to death from respiratory paralysis; the median survival is from 3–5 years. There are very rare reports of stabilization or even regression of ALS. In most societies, there is an incidence of 1–3 per 100,000 and a prevalence of 3–5 per 100,000. It is striking that about 1 in 1000 adult deaths in North America and Western Europe (and probably elsewhere) are due to ALS; this finding predicts that some 300,000 individuals now alive in the United States will die of ALS. Several endemic foci of higher prevalence exist in the western Pacific (e.g., in specific regions of Guam or Papua New Guinea). In the United States and Europe, males are somewhat more frequently affected than females. Epidemiologic studies have incriminated risk factors for this disease including exposure to pesticides and insecticides, smoking, and, in one report, service in the military. Although ALS is overwhelmingly a sporadic disorder, some 5–10% of cases are inherited

1	disease including exposure to pesticides and insecticides, smoking, and, in one report, service in the military. Although ALS is overwhelmingly a sporadic disorder, some 5–10% of cases are inherited as an autosomal dominant trait.

1	Several forms of selective motor neuron disease are inheritable (Table 452-3). Familial ALS (FALS) involves both corticospinal and lower motor neurons. Apart from its inheritance as an autosomal dominant trait, it is clinically indistinguishable from sporadic ALS. Genetic studies have identified mutations in multiple genes, including those encoding the protein C9orf 72 (open reading frame 72 on chromosome 9), cytosolic enzyme SOD1 (superoxide dismutase), the RNA binding proteins TDP43 (encoded by the TAR DNA binding protein gene), and FUS/TLS (fused in sarcoma/translocated in liposarcoma), as the most common causes of FALS. Mutations in C9orf72 account for ~45–50% of FALS and perhaps 4–5% of sporadic ALS cases. Mutations in SOD1 explain another 20% of cases of FALS, whereas TDP43 and FUS/TLS each represent about 5% of familial cases. It has recently been reported that ~1–2% of cases are caused by mutations in genes encoding the proteins optineuron and profilin-1 as well.

1	Rare mutations in other genes are also clearly implicated in ALS-like diseases. Thus, a familial, dominantly inherited motor disorder that in some individuals closely mimics the ALS phenotype arises from mutations in a gene that encodes a vesicle-binding protein. A predominantly lower motor neuron disease with early hoarseness due to laryngeal dysfunction has been ascribed to mutations in the gene encoding the cellular accessory motor protein dynactin. Mutations in senataxin, a helicase, cause an early adult-onset, slowly evolving ALS variant. Kennedy’s syndrome is an X-linked, adult-onset disorder that may mimic ALS, as described below.

1	Genetic analyses are also beginning to illuminate the pathogenesis of some childhood-onset motor neuron diseases. For example, a slowly disabling degenerative, predominantly upper motor neuron disease that starts in the first decade is caused by mutations in a gene that expresses a novel signaling molecule with properties of a guanine-exchange factor, termed alsin.

1	Because ALS is currently untreatable, it is imperative that potentially remediable causes of motor neuron dysfunction be excluded (Table 452-1). This is particularly true in cases that are atypical by virtue of (1) restriction to either upper or lower motor neurons, (2) involvement of neurons other than motor neurons, and (3) evidence of motor neuronal conduction block on electrophysiologic testing. Compression of the cervical spinal cord or cervicomedullary junction from tumors in the cervical regions or at the foramen magnum or from cervical spondylosis with osteophytes projecting into the vertebral canal can produce weakness, wasting, and fasciculations in the upper limbs and spasticity in the legs, closely resembling ALS. The absence of cranial nerve involvement may be helpful in differentiation, although some foramen magnum lesions may compress the twelfth cranial (hypoglossal) nerve, with resulting paralysis of the tongue. Absence of pain or of sensory changes, normal bowel and

1	although some foramen magnum lesions may compress the twelfth cranial (hypoglossal) nerve, with resulting paralysis of the tongue. Absence of pain or of sensory changes, normal bowel and bladder function, nor-2633 mal roentgenographic studies of the spine, and normal cerebrospinal fluid (CSF) all favor ALS. Where doubt exists, magnetic resonance imaging (MRI) scans and contrast myelography should be performed to visualize the cervical spinal cord.

1	Another important entity in the differential diagnosis of ALS is multifocal motor neuropathy with conduction block (MMCB), discussed below. A diffuse, lower motor axonal neuropathy mimicking ALS sometimes evolves in association with hematopoietic disorders such as lymphoma or multiple myeloma. In this clinical setting, the presence of an M-component in serum should prompt consideration of a bone marrow biopsy. Lyme disease (Chap. 210) may also cause an axonal, lower motor neuropathy, although typically with intense proximal limb pain and a CSF pleocytosis.

1	Other treatable disorders that occasionally mimic ALS are chronic lead poisoning and thyrotoxicosis. These disorders may be suggested by the patient’s social or occupational history or by unusual clinical features. When the family history is positive, disorders involving the genes encoding C9orf72, cytosolic SOD1, TDP43, FUS/TLS, and adult hexosaminidase A or α-glucosidase deficiency must be excluded (Chap. 432e). These are readily identified by appropriate laboratory tests. Benign fasciculations are occasionally a source of concern because on inspection they resemble the fascicular twitchings that accompany motor neuron degeneration. The absence of weakness, atrophy, or denervation phenomena on electrophysiologic examination usually excludes ALS or other serious neurologic disease. Patients who have recovered from poliomyelitis may experience a delayed deterioration of motor neurons that presents clinically with progressive weakness, atrophy, and fasciculations. Its cause is unknown,

1	who have recovered from poliomyelitis may experience a delayed deterioration of motor neurons that presents clinically with progressive weakness, atrophy, and fasciculations. Its cause is unknown, but it is thought to reflect sublethal prior injury to motor neurons by poliovirus (Chap. 228).

1	Rarely, ALS develops concurrently with features indicative of more widespread neurodegeneration. Thus, one infrequently encounters otherwise typical ALS patients with a parkinsonian movement disorder or frontotemporal dementia, particularly in instances of C9orf72 mutations, which strongly suggests that the simultaneous occurrence of two disorders is a direct consequence of the gene mutation. As another example, prominent amyotrophy has been described as a dominantly inherited disorder in individuals with bizarre behavior and a movement disorder suggestive of parkinsonism; many such cases have now been ascribed to mutations that alter the expression of tau protein in brain (Chap. 448). In other cases, ALS develops simultaneously with a striking frontotemporal dementia. An ALS-like disorder has also been described in some individuals with chronic traumatic encephalopathy, associated with deposition of TDP43 and neurofibrillary tangles in motor neurons.

1	The cause of sporadic ALS is not well defined. Several mechanisms that impair motor neuron viability have been elucidated in mice and rats induced to develop motor neuron disease by SOD1 transgenes with ALS-associated mutations. One may loosely group the genetic causes of ALS into three categories. In one group, the primarily problem is inherent instability of the mutant proteins, with subsequent perturbations in protein degradation (SOD1, ubiquilin-1 and -2, p62). In the second, most rapidly growing category, the causative mutant genes perturb RNA processing, transport, and metabolism (C9orf73, TDP43, FUS). In the case of C9orf72, the molecular pathology is an expansion of an intronic hexanucleotide repeat (-GGGGCC-) beyond an upper normal of 30 repeats to hundreds or more repeats. As observed in other intronic repeat disorders such as myotonic dystrophy (Chap. 462e) and spinocerebellar atrophy type 8 (Chap. 450), data suggest that the expanded intronic repeats generate expanded RNA

1	in other intronic repeat disorders such as myotonic dystrophy (Chap. 462e) and spinocerebellar atrophy type 8 (Chap. 450), data suggest that the expanded intronic repeats generate expanded RNA repeats that form intranuclear foci and confer toxicity by sequestering transcription factors or by undergoing noncanonical protein translation across all possible reading frames of the expanded RNA tracts. TDP43 and FUS are multifunctional proteins that bind RNA and DNA and shuttle between the nucleus and the cytoplasm, playing multiple roles in the control of cell proliferation, DNA repair and transcription, esterase SPG44 1q Connexin 47 AR Childhood Gap junction protein Possible mild CNS features SPG46 9p β-Glucosidase 2 AR Childhood Glycoside hydrolase Thin corpus callosum, mental retardation SPG2 Xq Proteolipid protein XR Early childhood Myelin protein Sometimes multiple

1	Xq Adrenoleukodystrophy XR Early adulthood ATP binding transporter protein Possible adrenal insufficiency, CNS inflammation IV. ALS-Plus Syndromes Abbreviations: ALS, amyotrophic lateral sclerosis; BSCL2, Bernadelli-Seip congenital lipodystrophy 2B; AD, autosomal dominant; AR, autosomal recessive; CNS, central nervous system; FSP, familial spastic paraplegia; FUS/TLS, fused in sarcoma/translocated in liposarcoma; TDP43, Tar DNA binding protein 43 kd; XR, X-linked recessive.

1	and gene translation, both in the cytoplasm and locally in dendritic spines in response to electrical activity. How mutations in FUS/TLS provoke motor neuron cell death is not clear, although this may represent loss of function of FUS/TLS in the nucleus or an acquired, toxic function of the mutant proteins in the cytosol. In the third group of ALS genes, the primary problem is defective axonal cytoskeleton and transport (dynactin, profilin-1). It is striking that variants in other genes (e.g., EphA4) influence survival in ALS but not ALS susceptibility. Beyond the upstream, primary defects, it is also evident that the ultimate neuronal cell death process is complex involving multiple cellular processes that accelerate cell death. These include but are not limited to excitotoxicity, impairment of axonal transport, oxidative stress, activation of endoplasmic reticulum stress and the unfolded protein response, and mitochondrial dysfunction.

1	Multiple recent studies have convincingly demonstrated that non-neuronal cells importantly influence the disease course, at least in ALS transgenic mice. A striking additional finding in neurodegenerative disorders is that miscreant proteins arising from gene defects in familial forms of these diseases are often implicated in sporadic forms of the same disorder. For example, germline mutations in the genes encoding β-amyloid and α-synuclein cause familial forms of Alzheimer’s and Parkinson’s diseases, and posttranslational, noninherited abnormalities in these proteins are also central to sporadic Alzheimer’s and Parkinson’s diseases. Analogously, recent reports propose that nonheritable, posttranslational modifications in SOD1 are pathogenic in sporadic ALS.

1	No treatment arrests the underlying pathologic process in ALS. The drug riluzole (100 mg/d) was approved for ALS because it produces a modest lengthening of survival. In one trial, the survival rate at 18 months with riluzole was similar to placebo at 15 months. The mechanism of this effect is not known with certainty; riluzole may reduce excitotoxicity by diminishing glutamate release. Riluzole is generally well tolerated; nausea, dizziness, weight loss, and elevated liver enzymes occur occasionally. Pathophysiologic studies of mutant SOD1–related ALS in mice have disclosed diverse targets for therapy; consequently, multiple therapies are presently in clinical trials for ALS including experimental trials of small molecules, mesenchymal stem cells, and immunosuppression. Interventions such as antisense oligonucleotides (ASO) that diminish expression of mutant SOD1 protein prolong survival in transgenic ALS mice and rats and are also nearing trial now for SOD1-mediated ALS.

1	In the absence of a primary therapy for ALS, a variety of rehabilitative aids may substantially assist ALS patients. Foot-drop splints facilitate ambulation by obviating the need for excessive hip flexion and by preventing tripping on a floppy foot. Finger extension splints can potentiate grip. Respiratory support may be life-sustaining. For patients electing against long-term ventilation by tracheostomy, positive-pressure ventilation by mouth or nose provides transient (several weeks) relief from hypercarbia and hypoxia. Also extremely beneficial for some patients is a respiratory device (Cough Assist Device) that produces an artificial cough. This is highly effective in clearing airways and preventing aspiration pneumonia. When bulbar disease prevents normal chewing and swallowing, gastrostomy is uniformly helpful, restoring normal nutrition and hydration. Fortunately, an increasing variety of speech synthesizers are now available to augment speech when there is advanced bulbar

1	gastrostomy is uniformly helpful, restoring normal nutrition and hydration. Fortunately, an increasing variety of speech synthesizers are now available to augment speech when there is advanced bulbar palsy. These facilitate oral communication and may be effective for telephone use.

1	In contrast to ALS, several of the disorders (Tables 452-1 and 452-3) that bear some clinical resemblance to ALS are treatable. For this reason, a careful search for causes of secondary motor neuron disease is warranted. In these motor neuron diseases, the peripheral motor neurons are affected without evidence of involvement of the corticospinal motor system (Tables 452-1, 452-2, and 452-3).

1	2636 X-Linked Spinobulbar Muscular Atrophy (Kennedy’s Disease) This is an X-linked lower motor neuron disorder in which progressive weakness and wasting of limb and bulbar muscles begins in males in mid-adult life and is conjoined with androgen insensitivity manifested by gynecomastia and reduced fertility (Chap. 411). In addition to gynecomastia, which may be subtle, two findings distinguishing this disorder from ALS are the absence of signs of pyramidal tract disease (spasticity) and the presence of a subtle sensory neuropathy in some patients. The underlying molecular defect is an expanded trinucleotide repeat (-CAG-) in the first exon of the androgen receptor gene on the X chromosome. DNA testing is available. An inverse correlation appears to exist between the number of -CAGrepeats and the age of onset of the disease.

1	Adult Tay-Sachs Disease Several reports have described adult-onset, predominantly lower motor neuropathies arising from deficiency of the enzymeβ-hexosaminidase (hex A). These tend to be distinguishable from ALS because they are very slowly progressive; dysarthria and radiographically evident cerebellar atrophy may be prominent. In rare cases, spasticity may also be present, although it is generally absent (Chap. 432e).

1	Spinal Muscular Atrophy The SMAs are a family of selective lower motor neuron diseases of early onset. Despite some phenotypic variability (largely in age of onset), the defect in the majority of families with SMA maps to a locus on chromosome 5 encoding a putative motor neuron survival protein (SMN, for survival motor neuron) that is important in the formation and trafficking of RNA complexes across the nuclear membrane. Neuropathologically these disorders are characterized by extensive loss of large motor neurons; muscle biopsy reveals evidence of denervation atrophy. Several clinical forms exist.

1	Infantile SMA (SMA I, Werdnig-Hoffmann disease) has the earliest onset and most rapidly fatal course. In some instances it is apparent even before birth, as indicated by decreased fetal movements late in the third trimester. Though alert, afflicted infants are weak and floppy (hypotonic) and lack muscle stretch reflexes. Death generally ensues within the first year of life. Chronic childhood SMA (SMA II) begins later in childhood and evolves with a more slowly progressive course. Juvenile SMA (SMA III, Kugelberg-Welander disease) manifests during late childhood and runs a slow, indolent course. Unlike most denervating diseases, in this chronic disorder, weakness is greatest in the proximal muscles; indeed, the pattern of clinical weakness can suggest a primary myopathy such as limb-girdle dystrophy. Electrophysiologic and muscle biopsy evidence of denervation distinguish SMA III from the myopathic syndromes. There is no primary therapy for SMA, although remarkable recent experimental

1	dystrophy. Electrophysiologic and muscle biopsy evidence of denervation distinguish SMA III from the myopathic syndromes. There is no primary therapy for SMA, although remarkable recent experimental data indicate that it may be possible to deliver the missing SMN gene to motor neurons using intravenously or intrathecally delivered adeno-associated viruses (e.g., AAV9) immediately after birth.

1	Multifocal Motor Neuropathy with Conduction Block In this disorder lower motor neuron function is regionally and chronically disrupted by remarkably focal blocks in conduction. Many cases have elevated serum titers of monoand polyclonal antibodies to ganglioside GM1; it is hypothesized that the antibodies produce selective, focal, paranodal demyelination of motor neurons. MMCB is not typically associated with corticospinal signs. In contrast with ALS, MMCB may respond dramatically to therapy such as IV immunoglobulin or chemotherapy; thus, it is imperative that MMCB be excluded when considering a diagnosis of ALS.

1	Other Forms of Lower Motor Neuron Disease In individual families, other syndromes characterized by selective lower motor neuron dysfunction in an SMA-like pattern have been described. There are rare X-linked and autosomal dominant forms of apparent SMA. There is an ALS variant of juvenile onset, the Fazio-Londe syndrome, that involves mainly the musculature innervated by the brainstem. A component of lower motor neuron dysfunction is also found in degenerative disorders such as Machado-Joseph disease and the related olivopontocerebellar degenerations (Chap. 450).

1	SELECTED DISORDERS OF THE UPPER MOTOR NEURON Primary Lateral Sclerosis This exceedingly rare disorder arises sporadically in adults in mid to late life. Clinically PLS is characterized by progressive spastic weakness of the limbs, preceded or followed by spastic dysarthria and dysphagia, indicating combined involvement of the corticospinal and corticobulbar tracts. Fasciculations, amyotrophy, and sensory changes are absent; neither electromyography nor muscle biopsy shows denervation. On neuropathologic examination, there is selective loss of the large pyramidal cells in the precentral gyrus and degeneration of the corticospinal and corticobulbar projections. The peripheral motor neurons and other neuronal systems are spared. The course of PLS is variable; although long-term survival is documented, the course may be as aggressive as in ALS, with ~3-year survival from onset to death. Early in its course, PLS raises the question of multiple sclerosis or other demyelinating diseases such

1	the course may be as aggressive as in ALS, with ~3-year survival from onset to death. Early in its course, PLS raises the question of multiple sclerosis or other demyelinating diseases such as adrenoleukodystrophy as diagnostic considerations (Chap. 458). A myelopathy suggestive of PLS is infrequently seen with infection with the retrovirus human T cell lymphotropic virus 1 (HTLV-1) (Chap. 456). The clinical course and laboratory testing will distinguish these possibilities.

1	Familial Spastic Paraplegia In its pure form, FSP is usually transmitted as an autosomal trait; most adult-onset cases are dominantly inherited. Symptoms usually begin in the third or fourth decade, presenting as progressive spastic weakness beginning in the distal lower extremities; however, there are variants with onset so early that the differential diagnosis includes cerebral palsy. FSP typically has a long survival, presumably because respiratory function is spared. Late in the illness, there may be urinary urgency and incontinence and sometimes fecal incontinence; sexual function tends to be preserved. In pure forms of FSP, the spastic leg weakness is often accompanied by posterior column (vibration and position) abnormalities and disturbance of bowel and bladder function. Some family members may have spasticity without clinical symptoms.

1	By contrast, particularly when recessively inherited, FSP may have complex or complicated forms in which altered corticospinal and dorsal column function is accompanied by significant involvement of other regions of the nervous system, including amyotrophy, mental retardation, optic atrophy, and sensory neuropathy. Neuropathologically, in FSP, there is degeneration of the corticospinal tracts, which appear nearly normal in the brainstem but show increasing atrophy at more caudal levels in the spinal cord; in effect, the pathologic picture is of a dying-back or distal axonopathy of long neuronal fibers within the CNS.

1	Defects at numerous loci underlie both dominantly and recessively inherited forms of FSP (Table 452-3). More than 30 FSP genes have now been identified. The gene most commonly implicated in dominantly inherited FSP is spastin, which encodes a microtubule interacting protein. The most common childhood-onset dominant form arises from mutations in the atlastin gene. A kinesin heavy-chain protein implicated in microtubule motor function was found to be defective in a family with dominantly inherited FSP of variable-onset age. An infantile-onset form of X-linked, recessive FSP arises from mutations in the gene for myelin proteolipid protein. This is an example of rather striking allelic variation, as most other mutations in the same gene cause not FSP but Pelizaeus-Merzbacher disease, a widespread disorder of CNS myelin. Another recessive variant is caused by defects in the paraplegin gene. Paraplegin has homology to metalloproteases that are important in mitochondrial function in yeast.

1	Several websites provide valuable information on ALS including those offered by the Muscular Dystrophy Association (www.mdausa.org), the Amyotrophic Lateral Sclerosis Association (www.alsa.org), and the World Federation of Neurology and the Neuromuscular Unit at Washington University in St. Louis (www.neuro.wustl.edu). Stanley B. Prusiner, Bruce L. Miller

1	Prions are proteins that adopt an alternative conformation, which becomes self-propagating. Some prions cause degeneration of the central nervous system (CNS). Once relegated to causing a group of rare disorders of the CNS such as Creutzfeldt-Jakob disease (CJD), prions—as mounting evidence shows—also appear to play a key role in more common illnesses such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). While CJD is caused by the accumulation of PrPSc, increasing data argue that Aβ prions cause AD, α-synuclein prions cause PD, and tau prions cause the frontotemporal dementias (FTDs). In this chapter, we confine our discussion to CJD, which typically presents with a rapidly progressive dementia as well as motor abnormalities. The illness is relentlessly progressive and generally causes death within 9 months of onset. Most CJD patients are between 50 and 75 years of age; however, patients as young as 17 and as old as 83 have been recorded.

1	CJD is one malady in a group of disorders caused by prions composed of the prion protein (PrP). PrP prions reproduce by binding to the normal, cellular isoform of the prion protein (PrPC) and stimulating conversion of PrPC into the disease-causing isoform PrPSc. PrPC is rich in α-helix and has little β-structure, whereas PrPSc has less α-helix and a high amount of β-structure (Fig. 453e-1). This α-to-β structural transition in PrP is the fundamental event underlying this group of prion diseases (Table 453e-1).

1	Four new concepts have emerged from studies of prions: (1) Prions are the only known transmissible pathogens that are devoid of nucleic acid; all other infectious agents possess genomes composed of either RNA or DNA that direct the synthesis of their progeny. (2) Prion diseases may be manifest as infectious, genetic, and sporadic disorders; no other group of illnesses with a single etiology presents with such a wide spectrum of clinical manifestations. (3) Prion diseases result from the accumulation of PrPSc, the conformation of which differs substantially from that of its precursor, PrPC. (4) Distinct strains of prions exhibit different biologic properties, which are epigenetically inherited. In other words, PrPSc can exist in a variety of different conformations, many of which seem to specify particular disease phenotypes.

1	How a specific conformation of a PrPSc molecule is imparted to PrPC during prion replication to produce nascent PrPSc with the same conformation is unknown. Additionally, it is unclear what factors determine where in the CNS a particular PrPSc molecule will be deposited. FIgURE 453e-1 Structures of prion proteins. A. NMR structure of Syrian hamster recombinant (rec) PrP(90–231). Presumably, the structure of the α-helical form of recPrP(90–231) resembles that of PrPC. recPrP(90–231) is viewed from the interface where PrPSc is thought to bind to PrPC. Shown are: α-helices A (residues 144–157), B (172–193), and C (200–227). Flat ribbons depict β-strands S1 (129–131) and S2 (161–163). B. Structural model of PrPSc. The 90–160 region has been modeled onto a β-helical architecture while the COOH terminal helices B and C are preserved as in PrPC. GLossary of prion terminoLoGy

1	GLossary of prion terminoLoGy Prion Proteinaceous infectious particle that lacks nucleic acid. Prions are composed entirely of alternatively folded proteins that undergo self-propagation. Distinct strains of prions exhibit different biologic properties, which are epigenetically heritable. PrP prions cause scrapie in sheep and goats, mad cow disease, and related neurodegenerative diseases of humans such as Creutzfeldt-Jakob disease (CJD). PrPSc Disease-causing isoform of the prion protein. This protein is the only identifiable macromolecule in purified preparations of scrapie prions. PrPC Cellular isoform of the prion protein. PrPC is the precursor of PrPSc. PrP 27-30 A fragment of PrPSc, generated by truncation of the NH2terminus by limited digestion with proteinase K. PrP 27-30 retains prion infectivity and polymerizes into amyloid. PRNP PrP gene located on human chromosome 20.

1	PRNP PrP gene located on human chromosome 20. Prion rod An aggregate of prions composed largely of PrP 27-30 molecules. Created by detergent extraction and limited proteolysis of PrPSc. Morphologically and histochemically indistinguishable from many amyloids. PrP amyloid Amyloid containing PrP in the brains of animals or humans with prion disease; often accumulates as plaques. The sporadic form of CJD is the most common prion disorder in humans. Sporadic CJD (sCJD) accounts for ~85% of all cases of human PrP prion disease, whereas inherited prion diseases account for 10–15% of all cases (Table 453e-2). Familial CJD (fCJD), GerstmannSträussler-Scheinker (GSS) disease, and fatal familial insomnia (FFI) are all dominantly inherited prion diseases that are caused by mutations in the PrP gene. Although infectious PrP prion diseases account for <1% of all cases and infection does not seem to play an important role in the natural history of these illnesses, the transmissibility of

1	Although infectious PrP prion diseases account for <1% of all cases and infection does not seem to play an important role in the natural history of these illnesses, the transmissibility of Abbreviations: BSE, bovine spongiform encephalopathy; CJD, Creutzfeldt-Jakob disease; CWD, chronic wasting disease; fCJD, familial Creutzfeldt-Jakob disease; FFI, fatal familial insomnia; FSE, feline spongiform encephalopathy; GSS, Gerstmann-Sträussler-Scheinker disease; hGH, human growth hormone; iCJD, iatrogenic Creutzfeldt-Jakob disease; MBM, meat and bone meal; sCJD, sporadic Creutzfeldt-Jakob disease; sFI, sporadic fatal insomnia; TME, transmissible mink encephalopathy; vCJD, variant Creutzfeldt-Jakob disease.

1	prions is an important biologic feature. Kuru of the Fore people of New Guinea is thought to have resulted from the consumption of brains from dead relatives during ritualistic cannibalism. With the cessation of ritualistic cannibalism in the late 1950s, kuru has nearly disappeared, with the exception of a few recent patients exhibiting incubation periods of >40 years. Iatrogenic CJD (iCJD) seems to be the result of the accidental inoculation of patients with prions. Variant CJD (vCJD) in teenagers and young adults in Europe is the result of exposure to tainted beef from cattle with bovine spongiform encephalopathy (BSE). Although occasional cases of iatrogenic CJD still occur, this form of CJD is currently on the decline due to public health measures aimed at preventing the spread of PrP prions.

1	Six diseases of animals are caused by prions (Table 453e-2). Scrapie of sheep and goats is the prototypic prion disease. Mink encephalopathy, BSE, feline spongiform encephalopathy, and exotic ungulate encephalopathy are all thought to occur after the consumption of prion-infected foodstuffs. The BSE epidemic emerged in Britain in the late 1980s and was shown to be due to industrial cannibalism. Whether BSE began as a sporadic case of BSE in a cow or started with scrapie in sheep is unknown. The origin of chronic wasting disease (CWD), a prion disease endemic in deer and elk in regions of North America, is uncertain. In contrast to other prion diseases, CWD is highly communicable. Feces from asymptomatic, infected cervids contain prions that are likely to be responsible for the spread of CWD.

1	CJD is found throughout the world. The incidence of sCJD is approximately one case per million population, and thus it accounts for approximately 1 in every 10,000 deaths. Because sCJD is an age-dependent neurodegenerative disease, its incidence is expected to increase steadily as older segments of populations in developed and developing countries continue to expand. Although many geographic clusters of CJD have been reported, each has been shown to segregate with a PrP gene mutation. Attempts to identify common exposure to some etiologic agent have been unsuccessful for both the sporadic and familial cases. Ingestion of scrapie-infected sheep or goat meat as a cause of CJD in humans has not been demonstrated by epidemiologic studies, although speculation about this potential route of inoculation continues. Of particular interest are deer hunters who develop CJD, because up to 90% of culled deer in some game herds have been shown to harbor CWD prions. Whether prion disease in deer or

1	continues. Of particular interest are deer hunters who develop CJD, because up to 90% of culled deer in some game herds have been shown to harbor CWD prions. Whether prion disease in deer or elk has passed to cows, sheep, or directly to humans remains unknown. Studies with rodents demonstrate that oral infection with prions can occur, but the process is inefficient compared to intracerebral inoculation.

1	The human prion diseases were initially classified as neurodegenerative disorders of unknown etiology on the basis of pathologic changes being confined to the CNS. With the transmission of kuru and CJD to apes, investigators began to view these diseases as infectious CNS illnesses caused by slow viruses. Even though the famil- FIgURE 453e-2 Prion protein isoforms. Bar diagram of Syrian hamster PrP, which consists of 254 amino acids. After processing of the NH2 and COOH termini, both PrPC and PrPSc consist of 209 residues. After limited proteolysis, the NH2 terminus of PrPSc is truncated to form PrP 27–30 composed of ~142 amino acids. GPI, glycosylphosphatidyl inositol anchor attachment site; S—S, disulfide bond; CHO, N-linked sugars.

1	completely hydrolyzed under the same conditions (Fig. 453e-2). In the presence of detergent, PrP 27-30 polymerizes into amyloid. Prion rods formed by limited proteolysis and detergent extraction are indistinguishable from the filaments that aggregate to form PrP amyloid plaques in the CNS. Both the rods and the PrP amyloid filaments found in brain tissue exhibit similar ultrastructural morphology and green-gold birefringence after staining with Congo red dye.

1	Prion Strains Distinct strains of prions exhibit different biologic properties, which are epigenetically heritable. The existence of prion strains raised the question of how heritable biologic information can be enciphered in a molecule other than nucleic acid. Various strains of prions have been defined by incubation times and the distribution of neuronal vacuolation. Subsequently, the patterns of PrPSc deposition were found to correlate with vacuolation profiles, and these patterns were also used to characterize prion strains.

1	Persuasive evidence that strain-specific information is enciphered in the tertiary structure of PrPSc comes from transmission of two different inherited human prion diseases to mice expressing a chimeric human-mouse PrP transgene. In FFI, the protease-resistant fragment of PrPSc after deglycosylation has a molecular mass of 19 kDa, whereas in fCJD and most sporadic prion diseases, it is 21 kDa (Table 453e-3). This difference in molecular mass was shown to be due to different sites of proteolytic cleavage at the NH2 termini of the two human PrPSc molecules, reflecting different tertiary structures. These distinct conformations were not unexpected because the amino acid sequences of the PrPs differ.

1	Extracts from the brains of patients with FFI transmitted disease into mice expressing a chimeric human-mouse PrP transgene and induced formation of the 19-kDa PrPSc, whereas brain extracts from ial nature of a subset of CJD cases was well described, the significance of this observation became more obscure with the transmission of CJD to animals. Eventually the meaning of heritable CJD became Inoculum Species Genotype ± SEM] (n/n0) PrPSc (kDa) clear with the discovery of mutations in the PRNP gene of these patients. The prion concept explains how a disease can manifest as a heritable as well as an infectious illness. Moreover, the hallmark of all prion diseases, whether sporadic, dominantly inherited, or acquired by infection, is that they involve the aberrant metabolism of PrP. A major feature that distinguishes prions from viruses is the finding that both PrP isoforms are Human FFI(D178N, M129) aTg(MHu2M) mice express a chimeric mouse-human PrP gene.

1	Human FFI(D178N, M129) aTg(MHu2M) mice express a chimeric mouse-human PrP gene. encoded by a chromosomal gene. In humans, the PrP gene is designated PRNP and is located on the Notes: Clinicopathologic phenotype is determined by the conformation of PrPSc in accord with the results of short arm of chromosome 20. Limited proteolysis of the transmission of human prions from patients with FFI to transgenic mice. PrPSc produces a smaller, protease-resistant molecule Abbreviations: fCJD, familial Creutzfeldt-Jakob disease; FFI, fatal familial insomnia; SEM, standard error of the of ~142 amino acids designated PrP 27-30; PrPC is mean. fCJD and sCJD patients produced the 21-kDa PrPSc in mice expressing the same transgene. On second passage, these differences were maintained, demonstrating that chimeric PrPSc can exist in two different conformations based on the sizes of the protease-resistant fragments, even though the amino acid sequence of PrPSc is invariant.

1	This analysis was extended when patients with sporadic fatal insomnia (sFI) were identified. Although they did not carry a PRNP gene mutation, the patients demonstrated a clinical and pathologic phenotype that was indistinguishable from that of patients with FFI. Furthermore, 19-kDa PrPSc was found in their brains, and on passage of prion disease to mice expressing a chimeric human-mouse PrP transgene, 19-kDa PrPSc was also found. These findings indicate that the disease phenotype is dictated by the conformation of PrPSc and not the amino acid sequence. PrPSc acts as a template for the conversion of PrPC into nascent PrPSc. On the passage of prions into mice expressing a chimeric hamster-mouse PrP transgene, a change in the conformation of PrPSc was accompanied by the emergence of a new strain of prions.

1	Many new strains of prions were generated using recombinant (rec) PrP produced in bacteria; recPrP was polymerized into amyloid fibrils and inoculated into transgenic mice expressing high levels of wild-type mouse PrPC; approximately 500 days later, the mice died of prion disease. The incubation times of the “synthetic prions” in mice were dependent on the conditions used for polymerization of the amyloid fibrils. Highly stable amyloids gave rise to stable prions with long incubation times; low-stability amyloids led to prions with short incubation times. Amyloids of intermediate stability gave rise to prions with intermediate stabilities and intermediate incubation times. Such findings are consistent with earlier studies showing that the incubation times of synthetic and naturally occurring prions are directly proportional to the stability of the prion.

1	Species Barrier Studies on the role of the primary and tertiary structures of PrP in the transmission of prion disease have given new insights into the pathogenesis of these maladies. The amino acid sequence of PrP encodes the species of the prion, and the prion derives its PrPSc sequence from the last mammal in which it was passaged. While the primary structure of PrP is likely to be the most important or even sole determinant of the tertiary structure of PrPC, PrPSc seems to function as a template in determining the tertiary structure of nascent PrPSc molecules as they are formed from PrPC. In turn, prion diversity appears to be enciphered in the conformation of PrPSc, and thus prion strains seem to represent different conformers of PrPSc.

1	In general, transmission of PrP prion disease from one species to another is inefficient, in that not all intracerebrally inoculated animals develop disease, and those that fall ill do so only after long incubation times that can approach the natural life span of the animal. This “species barrier” to transmission is correlated with the degree of similarity between the amino acid sequences of PrPC in the inoculated host and of PrPSc in the prion inoculum. The importance of sequence similarity between the host and donor PrP argues that PrPC directly interacts with PrPSc in the prion conversion process.

1	Several different scenarios might explain the initiation of sporadic prion disease: (1) A somatic mutation may be the cause and thus follow a path similar to that for germline mutations in inherited disease. In this situation, the mutant PrPSc must be capable of targeting wild-type PrPC, a process known to be possible for some mutations but less likely for others. (2) The activation energy barrier separating wild-type PrPC from PrPSc could be crossed on rare occasions when viewed in the context of a population. Most individuals would be spared, while presentations in the elderly with an incidence of ~1 per million would be seen. (3) PrPSc may be present at low levels in some normal cells, where it performs some important, as yet unknown, function. The level of PrPSc in such cells is hypothesized to be sufficiently low as to be not detected by routine bioassay. In some altered metabolic states, the cellular mechanisms for clearing PrPSc might become compromised, and the rate of PrPSc

1	to be sufficiently low as to be not detected by routine bioassay. In some altered metabolic states, the cellular mechanisms for clearing PrPSc might become compromised, and the rate of PrPSc formation would then begin to exceed the capacity of the cell to clear it. The third possible mechanism is attractive because 453e-3 it suggests PrPSc is not simply a misfolded protein, as proposed for the first and second mechanisms, but that it is an alternatively folded molecule with a function. Moreover, the multitude of conformational states that PrPSc can adopt, as described above, raises the possibility that PrPSc or another prion-like protein might function in a process like short-term memory where information storage occurs in the absence of new protein synthesis.

1	More than 40 different mutations resulting in nonconservative substitutions in the human PRNP gene have been found to segregate with inherited human prion diseases. Missense mutations and expansions in the octapeptide repeat region of the gene are responsible for familial forms of prion disease. Five different mutations of the PRNP gene have been linked genetically to heritable prion disease.

1	Although phenotypes may vary dramatically within families, specific phenotypes tend to be observed with certain mutations. A clinical phenotype indistinguishable from typical sCJD is usually seen with substitutions at codons 180, 183, 200, 208, 210, and 232. Substitutions at codons 102, 105, 117, 198, and 217 are associated with the GSS variant of prion disease. The normal human PrP sequence contains five repeats of an eight-amino-acid sequence. Insertions from two to nine extra octarepeats frequently cause variable phenotypes ranging from a condition indistinguishable from sCJD to a slowly progressive dementing illness of many years in duration to an early-age-of-onset disorder that is similar to AD. A mutation at codon 178 resulting in substitution of asparagine for aspartic acid produces FFI if a methionine is encoded at the polymorphic residue 129 on the same allele. Typical CJD is seen if the D178N mutation occurs with a valine encoded at position 129 of the same allele.

1	Polymorphisms influence the susceptibility to sporadic, inherited, and infectious forms of prion disease. The methionine/valine polymorphism at position 129 not only modulates the age of onset of some inherited prion diseases but can also determine the clinical phenotype. The finding that homozygosity at codon 129 predisposes to sCJD supports a model of prion production that favors PrP interactions between homologous proteins. Substitution of the basic residue lysine at position 218 in mouse PrP produced dominant-negative inhibition of prion replication in transgenic mice. This same lysine at position 219 in human PrP has been found in 12% of the Japanese population, and this group appears to be resistant to prion disease. Dominant-negative inhibition of prion replication was also found with substitution of the basic residue arginine at position 171; sheep with arginine were resistant to scrapie prions but were susceptible to BSE prions that were inoculated intracerebrally.

1	Accidental transmission of CJD to humans appears to have occurred with corneal transplantation, contaminated electroencephalogram (EEG) electrode implantation, and surgical procedures. Corneas from donors with unsuspected CJD have been transplanted to apparently healthy recipients who developed CJD after variable incubation periods. The same improperly decontaminated EEG electrodes that caused CJD in two young patients with intractable epilepsy caused CJD in a chimpanzee 18 months after their experimental implantation. Surgical procedures may have resulted in accidental inoculation of patients with prions, presumably because some instrument or apparatus in the operating theater became contaminated when a CJD patient underwent surgery. Although the epidemiology of these studies is highly suggestive, no proof for such episodes exists.

1	Dura Mater grafts More than 160 cases of CJD after implantation of dura mater grafts have been recorded. All of the grafts appear to have been acquired from a single manufacturer whose preparative procedures were inadequate to inactivate human prions. One case of CJD occurred after repair of an eardrum perforation with a pericardium graft.

1	Human growth Hormone and Pituitary gonadotropin Therapy The transmission of CJD prions from contaminated human growth hormone (hGH) preparations derived from human pituitaries has been responsible for fatal cerebellar disorders with dementia in >180 patients ranging in age from 10 to 41 years. These patients received injections of hGH every 2–4 days for 4–12 years. If it is thought that these patients developed CJD from injections of prion-contaminated hGH preparations, the possible incubation periods range from 4 to 30 years. Only recombinant hGH is now used therapeutically so that possible contamination with prions is no longer an issue. Four cases of CJD have occurred in women receiving human pituitary gonadotropin.

1	The restricted geographic occurrence and chronology of vCJD raised the possibility that BSE prions had been transmitted to humans through the consumption of tainted beef. More than 190 cases of vCJD have occurred, with >90% of these in Britain. vCJD has also been reported in people either living in or originating from France, Ireland, Italy, Netherlands, Portugal, Spain, Saudi Arabia, United States, Canada, and Japan. The steady decline in the number of vCJD cases over the past decade argues that there will not be a prion disease epidemic in Europe, similar to those seen for BSE and kuru. What is certain is that prion-tainted meat should be prevented from entering the human food supply.

1	The most compelling evidence that vCJD is caused by BSE prions was obtained from experiments in mice expressing the bovine PrP transgene. Both BSE and vCJD prions were efficiently transmitted to these transgenic mice and with similar incubation periods. In contrast to sCJD prions, vCJD prions did not transmit disease efficiently to mice expressing a chimeric human-mouse PrP transgene. Earlier studies with nontransgenic mice suggested that vCJD and BSE might be derived from the same source because both inocula transmitted disease with similar but very long incubation periods.

1	Attempts to determine the origin of BSE and vCJD prions have relied on passaging studies in mice, some of which are described above, as well as studies of the conformation and glycosylation of PrPSc. One scenario suggests that a particular conformation of bovine PrPSc was selected for heat resistance during the rendering process and was then reselected multiple times as cattle infected by ingesting prioncontaminated meat and bone meal (MBM) were slaughtered and their offal rendered into more MBM. Variant CJD cases have virtually disappeared with protection of the beef supply in Europe. Frequently the brains of patients with CJD have no recognizable abnormalities on gross examination. Patients who survive for several years have variable degrees of cerebral atrophy.

1	Frequently the brains of patients with CJD have no recognizable abnormalities on gross examination. Patients who survive for several years have variable degrees of cerebral atrophy. On light microscopy, the pathologic hallmarks of CJD are spongiform degeneration and astrocytic gliosis. The lack of an inflammatory response in CJD and other prion diseases is an important pathologic feature of these degenerative disorders. Spongiform degeneration is characterized by many 1to 5-μm vacuoles in the neuropil between nerve cell bodies. Generally the spongiform changes occur in the cerebral cortex, putamen, caudate nucleus, thalamus, and molecular layer of the cerebellum. Astrocytic gliosis is a constant but nonspecific feature of prion diseases. Widespread proliferation of fibrous astrocytes is found throughout the gray matter of brains infected with CJD prions. Astrocytic processes filled with glial filaments form extensive networks.

1	Amyloid plaques have been found in ~10% of CJD cases. Purified CJD prions from humans and animals exhibit the ultrastructural and histochemical characteristics of amyloid when treated with detergents during limited proteolysis. In first passage from some human Japanese CJD cases, amyloid plaques have been found in mouse brains. These plaques stain with antibodies raised against PrP. The amyloid plaques of GSS disease are morphologically distinct from those seen in kuru or scrapie. GSS plaques consist of a central dense core of amyloid surrounded by smaller globules of amyloid. Ultrastructurally, they consist of a radiating fibrillar network of amyloid fibrils, with scant or no neuritic degeneration. The plaques can be distributed throughout the brain but are most frequently found in the cerebellum. They are often located adjacent to blood vessels. Congophilic angiopathy has been noted in some cases of GSS disease.

1	In vCJD, a characteristic feature is the presence of “florid plaques.” These are composed of a central core of PrP amyloid, surrounded by vacuoles in a pattern suggesting petals on a flower.

1	Nonspecific prodromal symptoms occur in approximately a third of patients with CJD and may include fatigue, sleep disturbance, weight loss, headache, anxiety, vertigo, malaise, and ill-defined pain. Most patients with CJD present with deficits in higher cortical function. Similarly, psychiatric symptoms, such as depression, psychosis, and visual hallucinations, are often the defining features of the illness. These deficits almost always progress over weeks or months to a state of profound dementia characterized by memory loss, impaired judgment, and a decline in virtually all aspects of intellectual function. A few patients present with either visual impairment or cerebellar gait and coordination deficits. Frequently the cerebellar deficits are rapidly followed by progressive dementia. Visual problems often begin with blurred vision and diminished acuity, rapidly followed by dementia.

1	Other symptoms and signs include extrapyramidal dysfunction manifested as rigidity, masklike facies, or (less commonly) choreoathetoid movements; pyramidal signs (usually mild); seizures (usually major motor) and, less commonly, hypoesthesia; supranuclear gaze palsy; optic atrophy; and vegetative signs such as changes in weight, temperature, sweating, or menstruation.

1	Myoclonus Most patients (~90%) with CJD exhibit myoclonus that appears at various times throughout the illness. Unlike other involuntary movements, myoclonus persists during sleep. Startle myoclonus elicited by loud sounds or bright lights is frequent. It is important to stress that myoclonus is neither specific nor confined to CJD and tends to occur later in the course of CJD. Dementia with myoclonus can also be due to AD (Chap. 448), dementia with Lewy bodies (Chap. 448), corticobasal degeneration (Chap. 448) cryptococcal encephalitis (Chap. 239), or the myoclonic epilepsy disorder Unverricht-Lundborg disease (Chap. 445). Clinical Course In documented cases of accidental transmission of CJD to humans, an incubation period of 1.5–2 years preceded the development of clinical disease. In other cases, incubation periods of up to 40 years have been suggested. Most patients with CJD live 6–12 months after the onset of clinical signs and symptoms, whereas some live for up to 5 years.

1	The constellation of dementia, myoclonus, and periodic electrical bursts in an afebrile 60-year-old patient generally indicates CJD. Clinical abnormalities in CJD are confined to the CNS. Fever, elevated sedimentation rate, leukocytosis in blood, or a pleocytosis in cerebrospinal fluid (CSF) should alert the physician to another etiology to explain the patient’s CNS dysfunction, although there are rare cases of CJD in which mild CSF pleocytosis is observed.

1	Variations in the typical course appear in inherited and transmitted forms of the disease. fCJD has an earlier mean age of onset than sCJD. In GSS disease, ataxia is usually a prominent and presenting feature, with dementia occurring late in the disease course. GSS disease presents earlier than CJD (mean age 43 years) and is typically more slowly progressive than CJD; death usually occurs within 5 years of onset. FFI is characterized by insomnia and dysautonomia; dementia occurs only in the terminal phase of the illness. Rare sporadic cases have been identified. vCJD has an unusual clinical course, with a prominent psychiatric prodrome that may include visual hallucinations and early ataxia, whereas frank dementia is usually a late sign of vCJD.

1	Many conditions mimic CJD. Dementia with Lewy bodies (Chap. 448) is the most common disorder to be mistaken for CJD. It can present in a subacute fashion with delirium, myoclonus, and extrapyramidal features. Other neurodegenerative disorders (Chap. 448) to consider include AD, FTD, corticobasal degeneration, progressive supranuclear palsy, ceroid lipofuscinosis, and myoclonic epilepsy with Lafora bodies (Chap. 445). The absence of abnormalities on diffusion-weighted and fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging (MRI) will almost always distinguish these dementing conditions from CJD.

1	Hashimoto’s encephalopathy, which presents as a subacute progressive encephalopathy with myoclonus and periodic triphasic complexes on the EEG, should be excluded in every case of suspected CJD. It is diagnosed by the finding of high titers of antithyroglobulin or antithyroid peroxidase (antimicrosomal) antibodies in the blood and improves with glucocorticoid therapy. Unlike CJD, fluctuations in severity typically occur in Hashimoto’s encephalopathy. Intracranial vasculitides (Chap. 385) may produce nearly all of the symptoms and signs associated with CJD, sometimes without systemic abnormalities. Myoclonus is exceptional with cerebral vasculitis, but focal seizures may confuse the picture. Prominent headache, absence of myoclonus, stepwise change in deficits, abnormal CSF, and focal white matter changes on MRI or angiographic abnormalities all favor vasculitis.

1	Paraneoplastic conditions (Chap. 122), particularly limbic encephalitis and cortical encephalitis, can also mimic CJD. In many of these patients, dementia appears prior to the diagnosis of a tumor, and in some, no tumor is ever found. Detection of the paraneoplastic antibodies is often the only way to distinguish these cases from CJD. Other diseases that can simulate CJD include neurosyphilis (Chap. 206), AIDS dementia complex (Chap. 226), progressive multifocal leukoencephalopathy (Chap. 164), subacute sclerosing panencephalitis, progressive rubella panencephalitis, herpes simplex encephalitis (Chap. 164), diffuse intracranial tumor (gliomatosis cerebri; Chap. 118), anoxic encephalopathy, dialysis dementia, uremia, hepatic encephalopathy, voltage-gated potassium channel (VGkC) autoimmune encephalopathy, and lithium or bismuth intoxication.

1	The only specific diagnostic tests for CJD and other human prion diseases measure PrPSc. The most widely used method involves limited proteolysis that generates PrP 27-30, which is detected by immunoassay after denaturation. The conformation-dependent immunoassay (CDI) is based on immunoreactive epitopes that are exposed in PrPC but buried in PrPSc. In humans, the diagnosis of CJD can be established by brain biopsy if PrPSc is detected. If no attempt is made to measure PrPSc, but the constellation of pathologic changes frequently found in CJD is seen in a brain biopsy, then the diagnosis is reasonably secure (see “Neuropathology,” above). The high sensitivity and specificity of cortical ribboning and basal ganglia hyperintensity on FLAIR and diffusion-weighted MRI for the diagnosis of CJD have greatly diminished the need for brain biopsy in patients with suspected CJD. Because PrPSc is not uniformly distributed throughout the CNS, the apparent absence of PrPSc in a limited sample such

1	have greatly diminished the need for brain biopsy in patients with suspected CJD. Because PrPSc is not uniformly distributed throughout the CNS, the apparent absence of PrPSc in a limited sample such as a biopsy does not rule out prion disease. At autopsy, sufficient brain samples should be taken for both PrPSc immunoassay, preferably by CDI, and immunohistochemistry of tissue sections.

1	To establish the diagnosis of either sCJD or familial prion disease, sequencing the PRNP gene must be performed. Finding the wild-type PRNP gene sequence permits the diagnosis of sCJD if there is no history to suggest infection from an exogenous source of prions. The identification of a mutation in the PRNP gene sequence that encodes a nonconservative amino acid substitution argues for familial prion disease. CT may be normal or show cortical atrophy. MRI is valuable for distinguishing sCJD from most other conditions. On FLAIR sequences and diffusion-weighted imaging, ~90% of patients show increased intensity in the basal ganglia and cortical ribboning (Fig. 453e-3). This pattern is not seen with other neurodegenerative disorders but has been seen infrequently with viral encephalitis, paraneoplastic syndromes, or seizures. When the typical MRI pattern is present, in the

1	FIgURE 453e-3 T2-weighted (fluid-attenuated inversion recovery) magnetic resonance imaging showing hyperintensity in the cortex in a patient with sporadic CJD. This so-called “cortical ribboning” along with increased intensity in the basal ganglia on T2or diffusion-weighted imaging can aid in the diagnosis of Creutzfeldt-Jakob disease. proper clinical setting, diagnosis is facilitated. However, some cases of sCJD do not show this typical pattern, and other early diagnostic approaches are still needed. CSF is nearly always normal but may show protein elevation and, rarely, mild pleocytosis. Although the stress protein 14-3-3 is elevated in the CSF of some patients with CJD, similar elevations of 14-3-3 are found in patients with other disorders; thus this elevation is not specific. Similarly, elevations of CSF neuron-specific enolase and tau occur in CJD but lack specificity for diagnosis.

1	The EEG is often useful in the diagnosis of CJD, although only approximately 60% of individuals show the typical pattern. During the early phase of CJD, the EEG is usually normal or shows only scattered theta activity. In most advanced cases, repetitive, high-voltage, triphasic, and polyphasic sharp discharges are seen, but in many cases their presence is transient. The presence of these stereotyped periodic bursts of <200 ms in duration, occurring every 1–2 s, makes the diagnosis of CJD very likely. These discharges are frequently but not always symmetric; there may be a one-sided predominance in amplitude. As CJD progresses, normal background rhythms become fragmentary and slower.

1	Although CJD should not be considered either contagious or communicable, it is transmissible. The risk of accidental inoculation by aerosols is very small; nonetheless, procedures producing aerosols should be performed in certified biosafety cabinets. Biosafety level 2 practices, containment equipment, and facilities are recommended by the Centers for Disease Control and Prevention and the National Institutes of Health. The primary problem in caring for patients with CJD is the inadvertent infection of health care workers by needle and stab wounds. Electroencephalographic and electromyographic needles should not be reused after studies on patients with CJD have been performed.

1	There is no reason for pathologists or other morgue employees to resist performing autopsies on patients whose clinical diagnosis was CJD. Standard microbiologic practices outlined here, along with specific recommendations for decontamination, seem to be adequate precautions for the care of patients with CJD and the handling of infected specimens.

1	Prions are extremely resistant to common inactivation procedures, and there is some disagreement about the optimal conditions for sterilization. Some investigators recommend treating CJD-contaminated materials once with 1 N NaOH at room temperature, but we believe this procedure may be inadequate for sterilization. Autoclaving at 134°C for 5 h or treatment with 2 N NaOH for several hours is recommended for sterilization of prions. The term sterilization implies complete destruction of prions; any residual infectivity can be hazardous. Recent studies show that sCJD prions bound to stainless steel surfaces are resistant to inactivation by autoclaving at 134°C for 2 h; exposure of bound prions to an acidic detergent solution prior to autoclaving rendered prions susceptible to inactivation.

1	There is no known effective therapy for preventing or treating CJD. The finding that phenothiazines and acridines inhibit PrPSc formation in cultured cells led to clinical studies of quinacrine in CJD patients. Unfortunately, quinacrine failed to slow the rate of cognitive decline in CJD, possibly because therapeutic concentrations in the brain were not achieved. Although inhibition of the P-glycoprotein (Pgp) transport system resulted in substantially increased quinacrine levels in the brains of mice, the prion incubation times were not extended by treatment with the drug. Whether such an approach can be used to treat CJD remains to be established.

1	Like the acridines, anti-PrP antibodies have been shown to eliminate PrPSc from cultured cells. Additionally, such antibodies in mice, either administered by injection or produced from a transgene, have been shown to prevent prion disease when prions are introduced by a peripheral route, such as intraperitoneal inoculation. Unfortunately, the antibodies were ineffective in mice inoculated intracerebrally with prions. Several drugs, including pentosan polysulfate as well as porphyrin and phenylhydrazine derivatives, delay the onset of disease in animals inoculated intracerebrally with prions if the drugs are given intracerebrally beginning soon after inoculation. There is a rapidly expanding body of literature demonstrating that in addition to PrP, other proteins including amyloid beta (Aβ), tau, α-synuclein, and huntingtin can all becomes prions (Chap. 444e).

1	Experimental studies have shown that transgenic mice expressing mutant amyloid precursor protein (APP) develop amyloid plaques containing fibrils composed of the Aβ peptide approximately a year after inoculation with synthetic Aβ peptides polymerized into amyloid fibrils or extracts prepared from the brains of patients with AD. Mutant tau aggregates in transgenic mice and cultured cells can trigger the aggregation of tau into fibrils that resemble those found in neurofibrillary tangles and Pick bodies. Such tangles have been found in AD, FTDs, Pick’s disease, and some cases of posttraumatic brain injury, all of which are likely to be caused by the prion isoforms of Aβ and/or tau.

1	In patients with advanced PD who received grafts of fetal substantia nigral neurons, Lewy bodies containing β-sheet–rich α-synuclein were identified in grafted cells approximately 10 years after transplantation, arguing for the axonal transport of misfolded α-synuclein crossing into grafted neurons, where it initiated aggregation of nascent α-synuclein into fibrils that coalesced into Lewy bodies. These findings combined with studies of multiple system atrophy (MSA) argue that the synucleinopathies are caused by prions. Brain homogenates from MSA patients injected into transgenic mice transmitted lethal neurodegeneration in approximately 3 months; moreover, recombinant synuclein injected into wild-type mice initiated the deposition of synuclein fibrils.

1	In summary, a wealth of evidence continues to accumulate arguing that proteins causing AD, PD, FTDs, ALS, and even HD acquire alternative conformations that become self-propagating. Each of these neurodegenerative diseases is caused by a different protein that undergoes a conformational transformation to become a prion. Prions explain many of the features that the neurodegenerative diseases have in common: (1) incidence increases with age, (2) steady progression over years, (3) spread from one region of the CNS to another, (4) protein deposits consisting of amyloid fibrils, and (5) late onset of inherited forms of the neurodegenerative diseases. Notably, amyloid plaques containing PrPSc are a nonobligatory feature of PrP prion disease in humans and animals. Furthermore, amyloid plaques in AD do not correlate with the level of dementia; however, the level of soluble (oligomeric) Aβ peptide does correlate with memory loss and other intellectual deficits.

1	Disorders of the autonomic Nervous system Phillip A. Low, John W. Engstrom The autonomic nervous system (ANS) innervates the entire neuraxis and influences all organ systems. It regulates blood pressure (BP), 454 heart rate, sleep, and bladder and bowel function. It operates automatically; its full importance becomes recognized only when ANS function is compromised, resulting in dysautonomia. Hypothalamic disorders that cause disturbances in homeostasis are discussed in Chaps. 23 and 401e. The activity of the ANS is regulated by central neurons responsive to diverse afferent inputs. After central integration of afferent information, autonomic outflow is adjusted to permit the functioning of the major organ systems in accordance with the needs of the whole organism. Connections between the cerebral cortex and the autonomic centers in the brainstem coordinate autonomic outflow with higher mental functions.

1	The preganglionic neurons of the parasympathetic nervous system leave the central nervous system (CNS) in the third, seventh, ninth, and tenth cranial nerves as well as the second and third sacral nerves, while the preganglionic neurons of the sympathetic nervous system exit the spinal cord between the first thoracic and the second lumbar segments (Fig. 454-1). These are thinly myelinated. The postganglionic neurons, located in ganglia outside the CNS, give rise to the postganglionic unmyelinated autonomic nerves that innervate organs and tissues throughout the body. Responses to sympathetic and parasympathetic stimulation are frequently antagonistic (Table 454-1), reflecting highly coordinated interactions within the CNS; the resultant changes in parasympathetic and sympathetic activity provide more precise control of autonomic responses than could be achieved by the modulation of a single system.

1	Acetylcholine (ACh) is the preganglionic neurotransmitter for both divisions of the ANS as well as the postganglionic neurotransmitter from cranial nerves III, VII, IX, X from T1-L2 and from sacral nerves 2 and 3 Preganglionic fibers Postganglionic fibers in the heart wall FIGURE 454-1 Schematic representation of the autonomic nervous system. (From M Moskowitz: Clin Endocrinol Metab 6:77, 1977.) of the parasympathetic neurons; the preganglionic receptors are nicotinic, and the postganglionic are muscarinic in type. Norepinephrine (NE) is the neurotransmitter of the postganglionic sympathetic neurons, except for cholinergic neurons innervating the eccrine sweat glands.

1	Disorders of the ANS may result from pathology of either the CNS or the peripheral nervous system (PNS) (Table 454-2). Signs and symptoms may result from interruption of the afferent limb, CNS tone tone) Bowel motility Decreased motility Increased Lung Bronchodilation Bronchoconstriction Sweat glands Sweating — Pupils Dilation Constriction Adrenal glands Catecholamine release — Sexual function Ejaculation, orgasm Erection Lacrimal glands — Tearing Parotid glands — Salivation processing centers, or efferent limb of reflex arcs controlling autonomic responses. For example, a lesion of the medulla produced by a posterior fossa tumor can impair BP responses to postural changes and result in orthostatic hypotension (OH). OH can also be caused by lesions of the spinal cord or peripheral vasomotor nerve fibers (e.g., diabetic autonomic neuropathy). Lesions of the efferent limb cause the most consistent and severe OH. The site of reflex interruption is usually established by the clinical

1	nerve fibers (e.g., diabetic autonomic neuropathy). Lesions of the efferent limb cause the most consistent and severe OH. The site of reflex interruption is usually established by the clinical context in which the dysautonomia arises, combined with judicious use of ANS testing and neuroimaging studies. The presence or absence of CNS signs, association with sensory or motor polyneuropathy, medical illnesses, medication use, and family history are often important considerations. Some syndromes do not fit easily into any classification scheme.

1	Clinical manifestations can result from loss of function, overactivity, or dysregulation of autonomic circuits. Disorders of autonomic function should be considered in patients with unexplained OH, syncope, sleep dysfunction, altered sweating (hyperhidrosis or hypohidrosis), impotence, constipation or other gastrointestinal symptoms (bloating, I. Autonomic disorders with brain involvement A. Associated with multisystem degeneration 1. Multisystem degeneration: autonomic failure clinically prominent a. b. Parkinson’s disease with autonomic failure c. 2. Multisystem degeneration: autonomic failure clinically not usually prominent a. b. Other extrapyramidal disorders (inherited spinocerebellar atrophies, progressive supranuclear palsy, corticobasal degeneration, Machado-Joseph disease, fragile X syndrome [FXTAS]) B. Unassociated with multisystem degeneration (focal CNS disorders) 1. Disorders mainly due to cerebral cortex involvement a. b. c.

1	B. Unassociated with multisystem degeneration (focal CNS disorders) 1. Disorders mainly due to cerebral cortex involvement a. b. c. Cerebral infarction of the insula 2. Disorders of the limbic and paralimbic circuits a. Shapiro’s syndrome (agenesis of corpus callosum, hyperhidrosis, hypothermia) b. c. 3. Disorders of the hypothalamus a. b. c. d. e. f. Antidiuretic hormone (ADH) syndromes (diabetes insipidus, inappropriate ADH secretion) g. Disturbances of temperature regulation (hyperthermia, hypothermia) h. Disturbances of sexual function i. Disturbances of appetite j. Disturbances of BP/HR and gastric function k. 4. Disorders of the brainstem and cerebellum a. b. c. Disorders of BP control (hypertension, hypotension) d. e. f. g. h. i. II. Autonomic disorders with spinal cord involvement A. B. C. D. E. Amyotrophic lateral sclerosisF. Tetanus G. H. III. A. 1. Subacute autoimmune autonomic ganglionopathy (AAG) a. b. c. d. e.

1	A. B. C. D. E. Amyotrophic lateral sclerosisF. Tetanus G. H. III. A. 1. Subacute autoimmune autonomic ganglionopathy (AAG) a. b. c. d. e. Drug induced autonomic neuropathies-stimulants, drug withdrawal, vasoconstrictor, vasodilators, beta-receptor antagonists, betaagonists f. g. B. 1. 2. a. b. c. d. Sensory neuronopathy with autonomic failure e. f. Diabetic, uremic, or nutritional deficiency g. Dysautonomia of old age 3. Disorders of reduced orthostatic intolerance: reflex syncope, POTS, associated with prolonged bed rest, associated with space flight, chronic fatigue Abbreviations: BP, blood pressure; CNS, central nervous system; HR, heart rate; POTS, postural orthostatic tachycardia syndrome. Source: PA Low et al: Mayo Clin Proc 70:617, 1995.

1	nausea, vomiting of old food, diarrhea), or bladder disorders (urinary frequency, hesitancy, or incontinence). Symptoms may be widespread or regional in distribution. An autonomic history focuses on systemic functions (BP, heart rate, sleep, fever, sweating) and involvement of individual organ systems (pupils, bowel, bladder, sexual function). The autonomic symptom profile is a self-report questionnaire that can be used for formal assessment. It is also important to recognize the modulating effects of age. For example, OH typically produces lightheadedness in the young, whereas cognitive slowing is more common in the elderly. Specific symptoms of orthostatic intolerance are diverse (Table 454-3). Autonomic symptoms may vary dramatically, reflecting the dynamic nature of autonomic control over homeostatic function. For example, OH might be manifest only in the early morning, following a meal, with exercise, or with raised ambient temperature, depending on the regional vascular bed

1	over homeostatic function. For example, OH might be manifest only in the early morning, following a meal, with exercise, or with raised ambient temperature, depending on the regional vascular bed affected by the dysautonomia.

1	Early symptoms may be overlooked. Impotence, although not specific for autonomic failure, often heralds autonomic failure in men and may precede other symptoms by years (Chap. 67). A decrease in the frequency of spontaneous early morning erections may occur months before loss of nocturnal penile tumescence and development of total impotence. Bladder dysfunction may appear early in men and women, particularly in those with a CNS etiology. Cold feet may indicate increased peripheral vasomotor constriction. Brain and spinal cord disease above the level of the lumbar spine results first in urinary frequency and small bladder volumes and eventually in incontinence (upper motor neuron or spastic bladder). By contrast, PNS disease of autonomic nerve fibers results in large bladder volumes, urinary frequency, and overflow incontinence (lower motor neuron flaccid bladder). Measurement of bladder volume (postvoid residual) is a useful bedside test for distinguishing between upper and lower

1	frequency, and overflow incontinence (lower motor neuron flaccid bladder). Measurement of bladder volume (postvoid residual) is a useful bedside test for distinguishing between upper and lower motor neuron bladder dysfunction in the early stages of dysautonomia. Gastrointestinal autonomic dysfunction typically presents as severe constipation. Diarrhea may develop (typically in diabetes mellitus) due to rapid transit of contents or uncoordinated small-bowel motor activity, or on an osmotic basis from bacterial overgrowth associated with small-bowel stasis. Impaired glandular secretory function may cause difficulty with food intake due to decreased salivation or eye irritation due to decreased lacrimation. Occasionally, temperature elevation and vasodilation can result from anhidrosis because sweating is normally important for heat dissipation (Chap. 23). Lack of sweating after a hot bath, during exercise, or on a hot day can suggest sudomotor failure.

1	OH (also called orthostatic or postural hypotension) is perhaps the most disabling feature of autonomic dysfunction. The prevalence of OH is relatively high, especially when OH associated with aging and diabetes mellitus is included (Table 454-4). OH can cause a variety of symptoms, including dimming or loss of vision, lightheadedness, diaphoresis, diminished hearing, pallor, and weakness. Syncope results when the drop in BP impairs cerebral perfusion. Other manifestations of impaired baroreflexes are supine hypertension, a heart rate that is fixed regardless of posture, postprandial hypotension, and an excessively high nocturnal BP. Many patients with OH have a preceding

1	Aging 14–20% Diabetic neuropathy 10% Other autonomic neuropathies 10–50 per 100,000 Multiple system atrophy 5–15 per 100,000 Pure autonomic failure 10–30 per 100,000 diagnosis of hypertension or have concomitant supine hypertension, reflecting the great importance of baroreflexes in maintaining postural and supine normotension. The appearance of OH in patients receiving antihypertensive treatment may indicate overtreatment or the onset of an autonomic disorder. The most common causes of OH are not neurologic in origin; these must be distinguished from the neurogenic causes (Table 454-5). Neurocardiogenic and cardiac causes of syncope are considered in Chap. 27. APPROACH TO THE PATIENT:

1	APPROACH TO THE PATIENT: The first step in the evaluation of symptomatic OH is the exclusion of treatable causes. The history should include a review of medications that may affect the ANS (Table 454-6). The main classes of drugs that may cause OH are diuretics, antihypertensives, antidepressants, ethanol, narcotics, insulin, dopamine agonists, barbiturates, and calcium channel-blocking agents. However, the precipitation of OH by medications may also be the first sign of an underlying autonomic disorder. The history may reveal an underlying cause for symptoms (e.g., diabetes, Parkinson’s disease) or specific underlying mechanisms (e.g., cardiac pump failure, reduced intravascular volume). The relationship of symptoms to meals (splanchnic pooling), standing on awakening in the morning (intravascular volume depletion), ambient warming (vasodilatation), or exercise (muscle arteriolar vasodilatation) NoNNeuroGeNiC Causes of orthostatiC hypoteNsioN

1	NoNNeuroGeNiC Causes of orthostatiC hypoteNsioN Myocarditis Postprandial dilation of splanchnic vessel beds Constrictive pericarditis Vigorous exercise with dilation of Tachyarrhythmias Heat: hot environment, hot show-Bradyarrhythmias ers and baths, fever Salt-losing nephropathy Reduced intravascular volume Antihypertensives Straining or heavy lifting, urination, Diuretics defecation Vasodilators: nitrates, hydralazine Dehydration Alphaand beta-blocking agents Diarrhea, emesis Central nervous system sedatives: Hemorrhage barbiturates, opiates Burns Tricyclic antidepressants Metabolic Phenothiazines Adrenocortical insufficiency Hypoaldosteronism Pheochromocytoma Severe potassium depletion Disorders of the Autonomic Nervous System Abbreviations: CCBs, calcium channel blockers; HCTZ, hydrochlorothiazide; SSRIs, selective serotonin reuptake inhibitors. should be sought. Standing time to first symptom and to presyncope (Chap. 27) should be followed for management.

1	Physical examination includes measurement of supine and standing pulse and BP. OH is defined as a sustained drop in systolic (≥20 mmHg) or diastolic (≥10 mmHg) BP after 2–3 min of standing. In nonneurogenic causes of OH (such as hypovolemia), the BP drop is accompanied by a compensatory increase in heart rate of >15 beats/min. A clue that the patient has neurogenic OH is the aggravation or precipitation of OH by autonomic stressors (a meal, hot bath, or exercise). Neurologic examination should include mental status (neurodegenerative disorders), cranial nerves (impaired downgaze with progressive supranuclear palsy; abnormal pupils with Horner’s or Adie’s syndrome), motor tone (Parkinson’s disease and parkinsonism), and sensation (polyneuropathies). In patients without a clear diagnosis initially, follow-up evaluations every few months or whenever symptoms worsen may reveal the underlying cause.

1	Disorders of autonomic function should be considered in patients with symptoms of altered sweating (hyperhidrosis or hypohidrosis), gastroparesis (bloating, nausea, vomiting of old food), impotence, constipation, or bladder disturbances (urinary frequency, hesitancy, or incontinence). Autonomic function tests are helpful to document abnormalities when findings on history and examination are inconclusive; to detect subclinical involvement; or to follow the course of an autonomic disorder.

1	Heart Rate Variation with Deep Breathing This tests the parasympathetic component of cardiovascular reflexes via the vagus nerve. Results are influenced by multiple factors including the subject’s position (recumbent, sitting, or standing), rate and depth of respiration (6 breaths per minute and a forced vital capacity [FVC] >1.5 L are optimal), age, medications, weight, and degree of hypocapnia. Interpretation of results requires comparison of test data with results from age-matched controls collected under identical test conditions. For example, the lower limit of normal heart rate variation with deep breathing in persons <20 years is >15–20 beats/min, but for persons over age 60 it is 5–8 beats/min. Heart rate variation with deep breathing (respiratory sinus arrhythmia) is abolished by the muscarinic ACh receptor antagonist atropine but is unaffected by sympathetic postganglionic blockade (e.g., propranolol).

1	Valsalva Response This response (Table 454-7) assesses the integrity of the baroreflex control of heart rate (parasympathetic) and BP (adrenergic). Under normal conditions, increases in BP at the carotid bulb trigger a reduction in heart rate (increased vagal tone), and decreases in BP trigger an increase in heart rate (reduced vagal tone). The Valsalva response is tested in the supine position. The subject exhales against a closed glottis (or into a manometer maintaining a constant expiratory pressure of 40 mmHg) for 15 s while measuring changes in heart rate and beat-to-beat BP. There are four phases of the BP and heart rate response to the Valsalva maneuver. Phases I and III are mechanical and related to changes in intrathoracic and intraabdominal pressure. In early phase II, reduced venous return results in a fall in stroke volume and BP, counteracted by a combination of reflex tachycardia and increased total peripheral resistance. Increased total peripheral resistance arrests the

1	return results in a fall in stroke volume and BP, counteracted by a combination of reflex tachycardia and increased total peripheral resistance. Increased total peripheral resistance arrests the BP drop ~5–8 s after the onset of the maneuver. Late phase II begins with a progressive rise in BP toward or above baseline. Venous return and cardiac output return to normal in phase

1	IV. Persistent peripheral arteriolar vasoconstriction and increased cardiac adrenergic tone result in a temporary BP overshoot and phase IV bradycardia (mediated by the baroreceptor reflex). Autonomic function during the Valsalva maneuver can be measured using beat-to-beat BP or heart rate changes. The Valsalva ratio is defined as the maximum phase II tachycardia divided by the minimum phase IV bradycardia (Table 454-8). The ratio reflects the integrity of the entire baroreceptor reflex arc and of sympathetic efferents to blood vessels.

1	Sudomotor Function Sweating is induced by release of ACh from sympathetic postganglionic fibers. The quantitative sudomotor axon reflex test (QSART) is a measure of regional autonomic function mediated by ACh-induced sweating. A reduced or absent response indicates a lesion of the postganglionic sudomotor axon. For example, sweating may be reduced in the feet as a result of distal polyneuropathy (e.g., diabetes). The thermoregulatory sweat test (TST) is a qualitative measure of regional sweat production in response to an elevation of body temperature under controlled conditions. An indicator powder placed on the anterior surface of the body changes color with sweat production during temperature Abbreviations: BPBB, beat-to-beat blood pressure; HRDB, heart rate response to deep breathing; HUT, head-up tilt; QSART, quantitative sudomotor axon reflex test; VM, Valsalva maneuver.

1	Abbreviations: BPBB, beat-to-beat blood pressure; HRDB, heart rate response to deep breathing; HUT, head-up tilt; QSART, quantitative sudomotor axon reflex test; VM, Valsalva maneuver. elevation. The pattern of color change is a measure of regional sweat secretion. A postganglionic lesion is present if both QSART and TST show absent sweating. In a preganglionic lesion, the QSART is normal but TST shows anhidrosis. Orthostatic BP Recordings Beat-to-beat BP measurements determined in supine, 70° tilt, and tilt-back positions are useful to quantitate orthostatic failure of BP control. Allow a 20-min period of rest in the supine position before assessing changes in BP during tilting. The BP change combined with heart rate monitoring is useful for the evaluation of patients with suspected OH or unexplained syncope.

1	Tilt Table Testing for Syncope The great majority of patients with syncope do not have autonomic failure. Tilt table testing can be used to make the diagnosis of vasovagal syncope with sensitivity, specificity, and reproducibility. A standardized protocol is used that specifies the tilt apparatus, angle and duration of tilt, and procedure for provocation of vasodilation (e.g., sublingual or spray nitroglycerin). A positive nitroglycerin-stimulated test predicts recurrence of syncope. Recommendations for the performance of tilt studies for syncope have been incorporated in consensus guidelines. MULTIPLE SYSTEM ATROPHY (CHAP. 449)

1	Multiple system atrophy (MSA) is an entity that comprises autonomic failure (OH or a neurogenic bladder) and either parkinsonism (MSA-p) or a cerebellar syndrome (MSA-c). MSA-p is the more common form; the parkinsonism is atypical in that it is usually unassociated with significant tremor or a response to levodopa. Symptomatic OH within 1 year of onset of parkinsonism predicts eventual development of MSA-p in 75% of patients. There is a very high frequency of impotence in men. Although autonomic abnormalities are common in advanced Parkinson’s disease (Chap. 449), the severity and distribution of autonomic failure are more severe and generalized in MSA. Brain magnetic resonance imaging (MRI) is a useful diagnostic adjunct: in MSA-p, iron deposition in the striatum may be evident as T2 hypointensity, and in MSA-c, cerebellar atrophy is present with a characteristic T2 hyperintense signal (“hot cross buns sign”) in the pons (Fig. 454-2). Cardiac postganglionic adrenergic innervation,

1	and in MSA-c, cerebellar atrophy is present with a characteristic T2 hyperintense signal (“hot cross buns sign”) in the pons (Fig. 454-2). Cardiac postganglionic adrenergic innervation, measured by uptake of fluorodopamine on positron emission tomography, is markedly impaired in the dysautonomia of Parkinson’s disease (PD) but is usually normal in MSA. Neuropathologic changes include neuronal loss and gliosis in many CNS regions, including the brainstem, cerebellum, striatum, and intermediolateral cell column of the thoracolumbar spinal cord.

1	FIGURE 454-2 Multiple system atrophy, cerebellar type (MSA-c). Axial T2-weighted magnetic resonance image at the level of the pons shows a characteristic hyperintense signal, the “hot cross buns” sign. This appearance can also be seen in some spinocerebellar atrophies, as well as other neurodegenerative conditions affecting the brainstem.

1	MSA is uncommon, with a prevalence estimated at 2–5 per 100,000 individuals. Onset is typically in the mid-fifties, men are slightly more often affected than women, and most cases are sporadic. The diagnosis should be considered in adults over the age of 30 years who present with OH or urinary incontinence and either parkinsonism that is poorly responsive to dopamine replacement or a cerebellar syndrome. MSA generally progresses relentlessly to death 7–10 years after onset, but survival beyond 15 years has been reported. Factors that predict a worse prognosis include rapid progression of disability, bladder dysfunction, female gender, the MSA-p subtype, and an older age at onset. Attempts to slow the progression of MSA have thus far been unsuccessful, including trials of lithium, growth hormone, riluzole, rasagiline, minocycline, and a recent trail of rifampicin.

1	Management is symptomatic for neurogenic OH (see below), sleep disorders including laryngeal stridor, and gastrointestinal (GI) and urinary dysfunction. GI management includes frequent small meals, soft diet, stool softeners, and bulk agents. Gastroparesis is difficult to treat; metoclopramide stimulates gastric emptying but worsens parkinsonism by blocking central dopamine receptors. The peripheral dopamine (D2 and D3) receptor antagonist domperidone has been used patients with various GI conditions in many countries and is now available in the United States through the U.S. Food and Drug Administration’s (FDA) Expanded Access to Investigational Drugs program. Autonomic dysfunction is also a common feature in dementia with Lewy bodies (Chap. 448); the severity is usually less than that found in MSA or PD. In multiple sclerosis (MS; Chap. 458), autonomic complications reflect the CNS location of MS involvement and generally worsen with disease duration and disability.

1	Spinal cord lesions from any cause may result in focal autonomic deficits or autonomic hyperreflexia (e.g., spinal cord transection or hemisection) affecting bowel, bladder, sexual, temperature-regulation, or cardiovascular functions. Quadriparetic patients exhibit both supine hypertension and OH after upward tilting. Autonomic dysreflexia describes a dramatic increase in BP in patients with traumatic spinal cord lesions above the T6 level, often in response to stimulation of the bladder, skin, or muscles. A distended or obstructed bladder, suprapubic palpation, catheter insertion, and urinary infection are common triggers. Associated symptoms can include facial flushing, headache, hypertension, or piloerection. Potential complications

1	Disorders of the Autonomic Nervous System 2642 include intracranial vasospasm or hemorrhage, cardiac arrhythmia, and death. Awareness of the syndrome, identifying the trigger, and careful monitoring of BP during procedures in patients with acute or chronic spinal cord injury are essential. In patients with supine hypertension, BP can be lowered by tilting the head upward or sitting the patient up. Vasodilator drugs may be used to treat acute elevations in BP. Clonidine can be used prophylactically to reduce the hypertension resulting from bladder stimulation. Dangerous increases or decreases in body temperature may result from an inability to experience the sensory accompaniments of heat or cold exposure or control peripheral vasoconstriction or sweating below the level of the spinal cord injury.

1	Peripheral neuropathies (Chap. 459) are the most common cause of chronic autonomic insufficiency. Polyneuropathies that affect small myelinated and unmyelinated fibers of the sympathetic and parasympathetic nerves commonly occur in diabetes mellitus, amyloidosis, chronic alcoholism, porphyria, and Guillain-Barré syndrome. Neuromuscular junction disorders with autonomic involvement include botulism and Lambert-Eaton syndrome (Chap. 461).

1	Diabetes Mellitus Autonomic neuropathy in patients with diabetes increases the mortality rate 1.5to 3-fold, even after adjusting for other cardiovascular risk factors. Estimates of 5-year mortality risk among these patients range from 15 to 53%. Although many deaths are due to secondary vascular disease, there are patients who specifically suffer cardiac arrest due to autonomic neuropathy. The autonomic involvement is also predictive of other complications including renal disease, stroke, and sleep apnea. Diabetes mellitus is discussed in Chaps. 417–419.

1	Amyloidosis Autonomic neuropathy occurs in both sporadic and familial forms of amyloidosis (Chap. 137). The AL (immunoglobulin light chain) type is associated with primary amyloidosis or amyloidosis secondary to multiple myeloma. The ATTR type, with transthyretin as the primary protein component, is responsible for the most common form of inherited amyloidosis. Although patients usually present with a distal painful polyneuropathy accompanied by sensory loss, autonomic insufficiency can precede the development of the polyneuropathy or occur in isolation. The diagnosis can be made by protein electrophoresis of blood and urine, tissue biopsy (abdominal fat pad, rectal mucosa, or sural nerve) to search for amyloid deposits, and genetic testing for transthyretin mutations in familial cases. Treatment of familial cases with liver transplantation can be successful. The response of primary amyloidosis to melphalan and stem cell transplantation has been mixed. Death is usually due to cardiac

1	of familial cases with liver transplantation can be successful. The response of primary amyloidosis to melphalan and stem cell transplantation has been mixed. Death is usually due to cardiac or renal involvement. Postmortem studies reveal amyloid deposition in many organs, including two sites that contribute to autonomic failure: intraneural blood vessels and autonomic ganglia. Pathologic examination reveals a loss of both unmyelinated and myelinated nerve fibers.

1	Alcoholic Neuropathy Abnormalities in parasympathetic vagal and efferent sympathetic function are usually mild in alcoholic polyneuropathy. OH is usually due to brainstem involvement, rather than injury to the PNS. Impotence is a major problem, but concurrent gonadal hormone abnormalities may play a role in this symptom. Clinical symptoms of autonomic failure generally appear only when the stocking-glove polyneuropathy is severe, and there is usually coexisting Wernicke’s encephalopathy (Chap. 330). Autonomic involvement may contribute to the high mortality rates associated with alcoholism (Chap. 467).

1	Porphyria (Chap. 430) Autonomic dysfunction is most extensively documented in acute intermittent porphyria but can also occur with variegate porphyria and hereditary coproporphyria. Autonomic symptoms include tachycardia, sweating, urinary retention, abdominal pain, nausea and vomiting, insomnia, hypertension, and (less commonly) hypotension. Another prominent symptom is anxiety. Abnormal autonomic function can occur both during acute attacks and during remissions. Elevated catecholamine levels during acute attacks correlate with the degree of tachycardia and hypertension that is present.

1	Guillain-Barré Syndrome (Chap. 460) BP fluctuations and arrhythmias from autonomic instability can be severe. It is estimated that between 2 and 10% of patients with severe Guillain-Barré syndrome suffer fatal cardiovascular collapse. GI autonomic involvement, sphincter disturbances, abnormal sweating, and pupillary dysfunction can also occur. Demyelination has been described in the vagus and glossopharyngeal nerves, the sympathetic chain, and the white rami communicantes. Interestingly, the degree of autonomic involvement appears to be independent of the severity of motor or sensory neuropathy. Acute autonomic and sensory neuropathy is a variant that spares the motor system and presents with neurogenic OH and varying degrees of sensory loss. It is treated similarly to Guillain-Barré syndrome, but prognosis is less favorable, with persistent severe sensory deficits and variable degrees of OH in many patients.

1	Autoimmune Autonomic Ganglionopathy (AAG) This disorder presents with the subacute development of autonomic disturbances including OH, enteric neuropathy (gastroparesis, ileus, constipation/diarrhea), flaccid bladder, and cholinergic failure (e.g., loss of sweating, sicca complex, and a tonic pupil). A chronic form of AAG resembles pure autonomic failure (see below). Autoantibodies against the ganglionic ACh receptor (A3 AChR), which are present in approximately half of patients, are considered diagnostic of AAG. Pathology shows preferential involvement of small unmyelinated nerve fibers, with sparing of larger myelinated ones. Onset of the neuropathy follows a viral infection in approximately half of cases. Up to one-third of untreated patients experience significant functional improvement over time. Immunotherapies that have been reported to be helpful include plasmapheresis, intravenous immune globulin, glucocorticoids, azathioprine, rituximab, and mycophenolate mofetil. OH,

1	over time. Immunotherapies that have been reported to be helpful include plasmapheresis, intravenous immune globulin, glucocorticoids, azathioprine, rituximab, and mycophenolate mofetil. OH, gastroparesis, and sicca symptoms can be managed symptomatically.

1	AAG can also occur on a paraneoplastic basis, with adenocarcinoma or small-cell carcinoma of the lung, lymphoma, or thymoma being the most common (Chap. 122). In the paraneoplastic cases, distinctive additional features, such as cerebellar involvement or dementia, may be present (see Tables 122-1, 122-2, and 122-3). The neoplasm may be occult and possibly suppressed by the autoantibody. Botulism Botulinum toxin binds presynaptically to cholinergic nerve terminals and, after uptake into the cytosol, blocks ACh release. This acute cholinergic neuropathy presents as motor paralysis and autonomic disturbances that include blurred vision, dry mouth, nausea, unreactive or sluggishly reactive pupils, constipation, and urinary retention (Chap. 178).

1	This sporadic syndrome consists of postural hypotension, impotence, bladder dysfunction, and impaired sweating. The disorder begins in midlife and occurs in women more often than men. The symptoms can be disabling, but the disease does not shorten life span. The clinical and pharmacologic characteristics suggest primary involvement of postganglionic sympathetic neurons. A severe reduction in the density of neurons within sympathetic ganglia results in low supine plasma NE levels and noradrenergic supersensitivity. Some patients who are initially labeled with this diagnosis subsequently go on to develop AAG or MSA. Skin biopsies can demonstrate phosphorylated α-synuclein inclusions in postganglionic sympathetic adrenergic and cholinergic nerve fibers from some individuals with PAF, distinguishing them from AAG and suggesting that PAF is a synucleinopathy; patients with PD also have α-synuclein inclusions in sympathetic nerve biopsies.

1	This syndrome is characterized by symptomatic orthostatic intolerance without OH, accompanied by either an increase in heart rate to >120 beats/min or an increase of 30 beats/min with standing that subsides on sitting or lying down. Women are affected approximately five times more often than men, and most develop the syndrome between the ages of 15 and 50. Presyncopal symptoms (lightheadedness, weakness, blurred vision) combined with symptoms of autonomic overactivity (palpitations, tremulousness, nausea) are common. Recurrent unexplained episodes of dysautonomia and fatigue also occur. The pathogenesis is unclear, but there is increasing evidence for sympathetic denervation distally in the legs with preserved cardiovascular function. Hypovolemia, venous pooling, impaired brainstem regulation, or increased sympathetic activity may play a role. Optimal treatment is uncertain, but expansion of fluid volume with water, salt, and fludrocortisone can be helpful as initial interventions. If

1	or increased sympathetic activity may play a role. Optimal treatment is uncertain, but expansion of fluid volume with water, salt, and fludrocortisone can be helpful as initial interventions. If this approach is inadequate, then midodrine, pyridostigmine, phenobarbital, beta blockers, or clonidine can be tried. Reconditioning and a sustained exercise program are important adjuncts to treatment.

1	There are five known hereditary sensory and autonomic neuropathies (HSAN I–V). The most important autonomic variants are HSAN I and HSAN III. HSAN I is dominantly inherited and often presents as a distal small-fiber neuropathy (burning feet syndrome) associated with sensory loss and foot ulcers. The most common responsible gene, on chromosome 9q, is SPTLC1. SPTLC is an important enzyme in the regulation of ceramide. Cells from HSAN I patients with the mutation produce higher-than-normal levels of glucosyl ceramide, perhaps triggering apoptosis. HSAN III (Riley-Day syndrome; familial dysautonomia) is an autosomal recessive disorder of Ashkenazi Jewish children and adults and is much less prevalent than HSAN I. Decreased tearing, hyperhidrosis, reduced sensitivity to pain, areflexia, absent fungiform papillae on the tongue, and labile BP may be present. Episodic abdominal crises and fever are common. Pathologic examination of nerves reveals a loss of sympathetic, parasympathetic, and

1	fungiform papillae on the tongue, and labile BP may be present. Episodic abdominal crises and fever are common. Pathologic examination of nerves reveals a loss of sympathetic, parasympathetic, and sensory neurons. The defective gene, IKBKAP, may prevent normal transcription of important molecules in neural development.

1	This syndrome presents with excess sweating of the palms of the hands and soles of the feet beginning in childhood or early adulthood. The condition tends to improve with age. The disorder affects 0.6–1.0% of the population. The etiology is unclear, but there may be a genetic component because 25% of patients have a positive family history. The condition can be socially embarrassing (e.g., shaking hands) or even disabling (e.g., inability to write without soiling the paper). Topical antiperspirants are occasionally helpful. More useful are potent anticholinergic drugs such as glycopyrrolate (1–2 mg PO tid). T2 ganglionectomy or sympathectomy is successful in >90% of patients with palmar hyperhidrosis. The advent of endoscopic transaxillary T2 sympathectomy has lowered the complication rate of the procedure. The most common postoperative complication is compensatory hyperhidrosis, which improves spontaneously over months. Other potential complications include recurrent hyperhidrosis

1	of the procedure. The most common postoperative complication is compensatory hyperhidrosis, which improves spontaneously over months. Other potential complications include recurrent hyperhidrosis (16%), Horner’s syndrome (<2%), gustatory sweating, wound infection, hemothorax, and intercostal neuralgia. Local injection of botulinum toxin has also been used to block cholinergic, postganglionic sympathetic fibers to sweat glands in patients with palmar hyperhidrosis. This approach is limited by the need for repetitive injections (the effect usually lasts 4 months before waning).

1	The physician may be confronted occasionally with an acute state of sympathetic overactivity.

1	An autonomic storm is an acute state of sustained sympathetic surge that results in variable combinations of alterations in BP and heart rate, body temperature, respiration, and sweating. Causes of autonomic storm include brain and spinal cord injury, toxins and drugs, autonomic neuropathy, and chemodectomas (e.g., pheochromocytoma). Brain injury is the most common cause of autonomic storm and typically follows severe head trauma and postresuscitation encephalopathy anoxic-ischemic brain injury. Autonomic storm can also occur with other acute intracranial lesions such as hemorrhage, cerebral infarction, rapidly expanding tumors, subarachnoid hemor-2643 rhage, hydrocephalus, or (less commonly) an acute spinal cord lesion. The most consistent setting is that of an acute intracranial catastrophe of sufficient size and rapidity to produce a massive catecholaminergic surge. The surge can cause seizures, neurogenic pulmonary edema, and myocardial injury. Manifestations include fever,

1	of sufficient size and rapidity to produce a massive catecholaminergic surge. The surge can cause seizures, neurogenic pulmonary edema, and myocardial injury. Manifestations include fever, tachycardia, hypertension, tachypnea, hyperhidrosis, pupillary dilatation, and flushing. Lesions of the afferent limb of the baroreflex can result in milder recurrent autonomic storms; many of these follow neck irradiation.

1	Drugs and toxins may also be responsible, including sympathomimetics such as phenylpropanolamine, cocaine, amphetamines, and tricyclic antidepressants; tetanus; and, less often, botulinum toxin. Cocaine, including “crack,” can cause a hypertensive state with CNS hyperstimulation. Tricyclic overdose, such as from amitriptyline, can cause flushing, hypertension, tachycardia, fever, mydriasis, anhidrosis, and a toxic psychosis. The hyperadrenergic state associated with Guillain-Barré syndrome can produce a moderate autonomic storm. Pheochromocytoma presents with a paroxysmal or sustained hyperadrenergic state, headache, hyperhidrosis, palpitations, anxiety, tremulousness, and hypertension. Neuroleptic malignant syndrome refers to a syndrome of muscle rigidity, hyperthermia, and hypertension in psychotic patients treated with phenothiazines (Chap. 449). Management of autonomic storm includes ruling out other causes of autonomic instability, including malignant hyperthermia, porphyria, and

1	psychotic patients treated with phenothiazines (Chap. 449). Management of autonomic storm includes ruling out other causes of autonomic instability, including malignant hyperthermia, porphyria, and seizures. Sepsis and encephalitis need to be excluded with appropriate studies. An electroencephalogram (EEG) should be done to search for seizure activity; MRI of the brain and spine is often necessary. The patient should be managed in an intensive care unit. Management with morphine sulphate (10 mg every 4 h) and labetalol (100–200 mg twice daily) may be helpful. Supportive treatment may need to be maintained for several weeks. For chronic and milder autonomic storm, propranolol and/or clonidine can be effective.

1	Other conditions associated with autonomic failure include infections, malignancy, poisoning (organophosphates), and aging. Disorders of the hypothalamus can affect autonomic function and produce abnormalities in temperature control, satiety, sexual function, and circadian rhythms (Chap. 403). The failure to identify a primary role of the ANS in the pathogenesis of these disorders has resulted in a change of nomenclature. The terms complex regional pain syndrome (CRPS) types I and II are now used in place of reflex sympathetic dystrophy (RSD) and causalgia.

1	CRPS type I is a regional pain syndrome that often develops after tissue injury and most commonly affects one limb. Examples of associated injury include minor shoulder or limb trauma, fractures, myocardial infarction, or stroke. Allodynia (the perception of a nonpainful stimulus as painful), hyperpathia (an exaggerated pain response to a painful stimulus), and spontaneous pain occur. The symptoms are unrelated to the severity of the initial trauma and are not confined to the distribution of a single peripheral nerve. CRPS type II is a regional pain syndrome that develops after injury to a specific peripheral nerve, often a major nerve trunk. Spontaneous pain initially develops within the territory of the affected nerve but eventually may spread outside the nerve distribution.

1	Pain (usually burning or electrical in quality) is the primary clinical feature of CRPS. Vasomotor dysfunction, sudomotor abnormalities, or focal edema may occur alone or in combination but must be present for diagnosis. Limb pain syndromes that do not meet these criteria are best classified as “limb pain—not otherwise specified.” In CRPS, localized sweating (increased resting sweat output) and changes in blood flow may produce temperature differences between affected and unaffected limbs. CRPS type I (RSD) has been classically divided into three clinical phases. Phase I consists of pain and swelling in the distal extremity occurring within weeks to 3 months after the precipitating event. The

1	Disorders of the Autonomic Nervous System 2644 pain is diffuse, spontaneous, and either burning, throbbing, or aching in quality. The involved extremity is warm and edematous, and the joints are tender. Increased sweating and hair growth develop. In phase II (3–6 months after onset), thin, shiny, cool skin appears. After an additional 3–6 months (phase III), atrophy of the skin and subcutaneous tissue plus flexion contractures complete the clinical picture. Autonomic testing or bone scans are occasionally useful when the diagnosis is in doubt. The natural history of typical CRPS may be more benign and more variable than previously recognized. A variety of surgical and medical treatments have been developed, with conflicting reports of efficacy. Clinical trials suggest that early mobilization with physical therapy or a brief course of glucocorticoids may be helpful for CRPS type I or II. Other medical treatments include the use of adrenergic blockers, nonsteroidal anti-inflammatory

1	with physical therapy or a brief course of glucocorticoids may be helpful for CRPS type I or II. Other medical treatments include the use of adrenergic blockers, nonsteroidal anti-inflammatory drugs, calcium channel blockers, phenytoin, opioids, and calcitonin. Stellate ganglion blockade is a commonly used invasive technique that often provides temporary pain relief, but the efficacy of repetitive blocks is uncertain.

1	Management of autonomic failure is aimed at specific treatment of the cause and alleviation of symptoms. Of particular importance is the removal of drugs or amelioration of underlying conditions that cause or aggravate the autonomic symptoms, especially in the elderly. For example, OH can be caused or aggravated by angiotensin-converting enzyme inhibitors, calcium channel-blocking agents, tricyclic antidepressants, levodopa, alcohol, or insulin. A summary of drugs that can cause OH by class, putative mechanism, and magnitude of the BP drop is shown in Table 454-6.

1	Only a minority of patients with OH require drug treatment. All patients should be taught the mechanisms of postural normotension (volume status, resistance and capacitance bed, autoregulation) and the nature of orthostatic stressors (time of day and the influence of meals, heat, standing, and exercise). Patients should learn to recognize orthostatic symptoms early (especially subtle cognitive symptoms, weakness, and fatigue) and to modify or avoid activities that provoke episodes. Other helpful measures may include keeping a BP log and dietary education (salt/fluids). Learning physical counter-maneuvers that reduce standing OH and practicing postural and resistance training are helpful measures.

1	Nonpharmacologic approaches are summarized in Table 454-9. Adequate intake of salt and fluids to produce a voiding volume between 1.5 and 2.5 L of urine (containing >170 meq/L of Na+) each 24 h is essential. Sleeping with the head of the bed elevated will minimize the effects of supine nocturnal hypertension. Prolonged recumbency should be avoided when possible. Patients are advised to sit with legs dangling over the edge of the bed for several minutes before attempting to stand in the morning; other postural stresses should be similarly approached in a gradual manner. One iNitiaL treatmeNt of orthostatiC hypoteNsioN (oh)

1	Patient education: mechanisms and stressors of OH High-salt diet (10–20 g/d) High-fluid intake (2 L/d) Elevate head of bed 10 cm (4 in.) to minimize supine hypertension Maintain postural stimuli Learn physical counter-maneuvers Compression garments Correct anemia maneuver that can reduce OH is leg-crossing with maintained contraction of leg muscles for 30 s; this compresses leg veins and increases systemic resistance. Compressive garments, such as compression stockings or abdominal binders, are helpful on occasion but uncomfortable for many patients. For transient worsening of OH, drinking two 250-mL (8-oz) glasses of water can raise standing BP 20–30 mmHg for about 2 h, beginning ~20 min after the fluid load. The patient can increase intake of salt and fluids (bouillon treatment), increase use of physical counter-maneuvers (elevate the legs when supine), or temporarily resort to a full-body stocking (compression pressure 30–40 mmHg).

1	Anemia should be corrected with erythropoietin, administered subcutaneously at doses of 25–75 U/kg three times per week. The hematocrit increases after 2–6 weeks. A weekly maintenance dose is usually necessary. However, the increased intravascular volume that accompanies the rise in hematocrit can exacerbate supine hypertension.

1	If these measures are not sufficient, pharmacologic treatment may be necessary. Midodrine, a directly acting α1-agonist that does not cross the blood-brain barrier, is effective. It has a duration of action of 2–4 h. The usual dose is 5–10 mg orally tid, but some patients respond best to a decremental dose (e.g., 15 mg on awakening, 10 mg at noon, and 5 mg in the afternoon). Midodrine should not be taken after 6:00 P.M. Side effects include pruritus, uncomfortable piloerection, and supine hypertension especially at higher doses. Droxidopa (Northera) was recently approved by the FDA for treatment of neurogenic OH associated with PAF, PD, or MSA; oral droxidopa is converted to NE and in short-term clinical trails was effective in decreasing symptoms of OH. Pyridostigmine (Mestinon) appears to improve OH without aggravating supine hypertension by enhancing ganglionic transmission (maximal when orthostatic, minimal when supine). Fludrocortisone will reduce OH but aggravates supine

1	to improve OH without aggravating supine hypertension by enhancing ganglionic transmission (maximal when orthostatic, minimal when supine). Fludrocortisone will reduce OH but aggravates supine hypertension. At doses between 0.1 mg/d and 0.3 mg bid orally, it enhances renal sodium conservation and increases the sensitivity of arterioles to NE. Susceptible patients may develop fluid overload, congestive heart failure, supine hypertension, or hypokalemia. Potassium supplements are often necessary with chronic administration of fludrocortisone. Sustained elevations of supine BP >180/110 mmHg should be avoided. Supine hypertension (>180/110 mmHg) can be self-treated by avoiding the supine position (e.g., sleeping in a recumbent chair) and reducing fludrocortisone. A daily glass of wine, if requested by the patient, can be taken shortly before bedtime. If these simple measures are not adequate, drugs to be considered include oral hydralazine (25 mg qhs), oral nifedipine (Procardia; 10 mg

1	by the patient, can be taken shortly before bedtime. If these simple measures are not adequate, drugs to be considered include oral hydralazine (25 mg qhs), oral nifedipine (Procardia; 10 mg qhs), or a nitroglycerin patch.

1	A promising drug combination (atomoxetine and yohimbine) has been studied for use in human subjects with severe OH not responsive to other agents, as can occur is some patients with diabetes and severe autonomic neuropathy not responsive to other medications. The atomoxetine blocks the NE reuptake transporter, and yohimbine blocks α2 receptors that mediate the sympathetic feedback loop for downregulation of BP in response to atomoxetine. The result is a dramatic increase in BP and standing tolerance. This combination is not FDA approved for this purpose. It is possible that the limited drug duration of action can be used to withdraw drug treatment when the patient anticipates becoming supine (e.g., before sleep).

1	Postprandial OH may respond to several measures. Frequent, small, low-carbohydrate meals may diminish splanchnic shunting of blood after meals and reduce postprandial OH. Prostaglandin inhibitors (ibuprofen or indomethacin) taken with meals or midodrine (10 mg with the meal) can be helpful. The somatostatin analogue octreotide can be useful in the treatment of postprandial syncope by inhibiting the release of GI peptides that have vasodilator and hypotensive effects. The subcutaneous dose ranges from 25 μg bid to 200 μg tid.

1	trigeminal Neuralgia, Bell’s palsy, and other Cranial Nerve Disorders M. Flint Beal, Stephen L. Hauser Symptoms and signs of cranial nerve pathology are common in inter-nal medicine. They often develop in the context of a widespread neu-rologic disturbance, and in such situations, cranial nerve involvement 455 may represent the initial manifestation of the illness. In other disor ders, involvement is largely restricted to one or several cranial nerves; these distinctive disorders are reviewed in this chapter. Disorders of ocular movement are discussed in Chap. 39, disorders of hearing in Chap. 43, and vertigo and disorders of vestibular function in

1	The trigeminal (fifth cranial) nerve supplies sensation to the skin of the face and anterior half of the head (Fig. 455-1). The motor part innervates the muscles involved in chewing (including masseters and pterygoids) as well as the tensor tympani of the middle ear (hearing especially for high-pitched tones). It is the largest of the cranial nerves. It exits in the lateral midpons and traverses the middle cranial fossa to the semilunar (gasserian, trigeminal) ganglion in Meckel’s cave, where the nerve divides into three divisions (ophthalmic [V1], maxillary [V2], and mandibular [V3]). V1 and V2 traverse the cavernous sinus to exit in the superior orbital fissure and foramen rotundum, located above and below the eye socket respectively; V3 exits through the foramen ovale. The trigeminal nerve is predominantly sensory, and motor innervation is exclusively carried in V3. The cornea is primar- Chap. 28. ily innervated by V1, although an inferior crescent may be V2. Upon

1	Chap. 28. ily innervated by V1, although an inferior crescent may be V2. Upon Frontal branch of frontal nerve Mesencephalic nucleus of V nerve Main sensory nucleus of V ganglion Main motor nucleus of Vnerve External Nucleus of spinal tract of V rami of infraorbital Mylohyoid nerve and sublingual glands Anterior belly of Trigeminal Neuralgia, Bell’s Palsy, and Other Cranial Nerve Disorders

1	FIGURE 455-1 The trigeminal nerve and its branches and sensory distribution on the face. The three major sensory divisions of the trigeminal nerve consist of the ophthalmic, maxillary, and mandibular nerves. (Adapted from Waxman SG: Clinical Neuroanatomy, 26th ed. http://www.accessmedicine.com. Copyright © The McGraw-Hill Companies, Inc. All rights reserved.) 2646 entering the pons, pain and temperature fibers descend ipsilaterally to the upper cervical spinal cord as the spinal tract of V, before synapsing with the spinal nucleus of V; this accounts for the facial numbness that can occur with spinal cord lesions above C2. In the brainstem, the spinal tract of V is also located adjacent to crossed ascending fibers of the spinothalamic tract, producing a “crossed” sensory loss for pain and temperature (ipsilateral face, contralateral arm/trunk/leg) with lesions of the lateral lower brainstem. CN V is also ensheathed by oligodendrocyte-derived, rather than Schwann cell–derived, myelin

1	temperature (ipsilateral face, contralateral arm/trunk/leg) with lesions of the lateral lower brainstem. CN V is also ensheathed by oligodendrocyte-derived, rather than Schwann cell–derived, myelin for up to 7 mm after it leaves the brainstem, unlike just a few millimeters for other cranial and spinal nerves; this may explain the high frequency of trigeminal neuralgia in multiple sclerosis (Chap. 458), a disorder of oligodendrocyte myelin.

1	TRIGEMINAL NEURALGIA (TIC DOULOUREUX) Clinical Manifestations Trigeminal neuralgia is characterized by excruciating paroxysms of pain in the lips, gums, cheek, or chin and, very rarely, in the distribution of the ophthalmic division of the fifth nerve. The pain seldom lasts more than a few seconds or a minute or two but may be so intense that the patient winces, hence the term tic. The paroxysms, experienced as single jabs or clusters, tend to recur frequently, both day and night, for several weeks at a time. They may occur spontaneously or with movements of affected areas evoked by speaking, chewing, or smiling. Another characteristic feature is the presence of trigger zones, typically on the face, lips, or tongue, that provoke attacks; patients may report that tactile stimuli—e.g., washing the face, brushing the teeth, or exposure to a draft of air—generate excruciating pain. An essential feature of trigeminal neuralgia is that objective signs of sensory loss cannot be demonstrated

1	the face, brushing the teeth, or exposure to a draft of air—generate excruciating pain. An essential feature of trigeminal neuralgia is that objective signs of sensory loss cannot be demonstrated on examination.

1	Trigeminal neuralgia is relatively common, with an estimated annual incidence of 4–8 per 100,000 individuals. Middle-aged and elderly persons are affected primarily, and ~60% of cases occur in women. Onset is typically sudden, and bouts tend to persist for weeks or months before remitting spontaneously. Remissions may be long-lasting, but in most patients, the disorder ultimately recurs.

1	Pathophysiology Symptoms result from ectopic generation of action potentials in pain-sensitive afferent fibers of the fifth cranial nerve root just before it enters the lateral surface of the pons. Compression or other pathology in the nerve leads to demyelination of large myelinated fibers that do not themselves carry pain sensation but become hyperexcitable and electrically coupled with smaller unmyelinated or poorly myelinated pain fibers in close proximity; this may explain why tactile stimuli, conveyed via the large myelinated fibers, can stimulate paroxysms of pain. Compression of the trigeminal nerve root by a blood vessel, most often the superior cerebellar artery or on occasion a tortuous vein, is now believed to be the source of trigeminal neuralgia in most patients. In cases of vascular compression, age-related brain sagging and increased vascular thickness and tortuosity may explain the prevalence of trigeminal neuralgia in later life.

1	Differential Diagnosis Trigeminal neuralgia must be distinguished from other causes of face and head pain (Chap. 21) and from pain arising from diseases of the jaw, teeth, or sinuses. Pain from migraine or cluster headache tends to be deep-seated and steady, unlike the superficial stabbing quality of trigeminal neuralgia; rarely, cluster headache is associated with trigeminal neuralgia, a syndrome known as cluster-tic. In temporal arteritis, superficial facial pain is present but is not typically shock-like, the patient frequently complains of myalgias and other systemic symptoms, and an elevated erythrocyte sedimentation rate (ESR) is usually present (Chap. 385). When trigeminal neuralgia develops in a young adult or is bilateral, multiple sclerosis (MS) is a key consideration, and in such cases, the cause is a demyelinating plaque near the root entry zone of the fifth nerve in the pons; often, evidence of facial sensory loss can be found on careful examination. Cases that are

1	in such cases, the cause is a demyelinating plaque near the root entry zone of the fifth nerve in the pons; often, evidence of facial sensory loss can be found on careful examination. Cases that are secondary to mass lesions—such as aneurysms, neurofibromas, acoustic schwannomas, or meningiomas—usually produce objective signs of sensory loss in the trigeminal nerve distribution (trigeminal neuropathy, see below).

1	Laboratory Evaluation An ESR is indicated if temporal arteritis is suspected. In typical cases of trigeminal neuralgia, neuroimaging studies are usually unnecessary but may be valuable if MS is a consideration or in assessing overlying vascular lesions in order to plan for decompression surgery.

1	Drug therapy with carbamazepine is effective in ~50–75% of patients. Carbamazepine should be started as a single daily dose of 100 mg taken with food and increased gradually (by 100 mg daily in divided doses every 1–2 days) until substantial (>50%) pain relief is achieved. Most patients require a maintenance dose of 200 mg qid. Doses >1200 mg daily provide no additional benefit. Dizziness, imbalance, sedation, and rare cases of agranulocytosis are the most important side effects of carbamazepine. If treatment is effective, it is usually continued for 1 month and then tapered as tolerated. Oxcarbazepine (300–1200 mg bid) is an alternative to carbamazepine that has less bone marrow toxicity and probably is equally efficacious. If these agents are not well tolerated or are ineffective, lamotrigine, 400 mg daily, and phenytoin, 300–400 mg daily, are other options. Baclofen may also be tried, either alone or in combination with an anticonvulsant. The initial dose is 5–10 mg tid, gradually

1	400 mg daily, and phenytoin, 300–400 mg daily, are other options. Baclofen may also be tried, either alone or in combination with an anticonvulsant. The initial dose is 5–10 mg tid, gradually increasing as needed to 20 mg qid.

1	If drug treatment fails, surgical therapy should be offered. The most widely used method is currently microvascular decompression to relieve pressure on the trigeminal nerve as it exits the pons. This procedure requires a suboccipital craniotomy. This procedure appears to have a >70% efficacy rate and a low rate of pain recurrence in responders; the response is better for classic tic-like symptoms than for nonlancinating facial pains. In a small number of cases, there is perioperative damage to the eighth or seventh cranial nerves or to the cerebellum or a postoperative cerebrospinal fluid leak syndrome. High-resolution magnetic resonance angiography is useful preoperatively to visualize the relationships between the fifth cranial nerve root and nearby blood vessels.

1	Gamma knife radiosurgery of the trigeminal nerve root is also used for treatment and results in complete pain relief, sometimes delayed in onset, in more than two-thirds of patients and a low risk of persistent facial numbness; the response is sometimes long-lasting, but recurrent pain develops over 2–3 years in half of patients. Compared with surgical decompression, gamma knife surgery appears to be somewhat less effective but has few serious complications.

1	Another procedure, radiofrequency thermal rhizotomy, creates a heat lesion of the trigeminal ganglion or nerve. It is used less often now than in the past. Short-term relief is experienced by >95% of patients; however, long-term studies indicate that pain recurs in up to one-third of treated patients. Postoperatively, partial numbness of the face is common, masseter (jaw) weakness may occur especially following bilateral procedures, and corneal denervation with secondary keratitis can follow rhizotomy for first-division trigeminal neuralgia.

1	A variety of diseases can affect the trigeminal nerve (Table 455-1). Most present with sensory loss on the face or with weakness of the jaw muscles. Deviation of the jaw on opening indicates weakness of the pterygoids on the side to which the jaw deviates. Some cases are due to Sjögren’s syndrome or a collagen-vascular disease such as systemic lupus erythematosus, scleroderma, or mixed connective tissue disease. Among infectious causes, herpes zoster (acute or postherpetic) and leprosy should be considered. Tumors of the middle cranial fossa (meningiomas), of the trigeminal nerve (schwannomas), or of the base of the skull (metastatic tumors) may cause a combination of motor and sensory signs. Lesions in the cavernous sinus can affect the first and second divisions of the trigeminal nerve, and lesions of the superior orbital fissure can affect the first (ophthalmic) division; the accompanying corneal anesthesia increases the risk of ulceration (neurokeratitis).

1	Isolated sensory loss over the chin (mental neuropathy) can be the only manifestation of systemic malignancy. Rarely, an idiopathic form of trigeminal neuropathy is observed. It is characterized by numbness Nasopharyngeal carcinoma Trauma Guillain-Barré syndrome Sjögren’s syndrome Collagen-vascular diseases Sarcoidosis Leprosy Drugs (stilbamidine, trichloroethylene) Idiopathic trigeminal neuropathy and paresthesias, sometimes bilaterally, with loss of sensation in the territory of the trigeminal nerve but without weakness of the jaw. Gradual recovery is the rule. Tonic spasm of the masticatory muscles, known as trismus, is symptomatic of tetanus (Chap. 177) or may occur in patients treated with phenothiazines. (Fig. 455-2) The seventh cranial nerve supplies all the muscles concerned with facial expression. The sensory component is small (the nervus intermedius); it conveys taste sensation from the anterior Trigeminal Neuralgia, Bell’s Palsy, and Other Cranial Nerve Disorders

1	Trigeminal Neuralgia, Bell’s Palsy, and Other Cranial Nerve Disorders Lacrimal gland Sublingual gland Submandibular ganglion Nucleus fasciculus solitarius Motor nucleus VII n. Motor nucleus VI n. Superior salivatory nucleus Fasciculus solitarius Geniculate ganglion Trigeminal ganglion Major superficial petrosal nerve Lingual nerve Chorda tympani To nasal and palatine glands VII n. V n. 123BCA

1	FIGURE 455-2 The facial nerve. A, B, and C denote lesions of the facial nerve at the a rule) before regeneration occurs, and that it may be stylomastoid foramen, distal and proximal to the geniculate ganglion, respectively. incomplete. The presence of incomplete paralysis in Green lines indicate the parasympathetic fibers, red line indicates motor fibers, and the first week is the most favorable prognostic sign. purple lines indicate visceral afferent fibers (taste). (Adapted from MB Carpenter: Core Text Recurrences are reported in approximately 7% of of Neuroanatomy, 2nd ed. Williams & Wilkins, 1978.) cases.

1	two-thirds of the tongue and probably cutaneous impulses from the 2647 anterior wall of the external auditory canal. The motor nucleus of the seventh nerve lies anterior and lateral to the abducens nucleus. After leaving the pons, the seventh nerve enters the internal auditory meatus with the acoustic nerve. The nerve continues its course in its own bony channel, the facial canal, and exits from the skull via the stylomastoid foramen. It then passes through the parotid gland and subdivides to supply the facial muscles.

1	A complete interruption of the facial nerve at the stylomastoid foramen paralyzes all muscles of facial expression. The corner of the mouth droops, the creases and skinfolds are effaced, the forehead is unfurrowed, and the eyelids will not close. Upon attempted closure of the lids, the eye on the paralyzed side rolls upward (Bell’s phenomenon). The lower lid sags and falls away from the conjunctiva, permitting tears to spill over the cheek. Food collects between the teeth and lips, and saliva may dribble from the corner of the mouth. The patient complains of a heaviness or numbness in the face, but sensory loss is rarely demonstrable and taste is intact.

1	If the lesion is in the middle-ear portion, taste is lost over the anterior two-thirds of the tongue on the same side. If the nerve to the stapedius is interrupted, there is hyperacusis (sensitivity to loud sounds). Lesions in the internal auditory meatus may affect the adjacent auditory and vestibular nerves, causing deafness, tinnitus, or dizziness. Intrapontine lesions that paralyze the face usually affect the abducens nucleus as well, and often the corticospinal and sensory tracts.

1	If the peripheral facial paralysis has existed for some time and recovery of motor function is incomplete, a continuous diffuse contraction of facial muscles may appear. The palpebral fissure becomes narrowed, and the nasolabial fold deepens. Attempts to move one group of facial muscles may result in contraction of all (associated movements, or synkinesis). Facial spasms, initiated by movements of the face, may develop (hemifacial spasm). Anomalous regeneration of seventh nerve fibers may result in other troublesome phenomena. If fibers originally connected with the orbicularis oculi come to innervate the orbicularis oris, closure of the lids may cause a retraction of the mouth, or if fibers originally connected with muscles of the face later innervate the lacrimal gland, anomalous tearing (“crocodile tears”) may occur with any activity of the facial muscles, such as eating. Another facial synkinesia is triggered by jaw opening, causing closure of the eyelids on the side of the facial

1	(“crocodile tears”) may occur with any activity of the facial muscles, such as eating. Another facial synkinesia is triggered by jaw opening, causing closure of the eyelids on the side of the facial palsy (jaw-winking).

1	The most common form of facial paralysis is Bell’s palsy. The annual incidence of this idiopathic disorder is ~25 per 100,000 annually, or about 1 in 60 persons in a lifetime. Risk factors include pregnancy and diabetes mellitus.

1	Pterygopalatine Clinical Manifestations The onset of Bell’s palsy is ganglion fairly abrupt, with maximal weakness being attained by 48 h as a general rule. Pain behind the ear may precede the paralysis for a day or two. Taste sensation may be lost unilaterally, and hyperacusis may be present. In some cases, there is mild cerebrospinal fluid lymphocytosis. Magnetic resonance imaging (MRI) may reveal swelling and uniform enhancement of the geniculate ganglion and facial nerve and, in some cases, entrapment of the swollen nerve in the temporal bone. Approximately 80% of patients recover within a few weeks or months. Electromyography may be of some prognostic value; evidence of denervation after 10 days indicates there has been axonal degeneration, that there will be a long delay (3 months as 2648 Pathophysiology In acute Bell’s palsy, there is inflammation of the facial nerve with mononuclear cells, consistent with an infectious or immune cause. Herpes simplex virus (HSV) type 1 DNA was

1	2648 Pathophysiology In acute Bell’s palsy, there is inflammation of the facial nerve with mononuclear cells, consistent with an infectious or immune cause. Herpes simplex virus (HSV) type 1 DNA was frequently detected in endoneurial fluid and posterior auricular muscle, suggesting that a reactivation of this virus in the geniculate ganglion may be responsible for most cases. Reactivation of varicella-zoster virus is associated with Bell’s palsy in up to one-third of cases and may represent the second most frequent cause. A variety of other viruses have also been implicated less commonly. An increased incidence of Bell’s palsy was also reported among recipients of inactivated intranasal influenza vaccine, and it was hypothesized that this could have resulted from the Escherichia coli enterotoxin used as adjuvant or reactivation of latent virus.

1	Differential Diagnosis There are many other causes of acute facial palsy that must be considered in the differential diagnosis of Bell’s palsy. Lyme disease can cause unilateral or bilateral facial palsies; in endemic areas, 10% or more of cases of facial palsy are likely due to infection with Borrelia burgdorferi (Chap. 210). The Ramsay Hunt syndrome, caused by reactivation of herpes zoster in the geniculate ganglion, consists of a severe facial palsy associated with a vesicular eruption in the external auditory canal and sometimes in the pharynx and other parts of the cranial integument; often the eighth cranial nerve is affected as well. Facial palsy that is often bilateral occurs in sarcoidosis (Chap.

1	390) and in Guillain-Barré syndrome (Chap. 460). Leprosy frequently involves the facial nerve, and facial neuropathy may also occur in diabetes mellitus, connective tissue diseases including Sjögren’s syndrome, and amyloidosis. The rare Melkersson-Rosenthal syndrome consists of recurrent facial paralysis; recurrent—and eventually permanent—facial (particularly labial) edema; and, less constantly, plication of the tongue. Its cause is unknown. Acoustic neuromas frequently involve the facial nerve by local compression. Infarcts, demyelinating lesions of MS, and tumors are the common pontine lesions that interrupt the facial nerve fibers; other signs of brainstem involvement are usually present. Tumors that invade the temporal bone (carotid body, cholesteatoma, dermoid) may produce a facial palsy, but the onset is insidious and the course progressive.

1	All these forms of nuclear or peripheral facial palsy must be distinguished from the supranuclear type. In the latter, the frontalis and orbicularis oculi muscles of the forehead are involved less than those of the lower part of the face, since the upper facial muscles are innervated by corticobulbar pathways from both motor cortices, whereas the lower facial muscles are innervated only by the opposite hemisphere. In supranuclear lesions, there may be a dissociation of emotional and voluntary facial movements, and often some degree of paralysis of the arm and leg or an aphasia (in dominant hemisphere lesions) is present.

1	Laboratory Evaluation The diagnosis of Bell’s palsy can usually be made clinically in patients with (1) a typical presentation, (2) no risk factors or preexisting symptoms for other causes of facial paralysis, (3) absence of cutaneous lesions of herpes zoster in the external ear canal, and (4) a normal neurologic examination with the exception of the facial nerve. Particular attention to the eighth cranial nerve, which courses near to the facial nerve in the pontomedullary junction and in the temporal bone, and to other cranial nerves is essential. In atypical or uncertain cases, an ESR, testing for diabetes mellitus, a Lyme titer, angiotensin-converting enzyme and chest imaging studies for possible sarcoidosis, a lumbar puncture for possible Guillain-Barré syndrome, or MRI scanning may be indicated. MRI often shows swelling and enhancement of the facial nerve in idiopathic Bell’s palsy (Fig. 455-3).

1	Symptomatic measures include (1) the use of paper tape to depress the upper eyelid during sleep and prevent corneal drying, and (2) massage of the weakened muscles. A course of glucocorticoids, given as prednisone 60–80 mg daily during the first 5 days and then tapered over the next 5 days, modestly shortens the recovery period and improves the functional outcome. Although large and well-controlled randomized trials found no added benefit of the antiviral agents valacyclovir (1000 mg daily for 5–7 days) or acyclovir (400 mg five times daily for 10 days) compared to glucocorticoids alone, some earlier data suggested that combination therapy with prednisone plus valacyclovir might be marginally better than prednisolone alone, especially in patients with severe clinical presentations. For patients with permanent paralysis from Bell’s palsy, a number of cosmetic surgical procedures have been used to restore a relatively symmetric appearance to the face.

1	Hemifacial spasm consists of painless irregular involuntary contractions on one side of the face. Most cases appear related to vascular compression of the exiting facial nerve in the pons. Other cases develop as a sequela to Bell’s palsy or are secondary to compression and/or demyelination of the nerve by tumor, infection, or MS. Mild cases can be treated with carbamazepine, gabapentin, or, if these drugs fail, baclofen. Local injections of botulinum toxin into affected muscles can relieve spasms for 3–4 months, and the injections can be repeated. Refractory cases due to vascular compression usually respond to surgical decompression of the facial nerve. Blepharospasm is an involuntary recurrent spasm of both eyelids that usually occurs in elderly persons as an isolated phenomenon or with varying degrees of spasm of other facial muscles. Severe, persistent cases of blepharospasm can be treated by local injection of botulinum toxin into the orbicularis oculi. Facial myokymia refers to a

1	degrees of spasm of other facial muscles. Severe, persistent cases of blepharospasm can be treated by local injection of botulinum toxin into the orbicularis oculi. Facial myokymia refers to a fine rippling activity of the facial muscles; it may be caused by MS or follow Guillain-Barré syndrome (Chap. 460).

1	FIGURE 455-3 Axial and coronal T1-weighted images after gadolinium with fat suppression demonstrate diffuse smooth linear enhancement of the left facial nerve, involving the genu, tympanic, and mastoid segments within the temporal bone (arrows), without evidence of mass lesion. Although highly suggestive of Bell’s palsy, similar findings may be seen with other etiologies such as Lyme disease, sarcoidosis, and perineural malignant spread.

1	Facial hemiatrophy occurs mainly in women and is characterized by a disappearance of fat in the dermal and subcutaneous tissues on one side of the face. It usually begins in adolescence or the early adult years and is slowly progressive. In its advanced form, the affected side of the face is gaunt, and the skin is thin, wrinkled, and brown. The facial hair may turn white and fall out, and the sebaceous glands become atrophic. Bilateral involvement may occur. A limited form of systemic sclerosis (scleroderma) may be the cause of some cases. Treatment is cosmetic, consisting of transplantation of skin and subcutaneous fat.

1	This form of neuralgia involves the ninth (glossopharyngeal) and sometimes portions of the tenth (vagus) cranial nerves. It resembles trigeminal neuralgia in many respects but is much less common. The pain is intense and paroxysmal; it originates on one side of the throat, approximately in the tonsillar fossa. In some cases, the pain is localized in the ear or may radiate from the throat to the ear because of involvement of the tympanic branch of the glossopharyngeal nerve. Spasms of pain may be initiated by swallowing or coughing. There is no demonstrable motor or sensory deficit; the glossopharyngeal nerve supplies taste sensation to the posterior third of the tongue and, together with the vagus nerve, sensation to the posterior pharynx. Cardiac symptoms—bradycardia or asystole, hypotension, and fainting—have been reported. Glossopharyngeal neuralgia can result from vascular compression, MS, or tumors, but many cases are idiopathic. Medical therapy is similar to that for trigeminal

1	and fainting—have been reported. Glossopharyngeal neuralgia can result from vascular compression, MS, or tumors, but many cases are idiopathic. Medical therapy is similar to that for trigeminal neuralgia, and carbamazepine is generally the first choice. If drug therapy is unsuccessful, surgical procedures—including microvascular decompression if vascular compression is evident—or rhizotomy of glossopharyngeal and vagal fibers in the jugular bulb is frequently successful.

1	Glossopharyngeal neuropathy in conjunction with vagus and accessory nerve palsies may occur with herpes zoster infection or with a tumor or aneurysm in the posterior fossa or in the jugular foramen. Hoarseness due to vocal cord paralysis, some difficulty in swallowing, deviation of the soft palate to the intact side, anesthesia of the posterior wall of the pharynx, and weakness of the upper part of the trapezius and sternocleidomastoid muscles make up the jugular foramen syndrome (Table 455-2).

1	When the intracranial portion of one vagus (tenth cranial) nerve is interrupted, the soft palate droops ipsilaterally and does not rise in phonation. There is loss of the gag reflex on the affected side, as well as of the “curtain movement” of the lateral wall of the pharynx, whereby the faucial pillars move medially as the palate rises in saying “ah.” The voice is hoarse and slightly nasal, and the vocal cord lies immobile midway between abduction and adduction. Loss of sensation at the external auditory meatus and the posterior pinna may also be present. The pharyngeal branches of both vagal nerves may be affected in diphtheria; the voice has a nasal quality, and regurgitation of liquids through the nose occurs during swallowing.

1	The pharyngeal branches of both vagal nerves may be affected in diphtheria; the voice has a nasal quality, and regurgitation of liquids through the nose occurs during swallowing. Injury to the vagus nerve in the carotid sheath can also occur with carotid dissection or following endarterectomy. The vagus nerve may be involved at the meningeal level by neoplastic and infectious processes and within the medulla by tumors, vascular lesions (e.g., the lateral medullary syndrome), and motor neuron disease. This nerve may be involved by infection with varicella zoster virus. Polymyositis and dermatomyositis, which cause hoarseness and dysphagia by direct involvement of laryngeal and pharyngeal muscles, may be confused with diseases of the vagus nerves. Dysphagia is also a symptom in some patients with myotonic dystrophy. Nonneurologic causes of dysphagia are discussed in Chap. 53.

1	The recurrent laryngeal nerves, especially the left, are most often damaged as a result of intrathoracic disease. Aneurysm of the aortic arch, an enlarged left atrium, and tumors of the mediastinum and bronchi are much more frequent causes of an isolated vocal cord palsy than are intracranial disorders. However, a substantial number of cases of recurrent laryngeal palsy remain idiopathic.

1	When confronted with a case of laryngeal palsy, the physician must attempt to determine the site of the lesion. If it is intramedullary, there are usually other signs, such as ipsilateral cerebellar dysfunction, loss of pain and temperature sensation over the ipsilateral face and contralateral arm and leg, and an ipsilateral Horner’s syndrome. If the lesion is extramedullary, the glossopharyngeal and spinal accessory nerves are frequently involved (jugular foramen syndrome). If it is extracranial in the posterior laterocondylar or retroparotid space, there may be a combination of ninth, tenth, eleventh, and twelfth cranial nerve palsies and a Horner’s syndrome (Table 455-2). If there is no sensory loss over the palate and pharynx and no palatal weakness or dysphagia, the lesion is below the origin of the pharyngeal branches, which leave the vagus nerve high in the cervical region; the usual site of disease is then the mediastinum.

1	Isolated involvement of the accessory (eleventh cranial) nerve can occur anywhere along its route, resulting in partial or complete paralysis of the sternocleidomastoid and trapezius muscles. More commonly, involvement occurs in combination with deficits of the ninth and tenth cranial nerves in the jugular foramen or after exit from the skull (Table 455-2). An idiopathic form of accessory neuropathy, akin to Bell’s palsy, has been described, and it may be recurrent in some cases. Most but not all patients recover.

1	The hypoglossal (twelfth cranial) nerve supplies the ipsilateral muscles of the tongue. The nucleus of the nerve or its fibers of exit may be involved by intramedullary lesions such as tumor, poliomyelitis, or most often motor neuron disease. Lesions of the basal meninges and the occipital bones (platybasia, invagination of occipital condyles, Paget’s disease) may compress the nerve in its extramedullary course or in the hypoglossal canal. Isolated lesions of unknown cause can occur. Atrophy and fasciculation of the tongue develop weeks to months after interruption of the nerve. s Palsy, and Other Cranial Nerve Disorders

1	s Palsy, and Other Cranial Nerve Disorders Several cranial nerves may be affected by the same disease process. In this situation, the main clinical problem is to determine whether the lesion lies within the brainstem or outside it. Lesions that lie on the surface of the brainstem are characterized by involvement of adjacent cranial nerves (often occurring in succession) and late and rather slight involvement of the long sensory and motor pathways and segmental structures lying within the brainstem. The opposite is true of primary lesions within the brainstem. The extramedullary lesion is more likely to cause bone erosion or enlargement of the foramens of exit of cranial nerves. The intramedullary lesion involving cranial nerves often produces a crossed sensory or motor paralysis (cranial nerve signs on one side of the body and tract signs on the opposite side).

1	Involvement of multiple cranial nerves outside the brainstem is frequently the result of trauma, localized infections including varicellazoster virus, infectious and noninfectious (especially carcinomatous) causes of meningitis (Chaps. 164 and 165), granulomatous diseases such as Wegener’s granulomatosis, Behçet’s disease, vascular disorders including those associated with diabetes, enlarging aneurysms, or locally infiltrating tumors. Among the tumors, nasopharyngeal cancers, lymphomas, neurofibromas, meningiomas, chordomas, cholesteatomas, carcinomas, and sarcomas have all been observed to involve a succession of lower cranial nerves. Owing to their anatomic relationships, the multiple cranial nerve palsies form a number of distinctive syndromes, listed in Table 455-2. Sarcoidosis is the cause of some cases of multiple cranial neuropathy; tuberculosis, the Chiari malformation, platybasia, and basilar invagination of the skull are additional causes. A purely motor disorder without

1	the cause of some cases of multiple cranial neuropathy; tuberculosis, the Chiari malformation, platybasia, and basilar invagination of the skull are additional causes. A purely motor disorder without atrophy always raises the question of myasthenia gravis (Chap. 461). As noted above, Guillain-Barré syndrome commonly affects the facial nerves bilaterally. In the Fisher variant of the Guillain-Barré syndrome, oculomotor paresis occurs with ataxia and areflexia in the limbs (Chap. 460). Wernicke’s encephalopathy can cause a severe ophthalmoplegia combined with other brainstem signs (Chap. 330).

1	The cavernous sinus syndrome (Fig. 455-4) is a distinctive and frequently life-threatening disorder. It often presents as orbital or facial pain; orbital swelling and chemosis due to occlusion of the ophthalmic veins; fever; oculomotor neuropathy affecting the third, fourth, and sixth cranial nerves; and trigeminal neuropathy affecting the ophthalmic (V1) and occasionally the maxillary (V2) divisions of the trigeminal nerve. Cavernous sinus thrombosis, often secondary to infection from orbital cellulitis (frequently Staphylococcus aureus), a cutaneous source on the face, or sinusitis (especially with mucormycosis in diabetic patients), is the most frequent cause; other etiologies include Ant. cerebral a. Int. carotid a. Ant. clinoid process Subarachnoid Oculomotor (III) n. Trochlear (IV) n. Ophthalmic (VI) n. Abducens (VI) n. Maxillary (V2) n. Pia Arachnoid Sphenoid sinus

1	Ant. cerebral a. Int. carotid a. Ant. clinoid process Subarachnoid Oculomotor (III) n. Trochlear (IV) n. Ophthalmic (VI) n. Abducens (VI) n. Maxillary (V2) n. Pia Arachnoid Sphenoid sinus FIGURE 455-4 Anatomy of the cavernous sinus in coronal section, illustrating the location of the cranial nerves in relation to the vascular sinus, internal carotid artery (which loops anteriorly to the section), and surrounding structures. aneurysm of the carotid artery, a carotid-cavernous fistula (orbital bruit may be present), meningioma, nasopharyngeal carcinoma, other tumors, or an idiopathic granulomatous disorder (Tolosa-Hunt syndrome). The two cavernous sinuses directly communicate via intercavernous channels; thus, involvement on one side may extend to become bilateral. Early diagnosis is essential, especially when due to infection, and treatment depends on the underlying etiology.

1	In infectious cases, prompt administration of broad-spectrum antibiotics, drainage of any abscess cavities, and identification of the offending organism are essential. Anticoagulant therapy may benefit cases of primary thrombosis. Repair or occlusion of the carotid artery may be required for treatment of fistulas or aneurysms. The Tolosa-Hunt syndrome generally responds to glucocorticoids. A dramatic improvement in pain is usually evident within a few days; oral prednisone (60 mg daily) is usually continued for 2 weeks and then gradually tapered over a month, or longer if pain recurs. Occasionally an immunosuppressive medication, such as azathioprine or methotrexate, needs to be added to maintain an initial response to glucocorticoids.

1	An idiopathic form of multiple cranial nerve involvement on one or both sides of the face is occasionally seen. The syndrome consists of a subacute onset of boring facial pain, followed by paralysis of motor cranial nerves. The clinical features overlap those of the Tolosa-Hunt syndrome and appear to be due to idiopathic inflammation of the dura mater, which may be visualized by MRI. The syndrome is usually responsive to glucocorticoids. Diseases of the spinal Cord Stephen L. Hauser, Allan H. Ropper

1	Diseases of the spinal Cord Stephen L. Hauser, Allan H. Ropper Diseases of the spinal cord are frequently devastating. They produce quadriplegia, paraplegia, and sensory deficits far beyond the damage they would inflict elsewhere in the nervous system because the spinal cord contains, in a small cross-sectional area, almost the entire motor output and sensory input of the trunk and limbs. Many spinal cord diseases are reversible if recognized and treated at an early stage (Table 456-1); thus, they are among the most critical of neurologic emergencies. The efficient use of diagnostic procedures, guided by knowledge of the anatomy and the clinical features of spinal cord diseases, is required to maximize the likelihood of a successful outcome. APPROACH TO THE PATIENT:

1	The spinal cord is a thin, tubular extension of the central nervous system contained within the bony spinal canal. It originates at the medulla and continues caudally to the conus medullaris at the lumbar level; its fibrous extension, the filum terminale, terminates at the coccyx. The adult spinal cord is ~46 cm (18 in.) long, oval in shape, and enlarged in the cervical and lumbar regions, where neurons that innervate the upper and lower extremities, respectively, are located. The white matter tracts containing ascending sensory and descending motor pathways are located peripherally, whereas nerve cell bodies are clustered in an inner region of gray matter shaped like a four-leaf clover that surrounds the central canal (anatomically an extension of the fourth ventricle). The membranes that cover the spinal cord—the pia, arachnoid, and dura—are continuous with those of the brain, and the cerebrospinal fluid is contained within the subarachnoid space between the pia and arachnoid.

1	The spinal cord has 31 segments, each defined by an exiting ventral motor root and entering dorsal sensory root. During Epidural, intradural, or intramedullary neoplasm Epidural abscess Epidural hemorrhage Cervical spondylosis Herniated disk Posttraumatic compression by fractured or displaced vertebra or Viral: VZV, HSV-1 and -2, CMV, HIV, HTLV-1, others Bacterial and mycobacterial: Borrelia, Listeria, syphilis, others Mycoplasma pneumoniae Parasitic: schistosomiasis, toxoplasmosis Vitamin B12 deficiency (subacute combined degeneration) Copper deficiency Abbreviations: CMV, cytomegalovirus; HSV, herpes simplex virus; HTLV, human T cell lymphotropic virus; VZV, varicella-zoster virus.

1	embryologic development, growth of the cord lags behind that of the vertebral column, and the mature spinal cord ends at approximately the first lumbar vertebral body. The lower spinal nerves take an increasingly downward course to exit via intervertebral foramina. The first seven pairs of cervical spinal nerves exit above the same-numbered vertebral bodies, whereas all the subsequent nerves exit below the same-numbered vertebral bodies because of the presence of eight cervical spinal cord segments but only seven cervical vertebrae. The relationship between spinal cord segments and the corresponding vertebral bodies is shown in Table 456-2. These relationships assume particular importance for localization of lesions that cause spinal cord compression. Sensory loss below the circumferential level of the umbilicus, for example, corresponds to the T10 cord segment but indicates involvement of the cord adjacent to the seventh or eighth thoracic vertebral body (see Figs. 31-2 and 31-3). In

1	level of the umbilicus, for example, corresponds to the T10 cord segment but indicates involvement of the cord adjacent to the seventh or eighth thoracic vertebral body (see Figs. 31-2 and 31-3). In addition, at every level, the main ascending and descending tracts are somatotopically organized with a laminated distribution that reflects the origin or destination of nerve fibers.

1	Determining the Level of the Lesion The presence of a horizontally defined level below which sensory, motor, and autonomic function is impaired is a hallmark of a lesion of the spinal cord. This sensory level is sought by asking the patient to identify a pinprick or cold stimulus applied to the proximal legs and lower trunk and successively moved up toward the neck on each side. Sensory loss below this level is the result of damage to the spinothalamic tract on the opposite side, one to two segments higher in the case of a unilateral spinal cord lesion, and at the level of a bilateral lesion. The discrepancy in the level of a unilateral lesion is the result of the course of the second-order sensory fibers, which originate in the dorsal horn, and ascend for one or two levels as they cross anterior to the central canal to join the opposite spinothalamic tract. Lesions that transect the descending corticospinal and other motor tracts cause paraplegia or quadriplegia with heightened deep

1	anterior to the central canal to join the opposite spinothalamic tract. Lesions that transect the descending corticospinal and other motor tracts cause paraplegia or quadriplegia with heightened deep tendon reflexes, Babinski signs, and eventual spasticity (the upper motor neuron syndrome). Transverse damage to the cord also produces autonomic disturbances consisting of absent sweating below the implicated cord level and bladder, bowel, and sexual dysfunction.

1	The uppermost level of a spinal cord lesion can also be localized by attention to the segmental signs corresponding to disturbed motor or sensory innervation by an individual cord segment. A band of altered sensation (hyperalgesia or hyperpathia) at the upper end of the sensory disturbance, fasciculations or atrophy in muscles innervated by one or several segments, or a muted or absent deep tendon reflex may be noted at this level. These signs also can occur with focal root or peripheral nerve disorders; thus, they are most useful when they occur together with signs of long tract damage. With severe and acute transverse lesions, the limbs initially may be flaccid rather than spastic. This state of “spinal shock” lasts for several days, rarely for weeks, and may be mistaken for extensive damage to the anterior horn cells over many segments of the cord or for an acute polyneuropathy. The main features of transverse damage at each level of the spinal cord are summarized below.

1	The main features of transverse damage at each level of the spinal cord are summarized below. cerVicAl corD Upper cervical cord lesions produce quadriplegia and weakness of the diaphragm. The uppermost level of weakness and reflex loss with lesions at C5-C6 is in the biceps; at C7, in finger and wrist extensors and triceps; and at C8, finger and wrist flexion. Horner’s syndrome (miosis, ptosis, and facial hypohidrosis) may accompany a cervical cord lesion at any level. thorAcic corD Lesions here are localized by the sensory level on the trunk and, if present, by the site of midline back pain. Useful markers of the sensory level on the trunk are the nipples (T4) and umbilicus (T10). Leg weakness and disturbances of bladder and bowel function accompany the paralysis. Lesions at T9-T10 paralyze the lower—but not the upper—abdominal muscles, resulting in upward movement of the umbilicus when the abdominal wall contracts (Beevor’s sign).

1	lumbAr corD Lesions at the L2-L4 spinal cord levels paralyze flex-ion and adduction of the thigh, weaken leg extension at the knee, and abolish the patellar reflex. Lesions at L5-S1 paralyze only movements of the foot and ankle, flexion at the knee, and extension of the thigh, and abolish the ankle jerks (S1).

1	SAcrAl corD/conuS meDullAriS The conus medullaris is the tapered caudal termination of the spinal cord, comprising the sacral and single coccygeal segments. The distinctive conus syndrome consists of bilateral saddle anesthesia (S3-S5), prominent bladder and bowel dysfunction (urinary retention and incontinence with lax anal tone), and impotence. The bulbocavernosus (S2-S4) and anal (S4-S5) reflexes are absent (Chap. 437). Muscle strength is largely preserved. By contrast, lesions of the cauda equina, the nerve roots derived from the lower cord, are characterized by low back and radicular pain, asymmetric leg weakness and sensory loss, variable areflexia in the lower extremities, and relative sparing of bowel and bladder function. Mass lesions in the lower spinal Diseases of the Spinal Cord canal often produce a mixed clinical picture with elements of both cauda equina and conus medullaris syndromes. Cauda equina syndromes are also discussed in Chap. 22.

1	Diseases of the Spinal Cord canal often produce a mixed clinical picture with elements of both cauda equina and conus medullaris syndromes. Cauda equina syndromes are also discussed in Chap. 22. Special Patterns of Spinal Cord Disease The location of the major ascending and descending pathways of the spinal cord are shown in Fig. 456-1. Most fiber tracts—including the posterior columns and the spinocerebellar and pyramidal tracts—are situated on the side of the body they innervate. However, afferent fibers mediating pain and temperature sensation ascend in the spinothalamic tract contralateral to the side they supply. The anatomic configurations of these tracts produce characteristic syndromes that provide clues to the underlying disease process.

1	brown-SequArD hemicorD SynDrome This consists of ipsilateral weakness (corticospinal tract) and loss of joint position and vibratory sense (posterior column), with contralateral loss of pain and temperature sense (spinothalamic tract) one or two levels below the lesion. Segmental signs, such as radicular pain, muscle atrophy, or loss of a deep tendon reflex, are unilateral. Partial forms are more common than the fully developed syndrome.

1	centrAl corD SynDrome This syndrome results from selective damage to the gray matter nerve cells and crossing spinothalamic tracts surrounding the central canal. In the cervical cord, the central cord syndrome produces arm weakness out of proportion to leg weakness and a “dissociated” sensory loss, meaning loss of pain and temperature sensations over the shoulders, lower neck, and upper trunk (cape distribution), in contrast to preservation of light touch, joint position, and vibration sense in these regions. Spinal trauma, syringomyelia, and intrinsic cord tumors are the main causes. Anterior SpinAl Artery SynDrome Infarction of the cord is generally the result of occlusion or diminished flow in this artery. The result is bilateral tissue destruction at several contiguous levels that spares the posterior columns. All spinal cord functions—motor, sensory, and autonomic—are lost below the level of the lesion, with the striking exception of retained vibration and position sensation.

1	Anterior horn (motor neurons) Lateral corticospinal (pyramidal) tract Dorsal root Dorsal spinocerebellar tract Ventral spinocerebellar tract Lateral spinothalamic tract C T L S Ventral spinothalamic tract Pressure, touch (minor role) Ventral (uncrossed) corticospinal tract Tectospinal tract S L T C C T L S Fasciculus cuneatus Rubrospinal tract Lateral reticulospinal tract Vestibulospinal tract Ventral root (Joint Position, Vibration, Pressure) Posterior Columns Pain, temperature Ventral reticulospinal tract Fasciculus gracilis S L T CL/S L/S P E D F forAmen mAgnum SynDrome Lesions in this area interrupt decussating pyramidal tract fibers destined for the legs, which cross caudal to those of the arms, resulting in weakness of the legs (crural paresis). Compressive lesions near the foramen magnum may produce weakness of the ipsilateral shoulder and arm followed by weakness of the ipsilateral leg, then the contralateral leg, and finally the contralateral arm, an “around the clock”

1	magnum may produce weakness of the ipsilateral shoulder and arm followed by weakness of the ipsilateral leg, then the contralateral leg, and finally the contralateral arm, an “around the clock” pattern that may begin in any of the four limbs. There is typically suboccipital pain spreading to the neck and shoulders.

1	intrAmeDullAry AnD extrAmeDullAry SynDromeS It is useful to differentiate intramedullary processes, arising within the substance of the cord, from extramedullary ones that lie outside the cord and compress the spinal cord or its vascular supply. The differentiating features are only relative and serve as clinical guides. With extramedullary lesions, radicular pain is often prominent, and there is early sacral sensory loss and spastic weakness in the legs with incontinence due to the superficial location of the corresponding sensory and motor fibers in the spinothalamic and corticospinal tracts (Fig. 456-1). Intramedullary lesions tend to produce poorly localized burning pain rather than radicular pain and to spare sensation in the perineal and sacral areas (“sacral sparing”), reflecting the laminated configuration of the spinothalamic tract with sacral fibers outermost; corticospinal tract signs appear later. Regarding extramedullary lesions, a further distinction is made between

1	the laminated configuration of the spinothalamic tract with sacral fibers outermost; corticospinal tract signs appear later. Regarding extramedullary lesions, a further distinction is made between extradural and intradural masses, as the former are generally malignant and the latter benign (neurofibroma being a common cause). Consequently, a long duration of symptoms favors an intradural origin.

1	FIGURE 456-1 Transverse section through the spinal cord, composite representation, illustrating the principal ascending (left) and descending (right) pathways. The lateral and ventral spinothalamic tracts ascend contralateral to the side of the body that is innervated. C, cervical; D, distal; E, extensors; F, flexors; L, lumbar; P, proximal; S, sacral; T, thoracic.

1	The initial symptoms of structural diseases of the cord that evolve over days or weeks are focal neck or back pain, followed by various combinations of paresthesias, sensory loss, motor weakness, and sphincter disturbance. There may be only mild sensory symptoms or a devastating functional transection of the cord. Partial lesions selectively involve the posterior columns or anterior spinothalamic tracts or are limited to one side of the cord. Paresthesias or numbness typically begins in the feet and ascend symmetrically or asymmetrically. These symptoms simulate a polyneuropathy, but a sharply demarcated spinal cord level indicates the myelopathic nature of the process. In severe and abrupt cases, areflexia reflecting spinal shock may be present, but hyperreflexia supervenes over days or weeks; persistent areflexic paralysis with a sensory level usually indicates necrosis over multiple segments of the spinal cord. APPROACH TO THE PATIENT:

1	The first priority is to exclude a treatable compression of the cord by a mass that may be amenable to treatment. The common causes are tumor, epidural abscess or hematoma, herniated disk, and vertebral pathology. Epidural compression due to malignancy or abscess often causes warning signs of neck or back pain, bladder disturbances, and sensory symptoms that precede the development of paralysis. Spinal subluxation, hemorrhage, and noncompressive etiologies such as infarction are more likely to produce myelopathy without antecedent symptoms. Magnetic resonance imaging (MRI) with gadolinium, centered on the clinically suspected level, is the initial diagnostic procedure if it is available; in some cases, it is appropriate to image the entire spine (cervical through sacral regions) to search for additional clinically inapparent lesions. Once compressive lesions have been excluded, noncompressive causes of acute myelopathy that are intrinsic to the cord are considered, primarily vascular,

1	for additional clinically inapparent lesions. Once compressive lesions have been excluded, noncompressive causes of acute myelopathy that are intrinsic to the cord are considered, primarily vascular, inflammatory, and infectious etiologies.

1	COMPRESSIVE MYELOPATHIES Neoplastic Spinal Cord Compression In adults, most neoplasms are epidural in origin, resulting from metastases to the adjacent vertebral column. The propensity of solid tumors to metastasize to the vertebral column probably reflects the high proportion of bone marrow located in the axial skeleton. Almost any malignant tumor can metastasize to the spinal column, with breast, lung, prostate, kidney, lymphoma, and myeloma being particularly frequent. The thoracic spinal column is most commonly involved; exceptions are metastases from prostate and ovarian cancer, which occur disproportionately in the sacral and lumbar vertebrae, probably from spread through Batson’s plexus, a network of veins along the anterior epidural space. Retroperitoneal neoplasms (especially lymphomas or sarcomas) enter the spinal canal laterally through the intervertebral foramina and produce radicular pain with signs of weakness that corresponds to the level of involved nerve roots.

1	Pain is usually the initial symptom of spinal metastasis; it may be aching and localized or sharp and radiating in quality and typically worsens with movement, coughing, or sneezing and characteristically awakens patients at night. A recent onset of persistent back pain, particularly if in the thoracic spine (which is uncommonly involved by spondylosis), should prompt consideration of vertebral metastasis. Rarely, pain is mild or absent. Plain radiographs of the spine and radionuclide bone scans have a limited role in diagnosis because they do not identify 15–20% of metastatic vertebral lesions and fail to detect paravertebral masses that reach the epidural space through the inter-vertebral foramina. MRI provides excellent anatomic resolution of the extent of spinal tumors (Fig. 456-2) and is able to distinguish between malignant lesions and other masses—epidural abscess, tuberculoma,

1	FIGURE 456-2 Epidural spinal cord compression due to breast carcinoma. Sagittal T1-weighted (A) and T2-weighted (B) magnetic resonance imaging scans through the cervicothoracic junction reveal an infiltrated and collapsed second thoracic vertebral body with posterior displacement and compression of the upper thoracic spinal cord. The low-intensity bone marrow signal in A signifies replacement by tumor. lipoma, or epidural hemorrhage, among others—that present in a similar fashion. Vertebral metastases are usually hypointense relative to a normal bone marrow signal on T1-weighted MRI; after the administration of gadolinium, contrast enhancement may deceptively “normalize” the appearance of the tumor by increasing its intensity to that of normal bone marrow. Infections of the spinal column (osteomyelitis and related disorders) are distinctive in that, unlike tumor, they often cross the disk space to involve the adjacent vertebral body.

1	If spinal cord compression is suspected, imaging should be obtained promptly. If there are radicular symptoms but no evidence of myelopathy, it may be safe to defer imaging for 24–48 h. Up to 40% of patients who present with cord compression at one level are found to have asymptomatic epidural metastases elsewhere; thus, the length of the spine is often imaged when epidural malignancy is in question.

1	Management of cord compression includes glucocorticoids to reduce cord edema, local radiotherapy (initiated as early as possible) to the symptomatic lesion, and specific therapy for the underlying tumor type. Glucocorticoids (dexamethasone, up to 40 mg daily) can be administered before an imaging study if there is clinical suspicion of cord compression and the medication is continued at a lower dose until definitive treatment with radiotherapy (generally 3000 cGy administered in 15 daily fractions) and/or surgical decompression is completed. In one randomized controlled trial, initial management with surgery followed by radiotherapy was more effective than radiotherapy alone for patients with a single area of spinal cord compression by extradural tumor; however, patients with recurrent cord compression, brain metastases, radiosensitive tumors, or severe motor symptoms of >48 h in duration were excluded from this study. Radiotherapy alone may be effective even for some typically

1	cord compression, brain metastases, radiosensitive tumors, or severe motor symptoms of >48 h in duration were excluded from this study. Radiotherapy alone may be effective even for some typically radio-resistant metastases. A good response to therapy can be expected in individuals who are ambulatory at presentation. Treatment usually prevents new weakness, and some recovery of motor function occurs in up to one-third of patients. Motor deficits (paraplegia or quadriplegia), once established for >12 h, do not usually improve, and beyond 48 h the prognosis for substantial motor recovery is poor. Although most patients do not experience recurrences in the months following radiotherapy, with survival beyond 2 years, recurrence becomes

1	Diseases of the Spinal Cord 2654 increasingly likely and can be managed with additional radiotherapy. Newer techniques such as stereotactic radiosurgery can deliver high doses of focused radiation and with similar rates of response compared to traditional radiotherapy. Biopsy of the epidural mass is unnecessary in patients with known primary cancer, but it is indicated if a history of underlying cancer is lacking. Surgery, either decompression by laminectomy or vertebral body resection, is also indicated when signs of cord compression worsen despite radiotherapy, when the maximum-tolerated dose of radiotherapy has been delivered previously to the site, or when a vertebral compression fracture or spinal instability contributes to cord compression.

1	In contrast to tumors of the epidural space, most intradural mass lesions are slow-growing and benign. Meningiomas and neurofibromas account for most of these, with occasional cases caused by chordoma, lipoma, dermoid, or sarcoma. Meningiomas (Fig. 456-3) are often located posterior to the thoracic cord or near the foramen magnum, although they can arise from the meninges anywhere along the spinal canal. Neurofibromas are benign tumors of the nerve sheath that typically arise from the posterior root; when multiple, neurofibromatosis is the likely etiology. Symptoms usually begin with radicular sensory symptoms followed by an asymmetric, progressive spinal cord syndrome. Therapy is surgical resection.

1	Primary intramedullary tumors of the spinal cord are uncommon. They present as central cord or hemicord syndromes, often in the cervical region. There may be poorly localized burning pain in the extremities and sparing of sacral sensation. In adults, these lesions are ependymomas, hemangioblastomas, or low-grade astrocytomas (Fig. 456-4). Complete resection of an intramedullary ependymoma is often possible with microsurgical techniques. Debulking of an intramedullary astrocytoma can also be helpful, as these are often slowly growing lesions; the value of adjunctive radiotherapy and chemotherapy is uncertain. Secondary (metastatic) intramedullary tumors also occur, especially in patients with advanced metastatic disease (Chap. 118), although these are not nearly as frequent as brain metastases.

1	FIGURE 456-3 Magnetic resonance imaging of a thoracic meningioma. Coronal T1-weighted postcontrast image through the thoracic spinal cord demonstrates intense and uniform enhancement of a well-circumscribed extramedullary mass (arrows) that displaces the spinal cord to the left. FIGURE 456-4 Magnetic resonance imaging of an intramedullary astrocytoma. Sagittal T1-weighted postcontrast image through the cervical spine demonstrates expansion of the upper cervical spine by a mass lesion emanating from within the spinal cord at the cervicomedullary junction. Irregular peripheral enhancement occurs within the mass (arrows).

1	Spinal Epidural Abscess Spinal epidural abscess presents with midline back or neck pain, fever, and progressive limb weakness. Prompt recognition of this distinctive process may prevent permanent sequelae. Aching pain is almost always present, either over the spine or in a radicular pattern. The duration of pain prior to presentation is generally ≤2 weeks but may on occasion be several months or longer. Fever is typically but not invariably present, accompanied by elevated white blood cell count, sedimentation rate, and C-reactive protein. As the abscess expands, further spinal cord damage results from venous congestion and thrombosis. Once weakness and other signs of myelopathy appear, progression may be rapid and irreversible. A more chronic sterile granulomatous form of abscess is also known, usually after treatment of an acute epidural infection.

1	Risk factors include an impaired immune status (HIV, diabetes mellitus, renal failure, alcoholism, malignancy), intravenous drug abuse, and infections of the skin or other tissues. Two-thirds of epidural infections result from hematogenous spread of bacteria from the skin (furunculosis), soft tissue (pharyngeal or dental abscesses; sinusitis), or deep viscera (bacterial endocarditis). The remainder arises from direct extension of a local infection to the subdural space; examples of local predisposing conditions are vertebral osteomyelitis, decubitus ulcers, lumbar puncture, epidural anesthesia, or spinal surgery. Most cases are due to Staphylococcus aureus; gram-negative bacilli, Streptococcus, anaerobes, and fungi can also cause epidural abscesses. Tuberculosis from an adjacent vertebral source (Pott’s disease) remains an important cause in the developing world.

1	MRI (Fig. 456-5) localizes the abscess and excludes other causes of myelopathy. Blood cultures are positive in more than half of cases, but direct aspiration of the abscess at surgery is often required for a micro-biologic diagnosis. Lumbar puncture is only required if encephalopathy or other clinical signs raise the question of associated meningitis, a feature that is found in <25% of cases. The level of the puncture should be planned to minimize the risk of meningitis due to passage of the needle through infected tissue. A high cervical tap is sometimes the safest approach. Cerebrospinal fluid (CSF) abnormalities in epidural and subdural abscess consist of pleocytosis with a preponderance of polymorphonuclear cells, an elevated protein level, and a reduced glucose level, but the responsible organism is not cultured unless there is associated meningitis.

1	FIGURE 456-5 Magnetic resonance (MR) imaging of a spinal epidural abscess due to tuberculosis. A. Sagittal T2-weighted free spin-echo MR sequence. A hypointense mass replaces the posterior elements of C3 and extends epidurally to compress the spinal cord (arrows). B. Sagittal T1-weighted image after contrast administration reveals a diffuse enhancement of the epidural process (arrows) with extension into the epidural space.

1	Treatment is by decompressive laminectomy with debridement combined with long-term antibiotic treatment. Surgical evacuation prevents development of paralysis and may improve or reverse paralysis in evolution, but it is unlikely to improve deficits of more than several days in duration. Broad-spectrum antibiotics should be started empirically before surgery and then modified on the basis of culture results; medication is generally continued for at least 6 weeks. If surgery is contraindicated or if there is a fixed paraplegia or quadriplegia that is unlikely to improve following surgery, longterm administration of systemic and oral antibiotics can be used; in such cases, the choice of antibiotics may be guided by results of blood cultures. Surgical management remains the treatment of choice unless the abscess is limited in size and causes few or no neurologic signs.

1	With prompt diagnosis and treatment of spinal epidural abscess, up to two-thirds of patients experience significant recovery. Spinal Epidural Hematoma Hemorrhage into the epidural (or subdural) space causes acute focal or radicular pain followed by variable signs of a spinal cord or conus medullaris disorder. Therapeutic anticoagulation, trauma, tumor, or blood dyscrasias are predisposing conditions. Rare cases complicate lumbar puncture or epidural anesthesia. MRI and computed tomography (CT) confirm the clinical suspicion and can delineate the extent of the bleeding. Treatment consists of prompt reversal of any underlying clotting disorder and surgical decompression. Surgery may be followed by substantial recovery, especially in patients with some preservation of motor function preoperatively. Because of the risk of hemorrhage, lumbar puncture should be avoided whenever possible in patients with severe thrombocytopenia or other coagulopathies.

1	Hematomyelia Hemorrhage into the substance of the spinal cord is a rare result of trauma, intraparenchymal vascular malformation (see below), vasculitis due to polyarteritis nodosa or systemic lupus erythematosus (SLE), bleeding disorders, or a spinal cord neoplasm. Hematomyelia presents as an acute painful transverse myelopathy. With large lesions, extension into the subarachnoid space results in subarachnoid hemorrhage (Chap. 330). Diagnosis is by MRI or CT. evaLuatioN of aCute traNsverse myeLopathy 1. MRI of spinal cord with and without contrast (exclude compressive causes). 2. CSF studies: Cell count, protein, glucose, IgG index/synthesis rate, oligoclonal bands, VDRL; Gram’s stain, acid-fast bacilli, and India ink stains; PCR for VZV, HSV-2, HSV-1, EBV, CMV, HHV-6, enteroviruses, HIV; antibody for HTLV-1, Borrelia burgdorferi, Mycoplasma pneumoniae, and Chlamydia pneumoniae; viral, bacterial, mycobacterial, and fungal cultures. 3.

1	3. Blood studies for infection: HIV; RPR; IgG and IgM enterovirus antibody; IgM mumps, measles, rubella, group B arbovirus, Brucella melitensis, Chlamydia psittaci, Bartonella henselae, schistosomal antibody; cultures for B. melitensis. Also consider nasal/pharyngeal/anal cultures for enteroviruses; stool O&P for Schistosoma ova. 4. Immune-mediated disorders: ESR; ANA; ENA; dsDNA; rheumatoid factor; anti-SSA; anti-SSB, complement levels; antiphospholipid and anticardiolipin antibodies; p-ANCA; antimicrosomal and antithyroglobulin antibodies; if Sjögren’s syndrome suspected, Schirmer test, salivary gland scintigraphy, and salivary/lacrimal gland biopsy. 5. Sarcoidosis: Serum angiotensin-converting enzyme; serum Ca; 24-h urine Ca; chest x-ray; chest CT; total-body gallium scan; lymph node biopsy. 6. Demyelinating disease: Brain MRI scan, evoked potentials, CSF oligoclonal bands, neuromyelitis optica antibody (anti-aquaporin-4 [NMO] antibody). 7.

1	6. Demyelinating disease: Brain MRI scan, evoked potentials, CSF oligoclonal bands, neuromyelitis optica antibody (anti-aquaporin-4 [NMO] antibody). 7. Vascular causes: MRI, CT myelogram; spinal angiogram. Abbreviations: ANA, antinuclear antibodies; CMV, cytomegalovirus; CSF, cerebrospinal fluid; CT, computed tomography; EBV, Epstein-Barr virus; ENA, epithelial neutrophil-activating peptide; ESR, erythrocyte sedimentation rate; HHV, human herpes virus; HSV, herpes simplex virus; HTLV, human T cell leukemia/lymphoma virus; MRI, magnetic resonance imaging; O&P, ova and parasites; p-ANCA, perinuclear antineutrophilic cytoplasmic antibodies; PCR, polymerase chain reaction; RPR, rapid plasma reagin (test); VDRL, Venereal Disease Research Laboratory; VZV, varicella-zoster virus.

1	Therapy is supportive, and surgical intervention is generally not useful. An exception is hematomyelia due to an underlying vascular malformation, for which spinal angiography and endovascular occlusion may be indicated, or surgery to evacuate the clot and remove the underlying vascular lesion. The most frequent causes of noncompressive acute transverse myelopathy are spinal cord infarction; systemic inflammatory disorders, including SLE and sarcoidosis; demyelinating diseases, including multiple sclerosis (MS); neuromyelitis optica (NMO); postinfectious or idiopathic transverse myelitis, which is presumed to be an immune condition related to acute disseminated encephalomyelitis (Chap. 458); and infectious (primarily viral) causes. After spinal cord compression is excluded, the evaluation generally requires a lumbar puncture and a search for underlying systemic disease (Table 456-3).

1	Spinal Cord Infarction The cord is supplied by three arteries that course vertically over its surface: a single anterior spinal artery and paired posterior spinal arteries. The anterior spinal artery originates in paired branches of the vertebral arteries at the cranciocervical junction and is fed by additional radicular vessels that arise at C6, at an upper thoracic level, and, most consistently, at T11-L2 (artery of Adamkiewicz). At each spinal cord segment, paired penetrating vessels branch from the anterior spinal artery to supply the anterior two-thirds of the cord; the posterior spinal arteries, which often become less distinct below the midthoracic level, supply the posterior columns.

1	Spinal cord ischemia can occur at any level; however, the presence of the artery of Adamkiewicz below, and the anterior spinal artery circulation above, creates a region of marginal blood flow in the upper thoracic segments. With hypotension or cross-clamping of the aorta, cord infarction typically occurs at the level of T3-T4, and also at boundary zones between the anterior and posterior spinal artery territories. The latter may result in a rapidly progressive syndrome over hours of weakness and spasticity with little sensory change. Acute infarction in the territory of the anterior spinal artery produces paraplegia or quadriplegia, dissociated sensory loss affecting pain and temperature sense but sparing vibration and position sense, and loss of sphincter control (“anterior cord syndrome”). Onset may be sudden but more typically is progressive over minutes or a few hours, quite unlike stroke in the cerebral hemispheres. Sharp midline

1	Diseases of the Spinal Cord 2656 or radiating back pain localized to the area of ischemia is frequent. Areflexia due to spinal shock is often present initially; with time, hyperreflexia and spasticity appear. Less common is infarction in the territory of the posterior spinal arteries, resulting in loss of posterior column function either on one side or bilaterally. Causes of spinal cord infarction include aortic atherosclerosis, dissecting aortic aneurysm, vertebral artery occlusion or dissection in the neck, aortic surgery, or profound hypotension from any cause. A “surfer’s myelopathy” in the cervical region is probably vascular in origin. Cardiogenic emboli, vasculitis (Chap. 385), and collagen vascular disease (particularly SLE [Chap. 378], Sjögren’s syndrome [Chap. 383], and the antiphospholipid antibody syndrome [Chap. 379]) are other etiologies. Occasional cases develop from embolism of nucleus pulposus material into spinal vessels, usually from local spine trauma. In a

1	antiphospholipid antibody syndrome [Chap. 379]) are other etiologies. Occasional cases develop from embolism of nucleus pulposus material into spinal vessels, usually from local spine trauma. In a substantial number of cases, no cause can be found, and thromboembolism in arterial feeders is suspected. MRI may fail to demonstrate infarctions of the cord, especially in the first day, but often the imaging becomes abnormal at the affected level. In cord infarction due to presumed thromboembolism, acute anticoagulation is not indicated, with the possible exception of the unusual transient ischemic attack or incomplete infarction with a stuttering or progressive course. The antiphospholipid antibody syndrome is treated with anticoagulation (Chap. 379). Lumbar drainage of spinal fluid has reportedly been successful in some cases of cord infarction and has been used prophylactically during aortic surgery, but it has not been studied systematically.

1	Inflammatory and Immune Myelopathies (Myelitis) This broad category includes the demyelinating conditions MS, NMO, and postinfectious myelitis, as well as sarcoidosis and systemic autoimmune disease. In approximately one-quarter of cases of myelitis, no underlying cause can be identified. Some will later manifest additional symptoms of an immune-mediated disease. Recurrent episodes of myelitis are usually due to one of the immune-mediated diseases or to infection with herpes simplex virus (HSV) type 2 (below).

1	multiple ScleroSiS MS may present with acute myelitis, particularly in individuals of Asian or African ancestry. In Caucasians, MS attacks rarely cause a transverse myelopathy (i.e., attacks of bilateral sensory disturbances, unilateral or bilateral weakness, and bladder or bowel symptoms), but it is among the most common causes of a partial cord syndrome. MRI findings in MS-associated myelitis typically consist of mild swelling of the cord and diffuse or multifocal “shoddy” areas of abnormal signal on T2-weighted sequences. Contrast enhancement, indicating disruption in the blood-brain barrier associated with inflammation, is present in many acute cases. A brain MRI is most helpful in gauging the likelihood that a case of myelitis represents an initial attack of MS. A normal scan indicates that the risk of evolution to MS is low, ~10–15% over 5 years; in contrast, the finding of multiple periventricular T2-bright lesions indicates a much higher risk, >50% over 5 years and >90% by 14

1	that the risk of evolution to MS is low, ~10–15% over 5 years; in contrast, the finding of multiple periventricular T2-bright lesions indicates a much higher risk, >50% over 5 years and >90% by 14 years. The CSF may be normal, but more often there is a mild mononuclear cell pleocytosis, with normal or mildly elevated CSF protein levels; the presence of oligoclonal bands is variable, but when they are found, a diagnosis of MS is more likely.

1	There are no adequate trials of therapy for MS-associated transverse myelitis. Intravenous methylprednisolone (500 mg qd for 3 days) followed by oral prednisone (1 mg/kg per day for several weeks, then gradual taper) has been used as initial treatment. A course of plasma exchange may be indicated for severe cases if glucocorticoids are ineffective. MS is discussed in Chap. 458.

1	neuromyelitiS opticA NMO is an immune-mediated demyelinating disorder consisting of a severe myelopathy that is typically longitudinally extensive, meaning that the lesion spans three or more vertebral segments. NMO is associated with optic neuritis that is often bilateral and may precede or follow myelitis by weeks or months, and also by brainstem and, in some cases, hypothalamic involvement. Recurrent myelitis without optic nerve involvemement can also occur in NMO; affected individuals are usually female and often of Asian ancestry. CSF studies reveal a variable mononuclear pleocytosis of up to several hundred cells per microliter; unlike MS, oligoclonal bands are generally absent. Diagnostic serum autoantibodies against the water channel protein aquaporin-4 are present in 60–70% of patients with NMO. NMO has also been associated with SLE and antiphospholipid antibodies (see below) as well as with other systemic autoimmune diseases; rare cases are paraneoplastic in origin.

1	of patients with NMO. NMO has also been associated with SLE and antiphospholipid antibodies (see below) as well as with other systemic autoimmune diseases; rare cases are paraneoplastic in origin. Treatment is with glucocorticoids and, for refractory cases, plasma exchange (as for MS, above). Preliminary studies suggest that treatment with azathioprine, mycophenylate, or anti-CD20 (anti–B cell) monoclonal antibody may protect against subsequent relapses; treatment for 5 years or longer is generally recommended. NMO is discussed in Chap. 458.

1	SyStemic immune-meDiAteD DiSorDerS Myelitis occurs in a small number of patients with SLE, many cases of which are associated with antibodies to antiphospholipids and/or to aquaporin-4. Patients with aquaporin-4 antibodies are likely to have longitudinally extensive myelitis by MRI, are considered to have an NMO-spectrum disorder, and are at high risk of developing future episodes of myelitis and/or optic neuritis. The CSF in SLE myelitis is usually normal or shows a mild lymphocytic pleocytosis; oligoclonal bands are a variable finding. Although there are no systematic trials of therapy for SLE myelitis, based on limited data, high-dose glucocorticoids followed by cyclophosphamide have been recommended. Acute severe episodes of transverse myelitis that do not initially respond to glucocorticoids are often treated with a course of plasma exchange. Sjögren’s syndrome (Chap. 383) can also be associated with NMO spectrum disorder and also with cases of acute transverse or chronic

1	glucocorticoids are often treated with a course of plasma exchange. Sjögren’s syndrome (Chap. 383) can also be associated with NMO spectrum disorder and also with cases of acute transverse or chronic progressive myelopathy. Other immune-mediated myelitides include antiphospholipid antibody syndrome (Chap. 379), mixed connective tissue disease (Chap. 382), Behçet’s syndrome (Chap. 387), and vasculitis related to polyarteritis nodosa, perinuclear antineutrophilic cytoplasmic (p-ANCA) antibodies, or primary central nervous system vasculitis (Chap. 385).

1	Another important consideration in this group is sarcoid myelopathy that may present as a slowly progressive or relapsing disorder. MRI reveals an edematous swelling of the spinal cord that may mimic tumor; there is almost always gadolinium enhancement of active lesions and in some cases nodular enhancement of the adjacent surface of the cord; lesions may be single or multiple, and on axial images, enhancement of the central cord is usually present. The typical CSF profile consists of a mild lymphocytic pleocytosis and mildly elevated protein level; in a minority of cases, reduced glucose and oligoclonal bands are found. The diagnosis is particularly difficult when systemic manifestations of sarcoid are minor or absent (nearly 50% of cases) or when other typical neurologic manifestations of the disease—such as cranial neuropathy, hypothalamic involvement, or meningeal enhancement visualized by MRI—are lacking. A slit-lamp examination of the eye to search for uveitis, chest x-ray and

1	of the disease—such as cranial neuropathy, hypothalamic involvement, or meningeal enhancement visualized by MRI—are lacking. A slit-lamp examination of the eye to search for uveitis, chest x-ray and CT to assess pulmonary involvement and mediastinal lymphadenopathy, serum or CSF angiotensin-converting enzyme (ACE; CSF values elevated in only a minority of cases), serum calcium, and a gallium scan may assist in the diagnosis. The usefulness of spinal fluid ACE is uncertain. Initial treatment is with oral glucocorticoids; immunosuppressant drugs, including the tumor necrosis factor α inhibitor infliximab, have been used for resistant cases. Sarcoidosis is discussed in Chap. 390.

1	poStinfectiouS myelitiS Many cases of myelitis, termed postinfectious or postvaccinal, follow an infection or vaccination. Numerous organisms have been implicated, including Epstein-Barr virus (EBV), cytomegalovirus (CMV), mycoplasma, influenza, measles, varicella, rubeola, and mumps. As in the related disorder acute disseminated encephalomyelitis (Chap. 458), postinfectious myelitis often begins as the patient appears to be recovering from an acute febrile infection, or in the subsequent days or weeks, but an infectious agent cannot be isolated from the nervous system or CSF. The presumption is that the myelitis represents an autoimmune disorder triggered by infection and is not due to direct infection of the spinal cord. No randomized controlled trials of therapy exist; treatment is usually with glucocorticoids or, in fulminant cases, plasma exchange.

1	Acute infectiouS myelitiS Many viruses have been associated with an acute myelitis that is infectious in nature rather than postinfectious. Nonetheless, the two processes are often difficult to distinguish. Herpes zoster is the best characterized viral myelitis, but HSV types 1 and 2, EBV, CMV, and rabies virus are other well-described causes. HSV-2 (and less commonly HSV-1) produces a distinctive syndrome of recurrent sacral cauda equina neuritis in association with outbreaks of genital herpes (Elsberg’s syndrome). Poliomyelitis is the prototypic viral myelitis, but it is more or less restricted to the anterior gray matter of the cord containing the spinal motoneurons. A polio-like syndrome can also be caused by a large number of enteroviruses (including enterovirus 71 and coxsackie), and with West Nile virus and other flaviviruses. Recently, cases of paralysis in children and adolescents were associated with enterovirus D-68 infection but a causal role for this virus has not been

1	and with West Nile virus and other flaviviruses. Recently, cases of paralysis in children and adolescents were associated with enterovirus D-68 infection but a causal role for this virus has not been established. Chronic viral myelitic infections, such as those due to HIV or human T cell lymphotropic virus type 1 (HTLV-1), are discussed below.

1	Bacterial and mycobacterial myelitis (most are essentially abscesses) are less common than viral causes and much less frequent than cerebral bacterial abscess. Almost any pathogenic species may be responsible, including Borrelia burgdorferi (Lyme disease), Listeria monocytogenes, Mycobacterium tuberculosis, and Treponema pallidum (syphilis). Mycoplasma pneumoniae may be a cause of myelitis, but its status is uncertain because many cases are more properly classified as postinfectious. Schistosomiasis (Chap. 259) is an important cause of parasitic myelitis in endemic areas. The process is intensely inflammatory and granulomatous, caused by a local response to tissue-digesting enzymes from the ova of the parasite, typically Schistosoma mansoni. Toxoplasmosis (Chap. 253) can occasionally cause a focal myelopathy, and this diagnosis should especially be considered in patients with AIDS (Chap. 226).

1	In cases of suspected viral myelitis, it may be appropriate to begin specific therapy pending laboratory confirmation. Herpes zoster, HSV, and EBV myelitis are treated with intravenous acyclovir (10 mg/kg q8h) or oral valacyclovir (2 g tid) for 10–14 days; CMV is treated with ganciclovir (5 mg/kg IV bid) plus foscarnet (60 mg/kg IV tid) or cidofovir (5 mg/kg per week for 2 weeks). High-Voltage Electrical Injury Spinal cord injuries are prominent following electrocution from lightning strikes or other accidental electrical exposures. The syndrome consists of transient weakness acutely (often with an altered sensorium and focal cerebral disturbances), followed several days or even weeks later by a myelopathy that can be severe and permanent. This is a rare injury type, and limited data incriminate a vascular pathology involving the anterior spinal artery and its branches in some cases. Therapy is supportive.

1	Spondylotic myelopathy is one of the most common causes of chronic cord compression and of gait difficulty in the elderly. Neck and shoulder pain with stiffness are early symptoms; impingement of bone and soft tissue overgrowth on nerve roots results in radicular arm pain, most often in a C5 or C6 distribution. Compression of the cervical cord, which occurs in fewer than one-third of cases, produces a slowly progressive spastic paraparesis, at times asymmetric and often accompanied by paresthesias in the feet and hands. Vibratory sense is diminished in the legs, there is a Romberg sign, and occasionally there is a sensory level for vibration or pinprick on the upper thorax. In some cases, coughing or straining produces leg weakness or radiating arm or shoulder pain. Dermatomal sensory loss in the arms, atrophy of intrinsic hand muscles, increased deep-tendon reflexes in the legs, and extensor plantar responses are common. Urinary urgency or incontinence occurs in advanced cases, but

1	in the arms, atrophy of intrinsic hand muscles, increased deep-tendon reflexes in the legs, and extensor plantar responses are common. Urinary urgency or incontinence occurs in advanced cases, but there are many alternative causes of these problems in older individuals. A tendon reflex in the arms is often diminished at some level; most often at the biceps (C5-C6). In individual cases, radicular, myelopathic, or combined signs may predominate. The diagnosis should be considered in appropriate cases of progressive cervical myelopathy, paresthesias of the feet and hands, or wasting of the hands.

1	Diagnosis is usually made by MRI and may be suspected from CT images; plain x-rays are less helpful. Extrinsic cord compression and deformation are appreciated on axial MRI views, and T2-weighted 2657 sequences may reveal areas of high signal intensity within the cord adjacent to the site of compression. A cervical collar may be helpful in milder cases, but definitive therapy consists of surgical decompression. Posterior laminectomy or an anterior approach with resection of the protruded disk and bony material may be required. Cervical spondylosis and related degenerative diseases of the spine are discussed in Chap. 22.

1	Vascular malformations of the cord and overlying dura are treatable causes of progressive myelopathy. Most common are fistulas located within the dura or posteriorly along the surface of the cord. Most dural arteriovenous (AV) fistulas are located at or below the midthoracic level, usually consisting of a direct connection between a radicular feeding artery in the nerve root sleeve with dural veins. The typical presentation is a middle-aged man with a progressive myelopathy that worsens slowly or intermittently and may have periods of remission, sometimes mimicking MS. Acute deterioration due to hemorrhage into the spinal cord (hematomyelia) or subarachnoid space may also occur but is rare. A saltatory progression is most common and appears to be the result of local ischemia and edema from venous congestion. Most patients have incomplete sensory, motor, and bladder disturbances. The motor disorder may predominate and produce a mixture of upper and restricted lower motor neuron signs,

1	venous congestion. Most patients have incomplete sensory, motor, and bladder disturbances. The motor disorder may predominate and produce a mixture of upper and restricted lower motor neuron signs, simulating amyotrophic lateral sclerosis (ALS). Pain over the dorsal spine, dysesthesias, or radicular pain may be present. Other symptoms suggestive of AV malformation (AVM) or dural fistula include intermittent claudication; symptoms that change with posture, exertion, Valsalva maneuver, or menses; and fever.

1	Less commonly, AVM disorders are intramedullary rather than dural. One unusual disorder is a progressive thoracic myelopathy with paraparesis developing over weeks or months, characterized pathologically by abnormally thick, hyalinized vessels within the cord (subacute necrotic myelopathy, or Foix-Alajouanine syndrome).

1	Spinal bruits are infrequent but may be sought at rest and after exercise in suspected cases. A vascular nevus on the overlying skin may indicate an underlying vascular malformation as occurs with Klippel-Trenaunay-Weber syndrome. High-resolution MRI with contrast administration detects the draining vessels of many but not all AVMs (Fig. 456-6). An uncertain proportion may be visualized by CT myelography as enlarged vessels along the surface of the cord. Definitive diagnosis requires selective spinal angiography, which defines the feeding vessels and the extent of the malformation. Endovascular embolization of the major feeding vessels may stabilize a progressive neurologic deficit or allow for gradual recovery. Some lesions, especially small dural fistulas, can be resected surgically.

1	The myelopathy associated with HTLV-1, formerly called tropical spastic paraparesis, is a slowly progressive spastic syndrome with variable sensory and bladder disturbance. Approximately half of patients have mild back or leg pain. The neurologic signs may be asymmetric, often lacking a well-defined sensory level; the only sign in the arms may be hyperreflexia after several years of illness. The onset is insidious, and the illness is slowly progressive at a variable rate; most patients are unable to walk within 10 years of onset. This presentation may resemble primary progressive MS or a thoracic AVM. Diagnosis is made by demonstration of HTLV-1-specific antibody in serum by enzyme-linked immunosorbent assay (ELISA), confirmed by radioimmunoprecipitation or Western blot analysis. Especially in endemic areas, a finding of HTLV-1 seropositivity in a patient with myelopathy does not necessarily prove that HTLV-1 is causative. The CSF/serum antibody index may provide support by

1	Especially in endemic areas, a finding of HTLV-1 seropositivity in a patient with myelopathy does not necessarily prove that HTLV-1 is causative. The CSF/serum antibody index may provide support by establishing intrathecal synthesis of antibodies favoring HTVL-1 myelopathy over asymptomatic carriage. Measuring proviral DNA by polymerase chain reaction (PCR) in serum and CSF cells can be useful as an ancillary part of diagnosis, because proviral DNA levels may be higher in patients with myelopathy. The myelopathy appears to result from an

1	Diseases of the Spinal Cord FIGURE 456-6 Arteriovenous malformation. Sagittal magnetic resonance scans of the thoracic spinal cord: T2 fast spin-echo technique (left) and T1 postcontrast image (right). On the T2-weighted image (left), abnormally high signal intensity is noted in the central aspect of the spinal cord (arrowheads). Numerous punctate flow voids indent the dorsal and ventral spinal cord (arrow). These represent the abnormally dilated venous plexus supplied by a dural arteriovenous fistula. After contrast administration (right), multiple, serpentine, enhancing veins (arrows) on the ventral and dorsal aspect of the thoracic spinal cord are visualized, diagnostic of arteriovenous malformation. This patient was a 54-year-old man with a 4-year history of progressive paraparesis.

1	immune-mediated attack on the spinal cord rather than the result of direct viral infection. There is no effective treatment, but symptomatic therapy for spasticity and bladder symptoms may be helpful. A progressive myelopathy may also result from HIV infection (Chap. 226). It is characterized by vacuolar degeneration of the posterior and lateral tracts, resembling subacute combined degeneration (see below).

1	Syringomyelia is a developmental cavity of the cervical cord that may enlarge and produce progressive myelopathy or may remain asymptomatic. Symptoms begin insidiously in adolescence or early adulthood, progress irregularly, and may undergo spontaneous arrest for several years. Many young patients acquire a cervical-thoracic scoliosis. More than half of all cases are associated with Chiari type 1 malformations in which the cerebellar tonsils protrude through the foramen magnum and into the cervical spinal canal. The pathophysiology of syrinx expansion is controversial, but some interference with the normal flow of CSF seems likely, perhaps by the Chiari malformation. Acquired cavitations of the cord in areas of necrosis are also termed syrinx cavities; these follow trauma, myelitis, necrotic spinal cord tumors, and chronic arachnoiditis due to tuberculosis and other etiologies.

1	The presentation is a central cord syndrome consisting of a regional dissociated sensory loss (loss of pain and temperature sensation with sparing of touch and vibration) and areflexic weakness in the upper limbs. The sensory deficit has a distribution that is “suspended” over the nape of the neck, shoulders, and upper arms (cape distribution) or in the hands. Most cases begin asymmetrically with unilateral sensory loss in the hands that leads to injuries and burns that are not appreciated by the patient. Muscle wasting in the lower neck, shoulders, arms, and hands with asymmetric or absent reflexes in the arms reflects expansion of the cavity in the gray matter of the cord. As the cavity

1	FIGURE 456-7 Magnetic resonance imaging of syringomyelia associated with a Chiari malformation. Sagittal T1-weighted image through the cervical and upper thoracic spine demonstrates descent of the cerebellar tonsils below the level of the foramen magnum (black arrows). Within the substance of the cervical and thoracic spinal cord, a cerebrospinal fluid collection dilates the central canal (white arrows).

1	enlarges and compresses the long tracts, spasticity and weakness of the legs, bladder and bowel dysfunction, and a Horner’s syndrome appear. Some patients develop facial numbness and sensory loss from damage to the descending tract of the trigeminal nerve (C2 level or above). In cases with Chiari malformations, cough-induced headache and neck, arm, or facial pain may be reported. Extension of the syrinx into the medulla, syringobulbia, causes palatal or vocal cord paralysis, dysarthria, horizontal or vertical nystagmus, episodic dizziness or vertigo, and tongue weakness with atrophy. MRI accurately identifies developmental and acquired syrinx cavities and their associated spinal cord enlargement (Fig. 456-7). Images of the brain and the entire spinal cord should be obtained to delineate the full longitudinal extent of the syrinx, assess posterior fossa structures for the Chiari malformation, and determine whether hydrocephalus is present.

1	Treatment of syringomyelia is generally unsatisfactory. The Chiari tonsillar herniation may be decompressed, generally by suboccipital craniectomy, upper cervical laminectomy, and placement of a dural graft. Fourth ventricular outflow is reestablished by this procedure. If the syrinx cavity is large, some surgeons recommend direct decompression or drainage by one of a number of methods, but the added benefit of this procedure is uncertain, and complications are common. With Chiari malformations, shunting of hydrocephalus generally precedes any attempt to correct the syrinx. Surgery may stabilize the neurologic deficit, and some patients improve. Patients with few symptoms and signs from the syrinx do not require surgery and are followed by serial clinical and imaging examinations.

1	Syrinx cavities secondary to trauma or infection, if symptomatic, are treated with a decompression and drainage procedure in which a small shunt is inserted between the cavity and subarachnoid space; alternatively, the cavity can be fenestrated. Cases due to intramedullary spinal cord tumor are generally managed by resection of the tumor.

1	A chronic progressive myelopathy is the most frequent cause of disability in both primary progressive and secondary progressive forms of MS. Involvement is typically bilateral but asymmetric and produces motor, sensory, and bladder/bowel disturbances. Fixed motor disability appears to result from extensive loss of axons in the corticospinal tracts. Diagnosis is facilitated by identification of earlier attacks such as optic neuritis. MRI, CSF, and evoked response testing are confirmatory. Disease-modifying therapy is indicated for patients with progressive myelopathy who also have coexisting MS relapses. Therapy is sometimes offered to patients who have a progressive course without relapses but with “active” MRI scans (e.g., the presence of new focal demyelinating lesions) despite the lack of evidence supporting the value of treatment in this setting. MS is discussed in Chap. 458.

1	This treatable myelopathy presents with subacute paresthesias in the hands and feet, loss of vibration and position sensation, and a progressive spastic and ataxic weakness. Loss of reflexes due to an associated peripheral neuropathy in a patient who also has Babinski signs is an important diagnostic clue. Optic atrophy and irritability or other cognitive changes may be prominent in advanced cases and are occasionally the presenting symptoms. The myelopathy of subacute combined degeneration tends to be diffuse rather than focal; signs are generally symmetric and reflect predominant involvement of the posterior and lateral tracts, including Romberg’s sign. The diagnosis is confirmed by the finding of macrocytic red blood cells, a low serum B12 concentration, elevated serum levels of homocysteine and methylmalonic acid, and in uncertain cases, testing for anti–parietal cell antibodies and a Schilling test. Treatment is by replacement therapy, beginning with 1000 μg of intramuscular

1	and methylmalonic acid, and in uncertain cases, testing for anti–parietal cell antibodies and a Schilling test. Treatment is by replacement therapy, beginning with 1000 μg of intramuscular vitamin B12 repeated at regular intervals or by subsequent oral treatment (Chap. 128).

1	This myelopathy is similar to subacute combined degeneration (described above), except there is no neuropathy, and explains cases with normal serum levels of B12. Low levels of serum copper are found, and often there is also a low level of serum ceruloplasmin. Some cases follow gastrointestinal procedures, particularly bariatric surgery, that result in impaired copper absorption; others have been associated with excess zinc from health food supplements or, until recently, zinc-containing denture creams, all of which impair copper absorption via induction of metallothionein, a copper-binding protein. Many cases are idiopathic. Improvement or at least stabilization may be expected with reconstitution of copper stores by oral supplementation. There is microcytic or macrocytic anemia. The pathophysiology and pathology of the idiopathic form are not known.

1	The classic syphilitic syndromes of tabes dorsalis and meningovascular inflammation of the spinal cord are now less frequent than in the past but must be considered in the differential diagnosis of spinal cord disorders. The characteristic symptoms of tabes are fleeting and repetitive lancinating pains, primarily in the legs or less often in the back, thorax, abdomen, arms, and face. Ataxia of the legs and gait due to loss of position sense occurs in half of patients. Paresthesias, bladder disturbances, and acute abdominal pain with vomiting (visceral crisis) occur in 15–30% of patients. The cardinal signs of tabes are loss of reflexes in the legs; impaired position and vibratory sense; Romberg’s sign; and, in almost all cases, bilateral Argyll Robertson pupils, which fail to constrict to light but accommodate. Diabetic polyradiculopathy may simulate tabes.

1	Many cases of slowly progressive myelopathy are genetic in origin (Chap. 452). More than 30 different causative loci have been identified, including autosomal dominant, autosomal recessive, and X-linked forms. Especially for the recessive and X-linked forms, a family history of myelopathy may be lacking. Most patients present 2659 with almost imperceptibly progressive spasticity and weakness in the legs, usually but not always symmetrical. Sensory symptoms and signs are absent or mild, but sphincter disturbances may be present. In some families, additional neurologic signs are prominent, including nystagmus, ataxia, or optic atrophy. The onset may be as early as the first year of life or as late as middle adulthood. Only symptomatic therapies are available.

1	This X-linked disorder is a variant of adrenoleukodystrophy. Most affected males have a history of adrenal insufficiency and then develop a progressive spastic (or ataxic) paraparesis beginning in early or sometimes middle adulthood; some patients also have a mild peripheral neuropathy. Female heterozygotes may develop a slower, insidiously progressive spastic myelopathy beginning later in adulthood and without adrenal insufficiency. Diagnosis is usually made by demonstration of elevated levels of very-long-chain fatty acids in plasma and in cultured fibroblasts. The responsible gene encodes the adrenoleukodystrophy protein (ADLP), a peroxisomal membrane transporter involved in carrying long-chain fatty acids to peroxisomes for degradation. Corticosteroid replacement is indicated if hypoadrenalism is present, and bone marrow transplantation and nutritional supplements have been attempted for this condition without clear evidence of efficacy.

1	Primary lateral sclerosis (Chap. 452) is a degenerative disorder characterized by progressive spasticity with weakness, eventually accompanied by dysarthria and dysphonia; bladder symptoms occur in approximately half of patients. Sensory function is spared. The disorder resembles ALS and is considered a variant of the motor neuron degenerations, but without the characteristic lower motor neuron disturbance. Some cases may represent familial spastic paraplegia, particularly autosomal recessive or X-linked varieties in which a family history may be absent.

1	Tethered cord syndrome is a developmental disorder of the lower spinal cord and nerve roots that rarely presents in adulthood as low back pain accompanied by a progressive lower spinal cord and/or nerve root syndrome. Some patients have a small leg or foot deformity indicating a long-standing process, and in others, a dimple, patch of hair, or sinus tract on the skin overlying the lower back is the clue to a congenital lesion. Diagnosis is made by MRI, which demonstrates a low-lying conus medullaris and thickened filum terminale. The MRI may also reveal diastematomyelia (division of the lower spinal cord into two halves), lipomas, cysts, or other congenital abnormalities of the lower spine coexisting with the tethered cord. Treatment is with surgical release.

1	There are a number of rare toxic causes of spastic myelopathy, including lathyrism due to ingestion of chickpeas containing the excitotoxin β-N-oxalylamino-l-alanine (BOAA), seen primarily in the developing world, and nitrous oxide inhalation producing a myelopathy identical to subacute combined degeneration. SLE, Sjögren’s syndrome, and sarcoidosis may each cause a myelopathy without overt evidence of systemic disease. Cancer-related causes of chronic myelopathy, besides the common neoplastic compressive myelopathy discussed earlier, include radiation injury (Chap. 118) and rare paraneoplastic myelopathies. The last of these are most often associated with lung or breast cancer and anti-Hu antibodies (Chap. 122) or with lymphoma that causes a syndrome of destruction of anterior horn cells; NMO (Chap. 458) can also rarely be paraneoplastic in origin. Metastases to the cord are probably more common than either of these in patients with cancer. Often, a cause of intrinsic myelopathy can

1	NMO (Chap. 458) can also rarely be paraneoplastic in origin. Metastases to the cord are probably more common than either of these in patients with cancer. Often, a cause of intrinsic myelopathy can be identified only through periodic reassessment.

1	The prospects for recovery from an acute destructive spinal cord lesion fade after ~6 months. There are currently no effective means to Diseases of the Spinal Cord Low quadriplegia (C5-C8) Partially independent with adaptive May be dependent or independent May use manual wheelchair, drive an equipment automobile with adaptive equipment Paraplegia (below T1) Independent Independent Ambulates short distances with aids Source: Adapted from JF Ditunno, CS Formal: Chronic spinal cord injury. N Engl J Med 330:550, 1994; with permission.

1	promote repair of injured spinal cord tissue; promising but entirely experimental approaches include the use of factors that influence reinnervation by axons of the corticospinal tract, nerve and neural sheath graft bridges, forms of electrical stimulation at the site of injury, and the local introduction of stem cells. The disability associated with irreversible spinal cord damage is determined primarily by the level of the lesion and by whether the disturbance in function is complete or incomplete (Table 456-4). Even a complete high cervical cord lesion may be compatible with a productive life. The primary goals are development of a rehabilitation plan framed by realistic expectations and attention to the neurologic, medical, and psychological complications that commonly arise.

1	Many of the usual symptoms associated with medical illnesses, especially somatic and visceral pain, may be lacking because of the destruction of afferent pain pathways. Unexplained fever, worsening of spasticity, or deterioration in neurologic function should prompt a search for infection, thrombophlebitis, or an intraabdominal pathology. The loss of normal thermoregulation and inability to maintain normal body temperature can produce recurrent fever (quadriplegic fever), although most episodes of fever are due to infection of the urinary tract, lung, skin, or bone.

1	Bladder dysfunction generally results from loss of supraspinal innervation of the detrusor muscle of the bladder wall and the sphincter musculature. Detrusor spasticity is treated with anticholinergic drugs (oxybutynin, 2.5–5 mg qid) or tricyclic antidepressants with anticholinergic properties (imipramine, 25–200 mg/d). Failure of the sphincter muscle to relax during bladder emptying (urinary dyssynergia) may be managed with the α-adrenergic blocking agent terazosin hydrochloride (1–2 mg tid or qid), with intermittent catheterization, or, if that is not feasible, by use of a condom catheter in men or a permanent indwelling catheter. Surgical options include the creation of an artificial bladder by isolating a segment of intestine that can be catheterized intermittently (enterocystoplasty) or can drain continuously to an external appliance (urinary conduit). Bladder areflexia due to acute spinal shock or conus lesions is best treated by catheterization. Bowel regimens and disimpaction

1	or can drain continuously to an external appliance (urinary conduit). Bladder areflexia due to acute spinal shock or conus lesions is best treated by catheterization. Bowel regimens and disimpaction are necessary in most patients to ensure at least biweekly evacuation and avoid colonic distention or obstruction.

1	Patients with acute cord injury are at risk for venous thrombosis and pulmonary embolism. Use of calf-compression devices and anticoagulation with low-molecular-weight heparin is recommended. In cases of persistent paralysis, anticoagulation should probably be continued for 3 months. Prophylaxis against decubitus ulcers should involve frequent changes in position in a chair or bed, the use of special mattresses, and cushioning of areas where pressure sores often develop, such as the sacral prominence and heels. Early treatment of ulcers with careful cleansing, surgical or enzyme debridement of necrotic tissue, and appropriate dressing and drainage may prevent infection of adjacent soft tissue or bone.

1	Spasticity is aided by stretching exercises to maintain mobility of joints. Drug treatment is effective but may result in reduced function, as some patients depend on spasticity as an aid to stand, transfer, or walk. Baclofen (up to 240 mg/d in divided doses) is effective; it acts by facilitating γ-aminobutyric acid–mediated inhibition of motor reflex arcs. Diazepam acts by a similar mechanism and is useful for leg spasms that interrupt sleep (2–4 mg at bedtime). Tizanidine (2–8 mg tid), an α2 adrenergic agonist that increases presynaptic inhibition of motor neurons, is another option. For nonambulatory patients, the direct muscle inhibitor dantrolene (25–100 mg qid) may be used, but it is potentially hepatotoxic. In refractory cases, intrathecal baclofen administered via an implanted pump, botulinum toxin injections, or dorsal rhizotomy may be required to control spasticity.

1	Despite the loss of sensory function, many patients with spinal cord injury experience chronic pain sufficient to diminish their quality of life. Randomized controlled studies indicate that gabapentin or pregabalin is useful in this setting. Epidural electrical stimulation and intrathecal infusion of pain medications have been tried with some success. Management of chronic pain is discussed in Chap. 18.

1	A paroxysmal autonomic hyperreflexia may occur following lesions above the major splanchnic sympathetic outflow at T6. Headache, flushing, and diaphoresis above the level of the lesion, as well as hypertension with bradycardia or tachycardia, are the major symptoms. The trigger is typically a noxious stimulus—for example, bladder or bowel distention, a urinary tract infection, or a decubitus ulcer—below the level of the cord lesion. Treatment consists of removal of offending stimuli; ganglionic blocking agents (mecamylamine, 2.5–5 mg) or other short-acting antihypertensive drugs are useful in some patients. Attention to these details allows longevity and a productive life for patients with complete transverse myelopathies. Allan H. Ropper this is a digital-only chapter. it is available on the DvD that accompanies this book, as well as on access medicine/harrison’s online, and the eBook and “app” editions of hpim 19e.

1	Allan H. Ropper this is a digital-only chapter. it is available on the DvD that accompanies this book, as well as on access medicine/harrison’s online, and the eBook and “app” editions of hpim 19e. Almost 10 million head injuries occur annually in the United States, about 20% of which are serious enough to cause brain damage. Among men <35 years, accidents, usually motor vehicle collisions, are the chief cause of death and >70% of these involve head injury. Furthermore, minor head injuries are so common that almost all physicians will be called upon to provide immediate care or to see patients who are suffering from various sequelae.

1	Medical personnel caring for head injury patients should be aware that (1) spinal injury often accompanies head injury, and care must be taken in handling the patient to prevent compression of the spinal cord due to instability of the spinal column; (2) intoxication is frequently associated with traumatic brain injury, and thus testing for drugs and alcohol should be carried out when appropriate; and (3) additional injuries, including rupture of abdominal organs, may produce vascular collapse, shock, or respiratory distress that requires immediate attention.

1	Concussion and Other traumatic Brain Injuries Allan H. Ropper Almost 10 million head injuries occur annually in the United States, about 20% of which are serious enough to cause brain damage. Among 457e men <35 years, accidents, usually motor vehicle collisions, are the chief cause of death and >70% of these involve head injury. Furthermore, minor head injuries are so common that almost all physicians will be called upon to provide immediate care or to see patients who are suffering from various sequelae.

1	Medical personnel caring for head injury patients should be aware that (1) spinal injury often accompanies head injury, and care must be taken in handling the patient to prevent compression of the spinal cord due to instability of the spinal column; (2) intoxication is frequently associated with traumatic brain injury, and thus testing for drugs and alcohol should be carried out when appropriate; and (3) additional injuries, including rupture of abdominal organs, may produce vascular collapse, shock, or respiratory distress that requires immediate attention.

1	This form of minor head injury had in the past referred to an immediate and transient loss of consciousness that was associated with a short period of amnesia. Many patients, however, do not lose consciousness after a minor head injury but instead are dazed or confused, or feel stunned or “star struck,” and the term concussion is now applied to all such cognitive and perceptual changes experienced after a blow to the head. Severe concussion may precipitate a brief convulsion or autonomic signs such as facial pallor, bradycardia, faintness with mild hypotension, or sluggish pupillary reaction, but most patients quickly return to a neurologically normal state.

1	The mechanics of a typical concussion involve sudden deceleration of the head when hitting a blunt stationary object. This creates an anterior-posterior movement of the brain within the skull due to inertia and rotation of the cerebral hemispheres on the fulcrum of the relatively fixed upper brainstem. Loss of consciousness in concussion is believed to result from a transient electrophysiologic dysfunction of the reticular activating system in the upper midbrain that is at the site of rotation (Chap. 328). The transmission of a wave of kinetic energy throughout the brain is an alternative explanation for the disruption in consciousness.

1	Gross and light-microscopic changes in the brain are usually absent following concussion, but biochemical and ultrastructural changes, such as mitochondrial ATP depletion and local disruption of the blood-brain barrier, may be transient abnormalities. Computed tomography (CT) and magnetic resonance imaging (MRI) scans are usually normal; however, a small number of patients will be found to have a skull fracture, an intracranial hemorrhage, or a brain contusion.

1	A brief period of both retrograde and anterograde amnesia is characteristic of concussion, and it recedes rapidly in alert patients. Memory loss spans the moments before impact but may encompass the previous days or weeks (rarely months). With severe injuries, the extent of retrograde amnesia roughly correlates with the severity of injury. Memory is regained erratically from the most distant to more recent memories, with islands of amnesia occasionally remaining. The mechanism of amnesia is not known. Hysterical posttraumatic amnesia is not uncommon after head injury and should be suspected when inexplicable behavioral abnormalities occur, such as recounting events that cannot be recalled on later testing, a bizarre affect, forgetting one’s own name, or a persistent anterograde deficit that is excessive in comparison with the degree of injury. Amnesia is discussed in Chap. 36.

1	A single, uncomplicated concussion only infrequently produces permanent neurobehavioral changes in patients who are free of preexisting psychiatric and neurologic problems. Nonetheless, residual 457e-1 problems in memory and concentration may have an anatomic correlate in microscopic cerebral lesions (see below). The mechanisms by which a blast injury affects the brain and causes symptoms that are associated with concussion, a problem mainly in military medicine, are not known. The energy of a blast wave can enter the cranium through the openings of the orbits, auditory canals, and foramen magnum. There are not consistent changes in cerebral imaging studies but more subtle indications of tissue disruption have been found, comparable to those of mild concussion. It has been difficult to separate the direct effects of the blast from the consequences of being thrown against fixed objects or injured by flying debris. CONTUSION, BRAIN HEMORRHAGE, AND AXONAL SHEARING LESIONS

1	These pathologic changes are the result of severe cranial trauma. A surface bruise of the brain, or contusion, consists of varying degrees of petechial hemorrhage, edema, and tissue destruction. Contusions and deeper hemorrhages result from mechanical forces that displace and compress the hemispheres forcefully and by deceleration of the brain against the inner skull, either under a point of impact (coup lesion) or, as the brain swings back, in the antipolar area (contrecoup lesion). Trauma sufficient to cause prolonged unconsciousness usually produces some degree of contusion. Blunt deceleration impact, as occurs against an automobile dashboard or from falling forward onto a hard surface, causes contusions on the orbital surfaces of the frontal lobes and the anterior and basal portions of the temporal lobes. With lateral forces, as from impact on an automobile door frame, contusions are situated on the lateral convexity of the hemisphere. The clinical signs of contusion are

1	of the temporal lobes. With lateral forces, as from impact on an automobile door frame, contusions are situated on the lateral convexity of the hemisphere. The clinical signs of contusion are determined by the location and size of the lesion; often, there are no focal neurologic abnormalities, but these injured regions are later the sites of gliotic scars that may produce seizures. A hemiparesis or gaze preference is fairly typical of moderately sized contusions. Large bilateral contusions produce stupor with extensor posturing, while those limited to the frontal lobes cause a taciturn state. Contusions in the temporal lobe may cause delirium or an aggressive, combative syndrome.

1	Acute contusions are easily visible on CT and MRI scans, appearing as inhomogeneous hyperdensities on CT and as hyperintensities on T2 and fluid-attenuated inversion recovery (FLAIR) MRI sequences; there is usually surrounding localized brain edema (Fig. 457e-1) and some subarachnoid bleeding. Blood in the cerebrospinal fluid (CSF) due to trauma may provoke a mild inflammatory reaction. Over a few days, contusions acquire a surrounding contrast enhancement and edema that may be mistaken for tumor or abscess. Glial and macrophage reactions result in chronic, scarred, hemosiderin-stained depressions on the cortex (plaques jaunes) that are the main source of posttraumatic epilepsy. FIGURE 457e-1 Traumatic cerebral contusion. Noncontrast computed tomography scan demonstrating a hyperdense hemorrhagic region in the anterior temporal lobe.

1	FIGURE 457e-1 Traumatic cerebral contusion. Noncontrast computed tomography scan demonstrating a hyperdense hemorrhagic region in the anterior temporal lobe. FIGURE 457e-2 Multiple small areas of hemorrhage and tissue disruption in the white matter of the frontal lobes on noncontrast computed tomography scan. These appear to reflect an extreme type of the diffuse axonal shearing lesions that occur with closed head injury. Torsional or shearing forces within the brain cause hemorrhages of the basal ganglia and other deep regions. Large hemorrhages after minor trauma suggest that there is a bleeding diathesis or cerebrovascular amyloidosis. For unexplained reasons, deep cerebral hemorrhages may not develop until several days after injury. Sudden neurologic deterioration in a comatose patient or a sudden rise in intracranial pressure (ICP) suggests this complication has occurred and should therefore prompt investigation with a CT scan.

1	A special type of deep white matter lesion consists of widespread mechanical disruption, or shearing, of axons at the time of impact. Most characteristic are small areas of tissue injury in the corpus callosum and dorsolateral pons. The presence of widespread multifocal axonal damage in both hemispheres, a state called diffuse axonal injury (DAI), has been proposed to explain persistent coma and the vegetative state after closed head injury (Chap. 328), but small ischemichemorrhagic lesions in the midbrain and thalamus are an alternative explanation. Only severe shearing lesions that contain blood are visualized by CT, usually in the corpus callosum and centrum semiovale (Fig. 457e-2); however, special MRI sequences that detect small amounts of blood and diffusion tensor imaging can demonstrate numerous such lesions throughout the white matter.

1	A blow to the skull that exceeds the elastic tolerance of the bone causes a fracture. Intracranial lesions accompany roughly two-thirds of skull fractures, and the presence of a fracture increases many-fold the chances of an underlying subdural or epidural hematoma. Consequently, fractures are primarily markers of the site and severity of injury. If the underlying arachnoid membrane has been torn, fractures also provide potential pathways for entry of bacteria to the CSF with a risk of meningitis and for leakage of CSF outward through the dura. If there is leakage of CSF, severe orthostatic headache results from lowered pressure in the spinal fluid compartment.

1	Most fractures are linear and extend from the point of impact toward the base of the skull. Basilar skull fractures are often extensions of adjacent linear fractures over the convexity of the skull but may occur independently owing to stresses on the floor of the middle cranial fossa or occiput. Basilar fractures are usually parallel to the petrous bone or along the sphenoid bone and directed toward the sella turcica and ethmoidal groove. Although most basilar fractures are uncomplicated, they can cause CSF leakage, pneumocephalus, and delayed cavernous-carotid fistulas. Hemotympanum (blood behind the tympanic membrane), ecchymosis over the mastoid process (Battle sign), and periorbital ecchymosis (“raccoon sign”) are associated with basilar fractures. Because routine x-ray examination may fail to disclose basilar fractures, they should be suspected if these clinical signs are present.

1	CSF may leak through the cribriform plate or the adjacent sinus and cause CSF rhinorrhea (a watery discharge from the nose). Persistent rhinorrhea and recurrent meningitis usually require surgical repair of torn dura underlying the fracture. The site of the leak is often difficult to determine, but useful diagnostic tests include the instillation of water-soluble contrast into the CSF followed by CT with the patient in various positions, or injection of radionuclide compounds or fluorescein into the CSF and the insertion of absorptive nasal pledgets. The location of an intermittent leak is infrequently delineated, and many resolve spontaneously. Sellar fractures, even those associated with serious neuroendocrine dysfunction, may be radiologically occult or evident only by an air-fluid level in the sphenoid sinus. Fractures of the dorsum sella cause sixth or seventh nerve palsies or optic nerve damage.

1	Petrous bone fractures, especially those oriented along the long axis of the bone, may be associated with facial palsy, disruption of ear ossicles, and CSF otorrhea. Transverse petrous fractures are less common; they almost always damage the cochlea or labyrinths and often the facial nerve as well. External bleeding from the ear is usually from local abrasion of the external canal but can also result from petrous fracture. Fractures of the frontal bone are usually depressed, involving the frontal and paranasal sinuses and the orbits. Depressed skull fractures are typically compound, but they may be asymptomatic because the impact energy is dissipated in breaking the bone; some have underlying brain contusions. Debridement and exploration of compound fractures are required in order to avoid infection; simple fractures usually do not require surgery.

1	The cranial nerves most often injured with head trauma are the olfactory, optic, oculomotor, and trochlear; the first and second branches of the trigeminal nerve; and the facial and auditory nerves. Anosmia and an apparent loss of taste (actually a loss of perception of aromatic flavors, with retained elementary taste perception) occur in ~10% of persons with serious head injuries, particularly from falls on the back of the head. This is the result of displacement of the brain and shearing of the fine olfactory nerve filaments that course through the cribriform bone. At least partial recovery of olfactory and gustatory function is expected, but if bilateral anosmia persists for several months, the prognosis is poor. Partial optic nerve injuries from closed trauma result in blurring of vision, central or paracentral scotomas, or sector defects. Direct orbital injury may cause short-lived blurred vision for close objects due to reversible iridoplegia. Diplopia limited to downward gaze

1	central or paracentral scotomas, or sector defects. Direct orbital injury may cause short-lived blurred vision for close objects due to reversible iridoplegia. Diplopia limited to downward gaze and corrected when the head is tilted away from the side of the affected eye indicates trochlear (fourth nerve) nerve damage. It occurs frequently as an isolated problem after minor head injury or may develop for unknown reasons after a delay of several days. Facial nerve injury caused by a basilar fracture is present immediately in up to 3% of severe injuries; it may also be delayed for 5–7 days. Fractures through the petrous bone, particularly the less common transverse type, are liable to produce facial palsy. Delayed facial palsy occurring up to a week after injury, the mechanism of which is unknown, has a good prognosis. Injury to the eighth cranial nerve from a fracture of the petrous bone causes loss of hearing, vertigo, and nystagmus immediately after injury. Deafness from eighth nerve

1	has a good prognosis. Injury to the eighth cranial nerve from a fracture of the petrous bone causes loss of hearing, vertigo, and nystagmus immediately after injury. Deafness from eighth nerve injury is rare and must be distinguished from blood in the middle ear or disruption of the middle ear ossicles. Dizziness, tinnitus, and high-tone hearing loss occur from cochlear concussion, most typically after blast injury.

1	Convulsions are surprisingly uncommon immediately after a head injury, but a brief period of tonic extensor posturing or a few clonic movements of the limbs just after the moment of impact can occur. However, the cortical scars that evolve from contusions are highly epileptogenic and may later manifest as seizures, even after many months or years (Chap. 445). The severity of injury roughly determines the risk of future seizures. It has been estimated that 17% of individuals with brain contusion, subdural hematoma, or prolonged loss of consciousness will develop a seizure disorder and that this risk extends for an indefinite period of time, whereas the risk is ≤2% after mild injury. The majority of convulsions in the latter group occur within 5 years of injury but may be delayed for decades. Penetrating injuries have a much higher rate of subsequent epilepsy.

1	Hemorrhages beneath the dura (subdural) or between the dura and skull (epidural) have characteristic clinical and imaging features. They are sometimes associated with underlying contusions and other injuries, often making it difficult to determine the relative contribution of each component to the clinical state. The mass effect and raised ICP caused by these hematomas can be life threatening, making it imperative to identify them rapidly by CT or MRI scan and to remove them when appropriate.

1	Acute Subdural Hematoma (Fig. 457e-3) Direct cranial trauma may be minor and is not required for acute subdural hemorrhage to occur, especially in the elderly and those taking anticoagulant medications. Acceleration forces alone, as from whiplash, are sometimes sufficient to produce subdural hematoma. Up to one-third of patients have a lucid interval lasting minutes to hours before coma supervenes, but most are drowsy or comatose from the moment of injury. A unilateral headache and slightly enlarged pupil on the side of the hematoma are frequently, but not invariably, present. Stupor or coma, hemiparesis, and unilateral pupillary enlargement are signs of larger hematomas. In an acutely deteriorating patient, burr (drainage) holes or an emergency craniotomy are required. Small subdural hematomas may be asymptomatic and usually do not require evacuation if they do not enlarge.

1	A subacutely evolving syndrome due to subdural hematoma occurs days or weeks after injury with drowsiness, headache, confusion, or mild hemiparesis, usually in alcoholics and in the elderly and often after only minor trauma. On imaging studies, subdural hematomas appear as crescentic collections over the convexity of one or both hemispheres, most commonly in the frontotemporal region, and less often in the inferior middle fossa or over the occipital poles (Fig. 457e-3). Interhemispheric, posterior fossa, or bilateral convexity hematomas are less frequent and are difficult to diagnose clinically, although drowsiness and the neurologic signs expected from damage in each region can usually be detected. The bleeding that causes larger hematomas is primarily venous in origin, although additional arterial bleeding sites are sometimes found at operation, and a few large hematomas have a purely arterial origin.

1	FIGURE 457e-3 Acute subdural hematoma. Noncontrast computed tomography scan reveals a hyperdense clot that has an irregular border with the brain and causes more horizontal displacement (mass effect) than might be expected from its thickness. The disproportionate mass effect is the result of the large rostral-caudal extent of these hematomas. Compare to Fig. 457e-4. FIGURE 457e-4 Acute epidural hematoma. The tightly attached dura is stripped from the inner table of the skull, producing a characteristic lenticular-shaped hemorrhage on noncontrast computed tomography scan. Epidural hematomas are usually caused by tearing of the middle meningeal artery following fracture of the temporal bone.

1	Epidural Hematoma (Fig. 457e-4) These usually evolve more rapidly than subdural hematomas and are correspondingly more treacherous. They occur in up to 10% of cases of severe head injury but are associated with underlying cortical damage less often than for subdural hematomas. Most patients are unconscious when first seen. A “lucid interval” of several minutes to hours before coma supervenes is most characteristic of epidural hemorrhage, but it is still uncommon, and epidural hemorrhage is not the only cause of this temporal sequence. Rapid surgical evacuation and ligation or cautery of the damaged vessel is indicated, usually the middle meningeal artery that has been lacerated by an overlying skull fracture.

1	Chronic Subdural Hematoma (Fig. 457e-5) A subacutely evolving syndrome due to subdural hematoma occurs days or weeks after injury with drowsiness, headache, confusion, or mild hemiparesis, usually in alcoholics and in the elderly and often after only minor or unnoticed trauma. On imaging studies, chronic subdural hematomas appear as crescentic clots over the convexity of one or both hemispheres, most commonly in the frontotemporal region (Fig. 457e-3). A history of FIGURE 457e-5 Computed tomography scan of chronic bilateral subdural hematomas of different ages. The collections began as acute hematomas and have become hypodense in comparison to the adjacent brain after a period during which they were isodense and difficult to appreciate. Some areas of resolving blood are contained on the more recently formed collection on the left (arrows).

1	trauma may or may not be elicited in relation to chronic subdural hematoma; the injury may have been trivial and forgotten, particularly in the elderly and those with clotting disorders. Headache is common but not invariable. Additional features that may appear weeks later include slowed thinking, vague change in personality, seizure, or a mild hemiparesis. Headache fluctuates in severity, sometimes with changes in head position.

1	Bilateral chronic subdural hematomas produce perplexing clinical syndromes, and the initial clinical impression may be of a stroke, brain tumor, drug intoxication, depression, or a dementing illness. Drowsiness, inattentiveness, and incoherence of thought are generally more prominent than focal signs such as hemiparesis. Rarely, chronic hematomas cause brief episodes of hemiparesis or aphasia that are indistinguishable from transient ischemic attacks. Patients with undetected bilateral subdural hematomas have a low tolerance for surgery, anesthesia, and drugs that depress the nervous system; drowsiness or confusion persists for long periods postoperatively.

1	CT without contrast initially shows a low-density mass over the convexity of the hemisphere (Fig. 457e-5). Between 2 and 6 weeks after the initial bleeding, the clot becomes isodense compared to adjacent brain and may be inapparent. Many subdural hematomas that are several weeks in age contain areas of blood and intermixed serous fluid. Bilateral chronic hematomas may fail to be detected because of the absence of lateral tissue shifts; this circumstance in an older patient is suggested by a “hypernormal” CT scan with fullness of the cortical sulci and small ventricles. Infusion of contrast material demonstrates enhancement of the vascular fibrous capsule surrounding the collection. MRI reliably identifies subacute and chronic hematomas.

1	Clinical observation coupled with serial imaging is a reasonable approach to patients with few symptoms, such as headache alone, and in those with small chronic subdural collections. Treatment of minimally symptomatic chronic subdural hematoma with glucocorticoids is favored by some clinicians, but surgical evacuation is more often successful. The fibrous membranes that grow from the dura and encapsulate the collection require removal to prevent recurrent fluid accumulation. Small hematomas are resorbed, leaving only the organizing membranes. On imaging studies, very chronic subdural hematomas are difficult to distinguish from hygromas, which are collections of CSF from a rent in the arachnoid membrane.

1	The patient who has briefly lost consciousness or been stunned after a minor head injury usually becomes fully alert and attentive within minutes but may complain of headache, dizziness, faintness, nausea, a single episode of emesis, difficulty with concentration, a brief amnestic period, or slight blurring of vision. This typical concussion syndrome has a good prognosis with little risk of subsequent deterioration. Children are particularly prone to drowsiness, vomiting, and irritability, symptoms that are sometimes delayed for several hours after apparently minor injuries. Vasovagal syncope that follows injury may cause undue concern. Generalized or frontal headache is common in the following days. It may be migrainous (throbbing and hemicranial) in nature or aching and bilateral. After several hours of observation, patients with minor injury may be accompanied home and observed for a day by a family member or friend, with written instructions to return if symptoms worsen.

1	Persistent severe headache and repeated vomiting in the context of normal alertness and no focal neurologic signs is usually benign, but CT should be obtained and a longer period of observation is appropriate. The decision to perform imaging tests also depends on clinical signs that indicate that the impact was severe (e.g., persistent confusion, periorbital or mastoid hematoma, repeated vomiting, palpable skull fracture), on the seriousness of other bodily injuries, and on the degree of surveillance that can be anticipated after discharge. Two studies have indicated that older age, two or more episodes of vomiting, >30 min of retrograde or persistent anterograde amnesia, seizure, and concurrent drug or alcohol intoxication are sensitive (but not specific) indicators of intracranial hemorrhage that justify CT scanning. It may be appropriate to be more liberal in obtaining CT scans in children because a small number, even without loss of consciousness, will have intracranial traumatic

1	that justify CT scanning. It may be appropriate to be more liberal in obtaining CT scans in children because a small number, even without loss of consciousness, will have intracranial traumatic lesions but this exposes the child to radiation.

1	Concussion in Sports In the current absence of adequate data, a common sense approach to athletic concussion has been to remove the individual from play immediately and avoid contact sports for at least several days after a mild injury and for a longer period if there are more severe injuries or if there are protracted neurologic symptoms such as headache and difficulty concentrating. No individual should return to play unless all symptoms have resolved and an assessment has been made by a health care professional who has experience with treatment of concussion. Once cleared, the individual can then begin a graduated program of increasing activity. Younger athletes are particularly likely to experience protracted concussive symptoms, and a slower return to play in this age group may be reasonable. These guidelines are designed in part to avoid a perpetuation of symptoms but also to prevent the rare second impact syndrome, in which diffuse and fatal cerebral swelling follows a second

1	These guidelines are designed in part to avoid a perpetuation of symptoms but also to prevent the rare second impact syndrome, in which diffuse and fatal cerebral swelling follows a second minor head injury.

1	In the past, mental decline in boxers late in their careers had been called dementia pugilistica. There is some evidence that repeated concussions from other sports are associated with a similar delayed and progressive cognitive disorder that is due mainly to the deposition of tau protein in cortical neurons. The brains of these patients display deposition of tau protein in the superficial cortical layers, and particularly in the depths of sulci within the frontal cortices, a pattern named chronic traumatic encephalopathy (CTE) that is quite unlike other degenerative conditions. CTE is an intensively studied and provocative entity. Its contribution, if any, to late-life dementia and parkinsonism in former athletes, soldiers, or others who have sustained repeated concussive injuries is unknown. CTE is also discussed in Chap. 444e.

1	Patients who are not fully alert or have persistent confusion, behavioral changes, extreme dizziness, or focal neurologic signs such as hemiparesis should be admitted to the hospital and have cerebral imaging. A cerebral contusion or hematoma will usually be found. Common syndromes include: (1) delirium with a disinclination to be examined or moved, expletive speech, and resistance if disturbed (anterior temporal lobe contusions); (2) a quiet, disinterested, slowed mental state (abulia) alternating with irascibility (inferior frontal and frontopolar contusions); (3) a focal deficit such as aphasia or mild hemiparesis (due to subdural hematoma or convexity contusion or, less often, carotid artery dissection); (4) confusion and inattention, poor performance on simple mental tasks, and fluctuating orientation (associated with several types of injuries, including those described above, and with medial frontal contusions and interhemispheric subdural hematoma); (5) repetitive vomiting,

1	orientation (associated with several types of injuries, including those described above, and with medial frontal contusions and interhemispheric subdural hematoma); (5) repetitive vomiting, nystagmus, drowsiness, and unsteadiness (labyrinthine concussion, but occasionally due to a posterior fossa subdural hematoma or vertebral artery dissection); and (6) diabetes insipidus (damage to the median eminence or pituitary stalk). Injuries of this degree are often complicated by drug or alcohol intoxication, and clinically inapparent cervical spine injury may be present. Blast injuries are often accompanied by rupture of the tympanic membranes.

1	After surgical removal of hematomas, most patients in this category improve over weeks. During the first week, the state of alertness, memory, and other cognitive functions often fluctuate, and agitation and somnolence are common. Behavioral changes tend to be worse at night, as with many other encephalopathies, and may be treated with small doses of antipsychotic medications. Subtle abnormalities of attention, intellect, spontaneity, and memory return toward normal weeks or months after the injury, sometimes abruptly. Persistent cognitive problems are discussed below.

1	Patients who are comatose from the moment of injury require immediate neurologic attention and resuscitation. After intubation, with care taken to immobilize the cervical spine, the depth of coma, pupillary size and reactivity, limb movements, and Babinski responses are assessed. As soon as vital functions permit and cervical spine x-rays and a CT scan have been obtained, the patient should be transported to a critical care unit. Hypoxia should be reversed, and normal saline used as the resuscitation fluid in preference to albumin. The finding of an epidural or subdural hematoma or large intracerebral hemorrhage is usually an indication for prompt surgery and intracranial decompression in an otherwise salvageable patient. Measurement of ICP with a ventricular catheter or fiberoptic device in order to guide treatment has been favored by many units but has not improved outcome. Hyperosmolar intravenous solutions are used in various regimens to limit intracranial pressure. The inherently

1	in order to guide treatment has been favored by many units but has not improved outcome. Hyperosmolar intravenous solutions are used in various regimens to limit intracranial pressure. The inherently appealing approach of removing portions of the skull in order to decompress the intracranial contents, as has been successful for brain swelling after cerebral infarction, has so far not proven effective for traumatic brain injury. The use of prophylactic antiepileptic medications has been recommended, but there is little supportive data. Management of raised ICP, a frequent feature of severe head injury, is discussed in Chap. 330.

1	In severe head injury, the clinical features of eye opening, motor responses of the limbs, and verbal output have been found to be generally predictive of outcome. These three responses are assessed by the Glasgow Coma Scale; a score between 3 and 15 is assigned (Table 457e-1). Over 85% of patients with aggregate scores of <5 die within 24 h. However, a number of patients with slightly higher scores, including a few without pupillary light responses, survive, suggesting that an initially aggressive approach is justified in most patients. Patients <20 years old, particularly children, may make remarkable recoveries after having grave early neurologic signs. In one large study of severe head injury, 55% of children had a good outcome at 1 year, compared with 21% of adults. Older age, increased ICP, early hypoxia or hypotension, compression of the brainstem on CT or MRI, and a delay in the evacuation of large intracranial hemorrhages are indicators of a poor prognosis.

1	The postconcussion syndrome refers to a state following minor head injury consisting of combinations of fatigue, dizziness, headache, and difficulty in concentration. The syndrome simulates asthenia and anxious depression. Based on experimental models, it has been proposed that subtle axonal shearing lesions or as yet undefined biochemical alterations account for the cognitive symptoms. In moderate and severe trauma, neuropsychological changes such as difficulty with attention and memory and other cognitive deficits are undoubtedly present, sometimes severe, but many problems identified by formal 5 To loud voice 3 Confused, disoriented Note: Coma score = E + M + V. Patients scoring 3 or 4 have an 85% chance of dying or remaining vegetative, whereas scores >11 indicate only a 5–10% likelihood of death or vegetative state and 85% chance of moderate disability or good recovery. Intermediate scores correlate with proportional chances of recovery.

1	testing do not affect daily functioning. Test scores tend to improve rapidly during the first 6 months after injury and then more slowly for years.

1	Management of the postconcussive syndrome requires the identification and treatment of each separate element of depression, sleeplessness, anxiety, persistent headache, and dizziness. A clear explanation of the problems that may follow concussion has been shown to reduce subsequent complaints. Care is taken to avoid prolonged use of drugs that produce dependence. Headache may initially be treated with acetaminophen and small doses of amitriptyline. Vestibular exercises (Chap. 28) and small doses of vestibular suppressants such as promethazine (Phenergan) may be helpful when dizziness is the main problem. Patients who after minor or moderate injury have difficulty with memory or with complex cognitive tasks at work may be reassured that these problems usually improve over 6–12 months, and workload may be reduced in the interim. It is sometimes helpful to obtain serial and quantified neuropsychological testing in order to adjust the work environment to the patient’s abilities and to

1	workload may be reduced in the interim. It is sometimes helpful to obtain serial and quantified neuropsychological testing in order to adjust the work environment to the patient’s abilities and to document improvement over time. Whether cognitive exercises are useful in contrast to rest and a reduction in mental challenges is uncertain. Previously energetic and resilient individuals usually have the best recoveries. In patients with persistent symptoms, the possibility exists of malingering or prolongation as a result of litigation.

1	multiple sclerosis and other Demyelinating Diseases Stephen L. Hauser, Douglas S. Goodin Demyelinating disorders are immune-mediated conditions charac-terized by preferential destruction of central nervous system (CNS) 458 myelin. The peripheral nervous system (PNS) is spared, and most patients have no evidence of an associated systemic illness. Multiple sclerosis, the most common disease in this category, is second only to trauma as a cause of neurologic disability beginning in early to middle adulthood.

1	Multiple sclerosis (MS) is an autoimmune disease of the CNS characterized by chronic inflammation, demyelination, gliosis (scarring), and neuronal loss; the course can be relapsing-remitting or progressive. Lesions of MS typically develop at different times and in different CNS locations (i.e., MS is said to be disseminated in time and space). Approximately 350,000 individuals in the United States and 2.5 million individuals worldwide are affected. The clinical course can be extremely variable, ranging from a benign condition to a rapidly evolving and incapacitating disease requiring profound lifestyle adjustments.

1	PATHOGENESIS Pathology New MS lesions begin with perivenular cuffing by inflammatory mononuclear cells, predominantly T cells and macrophages, which also infiltrate the surrounding white matter. At sites of inflammation, the blood-brain barrier (BBB) is disrupted, but unlike vasculitis, the vessel wall is preserved. Involvement of the humoral immune system is also evident; small numbers of B lymphocytes also infiltrate the nervous system, myelin-specific autoantibodies are present on degenerating myelin sheaths, and complement is activated. Demyelination is the hallmark of the pathology, and evidence of myelin degeneration is found at the earliest time points of tissue injury. A remarkable feature of MS plaques is that oligodendrocyte precursor cells survive—and in many lesions are present in even greater numbers than in normal tissue—but these cells fail to differentiate into mature myelin-producing cells. In some lesions, surviving oligodendrocytes or those that differentiate from

1	in even greater numbers than in normal tissue—but these cells fail to differentiate into mature myelin-producing cells. In some lesions, surviving oligodendrocytes or those that differentiate from precursor cells partially remyelinate the surviving naked axons, producing so-called shadow plaques. As lesions evolve, there is prominent astrocytic proliferation (gliosis). Over time, ectopic lymphocyte follicle-like structures, consisting of aggregates of T and B cells resembling secondary lymphoid tissue, appear in the meninges and especially overlying deep cortical sulci and also in perivascular spaces. Although relative sparing of axons is typical of MS, partial or total axonal destruction can also occur, especially within highly inflammatory lesions. Thus MS is not solely a disease of myelin, and neuronal pathology is increasingly recognized as a major contributor to irreversible neurologic disability. Inflammation, demyelination, and plaque formation are also present in the cerebral

1	and neuronal pathology is increasingly recognized as a major contributor to irreversible neurologic disability. Inflammation, demyelination, and plaque formation are also present in the cerebral cortex, and significant axon loss indicating death of neurons is widespread, especially in advanced cases (see “Neurodegeneration,” below).

1	Physiology Nerve conduction in myelinated axons occurs in a saltatory manner, with the nerve impulse jumping from one node of Ranvier to the next without depolarization of the axonal membrane underlying the myelin sheath between nodes (Fig. 458-1). This produces considerably faster conduction velocities (∼70 m/s) than the slow velocities (∼1 m/s) produced by continuous propagation in unmyelinated nerves. Conduction block occurs when the nerve impulse is unable to traverse the demyelinated segment. This can happen when the resting axon membrane becomes hyperpolarized due to the exposure of voltage-dependent potassium channels that are normally buried underneath the myelin sheath. A temporary conduction block often follows a demyelinating event before sodium channels (originally concentrated at the nodes) redistribute along the naked Na+ channels Node of Ranvier

1	Na+ channels Node of Ranvier FIGURE 458-1 Nerve conduction in myelinated and demyelinated axons. A. Saltatory nerve conduction in myelinated axons occurs with the nerve impulse jumping from one node of Ranvier to the next. Sodium channels (shown as breaks in the solid black line) are concentrated at the nodes where axonal depolarization occurs. B. Following demyelination, additional sodium channels are redistributed along the axon itself, thereby allowing continuous propagation of the nerve action potential despite the absence of myelin.

1	axon (Fig. 458-1). This redistribution ultimately allows continuous propagation of nerve action potentials through the demyelinated segment. Conduction block may be incomplete, affecting highbut not low-frequency volleys of impulses. Variable conduction block can occur with raised body temperature or metabolic alterations and may explain clinical fluctuations that vary from hour to hour or appear with fever or exercise. Conduction slowing occurs when the demyelinated segments of the axonal membrane are reorganized to support continuous (slow) nerve impulse propagation. Epidemiology MS is approximately threefold more common in women than men. The age of onset is typically between 20 and 40 years (slightly later in men than in women), but the disease can present across the lifespan. Approximately 10% of cases begin before age 18 years of age, and a small percentage of cases begin before the age of 10 years.

1	Geographical gradients have been repeatedly observed in MS, with the highest known prevalence for MS (250 per 100,000) in the Orkney Islands, located north of Scotland. In other temperate zone areas (e.g., northern North America, northern Europe, southern Australia, and southern New Zealand), the prevalence of MS is 0.1–0.2%. By contrast, in the tropics (e.g., Asia, equatorial Africa, and the Middle East), the prevalence is often 10to 20-fold less. The prevalence of MS has increased steadily (and dramatically) in several regions around the world over the past half-century, presumably reflecting the impact of some environmental shift. Moreover, the fact that this increase has occurred primarily (or exclusively) in women indicates that women are more responsive to this environmental change. Well-established risk factors for MS include vitamin D deficiency, exposure to Epstein-Barr virus (EBV) after early childhood, and cigarette smoking.

1	Vitamin D deficiency is associated with an increase in MS risk, and data suggest that ongoing deficiency may also increase disease activity after MS begins. Immunoregulatory effects of vitamin D could explain these apparent relationships. Exposure of the skin to ultraviolet-B (UVB) radiation from the sun is essential for the biosynthesis of vitamin D, and this endogenous production is the most important source of vitamin D in most individuals; a diet rich in fatty fish represents another source of vitamin D. At high latitudes, the amount of UVB radiation reaching the earth’s surface is often insufficient, particularly during winter months, and consequently, low serum levels of vitamin D are common in temperate zones. The common practice to avoid direct 2662 sun exposure and the widespread use of sun block, which (at sun protection factor [SPF] 15) blocks 94% of the incoming UVB radia tion, would be expected to exacerbate any population-wide vitamin D deficiency.

1	Evidence of a remote EBV infection playing some role in MS is supported by numerous epidemiologic and laboratory studies. A higher risk of infectious mononucleosis (associated with relatively late EBV infection) and higher antibody titers to latency-associated EBV nuclear antigen have been repeatedly associated with MS risk, although a causal role for EBV has not been established. A history of cigarette smoking has also been associated with MS risk. Interestingly, in an animal model of MS, the lung was identified as a critical site for activation of pathogenic T lymphocytes responsible for autoimmune demyelination. Recent data in MS models have also shown that high levels of dietary sodium activate pathogenic autoreactive T lymphocytes, suggesting that consumption of a high-salt diet, now widespread in the Western world, might be part of the explanation for the observed increase in the prevalence of MS in recent years.

1	Whites are inherently at higher risk for MS than Africans or Asians, even when residing in a similar environment. MS also aggregates within some families, and adoption, half-sibling, twin, and spousal studies indicate that familial aggregation is due to genetic, and not environmental, factors (Table 458-1).

1	Susceptibility to MS is polygenic, with each gene contributing a relatively small amount to the overall risk. The strongest susceptibility signal in genome-wide studies maps to the HLA-DRB1 gene in the class II region of the major histocompatibility complex (MHC), and this association accounts for approximately 10% of the disease risk. This HLA association, which was first described several decades ago, suggests that MS, at its core, is an antigen-specific autoimmune disease. Whole-genome association studies have now identified approximately 110 other MS susceptibility variants, each of which individually has only a modest effect on MS risk. Most of these MS-associated genes have known roles in the adaptive immune system, for example the genes for the interleukin (IL) 7 receptor (CD127), IL-2 receptor (CD25), and T cell costimulatory molecule LFA-3 (CD58); some variants also influence susceptibility to other autoimmune diseases in addition to MS. The variants identified so far all

1	IL-2 receptor (CD25), and T cell costimulatory molecule LFA-3 (CD58); some variants also influence susceptibility to other autoimmune diseases in addition to MS. The variants identified so far all lack specificity and sensitivity for MS; thus, at present, they are not useful for diagnosis or to predict the future course of the disease.

1	immunology A proinflammatory autoimmune response directed against a component of CNS myelin, and perhaps other neural elements as well, remains the cornerstone of current concepts of MS pathogenesis.

1	AutoreActiVe t lymphocyteS Myelin basic protein (MBP), an intracellular protein involved in myelin compaction, is an important T cell antigen in experimental allergic encephalomyelitis (EAE), a laboratory model, and probably also in human MS. Activated MBP-reactive T cells have been identified in the blood, in cerebrospinal fluid (CSF), and within MS lesions. Moreover, DRB1*15:01 may influence the autoimmune response because it binds with high affinity to a fragment of MBP (spanning amino acids 89–96), stimulating T cell responses to this self-protein. Two different populations of proinflammatory T cells are likely to mediate autoimmunity in MS. T-helper type 1 (TH1) cells producing interferon γ (IFN-γ) are one key effector population, and more recently, a role for highly proinflammatory TH17 T cells has been established. TH17 cells are induced by transforming growth factor β (TGF-β) and IL-6 and are amplified by IL-21 and IL-23. TH17 cells, and levels of their corresponding cytokine

1	TH17 T cells has been established. TH17 cells are induced by transforming growth factor β (TGF-β) and IL-6 and are amplified by IL-21 and IL-23. TH17 cells, and levels of their corresponding cytokine IL-17, are increased in MS lesions and also in the circulation of people with active MS. High circulating levels of IL-17 may also be a marker of a more severe course of MS. TH1 cytokines, including IL-2, tumor necrosis factor (TNF)-α, and IFN-γ, also play key roles in activating and maintaining autoimmune responses, and TNF-α and IFN-γ may directly injure oligodendrocytes or the myelin membrane.

1	humorAl Autoimmunity B cell activation and antibody responses also appear to be necessary for the full development of demyelinating lesions to occur, both in experimental models and in human MS. Clonally restricted populations of activated, antigen-experienced, memory B cells and plasma cells are present in MS lesions, in lymphoid follicle-like structures in the meninges overlying the cerebral cortex, and in the CSF. Similar or identical clonal populations are found in each compartment, indicating that a highly focused B cell response is occurring locally within the CNS in MS. Myelin-specific autoantibodies, some directed against an extracellular myelin protein, myelin oligodendrocyte glycoprotein (MOG), have been detected bound to vesiculated myelin debris in MS plaques. In the CSF, elevated levels of locally synthesized immunoglobulins and oligoclonal antibodies, derived from clonally restricted CNS B cells and plasma cells, are also characteristic of MS. The pattern of oligoclonal

1	levels of locally synthesized immunoglobulins and oligoclonal antibodies, derived from clonally restricted CNS B cells and plasma cells, are also characteristic of MS. The pattern of oligoclonal banding is unique to each individual, and attempts to identify the targets of these antibodies have been largely unsuccessful.

1	triggerS Serial magnetic resonance imaging (MRI) studies in early relapsing-remitting MS reveal that bursts of focal inflammatory disease activity occur far more frequently than would have been predicted by the frequency of relapses. Thus, early in MS, most disease activity is clinically silent. Although the triggers causing these bursts are unknown, molecular mimicry between environmental agents, presumably pathogens, and myelin antigens activating pathogenic T cells may be responsible (Chap. 377e).

1	Neurodegeneration Axonal damage occurs in every newly formed MS lesion, and cumulative axonal loss is considered to be one important cause of irreversible neurologic disability in MS. As many as 70% of axons are lost from the lateral corticospinal (e.g., motor) tracts in patients with advanced paraparesis from MS, and longitudinal MRI studies suggest there is progressive axonal loss over time within established, inactive lesions. Demyelination can result in reduced trophic support for axons, redistribution of ion channels, and destabilization of action potential membrane potentials. Axons can adapt initially to these injuries, but over time, distal and retrograde degeneration often occurs. Therefore, promoting remyelination remains an important therapeutic goal.

1	In progressive MS, a key unresolved question is whether the primary neurodegenerative process occurs primarily in the cerebral cortex, the white matter, or in some combination of the two sites. As noted above, meningeal infiltrates of B and T cells are particularly prominent in progressive MS cases, and these “lymphoid follicles” are associated with underlying microglial activation, gray matter plaques, and loss of cortical neurons. White matter lesions may also contribute to late progressive MS; inactive plaques are often noninflammatory at the center, but at the edges, microglia and macrophages and evidence of ongoing axonal injury can be found. This suggests that a simmering, and possibly concentrically expanding, axonopathy may be present, even in the most chronic cases. In addition, a diffuse low-grade inflammation across large areas of white matter may be present, associated with reduced myelin staining and axonal injury (“dirty white matter”). Another characteristic of

1	a diffuse low-grade inflammation across large areas of white matter may be present, associated with reduced myelin staining and axonal injury (“dirty white matter”). Another characteristic of progressive MS is that inflammation is often present without a concomitant disruption of the BBB; possibly, this feature might explain the failure of immunotherapies not capable of crossing the BBB to benefit patients with progressive MS.

1	Evidence supports a role of one, or more likely several, of the following mechanisms in progressive MS. Axonal and neuronal death may result from glutamate-mediated excitotoxicity, oxidative injury, iron accumulation, and/or mitochondrial failure either occurring as a consequence of free-radical damage or due to accumulation of deletions in mitochondrial DNA.

1	The onset of MS may be abrupt or insidious. Symptoms may be severe or seem so trivial that a patient may not seek medical attention for months or years. Indeed, at autopsy, approximately 0.1% of individuals who were asymptomatic during life will be found, unexpectedly, to have pathologic evidence of MS. Similarly, in the modern era, an MRI scan obtained for an unrelated reason may show evidence of asymptomatic MS. Symptoms of MS are extremely varied and depend on the location and severity of lesions within the CNS (Table 458-2). Examination often reveals evidence of neurologic dysfunction, often in asymptomatic locations. For example, a patient may present with symptoms in one leg but signs in both.

1	Weakness of the limbs may manifest as loss of strength, speed, or dexterity, as fatigue, or as a disturbance of gait. Exercise-induced weakness is a characteristic symptom of MS. The weakness is of the upper motor neuron type (Chap. 30) and is usually accompanied by other pyramidal signs such as spasticity, hyperreflexia, and Babinski signs. Occasionally a tendon reflex may be lost (simulating a lower motor neuron lesion) if an MS lesion disrupts the afferent reflex fibers in the spinal cord (see Fig. 30-2). Spasticity (Chap. 30) is commonly associated with spontaneous and movement-induced muscle spasms. More than 30% of MS patients have moderate to severe spasticity, especially in the legs. This is often accompanied by painful spasms interfering with ambulation, work, or self-care. Occasionally spasticity provides support for the body weight during ambulation, and in these cases, treatment of spasticity may actually do more harm than good.

1	Optic neuritis (ON) presents as diminished visual acuity, dimness, or decreased color perception (desaturation) in the central field of vision. These symptoms can be mild or may progress to severe visual loss. Rarely, there is complete loss of light perception. Visual symptoms are generally monocular but may be bilateral. Periorbital pain (aggravated by eye movement) often precedes or accompanies the visual loss. An afferent pupillary defect (Chap. 39) is usually present. Funduscopic examination may be normal or reveal optic disc swelling (papillitis). Pallor of the optic disc (optic atrophy) commonly follows ON. Uveitis is uncommon and should raise the possibility of alternative diagnoses such as sarcoid or lymphoma. Visual blurring in MS may result from ON or diplopia (double vision); if the symptom resolves when either eye is covered, the cause is diplopia.

1	Visual blurring in MS may result from ON or diplopia (double vision); if the symptom resolves when either eye is covered, the cause is diplopia. Diplopia may result from internuclear ophthalmoplegia (INO) or from palsy of the sixth cranial nerve (rarely the third or fourth). An INO consists of impaired adduction of one eye due to a lesion in the ipsilateral medial longitudinal fasciculus (Chaps. 41e and 42). Percentage Symptom of Cases Symptom of Cases Source: After WB Matthews et al: McAlpine’s Multiple Sclerosis. New York, Churchill Livingstone, 1991. Prominent nystagmus is often observed in the abducting eye, along 2663 with a small skew deviation. A bilateral INO is particularly suggestive of MS. Other common gaze disturbances in MS include (1) a horizontal gaze palsy, (2) a “one and a half” syndrome (horizontal gaze palsy plus an INO), and (3) acquired pendular nystagmus.

1	Sensory symptoms are varied and include both paresthesias (e.g., tingling, prickling sensations, formications, “pins and needles,” or painful burning) and hypesthesia (e.g., reduced sensation, numbness, or a “dead” feeling). Unpleasant sensations (e.g., feelings that body parts are swollen, wet, raw, or tightly wrapped) are also common. Sensory impairment of the trunk and legs below a horizontal line on the torso (a sensory level) indicates that the spinal cord is the origin of the sensory disturbance. It is often accompanied by a bandlike sensation of tightness around the torso. Pain is a common symptom of MS, experienced by >50% of patients. Pain can occur anywhere on the body and can change locations over time. Ataxia usually manifests as cerebellar tremors (Chap. 450). Ataxia may also involve the head and trunk or the voice, producing a characteristic cerebellar dysarthria (scanning speech).

1	Ataxia usually manifests as cerebellar tremors (Chap. 450). Ataxia may also involve the head and trunk or the voice, producing a characteristic cerebellar dysarthria (scanning speech). Bladder dysfunction is present in >90% of MS patients, and in a third of patients, dysfunction results in weekly or more frequent episodes of incontinence. During normal reflex voiding, relaxation of the bladder sphincter (α-adrenergic innervation) is coordinated with contraction of the detrusor muscle in the bladder wall (muscarinic cholinergic innervation). Detrusor hyperreflexia, due to impairment of suprasegmental inhibition, causes urinary frequency, urgency, nocturia, and uncontrolled bladder emptying. Detrusor sphincter dyssynergia, due to loss of synchronization between detrusor and sphincter muscles, causes difficulty in initiating and/or stopping the urinary stream, producing hesitancy, urinary retention, overflow incontinence, and recurrent infection.

1	Constipation occurs in >30% of patients. Fecal urgency or bowel incontinence is less common (<15%) but can be socially debilitating. Cognitive dysfunction can include memory loss; impaired attention; difficulties in executive functioning, memory, and problem solving; slowed information processing; and problems shifting between cognitive tasks. Euphoria (elevated mood) was once thought to be characteristic of MS but is actually uncommon, occurring in <20% of patients. Cognitive dysfunction sufficient to impair activities of daily living is rare. Depression, experienced by approximately half of patients, can be reactive, endogenous, or part of the illness itself and can contribute to fatigue.

1	Depression, experienced by approximately half of patients, can be reactive, endogenous, or part of the illness itself and can contribute to fatigue. Fatigue (Chap. 29) is experienced by 90% of patients; this symptom is the most common reason for work-related disability in MS. Fatigue can be exacerbated by elevated temperatures, depression, expending exceptional effort to accomplish basic activities of daily living, or sleep disturbances (e.g., from frequent nocturnal awakenings to urinate). Sexual dysfunction may manifest as decreased libido, impaired genital sensation, impotence in men, and diminished vaginal lubrication or adductor spasms in women. Facial weakness due to a lesion in the pons may resemble idiopathic Bell’s palsy (Chap. 455). Unlike Bell’s palsy, facial weakness in MS is usually not associated with ipsilateral loss of taste sensation or retro-auricular pain.

1	Vertigo may appear suddenly from a brainstem lesion, superficially resembling acute labyrinthitis (Chap. 28). Hearing loss (Chap. 43) may also occur in MS but is uncommon. Ancillary Symptoms Heat sensitivity refers to neurologic symptoms produced by an elevation of the body’s core temperature. For example, unilateral visual blurring may occur during a hot shower or with physical exercise (Uhthoff ’s symptom). It is also common for MS symptoms to worsen transiently, sometimes dramatically, during febrile illnesses (see “Acute Attacks or Initial Demyelinating Episodes,” below). Such heat-related symptoms probably result from transient conduction block (see above).

1	Lhermitte’s symptom is an electric shock–like sensation (typically induced by flexion or other movements of the neck) that radiates down 2664 the back into the legs. Rarely, it radiates into the arms. It is generally self-limited but may persist for years. Lhermitte’s symptom can also occur with other disorders of the cervical spinal cord (e.g., cervical spondylosis). Paroxysmal symptoms are distinguished by their brief duration (10 s to 2 min), high frequency (5–40 episodes per day), lack of any alteration of consciousness or change in background electroencephalogram during episodes, and a self-limited course (generally lasting weeks to months). They may be precipitated by hyperventilation or movement. These syndromes may include Lhermitte’s symptom; tonic contractions of a limb, face, or trunk (tonic seizures); paroxysmal dysarthria and ataxia; paroxysmal sensory disturbances; and several other less well-characterized syndromes. Paroxysmal symptoms probably result from spontaneous

1	or trunk (tonic seizures); paroxysmal dysarthria and ataxia; paroxysmal sensory disturbances; and several other less well-characterized syndromes. Paroxysmal symptoms probably result from spontaneous discharges, arising at the edges of demyelinated plaques and spreading to adjacent white matter tracts. Trigeminal neuralgia, hemifacial spasm, and glossopharyngeal neuralgia (Chap. 455) can occur when the demyelinating lesion involves the root entry (or exit) zone of the fifth, seventh, and ninth cranial nerve, respectively. Trigeminal neuralgia (tic douloureux) is a very brief lancinating facial pain often triggered by an afferent input from the face or teeth. Most cases of trigeminal neuralgia are not MS related; however, atypical features such as onset before age 50 years, bilateral symptoms, objective sensory loss, or nonparoxysmal pain should raise the possibility that MS could be responsible. Facial myokymia consists of either persistent rapid flickering contractions of the facial

1	objective sensory loss, or nonparoxysmal pain should raise the possibility that MS could be responsible. Facial myokymia consists of either persistent rapid flickering contractions of the facial musculature (especially the lower portion of the orbicularis oculus) or a contraction that slowly spreads across the face. It results from lesions of the corticobulbar tracts or brainstem course of the facial nerve.

1	Four clinical types of MS exist (Fig. 458-2): 1. Relapsing/remitting MS (RRMS) accounts for 85% of MS cases at onset and is characterized by discrete attacks that generally evolve over days to weeks (rarely over hours). With initial attacks, there is often substantial or complete recovery over the ensuing weeks to months, but as attacks continue over time recovery may be less evident (Fig. 458-2A). Between attacks, patients are neurologically stable. 2.

1	2. Secondary progressive MS (SPMS) always begins as RRMS (Fig. 458-2B). At some point, however, the clinical course changes so that the patient experiences a steady deterioration in function unassociated with acute attacks (which may continue or cease during the progressive phase). SPMS produces a greater amount of fixed neurologic disability than RRMS. For a patient with RRMS, the risk of developing SPMS is ∼2% each year, meaning that the great majority of RRMS ultimately evolves into SPMS. SPMS appears to represent a late stage of the same underlying illness as RRMS. 3.

1	3. Primary progressive MS (PPMS) accounts for ∼15% of cases. These patients do not experience attacks but only a steady functional decline from disease onset (Fig. 458-2C). Compared to RRMS, the sex distribution is more even, the disease begins later in life (mean age ∼40 years), and disability develops faster (at least relative to the onset of the first clinical symptom). Despite these differences, PPMS appears to represent the same underlying illness as RRMS. 4. Progressive/relapsing MS (PRMS) overlaps PPMS and SPMS and accounts for ∼5% of MS patients. Like patients with PPMS, these patients experience a steady deterioration in their condition from disease onset. However, like SPMS patients, they experience occasional attacks superimposed upon their progressive course (Fig. 458-2D).

1	There is no definitive diagnostic test for MS. Diagnostic criteria for clinically definite MS require documentation of two or more episodes of symptoms and two or more signs that reflect pathology in anatomically noncontiguous white matter tracts of the CNS (Table 458-3). Symptoms must last for >24 h and occur as distinct episodes that are separated by a month or more. In patients who have only one of the two required signs on neurologic examination, the second may be documented by abnormal tests such as MRI or evoked potentials (EPs). Similarly, in the most recent diagnostic scheme, the second clinical event (in time) may be supported solely by MRI findings, consisting of either the development of new focal white matter lesions on MRI or the simultaneous presence of both an enhancing lesion and a nonenhancing lesion in an asymptomatic location. In patients whose course is progressive from onset for ≥6 months without superimposed relapses, documentation of intrathecal IgG synthesis

1	and a nonenhancing lesion in an asymptomatic location. In patients whose course is progressive from onset for ≥6 months without superimposed relapses, documentation of intrathecal IgG synthesis may be used to support a diagnosis of PPMS.

1	DIAGNOSTIC TESTS Magnetic Resonance Imaging MRI has revolutionized the diagnosis and management of MS (Fig. 458-3); characteristic abnormalities are found in >95% of patients, although more than 90% of the lesions visualized by MRI are asymptomatic. An increase in vascular permeability from a breakdown of the BBB is detected by leakage of intravenous gadolinium (Gd) into the parenchyma. Such leakage occurs early in the development of an MS lesion and serves as a useful marker of inflammation. Gd enhancement typically persists for approximately 1 month, and the residual MS plaque remains visible indefinitely as a focal area of hyperintensity (a lesion) on spin-echo (T2-weighted) and proton-density images. Lesions are frequently oriented perpendicular to the ventricular surface, corresponding to the pathologic pattern of perivenous demyelination (Dawson’s fingers). Lesions are multifocal within the brain, brainstem, and spinal cord. Lesions larger than 6 mm located in the corpus

1	to the pathologic pattern of perivenous demyelination (Dawson’s fingers). Lesions are multifocal within the brain, brainstem, and spinal cord. Lesions larger than 6 mm located in the corpus callosum, periventricular white matter, brain-stem, cerebellum, or spinal cord are particularly helpful diagnostically. Current criteria for the use of MRI in the diagnosis of MS are shown in Table 458-3.

1	The total volume of T2-weighted signal abnormality (the “burden of disease”) shows a significant (albeit weak) correlation with clinical disability, as do measures of brain atrophy. Approximately one-third of T2-weighted lesions appear as hypointense lesions (black holes) on T1-weighted imaging. Black holes may be a marker of irreversible demyelination and axonal loss, although even this measure depends on the timing of the image acquisition (e.g., most acute Gd-enhancing T2 lesions are T1 dark). Newer MRI methods such as magnetization transfer ratio (MTR) FIGURE 458-2 Clinical course of multiple sclerosis (MS). imaging and proton magnetic resonance spectroscopic imaging A. Relapsing/remitting MS (RRMS). B. Secondary progressive MS (SPMS). (MRSI) may ultimately serve as surrogate markers of clinical disabil-

1	C. Primary progressive MS (PPMS). D. Progressive/relapsing MS (PRMS). ity. MRSI can quantitate molecules such as N-acetyl aspartate, which 2 or more attacks; objective None clinical evidence of 2 or more lesions or objective clinical evidence of 1 lesion with reasonable historical evidence of a prior attack 2 or more attacks; objec-Dissemination in space, demonstrated by tive clinical evidence of • ≥1 T2 lesion on MRI in at least 2 out of 4 1 lesion MS-typical regions of the CNS (periventricular, juxtacortical, infratentorial, or spinal cord) 1 attack; objective clini-Dissemination in time, demonstrated by cal evidence of 2 or more • Simultaneous presence of asymptomatic lesion(s) on follow-up MRI, irrespective of its timing with reference to a baseline scan 1 attack; objective clinical Dissemination in space and time, demonstrated evidence of 1 lesion (clini-by: cally isolated syndrome) • ≥1 T2 lesion in at least 2 out of 4 MS-typical regions of the CNS (periventricular,

1	clinical Dissemination in space and time, demonstrated evidence of 1 lesion (clini-by: cally isolated syndrome) • ≥1 T2 lesion in at least 2 out of 4 MS-typical regions of the CNS (periventricular, juxtacortical, infratentorial, or spinal cord) • Simultaneous presence of asymptomatic lesion(s) on follow-up MRI, irrespective of its timing with reference to a baseline scan

1	Insidious neurologic 1 year of disease progression (retrospectively or progression suggestive of prospectively determined) MS (PPMS) 2 out of the 3 following criteria Evidence for dissemination in space in the brain based on ≥1 T2+ lesions in the MS-characteristic periventricular, juxtacortical, or infratentorial regions Evidence for dissemination in space in the spinal cord based on ≥2 T2+ lesions in the cord Positive CSF (isoelectric focusing evidence of oligoclonal bands and/or elevated IgG index) Abbreviations: CNS, central nervous system; CSF, cerebrospinal fluid; MRI, magnetic resonance imaging; PPMS, primary progressive multiple sclerosis. Source: From CH Polman et al: Diagnostic Criteria for Multiple Sclerosis: 2010 Revisions to the “McDonald Criteria.” Ann Neurol 69:292, 2011. is a marker of axonal integrity, and MTR may be able to distinguish demyelination from edema.

1	is a marker of axonal integrity, and MTR may be able to distinguish demyelination from edema. Evoked Potentials EP testing assesses function in afferent (visual, auditory, and somatosensory) or efferent (motor) CNS pathways. EPs use computer averaging to measure CNS electric potentials evoked by repetitive stimulation of selected peripheral nerves or of the brain. 2665

1	These tests provide the most information when the pathways studied are clinically uninvolved. For example, in a patient with a remitting and relapsing spinal cord syndrome with sensory deficits in the legs, an abnormal somatosensory EP following posterior tibial nerve stimulation provides little new information. By contrast, an abnormal visual EP in this circumstance would permit a diagnosis of clinically definite MS (Table 458-3). Abnormalities on one or more EP modalities occur in 80–90% of MS patients. EP abnormalities are not specific to MS, although a marked delay in the latency of a specific EP component (as opposed to a reduced amplitude or distorted wave-shape) is suggestive of demyelination.

1	Cerebrospinal Fluid CSF abnormalities found in MS include a mononuclear cell pleocytosis and an increased level of intrathecally synthesized IgG. The total CSF protein is usually normal. Various formulas distinguish intrathecally synthesized IgG from IgG that may have entered the CNS passively from the serum. One formula, the CSF IgG index, expresses the ratio of IgG to albumin in the CSF divided by the same ratio in the serum. The IgG synthesis rate uses serum and CSF IgG and albumin measurements to calculate the rate of CNS IgG synthesis. The measurement of oligoclonal bands (OCBs) by agarose gel electrophoresis in the CSF also assesses intrathecal production of IgG. Two or more discrete OCBs, not present in a paired serum sample, are found in >75% of patients with MS. OCBs may be absent at the onset of MS, and in individual patients, the number of bands may increase with time.

1	A mild CSF pleocytosis (>5 cells/μL) is present in ∼25% of cases, usually in young patients with RRMS. A pleocytosis of >75 cells/μL, the presence of polymorphonuclear leukocytes, or a protein concentration >1 g/L (>100 mg/dL) in CSF should raise concern that the patient may not have MS.

1	No single clinical sign or test is diagnostic of MS. The diagnosis is readily made in a young adult with relapsing and remitting symptoms involving different areas of CNS white matter. The possibility of an alternative diagnosis should always be considered (Table 458-4), particularly when (1) symptoms are localized exclusively to the posterior fossa, craniocervical junction, or spinal cord; (2) the patient is <15 or >60 years of age; (3) the clinical course is progressive from onset; (4) the patient has never experienced visual, sensory, or bladder symptoms; or (5) laboratory findings (e.g., MRI, CSF, or EPs) are atypical. Similarly, uncommon or rare symptoms in MS (e.g., aphasia, parkinsonism, chorea, isolated dementia, severe muscular atrophy, peripheral neuropathy, episodic loss of consciousness, fever, headache, seizures, or coma) should increase concern about an alternative diagnosis. Diagnosis is also difficult in patients with a rapid or explosive (stroke-like) onset or with

1	fever, headache, seizures, or coma) should increase concern about an alternative diagnosis. Diagnosis is also difficult in patients with a rapid or explosive (stroke-like) onset or with mild symptoms and a normal neurologic examination. Rarely, intense inflammation and swelling may produce a mass lesion that mimics a primary or metastatic tumor. In the current era, the disorders most likely to be mistaken for MS are neuromyelitis optica (see below), sarcoid, vascular disorders (including antiphospholipid syndrome and vasculitis), and rarely CNS lymphoma. The specific tests required to exclude alternative diagnoses will vary with each clinical situation; however, an erythrocyte sedimentation rate, serum B12 level, ANA, and treponemal antibody should probably be obtained in all patients with suspected MS.

1	Most patients with clinically evident MS ultimately experience progressive neurologic disability. In older studies conducted mostly before disease-modifying therapies for MS were widely available, 15 years after onset, only 20% of patients had no functional limitation, and between one-third and one-half progressed to SPMS and required assistance with ambulation; furthermore, 25 years after onset, ∼80% of MS patients reached this level of disability. The long-term prognosis for untreated MS appears to have improved in recent years, at least in part because of the development of therapies for the early relapsing form of the disease. Although the prognosis in an individual is

1	FIGURE 458-3 Magnetic resonance imaging findings in multiple sclerosis (MS). A. Axial first-echo image from T2-weighted sequence demonstrates multiple bright signal abnormalities in white matter, typical for MS. B. Sagittal T2-weighted fluid-attenuated inversion recovery (FLAIR) image in which the high signal of cerebrospinal fluid (CSF) has been suppressed. CSF appears dark, whereas areas of brain edema or demyelination appear high in signal as shown here in the corpus callosum (arrows). Lesions in the anterior corpus callosum are frequent in MS and rare in vascular disease. C. Sagittal T2-weighted fast spin echo image of the thoracic spine demonstrates a fusiform high-signal-intensity lesion in the midthoracic spinal cord. D. Sagittal T1-weighted image obtained after the intravenous administration of gadolinium DTPA reveals focal areas of blood-brain barrier disruption, identified as high-signal-intensity regions (arrows).

1	difficult to establish, certain clinical features suggest a more favorable prognosis. These include ON or sensory symptoms at onset; fewer than two relapses in the first year of illness; and minimal impairment after 5 years. By contrast, patients with truncal ataxia, action tremor, pyramidal symptoms, or a progressive disease course are more likely to become disabled. Patients with a long-term favorable course are likely to have developed fewer MRI lesions during the early years of disease, and vice versa. Importantly, some MS patients have a benign variant of MS and never develop neurologic disability. The likelihood of having benign MS is thought to be <20%. Patients with benign MS 15 years after onset who have entirely normal neurologic examinations are likely to maintain their benign course

1	In patients with their first demyelinating event (i.e., a clinically isolated syndrome), the brain MRI provides prognostic information. With three or more typical T2-weighted lesions, the risk of developing MS after 20 years is ∼80%. Conversely, with a normal brain MRI, the likelihood of developing MS is <20%. Similarly, the presence of two or more Gd-enhancing lesions at baseline is highly predictive of future MS, as is the appearance of either new T2-weighted lesions or new Gd enhancement ≥3 months after the initial episode. Mortality as a direct consequence of MS is uncommon, although it has been estimated that the 25-year survival is only 85% of expected. Death can occur during an acute MS attack, although this is distinctly rare. More commonly, death occurs as a complication of MS (e.g., pneumonia in a debilitated individual). Death can also result from suicide. Early disease-modifying therapy seems to reduce this excess mortality.

1	Effect of Pregnancy Pregnant MS patients experience fewer attacks than expected during gestation (especially in the last trimester), but more attacks than expected in the first 3 months postpartum. When considering the pregnancy year as a whole (i.e., 9 months of pregnancy plus 3 months postpartum), the overall disease course is unaffected. Decisions about childbearing should thus be made based on (1) the mother’s physical state, (2) her ability to care for the child, and (3) the availability of social support. Disease-modifying therapy is Acute disseminated encephalomyelitis (ADEM) Antiphospholipid antibody syndrome Behçet’s disease Cerebral autosomal dominant arteriopathy, subcortical infarcts, and leukoen

1	Acute disseminated encephalomyelitis (ADEM) Antiphospholipid antibody syndrome Behçet’s disease Cerebral autosomal dominant arteriopathy, subcortical infarcts, and leukoen Congenital leukodystrophies (e.g., adrenoleukodystrophy, metachromatic leukodystrophy) Human immunodeficiency virus (HIV) infection Ischemic optic neuropathy (arteritic and nonarteritic) Lyme disease Mitochondrial encephalopathy with lactic acidosis and stroke (MELAS) Neoplasms (e.g., lymphoma, glioma, meningioma) Sarcoid Sjögren’s syndrome Stroke and ischemic cerebrovascular disease Syphilis Systemic lupus erythematosus and related collagen vascular disorders Tropical spastic paraparesis (HTLV-1/2 infection) Vascular malformations (especially spinal dural AV fistulas) Vasculitis (primary CNS or other) Vitamin B12 deficiency Abbreviations: AV, arteriovenous; CNS, central nervous system; HTLV, human T cell lymphotropic virus.

1	Abbreviations: AV, arteriovenous; CNS, central nervous system; HTLV, human T cell lymphotropic virus. generally discontinued during pregnancy, although the actual risk from the interferons and glatiramer acetate (see below) appears to be low. Therapy for MS can be divided into several categories: (1) treatment of acute attacks, (2) treatment with disease-modifying agents that reduce the biologic activity of MS, and (3) symptomatic therapy. Treatments that promote remyelination or neural repair do not currently exist, but several promising approaches are being actively investigated. The Expanded Disability Status Score (EDSS) is a widely used measure of neurologic impairment in MS (Table 458-5). Most patients with EDSS scores <3.5 have RRMS, walk normally, and are generally not disabled; by contrast, patients with EDSS scores >5.5 have progressive MS (SPMS or PPMS), are gait-impaired, and, typically, are occupationally disabled.

1	When patients experience acute deterioration, it is important to consider whether this change reflects new disease activity or a “pseudoexacerbation” resulting from an increase in ambient temperature, fever, or an infection. When the clinical change is thought to reflect a pseudoexacerbation, glucocorticoid treatment is inappropriate. Glucocorticoids are used to manage either first attacks or acute exacerbations. They provide short-term clinical benefit by reducing the severity and shortening the duration of attacks. Whether treatment provides any long-term benefit on the course of the illness is less clear. Therefore, mild attacks are often not treated. Physical and occupational therapy can help with mobility and manual dexterity.

1	Glucocorticoid treatment is usually administered as intravenous methylprednisolone, 500–1000 mg/d for 3–5 days, either without a taper or followed by a course of oral prednisone beginning at a dose of 60–80 mg/d and gradually tapered over 2 weeks. Orally administered methylprednisolone or dexamethasone (in equivalent dosages) can be substituted for the intravenous portion of the therapy, although gastrointestinal complications are more common by this route. Outpatient treatment is almost always possible.

1	Side effects of short-term glucocorticoid therapy include fluid retention, potassium loss, weight gain, gastric disturbances, acne, and emotional lability. Concurrent use of a low-salt, potassium-2667 rich diet and avoidance of potassium-wasting diuretics are advisable. Lithium carbonate (300 mg orally bid) may help to manage emotional lability and insomnia associated with glucocorticoid therapy. Patients with a history of peptic ulcer disease may require cimetidine (400 mg bid) or ranitidine (150 mg bid). Proton pump inhibitors such as pantoprazole (40 mg orally bid) may reduce the likelihood of gastritis, especially when large doses are administered orally. Plasma exchange (five to seven exchanges: 40–60 mL/kg per exchange, every other day for 14 days) may benefit patients with fulminant attacks of demyelination that are unresponsive to glucocorticoids. However, the cost is high, and conclusive evidence of efficacy is lacking.

1	DISEASE-MODIFYING THERAPIES FOR RELAPSING FORMS OF MS (RRMS, SPMS WITH EXACERBATIONS) Ten such agents are approved by the U.S. Food and Drug Administration (FDA): (1) IFN-β-1a (Avonex), (2) IFN-β-1a (Rebif),

1	IFN-β-1b (Betaseron or Extavia), (4) glatiramer acetate (Copaxone), (5) natalizumab (Tysabri), (6) fingolimod (Gilenya), dimethyl fumarate (Tecfidera), (8) teriflunomide (Aubagio), mitoxantrone (Novantrone), and (10) alemtuzumab (Lemtrada). Several other promising agents are in varying stages of product development. Each of these treatments can also be used in SPMS patients who continue to experience attacks, both because SPMS can be difficult to distinguish from RRMS and because the available clinical trials, although not definitive, suggest that such patients may sometimes derive therapeutic benefit. Thus, in several phase 3 clinical trials, recipients of each of these agents experienced fewer clinical exacerbations and fewer new MRI lesions compared to placebo recipients (Table 458-6). Because of its potential toxicity as an immunosuppressant, mitoxantrone is generally reserved for patients with progressive disability who have failed other treatments. When considering the data in

1	Because of its potential toxicity as an immunosuppressant, mitoxantrone is generally reserved for patients with progressive disability who have failed other treatments. When considering the data in Table 458-6, however, it is important to note that the relative efficacy of the different agents cannot be determined by cross-trial comparisons. Relative efficacy can only be assessed from nonbiased head-to-head clinical trials.

1	Interferon-β IFN-β is a class I interferon originally identified by its antiviral properties. Efficacy in MS probably results from immunomodulatory properties including (1) downregulating expression of MHC molecules on antigen-presenting cells, (2) reducing proinflammatory and increasing regulatory cytokine levels, (3) inhibiting T cell proliferation, and (4) limiting the trafficking of inflammatory cells in the CNS. IFN-β reduces the attack rate and improves disease severity measures such as EDSS progression and MRI-documented disease burden. IFN-β should be considered in patients with either RRMS or SPMS with superimposed relapses. In patients with SPMS but without relapses, efficacy has not been established. Head-to-head trials suggest that dosing IFN-β more frequently and at higher doses has better efficacy but is also more likely to induce neutralizing antibodies (see below). IFN-β-1a (Avonex), 30 μg, is administered by intramuscular injection once every week. IFN-β-1a (Rebif),

1	has better efficacy but is also more likely to induce neutralizing antibodies (see below). IFN-β-1a (Avonex), 30 μg, is administered by intramuscular injection once every week. IFN-β-1a (Rebif), 44 μg, is administered by subcutaneous injection three times per week. IFN-β-1b (Betaseron or Extavia), 250 μg, is administered by subcutaneous injection every other day.

1	Common side effects of IFN-β therapy include flulike symptoms (e.g., fevers, chills, and myalgias) and mild abnormalities on routine laboratory evaluation (e.g., elevated liver function tests or lymphopenia). Rarely, more severe hepatotoxicity may occur. Subcutaneous IFN-β also causes reactions at the injection site (e.g., pain, redness, induration, or, rarely, skin necrosis). Side effects can usually be managed with concomitant nonsteroidal anti-inflammatory medications. Depression, increased spasticity, and cognitive changes have been reported, although these symptoms can also be due to the underlying disease. In any event, side effects due to IFN-β therapy usually subside over time.

1	0.0 1.0 = No disability, minimal signs in one FS (i.e., grade 1) 1.5 = No disability, minimal signs in more than one FS (more than one grade 1) 2.0 = Minimal disability in one FS (one FS grade 2, others 0 or 1) 2.5 = Minimal disability in two FS (two FS grade 2, others 0 or 1) 3.0 = Moderate disability in one FS (one FS grade 3, others 0 or 1) or mild disability in three or four FS (three/four FS grade 2, others 0 or 1) although fully ambulatory 3.5 = Fully ambulatory but with moderate disability in one FS (one grade 3) and one or two FS grade 2; or two FS grade 3; or five FS grade 2 (others 0 or 1) 4.0 4.5 5.0 5.5 = Ambulatory without aid or rest for ∼100 m 6.0 = Unilateral assistance required to walk about 100 m with or without resting 6.5 = Constant bilateral assistance required to walk about 20 m without resting 7.0 = Unable to walk beyond about 5 m even with aid; essentially restricted to wheelchair; wheels self and transfers alone 7.5 = Unable to take more than a few steps;

1	walk about 20 m without resting 7.0 = Unable to walk beyond about 5 m even with aid; essentially restricted to wheelchair; wheels self and transfers alone 7.5 = Unable to take more than a few steps; restricted to wheelchair; may need aid to transfer 8.0 = Essentially restricted to bed or chair or perambulated in wheelchair, but out of bed most of day; retains many self-care functions; generally has effective use of arms 8.5 = Essentially restricted to bed much of the day; has some effective use of arm(s); retains some self-care functions 9.0 9.5 = Totally helpless bed patient; unable to communicate or eat 10.0 = Death due to MS

1	A. Pyramidal functions 0 = Normal 1 = Abnormal signs without disability 2 = Minimal disability 3 = Mild or moderate paraparesis or hemiparesis, or severe monoparesis 4 = Marked paraparesis or hemiparesis, moderate quadriparesis, or 5 = Paraplegia, hemiplegia, or marked quadriparesis B. Cerebellar functions 0 = Normal 1 = Abnormal signs without disability 2 = Mild ataxia 3 = Moderate truncal or limb ataxia 4 = Severe ataxia all limbs 5 = Unable to perform coordinated movements due to ataxia C. Brainstem functions 0 = Normal 1 = Signs only 2 = Moderate nystagmus or other mild disability 3 = Severe nystagmus, marked extraocular weakness, or moderate disability of other cranial nerves 5 = Inability to swallow or speak

1	D. Sensory functions 1 = Vibration or figure-writing decrease only, in 1 or 2 limbs 2 = Mild decrease in touch or pain or position sense, and/or moderate decrease in vibration in 1 or 2 limbs, or vibratory decrease alone in 3 or 4 limbs 3 = Moderate decrease in touch or pain or position sense, and/or essentially lost vibration in 1 or 2 limbs, or mild decrease in touch or pain, and/or moderate decrease in all proprioceptive tests in 3 or 4 limbs 4 = Marked decrease in touch or pain or loss of proprioception, alone or combined, in 1 or 2 limbs or moderate decrease in touch or pain and/or severe proprioceptive decrease in more than 2 limbs 5 = Loss (essentially) of sensation in 1 or 2 limbs or moderate decrease in touch or pain and/or loss of proprioception for most of the body below the head 6 = Sensation essentially lost below the head

1	E. Bowel and bladder functions 0 = Normal 1 = Mild urinary hesitancy, urgency, or retention 2 = Moderate hesitancy, urgency, retention of bowel or bladder, or rare urinary 4 = In need of almost constant catheterization 5 = Loss of bladder function 6 = Loss of bowel and bladder function F. Visual (or optic) functions 0 = Normal 1 = Scotoma with visual acuity (corrected) better than 20/30 2 = Worse eye with scotoma with maximal visual acuity (corrected) of 20/30 to 3 = Worse eye with large scotoma, or moderate decrease in fields, but with maximal visual acuity (corrected) of 20/60 to 20/99 4 = Worse eye with marked decrease of fields and maximal acuity (corrected) of 20/100 to 20/200; grade 3 plus maximal acuity of better eye of 20/60 or less 5 = Worse eye with maximal visual acuity (corrected) less than 20/200; grade 4 plus maximal acuity of better eye of 20/60 or less 6 = Grade 5 plus maximal visual acuity of better eye of 20/60 or less G. Cerebral (or mental) functions

1	G. Cerebral (or mental) functions Source: Adapted from JF Kurtzke: Rating neurologic impairment in multiple sclerosis: An expanded disability status scale (EDSS). Neurology 33:1444, 1983.

1	Approximately 2–10% of IFN-β-1a (Avonex) recipients, 15–25% of IFN-β-1a (Rebif) recipients, and 30–40% of IFN-β-1b (Betaseron/ Extavia) recipients develop neutralizing antibodies to IFN-β, which may disappear over time. Two very large randomized trials (one with >2000 patients) provide unequivocal evidence that neutralizing antibodies reduce efficacy as determined by several MRI outcomes. Paradoxically, however, these same trials, despite abundant statistical power, failed to demonstrate any concomitant impact on the clinical outcomes of disability and relapse rate. The reason for this clinical-radiologic dissociation is unresolved. For a patient doing well on therapy, the presence of antibodies should not affect treatment. Conversely, for a patient doing poorly on therapy, alternative treatment should be considered, even if there are no detectable antibodies.

1	Glatiramer Acetate Glatiramer acetate is a synthetic, random polypeptide composed of four amino acids (L-glutamic acid, L-lysine, L-alanine, and L-tyrosine). Its mechanism of action may include (1) induction of antigen-specific suppressor T cells; (2) binding to MHC molecules, thereby displacing bound MBP; or (3) altering

1	Dose, Route, and Schedule Attack Rate, Mean Change in Disease Severity New T2 Lesionsd Total Burden of Disease aPercentage reductions (or increases) have been calculated by dividing the reported rates in the treated group by the comparable rates in the placebo group, except for magnetic resonance imaging (MRI) disease burden, which was calculated as the difference in the median percent change between the treated and placebo groups. bSeverity = 1 point Expanded Disability Status Score progression, sustained for 3 months (in the IFN-β-1a 30 μg qw trial, this change was sustained for 6 months; in the IFN-β-1b trial, this was over 3 years). cDifferent studies measured these MRI measures differently, making comparisons difficult (numbers for new T2 represent the best case scenario for each trial). dNew lesions seen on T2-weighted MRI. ep = .001. fp = .01. gp = .05.

1	Abbreviations: DMF, dimethyl fumarate; FDA, U.S. Food and Drug Administration; FGM, fingolimod; GA, glatiramer acetate; IFN-β, interferon β; IM, intramuscular; IV, intravenous; MTX, mitoxantrone; NR, not reported; NS, not significant; NTZ, natalizumab; PO, oral; q3mo, once every 3 months; qd, daily; qmo, once per month; qod, every other day; qw, once per week; qyr, once per year; SC, subcutaneous; TF, teriflunomide; tiw, three times per week.

1	the balance between proinflammatory and regulatory cytokines. Glatiramer acetate reduces the attack rate (whether measured clinically or by MRI) in RRMS. Glatiramer acetate also benefits disease severity measures, although, for clinical disability, this is less well established than for IFN-β. Nevertheless, two very large head-tohead trials demonstrated that the impact of glatiramer acetate on clinical relapse rates and disability was comparable to high-dose, high-frequency IFN-β. Therefore, glatiramer acetate should be considered as an equally effective alternative to IFN-β in RRMS patients. Its usefulness in progressive disease is unknown. Glatiramer acetate is administered by subcutaneous injection of either 20 mg every day or 40 mg thrice weekly. Injection-site reactions also occur with glatiramer acetate. Initially, these were thought to be less severe than with IFN-β, although two recent head-to-head comparisons of high-dose, high-frequency IFN-β to daily glatiramer acetate did

1	glatiramer acetate. Initially, these were thought to be less severe than with IFN-β, although two recent head-to-head comparisons of high-dose, high-frequency IFN-β to daily glatiramer acetate did not bear out this impression. In addition, approximately 15% of patients experience one or more episodes of flushing, chest tightness, dyspnea, palpitations, and anxiety after injection. This systemic reaction is unpredictable, brief (duration <1 h), and tends not to recur. Finally, some patients experience lipoatrophy, which, on occasion, can be disfiguring and require cessation of treatment.

1	Natalizumab Natalizumab is a humanized monoclonal antibody directed against the α4 subunit of α4β1 integrin, a cellular adhesion molecule expressed on the surface of lymphocytes. It prevents lymphocytes from binding to endothelial cells, thereby preventing lymphocytes from penetrating the BBB and entering the CNS. Natalizumab is highly effective in reducing the attack rate and significantly improves all measures of disease severity in MS (both clinical and MRI). Moreover, it is well-tolerated, and the dosing schedule of monthly intravenous infusions makes it very convenient for patients. However, progressive multifocal leukoencephalopathy (PML), a life-threatening condition resulting from infection by the John Cunningham (JC) virus, has occurred in approximately 0.3% of patients treated with natalizumab. The incidence of PML is very low in the first year of treatment but then rises by the second year to reach a level of about 2 cases per 1000 patients per year. Nevertheless, the

1	with natalizumab. The incidence of PML is very low in the first year of treatment but then rises by the second year to reach a level of about 2 cases per 1000 patients per year. Nevertheless, the measurement of antibodies against the JC virus in the serum can be used to stratify this risk. Thus, in patients who do not have these antibodies, the risk of PML is either minimal or nonexistent (as long as they remain JC antibody free). Conversely, in patients who have these antibodies (especially those who have them in high titer), the risk may be as high as 0.6% or greater. The risk is also high in patients who have previously received immunosuppressive therapy. Natalizumab is currently recommended only for JC antibody–negative patients, unless they have failed alternative therapies or if they have a particularly aggressive disease course. Head-to-head data show that natalizumab is superior to low-dose (weekly) IFN-β-1a in RRMS. However, its relative efficacy compared to other agents has

1	have a particularly aggressive disease course. Head-to-head data show that natalizumab is superior to low-dose (weekly) IFN-β-1a in RRMS. However, its relative efficacy compared to other agents has not been established conclusively.

1	Natalizumab, 300 mg, is administered by IV infusion each month. Treatment with natalizumab is, in general, well tolerated. A small percentage (<10%) of patients experience hypersensitivity reactions (including anaphylaxis), and ∼6% develop neutralizing antibodies to the molecule (only half of which persist).

1	The major concern with long-term treatment is the risk of PML. Approximately half of the adult population is JC antibody positive, indicating that they experienced an asymptomatic infection with the JC virus at some time in the past. Nevertheless, because the risk is extremely low during the first year of treatment with natalizumab (regardless of antibody status), natalizumab can still be used safely in JC antibody–positive patients for a period of 12 months. After this time, in antibody-positive patients, a change to another disease-modifying therapy should be strongly considered. By contrast, persistently antibody-negative patients can be continued on treatment indefinitely. Up to 2% of seronegative MS patients undergoing treatment with natalizumab seroconvert annually; thus it is recommended that JC antibody status be assessed at 6-month intervals in all patients receiving treatment with this agent.

1	Fingolimod Fingolimod is a sphingosine-1-phosphate (S1P) inhibitor that prevents the egress of lymphocytes from the secondary lymphoid organs such as the lymph nodes and spleen. Its mechanism of action is probably due, in part, to the trapping of lymphocytes in the periphery and inhibiting their trafficking to the CNS. Fingolimod reduces the attack rate and significantly improves all measures of disease severity in MS. It is well tolerated, and the daily oral dosing schedule makes it very convenient for patients. A large head-to-head phase 3 randomized study demonstrated the superiority of fingolimod over low-dose (weekly) IFN-β-1a. However, its relative efficacy compared to other agents has not been established conclusively.

1	Fingolimod, 0.5 mg, is administered orally each day. Treatment with fingolimod is also, in general, well tolerated. Mild abnormalities on routine laboratory evaluation (e.g., elevated liver function tests or lymphopenia) are more common than in controls, sometimes requiring discontinuation of the medication. Firstand second-degree heart block and bradycardia can also occur when fingolimod therapy is initiated. A 6-h period of observation (including electrocardiogram monitoring) is recommended for all patients receiving their first dose, and individuals with preexisting cardiac disease should probably not be treated with this agent. Other side effects 2670 include macular edema and, rarely, disseminated varicella-zoster virus (VZV) infection; prior to initiating therapy with fingolimod, an ophthalmic exam and VZV vaccination for seronegative individuals are indicated.

1	Dimethyl Fumarate (DMF) Although the precise mechanisms of action of DMF are not fully understood, it seems to have anti-inflammatory effects through its modulation of the expression of proinflammatory and anti-inflammatory cytokines. Also, DMF inhibits the ubiquitylation and degradation of nuclear factor E2-related factor 2 (Nrf2)—a transcription factor that binds to the antioxidant response elements (AREs) located on the DNA and thereby induces the transcription of several antioxidant proteins. DMF reduces the attack rate and significantly improves all measures of disease severity in MS patients. However, its twice-daily oral dosing schedule makes it somewhat less convenient for patients than daily oral therapies. In addition, compliance is likely to be less with a twice-daily dosing regimen—a factor that could be of concern given the observation (in a small clinical trial) that once-daily DMF lacks efficacy. A head-to-head trial provided evidence that DMF was superior to glatiramer

1	factor that could be of concern given the observation (in a small clinical trial) that once-daily DMF lacks efficacy. A head-to-head trial provided evidence that DMF was superior to glatiramer acetate on some outcome measures.

1	DMF, 240 mg, is administered orally twice each day. Gastrointestinal side effects (abdominal discomfort, nausea, vomiting, flushing, and diarrhea) are common at the start of therapy but generally subside with continued administration. Other adverse events included mild decreases in neutrophil and lymphocyte counts and mild elevations in liver enzymes. Nevertheless, in general, treatment with DMF is well tolerated after an initial period of adjustment. Following the release of DMF, four cases of PML were reported in patients receiving other products (not Tecfidera) that contained DMF. Each of these patients was lymphocytopenic, and most had received previous immunosuppressant therapy so that the relationship of DMF to the PML (if any) in these cases is uncertain. Nevertheless, these reports underscore the fact, stated previously, that long-term safety can never be guaranteed by the results of short-term trials. In the case of DMF for MS, only time and experience will tell us whether or

1	underscore the fact, stated previously, that long-term safety can never be guaranteed by the results of short-term trials. In the case of DMF for MS, only time and experience will tell us whether or not there is any cause for concern.

1	Teriflunomide Teriflunomide inhibits the mitochondrial enzyme dihydro-orotate dehydrogenase, which is a key part of the pathway for de novo pyrimidine biosynthesis from carbamoyl phosphate and aspartate. It is the active metabolite of the drug leflunomide (FDA-approved for rheumatoid arthritis), and it exerts its anti-inflammatory effects by limiting the proliferation of rapidly dividing T and B cells. This enzyme is not involved in the so-called “salvage pathway,” by which existing pyrimidine pools are recycled for DNA and RNA synthesis in resting and homeostatically proliferating cells. Consequently, teriflunomide is considered to be cytostatic rather than cytotoxic. Teriflunomide reduces the attack rate and significantly improves all measures of disease severity in MS patients. It is well tolerated, and its daily oral dosing schedule makes it very convenient for patients. A head-to-head trial suggested the equivalence, but not superiority, of teriflunomide and high-dose

1	It is well tolerated, and its daily oral dosing schedule makes it very convenient for patients. A head-to-head trial suggested the equivalence, but not superiority, of teriflunomide and high-dose (thriceweekly) IFN-β-1a. Teriflunomide, either 7 or 14 mg, is administered orally each day. In the pivotal clinical trials, mild hair thinning and gastrointestinal symptoms (nausea and diarrhea) were more common than in controls, but in general, treatment with teriflunomide was well tolerated. As with any new agent, the long-term safety is not guaranteed by the results of short-term trials. A major limitation, especially in women of childbearing age, is its possible teratogenicity (pregnancy category X); teriflunomide can remain in the bloodstream for 2 years, and it is recommended that exposed men and women who wish to conceive receive cholestyramine or activated charcoal to eliminate residual drug.

1	Mitoxantrone Hydrochloride Mitoxantrone, an anthracenedione, exerts its antineoplastic action by (1) intercalating into DNA and producing both strand breaks and interstrand cross-links, (2) interfering with RNA synthesis, and (3) inhibiting topoisomerase II (involved in DNA repair). The FDA approved mitoxantrone on the basis of a single (relatively small) phase 3 clinical trial in Europe, in addition to an even smaller phase 2 study completed earlier. Mitoxantrone received (from the FDA) the broadest indication of any current treatment for MS. Thus, mitoxantrone is indicated for use in SPMS, in PRMS, and in patients with worsening RRMS (defined as patients whose neurologic status remains significantly abnormal between MS attacks). Despite this broad indication, however, the data supporting its efficacy are weaker than for other approved therapies.

1	Mitoxantrone can be cardiotoxic (e.g., cardiomyopathy, reduced left ventricular ejection fraction, and irreversible congestive heart failure). As a result, a cumulative dose <140 mg/m2 is not recommended. At currently approved doses (12 mg/m2 every 3 months), the maximum duration of therapy can be only 2–3 years. Furthermore, >40% of women will experience amenorrhea, which may be permanent. Finally, there is risk of acute leukemia from mitoxantrone, estimated as at least a 1% lifetime risk, and this complication has been reported in several mitoxantrone-treated MS patients. Because of these risks, and a growing list of alternative therapies, mitoxantrone is now only rarely used for MS. It should not be used as a first-line agent in either RRMS or relapsing SPMS, but might be considered in selected patients with a progressive course who have failed other therapies.

1	Alemtuzumab Alemtuzumab is a humanized monoclonal antibody directed against the CD52 antigen, which is expressed on both monocytes and lymphocytes. It causes lymphocyte depletion (of both B and T cells) and a change in the composition of lymphocyte subsets. Both of these changes, particularly the impact on lymphocyte subsets, are long lasting. In preliminary trials, alemtuzumab markedly reduced the attack rate and significantly improved all measures of disease severity in MS patients. In two phase 3 trials, however, its impact on clinical disability was less convincing. Notably, both trials used the active comparator of thrice-weekly, high-dose IFN-β-1a. The European and Canadian drug agencies were the first to approve this agent for use in RRMS; the FDA has also approved alemtuzumab, but only after an appeal following initial disapproval. The reasons for the initial disapproval were based on a perceived lack of a convincing disability effect and concerns over potential toxicity. The

1	only after an appeal following initial disapproval. The reasons for the initial disapproval were based on a perceived lack of a convincing disability effect and concerns over potential toxicity. The toxicities of concern were the occurrence (during the trial or thereafter) of (1) autoimmune diseases including thyroiditis, Graves’ disease, thrombocytopenia, hemolytic anemia, pancytopenia, antiglomerular basement membrane disease, and membranous glomerulonephritis; (2) malignancies including thyroid cancer, melanoma, breast cancer, and human papillomavirus (HPV)–related cancers; (3) serious infections; and (4) infusion reactions.

1	Initiating and Changing Treatment Previously, most patients with relapsing forms of MS received injectable agents (IFN-β or glatiramer acetate) as first-line therapy. However, with the introduction of effective and probably safe oral agents, including DMF, fingolimod, and teriflunomide, this has begun to change. In addition, the monthly infusion therapy natalizumab, which is highly effective, well tolerated, and apparently safe in JC antibody–negative patients, provides an attractive option in many cases. As noted above, with the exception of the first-generation injectable agents, long-term safety data are not available, and for the most part, comparative data are lacking. The value of combination therapy is also largely unknown, although a recent clinical trial demonstrated no added benefit to the combination of glatiramer acetate with low-dose, once-weekly IFN-β-1a.

1	Despite these unknowns, clinicians need to make decisions based on the best available evidence, coupled with practical considerations. One reasonable approach stratifies initial decision-making based on two levels of disease aggressiveness (Fig. 458-4). milD initiAl courSe In the case of recent onset, normal exam or minimal impairment (EDSS ≤2.5 or less), or low disease activity, either an injectable (IFN-β or glatiramer acetate) or an oral (DMF,

1	milD initiAl courSe In the case of recent onset, normal exam or minimal impairment (EDSS ≤2.5 or less), or low disease activity, either an injectable (IFN-β or glatiramer acetate) or an oral (DMF, Continue therapy Good response Intolerant or poor response Continue periodic clinical/ MRI assessments No change Successive trials of alternatives* Identify and treat any underlying infection or trauma Exacerbation Pseudoexacerbation Initial course Mild Moderate or severe Acute neurologic change Stable Relapsing-Remitting MS Functional impairment No functional impairment ?Low attack frequency or single attack ?Normal neurologic exam ?Low disease burden by MRI No Yes Repeat clinical exam and MRI in 6 months Clinical or MRI change Options: 1. Injectable (GA or IFB-˜) 2. Oral (DMF, fingolimod, or (teriflunomide) 3. Natalizumab (if JCV-negative) Options: 1. Oral (DMF, fingolimod, or (teriflunomide) 2. Natalizumab (if JCV-negative) Methylprednisolone/ prednisone Symptomatic therapy

1	Multiple Sclerosis and Other Demyelinating DiseasesChapter 458Secondary progressive MS With relapses Without relapses Consider 1. IFN-˜1a, or2. IFN-˜1b Intolerant or poor response 1. Mitoxantrone 2. Azathioprine 3. Methotrexate 4. Pulse cyclophosphamide 5. IVIg 6. Pulse methylprednisolone No proven treatment Primary progressive MS Symptomatic therapy Progressive MS Consider Rx with one of the following: FIGURE 458-4 Therapeutic decision-making for multiple sclerosis (MS). *Can include trials of different preparations of interferon β (IFN-β, particularly advancing from once-weekly (Avonex) to a more frequent (e.g., Rebif, Betaseron/Extavia) dosing regimen. Options also include use of natalizumab in JC virus–positive patients. MRI, magnetic resonance imaging.

1	fingolimod, or teriflunomide) agent is reasonable. Although head-to-head comparisons are not available, natalizumab is thought to be more effective than these other agents, and therefore, this therapy can be considered even in minimally affected, JCV antibody–seronegative patients. The injectable agents (IFN-β and glatiramer acetate) have a superb track record for safety but have a high nuisance factor due to the need for frequent injections, as well as bothersome side effects that contribute to noncompliance. Some of the oral agents (DMF and fingolimod) are probably more effective than the injectables, but long-term risks are mostly unknown; DMF produces bothersome gastrointestinal symptoms in many patients at least initially (can be mitigated by beginning at one-quarter strength and gradually advancing to full dose), and fingolimod can lead to bradycardia and other cardiac disturbances of unclear clinical significance. Teriflunomide may be less effective than the other oral agents,

1	advancing to full dose), and fingolimod can lead to bradycardia and other cardiac disturbances of unclear clinical significance. Teriflunomide may be less effective than the other oral agents, and there are concerns about its possible long-lasting pregnancy risks. Nevertheless, its long-term safety has likely been established because of its extensive human exposure as the active metabolite of leflunomide—a drug long approved by the FDA.

1	moDerAte or SeVere initiAl courSe In highly active disease or moderate impairment (EDSS >2.5), either a highly effective oral agent (DMF or fingolimod) or, if the patient is JC virus antibody seronegative, infusion therapy with natalizumab is recommended. Regardless of which agent is chosen first, treatment should probably be changed in patients who continue to have relapses, progressive neurologic impairment or, arguably, ongoing evidence of subclinical MRI activity (Fig. 458-4).

1	The long-term impact of these treatments on the disease course remains controversial, although several recent studies have shown that these agents improve the long-term outcome of MS including a prolongation of the time to reach certain disability outcomes (e.g., SPMS and requiring assistance to ambulate) and reduction in MS-related mortality. These benefits seem most conspicuous when treatment begins early in the RRMS stage of the illness. Unfortunately, however, already established progressive symptoms do not respond well to treatment with these disease-modifying therapies. Because progressive symptoms are likely to result from accumulated axonal and neuronal loss, many experts now believe that very early treatment with a disease-modifying drug is appropriate for most MS patients. It may also be reasonable to delay initiating treatment in patients with (1) normal neurologic exams, (2) a single attack or a low attack frequency, and (3) a low burden of disease as assessed by brain

1	may also be reasonable to delay initiating treatment in patients with (1) normal neurologic exams, (2) a single attack or a low attack frequency, and (3) a low burden of disease as assessed by brain MRI. Untreated patients, however, should be followed closely with periodic brain MRI scans; the need for therapy is reassessed if scans reveal evidence of ongoing, subclinical disease. Finally, vitamin D deficiency should be corrected in all patients with MS, and generally this requires oral supplementation with vitamin D3, 4000 to 5000 IU daily.

1	DISEASE-MODIFYING THERAPIES FOR PROGRESSIVE MS SPMS High-dose IFN-β probably has a beneficial effect in patients with SPMS who are still experiencing acute relapses. IFN-β is probably ineffective in patients with SPMS who are not having acute attacks. All of the other agents have not yet been studied in this patient population. Although mitoxantrone has been approved for patients with progressive MS, this is not the population studied in the pivotal trial. Therefore no evidence-based recommendation can be made with regard to its use in this setting.

1	PPMS No therapies have been convincingly shown to modify the course of PPMS. A phase 3 clinical trial of glatiramer acetate in PPMS was stopped because of lack of efficacy. A phase 2/3 trial of the monoclonal antibody rituximab (anti-CD20) in PPMS was 2672 also negative, but in a preplanned secondary analysis, treatment appeared to modestly slow disability progression in patients with Gd-enhancing lesions at entry; the results of a follow-up trial with a fully humanized monoclonal anti-CD20 therapy (ocrelizumab) will soon be available. Azathioprine (2–3 mg/kg per day) has been used primarily in SPMS. Meta-analysis of published trials suggests that azathioprine is marginally effective at lowering relapse rates, although a benefit on disability progression has not been demonstrated.

1	Methotrexate (7.5–20 mg/week) was shown in one study to slow the progression of upper extremity dysfunction in SPMS. Because of the possibility of developing irreversible liver damage, some experts recommend a blind liver biopsy after 2 years of therapy. Cyclophosphamide (700 mg/m2, every other month) may be helpful for treatment-refractory patients who are (1) otherwise in good health, (2) ambulatory, and (3) <40 years of age. Because cyclophosphamide can be used for periods in excess of 3 years, it may be preferable to mitoxantrone in these circumstances. Intravenous immunoglobulin (IVIg), administered in monthly pulses (up to 1 g/kg) for up to 2 years, appears to reduce annual exacerbation rates. However, its use is limited because of its high cost, questions about optimal dose, and uncertainty about its having any impact on long-term disability. Methylprednisolone, administered in one study as monthly high-dose intravenous pulses, reduced disability progression (see above).

1	Methylprednisolone, administered in one study as monthly high-dose intravenous pulses, reduced disability progression (see above). Many purported treatments for MS have never been subjected to scientific scrutiny. These include dietary therapies (e.g., the Swank diet, in addition to others), megadose vitamins, calcium orotate, bee stings, cow colostrum, hyperbaric oxygen, procarin (a combination of histamine and caffeine), chelation, acupuncture, acupressure, various Chinese herbal remedies, and removal of mercury-amalgam tooth fillings, among many others. Patients should avoid costly or potentially hazardous unproven treatments. Many such treatments lack biologic plausibility. For example, no reliable case of mercury poisoning resembling typical MS has ever been described. Although potential roles for EBV, human herpesvirus (HHV) 6, or chlamydia have been suggested for MS, these reports are unconfirmed, and treatment with antiviral agents or antibiotics is not recommended.

1	Most recently, chronic cerebrospinal insufficiency (CCSVI) has been proposed as a cause of MS, and vascular-surgical intervention is recommended. However, the failure of independent investigators to even approximate the initial claims of 100% sensitivity and 100% specificity for the diagnostic procedure have raised considerable doubt that CCSVI is a real entity. Certainly, any potentially dangerous surgery should be avoided until more rigorous science is available.

1	For all patients, it is useful to encourage attention to a healthy lifestyle, including maintaining an optimistic outlook, a healthy diet, and regular exercise as tolerated (swimming is often well-tolerated because of the cooling effect of cold water). It is reasonable also to correct vitamin D deficiency with oral vitamin D and to recommend dietary supplementation with long-chain (omega-3) unsaturated fatty acids (present in oily fish such as salmon), because of their biologic plausibility for MS pathogenesis, safety, and general health benefits. Ataxia/tremor is often intractable. Clonazepam, 1.5–20 mg/d; primidone, 50–250 mg/d; propranolol, 40–200 mg/d; or ondansetron, 8–16 mg/d, may help. Wrist weights occasionally reduce tremor in the arm or hand. Thalamotomy or deep-brain stimulation has been tried with mixed success.

1	Spasticity and spasms may improve with physical therapy, regular exercise, and stretching. Avoidance of triggers (e.g., infections, fecal impactions, bed sores) is extremely important. Effective medications include baclofen (20–120 mg/d), diazepam (2–40 mg/d), tizanidine (8–32 mg/d), dantrolene (25–400 mg/d), and cyclobenzaprine hydrochloride (10–60 mg/d). For severe spasticity, a baclofen pump (delivering medication directly into the CSF) can provide substantial relief.

1	Weakness can sometimes be improved with the use of potassium channel blockers such as 4-aminopyridine (10–40 mg/d) and 3,4-diaminopyridine (40–80 mg/d), particularly in the setting where lower extremity weakness interferes with the patient’s ability to ambulate. The FDA has approved 4-aminopyridine (at 20 mg/d), and this can be obtained either as dalfampridine (Ampyra) or, more cheaply, through a compounding pharmacy. The principle concern with the use of these agents is the possibility of inducing seizures at high doses. Pain is treated with anticonvulsants (carbamazepine, 100–1000 mg/d; phenytoin, 300–600 mg/d; gabapentin, 300–3600 mg/d; or pregabalin, 50–300 mg/d), antidepressants (amitriptyline, 25–150 mg/d; nortriptyline, 25–150 mg/d; desipramine, 100–300 mg/d; or venlafaxine, 75–225 mg/d), or antiarrhythmics (mexiletine, 300–900 mg/d). If these approaches fail, patients should be referred to a comprehensive pain management program.

1	Bladder dysfunction management is best guided by urodynamic testing. Evening fluid restriction or frequent voluntary voiding may help detrusor hyperreflexia. If these methods fail, propantheline bromide (10–15 mg/d), oxybutynin (5–15 mg/d), hyoscyamine sulfate (0.5–0.75 mg/d), tolterodine tartrate (2–4 mg/d), or solifenacin (5–10 mg/d) may help. Coadministration of pseudoephedrine (30–60 mg) is sometimes beneficial. Detrusor/sphincter dyssynergia may respond to phenoxybenzamine (10–20 mg/d) or terazosin hydrochloride (1–20 mg/d). Loss of reflex bladder wall contraction may respond to bethanechol (30– 150 mg/d). However, both conditions often require catheterization.

1	Urinary tract infections should be treated promptly. Patients with large postvoid residual urine volumes are predisposed to infections. Prevention by urine acidification (with cranberry juice or vitamin C) inhibits some bacteria. Prophylactic administration of antibiotics is sometimes necessary but may lead to colonization by resistant organisms. Intermittent catheterization may help to prevent recurrent infections. Treatment of constipation includes high-fiber diets and fluids. Natural or other laxatives may help. Fecal incontinence may respond to a reduction in dietary fiber. Depression should be treated. Useful drugs include the selective serotonin reuptake inhibitors (fluoxetine, 20–80 mg/d, or sertraline, 50–200 mg/d), the tricyclic antidepressants (amitriptyline, 25–150 mg/d; nortriptyline, 25–150 mg/d; or desipramine, 100–300 mg/d), and the nontricyclic antidepressants (venlafaxine, 75–225 mg/d).

1	Fatigue may improve with assistive devices, help in the home, or successful management of spasticity. Patients with frequent nocturia may benefit from anticholinergic medication at bedtime. Primary MS fatigue may respond to amantadine (200 mg/d), methylphenidate (5–25 mg/d), or modafinil (100–400 mg/d). Cognitive problems may respond to the cholinesterase inhibitor donepezil hydrochloride (10 mg/d). Paroxysmal symptoms respond dramatically to low-dose anticonvulsants (acetazolamide, 200–600 mg/d; carbamazepine, 50–400 mg/d; phenytoin, 50–300 mg/d; or gabapentin, 600–1800 mg/d). Heat sensitivity may respond to heat avoidance, air-conditioning, or cooling garments.

1	Heat sensitivity may respond to heat avoidance, air-conditioning, or cooling garments. Sexual dysfunction may be helped by lubricants to aid in genital stimulation and sexual arousal. Management of pain, spasticity, fatigue, and bladder/bowel dysfunction may also help. Sildenafil (50–100 mg), tadalafil (5–20 mg), or vardenafil (5–20 mg), taken 1–2 h before sex, is now the standard treatment for maintaining erections. Numerous clinical trials are currently under way. These include studies on (1) monoclonal antibodies against CD20 to deplete B cells and against the IL-2 receptor; (2) selective oral sphingosine-1-phosphate receptor antagonists to sequester lymphocytes in secondary lymphoid organs; (3) estriol to induce a pregnancy-like state; (4) molecules to promote remyelination; and (4) bone marrow transplantation.

1	Acute MS (Marburg’s variant) is a fulminant demyelinating process that in some cases progresses inexorably to death within 1–2 years. Typically, there are no remissions. When acute MS presents as a solitary, usually cavitary, lesion, a brain tumor is often suspected. In such cases, a brain biopsy is usually required to establish the diagnosis. An antibody-mediated process appears to be responsible for most cases. Marburg’s variant does not seem to follow infection or vaccination, and it is unclear whether this syndrome represents an extreme form of MS or another disease altogether. No controlled trials of therapy exist; high-dose glucocorticoids, plasma exchange, and cyclophosphamide have been tried, with uncertain benefit.

1	Neuromyelitis optica (NMO; Devic’s disease) is an aggressive inflammatory disorder characterized by recurrent attacks of ON and myelitis (Table 458-7). NMO is more frequent in women than men (>3:1), typically begins in childhood or early adulthood but can arise at any age, and is uncommon in whites compared with individuals of Asian and African ancestry. Attacks of ON can be bilateral (rare in MS) or unilateral; myelitis can be severe and transverse (rare in MS) and is typically longitudinally extensive, involving three or more contiguous vertebral segments. Also in contrast to MS, progressive symptoms do not occur in NMO. The brain MRI was earlier thought to be normal in NMO, but it is now recognized that in approximately half of cases, there are lesions involving the hypothalamus causing an endocrinopathy; the lower brainstem presenting as intractable hiccoughs or vomiting from involvement of the area postrema in the lower medulla; or the cerebral hemispheres producing focal

1	causing an endocrinopathy; the lower brainstem presenting as intractable hiccoughs or vomiting from involvement of the area postrema in the lower medulla; or the cerebral hemispheres producing focal symptoms, encephalopathy, or seizures. Large MRI lesions in the cerebral hemispheres can be asymptomatic, sometimes have a “cloud-like” appearance and, unlike MS lesions, are often not destructive, and can resolve completely. Spinal cord MRI lesions typically consist of focal enhancing areas of swelling and tissue destruction, extending over three or more spinal cord segments, and on axial sequences, these are centered on the gray matter of the cord. CSF findings include pleocytosis greater than that observed in MS, with neutrophils and eosinophils present in some cases; OCBs are uncommon, occurring in fewer than 30% of NMO patients. The pathology of NMO is a distinctive astrocytopathy with inflammation, a loss of astrocytes, and an absence of staining of the water channel protein

1	occurring in fewer than 30% of NMO patients. The pathology of NMO is a distinctive astrocytopathy with inflammation, a loss of astrocytes, and an absence of staining of the water channel protein aquaporin-4 by immunohistochemistry, plus thickened blood vessel walls, demyelination, and deposition of antibody and complement.

1	NMO is best understood as a syndrome with diverse causes. Up to 40% of patients have a systemic autoimmune disorder, often systemic lupus erythematosus, Sjögren’s syndrome, perinuclear antineutrophil cytoplasmic antibody (p-ANCA)–associated vasculitis, myasthenia gravis, Hashimoto’s thyroiditis, or mixed connective tissue disease. In others, onset may be associated with acute infection with VZV, EBV, HIV, or tuberculosis. Rare cases appear to be paraneoplastic and Required: 1. 2. Supportive (2 of 3 criteria required): 1. 2. 3. Aquaporin-4 seropositivity Source: Adapted from DM Wingerchuk et al: Neurology 66:1485, 2006.

1	associated with breast, lung, or other cancers. NMO is often idio-2673 pathic, however. NMO is usually disabling over time; in one series, respiratory failure from cervical myelitis was present in one-third of patients, and 8 years after onset, 60% of patients were blind and more than half had permanent paralysis of one or more limbs. A highly specific autoantibody directed against aquaporin-4 is present in the sera of approximately two-thirds of patients with a clinical diagnosis of NMO. Seropositive patients have a very high risk for future relapses; more than half will relapse within 1 year if untreated. Aquaporin-4 is localized to the foot processes of astrocytes in close apposition to endothelial surfaces, as well as at paranodal regions near nodes of Ranvier. It is likely that aquaporin-4 antibodies are pathogenic, as passive transfer of antibodies from NMO patients into laboratory animals reproduce histologic features of the disease.

1	When MS affects individuals of African or Asian ancestry, there is a propensity for demyelinating lesions to involve predominantly the optic nerve and spinal cord, an MS subtype termed opticospinal MS. Interestingly, some individuals with opticospinal MS are seropositive for aquaporin-4 antibodies, suggesting that such cases represent an NMO spectrum disorder.

1	Disease-modifying therapies have not been rigorously studied in NMO. Acute attacks of NMO are usually treated with high-dose glucocorticoids (solumedrol 1–2 g/d for 5–10 days followed by a prednisone taper). Plasma exchange (typically 7 qod exchanges of 1.5 plasma volumes) has also been used empirically for acute episodes that fail to respond to glucocorticoids. Given the unfavorable natural history of untreated NMO, prophylaxis against relapses is recommended for most patients using one of the following regimens: mycophenylate mofetil (250 mg bid gradually increasing to 1000 mg bid); B cell depletion with anti-CD20 monoclonal antibody (rituximab); or a combination of glucocorticoids (500 mg IV methylprednisolone daily for 5 days; then oral prednisone 1 mg/kg per day for 2 months, followed by slow taper) plus azathioprine (2 mg/kg per day started on week 3). Available evidence suggests that use of IFN-β is ineffective and paradoxically may increase the risk of NMO relapses.

1	Acute disseminated encephalomyelitis (ADEM) has a monophasic course and is most frequently associated with an antecedent infection (postinfectious encephalomyelitis); approximately 5% of ADEM cases follow immunization (postvaccinal encephalomyelitis). ADEM is far more common in children than adults, and many adult cases initially thought to represent ADEM subsequently experience late relapses qualifying as either MS or another chronic inflammatory disorder such as vasculitis, sarcoid, or lymphoma. The hallmark of ADEM is the presence of widely scattered small foci of perivenular inflammation and demyelination, in contrast to larger confluent demyelinating lesions typical of MS. In the most explosive form of ADEM, acute hemorrhagic leukoencephalitis, the lesions are vasculitic and hemorrhagic, and the clinical course is devastating.

1	Postinfectious encephalomyelitis is most frequently associated with the viral exanthems of childhood. Infection with measles virus is the most common antecedent (1 in 1000 cases). Worldwide, measles encephalomyelitis is still common, although use of the live measles vaccine has dramatically reduced its incidence in developed countries. An ADEM-like illness rarely follows vaccination with live measles vaccine (1–2 in 106 immunizations). ADEM is now most frequently associated with varicella (chickenpox) infections (1 in 4000–10,000 cases). It may also follow infection with rubella, mumps, influenza, parainfluenza, EBV, HHV-6, HIV, other viruses, and Mycoplasma pneumoniae. Some patients may have a nonspecific upper respiratory infection or no known antecedent illness. In addition to measles, postvaccinal encephalomyelitis may also follow the administration 2674 of vaccines for smallpox (5 cases per million), the Semple rabies, and with predominantly cerebral involvement, acute

1	measles, postvaccinal encephalomyelitis may also follow the administration 2674 of vaccines for smallpox (5 cases per million), the Semple rabies, and with predominantly cerebral involvement, acute encephalitis due to Japanese encephalitis. Modern vaccines that do not require viral cul-infection with herpes simplex or other viruses including HIV may beture in CNS tissue have reduced the ADEM risk. difficult to exclude (Chap. 164); other considerations include hyper-All forms of ADEM presumably result from a cross-reactive coagulable states including the antiphospholipid antibody syndrome,immune response to the infectious agent or vaccine that then triggers vasculitis, neurosarcoid, primary CNS lymphoma, or metastatic can-an inflammatory demyelinating response. Autoantibodies to MBP and cer. An explosive presentation of MS can mimic ADEM, and especially to other myelin antigens have been detected in the CSF from some in adults, it may not be possible to distinguish these conditions

1	cer. An explosive presentation of MS can mimic ADEM, and especially to other myelin antigens have been detected in the CSF from some in adults, it may not be possible to distinguish these conditions atpatients with ADEM. Attempts to demonstrate direct viral invasion of onset. The simultaneous onset of disseminated symptoms and signs isthe CNS have been unsuccessful. common in ADEM and rare in MS. Similarly, meningismus, drowsi ness, coma, and seizures suggest ADEM rather than MS. Unlike MS, CLINICAL MANIFESTATIONS in ADEM, optic nerve involvement is generally bilateral and transverse In severe cases, onset is abrupt and progression rapid (hours to days). myelopathy complete. MRI findings that favor ADEM include exten-In postinfectious ADEM, the neurologic syndrome generally begins sive and relatively symmetric white matter abnormalities, basal ganglialate in the course of the viral illness as the exanthem is fading. Fever or cortical gray matter lesions, and Gd enhancement of all

1	and relatively symmetric white matter abnormalities, basal ganglialate in the course of the viral illness as the exanthem is fading. Fever or cortical gray matter lesions, and Gd enhancement of all abnormalreappears, and headache, meningismus, and lethargy progressing to areas. By contrast, OCBs in the CSF are more common in MS. In onecoma may develop. Seizures are common. Signs of disseminated study of adult patients initially thought to have ADEM, 30% experineurologic disease are consistently present (e.g., hemiparesis or enced additional relapses over a follow-up period of 3 years, and they quadriparesis, extensor plantar responses, lost or hyperactive ten-were reclassified as having MS. Occasional patients with “recurrent don reflexes, sensory loss, and brainstem involvement). In ADEM ADEM” have also been reported, especially children; however, it is not due to chickenpox, cerebellar involvement is often conspicuous. possible to distinguish this entity from atypical MS. CSF

1	ADEM ADEM” have also been reported, especially children; however, it is not due to chickenpox, cerebellar involvement is often conspicuous. possible to distinguish this entity from atypical MS. CSF protein is modestly elevated (0.5–1.5 g/L [50–150 mg/dL]). Lymphocytic pleocytosis, generally 200 cells/μL or greater, occurs in 80% of patients. Occasional patients have higher counts or a mixed polymorphonuclear-lymphocytic pattern during the initial days of the

1	Initial treatment is with high-dose glucocorticoids as for exacerba illness. Transient CSF oligoclonal banding has been reported. MRI usu tions of NMO (see above); depending on the response, treatment ally reveals extensive changes in the brain and spinal cord, consisting may need to be continued for 8 weeks. Patients who fail to respond within a few days may benefit from a course of plasma exchange recovery sequences with Gd enhancement on T1-weighted sequences. or intravenous immunoglobulin. The prognosis reflects the severity of the underlying acute illness. In recent case series of presumptive ADEM in adults, mortality rates of 5–20% are reported, and many The diagnosis is most reliably established when there is a history of survivors have permanent neurologic sequelae. recent vaccination or viral exanthematous illness. In severe cases In approaching a patient with a neuropathy, the clinician has three main

1	Anthony A. Amato, Richard J. Barohn goals: (1) identify where the lesion is, (2) identify the cause, and (3) deter mine the proper treatment. The first goal is accomplished by obtaining a thorough history, neurologic examination, and electrodiagnostic and Peripheral nerves are composed of sensory, motor, and autonomic other laboratory studies (Fig. 459-1). While gathering this information, elements. Diseases can affect the cell body of a neuron or its periph-seven key questions are asked (Table 459-1), the answers to which can eral processes, namely the axons or the encasing myelin sheaths. usually identify the category of pathology that is present (Table 459-2). Most peripheral nerves are mixed and contain sensory and motor as Despite an extensive evaluation, in approximately half of patients, no well as autonomic fibers. Nerves can be subdivided into three major etiology is ever found; these patients typically have a predominately classes: large myelinated, small myelinated, and

1	patients, no well as autonomic fibers. Nerves can be subdivided into three major etiology is ever found; these patients typically have a predominately classes: large myelinated, small myelinated, and small unmyelinated. sensory polyneuropathy and have been labeled as having idiopathic or Motor axons are usually large myelinated fibers that conduct rapidly cryptogenic sensory polyneuropathy (CSPN). (approximately 50 m/s). Sensory fibers may be any of the three types. Large-diameter sensory fibers conduct proprioception and vibratory sensation to the brain, while the smaller-diameter myelinated INFORMATION FROM THE HISTORY AND PHYSICAL EXAMINATION: SEVEN KEY and unmyelinated fibers transmit pain and temperature sensation. QUESTIONS (TABLE 459-1) Autonomic nerves are also small in diameter. Thus, peripheral neu-1. What Systems are Involved? It is important to determine if the ropathies can impair sensory, motor, or autonomic function, either patient’s symptoms and signs are motor,

1	Thus, peripheral neu-1. What Systems are Involved? It is important to determine if the ropathies can impair sensory, motor, or autonomic function, either patient’s symptoms and signs are motor, sensory, autonomic, or a com-singly or in combination. Peripheral neuropathies are further classi-bination of these. If the patient has only weakness without any evidence fied into those that primarily affect the cell body (e.g., neuronopathy of sensory or autonomic dysfunction, a motor neuropathy, neuromusor ganglionopathy), myelin (myelinopathy), and the axon (axonopathy). cular junction abnormality, or myopathy should be considered. Some These different classes of peripheral neuropathies have distinct clinical peripheral neuropathies are associated with significant autonomic nerand electrophysiologic features. This chapter discusses the clinical vous system dysfunction. Symptoms of autonomic involvement include approach to a patient suspected of having a peripheral neuropathy, fainting

1	features. This chapter discusses the clinical vous system dysfunction. Symptoms of autonomic involvement include approach to a patient suspected of having a peripheral neuropathy, fainting spells or orthostatic lightheadedness; heat intolerance; or any as well as specific neuropathies, including hereditary and acquired bowel, bladder, or sexual dysfunction (Chap. 454). There will typically neuropathies. The inflammatory neuropathies are discussed in be an orthostatic fall in blood pressure without an appropriate increase Chap. 460. in heart rate. Autonomic dysfunction in the absence of diabetes should

1	Peripheral NeuropathyChapter 459Patient Complaint: ? Neuropathy History and examination compatible with neuropathy? Mononeuropathy Mononeuropathy multiplex Polyneuropathy Evaluation of other ˜disorder or ˜reassurance and follow-up EDx Is the lesion axonal ˜or demyelinating? Is entrapment or ˜compression present? Is a contributing systemic disorder present? Axonal Demyelinating ˜with focal ˜conduction block Axonal Demyelinating Consider ˜vasculitis or other multifocal ˜process Consider ˜multifocal ˜form of ˜CIDP Subacute ˜course (months) Chronic ˜course (years) Uniform slowing, ˜chronic Nonuniform slowing, conduction block Decision on need ˜for surgery (nerve repair, ˜transposition, or release ˜procedure) Possible ˜nerve ˜biopsy Test for paraprotein, ˜HIV, Lyme disease Review history for toxins; test for associated systemic disease or intoxication Test for paraprotein, if negative Review family history; examine family members; genetic testing If chronic or ˜subacute: CIDP If

1	for toxins; test for associated systemic disease or intoxication Test for paraprotein, if negative Review family history; examine family members; genetic testing If chronic or ˜subacute: CIDP If acute: GBS Treatment appropriate ˜for specific diagnosis If tests are ˜negative, consider treatment for CIDP Treatment appropriate ˜for specific diagnosis Genetic counseling if appropriate Treatment for CIDP; see Ch. 460 IVIg or ˜plasmapheresis; ˜supportive ˜care including ˜respiratory assistance EDx EDx NoYes FIGURE 459-1 Approach to the evaluation of peripheral neuropathies. CIDP, chronic inflammatory demyelinating polyradiculoneuropathy; EDx, electrodiagnostic; GBS, Guillain-Barré syndrome; IVIg, intravenous immunoglobulin.

1	alert the clinician to the possibility of amyloid polyneuropathy. Rarely, a pandysautonomic syndrome can be the only manifestation of a peripheral neuropathy without other motor or sensory findings. The majority of neuropathies are predominantly sensory in nature. approaCh to NeuropathiC DisorDers: seveN key questioNs 1. What systems are involved? Motor, sensory, autonomic, or combinations 2. What is the distribution of weakness? 3. What is the nature of the sensory involvement? -Temperature loss or burning or stabbing pain (e.g., small fiber) -Vibratory or proprioceptive loss (e.g., large fiber) 4. Is there evidence of upper motor neuron involvement? 5. What is the temporal evolution? Acute (days to 4 weeks) Monophasic, progressive, or relapsing-remitting 6. Is there evidence for a hereditary neuropathy? Family history of neuropathy Lack of sensory symptoms despite sensory signs 7. Are there any associated medical conditions?

1	Family history of neuropathy Lack of sensory symptoms despite sensory signs 7. Are there any associated medical conditions? -Cancer, diabetes mellitus, connective tissue disease or other autoimmune diseases, infection (e.g., HIV, Lyme disease, leprosy) Preceding events, drugs, toxins 2.

1	Preceding events, drugs, toxins 2. What is the Distribution of Weakness? Delineating the pattern of weakness, if present, is essential for diagnosis, and in this regard two additional questions should be answered: (1) Does the weakness only involve the distal extremity, or is it both proximal and distal? and (2) Is the weakness focal and asymmetric, or is it symmetric? Symmetric proximal and distal weakness is the hallmark of acquired immune demyelinating polyneuropathies, both the acute form (acute inflammatory demyelinating polyneuropathy [AIDP], also known as Guillain-Barré syndrome [GBS]) and the chronic form (chronic inflammatory demyelinating polyneuropathy [CIDP]). The importance of finding symmetric proximal and distal weakness in a patient who presents with both motor and sensory symptoms cannot be overemphasized because this identifies the important subset of patients who may have a treatable acquired demyelinating neuropathic disorder (i.e., AIDP or CIDP).

1	Findings of an asymmetric or multifocal pattern of weakness narrow the differential diagnosis. Some neuropathic disorders may present with unilateral extremity weakness. In the absence of sensory symptoms and signs, such weakness evolving over weeks or months would be worrisome for motor neuron disease (e.g., amyotrophic lateral sclerosis [ALS]), but it would be important to exclude multifocal motor neuropathy that may be treatable (Chap. 452). In a patient presenting with asymmetric subacute or acute sensory and motor symptoms and signs, radiculopathies, plexopathies, compressive mononeuropathies, or multiple mononeuropathies (e.g., mononeuropathy multiplex) must be considered. 3.

1	3. What is the Nature of the Sensory Involvement? The patient may have loss of sensation (numbness), altered sensation to touch (hyperpathia or allodynia), or uncomfortable spontaneous sensations (tingling, burning, or aching) (Chap. 31). Neuropathic pain can be burning, dull, and poorly localized (protopathic pain), presumably transmitted by polymodal C nociceptor fibers, or sharp and lancinating (epicritic pain), relayed by A-delta fibers. If pain and temperature perception are lost, while vibratory and position sense are preserved along with patterNs of NeuropathiC DisorDers Pattern 1: Symmetric proximal and distal weakness with sensory loss Consider: inflammatory demyelinating polyneuropathy (GBS and CIDP) Pattern 2: Symmetric distal sensory loss with or without distal weakness Consider: cryptogenic or idiopathic sensory polyneuropathy (CSPN), diabetes mellitus and other metabolic disorders, drugs, toxins, familial (HSAN), CMT, amyloidosis, and others

1	Consider: cryptogenic or idiopathic sensory polyneuropathy (CSPN), diabetes mellitus and other metabolic disorders, drugs, toxins, familial (HSAN), CMT, amyloidosis, and others Pattern 3: Asymmetric distal weakness with sensory loss With involvement of multiple nerves Consider: multifocal CIDP, vasculitis, cryoglobulinemia, amyloidosis, sarcoid, infectious (leprosy, Lyme, hepatitis B, C, or E, HIV, CMV), HNPP, tumor infiltration With involvement of single nerves/regions Consider: may be any of the above but also could be compressive mononeuropathy, plexopathy, or radiculopathy Pattern 4: Asymmetric proximal and distal weakness with sensory loss Consider: polyradiculopathy or plexopathy due to diabetes mellitus, meningeal carcinomatosis or lymphomatosis, hereditary plexopathy (HNPP, HNA), idiopathic Pattern 5: Asymmetric distal weakness without sensory loss

1	Pattern 5: Asymmetric distal weakness without sensory loss With upper motor neuron findings Consider: motor neuron disease Without upper motor neuron findings Consider: progressive muscular atrophy, juvenile monomelic amyotrophy (Hirayama’s disease), multifocal motor neuropathy, multifocal acquired motor axonopathy Pattern 6: Symmetric sensory loss and distal areflexia with upper motor neuron findings Consider: Vitamin B12, vitamin E, and copper deficiency with combined system degeneration with peripheral neuropathy, hereditary leukodystrophies (e.g., adrenomyeloneuropathy) Pattern 7: Symmetric weakness without sensory loss With proximal and distal weakness Consider: SMA With distal weakness Consider: hereditary motor neuropathy (“distal” SMA) or atypical CMT Pattern 8: Asymmetric proprioceptive sensory loss without weakness

1	Pattern 8: Asymmetric proprioceptive sensory loss without weakness Consider causes of a sensory neuronopathy (ganglionopathy): Cancer (paraneoplastic) Sjögren’s syndrome Idiopathic sensory neuronopathy (possible GBS variant) Cisplatin and other chemotherapeutic agents Vitamin B6 toxicity HIV-related sensory neuronopathy Pattern 9: Autonomic symptoms and signs Consider neuropathies associated with prominent autonomic dysfunction: Hereditary sensory and autonomic neuropathy Amyloidosis (familial and acquired) Diabetes mellitus Idiopathic pandysautonomia (may be a variant of Guillain-Barré syndrome) Porphyria HIV-related autonomic neuropathy Vincristine and other chemotherapeutic agents

1	Abbreviations: CIDP, chronic inflammatory demyelinating polyneuropathy; CMT, Charcot-Marie-Tooth disease; CMV, cytomegalovirus; GBS, Guillain-Barré syndrome; HIV, human immunodeficiency virus; HNA, hereditary neuralgic amyotrophy; HNPP, hereditary neuropathy with liability to pressure palsies; HSAN, hereditary sensory and autonomic neuropathy; SMA, spinal muscular atrophy. muscle strength, deep tendon reflexes, and normal nerve conduction studies, a small-fiber neuropathy is likely. This is important, because the most likely cause of small-fiber neuropathies, when one is identified, is diabetes mellitus or glucose intolerance. Amyloid neuropathy should be considered as well in such cases, but most of these small-fiber neuropathies remain idiopathic in nature despite extensive evaluation.

1	Severe proprioceptive loss also narrows the differential diagnosis. Affected patients will note imbalance, especially in the dark. A neurologic examination revealing a dramatic loss of proprioception with vibration loss and normal strength should alert the clinician to consider a sensory neuronopathy/ganglionopathy (Table 459-2, Pattern 8). In particular, if this loss is asymmetric or affects the arms more than the legs, this pattern suggests a non-length-dependent process as seen in sensory neuronopathies.

1	4. Is There Evidence of Upper Motor Neuron Involvement? If the patient presents with symmetric distal sensory symptoms and signs suggestive of a distal sensory neuropathy, but there is additional evidence of symmetric upper motor neuron involvement (Chap. 30), the physician should consider a disorder such as combined system degeneration with neuropathy. The most common cause for this pattern is vitamin deficiency, but other causes of combined system degeneration with neuropathy should be considered (e.g., copper deficiency, HIV infection, severe hepatic disease, adrenomyeloneuropathy). 5.

1	5. What is the Temporal Evolution? It is important to determine the onset, duration, and evolution of symptoms and signs. Does the disease have an acute (days to 4 weeks), subacute (4–8 weeks), or chronic (>8 weeks) course? Is the course monophasic, progressive, or relapsing? Most neuropathies are insidious and slowly progressive in nature. Neuropathies with acute and subacute presentations include GBS, vasculitis, and radiculopathies related to diabetes or Lyme disease. A relapsing course can be present in CIDP and porphyria. 6.

1	6. Is There Evidence for a Hereditary Neuropathy? In patients with slowly progressive distal weakness over many years with very little in the way of sensory symptoms yet with significant sensory deficits on clinical examination, the clinician should consider a hereditary neuropathy (e.g., Charcot-Marie-Tooth disease [CMT]). On examination, the feet may show arch and toe abnormalities (high or flat arches, hammertoes); scoliosis may be present. In suspected cases, it may be necessary to perform both neurologic and electrophysiologic studies on family members in addition to the patient. 7.

1	7. Does the Patient Have Any Other Medical Conditions? It is important to inquire about associated medical conditions (e.g., diabetes mellitus, systemic lupus erythematosus); preceding or concurrent infections (e.g. diarrheal illness preceding GBS); surgeries (e.g., gastric bypass and nutritional neuropathies); medications (toxic neuropathy), including over-the-counter vitamin preparations (B6); alcohol; dietary habits; and use of dentures (e.g., fixatives contain zinc that can lead to copper deficiency). Based on the answers to the seven key questions, neuropathic disorders can be classified into several patterns based on the distribution or pattern of sensory, motor, and autonomic involvement (Table 459-2). Each pattern has a limited differential diagnosis. A final diagnosis is established by using other clues such as the temporal course, presence of other disease states, family history, and information from laboratory studies.

1	The electrodiagnostic (EDx) evaluation of patients with a suspected peripheral neuropathy consists of nerve conduction studies (NCS) and needle electromyography (EMG). In addition, studies of autonomic function can be valuable. The electrophysiologic data provide additional information about the distribution of the neuropathy that will support or refute the findings from the history and physical examination; they can confirm whether the neuropathic disorder is a mononeuropathy, multiple mononeuropathy (mononeuropathy multiplex), radiculopathy, plexopathy, or generalized polyneuropathy. Similarly, EDx evaluation can ascertain whether the process involves only sensory fibers, motor fibers, autonomic fibers, or a combination of these. Finally, the electrophysiologic data can help distinguish axonopathies from myelinopathies as well as axonal degeneration secondary to ganglionopathies from the more common length-dependent axonopathies.

1	Abbreviations: CB, conduction block; CMAP, compound motor action potential; EMG, electromyography; SNAP, sensory nerve action potential. NCS are most helpful in classifying a neuropathy as being due to axonal degeneration or segmental demyelination (Table 459-3). In general, low-amplitude potentials with relatively preserved distal latencies, conduction velocities, and late potentials, along with fibrillations on needle EMG, suggest an axonal neuropathy. On the other hand, slow conduction velocities, prolonged distal latencies and late potentials, relatively preserved amplitudes, and the absence of fibrillations on needle EMG imply a primary demyelinating neuropathy. The presence of nonuniform slowing of conduction velocity, conduction block, or temporal dispersion further suggests an acquired demyelinating neuropathy (e.g., GBS or CIDP) as opposed to a hereditary demyelinating neuropathy (e.g., CMT type 1).

1	Autonomic studies are used to assess small myelinated (A-delta) or unmyelinated (C) nerve fiber involvement. Such testing includes heart rate response to deep breathing, heart rate, and blood pressure response to both the Valsalva maneuver and tilt-table testing and quantitative sudomotor axon reflex testing (Chap. 454). These studies are particularly useful in patients who have pure small-fiber neuropathy or autonomic neuropathy in which routine NCS are normal.

1	In patients with generalized symmetric peripheral neuropathy, a standard laboratory evaluation should include a complete blood count, basic chemistries including serum electrolytes and tests of renal and hepatic function, fasting blood glucose (FBS), HbA1c, urinalysis, thyroid function tests, B12, folate, erythrocyte sedimentation rate (ESR), rheumatoid factor, antinuclear antibodies (ANA), serum protein electrophoresis (SPEP) and immunoelectrophoresis or immunofixation, and urine for Bence Jones protein. Quantification of the concentration of serum free light chains and the kappa/lambda ratio is more sensitive than SPEP, immunoelectrophoresis, or immunofixation in looking for a monoclonal gammopathy and therefore should be done if one suspects amyloidosis. A skeletal survey should be performed in patients with acquired demyelinating neuropathies and M-spikes to look for osteosclerotic or lytic lesions. Patients with monoclonal gammopathy should also be referred to a hematologist for

1	in patients with acquired demyelinating neuropathies and M-spikes to look for osteosclerotic or lytic lesions. Patients with monoclonal gammopathy should also be referred to a hematologist for consideration of a bone marrow biopsy. An oral glucose tolerance test is indicated in patients with painful sensory neuropathies even if FBS and HbA1c are normal, 2677 as the test is abnormal in about one-third of such patients. In addition to the above tests, patients with a mononeuropathy multiplex pattern of involvement should have a vasculitis workup, including antineutrophil cytoplasmic antibodies (ANCA), cryoglobulins, hepatitis serology, Western blot for Lyme disease, HIV, and occasionally a cytomegalovirus (CMV) titer.

1	There are many autoantibody panels (various antiganglioside antibodies) marketed for screening routine neuropathy patients for a treatable condition. These autoantibodies have no proven clinical util ity or added benefit beyond the information obtained from a complete clinical examination and detailed EDx. A heavy metal screen is also not necessary as a screening procedure, unless there is a history of possible exposure or suggestive features on examination (e.g., severe painful sensorimotor and autonomic neuropathy and alopecia—thallium; severe painful sensorimotor neuropathy with or without gastrointestinal [GI] disturbance and Mee’s lines—arsenic; wrist or finger extensor weakness and anemia with basophilic stippling of red blood cells—lead).

1	In patients with suspected GBS or CIDP, a lumbar puncture is indicated to look for an elevated cerebral spinal fluid (CSF) protein. In idiopathic cases of GBS and CIDP, there should not be pleocytosis in the CSF. If cells are present, one should consider HIV infection, Lyme disease, sarcoidosis, or lymphomatous or leukemic infiltration of nerve roots. Some patients with GBS and CIDP have abnormal liver function tests. In these cases, it is important to also check for hepatitis B and C, HIV, CMV, and Epstein-Barr virus (EBV) infection. In patients with an axonal GBS (by EMG/NCS) or those with a suspicious coinciding history (e.g., unexplained abdominal pain, psychiatric illness, significant autonomic dysfunction), it is reasonable to screen for porphyria.

1	In patients with a severe sensory ataxia, a sensory ganglionopathy or neuronopathy should be considered. The most common causes of sensory ganglionopathies are Sjögren’s syndrome and a paraneoplastic neuropathy. Neuropathy can be the initial manifestation of Sjögren’s syndrome. Thus, one should always inquire about dry eyes and mouth in patients with sensory signs and symptoms. Further, some patients can manifest sicca complex without full-blown Sjögren’s syndrome. Thus, patients with sensory ataxia should have a senile systemic amyloidosis (SSA) and single strand binding (SSB) in addition to the routine ANA. To work up a possible paraneoplastic sensory ganglionopathy, antineuronal nuclear antibodies (e.g., anti-Hu antibodies) should be obtained (Chap. 122). These antibodies are most commonly seen in patients with small-cell carcinoma of the lung but are seen also in breast, ovarian, lymphoma, and other cancers. Importantly, the paraneoplastic neuropathy can precede the detection of

1	seen in patients with small-cell carcinoma of the lung but are seen also in breast, ovarian, lymphoma, and other cancers. Importantly, the paraneoplastic neuropathy can precede the detection of the cancer, and detection of these autoantibodies should lead to a search for malignancy.

1	Nerve biopsies are now rarely indicated for evaluation of neuropathies. The primary indication for nerve biopsy is suspicion for amyloid neuropathy or vasculitis. In most instances, the abnormalities present on biopsies do not help distinguish one form of peripheral neuropathy from another (beyond what is already apparent by clinical examination and the NCS). Nerve biopsies should only be done if the NCS are abnormal. The sural nerve is most commonly biopsied because it is a pure sensory nerve and biopsy will not result in loss of motor function. In suspected vasculitis, a combination biopsy of a superficial peroneal nerve (pure sensory) and the underlying peroneus brevis muscle obtained from a single small incision increases the diagnostic yield. Tissue can be analyzed by frozen section and paraffin section to assess the supporting structures for evidence of inflammation, vasculitis, or amyloid deposition. Semithin plastic sections, teased fiber preparations, and electron microscopy

1	and paraffin section to assess the supporting structures for evidence of inflammation, vasculitis, or amyloid deposition. Semithin plastic sections, teased fiber preparations, and electron microscopy are used to assess the morphology of the nerve fibers and to distinguish axonopathies from myelinopathies.

1	Skin biopsies are sometimes used to diagnose a small-fiber neuropathy. Following a punch biopsy of the skin in the distal lower extremity, immunologic staining can be used to measure the density of small 2678 unmyelinated fibers. The density of these nerve fibers is reduced in patients with small-fiber neuropathies in whom NCS and routine nerve biopsies are often normal. This technique may allow for an objective measurement in patients with mainly subjective symptoms. However, it adds little to what one already knows from the clinical examination and EDx.

1	Charcot-Marie-Tooth (CMT) disease is the most common type of hereditary neuropathy. Rather than one disease, CMT is a syndrome of several genetically distinct disorders (Table 459-4). The various subtypes of CMT are classified according to the nerve conduction velocities and predominant pathology (e.g., demyelination or axonal degeneration), inheritance pattern (autosomal dominant, recessive, or X-linked), and the specific mutated genes. Type 1 CMT (or CMT1) refers to inherited demyelinating sensorimotor neuropathies, whereas the axonal sensory neuropathies are classified as CMT2. By definition, motor conduction velocities in the arms are slowed to less than 38 m/s in CMT1 and are greater than 38 m/s in CMT2. However, most cases of CMT1 actually have motor nerve conduction velocities (NCVs) between 20 and 25 m/s. CMT1 and CMT2 usually begin in childhood or early adult life; however, onset later in life can occur, particularly in CMT2. Both are associated with autosomal dominant

1	(NCVs) between 20 and 25 m/s. CMT1 and CMT2 usually begin in childhood or early adult life; however, onset later in life can occur, particularly in CMT2. Both are associated with autosomal dominant inheritance, with a few exceptions. CMT3 is an autosomal dominant neuropathy that appears in infancy and is associated with severe demyelination or hypomyelination. CMT4 is an autosomal recessive neuropathy that typically begins in childhood or early adult life. There are no medical therapies for any of the CMTs, but physical and occupational therapy can be beneficial, as can bracing (e.g., ankle-foot orthotics for foot-drop) and other orthotic devices.

1	CMT1 CMT1 is the most common form of hereditary neuropathy, with the ratio of CMT1:CMT2 being approximately 2:1. Affected individuals usually present in the first to third decade of life with distal leg weakness (e.g., footdrop), although patients may remain asymptomatic even late in life. People with CMT generally do not complain of numbness or tingling, which can be helpful in distinguishing CMT from acquired forms of neuropathy in which sensory symptoms usually predominate. Although usually asymptomatic in this regard, reduced sensation to all modalities is apparent on examination. Muscle stretch reflexes are unobtainable or reduced throughout. There is often atrophy of the muscles below the knee (particularly the anterior compartment), leading to so-called inverted champagne bottle legs.

1	Motor NCVs are usually in the 20–25 m/s range. Nerve biopsies usually are not performed on patients suspected of having CMT1, because the diagnosis usually can be made by less invasive testing (e.g., NCS and genetic studies). However, when done, the biopsies reveal reduction of myelinated nerve fibers with a predilection for the loss of the large-diameter fibers and Schwann cell proliferation around thinly or demyelinated fibers, forming so-called onion bulbs.

1	CMT1A is the most common subtype of CMT1, representing 70% of cases, and is caused by a 1.5-megabase (Mb) duplication within chromosome 17p11.2-12 wherein the gene for peripheral myelin protein-22 (PMP-22) lies. This results in patients having three copies of the PMP-22 gene rather than two. This protein accounts for 2–5% of myelin protein and is expressed in compact portions of the peripheral myelin sheath. Approximately 20% of patients with CMT1 have CMT1B, which is caused by mutations in the myelin protein zero (MPZ). CMT1B is for the most part clinically, electrophysiologically, and histologically indistinguishable from CMT1A. MPZ is an integral myelin protein and accounts for more than half of the myelin protein in peripheral nerves. Other forms of CMT1 are much less common and again indistinguishable from one another clinically and electrophysiologically.

1	CMT2 CMT2 tends to present later in life compared to CMT1. Affected individuals usually become symptomatic in the second decade of life; some cases present earlier in childhood, whereas others remain asymptomatic into late adult life. Clinically, CMT2 is for the most part indistinguishable from CMT1. NCS are helpful in this regard; in contrast to CMT1, the velocities are normal or only slightly slowed. The most common cause of CMT2 is a mutation in the gene for mitofusin 2 (MFN2), which accounts for one-third of CMT2 cases overall. MFN2 localizes to the outer mitochondrial membrane, where it regulates the mitochondrial network architecture by fusion of mitochondria. The other genes associated with CMT2 are much less common. CMTDI In dominant-intermediate CMTs (CMTDIs), the NCVs are faster than usually seen in CMT1 (e.g., >38 m/s) but slower than in CMT2.

1	CMTDI In dominant-intermediate CMTs (CMTDIs), the NCVs are faster than usually seen in CMT1 (e.g., >38 m/s) but slower than in CMT2. CMT3 CMT3 was originally described by Dejerine and Sottas as a hereditary demyelinating sensorimotor polyneuropathy presenting in infancy or early childhood. Affected children are severely weak. Motor NCVs are markedly slowed, typically 5–10 m/s or less. Most cases of CMT3 are caused by point mutations in the genes for PMP-22, MPZ, or ERG-2, which are also the genes responsible for CMT1. CMT4 CMT4 is extremely rare and is characterized by a severe, childhood-onset sensorimotor polyneuropathy that is usually inherited in an autosomal recessive fashion. Electrophysiologic and histologic evaluations can show demyelinating or axonal features. CMT4 is genetically heterogenic (Table 459-4).

1	CMT1X CMT1X is an X-linked dominant disorder with clinical features similar to CMT1 and CMT2, except that the neuropathy is much more severe in men than in women. CMT1X accounts for approximately 10–15% of CMT overall. Men usually present in the first two decades of life with atrophy and weakness of the distal arms and legs, areflexia, pes cavus, and hammertoes. Obligate women carriers are frequently asymptomatic, but can develop signs and symptoms. Onset in women is usually after the second decade of life, and the neuropathy is milder in severity.

1	NCS reveal features of both demyelination and axonal degeneration that are more severe in men compared to women. In men, motor NCVs in the arms and legs are moderately slowed (in the low to mid 30-m/s range). About 50% of men with CMT1X have motor NCVs between 15 and 35 m/s with about 80% of these falling between 25 and 35 m/s (intermediate slowing). In contrast, about 80% of women with CMT1X have NCVs in the normal range and 20% have NCVs in the intermediate range. CMT1X is caused by mutations in the connexin 32 gene. Connexins are gap junction structural proteins that are important in cell-to-cell communication.

1	Hereditary Neuropathy with Liability to Pressure Palsies (HNPP) HNPP is an autosomal dominant disorder related to CMT1A. Although CMT1A is usually associated with a 1.5-Mb duplication in chromosome 17p11.2 that results in an extra copy of PMP-22 gene, HNPP is caused by inheritance of the chromosome with the corresponding 1.5-Mb deletion of this segment, and thus affected individuals have only one copy of the PMP-22 gene. Patients usually manifest in the second or third decade of life with painless numbness and weakness in the distribution of single peripheral nerves, although multiple mononeuropathies can occur. Symptomatic mononeuropathy or multiple mononeuropathies are often precipitated by trivial compression of nerve(s) as can occur with wearing a backpack, leaning on the elbows, or crossing one’s legs for even a short period of time. These pressure-related mononeuropathies may take weeks or months to resolve. In addition, some affected individuals manifest with a progressive or

1	crossing one’s legs for even a short period of time. These pressure-related mononeuropathies may take weeks or months to resolve. In addition, some affected individuals manifest with a progressive or relapsing, generalized and symmetric, sensorimotor peripheral neuropathy that resembles CMT.

1	Hereditary Neuralgic Amyotrophy (HNA) HNA is an autosomal dominant disorder characterized by recurrent attacks of pain, weakness, and sensory loss in the distribution of the brachial plexus often beginning in childhood. These attacks are similar to those seen with idiopathic brachial plexitis (see below). Attacks may occur in the postpartum period, following surgery, or at other times of stress. Most patients recover over several weeks or months. Slightly dysmorphic features, including hypotelorism, epicanthal folds, cleft palate, syndactyly, micrognathia, and facial asymmetry, are evident in some individuals. EDx demonstrate an axonal process. HNA is caused by mutations in septin 9 (SEPT9). Septins may be important in formation of the

1	CMT1 CMT1A AD 17p11.2 PMP-22 (usually duplication of gene) CMT1B AD 1q21-23 MPZ CMT1C AD 16p13.1-p12.3 LITAF CMT1D AD 10q21.1-22.1 ERG2 CMT1E (with deafness) AD 17p11.2 Point mutations in PMP 22 gene CMT1F AD 8p13-21 Neurofilament light chain CMT1G AD 14q32.33 INF2 CMT1X X-linked dominant Xq13 Connexin-32 HNPP AD 17p11.2 PMP-22 1q21-23 MPZ CMT dominant-intermediate (CMTD1) CMTD1A AD 10q24.1-25.1 ? CMTD1B AD 19.p12-13.2 Dynamin 2 CMTD1C AD 1p35 YARS CMTD1D AD 1q22 MPZ

1	HNPP AD 17p11.2 PMP-22 1q21-23 MPZ CMT dominant-intermediate (CMTD1) CMTD1A AD 10q24.1-25.1 ? CMTD1B AD 19.p12-13.2 Dynamin 2 CMTD1C AD 1p35 YARS CMTD1D AD 1q22 MPZ CMT2 CMT2A2 (allelic to HMSN VI with optic atrophy) AD 1p36.2 MFN2 CMT2B AD 3q13-q22 RAB7 CMT2B1 (allelic to LGMD 1B) AR 1q21.2 Lamin A/C CMT2B2 AR and AD 19q13 MED25 for AR Unknown for AD CMT2C (with vocal cord and diaphragm paralysis) AD 12q23-24 TRPV4 CMT2D (allelic to distal SMA5) AD 7p14 Glycine tRNA synthetase CMT2E (allelic to CMT 1F) AD 8p21 Neurofilament light chain CMT2F AD 7q11-q21 Heat-shock 27-kDa protein-1 CMT2G AD 12q23 Unknown CMT2I (allelic to CMT1B) AD 1q22 MPZ CMT2J AD 1q22 MPZ CMT2H, CMT2K (allelic to CMT4A) AD 8q13-q21 GDAP1 CMT2L (allelic to distal hereditary motor neuropathy type 2) AD 12q24 Heat-shock protein 8 CMT2M AD 16q22 Dynamin-2 CMT2N AD 16q22.1 AARS CMT2O AD 14q32.31 DYNC1H1 CMT2P AD 9q34.13 LRSAM1 CMT2P-Okinawa (HSMN2P) AD 3q13-q14 TFG CMT2X X-linked Xq22-24 PRPS1

1	CMT3 AD 17p11.2 PMP-22 (Dejerine-Sottas disease, congenital hypomyelinating neuropathy) AD 1q21-23 MPZ AR 10q21.1-22.1 ERG2 AR 19q13 Periaxon CMT4 CMT4A AR 8q13-21.1 GDAP1 CMT4B1 AR 11q23 MTMR2 CMT4B2 AR 11p15 MTMR13 CMT4C AR 5q23-33 SH3TC2 CMT4D (HMSN-Lom) AR 8q24 NDRG1 CMT4E (congenital hypomyelinating neuropathy) AR Multiple Includes PMP22, MPZ, and ERG-2 CMT4F AR 19q13.1-13.3 Periaxin CMT4G AR 10q23.2 HKI CMT4H AR 12q12-q13 Frabin CMT4J AR 6q21 FIG4 HNA AD 17q24 SEPT9 HSAN1A AD 9q22 SPTLC1 HSAN1B AD 3q21 RAB7 HSAN1C AD 14q24.3 SPTLC2 HSAN1D AD 14q21.3 ATL1 HSAN1E AD 19p13.2 DNMT1

1	Abbreviations: AARS, alanyl-tRNA synthetase; AD, autosomal dominant; AR, autosomal recessive; ATL, atlastin; CMT, Charcot-Marie-Tooth; DNMT1, DNA methyltransferase 1; DYNC1HI, cytoplasmic dynein 1 heavy chain 1; ERG2, early growth response-2 protein; FAM134B, family with sequence similarity 134, member B; FIG4, FDG1-related F actin-binding protein; GDAP1, ganglioside-induced differentiation-associated protein-1; HK1, hexokinase 1; HMSN-P, hereditary motor and sensory neuropathyproximal; HNA, hereditary neuralgic amyotrophy; HNPP, hereditary neuropathy with liability to pressure palsies; HSAN; hereditary sensory and autonomic neuropathy; IFN2, inverted formin-2; IKAP, kB kinase complex-associated protein; LGMD, limb girdle muscular dystrophy; LITAF, lipopolysaccharide-induced tumor necrosis factor α factor; LRSAM1, E3 ubiquitin-protein ligase; MED25, mediator 25; MFN2, mitochondrial fusion protein mitofusin 2 gene; MPZ, myelin protein zero protein; MTMR2, myotubularin-related

1	necrosis factor α factor; LRSAM1, E3 ubiquitin-protein ligase; MED25, mediator 25; MFN2, mitochondrial fusion protein mitofusin 2 gene; MPZ, myelin protein zero protein; MTMR2, myotubularin-related protein-2; NDRG1, N-myc downstream regulated 1; PMP-22, peripheral myelin protein-22; PRKWNK1, protein kinase, lysine deficient 1; PRPS1, phosphoribosylpyrophosphate synthetase 1; RAB7, Ras-related protein 7; SEPT9, Septin 9; SH3TC2, SH3 domain and tetratricopeptide repeats 2; SMA, spinal muscular atrophy; SPTLC, serine palmitoyltransferase long-chain base; TFG, TRK-fused gene; TrkA/NGF, tyrosine kinase A/nerve growth factor; tRNA, transfer ribonucleic acid; TRPV4, transient receptor potential cation channel, subfamily V, member 4; WNK1, WNK lysine deficient; YARS, tyrosyl-tRNA synthetase.

1	Source: Modified from AA Amato, J Russell: Neuromuscular Disease. New York, McGraw-Hill, 2008. neuronal cytoskeleton and have a role in cell division, but the mechanism of causing HNA is unclear. Hereditary Sensory and Autonomic Neuropathy (HSAN) The HSANs are a very rare group of hereditary neuropathies in which sensory and autonomic dysfunction predominates over muscle weakness, unlike CMT, in which motor findings are most prominent (Table 459-4). Nevertheless, affected individuals can develop motor weakness and there can be overlap with CMT. There are no medical therapies available to treat these neuropathies, other than prevention and treatment of mutilating skin and bone lesions.

1	Of the HSANs, only HSAN1 typically presents in adults. HSAN1 is the most common of the HSANs and is inherited in an autosomal dominant fashion. Affected individuals with HSAN1 usually manifest in the second through fourth decades of life. HSAN1 is associated with the degeneration of small myelinated and unmyelinated nerve fibers leading to severe loss of pain and temperature sensation, deep dermal ulcerations, recurrent osteomyelitis, Charcot joints, bone loss, gross foot and hand deformities, and amputated digits. Although most people with HSAN1 do not complain of numbness, they often describe burning, aching, or lancinating pains. Autonomic neuropathy is not a prominent feature, but bladder dysfunction and reduced sweating in the feet may occur. HSAN1A, which is most common, is caused by mutations in the serine palmitoyltransferase long-chain base 1 (SPTLC1) gene.

1	Fabry’s disease (angiokeratoma corporis diffusum) is an X-linked dominant disorder. Although men are more commonly and severely affected, women can also show severe signs of the disease. Angiokeratomas are reddish-purple maculopapular lesions that are usually found around the umbilicus, scrotum, inguinal region, and perineum. Burning or lancinating pain in the hands and feet often develops in males in late childhood or early adult life. However, the neuropathy is usually overshadowed by complications arising from the associated premature atherosclerosis (e.g., hypertension, renal failure, cardiac disease, and stroke) that often lead to death by the fifth decade of life. Some patients also manifest primarily with a dilated cardiomyopathy.

1	Fabry’s disease is caused by mutations in the a-galactosidase gene that leads to the accumulation of ceramide trihexoside in nerves and blood vessels. A decrease in a-galactosidase activity is evident in leukocytes and cultured fibroblasts. Glycolipid granules may be appreciated in ganglion cells of the peripheral and sympathetic nervous systems and in perineurial cells. Enzyme replacement therapy with a-galactosidase B can improve the neuropathy if patients are treated early, before irreversible nerve fiber loss.

1	Adrenoleukodystrophy (ALD) and adrenomyeloneuropathy (AMN) are allelic X-linked dominant disorders caused by mutations in the peroxisomal transmembrane adenosine triphosphate-binding cassette (ABC) transporter gene. Patients with ALD manifest with central nervous system (CNS) abnormalities. However, 30% with mutations in this gene present with the AMN phenotype that typically manifests in the third to fifth decade of life with mild to moderate peripheral neuropathy combined with progressive spastic paraplegia (Chap. 456). Rare patients present with an adult-onset spinocerebellar ataxia or only with adrenal insufficiency.

1	EDx is suggestive of a primary axonopathy with secondary demyelination. Nerve biopsies demonstrate a loss of myelinated and unmyelinated nerve fibers with lamellar inclusions in the cytoplasm of Schwann cells. Very long chain fatty acid (VLCFA) levels (C24, C25, and C26) are increased in the urine. Laboratory evidence of adrenal insufficiency is evident in approximately two-thirds of patients. The diagnosis can be confirmed by genetic testing. Adrenal insufficiency is managed by replacement therapy; however, there is no proven effective therapy for the neurologic manifestations Hereditary Disorders of Lipid Metabolism Hereditary Ataxias with Neuropathy Disorders of Defective DNA Repair of ALD/AMN. Diets low in VLCFAs and supplemented with Lorenzo’s oil (erucic and oleic acids) reduce the levels of VLCFAs and increase the levels of C22 in serum, fibroblasts, and liver; however, several large, open-label trials of Lorenzo’s oil failed to demonstrate efficacy.

1	Refsum’s disease can manifest in infancy to early adulthood with the classic tetrad of (1) peripheral neuropathy, (2) retinitis pigmentosa, (3) cerebellar ataxia, and (4) elevated CSF protein concentration. Most affected individuals develop progressive distal sensory loss and weakness in the legs leading to footdrop by their 20s. Subsequently, the proximal leg and arm muscles may become weak. Patients may also develop sensorineural hearing loss, cardiac conduction abnormalities, ichthyosis, and anosmia. Serum phytanic acid levels are elevated. Sensory and motor NCS reveal reduced amplitudes, prolonged latencies, and slowed conduction velocities. Nerve biopsy demonstrates a loss of myelinated nerve fibers, with remaining axons often thinly myelinated and associated with onion bulb formation.

1	Refsum disease is genetically heterogeneous but autosomal recessive in nature. Classical Refsum disease with childhood or early adult onset is caused by mutations in the gene that encodes for phytanoyl-CoA α-hydroxylase (PAHX). Less commonly, mutations in the gene encoding peroxin 7 receptor protein (PRX7) are responsible. These mutations lead to the accumulation of phytanic acid in the central and peripheral nervous systems. Refsum’s disease is treated by removing phytanic precursors (phytols: fish oils, dairy products, and ruminant fats) from the diet.

1	Tangier disease is a rare autosomal recessive disorder that can present as (1) asymmetric multiple mononeuropathies, (2) a slowly progressive symmetric polyneuropathy predominantly in the legs, or (3) a pseudo-syringomyelia pattern with dissociated sensory loss (i.e., abnormal pain/temperature perception but preserved position/vibration in the arms [Chap. 456]). The tonsils may appear swollen and yellowish-orange in color, and there may also be splenomegaly and lymphadenopathy. Tangier disease is caused by mutations in the ATP-binding cassette transporter 1 (ABC1) gene, which leads to markedly reduced levels of high-density lipoprotein (HDL) cholesterol levels, whereas triacylglycerol levels are increased. Nerve biopsies reveal axonal degeneration with demyelination and remyelination. Electron microscopy demonstrates abnormal accumulation of lipid in Schwann cells, particularly those encompassing umyelinated and small myelinated nerves. There is no specific treatment.

1	Porphyria is a group of inherited disorders caused by defects in heme biosynthesis (Chap. 430). Three forms of porphyria are associated with peripheral neuropathy: acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), and variegate porphyria (VP). The acute neurologic manifestations are similar in each, with the exception that a photosensitive rash is seen with HCP and VP but not in AIP. Attacks of porphyria can be precipitated by certain drugs (usually those metabolized by the P450 system), hormonal changes (e.g., pregnancy, menstrual cycle), and dietary restrictions.

1	An acute attack of porphyria may begin with sharp abdominal pain. Subsequently, patients may develop agitation, hallucinations, or seizures. Several days later, back and extremity pain followed by weakness ensues, mimicking GBS. Weakness can involve the arms or the legs and can be asymmetric, proximal, or distal in distribution, as well as affecting the face and bulbar musculature. Dysautonomia and signs of sympathetic overactivity are common (e.g., pupillary dilation, tachycardia, and hypertension). Constipation, urinary retention, and incontinence can also be seen.

1	The CSF protein is typically normal or mildly elevated. Liver function tests and hematologic parameters are usually normal. Some patients are hyponatremic due to inappropriate secretion of antidiuretic hormone (Chap. 401e). The urine may appear brownish in color secondary to 2681 the high concentration of porphyrin metabolites. Accumulation of intermediary precursors of heme (i.e., d-aminolevulinic acid, porphobilinogen, uroporphobilinogen, coproporphyrinogen, and protoporphyrinogen) is found in urine. Specific enzyme activities can also be measured in erythrocytes and leukocytes. The primary abnormalities on EDx are marked reductions in compound motor action potential (CMAP) amplitudes and signs of active axonal degeneration on needle EMG. The porphyrias are inherited in an autosomal dominant fashion.

1	The porphyrias are inherited in an autosomal dominant fashion. AIP is associated with porphobilinogen deaminase deficiency, HCP is caused by defects in coproporphyrin oxidase, and VP is associated with protoporphyrinogen oxidase deficiency. The pathogenesis of the neuropathy is not completely understood. Treatment with glucose and hematin may reduce the accumulation of heme precursors. Intravenous glucose is started at a rate of 10–20 g/h. If there is no improvement within 24 h, intravenous hematin 2–5 mg/kg per day for 3–14 days should be given.

1	Familial amyloid polyneuropathy (FAP) is phenotypically and genetically heterogeneous and is caused by mutations in the genes for transthyretin (TTR), apolipoprotein A1, or gelsolin (Chap. 137). The majority of patients with FAP have mutations in the TTR gene. Amyloid deposition may be evident in abdominal fat pad, rectal, or nerve biopsies. The clinical features, histopathology, and EDx reveal abnormalities consistent with a generalized or multifocal, predominantly axonal but occasionally demyelinating, sensorimotor polyneuropathy.

1	Patients with TTR-related FAP usually develop insidious onset of numbness and painful paresthesias in the distal lower limbs in the third to fourth decade of life, although some patients develop the disorder later in life. Carpal tunnel syndrome (CTS) is common. Autonomic involvement can be severe, leading to postural hypotension, constipation or persistent diarrhea, erectile dysfunction, and impaired sweating. Amyloid deposition also occurs in the heart, kidneys, liver, and corneas. Patients usually die 10–15 years after the onset of symptoms from cardiac failure or complications from malnutrition. Because the liver produces much of the body’s TTR, liver transplantation has been used to treat FAP related to TTR mutations. Serum TTR levels decrease after transplantation, and improvement in clinical and EDx features has been reported.

1	Patients with apolipoprotein A1-related FAP (Van Allen type) usually present in the fourth decade with numbness and painful dysesthesias in the distal limbs. Gradually, the symptoms progress, leading to proximal and distal weakness and atrophy. Although autonomic neuropathy is not severe, some patients develop diarrhea, constipation, or gastroparesis. Most patients die from systemic complications of amyloidosis (e.g., renal failure) 12–15 years after the onset of the neuropathy. Gelsolin-related amyloidosis (Finnish type) is characterized by the combination of lattice corneal dystrophy and multiple cranial neuropathies that usually begin in the third decade of life. Over time, a mild generalized sensorimotor polyneuropathy develops. Autonomic dysfunction does not occur. PRIMARY OR AL AMYLOIDOSIS (SEE CHAP. 137)

1	PRIMARY OR AL AMYLOIDOSIS (SEE CHAP. 137) Besides FAP, amyloidosis can also be acquired. In primary or AL amyloidosis, the abnormal protein deposition is composed of immunoglobulin light chains. AL amyloidosis occurs in the setting of multiple myeloma, Waldenström’s macroglobulinemia, lymphoma, other plasmacytomas, or lymphoproliferative disorders, or without any other identifiable disease.

1	Approximately 30% of patients with AL primary amyloidosis present with a polyneuropathy, most typically painful dysesthesias and burning sensations in the feet. However, the trunk can be involved, and some patients manifest with a mononeuropathy multiplex pattern. CTS occurs in 25% of patients and may be the initial manifestation. The neuropathy is slowly progressive, and eventually weakness 2682 develops along with large-fiber sensory loss. Most patients develop autonomic involvement with postural hypertension, syncope, bowel and bladder incontinence, constipation, impotence, and impaired sweating. Patients generally die from their systemic illness (renal failure, cardiac disease). The monoclonal protein may be composed of IgG, IgA, IgM, or only free light chain. Lambda (λ) is more common than κ light chain (>2:1) in AL amyloidosis. The CSF protein is often increased (with normal cell count), and thus the neuropathy may be mistaken for CIDP (Chap. 460). Nerve biopsies reveal axonal

1	than κ light chain (>2:1) in AL amyloidosis. The CSF protein is often increased (with normal cell count), and thus the neuropathy may be mistaken for CIDP (Chap. 460). Nerve biopsies reveal axonal degeneration and amyloid deposition in either a globular or diffuse pattern infiltrating the perineurial, epineurial, and endoneurial connected tissue and in blood vessel walls. The median survival of patients with primary amyloidosis is less than 2 years, with death usually from progressive congestive heart failure or renal failure. Chemotherapy with melphalan, prednisone, and colchicine, to reduce the concentration of monoclonal proteins, and autologous stem cell transplantation may prolong survival, but whether the neuropathy improves is controversial.

1	Diabetes mellitus (DM) is the most common cause of peripheral neuropathy in developed countries. DM is associated with several types of polyneuropathy: distal symmetric sensory or sensorimotor polyneuropathy, autonomic neuropathy, diabetic neuropathic cachexia, polyradiculoneuropathies, cranial neuropathies, and other mononeuropathies. Risk factors for the development of neuropathy include long-standing, poorly controlled DM and the presence of retinopathy and nephropathy.

1	DSPN is the most common form of diabetic neuropathy and manifests as sensory loss beginning in the toes that gradually progresses over time up the legs and into the fingers and arms. When severe, a patient may develop sensory loss in the trunk (chest and abdomen), initially in the midline anteriorly and later extending laterally. Tingling, burning, deep aching pains may also be apparent. NCS usually show reduced amplitudes and mild to moderate slowing of conduction velocities (CVs). Nerve biopsy reveals axonal degeneration, endothelial hyperplasia, and, occasionally, perivascular inflammation. Tight control of glucose can reduce the risk of developing neuropathy or improve the underlying neuropathy. A variety of medications have been used with variable success to treat painful symptoms associated with DSPN, including antiepileptic medications, antidepressants, sodium channel blockers, and other analgesics (Table 459-6).

1	Diabetic Autonomic Neuropathy Autonomic neuropathy is typically seen in combination with DSPN. The autonomic neuropathy can manifest as abnormal sweating, dysfunctional thermoregulation, dry eyes and mouth, pupillary abnormalities, cardiac arrhythmias, postural hypotension, GI abnormalities (e.g., gastroparesis, postprandial bloating, chronic diarrhea or constipation), and genitourinary dysfunction (e.g., impotence, retrograde ejaculation, incontinence). Tests of autonomic function are generally abnormal, including sympathetic skin responses and quantitative sudomotor axon reflex testing. Sensory and motor NCS generally demonstrate features described above with DSPN.

1	Diabetic Radiculoplexus Neuropathy (Diabetic Amyotrophy or Bruns-Garland Syndrome) Diabetic radiculoplexus neuropathy is the presenting manifestation of DM in approximately one-third of patients. Typically, patients present with severe pain in the low back, hip, and thigh in one leg. Rarely, the diabetic polyradiculoneuropathy begins in both legs at the same time. Atrophy and weakness of proximal and distal muscles in the affected leg become apparent within a few days or weeks. The neuropathy is often accompanied or heralded by severe weight loss. Weakness usually progresses over several weeks or months, but can continue to progress for 18 months or more. Subsequently, there is slow recovery but many are left with residual weakness, sensory loss, and pain. In contrast to the more typical lumbosacral radiculoplexus neuropathy, some patients develop thoracic radiculopathy or, even less commonly, a cervical polyradiculoneuropathy. CSF protein is usually elevated, while the cell count is

1	lumbosacral radiculoplexus neuropathy, some patients develop thoracic radiculopathy or, even less commonly, a cervical polyradiculoneuropathy. CSF protein is usually elevated, while the cell count is normal. ESR is often increased. EDx reveals evidence of active denervation in affected proximal and distal muscles in the affected limbs and in paraspinal muscles. Nerve biopsies may demonstrate axonal degeneration along with perivascular inflammation. Patients with severe pain are sometimes treated in the acute period with glucocorticoids, although a randomized controlled trial has yet to be performed, and the natural history of this neuropathy is gradual improvement.

1	Diabetic Mononeuropathies or Multiple Mononeuropathies The most common mononeuropathies are median neuropathy at the wrist and ulnar neuropathy at the elbow, but peroneal neuropathy at the fibular head, and sciatic, lateral femoral, cutaneous, or cranial neuropathies also occur. In regard to cranial mononeuropathies, seventh nerve palsies are relatively common but may have other, nondiabetic etiologies. In diabetics, a third nerve palsy is most common, followed by sixth nerve, and, less frequently, fourth nerve palsies. Diabetic third nerve palsies are characteristically pupil-sparing (Chap. 39). Hypothyroidism is more commonly associated with a proximal myopathy, but some patients develop a neuropathy, most typically CTS. Rarely, a generalized sensory polyneuropathy characterized by painful paresthesias and numbness in both the legs and hands can occur. Treatment is correction of the hypothyroidism.

1	Sjögren’s syndrome, characterized by the sicca complex of xerophthalmia, xerostomia, and dryness of other mucous membranes, can be complicated by neuropathy (Chap. 383). Most common is a length-dependent axonal sensorimotor neuropathy characterized mainly by sensory loss in the distal extremities. A pure small-fiber neuropathy or a cranial neuropathy, particularly involving the trigeminal nerve, can also be seen. Sjögren’s syndrome is also associated with sensory neuronopathy/ganglionopathy. Patients with sensory ganglionopathies develop progressive numbness and tingling of the limbs, trunk, and face in a non-length-dependent manner such that symptoms can involve the face or arms more than the legs. The onset can be acute or insidious. Sensory examination demonstrates severe vibratory and proprioceptive loss leading to sensory ataxia.

1	Patients with neuropathy due to Sjögren’s syndrome may have ANAs, SS-A/Ro, and SS-B/La antibodies in the serum, but most do not. NCS demonstrate reduced amplitudes of sensory studies in the affected limbs. Nerve biopsy demonstrates axonal degeneration. Nonspecific perivascular inflammation may be present, but only rarely is there necrotizing vasculitis. There is no specific treatment for neuropathies related to Sjögren’s syndrome. When vasculitis is suspected, immunosuppressive agents may be beneficial. Occasionally, the sensory neuronopathy/ganglionopathy stabilizes or improves with immunotherapy, such as IVIg.

1	Peripheral neuropathy occurs in at least 50% of patients with rheumatoid arthritis (RA) and may be vasculitic in nature (Chap. 380). Vasculitic neuropathy can present with a mononeuropathy multiplex, a generalized symmetric pattern of involvement, or a combination of these patterns (Chap. 385). Neuropathies may also be due to drugs used to treat the RA (e.g., tumor necrosis blockers, leflunomide). Nerve biopsy often reveals thickening of the epineurial and endoneurial blood vessels as well as perivascular inflammation or vasculitis, with transmural inflammatory cell infiltration and fibrinoid necrosis of vessel walls. The neuropathy often is responsive to immunomodulating therapies.

1	Between 2 and 27% of individuals with SLE develop a peripheral neuropathy (Chap. 378). Affected patients typically present with a slowly progressive sensory loss beginning in the feet. Some patients develop burning pain and paresthesias with normal reflexes, and NCS suggest a pure small-fiber neuropathy. Less common are multiple mononeuropathies presumably secondary to necrotizing vasculitis. Rarely, a generalized sensorimotor polyneuropathy meeting clinical, laboratory, electrophysiologic, and histologic criteria for either GBS or CIDP may occur. Immunosuppressive therapy is beneficial in SLE patients with neuropathy due to vasculitis. Immunosuppressive agents are less likely to be effective in patients with a generalized sensory or sensorimotor 2683 polyneuropathy without evidence of vasculitis. Patients with a GBS or CIDP-like neuropathy should be treated accordingly (Chap. 385).

1	A distal symmetric, mainly sensory, polyneuropathy complicates 5–67% of scleroderma cases (Chap. 382). Cranial mononeuropathies can also develop, most commonly of the trigeminal nerve, producing numbness and dysesthesias in the face. Multiple mononeuropathies also occur. The EDx and histologic features of nerve biopsy are those of an axonal sensory greater than motor polyneuropathy. A mild distal axonal sensorimotor polyneuropathy occurs in approximately 10% of patients with MCTD.

1	A mild distal axonal sensorimotor polyneuropathy occurs in approximately 10% of patients with MCTD. The peripheral or central nervous system is involved in about 5% of patients with sarcoidosis (Chap. 390). The most common cranial nerve involved is the seventh nerve, which can be affected bilaterally. Some patients develop radiculopathy or polyradiculopathy. With a generalized root involvement, the clinical presentation can mimic GBS or CIDP. Patients can also present with multiple mononeuropathies or a generalized, slowly progressive, sensory greater than motor polyneuropathy. Some have features of a pure small-fiber neuropathy. EDx reveals an axonal neuropathy. Nerve biopsy can reveal noncaseating granulomas infiltrating the endoneurium, perineurium, and epineurium along with lymphocytic necrotizing angiitis. Neurosarcoidosis may respond to treatment with glucocorticoids or other immunosuppressive agents.

1	Hypereosinophilic syndrome is characterized by eosinophilia associated with various skin, cardiac, hematologic, and neurologic abnormalities. A generalized peripheral neuropathy or a mononeuropathy multiplex occurs in 6–14% of patients. Neurologic complications, particularly ataxia and peripheral neuropathy, are estimated to occur in 10% of patients with celiac disease (Chap. 349). A generalized sensorimotor polyneuropathy, pure motor neuropathy, multiple mononeuropathies, autonomic neuropathy, small-fiber neuropathy, and neuromyotonia have all been reported in association with celiac disease or antigliadin/antiendomysial antibodies. Nerve biopsy may reveal a loss of large myelinated fibers. The neuropathy may be secondary to malabsorption of vitamins B12 and

1	E. However, some patients have no appreciable vitamin deficiencies. The pathogenic basis for the neuropathy in these patients is unclear but may be autoimmune in etiology. The neuropathy does not appear to respond to a gluten-free diet. In patients with vitamin B12 or vitamin E deficiency, replacement therapy may improve or stabilize the neuropathy. Ulcerative colitis and Crohn’s disease may be complicated by GBS, CIDP, generalized axonal sensory or sensorimotor polyneuropathy, small-fiber neuropathy, or mononeuropathy (Chap. 351). These neuropathies may be autoimmune, nutritional (e.g., vitamin B12 deficiency), treatment related (e.g., metronidazole), or idiopathic in nature. An acute neuropathy with demyelination resembling GBS, CIDP, or multifocal motor neuropathy may occur in patients treated with tumor necrosis factor α blockers.

1	Approximately 60% of patients with renal failure develop a polyneuropathy characterized by length-dependent numbness, tingling, allodynia, and mild distal weakness. Rarely, a rapidly progressive weakness and sensory loss very similar to GBS can occur that improves with an increase in the intensity of renal dialysis or with transplantation. Mononeuropathies can also occur, the most common of which is CTS. 2684 Ischemic monomelic neuropathy (see below) can complicate arteriovenous shunts created in the arm for dialysis. EDx in uremic patients reveals features of a length-dependent, primarily axonal, sensorimotor polyneuropathy. Sural nerve biopsies demonstrate a loss of nerve fibers (particularly large myelinated nerve fibers), active axonal degeneration, and segmental and paranodal demyelination. The sensorimotor polyneuropathy can be stabilized by hemodialysis and improved with successful renal transplantation.

1	A generalized sensorimotor neuropathy characterized by numbness, tingling, and minor weakness in the distal aspects of primarily the lower limbs commonly occurs in patients with chronic liver failure. EDx studies are consistent with a sensory greater than motor axonopathy. Sural nerve biopsy reveals both segmental demyelination and axonal loss. It is not known if hepatic failure in isolation can cause peripheral neuropathy, as the majority of patients have liver disease secondary to other disorders, such as alcoholism or viral hepatitis, which can also cause neuropathy.

1	The most common causes of acute generalized weakness leading to admission to a medical intensive care unit (ICU) are GBS and myasthenia gravis (Chap. 461). However, weakness developing in critically ill patients while in the ICU is usually caused by critical illness polyneuropathy (CIP) or critical illness myopathy (CIM) or, much less commonly, by prolonged neuromuscular blockade. From a clinical and EDx standpoint, it can be quite difficult to distinguish these disorders. Most specialists suggest that CIM is more common. Both CIM and CIP develop as a complication of sepsis and multiple organ failure. They usually present as an inability to wean a patient from a ventilator. A coexisting encephalopathy may limit the neurologic exam, in particular the sensory examination. Muscle stretch reflexes are absent or reduced.

1	Serum creatine kinase (CK) is usually normal; an elevated serum CK would point to CIM as opposed to CIP. NCS reveal absent or markedly reduced amplitudes of motor and sensory studies in CIP, whereas sensory studies are relatively preserved in CIM. Needle EMG usually reveals profuse positive sharp waves and fibrillation potentials, and it is not unusual in patients with severe weakness to be unable to recruit motor unit action potentials. The pathogenic basis of CIP is not known. Perhaps circulating toxins and metabolic abnormalities associated with sepsis and multiorgan failure impair axonal transport or mitochondrial function, leading to axonal degeneration.

1	Leprosy, caused by the acid-fast bacteria Mycobacterium leprae, is the most common cause of peripheral neuropathy in Southeast Asia, Africa, and South America (Chap. 203). Clinical manifestations range from tuberculoid leprosy at one end to lepromatous leprosy at the other end of the spectrum, with borderline leprosy in between. Neuropathies are most common in patients with borderline leprosy. Superficial cutaneous nerves of the ears and distal limbs are commonly affected. Mononeuropathies, multiple mononeuropathies, or a slowly progressive symmetric sensorimotor polyneuropathy may develop. Sensory NCS are usually absent in the lower limb and are reduced in amplitude in the arms. Motor NCS may demonstrate reduced amplitudes in affected nerves but occasionally can reveal demyelinating features. Leprosy is usually diagnosed by skin lesion biopsy. Nerve biopsy can also be diagnostic, particularly when there are no apparent skin lesions. The tuberculoid form is characterized by

1	features. Leprosy is usually diagnosed by skin lesion biopsy. Nerve biopsy can also be diagnostic, particularly when there are no apparent skin lesions. The tuberculoid form is characterized by granulomas, and bacilli are not seen. In contrast, with lepromatous leprosy, large numbers of infiltrating bacilli, TH2 lymphocytes, and organism-laden, foamy macrophages with minimal granulomatous infiltration are evident. The bacilli are best appreciated using the Fite stain, where they can be seen as red-staining rods often in clusters free in the endoneurium, within macrophages, or within Schwann cells.

1	Patients are generally treated with multiple drugs: dapsone, rifampin, and clofazimine. Other medications that are used include thalidomide, pefloxacin, ofloxacin, sparfloxacin, minocycline, and clarithromycin. Patients are generally treated for 2 years. Treatment is sometimes complicated by the so-called reversal reaction, particularly in borderline leprosy. The reversal reaction can occur at any time during treatment and develops because of a shift to the tuberculoid end of the spectrum, with an increase in cellular immunity during treatment. The cellular response is upregulated as evidenced by an increased release of tumor necrosis factor α, interferon γ, and interleukin 2, with new granuloma formation. This can result in an exacerbation of the rash and the neuropathy as well as in appearance of new lesions. High-dose glucocorticoids blunt this adverse reaction and may be used prophylactically at treatment onset in high-risk patients. Erythema nodosum leprosum (ENL) is also treated

1	of new lesions. High-dose glucocorticoids blunt this adverse reaction and may be used prophylactically at treatment onset in high-risk patients. Erythema nodosum leprosum (ENL) is also treated with glucocorticoids or thalidomide.

1	Lyme disease is caused by infection with Borrelia burgdorferi, a spirochete usually transmitted by the deer tick Ixodes dammini (Chap. 210). Neurologic complications may develop during the second and third stages of infection. Facial neuropathy is most common and is bilateral in about half of cases, which is rare for idiopathic Bell’s palsy. Involvement of nerves is frequently asymmetric. Some patients present with a polyradiculoneuropathy or multiple mononeuropathies. EDx is suggestive of a primary axonopathy. Nerve biopsies can reveal axonal degeneration with perivascular inflammation. Treatment is with antibiotics (Chap. 210).

1	Diphtheria is caused by the bacteria Corynebacterium diphtheriae (Chap. 175). Infected individuals present with flulike symptoms of generalized myalgias, headache, fatigue, low-grade fever, and irritability within a week to 10 days of the exposure. About 20–70% of patients develop a peripheral neuropathy caused by a toxin released by the bacteria. Three to 4 weeks after infection, patients may note decreased sensation in their throat and begin to develop dysphagia, dysarthria, hoarseness, and blurred vision due to impaired accommodation. A generalized polyneuropathy may manifest 2 or 3 months following the initial infection, characterized by numbness, paresthesias, and weakness of the arms and legs and occasionally ventilatory failure. CSF protein can be elevated with or without lymphocytic pleocytosis. EDx suggests a diffuse axonal sensorimotor polyneuropathy. Antitoxin and antibiotics should be given within 48 h of symptom onset. Although early treatment reduces the incidence and

1	pleocytosis. EDx suggests a diffuse axonal sensorimotor polyneuropathy. Antitoxin and antibiotics should be given within 48 h of symptom onset. Although early treatment reduces the incidence and severity of some complications (i.e., cardiomyopathy), it does not appear to alter the natural history of the associated peripheral neuropathy. The neuropathy usually resolves after several months.

1	HIV infection can result in a variety of neurologic complications, including peripheral neuropathies (Chap. 226). Approximately 20% of HIV-infected individuals develop a neuropathy either as a direct result of the virus itself, other associated viral infections (e.g., CMV), or neurotoxicity secondary to antiviral medications (see below). The major presentations of peripheral neuropathy associated with HIV infection include (1) distal symmetric polyneuropathy, (2) inflammatory demyelinating polyneuropathy (including both GBS and CIDP), (3) multiple mononeuropathies (e.g., vasculitis, CMV-related), (4) polyradiculopathy (usually CMV-related), (5) autonomic neuropathy, and (6) sensory ganglionitis.

1	HIV-Related Distal Symmetric Polyneuropathy (DSP) DSP is the most common form of peripheral neuropathy associated with HIV infection and usually is seen in patients with AIDS. It is characterized by numbness and painful paresthesias involving the distal extremities. The pathogenic basis for DSP is unknown but is not due to actual infection of the peripheral nerves. The neuropathy may be immune mediated, perhaps caused by the release of cytokines from surrounding inflammatory cells. Vitamin B12 deficiency may contribute in some instances but is not a major cause of most cases of DSP. Some antiretroviral agents (e.g., dideoxycytidine, dideoxyinosine, stavudine) are also neurotoxic and can cause a painful sensory neuropathy.

1	HIV-Related Inflammatory Demyelinating Polyradiculoneuropathy Both AIDP and CIDP can occur as a complication of HIV infection. AIDP usually develops at the time of seroconversion, whereas CIDP can occur any time in the course of the infection. Clinical and EDx features are indistinguishable from idiopathic AIDP or CIDP (discussed in Chap. 460). In addition to elevated protein levels, lymphocytic pleocytosis is evident in the CSF, a finding that helps distinguish this HIV-associated polyradiculoneuropathy from idiopathic AIDP/CIDP.

1	HIV-Related Progressive Polyradiculopathy An acute, progressive lumbosacral polyradiculoneuropathy usually secondary to CMV infection can develop in patients with AIDS. Patients present with severe radicular pain, numbness, and weakness in the legs, which is usually asymmetric. CSF is abnormal, demonstrating an increased protein along with reduced glucose concentration and notably a neutrophilic pleocytosis. EDx studies reveal features of active axonal degeneration. The polyradiculoneuropathy may improve with antiviral therapy. HIV-Related Multiple Mononeuropathies Multiple mononeuropathies can also develop in patients with HIV infection, usually in the context of AIDS. Weakness, numbness, paresthesias, and pain occur in the distribution of affected nerves. Nerve biopsies can reveal axonal degeneration with necrotizing vasculitis or perivascular inflammation. Glucocorticoid treatment is indicated for vasculitis directly due to HIV infection.

1	HIV-Related Sensory Neuronopathy/Ganglionopathy Dorsal root ganglionitis is a very rare complication of HIV infection, and neuronopathy can be the presenting manifestation. Patients develop sensory ataxia similar to idiopathic sensory neuronopathy/ganglionopathy. NCS reveal reduced amplitudes or absence of sensory nerve action potentials (SNAPs).

1	Peripheral neuropathy from herpes varicella-zoster (HVZ) infection results from reactivation of latent virus or from a primary infection (Chap. 217). Two-thirds of infections in adults are characterized by dermal zoster in which severe pain and paresthesias develop in a dermatomal region followed within a week or two by a vesicular rash in the same distribution. Weakness in muscles innervated by roots corresponding to the dermatomal distribution of skin lesions occurs in 5–30% of patients. Approximately 25% of affected patients have continued pain (postherpetic neuralgia [PHN]). A large clinical trial demonstrated that vaccination against zoster reduces the incidence of HVZ among vaccine recipients by 51% and reduces the incidence of PHN by 67%. Treatment of PHN is symptomatic (Table 459-6). CMV can cause an acute lumbosacral polyradiculopathy and multiple mononeuropathies in patients with HIV infection and in other immune deficiency conditions (Chap. 219).

1	CMV can cause an acute lumbosacral polyradiculopathy and multiple mononeuropathies in patients with HIV infection and in other immune deficiency conditions (Chap. 219). EBV infection has been associated with GBS, cranial neuropathies, mononeuropathy multiplex, brachial plexopathy, lumbosacral radiculoplexopathy, and sensory neuronopathies (Chap. 218). Hepatitis B and C can cause multiple mononeuropathies related to vasculitis, AIDP, or CIDP (Chap. 362). Patients with malignancy can develop neuropathies due to (1) a direct effect of the cancer by invasion or compression of the nerves, (2) remote or paraneoplastic effect, (3) a toxic effect of treatment, or (4) as a consequence of immune compromise caused by immunosuppressive medications. The most common associated malignancy is lung cancer, but neuropathies also complicate carcinoma of the breast, ovaries, stomach, colon, rectum, and other organs, including the lymphoproliferative system.

1	Paraneoplastic encephalomyelitis/sensory neuronopathy (PEM/SN) usually complicates small-cell lung carcinoma (Chap. 122). Patients usually present with numbness and paresthesias in the distal extremities that are often asymmetric. The onset can be acute or insidiously progressive. Prominent loss of proprioception leads to sensory ataxia. Weakness can be present, usually secondary to an associated myelitis, motor neuronopathy, or concurrent Lambert-Eaton myasthenic syndrome (LEMS). Many patients also develop confusion, memory loss, depression, hallucinations or seizures, or cerebellar ataxia. Polyclonal antineuronal antibodies (IgG) directed against a 35to 40-kDa protein or complex of proteins, the so-called Hu antigen, are found in the sera or CSF in the majority of patients with paraneoplastic PEM/SN. CSF may be normal or may demonstrate mild lymphocytic pleocytosis and elevated protein. PEM/SN is probably the result of antigenic similarity between proteins expressed in the tumor

1	PEM/SN. CSF may be normal or may demonstrate mild lymphocytic pleocytosis and elevated protein. PEM/SN is probably the result of antigenic similarity between proteins expressed in the tumor cells and neuronal cells, leading to an immune response directed against both cell types. Treatment of the underlying cancer generally does not affect the course of PEM/ SN. However, occasional patients may improve following treatment of the tumor. Unfortunately, plasmapheresis, intravenous immunoglobulin, and immunosuppressive agents have not shown benefit.

1	Malignant cells, in particular leukemia and lymphoma, can infiltrate cranial and peripheral nerves, leading to mononeuropathy, mononeuropathy multiplex, polyradiculopathy, plexopathy, or even a generalized symmetric distal or proximal and distal polyneuropathy. Neuropathy related to tumor infiltration is often painful; it can be the presenting manifestation of the cancer or the heralding symptom of a relapse. The neuropathy may improve with treatment of the underlying leukemia or lymphoma or with glucocorticoids.

1	Neuropathies may develop in patients who undergo bone marrow transplantation (BMT) because of the toxic effects of chemotherapy, radiation, infection, or an autoimmune response directed against the peripheral nerves. Peripheral neuropathy in BMT is often associated with graft-versus-host disease (GVHD). Chronic GVHD shares many features with a variety of autoimmune disorders, and it is possible that an immune-mediated response directed against peripheral nerves is responsible. Patients with chronic GVHD may develop cranial neuropathies, sensorimotor polyneuropathies, multiple mononeuropathies, and severe generalized peripheral neuropathies resembling AIDP or CIDP. The neuropathy may improve by increasing the intensity of immunosuppressive or immunomodulating therapy and resolution of the GVHD.

1	Lymphomas may cause neuropathy by infiltration or direct compression of nerves or by a paraneoplastic process. The neuropathy can be purely sensory or motor, but most commonly is sensorimotor. The pattern of involvement may be symmetric, asymmetric, or multifocal, and the course may be acute, gradually progressive, or relapsing and remitting. EDx can be compatible with either an axonal or demyelinating process. CSF may reveal lymphocytic pleocytosis and an elevated protein. Nerve biopsy may demonstrate endoneurial inflammatory cells in both the infiltrative and the paraneoplastic etiologies. A monoclonal population of cells favors lymphomatous invasion. The neuropathy may respond to treatment of the underlying lymphoma or immunomodulating therapies.

1	Multiple myeloma (MM) usually presents in the fifth to seventh decade of life with fatigue, bone pain, anemia, and hypercalcemia (Chap. 136). Clinical and EDx features of neuropathy occur in as many as 40% of patients. The most common pattern is that of a distal, axonal, sensory, or sensorimotor polyneuropathy. Less frequently, a chronic demyelinating polyradiculoneuropathy may develop (see POEMS, Chap. 460). MM can be complicated by amyloid polyneuropathy and should be considered in patients with painful paresthesias, loss of pinprick and 2686 temperature discrimination, and autonomic dysfunction (suggestive of a small-fiber neuropathy) and CTS. Expanding plasmacytomas can compress cranial nerves and spinal roots as well. A monoclonal protein, usually composed of γ or μ heavy chains or κ light chains, may be identified in the serum or urine. EDx usually shows reduced amplitudes with normal or only mildly abnormal distal latencies and conduction velocities. A superimposed median

1	or κ light chains, may be identified in the serum or urine. EDx usually shows reduced amplitudes with normal or only mildly abnormal distal latencies and conduction velocities. A superimposed median neuropathy at the wrist is common. Abdominal fat pad, rectal, or sural nerve biopsy can be performed to look for amyloid deposition. Unfortunately, the treatment of the underlying MM does not usually affect the course of the neuropathy.

1	NEUROPATHIES ASSOCIATED WITH MONOCLONAL GAMMOPATHY OF UNCERTAIN SIGNIFICANCE (SEE CHAP. 460) Toxic Neuropathies Secondary to Chemotherapy Many of the commonly used chemotherapy agents can cause a toxic neuropathy (Table 459-7). The mechanisms by which these agents cause toxic neuropathies vary, as does the specific type of neuropathy produced. The risk of developing a toxic neuropathy or more severe neuropathy appears to be greater in patients with a preexisting neuropathy (e.g., Charcot-Marie-Tooth disease, diabetic neuropathy) and those who also take other potentially neurotoxic drugs (e.g., nitrofurantoin, isoniazid, disulfiram, pyridoxine). Chemotherapeutic agents usually cause a sensory greater than motor length-dependent axonal neuropathy or neuronopathy/ganglionopathy. Neuropathies can develop as complications of toxic effects of various drugs and other environmental exposures (Table 459-8). The more common neuropathies associated with these agents are discussed here.

1	Chloroquine and hydroxychloroquine can cause a toxic myopathy characterized by slowly progressive, painless, proximal weakness and atrophy, which is worse in the legs than the arms. In addition, neuropathy can also develop with or without the myopathy leading to sensory loss and distal weakness. The “neuromyopathy” usually appears in patients taking 500 mg daily for a year or more but has been reported with doses as low as 200 mg/d. Serum CK levels are usually elevated due to the superimposed myopathy. NCS reveal mild slowing of motor and sensory NCVs with a mild to moderate reduction in the amplitudes, although NCS may be normal in patients with only the myopathy. EMG demonstrates myopathic muscle action potentials (MUAPs), increased insertional activity in the form of positive sharp waves, fibrillation potentials, and occasionally myotonic potentials, particularly in the proximal muscles. Neurogenic MUAPs and reduced recruitment are found in more distal muscles. Nerve biopsy

1	waves, fibrillation potentials, and occasionally myotonic potentials, particularly in the proximal muscles. Neurogenic MUAPs and reduced recruitment are found in more distal muscles. Nerve biopsy demonstrates autophagic vacuoles within Schwann cells. Vacuoles may also be evident in muscle biopsies. The pathogenic basis of the neuropathy is not known but may be related to the amphiphilic properties of the drug. These agents contain both hydrophobic and hydrophilic regions

1	Drug Mechanism of Neurotoxicity Clinical Features Nerve Histopathology EMG/NCS Vinca alkaloids (vincristine, vinblastine, vindesine, vinorelbine) Taxanes (paclitaxel, docetaxel) Bortezomib (Velcade) Interfere with axonal microtubule assembly; impairs axonal transport Preferential damage to dorsal root ganglia: ? binds to and cross-links DNA ? inhibits protein synthesis ? impairs axonal transport Promotes axonal microtubule assembly; interferes with axonal transport Unknown; ? inhibition of neurotrophic growth factor binding; ? neuronal lysosomal storage Unknown; ? immunomodulating effects Unknown; ? selective Schwann cell toxicity; ? immunomodulating effects Unknown; ? selective dorsal root ganglia toxicity Unknown Symmetric, S-M, large-/ small-fiber PN; autonomic symptoms common; infrequent cranial neuropathies Symmetric, predominantly sensory PN; large-fiber modalities affected more than small-fiber Symmetric, length-dependent, sensory-predominant PN

1	Symmetric, predominantly sensory PN; large-fiber modalities affected more than small-fiber Symmetric, length-dependent, sensory-predominant PN Subacute, S-M PN with diffuse proximal and distal weakness; areflexia; increased CSF protein Length-dependent, sensory-predominant PN; autonomic neuropathy Length-dependent, sensory, predominantly small-fiber PN Axonal degeneration of myelinated and unmyelinated fibers; regenerating clusters, minimal segmental demyelination Loss of large > small myelinated and unmyelinated fibers; axonal degeneration with small clusters of regenerating fibers; secondary segmental demyelination Loss of large > small myelinated and unmyelinated fibers; axonal degeneration with small clusters of regenerating fibers; secondary segmental demyelination Loss of large and small myelinated fibers with primary demyelination and secondary axonal degeneration; occasional epiand endoneurial inflammatory cell infiltrates

1	Loss of large and small myelinated fibers with primary demyelination and secondary axonal degeneration; occasional epiand endoneurial inflammatory cell infiltrates Loss of myelinated nerve fibers; axonal degeneration; segmental demyelination; no inflammation Axonal sensorimotor PN; distal denervation on EMG; abnormal QST, particularly vibratory perception Low-amplitude or unobtainable SNAPs with normal CMAPs and EMG; abnormal QST, particularly vibratory perception Axonal sensorimotor PN; distal denervation on EMG; abnormal QST, particularly vibratory perception Abnormalities consistent with an axonal S-M PN Features suggestive of an acquired demyelinating sensorimotor PN (e.g., slow CVs, prolonged distal latencies and F-wave latencies, conduction block, temporal dispersion) Axonal, demyelinating, or mixed S-M PN; denervation on EMG Abnormalities consistent with an axonal S-M PN

1	Axonal, demyelinating, or mixed S-M PN; denervation on EMG Abnormalities consistent with an axonal S-M PN Abnormalities consistent with an axonal sensory neuropathy with early small-fiber involvement (abnormal autonomic studies) Abbreviations: CMAP, compound motor action potential; CSF, cerebrospinal fluid; CVs, conduction velocities; EMG, electromyography; GBS, Guillain-Barré syndrome; NCS, nerve conduction studies; PN, polyneuropathy; QST, quantitative sensory testing; S-M, sensorimotor; SNAP, sensory nerve action potential. Source: From AA Amato, J Russell: Neuromuscular Disease. New York, McGraw-Hill, 2008. Drug Mechanism of Neurotoxicity Clinical Features Nerve Histopathology EMG/NCS

1	Source: From AA Amato, J Russell: Neuromuscular Disease. New York, McGraw-Hill, 2008. Drug Mechanism of Neurotoxicity Clinical Features Nerve Histopathology EMG/NCS Misonidazole Unknown Painful paresthesias and loss of Axonal degeneration of large Low-amplitude or unobtainable largeand small-fiber sensory myelinated fibers; axonal SNAPs with normal or only slightly modalities and sometimes distal swellings; segmental demy-reduced CMAPs amplitudes weakness in length-dependent elination pattern Metronidazole Unknown Painful paresthesias and loss of Axonal degeneration Low-amplitude or unobtainable largeand small-fiber sensory SNAPs with normal CMAPs

1	Metronidazole Unknown Painful paresthesias and loss of Axonal degeneration Low-amplitude or unobtainable largeand small-fiber sensory SNAPs with normal CMAPs Chloroquine and Amphiphilic properties may Loss of largeand small-fiber sen-Axonal degeneration with Low-amplitude or unobtainable hydroxychloroquine lead to drug-lipid complexes sory modalities and distal weak-autophagic vacuoles in nerves SNAPs with normal or reduced that are indigestible and result ness in length-dependent pattern; as well as muscle fibers CMAP amplitudes; distal denervain accumulation of autopha-superimposed myopathy may tion on EMG; irritability and myogic vacuoles lead to proximal weakness pathic-appearing MUAPs proximally in patients with superimposed toxic myopathy

1	Amiodarone Amphiphilic properties may Paresthesias and pain with loss Axonal degeneration and Low-amplitude or unobtainable lead to drug-lipid complexes of largeand small-fiber sensory segmental demyelination with SNAPs with normal or reduced that are indigestible and result modalities and distal weakness in myeloid inclusions in nerves CMAP amplitudes; can also have in accumulation of autopha-length-dependent pattern; super-and muscle fibers prominent slowing of CVs; distal gic vacuoles imposed myopathy may lead to and myopathic-appearing MUAPs proximally in patients with superimposed toxic myopathy

1	Colchicine Inhibits polymerization of Numbness and paresthesias with Nerve biopsy demonstrates Low-amplitude or unobtainable tubulin in microtubules and loss of large-fiber modalities in a axonal degeneration; muscle SNAPs with normal or reduced impairs axoplasmic flow length-dependent fashion; super-biopsy reveals fibers with CMAP amplitudes; irritability and imposed myopathy may lead vacuoles myopathic-appearing MUAPs to proximal in addition to distal proximally in patients with super-weakness Podophyllin Binds to microtubules and Sensory loss, tingling, muscle Axonal degeneration Low-amplitude or unobtainable impairs axoplasmic flow weakness, and diminished SNAPs with normal or reduced muscle stretch reflexes in length- Thalidomide Unknown Numbness, tingling, and burning Axonal degeneration; autopsy Low-amplitude or unobtainable pain and weakness in a length-studies reveal degeneration of SNAPs with normal or reduced dependent pattern dorsal root ganglia CMAP amplitudes

1	Disulfiram Accumulation of neurofila-Numbness, tingling, and burn-Axonal degeneration with Low-amplitude or unobtainable ments and impaired axoplas-ing pain in a length-dependent accumulation of neurofila-SNAPs with normal or reduced mic flow pattern ments in the axons CMAP amplitudes Dapsone Unknown Distal weakness that may progress Axonal degeneration and seg-Low-amplitude or unobtainable to proximal muscles; sensory loss mental demyelination CMAPs with normal or reduced SNAP amplitudes SNAPs with normal or reduced CMAP amplitudes Nitrofurantoin Unknown Numbness, painful paresthesias, Axonal degeneration; autopsy Low-amplitude or unobtainable and severe weakness that may studies reveal degeneration SNAPs with normal or reduced resemble GBS of dorsal root ganglia and CMAP amplitudes anterior horn cells Pyridoxine Unknown Dysesthesias and sensory ataxia; Marked loss of sensory axons Reduced amplitudes or absent (vitamin B6)

1	Pyridoxine Unknown Dysesthesias and sensory ataxia; Marked loss of sensory axons Reduced amplitudes or absent (vitamin B6) Isoniazid Inhibits pyridoxal phospho-Dysesthesias and sensory ataxia; Marked loss of sensory axons Reduced amplitudes or absent kinase leading to pyridoxine impaired large-fiber sensory and cell bodies in dorsal root SNAPs and, to a lesser extent, deficiency modalities on examination ganglia and degeneration of CMAPs the dorsal columns Ethambutol Unknown Numbness with loss of large-fiber Axonal degeneration Reduced amplitudes or absent modalities on examination SNAPs Phenytoin Unknown Numbness with loss of large-fiber Axonal degeneration and Low-amplitude or unobtainable modalities on examination segmental demyelination SNAPs with normal or reduced CMAP amplitudes Lithium Unknown Numbness with loss of large-fiber Axonal degeneration Low-amplitude or unobtainable modalities on examination SNAPs with normal or reduced CMAP amplitudes

1	Lithium Unknown Numbness with loss of large-fiber Axonal degeneration Low-amplitude or unobtainable modalities on examination SNAPs with normal or reduced CMAP amplitudes Drug Mechanism of Neurotoxicity Clinical Features Nerve Histopathology EMG/NCS Unknown; may act as alkylating agent and bind DNA Unknown; may lead to covalent cross-linking between neurofilaments Unknown; may interfere with mitochondria Unknown; may combine with sulfhydryl groups Unknown; may combine with sulfhydryl groups Numbness with loss of large-fiber modalities on examination; sensory ataxia; mild distal weakness Length-dependent numbness and tingling with mild distal weakness Early features are those of neuromuscular blockade with generalized weakness; later axonal sensorimotor PN ensues Acute, severe sensorimotor PN that may resemble GBS Encephalopathy; motor neuropathy (often resembles radial neuropathy with wrist and finger drop); autonomic neuropathy; bluish-black discoloration of gums

1	Encephalopathy; motor neuropathy (often resembles radial neuropathy with wrist and finger drop); autonomic neuropathy; bluish-black discoloration of gums Encephalopathy; painful sensory symptoms; mild loss of vibration; distal or generalized weakness may also develop; autonomic neuropathy; alopecia Abdominal discomfort, burning pain, and paresthesias; generalized weakness; autonomic insufficiency; can resemble GBS Distal paresthesias and reduction of all sensory modalities Degeneration of sensory axons in peripheral nerves and posterior columns, spinocerebellar tracts, mammillary bodies, optic tracts, and corticospinal tracts in the CNS Axonal swellings with accumulation of neurofilaments Axonal degeneration along with degeneration of gracile fasciculus and corticospinal tracts Axonal degeneration and giant axons swollen with neurofilaments Axonal degeneration of motor axons Axonal degeneration; degeneration of dorsal root ganglia, calcarine, and cerebellar cortex

1	Axonal degeneration and giant axons swollen with neurofilaments Axonal degeneration of motor axons Axonal degeneration; degeneration of dorsal root ganglia, calcarine, and cerebellar cortex Axonal degeneration Low-amplitude or unobtainable SNAPs with normal or reduced CMAP amplitudes Low-amplitude or unobtainable SNAPs with normal or reduced CMAP amplitudes Low-amplitude or unobtainable SNAPs with normal or reduced CMAP amplitudes Early: repetitive firing of CMAPs and decrement with repetitive nerve stimulation; late: axonal sensorimotor PN Features of a mixed axonal and/or demyelinating sensorimotor axonal PN—reduced amplitudes, prolonged distal latencies, conduction block, and slowing of CVs Reduction of CMAP amplitudes with active denervation on EMG Low-amplitude or unobtainable SNAPs with normal or reduced CMAP amplitudes Low-amplitude or unobtainable SNAPs with normal or reduced CMAP amplitudes

1	Low-amplitude or unobtainable SNAPs with normal or reduced CMAP amplitudes Low-amplitude or unobtainable SNAPs with normal or reduced CMAP amplitudes Low-amplitude or unobtainable SNAPs with normal or reduced CMAP amplitudes; may have demyelinating features: prolonged distal latencies and slowing of CVs Abbreviations: CMAP, compound motor action potential; CVs, conduction velocities; EMG, electromyography; GBS, Guillain-Barré syndrome; MUAP, muscle action potential; NCS, nerve conduction studies; PN, polyneuropathy; S-M, sensorimotor; SNAP, sensory nerve action potential Source: From AA Amato, J Russell: Neuromuscular Disease. New York, McGraw-Hill, 2008.

1	that allow them to interact with the anionic phospholipids of cell axonal degeneration. Colchicine inhibits the polymerization of tubulin membranes and organelles. The drug-lipid complexes may be resis-into microtubules. The disruption of the microtubules probably leads to tant to digestion by lysosomal enzymes, leading to the formation of defective intracellular movement of important proteins, nutrients, and autophagic vacuoles filled with myeloid debris that may in turn cause waste products in muscle and nerves. degeneration of nerves and muscle fibers. The signs and symptoms of the neuropathy and myopathy are usually reversible following discon-THALIDOMIDE tinuation of medication. Thalidomide is an immunomodulating agent used to treat mul tiple myeloma, GVHD, leprosy, and other autoimmune disorders. AMIODARONE Thalidomide is associated with severe teratogenic effects as well as Amiodarone can cause a neuromyopathy similar to chloroquine peripheral neuropathy that can be

1	autoimmune disorders. AMIODARONE Thalidomide is associated with severe teratogenic effects as well as Amiodarone can cause a neuromyopathy similar to chloroquine peripheral neuropathy that can be dose-limiting. Patients develop and hydroxychloroquine. The neuromyopathy typically appears numbness, painful tingling, and burning discomfort in the feet and after patients have taken the medication for 2–3 years. Nerve biopsy hands and less commonly muscle weakness and atrophy. Even after demonstrates a combination of segmental demyelination and axonal stopping the drug for 4–6 years, as many as 50% patients continue loss. Electron microscopy reveals lamellar or dense inclusions in to have significant symptoms. NCS demonstrate reduced amplitudes Schwann cells, pericytes, and endothelial cells. The inclusions in or complete absence of SNAPs, with preserved conduction velocities muscle and nerve biopsies have persisted as long as 2 years following when obtainable. Motor NCS are usually

1	The inclusions in or complete absence of SNAPs, with preserved conduction velocities muscle and nerve biopsies have persisted as long as 2 years following when obtainable. Motor NCS are usually normal. Nerve biopsy reveals discontinuation of the medication. a loss of large-diameter myelinated fibers and axonal degeneration.

1	Degeneration of dorsal root ganglion cells has been reported at autopsy. COLCHICINE Colchicine can also cause a neuromyopathy. Patients usually present PYRIDOXINE (VITAMIN B6) TOXICITY with proximal weakness and numbness and tingling in the distal extrem-Pyridoxine is an essential vitamin that serves as a coenzyme for transities. EDx reveals features of an axonal polyneuropathy. Muscle biopsy amination and decarboxylation. However, at high doses (116 mg/d), reveals a vacuolar myopathy, whereas sensory nerves demonstrate patients can develop a severe sensory neuropathy with dysesthesias and sensory ataxia. NCS reveal absent or markedly reduced SNAP amplitudes with relatively preserved CMAPs. Nerve biopsy reveals axonal loss of fiber at all diameters. Loss of dorsal root ganglion cells with subsequent degeneration of both the peripheral and central sensory tracts have been reported in animal models.

1	One of the most common side effects of isoniazid (INH) is peripheral neuropathy. Standard doses of INH (3–5 mg/kg per day) are associated with a 2% incidence of neuropathy, whereas neuropathy develops in at least 17% of patients taking in excess of 6 mg/kg per day. The elderly, malnourished, and “slow acetylators” are at increased risk for developing the neuropathy. INH inhibits pyridoxal phosphokinase, resulting in pyridoxine deficiency and the neuropathy. Prophylactic administration of pyridoxine 100 mg/d can prevent the neuropathy from developing.

1	The nucleoside analogues zalcitabine (dideoxycytidine or ddC), didanosine (dideoxyinosine or ddI), stavudine (d4T), lamivudine (3TC), and antiretroviral nucleoside reverse transcriptase inhibitor (NRTI) are used to treat HIV infection. One of the major dose-limiting side effects of these medications is a predominantly sensory, length-dependent, symmetrically painful neuropathy. Zalcitabine (ddC) is the most extensively studied of the nucleoside analogues, and at doses greater than 0.18 mg/kg per day, it is associated with a subacute onset of severe burning and lancinating pains in the feet and hands. NCS reveal decreased amplitudes of the SNAPs with normal motor studies. The nucleoside analogues inhibit mitochondrial DNA polymerase, which is the suspected pathogenic basis for the neuropathy. Because of a “coasting effect,” patients can continue to worsen even 2–3 weeks after stopping the medication. Following dose reduction, improvement in the neuropathy is seen in most patients after

1	Because of a “coasting effect,” patients can continue to worsen even 2–3 weeks after stopping the medication. Following dose reduction, improvement in the neuropathy is seen in most patients after several months (mean time about 10 weeks).

1	HEXACARBONS (n-HEXANE, METHYL n-BUTYL KETONE)/GLUE SNIFFER’S NEUROPATHY n-Hexane and methyl n-butyl ketone are water-insoluble industrial organic solvents that are also present in some glues. Exposure through inhalation, accidentally or intentionally (glue sniffing), or through skin absorption can lead to a profound subacute sensory and motor polyneuropathy. NCS demonstrate decreased amplitudes of the SNAPs and CMAPs with slightly slow CVs. Nerve biopsy reveals a loss of myelinated fibers and giant axons that are filled with 10-nm neurofilaments. Hexacarbon exposure leads to covalent cross-linking between axonal neurofilaments that result in their aggregation, impaired axonal transport, swelling of the axons, and eventual axonal degeneration.

1	Lead neuropathy is uncommon, but it can be seen in children who accidentally ingest lead-based paints in older buildings and in industrial workers exposed to lead-containing products. The most common presentation of lead poisoning is an encephalopathy; however, symptoms and signs of a primarily motor neuropathy can also occur. The neuropathy is characterized by an insidious and progressive onset of weakness usually beginning in the arms, in particular involving the wrist and finger extensors, resembling a radial neuropathy. Sensation is generally preserved; however, the autonomic nervous system can be affected. Laboratory investigation can reveal a microcytic hypo-chromic anemia with basophilic stippling of erythrocytes, an elevated serum lead level, and an elevated serum coproporphyrin level. A 24-h urine collection demonstrates elevated levels of lead excretion. The NCS may reveal reduced CMAP amplitudes, while the SNAPs are typically normal. The pathogenic basis may be related to

1	level. A 24-h urine collection demonstrates elevated levels of lead excretion. The NCS may reveal reduced CMAP amplitudes, while the SNAPs are typically normal. The pathogenic basis may be related to abnormal porphyrin metabolism. The most important principle of management is to remove the source of the exposure. Chelation therapy with calcium disodium ethylene-diaminetetraacetic acid (EDTA), British anti-Lewisite (BAL), and penicillamine also demonstrates variable efficacy.

1	Mercury toxicity may occur as a result of exposure to either organic or inorganic mercurials. Mercury poisoning presents with paresthesias in hands and feet that progress proximally and may involve the face and tongue. Motor weakness can also develop. CNS symptoms often overshadow the neuropathy. EDx shows features of a primarily axonal sensorimotor polyneuropathy. The primary site of neuromuscular pathology appears to be the dorsal root ganglia. The mainstay of treatment is removing the source of exposure.

1	Thallium can exist in a monovalent or trivalent form and is primarily used as a rodenticide. The toxic neuropathy usually manifests as burning paresthesias of the feet, abdominal pain, and vomiting. Increased thirst, sleep disturbances, and psychotic behavior may be noted. Within the first week, patients develop pigmentation of the hair, an acne-like rash in the malar area of the face, and hyperreflexia. By the second and third week, autonomic instability with labile heart rate and blood pressure may be seen. Hyporeflexia and alopecia also occur but may not be evident until the third or fourth week following exposure. With severe intoxication, proximal weakness and involvement of the cranial nerves can occur. Some patients require mechanical ventilation due to respiratory muscle involvement. The lethal dose of thallium is variable, ranging from 8 to 15 mg/kg body weight. Death can result in less than 48 h following a particularly large dose. NCS demonstrate features of a primarily

1	The lethal dose of thallium is variable, ranging from 8 to 15 mg/kg body weight. Death can result in less than 48 h following a particularly large dose. NCS demonstrate features of a primarily axonal sensorimotor polyneuropathy. With acute intoxication, potassium ferric ferrocyanide II may be effective in preventing absorption of thallium from the gut. However, there may be no benefit once thallium has been absorbed. Unfortunately, chelating agents are not very efficacious. Adequate diuresis is essential to help eliminate thallium from the body without increasing tissue availability from the serum.

1	Arsenic is another heavy metal that can cause a toxic sensorimotor polyneuropathy. The neuropathy manifests 5–10 days after ingestion of arsenic and progresses for several weeks, sometimes mimicking GBS. The presenting symptoms are typically an abrupt onset of abdominal discomfort, nausea, vomiting, pain, and diarrhea followed within several days by burning pain in the feet and hands. Examination of the skin can be helpful in the diagnosis as the loss of the superficial epidermal layer results in patchy regions of increased or decreased pigmentation on the skin several weeks after an acute exposure or with chronic low levels of ingestion. Mee’s lines, which are transverse lines at the base of the fingernails and toenails, do not become evident until 1 or 2 months after the exposure. Multiple Mee’s lines may be seen in patients with long fingernails who have had chronic exposure to arsenic. Mee’s lines are not specific for arsenic toxicity as they can also be seen following thallium

1	Mee’s lines may be seen in patients with long fingernails who have had chronic exposure to arsenic. Mee’s lines are not specific for arsenic toxicity as they can also be seen following thallium poisoning. Because arsenic is cleared from blood rapidly, the serum concentration of arsenic is not diagnostically helpful. However, arsenic levels are increased in the urine, hair, and fingernails of patients exposed to arsenic. Anemia with stippling of erythrocytes is common, and occasionally pancytopenia and aplastic anemia can develop. Increased CSF protein levels without pleocytosis can be seen; this can lead to misdiagnosis as GBS. NCS are usually suggestive of an axonal sensorimotor polyneuropathy; however, demyelinating features can be present. Chelation therapy with BAL has yielded inconsistent results; therefore, it is not generally recommended.

1	Pernicious anemia is the most common cause of cobalamin deficiency. Other causes include dietary avoidance (vegetarians), gastrectomy, gastric bypass surgery, inflammatory bowel disease, pancreatic insufficiency, bacterial overgrowth, and possibly histamine-2 blockers and proton pump inhibitors. An underappreciated cause of cobalamin deficiency is food-cobalamin malabsorption. This typically occurs in older individuals and results from an inability to adequately absorb 2690 cobalamin in food protein. No apparent cause of deficiency is identified in a significant number of patients with cobalamin deficiency. The use of nitrous oxide as an anesthetic agent or as a recreational drug can produce acute cobalamin deficiency neuropathy and subacute combined degeneration. Complaints of numb hands typically appear before lower extremity paresthesias are noted. A preferential large-fiber sensory loss affecting proprioception and vibration with sparing of small-fiber modalities is present; an

1	typically appear before lower extremity paresthesias are noted. A preferential large-fiber sensory loss affecting proprioception and vibration with sparing of small-fiber modalities is present; an unsteady gait reflects sensory ataxia. These features, coupled with diffuse hyperreflexia and absent Achilles reflexes, should always focus attention on the possibility of cobalamin deficiency. Optic atrophy and, in severe cases, behavioral changes ranging from mild irritability and forgetfulness to severe dementia and frank psychosis may appear. The full clinical picture of subacute combined degeneration is uncommon. CNS manifestations, especially pyramidal tract signs, may be missing, and in fact some patients may only exhibit symptoms of peripheral neuropathy. EDx shows an axonal sensorimotor neuropathy. CNS involvement produces abnormal somatosensory and visual evoked potential latencies. The diagnosis is confirmed by finding reduced serum cobalamin levels. In up to 40% of patients,

1	neuropathy. CNS involvement produces abnormal somatosensory and visual evoked potential latencies. The diagnosis is confirmed by finding reduced serum cobalamin levels. In up to 40% of patients, anemia and macrocytosis are lacking. Serum methylmalonic acid and homocysteine, the metabolites that accumulate when cobalamin-dependent reactions are blocked, are elevated. Antibodies to intrinsic factor are present in approximately 60%, and antiparietal cell antibodies in about 90%, of individuals with pernicious anemia. Cobalamin deficiency can be treated with various regimens of cobalamin. One typical regimen consists of 1000 μg cyanocobalamin IM weekly for 1 month and monthly thereafter. Patients with food cobalamin malabsorption can absorb free cobalamin and therefore can be treated with oral cobalamin supplementation. An oral cobalamin dose of 1000 μg per day should be sufficient. Treatment for cobalamin deficiency usually does not completely reverse the clinical manifestations, and at

1	cobalamin supplementation. An oral cobalamin dose of 1000 μg per day should be sufficient. Treatment for cobalamin deficiency usually does not completely reverse the clinical manifestations, and at least 50% of patients exhibit some permanent neurologic deficit.

1	Thiamine (vitamin B1) deficiency is an uncommon cause of peripheral neuropathy in developed countries. It is now most often seen as a consequence of chronic alcohol abuse, recurrent vomiting, total parenteral nutrition, and bariatric surgery. Thiamine deficiency polyneuropathy can occur in normal, healthy young adults who do not abuse alcohol but who engage in inappropriately restrictive diets. Thiamine is water-soluble. It is present in most animal and plant tissues, but the greatest sources are unrefined cereal grains, wheat germ, yeast, soybean flour, and pork. Beriberi means “I can’t, I can’t” in Singhalese, the language of natives of what was once part of the Dutch East Indies (now Sri Lanka). Dry beriberi refers to neuropathic symptoms. The term wet beriberi is used when cardiac manifestations predominate (in reference to edema). Beriberi was relatively uncommon until the late 1800s when it became widespread among people for whom rice was a dietary mainstay. This epidemic was

1	predominate (in reference to edema). Beriberi was relatively uncommon until the late 1800s when it became widespread among people for whom rice was a dietary mainstay. This epidemic was due to a new technique of processing rice that removed the germ from the rice shaft, rendering the so-called polished rice deficient in thiamine and other essential nutrients.

1	Symptoms of neuropathy follow prolonged deficiency. These begin with mild sensory loss and/or burning dysesthesias in the toes and feet and aching and cramping in the lower legs. Pain may be the predominant symptom. With progression, patients develop features of a nonspecific generalized polyneuropathy, with distal sensory loss in the feet and hands.

1	Blood and urine assays for thiamine are not reliable for diagnosis of deficiency. Erythrocyte transketolase activity and the percentage increase in activity (in vitro) following the addition of thiamine pyrophosphate (TPP) may be more accurate and reliable. EDx shows nonspecific findings of an axonal sensorimotor polyneuropathy. When a diagnosis of thiamine deficiency is made or suspected, thiamine replacement should be provided until proper nutrition is restored. Thiamine is usually given intravenously or intramuscularly at a dose of 100 mg/d. Although cardiac manifestations show a striking response to thiamine replacement, neurologic improvement is usually more variable and less dramatic.

1	The term vitamin E is usually used for a-tocopherol, the most active of the four main types of vitamin E. Because vitamin E is present in animal fat, vegetable oils, and various grains, deficiency is usually due to factors other than insufficient intake. Vitamin E deficiency usually occurs secondary to lipid malabsorption or in uncommon disorders of vitamin E transport. One hereditary disorder is abetalipoproteinemia, a rare autosomal dominant disorder characterized by steatorrhea, pigmentary retinopathy, acanthocytosis, and progressive ataxia. Patients with cystic fibrosis may also have vitamin E deficiency secondary to steatorrhea. There are genetic forms of isolated vitamin E deficiency not associated with lipid malabsorption. Vitamin E deficiency may also occur as a consequence of various cholestatic and hepatobiliary disorders as well as short-bowel syndromes resulting from the surgical treatment of intestinal disorders.

1	Clinical features may not appear until many years after the onset of deficiency. The onset of symptoms tends to be insidious, and progression is slow. The main clinical features are spinocerebellar ataxia and polyneuropathy, thus resembling Friedreich’s ataxia or other spinocerebellar ataxias. Patients manifest progressive ataxia and signs of posterior column dysfunction, such as impaired joint position and vibratory sensation. Because of the polyneuropathy, there is hyporeflexia, but plantar responses may be extensor as a result of the spinal cord involvement. Other neurologic manifestations may include ophthalmoplegia, pigmented retinopathy, night blindness, dysarthria, pseudoathetosis, dystonia, and tremor. Vitamin E deficiency may present as an isolated polyneuropathy, but this is very rare. The yield of checking serum vitamin E levels in patients with isolated polyneuropathy is extremely low, and this test should not be part of routine practice.

1	Diagnosis is made by measuring a-tocopherol levels in the serum. EDx shows features of an axonal neuropathy. Treatment is replacement with oral vitamin E, but high doses are not needed. For patients with isolated vitamin E deficiency, treatment consists of 1500–6000 IU/d in divided doses. Vitamin B6, or pyridoxine, can produce neuropathic manifestations from both deficiency and toxicity. Vitamin B6 toxicity was discussed above. Vitamin B6 deficiency is most commonly seen in patients treated with isoniazid or hydralazine. The polyneuropathy of vitamin B6 is nonspecific, manifesting as a generalized axonal sensorimotor polyneuropathy. Vitamin B6 deficiency can be detected by direct assay. Vitamin B6 supplementation with 50–100 mg/d is suggested for patients being treated with isoniazid or hydralazine. This same dose is appropriate for replacement in cases of nutritional deficiency.

1	Pellagra is produced by deficiency of niacin. Although pellagra may be seen in alcoholics, this disorder has essentially been eradicated in most Western countries by means of enriching bread with niacin. Nevertheless, pellagra continues to be a problem in a number of underdeveloped regions, particularly in Asia and Africa, where corn is the main source of carbohydrate. Neurologic manifestations are variable; abnormalities can develop in the brain and spinal cord as well as peripheral nerves. When peripheral nerves are involved, the neuropathy is usually mild and resembles beriberi. Treatment is with niacin 40–250 mg/d.

1	A syndrome that has only recently been described is myeloneuropathy secondary to copper deficiency. Most patients present with lower limb paresthesias, weakness, spasticity, and gait difficulties. Large-fiber sensory function is impaired, reflexes are brisk, and plantar responses are extensor. In some cases, light touch and pinprick sensation are affected, and NCS indicate sensorimotor axonal polyneuropathy in addition to myelopathy.

1	Hematologic abnormalities are a known complication of copper deficiency; these can include microcytic anemia, neutropenia, and occasionally pancytopenia. Because copper is absorbed in the stomach and proximal jejunum, many cases of copper deficiency are in the setting of prior gastric surgery. Excess zinc is an established cause of copper deficiency. Zinc upregulates enterocyte production of metallothionine, which results in decreased absorption of copper. Excessive dietary zinc supplements or denture cream containing zinc can produce this clinical picture. Other potential causes of copper deficiency include malnutrition, prematurity, total parenteral nutrition, and ingestion of copper-chelating agents.

1	Following oral or IV copper replacement, some patients show neurologic improvement, but this may take many months or not occur at all. Replacement consists of oral copper sulfate or gluconate 2 mg one to three times a day. If oral copper replacement is not effective, elemental copper in the copper sulfate or copper chloride forms can be given as 2 mg IV daily for 3–5 days, then weekly for 1–2 months until copper levels normalize. Thereafter, oral daily copper therapy can be resumed. In contrast to the neurologic manifestations, most of the hematologic indices completely normalize in response to copper replacement therapy.

1	Polyneuropathy may occur following gastric surgery for ulcer, cancer, or weight reduction. This usually occurs in the context of rapid, significant weight loss and recurrent, protracted vomiting. The clinical picture is one of acute or subacute sensory loss and weakness. Neuropathy following weight loss surgery usually occurs in the first several months after surgery. Weight reduction surgical procedures include gastrojejunostomy, gastric stapling, vertical banded gastroplasty, and gastrectomy with Roux-en-Y anastomosis. The initial manifestations are usually numbness and paresthesias in the feet. In many cases, no specific nutritional deficiency factor is identified.

1	Management consists of parenteral vitamin supplementation, especially including thiamine. Improvement has been observed following supplementation, parenteral nutritional support, and reversal of the surgical bypass. The duration and severity of deficits before identification and treatment of neuropathy are important predictors of final outcome.

1	CSPN is a diagnosis of exclusion, established after a careful medical, family, and social history; neurologic examination; and directed laboratory testing. Despite extensive evaluation, the cause of polyneuropathy in as many as 50% of all patients is idiopathic. CSPN should be considered a distinct diagnostic subset of peripheral neuropathy. The onset of CSPN is predominantly in the sixth and seventh decades. Patients complain of distal numbness, tingling, and often burning pain that invariably begins in the feet and may eventually involve the fingers and hands. Patients exhibit a distal sensory loss to pinprick, touch, and vibration in the toes and feet, and occasionally in the fingers. It is uncommon to see significant proprioception deficits, even though patients may complain of gait unsteadiness. However, tandem gait may be abnormal in a minority of cases. Neither subjective nor objective evidence of weakness is a prominent feature. Most patients have evidence of both largeand

1	unsteadiness. However, tandem gait may be abnormal in a minority of cases. Neither subjective nor objective evidence of weakness is a prominent feature. Most patients have evidence of both largeand small-fiber loss on neurologic exam and EDx. Approximately 10% of patients have only evidence of small-fiber involvement. The ankle muscle stretch reflex is frequently absent, but in cases with predominantly small-fiber loss, this may be preserved. The EDx findings range from isolated sensory nerve action potential abnormalities (usually with loss of amplitude), to evidence for an axonal sensorimotor neuropathy, to a completely normal study (if primarily small fibers are involved). Therapy primarily involves the control of neuropathic pain (Table 459-6) if present. These drugs should not be used if the patient has only numbness and tingling but no pain.

1	Although no treatment is available that can reverse an idiopathic distal peripheral neuropathy, the prognosis is good. Progression often does not occur or is minimal, with sensory symptoms and signs pro-2691 gressing proximally up to the knees and elbows. The disorder does not lead to significant motor disability over time. The relatively benign course of this disorder should be explained to patients. CTS is a compression of the median nerve in the carpal tunnel at the wrist. The median nerve enters the hand through the carpal tunnel by coursing under the transverse carpal ligament. The symptoms of

1	CTS consist of numbness and paresthesias variably in the thumb, index, middle, and half of the ring finger. At times, the paresthesias can include the entire hand and extend into the forearm or upper arm or can be isolated to one or two fingers. Pain is another common symptom and can be located in the hand and forearm and, at times, in the proximal arm. CTS is common and often misdiagnosed as thoracic outlet syndrome. The signs of CTS are decreased sensation in the median nerve distribution; reproduction of the sensation of tingling when a percussion hammer is tapped over the wrist (Tinel’s sign) or the wrist is flexed for 30–60 s (Phalen’s sign); and weakness of thumb opposition and abduction. EDx is extremely sensitive and shows slowing of sensory and, to a lesser extent, motor median potentials across the wrist. Treatment options consist of avoidance of precipitating activities; control of underlying systemic-associated conditions if present; nonsteroidal anti-inflammatory

1	median potentials across the wrist. Treatment options consist of avoidance of precipitating activities; control of underlying systemic-associated conditions if present; nonsteroidal anti-inflammatory medications; neutral (volar) position wrist splints, especially for night use; glucocorticoid/anesthetic injection into the carpal tunnel; and surgical decompression by dividing the transverse carpal ligament. The surgical option should be considered if there is a poor response to nonsurgical treatments; if there is thenar muscle atrophy and/or weakness; and if there are significant denervation potentials on EMG.

1	Other proximal median neuropathies are very uncommon and include the pronator teres syndrome and anterior interosseous neuropathy. These often occur as a partial form of brachial plexitis.

1	The ulnar nerve passes through the condylar groove between the medial epicondyle and the olecranon. Symptoms consist of paresthesias, tingling, and numbness in the medial hand and half of the fourth and the entire fifth fingers, pain at the elbow or forearm, and weakness. Signs consist of decreased sensation in an ulnar distribution, Tinel’s sign at the elbow, and weakness and atrophy of ulnar-innervated hand muscles. The Froment sign indicates thumb adductor weakness and consists of flexion of the thumb at the interphalangeal joint when attempting to oppose the thumb against the lateral border of the second digit. EDx may show slowing of ulnar motor NCV across the elbow with prolonged ulnar sensory latencies. Treatment consists of avoiding aggravating factors, using elbow pads, and surgery to decompress the nerve in the cubital tunnel. Ulnar neuropathies can also rarely occur at the wrist in the ulnar (Guyon) canal or in the hand, usually after trauma.

1	The radial nerve winds around the proximal humerus in the spiral groove and proceeds down the lateral arm and enters the forearm, dividing into the posterior interosseous nerve and superficial nerve. The symptoms and signs consist of wristdrop; finger extension weakness; thumb abduction weakness; and sensory loss in the dorsal web between the thumb and index finger. Triceps and brachioradialis strength is often normal, and triceps reflex is often intact. Most cases of radial neuropathy are transient compressive (neuropraxic) injuries that recover spontaneously in 6–8 weeks. If there has been prolonged compression and severe axonal damage, it may take several months to recover. Treatment consists of cock-up wrist and finger splints, avoiding further compression, and physical therapy to avoid flexion contracture. If there is no improvement in 2–3 weeks, an EDx study is recommended to confirm the clinical diagnosis and determine the degree of severity.

1	2692 LATERAL FEMORAL CUTANEOUS NEUROPATHY (MERALGIA PARESTHETICA) The lateral femoral cutaneous nerve arises from the upper lumbar plexus (spinal levels L2/3), crosses through the inguinal ligament near its attachment to the iliac bone, and supplies sensation to the anterior lateral thigh. The neuropathy affecting this nerve is also known as meralgia paresthetica. Symptoms and signs consist of paresthesias, numbness, and occasionally pain in the lateral thigh. Symptoms are increased by standing or walking and are relieved by sitting. There is normal strength, and knee reflexes are intact. The diagnosis is clinical, and further tests usually are not performed. EDx is only needed to rule out lumbar plexopathy, radiculopathy, or femoral neuropathy. If the symptoms and signs are classic, EMG is not necessary. Symptoms often resolve spontaneously over weeks or months, but the patient may be left with permanent numbness. Treatment consists of weight loss and avoiding tight belts. Analgesics

1	not necessary. Symptoms often resolve spontaneously over weeks or months, but the patient may be left with permanent numbness. Treatment consists of weight loss and avoiding tight belts. Analgesics in the form of a lidocaine patch, nonsteroidal agents, and occasionally medications for neuropathic pain can be used (Table 459-6). Rarely, locally injecting the nerve with an anesthetic can be tried. There is no role for surgery.

1	Femoral neuropathies can arise as complications of retroperitoneal hematoma, lithotomy positioning, hip arthroplasty or dislocation, iliac artery occlusion, femoral arterial procedures, infiltration by hematogenous malignancy, penetrating groin trauma, pelvic surgery including hysterectomy and renal transplantation, and diabetes (a partial form of lumbosacral diabetic plexopathy); some cases are idiopathic. Patients with femoral neuropathy have difficulty extending their knee and flexing the hip. Sensory symptoms occurring either on the anterior thigh and/or medial leg occur in only half of reported cases. A prominent painful component is the exception rather than the rule, may be delayed, and is often self-limited in nature. The quadriceps (patellar) reflex is diminished.

1	Sciatic neuropathies commonly complicate hip arthroplasty, pelvic procedures in which patients are placed in a prolonged lithotomy position, trauma, hematomas, tumor infiltration, and vasculitis. In addition, many sciatic neuropathies are idiopathic. Weakness may involve all motions of the ankles and toes as well as flexion of the leg at the knee; abduction and extension of the thigh at the hip are spared. Sensory loss occurs in the entire foot and the distal lateral leg. The ankle jerk and on occasion the internal hamstring reflex are diminished or more typically absent on the affected side. The peroneal subdivision of the sciatic nerve is typically involved disproportionately to the tibial counterpart. Thus, patients may have only ankle dorsiflexion and eversion weakness with sparing of knee flexion, ankle inversion, and plantar flexion; these features can lead to misdiagnosis of a common peroneal neuropathy.

1	The sciatic nerve divides at the distal femur into the tibial and peroneal nerve. The common peroneal nerve passes posterior and laterally around the fibular head, under the fibular tunnel. It then divides into the superficial peroneal nerve, which supplies the ankle evertor muscles and sensation over the anterolateral distal leg and dorsum of the foot, and the deep peroneal nerve, which supplies ankle dorsiflexors and toe extensor muscles and a small area of sensation dorsally in the area of the first and second toes.

1	Symptoms and signs consist of footdrop (ankle dorsiflexion, toe extension, and ankle eversion weakness) and variable sensory loss, which may involve the superficial and deep peroneal pattern. There is usually no pain. Onset may be on awakening in the morning. Peroneal neuropathy needs to be distinguished from L5 radiculopathy. In L5 radiculopathy, ankle invertors and evertors are weak and needle EMG reveals denervation. EDx can help localize the lesion. Peroneal motor conduction velocity shows slowing and amplitude drop across the fibular head. Management consists of rapid weight loss and avoiding leg crossing. Footdrop is treated with an ankle brace. A knee pad can be worn over the lateral knee to avoid further compression. Most cases spontaneously resolve over weeks or months.

1	Radiculopathies are most often due to compression from degenerative joint disease and herniated disks, but there are a number of unusual etiologies (Table 459-9). Degenerative spine disease affects a number of different structures, which narrow the diameter of the neural foramen or canal of the spinal column and compromise nerve root integrity; these are discussed in detail in Chap. 22. The brachial plexus is composed of three trunks (upper, middle, and lower), with two divisions (anterior and posterior) per trunk (Fig. 459-2). Subsequently, the trunks divide into three cords (medial, lateral, and posterior), and from these arise the multiple terminal nerves innervating the arm. The anterior primary rami of C5 and C6 fuse to form the upper trunk; the anterior primary ramus of C7 continues as the middle trunk, while the anterior rami of C8 and T1 join to form the lower trunk. There are several disorders commonly associated with brachial plexopathy.

1	Immune-Mediated Brachial Plexus Neuropathy Immune-mediated brachial plexus neuropathy (IBPN) goes by various terms, including acute brachial plexitis, neuralgic amyotrophy, and Parsonage-Turner syndrome. IBPN usually presents with an acute onset of severe pain in the shoulder region. The intense pain usually lasts several days to a few weeks, but a dull ache can persist. Individuals who are affected may not appreciate weakness of the arm early in the course because the pain limits movement. However, as the pain dissipates, weakness and often sensory loss are appreciated. Attacks can occasionally recur.

1	Clinical findings are dependent on the distribution of involvement (e.g., specific trunk, divisions, cords, or terminal nerves). The most common pattern of IBPN involves the upper trunk or a single or multiple mononeuropathies primarily involving the suprascapular, long thoracic, or axillary nerves. Additionally, the phrenic and anterior interosseous nerves may be concomitantly affected. Any of these nerves may also be affected in isolation. EDx is useful to confirm and localize the site(s) of involvement. Empirical treatment of severe pain with glucocorticoids is often used in the acute period. Brachial Plexopathies Associated with Neoplasms Neoplasms involving the brachial plexus may be primary nerve tumors, local cancers expanding into the plexus (e.g., Pancoast lung tumor or lymphoma),

1	Brachial Plexopathies Associated with Neoplasms Neoplasms involving the brachial plexus may be primary nerve tumors, local cancers expanding into the plexus (e.g., Pancoast lung tumor or lymphoma), Causes of raDiCuLopathy by extradural mass (e.g., meningioma, metastatic tumor, hematoma, abscess) nerve tumor (e.g., neurofibroma, schwannoma, neurinoma) spread of tumor (e.g., prostate cancer) (Lyme disease, herpes zoster, cytomegalovirus, syphilis, schistosomiasis, strongyloides) FIGURE 459-2 Brachial plexus anatomy. L, lateral; M, medial; P, posterior. (From J Goodgold: Anatomical Correlates of Clinical Electromyography.

1	Baltimore, Williams and Wilkins, 1974, p. 126, with permission.) and metastatic tumors. Primary brachial plexus tumors are less common than the secondary tumors and include schwannomas, neurinomas, and neurofibromas. Secondary tumors affecting the brachial plexus are more common and are always malignant. These may arise from local tumors, expanding into the plexus. For example, a Pancoast tumor of the upper lobe of the lung may invade or compress the lower trunk, whereas a primary lymphoma arising from the cervical or axillary lymph nodes may also infiltrate the plexus. Pancoast tumors typically present as an insidious onset of pain in the upper arm, sensory disturbance in the medial aspect of the forearm and hand, and weakness and atrophy of the intrinsic hand muscles along with an ipsilateral Horner’s syndrome. Chest computed tomography (CT) scans or magnetic resonance imaging (MRI) can demonstrate extension of the tumor into the plexus. Metastatic involvement of the brachial plexus

1	Horner’s syndrome. Chest computed tomography (CT) scans or magnetic resonance imaging (MRI) can demonstrate extension of the tumor into the plexus. Metastatic involvement of the brachial plexus may occur with spread of breast cancer into the axillary lymph nodes with local spread into the nearby nerves.

1	Perioperative Plexopathies (Median Sternotomy) The most common surgical procedures associated with brachial plexopathy as a complication are those that involve median sternotomies (e.g., open-heart surgeries and thoracotomies). Brachial plexopathies occur in as many as 5% of patients following a median sternotomy and typically affect the lower trunk. Thus, individuals manifest with sensory disturbance affecting the medial aspect of forearm and hand along with weakness of the intrinsic hand muscles. The mechanism is related to the stretch of the lower trunk, so most individuals who are affected recover within a few months.

1	Lumbosacral Plexus The lumbar plexus arises from the ventral primary rami of the first to the fourth lumbar spinal nerves (Fig. 459-3). These nerves pass downward and laterally from the vertebral column within the psoas major muscle. The femoral nerve derives from the dorsal branches of the second to the fourth lumbar ventral rami. The obturator nerve arises from the ventral branches of the same lumbar rami. The lumbar plexus communicates with the sacral plexus by the lumbosacral trunk, which contains some fibers from the fourth and all of the fibers from the fifth lumbar ventral rami (Fig. 459-4).

1	The sacral plexus is the part of the lumbosacral plexus that is formed by the union of the lumbosacral trunk with the ventral rami of the first to fourth sacral nerves. The plexus lies on the posterior and posterolateral wall of the pelvis with its components converging toward the sciatic notch. The lateral trunk of the sciatic nerve (which forms the common peroneal nerve) arises from the union of the dorsal branches of the lumbosacral trunk (L4, L5) and the dorsal branches of the S1 and S2 spinal nerve ventral rami. The medial trunk of the sciatic nerve (which forms the tibial nerve) derives from the ventral branches of the same ventral rami (L4-S2).

1	Plexopathies are typically recognized when motor, sensory, and if applicable, reflex deficits occur in multiple nerve and segmental distributions confined to one extremity. If localization within the lumbosacral plexus can be accomplished, designation as a lumbar plexopathy, a sacral plexopathy, a lumbosacral trunk lesion, or a panplexopathy is the best localization that can be expected. Although lumbar plexopathies may be bilateral, usually occurring in a stepwise and chronologically dissociated manner, sacral plexopathies are more likely to behave in this manner due to their closer anatomic proximity. The differential diagnosis of plexopathy includes disorders of the conus medullaris and cauda equina (polyradiculopathy). If there is a paucity of pain and sensory involvement, motor neuron disease should be considered as well.

1	The causes of lumbosacral plexopathies are listed in Table 459-10. Diabetic radiculopathy (discussed above) is a fairly common cause of painful leg weakness. Lumbosacral plexopathies are a well-recognized T12 Lateral femoral cutaneous n. Femoral n. T12 Iliohypogastric n. Ilioinguinal n. Genitofemoral n. Obturator n. Lumbosacral trunk L1 L2 L3 L4 L5

1	T12 Lateral femoral cutaneous n. Femoral n. T12 Iliohypogastric n. Ilioinguinal n. Genitofemoral n. Obturator n. Lumbosacral trunk L1 L2 L3 L4 L5 FIGURE 459-3 Lumbar plexus. Posterior divisions are in orange, and anterior divisions are in yellow. (From J Goodgold: Anatomical Correlates of Clinical Electromyography.Baltimore, Williams and Wilkins, 1974, p. 126, with permission.) complication of retroperitoneal hemorrhage. Various primary and metastatic malignancies can affect the lumbosacral plexus as well; these include carcinoma of the cervix, endometrium, and ovary; osteosarcoma; testicular cancer; multiple myeloma; lymphoma; acute myelogenous leukemia; colon cancer; squamous cell carcinoma of the rectum; adenocarcinoma of unknown origin; and intraneural spread of prostate cancer.

1	The treatment for various malignancies is often radiation therapy, the field of which may include parts of the brachial plexus. It can be difficult in such situations to determine if a new brachial or lumbosacral plexopathy is related to tumor within the plexus or from radiation-induced nerve damage. Radiation can be associated with LumBosaCraL pLexopathies: etioLoGies

1	LumBosaCraL pLexopathies: etioLoGies L4 L5 S1 S2 S3 S4 To sphincter ani externus Pudendal Inferior gluteal Superior gluteal Common peroneal Tibial FIGURE 459-4 Lumbosacral plexus. Posterior divisions are in orange, and anterior divisions are in yellow. (From J Goodgold: Anatomical Correlates of Clinical Electromyography. Baltimore, Williams and Wilkins, 1974, p. 126, with permission.) Guillain-Barré syndrome and other immune-mediated Neuropathies Stephen L. Hauser, Anthony A. Amato GUILLAIN-BARRÉ SYNDROME 460 microvascular abnormalities and fibrosis of surrounding tissues, which can damage the axons and the Schwann cells. Radiation-induced plexopathy can develop months or years following therapy and is dose dependent.

1	Tumor invasion is usually painful and more commonly affects the lower trunk, whereas radiation injury is often painless and affects the upper trunk. Imaging studies such as MRI and CT scans are useful but can be misleading with small microscopic invasion of the plexus. EMG can be informative if myokymic discharges are appreciated, as this finding strongly suggests radiation-induced damage. Most patients with plexopathies will undergo both imaging with MRI and EDx evaluations. Severe pain from acute idiopathic lumbosacral plexopathy may respond to a short course of glucocorticoids. Guillain-Barré syndrome (GBS) is an acute, frequently severe, and fulminant polyradiculoneuropathy that is autoimmune in nature. It occurs year-round at a rate of between 1 and 4 cases per 100,000 annually; in the United States, ~5000–6000 cases occur per year. Males are at slightly higher risk for GBS than females, and in Western countries, adults are more frequently affected than children.

1	Clinical Manifestations GBS manifests as a rapidly evolving areflexic motor paralysis with or without sensory disturbance. The usual pattern is an ascending paralysis that may be first noticed as rubbery legs. Weakness typically evolves over hours to a few days and is frequently accompanied by tingling dysesthesias in the extremities. The legs are usually more affected than the arms, and facial diparesis is present in 50% of affected individuals. The lower cranial nerves are also frequently involved, causing bulbar weakness with difficulty handling secretions and maintaining an airway; the diagnosis in these patients may initially be mistaken for brainstem ischemia. Pain in the neck, shoulder, back, or diffusely over the spine is also common in the early stages of GBS, occurring in ~50% of patients. Most patients require hospitalization, and in different series, up to 30% require ventilatory assistance at some time during the illness. The need for mechanical ventilation is associated

1	patients. Most patients require hospitalization, and in different series, up to 30% require ventilatory assistance at some time during the illness. The need for mechanical ventilation is associated with more severe weakness on admission, a rapid tempo of progression, and the presence of facial and/or bulbar weakness during the first week of symptoms. Fever and constitutional symptoms are absent at the onset and, if present, cast doubt on the diagnosis. Deep tendon reflexes attenuate or disappear within the first few days of onset. Cutaneous sensory deficits (e.g., loss of pain and temperature sensation) are usually relatively mild, but functions subserved by large sensory fibers, such as deep tendon reflexes and proprioception, are more severely affected. Bladder dysfunction may occur in severe cases but is usually transient. If bladder dysfunction is a prominent feature and comes early in the course, diagnostic possibilities other than GBS should be considered, particularly spinal

1	severe cases but is usually transient. If bladder dysfunction is a prominent feature and comes early in the course, diagnostic possibilities other than GBS should be considered, particularly spinal cord disease. Once clinical worsening stops and the patient reaches a plateau (almost always within 4 weeks of onset), further progression is unlikely.

1	Autonomic involvement is common and may occur even in patients whose GBS is otherwise mild. The usual manifestations are loss of vasomotor control with wide fluctuation in blood pressure, postural hypotension, and cardiac dysrhythmias. These features require close monitoring and management and can be fatal. Pain is another common feature of GBS; in addition to the acute pain described above, a deep aching pain may be present in weakened muscles that patients liken to having overexercised the previous day. Other pains in GBS include dysesthetic pain in the extremities as a manifestation of sensory nerve fiber involvement. These pains are self-limited and often respond to standard analgesics (Chap. 18).

1	Several subtypes of GBS are recognized, as determined primarily by electrodiagnostic (Edx) and pathologic distinctions (Table 460-1). The most common variant is acute inflammatory demyelinating polyneuropathy (AIDP). Additionally, there are two axonal variants, which are often clinically severe—the acute motor axonal neuropathy (AMAN) and acute motor sensory axonal neuropathy (AMSAN) subtypes. In addition, a range of limited or regional GBS syndromes are also encountered. Notable among these is the Miller Fisher syndrome (MFS), which presents as rapidly evolving ataxia and areflexia of limbs without weakness, and ophthalmoplegia, often with pupillary paralysis. The MFS variant accounts for ~5% of all cases and is strongly associated with antibodies to the ganglioside GQ1b (see “Immunopathogenesis,” below). Other regional variants of GBS include (1) pure sensory forms; (2) ophthalmoplegia with anti-GQ1b antibodies as part of severe motor-sensory GBS; (3) GBS with severe bulbar and

1	below). Other regional variants of GBS include (1) pure sensory forms; (2) ophthalmoplegia with anti-GQ1b antibodies as part of severe motor-sensory GBS; (3) GBS with severe bulbar and facial paralysis, sometimes associated with antecedent cytomegalovirus (CMV) infection and anti-GM2 antibodies; and (4) acute pandysautonomia (Chap. 454).

1	Antecedent Events Approximately 70% of cases of GBS occur 1–3 weeks after an acute infectious process, usually respiratory or gastrointestinal. Culture and seroepidemiologic techniques show that 20–30% of all cases occurring in North America, Europe, and Australia are preceded by infection or reinfection with Campylobacter jejuni. A similar proportion is preceded by a human herpes virus infection, often CMV or Epstein-Barr virus. Other viruses (e.g., HIV, hepatitis E) and also Mycoplasma pneumoniae have been identified as agents involved in antecedent infections, as have recent immunizations. The swine influenza vaccine, administered widely in the United States in 1976, is the most notable example. Influenza vaccines in use from 1992 to 1994, however, resulted in only one additional case of GBS per million persons vaccinated, and the more recent seasonal influenza vaccines appear to confer a GBS risk of <1 per million. Epidemiologic studies looking at H1N1 vaccination demonstrated at

1	of GBS per million persons vaccinated, and the more recent seasonal influenza vaccines appear to confer a GBS risk of <1 per million. Epidemiologic studies looking at H1N1 vaccination demonstrated at most only a slight increased risk of GBS. Meningococcal vaccinations (Menactra) does not appear to carry an increased risk. Older-type rabies vaccine, prepared in nervous system tissue, is implicated as a trigger of GBS in developing countries where it is still used; the mechanism is presumably immunization against neural antigens. GBS also occurs more frequently than can be attributed to chance alone in patients with lymphoma (including Hodgkin’s disease), in HIV-seropositive individuals, and in patients with systemic lupus erythematosus (SLE). C. jejuni has also been implicated in summer outbreaks of AMAN among children and young adults exposed to chickens in rural China.

1	Immunopathogenesis Several lines of evidence support an autoimmune basis for acute inflammatory demyelinating polyneuropathy (AIDP), the most common and best-studied type of GBS; the concept extends to all of the subtypes of GBS (Table 460-1).

1	It is likely that both cellular and humoral immune mechanisms contribute to tissue damage in AIDP. T cell activation is suggested by the finding that elevated levels of cytokines and cytokine receptors are present in serum (interleukin [IL] 2, soluble IL-2 receptor) and in cerebrospinal fluid (CSF) (IL-6, tumor necrosis factor α, interferon γ). AIDP is also closely analogous to an experimental T cell–mediated immunopathy designated experimental allergic neuritis (EAN). EAN is induced in laboratory animals by immune sensitization against protein fragments derived from peripheral nerve proteins, and in particular against the P2 protein. Based on analogy to EAN, it was initially thought that AIDP was likely to be primarily a T cell–mediated disorder; however, abundant data now suggest that autoantibodies directed against nonprotein determinants may be central to many cases.

1	Circumstantial evidence suggests that all GBS results from immune responses to nonself antigens (infectious agents, vaccines) that misdirect to host nerve tissue through a resemblance-of-epitope (molecular mimicry) mechanism (Fig. 460-1). The neural targets are likely to be glycoconjugates, specifically gangliosides (Table 460-2; Fig. 460-2). Gangliosides are complex glycosphingolipids that contain one or more sialic acid residues; various gangliosides participate in cell-cell interactions (including those between axons and glia), modulation of receptors, and regulation of growth. They are typically exposed on the plasma membrane of cells, rendering them susceptible to an antibody-mediated attack. Gangliosides and other glycoconjugates are present in large quantity in human nervous tissues and in key sites, such as nodes of Ranvier. Antiganglioside antibodies, most frequently to GM1, are common in GBS (20–50% of cases), particularly in AMAN and AMSAN and in those cases preceded by C.

1	and in key sites, such as nodes of Ranvier. Antiganglioside antibodies, most frequently to GM1, are common in GBS (20–50% of cases), particularly in AMAN and AMSAN and in those cases preceded by C. jejuni infection. Furthermore, isolates of C. jejuni from stool cultures of patients with GBS have surface glycolipid structures that antigenically cross react with gangliosides, including GM1, concentrated in human nerves. Sialic acid residues from pathogenic C. jejuni strains can also trigger activation of dendritic cells via signaling through a toll-like receptor (TLR4), promoting B cell differentiation and further amplifying humoral autoimmunity. Another line of evidence is derived from experience in Europe with parenteral use of purified bovine brain gangliosides for treatment of various neuropathic disorders. Between 5 and 15 days after injection, some recipients developed acute motor axonal GBS with high titers of anti-GM1 antibodies that recognized epitopes at nodes of Ranvier and

1	neuropathic disorders. Between 5 and 15 days after injection, some recipients developed acute motor axonal GBS with high titers of anti-GM1 antibodies that recognized epitopes at nodes of Ranvier and motor endplates. Experimentally, anti-GM1 antibodies can trigger complement-mediated injury at paranodal axon-glial junctions, disrupting the clustering of sodium channels and likely contributing to conduction block (see “Pathophysiology,” below).

1	CjCjCjjCIL 3,4,5,10 CD4 Antigen presenting cell B cell TCR MHC II B cell Schwann cell plasmalemma Blood/nerve barrier Ganglioside (GM-1 and others) Myelin sheath Plasma cell lgG Cj Cj Cj O A FIGURE 460-1 Postulated immunopathogenesis of Guillain-Barré syndrome (GBS) associated with Campylobacter jejuni infection.

1	B cells recognize glycoconjugates on C. jejuni (Cj) (triangles) that cross-react with ganglioside present on Schwann cell surface and subjacent peripheral nerve myelin. Some B cells, activated via a T cell–independent mechanism, secrete primarily IgM (not shown). Other B cells (upper left side) are activated via a partially T cell–dependent route and secrete primarily IgG; T cell help is provided by CD4 cells activated locally by fragments of Cj proteins that are presented on the surface of antigen-presenting cells (APCs). A critical event in the development of GBS is the escape of activated B cells from Peyer’s patches into regional lymph nodes. Activated T cells probably also function to assist in opening of the blood-nerve barrier, facilitating penetration of pathogenic autoantibodies. The earliest changes in myelin (right) consist of edema between myelin lamellae and vesicular disruption (shown as circular blebs) of the outermost myelin layers. These effects are associated with

1	The earliest changes in myelin (right) consist of edema between myelin lamellae and vesicular disruption (shown as circular blebs) of the outermost myelin layers. These effects are associated with activation of the C5b-C9 membrane attack complex and probably mediated by calcium entry; it is possible that the macrophage cytokine tumor necrosis factor (TNF) also participates in myelin damage. A, axon; B, B cell; MHC II, class II major histocompatibility complex molecule; O, oligodendrocyte; TCR, T cell receptor.

1	Abbreviations: CIDP-M, CIDP with a monoclonal gammopathy; MAG, myelin-associated glycoprotein; MGUS, monoclonal gammopathy of undetermined significance. Source: Modified from HJ Willison, N Yuki: Brain 125:2591, 2002. FIGURE 460-2 Glycolipids implicated as antigens in immune-mediated neuropathies. (Modified from HJ Willison, N Yuki: Brain 125: 2591, 2002.) Anti-GQ1b IgG antibodies are found in >90% of patients with MFS (Table 460-2; Fig. 460-2), and titers of IgG are highest early in the course. Anti-GQ1b antibodies are not found in other forms of GBS unless there is extraocular motor nerve involvement. A possible explanation for this association is that extraocular motor nerves are enriched in GQ1b gangliosides in comparison to limb nerves. In addition, a monoclonal anti-GQ1b antibody raised against C. jejuni isolated from a patient with MFS blocked neuromuscular transmission experimentally.

1	Taken together, these observations provide strong but still inconclusive evidence that autoantibodies play an important pathogenic role in GBS. Although antiganglioside antibodies have been studied most intensively, other antigenic targets may also be important. One report identified IgG antibodies against Schwann cells and neurons (nerve growth cone region) in some GBS cases. Proof that these antibodies are pathogenic requires that they be capable of mediating disease following direct passive transfer to naïve hosts; this has not yet been demonstrated, although one case of possible maternal-fetal transplacental transfer of GBS has been described.

1	In AIDP, an early step in the induction of tissue damage appears to be complement deposition along the outer surface of the Schwann cell. Activation of complement initiates a characteristic vesicular disintegration of the myelin sheath and also leads to recruitment of activated macrophages, which participate in damage to myelin and axons. In AMAN, the pattern is different in that complement is deposited along with IgG at the nodes of Ranvier along large motor axons. Interestingly, in cases of AMAN, antibodies against GD1a appear to have a fine specificity that favors binding to motor rather than sensory nerve roots, even though this ganglioside is expressed on both fiber types.

1	Pathophysiology In the demyelinating forms of GBS, the basis for flaccid paralysis and sensory disturbance is conduction block. This finding, demonstrable electrophysiologically, implies that the axonal connections remain intact. Hence, recovery can take place rapidly as remyelination occurs. In severe cases of demyelinating GBS, secondary axonal degeneration usually occurs; its extent can be estimated electro-physiologically. More secondary axonal degeneration correlates with a slower rate of recovery and a greater degree of residual disability. When a severe primary axonal pattern is encountered electrophysiologically, the implication is that axons have degenerated and become disconnected from their targets, specifically the neuromuscular junctions, and must therefore regenerate for recovery to take place. In motor axonal cases in which recovery is rapid, the lesion is thought to be localized to preterminal motor branches, allowing regeneration and reinnervation to take place

1	recovery to take place. In motor axonal cases in which recovery is rapid, the lesion is thought to be localized to preterminal motor branches, allowing regeneration and reinnervation to take place quickly. Alternatively, in mild cases, collateral sprouting and reinnervation from surviving motor axons near the neuromuscular junction may begin to reestablish physiologic continuity with muscle cells over a period of several months.

1	Laboratory Features CSF findings are distinctive, consisting of an elevated CSF protein level (1–10 g/L [100–1000 mg/dL]) without accompanying pleocytosis. The CSF is often normal when symptoms have been present for ≤48 h; by the end of the first week, the level of protein is usually elevated. A transient increase in the CSF white cell count (10–100/μL) occurs on occasion in otherwise typical GBS; however, a sustained CSF pleocytosis suggests an alternative diagnosis (viral myelitis) or a concurrent diagnosis such as unrecognized HIV infection, leukemia or lymphoma with infiltration of nerves, or neurosarcoidosis. Edx features are mild or absent in the early stages of GBS and lag behind the clinical evolution. In AIDP, the earliest features are prolonged F-wave latencies, prolonged distal latencies, and reduced amplitudes of compound muscle action potentials (CMAPs), probably owing to the predilection for involvement of nerve roots and distal motor nerve terminals early in the course.

1	latencies, and reduced amplitudes of compound muscle action potentials (CMAPs), probably owing to the predilection for involvement of nerve roots and distal motor nerve terminals early in the course. Later, slowing of conduction velocity, conduction block, and temporal dispersion may be appreciated (Table 460-1). Occasionally, sensory nerve action potentials (SNAPs) may be normal in the feet (e.g., sural nerve) when abnormal in the arms. This is also a sign that the patient does not have one of the more typical “length-dependent” polyneuropathies. In cases with primary axonal pathology, the principal Edx finding is reduced ampli-2697 tude of CMAPs (and also SNAPS with AMSAN) without conduction slowing or prolongation of distal latencies. Diagnosis GBS is a descriptive entity. The diagnosis of AIDP is made by recognizing the pattern of rapidly evolving paralysis with areflexia, absence of fever or other systemic symptoms, and characteristic antecedent events. In 2011, the Brighton

1	of AIDP is made by recognizing the pattern of rapidly evolving paralysis with areflexia, absence of fever or other systemic symptoms, and characteristic antecedent events. In 2011, the Brighton Collaboration developed a new set of case definitions for GBS in response to needs of epidemiologic studies of vaccination and assessing risks of GBS (Table 460-3). These criteria have subsequently been validated. Other disorders that may enter into the differential diagnosis include acute myelopathies (especially with prolonged back pain and sphincter disturbances); diphtheria (early oropharyngeal disturbances); Lyme polyradiculitis and other tick-borne paralyses; porphyria (abdominal pain, seizures, psychosis); vasculitic neuropathy (check erythrocyte sedimentation rate, described below); poliomyelitis (fever and meningismus common); West Nile virus; CMV polyradiculitis (in immunocompromised patients); critical illness neuropathy or myopathy; neuromuscular junction disorders such as

1	poliomyelitis (fever and meningismus common); West Nile virus; CMV polyradiculitis (in immunocompromised patients); critical illness neuropathy or myopathy; neuromuscular junction disorders such as myasthenia gravis and botulism (pupillary reactivity lost early); poisonings with organophosphates, thallium, or arsenic; paralytic shellfish poisoning; or severe hypophosphatemia (rare). Laboratory tests are helpful primarily to exclude mimics of GBS. Edx features may be minimal, and the CSF protein level may not rise until the end of the first week. If the diagnosis is strongly suspected, treatment should be initiated without waiting for evolution of the characteristic Edx and CSF findings to occur. GBS patients with risk factors for HIV or with CSF pleocytosis should have a serologic test for HIV.

1	In the vast majority of patients with GBS, treatment should be initiated as soon after diagnosis as possible. Each day counts; ~2 weeks after the first motor symptoms, it is not known whether immunotherapy is still effective. If the patient has already reached the plateau stage, then treatment probably is no longer indicated, unless the patient has severe motor weakness and one cannot exclude the possibility that an immunologic attack is still ongoing. Either high-dose intravenous immune globulin (IVIg) or plasmapheresis can be initiated, as they are equally effective for typical GBS. A combination of the two therapies is not significantly better than either alone. IVIg is often the initial therapy chosen because of its ease of administration and good safety record. Anecdotal data have also suggested that IVIg may be preferable to plasma exchange (PE) for the AMAN and MFS variants of GBS. IVIg is administered as five daily infusions for a total dose of 2 g/kg body weight. There is

1	also suggested that IVIg may be preferable to plasma exchange (PE) for the AMAN and MFS variants of GBS. IVIg is administered as five daily infusions for a total dose of 2 g/kg body weight. There is some evidence that GBS autoantibodies are neutralized by anti-idiotypic antibodies present in IVIg preparations, perhaps accounting for the therapeutic effect. A course of plasmapheresis usually consists of ~40–50 mL/kg PE four to five times over a week. Meta-analysis of randomized clinical trials indicates that treatment reduces the need for mechanical ventilation by nearly half (from 27% to 14% with PE) and increases the likelihood of full recovery at 1 year (from 55% to 68%). Functionally significant improvement may occur toward the end of the first week of treatment or may be delayed for several weeks. The lack of noticeable improvement following a course of IVIg or PE is not an indication to treat with the alternate treatment. However, there are occasional patients who are treated

1	several weeks. The lack of noticeable improvement following a course of IVIg or PE is not an indication to treat with the alternate treatment. However, there are occasional patients who are treated early in the course of GBS and improve, who then relapse within a month. Brief retreatment with the original therapy is usually effective in such cases. Glucocorticoids have not been found to be effective in GBS. Occasional patients with very mild forms of GBS, especially those who appear to have already reached a plateau when initially seen, may be managed conservatively without IVIg or PE.

1	In the worsening phase of GBS, most patients require monitoring in a critical care setting, with particular attention to vital capacity, heart rhythm, blood pressure, nutrition, deep vein thrombosis prophylaxis, cardiovascular status, early consideration (after 2 weeks BriGhtoN Criteria for DiaGNosis of GuiLLaiN-Barré syNDrome (GBs) aND miLLer fisher syNDrome Clinical case definitions for diagnosis of GBS Level 1 of diagnostic certainty Bilateral AND flaccid weakness of the limbs Monophasic illness pattern and interval between onset and nadir of weakness between 12 h and 28 days and subsequent clinical plateau Electrophysiologic findings consistent with GBS Cytoalbuminologic dissociation (i.e., elevation of CSF protein level above laboratory normal value AND CSF total white cell count <50 cells/μL) Absence of an identified alternative diagnosis for weakness Level 2 of diagnostic certainty Bilateral AND flaccid weakness of the limbs

1	Absence of an identified alternative diagnosis for weakness Level 2 of diagnostic certainty Bilateral AND flaccid weakness of the limbs Monophasic illness pattern and interval between onset and nadir of weakness between 12 h and 28 days and subsequent clinical plateau If CSF not collected or results not available, electrophysiologic studies consistent with GBS Absence of identified alternative diagnosis for weakness Level 3 of diagnostic certainty Bilateral and flaccid weakness of the limbs Monophasic illness pattern and interval between onset and nadir of weakness between 12 h and 28 days and subsequent clinical plateau Absence of identified alternative diagnosis for weakness Clinical case definitions for diagnosis of Miller Fisher syndrome Level 1 of diagnostic certainty Bilateral ophthalmoparesis and bilateral reduced or absent tendon reflexes, and ataxia Abbreviation: CSF, cerebrospinal fluid. Absence of limb weakness

1	Level 1 of diagnostic certainty Bilateral ophthalmoparesis and bilateral reduced or absent tendon reflexes, and ataxia Abbreviation: CSF, cerebrospinal fluid. Absence of limb weakness Monophasic illness pattern and interval between onset and nadir of weakness between 12 h and 28 days and subsequent clinical plateau Cytoalbuminologic dissociation (i.e., elevation of cerebrospinal protein above the laboratory normal and total CSF white cell count <50 cells/μL) Nerve conduction studies are normal, OR indicate involvement of sensory nerves only Absence of identified alternative diagnosis Level 2 of diagnostic certainty Absence of limb weakness Monophasic illness pattern and interval between onset and nadir of weakness between 12 h and 28 days and subsequent clinical plateau CSF with a total white cell count <50 cells/μL) (with or without CSF protein elevation above laboratory normal value) Nerve conduction studies are normal, OR indicate involvement of sensory nerves only

1	Nerve conduction studies are normal, OR indicate involvement of sensory nerves only Absence of identified alternative diagnosis Level 3 of diagnostic certainty Absence of limb weakness Monophasic illness pattern and interval between onset and nadir of weakness between 12 h and 28 days and subsequent clinical plateau Absence of identified alternative diagnosis Source: From JJ Sejvar et al: Guillain-Barré syndrome and Fisher syndrome: case definitions and guidelines for collection, analysis, and presentation of immunization safety data. Vaccine 29:599, 2011. Validation study published by C Fokke et al: Diagnosis of Guillain-Barré syndrome and validation of Brighton criteria. Brain 137:33, 2014.

1	of intubation) of tracheotomy, and chest physiotherapy. As noted, ~30% of patients with GBS require ventilatory assistance, sometimes for prolonged periods of time (several weeks or longer). Frequent turning and assiduous skin care are important, as are daily range-ofmotion exercises to avoid joint contractures and daily reassurance as to the generally good outlook for recovery.

1	Prognosis and Recovery Approximately 85% of patients with GBS achieve a full functional recovery within several months to a year, although minor findings on examination (such as areflexia) may persist and patients often complain of continued symptoms, including fatigue. The mortality rate is <5% in optimal settings; death usually results from secondary pulmonary complications. The outlook is worst in patients with severe proximal motor and sensory axonal damage. Such axonal damage may be either primary or secondary in nature (see “Pathophysiology,” above), but in either case successful regeneration cannot occur. Other factors that worsen the outlook for recovery are advanced age, a fulminant or severe attack, and a delay in the onset of treatment. Between 5 and 10% of patients with typical GBS have one or more late relapses; such cases are then classified as chronic inflammatory demyelinating polyneuropathy (CIDP).

1	CIDP is distinguished from GBS by its chronic course. In other respects, this neuropathy shares many features with the common demyelinating form of GBS, including elevated CSF protein levels and the Edx findings of acquired demyelination. Most cases occur in adults, and males are affected slightly more often than females. The incidence of CIDP is lower than that of GBS, but due to the protracted course, the prevalence is greater.

1	Clinical Manifestations Onset is usually gradual over a few months or longer, but in a few cases, the initial attack is indistinguishable from that of GBS. An acute-onset form of CIDP should be considered when GBS deteriorates >9 weeks after onset or relapses at least three times. Symptoms are both motor and sensory in most cases. Weakness of the limbs is usually symmetric but can be strikingly asymmetric in multifocal acquired demyelinating sensory and motor (MADSAM) neuropathy variant (Lewis-Sumner syndrome) in which discrete peripheral nerves are involved. There is considerable variability from case to case. Some patients experience a chronic progressive course, whereas others, usually younger patients, have a relapsing and remitting course. Some have only motor findings, and a small proportion present with a relatively pure syndrome of sensory ataxia. Tremor occurs in ~10% and may become more prominent during periods of subacute worsening or improvement. A small proportion have

1	proportion present with a relatively pure syndrome of sensory ataxia. Tremor occurs in ~10% and may become more prominent during periods of subacute worsening or improvement. A small proportion have cranial nerve findings, including external ophthalmoplegia. CIDP tends to ameliorate over time with treatment; the result is that many years after onset, nearly 75% of patients have reasonable functional status. Death from CIDP is uncommon.

1	Diagnosis The diagnosis rests on characteristic clinical, CSF, and electrophysiologic findings. The CSF is usually acellular with an elevated protein level, sometimes several times normal. As with GBS, a CSF pleocytosis should lead to the consideration of HIV infection, leukemia or lymphoma, and neurosarcoidosis. Edx findings reveal variable degrees of conduction slowing, prolonged distal latencies, distal and temporal dispersion of CMAPs, and conduction block as the principal features. In particular, the presence of conduction block is a certain sign of an acquired demyelinating process. Evidence of axonal loss, presumably secondary to demyelination, is present in >50% of patients. Serum protein electrophoresis with immunofixation is indicated to search for monoclonal gammopathy and associated conditions (see “Monoclonal Gammopathy of Undetermined Significance,” below). In all patients with presumptive CIDP, it is also reasonable to exclude vasculitis, collagen vascular disease

1	associated conditions (see “Monoclonal Gammopathy of Undetermined Significance,” below). In all patients with presumptive CIDP, it is also reasonable to exclude vasculitis, collagen vascular disease (especially SLE), chronic hepatitis, HIV infection, amyloidosis, and diabetes mellitus. Other associated conditions include inflammatory bowel disease and lymphoma.

1	Pathogenesis Although there is evidence of immune activation in CIDP, the precise mechanisms of pathogenesis are unknown. Biopsy typically reveals little inflammation and onion-bulb changes (imbricated layers of attenuated Schwann cell processes surrounding an axon) that result from recurrent demyelination and remyelination (Fig. 460-1). The response to therapy suggests that CIDP is immune-mediated; CIDP responds to glucocorticoids, whereas GBS does not. Passive transfer of demyelination into experimental animals has been accomplished using IgG purified from the serum of some patients with CIDP, lending support for a humoral autoimmune pathogenesis. A minority of patients have serum antibodies against P0, myelin P2 protein, PMP22, or neurofascin. It is also of interest that a CIDP-like illness developed spontaneously in the nonobese diabetic (NOD) mouse when the immune co-stimulatory molecule B7-2 (CD86) was genetically deleted; this suggests that CIDP can result from altered

1	illness developed spontaneously in the nonobese diabetic (NOD) mouse when the immune co-stimulatory molecule B7-2 (CD86) was genetically deleted; this suggests that CIDP can result from altered triggering of T cells by antigen-presenting cells.

1	Approximately 25% of patients with clinical features of CIDP also have a monoclonal gammopathy of undetermined significance (MGUS). Cases associated with monoclonal IgA or IgG kappa usually respond to treatment as favorably as cases without a monoclonal gammopathy. Patients with IgM monoclonal gammopathy and antibodies directed against myelin-associated glycoprotein (MAG) have a distinct polyneuropathy, tend to have more sensory findings and a more protracted course, and usually have a less satisfactory response to treatment.

1	Most authorities initiate treatment for CIDP when progression is rapid or walking is compromised. If the disorder is mild, management can be expectant, awaiting spontaneous remission. Controlled studies have shown that high-dose IVIg, PE, and glucocorticoids are all more effective than placebo. Initial therapy is usually with IVIg, administered as 2.0 g/kg body weight given in divided doses over 2–5 days; three monthly courses are generally recommended before concluding a patient is a treatment failure. If the patient responds, the infusion intervals can be gradually increased or the dosage decreased (e.g., 1 g/kg per month). PE, which appears to be as effective as IVIg, is initiated at two to three treatments per week for 6 weeks; periodic re-treatment may also be required. Treatment with glucocorticoids is another option (60–80 mg prednisone PO daily for 1–2 months, followed by a gradual dose reduction of 10 mg per month as tolerated), but long-term adverse effects including bone

1	glucocorticoids is another option (60–80 mg prednisone PO daily for 1–2 months, followed by a gradual dose reduction of 10 mg per month as tolerated), but long-term adverse effects including bone demineralization, gastrointestinal bleeding, and cushingoid changes are problematic. As many as one-third of patients with CIDP fail to respond adequately to the initial therapy chosen; a different treatment should then be tried. Patients who fail therapy with IVIg, PE, and glucocorticoids may benefit from treatment with immunosuppressive agents such as azathioprine, methotrexate, cyclosporine, and cyclophosphamide, either alone or as adjunctive therapy. Early experience with anti-CD20 (rituximab) has also shown promise. Use of these therapies requires periodic reassessment of their risks and benefits. In patients with a CIDP-like neuropathy who fail to respond to treatment, it is important to evaluate for POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,

1	In patients with a CIDP-like neuropathy who fail to respond to treatment, it is important to evaluate for POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes; see below).

1	Multifocal motor neuropathy (MMN) is a distinctive but uncommon neuropathy that presents as slowly progressive motor weakness and atrophy evolving over years in the distribution of selected nerve trunks, associated with sites of persistent focal motor conduction block in the same nerve trunks. Sensory fibers are relatively spared. The arms are affected more frequently than the legs, and >75% of all patients are male. Some cases have been confused with lower motor neuron forms of amyotrophic lateral sclerosis (Chap. 452). Less than 50% of patients present with high titers of polyclonal IgM antibody to the ganglioside GM1. It is uncertain how this finding relates to the discrete foci of persistent motor conduction block, but high concentrations of GM1 gangliosides are normal constituents of nodes of Ranvier in peripheral nerve fibers. Pathology reveals demyelination and mild inflammatory changes at the sites of conduction block.

1	Most patients with MMN respond to high-dose IVIg (dosages as for CIDP, above); periodic re-treatment is required (usually at least monthly) to maintain the benefit. Some refractory patients have responded to rituximab or cyclophosphamide. Glucocorticoids and PE are not effective. Clinically overt polyneuropathy occurs in ~5% of patients with the commonly encountered type of multiple myeloma, which exhibits either lytic or diffuse osteoporotic bone lesions. These neuropathies are sensorimotor, are usually mild and slowly progressive but may be severe, and generally do not reverse with successful suppression of the myeloma. In most cases, Edx and pathologic features are consistent with a process of axonal degeneration.

1	In contrast, myeloma with osteosclerotic features, although representing only 3% of all myelomas, is associated with polyneuropathy in one-half of cases. These neuropathies, which may also occur with solitary plasmacytoma, are distinct because they (1) are usually 2700 demyelinating in nature and resemble CIDP; (2) often respond to radiation therapy or removal of the primary lesion; (3) are associated with different monoclonal proteins and light chains (almost always lambda as opposed to primarily kappa in the lytic type of multiple myeloma); (4) are typically refractory to standard treatments of CIDP; and (5) may occur in association with other systemic findings including thickening of the skin, hyperpigmentation, hypertrichosis, organomegaly, endocrinopathy, anasarca, and clubbing of fingers. These are features of the POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes). Levels of vascular endothelial growth factor (VEGF) are

1	of fingers. These are features of the POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes). Levels of vascular endothelial growth factor (VEGF) are increased in the serum, and this factor is felt to somehow play a pathogenic role in this syndrome. Treatment of the neuropathy is best directed at the osteosclerotic myeloma using surgery, radiotherapy, chemotherapy, or autologous peripheral blood stem cell transplantation. Neuropathies are also encountered in other systemic conditions with gammopathy, including Waldenström’s macroglobulinemia, primary systemic amyloidosis, and cryoglobulinemic states (mixed essential cryoglobulinemia, some cases of hepatitis C).

1	Chronic polyneuropathies occurring in association with MGUS are usually associated with the immunoglobulin isotypes IgG, IgA, and IgM. Most patients present with isolated sensory symptoms in their distal extremities and have Edx features of an axonal sensory or sensorimotor polyneuropathy. These patients otherwise resemble idiopathic sensory polyneuropathy, and the MGUS might just be coincidental. They usually do not respond to immunotherapies designed to reduce the concentration of the monoclonal protein. Some patients, however, present with generalized weakness and sensory loss and Edx studies indistinguishable from CIDP without monoclonal gammopathy (see “Chronic Inflammatory Demyelinating Polyneuropathy,” above), and their response to immunosuppressive agents is also similar. An exception is the syndrome of IgM kappa monoclonal gammopathy associated with an indolent, longstanding, sometimes static sensory neuropathy, frequently with tremor and sensory ataxia. Most patients are

1	is the syndrome of IgM kappa monoclonal gammopathy associated with an indolent, longstanding, sometimes static sensory neuropathy, frequently with tremor and sensory ataxia. Most patients are male and older than age 50 years. In the majority, the monoclonal IgM immunoglobulin binds to a normal peripheral nerve constituent, MAG, found in the paranodal regions of Schwann cells. Binding appears to be specific for a polysaccharide epitope that is also found in other normal peripheral nerve myelin glycoproteins, P0 and PMP22, and also in other normal nerve-related glycosphingolipids (Fig. 460-1). In the MAG-positive cases, IgM paraprotein is incorporated into the myelin sheaths of affected patients and widens the spacing of the myelin lamellae, thus producing a distinctive ultrastructural pattern. Demyelination and remyelination are the hallmarks of the lesions, but axonal loss develops over time. These anti-MAG polyneuropathies are typical refractory to immunotherapy. In a small

1	pattern. Demyelination and remyelination are the hallmarks of the lesions, but axonal loss develops over time. These anti-MAG polyneuropathies are typical refractory to immunotherapy. In a small proportion of patients (30% at 10 years), MGUS will in time evolve into frankly malignant conditions such as multiple myeloma or lymphoma.

1	Peripheral nerve involvement is common in polyarteritis nodosa (PAN), appearing in half of all cases clinically and in 100% of cases at postmortem studies (Chap. 385). The most common pattern is multifocal (asymmetric) motor-sensory neuropathy (mononeuropathy multiplex) due to ischemic lesions of nerve trunks and roots; however, some cases of vasculitic neuropathy present as a distal, symmetric sensorimotor polyneuropathy. Symptoms of neuropathy are a common presenting complaint in patients with PAN. The Edx findings are those of an axonal process. Smallto medium-sized arteries of the vasa nervorum, particularly the epineural vessels, are affected in PAN, resulting in a widespread ischemic neuropathy. A high frequency of neuropathy occurs in allergic angiitis and granulomatosis (Churg-Strauss syndrome [CSS]).

1	Systemic vasculitis should always be considered when a subacute or chronically evolving mononeuropathy multiplex occurs in conjunction with constitutional symptoms (fever, anorexia, weight loss, loss of energy, malaise, and nonspecific pains). Diagnosis of suspected vasculitic neuropathy is made by a combined nerve and muscle biopsy, with serial section or skip-serial techniques. Approximately one-third of biopsy-proven cases of vasculitic neuropathy are “nonsystemic” in that the vasculitis appears to affect only peripheral nerves. Constitutional symptoms are absent, and the course is more indolent than that of PAN. The erythrocyte sedimentation rate may be elevated, but other tests for systemic disease are negative. Nevertheless, clinically silent involvement of other organs is likely, and vasculitis is frequently found in muscle biopsied at the same time as nerve.

1	Vasculitic neuropathy may also be seen as part of the vasculitis syndrome occurring in the course of other connective tissue disorders (Chap. 385). The most frequent is rheumatoid arthritis, but ischemic neuropathy due to involvement of vasa nervorum may also occur in mixed cryoglobulinemia, Sjögren’s syndrome, granulomatosis with polyangiitis (Wegener’s), hypersensitivity angiitis, systemic lupus erythematosus, and progressive systemic sclerosis. Some vasculitides are associated with antineutrophil cytoplasmic antibodies (ANCAs), which in turn, are subclassified as cytoplasmic (cANCA) or perinuclear (pANCA). cANCAs are directed against proteinase 3 (PR3), whereas pANCAs target myeloperoxidase (MPO). PR3/cANCAs are associated with granulomatosis with polyangiitis (Wegener’s), whereas MPO/pANCAs are typically associated with microscopic polyangiitis, CSS, and less commonly PAN. Of note, MPO/pANCA has also been seen in minocycline-induced vasculitis.

1	Management of these neuropathies, including the “nonsystemic” vasculitic neuropathy, consists of treatment of the underlying condition as well as the aggressive use of glucocorticoids and cyclophosphamide. Use of these immunosuppressive agents has resulted in dramatic improvements in outcome, with 5-year survival rates now greater than 80%. Recent clinical trials found that the combination of rituximab and glucocorticoids is not inferior to cyclophosphamide and glucocorticoids. Thus, combination therapy with glucocorticoids and rituximab is increasingly recommended as the standard initial treatment, particularly for ANCA-associated vasculitis. ANTI-Hu PARANEOPLASTIC NEUROPATHY (CHAP. 122)

1	This uncommon immune-mediated disorder manifests as a sensory neuronopathy (i.e., selective damage to sensory nerve bodies in dorsal root ganglia). The onset is often asymmetric with dysesthesias and sensory loss in the limbs that soon progress to affect all limbs, the torso, and the face. Marked sensory ataxia, pseudoathetosis, and inability to walk, stand, or even sit unsupported are frequent features and are secondary to the extensive deafferentation. Subacute sensory neuronopathy may be idiopathic, but more than half of cases are paraneoplastic, primarily related to lung cancer, and most of those are small-cell lung cancer (SCLC). Diagnosis of the underlying SCLC requires awareness of the association, testing for the paraneoplastic antibody, and often positron emission tomography (PET) scanning for the tumor. The target antigens are a family of RNA-binding proteins (HuD, HuC, and Hel-N1) that in normal tissues are only expressed by neurons. The same proteins are usually expressed

1	scanning for the tumor. The target antigens are a family of RNA-binding proteins (HuD, HuC, and Hel-N1) that in normal tissues are only expressed by neurons. The same proteins are usually expressed by SCLC, triggering in some patients an immune response characterized by antibodies and cytotoxic T cells that cross-react with the Hu proteins of the dorsal root ganglion neurons, resulting in immune-mediated neuronal destruction. An encephalomyelitis may accompany the sensory neuronopathy and presumably has the same pathogenesis. Neurologic symptoms usually precede, by ≤6 months, the identification of SCLC. The sensory neuronopathy runs its course in a few weeks or months and stabilizes, leaving the patient disabled. Most cases are unresponsive to treatment with glucocorticoids, IVIg, PE, or immunosuppressant drugs.

1	myasthenia Gravis and other Diseases of the Neuromuscular junction Daniel B. Drachman, Anthony A. Amato Myasthenia gravis (MG) is a neuromuscular disorder characterized by 461 weakness and fatigability of skeletal muscles. The underlying defect is a decrease in the number of available acetylcholine receptors (AChRs) at neuromuscular junctions due to an antibody-mediated autoimmune attack. Treatment now available for MG is highly effective, although a specific cure has remained elusive.

1	At the neuromuscular junction (Fig. 461-1, Video 461-1), acetylcholine (ACh) is synthesized in the motor nerve terminal and stored in vesicles (quanta). When an action potential travels down a motor nerve and reaches the nerve terminal, ACh from 150 to 200 vesicles is released and combines with AChRs that are densely packed at the peaks of postsynaptic folds. The AChR consists of five subunits (2α, 1β, 1δ, and 1γ or ε) arranged around a central pore. When ACh combines with the binding sites on the α subunits of the AChR, the channel in the AChR opens, permitting the rapid entry of cations, chiefly sodium, which produces depolarization at the end-plate region of the muscle fiber. If the depolarization is sufficiently large, it initiates an action potential that is propagated along the muscle fiber, triggering muscle contraction. This process is rapidly terminated by hydrolysis of ACh by acetylcholinesterase (AChE), which is present within the synaptic folds, and by diffusion of ACh

1	fiber, triggering muscle contraction. This process is rapidly terminated by hydrolysis of ACh by acetylcholinesterase (AChE), which is present within the synaptic folds, and by diffusion of ACh away from the receptor.

1	In MG, the fundamental defect is a decrease in the number of available AChRs at the postsynaptic muscle membrane. In addition, the postsynaptic folds are flattened, or “simplified.” These changes result in decreased efficiency of neuromuscular transmission. Therefore, although ACh is released normally, it produces small end-plate potentials that may fail to trigger muscle action potentials. Failure of transmission at many neuromuscular junctions results in weakness of muscle contraction. The amount of ACh released per impulse normally declines on repeated activity (termed presynaptic rundown). In the myasthenic patient, the decreased efficiency of neuromuscular transmission com-2701 bined with the normal rundown results in the activation of fewer and

1	Axon Release site Small electrical response AChR A Normal Vesicle Nerve terminal Strong electrical response Mitochondria AChE Nerve impulse MuscleMuscle B MG End plate potential End plate potential FIGURE 461-1 Diagrams of (A) normal and (B) myasthenic neuromuscular junctions. AChE, acetylcholinesterase. See text for description of normal neuromuscular transmission. The myasthenia gravis (MG) junction demonstrates a normal nerve terminal; a reduced num-ber of acetylcholine receptors (AChRs) (stippling); flattened, simplified postsynaptic folds; and a widened synaptic space. See Video 461-1 also. (Modified from DB Drachman: N Engl J Med 330:1797, 1994; with permission.) fewer muscle fibers by successive nerve impulses and hence increasing weakness, or myasthenic fatigue. This mechanism also accounts for the decremental response to repetitive nerve stimulation seen on electro-diagnostic testing.

1	The neuromuscular abnormalities in MG are caused by an autoimmune response mediated by specific anti-AChR antibodies. The anti-AChR antibodies reduce the number of available AChRs at neuromuscular junctions by three distinct mechanisms: (1) accelerated turnover of AChRs by a mechanism involving cross-linking and rapid endocytosis of the receptors; (2) damage to the postsynaptic muscle membrane by the antibody in collaboration with complement; and (3) blockade of the active site of the AChR, i.e., the site that normally binds ACh. An immune response to muscle-specific kinase (MuSK), a protein involved in AChR clustering at neuromuscular junctions, can also result in MG, with reduction of AChRs demonstrated experimentally. Anti-MuSK antibody occurs in about 40% of patients without AChR antibody. A small proportion of patients whose sera are negative for both AChR and MuSK antibodies have antibodies to another protein at the neuromuscular junction—low-density lipoprotein receptor-related

1	A small proportion of patients whose sera are negative for both AChR and MuSK antibodies have antibodies to another protein at the neuromuscular junction—low-density lipoprotein receptor-related protein 4 (lrp4)—that is important for clustering of AChRs. The pathogenic antibodies are IgG and are T cell dependent. Thus, immunotherapeutic strategies directed against either the antibody-producing B cells or helper T cells are effective in this antibody-mediated disease.

1	How the autoimmune response is initiated and maintained in MG is not completely understood, but the thymus appears to play a role in this process. The thymus is abnormal in ~75% of patients with AChR antibody–positive MG; in ~65% the thymus is “hyperplastic,” with the presence of active germinal centers detected histologically, although the hyperplastic thymus is not necessarily enlarged. An additional 10% of patients have thymic tumors (thymomas). Muscle-like cells within the thymus (myoid cells), which express AChRs on their surface, may serve as a source of autoantigen and trigger the autoimmune reaction within the thymus gland. MG is not rare, having a prevalence as high as 2–7 in 10,000. It affects individuals in all age groups, but peaks of incidence occur in women in their twenties and thirties and in men in their fifties and sixties. Overall, women are affected more frequently than men, in a ratio of ~3:2. The cardinal features are weakness and fatigability of muscles.

1	The weakness increases during repeated use (fatigue) or late in the day and may improve following rest or sleep. The course of MG is often variable. Exacerbations and remissions may occur, particularly during the first few years after the onset of the disease. Remissions are rarely complete or permanent. Unrelated infections or systemic disorders can lead to increased myasthenic weakness and may precipitate “crisis” (see below).

1	The distribution of muscle weakness often has a characteristic pattern. The cranial muscles, particularly the lids and extraocular muscles, are typically involved early in the course of MG; diplopia and ptosis are common initial complaints. Facial weakness produces a “snarling” expression when the patient attempts to smile. Weakness in chewing is most noticeable after prolonged effort, as in chewing meat. Speech may have a nasal timbre caused by weakness of the palate or a dysarthric “mushy” quality due to tongue weakness. Difficulty in swallowing may occur as a result of weakness of the palate, tongue, or pharynx, giving rise to nasal regurgitation or aspiration of liquids or food. Bulbar weakness is especially prominent in MuSK antibody–positive MG. In ~85% of Myasthenia Gravis and Other Diseases of the Neuromuscular Junction DiaGNosis of myastheNia Gravis (mG) Diplopia, ptosis, dysarthria, dysphagia, dyspnea

1	Myasthenia Gravis and Other Diseases of the Neuromuscular Junction DiaGNosis of myastheNia Gravis (mG) Diplopia, ptosis, dysarthria, dysphagia, dyspnea Weakness in characteristic distribution: proximal limbs, neck extensors, generalized Fluctuation and fatigue: worse with repeated activity, improved by rest Effects of previous treatments Ptosis, diplopia Motor power survey: quantitative testing of muscle strength Absence of other neurologic signs Anti-AChR radioimmunoassay: ~85% positive in generalized MG; 50% in ocular MG; definite diagnosis if positive; negative result does not exclude MG; ~40% of AChR antibody–negative patients with generalized MG have anti-MuSK antibodies Repetitive nerve stimulation: decrement of >15% at 3 Hz: highly probable Single-fiber electromyography: blocking and jitter, with normal fiber density; confirmatory, but not specific For ocular or cranial MG: exclude intracranial lesions by CT or MRI

1	Single-fiber electromyography: blocking and jitter, with normal fiber density; confirmatory, but not specific For ocular or cranial MG: exclude intracranial lesions by CT or MRI Abbreviations: AChR, acetylcholine receptor; CT, computed tomography; MRI, magnetic resonance imaging; MuSK, muscle-specific tyrosine kinase. patients, the weakness becomes generalized, affecting the limb muscles as well. If weakness remains restricted to the extraocular muscles for 3 years, it is likely that it will not become generalized, and these patients are said to have ocular MG. The limb weakness in MG is often proximal and may be asymmetric. Despite the muscle weakness, deep tendon reflexes are preserved. If weakness of respiration becomes so severe as to require respiratory assistance, the patient is said to be in crisis.

1	(Table 461-1) The diagnosis is suspected on the basis of weakness and fatigability in the typical distribution described above, without loss of reflexes or impairment of sensation or other neurologic function. The suspected diagnosis should always be confirmed definitively before treatment is undertaken; this is essential because (1) other treatable conditions may closely resemble MG and (2) the treatment of MG may involve surgery and the prolonged use of drugs with potentially adverse side effects.

1	Antibodies to AChR, MuSK, or lpr4 As noted above, anti-AChR antibodies are detectable in the serum of ~85% of all myasthenic patients but in only about 50% of patients with weakness confined to the ocular muscles. The presence of anti-AChR antibodies is virtually diagnostic of MG, but a negative test does not exclude the disease. The measured level of anti-AChR antibody does not correspond well with the severity of MG in different patients. However, in an individual patient, a treatment-induced fall in the antibody level often correlates with clinical improvement, whereas a rise in the level may occur with exacerbations. Antibodies to MuSK have been found to be present in ~40% of AChR antibody–negative patients with generalized MG, and their presence is a useful diagnostic test in these patients. MuSK antibodies are rarely present in AChR antibody–positive patients or in patients with MG limited to ocular muscles. These antibodies may interfere with clustering of AChRs at

1	in these patients. MuSK antibodies are rarely present in AChR antibody–positive patients or in patients with MG limited to ocular muscles. These antibodies may interfere with clustering of AChRs at neuromuscular junctions, as MuSK is known to do during early development. A small proportion of MG patients without antibodies to AChR or MuSK may have antibodies to lrp4, although a test for lrp4 antibodies is not yet commercially available. Finally, antibodies against agrin have recently been found in some patients with MG. Agrin is a protein derived from motor nerves that normally binds to lrp4 and thus may also interfere with clustering of AChRs at neuromuscular junctions. There may well be other—as yet undefined—antibodies that impair neuromuscular transmission.

1	Electrodiagnostic Testing Repetitive nerve stimulation may provide helpful diagnostic evidence of MG. Anti-AChE medication is stopped 6–24 h before testing. It is best to test weak muscles or proximal muscle groups. Electric shocks are delivered at a rate of two or three per second to the appropriate nerves, and action potentials are recorded from the muscles. In normal individuals, the amplitude of the evoked muscle action potentials does not change at these rates of stimulation. However, in myasthenic patients, there is a rapid reduction of >10–15% in the amplitude of the evoked responses.

1	Anticholinesterase Test Drugs that inhibit the enzyme AChE allow ACh to interact repeatedly with the limited number of AChRs in MG, producing improvement in muscle strength. Edrophonium is used most commonly for diagnostic testing because of the rapid onset (30 s) and short duration (~5 min) of its effect. An objective end point must be selected to evaluate the effect of edrophonium, such as weakness of extraocular muscles, impairment of speech, or the length of time that the patient can maintain the arms in forward abduction. An initial IV dose of 2 mg of edrophonium is given. If definite improvement occurs, the test is considered positive and is terminated. If there is no change, the patient is given an additional 8 mg IV. The dose is administered in two parts because some patients react to edrophonium with side effects such as nausea, diarrhea, salivation, fasciculations, and rarely with severe symptoms of syncope or bradycardia. Atropine (0.6 mg) should be drawn up in a syringe

1	to edrophonium with side effects such as nausea, diarrhea, salivation, fasciculations, and rarely with severe symptoms of syncope or bradycardia. Atropine (0.6 mg) should be drawn up in a syringe and ready for IV administration if these symptoms become troublesome. The edrophonium test is now reserved for patients with clinical findings that are suggestive of MG but who have negative antibody and electrodiagnostic test results. False-positive tests occur in occasional patients with other neurologic disorders, such as amyotrophic lateral sclerosis, and in placebo-reactors. False-negative or equivocal tests may also occur. In some cases, it is helpful to use a longer-acting drug such as neostigmine (15 mg PO), because this permits more time for detailed evaluation of strength.

1	Inherited Myasthenic Syndromes The congenital myasthenic syndromes (CMS) comprise a heterogeneous group of disorders of the neuromuscular junction that are not autoimmune but rather are due to genetic mutations in which virtually any component of the neuromuscular junction may be affected. Alterations in function of the presynaptic nerve terminal, in the various subunits of the AChR, AChE, or the other molecules involved in end-plate development or maintenance, have been identified in the different forms of CMS. These disorders share many of the clinical features of autoimmune MG, including weakness and fatigability of skeletal muscles, in some cases involving extraocular muscles (EOMs), lids, and proximal muscles, similar to the distribution in autoimmune MG. CMS should be suspected when symptoms of myasthenia have begun in infancy or childhood and AChR antibody tests are consistently negative. By far the most common genetic defects occur in the AChR or other postsynaptic molecules

1	symptoms of myasthenia have begun in infancy or childhood and AChR antibody tests are consistently negative. By far the most common genetic defects occur in the AChR or other postsynaptic molecules (67% in the Mayo Clinic series of 350 CMS patients), with about equal frequencies of abnormalities in AChE (13%) and the various maintenance molecules (DOK7, GFPT, etc.; ~14%). In the forms that involve the AChR, a wide variety of mutations have been identified in each of the subunits, but the ε subunit is affected in ~75% of these cases. In most of the recessively inherited forms of CMS, the mutations are heteroallelic; that is, different mutations affecting each of the two alleles are present. Features of the four most common forms of CMS are summarized in Table 461-2. Although clinical features and electrodiagnostic and pharmacologic tests may suggest the correct diagnosis, molecular analysis is required for precise elucidation of the defect; this may lead to helpful treatment as well as

1	and electrodiagnostic and pharmacologic tests may suggest the correct diagnosis, molecular analysis is required for precise elucidation of the defect; this may lead to helpful treatment as well as genetic counseling.

1	Differential Diagnosis Other conditions that cause weakness of the cranial and/or somatic musculature include the nonautoimmune CMS discussed above, drug-induced myasthenia, Lambert-Eaton myasthenic syndrome (LEMS), neurasthenia, hyperthyroidism (Graves’ disease), botulism, intracranial mass lesions, oculopharyngeal dystrophy, and mitochondrial myopathy (Kearns-Sayre syndrome, progressive external ophthalmoplegia). Treatment with penicillamine (used for scleroderma or rheumatoid arthritis) may result in true autoimmune Abbreviations: AChE, acetylcholinesterase; AChR, acetylcholine receptor; EOM, extraocular muscles; MEPP, miniature end-plate potentials; MG, myasthenia gravis; 3,4-DAP, 3,4-diaminopyridine.

1	Abbreviations: AChE, acetylcholinesterase; AChR, acetylcholine receptor; EOM, extraocular muscles; MEPP, miniature end-plate potentials; MG, myasthenia gravis; 3,4-DAP, 3,4-diaminopyridine. MG, but the weakness is usually mild, and recovery occurs within weeks or months after discontinuing its use. Aminoglycoside antibiotics or procainamide can cause exacerbation of weakness in myasthenic patients; very large doses can cause neuromuscular weakness in normal individuals.

1	LEMS is a presynaptic disorder of the neuromuscular junction that can cause weakness similar to that of MG. The proximal muscles of the lower limbs are most commonly affected, but other muscles may be involved as well. Cranial nerve findings, including ptosis of the eyelids and diplopia, occur in up to 70% of patients and resemble features of MG. However, the two conditions are usually readily distinguished, because patients with LEMS have depressed or absent reflexes and experience autonomic changes such as dry mouth and impotence. Nerve stimulation produces an initial low-amplitude response and, at low rates of repetitive stimulation (2–3 Hz), decremental responses like those of MG; however, at high rates (50 Hz), or following exercise, incremental responses occur. LEMS is caused by autoantibodies directed against P/Q-type calcium channels at the motor nerve terminals, which can be detected in ~85% of LEMS patients by radioimmunoassay. These autoantibodies result in impaired release

1	directed against P/Q-type calcium channels at the motor nerve terminals, which can be detected in ~85% of LEMS patients by radioimmunoassay. These autoantibodies result in impaired release of ACh from nerve terminals. Many patients with LEMS have an associated malignancy, most commonly small-cell carcinoma of the lung, which may express calcium channels that stimulate the autoimmune response. The diagnosis of LEMS may signal the presence of a tumor long before it would otherwise be detected, permitting early removal. Treatment of LEMS involves plasmapheresis and immunosuppression, as for MG. 3,4-Diaminopyridine (3,4-DAP) and pyridostigmine may also be symptomatically helpful. 3,4-DAP acts by blocking potassium channels, which results in prolonged depolarization of the motor nerve terminals and thus enhances ACh release. Pyridostigmine prolongs the action of ACh, allowing repeated interactions with AChRs.

1	Botulism (Chap. 178) is due to potent bacterial toxins produced by any of eight different strains of Clostridium botulinum. The toxins enzymatically cleave specific proteins essential for the release of ACh from the motor nerve terminal, thereby interfering with neuromuscular transmission. Most commonly, botulism is caused by ingestion of improperly prepared food containing toxin. Rarely, the nearly ubiquitous spores of C. botulinum may germinate in wounds. In infants, the spores may germinate in the gastrointestinal (GI) tract and release toxin, causing muscle weakness. Patients present with myasthenia-like bulbar weakness (e.g., diplopia, dysarthria, dysphagia) and lack sensory symptoms and signs. Weakness may generalize to the limbs and may result in respiratory failure. Reflexes are present early, but they may be diminished as the disease progresses. Mentation is normal. Autonomic findings include paralytic ileus, constipation, urinary retention, dilated or poorly reactive pupils,

1	early, but they may be diminished as the disease progresses. Mentation is normal. Autonomic findings include paralytic ileus, constipation, urinary retention, dilated or poorly reactive pupils, and dry mouth. The demonstration of toxin in serum by bioassay is definitive, but the results usually take a relatively long time to be completed and may be negative. Nerve stimulation studies reveal findings of presynaptic neuromuscular blockade with reduced compound muscle action potentials (CMAPs) that increase in amplitude following high-frequency repetitive stimulation. Treatment includes ventilatory support and aggressive inpatient supportive care (e.g., nutrition, deep vein thrombosis prophylaxis) as needed. Antitoxin should be given as early as possible to be effective and can be obtained through the Centers for Disease Control and Prevention. A preventive vaccine is available for laboratory workers or other highly exposed individuals.

1	Neurasthenia is the historic term for a myasthenia-like fatigue syndrome without an organic basis. These patients may present with subjective symptoms of weakness and fatigue, but muscle testing usually reveals the “give-away weakness” characteristic of nonorganic disorders; the complaint of fatigue in these patients means tiredness or apathy rather than decreasing muscle power on repeated effort. Hyperthyroidism is readily diagnosed or excluded by tests of thyroid function, which should be carried out routinely in patients with suspected MG. Abnormalities of thyroid function (hyperor hypothyroidism) may increase myasthenic weakness. Diplopia resembling that in MG may occasionally be due to an intracranial mass lesion that compresses nerves to the EOMs (e.g., sphenoid ridge meningioma), but magnetic resonance imaging (MRI) of the head and orbits usually reveals the lesion.

1	Progressive external ophthalmoplegia is a rare condition resulting in weakness of the EOMs, which may be accompanied by weakness of the proximal muscles of the limbs and other systemic features. Most patients with this condition have mitochondrial disorders that can be detected on muscle biopsy (Chap. 462e).

1	Search for Associated Conditions (Table 461-3) Myasthenic patients have an increased incidence of several associated disorders. Thymic abnormalities occur in ~75% of AChR antibody–positive patients, as noted above. Neoplastic change (thymoma) may produce enlargement of the thymus, which is detected by computed tomography (CT) scanning of the anterior mediastinum. A thymic shadow on CT scan may normally be present through young adulthood, but enlargement of the thymus in a patient age >40 years is highly suspicious of thymoma. Hyperthyroidism occurs in 3–8% of patients and may aggravate the myasthenic weakness. Thyroid function tests should be obtained in all patients with suspected MG. Because of the association of MG with other autoimmune disorders, blood tests for rheumatoid factor and antinuclear antibodies should also be carried out. Chronic infection of any kind can exacerbate MG and should be sought carefully. Finally, measurements of ventilatory function are valuable because of

1	antibodies should also be carried out. Chronic infection of any kind can exacerbate MG and should be sought carefully. Finally, measurements of ventilatory function are valuable because of the frequency and seriousness of respiratory impairment in myasthenic patients.

1	Because of the side effects of glucocorticoids and other immunosuppressive agents used in the treatment of MG, a thorough medical Myasthenia Gravis and Other Diseases of the Neuromuscular Junction Associated disorders Disorders of the thymus: thymoma, hyperplasia Other autoimmune disorders: Hashimoto’s thyroiditis, Graves’ disease, rheu matoid arthritis, lupus erythematosus, skin disorders, family history of autoimmune disorder Disorders or circumstances that may exacerbate myasthenia gravis: hyperthyroidism or hypothyroidism, occult infection, medical treatment for other conditions (see Table 461-4) Disorders that may interfere with therapy: tuberculosis, diabetes, peptic ulcer, gastrointestinal bleeding, renal disease, hypertension, asthma, osteoporosis, obesity

1	Disorders that may interfere with therapy: tuberculosis, diabetes, peptic ulcer, gastrointestinal bleeding, renal disease, hypertension, asthma, osteoporosis, obesity Recommended laboratory tests or procedures CT or MRI of chest Tests for lupus erythematosus, antinuclear antibody, rheumatoid factor, antithyroid antibodies Thyroid function tests PPD skin test Fasting blood glucose, hemoglobin A1c Pulmonary function tests Bone densitometry Abbreviations: CT, computed tomography; MRI, magnetic resonance imaging; PPD, purified protein derivative. investigation should be undertaken, searching specifically for evidence of chronic or latent infection (such as tuberculosis or hepatitis), hypertension, diabetes, renal disease, and glaucoma.

1	investigation should be undertaken, searching specifically for evidence of chronic or latent infection (such as tuberculosis or hepatitis), hypertension, diabetes, renal disease, and glaucoma. The prognosis has improved strikingly as a result of advances in treatment. Nearly all myasthenic patients can be returned to full productive lives with proper therapy. The most useful treatments for MG include anticholinesterase medications, immunosuppressive agents, thymectomy, and plasmapheresis or intravenous immunoglobulin (IVIg) (Fig. 461-2).

1	Anticholinesterase medication produces at least partial improvement in most myasthenic patients, although improvement is complete in only a few. Patients with anti-MuSK MG generally obtain less benefit from anticholinesterase agents than those with AChR antibodies. Pyridostigmine is the most widely used anticholinesterase drug. The beneficial action of oral pyridostigmine begins within 15–30 min and lasts for 3–4 h, but individual responses vary. Treatment is begun with a moderate dose, e.g., 30–60 mg three to four times daily. The frequency and amount of the dose should be tailored to the patient’s individual requirements throughout the day. For example, patients with weakness in chewing and swallowing may benefit by taking the medication before meals so that peak strength coincides with mealtimes. Long-acting pyridostigmine may occasionally be useful to get the patient through the night but should not be used for daytime medication because of variable absorption. The maximum useful

1	mealtimes. Long-acting pyridostigmine may occasionally be useful to get the patient through the night but should not be used for daytime medication because of variable absorption. The maximum useful dose of pyridostigmine rarely exceeds 120 mg every 4–6 h during daytime. Overdosage with anticholinesterase medication may cause increased weakness and other side effects. In some patients, muscarinic side effects of the anticholinesterase medication (diarrhea, abdominal cramps, salivation, nausea) may limit the dose tolerated. Atropine/diphenoxylate or loperamide is useful for the treatment of GI symptoms.

1	Two separate issues should be distinguished: (1) surgical removal of thymoma, and (2) thymectomy as a treatment for MG. Surgical removal of a thymoma is necessary because of the possibility of local tumor spread, although most thymomas are histologically benign. Establish diagnosis unequivocally (see Table 461-1) Search for associated conditions (see Table 461-3) Ocular only MRI of brain (if positive, reassess) Anticholinesterase (pyridostigmine) Anticholinesterase (pyridostigmine) Evaluate for thymectomy (indications: thymoma or generalized MG); evaluate surgical risk, FVC Crisis Intensive care (tx respiratory infection; fluids) Generalized If unsatisfactory Thymectomy Good risk (good FVC) Poor risk (low FVC) If not improved Immunosuppression Evaluate clinical status; if indicated, go to immunosuppression Improved See text for short-term, intermediate, and long-term treatments Plasmapheresis or intravenous Ig then

1	FIGURE 461-2 Algorithm for the management of myasthenia gravis. FVC, forced vital capacity; MRI, magnetic resonance imaging.

1	In the absence of a tumor, the available evidence suggests that up to 85% of patients experience improvement after thymectomy; of these, ~35% achieve drug-free remission. However, the improvement is typically delayed for months to years. The advantage of thymectomy is that it offers the possibility of long-term benefit, in some cases diminishing or eliminating the need for continuing medical treatment. Review of the published studies showed that following thymectomy, MG patients were 1.7 times as likely to improve and twice as likely to attain remission as those who did not have surgical thymectomy. In view of these potential benefits and of the negligible risk in skilled hands, thymectomy has gained widespread acceptance in the treatment of MG. It is the consensus that thymectomy should be carried out in all patients with generalized MG who are between the ages of puberty and at least 55 years. Whether thymectomy should be recommended in children, in adults >55 years of age, and in

1	be carried out in all patients with generalized MG who are between the ages of puberty and at least 55 years. Whether thymectomy should be recommended in children, in adults >55 years of age, and in patients with weakness limited to the ocular muscles is still a matter of debate. There is also evidence that patients with MuSK antibody–positive MG respond less well to thymectomy than those with AChR antibody. Thymectomy must be carried out in a hospital where it is performed regularly and where the staff is experienced in the preand postoperative management, anesthesia, and surgical techniques of total thymectomy. Thymectomy should never be carried out as an emergency procedure, but only when the patient is adequately prepared. If necessary, treatment with IVIg or plasmapheresis may be used before surgery, but it is helpful to try to avoid immunosuppressive agents because of the risk of infection.

1	Immunosuppression using one or more of the available agents is effective in nearly all patients with MG. The choice of drugs or other immunomodulatory treatments should be guided by the relative benefits and risks for the individual patient and the urgency of treatment. It is helpful to develop a treatment plan based on short-term, intermediate-term, and long-term objectives. For example, if immediate improvement is essential either because of the severity of weakness or because of the patient’s need to return to activity as soon as possible, IVIg should be administered or plasmapheresis should be undertaken. For the intermediate term, glucocorticoids and cyclosporine or tacrolimus generally produce clinical improvement within a period of 1–3 months. The beneficial effects of azathioprine and mycophenolate mofetil usually begin after many months (as long as a year), but these drugs have advantages for the long-term treatment of patients with MG. There is a growing body of evidence

1	and mycophenolate mofetil usually begin after many months (as long as a year), but these drugs have advantages for the long-term treatment of patients with MG. There is a growing body of evidence that rituximab is effective in many MG patients, especially those with MuSK antibody.

1	Glucocorticoid Therapy Glucocorticoids, when used properly, produce improvement in myasthenic weakness in the great majority of patients. To minimize adverse side effects, prednisone should be given in a single dose rather than in divided doses throughout the day. The initial dose should be relatively low (15–25 mg/d) to avoid the early weakening that occurs in up to one-third of patients treated initially with a high-dose regimen. The dose is increased stepwise, as tolerated by the patient (usually by 5 mg/d at 2to 3-day intervals), until there is marked clinical improvement or a dose of 50–60 mg/d is reached. This dose is maintained for 1–3 months and then is gradually modified to an alternate-day regimen over the course of an additional 1–3 months; the goal is to reduce the dose on the “off day” to zero or to a minimal level. Generally, patients begin to improve within a few weeks after reaching the maximum dose, and improvement continues to progress for months or years. The

1	on the “off day” to zero or to a minimal level. Generally, patients begin to improve within a few weeks after reaching the maximum dose, and improvement continues to progress for months or years. The prednisone dosage may gradually be reduced, but usually months or years may be needed to determine the minimum effective dose, and close monitoring is required. Few patients are able to do without immunosuppressive agents entirely. Patients on longterm glucocorticoid therapy must be followed carefully to prevent or treat adverse side effects. The most common errors in glucocorticoid treatment of myasthenic patients include (1) insufficient persistence—improvement may be delayed and gradual; (2) tapering the dosage too early, too rapidly, or excessively; and (3) lack of attention to prevention and treatment of side effects.

1	The management of patients treated with glucocorticoids is discussed in Chap. 406. Other Immunosuppressive Drugs Mycophenolate mofetil, azathioprine, cyclosporine, tacrolimus, rituximab, and occasionally cyclophosphamide are effective in many patients, either alone or in various combinations.

1	Mycophenolate mofetil has become one of the most widely used drugs in the treatment of MG because of its effectiveness and relative lack of side effects. A dose of 1–1.5 g bid is recommended. Its mechanism of action involves inhibition of purine synthesis by the de novo pathway. Since lymphocytes have only the de novo pathway, but lack the alternative salvage pathway that is present in all other cells, mycophenolate inhibits proliferation of lymphocytes but not proliferation of other cells. It does not kill or eliminate preexisting autoreactive lymphocytes, and therefore clinical improvement may be delayed for many months to a year, until the preexisting autoreactive lymphocytes die spontaneously. The advantage of mycophenolate lies in its relative lack of adverse side effects, with only occasional production of GI symptoms, rare development of leukopenia, and very small risks of malignancy or progressive multifocal leukoencephalopathy inherent in nearly all immunosuppressive

1	only occasional production of GI symptoms, rare development of leukopenia, and very small risks of malignancy or progressive multifocal leukoencephalopathy inherent in nearly all immunosuppressive treatments. Although two published studies did not show positive outcomes, most experts attribute the negative results to flaws in the trial designs, and mycophenolate is widely used for long-term treatment of myasthenic patients.

1	Until recently, azathioprine has been the most commonly used 2705 immunosuppressive agent for MG because of its relative safety in most patients and long track record. Its therapeutic effect may add to that of glucocorticoids and/or allow the glucocorticoid dose to be reduced. However, up to 10% of patients are unable to tolerate azathioprine because of idiosyncratic reactions consisting of flu-like symptoms of fever and malaise, bone marrow suppression, or abnormalities of liver function. An initial dose of 50 mg/d should be used for several days to test for these side effects. If this dose is tolerated, it is increased gradually to about 2–3 mg/kg of total body weight, or until the white blood count falls to 3000–4000/μL. The beneficial effect of azathioprine takes 3–6 months to begin and even longer to peak. In patients taking azathioprine, allopurinol should never be used to treat hyperuricemia. Because the two drugs share a common degradation pathway; the result may be severe

1	and even longer to peak. In patients taking azathioprine, allopurinol should never be used to treat hyperuricemia. Because the two drugs share a common degradation pathway; the result may be severe bone marrow suppression due to increased effects of the azathioprine.

1	The calcineurin inhibitors cyclosporine and tacrolimus (FK506) are approximately as effective as azathioprine and are being used increasingly in the management of MG. Their beneficial effect appears more rapidly than that of azathioprine. Either drug may be used alone, but they are usually used as an adjunct to glucocorticoids to permit reduction of the glucocorticoid dose. The usual dose of cyclosporine is 4–5 mg/kg per day, and the average dose of tacrolimus is 0.07–0.1 mg/kg per day, given in two equally divided doses (to minimize side effects). Side effects of these drugs include hypertension and nephrotoxicity, which must be closely monitored. “Trough” blood levels are measured 12 h after the evening dose. The therapeutic range for the trough level of cyclosporine is 150–200 ng/L, and for tacrolimus, it is 5–15 ng/L.

1	Rituximab (Rituxan) is a monoclonal antibody that binds to the CD20 molecule on B lymphocytes. It has been widely used for the treatment of B cell lymphomas and has also proven successful in the treatment of several autoimmune diseases including rheumatoid arthritis, pemphigus, and some IgM-related neuropathies. There is now an extensive literature on the benefit of rituximab in MG. It is particularly effective in MuSK antibody–positive MG, although some patients with AChR antibody MG respond to it as well. The usual dose is 375 mg/m2, given IV in 4 weekly infusions, or 1 g, given IV on two occasions 2 weeks apart.

1	For the occasional patient with MG that is genuinely refractory to optimal treatment with conventional immunosuppressive agents, a course of high-dose cyclophosphamide may induce long-lasting benefit by “rebooting” the immune system. At high doses, cyclophosphamide eliminates mature lymphocytes but spares hematopoietic precursors (stem cells), because they express the enzyme aldehyde dehydrogenase, which hydrolyzes cyclophosphamide. At present, this procedure is reserved for refractory patients and should be administered only in a facility fully familiar with this approach. Maintenance immunotherapy after rebooting is usually required to sustain the beneficial effect.

1	Plasmapheresis has been used therapeutically in MG. Plasma, which contains the pathogenic antibodies, is mechanically separated from the blood cells, which are returned to the patient. A course of five exchanges (3–4 L per exchange) is generally administered over a 10to 14-day period. Plasmapheresis produces a short-term reduction in anti-AChR antibodies, with clinical improvement in many patients. It is useful as a temporary expedient in seriously affected patients or to improve the patient’s condition prior to surgery (e.g., thymectomy).

1	The indications for the use of IVIg are the same as those for plasma exchange: to produce rapid improvement to help the patient through a difficult period of myasthenic weakness or prior to surgery. This treatment has the advantages of not requiring special equipment or large-bore venous access. The usual dose is 2 g/ kg, which is typically administered over 5 days (400 mg/kg per day). If tolerated, the total dose of IVIg can be given over a 3to 4-day period. Improvement occurs in ~70% of patients, beginning during

1	Myasthenia Gravis and Other Diseases of the Neuromuscular Junction 2706 treatment, or within a week, and continuing for weeks to months. The mechanism of action of IVIg is not known; the treatment has no consistent effect on the measurable amount of circulating AChR antibody. Adverse reactions are generally not serious but may include headache, fluid overload, and rarely aseptic meningitis or renal failure. IVIg should rarely be used as a long-term treatment in place of rationally managed immunosuppressive therapy. Unfortunately, there is a tendency for physicians unfamiliar with immunosuppressive treatments to rely on repeated IVIg infusions, which usually produce only intermittent benefit, do not reduce the underlying autoimmune response, and are very costly. The intermediate and long-term treatment of myasthenic patients requires other methods of therapy outlined earlier in this chapter.

1	Myasthenic crisis is defined as an exacerbation of weakness sufficient to endanger life; it usually consists of respiratory failure caused by diaphragmatic and intercostal muscle weakness. Crisis rarely occurs in properly managed patients. Treatment should be carried out in intensive care units staffed with teams experienced in the management of MG, respiratory insufficiency, infectious disease, and fluid and electrolyte therapy. The possibility that deterioration could be due to excessive anticholinesterase medication (“cholinergic crisis”) is best excluded by temporarily stopping anticholinesterase drugs. The most common cause of crisis is intercurrent infection. This should be treated immediately, because the mechanical and immunologic defenses of the patient can be assumed to be compromised. The myasthenic patient with fever and early infection should be treated like other immunocompromised patients. Early and effective antibiotic therapy, respiratory assistance (preferably

1	compromised. The myasthenic patient with fever and early infection should be treated like other immunocompromised patients. Early and effective antibiotic therapy, respiratory assistance (preferably noninvasive, using bilevel positive airway pressure), and pulmonary physiotherapy are essentials of the treatment program. As discussed above, plasmapheresis or IVIg is frequently helpful in hastening recovery.

1	Many drugs have been reported to exacerbate weakness in patients with MG (Table 461-4), but not all patients react adversely to all of Aminoglycosides: e.g., streptomycin, tobramycin, kanamycin Quinolones: e.g., ciprofloxacin, levofloxacin, ofloxacin, gatifloxacin Macrolides: e.g., erythromycin, azithromycin D-Tubocurarine (curare), pancuronium, vecuronium, atracurium Propranolol, atenolol, metoprolol Procaine, Xylocaine in large amounts Procainamide (for arrhythmias) Quinine, quinidine, chloroquine, mefloquine (Lariam) Drugs with Important Interactions in MG Broad range of drug interactions, which may raise or lower cyclosporine levels. Avoid allopurinol—combination may result in myelosuppression. FIGURE 461-3 Abbreviated interval assessment form for use in evaluating treatment for myasthenia gravis.

1	Avoid allopurinol—combination may result in myelosuppression. FIGURE 461-3 Abbreviated interval assessment form for use in evaluating treatment for myasthenia gravis. these. Conversely, not all “safe” drugs can be used with impunity in patients with MG. As a rule, the listed drugs should be avoided whenever possible, and myasthenic patients should be followed closely when any new drug is introduced.

1	To evaluate the effectiveness of treatment as well as drug-induced side effects, it is important to assess the patient’s clinical status systematically at baseline and on repeated interval examinations. Because of the variability of symptoms of MG, the interval history and physical findings on examination must be taken into account. The most useful clinical tests include forward arm abduction time (up to a full 5 min), spirometry with determination of forced vital capacity, range of eye movements, and time to development of ptosis on upward gaze. Manual muscle testing or, preferably, quantitative dynamometry of limb muscles, especially proximal muscles, is also important. An interval form can provide a succinct summary of the patient’s status and a guide to treatment results; an abbreviated form is shown in Fig. 461-3. A progressive reduction in the patient’s AChR antibody level also provides clinically valuable confirmation of the effectiveness of treatment; conversely, a rise in

1	form is shown in Fig. 461-3. A progressive reduction in the patient’s AChR antibody level also provides clinically valuable confirmation of the effectiveness of treatment; conversely, a rise in AChR antibody levels during tapering of immunosuppressive medication may predict clinical exacerbation. For reliable quantitative measurement of AChR antibody levels, it is best to compare antibody levels from prior frozen serum aliquots with current serum samples in simultaneously run assays.

1	Muscular Dystrophies and Other Muscle Diseases Anthony A. Amato, Robert H. Brown, Jr. Skeletal muscle diseases, or myopathies, are disorders with structural changes or functional impairment of muscle. These conditions can be 462e

1	Variable weakness includes EOMs, ptosis, bulbar and limb muscles Yes No Exam normal between attacks Proximal > distal weakness during attacks Exam usually normal between attacks Proximal > distal weakness during attacks Forearm exercise DNA test confirms diagnosis Low potassium level Normal or elevated potassium level Hypokalemic PP Hyperkalemic PP Paramyotonia congenita Muscle biopsy defines specific defect Reduced lactic acid rise Consider glycolytic defect Normal lactic acid rise Consider CPT deficiency or other fatty acid metabolism disorders No Yes AChR or Musk AB positive Acquired seropositive MG Check chest CT for thymoma Lambert-Eaton myasthenic syndrome Check: Voltage gated Ca channel Abs Chest CT for lung Ca Yes No Yes No Decrement on 2–3 Hz repetitive nerve stimulation (RNS) or increased jitter on single fiber EMG (SFEMG) Consider: Seronegative MG Congenital MG* Psychosomatic weakness** *Genetic testing (Chap. 461) **If Abs, RNS, SFEMG are all normal or negative EKG

1	or increased jitter on single fiber EMG (SFEMG) Consider: Seronegative MG Congenital MG* Psychosomatic weakness** *Genetic testing (Chap. 461) **If Abs, RNS, SFEMG are all normal or negative EKG Abnormal Check for dysmorphic features Genetic testing for Anderson-Tawil syndrome Normal Myotonia on exam FIgURE 462e-1 Diagnostic evaluation of intermittent weakness. AChR AB, acetylcholine receptor antibody; CPT, carnitine palmitoyltransfer-ase; EOMs, extraocular muscles; MG, myasthenia gravis; PP, periodic paralysis.

1	differentiated from other diseases of the motor unit (e.g., lower motor neuron or neuromuscular junction pathologies) by characteristic clinical and laboratory findings. Myasthenia gravis and related disorders are discussed in Chap. 461; dermatomyositis, polymyositis, and inclusion body myositis are discussed in Chap. 388. Most myopathies present with proximal, symmetric limb weakness (arms or legs) with preserved reflexes and sensation. However, asymmetric and predominantly distal weakness can be seen in some myopathies. An associated sensory loss suggests injury to a peripheral nerve or the central nervous system (CNS) rather than myopathy. On occasion, disorders affecting the motor nerve cell bodies in the spinal cord (anterior horn cell disease), the neuromuscular junction, or peripheral nerves can mimic findings of myopathy.

1	Muscle Weakness Symptoms of muscle weakness can be either intermittent or persistent. Disorders causing intermittent weakness (Fig. 462e-1) include myasthenia gravis, periodic paralyses (hypokalemic, hyperkalemic, and paramyotonia congenita), and metabolic energy deficiencies of glycolysis (especially myophosphorylase deficiency), fatty acid utilization (carnitine palmitoyltransferase deficiency), and some mitochondrial myopathies. The states of energy deficiency cause activity-related muscle breakdown accompanied by myoglobinuria, appearing as light-brownto dark-brown-colored urine.

1	Most muscle disorders cause persistent weakness (Fig. 462e-2). In the majority of these, including most types of muscular dystrophy, polymyositis, and dermatomyositis, the proximal muscles are weaker than the distal and are symmetrically affected, and the facial muscles are spared, a pattern referred to as limb-girdle. The differential diagnosis is more restricted for other patterns of weakness. Facial weakness (difficulty with eye closure and impaired smile) and scapular winging (Fig. 462e-3) are characteristic of facioscapulohumeral dystrophy 462e-1 (FSHD). Facial and distal limb weakness associated with hand grip myotonia is virtually diagnostic of myotonic dystrophy type 1. When other cranial nerve muscles are weak, causing ptosis or extraocular muscle weakness, the most important disorders to consider include neuromuscular junction disorders, oculopharyngeal muscular dystrophy, mitochondrial myopathies, or some of the congenital myopathies (Table 462e-1). A pathognomonic pattern

1	to consider include neuromuscular junction disorders, oculopharyngeal muscular dystrophy, mitochondrial myopathies, or some of the congenital myopathies (Table 462e-1). A pathognomonic pattern characteristic of inclusion body myositis is atrophy and weakness of the flexor forearm (e.g., wrist and finger flexors) and quadriceps muscles that is often asymmetric. Less frequently, but important diagnostically, is the presence of a dropped head syndrome indicative of selective neck extensor muscle weakness. The most important neuromuscular diseases associated with this pattern of weakness include myasthenia gravis, amyotrophic lateral sclerosis, late-onset nemaline myopathy, hyperparathyroidism, focal myositis, and some forms of inclusion body myopathy. A final pattern, recognized because of preferential distal extremity weakness, is typical of a unique category of muscular dystrophy, the distal myopathies.

1	It is important to examine functional capabilities to help disclose certain patterns of weakness (Table 462e-2). The Gowers’ sign (Fig. 462e-4) is particularly useful. Observing the gait of an individual may disclose a lordotic posture caused by combined trunk and hip weakness, frequently exaggerated by toe walking (Fig. 462e-5). A waddling gait is caused by the inability of weak hip muscles to prevent hip drop or hip dip. Hyperextension of the knee (genu recurvatum or back-kneeing) is characteristic of quadriceps muscle weakness; and a steppage gait, due to footdrop, accompanies distal weakness.

1	Any disorder causing muscle weakness may be accompanied by fatigue, referring to an inability to maintain or sustain a force (pathologic fatigability). This condition must be differentiated from asthenia, a type of fatigue caused by excess tiredness or lack of energy. Associated symptoms may help differentiate asthenia and pathologic fatigability. Asthenia is often accompanied by a tendency to avoid physical activities, complaints of daytime sleepiness, necessity for frequent naps, and difficulty concentrating on activities such as reading. There may be feelings of overwhelming stress and depression. Thus, asthenia is not a myopathy. In contrast, pathologic fatigability occurs in disorders of neuromuscular transmission and in disorders altering energy production, including defects in glycolysis, lipid metabolism, or mitochondrial energy production. Pathologic fatigability also occurs in chronic myopathies because of difficulty accomplishing a task with less

1	FIgURE 462e-2 Diagnostic evaluation of persistent weakness. Examination reveals one of seven patterns of weakness. The pattern of weakness in combination with the laboratory evaluation leads to a diagnosis. ALS, amyotrophic lateral sclerosis; CK, creatine kinase; DM, dermatomyositis; EMG, electromyography; EOMs, extraocular muscles; FSHD, facioscapulohumeral dystrophy; IBM, inclusion body myositis; MG, myasthenia gravis; OPMD, oculopharyngeal muscular dystrophy; PM, polymyositis. muscle. Pathologic fatigability is accompanied by abnormal clinical or laboratory findings. Fatigue without those supportive features almost never indicates a primary muscle disease.

1	muscle. Pathologic fatigability is accompanied by abnormal clinical or laboratory findings. Fatigue without those supportive features almost never indicates a primary muscle disease. Muscle Pain (Myalgias), Cramps, and Stiffness Muscle pain can be associated with cramps, spasms, contractures, and stiff or rigid muscles. In distinction, true myalgia (muscle aching), which can be localized or generalized, may be accompanied by weakness, tenderness to palpation, or swelling. Certain drugs cause true myalgia (Table 462e-3).

1	There are two painful muscle conditions of particular importance, neither of which is associated with muscle weakness. Fibromyalgia is a common, yet poorly understood, type of myofascial pain syndrome. Patients complain of severe muscle pain and tenderness and have specific painful trigger points, sleep disturbances, and easy fatigability. Serum creatine kinase (CK), erythrocyte sedimentation rate (ESR), electromyography (EMG), and muscle biopsy are normal (Chap. 396). Polymyalgia rheumatica occurs mainly in patients >50 years and is characterized by stiffness and pain in the shoulders, lower back, hips, and thighs (Chap. 385). The ESR is elevated, while serum CK, EMG, and muscle biopsy are normal. Temporal arteritis, an inflammatory FIgURE 462e-3 Facioscapulohumeral dystrophy with prominent scapular winging.

1	FIgURE 462e-3 Facioscapulohumeral dystrophy with prominent scapular winging. disorder of mediumand large-sized arteries, usually involving one or more branches of the carotid artery, may accompany polymyalgia rheumatica. Vision is threatened by ischemic optic neuritis. Glucocorticoids can relieve the myalgias and protect against visual loss. Localized muscle pain is most often traumatic. A common cause of sudden abrupt-onset pain is a ruptured tendon, which leaves the muscle belly appearing rounded and shorter in appearance compared to the normal side. The biceps brachii and Achilles tendons are particularly vulnerable to rupture. Infection or neoplastic infiltration of the muscle is a rare cause of localized muscle pain.

1	A muscle cramp or spasm is a painful, involuntary, localized, muscle contraction with a visible or palpable hardening of the muscle. Cramps are abrupt in onset, short in duration, and may cause abnormal posturing of the joint. The EMG shows firing of motor units, reflecting an origin from spontaneous neural discharge. Muscle cramps often occur Inability to forcibly close eyes Upper facial muscles Inability to raise head from prone Neck extensor muscles position Inability to raise head from supine Neck flexor muscles position Inability to raise arms above head Proximal arm muscles (may be only scapular stabilizing muscles) Inability to walk without hyperex-Knee extensor muscles tending knee (back-kneeing or genu recurvatum) Inability to walk with heels touching Shortening of the Achilles tendon the floor (toe walking) Inability to lift foot while walking Anterior compartment of leg (steppage gait or footdrop) Inability to walk without a waddling Hip muscles gait

1	Inability to lift foot while walking Anterior compartment of leg (steppage gait or footdrop) Inability to walk without a waddling Hip muscles gait Inability to get up from the floor Hip, thigh, and trunk muscles without climbing up the extremities (Gowers’ sign) Inability to get up from a chair Hip muscles without using arms in neurogenic disorders, especially motor neuron disease (Chap. 452), radiculopathies, and polyneuropathies (Chap. 459), but are not a feature of most primary muscle diseases. Duchenne muscular dystrophy is an exception because calf muscle complaints are a common complaint. Muscle cramps are also common during pregnancy.

1	A muscle contracture is different from a muscle cramp. In both conditions, the muscle becomes hard, but a contracture is associated with energy failure in glycolytic disorders. The muscle is unable to relax after an active muscle contraction. The EMG shows electrical silence. Confusion is created because contracture also refers to a muscle that cannot be passively stretched to its proper length (fixed contracture) because of fibrosis. In some muscle disorders, especially in Emery-Dreifuss muscular dystrophy and Bethlem myopathy, fixed contractures occur early and represent distinctive features of the disease.

1	Muscle stiffness can refer to different phenomena. Some patients with inflammation of joints and periarticular surfaces feel stiff. This condition is different from the disorders of hyperexcitable motor nerves causing stiff or rigid muscles. In stiff-person syndrome, spontaneous discharges of the motor neurons of the spinal cord cause involuntary muscle contractions mainly involving the axial (trunk) and proximal lower extremity muscles. The gait becomes stiff and labored, with hyperlordosis of the lumbar spine. Superimposed episodic muscle spasms are precipitated by sudden movements, unexpected noises, and emotional upset. The muscles relax during sleep. Serum antibodies against glutamic acid decarboxylase are present in approximately two-thirds of cases. In neuromyotonia (Isaacs’ syndrome), there is hyper-excitability of the peripheral nerves manifesting as continuous muscle fiber activity. Myokymia (groups of fasciculations associated with continuous undulations of muscle) and

1	there is hyper-excitability of the peripheral nerves manifesting as continuous muscle fiber activity. Myokymia (groups of fasciculations associated with continuous undulations of muscle) and impaired muscle relaxation are the result. Muscles of the leg are stiff, and the constant contractions of the muscle cause increased sweating of the extremities. This peripheral nerve hyperexcitability is mediated by antibodies that target voltage-gated potassium channels. The site of origin of the spontaneous nerve discharges is principally in the distal portion of the motor nerves.

1	Myotonia is a condition of prolonged muscle contraction followed by slow muscle relaxation. It always follows muscle activation (action myotonia), usually voluntary, but may be elicited by mechanical stimulation (percussion myotonia) of the muscle. Myotonia typically causes difficulty in releasing objects after a firm grasp. In myotonic muscular dystrophy type 1 (DM1), distal weakness usually accompanies myotonia, whereas in DM2, proximal muscles are more affected; thus FIgURE 462e-4 Gowers’ sign showing a patient using his arms to climb up the legs in attempting to get up from the floor.

1	FIgURE 462e-4 Gowers’ sign showing a patient using his arms to climb up the legs in attempting to get up from the floor. the related term proximal myotonic myopathy (PROMM) is used to describe this condition. Myotonia also occurs with myotonia congenita (a chloride channel disorder), but in this condition muscle weakness is not prominent. Myotonia may also be seen in individuals with sodium channel mutations (hyperkalemic periodic paralysis or potassium-sensitive myotonia). Another sodium channelopathy, paramyotonia congenita, also is associated with muscle stiffness. In contrast to other disorders associated with myotonia in which the myotonia is eased by repetitive activity, paramyotonia congenita is named for a paradoxical phenomenon whereby the myotonia worsens with repetitive activity.

1	Muscle Enlargement and Atrophy In most myopathies muscle tissue is replaced by fat and connective tissue, but the size of the muscle is usually not affected. However, in many limb-girdle muscular dystrophies (and particularly the dystrophinopathies), enlarged calf muscles are typical. The enlargement represents true muscle hypertrophy; thus the term pseudohypertrophy should be avoided when referring to these patients. The calf muscles remain very strong even late in the course of these disorders. Muscle enlargement can also result from infiltration by sarcoid granulomas, amyloid deposits, bacterial and parasitic infections, and focal myositis. In contrast, muscle atrophy is characteristic of other myopathies. In dysferlinopathies (LGMD2B) FIgURE 462e-5 Lordotic posture, exaggerated by standing on toes, associated with trunk and hip weakness.

1	FIgURE 462e-5 Lordotic posture, exaggerated by standing on toes, associated with trunk and hip weakness. and anoctaminopathies (LGMD2L), there is a predilection for early atrophy of the gastrocnemius muscles, particularly the medial aspect. Atrophy of the humeral muscles is characteristic of FSHD. A limited battery of tests can be used to evaluate a suspected myopathy. Nearly all patients require serum enzyme level measurements and electrodiagnostic studies as screening tools to differentiate muscle disorders from other motor unit diseases. The other tests described— DNA studies, the forearm exercise test, and muscle biopsy—are used to diagnose specific types of myopathies.

1	Serum Enzymes CK is the preferred muscle enzyme to measure in the evaluation of myopathies. Damage to muscle causes the CK to leak from the muscle fiber to the serum. The MM isoenzyme predominates in skeletal muscle, whereas creatine kinase-myocardial bound (CKMB) is the marker for cardiac muscle. Serum CK can be elevated in normal individuals without provocation, presumably on a genetic basis or after strenuous activity, minor trauma (including the EMG needle), a prolonged muscle cramp, or a generalized seizure. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), aldolase, and lactic dehydrogenase (LDH) are enzymes sharing an origin in both muscle and liver. Problems arise when the levels of these enzymes are found to be elevated in a routine screening battery, leading to the erroneous assumption that liver disease is present when in fact muscle could be the cause. An elevated γ-glutamyl transferase (GGT) helps to establish a liver origin because this enzyme is not

1	to the erroneous assumption that liver disease is present when in fact muscle could be the cause. An elevated γ-glutamyl transferase (GGT) helps to establish a liver origin because this enzyme is not found in muscle.

1	Electrodiagnostic Studies EMG, repetitive nerve stimulation, and nerve conduction studies (Chap. 442e) are essential methods for evaluation of the patient with suspected muscle disease. In combination, they provide the information necessary to differentiate myopathies from neuropathies and neuromuscular junction diseases. Routine nerve conduction studies are typically normal in myopathies but reduced amplitudes of compound muscle action potentials may be seen in atrophied muscles. The needle EMG may reveal irritability on needle placement suggestive of a necrotizing myopathy (inflammatory myopathies, dystrophies, toxic myopathies, myotonic myopathies), whereas a lack of irritability is characteristic of long-standing myopathic disorders (muscular dystrophies, endocrine myopathies, disuse atrophy, and many of the metabolic myopathies). In addition, the EMG may demonstrate myotonic discharges that will narrow the differential diagnosis (Table 462e-4). Another important EMG finding is

1	atrophy, and many of the metabolic myopathies). In addition, the EMG may demonstrate myotonic discharges that will narrow the differential diagnosis (Table 462e-4). Another important EMG finding is the presence of short-duration, small-amplitude, polyphasic motor unit action potentials (MUAPs). Such MUAPs can be seen in both myopathic and neuropathic disorders; however, the recruitment or firing pattern is different. In myopathies, the MUAPs fire early but at a normal rate to compensate for the loss of individual muscle fibers, whereas in neurogenic disorders the MUAPs fire faster. The EMG is usually normal in steroid or disuse myopathy, both of which are associated with type 2 fiber atrophy; this is because the EMG preferentially assesses the physiologic function of type 1 fibers. The EMG can also be invaluable in helping to choose an appropriately affected muscle to sample for biopsy.

1	DNA Analysis This serves as an important tool for the definitive diagnosis of many muscle disorders. Nevertheless, there are a number of limitations in currently available molecular diagnostics. For example, in Duchenne and Becker dystrophies, two-thirds of patients have deletion or duplication mutations in the dystrophin gene that are easy to detect, while the remainder have point mutations that are much more difficult to find. For patients without identifiable gene defects, the muscle biopsy remains the main diagnostic tool. Cholesterol-lowering agents (statin medications, fibrates) Cyclosporine Chloroquine Glycogen storage disordersa (Pompe’s disease, branching enzyme deficiency, debranching enzyme deficiency) Myofibrillar myopathies (MFM)a aAssociated with myotonic discharges on electromyography but no clinical myotonia.

1	Forearm Exercise Test In myopathies with intermittent symptoms, and especially those associated with myoglobinuria, there may be a defect in glycolysis. Many variations of the forearm exercise test exist. For safety, the test should not be performed under ischemic conditions to avoid an unnecessary insult to the muscle, causing rhabdomyolysis. The test is performed by placing a small indwelling catheter into an antecubital vein. A baseline blood sample is obtained for lactic acid and ammonia. The forearm muscles are exercised by asking the patient to vigorously open and close the hand for 1 min. Blood is then obtained at intervals of 1, 2, 4, 6, and 10 min for comparison with the baseline sample. A threeto fourfold rise of lactic acid is typical. The simultaneous measurement of ammonia serves as a control, because it should also rise with exercise. In patients with myophosphorylase deficiency or other glycolytic defects, the lactic acid rise will be absent or below normal, while the

1	as a control, because it should also rise with exercise. In patients with myophosphorylase deficiency or other glycolytic defects, the lactic acid rise will be absent or below normal, while the rise in ammonia will reach control values. If there is lack of effort, neither lactic acid nor ammonia will rise. Patients with selective failure to increase ammonia may have myoadenylate deaminase deficiency. This condition has been reported to be a cause of myoglobinuria, but deficiency of this enzyme in asymptomatic individuals makes interpretation controversial.

1	Muscle Biopsy Muscle biopsy is an important step in establishing the diagnosis of a suspected myopathy. The biopsy is usually obtained from a quadriceps or biceps brachii muscle, less commonly from a deltoid muscle. Evaluation includes a combination of techniques—light microscopy, histochemistry, immunocytochemistry with a battery of antibodies, and electron microscopy. Not all techniques are needed for every case. A specific diagnosis can be established in many disorders. Endomysial inflammatory cells surrounding and invading muscle fibers are seen in polymyositis; similar endomysial infiltrates associated 462e-5 with muscle fibers containing rimmed vacuoles and amyloid deposits consisting of SMI-31-, p62-, and TDP-43-positive inclusions within fibers are characteristic of inclusion body myositis; and perivascular, perimysial inflammation associated with perifascicular atrophy is a feature of dermatomyositis. In addition, the congenital myopathies have distinctive light and electron

1	myositis; and perivascular, perimysial inflammation associated with perifascicular atrophy is a feature of dermatomyositis. In addition, the congenital myopathies have distinctive light and electron microscopy features essential for diagnosis. Mitochondrial and metabolic (e.g., glycogen and lipid storage diseases) myopathies also demonstrate distinctive histochemical and electron-microscopic profiles. Biopsied muscle tissue can be sent for metabolic enzyme or mitochondrial DNA analyses. A battery of antibodies is available for the identification of abnormal proteins to help diagnose specific types of muscular dystrophies. Western blot analysis on muscle specimens can be performed to determine whether specific muscle proteins are reduced in quantity or are of abnormal size.

1	Muscular dystrophy refers to a group of hereditary progressive diseases each with unique phenotypic and genetic features (Tables 462e-5, 462e-6, and 462e-7). This X-linked recessive disorder, sometimes also called pseudohypertrophic muscular dystrophy, has an incidence of ~1 per 5200 live-born males. Clinical Features Duchenne dystrophy is present at birth, but the disorder usually becomes apparent between ages 3 and 5 years. The boys fall frequently and have difficulty keeping up with friends when Myotonica AD DM1: Expansion Childhood to adult; pos-Slowly progressive weakness of face, shoulder Cardiac conduction (DM1, DM2) CTG repeat sibly infancy if mother girdle, and foot dorsiflexion defects affected (DM1 only) DM2: Expansion FSHD1 AD DUX4 4q Childhood to adult Slowly progressive weakness of face, shoulder Deafness girdle, and foot dorsiflexion

1	DM2: Expansion FSHD1 AD DUX4 4q Childhood to adult Slowly progressive weakness of face, shoulder Deafness girdle, and foot dorsiflexion Oculopharyngeal AD Expansion, poly-Fifth to sixth decade Slowly progressive weakness of extraocular, — A RNA binding pharyngeal, and limb muscles protein aTwo forms of myotonic dystrophy, DM1 and DM2, have been identified. Many features overlap (see text). Abbreviations: AD, autosomal dominant; AR, autosomal recessive; CNS, central nervous system; XR, X-linked recessive.

1	LGMD1A Onset second to eighth decade Serum CK 2× normal Myotilin playing. Running, jumping, and hopping are invariably abnormal. By age 5 years, muscle weakness is obvious by muscle testing. On getting up from the floor, the patient uses his hands to climb up himself (Gowers’ maneuver [Fig. 462e-4]). Contractures of the heel cords and iliotibial bands become apparent by age 6 years, when toe walking is associated with a lordotic posture. Loss of muscle strength is progressive, with predilection for proximal limb muscles and the neck flexors; leg involvement is more severe than arm involvement. Between ages 8 and 10 years, walking may require the use of braces; joint contractures and limitations of hip flexion, knee, elbow, and wrist extension are made worse by prolonged sitting. Prior to the use of glucocorticoids, most boys became wheelchair dependent by 12 years of age. Contractures become fixed, and a progressive scoliosis often develops that may be associated with pain. The chest

1	the use of glucocorticoids, most boys became wheelchair dependent by 12 years of age. Contractures become fixed, and a progressive scoliosis often develops that may be associated with pain. The chest deformity with scoliosis impairs pulmonary function, which is already diminished by muscle weakness. By age 16–18 years, patients are predisposed to serious, sometimes fatal pulmonary infections. Other causes of death include aspiration of food and acute gastric dilation.

1	A cardiac cause of death is uncommon despite the presence of a cardiomyopathy in almost all patients. Congestive heart failure seldom occurs except with severe stress such as pneumonia. Cardiac arrhythmias are rare. The typical electrocardiogram (ECG) shows an increased net RS in lead V1; deep, narrow Q waves in the precordial leads; and tall right precordial R waves in V1. Intellectual impairment in Duchenne dystrophy is common; the average intelligence quotient (IQ) is ~1 standard deviation (SD) below the mean. Impairment of intellectual function appears to be nonprogressive and affects verbal ability more than performance.

1	Laboratory Features Serum CK levels are invariably elevated to between 20 and 100 times normal. The levels are abnormal at birth but decline late in the disease because of inactivity and loss of muscle mass. EMG demonstrates features typical of myopathy. The muscle biopsy shows muscle fibers of varying size as well as small groups of necrotic and regenerating fibers. Connective tissue and fat replace lost muscle fibers. A definitive diagnosis of Duchenne dystrophy can be established on the basis of dystrophin deficiency in a biopsy of muscle tissue or mutation analysis on peripheral blood leukocytes, as discussed below.

1	Duchenne dystrophy is caused by a mutation of the gene that encodes dystrophin, a 427-kDa protein localized to the inner surface of the sarcolemma of the muscle fiber. The dystrophin gene is >2000 kb in size and thus is one of the largest identified human genes. It is localized to the short arm of the X chromosome at Xp21. The most common gene mutation is a deletion. The size varies but does not correlate with disease severity. Deletions are not uniformly distributed over the gene but rather are most common near the beginning (5′ end) and middle of the gene. Less often, Duchenne dystrophy is caused by a gene duplication or point mutation. Identification of a specific mutation allows for an unequivocal diagnosis, makes possible accurate testing of potential carriers, and is useful for prenatal diagnosis.

1	A diagnosis of Duchenne dystrophy can also be made by Western blot analysis of muscle biopsy specimens, revealing abnormalities on the quantity and molecular weight of dystrophin protein. In addition, immunocytochemical staining of muscle with dystrophin antibodies can be used to demonstrate absence or deficiency of dystrophin localizing to the sarcolemmal membrane. Carriers of the disease may demonstrate a mosaic pattern, but dystrophin analysis of muscle biopsy specimens for carrier detection is not reliable.

1	Pathogenesis Dystrophin is part of a large complex of sarcolemmal proteins and glycoproteins (Fig. 462e-6). Dystrophin binds to F-actin at its amino terminus and to β-dystroglycan at the carboxyl terminus. β-Dystroglycan complexes to α-dystroglycan, which binds to laminin in the extracellular matrix (ECM). Laminin has a heterotrimeric molecular structure arranged in the shape of a cross with one heavy chain and two light chains, β1 and γ1. The laminin heavy chain of skeletal muscle is designated laminin α2. Collagen proteins IV and VI are also found in the ECM. Like β-dystroglycan, the transmembrane sarcoglycan proteins also bind to dystrophin; these five proteins (designated αthrough ε-sarcoglycan) complex tightly with each other. More recently, other membrane proteins implicated in muscular dystrophy have been found to be loosely affiliated with constituents of the dystrophin complex. These include caveolin-3, α7 integrin, and collagen VI.

1	aUdd’s type distal myopathy is a form of titin deficiency with only distal muscle weakness (see Table 462e-9). Abbreviations: CK, creatine kinase; EMG, electromyography; NCS, nerve conduction studies; POMT1, protein-O-mannosyltransferase 1; POMT2, protein-O-mannosyltransferase 2; POMGNT1, O-linked mannose beta 1,2-N-acetylglucosaminyltransferase; TNPO3, transportin 3; TRAPC11, transport (trafficking) protein particle complex, subunit 11. in the cell membrane and Golgi complex.

1	in the cell membrane and Golgi complex. Dystrophin localizes to the cytoplasmic face of the muscle cell membrane. It complexes with two transmembrane protein complexes, the dystroglycans and the sarcoglycans. The dystroglycans bind to the extracellular matrix protein merosin, which is also complexed with β1 and α7 integrins (Tables 462e-5, 462e-6, and 462e-7). Dysferlin complexes with caveolin-3 (which binds to neuronal nitric oxide synthase, or nNOS) but not with the dystrophin-associated proteins or the integrins. In some of the congenital dystrophies and limb-girdle muscular dystrophies (LGMDs), there is loss of function of different enzymes that glycosylate α-dystroglycan, which thereby inhibits proper binding to merosin: POMT1, POMT2, POMGnT1, Fukutin, Fukutin-related protein, and LARGE.

1	The dystrophin-glycoprotein complex appears to confer stability to the sarcolemma, although the function of each individual component of the complex is incompletely understood. Deficiency of one member of the complex may cause abnormalities in other components. For example, a primary deficiency of dystrophin (Duchenne dystrophy) may lead to secondary loss of the sarcoglycans and dystroglycan. The primary loss of a single sarcoglycan (see “Limb-Girdle Muscular Dystrophy,” below) results in a secondary loss of other sarcoglycans in the membrane without uniformly affecting dystrophin. In either instance, disruption of the dystrophin-glycoprotein complexes weakens the sarcolemma, causing membrane tears and a cascade of events leading to muscle fiber necrosis. This sequence of events occurs repeatedly during the life of a patient with muscular dystrophy.

1	Glucocorticoids, administered as prednisone in a dose of 0.75 mg/kg per day, significantly slow progression of Duchenne dystrophy for up to 3 years. Some patients cannot tolerate glucocorticoid therapy; weight gain and increased risk of fractures in particular represent a significant deterrent for some boys. As in other recessively inherited dystrophies presumed to arise from loss of function of a critical muscle gene, there is optimism that Duchenne disease may benefit from novel therapies that either replace the defective gene or missing protein or implement downstream corrections (e.g., skipping mutated exons or reading through mutations that introduce stop codons). This less severe form of X-linked recessive muscular dystrophy results from allelic defects of the same gene responsible for Duchenne dystrophy. Becker muscular dystrophy is ~10 times less frequent than Duchenne.

1	Clinical Features The pattern of muscle wasting in Becker muscular dystrophy closely resembles that seen in Duchenne. Proximal muscles, especially of the lower extremities, are prominently involved. As the disease progresses, weakness becomes more generalized. Significant facial muscle weakness is not a feature. Hypertrophy of muscles, particularly in the calves, is an early and prominent finding. Most patients with Becker dystrophy first experience difficulties between ages 5 and 15 years, although onset in the third or fourth decade or even later can occur. By definition, patients with Becker dystrophy walk beyond age 15, whereas patients with Duchenne dystrophy are typically in a wheelchair by the age of 12. Patients with Becker dystrophy have a reduced life expectancy, but most survive into the fourth or fifth decade.

1	Mental retardation may occur in Becker dystrophy, but it is not as common as in Duchenne. Cardiac involvement occurs in Becker dystrophy and may result in heart failure; some patients manifest with only heart failure. Other less common presentations are asymptomatic hyper-CK-emia, myalgias without weakness, and myoglobinuria.

1	Laboratory Features Serum CK levels, results of EMG, and muscle biopsy findings closely resemble those in Duchenne dystrophy. The diagnosis of Becker muscular dystrophy requires Western blot analysis of muscle biopsy samples, demonstrating a reduced amount or abnormal size of dystrophin or mutation analysis of DNA from peripheral blood leukocytes. Genetic testing reveals deletions or duplications of the dystrophin gene in 65% of patients with Becker dystrophy, approximately the same percentage as in Duchenne dystrophy. In both Becker and Duchenne dystrophies, the size of the DNA deletion does not predict clinical severity; however, in ~95% of patients with Becker dystrophy, the DNA deletion does not alter the translational reading frame of messenger RNA. These “in-frame” mutations allow for production of some dystrophin, which accounts for the presence of altered rather than absent dystrophin on Western blot analysis.

1	The use of glucocorticoids has not been adequately studied in Becker dystrophy. The syndrome of LGMD represents more than one disorder. Both males and females are affected, with onset ranging from late in the first decade to the fourth decade. The LGMDs typically manifest with progressive weakness of pelvic and shoulder girdle musculature. Respiratory insufficiency from weakness of the diaphragm may occur, as may cardiomyopathy.

1	A systematic classification of LGMD is based on autosomal dominant (LGMD1) and autosomal recessive (LGMD2) inheritance. Superimposed on the backbone of LGMD1 and LGMD2, the classification uses a sequential alphabetical lettering system (LGMD1A, LGMD2A, etc.). Disorders receive letters in the order in which they are found to have chromosomal linkage. This results in an ever-expanding list of conditions summarized in Tables 462e-6 and 462e-7. None of the conditions is as common as the dystrophinopathies; however, prevalence data for the LGMDs have not been systematically gathered for any large heterogeneous population. In referral-based clinical populations, Fukutin-related protein (FKRP) deficiency (LGMD2I), calpainopathy (LGMD2A), anoctaminopathy (LGMD2L), and to a lesser extent dysferlinopathy (LGMD2B) have emerged as the most common disorders.

1	There are at least five genetically distinct forms of Emery-Dreifuss muscular dystrophy (EDMD). Emerin mutations are the most common cause of X-linked EDMD, although mutations in FHL1 may also be associated with a similar phenotype, which is X-linked as well. Mutations involving the gene for lamin A/C are the most common cause of autosomal dominant EDMD (also known as LGMD1B) and are also a common cause of hereditary cardiomyopathy. Less commonly, autosomal dominant EDMD has been reported with mutations in nesprin-1, nesprin-2, and TMEM43.

1	Clinical Features Prominent contractures can be recognized in early childhood and teenage years, often preceding muscle weakness. The contractures persist throughout the course of the disease and are present at the elbows, ankles, and neck. Muscle weakness affects humeral and peroneal muscles at first and later spreads to a limb-girdle distribution. The cardiomyopathy is potentially life threatening and may result in sudden death. A spectrum of atrial rhythm and conduction defects includes atrial fibrillation and paralysis and atrioventricular heart block. Some patients have a dilated cardiomyopathy. Female carriers of the X-linked variant may have cardiac manifestations that become clinically significant.

1	Laboratory Features Serum CK may be elevated twoto tenfold. EMG is myopathic. Muscle biopsy usually shows nonspecific dystrophic features, although cases associated with FHL1 mutations have features of myofibrillar myopathy. Immunohistochemistry reveals absent emerin staining of myonuclei in X-linked EDMD due to emerin mutations. ECGs demonstrate atrial and atrioventricular rhythm disturbances.

1	X-linked EDMD usually arises from defects in the emerin gene encoding a nuclear envelope protein. FHL1 mutations are also a cause of X-linked scapuloperoneal dystrophy, but can also present with an X-linked form of EDMD. The autosomal dominant disease can be caused by mutations in the LMNA gene encoding lamin A and C; in the synaptic nuclear envelope protein 1 (SYNE1) or 2 (SYNE2) encoding nesprin-1 and nesprin-2, respectively; and most recently in TMEM43 encoding LUMA. These proteins are essential components of the filamentous network underlying the inner nuclear membrane. Loss of structural integrity of the nuclear envelope from defects in emerin, lamin A/C, nesprin-1, nesprin-2, and LUMA accounts for overlapping phenotypes (Fig. 462e-7).

1	Supportive care should be offered for neuromuscular disability, including ambulatory aids, if necessary. Stretching of contractures is difficult. Management of cardiomyopathy and arrhythmias (e.g., early use of a defibrillator or cardiac pacemaker) may be life saving. This is not one entity but rather a group of disorders with varying degrees of muscle weakness, CNS impairment, and eye abnormalities. Clinical Features As a group, CMDs present at birth or in the first few months of life with hypotonia and proximal or generalized muscle weakness. Calf muscle hypertrophy is seen in some patients. Facial muscles may be weak, but other cranial nerve–innervated muscles are spared (e.g., extraocular muscles are normal). Most patients have joint contractures of varying degrees at elbows, hips, knees, and ankles. Contractures present at birth are referred to as arthrogryposis. Respiratory failure may be seen in some cases.

1	The CNS is affected in some forms of CMD. In merosin and FKRP deficiency, cerebral hypomyelination may be seen by magnetic resonance imaging (MRI), although only a small number of patients have mental retardation and seizures. Three forms of congenital muscular dystrophy have severe brain impairment. These include Fukuyama’s congenital muscular dystrophy (FCMD), muscle-eye-brain (MEB) disease, and Walker-Warburg syndrome (WWS). Patients are severely disabled in all three of these conditions. In MEB disease and WWS, but in the nuclear membrane and sarcomere. As shown in the exploded view, emerin and lamin A/C are constituents of the inner nuclear membrane. Several dystrophy-associated proteins are represented in the sarcomere including titin, nebulin, calpain, telethonin, actinin, and myotilin. The position of the dystrophin-dystroglycan complex is also illustrated.

1	not in FCMD, ocular abnormalities impair vision. WWS is the most severe congenital muscular dystrophy, causing death by 1 year of age. Laboratory Features Serum CK is markedly elevated in all of these conditions. The EMG is myopathic and muscle biopsies show nonspecific dystrophic features. Merosin, or laminin α2 chain (a basal lamina protein), is deficient in surrounding muscle fibers in merosin deficiency. Skin biopsies can also demonstrate defects in laminin α2 chain. In the other disorders (FKRP deficiency, FCMD, MEB disease, WWS), there is abnormal α-dystroglycan staining in muscle. In merosin deficiency, cerebral hypomyelination is common, and a host of brain malformations are seen in FCMD, MEB disease, and WWS.

1	All forms of CMD are inherited as autosomal recessive disorders. Chromosomal linkage and specific gene defects are presented in Table 462e-8. With the exception of merosin, the other gene defects affect posttranslational glycosylation of α-dystroglycan. This abnormality is thought to impair binding with merosin and leads to weakening of the dystrophin-glycoprotein complex, instability of the muscle membrane, and/or abnormalities in muscle contraction. CMDs with brain and eye phenotypes probably involve defective glycosylation of additional proteins, accounting for the more extensive phenotypes. There is no specific treatment for CMD. Proper wheelchair seating is important. Management of epilepsy and cardiac manifestations is necessary for some patients.

1	There is no specific treatment for CMD. Proper wheelchair seating is important. Management of epilepsy and cardiac manifestations is necessary for some patients. Myotonic dystrophy is also known as dystrophia myotonica (DM). The condition is composed of at least two clinical disorders with overlapping phenotypes and distinct molecular genetic defects: myotonic dystrophy type 1 (DM1), the classic disease originally described by Steinert, and myotonic dystrophy type 2 (DM2), also called proximal myotonic myopathy (PROMM). Merosin deficiency Onset at birth with hypotonia, joint contractures, delayed milestones, generalized muscle weakness Cerebral hypomyelination, less often cortical dysplasia Normal intelligence usually, some with MR (~6%) and seizures (~8%) Partial deficiency leads to milder phenotype (LGMD picture) Weakness of proximal muscles, especially shoulder girdles Hypertrophy of leg muscles Joint contractures Cognition normal

1	Partial deficiency leads to milder phenotype (LGMD picture) Weakness of proximal muscles, especially shoulder girdles Hypertrophy of leg muscles Joint contractures Cognition normal Hypotonia, joint contractures Generalized muscle weakness Hypertrophy of calf muscles Seizures, MR Cardiomyopathy Muscle-eye-brain disease Onset at birth, hypotonia Eye abnormalities include: progressive myopia, cataracts, and optic nerve, glaucoma, retinal pigmentary changes Progressive muscle weakness Joint contractures Seizures, MR Walker-Warburg syndromeb Onset at birth, hypotonia Generalized muscle weakness Joint contractures Microphthalmos, retinal dysplasia, buphthalmos, glaucoma, cataracts Seizures, MR Serum CK 5–35× normal EMG myopathic NCS abnormal in some cases MRI shows hydrocephalus, cobblestone lissencephaly, corpus callosum and cerebellar hypoplasia, cerebral hypomyelination

1	MRI shows hydrocephalus, cobblestone lissencephaly, corpus callosum and cerebellar hypoplasia, cerebral hypomyelination MRI shows cobblestone lissencephaly, hydrocephalus, encephalocele, absent corpus callosum aAll are inherited as recessive traits. bThere is phenotypic overlap between disorders related to defective glycosylation. In muscle, this is a consequence of altered glycosylation of dystroglycans; in brain/eye, other glycosylated proteins are involved. Clinically, Walker-Warburg syndrome is more severe, with death by 1 year. Abbreviations: CK, creatine kinase; EMG, electromyography; LGMD, limb-girdle muscular dystrophy; MR, mental retardation; MRI, magnetic resonance imaging; NCS, nerve conduction studies.

1	Clinical Features The clinical expression of DM1 varies widely and involves many systems other than muscle. Affected patients have a typical “hatchet-faced” appearance due to temporalis, masseter, and facial muscle atrophy and weakness. Frontal baldness is also characteristic of the disease. Neck muscles, including flexors and sternocleidomastoids, and distal limb muscles are involved early. Weakness of wrist extensors, finger extensors, and intrinsic hand muscles impairs function. Ankle dorsiflexor weakness may cause footdrop. Proximal muscles remain stronger throughout the course, although preferential atrophy and weakness of quadriceps muscles occur in many patients. Palatal, pharyngeal, and tongue involvement produce a dysarthric speech, nasal voice, and swallowing problems. Some patients have diaphragm and intercostal muscle weakness, resulting in respiratory insufficiency.

1	Myotonia, which usually appears by age 5 years, is demonstrable by percussion of the thenar eminence, the tongue, and wrist extensor muscles. Myotonia causes a slow relaxation of hand grip after a forced voluntary closure. Advanced muscle wasting makes myotonia more difficult to detect. Cardiac disturbances occur commonly in patients with DM1. ECG abnormalities include first-degree heart block and more extensive conduction system involvement. Complete heart block and sudden death can occur. Congestive heart failure occurs infrequently but may result from cor pulmonale secondary to respiratory failure. Mitral valve prolapse also occurs commonly. Other associated features include intellectual impairment, hypersomnia, posterior subcapsular cataracts, gonadal atrophy, insulin resistance, and decreased esophageal and colonic motility.

1	Congenital myotonic dystrophy is a more severe form of DM1 and occurs in ~25% of infants of affected mothers. It is characterized by severe facial and bulbar weakness, transient neonatal respiratory insufficiency, and mental retardation. DM2, or PROMM, has a distinct pattern of muscle weakness affecting mainly proximal muscles. Other features of the disease overlap with DM1, including cataracts, testicular atrophy, insulin resistance, constipation, hypersomnia, and cognitive defects. Cardiac conduction defects occur but are less common, and the hatchet face and frontal baldness are less consistent features. A very striking difference is the failure to clearly identify a congenital form of DM2.

1	Laboratory Features The diagnosis of myotonic dystrophy can usually be made on the basis of clinical findings. Serum CK levels may be normal or mildly elevated. EMG evidence of myotonia is present in most cases of DM1 but may be more patchy in DM2. Muscle biopsy shows muscle atrophy, which selectively involves type 1 fibers in 50% of cases, and ringed fibers in DM1 but not in DM2. Typically, numerous internalized nuclei can be seen in individual muscle fibers as well as atrophic fibers with pyknotic nuclear clumps in both DM1 and DM2. Necrosis of muscle fibers and increased connective tissue, common in other muscular dystrophies, are less apparent in myotonic dystrophy.

1	DM1 and DM2 are both autosomal dominant disorders. New mutations do not appear to contribute to the pool of affected individuals. DM1 is transmitted by an intronic mutation consisting of an unstable expansion of a CTG trinucleotide repeat in a serine-threonine protein kinase gene (named DMPK) on chromosome 19q13.3. An increase in the severity of the disease phenotype in successive generations (genetic anticipation) is accompanied by an increase in the number of trinucleotide repeats. A similar type of mutation has been identified in fragile X syndrome (Chap. 451e). The unstable triplet repeat in myotonic dystrophy can be used for prenatal diagnosis. Congenital disease occurs almost exclusively in infants born to affected mothers; it is possible that sperm with greatly expanded triplet repeats do not function well.

1	DM2 is caused by a DNA expansion mutation consisting of a CCTG repeat in intron 1 of the ZNF9 gene located at chromosome 3q13.3q24. The gene is believed to encode an RNA-binding protein expressed in many different tissues, including skeletal and cardiac muscle. The DNA expansions in DM1 and DM2 almost certainly impair muscle function by a toxic gain of function of the mutant mRNA. In both DM1 and DM2, the mutant RNA appears to form intranuclear inclusions composed of aberrant RNA. These RNA inclusions sequester RNA-binding proteins essential for proper splicing of a variety of other mRNAs. This leads to abnormal transcription of multiple proteins in a variety of tissues/organ systems, in turn causing the systemic manifestations of DM1 and DM2.

1	The myotonia in DM1 rarely warrants treatment, although some patients with DM2 are significantly bothered by the discomfort related to the associated muscle stiffness. Phenytoin and mexiletine are the preferred agents for the occasional patient who requires an antimyotonia drug; other agents, particularly quinine and procainamide, may worsen cardiac conduction. A cardiac pacemaker should be considered for patients with unexplained syncope, advanced conduction system abnormalities with evidence of second-degree heart block, or trifascicular conduction disturbances with marked prolongation of the PR interval. Molded ankle-foot orthoses help stabilize gait in patients with foot drop. Excessive daytime somnolence with or without sleep apnea is not uncommon. Sleep studies, noninvasive respiratory support (biphasic positive airway pressure [BiPAP]), and treatment with modafinil may be beneficial.

1	This form of muscular dystrophy has a prevalence of ~1 in 20,000. There are two forms of FSHD that have similar pathogenesis, as will be discussed. Most patients have FSHD type 1 (95%), whereas approximately 5% have FSHD2. FSHD1 and FSHD2 are clinically and histopathologically identical. FSHD is not to be confused with the genetically distinct scapuloperoneal dystrophies.

1	Clinical Features The condition typically has an onset in childhood or young adulthood. In most cases, facial weakness is the initial manifestation, appearing as an inability to smile, whistle, or fully close the eyes. Weakness of the shoulder girdles, rather than the facial muscles, usually brings the patient to medical attention. Loss of scapular stabilizer muscles makes arm elevation difficult. Scapular winging (Fig. 462e-3) becomes apparent with attempts at abduction and forward movement of the arms. Biceps and triceps muscles may be severely affected, with relative sparing of the deltoid muscles. Weakness is invariably worse for wrist extension than for wrist flexion, and weakness of the anterior compartment muscles of the legs may lead to footdrop.

1	In most patients, the weakness remains restricted to facial, upper extremity, and distal lower extremity muscles. In 20% of patients, weakness progresses to involve the pelvic girdle muscles, and severe functional impairment and possible wheelchair dependency result. Characteristically, patients with FSHD do not have involvement of other organ systems, although labile hypertension is common, and there is an increased incidence of nerve deafness. Coats’ disease, a disorder consisting of telangiectasia, exudation, and retinal detachment, also occurs. Laboratory Features The serum CK level may be normal or mildly elevated. EMG usually indicates a myopathic pattern. The muscle biopsy shows nonspecific features of a myopathy. A prominent inflammatory 462e-11 infiltrate, which is often multifocal in distribution, is present in some biopsy samples. The cause or significance of this finding is unknown.

1	An autosomal dominant inheritance pattern with almost complete penetrance has been established, but each family member should be examined for the presence of the disease, since ~30% of those affected are unaware of involvement. FSHD1 is associated with deletions of tandem 3.3-kb repeats at 4q35. The deletion reduces the number of repeats to a fragment of <35 kb in most patients. Within these repeats lies the DUX4 gene, which usually is not expressed. In patients with FSHD1 these deletions in the setting of a specific polymorphism leads to hypomethylation of the region and toxic expression of the DUX4 gene. In patients with FSHD2, there is no deletion, but a mutation in SMCHD1. Interestingly, in the setting of the same polymorphism, there again is seen hypomethylation of the region and the permissive expression of the DUX4 gene. In both FSHD1 and FSHD2, there is overexpression of the DUX4 transcript.

1	No specific treatment is available; ankle-foot orthoses are helpful for footdrop. Scapular stabilization procedures improve scapular winging but may not improve function. This form of muscular dystrophy represents one of several disorders characterized by progressive external ophthalmoplegia, which consists of slowly progressive ptosis and limitation of eye movements with sparing of pupillary reactions for light and accommodation. Patients usually do not complain of diplopia, in contrast to patients having conditions with a more acute onset of ocular muscle weakness (e.g., myasthenia gravis).

1	Clinical Features Oculopharyngeal muscular dystrophy has a late onset; it usually presents in the fourth to sixth decade with ptosis and/ or dysphagia. The extraocular muscle impairment is less prominent in the early phase but may be severe later. The swallowing problem may become debilitating and result in pooling of secretions and repeated episodes of aspiration. Mild weakness of the neck and extremities also occurs. Laboratory Features The serum CK level may be two to three times normal. Myopathic EMG findings are typical. On biopsy, muscle fibers are found to contain rimmed vacuoles, which by electron microscopy are shown to contain membranous whorls, accumulation of glycogen, and other nonspecific debris related to lysosomes. A distinct feature of oculopharyngeal dystrophy is the presence of tubular filaments, 8.5 nm in diameter, in muscle cell nuclei.

1	Oculopharyngeal dystrophy has an autosomal dominant inheritance pattern with complete penetrance. The incidence is high in French-Canadians and in Spanish-American families of the southwestern United States. Large kindreds of Italian and of eastern European Jewish descent have been reported. The molecular defect in oculopharyngeal muscular dystrophy is a subtle expansion of a modest polyalanine repeat tract in a poly-RNA-binding protein (PABP2) in muscle. Dysphagia can lead to significant undernourishment and inanition, making oculopharyngeal muscular dystrophy a potentially life-threatening disease. Cricopharyngeal myotomy may improve swallowing, although it does not prevent aspiration. Eyelid crutches can improve vision when ptosis obstructs vision; candidates for ptosis surgery must be carefully selected—those with severe facial weakness are not suitable.

1	A group of muscle diseases, the distal myopathies, are notable for their preferential distal distribution of muscle weakness in contrast to most muscle conditions associated with proximal weakness. The major distal myopathies are summarized in Table 462e-9.

1	Clinical Features Welander’s, Udd’s, and Markesbery-Griggs type distal myopathies are all late-onset, dominantly inherited disorders of distal limb muscles, usually beginning after age 40 years. Welander’s distal myopathy preferentially involves the wrist and finger extensors, whereas the others are associated with anterior tibial weakness leading to progressive footdrop. Laing’s distal myopathy is also a dominantly inherited disorder heralded by tibial weakness; however, it is distinguished by onset in childhood or early adult life. Nonaka’s distal myopathy and Miyoshi’s myopathy are distinguished by autosomal recessive inheritance and onset in the late teens or twenties. Nonaka’s and Williams’ myopathy entails anterior tibial weakness, whereas Miyoshi’s myopathy is unique in that gastrocnemius muscles are preferentially affected at onset. Finally, the myofibrillar myopathies (MFMs) are a clinically and genetically heterogeneous group of disorders that can be associated with

1	gastrocnemius muscles are preferentially affected at onset. Finally, the myofibrillar myopathies (MFMs) are a clinically and genetically heterogeneous group of disorders that can be associated with prominent distal weakness; they can be inherited in an autosomal dominant or recessive pattern. Of note, Markesbery-Griggs myopathy (caused by mutations in ZASP) and LGMD1B (caused by mutations in myotilin) are in fact subtypes of myofibrillar myopathy.

1	Laboratory Features Serum CK level is particularly helpful in diagnosing Miyoshi’s myopathy because it is very elevated. In the other conditions, serum CK is only slightly increased. EMGs are myopathic. In the MFMs, myotonic or pseudomyotonic discharges are common. Muscle biopsy shows nonspecific dystrophic features and, with the exception of Laing’s and Miyoshi’s myopathies, often shows rimmed vacuoles. MFM is associated with the accumulation of dense inclusions, as well as amorphous material best seen on Gomori trichrome and myofibrillar disruption on electron microscopy. Immune staining sometimes demonstrates accumulation of desmin and other proteins in MFM, large deposits of myosin heavy chain in the subsarcolemmal region of type 1 muscle fibers in Laing’s myopathy, and reduced or absent dysferlin in Miyoshi’s myopathy. The affected genes and their gene products are listed in Table 462e-9.

1	The affected genes and their gene products are listed in Table 462e-9. Occupational therapy is offered for loss of hand function; ankle-foot orthoses can support distal lower limb muscles. The MFMs can be associated with cardiomyopathy (congestive heart failure or arrhythmias) and respiratory failure that may require medical management. Laing’s-type distal myopathy can also be associated with a cardiomyopathy. These rare disorders are distinguished from muscular dystrophies by the presence of specific histochemical and structural abnormalities in muscle. Although primarily disorders of infancy or childhood, three forms that may present in adulthood are described here: central core disease, nemaline (rod) myopathy, and centronuclear (myotubular) myopathy. Sarcotubular myopathy is caused by mutations in TRIM-32 and is identical to LGMD2H. Other types, such as minicore myopathy (multi-minicore disease), fingerprint body myopathy, and cap myopathy, are not discussed.

1	Patients with central core disease may have decreased fetal movements and breech presentation. Hypotonia and delay in motor milestones, particularly in walking, are common. Later in childhood, patients develop problems with stair climbing, running, and getting up from the floor. On examination, there is mild facial, neck-flexor, and proximal-extremity muscle weakness. Legs are more affected than arms. Skeletal abnormalities include congenital hip dislocation, scoliosis, and pes cavus; clubbed feet also occur. Most cases are nonprogressive, but exceptions are well documented. Susceptibility to malignant hyperthermia must be considered as a potential risk factor for patients with central core disease. Recent series have demonstrated that many cases of late-onset axial myopathy in which patients manifest with bent spine (camptocormia) or neck extensor weakness (neck extensor myopathy) are caused by mutations in the ryanodine receptor gene (RYR1). This illustrates the interesting spectrum

1	manifest with bent spine (camptocormia) or neck extensor weakness (neck extensor myopathy) are caused by mutations in the ryanodine receptor gene (RYR1). This illustrates the interesting spectrum of RYR1 mutations.

1	The serum CK level is usually normal. Needle EMG demonstrates a myopathic pattern. Muscle biopsy shows fibers with single or multiple central or eccentric discrete zones (cores) devoid of oxidative enzymes. Cores occur preferentially in type 1 fibers and represent poorly aligned sarcomeres associated with Z disk streaming. Autosomal dominant inheritance is characteristic; sporadic cases also occur. As alluded above, this myopathy is caused by point mutations of RYR1, encoding the calcium-release channel of the sarcoplasmic reticulum of skeletal muscle; mutations of this gene also account for some cases of inherited malignant hyperthermia (Chap. 23). Malignant hyperthermia is an allelic condition; C-terminal mutations of the RYR1 gene predispose to this complication. Specific treatment is not required, but establishing a diagnosis of central core disease is extremely important because these patients have a known predisposition to malignant hyperthermia during anesthesia.

1	The term nemaline refers to the distinctive presence in muscle fibers of rods or threadlike structures (Greek nema, “thread”). Nemaline myopathy is clinically heterogeneous. A severe neonatal form presents with hypotonia and feeding and respiratory difficulties, leading to early death. Nemaline myopathy usually presents in infancy or childhood with delayed motor milestones. The course is nonprogressive or slowly progressive. The physical appearance is striking because of the long, narrow facies, high-arched palate, and open-mouthed appearance due to a prognathous jaw. Other skeletal abnormalities include pectus excavatum, kyphoscoliosis, pes cavus, and clubfoot deformities. Facial and generalized muscle weakness, including respiratory muscle weakness, is common. An adult-onset disorder with progressive proximal or distal weakness may be seen. Myocardial involvement is occasionally present in both the childhood and adult-onset forms. The serum CK level is usually normal or slightly

1	with progressive proximal or distal weakness may be seen. Myocardial involvement is occasionally present in both the childhood and adult-onset forms. The serum CK level is usually normal or slightly elevated. The EMG demonstrates a myopathic pattern. Muscle biopsy shows clusters of small rods (nemaline bodies), which occur preferentially, but not exclusively, in the sarcoplasm of type 1 muscle fibers. Occasionally, the rods are also apparent in myonuclei. The muscle often shows type 1 muscle fiber predominance. Rods originate from the Z disk material of the muscle fiber.

1	Six genes have been associated with nemaline myopathy. Five of these code for thin filament–associated proteins, suggesting disturbed assembly or interplay of these structures as a pivotal mechanism. Mutations of the nebulin (NEB) gene account for most cases, including both severe neonatal and early childhood forms, inherited as autosomal recessive disorders. Neonatal and childhood cases, inherited as predominantly autosomal dominant disorders, are caused by mutations of the skeletal muscle a-actinin (ACTA1) gene. In milder forms of the disease with autosomal dominant inheritance, mutations have been identified in both the slow a-tropomyosin (TPM3) and β;-tropomyosin (TPM2) genes accounting for <3% of cases. Muscle troponin T (TNNT1) gene mutations appear to be limited to the Amish population in North America. Mutations may also be seen in NEM6 that encodes a putative BTB/Kelch protein. No specific treatment is available.

1	Three distinct variants of centronuclear myopathy occur. A neonatal form, also known as myotubular myopathy, presents with severe hypotonia and weakness at birth. The late infancy–early childhood form presents with delayed motor milestones. Later, difficulty with running and stair climbing becomes apparent. A marfanoid, slender body habitus, long narrow face, and high-arched palate are typical. Scoliosis and Myofibrillar myopathies Onset in fifth decade Weakness begins in hands Slow progression with spread to distal lower extremities Lifespan normal Onset fourth to eighth decade Distal lower extremity weakness (tibial Onset fourth to eighth decade Distal lower extremity weakness (tibial distribution) with progression to distal arms and proximal muscles Onset childhood to third decade Onset: second to third decade Progression to other muscles sparing quadriceps Ambulation may be lost in 10–15 y Onset: second to third decade Lower extremity weakness in posterior

1	Onset: second to third decade Progression to other muscles sparing quadriceps Ambulation may be lost in 10–15 y Onset: second to third decade Lower extremity weakness in posterior Progression leads to weakness in other muscle groups Ambulation lost after 10–15 y in about one-third of cases Distal lower extremity weakness (anterior tibial distribution) Onset from early childhood to late adult life Weakness may be distal, proximal, or generalized Cardiomyopathy and respiratory involvement is not uncommon features and rimmed vacuoles Titin absent in M-line of muscle Serum CK is usually mildly elevated EMG reveals irritative myopathy Muscle biopsies demonstrate rimmed vacuoles and features of MFM Serum CK is normal or slightly elevated Muscle biopsies do not typically show rimmed vacuoles, but may show hyaline bodies with accumulation of myosin Large deposits of myosin heavy chain are seen in type 1 muscle fibers Serum CK 3–10× normal EMG myopathic

1	Large deposits of myosin heavy chain are seen in type 1 muscle fibers Serum CK 3–10× normal EMG myopathic Muscle biopsy shows nonspecific dystrophic features often with prominent inflammatory cell infiltration; no rimmed vacuoles Muscle biopsy may show rimmed vacuoles and features of MFM EMG is myopathic and often associated with myotonic discharges Muscle biopsy demonstrates abnormal accumulation of desmin and other proteins, rimmed vacuoles, and myofibrillar degeneration AD Titin AR (associated with more proximal GNE gene: UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase Allelic to hereditary inclusion body myopathy AR Allelic to LGMD2B (see Table 462e-7) Dysferlin Myotilin (also known as LGMD 1A) aMiyoshi’s myopathy phenotype may also be seen with mutations in ANO-5 that encodes for anoctamin 5 (allelic to LGMD2L).

1	Myotilin (also known as LGMD 1A) aMiyoshi’s myopathy phenotype may also be seen with mutations in ANO-5 that encodes for anoctamin 5 (allelic to LGMD2L). Abbreviations: AD, autosomal dominant; AR, autosomal recessive; CK, creatine kinase; EMG, electromyography; MFM, myofibrillar myopathy; NCS, nerve conduction studies. clubbed feet may be present. Most patients exhibit progressive weakness, some requiring wheelchairs. Progressive external ophthalmoplegia with ptosis and varying degrees of extraocular muscle impairment are characteristic of both the neonatal and the late-infantile forms. A third variant, the late childhood–adult form, has an onset in the second or third decade. Patients have full extraocular muscle movements and rarely exhibit ptosis. There is mild, slowly progressive limb weakness that may be distally predominant (some of these patients have been classified as having Charcot-Marie-Tooth disease type 2 [CMT2; Chap. 459]).

1	Normal or slightly elevated CK levels occur in each of the forms. Nerve conduction studies may reveal reduced amplitudes of distal compound muscle action potentials, in particular in adult-onset cases that resemble CMT2. EMG studies often give distinctive results, showing positive sharp waves and fibrillation potentials, complex and repetitive discharges, and rarely myotonic discharges. Muscle biopsy specimens in longitudinal section demonstrate rows of central nuclei, often surrounded by a halo. In transverse sections, central nuclei are found in 25–80% of muscle fibers.

1	A gene for the neonatal form of centronuclear myopathy has been localized to Xq28; this gene encodes myotubularin, a protein tyrosine phosphatase. Missense, frameshift, and splice-site mutations predict loss of myotubularin function in affected individuals. Carrier identification and prenatal diagnosis are possible. Autosomal recessive forms are caused by mutations in BIB1 that encodes for amphyphysin-2, whereas some autosomal dominant cases, which are allelic to a form of CMT2, are associated with mutations in the gene that encodes dynamin-2. No specific medical treatments are available at this time. There are two principal sources of energy for skeletal muscle—fatty acids and glucose. Abnormalities in either glucose or lipid utilization can be associated with distinct clinical presentations that can range from an acute, painful syndrome with rhabdomyolysis and myoglobinuria to a chronic, progressive muscle weakness simulating muscular dystrophy.

1	gLYCOgEN STORAgE AND gLYCOLYTIC DEFECTS Disorders of glycogen Storage Causing Progressive Weakness • α-Glucosidase, or acid maltase, deficiency (PomPe’s disease) Three clinical forms of α-glucosidase, or acid maltase, deficiency (type II glycogenosis) can be distinguished. The infantile form is the most common, with onset of symptoms in the first 3 months of life. Infants develop severe muscle weakness, cardiomegaly, hepatomegaly, and respiratory insufficiency. Glycogen accumulation in motor neurons of the spinal cord and brainstem contributes to muscle weakness. Death usually occurs by 1 year of age. In the childhood form, the picture resembles muscular dystrophy. Delayed motor milestones result from proximal limb muscle weakness and involvement of respiratory muscles. The heart may be involved, but the liver and brain are unaffected. The adult form usually begins in the third or fourth decade but can present as late as the seventh decade. Respiratory failure and diaphragmatic

1	be involved, but the liver and brain are unaffected. The adult form usually begins in the third or fourth decade but can present as late as the seventh decade. Respiratory failure and diaphragmatic weakness are often initial manifestations, heralding progressive proximal muscle weakness. The heart and liver are not involved.

1	The serum CK level is 2–10 times normal in infantile or childhood-onset Pompe’s disease but can be normal in adult-onset cases. EMG examination demonstrates a myopathic pattern, but other features are especially distinctive, including myotonic discharges, trains of fibrillation and positive waves, and complex repetitive discharges. EMG discharges are very prominent in the paraspinal muscles. The muscle biopsy in infants typically reveals vacuoles containing glycogen and the lysosomal enzyme acid phosphatase. Electron microscopy reveals membrane-bound and free tissue glycogen. However, muscle biopsies in late-onset Pompe’s disease may demonstrate only nonspecific abnormalities. Enzyme analysis of dried blood spots is a sensitive technique to screen for Pompe’s disease. A definitive diagnosis is established by enzyme assay in muscle or cultured fibroblasts or by genetic testing.

1	Pompe’s disease is inherited as an autosomal recessive disorder caused by mutations of the α-glucosidase gene. Enzyme replacement therapy (ERT) with IV recombinant human α-glucosidase has been shown to be beneficial in infantile-onset Pompe’s disease. Clinical benefits in the infantile disease include reduced heart size, improved muscle function, reduced need for ventilatory support, and longer life. In late-onset cases, ERT has not been associated with the dramatic response that can be seen in classic infantile Pompe’s disease, yet it appears to stabilize the disease process.

1	other GlycoGen storaGe diseases with ProGressive weakness In debranching enzyme deficiency (type III glycogenosis), a slowly progressive form of muscle weakness can develop after puberty. Rarely, myoglobinuria may be seen. Patients are usually diagnosed in infancy, however, because of hypotonia and delayed motor milestones, hepatomegaly, growth retardation, and hypoglycemia. Branching enzyme deficiency (type IV glycogenosis) is a rare and fatal glycogen storage disease characterized by failure to thrive and hepatomegaly. Hypotonia and muscle wasting may be present, but the skeletal muscle manifestations are minor compared to liver failure.

1	Disorders of glycolysis Causing Exercise Intolerance Several glycolytic defects are associated with recurrent myoglobinuria: myophosphorylase deficiency (type V glycogenosis), phosphofructokinase deficiency (type VII glycogenosis), phosphoglycerate kinase deficiency, phosphorylase kinase deficiency (type IX glycogenosis), phosphoglycerate mutase deficiency (type X glycogenosis), lactate dehydrogenase deficiency (glycogenosis type XI), and β-enolase deficiency. Myophosphorylase deficiency, also known as McArdle’s disease, is by far the most common of the glycolytic defects associated with exercise intolerance. These glycolytic defects result in a common failure to support energy production at the initiation of exercise, although the exact site of energy failure remains controversial.

1	Clinical muscle manifestations in these conditions usually begin in adolescence. Symptoms are precipitated by brief bursts of high-intensity exercise such as running or lifting heavy objects. A history of myalgia and muscle stiffness usually precedes the intensely painful muscle contractures, which may be followed by myoglobinuria. Acute renal failure accompanies significant pigmenturia. Certain features help distinguish some enzyme defects. In McArdle’s disease, exercise tolerance can be enhanced by a slow induction phase (warm-up) or brief periods of rest, allowing for the start of the “second-wind” phenomenon (switching to utilization of fatty acids). Varying degrees of hemolytic anemia accompany deficiencies of both phosphofructokinase (mild) and phosphoglycerate kinase (severe). In phosphoglycerate kinase deficiency, the usual clinical presentation is a seizure disorder associated with mental retardation; exercise intolerance is an infrequent manifestation.

1	In all of these conditions, the serum CK levels fluctuate widely and may be elevated even during symptom-free periods. CK levels >100 times normal are expected, accompanying myoglobinuria. All patients with suspected glycolytic defects leading to exercise intolerance should undergo a forearm exercise test. An impaired rise in venous lactate is highly indicative of a glycolytic defect. In lactate dehydrogenase deficiency, venous levels of lactate do not increase, but pyruvate rises to normal. A definitive diagnosis of glycolytic disease is made by muscle biopsy and subsequent enzyme analysis or by genetic testing. Myophosphorylase deficiency, phosphofructokinase deficiency, and phosphoglycerate mutase deficiency are inherited as autosomal recessive disorders. Phosphoglycerate kinase deficiency is X-linked recessive. Mutations can be found in the respective genes encoding the abnormal proteins in each of these disorders.

1	Training may enhance exercise tolerance, perhaps by increasing perfusion to muscle. Dietary intake of free glucose or fructose prior to activity may improve function but care must be taken to avoid obesity from ingesting too many calories.

1	Lipid is an important muscle energy source during rest and during prolonged, submaximal exercise. Fatty acids are derived from circulating very-low-density lipoprotein (VLDL) in the blood or from triglycerides stored in muscle fibers. Oxidation of fatty acids occurs in the mitochondria. To enter the mitochondria, a fatty acid must first be converted to an “activated fatty acid,” acyl-CoA. The acyl-CoA must be linked with carnitine by the enzyme carnitine palmitoyltransferase (CPT) I for transport into the mitochondria. CPT I is present on the inner side of the outer mitochondrial membrane. Carnitine is removed by CPT II, an enzyme attached to the inside of the inner mitochondrial membrane, allowing transport of acyl-CoA into the mitochondrial matrix for β-oxidation.

1	Carnitine Palmitoyltransferase Deficiency CPT II deficiency is the most common recognizable cause of recurrent myoglobinuria, more common than the glycolytic defects. Onset is usually in the teenage years or early twenties. Muscle pain and myoglobinuria typically occur after prolonged exercise but can also be precipitated by fasting or infections; up to 20% of patients do not exhibit myoglobinuria, however. Strength is normal between attacks. In contrast to disorders caused by defects in glycolysis, in which muscle cramps follow short, intense bursts of exercise, the muscle pain in CPT II deficiency does not occur until the limits of utilization have been exceeded and muscle breakdown has already begun. Episodes of rhabdomyolysis may produce severe weakness. In young children and newborns, CPT II deficiency can present with a very severe clinical picture including hypoketotic hypoglycemia, cardiomyopathy, liver failure, and sudden death.

1	Serum CK levels and EMG findings are both usually normal between episodes. A normal rise of venous lactate during forearm exercise distinguishes this condition from glycolytic defects, especially myophosphorylase deficiency. Muscle biopsy does not show lipid accumulation and is usually normal between attacks. The diagnosis requires direct measurement of muscle CPT or genetic testing. CPT II deficiency is much more common in men than women (5:1); nevertheless, all evidence indicates autosomal recessive inheritance. A mutation in the gene for CPT II (chromosome 1p36) causes the disease in some individuals. Attempts to improve exercise tolerance with frequent meals and a low-fat, high-carbohydrate diet, or by substituting medium-chain triglycerides in the diet, have not proven to be beneficial.

1	Myoadenylate Deaminase Deficiency The muscle enzyme myoadenylate deaminase converts adenosine-5′-monophosphate (5′-AMP) to inosine monophosphate (IMP) with liberation of ammonia. Myoadenylate deaminase may play a role in regulating adenosine triphosphate (ATP) levels in muscles. Most individuals with myoadenylate deaminase deficiency have no symptoms. There have been a few reports of patients with this disorder who have exercise-exacerbated myalgia and myoglobinuria. Many questions have been raised about the clinical effects of myoadenylate deaminase deficiency, and, specifically, its relationship to exertional myalgia and fatigability, but there is no consensus.

1	In 1972, Olson and colleagues recognized that muscle fibers with significant numbers of abnormal mitochondria could be highlighted with the modified trichrome stain; the term ragged red fibers was coined. By electron microscopy, the mitochondria in ragged red fibers are enlarged and often bizarrely shaped and have crystalline inclusions. Since that seminal observation, the understanding of these disorders of muscle and other tissues has expanded (Chap. 82). Mitochondria play a key role in energy production. Oxidation of the major nutrients derived from carbohydrate, fat, and protein leads to the generation of reducing equivalents. The latter are transported through the respiratory chain in the process known as oxidative phosphorylation. The energy generated by the oxidation-reduction reactions of the respiratory chain is stored in an electrochemical gradient coupled to ATP synthesis.

1	A novel feature of mitochondria is their genetic composition. Each mitochondrion possesses a DNA genome that is distinct from that of the nuclear DNA. Human mitochondrial DNA (mtDNA) consists of a double-strand, circular molecule comprising 16,569 base pairs. It codes for 22 transfer RNAs, 2 ribosomal RNAs, and 13 polypeptides 462e-15 of the respiratory chain enzymes. The genetics of mitochondrial diseases differ from the genetics of chromosomal disorders. The DNA of mitochondria is directly inherited from the cytoplasm of the gametes, mainly from the oocyte. The sperm contributes very little of its mitochondria to the offspring at the time of fertilization. Thus, mitochondrial genes are derived almost exclusively from the mother, accounting for maternal inheritance of some mitochondrial disorders.

1	Patients with mitochondrial myopathies have clinical manifestations that usually fall into three groups: chronic progressive external ophthalmoplegia (CPEO), skeletal muscle–CNS syndromes, and pure myopathy simulating muscular dystrophy or metabolic myopathy. The single most common sign of a mitochondrial myopathy is CPEO, occurring in >50% of all mitochondrial myopathies. Varying degrees of ptosis and weakness of extraocular muscles are seen, usually in the absence of diplopia, a point of distinction from disorders with fluctuating eye weakness (e.g., myasthenia gravis).

1	KSS is a widespread multiorgan system disorder with a defined triad of clinical findings: onset before age 20, CPEO, and pigmentary retinopathy, plus one or more of the following features: complete heart block, cerebrospinal fluid (CSF) protein >1 g/L (100 mg/dL), or cerebellar ataxia. Some patients with CPEO and ragged red fibers may not fulfill all of the criteria for KSS. The cardiac disease includes syncopal attacks and cardiac arrest related to the abnormalities in the cardiac conduction system: prolonged intraventricular conduction time, bundle branch block, and complete atrioventricular block. Death attributed to heart block occurs in ~20% of the patients. Varying degrees of progressive limb muscle weakness and easy fatigability affect activities of daily living. Endocrine abnormalities are common, including gonadal dysfunction in both sexes with delayed puberty, short stature, and infertility. Diabetes mellitus is a cardinal sign of mitochondrial disorders and is estimated to

1	are common, including gonadal dysfunction in both sexes with delayed puberty, short stature, and infertility. Diabetes mellitus is a cardinal sign of mitochondrial disorders and is estimated to occur in 13% of KSS patients. Other less common endocrine disorders include thyroid disease, hyperaldosteronism, Addison’s disease, and hypoparathyroidism. Both mental retardation and dementia are common accompaniments to this disorder. Serum CK levels are normal or slightly elevated. Serum lactate and pyruvate levels may be elevated. EMG is myopathic. Nerve conduction studies may be abnormal related to an associated neuropathy. Muscle biopsies reveal ragged red fibers, highlighted in oxidative enzyme stains, many showing defects in cytochrome oxidase. By electron microscopy, there are increased numbers of mitochondria that often appear enlarged and contain paracrystalline inclusions.

1	KSS is a sporadic disorder. The disease is caused by single mtDNA deletions presumed to arise spontaneously in the ovum or zygote. The most common deletion, occurring in about one-third of patients, removes 4977 bp of contiguous mtDNA. Monitoring for cardiac conduction defects is critical. Prophylactic pacemaker implantation is indicated when ECGs demonstrate a bifascicular block. In KSS, no benefit has been shown for supplementary therapies, including multivitamins or coenzyme Q10. Of all the proposed options, exercise might be the most applicable but must be approached cautiously because of defects in the cardiac conduction system.

1	This condition is caused by nuclear DNA mutations affecting mtDNA copy number and integrity and is thus inherited in a Mendelian fashion. Onset is usually after puberty. Fatigue, exercise intolerance, and complaints of muscle weakness are typical. Some patients notice swallowing problems. The neurologic examination confirms the ptosis and ophthalmoplegia, usually asymmetric in distribution. A sensorineural hearing loss may be encountered. Mild facial, neck flexor, and proximal weakness are typical. Rarely, respiratory muscles may be progressively affected and may be the direct cause of death. Serum CK is normal or mildly elevated. The resting lactate level is normal or slightly elevated but may rise excessively after exercise. CSF protein is normal. The EMG is myopathic, and nerve conduction studies are usually normal. Ragged red fibers are prominently displayed in the muscle biopsy. Southern blots of muscle reveal a normal mtDNA band at 16.6 kb and several additional mtDNA deletion

1	studies are usually normal. Ragged red fibers are prominently displayed in the muscle biopsy. Southern blots of muscle reveal a normal mtDNA band at 16.6 kb and several additional mtDNA deletion bands with genomes varying from 0.5 to 10 kb.

1	This autosomal dominant form of CPEO has been linked to loci on three chromosomes: 4q35, 10q24, and 15q22–26. In the chromosome 4q-related form of disease, mutations of the gene encoding the heart and skeletal muscle–specific isoform of the adenine nucleotide translocator 1 (ANT1) gene are found. This highly abundant mitochondrial protein forms a homodimeric inner mitochondrial channel through which adenosine diphosphate (ADP) enters and ATP leaves the mitochondrial matrix. In the chromosome 10q–related disorder, mutations of the gene C10orf2 are found. Its gene product, twinkle, co-localizes with the mtDNA and is named for its punctate, starlike staining properties. The function of twinkle is presumed to be critical for lifetime maintenance of mitochondrial integrity. In the cases mapped to chromosome 15q, a mutation affects the gene encoding mtDNA polymerase (POLG), an enzyme important in mtDNA replication. Autosomal recessive PEO has also been described with mutations in the POLG

1	to chromosome 15q, a mutation affects the gene encoding mtDNA polymerase (POLG), an enzyme important in mtDNA replication. Autosomal recessive PEO has also been described with mutations in the POLG gene. Point mutations have been identified within various mitochondrial tRNA (Leu, Ile, Asn, Trp) genes in families with maternal inheritance of PEO.

1	Exercise may improve function but will depend on the patient’s ability to participate. MITOCHONDRIAL DNA SKELETAL MUSCLE–CENTRAL NERVOUS SYSTEM SYNDROMES Myoclonic Epilepsy with Ragged Red Fibers (MERRF) The onset of MERRF is variable, ranging from late childhood to middle adult life. Characteristic features include myoclonic epilepsy, cerebellar ataxia, and progressive muscle weakness. The seizure disorder is an integral part of the disease and may be the initial symptom. Cerebellar ataxia precedes or accompanies epilepsy. It is slowly progressive and generalized. The third major feature of the disease is muscle weakness in a limb-girdle distribution. Other more variable features include dementia, peripheral neuropathy, optic atrophy, hearing loss, and diabetes mellitus.

1	Serum CK levels are normal or slightly increased. The serum lactate may be elevated. EMG is myopathic, and in some patients nerve conduction studies show a neuropathy. The electroencephalogram is abnormal, corroborating clinical findings of epilepsy. Typical ragged red fibers are seen on muscle biopsy. MERRF is caused by maternally inherited point mutations of mitochondrial tRNA genes. The most common mutation found in 80% of MERRF patients is an A to G substitution at nucleotide 8344 of tRNA lysine (A8344G tRNAlys). Other tRNAlys mutations include base-pair substitutions T8356C and G8363A. Only supportive treatment is possible, with special attention to epilepsy.

1	Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Strokelike Episodes (MELAS) MELAS is the most common mitochondrial encephalomyopathy. The term strokelike is appropriate because the cerebral lesions do not conform to a strictly vascular distribution. The onset in the majority of patients is before age 20. Seizures, usually partial motor or generalized, are common and may represent the first clearly recognizable sign of disease. The cerebral insults that resemble strokes cause hemiparesis, hemianopia, and cortical blindness. A presumptive stroke occurring before age 40 should place this mitochondrial encephalomyopathy high in the differential diagnosis. Associated conditions include hearing loss, diabetes mellitus, hypothalamic pituitary dysfunction causing growth hormone deficiency, hypothyroidism, and absence of secondary sexual characteristics. In its full expression, MELAS leads to dementia, a bedridden state, and a fatal outcome. Serum lactic acid is typically

1	deficiency, hypothyroidism, and absence of secondary sexual characteristics. In its full expression, MELAS leads to dementia, a bedridden state, and a fatal outcome. Serum lactic acid is typically elevated. The CSF protein is also increased but is usually ≤1 g/L (100 mg/dL). Muscle biopsies show ragged red fibers. Neuroimaging demonstrates basal ganglia calcification in a high percentage of cases. Focal lesions that mimic infarction are present predominantly in the occipital and parietal lobes. Strict vascular territories are not respected, and cerebral angiography fails to demonstrate lesions of the major cerebral blood vessels.

1	MELAS is caused by maternally inherited point mutations of mitochondrial tRNA genes. Most of the tRNA mutations are lethal, accounting for the paucity of multigeneration families with this syndrome. The A3243G point mutation in tRNALeu(UUR) is the most common, occurring in ~80% of MELAS cases. About 10% of MELAS patients have other mutations of the tRNALeu(UUR) gene, including 3252G, 3256T, 3271C, and 3291C. Other tRNA gene mutations have also been reported in MELAS, including G583A tRNAPhe, G1642A tRNAVal, G4332A tRNAGlu, and T8316C tRNALys. Mutations have also been reported in mtDNA polypeptide-coding genes. Two mutations were found in the ND5 subunit of complex I of the respiratory chain. A missense mutation has been reported at mtDNA position 9957 in the gene for subunit III of cytochrome C oxidase. No specific treatment is available. Supportive treatment is essential for the strokelike episodes, seizures, and endocrinopathies.

1	Muscle weakness and fatigue can be the predominant manifestations of mtDNA mutations. When the condition affects exclusively muscle (pure myopathy), the disorder becomes difficult to recognize. Occasionally, mitochondrial myopathies can present with recurrent myoglobinuria without fixed weakness and thus resemble a glycogen storage disorder or CPT deficiency.

1	Mitochondrial DNA Depletion Syndromes Mitochondrial DNA depletion syndrome (MDS) is a heterogeneous group of disorders that are inherited in an autosomal recessive fashion and can present in infancy or adults. MDS can be caused by mutations in genes (TK2, DGUOK, RRM2B, TYMP, SUCLA1, and SUCLA2) that lead to depletion of pools of mitochondrial deoxyribonucleotide (dNTP) pools necessary for mtDNA replication The other major cause of MDS is a set of mutations in genes essential for mtDNA replication (e.g., POLG1 and C10orf2). The clinical phenotypes associated with MDS vary. Patients may develop a severe encephalopathy (e.g., Leigh’s syndrome), PEO, an isolated myopathy, myo-neuro-gastrointestinal-encephalopathy (MNGIE), and a sensory neuropathy with ataxia. Muscle membrane excitability is affected in a group of disorders referred to as channelopathies. The heart may also be involved, resulting in life-threatening complications (Table 462e-10).

1	CALCIUM CHANNEL DISORDERS OF MUSCLE Hypokalemic Periodic Paralysis (HypoKPP) Onset occurs at adolescence. Men are more often affected because of decreased penetrance in women. Episodic weakness with onset after age 25 is almost never due to periodic paralyses, with the exception of thyrotoxic periodic paralysis (see below). Attacks are often provoked by meals high in carbohydrates or sodium and may accompany rest following prolonged exercise. Weakness usually affects proximal limb muscles more than distal. Ocular and bulbar muscles are less likely to be affected. Respiratory muscles are usually spared, but when they are involved, the condition may prove fatal. Weakness may take as long as 24 h to resolve. Life-threatening cardiac arrhythmias related to hypokalemia may occur during attacks. As a late complication, patients commonly develop severe, disabling proximal lower extremity weakness.

1	Attacks of thyrotoxic periodic paralysis resemble those of primary HypoKPP. Despite a higher incidence of thyrotoxicosis in women, men, particularly those of Asian descent, are more likely to manifest this complication. Attacks abate with treatment of the underlying thyroid condition. A low serum potassium level during an attack, excluding secondary causes, establishes the diagnosis. Interattack muscle biopsies show the presence of single or multiple centrally placed vacuoles or tubular aggregates. Provocative tests with glucose and insulin to establish a diagnosis are usually not necessary and are potentially hazardous.

1	type 1, the most common form, is inherited as an autosomal dominant disorder with incomplete penetrance. These patients have mutations in the voltage-sensitive, skeletal muscle calcium channel gene, CALCL1A3 (Fig. 462e-8). Approximately 10% of cases are HypoKPP type 2, arising from mutations in the voltage-sensitive sodium channel gene (SCN4A). In either instance, the mutations lead to an abnormal gating pore current that predisposes the muscle cell to depolarize when potassium levels are low. It is also now recognized that some cases of thyrotoxic HypoKPP are caused by genetic variants in a potassium channel (Kir 2.6), whose expression is regulated by thyroid hormone. The chloride channel is envisioned to have 10 membrane-spanning domains. The positions of mutations causing dominantly and recessively inherited myotonia congenita are indicated, along with mutations that cause this disease in mice and goats.

1	The acute paralysis improves after the administration of potassium. Muscle strength and ECG should be monitored. Oral KCl (0.2–0.4 mmol/kg) should be given every 30 min. Only rarely is IV therapy necessary (e.g., when swallowing problems or vomiting is present). Administration of potassium in a glucose solution should be avoided because it may further reduce serum potassium levels. Mannitol is the preferred vehicle for administration of IV potassium. The long-term goal of therapy is to avoid attacks. This may reduce late-onset, fixed weakness. Patients should be made aware of the importance of a low-carbohydrate, low-sodium diet and consequences of intense exercise. Prophylactic administration of acetazolamide (125–1000 mg/d in divided doses) reduces or may abolish attacks in HypoKPP type 1. Paradoxically the potassium is lowered, but this is offset by the beneficial effect of metabolic acidosis. If attacks persist on acetazolamide, oral KCl should be added. Some patients require

1	type 1. Paradoxically the potassium is lowered, but this is offset by the beneficial effect of metabolic acidosis. If attacks persist on acetazolamide, oral KCl should be added. Some patients require treatment with triamterene (25–100 mg/d) or spironolactone (25–100 mg/d). However, in patients with HypoKPP type 2, attacks of weakness can be exacerbated with acetazolamide.

1	SODIUM CHANNEL DISORDERS OF MUSCLE Hyperkalemic Periodic Paralysis (HyperKPP) The term hyperkalemic is misleading because patients are often normokalemic during attacks. FIgURE 462e-8 The sodium and calcium channels are depicted here as containing four homologous domains, each with six membrane-spanning segments. The fourth segment of each domain bears positive charges and acts as the “voltage sensor” for the channel. The association of the four domains is thought to form a pore through which ions pass. Sodium channel mutations are shown along with the phenotype that they confer. HyperKPP, hyperkalemic periodic paralysis; PC, paramyotonia congenita; PAM, potassium-aggravated myotonia. See text for details.

1	The fact that attacks are precipitated by potassium administration best defines the disease. The onset is in the first decade; males and females are affected equally. Attacks are brief and mild, usually lasting 30 min to 4 ho. Weakness affects proximal muscles, sparing bulbar muscles. Attacks are precipitated by rest following exercise and fasting. In a variant of this disorder, the predominant symptom is myotonia without weakness (potassium-aggravated myotonia). The symptoms are aggravated by cold, and myotonia makes the muscles stiff and painful. This disorder can be confused with paramyotonia congenita, myotonia congenita, and proximal myotonic myopathy (DM2).

1	Potassium may be slightly elevated but may also be normal during an attack. As in HypoKPP, nerve conduction studies in HyperKPP muscle may demonstrate reduced motor amplitudes and the EMG may be silent in very weak muscles. In between attacks of weakness, the conduction studies are normal. The EMG will often demonstrate myotonic discharges during and between attacks. The muscle biopsy shows vacuoles that are smaller, less numerous, and more peripheral compared to the hypokalemic form or tubular aggregates. Provocative tests by administration of potassium can induce weakness but are usually not necessary to establish the diagnosis. HyperKPP and potassium-aggravated myotonia are inherited as autosomal dominant disorders. Mutations of the voltage-gated sodium channel SCN4A gene (Fig. 462e-8) cause these conditions. For patients with frequent attacks, acetazolamide (125–1000 mg/d) is helpful. We have found mexiletine to be helpful in patients with significant myotonia.

1	Paramyotonia Congenita In paramyotonia congenita (PC), the attacks of weakness are cold-induced or occur spontaneously and are mild. Myotonia is a prominent feature but worsens with muscle activity (paradoxical myotonia). This is in contrast to classic myotonia in which exercise alleviates the condition. Attacks of weakness are seldom severe enough to require emergency room treatment. Over time patients develop interattack weakness as they do in other forms of periodic paralysis. PC is usually associated with normokalemia or hyperkalemia. Serum CK is usually mildly elevated. Routine sensory and motor nerve conduction studies are normal. Short exercise test may be abnormal however, and cooling of the muscle often dramatically reduces the amplitude of the compound muscle action potentials. EMG reveals diffuse myotonic potentials in PC. Upon local cooling of the muscle, the myotonic discharges disappear as the patient becomes unable to activate MUAPs.

1	PC is inherited as an autosomal dominant condition; voltage-gated sodium channel mutations (Fig. 462e-8) are responsible, and thus this disorder is allelic with HyperKPP and potassium-aggravated myotonia. Patients with PC seldom seek treatment during attacks. Oral administration of glucose or other carbohydrates hastens recovery. Because interattack weakness may develop after repeated episodes, prophylactic treatment is usually indicated. Thiazide diuretics (e.g., chlorothiazide, 250–1000 mg/d) and mexiletine (slowly increase dose from 450 mg/d) are reported to be helpful. Patients should be advised to increase carbohydrates in their diet.

1	POTASSIUM CHANNEL DISORDERS Andersen-Tawil Syndrome This rare disease is characterized by episodic weakness, cardiac arrhythmias, and dysmorphic features (short stature, scoliosis, clinodactyly, hypertelorism, small or prominent low-set ears, micrognathia, and broad forehead). The cardiac arrhythmias are potentially serious and life threatening. They include long QT, ventricular ectopy, bidirectional ventricular arrhythmias, and tachycardia. For many years, the classification of this disorder was uncertain because episodes of weakness are associated with elevated, normal, or reduced levels of potassium during an attack. In addition, the potassium levels differ among kindreds but are consistent within a family. Inheritance is autosomal dominant, with incomplete penetrance and variable expressivity. The disease is caused by mutations of the inwardly rectifying potassium channel (Kir 2.1) gene that heighten muscle cell excitability. The treatment is similar to that for other forms of

1	The disease is caused by mutations of the inwardly rectifying potassium channel (Kir 2.1) gene that heighten muscle cell excitability. The treatment is similar to that for other forms of periodic paralysis and must include cardiac monitoring. The episodes of weakness may differ between patients because of potassium variability. Acetazolamide may decrease the attack frequency and severity.

1	Two forms of this disorder, autosomal dominant (Thomsen’s disease) and autosomal recessive (Becker disease), are related to the same gene abnormality. Symptoms are noted in infancy and early childhood. The severity lessens in the third to fourth decade. Myotonia is worsened by cold and improved by activity. The gait may appear slow and labored at first but improves with walking. In Thomsen’s disease, muscle strength is normal, but in Becker disease, which is usually more severe, there may be muscle weakness. Muscle hypertrophy is usually present. Myotonic discharges are prominently displayed by EMG recordings.

1	Serum CK is normal or mildly elevated. The muscle biopsy shows hypertrophied fibers. The disease is inherited as dominant or recessive and is caused by mutations of the chloride channel gene (Fig. 462e-8) that increase muscle cell excitability. Many patients will not require treatment and learn that the symptoms improve with activity. Medications that can be used to decrease myotonia include quinine, phenytoin, and mexiletine. Many endocrine disorders cause weakness. Muscle fatigue is more common than true weakness. The cause of weakness in these disorders is not well defined. It is not even clear that weakness results from disease of muscle as opposed to another part of the motor unit, since the serum CK level is often normal (except in hypothyroidism) and the muscle histology is characterized by atrophy rather than destruction of muscle fibers. Nearly all endocrine myopathies respond to treatment.

1	(See also Chap. 405) Abnormalities of thyroid function can cause a wide array of muscle disorders. These conditions relate to the important role of thyroid hormones in regulating the metabolism of carbohydrates and lipids as well as the rate of protein synthesis and enzyme production. Thyroid hormones also stimulate calorigenesis in muscle, increase muscle demand for vitamins, and enhance muscle sensitivity to circulating catecholamines.

1	Hypothyroidism Patients with hypothyroidism have frequent muscle complaints, and proximal muscle weakness occurs in about one-third of them. Muscle cramps, pain, and stiffness are common. Some patients have enlarged muscles. Features of slow muscle contraction and relaxation occur in 25% of patients; the relaxation phase of muscle stretch reflexes is characteristically prolonged and best observed at the ankle or biceps brachii reflexes. The serum CK level is often elevated (up to 10 times normal), even when there is minimal clinical evidence of muscle disease. EMG is typically normal. The cause of muscle enlargement has not been determined, and muscle biopsy shows no distinctive morphologic abnormalities.

1	Hyperthyroidism Patients who are thyrotoxic commonly have proximal muscle weakness and atrophy on examination, but they rarely complain of myopathic symptoms. Activity of deep tendon reflexes may be enhanced. Bulbar, respiratory, and even esophageal muscles may occasionally be affected, causing dysphagia, dysphonia, and aspiration. When bulbar involvement occurs, it is usually accompanied by chronic proximal limb weakness, but occasionally it presents in the absence of generalized thyrotoxic myopathy. Fasciculations may be apparent and, when coupled with increased muscle stretch reflexes, may lead to an erroneous diagnosis of amyotrophic lateral sclerosis. A form hypokalemic periodic paralysis can occur in patients who are thyrotoxic. Recently, mutations in the KCNJ18 gene that encodes for the inwardly rectifying potassium channel, Kir 2.6, have been discovered in up to a third of cases. Other neuromuscular disorders that occur in association with hyperthyroidism include myasthenia

1	for the inwardly rectifying potassium channel, Kir 2.6, have been discovered in up to a third of cases. Other neuromuscular disorders that occur in association with hyperthyroidism include myasthenia gravis (Chap.

1	461) and a progressive ocular myopathy associated with proptosis (Graves’ ophthalmopathy). Serum CK levels are not elevated in thyrotoxic myopathy, the EMG is normal, and muscle histology usually shows only atrophy of muscle fibers. (See also Chap. 424) Hyperparathyroidism Muscle weakness is an integral part of primary and secondary hyperparathyroidism. Proximal muscle weakness, muscle wasting, and brisk muscle stretch reflexes are the main features of this endocrinopathy. Some patients develop neck extensor weakness (part of the dropped head syndrome). Serum CK levels are usually normal or slightly elevated. Serum parathyroid hormone levels are elevated. Serum calcium and phosphorus levels show no correlation with the clinical neuromuscular manifestations. Muscle biopsies show only varying degrees of atrophy without muscle fiber degeneration.

1	Hypoparathyroidism An overt myopathy due to hypocalcemia rarely occurs. Neuromuscular symptoms are usually related to localized or generalized tetany. Serum CK levels may be increased secondary to muscle damage from sustained tetany. Hyporeflexia or areflexia is usually present and contrasts with the hyperreflexia in hyperparathyroidism.

1	(See also Chap. 406) Conditions associated with glucocorticoid excess cause a myopathy; in fact, steroid myopathy is the most commonly diagnosed endocrine muscle disease. Glucocorticoid excess, either endogenous or exogenous (see “Drug-Induced Myopathies,” below), produces various degrees of proximal limb weakness. Muscle wasting may be striking. A cushingoid appearance usually accompanies clinical signs of myopathy. Histologic sections demonstrate muscle fiber atrophy, preferentially affecting type 2b fibers, rather than degeneration or necrosis of muscle fibers. Adrenal insufficiency commonly causes muscle fatigue. The degree of weakness may be difficult to assess but is typically mild. In primary hyperaldosteronism (Conn’s syndrome), neuromuscular complications are due to potassium depletion. The clinical picture is one of persistent muscle weakness. Long-standing hyperaldosteronism may lead to proximal limb weakness and wasting. Serum CK levels may be elevated, and a muscle biopsy

1	The clinical picture is one of persistent muscle weakness. Long-standing hyperaldosteronism may lead to proximal limb weakness and wasting. Serum CK levels may be elevated, and a muscle biopsy may demonstrate degenerating fibers, some with vacuoles. These changes relate to hypokalemia and are not a direct effect of aldosterone on skeletal muscle.

1	(See also Chap. 403) Patients with acromegaly usually have mild proximal weakness without muscle atrophy. Muscles often appear enlarged but exhibit decreased force generation. The duration of acromegaly, rather than the serum growth hormone levels, correlates with the degree of myopathy. (See also Chap. 417) Neuromuscular complications of diabetes mellitus are most often related to neuropathy, with cranial and peripheral nerve palsies or distal sensorimotor polyneuropathy. Diabetic amyotrophy is a clumsy term because the condition represents a neuropathy affecting the proximal major nerve trunks and lumbosacral plexus. More appropriate terms for this disorder include diabetic proximal neuropathy and lumbosacral radiculoplexus neuropathy.

1	The only notable myopathy of diabetes mellitus is ischemic infarction of leg muscles, usually involving one of the thigh muscles but on occasion affecting the distal leg. This condition occurs in patients with poorly controlled diabetes and presents with abrupt onset of pain, tenderness, and edema of one thigh. The area of muscle infarction is hard and indurated. The muscles most often affected include the vastus lateralis, thigh adductors, and biceps femoris. Computed tomography (CT) or MRI can demonstrate focal abnormalities in the affected muscle. Diagnosis by imaging is preferable to muscle biopsy, if possible, as hemorrhage into the biopsy site can occur.

1	Vitamin D deficiency (Chap. 96e) due to decreased intake, decreased absorption, or impaired vitamin D metabolism (as occurs in renal disease) may lead to chronic muscle weakness. Pain reflects the underlying bone disease (osteomalacia). Vitamin E deficiency may result from malabsorption. Clinical manifestations include ataxic neuropathy due to loss of proprioception and myopathy with proximal weakness. Progressive external ophthalmoplegia is a distinctive finding. It has not 462e-19 been established that deficiency of other vitamins causes a myopathy. Systemic illnesses such as chronic respiratory, cardiac, or hepatic failure are frequently associated with severe muscle wasting and complaints of weakness. Fatigue is usually a more significant problem than weakness, which is typically mild.

1	Myopathy may be a manifestation of chronic renal failure (CRF), independent of the better known uremic polyneuropathy. Abnormalities of calcium and phosphorus homeostasis and bone metabolism in chronic renal failure result from a reduction in 1,25-dihydroxyvitamin D, leading to decreased intestinal absorption of calcium. Hypocalcemia, further accentuated by hyperphosphatemia due to decreased renal phosphate clearance, leads to secondary hyperparathyroidism. Renal osteodystrophy results from the compensatory hyperparathyroidism, which leads to osteomalacia from reduced calcium availability and to osteitis fibrosa from the parathyroid hormone excess. The clinical picture of the myopathy of CRF is identical to that of primary hyperparathyroidism and osteomalacia. There is proximal limb weakness with bone pain.

1	Gangrenous calcification represents a separate, rare, and sometimes fatal complication of CRF. In this condition, widespread arterial calcification occurs and results in ischemia. Extensive skin necrosis may occur, along with painful myopathy and even myoglobinuria. Drug-induced myopathies are relatively uncommon in clinical practice with the exception of those caused by the cholesterol-lowering agents and glucocorticoids. Others impact practice to a lesser degree but are important to consider in specific situations. Table 462e-11 provides a comprehensive list of drug-induced myopathies with their distinguishing features.

1	All classes of lipid-lowering agents have been implicated in muscle toxicity, including fibrates (clofibrate, gemfibrozil), HMG-CoA reductase inhibitors (referred to as statins), niacin (nicotinic acid), and ezetimibe. Myalgia, malaise, and muscle tenderness are the most common manifestations. Muscle pain may be related to exercise. Patients may exhibit proximal weakness. Varying degrees of muscle necrosis are seen, and in severe reactions rhabdomyolysis and myoglobinuria occur. Concomitant use of statins with fibrates and cyclosporine is more likely to cause adverse reactions than use of one agent alone. Elevated serum CK is an important indication of toxicity. Muscle weakness is accompanied by a myopathic EMG, and muscle necrosis is observed by muscle biopsy. Severe myalgias, muscle weakness, significant elevations in serum CK (>three times baseline), and myoglobinuria are indications for stopping the drug. Patients usually improve with drug cessation, although this may take several

1	significant elevations in serum CK (>three times baseline), and myoglobinuria are indications for stopping the drug. Patients usually improve with drug cessation, although this may take several weeks. Rare cases continue to progress after the offending agent is discontinued. It is possible that in such cases the statin may have triggered an immune-mediated necrotizing myopathy, as these individuals require aggressive immunotherapy (e.g., prednisone and sometimes other agents) to improve and often relapse when these therapies are discontinued. Interestingly, antibodies directed against the 100-kDa HMG-CoA reductase receptor on muscle fibers have been identified in many of these cases.

1	Glucocorticoid myopathy occurs with chronic treatment or as “acute quadriplegic” myopathy secondary to high-dose IV glucocorticoid use. Chronic administration produces proximal weakness accompanied by cushingoid manifestations, which can be quite debilitating; the chronic use of prednisone at a daily dose of ≥30 mg/d is most often associated with toxicity. Patients taking fluorinated glucocorticoids (triamcinolone, betamethasone, dexamethasone) appear to be at especially high risk for myopathy. In chronic steroid myopathy, the serum CK is Lipid-lowering agents Drugs belonging to all three of the major classes of lipid-lowering agents Fibric acid derivatives can produce a spectrum of toxicity: HMG-CoA reductase inhibitors asymptomatic serum creatine kinase Niacin (nicotinic acid) elevation, myalgias, exercise-induced pain, rhabdomyolysis, and myoglobinuria.

1	Niacin (nicotinic acid) elevation, myalgias, exercise-induced pain, rhabdomyolysis, and myoglobinuria. Glucocorticoids Acute, high-dose glucocorticoid treatment can cause acute quadriplegic myopathy. These high doses of steroids are often combined with nondepolarizing neuromuscular blocking agents but the weakness can occur without their use. Chronic steroid administration produces predominantly proximal weakness. Nondepolarizing neuromuscular Acute quadriplegic myopathy can blocking agents occur with or without concomitant glucocorticoids. Zidovudine Mitochondrial myopathy with ragged red fibers Drugs of abuse All drugs in this group can lead to widespread muscle breakdown, rhab- Alcohol domyolysis, and myoglobinuria. necrosis, skin induration, and limb Heroin contractures. Autoimmune toxic myopathy Use of this drug may cause polymyositis and myasthenia gravis. Amphophilic cationic drugs All amphophilic drugs have the potential to produce painless,

1	Autoimmune toxic myopathy Use of this drug may cause polymyositis and myasthenia gravis. Amphophilic cationic drugs All amphophilic drugs have the potential to produce painless, Amiodarone proximal weakness associated with Chloroquine autophagic vacuoles in the muscle Hydroxychloroquine biopsy. Antimicrotubular drugs This drug produces painless, proximal weakness especially in the setting of Colchicine renal failure. Muscle biopsy shows autophagic vacuoles. usually normal. Serum potassium may be low. The muscle biopsy in chronic cases shows preferential type 2 muscle fiber atrophy; this is not reflected in the EMG, which is usually normal.

1	Patients receiving high-dose IV glucocorticoids for status asthmaticus, chronic obstructive pulmonary disease, organ transplantation, or other indications may develop severe generalized weakness (critical illness myopathy). This myopathy, also known as acute quadriplegic myopathy, can also occur in the setting of sepsis. Involvement of the diaphragm and intercostal muscles causes respiratory failure and requires ventilatory support. In these settings, the use of glucocorticoids in combination with nondepolarizing neuromuscular blocking agents potentiates this complication. In critical illness myopathy, the muscle biopsy is abnormal, showing a distinctive loss of thick filaments (myosin) by electron microscopy. By light microscopy, there is focal loss of ATPase staining in central or paracentral areas of the muscle fiber. Calpain stains show diffusely reactive atrophic fibers. Withdrawal of glucocorticoids will improve the chronic myopathy. In acute quadriplegic myopathy, recovery is

1	areas of the muscle fiber. Calpain stains show diffusely reactive atrophic fibers. Withdrawal of glucocorticoids will improve the chronic myopathy. In acute quadriplegic myopathy, recovery is slow. Patients require supportive care and rehabilitation.

1	Zidovudine, used in the treatment of HIV infection, is a thymidine analogue that inhibits viral replication by interrupting reverse transcriptase. Myopathy is a well-established complication of this agent. Patients present with myalgias, muscle weakness, and atrophy affecting the thigh and calf muscles. The complication occurs in about 17% of patients treated with doses of 1200 mg/d for 6 months. The introduction of protease inhibitors for treatment of HIV infection has led to lower doses of zidovudine therapy and a decreased incidence of myopathy. Serum CK is elevated and EMG is myopathic. Muscle biopsy shows ragged red fibers with minimal inflammation; the lack of inflammation serves to distinguish zidovudine toxicity from HIV-related myopathy. If the myopathy is thought to be drug related, the medication should be stopped or the dosage reduced.

1	Myotoxicity is a potential consequence of addiction to alcohol and illicit drugs. Ethanol is one of the most commonly abused substances with potential to damage muscle. Other potential toxins include cocaine, heroin, and amphetamines. The most deleterious reactions occur from overdosing leading to coma and seizures, causing rhabdomyolysis, myoglobinuria, and renal failure. Direct toxicity can occur from cocaine, heroin, and amphetamines causing muscle breakdown and varying degrees of weakness. The effects of alcohol are more controversial. Direct muscle damage is less certain, since toxicity usually occurs in the setting of poor nutrition and possible contributing factors such as hypokalemia and hypophosphatemia. Alcoholics are also prone to neuropathy (Chap. 467).

1	Focal myopathies from self-administration of meperidine, heroin, and pentazocine can cause pain, swelling, muscle necrosis, and hemorrhage. The cause is multifactorial; needle trauma, direct toxicity of the drug or vehicle, and infection may all play a role. When severe, there may be overlying skin induration and contractures with replacement of muscle by connective tissue. Elevated serum CK and myopathic EMG are characteristic of these reactions. The muscle biopsy shows widespread or focal areas of necrosis. In conditions leading to rhabdomyolysis, patients need adequate hydration to reduce serum myoglobin and protect renal function. In all of these conditions, counseling is essential to limit drug abuse.

1	As mentioned previously, an autoimmune necrotizing myopathy associated with autoantibodies directed against HMG-CoA rarely occurs in the setting of statin use. An inflammatory myopathy also may occur with d-penicillamine, sometimes used in the treatment of Wilson’s disease scleroderma, rheumatoid arthritis, and primary biliary cirrhosis. The incidence of this inflammatory muscle disease is about 1%. Myasthenia gravis is also induced by d-penicillamine, with a higher incidence estimated at 7%. These disorders resolve with drug withdrawal, although immunosuppressive therapy may be warranted in severe cases.

1	Scattered reports of other drugs causing an inflammatory myopathy are rare and include a heterogeneous group of agents: cimetidine, phenytoin, procainamide, and propylthiouracil. In most cases, a causeand-effect relationship is uncertain. A complication of interest was related to l-tryptophan. In 1989 an epidemic of eosinophilia-myalgia syndrome (EMS) in the United States was caused by a contaminant in the product from one manufacturer. The product was withdrawn, and incidence of EMS diminished abruptly following this action. Certain drugs produce painless, largely proximal, muscle weakness. These drugs include the amphophilic cationic drugs (amiodarone, chloroquine, hydroxychloroquine) and antimicrotubular drugs (colchicine) (Table 462e-11). Muscle biopsy can be useful in the identification of toxicity because autophagic vacuoles are prominent pathologic features of these toxins.

1	Special Issues in Inpatient Neurologic Consultation S. Andrew Josephson, Martin A. Samuels Inpatient neurologic consultations usually involve questions regard-ing specific disease processes or prognostication after various cerebral 463e injuries. Common reasons for neurologic consultation include stroke (Chap. 446), seizures (Chap. 445), altered mental status (Chap. 34), headache (Chap. 21), and management of coma and other neurocritical care conditions (Chaps. 328 and 330). This chapter focuses on additional common reasons for consultation that are not addressed elsewhere in the text.

1	A group of neurologic disorders shares the common feature of hyper-perfusion, probably related to endothelial dysfunction, playing a key role in pathogenesis. These seemingly diverse syndromes include hypertensive encephalopathy, eclampsia, postcarotid endarterectomy syndrome, and toxicity from calcineurin-inhibitor and other medications. Modern imaging techniques and experimental models suggest that vasogenic edema is typically the primary process leading to neurologic dysfunction; therefore, prompt recognition and management of this condition should allow for clinical recovery as long as superimposed hemorrhage or infarction has not occurred.

1	The brain’s autoregulatory capability successfully maintains a fairly stable cerebral blood flow in adults despite alterations in systemic mean arterial pressure (MAP) ranging from 50 to 150 mmHg (Chap. 330). In patients with chronic hypertension, this cerebral autoregulation curve is shifted, resulting in autoregulation working over a much higher range of pressures (e.g., 70–175 mmHg). In these hypertensive patients, cerebral blood flow is kept steady at higher MAP, but a rapid lowering of pressure can lead to ischemia on the lower end of the autoregulatory curve, even at values typically thought of as normotensive. This autoregulatory phenomenon is achieved through both myogenic and neurogenic influences causing small arterioles to contract and dilate. When the systemic blood pressure exceeds the limits of this mechanism, breakthrough of autoregulation occurs, resulting in hyperperfusion via increased cerebral blood flow, capillary leakage into the interstitium, and resulting edema.

1	the limits of this mechanism, breakthrough of autoregulation occurs, resulting in hyperperfusion via increased cerebral blood flow, capillary leakage into the interstitium, and resulting edema. The predilection of all of the hyperperfusion disorders to affect the posterior rather than anterior portions of the brain may be due to a lower threshold for autoregulatory breakthrough in the posterior circulation or a vasculopathy that is more common in these blood vessels.

1	Although elevated or relatively elevated blood pressure is common in many of these disorders, some hyperperfusion states such as calcineurin-inhibitor toxicity occur with no apparent pressure rise. In these cases, vasogenic edema is likely due primarily to dysfunction of the capillary endothelium itself, leading to breakdown of the blood-brain barrier. It is useful to separate disorders of hyperperfusion into those caused primarily by increased pressure and those due mostly to endothelial dysfunction from a toxic or autoimmune etiology (Table 463e-1). In reality, both of these pathophysiologic processes likely play some role in each of these disorders.

1	The clinical presentation of all of the hyperperfusion syndromes is similar with prominent headaches, seizures, or focal neurologic deficits. Headaches have no specific characteristics, range from mild to severe, and may be accompanied by alterations in consciousness ranging from confusion to coma. Seizures may be present, and these can be of multiple types depending on the severity and location of the edema. Nonconvulsive seizures have been described in hyperperfusion states; therefore, a low threshold for obtaining an electroencephalogram (EEG) in these patients should be maintained. The typical focal deficit Some CommoN etIoLogIeS of hyperperfuSIoN SyNdrome Disorders in which increased capillary pressure dominates the pathophysiology Hypertensive encephalopathy, including secondary causes such as reno vascular hypertension, pheochromocytoma, cocaine use, etc. Disorders in which endothelial dysfunction dominates the pathophysiology

1	Disorders in which endothelial dysfunction dominates the pathophysiology Chemotherapeutic agent toxicity (e.g., cytarabine, azathioprine, 5-fluoro uracil, cisplatin, methotrexate, tumor necrosis factor α antagonists) HELLP syndrome (hemolysis, elevated liver enzyme levels, low platelet Granulomatosis with polyangiitis (Wegener’s) in hyperperfusion states is cortical visual loss, given the tendency of the process to involve the occipital lobes. However, any focal deficit can occur depending on the area affected, as evidenced by patients who, after carotid endarterectomy, exhibit neurologic dysfunction referable to the ipsilateral newly reperfused hemisphere. In conditions where increased cerebral blood flow plays a role, examination of the inpatient vital signs record will usually reveal a systemic blood pressure that is increased above the patient’s baseline. It appears as if the rapidity of rise, rather than the absolute value of pressure, is the most important risk factor.

1	The diagnosis in all of these conditions is clinical. The symptoms of these disorders are common and nonspecific, so a long differential diagnosis should be entertained, including consideration of other causes of confusion, focal neurologic deficits, headache, and seizures. Magnetic resonance imaging (MRI) has improved the ability of clinicians to diagnose hyperperfusion syndromes, although cases have been reported with normal imaging. Patients classically exhibit the high T2 signal of edema primarily in the posterior occipital lobes, not respecting any single vascular territory (Fig. 463e-1). Diffusion-weighted images are typically normal, emphasizing the vasogenic rather than cytotoxic nature of this edema. Imaging with computed tomography (CT) is less sensitive but may show a pattern of patchy hypodensity in the involved territory. Previously this classic radiographic appearance had been termed reversible posterior leukoencephalopathy (RPLE). However, this term has fallen out of

1	of patchy hypodensity in the involved territory. Previously this classic radiographic appearance had been termed reversible posterior leukoencephalopathy (RPLE). However, this term has fallen out of favor because none of its elements are completely accurate. The radiographic and clinical changes are not always reversible; the territory involved is not uniquely posterior; and gray matter may be affected as well, rather than purely white matter as the term “leukoencephalopathy” intimates. The now more commonly used radiologic term posterior reversible encephalopathy syndrome (PRES) suffers from many of these same limitations. Vessel imaging may demonstrate narrowing of the cerebral vasculature, especially in the posterior circulation; whether this noninflammatory vasculopathy is a primary cause of the edema or occurs as a secondary phenomenon remains unclear. Other ancillary studies such as cerebrospinal fluid (CSF) analysis often yield nonspecific results. It should be noted that many

1	of the edema or occurs as a secondary phenomenon remains unclear. Other ancillary studies such as cerebrospinal fluid (CSF) analysis often yield nonspecific results. It should be noted that many of the substances that have been implicated, such as cyclosporine, can cause this syndrome even at low doses or after years of treatment. Therefore, normal serum levels of these medications do not exclude them as inciting agents.

1	In cases of hyperperfusion syndromes, treatment should commence urgently once the diagnosis is considered. Hypertension plays a key role commonly, and judicious lowering of the blood pressure with IV agents such as labetalol or nicardipine is advised along with continuous cardiac and blood pressure monitoring, often through an arterial line. It is reasonable to lower MAP by ~20% initially, as further FIGURE 463e-1 Axial fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging (MRI) of the brain in a patient taking cyclosporine after liver transplantation, who presented with seizures, headache, and cortical blindness. Increased signal is seen bilaterally in the occipital lobes predominantly involving the white matter, consistent with a hyperperfusion state secondary to calcineurin-inhibitor exposure.

1	lowering of the pressure may cause secondary ischemia and possibly infarction as pressure drops below the lower range of the patient’s autoregulatory capability. In cases where there is an identified cause of the syndrome, these etiologies should be treated promptly, including discontinuation of offending substances such as calcineurin inhibitors in toxic processes, treatment of immune-mediated disorders such as thrombotic thrombocytopenic purpura (TTP), and prompt delivery of the fetus in eclampsia. Seizures must be identified and controlled, often necessitating continuous EEG monitoring. Anticonvulsants are effective when seizure activity is identified, but in the special case of eclampsia, there is evidence to support the use of magnesium sulfate for seizure control.

1	Central nervous system (CNS) injuries following open heart or coronary artery bypass grafting (CABG) surgery are common and include acute encephalopathy, stroke, and a chronic syndrome of cognitive impairment. Hypoperfusion and embolic disease are frequently involved in the pathogenesis of these syndromes, although multiple mechanisms may be involved in these critically ill patients who are at risk for various metabolic and polypharmaceutical complications.

1	The frequency of hypoxic injury secondary to inadequate blood flow intraoperatively has been markedly decreased by the use of modern surgical and anesthetic techniques. Despite these advances, some patients still experience neurologic complications from cerebral hypoperfusion or may suffer focal ischemia from carotid or focal intra-cranial stenoses in the setting of regional hypoperfusion. Postoperative infarcts in the border zones between vascular territories commonly are blamed on systemic hypotension, although these infarcts can also result from embolic disease (Fig. 463e-2). Embolic disease is likely the predominant mechanism of cerebral injury during cardiac surgery as evidenced by diffusion-weighted MRI and intraoperative transcranial Doppler studies. It should be noted that some of the emboli that are found histologically in these

1	FIGURE 463e-2 Coronal fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging (MRI) of the brain in a patient presenting with altered mental status after an episode of hypotension during coronary artery bypass grafting (CABG). Increased signal is seen in the border zones bilaterally between the middle cerebral artery and anterior cerebral artery territories. Diffusion-weighted MRI sequences demonstrated restricted diffusion in these same locations, suggesting acute infarction.

1	patients are too small to be detected by standard imaging sequences; therefore, a negative MRI after surgery does not exclude the diagnosis of emboli-related complications. Thrombus in the heart itself as well as atheromas in the aortic arch can become dislodged during cardiac surgeries, releasing a shower of particulate matter into the cerebral circulation. Cross-clamping of the aorta, manipulation of the heart, extracorporeal circulation techniques (“bypass”), arrhythmias such as atrial fibrillation, and introduction of air through suctioning have all been implicated as potential sources of emboli. Histologic studies indicate that literally millions of tiny emboli may be released, even using modern surgical techniques.

1	This shower of microemboli results in a number of clinical syndromes. Occasionally, a single large embolus leads to an isolated large-vessel stroke that presents with obvious clinical focal deficits. More commonly, the emboli released are multiple and smaller. When there is a high burden of these small emboli, an acute encephalopathy can occur postoperatively, presenting as either a hyperactive or hypoactive confusional state, the latter of which is frequently and incorrectly ascribed to depression or a sedative-induced delirium. When the burden of microemboli is lower, no acute syndrome is recognized, but the patient may suffer a chronic cognitive deficit. Cardiac surgery can be viewed, like delirium, as a “stress test for the brain.” Some patients with a low cerebral reserve due to underlying cerebrovascular disease or an early neurodegenerative process will develop a chronic, cognitive deficit, whereas others with higher reserves may remain asymptomatic despite a similar dose of

1	cerebrovascular disease or an early neurodegenerative process will develop a chronic, cognitive deficit, whereas others with higher reserves may remain asymptomatic despite a similar dose of microemboli. In this manner, cardiac surgery may serve to unmask the early manifestations of neurodegenerative disorders such as Alzheimer’s disease.

1	Since modern techniques have successfully minimized hypoperfusion complications during these surgeries, much attention is now focused on reducing this inevitable shower of microemboli. Off-pump CABG surgeries have the advantages of reducing length of stay and perioperative complications; however, off-pump CABG probably does not preserve cognitive function compared with on-pump CABG. Filters placed in the aortic arch may have some promise in capturing these emboli, although convincing evidence is lacking. Development of successful endovascular operative approaches may provide a reasonable alternative to conventional CABG procedures, especially for patients at high risk of developing cognitive dysfunction after surgery due to advanced age, previous stroke, underlying neurodegenerative disorders, or severe atheromatous disease of the carotid arteries or aortic arch.

1	Patients who have undergone solid organ transplantation are at risk for neurologic injury in the postoperative period and for months to years thereafter. Neurologic consultants should view these patients as a special population at risk for both unique neurologic complications as well as for the usual disorders found in any critically ill inpatient.

1	Immunosuppressive medications are administered in high doses to patients after solid organ transplant, and many of these compounds have well-described neurologic complications. In patients with headache, seizures, or focal neurologic deficits taking calcineurin inhibitors, the diagnosis of hyperperfusion syndrome should be considered, as discussed above. This neurotoxicity occurs mainly with cyclosporine and tacrolimus and can present even in the setting of normal serum drug levels. Treatment primarily involves lowering the drug dosage or discontinuing the drug. Sirolimus has very few recorded cases of neurotoxicity and may be a reasonable alternative for some patients. Other examples of immunosuppressive medications and their neurologic complications include OKT3-associated akinetic mutism and the leukoencephalopathy seen with methotrexate, especially when it is administered intrathecally or with concurrent radiotherapy. In any solid organ transplant patient with neurologic

1	mutism and the leukoencephalopathy seen with methotrexate, especially when it is administered intrathecally or with concurrent radiotherapy. In any solid organ transplant patient with neurologic complaints, a careful examination of the medication list is required to search for these possible drug effects.

1	Cerebrovascular complications of solid organ transplant are often first recognized in the immediate postoperative period. Border zone territory infarctions can occur, especially in the setting of systemic hypotension during cardiac transplant surgery. Embolic infarctions classically complicate cardiac transplantation, but all solid organ transplant procedures place patients at risk for systemic emboli. When cerebral embolization accompanies renal or liver transplantation surgery, a careful search for right-to-left shunting should include evaluation of the heart with agitated saline echocardiography (i.e., “bubble study”), as well as looking for intrapulmonary shunting. Renal and some cardiac transplant patients often have advanced atherosclerosis, providing yet another mechanism for stroke. Imaging with CT or MRI with diffusion is advised when cerebrovascular complications are suspected to confirm the diagnosis and to exclude intracerebral hemorrhage, which most often occurs in the

1	Imaging with CT or MRI with diffusion is advised when cerebrovascular complications are suspected to confirm the diagnosis and to exclude intracerebral hemorrhage, which most often occurs in the setting of coagulopathy secondary to liver failure or after cardiac bypass procedures.

1	Given that patients with solid organ transplants are chronically immunosuppressed, infections are a common concern (Chap. 169). In any transplant patient with new CNS signs or symptoms such as seizure, confusion, or focal deficit, the diagnosis of a CNS infection should be considered and evaluated through imaging (usually MRI) and possibly lumbar puncture. The most common pathogens responsible for CNS infections in these patients vary based on time since transplant. In the first month posttransplant, common pathogens include the usual bacterial organisms associated with surgical procedures and indwelling catheters. Starting in the second month posttransplant, opportunistic infections of the CNS become more common, including Nocardia and Toxoplasma species as well as fungal infections such as aspergillosis. Viral infections that can affect the brain of the immunosuppressed patient, such as herpes simplex virus, cytomegalovirus, human herpesvirus type 6 (HHV-6), and varicella, also

1	such as aspergillosis. Viral infections that can affect the brain of the immunosuppressed patient, such as herpes simplex virus, cytomegalovirus, human herpesvirus type 6 (HHV-6), and varicella, also become more common after the first month posttransplant. After 6 months posttransplant, immunosuppressed patients still remain at risk for these opportunistic bacterial, fungal, and viral infections but can also suffer late CNS infectious complications such as progressive multifocal leukoencephalopathy (PML) associated with JC virus and Epstein-Barr virus–driven clonal expansions of B cells resulting in posttransplant lymphoproliferative disorder or CNS lymphoma.

1	A wide variety of neurologic conditions can result from abnormalities in serum electrolytes, and consideration of electrolyte disturbances should be part of any inpatient neurologic consultation. A complete general discussion of fluid and electrolyte imbalance and homeostasis can be found in Chap. 63.

1	The normal range of serum osmolality is around 275–295 mOsm/kg, but neurologic manifestations are usually seen only at levels >325 mOsm/kg. Hyperosmolality is usually due to hypernatremia, hyperglycemia, azotemia, or the addition of extrinsic osmoles such as mannitol, which is commonly used in critically ill neurologic patients. Hyperosmolality itself can lead to a generalized encephalopathy that is nonspecific and without focal findings; however, an underlying lesion such as a mass can become symptomatic under the metabolic stress of a hyperosmolar state, producing focal neurologic signs. Some patients with hyperosmolality from severe hyperglycemia can present, for unclear reasons, with generalized seizures or unilateral movement disorders, which usually respond to lowering of the serum glucose. The treatment of all forms of hyperosmolality involves calculation of apparent water losses and slow replacement so that the serum sodium declines no faster than 2 mmol/L (2 meq/L) per hour.

1	Hypernatremia leads to the loss of intracellular water, leading to cell shrinkage. In the cells of the brain, solutes such as glutamine and urea are generated under these conditions in order to minimize this shrinkage. Despite this corrective mechanism, when hypernatremia is severe (serum sodium >160 mmol/L [>160 meq/L]) or occurs rapidly, cellular metabolic processes fail and encephalopathy will result. There are many etiologies of hypernatremia including, most commonly, renal and extrarenal losses of water. Causes of neurologic relevance include central diabetes insipidus, where hyperosmolality is accompanied by submaximal urinary concentration due to inadequate release of arginine vasopressin (AVP) from the posterior pituitary, resulting often from pituitary injury in the setting of surgery, hemorrhage, infiltrative processes, or cerebral herniation.

1	Hyponatremia is commonly defined as a serum sodium <135 mmol/L (<135 meq/L). Neurologic symptoms occur at different levels of low sodium, depending not only on the absolute value but also on the rate of fall. In patients with hyponatremia that develops over hours, life-threatening seizures and cerebral edema may occur at values as high as 125 mmol/L. In contrast, some patients with more chronic hyponatremia that has slowly developed over months to years may be asymptomatic even with serum levels <110 mmol/L. Correction of hyponatremia, especially when chronic, must take place slowly in order to avoid additional neurologic complications. Cells in the brain swell in hypotonic hyponatremic states but may compensate over time by excreting solute into the extracellular space, leading to restoration of cell volume when water follows the solute out of the cells. If treatment of hyponatremia results in a rapid rise in serum sodium, cells in the brain may quickly shrink, leading to osmotic

1	of cell volume when water follows the solute out of the cells. If treatment of hyponatremia results in a rapid rise in serum sodium, cells in the brain may quickly shrink, leading to osmotic demyelination, a process that previously was thought to be limited exclusively to the brainstem (central pontine myelinolysis; see Fig. 330-6), but now has been described elsewhere in the CNS.

1	Treatment of hyponatremia is dependent on the cause. Hypertonic hyponatremia treatment focuses on correcting the underlying condition, such as hyperglycemia. Isovolemic hyponatremia (syndrome of inappropriate antidiuretic hormone [SIADH]) is managed with water restriction or administration of AVP antagonists. The management of choice for patients with hypervolemic hypotonic hyponatremia is free-water restriction and treatment of the underlying edematous disorder, such as hepatic failure, nephrotic syndrome, or congestive heart failure. Finally, in hypovolemic hypotonic hyponatremia, volume is replaced with isotonic saline while underlying conditions of the kidneys, adrenals, and gastrointestinal tract are addressed.

1	One neurologic cause of hypovolemic hypotonic hyponatremia is the cerebral salt-wasting syndrome that accompanies subarachnoid hemorrhage and, less commonly, other cerebral processes such as meningitis, stroke, or traumatic brain injury. In these cases, the degree of renal sodium excretion can be remarkable, and large amounts of saline, hypertonic saline, or oral sodium may need to be given in a judicious fashion in order to avoid complications from cerebral edema.

1	Hypokalemia, defined as a serum potassium level < 3.5 mmol/L (<3.5 meq/L), occurs either because of excessive potassium losses (from the kidneys or gut) or due to an abnormal potassium distribution between the intracellular and extracellular spaces. At very low levels (<1.5 mmol/L), hypokalemia may be life threatening due to the risk of cardiac arrhythmia and may present neurologically with severe muscle weakness and paralysis. Hypokalemic periodic paralysis is a rare disorder caused by excessive intracellular potassium uptake in the setting of a calcium or sodium channel mutation. Treatment of hypokalemia is dependent on the etiology but usually includes replacement of potassium through oral or IV routes as well as correcting the cause of potassium balance problems (e.g., eliminating β2-adrenergic agonist medications or treating the underlying cause of severe diarrhea).

1	Hyperkalemia is defined as a serum potassium level >5.5 mmol/L (>5.5 meq/L) and can neurologically present as muscle weakness with or without paresthesias. Hyperkalemia becomes life threatening when it produces electrocardiographic abnormalities such as peaked T waves or a widened QRS complex. In these cases, prompt treatment is essential and consists of strategies that protect the heart against arrhythmias (calcium gluconate administration); promote potassium redistribution into cells (with glucose, insulin, and β2-agonist medications); and increase potassium removal (through sodium polystyrene sulfonate, loop diuretics, or hemodialysis). Hypercalcemia usually occurs in the setting of either hyperparathyroidism or systemic malignancy. Neurologic manifestations include encephalopathy as well as muscle weakness due to reduced neuromuscular excitability. Seizures can occur but are more common in states of low calcium.

1	Hypocalcemia in adults often follows surgical treatment of the thyroid or parathyroid. Seizures and altered mental status dominate the neurologic picture and usually resolve with calcium repletion. Tetany is due to spontaneous, repetitive action potentials in peripheral nerves and remains the classic sign of symptomatic hypocalcemia. Disorders of magnesium are difficult to correlate with serum levels because a very small amount of total-body magnesium is located in the extracellular space. Hypomagnesemia presents neurologically with seizures, tremor, and myoclonus. When intractable seizures occur in the setting of hypomagnesemia, only administration of magnesium will lead to their resolution. High levels of magnesium, in contrast, lead to CNS depression. Hypermagnesemia usually only occurs in the setting of renal failure or magnesium administration and can lead to confusion and muscular paralysis when severe.

1	Polyneuropathy is a common cause of outpatient neurologic consultation (Chap. 459). In the inpatient setting, however, mononeuropathies are more frequent, especially the entrapment neuropathies that complicate many surgical procedures and medical conditions. Median neuropathy at the wrist (carpal tunnel syndrome) is the most frequent entrapment neuropathy by far, but it is rarely a cause for inpatient consultation. Mechanisms for perioperative mononeuropathy include traction, compression, and ischemia of the nerve. Imaging with MRI, including neurography, may allow these causes to be distinguished. In all cases of mononeuropathy, the diagnosis can be made through the clinical examination and then confirmed with electrodiagnostic studies in the subacute period, if necessary. Treatment consists mainly of avoidance of repetitive trauma but may also include surgical approaches to relieve pressure or repair the nerve.

1	Radial nerve injury classically presents with weakness of extension of the wrist and fingers (“wrist drop”) with or without more proximal weakness of extensor muscles of the upper extremity, depending on the site of injury. Sensory loss is in the distribution of the radial nerve, which includes the dorsum of the hand (Fig. 463e-3A). Compression at the level of the axilla, e.g., resulting from use of crutches, leads to weakness of the triceps, brachioradialis, and supinator muscles in addition to wrist drop. A more common site of compression occurs in the spiral groove of the upper arm in the setting of a humerus fracture or from sleeping with the arm draped over a bench or chair (“Saturday night palsy”). Sparing of the triceps is the rule when the nerve is injured in this location. Because extensors of the upper extremity are injured preferentially in radial nerve injury, these lesions may be mistaken for the pyramidal distribution of weakness that accompanies upper motor neuron

1	extensors of the upper extremity are injured preferentially in radial nerve injury, these lesions may be mistaken for the pyramidal distribution of weakness that accompanies upper motor neuron lesions from brain or spinal cord processes, prompting neuroimaging to exclude acute stroke or mass lesion.

1	Compression of the ulnar nerve is the second most common entrapment neuropathy after carpal tunnel syndrome. The most frequent site of compression is at the elbow where the nerve passes superficially in the ulnar groove. Symptoms usually begin with tingling in the ulnar distribution, including the fourth and fifth digits of the hand (Fig. 463e-3B). Sensory symptoms may be worsened by elbow flexion due to increased pressure on the nerve, hence the tendency of patients to complain of increasing paresthesias at night when the arm is flexed at the elbow during sleep. Motor dysfunction can be disabling and involves most of the intrinsic hand muscles, limiting dexterity and strength of grasp and pinch. Etiologies of ulnar entrapment include trauma to the nerve (hitting the “funny bone”), malpositioning during anesthesia for surgical procedures, and chronic arthritis of the elbow. When a perioperative ulnar nerve injury is considered, stretch injury or trauma to the lower trunk of the

1	malpositioning during anesthesia for surgical procedures, and chronic arthritis of the elbow. When a perioperative ulnar nerve injury is considered, stretch injury or trauma to the lower trunk of the brachial plexus should be entertained as well since its symptoms can mimic those of an ulnar neuropathy. If the clinical examination is equivocal, electrodiagnostic studies can definitively distinguish between plexus and ulnar nerve lesions a few weeks after the injury. Conservative methods of treatment are often the first step including bracing of the elbow to prevent flexion while sleeping. A variety of surgical approaches may also be effective in refractory or recurrent cases, including anterior ulnar nerve transposition and release of the flexor carpi ulnaris aponeurosis.

1	The peroneal (also known as the fibular) nerve winds around the head of the fibula in the leg below the lateral aspect of the knee, and its superficial location at this site makes it vulnerable to trauma. Patients present with weakness of foot dorsiflexion (“foot drop”) as well as with weakness in eversion but not inversion at the ankle. Sparing of inversion, which is a function of muscles innervated by the tibial nerve, helps to distinguish peroneal neuropathies from L5 radiculopathies. Sensory loss involves the lateral aspect of the leg as well as the dorsum of the foot (Fig. 463e-3C). Fractures of the fibular head may be responsible for peroneal neuropathies, but in the perioperative setting, poorly applied braces exerting pressure on the nerve while the patient is unconscious, often in lithotomy position, are more often responsible. Tight-fitting stockings or casts of the upper leg can also cause a peroneal neuropathy, and thin individuals and those with recent weight loss are at

1	lithotomy position, are more often responsible. Tight-fitting stockings or casts of the upper leg can also cause a peroneal neuropathy, and thin individuals and those with recent weight loss are at increased risk.

1	Lateral cutaneous nerve of arm Sensory distribution of the radial nerve Posterior cutaneous nerve of arm Posterior cutaneous nerve of forearm FIGURE 463e-3 Sensory distribution of peripheral nerves commonly affected by entrapment neuropathies. A. Radial nerve. B. Ulnar nerve. C. Peroneal nerve. D. Femoral nerve. E. Lateral femoral cutaneous nerve. Sensory distribution of the peroneal nerve Lateral cutaneous nerve of calf Sensory distribution of the ulnar nerve of the femoral nerve Lateral femoral cutaneus nerve

1	Lesions of the proximal femoral nerve are relatively uncommon but may present dramatically with weakness of hip flexion, quadriceps atrophy, weakness of knee extension (often manifesting with leg-buckling falls), and an absent patellar reflex. Adduction of the thigh is spared as these muscles are supplied by the obturator nerve, thereby distinguishing a femoral neuropathy from a more proximal lumbosacral plexus lesion. The sensory loss found is in the distribution of the femoral nerve sensory branches including the anterior part of the thigh (Fig. 463e-3D). Compressive lesions from retroperitoneal hematomas or masses are common, and a CT of the pelvis should be obtained in all cases of femoral neuropathy to exclude these conditions. Bleeding into the pelvis resulting in hematoma can occur spontaneously, following trauma, or after intrapelvic surgeries such as renal transplantation. In intoxicated or comatose patients, stretch injuries to the femoral nerve are seen following prolonged,

1	spontaneously, following trauma, or after intrapelvic surgeries such as renal transplantation. In intoxicated or comatose patients, stretch injuries to the femoral nerve are seen following prolonged, extreme hip flexion or extension. Rarely, attempts at femoral vein or arterial puncture can be complicated by injury to this nerve.

1	The symptoms of lateral femoral cutaneous nerve entrapment, commonly known as “meralgia paresthetica,” include sensory loss, pain, and dysesthesia in part of the area supplied by the nerve (Fig. 463e-3E). There is no motor component to the nerve, and therefore weakness is not a part of this syndrome. Symptoms often are worsened by standing or walking. Compression of the nerve occurs where it enters the leg near the inguinal ligament, usually in the setting of tight-fitting belts, pants, corsets, or recent weight gain, including that of pregnancy. The differential diagnosis of these symptoms includes hip problems such as trochanteric bursitis.

1	Pregnancy and delivery place women at special risk for a variety of nerve injuries. Radiculopathy due to a herniated lumbar disc is not common during pregnancy, but compressive injuries of the lumbosacral plexus do occur secondary to either the fetal head passing through the pelvis or the use of forceps during delivery. These plexus injuries are more frequent with cephalopelvic disproportion and often present with a painless unilateral foot drop which must be distinguished from a peroneal neuropathy caused by pressure on the nerve while in lithotomy position during delivery. Other compressive mononeuropathies of pregnancy include meralgia paresthetica, carpal tunnel syndrome, femoral neuropathy when the thigh is abducted severely in an effort to facilitate delivery of the fetal shoulder, and isolated obturator neuropathy during lithotomy positioning. The latter presents with medial thigh pain that may be accompanied by weakness of thigh adduction. There is also a clear association

1	and isolated obturator neuropathy during lithotomy positioning. The latter presents with medial thigh pain that may be accompanied by weakness of thigh adduction. There is also a clear association between pregnancy and an increased frequency of idiopathic facial palsy (Bell’s palsy).

1	Chronic Fatigue Syndrome Gijs Bleijenberg, Jos W. M. van der Meer DEFINITION Chronic fatigue syndrome (CFS) is a disorder characterized by persis-tent and unexplained fatigue resulting in severe impairment in daily functioning. Besides intense fatigue, most patients with CFS report 464e SECTion 4 concomitant symptoms such as pain, cognitive dysfunction, and unrefreshing sleep. Additional symptoms can include headache, sore throat, tender lymph nodes, muscle aches, joint aches, feverishness, difficulty sleeping, psychiatric problems, allergies, and abdominal cramps. Criteria for the diagnosis of CFS have been developed by the U.S. Centers for Disease Control and Prevention (Table 464e-1).

1	U.S. Centers for Disease Control and Prevention (Table 464e-1). CFS is seen worldwide, with adult prevalence rates varying between 0.2% and 0.4%. In the United States, the prevalence is higher among women (∼75% of cases), members of minority groups (African and Native Americans), and individuals with lower levels of education and occupational status. The mean age of onset is between 29 and 35 years. Many patients probably go undiagnosed and/ or do not seek help. There are numerous hypotheses about the etiology of CFS; there is no definitively identified cause. Distinguishing between predisposing, precipitating, and perpetuating factors in CFS helps to provide a framework for understanding this complex condition (Table 464e-2).

1	Predisposing Factors Physical inactivity and trauma in childhood tend to increase the risk of CFS in adults. Neuroendocrine dysfunction may be associated with childhood trauma, reflecting a biological correlate of vulnerability. Psychiatric illness and physical hyperactivity in adulthood raise the risk of CFS in later life. Twin studies suggest a familial predisposition to CFS, but no causative genes have been identified. Precipitating Factors Physical or psychological stress may elicit the onset of CFS. Most patients report an infection (usually a flulike illness or infectious mononucleosis) as the trigger of their fatigue. Relatively high percentages of CFS cases follow Q fever and Lyme disease.

1	Fatigue lasts for at least 6 months. Fatigue is of new or definite onset. Fatigue is not the result of an organic disease or of continuing exertion. Fatigue is not alleviated by rest. Fatigue results in a substantial reduction in previous occupational, educa tional, social, and personal activities. Four or more of the following symptoms are concurrently present for 6 months: impaired memory or concentration, sore throat, tender cervical or axillary lymph nodes, muscle pain, pain in several joints, new headaches, unrefreshing sleep, or malaise after exertion. Medical condition explaining fatigue Major depressive disorder (psychotic features) or bipolar disorder Schizophrenia, dementia, or delusional disorder Anorexia nervosa, bulimia nervosa Alcohol or substance abuse Severe obesity (body mass index >40) TABLE 464e-2 PrEDiSPoSing, PrECiPiTATing, AnD PErPETuATing FACTorS in ChroniC FATiguE SynDromE

1	TABLE 464e-2 PrEDiSPoSing, PrECiPiTATing, AnD PErPETuATing FACTorS in ChroniC FATiguE SynDromE Childhood trauma (sexual, physical, emotional abuse; emotional and physical neglect) Physical inactivity during childhood Premorbid psychiatric illness or psychopathology Premorbid hyperactivity Somatic events: infection (e.g., mononucleosis, Q fever, Lyme disease), surgery, pregnancy Psychosocial stress, life events Fear of fatigue Lack of social support

1	Somatic events: infection (e.g., mononucleosis, Q fever, Lyme disease), surgery, pregnancy Psychosocial stress, life events Fear of fatigue Lack of social support However, no differences in Epstein-Barr virus load and immunologic reactivity were found between individuals who developed CFS and those who did not. While antecedent infections are associated with CFS, a direct microbial causality is unproven and unlikely. One study identified a murine leukemia virus–related retrovirus (XMRV); however, several subsequent studies have established this virus as a laboratory artifact. Patients also often report other precipitating somatic events such as serious injury, surgery, pregnancy, or childbirth. Serious life events, such as the loss of a loved one or a job, military combat, and other stressful situations, may also precipitate CFS. One-third of all patients cannot recall a trigger.

1	Perpetuating Factors Once CFS has developed, numerous factors may impede recovery. Physicians may contribute to chronicity by ordering unnecessary diagnostic procedures, by persistently suggesting psychological causes, and by not acknowledging CFS as a diagnosis. A patient’s focus on illness and avoidance of activities may perpetuate symptoms. A firm belief in a physical cause, a strong focus on bodily sensations, and a poor sense of control over symptoms may also prolong or exacerbate the fatigue and functional impairment. In most patients, inactivity is caused by negative illness perceptions rather than by poor physical fitness. Solicitous behavior of others may reinforce a patient’s illness-related perceptions and behavior. A lack of social support is another known perpetuating factor.

1	The pathophysiology of CFS is unclear. Neuroimaging studies have found that CFS is associated with reduced gray matter volume, which in turn is associated with a decline in physical activity; these changes have been partially reversed following cognitive behavioral therapy (CBT). In addition, functional MRI data have suggested that abnormal patterns of activation correlate with self-reported problems with information processing. Neurophysiologic studies have shown altered CNS activation patterns during muscle contraction. Evidence for immunologic dysfunction is inconsistent. Modest elevations in titers of antinuclear antibodies, reductions in immunoglobulin subclasses, deficiencies in mitogen-driven lymphocyte proliferation, reductions in natural killer cell activity, disturbances How have you felt during the last two weeks? Please rate all four statements and per statement check the box that reflects your situation best.

1	How have you felt during the last two weeks? Please rate all four statements and per statement check the box that reflects your situation best. No, 1. I feel tired Yes, that is not true that is true No, 2. I tire easily Yes, that is true that is not true No, 3. I feel fit Yes, that is true that is not true No, 4. Physically I feel exhausted Yes, that is true that is not true Scoring: Yes, 1, 2 and 4: 7 6 5 4 that is true FIGurE 464e-1 Shortened fatigue questionnaire.

1	No, 4. Physically I feel exhausted Yes, that is true that is not true Scoring: Yes, 1, 2 and 4: 7 6 5 4 that is true FIGurE 464e-1 Shortened fatigue questionnaire. in cytokine production, and shifts in lymphocyte subsets have been described. None of these immune findings appears in most patients, nor does any correlate with the severity of CFS. In theory, symptoms of CFS could result from excessive production of a cytokine, such as interleukin 1, that induces asthenia and other flulike symptoms; however, compelling data in support of this hypothesis are lacking. There is some evidence that CFS patients have mild hypocortisolism, the degree of which is associated with a poorer response to CBT. Discrepancies in perceived and actual cognitive performance are consistent findings in patients with CFS.

1	In addition to a thorough history, a systematic physical examination is warranted to exclude disorders causing fatigue (e.g., endocrine disorders, neoplasms, heart failure). The heart rate of CFS patients is often slightly above normal. Laboratory tests serve primarily to exclude other diagnoses; no test can diagnose CFS. The following laboratory screen usually suffices: complete blood count; erythrocyte sedimentation rate; C-reactive protein; serum creatinine, electrolytes, calcium, and iron; blood glucose; creatine kinase; liver function tests; thyroid-stimulating hormone; anti-gliadin antibodies; and urinalysis. Serology for viral or bacterial infections usually is not helpful. No specific abnormalities have been identified on MRI or CT scans. The decrease in gray matter volume observed at a population level by MRI is not useful for diagnosis in the individual patient. Extensive, unfocused, and expensive testing in a search for the “hidden” cause of the fatigue is not productive.

1	at a population level by MRI is not useful for diagnosis in the individual patient. Extensive, unfocused, and expensive testing in a search for the “hidden” cause of the fatigue is not productive. CFS is a constellation of symptoms with no pathognomonic features and remains a diagnosis of exclusion.

1	Bipolar disorders, schizophrenia, and substance abuse exclude a diagnosis of CFS, as do eating disorders, unless these issues have been resolved ≥5 years before symptom onset. In addition, CFS is excluded if the chronic fatigue developed immediately after a depressive episode. Depression developing in the course of the fatigue, however, does not preclude CFS. Concurrent psychiatric disorders, especially anxiety and mood disorders, are present in 30–60% of cases. In cases of suspected CFS, the clinician should acknowledge the impact of the patient’s symptoms on daily functioning. Disbelief or denial can provoke an exacerbation of genuine symptoms, which in turn strengthens the clinician’s disbelief, leading to an unfortunate cycle of miscommunication. The possibility of CFS should be considered if No, 3 2 1 3: Reversed that is not true a patient fulfils all criteria (Table 464e-1) and if other diagnoses have been excluded.

1	No, 3 2 1 3: Reversed that is not true a patient fulfils all criteria (Table 464e-1) and if other diagnoses have been excluded. The patient should be asked to describe the symptoms (fatigue and accompanying symptoms) and their duration as well as their consequences (reduction in daily activities). To assess symptom severity and the extent of daily-life impairment, the patient should describe a typical day, from waking to retiring, and, for comparison, an average day prior to symptom onset. Next, potential fatigue-precipitating factors are sought. The severity of fatigue is commonly difficult to assess quantitatively; a brief questionnaire is often helpful (Fig. 464e-1).

1	The patient should be informed of the current understanding of precipitating and perpetuating factors and effective treatments and should be offered general advice about disease management. If CBT for CFS is not available as an initial option (see below) and depression and anxiety are present, these symptoms should be treated. For patients with headache, diffuse pain, and feverishness, nonsteroidal anti-inflammatory drugs may be helpful. Even modest improvements in symptoms can make an important difference in the patient’s degree of self-sufficiency and ability to appreciate life’s pleasures. Controlled therapeutic trials have established that acyclovir, fludrocortisone, galantamine, modafinil, and IV immunoglobulin, among other agents, offer no significant benefit in CFS. Countless anecdotes circulate regarding other traditional and nontraditional therapies. It is important to guide patients away from those therapeutic modalities that are toxic, expensive, or unreasonable.

1	The patient should be encouraged to maintain regular sleep patterns, to remain as active as possible, and to gradually return to previous levels of exercise and other activity (work).

1	CBT and graded exercise therapy (GET) have been found to be the only beneficial interventions in CFS. Some patient groups argue against these approaches because of the implication that CFS is a purely mental disorder. CBT is a psychotherapeutic approach directed at changing unhealthy disease-perpetuating patterns of thoughts and behaviors. It includes educating the patient about the etiologic model, setting goals, restoring fixed bedtimes and wakeup times, challenging and changing fatigueand activity-related concerns, reducing a focus on symptoms, spreading activities evenly throughout the day, gradually increasing physical activity, planning a return to work, and resuming other activities. The intervention, which typically consists of 12–14 sessions spread over 6 months, helps CFS patients gain control over their symptoms.

1	GET targets deconditioning and exercise intolerance and usually involves a home exercise program that continues for 3–5 months. Walking or cycling is systematically increased, with set goals for maximal heart rates. Evidence that deconditioning is the basis for symptoms in CFS is lacking, however. CBT and GET appear to improve fatigue primarily by changing the patient’s perception of the fatigue and also by reducing the focus on symptoms. In general, CBT is the more multifaceted treatment, which may explain why CBT studies tend to yield better improvement rates than GET trials. Not all patients benefit from CBT or GET. Predictors of poor outcome are medical (including psychiatric) comorbidities, current disability claims, and severe pain. CBT offered in an early stage of the illness reduces the burden of CFS for the patient as well as for society in terms of decreased medical and disability-related costs.

1	Full recovery from untreated CFS is rare: the median annual recovery rate is 5% (range, 0–31%), and the median improvement rate is 39% (range, 8–63%). Patients with an underlying psychiatric disorder and those who continue to attribute their symptoms to an undiagnosed medical condition have poorer outcomes. Biology of psychiatric Disorders Robert O. Messing, Eric J. Nestler Psychiatric disorders are central nervous system diseases characterized by disturbances in emotion, cognition, motivation, and socialization.

1	Robert O. Messing, Eric J. Nestler Psychiatric disorders are central nervous system diseases characterized by disturbances in emotion, cognition, motivation, and socialization. They are highly heritable, with genetic risk comprising 20–90% of disease vulnerability. As a result of their prevalence, early onset, and persistence, they contribute substantially to the burden of illness world wide. All psychiatric disorders are broad heterogeneous syndromes markers. Therefore, diagnoses continue to be made solely from clini cal observations using criteria in the Diagnostic and Statistical Manual of Mental Disorders (DSM) of the American Psychiatric Association, which is in its fifth edition as of 2013.

1	There is increasing agreement that the classification of psychiatric illnesses in DSM does not accurately reflect the underlying biology of these disorders. Uncertainties in diagnosis make it extremely difficult to study the neurobiologic and genetic basis of mental illness. This has led to the development of an alternative diagnostic scheme, termed Research Domain Criteria (RDoCs), which classifies mental illness on the basis of core abnormalities—e.g., psychosis (loss of reality) or anhedonia (decreased ability to experience pleasure), which are common symptoms of several illnesses—with the idea that such classifications will assist in defining the biologic basis of at least key symptoms. Other factors that have impeded progress in understanding mental illness include the lack of access to pathologic brain tissue except upon death and the inherent limitations of animal models for disorders defined largely by behavioral abnormalities (e.g., hallucinations, delusions, guilt,

1	of access to pathologic brain tissue except upon death and the inherent limitations of animal models for disorders defined largely by behavioral abnormalities (e.g., hallucinations, delusions, guilt, suicidality) that are inaccessible in animals.

1	Despite these limitations, the past decade has seen significant advances. Neuroimaging methods are beginning to provide evidence of brain pathology, genome-wide association studies and high-throughput sequencing are at last revealing genes that confer risk for severe forms of mental illness, and investigations using better validated animal models are offering new insight into the molecular, cellular, and circuit mechanisms of disease pathogenesis. There is also excitement in the potential utility of neuron-like cells induced in vitro from a patient’s peripheral tissues (e.g., fibroblasts) providing novel ways of studying disease pathophysiology and screening for new treatments. There is consequently justified optimism that the field of psychiatry will transition from behaviorally defined syndromes to true biologic disease entities and that such advances will drive the development of improved treatments and eventually cures and preventive measures. This chapter describes several

1	syndromes to true biologic disease entities and that such advances will drive the development of improved treatments and eventually cures and preventive measures. This chapter describes several examples of recent discoveries in basic neuroscience that have informed our current understanding of disease mechanisms in psychiatry.

1	Because the human brain can only be examined indirectly during life, genome analyses have been extremely important for obtaining molecular clues about the pathogenesis of psychiatric disorders. A wealth of new information has been made possible by recent technological developments that have permitted affordable, large-scale genome-wide association studies and fine-scale sequencing. As an example, significant progress has been made in the genetics of autism spectrum disorders (ASDs), which are a heterogeneous group of neurodevelopmental diseases that share clinical features of impaired reciprocal social communication and interaction and restricted, repetitive patterns of behavior. ASDs are highly heritable; concordance rates in monozygotic twins (~60–90%) are roughly 10-fold higher than in dizygotic twins and siblings, whereas first-degree relatives show about 50-fold increased risk compared with the general population. ASDs are also genetically heterogeneous. More than 100 known

1	in dizygotic twins and siblings, whereas first-degree relatives show about 50-fold increased risk compared with the general population. ASDs are also genetically heterogeneous. More than 100 known mutations account for up to 20% of cases, although none individually accounts for more than 1% (Table 465e-1). It appears that most cases result from complex genetic mechanisms, including inheritance of multiple genetic risk variants and epigenetic modifications. For example, up to 10% of patients with autism have large (>500 kb) de novo copy number variations scattered across the genome, suggesting that hundreds of different genes can influence autism risk.

1	Amid this genetic heterogeneity, however, some common themes have emerged that inform pathogenesis of ASDs. For instance, many identified mutations are in genes that encode proteins involved in synaptic function and early transcriptional regulation (Table 465e-1) and have a clear relationship to activity-dependent neural responses that can affect the development of neural systems underlying cognition and social behaviors. Mutations in these genes may be detrimental by altering the balance of excitatory versus inhibitory synaptic signaling in local and extended circuits and by altering mechanisms that control brain growth. Some mutations affect genes (e.g., PTEN, TSC1, and TSC2) that negatively regulate signaling from several types of extracellular stimuli, including those transduced by receptor tyrosine kinases. Their dysregulation can alter neuronal growth as well as synaptic development and function.

1	With further understanding of pathogenesis and the definition of specific ASD subtypes, there is reason to believe that effective therapies will be identified. Work in mouse models has already demonstrated that some autism-like behaviors can be reversed, even in fully developed adult animals, by modifying the underlying pathology; these results encourage hope for many affected individuals. Treatments that target excitation-inhibition imbalance or altered mRNA translation appear to offer early promise. For example, the genes TSC1, TSC2, and PTEN are negative regulators of signaling through the target of rapamycin complex 1 (TORC1), which regulates protein synthesis. Rapamycin, a selective inhibitor of TORC1, can reverse several behavioral and synaptic defects in mice carrying null mutations in these genes. Another example is fragile X syndrome, which is the leading cause of inherited autism and mental disability and is due to mutations in FMR1 that result in loss of the encoded fragile

1	in these genes. Another example is fragile X syndrome, which is the leading cause of inherited autism and mental disability and is due to mutations in FMR1 that result in loss of the encoded fragile X mental retardation protein (FMRP). FMRP is a polyribosome-associated mRNA-binding protein that represses the translation of a subset (~5%) of all mRNAs, several of which encode proteins that comprise the postsynaptic density, including the metabotropic glutamate receptor 5 (mGluR5). Treatment of Fmr1 knockout mice with mGluR5 antagonists reduces several behavioral and morphologic abnormalities in these mice; these promising preclinical results have led to ongoing trials of mGluR5 antagonists in humans with fragile X syndrome.

1	The ability to catalog common genetic variants and assay them on array-based platforms and, more recently, to carry out whole-exome sequencing has allowed investigators to collect sample sizes sufficient to detect genetic risk loci for schizophrenia with genome-wide significance. Several of the identified genes are parts of molecular complexes, such as voltage-gated calcium channels (in particular, CACNA1C and CACNB2) and the postsynaptic density of excitatory synapses. As in ASDs, copy number variants, single-nucleotide polymorphisms, and small insertions and deletions are common in schizophrenia. Genes that promote risk for drug addiction have also begun to emerge from large family and population studies. The best-established susceptibility loci are regions on chromosomes 4 and 5 containing GABAA receptor gene clusters linked to alcoholism and the CHRNA5-A3-B4 nicotinic acetylcholine receptor gene cluster on chromosome 15 associated with nicotine and alcohol addiction.

1	A recurrent theme that has emerged from genetic studies of psychiatric disorders is pleiotropy, namely, that many genes are associated Chapter 465e Biology of Psychiatric Disorders Sodium channel, voltage-gated, type I, alpha subunit Sodium channel, voltage-gated, type II, alpha subunit exchanger Glutamate receptor, ionotropic, N-methyl-Daspartate 2B

1	T-box, brain 1 Signal transduction Synaptic function Signal transduction Translation and protein stability Synaptic function Signal transduction Translation and protein stability Synaptic function Signal transduction with multiple psychiatric syndromes. For example, mutations in MECP2, FMR1, and TSC1 and TSC2 (see Table 465e-1 for abbreviations) can cause mental retardation without ASD, others in MECP2 can cause obsessive-compulsive and attention-deficit hyperactivity disorders, some alleles of NRXN1 are associated with symptoms of both ASD and schizophrenia, and common polymorphisms in CACNA1C are strongly associated with both schizophrenia and bipolar disorder. Likewise, duplication of chromosome 16p is associated with both schizophrenia and autism, whereas DiGeorge’s (velocardiofacial) syndrome region deletions and the DISC1 (disrupted in schizophrenia 1) locus on chromosome 22 are associated with schizophrenia, autism, and bipolar disorder. The association of genes with multiple

1	syndrome region deletions and the DISC1 (disrupted in schizophrenia 1) locus on chromosome 22 are associated with schizophrenia, autism, and bipolar disorder. The association of genes with multiple syndromes attests to the complexity of psychiatric disorders and the influence of additional factors that combine to specify the ultimate phenotype, including regulatory variants that determine cell-type specificity and timing of gene expression, protective variants, and epigenetic effects.

1	Studies of signal transduction have revealed numerous intracellular signaling pathways that are perturbed in psychiatric disorders, and such research has provided insight into development of new therapeutic agents. For example, lithium is a highly effective drug for bipolar disorder and competes with magnesium to inhibit numerous magnesium-dependent enzymes, including the enzyme GSK3β and several enzymes involved in phosphoinositide signaling that lead to activation of protein kinase C. These findings have led to discovery programs focused on developing GSK3β or protein kinase C inhibitors as potential novel treatments for mood disorders.

1	The observations that tricyclic antidepressants (e.g., imipramine) inhibit serotonin and/or norepinephrine reuptake and that monoamine oxidase inhibitors (e.g., tranylcypromine) are effective antidepressants initially led to the view that depression is caused by a deficiency of these monoamines. However, this hypothesis has not been substantiated. A cardinal feature of these drugs is that longterm administration is needed for their antidepressant effects. This means that their short-term actions, namely promotion of serotonin or norepinephrine function, are not per se antidepressant but rather induce a cascade of adaptations in the brain that underlie their clinical effects. The nature of these therapeutic drug-induced adaptations has not been identified with certainty. One theory holds that, in a subset of depressed patients who display upregulation of the hypothalamic-pituitary-adrenal (HPA) axis characterized by increased secretion of corticotropin-releasing factor (CRF) and

1	that, in a subset of depressed patients who display upregulation of the hypothalamic-pituitary-adrenal (HPA) axis characterized by increased secretion of corticotropin-releasing factor (CRF) and glucocorticoids, excessive glucocorticoids cause atrophy of hippocampal neurons, which is associated with reduced hippocampal volumes seen clinically. Chronic antidepressant administration might reverse this atrophy by increasing brain-derived neurotrophic factor (BDNF) in hippocampus. A role for stress-induced decreases in the generation of newly born hippocampal granule cell neurons, and its reversal by antidepressants through BDNF and other growth factors, has also been suggested.

1	A major advance in recent years has been the identification of several rapidly acting antidepressants with non–monoamine-based mechanisms of action. The best established is ketamine, a noncompetitive antagonist of N-methyl-d-aspartate (NMDA) glutamate receptors, which exerts rapid (hours) and robust antidepressant effects in severely depressed patients who have not responded to other treatments. Ketamine, which at higher doses is psychotomimetic and anesthetic, exerts these antidepressant effects at low doses with minimal side effects. However, the response to ketamine is transient, which has led to several approaches to maintain treatment response, such as repeated ketamine delivery. The mechanism underlying ketamine’s antidepressant action is not known, but its striking clinical efficacy has stimulated animal research on the role of glutamate neurotransmission and synaptic plasticity in key limbic regions. Recent evidence supports a role for TORC1 activation because administration

1	has stimulated animal research on the role of glutamate neurotransmission and synaptic plasticity in key limbic regions. Recent evidence supports a role for TORC1 activation because administration of rapamycin blocks the antidepressant-like effects of ketamine in animal models. Mechanisms by which ketamine activates TORC1 are currently an active area of investigation.

1	A major goal in the field of drug abuse has been to identify neuroadaptive mechanisms that lead from recreational use to addiction. Such research has determined that repeated intake of abused drugs induces specific changes in cellular signal transduction, leading to changes in synaptic strength (long-term potentiation or depression) and neuronal structure (altered dendritic branching or cell soma size) within the brain’s reward circuitry. These modifications are mediated in part by changes in gene expression, achieved by drug regulation of transcription factors (e.g., CREB [cAMP response element-binding protein] and ΔFosB [a Fos family protein]) and their target genes. Such alterations in gene expression are associated with lasting alterations in epigenetic modifications, including histone acetylation and methylation and DNA methylation. These adaptations provide opportunities for developing treatments targeted to drug-addicted individuals. The fact that the spectrum of these

1	histone acetylation and methylation and DNA methylation. These adaptations provide opportunities for developing treatments targeted to drug-addicted individuals. The fact that the spectrum of these adaptations partly differs depending on the particular addictive substance used creates opportunities for treatments that are specific for different classes of addictive drugs and that may, therefore, be less likely to disturb basic mechanisms that govern normal motivation and reward.

1	Increasingly, causal relationships are being established between individual molecular and cellular adaptations and specific behavioral abnormalities that characterize the addicted state. For example, acute activation of μ-opioid receptors by morphine or other opiates activates Gi/o proteins, leading to inhibition of adenylyl cyclase, resulting in reduced cyclic AMP (cAMP) production, protein kinase A (PKA) activation, and activation of the transcription factor CREB. Repeated administration of these drugs (Fig. 465e-1) evokes a homeostatic response involving upregulation of adenylyl cyclases and PKA and increased activation of CREB. Such upregulation of cAMP-CREB signaling has been identified in the locus coeruleus, periaqueductal gray, ventral tegmental area (VTA), nucleus accumbens, and several other CNS regions, and contributes to opiate craving and signs of opiate withdrawal. The fact that endogenous opioid peptides do not produce tolerance and dependence, while morphine and heroin

1	other CNS regions, and contributes to opiate craving and signs of opiate withdrawal. The fact that endogenous opioid peptides do not produce tolerance and dependence, while morphine and heroin do, may relate to the observation that, unlike endogenous opioids, morphine and heroin are weak inducers of μ-opioid receptor desensitization and endocytosis. Therefore, these drugs cause prolonged receptor activation and inhibition of adenylyl cyclases, which provides a powerful stimulus for the upregulation of cAMP-CREB signaling that characterizes the opiate-dependent state.

1	The study of interconnected brain circuits that drive behavior has been greatly advanced through newer methods in brain imagining that have documented abnormalities in neural function and connectivity in psychiatric disorders. The past decade has also witnessed the development of revolutionary new techniques—optogenetics and designer receptors and ligands—that provide unprecedented temporal and spatial control of neural circuits and permit detection of neural activity in real time in awake, behaving animals.

1	Positron emission tomography (PET), diffusion tensor imaging (DTI), and functional magnetic resonance imaging (fMRI) have identified neural circuits that contribute to psychiatric disorders, for example, defining the neural circuitry of mood within the brain’s limbic system (Fig. 465e-2). Integral to this system are the nucleus accumbens (important also for brain reward—see below), amygdala, hippocampus, and regions of prefrontal cortex. Recent optogenetic research in animals, where the activity of specific types of neurons in defined circuits can be controlled with light, has confirmed the importance of this limbic circuitry in controlling depression-related behavioral abnormalities. Given that many symptoms of depression (so-called neurovegetative symptoms) involve physiologic functions, a key role for the hypothalamus is also presumed. A subset of depressed individuals shows a small reduction in hippocampal size, as noted above. In addition, brain imaging investigations have

1	a key role for the hypothalamus is also presumed. A subset of depressed individuals shows a small reduction in hippocampal size, as noted above. In addition, brain imaging investigations have revealed increased activation of the amygdala by negative stimuli and reduced activation of the nucleus accumbens by rewarding stimuli. There is also evidence for altered activity in prefrontal cortex, such as hyperactivity of subgenual area 25 in anterior cingulate cortex. Such findings

1	FIGURE 465e-1 Opiate action in the locus coeruleus (LC). Binding of opiate agonists to μ-opioid receptors catalyzes nucleotide exchange on Gi and Go proteins, leading to inhibition of adenylyl cyclase, neuronal hyperpolarization via activation of K+ channels, and inhibition of neurotransmitter release via inhibition of Ca2+ channels. Inhibition of adenylyl cyclase (AC) reduces protein kinase A (PKA) activity and phosphorylation of several PKA substrate proteins, thereby altering their function. For example, opiates reduce phosphorylation of the cAMP response element-binding protein (CREB), which appears to initiate longer term changes in neuronal function. Chronic administration of opiates increases levels of AC isoforms, PKA catalytic (C) and regulatory (R) subunits, and the phosphorylation of several proteins, including CREB (indicated by red arrows). These changes contribute to the altered phenotype of the drug-addicted state. For example, the excitability of LC neurons is

1	of several proteins, including CREB (indicated by red arrows). These changes contribute to the altered phenotype of the drug-addicted state. For example, the excitability of LC neurons is increased by enhanced cAMP signaling, although the ionic basis of this effect remains unknown. Activation of CREB causes upregulation of AC isoforms and tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis.

1	have led to trials of deep brain stimulation (DBS) of either the nucleus accumbens or subgenual area 25, which appears to be therapeutic in some severely depressed individuals.

1	In schizophrenia, structural and functional imaging studies have identified a 3% loss of brain volume, most of which is in gray matter. This loss is progressive, and cortical gray matter appears to be particularly affected over time. The temporal lobes, particularly the left superior temporal gyrus, Heschl gyrus, and planum temporale, are often the most severely affected. The rate of loss in these regions as well as in frontal and parietal lobes appears to be greatest early in the course of the disease. Functional imaging studies provide evidence of reduced metabolic (presumably neural) activity in the dorsolateral prefrontal cortex at rest and when performing tests of executive function, including working memory. There is also evidence for impaired structural and task-related functional connectivity, mainly in frontal and temporal lobes. These neuroimaging findings in schizophrenia have been confirmed in pathologic studies that show enlargement of the ventricular

1	These neuroimaging findings in schizophrenia have been confirmed in pathologic studies that show enlargement of the ventricular Chapter 465e Biology of Psychiatric Disorders the rewards (nucleus accumbens), memories of reward-related cues (amygdala, hippocampus), and executive control of obtaining rewards (prefrontal cortex).

1	Drugs of abuse alter neurotransmission through initial actions at different classes of ion channels, neurotransmitter receptors, or neurotransmitter transporters (Table 465e-2). Studies in animal models have demonstrated that although the initial targets differ, the actions of these drugs converge on the brain’s reward circuitry by promoting dopamine neurotransmission in the nucleus accumbens and other limbic targets of the VTA. In addition, some drugs promote activation of opioid and cannabinoid receptors, which modulate this reward circuitry. By these mechanisms, drugs of abuse produce powerful rewarding signals, which, after repeated drug administration, corrupt a vulnerable brain’s reward circuitry in ways that promote addiction. Three major pathologic adaptations have been described. First, drugs produce tolerance and dependence in reward circuits, which promote escalating drug intake and a negative emotional state during drug withdrawal that promotes relapse. Second,

1	described. First, drugs produce tolerance and dependence in reward circuits, which promote escalating drug intake and a negative emotional state during drug withdrawal that promotes relapse. Second, sensitization to the

1	FIGURE 465e-2 Neural circuitry of depression and addiction. The figure shows a simplified rewarding effects of the drugs and associ summary of a series of limbic circuits in brain that regulate mood and motivation and are impli ated cues is seen during prolonged absti cated in depression and addiction. Shown in the figure are the hippocampus (HP) and amyg nence and also triggers relapse. Third, dala (Amy) in the temporal lobe, regions of prefrontal cortex, nucleus accumbens (NAc), and executive function is impaired in such a hypothalamus (Hyp). Only a subset of the known interconnections among these brain regions is way as to increase impulsivity and com- shown. Also shown is the innervation of several of these brain regions by monoaminergic neurons. pulsivity, both of which promote relapse. The ventral tegmental area (VTA) provides dopaminergic input to each of the limbic structures.

1	pulsivity, both of which promote relapse. The ventral tegmental area (VTA) provides dopaminergic input to each of the limbic structures. Norepinephrine (from the locus coeruleus [LC]) and serotonin (from the dorsal raphe [DR] and that addictive drugs, as well as craving for other raphe nuclei) innervate all of the regions shown. In addition, there are strong connections them, activate the brain’s reward circuitry. between the hypothalamus and the VTA-NAc pathway. Important peptidergic projections from In addition, patients who abuse alcohol the hypothalamus include those from the arcuate nucleus that release β-endorphin and melano cortin and from the lateral hypothalamus that release orexin.

1	system and reduction of cortical and subcortical gray matter in frontal and temporal lobes and in the limbic system. The reduction in cortical thickness is associated with increased cell packing density and reduced neuropil (defined as axons, dendrites, and glial cell processes) without an apparent change in neuronal cell number. Specific classes of inter-neurons in prefrontal cortex consistently show reduced expression of the gene encoding the enzyme glutamic acid decarboxylase 1 (GAD1), which synthesizes γ-aminobutyric acid (GABA), the principal inhibitory neurotransmitter in the brain. Neuregulin 1 (NRG1), a member of the epidermal growth factor (EGF) family of growth factors, and its receptor ERBB4, have been implicated in schizophrenia, and they serve important roles in the maturation of GABAergic interneurons in cerebral cortex; loss of NRG1-ERBB4 in mice leads to a reduced neuropil, thus phenocopying a pathologic finding in schizophrenia. These findings are consistent with one

1	of GABAergic interneurons in cerebral cortex; loss of NRG1-ERBB4 in mice leads to a reduced neuropil, thus phenocopying a pathologic finding in schizophrenia. These findings are consistent with one working hypothesis of schizophrenia as a developmental neurodegenerative disorder due in part to loss of cortical interneurons in frontal and temporal lobes.

1	Work in rodent and nonhuman primate models of addiction has established the brain’s reward regions as key neural substrates for the acute actions of drugs of abuse and for addiction induced by repeated drug administration (Fig. 465e-2). Midbrain dopamine neurons in the VTA function normally as rheostats of reward: they are activated by natural rewards (food, sex, social interaction) or even by the expectation of such rewards, and many are suppressed by the absence of an expected reward or by aversive stimuli. These neurons thereby transmit crucial survival signals to the rest of the limbic brain to promote reward-related behavior, including motor responses to seek and obtain matter in the prefrontal cortex as well as reduced activity in anterior cingulate and orbitofrontal cortex during tasks of attention and inhibitory control. It is thought that damage to these cortical areas contributes to addiction by impairing decision-making and increasing impulsivity.

1	There is increasing evidence for the involvement of inflammatory mechanisms in a subset of depressed patients. These individuals display elevated blood levels of interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and other cytokines. Moreover, rodents exposed to chronic stress exhibit similar increases in peripheral cytokines, and peripheral or central delivery of those cytokines to normal rodents increases their susceptibility to chronic stress. These findings have led to the novel idea of using peripheral cytokines as biomarkers of a subtype of depression and the potential utility of developing new antidepressants that oppose cytokine action.

1	Recent evidence has also linked proinflammatory signaling in the brain to addiction, particularly to alcohol. Human alcoholism is associated with impaired innate immunity, increases in circulating proinflammatory cytokines, and an increase in brain levels of the cytokine monocyte chemotactic protein-1 (MCP-1, also referred to as CCL2). Many of these cytokines are produced by astrocytes and microglia, and by neurons under certain pathologic conditions, where they play important roles in modifying neuronal function and plasticity. For example MCP-1 modulates the release of certain neurotransmitters and, when administered into the VTA, increases neuronal excitability, promotes dopamine release, and increases locomotor activity. Recent gene expression array studies of alcohol drinking in mice have Opiates Endorphins, enkephalins Psychostimulants Dopamine (cocaine, amphetamine, methamphetamine) Marijuana Endocannabinoids (anandamide, 2arachidonoylglycerol)

1	Opiates Endorphins, enkephalins Psychostimulants Dopamine (cocaine, amphetamine, methamphetamine) Marijuana Endocannabinoids (anandamide, 2arachidonoylglycerol) Dopamine transporter (antagonist—cocaine; reverse transport—amphetamine, methamphetamine) identified a network of regulated cytokines in brain, and a role in regu-465e-5 lation of alcohol consumption has been recently validated for several of them, including IL-6. A major focus of current research is to define the site and mechanism by which proinflammatory cytokines impair brain function to elicit a depressive episode or promote drug abuse. This brief narrative illustrates the substantial progress that is being made in understanding the genetic and neurobiologic basis of mental illness. It is anticipated that biologic measures will be used increasingly to diagnosis and subtype a psychiatric disorder and that targeted therapeutics will become available to treat them. Chapter 465e Biology of Psychiatric Disorders

1	Chapter 465e Biology of Psychiatric Disorders Victor I. Reus Mental disorders are common in medical practice and may present either as a primary disorder or as a comorbid condition. The prevalence of mental or substance use disorders in the United States is approximately 30%, but only one-third of affected individuals are currently receiving treatment. Global burden of disease statistics indicate that 4 of the 10 most important causes of morbidity and attendant health care costs worldwide are psychiatric in origin.

1	Changes in health care delivery underscore the need for primary care physicians to assume responsibility for the initial diagnosis and treatment of the most common mental disorders. Prompt diagnosis is essential to ensure that patients have access to appropriate medical services and to maximize the clinical outcome. Validated patient-based questionnaires have been developed that systematically probe for signs and symptoms associated with the most prevalent psychiatric diagnoses and guide the clinician into targeted assessment. The Primary Care Evaluation of Mental Disorders (PRIME-MD; and a self-report form, the Patient Health Questionnaire) and the Symptom-Driven Diagnostic System for Primary Care (SDDS-PC) are inventories that require only 10 min to complete and link patient responses to the formal diagnostic criteria of anxiety, mood, somatoform, and eating disorders and to alcohol abuse or dependence.

1	A physician who refers patients to a psychiatrist should know not only when doing so is appropriate but also how to refer, because societal misconceptions and the stigma of mental illness impede the process. Primary care physicians should base referrals to a psychiatrist on the presence of signs and symptoms of a mental disorder and not simply on the absence of a physical explanation for a patient’s complaint. The physician should discuss with the patient the reasons for requesting the referral or consultation and provide reassurance that he or she will continue to provide medical care and work collaboratively with the mental health professional. Consultation with a psychiatrist or transfer of care is appropriate when physicians encounter evidence of psychotic symptoms, mania, severe depression, or anxiety; symptoms of posttraumatic stress disorder (PTSD); suicidal or homicidal preoccupation; or a failure to respond to first-order treatment. This chapter reviews the clinical

1	depression, or anxiety; symptoms of posttraumatic stress disorder (PTSD); suicidal or homicidal preoccupation; or a failure to respond to first-order treatment. This chapter reviews the clinical assessment and treatment of some of the most common mental disorders presenting in primary care and is based on the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5), the framework for categorizing psychiatric illness used in the United States. Eating disorders are discussed later in this chapter, and the biology of psychiatric and addictive disorders is discussed in Chap. 465e.

1	The DSM-5 and the tenth revision of the International

1	Classification of Diseases (ICD-10), which is used more com monly worldwide, have taken somewhat differing approaches to the diagnosis of mental illness, but considerable effort has been expended to provide an operational translation between the two nosologies. Both systems are in essence purely descriptive and emphasize clinical pragmatism, in distinction to the Research Domain Criteria (RDOC) proposed by National Institute of Mental Health, which aspires to provide a causal framework for classification of behavioral disturbance. None of these diagnostic systems has as yet achieved adequate validation. The Global Burden of Disease Study 2010, using available epidemiologic data, nevertheless has reinforced the conclusion that, regardless of nosologic differences, mental and substance abuse disorders are the major cause of life-years lost to disability among all medical illnesses. There is general agreement that high-income countries will need to build capacity in professional training

1	disorders are the major cause of life-years lost to disability among all medical illnesses. There is general agreement that high-income countries will need to build capacity in professional training in lowand middle-income countries in order to provide an adequate balanced care model for the delivery of evidence-based therapies for mental disorders. Recent surveys that indicate a dramatic increase in mental disorder prevalence in rapidly developing countries, such as China, may reflect both an increased recognition of the issue, but also the consequence of social turmoil, stigma, and historically inadequate resources. The need for improved prevention strategies and for more definitive and effective interventional treatments remains a global concern.

1	Anxiety disorders, the most prevalent psychiatric illnesses in the general community, are present in 15–20% of medical clinic patients. Anxiety, defined as a subjective sense of unease, dread, or foreboding, can indicate a primary psychiatric condition or can be a component of, or reaction to, a primary medical disease. The primary anxiety disorders are classified according to their duration and course and the existence and nature of precipitants. When evaluating the anxious patient, the clinician must first determine whether the anxiety antedates or postdates a medical illness or is due to a medication side effect. Approximately one-third of patients presenting with anxiety have a medical etiology for their psychiatric symptoms, but an anxiety disorder can also present with somatic symptoms in the absence of a diagnosable medical condition.

1	PANIC DISORDER Clinical Manifestations Panic disorder is defined by the presence of recurrent and unpredictable panic attacks, which are distinct episodes of intense fear and discomfort associated with a variety of physical symptoms, including palpitations, sweating, trembling, shortness of breath, chest pain, dizziness, and a fear of impending doom or death. Paresthesias, gastrointestinal distress, and feelings of unreality are also common. Diagnostic criteria require at least 1 month of concern or worry about the attacks or a change in behavior related to them. The lifetime prevalence of panic disorder is 2–3%. Panic attacks have a sudden onset, developing within 10 min and usually resolving over the course of an hour, and they occur in an unexpected fashion. Some may occur when waking from sleep. The frequency and severity of panic attacks vary, ranging from once a week to clusters of attacks separated by months of well-being. The first attack is usually outside the home, and onset

1	sleep. The frequency and severity of panic attacks vary, ranging from once a week to clusters of attacks separated by months of well-being. The first attack is usually outside the home, and onset is typically in late adolescence to early adulthood. In some individuals, anticipatory anxiety develops over time and results in a generalized fear and a progressive avoidance of places or situations in which a panic attack might recur. Agoraphobia, which occurs commonly in patients with panic disorder, is an acquired irrational fear of being in places where one might feel trapped or unable to escape. It may, however, be diagnosed even if panic disorder is not present. Typically, it leads the patient into a progressive restriction in lifestyle and, in a literal sense, in geography. Frequently, patients are embarrassed that they are housebound and dependent on the company of others to go out into the world and do not volunteer this information; thus, physicians will fail to recognize the

1	patients are embarrassed that they are housebound and dependent on the company of others to go out into the world and do not volunteer this information; thus, physicians will fail to recognize the syndrome if direct questioning is not pursued.

1	Differential Diagnosis A diagnosis of panic disorder is made after a medical etiology for the panic attacks has been ruled out. A variety of cardiovascular, respiratory, endocrine, and neurologic conditions can present with anxiety as the chief complaint. Patients with true panic disorder will often focus on one specific feature to the exclusion of others. For example, 20% of patients who present with syncope as a primary medical complaint have a primary diagnosis of a mood, anxiety, or substance abuse disorder, the most common being panic disorder. The differential diagnosis of panic disorder is complicated by a high rate of comorbidity with other psychiatric conditions, especially alcohol and benzodiazepine abuse, which patients initially use in an attempt at self-medication. Some 75% of panic disorder patients will also satisfy criteria for major depression at some point in their illness.

1	When the history is nonspecific, physical examination and focused laboratory testing must be used to rule out anxiety states resulting from medical disorders such as pheochromocytoma, thyrotoxicosis, or hypoglycemia. Electrocardiogram (ECG) and echocardiogram may detect some cardiovascular conditions associated with panic such as paroxysmal atrial tachycardia and mitral valve prolapse. In two studies, panic disorder was the primary diagnosis in 43% of patients with chest pain who had normal coronary angiograms and was present in 9% of all outpatients referred for cardiac evaluation. Panic disorder has also been diagnosed in many patients referred for pulmonary function testing or with symptoms of irritable bowel syndrome.

1	Etiology and Pathophysiology The etiology of panic disorder is unknown but appears to involve a genetic predisposition, altered autonomic responsivity, and social learning. Panic disorder shows familial aggregation; the disorder is concordant in 30–45% of monozygotic twins, and genome-wide screens have identified suggestive risk loci. Acute panic attacks appear to be associated with increased noradrenergic discharges in the locus coeruleus. Intravenous infusion of sodium lactate evokes an attack in two-thirds of panic disorder patients, as do the α2-adrenergic antagonist yohimbine, cholecystokinin tetrapeptide (CCK-4), and carbon dioxide inhalation. It is hypothesized that each of these stimuli activates a pathway involving noradrenergic neurons in the locus coeruleus and serotonergic neurons in the dorsal raphe. Agents that block serotonin reuptake can prevent attacks. Patients with panic disorder have a heightened sensitivity to somatic symptoms, which triggers increasing arousal,

1	in the dorsal raphe. Agents that block serotonin reuptake can prevent attacks. Patients with panic disorder have a heightened sensitivity to somatic symptoms, which triggers increasing arousal, setting off the panic attack; accordingly, therapeutic intervention involves altering the patient’s cognitive interpretation of anxiety-producing experiences as well as preventing the attack itself.

1	Achievable goals of treatment are to decrease the frequency of panic attacks and to reduce their intensity. The cornerstone of drug therapy is antidepressant medication (Tables 466-1 through 466-3). Selective serotonin reuptake inhibitors (SSRIs) benefit the majority of panic disorder patients and do not have the adverse effects of tricyclic antidepressants (TCAs). Fluoxetine, paroxetine, sertraline, and the selective serotonin-norepinephrine reuptake inhibitor (SNRI) venlafaxine have received approval from the U.S. Food and Drug Administration (FDA) for this indication. These drugs should be started at one-third to one-half of their usual antidepressant dose (e.g., 5–10 mg fluoxetine, 25–50 mg sertraline, 10 mg paroxetine, venlafaxine 37.5 2709 mg). Monoamine oxidase inhibitors (MAOIs) are also effective and may specifically benefit patients who have comorbid features of atypical depression (i.e., hypersomnia and weight gain). Insomnia, orthostatic hypotension, and the need to

1	are also effective and may specifically benefit patients who have comorbid features of atypical depression (i.e., hypersomnia and weight gain). Insomnia, orthostatic hypotension, and the need to maintain a low-tyramine diet (avoidance of cheese and wine) have limited their use, however. Antidepressants typically take 2–6 weeks to become effective, and doses may need to be adjusted based on the clinical response.

1	Because of anticipatory anxiety and the need for immediate relief of panic symptoms, benzodiazepines are useful early in the course of treatment and sporadically thereafter (Table 466-4). For example, alprazolam, starting at 0.5 mg qid and increasing to 4 mg/d in divided doses, is effective, but patients must be monitored closely, as some develop dependence and begin to escalate the dose of this medication. Clonazepam, at a final maintenance dose of 2–4 mg/d, is also helpful; its longer half-life permits twice-daily dosing, and patients appear less likely to develop dependence on this agent.

1	Early psychotherapeutic intervention and education aimed at symptom control enhance the effectiveness of drug treatment. Patients can be taught breathing techniques, be educated about physiologic changes that occur with panic, and learn to expose themselves voluntarily to precipitating events in a treatment program spanning 12–15 sessions. Homework assignments and monitored compliance are important components of successful treatment. Once patients have achieved a satisfactory response, drug treatment should be maintained for 1–2 years to prevent relapse. Controlled trials indicate a success rate of 75–85%, although the likelihood of complete remission is somewhat lower.

1	GENERALIZED ANXIETY DISORDER Clinical Manifestations Patients with generalized anxiety disorder (GAD) have persistent, excessive, and/or unrealistic worry associated with muscle tension, impaired concentration, autonomic arousal, feeling “on edge” or restless, and insomnia (Table 466-5). Onset is usually before age 20 years, and a history of childhood fears and social inhibition may be present. The lifetime prevalence of GAD is 5–6%; the risk is higher in first-degree relatives of patients with the diagnosis. Interestingly, family studies indicate that GAD and panic disorder segregate independently. More than 80% of patients with GAD also suffer from major depression, dysthymia, or social phobia. Comorbid substance abuse is common in these patients, particularly alcohol and/or sedative/hypnotic abuse. Patients with GAD worry excessively over minor matters, with life-disrupting effects; unlike in panic disorder, complaints of shortness of breath, palpitations, and tachycardia are

1	abuse. Patients with GAD worry excessively over minor matters, with life-disrupting effects; unlike in panic disorder, complaints of shortness of breath, palpitations, and tachycardia are relatively rare.

1	Etiology and Pathophysiology All anxiogenic agents act on the γ-aminobutyric acid (GABA)A receptor/chloride ion channel complex, implicating this neurotransmitter system in the pathogenesis of anxiety and panic attacks. Benzodiazepines are thought to bind two separate GABAA receptor sites: type I, which has a broad neuroanatomic distribution, and type II, which is concentrated in the hippocampus, striatum, and neocortex. The antianxiety effects of the various benzodiazepines are influenced by their relative binding to alpha 2 and 3 subunits of the GABAA receptor, and sedation and memory impairment to the alpha 1 subunit, Serotonin (5-hydroxytryptamine [5-HT]) and 3α-reduced neuroactive steroids (allosteric modulators of GABAA) also appear to have a role in anxiety, and buspirone, a partial 5-HT1A receptor agonist, and certain 5-HT2A and 5-HT2C receptor antagonists (e.g., nefazodone) may have beneficial effects.

1	A combination of pharmacologic and psychotherapeutic interventions is most effective in GAD, but complete symptomatic relief is rare. A short course of a benzodiazepine is usually indicated, preferably lorazepam, oxazepam, or alprazolam. (The first two of these agents are metabolized via conjugation rather than oxidation and thus do not accumulate if hepatic function is impaired; the latter Once-daily dosing, usually qhs; blood levels of most TCAs available; can be lethal in overdose (lethal dose = 2 g); nortriptyline best tolerated, especially by elderly Nausea, dizziness, insomnia Nausea, dizziness, headache, insomnia, constipation Somnolence, weight gain; neutropenia rare Nausea, diarrhea, headache; dosage adjustment if given with CYP3A4 inhibitor/stimulator Nausea, diarrhea, sweating, headache; low incidence of sedation or weight gain Nausea, constipation, sweating; rare increase in blood pressure/pulse

1	Nausea, diarrhea, sweating, headache; low incidence of sedation or weight gain Nausea, constipation, sweating; rare increase in blood pressure/pulse Bid-tid dosing (extended release available); lower potential for drug interactions than SSRIs; contraindicated with MAOIs Primary metabolite of venlafaxine; no increased efficacy with higher dosing May have utility in treatment of neuropathic pain and stress incontinence No specific p450 effects; 5-HT3a and 5-HT7 receptor antagonist, 5-HT1b partial agonist, and 5-HT1a agonist Most noradrenergic of SNRIs Sedation; dry mouth; ventricular irritability; postural hypotension; priapism rare Daytime somnolence, dizziness, nausea Sexual dysfunction Tid dosing, but sustained release also available; fewer sexual side effects than SSRIs or TCAs; may be useful for adult ADD Useful in low doses for sleep because of sedating effects with no anticholinergic side effects

1	Useful in low doses for sleep because of sedating effects with no anticholinergic side effects Insomnia; hypotension; edema; anorgasmia; weight gain; neuropathy; hypertensive crisis; toxic reactions with SSRIs; narcotics Local skin reaction hypertension May be more effective in patients with atypical features or treatment-refractory depression Less weight gain and hypotension than phenelzine No dietary restrictions with 6 mg dose Abbreviations: ADD, attention deficit disorder; EPS, extrapyramidal symptoms; FDA, U.S. Food and Drug Administration; GI, gastrointestinal; MAOIs, monoamine oxidase inhibitors; OCD, obsessive-compulsive disorder; SSRIs, selective serotonin reuptake inhibitors; TCAs, tricyclic antidepressants.

1	also has limited active metabolites.) Treatment should be initiated at the lowest dose possible and prescribed on an as-needed basis as symptoms warrant. Benzodiazepines differ in their milligram per kilogram potency, half-life, lipid solubility, metabolic pathways, and presence of active metabolites. Agents that are absorbed rapidly and are lipid soluble, such as diazepam, have a rapid onset of action and a higher abuse potential. Benzodiazepines should generally not be prescribed for >4–6 weeks because of the development of tolerance and the risk of abuse and dependence. Withdrawal must be closely monitored as relapses can occur. It is important to warn patients that concomitant use of alcohol or other sedating drugs may exacerbate side effects and impair their ability to function. An optimistic approach that encourages the patient to clarify environmental precipitants, anticipate his or her reactions, and plan effective response strategies is an essential element of therapy.

1	Adverse effects of benzodiazepines generally parallel their relative half-lives. Longer-acting agents, such as diazepam, chlordiazepoxide, flurazepam, and clonazepam, tend to accumulate active metabolites, with resultant sedation, impairment of cognition, and poor psychomotor performance. Shorter-acting compounds, such as alprazolam, lorazepam, and oxazepam, can produce daytime anxiety, early morning insomnia, and, with discontinuation, rebound anxiety and insomnia. Although patients develop tolerance to the sedative effects of benzodiazepines, they are less likely to habituate to the adverse psychomotor effects. Withdrawal from the longer half-life benzodiazepines can be accomplished through gradual, stepwise dose reduction (by 10% every 1–2 weeks) over 6–12 weeks. It is usually more difficult to taper patients off shorter-acting benzodiazepines. Physicians may need to switch the patient to a benzodiazepine with a longer half-life or use an

1	Nausea, loss of Usually short-lived and dose-related; consider appetite temporary dose reduction or administration with food and antacids Diarrhea Famotidine, 20–40 mg/d Constipation Wait for tolerance; try diet change, stool softener, exercise; avoid laxatives Anorgasmia/ Bethanechol, 10–20 mg, 2 h before activity, or impotence; impaired cyproheptadine, 4–8 mg 2 h before activity, or ejaculation bupropion, 100 mg bid, or amantadine, 100 mg Orthostasis Tolerance unlikely; increase fluid intake, use calf exercises/support hose; fludrocortisone, 0.025 mg/d Dry mouth, eyes Maintain good oral hygiene; use artificial tears, sugar-free gum Tremor/jitteriness Antiparkinsonian drugs not effective; use dose reduction/slow increase; lorazepam, 0.5 mg bid, or propranolol, 10–20 mg bid Insomnia Schedule all doses for the morning; trazodone, 50–100 mg qhs Sedation Caffeine; schedule all dosing for bedtime; bupropion, 75–100 mg in afternoon

1	Insomnia Schedule all doses for the morning; trazodone, 50–100 mg qhs Sedation Caffeine; schedule all dosing for bedtime; bupropion, 75–100 mg in afternoon Headache Evaluate diet, stress, other drugs; try dose reduction; amitriptyline, 50 mg/d Loss of therapeutic ben-Related to tolerance? Increase dose or drug holiefit over time day; add amantadine, 100 mg bid, buspirone, 10 mg tid, or pindolol, 2.5 mg bid adjunctive medication such as a beta blocker or carbamazepine, before attempting to discontinue the benzodiazepine. Withdrawal reactions vary in severity and duration; they can include depression, anxiety, lethargy, diaphoresis, autonomic arousal, and, rarely, seizures. Serotonergic agonists, e.g., tryptophan, fenfluramine, tryptans Drugs that are metabolized by P450 isoenzymes: tricyclics, other SSRIs, antipsychotics, beta blockers, codeine, triazolobenzodiazepines, calcium channel blockers Drugs that are bound tightly to plasma proteins, e.g., warfarin

1	Drugs that are bound tightly to plasma proteins, e.g., warfarin Drugs that inhibit the metabolism of SSRIs by P450 isoenzymes, e.g., quinidine Serotonin syndrome—absolute contraindication Increased bleeding secondary to displacement Abbreviation: SSRIs, selective serotonin reuptake inhibitors.

1	Increased bleeding secondary to displacement Abbreviation: SSRIs, selective serotonin reuptake inhibitors. Buspirone is a nonbenzodiazepine anxiolytic agent. It is non-sedating, does not produce tolerance or dependence, does not interact with benzodiazepine receptors or alcohol, and has no abuse or disinhibition potential. However, it requires several weeks to take effect and requires thrice-daily dosing. Patients who were previously responsive to a benzodiazepine are unlikely to rate buspirone as equally effective, but patients with head injury or dementia who have symptoms of anxiety and/or agitation may do well with this agent. Escitalopram, paroxetine, and venlafaxine are FDA approved for the treatment of GAD, usually at doses that are comparable to their efficacy in major depression, and may be preferable to usage of benzodiazepines in the treatment of chronic anxiety. Benzodiazepines are contraindicated during pregnancy and breast-feeding.

1	Anticonvulsants with GABAergic properties may also be effective against anxiety. Gabapentin, oxcarbazepine, tiagabine, pregabalin, and divalproex have all shown some degree of benefit in a variety of anxiety-related syndromes in off-label usage. Agents that selectively target GABAA receptor subtypes are currently under development, and it is hoped that these will lack the sedating, memory-impairing, and addicting properties of benzodiazepines. Equivalent PO Name Dose, mg Onset of Action Half-Life, h Comments

1	Benzodiazepines Diazepam (Valium) 5 Fast 20–70 Active metabolites; quite sedating Flurazepam (Dalmane) 15 Fast 30–100 Flurazepam is a prodrug; metabolites are active; quite sedating Triazolam (Halcion) 0.25 Intermediate 1.5–5 No active metabolites; can induce confusion and delirium, especially in elderly Lorazepam (Ativan) 1 Intermediate 10–20 No active metabolites; direct hepatic glucuronide conjugation; quite sedating; FDA approved for anxiety with depression Alprazolam (Xanax) 0.5 Intermediate 12–15 Active metabolites; not too sedating; FDA approved for panic disorder and anxiety with depression; tolerance and dependence develop easily; difficult to withdraw Chlordiazepoxide (Librium) 10 sedating Temazepam (Restoril) 15 Slow 9–12 No active metabolites; moderately sedating Clonazepam (Klonopin) 0.5 Slow 18–50 No active metabolites; moderately sedating; FDA approved for panic disorder Clorazepate (Tranxene) 15 Fast 40–200 Low sedation; unreliable absorption Nonbenzodiazepines

1	(Klonopin) 0.5 Slow 18–50 No active metabolites; moderately sedating; FDA approved for panic disorder Clorazepate (Tranxene) 15 Fast 40–200 Low sedation; unreliable absorption Nonbenzodiazepines Buspirone (BuSpar) 7.5 2 weeks 2–3 Active metabolites; tid dosing—usual daily dose 10–20 mg tid; nonsedating; no additive effects with alcohol; useful for controlling agitation in demented or brain-injured patients Abbreviation: FDA, U.S. Food and Drug Administration.

1	A. Excessive anxiety and worry (apprehensive expectation), occurring more days than not for at least 6 months, about a number of events or activities (such as work or school performance). B. The individual finds it difficult to control the worry. C. The anxiety and worry are associated with three (or more) of the following six symptoms (with at least some symptoms present for more days than not for the past 6 months): (1) restlessness or feeling keyed up or on edge; irritability; (5) muscle tension; (6) sleep disturbance (difficulty falling or staying asleep, or restless, unsatisfying sleep). D. The anxiety, worry, or physical symptoms cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. E. The disturbance is not attributable to the physiologic effects of a substance (e.g., a drug of abuse, a medication) or another medical condition (e.g., hyperthyroidism). F.

1	E. The disturbance is not attributable to the physiologic effects of a substance (e.g., a drug of abuse, a medication) or another medical condition (e.g., hyperthyroidism). F. The disturbance is not better explained by another mental disorder (e.g., anxiety or worry about having panic attacks in panic disorder, negative evaluation in social anxiety disorder [social phobia], contamination or other obsessions in obsessive-compulsive disorder, separation from attachment figures in separation anxiety disorder, reminders of traumatic events in posttraumatic stress disorder, gaining weight in anorexia nervosa, physical complaints in somatic symptom disorder, perceived appearance flaws in body dysmorphic disorder, having a serious illness in illness anxiety disorder, or the content of delusional beliefs in schizophrenia or delusional disorder). Source: Diagnostic and Statistical Manual of Mental Disorders, 5th ed. Washington, DC, American Psychiatric Association, 2013.

1	PHOBIC DISORDERS Clinical Manifestations The cardinal feature of phobic disorders is a marked and persistent fear of objects or situations, exposure to which results in an immediate anxiety reaction. The patient avoids the phobic stimulus, and this avoidance usually impairs occupational or social functioning. Panic attacks may be triggered by the phobic stimulus or may occur spontaneously. Unlike patients with other anxiety disorders, individuals with phobias usually experience anxiety only in specific situations. Common phobias include fear of closed spaces (claustrophobia), fear of blood, and fear of flying. Social phobia is distinguished by a specific fear of social or performance situations in which the individual is exposed to unfamiliar individuals or to possible examination and evaluation by others. Examples include having to converse at a party, use public restrooms, and meet strangers. In each case, the affected individual is aware that the experienced fear is excessive and

1	by others. Examples include having to converse at a party, use public restrooms, and meet strangers. In each case, the affected individual is aware that the experienced fear is excessive and unreasonable given the circumstance. The specific content of a phobia may vary across gender, ethnic, and cultural boundaries.

1	Phobic disorders are common, affecting ~7–9% of the population. Twice as many females are affected than males. Full criteria for diagnosis are usually satisfied first in early adulthood, but behavioral avoidance of unfamiliar people, situations, or objects dating from early childhood is common. In one study of female twins, concordance rates for agoraphobia, social phobia, and animal phobia were found to be 23% for monozygotic twins and 15% for dizygotic twins. A twin study of fear conditioning, a model for the acquisition of phobias, demonstrated a heritability of 35–45%. Animal studies of fear conditioning have indicated that processing of the fear stimulus occurs through the lateral nucleus of the amygdala, extending through the central nucleus and projecting to the periaqueductal gray region, lateral hypothalamus, and paraventricular hypothalamus.

1	Beta blockers (e.g., propranolol, 20–40 mg orally 2 h before the event) are particularly effective in the treatment of “performance anxiety” (but not general social phobia) and appear to work by blocking the peripheral manifestations of anxiety such as perspiration, tachycardia, palpitations, and tremor. MAOIs alleviate social phobia independently of their antidepressant activity, and paroxetine, sertraline, and venlafaxine have received FDA approval for treatment of social anxiety. Benzodiazepines can be helpful in reducing fearful avoidance, but the chronic nature of phobic disorders limits their usefulness.

1	Behaviorally focused psychotherapy is an important component of treatment because relapse rates are high when medication is used as the sole treatment. Cognitive-behavioral strategies are based on the finding that distorted perceptions and interpretations of fear-producing stimuli play a major role in perpetuation of phobias. Individual and group therapy sessions teach the patient to identify specific negative thoughts associated with the anxiety-producing situation and help to reduce the patient’s fear of loss of control. In desensitization therapy, hierarchies of feared situations are constructed, and the patient is encouraged to pursue and master gradual exposure to the anxiety-producing stimuli. Patients with social phobia, in particular, have a high rate of comorbid alcohol abuse, as well as of other psychiatric conditions (e.g., eating disorders), necessitating the need for parallel management of each disorder if anxiety reduction is to be achieved.

1	STRESS DISORDERS Clinical Manifestations Patients may develop anxiety after exposure to extreme traumatic events such as the threat of personal death or injury or the death of a loved one. The reaction may occur shortly after the trauma (acute stress disorder) or be delayed and subject to recurrence (PTSD) (Table 466-6). In both syndromes, individuals experience associated symptoms of detachment and loss of emotional responsivity. The patient may feel depersonalized and unable to recall specific aspects of the trauma, although typically it is reexperienced through intrusions in thought, dreams, or flashbacks, particularly when cues of the original event are present. Patients often actively avoid stimuli that precipitate recollections of the trauma and demonstrate a resulting increase in vigilance, arousal, and startle response. Patients with stress disorders are at risk for the development of other disorders related to anxiety, mood, and substance abuse (especially alcohol). Between 5

1	arousal, and startle response. Patients with stress disorders are at risk for the development of other disorders related to anxiety, mood, and substance abuse (especially alcohol). Between 5 and 10% of Americans will at some time in their life satisfy criteria for PTSD, with women more likely to be affected than men.

1	Risk factors for the development of PTSD include a past psychiatric history and personality characteristics of high neuroticism and extroversion. Twin studies show a substantial genetic influence on all symptoms associated with PTSD, with less evidence for an environmental effect. Etiology and Pathophysiology It is hypothesized that in PTSD there is excessive release of norepinephrine from the locus coeruleus in response to stress and increased noradrenergic activity at projection sites in the hippocampus and amygdala. These changes theoretically facilitate the encoding of fear-based memories. Greater sympathetic responses to cues associated with the traumatic event occur in PTSD, although pituitary adrenal responses are blunted.

1	Acute stress reactions are usually self-limited, and treatment typically involves the short-term use of benzodiazepines and supportive/expressive psychotherapy. The chronic and recurrent nature of PTSD, however, requires a more complex approach using drug and behavioral treatments. PTSD is highly correlated with peritraumatic dissociative symptoms and the development of an acute stress disorder at the time of the trauma. The SSRIs (paroxetine and sertraline are FDA approved for PTSD), venlafaxine, and topiramate can all reduce anxiety, symptoms of intrusion, and avoidance behaviors, as can prazosin, an α1 antagonist. Propranolol and opiates such as morphine, given during the acute stress period, may have beneficial effects in preventing the development of PTSD, and adjunctive naltrexone can be effective when comorbid alcoholism is present. Trazodone, a sedating antidepressant, is frequently used at night

1	A. Exposure to actual or threatened death, serious injury, or sexual violence in one (or more) of the following ways: 1. Directly experiencing the traumatic event(s). 2. Witnessing, in person, the event(s) as it occurred to others. 3. Learning that the traumatic event(s) occurred to a close family member or close friend. In cases of actual or threatened death of a family member or friend, the event(s) must have been violent or accidental. 4. Experiencing repeated or extreme exposure to aversive details of the traumatic event(s) (e.g., first responders collecting human remains; police officers repeatedly exposed to details of child abuse). B. Presence of one (or more) of the following intrusion symptoms associated with the traumatic event(s), beginning after the traumatic event(s) occurred: 1. Recurrent, involuntary, and intrusive distressing memories of the traumatic event(s). 2.

1	Recurrent, involuntary, and intrusive distressing memories of the traumatic event(s). 2. Recurrent distressing dreams in which the content and/or affect of the dream are related to the traumatic event(s). 3. Dissociative reactions (e.g., flashbacks) in which the individual feels or acts as if the traumatic event(s) were recurring. (Such reactions may occur on a continuum, with the most extreme expression being a complete loss of awareness of present surroundings.) 4. Intense or prolonged psychological distress at exposure to internal or external cues that symbolize or resemble an aspect of the traumatic event(s). 5. Marked physiologic reactions to internal or external cues that symbolize or resemble an aspect of the traumatic event(s). C. Persistent avoidance of stimuli associated with the traumatic event(s), beginning after the traumatic event(s) occurred, as evidenced by one or both of the following: 1.

1	C. Persistent avoidance of stimuli associated with the traumatic event(s), beginning after the traumatic event(s) occurred, as evidenced by one or both of the following: 1. Avoidance of or efforts to avoid distressing memories, thoughts, or feelings about or closely associated with the traumatic event(s). 2. Avoidance of or efforts to avoid external reminders (people, places, conversations, activities, objects, situations) that arouse distressing memories, thoughts, or feelings about or closely associated with the traumatic event(s). D. Negative alterations in cognitions and mood associated with the traumatic event(s), beginning or worsening after the traumatic event(s) occurred as evidenced by two (or more) of the following: 1. Inability to remember an important aspect of the traumatic event(s) (typically due to dissociative amnesia and not to other factors such as head injury, alcohol, or drugs). 2.

1	Inability to remember an important aspect of the traumatic event(s) (typically due to dissociative amnesia and not to other factors such as head injury, alcohol, or drugs). 2. Persistent and exaggerated negative beliefs or expectations about oneself, others, or the world (e.g., “I am bad,”“No one can be trusted,”“The world is completely dangerous,”“My whole nervous system is permanently ruined”). 3. Persistent, distorted cognitions about the cause or consequences of the traumatic event(s) that lead the individual to blame himself/herself or others. 4. Persistent negative emotional state (e.g., fear, horror, anger, guilt, or shame). 5. Markedly diminished interest or participation in significant activities. 6. Feelings of detachment or estrangement from others. 7. Persistent inability to experience positive emotions (e.g., inability to experience happiness, satisfaction, or loving feelings).

1	6. Feelings of detachment or estrangement from others. 7. Persistent inability to experience positive emotions (e.g., inability to experience happiness, satisfaction, or loving feelings). E. Marked alterations in arousal and reactivity associated with the traumatic event(s), beginning or worsening after the traumatic event(s) occurred, as evidenced by two (or more) of the following: 1. Irritable behavior and angry outbursts (with little or no provocation) typically expressed as verbal or physical aggression toward people or objects. 2. Reckless or self-destructive behavior. 3. Hypervigilance. 4. Exaggerated startle response. 5. Problems with concentration. 6. Sleep disturbance (e.g., difficulty falling or staying asleep or restless sleep). F. Duration of the disturbance (criteria B, C, D, and E) is more than 1 month. G. The disturbance causes clinically significant distress or impairment in social, occupational, or other important areas of functioning.

1	G. The disturbance causes clinically significant distress or impairment in social, occupational, or other important areas of functioning. H. The disturbance is not attributable to the physiologic effects of a substance (e.g., medication, alcohol) or another medical condition. Source: Diagnostic and Statistical Manual of Mental Disorders, 5th ed. Washington, DC, American Psychiatric Association, 2013.

1	to help with insomnia (50–150 mg qhs). Carbamazepine, valproic undertaken to relieve the anxiety triggered by the core fear. Patients acid, and alprazolam have also independently produced improve-often conceal their symptoms, usually because they are embarrassed by ment in uncontrolled trials. Psychotherapeutic strategies for PTSD the content of their thoughts or the nature of their actions. Physicians help the patient overcome avoidance behaviors and demoraliza-must ask specific questions regarding recurrent thoughts and behaviors, tion and master fear of recurrence of the trauma; therapies that particularly if physical clues such as chafed and reddened hands or encourage the patient to dismantle avoidance behaviors through patchy hair loss (from repetitive hair pulling, or trichotillomania) are stepwise focusing on the experience of the traumatic event, such present. Comorbid conditions are common, the most frequent being as trauma-focused cognitive-behavioral therapy, exposure

1	are stepwise focusing on the experience of the traumatic event, such present. Comorbid conditions are common, the most frequent being as trauma-focused cognitive-behavioral therapy, exposure therapy, depression, other anxiety disorders, eating disorders, and tics. OCD and eye movement desensitization and reprocessing, are the most has a lifetime prevalence of 2–3% worldwide. Onset is usually gradual, effective. beginning in early adulthood, but childhood onset is not rare. The disorder usually has a waxing and waning course, but some cases may show a steady deterioration in psychosocial functioning.

1	OBSESSIVE-COMPULSIVE DISORDER Etiology and Pathophysiology A genetic contribution to OCD is sug-Clinical Manifestations Obsessive-compulsive disorder (OCD) is char-gested by twin studies, but no susceptibility gene for OCD has been acterized by obsessive thoughts and compulsive behaviors that impair identified to date. Family studies show an aggregation of OCD with everyday functioning. Fears of contamination and germs are com-Tourette’s disorder, and both are more common in males and in firstmon, as are handwashing, counting behaviors, and having to check born children. and recheck such actions as whether a door is locked. The degree to The anatomy of obsessive-compulsive behavior is thought to which the disorder is disruptive for the individual varies, but in all include the orbital frontal cortex, caudate nucleus, and globus pal-cases, obsessive-compulsive activities take up >1 h per day and are lidus. The caudate nucleus appears to be involved in the acquisition 2714 and

1	frontal cortex, caudate nucleus, and globus pal-cases, obsessive-compulsive activities take up >1 h per day and are lidus. The caudate nucleus appears to be involved in the acquisition 2714 and maintenance of habit and skill learning, and interventions that are successful in reducing obsessive-compulsive behaviors also decrease metabolic activity measured in the caudate.

1	Clomipramine, fluoxetine, fluvoxamine, and sertraline are approved for the treatment of OCD in adults (fluvoxamine is also approved for children). Clomipramine is a TCA that is often tolerated poorly owing to anticholinergic and sedative side effects at the doses required to treat the illness (25–250 mg/d); its efficacy in OCD is unrelated to its antidepressant activity. Fluoxetine (5–60 mg/d), fluvoxamine (25–300 mg/d), and sertraline (50–150 mg/d) are as effective as clomipramine and have a more benign side effect profile. Only 50–60% of patients with OCD show adequate improvement with pharmacotherapy alone. In treatment-resistant cases, augmentation with other serotonergic agents such as buspirone, or with a neuroleptic or benzodiazepine, may be beneficial, and in severe cases, deep brain stimulation has been found to be effective. When a therapeutic response is achieved, long-duration maintenance therapy is usually indicated.

1	For many individuals, particularly those with time-consuming compulsions, behavior therapy will result in as much improvement as that afforded by medication. Effective techniques include the gradual increase in exposure to stressful situations, maintenance of a diary to clarify stressors, and homework assignments that substitute new activities for compulsive behaviors.

1	Mood disorders are characterized by a disturbance in the regulation of mood, behavior, and affect. Mood disorders are subdivided into (1) depressive disorders, (2) bipolar disorders, and (3) depression in association with medical illness or alcohol and substance abuse (Chaps. 467 through 471e). Major depressive disorder (MDD) is differentiated from bipolar disorder by the absence of a manic or hypo-manic episode. The relationship between pure depressive syndromes and bipolar disorders is not well understood; MDD is more frequent in families of bipolar individuals, but the reverse is not true. In the Global Burden of Disease Study conducted by the World Health Organization, unipolar major depression ranked fourth among all diseases in terms of disability-adjusted life-years and was projected to rank second by the year 2020. In the United States, lost productivity directly related to mood disorders has been estimated at $55.1 billion per year.

1	Depression occurring in the context of medical illness is difficult to evaluate. Depressive symptomatology may reflect the psychological stress of coping with the disease, may be caused by the disease process itself or by the medications used to treat it, or may simply coexist in time with the medical diagnosis. Virtually every class of medication includes some agent that can induce depression. Antihypertensive drugs, anticholesterolemic agents, and antiarrhythmic agents are common triggers of depressive symptoms. Iatrogenic depression should also be considered in patients receiving glucocorticoids, antimicrobials, systemic analgesics, antiparkinsonian medications, and anticonvulsants. To decide whether a causal relationship exists between pharmacologic therapy and a patient’s change in mood, it may sometimes be necessary to undertake an empirical trial of an alternative medication.

1	Between 20 and 30% of cardiac patients manifest a depressive disorder; an even higher percentage experience depressive symptomatology when self-reporting scales are used. Depressive symptoms following unstable angina, myocardial infarction, cardiac bypass surgery, or heart transplant impair rehabilitation and are associated with higher rates of mortality and medical morbidity. Depressed patients often show decreased variability in heart rate (an index of reduced parasympathetic nervous system activity), which may predispose individuals to ventricular arrhythmia and increased morbidity. Depression also appears to increase the risk of developing coronary heart disease, possibly through increased platelet aggregation. TCAs are contraindicated in patients with bundle branch block, and TCA-induced tachycardia is an additional concern in patients with congestive heart failure. SSRIs appear not to induce ECG changes or adverse cardiac events and thus are reasonable first-line drugs for

1	tachycardia is an additional concern in patients with congestive heart failure. SSRIs appear not to induce ECG changes or adverse cardiac events and thus are reasonable first-line drugs for patients at risk for TCA-related complications. SSRIs may interfere with hepatic metabolism of anticoagulants, however, causing increased anticoagulation.

1	In patients with cancer, the mean prevalence of depression is 25%, but depression occurs in 40–50% of patients with cancers of the pancreas or oropharynx. This association is not due to the effect of cachexia alone, as the higher prevalence of depression in patients with pancreatic cancer persists when compared to those with advanced gastric cancer. Initiation of antidepressant medication in cancer patients has been shown to improve quality of life as well as mood. Psychotherapeutic approaches, particularly group therapy, may have some effect on short-term depression, anxiety, and pain symptoms.

1	Depression occurs frequently in patients with neurologic disorders, particularly cerebrovascular disorders, Parkinson’s disease, dementia, multiple sclerosis, and traumatic brain injury. One in five patients with left-hemisphere stroke involving the dorsolateral frontal cortex experiences major depression. Late-onset depression in otherwise cognitively normal individuals increases the risk of a subsequent diagnosis of Alzheimer’s disease. All classes of antidepressant agents are effective against these depressions, as are, in some cases, stimulant compounds.

1	The reported prevalence of depression in patients with diabetes mellitus varies from 8 to 27%, with the severity of the mood state correlating with the level of hyperglycemia and the presence of diabetic complications. Treatment of depression may be complicated by effects of antidepressive agents on glycemic control. MAOIs can induce hypoglycemia and weight gain, whereas TCAs can produce hyperglycemia and carbohydrate craving. SSRIs and SNRIs, like MAOIs, may reduce fasting plasma glucose, but they are easier to use and may also improve dietary and medication compliance.

1	Hypothyroidism is frequently associated with features of depression, most commonly depressed mood and memory impairment. Hyperthyroid states may also present in a similar fashion, usually in geriatric populations. Improvement in mood usually follows normalization of thyroid function, but adjunctive antidepressant medication is sometimes required. Patients with subclinical hypothyroidism can also experience symptoms of depression and cognitive difficulty that respond to thyroid replacement. The lifetime prevalence of depression in HIV-positive individuals has been estimated at 22–45%. The relationship between depression and disease progression is multifactorial and likely to involve psychological and social factors, alterations in immune function, and central nervous system (CNS) disease. Chronic hepatitis C infection is also associated with depression, which may worsen with interferon-α treatment.

1	Some chronic disorders of uncertain etiology, such as chronic fatigue syndrome (Chap. 464e) and fibromyalgia (Chap. 396), are strongly associated with depression and anxiety; patients may benefit from antidepressant treatment or anticonvulsant agents such as pregabalin.

1	DEPRESSIVE DISORDERS Clinical Manifestations Major depression is defined as depressed mood on a daily basis for a minimum duration of 2 weeks (Table 466-7). An episode may be characterized by sadness, indifference, apathy, or irritability and is usually associated with changes in sleep patterns, appetite, and weight; motor agitation or retardation; fatigue; impaired concentration and decision making; feelings of shame or guilt; and thoughts of death or dying. Patients with depression have a profound loss of pleasure in all enjoyable activities, exhibit early morning awakening, feel that the dysphoric mood state is qualitatively different from sadness, and often notice a diurnal variation in mood (worse in morning hours). Patients experiencing bereavement or grief may exhibit many of the same signs and symptoms of major depression, although the emphasis is usually on feelings of emptiness and loss, rather than

1	A. Five (or more) of the following symptoms have been present during the same 2-week period and represent a change from previous functioning; at least one of the symptoms is either (1) depressed mood or (2) loss of interest or pleasure. Note: Do not include symptoms that are clearly attributable to another medical condition. 1. Depressed mood most of the day, nearly every day, as indicated by either subjective report (e.g., feels sad, empty, hopeless) or observation made by others (e.g., appears tearful). 2. Markedly diminished interest or pleasure in all, or almost all, activities most of the day, nearly every day (as indicated by either subjective account or observation). 3. Significant weight loss when not dieting or weight gain (e.g., a change of >5% of body weight in a month), or decrease or increase in appetite nearly every day. 4. Insomnia or hypersomnia nearly every day. 5.

1	4. Insomnia or hypersomnia nearly every day. 5. Psychomotor agitation or retardation nearly every day (observable by others, not merely subjective feelings of restlessness or being slowed down). 6. Fatigue or loss of energy nearly every day. 7. Feelings of worthlessness or excessive or inappropriate guilt (which may be delusional) nearly every day (not merely self-reproach or guilt about being sick). 8. Diminished ability to think or concentrate, or indecisiveness, nearly every day (either by subjective account or as observed by others). 9. Recurrent thoughts of death (not just fear of dying), recurrent suicidal ideation without a specific plan, or a suicide attempt or a specific plan for committing suicide B. The symptoms cause clinically significant distress or impairment in social, occupational, or other important areas of functioning C. The episode is not attributable to the physiologic effects of a substance or to another medical condition.

1	C. The episode is not attributable to the physiologic effects of a substance or to another medical condition. D. The occurrence of the major depressive episode is not better explained by seasonal affective disorder, schizophrenia, schizophreniform disorder, delusional disorder, or other specified and unspecified schizophrenia spectrum and other psychotic disorders. E. There has never been a manic episode or a hypomanic episode. Source: Diagnostic and Statistical Manual of Mental Disorders, 5th ed. Washington, DC, American Psychiatric Association, 2013. anhedonia and loss of self-esteem, and the duration is usually limited. In certain cases, however, the diagnosis of major depression may be warranted even in the context of a significant loss.

1	Approximately 15% of the population experiences a major depressive episode at some point in life, and 6–8% of all outpatients in primary care settings satisfy diagnostic criteria for the disorder. Depression is often undiagnosed, and even more frequently, it is treated inadequately. If a physician suspects the presence of a major depressive episode, the initial task is to determine whether it represents unipolar or bipolar depression or is one of the 10–15% of cases that are secondary to general medical illness or substance abuse. Physicians should also assess the risk of suicide by direct questioning, as patients are often reluctant to verbalize such thoughts without prompting. If specific plans are uncovered or if significant risk factors exist (e.g., a past history of suicide attempts, profound hopelessness, concurrent medical illness, substance abuse, or social isolation), the patient must be referred to a mental health specialist for immediate care. The physician should

1	attempts, profound hopelessness, concurrent medical illness, substance abuse, or social isolation), the patient must be referred to a mental health specialist for immediate care. The physician should specifically probe each of these areas in an empathic and hopeful manner, being sensitive to denial and possible minimization of distress. The presence of anxiety, panic, or agitation significantly increases near-term suicidal risk. Approximately 4–5% of all depressed patients will commit suicide; most will have sought help from physicians within 1 month of their deaths.

1	In some depressed patients, the mood disorder does not appear to be episodic and is not clearly associated with either psychosocial dysfunction or change from the individual’s usual experience in life. Persistent depressive disorder (dysthymic disorder) consists of a pattern of chronic (at least 2 years), ongoing depressive symptoms that are usually less severe and/or less numerous than those found in major depression, but the functional consequences may be equivalent to or 2715 even greater; the two conditions are sometimes difficult to separate and can occur together (“double depression”). Many patients who exhibit a profile of pessimism, disinterest, and low self-esteem respond to antidepressant treatment. Persistent and chronic depressive disorders occur in approximately 2% of the general population.

1	Depression is approximately twice as common in women as in men, and the incidence increases with age in both sexes. Twin studies indicate that the liability to major depression of early onset (before age 25) is largely genetic in origin. Negative life events can precipitate and contribute to depression, but genetic factors influence the sensitivity of individuals to these stressful events. In most cases, both biologic and psychosocial factors are involved in the precipitation and unfolding of depressive episodes. The most potent stressors appear to involve death of a relative, assault, or severe marital or relationship problems.

1	Unipolar depressive disorders usually begin in early adulthood and recur episodically over the course of a lifetime. The best predictor of future risk is the number of past episodes; 50–60% of patients who have a first episode have at least one or two recurrences. Some patients experience multiple episodes that become more severe and frequent over time. The duration of an untreated episode varies greatly, ranging from a few months to ≥1 year. The pattern of recurrence and clinical progression in a developing episode are also variable. Within an individual, the nature of episodes (e.g., specific presenting symptoms, frequency and duration) may be similar over time. In a minority of patients, a severe depressive episode may progress to a psychotic state; in elderly patients, depressive symptoms may be associated with cognitive deficits mimicking dementia (“pseudodementia”). A seasonal pattern of depression, called seasonal affective disorder, may manifest with onset and remission of

1	may be associated with cognitive deficits mimicking dementia (“pseudodementia”). A seasonal pattern of depression, called seasonal affective disorder, may manifest with onset and remission of episodes at predictable times of the year. This disorder is more common in women, whose symptoms are anergy, fatigue, weight gain, hypersomnia, and episodic carbohydrate craving. The prevalence increases with distance from the equator, and improvement may occur by altering light exposure.

1	Etiology and Pathophysiology Although evidence for genetic transmission of unipolar depression is not as strong as in bipolar disorder, monozygotic twins have a higher concordance rate (46%) than dizygotic siblings (20%), with little support for any effect of a shared family environment. Neuroendocrine abnormalities that reflect the neurovegetative signs and symptoms of depression include: (1) increased cortisol and corticotropin-releasing hormone (CRH) secretion, (2) an increase in adrenal size, (3) a decreased inhibitory response of glucocorticoids to dexamethasone, and (4) a blunted response of thyroid-stimulating hormone (TSH) level to infusion of thyroid-releasing hormone (TRH). Antidepressant treatment leads to normalization of these abnormalities. Major depression is also associated with changes in levels of pro-inflammatory cytokines and neurotrophins.

1	Diurnal variations in symptom severity and alterations in circadian rhythmicity of a number of neurochemical and neurohumoral factors suggest that biologic differences may be secondary to a primary defect in regulation of biologic rhythms. Patients with major depression show consistent findings of a decrease in rapid eye movement (REM) sleep onset (REM latency), an increase in REM density, and, in some subjects, a decrease in stage IV delta slow-wave sleep. Although antidepressant drugs inhibit neurotransmitter uptake within hours, their therapeutic effects typically emerge over several weeks, implicating adaptive changes in second messenger systems and transcription factors as possible mechanisms of action. The pathogenesis of depression is discussed in detail in Chap. 465e. Treatment planning requires coordination of short-term strategies to induce remission combined with longer term maintenance designed to prevent recurrence. The most effective intervention

1	Treatment planning requires coordination of short-term strategies to induce remission combined with longer term maintenance designed to prevent recurrence. The most effective intervention Determine whether there is a history of good response to a medication in the patient or a first-degree relative; if yes, consider treatment with this agent if compatible with considerations in step 2. Evaluate patient characteristics and match to drug; consider health status, side effect profile, convenience, cost, patient preference, drug interaction risk, suicide potential, and medication compliance history. Begin new medication at 1/3 to 1/2 target dose if drug is a TCA, bupropion, venlafaxine, or mirtazapine, or full dose as tolerated if drug is an SSRI. If problem side effects occur, evaluate possibility of tolerance; consider temporary decrease in dose or adjunctive treatment.

1	If problem side effects occur, evaluate possibility of tolerance; consider temporary decrease in dose or adjunctive treatment. If unacceptable side effects continue, taper drug over 1 week and initiate new trial; consider potential drug interactions in choice. Evaluate response after 6 weeks at target dose; if response is inadequate, increase dose in stepwise fashion as tolerated. If inadequate response after maximal dose, consider tapering and switching to a new drug vs adjunctive treatment; if drug is a TCA, obtain plasma level to guide further treatment. FIGURE 466-1 A guideline for the medical management of major depressive disorder. SSRI, selective serotonin reuptake inhibitor; TCA, tricyclic antidepressant.

1	for achieving remission and preventing relapse is medication, but combined treatment, incorporating psychotherapy to help the patient cope with decreased self-esteem and demoralization, improves outcome (Fig. 466-1). Approximately 40% of primary care patients with depression drop out of treatment and discontinue medication if symptomatic improvement is not noted within a month, unless additional support is provided. Outcome improves with (1) increased intensity and frequency of visits during the first 4–6 weeks of treatment, (2) supplemental educational materials, and (3) psychiatric consultation as indicated. Despite the widespread use of SSRIs and other second-generation antidepressant drugs, there is no convincing evidence that these classes of antidepressants are more efficacious than TCAs. Between 60 and 70% of all depressed patients respond to any drug chosen, if it is given in a sufficient dose for 6–8 weeks.

1	A rational approach to selecting which antidepressant to use involves matching the patient’s preference and medical history with the metabolic and side effect profile of the drug (Tables 466-4 and 466-5). A previous response, or a family history of a positive response, to a specific antidepressant often suggests that that drug be tried first. Before initiating antidepressant therapy, the physician should evaluate the possible contribution of comorbid illnesses and consider their specific treatment. In individuals with suicidal ideation, particular attention should be paid to choosing a drug with low toxicity if taken in overdose. Newer antidepressant drugs are distinctly safer in this regard; nevertheless, the advantages of TCAs have not been completely superseded. The existence of generic equivalents makes TCAs relatively cheap, and for secondary tricyclics, particularly nortriptyline and desipramine, well-defined relationships among dose, plasma level, and therapeutic response

1	equivalents makes TCAs relatively cheap, and for secondary tricyclics, particularly nortriptyline and desipramine, well-defined relationships among dose, plasma level, and therapeutic response exist. The steady-state plasma level achieved for a given drug dose can vary more than 10-fold between individuals, and plasma levels may help in interpreting apparent resistance to treatment and/or unexpected drug toxicity. The principal side effects of TCAs are antihistamine (sedation) and anticholinergic (constipation, dry mouth, urinary hesitancy, blurred vision). TCAs are contraindicated in patients with serious cardiovascular risk factors, and overdoses of tricyclic agents can be lethal, with desipramine carrying the greatest risk. It is judicious to prescribe only a 10-day supply when suicide is a risk. Most patients require a daily dose of 150–200 mg of imipramine or amitriptyline or its equivalent to achieve a therapeutic blood level of 150–300 ng/mL and a satisfactory remission; some

1	is a risk. Most patients require a daily dose of 150–200 mg of imipramine or amitriptyline or its equivalent to achieve a therapeutic blood level of 150–300 ng/mL and a satisfactory remission; some patients show a partial effect at lower doses. Geriatric patients may require a low starting dose and slow escalation. Ethnic differences in drug metabolism are significant, with Hispanic, Asian, and black patients generally requiring lower doses than whites to achieve a comparable blood level. P450 profiling using genetic chip technology may be clinically useful in predicting individual sensitivity.

1	Second-generation antidepressants are similar to tricyclics in their effect on neurotransmitter reuptake, although some also have specific actions on catecholamine and indolamine receptors as well. Amoxapine is a dibenzoxazepine derivative that blocks norepinephrine and serotonin reuptake and has a metabolite that shows a degree of dopamine blockade. Long-term use of this drug carries a risk of tardive dyskinesia. Maprotiline is a potent noradrenergic reuptake blocker that has little anticholinergic effect but may produce seizures. Bupropion is a novel antidepressant whose mechanism of action is thought to involve enhancement of noradrenergic function. It has no anticholinergic, sedating, or orthostatic side effects and has a low incidence of sexual side effects. It may, however, be associated with stimulant-like side effects, may lower seizure threshold, and has an exceptionally short half-life, requiring frequent dosing. An extended-release preparation is available.

1	SSRIs such as fluoxetine, sertraline, paroxetine, citalopram, and escitalopram cause a lower frequency of anticholinergic, sedating, and cardiovascular side effects but a possibly greater incidence of gastrointestinal complaints, sleep impairment, and sexual dysfunction than do TCAs. Akathisia, involving an inner sense of restlessness and anxiety in addition to increased motor activity, may also be more common, particularly during the first week of treatment. One concern is the risk of “serotonin syndrome,” which is thought to result from hyperstimulation of brainstem 5-HT1A receptors and characterized by myoclonus, agitation, abdominal cramping, hyperpyrexia, hypertension, and potentially death. Serotonergic agonists taken in combination should be monitored closely for this reason. Considerations such as half-life, compliance, toxicity, and drug-drug interactions may guide the choice of a particular SSRI. Fluoxetine and its principal active metabolite, norfluoxetine, for example,

1	such as half-life, compliance, toxicity, and drug-drug interactions may guide the choice of a particular SSRI. Fluoxetine and its principal active metabolite, norfluoxetine, for example, have a combined half-life of almost 7 days, resulting in a delay of 5 weeks before steady-state levels are achieved and a similar delay for complete drug excretion once its use is discontinued. All the SSRIs may impair sexual function, resulting in diminished libido, impotence, or difficulty in achieving orgasm. Sexual dysfunction frequently results in noncompliance and should be asked about specifically. Sexual dysfunction can sometimes be ameliorated by lowering the dose, by instituting weekend drug holidays (two or three times a month), or by treatment with amantadine (100 mg tid), bethanechol (25 mg tid), buspirone (10 mg tid), or bupropion (100–150 mg/d). Paroxetine appears to be more anticholinergic than either fluoxetine or sertraline, and sertraline carries a lower risk of producing an adverse

1	buspirone (10 mg tid), or bupropion (100–150 mg/d). Paroxetine appears to be more anticholinergic than either fluoxetine or sertraline, and sertraline carries a lower risk of producing an adverse drug interaction than the other two. Rare side effects of SSRIs include angina due to vasospasm and prolongation of the prothrombin time. Escitalopram is the most specific of currently available SSRIs and appears to have no specific inhibitory effects on the P450 system.

1	Venlafaxine, desvenlafaxine, duloxetine, vilazodone, vortioxetine, and levomilnacipran block the reuptake of both norepinephrine and serotonin but produce relatively little in the way of traditional tricyclic side effects. Unlike the SSRIs, venlafaxine and vortioxetine have relatively linear dose-response curves. Patients on immediate release venlafaxine should be monitored for a possible increase in diastolic blood pressure, and multiple daily dosing is required because of the drug’s short half-life. An extended-release form is available and has a somewhat lower incidence of gastrointestinal side effects. Mirtazapine is a TCA that has a unique spectrum of activity. It increases noradrenergic and serotonergic neurotransmission through a blockade of central α2-adrenergic receptors and postsynaptic 5-HT2 and 5-HT3 receptors. It is also strongly antihistaminic and, as such, may produce sedation. Levomilnacipran is the most noradrenergic of the SNRIs and theoretically may be appropriate

1	5-HT2 and 5-HT3 receptors. It is also strongly antihistaminic and, as such, may produce sedation. Levomilnacipran is the most noradrenergic of the SNRIs and theoretically may be appropriate for patients with more severe fatigue and anergia.

1	With the exception of citalopram and escitalopram, each of the SSRIs may inhibit one or more cytochrome P450 enzymes. Depending on the specific isoenzyme involved, the metabolism of a number of concomitantly administered medications can be dramatically affected. Fluoxetine and paroxetine, for example, by inhibiting 2D6, can cause dramatic increases in the blood level of type 1C antiarrhythmics, whereas sertraline, by acting on 3A4, may alter blood levels of carbamazepine or digoxin. Depending on drug specificity for a particular CYP enzyme for its own metabolism, concomitant medications or dietary factors, such as grapefruit juice, may in turn affect the efficacy or toxicity of the SSRI.

1	The MAOIs are highly effective, particularly in atypical depression, but the risk of hypertensive crisis following intake of tyraminecontaining food or sympathomimetic drugs makes them inappropriate as first-line agents. Transdermal selegiline may avert this risk at low dose. Common side effects include orthostatic hypotension, weight gain, insomnia, and sexual dysfunction. MAOIs should not be used concomitantly with SSRIs, because of the risk of serotonin syndrome, or with TCAs, because of possible hyperadrenergic effects.

1	Electroconvulsive therapy is at least as effective as medication, but its use is reserved for treatment-resistant cases and delusional depressions. Transcranial magnetic stimulation (TMS) is approved for treatment-resistant depression and has been shown to have efficacy in several controlled trials. Vagus nerve stimulation (VNS) has also recently been approved for treatment-resistant depression, but its degree of efficacy is controversial. Deep brain stimulation and ketamine, a glutamatergic antagonist, are experimental approaches for treatment-resistant cases.

1	Regardless of the treatment undertaken, the response should be evaluated after ~2 months. Three-quarters of patients show improvement by this time, but if remission is inadequate, the patient should be questioned about compliance, and an increase in medication dose should be considered if side effects are not troublesome. If this approach is unsuccessful, referral to a mental health specialist is advised. Strategies for treatment then include selection of an alternative drug, combinations of antidepressants, and/or adjunctive treatment with other classes of drugs, including lithium, thyroid hormone, atypical antipsychotic agents, and dopamine agonists. A large randomized trial (STAR-D) was unable to show preferential efficacy, but the addition of certain atypical antipsychotic drugs (quetiapine extended-release; aripiprazole) has received FDA approval, as has usage of a combined medication, olanzapine and fluoxetine (Symbyax). Patients whose response to an SSRI wanes over time may

1	extended-release; aripiprazole) has received FDA approval, as has usage of a combined medication, olanzapine and fluoxetine (Symbyax). Patients whose response to an SSRI wanes over time may benefit from the addition of buspirone (10 mg tid) or pindolol (2–5 mg tid) or small amounts of a TCA such as desipramine (25 mg bid or tid). Most patients will show some degree of response, but aggressive treatment should be pursued until remission is achieved, and drug treatment should be continued for at least 6–9 more months to prevent relapse. In patients who have had two or more episodes of depression, indefinite maintenance treatment should be considered.

1	It is essential to educate patients both about depression and the benefits and side effects of medications they are receiving. Advice about stress reduction and cautions that alcohol may exacerbate depressive symptoms and impair drug response are helpful. Patients should be given time to describe their experience, their outlook, and the impact of the depression on them and their families. Occasional empathic silence may be as helpful for the treatment alliance as verbal reassurance. Controlled trials have shown that cognitive-behavioral and interpersonal therapies are effective in improving psychological and social adjustment and that a com-2717 bined treatment approach is more successful than medication alone for many patients.

1	BIPOLAR DISORDER Clinical Manifestations Bipolar disorder is characterized by unpredictable swings in mood from mania (or hypomania) to depression. Some patients suffer only from recurrent attacks of mania, which in its pure form is associated with increased psychomotor activity; excessive social extroversion; decreased need for sleep; impulsivity and impairment in judgment; and expansive, grandiose, and sometimes irritable mood (Table 466-8). In severe mania, patients may experience delusions and paranoid thinking indistinguishable from schizophrenia. One-half of patients with bipolar disorder present with a mixture of psychomotor agitation and activation with dysphoria, anxiety, and irritability. It may be difficult to distinguish mixed mania from agitated depression. In some bipolar patients (bipolar II disorder), the full criteria for mania are lacking, and the requisite recurrent depressions are separated by periods of mild activation and increased energy (hypomania). In

1	patients (bipolar II disorder), the full criteria for mania are lacking, and the requisite recurrent depressions are separated by periods of mild activation and increased energy (hypomania). In cyclothymic disorder, there are numerous hypomanic periods, usually of relatively short duration, alternating with clusters of depressive symptoms that fail, either in severity or duration, to meet the criteria of major depression. The mood fluctuations are chronic and should be present for at least 2 years before the diagnosis is made.

1	Manic episodes typically emerge over a period of days to weeks, but onset within hours is possible, usually in the early morning hours. An untreated episode of either depression or mania can be as short as several weeks or last as long as 8–12 months, and rare patients have an unremitting chronic course. The term rapid cycling is used for patients who have four or more episodes of either depression or mania in a given year. This pattern occurs in 15% of all patients, almost all of whom are women. In some cases, rapid cycling is linked to an underlying thyroid dysfunction, and in others, it is iatrogenically triggered by prolonged antidepressant treatment. Approximately one-half of patients have sustained difficulties in work performance and psychosocial functioning, with depressive phases being more responsible for impairment than mania.

1	A. A distinct period of abnormally and persistently elevated, expansive, or irritable mood and abnormally and persistently increased goal-directed activity or energy, lasting at least 1 week and present most of the day, nearly every day (or any duration if hospitalization is necessary). B. During the period of the mood disturbance and increased energy or activity, three (or more) of the following symptoms (four if the mood is only irritable) are present to a significant degree and represent a noticeable change from usual behavior: 1. Inflated self-esteem or grandiosity. 2. Decreased need for sleep (e.g., feels rested after only 3 h of sleep). 3. More talkative than usual or pressure to keep talking. 4. Flight of ideas or subjective experience that thoughts are racing. 5. Distractibility (i.e., attention too easily drawn to unimportant or irrelevant external stimuli), as reported or observed. 6.

1	Flight of ideas or subjective experience that thoughts are racing. 5. Distractibility (i.e., attention too easily drawn to unimportant or irrelevant external stimuli), as reported or observed. 6. Increase in goal-directed activity (either socially, at work or school, or sexually) or psychomotor agitation (i.e., purposeless non-goal-directed activity). 7. Excessive involvement in activities that have a high potential for painful consequences (e.g., engaging in unrestrained buying sprees, sexual indiscretions, or foolish business investments). C. The mood disturbance is sufficiently severe to cause marked impairment in social or occupational functioning or to necessitate hospitalization to prevent harm to self or others, or there are psychotic features. D. The episode is not attributable to the physiologic effects of a substance (e.g., a drug of abuse, a medication, or other treatment) or another medical condition.

1	D. The episode is not attributable to the physiologic effects of a substance (e.g., a drug of abuse, a medication, or other treatment) or another medical condition. Source: Diagnostic and Statistical Manual of Mental Disorders, 5th ed. Washington, DC, American Psychiatric Association, 2013. 2718 Bipolar disorder is common, affecting ~1.5% of the population in the United States. Onset is typically between 20 and 30 years of age, but many individuals report premorbid symptoms in late childhood or early adolescence. The prevalence is similar for men and women; women are likely to have more depressive and men more manic episodes over a lifetime.

1	Differential Diagnosis The differential diagnosis of mania includes secondary mania induced by stimulant or sympathomimetic drugs, hyperthyroidism, AIDS, and neurologic disorders such as Huntington’s or Wilson’s disease and cerebrovascular accidents. Comorbidity with alcohol and substance abuse is common, either because of poor judgment and increased impulsivity or because of an attempt to self-treat the underlying mood symptoms and sleep disturbances. Etiology and Pathophysiology Genetic predisposition to bipolar disorder is evident from family studies; the concordance rate for monozygotic twins approaches 80%. Patients with bipolar disorder also appear to have altered circadian rhythmicity, and lithium may exert its therapeutic benefit through a resynchronization of intrinsic rhythms keyed to the light/dark cycle.

1	(Table 466-9) Lithium carbonate is the mainstay of treatment in bipolar disorder, although sodium valproate and carbamazepine, as well as a number of second-generation antipsychotic agents (aripiprazole, asenapine, olanzapine, quetiapine, risperidone, ziprasidone), also have FDA approval for the treatment of acute mania. Oxcarbazepine is not FDA approved, but appears to enjoy carbamazepine’s spectrum of efficacy. The response rate to lithium carbonate Starting dose: 300 mg bid Nausea/anorexia/diarrhea, fine tremor, or tid thirst, polyuria, fatigue, weight gain, acne, folliculitis, neutrophilia, hypothyroidism Therapeutic blood level: 0.8–1.2 meq/L Blood level is increased by thiazides, tetracyclines, and NSAIDs Blood level is decreased by bronchodilators, verapamil, and carbonic anhydrase inhibitors Rare side effects: Neurotoxicity, renal toxicity, hypercalcemia, ECG changes Starting dose: 250 mg tid Nausea/anorexia, weight gain, sedation, tremor, rash, alopecia

1	Rare side effects: Neurotoxicity, renal toxicity, hypercalcemia, ECG changes Starting dose: 250 mg tid Nausea/anorexia, weight gain, sedation, tremor, rash, alopecia Therapeutic blood level: 50–125 μg/mL Inhibits hepatic metabolism of other medications Rare side effects: Pancreatitis, hepatotoxicity, Stevens-Johnson syndrome Starting dose: 200 mg bid for Nausea/anorexia, sedation, rash, dizziness/ carbamazepine, 150 mg bid for ataxia oxcarbazepine Therapeutic blood level: Carbamazepine, but not oxcarbazepine, 4–12 μg/mL for induces hepatic metabolism of other carbamazepine medications Rare side effects: Hyponatremia, agranulocytosis, Stevens-Johnson syndrome Starting dose: 25 mg/d Rash, dizziness, headache, tremor, sedation, nausea Rare side effect: Stevens-Johnson syndrome Abbreviations: ECG, electrocardiogram; NSAIDs, nonsteroidal anti-inflammatory drugs.

1	is 70–80% in acute mania, with beneficial effects appearing in 1–2 weeks. Lithium also has a prophylactic effect in prevention of recurrent mania and, to a lesser extent, in the prevention of recurrent depression. A simple cation, lithium is rapidly absorbed from the gastrointestinal tract and remains unbound to plasma or tissue proteins. Some 95% of a given dose is excreted unchanged through the kidneys within 24 h.

1	Serious side effects from lithium are rare, but minor complaints such as gastrointestinal discomfort, nausea, diarrhea, polyuria, weight gain, skin eruptions, alopecia, and edema are common. Over time, urine-concentrating ability may be decreased, but significant nephrotoxicity does not usually occur. Lithium exerts an antithyroid effect by interfering with the synthesis and release of thyroid hormones. More serious side effects include tremor, poor concentration and memory, ataxia, dysarthria, and incoordination. There is suggestive, but not conclusive, evidence that lithium is teratogenic, inducing cardiac malformations in the first trimester.

1	In the treatment of acute mania, lithium is initiated at 300 mg bid or tid, and the dose is then increased by 300 mg every 2–3 days to achieve blood levels of 0.8–1.2 meq/L. Because the therapeutic effect of lithium may not appear until after 7–10 days of treatment, adjunctive usage of lorazepam (1–2 mg every 4 h) or clonazepam (0.5–1 mg every 4 h) may be beneficial to control agitation. Antipsychotics are indicated in patients with severe agitation who respond only partially to benzodiazepines. Patients using lithium should be monitored closely, since the blood levels required to achieve a therapeutic benefit are close to those associated with toxicity. Valproic acid may be better than lithium for patients who experience rapid cycling (i.e., more than four episodes a year) or who present with a mixed or dysphoric mania. Tremor and weight gain are the most common side effects; hepatotoxicity and pancreatitis are rare toxicities.

1	The recurrent nature of bipolar mood disorder necessitates maintenance treatment. A sustained blood lithium level of at least 0.8 meq/L is important for optimal prophylaxis and has been shown to reduce the risk of suicide, a finding not yet apparent for other mood stabilizers. Combinations of mood stabilizers together or with atypical antipsychotic drugs are sometimes required to maintain mood stability. Quetiapine extended release, olanzapine, risperidone, and lamotrigine have been approved for maintenance treatment as sole agents, in combination with lithium and with aripiprazole and ziprasidone as adjunctive drugs. Lurasidone, olanzapine/fluoxetine, and quetiapine are also approved to treat acute depressive episodes in bipolar disorder. Compliance is frequently an issue and often requires enlistment and education of concerned family members. Efforts to identify and modify psychosocial factors that may trigger episodes are important, as is an emphasis on lifestyle regularity.

1	requires enlistment and education of concerned family members. Efforts to identify and modify psychosocial factors that may trigger episodes are important, as is an emphasis on lifestyle regularity. Antidepressant medications are sometimes required for the treatment of severe breakthrough depressions, but their use should generally be avoided during maintenance treatment because of the risk of precipitating mania or accelerating the cycle frequency. Loss of efficacy over time may be observed with any of the mood-stabilizing agents. In such situations, an alternative agent or combination therapy is usually helpful.

1	Many patients presenting in general medical practice, perhaps as many as 5–7%, will experience a somatic symptom(s) as particularly distressing and preoccupying, to the point that it comes to dominate their thoughts, feelings, and beliefs and interferes to a varying degree with everyday functioning. Although the absence of a medical explanation for these complaints was historically emphasized as a diagnostic element, it has been recognized that the patient’s interpretation and elaboration of the experience is the critical defining factor and that patients with well-established medical causation may qualify for the diagnosis. Multiple complaints are typical, but severe single symptoms can occur as well. Comorbidity with depressive and anxiety disorders is common and may affect the severity of the experience and its functional consequences. Personality factors may be a significant risk factor, as may a low level of educational or socioeconomic status or a history of recent stressful

1	of the experience and its functional consequences. Personality factors may be a significant risk factor, as may a low level of educational or socioeconomic status or a history of recent stressful life events. Cultural factors are relevant as well and should be incorporated into the evaluation. Individuals who have persistent preoccupations about having or acquiring a serious illness, but who do not have a specific somatic complaint, may qualify for a related diagnosis—illness anxiety disorder. The diagnosis of conversion disorder (functional neurologic symptom disorder) is used to specifically identify those individuals whose somatic complaints involve one or more symptoms of altered voluntary motor or sensory function that cannot be medically explained and that causes significant distress or impairment or requires medical evaluation.

1	In factitious illnesses, the patient consciously and voluntarily produces physical symptoms of illness. The term Munchausen’s syndrome is reserved for individuals with particularly dramatic, chronic, or severe factitious illness. In true factitious illness, the sick role itself is gratifying. A variety of signs, symptoms, and diseases have been either simulated or caused by factitious behavior, the most common including chronic diarrhea, fever of unknown origin, intestinal bleeding or hematuria, seizures, and hypoglycemia. Factitious disorder is usually not diagnosed until 5–10 years after its onset, and it can produce significant social and medical costs. In malingering, the fabrication derives from a desire for some external reward such as a narcotic medication or disability reimbursement.

1	Patients with somatic symptom disorder are frequently subjected to many diagnostic tests and exploratory surgeries in an attempt to find their “real” illness. Such an approach is doomed to failure and does not address the core issue. Successful treatment is best achieved through behavior modification, in which access to the physician is tightly regulated and adjusted to provide a sustained and predictable level of support that is less clearly contingent on the patient’s level of presenting distress. Visits can be brief and should not be associated with a need for a diagnostic or treatment action. Although the literature is limited, some patients may benefit from antidepressant treatment.

1	Any attempt to confront the patient usually creates a sense of humiliation and causes the patient to abandon treatment from that caregiver. A better strategy is to introduce psychological causation as one of a number of possible explanations in the differential diagnoses that are discussed. Without directly linking psychotherapeutic intervention to the diagnosis, the patient can be offered a face-saving means by which the pathologic relationship with the health care system can be examined and alternative approaches to life stressors developed. Specific medical treatments also may be indicated and effective in treating some of the functional consequences of conversion disorder.

1	Feeding and eating disorders constitute a group of conditions in which there is a persistent disturbance of eating or associated behaviors that significantly impair an individual’s physical health or psychosocial functioning. In DSM-5 the described categories (with the exception of pica) are defined to be mutually exclusive in a given episode, based on the understanding that although they are phenotypically similar in some ways, they differ in course, prognosis, and effective treatment interventions. Compared with DSM-IV-TR, three disorders (i.e., avoidant/restrictive food intake disorder, rumination disorder, pica) that were previously classified as disorders of infancy or childhood have been grouped together with the disorders of anorexia and bulimia nervosa. Binge-eating disorder is also now included as a formal diagnosis; the intent of each of these modifications is to encourage clinicians to be more specific in their codification of eating and feeding pathology.

1	Pica is diagnosed when the individual, over age 2, eats one or more nonnutritive, nonfood substances for a month or more and requires medical attention as a result. There is usually no specific aversion to food in general but a preferential choice to ingest substances such as clay, starch, soap, paper, or ash. The diagnosis requires the exclusion of specific culturally approved practices and has not been commonly found to be caused by a specific nutritional deficiency. Onset is most common in childhood but the disorder can occur in association with other major psychiatric conditions in adults. An association with preg nancy has been observed, but the condition is only diagnosed when medical risks are increased by the behavior.

1	In this condition, individuals who have no demonstrable associated gastrointestinal or other medical condition repeatedly regurgitate their food after eating and then either rechew or swallow it or spit it out. The behavior typically occurs on a daily basis and must persist for at least 1 month. Weight loss and malnutrition are common sequelae, and individuals may attempt to conceal their behavior, either by covering their mouth or through social avoidance while eating. In infancy, the onset is typically between 3 to 12 months, and the behavior may remit spontaneously, although in some it appears to be recurrent.

1	The cardinal feature of this disorder is avoidance or restriction of food intake, usually stemming from a lack of interest in or distaste of food and associated with weight loss, nutritional deficiency, dependency on nutritional supplementation, or marked impairment in psychosocial functioning, either alone or in combination. Culturally approved practices, such as fasting, or a lack of available food must be excluded as possible causes. The disorder is distinguished from anorexia nervosa by the presence of emotional factors, such as a fear of gaining weight and distortion of body image in the latter condition. Onset is usually in infancy or early childhood, but avoidant behaviors may persist into adulthood. The disorder is equally prevalent in males and females and is frequently comorbid with anxiety and cognitive and attention-deficit disorders and situations of familial stress. Developmental delay and functional deficits may be significant if the disorder is long-standing and

1	with anxiety and cognitive and attention-deficit disorders and situations of familial stress. Developmental delay and functional deficits may be significant if the disorder is long-standing and unrecognized.

1	Individuals are diagnosed with anorexia nervosa if they restrict their caloric intake to a degree that their body weight deviates significantly from age, gender, health, and developmental norms and if they also exhibit a fear of gaining weight and an associated disturbance in body image. The condition is further characterized by differentiating those who achieve their weight loss predominantly through restricting intake or by excessive exercise (restricting type) from those who engage in recurrent binge eating and/or subsequent purging, self-induced vomiting, and usage of enemas, laxatives, or diuretics (bingeeating/purging type). Such subtyping is more state than trait specific, as individuals may transition from one profile to the other over time. Determination of whether an individual satisfies the primary criterion of significant low weight is complex and must be individualized, using all available historical information and comparison of body habitus to international body mass

1	satisfies the primary criterion of significant low weight is complex and must be individualized, using all available historical information and comparison of body habitus to international body mass norms and guidelines.

1	Individuals with anorexia nervosa frequently lack insight into their condition and are in denial about possible medical consequences; they often are not comforted by their achieved weight loss and persist in their behaviors despite having met previously self-designated weight goals. Recent research has identified alterations in the circuitry of reward sensitivity and executive function in anorexia and implicated disturbances in frontal cortex and anterior insula regulation of interoceptive awareness of satiety and hunger. Neurochemical findings, including the role of ghrelin, remain controversial.

1	Onset is most common in adolescence, although onset in later life can occur. Many more females than males are affected, with a lifetime prevalence in women of up to 4%. The disorder appears 2720 most prevalent in postindustrialized and urbanized countries and is frequently comorbid with preexisting anxiety disorders. The medical consequences of prolonged anorexia nervosa are multisystemic and can be life-threatening in severe presentations. Changes in blood chemistry include leukopenia with lymphocytosis, elevations in blood urea nitrogen, and metabolic alkalosis and hypokalemia when purging is present. History and physical examination may reveal amenorrhea in females, skin abnormalities (petechiae, lanugo hair, dryness), and signs of hypometabolic function, including hypotension, hypothermia, and sinus bradycardia. Endocrine effects include hypogonadism, growth hormone resistance, and hypercortisolemia. Osteoporosis is a longer-term concern. The course of the disorder is variable,

1	and sinus bradycardia. Endocrine effects include hypogonadism, growth hormone resistance, and hypercortisolemia. Osteoporosis is a longer-term concern. The course of the disorder is variable, with some individuals recovering after a single episode, while others exhibit recurrent episodes or a chronic course. Untreated anorexia has a mortality of 5.1/1000, the highest among psychiatric conditions. Maudsley family-based therapy has proven to be an effective therapy in younger individuals, with strict behavioral contingencies used when weight loss becomes critical. No pharmacologic intervention has proven to be specifically beneficial, but comorbid depression and anxiety should be treated. Weight gain should be undertaken gradually with a goal of 0.5 to 1 pound per week to prevent refeeding syndrome. Most individuals are able to achieve remission within 5 years of the original diagnosis.

1	Bulimia nervosa describes individuals who engage in recurrent and frequent (at least once a week for 3 months) periods of binge eating and who then resort to compensatory behaviors, such as self-induced purging, enemas, use of laxatives, or excessive exercise to avoid weight gain. Binge eating itself is defined as excessive food intake in a prescribed period of time, usually <2 h. As in anorexia nervosa, disturbances in body image occur and promote the behavior, but unlike in anorexia, individuals are of normal weight or even somewhat overweight. Subjects typically describe a loss of control and express shame about their actions, and often relate that their episodes are triggered by feelings of negative self-esteem or social stresses. The lifetime prevalence in women is approximately 2%, with a 10:1 female-to-male ratio. The disorder typically begins in adolescence and may be persistent over a number of years. Transition to anorexia occurs in only 10–15% of cases. Many of the medical

1	with a 10:1 female-to-male ratio. The disorder typically begins in adolescence and may be persistent over a number of years. Transition to anorexia occurs in only 10–15% of cases. Many of the medical risks associated with bulimia nervosa parallel those of anorexia nervosa and are a direct consequence of purging, including fluid and electrolyte disturbances and conduction abnormalities. Physical examination often results in no specific findings, but dental erosion and parotid gland enlargement may be present. Effective treatment approaches include SSRI antidepressants, usually in combination with cognitive-behavioral, emotion regulation, or interpersonal-based psychotherapies.

1	Binge-eating disorder is distinguished from bulimia nervosa by the absence of compensatory behaviors to prevent weight gain after an episode and by a lack of effort to restrict weight gain between episodes. Other features are similar, including distress over the behavior and the experience of loss of control, resulting in eating more rapidly or in greater amounts than intended or eating when not hungry. The 12-month prevalence in females is 1.6%, with a much lower female-tomale ratio than bulimia nervosa. Little is known about the course of the disorder, given its recent categorization, but its prognosis is markedly better than for other eating disorders, both in terms of its natural course and response to treatment. Transition to other eating disorder conditions is thought to be rare.

1	Personality disorders are characteristic patterns of thinking, feeling, and interpersonal behavior that are relatively inflexible and cause significant functional impairment or subjective distress for the individual. The observed behaviors are not secondary to another mental disorder, nor are they precipitated by substance abuse or a general medical condition.

1	This distinction is often difficult to make in clinical practice, because personality change may be the first sign of serious neurologic, endocrine, or other medical illness. Patients with frontal lobe tumors, for example, can present with changes in motivation and personality while the results of the neurologic examination remain within normal limits. Individuals with personality disorders are often regarded as “difficult patients” in clinical medical practice because they are seen as excessively demanding and/or unwilling to follow recommended treatment plans. Although DSM-5 portrays personality disorders as qualitatively distinct categories, there is an alternative perspective that personality characteristics vary as a continuum between normal functioning and formal mental disorder.

1	Personality disorders have been grouped into three overlapping clusters. Cluster A includes paranoid, schizoid, and schizotypal personality disorders. It includes individuals who are odd and eccentric and who maintain an emotional distance from others. Individuals have a restricted emotional range and remain socially isolated. Patients with schizotypal personality disorder frequently have unusual perceptual experiences and express magical beliefs about the external world. The essential feature of paranoid personality disorder is a pervasive mistrust and suspiciousness of others to an extent that is unjustified by available evidence. Cluster B disorders include antisocial, borderline, histrionic, and narcissistic types and describe individuals whose behavior is impulsive, excessively emotional, and erratic. Cluster C incorporates avoidant, dependent, and obsessive-compulsive personality types; enduring traits are anxiety and fear. The boundaries between cluster types are to some extent

1	and erratic. Cluster C incorporates avoidant, dependent, and obsessive-compulsive personality types; enduring traits are anxiety and fear. The boundaries between cluster types are to some extent artificial, and many patients who meet criteria for one personality disorder also meet criteria for aspects of another. The risk of a comorbid major mental disorder is increased in patients who qualify for a diagnosis of personality disorder.

1	Dialectical behavior therapy (DBT) is a cognitive-behavioral approach that focuses on behavioral change while providing acceptance, compassion, and validation of the patient. Several randomized trials have demonstrated the efficacy of DBT in the treatment of personality disorders. Antidepressant medications and low-dose antipsychotic drugs have some efficacy in cluster A personality disorders, whereas anticonvulsant mood-stabilizing agents and MAOIs may be considered for patients with cluster B diagnoses who show marked mood reactivity, behavioral dyscontrol, and/or rejection hypersensitivity. Anxious or fearful cluster C patients often respond to medications used for axis I anxiety disorders (see above). It is important that the physician and the patient have reasonable expectations vis-à-vis the possible benefit of any medication used and its side effects. Improvement may be subtle and observable only over time.

1	Schizophrenia is a heterogeneous syndrome characterized by perturbations of language, perception, thinking, social activity, affect, and volition. There are no pathognomonic features. The syndrome commonly begins in late adolescence, has an insidious (and less commonly acute) onset, and, often, a poor outcome, progressing from social withdrawal and perceptual distortions to recurrent delusions and hallucinations. Patients may present with positive symptoms (such as conceptual disorganization, delusions, or hallucinations) or negative symptoms (loss of function, anhedonia, decreased emotional expression, impaired concentration, and diminished social engagement) and must have at least two of these for a 1-month period and continuous signs for at least 6 months to meet formal diagnostic criteria. Disorganized thinking or speech and grossly disorganized motor behavior, including catatonia, may also be present. As individuals age, positive psychotic symptoms tend to attenuate, and some

1	Disorganized thinking or speech and grossly disorganized motor behavior, including catatonia, may also be present. As individuals age, positive psychotic symptoms tend to attenuate, and some measure of social and occupational function may be regained. “Negative” symptoms predominate in one-third of the schizophrenic population and are associated with a poor long-term outcome and a poor response to drug treatment. However, marked variability in the course and individual character of symptoms is typical.

1	The term schizophreniform disorder describes patients who meet the symptom requirements but not the duration requirements for schizophrenia, and schizoaffective disorder is used for those who manifest symptoms of schizophrenia and independent periods of mood disturbance. The terms “schizotypal” and “schizoid” refer to specific personality disorders and are discussed in that section. The diagnosis of delusional disorder is used for individuals who have delusions of various content for at least 1 month but who otherwise do not meet criteria for schizophrenia. Patients who experience a sudden onset of a brief (<1 month) alteration in thought processing, characterized by delusions, hallucinations, disorganized speech, or gross motor behavior, are most appropriately designated as having a brief psychotic disorder. Catatonia is recognized as a nonspecific syndrome that can occur as a consequence of other severe psychiatric/medical disorders and is diagnosed by the documentation of three or

1	psychotic disorder. Catatonia is recognized as a nonspecific syndrome that can occur as a consequence of other severe psychiatric/medical disorders and is diagnosed by the documentation of three or more of a cluster of motor and behavioral symptoms, including stupor, cataplexy, mutism, waxy flexibility, and stereotypy, among others. Prognosis depends not on symptom severity but on the response to antipsychotic medication. A permanent remission without recurrence does occasionally occur. About 10% of schizophrenic patients commit suicide.

1	Schizophrenia is present in 0.85% of individuals worldwide, with a lifetime prevalence of ~1–1.5%. An estimated 300,000 episodes of acute schizophrenia occur annually in the United States, resulting in direct and indirect costs of $62.7 billion. The diagnosis is principally one of exclusion, requiring the absence of significant associated mood symptoms, any relevant medical condition, and substance abuse. Drug reactions that cause hallucinations, paranoia, confusion, or bizarre behavior may be dose-related or idiosyncratic; parkinsonian medications, clonidine, quinacrine, and procaine derivatives are the most common prescription medications associated with these symptoms. Drug causes should be ruled out in any case of newly emergent psychosis. The general neurologic examination in patients with schizophrenia is usually normal, but motor rigidity, tremor, and dyskinesias are noted in one-quarter of untreated patients.

1	Epidemiologic surveys identify several risk factors for schizophrenia, including genetic susceptibility, early developmental insults, winter birth, and increasing parental age. Genetic factors are involved in at least a subset of individuals who develop schizophrenia. Schizophrenia is observed in ~6.6% of all first-degree relatives of an affected pro-band. If both parents are affected, the risk for offspring is 40%. The concordance rate for monozygotic twins is 50%, compared to 10% for dizygotic twins. Schizophrenia-prone families are also at risk for other psychiatric disorders, including schizoaffective disorder and schizotypal and schizoid personality disorders, the latter terms designating individuals who show a lifetime pattern of social and interpersonal deficits characterized by an inability to form close interpersonal relationships, eccentric behavior, and mild perceptual distortions.

1	Antipsychotic agents (Table 466-10) are the cornerstone of acute and maintenance treatment of schizophrenia and are effective in the treatment of hallucinations, delusions, and thought disorders, regardless of etiology. The mechanism of action involves, at least in part, binding to dopamine D2/D3 receptors in the ventral striatum; the clinical potencies of traditional antipsychotic drugs parallel their affinities for the D2 receptor, and even the newer “atypical” agents exert some degree of D2 receptor blockade. All neuroleptics induce expression of the immediate-early gene c-fos in the nucleus accumbens, a dopaminergic site connecting prefrontal and limbic cortices. The clinical efficacy of newer atypical neuroleptics, however, may involve N-methyl-D-aspartate (NMDA) receptor blockade, α1-and α2-2721 noradrenergic activity, altering the relationship between 5-HT2 and D2 receptor activity, and faster dissociation of D2 binding and effects on neuroplasticity.

1	Conventional neuroleptics differ in their potency and side effect profile. Older agents, such as chlorpromazine and thioridazine, are more sedating and anticholinergic and more likely to cause orthostatic hypotension, whereas higher potency antipsychotics, such as haloperidol, perphenazine, and thiothixene, are more likely to induce extrapyramidal side effects. The model “atypical” antipsy chotic agent is clozapine, a dibenzodiazepine that has a greater potency in blocking the 5-HT2 than the D2 receptor and a much higher affinity for the D4 than the D2 receptor. Its principal disadvantage is a risk of blood dyscrasias. Paliperidone is a recently approved agent that is a metabolite of risperidone and shares many of its properties. Unlike other antipsychotics, clozapine does not cause a rise in prolactin level. Approximately 30% of patients who do not benefit from conventional antipsychotic agents will have a better response to this drug, which also has a demonstrated superiority to

1	rise in prolactin level. Approximately 30% of patients who do not benefit from conventional antipsychotic agents will have a better response to this drug, which also has a demonstrated superiority to other antipsychotic agents in preventing suicide; however, its side effect profile makes it most appropriate for treatment-resistant cases. Risperidone, a benzisoxazole derivative, is more potent at 5-HT2 than D2 receptor sites, like clozapine, but it also exerts significant α2 antagonism, a property that may contribute to its perceived ability to improve mood and increase motor activity. Risperidone is not as effective as clozapine in treatment-resistant cases but does not carry a risk of blood dyscrasias. Olanzapine is similar neurochemically to clozapine but has a significant risk of inducing weight gain. Quetiapine is distinct in having a weak D2 effect but potent α1 and histamine blockade. Ziprasidone causes minimal weight gain and is unlikely to increase prolactin but may increase

1	weight gain. Quetiapine is distinct in having a weak D2 effect but potent α1 and histamine blockade. Ziprasidone causes minimal weight gain and is unlikely to increase prolactin but may increase QT prolongation. Aripiprazole also has little risk of weight gain or prolactin increase but may increase anxiety, nausea, and insomnia as a result of its partial agonist properties. Asenapine is associated with minimal weight gain and anticholinergic effect but may have a higher than expected risk of extrapyramidal symptoms.

1	Antipsychotic agents are effective in 70% of patients presenting with a first episode. Improvement may be observed within hours or days, but full remission usually requires 6–8 weeks. The choice of agent depends principally on the side effect profile and cost of treatment or on a past personal or family history of a favorable response to the drug in question. Atypical agents appear to be more effective in treating negative symptoms and improving cognitive function. An equivalent treatment response can usually be achieved with relatively low doses of any drug selected (i.e., 4–6 mg/d of haloperidol, 10–15 mg of olanzapine, or 4–6 mg/d of risperidone). Doses in this range result in >80% D2 receptor blockade, and there is little evidence that higher doses increase either the rapidity or degree of response. Maintenance treatment requires careful attention to the possibility of relapse and monitoring for the development of a movement disorder. Intermittent drug treatment is less effective

1	of response. Maintenance treatment requires careful attention to the possibility of relapse and monitoring for the development of a movement disorder. Intermittent drug treatment is less effective than regular dosing, but gradual dose reduction is likely to improve social functioning in many schizophrenic patients who have been maintained at high doses. If medications are completely discontinued, however, the relapse rate is 60% within 6 months. Long-acting injectable preparations (risperidone, paliperidone, olanzapine, aripiprazole) are considered when noncompliance with oral therapy leads to relapses but should not be considered interchangeable, because the agents differ in their indications, injection intervals and sites/volumes, and possible adverse reactions, among other factors. In treatment-resistant patients, a transition to clozapine usually results in rapid improvement, but a prolonged delay in response in some cases necessitates a 6to 9-month trial for maximal benefit to

1	treatment-resistant patients, a transition to clozapine usually results in rapid improvement, but a prolonged delay in response in some cases necessitates a 6to 9-month trial for maximal benefit to occur.

1	Antipsychotic medications can cause a broad range of side effects, including lethargy, weight gain, postural hypotension, constipation, and dry mouth. Extrapyramidal symptoms such as dystonia, akathisia, and akinesia are also frequent with first-generation agents and may contribute to poor adherence if not specifically Usual PO Daily Name Dose, mg Side Effects Sedation Comments Lurasidone (Latuda) 40–80 Nausea, EPSEs Abbreviations: EPSEs, extrapyramidal side effects; WBC, white blood cell.

1	Usual PO Daily Name Dose, mg Side Effects Sedation Comments Lurasidone (Latuda) 40–80 Nausea, EPSEs Abbreviations: EPSEs, extrapyramidal side effects; WBC, white blood cell. addressed. Anticholinergic and parkinsonian symptoms respond well to trihexyphenidyl, 2 mg bid, or benztropine mesylate, 1–2 mg bid. Akathisia may respond to beta blockers. In rare cases, more serious and occasionally life-threatening side effects may emerge, including hyperprolactinemia, ventricular arrhythmias, gastrointestinal obstruction, retinal pigmentation, obstructive jaundice, and neuroleptic malignant syndrome (characterized by hyperthermia, autonomic dysfunction, muscular rigidity, and elevated creatine phosphokinase levels). The most serious adverse effects of clozapine are agranulocytosis, which has an incidence of 1%, and induction of seizures, which has an incidence of 10%. Weekly white blood cell counts are required, particularly during the first 3 months of treatment.

1	The risk of type 2 diabetes mellitus appears to be increased in schizophrenia, and second-generation agents as a group produce greater adverse effects on glucose regulation, independent of effects on obesity, than traditional agents. Clozapine, olanzapine, and quetiapine seem more likely to cause hyperglycemia, weight gain, and hypertriglyceridemia than other atypical antipsychotic drugs. Close monitoring of plasma glucose and lipid levels are indicated with the use of these agents.

1	A serious side effect of long-term use of first-generation antipsychotic agents is tardive dyskinesia, characterized by repetitive, involuntary, and potentially irreversible movements of the tongue and lips (bucco-linguo-masticatory triad) and, in approximately half of cases, choreoathetosis. Tardive dyskinesia has an incidence of 2–4% per year of exposure and a prevalence of 20% in chronically treated patients. The prevalence increases with age, total dose, and duration of drug administration. The risk associated with second-generation agents appears to be much lower. The cause may involve formation of free radicals and perhaps mitochondrial energy failure. Vitamin E may reduce abnormal involuntary movements if given early in the syndrome.

1	The CATIE study, a large-scale investigation of the effectiveness of antipsychotic agents in “real-world” patients, revealed a high rate of discontinuation of treatment over 18 months. Olanzapine showed greater effectiveness than quetiapine, risperidone, perphenazine, or ziprasidone but also a higher discontinuation rate due to weight gain and metabolic effects. Surprisingly, perphenazine, a first-generation agent, showed little evidence of inferiority to newer drugs. Drug treatment of schizophrenia is by itself insufficient. Educational efforts directed toward families and relevant community resources have proved to be necessary to maintain stability and optimize outcome. A treatment model involving a multidisciplinary case-management team that seeks out and closely follows the patient in the community has proved particularly effective.

1	Primary care physicians may encounter situations in which family, domestic, or societal violence is discovered or suspected. Such an awareness can carry legal and moral obligations; many state laws mandate reporting of child, spousal, and elder abuse. Physicians are frequently the first point of contact for both victim and abuser. Approximately 2 million older Americans and 1.5 million U.S. children are thought to experience some form of physical maltreatment each year. Spousal abuse is thought to be even more prevalent. An interview study of 24,000 women in 10 countries found a lifetime prevalence of physical or sexual violence that ranged from 15 to 71%; these individuals are more likely to suffer from depression, anxiety, and substance abuse and to have attempted suicide. In addition, abused individuals frequently express low self-esteem, vague somatic symptomatology, social isolation, and a passive feeling of loss of control. Although it is essential to treat these elements in the

1	individuals frequently express low self-esteem, vague somatic symptomatology, social isolation, and a passive feeling of loss of control. Although it is essential to treat these elements in the victim, the first obligation is to ensure that the perpetrator has taken responsibility for preventing any further violence. Substance abuse and/or dependence and serious mental illness in the abuser may contribute to the risk of harm and require direct intervention. Depending on the situation, law enforcement agencies, community resources such as support groups and shelters, and individual and family counseling can be appropriate components of a treatment plan. A safety plan should be formulated with the victim, in addition to providing information about abuse, its likelihood of recurrence, and its tendency to increase in severity and frequency. Antianxiety and antidepressant medications may sometimes be useful in treating the acute symptoms, but only if independent evidence for an appropriate

1	tendency to increase in severity and frequency. Antianxiety and antidepressant medications may sometimes be useful in treating the acute symptoms, but only if independent evidence for an appropriate psychiatric diagnosis exists.

1	There is a high prevalence of mental disorders and substance abuse among homeless and impoverished individuals. Depending on the definition used, estimates of the total number of homeless individuals in the United States range from 800,000 to 2 million, one-third of whom qualify as having a serious mental disorder. Poor hygiene and nutrition, substance abuse, psychiatric illness, physical trauma, and exposure to the elements combine to make the provision of medical care challenging. Only a minority of these individuals receives formal mental health care; the main points of contact are outpatient medical clinics and emergency departments. Primary care settings represent a critical site in which housing needs, treatment of substance dependence, and evaluation and treatment of psychiatric illness can most efficiently take place. Successful intervention is dependent on breaking down traditional administrative barriers to health care and recognizing the physical constraints and emotional

1	can most efficiently take place. Successful intervention is dependent on breaking down traditional administrative barriers to health care and recognizing the physical constraints and emotional costs imposed by homelessness. Simplifying health care instructions and follow-up, allowing frequent visits, and dispensing medications in limited amounts that require ongoing contact are possible techniques for establishing a successful therapeutic relationship.

1	Marc A. Schuckit Alcohol (beverage ethanol) distributes throughout the body, affecting almost all systems and altering nearly every neurochemical process in the brain. This drug is likely to exacerbate most medical problems, affect medications metabolized in the liver, and temporarily mimic many medical (e.g., diabetes) and psychiatric (e.g., depression) conditions. The lifetime risk for repetitive alcohol problems is almost 20% for men and 10% for women, regardless of a person’s education or income. Although low doses of alcohol might have healthful benefits, greater than three standard drinks per day enhances the risk for cancer and vascular disease, and alcohol use disorders decrease the life span by about 10 years. Unfortunately, most clinicians have had only limited training regarding alcohol-related disorders. This chapter presents a brief overview of clinically useful information about alcohol use and problems.

1	Ethanol blood levels are expressed as milligrams or grams of ethanol per deciliter (e.g., 100 mg/dL = 0.10 g/dL), with values of ~0.02 g/dL resulting from the ingestion of one typical drink. In round figures, a 2723 standard drink is 10–12 g, as seen in 340 mL (12 oz) of beer, 115 mL (4 oz) of nonfortified wine, and 43 mL (1.5 oz) (a shot) of 80-proof beverage (e.g., whisky); 0.5 L (1 pint) of 80-proof beverage contains ~160 g of ethanol (about 16 standard drinks), and 750 mL of wine contains ~60 g of ethanol. These beverages also have additional components (congeners) that affect the drink’s taste and might contribute to adverse effects on the body. Congeners include methanol, butanol, acetaldehyde, histamine, tannins, iron, and lead. Alcohol acutely decreases neuronal activity and has similar behavioral effects and cross-tolerance with other depressants, including benzodiazepines and barbiturates.

1	Alcohol is absorbed from mucous membranes of the mouth and esophagus (in small amounts), from the stomach and large bowel (in modest amounts), and from the proximal portion of the small intestine (the major site). The rate of absorption is increased by rapid gastric emptying (as seen with carbonation); by the absence of proteins, fats, or carbohydrates (which interfere with absorption); and by dilution to a modest percentage of ethanol (maximum at ~20% by volume).

1	Between 2% (at low blood alcohol concentrations) and 10% (at high blood alcohol concentrations) of ethanol is excreted directly through the lungs, urine, or sweat, but most is metabolized to acetaldehyde, primarily in the liver. The most important pathway occurs in the cell cytosol where alcohol dehydrogenase (ADH) produces acetaldehyde, which is then rapidly destroyed by aldehyde dehydrogenase (ALDH) in the cytosol and mitochondria (Fig. 467-1). A second pathway occurs in the microsomes of the smooth endoplasmic reticulum (the microsomal ethanol-oxidizing system, or MEOS) that is responsible for ≥10% of ethanol oxidation at high blood alcohol concentrations.

1	Although a drink contains ~300 kJ, or 70–100 kcal, these are devoid of minerals, proteins, and vitamins. In addition, alcohol interferes with absorption of vitamins in the small intestine and decreases their storage in the liver with modest effects on folate (folacin or folic acid), pyridoxine (B6), thiamine (B1), nicotinic acid (niacin, B3), and vitamin A.

1	Heavy drinking in a fasting, healthy individual can produce transient hypoglycemia within 6–36 h, secondary to the acute actions of ethanol on gluconeogenesis. This can result in temporary abnormal glucose tolerance tests (with a resulting erroneous diagnosis of diabetes mellitus) until the alcoholic has abstained for 2–4 weeks. Alcohol ketoacidosis, probably reflecting a decrease in fatty acid oxidation coupled with poor diet or recurrent vomiting, can be misdiagnosed as diabetic ketosis. With the former, patients show an increase in serum ketones along with a mild increase in glucose but a large anion gap, a mild to moderate increase in serum lactate, and a β-hydroxybutyrate/ lactate ratio of between 2:1 and 9:1 (with normal being 1:1).

1	In the brain, alcohol affects almost all neurotransmitter systems, with acute effects that are often the opposite of those seen following desistance after a period of heavy drinking. The most prominent actions relate to boosting γ-aminobutyric acid (GABA) activity, FIGURE 467-1 The metabolism of alcohol. CoA, coenzyme A; MEOS, microsomal ethanoloxidizing system.

1	2724 especially at GABAA receptors. Enhancement of this complex chloride channel system contributes to anticonvulsant, sleep-inducing, antianxiety, and muscle relaxation effects of all GABA-boosting drugs. Acutely administered alcohol produces a release of GABA, and continued use increases density of GABAA receptors, whereas alcohol withdrawal states are characterized by decreases in GABA-related activity. Equally important is the ability of acute alcohol to inhibit postsynaptic N-methyl-d-aspartate (NMDA) excitatory glutamate receptors, whereas chronic drinking and desistance are associated with an upregulation of these excitatory receptor subunits. The relationships between greater GABA and diminished NMDA receptor activity during acute intoxication and diminished GABA with enhanced NMDA actions during alcohol withdrawal explain much of intoxication and withdrawal phenomena. As with all pleasurable activities, alcohol acutely increases dopamine levels in the ventral tegmentum and

1	actions during alcohol withdrawal explain much of intoxication and withdrawal phenomena. As with all pleasurable activities, alcohol acutely increases dopamine levels in the ventral tegmentum and related brain regions, and this effect plays an important role in continued alcohol use, craving, and relapse. The changes in dopamine pathways are also linked to increases in “stress hormones,” including cortisol and adrenocorticotropic hormone (ACTH) during intoxication and withdrawal. Such alterations are likely to contribute to both feelings of reward during intoxication and depression during falling blood alcohol concentrations. Also closely linked to alterations in dopamine (especially in the nucleus accumbens) are alcohol-induced changes in opioid receptors, with acute alcohol causing release of beta endorphins. Additional neurochemical changes include increases in synaptic levels of serotonin during acute intoxication and subsequent upregulation of serotonin receptors. Acute increases

1	of beta endorphins. Additional neurochemical changes include increases in synaptic levels of serotonin during acute intoxication and subsequent upregulation of serotonin receptors. Acute increases in nicotinic acetylcholine systems contribute to the impact of alcohol in the ventral tegmental region, which occurs in concert with enhanced dopamine activity. In the same regions, alcohol impacts on cannabinol receptors, with resulting release of dopamine, GABA, and glutamate as well as subsequent effects on brain reward circuits.

1	BEHAVIORAL EFFECTS, TOLERANCE, AND WITHDRAWAL The acute effects of a drug depend on the dose, the rate of increase in plasma, the concomitant presence of other drugs, and past experience with the agent. “Legal intoxication” with alcohol in most states requires a blood alcohol concentration of 0.08 g/dL, but levels of 0.04 are cited in some other countries. However, behavioral, psychomotor, and cognitive changes are seen at 0.02–0.04 g/dL (i.e., after one to two drinks) (Table 467-1). Deep but disturbed sleep can be seen at twice the legal intoxication level, and death can occur with levels between 0.30 and 0.40 g/dL. Beverage alcohol is probably responsible for more overdose deaths than any other drug.

1	Repeated use of alcohol contributes to acquired tolerance, a phenomenon involving at least three compensatory mechanisms. (1) After 1–2 weeks of daily drinking, metabolic or pharmacokinetic tolerance can be seen, with up to 30% increases in the rate of hepatic ethanol metabolism. This alteration disappears almost as rapidly as it develops. (2) Cellular or pharmacodynamic tolerance develops through neurochemical changes that maintain relatively normal physiologic functioning despite the presence of alcohol. Subsequent decreases in blood levels contribute to symptoms of withdrawal. (3) Individuals learn to adapt their behavior so that they can function better than expected under influence of the drug (learned or behavioral tolerance). Blood Level, g/dL Usual Effect 0.02 Decreased inhibitions, a slight feeling of intoxication 0.08 0.20 Obvious slurred speech, motor incoordination, irritability, and poor judgment 0.30 Light coma and depressed vital signs 0.40 Death

1	The cellular changes caused by chronic ethanol exposure may not resolve for several weeks or longer following cessation of drinking. Rapid decreases in blood alcohol levels before that time can produce a withdrawal syndrome, which is most intense during the first 5 days, but some symptoms (e.g., disturbed sleep and anxiety) can take up to 4–6 months to resolve.

1	Relatively low doses of alcohol (one or two drinks per day) have potential beneficial effects of increasing high-density lipoprotein cholesterol and decreasing aggregation of platelets, with a resulting decrease in risk for occlusive coronary disease and embolic strokes. Red wine has additional potential health-promoting qualities at relatively low doses due to flavinols and related substances. Modest drinking might also decrease the risk for vascular dementia and, possibly, Alzheimer’s disease. However, any potential healthful effects disappear with the regular consumption of three or more drinks per day, and knowledge about the deleterious effects of alcohol can both help the physician to identify patients with an alcohol use disorder and to supply them with information that might help motivate a change in behavior.

1	Approximately 35% of drinkers (and a much higher proportion of alcoholics) experience a blackout, an episode of temporary anterograde amnesia, in which the person forgets all or part of what occurred during a drinking evening. Another common problem, one seen after as few as one or two drinks shortly before bedtime, is disturbed sleep. Although alcohol might initially help a person fall asleep, it disrupts sleep throughout the rest of the night. The stages of sleep are altered, and time spent in rapid eye movement (REM) and deep sleep is reduced. Alcohol relaxes muscles in the pharynx, which can cause snoring and exacerbate sleep apnea; symptoms of the latter occur in 75% of alcoholic men older than age 60 years. Patients may also experience prominent and sometimes disturbing dreams later in the night. All of these sleep problems are more pronounced in alcoholics, and their persistence may contribute to relapse.

1	Another common consequence of alcohol use is impaired judgment and coordination, increasing the risk of injury. In the United States, ~40% of drinkers have at some time driven while intoxicated. Heavy drinking can also be associated with headache, thirst, nausea, vomiting, and fatigue the following day, a hangover syndrome that is responsible for much missed time and temporary cognitive deficits at work and school.

1	Chronic high doses cause peripheral neuropathy in ~10% of alcoholics: similar to diabetes, patients experience bilateral limb numbness, tingling, and paresthesias, all of which are more pronounced distally. Approximately 1% of alcoholics develop cerebellar degeneration or atrophy, producing a syndrome of progressive unsteady stance and gait often accompanied by mild nystagmus; neuroimaging studies reveal atrophy of the cerebellar vermis. Fortunately, very few alcoholics (perhaps as few as 1 in 500 for the full syndrome) develop Wernicke’s (ophthalmoparesis, ataxia, and encephalopathy) and Korsakoff’s (retrograde and anterograde amnesia) syndromes, although a higher proportion have one or more neuropathologic findings related to these conditions. These result from low levels of thiamine, especially in predisposed individuals with transketolase deficiencies. Alcoholics can manifest cognitive problems and temporary memory impairment lasting for weeks to months after drinking heavily for

1	in predisposed individuals with transketolase deficiencies. Alcoholics can manifest cognitive problems and temporary memory impairment lasting for weeks to months after drinking heavily for days or weeks. Brain atrophy, evident as ventricular enlargement and widened cortical sulci on magnetic resonance imaging (MRI) and computed tomography (CT) scans, occurs in ~50% of chronic alcoholics; these changes are usually reversible if abstinence is maintained. There is no single alcoholic dementia syndrome; rather, this label describes patients who have irreversible cognitive changes (possibly from diverse causes) in the context of chronic alcoholism.

1	Psychiatric Comorbidity As many as two-thirds of individuals with alcohol use disorders meet the criteria for another psychiatric syndrome in the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) of the American Psychiatric Association (Chap. 466). Half of these relate to a preexisting antisocial personality manifesting as impulsivity and disinhibition that contribute to both alcohol and drug use disorders. The lifetime risk is 3% in males, and ≥80% of such individuals demonstrate alcoholand/or drug-related conditions. Another common comorbidity occurs with problems regarding illicit substances. The remainder of alcoholics with psychiatric syndromes have preexisting conditions such as schizophrenia or manic-depressive disease and anxiety syndromes such as panic disorder. The comorbidities of alcoholism with independent psychiatric disorders might represent an overlap in genetic vulnerabilities, impaired judgment in the use of alcohol from the

1	such as panic disorder. The comorbidities of alcoholism with independent psychiatric disorders might represent an overlap in genetic vulnerabilities, impaired judgment in the use of alcohol from the independent psychiatric condition, or an attempt to use alcohol to alleviate symptoms of the disorder or side effects of medications.

1	Many psychiatric syndromes can be seen temporarily during heavy drinking and subsequent withdrawal. These alcohol-induced conditions include an intense sadness lasting for days to weeks in the midst of heavy drinking seen in 40% of alcoholics, which tends to disappear over several weeks of abstinence (alcohol-induced mood disorder); temporary severe anxiety in 10–30% of alcoholics, often beginning during alcohol withdrawal, which can persist for a month or more after cessation of drinking (alcohol-induced anxiety disorder); and auditory hallucinations and/or paranoid delusions in a person who is alert and oriented, seen in 3–5% of alcoholics (alcohol-induced psychotic disorder).

1	Treatment of all forms of alcohol-induced psychopathology includes helping patients achieve abstinence and offering supportive care, as well as reassurance and “talk therapy” such as cognitive-behavioral approaches. However, with the exception of short-term antipsychotics or similar drugs for substance-induced psychoses, substance-induced psychiatric conditions only rarely require medications. Recovery is likely within several days to 4 weeks of abstinence. Conversely, because alcohol-induced conditions are temporary and do not indicate a need for long-term pharmacotherapy, a history of alcohol intake is an important part of the workup for any patient with one of these psychiatric symptoms.

1	THE GASTROINTESTINAL SYSTEM Esophagus and Stomach Alcohol can cause inflammation of the esophagus and stomach causing epigastric distress and gastrointestinal bleeding, making alcohol one of the most common causes of hemorrhagic gastritis. Violent vomiting can produce severe bleeding through a Mallory-Weiss lesion, a longitudinal tear in the mucosa at the gastroesophageal junction.

1	Pancreas and Liver The incidence of acute pancreatitis (~25 per 1000 per year) is almost threefold higher in alcoholics than in the general population, accounting for an estimated 10% or more of the total cases. Alcohol impairs gluconeogenesis in the liver, resulting in a fall in the amount of glucose produced from glycogen, increased lactate production, and decreased oxidation of fatty acids. This contributes to an increase in fat accumulation in liver cells. In healthy individuals these changes are reversible, but with repeated exposure to ethanol, especially daily heavy drinking, more severe changes in the liver occur, including alcohol-induced hepatitis, perivenular sclerosis, and cirrhosis, with the latter observed in an estimated 15% of alcoholics (Chap. 363). Perhaps through an enhanced vulnerability to infections, alcoholics have an elevated rate of hepatitis C, and drinking in the context of that disease is associated with more severe liver deterioration.

1	As few as 1.5 drinks per day increases a woman’s risk of breast cancer 1.4-fold. For both genders, four drinks per day increases the risk for oral and esophageal cancers approximately threefold and rectal cancers by a factor of 1.5; seven to eight or more drinks per day produces an approximately fivefold increased risk for many cancers. These consequences may result directly from cancer-promoting effects of alcohol and acetaldehyde or indirectly by interfering with immune homeostasis.

1	Ethanol causes an increase in red blood cell size (mean corpuscular volume [MCV]), which reflects its effects on stem cells. If heavy drinking is accompanied by folic acid deficiency, there can also be 2725 hypersegmented neutrophils, reticulocytopenia, and a hyperplastic bone marrow; if malnutrition is present, sideroblastic changes can be observed. Chronic heavy drinking can decrease production of white blood cells, decrease granulocyte mobility and adherence, and impair delayed-hypersensitivity responses to novel antigens (with a possible false-negative tuberculin skin test). Associated immune deficiencies can contribute to vulnerability toward infections, including hepatitis and HIV, and interfere with their treatment. Finally, many alcoholics have mild thrombocytopenia, which usually resolves within a week of abstinence unless there is hepatic cirrhosis or congestive splenomegaly.

1	Acutely, ethanol decreases myocardial contractility and causes peripheral vasodilation, with a resulting mild decrease in blood pressure and a compensatory increase in cardiac output. Exercise-induced increases in cardiac oxygen consumption are higher after alcohol intake. These acute effects have little clinical significance for the average healthy drinker but can be problematic when persisting cardiac disease is present.

1	The consumption of three or more drinks per day results in a dose-dependent increase in blood pressure, which returns to normal within weeks of abstinence. Thus, heavy drinking is an important factor in mild to moderate hypertension. Chronic heavy drinkers also have a sixfold increased risk for coronary artery disease, related, in part, to increased low-density lipoprotein cholesterol, and carry an increased risk for cardiomyopathy through direct effects of alcohol on heart muscle. Symptoms of the latter include unexplained arrhythmias in the presence of left ventricular impairment, heart failure, hypocontractility of heart muscle, and dilation of all four heart chambers with associated mural thrombi and mitral valve regurgitation. Atrial or ventricular arrhythmias, especially paroxysmal tachycardia, can also occur temporarily after heavy drinking in individuals showing no other evidence of heart disease—a syndrome known as the “holiday heart.”

1	GENITOURINARY SYSTEM CHANGES, SEXUAL FUNCTIONING, AND FETAL DEVELOPMENT Drinking in adolescence can affect normal sexual development and reproductive onset. At any age, modest ethanol doses (e.g., blood alcohol concentrations of 0.06 g/dL) can increase sexual drive but also decrease erectile capacity in men. Even in the absence of liver impairment, a significant minority of chronic alcoholic men show irreversible testicular atrophy with shrinkage of the seminiferous tubules, decreases in ejaculate volume, and a lower sperm count (Chap. 411).

1	The repeated ingestion of high doses of ethanol by women can result in amenorrhea, a decrease in ovarian size, absence of corpora lutea with associated infertility, and an increased risk of spontaneous abortion. Heavy drinking during pregnancy results in the rapid placental transfer of both ethanol and acetaldehyde, which may contribute to a range of consequences known as fetal alcohol spectrum disorder (FASD). One severe result is the fetal alcohol syndrome (FAS), seen in ~5% of children born to heavy-drinking mothers, which can include any of the following: facial changes with epicanthal eye folds; poorly formed ear concha; small teeth with faulty enamel; cardiac atrial or ventricular septal defects; an aberrant palmar crease and limitation in joint movement; and microcephaly with mental retardation. Less pervasive FASD conditions include combinations of low birth weight, a lower intelligence quotient (IQ), hyperactive behavior, and some modest cognitive deficits. The amount of

1	retardation. Less pervasive FASD conditions include combinations of low birth weight, a lower intelligence quotient (IQ), hyperactive behavior, and some modest cognitive deficits. The amount of ethanol required and the time of vulnerability during pregnancy have not been defined, making it advisable for pregnant women to abstain completely.

1	Between one-half and two-thirds of alcoholics have skeletal muscle weakness caused by acute alcoholic myopathy, a condition that improves but which might not fully remit with abstinence. Effects of repeated heavy drinking on the skeletal system include changes in calcium metabolism, lower bone density, and decreased growth in the epiphyses, leading to an increased risk for fractures and osteonecrosis of the femoral head. Hormonal changes include an increase in cortisol 2726 levels, which can remain elevated during heavy drinking; inhibition of vasopressin secretion at rising blood alcohol concentrations and enhanced secretion at falling blood alcohol concentrations (with the final result that most alcoholics are likely to be slightly overhydrated); a modest and reversible decrease in serum thyroxine (T4); and a more marked decrease in serum triiodothyronine (T3). Hormone irregularities should be reevaluated because they may disappear after a month of abstinence.

1	Because many drinkers occasionally imbibe to excess, temporary alcohol-related problems are common in nonalcoholics, especially in the late teens to the late twenties. However, repeated problems in multiple life areas can indicate an alcohol use disorder as defined in DSM-5. An alcohol use disorder is defined as repeated alcohol-related difficulties in at least 2 of 11 life areas that cluster together in the same 12-month period (Table 467-2). Ten of the 11 items were taken directly from the 7 dependence and 4 abuse criteria in DSM-IV, after deleting legal problems and adding craving. Severity of an alcohol use disorder is based on the number of items endorsed: mild is two or three items; moderate is four or five; and severe is six or more of the criterion items. The new diagnostic approach is similar enough to DSM-IV that the following descriptions of associated phenomena are still accurate.

1	The lifetime risk for an alcohol use disorder in most Western countries is about 10–15% for men and 5–8% for women. Rates are similar in the United States, Canada, Germany, Australia, and the United Kingdom, tend to be lower in most Mediterranean countries, such as Italy, Greece, and Israel, and may be higher in Ireland, France, and Scandinavia. An even higher lifetime prevalence has been reported for most native cultures, including American Indians, Eskimos, Maori groups, and aboriginal tribes of Australia. These differences reflect both cultural and genetic influences, as described below. In Western countries, the typical alcoholic is more often a blueor white-collar worker or homemaker. The lifetime risk for alcoholism among physicians is similar to that of the general population.

1	Approximately 60% of the risk for alcohol use disorders is attributed to genes, as indicated by the fourfold higher risk in children of alcoholics (even if adopted early in life and raised by nonalcoholics) and a higher risk in identical twins compared to fraternal twins of alcoholics. The genetic variations operate primarily through intermediate DIAGNOSTIC AND STATISTICAL MANUAL OF MENTAL DISORDERS, fifth eDitioN, CLassifiCatioN of aLCohoL use DisorDer (auD) Two or more of the following items occurring in the same 12-month period must be endorsed for the diagnosis of an alcohol use disordera: Drinking resulting in recurrent failure to fulfill role obligations

1	Tolerance Withdrawal, or substance use for relief/avoidance of withdrawal Drinking in larger amounts or for longer than intended Persistent desire/unsuccessful attempts to stop or reduce drinking Great deal of time spent obtaining, using, or recovering from alcohol Important activities given up/reduced because of drinking Continued drinking despite knowledge of physical or psychological problems caused by alcohol Alcohol craving aMild AUD: 2–3 criteria required; Moderate AUD: 4–5 items endorsed; severe AUD: 6 or more items endorsed.

1	characteristics that subsequently combine with environmental influences to alter the risk for heavy drinking and alcohol problems. These include genes relating to a high risk for all substance use disorders that operate through impulsivity, schizophrenia, and bipolar disorder. Another characteristic, an intense flushing response when drinking, decreases the risk for only alcohol use disorders through gene variations for several alcohol-metabolizing enzymes, especially aldehyde dehydrogenase (a mutation only seen in Asians), and to a lesser extent, variations in ADH.

1	An additional genetically influenced characteristic, a low sensitivity to alcohol, affects the risk for heavy drinking and may operate, in part, through variations in genes relating to calcium and potassium channels, GABA, nicotinic, and serotonin systems. A low response per drink is observed early in the drinking career and before alcohol use disorders develop. All follow-up studies have demonstrated that this need for higher doses of alcohol to achieve effects predicts future heavy drinking, alcohol problems, and alcohol use disorders. The impact of a low response to alcohol on adverse drinking outcomes is partially mediated by a range of environmental influences, including the selection of heavier-drinking friends, more positive expectations of the effects of high doses of alcohol, and suboptimal ways of coping with stress.

1	Although the age of the first drink (~15 years) is similar in most alcoholics and nonalcoholics, a slightly earlier onset of regular drinking and drunkenness, especially in the context of conduct problems, is associated with a higher risk for later alcohol use disorders. By the mid-twenties, most nonalcoholic men and women moderate their drinking (perhaps learning from problems), whereas alcoholics are likely to escalate their patterns of drinking despite difficulties. The first major life problem from alcohol often appears in the late teens to early twenties, and a pattern of multiple alcohol difficulties by the midtwenties. Once established, the course of alcoholism is likely to include exacerbations and remissions, with little difficulty in temporarily stopping or controlling alcohol use when problems develop, but without help, desistance usually gives way to escalations in alcohol intake and subsequent problems. Following treatment, between half and two-thirds of alcoholics

1	use when problems develop, but without help, desistance usually gives way to escalations in alcohol intake and subsequent problems. Following treatment, between half and two-thirds of alcoholics maintain abstinence for years, and often permanently. Even without formal treatment or self-help groups, there is at least a 20% chance of spontaneous remission with long-term abstinence. However, should the alcoholic continue to drink heavily, the life span is shortened by ~10 years on average, with the leading causes of death being heart disease, cancer, accidents, and suicide.

1	The approach to treating alcohol-related conditions is relatively straightforward: (1) recognize that at least 20% of all patients have an alcohol use disorder; (2) learn how to identify and treat acute alcohol-related conditions; (3) know how to help patients begin to address their alcohol problems; and (4) know enough about treating alcoholism to appropriately refer patients for additional help.

1	Even in affluent locales, ~20% of patients have an alcohol use disorder. These men and women can be identified by asking questions about alcohol problems and noting laboratory test results that can reflect regular consumption of six to eight or more drinks per day. The two blood tests with ≥60% sensitivity and specificity for heavy alcohol consumption are γ-glutamyl transferase (GGT) (>35 U) and carbohydrate-deficient transferrin (CDT) (>20 U/L or >2.6%); the combination of the two is likely to be more accurate than either alone. The values for these serologic markers are likely to return toward normal within several weeks of abstinence. Other useful blood tests include high-normal MCVs (≥91 μm3) and serum uric acid (>416 mol/L, or 7 mg/dL).

1	The diagnosis of an alcohol use disorder ultimately rests on the documentation of a pattern of repeated difficulties associated with alcohol (Table 467-2). Thus, in screening, it is important to probe for marital or job problems, legal difficulties, histories of accidents, medical problems, evidence of tolerance, and so on, and then attempt to tie in use 5-Point Scale Item (Least to Most) 1. How often do you have a drink containing alcohol? 2. How many drinks containing alcohol do you have on a typical day? 3. How often do you have six or more drinks on one occasion? 4. How often during the last year have you found that you were not able to stop drinking once you had started? 5. How often during the last year have you failed to do what was normally expected from you because of drinking? 6. How often during the last year have you needed a first drink in the morning to get yourself going after a heavy drinking session? 7.

1	6. How often during the last year have you needed a first drink in the morning to get yourself going after a heavy drinking session? 7. How often during the last year have you had a feeling of guilt or remorse after drinking? 8. How often during the last year have you been unable to remember what happened the night before because you had been drinking? 9. Have you or someone else been injured as a result of your drinking? 10. Has a relative, friend, doctor or other health worker been concerned about your drinking or suggested that you should cut down? Never (0) to 4+ per week (4) 1 or 2 (0) to 10+ (4) Never (0) to daily or almost daily (4) Never (0) to daily or almost daily (4) Never (0) to daily or almost daily (4) Never (0) to daily or almost daily (4) Never (0) to daily or almost daily (4) Never (0) to daily or almost daily (4) No (0) to yes, during the last year (4)

1	Never (0) to daily or almost daily (4) Never (0) to daily or almost daily (4) Never (0) to daily or almost daily (4) No (0) to yes, during the last year (4) No (0) to yes, during the last year (4) aThe AUDIT is scored by simply summing the values associated with the endorsed response. A score ≥8 may indicate harmful alcohol use. of alcohol or another substance. Some standardized questionnaires can be helpful, including the 10-item Alcohol Use Disorders Identification Test (AUDIT) (Table 467-3), but these are only screening tools, and a face-to-face interview is still required for a meaningful diagnosis.

1	The first priority in treating severe intoxication is to assess vital signs and manage respiratory depression, cardiac arrhythmias, or blood pressure instability, if present. The possibility of intoxication with other drugs should be considered by obtaining toxicology screens for other central nervous system (CNS) depressants such as benzodiazepines and for opioids. Aggressive behavior should be handled by offering reassurance but also by considering a possible show of force with an intervention team. If the aggressive behavior continues, relatively low doses of a short-acting benzodiazepine such as lorazepam (e.g., 1–2 mg PO or IV) may be used and can be repeated as needed, but care must be taken not to destabilize vital signs or worsen confusion. An alternative approach is to use an anti-psychotic medication (e.g., 0.5–5 mg of haloperidol PO or IM every 4–8 h as needed, or olanzapine 2.5–10 mg IM repeated at 2 and 6 h, if needed).

1	There are two main elements to intervention in a person with alcoholism: motivational interviewing and brief interventions. During motivational interviewing, the clinician helps the patient to think through the assets (e.g., comfort in social situations) and liabilities (e.g., healthand interpersonal-related problems) of the current pattern of drinking. The patient’s responses are key, and the clinician should listen empathetically, helping to weigh options and encouraging the patient to take responsibility for needed changes. Patients should be reminded that only they can decide to avoid the consequences that will occur without changes in drinking. The 2727 process of brief intervention has been summarized by the acronym FRAMES: Feedback to the patient; Responsibility to be taken by the patient; Advice, rather than orders, on what needs to be done; Menus of options that might be considered; Empathy for understanding the patient’s thoughts and feelings; and Self-efficacy, i.e.,

1	the patient; Advice, rather than orders, on what needs to be done; Menus of options that might be considered; Empathy for understanding the patient’s thoughts and feelings; and Self-efficacy, i.e., offering support for the capacity of the patient to make changes.

1	Once the patient begins to consider change, the emphasis shifts to brief interventions designed to help them understand more about potential actions. Discussions focus on consequences of high alcohol consumption, suggested approaches to stopping drinking, and help in recognizing and avoiding situations likely to lead to heavy drinking. Both motivational interviewing and brief interventions can be carried out in 15-min sessions, but because patients do not always change behavior immediately, multiple meetings are often required to explain the problem, discuss optimal treatments, and explain the benefits of abstinence.

1	If the patient agrees to stop drinking, sudden decreases in alcohol intake can produce withdrawal symptoms, many of which are the opposite of those produced by intoxication. Features include tremor of the hands (shakes); agitation and anxiety; autonomic nervous system overactivity including an increase in pulse, respiratory rate, sweating, and body temperature; and insomnia. These symptoms usually begin within 5–10 h of decreasing ethanol intake, peak on day 2 or 3, and improve by day 4 or 5, although mild levels of these problems may persist for 4–6 months as a protracted abstinence syndrome.

1	About 2% of alcoholics experience a withdrawal seizure, with the risk increasing in the context of concomitant medical problems, misuse of additional drugs, and higher alcohol quantities. The same risk factors also contribute to a similar rate of delirium tremens (DTs), where the withdrawal includes delirium (mental confusion, agitation, and fluctuating levels of consciousness) associated with a tremor and autonomic overactivity (e.g., marked increases in pulse, blood pressure, and respirations). The risks for seizures and DTs can be diminished by identifying and treating any underlying medical conditions early in the course of withdrawal.

1	Thus, the first step is a thorough physical examination in all alcoholics considering abstinence, including a search for evidence of liver failure, gastrointestinal bleeding, cardiac arrhythmia, infection, and glucose or electrolyte imbalances. It is also important to offer adequate nutrition and oral multiple B vitamins, including 50–100 mg of thiamine daily for a week or more. Because most alcoholics who enter withdrawal are either normally hydrated or mildly overhydrated, IV fluids should be avoided unless there is a relevant medical problem or significant recent bleeding, vomiting, or diarrhea.

1	The next step is to recognize that because withdrawal symptoms reflect the rapid removal of a CNS depressant, alcohol, the symptoms can be controlled by administering any depressant in doses that decrease symptoms (e.g., a rapid pulse and tremor) and then tapering the dose over 3–5 days. Although most depressants are effective, benzodiazepines (Chap. 466) have the highest margin of safety and lowest cost and are, therefore, the preferred class of drugs. Short-half-life benzodiazepines can be considered for patients with serious liver impairment or evidence of significant brain damage, but they must be given every 4 h to avoid abrupt blood-level fluctuations that may increase the risk for seizures. Therefore, most clinicians use drugs with longer half-lives (e.g., chlordiazepoxide), adjusting the dose if signs of withdrawal escalate, and withholding the drug if the patient is sleeping or has orthostatic hypotension. The average patient requires 25–50 mg of chlordiazepoxide or 10 mg of

1	the dose if signs of withdrawal escalate, and withholding the drug if the patient is sleeping or has orthostatic hypotension. The average patient requires 25–50 mg of chlordiazepoxide or 10 mg of diazepam given PO every 4–6 h on the first day, with doses then decreased to zero over the next 5 days. Although alcohol withdrawal can be treated in a hospital, patients in good physical condition who demonstrate mild signs of withdrawal despite low blood alcohol concentrations and who have no prior history of DTs or withdrawal seizures can be considered for outpatient detoxification. For the next 2728 4 or 5 days, these patients should return daily for evaluation of vital signs and can be hospitalized if signs and symptoms of withdrawal escalate.

1	Treatment of patient with DTs can be challenging, and the condition is likely to run a course of 3–5 days regardless of the therapy used. The focus of care is to identify and correct medical problems and to control behavior and prevent injuries. Many clinicians recommend the use of high doses of a benzodiazepine (as much as 800 mg/d of chlordiazepoxide has been reported), a treatment that will decrease agitation and raise the seizure threshold but probably does little to improve the confusion. Other clinicians recommend the use of antipsychotic medications, such as haloperidol or olanzapine as discussed above, although these drugs have not been directly evaluated for DTs. Antipsychotics are less likely to exacerbate confusion but may increase the risk of seizures; they have no place in the treatment of mild withdrawal symptoms.

1	Generalized withdrawal seizures rarely require more than giving an adequate dose of benzodiazepines. There is little evidence that anticonvulsants such as phenytoin or gabapentin are more effective in drug-withdrawal seizures, and the risk of seizures has usually passed by the time effective drug levels are reached. The rare patient with status epilepticus must be treated aggressively (Chap. 445).

1	REHABILITATION OF ALCOHOLICS An Overview After completing alcoholic rehabilitation, ≥60% of alcoholics, especially middle-class patients, maintain abstinence for at least a year, and many achieve lifetime sobriety. The core of treatment uses cognitive-behavioral approaches to help patients recognize the need to change, while working with them to alter their behaviors to enhance compliance. A key step is to optimize motivation toward abstinence through education about alcoholism and instructions to family members to stop protecting the patient from problems caused by alcohol. After years of heavy drinking, many patients also need counseling, some require vocational or avocational help to structure their days, and all should try self-help groups such as Alcoholics Anonymous (AA) to help them develop a sober peer group and learn how to deal with life’s stresses while sober. A third component, relapse prevention, helps the patient identify situations in which a return to drinking is

1	them develop a sober peer group and learn how to deal with life’s stresses while sober. A third component, relapse prevention, helps the patient identify situations in which a return to drinking is likely, formulate ways of managing these risks, and develop coping strategies that increase the chances of a return to abstinence if a slip occurs.

1	Although many can be treated as outpatients, more intense interventions are more effective, and some alcoholics do not respond to AA or outpatient groups. Whatever the setting, subsequent contact with outpatient treatment staff should be maintained for at least 6 months and preferably a year after abstinence. Counseling focuses on areas of improved functioning in the absence of alcohol (i.e., why it is a good idea to continue abstinence) and helping the patient to manage free time without alcohol, develop a nondrinking peer group, and handle stresses.

1	The physician serves an important role in identifying the alcoholic, diagnosing and treating associated medical and psychiatric syndromes, overseeing detoxification, referring the patient to rehabilitation programs, providing counseling, and, if appropriate, selecting which (if any) medication might be needed. For insomnia, patients should be reassured that troubled sleep is normal after alcohol withdrawal and will improve over subsequent weeks. They should be taught the elements of “sleep hygiene” including maintaining consistent schedules for bedtime and awakening. Sleep medications have the danger of being misused and of rebound insomnia when stopped. Sedating antidepressants (e.g., trazodone) should not be used because they interfere with cognitive functioning the next morning and disturb the normal sleep architecture, but occasional use of over-the-counter sleeping medications (sedating antihistamines) can be considered. Anxiety can be addressed by increasing the patient’s

1	disturb the normal sleep architecture, but occasional use of over-the-counter sleeping medications (sedating antihistamines) can be considered. Anxiety can be addressed by increasing the patient’s insight into the temporary nature of the symptoms and helping the patient to develop strategies to achieve relaxation by using forms of cognitive therapy.

1	Medications for Rehabilitation Several medications have modest benefits when used for the first 6 months of recovery. The opioid antagonist, naltrexone, 50–150 mg/d orally, may shorten subsequent relapses, whether used in the oral form or as a once-per-month 380-mg injection, especially in individuals with the G allele of the AII8G polymorphism of the μ opioid receptor. By blocking opioid receptors, naltrexone decreases activity in the dopamine-rich ventral tegmental reward system and decreases the feeling of pleasure if alcohol is imbibed. A second medication, acamprosate (Campral) at ~2 g/d divided into three oral doses, has similar modest effects; acamprosate inhibits NMDA receptors, decreasing mild symptoms of protracted withdrawal. Several trials of combined naltrexone and acamprosate have reported that the combination may be superior to either drug alone, although not all studies agree.

1	It is more difficult to establish the asset-to-liability ratio of a third drug, disulfiram, an ALDH inhibitor, used at doses of 250 mg/d. This drug produces vomiting and autonomic nervous system instability in the presence of alcohol as a result of rapidly rising blood levels of acetaldehyde. This reaction can be dangerous, especially for patients with heart disease, stroke, diabetes mellitus, or hypertension. The drug itself carries potential risks of depression, psychotic symptoms, peripheral neuropathy, and liver damage. Disulfiram is best given under supervision by someone (such as a spouse), especially during high-risk drinking situations (such as the Christmas holiday). Other drugs under investigation include another opioid antagonist nalmefene, the nicotinic receptor agonist varenicline, the serotonin antagonist ondansetron, the α-adrenergic agonist prazosin, the GABAB receptor agonist baclofen, the anticonvulsant topiramate, and cannabinol receptor antagonists. At present,

1	the serotonin antagonist ondansetron, the α-adrenergic agonist prazosin, the GABAB receptor agonist baclofen, the anticonvulsant topiramate, and cannabinol receptor antagonists. At present, there are insufficient data to determine the asset-to-liability ratio for these medications in treating alcoholism and, therefore, no data to offer solid support for their use in routine clinical settings.

1	As described above, rates of alcohol use disorders differ across sex, age, ethnicity, and country. There are also differences across countries regarding the definition of a standard drink (e.g., 10–12 g of ethanol in the United States and 8 g in the United Kingdom) and the definition of being legally drunk. The preferred alcoholic beverage also varies across groups, even within countries. That said, regardless of sex, ethnicity, or country, the actual drug in the drink is still ethanol, and the risks for problems, course of alcohol use disorders, and approaches to treatment are similar across the world. Thomas R. Kosten, Colin N. Haile this is a digital-only chapter. it is available on the DvD that accompanies this book, as well as on access medicine/harrison’s online, and the eBook and “app” editions of hpim 19e.

1	Opiate analgesics have been abused since at least 300 b.c. Nepenthe (Greek “free from sorrow”) helped the hero of the Odyssey, but widespread opium smoking in China and the Near East has caused harm for centuries. Since the first chemical isolation of opium and codeine 200 years ago, a wide range of synthetic opioids have been developed, and opioid receptors were cloned in the 1990s. Two of the most important adverse effects of all these agents are the development of opioid use disorder and overdose. The 0.1% annual prevalence of heroin dependence in the United States is only about one-third the rate of prescription opiate use and is substantially lower than the 2% rate of morphine users in Southeast and Southwest Asia.

1	Opioid-related Disorders Thomas R. Kosten, Colin N. Haile Opiate analgesics have been abused since at least 300 b.c. Nepenthe (Greek “free from sorrow”) helped the hero of the Odyssey, but widespread opium smoking in China and the Near East has caused harm for centuries. Since the first chemical isola-468e tion of opium and codeine 200 years ago, a wide range of synthetic opioids have been developed, and opioid receptors were cloned in the 1990s. Two of the most important adverse effects of all these agents are the development of opioid use disorder and overdose. The 0.1% annual prevalence of heroin dependence in the United States is only about one-third the rate of prescription opiate use and is substantially lower than the 2% rate of morphine users in Southeast and Southwest Asia. Prescription opiates are primarily used for pain management, but due to ease of availability, adolescents procure and use these drugs with dire consequences. In 2011, for example, 11 million individuals in

1	opiates are primarily used for pain management, but due to ease of availability, adolescents procure and use these drugs with dire consequences. In 2011, for example, 11 million individuals in the United States used nonmedically prescribed pain killers that were linked to over 420,000 emergency department visits and nearly 17,000 overdose deaths. Although these rates are low relative to other abused substances, their disease burden is substantial, with high rates of morbidity and mortality; disease transmission; increased health care, crime, and law enforcement costs; and less tangible costs of family distress and lost productivity.

1	The terms “dependence” and “addiction” are no longer used to describe substance use disorders. Opioid-related disorders encompass opioid use disorder, opioid intoxication, and opioid withdrawal. The diagnosis of opioid use disorder as defined in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) requires the repeated use of the opiate while producing problems in two or more areas in a 12-month period. The areas include tolerance, withdrawal, use of greater amounts of opiates than intended, craving, and use despite adverse consequences. This new definition of opiate use disorder, reducing the criteria for diagnosis from three problem areas to two, is not expected to change the rates of these disorders because most individuals using these substances meet more than three criteria.

1	A striking recent aspect of illicit opiate use has been its marked increase as the gateway to illicit drugs in the United States. Since 2007, prescription opiates have surpassed marijuana as the most common illicit drug that adolescents initially use, although overall rates of opiate dependence are far lower than marijuana. The most commonly used opiates are diverted prescriptions for oxycodone and hydrocodone, followed by heroin and morphine, and—among health professionals—meperidine and fentanyl. Heroin is derived from morphine and acts as a prodrug that more readily penetrates the brain and is converted rapidly to morphine in the body. Two opiate maintenance treatment agents—methadone and buprenorphine—are also misused, but at substantially lower rates, and the partial opiate agonists such as butorphanol, tramadol, and pentazocine are misused even less frequently. Because the chemistry and general pharmacology of these agents are covered in major pharmacology texts, this chapter

1	such as butorphanol, tramadol, and pentazocine are misused even less frequently. Because the chemistry and general pharmacology of these agents are covered in major pharmacology texts, this chapter focuses on the neurobiology and pharmacology relevant to dependence and its treatments. Although the neurobiology of abuse involves all four of the known opiate receptors—mu, kappa, delta, and nociceptin/orphanin—this discussion focuses on the mu receptor, at which most of the clinically used opiates are active.

1	The neurobiology of opiates and their effects not only include opiate receptors, but also the downstream intracellular messenger systems and ion channels that the receptors regulate. The different functional activities of opiate receptors are summarized in Table 468e-1. Abuse liability of opiates is primarily associated with the mu receptor. All opiate receptors are G protein–linked and coupled to the cyclic adenosine monophosphate (cAMP) second messenger system and to G protein– coupled, inwardly rectifying potassium channels (GIRKs). Opiates activate GIRKs, increasing permeability to potassium ions to cause Mu (μ) (e.g., morphine, Analgesia, reinforcement euphoria, cough and buprenorphine) appetite suppression, decreased respirations, decreased GI motility, sedation, hormone changes, dopamine and acetylcholine release Kappa (κ) (e.g., butorphanol) Dysphoria, decreased GI motility, decreased appetite, decreased respiration, psychotic symptoms, sedation, diuresis, analgesia

1	Kappa (κ) (e.g., butorphanol) Dysphoria, decreased GI motility, decreased appetite, decreased respiration, psychotic symptoms, sedation, diuresis, analgesia Delta (δ) (e.g., etorphine) Analgesia, euphoria, physical dependence, Hormone changes, appetite suppression, Nociceptin/orphanin (e.g., Analgesia, appetite, anxiety, tolerance to opi buprenorphine) oids, hypotension, decreased GI motility, 5-HT Abbreviations: GI, gastrointestinal; 5-HT, serotonin; NE, norepinephrine. hyperpolarization, which inhibits the production of action potentials. Thus, opiates inhibit the activity of diverse and widely distributed neuronal types. The major effects of opiates, such as analgesia, sedation, and drug reinforcement are produced through this inhibition of neurons that belong to specific brain pathways.

1	Many opiate actions are related to the specific neuroanatomic locations of mu receptors. Reinforcing and euphoric effects of opiates occur in the mesolimbic dopaminergic pathway from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), where opiates increase synaptic levels of dopamine. This increase is due to inhibition of GABAergic neurons that inhibit both the activity of neurons within the VTA and the NAc. The positive subjective effects of opioid drugs also include mu receptor desensitization and internalization, potentially related to stimulation of beta-arrestin signalizing pathways. However, the “high” only occurs when the rate of change in dopamine is fast. Large, rapidly administered doses of opiates block γ-aminobutyric acid (GABA) inhibition and produce a burst of VTA dopamine neuron activity that is associated with “high” in all abused drugs. Therefore, routes of administration that slowly increase opiate blood and brain levels, such as oral and transdermal

1	VTA dopamine neuron activity that is associated with “high” in all abused drugs. Therefore, routes of administration that slowly increase opiate blood and brain levels, such as oral and transdermal routes, are effective for analgesia and sedation but do not produce an opiate “high” that follows smoking and intravenous routes. Other acute effects such as analgesia and respiratory depression involve opiate receptors located in other brain areas such as the locus coeruleus (LC).

1	Opiate tolerance and withdrawal are chronic effects related to the cAMP-protein kinase A (PKA)-cAMP response-element binding protein (CREB) intracellular cascade (Fig. 468e-1). These effects are also reflective of genetic risk factors for developing opiate use disorder, with estimates of up to 50% of the risk for dependence due to polygenic inheritance. Specific functional polymorphisms in the mu opiate receptor gene appear to be associated with this risk for opiate abuse, including one producing a threefold increase in this receptor’s affinity for opiates and the endogenous ligand beta endorphin. Epigenetic methylation changes also occur on the DNA of the mu receptor gene of opiate addicts, inhibiting gene transcription. This molecular cascade links acute intoxication and sedation to opiate tolerance and withdrawal mediated by the LC. Noradrenergic neurons in the LC mediate activation of the cortical hemispheres. When large opiate doses saturate and activate all of its mu receptors,

1	tolerance and withdrawal mediated by the LC. Noradrenergic neurons in the LC mediate activation of the cortical hemispheres. When large opiate doses saturate and activate all of its mu receptors, action potentials cease. When this direct inhibitory effect is sustained over weeks and months of opiate use, a secondary set of adaptive changes occur that lead to tolerance and withdrawal symptoms (Fig. 468e-1). Withdrawal symptoms reflect, in part, overactivity of norepinephrine (NE) neurons in the LC. This molecular model of NE neuronal activation during withdrawal has had important treatment implications, such as the use of the alpha-2 agonist clonidine to treat opioid withdrawal. Other contributors to withdrawal include deficits within the dopamine reward system.

1	Tolerance and withdrawal commonly occur with chronic daily use, developing as quickly as 6–8 weeks depending on dose concentration B-endorphineenkephalinsK+Na+Na+K+˜˜Gi/oGi/oACcAMPcAMPNucleusNucleusPKAPKABDNFBDNFTHTHCREBCREBNCH3HHHOOHOABAC Modified gene expression, neuroplasticity, genetic effects

1	FIGURE 468e-1 Normal mu-receptor activation by endogenous opioids inhibits the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response-element binding protein (CREB) cascade in noradrenergic neurons within the locus coeruleus (A) through inhibitory Gi/o protein influence on adenylyl cyclase (AC). Similarly, acute exposure to opiates (e.g., morphine) inhibits this system, whereas chronic exposure to opiates (B) leads to upregulation of the cAMP pathway in an attempt to oppose opiate-induced inhibitory influence. Upregulation of this system is involved in opiate tolerance, and when the opiate is removed, unopposed noradrenergic neurotransmission is involved in opiate withdrawal. Upregulated PKA phosphorylates CREB, initiating the expression of various genes such as tyrosine hydroxylase (TH) and brain-derived neurotrophic factor (BDNF). BDNF is implicated in long-term neuroplastic changes in response to chronic opiates.

1	and dosing frequency. Tolerance appears to be primarily a pharmacodynamic rather than pharmacokinetic effect, with relatively limited induction of cytochrome P450 or other liver enzymes. The metabolism of opiates occurs in the liver primarily through the cytochrome P450 systems of 2D6 and 3A4. They then are conjugated to glucuronic acid and excreted in small amounts in feces. The plasma half-lives generally range from 2.5 to 3 h for morphine and more than 22 h for methadone. The shortest half-lives of several minutes are for fentanyl-related opiates and the longest are for buprenorphine and its active metabolites, which can block opiate withdrawal for up to 3 days after a single dose. Tolerance to opioids leads to the need for increasing amounts of drugs to sustain the desired euphoric effects—as well as to avoid the discomfort of withdrawal. This combination has the expected consequence of strongly reinforcing dependence once it has started. Methadone taken chronically at maintenance

1	well as to avoid the discomfort of withdrawal. This combination has the expected consequence of strongly reinforcing dependence once it has started. Methadone taken chronically at maintenance doses is stored in the liver, which may reduce the occurrence of withdrawal between daily doses. The role of endogenous opioid peptides in tolerance and withdrawal is uncertain.

1	The clinical features of abuse are tied to route of administration and the rapidity of an opiate bolus in reaching the brain. Intravenous and smoked administration rapidly produces a bolus of high drug concentration in the brain. This bolus produces a “rush,” followed by euphoria, a feeling of tranquility, and sleepiness (“the nod”). Heroin produces effects that last 3–5 h, and several doses a day are required to forestall manifestations of withdrawal in chronic users. Symptoms of opioid withdrawal begin 8–10 h after the last dose; lacrimation, rhinorrhea, yawning, and sweating appear first. Restless sleep followed by weakness, chills, gooseflesh (“cold turkey”), nausea and vomiting, muscle aches, and involuntary movements (“kicking the habit”), hyperpnea, hyperthermia, and hypertension occur in later stages of the withdrawal syndrome. The acute course of withdrawal may last 7–10 days. A secondary phase of protracted abstinence lasts for 26–30 weeks and is characterized by

1	occur in later stages of the withdrawal syndrome. The acute course of withdrawal may last 7–10 days. A secondary phase of protracted abstinence lasts for 26–30 weeks and is characterized by hypotension, bradycardia, hypothermia, mydriasis, and decreased responsiveness of the respiratory center to carbon dioxide.

1	Besides the brain effects of opioids on sedation and euphoria and the combined brain and peripheral nervous system effects on analgesia, a wide range of other organs can be affected. The cough reflex is inhibited through the brain, leading to the use of some opiates as an antitussive, and nausea and vomiting are due to effects on the medulla. The release of several pituitary hormones is inhibited, including corticotropin-releasing factor (CRF) and luteinizing hormone, which reduces levels of cortisol and sex hormones and can lead to impaired stress responses and reduced libido. An increase in prolactin also contributes to the reduced sex drive in males. Two other hormones affected are thyrotropin, which is reduced, and growth hormone, which is increased. Respiratory depression results from opiate-induced insensitivity of brainstem neurons to increases in carbon dioxide, and in patients with pulmonary disease, this can result in clinically significant complications. In overdoses,

1	from opiate-induced insensitivity of brainstem neurons to increases in carbon dioxide, and in patients with pulmonary disease, this can result in clinically significant complications. In overdoses, aspiration pneumonia is common due to loss of the gag reflex. Opiates reduce gut motility, which is helpful for treating diarrhea, but can lead to nausea, constipation, and anorexia with weight loss. Deaths occurred in early methadone maintenance programs due to severe constipation and toxic megacolon. Opiates such as methadone may prolong QT intervals and lead to sudden death in some patients. Orthostatic hypotension may occur due to histamine release and peripheral blood vessel dilation, which is an opiate effect usefully applied to managing acute myocardial infarction. During opiate maintenance, interactions with other medications are of concern; these include inducers of the cytochrome P450 system (usually CYP3A4) such as rifampin and carbamazepine.

1	Heroin users in particular tend to use opiates intravenously and are likely to be polydrug users, also using alcohol, sedatives, cannabinoids, and stimulants. None of these other drugs are substitutes for opioids, but they have desired additive effects. Therefore, one needs to be sure that the person undergoing a withdrawal reaction is not also withdrawing from alcohol or sedatives, which might be more dangerous and more difficult to manage.

1	Intravenous opiate use carries with it the risk of serious complications. The common sharing of hypodermic syringes can lead to infections with hepatitis B and HIV/AIDS, among others. Bacterial infections can lead to septic complications such as meningitis, osteomyelitis, and abscesses in various organs. Off-target effects of opiates synthesized in illicit drug labs can lead to serious toxicity. For example, attempts to illicitly manufacture meperidine in the 1980s resulted in the production of a highly specific neurotoxin, MPTP, which produced parkinsonism in users (Chap. 449).

1	Lethal overdose is a relatively common complication of opiate use disorder. Rapid recognition and treatment with naloxone, a highly specific reversal agent that is relatively free of complications, is essential. The diagnosis is based on recognition of characteristic signs and symptoms, including shallow and slow respirations, pupillary miosis (mydriasis does not occur until significant brain anoxia supervenes), bradycardia, hypothermia, and stupor or coma. Blood or urine toxicology studies can confirm a suspected diagnosis, but immediate management must be based on clinical criteria. If naloxone is not administered, progression to respiratory and cardiovascular collapse leading to death occurs. At autopsy, cerebral edema and sometimes frothy pulmonary edema are generally found. Opiates generally do not produce seizures except for unusual cases of polydrug use with the opiate meperidine, with high doses of tramadol, or in the newborn.

1	Beyond the acute treatment of opiate overdose with naloxone, clinicians have two general treatment options: opioid maintenance or detoxification. Opioid agonist and partial agonist medications are commonly used for both maintenance and detoxification purposes. Alpha-2-adrenergic agonists are primarily used for detoxification. Antagonists are used to accelerate detoxification and then continued after detoxification to prevent relapse. Only the residential medication-free programs have had success that comes close to matching that of the medication-based programs. Success of the various treatment approaches is assessed as retention in treatment and reduced opioid and other drug use; secondary outcomes, such as reduced HIV risk behaviors, crime, psychiatric symptoms, and medical comorbidity, also indicate successful treatment.

1	Stopping opiates is much easier than preventing relapse. Longterm relapse prevention for opioid-dependent persons requires combined pharmacologic and psychosocial approaches. Chronic users tend to prefer pharmacologic approaches; those with shorter histories of drug abuse are more amenable to detoxification and psychosocial interventions.

1	Managing overdose requires naloxone and support of vital functions, including intubation if needed (Table 468e-2). If the overdose is due to buprenorphine, then naloxone might be required at total doses of 10 mg or greater, but primary buprenorphine overdose is nearly impossible because this agent is a partial opiate agonist, meaning that as the dose of buprenorphine is increased it has greater opiate antagonist than agonist activity. Thus, a 0.2-mg buprenorphine dose leads to analgesia and sedation, while a hundred times greater 20-mg dose produces profound opiate antagonism, precipitating opiate withdrawal in a person who was opiate dependent on morphine or methadone. It is important to recognize that the goal is to reverse the respiratory depression and not to administer so much naloxone that it precipitates opiate withdrawal. Because naloxone only lasts a few hours and most opiates last considerably longer, an IV naloxone drip with close monitoring is frequently employed to

1	naloxone that it precipitates opiate withdrawal. Because naloxone only lasts a few hours and most opiates last considerably longer, an IV naloxone drip with close monitoring is frequently employed to provide a continuous level of antagonism for 24–72 h depending on the opiate used in the overdose (e.g., morphine vs methadone). Whenever naloxone has only a limited effect, other sedative drugs that produce significant overdoses must be considered. The most common are benzodiazepines, which have produced overdoses and deaths in combination with buprenorphine. A specific antagonist for benzodiazepines—flumazenil at 0.2 mg/min—can be given to a maximum of 3 g/h, but it may precipitate seizures and increase intracranial pressure. Like naloxone, administration for a prolonged period is usually required because most benzodiazepines remain active for considerably longer than flumazenil. Support of vital functions may include oxygen and positive-pressure breathing, IV fluids, pressor agents for

1	because most benzodiazepines remain active for considerably longer than flumazenil. Support of vital functions may include oxygen and positive-pressure breathing, IV fluids, pressor agents for hypotension, and cardiac monitoring to detect QT prolongation, which might require specific treatment. Activated

1	Establish airway. Intubation and mechanical ventilation may be necessary. Naloxone 0.4–2.0 mg (IV, IM, or endotracheal tube). Onset of action with IV is approximately 1–2 min. Repeat doses of naloxone if needed to restore adequate respiration or a continuous infusion of naloxone can be used. One-half to two-thirds of the initial naloxone dose that reversed the respiratory depression is administered on an hourly basis (note: naloxone dosing is not necessary if the patient has been intubated). charcoal and gastric lavage may be helpful for oral ingestions, but 468e-3 intubation will be needed if the patient is stuporous.

1	charcoal and gastric lavage may be helpful for oral ingestions, but 468e-3 intubation will be needed if the patient is stuporous. The principles of detoxification are the same for all drugs: to substitute a longer-acting, orally active, pharmacologically equivalent medication for the substance being used, stabilize the patient on that medication, and then gradually withdraw the substituted medication. Methadone or buprenorphine are the two medications used to treat opioid use disorder. Clonidine, a centrally acting sympatholytic agent, has also been used for detoxification in the United States. By reducing central sympathetic outflow, clonidine mitigates many of the signs of sympathetic overactivity but typically requires augmentation with other agents. Clonidine has no narcotic action and is not addictive. Lofexidine, a clonidine analogue with less hypotensive effect, is not yet approved in the United States.

1	Methadone for Detoxification Dose-tapering regimens for detoxification using methadone range from 2–3 weeks to as long as 180 days, but this approach is controversial given the relative effectiveness of methadone maintenance and the low success rates of detoxification. Unfortunately, the vast majority of patients tend to relapse to heroin or other opiates during or after the detoxification period, indicative of the chronic and relapsing nature of opioid use disorder. Buprenorphine for Detoxification Buprenorphine does not appear to lead to better outcomes than methadone but is superior to clonidine in reducing symptoms of withdrawal, retaining patients in a withdrawal protocol, and in completing treatment.

1	Alpha-2-Adrenergic Agonists for Detoxification Several alpha-2-adrenergic agonists have relieved opioid withdrawal by suppressing brain noradrenergic hyperactivity. Clonidine relieves some signs and symptoms of opiate withdrawal such as lacrimation, rhinorrhea, muscle pain, joint pain, restlessness, and gastrointestinal symptoms. Related agents are lofexidine, guanfacine, and guanabenz acetate. Lofexidine can be dosed up to ~2 mg/d and appears to be associated with fewer adverse effects. Clonidine or lofexidine is typically administered orally, in three or four doses per day, with dizziness, sedation, lethargy, and dry mouth as the primary adverse side effects. Outpatient-managed withdrawal will require close follow-up often with naltrexone maintenance to prevent relapse.

1	Rapid and Ultrarapid Opiate Detoxification The opioid antagonist naltrexone typically combined with an alpha-2-adrenergic agonist has been purported to shorten the duration of withdrawal without significantly increasing patient discomfort. Completion rates using naltrexone and clonidine range from 75 to 81% compared to 40 to 65% for methadone or clonidine alone. Ultrarapid opiate detoxification is an extension of this approach using anesthetics but is highly controversial due to the medical risks and mortality associated with it.

1	Opioid Agonist Medications for Maintenance Methadone maintenance substitutes a once-daily oral opioid dose for threeto four-times daily heroin. Methadone saturates the opioid receptors and, by inducing a high level of opiate tolerance, blocks the euphoria from additional opiates. Buprenorphine, a partial opioid agonist, also can be given once daily at sublingual doses of 4–32 mg daily, and in contrast to methadone, it can be given in an office-based primary care setting.

1	METHADONE MAINTENANCE Methadone’s slow onset of action when taken orally, long elimination half-life (24–36 h), and production of cross-tolerance at doses from 80 to 150 mg are the basis for its efficacy in treatment retention and reductions in IV drug use, criminal activity, and HIV risk behaviors and mortality. Methadone can prolong the QT interval at rates as high as 16% above the rates in non-methadonemaintained, drug-injecting patients, but it has been used safely in the treatment of opioid dependence for 40 years.

1	BUPRENORPHINE MAINTENANCE While France and Australia have had sublingual buprenorphine maintenance since 1996, it was first approved by the U.S. Food and Drug Administration (FDA) in 2002 as a Schedule III drug for managing opioid use disorder. Unlike the full agonist methadone, buprenorphine is a partial agonist of mu-opioid receptors with a slow onset and long duration of action. Its partial agonism reduces the risk of unintentional overdose but limits its efficacy to patients who need the equivalent of only 60–70 mg of methadone, and many patients in methadone maintenance require higher doses up to 150 mg daily. Buprenorphine is combined with naloxone at a 4:1 ratio in order to reduce its abuse liability. Because of pediatric exposures and diversion of buprenorphine to illicit use, a new formulation, using mucosal films rather than sublingual pills that were crushed and snorted, is now marketed. A subcutaneous buprenorphine implant that lasts up to 6 months has also been tested and

1	formulation, using mucosal films rather than sublingual pills that were crushed and snorted, is now marketed. A subcutaneous buprenorphine implant that lasts up to 6 months has also been tested and is pending FDA approval as a formulation improvement to prevent pediatric exposures and illicit diversion and enhance compliance.

1	In the United States, the ability of primary care physicians to prescribe buprenorphine for opioid use disorder represents an important opportunity to improve access and quality of treatment as well as reduce social harm. Europe, Asia, and Australia have found reduced opioid-related deaths and drug-injection-related medical morbidity with buprenorphine available in primary care. Retention in office-based buprenorphine treatment has been high as 70% at 6-month follow-ups.

1	Opioid Antagonist Medications The rationale for using narcotic antagonist therapy is that blocking the action of self-administered opioids should eventually extinguish the habit, but this therapy is poorly accepted by patients. Naltrexone, a long-acting orally active pure opioid antagonist, can be given three times a week at doses of 100–150 mg. Because it is an antagonist, the patient must first be detoxified from opioid dependence before starting naltrexone. It is safe even when taken chronically for years, is associated with few side effects (headache, nausea, abdominal pain), and can be given to patients infected with hepatitis B or C without producing hepatotoxicity. However, most providers refrain from prescribing naltrexone if liver function tests are three times above normal levels. Naltrexone maintenance combined with psychosocial therapy is effective in reducing heroin use, but medication adherence is low. Depot injection formulations lasting up to 4 weeks markedly improve

1	Naltrexone maintenance combined with psychosocial therapy is effective in reducing heroin use, but medication adherence is low. Depot injection formulations lasting up to 4 weeks markedly improve adherence, retention, and drug use. Subcutaneous naltrexone implants in Russia, China, and Australia have doubled treatment retention and reduced relapse to half that of oral naltrexone. In the United States, a depot naltrexone formulation is available for monthly use and maintains blood levels equivalent to 25 mg of daily oral use.

1	Medication-Free Treatment Most opiate addicts enter medication-free treatments in inpatient, residential, or outpatient settings, but 1to 5-year outcomes are very poor compared to pharmacotherapy except for residential settings lasting 6 to 18 months. The residential programs require full immersion in a regimented system with progressively increasing levels of independence and responsibility within a controlled community of fellow drug abusers. These medication-free programs, as well as the pharmacotherapy programs, also include counseling and behavioral treatments designed to teach interpersonal and cognitive skills for coping with stress and for avoiding situations leading to easy access to drugs or to craving. Relapse is prevented by having the individual very gradually reintroduced to greater responsibilities and to the working environment outside of the protected therapeutic community.

1	Preventing opiate abuse represents a critically important challenge for physicians. Opiate prescriptions are the most common source of drugs accessed by adolescents who begin a pattern of illicit drug use. The major sources of these drugs are family members, not drug dealers or the Internet. Pain management involves providing sufficient opiates to relieve the pain over as short a period of time as the pain warrants (Chap. 18). The patient then needs to dispose of any remaining opiates, not save them in the medicine cabinet, because this behavior leads to diversion by adolescents. Finally, physicians should never prescribe opiates for themselves.

1	Cocaine and Other Commonly Abused Drugs Nancy K. Mello1, Jack H. Mendelson1 The abuse of cocaine and other psychostimulants reflects a complex interaction between the pharmacology of the drug, the personality and 469e expectations of the user, and the environmental context in which the drug is used. Polydrug abuse involving the concurrent use of several drugs with different pharmacologic effects is increasingly common. Sometimes one drug is used to enhance the effects of another, as with the combined use of cocaine and nicotine, benzodiazepines and methadone, or cocaine and heroin in methadone-maintained patients. Some forms of polydrug abuse, such as the combined use of IV heroin and cocaine, are especially dangerous and account for many hospital emergency room visits.

1	number of adverse health consequences and may exacerbate preexisting disorders such as hypertension and cardiac disease. The combined use of two or more drugs may accentuate medical complications associated with abuse of one drug. Chronic drug abuse is often associated with immune system dysfunction and increased vulnerability to infections, including risk for HIV infection. In addition, concurrent use of cocaine and opiates (the “speedball”) is frequently associated with needle sharing by IV drug users. IV drug abusers continue to be the largest single group of persons with HIV infection in several major metropolitan areas in the United States as well as in many parts of Europe and Asia.

1	Stimulants and hallucinogens have been used to induce euphoria and alter consciousness for centuries. Cocaine and marijuana are two of the most commonly abused drugs today. Synthetic variations of marijuana and a variety of hallucinogens have become popular recently, and new drugs are continually being developed. This chapter describes the subjective and adverse medical effects of cocaine, marijuana, and lysergic acid diethylamide (LSD), as well as methamphetamine, 3,4-methylenedioxyN-methamphetamine (MDMA), synthetic cathinones (bath salts), phencyclidine (PCP), Salvia divinorum, and other drugs of abuse (flunitrazepam, γ-hydroxybutyric acid [GHB], ketamine). Some options for medical management of severe adverse effects are also described.

1	Cocaine is a stimulant and a local anesthetic with potent vasoconstrictor properties. The leaves of the coca plant (Erythroxylum coca) contain ~0.5–1% cocaine. The drug produces physiologic and behavioral effects after oral, intranasal, IV, or inhalation/smoking routes of administration. The reinforcing effects of cocaine are related to activation of dopaminergic neurons in the mesolimbic system (Chap. 465e). Cocaine increases synaptic concentrations of the monoamine neurotransmitters dopamine, norepinephrine, and serotonin by binding to transporter proteins in presynaptic neurons and blocking reuptake.

1	Cocaine is widely available and is abused in virtually all social and economic strata of society. In 2012, an estimated 1.6 million persons in the United States used cocaine, and 1.1 million abused or were dependent on cocaine. Emergency room admissions involving cocaine totaled 505,224 in 2011. Cocaine abuse is prevalent in the general population and in heroin-dependent persons, including those in methadone maintenance programs. IV cocaine is often used concurrently with IV heroin in a combination called a “speedball.” This combination purportedly attenuates the postcocaine “crash” and substitutes a cocaine “high” for the heroin “high” blocked by methadone.

1	There has been an increase in both IV administration and inhalation of pyrolyzed cocaine via smoking. Following intranasal administration, changes in mood and sensation are perceived within 3–5 min, and peak effects occur at 10–20 min. These effects rarely last more than 1 h. Inhalation of pyrolyzed materials includes inhaling crack/cocaine or smoking coca paste, a product made by extracting cocaine preparations with flammable solvents, and cocaine free-base smoking. Freebase cocaine, including the free-base prepared with sodium bicarbonate (crack), has become increasingly popular because of its relative high potency and rapid onset of action (8–10 seconds following smoking).

1	Cocaine produces a brief, dose-related stimulation and euphoria and an increase in cardiac rate and blood pressure. Body temperature usually increases following cocaine administration, and high doses of cocaine may induce lethal pyrexia or hypertension. Because cocaine inhibits reuptake of catecholamines at adrenergic nerve endings, it potentiates sympathetic nervous system activity. Cocaine has a short plasma half-life of approximately 45–60 min. Cocaine is metabolized by plasma esterases, and cocaine metabolites are excreted in urine. The brief duration of the euphorigenic effects of cocaine reported by chronic abusers is probably due to both acute and chronic tolerance. Cocaine may be used as often as two to three times per hour. Alcohol is often used to modulate both the cocaine high and the dysphoria associated with the abrupt disappearance of cocaine’s effects. A metabolite of cocaine, cocaethylene, has been detected in blood and urine of persons who concurrently abuse alcohol

1	and the dysphoria associated with the abrupt disappearance of cocaine’s effects. A metabolite of cocaine, cocaethylene, has been detected in blood and urine of persons who concurrently abuse alcohol and cocaine. Cocaethylene induces changes in cardiovascular function similar to those of cocaine alone, and the pathophysiologic consequences of the concurrent abuse of alcohol plus cocaine may be additive.

1	Cocaine may cause serious medical consequences by any route of administration. The prevalent assumption that cocaine inhalation or IV administration is relatively safe is contradicted by reports of death from respiratory depression, cardiac arrhythmias, and convulsions associated with cocaine use. In addition to generalized seizures, neurologic complications may include headache, ischemic or hemorrhagic stroke, or subarachnoid hemorrhage. Disorders of cerebral blood flow and perfusion in cocaine-dependent persons have been detected with magnetic resonance spectroscopy (MRS). Inhalation of crack cocaine may lead to severe pulmonary disease due to the direct effects of cocaine and to residual contaminants in the smoked material. Hepatic necrosis may occur following chronic crack/cocaine use. Protracted cocaine abuse may also cause paranoid ideation and visual and auditory hallucinations, a state that resembles alcoholic hallucinosis.

1	Although men and women who abuse cocaine may report that the drug enhances libidinal drive, chronic cocaine use causes significant loss of libido and adversely affects sexual function. Impotence and gynecomastia have been observed in male cocaine abusers, and these abnormalities often persist for long periods following cessation of drug use. Cocaine abuse may produce major derangements in menstrual cycle function including galactorrhea, amenorrhea, and infertility in women and in a rhesus monkey model of cocaine self-administration. Chronic cocaine abuse may cause persistent hyperprolactinemia as a consequence of disordered dopaminergic inhibition of prolactin secretion by the anterior pituitary. Cocaine abuse by pregnant women, particularly crack smoking, has been associated with both an increased risk of congenital malformations in the fetus and perinatal cardiovascular and cerebrovascular disease in the mother. However, cocaine abuse per se is probably not the sole cause of these

1	increased risk of congenital malformations in the fetus and perinatal cardiovascular and cerebrovascular disease in the mother. However, cocaine abuse per se is probably not the sole cause of these perinatal disorders, because maternal cocaine abuse is often associated with poor nutrition and prenatal health care as well as polydrug abuse that may contribute to the risk for perinatal disease.

1	Psychological dependence on cocaine, indicated by inability to abstain from frequent compulsive use, has been reported. Although the occurrence of withdrawal syndromes involving psychomotor agitation and autonomic hyperactivity remains controversial, severe depression (“crashing”) following cocaine intoxication may accompany drug withdrawal.

1	Treatment of cocaine overdose is a medical emergency that is best managed in an intensive care unit. Cocaine toxicity produces a hyperadrenergic state characterized by hypertension, tachycardia, tonic-clonic seizures, dyspnea, and ventricular arrhythmias. IV diazepam in doses up to 0.5 mg/kg administered over an 8-h period has been shown to be effective for control of seizures. Ventricular arrhythmias have been managed successfully by administration of 0.5–1.0 mg of propranolol IV. Because many instances of cocaine-related mortality have been associated with concurrent use of other illicit drugs (particularly heroin), the physician must be prepared to institute effective emergency treatment for multiple drug toxicities.

1	Treatment of chronic cocaine abuse requires the combined efforts of primary care physicians, psychiatrists, and psychosocial care providers. Early abstinence from cocaine use is often complicated by symptoms of depression and guilt, insomnia, and anorexia, which may be as severe as those observed in major affective disorders. Individual and group psychotherapy, family therapy, and peer group assistance programs are often useful for inducing prolonged remission from drug use. Although psychotherapy may be helpful, no specific form of psychotherapy or behavioral modification is uniquely beneficial.

1	A number of medications used for the treatment of various medical and psychiatric disorders have been administered to reduce the duration and severity of cocaine abuse and dependence. The search for a medication that is both safe and highly effective for cocaine detoxification or maintenance of abstinence is continuing. Clinical trials of buspirone (BuSpar), a nonbenzodiazepine anxiolytic with dopamine D3 and D4 receptor antagonist properties, are ongoing. Buspirone reduced use of cocaine, nicotine, and cocaine plus nicotine in combination in a nonhuman primate model of stimulant addiction.

1	Another approach to reducing cocaine abuse is the development of vaccines to actively immunize against cocaine or to functionally antagonize cocaine by preventing it from reaching the brain. Cocaine is converted into inactive metabolites by the plasma enzyme, butyrylcholinesterase (BChE). When this enzyme is modified to increase its catalytic efficiency and accelerate cocaine metabolism, it can both prevent and reverse cocaine-induced toxicity in animals. Importantly, it remains effective even when high doses of cocaine are administered. Ongoing development of this approach includes cocaine hydrolase gene therapy. Vaccines for both cocaine and nicotine have been designed and shown to be safe and somewhat effective in clinical trials. Individual variability in antibody titers and difficulties in determining the optimally effective antibody titer that will neutralize responses to increasing doses of cocaine and have a relatively long duration of action are among the challenges that

1	in determining the optimally effective antibody titer that will neutralize responses to increasing doses of cocaine and have a relatively long duration of action are among the challenges that remain to be resolved.

1	Cannabis sativa contains >400 compounds in addition to the psychoactive substance, delta-9-tetrahydrocannabinol (THC). Marijuana cigarettes are prepared from the leaves and flowering tops of the plant, and a typical marijuana cigarette contains 0.5–1 g of plant material. The usual THC concentration varies between 10 and 40 mg, but concentrations <100 mg per cigarette have been detected. Hashish is prepared from concentrated resin of C. sativa and contains a THC concentration of between 8 and 12% by weight. “Hash oil,” a lipid-soluble plant extract, may contain THC between 25 and 60% and may be added to marijuana or hashish to enhance its THC concentration. Smoking is the most common mode of marijuana or hashish use. During pyrolysis, <150 compounds in addition to THC are released in the smoke. Although most of these compounds do not have psychoactive properties, they may have physiologic effects.

1	THC is quickly absorbed from the lungs into blood and then rapidly sequestered in tissues. THC is metabolized primarily in the liver, where it is converted to 11-hydroxy-THC, a psychoactive compound, and >20 other metabolites. Many THC metabolites are excreted through the feces at a relatively slow rate of clearance compared with most other psychoactive drugs. Specific cannabinoid receptors (CB1 and CB2) have been identified in the central and peripheral nervous system. High densities of cannabinoid receptors have been found in the cerebral cortex, basal ganglia, and hippocampus. T and B lymphocytes also contain cannabinoid receptors, and these appear to mediate the anti-inflammatory and immunoregulatory properties of cannabinoids. A naturally occurring THC-like ligand has been identified and is widely distributed in the nervous system.

1	Herbal marijuana alternatives are also available. These are usually a combination of several herbs and synthetic cannabinoids. “Spice” and “K2” are among the best known, but many formulations exist, and marijuana is undetectable by the usual methods. These compounds are marketed on the Internet as containing no illegal ingredients. However a number of synthetic cannabinoids are now classified as Schedule I by the Drug Enforcement Administration due to reports of toxicity.

1	Marijuana is the most commonly used illegal drug in the United States. In 2012, an estimated 18.9 million people reported using marijuana within the past month. An estimated 7.2% of adolescents age 12 to 17 years reported current use of marijuana. Marijuana is relatively inexpensive and is often considered to be less hazardous than other controlled drugs and substances. Very potent forms of marijuana (sinsemilla) are widely available, and concurrent use of marijuana with other drugs such as cocaine is not uncommon. Due in part to the difficulty of detecting herbal marijuana alternatives, the prevalence of use is unknown.

1	Acute intoxication from marijuana and cannabis compounds is related to both the dose of THC and the route of administration. THC is absorbed more rapidly from marijuana smoking than from orally ingested cannabis compounds. Acute marijuana intoxication may produce a perception of relaxation and mild euphoria resembling mild to moderate alcohol intoxication. This condition is usually accompanied by some impairment in thinking, concentration, and perceptual and psychomotor function. Higher doses of cannabis may produce more pronounced impairment in concentration and perception, as well as greater sedation. Although the acute effects of marijuana intoxication are relatively benign in normal users, the drug can precipitate severe emotional disorders in individuals who have antecedent psychotic or neurotic problems. Like other psychoactive compounds, both the user’s expectations and the environmental context are important determinants of the type and severity of the effects of marijuana

1	or neurotic problems. Like other psychoactive compounds, both the user’s expectations and the environmental context are important determinants of the type and severity of the effects of marijuana intoxication.

1	As with abuse of cocaine, opioids, and alcohol, chronic marijuana abusers may lose interest in common socially desirable goals and devote progressively more time to drug acquisition and use. However, THC does not cause a specific and unique “amotivational syndrome.” The range of symptoms sometimes attributed to marijuana use is difficult to distinguish from mild to moderate depression and the maturational dysfunctions often associated with protracted adolescence. Chronic marijuana use has also been reported to increase the risk of psychotic symptoms in individuals with a past history of schizophrenia. Persons who begin marijuana smoking before the age of 17 may have more pronounced cognitive deficits and also may be at higher risk for polydrug and alcohol abuse problems in later life, but the role of marijuana in this sequence is uncertain.

1	The acute effects of herbal marijuana alternatives are based primarily on case reports and include anxiety, agitation, delusions, paranoia, and psychosis. The extent to which these symptoms reflect drug effects or exacerbation of an underlying psychiatric disorder is often difficult to determine. Conjunctival injection and tachycardia are the most frequent immediate physical concomitants of smoking marijuana. Tolerance for marijuana-induced tachycardia develops rapidly among regular users. However, marijuana smoking may precipitate angina in persons with a history of coronary insufficiency. Exercise-induced angina may increase after marijuana use to a greater extent than after tobacco cigarette smoking. Patients with cardiac disease should be strongly advised not to smoke marijuana or use cannabis compounds.

1	Significant decrements in pulmonary vital capacity have been found in regular daily marijuana smokers. Because marijuana smoking typically involves deep inhalation and prolonged retention of marijuana smoke, chronic bronchial irritation may develop. Impairment of single-breath carbon monoxide diffusion capacity (DlCO) is greater in persons who smoke both marijuana and tobacco than in tobacco smokers.

1	Although marijuana has also been associated with a number of other adverse effects, many of these studies await replication and confirmation. A reported correlation between chronic marijuana use and decreased testosterone levels in males has not been confirmed. Decreased sperm count and sperm motility and morphologic abnormalities of spermatozoa following marijuana use have been reported. Prospective studies found a correlation between impaired fetal growth and development and heavy marijuana use during pregnancy. Marijuana has also been implicated in derangements of the immune system; in chromosomal abnormalities; and in inhibition of DNA, RNA, and protein synthesis; however, these findings have not been confirmed or related to any specific physiologic effect in humans. Herbal marijuana alternatives produce many of the effects of marijuana including conjunctival injection and tachycardia.

1	Habitual marijuana users may develop tolerance to the psychoactive effects of marijuana, and then smoke more frequently and try to acquire more potent cannabis compounds. Tolerance for the physiologic effects of marijuana develops at different rates; e.g., tolerance develops rapidly for marijuana-induced tachycardia but more slowly for marijuana-induced conjunctival injection. Tolerance for both behavioral and physiologic effects of marijuana decreases rapidly upon cessation of marijuana use.

1	A distinct withdrawal syndrome has been documented in chronic cannabis users, and the severity of symptoms is related to dosage and duration of use. These symptoms typically reach their peak several days after cessation of chronic use and include irritability, anorexia, and sleep disturbances. Withdrawal signs and symptoms observed in chronic marijuana users are usually relatively mild in comparison to those observed in heavy opioid or alcohol users and rarely require medical or pharmacologic intervention. However, more severe and protracted abstinence syndromes may occur after sustained use of high-potency cannabis compounds. As yet there have been no systematic studies of tolerance and physical dependence to the herbal marijuana alternatives. The large number of synthetic cannabinoids available for combination with about 20 herbs presents a daunting challenge for analysis.

1	Marijuana, administered as cigarettes or as a synthetic oral cannabinoid (dronabinol), is thought to have a number of clinically useful medicinal properties. These include antiemetic effects in chemotherapy recipients, appetite-promoting effects in AIDS patients, reduction of intraocular pressure in glaucoma, and reduction of spasticity in multiple sclerosis and other neurologic disorders. With the possible exception of AIDS-related cachexia, none of these attributes of marijuana compounds is clearly superior to other readily available therapies.

1	Methamphetamine is also referred to as “meth,” “speed,” “crank,” “chalk,” “ice,” “glass,” or “crystal.” Methamphetamine is a mixed-action monoamine releaser with activity at dopamine, serotonin, and norepinephrine systems. Methamphetamine was considered second only to cocaine as a drug threat to society by the U.S. Department of Justice in 2009. Hospital admissions for methamphetamine treat-469e-3 ment more than doubled between 1998 and 2007, and young adults (age 18–25) have the highest use rates. In 2011, an estimated 439,000 people reported current use of methamphetamine in the United States, and emergency room admissions involving amphetamines/methamphetamine totaled 160,000. Persistent abuse of methamphetamine continues despite drug seizures, closures of clandestine laboratories that produce methamphetamine illegally, and an increase in methamphetamine abuse prevention programs.

1	Methamphetamine can be used by smoking, snorting, IV injection, or oral administration. Methamphetamine abusers report that drug use induces feelings of euphoria and decreased fatigue. Adverse consequences of methamphetamine use include headache, difficulty concentrating, diminished appetite, abdominal pain, vomiting or diarrhea, disordered sleep, paranoid or aggressive behavior, and psychosis. Chronic methamphetamine abuse can result in severe dental caries, described as blackened, rotting, crumbling teeth. Severe, life-threatening methamphetamine toxicity may include hypertension, cardiac arrhythmia or cardiac failure, subarachnoid hemorrhage, ischemic stroke, intracerebral hemorrhage, convulsions, or coma.

1	Methamphetamines increase the release of monoamine neurotransmitters (dopamine, norepinephrine, and serotonin) from presynaptic neurons. It is thought that the euphoric and reinforcing effects of this class of drugs are mediated through dopamine and the mesolimbic system, whereas the cardiovascular effects are related to norepinephrine. MRS studies of the brain suggest that chronic abusers have neuronal damage in the frontal areas and basal ganglia. Treatment of acute methamphetamine overdose is largely symptomatic. Ammonium chloride may be useful to acidify the urine and enhance clearance of the drug. Hypertension may respond to sodium nitroprusside or α-adrenergic antagonists. Sedatives may reduce agitation and other signs of central nervous system hyperactivity. Treatment of chronic methamphetamine dependence may be accomplished in either an inpatient or outpatient setting using strategies similar to those described earlier for cocaine abuse.

1	MDMA is a derivative of methamphetamine also called Ecstasy or Molly. Reported use of MDMA in the United States has increased from 615,000 persons in 2005, to an estimated 869,000 people in 2012. Emergency ward admissions involving MDMA totaled more than 22,000 in 2011. Ecstasy is usually taken orally but may be injected or inhaled, and its effects last for 3–6 h. MDMA has amphetamine-like effects including vivid visual and auditory hallucinations and other perceptual distortions. Recent studies indicate that MDMA use is associated with cognitive and memory impairment. MDMA can induce hyperthermia and elevated blood pressure, seizures, comma, and death. Withdrawal symptoms after cessation of use may include teeth grinding, anxiety, loss of appetite, insomnia, and fever. The longterm consequences of recreational use of MDMA by young persons are poorly understood.

1	The rapid emergence of synthetic cathinone abuse during 2010 was accompanied by numerous reports of adverse medical and psychiatric effects, suicides, and deaths. Reports to poison centers and health agencies increased from about 300 in 2010 to over 6000 in 2011. In 2011, the Drug Enforcement Administration classified three commonly abused synthetic cathinones (mephedrone [4-methyl methcathinone], MDPV [3,4-methylenedioxy pyrovalerone], and methylone) as Schedule I compounds with no accepted medical use and a high potential for abuse. However, synthetic cathinones are readily available on the Internet as well as in convenience stores, gas stations, and head shops. These drugs are merchandised under a variety of names such as Vanilla Sky, Purple Wave, Blue Silk, White Lightening, and Snow Leopard. Regulatory constraints are evaded by labeling the products as plant food, insecticides, pond cleaner, and bath salts with the qualifier, “not for human consumption.”

1	Cathinone is the primary psychoactive ingredient in khat leaves. Chewing the leaves of the khat shrub (Catha edulis) produces mild stimulant and euphoric effects and remains a common practice in east Africa that has persisted for centuries. Cathinone is structurally similar to amphetamine, and mephedrone is structurally similar to methamphetamine. Cathinones, like amphetamines, inhibit dopamine, serotonin, and norepinephrine transporters to varying degrees, and this probably accounts for variations in the behavioral effects observed. The effects of cathinone derivatives are often described as similar to the effects of MDMA or Ecstasy. Synthetic cathinones can be inhaled, snorted, injected, or taken orally. These drugs may be taken repeatedly over several hours in episodes lasting for hours or days. The onset of effects after oral ingestion is relatively rapid for MDPV (15–30 min) and slightly slower for mephedrone and methylone (30–45 min). The duration of action varies from 2 to 5 or

1	or days. The onset of effects after oral ingestion is relatively rapid for MDPV (15–30 min) and slightly slower for mephedrone and methylone (30–45 min). The duration of action varies from 2 to 5 or 7 h. After mephedrone inhalation, effects occur within minutes and only last for 1 h or less, but mood changes may persist for several days. Evaluation of the neurotoxic effects of prolonged synthetic cathinone abuse is just beginning, and the long-term consequences are unknown.

1	The reported positive subjective effects of synthetic cathinones include euphoria, improved energy, alertness, sociability, and increased sensitivity to music and other sensory experiences. The reported negative subjective effects include agitation, visual and auditory hallucinations, anxiety and panic attacks, paranoid delusions, disorientation, depression, and suicidal ideation. Observers report irritability, aggression, violent behavior, tremors, and seizures. Medical evidence of adverse effects includes cardiovascular dysfunction and cardiac arrest, hypertension, hyperthermia, nausea and vomiting, and anorexia. There is no specific antagonist for synthetic cathinone intoxication. Patients with severe hyperthermia, seizures, and arrhythmia are medical emergencies and should be treated in a hospital. Sedation with benzodiazepines can be useful for managing agitation, seizures, aggression, and other related symptoms. Antipsychotic medications may be necessary for management of severe

1	a hospital. Sedation with benzodiazepines can be useful for managing agitation, seizures, aggression, and other related symptoms. Antipsychotic medications may be necessary for management of severe and persistent psychiatric symptoms.

1	Discovery of the psychedelic effects of LSD led to an epidemic of LSD abuse during the 1960s. Imposition of stringent constraints on the manufacture and distribution of LSD (classified as a Schedule I substance by the U.S. Food and Drug Administration [FDA]) and public recognition that psychedelic experiences induced by LSD were a health hazard have resulted in a reduction in LSD abuse. LSD still remains popular among adolescents and young adults, and there are indications that LSD use among young persons has been increasing in some areas in the United States. In 2011, an estimated 358,000 persons used LSD, whereas 200,000 and 271,000 persons reported LSD use in 2003 and 2007, respectively.

1	LSD is a very potent hallucinogen; oral doses as low as 20 μg may induce profound psychological and physiologic effects. Tachycardia, hypertension, pupillary dilation, tremor, and hyperpyrexia occur within minutes following oral administration of 0.5–2 μg/kg. A variety of bizarre and often conflicting perceptual and mood changes, including visual illusions, synesthesias, and extreme lability of mood, usually occur within 30 min after LSD intake. These effects of LSD may persist for 12–18 h, even though the half-life of the drug is only 3 h.

1	Emergency ward visits involving LSD totaled nearly 5000 in 2011. The most frequent acute medical emergency associated with LSD use is a panic episode (the “bad trip”), which may persist up to 24 h. Management of this problem is best accomplished by supportive reassurance (“talking down”) and, if necessary, administration of small doses of anxiolytic drugs. Adverse consequences of chronic LSD use include an enhanced risk for schizophreniform psychosis and derangements in memory function, problem solving, and abstract thinking. Treatment of these disorders is best carried out in specialized psychiatric facilities. Tolerance develops rapidly for LSD-induced changes in psychological function when the drug is used one or more times per day for >4 days. Abrupt abstinence following continued use does not produce withdrawal signs or symptoms. There have been no clinical reports of death caused by the direct effects of LSD.

1	PCP, a cyclohexylamine derivative, is widely used in veterinary medicine to briefly immobilize large animals and is sometimes described as a dissociative anesthetic. PCP binds to ionotropic N-methyl-daspartate (NMDA) receptors in the nervous system, blocking ion current through these channels. PCP is easily synthesized and is abused primarily by young people and polydrug users. It is used orally, by smoking, by snorting, or by IV injection. It is also used as an adulterant in THC, LSD, amphetamine, or cocaine. The most common street preparation, angel dust, is a white granular powder that contains 50–100% of the drug. Low doses (5 mg) produce agitation, excitement, impaired motor coordination, dysarthria, and analgesia. Physical signs of intoxication may include horizontal or vertical nystagmus, flushing, diaphoresis, and hyperacusis. Behavioral changes include distortions of body image, disorganization of thinking, and feelings of estrangement. Higher doses of PCP (5–10 mg) may

1	nystagmus, flushing, diaphoresis, and hyperacusis. Behavioral changes include distortions of body image, disorganization of thinking, and feelings of estrangement. Higher doses of PCP (5–10 mg) may produce profuse salivation, vomiting, myoclonus, fever, stupor, or coma. PCP doses of ≥10 mg cause convulsions, opisthotonus, and decerebrate posturing that may be followed by prolonged coma.

1	In 2011, more than 75,000 emergency ward admissions involved PCP. The diagnosis of PCP overdose is difficult because the patient’s initial symptoms (anxiety, paranoia, delusions, and hallucinations) may suggest an acute schizophrenic reaction. Confirmation of PCP use is possible by determination of PCP levels in serum or urine. PCP assays are available at most toxicologic centers. PCP remains in urine for 1–5 days following high-dose intake. PCP overdose requires emergency life-support measures that may involve treatment of coma, convulsions, and respiratory depression in an intensive care unit. There is no specific antidote or antagonist for PCP. PCP excretion from the body can be enhanced by gastric lavage and acidification of urine. Death from PCP overdose may occur as a consequence of some combination of pharyngeal hypersecretion, hyperthermia, respiratory depression, severe hypertension, seizures, hypertensive encephalopathy, and intracerebral hemorrhage.

1	Acute psychosis associated with PCP use is a psychiatric emergency because patients may be at high risk for suicide or extreme violence toward others. Phenothiazines should not be used for treatment because these drugs potentiate PCP’s anticholinergic effects. Haloperidol (5 mg IM) has been administered on an hourly basis to induce suppression of psychotic behavior. PCP, like LSD and mescaline, produces vasospasm of cerebral arteries at relatively low doses. Chronic PCP use has been shown to induce insomnia, anorexia, severe changes in behavior, and, in some cases, chronic schizophrenia.

1	This naturally occurring herb is a recent entry into the spectrum of hallucinogens. Like PCP and ecstasy, this drug can produce profound alterations in mood, hallucinations, and distorted perceptions. This drug is available on the Internet and is known by a variety of names including magic mint, mystic sage, Mariana Pastora, and purple sticky. The drug was first added to the annual National Surveys on Drug Use and Health in 2006, and its use is increasing. Between 2006 and 2011, the number of estimated users in the United States nearly tripled to more than 5000.

1	The active ingredient is salvinorin A, a selective kappa opioid receptor agonist that has a range of effects including hallucinations, sedation, analgesia, and depression. The hallucinatory symptoms may be associated with intense anxiety and severe agitation that can be managed with benzodiazepines. Importantly, this kappa opioid receptor agonist does not produce respiratory depression, and no significant change in blood pressure or heart rate was reported in a clinical study with healthy subjects.

1	Salvinorin A extract or crushed leaves of the Salvia divinorum plant can be chewed and absorbed through the buccal membrane or inhaled during smoking. The onset of the acute “high” is within 5–10 min after chewing and 30 s after inhalation. The duration of the effect is relatively brief, usually 15–20 min. However, if the drug is taken with alcohol or other hallucinogens, the duration and intensity of adverse effects may be increased. The effects of the drug are reported to be similar to those of ketamine, LSD, and marijuana.

1	A number of other pharmacologically diverse drugs of abuse are often referred to as “club drugs” because these are frequently used in bars, at concerts, and at rave parties. Commonly abused club drugs include flunitrazepam, GHB, and ketamine and are described below. Methamphetamine, MDMA, and LSD are also considered club drugs and were described earlier in this chapter. Abuse of club drugs at high doses, especially in combination with alcohol, can be lethal and should be treated as a medical emergency. GHB and ketamine can be identified in blood, and flunitrazepam can be identified in urine and hair samples. Flunitrazepam and GHB toxicity can be treated with antagonists at benzodiazepine and γ-aminobutyric acid B (GABAB) receptors, respectively.

1	Flunitrazepam (Rohypnol) is a benzodiazepine derivative primarily used to treat insomnia, but it has significant abuse potential because of its strong hypnotic, anxiolytic, and amnesia-producing effects. It is a club drug commonly referred to as a “date-rape drug” or “roofies.” The drug enhances GABAA receptor activity, and overdose can be treated with flumazenil, a benzodiazepine receptor antagonist. Flunitrazepam is typically used orally but can be snorted or injected. Concomitant use of alcohol or opioids is common, and this enhances the sedative and hypnotic effects of flunitrazepam and also the risk of motor vehicle accidents. Overdose can produce life-threatening respiratory depression and coma. Abrupt cessation after chronic use may result in a benzodiazepine withdrawal syndrome consisting of anxiety, insomnia, disordered thinking, and seizures.

1	GHB (Xyrem) is a sedative drug that is approved by the FDA for the treatment of narcolepsy. It is classified as a club drug, is sometimes used in combination with alcohol or other drugs of abuse, and has been implicated in cases of date rape. It is also used by body builders as a growth hormone stimulant. GHB is usually available as a liquid, is taken orally, and has no distinctive color or odor. Its stimulant properties are attributed to agonist activity at the GHB receptor, but it also has sedative effects at high doses that reflect its activity at GABAB receptors. GABAB antagonists can reverse GHB’s sedative effects, and opioid antagonists (naloxone, naltrexone) can attenuate GHB effects on dopamine release. Low doses of GHB may produce euphoria and disinhibition, whereas high doses result in nausea, agitation, convulsions, and sedation that can lead to unconsciousness and death from respiratory depression. In 2011, more than 2400 emergency ward admissions involved GHB.

1	Ketamine (Ketaset, Ketalar) is a dissociative anesthetic, similar to PCP. In veterinary medicine, it is used for brief immobilization. In clinical medicine, it is used for sedation, analgesia, and to supplement anesthesia. Ketamine increases heart rate and blood pressure, with less respiratory depression than other anesthetics. Ketamine’s popularity as a club drug appears to reflect its ability to induce a dissociative state and feelings of depersonalization, accompanied by intense hallucinations and subsequent amnesia. It can be administered orally, by smoking (usually in combination with tobacco and/or marijuana), or by IV or IM injection. Like PCP, it binds to NMDA receptors and acts as a noncompetitive NMDA antagonist. In 2011, ketamine accounted for 1550 emergency ward admissions. Ketamine has a complex profile of action and appears to be useful as an antidepressant in treatment-resistant patients and as an analgesic in patients with chronic pain.

1	The extent to which chronic recreational use leads to memory impair-469e-5 ment remains controversial.

1	Although some drug abusers may prefer a particular drug, the concurrent use of multiple drugs is common. Polydrug abuse often involves substances that may have different pharmacologic effects from the preferred drug. For example, concurrent use of such dissimilar compounds as stimulants and opioids or stimulants and alcohol is common. The diversity of reported drug use combinations suggests that achieving a change in subjective state, rather than any particular direction of change (stimulation or sedation), may be the primary reinforcer in polydrug abuse. There is also evidence that intoxication with alcohol, opiates, and cocaine is associated with increased tobacco smoking. Nicotine and cocaine enhance each other’s effects in clinical laboratory studies, and this drug combination maintains significantly higher levels of self-administration than either drug alone in preclinical models of addiction. There are relatively few controlled studies of multiple drug interactions. However, the

1	significantly higher levels of self-administration than either drug alone in preclinical models of addiction. There are relatively few controlled studies of multiple drug interactions. However, the combined use of cocaine, heroin, and alcohol increases the risk for toxic effects and adverse medical consequences. Similarly, some hallucinogens (MDMA, LSD) and club drugs (GHB, ketamine, flunitrazepam) are used in various combinations with an associated increase in toxic consequences.

1	One determinant of polydrug use patterns is the relative availability and cost of the drugs. For example, alcohol abuse, with its attendant medical complications, is one of the most serious problems encountered in former heroin addicts participating in methadone maintenance programs. Cocaine abuse often increases during methadone maintenance. The physician must recognize that perpetuation of polydrug abuse and drug dependence is not necessarily a symptom of an underlying emotional disorder. Neither alleviation of anxiety nor reduction of depression accounts for initiation and perpetuation of polydrug abuse. Severe depression and anxiety are the consequences of polydrug abuse as frequently as they are the antecedents. Interestingly, some adverse consequences of drug use may be reinforcing and contribute to the continuation of polydrug abuse.

1	Adequate treatment of polydrug abuse, as well as other forms of drug abuse, requires innovative intervention programs. The first step in successful treatment is detoxification, a process that may be difficult when several drugs with different pharmacologic actions (e.g., alcohol, opiates, and cocaine) have been abused. Because patients may not recall or may deny simultaneous multiple drug use, diagnostic evaluation should always include urinalysis for qualitative detection of psychoactive substances and their metabolites. Treatment of polydrug abuse often requires hospitalization or inpatient residential care during detoxification and the initial phase of drug abstinence. When possible, specialized facilities for the care and treatment of drug-dependent persons should be used. Outpatient detoxification of polydrug abuse patients is unlikely to be effective and may increase risk for dangerous medical consequences.

1	Drug abuse disorders often respond to effective treatment, but periods of relapse may occur unpredictably. The physician should continue to assist patients during episodes of relapse with compassion and understanding. The physician and the patient must recognize that occasional recurrent drug use is not unusual in this complex behavioral disorder. The aggregate of particulate matter, after subtracting nicotine and 2729 moisture, is referred to as tar.

1	The alkaline pH of smoke from blends of tobacco used for pipes and cigars allows sufficient absorption of nicotine across the oral mucosa to satisfy the smoker’s need for this drug. Therefore, smokers of pipes and cigars tend not to inhale the smoke into the lung, confining the toxic and carcinogenic exposure (and the increased rates of disease) largely to the upper airway for most users of these products. The acidic pH of smoke generated by the tobacco used in cigarettes dramatically reduces absorption of nicotine in the mouth, necessitating inhalation of the smoke into the larger surface of the lungs in order to absorb quantities of nicotine sufficient to satisfy the smoker’s addiction. The shift to using tobacco as cigarettes, with resultant increased deposition of smoke in the lung, has created the epidemic of heart disease, lung disease, and lung cancer that dominates the current disease manifestations of tobacco use.

1	Several genes have been associated with nicotine addiction. Some reduce the clearance of nicotine, and others have been associated with an increased likelihood of becoming dependent on tobacco and other drugs as well as a higher incidence of depression. Rates of smoking cessation have increased, and rates of nicotine addiction have decreased dramatically, since the mid-1950s, suggesting that factors other than genetics are important. It is likely that genetic susceptibility can influence the probability that adolescent experimentation with tobacco will lead to addiction as an adult.

1	Adult cigarette smoking prevalence has declined to about 19% in the United States, with 20–40% of those smokers not smoking every day. Male smoking prevalence is falling but remains high in most Asian countries, with increasing smoking prevalence among women in those countries. The highest rates of smoking and least cessation are observed in eastern European countries. Of particular concern is the rapidly rising smoking rate observed in the developing world. The World Health Organization Framework Convention on Tobacco Control is encouraging effective tobacco control approaches in these countries with the hope of preventing a future epidemic of tobacco-related illness. More than 400,000 individuals die prematurely each year in the United States from cigarette use; this represents almost one of every five deaths in the United States. Approximately 40% of cigarette smokers will die prematurely due to cigarette smoking unless they are able to quit.

1	The major diseases caused by cigarette smoking are listed in Table 470-1. The ratio of smoking-related disease rates in smokers compared to never smokers (relative risk) is greater at younger ages, particularly for coronary artery disease and stroke. At older ages, the background rate of disease in nonsmokers increases, diminishing the fractional contribution of smoking and the relative risk; however, absolute excess rates of disease mortality found in smokers compared to nonsmokers increase with increasing age. The organ damage caused by smoking and the number of smokers who die from smoking are both greater among the elderly, as one would expect from a process of cumulative injury.

1	Cigarette smokers are more likely than nonsmokers to develop both large-vessel atherosclerosis and small-vessel disease. Approximately 90% of peripheral vascular disease in the nondiabetic population can be attributed to cigarette smoking, as can ~50% of aortic aneurysms. In contrast, 20–30% of coronary artery disease and ~10% of ischemic and hemorrhagic strokes are caused by cigarette smoking. There is a multiplicative interaction between cigarette smoking and other cardiac risk factors such that the increment in risk produced by smoking among individuals with hypertension or elevated serum lipids is substantially greater than the increment in risk produced by smoking for individuals without these risk factors. In addition to its role in promoting atherosclerosis, cigarette smoking also increases the likelihood of myocardial infarction and sudden cardiac death by promoting platelet aggregation and vascular occlusion. Reversal of these effects on coagulation may explain the rapid

1	Nicotine addiction David M. Burns The use of tobacco leaf to create and satisfy nicotine addiction was introduced to Columbus by Native Americans and spread rapidly to Europe. Use of tobacco as cigarettes, however, only became popular in the twentieth century and so is a modern phenomenon, as is the 470 epidemic of disease caused by this form of tobacco use. Nicotine is the principal constituent of tobacco responsible for its addictive character, but other smoke constituents and behavioral associations contribute to the strength of the addiction. Addicted smokers regulate their nicotine intake by adjusting the frequency and intensity of their tobacco use both to obtain the desired psychoactive effects and avoid withdrawal.

1	Unburned cured tobacco used orally contains nicotine, carcinogens, and other toxicants capable of causing gum disease, oral and pancreatic cancers, and an increase in the risk of heart disease. When tobacco is burned, the resultant smoke contains, in addition to nicotine, more than 7000 other compounds that result from volatilization, pyrolysis, and pyrosynthesis of tobacco and various chemical additives used in making different tobacco products. The smoke is composed of a fine aerosol and a vapor phase; aerosolized particles are of a size range that results in deposition in the airways and alveolar surfaces of the lungs. Age 35–64 2.8 3.1 Age ≥65 1.5 1.6 Age 35–64 3.3 4 Age ≥65 1.6 1.5 Aortic aneurysm 6.2 7.1 Chronic airway obstruction 10.6 13.1 Lung 23.3 12.7 Larynx 14.6 13 Lip, oral cavity, pharynx 10.9 5.1 Esophagus 6.8 7.8 Bladder, other urinary organs 3.3 2.2 Kidney 2.7 1.3 Pancreas 2.3 2.3 Stomach 2 1.4 Liver 1.7 1.7

1	Lung 23.3 12.7 Larynx 14.6 13 Lip, oral cavity, pharynx 10.9 5.1 Esophagus 6.8 7.8 Bladder, other urinary organs 3.3 2.2 Kidney 2.7 1.3 Pancreas 2.3 2.3 Stomach 2 1.4 Liver 1.7 1.7 Colorectal 1.2 1.2 1.6 Acute myeloid leukemia 1.4 1.4 Sudden infant death syndrome 2.3 Infant respiratory distress syndrome 1.3 Low birth weight at delivery 1.8 benefit of smoking cessation for a new coronary event demonstrable among those who have survived a first myocardial infarction. This effect may also explain the substantially higher rates of graft occlusion among continuing smokers following vascular bypass surgery for cardiac or peripheral vascular disease.

1	Cessation of cigarette smoking reduces the risk of a second coronary event within 6–12 months; rates of first myocardial infarction and death from coronary heart disease also decline within the first few years following cessation among those with no prior cardiovascular history. After 15 years of abstinence, the risk of a new myocardial infarction or death from coronary heart disease in former smokers is similar to that for those who have never smoked.

1	Tobacco smoking causes cancer of the lung; oral cavity; naso-, oro-, and hypopharynx; nasal cavity and paranasal sinuses; larynx; esophagus; stomach; pancreas; liver (hepatocellular); colon and rectum; kidney (body and pelvis); ureter; urinary bladder; and uterine cervix, and also causes myeloid leukemia. There is evidence suggesting that cigarette smoking may play a role in increasing the risk of breast cancer. There does not appear to be a causal link between cigarette smoking and cancer of the endometrium, and there is a lower risk of uterine cancer among postmenopausal women who smoke. The risks of cancer increase with the increasing number of cigarettes smoked per day and with increasing duration of smoking. Additionally, there are synergistic interactions between cigarette smoking and alcohol use for cancer of the oral cavity and esophagus. Several occupational exposures synergistically increase lung cancer risk among cigarette smokers, most notably occupational asbestos and

1	and alcohol use for cancer of the oral cavity and esophagus. Several occupational exposures synergistically increase lung cancer risk among cigarette smokers, most notably occupational asbestos and radon exposure.

1	Cessation of cigarette smoking reduces the risk of developing cancer relative to continuing smoking, but even 20 years after cessation, there is a modest persistent increased risk of developing lung cancer.

1	Cigarette smoking is responsible for 90% of chronic obstructive pulmonary disease. Within 1–2 years of beginning to smoke regularly, many young smokers will develop inflammatory changes in their small airways, although lung function measures of these changes do not predict development of chronic airflow obstruction. After 20 years of smoking, pathophysiologic changes in the lungs develop and progress proportional to smoking intensity and duration. Chronic mucous hyperplasia of the larger airways results in a chronic productive cough in as many as 80% of smokers >60 years of age. Chronic inflammation and narrowing of the small airways and/or enzymatic digestion of alveolar walls resulting in pulmonary emphysema can result in reduced expiratory airflow sufficient to produce clinical symptoms of respiratory limitation in ~15–25% of smokers.

1	Changes in the small airways of young smokers will reverse after 1–2 years of cessation. There may also be a small increase in measures of expiratory airflow following cessation among individuals who have developed chronic airflow obstruction, but the major change following cessation is a slowing of the rate of decline in lung function with advancing age rather than a return of lung function toward normal.

1	Cigarette smoking is associated with several maternal complications of pregnancy: premature rupture of membranes, abruptio placentae, and placenta previa; there is also a small increase in the risk of spontaneous abortion among smokers. Infants of smoking mothers are more likely to experience preterm delivery, have a higher perinatal mortality rate, be small for their gestational age, and have higher rates of infant respiratory distress syndrome; they are more likely to die of sudden infant death syndrome and appear to have a developmental lag for at least the first several years of life. Smoking delays healing of peptic ulcers; increases the risk of developing diabetes, active tuberculosis, rheumatoid arthritis, osteoporosis, senile cataracts, and neovascular and atrophic forms of macular degeneration; and results in premature menopause, wrinkling of the skin, gallstones and cholecystitis in women, and male impotence.

1	Long-term exposure to environmental tobacco smoke increases the risk of lung cancer and coronary artery disease among nonsmokers. It also increases the incidence of respiratory infections, chronic otitis media, and asthma in children and causes exacerbation of asthma in children. Some evidence suggests that environmental tobacco smoke exposure may increase the risk of premenopausal breast cancer. Patients who continue to smoke during treatment for cancer with chemotherapy or radiation have poorer outcomes and reduced survival.

1	Cigarette smoking may interact with a variety of other drugs (Table 470-2). Cigarette smoking induces the cytochrome P450 system, which may alter the metabolic clearance of drugs such as theophylline. This may result in inadequate serum levels in smokers as outpatients when the dosage is established in the hospital under nonsmoking conditions. Correspondingly, serum levels may rise when smokers are hospitalized and not allowed to smoke. Smokers may also have higher first-pass clearance for drugs such as lidocaine, and the stimulant effects of nicotine may reduce the effect of benzodiazepines or beta blockers.

1	Other major forms of tobacco use are moist snuff deposited between the cheek and gum, chewing tobacco, pipes and cigars, and recently bidi (tobacco wrapped in tendu or temburni leaf; commonly used in India), clove cigarettes, and water pipes. Oral tobacco use leads to gum disease and can result in oral and pancreatic cancer as well as heart disease, with dramatic differences in the risks evident for products used in Africa and Asia as compared to those in the United States and Europe. aClinical implications uncertain.

1	aClinical implications uncertain. All forms of burned tobacco generate toxic and carcinogenic smoke similar to that of cigarette smoke. The differences in disease consequences of use relate to frequency of use and depth of inhalation. The risk of upper airway cancers is similar among cigarette, pipe, and cigar smokers, whereas those who have smoked only pipes and cigars have a much lower risk of lung cancer, heart disease, and chronic obstructive pulmonary disease. However, cigarette smokers who switch to pipes or cigars do tend to inhale the smoke, increasing their risk; and it is likely that comparable inhalation and frequency of exposure to tobacco smoke from any of these forms of tobacco use will lead to comparable disease outcomes.

1	A resurgence of cigar, bidi, and water pipe use among adolescents of both genders has raised concerns that these older forms of tobacco use are once again causing a public health problem. A variety of devices are currently sold that deliver nicotine by electronically heating materials containing nicotine, the so-called electronic cigarettes. Although these devices are marketed as substitutes for cigarettes and as cessation tools, the composition of the vapor and nicotine delivery varies widely from product to product, raising questions of both safety and efficacy in the absence of regulatory oversight.

1	Filtered cigarettes with lower machine-measured yields of tar and nicotine commonly use ventilation holes in the filters and other engineering designs to artificially lower the machine measurements. Smokers compensate for the lowered nicotine delivery by changing the manner in which they puff on the cigarette or the number of cigarettes smoked per day, and tar and nicotine deliveries are not reduced with use of these products. Cigarette design changes that reduce machine-measured tar and nicotine lead to deeper inhalation of the smoke and an increase in 2731 the carcinogenicity of the smoke inhaled by smokers. The presentation of more carcinogenic smoke to the alveolar portions of the lung has resulted in an increase in the risk of lung cancer, and possibly chronic obstructive pulmonary disease, among smokers over the past six decades. This change in cigarette product is also one cause of the dramatic rise in rates of adenocarcinoma of the lung observed over the past half century.

1	disease, among smokers over the past six decades. This change in cigarette product is also one cause of the dramatic rise in rates of adenocarcinoma of the lung observed over the past half century. There has been no increase in risk of lung cancer or adenocarcinoma of the lung in never smokers over time.

1	The process of stopping smoking is commonly a cyclical one, with the smoker sometimes making multiple attempts to quit and failing before finally being successful. Approximately 70–80% of smokers would like to quit smoking. More than one-half of current smokers attempted to quit in the last year, but only 6% quit for 6 months, and only 3% remain abstinent for 2 years. Clinician-based smoking interventions should repeatedly encourage smokers to try to quit and to use different forms of cessation assistance with each new cessation attempt rather than focusing exclusively on immediate cessation at the time of the first visit. Advice from a physician to quit smoking, particularly at the time of an acute illness, is a powerful trigger for cessation attempts, with up to half of patients who are advised to quit making a cessation effort. Other triggers include the cost of cigarettes, media campaigns, and changes in rules to restrict smoking in the workplace.

1	All patients should be asked whether they smoke, how much they smoke, how long they have smoked, their past experience with quitting, and whether they are currently interested in quitting. Intensity of smoking and smoking within 30 min of waking are useful measures of the intensity of nicotine addiction. Even those who are not interested in quitting should be encouraged and motivated to quit; provided a clear, strong, and personalized message by the clinician that smoking Ask: Systematically identify all tobacco users at every visit Advise: Strongly urge all smokers to quit Identify smokers willing to quit Assist the patient in quitting Arrange follow-up contact First-line therapies Nicotine gum (1.5) Nicotine patch (1.9) Nicotine nasal inhaler (2.7) Nicotine oral inhaler (2.5) Nicotine lozenge (2.0) Bupropion (2.1) Varenicline (2.7) Second-line therapies Clonidine (2.1) Nortriptyline (3.2) Physician or other medical personnel counseling (10 min) (1.3)

1	Second-line therapies Clonidine (2.1) Nortriptyline (3.2) Physician or other medical personnel counseling (10 min) (1.3) Intensive smoking cessation programs (at least 4–7 sessions of 20to 30-min duration lasting at least 2 and preferably 8 weeks) (2.3) Clinic-based smoking status identification system (3.1) Counseling by nonclinicians and social support by family and friends Telephone counseling (1.2) aNumerical value following the intervention is the multiple for cessation success compared to no intervention.

1	22 is an important health concern; and offered assistance if they become interested in quitting in the future. Many of those not currently expressing an interest in quitting may nevertheless make an attempt to quit in the subsequent year. For those interested in quitting, a quit date should be negotiated, usually not the day of the visit but within the next few weeks, and a follow-up contact by office staff around the time of the quit date should be provided. There is a relationship between the amount of assistance a patient is willing to accept and the success of the cessation attempt. There are a variety of nicotine-replacement products, including over-the-counter nicotine patches, gum, and lozenges, as well as nicotine nasal and oral inhalers available by prescription. These products can be used for up to 3–6 months, and some products are formulated to allow a gradual step-down in dosage with increasing duration of abstinence. Antidepressants such as bupropion (300 mg in divided

1	can be used for up to 3–6 months, and some products are formulated to allow a gradual step-down in dosage with increasing duration of abstinence. Antidepressants such as bupropion (300 mg in divided doses for up to 6 months) have also been shown to be effective, as has varenicline, a partial agonist for the nicotinic acetylcholine receptor (initial dose 0.5 mg daily increasing to 1 mg twice daily at day 8; treatment duration up to 6 months). Severe psychiatric symptoms, including suicidal ideation, have been reported with varenicline, resulting in a U.S. Food and Drug Administration–mandated warning and a recommendation for closer therapeutic supervision, but evidence to establish the frequency of these responses and the specificity of their association with varenicline remains unclear. Some evidence supports the combined use of nicotine-replacement therapy (NRT) and antidepressants as well as the use of gum or lozenges for acute cravings in patients using patches. Pretreatment with

1	Some evidence supports the combined use of nicotine-replacement therapy (NRT) and antidepressants as well as the use of gum or lozenges for acute cravings in patients using patches. Pretreatment with antidepressants or varenicline is recommended for 1–2 weeks prior to the quit date, and pretreatment with nicotine-replacement products is also being explored, as is longer duration of nicotine replacement as a maintenance therapy for those who are unsuccessful in quitting with a shorter duration of use. NRT is provided in different dosages, with higher doses being recommended for more intense smokers. Clonidine or nortriptyline may be useful for patients who have failed on first-line pharmacologic treatment or who are unable to use other therapies. Antidepressants are more effective in those with a history of depression symptoms. Current recommendations are to offer pharmacologic treatment, usually with NRT or varenicline, to all who will accept it and to provide counseling and other

1	with a history of depression symptoms. Current recommendations are to offer pharmacologic treatment, usually with NRT or varenicline, to all who will accept it and to provide counseling and other support as a part of the cessation attempt. There are some data to suggest that longer term use of NRT may enable cessation in some smokers who are unable to quit with shorter duration use and that some individuals are able to achieve abstinence from tobacco through use of NRT chronically. Cessation advice alone by a physician or his or her staff is likely to increase success compared with no intervention; a more comprehensive approach with advice, pharmacologic assistance, and counseling can increase cessation success nearly threefold. Incorporation of cessation assistance into a practice requires a change of the care delivery infrastructure. Simple changes include (1) adding questions about smoking and interest in cessation on patient-intake questionnaires, (2) asking patients whether they

1	a change of the care delivery infrastructure. Simple changes include (1) adding questions about smoking and interest in cessation on patient-intake questionnaires, (2) asking patients whether they smoke as part of the initial vital sign measurements made by office staff, (3) listing smoking as a problem in the medical record, and (4) automating follow-up contact with the patient on the quit date. These changes are essential to institutionalizing smoking intervention within the practice setting; without this institutionalization, the best intentions of physicians to intervene with their patients who smoke are often lost in the time crush of a busy practice.

1	Approximately 85% of individuals who become cigarette smokers initiate the behavior during adolescence. Factors that promote adolescent initiation are parental or older-sibling cigarette smoking, tobacco advertising and promotional activities, the availability of cigarettes, and the social acceptability of smoking. The need for an enhanced self-image and to imitate adult behavior is greatest for those adolescents who have the least external validation of their self-worth, which may explain in part the enormous differences in adolescent smoking prevalence by socioeconomic and school performance strata.

1	Prevention of smoking initiation must begin early, preferably in the elementary school years. Physicians who treat adolescents should be sensitive to the prevalence of this problem even in the pre-teen population. Physicians should ask all adolescents whether they have experimented with tobacco or currently use tobacco, reinforce the fact that most adolescents and adults do not smoke, and explain that all forms of tobacco are both addictive and harmful.

1	Neuropsychiatric Illnesses in War Veterans Charles W. Hoge Neuropsychiatric sequelae are common in combat veterans. Advances in personal protective body armor, armored vehicles, battlefield resus-471e citation, and the speed of evacuation to tertiary care have considerably improved the survivability of battlefield injuries, resulting in a greater awareness of the “silent wounds” associated with service in a combat zone. Although psychiatric and neurologic problems have been well documented in veterans of prior wars, the conflicts in Iraq and Afghanistan that began after September 11, 2001, were unique in terms of the level of commitment by the U.S. Department of Defense (DoD) and Department of Veterans Affairs (VA), Veterans Health Administration (VHA) to support research as the wars unfolded and to use that knowledge to guide population-level screening, evaluation, and treatment initiatives.

1	The Iraq and Afghanistan conflicts produced over 2.5 million combat veterans, many of whom have received or will need care in government and civilian medical facilities in the future. Studies clearly showed that service in the Iraq and Afghanistan theaters was associated with significantly elevated rates of mental disorders. Two conditions in particular have been labeled the signature injuries related to these wars: posttraumatic stress disorder (PTSD) and mild traumatic brain injury (mTBI)—also known as concussion. Although particular emphasis will be given in this chapter to PTSD and concussion/mTBI, it is important to understand that the neuropsychiatric sequelae of war are much broader than these two conditions. Wartime service is associated with a number of health concerns that coexist and overlap, and a multidisciplinary patient-centered approach to care is necessary.

1	Service members involved in the Iraq and Afghanistan wars faced multiple deployments to two very different high-intensity combat theaters, and for many veterans, the cumulative strain negatively impacted health, marriages, parenting, educational goals, and civilian occupations. The stresses of service in these conflicts also led to a significant increase in rates of suicide in personnel from the two branches of service involved in the greatest level of ground combat (U.S. Army, Marines).

1	Service in a war zone can involve extreme physical stress in austere environments, prolonged sleep deprivation, physical injury, exposure to highly life-threatening events, and hazards such as explosive devices, sniper fire, ambushes, indirect fire from rockets and mortars, and chemical pollutants. Certain events, such as loss of a close friend in combat, leave indelible scars. All of these experiences have additive effects on health, likely mediated through physiologic mechanisms involving dysregulation of neuroendocrine and autonomic nervous system (ANS) functions.

1	Veterans of virtually all wars have reported elevated rates of generalized and multisystem physical, cognitive, and psychological health concerns that often become the focus of treatment months or years after returning home. These multisystem health concerns include sleep disturbance, memory and concentration problems, headaches, musculoskeletal pain, gastrointestinal symptoms (including gastroesophageal reflux), residual effects of wartime injuries, fatigue, anger, hyper-arousal symptoms, high blood pressure, rapid heart rate (sometimes associated with panic symptoms), sexual problems, and symptoms associated with PTSD and depression. In order to provide optimal care to veterans with these symptoms, it is important to understand how the symptoms interrelate and to consider the possibility that there may be underlying combat-related physiologic effects.

1	The overlapping and multisystem health concerns reported by warriors from every generation have been given different labels and have led to debates among medical professionals as to whether these are mediated primarily by physical or psychological causes. For example, World War I produced extensive debate about whether “shell shock,” diagnosed in more than 80,000 British soldiers, was neurologic (“commotional” from the brain being shaken in the skull by concussive blasts) or psychological (“emotional” or “neurasthenia”) in origin. World War II veterans were said to suffer from “battle fatigue,” Korean War veterans developed “combat stress reactions,” and Vietnam veterans developed the “post-Vietnam syndrome.” The role of environmental exposure (e.g., Agent Orange) and psychological causes (e.g., PTSD, depression, substance use disorders) continue to be debated.

1	Gulf War I (Operation Desert Storm), following the Iraqi invasion of Kuwait in 1990, led to extensive debates as to whether Gulf War syndrome, also known as multisystem illness, was best explained by environmental exposures (e.g., oil fires, depleted uranium, nerve gas, pesticides, multiple vaccinations) or the psychological stress of deployment to a war zone where there was anticipation of high casualty rates from chemical and biologic weapons, repeated stressful alerts, and training exercises involving the use of impermeable full-body protective uniforms (made from rubber, vinyl, charcoal-impregnated polyurethane, and other materials) in desert conditions under extreme temperatures. Although no clinical syndrome was ever definitively confirmed among the nearly 1 million service members who deployed in 1990–1991, studies consistently found that military personnel who served in the Gulf experienced elevations in generalized symptoms across all health domains (e.g., physical,

1	members who deployed in 1990–1991, studies consistently found that military personnel who served in the Gulf experienced elevations in generalized symptoms across all health domains (e.g., physical, cognitive, neurologic, psychological) compared with service members who deployed elsewhere or did not deploy. In addition, there is good evidence that deployment to the Persian Gulf region during this period was associated with subsequent development of PTSD; other psychiatric disorders including generalized anxiety disorder, depression, and substance use disorders (Chap. 467); functional gastrointestinal symptoms such as irritable bowel syndrome (Chap. 352); and chronic fatigue syndrome (Chap. 464e).

1	The conflicts in Iraq and Afghanistan led to similar debates as to whether postwar symptoms such as headaches, irritability, sleep disturbance, dizziness, and concentration problems are best attributed to concussion/mTBI or to PTSD. Numerous studies showed that either PTSD or depression explained the majority of the postdeployment “postconcussive” symptoms attributed to concussion/mTBI, a finding not well received by many experts in traumatic brain injury (TBI) but consistent with civilian studies on risk factors for developing persistent symptoms after concussion. As in past wars, the polarized nature of the debate largely focused on only the two conditions of PTSD and concussion/mTBI, which has interfered with full appreciation of how the large spectrum of deployment-related health concerns interrelate and of the clinical implications for designing effective evaluation and treatment strategies.

1	Veterans understandably may become angry at the suggestion that their postwar health concerns could be “stress-related” or psychological, and thus it is necessary for primary care professionals to be able to discuss the physical toll that war-zone service has on the body, the generalized nature of war-related health concerns, and the likely underlying physiologic neuroendocrine and ANS contributors. Mental health specialists also need to be able to reinforce this message and understand the important role they have in promoting physical health through addressing comorbid health concerns.

1	PTSD (Chap. 466) is the most common mental disorder documented following war-zone service. Studies from the conflicts in Iraq and Afghanistan found PTSD prevalence rates of 2–6% before deployment (comparable to civilian general population samples) and rates of 6–20% after deployment, depending primarily on the level of combat frequency and intensity. Many other veterans experience subclinical PTSD symptoms after war-zone service, sometimes termed posttraumatic stress (PTS) or combat stress. These subclinical symptoms can contribute to distress and affect health, even if overall functioning is not as impaired as in the full disorder.

1	The definition of PTSD was modified in the fifth edition of the American Psychiatric Association’s Diagnostic and Statistical Manual of Mental Disorders (2013), although most individuals who had PTSD diagnosed according to the previous criteria also meet the definition under the new criteria. PTSD is defined as persistent (>1 month) symptoms occurring after a traumatic event (involving exposure to actual or threatened death, serious injury, or sexual assault). The symptoms must be associated with significant distress or impairment in social or occupational functioning. Symptoms are grouped into four categories: (1) intrusion/reexperiencing symptoms in which the person has nightmares, flashbacks, or intrusive (often involuntary) memories connected with the traumatic event; (2) avoidance symptoms where the person avoids distressing memories or people, places, situations, or other stimuli that serve as reminders of the traumatic event (for example, a crowded mall that triggers heightened

1	where the person avoids distressing memories or people, places, situations, or other stimuli that serve as reminders of the traumatic event (for example, a crowded mall that triggers heightened alertness to threat); (3) negative alterations of cognitions or mood (for example, feeling detached or losing interest in things that previously brought enjoyment); and (4) hyperarousal symptoms in which the person is physiologically revved up, hyperalert, startles easily, and experiences sleep disturbance, anger, and/or concentration problems. Although PTSD is a clinical symptom-based case definition, it is best to think of PTSD not as an emotional or psychological/psychiatric condition, but rather as a physiologically-based response to life-threatening trauma that is associated with physical, cognitive, emotional, and psychological symptoms.

1	PTSD has strong biologic correlates, based in fear-conditioning responses to threat and responses to extreme stress involving neuroendocrine dysregulation and ANS reactivity. Numerous studies have shown that PTSD is highly correlated with generalized physical and cognitive symptoms—including hypertension, chronic pain, and cardiovascular disease—as well as cell-mediated immune dysfunction and shortened life expectancy. PTSD is frequently comorbid with other mental disorders such as major depressive disorder, generalized anxiety, substance use disorders, and risky behaviors (e.g., aggression, accidents); it has been estimated that up to 80% of patients with PTSD exhibit one or more comorbid conditions. Misuse of alcohol or substances is most prevalent, often reflecting self-medication. PTSD is also associated with tolerance and withdrawal symptoms related to prescription pain and sleep medications, as well as nicotine dependence (Chap. 470).

1	Clinicians should understand how to provide meaningful psychological education in a way that resonates with veterans who may have PTSD symptoms as a result of their military service. There is an important occupational context to consider, which is also applicable to trauma exposures that occur in other first responder professions, such as law enforcement officers and firefighters. Service members and other first responders are trained to respond to traumatic events and effectively learn to override automatic fight-or-flight reflexes in order to carry out their duties. Reactions that are labeled as symptoms of PTSD are based on adaptive survival responses that are beneficial in a combat environment. For example, physiologic hyperarousal, use of anger, and being able to shut down other emotions are very useful skills in combat and can be present even prior to traumatic events during tough realistic training. It is natural for these responses to persist after returning home, and the

1	emotions are very useful skills in combat and can be present even prior to traumatic events during tough realistic training. It is natural for these responses to persist after returning home, and the label of a “disorder” only gets applied when the responses that persist significantly impair functioning.

1	TBI (Chap. 457e) gained increased recognition during the conflicts in Iraq and Afghanistan because of the widespread exposure of troops to improvised explosive devices. Many veterans of Iraq and Afghanistan reported experiencing multiple concussions during deployments, and many also reported ignoring concussions and not seeking treatment at the time of injury in order to remain with their unit. However, these legitimate concerns were also counterbalanced and challenged by high prevalence estimates of deployment-related TBI that did not distinguish concussion/mTBI from moderate or severe TBI; data from animal models of blast exposure that did not necessarily extrapolate to human experiences on the battlefield; neuroimaging studies (e.g., diffusion tensor imaging) that attributed putative abnormalities to blast exposure but lacked adequate control comparisons; and fear-provoking speculation that repetitive blast exposure may lead to future dementia, based largely on case series of

1	abnormalities to blast exposure but lacked adequate control comparisons; and fear-provoking speculation that repetitive blast exposure may lead to future dementia, based largely on case series of professional athletes (e.g., boxers, football players) exposed to highly repetitive injuries linked to chronic traumatic encephalopathy (previously termed dementia pugilistica) (Chap. 444e).

1	TBI includes closed and penetrating head injuries; closed head injuries are categorized as mild (mTBI or concussion), moderate, or severe based on the duration of loss of consciousness, duration of posttraumatic amnesia, and the Glasgow coma score (GCS) (see Table 457e-2). Several studies have estimated that 10–20% of all military personnel deployed to Iraq or Afghanistan sustained one or more concussion/ mTBI events during deployment, most commonly from exposure to blasts; however, concussion injuries are also common in nondeployed environments from sports, training (e.g., hand-to-hand combatives), and accidents.

1	Although there is a neurophysiologic continuum of injury, there are stark clinical and epidemiologic distinctions between concussion/ mTBI and moderate or severe TBI (Table 471e-1). Concussion/mTBI is defined as a blow or jolt to the head that results in brief loss of consciousness (LOC) for <30 min (most commonly, only a few seconds to minutes), posttraumatic amnesia (PTA) of <24 h (most commonly <1 h), or transient alteration in consciousness (AOC) without LOC. The majority of concussions in Iraq or Afghanistan involved AOC without LOC or PTA (which soldiers may refer to as getting their “bell rung”). GCSs in concussion/mTBI are usually normal (15 out of 15). Concussion is treated with rest to allow the brain time to heal, and Loss of consciousness <30 min (usually a few ≥30 min to seconds to minutes) indefinite Altered consciousness <24 h (usually <30 min) ≥24 h to indefinite Posttraumatic amnesia <24 h (usually <30 min) ≥24 h to indefinite

1	Altered consciousness <24 h (usually <30 min) ≥24 h to indefinite Posttraumatic amnesia <24 h (usually <30 min) ≥24 h to indefinite Glasgow coma score 13–15 (usually 15) As low as 3 Clinical usefulness of neu-Usually inconclusive Essential and rocognitive testing after Neuronal cell damage Metabolic/ionic processes Direct injury effects associated with axonal plus metabolic/ionic swelling, which can lead effects to disconnection Sequelae, natural history, Full recovery expected Based directly on and recovery in majority of individuals; injury characteristics; no consensus on natural may be severely history; the percentage disabling who develop persistent symptoms is debated

1	Predictors of persistent Intensely debated; risk Not debated; prepostconcussive factors found to be most dictors are directly symptoms or disability predictive include psy-related to injury chiatric conditions (e.g., severity and clinical depression, PTSD) and progress with rehanegative expectations bilitation treatment Abbreviations: CT, computed tomography; MRI, magnetic resonance imaging; PTSD, post-traumatic stress disorder. it almost never resulted in air evacuation from Iraq or Afghanistan unless there were other associated injuries.

1	it almost never resulted in air evacuation from Iraq or Afghanistan unless there were other associated injuries. In contrast, moderate, severe, or penetrating TBI, which is estimated to account for <1% of all battlefield head injuries in Iraq and Afghanistan, is characterized by LOC ≥30 min (up to permanent coma), PTA ≥24 h (also may be permanent), and GCSs as low as 3 (the minimum value). These virtually always result in air evacuation from the battlefield and carry a significant risk of severe long-term neurologic impairment and requirement for rehabilitative care.

1	Symptoms following concussion/mTBI can include headache; fatigue; concentration, memory, or attention problems; sleep disturbance; irritability; balance difficulties; and tinnitus, among other symptoms. Recovery is usually rapid, with symptoms usually resolving in a few hours to days, but in a small percentage of patients, symptoms may persist for a longer period or become chronic (referred to as persistent “postconcussive symptoms” [PCS]).

1	Establishing a clear causal connection between a deployment concussion injury and persistent PCS months or years after return from deployment has been difficult and often confounded by other postwar conditions that are associated with the same symptoms, including injuries not involving the head, other medical disorders, sleep disorders, PTSD, depression, grief, substance use disorders, chronic pain, and the generalized physiologic effects of wartime service. Contributing to the difficulty in establishing causation is the fact that the concussion/ mTBI case definition refers only to the acute injury event and lacks symptoms, time course, or impairment; case definitions for persistent postconcussion syndrome have failed tests of validation. Numerous studies found that PTSD and depression were much stronger predictors of PCS and objective neuropsychological impairment after combat deployment than concussions/mTBIs, and one study even found that bereavement (particularly related to the

1	were much stronger predictors of PCS and objective neuropsychological impairment after combat deployment than concussions/mTBIs, and one study even found that bereavement (particularly related to the death of a team member) was as strong a predictor of postdeployment symptoms and poor general health as were symptoms of depression or PTSD. These data do not minimize the importance of concussion/mTBI per se, but highlight the complex interrelationships of war-related health problems and the relatively lower importance of concussion/mTBI in overall postdeployment health than is generally thought.

1	Studies of veterans who sustained concussions in Iraq or Afghanistan have suggested that blast mechanisms produce similar clinical outcomes as nonblast mechanisms, in contrast to expectations based on some animal models. An explosion can produce serious injury from rapid atmospheric pressure changes (primary blast wave mechanism), as well as from munition fragments/flying debris (secondary blast mechanism) or being thrown into a hard object (tertiary blast mechanism). Secondary and tertiary mechanisms are similar to other mechanical mechanisms of concussions sustained during accidents. It is likely that blast physics explains differences between human clinical studies and experimental animal studies. Because the distribution of munition fragments usually extends well beyond the distribution of the primary blast wave in most explosions, the possibility of a unique head injury solely from the primary blast wave in otherwise uninjured service members appears to be very low.

1	Multisystem health problems that lack clear case definitions do not lend themselves well to uniform public health strategies such as screening. Nevertheless, mass population screening for concussion/ mTBI was mandated for all U.S. service members returning from Iraq or Afghanistan and all veterans presenting for care at VA health care facilities. These screening processes attempt to apply the acute concussion case definition (lacking symptoms, time course, or impairment) months or years after injury, and often involve questions that encourage patients and clinicians to make a direct link between current symptoms and past head injuries that likely have very little to do with the current symptoms. These screening approaches led to sharp criticism that they were encouraging clinicians to misattribute common postwar symptoms to concussion/mTBI. Nevertheless, the screening processes have persisted and are part of an extensive specialty structure of care erected in both the DoD and VA to

1	common postwar symptoms to concussion/mTBI. Nevertheless, the screening processes have persisted and are part of an extensive specialty structure of care erected in both the DoD and VA to address health concerns attributed to concussions/mTBIs.

1	Management of postwar physical and cognitive health concerns 471e-3 is largely symptom focused and ideally carried out within primary care–based structures of care. Studies suggest that optimal strategies for treatment of multisymptom health concerns include regularly scheduled primary care visits with brief physical exam at each visit, protecting patients from unnecessary diagnostic tests and nonevidence-based interventions, judicious use of consultations that protects patients from unnecessary specialty referrals, care/case management, and communication that enhances positive expectations for recovery. Concussion research has shown that negative expectations are one of the most important risk factors for persistent symptoms.

1	Although many questions remain regarding the long-term health effects of concussions (particularly multiple concussions) sustained during deployment, these are important battlefield injuries that require careful attention. However, they need to be addressed within the context of a much broader approach to other war-related health concerns.

1	Stigma and other barriers to care add to the complexity of treating veterans. Despite extensive education efforts among military leaders and service members, perceptions of stigma showed little change over the many years of war; warriors are often concerned that they will be perceived as weak by peers or leaders if they seek care. Studies have shown that less than one-half of service members and veterans with serious mental health problems receive needed care, and upwards of half of those who begin treatment drop out before receiving an adequate number of encounters. Many factors contribute to this, including the pervasive nature of stigma in society in general (particularly among men), the critical importance of group cohesiveness of military teams, the nature of avoidance symptoms in PTSD, perceptions of self-sufficiency (e.g., “I can handle problems on my own”), and sometimes negative perceptions of mental health care and skepticism that mental health professionals will be able to

1	perceptions of self-sufficiency (e.g., “I can handle problems on my own”), and sometimes negative perceptions of mental health care and skepticism that mental health professionals will be able to help.

1	APPROACH TO THE PATIENT: evaluation of Veterans with Neuropsychiatric health Concerns

1	Evaluation should begin with a careful occupational history as part of the routine medical evaluation; this includes the number of years served, military occupation, deployment locations and dates, illnesses or injuries resulting from service, and significant combat traumatic experiences that may be continuing to affect the individual (Table 471e-2). The clinician should evaluate the degree to which the patient’s current difficulties reflect the normal course of readjusting after the intense occupational experience of combat. It is helpful to reinforce the many strengths associated with being a professional in the military: courage, honor, service to country, resiliency in combat, leadership, ability to work in a cohesive workgroup with peers, and demonstrated skills in handling extreme stress, as well as the fact that reactions that interfere with functioning back home may have their roots in beneficial adaptive physiologic processes.

1	One of the challenges with current medical practice is that there may be multiple providers with different clinical perspectives. Care should be coordinated through the primary care clinician, with the assistance of a care manager if needed. It is particularly important to continually evaluate all medications prescribed by other practitioners and assess each for possible long-term side effects, dependency, or drug-drug interactions. Particular attention should be given to the level of chronic pain and sleep disturbance, self-medication with alcohol or substances, chronic use of nonsteroidal anti-inflammatory agents (which can contribute to rebound headaches or pain), chronic use of sedative-hypnotic agents, chronic use of narcotic pain medications, and the impact of war-related health concerns on social and occupational functioning.

1	Occupational Deployment locations and dates, combat experiences or context of health other deployment stressors, frequent moves, separations concerns from family, impact of deployment on civilian occupa Medical problems History of deployment-related injuries (including con-during deploy-cussions), environmental exposures, sleep pattern durment ing deployment, use of caffeine/energy drinks, use of Current medical Current symptoms, level of chronic pain, sleep problems, history evidence of persistent physiologic hyperarousal (hypertension, tachycardia, panic symptoms, concentration/ memory problems, irritability/anger, sleep disturbance), chronic use of caffeine or energy drinks, chronic use of nonsteroidal anti-inflammatory medications, chronic use of narcotic pain medications, chronic use of nonbenzodiazepine sedative-hypnotic medications, chronic use of benzodiazepines for sleep or anxiety

1	Mental health Screen for PTSD, major depressive disorder; ask about assessment suicidal or homicidal ideation, intent, or plans, as well as access to firearms Alcohol/substance Screen for alcohol and substance use disorders, quantity use and frequency of use, and evidence of tolerance; inquire about “self-medication” (e.g., use of alcohol to sleep, “calm down,” or “forget” war-zone experiences) Functional impair-Impact of current symptoms on social and occupational ment functioning; high-risk behaviors (e.g., drinking and driving, reckless driving, aggression) Social support, Level of social support; readjustment stress on spouse, impact of military children, or other family members service on marriage and family Abbreviation: PTSD, posttraumatic stress disorder.

1	Abbreviation: PTSD, posttraumatic stress disorder. Screening for PTSD, depression, and alcohol misuse should be performed routinely in all combat veterans. Three screening tools, which are in the public domain, have been validated for use in primary care, and have been used frequently in veterans: the four-question Primary Care PTSD Screen (PC-PTSD), the two-question Patient Health Questionnaire (PHQ-2), and the three-question Alcohol Use Disorders Identification Test-Consumption module (AUDIT-C) (Table 471e-3).

1	Because the clinical definition of an acute concussion/mTBI does not include symptoms, time course, or impairment, there is currently no clinically validated screening process for use months or years after injury. However, it is important to gather information about all injuries sustained during deployment, including any that resulted in loss or alteration of consciousness or loss of memory around the time of the event. If concussion injuries have occurred, the clinician should assess the number of such injuries, the duration of time unconscious, and injury mechanisms. This should be followed by an assessment of any PCS immediately following the injury event (e.g., headaches, dizziness, tinnitus, nausea, irritability, insomnia, and concentration or memory problems) and the severity and duration of such symptoms.

1	Given the interrelationship of postwar health concerns, care needs to be carefully coordinated. Specific techniques that have been found to be helpful include scheduling regular primary care visits instead of as-needed visits, establishing care management, using good risk-communication principles, establishing a consultative step care approach that draws on the expertise of specialists in a collaborative manner (instead of immediately referring the patient to a specialist and relying on the specialist to provide care), and having behavioral health support directly within primary care clinics (both for referrals and to provide education and support to primary care professionals prescribing treatment for depression or PTSD).

1	It is important not to implicitly or explicitly convey the message that physical or cognitive symptoms are psychological or due to “stress.” Even if depression or anxiety plays a large role in the etiology of physical health symptoms, the treatment approach should be designed within a patient-centered primary care structure, and referrals should be managed from within this framework. For example, it might help to explain that the primary goal of referral to a mental health professional is to improve sleep and reduce physiologic hyperarousal, which in turn will help with treatment of war-related chronic headaches, concentration problems, or chronic fatigue. If, however, the primary care professional conveys the message that the cause of headaches or concentration problems is anxiety or depression, and this conflicts with the patient’s own viewpoint, then this could damage therapeutic rapport and in turn exacerbate the symptoms.

1	Specific questions related to military service (Table 471e-2) combined with screening for depression, PTSD, and alcohol use disorders (Table 471e-3) should be a routine part of care for all veterans. A positive screen for depression or PTSD should prompt follow-up questions related to these disorders (or use of a longer screening tool such as the nine-question Patient Health Questionnaire or National Center for PTSD Checklist), as well as risk assessment for suicide or homicide. It is important to assess the impact of depression or PTSD symptoms on occupational functioning and interpersonal relationships.

1	A positive screen for alcohol misuse should prompt a brief motivational intervention that includes bringing attention to the elevated level of drinking, informing the veteran about the effects of alcohol on health, recommending limiting use or abstaining, exploring and setting goals related to drinking behavior, and follow-up and referral to specialty care if needed. This type of brief primary care intervention has been found to be effective and should be incorporated into routine practice. One way to facilitate dialogue about this topic with veterans is to point out how hyperarousal associated with combat service can lead to increased craving for alcohol as the body searches for ways to modulate this. Veterans may consciously or unconsciously drink more to help with sleep, reduce arousal, or avoid thinking about events that happened “downrange.” A key educational strategy is to help the veteran to learn that drinking to get to sleep actually damages sleep architecture and makes sleep

1	or avoid thinking about events that happened “downrange.” A key educational strategy is to help the veteran to learn that drinking to get to sleep actually damages sleep architecture and makes sleep worse (e.g., reduces rapid eye movement [REM] sleep initially followed by rebound REM activity and early morning wakening).

1	PTSD and depression are highly comorbid in combat veterans, and the evidence-based treatments are similar, involving antidepressant medications, cognitive behavioral therapy (CBT), or both. Psychoeducation that assists veterans to understand that their symptoms of PTSD have a basis in adaptive survival mechanisms and skills they exhibited in combat can facilitate therapeutic rapport. Remaining hypervigilant to threat, being able to shut down emotions, being able to function on less sleep, and using anger to help focus and control fear are all adaptive beneficial survival skills in a combat environment. Therefore, PTSD for warriors is both a medical disorder and a set of reactions that have their roots in the physiologic adaptation and skills they successfully applied in combat.

1	It is important to know that combat is not the only important trauma in a war-zone environment. Rape, assault, and accidents also occur. Rape or assault by a fellow service member, which affects a greater number of women veterans, but also occurs in men, can be particularly devastating because it destroys the vital feeling of safety that individuals derive from their own unit peers in a war environment. The treatments for PTSD considered by most consensus guideline committees to have an A level of evidence include CBTs and medications, specifically selective serotonin reuptake inhibitors (SSRIs) and serotonin norepinephrine reuptake inhibitors (SNRIs), with the strongest evidence from double-blind, placebo-controlled studies Never (0) Less than Monthly (1) Monthly (2) Two to three times per Four or more times a week (4) week (3)

1	Never (0) Less than Monthly (1) Monthly (2) Two to three times per Four or more times a week (4) week (3) Note: A positive AUDIT-C screen is defined as a total score for men ≥4; for women ≥3. A report of drinking 6 or more drinks on one occasion should prompt an in-depth assessment of drinking. Source: K Bush et al: The AUDIT Alcohol Consumption Questions (AUDIT-C): An effective brief screening test for problem drinking. Arch Intern Med 158:1789, 1998. for sertraline, paroxetine, fluoxetine, and venlafaxine (of which paroxetine and sertraline received U.S. Food and Drug Administration approval for PTSD). (See Table 466-3 for recommended dosages.) Prazosin has also gained very strong evidence recently through randomized placebo-controlled studies for its effectiveness in controlling nightmares as well as global PTSD symptoms, through modulation of the physiologic processes associated with PTSD.

1	CBT interventions include narrative therapy (often called “imaginal exposure”), in vivo exposure focused on retraining the body not to react to stimuli related to traumatic reminders (e.g., a crowded mall), and techniques to modulate physiologic hyperarousal (e.g., diaphragmatic breathing, progressive muscle relaxation). A number of complementary alternative medicine approaches including acupuncture, mindfulness meditation, yoga, and massage are also being used in PTSD. Although not evidence-based treatments per se, if they facilitate a relaxation response and alleviation of hyper-arousal or sleep symptoms, they can be considered useful adjunctive modalities.

1	There have been no head-to-head comparisons of medication compared with psychotherapy for treatment of PTSD. It is reasonable for primary care clinicians to consider initiating treatment for mild to moderate PTSD symptoms with an SSRI and to refer patients to a mental health professional if there are more severe symptoms, significant comorbidity, safety concerns, or limited response to initial treatment. All PTSD treatments are associated with a sizable proportion of individuals who fail to respond adequately, and it is often necessary to add modalities or switch treatment. SNRIs may be useful alternatives to SSRIs if there has been nonresponse or side effects with SSRIs or if there is comorbid pain (duloxetine, in particular, has indications for pain). Both SSRIs and SNRIs can increase anxiety initially; patients should be warned about this possibility, and treatment should be initiated with the lowest recommended dose (or even one-half of the lowest dose for a few days) and

1	anxiety initially; patients should be warned about this possibility, and treatment should be initiated with the lowest recommended dose (or even one-half of the lowest dose for a few days) and gradually increased thereafter. Antidepressants also are likely to be useful in comorbid depression, which is common in veterans with PTSD. All antidepressants have potential drug-drug interactions that must be considered.

1	Many other medications have been used in PTSD, including tricyclic antidepressants, benzodiazepines, atypical antipsychotics, and anticonvulsants. In general, these should be prescribed in conjunction with psychiatric consultation because of their greater side effects and risks. Benzodiazepines, in particular, should be avoided in the treatment of PTSD. Studies have shown that they do not reduce core PTSD symptoms, are likely to exacerbate substance use disorders that are common in veterans with PTSD, and may produce significant rebound anxiety and anger. Individuals with PTSD often report symptomatic relief upon initiation of a benzodiazepine, but this is generally short lived and associated with a high risk of tolerance and dependence that can worsen recovery. Atypical antipsychotics, which have gained widespread popularity as adjunctive treatment for depression, anxiety, or sleep problems, have significant long-term side effects, including metabolic effects (e.g., glucose

1	which have gained widespread popularity as adjunctive treatment for depression, anxiety, or sleep problems, have significant long-term side effects, including metabolic effects (e.g., glucose dysregulation), weight gain, and cardiovascular risks.

1	Sleep disturbance should be addressed initially with sleep hygiene education, followed by consideration of an antihistamine, trazodone, low-dose mirtazapine, or nonbenzodiazepine sedative-hypnotic such as zolpidem, eszopiclone, or zaleplon. However, the nonbenzodiazepine sedative-hypnotics should be used with caution in veterans because they can lead to tolerance and rebound sleep problems similar to those seen with benzodiazepine use. Concussion/mTBI is best treated at the time of injury with education and rest to allow time for the brain to heal and protect against a second impact syndrome (a rare but life-threatening event involving brain swelling that can occur when a second concussion occurs before the brain has adequately healed from an initial event). Randomized trials have shown that education regarding concussion that informs the patient of what to expect and promotes the expectation of recovery is the most effective treatment in preventing persistent symptoms.

1	Once service members return from deployment and seek care for postwar health problems, treatment is largely symptom focused, following the principles of patient-centered and collaborative care models. Cognitive rehabilitation, which is very useful in moderate and severe TBI to improve memory, attention, and concentration, has generally not been shown to be effective for mTBI in randomized clinical studies, although consensus groups have supported its use.

1	General recommendations for the clinical management of persistent, chronic PCS include treating physical and cognitive health problems based on symptom presentation, coexisting health problems, and individual preferences; and addressing coexisting depression, PTSD, substance use disorders, or other factors that may be contributing to symptom persistence. Headache is the most common symptom associated with concussion/mTBI, and the evaluation and treatment of headache parallels that for other causes of headache (Chaps. 21 and 447). Stimulant medications for alleviating neurocognitive effects attributed to concussion/mTBI are not recommended. Clinicians should be aware of the potential for cognitive or sedative side effects of certain medications that may be prescribed for depression, anxiety, sleep, or chronic pain.

1	Treatment of neuropsychiatric problems must be coordinated with care for other war-related health concerns, with the goal of treatment to reduce the severity of symptoms, improve social and occupational functioning, and prevent long-term disability. Understanding the occupational context of war-related health concerns is important in communicating with veterans and developing a comprehensive treatment strategy. The opinions or assertions contained herein are the private views of the author and are not to be construed as official, or as reflecting the views of the Department of the Army or the Department of Defense. 472e-1 PART 18: Poisoning, Drug Overdose, and Envenomation that low-level arsenic may cause neurodevelopmental delays in chil-Heavy Metal Poisoning dren and possibly diabetes, but the evidence remains uneven. Serious cadmium poisoning from the contamination of food and water by mining effluents in Japan contributed to the 1946 outbreak

1	Metals pose a significant threat to health through low-level environmental as well as occupational exposures. One indication of their importance relative to other potential hazards is their ranking by the U.S. Agency for Toxic Substances and Disease Registry, which maintains an updated list of all hazards present in toxic waste sites according to their prevalence and the severity of their toxicity. The first, second, third, and seventh hazards on the list are heavy metals: lead, mercury, arsenic, and cadmium, respectively (http://www.atsdr.cdc.gov/spl/). Specific information pertaining to each of these metals, including sources and metabolism, toxic effects produced, diagnosis, and the appropriate treatment for poisoning, is summarized in Table 472e-1.

1	Metals are inhaled primarily as dusts and fumes (the latter defined as tiny particles generated by combustion). Metal poisoning can also result from exposure to vapors (e.g., mercury vapor in creating dental amalgams). When metals are ingested in contaminated food or drink or by hand-to-mouth activity (implicated especially in children), their gastrointestinal absorption varies greatly with the specific chemical form of the metal and the nutritional status of the host. Once a metal is absorbed, blood is the main medium for its transport, with the precise kinetics dependent on diffusibility, protein binding, rates of biotransformation, availability of intracellular ligands, and other factors. Some organs (e.g., bone, liver, and kidney) sequester metals in relatively high concentrations for years. Most metals are excreted through renal clearance and gastrointestinal excretion; some proportion is also excreted through salivation, perspiration, exhalation, lactation, skin exfoliation, and

1	years. Most metals are excreted through renal clearance and gastrointestinal excretion; some proportion is also excreted through salivation, perspiration, exhalation, lactation, skin exfoliation, and loss of hair and nails. The intrinsic stability of metals facilitates tracing and measurement in biologic material, although the clinical significance of the levels measured is not always clear.

1	Some metals, such as copper and selenium, are essential to normal metabolic function as trace elements (Chap. 96e) but are toxic at high levels of exposure. Others, such as lead and mercury, are xenobiotic and theoretically are capable of exerting toxic effects at any level of exposure. Indeed, much research is currently focused on the contribution of low-level xenobiotic metal exposure to chronic diseases and to subtle changes in health that may have significant public health consequences. Genetic factors, such as polymorphisms that encode for variant enzymes with altered properties in terms of metal binding, transport, and effects, also may modify the impact of metals on health and thereby account, at least in part, for individual susceptibility to metal effects.

1	The most important component of treatment for metal toxicity is the termination of exposure. Chelating agents are used to bind metals into stable cyclic compounds with relatively low toxicity and to enhance their excretion. The principal chelating agents are dimercaprol (British anti-Lewisite [BAL]), ethylenediamine tetraacetic acid (EDTA), succimer (dimercaptosuccinic acid [DMSA]), and penicillamine; their specific use depends on the metal involved and the clinical circumstances. Activated charcoal does not bind metals and thus is of limited usefulness in cases of acute metal ingestion. In addition to the information provided in Table 472e-1, several other aspects of exposure, toxicity, or management are worthy of discussion with respect to the four most hazardous toxicants (arsenic, cadmium, lead, and mercury).

1	Arsenic, even at moderate levels of exposure, has been clearly linked with increased risks for cancer of the skin, bladder, renal pelvis, ureter, kidney, liver, and lung. These risks appear to be modified by smoking, folate and selenium status, genetic traits (such as ability to methylate arsenic), and other factors. Studies in community-based populations are beginning to demonstrate that arsenic exposure is a risk factor for increased coronary heart disease and stroke. Evidence is also emerging of “itai-itai” (“ouch-ouch”) disease, so named because of cadmium-induced bone toxicity that led to painful bone fractures. Modest exposures from environmental contamination have recently been associated in some studies with a lower bone density, a higher incidence of fractures, and a faster decline in height in both men and women, effects that may be related to cadmium’s calciuric effect on the kidney. There is some evidence for synergy between the adverse impacts of cadmium and lead on

1	decline in height in both men and women, effects that may be related to cadmium’s calciuric effect on the kidney. There is some evidence for synergy between the adverse impacts of cadmium and lead on kidney function. Environmental exposures have also been linked to lower lung function (even after adjusting for smoking cigarettes, which contain cadmium) as well as increased risk of cardiovascular disease and mortality, stroke, and heart failure. Several studies have also raised concerns that cadmium may be carcinogenic and contribute to elevated risks of prostate, breast, and pancreatic cancer. Overall, this growing body of research indicates that cadmium exposure may be contributing significantly to morbidity and mortality rates in the general population.

1	Advances in our understanding of lead toxicity have recently benefited by the development of K x-ray fluorescence (KXRF) instruments for making safe in vivo measurements of lead levels in bone (which, in turn, reflect cumulative exposure over many years, as opposed to blood lead levels, which mostly reflect recent exposure). Higher bone lead levels measured by KXRF have been linked to increased risk of hypertension and accelerated declines in cognition in both men and women from an urban population. Upon reviewing these studies in conjunction with other epidemiologic and toxicologic studies, a recent federal expert panel concluded that the impact of lead exposure on hypertension and cognition in adults was causal. Prospective studies have also demonstrated that higher bone lead levels are a major risk factor for increased cardiovascular morbidity and mortality rates in both community-based and occupational-exposed populations. Lead exposure at community levels has also been recently

1	a major risk factor for increased cardiovascular morbidity and mortality rates in both community-based and occupational-exposed populations. Lead exposure at community levels has also been recently associated with increased risks of hearing loss, Parkinson’s disease, and amyotrophic lateral sclerosis. With respect to pregnancy-associated risks, high maternal bone lead levels were found to predict lower birth weight, head circumference, birth length, and neurodevelopmental performance in offspring by age 2 years. In a randomized trial, calcium supplementation (1200 mg daily) was found to significantly reduce the mobilization of lead from maternal bone into blood during pregnancy.

1	The toxicity of low-level organic mercury exposure (as manifested by neurobehavioral performance) is of increasing concern based on studies of the offspring of mothers who ingested mercury-contaminated fish. With respect to whether the consumption of fish by women during pregnancy is good or bad for offspring neurodevelopment, balancing the trade-offs of the beneficial effects of the omega-3-fatty acids (FAs) in fish versus the adverse effects of mercury contamination in fish has led to some confusion and inconsistency in public health recommendations. Overall, it would appear that it would be best for pregnant women to either limit fish consumption to those species known to be low in mercury contamination but high in omega-3-FAs (such as sardines or mackerel) or to avoid fish and obtain omega-3-FAs through supplements or other dietary sources. Current evidence has not supported the recent contention that ethyl mercury, used as a preservative in multiuse vaccines administered in early

1	through supplements or other dietary sources. Current evidence has not supported the recent contention that ethyl mercury, used as a preservative in multiuse vaccines administered in early childhood, has played a significant role in causing neurodevelopmental problems such as autism. With regard to adults, there is conflicting evidence as to whether mercury exposure is associated with increased risk of hypertension and cardiovascular disease. At this point, conclusions cannot be drawn.

1	Heavy metals pose risks to health that are especially burdensome in selected parts of the world. For example, arsenic exposure from natural contamination of shallow tube wells inserted for drinking water is a major environmental problem for millions of Smelting and microelectronics industries; wood preservatives, pesticides, herbicides, fungicides; contaminant of deep-water wells; folk remedies; and coal; incineration of these products. Organic arsenic (arsenobetaine, arsenocholine) is ingested in seafood and fish, but is nontoxic; inorganic arsenic is readily absorbed (lung and GI); sequesters in liver, spleen, kidneys, lungs, and GI tract; residues persist in skin, hair, and nails; biomethylation results in detoxification, but this process saturates. Acute arsenic poisoning results in necrosis of intestinal mucosa with hemorrhagic gastroenteritis, fluid loss, hypotension, delayed cardiomyopathy, acute tubular necrosis, and hemolysis.

1	Acute arsenic poisoning results in necrosis of intestinal mucosa with hemorrhagic gastroenteritis, fluid loss, hypotension, delayed cardiomyopathy, acute tubular necrosis, and hemolysis. Chronic arsenic exposure causes diabetes, vasospasm, peripheral vascular insufficiency and gangrene, peripheral neuropathy, and cancer of skin, lung, liver (angiosarcoma), bladder, and kidney. Lethal dose: 120–200 mg (adults); 2 mg/kg (children). Nausea, vomiting, diarrhea, abdominal pain, delirium, coma, seizures; garlicky odor on breath; hyperkeratosis, hyperpigmentation, exfoliative dermatitis, and Mees’ lines (transverse white striae of the fingernails); sensory and motor polyneuritis, distal weakness. Radiopaque sign on abdominal x-ray; ECG–QRS broadening, QT prolongation, ST depression, T-wave flattening; 24-h urinary arsenic >67 μmol/d or 50 μg/d; (no seafood × 24 h); if recent exposure, serum arsenic >0.9 μmol/L (7 μg/dL). High arsenic in hair or nails.

1	If acute ingestion, ipecac to induce vomiting, gastric lavage, activated charcoal with a cathartic. Supportive care in ICU. Dimercaprol 3–5 mg/kg IM q4h × 2 days; q6h × 1 day, then q12h × 10 days; alternative: oral succimer. PART 18 Poisoning, Drug Overdose, and Envenomation Metal-plating, pigment, smelting, battery, and plastics industries; tobacco; incineration of these products; ingestion of food that concentrates cadmium (grains, cereals). Absorbed through ingestion or inhalation; bound by metallothionein, filtered at the glomerulus, but reabsorbed by proximal tubules (thus, poorly excreted). Biologic half-life: 10–30 y. Binds cellular sulfhydryl groups, competes with zinc, calcium for binding sites. Concentrates in liver and kidneys. Acute cadmium inhalation causes pneumonitis after 4–24 h; acute ingestion causes gastroenteritis.

1	Acute cadmium inhalation causes pneumonitis after 4–24 h; acute ingestion causes gastroenteritis. Chronic exposure causes anosmia, yellowing of teeth, emphysema, minor LFT elevations, microcytic hypochromic anemia unresponsive to iron therapy, proteinuria, increased urinary β2microglobulin, calciuria, leading to chronic renal failure, osteomalacia, and fractures. Possible risks of cardiovascular disease and cancer. With inhalation: pleuritic chest pain, dyspnea, cyanosis, fever, tachycardia, nausea, noncardiogenic pulmonary edema. With ingestion: nausea, vomiting, cramps, diarrhea. Bone pain, fractures with osteomalacia. If recent exposure, serum cadmium >500 nmol/L (5 μg/dL). Urinary cadmium >100 nmol/L (10 μg/g creatinine) and/or urinary β2-microglobulin >750 μg/g creatinine (but urinary β2-microglobulin also increased in other renal diseases such as pyelonephritis).

1	There is no effective treatment for cadmium poisoning (chelation not useful; dimercaprol can exacerbate nephrotoxicity). Avoidance of further exposure, supportive therapy, vitamin D for osteomalacia. Manufacturing of auto batteries, lead crystal, ceramics, fishing weights, etc.; demolition or sanding of lead-painted houses, bridges; stained glass–making, plumbing, soldering; environmental exposure to paint chips, house dust (in homes built <1975), firing ranges (from bullet dust), food or water from improperly glazed ceramics, lead pipes; contaminated herbal remedies, candies; exposure to the combustion of leaded fuels.

1	Absorbed through ingestion or inhalation; organic lead (e.g., tetraethyl lead) absorbed dermally. In blood, 95–99% sequestered in RBCs—thus, must measure lead in whole blood (not serum). Distributed widely in soft tissue, with half-life ~30 days; 15% of dose sequestered in bone with half-life of >20 years. Excreted mostly in urine, but also appears in other fluids including breast milk. Interferes with mitochondrial oxidative phosphorylation, ATPases, calcium-dependent messengers; enhances oxidation and cell apoptosis.

1	Acute exposure with blood lead levels (BPb) of >60–80 μg/dL can cause impaired neurotransmission and neuronal cell death (with central and peripheral nervous system effects); impaired hematopoiesis and renal tubular dysfunction. At higher levels of exposure (e.g., BPb >80–120 μg/dL), acute encephalopathy with convulsions, coma, and death may occur. Subclinical exposures in children (BPb 25–60 μg/dL) are associated with anemia; mental retardation; and deficits in language, motor function, balance, hearing, behavior, and school performance. Impairment of IQ appears to occur at even lower levels of exposure with no measurable threshold above the limit of detection in most assays of 1 μg/dL.

1	In adults, chronic subclinical exposures (BPb >40 μg/dL) are associated with an increased risk of anemia, demyelinating peripheral neuropathy (mainly motor), impairments of reaction time and hearing, accelerated declines in cognition, hypertension, ECG conduction delays, higher risk of cardiovascular disease and death, interstitial nephritis and chronic renal failure, diminished sperm counts, and spontaneous abortions. Abdominal pain, irritability, lethargy, anorexia, anemia, Fanconi’s syndrome, pyuria, azotemia in children with blood lead level (BPb) >80 μg/ dL; may also see epiphyseal plate “lead lines” on long bone x-rays. Convulsions, coma at BPb >120 μg/ dL. Noticeable neurodevelopmental delays at BPb of 40–80 μg/dL; may also see symptoms associated with higher BPb levels. Screening of all U.S.

1	U.S. children when they begin to crawl (~6 months) is recommended by the CDC; source identification and intervention is begun if the BPb >10 μg/dL. In adults, acute exposure causes similar symptoms as in children as well as headaches, arthralgias, myalgias, depression, impaired short-term memory, loss of libido. Physical exam may reveal a “lead line” at the gingiva-tooth border, pallor, wrist drop, and cognitive dysfunction (e.g., declines on the mini-mental state exam); lab tests may reveal a normocytic, normochromic anemia, basophilic stippling, an elevated blood protoporphyrin level (free erythrocyte or zinc), and motor delays on nerve conduction. U.S. OSHA requires regular testing of lead-exposed workers with removal if BPb >40 μg/dL. New guidelines have been proposed recommending that BPb be maintained at <10 μg/dL, removal of workers if BPb >20 μg/dL, and monitoring of cumulative exposure parameters.

1	Identification and correction of exposure sources is critical. In some U.S. states, screening and reporting to local health boards of children with BPb >10 μg/ dL and workers with BPb >40 μg/dL is required. In the highly exposed individual with symptoms, chelation is recommended with oral DMSA (succimer); if acutely toxic, hospitalization and IV or IM chelation with ethylenediamine tetraacetic acid calcium disodium (CaEDTA) may be required, with the addition of dimercaprol to prevent worsening of encephalopathy. It is uncertain whether children with asymptomatic lead exposure (e.g., BPb 20–40 μg/dL) benefit from chelation; a recent randomized trial showed no benefit. Correction of dietary deficiencies in iron, calcium, magnesium, and zinc will lower lead absorption and may also improve toxicity. Vitamin C is a weak but natural chelating agent. Calcium supplements (1200 mg at bedtime) have been shown to lower blood lead levels in pregnant women.

1	Abbreviations: ATPase, adenosine triphosphatase; BPb, blood lead; CDC, Centers for Disease Control and Prevention; CNS, central nervous system; DMSA, dimercaptosuccinic acid; ECG, electrocardiogram; GI, gastrointestinal; ICU, intensive care unit; IQ, intelligence quotient; LFT, liver function tests; OSHA, Occupational Safety and Health Administration; RBC, red blood cell.

1	residents in parts of Bangladesh and Western India. Contamination masked, expressionless face; tremor; and psychiatric symptoms. With was formerly considered only a problem with deep wells; however, the the introduction of methylcyclopentadienyl manganese tricarbonyl geology of this region allows most residents only a few alternatives for (MMT) as a gasoline additive, there is concern for the toxic potential potable drinking water. The combustion of leaded gasoline with result-of environmental manganese exposure. For example, a recent study ing contamination of air and soil with lead oxide remains a problem in found a high prevalence of parkinsonian disorders in a community some countries of Central Asia, Southeast Asia, Africa, and the Middle with risks proportionate to estimated manganese exposures emitted by East. Populations living in the Arctic have been shown to have particu-local ferroalloy industries. Epidemiologic studies have also suggested larly high exposures to mercury

1	exposures emitted by East. Populations living in the Arctic have been shown to have particu-local ferroalloy industries. Epidemiologic studies have also suggested larly high exposures to mercury due to long-range transport patterns that manganese may interfere with early childhood neurodevelopment that concentrate mercury in the polar regions, as well as the traditional in ways similar to that of lead. Nickel exposure induces an allergic dependence of Arctic peoples on the consumption of fish and other response, and inhalation of nickel compounds with low aqueous wildlife that bioconcentrate methylmercury. solubility (e.g., nickel subsulfide and nickel oxide) in occupational set-

1	A few additional metals deserve brief mention but are not covered tings is associated with an increased risk of lung cancer. Overexposure in Table 472e-1 because of the relative rarity of their being clinically to selenium may cause local irritation of the respiratory system and encountered or the uncertainty regarding their potential toxicities. eyes, gastrointestinal irritation, liver inflammation, loss of hair, depig-Aluminum contributes to the encephalopathy in patients with severe mentation, and peripheral nerve damage. Workers exposed to certain renal disease, who are undergoing dialysis (Chap. 424). High levels organic forms of tin (particularly trimethyl and triethyl derivatives) of aluminum are found in the neurofibrillary tangles in the cerebral have developed psychomotor disturbances, including tremor, convulcortex and hippocampus of patients with Alzheimer’s disease, as well sions, hallucinations, and psychotic behavior. as in the drinking water and soil of areas with an

1	including tremor, convulcortex and hippocampus of patients with Alzheimer’s disease, as well sions, hallucinations, and psychotic behavior. as in the drinking water and soil of areas with an unusually high inci-Thallium, which is a component of some insecticides, metal alloys, dence of Alzheimer’s. The experimental and epidemiologic evidence and fireworks, is absorbed through the skin as well as by ingestion and for the aluminum–Alzheimer’s disease link remains relatively weak, inhalation. Severe poisoning follows a single ingested dose of >1 g or however, and it cannot be concluded that aluminum is a causal agent >8 mg/kg. Nausea and vomiting, abdominal pain, and hematemesis or a contributing factor in neurodegenerative disease. Hexavalent precede confusion, psychosis, organic brain syndrome, and coma. chromium is corrosive and sensitizing. Workers in the chromate and Thallium is radiopaque. Induced emesis or gastric lavage is indicated chrome pigment production industries have

1	syndrome, and coma. chromium is corrosive and sensitizing. Workers in the chromate and Thallium is radiopaque. Induced emesis or gastric lavage is indicated chrome pigment production industries have consistently had a greater within 4–6 h of acute ingestion; Prussian blue prevents absorption risk of lung cancer. The introduction of cobalt chloride as a fortifier in and is given orally at 250 mg/kg in divided doses. Unlike other types beer led to outbreaks of fatal cardiomyopathy among heavy consum-of metal poisoning, thallium poisoning may be less severe when actiers. Occupational exposure (e.g., of miners, dry-battery manufacturers, vated charcoal is used to interrupt its enterohepatic circulation. Other and arc welders) to manganese can cause a parkinsonian syndrome measures include forced diuresis, treatment with potassium chloride within 1–2 years, including gait disorders; postural instability; a (which promotes renal excretion of thallium), and peritoneal dialysis.

1	Poisoning and Drug Overdose Mark B. Mycyk Poisoning refers to the development of dose-related adverse effects following exposure to chemicals, drugs, or other xenobiotics. To para-phrase Paracelsus, the dose makes the poison. In excessive amounts, substances that are usually innocuous, such as oxygen and water, 473e can cause toxicity. Conversely, in small doses, substances commonly regarded as poisons, such as arsenic and cyanide, can be consumed without ill effect. Although most poisons have predictable dose-related effects, individual responses to a given dose may vary because of genetic polymorphism, enzymatic induction or inhibition in the presence of other xenobiotics, or acquired tolerance. Poisoning may be local (e.g., skin, eyes, or lungs) or systemic depending on the route of exposure, the chemical and physical properties of the poison, and its mechanism of action. The severity and reversibility of poisoning also depend on the functional reserve of the individual or target

1	the chemical and physical properties of the poison, and its mechanism of action. The severity and reversibility of poisoning also depend on the functional reserve of the individual or target organ, which is influenced by age and preexisting disease.

1	More than 5 million poison exposures occur in the United States each year. Most are acute, are accidental (unintentional), involve a single agent, occur in the home, result in minor or no toxicity, and involve children <6 years of age. Pharmaceuticals are involved in 47% of exposures and in 84% of serious or fatal poisonings. Unintentional exposures can result from the improper use of chemicals at work or play; label misreading; product mislabeling; mistaken identification of unlabeled chemicals; uninformed self-medication; and dosing errors by nurses, pharmacists, physicians, parents, and the elderly. Excluding the recreational use of ethanol, attempted suicide (deliberate self-harm) is the most common reported reason for intentional poisoning. Recreational use of prescribed and over-the-counter drugs for psychotropic or euphoric effects (abuse) or excessive self-dosing (misuse) is increasingly common and may also result in unintentional self-poisoning.

1	About 20–25% of exposures require bedside health-professional evaluation, and 5% of all exposures require hospitalization. Poisonings account for 5–10% of all ambulance transports, emergency department visits, and intensive care unit admissions. Up to 30% of psychiatric admissions are prompted by attempted suicide via overdosage. Overall, the mortality rate is low: <1% of all exposures. It is much higher (1–2%) among hospitalized patients with intentional (suicidal) overdose, who account for the majority of serious poisonings. Acetaminophen is the pharmaceutical agent most often implicated in fatal poisoning. Overall, carbon monoxide is the leading cause of death from poisoning, but this prominence is not reflected in hospital or poison center statistics because patients with such poisoning are typically dead when discovered and are referred directly to medical examiners.

1	Although poisoning can mimic other illnesses, the correct diagnosis can usually be established by the history, physical examination, routine and toxicologic laboratory evaluations, and characteristic clinical course. The history should include the time, route, duration, and circumstances (location, surrounding events, and intent) of exposure; the name and amount of each drug, chemical, or ingredient involved; the time of onset, nature, and severity of symptoms; the time and type of first-aid measures provided; and the medical and psychiatric history.

1	In many cases the patient is confused, comatose, unaware of an exposure, or unable or unwilling to admit to one. Suspicious circumstances include unexplained sudden illness in a previously healthy person or a group of healthy people; a history of psychiatric problems (particularly depression); recent changes in health, economic status, or social relationships; and onset of illness during work with chemicals 473e-1 or after ingestion of food, drink (especially ethanol), or medications. When patients become ill soon after arriving from a foreign country or being arrested for criminal activity, “body packing” or “body stuffing” (ingesting or concealing illicit drugs in a body cavity) should be suspected. Relevant information may be available from family, friends, paramedics, police, pharmacists, physicians, and employers, who should be questioned regarding the patient’s habits, hobbies, behavioral changes, available medications, and antecedent events. A search of clothes, belongings, and

1	physicians, and employers, who should be questioned regarding the patient’s habits, hobbies, behavioral changes, available medications, and antecedent events. A search of clothes, belongings, and place of discovery may reveal a suicide note or a container of drugs or chemicals. The imprint code on pills and the label on chemical products may be used to identify the ingredients and potential toxicity of a suspected poison by consulting a reference text, a computerized database, the manufacturer, or a regional poison information center (800-222-1222). Occupational exposures require review of any available material safety data sheet (MSDS) from the worksite. Because of increasing globalization, unfamiliar poisonings may result in local emergency department evaluation. Pharmaceuticals, industrial chemicals, or drugs of abuse from foreign countries may be identified with the assistance of a regional poison center or via the World Wide Web.

1	The physical examination should focus initially on vital signs, the cardiopulmonary system, and neurologic status. The neurologic examination should include documentation of neuromuscular abnormalities such as dyskinesia, dystonia, fasciculations, myoclonus, rigidity, and tremors. The patient should also be examined for evidence of trauma and underlying illnesses. Focal neurologic findings are uncommon in poisoning, and their presence should prompt evaluation for a structural central nervous system (CNS) lesion. Examination of the eyes (for nystagmus and pupil size and reactivity), abdomen (for bowel activity and bladder size), and skin (for burns, bullae, color, warmth, moisture, pressure sores, and puncture marks) may reveal findings of diagnostic value. When the history is unclear, all orifices should be examined for the presence of chemical burns and drug packets. The odor of breath or vomitus and the color of nails, skin, or urine may provide important diagnostic clues.

1	The diagnosis of poisoning in cases of unknown etiology primarily relies on pattern recognition. The first step is to assess the pulse, blood pressure, respiratory rate, temperature, and neurologic status and to characterize the overall physiologic state as stimulated, depressed, discordant, or normal (Table 473e-1). Obtaining a complete set of vital signs and reassessing them frequently are critical. Measuring core temperature is especially important, even in difficult or combative patients, since temperature elevation is the most reliable prognosticator of poor outcome in poisoning or drug withdrawal. The next step is to consider the underlying causes of the physiologic state and to attempt to identify a pathophysiologic pattern or toxic syndrome (toxidrome) based on the observed findings. Assessing the severity of physiologic derangements (Table 473e-2) is useful in this regard and also for monitoring the clinical course and response to treatment. The final step is to attempt to

1	Assessing the severity of physiologic derangements (Table 473e-2) is useful in this regard and also for monitoring the clinical course and response to treatment. The final step is to attempt to identify the particular agent involved by looking for unique or relatively poison-specific physical or ancillary test abnormalities. Distinguishing among toxidromes on the basis of the physiologic state is summarized next.

1	The Stimulated Physiologic State Increased pulse, blood pressure, respiratory rate, temperature, and neuromuscular activity characterize the stimulated physiologic state, which can reflect sympathetic, antimuscarinic (anticholinergic), or hallucinogen poisoning or drug withdrawal (Table 473e-1). Other features are noted in (Table 473e-2). Mydriasis, a characteristic feature of all stimulants, is most marked in antimuscarinic (anticholinergic) poisoning since pupillary reactivity relies on muscarinic control. In sympathetic poisoning (e.g., due to cocaine), pupils are also enlarged, but some reactivity to light remains. The antimuscarinic (anticholinergic) toxidrome is also distinguished by hot, dry, flushed skin; decreased bowel sounds; and urinary retention. Other stimulant syndromes increase sympathetic activity and cause diaphoresis, pallor, and increased bowel activity with varying

1	Abbreviations: ACE, angiotensin-converting enzyme; AGMA, anion-gap metabolic acidosis; CNS, central nervous system; GABA, γ-aminobutyric acid; GHB, γ-hydroxybutyrate; GI, gastrointestinal; LSD, lysergic acid diethylamide; MAO, monoamine oxidase.

1	Grade 1 Awake, lethargic, or sleeping but arousable by voice or tactile stimulation; able to converse and follow commands; may be confused Grade 2 Responds to pain but not voice; can vocalize but not converse; spontaneous motor activity present; brainstem reflexes intact Grade 3 Unresponsive to pain; spontaneous motor activity absent; brainstem reflexes depressed; motor tone, respirations, and temperature decreased Grade 4 Unresponsive to pain; flaccid paralysis; brainstem reflexes and respirations absent; cardiovascular vital signs decreased degrees of nausea, vomiting, abnormal distress, and occasionally diarrhea. The absolute and relative degree of vital-sign changes and neuromuscular hyperactivity can help distinguish among stimulant toxidromes. Since sympathetics stimulate the peripheral nervous system more directly than do hallucinogens or drug withdrawal, markedly increased vital signs and organ ischemia suggest sympathetic poisoning. Findings helpful in suggesting the

1	peripheral nervous system more directly than do hallucinogens or drug withdrawal, markedly increased vital signs and organ ischemia suggest sympathetic poisoning. Findings helpful in suggesting the particular drug or class causing physiologic stimulation include reflex bradycardia from selective α-adrenergic stimulants (e.g., decongestants), hypotension from selective β-adrenergic stimulants (e.g., asthma therapeutics), limb ischemia from ergot alkaloids, rotatory nystagmus from phencyclidine and ketamine (the only physiologic stimulants that cause this finding), and delayed cardiac conduction from high doses of cocaine and some anticholinergic agents (e.g., antihistamines, cyclic antidepressants, and antipsychotics). Seizures suggest a sympathetic etiology, an anticholinergic agent with membrane-active properties (e.g., cyclic antidepressants, orphenadrine, phenothiazines), or a withdrawal syndrome. Close attention to core temperature is critical in patients with grade 4 physiologic

1	membrane-active properties (e.g., cyclic antidepressants, orphenadrine, phenothiazines), or a withdrawal syndrome. Close attention to core temperature is critical in patients with grade 4 physiologic stimulation (Table 473e-2).

1	The Depressed Physiologic State Decreased pulse, blood pressure, respiratory rate, temperature, and neuromuscular activity are indicative of the depressed physiologic state caused by “functional” sympatholytics (agents that decrease cardiac function and vascular tone as well as sympathetic activity), cholinergic (muscarinic and nicotinic) agents, opioids, and sedative-hypnotic γ-aminobutyric acid (GABA)-ergic agents (Table 473e-1 and 473e-2). Miosis is also common and is most pronounced in opioid and cholinergic poisoning. Miosis is distinguished from other depressant syndromes by muscarinic and nicotinic signs and symptoms (Table 473e-1). Pronounced cardiovascular depression in the absence of significant CNS depression suggests a direct or peripherally acting sympatholytic. In contrast, in opioid and sedative-hypnotic poisoning, vital-sign changes are secondary to depression of CNS cardiovascular and respiratory centers (or consequent hypoxemia), and significant abnormalities in

1	in opioid and sedative-hypnotic poisoning, vital-sign changes are secondary to depression of CNS cardiovascular and respiratory centers (or consequent hypoxemia), and significant abnormalities in these parameters do not occur until there is a marked decrease in the level of consciousness (grade 3 or 4 physiologic depression; [Table 473e-2]). Other clues that suggest the cause of physiologic depression include cardiac arrhythmias and conduction disturbances (due to antiarrhythmics, β-adrenergic antagonists, calcium channel blockers, digitalis glycosides, propoxyphene, and cyclic antidepressants), mydriasis (due to tricyclic antidepressants, some antiarrhythmics, meperidine, and diphenoxylate-atropine [Lomotil]), nystagmus (due to sedative-hypnotics), and seizures (due to cholinergic agents, propoxyphene, and cyclic antidepressants).

1	The Discordant Physiologic State The discordant physiologic state is characterized by mixed vital-sign and neuromuscular abnormalities, as observed in poisoning by asphyxiants, CNS syndromes, membrane-active agents, and anion-gap metabolic acidosis (AGMA) inducers (Table 473e-1). In these conditions, manifestations of physiologic stimulation and physiologic depression occur together or at different times during the clinical course. For example, membrane-active agents can cause simultaneous coma, seizures, hypotension, and tachyarrhythmias. Alternatively, vital signs may be normal while the patient has an altered mental status or is obviously sick or clearly symptomatic. Early, pronounced vital-sign and mental-status changes suggest asphyxiant or membrane-active agent poisoning; the lack of such abnormalities suggests an AGMA inducer; and marked neuromuscular dysfunction without significant vital-sign abnormalities suggests a CNS syndrome.

1	The Normal Physiologic State A normal physiologic status and physical examination may be due to a nontoxic exposure, psychogenic illness, or poisoning by “toxic time-bombs”: agents that are slowly absorbed, are slowly distributed to their sites of action, require metabolic activation, or disrupt metabolic processes (Table 473e-1). Because so many medications have now been reformulated into a once-a-day preparations for the patient’s convenience and adherence, toxic time-bombs are increasingly common. Diagnosing a nontoxic exposure requires that the identity of the exposure agent be known or that a toxic time-bomb exposure be excluded and the time since exposure exceed 473e-3 the longest known or predicted interval between exposure and peak toxicity. Psychogenic illness (fear of being poisoned, mass hysteria) may also follow a nontoxic exposure and should be considered when symptoms are inconsistent with exposure history. Anxiety reactions resulting from a nontoxic exposure can cause

1	mass hysteria) may also follow a nontoxic exposure and should be considered when symptoms are inconsistent with exposure history. Anxiety reactions resulting from a nontoxic exposure can cause mild physiologic stimulation (Table 473e-2) and be indistinguishable from toxicologic causes without ancillary testing or a suitable period of observation.

1	Laboratory assessment may be helpful in the differential diagnosis. Increased AGMA is most common in advanced methanol, ethylene glycol, and salicylate intoxication but can occur with any poisoning that results in hepatic, renal, or respiratory failure; seizures; or shock. The serum lactate concentration is more commonly low (less than the anion gap) in the former and high (nearly equal to the anion gap) in the latter. An abnormally low anion gap can be due to elevated blood levels of bromide, calcium, iodine, lithium, or magnesium. An increased osmolal gap—a difference of >10 mmol/L between serum osmolality (measured by freezing-point depression) and osmolality calculated from serum sodium, glucose, and blood urea nitrogen levels— suggests the presence of a low-molecular-weight solute such as acetone; an alcohol (benzyl, ethanol, isopropanol, methanol); a glycol (diethylene, ethylene, propylene); ether (ethyl, glycol); or an “unmeasured” cation (calcium, magnesium) or sugar

1	solute such as acetone; an alcohol (benzyl, ethanol, isopropanol, methanol); a glycol (diethylene, ethylene, propylene); ether (ethyl, glycol); or an “unmeasured” cation (calcium, magnesium) or sugar (glycerol, mannitol, sorbitol). Ketosis suggests acetone, isopropyl alcohol, salicylate poisoning, or alcoholic ketoacidosis. Hypoglycemia may be due to poisoning with β-adrenergic blockers, ethanol, insulin, oral hypoglycemic agents, quinine, and salicylates, whereas hyperglycemia can occur in poisoning with acetone, β-adrenergic agonists, caffeine, calcium channel blockers, iron, theophylline, or N-3-pyridylmethyl-N′-p-nitrophenylurea (PNU [Vacor]). Hypokalemia can be caused by barium, β-adrenergic agonists, caffeine, diuretics, theophylline, or toluene; hyperkalemia suggests poisoning with an α-adrenergic agonist, a β-adrenergic blocker, cardiac glycosides, or fluoride. Hypocalcemia may be seen in ethylene glycol, fluoride, and oxalate poisoning.

1	The electrocardiogram (ECG) can be useful for rapid diagnostic purposes. Bradycardia and atrioventricular block may occur in patients poisoned by α-adrenergic agonists, antiarrhythmic agents, beta blockers, calcium channel blockers, cholinergic agents (carbamate and organophosphate insecticides), cardiac glycosides, lithium, or tricyclic antidepressants. QRSand QT-interval prolongation may be caused by hyperkalemia, various antidepressants, and other membrane-active drugs (Table 473e-1). Ventricular tachyarrhythmias may be seen in poisoning with cardiac glycosides, fluorides, membrane-active drugs, methylxanthines, sympathomimetics, antidepressants, and agents that cause hyperkalemia or potentiate the effects of endogenous catecholamines (e.g., chloral hydrate, aliphatic and halogenated hydrocarbons).

1	Radiologic studies may occasionally be useful. Pulmonary edema (adult respiratory distress syndrome (ARDS) can be caused by poisoning with carbon monoxide, cyanide, an opioid, paraquat, phencyclidine, a sedative-hypnotic, or salicylate; by inhalation of irritant gases, fumes, or vapors (acids and alkali, ammonia, aldehydes, chlorine, hydrogen sulfide, isocyanates, metal oxides, mercury, phosgene, polymers); or by prolonged anoxia, hyperthermia, or shock. Aspiration pneumonia is common in patients with coma, seizures, and petroleum distillate aspiration. The presence of radiopaque densities on abdominal x-rays suggests the ingestion of calcium salts, chloral hydrate, chlorinated hydrocarbons, heavy metals, illicit drug packets, iodinated compounds, potassium salts, enteric-coated tablets, or salicylates.

1	Toxicologic analysis of urine and blood (and occasionally of gastric contents and chemical samples) can sometimes confirm or rule out suspected poisoning. Interpretation of laboratory data requires knowledge of the qualitative and quantitative tests used for screening and confirmation (enzyme-multiplied, fluorescence polarization, and radio-immunoassays; colorimetric and fluorometric assays; thin-layer, gas-liquid, or high-performance liquid chromatography; gas chromatography; mass spectrometry), their sensitivity (limit of detection) and specificity, the preferred biologic specimen for analysis, and the 473e-4 optimal time of specimen sampling. Personal communication with the hospital laboratory is essential to an understanding of institutional testing capabilities and limitations. Rapid qualitative hospital-based urine tests for drugs of abuse are only screening tests that cannot confirm the exact identity of the detected substance and should not be considered diagnostic or used for

1	qualitative hospital-based urine tests for drugs of abuse are only screening tests that cannot confirm the exact identity of the detected substance and should not be considered diagnostic or used for forensic purposes: False-positive and false-negative results are common. A positive screen may result from other pharmaceuticals that interfere with laboratory analysis (e.g., fluoroquinolones commonly cause “false-positive” opiate screens). Confirmatory testing with gas chromatography/mass spectrometry can be requested, but it often takes weeks to obtain a reported result. A negative screening result may mean that the responsible substance is not detectable by the test used or that its concentration is too low for detection at the time of sampling. For instance, recent new drugs of abuse that often result in emergency department evaluation for unexpected complications, such as synthetic cannabinoids (spice), cathinones (bath salts), and opiate substitutes (kratom), are not detectable by

1	often result in emergency department evaluation for unexpected complications, such as synthetic cannabinoids (spice), cathinones (bath salts), and opiate substitutes (kratom), are not detectable by hospital-based tests. In cases where a drug concentration is too low to be detected early during clinical evaluation, repeating the test at a later time may yield a positive result. Patients symptomatic from drugs of abuse often require immediate management based on the history, physical examination, and observed toxidrome without laboratory confirmation (e.g., apnea from opioid intoxication). When the patient is asymptomatic or when the clinical picture is consistent with the reported history, qualitative screening is neither clinically useful nor cost-effective. Thus, qualitative drug screens are of greatest value for the evaluation of patients with severe or unexplained toxicities, such as coma, seizures, cardiovascular instability, metabolic or respiratory acidosis, and nonsinus cardiac

1	are of greatest value for the evaluation of patients with severe or unexplained toxicities, such as coma, seizures, cardiovascular instability, metabolic or respiratory acidosis, and nonsinus cardiac rhythms. In contrast to qualitative drug screens, quantitative serum tests are useful for evaluation of patients poisoned with acetaminophen (Chap. 361), alcohols (including ethylene glycol and methanol), anticonvulsants, barbiturates, digoxin, heavy metals, iron, lithium, salicylate, and theophylline as well as for the presence of carboxyhemoglobin and methemoglobin. The serum concentration in these cases guides clinical management, and results are often available within an hour. The response to antidotes is sometimes useful for diagnostic purposes. Resolution of altered mental status and abnormal vital signs within minutes of IV administration of dextrose, naloxone, or flumazenil is virtually diagnostic of hypoglycemia, opioid poisoning, and benzodiazepine intoxication, respectively.

1	abnormal vital signs within minutes of IV administration of dextrose, naloxone, or flumazenil is virtually diagnostic of hypoglycemia, opioid poisoning, and benzodiazepine intoxication, respectively. The prompt reversal of dystonic (extrapyramidal) signs and symptoms following an IV dose of benztropine or diphenhydramine confirms a drug etiology. Although complete reversal of both central and peripheral manifestations of anticholinergic poisoning by physostigmine is diagnostic of this condition, physostigmine may cause some arousal in patients with CNS depression of any etiology.

1	PART 18 Poisoning, Drug Overdose, and Envenomation Treatment goals include support of vital signs, prevention of further poison absorption (decontamination), enhancement of poison elimination, administration of specific antidotes, and prevention of reexposure (Table 473e-3). Specific treatment depends on the identity of the poison, the route and amount of exposure, the time of presentation relative to the time of exposure, and the severity of poisoning. Knowledge of the offending agents’ pharmacokinetics and pharmacodynamics is essential.

1	During the pretoxic phase, prior to the onset of poisoning, decontamination is the highest priority, and treatment is based solely on the history. The maximal potential toxicity based on the greatest possible exposure should be assumed. Since decontamination is more effective when accomplished soon after exposure and when the patient is asymptomatic, the initial history and physical examination should be focused and brief. It is also advisable to establish IV access and initiate cardiac monitoring, particularly in patients with potentially serious ingestions or unclear histories. Prevention of Further Poison Absorption Enhancement of Poison Elimination Hemodialysis Alteration of urinary pH Administration of Antidotes Prevention of Reexposure Adult education Notification of regulatory agencies

1	Hemodialysis Alteration of urinary pH Administration of Antidotes Prevention of Reexposure Adult education Notification of regulatory agencies When an accurate history is not obtainable and a poison causing delayed toxicity (i.e., a toxic time-bomb) or irreversible damage is suspected, blood and urine should be sent for appropriate toxicologic screening and quantitative analysis. During poison absorption and distribution, blood levels may be greater than those in tissue and may not correlate with toxicity. However, high blood levels of agents whose metabolites are more toxic than the parent compound (acetaminophen, ethylene glycol, or methanol) may indicate the need for additional interventions (antidotes, dialysis). Most patients who remain asymptomatic or who become asymptomatic 6 h after ingestion are unlikely to develop subsequent toxicity and can be discharged safely. Longer observation will be necessary for patients who have ingested toxic time-bombs.

1	During the toxic phase—the interval between the onset of poisoning and its peak effects—management is based primarily on clinical and laboratory findings. Effects after an overdose usually begin sooner, peak later, and last longer than they do after a therapeutic dose. A drug’s published pharmacokinetic profile in standard references such as the Physician’s Desk Reference (PDR) is usually different from its toxicokinetic profile in overdose. Resuscitation and stabilization are the first priority. Symptomatic patients should have an IV line placed and should undergo oxygen saturation determination, cardiac monitoring, and continuous observation. Baseline laboratory, ECG, and x-ray evaluation may also be appropriate. Intravenous glucose (unless the serum level is documented to be normal), naloxone, and thiamine should be considered in patients with altered mental status, particularly those with coma or seizures. Decontamination should also be considered, but it is less likely to be

1	naloxone, and thiamine should be considered in patients with altered mental status, particularly those with coma or seizures. Decontamination should also be considered, but it is less likely to be effective during this phase than during the pretoxic phase.

1	Measures that enhance poison elimination may shorten the duration and severity of the toxic phase. However, they are not without risk, which must be weighed against the potential benefit. Diagnostic certainty (usually via laboratory confirmation) is generally a prerequisite. Intestinal (gut) dialysis with repetitive doses of activated charcoal (see “Multiple-Dose Activated Charcoal,” later) can enhance the elimination of selected poisons such as theophylline or carbamazepine. Urinary alkalinization may enhance the elimination of salicylates and a few other poisons. Chelation therapy can enhance the elimination of selected metals. Extracorporeal elimination methods are effective for many poisons, but their expense and risk make their use reasonable only in patients who would otherwise have an unfavorable outcome.

1	During the resolution phase of poisoning, supportive care and monitoring should continue until clinical, laboratory, and ECG abnormalities have resolved. Since chemicals are eliminated sooner from the blood than from tissues, blood levels are usually lower than tissue levels during this phase and again may not correlate with toxicity. This discrepancy applies particularly when extracorporeal elimination procedures are used. Redistribution from tissues may cause a rebound increase in the blood level after termination of these procedures. When a metabolite is responsible for toxic effects, continued treatment may be necessary in the absence of clinical toxicity or abnormal laboratory studies.

1	The goal of supportive therapy is to maintain physiologic homeostasis until detoxification is accomplished and to prevent and treat secondary complications such as aspiration, bedsores, cerebral and pulmonary edema, pneumonia, rhabdomyolysis, renal failure, sepsis, thromboembolic disease, coagulopathy, and generalized organ dysfunction due to hypoxemia or shock.

1	Admission to an intensive care unit is indicated for the following: patients with severe poisoning (coma, respiratory depression, hypo-tension, cardiac conduction abnormalities, cardiac arrhythmias, hypothermia or hyperthermia, seizures); those needing close monitoring, antidotes, or enhanced elimination therapy; those showing progressive clinical deterioration; and those with significant underlying medical problems. Patients with mild to moderate toxicity can be managed on a general medical service, on an intermediate care unit, or in an emergency department observation area, depending on the anticipated duration and level of monitoring needed (intermittent clinical observation versus continuous clinical, cardiac, and respiratory monitoring). Patients who have attempted suicide require continuous observation and measures to prevent self-injury until they are no longer suicidal.

1	Respiratory Care Endotracheal intubation for protection against the aspiration of gastrointestinal contents is of paramount importance in patients with CNS depression or seizures as this complication can increase morbidity and mortality rates. Mechanical ventilation may be necessary for patients with respiratory depression or hypoxemia and for facilitation of therapeutic sedation or paralysis of patients in order to prevent or treat hyperthermia, acidosis, and rhabdomyolysis associated with neuromuscular hyperactivity. Since clinical assessment of respiratory function can be inaccurate, the need for oxygenation and ventilation is best determined by continuous pulse oximetry or arterial blood-gas analysis. The gag reflex is not a reliable indicator of the need for intubation. A patient with CNS depression may maintain airway patency while being stimulated but not if left alone. Drug-induced pulmonary edema is usually noncardiac rather than cardiac in origin, although profound CNS

1	with CNS depression may maintain airway patency while being stimulated but not if left alone. Drug-induced pulmonary edema is usually noncardiac rather than cardiac in origin, although profound CNS depression and cardiac conduction abnormalities suggest the latter. Measurement of pulmonary artery pressure may be necessary to establish the cause and direct appropriate therapy. Extracorporeal measures (membrane oxygenation, venoarterial perfusion, cardiopulmonary bypass) and partial liquid (perfluorocarbon) ventilation may be appropriate for severe but reversible respiratory failure.

1	Cardiovascular Therapy Maintenance of normal tissue perfusion is critical for complete recovery to occur once the offending agent has been eliminated. If hypotension is unresponsive to volume expansion, treatment with norepinephrine, epinephrine, or high-dose dopamine may be necessary. Intraaortic balloon pump counterpulsation and venoarterial or cardiopulmonary perfusion techniques should be considered for severe but reversible cardiac failure. For patients with a return of spontaneous circulation after resuscitative treatment for 473e-5 cardiopulmonary arrest secondary to poisoning, therapeutic hypothermia should be used according to protocol. Bradyarrhythmias associated with hypotension generally should be treated as described in Chaps. 274 and 275. Glucagon, calcium, and high-dose insulin with dextrose may be effective in beta blocker and calcium channel blocker poisoning. Antibody therapy may be indicated for cardiac glycoside poisoning.

1	Supraventricular tachycardia associated with hypertension and CNS excitation is almost always due to agents that cause generalized physiologic excitation (Table 473e–1). Most cases are mild or moderate in severity and require only observation or nonspecific sedation with a benzodiazepine. In severe cases or those associated with hemodynamic instability, chest pain, or ECG evidence of ischemia, specific therapy is indicated. When the etiology is sympathetic hyperactivity, treatment with a benzodiazepine should be prioritized. Further treatment with a combined alpha and beta blocker (labetalol), a calcium channel blocker (verapamil or diltiazem), or a combination of a beta blocker and a vasodilator (esmolol and nitroprusside) may be considered for cases refractory to high doses of benzodiazepines. Treatment with an α-adrenergic antagonist (phentolamine) alone may sometimes be appropriate. If the cause is anticholinergic poisoning, physostigmine alone can be effective. Supraventricular

1	Treatment with an α-adrenergic antagonist (phentolamine) alone may sometimes be appropriate. If the cause is anticholinergic poisoning, physostigmine alone can be effective. Supraventricular tachycardia without hypertension is generally secondary to vasodilation or hypovolemia and responds to fluid administration.

1	For ventricular tachyarrhythmias due to tricyclic antidepressants and other membrane-active agents (Table 473e-1), sodium bicarbonate is indicated, whereas class IA, IC, and III antiarrhythmic agents are contraindicated because of similar electrophysiologic effects. Although lidocaine and phenytoin are historically safe for ventricular tachyarrhythmias of any etiology, sodium bicarbonate should be considered first for any ventricular arrhythmia suspected to have a toxicologic etiology. Intravenous emulsion therapy has shown benefit for treatment of arrhythmias and hemodynamic instability from various membrane-active agents. Beta blockers can be hazardous if the arrhythmia is due to sympathetic hyperactivity. Magnesium sulfate and overdrive pacing (by isoproterenol or a pacemaker) may be useful in patients with torsades des pointes and prolonged QT intervals. Magnesium and anti-digoxin antibodies should be considered in patients with severe cardiac glycoside poisoning. Invasive

1	be useful in patients with torsades des pointes and prolonged QT intervals. Magnesium and anti-digoxin antibodies should be considered in patients with severe cardiac glycoside poisoning. Invasive (esophageal or intracardiac) ECG recording may be necessary to determine the origin (ventricular or supraventricular) of wide-complex tachycardias (Chaps. 276 and 277). If the patient is hemodynamically stable, however, it is reasonable to simply observe him or her rather than to administer another potentially proarrhythmic agent. Arrhythmias may be resistant to drug therapy until underlying acid-base, electrolyte, oxygenation, and temperature derangements are corrected.

1	Central Nervous System Therapies Neuromuscular hyperactivity and seizures can lead to hyperthermia, lactic acidosis, and rhabdomyolysis and should be treated aggressively. Seizures caused by excessive stimulation of catecholamine receptors (sympathomimetic or hallucinogen poisoning and drug withdrawal) or decreased activity of GABA (isoniazid poisoning) or glycine (strychnine poisoning) receptors are best treated with agents that enhance GABA activity, such as benzodiazepine or barbiturates. Since benzodiazepines and barbiturates act by slightly different mechanisms (the former increases the frequency and the latter increases the duration of chloride channel opening in response to GABA), therapy with both may be effective when neither is effective alone. Seizures caused by isoniazid, which inhibits the synthesis of GABA at several steps by interfering with the cofactor pyridoxine (vitamin B6), may require high doses of supplemental pyridoxine. Seizures resulting from membrane

1	which inhibits the synthesis of GABA at several steps by interfering with the cofactor pyridoxine (vitamin B6), may require high doses of supplemental pyridoxine. Seizures resulting from membrane destabilization (beta blocker or cyclic antidepressant poisoning) require GABA enhancers (benzodiazepines first, barbiturates second). Phenytoin is contraindicated in toxicologic seizures: Animal and human data demonstrate worse outcomes after phenytoin loading, especially

1	PART 18 Poisoning, Drug Overdose, and Envenomation 473e-6 in theophylline overdose. For poisons with central dopaminergic effects (methamphetamine, phencyclidine) manifested by psychotic behavior, a dopamine receptor antagonist, such as haloperidol, may be useful. In anticholinergic and cyanide poisoning, specific antidotal therapy may be necessary. The treatment of seizures secondary to cerebral ischemia or edema or to metabolic abnormalities should include correction of the underlying cause. Neuromuscular paralysis is indicated in refractory cases. Electroencephalographic monitoring and continuing treatment of seizures are necessary to prevent permanent neurologic damage. Serotonergic receptor overstimulation in serotonin syndrome may be treated with cyproheptadine. Other Measures Temperature extremes, metabolic abnormalities, hepatic and renal dysfunction, and secondary complications should be treated by standard therapies.

1	PREVENTION OF POISON ABSORPTION Gastrointestinal Decontamination Whether or not to perform gastrointestinal decontamination and which procedure to use depends on the time since ingestion; the existing and predicted toxicity of the ingestant; the availability, efficacy, and contraindications of the procedure; and the nature, severity, and risk of complications. The efficacy of all decontamination procedures decreases with time, and data are insufficient to support or exclude a beneficial effect when they are used >1 h after ingestion. The average time from ingestion to presentation for treatment is >1 h for children and >3 h for adults. Most patients will recover from poisoning uneventfully with good supportive care alone, but complications of gastrointestinal decontamination, particularly aspiration, can prolong this process. Hence, gastrointestinal decontamination should be performed selectively, not routinely, in the management of overdose patients. It is clearly unnecessary when

1	aspiration, can prolong this process. Hence, gastrointestinal decontamination should be performed selectively, not routinely, in the management of overdose patients. It is clearly unnecessary when predicted toxicity is minimal or the time of expected maximal toxicity has passed without significant effect.

1	Activated charcoal has comparable or greater efficacy; has fewer contraindications and complications; and is less aversive and invasive than ipecac or gastric lavage. Thus it is the preferred method of gastrointestinal decontamination in most situations. Activated charcoal suspension (in water) is given orally via a cup, straw, or small-bore nasogastric tube. The generally recommended dose is 1 g/kg body weight because of its dosing convenience, although in vitro and in vivo studies have demonstrated that charcoal adsorbs ≥90% of most substances when given in an amount equal to 10 times the weight of the substance. Palatability may be increased by adding a sweetener (sorbitol) or a flavoring agent (cherry, chocolate, or cola syrup) to the suspension. Charcoal adsorbs ingested poisons within the gut lumen, allowing the charcoal-toxin complex to be evacuated with stool. Charged (ionized) chemicals such as mineral acids, alkalis, and highly dissociated salts of cyanide, fluoride, iron,

1	the gut lumen, allowing the charcoal-toxin complex to be evacuated with stool. Charged (ionized) chemicals such as mineral acids, alkalis, and highly dissociated salts of cyanide, fluoride, iron, lithium, and other inorganic compounds are not well adsorbed by charcoal. In studies with animals and human volunteers, charcoal decreases the absorption of ingestants by an average of 73% when given within 5 min of ingestant administration, 51% when given at 30 min, and 36% when given at 60 min. For this reason, charcoal given before hospital arrival increases the potential clinical benefit. Side effects of charcoal include nausea, vomiting, and diarrhea or constipation. Charcoal may also prevent the absorption of orally administered therapeutic agents. Complications include mechanical obstruction of the airway, aspiration, vomiting, and bowel obstruction and infarction caused by inspissated charcoal. Charcoal is not recommended for patients who have ingested corrosives because it obscures

1	of the airway, aspiration, vomiting, and bowel obstruction and infarction caused by inspissated charcoal. Charcoal is not recommended for patients who have ingested corrosives because it obscures endoscopy.

1	Gastric lavage should be considered for life-threatening poisons that cannot be treated effectively with other decontamination, elimination, or antidotal therapies (e.g., colchicine). Gastric lavage is performed by sequentially administering and aspirating ~5 mL of fluid per kilogram of body weight through a no. 40 French orogastric tube (no. 28 French tube for children). Except in infants, for whom normal saline is recommended, tap water is acceptable. The patient should be placed in Trendelenburg and left lateral decubitus positions to prevent aspiration (even if an endotracheal tube is in place). Lavage decreases ingestant absorption by an average of 52% if performed within 5 min of ingestion administration, 26% if performed at 30 min, and 16% if performed at 60 min. Significant amounts of ingested drug are recovered from <10% of patients. Aspiration is a common complication (occurring in up to 10% of patients), especially when lavage is performed improperly. Serious complications

1	of ingested drug are recovered from <10% of patients. Aspiration is a common complication (occurring in up to 10% of patients), especially when lavage is performed improperly. Serious complications (esophageal and gastric perforation, tube misplacement in the trachea) occur in ~1% of patients. For this reason, the physician should personally insert the lavage tube and confirm its placement, and the patient must be cooperative during the procedure. Gastric lavage is contraindicated in corrosive or petroleum distillate ingestions because of the respective risks of gastroesophageal perforation and aspiration pneumonitis. It is also contraindicated in patients with a compromised unprotected airway and those at risk for hemorrhage or perforation due to esophageal or gastric pathology or recent surgery. Finally, gastric lavage is absolutely contraindicated in combative patients or those who refuse, as most published complications involve patient resistance to the procedure.

1	Syrup of ipecac, an emetogenic agent that was once the substance most commonly used for decontamination, no longer has a role in poisoning management. Even the American Academy of Pediatrics—traditionally the strongest proponent of ipecac—issued a policy statement in 2003 recommending that ipecac should no longer be used in poisoning treatment. Chronic ipecac use (by patients with anorexia nervosa or bulimia) has been reported to cause electrolyte and fluid abnormalities, cardiac toxicity, and myopathy.

1	Whole-bowel irrigation is performed by administering a bowel-cleansing solution containing electrolytes and polyethylene glycol (Golytely, Colyte) orally or by gastric tube at a rate of 2 L/h (0.5 L/h in children) until rectal effluent is clear. The patient must be in a sitting position. Although data are limited, whole-bowel irrigation appears to be as effective as other decontamination procedures in volunteer studies. It is most appropriate for those who have ingested foreign bodies, packets of illicit drugs, and agents that are poorly adsorbed by charcoal (e.g., heavy metals). This procedure is contraindicated in patients with bowel obstruction, ileus, hemodynamic instability, and compromised unprotected airways.

1	Cathartics are salts (disodium phosphate, magnesium citrate and sulfate, sodium sulfate) or saccharides (mannitol, sorbitol) that historically have been given with activated charcoal to promote the rectal evacuation of gastrointestinal contents. However, no animal, volunteer, or clinical data have ever demonstrated any decontamination benefit from cathartics. Abdominal cramps, nausea, and occasional vomiting are side effects. Complications of repeated dosing include severe electrolyte disturbances and excessive diarrhea. Cathartics are contraindicated in patients who have ingested corrosives and in those with preexisting diarrhea. Magnesium-containing cathartics should not be used in patients with renal failure.

1	Dilution (i.e., drinking water, another clear liquid, or milk at a volume of 5 mL/kg of body weight) is recommended only after the ingestion of corrosives (acids, alkali). It may increase the dissolution rate (and hence absorption) of capsules, tablets, and other solid ingestants and should not be used in these circumstances. Endoscopic or surgical removal of poisons may be useful in rare situations, such as ingestion of a potentially toxic foreign body that fails to transit the gastrointestinal tract, a potentially lethal amount of a heavy metal (arsenic, iron, mercury, thallium), or agents that have coalesced into gastric concretions or bezoars (heavy metals, lithium, salicylates, sustained-release preparations). Patients who become toxic from cocaine due to its leakage from ingested drug packets require immediate surgical intervention.

1	Decontamination of Other Sites Immediate, copious flushing with water, saline, or another available clear, drinkable liquid is the initial treatment for topical exposures (exceptions include alkali metals, calcium oxide, phosphorus). Saline is preferred for eye irrigation. A triple wash (water, soap, water) may be best for dermal decontamination. Inhalational exposures should be treated initially with fresh air or oxygen. The removal of liquids from body cavities such as the vagina or rectum is best accomplished by irrigation. Solids (drug packets, pills) should be removed manually, preferably under direct visualization.

1	Although the elimination of most poisons can be accelerated by therapeutic interventions, the pharmacokinetic efficacy (removal of drug at a rate greater than that accomplished by intrinsic elimination) and clinical benefit (shortened duration of toxicity or improved outcome) of such interventions are often more theoretical than proven. Accordingly, the decision to use such measures should be based on the actual or predicted toxicity and the potential efficacy, cost, and risks of therapy.

1	Multiple-Dose Activated Charcoal Repetitive oral dosing with charcoal can enhance the elimination of previously absorbed substances by binding them within the gut as they are excreted in the bile, are secreted by gastrointestinal cells, or passively diffuse into the gut lumen (reverse absorption or enterocapillary exsorption). Doses of 0.5–1 g/kg of body weight every 2–4 h, adjusted downward to avoid regurgitation in patients with decreased gastrointestinal motility, are generally recommended. Pharmacokinetic efficacy approaches that of hemodialysis for some agents (e.g., phenobarbital, theophylline). Multiple-dose therapy should be considered only for selected agents (theophylline, phenobarbital, carbamazepine, dapsone, quinine). Complications include intestinal obstruction, pseudo-obstruction, and nonocclusive intestinal infarction in patients with decreased gut motility. Because of electrolyte and fluid shifts, sorbitol and other cathartics are absolutely contraindicated when

1	and nonocclusive intestinal infarction in patients with decreased gut motility. Because of electrolyte and fluid shifts, sorbitol and other cathartics are absolutely contraindicated when multiple doses of activated charcoal are administered.

1	Urinary Alkalinization Ion trapping via alteration of urine pH may prevent the renal reabsorption of poisons that undergo excretion by glomerular filtration and active tubular secretion. Since membranes are more permeable to non-ionized molecules than to their ionized counterparts, acidic (low-pK a) poisons are ionized and trapped in alkaline urine, whereas basic ones become ionized and trapped in acid urine. Urinary alkalinization (producing a urine pH ≥7.5 and a urine output of 3–6 mL/kg of body weight per hour by the addition of sodium bicarbonate to an IV solution) enhances the excretion of chlorophenoxyacetic acid herbicides, chlorpropamide, diflunisal, fluoride, methotrexate, phenobarbital, sulfonamides, and salicylates. Contraindications include congestive heart failure, renal failure, and cerebral edema. Acid-base, fluid, and electrolyte parameters should be monitored carefully. Although acid diuresis may make theoretical sense for some overdoses (amphetamines), it is never

1	and cerebral edema. Acid-base, fluid, and electrolyte parameters should be monitored carefully. Although acid diuresis may make theoretical sense for some overdoses (amphetamines), it is never indicated and is potentially harmful.

1	Extracorporeal Removal Hemodialysis, charcoal or resin hemoperfusion, hemofiltration, plasmapheresis, and exchange transfusion are capable of removing any toxin from the bloodstream. Agents most amenable to enhanced elimination by dialysis have low molecular mass (<500 Da), high water solubility, low protein binding, small volumes of distribution (<1 L/kg of body weight), prolonged elimination (long half-life), and high dialysis clearance relative to total-body clearance. Molecular weight, water solubility, and protein binding do not limit the efficacy of the other forms of extracorporeal removal.

1	Dialysis should be considered in cases of severe poisoning due to carbamazepine, ethylene glycol, isopropyl alcohol, lithium, methanol, theophylline, salicylates, and valproate. Although hemoperfusion may be more effective in removing some of these poisons, it does not correct associated acid-base and electrolyte abnormalities, and most hospitals no longer have hemoperfusion cartridges readily available. Fortunately, recent advances in hemodialysis technology make it as effective as hemoperfusion for removing poisons such as caffeine, carbamazepine, and theophylline. Both techniques require central venous access and systemic anticoagulation and may result in transient hypotension. Hemoperfusion may also cause hemolysis, hypocalcemia, and thrombocytopenia.

1	Peritoneal dialysis and exchange transfusion are less effective but 473e-7 may be used when other procedures are unavailable, contraindicated, or technically difficult (e.g., in infants). Exchange transfusion may be indicated in the treatment of severe arsineor sodium chlorate–induced hemolysis, methemoglobinemia, and sulfhemoglobinemia. Although hemofiltration can enhance elimination of aminoglycosides, vancomycin, and metal-chelate complexes, the roles of hemofiltration and plasmapheresis in the treatment of poisoning are not yet defined. Candidates for extracorporeal removal therapies include patients with severe toxicity whose condition deteriorates despite aggressive supportive therapy; those with potentially prolonged, irreversible, or fatal toxicity; those with dangerous blood levels of toxins; those who lack the capacity for self-detoxification because of liver or renal failure; and those with a serious underlying illness or complication that will adversely affect recovery.

1	Other Techniques The elimination of heavy metals can be enhanced by chelation, and the removal of carbon monoxide can be accelerated by hyperbaric oxygenation.

1	Antidotes counteract the effects of poisons by neutralizing them (e.g., antibody-antigen reactions, chelation, chemical binding) or by antagonizing their physiologic effects (e.g., activation of opposing nervous system activity, provision of a competitive metabolic or receptor substrate). Poisons or conditions with specific antidotes include acetaminophen, anticholinergic agents, anticoagulants, benzodiazepines, beta blockers, calcium channel blockers, carbon monoxide, cardiac glycosides, cholinergic agents, cyanide, drug-induced dystonic reactions, ethylene glycol, fluoride, heavy metals, hypoglycemic agents, isoniazid, membrane-active agents, methemoglobinemia, opioids, sympathomimetics, and a variety of envenomations. Intravenous lipid emulsion has been shown to be a successful antidote for poisoning from various anesthetics and membrane-active agents (e.g., cyclic antidepressants), but the exact mechanism of benefit is still under investigation. Antidotes can significantly reduce

1	for poisoning from various anesthetics and membrane-active agents (e.g., cyclic antidepressants), but the exact mechanism of benefit is still under investigation. Antidotes can significantly reduce morbidity and mortality rates but are potentially toxic if used for inappropriate reasons. Since their safe use requires correct identification of a specific poisoning or syndrome, details of antidotal therapy are discussed with the conditions for which they are indicated (Table 473e-4).

1	Poisoning is a preventable illness. Unfortunately, some adults and children are poison-prone, and recurrences are common. Unintentional polypharmacy poisoning has become especially common among adults with developmental delays, among the growing population of geriatric patients who are prescribed a large number of medications, and among adolescents and young adults experimenting with pharmaceuticals for recreational euphoria. Adults with unintentional exposures should be instructed regarding the safe use of medications and chemicals (according to labeling instructions). Confused patients may need assistance with the administration of their medications. Errors in dosing by health care providers may require educational efforts. Patients should be advised to avoid circumstances that result in chemical exposure or poisoning. Appropriate agencies and health departments should be notified in cases of environmental or workplace exposure. The best approach to young children and patients with

1	chemical exposure or poisoning. Appropriate agencies and health departments should be notified in cases of environmental or workplace exposure. The best approach to young children and patients with intentional overdose (deliberate self-harm or attempted suicide) is to limit their access to poisons. In households where children live or visit, alcoholic beverages, medications, household products (automotive, cleaning, fuel, pet-care, and toiletry products), inedible plants, and vitamins should be kept out of reach or in locked or child-proof cabinets. Depressed or psychotic patients should undergo psychiatric assessment, disposition, and follow-up. They should be given prescriptions for a limited supply of drugs with a limited number of refills and should be monitored for compliance and response to therapy.

1	Physiologic Condition, Causes Examples Mechanism of Action Clinical Features Specific Treatments PART 18 Poisoning, Drug Overdose, and Envenomation Sympathomimetics α1-Adrenergic agonists (decongestants): phenylephrine, phenylpropanolamine β2-Adrenergic agonists (bronchodilators): albuterol, terbutaline Nonspecific adrenergic agonists: amphetamines, cocaine, ephedrine Ergot alkaloids Ergotamine, methysergide, bromocriptine, pergolide Methylxanthines Caffeine, theophylline Monoamine oxidase Phenelzine, tranylcypromine, inhibitors selegiline Antihistamines Diphenhydramine, doxylamine, pyrilamine Antipsychotics Chlorpromazine, olanzapine, quetiapine, thioridazine Belladonna alkaloids Atropine, hyoscyamine, scopolamine Stimulation of central and peripheral sympathetic receptors directly or indirectly (by promoting release or inhibiting reuptake of norepinephrine and sometimes dopamine)

1	Stimulation and inhibition of serotonergic and α-adrenergic receptors; stimulation of dopamine receptors Inhibition of adenosine synthesis and adenosine receptor antagonism; stimulation of epinephrine and norepinephrine release; inhibition of phosphodiesterase resulting in increased intracellular cyclic adenosine and guanosine monophosphate Inhibition of monoamine oxidase resulting in impaired metabolism of endogenous catecholamines and exogenous sympathomimetic agents Inhibition of central and post-ganglionic parasympathetic muscarinic cholinergic receptors. At high doses, amantadine, diphenhydramine, orphenadrine, phenothiazines, and tricyclic antidepressants have additional nonanticholinergic activity (see below). Inhibition of α-adrenergic, dopaminergic, histaminergic, muscarinic, and serotonergic receptors. Some agents also inhibit sodium, potassium, and calcium channels.

1	Inhibition of α-adrenergic, dopaminergic, histaminergic, muscarinic, and serotonergic receptors. Some agents also inhibit sodium, potassium, and calcium channels. Inhibition of central and postganglionic parasympathetic muscarinic cholinergic receptors Physiologic stimulation (Table 473e-2). Reflex bradycardia can occur with selective α1 agonists; β agonists can cause hypotension and hypokalemia. Physiologic stimulation (Table 473e-2); formication; vasospasm with limb (isolated or generalized), myocardial, and cerebral ischemia progressing to gangrene or infarction. Hypotension, bradycardia, and involuntary movements can also occur. Physiologic stimulation (Table 473e-2); pronounced gastrointestinal symptoms and β agonist effects (see above). Toxicity occurs at lower drug levels in chronic poisoning than in acute poisoning.

1	Physiologic stimulation (Table 473e-2); pronounced gastrointestinal symptoms and β agonist effects (see above). Toxicity occurs at lower drug levels in chronic poisoning than in acute poisoning. Physiologic stimulation (Table 473e-2); dry skin and mucous membranes, decreased bowel sounds, flushing, and urinary retention; myoclonus and picking activity. Central effects may occur without significant autonomic dysfunction. Physiologic depression (Table 473e-2), miosis, anticholinergic effects (see above), extrapyramidal reactions (see below), tachycardia Physiologic stimulation (Table 473e-2); dry skin and mucous membranes, decreased bowel sounds, flushing, and urinary retention; myoclonus and picking activity. Central effects may occur without significant autonomic dysfunction.

1	Phentolamine, a nonselective α1-adrenergic receptor antagonist, for severe hypertension due to α1-adrenergic agonists; propranolol, a nonselective β blocker, for hypotension and tachycardia due to β2 agonists; either labetalol, a β blocker with α-blocking activity, or phentolamine with esmolol, metoprolol, or another cardioselective β blocker for hypertension with tachycardia due to non-selective agents (β blockers, if used alone, can exacerbate hypertension and vasospasm due to unopposed α stimulation.); benzodiazepines; propofol Nitroprusside or nitroglycerine for severe vasospasm; prazosin (an α1 blocker), captopril, nifedipine, and cyproheptadine (a serotonin receptor antagonist) for mild-to-moderate limb ischemia; dopamine receptor antagonists (antipsychotics) for hallucinations and movement disorders

1	Propranolol, a nonselective β blocker, for tachycardia with hypotension; any β blocker for supraventricular or ventricular tachycardia without hypotension; elimination enhanced by multiple-dose charcoal, hemoperfusion, and hemodialysis. Indications for hemoperfusion or hemodialysis include unstable vital signs, seizures, and a theophylline level of 80–100 μg/mL after an acute overdose and 40–60 μg/mL with chronic exposure. Short-acting agents (e.g., nitroprusside, esmolol) for severe hypertension and tachycardia; direct-acting sympathomimetics (e.g., norepinephrine, epinephrine) for hypotension and bradycardia Physostigmine, an acetylcholinesterase inhibitor (see below), for delirium, hallucinations, and neuromuscular hyperactivity. Contraindications include asthma and nonanticholinergic cardiovascular toxicity (e.g., cardiac conduction abnormalities, hypotension, and ventricular arrhythmias).

1	Sodium bicarbonate for ventricular tachydysrhythmias associated with QRS prolongation; magnesium, isoproterenol, and overdrive pacing for torsades des pointes. Avoid class IA, IC, and III antiarrhythmics. Physostigmine, an acetylcholinesterase inhibitor (see below), for delirium, hallucinations, and neuromuscular hyperactivity. Contraindications include asthma and nonanticholinergic cardiovascular toxicity (e.g., cardiac conduction abnormalities, hypotension, and ventricular arrhythmias). Physiologic Condition, Causes Examples Mechanism of Action Clinical Features Specific Treatments Cyclic antidepres-Amitriptyline, doxepin, sants imipramine Mushrooms and Amanita muscaria and plants A. pantherina, henbane, jimson weed, nightshade α2-Adrenergic Clonidine, guanabenz, agonists tetrahydrozoline and other imidazoline decongestants, tizanidine and other imidazoline muscle relaxants

1	Antipsychotics Chlorpromazine, clozapine, haloperidol, risperidone, thioridazine β-Adrenergic Cardioselective (β1) blockers: blockers atenolol, esmolol, metoprolol Nonselective (β1 and β2) blockers: nadolol, propranolol, timolol Partial β agonists: acebutolol, pindolol α1 Antagonists: carvedilol, labetalol Membrane-active agents: acebutolol, propranolol, sotalol Calcium channel Diltiazem, nifedipine and blockers other dihydropyridine derivatives, verapamil Cardiac glycosides Digoxin, endogenous cardioactive steroids, foxglove and other plants, toad skin secretions (Bufonidae spp.) Inhibition of α-adrenergic, dopaminergic, GABA-ergic, histaminergic, muscarinic, and serotonergic receptors; inhibition of sodium channels (see membrane-active agents); inhibition of norepinephrine and serotonin reuptake Inhibition of central and postganglionic parasympathetic muscarinic cholinergic receptors

1	Inhibition of central and postganglionic parasympathetic muscarinic cholinergic receptors Stimulation of α2-adrenergic receptors leading to inhibition of CNS sympathetic outflow. Activity at nonadrenergic imidazoline binding sites also contributes to CNS effects. Inhibition of α-adrenergic, dopaminergic, histaminergic, muscarinic, and serotonergic receptors. Some agents also inhibit sodium, potassium, and calcium channels. Inhibition of β-adrenergic receptors (class II antiarrhythmic effect). Some agents have activity at additional receptors or have membrane effects (see below). Inhibition of slow (type L) cardiovascular calcium channels (class IV antiarrhythmic effect) Inhibition of cardiac Na+, K+-ATPase membrane pump Physiologic depression (Table 473e-2), seizures, tachycardia, cardiac conduction delays (increased PR, QRS, JT, and QT intervals; terminal QRS right-axis deviation) with aberrancy and ventricular tachydysrhythmias; anticholinergic toxidrome (see above)

1	Physiologic stimulation (Table 473e-2); dry skin and mucous membranes, decreased bowel sounds, flushing, and urinary retention; myoclonus and picking activity. Central effects may occur without significant autonomic dysfunction. Physiologic depression (Table 473e-2), miosis. Transient initial hypertension may be seen. Physiologic depression (Table 473e-2), miosis, anticholinergic effects (see above), extrapyramidal reactions (see below), tachycardia. Cardiac conduction delays (increased PR, QRS, JT, and QT intervals) with ventricular tachydysrhythmias, including torsades des pointes, can sometimes develop. Physiologic depression (Table 473e-2), atrioventricular block, hypoglycemia, hyperkalemia, seizures. Partial agonists can cause hypertension and tachycardia. Sotalol can cause increased QT interval and ventricular tachydysrhythmias. Onset may be delayed after sotalol and sustained-release formulation overdose.

1	Physiologic depression (Table 473e-2), atrioventricular block, organ ischemia and infarction, hyperglycemia, seizures. Hypotension is usually due to decreased vascular resistance rather than to decreased cardiac output. Onset may be delayed for ≥12 h after overdose of sustained-release formulations. Physiologic depression (Table 473e-2); gastrointestinal, psychiatric, and visual symptoms; atrioventricular block with or without concomitant supra-ventricular tachyarrhythmia; ventricular tachyarrhythmias; hyperkalemia in acute poisoning. Toxicity occurs at lower drug levels in chronic poisoning than in acute poisoning. Hypertonic sodium bicarbonate (or hypertonic saline) for ventricular tachydysrhythmias associated with QRS prolongation. Use of phenytoin is controversial. Avoid class IA, IC, and III antiarrhythmics. IV emulsion therapy may be beneficial in some cases.

1	Physostigmine, an acetylcholinesterase inhibitor (see below), for delirium, hallucinations, and neuromuscular hyperactivity. Contraindications include asthma and nonanticholinergic cardiovascular toxicity (e.g., cardiac conduction abnormalities, hypo-tension, and ventricular arrhythmias). Sodium bicarbonate for ventricular tachydysrhythmias associated with QRS prolongation; magnesium, isoproterenol, and overdrive pacing for torsades des pointes. Avoid class IA, IC, and III antiarrhythmics. Glucagon for hypotension and symptomatic bradycardia. Atropine, isoproterenol, dopamine, dobutamine, epinephrine, and norepinephrine may sometimes be effective. High-dose insulin (with glucose and potassium to maintain euglycemia and normokalemia), electrical pacing, and mechanical cardiovascular support for refractory cases

1	Calcium and glucagon for hypotension and symptomatic bradycardia. Dopamine, epinephrine, norepinephrine, atropine, and isoproterenol are less often effective but can be used adjunctively. High-dose insulin (with glucose and potassium to maintain euglycemia and normokalemia), IV lipid emulsion therapy, electrical pacing, and mechanical cardiovascular support for refractory cases Digoxin-specific antibody fragments for hemodynamically compromising dysrhythmias, Mobitz II or third-degree atrioventricular block, hyperkalemia (>5.5 meq/L; in acute poisoning only). Temporizing measures include atropine, dopamine, epinephrine, and external cardiac pacing for bradydysrhythmias and magnesium, lidocaine, or phenytoin, for ventricular tachydysrhythmias. Internal cardiac pacing and cardioversion can increase ventricular irritability and should be reserved for refractory cases. Physiologic Condition, Causes Examples Mechanism of Action Clinical Features Specific Treatments

1	Physiologic Condition, Causes Examples Mechanism of Action Clinical Features Specific Treatments PART 18 Poisoning, Drug Overdose, and Envenomation Cyclic antidepres-Amitriptyline, doxepin, sants imipramine esterase inhibitors carb, carbaryl, propoxur) and medicinals (neostigmine, physostigmine, tacrine); nerve gases (sarin, soman, tabun, VX); organophosphate insecticides (diazinon, chlorpyrifosethyl, malathion) Muscarinic agonists Bethanechol, mushrooms (Boletus, Clitocybe, Inocybe spp.), pilocarpine Nicotinic agonists Lobeline, nicotine (tobacco) Anticonvulsants Carbamazepine, ethosuximide, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, phenytoin, tiagabine, topiramate, valproate, zonisamide Barbiturates Short-acting: butabarbital, pentobarbital, secobarbital Long-acting: phenobarbital, primidone Benzodiazepines Ultrashort-acting: estazolam, midazolam, temazepam, triazolam Short-acting: alprazolam, flunitrazepam, lorazepam, oxazepam

1	Long-acting: phenobarbital, primidone Benzodiazepines Ultrashort-acting: estazolam, midazolam, temazepam, triazolam Short-acting: alprazolam, flunitrazepam, lorazepam, oxazepam Long-acting: chlordiazepoxide, clonazepam, diazepam, flurazepam Pharmacologically related agents: zaleplon, zolpidem GABA precursors γ-Hydroxybutyrate (sodium oxybate; GHB), γ-butyrolactone (GBL), 1,4-butanediol Muscle relaxants Baclofen, carisoprodol, cyclobenzaprine, etomidate, metaxalone, methocarbamol, orphenadrine, propofol, tizanidine and other imidazoline muscle relaxants Inhibition of α-adrenergic, dopaminergic, GABA-ergic, histaminergic, muscarinic, and serotonergic receptors; inhibition of sodium channels (see membrane-active agents); inhibition of norepinephrine and serotonin reuptake Inhibition of acetylcholinesterase leading to increased synaptic acetylcholine at muscarinic and nicotinic cholinergic receptor sites

1	Inhibition of acetylcholinesterase leading to increased synaptic acetylcholine at muscarinic and nicotinic cholinergic receptor sites Stimulation of CNS and post-ganglionic parasympathetic cholinergic (muscarinic) receptors Stimulation of preganglionic sympathetic and parasympathetic and striated muscle (neuromuscular junction) cholinergic (nicotine) receptors

1	Stimulation of preganglionic sympathetic and parasympathetic and striated muscle (neuromuscular junction) cholinergic (nicotine) receptors Potentiation of the inhibitory effects of GABA by binding to the neuronal GABA–A chloride channel receptor complex and increasing the frequency or duration of chloride channel opening in response to GABA stimulation. Baclofen and, to some extent, GHB act at the GABA–B receptor complex. Meprobamate, its metabolite carisoprodol, felbamate, and orphenadrine antagonize NDMA excitatory receptors. Ethosuximide, valproate, and zonisamide decrease conduction through T-type calcium channels. Valproate decreases GABA degradation, and tiagabine blocks GABA reuptake. Carbamazepine, lamotrigine, oxcarbazepine, phenytoin, topiramate, valproate, and zonisamide slow the rate of recovery of inactivated sodium channels. Some agents also have α2 agonist, anticholinergic, and sodium channel–blocking activity (see above and below).

1	Physiologic depression (Table 473e-2), seizures, tachycardia, cardiac conduction delays (increased PR, QRS, JT, and QT intervals; terminal QRS right-axis deviation) with aberrancy and ventricular tachydysrhythmias; anticholinergic toxidrome (see above) Physiologic depression (Table 473e-2). Muscarinic signs and symptoms: seizures, excessive secretions (lacrimation, salivation, bronchorrhea and wheezing, diaphoresis), and increased bowel and bladder activity with nausea, vomiting, diarrhea, abdominal cramps, and incontinence of feces and urine. Nicotinic signs and symptoms: hypertension, tachycardia, muscle cramps, fasciculations, weakness, and paralysis. Death is usually due to respiratory failure. Cholinesterase activity in plasma and red cells is <50% of normal in acetylcholinesterase inhibitor poisoning. Physiologic depression (Table 473e-2), nystagmus. Delayed absorption can occur with carbamazepine, phenytoin, and valproate.

1	Physiologic depression (Table 473e-2), nystagmus. Delayed absorption can occur with carbamazepine, phenytoin, and valproate. Myoclonus, seizures, hypertension, and tachyarrhythmias can occur with baclofen, carbamazepine, and orphenadrine. Tachyarrhythmias can also occur with chloral hydrate. AGMA, hypernatremia, hyperosmolality, hyperammonemia, chemical hepatitis, and hypoglycemia can be seen in valproate poisoning. Carbamazepine and oxcarbazepine may produce hyponatremia from SIADH. Some agents can cause anticholinergic and sodium channel (membrane) blocking effects (see above and below). Hypertonic sodium bicarbonate (or hypertonic saline) for ventricular tachydysrhythmias associated with QRS prolongation. Use of phenytoin is controversial. Avoid class IA, IC, and III antiarrhythmics. IV emulsion therapy may be beneficial in some cases.

1	Atropine for muscarinic signs and symptoms; 2-PAM, a cholinesterase reactivator, for nicotinic signs and symptoms due to organophosphates, nerve gases, or an unknown anticholinesterase Benzodiazepines, barbiturates, or propofol for seizures. Hemodialysis and hemoperfusion may be indicated for severe poisoning by some agents (see “Extracorporeal Removal,” in text). See above and below for treatment of anticholinergic and sodium channel (membrane)–blocking effects. Physiologic Condition, Causes Examples Mechanism of Action Clinical Features Specific Treatments Other agents Chloral hydrate, ethchlorvynol, glutethimide, meprobamate, methaqualone, methyprylon Cytochrome oxidase Cyanide, hydrogen sulfide inhibitors

1	Other agents Chloral hydrate, ethchlorvynol, glutethimide, meprobamate, methaqualone, methyprylon Cytochrome oxidase Cyanide, hydrogen sulfide inhibitors Inhibition of mitochondrial cytochrome oxidase, with consequent blockage of electron transport and oxidative metabolism. Carbon monoxide also binds to hemoglobin and myoglobin and prevents oxygen binding, transport, and tissue uptake. (Binding to hemoglobin shifts the oxygen dissociation curve to the left.) Oxidation of hemoglobin iron from ferrous (Fe2+) to ferric (Fe3+) state prevents oxygen binding, transport, and tissue uptake. (Methemoglobinemia shifts oxygen dissociation curve to the left.) Oxidation of hemoglobin protein causes hemoglobin precipitation and hemolytic anemia (manifesting as Heinz bodies and "bite cells" on peripheral-blood smear).

1	Ethylene glycol causes CNS depression and increased serum osmolality. Metabolites (primarily glycolic acid) cause AGMA, CNS depression, and renal failure. Precipitation of oxalic acid metabolite as calcium salt in tissues and urine results in hypocalcemia, tissue edema, and crystalluria. Hydration of ferric (Fe3+) ion generates H+. Non-transferrinbound iron catalyzes formation of free radicals that cause mitochondrial injury, lipid peroxidation, increased capillary permeability, vasodilation, and organ toxicity.

1	Signs and symptoms of hypoxemia with initial physiologic stimulation and subsequent depression (Table 473e-2); lactic acidosis; normal Po2 and calculated oxygen saturation but decreased oxygen saturation by cooximetry. (That measured by pulse oximetry is falsely elevated but is less than normal and less than the calculated value.) Headache and nausea are common with carbon monoxide. Sudden collapse may occur with cyanide and hydrogen sulfide exposure. A bitter almond breath odor may be noted with cyanide ingestion, and hydrogen sulfide smells like rotten eggs.

1	Signs and symptoms of hypoxemia with initial physiologic stimulation and subsequent depression (Table 473e-2), gray-brown cyanosis unresponsive to oxygen at methemoglobin fractions >15–20%, headache, lactic acidosis (at methemoglobin fractions >45%), normal Po2 and calculated oxygen saturation but decreased oxygen saturation and increased methemoglobin fraction by co-oximetry (Oxygen saturation by pulse oximetry may be falsely increased or decreased but is less than normal and less than the calculated value.) Initial ethanol-like intoxication, nausea, vomiting, increased osmolar gap, calcium oxylate crystalluria; delayed AGMA, back pain, renal failure; coma, seizures, hypotension, ARDS in severe cases Initial nausea, vomiting, abdominal pain, diarrhea; AGMA, cardiovascular and CNS depression, hepatitis, coagulopathy, and seizures in severe cases. Radiopaque iron tablets may be seen on abdominal x-ray.

1	High-dose oxygen; IV hydroxocobalamin or IV sodium nitrite and sodium thiosulfate (Lilly cyanide antidote kit) for coma, metabolic acidosis, and cardiovascular dysfunction in cyanide poisoning High-dose oxygen; IV methylene blue for methemoglobin fraction >30%, symptomatic hypoxemia, or ischemia (contraindicated in G6PD deficiency); exchange transfusion and hyperbaric oxygen for severe or refractory cases Sodium bicarbonate to correct acidemia; thiamine, folinic acid, magnesium, and high-dose pyridoxine to facilitate metabolism; ethanol or fomepizole for AGMA, crystalluria or renal dysfunction, ethylene glycol level >3 mmol/L (20 mg/ dL), and ethanol-like intoxication or increased osmolal gap if level not readily obtainable; hemodialysis for persistent AGMA, lack of clinical improvement, and renal dysfunction; hemodialysis also useful for enhancing ethylene glycol elimination and shortening duration of treatment when ethylene glycol level is >8 mmol/L (50 mg/dL)

1	Whole-bowel irrigation for large ingestions; endoscopy and gastrostomy if clinical toxicity and large number of tablets are still visible on x-ray; IV hydration; sodium bicarbonate for acidemia; IV deferoxamine for systemic toxicity, iron level >90 μmol/L (500 μg/dL) Physiologic Condition, Causes Examples Mechanism of Action Clinical Features Specific Treatments PART 18 Poisoning, Drug Overdose, and Envenomation Methanol Methanol causes ethanol-like CNS depression and increased serum osmolality. Formic acid metabolite causes AGMA and retinal toxicity. Initial ethanol-like intoxication, nausea, vomiting, increased osmolar gap; delayed AGMA, visual (clouding, spots, blindness) and retinal (edema, hyperemia) abnormalities; coma, seizures, cardiovascular depression in severe cases; possible pancreatitis

1	Initial nausea, vomiting, hyperventilation, alkalemia, alkaluria; subsequent alkalemia with both respiratory alkalosis and AGMA and paradoxical aciduria; late acidemia with CNS and respiratory depression; cerebral and pulmonary edema in severe cases. Hypoglycemia, hypocalcemia, hypokalemia, and seizures can occur. Akathisia, dystonia, parkinsonism Nausea, vomiting, agitation, confusion; coma, respiratory depression, seizures, lactic and ketoacidosis in severe cases Nausea, vomiting, diarrhea, ataxia, choreoathetosis, encephalopathy, hyperreflexia, myoclonus, nystagmus, nephrogenic diabetes insipidus, falsely elevated serum chloride with low anion gap, tachycardia; coma, seizures, arrhythmias, hyperthermia, and prolonged or permanent encephalopathy and movement disorders in severe cases; delayed onset after acute overdose, particularly with delayed-release formulations. Toxicity occurs at lower drug levels in chronic poisoning than in acute poisoning.

1	Gastric aspiration for recent ingestion; sodium bicarbonate to correct acidemia; high-dose folinic acid or folate to facilitate metabolism; ethanol or fomepizole for AGMA, visual symptoms, methanol level >6 mmol/L (20 mg/dL), and ethanol-like intoxication or increased osmolal gap if level not readily obtainable; hemodialysis for persistent AGMA, lack of clinical improvement, and renal dysfunction; hemodialysis also useful for enhancing methanol elimination and shortening duration of treatment when methanol level is >15 mmol/L (50 mg/dL) IV hydration and supplemental glucose; sodium bicarbonate to correct acidemia; urinary alkalinization for systemic toxicity; hemodialysis for coma, cerebral edema, seizures, pulmonary edema, renal failure, progressive acid-base disturbances or clinical toxicity, salicylate level >7 mmol/L (100 mg/dL) following acute overdose Oral or parenteral anticholinergic agent such as benztropine or diphenhydramine

1	Oral or parenteral anticholinergic agent such as benztropine or diphenhydramine High-dose IV pyridoxine (vitamin B6) for agitation, confusion, coma, and seizures; diazepam or barbiturates for seizures Whole-bowel irrigation for large ingestions; IV hydration; hemodialysis for coma, seizures, encephalopathy or neuromuscular dysfunction (severe, progressive, or persistent), peak lithium level >4 meq/L following acute overdose Physiologic Condition, Causes Examples Mechanism of Action Clinical Features Specific Treatments Serotonin syndrome Amphetamines, cocaine, dextromethorphan, meperidine, MAO inhibitors, selective serotonin (5-HT) reuptake inhibitors, tricyclic antidepressants, tramadol, triptans, tryptophan

1	Serotonin syndrome Amphetamines, cocaine, dextromethorphan, meperidine, MAO inhibitors, selective serotonin (5-HT) reuptake inhibitors, tricyclic antidepressants, tramadol, triptans, tryptophan Membrane-active Amantadine, anti-arrhythmics agents (class I and III agents; some beta blockers), antipsychotics (see above), antihistamines (particularly diphenhydramine), carbamazepine, local anesthetics (including cocaine), opioids (meperidine, propoxyphene), orphenadrine, quinoline antimalarials (chloroquine, hydroxychloroquine, quinine), cyclic antidepressants (see above) aSee above and Chap. 469e. bSee above and Chap. 468e. Promotion of serotonin release, inhibition of serotonin reuptake, or direct stimulation of CNS and peripheral serotonin receptors (primarily 5-HT-1a and 5-HT-2), alone or in combination

1	Promotion of serotonin release, inhibition of serotonin reuptake, or direct stimulation of CNS and peripheral serotonin receptors (primarily 5-HT-1a and 5-HT-2), alone or in combination Blockade of fast sodium membrane channels prolongs phase 0 (depolarization) of the cardiac action potential, which prolongs QRS duration and promotes reentrant (monomorphic) ventricular tachycardia. Class Ia, Ic, and III antiarrhythmics also block potassium channels during phases 2 and 3 (repolarization) of the action potential, prolonging the JT interval and promoting early after-depolarizations and polymorphic (tor-sades des pointes) ventricular tachycardia. Similar effects on neuronal membrane channels cause CNS dysfunction. Some agents also block α-adrenergic and cholinergic receptors or have opioid effects (see above and Chap. 468e).

1	Altered mental status (agitation, confusion, mutism, coma, seizures), neuromuscular hyperactivity (hyperreflexia, myoclonus, rigidity, tremors), and autonomic dysfunction (abdominal pain, diarrhea, diaphoresis, fever, flushing, labile hypertension, mydriasis, tearing, salivation, tachycardia). Complications include hyperthermia, lactic acidosis, rhabdomyolysis, and multisystem organ failure. QRS and JT prolongation (or both) with hypotension, ventricular tachyarrhythmias, CNS depression, seizures; anticholinergic effects with amantadine, antihistamines, carbamazepine, disopyramide, antipsychotics, and cyclic antidepressants (see above); opioid effects with meperidine and propoxyphene (see Chap. 468e); cinchonism (hearing loss, tinnitus, nausea, vomiting, vertigo, ataxia, headache, flushing, diaphoresis) and blindness with quinoline antimalarials Discontinue the offending agent(s); the serotonin receptor antagonist cyproheptadine may be helpful in severe cases.

1	Hypertonic sodium bicarbonate (or hypertonic saline) for cardiac conduction delays and monomorphic ventricular tachycardia; lidocaine for monomorphic ventricular tachycardia (except when due to class Ib antiarrhythmics); magnesium, isoproterenol, and overdrive pacing for polymorphic ventricular tachycardia; physostigmine for anticholinergic effects (see above); naloxone for opioid effects (see Chap. 468e); extra-corporeal removal for some agents (see text). Abbreviations: AGMA, anion-gap metabolic acidosis; ARDS, adult respiratory distress syndrome; CNS, central nervous system; GABA, γ-aminobutyric acid; GBL, γ-butyrolactone; GHB, γ-hydroxybutyrate; G6PD, glucose-6-phosphate dehydrogenase; MAO, monoamine oxidase; NDMA, N-methyl-D-aspartate; 2-PAM, pralidoxime; SIADH, syndrome of inappropirate antidiuretic hormone secretion.

1	Table 473e-4 summarizes the pathophysiology, clinical features, and treatment of toxidromes and poisonings that are common, produce life-threatening toxicity, or require unique therapeutic interventions. In all cases, treatment should include attention to the general principles discussed above and, in particular, supportive care. Poisonings not covered in this chapter are discussed in specialized texts. Alcohol, cocaine, hallucinogen, and opioid poisoning and alcohol and opioid withdrawal are discussed in Chaps. 467–469e; nicotine addiction is discussed in Chap. 470; acetaminophen poisoning is discussed in Chap. 361; the neuroleptic malignant syndrome is discussed in Chap. 449; and heavy metal poisoning is discussed in Chap. 472e. The author acknowledges the contributions of Christopher H. Linden and Michael J. Burns to this chapter in previous editions of this text. Part 18: Poisoning, Drug Overdose, and Envenomation Exposures Robert L. Norris 474

1	Part 18: Poisoning, Drug Overdose, and Envenomation Exposures Robert L. Norris 474 Charles Lei, Natalie J. Badowski, Paul S. Auerbach, This chapter outlines general principles for the evaluation and management of victims of envenomation and poisoning by venomous snakes and marine animals. Because the incidence of serious bites and stings is relatively low in developed nations, there is a paucity of relevant clinical research; as a result, therapeutic decision making often is based on anecdotal information.

1	The venomous snakes of the world belong to the families Viperidae (subfamily Viperinae: Old World vipers; subfamily Crotalinae: New World and Asian pit vipers), Elapidae (including cobras, coral snakes, sea snakes, kraits, and all Australian venomous snakes), Lamprophiidae (subfamily Atractaspidinae: burrowing asps), and Colubridae (a large family in which most species are nonvenomous and only a few are dangerously toxic to humans). Most snakebites occur in developing countries with temperate and tropical climates in which populations subsist on agriculture and fishing. Recent estimates indicate that somewhere between 1.2 million and 5.5 million snakebites occur worldwide each year, with 421,000–1,841,000 envenomations and 20,000–94,000 deaths. Such wide-ranging estimates reflect the challenges of collecting accurate data in the regions most affected by venomous snakes; many victims do not seek hospital treatment, and reporting and record keeping are generally poor.

1	The typical snake venom delivery apparatus consists of bilateral venom glands situated below and behind the eyes and connected by ducts to hollow anterior maxillary fangs. In viperids (vipers and pit vipers), these fangs are long and highly mobile; they are retracted against the roof of the mouth when the snake is at rest and brought to an upright position for a strike. In elapids, the fangs are smaller and are relatively fixed in an erect position. Approximately 20% of pit viper bites and higher percentages of other snakebites (up to 75% for sea snakes) are “dry” bites, meaning no venom is released. Significant envenomation probably occurs in ~50% of all venomous snakebites.

1	Differentiation of venomous from nonvenomous snake species can be difficult. Viperids are characterized by somewhat triangular heads (a feature shared with many harmless snakes), elliptical pupils (also seen in some nonvenomous snakes, such as boas and pythons), enlarged maxillary fangs, and, in pit vipers, heat-sensing pits (foveal organs) on each side of the head that assist with locating prey and aiming strikes. The New World rattlesnakes possess a series of interlocking keratin plates (the rattle) on the tip of the tail that emits a buzzing sound when the snake rapidly vibrates its tail; this sound serves as a warning signal to perceived threats. Identifying venomous snakes by color pattern is notoriously misleading, as many harmless snakes have color patterns that closely mimic those of venomous snakes found in the same region.

1	Snake venoms are highly variable and complex mixtures of enzymes, low-molecular-weight polypeptides, glycoproteins, and other constituents. Among the deleterious components are hemorrhagins that promote vascular leakage and cause both local and systemic bleeding.

1	2734 Proteolytic enzymes cause local tissue necrosis, affect the coagulation pathway at various steps, and impair organ function. Hyaluronidases promote the spread of venom through connective tissue. Myocardial depressant factors reduce cardiac output, and bradykinins cause vasodilation and hypotension. Neurotoxins act either preor postsynaptically to block transmission at the neuromuscular junction, causing muscle paralysis. Most snake venoms have multisystem effects on their victims. After a venomous snakebite, the time to symptom onset and clinical presentation can be quite variable and depend on the species involved, the anatomic location of the bite, and the amount of venom injected. Envenomations by most viperids and some elapids with necrotizing venoms cause progressive local pain, swelling, ecchymosis (Fig. 474-1), and (over a period of hours to days) hemorrhagic or serum-filled vesicles and bullae. In serious bites, tissue loss can be significant (Figs. 474-2 and 474-3).

1	swelling, ecchymosis (Fig. 474-1), and (over a period of hours to days) hemorrhagic or serum-filled vesicles and bullae. In serious bites, tissue loss can be significant (Figs. 474-2 and 474-3). Systemic findings are extremely variable and can include tachycardia or bradycardia, hypotension, generalized weakness, changes in taste, mouth numbness, muscle fasciculations, pulmonary edema, renal dysfunction, and spontaneous hemorrhage (from essentially any anatomic site). Envenomations by neurotoxic elapids such as kraits (Bungarus species), many Australian elapids (e.g., death adders [Acanthophis species] and tiger snakes [Notechis species]), some cobras (Naja species), and some viperids (e.g., the South American rattlesnake [Crotalus durissus] and some Indian Russell’s vipers [Daboia russelii]) cause neurologic dysfunction. Early findings may consist of nausea and vomiting, headache, paresthesias or numbness, and altered mental status. Victims may develop cranial nerve abnormalities

1	cause neurologic dysfunction. Early findings may consist of nausea and vomiting, headache, paresthesias or numbness, and altered mental status. Victims may develop cranial nerve abnormalities (e.g., ptosis, difficulty swallowing) followed by peripheral motor weakness. Severe envenomation may result in muscle paralysis, including the muscles of respiration, and lead to death from respiratory failure and aspiration. Sea snake envenomation results in

1	FIGUrE 474-1 Northern Pacific rattlesnake (Crotalus oreganus oreganus) envenomations. A. Moderately severe envenomation. Note edema and early ecchymosis 2 h after a bite to the finger. B. Severe envenomation. Note extensive ecchymosis 5 days after a bite to the ankle. FIGUrE 474-2 Early stages of severe, full-thickness necrosis 5 days after a Russell’s viper (Daboia russelii) bite in southwestern India. local pain (variable), generalized myalgias, trismus, rhabdomyolysis, and progressive flaccid paralysis; these manifestations can be delayed for several hours.

1	The most important aspect of prehospital care of a person bitten by a venomous snake is rapid transport to a medical facility equipped to provide supportive care (airway, breathing, and circulation) and antivenom therapy. Most of the first-aid measures recommended in the past are of little benefit, and some actually worsen outcome. It is reasonable to apply a splint to the bitten extremity to lessen bleeding and discomfort and, if possible, to keep the extremity at approximately heart level. In developing countries, indigenous people should be encouraged to seek immediate care at a health care facility equipped with antivenom instead of consulting traditional healers and thus incurring significant delays in reaching appropriate care. Attempting to capture and transport the offending snake, alive or dead, is not advised; instead, digital photographs of the snake taken from a safe distance may assist with snake identification and treatment decisions.

1	FIGUrE 474-3 Severe necrosis 10 days after a pit viper bite in a young child in Colombia. (Courtesy of Jay R. Stanka; with permission.) Incising and/or applying suction to the bite site should be avoided, as these measures are ineffective and exacerbate local tissue damage. Similarly ineffective and potentially harmful are the application of poultices, ice, and electric shock. Techniques or devices used in an effort to limit venom spread (e.g., lymphoocclusive bandages or tourniquets) are ineffective and may result in greater local tissue damage by restricting the spread of potentially necrotizing venom. Tourniquet use can result in loss of function and amputation even in the absence of envenomation.

1	Elapid venoms that are primarily neurotoxic and have no significant effects on local tissue may be localized by pressure-immobilization, a technique in which the entire limb is wrapped immediately with a bandage (e.g., crepe or elastic) and then immobilized. For this technique to be effective, the wrap pressure must be precise (40–70 mmHg in upper-extremity application and 55–70 mmHg in lower-extremity application) and the victim must be carried out of the field because walking generates muscle-pumping activity that— regardless of the anatomic site of the bite—will disperse venom into the systemic circulation. Pressure-immobilization should be used only in cases in which the offending snake is reliably identified and known to be primarily neurotoxic, the rescuer is skilled in pressure-wrap application, the necessary supplies are readily available, and the victim can be fully immobilized and carried to medical care—an uncommon combination of conditions, particularly in the regions of

1	application, the necessary supplies are readily available, and the victim can be fully immobilized and carried to medical care—an uncommon combination of conditions, particularly in the regions of the world where such bites are most common.

1	In the hospital, the victim should be closely monitored (vital signs, cardiac rhythm, oxygen saturation, urine output) while a history is quickly obtained and a rapid, thorough physical examination is performed. To objectively evaluate the progression of local envenomation, the level of swelling in the bitten extremity should be marked and limb circumference should be measured every 15 min until the swelling has stabilized. During this period of observation, the extremity should be positioned at approximately heart level. Measures applied in the field (such as bandages or wraps) should be removed once IV access has been obtained, with cognizance that the release of such ligatures may result in hypotension or dysrhythmias when stagnant acidotic blood containing venom is released into the systemic circulation. Two large-bore IV lines should be established in unaffected extremities. Because of the potential for coagulopathy, venipuncture attempts should be minimized, and noncompressible

1	circulation. Two large-bore IV lines should be established in unaffected extremities. Because of the potential for coagulopathy, venipuncture attempts should be minimized, and noncompressible sites (e.g., a subclavian vein) should be avoided. Early hypotension is due to pooling of blood in the pulmonary and splanchnic vascular beds. Later, systemic bleeding, hemolysis, and loss of intravascular volume into the soft tissues may play important roles. Fluid resuscitation with isotonic saline (20–40 mL/kg IV) should be initiated if there is any evidence of hemodynamic instability, and a trial of 5% albumin (10–20 mL/kg IV) may be given if the response to saline infusion is inadequate. Only after aggressive volume resuscitation and antivenom administration (see below) are accomplished should vasopressors (e.g., dopamine) be added. Invasive hemodynamic monitoring (central venous and/or continuous arterial pressures) can be helpful in such cases, although obtaining central vascular access is

1	(e.g., dopamine) be added. Invasive hemodynamic monitoring (central venous and/or continuous arterial pressures) can be helpful in such cases, although obtaining central vascular access is risky if coagulopathy has developed. Victims of neurotoxic envenomation should be watched carefully for evidence of cranial nerve dysfunction (e.g., ptosis) that may precede the onset of difficulty swallowing or respiratory insufficiency that necessitates definitive airway protection by endotracheal intubation.

1	Blood should be drawn for typing and cross-matching and for laboratory evaluation as soon as possible. Important studies include a complete blood count to determine the degree of hemorrhage or hemolysis and to identify thrombocytopenia; studies of renal and hepatic function; coagulation studies to diagnose consumptive 2735 coagulopathy; creatine kinase for suspected rhabdomyolysis; and testing of urine for blood or myoglobin. In developing regions, the 20-min whole-blood clotting test can be used to reliably diagnose coagulopathy. A few milliliters of fresh blood are placed in a new, clean, plain glass receptacle (e.g., a test tube) and left undisturbed for 20 min. The tube is then tipped once to 45° to determine whether a clot has formed. If it has not, coagulopathy is diagnosed. Arterial blood gas studies, electrocardiography, and chest radiography may be helpful in severe envenomations or when there is significant comorbidity. Any arterial puncture in the setting of coagulopathy

1	blood gas studies, electrocardiography, and chest radiography may be helpful in severe envenomations or when there is significant comorbidity. Any arterial puncture in the setting of coagulopathy requires great caution and must be performed at an anatomic site amenable to direct-pressure tamponade. After antivenom therapy (see below), laboratory values should be rechecked every 6 h until clinical stability is achieved. If initial laboratory values are normal, the complete blood count and coagulation studies should be repeated every hour until it is clear that no systemic envenomation has occurred.

1	The mainstay of treatment of a venomous snakebite resulting in significant envenomation is prompt administration of specific antivenom. Antivenoms are produced by injecting animals (generally horses or sheep) with venoms from medically important snakes. Once the stock animals develop antibodies to the venoms, their serum is harvested and the antibodies are isolated for antivenom preparation, which may involve varying degrees of digestion and purification of the IgG molecules. The goal of antivenom administration is to allow antibodies (or antibody fragments) to bind and deactivate circulating venom components before they can attach to target tissues and cause deleterious effects. Antivenoms may be monospecific (directed against a particular snake species) or polyspecific (covering several medically important species in the region) but rarely offer cross-protection against snake species other than those used in their production unless the species are known to have homologous venoms.

1	medically important species in the region) but rarely offer cross-protection against snake species other than those used in their production unless the species are known to have homologous venoms. Thus, antivenom selection must be specific for the offending snake; if the antivenom chosen does not contain antibodies to that snake’s venom components, it will provide no benefit and may lead to unnecessary complications (see below). In the United States, assistance in finding appropriate antivenom can be obtained from regional poison control centers.

1	For victims of bites by viperids or cytotoxic elapids, indications for antivenom administration include significant progressive local findings (e.g., soft tissue swelling crossing a joint or involving more than half the bitten limb) and any evidence of systemic envenomation (systemic symptoms or signs, laboratory abnormalities). Caution must be used in determining the significance of isolated soft tissue swelling after the bite of an unidentified snake because the saliva of some relatively harmless species can cause mild edema at the bite site; in such bites, antivenoms are useless and potentially harmful. Antivenoms have limited efficacy in preventing tissue damage caused by necrotizing venoms, as venom components bind to local tissues very quickly, before antivenom administration can be initiated. Nevertheless, antivenom should be administered as soon as the need for it is identified to limit further tissue damage and systemic effects. Antivenom administration after bites by

1	can be initiated. Nevertheless, antivenom should be administered as soon as the need for it is identified to limit further tissue damage and systemic effects. Antivenom administration after bites by neurotoxic elapids is indicated at the first sign of any evidence of neurotoxicity (e.g., cranial nerve dysfunction, peripheral neuropathy). In general, antivenom is effective only in reversing active venom toxicity; it is of no benefit in reversing effects that already have been established (e.g., renal failure, established paralysis) and that will improve only with time and other supportive therapies.

1	Specific comments related to the management of venomous snakebites in the United States and Canada appear in Table 474-1. The package insert for the selected antivenom can be consulted regarding species covered, method of administration, starting dose, and need (if any) for re-dosing. The information in antivenom package inserts, however, is not always accurate and reliable. Whenever possible, it is advisable for treating physicians to seek advice from experts in snakebite management regarding indications for and dosing of antivenom. Pit viper bites (rattlesnakes [Crotalus and Sistrurus spp.], cottonmouth water moccasins [Agkistrodon piscivorus], and copperheads [Agkistrodon contortrix]) airway, breathing, and circulation. monitoring (vital signs, cardiac rhythm, and oxygen saturation). two large-bore IV lines. the patient is hypotensive, administer a normal saline bolus (20–40 mL/kg IV). hypotension persists, consider 5% albumin (10–20 mL/kg IV).

1	two large-bore IV lines. the patient is hypotensive, administer a normal saline bolus (20–40 mL/kg IV). hypotension persists, consider 5% albumin (10–20 mL/kg IV). rapid history and perform thorough physical examination. offending snake if possible. and record circumference of bitten extremity every 15 min until swelling has stabilized. laboratory studies (CBC, metabolic panel, PT/INR/PTT, fibrinogen level, FDP, blood type and cross-matching, urinalysis). normal, repeat CBC and coagulation studies every hour until it is clear that no systemic envenomation has occurred. abnormal, repeat 6 h after antivenom administration (see below). severity of envenomation.

1	abnormal, repeat 6 h after antivenom administration (see below). severity of envenomation. • None: • Mild: local findings only (e.g., pain, ecchymosis, nonprogressive swelling) • Moderate: swelling that is clearly progressing, systemic symptoms or signs, and/or laboratory abnormalities • Severe: neurologic dysfunction, respiratory distress, and/or cardiovascular instability/shock regional poison control center. and administer antivenom as indicated: Crotalidae Polyvalent Immune Fab (CroFab) (Ovine) (BTG International Inc., West Conshohocken, PA). on severity of envenomation or mild: none • Moderate: • Severe: reconstituted vials in 250 mL of normal saline. IV over 1 h (with physician in close attendance). acute reaction to antivenom: infusion. with standard doses of epinephrine (IM or IV; latter route only in setting of severe hypotension), antihistamines (IV), and glucocorticoids (IV).

1	acute reaction to antivenom: infusion. with standard doses of epinephrine (IM or IV; latter route only in setting of severe hypotension), antihistamines (IV), and glucocorticoids (IV). reaction is controlled, restart antivenom as soon as possible (may further dilute in larger volume of normal saline). clinical status over 1 h. or improved: Admit to hospital. or unimproved: Repeat starting dose (and continue this pattern until patient’s condition is stable or improved). products are rarely needed; if required, they should be given only after antivenom administration. tetanus immunization as needed. antibiotics are unnecessary unless prehospital care included incision or mouth suction. management: acetaminophen and/or narcotics as needed; avoidance of salicylates and nonsteroidal anti-inflammatory agents to hospital. (If no evidence of envenomation, monitor for 8 h before discharge.) additional CroFab (2 vials every 6 h for 3 additional doses, with close monitoring).

1	for evidence of rising intracompartmental pressures (see text). wound care (see text). physical therapy (see text). discharge, warn patient of possible recurrent coagulopathy and symptoms/signs of delayed serum sickness. Coral snakebites (Micrurus spp. and Micruroides euryxanthus) airway, breathing, and circulation. monitoring (vital signs, cardiac rhythm, and oxygen saturation). one large-bore IV line and initiate normal saline infusion. rapid history and perform thorough physical examination. offending snake if possible. studies are unlikely to be helpful. regional poison control center. and administer antivenom as indicated: Antivenin (Micrurus fulvius) (equine) (commonly referred to as North American Coral Snake Antivenin; Wyeth Pharmaceuticals, New York, NY).b to antivenom package insert. 3–5 reconstituted vials of antivenom in 250 mL of normal saline. IV over 1 h (with physician in close attendance).

1	3–5 reconstituted vials of antivenom in 250 mL of normal saline. IV over 1 h (with physician in close attendance). signs of envenomation progress despite initial antivenom, repeat the starting dose (up to 10 vials total may be required). (Continued ) acute adverse reaction to antivenom: infusion. with standard doses of epinephrine (IM or IV; latter route only in setting of severe hypotension), antihistamines (IV), and glucocorticoids (IV). reaction is controlled, restart antivenom as soon as possible (may further dilute in larger volume of normal saline). any evidence of neurologic dysfunction (e.g., any cranial nerve abnormalities such as ptosis): any evidence of difficulty swallowing or breathing, proceed with endotracheal intubation and ventilatory support (may be required for days or weeks). tetanus immunization as needed. antibiotics are unnecessary unless prehospital care included incision or mouth suction.

1	tetanus immunization as needed. antibiotics are unnecessary unless prehospital care included incision or mouth suction. to hospital (intensive care unit) even if there is no evidence of envenomation; monitor for at least 24 h. aThese recommendations are specific to the care of victims of venomous snakebites in the United States and Canada and should not be applied to bites in other regions of the world. bAt the time of publication, a single lot of antivenom remains, with an extended expiration date of April 30, 2015. Abbreviations: CBC, complete blood count; FDP, fibrin degradation products; PT/INR/PTT, prothrombin time/international normalized ratio/partial thromboplastin time.

1	Antivenom should be administered only by the IV route, and the infusion should be started slowly, with the physician at the bedside ready to intervene immediately at the first signs of an acute adverse reaction. In the absence of an adverse reaction, the rate of infusion can be increased gradually until the full starting dose has been administered (over a total period of ~1 h). Further antivenom may be necessary if the patient’s acute clinical condition worsens or fails to stabilize or if venom effects that were initially controlled recur. The decision to administer further antivenom to a stabilized patient should be based on clinical evidence of persistent circulation of unbound venom components. For viperid bites, antivenom administration generally should be continued until the victim shows definite improvement (e.g., stabilized vital signs, reduced pain, restored coagulation). Neurotoxicity from elapid bites may be more difficult to reverse with antivenom. Once neurotoxicity is

1	shows definite improvement (e.g., stabilized vital signs, reduced pain, restored coagulation). Neurotoxicity from elapid bites may be more difficult to reverse with antivenom. Once neurotoxicity is established and endotracheal intubation is required, further doses of antivenom are unlikely to be beneficial. In such cases, the victim must be maintained on mechanical ventilation until recovery, which may take days or weeks.

1	Adverse reactions to antivenom administration include immediate (nonallergic and, less commonly, allergic anaphylaxis) and delayed-type hypersensitivity reactions (serum sickness). Clinical manifestations of immediate hypersensitivity include urticaria, laryngeal edema, bronchospasm, and hypotension. Skin testing for potential hypersensitivity, although recommended by some antivenom manufacturers, is insensitive and nonspecific and should be omitted. Worldwide, the quality of antivenoms is highly variable. Rates of acute nonallergic anaphylactic reactions to some of these products exceed 50%. For this reason, some authorities have recommended pretreatment with IV antihistamines (e.g., diphenhydramine, 1 mg/kg to a maximum of 100 mg; and cimetidine, 5–10 mg/kg to a maximum of 300 mg) or even a prophylactic SC or IM dose of epinephrine (0.01 mg/kg, up to 0.3 mg). Further research is necessary, however, to determine whether any pretreatment measures are truly beneficial. Modest expansion

1	a prophylactic SC or IM dose of epinephrine (0.01 mg/kg, up to 0.3 mg). Further research is necessary, however, to determine whether any pretreatment measures are truly beneficial. Modest expansion of the patient’s intra-vascular volume with crystalloids may blunt acute adverse blood pressure decline. Epinephrine and airway equipment should always be immediately available during antivenom infusion. An acute anaphylactic reaction may be heralded by a single hive or mild itching or may present as bronchospasm or acute cardiovascular collapse. If the patient develops an acute reaction to antivenom, the infusion should be temporarily stopped and the reaction immediately treated with IM epinephrine and IV antihistamines and glucocorticoids. Once the reaction has been controlled, if the severity of the envenomation warrants additional antivenom, the dose should be diluted further in isotonic saline and restarted as soon as possible. Rarely, in cases of recalcitrant hypotension, a

1	severity of the envenomation warrants additional antivenom, the dose should be diluted further in isotonic saline and restarted as soon as possible. Rarely, in cases of recalcitrant hypotension, a concomitant IV infusion of epinephrine may be initiated and titrated to clinical effect while antivenom is administered. The patient must be monitored very closely during such therapy, preferably in an intensive care setting. Serum sickness typically develops 1–2 weeks after antivenom administration and may present as fever, chills, urticaria, myalgias, arthralgias, lymphadenopathy, and renal or neurologic dysfunction. Treatment of serum sickness consists of systemic glucocorticoids (e.g., oral prednisone, 1–2 mg/kg daily) until all symptoms have resolved, followed by a taper over 1–2 weeks. Oral antihistamines and analgesics may provide additional relief of symptoms.

1	Blood products are rarely necessary in the management of an envenomed patient. The venoms of many snake species can deplete coagulation factors and cause a decrease in platelet count or hematocrit. Nevertheless, these components usually rebound within hours after administration of adequate antivenom. If the need for blood products is thought to be great (e.g., a dangerously low platelet count in a hemorrhaging patient), these products should be given only after adequate antivenom administration to avoid fueling ongoing consumptive coagulopathy.

1	Rhabdomyolysis and hemolysis should be managed in standard fashion. Victims who develop acute renal failure should be evaluated by a nephrologist and referred for hemodialysis or peritoneal dialysis as needed. Such renal failure, which usually is due to acute tubular necrosis, is frequently reversible. If bilateral cortical necrosis occurs, however, the prognosis for renal recovery is less favorable, and longterm dialysis with possible renal transplantation may be necessary.

1	Acetylcholinesterase inhibitors (e.g., edrophonium and neostigmine) may promote neurologic improvement in patients bitten by snakes with postsynaptic neurotoxins. Snakebite victims with objective evidence of neurologic dysfunction should receive a test dose of acetylcholinesterase inhibitors, as outlined in Table 474-2. If they exhibit improvement, additional doses of long-acting neostigmine can be administered as needed. Close monitoring is required to prevent aspiration if repetitive dosing of neostigmine is used in an 1. Patients with clear, objective evidence of neurotoxicity (e.g., ptosis or inability to maintain upward gaze) should receive a test dose of edrophonium (if available) or neostigmine. a. Pretreat with atropine: 0.6 mg IV (children, 0.02 mg/kg with a minimum of 0.1 mg) b. Treat with: Edrophonium: 10 mg IV (children, 0.25 mg/kg)

1	a. Pretreat with atropine: 0.6 mg IV (children, 0.02 mg/kg with a minimum of 0.1 mg) b. Treat with: Edrophonium: 10 mg IV (children, 0.25 mg/kg) Neostigmine: 1.5–2.0 mg IM (children, 0.025–0.08 mg/kg) 2. If objective improvement is evident after 5 min, treat with: a. Neostigmine: 0.5 mg IV or SC (children, 0.01 mg/kg) every 30 min as b. Atropine: 0.6 mg IV continuous infusion over 8 h (children, 0.02 mg/kg over 8 h) 3. Closely monitor the airway and perform endotracheal intubation as needed.

1	2738 attempt to obviate endotracheal intubation. Acetylcholinesterase inhibitors are not a substitute for administration of an appropriate antivenom when available. Care of the bite wound includes simple cleansing with soap and water; application of a dry, sterile dressing; and splinting of the affected extremity with padding between the digits. Once antivenom therapy has been initiated, the extremity should be elevated above heart level to reduce swelling. Tetanus immunization should be updated as appropriate. Prophylactic antibiotics are generally unnecessary after bites by North American snakes, as the incidence of secondary infection is low. In some regions, secondary bacterial infection is more common and the consequences are dire; in these regions, prophylactic antibiotics (e.g., cephalosporins) are used commonly. Antibiotics may also be considered if misguided first aid efforts have included incision or mouth suction of the bite site. Pain control should be achieved with

1	cephalosporins) are used commonly. Antibiotics may also be considered if misguided first aid efforts have included incision or mouth suction of the bite site. Pain control should be achieved with acetaminophen or narcotic analgesics. Salicylates and nonsteroidal anti-inflammatory agents should be avoided because of their effects on blood clotting. Most snake envenomations involve SC deposition of venom. On occasion, however, venom can be injected more deeply into muscle compartments, particularly if the offending snake was large and the bite occurred on the lower leg, forearm, or hand. Intramuscular swelling of the affected extremity may be accompanied by severe pain, decreased strength, altered sensation, cyanosis, and apparent pulselessness—signs suggesting a muscle compartment syndrome. If there is clinical concern that subfascial muscle edema may be impeding tissue perfusion, intracompartmental pressures should be measured by a minimally invasive technique (e.g., wick catheter or

1	If there is clinical concern that subfascial muscle edema may be impeding tissue perfusion, intracompartmental pressures should be measured by a minimally invasive technique (e.g., wick catheter or digital readout device). If the intracompartmental pressure is high (>30–40 mmHg), the extremity should be kept elevated while antivenom is administered. A dose of IV mannitol (1 g/kg) can be given in an effort to reduce muscle edema if the patient is hemodynamically stable. If the intracompartmental pressure remains elevated after 1 h of such therapy, a surgical consultation should be obtained for possible fasciotomy. Although evidence from animal studies suggests that fasciotomy may actually worsen myonecrosis, compartmental decompression is still necessary to preserve nerve function. Fortunately, the incidence of compartment syndrome is very low after a snakebite, with fasciotomies required in <1% of cases. Nevertheless, vigilance is essential. If a fasciotomy is deemed necessary, it

1	the incidence of compartment syndrome is very low after a snakebite, with fasciotomies required in <1% of cases. Nevertheless, vigilance is essential. If a fasciotomy is deemed necessary, it should be undertaken with the patient’s informed consent whenever possible. Wound care in the days after the bite should include careful aseptic debridement of clearly necrotic tissue once coagulation has been restored. Intact serum-filled vesicles or hemorrhagic blebs should be left undisturbed. If ruptured, they should be debrided with sterile technique. Any debridement of damaged muscle should be conservative because there is evidence that such muscle may recover to a significant degree after antivenom therapy. Physical therapy should be started as soon as possible so that the victim can return to a functional state. The incidence of long-term loss of function (e.g., reduced range of motion, impaired sensory function) is unclear but is probably quite high (>30%), particularly after viperid

1	to a functional state. The incidence of long-term loss of function (e.g., reduced range of motion, impaired sensory function) is unclear but is probably quite high (>30%), particularly after viperid bites. Any patient with signs of envenomation should be observed in the hospital for at least 24 h. In North America, a patient with an apparently “dry” viperid bite should be watched for at least 8 h before discharge, as significant toxicity occasionally develops after a delay of several hours. The onset of systemic symptoms commonly is delayed for a number of hours after bites by several of the elapids (including coral snakes, Micrurus species), some non–North American viperids (e.g., the hump-nosed pit viper [Hypnale hypnale]), and sea snakes. Patients bitten by these snakes should be observed in the hospital for at least 24 h. Patients whose condition is not stable should be admitted to an intensive care setting. At hospital discharge, victims of venomous snakebites should be warned

1	in the hospital for at least 24 h. Patients whose condition is not stable should be admitted to an intensive care setting. At hospital discharge, victims of venomous snakebites should be warned about symptoms and signs of wound infection, antivenomrelated serum sickness, and potential long-term sequelae, such as pituitary insufficiency from Russell’s viper (D. russelii) bites. If coagulopathy developed in the acute stages of envenomation, it can recur during the first 2–3 weeks after the bite. In such cases, victims should be warned to avoid elective surgery or activities posing a high risk of trauma during this period. Outpatient analgesic treatment, wound management, and physical therapy should be provided.

1	The overall mortality rates for victims of venomous snakebites are low in regions with rapid access to medical care and appropriate antivenoms. In the United States, for example, the mortality rate is <1% for victims who receive antivenom. Eastern and western diamondback rattlesnakes (Crotalus adamanteus and Crotalus atrox, respectively) are responsible for the majority of snakebite deaths in the United States. Snakes responsible for large numbers of deaths in other countries include cobras (Naja spp.), carpet and saw-scaled vipers (Echis spp.), Russell’s vipers (D. russelii), large African vipers (Bitis spp.), lancehead pit vipers (Bothrops spp.), and tropical rattlesnakes (C. durissus).

1	The incidence of morbidity—defined as permanent functional loss in a bitten extremity—is difficult to estimate but is substantial. Morbidity may be due to muscle, nerve, or vascular injury or to scar contracture. Such morbidity can have devastating consequences for victims in the developing world when they lose the ability to work and provide for their families. In the United States, functional loss tends to be more common and severe after rattlesnake bites than after bites by copperheads (Agkistrodon contortrix) or water moccasins (Agkistrodon piscivorus).

1	In many developing countries where snakebites are common, scarce access to medical care and antivenom resources con tributes to high rates of morbidity and mortality. In many countries, the available antivenoms are inappropriate and ineffective against the venoms of medically important indigenous snakes. In those regions, further research is necessary to determine the actual impact of venomous snakebites and the specific antivenom needs in terms of both quantity and spectrum of coverage. Without accurate statistics, it is difficult to persuade antivenom manufacturers to begin and sustain production of appropriate antisera in developing nations. There is evidence that antivenoms can be produced in much more cost-effective ways than those currently being used. Just as important as getting the correct antivenoms into underserved regions is the need to educate populations about snakebite prevention and to train medical care providers in proper management approaches. Local protocols

1	the correct antivenoms into underserved regions is the need to educate populations about snakebite prevention and to train medical care providers in proper management approaches. Local protocols written with significant input from experienced providers in the region of concern should be developed and distributed. Appropriate antivenoms must be available at the likely first point of medical contact for patients (e.g., primary health centers) in order to minimize the common practice of referring victims to more distant, higher levels of care for the initiation of antivenom therapy. Those who care for snakebite victims in these often-remote clinics must have the skills and confidence required to begin antivenom treatment (and to treat possible reactions) as soon as possible when needed.

1	Much of the management of envenomation by marine creatures is supportive in nature. A specific marine antivenom can be used when appropriate.

1	INVErtEBratES Cnidarians The Golgi apparatus of the cnidoblast cells within cnidarians, such as hydroids, fire coral, jellyfish, Portuguese men-of-war, and sea anemones, secretes specialized living stinging organelles called cnidae (also referred to as cnidocysts, a term that encompasses nematocysts, ptychocysts, and spirocysts). Within each organelle resides a stinging mechanism (“thread tube”) and venom. In the stinging process, cnidocysts are released and discharged upon mechanosensory stimulation. The venoms from these organisms contain bioactive substances such as tetramine, 5-hydroxytryptamine, histamine, serotonin, and high-molecular-weight toxins, all of which can, among other effects, change the permeability of cells to ions. Victims usually report immediate prickling or burning, pruritus, paresthesias, and painful throbbing with radiation. The skin becomes reddened, darkened, edematous, and blistered and may show signs of superficial necrosis. A legion of neurologic,

1	pruritus, paresthesias, and painful throbbing with radiation. The skin becomes reddened, darkened, edematous, and blistered and may show signs of superficial necrosis. A legion of neurologic, cardiovascular, respiratory, rheumatologic, gastrointestinal, renal, and ocular symptoms has been described, especially following stings from anemones, Physalia species, and scyphozoans. Anaphylaxis is possible. Hundreds of deaths have been reported, many of them caused by Chironex fleckeri, Stomolophus nomurai, Physalia physalis, and Chiropsalmus quadrumanus. Irukandji syndrome, associated with the Australian jellyfish Carukia barnesi and other species, is a potentially fatal condition that most commonly is characterized by hypertension; severe back, chest, and abdominal pain; nausea and vomiting; headache; sweating; and, in the most serious cases, myocardial troponin leak, pulmonary edema, and ultimately hypotension. This syndrome is thought to be mediated, at least in part, by the release of

1	headache; sweating; and, in the most serious cases, myocardial troponin leak, pulmonary edema, and ultimately hypotension. This syndrome is thought to be mediated, at least in part, by the release of endogenous catecholamines followed by cytokines and nitric oxide.

1	Rescuers should note that envenomations by different cnidarians (typified by jellyfish) may respond differently to similar topical therapies; thus, the recommendations in this chapter must be tailored to local species and clinical practices. During stabilization, the skin should be decontaminated immediately with a generous application of lidocaine (up to 4%), an all-purpose agent that appears to be useful for relieving pain caused by a large number of species. Vinegar (5% acetic acid), rubbing alcohol (40–70% isopropyl alcohol), baking soda (sodium bicarbonate, especially for sea nettle stings), papain (unseasoned meat tenderizer), fresh lemon or lime juice, olive oil, or sugar may be effective, depending on the species of stinging creature. Household ammonia may in and of itself cause skin irritation.

1	The pressure-immobilization technique is no longer recommended for venom containment in the setting of any jellyfish sting. For the sting of the venomous box jellyfish (C. fleckeri), vinegar should be used. Local application of heat (up to 45°C/113°F), commonly by immersion in hot water, may be as effective. A baking soda slurry (50% baking soda, 50% water) has been recommended for Cyanea and Chrysaora species. Commercial (chemical) cold packs or real ice packs applied over a thin dry cloth or a plastic membrane have been shown to be effective in alleviating mild or moderate Physalia utriculus (bluebottle jellyfish) stings but may be less effective than application of heat. Perfume, aftershave lotion, and high-proof ethanol are not efficacious and may be detrimental; formalin, ether, gasoline, and other organic solvents should not be used. Shaving the skin helps remove remaining nematocysts. Freshwater irrigation and rubbing lead to further stinging by adherent nematocysts and should

1	and other organic solvents should not be used. Shaving the skin helps remove remaining nematocysts. Freshwater irrigation and rubbing lead to further stinging by adherent nematocysts and should be avoided.

1	After decontamination, topical application of an anesthetic ointment (lidocaine, benzocaine), an antihistamine (diphenhydramine), or a glucocorticoid (hydrocortisone) may be helpful. Persistent severe pain after decontamination may be treated with morphine, meperidine, fentanyl, or another narcotic analgesic. Muscle spasms may respond to diazepam (2–5 mg, titrated upward as necessary) or 10% calcium gluconate (5–10 mL) given IV. An ovine-derived IgG antivenom is available from Commonwealth Serum Laboratories (see “Sources of Antivenoms and Other Assistance,” below) for stings from the box jellyfish found in Australian and Indo-Pacific waters. However, despite its reported clinical efficacy, one school of thought holds that perhaps the antivenom is unable to bind the venom rapidly enough to account for its effects. Until further notice, current recommendations for its use apply. Treatment for Irukandji syndrome may require administration of opioid analgesics and MgSO4 as well as

1	to account for its effects. Until further notice, current recommendations for its use apply. Treatment for Irukandji syndrome may require administration of opioid analgesics and MgSO4 as well as aggressive treatment (phentolamine, 5 mg IV) of hypertension. All victims with systemic reactions should be observed for at least 6–8 h for rebound from any therapy, and all elderly adults should be checked for cardiac arrhythmias. Patients may suffer postinflammatory hyperpigmentation and persistent cutaneous hypersensitivity in areas of skin contact.

1	Safe Sea, a “jellyfish-safe” sunblock (www.nidaria.com) applied to the skin before an individual enters the water, inactivates the recognition and discharge mechanisms of nematocysts, has been tested successfully against a number of marine stingers, and may prevent or 2739 diminish the effects of coelenterate stings. Whenever possible, a dive skin or wet suit should be worn when entering ocean waters.

1	Sea Sponges Many sponges produce crinotoxins that are present on their surface or in their internal secretions. As a result, touching a sea sponge may result in dermatitis or “sponge diver’s disease,” a necrotic skin reaction. Irritant dermatitis may result if small spicules of silica or calcium carbonate penetrate the skin. It is impossible to distinguish between the allergic and spicule reactions, so the treatment is the same for both. Afflicted skin should be gently dried and adhesive tape used to remove embedded spicules. Vinegar should be applied immediately and then for 10–30 min three or four times a day. Rubbing alcohol may be used if vinegar is unavailable. After spicule removal and skin decontamination, glucocorticoid or antihistamine cream may be applied to the skin. Severe vesiculation should be treated with a 2-week tapering course of systemic glucocorticoids. Mild reactions subside in 3–7 days, while involvement of large areas of the skin may result in systemic symptoms

1	should be treated with a 2-week tapering course of systemic glucocorticoids. Mild reactions subside in 3–7 days, while involvement of large areas of the skin may result in systemic symptoms of fever, dizziness, nausea, muscle cramps, and formication.

1	annelid Worms Annelid worms (bristleworms) possess rows of soft, cactus-like spines capable of inflicting painful stings. Contact results in symptoms similar to those of nematocyst envenomation. Without treatment, pain usually subsides over several hours, but inflammation may persist for up to a week (Fig. 474-4). Victims should resist the urge to scratch because scratching may fracture retrievable spines. Visible bristles should be removed with forceps and adhesive tape or a commercial facial peel; alternatively, a thin layer of rubber cement can be used to entrap and then peel away the spines. Use of vinegar or rubbing alcohol or a brief application of lidocaine or unseasoned meat tenderizer (papain) may provide additional relief. Local inflammation should be treated with topical or systemic glucocorticoids.

1	Sea Urchins Venomous sea urchins possess either hollow, venom-filled calcified spines or triple-jawed, globiferous pedicellariae with venom glands. Venom may also be found within the integumentary sheath on the external spine surface of certain species. The venom contains toxic components, including steroid glycosides, hemolysins, proteases, serotonin, and cholinergic substances. Contact with either venom apparatus produces immediate and intensely painful stings. One or more spines entering a joint can cause synovitis that may, over time, progress to arthritis if the spine(s) remain in or near the joint. If multiple spines penetrate the skin, the patient may develop systemic symptoms, including nausea, vomiting, numbness, muscular paralysis, and respiratory distress. A delayed hypersensitivity reaction 7–10 days after resolution of primary symptoms has been described.

1	The affected part should be immersed immediately in hot water to tolerance (up to 45°C/113°F). Pedicellariae should be removed by shaving so that envenomation cannot continue. Accessible embedded spines should be removed but may break off and remain lodged in

1	FIGUrE 474-4 Rash on the hand of a diver from the spines of a bristleworm. (Courtesy of Paul Auerbach, with permission.) 2740 the victim. Residual dye from the surface of a spine remaining after the spine’s removal may mimic a retained spine but is otherwise of no consequence. Soft tissue radiography or MRI can confirm the presence of retained spines, which may warrant referral for attempted surgical removal if the spines are near vital structures (e.g., joints, neurovascular bundles). Retained spines may cause the formation of granulomas that are amenable to excision or to intralesional injection with triamcinolone hexacetonide (5 mg/mL). Chronic granulomatous arthritis of the proximal interphalangeal joints has been treated with synovectomy and removal of granulation tissue. Erbium-YAG laser ablation has been deployed to destroy multiple sea urchin spines embedded in the foot and identified visually at the surface level without causing thermal necrosis of the adjacent tissues.

1	laser ablation has been deployed to destroy multiple sea urchin spines embedded in the foot and identified visually at the surface level without causing thermal necrosis of the adjacent tissues. Eosinophilic pneumonia and local and diffuse neuropathies have been observed separately after penetration by multiple spines of the black sea urchin (presumed Diadema species). The pathophysiologies of these phenomena have not been determined.

1	Starfish The crown-of-thorns Acanthaster planci produces venom in glandular tissue underneath the epidermis, which is released via its spiny surfaces (Fig. 474-5). Skin puncture causes pain, bleeding, and local edema, usually with remission over 30–180 min. Multiple punctures may result in reactions such as local muscle paralysis; retained fragments may cause granulomatous lesions and synovitis. There has also been a case report of elevated liver enzymes after A. planci envenomation. Envenomed persons benefit from acute immersion therapy in hot water, local anesthesia, wound cleansing, and possible exploration to remove foreign material.

1	Sea Cucumbers Sea cucumbers produce holothurin (a cantharin-like liquid toxin) in their body walls. This toxin is concentrated in the tentacular organs that are projected when the animal is threatened. Underwater, holothurin induces minimal contact dermatitis in the skin but can cause significant corneal and conjunctival irritation. A severe reaction can lead to blindness. Skin should be detoxified with 5% acetic acid (vinegar), papain, or isopropyl alcohol. The eye should be anesthetized with one or two drops of 0.5% proparacaine and irrigated with 100–250 mL of normal saline, with subsequent slit-lamp examination to identify corneal defects.

1	Cone Snails Cone snails use a detachable dartlike tooth to inject conotoxins into prey, inducing tetanus followed by paralysis. In an unknowing handler, stings result in small, burning punctate wounds followed by local ischemia, cyanosis, and numbness. Dysphagia, syncope, dysarthria, ptosis, blurred vision, and pruritus have also been documented. Some envenomations induce paralysis leading to respiratory failure, coma, and death. There is no antivenom. Pressure-immobilization (see “Octopuses,” below), hot-water soaks, and local anesthetics have been used empirically with success. The wound should be inspected for a foreign body. Edrophonium has been recommended as therapy for paralysis if a Tensilon test is positive.

1	Octopuses Serious envenomations and deaths have followed bites of Australian blue-ringed octopuses (Octopus maculosus and Octopus lunulata). Although these animals rarely exceed 20 cm in length, their salivary venom contains a potent neurotoxin (maculotoxin) that inhibits peripheral nerve transmission by blocking sodium conductance. Oral numbness and facial numbness develop within several minutes of a serious envenomation and rapidly progress to total flaccid paralysis, including failure of respiratory muscles. Immediately after envenomation, a circumferential pressure-immobilization dressing 15 cm wide should be applied over a gauze pad (~7 × 7 × 2 cm) that has been placed directly over the sting. The dressing should be applied at venous-lymphatic pressure, with the preservation of distal arterial pulses. The limb should then be splinted. Once the victim has been transported to the nearest medical facility, the bandage can be released. Because there is no antidote and passive

1	distal arterial pulses. The limb should then be splinted. Once the victim has been transported to the nearest medical facility, the bandage can be released. Because there is no antidote and passive immunotherapy (rabbit IgG antibody) has been proven effective only against tetrodotoxin in mice, treatment is supportive. Patients with respiratory failure may need to be mechanically ventilated. If respirations are assisted, the victim may remain awake although completely paralyzed. Even with serious envenomations, significant recovery often takes place within 4–10 h, although complete recovery may require 2–4 days. Sequelae are uncommon unless related to hypoxia.

1	FIGUrE 474-5 Spines on the crown-of-thorns sea star (Acanthaster planci).(Courtesy of Paul Auerbach, with permission.) As for all penetrating injuries, first-aid care should be undertaken. In addition, consideration must be given to local wound infection by marine Vibrio species and freshwater Aeromonas hydrophila as well as other “aquatic bacteria,” particularly if spines and needles remain embedded.

1	Stingrays A stingray injury is both an envenomation and a traumatic wound. Thoracic and cardiac penetration, major vessel laceration, and compartment syndrome have all been observed. The venom, which contains serotonin, 5′-nucleotidase, and phosphodiesterase, causes immediate, intense pain that may last up to 48 h. The wound is very painful (with the pain peaking at 30–60 min and lasting up to 48 h), often becomes ischemic in appearance, and heals poorly, with adjacent soft tissue swelling and prolonged disability. Systemic effects include weakness, diaphoresis, nausea, vomiting, diarrhea, dysrhythmias, syncope, hypotension, muscle cramps, fasciculations, paralysis, and (in rare cases) death. Because of differences in the toxins present on the tissues covering the stingers, freshwater stingrays may cause more severe injuries than marine stingrays.

1	Scorpionfish The designation scorpionfish encompasses members of the family Scorpaenidae and includes not only scorpionfish but also lionfish and stonefish. A complex venom with neuromuscular toxicity is delivered through 12 or 13 dorsal, 2 pelvic, and 3 anal spines. In general, the sting of a stonefish is regarded as the most serious (severe to life-threatening); that of the scorpionfish is of intermediate seriousness; and that of the lionfish is the least serious. Like that of a stingray, the sting of a scorpionfish is immediately and intensely painful. Pain from a stonefish envenomation may last for days. Systemic manifestations of scorpionfish stings are similar to those of stingray envenomations but may be more pronounced, particularly in the case of a stonefish sting, which may cause severe local tissue necrosis in addition to vital organ failure. The rare deaths that follow stonefish envenomation usually occur within 6–8 h. There is a commercially available stonefish antivenom.

1	Other Fish Three species of marine catfish—Plotosus lineatus (oriental catfish), Bagre marinus (sail catfish), and Galeichthys felis (common sea catfish)—as well as several species of freshwater catfish are capable of stinging humans. Venom is delivered through a single dorsal spine and two pectoral spines. Clinically, a catfish sting is comparable to that of a stingray, although marine catfish envenomations are generally more severe than those of their freshwater counterparts. Surgeonfish (doctorfish, tang), weeverfish, ratfish, and horned venomous sharks have also envenomed humans. Platypus The platypus is a venomous mammal. The male has a keratinous spur on each hind limb; the spur is connected to a venom gland within the upper thigh. Skin puncture causes soft tissue edema and pain that may last for days or weeks. Care is supportive, and hot-water therapy does not appear to benefit the victim.

1	The stings of all marine vertebrates are treated in a similar fashion. Except for stonefish and serious scorpionfish envenomations (see below), no antivenom is available. The affected part should be immersed immediately in nonscalding hot water (45°C/113°F) for 30–90 min or until there is significant relief from pain. Recurrent pain may respond to repeated hot-water treatment. Cryotherapy is contraindicated, and no data support the use of antihistamines or steroids. Opiates will help alleviate the pain, as will local wound infiltration or regional nerve block with 1% lidocaine, 0.5% bupivacaine, and sodium bicarbonate mixed in a 5:5:1 ratio. After soaking and anesthetic administration, the wound must be explored and debrided. Radiography (in particular, MRI) may be helpful in identification of foreign bodies. After exploration and debridement, the wound should be irrigated vigorously with warm sterile water, saline, or 1% povidone-iodine in solution. Bleeding usually can be controlled

1	of foreign bodies. After exploration and debridement, the wound should be irrigated vigorously with warm sterile water, saline, or 1% povidone-iodine in solution. Bleeding usually can be controlled by sustained local pressure for 10–15 min. In general, wounds should be left open to heal by secondary intention or treated by delayed primary closure. Tetanus immunization should be updated. Antibiotic treatment should be considered for serious wounds and for envenomation in immunocompromised hosts. The initial antibiotics should cover Staphylococcus and Streptococcus species. If the victim is immunocompromised, if a wound is primarily repaired and is more than minor, or if an infection develops, antibiotic coverage should be broadened to include Vibrio species. Infection with Aeromonas species is of similar concern for wounds associated with natural freshwater.

1	APPROACH TO THE PATIENT: It is useful to be familiar with the local marine fauna and to recognize patterns of injury. A large puncture wound or jagged laceration (particularly on the lower extremity) that is more painful than one would expect from the size and configuration of the wound is likely to be a stingray envenomation. Smaller punctures, as described above, represent the activity of a sea urchin (Fig. 474-6) or starfish. Stony corals cause rough abrasions and, in rare instances, lacerations or puncture wounds.

1	Coelenterate (marine invertebrate) stings sometimes create diagnostic skin patterns. A diffuse urticarial rash on exposed skin is often indicative of exposure to fragmented hydroids or larval anemones. A linear, whiplike print pattern appears where a jellyfish tentacle has contacted the skin. In the case of the dreaded box jellyfish, a cross-hatched appearance, followed by development of dark purple coloration within a few hours of the sting, heralds skin necrosis. A frosted appearance may be created by aluminum salt– based remedies applied to the wound. An encounter with fire coral causes immediate pain and swollen red skin irritation in the pattern of contact, similar to but more severe than the imprint left by exposure to an intact feather hydroid. Seabather’s eruption, caused by thimble jellyfishes and larval anemones, may produce a diffuse rash that consists of clusters of erythematous macules or raised papules and is accompanied by intense itching (Fig. 474-7). Toxic sponges

1	jellyfishes and larval anemones, may produce a diffuse rash that consists of clusters of erythematous macules or raised papules and is accompanied by intense itching (Fig. 474-7). Toxic sponges create a burning and painful red rash on exposed skin, which may blister and later desquamate. Because virtually all marine stingers invoke the sequelae of inflammation, local erythema, swelling, and adenopathy are fairly nonspecific.

1	The best way to locate a specific antivenom in the United States is to call a regional poison control center and ask for assistance. FIGUrE 474-6 Spiny sea urchins. (Courtesy of Dr. Paul Auerbach, with permission.) Divers Alert Network, a nonprofit organization designed to assist in the care of injured divers, also may help with the treatment of marine injuries. The network can be reached on the Internet at www .diversalertnetwork.org or by telephone 24 h a day at (919) 684-9111. An antivenom for the box jellyfish (C. fleckeri) and another for stonefish FIGUrE 474-7 Erythematous, papular rash typical of seabather’s eruption causedbythimblejellyfishandlarvalanemones.

1	FIGUrE 474-7 Erythematous, papular rash typical of seabather’s eruption causedbythimblejellyfishandlarvalanemones. 2742 (and severe scorpionfish) envenomation are made in Australia by the Commonwealth Serum Laboratories (CSL; 45 Poplar Road, Parkville, Victoria, Australia 3052; www.csl.com.au; 61-3-9389-1911). When administering the box jellyfish antivenom, time is of the essence. For cardiac or respiratory decompensation, give a minimum of one ampoule and up to six ampoules consecutively IV, preferably in a 1:10 dilution with normal saline. For stonefish (or severe scorpionfish) envenomation, give one ampoule of specific antivenom IM for every one or two punctures, to a maximum of three ampoules.

1	CIGUatEra Epidemiology and Pathogenesis Ciguatera poisoning is the most common nonbacterial food poisoning associated with fish in the United States; most U.S. cases occur in Florida and Hawaii, although, with transportation of imported fish nationwide, all clinicians need to be aware of ciguatera. The poisoning almost exclusively involves tropical and semitropical marine coral reef fish common in the Indian Ocean, the South Pacific, and the Caribbean Sea. Global estimates predict that 20,000–50,000 people may be affected by this poisoning each year. More than 400 different fish have been associated with ciguatera toxicity, but 75% of poisonings involve the reef-dwelling barracuda, snapper, jack, or grouper. Ciguatera toxin is created by warm-water ocean reef microalgae of the genus Gambierdiscus toxicus, whose consumption by grazing fish allows the toxin to bioaccumulate in the food chain. Three major ciguatoxins are found in the flesh and viscera of ciguateric fish: CTX-1, -2, and

1	toxicus, whose consumption by grazing fish allows the toxin to bioaccumulate in the food chain. Three major ciguatoxins are found in the flesh and viscera of ciguateric fish: CTX-1, -2, and -3. Recent research suggests that CTX-1 activates astrocytes and astroglia. In addition, TRPV1, a nonselective cation channel expressed in nociceptive neurons, may play a role in the neurologic disturbances unique to ciguatera poisoning. Most, if not all, ciguatoxins are unaffected by freeze-drying, heat, cold, and gastric acid. None of the toxins affects the odor, color, or taste of fish. Cooking methods may alter the relative concentrations of the various toxins.

1	Clinical Manifestations The onset of symptoms may come within 15–30 min of ingestion and typically takes place within 2–6 h. Symptoms increase in severity over the ensuing 4–6 h. Most victims develop symptoms within 12 h of ingestion, and virtually all are afflicted within 24 h. The more than 150 signs and symptoms reported include those shown in Table 474-3. Diarrhea, vomiting, and abdominal pain usually develop 3–6 h after ingestion of a ciguatoxic fish. Symptoms may persist for 48 h and then generally resolve (even without treatment). A pathognomonic symptom is the reversal of hot and cold tactile perception, which develops in some persons after 3–5 days and may last for months. More severe reactions tend to occur in persons previously stricken with the disease. Persons who have ingested parrotfish (scaritoxin) may develop classic ciguatera poisoning as well as a “second-phase” syndrome (after 5–10 days’ delay) of disequilibrium

1	Gastrointestinal Abdominal pain, nausea, vomiting, diarrhea Neurologic Paresthesias, pruritus, tongue and throat numbness or burning, sensation of “carbonation” during swallowing, odontalgia or dental dysesthesias, dysphagia, tremor, fasciculations, athetosis, meningismus, aphonia, ataxia, vertigo, pain and weakness in the lower extremities, visual blurring, transient blindness, hyporeflexia, seizures, coma Dermatologic Conjunctivitis, maculopapular rash, skin vesiculations, dermatographism Cardiovascular Bradycardia, heart block, hypotension, central respiratory failurea Other Chills, dysuria, dyspnea, dyspareunia, weakness, fatigue, nasal congestion and dryness, insomnia, sialorrhea, diaphoresis, headache, arthralgias, myalgias aTachycardia and hypertension may occur after potentially severe transient bradycardia and hypotension. Death is rare. with locomotor ataxia, dysmetria, and resting or kinetic tremor. This syndrome may persist for 2–6 weeks.

1	Diagnosis The differential diagnosis of ciguatera includes paralytic shellfish poisoning, eosinophilic meningitis, type E botulism, organophosphate insecticide poisoning, tetrodotoxin poisoning, and psychogenic hyperventilation. At present, the diagnosis of ciguatera poisoning is made on clinical grounds because no routinely used laboratory test detects ciguatoxin in human blood. Liquid chromatography–mass spectrometry is available for ciguatoxins but is of limited clinical value because most health care institutions do not have the equipment needed to perform the test. A ciguatoxin enzyme immunoassay or radioimmunoassay may be used to test small portions of the suspected fish, but even these tests may not detect the very small amount of toxin (0.1 ppb) necessary to render fish flesh toxic. A newer neuroblastoma assay may be sufficiently sensitive to detect small amounts of toxin but is not readily available for clinical use.

1	Therapy is supportive and based on symptoms. Nausea and vomiting may be controlled with an antiemetic such as ondansetron (4–8 mg IV). Syrup of ipecac and activated charcoal are not recommended for ciguatera poisoning. Hypotension may require the administration of IV crystalloid and, in rare cases, a pressor drug. Bradyarrhythmias that lead to cardiac insufficiency and hypotension generally respond well to atropine (0.5 mg IV, up to 2 mg). Goal-directed combination cardiovascular fluid and pressor therapy may be required. Cool showers or the administration of hydroxyzine (25 mg PO every 6–8 h) may relieve pruritus. Amitriptyline (25 mg PO twice a day) reportedly alleviates pruritus and dysesthesias. In three cases unresponsive to amitriptyline, tocainide has appeared to be efficacious. Nifedipine has been used to treat headache and poor circulation in order to prevent hypotension, but only after the initial acute phase of the poisoning has passed. IV infusion of mannitol may be

1	Nifedipine has been used to treat headache and poor circulation in order to prevent hypotension, but only after the initial acute phase of the poisoning has passed. IV infusion of mannitol may be beneficial in moderate or severe cases in fluid-repleted patients, particularly for the relief of distressing neurologic or cardiovascular symptoms, although the efficacy of this therapy has been challenged and has not been definitively proved. The infusion is rendered initially as 1 g/kg per day over 45–60 min during the acute phase (days 1–5). If symptoms improve, a second dose may be given within 3–4 h and a third dose may be administered the next day. Care must be taken to avoid dehydration in a treated patient. The mechanism of the benefit against ciguatera intoxication is perhaps hyperosmotic water-drawing action, which reverses ciguatoxin-induced Schwann cell edema. Mannitol may also act in some fashion as a “hydroxyl scavenger” or may competitively inhibit ciguatoxin at the cell

1	water-drawing action, which reverses ciguatoxin-induced Schwann cell edema. Mannitol may also act in some fashion as a “hydroxyl scavenger” or may competitively inhibit ciguatoxin at the cell membrane.

1	During recovery from ciguatera poisoning, the victim should exclude the following from the diet for 6 months: fish (fresh or preserved), fish sauces, shellfish, shellfish sauces, alcoholic beverages, nuts, and nut oils. Consumption of fish in ciguatera-endemic regions should be avoided. All oversized fish of any predacious reef species should be suspected of harboring ciguatoxin. Neither moray eels nor the viscera of tropical marine fish should ever be eaten.

1	Diarrhetic shellfish poisoning occurs with consumption of shellfish producing diarrheal illness. The first suspected incident, which occurred in the Netherlands in 1961, was followed by outbreaks in Japan, the United Kingdom, and (most recently) China. The causative agents are the lipophilic compound okadaic acid and the dinophysistoxins, which inhibit serine and threonine protein phosphatases, with consequent protein accumulation and continued secretion of fluid in intestinal cells leading to diarrhea. Shellfish acquire these toxins by feeding on dinoflagellates, particularly of the genera Dinophysis and Prorocentrum.

1	Symptoms include diarrhea, nausea, vomiting, abdominal pain, and chills. Onset occurs within 30 min to 12 h. The illness is usually self-limited; most patients recover in 3 or 4 days, and only a few require hospitalization. Treatment is supportive and focused on hydration. Toxins can be detected in food samples by a mouse bioassay, an immunoassay, and high-performance liquid chromatography with fluorometric detection (HPLC-FLD).

1	Paralytic shellfish poisoning is induced by ingestion of any of a variety of feral or aquacultured filter-feeding organisms, including clams, oysters, scallops, mussels, chitons, limpets, starfish, and sand crabs. The origin of their toxicity is the chemical toxin they accumulate and concentrate by feeding on various planktonic dinoflagellates (e.g., Protogonyaulax, Ptychodiscus, and Gymnodinium) and protozoan organisms. The unicellular phytoplanktonic organisms form the foundation of the food chain, and in warm summer months these organisms “bloom” in nutrient-rich coastal temperate and semitropical waters. In the United States, paralytic shellfish poisoning is acquired primarily from seafood harvested in the Northeast, the Pacific Northwest, and Alaska. These planktonic species can release massive amounts of toxic metabolites into the water and cause mortality in bird and marine populations. The paralytic shellfish toxins are water soluble as well as heat and acid stable; they

1	release massive amounts of toxic metabolites into the water and cause mortality in bird and marine populations. The paralytic shellfish toxins are water soluble as well as heat and acid stable; they cannot be destroyed by ordinary cooking or freezing. Contaminated seafood looks, smells, and tastes normal. The best-characterized, most potent, and most frequently identified paralytic shellfish toxin is saxitoxin, which takes its name from the Alaska butter clam Saxidomus giganteus. Saxitoxin appears to block sodium conductance, inhibiting neuromuscular transmission at the axonal and muscle membrane levels. A toxin concentration of >75 µg/100 g of foodstuff is considered hazardous to humans. In the 1972 New England “red tide,” the concentration of saxitoxin in blue mussels exceeded 9000 µg/100 g of foodstuff.

1	The onset of intraoral and perioral paresthesias (notably of the lips, tongue, and gums) comes within minutes to a few hours after ingestion of contaminated shellfish, and these paresthesias progress rapidly to involve the neck and distal extremities. The tingling or burning sensation later changes to numbness. Other symptoms rapidly develop and include light-headedness, disequilibrium, incoordination, weakness, hyperreflexia, incoherence, dysarthria, sialorrhea, dysphagia, thirst, diarrhea, abdominal pain, nausea, vomiting, nystagmus, dysmetria, headache, diaphoresis, loss of vision, chest pain, and tachycardia. Flaccid paralysis and respiratory insufficiency may follow 2–12 h after ingestion. In the absence of hypoxia, the victim often remains alert but paralyzed. Up to 12% of patients die.

1	Treatment is supportive and based on symptoms. If the victim comes to medical attention within the first few hours after poison ingestion, the stomach should be emptied by gastric lavage and then irrigated with 2 L (in 200-mL aliquots) of a solution of 2% sodium bicarbonate; this intervention has not been proved to be of benefit but is based on the notion that gastric acidity may enhance the potency of saxitoxin. Because breathing difficulty can be rapid in onset, induction of emesis is not advised. The administration of activated charcoal (50–100 g) and a cathartic (sorbitol, 20–50 g) makes empirical sense because these shellfish toxins are believed to bind well to charcoal. Some authors advise against administration of magnesium-based solutions (e.g., certain cathartics), cautioning that hypermagnesemia may contribute to suppression of nerve conduction.

1	The most serious problem is respiratory paralysis. The victim should be closely observed for respiratory distress for at least 24 h in a hospital. With prompt recognition of ventilatory failure, endotracheal intubation, and assisted ventilation, anoxic myocardial and brain injury may be prevented. If the patient survives for 18 h, the prognosis is good for a complete recovery. A direct human serum assay to identify the toxin responsible for paralytic shellfish poisoning is not yet clinically available; the mouse bioassay in widespread use may be replaced by an automated tissue-culture bioassay. A polyclonal enzyme-linked immunosor-2743 bent assay (ELISA) to measure specific toxins is under development, as is HPLC-FLD. In addition, an inhibition immunoassay that may be able to simultaneously detect paralytic shellfish, diarrhetic shellfish, and amnesic shellfish toxins is being investigated.

1	In late 1987 in eastern Canada, an outbreak of gastrointestinal and neurologic symptoms (amnestic shellfish poisoning) was documented in persons who had consumed mussels found to be contaminated with domoic acid. In this outbreak, the source of the toxin was Nitzschia pungens, a diatom ingested by the mussels. Since the Canadian outbreak, the toxin has been found in shellfish from the United States, the United Kingdom, and Spain. In 1991, an epidemic of domoic acid poisoning in the state of Washington was attributed to the consumption of razor clams. A water-soluble, heat-stable neuroexcitatory amino acid with biochemical analogues of kainic acid and glutamic acid, domoic acid binds to the kainate type of glutamate receptor with three times the affinity of kainic acid and is 20 times as powerful a toxin. Shellfish can be tested for domoic acid by mouse bioassay and HPLC. The regulatory limit for domoic acid in shellfish is 20 parts per million.

1	The abnormalities noted within 24 h of ingesting contaminated mussels (Mytilus edulis) include arousal, confusion, disorientation, and memory loss. The median time of onset is 5.5 h. Other prominent signs and symptoms include severe headache, nausea, vomiting, diarrhea, abdominal cramps, hiccups, arrhythmias, hypotension, seizures, ophthalmoplegia, pupillary dilation, piloerection, hemiparesis, mutism, grimacing, agitation, emotional lability, coma, copious bronchial secretions, and pulmonary edema. Histologic study of brain tissue taken at autopsy has shown neuronal necrosis or cell loss and astrocytosis, most prominently in the hippocampus and the amygdaloid nucleus—findings similar to those in animals poisoned with kainic acid. Several months after the primary intoxication, victims still display chronic residual memory deficits and motor neuronopathy or axonopathy. Nonneurologic illness does not persist.

1	Therapy is supportive and based on symptoms. Because kainic acid neuropathology seems to be nearly entirely seizure mediated, the emphasis should be on anticonvulsive therapy, for which diazepam appears to be as effective as any other drug. Scombroid fish poisoning may be the most common type of seafood poisoning worldwide. It follows consumption of scombroid (mackerellike) fish, which include albacore, bluefin, and yellowfin tuna; mackerel; saury; needlefish; wahoo; skipjack; and bonito, as well as nonscombroid fish, such as dolphinfish (Hawaiian mahimahi, Coryphaena hippurus), kahawai, sardine, black marlin, pilchard, anchovy, herring, amberjack, and Australian ocean salmon. In the northeastern and mid-Atlantic United States, bluefish (Pomatomus saltatrix) has been linked to scombroid poisoning. Because greater numbers of nonscombroid fish are being recognized as scombrotoxic, the syndrome may more appropriately be called pseudoallergic fish poisoning.

1	Under conditions of inadequate preservation or refrigeration, the musculature of these darkor red-fleshed fish undergoes decomposition by Proteus morganii and Klebsiella pneumoniae bacteria, with consequent decarboxylation of the amino acid l-histidine to histamine, histamine phosphate, and histamine hydrochloride. Histamine levels of 20–50 mg/100 g are noted in toxic fish, with levels >400 mg/100 g on occasion. However, it is possible that some other compound may be responsible for this intoxication, because large doses of oral histamine do not reproduce the affliction. It is proposed that this unknown agent works by inhibiting the metabolism of histamine, promoting degranulation of mast cells to release endogenous histamine, or acting as a histamine receptor agonist. Whatever toxin or toxins are involved 2744 are heat stable and are not destroyed by domestic or commercial cooking. Affected fish typically have a sharply metallic or peppery taste; however, they may be normal in

1	or toxins are involved 2744 are heat stable and are not destroyed by domestic or commercial cooking. Affected fish typically have a sharply metallic or peppery taste; however, they may be normal in appearance, color, and flavor. Not all persons who eat a contaminated fish necessarily become ill, perhaps because of uneven distribution of decay within the fish. Symptoms develop within 15–90 min of ingestion. Most cases are mild, with tingling of lips and mouth, mild abdominal discomfort, and nausea. The more severe and commonly described presentation includes flushing (sharply demarcated; exacerbated by ultraviolet exposure; particularly pronounced on the face, neck, and upper trunk), a sensation of warmth without elevated core temperature, conjunctival hyperemia, pruritus, urticaria, and angioneurotic edema. This syndrome may progress to bronchospasm, nausea, vomiting, diarrhea, epigastric pain, abdominal cramps, dysphagia, headache, thirst, pharyngitis, gingival burning, palpitations,

1	edema. This syndrome may progress to bronchospasm, nausea, vomiting, diarrhea, epigastric pain, abdominal cramps, dysphagia, headache, thirst, pharyngitis, gingival burning, palpitations, tachycardia, dizziness, and hypotension. Without treatment, the symptoms generally resolve within 8–12 h. Because of blockade of gastrointestinal tract histaminase, the reaction may be more severe in a person who is concurrently ingesting isoniazid.

1	Therapy is directed at reversing the histamine effect with antihistamines, either H-1 or H-2. If bronchospasm is severe, an inhaled bronchodilator—or in rare, extremely severe circumstances, injected epinephrine—may be used. Glucocorticoids are of no proven benefit. Protracted nausea and vomiting, which may empty the stomach of toxin, may be controlled with a specific antiemetic, such as ondansetron or prochlorperazine. The persistent headache of scombroid poisoning may respond to cimetidine or a similar antihistamine if standard analgesics are not effective.

1	Poisoning, Drug Overdose, and Envenomation Ectoparasite Infestations and Arthropod Injuries Richard J. Pollack, Scott A. Norton Ectoparasites include arthropods and creatures from other phyla that infest the skin or hair of animals; the host animals provide them with sustenance and shelter. The ectoparasites may penetrate within or beneath the surface of the host or may attach by mouthparts and spe-cialized claws. These organisms may inflict direct mechanical injury, consume blood or nutrients, induce hypersensitivity reactions, inocu-late toxins, transmit pathogens, and incite fear or disgust. Humans are the sole or obligate hosts for many kinds of ectoparasites and serve as facultative or paratenic (accidental) hosts for many others. Arthropods that are ectoparasitic or otherwise cause injury include insects (such as lice, fleas, bedbugs, wasps, ants, bees, and flies), arach-nids (spiders, scorpions, mites, and ticks), millipedes, and centipedes. Certain nematodes (helminths), such

1	injury include insects (such as lice, fleas, bedbugs, wasps, ants, bees, and flies), arach-nids (spiders, scorpions, mites, and ticks), millipedes, and centipedes. Certain nematodes (helminths), such as the hookworms (Chap. 256), are ectoparasitic in that they penetrate and migrate through the skin. Infrequently encountered ectoparasites in other phyla include the pen-tastomes (tongue worms) and leeches. Arthropods may also cause injury when they attempt to take a blood meal or as they defend themselves by biting, stinging, or exuding ven-oms. Various arachnids (spiders and scorpions), insects (bees, hornets, wasps, ants, flies, true bugs, caterpillars, and beetles), millipedes, and centipedes produce ill effects during these behaviors. Similarly, certain ectoparasites (e.g., ticks, biting mites, and fleas) that typically infest nonhuman animals can be medically significant. In the United States, lesions caused by arthropod bites and stings are so diverse and vari-able that it is

1	mites, and fleas) that typically infest nonhuman animals can be medically significant. In the United States, lesions caused by arthropod bites and stings are so diverse and vari-able that it is rarely possible to identify the precise causative organism without a bona fide specimen and taxonomic expertise. 475

1	The human itch mite, Sarcoptes scabiei var. hominis, is a common cause of itching dermatosis, infesting ~300 million persons worldwide at any one time. Gravid female mites (~0.3 mm in length) burrow superficially within the stratum corneum, depositing three or fewer eggs per day. Six-legged larvae mature to eight-legged nymphs and then to adults. Gravid adult females emerge to the surface of the skin about 8 days later and then (re)invade the skin of the same or another host. Newly fertilized female mites are transferred from person to person mainly by direct skin-to-skin contact; transfer is facilitated by crowding, poor hygiene, and sex with multiple partners. Generally, these mites die within a day or so in the absence of host contact. Transmission via sharing of contaminated bedding or clothing occurs far less frequently than is often thought. In the United States, scabies may account for up to 5% of visits to dermatologists. Outbreaks occur in preschools, hospitals, nursing

1	or clothing occurs far less frequently than is often thought. In the United States, scabies may account for up to 5% of visits to dermatologists. Outbreaks occur in preschools, hospitals, nursing homes, and other residential institutions.

1	The itching and rash associated with scabies derive from a sensitization reaction to the mites and their secretions/excretions. A person’s initial infestation remains asymptomatic for up to 6 weeks before the onset of intense pruritus, but a reinfestation produces a hypersensitivity reaction without delay. Burrows become surrounded by inflammatory infiltrates composed of eosinophils, lymphocytes, and histiocytes, and a generalized hypersensitivity rash later develops in remote sites. Immunity and associated scratching limit most infestations to <15 mites per person. Hyperinfestation with thousands of mites, a condition known as crusted scabies (formerly termed Norwegian scabies), may result from glucocorticoid use, immunodeficiency, and neurologic or psychiatric illnesses that limit the itch and/or the scratch response.

1	Pruritus typically intensifies at night and after hot showers. Classic burrows are often difficult to find because they are few in number and may be obscured by excoriations. Burrows appear as dark wavy lines in the upper epidermis and are 3–15 mm long. Scabetic lesions are most common on the volar wrists and along the digital web spaces. In males, the penis and scrotum become involved. Small papules and vesicles, often accompanied by eczematous plaques, pustules, or nodules, appear symmetrically at those sites; within intertriginous areas; around the navel and belt line; in the axillae; and on the buttocks and upper thighs. Except in infants, the face, scalp, neck, palms, and soles are usually spared. Crusted scabies often resembles psoriasis: both are characterized by widespread thick keratotic crusts, scaly plaques, and dystrophic nails. Characteristic burrows are not seen in crusted scabies, and patients usually do not itch, although their infestations are highly contagious and

1	keratotic crusts, scaly plaques, and dystrophic nails. Characteristic burrows are not seen in crusted scabies, and patients usually do not itch, although their infestations are highly contagious and have been responsible for outbreaks of classic scabies in hospitals.

1	Scabies should be considered in patients with pruritus and symmetric superficial, excoriated, papulovesicular skin lesions in characteristic locations, particularly if there is a history of household contact with an infested person. Burrows should be sought and unroofed with a sterile needle or scalpel blade, and the scrapings should be examined microscopically for mites, eggs, and fecal pellets. Examination of skin biopsies (including superficial cyanoacrylate biopsy) or scrapings, dermatoscopic imaging of papulovesicular lesions, and microscopic inspection of clear cellophane tape lifted from lesions also may be diagnostic. In the absence of identifiable mites or eggs, the diagnosis is based on a history of pruritus, a clinical examination, and an epidemiologic link. Diverse kinds of dermatitis from other causes frequently are misdiagnosed as scabies, particularly in presumed “outbreak” situations. Scabies mites of other animals may cause transient irritation, but they do not reside

1	from other causes frequently are misdiagnosed as scabies, particularly in presumed “outbreak” situations. Scabies mites of other animals may cause transient irritation, but they do not reside or reproduce in human hosts.

1	Permethrin cream (5%) is less toxic than 1% lindane preparations and is effective against lindane-tolerant infestations. Scabicides are applied thinly but thoroughly behind the ears and from the neck down after bathing—with careful application to interdigital spaces and the umbilicus and under the fingernails—and are removed 8–14 h later with soap and water. Successful treatment of crusted scabies requires preapplication of a keratolytic agent such as 6% salicylic acid and then of scabicides to the scalp, face, and ears. Repeated treatments or the sequential use of several agents may be necessary. Ivermectin has not been approved by the U.S. Food and Drug Administration (FDA) for treatment of any form of scabies, but a single oral dose (200 μg/kg) is effective in otherwise healthy persons; patients with crusted scabies may require two doses separated by an interval of 1–2 weeks. All FDA-approved scabicides are available solely by prescription.

1	Within 1 day of effective treatment, scabies infestations become noncommunicable, but the pruritic hypersensitivity dermatitis induced by the now-dead mites and their remnant products frequently persists for weeks. Unnecessary re-treatment with topical agents may provoke contact dermatitis. Antihistamines, salicylates, and calamine lotion relieve itching during treatment, and topical glucocorticoids are useful for pruritus that lingers after effective treatment. To prevent reinfestations, bedding and clothing should be washed and dried on high heat or heat-pressed. Close contacts of confirmed cases, even if asymptomatic, should be treated simultaneously.

1	Chiggers are the larvae of trombiculid (harvest) mites that normally feed on mice in grassy or brush-covered sites in tropical, subtropical, and (less frequently) temperate areas during warm months. They reside on low vegetation and attach themselves to passing mammalian hosts. While feeding, larvae secrete saliva with proteolytic enzymes to create a tube-like invagination in the host’s skin; this stylostome allows the mite to imbibe tissue fluids. The stylostomal saliva is highly antigenic and causes exceptionally pruritic papular, papulovesicular, or papulourticarial lesions (≤2 cm in diameter). In people previously sensitized to salivary antigens, the papules develop within hours of attachment. While attached, mites appear as tiny red vesicles on the skin. Generally, lesions vesicate and develop a hemorrhagic base. Scratching, however, invariably destroys the body of a mite. Itching and burning often persist for weeks. The rash is common on the ankles and areas where clothing

1	and develop a hemorrhagic base. Scratching, however, invariably destroys the body of a mite. Itching and burning often persist for weeks. The rash is common on the ankles and areas where clothing obstructs the further wanderings of the mites. Repellents are useful for preventing chigger bites.

1	Many kinds of mites that are associated with peridomestic birds and rodents are particularly bothersome when they invade homes and bite people. In North America, the northern fowl mite, chicken mite, tropical rat mite, and house mouse mite normally feed on poultry, various songbirds, and small mammals and are abundant in and around their hosts’ nests. After their natural hosts die or leave the nest, these mites frequently invade human habitations. Although the mites are rarely seen because of their small size, their bites can be painful and pruritic. Once confirmed as the cause of irritation, rodentand bird-associated mites are best eliminated by excluding their hosts, removing the nests, and cleaning and treating the nesting area with appropriate acaricides. Pyemotes and other mites that infest grain, straw, cheese, hay, or other products occasionally produce similar episodes of rash and discomfort and may produce a unique dermatologic “comet sign” lesion—a paisley-shaped urticarial

1	infest grain, straw, cheese, hay, or other products occasionally produce similar episodes of rash and discomfort and may produce a unique dermatologic “comet sign” lesion—a paisley-shaped urticarial plaque.

1	Diagnosis of mite-induced dermatitides (including those caused by chiggers) relies on confirmation of the mite’s identity or elicitation of a history of exposure to the mite’s source. Oral antihistamines or topical steroids may suppress mite-induced pruritus temporarily but do not eliminate the mites.

1	Ticks attach superficially to skin and feed painlessly; blood is their only food. Their salivary secretions are biologically active and can produce local reactions, induce fevers, and cause paralysis in addition to transmitting diverse pathogens. The two main families of ticks are the hard (ixodid) and soft (argasid) ticks. Generally, soft ticks attach for <1 h, leaving red macules after they drop off. Some species in Africa, the western United States, and Mexico produce painful hemorrhagic lesions. Hard ticks are much more common and transmit most of the 2745 tick-borne infections that are familiar to physicians and patients. Hard ticks attach to the host and feed for several days or sometimes for >1 week. At the site of hard-tick bites, small areas of induration, often purpuric, develop and may be surrounded by an erythematous rim. A necrotic eschar, called a tâche noire, occasionally develops. Chronic nodules (persistent tick-bite granulomas) can be several centimeters in diameter

1	and may be surrounded by an erythematous rim. A necrotic eschar, called a tâche noire, occasionally develops. Chronic nodules (persistent tick-bite granulomas) can be several centimeters in diameter and may linger for months after the feeding tick has been removed. These granulomas can be treated with injected intralesional glucocorticoids or by surgical excision. Tick-induced fever, unas sociated with transmission of any pathogen, is often accompanied by headache, nausea, and malaise but usually resolves ≤36 h after the tick is removed.

1	Tick paralysis, an acute ascending flaccid paralysis that resembles Guillain-Barré syndrome, is believed to be caused by one or more toxins in tick saliva that block neuromuscular transmission and decrease nerve conduction. This rare complication has followed the bites of more than 60 kinds of ticks, although in the United States dog and wood ticks (Dermacentor species) are most commonly involved.

1	Weakness begins symmetrically in the lower extremities ≤6 days after the tick’s attachment, ascends symmetrically during several days, and may culminate in complete paralysis of the extremities and cranial nerves. Deep tendon reflexes are diminished or absent, but sensory examination and findings on lumbar puncture are typically normal. Removal of the tick generally leads to rapid improvement within a few hours and complete recovery after several days, although the patient’s condition may continue to deteriorate for a full day. Failure to remove the tick may lead to dysarthria, dysphagia, and ultimately death from aspiration or respiratory paralysis. Diagnosis depends on finding the tick, which is often hidden beneath scalp hair. An antiserum to the saliva of Ixodes holocyclus, the usual cause of tick paralysis in Australia, effectively reverses paralysis caused by these ticks.

1	Removal of hard ticks during the first 36 h of attachment nearly always prevents transmission of the agents of Lyme disease, babesiosis, anaplasmosis, and ehrlichiosis, although several tick-borne viruses may be transmitted more quickly. Ticks should be removed by traction with fine-tipped forceps placed firmly around the tick’s mouthparts. Careful handling (to avoid rupture of ticks) and use of gloves may avert accidental contamination with pathogens contained in tick fluids. Use of occlusive dressings, heat, or other substances (in an attempt to induce the tick to detach) merely delay tick removal. Afterward, the site of attachment should be disinfected. Tick mouthparts sometimes remain in the skin but generally are shed spontaneously within days without excision. Although somewhat controversial, current guidelines from the Centers for Disease Control and Prevention suggest that, rather than awaiting the onset of erythema migrans, the results of tick testing, or seroconversion to

1	current guidelines from the Centers for Disease Control and Prevention suggest that, rather than awaiting the onset of erythema migrans, the results of tick testing, or seroconversion to antigens diagnostic for Lyme disease, administering prophylaxis with a single oral dose of doxycycline (200 mg) within 72 h of tick removal is appropriate in adult patients with bites thought to be associated with deer ticks (Fig. 475-1) in Lyme disease–endemic areas from Maryland to Maine and in Wisconsin and Minnesota.

1	Nymphs and adults of all three kinds of human lice feed at least once a day, ingesting human blood exclusively. Head lice (Pediculus capitis) infest mainly the hair of the scalp, body lice (Pediculus humanus) the clothing, and crab or pubic lice (Pthirus pubis) mainly the hair of the pubis. The saliva of lice produces a pruritic morbilliform or urticarial rash in some sensitized persons. Female head and pubic lice cement their eggs (nits) firmly to hair, whereas female body lice cement their eggs to clothing, particularly to threads along clothing seams. After ~10 days of development within the egg, a nymph hatches. Empty eggs may remain affixed for months thereafter.

1	In North America, head lice infest ~1% of elementary school-age children. Head lice are transmitted mainly by direct head-to-head contact rather than by fomites such as shared headgear, bed linens, hairbrushes, and other grooming implements. Chronic infestations by head lice tend to be asymptomatic. Pruritus, due mainly to hypersensitivity to the louse’s saliva, generally is transient and mild. Head lice FIGUrE 475-1 Deer ticks (Ixodes scapularis, black-legged ticks) on a U.S. penny: larva(below ear),nymph(right),adultmale(above),andadultfemale(left). removed from a person succumb to desiccation and starvation within ~1 day. Head lice are not known to serve as a natural vector for any pathogens.

1	Body lice remain on clothing except when feeding and generally succumb in ≤2 days if separated from their host. In most Western countries, body lice are generally found on a small proportion of indigent persons but may become increasingly prevalent after upheaval associated with natural or human-caused disasters, when homeless victims are in close contact with infested individuals with whom they share accommodations. Body lice are acquired by direct contact or by sharing of infested clothing and bedding. These lice are vectors for the agents of louse-borne (epidemic) typhus (Chap. 211), louse-borne relapsing fever (Chap. 209), and trench fever (Chap. 197). Pruritic lesions from their bites are particularly common around the neckline. Chronic infestations result in a postinflammatory hyperpigmentation and thickening of skin known as vagabond’s disease.

1	The crab or pubic louse is transmitted mainly by sexual contact. These lice occur predominantly on pubic hair and less frequently on axillary or facial hair, including the eyelashes. Children and adults may acquire pubic lice by sexual or close nonsexual contact. Intensely pruritic, bluish macules ~3 mm in diameter (maculae ceruleae) develop at the site of bites. Blepharitis commonly accompanies infestations of the eyelashes. Pediculiasis is often suspected upon the detection of nits firmly cemented to hairs or in clothing. Many bona fide nits, however, are dead or hatched relics of prior infestation, and pseudo-nits are frequently misconstrued to be signs of a louse infestation. Confirmation of a louse infestation, therefore, best relies on the discovery of a live louse.

1	Generally, treatment is warranted only if live lice are discovered. The presence of nits alone is evidence of a former—not necessarily current—infestation. Mechanical removal of lice and their eggs with a fine-toothed louse or nit comb (Fig. 475-2) often fails to eliminate infestations. Treatment of newly identified active infestations generally relies on a 10-min topical application of ~1% permethrin or pyrethrins, with a second application ~10 days later. Lice persisting after this treatment may be resistant to pyrethroids. Chronic infestations may be treated for ≤12 h with 0.5% malathion. Lindane is applied for just 4 min but seems less effective and may pose a greater risk of adverse reactions, particularly when misused. Resistance of head lice to permethrin, malathion, and lindane has been reported. Newer FDA-approved topical pediculicides contain benzyl alcohol, dimethicone, spinosad, and ivermectin. Although children infested by head lice—or those who simply have remnant nits

1	been reported. Newer FDA-approved topical pediculicides contain benzyl alcohol, dimethicone, spinosad, and ivermectin. Although children infested by head lice—or those who simply have remnant nits from a prior infestation—are frequently isolated or excluded a nit (louse-egg) comb.

1	from school, this practice increasingly is seen as unjustified and ineffective. Body lice usually are eliminated by bathing and by changing to laundered clothes. Application of topical pediculicides from head to foot may be necessary for hirsute patients. Clothes and bedding are effectively deloused by heating in a clothes dryer at ≥55°C (≥131°F) for 30 min or by heat-pressing. Emergency mass delousing of persons and clothing may be warranted during periods of civil strife and after natural disasters to reduce the risk of pathogen transmission by body lice. Pubic louse infestations are treated with topical pediculicides, except for eyelid infestations (pthiriasis palpebrum), which generally respond to a coating of petrolatum applied for 3–4 days. Myiasis refers to infestations by several kinds of fly larvae (maggots) that invade living or necrotic tissues or body cavities and produce different clinical syndromes, depending on the species of fly.

1	In forested parts of Central and South America, larvae of the human botfly (Dermatobia hominis) produce furuncular (boil-like) papules or subcutaneous nodules ≤3 cm in diameter. A gravid adult female botfly captures a mosquito or another bloodsucking insect and deposits her eggs on its abdomen. When the carrier insect attacks a human or bovine host several days later, the warmth and moisture of the host’s skin stimulate the eggs to hatch. The emerging larvae promptly penetrate intact skin. After 6–12 weeks of development, mature larvae emerge from the skin and drop to the ground to pupate and then become adults.

1	The African tumbu fly (Cordylobia anthropophaga) deposits its eggs on damp sand or leaf litter or on drying laundry, particularly that contaminated by urine or sweat. Larvae hatch from eggs upon contact with a host’s body and penetrate the skin, producing boil-like lesions from which mature larvae emerge ~9 days later. Furuncular myiasis is suggested by uncomfortable lesions with a central breathing pore that emits bubbles when submerged in water. A sensation of movement under the patient’s skin may cause severe emotional distress. Larvae that cause furuncular myiasis may be induced to emerge if the air pore is coated with petrolatum or another occlusive substance. Removal may be facilitated by injection of a local anesthetic into the surrounding tissue, but surgical excision is sometimes necessary because upward-pointing spines of some species hold the larvae firmly in place.

1	Other fly larvae cause nonfuruncular myiasis. For example, larvae of the horse botfly (Gasterophilus intestinalis) emerge from eggs deposited on the horse’s flanks and may come into contact with and infest human beings. After penetrating human skin, these larvae rarely mature but instead may migrate for weeks in the dermis. The resulting pruritic and serpiginous eruption resembles cutaneous larva migrans caused by canine or feline hookworms (Chap. 256). Larvae of rabbit and rodent botflies (Cuterebra species) occasionally cause dermal or tracheopulmonary myiasis.

1	Certain flies are attracted to blood and pus, laying their eggs on open or draining sores. Newly hatched larvae enter wounds or diseased skin. Larvae of several types of green bottle flies (Lucilia/Phaenicia species) usually remain superficial and confined to necrotic tissue. Specially raised, sterile “surgical maggots” are sometimes used intentionally for wound debridement. Larvae of screwworm flies, Cochliomyia, and the flesh fly invade viable tissues more deeply and produce large suppurating lesions. Larvae that infest wounds also may enter body cavities such as the mouth, nose, ears, sinuses, anus, vagina, and lower urinary tract, particularly in unconscious or otherwise debilitated patients. The consequences range from harmless colonization to destruction of the nose, meningitis, and deafness. Treatment involves removal of maggots and debridement of tissue.

1	The maggots responsible for furuncular and wound myiasis also may cause ophthalmomyiasis. Sequelae include nodules in the eyelid, retinal detachment, and destruction of the globe. Most instances in which maggots are found in human feces result from deposition of eggs or larvae by flies on recently passed stools, not from an intestinal maggot infestation.

1	Pentastomids (tongue worms) inhabit the respiratory passages of reptiles and carnivorous mammals. Human infestation by Linguatula serrata is common in the Middle East and results from the consumption of encysted larval stages in raw liver or lymph nodes of sheep and goats, which are true intermediate hosts for the tongue worms. Larvae migrate to the nasopharynx and produce an acute self-limiting syndrome—known as halzoun or marrara—characterized by pain and itching of the throat and ears, coughing, hoarseness, dysphagia, and dyspnea. Severe edema may cause obstruction that requires tracheostomy. In addition, ocular invasion has been described. Diagnostic larvae measuring ≤10 mm in length appear in copious nasal discharge or vomitus. Individuals become infected with another type of tongue worm, Armillifer armillatus, by consuming its eggs in contaminated food or drink or after handling the definitive host, the African python. Larvae encyst in various organs but rarely cause symptoms.

1	worm, Armillifer armillatus, by consuming its eggs in contaminated food or drink or after handling the definitive host, the African python. Larvae encyst in various organs but rarely cause symptoms. Cysts may require surgical removal as they enlarge during molting, but they usually are encountered as an incidental finding at autopsy. Parasite-induced lesions may be misinterpreted as a malignancy, with the correct diagnosis confirmed histopathologically. Cutaneous larva migrans–type syndromes of other pentastomes have been reported from Southeast Asia and Central America.

1	Medically important leeches are annelid worms that attach to their hosts with chitinous cutting jaws and draw blood through muscular suckers. The medicinal leech (Hirudo medicinalis) is still used occasionally for medical purposes to reduce venous congestion in surgical flaps or replanted body parts. This practice has been complicated by intractable bleeding, wound infections, myonecrosis, and sepsis due to Aeromonas hydrophila, which colonizes the gullets of commercially available leeches.

1	Ubiquitous aquatic leeches that parasitize fish, frogs, and turtles readily attach to the skin of human beings and avidly suck blood. More notorious are arboreal land leeches that live among moist vegetation of tropical rain forests. Attachment is usually painless, and the leeches will detach themselves when satiated with a blood meal. Hirudin, a powerful anticoagulant secreted by the leech, causes continued bleeding after the leech has detached. Healing of a leech-bite wound is slow, and bacterial infections are not uncommon. Several kinds of aquatic leeches in Africa, Asia, and southern Europe can enter the mouth, nose, and genitourinary tract and attach to mucosal surfaces at sites as deep as the esophagus and trachea. Externally attached leeches generally drop off after they have engorged, but removal is hastened by gentle scraping aside of the anterior and posterior suckers the leech uses for attachment and feeding. Some authorities dispute the wisdom of removing leeches with

1	but removal is hastened by gentle scraping aside of the anterior and posterior suckers the leech uses for attachment and feeding. Some authorities dispute the wisdom of removing leeches with alcohol, salt, vinegar, insect repellent, a flame or heated instrument, or applications of other noxious substances.

1	Internally attached leeches may detach on exposure to gargled saline 2747 or may be removed by forceps.

1	Of the more than 30,000 recognized species of spiders, only ~100 defend themselves aggressively and have fangs sufficiently long to penetrate human skin. The venom that some spiders use to immobilize and digest their prey can cause necrosis of skin and systemic toxicity. Whereas the bites of most spiders are painful but not harmful, envenomations by recluse or fiddleback spiders (Loxosceles species) and widow spiders (Latrodectus species) may be life-threatening. Identification of the offending spider is important because specific treatments exist for bites of widow spiders and because injuries attributed to spiders are frequently due to other causes. Except in cases where the patient actually observes a spider immediately associated with the bite or fleeing from the site, lesions reported as spider-bite reactions are most often due to other injuries or to infections with bacteria such as methicillin-resistant Staphylococcus aureus (MRSA).

1	recluse Spider Bites and Necrotic arachnidism Brown recluse spiders live mainly in the south-central United States and have close relatives in Central and South America, Africa, and the Middle East. Bites by brown recluse spiders usually cause only minor injuries, with edema and erythema. Envenomation, however, occasionally causes severe necrosis of skin and subcutaneous tissue and more rarely causes systemic hemolysis. These spiders are not aggressive toward humans and will bite only if threatened or pressed against the skin. They hide under rocks and logs or in caves and animal burrows. They invade homes and seek dark and undisturbed hiding spots in closets, in folds of clothing, or under furniture and rubbish in storage rooms, garages, and attics. Despite their impressive abundance in some homes, these spiders rarely bite humans. Bites tend to occur while the victim is dressing and are sustained primarily on the hands, arms, neck, and lower abdomen.

1	Initially, the bite is painless or may produce a stinging sensation. Within the next few hours, the site becomes painful and pruritic, with central induration surrounded by a pale ischemic zone that itself is encircled by a zone of erythema. In most cases, the lesion resolves without treatment in just a few days. In severe cases, the erythema spreads, and the center of the lesion becomes hemorrhagic or necrotic with an overlying bulla. A black eschar forms and sloughs several weeks later, leaving an ulcer that eventually may create a depressed scar. Healing usually takes place in ≤6 months but may take as long as 3 years if adipose tissue is involved. Local complications include injury to nerves and secondary bacterial infection. Fever, chills, weakness, headache, nausea, vomiting, myalgia, arthralgia, maculopapular rash, and leukocytosis may develop ≤72 h after the bite. Reports of deaths attributed to bites of North American brown recluse spiders have not been verified.

1	Initial management includes RICE (rest, ice, compression, elevation). Analgesics, antihistamines, antibiotics, and tetanus prophylaxis should be administered if indicated. Early debridement or surgical excision of the wound without closure delays healing. Routine use of antibiotics or dapsone is unnecessary. Patients should be monitored closely for signs of hemolysis, renal failure, and other systemic complications. Widow Spider Bites The black widow spider, common in the southeastern United States, measures ≤1 cm in body length and 5 cm in leg span and is shiny black with a red hourglass marking on the ventral abdomen. Other dangerous Latrodectus species occur elsewhere in temperate and subtropical parts of the world. The bites of the female widow spiders are notorious for their potent neurotoxins.

1	Widow spiders spin their webs under stones, logs, plants, or rock piles and in dark spaces in barns, garages, and outhouses. Bites are most common in the summer and early autumn and occur when a web is disturbed or a spider is trapped or provoked. The initial bite 2748 is perceived as a sharp pinprick or may go unnoticed. Fang-puncture marks are uncommon. The venom that is injected does not produce local necrosis, and some persons experience no other symptoms. α-Latrotoxin, the most active component of the venom, binds irreversibly to presynaptic nerve terminals and causes release and eventual depletion of acetylcholine, norepinephrine, and other neurotransmitters from those terminals. Painful cramps may spread within 60 min from the bite site to large muscles of the extremities and trunk. Extreme rigidity of the abdominal muscles and excruciating pain may suggest peritonitis, but the abdomen is not tender on palpation and surgery is not warranted. The pain begins to subside during

1	Extreme rigidity of the abdominal muscles and excruciating pain may suggest peritonitis, but the abdomen is not tender on palpation and surgery is not warranted. The pain begins to subside during the first 12 h but may recur during several days or weeks before resolving spontaneously. A wide range of other neurologic sequelae may include salivation, diaphoresis, vomiting, hypertension, tachycardia, labored breathing, anxiety, headache, weakness, fasciculations, paresthesia, hyperreflexia, urinary retention, uterine contractions, and premature labor. Rhabdomyolysis and renal failure have been reported, and respiratory arrest, cerebral hemorrhage, or cardiac failure may end fatally, especially in very young, elderly, or debilitated persons.

1	Treatment consists of RICE and tetanus prophylaxis. Hypertension that does not respond to analgesics and antispasmodics (e.g., benzodiazepines or methocarbamol) requires specific antihypertensive medication. The efficacy and safety of antivenoms for black widow and redback spiders are controversial because of concerns about potential anaphylaxis or serum sickness.

1	tarantulas and Other Spiders Tarantulas are hairy spiders of which 30 species are found in the United States, mainly in the Southwest. The tarantulas that have become popular household pets are usually imported from Central or South America. Tarantulas bite only when threatened and usually cause no more harm than a bee sting, but on occasion the venom causes deep pain and swelling. Several species of tarantulas are covered with urticating hairs that are brushed off in the thousands when a threatened spider rubs its hind legs across its dorsal abdomen. These hairs can penetrate human skin and produce pruritic papules that may persist for weeks. Failure to wear gloves or to wash the hands after handling the Chilean Rose tarantula, a popular pet spider, has resulted in transfer of hairs to the eye with subsequent devastating ocular inflammation. Treatment of bites includes local washing and elevation of the bitten area, tetanus prophylaxis, and analgesic administration. Antihistamines

1	the eye with subsequent devastating ocular inflammation. Treatment of bites includes local washing and elevation of the bitten area, tetanus prophylaxis, and analgesic administration. Antihistamines and topical or systemic glucocorticoids are given for exposure to urticating hairs.

1	Atrax robustus, a funnel-web spider of Australia, and Phoneutria species, the South American banana spiders, are among the most dangerous spiders in the world because of their aggressive behavior and potent neurotoxins. Envenomation by A. robustus causes a rapidly progressive neuromotor syndrome that can be fatal within 2 h. The bite of a banana spider causes severe local pain followed by profound systemic symptoms and respiratory paralysis that can lead to death within 2–6 h. Specific antivenoms for use after bites by each of these spiders are available. Yellow sac spiders (Cheiracanthium species) are common in homes worldwide. Their bites, though painful, generally lead to only minor erythema, edema, and pruritus.

1	Scorpions are arachnids that feed on ground-dwelling arthropods and small lizards. They paralyze their prey and defend themselves by injecting venom from a stinger on the tip of the tail. Painful but relatively harmless scorpion stings need to be distinguished from the potentially lethal envenomations that are produced by ~30 of the ~1000 known species and that cause more than 5000 deaths worldwide each year. Scorpions are nocturnal and remain hidden during the day in crevices or burrows or under wood, loose bark, or rocks. They occasionally enter houses and tents and may hide in shoes, clothing, or bedding. Scorpions sting humans only when threatened.

1	Of the 40 or so scorpion species in the United States, only bark scorpions—e.g., Centruroides sculpturatus (C. exilicauda) in the Southwest—produce venom that is potentially lethal to humans. This venom contains neurotoxins that cause sodium channels to remain open. Such envenomations usually are associated with little swelling, but prominent pain, paresthesia, and hyperesthesia can be accentuated by tapping on the affected area (the tap test). These symptoms soon spread to other locations; dysfunction of cranial nerves and hyperexcitability of skeletal muscles develop within hours. Patients present with restlessness, blurred vision, abnormal eye movements, profuse salivation, lacrimation, rhinorrhea, slurred speech, difficulty in handling secretions, diaphoresis, nausea, and vomiting. Muscle twitching, jerking, and shaking may be mistaken for a seizure. Complications include tachycardia, arrhythmias, hypertension, hyperthermia, rhabdomyolysis, and acidosis. Symptoms progress to

1	Muscle twitching, jerking, and shaking may be mistaken for a seizure. Complications include tachycardia, arrhythmias, hypertension, hyperthermia, rhabdomyolysis, and acidosis. Symptoms progress to maximal severity in ~5 h and subside within a day or two, although pain and paresthesia can last for weeks. Fatal respiratory arrest is most common among young children and the elderly.

1	Envenomations by Leiurus quinquestriatus in the Middle East and North Africa, by Mesobuthus tamulus in India, by Androctonus species along the Mediterranean littoral and in North Africa and the Middle East, and by Tityus serrulatus in Brazil cause massive release of endogenous catecholamines with hypertensive crises, arrhythmias, pulmonary edema, and myocardial damage. Acute pancreatitis occurs with stings of Tityus trinitatis in Trinidad, and central nervous toxicity complicates stings of Parabuthus and Buthotus scorpions of South Africa. Tissue necrosis and hemolysis may follow stings of the Iranian Hemiscorpius lepturus. Stings of most other species cause immediate sharp local pain followed by edema, ecchymosis, and a burning sensation. Symptoms typically resolve within a few hours, and skin does not slough. Allergic reactions to the venom sometimes develop.

1	Identification of the offending scorpion helps to determine the course of treatment. Stings of nonlethal species require at most ice packs, analgesics, or antihistamines. Because most victims experience only local discomfort, they can be managed at home with instructions to return to the emergency department if signs of cranial-nerve or neuromuscular dysfunction develop. Aggressive supportive care and judicious use of antivenom can reduce or eliminate deaths from more severe envenomations. Keeping the patient calm and applying pressure dressings and cold packs to the sting site are measures that decrease the absorption of venom. A continuous IV infusion of midazolam controls the agitation, flailing, and involuntary muscle movements produced by scorpion stings. Close monitoring during treatment with this drug and other sedatives or narcotics is necessary for persons with neuromuscular symptoms because of the risk of respiratory arrest. Hypertension and pulmonary edema respond to

1	treatment with this drug and other sedatives or narcotics is necessary for persons with neuromuscular symptoms because of the risk of respiratory arrest. Hypertension and pulmonary edema respond to nifedipine, nitroprusside, hydralazine, or prazosin. Dangerous bradyarrhythmias can be controlled with atropine.

1	Commercially prepared antivenoms are available in several countries for some of the most dangerous species. An FDA-approved C. sculpturatus antivenom in horse serum is now available. IV administration of antivenom rapidly reverses cranial-nerve dysfunction and muscular symptoms. Although effective, cost analyses suggest that antivenoms should be reserved for only the most severe envenomations. Insects that sting in defense or to subdue their prey belong to the order Hymenoptera, which includes bees, wasps, hornets, yellow jackets, and ants. Their venoms contain a wide array of amines, peptides, and enzymes that cause local and systemic reactions. Although the toxic effect of multiple stings can be fatal to a human, nearly all of the ≥100 deaths due to hymenopteran stings in the United States each year result from allergic reactions.

1	Bee and Wasp Stings The stinger of the honeybee (Apis mellifera) is unique in being barbed. When a bee stings a foe, its stinging apparatus and attached venom sac tear loose from its body. Muscular contraction of the venom sac continues to inject venom into the skin. Other kinds of bees, ants, and wasps have smooth stinging mechanisms and can sting numerous times in succession. Honeybees, bumblebees, and social wasps generally attack only when a colony is disturbed. Africanized honeybees (now present in South and Central America and the southern and western United States) respond to minimal intrusions more aggressively. Whereas the sting of an Africanized bee contains less venom than that of its non-Africanized relatives, victims tend to sustain far more stings and therefore to receive a far greater overall volume of venom. Most patients who report having sustained a “bee sting,” however, are more likely to have encountered stinging wasps instead.

1	The venoms of different species of hymenopterans are biochemically and immunologically distinct. Direct toxic effects are mediated by mixtures of low-molecular-weight compounds such as serotonin, histamine, acetylcholine, and several kinins. Polypeptide toxins in honeybee venom include mellitin that damages cell membranes, mast cell– degranulating protein that causes histamine release, the neurotoxin apamin, and the anti-inflammatory compound adolapin. Enzymes in venom include hyaluronidase and phospholipases. There appears to be little cross-sensitization between the venoms of honeybees and wasps.

1	Uncomplicated hymenopteran stings cause immediate pain, a wheal-and-flare reaction, and local edema, all of which usually subside in a few hours. Multiple stings can lead to vomiting, diarrhea, generalized edema, dyspnea, hypotension, and non-anaphylactic circulatory collapse. Rhabdomyolysis and intravascular hemolysis may cause renal failure. Death from the direct (nonallergic) effects of venom has followed stings of several hundred honeybees. Stings to the tongue or mouth may induce life-threatening edema of the upper airways. Large local reactions accompanied by erythema, edema, warmth, and tenderness that spread ≥10 cm around the sting site over 1–2 days are not uncommon. These reactions may resemble bacterial cellulitis but are caused by hypersensitivity rather than by secondary infection. Such reactions tend to recur on subsequent exposure but are seldom accompanied by anaphylaxis and are not prevented by venom immunotherapy.

1	An estimated 0.4–4.0% of the U.S. population exhibits clinical immediate-type hypersensitivity to hymenopteran stings, and 15% may have asymptomatic sensitization manifested by positive skin tests. Persons who experience severe allergic reactions are likely to have similar reactions after subsequent stings by the same or closely related species. Occasionally, persons who have had mild reactions earlier in life will experience more serious reactions to subsequent stings. Mild anaphylactic reactions from insect stings, as from other causes, consist of nausea, abdominal cramping, generalized urticaria, flushing, and angioedema. Serious reactions, including upper airway edema, bronchospasm, hypotension, and shock, may be rapidly fatal. Severe reactions usually begin within 10 min of the sting and only rarely develop after 5 h.

1	Honeybee stingers embedded in the skin should be removed as soon as possible to limit the quantity of venom delivered. The stinger and venom sac may be scraped off with a blade, the edge of a credit card, or a fingernail or may be removed with forceps. The site should be cleansed and disinfected and ice packs applied to slow the spread of venom. Elevation of the affected site and administration of analgesics, oral antihistamines, and topical calamine lotion help relieve symptoms. Large local reactions may require a short course of oral therapy with glucocorticoids. Patients with numerous stings should be monitored for 24 h for evidence of renal failure or coagulopathy.

1	Anaphylaxis is treated with SC injection of 0.3–0.5 mL of epinephrine hydrochloride in a 1:1000 dilution; treatment is repeated every 20–30 min as necessary. IV epinephrine (2–5 mL of a 1:10,000 solution administered by slow push) is indicated for profound shock. 2749 A tourniquet may slow the spread of venom. Parenteral antihistamines, fluid resuscitation, bronchodilators, supplemental oxygen, intubation, and vasopressors may be required. Patients should be observed for 24 h for recurrent anaphylaxis. Persons with a history of allergy to insect stings should carry an anaphylaxis kit with a preloaded syringe containing epinephrine for self-administration. These patients should seek medical attention immediately after using the kit.

1	Repeated injections of purified venom produce a blocking IgG antibody response to venom and reduce the incidence of recurrent anaphylaxis. Honeybee, wasp, and yellow jacket venoms are commercially available for desensitization and for skin testing. Results of skin tests and venom-specific radioallergosorbent tests (RASTs) aid in the selection of patients for immunotherapy and guide the design of such treatment. Stinging fire ants are an important medical problem in the United States. Imported fire ants (Solenopsis species) infest southern states from Texas to North Carolina, with colonies now established in California, New Mexico, Arizona, and Virginia. Slight disturbances of their mound nests have provoked massive outpourings of ants and as many as 10,000 stings on a single person. Elderly and immobile persons are at high risk for attacks when fire ants invade dwellings.

1	Fire ants attach to skin with powerful mandibles and rotate their bodies while repeatedly injecting venom with posteriorly situated stingers. The alkaloid venom consists of cytotoxic and hemolytic piperidines and several proteins with enzymatic activity. The initial wheal-and-flare reaction, burning, and itching resolve in ~30 min, and a sterile pustule develops within 24 h. The pustule ulcerates over the next 48 h and then heals in ≥1 week. Large areas of erythema and edema lasting several days are not uncommon and in extreme cases may compress nerves and blood vessels. Anaphylaxis occurs in fewer than 2% of victims; seizures and mononeuritis have been reported. Stings are treated with ice packs, topical glucocorticoids, and oral antihistamines. Pustules should be cleansed and then covered with bandages and antibiotic ointment to prevent bacterial infection. Epinephrine administration and supportive measures are indicated for anaphylactic reactions. Fire ant whole-body extracts are

1	with bandages and antibiotic ointment to prevent bacterial infection. Epinephrine administration and supportive measures are indicated for anaphylactic reactions. Fire ant whole-body extracts are available for skin testing and immunotherapy, which appears to lower the rate of anaphylactic reactions.

1	European fire (red) ants (Myrmica rubra) have recently become public health pests in the northeastern United States and southern Canada. The western United States is home to harvester ants (Pogonomyrmex species). The painful local reaction that follows harvester ant stings often extends to lymph nodes and may be accompanied by anaphylaxis. The bullet or conga ant (Paraponera clavata) of South America is known locally as hormiga veinticuatro (“24-hour ant”), a designation that refers to the 24 h of throbbing, excruciating pain following a sting that delivers the potent paralyzing neurotoxin poneratoxin.

1	In the process of feeding on vertebrate blood and tissue fluids, adults of certain flies inflict painful bites, produce local allergic reactions, and may transmit pathogenic agents. Bites of mosquitoes, tiny “no-seeum” (ceratopogonid) midges, and phlebotomine sand flies typically produce a wheal and a pruritic papule. Small humpbacked black flies (simuliids) lacerate skin, resulting in a lesion with serosanguineous discharge that is often painful and pruritic. Regional lymphadenopathy, fever, or anaphylaxis occasionally ensues. The widely distributed deerflies and horseflies as well as the tsetse flies of Africa are stout flies measuring ≤25 mm in length that attack during the day and produce large and painful bleeding punctures. Houseflies (Musca domestica) do not consume blood but use rasping mouthparts to scarify skin and feed upon tissue fluids and salt. Beyond direct injury from bites of any kind 2750 of fly, risks include transmission of diverse pathogens and secondary infection

1	mouthparts to scarify skin and feed upon tissue fluids and salt. Beyond direct injury from bites of any kind 2750 of fly, risks include transmission of diverse pathogens and secondary infection of the lesion.

1	Treatment of fly bites is symptom based. Topical application of antipruritic agents, glucocorticoids, or antiseptic lotions may relieve itching and pain. Allergic reactions may require oral antihistamines. Antibiotics may be necessary for the treatment of large bite wounds that become secondarily infected. Common human-biting fleas include the dog and cat fleas (Ctenocephalides species) and the rat flea (Xenopsylla cheopis), which infest their respective hosts and the hosts’ nests and resting sites. Sensitized persons develop erythematous pruritic papules (papular urticaria) and occasionally vesicles and bacterial superinfection at the site of the bite. Symptom-based treatment consists of antihistamines, topical glucocorticoids, and topical antipruritic agents.

1	Flea infestations are eliminated by removal and treatment of animal nests, frequent cleaning of pet bedding, and application of contact and systemic insecticides to pets and the dwelling. Flea infestations in the home may be abated or prevented if pets are regularly treated with veterinary antiparasitic agents and insect growth regulators.

1	Tunga penetrans, like other fleas, is a wingless, laterally flattened insect that feeds on blood. Also known as the chigoe flea, sand flea, or jigger (not to be confused with the chigger), it occurs in tropical regions of Africa and the Americas. Adult female chigoes live in sandy soil and burrow under the skin, usually between toes, under nails, or on the soles of bare feet. Gravid chigoes engorge on the host’s blood and grow from pinpoint to pea size during a 2-week interval. They produce lesions that resemble a white pustule with a central black depression and that may be pruritic or painful. Occasional complications include tetanus, bacterial infections, and autoamputation of toes (ainhum). Tungiasis is treated by removal of the intact flea with a sterile needle or scalpel, tetanus vaccination, and topical application of antibiotics.

1	Several true bugs of the family Reduviidae inflict bites that produce allergic reactions and are sometimes painful. The cone-nose bugs, so called because of their elongated heads, include the assassin and wheel bugs, which feed on other insects and bite vertebrates only in self-defense, and the kissing bugs, which routinely feed on vertebrate blood. The bites of the night-feeding kissing bugs are painless. Reactions to such bites depend on prior sensitization and include tender and pruritic papules, vesicular or bullous lesions, extensive urticaria, fever, lymphadenopathy, and (rarely) anaphylaxis. Bug bites are treated with topical antipruritics or oral antihistamines. Persons with anaphylactic reactions to reduviid bites should keep an epinephrine kit available. Some reduviids transmit Trypanosoma cruzi, the agent of New World trypanosomiasis (also called Chagas disease) (Chap. 252).

1	The cosmopolitan bedbugs (Cimex species) hide in crevices of mattresses, bed frames and other furniture, walls, and picture frames and under loose wallpaper. Bedbug populations have resurged, recently attaining levels and spreading to an extent not encountered since the mid-twentieth century. These bugs are now a common pest in homes, dormitories, and hotels; on cruise ships; and even in medical facilities. Generally, the bugs hide during the day and take blood meals at night. Their bite is painless, but minutes to days later, sensitized persons develop erythema, itching, and wheals around a central hemorrhagic punctum. Bedbugs are not known to transmit pathogens.

1	The fangs of centipedes of the genus Scolopendra can penetrate human skin and deliver a venom that produces intense burning pain, swelling, erythema, and sterile lymphangitis. Dizziness, nausea, and anxiety are described occasionally, and rhabdomyolysis and renal failure have been reported. Treatment includes washing of the site, application of cold dressings, oral analgesic administration or local lidocaine infiltration, and tetanus prophylaxis. Millipedes are docile and do not bite, but some secrete defensive fluids that may burn and discolor human skin. Affected skin turns brown overnight and may blister and exfoliate. Secretions in the eye cause intense pain and inflammation that can result in corneal ulcers and even blindness. Management includes irrigation with copious amounts of water or saline, use of analgesics, and local care of denuded skin.

1	Caterpillars of several moth species are covered with hairs or spines that produce mechanical irritation and may contain or be coated with venom. Contact with these caterpillars or their hairs may lead to lepidopterism or caterpillar envenomation. The response typically consists of an immediate burning sensation followed by local swelling and erythema and occasionally by regional lymphadenopathy, nausea, vomiting, and headache. A rare reaction to a South American caterpillar, Lonomia obliqua, can cause disseminated coagulopathy and fatal hemorrhagic shock. In the United States, dermatitis is most often associated with caterpillars of the io, puss, saddleback, and brown-tail moths. Even contact with detached hairs of other caterpillars, such as gypsy moth larvae, can later produce a pruritic urticarial or papular rash called erucism. Spines may be deposited on tree trunks or drying laundry or may be airborne and cause irritation of the eyes and upper airways. Treatment of caterpillar

1	urticarial or papular rash called erucism. Spines may be deposited on tree trunks or drying laundry or may be airborne and cause irritation of the eyes and upper airways. Treatment of caterpillar stings consists of repeated application of adhesive or cellophane tape to remove the hairs, which can then be identified microscopically. Local ice packs, topical glucocorticoids, and oral antihistamines relieve symptoms.

1	Several families of beetles have independently developed the ability to produce chemically unrelated vesicating toxins. When disturbed, blister beetles (family Meloidae) extrude cantharidin, a low-molecularweight toxin that produces thin-walled blisters (≤5 cm in diameter) 2–5 h after contact. The blisters are not painful or pruritic unless broken and resolve without treatment in ≤10 days. Nephritis may follow unusually heavy cantharidin exposure. Contact occurs when individuals sit on the ground, work in the garden, or deliberately handle the beetles. The hemolymph of certain rove beetles (Staphylinidae) contains pederin, a potent vesicant. When these beetles are crushed or brushed against the skin, the released fluid causes painful, red, flaccid bullae. These beetles occur worldwide but are most numerous and problematic in parts of Africa (where they are called “Nairobi fly”) and southwestern Asia. Ocular lesions may develop after impact with flying beetles at night or unintentional

1	are most numerous and problematic in parts of Africa (where they are called “Nairobi fly”) and southwestern Asia. Ocular lesions may develop after impact with flying beetles at night or unintentional transfer of the vesicant on the fingers. Treatment is rarely necessary, although ruptured blisters should be kept clean and bandaged.

1	Larvae of common carpet beetles are adorned with dense arrays of ornate hairs called hastisetae. Contact with these larvae or their setae results in delayed dermal reactions in sensitized individuals. The lesions are commonly mistaken as bites of bedbugs.

1	The groundless conviction that one is infested with arthropods or other parasites (Ekbom’s syndrome, delusory parasitosis, delusions of parasitosis, and perhaps Morgellons syndrome) is extremely difficult to treat and, unfortunately, is not uncommon. Patients describe uncomfortable sensations of something moving in or on their skin. Excoriations and self-induced ulcerations typically accompany the pruritus, dysesthesias, and imaginary insect bites. Patients often believe that some invisible or as yet undescribed creatures are infesting their skin, clothing, homes, or environment in general. Frequently, patients submit as evidence of infestation specimens that consist of plant-feeding and nonbiting peridomestic arthropods, pieces of skin, vegetable matter, lint, and other inanimate detritus. When evaluating a patient with possible delusional parasitosis, it is imperative to rule out true infestations and bites by arthropods, endocrinopathies, sensory disorders due to neuropathies,

1	When evaluating a patient with possible delusional parasitosis, it is imperative to rule out true infestations and bites by arthropods, endocrinopathies, sensory disorders due to neuropathies, opiate and other drug use, environmental irritants (e.g., fiberglass threads), and other causes of tingling or prickling infested by a previously unknown pathogen, while their personal lives, 2751 sensations. Frequently, such patients repeatedly seek medical consulta-family support, and employment collapse around them. tions, resist alternative explanations for their symptoms, and exacerbate their discomfort by self-treatment. Long-term pharmacotherapy with pimozide or other psychotropic agents has been more helpful Acknowledgmentthan psychotherapy in treating this disorder. Patients with delusional The substantial contributions of Andrew Spielman and James H. parasitosis often develop the unshakeable conviction that they are Maguire to this chapter in previous editions are gratefully

1	delusional The substantial contributions of Andrew Spielman and James H. parasitosis often develop the unshakeable conviction that they are Maguire to this chapter in previous editions are gratefully acknowledged.

1	476e-1 altitude Illness Buddha Basnyat, Geoffrey Tabin EPIDEMIOLOGY Mountains cover one-fifth of the earth’s surface; 38 million people live permanently at altitudes ≥2400 m, and 100 million people travel to high-altitude locations each year. Skiers in the Alps or Aspen; religious pilgrims to Lhasa or Kailash; trekkers and climbers to Kilimanjaro, Aconcagua, or Everest; and military personnel deployed ACUTE MOUNTAIN SICKNESS AND HIGH-ALTITUDE CEREBRAL EDEMA AMS is a neurologic syndrome characterized by nonspecific symp-toms (headache, nausea, fatigue, and dizziness), with a paucity of physical findings, developing 6–12 h after ascent to a high altitude. AMS is a clinical diagnosis. For uniformity in research studies, the Lake Louise Scoring System, created at the 1991 International Hypoxia Symposium, is generally used. AMS must be distinguished from exhaustion, dehydration, hypothermia, alcoholic hangover, and hyponatremia. AMS and HACE are thought to represent oppo-site ends of a

1	Symposium, is generally used. AMS must be distinguished from exhaustion, dehydration, hypothermia, alcoholic hangover, and hyponatremia. AMS and HACE are thought to represent oppo-site ends of a continuum of altitude-related neurologic disorders. HACE (but not AMS) is an encephalopathy whose hallmarks are ataxia and altered consciousness with diffuse cerebral involvement 476e PART 19: Disorders Associated with Environmental Exposures to high-altitude locations are all at risk of developing acute mountain sickness (AMS), high-altitude cerebral edema (HACE), high-altitude pulmonary edema (HAPE), and other altitude-related problems. AMS is the benign form of altitude illness, whereas HACE and HAPE are life-threatening. Altitude illness is likely to occur above 2500 m but has been documented even at 1500–2500 m. In the Mount Everest region of Nepal, ~50% of trekkers who walk to altitudes >4000 m over ≥5 days develop AMS, as do 84% of people who fly directly to 3860 m. The incidences of

1	even at 1500–2500 m. In the Mount Everest region of Nepal, ~50% of trekkers who walk to altitudes >4000 m over ≥5 days develop AMS, as do 84% of people who fly directly to 3860 m. The incidences of HACE and HAPE are much lower than that of AMS, with estimates in the range of 0.1–4%.

1	Ascent to a high altitude subjects the body to a decrease in barometric pressure that results in a decreased partial pressure of oxygen in the inspired gas in the lungs. This change leads in turn to less pressure driving oxygen diffusion from the alveoli and throughout the oxygen cascade. A normal initial “struggle response” to such an ascent includes increased ventilation—the cornerstone of acclimation—mediated by the carotid bodies. Hyperventilation may cause respiratory alkalosis and dehydration. Alkalosis may depress the ventilatory drive during sleep, with consequent periodic breathing and hypoxemia. During early acclimation, renal suppression of carbonic anhydrase and excretion of dilute alkaline urine combat alkalosis and tend to bring the pH of the blood to normal. Other physiologic changes during normal acclimation include increased sympathetic tone; increased erythropoietin levels, leading to increased hemoglobin levels and red blood cell mass; increased tissue capillary

1	changes during normal acclimation include increased sympathetic tone; increased erythropoietin levels, leading to increased hemoglobin levels and red blood cell mass; increased tissue capillary density and mitochondrial numbers; and higher levels of 2,3-bisphosphoglycerate, enhancing oxygen utilization. Even with normal acclimation, however, ascent to a high altitude decreases maximal exercise capacity (by ~1% for every 100 m gained above 1500 m) and increases susceptibility to cold injury due to peripheral vasoconstriction. Finally, if the ascent is made faster than the body can adapt to the stress of hypobaric hypoxemia, altitude-related disease states can result.

1	Hypoxia-inducible factor, which is important in high-altitude adaptation, controls transcriptional responses to hypoxia throughout the body and is involved in the release of vascular endothelial growth factor (VEGF) in the brain, erythropoiesis, and other pulmonary and cardiac functions at high altitudes. In particular, the gene EPAS1, which codes for transcriptional regulator hypoxia-inducible factor 2α, appears to play an important role in the adaptation of Tibetans living at high altitude, resulting in lower hemoglobin concentrations than are found in the Han Chinese. For acute altitude illness, a single gene variant is unlikely to be found, but the differences in the susceptibility of individuals and populations, familial clustering of cases, and a positive association of some genetic variants all clearly support a role for genetics. Approximately 58 candidate genes have been tested, and at least one variant from 17 of these genes is associated with altitude illness.

1	but generally without focal neurologic deficits. Progression to these signal manifestations can be rapid. Papilledema and, more commonly, retinal hemorrhages may also develop. In fact, retinal hemorrhages occur frequently at ≥5000 m, even in individuals without clinical symptoms of AMS or HACE. It is unclear whether retinal hemorrhage and cerebral hemorrhage at high altitude are caused by the same mechanism.

1	Risk Factors The most important risk factors for the development of altitude illness are the rate of ascent and a prior history of high-altitude illness. Exertion is a risk factor, but lack of physical fitness is not. An attractive but still speculative hypothesis proposes that AMS develops in people who have inadequate cerebrospinal capacity to buffer the brain swelling that occurs at high altitude. Children and adults seem to be equally affected, but people >50 years of age may be less likely to develop AMS than younger people. Aging appears to be associated with blunting of cardiac chronotropic function and an increase in ventilatory response leading to maintenance of arterial oxygen saturation in hypoxia. Most studies reveal no gender difference in AMS incidence. A recent study showed that, in women, adaptive responses to hypoxia with aging are blunted by menopause but can be maintained with endurance training. Sleep desaturation—a common phenomenon at high altitude—is associated

1	that, in women, adaptive responses to hypoxia with aging are blunted by menopause but can be maintained with endurance training. Sleep desaturation—a common phenomenon at high altitude—is associated with AMS. Debilitating fatigue consistent with severe AMS on descent from a summit is also an important risk factor for death in mountaineers. A recently published prospective study involving trekkers and climbers who ascended to altitudes between 4000 m and 8848 m showed that high oxygen desaturation and low ventilatory response to hypoxia during exercise are independent predictors of severe altitude illness. However, because there may be overlap between groups of susceptible and nonsusceptible individuals, accurate cutoff values are hard to define. Prediction is made more difficult because the pretest probabilities of HAPE and HACE are low. Neck irradiation or surgery damaging the carotid bodies, respiratory tract infections, and dehydration appear to be other potential risk factors for

1	pretest probabilities of HAPE and HACE are low. Neck irradiation or surgery damaging the carotid bodies, respiratory tract infections, and dehydration appear to be other potential risk factors for altitude illness.

1	Pathophysiology The exact mechanisms causing AMS and HACE are unknown. Evidence points to a central nervous system process. MRI studies have suggested that vasogenic (interstitial) cerebral edema is a component of the pathophysiology of HACE. In the setting of high-altitude illness, the MRI findings shown in Fig. 476e-1 are confirmatory of HACE, with increased signal in the white matter and particularly in the splenium of the corpus callosum. Quantitative analysis in a 3-tesla MRI study revealed that hypoxia is associated with mild vasogenic cerebral edema irrespective of AMS. This finding is in keeping with case reports of suddenly symptomatic brain tumors and of cranial nerve palsies without AMS at high altitudes. Vasogenic edema may become cytotoxic (intracellular) in severe HACE.

1	Impaired cerebral autoregulation in the presence of hypoxic cerebral vasodilation and altered permeability of the blood-brain barrier due to hypoxia-induced chemical mediators like histamine, arachidonic acid, and VEGF may all contribute to brain edema. In 1995, VEGF was first proposed as a potent promoter of capillary leakage FIGURE 476e-1 T2 magnetic resonance image of the brain of a patient with high-altitude cerebral edema (HACE) shows marked swelling and a hyperintense posterior body and splenium of the corpus callosum (area with dense opacity). The patient, a climber, went on to climb Mount Everest about 9 months after this episode of HACE.

1	(With permission from Wilderness Environ Med 15:53–55, 2004.) in the brain at high altitude, and studies in mice have borne out this role. Although preliminary studies of VEGF in climbers have yielded inconsistent results regarding its association with altitude illness, indirect evidence of a role for this growth factor in AMS and HACE comes from the observation that dexamethasone, when used in the prevention and treatment of these conditions, blocks hypoxic upregulation of VEGF. Other factors in the development of cerebral edema may be the release of calcium-mediated nitric oxide and neuronally mediated adenosine, which may promote cerebral vasodilation.

1	Increased sympathetic activity triggered by hypoxia may also contribute to blood-brain barrier leakage. Enhanced optic-nerve sheath diameter with increasing severity of AMS has been noted and suggests an important role for increased intracranial pressures in the pathophysiology of AMS. Microhemorrhage formation caused by cytokines or damage through increased hydrostatic pressure is an important feature of HACE. Lesions in the globus pallidum (which is sensitive to hypoxia) leading to Parkinson’s disease have also been reported to be complications of HACE. Finally, the effect of hypoxia on reactive oxygen species and the role of these species in clinical AMS are unclear.

1	The pathophysiology of the most common and prominent symptom of AMS—headache—remains unclear because the brain itself is an insensate organ; only the meninges contain trigeminal sensory nerve fibers. The cause of high-altitude headache is multifactorial. Various chemicals and mechanical factors activate a final common pathway, the trigeminovascular system. In the genesis of high-altitude headache, the response to nonsteroidal anti-inflammatory drugs and glucocorticoids provides indirect evidence for involvement of the arachidonic acid pathway and inflammation. Although the International Headache Society acknowledges that high altitude may be a trigger for migraine, it is unclear whether high-altitude headache and migraine share the same pathophysiology.

1	Prevention and Treatment (Table 476e-1) Gradual ascent, with adequate time for acclimation, is the best method for the prevention of altitude illness. Even though there may be individual variation in the rate of acclimation, a graded ascent of ≤400 m from the previous day’s sleeping altitude is recommended above 3000 m, and taking every third day of gain in sleeping altitude as an extra day for acclimation is helpful. Spending one night at an intermediate altitude before proceeding to a higher altitude may enhance acclimation and attenuate the risk of AMS. Another protective factor in AMS is high-altitude exposure Acute mountain sickness Discontinuation of ascent (AMS), milda Treatment with acetazolamide (250 mg q12h) AMS, moderatea Immediate descent for worsening symptoms Use of low-flow oxygen if available Treatment with acetazolamide (250 mg q12h) and/or dexamethasone (4 mg q6h)c Administration of oxygen (2–4 L/min)

1	Use of low-flow oxygen if available Treatment with acetazolamide (250 mg q12h) and/or dexamethasone (4 mg q6h)c Administration of oxygen (2–4 L/min) Treatment with dexamethasone (8 mg PO/IM/IV; then 4 mg q6h) Hyperbaric therapy if descent is not possible Minimization of exertion while patient is kept warm Administration of oxygen (4–6 L/min) to bring O2 saturation to >90% Adjunctive therapy with nifedipinee (30 mg, extended-release, q12h)

1	Adjunctive therapy with nifedipinee (30 mg, extended-release, q12h) Hyperbaric therapy if descent is not possible aCategorization of cases as mild or moderate is a subjective judgment based on the severity of headache and the presence and severity of other manifestations (nausea, fatigue, dizziness, insomnia). bNo fixed altitude is specified; the patient should descend to a point below that at which symptoms developed. cAcetazolamide treats and dexamethasone masks symptoms. For prevention (as opposed to treatment), 125–250 mg of acetazolamide q12h or (when acetazolamide is contraindicated—e.g., in people with sulfa allergy) 4 mg of dexamethasone q12h may be used. dIn hyperbaric therapy, the patient is placed in a portable altitude chamber or bag to simulate descent. eNifedipine at this dose is also effective for the prevention of HAPE, as is salmeterol (125 mg inhaled twice daily), tadalafil (10 mg twice daily), or dexamethasone (8 mg twice daily).

1	during the preceding 2 months; for example, the incidence and severity of AMS at 4300 m are reduced by 50% with an ascent after 1 week at an altitude of ≥2000 m rather than with an ascent from sea level. Studies have examined whether exposure to a normobaric hypoxic environment (in a room or a tent) before an ascent can provide protection against AMS. In double-blind placebo-controlled trials, repeated intermittent exposure (60–90 min) to normobaric hypoxia (up to 4500 m) or continuous exposure to 3000 m during 8 h of sleep for 7 consecutive days failed to reduce the incidence of AMS at altitudes of 4300–4559 m.

1	Clearly, a flexible itinerary that permits additional rest days will be helpful. Sojourners to high-altitude locations must be aware of the symptoms of altitude illness and should be encouraged not to ascend further if these symptoms develop. Any hint of HAPE (see below) or HACE mandates descent. Finally, proper hydration (but not overhydration) in high-altitude trekking and climbing, aimed at countering fluid loss due to hyperventilation and sweating, may also play a role in avoiding AMS. Pharmacologic prophylaxis at the time of travel to high altitudes is warranted for people with a history of AMS or when a graded ascent and acclimation are not possible—e.g., when rapid ascent is necessary for rescue purposes or when flight to a high-altitude location is required. Acetazolamide is the drug of choice for AMS prevention. It inhibits renal carbonic anhydrase, causing a prompt bicarbonate diuresis that leads to metabolic acidosis and hyperventilation. Acetazolamide (125–250 mg twice a

1	drug of choice for AMS prevention. It inhibits renal carbonic anhydrase, causing a prompt bicarbonate diuresis that leads to metabolic acidosis and hyperventilation. Acetazolamide (125–250 mg twice a day), administered for 1 day before ascent and continued for 2 or 3 days, is effective. Higher doses are not required. A meta-analysis limited to randomized controlled trials revealed that 125 mg of acetazolamide twice daily was effective in the prevention of AMS, with a relative-risk reduction of ~48% from values obtained with placebo. Paresthesia and a tingling sensation are common side effects of acetazolamide. This drug is a nonantibiotic sulfonamide that has low-level cross-reactivity with sulfa antibiotics; as a result, severe reactions are rare. Dexamethasone (8 mg/d in divided doses) is also effective. A large-scale, randomized, double-blind, placebo-controlled trial in partially acclimated trekkers has clearly shown that Ginkgo biloba is ineffective in the prevention of AMS.

1	is also effective. A large-scale, randomized, double-blind, placebo-controlled trial in partially acclimated trekkers has clearly shown that Ginkgo biloba is ineffective in the prevention of AMS. Recently, ibuprofen (600 mg three times daily) was shown to be beneficial in the prevention of AMS, but more definitive studies and proper gastrointestinal-bleeding risk assessment need to be conducted before ibuprofen can be routinely recommended for AMS prevention. Many drugs, including spironolactone, medroxyprogesterone, magnesium, calcium channel blockers, and antacids, confer no benefit in the prevention of AMS. Similarly, no efficacy studies are available for coca leaves (a weak form of cocaine), which are offered to high-altitude travelers in the Andes, or for soroche pills, which contain aspirin, caffeine, and acetaminophen and are sold over the counter in Bolivia and Peru. Finally, a word of caution applies in the pharmacologic prevention of altitude illness. A fast-growing

1	contain aspirin, caffeine, and acetaminophen and are sold over the counter in Bolivia and Peru. Finally, a word of caution applies in the pharmacologic prevention of altitude illness. A fast-growing population of climbers in pursuit of a summit are using prophylactic drugs such as glucocorticoids in an attempt to improve their performance; the outcome can be tragic because of potentially severe side effects of these drugs.

1	For the treatment of mild AMS, rest alone with analgesic use may be adequate. Descent and the use of acetazolamide and (if available) oxygen are sufficient to treat most cases of moderate AMS. Even a minor descent (400–500 m) may be adequate for symptom relief. For moderate AMS or early HACE, dexamethasone (4 mg orally or parenterally) is highly effective. For HACE, immediate descent is mandatory. When descent is not possible because of poor weather conditions or darkness, a simulation of descent in a portable hyperbaric chamber is effective and, like dexamethasone administration, “buys time.” Thus, in certain high-altitude locations (e.g., remote pilgrimage sites), the decision to bring along the light-weight hyperbaric chamber may prove lifesaving. Like nifedipine, phosphodiesterase-5 inhibitors have no role in the treatment of AMS or HACE.

1	HIGH-ALTITUDE PULMONARY EDEMA Risk Factors and Manifestations Unlike HACE (a neurologic disorder), HAPE is primarily a pulmonary problem and therefore is not necessarily preceded by AMS. HAPE develops within 2–4 days after arrival at high altitude; it rarely occurs after more than 4 or 5 days at the same altitude, probably because of remodeling and adaptation that render the pulmonary vasculature less susceptible to the effects of hypoxia. A rapid rate of ascent, a history of HAPE, respiratory tract infections, and cold environmental temperatures are risk factors. Men are more susceptible than women. People with abnormalities of the cardiopulmonary circulation leading to pulmonary hypertension—e.g., large patent foramen ovale, mitral stenosis, primary pulmonary hypertension, and unilateral absence of the pulmonary artery—are at increased risk of HAPE, even at moderate altitudes. For example, patent foramen ovale is four times more common among HAPE-susceptible individuals than in the

1	absence of the pulmonary artery—are at increased risk of HAPE, even at moderate altitudes. For example, patent foramen ovale is four times more common among HAPE-susceptible individuals than in the general population. Echocardiography is recommended when HAPE develops at relatively low altitudes (<3000 m) and whenever cardiopulmonary abnormalities predisposing to HAPE are suspected.

1	The initial manifestation of HAPE may be a reduction in exercise tolerance greater than that expected at the given altitude. Although a dry, persistent cough may presage HAPE and may be followed by the production of blood-tinged sputum, cough in the mountains is almost universal and the mechanism is poorly understood. Tachypnea and tachycardia, even at rest, are important markers as illness progresses. Crackles may be heard on auscultation but are not diagnostic. HAPE may be accompanied by signs of HACE. Patchy or localized opacities (Fig. 476e-2) or streaky interstitial edema may be noted on chest radiography. In the past, HAPE was mistaken for pneumonia due to the cold or for heart failure due to hypoxia and exertion. Kerley B lines or a bat-wing appearance are not seen on radiography. Electrocardiography may reveal right ventricular strain or even hypertrophy. Hypoxemia and respiratory alkalosis are consistently present unless the patient is taking acetazolamide, in which case

1	Electrocardiography may reveal right ventricular strain or even hypertrophy. Hypoxemia and respiratory alkalosis are consistently present unless the patient is taking acetazolamide, in which case metabolic acidosis may supervene. Assessment of arterial blood gases is not necessary in the evaluation of HAPE; an oxygen saturation reading with a pulse oximeter is generally adequate. The existence of a subclinical form of HAPE has been suggested by an increased alveolar-arterial oxygen gradient in Everest

1	FIGURE 476e-2 Chest radiograph of a patient with high-altitude pulmonary edema shows opacity in the right middle and lower zones simulating pneumonic consolidation. The opacity cleared almost completely in 2 days with descent and supplemental oxygen. climbers near the summit, but hard evidence correlating this abnormality with the development of clinically relevant HAPE is lacking.

1	Pathophysiology HAPE is a noncardiogenic pulmonary edema characterized by patchy pulmonary vasoconstriction that leads to over-perfusion in some areas. This abnormality leads in turn to increased pulmonary capillary pressure (>18 mmHg) and capillary “stress” failure. The exact mechanism for the vasoconstriction is unknown. Endothelial dysfunction due to hypoxia may play a role by impairing the release of nitric oxide, an endothelium-derived vasodilator. At high altitude, HAPE-prone persons have reduced levels of exhaled nitric oxide. The effectiveness of phosphodiesterase-5 inhibitors in alleviating altitude-induced pulmonary hypertension, decreased exercise tolerance, and hypoxemia supports the role of nitric oxide in the pathogenesis of HAPE. One study demonstrated that prophylactic use of tadalafil, a phosphodiesterase-5 inhibitor, decreases the risk of HAPE by 65%. In contrast, the endothelium also synthesizes endothelin-1, a potent vasoconstrictor whose concentrations are higher

1	of tadalafil, a phosphodiesterase-5 inhibitor, decreases the risk of HAPE by 65%. In contrast, the endothelium also synthesizes endothelin-1, a potent vasoconstrictor whose concentrations are higher than average in HAPE-prone mountaineers. Bosentan, an endothelin receptor antagonist, attenuates hypoxia-induced pulmonary hypertension, but further field studies with this drug are necessary.

1	Exercise and cold lead to increased pulmonary intravascular pressure and may predispose to HAPE. In addition, hypoxia-triggered increases in sympathetic drive may lead to pulmonary venoconstriction and extravasation into the alveoli from the pulmonary capillaries. Consistent with this concept, phentolamine, which elicits α-adrenergic blockade, improves hemodynamics and oxygenation in HAPE more than do other vasodilators. The study of tadalafil cited above also investigated dexamethasone in the prevention of HAPE. Surprisingly, dexamethasone reduced the incidence of HAPE by 78%—a greater decrease than with tadalafil. Besides possibly increasing the availability of endothelial nitric oxide, dexamethasone may have altered the excessive sympathetic activity associated with HAPE: the heart rate of participants in the dexamethasone arm of the study was significantly lowered. Finally, people susceptible to HAPE also display enhanced sympathetic activity during short-term hypoxic breathing at

1	of participants in the dexamethasone arm of the study was significantly lowered. Finally, people susceptible to HAPE also display enhanced sympathetic activity during short-term hypoxic breathing at low altitudes.

1	Because many patients with HAPE have fever, peripheral leukocytosis, and an increased erythrocyte sedimentation rate, inflammation has been considered an etiologic factor in HAPE. However, strong evidence suggests that inflammation in HAPE is an epiphenomenon rather than the primary cause. Nevertheless, inflammatory processes (e.g., those elicited by viral respiratory tract infections) do predispose persons to HAPE—even those who are constitutionally resistant to its development.

1	Another proposed mechanism for HAPE is impaired transepithelial clearance of sodium and water from the alveoli. β-Adrenergic agonists upregulate the clearance of alveolar fluid in animal models. In a double-blind, randomized, placebo-controlled study of HAPE-susceptible mountaineers, prophylactic inhalation of the β-adrenergic agonist salmeterol reduced the incidence of HAPE by 50%. Other effects of β agonists may also contribute to the prevention of HAPE, but these findings are in keeping with the concept that alveolar fluid clearance may play a pathogenic role in this illness.

1	Prevention and Treatment (Table 476e-1) Allowing sufficient time for acclimation by ascending gradually (as discussed above for AMS and HACE) is the best way to prevent HAPE. Sustained-release nifedipine (30 mg), given once or twice daily, prevents HAPE in people who must ascend rapidly or who have a history of HAPE. Other drugs for the prevention of HAPE are listed in Table 476e-1 (footnote e). Although dexamethasone is listed for prevention, its adverse-effect profile requires close monitoring. Acetazolamide has been shown to blunt hypoxic pulmonary vasoconstriction in animal models, and this observation warrants further study in HAPE prevention. However, one large study failed to show a decrease in pulmonary vasoconstriction in partially acclimated individuals given acetazolamide.

1	Early recognition is paramount in the treatment of HAPE, especially when it is not preceded by the AMS symptoms of headache and nausea. Fatigue and dyspnea at rest may be the only initial manifestations. Descent and the use of supplementary oxygen (aimed at bringing oxygen saturation to >90%) are the most effective therapeutic interventions. Exertion should be kept to a minimum, and the patient should be kept warm. Hyperbaric therapy in a portable altitude chamber may be used if descent is not possible and oxygen is not available. Oral sustained-release nifedipine (30 mg once or twice daily) can be used as adjunctive therapy. Inhaled β agonists, which are safe and convenient to carry, are useful in the prevention of HAPE and may be effective in its treatment, although no trials have yet been carried out. Inhaled nitric oxide and expiratory positive airway pressure may also be useful therapeutic measures but may not be available in high-altitude settings. No studies have investigated

1	been carried out. Inhaled nitric oxide and expiratory positive airway pressure may also be useful therapeutic measures but may not be available in high-altitude settings. No studies have investigated phosphodiesterase-5 inhibitors in the treatment of HAPE, but reports have described their use in clinical practice. The mainstays of treatment remain descent and (if available) oxygen.

1	In AMS, if symptoms abate (with or without acetazolamide), the patient may reascend gradually to a higher altitude. Unlike that in acute respiratory distress syndrome (another noncardiogenic pulmonary edema), the architecture of the lung in HAPE is usually well preserved, with rapid reversibility of abnormalities (Fig. 476e-2). This fact has allowed some people with HAPE to reascend slowly after a few days of descent and rest. In HACE, reascent after a few days may not be advisable during the same trip.

1	OTHER HIGH-ALTITUDE PROBLEMS Sleep Impairment The mechanisms underlying sleep problems, which are among the most common adverse reactions to high altitude, include increased periodic breathing; changes in sleep architecture, with increased time in lighter sleep stages; and changes in rapid eye movement sleep. Sojourners should be reassured that sleep quality improves with acclimation. In cases where drugs do need to be used, acetazolamide (125 mg before bedtime) is especially useful because this agent decreases hypoxemic episodes and alleviates sleeping disruptions caused by excessive periodic breathing. Whether combining acetazolamide with temazepam or zolpidem is more effective than administering acetazolamide alone is unknown. In combinations, the doses of temazepam and zolpidem should not be increased by >10 mg at high altitudes. Limited evidence suggests that diazepam causes hypoventilation at high altitudes and therefore is contraindicated. For trekkers with obstructive sleep

1	not be increased by >10 mg at high altitudes. Limited evidence suggests that diazepam causes hypoventilation at high altitudes and therefore is contraindicated. For trekkers with obstructive sleep apnea who are using a continuous positive airway pressure (CPAP) machine, the addition of acetazolamide, which will decrease centrally mediated sleep apnea, may be helpful. There is evidence to show that obstructive sleep apnea at high altitude may decrease and “convert” to central sleep apnea.

1	Gastrointestinal Issues High-altitude exposure may be associated with increased gastric and duodenal bleeding, but further studies are required to determine whether there is a causal effect. Because of decreased atmospheric pressure and consequent intestinal gas expansion at high altitudes, many sojourners experience abdominal bloating and distension as well as excessive flatus expulsion. In the absence of diarrhea, these phenomena are normal, if sometimes uncomfortable. Accompanying diarrhea, however, may indicate the involvement of bacteria or Giardia parasites, which are common at many high-altitude locations in the developing world. Prompt treatment with fluids and empirical antibiotics may be required to combat dehydration in the mountains. Finally, hemorrhoids are common on high-altitude treks; treatment includes hot soaks, application of hydrocortisone ointment, and measures to avoid constipation.

1	High-Altitude Cough High-altitude cough can be debilitating and is sometimes severe enough to cause rib fracture, especially at >5000 m. The etiology is probably multifactorial. Although high-altitude cough has been attributed to inspiration of cold dry air, this explanation appears not to be sufficient by itself; in long-duration studies in hypobaric chambers, cough has occurred despite controlled temperature and humidity. The implication is that hypoxia also plays a role. Exercise can precipitate cough at high altitudes, possibly because of water loss from the respiratory tract. Long-acting β agonists and glucocorticoids prevent bronchoconstriction that otherwise may be brought on by cold and exercise. In general, infection does not seem to be a common etiology. Anecdotal reports have described the efficacy of an inhaled combination of fluticasone and salmeterol in the treatment of high-altitude cough; however, a placebo-controlled, randomized trial failed to support this beneficial

1	the efficacy of an inhaled combination of fluticasone and salmeterol in the treatment of high-altitude cough; however, a placebo-controlled, randomized trial failed to support this beneficial effect. Furthermore, anecdotal evidence supports the utility of the proton pump inhibitor omeprazole in preventing gastroesophageal reflux in some trekkers and climbers. In most situations, cough resolves upon descent.

1	High-Altitude Neurologic Events Unrelated to “Altitude Illness” Transient ischemic attacks (TIAs) and strokes have been well described in high-altitude sojourners outside the setting of altitude sickness. However, these descriptions are not based on cause (hypoxia) and effect. In general, symptoms of AMS present gradually, whereas many of these neurologic events happen suddenly. The population that suffers strokes and TIAs at sea level is generally an older age group with other risk factors, whereas those so afflicted at high altitudes are generally younger and probably have fewer risk factors for atherosclerotic vascular disease. Other mechanisms (e.g., migraine, vasospasm, focal edema, hypocapneic vasoconstriction, hypoxia in the watershed zones of minimal cerebral blood flow, or cardiac right-to-left shunt) may be operative in TIAs and strokes at high altitude.

1	Subarachnoid hemorrhage, transient global amnesia, delirium, and cranial nerve palsies (e.g., lateral rectus palsy) occurring at high altitudes but outside the setting of altitude sickness have also been well described. Syncope is common at moderately high altitudes, generally occurs shortly after ascent, usually resolves without descent, and appears to be a vasovagal event related to hypoxemia. Seizures occur rarely with HACE, but hypoxemia and hypocapnia, which are prevalent at high altitudes, are well-known triggers that may contribute to new or breakthrough seizures in predisposed individuals. Nevertheless, the consensus among experts is that sojourners with well-controlled seizure disorders can ascend to high altitudes. Ophthalmologic problems, such as cortical blindness, amaurosis fugax, and retinal hemorrhage with macular involvement and compromised vision, are well recognized. Visual problems from previous refractive surgery and blurred monocular vision—due either to the use

1	fugax, and retinal hemorrhage with macular involvement and compromised vision, are well recognized. Visual problems from previous refractive surgery and blurred monocular vision—due either to the use of a transdermal scopolamine patch (touching the eye after touching the patch) or to dry-eye syndrome—may also occur in the field at high altitudes and may be confused with neurologic conditions. Finally, persons with hypercoagulable conditions (e.g., antiphospholipid syndrome, protein C deficiency) who are asymptomatic at sea level may experience cerebral venous thrombosis (possibly due to the enhanced blood viscosity triggered by polycythemia and dehydration) at high altitudes. Proper history taking, examination, and prompt investigations where possible will help define these conditions as entities separate from altitude sickness. Administration of oxygen (where available) and prompt descent are the cornerstones of treatment of most of these neurologic conditions.

1	Psychological/Psychiatric Problems Delirium characterized by a sudden change in mental status, a short attention span, disorganized thinking, and an agitated state during the period of confusion has been well described in mountain climbers and trekkers without a prior history. In addition, anxiety attacks, often triggered at night by excessive periodic breathing, are well documented. The contribution of hypoxia to these conditions is unknown. Expedition medical kits need to include antipsychotic injectable drugs to control psychosis in patients in remote high-altitude locations.

1	Because travel to high altitudes is increasingly popular, common conditions such as hypertension, coronary artery disease, and diabetes are more frequently encountered among high-altitude sojourners. This situation is of particular concern for the thousands of elderly pilgrims with medical problems who visit high-altitude sacred areas (e.g., in the Himalayas) each year. In recent years, high-altitude travel has attracted intrepid trekkers who are taking immunosuppressive medications (e.g., kidney transplant recipients or patients undergoing chemotherapy). Recommended vaccinations and other precautions (e.g., hand washing) may be especially important for this group. Although most of these medical conditions do not appear to influence susceptibility to altitude illness, they may be exacerbated by ascent to altitude, exertion in cold conditions, and hypoxemia. Advice regarding the advisability of high-altitude travel and the impact of high-altitude hypoxia on these preexisting conditions

1	by ascent to altitude, exertion in cold conditions, and hypoxemia. Advice regarding the advisability of high-altitude travel and the impact of high-altitude hypoxia on these preexisting conditions is becoming increasingly relevant, but there are no evidence-based guidelines. In addition, recommendations made for relatively low altitudes (~3000 m) may not hold true for higher altitudes (>4000 m), where hypoxic stress is greater. Personal risks and benefits must be clearly thought through before ascent.

1	Hypertension At high altitudes, enhanced sympathetic activity may lead to a transient rise in blood pressure. Occasionally, nonhypertensive, healthy, asymptomatic trekkers have pathologically high blood pressure at high altitude that rapidly normalizes without medicines on descent. Sojourners should continue to take their antihypertensive medications at high altitudes. Hypertensive patients are not more likely than others to develop altitude illness. Because the probable mechanism of high-altitude hypertension is α-adrenergic activity, antiα-adrenergic drugs like prazosin have been suggested for symptomatic patients and those with labile hypertension. It is best to start taking the drug several weeks before the trip and to carry a sphygmomanometer if a trekker has labile hypertension. Sustained-release nifedipine may also be useful. For a common problem like hypertension, there is clearly inadequate knowledge on which to base appropriate recommendations.

1	Coronary Artery Disease Myocardial oxygen demand and maximal heart rate are reduced at high altitudes because the VO2 max (maximal oxygen consumption) decreases with increasing altitude. This effect may explain why signs of cardiac ischemia or dysfunction usually are not seen in healthy persons at high altitudes. Asymptomatic, fit individuals with no risk factors need not undergo any tests for coronary artery disease before ascent. For persons with ischemic heart disease, previous myocardial infarction, angioplasty, and/or bypass surgery, an exercise treadmill test is indicated. A strongly positive treadmill test is a contraindication for high-altitude trips. Patients with poorly controlled arrhythmias should avoid high-altitude travel, but patients with arrhythmias that are well controlled with antiarrhythmic medications do not seem to be at increased risk. Sudden cardiac deaths are not noted with a greater frequency in the Alps than at lower altitudes; although sudden cardiac deaths

1	with antiarrhythmic medications do not seem to be at increased risk. Sudden cardiac deaths are not noted with a greater frequency in the Alps than at lower altitudes; although sudden cardiac deaths are encountered every trekking season in the higher Himalayan range, accurate documentation is lacking.

1	Asthma Although cold air and exercise may provoke acute bronchoconstriction, asthmatic patients usually have fewer problems at high than at low altitudes, possibly because of decreased allergen levels and increased circulating catecholamine levels. Nevertheless, asthmatic individuals should carry all their medications, including oral glucocorticoids, with proper instructions for use in case of an exac-476e-5 erbation. Severely asthmatic persons should be cautioned against ascending to high altitudes. Pregnancy In general, low-risk pregnant women ascending to 3000 m are not at special risk except for the relative unavailability of medical care in many high-altitude locations, especially in developing countries. Despite the lack of firm data on this point, venturing higher than 3000 m to altitudes at which oxygen saturation drops steeply seems unadvisable for pregnant women.

1	Obesity Although living at a high altitude has been suggested as a means of controlling obesity, obesity has also been reported to be a risk factor for AMS, probably because nocturnal hypoxemia is more pronounced in obese individuals. Hypoxemia may also lead to greater pulmonary hypertension, thus possibly predisposing the trekker to HAPE. Sickle Cell Disease High altitude is one of the rare environmental exposures that occasionally provokes a crisis in persons with the sickle cell trait. Even when traversing mountain passes as low as 2500 m, people with sickle cell disease have been known to have a vasoocclusive crisis. Sickle cell disease needs to be considered when persons traveling to high altitudes become unwell and develop left-upper-quadrant pain. Patients with known sickle cell disease who need to travel to high altitudes should use supplemental oxygen and travel with caution.

1	Diabetes Mellitus Trekking at high altitudes may enhance sugar uptake. Thus, high-altitude travel may not pose problems for persons with diabetes that is well controlled with oral hypoglycemic agents. An eye examination before travel may be useful. Patients taking insulin may require lower doses on trekking/climbing days than on rest days. Because of these variations, diabetic patients need to carry a reliable glucometer and use fast-acting insulin. Ready access to sweets is also essential. It is important for companions of diabetic trekkers to be fully aware of potential problems like hypoglycemia.

1	Chronic Lung Disease Depending on disease severity and access to medical care, preexisting lung disease may not always preclude high-altitude travel. A proper pretravel evaluation must be conducted. Supplemental oxygen may be required if the predicted PaO2 for the altitude is <50–55 mmHg. Preexisting pulmonary hypertension may also need to be assessed in these patients. If the result is positive, patients should be discouraged from ascending to high altitudes; if such travel is necessary, treatment with sustained-release nifedipine (20 mg twice a day) should be considered. Small-scale studies have revealed that when patients with bullous disease reach ~5000 m, bullous expansion and pneumothorax are not noted. Compared with information on chronic obstructive pulmonary disease, fewer data exist about the safety of travel to high altitude for people with pulmonary fibrosis, but acute exacerbation of pulmonary fibrosis has been seen at high altitude. A handheld pulse oximeter can be

1	exist about the safety of travel to high altitude for people with pulmonary fibrosis, but acute exacerbation of pulmonary fibrosis has been seen at high altitude. A handheld pulse oximeter can be useful to check for oxygen saturation.

1	Chronic Kidney Disease Patients with chronic kidney disease can tolerate short-term stays at high altitudes, but theoretical concern persists about progression to end-stage renal disease. Acetazolamide, the drug most commonly used for altitude sickness, should be avoided by anyone with preexisting metabolic acidosis, which can be exacerbated by this drug. In addition, the acetazolamide dosage should be adjusted when the glomerular filtration rate falls below 50 mL/min, and the drug should not be used at all if this value falls below 10 mL/min.

1	Chronic mountain sickness (Monge’s disease) is a disease of long-term residents of altitudes above 2500 m that is characterized by excessive erythrocytosis with moderate to severe pulmonary hypertension leading to cor pulmonale. This condition was originally described in South America and has also been documented in Colorado and in the Han Chinese population in Tibet. Migration to a low altitude results in the resolution of chronic mountain illness. Venesection and acetazolamide are helpful.

1	High-altitude pulmonary hypertension is also a subacute disease in Tibet have presented with the adult and infantile forms, respectively. of long-term high-altitude residents. Unlike Monge’s disease, this High-altitude pulmonary hypertension bears a striking pathophysisyndrome is characterized primarily by pulmonary hypertension ologic resemblance to brisket disease in cattle. Descent to a lower alti(not erythrocytosis) leading to heart failure. Indian soldiers living at tude is curative. extreme altitudes for prolonged periods and Han Chinese infants born body exposure to pressures greater than 101.3 kPa (1 atmosphere or 760 mmHg). In practice, this almost always means the administration of hyperbaric oxygen therapy (HBO2T). The Undersea and Hyperbaric Medical Society (UHMS) defines HBO2T as: “a treatment in which a patient breathes 100% oxygen … while inside a treatment chamber at a pressure higher than sea level pressure (i.e., >1 atmosphere absolute or ATA).” The treatment chamber

1	as: “a treatment in which a patient breathes 100% oxygen … while inside a treatment chamber at a pressure higher than sea level pressure (i.e., >1 atmosphere absolute or ATA).” The treatment chamber is an airtight vessel variously called a hyperbaric chamber, recompression chamber, or decompression chamber, depending on the clinical and historical context. Such chambers may be capable of compressing a single patient (a monoplace chamber) or multiple patients and attendants as required (a multiplace chamber) (Figs. 477e-1 and 477e-2). Historically, these compression chambers were first used for the treatment of divers and compressed air workers suffering decompression sickness (DCS; “the bends”). Although the prevention and treatment of disorders arising after decompression in diving, aviation, and space flight has developed into a specialized field of its own, it remains closely linked to the broader practice of hyperbaric medicine.

1	hyperbaric and Diving Medicine Michael H. Bennett, Simon J. Mitchell WHAT IS HYPERBARIC AND DIVING MEDICINE? Hyperbaric medicine is the treatment of health disorders using whole-477e

1	Despite an increased understanding of mechanisms and an improving evidence basis, hyperbaric medicine has struggled to achieve widespread recognition as a “legitimate” therapeutic measure. There are several contributing factors, but high among them are a poor grounding in general oxygen physiology and oxygen therapy at medical schools and a continuing tradition of charlatans advocating hyperbaric therapy (often using air) as a panacea. Funding for both basic and clinical research has been difficult in an environment where the pharmacologic agent under study is abundant, cheap, and unpatentable. Recently, however, there are signs of an improved appreciation of the potential importance of HBO2T with significant National Institutes of Health (NIH) funding for mechanisms research and from the U.S. military for clinical investigation.

1	Increased hydrostatic pressure will reduce the volume of any bubbles present within the body (see “Diving Medicine”), and this is partly responsible for the success of prompt recompression in DCS and FIGuRE 477e-1 A monoplace chamber. (Prince of Wales Hospital, Sydney.) FIGuRE 477e-2 A chamber designed to treat multiple patients.

1	FIGuRE 477e-1 A monoplace chamber. (Prince of Wales Hospital, Sydney.) FIGuRE 477e-2 A chamber designed to treat multiple patients. (Karolinska University Hospital.) arterial gas embolism. Supplemental oxygen breathing has a dose-dependent effect on oxygen transport, ranging from improvement in hemoglobin oxygen saturation when a few liters per minute are delivered by simple mask at 101.3 kPa (1 ATA) to raising the dissolved plasma oxygen sufficiently to sustain life without the need for hemoglobin at all when 100% oxygen is breathed at 303.9 kPa (3 ATA). Most HBO2T regimens involve oxygen breathing at between 202.6 kPa and 283.6 kPa (2 and 2.8 ATA), and the resultant increase in arterial oxygen tensions to >133.3 kPa (1000 mmHg) has widespread physiologic and pharmacologic consequences (Fig. 477e-3).

1	One direct consequence of such high intravascular tension is to increase greatly the effective capillary-tissue diffusion distance for oxygen such that oxygen-dependent cellular processes can resume in hypoxic tissues. Important as this may be, the mechanism of action is not limited to this restoration of oxygenation in hypoxic tissue. Indeed, there are pharmacologic effects that are profound and long-lasting. Although removal from the hyperbaric chamber results in a rapid return of poorly vascularized tissues to their hypoxic state, even a single dose of HBO2T produces changes in fibroblast, leukocyte, and angiogenic functions and antioxidant defenses that persist many hours after oxygen tensions are returned to pretreatment levels.

1	It is widely accepted that oxygen in high doses produces adverse effects due to the production of reactive oxygen species (ROS) such as superoxide (O2 -) and hydrogen peroxide (H2O2). It has become increasingly clear over the last decade that both ROS and reactive nitrogen species (RNS) such as nitric oxide (NO) participate in a wide range of intracellular signaling pathways involved in the production of a range of cytokines, growth factors, and other inflammatory and repair modulators. Such mechanisms are complex and at times apparently paradoxical. For example, when used to treat chronic hypoxic wounds, HBO2T has been shown to enhance the clearance of cellular debris and bacteria by providing the substrate for macrophage phagocytosis; stimulate growth factor synthesis by increased production and stabilization of hypoxia-inducible factor 1 (HIF-1); inhibit leukocyte activation and adherence to damaged endothelium; and mobilize CD34+ pluripotent vasculogenic progenitor cells from the

1	and stabilization of hypoxia-inducible factor 1 (HIF-1); inhibit leukocyte activation and adherence to damaged endothelium; and mobilize CD34+ pluripotent vasculogenic progenitor cells from the bone marrow. The interactions between these mechanisms remain a very active field of investigation. One exciting development is the concept of hyperoxic preconditioning in which a short exposure to HBO2 can induce tissue protection against future hypoxic/ ischemic insult, most likely through an inhibition of mitochondrial permeability transition pore (MPTP) opening and the release of cytochrome c. By targeting these mechanisms of cell death during reperfusion events, HBO2 has potential applications in a variety of settings including organ transplantation. One randomized clinical trial suggested that HBO2T prior to coronary artery bypass grafting reduces biochemical markers of ischemic stress and improves neurocognitive outcomes.

1	Hyperbaric oxygen Restoration of tissue normoxia Edema reduction Hyperoxic vasoconstriction ˜Wound growth factors Stem cell mobilization °˛2 integrin function Enhanced phagocytosis, angiogenesis, and fibroblast activity Ischemic preconditioning, e.g., HIF-1 HO-1 Wound healing, radiation tissue injury Threatened grafts/flaps cadaveric organ preservation Enhanced inert gas diffusion gradients between bubble, tissue, and lungs High arterial PO2 Hydrostatic compression Bubble volume reduction DCS CAGE Enhanced O2 diffusion Generation of ROS and RNS Osmotic effect Crush injury

1	FIGuRE 477e-3 Mechanisms of action of hyperbaric oxygen. There are many consequences of compression and oxygen breathing. The cell-signaling effects of HBO2T are the least understood but potentially most important. Examples of indications for use are shown in the shaded boxes. CAGE, cerebral arterial gas embolism; DCS, decompression sickness; HIF-1, hypoxia inducible factor-1; HO-1, hemoxygenase 1; RNS, reactive nitrogen species; ROS, reactive oxygen species. HBO2T is generally well tolerated and safe in clinical practice. Adverse effects are associated with both alterations in pressure (barotrauma) and the administration of oxygen.

1	Barotrauma occurs when any noncompliant gas-filled space within the body does not equalize with environmental pressure during compression or decompression. About 10% of patients complain of some difficulty equalizing middle-ear pressure early in compression, and although most of these problems are minor and can be overcome with training, 2–5% of conscious patients require middle-ear ventilation tubes or formal grommets across the tympanic membrane. Unconscious patients cannot equalize and should have middle-ear ventilation tubes placed prior to compression if possible. Other less common sites for barotrauma of compression include the respiratory sinuses and dental caries. The lungs are potentially vulnerable to barotrauma of decompression as described below in the section on diving medicine, but the decompression following HBO2T is so slow that pulmonary gas trapping is extremely rare in the absence of an undrained pneumothorax or lesions such as bullae.

1	The practical limit to the dose of oxygen, either in a single treatment session or in a series of daily sessions, is oxygen toxicity. The most common acute manifestation is a seizure, often preceded by anxiety and agitation, during which time a switch from oxygen to air breathing may avoid the convulsion. Hyperoxic seizures are typically generalized tonic-clonic seizures followed by a variable postictal period. The cause is an overwhelming of the antioxidant defense systems within the brain. Although clearly dose-dependent, onset is very variable both between individuals and within the same individual on different days. In routine clinical hyperbaric practice, the incidence is about 1:1500 to 1:2000 compressions.

1	Chronic oxygen poisoning most commonly manifests as myopic shift. This is due to alterations in the refractive index of the lens following oxidative damage that reduces the solubility of lenticular proteins in a process similar to that associated with senescent cataract formation. Up to 75% of patients show deterioration in visual acuity after a course of 30 treatments at 202.6 kPa (2 ATA). Although most return to pretreatment values 6–12 weeks after cessation of treatment, a small proportion do not recover. A more rapid maturation of preexisting cataracts has occasionally been associated with HBO2T. Although a theoretical problem, the development of pulmonary oxygen toxicity over time does not seem to be problematic in practice—probably due to the intermittent nature of the exposure.

1	There are few absolute contraindications to HBO2T. The most commonly encountered is an untreated pneumothorax. A pneumothorax may expand rapidly on decompression and come under tension. Prior to any compression, patients with a pneumothorax should have a patent chest drain in place. The presence of other obvious risk factors for pulmonary gas trapping such as bullae should trigger a very cautious analysis of the risks of treatment versus benefit. Prior bleomycin treatment deserves special mention because of its association with a partially dose-dependent pneumonitis in about 20% of people. These individuals appear to be at particular risk for rapid deterioration of ventilatory function following exposure to high oxygen tensions. The relationship between distant bleomycin exposure and subsequent risk of pulmonary oxygen toxicity is uncertain, however late pulmonary fibrosis is a potential complication of bleomycin, and any patient with a history of receiving this drug should be

1	subsequent risk of pulmonary oxygen toxicity is uncertain, however late pulmonary fibrosis is a potential complication of bleomycin, and any patient with a history of receiving this drug should be carefully counseled prior to exposure to HBO2T. For those recently exposed to doses above 300,000 IU (200 mg) and whose course was complicated by a respiratory reaction to bleomycin, compression should be avoided except in a life-threatening situation.

1	The appropriate indications for HBO2T are controversial and evolving. Practitioners in this area are in an unusual position. Unlike most branches of medicine, hyperbaric physicians do not deal with a range of disorders within a defined organ system (e.g., cardiology), nor are they masters of a therapy specifically designed for a single category of disorders. Inevitably, the encroachment of hyperbaric physicians into other medical fields generates suspicion from specialist practitioners in those fields. At the same time this relatively benign therapy, the prescription and delivery of which requires no medical license in most jurisdictions (including the United States), attracts both charlatans and well-motivated proselytizers who tout the benefits of oxygen for a plethora of chronic incurable diseases. This battle on two fronts has meant that mainstream hyperbaric physicians have been particularly careful to claim effectiveness only for those conditions where there is a reasonable body

1	diseases. This battle on two fronts has meant that mainstream hyperbaric physicians have been particularly careful to claim effectiveness only for those conditions where there is a reasonable body of supporting evidence.

1	In 1977, the UHMS systematically examined claims for the use of HBO2T in more than 100 disorders and found sufficient evidence to support routine use in only 12. The Hyperbaric Oxygen Therapy Committee of that organization has continued to update this list periodically with an increasingly formalized system of appraisal for new indications and emerging evidence (Table 477e-1). Around the world, other relevant medical organizations have generally taken a similar approach, although indications vary considerably—particularly those recommended by hyperbaric medical societies in Russia and China where HBO2T has gained much wider support than in the United States, Europe, and Australasia. Recently, several Cochrane reviews have examined the randomized trial evidence for many putative indications, including attempts to examine the cost-effectiveness of HBO2T. Table 477e-2 is a synthesis of these two approaches and lists the estimated cost of attaining health outcomes with the use of HBO2T.

1	including attempts to examine the cost-effectiveness of HBO2T. Table 477e-2 is a synthesis of these two approaches and lists the estimated cost of attaining health outcomes with the use of HBO2T. Any savings associated with alternative treatment strategies avoided as a result of HBO2T are not accounted for in these estimates (e.g., the avoidance of lower leg amputation in diabetic foot ulcers). Following are short reviews of three important indications currently accepted by the UHMS.

1	Radiotherapy is a well-established treatment for suitable malignancies. In the United States alone, approximately 300,000 individuals annually will become long-term survivors of cancer treated by irradiation. Serious radiation-related complications developing months or years after treatment (late radiation tissue injury [LRTI]) will significantly affect between 5 and 15% of those long-term survivors, although inci Current List of inDiCations for hyperBariC oxygen therapy 1. Air or gas embolism (includes diving-related, iatrogenic, and accidental causes) 2. 3. 4. Crush injury, compartment syndrome, and acute traumatic ischemias 5. 6. Arterial insufficiency Central retinal artery occlusion Enhancement of healing in selected problem wounds 7. Exceptional blood loss (where transfusion is refused or impossible) 8. 9. Necrotizing soft tissue infections (e.g., Fournier’s gangrene) 10. Osteomyelitis (refractory to other therapy) 11. 12. 13. 14.

1	9. Necrotizing soft tissue infections (e.g., Fournier’s gangrene) 10. Osteomyelitis (refractory to other therapy) 11. 12. 13. 14. Sudden sensorineural hearing loss Source: The Undersea and Hyperbaric Medical Society (2013). dence varies widely with dose, age, and site. LRTI is most common in 477e-3 the head and neck, chest wall, breast, and pelvis.

1	Pathology and Clinical Course With time, tissues undergo a progressive deterioration characterized by a reduction in the density of small blood vessels (reduced vascularity) and the replacement of normal tissue with dense fibrous tissue (fibrosis). An alternative model of pathogenesis suggests that rather than a primary hypoxia, the principle trigger is an overexpression of inflammatory cytokines that promote fibrosis, probably through oxidative stress and mitochondrial dysfunction, and a secondary tissue hypoxia. Ultimately, and often triggered by a further physical insult such as surgery or infection, there may be insufficient oxygen to sustain normal function, and the tissue becomes necrotic (radiation necrosis). LRTI may be life-threatening and significantly reduce quality of life. Historically, the management of these injuries has been unsatisfactory. Conservative treatment is usually restricted to symptom management, whereas definitive treatment traditionally entails surgery to

1	the management of these injuries has been unsatisfactory. Conservative treatment is usually restricted to symptom management, whereas definitive treatment traditionally entails surgery to remove the affected part and extensive repair. Surgical intervention in an irradiated field is often disfiguring and associated with an increased incidence of delayed healing, breakdown of a surgical wound, or infection. HBO2T may act by several mechanisms to improve this situation, including edema reduction, vasculogenesis, and enhancement of macrophage activity (Fig. 477e-3). The intermittent application of HBO2 is the only intervention shown to increase the microvascular density in irradiated tissue.

1	Clinical Evidence The typical course of HBO2T consists of 30 once-daily compressions to 202.6–243.1 kPa (2–2.4 ATA) for 1.5–2 h each session, often bracketed around surgical intervention if required. Although HBO2T has been used for LRTI since at least 1975, most clinical studies have been limited to small case series or individual case reports. In a review, Feldmeier and Hampson located 71 such reports involving a total of 1193 patients across eight different tissues. There were clinically significant improvements in the majority of patients, and only 7 of 71 reports indicated a generally poor response to HBO2T. A Cochrane systematic review with meta-analysis included six randomized trials published since 1985 and drew the following conclusions (see Table 477e-2 for numbers needed to treat): HBO2T improves healing in radiation proctitis (relative risk [RR] of healing with HBO2T 2.7; 95% confidence interval [CI] 1.2–6) and after hemimandibulectomy and reconstruction of the mandible

1	HBO2T improves healing in radiation proctitis (relative risk [RR] of healing with HBO2T 2.7; 95% confidence interval [CI] 1.2–6) and after hemimandibulectomy and reconstruction of the mandible (RR 1.4; 95% CI 1.1–1.8); HBO2T improves the probability of achieving mucosal coverage (RR 1.4; 95% CI 1.2–1.6) and the restoration of bony continuity with osteoradionecrosis (ORN) (RR 1.4; 95% CI 1.1–1.8); HBO2T prevents the development of ORN following tooth extraction from a radiation field (RR 1.4; 95% CI 1.08–1.7) and reduces the risk of wound dehiscence following grafts and flaps in the head and neck (RR 4.2; 95% CI 1.1–16.8). Conversely, there was no evidence of benefit in established radiation brachial plexus lesions or brain injury.

1	A problem wound is any cutaneous ulceration that requires a prolonged time to heal, does not heal, or recurs. In general, wounds referred to hyperbaric facilities are those where sustained attempts to heal by other means have failed. Problem wounds are common and constitute a significant health problem. It has been estimated that 1% of the population of industrialized countries will experience a leg ulcer at some time. The global cost of chronic wound care may be as high as U.S. $25 billion per year.

1	U.S. $25 billion per year. Pathology and Clinical Course By definition, chronic wounds are indolent or progressive and resistant to the wide array of treatments applied. Although there are many contributing factors, most commonly these wounds arise in association with one or more comorbidities such as diabetes, peripheral venous or arterial disease, or prolonged pressure (decubitus ulcers). First-line treatments are aimed at correction of the underlying pathology (e.g., vascular reconstruction, compression bandaging, or normalization of blood glucose level), and HBO2T is an adjunctive therapy to good general wound care practice to maximize the chance of healing. Estimated Cost to Produce Outcome (number of

1	Estimated Cost to Produce Outcome (number of Radiation tissue injury More information is required on the subset of disease severity, the affected tissue type that is most likely to benefit, and the time over which benefit may persist. Resolved proctitis (30) 3 22,392 Large ongoing multicenter trial 2–11 14,928–82,104 Healed mandible (30) 4 29,184 Based on one poorly reported study 2–8 14,592–58,368 Mucosal cover in ORN (30) 3 29,888 Based on one poorly reported study 2–4 14,592–29,184 Bony continuity in ORN (30) 4 29,184 Based on one poorly reported study 2–8 14,592–58,368 Prevention of ORN after dental 4 29,184 Based on a single study extraction (30) 2–13 14,592–94,848 Prevention of dehiscence (30) 5 36,480 Based on one poorly reported study 3–8 21,888–58,368

1	Prevention of dehiscence (30) 5 36,480 Based on one poorly reported study 3–8 21,888–58,368 Chronic wounds More information is required on the subset of disease severity or classification most likely to benefit, the time over which benefit may persist, and the most appropriate oxygen dose. Economic analysis is required. Diabetic ulcer healed at 2 14,928 Based on one small study, more 1 year (30) 1–5 7464–37,320 Diabetic ulcer, major 4 29,856 Three small studies; outcome over a amputation avoided (30) 3–11 22,392–82,104 ISSNHL No evidence of benefit more than 2 weeks after onset. More research is required to define the role (if any) of HBO2T in routine therapy. Improvement of 25% in hearing 5 18,240 Some improvement in hearing, but loss within 2 weeks of onset 3–20 10,944–72,960

1	Improvement of 25% in hearing 5 18,240 Some improvement in hearing, but loss within 2 weeks of onset 3–20 10,944–72,960 Acute coronary More information is required on the subset of disease severity and timing of therapy most likely to result in benefit. Given syndrome the potential of HBO2T in modifying ischemia-reperfusion injury, attention should be given to the combination of HBO2T and thrombolysis in early management and in the prevention of restenosis after stent placement. Episode of MACE (5) 4 4864 Based on a single small study; more research required 3–10 3648–12,160 Incidence of significant 3–24 3648–29,184 Based on a single moderately powered dysrhythmia (5) study in the 1970s Traumatic brain injury Limited evidence that for acute injury HBO2T reduces mortality but not functional morbidity. Routine use not yet justified. Mortality (15) 7 34,104 Based on four heterogeneous studies 4–22 19,488–58,464

1	Enhancement of There is some evidence that HBO2T improves local tumor control, reduces mortality for cancers of the head and neck, and radiotherapy reduces the chance of local tumor recurrence in cancers of the head, neck, and uterine cervix. Decompression illnessa Reasonable evidence for reduced number of HBO2T sessions but similar outcomes when NSAID added. Abbreviations: CI, confidence interval; HBO2T, hyperbaric oxygen therapy; ISSNHL, idiopathic sudden sensorineural hearing loss; MACE, major adverse cardiac events; N/R, not remarkable; NNT, number needed to treat; NSAID, nonsteroidal anti-inflammatory drug; ORN, osteoradionecrosis; USD, U.S. dollars. Source: M Bennett: The evidence-basis of diving and hyperbaric medicine—a synthesis of the high level evidence with meta-analysis. http://unsworks.unsw.edu.au/vital/access/manager/ Repository/unsworks:949.

1	For most indolent wounds, hypoxia is a major contributor to failure to heal. Many guidelines to patient selection for HBO2T include the interpretation of transcutaneous oxygen tensions around the wound while breathing air and oxygen at pressure (Fig. 477e-4). Wound healing is a complex and incompletely understood process. While it appears that in acute wounds healing is stimulated by the initial hypoxia, low pH, and high lactate concentrations found in freshly injured tissue, some elements of tissue repair are extremely oxygen dependent, for example, collagen elaboration and deposition by fibroblasts, and bacterial killing by macrophages. In this complicated interaction between wound hypoxia and peri-wound oxygenation, successful healing relies on adequate tissue oxygenation in the area surrounding the fresh wound. Certainly, wounds that lie in hypoxic tissue beds are those that most often display poor or absent healing. Some causes of tissue hypoxia will be reversible with HBO2T,

1	surrounding the fresh wound. Certainly, wounds that lie in hypoxic tissue beds are those that most often display poor or absent healing. Some causes of tissue hypoxia will be reversible with HBO2T, whereas some will not (e.g., in the presence of severe large vessel disease). When tissue hypoxia can be overcome by a high driving pressure of oxygen in the arterial blood, this can be demonstrated by measuring the tissue partial pressure of oxygen using an implantable oxygen electrode or, more commonly, a modified transcutaneous Clarke electrode.

1	The intermittent presentation of oxygen to those hypoxic tissues facilitates a resumption of healing. These short exposures to high oxygen tensions have long-lasting effects (at least 24 h) on a wide range of healing processes (Fig. 477e-3). The result is a gradual improvement in oxygen tension around the wound that reaches a plateau in experimental studies at about 20 treatments over 4 weeks. Improvements in oxygenation are associated with an eightto ninefold increase in vascular density over both normobaric oxygen and air-breathing controls.

1	Clinical Evidence The typical course of HBO2T consists of 20–30 once-daily compressions to 2–2.4 ATA for 1.5–2 h each session, but is highly dependent on the clinical response. There are many case series in the literature supporting the use of HBO2T for a wide range of problem wounds. Both retrospective and prospective cohort studies suggest that 6 months after a course of therapy, about 70% of indolent ulcers will be substantially improved or healed. Often these ulcers have been present for many months or years, suggesting that the application of HBO2T has a profound effect, either primarily or as a facilitator of other strategies. A recent Cochrane review included nine randomized controlled trials (RCTs) and concluded that the chance of ulcer healing improved about fivefold with HBO2T (RR 5.20; 95% CI 1.25–21.66; p = .02).

1	Although there was a trend to benefit with HBO2T, there was no sta-477e-5 tistically significant difference in the rate of major amputations (RR 0.36; 95% CI 0.11–1.18). Carbon monoxide (CO) is a colorless, odorless gas formed during incomplete hydrocarbon combustion. Although CO is an essential endogenous neurotransmitter linked to NO metabolism and activity, it is also a leading cause of poisoning death, and in the United States alone results in more than 50,000 emergency department visits per year and about 2000 deaths. Although there are large variations from country to country, about half of nonlethal exposures are due to self-harm. Accidental poisoning is commonly associated with defective or improperly installed heaters, house fires, and industrial exposures. The motor vehicle is by far the most common source of intentional poisoning.

1	Pathology and Clinical Course The pathophysiology of carbon monoxide exposure is incompletely understood. CO binds to hemoglobin with an affinity more than 200 times that of oxygen, directly reducing the oxygen-carrying capacity of blood, and further promoting tissue hypoxia by shifting the oxyhemoglobin dissociation curve to the left. CO is also an anesthetic agent that inhibits evoked responses and narcotizes experimental animals in a dose-dependent manner. The associated loss of airway patency together with reduced oxygen carriage in blood may cause death from acute arterial hypoxia in severe poisoning. CO may also cause harm by other mechanisms including direct disruption of cellular oxidative processes, binding to myoglobin and hepatic cytochromes, and peroxidation of brain lipids.

1	The brain and heart are the most sensitive target organs due to their high blood flow, poor tolerance of hypoxia, and high oxygen requirements. Minor exposure may be asymptomatic or present with vague constitutional symptoms such as headache, lethargy, and nausea, whereas higher doses may present with poor concentration and cognition, short-term memory loss, confusion, seizures, and loss of consciousness. While carboxyhemoglobin (COHb) levels on admission do not necessarily reflect the severity or the prognosis of CO poisoning, cardiorespiratory arrest carries a very poor prognosis. Over the longer term, surviving patients commonly have neuropsychological sequelae. Motor disturbances, peripheral neuropathy, hearing loss, vestibular abnormalities, dementia, and psychosis have all been reported. Risk One schema for using transcutaneous oximetry to assist in patient selection for HBO2T. If the wound area is Suitable for compression?

1	One schema for using transcutaneous oximetry to assist in patient selection for HBO2T. If the wound area is Suitable for compression? hypoxic and responds to the administration of oxygen at 1 ATA or 2.4 ATA, treatment may be justified. Contraindication, critical major vessel disease or surgical option available HBO2T indicated on a case by case basis. Consider alternatives Transcutaneous mapping on 100% oxygen 2.4 ATA FIGuRE 477e-4 Determining suitability for hyperbaric oxygen therapy (HBO2T) guided by transcutaneous oximetry around the wound bed. *In diabetic patients, <50 mmHg may be more appropriate. PtcO2, transcutaneous oxygen pressure. factors for poor outcome are age >35 years, exposure for >24 h, acidosis, and loss of consciousness.

1	Clinical Evidence The typical course of HBO2T consists of two to three compressions to 2–2.8 ATA for 1.5–2 h each session. It is common for the first two compressions to be delivered within 24 h of the exposure. CO poisoning is one of the longest-standing indications for HBO2T— based largely on the obvious connection between exposure, tissue hypoxia, and the ability of HBO2T rapidly to overcome this hypoxia. CO is eliminated rapidly via the lungs on application of HBO2T, with a half-life of about 21 min at 2.0 ATA versus 5.5 h breathing air and 71 min breathing oxygen at sea level. In practice, however, it seems unlikely that HBO2T can be delivered in time to prevent either acute hypoxic death or irreversible global cerebral hypoxic injury. If HBO2T is beneficial in CO poisoning, it must reduce the likelihood of persisting and/or delayed neurocognitive deficit through a mechanism other than the simple reversal of arterial hypoxia due to high levels of COHb. The difficulty in

1	it must reduce the likelihood of persisting and/or delayed neurocognitive deficit through a mechanism other than the simple reversal of arterial hypoxia due to high levels of COHb. The difficulty in accurately assessing neurocognitive deficit has been one of the primary sources of controversy surrounding the clinical evidence in this area. To date there have been six randomized controlled trials of HBO2T for CO poisoning, although only four have been reported in full. While a Cochrane review suggested that overall there is insufficient evidence to confirm a beneficial effect of HBO2T on the chance of persisting neurocognitive deficit following poisoning (34% of patients treated with oxygen at 1 atmosphere vs 29%, of those treated with HBO2T; odds ratio [OR] 0.78; 95% CI 0.54–1.1), this may have more to do with poor reporting and inadequate follow-up than with evidence that HBO2T is not effective. The interpretation of the literature has much to do with how one defines neurocognitive

1	have more to do with poor reporting and inadequate follow-up than with evidence that HBO2T is not effective. The interpretation of the literature has much to do with how one defines neurocognitive deficit. In the most methodologically rigorous of these studies (Weaver et al.), a professionally administered battery of validated neuropsychological tests and a definition based on the deviation of individual subtest scores from the age-adjusted normal values was used; if the patient complained of memory, attention, or concentration difficulties, the required decrement was decreased. Using this approach, 6 weeks after poisoning, 46% of patients treated with normobaric oxygen alone had cognitive sequelae compared to 25% of those who received HBO2T (p = .007; number needed to treat [NNT] = 5; 95% CI 3–16). At 12 months, the difference remained significant (32% vs 18%; p = .04; NNT = 7; 95% CI 4–124) despite considerable loss to follow-up.

1	On this basis, HBO2T remains widely advocated for the routine treatment of patients with moderate to severe poisoning—in particular in those older than 35 years, presenting with a metabolic acidosis on arterial blood-gas analysis, exposed for lengthy periods, or with a history of unconsciousness. Conversely, many toxicologists remain unconvinced about the place of HBO2T in this situation and call for further well-designed studies.

1	Underwater diving is both a popular recreational activity and a means of employment in a range of tasks from underwater construction to military operations. It is a complex activity with unique hazards and medical complications arising mainly as a consequence of the dramatic changes in pressure associated with both descent and ascent through the water column. For every 10.13 m increase in depth of seawater, the ambient pressure (Pamb) increases by 101.3 kPa (1 atmosphere) so that a diver at 20 m depth is exposed to a Pamb of approximately 303.9 kPa (3 ATA), made up of 1 ATA due to atmospheric pressure and 2 ATA generated by the water column.

1	Most diving is undertaken using a self-contained underwater breathing apparatus (scuba) consisting of one or more cylinders of compressed gas connected to a pressure-reducing regulator and a demand valve activated by inspiratory effort. Some divers use “rebreathers,” which are scuba devices that are closed or semiclosed circle systems with a carbon dioxide scrubber and a system designed to maintain a safe inspired PO2. Exhaled gas is recycled, and gas consumption is limited to little more than the oxygen metabolized by the diver. Rebreathers are therefore popular for deep dives where expensive helium is included in the respired mix (see below). Occupational divers frequently use “surface supply” equipment where gas, along with other utilities such as communications and power, is supplied via an umbilical from the surface.

1	All these systems must supply gas to the diver at the Pamb of the surrounding water or inspiration would be impossible against the surrounding water pressure. For most recreational diving, the respired gas is air. Pure oxygen is rarely used because oxygen may provoke seizures above an inspired PO2 of 162 kPa (1.6 ATA) in aquatic environments, limiting the practical safe depth to 6 m. This is a conspicuously lower PO2 than routinely used for hyperbaric therapy, reflecting a higher risk of both seizures and pulmonary toxicity during immersion. For the same reason, very deep diving requires the use of oxygen fractions lower than in air (FO2 0.21). This is because breathing air at 66 m means inspiring 1.6 ATA of oxygen, the maximum allowable pressure. To dive any deeper, breathing gases must contain less oxygen than air. Deep-diving gases often include helium instead of nitrogen to reduce both the narcotic effect and high gas density that result from breathing nitrogen at high pressures.

1	The most common reason for physician consultation in relation to diving is for the evaluation of suitability for diver training or after a health event. Occupational diver candidates are usually compelled to see doctors with specialist training in the field, both at entry to the industry and periodically thereafter, and their medical evaluations are usually conducted according to legally mandated standards. In contrast, in most jurisdictions prospective recreational diver candidates simply complete a self-assessment medical questionnaire prior to diver training. If there are no positive responses, the candidate proceeds directly to training, but positive responses mandate the candidate see a doctor for evaluation of the identified medical issue. Prospective divers will often present to their family medicine practitioner for this purpose. In the modern era, such consultations have evolved from a simple proscriptive exercise of excluding those with potential contraindications to a more

1	family medicine practitioner for this purpose. In the modern era, such consultations have evolved from a simple proscriptive exercise of excluding those with potential contraindications to a more “risk analysis” approach in which each case is evaluated on its own merits. Such analyses require integration of diving physiology, the impact of associated medical problems, and a detailed knowledge of the specific medical condition of the candidate. A detailed discussion of the subject is beyond the scope of this chapter, but a few important principles are outlined below.

1	There are three primary questions that should be answered: (1) Could the underlying condition be exacerbated by diving? (2) Could the condition make a diving medical problem more likely? (3) Could the condition prevent the diver from meeting the functional requirements of diving? As examples, epilepsy is usually considered a contraindication because there are epileptogenic stimuli encountered in diving that could make a seizure more likely (such as thermal stress and exercise). Active asthma is a relative contraindication because it could predispose to pulmonary barotrauma (see below), and untreated ischemic heart disease is a contraindication because it could prevent a diver from exercising sufficiently to get out of a difficult situation such as being caught in a current. It can be a complex matter to recognize the relevant interactions between diving and medical conditions and to determine the impact on suitability for diving. Physicians interested in regularly conducting such

1	a complex matter to recognize the relevant interactions between diving and medical conditions and to determine the impact on suitability for diving. Physicians interested in regularly conducting such evaluations should obtain relevant training. Short courses providing relevant training are offered by specialist groups in most countries.

1	The problem of middle-ear barotrauma (MEBT) with diving is similar to the problem that may occur during descent from altitude in an airplane, but difficulties with equalizing pressure in the middle ear are exaggerated underwater by both the rapidity and magnitude of pressure change as a diver descends or ascends. Failure to periodically insufflate the middle-ear spaces via the eustachian tubes during descent results in increasing pain. As the Pamb increases, the tympanic membrane (TM) may be bruised or even ruptured as it is pushed inward. Negative pressure in the middle ear results in engorgement of blood vessels in the mucous membranes and leads to effusion or bleeding, which can be associated with a conductive hearing loss. MEBT is much less common during ascent because expanding gas in the middle-ear space tends to open the eustachian tube easily and “automatically.” Barotrauma may also affect the respiratory sinuses, although the sinus ostia are usually widely patent and allow

1	in the middle-ear space tends to open the eustachian tube easily and “automatically.” Barotrauma may also affect the respiratory sinuses, although the sinus ostia are usually widely patent and allow automatic pressure equalization without the need for specific maneuvers. If equalization fails, pain usually results in termination of the dive. Difficulty with equalizing ears or sinuses may respond to oral or nasal decongestants.

1	Much less commonly the inner ear may suffer barotrauma (IEBT). Several explanations have been proposed, of which the most favored holds that forceful attempts to insufflate the middle-ear space by Valsalva maneuvers during descent cause sudden transmission of pressure to the perilymph via the cochlear aqueduct and outward rupture of the round window already under tension because of negative middle ear pressure. The clinician should be alerted to possible IEBT after diving by a sensorineural hearing loss or true vertigo (which is often accompanied by nausea, vomiting, nystagmus, and ataxia). These manifestations can also occur in vestibulocochlear DCS (see below) but should never be attributed to MEBT. Immediate review by an expert diving physician is recommended, and urgent referral to an otologist will often follow.

1	The lungs are also vulnerable to barotrauma but are at most risk during ascent. If expanding gas becomes trapped in the lungs as Pamb falls, this may rupture alveoli and associated vascular tissue. Gas trapping may occur if divers intentionally or involuntarily hold their breath during ascent or if there are bullae. The extent to which asthma predisposes to pulmonary barotrauma is debated, but the presence of active bronchoconstriction must increase risk. For this reason, asthmatics who regularly require bronchodilator medications or whose airways are sensitive to exercise or cold air are usually discouraged from diving. While possible consequences of pulmonary barotrauma include pneumothorax and mediastinal emphysema, the most feared is the introduction of gas into the pulmonary veins leading to cerebral arterial gas embolism (CAGE). Manifestations of CAGE include loss of consciousness, confusion, hemiplegia, visual disturbances, and speech difficulties, appearing immediately or

1	leading to cerebral arterial gas embolism (CAGE). Manifestations of CAGE include loss of consciousness, confusion, hemiplegia, visual disturbances, and speech difficulties, appearing immediately or within minutes after surfacing. The management is the same as for DCS described below. It is notable that the natural history of CAGE often includes substantial or complete resolution of symptoms early after the event. This is probably the clinical correlate of bubble involution and redistribution with consequent restoration of flow. Patients exhibiting such remissions should still be reviewed at specialist diving medical centers because secondary deterioration or reembolization can occur. Unsurprisingly, these events can be misdiagnosed as typical strokes or transient ischemic attacks (TIAs) (Chap. 455) when patients are seen by those unfamiliar with diving medicine. All patients presenting with neurologic symptoms after diving should have their symptoms discussed with a specialist in

1	(Chap. 455) when patients are seen by those unfamiliar with diving medicine. All patients presenting with neurologic symptoms after diving should have their symptoms discussed with a specialist in diving medicine and be considered for recompression therapy.

1	DCS is caused by the formation of bubbles from dissolved inert gas (usually nitrogen) during or after ascent (decompression) from a compressed gas dive. Bubble formation is also possible following decompression for extravehicular activity during space flight and with ascent to altitude in unpressurized aircraft. DCS in the latter scenarios is probably rare in comparison with diving, where the incidence is approximately 1:10,000 recreational dives.

1	Breathing at elevated Pamb results in increased uptake of inert gas into blood and then into tissues. The rate at which tissue inert gas equilibrates with the inspired inert gas pressure is proportional to tissue blood flow and the blood-tissue partition coefficient for the gas. Similar factors dictate the kinetics of inert gas washout during ascent. If the rate of gas washout from tissues does not match the rate of decline in Pamb, then the sum of dissolved gas pressures in the tissue will exceed Pamb, a condition referred to as “supersaturation.” This is the prerequisite for bubbles to form during decompression, although other less well-understood factors are also involved. Deeper and longer dives 477e-7 result in greater inert gas absorption and greater likelihood of tissue supersaturation during ascent. Divers control their ascent for a given depth and time exposure using algorithms that often include periods where ascent is halted for a prescribed period at different depths to

1	during ascent. Divers control their ascent for a given depth and time exposure using algorithms that often include periods where ascent is halted for a prescribed period at different depths to allow time for gas washout (“decompression stops”). Although a breach of these protocols increases the risk of DCS, adherence does not guarantee against it. DCS should be considered in any diver manifesting symptoms not readily explained by an alternative mechanism.

1	Bubbles may form within tissues themselves, where they cause symptoms by mechanical distraction of pain-sensitive or functionally important structures. They also appear in the venous circulation as blood passes through supersaturated tissues. Some venous bubbles are tolerated without symptoms and are filtered from the circulation in the pulmonary capillaries. However, in sufficiently large numbers, these bubbles are capable of inciting inflammatory and coagulation cascades, damaging endothelium, activating formed elements of blood such as platelets, and causing symptomatic pulmonary vascular obstruction. Moreover, if there is a right-to-left shunt such as through a patent foramen ovale (PFO) or an intrapulmonary shunt, then venous bubbles may enter the arterial circulation (25% of adults have a probe-patent PFO). The risk of cerebral, spinal cord, inner ear, and skin manifestations appears higher in the presence of significant shunts, suggesting that these “arterialized” venous

1	have a probe-patent PFO). The risk of cerebral, spinal cord, inner ear, and skin manifestations appears higher in the presence of significant shunts, suggesting that these “arterialized” venous bubbles can cause harm, perhaps by disrupting flow in the microcirculation of target organs. Circulating endothelial microparticles, which are elevated in number and size after diving, are currently under investigation as indicators of decompression stress or possibly as injurious agents in their own right. How they arise, and their role in DCS, remain unclear.

1	Table 477e-3 lists manifestations of DCS grouped according to organ system. The majority of cases present with mild symptoms, including musculoskeletal pain, fatigue, and minor neurologic manifestations such as patchy paresthesias. Serious presentations are much less common. Pulmonary and cardiovascular manifestations can be life-threatening, and spinal cord involvement frequently results in permanent disability. Latency is variable. Serious DCS usually manifests within minutes of surfacing, but mild symptoms may not appear for several hours. Symptoms arising more than 24 h after diving are very unlikely to be DCS. The presentation may be confusing and nonspecific, and there are as yet no useful diagnostic investigations. Diagnosis is based on integration of findings from examination of the dive profile, the nature and temporal relationship of symptoms, and the clinical examination. Some DCS presentations may be difficult to separate from CAGE following pulmonary barotrauma, but from

1	dive profile, the nature and temporal relationship of symptoms, and the clinical examination. Some DCS presentations may be difficult to separate from CAGE following pulmonary barotrauma, but from a clinical perspective the distinction is unimportant because the first aid and definitive management of both conditions are the same.

1	First aid includes horizontal positioning (especially if there are cerebral manifestations), intravenous fluids if available, and sustained 100% oxygen administration. The latter accelerates inert gas washout from tissues and promotes resolution of bubbles. Definitive treatment of DCS or CAGE with recompression and hyperbaric oxygen is justified in most instances, although some mild or marginal DCS cases may be managed with first aid measures, an option that may be invoked under various circumstances, but especially if evacuation for recompression is hazardous or extremely difficult. Long-distance evacuations are usually undertaken using a helicopter flying at low altitude or a fixed wing air ambulance pressurized to 1 atmosphere pressure.

1	Recompression reduces bubble volume in accordance with Boyle’s law and increases the inert gas partial pressure difference between a bubble and surrounding tissue. At the same time, oxygen administration markedly increases the inert gas partial pressure difference between alveoli and tissue. The net effect is to significantly increase the rate of inert gas diffusion from bubble to tissue and tissue to blood, thus accelerating bubble resolution. Hyperbaric oxygen also helps oxygenate compromised tissues and appears to ameliorate some of the proinflammatory effects of bubbles. Various recompression protocols have been advocated, but there are no data that define the optimum approach. It typically begins with oxygen administered at 2.8 atmospheres absolute, the maximum pressure at which the risk of oxygen toxicity remains acceptable in a hyperbaric chamber. There follows a stepwise decompression over variable periods adjusted to symptom response. The most widely used algorithm is the

1	risk of oxygen toxicity remains acceptable in a hyperbaric chamber. There follows a stepwise decompression over variable periods adjusted to symptom response. The most widely used algorithm is the U.S. Navy Table 6, whose shortest format lasts 4 h and 45 min. Typically, shorter “follow-up” recompressions are repeated daily while symptoms persist and appear responsive to treatment. Adjuncts to recompression include intravenous fluids and other supportive care as necessary. Occasionally, very sick divers require high-level intensive care.

1	hypothermia and Frostbite Daniel F. Danzl HYPOTHERMIA Accidental hypothermia occurs when there is an unintentional drop in the body’s core temperature below 35°C (95°F). At this temperature, many of the compensatory physiologic mechanisms that conserve heat 478e begin to fail. Primary accidental hypothermia is a result of the direct exposure of a previously healthy individual to the cold. The mortality rate is much higher for patients who develop secondary hypothermia as a complication of a serious systemic disorder.

1	Primary accidental hypothermia is geographically and seasonally pervasive. Although most cases occur in the winter months and in colder climates, this condition is surprisingly common in warmer regions as well. Multiple variables render individuals at the extremes of age— both the elderly and neonates—particularly vulnerable to hypothermia (Table 478e-1). The elderly have diminished thermal perception and are more susceptible to immobility, malnutrition, and systemic illnesses that interfere with heat generation or conservation. Dementia, psychiatric illness, and socioeconomic factors often compound these problems by impeding adequate measures to prevent hypothermia. Neonates have high rates of heat loss because of their increased surface-to-mass ratio and their lack of effective shivering and adaptive behavioral responses. At all ages, malnutrition can contribute to heat loss because of diminished subcutaneous fat and as a result of depleted energy stores used for thermogenesis.

1	Individuals whose occupations or hobbies entail extensive exposure to cold weather are at increased risk for hypothermia. Military history is replete with hypothermic tragedies. Hunters, sailors, skiers, and climbers also are at great risk of exposure, whether it involves injury, changes in weather, or lack of preparedness. Ethanol causes vasodilation (which increases heat loss), reduces thermogenesis and gluconeogenesis, and may impair judgment or lead to obtundation. Phenothiazines, barbiturates, benzodiazepines, heterocyclic antidepressants, and many other medications reduce centrally mediated vasoconstriction. Up to 25% of patients admitted to an intensive care unit because of drug overdose are hypothermic. Anesthetics can block shivering responses; their effects are compounded when patients are not insulated adequately in the operating or recovery units.

1	Several types of endocrine dysfunction can lead to hypothermia. 478e-1 Hypothyroidism—particularly when extreme, as in myxedema coma— reduces the metabolic rate and impairs thermogenesis and behavioral responses. Adrenal insufficiency and hypopituitarism also increase susceptibility to hypothermia. Hypoglycemia, most commonly caused by insulin or oral hypoglycemic drugs, is associated with hypothermia, in part because of neuroglycopenic effects on hypothalamic function. Increased osmolality and metabolic derangements associated with uremia, diabetic ketoacidosis, and lactic acidosis can lead to altered hypothalamic thermoregulation.

1	Neurologic injury from trauma, cerebrovascular accident, subarachnoid hemorrhage, and hypothalamic lesion increases susceptibility to hypothermia. Agenesis of the corpus callosum (Shapiro syndrome) is one cause of episodic hypothermia; in this syndrome, profuse perspiration is followed by a rapid fall in temperature. Acute spinal cord injury disrupts the autonomic pathways that lead to shivering and prevents cold-induced reflex vasoconstrictive responses. Hypothermia associated with sepsis is a poor prognostic sign. Hepatic failure causes decreased glycogen storage and gluconeogenesis as well as a diminished shivering response. In acute myocardial infarction associated with low cardiac output, hypothermia may be reversed after adequate resuscitation. With extensive burns, psoriasis, erythrodermas, and other skin diseases, increased peripheral-blood flow leads to excessive heat loss.

1	Heat loss occurs through five mechanisms: radiation (55–65% of heat loss), conduction (10–15% of heat loss, much increased in cold water), convection (increased in the wind), respiration, and evaporation; both of the latter two mechanisms are affected by the ambient temperature and the relative humidity. The preoptic anterior hypothalamus normally orchestrates thermo-regulation (Chap. 23). The immediate defense of thermoneutrality is via the autonomic nervous system, whereas delayed control is mediated by the endocrine system. Autonomic nervous system responses include the release of norepinephrine, increased muscle tone, and shivering, leading to thermogenesis and an increase in the basal metabolic rate. Cutaneous cold thermoreception causes direct reflex vasoconstriction to conserve heat. Prolonged exposure to cold also stimulates the thyroid axis, leading to an increased metabolic rate.

1	In most cases of hypothermia, the history of exposure to environmental factors (e.g., prolonged exposure to the outdoors without adequate clothing) makes the diagnosis straightforward. In urban settings, however, the presentation is often more subtle, and other disease processes, toxin exposures, or psychiatric diagnoses should be considered.

1	After initial stimulation by hypothermia, there is progressive depression of all organ systems. The timing of the appearance of these clinical manifestations varies widely (Table 478e-2). Without knowing the core temperature, it can be difficult to interpret other vital signs. For example, tachycardia disproportionate to the core temperature suggests secondary hypothermia resulting from hypoglycemia, hypovolemia, or a toxin overdose. Because carbon dioxide production declines progressively, the respiratory rate should be low; persistent hyperventilation suggests a central nervous system (CNS) lesion or one of the organic acidoses. A markedly depressed level of consciousness in a patient with mild hypothermia raises suspicion of an overdose or CNS dysfunction due to infection or trauma.

1	Physical examination findings can also be altered by hypothermia. For instance, the assumption that areflexia is solely attributable to hypothermia can obscure and delay the diagnosis of a spinal cord lesion. Patients with hypothermia may be confused or combative; these symptoms abate more rapidly with rewarming than with chemical or physical restraint. A classic example of maladaptive behavior in patients with hypothermia is paradoxical undressing, which involves the inappropriate removal of clothing in response to a cold stress. The cold-induced ileus and abdominal rectus spasm can mimic or mask the presentation of an acute abdomen (Chap. 20). Mild 35°C (95°F)– 32.2°C (90°F) Moderate <32.2°C (90°F)–28°C (82.4°F) Severe <28°C (<82.4°F)

1	Mild 35°C (95°F)– 32.2°C (90°F) Moderate <32.2°C (90°F)–28°C (82.4°F) Severe <28°C (<82.4°F) Linear depression of Tachycardia, then pro-Tachypnea, then Diuresis; increase in cerebral metabolism; gressive bradycardia; progressive decrease catecholamines, adreamnesia; apathy; dysar-cardiac cycle prolonga-in respiratory minute nal steroids, triiodothythria; impaired judgment; tion; vasoconstriction; volume; declining ronine, and thyroxine; maladaptive behavior increase in cardiac out-oxygen consumption; increase in metabolism

1	EEG abnormalities; pro-Progressive decrease Hypoventilation; 50% 50% increase in renal gressive depression of in pulse and cardiac decrease in carbon blood flow; renal level of consciousness; output; increased atrial dioxide production autoregulation intact; pupillary dilation; para-and ventricular arrhyth-per 8°C (46°F) drop in impaired insulin action doxical undressing; hal-mias; suggestive (Jtemperature; absence lucinations wave) ECG changes of protective airway Loss of cerebrovascular Progressive decrease in Pulmonic conges-Decrease in renal blood autoregulation; decline blood pressure, heart tion and edema; 75% flow that parallels in cerebral blood flow; rate, and cardiac out-decrease in oxygen decrease in cardiac coma; loss of ocular put; reentrant dysrhyth-consumption; apnea output; extreme olireflexes; progressive mias; maximal risk of Abbreviations: ECG, electrocardiogram; EEG, electroencephalogram. Source: Modified from DF Danzl, RS Pozos: N Engl J Med 331:1756, 1994.

1	When a patient in hypothermic cardiac arrest is first discovered, cardiopulmonary resuscitation is indicated unless (1) a do-not-resuscitate status is verified, (2) obviously lethal injuries are identified, or (3) the depression of a frozen chest wall is not possible. As the resuscitation proceeds, the prognosis is grave if there is evidence of widespread cell lysis, as reflected by potassium levels >10–12 mmol/L (10–12meq/L). Other findings that may preclude continuing resuscitation include a core temperature <10–12°C (<50–54°F), a pH <6.5, and evidence of intravascular thrombosis with a fibrinogen value <0.5 g/L (<50 mg/ dL). The decision to terminate resuscitation before rewarming the patient past 33°C (91°F) should be predicated on the type and severity of the precipitants of hypothermia. There are no validated prognostic indicators for recovery from hypothermia. A history of asphyxia with secondary cooling is the most important negative predictor of survival.

1	Hypothermia is confirmed by measurement of the core temperature, preferably at two sites. Rectal probes should be placed to a depth of 15 cm and not adjacent to cold feces. A simultaneous esophageal probe should be placed 24 cm below the larynx; it may read falsely high during heated inhalation therapy. Relying solely on infrared tympanic thermography is not advisable. After a diagnosis of hypothermia is established, cardiac monitoring should be instituted, along with attempts to limit further heat loss. If the patient is in ventricular fibrillation, it is unclear at what core temperature ventricular defibrillation (2 J/kg) should first be attempted. One attempt below 30°C is warranted. Further defibrillation attempts should be deferred until some rewarming (1°–2°C) is achieved and ventricular fibrillation is coarser. Although cardiac pacing for hypothermic bradydysrrhythmias is rarely indicated, the transthoracic technique is preferable.

1	Supplemental oxygenation is always warranted, since tissue oxygenation is affected adversely by the leftward shift of the oxyhemoglobin dissociation curve. Pulse oximetry may be unreliable in patients with vasoconstriction. If protective airway reflexes are absent, gentle endotracheal intubation should be performed. Adequate preoxygenation will prevent ventricular arrhythmias. Insertion of a gastric tube prevents dilation secondary to decreased bowel motility. Indwelling bladder catheters facilitate monitoring of cold-induced diuresis. Dehydration is encountered commonly with chronic hypothermia, and most patients benefit from an intravenous or intraosseous bolus of crystalloid. Normal saline is preferable to

1	Increased preshivering muscle tone, then fatiguing lactated Ringer’s solution, as the liver in hypothermic patients inefficiently metabolizes lactate. The placement of a pulmonary artery catheter can cause perforation of the less compliant pulmonary artery. Insertion of a central venous catheter deeply into the cold right atrium should be avoided since this procedure can precipitate arrhythmias. Arterial blood gases should not be corrected for temperature (Chap. 66). An uncorrected pH of 7.42 and a Pco2 of 40 mmHg reflect appropriate alveolar ventilation and acid-base balance at any core temperature. Acid-base imbalances should be corrected gradually, since the bicarbonate buffering system is inefficient. A common error is overzealous hyperventilation in the setting of depressed CO2 production. When the Pco2 decreases by 10 mmHg at 28°C (82°F), it doubles the pH increase of 0.08 that occurs at 37°C (99°F).

1	The severity of anemia may be underestimated because the hematocrit increases 2% for each 1°C drop in temperature. White blood cell sequestration and bone marrow suppression are common, potentially masking an infection. Although hypokalemia is more common in chronic hypothermia, hyperkalemia also occurs; the expected electrocardiographic changes can be obscured by hypothermia. Patients with renal insufficiency, metabolic acidoses, or rhabdomyolysis are at greatest risk for electrolyte disturbances.

1	Coagulopathies are common because cold inhibits the enzymatic reactions required for activation of the intrinsic cascade. In addition, thromboxane B2 production by platelets is temperature dependent, and platelet function is impaired. The administration of platelets and fresh-frozen plasma is therefore not effective. The prothrombin or partial thromboplastin times or the international normalized ratio can be deceptively normal and contrast with the observed in vivo coagulopathy. This contradiction occurs because all coagulation tests are routinely performed at 37°C (99°F), and the enzymes are thus rewarmed.

1	The key initial decision is whether to rewarm the patient passively or actively. Passive external rewarming simply involves covering and insulating the patient in a warm environment. With the head also covered, the rate of rewarming is usually 0.5°–2°C (1.10°–4.4°F) per hour. This technique is ideal for previously healthy patients who develop acute, mild primary accidental hypothermia. The patient must have sufficient glycogen to support endogenous thermogenesis. The application of heat directly to the extremities of patients with chronic severe hypothermia should be avoided because it can induce peripheral vasodilation and precipitate core temperature “afterdrop,” a response characterized by a continual decline in the core temperature after removal of the patient from the cold. Truncal heat application reduces the risk of afterdrop.

1	Active rewarming is necessary under the following circumstances: core temperature <32°C (<90°F) (poikilothermia), cardiovascular instability, age extremes, CNS dysfunction, hormone insufficiency, and suspicion of secondary hypothermia. Active external rewarming is best accomplished with forced-air heating blankets. Other options include devices that circulate water through external heat exchange pads, radiant heat sources, and hot packs. Monitoring a patient with hypothermia in a heated tub is extremely difficult. Electric blankets should be avoided because vasoconstricted skin is easily burned.

1	There are numerous widely available options for active core rewarming. Airway rewarming with heated humidified oxygen (40°–45°C [104°–113°F]) via mask or endotracheal tube is a convenient option. Although airway rewarming provides less heat than do some other forms of active core rewarming, it eliminates respiratory heat loss and adds 1°–2°C (1.1°–4.4°F) to the overall rewarming rate. Crystalloids should be heated to 40°–42°C (104°–108°F), but the quantity of heat provided is significant only during massive volume resuscitation. The most efficient method for heating and delivering fluid or blood is with a countercurrent in-line heat exchanger. Heated irrigation of the gastrointestinal tract or bladder transfers minimal heat because of the limited available surface area. These methods should be reserved for patients in cardiac arrest and then used in combination with all available active rewarming techniques.

1	Closed thoracic lavage is far more efficient in severely hypothermic patients with cardiac arrest. The hemithoraxes are irrigated through two inserted large-bore thoracostomy tubes. Thoracostomy tubes should not be placed in the left chest of a spontaneously perfusing patient for purposes of rewarming. Peritoneal lavage with the dialysate at 40°–45°C (104°–113°F) efficiently transfers heat when delivered through two catheters with outflow suction. Like peritoneal dialysis, standard hemodialysis is especially useful for patients with electrolyte abnormalities, rhabdomyolysis, or toxin ingestion. Another option involves the central venous insertion of a rapid endovascular warming device.

1	Extracorporeal blood rewarming options (Table 478e-3) should be considered in severely hypothermic patients, especially those with primary accidental hypothermia. Cardiopulmonary bypass should be considered in nonperfusing patients without documented contraindications to resuscitation. Circulatory support may be the only effective option in patients with completely frozen extremities or those with significant tissue destruction coupled with rhabdomyolysis. There is no evidence that extremely rapid rewarming improves survival in per-fusing patients. The best strategy is usually a combination of passive, truncal active, and active core rewarming techniques.

1	When a patient is hypothermic, target organs and the cardiovascular system respond minimally to most medications. Generally, IV medications are withheld below 30°C (86°F). In contrast to antiarrhythmics, low-dose vasopressor medications may improve the intra-arrest rates of return of spontaneous circulation. Because of increased binding of drugs to proteins as well as impaired metabolism and excretion, either a lower dose or a longer interval between doses should be used to avoid toxicity. As an example, the administration of repeated doses of digoxin or insulin would be ineffective while the patient is hypothermic, and the residual drugs would be potentially toxic during rewarming.

1	Achieving a mean arterial pressure of at least 60 mmHg should be an early objective. If the hypotension does not respond to crystalloid/ colloid infusion and rewarming, low-dose dopamine support (2–5 μg/ kg per min) should be considered. Perfusion of the vasoconstricted cardiovascular system also may be improved with low-dose IV nitroglycerin. Atrial arrhythmias should be monitored initially without intervention, as the ventricular response should be slow and, unless preexistent, most will convert spontaneously during rewarming. The role of Continuous venovenous Circuit: CV catheter to CV, dual-lumen CV, or Hemodialysis Circuit: singleor dual-vessel cannulation Continuous arteriove-Circuit: percutaneous 8.5-Fr femoral catheters nous rewarming (CAVR) Requires systolic blood pressure of 60 mmHg Cardiopulmonary Circuit: full circulatory support with pump and bypass (CPB) oxygenator ROR up to 9.5°C (49°F)/h

1	Requires systolic blood pressure of 60 mmHg Cardiopulmonary Circuit: full circulatory support with pump and bypass (CPB) oxygenator ROR up to 9.5°C (49°F)/h Extracorporeal Decreased risk of post-rewarming cardiorespiratory membrane oxygenation failure (ECMO) Abbreviations: CV, central venous; ROR, rate of rewarming. prophylaxis and treatment of ventricular arrhythmias is problematic. Preexisting ventricular ectopy may be suppressed by hypothermia and reappear during rewarming. None of the class I agents has proved to be safe and efficacious. There is also insufficient evidence that the class III ventricular antiarrhythmic amiodarone is safe.

1	Initiating empirical therapy for adrenal insufficiency usually is not warranted unless the history suggests steroid dependence or hypoadrenalism or efforts to rewarm with standard therapy fail. The administration of parenteral levothyroxine to euthyroid patients with hypothermia, however, is potentially hazardous. Because laboratory results can be delayed and confounded by the presence of the sick euthyroid syndrome (Chap. 405), historic clues or physical findings suggestive of hypothyroidism should be sought. When myxedema is the cause of hypothermia, the relaxation phase of the Achilles reflex is prolonged more than is the contraction phase.

1	Hypothermia obscures most of the symptoms and signs of infection, notably fever and leukocytosis. Shaking rigors from infection may be mistaken for shivering. Except in mild cases, extensive cultures and repeated physical examinations are essential. Unless an infectious source is identified, empirical antibiotic prophylaxis is most warranted in the elderly, neonates, and immunocompromised patients. Preventive measures should be discussed with high-risk individuals, such as the elderly and people whose work frequently exposes them to extreme cold. The importance of layered clothing and headgear, adequate shelter, increased caloric intake, and the avoidance of ethanol should be emphasized, along with access to rescue services.

1	Peripheral cold injuries include both freezing and nonfreezing injuries to tissue. Tissue freezes quickly when in contact with thermal conductors such as metal and volatile solutions. Other predisposing factors include constrictive clothing or boots, immobility, and vasoconstrictive medications. Frostbite occurs when the tissue temperature drops below 0°C (32°F). Ice-crystal formation subsequently distorts and destroys the cellular architecture. Once the vascular endothelium is damaged, stasis progresses rapidly to microvascular thrombosis. After the tissue thaws, there is progressive dermal ischemia. The microvasculature begins to collapse, arteriovenous shunting increases tissue pressures, and edema forms. Finally, thrombosis, ischemia, and superficial necrosis appear. The development of mummification and demarcation may take weeks to months.

1	The initial presentation of frostbite can be deceptively benign. The symptoms always include a sensory deficiency affecting light touch, pain, and temperature perception. The acral areas and distal extremities are the most common insensate areas. Some patients describe a clumsy or “chunk of wood” sensation in the extremity. Deep frostbitten tissue can appear waxy, mottled, yellow, or violaceous-white. Favorable presenting signs include some warmth or sensation with normal color. The injury is often superficial if the subcutaneous tissue is pliable or if the dermis can be rolled over bony prominences. Frostnip may precede frostbite. Frostnip is a nonfreezing cold injury resulting from intense vasoconstriction of exposed acral skin.

1	Frostnip may precede frostbite. Frostnip is a nonfreezing cold injury resulting from intense vasoconstriction of exposed acral skin. Clinically, it is most practical to classify frostbite as superficial or deep. Superficial frostbite does not entail tissue loss but rather causes only anesthesia and erythema. The appearance of vesiculation surrounded by edema and erythema implies deeper involvement (Fig. 478e-1). Hemorrhagic vesicles reflect a serious injury to the microvasculature and indicate severe frostbite. Damages in subcuticular, muscular, or osseous tissues may result in amputation. The two most common nonfreezing peripheral cold injuries are chilblain (pernio) and immersion (trench) foot. Chilblain results from neuronal and endothelial damage induced by repetitive exposure to FIGURE 478e-1 Frostbite with vesiculation, surrounded by edema and erythema.

1	FIGURE 478e-1 Frostbite with vesiculation, surrounded by edema and erythema. dry cold. Young females, particularly those with a history of Raynaud’s phenomenon, are at greatest risk. Persistent vasospasticity and vasculitis can cause erythema, mild edema, and pruritus. Eventually plaques, blue nodules, and ulcerations develop. These lesions typically involve the dorsa of the hands and feet. In contrast, immersion foot results from repetitive exposure to wet cold above the freezing point. The feet initially appear cyanotic, cold, and edematous. The subsequent development of bullae is often indistinguishable from frostbite. This vesiculation rapidly progresses to ulceration and liquefaction gangrene. Patients with milder cases report hyperhidrosis, cold sensitivity, and painful ambulation for many years.

1	When frostbite accompanies hypothermia, hydration may improve vascular stasis. Frozen tissue should be thawed rapidly and completely by immersion in circulating water at 37°–40°C (99°–104°F). Rapid rewarming often produces an initial hyperemia. The early formation of large clear distal blebs is more favorable than that of smaller proximal dark hemorrhagic blebs. A common error is the premature termination of thawing, since the reestablishment of perfusion is intensely painful. Parenteral narcotics will be necessary with deep frostbite. If cyanosis persists after rewarming, the tissue compartment pressures should be monitored carefully. Many antithrombotic and vasodilatory primary and adjunctive treatment regimens have been evaluated. The prostacyclin analogue iloprost in combination with aspirin may prove useful. There is no conclusive evidence that sympathectomy, steroids, calcium channel blockers, or hyperbaric oxygen salvages tissue.

1	Patients who have deep frostbite injuries with the potential for significant morbidity should be considered for intravenous or intraarterial thrombolytic therapy. Angiography or pyrophosphate scanning should help evaluate the injury and monitor the progress of tissue plasminogen activator therapy. Heparin is recommended as adjunctive therapy. Intraarterial thrombolysis may reduce the need for digital and more proximal amputations when administered within 24 h of severe injuries. A treatment protocol for frostbite is summarized in (Table 478e-4). Unless infection develops, any decision regarding debridement or amputation should generally be deferred. Angiography or Remove from environment. Prevent partial thawing and refreezing. Stabilize core temperature and treat hypothermia. Protect frozen part—no friction or massage. Consider parenteral analgesia and ketorolac. Administer ibuprofen (400 mg PO).

1	Stabilize core temperature and treat hypothermia. Protect frozen part—no friction or massage. Consider parenteral analgesia and ketorolac. Administer ibuprofen (400 mg PO). Immerse part in 37°–40°C (99°–104°F) (thermometermonitored) circulating water containing an antiseptic soap until distal flush (10–45 min). Encourage patient to gently move part. Gently dry and protect part; elevate; place pledgets between toes, if macerated. If clear vesicles are intact, aspirate sterilely; if broken, debride and dress with antibiotic or sterile aloe vera ointment. Leave hemorrhagic vesicles intact to prevent desiccation and infection. Continue ibuprofen (400 mg PO [12 mg/kg per day] q12h). Address medical or If pain is refractory, reduce Consider tetanus and surgical conditions. water temperature to streptococcal prophylaxis; 35°–37°C (95°–99°F) and elevate part. administer parenteral Administer hydrotherapy narcotics. at 37°C (99°F).

1	Administer hydrotherapy narcotics. at 37°C (99°F). Consider dextran or phenoxybenzamine or, in severe cases, thrombolysis heat-related Illnesses Daniel F. Danzl Heat-related illnesses include a spectrum of disorders ranging from heat syncope, muscle cramps, and heat exhaustion to medical emer-gencies such as heatstroke. The core body temperature is normally maintained within a very narrow range. Although significant levels 479e of hypothermia are tolerated (Chap. 478e), multiorgan dysfunction occurs rapidly at temperatures >41°–43°C. In contrast to severe hyperthermia, the far more common sign of fever reflects intact thermoregulation.

1	Humans are capable of significant heat generation. Strenuous exercise can increase heat generation twentyfold. The heat load from metabolic heat production and environmental heat absorption is balanced by a variety of heat dissipation mechanisms. These dissipation pathways are orchestrated by the central thermostat, which is located in the preoptic nucleus of the anterior hypothalamus. Efferent signals sent via the autonomic nervous system trigger cutaneous vasodilation and diaphoresis to facilitate heat loss.

1	Normally, the body dissipates heat into the environment via four mechanisms. The evaporation of skin moisture is the single most efficient mechanism of heat loss but becomes progressively ineffective as the relative humidity rises to >70%. The radiation of infrared electromagnetic energy directly into the surrounding environment occurs continuously. (Conversely, radiation is a major source of heat gain in hot climates.) Conduction— the direct transfer of heat to a cooler object—and convection—the loss of heat to air currents—become ineffective when the environmental temperature exceeds the skin temperature.

1	Factors that interfere with the evaporation of diaphoresis significantly increase the risk of heat illness. Examples include dripping of sweat off the skin, constrictive or occlusive clothing, dehydration, and excessive humidity. While air is an effective insulator, the thermal conductivity of water is 25 times greater than that of air at the same temperature. The wet-bulb globe temperature is a commonly used index to assess the environmental heat load. This calculation considers the ambient air temperature, the relative humidity, and the degree of radiant heat.

1	The regulation of this heat load is complex and involves the central nervous system (CNS), thermosensors, and thermoregulatory effectors. The central thermostat activates the effectors that produce peripheral vasodilation and sweating. The skin surface is in effect the radiator and the principal location of heat loss, since skin blood flow can increase 25–30 times over the basal rate. This dramatic increase in skin blood flow, coupled with the maintenance of peripheral vasodilation, efficiently radiates heat. At the same time, there is a compensatory vasoconstriction of the splanchnic and renal beds.

1	Acclimatization to heat reflects a constellation of physiologic adaptations that permit the body to lose heat more efficiently. This process often requires one to several weeks of exposure and work in a hot environment. During acclimatization, the thermoregulatory set point is altered, and this alteration affects the onset, volume, and content of diaphoresis. The threshold for the initiation of sweating is lowered, and the amount of sweat increases, with a lowered salt concentration. Sweating rates can be 1–2 L/h in acclimated individuals during heat stress. Plasma volume expansion also occurs and improves cutaneous vascular flow. The heart rate lowers, with a higher stroke volume. After the individual leaves the hot environment, improved tolerance to heat stress dissipates rapidly, the plasma volume decreases, and de-acclimatization occurs within weeks.

1	When there is an excessive heat load, unacclimated individuals can develop a variety of heat-related illnesses. Heat waves exacerbate the mortality rate, particularly among the elderly and poor and among 479e-1 persons lacking adequate nutrition and access to air-conditioned environments. Secondary vascular events, including cerebrovascular accidents and myocardial infarctions, occur at least 10 times more often in conditions of extreme heat. Exertional heat illness continues to occur when laborers, military personnel, or athletes exercise strenuously in the heat. A variety of common factors predispose to heat illness. In addition to the very young and very old, preadolescents and teenagers are at risk since they may use poor judgment when vigorously exercising in high humidity and heat. Other risk factors include obesity, poor conditioning and lack of acclimatization, and mild dehydration.

1	Cardiovascular inefficiency is a common feature of heat illness. Any physiologic or pharmacologic impediment to cutaneous perfusion will impair heat loss. Many patients are unaware of the heat risk associated with their medications. Anticholinergic agents impair sweating and blunt the normal cardiovascular response to heat. Phenothiazines also have anticholinergic properties that interfere with the function of the preoptic nucleus of the anterior hypothalamus due to central depletion of dopamine.

1	Calcium channel blockers, beta blockers, and various stimulants also inhibit sweating by reducing peripheral blood flow. To maintain the mean arterial blood pressure, increased cardiac output must be capable of compensating for progressive dehydration. A variety of stimulants and substances of abuse also increase muscle activity and heat production. Careful consideration of the differential diagnosis is important in the evaluation of a patient for a potential heat-related illness. The clinical setting may suggest other etiologies, such as malignant hyperthermia after general anesthesia or neuroleptic malignant syndrome in a patient taking certain antipsychotic medications. A variety of infectious and endocrine disorders as well as conditions with toxicologic or CNS etiologies may initially mimic heatstroke (Table 479e-1).

1	Heat edema is characterized by mild swelling of the hands, feet, and ankles during the first few days of significant heat exposure. The principal mechanism involves cutaneous vasodilation and pooling of interstitial fluid in response to heat stress. Heat also increases the secretion of antidiuretic hormone and aldosterone. Systemic causes of edema, including cirrhosis, nephrotic syndrome, and congestive heart failure, can usually be excluded by the history and physical examination. Heat edema generally resolves without treatment in several days. Diuretics are not effective and, in fact, predispose to volume depletion and the development of more serious heat-related illnesses.

1	Prickly heat (miliaria rubra, lichen tropicus) is a maculopapular, pruritic, erythematous rash that commonly occurs in clothed areas. Blockage of the sweat pores by debris from macerated stratum corneum causes inflammation in the sweat ducts. As the ducts dilate, they rupture and produce superficial vesicles. The predominant symptom is pruritus. In addition to antihistamines, chlorhexidine may provide some relief. Localized areas may benefit from 1% salicylic acid, with caution taken to avoid salicylate intoxication. Clothing should be clean and loose fitting, and activities or environments that induce diaphoresis should be avoided.

1	Heat syncope (exercise-associated collapse) can follow endurance exercise or can occur in the elderly. Other common clinical scenarios include prolonged standing while stationary in the heat and sudden standing after prolonged exposure to heat. Heat stress routinely causes relative volume depletion, decreased vasomotor tone, and peripheral vasodilation. The cumulative effect of this decrease in venous return is postural hypotension, especially in nonacclimated elderly individuals. Many of those affected also have comorbidities. Therefore, other cardiovascular, neurologic, and metabolic causes of syncope should be considered. After removal from the heat source, most patients will recover promptly with cooling and rehydration.

1	Hyperventilation tetany occurs in some individuals when exposure to heat stimulates hyperventilation, producing respiratory alkalosis, paresthesias, and carpopedal spasm. Unlike heat cramps, heat tetany causes very little muscle-compartment pain. Treatment includes providing reassurance, moving the patient out of the heat, and addressing the hyperventilation. Heat cramps (exercise-associated muscle cramps) are intermittent, painful, and involuntary spasmodic contractions of skeletal muscles. They typically occur in an unacclimated individual who is at rest after vigorous exertion in a humid, hot environment. In contrast, cramps that occur in athletes during exercise last longer, are relieved by stretching and massage, and resolve spontaneously. Of note, not all muscle cramps are related to exercise, and the differential diagnosis includes many other disorders. A variety of medications, myopathies, endocrine disorders, and sickle cell trait are other possible causes.

1	The typical patient with heat cramps is usually profusely diaphoretic and has been replacing fluid losses with copious water or other hypotonic fluids. Roofers, firefighters, military personnel, athletes, steel workers, and field workers are commonly affected. Other predisposing factors include insufficient sodium intake before intense activity in the heat and lack of heat acclimatization, resulting in sweat with a high salt concentration. The precise pathogenesis of heat cramps is unclear but appears to involve a relative deficiency of sodium, potassium, and fluid at the intracellular level. Coupled with copious hypotonic fluid ingestion, large amounts of sodium in the diaphoresis cause hyponatremia and hypochloremia, resulting in muscle cramps due to calcium-dependent muscle relaxation. Total-body depletion of potassium may be observed during the period of heat acclimatization. Rhadomyolysis is very rare with routine exercise-associated muscle cramps.

1	Heat cramps that are not accompanied by significant dehydration can be treated with commercially available electrolyte solutions. Although the flavored electrolyte solutions are far more palatable, two 650-mg salt tablets dissolved in 1 quart of water produces a 0.1% saline solution. Individuals should avoid the ingestion of undissolved salt tablets, which are a gastric irritant and may induce vomiting.

1	The physiologic hallmarks of heat exhaustion—in contrast to heatstroke—are the maintenance of thermoregulatory control and CNS function. The core temperature is usually elevated but is generally <40.5°C (<105°F). The two physiologic precipitants are water depletion and sodium depletion, which often occur in combination. Laborers, athletes, and elderly individuals exerting themselves in hot environments, without adequate fluid intake, tend to develop water-depletion heat exhaustion. Persons working in the heat frequently consume only two-thirds of their net water loss and are voluntarily dehydrated. In contrast, salt-depletion heat exhaustion occurs more slowly in unacclimated persons who have been consuming large quantities of hypotonic solutions.

1	Heat exhaustion is usually a diagnosis of exclusion because of the multitude of nonspecific symptoms. If any signs of heatstroke are present, rapid cooling and crystalloid resuscitation should be initiated immediately during stabilization and evaluation. Mild neurologic and gastrointestinal influenza-like symptoms are common. These symptoms may include headache, vertigo, ataxia, impaired judgment, malaise, dizziness, nausea, and muscle cramps. Orthostatic hypotension and sinus tachycardia develop frequently. More significant CNS impairment suggests heatstroke or other infectious, neurologic, or toxicologic diagnoses.

1	Hemoconcentration does not always develop, and rapid infusion of isotonic IV fluids should be guided by frequent electrolyte determinations and perfusion requirements. Most cases of heat exhaustion reflect mixed sodium and water depletion. Sodium-depletion heat exhaustion is characterized by hyponatremia and hypochloremia. Hepatic aminotransferases are mildly elevated in both types of heat exhaustion. Urinary sodium and chloride concentrations are usually low. Some patients with heat exhaustion develop heatstroke after removal from the heat-stress environment. Aggressive cooling of non-responders is indicated until their core temperature is 39°C (102.2°F). Except in mild cases, free water deficits should be replaced slowly over 24–48 h to avoid a decrease of serum osmolality by >2 mOsm/h.

1	The disposition of younger, previously healthy heat-exhaustion patients who have no major laboratory abnormalities may include hospital observation and discharge after IV rehydration. Older patients with comorbidities (including cardiovascular disease) or predisposing factors often require inpatient fluid and electrolyte replacement, monitoring, and reassessment. The clinical manifestations of heatstroke reflect a total loss of thermo-regulatory function. Typical vital-sign abnormalities include tachypnea, various tachycardias, hypotension, and a widened pulse pressure. Although there is no single specific diagnostic test, the historical and physical triad of exposure to a heat stress, CNS dysfunction, and a core temperature >40.5°C helps establish the preliminary diagnosis.

1	The definitive diagnosis should be reserved until the other potential causes of hyperthermia are excluded. Many of the usual laboratory abnormalities seen with heatstroke overlap with other conditions. If the patient's mental status does not improve with cooling, toxicologic screening may be indicated, and cranial CT and spinal fluid analysis can be considered.

1	The premonitory clinical characteristics may be nonspecific and include weakness, dizziness, disorientation, ataxia, and gastrointestinal or psychiatric symptoms. These prodromal symptoms often resemble heat exhaustion. The sudden onset of heatstroke occurs when the maintenance of adequate perfusion requires peripheral vasoconstriction to stabilize the mean arterial blood pressure. As a result, the cutaneous radiation of heat ceases. At this juncture, the core temperature rises dramatically. Since many patients with heatstroke also meet the criteria for systemic inflammatory response syndrome and have a broad differential diagnosis, rapid cooling is essential during the extensive diagnostic evaluation (Table 479e-1).

1	There are two forms of heatstroke with significantly different manifestations (Table 479e-2). Classic (epidemic) heatstroke (CHS) usually occurs during long periods of high ambient temperature and humidity, as during summer heat waves. Patients with CHS commonly have chronic diseases that predispose to heat-related illness, and they may have limited access to oral fluids. Heat dissipation mechanisms are overwhelmed by both endogenous heat production and exogenous heat stress. Patients with CHS are often compliant with prescribed medications that can impair tolerance to a heat stress. In many of these dehydrated CHS patients, sweating has ceased and the skin is hot and dry. If cooling is delayed, severe hepatic dysfunction, renal failure, disseminated intravascular coagulation, and fulminant multisystem organ failure may occur. Hepatocytes are very heat sensitive. On presentation, the serum level of aspartate aminotransferase (AST) is routinely elevated. Eventually, levels of both AST

1	multisystem organ failure may occur. Hepatocytes are very heat sensitive. On presentation, the serum level of aspartate aminotransferase (AST) is routinely elevated. Eventually, levels of both AST and alanine aminotransferase (ALT) often increase to >100 times the normal values. Coagulation studies commonly demonstrate decreased platelets, fibrinogen, and prothrombin. Most patients with CHS require cautious crystalloid resuscitation, electrolyte monitoring, and—in certain refractory cases—consideration of central venous pressure (CVP) measurements. Hypernatremia is secondary to dehydration in CHS. Many patients exhibit significant stress leukocytosis, even in the absence of infection.

1	Patients with exertional heatstroke (EHS), in contrast to those with CHS, are often young and previously healthy, and their diagnosis is usually more obvious from the history. Athletes, laborers, and military recruits are common victims. Unlike those with CHS, many EHS patients present profusely diaphoretic despite significant dehydration. As a result of muscular exertion, rhabdomyolysis and acute renal failure are more common in EHS. Studies to detect rhabdomyolysis and its complications, including hypocalcemia and hyperphosphatemia, should be considered. Hyponatremia, hypoglycemia, and coagulopathies are frequent findings. Elevated creatine kinase and lactate dehydrogenase 479e-3 levels also suggest EHS. Oliguria is a common finding. Renal failure can result from direct thermal injury, untreated rhabdomyolysis, or volume depletion. Common urinalysis findings include microscopic hematuria, myoglobinuria, and granular or red cell casts.

1	With both CHS and EHS, heat-related reversible increases in cardiac biomarker levels are often present. Heatstroke often causes thermal cardiomyopathy. As a result, the CVP may be elevated despite significant dehydration. In addition, the patient often presents with potentially deceptive noncardiogenic pulmonary edema and basilar rales despite being significantly hypovolemic. The electrocardiogram commonly displays a variety of tachyarrhythmias, nonspecific ST-T wave changes, and heat-related ischemia or infarction. Rapid cooling—not the administration of antiarrhythmic medications—is essential.

1	Above 42°C (107.6° F), heat can rapidly produce direct cellular injury. Thermosensitive enzymes become nonfunctional, and eventually there is irreversible uncoupling of oxidative phosphorylation. The production of heat-shock proteins increases, and cytokines mediate a systemic inflammatory response. The vascular endothelium is also damaged, and this injury activates the coagulation cascade. Significant shunting away from the splanchnic circulation produces gastrointestinal ischemia. Endotoxins further impair normal thermoregulation. As a result, if cooling is delayed, severe hepatic dysfunction, permanent renal failure, disseminated intravascular coagulation, and fulminant multisystem organ failure may occur.

1	Before cooling is initiated, endotracheal intubation, CVP determination, and continuous core-temperature monitoring should be considered. Hypoglycemia is a frequent finding and can be addressed by glucose infusion. Since peripheral vasoconstriction delays heat dissipation, repeated administration of discrete boluses of isotonic crystalloid for hypotension is preferable to the administration of α-adrenergic agonists.

1	Evaporative cooling is usually the most practical and effective technique. Rapid cooling is essential in both CHS and EHS, and an immediate improvement in vital signs and mental status may prove valuable for diagnostic purposes. Cool water (15°C [60° F]) is sprayed on the exposed skin while fans direct continuous airflow over the moistened skin. Cold packs applied to the axillae and groin are a useful cooling adjunct. If cardiac electrodes will not adhere, they can be applied to the patient's back. To avoid “overshoot hypothermia,” active cooling should be terminated at ~38°–39°C (100.4°–102.2°F). Immersion cooling in cold water is an alternative option in EHS but induces peripheral vasoconstriction and intense shivering. This technique presents significant monitoring and resuscitation challenges in most clinical settings. The safety of immersion cooling is best established for young, previously healthy patients with EHS (but not for those with CHS).

1	Cooling with commercially available cooling blankets should not be the sole technique used, since the rate of cooling is far too slow. Other methods are less efficacious and rarely indicated, such as IV infusion of cold fluids and cold irrigation of the bladder or gastrointestinal tract. Cold thoracic and peritoneal lavage are efficient maneuvers but are invasive and rarely necessary. Cardiopulmonary bypass provides fast and effective cooling but is labor intensive and is rarely available on a stat basis. Aspiration and seizures commonly occur in heatstroke, and endotracheal intubation is usually necessary. The metabolic demands are high, and supplemental oxygenation is essential due to hypoxemia induced by thermal stress and pulmonary dysfunction. Pneumonitis, pulmonary infarction, hemorrhage, edema, and acute respiratory distress syndrome occur frequently in heatstroke patients.

1	The circulatory fluid requirements, particularly in CHS, may be deceptively modest. Aggressive cooling and modest volume repletion usually elevate the CVP to 12–14 mmHg. The reading, however, may be deceptive. Many patients present with a thermally induced hyperdynamic circulation accompanied by a high cardiac index, low peripheral vascular resistance, and an elevated CVP caused by right-sided heart failure. Rarely, wedge pressures measured via a pulmonary artery catheter may be necessary to guide resuscitation. In contrast, most patients with EHS require far more zealous isotonic crystalloid resuscitation.

1	The hypotension that is initially common among patients with heatstroke results from both dehydration and high-output cardiac failure caused by peripheral vasodilation. Inotropes causing α-adrenergic stimulation (e.g., norepinephrine) can impede cooling by causing significant vasoconstriction. Vasoactive catecholamines such as dopamine or dobutamine may be necessary if the cardiac output remains depressed despite an elevated CVP, particularly in patients with a hyperdynamic circulation. A wide variety of tachyarrhythmias are routinely observed on presentation and usually resolve during cooling. The administration of atrial or ventricular antiarrhythmic medications is rarely indicated during cooling. Anticholinergic medications (including atropine) that inhibit sweating should not be given, and electrical cardioversion of the hyperthermic myocardium should be avoided except when there is ventricular fibrillation.

1	Significant shivering, discomfort, or extreme agitation is preferably mitigated with short-acting benzodiazepines, which are ideal due to their renal clearance. On the other hand, chlorpromazine may lower the seizure threshold, has anticholinergic properties, and can exacerbate the hypotension or cause neuroleptic malignant syndrome. Because of likely hepatic dysfunction, barbiturates should be avoided and seizures should be treated with benzodiazepines. Coagulopathies more commonly occur after the first day of illness. After cooling, the patient should be monitored for laboratory signs of disseminated intravascular coagulation, and replacement therapy with fresh-frozen plasma and platelets should be considered.

1	There is no therapeutic role for antipyretics in the control of environmentally induced hyperthermia; these drugs block the actions of pyrogens at hypothalamic receptor sites. Salicylates can further uncouple oxidative phosphorylation in heatstroke and exacerbate coagulopathies. Acetaminophen may further stress hepatic function. The safety and efficacy of other medications, including dantrolene and aminocaproic acid, are not established. Most patients with minor heat-emergency syndromes (including heat edema, heat syncope, and heat cramps) require only stabilization and treatment with outpatient follow-up. Although there are no decision rules to guide disposition choices in heat exhaustion, many of these patients have multiple predisposing factors and comorbidities that will require prolonged observation or hospital admission.

1	Essentially all patients with actual heatstroke require admission to a monitored setting, and most require intensive care. Many of these patients also require prolonged tracheal intubation, invasive hemodynamic monitoring, and support for various degrees of multiorgan dysfunction syndrome. The prognosis worsens if the initial core temperature exceeds 42°C (107.6°F) or if there was a prolonged period during which the core temperature exceeded this level. Other features of a negative prognosis include acute renal failure, massively elevated liver enzymes, and significant hyperkalemia. As expected, the number of dysfunctional organ systems also correlates directly with mortality risk.

1	2754 appeNDIX: Laboratory Values of Clinical Importance Alexander Kratz, Michael A. Pesce, Robert C. Basner, Andrew J. Einstein This Appendix contains tables of reference values for common labo-ratory tests. A variety of factors can influence reference values. Such variables include the population studied, the duration and means of specimen transport, laboratory methods and instrumentation, and even the type of container used for the collection of the specimen. The reference or “normal” ranges given in this appendix may therefore not be appropriate for all laboratories, and these values should only be used as general guidelines. Whenever possible, reference values provided by the laboratory performing the testing should be used in the interpretation of laboratory data. Values supplied in this Appendix reflect typical reference ranges in nonpregnant adults. Pediatric reference ranges and values in pregnant patients may vary significantly from the data presented in the Appendix.

1	In preparing the Appendix, the authors have taken into account the fact that the system of international units (SI, système international d’unités) is used in most countries and in some medical journals. However, clinical laboratories may continue to report values in “traditional” or conventional units. Therefore, both systems are provided in the Appendix. The dual system is also used in the text except for those instances in which the numbers remain the same and only the terminology is changed (mmol/L for meq/L or IU/L for mIU/mL), when only the SI units are given. Laboratory Values of Clinical Importance Laboratory Values of Clinical Importance

1	B-type natriuretic peptide (BNP) P Age and gender specific: <100 ng/L Age and gender specific: <100 pg/mL Bence Jones protein, serum qualitative S Not applicable None detected Bence Jones protein, serum quantitative S 3.3–19.4 mg/L 0.33–1.94 mg/dL Free lambda 5.7–26.3 mg/L 0.57–2.63 mg/dL K/L ratio 0.26–1.65 0.26–1.65 Beta-2-microglobulin S 1.1–2.4 mg/L 1.1–2.4 mg/L Bile acids S 0–1.9 μmol/L 0–1.9 μmol/L Chenodeoxycholic acid 0–3.4 μmol/L 0–3.4 μmol/L Deoxycholic acid 0–2.5 μmol/L 0–2.5 μmol/L Ursodeoxycholic acid 0–1.0 μmol/L 0–1.0 μmol/L Total 0–7.0 μmol/L 0–7.0 μmol/L Bilirubin S Total 5.1–22 μmol/L 0.3–1.3 mg/dL Direct 1.7–6.8 μmol/L 0.1–0.4 mg/dL Indirect 3.4–15.2 μmol/L 0.2–0.9 mg/dL C peptide S 0.27–1.19 nmol/L 0.8–3.5 ng/mL C1-esterase-inhibitor protein S 210–390 mg/L 21–39 mg/dL CA 125 S <35 kU/L <35 U/mL CA 19-9 S <37 kU/L <37 U/mL CA 15-3 S <33 kU/L <33 U/mL CA 27-29 S 0–40 kU/L 0–40 U/mL Calcitonin S 0–7.5 ng/L 0–7.5 pg/mL Female 0–5.1 ng/L 0–5.1 pg/mL Calcium S 2.2–2.6

1	CA 125 S <35 kU/L <35 U/mL CA 19-9 S <37 kU/L <37 U/mL CA 15-3 S <33 kU/L <33 U/mL CA 27-29 S 0–40 kU/L 0–40 U/mL Calcitonin S 0–7.5 ng/L 0–7.5 pg/mL Female 0–5.1 ng/L 0–5.1 pg/mL Calcium S 2.2–2.6 mmol/L 8.7–10.2 mg/dL Calcium, ionized WB 1.12–1.32 mmol/L 4.5–5.3 mg/dL Carbon dioxide content (TCO2) P (sea level) 22–30 mmol/L 22–30 meq/L Carboxyhemoglobin (carbon monoxide content) WB 0.0–0.025 0–2.5% of total hemoglobin (Hgb) value Smokers 0.04–0.09 4–9% of total Hgb value Loss of consciousness and death >0.50 >50% of total Hgb value Carcinoembryonic antigen (CEA) S Nonsmokers 0.0–3.0 μg/L 0.0–3.0 ng/mL Smokers 0.0–5.0 μg/L 0.0–5.0 ng/mL Ceruloplasmin S 250–630 mg/L 25–63 mg/dL Chloride S 102–109 mmol/L 102–109 meq/L

1	Cholesterol (LCL, Total, HDL): Ranges depend on individual patient factors; see 2013 ACC/AHA Guideline on the Treatment of Blood Cholesterol Cholinesterase S 5–12 kU/L 5–12 U/mL Chromogranin A S 0–95 μg/L 0–95 ng/mL Complement S 0.83–1.77 g/L 83–177 mg/dL C4 0.16–0.47 g/L 16–47 mg/dL Complement total Cortisol Fasting, 8 A.M.–12 noon S 138–690 nmol/L 5–25 μg/dL 12 noon–8 P.M. 138–414 nmol/L 5–15 μg/dL 8 P.M.–8 A.M. 0–276 nmol/L 0–10 μg/dL C-reactive protein S <10 mg/L <10 mg/L C-reactive protein, high sensitivity S Cardiac risk Cardiac risk Low: <1.0 mg/L Low: <1.0 mg/L Average: 1.0–3.0 mg/L Average: 1.0–3.0 mg/L High: >3.0 mg/L High: >3.0 mg/L Laboratory Values of Clinical Importance Laboratory Values of Clinical Importance Laboratory Values of Clinical Importance Laboratory Values of Clinical Importance

1	Aluminum S <0.2 μmol/L <5.41 μg/L Arsenic WB 0.0–0.17 μmol/L 0–13 μg/L Cadmium WB <44.5 nmol/L <5.0 μg/L Coenzyme Q10 (ubiquinone) P 433–1532 μg/L 433–1532 μg/L β-Carotene S 0.07–1.43 μmol/L 4–77 μg/dL Copper S 11–22 μmol/L 70–140 μg/dL Folic acid RC 340–1020 nmol/L cells 150–450 ng/mL cells Folic acid S 12.2–40.8 nmol/L 5.4–18.0 ng/mL Lead S <0.5 μmol/L <10 μg/dL Children <0.25 μmol/L <5 μg/dL Mercury WB 0–50 μmol/L 0–10 μg/L Selenium S 0.8–2.0 μmol/L 63–160 μg/L Vitamin A S 0.7–3.5 μmol/L 20–100 μg/dL Vitamin B1 (thiamine) S 0–75 nmol/L 0–2 μg/dL Vitamin B2 (riboflavin) S 106–638 nmol/L 4–24 μg/dL Vitamin B6 P 20–121 nmol/L 5–30 ng/mL Vitamin B12 S 206–735 pmol/L 279–996 pg/mL Vitamin C (ascorbic acid) S 23–57 μmol/L 0.4–1.0 mg/dL Vitamin D 1,25-dihydroxy, total S, P 36–180 pmol/L 15–75 pg/mL Vitamin D 25-hydroxy, total P 75–250 nmol/L 30–100 ng/mL Vitamin E S 12–42 μmol/L 5–18 μg/mL Vitamin K S 0.29–2.64 nmol/L 0.13–1.19 ng/mL Zinc S 11.5–18.4 μmol/L 75–120 μg/dL

1	Abbreviations: P, plasma; RC, red cells; S, serum; WB, whole blood. Electrolytes Sodium 137–145 mmol/L 137–145 meq/L Potassium 2.7–3.9 mmol/L 2.7–3.9 meq/L Calcium 1.0–1.5 mmol/L 2.1–3.0 meq/L Magnesium 1.0–1.2 mmol/L 2.0–2.5 meq/L Chloride 116–122 mmol/L 116–122 meq/L CO2 content 20–24 mmol/L 20–24 meq/L PCO2 6–7 kPa 45–49 mmHg pH 7.31–7.34 Glucose 2.22–3.89 mmol/L 40–70 mg/dL Lactate 1–2 mmol/L 10–20 mg/dL Total protein: Lumbar 0.15–0.5 g/L 15–50 mg/dL Cisternal 0.15–0.25 g/L 15–25 mg/dL Ventricular 0.06–0.15 g/L 6–15 mg/dL Albumin 0.066–0.442 g/L 6.6–44.2 mg/dL IgG 0.009–0.057 g/L 0.9–5.7 mg/dL IgG indexb 0.29–0.59 Oligoclonal bands (OGB) <2 bands not present in matched serum sample

1	Ammonia 15–47 μmol/L 25–80 μg/dL Creatinine 44–168 μmol/L 0.5–1.9 mg/dL Myelin basic protein <4 μg/L Cerebrospinal fluid aSince cerebrospinal fluid (CSF) concentrations are equilibrium values, measurements of the same parameters in blood plasma obtained at the same time are recommended. However, there is a time lag in attainment of equilibrium, and cerebrospinal levels of plasma constituents that can fluctuate rapidly (such as plasma glucose) may not achieve stable values until after a significant lag phase. bIgG index = CSF IgG (mg/dL) × serum albumin (g/dL)/serum IgG (g/dL) × CSF albumin (mg/dL). Laboratory Values of Clinical Importance The contributions of Drs. Daniel J. Fink, Patrick M. Sluss, James L. Januzzi, Kent B. Lewandrowski, Amudha Palanisamy, and Scott Fink to this chapter in previous editions are gratefully acknowledged. We also express our gratitude to Drs. Alex Rai and Jeffrey Jhang for helpful suggestions.

1	Laboratory Values of Clinical Importance the Clinical Laboratory in Modern health Care Anthony A. Killeen Modern medicine relies extensively on the clinical laboratory as a 480e key component of health care. It is estimated that, in current practice, at least 60–70% of all clinical decisions rely to some extent on a laboratory result. For many diseases, the clinical laboratory provides essential diagnostic information. As an example, histopathologic analysis provides basic information about histologic type and classification of tumors and their degree of invasion into adjacent tissues. Microbiologic testing is required to identify infectious organisms and determine antibiotic susceptibility. For many common diseases, expert groups have produced standard guidelines for diagnosis that rely on defined clinical laboratory values, e.g., blood glucose or hemoglobin A1C levels form the basis for diagnosis of diabetes mellitus; the presence of specific serum antibodies is required for

1	rely on defined clinical laboratory values, e.g., blood glucose or hemoglobin A1C levels form the basis for diagnosis of diabetes mellitus; the presence of specific serum antibodies is required for diagnosis of many rheumatologic diseases; and serum levels of cardiac markers are a mainstay in diagnosis of acute coronary syndromes.

1	With their ever-increasing number and scope, clinical laboratory tests provide the clinician with a powerful set of tools but pose challenges in terms of appropriate selection and judicious, cost-effective use to deliver effective patient care.

1	One of the most frequent reasons for performing clinical laboratory tests is to support, confirm, or refute a diagnosis of disease that is suspected on the basis of other information sources, such as history, physical examination findings, and imaging studies. The following questions need to be considered: Which clinical laboratory tests may be of value in supporting, confirming, or excluding the clinical impression? What is the most efficient test-ordering strategy? Will a positive test result confirm the clinical impression or even definitively establish the diagnosis? Will a negative result disprove the clinical suspicion, and, if so, what further testing or alternative approach will be needed? What are the known sources of false-positive and false-negative results, and how are these misleading results recognized?

1	Another reason for ordering clinical laboratory tests is to screen for disease in asymptomatic individuals (Chap. 4). Perhaps the most common examples of this application are the newborn screening programs now routinely used in most developed countries. Their purpose is to identify newborns with treatable conditions for which early intervention—even before clinical symptoms develop—is known to be beneficial. In adults, screening tests for diabetes mellitus, renal disease, prostate cancer (measurement of serum prostate-specific antigen [PSA] levels), and colorectal cancer (testing for occult blood in stool), for example, are widely applied to apparently healthy individuals on the grounds that early diagnosis and intervention lead to improved long-term outcomes.

1	Differences between Screening Tests and Confirmatory Tests It is important to distinguish between clinical laboratory tests that can be used to screen for disease and those that offer a confirmatory result. Screening tests are generally less expensive and more widely available than are confirmatory tests, which often require more specialized equipment or personnel. As a general principle, screening tests are designed to identify all individuals who have the disease of interest, even if, in the process, some healthy individuals are misidentified as possibly having the disease. In other words, maximization of the diagnostic sensitivity of screening tests inevitably comes at the expense of reduced diagnostic specificity. Confirmatory testing is intended to correctly separate those individuals who have a disease from those who do not.

1	These principles are exemplified by screening for hepatitis C virus 480e-1 (HCV) infection. A common approach is to test first for the presence of antibodies to HCV in serum. A positive result generally indicates either a current infection or a previous infection that the patient’s immune system has successfully cleared. In the latter situation, antibodies may persist at detectable levels for life. However, a small proportion of patients have false-positive results in the serologic screening test for HCV. To resolve uncertainty in these instances, a positive serologic screening test should be followed by confirmatory identification of HCV RNA with molecular techniques. This confirmatory testing can provide evidence of current viral infection and can identify patients who are not infected.

1	Another reason for clinical laboratory testing is to assess a patient’s risk of developing disease in the future. A number of diseases are associated with well-established clinical laboratory–defined risk factors that, if present, indicate the need for more frequent monitoring for the disease in question. The need for risk assessment is even clearer if there are useful interventions that decrease the risk of developing disease. For example, hypercholesterolemia is a well-established risk factor for coronary artery disease that may be modified by pharmacologic intervention (Chap. 291e). Many genetic mutations are known to be associated with increased risk of cancer. For example, hereditary mutations in the BRCA1 and BRCA2 genes predispose to breast and/ or ovarian cancer. Individuals who are known to carry these mutations require more vigilant monitoring for early signs of cancer and may even opt for prophylactic surgery in an attempt to prevent cancer (Chap. 84). Individuals with

1	are known to carry these mutations require more vigilant monitoring for early signs of cancer and may even opt for prophylactic surgery in an attempt to prevent cancer (Chap. 84). Individuals with factor V Leiden are at increased risk of developing deep venous thrombosis and may benefit from prophylactic anticoagulation in the perioperative period.

1	Many clinical laboratory tests offer useful information on the progress of disease and the response to therapy. One example is the measurement of viral load in HIV-1-infected patients who are taking antiretroviral agents. According to current guidelines from the Centers for Disease Control and Prevention, a successful antiretroviral response is defined by a fall in plasma HIV-1 levels of 0.5 log10 copies/mL, and a key goal of treatment is a reduction in the viral load to below the level of detection, which is typically in the range of 40–75 copies/mL. Other examples of the use of clinical laboratory testing for monitoring disease include measurement of tumor markers such as PSA, especially following surgical removal of tumors. In this situation, the expectation is that successful treatment of a tumor will cause a decrease in the level of the tumor marker. A later increase in the level of the tumor marker suggests a recurrence of the disease. Finally, the clinical laboratory offers

1	of a tumor will cause a decrease in the level of the tumor marker. A later increase in the level of the tumor marker suggests a recurrence of the disease. Finally, the clinical laboratory offers direct monitoring of levels of some therapeutic agents, such as drugs. This monitoring is important if a drug has a defined therapeutic concentration range above which it is toxic and below which it is ineffective. Monitoring of drug levels in this situation facilitates optimal dosing and avoidance of toxicity.

1	It is common practice for clinical laboratories to establish a list of “critical values.” These are values of test results that indicate immediate risk to the health or life of the patient and therefore require urgent communication with the patient’s physician so that appropriate medical intervention may be initiated. Critical values are reported regardless of whether the test was ordered as a “stat” or a routine test. The critical values themselves are generally created by the clinical laboratory medical director in conjunction with the medical staff. A representative list of critical values is shown in Table 480e-1. Tests that are ordered on a “stat” basis receive priority in the clinical laboratory’s testing queue; testing of specimens from other patients may be delayed while a stat specimen is analyzed. Ordering a test stat Chapter 480e The Clinical Laboratory in Modern Health Care

1	Chapter 480e The Clinical Laboratory in Modern Health Care Malarial smear aAny positive result is critical. Abbreviations: CO2, carbon dioxide; CSF, cerebrospinal fluid; INR, international normalized ratio; PCO2, partial pressure of carbon dioxide; PO2, partial pressure of oxygen; PTT, partial thromboplastin time; WBCs, white blood cells. should be reserved for situations in which a result is needed for urgent medical care—a judgment that must be made by the ordering physician. Stat testing should not be used merely for the convenience of either the patient or the health care provider.

1	The commonly used metrics of a clinical laboratory test are the diagnostic sensitivity, specificity, and positive and negative predictive values. These concepts are discussed in Chap. 3. In the clinical laboratory, the terms sensitivity and specificity have alternative meanings that are applied to tests, and the different uses of these terms may cause confusion. Analytic sensitivity can refer to the lowest detectable concentration of analyte that can be measured with some defined certainty or to the rate of change of signal intensity as analyte concentration changes. For example, newer generations of laboratory assays frequently exhibit improved sensitivity over that of earlier generations, i.e., they can detect lower concentrations of the analyte—a feature that is often of value in disease diagnosis. Analytic specificity refers to the extent to which other substances in the test system interfere with measurement of the analyte of interest. This concept is frequently applied to

1	disease diagnosis. Analytic specificity refers to the extent to which other substances in the test system interfere with measurement of the analyte of interest. This concept is frequently applied to immunoassays, in which a detection antibody may also bind with compounds that have a structure similar to the substance sought. For instance, immunoassays for drugs may show cross-reactivity with drug metabolites, and immunoassays for glucocorticoids may show cross-reactivity with other glucocorticoids of similar structure. Certain chemical assays are also subject to nonspecificity. For example, the Jaffe reaction, a chemical method commonly used to measure creatinine, is subject to positive interference from a number of other compounds, including glucose, certain ketones, and cephalosporin antibiotics. Elevated concentrations of bilirubin, free hemoglobin, or turbidity in plasma or serum specimens may also interfere with some assays. The clinical laboratory should be able to provide

1	antibiotics. Elevated concentrations of bilirubin, free hemoglobin, or turbidity in plasma or serum specimens may also interfere with some assays. The clinical laboratory should be able to provide advice about the presence or magnitude of these effects in the assays it performs.

1	Clinical laboratory diagnosis, like all diagnosis, is based on observation of disease-related changes from normality. 1.

1	1. Tissue injury or necrosis allows leakage of intracellular components into the circulation, with consequent detectable rises in blood levels of these components. Many intracellular molecules are common across tissue types and are therefore not indicative of injury to a specific tissue. Other constituents are selectively expressed in relatively high concentrations—or are even uniquely present—in certain tissues. Therefore, their presence in the blood is evidence of injury to that tissue. This principle forms the rationale for measurement of blood levels of, for example, liver enzymes in evaluating liver disease (Chap. 358), cardiac troponins in acute coronary syndromes (Chap. 295), and myoglobulin in muscle injury. The extent of the rise in blood levels of these markers generally correlates with the extent of tissue damage, although there are exceptions; for example, liver enzyme levels may fall in end-stage liver disease. 2.

1	2. An increase in blood levels of some analytes indicates failure of normal excretory processes. This principle is illustrated by elevations in conjugated bilirubin that accompany obstruction of the biliary system, by elevations in ammonia in advanced or metabolic liver disease, by rises in creatinine and potassium levels in renal failure, and by increases in Pco2 in some pulmonary diseases. 3.

1	3. Increases in the blood concentration of tissue-specific markers may result from expansion of the total volume of that tissue. This principle forms the basis for the measurement of levels of many tumor markers such as PSA (prostate cancer), CA-125 (ovarian cancer), CEA (colon cancer), and CA-19–9 (pancreatic cancer). In practice, the usefulness of these markers varies with the degree to which they are produced by a tumor and by the tumor’s size. Small colon cancers, for example, may not produce a significant rise in CEA levels, whereas small prostate cancers often produce detectable rises in PSA concentrations. 4.

1	4. Disease processes often manifest characteristic patterns of coincident changes in levels of several analytes. These patterns of change can be understood by consideration of the underlying pathophysiology. For example, acute intravascular hemolysis is characterized by a fall in levels of hemoglobin and haptoglobin and by a rise in unconjugated bilirubin. In endocrine diseases, there are often changes in concentrations of several hormones because of disturbance of feedback loops. Primary hyperthyroidism, as an example, is characterized by increases in thyroxine and by suppression of thyroid-stimulating hormone. In diabetic ketoacidosis caused by insulin deficiency, there are concomitant elevations of plasma glucose, ketones, and (frequently) potassium. In response to metabolic acidosis, levels of bicarbonate are typically reduced. 5.

1	Genetic changes underlie many diseases, both inherited and acquired. In the era of molecular medicine, there is increasing recognition of the contribution of hereditary factors to many common diseases. Often, the epidemiology of common diseases such as hypertension is characterized by a minority of families that have mutations in recognized genes, whereas the genetic basis of the same disease phenotype in the larger population is unclear. The search for the genetic factors that contribute to many common diseases remains a topic of intense research interest. It is now clear that essentially all tumors have genetic abnormalities. Although there is an inherited predisposition in some families, most of these genetic changes are acquired. Identification of the genetic abnormalities in cancer offers new tools for clinical laboratory diagnosis and classification of tumors in ways that surpass traditional histopathology and also provides insights into cellular processes that may be targets

1	offers new tools for clinical laboratory diagnosis and classification of tumors in ways that surpass traditional histopathology and also provides insights into cellular processes that may be targets for treatment.

1	6. Clinical laboratory results should always be interpreted in the context of the patient’s history and physical examination as well as any other relevant information (e.g., imaging studies). The clinician should avoid treating laboratory results rather than the patient. 7.

1	Recommended clinical laboratory tests change with time. As new markers of disease emerge, they may replace older markers. For example, measurement of serum creatine kinase (CK) levels was introduced for diagnosis of acute myocardial infarction in the 1980s. Use of the cardiac-specific isoenzyme CK-MB later became widespread in clinical practice. Today, cardiac troponins are replacing CK (or CK-MB) measurements in recommended guidelines. Many other assays have fallen out of use as better assays have become available. Measurement of urine 17-ketosteroids (arising from androgens) and of urine 17-hydroxycorticosteroids (arising from glucocorticoids) has been supplanted by immunoassays or mass spectroscopy determinations of specific steroid hormones. Today, many steroid hormones are measured by mass spectrometry, often with better analytic specificity than is provided by immunoassays. As new tests are introduced, it is essential that they be evaluated critically before adoption for

1	by mass spectrometry, often with better analytic specificity than is provided by immunoassays. As new tests are introduced, it is essential that they be evaluated critically before adoption for clinical use. At a minimum, consideration needs to be given to questions of clinical validation, specimen stability, diagnostic sensitivity and specificity, positive and negative predictive values, analytic accuracy and precision, and relative costs.

1	In the interpretation of clinical laboratory results, comparison is usually made to a reference range (sometimes called a normal range) that defines the values seen in health or considered to be desirable for health. Several common methods are used to describe reference ranges in the clinical laboratory.

1	1. For many quantitative clinical laboratory tests, the range of observed values in a healthy population shows an approximately Gaussian distribution. The factors that contribute to this range include the interand intraindividual variation in the concentration of the analyte and the analytic imprecision. When there is an approximately Gaussian distribution of values in the population, the reference range is commonly defined as being the central 95% of the range of distribution of those values. According to this method, 2.5% of the population will have a measured value that is below the reference range for the analyte, and 2.5% will have a value that is above the reference range. The fact that 5% of healthy individuals will have a test value that is outside the reference range has important implications when multiple tests are ordered. If N independent tests are performed on a specimen, then the probability that at least one result will be outside the reference range is (1–0.95N). The

1	implications when multiple tests are ordered. If N independent tests are performed on a specimen, then the probability that at least one result will be outside the reference range is (1–0.95N). The greater the number of tests ordered (even for a healthy individual), the greater is the likelihood of an abnormal result (Fig. 480e-1). If 20 independent tests are performed on a healthy subject, the probability of at least one abnormal result is almost two-thirds.

1	In some settings, a narrower range of values is considered to be abnormal. For example, current American Heart Association guidelines recommend the use of a serum level of cardiac troponins that is greater than the 99th percentile of values found in a healthy population as evidence of acute myocardial infarction. Number of tests performed FIGURE 480e-1 Probability that at least one laboratory result will be abnormal in a healthy individual as an increasing number of independent tests are performed. The reference range is the central 95% of values measured in a healthy population.

1	2. An alternative approach to using population means and standard deviations is to define a range of analyte values that is judged to be consistent with health on the basis of expert consensus opinion. These ranges are often referred to as decision limits. Examples of reference ranges established in this way include those for total, high-density, and low-density cholesterol (Table 480e-2). Such ranges may deviate considerably from those that would be established if the analyte concentrations of the population (mean ± 2 standard deviations) were used as a basis for establishing the reference range. For example, the “desirable” total cholesterol value according to the National Cholesterol Education Program is <200 mg/dL. This value is actually very close to the mean concentration among U.S. adults; in fact, almost one-half of

1	U.S. adults have a total cholesterol concentration that is above the “desirable” range. If the central 95% of cholesterol concentrations in the population were taken as the reference range, the upper end of that range would be ~240 mg/dL, well beyond what is considered desirable. Reference ranges may vary with age, gender, ethnic background, and physiologic state (e.g., pregnancy, high-altitude adaptation). Some examples of these variations are shown in the Appendix. The existence of different reference ranges poses challenges for interpretation of results. In particular, creatinine stands out as an analyte for which conventional reference ranges are not always easy to apply in clinical practice. Plasma levels of creatinine vary with age, gender, and ethnic group. This fact makes it difficult in practice to use a simple reference Probability of ˜1 result outside the reference range (%) Chapter 480e The Clinical Laboratory in Modern Health Care

1	Probability of ˜1 result outside the reference range (%) Chapter 480e The Clinical Laboratory in Modern Health Care High ≥60 Abbreviations: HDL, high-density lipoprotein; LDL, low-density lipoprotein. GFR (mL/min/1.73 m2) 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 Plasma creatinine (mg/dL) FIGURE 480e-2 Relationship between plasma creatinine and estimated glomerular filtration rate (eGFR) using the 4-parameter Modification of Diet in Renal Disease (MDRD) equation. IDMS, isotope dilution mass spectrometry.

1	range for this analyte when attempting to gauge a patient’s renal function. A large decrease in glomerular filtration rate (GFR) is associated with slight increases in the plasma creatinine concentration within the typical reference range provided by many laboratories (Fig. 480e-2). A 60-year-old white woman with a serum creatinine level of 1.00 mg/dL, which is well within the typical reference range, has an estimated GFR of only 57 mL/min per 1.73 m2, whereas the same creatinine concentration in a 20-year-old African-American male is consistent with normal renal function. To better estimate the GFR, which is widely considered to be the most useful index of overall renal function, it has become customary to use equations that incorporate plasma creatinine with other parameters. The most widely used of these equations in current practice is the 4-parameter Modification of Diet in Renal Disease (MDRD) equation that incorporates plasma creatinine, age, gender, and ethnic group (African

1	widely used of these equations in current practice is the 4-parameter Modification of Diet in Renal Disease (MDRD) equation that incorporates plasma creatinine, age, gender, and ethnic group (African American or not African American). The more recent CKD-EPI equation, which uses the same 4 parameters, is beginning to replace the MDRD equation. Recommended clinical laboratory practice is to report the estimated GFR with all creatinine measurements in adults. This addition provides more useful information than would a creatinine reference range alone.

1	Errors can arise at all stages of the testing process, from specimen collection to result interpretation. An error arising at any stage may adversely affect patient care. In clinical laboratory practice, it is customary to divide the testing process into three phases: preanalytic, analytic, and postanalytic. Examples of each type of error are shown in Table 480e-3. The most common error in the testing process is specimen mislabeling, in which a specimen from one patient is placed in a container labeled with another patient’s name or identifiers. Specimen mislabeling errors may have very serious consequences for a patient. For example, if erroneous typing of a patient’s blood group results from specimen mislabeling and is followed by transfusion of a mismatched unit of blood, the outcome may be fatal. A mislabeled biopsy specimen can lead either to an erroneous diagnosis and inappropriate therapy or to a failure to make a diagnosis and institute appropriate therapy.

1	In addition to errors, many preanalytic factors can influence clinical laboratory results. Posture (i.e., recumbent versus upright), exercise, diet, recently ingested food, and use of prescribed or recreational drugs (including tobacco, alcohol, caffeine, and herbal supplements) can influence a variety of analyte concentrations. After blood has been collected, certain analytes undergo changes in their concentration during storage or transportation. Glucose levels fall as a result of red cell metabolism. Ammonia levels rise as a result of protein breakdown. Increasing permeability and breakdown of red cell membranes leads to increases in plasma potassium and free hemoglobin levels. Bacterial contamination can lead to overgrowth of specimens. To minimize tabLe 480e-3 exaMpLeS of preaNaLytiC, aNaLytiC, aNd poStaNaLytiC errorS duriNg the Laboratory teStiNg proCeSS Preanalytic Sources of Error

1	Preanalytic Sources of Error Test selection Inappropriate test for the clinical need Lack of clinical usefulness, regardless of possible results Test order misunderstood or not communicated Specimen collection Incorrect time of collection Patient not prepared for collection (e.g., not fasting) Incorrect specimen type (e.g., wrong anticoagulant, wrong tissue fixative) Use of incorrect specimen container Insufficient specimen collected Contamination of specimen by IV fluids, drugs, or bacteria Specimen mislabeled or unlabeled Important clinical information not provided Delays in transportation to the lab, leading to alterations in specimen constituents Analytic Sources of Error Incorrect storage conditions prior to analysis Specimen misidentification in the laboratory Wrong test performed Assay interferences Assay failure (e.g., assay out of control) Postanalytic Sources of Error

1	Postanalytic Sources of Error Delay in communication of assay results Results not communicated to correct person Incorrect result communicated Misinterpretation of result these precollection alterations, specimens should be processed or transported to the clinical laboratory as soon as possible after collection. The list of known preanalytic variables and their effects is extensive, and the reader is referred to the compendium on this subject (see Young DS: Effects of Preanalytical Variables on Clinical Laboratory Tests, 3rd ed. Washington, DC, AACC Press, 2007).

1	The great majority of tests continue to be performed in dedicated clinical laboratory facilities, but for several decades there has been a trend toward point-of-care testing. This change has been made possible by the development of portable analytic devices, including single-purpose instruments such as glucometers and oxygen saturation monitors, and multifunction instruments that can perform a wider variety of analyses, particularly in chemistry and hematology but also in some areas of microbiology. The use of these devices is driven largely by the convenience of faster result availability. In some settings (e.g., in rural areas and developing countries), there may be no easily accessible clinical laboratory and a point-of-care device may be the best or only option for testing. However, the per-specimen cost of point-of-care testing, in terms both of reagents and supplies and of personnel, is often greater than that of centralized testing. Other concerns relate to the adequacy of

1	the per-specimen cost of point-of-care testing, in terms both of reagents and supplies and of personnel, is often greater than that of centralized testing. Other concerns relate to the adequacy of personnel training for point-of-care testing, the quality of the results, and the incorporation of results into the medical record.

1	One of the largest markets for point-of-care testing is home testing by patients, which has long been an important element in the management of persons with diabetes who monitor their own blood glucose levels. Over-the-counter kits for home pregnancy testing have been available for decades. More recently, kits have become available for home testing of the international normalized ratio or prothrombin time by patients taking oral anticoagulants. Kits are also available for cholesterol monitoring, fecal occult-blood detection, and hemoglobin measurement. In these areas, there is often little information on the quality of test performance, the accuracy of the results, or the correctness of result interpretation. The principles of genetic medicine in clinical practice are dis cussed in Chaps. 82–84. Here we will concentrate on issues related to clinical laboratory testing for genetic disease.

1	The principles of genetic medicine in clinical practice are dis cussed in Chaps. 82–84. Here we will concentrate on issues related to clinical laboratory testing for genetic disease. The distinction between genetic testing for inherited disorders and that for acquired disorders affects the type of tissue that should be obtained for analysis. In inherited disorders, all nucleated cells are expected to carry the inherited mutation; thus white blood cells or buccal cells (obtained by scraping the inside of the cheek) are convenient sources of DNA for clinical laboratory testing. For prenatal testing of the fetus, chorionic villi or amniocytes are commonly used. In tests for acquired genetic disorders (e.g., in tumors), the tissue of interest that contains a suspected mutation must be sampled. It is often useful to compare tumor DNA with the patient’s normal DNA in order to identify acquired mutations (e.g., testing for microsatellite instability in colorectal cancer; Chap. 101e).

1	Although it is assumed that all clinical laboratory testing is performed with the consent of the patient (or, in the case of minors, the parents), regulations may require formal written consent for genetic testing. Such regulations vary among jurisdictions, and the practicing clinician should be aware of local regulations. In some jurisdictions, there are regulations on the storage and use of genetic information and on the maximal period for which genetic specimens may be stored.

1	For some late-onset genetic diseases, such as Huntington’s disease (Chap. 449), genetic testing allows a prediction about the future development of the disease. The degree of certainty that is possible on the basis of this testing surpasses that associated with identifica-480e-5 tion of more traditional disease risk factors (e.g., hyperlipidemia as a risk factor for future myocardial infarction). When deciding to undertake predictive genetic testing, it is important for the patient to consider the broad implications of a positive or negative test result, to be made aware of any support and counseling that is available, and to understand the implications of a result for other family members. In dealing with these issues, genetic counselors play an important role (Chap. 84). Their expertise includes the ability to explain genetic disorders at an understandable level to patients and their families, to arrange for support services, and to provide genetic risk assessments to members of

1	includes the ability to explain genetic disorders at an understandable level to patients and their families, to arrange for support services, and to provide genetic risk assessments to members of families with genetic disorders.

1	When testing for genetic disorders, the clinical laboratory will use different analytic approaches according to the disease of interest. Some disorders, such as sickle cell anemia, are caused by single-point mutations. Testing for these disorders involves mere assessment for one or a few mutations in a single gene. Other disorders (e.g., hyperphenylalaninemias) may be caused by numerous mutations in a single gene. Still others (e.g., hereditary breast cancer) may be caused by mutations in many genes. The number of possible mutations and genes that underlie a clinical phenotype affects the cost of and time required for clinical laboratory testing as well as the likelihood of finding a disease-causing mutation.

1	If a disease phenotype can be caused by many mutations, a clinical laboratory result that is negative should be interpreted with care. For example, it is common to screen healthy pregnant women (and their partners) for mutations in the CFTR gene, which is mutated in patients with cystic fibrosis (CF). The goal of this screening is to identify women who are carriers of a CFTR mutation and therefore are at increased risk of having a baby with CF. Because CF is an autosomal recessive disorder, a fetus has a 1:4 chance of being affected if both parents are carriers of disease-causing CFTR mutations. The screening test approach that is commonly used to identify mutations in carriers detects 80–85% of all known disease-causing CFTR mutations in Caucasians and up to 97% of mutations among Ashkenazi Jews. A negative screening result therefore does not completely eliminate the possibility that a woman (or her partner) actually has a mutation. What can be inferred from a negative test result is

1	Jews. A negative screening result therefore does not completely eliminate the possibility that a woman (or her partner) actually has a mutation. What can be inferred from a negative test result is that the risk of having a CF-affected baby has decreased significantly to an extent that depends on the woman’s ethnic group and the mutations that were examined. The clinical laboratory should calculate and report the woman’s new risk of being a carrier if the screening result is negative.

1	The increasing availability of large-scale (next-generation) sequencing of a patient’s whole genome or exome will greatly affect genetic testing over the next decade, with implications for the number of mutations that can be detected and the increased complexity of result interpretation.

1	Genetic testing has limitations that are often unique to this field. Results may be inconclusive. For example, a search for mutations in a gene that is suspected of causing a disease may fail to reveal any known disease-causing mutations. A mutation may be discovered that is of unknown clinical significance. In this situation, consideration of the predicted change in the amino acid sequence of the encoded protein may suggest a biologic effect—e.g., replacement of a charged amino acid by one of the opposite charge or by a neutral amino acid; replacement of an amino acid by one of a different size; or replacement of an amino acid that is conserved across multiple species. Further information may be obtained by determining whether the mutation is found in healthy individuals. Even with all of these considerations, it is not uncommon that the biologic significance of an identified mutation remains uncertain, and further research may be needed to assess its significance.

1	It is also important to understand the limitations of the clinical laboratory approach used to detect mutations. At this time, next-generation sequencing remains an impractical undertaking for financial reasons, although extensive sequence analysis has become the Chapter 480e The Clinical Laboratory in Modern Health Care standard of care for a few genes (e.g., analysis of BRCA1 and BRCA2 in assessing the risk of breast and ovarian cancer in individuals with a strong family history of these disorders). As sequencing technologies become less expensive, they can be expected to be more commonly used for both identifying mutations in patients with genetic disorders and screening asymptomatic individuals at risk of genetic disease.

1	Another unique aspect of genetic testing is the concern that genetic information about individuals may be used to discriminate against them by employers or by insurance companies. In the United States, the Genetic Information Nondiscrimination Act of 2008 (GINA) prohibits the use of genetic information by employers in making decisions related to employment and by health insurance companies in issuing insurance policies or setting premium rates based on knowledge of the applicant’s genetic status. GINA does not cover disability insurance, long-term care insurance, or life insurance policies.

1	Although public attention has been most closely focused on DNA testing, other clinical laboratory investigations that are not usually thought of as genetic may provide important genetic information about the person being tested. For example, serum protein electrophoresis may reveal α-1 antitrypsin deficiency. Depending on the clinical laboratory technology used, measurement of hemoglobin A1C, commonly used for monitoring diabetes control, may reveal a hemoglobin variant such as HbS (sickle cell). Measurement of cholesterol and triglyceride levels may reveal any of a number of hereditary disorders. All of these results are types of genetic information.

1	In the United States, all clinical laboratory testing performed for clinical purposes (but not for research purposes) is regulated by the federal Clinical Laboratory Improvement Amendments Act of 1988 (CLIA). Home monitoring by patients who are testing their own specimens is not covered by CLIA. The statute and the regulations, which are administered by the Centers for Medicare and Medicaid Services, apply to all laboratories, whether located in a physician’s office, a large hospital, or a reference laboratory; and all laboratories are required to hold a valid CLIA certificate that is appropriate for the highest complexity level of the tests they perform. The U.S. Food and Drug Administration is responsible for assigning the complexity level of commercial tests. The lowest category of complexity is the “waived” category, which is followed (in order of increasing complexity) by the categories of “provider-performed microscopy,” “moderatecomplexity testing,” and “high-complexity

1	of complexity is the “waived” category, which is followed (in order of increasing complexity) by the categories of “provider-performed microscopy,” “moderatecomplexity testing,” and “high-complexity testing.” The category of provider-performed microscopy is used to cover tests such as the use of potassium hydroxide preparations on skin scrapings to examine for fungi, fern tests, and sperm motility tests; it does not encompass histopathology that falls into the high-complexity category. Even if a clinical laboratory is performing only testing in the “waived” category, it must still hold a valid CLIA certificate. Laboratories that hold certificates for nonwaived tests are required to participate in proficiency testing and are regularly inspected to monitor their performance.

1	Placement Gyorgy Frendl, Kurt Fink 481e Clinical Procedure Tutorial: Central Venous Catheter Maria A. Yialamas, William E. Corcoran,

1	Maria A. Yialamas, William E. Corcoran, Clinical procedures are an important component of medical student and resident training, and some are required for board and hospital certification. In these Harrison’s Chaps. 481e–486e, video tutorials are presented for performing abdominal paracentesis, thoracentesis, endotracheal intubation, central venous catheter placement, percutaneous arterial blood gas sampling, and lumbar puncture. These videos have been created specifically for Harrison’s. Each includes the indications, contraindications, equipment, potential complications, and related patient safety considerations. Additional video tutorials covering clinical procedures such as breast biopsy, IV line insertion, phlebotomy, arterial line insertion, arthrocentesis, bone marrow biopsy, pelvic examination, thyroid aspiration, basic suturing, and urethral catheterization are available to subscribers of Harrison’s Online and AccessMedicine (available at www.accessmedicine.com).

1	CHAPTER 481e Clinical Procedure Tutorial: Central Venous Catheter Placement

1	Clinical Procedure Tutorial: Thoracentesis Charles A. Morris, Andrea Wolf Clinical procedures are an important component of medical student and resident training, and some are required for board and hospital 482e certification. In these new Harrison’s Chaps. 481e–486e, video tutorials are presented for performing abdominal paracentesis, thoracentesis, endotracheal intubation, central venous catheter placement, percutaneous arterial blood gas sampling, and lumbar puncture. These videos have been created specifically for Harrison’s. Each includes the indications, contraindications, equipment, potential complications, and related patient safety considerations. Additional video tutorials covering clinical procedures such as breast biopsy, IV line insertion, phlebotomy, arterial line insertion, arthrocentesis, bone marrow biopsy, pelvic examination, thyroid aspiration, basic suturing, and urethral catheterization are available to subscribers of Harrison’s Online and AccessMedicine

1	arthrocentesis, bone marrow biopsy, pelvic examination, thyroid aspiration, basic suturing, and urethral catheterization are available to subscribers of Harrison’s Online and AccessMedicine (available at www.accessmedicine.com).

1	CHAPTER 482e Clinical Procedure Tutorial: Thoracentesis

1	Clinical Procedure Tutorial: Abdominal Paracentesis Maria A. Yialamas, Anna E. Rutherford, Lindsay King Clinical procedures are an important component of medical student and resident training, and some are required for board and hospital cer-483e tification. In these new Harrison’s Chaps. 481e–486e video tutorials are presented for performing abdominal paracentesis, thoracentesis, endotracheal intubation, central venous catheter placement, percutaneous arterial blood gas sampling, and lumbar puncture. These videos have been created specifically for Harrison’s. Each includes the indications, contraindications, equipment, potential complications, and related patient safety considerations. Additional video tutorials covering clinical procedures such as breast biopsy, IV line insertion, phlebotomy, arterial line insertion, arthrocentesis, bone marrow biopsy, pelvic examination, thyroid aspiration, basic suturing, and urethral catheterization are available to subscribers of Harrison’s

1	arterial line insertion, arthrocentesis, bone marrow biopsy, pelvic examination, thyroid aspiration, basic suturing, and urethral catheterization are available to subscribers of Harrison’s Online and AccessMedicine (available at www.accessmedicine.com).

1	CHAPTER 483e Clinical Procedure Tutorial: Abdominal Paracentesis

1	Clinical Procedure Tutorial: Endotracheal Intubation Charles A. Morris, Emily Page Nelson Clinical procedures are an important component of medical student and resident training, and some are required for board and hospital 484e certification. In these new Harrison’s Chaps. 481e–486e, video tutorials are presented for performing abdominal paracentesis, thoracentesis, endotracheal intubation, central venous catheter placement, percutaneous arterial blood gas sampling, and lumbar puncture. These videos have been created specifically for Harrison’s. Each includes the indications, contraindications, equipment, potential complications, and related patient safety considerations. Additional video tutorials covering clinical procedures such as breast biopsy, IV line insertion, phlebotomy, arterial line insertion, arthrocentesis, bone marrow biopsy, pelvic examination, thyroid aspiration, basic suturing, and urethral catheterization are available to subscribers of Harrison’s Online and

1	arterial line insertion, arthrocentesis, bone marrow biopsy, pelvic examination, thyroid aspiration, basic suturing, and urethral catheterization are available to subscribers of Harrison’s Online and AccessMedicine (available at www.accessmedicine.com).

1	CHAPTER 484e Clinical Procedure Tutorial: Endotracheal Intubation Gas Sampling Christian D. Becker 485e Clinical Procedures Tutorial: Percutaneous Arterial Blood Medical Editors: Sean Sadikot, Jeremy Matloff

1	Medical Editors: Sean Sadikot, Jeremy Matloff Clinical procedures are an important component of medical student and resident training, and some are required for board and hospital certification. In these Harrison’s Chaps. 481e–486e, video tutorials are presented for performing abdominal paracentesis, thoracentesis, endotracheal intubation, central venous catheter placement, percutaneous arterial blood gas sampling, and lumbar puncture. These videos have been created specifically for Harrison’s. Each includes the indications, contraindications, equipment, potential complications, and related patient safety considerations. Additional video tutorials covering clinical procedures such as breast biopsy, IV line insertion, phlebotomy, arterial line insertion, arthrocentesis, bone marrow biopsy, pelvic examination, thyroid aspiration, basic suturing, and urethral catheterization are available to subscribers of Harrison’s Online and AccessMedicine (available at www.accessmedicine.com).

1	CHAPTER 485e Clinical Procedures Tutorial: Percutaneous Arterial Blood Gas Sampling

1	Clinical Procedures Tutorial: Lumbar Puncture Beth Rapaport, Stephen Krieger, Corey McGraw Medical Editors: Sean Sadikot, Jeremy Matloff Clinical procedures are an important component of medical student 486e and resident training, and some are required for board and hospital certification. In these Harrison’s Chaps. 481e–486e, video tutorials are presented for performing abdominal paracentesis, thoracentesis, endotracheal intubation, central venous catheter placement, percutaneous arterial blood gas sampling, and lumbar puncture. These videos have been created specifically for Harrison’s. Each includes the indications, contraindications, equipment, potential complications, and related patient safety considerations. Additional video tutorials covering clinical procedures such as breast biopsy, IV line insertion, phlebotomy, arterial line insertion, arthrocentesis, bone marrow biopsy, pelvic examination, thyroid aspiration, basic suturing, and urethral catheterization are available to

1	IV line insertion, phlebotomy, arterial line insertion, arthrocentesis, bone marrow biopsy, pelvic examination, thyroid aspiration, basic suturing, and urethral catheterization are available to subscribers of Harrison’s Online and AccessMedicine (available at www.accessmedicine.com).

1	CHAPTER 486e Clinical Procedures Tutorial: Lumbar Puncture

1	Growth Adaptations, Cellular Injury, and Cell Death I. BASIC PRINCIPLES A. An organ is in homeostasis with the physiologic stress placed on it. B. An increase, decrease, or change in stress on an organ can result in growth adaptations. II. HYPERPLASIA AND HYPERTROPHY A. An increase in stress leads to an increase in organ size. 1. Occurs via an increase in the size (hypertrophy) and/or the number (hyperplasia) of cells B. Hypertrophy involves gene activation, protein synthesis, and production of organelles. C. Hyperplasia involves the production of new cells from stem cells. D. Hyperplasia and hypertrophy generally occur together (e.g., uterus during pregnancy). 1. Permanent tissues (e.g., cardiac muscle, skeletal muscle, and nerve), however, cannot make new cells and undergo hypertrophy only. 2. For example, cardiac myocytes undergo hypertrophy, not hyperplasia, in response to systemic hypertension (Fig. 1.1).

1	2. For example, cardiac myocytes undergo hypertrophy, not hyperplasia, in response to systemic hypertension (Fig. 1.1). E. Pathologic hyperplasia (e.g., endometrial hyperplasia) can progress to dysplasia and, eventually, cancer. 1. A notable exception is benign prostatic hyperplasia (BPH), which does not increase the risk for prostate cancer. III. ATROPHY A. A decrease in stress (e.g., decreased hormonal stimulation, disuse, or decreased nutrients/blood supply) leads to a decrease in organ size (atrophy). 1. Occurs via a decrease in the size and number of cells B. Decrease in cell number occurs via apoptosis. C. Decrease in cell size occurs via ubiquitin-proteosome degradation of the cytoskeleton and autophagy of cellular components. 1. In ubiquitin-proteosome degradation, intermediate filaments of the cytoskeleton are "tagged" with ubiquitin and destroyed by proteosomes. 2.

1	1. In ubiquitin-proteosome degradation, intermediate filaments of the cytoskeleton are "tagged" with ubiquitin and destroyed by proteosomes. 2. Autophagy of cellular components involves generation of autophagic vacuoles. These vacuoles fuse with lysosomes whose hydrolytic enzymes breakdown cellular components. IV. METAPLASIA A. A change in stress on an organ leads to a change in cell type (metaplasia). 1. Most commonly involves change of one type of surface epithelium (squamous, columnar, or urothelial) to another 2. Metaplastic cells are better able to handle the new stress. B. Barrett esophagus is a classic example. pathoma.com 1. Esophagus is normally lined by nonkeratinizing squamous epithelium (suited to handle friction of a food bolus). 2. Acid reflux from the stomach causes metaplasia to nonciliated, mucin-producing columnar cells (better able to handle the stress of acid, Fig. 1.2).

1	2. Acid reflux from the stomach causes metaplasia to nonciliated, mucin-producing columnar cells (better able to handle the stress of acid, Fig. 1.2). C. Metaplasia occurs via reprogramming ofstem cells, which then produce the new cell type. 1. Metaplasia is reversible, in theory, with removal of the driving stressor. 2. For example, treatment of gastroesophageal reflux may reverse Barrett esophagus. D. Under persistent stress, metaplasia can progress to dysplasia and eventually result in cancer. 1. For example, Barrett esophagus may progress to adenocarcinoma of the esophagus. 2. A notable exception is apocrine metaplasia of breast, which carries no increased risk for cancer. E. Vitamin A deficiency can also result in metaplasia. 1. Vitamin A is necessary for differentiation of specialized epithelial surfaces such as the conjunctiva covering the eye. 2.

1	E. Vitamin A deficiency can also result in metaplasia. 1. Vitamin A is necessary for differentiation of specialized epithelial surfaces such as the conjunctiva covering the eye. 2. In vitamin A deficiency, the thin squamous lining of the conjunctiva undergoes metaplasia into stratified keratinizing squamous epithelium. This change is called keratomalacia (Fig. 1.3). F. Mesenchymal (connective) tissues can also undergo metaplasia. 1. A classic example is myositis ossificans in which connective tissue within muscle changes to bone during healing after trauma (Fig. 1.4). V. DYSPLASIA A. Disordered cellular growth B. Most often refers to proliferation of precancerous cells 1. For example, cervical intraepithelial neoplasia (CIN) represents dysplasia and is a precursor to cervical cancer. C. Often arises from longstanding pathologic hyperplasia (e.g., endometrial hyperplasia) or metaplasia (e.g., Barrett esophagus)

1	C. Often arises from longstanding pathologic hyperplasia (e.g., endometrial hyperplasia) or metaplasia (e.g., Barrett esophagus) D. Dysplasia is reversible, in theory, with alleviation of inciting stress. 1. Ifstress persists, dysplasia progresses to carcinoma (irreversible). VI. APLASIA AND HYPOPLASIA A. Aplasia is failure of cell production during embryogenesis (e.g., unilateral renal a genesis). B. Hypoplasia is a decrease in cell production during embryogenesis, resulting in a relatively small organ (e.g., streak ovary in Turner syndrome). Fig. 1.1 Left ventricular hypertrophy. (Courtesy of Fig. 1.2 Barrett esophagus. Aliya Husa in, MD) ------Growth Adaptations, Cellular Injury, and Cell Death I. BASIC PRINCIPLES A. Cellular injury occurs when a stress exceeds the cell's ability to adapt. B. The likelihood ofinjury depends on the type of stress, its severity, and the type of cell affected. 1.

1	A. Cellular injury occurs when a stress exceeds the cell's ability to adapt. B. The likelihood ofinjury depends on the type of stress, its severity, and the type of cell affected. 1. Neurons are highly susceptible to ischemic injury; whereas, skeletal muscle is relatively more resistant. 2. Slowly developing ischemia (e.g., renal artery atherosclerosis) results in atrophy; whereas, acute ischemia (e.g., renal artery embolus) results in injury. C. Common causes of cellular injury include inflammation, nutritional deficiency or excess, hypoxia, trauma, and genetic mutations. II. HYPOXIA A. Low oxygen delivery to tissue; important cause ofcellular injury 1. Oxygen is the final electron acceptor in the electron transport chain of oxidative phosphorylation. 2. Decreased oxygen impairs oxidative phosphorylation, resulting in decreased ATP production. 3. Lack of ATP (essential energy source) leads to cellular injury.

1	2. Decreased oxygen impairs oxidative phosphorylation, resulting in decreased ATP production. 3. Lack of ATP (essential energy source) leads to cellular injury. B. Causes of hypoxia include ischemia, hypoxemia, and decreased 0 2-carrying capacity of blood. C. Ischemia is decreased blood flow through an organ. Arises with 1. Decreased arterial perfusion (e.g., atherosclerosis) 2. Decreased venous drainage (e.g., Budd-Chiari syndrome) 3. D. Hypoxemia is a low partial pressure ofoxygen in the blood (Pao2 < 60 mm Hg, Sao2 < 90%). Arises with 1. 2. 3. Diffusion defect-PAo2 not able to push as much 0 2 into the blood due to a thicker diffusion barrier (e.g., interstitial pulmonary fibrosis) 4. V/Q mismatch-Blood bypasses oxygenated lung (circulation problem, e.g., right-to-left shunt), or oxygenated air cannot reach blood (ventilation problem, e.g., atelectasis). E. Decreased 0 -carrying capacity arises with hemoglobin (Hb) loss or dysfunction. 1. 2.

1	E. Decreased 0 -carrying capacity arises with hemoglobin (Hb) loss or dysfunction. 1. 2. Fig.1.3 Keratomalacia. (Courtesy of Fig. 1.4 Myositis Ossificans. (Reprinted with motherchildnutrition.org) permission from orthopaedia.com) i. CO binds hemoglobin more avidly than oxygen-Pao2 normal; Sao2 decreased ii. Exposures include smoke from fires and exhaust from cars or gas heaters. iii. Classic finding is cherry-red appearance of skin. iv. Early sign of exposure is headache; significant exposure leads to coma and death. 3. Methemoglobinemia i. Iron in heme is oxidized to FeH, which cannot bind oxygen-Pao normal; 11. Seen with oxidant stress (e.g., sulfa and nitrate drugs) or in newborns 111. Classic finding is cyanosis with chocolate-colored blood. iv. Treatment is intravenous methylene blue, which helps reduce Fe3+ back to Fe2+ state. III. REVERSIBLE AND IRREVERSIBLE CELLULAR INJURY A. Hypoxia impairs oxidative phosphorylation resulting in decreased ATP.

1	III. REVERSIBLE AND IRREVERSIBLE CELLULAR INJURY A. Hypoxia impairs oxidative phosphorylation resulting in decreased ATP. B. Low ATP disrupts key cellular functions including 1. Na+-K+ pump, resulting in sodium and water buildup in the cell 2. Ca2+ pump, resulting in Ca2+ buildup in the cytosol of the cell 3. Aerobic glycolysis, resulting in a switch to anaerobic glycolysis. Lactic acid buildup results in low pH, which denatures proteins and precipitates DNA. C. The initial phase of injury is reversible. The hallmark of reversible injury is cellular swelling. 1. Cytosol swelling results in loss of microvilli and membrane blebbing. 2. Swelling of the rough endoplasmic reticulum (RER) results in dissociation of ribosomes and decreased protein synthesis. D. Eventually, the damage becomes irreversible. The hallmark of irreversible injury is membrane damage.

1	D. Eventually, the damage becomes irreversible. The hallmark of irreversible injury is membrane damage. 1. Plasma membrane damage results in i. Cytosolic enzymes leaking into the serum (e.g., cardiac troponin) ii. Additional calcium entering into the cell 2. Mitochondrial membrane damage results in i. Loss of the electron transport chain (inner mitochondrial membrane) ii. Cytochrome c leaking into cytosol (activates apoptosis) 3. Lysosome membrane damage results in hydrolytic enzymes leaking into the cytosol, which, in turn, are activated by the high intracellular calcium. E. The end result of irreversible injury is cell death. Fig. 1.5 Coagulative necrosis of kidney. A, Gross appearance. B, Microscopic appearance. C, Normal kidney histology for comparison. {A, Courtesy of Aliya Husain, MD) Growth Adaptations, Cellular Injury, and Cell Death I. BASIC PRINCIPLES

1	Growth Adaptations, Cellular Injury, and Cell Death I. BASIC PRINCIPLES A. The morphologic hallmark of cell death is loss of the nucleus, which occurs via nuclear condensation (pyknosis), fragmentation (karyorrhexis), and dissolution (karyolysis). B. The two mechanisms of cell death are necrosis and apoptosis. II. NECROSIS A. Death oflarge groups of cells followed by acute inflammation B. Due to some underlying pathologic process; never physiologic C. Divided into several types based on gross features III. GROSS PATTERNS OF NECROSIS A. Coagulative necrosis 1. Necrotic tissue that remains firm (Fig. l.SA); cell shape and organ structure are preserved by coagulation of proteins, but the nucleus disappears (Fig. l.SB). 2. Characteristic of ischemic infarction of any organ except the brain 3. Area of infarcted tissue is often wedge-shaped (pointing to focus of vascular occlusion) and pale. 4.

1	2. Characteristic of ischemic infarction of any organ except the brain 3. Area of infarcted tissue is often wedge-shaped (pointing to focus of vascular occlusion) and pale. 4. Red infarction arises if blood re-enters a loosely organized tissue (e.g., pulmonary or testicular infarction, Fig. 1.6). B. Liquefactive necrosis 1. Necrotic tissue that becomes liquefied; enzymatic lysis of cells and protein results in liquefaction. 2. Characteristic of i. Brain infarction-Proteolytic enzymes from microglial cells liquefy the brain. ii. Abscess-Proteolytic enzymes from neutrophils liquefy tissue. u1. Pancreatitis-Proteolytic enzymes from pancreas liquefy parenchyma. C. Gangrenous necrosis 1. Coagulative necrosis that resembles mummified tissue (dry gangrene, Fig. 1.7) 2. Characteristic of ischemia of lower limb and GI tract 3. If superimposed infection of dead tissues occurs, then liquefactive necrosis ensues (wet gangrene). D. Caseous necrosis 1.

1	Characteristic of ischemia of lower limb and GI tract 3. If superimposed infection of dead tissues occurs, then liquefactive necrosis ensues (wet gangrene). D. Caseous necrosis 1. Soft and friable necrotic tissue with "cottage cheese-like" appearance (Fig. 1.8) 2. Combination of coagulative and liquefactive necrosis 3. Characteristic of granulomatous inflammation due to tuberculous or fungal infection Fig. 1.8 Caseous necrosis of lung. (Courtesy of Fig. 1 .6 Hemorrhagic infarction of testicle. Fig. 1.7 Dry gangrene. Yale Rosen, MD) (Courtesy of humpath.com) E. Fat necrosis 1. Necrotic adipose tissue with chalky-white appearance due to deposition of calcium (Fig. 1.9) 2. Characteristic of trauma to fat (e.g., breast) and pancreatitis-mediated damage of peripancreatic fat 3. Fatty acids released by trauma (e.g., to breast) or lipase (e.g., pancreatitis) join with calcium via a process called saponification.

1	Fatty acids released by trauma (e.g., to breast) or lipase (e.g., pancreatitis) join with calcium via a process called saponification. i. Saponification is an example of dystrophic calcification in which calcium deposits on dead tissues. In dystrophic calcification, the necrotic tissue acts as a nidus for calcification in the setting of normal serum calcium and phosphate. ii. Metastatic calcification, as opposed to dystrophic calcification, occurs when high serum calcium or phosphate levels lead to calcium deposition in normal tissues (e.g., hyperparathyroidism leading to nephrocalcinosis). F. Fibrinoid necrosis 1. Necrotic damage to blood vessel wall 2. Leaking of proteins (including fibrin) into vessel wall results in bright pink staining of the wall microscopically (Fig. 1.10). 3. Characteristic of malignant hypertension and vasculitis IV. APOPTOSIS

1	3. Characteristic of malignant hypertension and vasculitis IV. APOPTOSIS A. Energy (ATP) -dependent, genetically programmed cell death involving single cells or small groups of cells. Examples include 1. 2. Removal of cells during embryogenesis 3. CDs+ T cell-mediated killing of virally infected cells B. Morphology 1. Dying cell shrinks, leading cytoplasm to become more eosinophilic (pink, Fig. l.ll). 2. Nucleus condenses and fragments in an organized manner. 3. Apoptotic bodies fa ll from the cell and are removed by macrophages; apoptosis is not followed by inflammation. C. Apoptosis is mediated by caspases that activate proteases and endonucleases. 1. Proteases break down the cytoskeleton. 2. Endonucleases break down DNA. D. Caspases are activated by multiple pathways. 1. Intrinsic mitochondrial pathway i. Cellular injury, DNA damage, or decreased hormonal stimulation leads to inactivation of Bcl2.

1	D. Caspases are activated by multiple pathways. 1. Intrinsic mitochondrial pathway i. Cellular injury, DNA damage, or decreased hormonal stimulation leads to inactivation of Bcl2. ii. Lack of Bcl2 allows cytochrome c to leak from the inner mitochondrial matrix into the cytoplasm and activate caspases. Fig. 1.9 Fat necrosis of peri-pancreatic adipose Fig. 1.10 Fibrinoid necrosis of vessel. Fig. 1.11 Apoptosis. tissue. (Courtesy of humpath.com) Growth Adaptations, Cellular Injury, and Cell Death 2. Extrinsic receptor-ligand pathway i. FAS ligand binds FAS death receptor (CD95) on the target cell, activating caspases (e.g., negative selection of thymocytes in thymus). ii. Tumor necrosis factor (TNF) binds TNF receptor on the target cell, activating caspases. 3. Cytotoxic CDs+ T cell-mediated pathway i. Perforins secreted by CDs+ T cell create pores in membrane of target cell. ii. Granzyme from CDS+ T cell enters pores and activates caspases.

1	3. Cytotoxic CDs+ T cell-mediated pathway i. Perforins secreted by CDs+ T cell create pores in membrane of target cell. ii. Granzyme from CDS+ T cell enters pores and activates caspases. iii. CDs+ T-cell killing ofvirally infected cells is an example. I. BASIC PRINCIPLES A. Free radicals are chemical species with an unpaired electron in their outer orbit. B. Physiologic generation of free radicals occurs during oxidative phosphorylation. 1. Cytochrome c oxidase (complex IV) transfers electrons to oxygen. 2. Partial reduction of 0 2 yields superoxide (0:), hydrogen peroxide (H2O), and hydroxyl radicals ("OH). C. Pathologic generation of free radicals arises with 1. Ionizing radiation-water hydrolyzed to hydroxyl free radical 2. Inflammation-NADPH oxidase generates superoxide ions during oxygendependent killing by neutrophils. 3. Metals (e.g., copper and iron)-Fe2+ generates hydroxyl free radicals (Fenton reaction). 4.

1	Inflammation-NADPH oxidase generates superoxide ions during oxygendependent killing by neutrophils. 3. Metals (e.g., copper and iron)-Fe2+ generates hydroxyl free radicals (Fenton reaction). 4. Drugs and chemicals-P450 system of liver metabolizes drugs (e.g., acetaminophen), generating free radicals. D. Free radicals cause cellular injury via peroxidation of lipids and oxidation of DNA and proteins; DNA damage is implicated in aging and oncogenesis. E. Elimination of free radicals occurs via multiple mechanisms. 1. Antioxidants (e.g., glutathione and vitamins A , C, and E) 2. i. Superoxide dismutase (in mitochondria)-Superoxide co:) ➔ HP2 ii. Glutathione peroxidase (in mitochondria)-2GSH + free radical ➔ GS-SG and H2O iii. Catalase (in peroxisomes)-H2O2 ➔ 0 2and H2O 3. Metal carrier proteins (e.g., transferrin and ceruloplasmin) II. EXAMPLES OF FREE RADICAL INJURY A. Carbon tetrachloride (CC1) 1. Organic solvent used in the dry cleaning industry 2.

1	Metal carrier proteins (e.g., transferrin and ceruloplasmin) II. EXAMPLES OF FREE RADICAL INJURY A. Carbon tetrachloride (CC1) 1. Organic solvent used in the dry cleaning industry 2. Converted to CC13 free radical by P450 system of hepatocytes 3. Results in cell injury with swelling of RER; consequently, ribosomes detach, impairing protein synthesis. 4. Decreased apolipoproteins lead to fatty change in the liver (Fig. 1.12). B. Reperfusion injury 1. Return of blood to ischemic tissue results in production ofO2-derived free radicals, which further damage tissue. 2. Leads to a continued rise in cardiac enzymes (e.g., troponin) after reperfusion of infarcted myocardial tissue I. BASIC PRINCIPLES A. Amyloid is a misfolded protein that deposits in the extracellular space, thereby damaging tissues. B. Multiple proteins can deposit as amyloid. Shared features include 1. 2.

1	A. Amyloid is a misfolded protein that deposits in the extracellular space, thereby damaging tissues. B. Multiple proteins can deposit as amyloid. Shared features include 1. 2. Congo red staining and apple-green birefringence when viewed microscopically under polarized light (Fig. 1.13) C. Deposition can be systemic or localized. II. SYSTEMIC AMYLOIDOSIS A. Amyloid deposition in multiple organs; divided into primary and secondary amyloidosis B. Primary amyloidosis is systemic deposition of AL amyloid, which is derived from immunoglobulin light chain. 1. Associated with plasma cell dyscrasias (e.g., multiple myeloma) C. Secondary amyloidosis is systemic deposition of AA amyloid, which is derived from serum amyloid-associated protein (SAA). 1. SAA is an acute phase reactant that is increased in chronic inflammatory states, malignancy, and Familial Mediterranean fever (FMF). 2.

1	1. SAA is an acute phase reactant that is increased in chronic inflammatory states, malignancy, and Familial Mediterranean fever (FMF). 2. FMF is due to a dysfunction of neutrophils (autosomal recessive) and occurs in persons of Mediterranean origin. i. Presents with episodes of fever and acute serosal inflammation (can mimic appendicitis, arthritis, or myocardial infarction) ii. High SAA during attacks deposits as AA amyloid in tissues. D. Clinical findings of systemic amyloidosis are diverse since almost any tissue can be involved. Classic findings include 1. Nephrotic syndrome; kidney is the most common organ involved. 2. 3. Tongue enlargement, malabsorption, and hepatosplenomegaly E. Diagnosis requires tissue biopsy. Abdominal fat pad and rectum are easily accessible biopsy targets. F. Damaged organs must be transplanted. Amyloid cannot be removed. III. LOCALIZED AMYLOIDOSIS A. Amyloid deposition usually localized to a single organ. B. Senile cardiac amyloidosis 1.

1	F. Damaged organs must be transplanted. Amyloid cannot be removed. III. LOCALIZED AMYLOIDOSIS A. Amyloid deposition usually localized to a single organ. B. Senile cardiac amyloidosis 1. Non-mutated serum transthyretin deposits in the heart. 2. Usually asymptomatic; present in 25% of individuals > 80 years of age C. Familial amyloid cardiomyopathy Fig. 1.12 Fatty change of liver. Fig. 1.13 Amyloid. A, Congo red. B, Apple-green birefringence. (Courtesy of Ed Uthman, MD) Growth Adaptations, Cellular Injury, and Cell Death 1. Mutated serum transthyretin deposits in the heart leading to restrictive cardiomyopathy. 2. 5% of African Americans carry the mutated gene. D. Non-insulin-dependent diabetes mellitus (type II) 1. Amylin (derived from insulin) deposits in the islets of the pancreas. E. Alzheimer disease 1. AP amyloid (derived from P-amyloid precursor protein) deposits in the brain forming amyloid plaques. 2.

1	E. Alzheimer disease 1. AP amyloid (derived from P-amyloid precursor protein) deposits in the brain forming amyloid plaques. 2. Gene for P-APP is present on chromosome 21. Most individuals with Down syndrome (trisomy 21) develop Alzheimer disease by the age of 40 (early-onset). F. Dialysis-associated amyloidosis 1. P2-microglobulin deposits in joints. G. Medullary carcinoma of the thyroid 1. Calcitonin (produced by tumor cells) deposits within the tumor ('tumor cells in an amyloid background').

1	G. Medullary carcinoma of the thyroid 1. Calcitonin (produced by tumor cells) deposits within the tumor ('tumor cells in an amyloid background'). Thank you for choosing Pathoma for your studies. We strive to provide the highest quality educational materials while keeping affordability in mind. A tremendous amount of time and effort has gone into developing these materials, so we appreciate your legitimate use of this program. It speaks to your integrity as a future physician and the high ethical standards that we all set forth for ourselves when taking the Hippocratic oath. Unauthorized use of Pathoma materials is contrary to the ethical standards of a training physician and is a violation of copyright. Pathoma videos are updated on a regular basis and the most current version, as well as a complete list of errata, can be accessed through your account at Pathoma.com. Sincerely, Dr. Sattar, MD Inflammation, Inflammatory Disorders, and Wound Healing I. INFLAMMATION

1	Sincerely, Dr. Sattar, MD Inflammation, Inflammatory Disorders, and Wound Healing I. INFLAMMATION A. Allows inflammatory cells, plasma proteins (e.g., complement), and fluid to exit blood vessels and enter the interstitial space B. Divided into acute and chronic inflammation I. BASIC PRINCIPLES A. Characterized by the presence of edema and neutrophils in tissue (Fig. 2.lA) B. Arises in response to infection (to eliminate pathogen) or tissue necrosis (to clear necrotic debris) C. Immediate response with limited specificity (innate immunity) II. MEDIATORS OF ACUTE INFLAMMATION A. Toll-like receptors (TLRs) 1. Present on cells of the innate immune system (e.g., macrophages and dendritic cells) 2. Activated by pathogen-associated molecular patterns (PAMPs) that are commonly shared by microbes i. CD14 (a co-receptor for TLR4) on macrophages recognizes lipopolysaccharide (a PAMP) on the outer membrane of gram-negative bacteria. 3.

1	3. TLR activation results in upregulation of NF-KB, a nuclear transcription factor that activates immune response genes leading to production of multiple immune mediators. 4. TLRs are also present on cells of adaptive immunity (e.g., lymphocytes) and, hence, play an important role in mediating chronic inflammation. B. Arachidonic acid (AA) metabolites 1. AA is released from the phospholipid cell membrane by phospholipase A2 and then acted upon by cyclooxygenase or 5-lipoxygenase. i. Cyclooxygenase produces prostaglandins (PG). a. PGI2, PGD2, and PGE2 mediate vasodilation and increased vascular permeability. b. PGE also mediates pain and fever. ii. 5-lipoxygenase produces leukotrienes (LT). a. LTB4 attracts and activates neutrophils. b. LTC , LTD , and LTE (slow reacting substances of anaphylaxis) mediate vasoconstriction, bronchospasm, and increased vascular permeability. C. Mast cells 1. 2.

1	b. LTC , LTD , and LTE (slow reacting substances of anaphylaxis) mediate vasoconstriction, bronchospasm, and increased vascular permeability. C. Mast cells 1. 2. Activated by (1) tissue trauma, (2) complement proteins C3a and CSa, or (3) cross-linking of cell-surface IgE by antigen pathoma.com i. Immediate response involves release of preformed histamine granules, which mediate vasodilation of arterioles and increased vascular permeability. ii. Delayed response involves production of arachidonic acid metabolites, particularly leukotrienes. D. Complement 1. 2. Circulate as inactive precursors; activation occurs via i. Classical pathway-Cl binds IgG or IgM that is bound to antigen. ii. Alternative pathway-Microbial products directly activate complement. iii. Mannose-binding lectin (MBL) pathway-MEL binds to mannose on microorganisms and activates complement. 3.

1	ii. Alternative pathway-Microbial products directly activate complement. iii. Mannose-binding lectin (MBL) pathway-MEL binds to mannose on microorganisms and activates complement. 3. All pathways result in production of C3 convertase (mediates C3 ➔ C3a and C3b), which, in turn, produces CS convertase (mediates CS ➔ CSa and CSb). CSb complexes with C6-C9 to form the membrane attack complex (MAC). 1. C3a and CSa (anaphylatoxins)-trigger mast cell degranulation, resulting in histamine-mediated vasodilation and increased vascular permeability ii. CSa-chemotactic for neutrophils iii. C3b-opsonin for phagocytosis iv. MAC-lyses microbes by creating a hole in the cell membrane E. Hageman factor (Factor XII) 1. 2.

1	E. Hageman factor (Factor XII) 1. 2. Activated upon exposure to subendothelial or tissue collagen; in turn, activates i. Coagulation and fibrinolytic systems ii. Complement iii. Kin in system-Kinin cleaves high-molecular-weight kininogen (HMWK) to bradykinin, which mediates vasodilation and increased vascular permeability (similar to histamine), as well as pain. III. CARDINAL SIGNS OF INFLAMMATION A. Redness (rubor) and warmth (calor) 1. Due to vasodilation, which results in increased blood flow 2. Occurs via relaxation of arteriolar smooth muscle; key mediators are histamine, prostaglandins, and bradykinin. B. Swelling (tumor) 1. Due to leakage of fluid from postcapillary venules into the interstitial space (exudate) 2. Key mediators are (1) histamine, which causes endothelial cell contraction and (2) tissue damage, resulting in endothelial cell disruption. C. Pain (dolor) 1. Bradykinin and PGE sensitize sensory nerve endings.

1	C. Pain (dolor) 1. Bradykinin and PGE sensitize sensory nerve endings. Fig. 2.1 Inflammation. A, Acute inflammation with neutrophils. B, Chronic inflammation with lymphocytes and plasma cells. Inflammation, Inflammatory Disorders, and Wound Healing D. Fever 1. Pyrogens (e.g., LPS from bacteria) cause macrophages to release IL-1 and TNF, which increase cyclooxygenase activity in perivascular cells of the hypothalamus. 2. Increased PGE2 raises temperature set point. IV. NEUTROPHIL ARRIVAL AND FUNCTION A. Step 1-Margination 1. Vasodilation slows blood flow in postcapillary venules. 2. Cells marginate from center of flow to the periphery. B. Step 2-Rolling 1. Selectin "speed bumps" are upregulated on endothelial cells. i. P-selectin release from Weibel-Palade bodies is mediated by histamine. ii. E-selectin is induced by TNF and IL-1. 2. Selectins bind sialyl Lewis X on leukocytes. 3. Interaction results in rolling of leukocytes along vessel wall. C. Step 3-Adhesion 1.

1	ii. E-selectin is induced by TNF and IL-1. 2. Selectins bind sialyl Lewis X on leukocytes. 3. Interaction results in rolling of leukocytes along vessel wall. C. Step 3-Adhesion 1. Cellular adhesion molecules (ICAM and VCAM) are upregulated on endothelium by TNF and IL-1. 2. Integrins are upregulated on leukocytes by C5a and LTB4 • 3. Interaction between CA Ms and integrins results in firm adhesion ofleukocytes to the vessel wall. 4. Leukocyte adhesion deficiency is most commonly due to an autosomal recessive defect of integrins (CD18 subunit). i. Clinical features include delayed separation of the umbilical cord, increased circulating neutrophils (due to impaired adhesion of marginated pool of leukocytes), and recurrent bacterial infections that lack pus formation. D. Step 4-Transmigration and Chemotaxis 1. Leukocytes transmigrate across the endothelium of postcapillary venules and move toward chemical attractants (chemotaxis). 2.

1	D. Step 4-Transmigration and Chemotaxis 1. Leukocytes transmigrate across the endothelium of postcapillary venules and move toward chemical attractants (chemotaxis). 2. Neutrophils are attracted by bacterial products, IL-8, C5a, and LTB • E. Step 5-Phagocytosis 1. Consumption of pathogens or necrotic tissue; phagocytosis is enhanced by opsonins (IgG and C3b). 2. Pseudopods extend from leukocytes to form phagosomes, which are internalized and merge with lysosomes to produce phagolysosomes. 3. Chediak-Higashi syndrome is a protein trafficking defect (autosomal recessive) characterized by impaired phagolysosome formation. Clinical features include i. Increased risk of pyogenic infections ii. Neutropenia (due to intramedullary death of neutrophils) iii. Giant granules in leukocytes (due to fusion of granules arising from the Golgi apparatus) iv. Defective primary hemostasis (due to abnormal dense granules in platelets) v. vi. Peripheral neuropathy

1	Defective primary hemostasis (due to abnormal dense granules in platelets) v. vi. Peripheral neuropathy F. Step 6-Destruction of phagocytosed material 1. 0 2-dependent killing is the most effective mechanism. 2. HOC! generated by oxidative burst in phagolysosomes destroys phagocytosed microbes. 1. 0 2 is converted too; by NADPH oxidase (oxidative burst). ii. o ; is converted to H 0 by superoxide dismutase (SOD). iii. Hp2 is converted to HOC! (bleach) by myeloperoxidase (MPO). 3. Chronic granulomatous disease (CGD) is characterized by poor 0 -dependent killing.

1	iii. Hp2 is converted to HOC! (bleach) by myeloperoxidase (MPO). 3. Chronic granulomatous disease (CGD) is characterized by poor 0 -dependent killing. i. Due to NADPH oxidase defect (X-linked or autosomal recessive) ii. Leads to recurrent infection and granuloma formation with catalase-positive organisms, particularly Staphylococcus aureus, Pseudomonas cepacia, Serratia marcescens, Nocardia, and Aspergillus iii. Nitroblue tetrazolium test is used to screen for CGD. Leukocytes are incubated with NBT dye, which turns blue ifNADPH oxidase can convert 0 too;, but remains colorless ifNADPH oxidase is defective. 4. MPO deficiency results in defective conversion ofH 0 to HOCL i. Increased risk for Candida infections; however, most patients are asymptomatic. ii. NBT is normal; respiratory burst (02 to Hp2) is intact.

1	ii. NBT is normal; respiratory burst (02 to Hp2) is intact. 5. 0 2-independent killing is less effective than 0 2 -dependent killing and occurs via enzymes present in leukocyte secondary granules (e.g., lysozyme in macrophages and major basic protein in eosinophils). G. Step ?-Resolution 1. Neutrophils undergo apoptosis and disappear within 24 hours after resolution of the inflammatory stimulus. V. MACROPHAGES A. Macrophages predominate after neutrophils and peak 2-3 days after inflammation begins. 1. Derived from monocytes in blood B. Arrive in tissue via the margination, rolling, adhesion, and transmigration sequence C. Ingest organisms via phagocytosis (augmented by opsonins) and destroy phagocytosed material using enzymes (e.g., lysozyme) in secondary granules (02independent killing) D. Manage the next step of the inflammatory process. Outcomes include 1. Resolution and healing-Anti-inflammatory cytokines (e.g., IL-10 and TGF-~) are produced by macrophages. 2.

1	D. Manage the next step of the inflammatory process. Outcomes include 1. Resolution and healing-Anti-inflammatory cytokines (e.g., IL-10 and TGF-~) are produced by macrophages. 2. Continued acute inflammation-marked by persistent pus formation; IL-8 from macrophages recruits additional neutrophils. 3. Abscess-acute inflammation surrounded by fibrosis; macrophages mediate fibrosis via fibrogenic growth factors and cytokines. 4. Chronic inflammation-Macrophages present antigen to activate CD4+ helper T cells, which secrete cytokines that promote chronic inflammation. I. BASIC PRINCIPLES A. Characterized by the presence oflymphocytes and plasma cells in tissue (Fig. 2.lB) B. Delayed response, but more specific (adaptive immunity) than acute inflammation C. Stimuli include (1) persistent infection (most common cause); (2) infection with viruses, mycobacteria, parasites, and fungi; (3) autoimmune disease; (4) foreign material; and (5) some cancers. II. T LYMPHOCYTES

1	II. T LYMPHOCYTES A. Produced in bone marrow as progenitor T cells B. Further develop in the thymus where the T-cell receptor (TCR) undergoes rearrangement and progenitor cells become CD4+ helper T cells or CDs+ cytotoxic T cells 1. T cells use TCR complex (TCR and CD3) for antigen surveillance. Inflammation, Inflammatory Disorders, and Wound Healing 2. TCR complex recognizes antigen presented on MHC molecules. i. CD4+ T cells-MHC class II ii. CDS+ T cells-MHC class I 3. Activation ofT cells requires (1) binding of antigen/MHC complex and (2) an additional 2nd signal. C. CD4 + helper T-cell activation 1. Extracellular antigen (e.g., foreign protein) is phagocytosed, processed, and presented on MHC class II, which is expressed by antigen presenting cells (APCs). 2. B7 on APC binds CD2S on CD4+ helper T cells providing 2nd activation signal. 3. Activated CD4 + helper T cells secrete cytokines that "help" inflammation and are divided into two subsets.

1	2. B7 on APC binds CD2S on CD4+ helper T cells providing 2nd activation signal. 3. Activated CD4 + helper T cells secrete cytokines that "help" inflammation and are divided into two subsets. i. T Hl subset secretes IFN-y (activates macrophage, promotes B-cell class switching from IgM to IgG, promotes T1) phenotype and inhibits TH2 phenotype). ii. TH2 subset secretes IL-4 (facilitates B-cell class switching to IgE), IL-5 (eosinophil chemotaxis and activation, and class switching to IgA), and IL-13 (function similar to IL-4). D. CDs+ cytotoxic T-cell activation 1. Intracellular antigen (derived from proteins in the cytoplasm) is processed and presented on MHC class I, which is expressed by all nucleated cells and platelets. 2. IL-2 from CD4+ THl cell provides 2nd activation signal. 3. Cytotoxic T cells are activated for killing. 4. 1. Secretion of perforin and granzyme; perforin creates pores that allow granzyme to enter the target cell, activating apoptosis.

1	3. Cytotoxic T cells are activated for killing. 4. 1. Secretion of perforin and granzyme; perforin creates pores that allow granzyme to enter the target cell, activating apoptosis. ii. Expression of FasL, which binds Fas on target cells, activating apoptosis III. B LYMPHOCYTES A. Immature B cells are produced in the bone marrow and undergo immunoglobulin rearrangements to become na'ive B cells that express surface IgM and IgD. B. B-cell activation occurs via 1. Antigen binding by surface IgM or IgD; results in maturation to IgM-or IgDsecreting plasma cells 2. B-cell antigen presentation to CD4+ helper T cells via MHC class II. i. CD40 receptor on B cell binds CD40L on helper T cell, providing 2nd activation signal. ii. Helper T cell then secretes IL-4 and IL-5 (mediate B-cell isotype switching, hypermutation, and maturation to plasma cells). IV. GRANULOMATOUS INFLAMMATION A. Subtype of chronic inflammation

1	ii. Helper T cell then secretes IL-4 and IL-5 (mediate B-cell isotype switching, hypermutation, and maturation to plasma cells). IV. GRANULOMATOUS INFLAMMATION A. Subtype of chronic inflammation B. Characterized by granuloma, which is a collection of epithelioid histiocytes (macrophages with abundant pink cytoplasm), usually surrounded by giant cells and a rim of lymphocytes C. Divided into noncaseating and caseating subtypes 1. Noncaseating granulomas lack central necrosis (Fig. 2.2A). Common etiologies include reaction to foreign material, sarcoidosis, beryllium exposure, Crohn disease, and cat scratch disease. 2. Caseating granulomas exhibit central necrosis and are characteristic of tuberculosis and fungal infections (Fig. 2.2B). D. Steps involved in granuloma formation 1. Macrophages process and present antigen via MHC class II to CD4+ helper T cells. 2. Interaction leads macrophages to secrete IL-12, inducing CD4+ helper T cells to differentiate into TH 1 subtype.

1	Macrophages process and present antigen via MHC class II to CD4+ helper T cells. 2. Interaction leads macrophages to secrete IL-12, inducing CD4+ helper T cells to differentiate into TH 1 subtype. 3. TH 1 cells secrete IFN-y, which converts macrophages to epithelioid histiocytes and giant cells. I. DIGEORGE SYNDROME A. Developmental failure of the third and fourth pharyngeal pouches 1. Due to 22qll microdeletion B. Presents with T-cell deficiency (lack of thymus); hypocalcemia (lack of parathyroids); and abnormalities of heart, great vessels, and face II. SEVERE COMBINED IMMUNODEFICIENCY (SCIO) A. Defective cell-mediated and humoral immunity B. Etiologies include 1. Cytokine receptor defects-Cytokine signaling is necessary for proliferation and maturation of B and T cells. 2. Adenosine deaminase (ADA) deficiency-ADA is necessary to deaminate adenosine and deoxyadenosine for excretion as waste products; buildup of adenosine and deoxyadenosine is toxic to lymphocytes. 3.

1	Adenosine deaminase (ADA) deficiency-ADA is necessary to deaminate adenosine and deoxyadenosine for excretion as waste products; buildup of adenosine and deoxyadenosine is toxic to lymphocytes. 3. MHC class II deficiency-MHC class II is necessary for CD4+ helper T cell activation and cytokine production. C. Characterized by susceptibility to fungal, viral, bacterial, and protozoa! infections, including opportunistic infections and live vaccines D. Treatment is sterile isolation ('bubble baby') and stem cell transplantation. III. X-LINKED AGAMMAGLOBULINEMIA A. Complete lack of immunoglobulin due to disordered B-cell maturation 1. Pre-and pro-B cells cannot mature. B. Due to mutated Bruton tyrosine kinase; X-linked C. Presents after 6 months of life with recurrent bacterial, enterovirus (e.g., polio and coxsackievirus), and Giardia lamblia infections; maternal antibodies present during the first 6 months oflife are protective. D. Live vaccines (e.g., polio) must be avoided.

1	D. Live vaccines (e.g., polio) must be avoided. IV. COMMON VARIABLE IMMUNODEFICIENCY (CVID) A. Low immunoglobulin due to B-cell or helper T-cell defects Fig. 2.2 Granuloma. A, Noncaseating. B, Caseating. Fig. 2.3 Angioedema. (Courtesy of James Heilman, MD, Wikipedia) Inflammation, Inflammatory Disorders, and Wound Healing B. Increased risk for bacterial, enterovirus, and Giardia lamblia infections, usually in late childhood C. Increased risk for autoimmune disease and lymphoma V. IgA DEFICIENCY A. Low serum and mucosa! IgA; most common immunoglobulin deficiency B. Increased risk for mucosa! infection, especially viral; however, most patients are asymptomatic. VI. HYPER-IgM SYNDROME A. Characterized by elevated IgM B. Due to mutated CD40L (on helper T cells) or CD40 receptor (on B cells) 1. Second signal cannot be delivered to helper T cells during B-cell activation. 2. Consequently, cytokines necessary for immunoglobulin class switching are not produced.

1	Second signal cannot be delivered to helper T cells during B-cell activation. 2. Consequently, cytokines necessary for immunoglobulin class switching are not produced. C. Low IgA, IgG, and IgE result in recurrent pyogenic infections (due to poor opsonization), especially at mucosa! sites. VII. WISKOTT-ALDRICH SYNDROME A. Characterized by thrombocytopenia, eczema, and recurrent infections (defective humoral and cellular immunity); bleeding is a major cause of death B. Due to mutation in the WASP gene; X-linked VIII. COMPLEMENT DEFICIENCIES A. C5-C9 deficiencies-increased risk for Neisseria infection (N gonorrhoeae and N meningitidis) B. Cl inhibitor deficiency-results in hereditary angioedema, which is characterized by edema of the skin (especially periorbital, Fig. 2.3) and mucosa! surfaces I. BASIC PRINCIPLES A. Characterized by immune-mediated damage of self tissues 1. US prevalence is 1%-2%. B. Involves loss of self-tolerance 1.

1	I. BASIC PRINCIPLES A. Characterized by immune-mediated damage of self tissues 1. US prevalence is 1%-2%. B. Involves loss of self-tolerance 1. Self-reactive lymphocytes are regularly generated but develop central (thymus and bone marrow) or peripheral tolerance. 2. Central tolerance in thymus leads to T-cell (thymocyte) apoptosis or generation of regulatory T cells. i. AIRE mutations result in autoimmune polyendocrine syndrome. 3. Central tolerance in bone marrow leads to receptor editing or B-cell apoptosis. 4. Peripheral tolerance leads to anergy or apoptosis ofT and B cells. 1. Fas apoptosis pathway mutations result in autoimmune lymphoproliferative syndrome (ALPS). 5. Regulatory T cells suppress autoimmunity by blocking T-cell activation and producing anti-inflammatory cytokines (IL-10 and TGF-~). i. CD25 polymorphisms are associated with autoimmunity (MS and type 1 DM).

1	i. CD25 polymorphisms are associated with autoimmunity (MS and type 1 DM). ii. FOXP3 mutations lead to IPEX syndrome (Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked). C. More common in women; classically affects women ofchildbearing age 1. Estrogen may reduce apoptosis of self-reactive B cells. D. Etiology is likely an environmental trigger in genetically-susceptible individuals. 1. Increased incidence in twins 2. Association with certain HLA types (e.g., HLA-B27) and PTPN22 polymorphisms 3. Environmental triggers lead to bystander activation or molecular mimicry. E. Autoimmune disorders are clinically progressive with relapses and remissions and often show overlapping features; partially explained by epitope spreading II. SYSTEMIC LUPUS ERYTHEMATOSUS A. Chronic, systemic autoimmune disease 1. Flares and remissions are common.

1	II. SYSTEMIC LUPUS ERYTHEMATOSUS A. Chronic, systemic autoimmune disease 1. Flares and remissions are common. B. Classically arises in middle-aged females, especially African American and Hispanic women 1. May also arise in children and older adults (less dramatic gender bias) C. Antigen-antibody complexes damage multiple tissues (type III HSR). 1. Poorly-cleared apoptotic debris (e.g., from UV damage) activates self-reactive lymphocytes, which then produce antibodies to host nuclear antigens. 2. Antigen-antibody complexes are generated at low levels and taken up by dendritic cells. 3. DNA antigens activate TLRs, amplifying immune response (IFN-a). 4. Antigen-antibody complexes are subsequently generated at higher levels and deposit in multiple tissues causing disease. 5. Deficiency of early complement proteins (Clq, C4, and C2) is associated with SLE. D. Almost any tissue can be involved. Classic findings include 1.

1	5. Deficiency of early complement proteins (Clq, C4, and C2) is associated with SLE. D. Almost any tissue can be involved. Classic findings include 1. Fever, weight loss, fatigue, lymphadenopathy, and Raynaud phenomenon 2. Malar 'butterfly' rash (Fig. 2.4A) or discoid rash (Fig. 2.4B), especially upon exposure to sunlight 3. 4. Arthritis (usually involving ?. 2 joints) 5. 6. 7. i. Diffuse proliferative glomerulonephritis is the most common and most severe form of injury. ii. Other patterns of injury (e.g., membranous glomerulonephritis) also occur. 8. Anemia, thrombocytopenia, or leukopenia (type II HSR) 9. 10. Antinuclear antibody (ANA; sensitive, but not specific) 11. E. Antiphospholipid antibody is associated with SLE (one-third of patients). 1. Autoantibody directed against proteins bound to phospholipids Fig. 2.4A Malar 'butterfly' rash, SLE. Fig. 2.4B Discoid rash, SLE.

1	1. Autoantibody directed against proteins bound to phospholipids Fig. 2.4A Malar 'butterfly' rash, SLE. Fig. 2.4B Discoid rash, SLE. Inflammation, Inflammatory Disorders, and Wound Healing 2. Important antiphospholipid antibodies include anticardiolipin (false-positive VDRL and RPR syphilis screening tests), anti-02-glycoprotein I, and lupus anticoagulant (falsely-elevated PTT). F. Antiphospholipid antibody syndrome is characterized by hypercoagulable state due to antiphospholipid antibodies (especially lupus anticoagulant). 1. Results in arterial and venous thrombosis including deep venous, hepatic vein, placental (recurrent pregnancy loss), and cerebral (stroke) thrombosis 2. 3. Associated with SLE; however, more commonly occurs as a primary disorder G. Antihistone antibody is characteristic of drug-induced lupus. 1. Procainamide, hydralazine, and isoniazid are common causes. 2. ANA is positive by definition. 3. CNS and renal involvement are rare. 4.

1	1. Procainamide, hydralazine, and isoniazid are common causes. 2. ANA is positive by definition. 3. CNS and renal involvement are rare. 4. Removal of drug usually results in remission. H. First-line treatment includes avoiding exposure to direct sunlight and glucocorticoids for flares; other immunosuppressive agents are useful in severe or refractory disease. I. 5-year survival is > 90%; renal failure, infection, and accelerated coronary atherosclerosis (occurs late) are common causes of death. III. SJOGREN SYNDROME A. Autoimmune destruction of lacrimal and salivary glands 1. Lymphocyte-mediated damage (type IV HSR) with fibrosis B. Classically presents as dry eyes (keratoconjunctivitis sicca), dry mouth (xerostomia), and recurrent dental caries in an older woman (50-60 years)-"Can't chew a cracker, dirt in my eyes" 1. May progress to ulceration of corneal epithelium and oral mucosa

1	C. Can be primary (sicca syndrome) or associated with another autoimmune disorder, especially rheumatoid arthritis 1. Rheumatoid factor is often present even when rheumatoid arthritis is absent. D. Characterized by ANA and anti-ribonucleoprotein antibodies (anti-SSA/Ro and anti-SSB/La) 1. Anti-SSA and anti-SSB are associated with extraglandular manifestations (e.g., neuropathy). 2. Pregnant women with anti-SSA are at risk for delivering babies with neonatal lupus and congenital heart block. 3. Anti-SSA and anti-SSB are also seen in a subset of patients with SLE (screen pregnant patients) E. Lymphocytic sialadenitis on lip biopsy (minor salivary glands) is an additional diagnostic criterion (Fig. 2.4C). F. Increased risk for B-cell (marginal zone) lymphoma, which presents as unilateral enlargement of the parotid gland late in disease course IV. SYSTEMIC SCLEROSIS (SCLERODERMA)

1	F. Increased risk for B-cell (marginal zone) lymphoma, which presents as unilateral enlargement of the parotid gland late in disease course IV. SYSTEMIC SCLEROSIS (SCLERODERMA) A. Autoimmune disorder characterized by sclerosis of skin and visceral organs 1. Classically presents in middle-aged females (30-50 years) B. Fibroblast activation leads to deposition of collagen. 1. Autoimmune damage to mesenchyme is possible initiating event. 2. Endothelial dysfunction leads to inflammation (increased adhesion molecules), vasoconstriction (increased endothelin and decreased NO), and secretion of growth factors (TGF-0 and PDGF). 3. Fibrosis, initially perivascular, progresses and causes organ damage. V. I. II. C. Limited type-Skin involvement is limited (hands and face) with late visceral involvement. 1. Prototype is CREST syndrome: Calcinosis/anti-Centromere antibodies, Raynaud phenomenon, Esophageal dysmotility, Sclerodactyly (Fig. 2.4D), and Telangiectasias of the skin.

1	1. Prototype is CREST syndrome: Calcinosis/anti-Centromere antibodies, Raynaud phenomenon, Esophageal dysmotility, Sclerodactyly (Fig. 2.4D), and Telangiectasias of the skin. D. Diffuse type-Skin involvement is diffuse with early visceral involvement. 1. Any organ can be involved. 2. i. Vessels (Raynaud phenomenon) ii. GI tract (esophageal dysmotility and reflux) iii. Lungs (interstitial fibrosis and pulmonary hypertension) iv. Kidneys (scleroderma renal crisis) 3. Highly associated with antibodies to DNA topoisomerase I (anti-Scl-70). A. Autoimmune-mediated tissue damage with mixed features ofSLE, systemic sclerosis, and polymyositis B. Characterized by ANA along with serum antibodies to Ul ribonucleoprotein A. Healing is initiated when inflammation begins. B. Occurs via a combination of regeneration and repair A. Replacement of damaged tissue with native tissue; dependent on regenerative capacity of tissue

1	B. Occurs via a combination of regeneration and repair A. Replacement of damaged tissue with native tissue; dependent on regenerative capacity of tissue B. Tissues are divided into three types based on regenerative capacity: labile, stable, and permanent. C. Labile tissues possess stem cells that continuously cycle to regenerate the tissue. 1. Small and large bowel (stem cells in mucosa] crypts, Fig. 2.5) 2. Skin (stem cells in basal layer, Fig. 2.6) 3. D. Stable tissues are comprised of cells that are quiescent (G ), but can reenter the cell cycle to regenerate tissue when necessary. 1. Classic example is regeneration of liver by compensatory hyperplasia after partial resection. Each hepatocyte produces additional cells and then reenters quiescence. Fig. 2.4C Lymphocytic sialadenitis, Sjogren Fig. 2.4D Sclerodactyly, scleroderma. Fig. 2.5 Intestinal crypts. syndrome. Inflammation, Inflammatory Disorders, and Wound Healing

1	Fig. 2.4C Lymphocytic sialadenitis, Sjogren Fig. 2.4D Sclerodactyly, scleroderma. Fig. 2.5 Intestinal crypts. syndrome. Inflammation, Inflammatory Disorders, and Wound Healing E. Permanent tissues lack significant regenerative potential (e.g., myocardium, skeletal muscle, and neurons). III. REPAIR A. Replacement of damaged tissue with fibrous scar B. Occurs when regenerative stem cells are lost (e.g., deep skin cut) or when a tissue lacks regenerative capacity (e.g., healing after a myocardial infarction, Fig. 2.7) C. Granulation tissue formation is the initial phase of repair (Fig. 2.8). 1. Consists of fibroblasts (deposit type III collagen), capillaries (provide nutrients), and myofibroblasts (contract wound) D. Eventually results in scar formation, in which type III collagen is replaced with type I collagen 1. Type III collagen is pliable and present in granulation tissue, embryonic tissue, uterus, and keloids. 2.

1	Type III collagen is pliable and present in granulation tissue, embryonic tissue, uterus, and keloids. 2. Type I collagen has high tensile strength and is present in skin, bone, tendons, and most organs. 3. Collagenase removes type III collagen and requires zinc as a cofactor. IV. A. Mediated by paracrine signaling via growth factors (e.g., macrophages secrete growth factors that target fibroblasts) B. Interaction of growth factors with receptors (e.g., epidermal growth factor with growth factor receptor) results in gene expression and cellular growth. C. Examples of mediators include 1. 2. 3. Platelet-derived growth factor-growth factor for endothelium, smooth muscle, and fibroblasts 4. 5. V. A. Cutaneous healing occurs via primary or secondary intention. 1. Primary intention-Wound edges are brought together (e.g., suturing of a surgical incision); leads to minimal scar formation 2.

1	A. Cutaneous healing occurs via primary or secondary intention. 1. Primary intention-Wound edges are brought together (e.g., suturing of a surgical incision); leads to minimal scar formation 2. Secondary intention-Edges are not approximated. Granulation tissue fills the defect; myofibroblasts then contract the wound, forming a scar. B. Delayed wound healing occurs in 1. Infection (most common cause; S aureus is the most common offender) Fig. 2.6 Basal layer of skin. Fig. 2.7 Myocardial scarring. (Courtesy of Aliya Fig. 2.8 Granulation tissue. Husa in, MD) 2. Vitamin C, copper, or zinc deficiency i. Vitamin C is an important cofactor in the hydroxylation of proline and lysine procollagen residues; hydroxylation is necessary for eventual collagen cross-linking. ii. Copper is a cofactor for lysyl oxidase, which cross-links lysine and hydroxylysine to form stable collagen.

1	ii. Copper is a cofactor for lysyl oxidase, which cross-links lysine and hydroxylysine to form stable collagen. iii. Zinc is a cofactor for collagenase, which replaces the type III collagen of granulation tissue with stronger type I collagen. 3. Other causes include foreign body, ischemia, diabetes, and malnutrition. C. Dehiscence is rupture of a wound; most commonly seen after abdominal surgery D. Hypertrophic scar is excess production of scar tissue that is localized to the wound (Fig. 2.9). E. Keloid is excess production of scar tissue that is out of proportion to the wound (Fig. 2.10). 1. 2. 3. Classically affects earlobes, face, and upper extremities Fig. 2.9 Hypertrophic scar. (Reprinted with Fig . 2.10 Keloid. permission, http://emedicine.medscape.com/ article/ 1128404-overview) Principles of Neoplasia I. BASIC PRINCIPLES A. Neoplasia is new tissue growth that is unregulated, irreversible, and monoclonal; these features distinguish it from hyperplasia and repair.

1	Principles of Neoplasia I. BASIC PRINCIPLES A. Neoplasia is new tissue growth that is unregulated, irreversible, and monoclonal; these features distinguish it from hyperplasia and repair. B. Monoclonal means that the neoplastic cells are derived from a single mother cell. C. Clonality was historically determined by glucose-6-phosphate dehydrogenase (G6PD) enzyme isoforms. 1. Multiple isoforms (e.g., G6PDA, G6PDB, and G6PDc) exist; only one isoform is inherited from each parent. 2. In females, one isoform is randomly inactivated in each cell by lyonization (G6PD is present on the X chromosome). 3. Normal ratio of active isoforms in cells of any tissue is 1:1 (e.g., 50% of cells have G6PDA, and 50% of cells have G6PD8) . 4. 1:1 ratio is maintained in hyperplasia, which is polyclonal (cells are derived from multiple cells). 5. Only one isoform is present in neoplasia, which is monoclonal. 6.

1	4. 1:1 ratio is maintained in hyperplasia, which is polyclonal (cells are derived from multiple cells). 5. Only one isoform is present in neoplasia, which is monoclonal. 6. Clonality can also be determined by androgen receptor isoforms, which are also present on the X chromosome. D. Clonality ofB lymphocytes is determined by immunoglobulin (Ig) light chain phenotype. 1. lg is comprised of heavy and light chains. 2. Each B cell expresses light chain that is either kappa or lambda. 3. Normal kappa to lambda light chain ratio is 3:1. 4. This ratio is maintained in hyperplasia, which is polyclonal. 5. Ratio increases to > 6:1 or is inverted (e.g., kappa to lambda ratio= 1:3) in lymphoma, which is monoclonal. E. Neoplastic tumors are benign or malignant. 1. Benign tumors remain localized and do not metastasize. 2. Malignant tumors (cancer) invade locally and have the potential to metastasize.

1	E. Neoplastic tumors are benign or malignant. 1. Benign tumors remain localized and do not metastasize. 2. Malignant tumors (cancer) invade locally and have the potential to metastasize. F. Tumor nomenclature is based on lineage of differentiation (type of tissue produced) and whether the tumor is benign or malignant (Table 3.1). Table 3.1: Examples of Tumor Nomenclature pathoma.com II. EPIDEMIOLOGY A. Cancer is the 2nd leading cause of death in both adults and children. 1. The leading causes of death in adults are (1) cardiovascular disease, (2) cancer, and (3) chronic respiratory disease. 2. The leading causes of death in children are (1) accidents, (2) cancer, and (3) congenital defects. B. The most common cancers by incidence in adults are (1) breast/prostate, (2) lung, and (3) colorectal. C. The most common causes of cancer mortality in adults are (1) lung, (2) breast/ prostate, and (3) colorectal. III. ROLE OF SCREENING A. Cancer begins as a single mutated cell.

1	C. The most common causes of cancer mortality in adults are (1) lung, (2) breast/ prostate, and (3) colorectal. III. ROLE OF SCREENING A. Cancer begins as a single mutated cell. B. Approximately 30 divisions occur before the earliest clinical symptoms arise. C. Each division (doubling time) results in increased mutations. 1. Cancers that do not produce symptoms until late in disease will have undergone additional divisions and, hence, additional mutations. 2. Cancers that are detected late tend to have a poor prognosis. 3. Screening seeks to catch dysplasia (precancerous change) before it becomes carcinoma or carcinoma before clinical symptoms arise; efficacy of screening, however, requires a decrease in cancer-specific mortality. D. Common screening methods include 1. 2. Mammography-detects in situ breast cancer (e.g., DCIS) before it invades or invasive carcinoma before it becomes clinically palpable 3. 4. I. BASIC PRINCIPLES

1	2. Mammography-detects in situ breast cancer (e.g., DCIS) before it invades or invasive carcinoma before it becomes clinically palpable 3. 4. I. BASIC PRINCIPLES A. Cancer formation is initiated by damage to DNA of stem cells. The damage overcomes DNA repair mechanisms, but is not lethal. 1. Carcinogens are agents that damage DNA, increasing the risk for cancer. Important carcinogens include chemicals, oncogenic viruses, and radiation (Table 3.2). B. DNA mutations eventually disrupt key regulatory systems, allowing for tumor promotion (growth) and progression (spread). 1. Disrupted systems include proto-oncogenes, tumor suppressor genes, and regulators of apoptosis. II. ONCOGENES A. Proto-oncogenes are essential for cell growth and differentiation; mutations of proto-oncogenes form oncogenes that lead to unregulated cellular growth.

1	II. ONCOGENES A. Proto-oncogenes are essential for cell growth and differentiation; mutations of proto-oncogenes form oncogenes that lead to unregulated cellular growth. B. Categories of oncogenes include growth factors, growth factor receptors, signal transducers, nuclear regulators, and cell cycle regulators (Table 3.3). 1. Growth factors induce cellular growth (e.g., PDGFB in astrocytoma). 2. Growth factor receptors mediate signals from growth factors (e.g., ERBB2 [HER2/neu] in breast cancer). 3. Signal transducers relay receptor activation to the nucleus (e.g., ras). Principles of Neoplasia Table 3.2: Important Carcinogens and Associated Cancers Nitrosamines Naphthylamine Vinyl chloride Nickel, chromium, beryllium, or silica HBVandHCV HTLV-1 High-risk HPV (e.g., subtypes 16, 18, 31, 33) sunlight is most common source) Squamous cell carcinoma of oropharynx and upper esophagus, and hepatocellular carcinoma

1	HBVandHCV HTLV-1 High-risk HPV (e.g., subtypes 16, 18, 31, 33) sunlight is most common source) Squamous cell carcinoma of oropharynx and upper esophagus, and hepatocellular carcinoma Squamous cell carcinoma of skin, lung cancer, and angiosarcoma of liver Carcinoma of oropharynx, esophagus, lung, kidney, bladder, and pancreas Urothelial carcinoma of bladder Angiosarcoma of liver Nasopharyngeal carcinoma, Burkitt lymphoma, and CNS lymphoma in AIDS Squamous cell carcinoma ofvulva, vagina, anus, and cervix; adenocarcinoma of cervix AML, CML, and papillary carcinoma of the thyroid Basal cell carcinoma, squamous cell carcinoma, and melanoma of skin Derived from Aspergillus, which can contaminate stored rice and grains Side effect of chemotherapy Arsenic is present in cigarette smoke. Exposure to asbestos is more likely to lead to lung cancer than mesothelioma. Most common carcinogen worldwide; polycyclic hydrocarbons are particularly carcinogen ic.

1	Exposure to asbestos is more likely to lead to lung cancer than mesothelioma. Most common carcinogen worldwide; polycyclic hydrocarbons are particularly carcinogen ic. Found in smoked foods; responsible for high rate of stomach carcinoma in Japan Occupational exposure; used to make polyvinyl chloride (PVC) for use in pipes Results in formation of pyrimidine dimers in DNA, which are normally excised by restriction endonuclease -,--~---------FUNDAMENTALS OF PA_T_H_O_L_O_G_Y_______ 1. Ras is associated with growth factor receptors in an inactive GDP-bound state. ii. Receptor binding causes GDP to be replaced with GTP, activating ras. iii. Activated ras sends growth signals to the nucleus. iv. Ras inactivates itself by cleaving GTP to GDP; this is augmented by GTPase activating protein. v. Mutated ras inhibits the activity of GTPase activating protein. This prolongs the activated state of ras, resulting in increased growth signals.

1	v. Mutated ras inhibits the activity of GTPase activating protein. This prolongs the activated state of ras, resulting in increased growth signals. 4. Cell cycle regulators mediate progression through the cell cycle (e.g., cyclin and cyclin-dependent kinase). i. Cyclins and cyclin-dependent kinases (CDKs) form a complex which phosphorylates proteins that drive the cell through the cell cycle. ii. For example, the cyclinD/CDK4 complex phosphorylates the retinoblastoma protein, which promotes progression through the G/ S checkpoint. III. TUMOR SUPPRESSOR GENES A. Regulate cell growth and, hence, decrease ("suppress") the risk of tumor formation; p53 and Rb (retinoblastoma) are classic examples. B. p53 regulates progression of the cell cycle from G to S phase. Table 3.3: Important Oncogenes and Associated Tumors PDGFB Platelet-derived growth factor Overexpression, autocrine loop Astrocytoma

1	Table 3.3: Important Oncogenes and Associated Tumors PDGFB Platelet-derived growth factor Overexpression, autocrine loop Astrocytoma Amplification Subset of breast carcinomas neu] receptor MEN 2A, MEN 2B and sporadic RET Neural growth factor receptor Point mutation medullary carcinoma of thyroid Carcinomas, melanoma, and ABL Tyrosine kinase t(9;22) with BCR CML and some types of ALL Principles of Neoplasia 1. In response to DNA damage, p53 slows the cell cycle and upregulates DNA repair enzymes. 2. IfDNA repair is not possible, p53 induces apoptosis. 1. p53 upregulates BAX, which disrupts Bcl2. ii. Cytochrome c leaks from the mitochondria activating apoptosis. 3. Both copies of the p53 gene must be knocked out for tumor formation (Knudson two-hit hypothesis). i. Loss is seen in > 50% of cancers. ii. Germline mutation results in Li-Fraumeni syndrome (2nd hit is somatic), characterized by the propensity to develop multiple types of carcinomas and sarcomas.

1	ii. Germline mutation results in Li-Fraumeni syndrome (2nd hit is somatic), characterized by the propensity to develop multiple types of carcinomas and sarcomas. C. Rb also regulates progression from G1 to S phase. 1. Rb "holds" the E2F transcription factor, which is necessary for transition to the S phase. 2. E2F is released when RB is phosphorylated by the cyclinD/cyclin-dependent kinase 4 (CDK4) complex. 3. Rb mutation results in constitutively free E2F, allowing progression through the cell cycle and uncontrolled growth of cells. 4. Both copies of Rb gene must be knocked out for tumor formation (Knudson twohit hypothesis). i. Sporadic mutation (both hits are somatic) is characterized by unilateral retinoblastoma (Fig. 3.1). 11. Germline mutation results in familial retinoblastoma (2nd hit is somatic), characterized by bilateral retinoblastoma and osteosarcoma. IV. REGULATORS OF APOPTOSIS

1	11. Germline mutation results in familial retinoblastoma (2nd hit is somatic), characterized by bilateral retinoblastoma and osteosarcoma. IV. REGULATORS OF APOPTOSIS A. Prevent apoptosis in normal cells, but promote apoptosis in mutated cells whose DNA cannot be repaired (e.g., Bcl2) l. Bcl2 normally stabilizes the mitochondrial membrane, blocking release of cytochrome c. 2. Disruption of Bcl2 allows cytochrome c to leave the mitochondria and activate apoptosis. B. Bcl2 is overexpressed in follicular lymphoma. 1. t(l4;18) moves Bcl2 (chromosome 18) to the lg heavy chain locus (chromosome 14), resulting in increased Bcl2. 2. Mitochondrial membrane is further stabilized, prohibiting apoptosis. 3. B cells that would normally undergo apoptosis during somatic hypermutation in the lymph node germinal center accumulate, leading to lymphoma.

1	3. B cells that would normally undergo apoptosis during somatic hypermutation in the lymph node germinal center accumulate, leading to lymphoma. Fig. 3.1 Retinoblastoma. (Courtesy of Jerome Fig. 3.2 Carcinoma involving lymph node. Fig. 3.3 Seeding of the omentum by carcinoma. Taxy, MD) (Courtesy of Jerome Taxy, MD) V. OTHER IMPORTANT FEATURES OF TUMOR DEVELOPMENT A. Telomerase is necessary for cell immortality. 1. Normally, telomeres shorten with serial cell divisions, eventually resulting in cellular senescence. 2. Cancers often have upregulated telomerase, which preserves telomeres. B. Angiogenesis (production of new blood vessels) is necessary for tumor survival and growth. 1. FGF and VEGF (angiogenic factors) are commonly produced by tumor cells. C. Avoiding immune surveillance is necessary for tumor survival. 1. Mutations often result in production of abnormal proteins, which are expressed on MHC class I. 2. CDS+ T cells detect and destroy such mutated cells. 3.

1	1. Mutations often result in production of abnormal proteins, which are expressed on MHC class I. 2. CDS+ T cells detect and destroy such mutated cells. 3. Tumor cells can evade immune surveillance by downregulating expression of MHC class I. 4. Immunodeficiency (both primary and secondary) increases risk for cancer. I. TUMOR INVASION AND SPREAD A. Accumulation of mutations eventually results in tumor invasion and spread. 1. Epithelial tumor cells are normally attached to one another by cellular adhesion molecules (e.g., E-cadherin). 2. Downregulation of E-cadherin leads to dissociation of attached cells. 3. Cells attach to laminin and destroy basement membrane (collagen type IV) via collagenase. 4. Cells attach to fibronectin in the extracellular matrix and spread locally. 5. Entrance into vascular or lymphatic spaces allows for metastasis (distant spread). II. ROUTES OF METASTASIS A. Lymphatic spread is characteristic of carcinomas.

1	5. Entrance into vascular or lymphatic spaces allows for metastasis (distant spread). II. ROUTES OF METASTASIS A. Lymphatic spread is characteristic of carcinomas. 1. Initial spread is to regional draining lymph nodes (Fig. 3.2). B. Hematogenous spread is characteristic of sarcomas and some carcinomas. 1. 2. 3. Follicular carcinoma of the thyroid 4. C. Seeding of body cavities is characteristic of ovarian carcinoma, which often involves the peritoneum ('omental caking', Fig. 3.3). Fig. 3.4 Histologic features of neoplasia. A, Well differentiated, follicular adenoma of thyroid. B, Fig. 3.5 lmmunohistochemical stain for keratin Poorly differentiated, anaplastic carcinoma of thyroid. marking epithelial cells (in brown). I. CLINICAL FEATURES A. Benign tumors tend to be slow growing, well circumscribed, distinct, and mobile. B. Malignant tumors are usually rapid growing, poorly circumscribed, infiltrative, and fixed to surrounding tissues and local structures.

1	B. Malignant tumors are usually rapid growing, poorly circumscribed, infiltrative, and fixed to surrounding tissues and local structures. C. Biopsy or excision is generally required before a tumor can be classified as benign or malignant with certainty. 1. Some benign tumors can grow in a malignant-like fashion, and some malignant tumors can grow in a benign-like fashion. A. Benign tumors are usually well differentiated (Fig. 3.4A). Characteristics include 1. 2. 3. Low nuclear to cytoplasmic ratio 4. 5. Lack of invasion (of basement membrane or local tissue) 6. B. Malignant tumors are classically poorly differentiated (anaplastic, Fig. 3.4B). Characteristics include 1. Disorganized growth (loss of polarity) 2. 3. High nuclear to cytoplasmic ratio 4. High mitotic activity with atypical mitosis 5. C. Metastatic potential is the hallmark of malignancy-benign tumors never metastasize. Table 3.4: Common lmmunohistochemical Stains and Target Cell Types

1	C. Metastatic potential is the hallmark of malignancy-benign tumors never metastasize. Table 3.4: Common lmmunohistochemical Stains and Target Cell Types Neuroendocrine cells (e.g., small cell Melanoma, Schwannoma and Langerhans cell histiocytosis IBll, ______________ F_U_N_D_A_M_E_N_T_A_L_S_O_F_P_A_T_H_O_L_O_G_Y_______ D. Immunohistochemistry is used to characterize tumors that are difficult to classify on histology (Fig. 3.5, Table 3.4). III. SERUM TUMOR MARKERS A. Proteins released by tumor into serum (e.g., PSA) B. Useful for screening, monitoring response to treatment, and monitoring recurrence C. Elevated levels require tissue biopsy for diagnosis of carcinoma (e.g., biopsy of prostate with elevated PSA). IV. A. Microscopic assessment of differentiation (i.e., how much a cancer resembles the tissue in which it grows); takes into account architectural and nuclear features 1. 2.

1	IV. A. Microscopic assessment of differentiation (i.e., how much a cancer resembles the tissue in which it grows); takes into account architectural and nuclear features 1. 2. B. Important for determining prognosis; well-differentiated cancers have better prognosis than poorly-differentiated cancers. V. A. Assessment of size and spread of a cancer B. Key prognostic factor; more important than grade C. Determined after final surgical resection ofthe tumor D. Utilizes TNM staging system 1. T-tumor (size and/or depth of invasion) 2. N-spread to regional lymph nodes; second most important prognostic factor 3. I. HEMOSTASIS A. Integrity of the blood vessel is necessary to carry blood to tissues. 1. Damage to the wall is repaired by hemostasis, which involves formation of a thrombus (clot) at the site of vessel injury. B. Hemostasis occurs in two stages: primary and secondary. 1.

1	1. Damage to the wall is repaired by hemostasis, which involves formation of a thrombus (clot) at the site of vessel injury. B. Hemostasis occurs in two stages: primary and secondary. 1. Primary hemostasis forms a weak platelet plug and is mediated by interaction between platelets and the vessel wall. 2. Secondary hemostasis stabilizes the platelet plug and is mediated by the coagulation cascade. I. PRIMARY HEMOSTASIS A. Step 1-Transient vasoconstriction of damaged vessel 1. Mediated by reflex neural stimulation and endothelin release from endothelial cells B. Step 2-Platelet adhesion to the surface of disrupted vessel 1. Von Willebrand factor (vWF) binds exposed subendothelial collagen. 2. Platelets bind vWF using the GPib receptor. 3. vWF is derived from the Weibel-Palade bodies of endothelial cells and a-granules of platelets. C. Step 3-Platelet degranulation 1. Adhesion induces shape change in platelets and degranulation with release of multiple mediators.

1	C. Step 3-Platelet degranulation 1. Adhesion induces shape change in platelets and degranulation with release of multiple mediators. i. ADP is released from platelet dense granules; promotes exposure of GPilb/ Illa receptor on platelets. 11. TXA2 is synthesized by platelet cyclooxygenase (COX) and released; promotes platelet aggregation D. Step 4-Platelet aggregation 1. Platelets aggregate at the site of injury via GPIIb/llla using fibrinogen (from plasma) as a linking molecule; results in formation of platelet plug 2. Platelet plug is weak; coagulation cascade (secondary hemostasis) stabilizes it. II. DISORDERS OF PRIMARY HEMOSTASIS A. Usually due to abnormalities in platelets; divided into quantitative or qualitative disorders B. Clinical features include mucosa! and skin bleeding. 1. Symptoms of mucosa! bleeding include epistaxis (most common overall symptom), hemoptysis, GI bleeding, hematuria, and menorrhagia. Intracranial bleeding occurs with severe thrombocytopenia.

1	1. Symptoms of mucosa! bleeding include epistaxis (most common overall symptom), hemoptysis, GI bleeding, hematuria, and menorrhagia. Intracranial bleeding occurs with severe thrombocytopenia. 2. Symptoms of skin bleeding include petechiae (1-2 mm, Fig. 4.1), purpura (> 3 mm), ecchymoses (> 1 cm), and easy bruising; petechiae are a sign of thrombocytopenia and are not usually seen with qualitative disorders. pathoma.com C. Useful laboratory studies include 1. Platelet count-normal 150-400 K/µL; < 50 K/µL leads to symptoms. 2. Bleeding time-normal 2-7 minutes; prolonged with quantitative and qualitative platelet disorders 3. Blood smear-used to assess number and size of platelets 4. Bone marrow biopsy-used to assess megakaryocytes, which produce platelets III. IMMUNE THROMBOCYTOPENIC PURPURA (ITP) A. Autoimmune production oflgG against platelet antigens (e.g., GPIIb/IIIa) 1. Most common cause of thrombocytopenia in children and adults

1	III. IMMUNE THROMBOCYTOPENIC PURPURA (ITP) A. Autoimmune production oflgG against platelet antigens (e.g., GPIIb/IIIa) 1. Most common cause of thrombocytopenia in children and adults B. Autoantibodies are produced by plasma cells in the spleen. C. Antibody-bound platelets are consumed by splenic macrophages, resulting in thrombocytopenia. D. Divided into acute and chronic forms 1. Acute form arises in children weeks after a viral infection or immunization; selflimited, usually resolving within weeks of presentation 2. Chronic form arises in adults, usually women of childbearing age. May be primary or secondary (e.g., SLE). May cause short-lived thrombocytopenia in offspring since antiplatelet IgG can cross the placenta. E. Laboratory findings include 1. ..J.. platelet count, often < 50 K/µL 2. Normal PT/PTT-Coagulation factors are not affected. 3. F. Initial treatment is corticosteroids. Children respond well; adults may show early response, but often relapse. 1.

1	Normal PT/PTT-Coagulation factors are not affected. 3. F. Initial treatment is corticosteroids. Children respond well; adults may show early response, but often relapse. 1. IVIG is used to raise the platelet count in symptomatic bleeding, but its effect is short-lived. 2. Splenectomy eliminates the primary source of antibody and the site of platelet destruction (performed in refractory cases). IV. MICROANGIOPATHIC HEMOLYTIC ANEMIA A. Pathologic formation of platelet microthrombi in small vessels 1. Platelets are consumed in the formation of microthrombi. 2. RBCs are "sheared" as they cross microthrombi, resulting in hemolytic anemia with schistocytes (Fig. 4.2). B. Seen in thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) C. TTP is due to decreased ADAMTS13, an enzyme that normally cleaves vWF multimers into smaller monomers for eventual degradation. Fig. 4.1 Petechiae involving skin. Fig. 4.2 Schistocyte. 1.

1	C. TTP is due to decreased ADAMTS13, an enzyme that normally cleaves vWF multimers into smaller monomers for eventual degradation. Fig. 4.1 Petechiae involving skin. Fig. 4.2 Schistocyte. 1. Large, uncleaved multimers lead to abnormal platelet adhesion, resulting in microthrombi. 2. Decreased ADAMTS13 is usually due to an acquired autoantibody; most commonly seen in adult females D. HUS is due to endothelial damage by drugs or infection. 1. Classically seen in children with E coli 0157:H? dysentery, which results from exposure to undercooked beef 2. E coli verotoxin damages endothelial cells resulting in platelet microthrombi. E. Clinical findings (HUS and TTP) include 1. 2. 3. 4. Renal insufficiency (more common in HUS)-Thrombi involve vessels of the kidney. 5. CNS abnormalities (more common in TTP)-Thrombi involve vessels of the CNS. F. Laboratory findings include 1. Thrombocytopenia with t bleeding time 2. Normal PT/PTT (coagulation cascade is not activated) 3.

1	F. Laboratory findings include 1. Thrombocytopenia with t bleeding time 2. Normal PT/PTT (coagulation cascade is not activated) 3. Anemia with schistocytes 4. G. Treatment involves plasmapheresis and corticosteroids, particularly in TTP. V. QUALITATIVE PLATELET DISORDERS A. Bernard-Soulier syndrome is due to a genetic GPib deficiency; platelet adhesion is impaired. 1. Blood smear shows mild thrombocytopenia with enlarged platelets. B. Glanzmann thrombasthenia is due to a genetic GPIIb/IIIa deficiency; platelet aggregation is impaired. C. Aspirin irreversibly inactivates cyclooxygenase; lack of TXA impairs aggregation. D. Uremia disrupts platelet function; both adhesion and aggregation are impaired. I. SECONDARY HEMOSTASIS A. Stabilizes the weak platelet plug via the coagulation cascade 1. Coagulation cascade generates thrombin, which converts fibrinogen in the platelet plug to fibrin. 2. Fibrin is then cross-linked, yielding a stable platelet-fibrin thrombus.

1	Coagulation cascade generates thrombin, which converts fibrinogen in the platelet plug to fibrin. 2. Fibrin is then cross-linked, yielding a stable platelet-fibrin thrombus. B. Factors of the coagulation cascade are produced by the liver in an inactive state. Activation requires 1. Exposure to an activating substance i. Tissue thromboplastin activates factor VII (extrinsic pathway). ii. Subendothelial collagen activates factor XII (intrinsic pathway). 2. Phospholipid surface of platelets 3. II. DISORDERS OF SECONDARY HEMOSTASIS A. Usually due to factor abnormalities B. Clinical features include deep tissue bleeding into muscles and joints (hemarthrosis) and rebleeding after surgical procedures (e.g., circumcision and wisdom tooth extraction). C. Laboratory studies include 1. Prothrombin time (PT)-measures extrinsic (factor VII) and common (factors II, V, X, and fibrinogen) pathways of the coagulation cascade 2.

1	C. Laboratory studies include 1. Prothrombin time (PT)-measures extrinsic (factor VII) and common (factors II, V, X, and fibrinogen) pathways of the coagulation cascade 2. Partial thromboplastin time (PTT)-measures intrinsic (factors XII, XI, IX, VIII) and common (factors II, V, X, and fibrinogen) pathways of the coagulation cascade III. HEMOPHILIA A A. Genetic factor VIII (FVIII) deficiency 1. 2. B. Presents with deep tissue, joint, and postsurgical bleeding 1. Clinical severity depends on the degree of deficiency. C. Laboratory findings include 1. 2. ,I, FVIII 3. D. Treatment involves recombinant FVIII. IV. A. Genetic factor IX deficiency 1. Resembles hemophilia A, except FIX levels are decreased instead of FVIII V. A. Acquired antibody against a coagulation factor resulting in impaired factor function; anti-FVIII is most common. 1. Clinical and lab findings are similar to hemophilia A. 2.

1	V. A. Acquired antibody against a coagulation factor resulting in impaired factor function; anti-FVIII is most common. 1. Clinical and lab findings are similar to hemophilia A. 2. PTT does not correct upon mixing normal plasma with patient's plasma (mixing study) due to inhibitor; PTT does correct in hemophilia A. VI. VON WILLEBRAND DISEASE A. Genetic vWF deficiency 1. Most common inherited coagulation disorder B. Multiple subtypes exist, causing quantitative and qualitative defects; the most common type is autosomal dominant with decreased vWF levels. C. Presents with mild mucosa! and skin bleeding; low vWF impairs platelet adhesion. D. Laboratory findings include 1. 2. t PTT; normal PT-Decreased FVIII half-life (vWF normally stabilizes FVIII); however, deep tissue, joint, and postsurgical bleeding are usually not seen. 3.

1	D. Laboratory findings include 1. 2. t PTT; normal PT-Decreased FVIII half-life (vWF normally stabilizes FVIII); however, deep tissue, joint, and postsurgical bleeding are usually not seen. 3. Abnormal ristocetin test-Ristocetin induces platelet agglutination by causing vWF to bind platelet GPib; lack of vWF ➔ impaired agglutination ➔ abnormal test. E. Treatment is desmopressin (ADH analog), which increases vWF release from Weibel-Palade bodies of endothelial cells. VII. VITAMIN K DEFICIENCY A. Disrupts function of multiple coagulation factors 1. Vitamin K is activated by epoxide reductase in the liver. 2. Activated vitamin K gamma carboxylates factors II, VII, IX, X, and proteins C and S; gamma carboxylation is necessary for factor function. B. Deficiency occurs in 1. Newborns-due to lack of GI colonization by bacteria that normally synthesize vitamin K; vitamin K injection is given prophylactically to all newborns at birth to prevent hemorrhagic disease of the newborn.

1	2. Long-term antibiotic therapy-disrupts vitamin K-producing bacteria in the GI tract 3. Malabsorption-leads to deficiency of fat-soluble vitamins, including vitamin K VIII. OTHER CAUSES OF ABNORMAL SECONDARY HEMOSTASIS A. Liver failure-decreased production of coagulation factors and decreased activation ofvitamin K by epoxide reductase; effect of liver failure on coagulation is followed using PT. B. Large-volume transfusion-dilutes coagulation factors, resulting in a relative deficiency I. HEPARIN-INDUCED THROMBOCYTOPENIA A. Platelet destruction that arises secondary to heparin therapy B. Fragments of destroyed platelets may activate remaining platelets, leading to thrombosis. II. DISSEMINATED INTRAVASCULAR COAGULATION A. Pathologic activation of the coagulation cascade 1. Widespread microthrombi result in ischemia and infarction. 2. Consumption of platelets and factors results in bleeding, especially from IV sites and mucosa! surfaces (bleeding from body orifices).

1	Widespread microthrombi result in ischemia and infarction. 2. Consumption of platelets and factors results in bleeding, especially from IV sites and mucosa! surfaces (bleeding from body orifices). B. Almost always secondary to another disease process 1. Obstetric complications-Tissue thromboplastin in the amniotic fluid activates coagulation. 2. Sepsis (especially with E Coli or N meningitidis)-Endotoxins from the bacterial wall and cytokines (e.g., TNF and IL-1) induce endothelial cells to make tissue factor. 3. Adenocarcinoma-Mucin activates coagulation. 4. Acute promyelocytic leukemia-Primary granules activate coagulation. 5. Rattlesnake bite-Venom activates coagulation. C. Laboratory findings include 1. j,. platelet count 2. 3. j,. fibrinogen 4. 5. Elevated fibrin split products, particularly D-dimer i. Elevated D-dimer is the best screening test for DIC. ii. Derived from splitting of cross-linked fibrin; D-dimer is not produced from splitting of fibrinogen.

1	ii. Derived from splitting of cross-linked fibrin; D-dimer is not produced from splitting of fibrinogen. D. Treatment involves addressing the underlying cause and transfusing blood products and cryoprecipitate (contains coagulation factors), as necessary. III. DISORDERS OF FIBRINOLYSIS A. Normal fibrinolysis removes thrombus after damaged vessel heals. 1. Tissue plasminogen activator (tPA) converts plasminogen to plasmin. 2. Plasmin cleaves fibrin and serum fibrinogen, destroys coagulation factors, and blocks platelet aggregation. 3. a2-antiplasmin inactivates plasmin. B. Disorders of fibrinolysis are due to plasmin overactivity resulting in excessive cleavage of serum fibrinogen. Examples include 1. Radical prostatectomy-Release of urokinase activates plasmin. 2. Cirrhosis ofliver-reduced production of a2-antiplasmin C. Presents with increased bleeding (resembles DIC) D. Laboratory findings include 1. t PT/PTT-Plasmin destroys coagulation factors. 2.

1	2. Cirrhosis ofliver-reduced production of a2-antiplasmin C. Presents with increased bleeding (resembles DIC) D. Laboratory findings include 1. t PT/PTT-Plasmin destroys coagulation factors. 2. t bleeding time with normal platelet count-Plasmin blocks platelet aggregation. 3. Increased fibrinogen split products without D-dimers-Serum fibrinogen is lysed; however, D-dimers are not formed because fibrin thrombi are absent. E. Treatment is aminocaproic acid, which blocks activation of plasminogen. I. BASIC PRINCIPLES A. Pathologic formation of an intravascular blood clot (thrombus) 1. 2. Most common location is the deep veins (DVT) of the leg below the knee. B. Characterized by (1) lines of Zahn (alternating layers of platelets/fibrin and RBCs, Fig. 4.3) and (2) attachment to vessel wall 1. Both features distinguish thrombus from postmortem clot.

1	B. Characterized by (1) lines of Zahn (alternating layers of platelets/fibrin and RBCs, Fig. 4.3) and (2) attachment to vessel wall 1. Both features distinguish thrombus from postmortem clot. C. Three major risk factors for thrombosis are disruption in blood flow, endothelial cell damage, and hypercoagulable state (Virchow triad). II. DISRUPTION IN NORMAL BLOOD FLOW A. Stasis and turbulence of blood flow increases risk for thrombosis. 1. Blood flow is normally continuous and laminar; keeps platelets and factors dispersed and inactivated B. Examples include 1. 2. Cardiac wall dysfunction (e.g., arrhythmia or myocardial infarction) 3. III. ENDOTHELIAL CELL DAMAGE A. Endothelial damage disrupts the protective function of endothelial cells, increasing the risk for thrombosis. B. Endothelial cells prevent thrombosis by several mechanisms. 1. Block exposure to subendothelial collagen and underlying tissue factor 2.

1	B. Endothelial cells prevent thrombosis by several mechanisms. 1. Block exposure to subendothelial collagen and underlying tissue factor 2. Produce prostacyclin (PGI ) and NO-vasodilation and inhibition of platelet Fig, 4.3 Lines of Zahn characterized by alternating layers of platelets/fibrin and RBCs. 3. Secrete heparin-like molecules-augment antithrombin III (ATIII), which inactivates thrombin and coagulation factors 4. Secrete tissue plasminogen activator (tPA)-converts plasminogen to plasmin, which (1) cleaves fibrin and serum fibrinogen, (2) destroys coagulation factors, and (3) blocks platelet aggregation 5. Secrete thrombomodulin-redirects thrombin to activate protein C, which inactivates factors V and VIII C. Causes of endothelial cell damage include atherosclerosis, vasculitis, and high levels of homocysteine. 1. Vitamin Bl2 and folate deficiency result in mildly elevated homocysteine levels, increasing the risk for thrombosis.

1	1. Vitamin Bl2 and folate deficiency result in mildly elevated homocysteine levels, increasing the risk for thrombosis. i. Folic acid (tetrahydrofolate, THF) circulates as methyl-THF in the serum. 11. Methyl is transferred to cobalamin (vitamin Bl2), allowing THF to participate in the synthesis of DNA precursors. iii. Cobalamin transfers methyl to homocysteine resulting in methionine. iv. Lack ofvitamin Bl2 or folate leads to decreased conversion of homocysteine to methionine resulting in buildup of homocysteine. 2. Cystathionine beta synthase (CBS) deficiency results in high homocysteine levels with homocystinuria. 1. CBS converts homocysteine to cystathionine; enzyme deficiency leads to homocysteine buildup. ii. Characterized by vessel thrombosis, mental retardation, lens dislocation, and long slender fingers. A. Due to excessive procoagulant proteins or defective anticoagulant proteins; may be inherited or acquired

1	A. Due to excessive procoagulant proteins or defective anticoagulant proteins; may be inherited or acquired B. Classic presentation is recurrent DVTs or DVT at a young age. 1. Usually occurs in the deep veins of the leg; other sites include hepatic and cerebral veins. C. Protein C or S deficiency (autosomal dominant) decreases negative feedback on the coagulation cascade. 1. Proteins C and S normally inactivate factors V and VIII. 2. i. Initial stage of warfarin therapy results in a temporary deficiency of proteins C and S (due to shorter half-life) relative to factors II, VII, IX, and X ii. In preexisting C or S deficiency, a severe deficiency is seen at the onset of warfarin therapy increasing risk for thrombosis, especially in the skin. D. Factor V Leiden is a mutated form of factor V that lacks the cleavage site for deactivation by proteins C and S. 1. Most common inherited cause of hypercoagulable state

1	D. Factor V Leiden is a mutated form of factor V that lacks the cleavage site for deactivation by proteins C and S. 1. Most common inherited cause of hypercoagulable state E. Prothrombin 20210A is an inherited point mutation in prothrombin that results in increased gene expression. 1. Increased prothrombin results in increased thrombin, promoting thrombus formation. F. A TIII deficiency decreases the protective effect of heparin-like molecules produced by the endothelium, increasing the risk for thrombus. 1. Heparin-like molecules normally activate ATIII, which inactivates thrombin and coagulation factors. 2. In ATIII deficiency, PTT does not rise with standard heparin dosing. 1. Pharmacologic heparin works by binding and activating ATIII. 11. High doses of heparin activate limited ATIII; coumadin is then given to maintain an anticoagulated state. I. II. G. Oral contraceptives are associated with a hypercoagulable state.

1	11. High doses of heparin activate limited ATIII; coumadin is then given to maintain an anticoagulated state. I. II. G. Oral contraceptives are associated with a hypercoagulable state. 1. Estrogen induces increased production of coagulation factors, thereby increasing the risk for thrombosis. A. Intravascular mass that travels and occludes downstream vessels; symptoms depend on the vessel involved. B. Thromboembolus is due to a thrombus that dislodges; most common type of embolus (>95%) C. Atherosclerotic embolus is due to an atherosclerotic plaque that dislodges. 1. Characterized by the presence of cholesterol clefts in the embolus (Fig. 4.4A) D. Fat embolus is associated with bone fractures, particularly long bones, and soft tissue trauma. 1. Develops while fracture is still present or shortly after repair 2. Characterized by dyspnea (fat, often with bone marrow elements, is seen in pulmonary vessels, Fig. 4.4B) and petechiae on the skin overlying the chest

1	Characterized by dyspnea (fat, often with bone marrow elements, is seen in pulmonary vessels, Fig. 4.4B) and petechiae on the skin overlying the chest E. Gas embolus is classically seen in decompression sickness. 1. Nitrogen gas precipitates out of blood due to rapid ascent by a diver. 2. Presents with joint and muscle pain ('bends') and respiratory symptoms ('chokes'). 3. Chronic form (Caisson disease) is characterized by multifocal ischemic necrosis of bone. 4. Gas embolus may also occur during laparoscopic surgery (air is pumped into the abdomen). F. Amniotic fluid embolus enters maternal circulation during labor or delivery 1. Presents with shortness of breath, neurologic symptoms, and DIC (due to the thrombogenic nature of amniotic fluid) 2. Characterized by squamous cells and keratin debris, from fetal skin, in embolus (Fig. 4.4C)

1	Characterized by squamous cells and keratin debris, from fetal skin, in embolus (Fig. 4.4C) A. Usually due to thromboembolus; the most common source is deep venous thrombus (DVT) of the lower extremity, usually involving the femoral, iliac, or popliteal veins. B. Most often clinically silent because (1) the lung has a dual blood supply (via pulmonary and bronchial arteries) and (2) the embolus is usually small (selfresolves) Fig. 4.4 Emboli. A, Atherosclerotic em bolus characterized by cholesterol clefts. B, Fat embolus with bone marrow elements. C, Amniotic fluid em bolus with squamous cells and keratin debris from fetal skin. (A, Courtesy of Professor A. Garfia. C, Courtesy of Shakuntala Teas, MD) C. Pulmonary infarction occurs if a large-or medium-sized artery is obstructed in patients with pre-existing cardiopulmonary compromise; only 10% of PEs cause infarction. 1. Presents with shortness of breath, hemoptysis, pleuritic chest pain, and pleural effusion 2.

1	1. Presents with shortness of breath, hemoptysis, pleuritic chest pain, and pleural effusion 2. V/Q lung scan shows mismatch; perfusion is abnormal. 3. Spiral CT shows a vascular filling defect in the lung. 4. Lower extremity Doppler ultrasound is useful to detect DVT. 5. D-dimer is elevated. 6. Gross examination reveals a hemorrhagic, wedge-shaped infarct. D. Sudden death occurs with a large saddle embolus that blocks both left and right pulmonary arteries or with significant occlusion of a large pulmonary artery (Fig. 4.5); death is due to electromechanical dissociation. E. Pulmonary hypertension may arise with chronic emboli that are reorganized over time. III. SYSTEMIC EMBOLISM A. Usually due to thromboembolus B. Most commonly arise in the left heart C. Travel down systemic circulation to occlude flow to organs, most commonly the lower extremities Fig. 4.5 Saddle em bolus involving pulmonary artery. (Courtesy of Yale Rosen, MD) I. BASIC PRINCIPLES

1	Fig. 4.5 Saddle em bolus involving pulmonary artery. (Courtesy of Yale Rosen, MD) I. BASIC PRINCIPLES A. Reduction in circulating red blood cell (RBC) mass B. Presents with signs and symptoms of hypoxia 1. Weakness, fatigue, and dyspnea 2. 3. 4. Angina, especially with preexisting coronary artery disease C. Hemoglobin (Hb), hematocrit (Hct), and RBC count are used as surrogates for RBC mass, which is difficult to measure. 1. Anemia is defined as Hb < 13.5 g/dL in males and < 12.5 g/dL in females (normal Hb is 13.5-17.5 g/dL in males and 12.5-16.0 g/dL in females). D. Based on mean corpuscular volume (MCV), anemia can be classified as microcytic (MCV < 80 µm3), normocytic (MCV = 80-100 µm 3), or macrocytic (MCV > 100 µm 3). I. BASIC PRINCIPLES A. Anemia with MCV < 80 µm 3 B. Microcytic anemias are due to decreased production of hemoglobin. 1.

1	I. BASIC PRINCIPLES A. Anemia with MCV < 80 µm 3 B. Microcytic anemias are due to decreased production of hemoglobin. 1. RBC progenitor cells in the bone marrow are large and normally divide multiple times to produce smaller mature cells (MCV = 80-100 µm 3). 2. Microcytosis is due to an "extra" division which occurs to maintain hemoglobin concentration. C. Hemoglobin is made of heme and globin; heme is composed of iron and protoporphyrin. A decrease in any of these components leads to microcytic anemia. D. Microcytic anemias include (1) iron deficiency anemia, (2) anemia of chronic disease, (3) sideroblastic anemia, and (4) thalassemia. II. IRON DEFICIENCY ANEMIA A. Due to decreased levels of iron 1. ..I.-iron ➔ ..I.-heme ➔ ..I.-hemoglobin ➔ microcytic anemia B. Most common type of anemia 1. Lack of iron is the most common nutritional deficiency in the world, affecting roughly 1/3 of world's population.

1	B. Most common type of anemia 1. Lack of iron is the most common nutritional deficiency in the world, affecting roughly 1/3 of world's population. C. Iron is consumed in heme (meat-derived) and non-heme (vegetable-derived) forms. 1. Absorption occurs in the duodenum. Enterocytes have heme and non-heme (DMTl) transporters; the heme form is more readily absorbed. 2. Enterocytes transport iron across the cell membrane into blood via ferroportin. 3. Transferrin transports iron in the blood and delivers it to liver and bone marrow macrophages for storage. 4. Stored intracellular iron is bound to ferritin, which prevents iron from forming free radicals via the Fenton reaction. pathoma.com D. Laboratory measurements of iron status 1. Serum iron-measure of iron in the blood 2. Total iron-binding capacity (TIBC)-measure of transferrin molecules in the blood 3. % saturation-percentage of transferrin molecules that are bound by iron (normal is 33%) 4.

1	Total iron-binding capacity (TIBC)-measure of transferrin molecules in the blood 3. % saturation-percentage of transferrin molecules that are bound by iron (normal is 33%) 4. Serum ferritin-reflects iron stores in macrophages and the liver E. Iron deficiency is usually caused by dietary lack or blood loss. 1. Infants-breast-feeding (human milk is low in iron) 2. 3. 4. Elderly-colon polyps/carcinoma in the Western world; hookworm (Ancylostoma duodenale and Necator americanus) in the developing world 5. Other causes include malnutrition, malabsorption, and gastrectomy (acid aids iron absorption by maintaining the Fe2+ state, which is more readily absorbed than Fe3+). F. Stages of iron deficiency 1. Storage iron is depleted--J.. ferritin; t TIBC 2. Serum iron is depleted--J.. serum iron; -J.. % saturation 3. Normocytic anemia-Bone marrow makes fewer, but normal-sized, RBCs. 4. Microcytic, hypochromic anemia-Bone marrow makes smaller and fewer RBCs.

1	Normocytic anemia-Bone marrow makes fewer, but normal-sized, RBCs. 4. Microcytic, hypochromic anemia-Bone marrow makes smaller and fewer RBCs. G. Clinical features of iron deficiency include anemia, koilonychia, and pica. H. Laboratory findings include 1. Microcytic, hypochromic RBCs with t red cell distribution width (RDW, Fig. 5.1) 2. -J.. ferritin; t TIBC; -J.. serum iron; -J.. % saturation 3. I. Treatment involves supplemental iron (ferrous sulfate). J. Plummer-Vinson syndrome is iron deficiency anemia with esophageal web and atrophic glossitis; presents as anemia, dysphagia, and beefy-red tongue III. ANEMIA OF CHRONIC DISEASE A. Anemia associated with chronic inflammation (e.g., endocarditis or autoimmune conditions) or cancer; most common type of anemia in hospitalized patients B. Chronic disease results in production of acute phase reactants from the liver, including hepcidin.

1	B. Chronic disease results in production of acute phase reactants from the liver, including hepcidin. 1. Hepcidin sequesters iron in storage sites by (1) limiting iron transfer from macrophages to erythroid precursors and (2) suppressing erythropoietin (EPO) Fig. 5.1 Microcytic, hypochromic RBCs of iron Fig. 5.2 Ringed sideroblasts (Prussian blue stain). deficiency anemia. production; aim is to prevent bacteria from accessing iron, which is necessary for their survival. 2. ..l.-available iron ➔ ..l.-heme ➔ ..l.-hemoglobin ➔ microcytic anemia C. Laboratory findings include 1. t ferritin, ..l.-TIBC, ..l.-serum iron, and ..l.-% saturation 2. D. Treatment involves addressing the underlying cause. IV. A. Anemia due to defective protoporphyrin synthesis 1. ..l.-protoporphyrin ➔ ..l.-heme ➔ ..l.-hemoglobin ➔ microcytic anemia B. Protoporphyrin is synthesized via a series of reactions. 1.

1	IV. A. Anemia due to defective protoporphyrin synthesis 1. ..l.-protoporphyrin ➔ ..l.-heme ➔ ..l.-hemoglobin ➔ microcytic anemia B. Protoporphyrin is synthesized via a series of reactions. 1. Aminolevulinic acid synthetase (ALAS) converts succinyl CoA to aminolevulinic acid (ALA) using vitamin B6 as a cofactor (rate-limiting step). 2. Aminolevulinic acid dehydratase (ALAD) converts ALA to porphobilinogen. 3. Additional reactions convert porphobilinogen to protoporphyrin. 4. Ferrochelatase attaches protoporphyrin to iron to make heme (final reaction; occurs in the mitochondria). C. Iron is transferred to erythroid precursors and enters the mitochondria to form heme. If protoporphyrin is deficient, iron remains trapped in mitochondria. 1. Iron-laden mitochondria form a ring around the nucleus of erythroid precursors; these cells are called ringed sideroblasts (hence, the term sideroblastic anemia, Fig. 5.2). D. Sideroblastic anemia can be congenital or acquired. 1.

1	D. Sideroblastic anemia can be congenital or acquired. 1. Congenital defect most commonly involves ALAS (rate-limiting enzyme). 2. 1. Alcoholism-mitochondrial poison ii. Lead poisoning-inhibits ALAD and ferrochelatase iii. Vitamin B6 deficiency-required cofactor for ALAS; most commonly seen as a side effect of isoniazid treatment for tuberculosis E. Laboratory findings include t ferritin, -1, TIBC, t serum iron, and t % saturation (iron-overloaded state). V. A. Anemia due to decreased synthesis of the globin chains of hemoglobin 1. -1, globin ➔ -1, hemoglobin ➔ microcytic anemia B. Inherited mutation; carriers are protected against Plasmodium falciparum malaria. C. Divided into a-and P-thalassemia based on decreased production of alpha or beta globin chains. 1. Normal types of hemoglobin are HbF (a2y), HbA (a2p), and HbA2 (a/\). Table 5.1: Laboratory Findings in Microcytic Anemia Anemia of Chronic Disease High Low Low Low D. E.

1	1. Normal types of hemoglobin are HbF (a2y), HbA (a2p), and HbA2 (a/\). Table 5.1: Laboratory Findings in Microcytic Anemia Anemia of Chronic Disease High Low Low Low D. E. a-Thalassemia is usually due to gene deletion; normally, 4 alpha genes are present on chromosome 16. 1. 2. Two genes deleted-mild anemia with t RBC count; cis deletion is associated with an increased risk of severe thalassemia in offspring. i. Cis deletion is when both deletions occur on the same chromosome; seen in Asians ii. Trans deletion is when one deletion occurs on each chromosome; seen in Africans, including African Americans 3. Three genes deleted-severe anemia; p chains form tetramers (HbH) that damage RBCs; HbH is seen on electrophoresis. 4. (Hb Barts) that damage RBCs; Hb Barts is seen on electrophoresis. P-Thalassemia is usually due to gene mutations (point mutations in promoter or splicing sites); seen in individuals of African and Mediterranean descent 1.

1	Two p genes are present on chromosome 11; mutations result in absent (P0) or diminished (p+) production of the p-globin chain. 2. P-Thalassemia minor (PIP+) is the mildest form of disease and is usually asymptomatic with an increased RBC count. i. Microcytic, hypochromic RBCs and target cells are seen on blood smear (Fig. 5.3). ii. Hemoglobin electrophoresis shows slightly decreased HbA with increased HbA (5%, normal 2.5%) and HbF (2%, normal 1%). 3. P-Thalassemia major (P 0!pa) is the most severe form of disease and presents with severe anemia a few months after birth; high HbF (a2y) at birth is temporarily protective. i. Unpaired a chains precipitate and damage RBC membrane, resulting in ineffective erythropoiesis and extravascular hemolysis (removal of circulating RBCs by the spleen).

1	i. Unpaired a chains precipitate and damage RBC membrane, resulting in ineffective erythropoiesis and extravascular hemolysis (removal of circulating RBCs by the spleen). ii. Massive erythroid hyperplasia ensues resulting in (1) expansion of hematopoiesis into the skull (reactive bone formation leads to 'crewcut' appearance on x-ray, Fig. 5.4) and facial bones ('chipmunk facies'), (2) extramedullary hematopoiesis with hepatosplenomegaly, and (3) risk of aplastic crisis with parvovirus Bl9 infection of erythroid precursors. iii. Chronic transfusions are often necessary; leads to risk for secondary hemochromatosis iv. Smear shows microcytic, hypochromic RBCs with target cells and nucleated red blood cells. v. Electrophoresis shows HbA and HbF with little or no HbA. Fig. 5.3 Target cells. Fig. 5.4 'Crewcut' appearance. (Reproduced with Fig. 5.5 Hypersegmented neutrophil in permission, www.orthopaedia.com/x/xgGvAQ) macrocytic anemia. I. BASIC PRINCIPLES

1	Fig. 5.3 Target cells. Fig. 5.4 'Crewcut' appearance. (Reproduced with Fig. 5.5 Hypersegmented neutrophil in permission, www.orthopaedia.com/x/xgGvAQ) macrocytic anemia. I. BASIC PRINCIPLES A. Anemia with MCV > 100 µm3; most commonly due to folate or vitamin Bl2 deficiency (megaloblastic anemia) B. Folate and vitamin Bl2 are necessary for synthesis of DNA precursors. 1. Folate circulates in the serum as methyltetrahydrofolate (methyl THF); removal of the methyl group allows for participation in the synthesis of DNA precursors. 2. Methyl group is transferred to vitamin Bl2 (cobalamin). 3. Vitamin Bl2 then transfers it to homocysteine, producing methionine. C. Lack of folate or vitamin Bl2 impairs synthesis of DNA precursors. 1. Impaired division and enlargement of RBC precursors leads to megaloblastic anemia. 2. Impaired division of granulocytic precursors leads to hypersegmented neutrophils. 3.

1	1. Impaired division and enlargement of RBC precursors leads to megaloblastic anemia. 2. Impaired division of granulocytic precursors leads to hypersegmented neutrophils. 3. Megaloblastic change is also seen in rapidly-dividing (e.g., intestinal) epithelial cells. D. Other causes of macrocytic anemia (without megaloblastic change) include alcoholism, liver disease, and drugs (e.g., 5-FU). II. FOLATE DEFICIENCY A. Dietary folate is obtained from green vegetables and some fruits. 1. Absorbed in the jejunum B. Folate deficiency develops within months, as body stores are minimal. C. Causes include poor diet (e.g., alcoholics and elderly), increased demand (e.g., pregnancy, cancer, and hemolytic anemia), and folate antagonists (e.g., methotrexate, which inhibits dihydrofolate reductase). D. Clinical and laboratory findings include 1. Macrocytic RBCs and hypersegmented neutrophils (> 5 lobes, Fig. 5.5) 2. 3. .,J., serum folate 4. 5. Ill. VITAMIN Bl2 DEFICIENCY

1	D. Clinical and laboratory findings include 1. Macrocytic RBCs and hypersegmented neutrophils (> 5 lobes, Fig. 5.5) 2. 3. .,J., serum folate 4. 5. Ill. VITAMIN Bl2 DEFICIENCY A. Dietary vitamin Bl2 is complexed to animal-derived proteins. 1. Salivary gland enzymes (e.g., amylase) liberate vitamin Bl 2, which is then bound by R-binder (also from the salivary gland) and carried through the stomach. 2. Pancreatic proteases in the duodenum detach vitamin Bl2 from R-binder. 3. Vitamin Bl2 binds intrinsic factor (made by gastric parietal cells) in the small bowel; the intrinsic factor-vitamin Bl2 complex is absorbed in the ileum. B. Vitamin Bl2 deficiency is less common than folate deficiency and takes years to develop due to large hepatic stores of vitamin Bl 2. C. Pernicious anemia is the most common cause of vitamin Bl2 deficiency. 1. Autoimmune destruction of parietal cells (body of stomach) leads to intrinsic factor deficiency

1	C. Pernicious anemia is the most common cause of vitamin Bl2 deficiency. 1. Autoimmune destruction of parietal cells (body of stomach) leads to intrinsic factor deficiency D. Other causes of vitamin Bl2 deficiency include pancreatic insufficiency and damage to the terminal ileum (e.g., Crohn disease or Diphyllobothrium latum [fish tapeworm]); dietary deficiency is rare, except in vegans. E. Clinical and laboratory findings include 1. Macrocytic RBCs with hypersegmented neutrophils 2. 3. Subacute combined degeneration of the spinal cord i. Vitamin B12 is a cofactor for the conversion of methylmalonic acid to succinyl CoA (important in fatty acid metabolism). ii. Vitamin B12 deficiency results in increased levels of methylmalonic acid, which impairs spinal cord myelinization. iii. Damage results in poor proprioception and vibratory sensation (posterior column) and spastic paresis (lateral corticospinal tract). 4. -.J, serum vitamin B12 5.

1	iii. Damage results in poor proprioception and vibratory sensation (posterior column) and spastic paresis (lateral corticospinal tract). 4. -.J, serum vitamin B12 5. t serum homocysteine (similar to folate deficiency), which increases risk for thrombosis 6. I. BASIC PRINCIPLES A. Anemia with normal-sized RBCs (MCV = 80-100 µm3) B. Due to increased peripheral destruction or underproduction 1. Reticulocyte count helps to distinguish between these two etiologies. II. RETICULOCYTES A. Young RBCs released from the bone marrow 1. Identified on blood smear as larger cells with bluish cytoplasm (due to residual RNA, Fig. 5.6) B. Normal reticulocyte count (RC) is 1-2%. 1. RBC lifespan is 120 days; each day roughly 1-2% ofRBCs are removed from circulation and replaced by reticulocytes. C. A properly functioning marrow responds to anemia by increasing the RC to > 3%. D. RC, however, is falsely elevated in anemia.

1	C. A properly functioning marrow responds to anemia by increasing the RC to > 3%. D. RC, however, is falsely elevated in anemia. 1. RC is measured as percentage of total RBCs; decrease in total RBCs falsely elevates percentage of reticulocytes. E. RC is corrected by multiplying reticulocyte count by Hct/45. 1. Corrected count > 3% indicates good marrow response and suggests peripheral destruction. 2. Corrected count < 3% indicates poor marrow response and suggests underproduction. III. PERIPHERAL RBC DESTRUCTION (HEMOLYSIS) A. Divided into extravascular and intravascular hemolysis; both result in anemia with a good marrow response. B. Extravascular hemolysis involves RBC destruction by the reticuloendothelial system (macrophages of the spleen, liver, and lymph nodes). Fig. 5.6 Reticulocyte. Fig. 5.7 Spherocytes. 1. Macrophages consume RBCs and break down hemoglobin. i. Globin is broken down into amino acids.

1	Fig. 5.6 Reticulocyte. Fig. 5.7 Spherocytes. 1. Macrophages consume RBCs and break down hemoglobin. i. Globin is broken down into amino acids. ii. Heme is broken down into iron and protoporphyrin; iron is recycled. 111. Protoporphyrin is broken down into unconjugated bilirubin, which is bound to serum albumin and delivered to the liver for conjugation and excretion into bile. 2. Clinical and laboratory findings include i. Anemia with splenomegaly, jaundice due to unconjugated bilirubin, and increased risk for bilirubin gallstones ii. Marrow hyperplasia with corrected reticulocyte count> 3% C. Intravascular hemolysis involves destruction of RBCs within vessels. 1. Clinical and laboratory findings include 1. Hemoglobinemia 11. Hemoglobinuria iii. Hemosiderinuria-Renal tubular cells pick up some of the hemoglobin that is filtered into the urine and break it down into iron, which accumulates as hemosiderin; tubular cells are eventually shed resulting in hemosiderinuria.

1	iv. Decreased serum haptoglobin I. HEREDITARY SPHEROCYTOSIS A. Inherited defect ofRBC cytoskeleton-membrane tethering proteins 1. Most commonly involves ankyrin, spectrin, or band 3 B. Membrane blebs are formed and lost over time. 1. Loss of membrane renders cells round (spherocytes) instead of disc-shaped. 2. Spherocytes are less able to maneuver through splenic sinusoids and are consumed by splenic macrophages, resulting in anemia. C. Clinical and laboratory findings include 1. Spherocytes with loss of central pallor (Fig. 5.7) 2. 3. Splenomegaly, jaundice with unconjugated bilirubin, and increased risk for bilirubin gallstones (extravascular hemolysis) 4. Increased risk for a plastic crisis with parvovirus Bl9 infection of erythroid precursors D. Diagnosed by osmotic fragility test, which reveals increased spherocyte fragility in hypotonic solution

1	Increased risk for a plastic crisis with parvovirus Bl9 infection of erythroid precursors D. Diagnosed by osmotic fragility test, which reveals increased spherocyte fragility in hypotonic solution E. Treatment is splenectomy; anemia resolves, but spherocytes persist and Howell-Jolly bodies (fragments of nuclear material in RBCs) emerge on blood smear (Fig. 5.8). II. SICKLE CELL ANEMIA A. Autosomal recessive mutation in~ chain of hemoglobin; a single amino acid change replaces normal glutamic acid (hydrophilic) with valine (hydrophobic). B. Gene is carried by 10% of individuals of African descent, likely due to protective role against falciparum malaria. C. Sickle cell disease arises when two abnormal ~ genes are present; results in >90% HbS in RBCs D. HbS polymerizes when deoxygenated; polymers aggregate into needle-like structures, resulting in sickle cells (Fig. 5.9). 1. Increased risk of sickling occurs with hypoxemia, dehydration, and acidosis. 2.

1	1. Increased risk of sickling occurs with hypoxemia, dehydration, and acidosis. 2. HbF protects against sickling; high HbF at birth is protective for the first few months of life. Treatment with hydroxyurea increases levels ofHbF. E. F. G. H. Cells continuously sickle and de-sickle while passing through the microcirculation, resulting in complications related to RBC membrane damage. 1. Extravascular hemolysis-Reticuloendothelial system removes RBCs with damaged membranes, leading to anemia, jaundice with unconjugated hyperbilirubinemia, and increased risk for bilirubin gallstones. 2. Intravascular hemolysis-RBCs with damaged membranes dehydrate, leading to hemolysis with decreased haptoglobin and target cells on blood smear. 3.

1	2. Intravascular hemolysis-RBCs with damaged membranes dehydrate, leading to hemolysis with decreased haptoglobin and target cells on blood smear. 3. i. Expansion of hematopoiesis into the skull ('crewcut' appearance on x-ray) and facia l bones ('chipmunk facies') 11. Extramedullary hematopoiesis with hepatomegaly iii. Risk of aplastic crisis with parvovirus B19 infection of erythroid precursors Extensive sickling leads to complications of vaso-occlusion. 1. Dactylitis-swollen hands and feet due to vaso-occlusive infarcts in bones; common presenting sign in infants 2. Autosplenectomy-shrunken, fibrotic spleen. Consequences include i. Increased risk of infection with encapsulated organisms such as Streptococcus pneumoniae and Haemophilus influenzae (most common cause of death in children); affected children should be vaccinated by 5 years of age. ii. Increased risk of Salmonella paratyphi osteomyelitis iii. Howell-Jolly bodies on blood smear 3.

1	ii. Increased risk of Salmonella paratyphi osteomyelitis iii. Howell-Jolly bodies on blood smear 3. i. Presents with chest pain, shortness of breath, and lung infiltrates ii. Often precipitated by pneumonia iii. Most common cause of death in adult patients 4. 5. Renal papillary necrosis-results in gross hematuria and proteinuria Sickle cell trait is the presence of one mutated and one normal pchain; results in < 50% HbS in RBCs (HbA is slightly more efficiently produced than HbS) 1. Generally asymptomatic with no anemia; RBCs with < 50% HbS do not sickle in vivo except in the renal medulla. 1. Extreme hypoxia and hypertonicity of the medulla cause sickling, which results in microinfarctions leading to microscopic hematuria and, eventually, decreased ability to concentrate urine. 1. Sickle cells and target cells are seen on blood smear in sickle cell disease, but not in sickle cell trait. 2.

1	1. Sickle cells and target cells are seen on blood smear in sickle cell disease, but not in sickle cell trait. 2. Metabisulfite screen causes cells with any amount of HbS to sickle; positive in both disease and trait 3. Hb electrophoresis confirms the presence and amount ofHbS. Fig. 5.8 Fragment of nuclear remnant (HowellFig. 5.9 Sickle cell disease. Fig. 5.10 Hemoglobin C crystal. Jolly body) within RBC. Red Blood Cell Disorders _,... i. Disease-90% HbS, 8% HbF, 2% HbA (no HbA) ii. Trait-55% HbA, 43% HbS, 2% HbA2 III. HEMOGLOBIN C A. Autosomal recessive mutation in ~ chain of hemoglobin 1. Normal glutamic acid is replaced by lysine. 2. B. Presents with mild anemia due to extravascular hemolysis C. Characteristic HbC crystals are seen in RBCs on blood smear (Fig. 5.10). I. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH) A. Acquired defect in myeloid stem cells resulting in absent glycosylphosphatidylinositol (GPI); renders cells susceptible to destruction by complement 1.

1	A. Acquired defect in myeloid stem cells resulting in absent glycosylphosphatidylinositol (GPI); renders cells susceptible to destruction by complement 1. Blood cells coexist with complement. 2. Decay accelerating factor (DAF) on the surface of blood cells protects against complement-mediated damage by inhibiting C3 convertase. 3. DAF is secured to the cell membrane by GPI (an anchoring glycolipid). 4. Absence of GPI leads to absence of DAF, rendering cells susceptible to complement-mediated damage. B. Intravascular hemolysis occurs episodically, often at night during sleep. 1. Mild respiratory acidosis develops with shallow breathing during sleep and activates complement. 2. RBCs, WBCs, and platelets are lysed. 3. Intravascular hemolysis leads to hemoglobinemia and hemoglobinuria (especially in the morning); hemosiderinuria is seen days after hemolysis.

1	2. RBCs, WBCs, and platelets are lysed. 3. Intravascular hemolysis leads to hemoglobinemia and hemoglobinuria (especially in the morning); hemosiderinuria is seen days after hemolysis. C. Sucrose test is used to screen for disease; confirmatory test is the acidified serum test or flow cytometry to detect lack of CDSS (DAF) on blood cells. D. Main cause of death is thrombosis of the hepatic, portal, or cerebral veins. 1. Destroyed platelets release cytoplasmic contents into circulation, inducing thrombosis. E. Complications include iron deficiency anemia (due to chronic loss of hemoglobin in the urine) and acute myeloid leukemia (AML), which develops in 10% of patients. II. GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) DEFICIENCY A. X-linked recessive disorder resulting in reduced half-life of G6PD; renders cells susceptible to oxidative stress 1. RBCs are normally exposed to oxidative stress, in particular H20 2• 2.

1	A. X-linked recessive disorder resulting in reduced half-life of G6PD; renders cells susceptible to oxidative stress 1. RBCs are normally exposed to oxidative stress, in particular H20 2• 2. Glutathione (an antioxidant) neutralizes H 0 , but becomes oxidized in the process. 3. NADPH, a by-product of G6PD, is needed to regenerate reduced glutathione. 4. ,J, G6PD ➔ ,J, NADPH ➔ ,J, reduced glutathione ➔ oxidative injury by H20 2 ➔ intravascular hemolysis B. G6PD deficiency has two major variants. 1. African variant-mildly reduced half-life of G6PD leading to mild intravascular hemolysis with oxidative stress 2. Mediterranean variant-markedly reduced half-life of G6PD leading to marked intravascular hemolysis with oxidative stress 3. High carrier frequency in both populations is likely due to protective role against falciparum malaria. C. Oxidative stress precipitates Hb as Heinz bodies. 1.

1	High carrier frequency in both populations is likely due to protective role against falciparum malaria. C. Oxidative stress precipitates Hb as Heinz bodies. 1. Causes of oxidative stress include infections, drugs (e.g., primaquine, sulfa drugs, and dapsone), and fava beans. 2. Heinz bodies are removed from RBCs by splenic macrophages, resulting in bite cells (Fig. 5.11). 3. Leads to predominantly intravascular hemolysis D. Presents with hemoglobinuria and back pain hours after exposure to oxidative stress E. Heinz preparation is used to screen for disease (precipitated hemoglobin can only be seen with a special Heinz stain, Fig. 5.12); enzyme studies confirm deficiency (performed weeks after hemolytic episode resolves). III. IMMUNE HEMOLYTIC ANEMIA (IHA) A. Antibody-mediated (IgG or IgM) destruction ofRBCs B. IgG-mediated disease usually involves extravascular hemolysis. 1.

1	III. IMMUNE HEMOLYTIC ANEMIA (IHA) A. Antibody-mediated (IgG or IgM) destruction ofRBCs B. IgG-mediated disease usually involves extravascular hemolysis. 1. IgG binds RBCs in the relatively warm temperature of the central body (warm agglutinin); membrane of antibody-coated RBC is consumed by splenic macrophages, resulting in spherocytes. 2. Associated with SLE (most common cause), CLL, and certain drugs (classically, penicillin and cephalosporins) i. Drug may attach to RBC membrane (e.g., penicillin) with subsequent binding of antibody to drug-membrane complex ii. Drug may induce production of autoantibodies (e.g., a-methyldopa) that bind self antigens on RBCs 3. Treatment involves cessation of the offending drug, steroids, IVIG, and, if necessary, splenectomy. C. IgM-mediated disease can lead to intravascular hemolysis. 1. IgM binds RBCs and fixes complement in the relatively cold temperature of the extremities (cold agglutinin). 2.

1	C. IgM-mediated disease can lead to intravascular hemolysis. 1. IgM binds RBCs and fixes complement in the relatively cold temperature of the extremities (cold agglutinin). 2. RBCs inactivate complement, but residual C3b serves as an opsonin for splenic macrophages resulting in spherocytes; extreme activation of complement can lead to intravascular hemolysis. 3. Associated with Mycoplasma pneumoniae and infectious mononucleosis. D. Coombs test is used to diagnose IHA; testing can be direct or indirect. 1. Direct Coombs test confirms the presence of antibody-or complement-coated RBCs. When anti-IgG/complement is added to patient RBCs, agglutination occurs if RBCs are already coated with IgG or complement. This is the most important test for IHA. 2. Indirect Coombs test confirms the presence of antibodies in patient serum. AntiIgG and test RBCs are mixed with the patient serum; agglutination occurs if serum antibodies are present.

1	2. Indirect Coombs test confirms the presence of antibodies in patient serum. AntiIgG and test RBCs are mixed with the patient serum; agglutination occurs if serum antibodies are present. Fig. 5.11 Bite cell. Fig. 5.12 Heinz bodies (Heinz preparation). Fig. 5.13 Schistocyte. IV. A. Intravascular hemolysis that results from vascular pathology; RBCs are destroyed as they pass through the circulation. 1. Iron deficiency anemia occurs with chronic hemolysis. B. Occurs with microthrombi (TTP-HUS, DIC, HELLP), prosthetic heart valves, and aortic stenosis; when present, microthrombi produce schistocytes on blood smear (Fig. 5.13). V. A. Infection ofRBCs and liver with Plasmodium (Fig. 5.14); transmitted by the female Anopheles mosquito B. RBCs rupture as a part of the Plasm odium life cycle, resulting in intravascular hemolysis and cyclical fever. 1. 2. C. Spleen also consumes some infected RBCs; results in mild extravascular hemolysis with splenomegaly I. BASIC PRINCIPLES

1	1. 2. C. Spleen also consumes some infected RBCs; results in mild extravascular hemolysis with splenomegaly I. BASIC PRINCIPLES A. Decreased production of RBCs by bone marrow; characterized by low corrected reticulocyte count B. Etiologies include 1. Causes of microcytic and macrocytic anemia 2. Renal failure-decreased production of EPO by peritubular interstitial cells 3. Damage to bone marrow precursor cells (may result in anemia or pancytopenia) II. PARVOVIRUS Bl9 A. Infects progenitor red cells and temporarily halts erythropoiesis; leads to significant anemia in the setting of preexisting marrow stress (e.g., sickle cell anemia). B. Treatment is supportive (infection is self-limited). III. APLASTIC ANEMIA A. Damage to hematopoietic stem cells, resulting in pancytopenia (anemia, thrombocytopenia, and leukopenia) with low reticulocyte count B. Etiologies include drugs or chemicals, viral infections, and autoimmune damage.

1	B. Etiologies include drugs or chemicals, viral infections, and autoimmune damage. C. Biopsy reveals an empty, fatty marrow (Fig. 5.15). Fig. 5.14 Erythrocytes infected with P falciparum. Fig. 5.15 Aplastic anemia. (Courtesy of Paulo Mourao, MD) D. Treatment includes cessation of any causative drugs and supportive care with transfusions and marrow-stimulating factors (e.g., erythropoietin, GM-CSF, and G-CSF). 1. Immunosuppression may be helpful as some idiopathic cases are due to abnormal T-cell activation with release of cytokines. 2. May require bone marrow transplantation as a last resort IV. MYELOPHTHISIC PROCESS A. Pathologic process (e.g., metastatic cancer) that replaces bone marrow; hematopoiesis is impaired, resulting in pancytopenia. I. BASIC PRINCIPLES A. Hematopoiesis occurs via a stepwise maturation of CD34+ hematopoietic stem cells (Fig. 6.1). B. Cells mature and are released from the bone marrow into the blood.

1	I. BASIC PRINCIPLES A. Hematopoiesis occurs via a stepwise maturation of CD34+ hematopoietic stem cells (Fig. 6.1). B. Cells mature and are released from the bone marrow into the blood. C. A normal white blood cell (WBC) count is approximately 5-10 K/µL. l. A low WBC count(< 5 K) is called leukopenia. 2. A high WBC count(> 10 K) is called leukocytosis. 3. A low or high WBC count is usually due to a decrease or increase in one particular cell lineage. II. LEUKOPENIA A. Neutropenia refers to a decreased number of circulating neutrophils. Causes include l. Drug toxicity (e.g., chemotherapy with alkylating agents)-Damage to stem cells results in decreased production of WBCs, especially neutrophils. 2. Severe infection (e.g., gram-negative sepsis)-Increased movement of neutrophils into tissues results in decreased circulating neutrophils. 3.

1	2. Severe infection (e.g., gram-negative sepsis)-Increased movement of neutrophils into tissues results in decreased circulating neutrophils. 3. As a treatment, GM-CSF or G-CSF may be used to boost granulocyte production, thereby decreasing risk of infection in neutropenic patients. B. Lymphopenia refers to a decreased number of circulating lymphocytes. Causes include l. Immunodeficiency (e.g., Di George syndrome or HIV) 2. High cortisol state (e.g., exogenous corticosteroids or Cushing syndrome)induces apoptosis of lymphocytes 3. Autoimmune destruction (e.g., systemic lupus erythematosus) 4. Whole body radiation-Lymphocytes are highly sensitive to radiation; lymphopenia is the earliest change to emerge after whole body radiation. III. LEUKOCYTOSIS

1	Whole body radiation-Lymphocytes are highly sensitive to radiation; lymphopenia is the earliest change to emerge after whole body radiation. III. LEUKOCYTOSIS A. Neutrophilic leukocytosis refers to increased circulating neutrophils. Causes include l. Bacterial infection or tissue necrosis-induces release of marginated pool and bone marrow neutrophils, including immature forms (left shift); immature cells are characterized by decreased Fe receptors (CD16). Fig. 6.1 Hematopoiesis. pathoma.com 2. High cortisol state-impairs leukocyte adhesion, leading to release of marginated pool of neutrophils B. Monocytosis refers to increased circulating monocytes. Causes include chronic inflammatory states (e.g., autoimmune and infectious) and malignancy. C. Eosinophilia refers to increased circulating eosinophils. Causes include allergic reactions (type I hypersensitivity), parasitic infections, and Hodgkin lymphoma. Eosinophilia is driven by increased eosinophil chemotactic factor.

1	D. Basophilia refers to increased circulating basophils; classically seen in chronic myeloid leukemia E. Lymphocytic leukocytosis refers to increased circulating lymphocytes. Causes include 1. Viral infections-T lymphocytes undergo hyperplasia in response to virally infected cells. 2. Bordetella pertussis infection-Bacteria produce lymphocytosis-promoting factor, which blocks circulating lymphocytes from leaving the blood to enter the lymph node. IV. INFECTIOUS MONONUCLEOSIS (IM) A. EBV infection that results in a lymphocytic leukocytosis comprised of reactive CDs+ T cells; CMV is a less common cause. 1. EBV is transmitted by saliva ("kissing disease"); classically affects teenagers B. EBV primarily infects 1. Oropharynx, resulting in pharyngitis 2. Liver, resulting in hepatitis with hepatomegaly and elevated liver enzymes 3. C. CDs+ T-cell response leads to 1. Generalized lymphadenopathy (LAD) due to T-cell hyperplasia in the lymph node paracortex 2.

1	C. CDs+ T-cell response leads to 1. Generalized lymphadenopathy (LAD) due to T-cell hyperplasia in the lymph node paracortex 2. Splenomegaly due to T-cell hyperplasia in the periarterial lymphatic sheath (PALS) 3. High WBC count with atypical lymphocytes (reactive CDs+ T cells) in the blood (Fig. 6.2) D. The monospot test is used for screening. 1. Detects IgM antibodies that cross-react with horse or sheep red blood cells (heterophile antibodies) 2. 3. A negative monospot test suggests CMV as a possible cause of IM. 4. Definitive diagnosis is made by serologic testing for the EBV viral capsid antigen. Fig. 6.2 Atypical lymphocyte, infectious Fig. 6.3 Splenic rupture. (Courtesy of K.V. mononucleosis. Santosh, MD) E. Complications 1. Increased risk for splenic rupture (Fig. 6.3); patients are generally advised to avoid contact sports for one month. 2. Rash if exposed to ampicillin 3.

1	E. Complications 1. Increased risk for splenic rupture (Fig. 6.3); patients are generally advised to avoid contact sports for one month. 2. Rash if exposed to ampicillin 3. Dormancy of virus in B cells leads to increased risk for both recurrence and B-cell lymphoma, especially if immunodeficiency (e.g., HIV) develops. I. BASIC PRINCIPLES A. Neoplastic proliferation of blasts; defined as the accumulation of > 20% blasts in the bone marrow. B. Increased blasts "crowd-out" normal hematopoiesis, resulting in an "acute" presentation with anemia (fatigue), thrombocytopenia (bleeding), or neutropenia (infection). C. Blasts usually enter the blood stream, resulting in a high WBC count. 1. Blasts are large, immature cells, often with punched out nucleoli (Fig. 6.4). D. Acute leukemia is subdivided into acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML) based on the phenotype of the blasts. II. ACUTE LYMPHOBLASTIC LEUKEMIA

1	D. Acute leukemia is subdivided into acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML) based on the phenotype of the blasts. II. ACUTE LYMPHOBLASTIC LEUKEMIA A. Neoplastic accumulation oflymphoblasts (> 20%) in the bone marrow 1. Lymphoblasts are characterized by positive nuclear staining for TdT, a DNA polymerase. 2. TdT is absent in myeloid blasts and mature lymphocytes. B. Most commonly arises in children; associated with Down syndrome (usually arises after the age of 5 years) C. Subclassified into B-ALL and T-ALL based on surface markers D. B-ALL is the most common type of ALL. 1. Usually characterized by lymphoblasts (TdT+) that express CDlO, CD19, and CD20. 2. Excellent response to chemotherapy; requires prophylaxis to scrotum and CSF (Fig. 6.5) 3. Prognosis is based on cytogenetic abnormalities. i. t(l2;21) has a good prognosis; more commonly seen in children ii. t(9;22) has a poor prognosis; more commonly seen in adults (Philadelphia+ ALL)

1	Prognosis is based on cytogenetic abnormalities. i. t(l2;21) has a good prognosis; more commonly seen in children ii. t(9;22) has a poor prognosis; more commonly seen in adults (Philadelphia+ ALL) E. T-ALL is characterized by lymphoblasts (TdT+) that express markers ranging from CD2 to CDS (e.g., CD3, CD4, CD7). The blasts do not express CDlO. Fig. 6.4 Blasts of acute leukemia. Fig. 6.5 Acute lymphoblastic leukemia involving meninges. 1. Usually presents in teenagers as a mediastinal (thymic) mass (called acute lymphoblastic lymphoma because the malignant cells form a mass) III. ACUTE MYELOID LEUKEMIA A. Neoplastic accumulation of immature myeloid cells (> 20%) in the bone marrow B. Myeloblasts are usually characterized by positive cytoplasmic staining for myeloperoxidase (MPO). 1. Crystal aggregates of MPO may be seen as Auer rods (Fig. 6.6). C. Most commonly arises in older adults (average age is 50-60 years)

1	1. Crystal aggregates of MPO may be seen as Auer rods (Fig. 6.6). C. Most commonly arises in older adults (average age is 50-60 years) D. Subclassification based on cytogenetic abnormalities, lineage of immature myeloid cells, and surface markers. High-yield subtypes include 1. Acute promyelocytic leukemia (APL) i. Characterized by t(l5;17), which involves translocation of the retinoic acid receptor (RAR) on chromosome 17 to chromosome 15; RAR disruption blocks maturation and promyelocytes (blasts) accumulate. ii. Abnormal promyelocytes contain numerous primary granules that increase the risk for DIC. iii. Treatment is with all-trans-retinoic acid (ATRA, a vitamin A derivative), which binds the altered receptor and causes the blasts to mature (and eventually die). 2. Acute monocytic leukemia i. Proliferation of monoblasts; usually lack MPO ii. Blasts characteristically infiltrate gums (Fig. 6.7).

1	2. Acute monocytic leukemia i. Proliferation of monoblasts; usually lack MPO ii. Blasts characteristically infiltrate gums (Fig. 6.7). 3. Acute megakaryoblastic leukemia i. Proliferation of megakaryoblasts; lack MPO ii. Associated with Down syndrome (usually arises before the age of 5) E. AML may also arise from pre-existing dysplasia (myelodysplastic syndromes), especially with prior exposure to alkylating agents or radiotherapy. 1. Myelodysplastic syndromes usually present with cytopenias, hypercellular bone marrow, abnormal maturation ofcells, and increased blasts(< 20%). 2. Most patients die from infection or bleeding, though some progress to acute leukemia. I. BASIC PRINCIPLES A. Neoplastic proliferation of mature circulating lymphocytes; characterized by a high WBC count B. Usually insidious in onset and seen in older adults II. CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) A. Neoplastic proliferation of naive B cells that co-express CDS and CD20; most common leukemia overall

1	II. CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) A. Neoplastic proliferation of naive B cells that co-express CDS and CD20; most common leukemia overall B. Increased lymphocytes and smudge cells are seen on blood smear (Fig. 6.8). C. Involvement of lymph nodes leads to generalized lymphadenopathy and is called small lymphocytic lymphoma. D. Complications include 1. Hypogammaglobulinemia-Infection is the most common cause of death in CLL. 2. 3. Transformation to diffuse large B-cell lymphoma (Richter transformation)marked clinically by an enlarging lymph node or spleen III. HAIRY CELL LEUKEMIA A. Neoplastic proliferation of mature B cells characterized by hairy cytoplasmic processes (Fig. 6.9) B. Cells are positive for tartrate-resistant acid phosphatase (TRAP). C. Clinical features include splenomegaly (due to accumulation of hairy cells in red pulp) and "dry tap" on bone marrow aspiration (due to marrow fibrosis). Lymphadenopathy is usually absent.

1	C. Clinical features include splenomegaly (due to accumulation of hairy cells in red pulp) and "dry tap" on bone marrow aspiration (due to marrow fibrosis). Lymphadenopathy is usually absent. D. Excellent response to 2-CDA (cladribine), an adenosine deaminase inhibitor; adenosine accumulates to toxic levels in neoplastic B cells. IV. A. Neoplastic proliferation of mature CD4+ T cells B. Associated with HTLV-1; most commonly seen in Japan and the Caribbean C. Clinical features include rash (skin infiltration), generalized lymphadenopathy with hepatosplenomegaly, and lytic (punched-out) bone lesions with hypercalcemia. V. A. Neoplastic proliferation of mature CD4+ T cells that infiltrate the skin, producing localized skin rash, plaques, and nodules. Aggregates of neoplastic cells in the epidermis are called Pautrier microabscesses. B. Cells can spread to involve the blood, producing Sezary syndrome.

1	B. Cells can spread to involve the blood, producing Sezary syndrome. 1. Characteristic lymphocytes with cerebriform nuclei (Sezary cells) are seen on blood smear (Fig. 6.10). Fig. 6.9 Hairy cell leukemia. (Courtesy of Paulo Fig. 6.10 Sezary cells. (Courtesy of Paulo Mourao, Mourao, MD) MD) I. BASIC PRINCIPLES A. Neoplastic proliferation of mature cells of myeloid lineage; disease of late adulthood (average age is 50-60 years) B. Results in high WBC count with hypercellular bone marrow 1. Cells of all myeloid lineages are increased; classified based on the dominant myeloid cell produced C. Complications include 1. Increased risk for hyperuricemia and gout due to high turnover of cells 2. Progression to marrow fibrosis or transformation to acute leukemia II. CHRONIC MYELOID LEUKEMIA (CML) A. Neoplastic proliferation of mature myeloid cells, especially granulocytes and their precursors; basophils are characteristically increased (Fig. 6.11).

1	II. CHRONIC MYELOID LEUKEMIA (CML) A. Neoplastic proliferation of mature myeloid cells, especially granulocytes and their precursors; basophils are characteristically increased (Fig. 6.11). B. Driven by t(9;22) (Philadelphia chromosome) which generates a BCR-ABL fusion protein with increased tyrosine kinase activity. 1. First line treatment is imatinib, which blocks tyrosine kinase activity. C. Splenomegaly is common. Enlarging spleen suggests progression to accelerated phase of disease; transformation to acute leukemia usually follows shortly thereafter. 1. Can transform to AML (2/3 of cases) or ALL (1/3 of cases) since mutation is in a pluripotent stem cell. D. CML is distinguished from a leukemoid reaction (reactive neutrophilic leukocytosis) by 1. Negative leukocyte alkaline phosphatase (LAP) stain (granulocytes in a leukemoid reaction are LAP positive) 2. Increased basophils (absent with leukemoid reaction) 3. III. POLYCYTHEMIA VERA (PV)

1	Negative leukocyte alkaline phosphatase (LAP) stain (granulocytes in a leukemoid reaction are LAP positive) 2. Increased basophils (absent with leukemoid reaction) 3. III. POLYCYTHEMIA VERA (PV) A. Neoplastic proliferation of mature myeloid cells, especially RBCs 1. Granulocytes and platelets are also increased. B. Associated with JAK2 kinase mutation C. Clinical symptoms are mostly due to hyperviscosity of blood. 1. 2. Increased risk of venous thrombosis (e.g., hepatic vein, portal vein, and dural sinus) 3. Flushed face due to congestion (plethora) 4. Itching, especially after bathing (due to histamine release from increased mast cells) D. Treatment is phlebotomy; second-line therapy is hydroxyurea. Fig. 6.11 Chronic myelogenous leukemia. Fig. 6.12 Essential thrombocythemia. 1. Without treatment, death usually occurs within one year. E. PV must be distinguished from reactive polycythemia. 1. In PV, erythropoietin (EPO) levels are decreased, and Sao is normal. 2.

1	1. Without treatment, death usually occurs within one year. E. PV must be distinguished from reactive polycythemia. 1. In PV, erythropoietin (EPO) levels are decreased, and Sao is normal. 2. In reactive polycythemia due to high altitude or lung disease, Sao2 is low, and EPO is increased. 3. In reactive polycythemia due to ectopic EPO production from renal cell carcinoma, EPO is high, and Sao2 is normal. IV. A. Neoplastic proliferation of mature myeloid cells, especially platelets (Fig. 6.12) 1. RBCs and granulocytes are also increased. B. Associated with JAK2 kinase mutation C. Symptoms are related to an increased risk of bleeding and/or thrombosis. 1. Rarely progresses to marrow fibrosis or acute leukemia 2. V. A. Neoplastic proliferation of mature myeloid cells, especially megakaryocytes 1. Associated with JAK2 kinase mutation (50% of cases) B. Megakaryocytes produce excess platelet-derived growth factor (PDGF) causing marrow fibrosis (Fig. 6.13).

1	B. Megakaryocytes produce excess platelet-derived growth factor (PDGF) causing marrow fibrosis (Fig. 6.13). C. Clinical features include 1. Splenomegaly due to extramedullary hematopoiesis 2. Leukoerythroblastic smear (tear-drop RBCs, nucleated RBCs, and immature granulocytes, Fig. 6.14) 3. Increased risk of infection, thrombosis, and bleeding I. BASIC PRINCIPLES A. LAD refers to enlarged lymph nodes. 1. Painful LAD is usually seen in lymph nodes that are draining a region of acute infection (acute lymphadenitis). 2. Painless LAD can be seen with chronic inflammation (chronic lymphadenitis), metastatic carcinoma, or lymphoma. B. In inflammation, lymph node enlargement is due to hyperplasia of particular regions of the lymph node (Fig. 6.15). 1. Follicular hyperplasia (B-cell region) is seen with rheumatoid arthritis and early stages of HIV infection, for example. Fig. 6.13 Myelofibrosis. Fig. 6.14 Tear-drop RBC. Fig. 6.15 Normal lymph node. 2.

1	Fig. 6.13 Myelofibrosis. Fig. 6.14 Tear-drop RBC. Fig. 6.15 Normal lymph node. 2. Paracortex hyperplasia (T-cell region) is seen with viral infections (e.g., infectious mononucleosis). 3. Hyperplasia of sinus histiocytes is seen in lymph nodes that are draining a tissue with cancer. I. BASIC PRINCIPLES A. Neoplastic proliferation oflymphoid cells that forms a mass; may arise in a lymph node or in extranodal tissue B. Divided into non-Hodgkin lymphoma (NHL, 60%) and Hodgkin lymphoma (HL, 40%) (Table 6.1) C. NHL is further classified based on cell type (e.g., B versus T), cell size, pattern of cell growth, expression of surface markers, and cytogenetic translocations. 1. Small B cells-follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, and small lymphocytic lymphoma (i.e., CLL cells that involve tissue) 2. 3. Table 6.1: Differences Between Non-Hodgkin Lymphoma and Hodgkin Lymphoma Composition of mass Lymphoid cells Painless lymphadenopathy, usually arises

1	3. Table 6.1: Differences Between Non-Hodgkin Lymphoma and Hodgkin Lymphoma Composition of mass Lymphoid cells Painless lymphadenopathy, usually arises Clinical with 'B' symptoms, usually arises in young Guides therapy; radiation is the mainstay of treatment. Fig. 6.16 Follicular lymphoma. A, Disruption of normal lymph node architecture by neoplastic follicles. B, Neoplastic follicles lacking tingible body macrophages. C, Reactive follicular hyperplasia with tingible body macrophages for comparison. II. FOLLICULAR LYMPHOMA A. Neoplastic proliferation of small B cells (CD20+) that form follicle-like nodules (Fig. 6.16A) B. Presents in late adulthood with painless lymphadenopathy C. Driven by t(l4;18) l. BCL2 on chromosome 18 translocates to the lg heavy chain locus on chromosome 14. 2. Results in overexpression of Bcl2, which inhibits apoptosis D. Treatment is reserved for patients who are symptomatic and involves low-dose chemotherapy or rituximab (anti-CD20 antibody).

1	2. Results in overexpression of Bcl2, which inhibits apoptosis D. Treatment is reserved for patients who are symptomatic and involves low-dose chemotherapy or rituximab (anti-CD20 antibody). E. Progression to diffuse large B-cell lymphoma is an important complication; presents as an enlarging lymph node F. Follicular lymphoma is distinguished from reactive follicular hyperplasia by 1. Disruption of normal lymph node architecture (maintained in follicular hyperplasia) 2. Lack of tingible body macrophages in germinal centers (tingible body macrophages are present in follicular hyperplasia, Fig. 6.16B,C) 3. 4. Monoclonality (follicular hyperplasia is polyclonal) III. MANTLE CELL LYMPHOMA A. Neoplastic proliferation of small B cells (CD20+) that expands the mantle zone B. Presents in late adulthood with painless lymphadenopathy C. Driven by t(ll;l4) 1. Cyclin Dl gene on chromosome 11 translocates to lg heavy chain locus on chromosome 14. 2.

1	B. Presents in late adulthood with painless lymphadenopathy C. Driven by t(ll;l4) 1. Cyclin Dl gene on chromosome 11 translocates to lg heavy chain locus on chromosome 14. 2. Overexpression of cyclin Dl promotes Gl/S transition in the cell cycle, facilitating neoplastic proliferation. IV. A. Neoplastic proliferation of small B cells (CD20+) that expands the marginal zone B. Associated with chronic inflammatory states such as Hashimoto thyroiditis, Sjogren syndrome, and H pylori gastritis 1. The marginal zone is formed by post-germinal center B cells. C. MALToma is marginal zone lymphoma in mucosal sites. 1. Gastric MALToma may regress with treatment of H Pylori. V. A. Neoplastic proliferation of intermediate-sized B cells (CD20+); associated with EBV B. Classically presents as an extranodal mass in a child or young adult Fig. 6.17 Burkitt lymphoma. A, Involvement of jaw. B, 'Starry-sky' appearance. (A, Courtesy Mike Blyth) 1.

1	B. Classically presents as an extranodal mass in a child or young adult Fig. 6.17 Burkitt lymphoma. A, Involvement of jaw. B, 'Starry-sky' appearance. (A, Courtesy Mike Blyth) 1. African form usually involves the jaw (Fig. 6.17A). 2. Sporadic form usually involves the abdomen. C. Driven by translocations of c-myc (chromosome 8) 1. t(8;14) is most common, resulting in translocation of c-myc to the lg heavy chain locus on chromosome 14. 2. Overexpression of c-myc oncogene promotes cell growth. D. Characterized by high mitotic index and 'starry-sky' appearance on microscopy (Fig. 6.17B) VI. DIFFUSE LARGE B-CELL LYMPHOMA A. Neoplastic proliferation oflarge B cells (CD20+) that grow diffusely in sheets 1. Most common form of NHL 2. B. Arises sporadically or from transformation of a low-grade lymphoma (e.g., follicular lymphoma) 1. Presents in late adulthood as an enlarging lymph node or an extranodal mass I. BASIC PRINCIPLES

1	B. Arises sporadically or from transformation of a low-grade lymphoma (e.g., follicular lymphoma) 1. Presents in late adulthood as an enlarging lymph node or an extranodal mass I. BASIC PRINCIPLES A. Neoplastic proliferation of Reed-Sternberg (RS) cells, which are large B cells with multilobed nuclei and prominent nucleoli ('owl-eyed nuclei', Fig. 6.18); classically positive for CD15 and CD30 B. RS cells secrete cytokines. 1. Occasionally results in 'B' symptoms (fever, chills, weight loss, and night sweats) 2. Attract reactive lymphocytes, plasma cells, macrophages, and eosinophils 3. May lead to fibrosis C. Reactive inflammatory cells make up a bulk of the tumor and form the basis for classification of HL. Subtypes include 1. 2. 3. 4. D. Nodular sclerosis is the most common subtype of HL (70% of all cases). 1. Classic presentation is an enlarging cervical or mediastinal lymph node in a young adult, usually female. 2.

1	3. 4. D. Nodular sclerosis is the most common subtype of HL (70% of all cases). 1. Classic presentation is an enlarging cervical or mediastinal lymph node in a young adult, usually female. 2. Lymph node is divided by bands of sclerosis (Fig. 6.19A); RS cells are present in lake-like spaces (lacunar cells, Fig. 6.19B). E. Important considerations regarding other subtypes of HL 1. Lymphocyte-rich has the best prognosis of all types. Fig. 6.18 Reed-Sternberg cell. Fig. 6.19 Hodgkin lymphoma, nodular sclerosis type. A, Sclerosis of lymph node. B, Reed-Sternberg cells in lacunar spaces. 2. Mixed cellularity is often associated with abundant eosinophils (RS cells produce IL-5). 3. Lymphocyte-depleted is the most aggressive of all types; usually seen in the elderly and HIV-positive individuals I. MULTIPLE MYELOMA A. Malignant proliferation of plasma cells in the bone marrow 1.

1	I. MULTIPLE MYELOMA A. Malignant proliferation of plasma cells in the bone marrow 1. Most common primary malignancy of bone; metastatic cancer, however, is the most common malignant lesion of bone overall. 2. B. Clinical features include 1. Bone pain with hypercalcemia-Neoplastic plasma cells activate the RANK receptor on osteoclasts, leading to bone destruction. Lytic, 'punched-out' skeletal lesions are seen on x-ray (Fig. 6.20A), especially in the vertebrae and skull; increased risk for fracture 2. Elevated serum protein-Neoplastic plasma cells produce immunoglobulin; M spike is present on serum protein electrophoresis (SPEP), most commonly due to monoclonal IgG or IgA. 3. Increased risk of infection-Monoclonal antibody lacks antigenic diversity; infection is the most common cause of death in multiple myeloma. 4. Rouleaux formation ofRBCs on blood smear-Increased serum protein decreases charge between RBCs (Fig. 6.20B). 5.

1	4. Rouleaux formation ofRBCs on blood smear-Increased serum protein decreases charge between RBCs (Fig. 6.20B). 5. Primary AL amyloidosis-Free light chains circulate in serum and deposit in tissues. 6. Proteinuria-Free light chain is excreted in the urine as Bence Jones protein; deposition in kidney tubules leads to risk for renal failure (myeloma kidney). II. MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE (MGUS) A. Increased serum protein with M spike on SPEP; other features of multiple myeloma are absent (e.g., no lytic bone lesions, hypercalcemia, AL amyloid, or Bence Jones proteinuria). B. Common in elderly (seen in 5% of 70-year-old individuals); 1 % of patients with MGUS develop multiple myeloma each year. III. WALDENSTROM MACROGLOBULINEMIA A. B-cell lymphoma with monoclonal IgM production B. Clinical features include

1	III. WALDENSTROM MACROGLOBULINEMIA A. B-cell lymphoma with monoclonal IgM production B. Clinical features include Fig. 6.20 Multiple myeloma. A, 'Punched-out' lesions involving skull, x-ray. B, Rouleaux formation of Fig. 6.21 Birbeck granule, electron microscopy. RBCs. (Courtesy of humpath.com) 1. Generalized lymphadenopathy; lytic bone lesions are absent. 2. Increased serum protein with M spike (comprised oflgM) 3. Visual and neurologic deficits (e.g., retinal hemorrhage or stroke)-IgM (large pentamer) causes serum hyperviscosity. 4. Bleeding-Viscous serum results in defective platelet aggregation. C. Acute complications are treated with plasmapheresis, which removes IgM from the serum. I. BASIC PRINCIPLES A. Langerhans cells are specialized dendritic cells found predominantly in the skin. 1. 2. Present antigen to nai:ve T cells B. Langerhans cell histiocytosis is a neoplastic proliferation of Langerhans cells.

1	1. 2. Present antigen to nai:ve T cells B. Langerhans cell histiocytosis is a neoplastic proliferation of Langerhans cells. 1. Characteristic Birbeck (tennis racket) granules are seen on electron microscopy (Fig. 6.21); cells are CDla+ and S-100+ by immunohistochemistry. II. LETTERER-SIWE DISEASE A. Malignant proliferation of Langerhans cells B. Classic presentation is skin rash and cystic skeletal defects in an infant(< 2 years old). C. Multiple organs may be involved; rapidly fatal III. EOSINOPHILIC GRANULOMA A. Benign proliferation of Langerhans cells in bone B. Classic presentation is pathologic fracture in an adolescent; skin is not involved. C. Biopsy shows Langerhans cells with mixed inflammatory cells, including numerous eosinophils. IV. HAND-SCHULLER-CHRISTIAN DISEASE A. Malignant proliferation of Langerhans cells B. Classic presentation is scalp rash, lytic skull defects, diabetes insipidus, and exophthalmos in a child. I. BASIC PRINCIPLES

1	A. Malignant proliferation of Langerhans cells B. Classic presentation is scalp rash, lytic skull defects, diabetes insipidus, and exophthalmos in a child. I. BASIC PRINCIPLES A. Inflammation of the blood vessel wall 1. Arterial wall is comprised of three layers: endothelial intima, smooth muscle media, and connective tissue adventitia (Fig. 7.1). B. Etiology is usually unknown; most cases are not infectious. C. Clinical features include 1. Nonspecific symptoms of inflammation (e.g., fever, fatigue, weight loss, and myalgias) 2. Symptoms of organ ischemia-due to luminal narrowing or thrombosis of the inflamed vessels D. Divided into large-, medium-, and small-vessel vasculitides 1. Large-vessel vasculitis involves the aorta and its major branches. 2. Medium-vessel vasculitis involves muscular arteries that supply organs. 3. Small-vessel vasculitis involves arterioles, capillaries, and venules. II. LARGE-VESSEL VASCULITIS A. Temporal (Giant Cell) Arteritis 1.

1	3. Small-vessel vasculitis involves arterioles, capillaries, and venules. II. LARGE-VESSEL VASCULITIS A. Temporal (Giant Cell) Arteritis 1. Granulomatous vasculitis that classically involves branches of the carotid artery 2. 3. Presents as headache (temporal artery involvement), visual disturbances (ophthalmic artery involvement), and jaw claudication. Flu-like symptoms with joint and muscle pain (polymyalgia rheumatica) are often present. ESR is elevated. 4. Biopsy reveals inflamed vessel wall with giant cells and intimal fibrosis (Fig. 7.2). i. Lesions are segmental; diagnosis requires biopsy of a long segment of vessel, and a negative biopsy does not exclude disease. 5. Treatment is corticosteroids; high risk of blindness without treatment B. Takayasu Arteritis 1. Granulomatous vasculitis that classically involves the aortic arch at branch points 2.

1	5. Treatment is corticosteroids; high risk of blindness without treatment B. Takayasu Arteritis 1. Granulomatous vasculitis that classically involves the aortic arch at branch points 2. Presents in adults < 50 years old (classically, young Asian females) as visual and neurologic symptoms with a weak or absent pulse in the upper extremity ('pulseless disease'). ESR is elevated. 3. Treatment is corticosteroids. III. MEDIUM-VESSEL VASCULITIS A. Polyarteritis Nodosa 1. Necrotizing vasculitis involving multiple organs; lungs are spared. 2. Classically presents in young adults as hypertension (renal artery involvement), abdominal pain with melena (mesenteric artery involvement), neurologic disturbances, and skin lesions. Associated with serum HBsAg pathoma.com 3. Lesions of varying stages are present. Early lesion consists of transmural inflammation with fibrinoid necrosis (Fig. 7.3); eventually heals with fibrosis, producing a 'string-of-pearls' appearance on imaging 4.

1	Treatment is corticosteroids and cyclophosphamide; fatal if not treated B. Kawasaki Disease 1. 2. Presents with nonspecific signs including fever, conjunctivitis, erythematous rash of palms and soles, and enlarged cervical lymph nodes 3. Coronary artery involvement is common and leads to risk for (1) thrombosis with myocardial infarction and (2) aneurysm with rupture. 4. Treatment is aspirin and IVIG; disease is self-limited. C. Buerger Disease 1. 2. Presents with ulceration, gangrene, and autoamputation of fingers and toes; Raynaud phenomenon is often present. 3. Highly associated with heavy smoking; treatment is smoking cessation. IV. SMALL-VESSEL VASCULITIS A. Wegener Granulomatosis 1. Necrotizing granulomatous vasculitis involving nasopharynx, lungs, and kidneys 2. Classic presentation is a middle-aged male with sinusitis or nasopharyngeal ulceration, hemoptysis with bilateral nodular lung infiltrates, and hematuria due to rapidly progressive glomerulonephritis.

1	3. Serum c-ANCA levels correlate with disease activity. 4. Biopsy reveals large necrotizing granulomas with adjacent necrotizing vasculitis (Fig. 7.4). 5. Treatment is cyclophosphamide and steroids; relapses are common. B. Microscopic Polyangiitis 1. Necrotizing vasculitis involving multiple organs, especially lung and kidney 2. Presentation is similar to Wegener granulomatosis, but nasopharyngeal involvement and granulomas are absent. 3. Serum p-ANCA levels correlate with disease activity. 4. Treatment is corticosteroids and cyclophosphamide; relapses are common. C. Churg-Strauss Syndrome 1. Necrotizing granulomatous inflammation with eosinophils involving multiple organs, especially lungs and heart 2. Asthma and peripheral eosinophilia are often present. 3. Serum p-ANCA levels correlate with disease activity. Fig. 7.1 Normal muscular artery. Fig. 7.2 Temporal (giant cell) arteritis. Fig. 7.3 Fibrinoid necrosis, polyarteritis nodosa. D. Henoch-Schonlein Purpura 1.

1	Fig. 7.1 Normal muscular artery. Fig. 7.2 Temporal (giant cell) arteritis. Fig. 7.3 Fibrinoid necrosis, polyarteritis nodosa. D. Henoch-Schonlein Purpura 1. Vasculitis due to IgA immune complex deposition; most common vasculitis in children 2. Presents with palpable purpura on buttocks and legs, GI pain and bleeding, and hematuria (IgA nephropathy); usually occurs following an upper respiratory tract infection 3. Disease is self-limited, but may recur; treated with steroids, if severe I. BASIC PRINCIPLES A. Increased blood pressure; may involve pulmonary (see chapter 9) or systemic circulation B. Systemic HTN is defined as pressure :2'. 140/90 mm Hg (normal ~ 120/80 mm Hg); divided into primary or secondary types based on etiology II. PRIMARY HTN A. HTN of unknown etiology (95% of cases) B. Risk factors include age, race (increased risk in African Americans, decreased risk in Asians), obesity, stress, lack of physical activity, and high-salt diet. III. SECONDARY HTN

1	B. Risk factors include age, race (increased risk in African Americans, decreased risk in Asians), obesity, stress, lack of physical activity, and high-salt diet. III. SECONDARY HTN A. HTN due to an identifiable etiology (5% ofcases) B. Renal artery stenosis is a common cause (renovascular hypertension). 1. Stenosis decreases blood flow to glomerulus. 2. Juxtaglomerular apparatus (JGA) responds by secreting renin, which converts angiotensinogen to angiotensin I. 3. Angiotensin I is converted to angiotensin II (ATII) by angiotensin converting enzyme (ACE). 4. ATII raises blood pressure by (1) contracting arteriolar smooth muscle, increasing total peripheral resistance and (2) promoting adrenal release of aldosterone, which increases resorption of sodium in the distal convoluted tubule (expanding plasma volume). 5. Leads to HTN with increased plasma renin and unilateral atrophy (due to low blood flow) of the affected kidney; neither feature is seen in primary hypertension.

1	5. Leads to HTN with increased plasma renin and unilateral atrophy (due to low blood flow) of the affected kidney; neither feature is seen in primary hypertension. C. Important causes of stenosis include atherosclerosis (elderly males) and fibromuscular dysplasia (young females). Fig. 7.4 Wegener granulomatosis. A, Necrotizing granuloma. B, Giant cells at edge of granuloma. Fig. 7.5 Atherosclerosis. 1. Fibromuscular dysplasia is a developmental defect of the blood vessel wall, resulting in irregular thickening of large-and medium-sized arteries, especially the renal artery. IV. BENIGN AND MALIGNANT HTN A. HTN can also be classified as benign or malignant. B. Benign HTN is a mild or moderate elevation in blood pressure; most cases of HTN are benign. 1. Clinically silent; vessels and organs are damaged slowly over time. C. Malignant HTN is severe elevation in blood pressure (> 180/120 mm Hg); comprises < 5% of cases 1. 2.

1	1. Clinically silent; vessels and organs are damaged slowly over time. C. Malignant HTN is severe elevation in blood pressure (> 180/120 mm Hg); comprises < 5% of cases 1. 2. Presents with acute end-organ damage (e.g., acute renal failure, headache, and papilledema) and is a medical emergency I. BASIC PRINCIPLES A. Literally, "hard arteries;" due to thickening of the blood vessel wall B. Three pathologic patterns-atherosclerosis, arteriolosclerosis, and Monckeberg medial calcific sclerosis II. ATHEROSCLEROSIS A. Intimal plaque that obstructs blood flow 1. Consists of a necrotic lipid core (mostly cholesterol) with a fibromuscular cap (Fig. 7.5); often undergoes dystrophic calcification B. Involves large-and medium-sized arteries; abdominal aorta, coronary artery, popliteal artery, and internal carotid artery are commonly affected. C. Risk factors for atherosclerosis are divided into modifiable and nonmodifiable. 1.

1	C. Risk factors for atherosclerosis are divided into modifiable and nonmodifiable. 1. Modifiable risk factors include hypertension, hypercholesterolemia (LDL increases risk; HDL decreases risk), smoking, and diabetes. 2. Nonmodifiable risk factors include age (number and severity of lesions increase with age), gender (increased risk in males and postmenopausal females; estrogen is protective), and genetics (multifactorial, but family history is highly predictive of risk). D. Pathogenesis 1. Damage to endothelium allows lipids to leak into the intima. 2. Lipids are oxidized and then consumed by macrophages via scavenger receptors, resulting in foam cells. Fig. 7.6 Atherosclerotic em bolus. (Courtesy of Fig. 7.7 Hyaline arteriolosclerosis. Fig. 7.8 Arteriolonephrosclerosis. (Courtesy of Professor A. Garfia) Jerome Taxy, MD) 3. Inflammation and healing leads to deposition of extracellular matrix and proliferation of smooth muscle. E. Morphologic stages 1.

1	E. Morphologic stages 1. Begins as fatty streaks (flat yellow lesions of the intima consisting oflipid-laden macrophages); arise early in life (present in most teenagers) 2. Progresses to atherosclerotic plaque F. Complications of atherosclerosis account for > 50% of disease in Western countries. 1. Stenosis of medium-sized vessels results in impaired blood flow and ischemia leading to i. Peripheral vascular disease (lower extremity arteries, e.g., popliteal) ii. Angina (coronary arteries) iii. Ischemic bowel disease (mesenteric arteries) 2. Plaque rupture with thrombosis results in myocardial infarction (coronary arteries) and stroke (e.g., middle cerebral artery). 3. Plaque rupture with embolization results in atherosclerotic emboli, characterized by cholesterol crystals within the embolus (Fig. 7.6). 4. Weakening of vessel wall results in aneurysm (e.g., abdominal aorta). III. ARTERIOLOSCLEROSIS

1	4. Weakening of vessel wall results in aneurysm (e.g., abdominal aorta). III. ARTERIOLOSCLEROSIS A. Narrowing of small arterioles; divided into hyaline and hyperplastic types B. Hyaline arteriolosclerosis is caused by proteins leaking into the vessel wall, producing vascular thickening; proteins are seen as pink hyaline on microscopy (Fig. 7.7). 1. 2. Results in reduced vessel caliber with end-organ ischemia; classically produces glomerular scarring (arteriolonephrosclerosis, Fig. 7.8) that slowly progresses to chronic renal failure C. Hyperplastic arteriolosclerosis involves thickening of vessel wall by hyperplasia of smooth muscle ('onion-skin' appearance, Fig. 7.9). 1. Consequence of malignant hypertension 2. Results in reduced vessel caliber with end-organ ischemia 3. May lead to fibrinoid necrosis of the vessel wall with hemorrhage; classically causes acute renal failure with a characteristic 'flea-bitten' appearance IV. MONCKEBERG MEDIAL CALCIFIC SCLEROSIS

1	May lead to fibrinoid necrosis of the vessel wall with hemorrhage; classically causes acute renal failure with a characteristic 'flea-bitten' appearance IV. MONCKEBERG MEDIAL CALCIFIC SCLEROSIS A. Calcification of the media of muscular (medium-sized) arteries; nonobstructive (Fig. 7.lOA) B. Not clinically significant; seen as an incidental finding on x-ray or mammography (Fig. 7.lOB) Fig. 7.9 Hyperplastic arteriolosclerosis. Fig. 7.10 Monckeberg medial calcific sclerosis. A, Microscopic appearance. B, Mammogram. I. AORTIC DISSECTION A. Intimal tear with dissection of blood through media of the aortic wall (Fig. 7.11) B. Occurs in the proximal 10 cm of the aorta (high stress region) with preexisting weakness of the media C. Most common cause is hypertension (older adults); also associated with inherited defects of connective tissue (younger individuals) 1. Hypertension results in hyaline arteriolosclerosis of the vasa vasorum; decreased flow causes atrophy of the media. 2.

1	Hypertension results in hyaline arteriolosclerosis of the vasa vasorum; decreased flow causes atrophy of the media. 2. Marfan syndrome and Ehlers-Danlos syndrome classically lead to weakness of the connective tissue in the media (cystic medial necrosis). D. Presents as sharp, tearing chest pain that radiates to the back E. Complications include pericardia! tamponade (most common cause of death), rupture with fatal hemorrhage, and obstruction of branching arteries (e.g., coronary or renal) with resultant end-organ ischemia. II. THORACIC ANEURYSM A. Balloon-like dilation of the thoracic aorta B. Due to weakness in the aortic wall. Classically seen in tertiary syphilis; endarteritis of the vasa vasorum results in luminal narrowing, decreased flow, and atrophy of the vessel wall. Results in a 'tree-bark' appearance of the aorta (Fig. 7.12) C. Major complication is dilation of the aortic valve root, resulting in aortic valve insufficiency.

1	C. Major complication is dilation of the aortic valve root, resulting in aortic valve insufficiency. 1. Other complications include compression of mediastinal structures (e.g., airway or esophagus) and thrombosis/embolism. III. ABDOMINAL AORTIC ANEURYSM A. Balloon-like dilation of the abdominal aorta; usually arises below the renal arteries, but above the aortic bifurcation (Fig. 7.13) B. Primarily due to atherosclerosis; classically seen in male smokers > 60 years old with hypertension 1. Atherosclerosis increases the diffusion barrier to the media, resulting in atrophy and weakness of the vessel wall. C. Presents as a pulsatile abdominal mass that grows with time D. Major complication is rupture, especially when > 5 cm in diameter; presents with triad of hypotension, pulsatile abdominal mass, and flank pain 1. Other complications include compression oflocal structures (e.g., ureter) and thrombosis/embolism.

1	Fig. 7.11 Aortic dissection. (Courtesy of humpath. Fig. 7.12 'Tree-bark' appearance of aorta due to Fig, 7.13 Abdominal aortic aneurysm. (Courtesy com) syphi lis. (Courtesy of Aliya Husain, MD) of Aliya Husain, MD) I. HEMANGIOMA A. Benign tumor comprised of blood vessels (Fig. 7. 14) B. Commonly present at birth; often regresses during childhood C. Most often involves skin and liver II. ANGIOSARCOMA A. Malignant proliferation of endothelial cells; highly aggressive B. Common sites include skin, breast, and liver. 1. Liver angiosarcoma is associated with exposure to polyvinyl chloride, arsenic, and Thorotrast. III. KAPOSI SARCOMA A. Low-grade malignant proliferation of endothelial cells; associated with HHV-8 B. Presents as purple patches, plaques, and nodules on the skin (Fig. 7.15); may also involve visceral organs C. Classically seen in 1. Older Eastern European males-tumor remains localized to skin; treatment involves surgical removal. 2.

1	C. Classically seen in 1. Older Eastern European males-tumor remains localized to skin; treatment involves surgical removal. 2. AIDS-tumor spreads early; treatment is antiretroviral agents (to boost immune system). 3. Transplant recipients-tumor spreads early; treatment involves decreasing immunosuppression. Fig. 7.14 Hemangioma. Fig. 7.15 Kaposi sarcoma. I. BASIC PRINCIPLES A. Group of syndromes related to myocardial ischemia; IHD is the leading cause of death in the US. B. Usually due to atherosclerosis of coronary arteries, which decreases blood flow to the myocardium 1. Risk factors for IHD are similar to those of atherosclerosis; incidence increases with age. II. ANGINA A. Stable angina is chest pain that arises with exertion or emotional stress. 1. Due to atherosclerosis of coronary arteries with > 70% stenosis; decreased blood flow is not able to meet the metabolic demands of the myocardium during exertion. 2.

1	1. Due to atherosclerosis of coronary arteries with > 70% stenosis; decreased blood flow is not able to meet the metabolic demands of the myocardium during exertion. 2. Represents reversible injury to myocytes (no necrosis) 3. Presents as chest pain (lasting < 20 minutes) that radiates to the left arm or jaw, diaphoresis, and shortness of breath 4. EKG shows ST-segment depression due to subendocardial ischemia. 5. B. Unstable angina is chest pain that occurs at rest. 1. Usually due to rupture of an atherosclerotic plaque with thrombosis and incomplete occlusion of a coronary artery (Fig. 8.lA). 2. Represents reversible injury to myocytes (no necrosis) 3. EKG shows ST-segment depression due to subendocardial ischemia. 4. 5. High risk of progression to myocardial infarction C. Prinzmetal angina is episodic chest pain unrelated to exertion. 1. Due to coronary artery vasospasm 2. Represents reversible injury to myocytes (no necrosis) 3.

1	C. Prinzmetal angina is episodic chest pain unrelated to exertion. 1. Due to coronary artery vasospasm 2. Represents reversible injury to myocytes (no necrosis) 3. EKG shows ST-segment elevation due to transmural ischemia. 4. III. MYOCARDIAL INFARCTION A. Necrosis of cardiac myocytes B. Usually due to rupture of an atherosclerotic plaque with thrombosis and complete occlusion of a coronary artery (Fig. 8.lB) 1. Other causes include coronary artery vasospasm (due to Prinzmetal angina or cocaine use), emboli, and vasculitis (e.g., Kawasaki disease). C. Clinical features include severe, crushing chest pain (lasting > 20 minutes) that radiates to the left arm or jaw, diaphoresis, and dyspnea; symptoms are not relieved by nitroglycerin. D. Infarction usually involves the left ventricle (LV); right ventricle (RV) and both atria are generally spared. pathoma.com 1.

1	D. Infarction usually involves the left ventricle (LV); right ventricle (RV) and both atria are generally spared. pathoma.com 1. Occlusion ofleft anterior descending artery (LAD) leads to infarction of the anterior wall and anterior septum of the LV; LAD is the most commonly involved artery in MI (45% of cases). 2. Occlusion of right coronary artery (RCA) leads to infarction of the posterior wall, posterior septum, and papillary muscles of the LV; RCA is the 2nd most commonly involved artery in MI. 3. Occlusion of left circumflex artery leads to infarction of lateral wall of the L V. E. Initial phase of infarction leads to subendocardial necrosis involving < 50% of the myocardial thickness (subendocardial infarction, Fig. 8.2); EKG shows ST-segment depression. 1. Continued or severe ischemia leads to transmural necrosis involving most of the myocardial wall (transmural infarction); EKG shows ST-segment elevation. F. Laboratory tests detect elevated cardiac enzymes. 1.

1	F. Laboratory tests detect elevated cardiac enzymes. 1. Troponin I is the most sensitive and specific marker (gold standard) for MI. Levels rise 2-4 hours after infarction, peak at 24 hours, and return to normal by 7-10 days. 2. CK-MB is useful for detecting reinfarction that occurs days after an initial Ml; creatine kinase MB (CK-MB) levels rise 4-6 hours after infarction, peak at 24 hours, and return to normal by 72 hours. G. Treatment includes 1. Aspirin and/or heparin-limits thrombosis 2. Supplemental 0 2-minimizes ischemia 3. 4. ~-blocker-slows heart rate, decreasing 0 demand and risk for arrhythmia 5. 6. i. Reperfusion of irreversibly-damaged cells results in calcium influx, leading to hypercontraction of myofibrils (contraction band necrosis, Fig. 8.3). ii. Return of oxygen and inflammatory cells may lead to free radical generation, further damaging myocytes (reperfusion injury).

1	ii. Return of oxygen and inflammatory cells may lead to free radical generation, further damaging myocytes (reperfusion injury). H. Complications of myocardial infarction are closely related to gross and microscopic changes (Table 8.1). Table 8.1: Morphologic Changes in Myocardial Infarction ~~~::c~;~N GROSS CHANGES MICROSCOPIC CHANGES COMPLICATIONS Cardiogenic shock (massive infarction), congestive heart failure, and arrhythmia Coagulative necrosis (Fig. 8.4A) Fibrinous pericarditis (Fig. 8.SA); presents as 1-3 days Yellow pallor Neutrophils (Fig. 8.4B) chest pain with friction rub Rupture of ventricular free wall (Fig. 8.SB; leads to cardiac tamponade), interventricular septum 4-7 days Yellow pallor Macrophages (leads to shunt), or papillary muscle (Fig. 8.SC; leads to mitral insufficiency) Red border emerges as Granulation tissue with 1-3 weeks granulation tissue enters plump fibroblasts, collagen, from edge of infarct. and blood vessels

1	Red border emerges as Granulation tissue with 1-3 weeks granulation tissue enters plump fibroblasts, collagen, from edge of infarct. and blood vessels Aneurysm (Fig. 8.7), mural thrombus, or Months White scar (Fig. 8.6A) Fibrosis (Fig. 8.6B) Fig. 8.3 Contraction band necrosis. Fig. 8.4 Myocardial infarction. A, Coagulative necrosis of myocardium. B, Neutrophilic infiltrate. Fig. 8.5 Complications of myocardial infarction. A, Fibrinous pericarditis. B, Rupture of ventricular free wall. C, Rupture of papillary muscle. (Courtesy of Aliya Husain, MD) Fig. 8.6 Myocardial scar. A, Gross appearance. B, Microscopic appearance. (A, Courtesy of Ajit Paintal, Fig. 8.7 Ventricular aneurysm. MD) IV. A. Unexpected death due to cardiac disease; occurs without symptoms or < 1 hour after symptoms arise 1. Usually due to fatal ventricular arrhythmia B. Most common etiology is acute ischemia; 90% of patients have preexisting severe atherosclerosis.

1	B. Most common etiology is acute ischemia; 90% of patients have preexisting severe atherosclerosis. 1. Less common causes include mitral valve prolapse, cardiomyopathy, and cocaine abuse. V. A. Poor myocardial function due to chronic ischemic damage (with or without infarction); progresses to congestive heart failure (CHF) I. BASIC PRINCIPLES A. Pump failure; divided into right-and left-sided failure II. LEFT-SIDED HEART FAILURE A. Causes include ischemia, hypertension, dilated cardiomyopathy, myocardial infarction, and restrictive cardiomyopathy. B. Clinical features are due to decreased forward perfusion and pulmonary congestion. 1. Pulmonary congestion leads to pulmonary edema.

1	B. Clinical features are due to decreased forward perfusion and pulmonary congestion. 1. Pulmonary congestion leads to pulmonary edema. i. Results in dyspnea, paroxysmal nocturnal dyspnea (due to increased venous return when lying flat), orthopnea, and crackles ii. Small, congested capillaries may burst, leading to intraalveolar hemorrhage; marked by hemosiderin-laden macrophages ('heart-failure' cells, Fig. 8.8) 2. Decreased flow to kidneys leads to activation of renin-angiotensin system. i. Fluid retention exacerbates CHF. C. Mainstay of treatment is ACE inhibitor. III. RIGHT-SIDED HEART FAILURE A. Most commonly due to left-sided heart failure; other important causes include leftto-right shunt and chronic lung disease (cor pulmonale). B. Clinical features are due to congestion. 1. 2. Painful hepatosplenomegaly with characteristic 'nutmeg' liver (Fig. 8.9); may lead to cardiac cirrhosis 3. Dependent pitting edema (due to increased hydrostatic pressure)

1	1. 2. Painful hepatosplenomegaly with characteristic 'nutmeg' liver (Fig. 8.9); may lead to cardiac cirrhosis 3. Dependent pitting edema (due to increased hydrostatic pressure) Fig. 8.8 Hemosiderin-laden macrophages ('heart Fig. 8.9 'Nutmeg' liver, congestive heart failure. Fig. 8.10 'Boot-shaped' heart, tetralogy of Fallot. failure' cells). I. BASIC PRINCIPLES A. Arise during embryogenesis (usually weeks 3 through 8); seen in 1% of live births 1. Most defects are sporadic. B. Often result in shunting between left (systemic) and right (pulmonary) circulations. C. Defects with left-to-right shunting may be relatively asymptomatic at birth, but the shunt can eventually reverse. 1. Increased flow through the pulmonary circulation results in hypertrophy of pulmonary vessels and pulmonary hypertension. 2.

1	1. Increased flow through the pulmonary circulation results in hypertrophy of pulmonary vessels and pulmonary hypertension. 2. Increased pulmonary resistance eventually results in reversal of shunt, leading to late cyanosis (Eisenmenger syndrome) with right ventricular hypertrophy, polycythemia, and clubbing. D. Defects with right-to-left shunting usually present as cyanosis shortly after birth. II. VENTRICULAR SEPT AL DEFECT (VSD) A. Defect in the septum that divides the right and left ventricles 1. 2. Associated with fetal alcohol syndrome B. Results in left-to-right shunt; size of defect determines extent of shunting and age at presentation. Small defects are often asymptomatic; large defects can lead to Eisenmenger syndrome. C. Treatment involves surgical closure; small defects may close spontaneously. III. ATRIAL SEPTAL DEFECT (ASD) A. Defect in the septum that divides right and left atria; most common type is ostium secundum (90% of cases).

1	III. ATRIAL SEPTAL DEFECT (ASD) A. Defect in the septum that divides right and left atria; most common type is ostium secundum (90% of cases). B. Ostium primum type is associated with Down syndrome. C. Results in left-to-right shunt and split S2 on auscultation (increased blood in right heart delays closure of pulmonary valve) D. Paradoxical emboli are an important complication. IV. A. Failure of ductus arteriosus to close; associated with congenital rubella B. Results in left-to-right shunt between the aorta and the pulmonary artery 1. During development, the ductus arteriosus normally shunts blood from the pulmonary artery to the aorta, bypassing the lungs. C. Asymptomatic at birth with continuous 'machine-like' murmur; may lead to Eisenmenger syndrome, resulting in lower extremity cyanosis D. Treatment involves indomethacin, which decreases PGE, resulting in PDA closure (PGE maintains patency of the ductus arteriosus). V.

1	D. Treatment involves indomethacin, which decreases PGE, resulting in PDA closure (PGE maintains patency of the ductus arteriosus). V. A. Characterized by (1) stenosis of the right ventricular outflow tract, (2) right ventricular hypertrophy, (3) VSD, and (4) an aorta that overrides the VSD B. Right-to-left shunt leads to early cyanosis; degree of stenosis determines the extent of shunting and cyanosis. C. Patients learn to squat in response to a cyanotic spell; increased arterial resistance decreases shunting and allows more blood to reach the lungs. D. 'Boot-shaped' heart on x-ray (Fig. 8.10) VI. TRANSPOSITION OF THE GREAT VESSELS A. Characterized by pulmonary artery arising from the left ventricle and aorta arising from the right ventricle B. Associated with maternal diabetes C. Presents with early cyanosis; pulmonary and systemic circuits do not mix. 1. Creation of shunt (allowing blood to mix) after birth is required for survival. 2.

1	C. Presents with early cyanosis; pulmonary and systemic circuits do not mix. 1. Creation of shunt (allowing blood to mix) after birth is required for survival. 2. PGE can be administered to maintain a PDA until definitive surgical repair is performed. D. Results in hypertrophy of the right ventricle and atrophy of the left ventricle VII. TRUNCUS ARTERIOSUS A. Characterized by a single large vessel arising from both ventricles 1. Truncus fails to divide. B. Presents with early cyanosis; deoxygenated blood from right ventricle mixes with oxygenated blood from left ventricle before pulmonary and aortic circulations separate. VIII. TRI CUSPID ATRESIA A. Tricuspid valve orifice fails to develop; right ventricle is hypoplastic. B. Often associated with ASD, resulting in a right-to-left shunt; presents with early cyanosis. IX. COARCTATION OF THE AORTA A. Narrowing of the aorta (Fig. 8.llA); classically divided into infantile and adult forms

1	IX. COARCTATION OF THE AORTA A. Narrowing of the aorta (Fig. 8.llA); classically divided into infantile and adult forms B. Infantile form is associated with a PDA; coarctation lies after (distal to) the aortic arch, but before (proximal to) the PDA. 1. Presents as lower extremity cyanosis in infants, often at birth 2. Associated with Turner syndrome C. Adult form is not associated with a PDA; coarctation lies after (distal to) the aortic arch. 1. Presents as hypertension in the upper extremities and hypotension with weak pulses in the lower extremities; classically discovered in adulthood 2. Collateral circulation develops across the intercostal arteries; engorged arteries cause 'notching' of ribs on x-ray (Fig. 8.llB), 3. Associated with bicuspid aortic valve I. BASIC PRINCIPLES A. The heart has four valves (tricuspid, pulmonary, mitral, and aortic) that prevent backflow.

1	Associated with bicuspid aortic valve I. BASIC PRINCIPLES A. The heart has four valves (tricuspid, pulmonary, mitral, and aortic) that prevent backflow. B. Valvular lesions generally result in stenosis (decreased caliber of the valve orifice) or regurgitation (backflow). Fig. 8.11 Coarctation of the aorta. A, Gross specimen. B, 'Notching of ribs' on x-ray. (A, Courtesy of Aliya Husain, MD. B, Courtesy of Images Paediatr Cardio/ 2009;38:7-9) II. ACUTE RHEUMATIC FEVER A. Systemic complication of pharyngitis due to group A ~-hemolytic streptococci; affects children 2-3 weeks after an episode ofstreptococcal pharyngitis ("strep throat") B. Caused by molecular mimicry; bacterial M protein resembles proteins in human tissue. C. Diagnosis is based on Jones criteria. 1. Evidence of prior group A ~-hemolytic streptococcal infection (e.g., elevated ASO or anti-DNase B titers) with the presence of major and minor criteria 2.

1	1. Evidence of prior group A ~-hemolytic streptococcal infection (e.g., elevated ASO or anti-DNase B titers) with the presence of major and minor criteria 2. Minor criteria are nonspecific and include fever and elevated ESR. 3. i. Migratory polyarthritis-swelling and pain in a large joint (e.g., wrist, knees, ankles) that resolves within days and "migrates" to involve another large joint 11. Pancarditis a. Endocarditis-Mitral valve is involved more commonly than the aortic valve. Characterized by small vegetations along lines of closure that lead to regurgitation (Fig. 8.12A) b. Myocarditis with Aschoff bodies that are characterized by foci of chronic inflammation, reactive histiocytes with slender, wavy nuclei (Anitschkow cells), giant cells, and fibrinoid material (Fig. 8.12B,C); myocarditis is the most common cause of death during the acute phase. c. Pericarditis-leads to friction rub and chest pain iii. Subcutaneous nodules iv.

1	c. Pericarditis-leads to friction rub and chest pain iii. Subcutaneous nodules iv. Erythema marginatum-annular, nonpruritic rash with erythematous borders, commonly involving trunk and limbs v. Sydenham chorea (rapid, involuntary muscle movements) D. Acute attack usually resolves, but may progress to chronic rheumatic heart disease; repeat exposure to group A ~-hemolytic streptococci results in relapse of the acute phase and increases risk for chronic disease. III. CHRONIC RHEUMATIC HEART DISEASE A. Valve scarring that arises as a consequence of rheumatic fever B. Results in stenosis with a classic 'fish-mouth ' appearance 1. Almost always involves the mitral valve; leads to thickening of chordae tendineae and cusps 2. Occasionally involves the aortic valve; leads to fusion of the commissures (Fig. 8.13) 3. Other valves are less commonly involved. C. Complications include infectious endocarditis.

1	Occasionally involves the aortic valve; leads to fusion of the commissures (Fig. 8.13) 3. Other valves are less commonly involved. C. Complications include infectious endocarditis. Fig. 8.12 Acute rheumatic heart disease. A, Mitra! valve vegetations. B, Aschoff body involving myocardium. C, Anitschkow cells. (A, Courtesy of Aliya Husain, MD. B, Courtesy of Ed Uthman, MD) IV. A. Narrowing of the aortic valve orifice (Fig. 8.14) B. Usually due to fibrosis and calcification from "wear and tear" 1. 2. Bicuspid aortic valve increases risk and hastens disease onset. A normal aortic valve has three cusps; fewer cusps results in increased "wear and tear" on each cusp. C. May also arise as a consequence of chronic rheumatic valve disease; coexisting mitral stenosis and fusion of the aortic valve commissures distinguish rheumatic disease from "wear and tear."

1	C. May also arise as a consequence of chronic rheumatic valve disease; coexisting mitral stenosis and fusion of the aortic valve commissures distinguish rheumatic disease from "wear and tear." D. Cardiac compensation leads to a prolonged asymptomatic stage during which a systolic ejection click followed by a crescendo-decrescendo murmur is heard. E. Complications include 1. Concentric left ventricular hypertrophy-may progress to cardiac failure 2. Angina and syncope with exercise-Limited ability to increase blood flow across the stenotic valve leads to decreased perfusion of the myocardium and brain. 3. Microangiopathic hemolytic anemia-RBCs are damaged (producing schistocytes) while crossing the calcified valve. F. Treatment is valve replacement after onset of complications. V. A. Backflow of blood from the aorta into the left ventricle during diastole

1	F. Treatment is valve replacement after onset of complications. V. A. Backflow of blood from the aorta into the left ventricle during diastole B. Arises due to aortic root dilation (e.g., syphilitic aneurysm and aortic dissection) or valve damage (e.g., infectious endocarditis); most common cause is isolated root dilation C. Clinical features include 1. Early, blowing diastolic murmur 2. Hyperdynamic circulation due to increased pulse pressure i. Pulse pressure is the difference between systolic and diastolic pressures. ii. Diastolic pressure decreases due to regurgitation, while systolic pressure increases due to increased stroke volume. iii. Presents with bounding pulse (water-hammer pulse), pulsating nail bed (Quincke pulse), and head bobbing 3. Results in LV dilation and eccentric hypertrophy (due to volume overload) D. Treatment is valve replacement once LV dysfunction develops. VI. MITRAL VALVE PROLAPSE

1	Results in LV dilation and eccentric hypertrophy (due to volume overload) D. Treatment is valve replacement once LV dysfunction develops. VI. MITRAL VALVE PROLAPSE A. Ballooning of mitral valve into left atrium during systole 1. Seen in 2-3% of US adults B. Due to myxoid degeneration (accumulation of ground substance) of the valve, making it floppy (Fig. 8.15) 1. Etiology is unknown; may be seen in Marfan syndrome or Ehlers-Dantos syndrome C. Presents with an incidental mid-systolic click followed by a regurgitation murmur; usually asymptomatic 1. Click and murmur become softer with squatting (increased systemic resistance decreases left ventricular emptying). D. Complications are rare, but include infectious endocarditis, arrhythmia, and severe mitral regurgitation. E. Treatment is valve replacement. VII. MITRAL REGURGITATION A. Reflux of blood from the left ventricle into the left atrium during systole

1	E. Treatment is valve replacement. VII. MITRAL REGURGITATION A. Reflux of blood from the left ventricle into the left atrium during systole B. Usually arises as a complication of mitral valve prolapse; other causes include LV dilatation (e.g., left-sided cardiac failure), infective endocarditis, acute rheumatic heart disease, and papillary muscle rupture after a myocardial infarction. C. Clinical features 1. Holosystolic "blowing" murmur; louder with squatting (increased systemic resistance decreases left ventricular emptying) and expiration (increased return to left atrium) 2. VIII.MITRAL STENOSIS A. Narrowing of the mitral valve orifice 1. Usually due to chronic rheumatic valve disease B. Clinical features 1. 2.

1	VIII.MITRAL STENOSIS A. Narrowing of the mitral valve orifice 1. Usually due to chronic rheumatic valve disease B. Clinical features 1. 2. Volume overload leads to dilatation of the left atrium, resulting in i. Pulmonary congestion with edema and alveolar hemorrhage ii. Pulmonary hypertension and eventual right-sided heart failure iii. Atrial fibrillation with associated risk for mural thrombi (Fig. 8.16) I. BASIC PRINCIPLES A. Inflammation of endocardium that lines the surface of cardiac valves; usually due to bacterial infection B. Streptococcus viridans is the most common overall cause. It is a low-virulence organism that infects previously damaged valves (e.g., chronic rheumatic heart Fig. 8.16 Mural t hrombus involving left atrium. Fig. 8.17A Large vegetations involving tricuspid Fig. 8.17B Janeway lesions. (Courtesy of Aliya Husain, MD) valve. (Courtesy of Aliya Husain, MD) I.

1	Fig. 8.16 Mural t hrombus involving left atrium. Fig. 8.17A Large vegetations involving tricuspid Fig. 8.17B Janeway lesions. (Courtesy of Aliya Husain, MD) valve. (Courtesy of Aliya Husain, MD) I. disease and mitral valve prolapse). Results in small vegetations that do not destroy the valve (subacute endocarditis) 1. Damaged endocardial surface develops thrombotic vegetations (platelets and fibrin). 2. Transient bacteremia leads to trapping of bacteria in the vegetations; prophylactic antibiotics decrease risk of endocarditis. C. Staphylococcus aureus is the most common cause in IV drug abusers. 1. High-virulence organism that infects normal valves, most commonly the tricuspid. 2. Results in large vegetations that destroy the valve (acute endocarditis, Fig. 8.17A) D. Staphylococcus epidermidis is associated with endocarditis of prosthetic valves. E. Streptococcus bovis is associated with endocarditis in patients with underlying colorectal carcinoma.

1	D. Staphylococcus epidermidis is associated with endocarditis of prosthetic valves. E. Streptococcus bovis is associated with endocarditis in patients with underlying colorectal carcinoma. F. HACEK organisms (Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, Kingella) are associated with endocarditis with negative blood cultures. G. Clinical features of bacterial endocarditis include 1. Fever-due to bacteremia 2. Murmur-due to vegetations on heart valve 3. Janeway lesions (erythematous nontender lesions on palms and soles, Fig. 8.17B), Osler nodes (tender lesions on fingers or toes), splinter hemorrhages in nail bed (Fig. 8.17C) and Roth spots (Fig. 8.17D)-due to embolization of septic vegetations 4. Anemia of chronic disease-due to chronic inflammation H. Laboratory findings 1. 2. Anemia of chronic disease(-!-Hb, -!-MCV; t ferritin,-!-TIBC,-!-serum iron, and -!-% saturation) 3. Transesophageal echocardiogram is useful for detecting lesions on valves.

1	2. Anemia of chronic disease(-!-Hb, -!-MCV; t ferritin,-!-TIBC,-!-serum iron, and -!-% saturation) 3. Transesophageal echocardiogram is useful for detecting lesions on valves. I. Nonbacterial thrombotic endocarditis is due to sterile vegetations that arise in association with a hypercoagulable state or underlying adenocarcinoma. Vegetations arise on the mitral valve along lines of closure and result in mitral regurgitation. J. Libman-Sacks endocarditis is due to sterile vegetations that arise in association with SLE. Vegetations are present on the surface and undersurface of the mitral valve and result in mitral regurgitation. A. Group of myocardial diseases that result in cardiac dysfunction Fig. 8.17C Splinter hemorrhages. Fig. 8.17D Roth spots. Fig. 8.18 Dilated cardiomyopathy. (Courtesy of Jamie Steinmetz, MD) ____________c_a_r_d_i_a_c_P_a_th_o_l_o...g...y_ ---~---~----~----- II. DILATED CARDIOMYOPATHY

1	II. DILATED CARDIOMYOPATHY A. Dilation of all four chambers of the heart (Fig. 8.18); most common form of cardiomyopathy B. Results in systolic dysfunction (ventricles cannot pump), leading to biventricular CHF; complications include mitral and tricuspid valve regurgitation and arrhythmia. C. Most commonly idiopathic; other causes include 1. 2. Myocarditis (usually due to coxsackie A or B)-characterized by a lymphocytic infiltrate in the myocardium (Fig. 8.19); results in chest pain, arrhythmia with sudden death, or heart failure. Dilated cardiomyopathy is a late complication. 3. 4. Drugs (e.g., doxorubicin) 5. Pregnancy-seen during late pregnancy or soon (weeks to months) after childbirth 6. D. Treatment is heart transplant. III. HYPERTROPHIC CARDIOMYOPATHY A. Massive hypertrophy of the left ventricle B. Usually due to genetic mutations in sarcomere proteins; most common form is autosomal dominant. C. Clinical features include 1.

1	A. Massive hypertrophy of the left ventricle B. Usually due to genetic mutations in sarcomere proteins; most common form is autosomal dominant. C. Clinical features include 1. Decreased cardiac output-Left ventricular hypertrophy leads to diastolic dysfunction (ventricle cannot fill). 2. Sudden death due to ventricular arrhythmias; hypertrophic cardiomyopathy is a common cause of sudden death in young athletes. 3. Syncope with exercise-Subaortic hypertrophy of the ventricular septum results in functional aortic stenosis. D. Biopsy shows myofiber hypertrophy with disarray (Fig. 8.20). IV. RESTRICTIVE CARDIOMYOPATHY A. Decreased compliance of the ventricular endomyocardium that restricts filling during diastole B. Causes include amyloidosis, sarcoidosis, endocardial fibroelastosis (children, Fig. 8.21), and Loeffler syndrome (endomyocardial fibrosis with an eosinophilic infiltrate and eosinophilia).

1	B. Causes include amyloidosis, sarcoidosis, endocardial fibroelastosis (children, Fig. 8.21), and Loeffler syndrome (endomyocardial fibrosis with an eosinophilic infiltrate and eosinophilia). C. Presents as congestive heart failure; classic finding is low-voltage EKG with diminished QRS amplitude. Fig. 8.19 Myocarditis. Fig. 8.20 Myofiber disarray, hypertrophic Fig. 8.21 Endocardial fibroelastosis. (Courtesy of cardiomyopathy. humpath.com) I. MYXOMA A. Benign mesenchymal tumor with a gelatinous appearance and abundant ground substance on histology 1. Most common primary cardiac tumor in adults B. Usually forms a pedunculated mass in the left atrium that causes syncope due to obstruction of the mitral valve II. RHABDOMYOMA A. Benign hamartoma of cardiac muscle 1. Most common primary cardiac tumor in children; associated with tuberous sclerosis B. Usually arises in the ventricle III. METASTASIS A. Metastatic tumors are more common in the heart than primary tumors.

1	B. Usually arises in the ventricle III. METASTASIS A. Metastatic tumors are more common in the heart than primary tumors. 1. Common metastases to the heart include breast and lung carcinoma, melanoma, and lymphoma. B. Most commonly involve the pericardium, resulting in a pericardia! effusion I. RHINITIS A. Inflammation of the nasal mucosa; rhinovirus is the most common cause. B. Presents with sneezing, congestion, and runny nose (common cold) C. Allergic rhinitis is a subtype of rhinitis due to a type I hypersensitivity reaction (e.g., to pollen) 1. Characterized by an inflammatory infiltrate with eosinophils 2. Associated with asthma and eczema II. NASAL POLYP A. Protrusion of edematous, inflamed nasal mucosa B. Usually secondary to repeated bouts of rhinitis; also occurs in cystic fibrosis and aspirin-intolerant asthma 1. Aspirin-intolerant asthma is characterized by the triad of asthma, aspirininduced bronchospasms, and nasal polyps; seen in 10% of asthmatic adults

1	III. ANGIOFIBROMA A. Benign tumor of nasal mucosa composed of large blood vessels and fibrous tissue; classically seen in adolescent males B. Presents with profuse epistaxis IV. NASO PHARYNGEAL CARCINOMA A. Malignant tumor of nasopharyngeal epithelium B. Associated with EBY; classically seen in African children and Chinese adults C. Biopsy usually reveals pleomorphic keratin-positive epithelial cells (poorly differentiated squamous cell carcinoma) in a background of lymphocytes. D. Often presents with involvement of cervical lymph nodes I. ACUTE EPIGLOTTITIS A. Inflammation of the epiglottis (Fig. 9.1); H influenzae type b is the most common cause, especially in nonimmunized children. B. Presents with high fever, sore throat, drooling with dysphagia, muffled voice, and inspiratory stridor; risk ofairway obstruction II. LARYNGOTRACHEOBRONCHITIS (CROUP) A. Inflammation of the upper airway; parainfluenza virus is the most common cause.

1	II. LARYNGOTRACHEOBRONCHITIS (CROUP) A. Inflammation of the upper airway; parainfluenza virus is the most common cause. B. Presents with a hoarse, "barking" cough and inspiratory stridor III. VOCAL CORD NODULE (SINGER'S NODULE) A. Nodule that arises on the true vocal cord B. Due to excessive use of vocal cords; usually bilateral (Fig. 9.2A) 1. Composed of degenerative (myxoid) connective tissue (Fig. 9.2B) pathoma.com C. Presents with hoarseness; resolves with resting ofvoice IV. A. Benign papillary tumor of the vocal cord B. Due to HPV 6 and 11; papillomas are usually single in adults and multiple in children. C. Presents with hoarseness V. A. Squamous cell carcinoma usually arising from the epithelial lining of the vocal cord B. Risk factors are alcohol and tobacco; can rarely arise from a laryngeal papilloma C. Presents with hoarseness; other signs include cough and stridor. I. PNEUMONIA A. Infection of the lung parenchyma

1	C. Presents with hoarseness; other signs include cough and stridor. I. PNEUMONIA A. Infection of the lung parenchyma B. Occurs when normal defenses are impaired (e.g., impaired cough reflex, damage to mucociliary escalator, or mucus plugging) or organism is highly virulent. C. Clinical features include fever and chills, productive cough with yellow-green (pus) or rusty (bloody) sputum, tachypnea with pleuritic chest pain, decreased breath sounds, crackles, dullness to percussion, and elevated WBC count. D. Diagnosis is made by chest x-ray, sputum gram stain and culture, and blood cultures. E. Three patterns are classically seen on chest x-ray: lobar pneumonia, bronchopneumonia, and interstitial pneumonia. Fig. 9.1 Acute epiglottitis. (Courtesy of Stephanie Fig. 9.2 Vocal cord nodules. A, Gross appearance. B, Microscopic appearance. (B, Reproduced from Rozell, MD) wikipedia.org, CCBY-SA 3.0) .( • ~;"I. ❖ ~:;: , • "' . • •~~ '• . •. r,;. ·:£;1« ·"fl:

1	·:£;1« ·"fl: Fig. 9.3 Lobar pneumonia. A, X-ray appearance. B, Red hepatization. C, Acute inflammation involving alveolar sacs. (A, Courtesy of James Heilman, MD, Wikipedia. B, Courtesy of Yale Rosen, MD) II. LOBAR PNEUMONIA A. Characterized by consolidation of an entire lobe of the lung (Fig. 9.3A) B. Usually bacterial; most common causes are Streptococcus pneumoniae (95%) and Klebsiella pneumoniae (Table 9.1) C. Classic gross phases oflobar pneumonia 1. Congestion-due to congested vessels and edema 2. Red hepatization-due to exudate, neutrophils, and hemorrhage filling the alveolar air spaces, giving the normally spongy lung a solid consistency (Fig. 9.3B,C) 3. Gray hepatization-due to degradation of red cells within the exudate 4. Ill. BRONCHOPNEUMONIA A. Characterized by scattered patchy consolidation centered around bronchioles; often multifocal and bilateral (Fig. 9.4) B. Caused by a variety of bacterial organisms (Table 9.2) IV.

1	A. Characterized by scattered patchy consolidation centered around bronchioles; often multifocal and bilateral (Fig. 9.4) B. Caused by a variety of bacterial organisms (Table 9.2) IV. A. Characterized by diffuse interstitial infiltrates (Fig. 9.5) B. Presents with relatively mild upper respiratory symptoms (minimal sputum and low fever); 'atypical' presentation C. Caused by bacteria or viruses (Table 9.3) V. A. Seen in patients at risk for aspiration (e.g., alcoholics and comatose patients) B. Most often due to anaerobic bacteria in the oropharynx (e.g., Bacteroides, Fusobacterium, and Peptococcus) Fig. 9.4 Bronchopneumonia. A, X-ray appearance. B, Gross appearance. (B, Courtesy of Yale Rosen, MD) Fig. 9.5 Interstitial pneumonia. A, X-ray appearance. B, Inflammatory infiltrate involving interstitium. Table 9.2: Causes of Bronchopneumonia 2nd most common cause of secondary pneumonia; often complicated by abscess or empyema

1	Table 9.2: Causes of Bronchopneumonia 2nd most common cause of secondary pneumonia; often complicated by abscess or empyema Common cause of secondary pneumonia and pneumonia superimposed on COPD (leads to exacerbation of COPD) Community-acquired pneumonia and pneumonia superimposed on COPD (leads to exacerbation of COPD) Community-acquired pneumonia, pneumonia superimposed on COPD, or pneumonia in immunocompromised states; transmitted from water source Intracellular organism that is best visualized by silver stain Table 9.3: Causes of Interstitial (Atypical) Pneumonia Most common cause of atypical pneumonia, usually affects young adults (classically, military recruits or college students living in a dormitory). Complications include autoimmune hemolytic anemia (IgM against I antigen on RBCs causes cold hemolytic anemia) and erythema multiforme. Not visible on gram stain due to lack ofcell wall Second most common cause of atypical pneumonia in young adults

1	Second most common cause of atypical pneumonia in young adults Most common cause of atypical pneumonia in infants Atypical pneumonia with posttransplant immunosuppression or chemotherapy Atypical pneumonia in the elderly, immunocompromised, and those with preexisting lung disease. Also increases the risk for superimposed S aureus or H influenzae bacterial pneumonia Atypical pneumonia with high fever (Q fever); seen in farmers and veterinarians (Coxiella spores are deposited on cattle by ticks or are present in cattle placentas). Coxiella is a rickettsial organism, but it is distinct from most rickettsiae because it (1) causes pneumonia, (2) does not require arthropod vector for transmission (survives as highly heat-resistant endospores), and (3) does not produce a skin rash. C. Classically results in a right lower lobe abscess 1. Anatomically, the right main stem bronchus branches at a less acute angle than the left. VI. TUBERCULOSIS (TB)

1	C. Classically results in a right lower lobe abscess 1. Anatomically, the right main stem bronchus branches at a less acute angle than the left. VI. TUBERCULOSIS (TB) A. Due to inhalation of aerosolized Mycobacterium tuberculosis B. Primary TB arises with initial exposure. 1. Results in focal, caseating necrosis in the lower lobe of the lung and hilar lymph nodes that undergoes fibrosis and calcification, forming a Ghon complex (Fig. 9.6A) 2. Primary TB is generally asymptomatic, but leads to a positive PPD. C. Secondary TB arises with reactivation ofMycobacterium tuberculosis. 1. Reactivation is commonly due to AIDS; may also be seen with aging 2. 3. Forms cavitary foci of caseous necrosis; may also lead to miliary pulmonary TB or tuberculous bronchopneumonia 4. Clinical features include fevers and night sweats, cough with hemoptysis, and weight loss. 5. Biopsy reveals caseating granulomas; AFB stain reveals acid-fast bacilli (Fig. 9.6B,C). 6.

1	Clinical features include fevers and night sweats, cough with hemoptysis, and weight loss. 5. Biopsy reveals caseating granulomas; AFB stain reveals acid-fast bacilli (Fig. 9.6B,C). 6. Systemic spread often occurs and can involve any tissue; common sites include meninges (meningitis), cervical lymph nodes, kidneys (sterile pyuria), and lumbar vertebrae (Pott disease). I. BASIC PRINCIPLES A. Group of diseases characterized by airway obstruction; lung does not empty, and air is trapped. 1. Volume ofair that can be forcefully expired is decreased (-.I, FVC), especially during the first second ofexpiration (-.l,-.l, FEV); results in -.I, FEV :FVC ratio 2. Total lung capacity (TLC) is usually increased due to air trapping. II. CHRONIC BRONCHITIS A. Chronic productive cough lasting at least 3 months over a minimum of 2 years; highly associated with smoking B. Characterized by hypertrophy of bronchial mucous glands (Fig. 9.7)

1	A. Chronic productive cough lasting at least 3 months over a minimum of 2 years; highly associated with smoking B. Characterized by hypertrophy of bronchial mucous glands (Fig. 9.7) Fig. 9.6 Tuberculosis. A, Ghon focus. B, Caseating granuloma. C, AFB staining of M tuberculosis. (A, Courtesy of Ya le Rosen, MD) 1. Leads to increased thickness of mucus glands relative to bronchial wall thickness (Reid index increases to > 50%; normal is < 40%). C. Clinical features 1. Productive cough due to excessive mucus production 2. Cyanosis ('blue bloaters')-Mucus plugs trap carbon dioxide; t Paco2 and ..I.-Pao2 3. Increased risk of infection and cor pulmonale III. EMPHYSEMA A. Destruction of alveolar air sacs (Fig. 9.8) 1. Loss of elastic recoil and collapse of airways during exhalation results in obstruction and air trapping. B. Due to imbalance of proteases and anti proteases 1. Inflammation in the lung normally leads to release of proteases by neutrophils and macrophages. 2.

1	B. Due to imbalance of proteases and anti proteases 1. Inflammation in the lung normally leads to release of proteases by neutrophils and macrophages. 2. a -anti trypsin (Al AT) neutralizes proteases. 3. Excessive inflammation or lack of AlAT leads to destruction of the alveolar air sacs. C. Smoking is the most common cause of emphysema. 1. Pollutants in smoke lead to excessive inflammation and protease-mediated damage. 2. Results in centriacinar emphysema that is most severe in the upper lobes D. AlAT deficiency is a rare cause of emphysema. 1. Lack of antiprotease leaves the air sacs vulnerable to protease-mediated damage. 2. Results in panacinar emphysema that is most severe in the lower lobes 3. Liver cirrhosis may also be present. i. Al AT deficiency is due to misfolding of the mutated protein. ii. Mutant AlAT accumulates in the endoplasmic reticulum ofhepatocytes, resulting in liver damage.

1	i. Al AT deficiency is due to misfolding of the mutated protein. ii. Mutant AlAT accumulates in the endoplasmic reticulum ofhepatocytes, resulting in liver damage. 111. Biopsy reveals pink, PAS-positive globules in hepatocytes (Fig. 9.9). 4. Disease severity is based on the degree of AlAT deficiency. i. PiM is the normal allele; two copies are usually expressed (PiMM). ii. PiZ is the most common clinically relevant mutation; results in significantly low levels of circulating AlAT iii. PiMZ heterozygotes are usually asymptomatic with decreased circulating levels of AlAT; however, significant risk for emphysema with smoking exists. iv. PiZZ homozygotes are at significant risk for panacinar emphysema and cirrhosis. Fig. 9.7 Chronic bronchitis. Fig. 9.8 Emphysema. A, Gross appearance. B, Microscopic appearance. (A, Courtesy of Yale Rosen, MD) E. Clinical features of emphysema include 1. Dyspnea and cough with minimal sputum 2.

1	E. Clinical features of emphysema include 1. Dyspnea and cough with minimal sputum 2. Prolonged expiration with pursed lips ('pink-puffer') 3. 4. Increased anterior-posterior diameter of chest ('barrel-chest,' Fig. 9.10) 5. Hypoxemia (due to destruction of capillaries in the alveolar sac) and cor pulmonale are late complications. IV. A. Reversible airway bronchoconstriction, most often due to allergic stimuli (atopic asthma) B. Presents in childhood; often associated with allergic rhinitis, eczema, and a family history of atopy C. Pathogenesis (type I hypersensitivity) 1. Allergens induce T H2 phenotype in CD4+ T cells of genetically susceptible individuals. 2. T H2 cells secrete IL-4 (mediates class switch to IgE), IL-5 (attracts eosinophils), and IL-10 (stimulates T H2 cells and inhibits T Hl). 3. Reexposure to allergen leads to IgE-mediated activation of mast cells.

1	3. Reexposure to allergen leads to IgE-mediated activation of mast cells. i. Release of preformed histamine granules and generation ofleukotrienes C4, D4, and E4 lead to bronchoconstriction, inflammation, and edema (earlyphase reaction). ii. Inflammation, especially major basic protein derived from eosinophils, damages cells and perpetuates bronchoconstriction (late-phase reaction). D. Clinical features are episodic and related to allergen exposure. 1. 2. Productive cough, classically with spiral-shaped mucus plugs (Curschmann spirals) and eosinophil-derived crystals (Charcot-Leyden crystals, Fig. 9.11). 3. Severe, unrelenting attack can result in status asthmaticus and death. E. Asthma may also arise from nonallergic causes (non-atopic asthma) such as exercise, viral infection, aspirin (e.g., aspirin intolerant asthma), and occupational exposures. V. A. Permanent dilatation of bronchi (Fig. 9.12); loss of airway tone results in air trapping.

1	V. A. Permanent dilatation of bronchi (Fig. 9.12); loss of airway tone results in air trapping. B. Due to severe inflammation with damage to airway walls. Causes include 1. 2. Primary ciliary dyskinesia-inherited defect of the dynein arm, which is necessary for ciliary movement. Associated with sinusitis, infertility (poor motility of sperm), and situs inversus (position of major organs is reversed, e.g., heart is on right side of thorax), which is called Kartagener syndrome Fig. 9.9 Al AT accumulation in hepatocytes. Fig. 9,10 Increased AP diameter, emphysema. Fig. 9.11 Charcot-Leyden crystals, asthma. (Courtesy of James Heilman, MD, Wikipedia) 3. 4. 5. Allergic bronchopulmonary aspergillosis-Hypersensitivity reaction to Aspergillus leads to chronic inflammatory damage; usually seen in individuals with asthma or cystic fibrosis C. Clinical features 1. Cough, dyspnea, and foul-smelling sputum 2.

1	C. Clinical features 1. Cough, dyspnea, and foul-smelling sputum 2. Complications include hypoxemia with cor pulmonale and secondary (AA) amyloidosis. I. BASIC PRINCIPLES A. Characterized by restricted filling of the lung; .,I.. TLC, .,I.. FEV , and .,1...,1.. FVC; FEV :FVC ratio is increased. B. Most commonly due to interstitial diseases of the lung; may also arise with chest wall abnormalities (e.g., massive obesity) II. IDIOPATHIC PULMONARY FIBROSIS A. Fibrosis oflung interstitium (Fig. 9.13) B. Etiology is unknown. Likely related to cyclical lung injury; TGF-~ from injured pneumocytes induces fibrosis. 1. Secondary causes of interstitial fibrosis such as drugs (e.g., bleomycin and amiodarone) and radiation therapy must be excluded. C. Clinical features 1. 2. Bilateral fibrosis on lung CT; initially seen in subpleural patches, but eventually results in extensive fibrosis with end-stage 'honeycomb' lung 3. 4. Definitive treatment is lung transplantation.

1	4. Definitive treatment is lung transplantation. III. PNEUMOCONIOSES A. Interstitial fibrosis due to occupational exposure; requires chronic exposure to small particles that are fibrogenic (Table 9.4) 1. Alveolar macrophages engulf foreign particles and induce fibrosis. IV. SARCOIDOSIS A. Systemic disease characterized by noncaseating granulomas in multiple organs; classically seen in African American females Fig. 9.12 Bronchiectasis. (Courtesy of Yale Rosen, Fig. 9.13 Interstitial fibrosis, idiopathic MD) pulmonary fibrosis. Table 9.4: Summary of Pneumoconioses Massive exposure leads to diffuse Coal Workers' fibrosis ('black lung'); associated Pneumoconiosis with rheumatoid arthritis (Caplan syndrome) silica miners the lung Beryllium; seen in beryllium Noncaseating granulomas in the Berylliosis miners and workers in the lung, hilar lymph nodes, and aerospace industry systemic organs

1	Beryllium; seen in beryllium Noncaseating granulomas in the Berylliosis miners and workers in the lung, hilar lymph nodes, and aerospace industry systemic organs Fibrosis oflung and pleura (plaques) with increased risk for Asbestos fibers; seen in lung carcinoma and mesothelioma;Asbestosis construction workers, plumbers, lung carcinoma is more commonand shipyard workers than mesothelioma in exposed individuals. B. Etiology is unknown; likely due to CD4 • helper T-cell response to an unknown antigen C. Granulomas most commonly involve the hilar lymph nodes and lung (Fig. 9.15A), leading to restrictive lung disease. 1. Characteristic stellate inclusions ('asteroid bodies') are often seen within giant cells of the granulomas (Fig. 9.15B). D. Other commonly involved tissues include the uvea (uveitis), skin (cutaneous nodules or erythema nodosum), and salivary and lacrimal glands (mimics Sjogren syndrome); almost any tissue can be involved. E. Clinical features 1. 2. 3.

1	E. Clinical features 1. 2. 3. Hypercalcemia (I-alpha hydroxylase activity of epithelioid histiocytes converts vitamin D to its active form) 4. Treatment is immunosuppression; often resolves spontaneously without treatment. Mild exposure to carbon (e.g., pollution) results in anthracosis (collections of carbon-laden macrophages); not clinically significant Increased risk for TB; silica impairs phagolysosome formation by macrophages. Lesions may contain long, goldenbrown fibers with associated iron (asbestos bodies, Fig. 9.14), which confirm exposure to asbestos Fig. 9.14 Asbestos bodies. (Courtesy of Nephron, Fig. 9.15 Sarcoidosis. A, Noncaseating granuloma involving lung. B, Asteroid body. GNU-FOL vl.3) V. HYPERSENSITIVITY PNEUMONITIS A. Granulomatous reaction to inhaled organic antigens (e.g., pigeon breeder's lung) B. Presents with fever, cough, and dyspnea hours after exposure; resolves with removal of the exposure C. Chronic exposure leads to interstitial fibrosis.

1	B. Presents with fever, cough, and dyspnea hours after exposure; resolves with removal of the exposure C. Chronic exposure leads to interstitial fibrosis. I. BASIC PRINCIPLES A. High pressure in the pulmonary circuit (mean arterial pressure > 25 mm Hg; normal is 10 mm Hg) B. Characterized by atherosclerosis of the pulmonary trunk, smooth muscle hypertrophy of pulmonary arteries, and intimal fibrosis; plexiform lesions are seen with severe, long-standing disease (Fig. 9.16). C. Leads to right ventricular hypertrophy with eventual cor pulmonale D. Presents with exertional dyspnea or right-sided heart failure E. Subclassified as primary or secondary based on etiology II. PRIMARY PULMONARY HYPERTENSION A. Classically seen in young adult females B. Etiology is unknown; some familial forms are related to inactivating mutations of BMPR2, leading to proliferation ofvascular smooth muscle. III. SECONDARY PULMONARY HYPERTENSION

1	B. Etiology is unknown; some familial forms are related to inactivating mutations of BMPR2, leading to proliferation ofvascular smooth muscle. III. SECONDARY PULMONARY HYPERTENSION A. Due to hypoxemia (e.g., COPD and interstitial lung disease) or increased volume in the pulmonary circuit (e.g., congenital heart disease); may also arise with recurrent pulmonary embolism I. ACUTE RESPIRATORY DISTRESS SYNDROME A. Diffuse damage to the alveolar-capillary interface (diffuse alveolar damage) B. Leakage of protein-rich fluid leads to edema that combines with necrotic epithelial cells to form hyaline membranes lining alveoli (Fig. 9.17A). C. Clinical features 1. Hypoxemia and cyanosis with respiratory distress-due to thickened diffusion barrier and collapse of air sacs (increased surface tension) 2. 'White-out' on chest x-ray (Fig. 9.17B)

1	Hypoxemia and cyanosis with respiratory distress-due to thickened diffusion barrier and collapse of air sacs (increased surface tension) 2. 'White-out' on chest x-ray (Fig. 9.17B) D. Secondary to a variety of disease processes including sepsis, infection, shock, trauma, aspiration, pancreatitis, DIC, hypersensitivity reactions, and drugs. Fig. 9.16 Plexiform lesion, primary pulmonary Fig. 9.17 ARDS. A, Hyaline membranes. B, 'White-out' of lung on x-ray. (B, Courtesy of Samir, GNUhypertension. FDL vl.2) 1. Activation of neutrophils induces protease-and free radical-mediated damage of type I and II pneumocytes. E. Treatment 1. 2. Ventilation with positive end-expiratory pressure (PEEP) F. Recovery may be complicated by interstitial fibrosis; damage and loss of type II pneumocytes leads to scarring and fibrosis. II. NEONATAL RESPIRATORY DISTRESS SYNDROME A. Respiratory distress due to inadequate surfactant levels 1.

1	II. NEONATAL RESPIRATORY DISTRESS SYNDROME A. Respiratory distress due to inadequate surfactant levels 1. Surfactant is made by type II pneumocytes; phosphatidylcholine (lecithin) is the major component. 2. Surfactant decreases surface tension in the lung, preventing collapse of alveolar air sacs after expiration. 3. Lack of surfactant leads to collapse of air sacs and formation of hyaline membranes. B. Associated with 1. Prematurity-Surfactant production begins at 28 weeks; adequate levels are not reached until 34 weeks. i. Amniotic fluid lecithin to sphingomyelin ratio is used to screen for lung maturity. ii. Phosphatidylcholine (lecithin) levels increase as surfactant is produced; sphingomyelin remains constant. iii. A ratio > 2 indicates adequate surfactant production. 2. Caesarian section delivery-due to lack of stress-induced steroids; steroids increase synthesis of surfactant. 3. Maternal diabetes-Insulin decreases surfactant production. C. Clinical features 1.

1	Caesarian section delivery-due to lack of stress-induced steroids; steroids increase synthesis of surfactant. 3. Maternal diabetes-Insulin decreases surfactant production. C. Clinical features 1. Increasing respiratory effort after birth, tachypnea with use of accessory muscles, and grunting 2. Hypoxemia with cyanosis 3. Diffuse granularity of the lung ('ground-glass' appearance) on x-ray (Fig. 9.18) D. Complications 1. Hypoxemia increases the risk for persistence of patent ductus arteriosus and necrotizing enterocolitis. 2. Supplemental oxygen increases the risk for free radical injury. Retinal injury leads to blindness; lung damage leads to bronchopulmonary dysplasia. I. BASIC PRINCIPLES A. Most common cause of cancer mortality in the US; average age at presentation is 60 years. B. Key risk factors are cigarette smoke, radon, and asbestos. 1. Cigarette smoke contains over 60 carcinogens; 85% oflung cancer occurs in smokers.

1	B. Key risk factors are cigarette smoke, radon, and asbestos. 1. Cigarette smoke contains over 60 carcinogens; 85% oflung cancer occurs in smokers. i. Polycyclic aromatic hydrocarbons and arsenic are particularly mutagenic. ii. Cancer risk is directly related to the duration and amount of smoking ('packyears'). 2. Radon is formed by radioactive decay of uranium, which is present in soil. i. Accumulates in closed spaces such as basements ii. Responsible for most of the public exposure to ionizing radiation; 2nd most frequent cause oflung carcinoma in US iii. Increased risk of lung cancer is also seen in uranium miners. 3. Environmental toxins include asbestos, coal fueled stoves, and metals (arsenic, chromium, and nickel) C. Presenting symptoms are nonspecific (e.g., cough, hemoptysis, dyspnea, weight loss, and post-obstructive pneumonia).

1	C. Presenting symptoms are nonspecific (e.g., cough, hemoptysis, dyspnea, weight loss, and post-obstructive pneumonia). D. Imaging usually reveals a large, spiculated mass; however, a solitary nodule ('coinlesion') can be seen. Biopsy is necessary for a diagnosis of cancer. 1. Benign lesions may also produce a 'coin-lesion,' especially in younger patients. Examples include i. Granuloma-often due to TB or fungus (especially Histoplasma in the Midwest) ii. Bronchial hamartoma-benign tumor composed oflung tissue and cartilage; often calcified on imaging E. Lung carcinoma is classically divided into 2 categories (Table 9.5). 1. 2. Table 9.5: Cancers of the Lung , , HISTOLOGY Poorly differentiated small cells (Fig. 9.19) differentiation, chromogranin positive Glands, mucin (Fig. 9.20A), or TTF- Keratin pearls, Squamous cell intercellular bridges carcinoma (Fig. 9.22A,B), or p40

1	Glands, mucin (Fig. 9.20A), or TTF- Keratin pearls, Squamous cell intercellular bridges carcinoma (Fig. 9.22A,B), or p40 Poorly differentiated Large cell large cells (no glands, neuroendocrine mucin, TTF-1, keratin carcinoma pearls, intercellular bridges, or p40) (nests); chromogranin positive (Fig. 9.23A,B) Most common sources Metastasis to lung are breast and colon carcinoma. Small cell carcinoma (15%)-usually not amenable to surgical resection (treated with chemotherapy and radiation) Non-small cell carcinoma (85%) i. Usually amenable to surgical resection ii. Major subtypes include adenocarcinoma (50%), squamous cell carcinoma (30%), large cell neuroendocrine carcinoma (5%), and carcinoid tumor (5%). Not significantly related to smoking Peripheral (Fig. 9.20B) Central (Fig. 9.22C) Central or peripheral; when central, classically forms a polyp-like mass in the bronchus (Fig. 9.23C)

1	Not significantly related to smoking Peripheral (Fig. 9.20B) Central (Fig. 9.22C) Central or peripheral; when central, classically forms a polyp-like mass in the bronchus (Fig. 9.23C) Rapid growth and early metastasis; may produce endocrine (e.g., ADH or ACTH) or nervous system (e.g., Lambert-Eaton myasthenic syndrome) paraneoplastic syndromes Adenocarcinoma in-situ exhibits columnar cells that grow along preexisting bronchioles and alveoli (Fig. 9.21); may present as pneumonia-like consolidation on imaging Diagnosis of exclusion Low-grade malignancy; rarely, can cause carcinoid syndrome Fig. 9.18 Granularity of lung, neonatal respiratory Fig. 9.19 Small cell carcinoma. distress syndrome. (Published with permission from LearningRadiology.com) I .. ·.°' Fig. 9.20 Adenocarcinoma. A, Gland formation and mucin production. B, Peripheral location. (B, Fig. 9.21 Adenocarcinoma in-situ. Courtesy ofThomas Krausz, MD)

1	I .. ·.°' Fig. 9.20 Adenocarcinoma. A, Gland formation and mucin production. B, Peripheral location. (B, Fig. 9.21 Adenocarcinoma in-situ. Courtesy ofThomas Krausz, MD) Fig . 9.22 Squamous cell carcinoma. A, Keratin pearls. B, lntercellular bridges. C, Central location. (B, Courtesy Thomas Krausz, MD. C, Courtesy of Yale Rosen, MD) Fig. 9.23 Carcinoid tumor. A, Microscopic appea rance. B, Chromogranin expression by immunohistochemistry. C, Polyp-like growth in the bronchus. (C, Courtesy of Ya le Rosen, MD) F. TNM staging 1. T-Tumor size and local extension i. Obstruction of SVC leads to distended head and neck veins with edema and blue discoloration of arms and face (superior vena cava syndrome).

1	F. TNM staging 1. T-Tumor size and local extension i. Obstruction of SVC leads to distended head and neck veins with edema and blue discoloration of arms and face (superior vena cava syndrome). ii. Involvement of recurrent laryngeal (hoarseness) or phrenic (diaphragmatic paralysis) nerve iii. Involvement of the sympathetic chain (ptosis, miosis, and anhidrosis; Horner syndrome) and brachia! plexus (shoulder pain and hand weakness) is seen with apical tumors involving the superior sulcus (Pancoast tumor) 2. N-spread to regional lymph nodes (hilar and mediastinal) 3. M-a unique site of distant metastasis is the adrenal gland. 4. Overall, 15% 5-year survival; lung carcinoma often presents late i. Screening by low-dose CT recommended for patients with long smoking history 5. i. EGFR mutations (erlotinib) or ALK translocation (crizotinib) may be present in adenocarcinoma; EGFR is especially common in Asian females who are non-smokers. 6.

1	i. EGFR mutations (erlotinib) or ALK translocation (crizotinib) may be present in adenocarcinoma; EGFR is especially common in Asian females who are non-smokers. 6. Testing for PD-Ll expression (pembrolizumab) guides immunotherapy in advanced disease; PD-Ll may be present in any non-small cell carcinoma. I. PNEUMOTHORAX A. Accumulation of air in the pleural space B. Spontaneous pneumothorax is due to rupture of an emphysematous bleb; seen in young adults 1. Results in collapse of a portion of the lung (Fig. 9.24); trachea shifts to the side of collapse. C. Tension pneumothorax arises with penetrating chest wall injury. 1. Air enters the pleural space, but cannot exit; trachea is pushed opposite to the side of injury. 2. Medical emergency; treated with insertion of a chest tube II. MESOTHELIOMA A. Malignant neoplasm of mesothelial cells; highly associated with occupational exposure to asbestos

1	2. Medical emergency; treated with insertion of a chest tube II. MESOTHELIOMA A. Malignant neoplasm of mesothelial cells; highly associated with occupational exposure to asbestos B. Presents with recurrent pleural effusions, dyspnea, and chest pain; tumor encases the lung (Fig. 9.25). Fig. 9.24 Spontaneous pneumothorax. (Courtesy Fig. 9.25 Mesothelioma. (Courtesy of Jamie of James Heilman, MD, Wikiped ia.) Steinmetz, MD) I. CLEFT LIP AND PALATE A. Full-thickness defect of lip or palate (Fig. 10.1) B. Due to failure of facial prominences to fuse 1. During early pregnancy, facial prominences (one from superior, two from the sides, and two from inferior) grow and fuse together to form the face. C. Cleft lip and palate usually occur together; isolated cleft lip or palate is less common. II. APHTHOUS ULCER A. Painful, superficial ulceration of the oral mucosa (Fig. 10.2) B. Arises in relation to stress and resolves spontaneously, but often recurs

1	II. APHTHOUS ULCER A. Painful, superficial ulceration of the oral mucosa (Fig. 10.2) B. Arises in relation to stress and resolves spontaneously, but often recurs C. Characterized by a grayish base surrounded by erythema III. BEH<;:ET SYNDROME A. Recurrent aphthous ulcers, genital ulcers, and uveitis B. Due to immune complex vasculitis involving small vessels C. Can be seen after viral infection, but etiology is unknown IV. A. Vesicles involving oral mucosa that rupture, resulting in shallow, painful, red ulcers B. Usually due to HSV-1 C. Primary infection occurs in childhood; lesions heal, but virus remains dormant in ganglia of the trigeminal nerve. D. Stress and sunlight cause reactivation of the virus, leading to vesicles that often arise on the lips (cold sore, Fig. 10.3). V. A. Malignant neoplasm of squamous cells lining the oral mucosa B. Tobacco and alcohol are major risk factors. C. Floor of mouth is the most common location.

1	V. A. Malignant neoplasm of squamous cells lining the oral mucosa B. Tobacco and alcohol are major risk factors. C. Floor of mouth is the most common location. D. Oral leukoplakia and erythroplakia are precursor lesions. 1. Leukoplakia is a white plaque that cannot be scraped away; often represents squamous cell dysplasia 2. Leukoplakia is distinct from oral candidiasis (thrush) and hairy leukoplakia. i. Oral candidiasis is a white deposit on the tongue, which is easily scraped away (Fig. 10.4); usually seen in immunocompromised states ii. Hairy leukoplakia is a white, rough ('hairy') patch that arises on the lateral tongue. It is usually seen in immunocompromised individuals (e.g., AIDS) and is due to EBY-induced squamous cell hyperplasia; not pre-malignant 3. Erythroplakia (red plaque) represents vascularized leukoplakia and is highly suggestive of squamous cell dysplasia. 4. Erythroplakia and leukoplakia are often biopsied to rule out carcinoma. pathoma.com

1	4. Erythroplakia and leukoplakia are often biopsied to rule out carcinoma. pathoma.com I. BASIC PRINCIPLES A. Salivary glands are exocrine glands that secrete saliva. B. Divided into major (parotid, submandibular, and sublingual glands) and minor glands (hundreds of microscopic glands distributed throughout the oral mucosa) II. MUMPS A. Infection with mumps virus resulting in bilateral inflamed parotid glands B. Orchitis, pancreatitis, and aseptic meningitis may also be present. 1. Serum amylase is increased due to salivary gland or pancreatic involvement. 2. Orchitis carries risk of sterility, especially in teenagers. III. SIALADENITIS A. Inflammation of the salivary gland B. Most commonly due to an obstructing stone (sialolithiasis) leading to Staphylococcus aureus infection; usually unilateral IV. A. Benign tumor composed of stromal (e.g., cartilage) and epithelial tissue; most common tumor of the salivary gland

1	IV. A. Benign tumor composed of stromal (e.g., cartilage) and epithelial tissue; most common tumor of the salivary gland B. Usually arises in parotid; presents as a mobile, painless, circumscribed mass at the angle of the jaw C. High rate of recurrence; extension of small islands of tumor through tumor capsule often leads to incomplete resection (Fig. 10.5). D. Rarely may transform into carcinoma, which presents with signs of facial nerve damage (facial nerve runs through parotid gland) V. A. Benign cystic tumor with abundant lymphocytes and germinal centers (lymph node-like stroma); 2nd most common tumor of the salivary gland B. Almost always arises in the parotid VI. MUCOEPIDERMOID CARCINOMA A. Malignant tumor composed of mucinous and squamous cells; most common malignant tumor of the salivary gland B. Usually arises in the parotid; commonly involves the facial nerve

1	A. Malignant tumor composed of mucinous and squamous cells; most common malignant tumor of the salivary gland B. Usually arises in the parotid; commonly involves the facial nerve Fig, 10,1 Cleft lip and palate. (Courtesy of James Fig. 10.2 Aphthous ulcer. (Reproduced from Fig, 10.3 Cold sore. (Courtesy of Dr. Herrmann, Heilman, MD, Wikipedia) wikipedia.org, CCBY-SA 3.0) CDC) I. TRACHEOESOPHAGEAL FISTULA A. Congenital defect resulting in a connection between the esophagus and trachea B. Most common variant consists of proximal esophageal atresia with the distal esophagus arising from the trachea (Fig. 10.6). 1. Presents with vomiting, polyhydramnios, abdominal distension, and aspiration II. ESOPHAGEAL WEB A. Thin protrusion of esophageal mucosa, most often in the upper esophagus B. Presents with dysphagia for poorly chewed food C. Increased risk for esophageal squamous cell carcinoma

1	A. Thin protrusion of esophageal mucosa, most often in the upper esophagus B. Presents with dysphagia for poorly chewed food C. Increased risk for esophageal squamous cell carcinoma D. Plummer-Vinson syndrome is characterized by severe iron deficiency anemia, esophageal web, and beefy-red tongue due to atrophic glossitis. III. ZENKER DIVERTICULUM A. Outpouching of pharyngeal mucosa through an acquired defect in the muscular wall (false diverticulum) B. Arises above the upper esophageal sphincter at the junction of the esophagus and pharynx C. Presents with dysphagia, obstruction, and halitosis (bad breath) IV. A. Longitudinal laceration of mucosa at the gastroesophageal (GE) junction B. Caused by severe vomiting, usually due to alcoholism or bulimia C. Presents with painful hematemesis D. Risk ofBoerhaave syndrome-rupture of esophagus leading to air in the mediastinum and subcutaneous emphysema V. A. Dilated submucosal veins in the lower esophagus

1	D. Risk ofBoerhaave syndrome-rupture of esophagus leading to air in the mediastinum and subcutaneous emphysema V. A. Dilated submucosal veins in the lower esophagus B. Arise secondary to portal hypertension 1. Distal esophageal vein normally drains into the portal vein via the left gastric vein. 2. In portal hypertension, the left gastric vein backs up into the esophageal vein, resulting in dilation (varices). C. Asymptomatic, but risk of rupture exists 1. Presents with painless hematemesis 2. Most common cause of death in cirrhosis Fig. 10.4 Oral candidiasis. (Courtesy of James Fig. 10.5 Pleomorphic adenoma. (Courtesy of Fig. 10.6 Tracheoesophageal fistula. (Courtesy of Heilman, MD, Wikipedia) Bu lent Celasun, MD) humpath.com) VI. ACHALASIA A. Disordered esophageal motility with inability to relax the lower esophageal sphincter (LES) B. Due to damaged ganglion cells in the myenteric plexus 1.

1	VI. ACHALASIA A. Disordered esophageal motility with inability to relax the lower esophageal sphincter (LES) B. Due to damaged ganglion cells in the myenteric plexus 1. Ganglion cells of myenteric plexus are located between the inner circular and outer longitudinal layers of the muscularis propria and are important for regulating bowel motility and relaxing the LES. 2. Damage to ganglion cells can be idiopathic or secondary to a known insult (e.g., Trypanosoma cruzi infection in Chagas disease). C. Clinical features 1. 2. 3. 4. 'Bird-beak' sign on barium swallow study (Fig. 10.7) 5. VII. GASTROESOPHAGEAL REFLUX DISEASE (GERD) A. Reflux of acid from the stomach due to reduced LES tone B. Risk factors include alcohol, tobacco, obesity, fat-rich diet, caffeine, and hiatal hernia. C. Clinical features 1. 2. 3. Damage to enamel ofteeth 4. Ulceration with stricture and Barrett esophagus are late complications. VIII. BARRETT ESOPHAGUS

1	C. Clinical features 1. 2. 3. Damage to enamel ofteeth 4. Ulceration with stricture and Barrett esophagus are late complications. VIII. BARRETT ESOPHAGUS A. Metaplasia of the lower esophageal mucosa from stratified squamous epithelium to nonciliated columnar epithelium with goblet cells (Fig. 10.8); seen in 10% of patients with GERD 1. Response oflower esophageal stem cells to acidic stress B. May progress to dysplasia and adenocarcinoma IX. ESOPHAGEAL CARCINOMA A. Subclassified as adenocarcinoma or squamous cell carcinoma B. Adenocarcinoma is a malignant proliferation of glands; most common type of esophageal carcinoma in the West 1. Arises from preexisting Barrett esophagus; usually involves the lower one-third of the esophagus Fig. 10.7 'Bird-beak' sign, achalasia. (Courtesy of Fig. 10.8 Barrett esophagus. F. Farrokhi, MD and M. Vaezi, MD)

1	Fig. 10.7 'Bird-beak' sign, achalasia. (Courtesy of Fig. 10.8 Barrett esophagus. F. Farrokhi, MD and M. Vaezi, MD) C. Squamous cell carcinoma is a malignant proliferation of squamous cells; most common esophageal cancer worldwide 1. Usually arises in upper or middle third of the esophagus; major risk factors include 1. Alcohol and tobacco (most common causes) ii. Very hot tea iii. Achalasia iv. Esophageal web (e.g., Plummer-Vinson syndrome) v. Esophageal injury (e.g., lye ingestion) D. Esophageal carcinoma presents late (poor prognosis). 1. Symptoms include progressive dysphagia (solids to liquids), weight loss, pain, and hematemesis. 2. Squamous cell carcinoma may additionally present with hoarse voice (recurrent laryngeal nerve involvement) and cough (tracheal involvement). E. Location oflymph node spread depends on the level of the esophagus that is involved. 1. 2. 3. I. GASTROSCHISIS

1	E. Location oflymph node spread depends on the level of the esophagus that is involved. 1. 2. 3. I. GASTROSCHISIS A. Congenital malformation of the anterior abdominal wall leading to exposure of abdominal contents (Fig. 10.9) II. OMPHALOCELE A. Persistent herniation of bowel into umbilical cord B. Due to failure of herniated intestines to return to the body cavity during development 1. Contents are covered by peritoneum and amnion of the umbilical cord (Fig. 10.10). III. PYLORIC STENOSIS A. Congenital hypertrophy of pyloric smooth muscle; more common in males B. Classically presents two weeks after birth as 1. 2. 3. Olive-like mass in the abdomen C. Treatment is myotomy. Fig. 10.9 Gastroschisis. (Courtesy of humpath. Fig.10.10 Omphalocele. (Courtesy of J.T. Stocker, Fig.10.11 Intestinal metaplasia, chronic gastritis. com) MD) IV. A. Acidic damage to the stomach mucosa

1	IV. A. Acidic damage to the stomach mucosa B. Due to imbalance between mucosa! defenses and acidic environment 1. Defenses include mucin layer produced by foveolar cells, bicarbonate secretion by surface epithelium, and normal blood supply (provides nutrients and picks up leaked acid). C. Risk factors 1. Severe burn (Curling ulcer)-Hypovolemia leads to decreased blood supply. 2. 3. 4. 5. Increased intracranial pressure (Cushing ulcer)-Increased stimulation ofvagus nerve leads to increased acid production. 6. Shock-Multiple (stress) ulcers may be seen in ICU patients. D. Acid damage results in superficial inflammation, erosion (loss of superficial epithelium), or ulcer (loss of mucosa! layer). V. A. Chronic inflammation of stomach mucosa B. Divided into two types based on underlying etiology: chronic autoimmune gastritis and chronic H pylori gastritis

1	V. A. Chronic inflammation of stomach mucosa B. Divided into two types based on underlying etiology: chronic autoimmune gastritis and chronic H pylori gastritis C. Chronic autoimmune gastritis is due to autoimmune destruction of gastric parietal cells, which are located in the stomach body and fundus. 1. Associated with antibodies against parietal cells and/or intrinsic factor; useful for diagnosis, but pathogenesis is mediated by T cells (type IV hypersensitivity) 2. i. Atrophy of mucosa with intestinal metaplasia (Fig. 10.11) ii. Achlorhydria with increased gastrin levels and antral G-cell hyperplasia iii. Megaloblastic (pernicious) anemia due to lack of intrinsic factor iv. Increased risk for gastric adenocarcinoma (intestinal type) D. Chronic H pylori gastritis is due to H pylori-induced acute and chronic inflammation; most common form of gastritis (90%) 1.

1	D. Chronic H pylori gastritis is due to H pylori-induced acute and chronic inflammation; most common form of gastritis (90%) 1. H pylori ureases and proteases along with inflammation weaken mucosa! defenses; antrum is the most common site (Fig. 10.12). 2. Presents with epigastric abdominal pain; increased risk for ulceration (peptic ulcer disease), gastric adenocarcinoma (intestinal type), and MALT lymphoma 3. Treatment involves triple therapy. i. Resolves gastritis/ulcer and reverses intestinal metaplasia ii. Negative urea breath test and lack of stool antigen confirm eradication of Hpylori. Fig. 10.12 H pylori. (Courtesy of Ed Uthman, MD) Fig. 10.13 Gastric ulcer. A, Peptic ulcer disease. B, Carcinoma. (Courtesy of Aliya Husain, MD) VI. PEPTIC ULCER DISEASE A. Solitary mucosa! ulcer involving proximal duodenum (90%) or distal stomach (10%) B. Duodenal ulcer is almost always due to H pylori (> 95%); rarely, may be due to ZE syndrome 1.

1	A. Solitary mucosa! ulcer involving proximal duodenum (90%) or distal stomach (10%) B. Duodenal ulcer is almost always due to H pylori (> 95%); rarely, may be due to ZE syndrome 1. Presents with epigastric pain that improves with meals 2. Diagnostic endoscopic biopsy shows ulcer with hypertrophy of Brunner glands. 3. Usually arises in anterior duodenum; when present in posterior duodenum, rupture may lead to bleeding from the gastroduodenal artery or acute pancreatitis C. Gastric ulcer is usually due to H pylori (75%); other causes include NSAIDs and bile reflux. 1. Presents with epigastric pain that worsens with meals 2. Ulcer is usually located on the lesser curvature of the antrum. 3. Rupture carries risk of bleeding from left gastric artery. D. Differential diagnosis of ulcers includes carcinoma. 1. Duodenal ulcers are almost never malignant (duodenal carcinoma is extremely rare). 2. Gastric ulcers can be caused by gastric carcinoma (intestinal subtype).

1	1. Duodenal ulcers are almost never malignant (duodenal carcinoma is extremely rare). 2. Gastric ulcers can be caused by gastric carcinoma (intestinal subtype). i. Benign peptic ulcers are usually small (< 3 cm), sharply demarcated ("punched-out"), and surrounded by radiating folds of mucosa (Fig. 10.13A). 11. Malignant ulcers are large and irregular with heaped up margins (Fig. 10.13B) iii. Biopsy is required for definitive diagnosis. VII. GASTRIC CARCINOMA A. Malignant proliferation of surface epithelial cells (adenocarcinoma) B. Subclassified into intestinal and diffuse types C. Intestinal type (more common) presents as a large, irregular ulcer with heaped up margins; most commonly involves the lesser curvature of the antrum (similar to gastric ulcer) 1. Risk factors include intestinal metaplasia (e.g., due to H pylori and autoimmune gastritis), nitrosamines in smoked foods (Japan), and blood type A.

1	D. Diffuse type is characterized by signet ring cells that diffusely infiltrate the gastric wall (Fig. 10.14B); desmoplasia results in thickening of stomach wall (linitis plastica, Fig. 10.14A). 1. Not associated with H pylori, intestinal metaplasia, or nitrosamines E. Gastric carcinoma presents late with weight loss, abdominal pain, anemia, and early satiety; rarely presents as acanthosis nigricans or Leser-Trelat sign F. Spread to lymph nodes can involve the left supraclavicular node (Virchow node). G. Distant metastasis most commonly involves liver; other sites include 1. Periumbilical region (Sister Mary Joseph nodule); seen with intestinal type Fig. 10.14 Gastric carcinoma, diffuse type. A, Linitis plastica. B, Signet-ring cells. (A, Courtesy of Ed Uthman, MD) 2. Bilateral ovaries (Krukenberg tumor); seen with diffuse type I. DUODENAL ATRESIA A. Congenital failure of duodenum to canalize; associated with Down syndrome B. Clinical features 1. 2.

1	I. DUODENAL ATRESIA A. Congenital failure of duodenum to canalize; associated with Down syndrome B. Clinical features 1. 2. Distension of stomach and blind loop of duodenum ('double bubble' sign, Fig. 10.15) 3. II. MECKEL DIVERTICULUM A. Outpouching of all three layers of the bowel wall (true diverticulum, Fig. 10.16) B. Arises due to failure of the vitelline duct to involute C. 'Rule of 2s' 1. Seen in 2% of the population (most common congenital anomaly of the GI tract) 2. 2 inches long and located in the small bowel within 2 feet of the ileocecal valve 3. Can present during the first 2 years of life with bleeding (due to heterotopic gastric mucosa), volvulus, intussusception, or obstruction (mimics appendicitis); however, most cases are asymptomatic. III. VOLVULUS A. Twisting of bowel along its mesentery B. Results in obstruction and disruption of the blood supply with infarction (Fig. 10.17)

1	III. VOLVULUS A. Twisting of bowel along its mesentery B. Results in obstruction and disruption of the blood supply with infarction (Fig. 10.17) C. Most common locations are sigmoid colon (elderly) and cecum (young adults). IV. A. Telescoping of proximal segment of bowel forward into distal segment 1. Telescoped segment is pulled forward by peristalsis, resulting in obstruction and disruption of blood supply with infarction. B. Associated with a leading edge (focus of traction) 1. In children, the most common cause is lymphoid hyperplasia (e.g., due to rotavirus); usually arises in the terminal ileum, leading to intussusception into the cecum 2. In adults, the most common cause is tumor. V. A. Small bowel is highly susceptible to ischemic injury. 1. Transmural infarction occurs with thrombosis/embolism of the superior mesenteric artery or thrombosis of the mesenteric vein. 2. Mucosa! infarction occurs with marked hypotension.

1	1. Transmural infarction occurs with thrombosis/embolism of the superior mesenteric artery or thrombosis of the mesenteric vein. 2. Mucosa! infarction occurs with marked hypotension. B. Clinical features include abdominal pain, bloody diarrhea, and decreased bowel sounds. VI. LACTOSE INTOLERANCE A. Decreased function of the lactase enzyme found in the brush border of enterocytes 1. Lactase normally breaks down lactose into glucose and galactose. B. Presents with abdominal distension and diarrhea upon consumption of milk products; undigested lactose is osmotically active. C. Deficiency may be congenital (rare autosomal recessive disorder) or acquired (often develops in late childhood); temporary deficiency is seen after small bowel infection (]actase is highly susceptible to injury). VII. CELIAC DISEASE A. Immune-mediated damage of small bowel villi due to gluten exposure; associated with HLA-DQ2 and DQS

1	VII. CELIAC DISEASE A. Immune-mediated damage of small bowel villi due to gluten exposure; associated with HLA-DQ2 and DQS B. Gluten is present in wheat and grains; its most pathogenic component is gliadin. 1. Once absorbed, gliadin is deamidated by tissue transglutaminase (tTG). 2. Deamidated gliadin is presented by antigen presenting cells via MHC class II. 3. Helper T cells mediate tissue damage. C. Clinical presentation 1. Children classically present with abdominal distension, diarrhea, and failure to thrive. 2. Adults classically present with chronic diarrhea and bloating. 3. Small, herpes-like vesicles may arise on skin (dermatitis herpetiformis). Due to IgA deposition at the tips of dermal papillae; resolves with gluten-free diet D. Laboratory findings 1.

1	3. Small, herpes-like vesicles may arise on skin (dermatitis herpetiformis). Due to IgA deposition at the tips of dermal papillae; resolves with gluten-free diet D. Laboratory findings 1. IgA antibodies against endomysium, tTG, or gliadin; IgG antibodies are also present and are useful for diagnosis in individuals with IgA deficiency (increased incidence ofigA deficiency is seen in celiac disease). 2. Duodenal biopsy reveals flattening of villi, hyperplasia of crypts, and increased intraepithelial lymphocytes (Fig. 10.18). Damage is most prominent in the duodenum; jejunum and ileum are less involved. E. Symptoms resolve with gluten-free diet. 1. Small bowel carcinoma and T-cell lymphoma are late complications that present as refractory disease despite good dietary control. Fig. 10.18 Celiac disease. A, Flattened villi. B, Normal villi for comparison. VIII.TROPICAL SPRUE A. Damage to small bowel villi due to an unknown organism resulting in malabsorption

1	Fig. 10.18 Celiac disease. A, Flattened villi. B, Normal villi for comparison. VIII.TROPICAL SPRUE A. Damage to small bowel villi due to an unknown organism resulting in malabsorption B. Similar to celiac disease except 1. Occurs in tropical regions (e.g., Caribbean) 2. Arises after infectious diarrhea and responds to antibiotics 3. Damage is most prominent in jejunum and ileum (secondary vitamin B12 or folate deficiency may ensue); duodenum is less commonly involved. IX. A. Systemic tissue damage characterized by macrophages loaded with Tropheryma whippelii organisms; partially destroyed organisms are present in macrophage lysosomes (positive for PAS). B. Classic site of involvement is the small bowel lamina propria (Fig. 10.19). 1. Macrophages compress lacteals. 2. Chylomicrons cannot be transferred from enterocytes to lymphatics. 3. C. Other common sites ofinvolvement include synovium ofjoints (arthritis), cardiac valves, lymph nodes, and CNS. X.

1	2. Chylomicrons cannot be transferred from enterocytes to lymphatics. 3. C. Other common sites ofinvolvement include synovium ofjoints (arthritis), cardiac valves, lymph nodes, and CNS. X. A. Autosomal recessive deficiency of apolipoprotein B-48 and B-100 B. Clinical features 1. Malabsorption-due to defective chylomicron formation (requires B-48) 2. XI. CARCINOID TUMOR A. Malignant proliferation of neuroendocrine cells; low-grade malignancy 1. Tumor cells contain neurosecretory granules that are positive for chromogranin. B. Can arise anywhere along the gut; small bowel is the most common site. 1. Grows as a submucosal polyp-like nodule (Fig. 10.20) C. Often secretes serotonin 1. Serotonin is released into the portal circulation and metabolized by liver monoamine oxidase (MAO) into 5-HIAA. 2. 5-HIAA is excreted in the urine. D. Metastasis of carcinoid tumor to the liver allows serotonin to bypass liver metabolism.

1	2. 5-HIAA is excreted in the urine. D. Metastasis of carcinoid tumor to the liver allows serotonin to bypass liver metabolism. 1. Serotonin is released into the hepatic vein and leaks into systemic circulation via hepato-systemic shunts, resulting in carcinoid syndrome and carcinoid heart disease. Fig. 10.19 Whipple disease. A, Macrophages within lamina propria of small bowel villi. B, Twhippelii Fig. 10.20 Carcinoid tumor. organisms highlighted by PAS stain. 2. Carcinoid syndrome is characterized by bronchospasm, diarrhea, and flushing of skin; symptoms can be triggered by alcohol or emotional stress, which stimulate serotonin release from the tumor. 3. Carcinoid heart disease is characterized by right-sided valvular fibrosis (increased collagen) leading to tricuspid regurgitation and pulmonary valve stenosis; left-sided valvular lesions are not seen due to presence of monoamine oxidase (metabolizes serotonin) in the lung. I. ACUTE APPENDICITIS

1	I. ACUTE APPENDICITIS A. Acute inflammation of the appendix; most common cause of acute abdomen B. Related to obstruction of the appendix by lymphoid hyperplasia (children) or a fecalith (adults) C. Clinical features include 1. Periumbilical pain, fever, and nausea; pain eventually localizes to right lower quadrant (McBurney point). 2. Rupture results in peritonitis that presents with guarding and rebound tenderness. 3. Periappendiceal abscess is a common complication. I. BASIC PRINCIPLES A. Chronic, relapsing inflammation of bowel B. Possibly due to abnormal immune response to enteric flora C. Classically presents in young women (teens to 30s) as recurrent bouts of bloody diarrhea and abdominal pain 1. More prevalent in the West, particularly in Caucasians and Eastern European Jews D. Diagnosis of exclusion; symptoms mimic other causes of bowel inflammation (e.g., infection). E. Subclassified as ulcerative colitis or Crohn disease (Table 10.1) I. HIRSCHSPRUNG DISEASE

1	D. Diagnosis of exclusion; symptoms mimic other causes of bowel inflammation (e.g., infection). E. Subclassified as ulcerative colitis or Crohn disease (Table 10.1) I. HIRSCHSPRUNG DISEASE A. Defective relaxation and peristalsis of rectum and distal sigmoid colon 1. Associated with Down syndrome B. Due to congenital failure of ganglion cells (neural crest-derived) to descend into myenteric and submucosal plexus 1. Myenteric (Auerbach) plexus is located between the inner circular and outer longitudinal muscle layers of the muscularis propria and regulates motility. 2. Submucosal (Meissner) plexus is located in the submucosa and regulates blood flow, secretions, and absorption. C. Clinical features are based on obstruction. 1. Failure to pass meconium 2. 3. Massive dilatation (megacolon) of bowel proximal to obstruction with risk for rupture D. Rectal suction biopsy reveals lack of ganglion cells.

1	1. Failure to pass meconium 2. 3. Massive dilatation (megacolon) of bowel proximal to obstruction with risk for rupture D. Rectal suction biopsy reveals lack of ganglion cells. E. Treatment involves resection of the involved bowel; ganglion cells are present in the bowel proximal to the diseased segment. Table 10.1: Main Features of Ulcerative Colitis and Crohn Disease Begins in rectum and can extend proximally up to the cecum (involvement is continuous, Fig. 10.21A); remainder of the GI tract is unaffected. Left lower quadrant pain (rectum) with bloody diarrhea Crypt abscesses with neutrophils (Fig. 10.21B) Pseudopolyps; loss ofhaustra ('lead pipe' sign on imaging, Fig. 10.21C) Toxic megacolon and carcinoma (risk is based on extent of colonic involvement and duration of disease; generally not a concern until > 10 years of disease) CROHN DISEASE Full-thickness inflammation with knife-like fissures

1	Full-thickness inflammation with knife-like fissures Anywhere from mouth to anus with skip lesions; terminal ileum is the most common site, rectum is least common. Right lower quadrant pain (ileum) with nonbloody diarrhea Lymphoid aggregates with granulomas (40% of cases) Cobblestone mucosa (Fig. 10.22A), creeping fat, and strictures ('string-sign' on imaging, Fig. 10.22B) Malabsorption with nutritional deficiency, calcium oxalate nephrolithiasis, fistula formation, and carcinoma, if colonic disease is present Arthritis (peripheral joints, ankylosing spondylitis, sacroiliitis, migratory polyarthritis), uveitis, erythema nodosum (Fig. 10.22C), pyoderma gangrenosum, primary sclerosing cholangitis, and p-ANCA Fig. 10.21 Ulcerative colitis. A, Gross appearance. B, Crypt abscess. C, 'Lead pipe' sign (A, Courtesy of Jamie Steinmetz, MD. C, Published with permission from LearningRadiology.com)

1	Fig. 10.21 Ulcerative colitis. A, Gross appearance. B, Crypt abscess. C, 'Lead pipe' sign (A, Courtesy of Jamie Steinmetz, MD. C, Published with permission from LearningRadiology.com) Fig. 10.22 Crohn disease. A, Cobblestone mucosa with stricture. B, 'String' sign. (A, Courtesy of Fig. 10.22C Erythema nodosum. humpath.com. B, Published with permission from LearningRadiology.com) II. COLONIC DIVERTICULA A. Outpouchings of mucosa and submucosa through the muscularis propria (Fig. 10.23, false diverticulum) B. Related to wall stress 1. Associated with constipation, straining, and low-fiber diet; commonly seen in older adults (risk increases with age) 2. Arise where the vasa recta traverse the muscularis propria (weak point in colonic wall); sigmoid colon is the most common location. C. Usually asymptomatic; complications include 1. 2. Diverticulitis-due to obstructing fecal material; presents with appendicitis-like symptoms in the left lower quadrant 3.

1	C. Usually asymptomatic; complications include 1. 2. Diverticulitis-due to obstructing fecal material; presents with appendicitis-like symptoms in the left lower quadrant 3. Fistula-Inflamed diverticulum ruptures and attaches to a local structure. Colovesicular fistula presents with air (or stool) in urine. III. ANGIODYSPLASIA A. Acquired malformation of mucosa! and submucosal capillary beds B. Usually arises in the cecum and right colon due to high wall tension C. Rupture classically presents as hematochezia in an older adult. IV. A. Autosomal dominant disorder resulting in thin-walled blood vessels, especially in the mouth and GI tract (Fig. 10.24) B. Rupture presents as bleeding. V. A. Ischemic damage to the colon, usually at the splenic flexure (watershed area of superior mesenteric artery [SMA]) B. Atherosclerosis of SMA is the most common cause. C. Presents with postprandial pain and weight loss; infarction results in pain and bloody diarrhea.

1	B. Atherosclerosis of SMA is the most common cause. C. Presents with postprandial pain and weight loss; infarction results in pain and bloody diarrhea. VI. IRRITABLE BOWEL SYNDROME A. Relapsing abdominal pain with bloating, flatulence, and change in bowel habits (diarrhea or constipation) that improves with defecation; classically seen in middleaged females B. Related to disturbed intestinal motility; no identifiable pathologic changes C. Increased dietary fiber may improve symptoms. Fig. 10.23 Colonic diverticula. A, Gross appearance. B, Microscopic appearance. Fig. 10.24 Hereditary hemorrhagic telangiectasia. (Courtesy of Ors. H. Fred and H. van Dijk, Images ofMemorable Cases) VII. COLONIC POLYPS A. Raised protrusions of colonic mucosa B. Most common types are hyperplastic and adenomatous polyps.

1	VII. COLONIC POLYPS A. Raised protrusions of colonic mucosa B. Most common types are hyperplastic and adenomatous polyps. 1. Hyperplastic polyps are due to hyperplasia of glands; classically show a 'serrated' appearance on microscopy i. Most common type of polyp; usually arise in the left colon (rectosigmoid) ii. Benign, with no malignant potential 2. Adenomatous polyps are due to neoplastic proliferation of glands (Fig. 10.25); 2nd most common type of colonic polyp i. Benign, but premalignant; may progress to adenocarcinoma via the adenoma-carcinoma sequence 3. Adenoma-carcinoma sequence describes the molecular progression from normal colonic mucosa to adenomatous polyp to carcinoma. i. APC (adenomatous polyposis coli gene) mutations (sporadic or germline) increase risk for formation of polyp. ii. K-ras mutation leads to formation of polyp.

1	i. APC (adenomatous polyposis coli gene) mutations (sporadic or germline) increase risk for formation of polyp. ii. K-ras mutation leads to formation of polyp. iii. p53 mutation and increased expression of COX allow for progression to carcinoma; aspirin impedes progression from adenoma to carcinoma. C. Screening for polyps is performed by colonoscopy and testing for fecal occult blood; polyps are usually clinically silent, but can bleed. 1. Goal is to remove adenomatous polyps before progression to carcinoma. D. On colonoscopy, hyperplastic and adenomatous polyps look identical. Hence, all polyps are removed and examined microscopically. 1. Greatest risk for progression from adenoma to carcinoma is related to size > 2 cm, sessile growth, and villous histology. VIII. FAMILIAL ADENOMATOUS POLYPOSIS (FAP) A. Autosomal dominant disorder characterized by 100s to 1000s of adenomatous colonic polyps (Fig. 10.26)

1	VIII. FAMILIAL ADENOMATOUS POLYPOSIS (FAP) A. Autosomal dominant disorder characterized by 100s to 1000s of adenomatous colonic polyps (Fig. 10.26) B. Due to inherited APC mutation (chromosome 5); increases propensity to develop adenomatous polyps throughout colon and rectum C. Colon and rectum are removed prophylactically; otherwise, almost all patients develop carcinoma by 40 years of age. D. Gardner syndrome is FAP with fibromatosis and osteomas. 1. Fibromatosis is a non-neoplastic proliferation of fibroblasts; arises in retroperitoneum (desmoid) and locally destroys tissue 2. Osteoma is a benign tumor of bone that usually arises in the skull. E. Turcot syndrome is FAP with CNS tumors (medulloblastoma and glial tumors). Fig. 10.25 Adenomatous polyp. Fig. 10.26 Familial adenomatous polyposis. (Courtesy of hum path.com) IX. A. Sporadic, hamartomatous (benign) polyp that arises in children(< 5 years) 1. Usually presents as a solitary rectal polyp that prolapses and bleeds

1	IX. A. Sporadic, hamartomatous (benign) polyp that arises in children(< 5 years) 1. Usually presents as a solitary rectal polyp that prolapses and bleeds B. Juvenile polyposis is characterized by multiple juvenile polyps in the stomach and colon; large numbers ofjuvenile polyps increase the risk of progression to carcinoma. X. A. Hamartomatous (benign) polyps throughout GI tract and mucocutaneous hyperpigmentation (freckle-like spots) on lips, oral mucosa, and genital skin; autosomal dominant disorder B. Increased risk for colorectal, breast, and gynecologic cancer XI. COLORECTAL CARCINOMA A. Carcinoma arising from colonic or rectal mucosa; 3rd most common site of cancer and 3rd most common cause of cancer-related death 1. Peak incidence is 60-70 years of age. B. Most commonly arises from adenoma-carcinoma sequence; a second important molecular pathway is microsatellite instability (MSI). 1.

1	B. Most commonly arises from adenoma-carcinoma sequence; a second important molecular pathway is microsatellite instability (MSI). 1. Microsatellites are repeating sequences of noncoding DNA; integrity of sequence (stability) is maintained during cell division. 2. Instability indicates defective DNA copy mechanisms (e.g., DNA mismatch repair enzymes). 3. Hereditary nonpolyposis colorectal carcinoma (HNPCC) is due to inherited mutations in DNA mismatch repair enzymes. i. Increased risk for colorectal, ovarian, and endometrial carcinoma ii. Colorectal carcinoma arises de novo (not from adenomatous polyps) at a relatively early age; usually right-sided C. Screening for colorectal carcinoma occurs via colonoscopy and fecal occult blood testing; begins at 50 years of age 1. Goal is to remove adenomatous polyps before carcinoma develops and to detect cancer early (before clinical symptoms arise). D. Carcinoma can develop anywhere along entire length of colon. 1.

1	D. Carcinoma can develop anywhere along entire length of colon. 1. Left-sided carcinoma usually grows as a 'napkin-ring' lesion; presents with decreased stool caliber, left lower quadrant pain, and blood-streaked stool 2. Right-sided carcinoma usually grows as a raised lesion; presents with irondeficiency anemia (occult bleeding) and vague pain. An older adult with iron deficiency anemia has colorectal carcinoma until proven otherwise. E. Colonic carcinoma is associated with an increased risk for Streptococcus bovis endocarditis. F. Staging 1. T-depth of invasion; tumors limited to the mucosa generally do not spread due to lack of lymphatics in the mucosa. 2. N-spread to regional lymph nodes 3. M-distant spread; most commonly involves the liver G. CEA is a serum tumor marker that is useful for assessing treatment response and detecting recurrence; not useful for screening Exocrine Pancreas, Gallbladder, and I. ANNULAR PANCREAS

1	G. CEA is a serum tumor marker that is useful for assessing treatment response and detecting recurrence; not useful for screening Exocrine Pancreas, Gallbladder, and I. ANNULAR PANCREAS A. Developmental malformation in which the pancreas forms a ring around the duodenum; risk of duodenal obstruction II. ACUTE PANCREATITIS A. Inflammation and hemorrhage of the pancreas B. Due to autodigestion of pancreatic parenchyma by pancreatic enzymes 1. Premature activation of trypsin leads to activation of other pancreatic enzymes. C. Results in liquefactive hemorrhagic necrosis of the pancreas and fat necrosis of the peripancreatic fat (Fig. 11.1) D. Most commonly due to alcohol and gallstones; other causes include trauma, hypercalcemia, hyperlipidemia, drugs, scorpion stings, mumps, and rupture of a posterior duodenal ulcer. E. Clinical features 1. Epigastric abdominal pain that radiates to the back 2. 3.

1	E. Clinical features 1. Epigastric abdominal pain that radiates to the back 2. 3. Periumbilical and flank hemorrhage (necrosis spreads into the periumbilical soft tissue and retroperitoneum) 4. Elevated serum lipase and amylase; lipase is more specific for pancreatic damage. 5. Hypocalcemia (calcium is consumed during saponification in fat necrosis) F. Complications 1. Shock-due to peripancreatic hemorrhage and fluid sequestration 2. i. Presents as an abdominal mass with persistently elevated serum amylase ii. Rupture is associated with release ofenzymes into the abdominal cavity and hemorrhage. 3. Pancreatic abscess-often due to E coli; presents with abdominal pain, high fever, and persistently elevated amylase 4. III. CHRONIC PANCREATITIS A. Fibrosis ofpancreatic parenchyma, most often secondary to recurrent acute pancreatitis 1. Most commonly due to alcohol (adults) and cystic fibrosis (children); however, many cases are idiopathic. B. Clinical features 1.

1	B. Clinical features 1. Epigastric abdominal pain that radiates to the back 2. Pancreatic insufficiency-results in malabsorption with steatorrhea and fatsoluble vitamin deficiencies. Amylase and lipase are not useful serologic markers ofchronic pancreatitis. 3. Dystrophic calcification of pancreatic parenchyma on imaging; contrast studies reveal a 'chain oflakes' pattern due to dilatation of pancreatic ducts. pathoma.com 4. Secondary diabetes mellitus-late complication due to destruction of islets 5. IV. PANCREATIC CARCINOMA A. Adenocarcinoma arising from the pancreatic ducts 1. Most commonly seen in the elderly (average age is 70 years) B. Major risk factors are smoking and chronic pancreatitis. C. Clinical features (usually occur late in disease) 1. 2. Obstructive jaundice with pale stools and palpable gallbladder; associated with tumors that arise in the head of the pancreas (most common location) 3.

1	2. Obstructive jaundice with pale stools and palpable gallbladder; associated with tumors that arise in the head of the pancreas (most common location) 3. Secondary diabetes mellitus; associated with tumors that arise in the body or tail 4. 5. Migratory thrombophlebitis (Trousseau syndrome); presents as swelling, erythema, and tenderness in the extremities (seen in 10% of patients) 6. Serum tumor marker is CA 19-9. D. Surgical resection involves en bloc removal of the head and neck of pancreas, proximal duodenum, and gallbladder (Whipple procedure). E. Very poor prognosis; I-year survival is < 10%. I. BILIARY ATRESIA A. Failure to form or early destruction of extrahepatic biliary tree B. Leads to biliary obstruction within the first 2 months of life C. Presents with jaundice and progresses to cirrhosis II. CHOLELITHIASIS (GALLSTONES) A. Solid, round stones in the gallbladder

1	B. Leads to biliary obstruction within the first 2 months of life C. Presents with jaundice and progresses to cirrhosis II. CHOLELITHIASIS (GALLSTONES) A. Solid, round stones in the gallbladder B. Due to precipitation of cholesterol (cholesterol stones) or bilirubin (bilirubin stones) in bile 1. Arises with (1) supersaturation of cholesterol or bilirubin, (2) decreased phospholipids (e.g., lecithin) or bile acids (normally increase solubility), or (3) stasis C. Cholesterol stones (yellow) are the most common type (90%), especially in the West (Fig. 11.2A). 1. Usually radiolucent (10% are radiopaque due to associated calcium) 2. Risk factors include age (40s), estrogen (female gender, obesity, multiple pregnancies and oral contraceptives), clofibrate, Native American ethnicity, Crohn disease, and cirrhosis.

1	Risk factors include age (40s), estrogen (female gender, obesity, multiple pregnancies and oral contraceptives), clofibrate, Native American ethnicity, Crohn disease, and cirrhosis. Fig. 11.1 Acute pancreatitis. (Courtesy of Fig. 11.2 Gallstones. A, Cholesterol stones. B, Bilirubin stones. (A, Courtesy of K.V. Santosh, MD. B, hum path.com) Courtesy of humpath.com) Exocrine Pancreas, Gallbladder, and Liver Pathology D. Bilirubin stones (pigmented) are composed ofbilirubin (Fig. 11.2B). 1. 2. Risk factors include extravascular hemolysis (increased bilirubin in bile) and biliary tract infection (e.g., E coli, Ascaris lumbricoides, and Clonorchis sinensis).

1	1. 2. Risk factors include extravascular hemolysis (increased bilirubin in bile) and biliary tract infection (e.g., E coli, Ascaris lumbricoides, and Clonorchis sinensis). i. Ascaris lumbricoides is a common roundworm that infects 25% of the world's population, especially in areas with poor sanitation (fecal-oral transmission); infects the biliary tract, increasing the risk for gallstones ii. Clonorchis sinensis is endemic in China, Korea, and Vietnam (Chinese liver fluke); infects the biliary tract, increasing the risk for gallstones, cholangitis, and cholangiocarcinoma E. Gallstones are usually asymptomatic; complications include biliary colic, acute and chronic cholecystitis, ascending cholangitis, gallstone ileus, and gallbladder cancer. III. BILIARY COLIC A. Waxing and waning right upper quadrant pain B. Due to the gallbladder contracting against a stone lodged in the cystic duct C. Symptoms are relieved if the stone passes.

1	III. BILIARY COLIC A. Waxing and waning right upper quadrant pain B. Due to the gallbladder contracting against a stone lodged in the cystic duct C. Symptoms are relieved if the stone passes. D. Common bile duct obstruction may result in acute pancreatitis or obstructive jaundice. IV. A. Acute inflammation of the gallbladder wall B. Impacted stone in the cystic duct results in dilatation with pressure ischemia, bacterial overgrowth (E coli), and inflammation. C. Presents with right upper quadrant pain, often radiating to right scapula, fever with t WBC count, nausea, vomiting, and t serum alkaline phosphatase (from duct damage) D. Risk of rupture ifleft untreated V. A. Chronic inflammation of the gallbladder B. Due to chemical irritation from longstanding cholelithiasis, with or without superimposed bouts of acute cholecystitis C. Characterized by herniation of gallbladder mucosa into the muscular wall (Rokitansky-Aschoff sinus, Fig. 11.3A)

1	C. Characterized by herniation of gallbladder mucosa into the muscular wall (Rokitansky-Aschoff sinus, Fig. 11.3A) D. Presents with vague right upper quadrant pain, especially after eating E. Porcelain gallbladder is a late complication (Fig. 11.3B). 1. Shrunken, hard gallbladder due to chronic inflammation, fibrosis, and dystrophic calcification :~7~/ ·'~{~. . . t1 ..•'-·.,_ :",A~?·'\),:~;,.:;,,;_ Fig. 11.3 Chronic cholecystitis. A, Rokitansky-Aschoff sinus. B, Porcelain ga ll bladder. (B, Courtesy of Drs. H. Fred and H. van Dijk, Images ofMemorable Cases) 2. Increased risk for carcinoma F. Treatment is cholecystectomy, especially if porcelain gallbladder is present. VI. ASCENDING CHOLANGITIS A. Bacterial infection of the bile ducts B. Usually due to ascending infection with enteric gram-negative bacteria C. Presents as sepsis (high fever and chills), jaundice, and abdominal pain D. Increased incidence with choledocholithiasis (stone in biliary ducts)

1	C. Presents as sepsis (high fever and chills), jaundice, and abdominal pain D. Increased incidence with choledocholithiasis (stone in biliary ducts) VII. GALLSTONE ILEUS A. Gallstone enters and obstructs the small bowel B. Due to cholecystitis with fistula formation between the gallbladder and small bowel VIII.GALLBLADDER CARCINOMA A. Adenocarcinoma arising from the glandular epithelium that lines the gallbladder wall (Fig. 11.4) B. Gallstones are a major risk factor, especially when complicated by porcelain gallbladder. C. Classically presents as cholecystitis in an elderly woman D. Poor prognosis I. JAUNDICE A. Yellow discoloration of the skin (Fig. 11.5); earliest sign is scleral icterus (yellow discoloration of the sclera). B. Due to t serum bilirubin, usually > 2.5 mg/dL C. Arises with disturbances in bilirubin metabolism (Table 11.1) D. Normal bilirubin metabolism 1. RBCs are consumed by macrophages of the reticuloendothelial system. 2.

1	C. Arises with disturbances in bilirubin metabolism (Table 11.1) D. Normal bilirubin metabolism 1. RBCs are consumed by macrophages of the reticuloendothelial system. 2. Protoporphyrin (from heme) is converted to unconjugated bilirubin (UCB). 3. Albumin carries UCB to the liver. 4. Uridine glucuronyl transferase (UGT) in hepatocytes conjugates bilirubin. 5. Conjugated bilirubin (CB) is transferred to bile canaliculi to form bile, which is stored in the gallbladder. 6. Bile is released into the small bowel to aid in digestion. 7. Intestinal flora convert CB to urobilinogen, which is oxidized to stercobilin (makes stool brown) and urobilin (partially reabsorbed into blood and filtered by kidney, making urine yellow). Fig.11 .4 Gallbladder carcinoma. (Courtesy of K.V. Fig. 11.5 Jaundice. (Courtesy of James Heilman, Fig. 11 .6 Acute hepatitis. Santosh, MD) MD, Wikipedia) Exocrine Pancreas, Gallbladder, and Liver Pathology Table 11.1: Causes of Jaundice

1	Exocrine Pancreas, Gallbladder, and Liver Pathology Table 11.1: Causes of Jaundice Physiologic jaundice of the newborn High levels of UCB overwhelm the conjugating ability of the liver. Newborn liver has transiently low UGT activity. Associated with gallstones, pancreatic carcinoma, cholangiocarcinoma, parasites, and liver fluke Inflammation disrupts hepatocytes and small bile ductules. t CB, ..J.. urine urobilinogen, and t alkaline phosphatase Table 11.2: Important Features of Hepatitis Viruses HAV is commonly acquired by travelers. HEV is commonly acquired from contaminated water or undercooked seafood. Parenteral transmission (e.g., childbirth, unprotected intercourse, intravenous drug abuse [IVDA], and needle stick) Parenteral transmission (e.g., IVDA, unprotected intercourse); risk from transfusion is almost nonexistent due to screening of the blood supply.

1	Parenteral transmission (e.g., IVDA, unprotected intercourse); risk from transfusion is almost nonexistent due to screening of the blood supply. Dark urine due to t urine urobilinogen (UCB is not water soluble and, thus, is absent from urine) Increased risk for pigmented bilirubin gallstones UCB is fat soluble and can deposit in the basal ganglia (kernicterus) leading to neurological deficits and death. Treatment is phototherapy (makes UCB water soluble). Jaundice during stress (e.g., severe infection); otherwise, not clinically significant Liver is dark; otherwise, not clinically significant Rotor syndrome is similar to Dubin-Johnson syndrome, but lacks liver discoloration. Dark urine (due to bilirubinuria) and pale stool Pruritus due to t plasma bile acids Hypercholesterolemia with xanthomas Steatorrhea with malabsorption of fat-soluble vitamins Dark urine due to t urine bilirubin; urine urobilinogen is normal or decreased.

1	Dark urine due to t urine bilirubin; urine urobilinogen is normal or decreased. Acute hepatitis; no chronic state. Anti-virus IgM marks active infection. Antivirus IgG is protective, and its presence indicates prior infection or immunization (immunization is available for HAV). HEV infection in pregnant women is associated with fulminant hepatitis (liver failure with massive liver necrosis). Results in acute hepatitis; chronic disease occurs in 20% of cases (Table 11.3). Results in acute hepatitis; chronic disease occurs in most cases. HCV-RNA test confirms infection; decreased RNA levels indicate recovery; persistence indicates chronic disease. Dependent on HBV for infection; superinfection upon existing HBV is more severe than coinfection (infection with HBV and HDV at the same time) f}~,. ,f: Fig.11 .7 Chronic hepatitis. Table 11.3: Serologic Markers of Hepatitis B Virus STAGE HBsAG ~!:AG AND HBV HBcAB HBsAB marker to rise) + (presence > 6 +/-; presence of

1	,f: Fig.11 .7 Chronic hepatitis. Table 11.3: Serologic Markers of Hepatitis B Virus STAGE HBsAG ~!:AG AND HBV HBcAB HBsAB marker to rise) + (presence > 6 +/-; presence of Chronic months defines HBeAg or HBV DNA IgG chronic state) indicates infectivity. II. VIRAL HEPATITIS A. Inflammation ofliver parenchyma, usually due to hepatitis virus (Table 11.2); other causes include EBV and CMV. B. Hepatitis virus causes acute hepatitis, which may progress to chronic hepatitis. C. Acute hepatitis presents as jaundice (mixed CB and UCB) with dark urine (due to CB), fever, malaise, nausea, and elevated liver enzymes (ALT > AST). 1. Inflammation involves lobules of the liver and portal tracts and is characterized by apoptosis of hepatocytes (Fig. 11.6). 2. Some cases may be asymptomatic with elevated liver enzymes. 3. Symptoms last < 6 months. D. Chronic hepatitis is characterized by symptoms that last > 6 months. 1. Inflammation predominantly involves portal tract (Fig. 11.7) 2.

1	3. Symptoms last < 6 months. D. Chronic hepatitis is characterized by symptoms that last > 6 months. 1. Inflammation predominantly involves portal tract (Fig. 11.7) 2. Risk of progression to cirrhosis III. CIRRHOSIS A. End-stage liver damage characterized by disruption of the normal hepatic parenchyma by bands of fibrosis and regenerative nodules of hepatocytes (Fig. 11.8) B. Fibrosis is mediated by TGF-~ from stellate cells which lie beneath the endothelial cells that line the sinusoids. C. Clinical features 1. Portal hypertension leads to i. Ascites (fluid in the peritoneal cavity) ii. Congestive splenomegaly/hypersplenism iii. Portosystemic shunts (esophageal varices, hemorrhoids, and caput medusae) Fig. 11.8 Cirrhosis. A, Microscopic appearance. B, Gross appearance. (8, Courtesy of humpath.com)

1	Fig. 11.8 Cirrhosis. A, Microscopic appearance. B, Gross appearance. (8, Courtesy of humpath.com) Exocrine Pancreas, Gallbladder, and Liver Pathology iv. Hepatorenal syndrome (rapidly developing renal failure secondary to cirrhosis) 2. Decreased detoxification results in i. Mental status changes, asterixis, and eventual coma (due tot serum ammonia); metabolic, hence reversible ii. Gynecomastia, spider angiomata, and palmar erythema due to hyperestrinism iii. Jaundice 3. Decreased protein synthesis leads to i. Hypoalbuminemia with edema 11. Coagulopathy due to decreased synthesis of clotting factors; degree of deficiency is followed by PT. IV. A. Damage to hepatic parenchyma due to consumption of alcohol 1. Most common cause ofliver disease in the West B. Fatty liver is the accumulation of fat in hepatocytes (Fig. 11.9A). 1. Results in a heavy, greasy liver; resolves with abstinence (Fig. 11.9B)

1	B. Fatty liver is the accumulation of fat in hepatocytes (Fig. 11.9A). 1. Results in a heavy, greasy liver; resolves with abstinence (Fig. 11.9B) C. Alcoholic hepatitis results from chemical injury to hepatocytes; generally seen with binge drinking 1. Acetaldehyde (metabolite of alcohol) mediates damage. 2. Characterized by swelling of hepatocytes with formation of Mallory bodies (damaged cytokeratin filaments, Fig. 11.10), necrosis, and acute inflammation 3. Presents with painful hepatomegaly and elevated liver enzymes (AST > ALT); may result in death D. Cirrhosis is a complication of long-term, chronic alcohol-induced liver damage; occurs in 10-20% of alcoholics V. A. Fatty change, hepatitis, and/or cirrhosis that develop without exposure to alcohol (or other known insult) B. Associated with obesity C. Diagnosis of exclusion; ALT > AST VI. HEMOCHROMATOSIS

1	A. Fatty change, hepatitis, and/or cirrhosis that develop without exposure to alcohol (or other known insult) B. Associated with obesity C. Diagnosis of exclusion; ALT > AST VI. HEMOCHROMATOSIS A. Excess body iron leading to deposition in tissues (hemosiderosis) and organ damage (hemochromatosis) 1. Tissue damage is mediated by generation of free radicals. B. Due to autosomal recessive defect in iron absorption (primary) or chronic transfusions (secondary) Fig.11.9 Fatty liver. A, Microscopic appearance. B, Gross appearance. (B, Courtesy of Jerome Taxy, Fig. 11.10 Mallory bodies, alcoholic hepatitis. MD) 1. Primary hemochromatosis is due to mutations in the HFE gene, usually C282Y (cysteine is replaced by tyrosine at amino acid 282). C. Presents in late adulthood 1. Classic triad is cirrhosis, secondary diabetes mellitus, and bronze skin; other findings include dilated cardiomyopathy, cardiac arrhythmias and gonadal dysfunction (due to testicular atrophy). 2.

1	Classic triad is cirrhosis, secondary diabetes mellitus, and bronze skin; other findings include dilated cardiomyopathy, cardiac arrhythmias and gonadal dysfunction (due to testicular atrophy). 2. Labs show t ferritin, -1, TIBC, t serum iron, and t % saturation. 3. Liver biopsy reveals accumulation of brown pigment in hepatocytes (Fig. 11.llA); Prussian blue stain distinguishes iron (blue) from lipofuscin (Fig. 11.llB). i. Lipofuscin is a brown pigment that is a by-product from the turnover ('wear and tear') of peroxidized lipids; it is commonly present in hepatocytes (Fig. 11.llC). D. Increased risk ofhepatocellular carcinoma E. Treatment is phlebotomy. VII. WILSON DISEASE A. Autosomal recessive defect (ATP7B gene) in ATP-mediated hepatocyte copper transport 1. Results in lack of copper transport into bile and lack of copper incorporation into ceruloplasmin B. Copper builds up in hepatocytes, leaks into serum, and deposits in tissues.

1	B. Copper builds up in hepatocytes, leaks into serum, and deposits in tissues. 1. Copper-mediated production of hydroxyl free radicals leads to tissue damage. C. Presents in childhood with 1. 2. Neurologic manifestations (behavioral changes, dementia, chorea, and Parkinsonian symptoms due to deposition of copper in basal ganglia) 3. Kayser-Fleisher rings in the cornea D. Labs show t urinary copper, -1, serum ceruloplasmin, and t copper on liver biopsy. E. Increased risk of hepatocellular carcinoma F. Treatment is D-penicillamine (chelates copper). VIII. PRIMARY BILIARY CIRRHOSIS A. Autoimmune granulomatous destruction of intrahepatic bile ducts 1. Classically arises in women (average age is 40 years) 2. Associated with other autoimmune diseases B. Etiology is unknown; antimitochondrial antibody is present. C. Presents with features of obstructive jaundice D. Cirrhosis is a late complication. IX. PRIMARY SCLEROSING CHOLANGITIS

1	B. Etiology is unknown; antimitochondrial antibody is present. C. Presents with features of obstructive jaundice D. Cirrhosis is a late complication. IX. PRIMARY SCLEROSING CHOLANGITIS A. Inflammation and fibrosis of intrahepatic and extrahepatic bile ducts Fig. 11.11 Hemochromatosis. A, Iron deposition in hepatocytes. B, Prussian blue stain. C, Lipofuscin in hepatocytes for comparison. Exocrine Pancreas, Gallbladder, and Liver Pathology 1. Periductal fibrosis with an 'onion-skin' appearance (Fig. 11.12) 2. Uninvolved regions are dilated resulting in a "beaded" appearance on contrast imaging. B. Etiology is unknown, but associated with ulcerative colitis; p-ANCA is often positive. C. Presents with obstructive jaundice; cirrhosis is a late complication. D. Increased risk for cholangiocarcinoma X. REYE SYNDROME A. Fulminant liver failure and encephalopathy in children with viral illness who take aspirin 1. Likely related to mitochondrial damage ofhepatocytes

1	X. REYE SYNDROME A. Fulminant liver failure and encephalopathy in children with viral illness who take aspirin 1. Likely related to mitochondrial damage ofhepatocytes B. Presents with hypoglycemia, elevated liver enzymes, and nausea with vomiting; may progress to coma and death XI. HEPATIC ADENOMA A. Benign tumor ofhepatocytes B. Associated with oral contraceptive use; regresses upon cessation of drug C. Risk of rupture and intraperitoneal bleeding, especially during pregnancy 1. Tumors are subcapsular and grow with exposure to estrogen. XII. HEPATOCELLULAR CARCINOMA A. Malignant tumor of hepatocytes B. Risk factors include 1. Chronic hepatitis (e.g., HBV and HCV) 2. Cirrhosis (e.g., alcohol, nonalcoholic fatty liver disease, hemochromatosis, Wilson disease, and AlAT deficiency) 3. C. Increased risk for Budd-Chiari syndrome 1. Liver infarction secondary to hepatic vein obstruction 2. Presents with painful hepatomegaly and ascites

1	C. Increased risk for Budd-Chiari syndrome 1. Liver infarction secondary to hepatic vein obstruction 2. Presents with painful hepatomegaly and ascites D. Tumors are often detected late because symptoms are masked by cirrhosis; poor prognosis E. Serum tumor marker is alpha-fetoprotein. XIII. METASTASIS TO LIVER A. More common than primary liver tumors; most common sources include colon, pancreas, lung, and breast carcinomas. B. Results in multiple nodules in the liver (Fig. 11.13) C. Clinically may be detected as hepatomegaly with a nodular free edge of the liver Fig.11 .12 'Onion-skin' fibrosis, primary sclerosing Fig. 11.13 Metastatic carcinoma involving liver. cholangitis. (Courtesy of Jerome Taxy, MD) I. HORSESHOE KIDNEY A. Conjoined kidneys usually connected at the lower pole (Fig. 12.1); most common congenital renal anomaly

1	I. HORSESHOE KIDNEY A. Conjoined kidneys usually connected at the lower pole (Fig. 12.1); most common congenital renal anomaly B. Kidney is abnormally located in the lower abdomen; horseshoe kidney gets caught on the inferior mesenteric artery root during its ascent from the pelvis to the abdomen. II. RENAL AGENESIS A. Absent kidney formation; may be unilateral or bilateral B. Unilateral agenesis leads to hypertrophy of the existing kidney; hyperfiltration increases risk of renal failure later in life. C. Bilateral agenesis leads to oligohydramnios with lung hypoplasia, flat face with low set ears, and developmental defects of the extremities (Potter sequence, Fig. 12.2); incompatible with life III. DYSPLASTIC KIDNEY A. Noninherited, congenital malformation of the renal parenchyma characterized by cysts and abnormal tissue (e.g., cartilage, Fig. 12.3) B. Usually unilateral; when bilateral, must be distinguished from inherited polycystic kidney disease IV.

1	B. Usually unilateral; when bilateral, must be distinguished from inherited polycystic kidney disease IV. A. Inherited defect leading to bilateral enlarged kidneys with cysts in the renal cortex and medulla (Fig. 12.4) B. Autosomal recessive form presents in infants as worsening renal failure and hypertension; newborns may present with Potter sequence. 1. Associated with congenital hepatic fibrosis (leads to portal hypertension) and hepatic cysts C. Autosomal dominant form presents in young adults as hypertension (due to increased renin), hematuria, and worsening renal failure. 1. Due to mutation in the APKDl or APKD2 gene; cysts develop over time. 2. Associated with berry aneurysm, hepatic cysts, and mitral valve prolapse V. A. Inherited (autosomal dominant) defect leading to cysts in the medullary collecting ducts B. Parenchymal fibrosis results in shrunken kidneys and worsening renal failure. I. BASIC PRINCIPLES

1	V. A. Inherited (autosomal dominant) defect leading to cysts in the medullary collecting ducts B. Parenchymal fibrosis results in shrunken kidneys and worsening renal failure. I. BASIC PRINCIPLES A. Acute, severe decrease in renal function (develops within days) B. Hallmark is azotemia (increased BUN and creatinine [Cr]), often with oliguria. pathoma.com C. Divided into prerenal, postrenal, and intrarenal azotemia based on etiology II. PRERENAL AZOTEMIA A. Due to decreased blood flow to kidneys (e.g., cardiac failure); common cause of ARF B. Decreased blood flow results in ,I, GFR, azotemia, and oliguria. C. Reabsorption of fluid and BUN ensues (serum BUN:Cr ratio > 15); tubular function remains intact (fractional excretion of sodium [FENa] < 1% and urine osmolality [osm] > 500 mOsm/kg). III. POSTRENAL AZOTEMIA A. Due to obstruction of urinary tract downstream from the kidney (e.g., ureters) B. Decreased outflow results in ,I, GFR, azotemia, and oliguria.

1	III. POSTRENAL AZOTEMIA A. Due to obstruction of urinary tract downstream from the kidney (e.g., ureters) B. Decreased outflow results in ,I, GFR, azotemia, and oliguria. C. During early stage of obstruction, increased tubular pressure "forces" BUN into the blood (serum BUN:Cr ratio > 15); tubular function remains intact (FENa < 1% and urine osm > 500 mOsm/kg). D. With long-standing obstruction, tubular damage ensues, resulting in decreased reabsorption of BUN (serum BUN:Cr ratio < 15), decreased reabsorption of sodium (FEN a > 2%), and inability to concentrate urine (urine osm < 500 mOsm/kg). IV. ACUTE TUBULAR NECROSIS A. Injury and necrosis of tubular epithelial cells (Fig. 12.5); most common cause of acute renal failure (intrarenal azotemia) B. Necrotic cells plug tubules; obstruction decreases GFR. 1. Brown, granular casts are seen in the urine.

1	B. Necrotic cells plug tubules; obstruction decreases GFR. 1. Brown, granular casts are seen in the urine. C. Dysfunctional tubular epithelium results in decreased reabsorption of BUN (serum BUN:Cr ratio < 15), decreased reabsorption of sodium (FEN a > 2%), and inability to concentrate urine (urine osm < 500 mOsm/kg). D. Etiology may be ischemic or nephrotoxic. 1. Ischemia-Decreased blood supply results in necrosis of tubules. i. Often preceded by prerenal azotemia ii. Proximal tubule and medullary segment of the thick ascending limb are particularly susceptible to ischemic damage. 2. Nephrotoxic-Toxic agents result in necrosis of tubules. i. Proximal tubule is particularly susceptible. ii. Causes include aminoglycosides (most common), heavy metals (e.g., lead), myoglobinuria (e.g., from crush injury to muscle), ethylene glycol (associated with oxalate crystals in urine), radiocontrast dye, and urate (e.g., tumor lysis syndrome).

1	iii. Hydration and allopurinol are used prior to initiation of chemotherapy to decrease risk of urate-induced ATN. :,j.U,,J .• /J',.// t~.. \ f , ',· ~-.,;i· ~4' , ♦ i". ~·t,)l ', : .. ti'•;',,1 . •..' !~,-~i >,,:' : ~··~t:, .·· / .:. :-~ l6 ,. .....,-.-·.\· ,:j~I 1 , ;' 1,'' I ~~ • ,f'•~,.~,,,1' •• ' ~••• I, Fig. 12.1 Horseshoe kidney. (Courtesy of Fig. 12.2 Potter sequence, (Courtesy of humpath, Fig. 12.3 Dysplastic kidney, (Courtesy of Aliya humpath,com) com) Husain, MD) E. Clinical features 1. Oliguria with brown, granular casts 2. 3. Hyperkalemia (due to decreased renal excretion) with metabolic acidosis F. Reversible, but often requires supportive dialysis since electrolyte imbalances can be fatal 1. Oliguria can persist for 2-3 weeks before recovery; tubular cells (stable cells) take time to reenter the cell cycle and regenerate. V. ACUTE INTERSTITIAL NEPHRITIS

1	V. ACUTE INTERSTITIAL NEPHRITIS A. Drug-induced hypersensitivity involving the interstitium and tubules (Fig. 12.6); results in acute renal failure (intra renal azotemia) B. Causes include NSAIDs, penicillin, and diuretics. C. Presents as oliguria, fever, and rash days to weeks after starting a drug; eosinophils may be seen in urine. D. Resolves with cessation of drug E. May progress to renal papillary necrosis VI. RENAL PAPILLARY NECROSIS A. Necrosis of renal papillae B. Presents with gross hematuria and flank pain C. Causes include 1. Chronic analgesic abuse (e.g., long-term phenacetin or aspirin use) 2. 3. 4. I. BASIC PRINCIPLES A. Glomerular disorders characterized by proteinuria (> 3.5 g/day) resulting in 1. 2. Hypogammaglobulinemia-increased risk of infection 3. Hypercoagulable state-due to loss of antithrombin III 4. II. MINIMAL CHANGE DISEASE (MCD) A. Most common cause of nephrotic syndrome in children

1	Hypercoagulable state-due to loss of antithrombin III 4. II. MINIMAL CHANGE DISEASE (MCD) A. Most common cause of nephrotic syndrome in children B. Usually idiopathic; may be associated with Hodgkin lymphoma Fig. 12.4 Polycystlc kidney disease. (Courtesy of Fig. 12.5 Acute tubular necrosis. Fig. 12.6 Acute interstitial nephritis. Jamie Steinmet z, MD) C. Normal glomeruli on H&E stain (Fig. 12.7A); lipid may be seen in proximal tubule cells. D. Effacement of foot processes on electron microscopy (EM, Fig. 12.7B) E. No immune complex deposits; negative immunofluorescence (IF) F. Selective proteinuria (loss of albumin, but not immunoglobulin) G. Excellent response to steroids (damage is mediated by cytokines from T cells) III. FOCAL SEGMENTAL GLOMERULOSCLEROSIS (FSGS) A. Most common cause of nephrotic syndrome in Hispanics and African Americans B. Usually idiopathic; may be associated with HIV, heroin use, and sickle cell disease

1	A. Most common cause of nephrotic syndrome in Hispanics and African Americans B. Usually idiopathic; may be associated with HIV, heroin use, and sickle cell disease C. Focal (some glomeruli) and segmental (involving only part of the glomerulus) sclerosis on H&E stain (Fig. 12.8) D. Effacement of foot processes on EM E. No immune complex deposits; negative IF F. Poor response to steroids; progresses to chronic renal failure IV. A. Most common cause of nephrotic syndrome in Caucasian adults B. Usually idiopathic; may be associated with hepatitis B or C, solid tumors, SLE, or drugs (e.g., NSAIDs and penicillamine) C. Thick glomerular basement membrane on H&E (Fig. 12.9A) D. Due to immune complex deposition (granular IF, Fig. 12.9B); subepithelial deposits with 'spike and dome' appearance on EM (Fig. 12.9C) E. Poor response to steroids; progresses to chronic renal failure V. A. Thick glomerular basement membrane on H&E, often with 'tram-track' appearance

1	E. Poor response to steroids; progresses to chronic renal failure V. A. Thick glomerular basement membrane on H&E, often with 'tram-track' appearance B. Due to immune complex deposition (granular IF) C. Divided into two types based on location of deposits 1. Type I-subendothelial (Fig. 12.10); associated with HBV and HCV 2. Type II (dense deposit disease)-intramembranous; associated with C3 nephritic factor (autoantibody that stabilizes C3 convertase, leading to overactivation of complement, inflammation, and low levels of circulating C3) D. Poor response to steroids; progresses to chronic renal failure VI. DIABETES MELLITUS A. High serum glucose leads to nonenzymatic glycosylation of the vascular basement membrane resulting in hyaline arteriolosclerosis. Fig. 12.7 Minimal change disease. A, Normal glomerulus. B, Effacement of foot processes on EM. Fig. 12.8 Focal segmental glomerulosclerosis. (Courtesy ofTony Chang, MD)

1	Fig. 12.7 Minimal change disease. A, Normal glomerulus. B, Effacement of foot processes on EM. Fig. 12.8 Focal segmental glomerulosclerosis. (Courtesy ofTony Chang, MD) B. Glomerular efferent arteriole is more affected than the afferent arteriole, leading to high glomerular filtration pressure. 1. Hyperfiltration injury leads to microalbuminuria. C. Eventually progresses to nephrotic syndrome 1. Characterized by sclerosis of the mesangium with formation of KimmelstielWilson nodules (Fig. 12.11) D. ACE inhibitors slow progression ofhyperfiltration-induced damage. VII. SYSTEMIC AMYLOIDOSIS A. Kidney is the most commonly involved organ in systemic amyloidosis. B. Amyloid deposits in the mesangium, resulting in nephrotic syndrome. C. Characterized by apple-green birefringence under polarized light after staining with Congo red I. BASIC PRINCIPLES A. Glomerular disorders characterized by glomerular inflammation and bleeding 1. Limited proteinuria (< 3.5 g/day) 2. 3.

1	I. BASIC PRINCIPLES A. Glomerular disorders characterized by glomerular inflammation and bleeding 1. Limited proteinuria (< 3.5 g/day) 2. 3. Salt retention with periorbital edema and hypertension 4. B. Biopsy reveals hypercellular, inflamed glomeruli (Fig. 12.12). Fig. 12.9 Membranous nephropathy. A, Thick glomerular basement membranes. B, Granular IF. C, Subepithelial deposits with 'spike and dome' appearance. (Courtesy ofTony Chang, MD) 1. Immune-complex deposition activates complement; CSa attracts neutrophils, which mediate damage. A. Nephritic syndrome that arises after group A 0-hemolytic streptococcal infection of the skin (impetigo) or pharynx 1. Occurs with nephritogenic strains 2. May occur after infection with nonstreptococcal organisms as well B. Presents 2-3 weeks after infection as hematuria (cola-colored urine), oliguria, hypertension, and periorbital edema 1. Usually seen in children, but may occur in adults C. Hypercellular, inflamed glomeruli on H&E

1	C. Hypercellular, inflamed glomeruli on H&E D. Mediated by immune complex deposition (granular IF); subepithelial 'humps' on EM (Fig. 12.13) E. Treatment is supportive. 1. Children rarely (1%) progress to renal failure. 2. Some adults (25%) develop rapidly progressive glomerulonephritis (RPGN). III. RAPIDLY PROGRESSIVE GLOMERULONEPHRITIS A. Nephritic syndrome that progresses to renal failure in weeks to months Table 12.1: lmmunotluorescence Findings in Rapidly Progressive Glomerulonephritis IMMUNOFI.UORESCENCE > . . • . ... l'A'ITERN I ISl:ASI·. COMMl:N IS antibody, Fig. 12.15) presents as hematuria and hemoptysis, classically in young, adult males Diffuse proliferative glomerulonephritis is Granular (immune complex PSGN (most common) or diffuse proliferative due to diffuse antigen-antibody complex deposition) glomerulonephritis deposition, usually sub-endothelial; most common type of renal disease in SLE

1	Wegener granulomatosis is associated with c-ANCA; microscopic polyangiitis and Wegener granulomatosis, microscopic Churg-Strauss are associated with p-ANCA. polyangiitis, and Churg-Strauss syndrome Granulomatous inflammation, eosinophilia, and asthma distinguish Churg-Strauss from microscopic polyangiitis. Fig. 12.13 Subepithelial 'humps,' PSGN. (Courtesy Fig. 12.14 Crescent formation, rapidly ofTony Chang, MD) progressive glomerulonephritis. B. Characterized by crescents in Bowman space (of glomeruli) on H&E stain; crescents are comprised of fibrin and macrophages (Fig. 12.14). C. Clinical picture and IF help resolve etiology (Table 12.1). IV. A. IgA immune complex deposition in mesangium of glomeruli; most common nephropathy worldwide B. Presents during childhood as episodic gross or microscopic hematuria with RBC casts, usually following mucosa! infections (e.g., gastroenteritis) 1. IgA production is increased during infection.

1	B. Presents during childhood as episodic gross or microscopic hematuria with RBC casts, usually following mucosa! infections (e.g., gastroenteritis) 1. IgA production is increased during infection. C. IgA immune complex deposition in the mesangium is seen on IF (Fig. 12.16). D. May slowly progress to renal failure V. A. Inherited defect in type IV collagen; most commonly X-linked B. Results in thinning and splitting of the glomerular basement membrane C. Presents as isolated hematuria, sensory hearing loss, and ocular disturbances I. BASIC PRINCIPLES A. Infection of urethra, bladder, or kidney B. Most commonly arises due to ascending infection; increased incidence in females C. Risk factors include sexual intercourse, urinary stasis, and catheters. II. CYSTITIS A. Infection of the bladder B. Presents as dysuria, urinary frequency, urgency, and suprapubic pain; systemic signs (e.g., fever) are usually absent. C. Laboratory findings 1.

1	II. CYSTITIS A. Infection of the bladder B. Presents as dysuria, urinary frequency, urgency, and suprapubic pain; systemic signs (e.g., fever) are usually absent. C. Laboratory findings 1. Urinalysis-cloudy urine with > 10 WBCs/high power field (hpf) 2. Dipstick-Positive leukocyte esterase (due to pyuria) and nitrites (bacteria convert nitrates to nitrites) 3. Culture-greater than 100,000 colony forming units (gold standard) D. Etiology 1. E coli (80%) 2. Staphylococcus saprophyticus-increased incidence in young, sexually active women (but E coli is still more common in this population) 3. Klebsiella pneumoniae 4. Proteus mirabilis-alkaline urine with ammonia scent Fig. 12.15 Linear IF, Goodpasture syndrome. Fig. 12.16 lgA nephropathy. (Courtesy ofTony (Courtesy ofTony Chang, MD) Chang, MD) 5. Enterococcus faecalis E. Sterile pyuria is the presence of pyuria (> 10 WBCs/hpf and leukocyte esterase) with a negative urine culture.

1	E. Sterile pyuria is the presence of pyuria (> 10 WBCs/hpf and leukocyte esterase) with a negative urine culture. 1. Suggests urethritis due to Chlamydia trachomatis or Neisseria gonorrhoeae (dominant presenting sign of urethritis is dysuria) III. PYELONEPHRITIS A. Infection of the kidney 1. Usually due to ascending infection; increased risk with vesicoureteral reflux B. Presents with fever, flank pain, WBC casts, and leukocytosis in addition to symptoms of cystitis C. Most common pathogens are 1. E coli (90%) 2. 3. IV. CHRONIC PYELONEPHRITIS A. Interstitial fibrosis and atrophy of tubules due to multiple bouts of acute pyelonephritis B. Due to vesicoureteral reflux (children) or obstruction (e.g., BPH or cervical carcinoma) C. Leads to cortical scarring with blunted calyces; scarring at upper and lower poles is characteristic of vesicoureteral reflux.

1	C. Leads to cortical scarring with blunted calyces; scarring at upper and lower poles is characteristic of vesicoureteral reflux. D. Atrophic tubules containing eosinophilic proteinaceous material resemble thyroid follicles ('thyroidization' of the kidney, Fig. 12.17); waxy casts may be seen in urine. I. BASIC PRINCIPLES A. Precipitation of a urinary solute as a stone (Table 12.2) B. Risk factors include high concentration of solute in the urinary filtrate and low urine volume. C. Presents as colicky pain with hematuria and unilateral flank tenderness 1. Stone is usually passed within hours; if not, surgical intervention may be required. I. BASIC PRINCIPLES A. End-stage kidney failure 1. May result from glomerular, tubular, inflammatory, or vascular insults 2. Most common causes are diabetes mellitus, hypertension, and glomerular disease. B. Clinical features 1.

1	May result from glomerular, tubular, inflammatory, or vascular insults 2. Most common causes are diabetes mellitus, hypertension, and glomerular disease. B. Clinical features 1. Uremia-Increased nitrogenous waste products in blood (azotemia) result in nausea, anorexia, pericarditis, platelet dysfunction, encephalopathy with asterixis, and deposition of urea crystals in skin. 2. Salt and water retention with resultant hypertension 3. Hyperkalemia with metabolic acidosis 4. Anemia due to decreased erythropoietin production by renal peritubular interstitial cells 5. Hypocalcemia due to decreased !-alpha-hydroxylation ofvitamin D by proximal renal tubule cells and hyperphosphatemia 6. Renal osteodystrophy due to secondary hyperparathyroidism, osteomalacia, and osteoporosis C. Treatment involves dialysis or renal transplant. 1. Cysts often develop within shrunken end-stage kidneys during dialysis, increasing risk for renal cell carcinoma. I. ANGIOMYOLIPOMA

1	C. Treatment involves dialysis or renal transplant. 1. Cysts often develop within shrunken end-stage kidneys during dialysis, increasing risk for renal cell carcinoma. I. ANGIOMYOLIPOMA A. Hamartoma comprised of blood vessels, smooth muscle, and adipose tissue B. Increased frequency in tuberous sclerosis II. RENAL CELL CARCINOMA A. Malignant epithelial tumor arising from kidney tubules Fig. 12.17 'Thyroidization' of kidney, chronic Fig. 12.18 Ammonium magnesium phosphate pyelonephritis. stone. (Courtesy of webpathology.com) B. Presents with classic triad of hematuria, palpable mass, and flank pain 1. All three symptoms rarely occur together; hematuria is the most common symptom. 2. Fever, weight loss, or paraneoplastic syndrome (e.g., EPO, renin, PTHrP, or ACTH) may also be present. 3. Rarely may present with left-sided varicocele i. Involvement of the left renal vein by carcinoma blocks drainage of the left spermatic vein leading to varicocele.

1	3. Rarely may present with left-sided varicocele i. Involvement of the left renal vein by carcinoma blocks drainage of the left spermatic vein leading to varicocele. 11. Right spermatic vein drains directly into the IVC; hence, right-sided varicocele is not seen. C. Gross exam reveals a yellow mass (Fig. 12.19A); microscopically, the most common variant exhibits clear cytoplasm (clear cell type, Fig. 12.19B). D. Pathogenesis involves loss of VHL (3p) tumor suppressor gene, which leads to increased IGF-1 (promotes growth) and increased HIF transcription factor (increases VEGF and PDGF). E. Tumors may be hereditary or sporadic. 1. Sporadic tumors classically arise in adult males (average age is 60 years) as a single tumor in the upper pole of the kidney; major risk factor for sporadic tumors is cigarette smoke. 2. Hereditary tumors arise in younger adults and are often bilateral.

1	2. Hereditary tumors arise in younger adults and are often bilateral. i. Von Hippel-Lindau disease is an autosomal dominant disorder associated with inactivation of the VHL gene leading to increased risk for hemangioblastoma of the cerebellum and renal cell carcinoma. F. Staging 1. T-based on size and involvement of the renal vein (occurs commonly and increases risk of hematogenous spread to the lungs and bone) 2. N-spread to retroperitoneal lymph nodes III. WILMS TUMOR A. Malignant kidney tumor comprised ofblastema (immature kidney mesenchyme), primitive glomeruli and tubules, and stromal cells (Fig. 12.20) 1. Most common malignant renal tumor in children; average age is 3 years. B. Presents as a large, unilateral flank mass with hematuria and hypertension (due to renin secretion) C. Most cases (90%) are sporadic; syndromic tumors may be seen with 1.

1	B. Presents as a large, unilateral flank mass with hematuria and hypertension (due to renin secretion) C. Most cases (90%) are sporadic; syndromic tumors may be seen with 1. WAGR syndrome-Wilms tumor, Aniridia, Genital abnormalities, and mental and motor Retardation; associated with deletion of WTl tumor suppressor gene (located at llpl3) 2. Denys-Drash syndrome-Wilms tumor, progressive renal (glomerular) disease, and male pseudohermaphroditism; associated with mutations of WTl Fig. 12.19 Renal cell carcinoma. A, Gross appearance. B, Microscopic appeara nce. Fig, 12,20 Wl lms tumor. 3. Beckwith-Wiedemann syndrome-Wilms tumor, neonatal hypoglycemia, muscular hemihypertrophy, and organomegaly (including tongue); associated with mutations in WT2 gene cluster (imprinted genes at llplS.5), particularly IGF-2 I. UROTHELIAL (TRANSITIONAL CELL) CARCINOMA

1	I. UROTHELIAL (TRANSITIONAL CELL) CARCINOMA A. Malignant tumor arising from the urothelial lining of the renal pelvis, ureter, bladder, or urethra 1. Most common type of lower urinary tract cancer; usually arises in the bladder B. Major risk factor is cigarette smoke; additional risk factors are naphthylamine, azo dyes, and long-term cyclophosphamide or phenacetin use. C. Generally seen in older adults; classically presents with painless hematuria D. Arises via two distinct pathways (Fig. 12.21) 1. Flat-develops as a high-grade flat tumor and then invades; associated with early p53 mutations 2. Papillary-develops as a low-grade papillary tumor that progresses to a highgrade papillary tumor and then invades; not associated with early p53 mutations E. Tumors are often multifocal and recur ("field defect"). II. SQUAMOUS CELL CARCINOMA A. Malignant proliferation of squamous cells, usually involving the bladder

1	E. Tumors are often multifocal and recur ("field defect"). II. SQUAMOUS CELL CARCINOMA A. Malignant proliferation of squamous cells, usually involving the bladder B. Arises in a background ofsquamous metaplasia (normal bladder surface is not lined by squamous epithelium) C. Risk factors include chronic cystitis (older woman), Schistosoma haematobium infection (Egyptian male), and long-standing nephrolithiasis. III. ADENOCARCINOMA A. Malignant proliferation of glands, usually involving bladder B. Arises from a urachal remnant (tumor develops at the dome of the bladder), cystitis glandularis, or exstrophy (congenital failure to form the caudal portion of the anterior abdominal and bladder walls) Fig. 12.21 Urothelial carcinoma. A, Flat. B, Papi llary. I. BASIC PRINCIPLES A. Anatomically includes the skin and mucosa of the female genitalia external to the hymen (labia majora, labia minora, mons pubis, and vestibule) B. Lined by squamous epithelium II. BARTHOLIN CYST

1	B. Lined by squamous epithelium II. BARTHOLIN CYST A. Cystic dilation of the Bartholin gland 1. One Bartholin gland is present on each side of the vaginal canal and produces mucus-like fluid that drains via ducts into the lower vestibule. B. Arises due to inflammation and obstruction of gland 1. Usually occurs in women of reproductive age C. Presents as a unilateral, painful cystic lesion at the lower vestibule adjacent to the vaginal canal III. CONDYLOMA A. Warty neoplasm of vulvar skin, often large B. Most commonly due to HPV types 6 or 11 (condyloma acuminatum, Fig. 13.lA); secondary syphilis (condyloma latum) is a less common cause. Both are sexually transmitted. C. Histologically, HPV-associated condylomas are characterized by koilocytes (hallmark ofHPV-infected cells, Fig. 13.lB). D. Condylomas rarely progress to carcinoma (6 and 11 are low-risk HPV types). IV. A. Characterized by thinning of the epidermis and fibrosis (sclerosis) of the dermis

1	D. Condylomas rarely progress to carcinoma (6 and 11 are low-risk HPV types). IV. A. Characterized by thinning of the epidermis and fibrosis (sclerosis) of the dermis B. Presents as a white patch (leukoplakia) with parchment-like vulvar skin C. Most commonly seen in postmenopausal women; possible autoimmune etiology D. Benign, but associated with a slightly increased risk for squamous cell carcinoma V. A. Characterized by hyperplasia of the vulvar squamous epithelium B. Presents as leukoplakia with thick, leathery vulvar skin C. Associated with chronic irritation and scratching D. Benign; no increased risk of squamous cell carcinoma VI. VULVAR CARCINOMA A. Carcinoma arising from squamous epithelium lining the vulva B. Relatively rare, accounting for only a small percentage of female genital cancers C. Presents as leukoplakia; biopsy may be required to distinguish carcinoma from other causes ofleukoplakia. D. Etiology may be HPV related or non-HPV related.

1	C. Presents as leukoplakia; biopsy may be required to distinguish carcinoma from other causes ofleukoplakia. D. Etiology may be HPV related or non-HPV related. E. HPV-related vulvar carcinoma is due to high-risk HPV types 16 and 18. pathoma.com 1. Risk factors are related to HPV exposure and include multiple partners and early first age of intercourse; generally occurs in women of reproductive age 2. Arises from vulvar intraepithelial neoplasia (VIN), a dysplastic precursor lesion characterized by koilocytic change, disordered cellular maturation, nuclear atypia, and increased mitotic activity F. Non-HPV related vulvar carcinoma arises, most often, from long-standing lichen sclerosis. 1. Chronic inflammation and irritation eventually lead to carcinoma. 2. Generally seen in elderly women (average age is > 70 years) VII. EXTRAMAMMARY PAGET DISEASE A. Characterized by malignant epithelial cells in the epidermis of the vulva (Fig. 13.2A)

1	2. Generally seen in elderly women (average age is > 70 years) VII. EXTRAMAMMARY PAGET DISEASE A. Characterized by malignant epithelial cells in the epidermis of the vulva (Fig. 13.2A) B. Presents as erythematous, pruritic, ulcerated vulvar skin C. Represents carcinoma in situ, usually with no underlying carcinoma 1. Paget disease of the nipple is also characterized by malignant epithelial cells in the epidermis of the nipple, but it is almost always associated with an underlying carcinoma. D. Must be distinguished from melanoma, which rarely can occur on the vulva 1. Paget cells are PAS+, keratin+, and S-100-. 2. Melanoma is PAS-, keratin-, and S-100+. I. BASIC PRINCIPLES A. Canal leading to the cervix B. Mucosa is lined by non-keratinizing squamous epithelium II. ADENOSIS

1	2. Melanoma is PAS-, keratin-, and S-100+. I. BASIC PRINCIPLES A. Canal leading to the cervix B. Mucosa is lined by non-keratinizing squamous epithelium II. ADENOSIS A. Focal persistence of columnar epithelium in the upper vagina (Fig. 13.2B) 1. During development, squamous epithelium from the lower 1/3 of the vagina (derived from the urogenital sinus) grows upward to replace the columnar epithelium lining of the upper 2/3 of the vagina (derived from the Miillerian ducts). B. Increased incidence in females who were exposed to diethylstilbestrol (DES) in utero Ill. CLEAR CELL ADENOCARCINOMA A. Malignant proliferation of glands with clear cytoplasm B. Rare, but feared, complication of DES-associated vaginal adenosis Fig. 13.1 Condyloma acuminata. A, Microscopic appearance. B, Koi iocytic change. Fig. 13.2A Extramammary Paget disease of the vulva. (Courtesy of webpathology.com)

1	Fig. 13.1 Condyloma acuminata. A, Microscopic appearance. B, Koi iocytic change. Fig. 13.2A Extramammary Paget disease of the vulva. (Courtesy of webpathology.com) C. Discovery of this complication (along with other DES-induced abnormalities of the gynecologic tract such as abnormal shape of the uterus) led to cessation of DES usage. IV. A. Malignant mesenchymal proliferation of immature skeletal muscle; rare B. Presents as bleeding and a grape-like mass protruding from the vagina or penis of a child (usually< 5 yrs of age); also known as sarcoma botryoides (Fig. 13.3) C. Rhabdomyoblast, the characteristic cell, exhibits cytoplasmic cross-striations and positive immunohistochemical staining for desmin and myogenin. V. A. Carcinoma arising from squamous epithelium lining the vaginal mucosa B. Usually related to high-risk HPV C. Precursor lesion is vaginal intraepithelial neoplasia (VAIN).

1	V. A. Carcinoma arising from squamous epithelium lining the vaginal mucosa B. Usually related to high-risk HPV C. Precursor lesion is vaginal intraepithelial neoplasia (VAIN). D. When spread to regional lymph nodes occurs, cancer from the lower 1/3 ofvagina goes to inguinal nodes, and cancer from the upper 2/3 goes to regional iliac nodes. I. BASIC PRINCIPLES A. Anatomically, comprises the "neck" of the uterus B. Divided into the exocervix (visible on vaginal exam) and endocervix 1. Exocervix is lined by nonkeratinizing squamous epithelium. 2. Endocervix is lined by a single layer of columnar cells. 3. Junction between the exocervix and endocervix is called the transformation zone (Fig. 13.4A). II. HPV A. Sexually transmitted DNA virus that infects the lower genital tract, especially the cervix in the transformation zone

1	II. HPV A. Sexually transmitted DNA virus that infects the lower genital tract, especially the cervix in the transformation zone B. Infection is usually eradicated by acute inflammation; persistent infection leads to an increased risk for cervical dysplasia (cervical intraepithelial neoplasia, CIN). C. Risk of CIN depends on HPV type, which is determined by DNA sequencing. 1. High-risk-HPV types 16, 18, 31, and 33 2. D. High-risk HPV produce E6 and E7 proteins which result in increased destruction of p53 and Rb, respectively. Loss of these tumor suppressor proteins increases the risk for CIN. Fig. 13.2B Vaginal adenosis. Fig. 13.3 Embryonal rhabdomyosarcoma. (Courtesy of hum path.corn) III. CERVICAL INTRAEPITHELIAL NEOPLASIA A. Characterized by koilocytic change, disordered cellular maturation, nuclear atypia, and increased mitotic activity within the cervical epithelium. B. Divided into grades based on the extent of epithelial involvement by immature dysplastic cells 1.

1	B. Divided into grades based on the extent of epithelial involvement by immature dysplastic cells 1. CIN I involves < 1/3 of the thickness of the epithelium. 2. CIN II involves < 2/3 of the thickness of the epithelium. 3. CIN III involves slightly less than the entire thickness of the epithelium (Fig. 13.4B). 4. Carcinoma in situ (CIS) involves the entire thickness of the epithelium. C. CIN classically progresses in a stepwise fashion through CIN I, CIN II, CIN III, and CIS to become invasive squamous cell carcinoma. 1. Progression is not inevitable (e.g., CINI often regresses). 2. The higher the grade of dysplasia, the more likely it is to progress to carcinoma and the less likely it is to regress to normal. IV. A. Invasive carcinoma that arises from the cervical epithelium B. Most commonly seen in middle-aged women (average age is 40-50 years) C. Presents as vaginal bleeding, especially postcoital bleeding, or cervical discharge

1	B. Most commonly seen in middle-aged women (average age is 40-50 years) C. Presents as vaginal bleeding, especially postcoital bleeding, or cervical discharge D. Key risk factor is high-risk HPV infection; secondary risk factors include smoking and immunodeficiency (e.g., cervical carcinoma is an AIDS-defining illness). E. Most common subtypes of cervical carcinoma are squamous cell carcinoma (80% of cases) and adenocarcinoma (15% of cases). Both types are related to HPV infection. F. Advanced tumors often invade through the anterior uterine wall into the bladder, blocking the ureters. Hydronephrosis with postrenal failure is a common cause of death in advanced cervical carcinoma. V. A. The goal of screening is to catch dysplasia (CIN) before it develops into carcinoma. 1. Progression from CIN to carcinoma, on average, takes 10-20 years. 2. Screening begins at age 21 and is initially performed every three years. B. Pap smear is the gold standard for screening. 1.

1	Progression from CIN to carcinoma, on average, takes 10-20 years. 2. Screening begins at age 21 and is initially performed every three years. B. Pap smear is the gold standard for screening. 1. Cells are scraped from the transformation zone using a brush and analyzed under a microscope. 2. Dysplastic cells are classified as low grade (CIN I) or high grade (CIN II and III). 3. High-grade dysplasia is characterized by cells with hyperchromatic (dark) nuclei and high nuclear to cytoplasmic ratios (Fig. 13.4C). C. Pap smear is the most successful screening test developed to date. Fig. 13.4 Cervix. A, Cervical transformation zone, normal. B, CIN Ill. C, High-grade dysplasia, Pap smear. (Courtesy of Ed Uthman, MD) 1. It is responsible for a significant reduction in the morbidity and mortality of cervical carcinoma (cervical carcinoma went from being the most common to one of the least common types of gynecologic carcinoma in the US). 2.

1	2. Women who develop invasive cervical carcinoma usually have not undergone screening. D. An abnormal Pap smear is followed by confirmatory colposcopy (visualization of cervix with a magnifying glass) and biopsy. E. Limitations of the Pap smear include inadequate sampling of the transformation zone (false negative screening) and limited efficacy in screening for adenocarcinoma. 1. Despite Pap smear screening, the incidence of adenocarcinoma has not decreased significantly. F. Immunization is effective in preventing HPV infections. 1. The quadrivalent vaccine covers HPV types 6, 11, 16, and 18. 2. Antibodies generated against types 6 and 11 protect against condylomas. 3. Antibodies generated against types 16 and 18 protect against CIN and carcinoma. 4. Pap smears are still necessary due to the limited number ofHPV types covered by the vaccine. I. BASIC PRINCIPLES A. Endometrium is the mucosa I lining of the uterine cavity.

1	4. Pap smears are still necessary due to the limited number ofHPV types covered by the vaccine. I. BASIC PRINCIPLES A. Endometrium is the mucosa I lining of the uterine cavity. B. Myometrium is the smooth muscle wall underlying the endometrium (Fig. 13.5). C. Endometrium is hormonally sensitive. 1. Growth of the endometrium is estrogen driven (proliferative phase). 2. Preparation of the endometrium for implantation is progesterone driven (secretory phase). 3. Shedding occurs with loss of progesterone support (menstrual phase). II. ASHERMAN SYNDROME A. Secondary amenorrhea due to loss of the basalis and scarring B. Result of overaggressive dilation and curettage (D&C) III. ANOVULATORY CYCLE A. Lack of ovulation B. Results in an estrogen-driven proliferative phase without a subsequent progesteronedriven secretory phase 1. Proliferative glands break down and shed resulting in uterine bleeding.

1	B. Results in an estrogen-driven proliferative phase without a subsequent progesteronedriven secretory phase 1. Proliferative glands break down and shed resulting in uterine bleeding. Fig. 13.5 Endometrium overlying myometrium, Fig. 13.6 Endometrial polyp. normal. 2. Represents a common cause of dysfunctional uterine bleeding, especially during menarche and menopause IV. A. Bacterial infection of the endometrium B. Usually due to retained products of conception (e.g., after delivery or miscarriage); retained products act as a nidus for infection. C. Presents as fever, abnormal uterine bleeding, and pelvic pain V. A. Chronic inflammation of the endometrium B. Characterized by lymphocytes and plasma cells 1. Plasma cells are necessary for the diagnosis of chronic endometritis given that lymphocytes are normally found in the endometrium. C. Causes include retained products of conception, chronic pelvic inflammatory disease (e.g., Chlamydia), IUD, and TB.

1	C. Causes include retained products of conception, chronic pelvic inflammatory disease (e.g., Chlamydia), IUD, and TB. D. Presents as abnormal uterine bleeding, pain, and infertility VI. ENDOMETRIAL POLYP A. Hyperplastic protrusion of endometrium (Fig. 13.6) B. Presents as abnormal uterine bleeding C. Can arise as a side effect of tamoxifen, which has anti-estrogenic effects on the breast but weak pro-estrogenic effects on the endometrium VII. ENDOMETRIOSIS A. Endometrial glands and stroma outside of the uterine endometrial lining 1. Most likely due to retrograde menstruation with implantation at an ectopic site B. Presents as dysmenorrhea (pain during menstruation) and pelvic pain; may cause infertility 1. Endometriosis cycles just like normal endometrium. C. Most common site of involvement is the ovary, which classically results in formation of a 'chocolate' cyst (Fig. 13.7A). 1.

1	C. Most common site of involvement is the ovary, which classically results in formation of a 'chocolate' cyst (Fig. 13.7A). 1. Other sites of involvement include the uterine ligaments (pelvic pain), pouch of Douglas (pain with defecation), bladder wall (pain with urination), bowel serosa (abdominal pain and adhesions), and fallopian tube mucosa (scarring increases risk for ectopic tubal pregnancy); implants classically appear as yellow-brown 'gun-powder' nodules (Fig. 13.7B). 2. Involvement of the uterine myometrium is called adenomyosis. D. There is an increased risk of carcinoma at the site ofendometriosis, especially in the ovary. Fig. 13.7 Endometriosis. A, 'Chocolate cyst' of ovary. B, 'Gun powder' nodules. (A, Courtesy of Bulent Fig. 13.8 Endometrial hyperplasia. (Courtesy of Celasun, MD. B, Courtesy of hum path.com) Ed Uthman, MD) VIII. ENDOMETRIAL HYPERPLASIA A. Hyperplasia ofendometrial glands relative to stroma (Fig. 13.8)

1	VIII. ENDOMETRIAL HYPERPLASIA A. Hyperplasia ofendometrial glands relative to stroma (Fig. 13.8) B. Occurs as a consequence of unopposed estrogen (e.g., obesity, polycystic ovary syndrome, and estrogen replacement) C. Classically presents as postmenopausal uterine bleeding D. Classified histologically based on architectural growth pattern (simple or complex) and the presence or absence of cellular atypia 1. Most important predictor for progression to carcinoma (major complication) is the presence of cellular atypia; simple hyperplasia with atypia often progresses to cancer (30%); whereas, complex hyperplasia without atypia rarely does (<5%). IX. A. Malignant proliferation of endometrial glands (Fig. 13.9A) 1. Most common invasive carcinoma of the female genital tract B. Presents as postmenopausal bleeding C. Arises via two distinct pathways: hyperplasia and sporadic D. In the hyperplasia pathway (75% of cases), carcinoma arises from endometrial hyperplasia. 1.

1	C. Arises via two distinct pathways: hyperplasia and sporadic D. In the hyperplasia pathway (75% of cases), carcinoma arises from endometrial hyperplasia. 1. Risk factors are related to estrogen exposure and include early menarche/late menopause, nulliparity, infertility with anovulatory cycles, and obesity. 2. Average age ofpresentation is 60 years. 3. Histology is endometrioid (i.e., normal endometrium-like, Fig. 13.9B). E. In the sporadic pathway (25% of cases), carcinoma arises in an atrophic endometrium with no evident precursor lesion. 1. Average age at presentation is 70 years. 2. Histology is usually serous and is characterized by papillary structures (Fig. 13.9C) with psammoma body formation; p53 mutation is common, and the tumor exhibits aggressive behavior. X. A. Benign neoplastic proliferation of smooth muscle arising from myometrium; most common tumor in females B. Related to estrogen exposure 1. 2. 3.

1	X. A. Benign neoplastic proliferation of smooth muscle arising from myometrium; most common tumor in females B. Related to estrogen exposure 1. 2. 3. C. Gross exam shows multiple, well-defined, white, whorled masses that may distort the uterus and impinge on pelvic structures (Fig. 13.10). D. Usually asymptomatic; when present, symptoms include abnormal uterine bleeding, infertility, and a pelvic mass. Fig. 13.9 Endometrial carcinoma. A, Gross appearance. B, Endometrioid type. C, Serous type. (A, Courtesy of www.anatomiapatologica.com.br) XI. LEIOMYOSARCOMA A. Malignant proliferation of smooth muscle arising from the myometrium B. Arises de novo; leiomyosarcomas do not arise from leiomyomas. C. Usually seen in postmenopausal women D. Gross exam often shows a single lesion with areas of necrosis and hemorrhage; histological features include necrosis, mitotic activity, and cellular atypia. I. BASIC PRINCIPLES A. The functional unit of the ovary is the follicle.

1	I. BASIC PRINCIPLES A. The functional unit of the ovary is the follicle. B. A follicle consists of an oocyte surrounded by granulosa and theca cells (Fig. 13.llA) 1. LH acts on theca cells to induce androgen production. 2. FSH stimulates granulosa cells to convert androgen to estradiol (drives the proliferative phase of the endometrial cycle). 3. Estradiol surge induces an LH surge, which leads to ovulation (marking the beginning of the secretory phase of the endometrial cycle). C. After ovulation, the residual follicle becomes a corpus luteum (Fig. 13.llB), which primarily secretes progesterone (drives the secretory phase which prepares the endometrium for a possible pregnancy). 1. Hemorrhage into a corpus luteum can result in a hemorrhagic corpus luteal cyst, especially during early pregnancy. D. Degeneration of follicles results in follicular cysts. Small numbers of follicular cysts are common in women and have no clinical significance.

1	D. Degeneration of follicles results in follicular cysts. Small numbers of follicular cysts are common in women and have no clinical significance. II. POLYCYSTIC OVARIAN DISEASE (PCOD) A. Multiple ovarian follicular cysts due to hormone imbalance 1. Affects roughly 5% of women of reproductive age B. Characterized by increased LH and low FSH (LH:FSH > 2) 1. Increased LH induces excess androgen production (from theca cells) resulting in hirsutism (excess hair in a male distribution). 2. Androgen is converted to estrone in adipose tissue. i. Estrone feedback decreases FSH resulting in cystic degeneration of follicles. ii. High levels of circulating estrone increase risk for endometrial carcinoma. C. Classic presentation is an obese young woman with infertility, oligomenorrhea, and hirsutism; some patients have insulin resistance and may develop type 2 diabetes mellitus 10-15 years later.

1	C. Classic presentation is an obese young woman with infertility, oligomenorrhea, and hirsutism; some patients have insulin resistance and may develop type 2 diabetes mellitus 10-15 years later. Fig. 13.10 Uterine leiomyomas. (Courtesy of Fig. 13.11 Ovarian follicle. A, Normal. B, Corpus luteum. (B, Courtesy of Ed Uthman, MD) Jamie Steinmetz, MD) I. BASIC PRINCIPLES A. Ovary is composed of three cell types: surface epithelium, germ cells, and sex cordstroma. B. Tumor can arise from any of these cell types or from metastases. II. SURFACE EPITHELIAL TUMORS A. Most common type of ovarian tumor (70% of cases) B. Derived from coelomic epithelium that lines the ovary; coelomic epithelium embryologically produces the epithelial lining of the fallopian tube (serous cells), endometrium, and endocervix (mucinous cells). C. The two most common subtypes of surface epithelial tumors are serous and mucinous; both are usually cystic. 1. Serous tumors are full ofwatery fluid. 2.

1	C. The two most common subtypes of surface epithelial tumors are serous and mucinous; both are usually cystic. 1. Serous tumors are full ofwatery fluid. 2. Mucinous tumors are full of mucus-like fluid. D. Mucinous and serous tumors can be benign, borderline, or malignant. 1. Benign tumors (cystadenomas) are composed of a single cyst with a simple, flat lining (Fig. 13.12); most commonly arise in premenopausal women (30 -40 years old) 2. Malignant tumors (cystadenocarcinomas) are composed of complex cysts with a thick, shaggy lining; most commonly arise in postmenopausal women (60-70 years old) 3. Borderline tumors have features in between benign and malignant tumors. i. Better prognosis than clearly malignant tumors, but still carry metastatic potential 4. BRCAl mutation carriers have an increased risk for serous carcinoma of the ovary and fallopian tube.

1	i. Better prognosis than clearly malignant tumors, but still carry metastatic potential 4. BRCAl mutation carriers have an increased risk for serous carcinoma of the ovary and fallopian tube. 1. BRCAl carriers often elect to have a prophylactic salpingo-oophorectomy (along with prophylactic mastectomy due to the increased risk for breast cancer). E. Less common subtypes of surface epithelial tumors include endometrioid and Brenner tumor. 1. Endometrioid tumors are composed of endometrial-like glands and are usually malignant. i. May arise from endometriosis ii. 15% of endometrioid carcinomas of the ovary are associated with an independent endometrial carcinoma (endometrioid type). 2. Brenner tumors are composed of bladder-like epithelium and are usually benign. F. Surface tumors clinically present late with vague abdominal symptoms (pain and fullness) or signs of compression (urinary frequency). Fig. 13.12 Ovarian cystadenoma. 1.

1	F. Surface tumors clinically present late with vague abdominal symptoms (pain and fullness) or signs of compression (urinary frequency). Fig. 13.12 Ovarian cystadenoma. 1. Prognosis is generally poor for surface epithelial carcinoma (worst prognosis of female genital tract cancers). 2. Epithelial carcinomas tend to spread locally, especially to the peritoneum. G. CA-125 is a useful serum marker to monitor treatment response and screen for recurrence. III. GERM CELL TUMORS A. 2nd most common type of ovarian tumor (15% of cases) B. Usually occur in women of reproductive age C. Tumor subtypes mimic tissues normally produced by germ cells. 1. 2. 3. 4. D. Cystic teratoma 1. Cystic tumor composed of fetal tissue derived from two or three embryo logic layers (e.g., skin, hair, bone, cartilage, gut, and thyroid, Fig. 13.13) i. Most common germ cell tumor in females; bilateral in 10% of cases 2.

1	Benign, but presence of immature tissue (usually neural) or somatic malignancy (usually squamous cell carcinoma of skin) indicates malignant potential. 3. Struma ovarii is a teratoma composed primarily of thyroid tissue. E. Dysgerminoma 1. Tumor composed oflarge cells with clear cytoplasm and central nuclei (resemble oocytes, Fig. 13.14); most common malignant germ cell tumor 2. Testicular counterpart is called seminoma, which is a relatively common germ cell tumor in males. 3. Good prognosis; responds to radiotherapy 4. Serum LDH may be elevated. F. Endodermal sinus tumor 1. Malignant tumor that mimics the yolk sac; most common germ cell tumor in children 2. Serum AFP is often elevated. 3. Schiller-Duval bodies (glomerulus-like structures) are classically seen on histology (Fig. 13.15). G. Choriocarcinoma 1. Malignant tumor composed of cytotrophoblasts and syncytiotrophoblasts; mimics placental tissue, but villi are absent 2.

1	G. Choriocarcinoma 1. Malignant tumor composed of cytotrophoblasts and syncytiotrophoblasts; mimics placental tissue, but villi are absent 2. Small, hemorrhagic tumor with early hematogenous spread 3. High P-hCG is characteristic (produced by syncytiotrophoblasts); may lead to thecal cysts in the ovary Fig. 13.13 Cystic teratoma. Fig. 13.14 Dysgermin oma. Fig. 13.15 Schiller-Duval body. (Courtesy webpathology.com) 4. Poor response to chemotherapy H. Embryonal carcinoma 1. 2. Aggressive with early metastasis IV. A. Tumors that resemble sex cord-stromal tissues of the ovary B. Granulosa-theca cell tumor 1. Neoplastic proliferation of granulosa and theca cells 2. Often produces estrogen; presents with signs of estrogen excess 1. Prior to puberty-precocious puberty 11. Reproductive age-menorrhagia or metrorrhagia iii. Postmenopause (most common setting for granulosa-theca cell tumors)endometrial hyperplasia with postmenopausal uterine bleeding 3.

1	Malignant, but minimal risk for metastasis C. Sertoli-Leydig cell tumor 1. Composed of Sertoli cells that form tubules and Leydig cells (between tubules) with characteristic Reinke crystals 2. May produce androgen; associated with hirsutism and virilization D. Fibroma 1. Benign tumor of fibroblasts (Fig. 13.16) 2. Associated with pleural effusions and ascites (Meigs syndrome); syndrome resolves with removal of tumor. V. A. Krukenberg tumor is a metastatic mucinous tumor that involves both ovaries; most commonly due to metastatic gastric carcinoma (diffuse type) 1. Bilaterality helps distinguish metastases from primary mucinous carcinoma of the ovary, which is usually unilateral. B. Pseudomyxoma peritonei is massive amounts of mucus in the peritoneum. 1. Due to a mucinous tumor of the appendix, usually with metastasis to the ovary I. ECTOPIC PREGNANCY

1	B. Pseudomyxoma peritonei is massive amounts of mucus in the peritoneum. 1. Due to a mucinous tumor of the appendix, usually with metastasis to the ovary I. ECTOPIC PREGNANCY A. Implantation of fertilized ovum at a site other than the uterine wall; most common site is the lumen of the fallopian tube (Fig. 13.17). B. Key risk factor is scarring (e.g., secondary to pelvic inflammatory disease or endometriosis). Fig. 13.16 Ovarian fibroma. Fig. 13.17 Ectopic tubal pregnancy. (Courtesy of humpath.com) C. Classic presentation is lower quadrant abdominal pain a few weeks after a missed period. 1. Surgical emergency; major complications are bleeding into fallopian tube (hematosalpinx) and rupture. II. SPONTANEOUS ABORTION A. Miscarriage of fetus occurring before 20 weeks gestation (usually during first trimester) 1. Common; occurs in up to 1/4 of recognizable pregnancies B. Presents as vaginal bleeding, cramp-like pain, and passage of fetal tissues

1	B. Presents as vaginal bleeding, cramp-like pain, and passage of fetal tissues C. Most often due to chromosomal anomalies (especially trisomy 16); other causes include hypercoagulable states (e.g., antiphospholipid syndrome), congenital infection, and exposure to teratogens (especially during the first 2 weeks of embryogenesis). 1. Effect of teratogens generally depends on the dose, agent, and time of exposure (Table 12.1). i. First two weeks of gestation-spontaneous abortion ii. Weeks 3-8-risk of organ malformation iii. Months 3-9-risk of organ hypoplasia III. PLACENTA PREVIA A. Implantation of the placenta in the lower uterine segment; placenta overlies cervical os (opening). B. Presents as third-trimester bleeding C. Often requires delivery of fetus by caesarian section IV. A. Separation of placenta from the decidua prior to delivery of the fetus (Fig. 13.18) B. Common cause of still birth C. Presents with third-trimester bleeding and fetal insufficiency V.

1	IV. A. Separation of placenta from the decidua prior to delivery of the fetus (Fig. 13.18) B. Common cause of still birth C. Presents with third-trimester bleeding and fetal insufficiency V. A. Improper implantation of placenta into the myometrium with little or no intervening decidua B. Presents with difficult delivery of the placenta and postpartum bleeding Table 12.1: Common Teratogens and Associated Effects Most common cause of mental retardation; also leads to facial lsotretinoin Spontaneous abortion, hearing and visual impairment C. Often requires hysterectomy VI. PREECLAMPSIA A. Pregnancy-induced hypertension, proteinuria, and edema, usually arising in the third trimester; seen in approximately 5% of pregnancies 1. Hypertension may be severe, leading to headaches and visual abnormalities. B. Due to abnormality of the maternal-fetal vascular interface in the placenta; resolves with delivery C. Eclampsia is preeclampsia with seizures.

1	B. Due to abnormality of the maternal-fetal vascular interface in the placenta; resolves with delivery C. Eclampsia is preeclampsia with seizures. D. HELLP is preeclampsia with thrombotic microangiopathy involving the liver; characterized by Hemolysis, Elevated Liver enzymes, and Low Platelets E. Both eclampsia and HELLP usually warrant immediate delivery. VII. SUDDEN INFANT DEATH SYNDROME A. Death of a healthy infant (1 month to 1 year old) without obvious cause B. Infants usually expire during sleep C. Risk factors include sleeping on stomach, exposure to cigarette smoke, and prematurity. VIII. HYDATIDIFORM MOLE A. Abnormal conception characterized by swollen and edematous villi with proliferation of trophoblasts Table 12.2: Features of Complete and Partial Moles Some villi are hydropic, and some Villous edema Most villi are hydropic (Fig. 13.19B). are normal.

1	Table 12.2: Features of Complete and Partial Moles Some villi are hydropic, and some Villous edema Most villi are hydropic (Fig. 13.19B). are normal. Trophoblastic Focal proliferation present around Diffuse, circumferential proliferation hydropic villi proliferation around hydropic villi Fig. 13.18 Placental abruption. (Courtesy of Fig. 13.19 Complete mole. A, Swollen, 'grape-like' villi. B, Hydropic villi. (A, Courtesy of Steven Jerome Taxy, MD) O'Connor, MD) B. Uterus expands as if a normal pregnancy is present, but the uterus is much larger and ~-hCG much higher than expected for date of gestation. C. Classically presents in the second trimester as passage of grape-like masses through the vaginal canal (Fig. 13.19A). 1. With prenatal care, moles are diagnosed by routine ultrasound in the early first trimester. Fetal heart sounds are absent, and a 'snowstorm' appearance is classically seen on ultrasound. D. Classified as complete or partial (Table 12.2)

1	D. Classified as complete or partial (Table 12.2) E. Treatment is suction curettage. 1. Subsequent p-hCG monitoring is important to ensure adequate mole removal and to screen for the development of choriocarcinoma. 1. Choriocarcinoma may arise as a complication of gestation (spontaneous abortion, normal pregnancy, or hydatidiform mole) or as a spontaneous germ cell tumor. ii. Choriocarcinomas that arise from the gestational pathway respond well to chemotherapy; those that arise from the germ cell pathway do not.

1	ii. Choriocarcinomas that arise from the gestational pathway respond well to chemotherapy; those that arise from the germ cell pathway do not. Thank you for choosing Pathoma for your studies. We strive to provide the highest quality educational materials while keeping affordability in mind. A tremendous amount of time and effort has gone into developing these materials, so we appreciate your legitimate use of this program. It speaks to your integrity as a future physician and the high ethical standards that we all set forth for ourselves when taking the Hippocratic oath. Unauthorized use of Pathoma materials is contrary to the ethical standards of a training physician and is a violation of copyright. Pathoma videos are updated on a regular basis and the most current version, as well as a complete list of errata, can be accessed through your account at Pathoma.com. Sincerely, Dr. Sattar, MD I. HYPOSPADIAS A. Opening of urethra on inferior surface of penis

1	Pathoma.com. Sincerely, Dr. Sattar, MD I. HYPOSPADIAS A. Opening of urethra on inferior surface of penis B. Due to failure of the urethral folds to close II. EPISPADIAS A. Opening of urethra on superior surface of penis B. Due to abnormal positioning of the genital tubercle C. Associated with bladder exstrophy III. CONDYLOMA ACUMINATUM A. Benign warty growth on genital skin B. Due to HPV type 6 or 11; characterized by koilocytic change (Fig. 14.1) IV. A. Necrotizing granulomatous inflammation of the inguinal lymphatics and lymph nodes B. Sexually transmitted disease caused by Chlamydia trachomatis (serotypes Ll-L3) C. Eventually heals with fibrosis; perianal involvement may result in rectal stricture. V. A. Malignant proliferation of squamous cells of penile skin B. Risk factors 1. High risk HPV (2/3 of cases) 2. Lack of circumcision-Foreskin acts as a nidus for inflammation and irritation if not properly maintained. C. Precursor in situ lesions 1.

1	B. Risk factors 1. High risk HPV (2/3 of cases) 2. Lack of circumcision-Foreskin acts as a nidus for inflammation and irritation if not properly maintained. C. Precursor in situ lesions 1. Bowen disease-in situ carcinoma of the penile shaft or scrotum that presents as leukoplakia 2. Erythroplasia of Queyrat-in situ carcinoma on the glans that presents as erythroplakia 3. Bowenoid papulosis-in situ carcinoma that presents as multiple reddish papules i. Seen in younger patients (40s) relative to Bowen disease and erythroplasia of Queyrat ii. Does not progress to invasive carcinoma I. CRYPTORCHIDISM A. Failure of testicle to descend into the scrotal sac 1. Testicles normally develop in the abdomen and then "descend" into the scrotal sac as the fetus grows. B. Most common congenital male reproductive abnormality; seen in 1% of male infants pathoma.com C. Most cases resolve spontaneously; otherwise, orchiopexy is performed before 2 years of age.

1	B. Most common congenital male reproductive abnormality; seen in 1% of male infants pathoma.com C. Most cases resolve spontaneously; otherwise, orchiopexy is performed before 2 years of age. D. Complications include testicular atrophy with infertility and increased risk for seminoma. II. ORCHITIS A. Inflammation of the testicle B. Causes 1. Chlamydia trachomatis (serotypes D-K) or Neisseria gonorrhoeae-Seen in young adults. Increased risk of sterility, but libido is not affected because Leydig cells are spared. 2. Escherichia coli and Pseudomonas-Seen in older adults; urinary tract infection pathogens spread into the reproductive tract. 3. Mumps virus (teenage males)-increased risk for infertility; testicular inflammation is usually not seen in children < 10 years old. 4. Autoimmune orchitis-characterized by granulomas involving the seminiferous tubules III. TESTICULAR TORSION

1	4. Autoimmune orchitis-characterized by granulomas involving the seminiferous tubules III. TESTICULAR TORSION A. Twisting of the spermatic cord; thin-walled veins become obstructed leading to congestion and hemorrhagic infarction (Fig. 14.2). B. Usually due to congenital failure of testes to attach to the inner lining of the scrotum (via the processus vaginalis) C. Presents in adolescents with sudden testicular pain and absent cremasteric reflex IV. A. Dilation of the spermatic vein due to impaired drainage B. Presents as scrotal swelling with a "bag of worms" appearance C. Usually left sided; left testicular vein drains into the left renal vein, while the right testicular vein drains directly into the IVC. 1. Associated with left-sided renal cell carcinoma; RCC often invades the renal vein. D. Seen in a large percentage of infertile males V.

1	1. Associated with left-sided renal cell carcinoma; RCC often invades the renal vein. D. Seen in a large percentage of infertile males V. A. Fluid collection within the tunica vaginalis 1. Tunica vaginalis is a serous membrane that covers the testicle as well as the internal surface of the scrotum. B. Associated with incomplete closure of the processus vaginalis leading to communication with the peritoneal cavity (infants) or blockage oflymphatic drainage (adults) Fig . 14.1 Koilocytic change. Fig. 14.2 Hemorrhagic infarction of testicle. Fig. 14.3 Hydrocele. (Courtesy of humpath.com) C. Presents as scrotal swelling that can be transilluminated (Fig. 14.3) I. BASIC PRINCIPLES A. Arise from germ cells or sex cord-stroma B. Present as a firm, painless testicular mass that cannot be transilluminated C. Usually not biopsied due to risk of seeding the scrotum; removed via radical orchiectomy 1. Most testicular tumors are malignant germ cell tumors. II. GERM CELL TUMORS

1	C. Usually not biopsied due to risk of seeding the scrotum; removed via radical orchiectomy 1. Most testicular tumors are malignant germ cell tumors. II. GERM CELL TUMORS A. Most common type of testicular tumor(> 95% of cases) B. Usually occur between 15-40 years of age C. Risk factors include cryptorchidism and Klinefelter syndrome. D. Divided into seminoma and nonseminoma 1. Seminomas (55% of cases) are highly responsive to radiotherapy, metastasize late, and have an excellent prognosis. 2. Nonseminomas (45% of cases) show variable response to treatment and often metastasize early. E. Seminoma is a malignant tumor comprised oflarge cells with clear cytoplasm and central nuclei (resemble spermatogonia, Fig. 14.4A); forms a homogeneous mass with no hemorrhage or necrosis (Fig. 14.4B) 1. 2. Rare cases may produce ~-hCG. 3. Good prognosis; responds to radiotherapy

1	2. Rare cases may produce ~-hCG. 3. Good prognosis; responds to radiotherapy F. Embryonal carcinoma is a malignant tumor comprised of immature, primitive cells that may produce glands (Fig. 14.SA); forms a hemorrhagic mass with necrosis (Fig. 14.SB) 1. Aggressive with early hematogenous spread 2. Chemotherapy may result in differentiation into another type of germ cell tumor (e.g., teratoma). 3. Increased AFP or ~-hCG may be present. G. Yolk sac (endodermal sinus) tumor is a malignant tumor that resembles yolk sac elements. 1. 2. Schiller-Duval bodies (glomerulus-like structures) are seen on histology (Fig. 14.6). 3. AFP is characteristically elevated. Fig. 14.4 Seminoma. A, Microscopic appearance. B, Gross appearance. (A, Courtesy of Ed Uthman, MD) H. Choriocarcinoma is a malignant tumor of syncytiotrophoblasts and cytotrophoblasts (placenta-like tissue, but villi are absent, Fig. 14.7). 1. 2.

1	H. Choriocarcinoma is a malignant tumor of syncytiotrophoblasts and cytotrophoblasts (placenta-like tissue, but villi are absent, Fig. 14.7). 1. 2. ~-hCG is characteristically elevated; may lead to hyperthyroidism or gynecomastia (a-subunit ofhCG is similar to that ofFSH, LH, and TSH) I. Teratoma is a tumor composed of mature fetal tissue derived from two or three embryonic layers. 1. Malignant in males (as opposed to females) 2. AFP or ~-hCG may be increased. J. Mixed germ cell tumors 1. Germ cell tumors are usually mixed. 2. Prognosis is based on the worst component. Ill. SEX CORD-STROMAL TUMORS A. Tumors that resemble sex cord-stromal tissues of the testicle (Fig. 14.8); usually benign B. Leydig cell tumor usually produces androgen, causing precocious puberty in children or gynecomastia in adults. 1. Characteristic Reinke crystals may be seen on histology. C. Sertoli cell tumor is comprised of tubules and is usually clinically silent. IV. LYMPHOMA

1	1. Characteristic Reinke crystals may be seen on histology. C. Sertoli cell tumor is comprised of tubules and is usually clinically silent. IV. LYMPHOMA A. Most common cause of a testicular mass in males > 60 years old; often bilateral B. Usually of diffuse large B-cell type Fig. 14.5 Embryonal carcinoma. A, Microscopic appearance. B, Gross appearance. (Courtesy of webpathology.com) Fig. 14.6 Schiller-Duval body, yolk sac tumor. Fig. 14.7 Choriocarcinoma. (Courtesy of Fig. 14.8 Normal testicle, microscopic (Courtesy of webpathology.com) web pathology.com) appearance. I. BASIC PRINCIPLES A. Small, round organ that lies at the base of the bladder encircling the urethra B. Sits anterior to the rectum; posterior aspect of prostate is palpable by digital rectal exam (DRE). C. Consists of glands and stroma (Fig. 14.9) 1.

1	B. Sits anterior to the rectum; posterior aspect of prostate is palpable by digital rectal exam (DRE). C. Consists of glands and stroma (Fig. 14.9) 1. Glands are composed of an inner layer ofluminal cells and an outer layer of basal cells; secrete alkaline, milky fluid that is added to sperm and seminal vesicle fluid to make semen. 2. Glands and stroma are maintained by androgens. II. ACUTE PROSTATITIS A. Acute inflammation of the prostate; usually due to bacteria 1. Chlamydia trachomatis and Neisseria gonorrhoeae are common causes in young adults. 2. Escherichia coli and Pseudomonas are common causes in older adults. B. Presents as dysuria with fever and chills C. Prostate is tender and boggy on digital rectal exam. D. Prostatic secretions show WBCs; culture reveals bacteria. III. CHRONIC PROSTATITIS A. Chronic inflammation of prostate B. Presents as dysuria with pelvic or low back pain C. Prostatic secretions show WBCs, but cultures are negative.

1	III. CHRONIC PROSTATITIS A. Chronic inflammation of prostate B. Presents as dysuria with pelvic or low back pain C. Prostatic secretions show WBCs, but cultures are negative. IV. BENIGN PROSTATIC HYPERPLASIA (BPH) A. Hyperplasia of prostatic stroma and glands B. Age-related change (present in most men by the age of 60 years); no increased risk for cancer C. Related to dihydrotestosterone (DHT) 1. Testosterone is converted to DHT by Sa-reductase in stromal cells. 2. DHT acts on the androgen receptor of stromal and epithelial cells resulting in hyperplastic nodules. D. Occurs in the central periurethral zone of the prostate E. Clinical features include 1. 2. Impaired bladder emptying with increased risk for infection and hydronephrosis (Fig. 14.10) 3. Fig. 14.9 Normal prostate, microscopic Fig. 14.10 Hydronephrosis. appearance. 4. Hypertrophy of bladder wall smooth muscle; increased risk for bladder diverticula 5. Microscopic hematuria may be present. 6.

1	4. Hypertrophy of bladder wall smooth muscle; increased risk for bladder diverticula 5. Microscopic hematuria may be present. 6. Prostate-specific antigen (PSA) is often slightly elevated (usually less than 10 ng/ mL) due to the increased number of glands; PSA is made by prostatic glands and liquefies semen. F. Treatment 1. a -antagonist (e.g., terazosin) to relax smooth muscle i. Also relaxes vascular smooth muscle lowering blood pressure ii. Selective a A -antagonists (e.g., tamsulosin) are used in normotensive individuals to avoid a effects on blood vessels. 2. 5a-reductase inhibitor i. Blocks conversion of testosterone to DHT ii. Takes months to produce results iii. Also useful for male pattern baldness iv. Side effects are gynecomastia and sexual dysfunction. V. PROSTATE ADENOCARCINOMA A. Malignant proliferation of prostatic glands B. Most common cancer in men; 2nd most common cause of cancer-related death

1	V. PROSTATE ADENOCARCINOMA A. Malignant proliferation of prostatic glands B. Most common cancer in men; 2nd most common cause of cancer-related death C. Risk factors include age, race (African Americans > Caucasians > Asians), and diet high in saturated fats . D. Prostatic carcinoma is most often clinically silent. 1. Usually arises in the peripheral, posterior region of the prostate and, hence, does not produce urinary symptoms early on (Fig. 14.llA) 2. Screening begins at the age of 50 years with DRE and PSA. i. Normal serum PSA increases with age due to BPH (2.5 ng/mL for ages 40-49 years vs. 7.5 ng/mL for ages 70-79 years) ii. PSA > 10 ng/mL is highly worrisome at any age. iii. Decreased% free-PSA is suggestive of cancer (cancer makes bound PSA). E. Prostatic biopsy is required to confirm the presence of carcinoma. 1. Shows small, invasive glands with prominent nucleoli (Fig. 14.llB) 2. Gleason grading system is based on architecture alone (and not nuclear atypia).

1	1. Shows small, invasive glands with prominent nucleoli (Fig. 14.llB) 2. Gleason grading system is based on architecture alone (and not nuclear atypia). i. Multiple regions of the tumor are assessed because architecture varies from area to area. ii. A score (1-5) is assigned for two distinct areas and then added to produce a final score (2-10). iii. Higher score suggests worse prognosis. Fig. 14.11 Prostatic adenocarcinoma. A, Gross appearance. B, Microscopic appearance. C, Osteoblastic metastasis involving lumbar spine. F. Spread to lumbar spine or pelvis is common (Fig. 14.llC); results in osteoblastic metastases that present as low back pain and increased serum alkaline phosphatase, PSA, and prostatic acid phosphatase (PAP) G. Prostatectomy is performed for localized disease; advanced disease is treated with hormone suppression to reduce testosterone and DHT. 1.

1	G. Prostatectomy is performed for localized disease; advanced disease is treated with hormone suppression to reduce testosterone and DHT. 1. Continuous GnRH analogs (e.g., leuprolide) shut down the anterior pituitary gonadotrophs (LH and FSH are reduced). 2. Flutamide acts as a competitive inhibitor at the androgen receptor. I. ENDOCRINE SYSTEM A. Group of glands that maintain body homeostasis B. Functions by release of hormones that travel via blood to distant organs C. "Feedback" mechanisms control hormone release. I. PITUITARY ADENOMA A. Benign tumor of anterior pituitary cells B. May be functional (hormone-producing) or nonfunctional (silent) 1. Nonfunctional tumors often present with mass effect. i. Bitemporal hemianopsia occurs due to compression of the optic chiasm. ii. Hypopituitarism occurs due to compression of normal pituitary tissue. iii. Headache 2. Functional tumors present with features based on the type of hormone produced.

1	ii. Hypopituitarism occurs due to compression of normal pituitary tissue. iii. Headache 2. Functional tumors present with features based on the type of hormone produced. C. Prolactinoma presents as galactorrhea and amenorrhea (females) or as decreased libido and headache (males); most common type of pituitary adenoma 1. Treatment is dopamine agonists (e.g., bromocriptine or cabergoline) to suppress prolactin production (shrinks tumor) or surgery for larger lesions. D. Growth hormone cell adenoma 1. Gigantism in children-increased linear bone growth (epiphyses are not fused) 2. 1. Enlarged bones of hands, feet, and jaw ii. Growth of visceral organs leading to dysfunction (e.g., cardiac failure) iii. Enlarged tongue 3. Secondary diabetes mellitus is often present (GH induces liver gluconeogenesis). 4. Diagnosed by elevated GH and insulin growth factor-1 (IGF-1) levels along with lack of GH suppression by oral glucose 5.

1	4. Diagnosed by elevated GH and insulin growth factor-1 (IGF-1) levels along with lack of GH suppression by oral glucose 5. Treatment is octreotide (somatostatin analog that suppresses GH release), GH receptor antagonists, or surgery. E. ACTH cell adenomas secrete ACTH leading to Cushing syndrome (see "Adrenal Cortex" below). F. TSH cell, LH-producing, and FSH-producing adenomas occur, but are rare. II. HYPOPITUITARISM A. Insufficient production of hormones by the anterior pituitary gland; symptoms arise when > 75% of the pituitary parenchyma is lost. B. Causes include 1. Pituitary adenomas (adults) or craniopharyngioma (children)-due to mass effect or pituitary apoplexy (bleeding into an adenoma) 2. Sheehan syndrome-pregnancy-related infarction of the pituitary gland i. Gland doubles in size during pregnancy, but blood supply does not increase significantly; blood loss during parturition precipitates infarction.

1	pathoma.com ii. Presents as poor lactation, loss of pubic hair, and fatigue 3. Empty sella syndrome-congenital defect of the sella i. Herniation of the arachnoid and CSF into the sella compresses and destroys the pituitary gland. ii. Pituitary gland is "absent" (empty sella) on imaging. I. BASIC PRINCIPLES A. Antidiuretic hormone (ADH) and oxytocin are made in the hypothalamus and then transported via axons to the posterior pituitary for release. 1. ADH acts on the distal tubules and collecting ducts of the kidney to promote free water retention. 2. Oxytocin mediates uterine contraction during labor and release of breast milk (let-down) in lactating mothers. II. CENTRAL DIABETES INSIPIDUS A. ADH deficiency B. Due to hypothalamic or posterior pituitary pathology (e.g., tumor, trauma, infection, or inflammation) C. Clinical features are based on loss of free water. 1. Polyuria and polydipsia with risk oflife-threatening dehydration 2. 3.

1	C. Clinical features are based on loss of free water. 1. Polyuria and polydipsia with risk oflife-threatening dehydration 2. 3. D. Water deprivation test fails to increase urine osmolality (useful for diagnosis). E. Treatment is desmopressin (ADH analog). III. NEPHROGENIC DIABETES INSIPIDUS A. Impaired renal response to ADH B. Due to inherited mutations or drugs (e.g., lithium and demeclocycline) C. Clinical features are similar to central diabetes insipidus, but there is no response to desmopressin. IV. SYNDROME OF INAPPROPRIATE ADH (SIADH) SECRETION A. Excessive ADH secretion B. Most often due to ectopic production (e.g., small cell carcinoma of the lung); other causes include CNS trauma, pulmonary infection, and drugs (e.g., cyclophosphamide). C. Clinical features are based on retention of free water. 1. 2. Mental status changes and seizures-Hyponatremia leads to neuronal swelling and cerebral edema. D. Treatment is free water restriction or demeclocycline.

1	1. 2. Mental status changes and seizures-Hyponatremia leads to neuronal swelling and cerebral edema. D. Treatment is free water restriction or demeclocycline. I. THYROGLOSSAL DUCT CYST A. Cystic dilation of thyroglossal duct remnant 1. Thyroid develops at the base of tongue and then travels along the thyroglossal duct to the anterior neck. 2. Thyroglossal duct normally involutes; a persistent duct, however, may undergo cystic dilation. B. Presents as an anterior neck mass II. LINGUAL THYROID A. Persistence of thyroid tissue at the base of tongue B. Presents as a base of tongue mass I. BASIC PRINCIPLES A. Increased level of circulating thyroid hormone 1. Increases basal metabolic rate (due to increased synthesis of Na+ -K+ ATPase) 2. Increases sympathetic nervous system activity (due to increased expression of ~ -adrenergic receptors) B. Clinical features include 1. 2. 3. Tachycardia with increased cardiac output 4.

1	Increases sympathetic nervous system activity (due to increased expression of ~ -adrenergic receptors) B. Clinical features include 1. 2. 3. Tachycardia with increased cardiac output 4. Arrhythmia (e.g., atrial fibrillation), especially in the elderly 5. Tremor, anxiety, insomnia, and heightened emotions 6. Staring gaze with lid lag 7. Diarrhea with malabsorption 8. 9. Bone resorption with hypercalcemia (risk for osteoporosis) 10. Decreased muscle mass with weakness 11. 12. Hyperglycemia (due to gluconeogenesis and glycogenolysis) II. GRAVES DISEASE A. Autoantibody (IgG) that stimulates TSH receptor (type II hypersensitivity) B. Leads to increased synthesis and release of thyroid hormone 1. Most common cause of hyperthyroidism 2. Classically occurs in women of childbearing age (20-40 years) C. Clinical features include 1. 2. Diffuse goiter-Constant TSH stimulation leads to thyroid hyperplasia and hypertrophy (Fig. 15.lA). 3.

1	C. Clinical features include 1. 2. Diffuse goiter-Constant TSH stimulation leads to thyroid hyperplasia and hypertrophy (Fig. 15.lA). 3. 1. Fibroblasts behind the orbit and overlying the shin express the TSH receptor. ii. TSH activation results in glycosaminoglycan (chondroitin sulfate and hyaluronic acid) buildup, inflammation, fibrosis, and edema leading to exophthalmos and pretibial myxedema. Fig. 15.1 Graves disease. A, Diffuse goiter. B, Microscopic appearance. (A, Courtesy of Ed Uthman, MD) D. Irregular follicles with scalloped colloid and chronic inflammation are seen on histology (Fig. 15.lB). E. Laboratory findings include 1. t total and free T ; ..l, TSH (free T downregulates TRH receptors in the anterior pituitary to decrease TSH release) 2. 3. F. Treatment involves P-blockers, thioamide, and radioiodine ablation. G. Thyroid storm is a potentially fatal complication. 1.

1	3. F. Treatment involves P-blockers, thioamide, and radioiodine ablation. G. Thyroid storm is a potentially fatal complication. 1. Due to elevated catecholamines and massive hormone excess, usually in response to stress (e.g., surgery or childbirth) 2. Presents as arrhythmia, hyperthermia, and vomiting with hypovolemic shock 3. Treatment is propylthiouracil (PTU), P-blockers, and steroids. i. PTU inhibits peroxidase-mediated oxidation, organification, and coupling steps of thyroid hormone synthesis, as well as peripheral conversion of T4 to T3. III. MULTINODULAR GOITER A. Enlarged thyroid gland with multiple nodules (Fig. 15.2) B. Due to relative iodine deficiency C. Usually nontoxic (euthyroid) D. Rarely, regions become TSH-independent leading to T release and hyperthyroidism ('toxic goiter'). I. CRETINISM A. Hypothyroidism in neonates and infants

1	C. Usually nontoxic (euthyroid) D. Rarely, regions become TSH-independent leading to T release and hyperthyroidism ('toxic goiter'). I. CRETINISM A. Hypothyroidism in neonates and infants B. Characterized by mental retardation, short stature with skeletal abnormalities, coarse facial features, enlarged tongue, and umbilical hernia 1. Thyroid hormone is required for normal brain and skeletal development. C. Causes include maternal hypothyroidism during early pregnancy, thyroid agenesis, dyshormonogenetic goiter, and iodine deficiency. 1. Dyshormonogenetic goiter is due to a congenital defect in thyroid hormone production; most commonly involves thyroid peroxidase II. MYXEDEMA A. Hypothyroidism in older children or adults B. Clinical features are based on decreased basal metabolic rate and decreased sympathetic nervous system activity. 1. Myxedema-accumulation of glycosaminoglycans in the skin and soft tissue; results in a deepening ofvoice and large tongue 2. 3.

1	1. Myxedema-accumulation of glycosaminoglycans in the skin and soft tissue; results in a deepening ofvoice and large tongue 2. 3. Slowing of mental activity 4. 5. Cold intolerance with decreased sweating 6. Bradycardia with decreased cardiac output, leading to shortness of breath and fatigue 7. 8. 9. C. Most common causes are iodine deficiency and Hashimoto thyroiditis; other causes include drugs (e.g., lithium) and surgical removal or radioablation of the thyroid. I. HASHIMOTO THYROIDITIS A. Autoimmune destruction of the thyroid gland; associated with HLA-DR5 1. Most common cause of hypothyroidism in regions where iodine levels are adequate B. Clinical features 1. Initially may present as hyperthyroidism (due to follicle damage) 2. Progresses to hypothyroidism; ,l., T4 and t TSH 3. Antithyroglobulin and antithyroid peroxidase antibodies are often present (sign of thyroid damage).

1	Progresses to hypothyroidism; ,l., T4 and t TSH 3. Antithyroglobulin and antithyroid peroxidase antibodies are often present (sign of thyroid damage). C. Chronic inflammation with germinal centers and Hurthle cells (eosinophilic metaplasia of cells that line follicles) is seen on histology (Fig. 15.3). D. Increased risk for B-cell (marginal zone) lymphoma; presents as an enlarging thyroid gland late in disease course II. SUBACUTE GRANULOMATOUS (DE QUERVAIN) THYROIDITIS A. Granulomatous thyroiditis that follows a viral infection B. Presents as a tender thyroid with transient hyperthyroidism C. Self-limited; rarely (15% of cases) may progress to hypothyroidism III. RIEDEL FIBROSING THYROIDITIS A. Chronic inflammation with extensive fibrosis of the thyroid gland B. Presents as hypothyroidism with a 'hard as wood,' non tender thyroid gland C. Fibrosis may extend to involve local structures (e.g., airway).

1	B. Presents as hypothyroidism with a 'hard as wood,' non tender thyroid gland C. Fibrosis may extend to involve local structures (e.g., airway). 1. Clinically mimics anaplastic carcinoma, but patients are younger (40s), and malignant cells are absent I. BASIC PRINCIPLES A. Usually presents as a distinct, solitary nodule 1. Thyroid nodules are more likely to be benign than malignant. B. 13 1I radioactive uptake studies are useful to further characterize nodules. 1. Increased uptake ('hot' nodule) is seen in Graves disease or nodular goiter. 2. Decreased uptake ('cold' nodule) is seen in adenoma and carcinoma; often warrants biopsy C. Biopsy is performed by fine needle aspiration (FNA). II. FOLLICULAR ADENOMA A. Benign proliferation of follicles surrounded by a fibrous capsule (Fig. 15.4) Fig. 15.2 Multinodular goiter. (Courtesy of Jamie Fig. 15.3 Hashimoto thyroiditis. Fig. 15.4 Follicular adenoma. Steinmetz, MD)

1	Fig. 15.2 Multinodular goiter. (Courtesy of Jamie Fig. 15.3 Hashimoto thyroiditis. Fig. 15.4 Follicular adenoma. Steinmetz, MD) B. Usually nonfunctional; less commonly, may secrete thyroid hormone III. PAPILLARY CARCINOMA A. Most common type of thyroid carcinoma (80% of cases) B. Exposure to ionizing radiation in childhood is a major risk factor. C. Comprised of papillae lined by cells with clear, 'Orphan Annie eye' nuclei and nuclear grooves (Fig. 15.5A); papillae are often associated with psammoma bodies (Fig. 15.5B). D. Often spreads to cervical (neck) lymph nodes, but prognosis is excellent (10-year survival > 95%) IV. A. Malignant proliferation of follicles surrounded by a fibrous capsule with invasion through the capsule (Fig. 15.6) 1. Invasion through the capsule helps distinguish follicular carcinoma from follicular adenoma. 2. Entire capsule must be examined microscopically. 3.

1	Invasion through the capsule helps distinguish follicular carcinoma from follicular adenoma. 2. Entire capsule must be examined microscopically. 3. FNA only examines cells and not the capsule; hence, a distinction between follicular adenoma and follicular carcinoma cannot be made by FNA. B. Metastasis generally occurs hematogenously. V. A. Malignant proliferation of parafollicular C cells; comprises 5% of thyroid carcinomas 1. C cells are neuroendocrine cells that secrete calcitonin. 2. Calcitonin lowers serum calcium by increasing renal calcium excretion but is inactive at normal physiologic levels. 3. High levels of calcitonin produced by tumor may lead to hypocalcemia. 4. Calcitonin often deposits within the tumor as amyloid. B. Biopsy reveals sheets of malignant cells in an amyloid stroma (Fig. 15.7). C. Familial cases are often due to multiple endocrine neoplasia (MEN) 2A and 2B, which are associated with mutations in the RET oncogene. 1.

1	C. Familial cases are often due to multiple endocrine neoplasia (MEN) 2A and 2B, which are associated with mutations in the RET oncogene. 1. MEN 2 results in medullary carcinoma, pheochromocytoma, and parathyroid adenomas (2A) or ganglioneuromas of the oral mucosa (2B). 2. Detection of the RET mutation warrants prophylactic thyroidectomy. VI. ANAPLASTIC CARCINOMA A. Undifferentiated malignant tumor of the thyroid (Fig. 15.8); usually seen in elderly B. Often invades local structures, leading to dysphagia or respiratory compromise C. Poor prognosis Fig. 15.5 Papillary carcinoma. A, Nuclear features. B, Psammoma bodies. Fig. 15.6 Follicular carcinoma. (Courtesy of Bu lent Celasun, MD) I. BASIC PRINCIPLES A. Chief cells regulate serum free (ionized) calcium via parathyroid hormone (PTH) secretion, which 1. Increases bone osteoclast activity, releasing calcium and phosphate 2. Increases small bowel absorption of calcium and phosphate (indirectly by activating vitamin D) 3.

1	Increases bone osteoclast activity, releasing calcium and phosphate 2. Increases small bowel absorption of calcium and phosphate (indirectly by activating vitamin D) 3. B. Increased serum ionized calcium levels provide negative feedback to decrease PTH secretion. II. PRIMARY HYPERPARATHYROIDISM A. Excess PTH due to a disorder of the parathyroid gland itself B. Most common cause is parathyroid adenoma (>80% of cases); sporadic parathyroid hyperplasia and parathyroid carcinoma are less common causes. C. Parathyroid adenoma is a benign neoplasm, usually involving one gland.

1	C. Parathyroid adenoma is a benign neoplasm, usually involving one gland. 1. Most often results in asymptomatic hypercalcemia; however, may present with consequences of increased PTH and hypercalcemia such as i. Nephrolithiasis (calcium oxalate stones) ii. Nephrocalcinosis-metastatic calcification of renal tubules (Fig. 15.9), potentially leading to renal insufficiency and polyuria 111. CNS disturbances (e.g., depression and seizures) 1v. Constipation, peptic ulcer disease, and acute pancreatitis v. Osteitis fibrosa cystica-resorption of bone leading to fibrosis and cystic spaces (Fig. 15.10) 2. Laboratory findings include t serum PTH, t serum calcium, J,. serum phosphate, t urinary cAMP, and t serum alkaline phosphatase. 3. Treatment involves surgical removal of the affected gland. III. SECONDARY HYPERPARATHYROIDISM A. Excess production of PTH due to a disease process extrinsic to the parathyroid gland B. Most common cause is chronic renal failure. 1.

1	III. SECONDARY HYPERPARATHYROIDISM A. Excess production of PTH due to a disease process extrinsic to the parathyroid gland B. Most common cause is chronic renal failure. 1. Renal insufficiency leads to decreased phosphate excretion. 2. t serum phosphate binds free calcium. 3. J,. free calcium stimulates all four parathyroid glands. 4. t PTH leads to bone resorption (contributing to renal osteodystrophy). 5. Lab findings include t PTH, J,. serum calcium, t serum phosphate, and t alkaline phosphatase. Fig. 15.7 Medullary carcinoma. Fig. 15.8 Ana plastic carcinoma. Fig. 15.9 Nephrocalcinosis. IV. HYPOPARATHYROIDISM A. LowPTH B. Causes include autoimmune damage to the parathyroids, surgical excision, and DiGeorge syndrome C. Presents with symptoms related to low serum calcium 1. 2. Muscle spasms (tetany)-may be elicited with filling of a blood pressure cuff (Trousseau sign) or tapping on the facial nerve (Chvostek sign)

1	2. Muscle spasms (tetany)-may be elicited with filling of a blood pressure cuff (Trousseau sign) or tapping on the facial nerve (Chvostek sign) D. Labs reveal .,J, PTH levels and .,J, serum calcium. E. Pseudohypoparathyroidism is due to end-organ resistance to PTH. 1. Labs reveal hypocalcemia with t PTH levels. 2. Autosomal dominant form is associated with short stature and short 4th and 5th digits. I. BASIC PRINCIPLES A. Composed of clusters of cells termed islets of Langerhans (Fig. 15.11) B. A single islet consists of multiple cell types, each producing one type of hormone. C. Insulin is secreted by beta cells, which lie in the center of the islets. 1. 2. Increased glucose uptake by tissues leads to increased glycogen synthesis, protein synthesis, and lipogenesis. D. Glucagon is secreted by alpha cells; it opposes insulin in order to increase blood glucose levels (e.g., in states of fasting) via glycogenolysis and lipolysis.

1	D. Glucagon is secreted by alpha cells; it opposes insulin in order to increase blood glucose levels (e.g., in states of fasting) via glycogenolysis and lipolysis. A. Insulin deficiency leading to a metabolic disorder characterized by hyperglycemia B. Due to autoimmune destruction of beta cells by T lymphocytes 1. Characterized by inflammation of islets 2. Associated with HLA-DR3 and HLA-DR4 3. Autoantibodies against insulin are often present (sign of damage) and may be seen years before clinical disease develops. C. Manifests in childhood with clinical features of insulin deficiency 1. High serum glucose-Lack of insulin leads to decreased glucose uptake by fat and skeletal muscle. Fig. 15.10 Osteitis fibrosa cystica. Fig. 15.11 Islets of Langerhans. Fig. 15.12 Amyloid in islets, type II DM. 2. Weight loss, low muscle mass, and polyphagia-Unopposed glucagon leads to gluconeogenesis, glycogenolysis and lipolysis, which further exacerbates hyperglycemia. 3.

1	2. Weight loss, low muscle mass, and polyphagia-Unopposed glucagon leads to gluconeogenesis, glycogenolysis and lipolysis, which further exacerbates hyperglycemia. 3. Polyuria, polydipsia, and glycosuria-Hyperglycemia exceeds renal ability to resorb glucose; excess filtered glucose leads to osmotic diuresis. 4. Treatment involves lifelong insulin. D. Risk for diabetic ketoacidosis 1. 2. Often arises with stress (e.g., infection); epinephrine stimulates glucagon secretion increasing lipolysis (along with gluconeogenesis and glycogenolysis). i. Increased lipolysis leads to increased free fatty acids (FFAs). ii. Liver converts FFAs to ketone bodies (~-hydroxybutyric acid and acetoacetic acid). 3. Results in hyperglycemia (> 300 mg/dL), anion gap metabolic acidosis, and hyperkalemia 4. Presents with Kussmaul respirations, dehydration, nausea, vomiting, mental status changes, and fruity smelling breath (due to acetone) 5.

1	Presents with Kussmaul respirations, dehydration, nausea, vomiting, mental status changes, and fruity smelling breath (due to acetone) 5. Treatment is fluids (corrects dehydration from polyuria), insulin, and replacement of electrolytes (e.g., potassium). Ill. TYPE 2 DIABETES MELLITUS A. End-organ insulin resistance leading to a metabolic disorder characterized by hyperglycemia 1. Most common type of diabetes (90% of cases); affects 5-10% of the US population 2. Incidence is rising. B. Arises in middle-aged, obese adults 1. Obesity leads to decreased numbers of insulin receptors. 2. Strong genetic predisposition exists. C. Insulin levels are increased early in disease, but later, insulin deficiency develops due to beta cell exhaustion; histology reveals amyloid deposition in the islets (Fig. 15.12). D. Clinical features include polyuria, polydipsia, and hyperglycemia, but disease is often clinically silent.

1	D. Clinical features include polyuria, polydipsia, and hyperglycemia, but disease is often clinically silent. E. Diagnosis is made by measuring glucose levels (normal is 70-120 mg/dL). 1. 2. 3. Glucose tolerance test with a serum glucose level > 200 mg/dL two hours after glucose loading F. Treatment involves weight loss (diet and exercise) initially; may require drug therapy to counter insulin resistance (e.g., sulfonylureas or metformin) or exogenous insulin after exhaustion of beta cells G. Risk for hyperosmolar non-ketotic coma 1. High glucose (> 500 mg/dL) leads to life-threatening diuresis with hypotension and coma. 2. Ketones are absent due to small amounts of circulating insulin. IV. LONG-TERM CONSEQUENCES OF DIABETES A. Nonenzymatic glycosylation (NEG) ofvascular basement membranes 1. NEG oflarge-and medium-sized vessels leads to atherosclerosis and its resultant complications. i. Cardiovascular disease is the leading cause of death among diabetics.

1	i. Cardiovascular disease is the leading cause of death among diabetics. ii. Peripheral vascular disease in diabetics is the leading cause of non traumatic amputations. 2. NEG of small vessels (arterioles) leads to hyaline arteriolosclerosis (Fig. 15.13A). i. Involvement of renal arterioles leads to glomerulosclerosis, resulting in small, scarred kidneys with a granular surface (Fig. 15.13B). ii. Preferential involvement of efferent arterioles leads to glomerular hyperfiltration injury with microalbuminuria that eventually progresses to nephrotic syndrome; characterized by Kimmelstiel-Wilson nodules in glomeruli 3. NEG of hemoglobin produces glycated hemoglobin (HbA1c), a marker of glycemic control. B. Osmotic damage 1. Glucose freely enters into Schwann cells (which myelinate peripheral nerves), pericytes of retinal blood vessels, and the lens. 2. Aldose reductase converts glucose to sorbitol, resulting in osmotic damage. 3.

1	2. Aldose reductase converts glucose to sorbitol, resulting in osmotic damage. 3. Leads to peripheral neuropathy, impotence, blindness, and cataracts; diabetes is the leading cause of blindness in the developed world. V. PANCREATIC ENDOCRINE NEOPLASMS A. Tumors of islet cells; account for < 5% of pancreatic neoplasms. 1. Often a component of MEN 1 along with parathyroid hyperplasia and pituitary adenomas B. Insulinomas present as episodic hypoglycemia with mental status changes that are relieved by administration of glucose. 1. Diagnosed by -l.-serum glucose levels (usually < 50 mg/dL), t insulin, and t C-peptide C. Gastrinomas present as treatment-resistant peptic ulcers (Zollinger-Ellison syndrome); ulcers may be multiple and can extend into the jejunum. D. Somatostatinomas present as achlorhydria (due to inhibition of gastrin) and cholelithiasis with steatorrhea (due to inhibition of cholecystokinin).

1	D. Somatostatinomas present as achlorhydria (due to inhibition of gastrin) and cholelithiasis with steatorrhea (due to inhibition of cholecystokinin). E. VIPomas secrete excessive vasoactive intestinal peptide leading to watery diarrhea, hypokalemia, and achlorhydria. I. BASIC PRINCIPLES A. Composed of three layers; each secretes predominantly one class of hormones. 1. Glomerulosa produces mineralocorticoids (e.g., aldosterone). 2. Fasciculata produces glucocorticoids (e.g., cortisol). 3. Reticularis produces weak androgens (e.g., DHEA). B. Cortical hormones are derived from cholesterol. Fig. 15.13 Complications of diabetes. A, Hyaline arteriolosclerosis. B, Glomerul osclerosis. 1. Sequential enzymatic modification produces mineralocorticoids, glucocorticoids, and androgens (Fig. 15.14). 2. Each layer secretes distinct hormones based on enzymes present in that layer. II. HYPERALDOSTERONISM A. Excess aldosterone

1	2. Each layer secretes distinct hormones based on enzymes present in that layer. II. HYPERALDOSTERONISM A. Excess aldosterone B. Classically presents as HTN, hypokalemia, and metabolic alkalosis 1. Aldosterone increases absorption of sodium and secretion of potassium and hydrogen ions in the distal tubule and collecting duct. 2. Increased sodium expands plasma volume leading to HTN. C. Primary hyperaldosteronism is most commonly due to bilateral adrenal hyperplasia (60%, Fig. 15.15) or adrenal adenoma (40%, Conn syndrome, Fig. 15.16); adrenal carcinoma is rare. 1. CT is useful to determine pathology 2. Treatment for bilateral adrenal hyperplasia is mineralocorticoid receptor antagonist (e.g., spironolactone or eplerenone); adenoma and carcinoma are surgically resected. D. Secondary hyperaldosteronism arises with activation of the renin-angiotensin system (e.g., renovascular hypertension or CHF). Fig. 15.14 Steroidogenesis, adrenal cortex.

1	D. Secondary hyperaldosteronism arises with activation of the renin-angiotensin system (e.g., renovascular hypertension or CHF). Fig. 15.14 Steroidogenesis, adrenal cortex. Fig. 15.15 Adrenal hyperplasia. Fig. 15.16 Adrena l adenoma. E. Primary and secondary hyperaldosteronism are distinguished by plasma renin and edema 1. Primary is characterized by low renin and no edema (aldosterone escape). 2. Secondary is characterized by high renin; edema is often present. F. Glucocorticoid-remediable aldosteronism (GRA) leads to familial hyperaldosteron ism 1. Aberrant expression of aldosterone synthase in the fasciculata due to genetic mutation; autosomal dominant 2. Classically presents as child with HTN and hypokalemia; aldosterone is high and renin is low. 3. Responds to dexamethasone; confirmed with genetic testing G. Liddle syndrome mimics hyperaldosteronism. 1. Decreased degradation of sodium channels in collecting tubules due to genetic mutation; autosomal dominant 2.

1	G. Liddle syndrome mimics hyperaldosteronism. 1. Decreased degradation of sodium channels in collecting tubules due to genetic mutation; autosomal dominant 2. Presents as child with HTN, hypokalemia, and metabolic alkalosis, but with low aldosterone and low renin 3. 4. Treatment is potassium-sparing diuretics (e.g., amiloride or triamterene), which block tubular sodium channels; spironolactone is not effective. H. Syndrome of apparent mineralocorticoid excess (SAME) also mimics hyperaldosteronism. 1. 11~-hydroxysteroid dehydrogenase 2 (ll~-HSD2) deficiency allows cortisol to activate renal aldosterone receptors; autosomal recessive 2. Presents as child with HTN, hypokalemia, and metabolic alkalosis, but with low aldosterone and low renin 3. 4. May also arise with licorice (glycyrrhetinic acid), which blocks ll~-HSD2 III. HYPERCORTISOLISM (CUSHING SYNDROME) A. Excess cortisol B. Clinical features include 1.

1	4. May also arise with licorice (glycyrrhetinic acid), which blocks ll~-HSD2 III. HYPERCORTISOLISM (CUSHING SYNDROME) A. Excess cortisol B. Clinical features include 1. Muscle weakness with thin extremities-Cortisol breaks down muscle to produce amino acids for gluconeogenesis. 2. Moon facies, buffalo hump, and truncal obesity-High insulin (due to high glucose) increases storage of fat centrally. 3. Abdominal striae-Impaired collagen synthesis results in thinning of skin 4. HTN often with hypokalemia and metabolic alkalosis 1. High cortisol increases sensitivity of peripheral vessels to catecholamines. ii. At very high levels, cortisol cross-reacts with mineralocorticoid receptors (aldosterone is not increased). 5. 6. C. Diagnosis is based on 24-hour urine cortisol level (increased), late night salivary cortisol level (increased), and low-dose dexamethasone suppression test.

1	5. 6. C. Diagnosis is based on 24-hour urine cortisol level (increased), late night salivary cortisol level (increased), and low-dose dexamethasone suppression test. 1. Low-dose dexamethasone suppresses cortisol in normal individuals but fails to suppress cortisol in all causes of Cushing syndrome. D. Plasma ACTH distinguishes ACTH-dependent causes of Cushing syndrome from ACTH-independent causes (table 15.1). 1. IfACTH-independent, next step is CT to look for an adrenal lesion. 2. IfACTH-dependent, next step is high-dose dexamethasone test. E. High-dose dexamethasone suppresses ACTH production by a pituitary adenoma (serum cortisol is lowered) but does not suppress ectopic ACTH production (serum cortisol remains high). _____________11111, Table 15.1 Cushing Syndrome Low NIA NIA Tapering of steroids, if possible

1	_____________11111, Table 15.1 Cushing Syndrome Low NIA NIA Tapering of steroids, if possible Transsphenoidal resection of pituitary ACTH-secreting adenoma; bilateral adrenalectomy in pituitary refractory cases can lead to enlargement adenoma (70%, of pituitary adenoma, resulting in may be present. Cushing disease) hyperpigmentation, headaches, and bitemporal hemianopsia (Nelson syndrome) Ectopic ACTH ACTH (e.g., small excess and No suppression Resection of ectopic source may be present. Adenomal Primary adrenal carcinoma with Resection of adenomalcarcinoma or adenoma (10%), contralateral Low NIA bilateral resection of hyperplasia with hyperplasia, or atrophy or F. Treatment generally involves surgical resection 1. Ketoconazole or metyrapone useful if surgery is not an option IV. CONGENITAL ADRENAL HYPERPLASIA A. Due to enzymatic defects in cortisol production; autosomal recessive 1. High ACTH (decreased negative feedback) leads to bilateral adrenal hyperplasia. 2.

1	IV. CONGENITAL ADRENAL HYPERPLASIA A. Due to enzymatic defects in cortisol production; autosomal recessive 1. High ACTH (decreased negative feedback) leads to bilateral adrenal hyperplasia. 2. Mineralocorticoids and androgens may be increased or decreased depending on the enzyme defect. B. 21-hydroxylase deficiency is the most common cause (90% of cases). 1. Aldosterone and cortisol are decreased; steroidogenesis is shunted towards androgens. 2. Classic form presents in neonates as hyponatremia and hyperkalemia with lifethreatening hypotension (salt-wasting type); females have clitoral enlargement (genital ambiguity). 3. Nonclassic form presents later in life with androgen excess leading to precocious puberty (males) or hirsutism with menstrual irregularities (females). C. 11-hydroxylase deficiency also leads to androgen excess, but weak mineralocorticoids (DOC) are increased. 1.

1	C. 11-hydroxylase deficiency also leads to androgen excess, but weak mineralocorticoids (DOC) are increased. 1. Deoxycorticosterone (DOC) leads to HTN (sodium retention) with mild hypokalemia; renin and aldosterone are low. 2. Clitoral enlargement is seen in females D. 17-hydroxylase deficiency leads to decreased cortisol and androgens. 1. Weak mineralocorticoids (DOC) are increased leading to HTN with mild hypokalemia; renin and aldosterone are low. 2. Decreased androgens lead to primary amenorrhea and lack of pubic hair (females) or ambiguous genitalia with undescended testes (males). E. Newborn screening for CAH via serum 17-hydroxyprogesterone is routine 1. 2. F. Treatment for CAH is glucocorticoids; mineralocorticoids (21-hydroxylase deficiency) or sex steroids (17-hydroxylase deficiency) given as necessary. V. ADRENAL INSUFFICIENCY A. Deficiency of adrenal hormones B. Acute insufficiency presents as weakness and shock. Causes include 1.

1	V. ADRENAL INSUFFICIENCY A. Deficiency of adrenal hormones B. Acute insufficiency presents as weakness and shock. Causes include 1. Abrupt withdrawal of glucocorticoids 2. Treatment of Cushing syndrome 3. Waterhouse-Friderichsen syndrome-hemorrhagic necrosis of the adrenal glands (Fig. 15.17), classically due to sepsis and DIC in young children with Neisseria meningitidis infection C. Chronic insufficiency (Addison disease) presents with vague, progressive symptoms such as hypotension, weakness, fatigue, nausea, vomiting, and weight loss. D. Addison disease most often arises with progressive adrenal damage. 1. Common causes include autoimmune destruction (most common cause in the West), TB (most common cause in the developing world), and metastatic carcinoma (e.g., from lung). 2. Autoimmune adrenalitis can be a component of autoimmune polyendocrine syndromes E. Addison disease may also arise with pituitary (secondary) or hypothalamic (tertiary) disease. 1.

1	2. Autoimmune adrenalitis can be a component of autoimmune polyendocrine syndromes E. Addison disease may also arise with pituitary (secondary) or hypothalamic (tertiary) disease. 1. Hyperpigmentation (high ACTH) and hyperkalemia (low aldosterone) suggest primary adrenal insufficiency; secondary/tertiary insufficiency has no hyperpigmentation (low ACTH) and normal potassium (normal aldosterone). 2. Lack of ACTH response with metyrapone stimulation test supports a secondary or tertiary cause. 3. ACTH response with CRH stimulation test suggests hypothalamic (tertiary) disease. F. Treatment is glucocorticoids and mineralocorticoids. I. BASIC PRINCIPLES A. Composed of neural crest-derived chromaffin cells B. Main physiologic source of catecholamines (epinephrine and norepinephrine) II. PHEOCHROMOCYTOMA A. Tumor of chromaffin cells (Fig. 15.18) Fig. 15.17 Waterhouse-Friderichsen syndrome. Fig. 15.18 Pheochromocytoma. (Courtesy of (Courtesy of hum path.com) humpath.com)

1	II. PHEOCHROMOCYTOMA A. Tumor of chromaffin cells (Fig. 15.18) Fig. 15.17 Waterhouse-Friderichsen syndrome. Fig. 15.18 Pheochromocytoma. (Courtesy of (Courtesy of hum path.com) humpath.com) B. Clinical features are due to episodic release of catecholamines. 1. Episodic HTN, headaches, palpitations, tachycardia, and sweating C. Diagnosed by increased metanephrines and catecholamines in serum and urine D. Treatment is adrenalectomy. 1. Catecholamines may leak into the bloodstream upon manipulation of the tumor. 2. Phenoxybenzamine (irreversible a-blocker) followed by ~-blocker is administered preoperatively to prevent a hypertensive crisis. E. Often follows the 'rule of 10s:' 10% bilateral, 10% familial, 10% malignant, and 10% located outside of the adrenal medulla (e.g., organ of Zuckerkandl at the inferior mesenteric artery root or bladder wall) 1. Malignancy is defined by metastatic spread; patients require long-term follow-up

1	F. Associated with MEN 2A and 2B (RET), von Hippel-Lindau disease, and neurofibromatosis type 1 I. BREAST A. Modified sweat gland embryologically derived from the skin 1. Breast tissue can develop anywhere along the milk line, which runs from the axilla to the vulva (e.g., supernumerary nipples). B. The terminal duct lobular unit is the functional unit of the breast (Fig. 16.1); lobules make milk that drains via ducts to the nipple. C. Lobules and ducts are lined by two layers of epithelium. 1. Luminal cell layer-inner cell layer lining the ducts and lobules; responsible for milk production in the lobules 2. Myoepithelial cell layer-outer cell layer lining ducts and lobules; contractile function propels milk towards the nipple. D. Breast tissue is hormone sensitive. 1. Before puberty, male and female breast tissue primarily consists oflarge ducts under the nipple. 2.

1	D. Breast tissue is hormone sensitive. 1. Before puberty, male and female breast tissue primarily consists oflarge ducts under the nipple. 2. Development after menarche is primarily driven by estrogen and progesterone; lobules and small ducts form and are present in highest density in the upper outer quadrant. 3. Breast tenderness during the menstrual cycle is a common complaint, especially prior to menstruation. 4. During pregnancy, breast lobules undergo hyperplasia. i. Hyperplasia is driven by estrogen and progesterone produced by the corpus luteum (early first trimester), fetus, and placenta (later in pregnancy) 5. After menopause, breast tissue undergoes atrophy. E. Galactorrhea refers to milk production outside oflactation. 1. It is not a symptom of breast cancer. 2. Causes include nipple stimulation (common physiologic cause), prolactinoma of the anterior pituitary (common pathologic cause), and drugs. I. ACUTE MASTITIS

1	2. Causes include nipple stimulation (common physiologic cause), prolactinoma of the anterior pituitary (common pathologic cause), and drugs. I. ACUTE MASTITIS A. Bacterial infection of the breast, usually due to Staphylococcus aureus B. Associated with breast-feeding; fissures develop in the nipple providing a route of entry for microbes. C. Presents as an erythematous breast with purulent nipple discharge; may progress to abscess formation D. Treatment involves continued drainage (e.g., feeding) and antibiotics (e.g., dicloxacillin). II. PERIDUCTAL MASTITIS A. Inflammation of the subareolar ducts B. Usually seen in smokers pathoma.com 1. Relative vitamin A deficiency results in squamous metaplasia oflactiferous ducts, producing duct blockage and inflammation. C. Clinically presents as a subareolar mass with nipple retraction III. MAMMARY DUCT ECTASIA

1	C. Clinically presents as a subareolar mass with nipple retraction III. MAMMARY DUCT ECTASIA A. Inflammation with dilation (ectasia) of the subareolar ducts 1. Rare; classically arises in multiparous postmenopausal women B. Presents as a periareolar mass with green-brown nipple discharge (inflammatory debris) 1. Chronic inflammation with plasma cells is seen on biopsy. IV. FAT NECROSIS A. Necrosis of breast fat B. Usually related to trauma; however, a history of trauma may not always be evident. C. Presents as a mass on physical exam or abnormal calcification on mammography (due to saponification) D. Biopsy shows necrotic fat with associated calcifications and giant cells. I. FIBROCYSTIC CHANGE A. Development of fibrosis and cysts in the breast 1. Most common change in the premenopausal breast; thought to be hormone mediated B. Presents as vague irregularity of the breast tissue ('lumpy breast'), usually in the upper outer quadrant

1	B. Presents as vague irregularity of the breast tissue ('lumpy breast'), usually in the upper outer quadrant C. Cysts have a blue-dome appearance on gross exam. D. Benign, but some fibrocystic-related changes are associated with an increased risk for invasive carcinoma (increased risk applies to both breasts) 1. Fibrosis, cysts, and apocrine metaplasia (Fig. 16.2)-no increased risk 2. 3. II. INTRADUCTAL PAPILLOMA A. Papillary growth, usually into a large duct B. Characterized by fibrovascular projections lined by epithelial (luminal) and myoepithelial cells C. Classically presents as bloody nipple discharge in a premenopausal woman D. Must be distinguished from papillary carcinoma, which also presents as bloody nipple discharge o•o. 0~ O)~",.. ; ... ·~"-0 0 o·.::>i; • ·••~:?.t,00 Fig. 16.1 Terminal duct lobular unit. Fig. 16.2 Fibrocystic change with apocrine Fig. 16.3 Fibroadenoma. metaplasia. 1.

1	O)~",.. ; ... ·~"-0 0 o·.::>i; • ·••~:?.t,00 Fig. 16.1 Terminal duct lobular unit. Fig. 16.2 Fibrocystic change with apocrine Fig. 16.3 Fibroadenoma. metaplasia. 1. Papillary carcinoma is characterized by fibrovascular projections lined by epithelial cells without underlying myoepithelial cells. 2. Risk of papillary carcinoma increases with age; thus, it is more commonly seen in postmenopausal women. III. FIBROADENOMA A. Tumor of fibrous tissue and glands (Fig. 16.3) B. Most common benign neoplasm of the breast; usually seen in premenopausal women C. Presents as a well-circumscribed, mobile marble-like mass D. Estrogen sensitive-grows during pregnancy and may be painful during the menstrual cycle E. Benign, with no increased risk of carcinoma IV. PHYLLODES TUMOR A. Fibroadenoma-like tumor with overgrowth of the fibrous component; characteristic 'leaf-like' projections are seen on biopsy (Fig. 16.4). B. Most commonly seen in postmenopausal women

1	A. Fibroadenoma-like tumor with overgrowth of the fibrous component; characteristic 'leaf-like' projections are seen on biopsy (Fig. 16.4). B. Most commonly seen in postmenopausal women C. Can be malignant in some cases I. BASIC PRINCIPLES A. Most common carcinoma in women by incidence (excluding skin cancer) B. 2nd most common cause of cancer mortality in women C. Risk factors are mostly related to estrogen exposure. 1. 2. Age-Cancer usually arises in postmenopausal women, with the notable exception of hereditary breast cancer. 3. 4. 5. 6. First-degree relative (mother, sister, or daughter) with breast cancer II. DUCTAL CARCINOMA IN SITU (DCIS) A. Malignant proliferation of cells in ducts with no invasion of the basement membrane B. Often detected as calcification on mammography; DCIS does not usually produce a mass.

1	A. Malignant proliferation of cells in ducts with no invasion of the basement membrane B. Often detected as calcification on mammography; DCIS does not usually produce a mass. 1. Mammographic calcifications can also be associated with benign conditions such as fibrocystic changes (especially sclerosing adenosis) and fat necrosis. Fig. 16.4 Phyllodes tumor. Fig. 16.5 Ductal carcinoma in situ, comedo type. 2. Biopsy of calcifications is often necessary to distinguish between benign and malignant conditions. C. Histologic subtypes are based on architecture; comedo type is characterized by high-grade cells with necrosis and dystrophic calcification in the center of ducts (Fig. 16.5). D. Paget disease of the breast is DCIS that extends up the ducts to involve the skin of the nipple (Fig. 16.6). 1. Presents as nipple ulceration and erythema 2. Paget disease of the breast is almost always associated with an underlying carcinoma. III. INVASIVE DUCTAL CARCINOMA

1	1. Presents as nipple ulceration and erythema 2. Paget disease of the breast is almost always associated with an underlying carcinoma. III. INVASIVE DUCTAL CARCINOMA A. Invasive carcinoma that classically forms duct-like structures B. Most common type of invasive carcinoma in the breast, accounting for > 80% of cases C. Presents as a mass detected by physical exam or by mammography 1. Clinically detected masses are usually 2 cm or greater. 2. Mammographically detected masses are usually 1 cm or greater. 3. Advanced tumors may result in dimpling of the skin or retraction of the nipple. D. Biopsy usually shows duct-like structures in a desmoplastic stroma; special subtypes of invasive ductal carcinoma include 1. Tubular carcinoma-characterized by well-differentiated tubules that lack myoepithelial cells (Fig. 16.7A); relatively good prognosis 2.

1	Tubular carcinoma-characterized by well-differentiated tubules that lack myoepithelial cells (Fig. 16.7A); relatively good prognosis 2. Mucinous carcinoma-characterized by carcinoma with abundant extracellular mucin ('tumor cells floating in a mucus pool', Fig. 16.7B) Fig. 16.6 Paget disease of the nipple. A, Clinical appearance. B, Microscopic appearance. (A, Courtesy of Jerome Taxy, MD) ,';,: . ~ i, ,, :1/ -~: . rt·.· r:. .~!t_ ·~ p.•'l Fig. 16.7 Invasive ductal carcinoma. A, Tubular carcinoma. B, Mucinous ca rcinoma.

1	rt·.· r:. .~!t_ ·~ p.•'l Fig. 16.7 Invasive ductal carcinoma. A, Tubular carcinoma. B, Mucinous ca rcinoma. i. Tends to occur in older women (average age is 70 years) 11. Relatively good prognosis 3. Medullary carcinoma-characterized by large, high-grade cells growing in sheets with associated lymphocytes and plasma cells i. Grows as a well-circumscribed mass that can mimic fibroadenoma on mammography ii. Relatively good prognosis 111. Increased incidence in BRCAl carriers 4. Inflammatory carcinoma-characterized by carcinoma in dermal lymphatics (Fig. 16.8) i. Presents classically as an inflamed, swollen breast (tumor cells block drainage of lymphatics) with no discrete mass; can be mistaken for acute mastitis ii. Poor prognosis IV. A. Malignant proliferation of cells in lobules with no invasion of the basement membrane B. LCIS does not produce a mass or calcifications and is usually discovered incidentally on biopsy.

1	IV. A. Malignant proliferation of cells in lobules with no invasion of the basement membrane B. LCIS does not produce a mass or calcifications and is usually discovered incidentally on biopsy. C. Characterized by dyscohesive cells lacking E-cadherin adhesion protein D. Often multifocal and bilateral E. Treatment is tamoxifen (to reduce the risk of subsequent carcinoma) and close follow-up; low risk of progression to invasive carcinoma V. A. Invasive carcinoma that characteristically grows in a single-file pattern (Fig. 16.9); cells may exhibit signet-ring morphology. 1. No duct formation due to lack ofE-cadherin. VI. PROGNOSTIC AND PREDICTIVE FACTORS A. Prognosis in breast cancer is based on TNM staging. 1. Metastasis is the most important factor, but most patients present before metastasis occurs. 2.

1	VI. PROGNOSTIC AND PREDICTIVE FACTORS A. Prognosis in breast cancer is based on TNM staging. 1. Metastasis is the most important factor, but most patients present before metastasis occurs. 2. Spread to axillary lymph nodes is the most useful prognostic factor (given that metastasis is not common at presentation); sentinel lymph node biopsy is used to assess axillary lymph nodes. B. Predictive factors predict response to treatment. 1. Most important factors are estrogen receptor (ER), progesterone receptor (PR), and HER2/neu gene amplification (overexpression) status. Fig. 16.8 Inflammatory carcinoma. A, Microscopic appearance. B, Clinical appearance. (8, Courtesy of Fig. 16.9 Invasive lobular carcinoma. Jerome Taxy, MD) 2. Presence of ER and PR is associated with response to antiestrogenic agents (e.g., tamoxifen); both receptors are located in the nucleus (Fig. 16.10). 3.

1	Presence of ER and PR is associated with response to antiestrogenic agents (e.g., tamoxifen); both receptors are located in the nucleus (Fig. 16.10). 3. HER2/neu amplification is associated with response to trastuzumab (Herceptin), a designer antibody directed against the HER2 receptor; HER2/neu is a growth factor receptor present on the cell surface (Fig. 16.11). 4. 'Triple-negative' tumors are negative for ER, PR, and HER2/neu and have a poor prognosis; African American women have an increased propensity to develop triple-negative carcinoma. VII. HEREDITARY BREAST CANCER A. Represents 10% of breast cancer cases B. Clinical features that suggest hereditary breast cancer include multiple first-degree relatives with breast cancer, tumor at an early age (premenopausal), and multiple tumors in a single patient. C. BRCAl and BRCA2 mutations are the most important single gene mutations associated with hereditary breast cancer. 1.

1	C. BRCAl and BRCA2 mutations are the most important single gene mutations associated with hereditary breast cancer. 1. BRCAl mutation is associated with breast and ovarian carcinoma. 2. BRCA2 mutation is associated with breast carcinoma in males. D. Women with a genetic propensity to develop breast cancer may choose to undergo removal of both breasts (bilateral mastectomy) to decrease the risk of developing carcinoma. 1. A small risk for cancer remains because breast tissue sometimes extends into the axilla or subcutaneous tissue of the chest wall. VIII. MALE BREAST CANCER A. Breast cancer is rare in males (represents 1 % of all breast cancers). B. Usually presents as a subareolar mass in older males 1. Highest density of breast tissue in males is underneath the nipple. 2. C. Most common histological subtype is invasive ductal carcinoma. 1. Lobular carcinoma is rare (the male breast develops very few lobules). D. Associated with BRCA2 mutations and Klinefelter syndrome

1	1. Lobular carcinoma is rare (the male breast develops very few lobules). D. Associated with BRCA2 mutations and Klinefelter syndrome Fig. 16.10 Estrogen receptor, Fig. 16.11 HER2/neu amplification, immunohistochemical stain. immunohistochemical stain. I. NEURAL TUBE DEFECTS A. Arise from incomplete closure of the neural tube 1. Neural plate invaginates early in gestation to form the neural tube, which runs along the cranial-caudal axis of the embryo. 2. The wall of the neural tube forms central nervous system tissue, the hollow lumen forms the ventricles and spinal cord canal, and the neural crest forms the peripheral nervous system. B. Associated with low folate levels prior to conception C. Detected during prenatal care by elevated alpha-fetoprotein (AFP) levels in the amniotic fluid and maternal blood D. Anencephaly is absence of the skull and brain (disruption of the cranial end of the neural tube). 1. Leads to a 'frog-like' appearance of the fetus (Fig. 17.1) 2.

1	D. Anencephaly is absence of the skull and brain (disruption of the cranial end of the neural tube). 1. Leads to a 'frog-like' appearance of the fetus (Fig. 17.1) 2. Results in maternal polyhydramnios since fetal swallowing of amniotic fluid is impaired E. Spina bifida is failure of the posterior vertebral arch to close, resulting in a vertebral defect (disruption of the caudal end of the neural tube). 1. Spina bifida occulta presents as a dimple or patch of hair overlying the vertebral defect. 2. Spina bifida presents with cystic protrusion of the underlying tissue through the vertebral defect. i. Meningocele-protrusion of meninges ii. Meningomyelocele-protrusion of meninges and spinal cord II. CEREBRAL AQUEDUCT STENOSIS A. Congenital stenosis of the channel that drains cerebrospinal fluid (CSF) from the 3rd ventricle into the 4th ventricle B. Leads to accumulation of CSF in the ventricular space; most common cause of hydrocephalus in newborns 1.

1	B. Leads to accumulation of CSF in the ventricular space; most common cause of hydrocephalus in newborns 1. CSF is produced by the choroid plexus lining the ventricles. 2. Flows from the lateral ventricles into the 3rd ventricle via the interventricular foramen of Monro 3. Flows from the 3rd ventricle into the 4th ventricle via the cerebral aqueduct 4. Flows from the 4th ventricle into the subarachnoid space via the foramina of Magendie and Luschka C. Presents with enlarging head circumference due to dilation of the ventricles (cranial suture lines are not fused) Ill. DANDY-WALKER MALFORMATION A. Congenital failure of the cerebellar vermis to develop B. Presents as a massively dilated 4th ventricle (posterior fossa) with an absent cerebellum (Fig. 17.2); often accompanied by hydrocephalus pathoma.com IV. ARNOLD-CHIARI MALFORMATION (TYPE II) A. Congenital downward displacement of cerebellar vermis and tonsils through the foramen magnum

1	IV. ARNOLD-CHIARI MALFORMATION (TYPE II) A. Congenital downward displacement of cerebellar vermis and tonsils through the foramen magnum B. Obstruction of CSF flow commonly results in hydrocephalus. C. Often associated with meningomyelocele I. SYRINGOMYELIA A. Cystic degeneration of the spinal cord B. Arises with trauma or in association with a type 1 Arnold-Chiari malformation C. Usually occurs at CS-Tl 1. Presents as sensory loss of pain and temperature with sparing of fine touch and position sense in the upper extremities ("cape like" distribution)-due to involvement of the anterior white commissure of the spinothalamic tract with sparing of the dorsal column {Table 17.1) D. Syrinx expansion results in involvement of other spinal tracts leading to 1. Muscle atrophy and weakness with decreased muscle tone and impaired reflexes-due to damage to lower motor neurons of the anterior horn 2.

1	Muscle atrophy and weakness with decreased muscle tone and impaired reflexes-due to damage to lower motor neurons of the anterior horn 2. Horner syndrome with ptosis {droopy eyelid), miosis (constricted pupil), and anhidrosis (decreased sweating)-due to disruption of the lateral horn of the hypothalamospinal tract {Table 17.1) II. POLIOMYELITIS A. Damage to the anterior motor horn due to poliovirus infection B. Presents with lower motor neuron signs-flaccid paralysis with muscle atrophy, fasciculations, weakness with decreased muscle tone, impaired reflexes, and negative Babinski sign (downgoing toes) III. WERDNIG-HOFFMAN DISEASE A. Inherited degeneration of the anterior motor horn; autosomal recessive B. Presents as a "floppy baby;" death occurs within a few years after birth. IV. AMYOTROPHIC LATERAL SCLEROSIS (ALS)

1	A. Inherited degeneration of the anterior motor horn; autosomal recessive B. Presents as a "floppy baby;" death occurs within a few years after birth. IV. AMYOTROPHIC LATERAL SCLEROSIS (ALS) A. Degenerative disorder of upper and lower motor neurons of the corticospinal tract (Table 17.1) 1. Anterior motor horn degeneration leads to lower motor neuron signs-flaccid paralysis with muscle atrophy, fasciculations, weakness with decreased muscle tone, impaired reflexes, and negative Babinski sign. Fig. 17.1 Anencephaly. (Courtesy of humpath. Fig. 17.2 Dandy-Walker malformation, MRI. Fig. 17.3 Bacterial meningitis, gross appearance. com) (Courtesy of Robert Heng, MD) 2. Lateral corticospinal tract degeneration leads to upper motor neuron signsspastic paralysis with hyperreflexia, increased muscle tone, and positive Babinski sign. B. Atrophy and weakness of hands is an early sign. 1. Lack of sensory impairment distinguishes ALS from syringomyelia.

1	B. Atrophy and weakness of hands is an early sign. 1. Lack of sensory impairment distinguishes ALS from syringomyelia. C. Most cases are sporadic, arising in middle age adults. 1. Zinc-copper superoxide dismutase mutation (SODJ) is present in some familial cases; leads to free radical injury in neurons V. FRIEDREICH ATAXIA A. Degenerative disorder of the cerebellum and spinal cord 1. Degeneration of the cerebellum leads to ataxia. 2. Degeneration of multiple spinal cord tracts leads to loss of vibratory sense and proprioception, muscle weakness in the lower extremities, and loss of deep tendon reflexes. B. Autosomal recessive; due to expansion of an unstable trinucleotide repeat (GAA) in the frataxin gene 1. Frataxin is essential for mitochondrial iron regulation; loss results in iron buildup with free radical damage. C. Presents in early childhood; patients are wheelchair bound within a few years. D. Associated with hypertrophic cardiomyopathy I. BASIC PRINCIPLES

1	C. Presents in early childhood; patients are wheelchair bound within a few years. D. Associated with hypertrophic cardiomyopathy I. BASIC PRINCIPLES A. Inflammation of the leptomeninges (Fig. 17.3) 1. Meninges consist of three layers (dura, arachnoid, and pia) that lie between the brain and the skull. 2. Pia and arachnoid together are termed leptomeninges. Table 17.1: Spinal Cord Tracts Arises from posterior horn, immediately crosses Peripheral nerves to posterior horn; cell body is in dorsal root Thalamus to cortex temperature sensation) commissure, and ascends via ganglion. the spinothalamic tract to thalamus Arises from medulla, crosses lemniscus (pressure, to medulla via dorsal column; over, and ascends via the Thalamus to cortex touch, vibration, and cell body is in dorsal root medial lemniscus to thalamus proprioception) ganglion. Pyramidal neurons in cortex descend, cross over Arises from the anterior motor

1	Pyramidal neurons in cortex descend, cross over Arises from the anterior motor Lateral corticospinal in medullary pyramids, and horn and synapses on muscle (None)(voluntary movement) synapse on the anterior motor (lower motor neuron) horn of the cord (upper motor neuron). Hypothalamospinal Arises from the hypothalamus Arises from lateral horn at Tl (sympathetic input of the and synapses on the lateral and synapses on the superior eyelids, pupil, and skin of face B. Most commonly due to an infectious agent 1. Group B streptococci, E coli, and Listeria monocytogenes (neonates) 2. N meningitidis (children and teenagers), Streptococcus pneumoniae (adults and elderly), and H influenza (nonvaccinated infants) 3. 4. C. Presents with classic triad of headache, nuchal rigidity, and fever; photophobia, vomiting, and altered mental status may also be present. D. Diagnosis is made by lumbar puncture (sampling of CSF). 1.

1	D. Diagnosis is made by lumbar puncture (sampling of CSF). 1. Performed by placing a needle between L4 and LS (level of the iliac crest). Spinal cord ends at L2, but subarachnoid space and cauda equina continue to S2. 2. Layers crossed include skin, ligaments, epidural space, dura, and arachnoid. E. CSF findings 1. Bacterial meningitis-neutrophils with 1-CSF glucose; gram stain and culture often identify the causative organism. 2. Viral meningitis-lymphocytes with normal CSF glucose 3. Fungal meningitis-lymphocytes with 1-CSF glucose F. Complications are usually seen with bacterial meningitis. 1. Death-herniation secondary to cerebral edema 2. Hydrocephalus, hearing loss, and seizures-sequelae related to fibrosis I. BASIC PRINCIPLES A. Neurologic deficit due to cerebrovascular compromise; major cause of morbidity and mortality

1	Hydrocephalus, hearing loss, and seizures-sequelae related to fibrosis I. BASIC PRINCIPLES A. Neurologic deficit due to cerebrovascular compromise; major cause of morbidity and mortality B. Due to ischemia (85% of cases) or hemorrhage (15% of cases) 1. Neurons are dependent on serum glucose as an essential energy source and are particularly susceptible to ischemia (undergo necrosis within 3-5 minutes). II. GLOBAL CEREBRAL ISCHEMIA A. Global ischemia to the brain B. Major etiologies 1. Low perfusion (e.g., atherosclerosis) 2. Acute decrease in blood flow (e.g., cardiogenic shock) 3. Chronic hypoxia (e.g., anemia) 4. Repeated episodes of hypoglycemia (e.g., insulinoma) C. Clinical features are based on duration and magnitude of the insult. 1. Mild global ischemia results in transient confusion with prompt recovery. Fig . 17.4 Pale infarct, cortex. (Courtesy of Robert Fig. 17.5 Lacunar infarcts. (Courtesy of Robert Wollmann, MD) Wollmann, MD) 2.

1	Fig . 17.4 Pale infarct, cortex. (Courtesy of Robert Fig. 17.5 Lacunar infarcts. (Courtesy of Robert Wollmann, MD) Wollmann, MD) 2. Severe global ischemia results in diffuse necrosis; survival leads to a 'vegetative state.' 3. Moderate global ischemia leads to infarcts in watershed areas (e.g., area lying between regions fed by the anterior and middle cerebral artery) and damage to highly vulnerable regions such as i. Pyramidal neurons of the cerebral cortex (layers 3, 5, and 6)-leads to laminar necrosis ii. Pyramidal neurons of the hippocampus (temporal lobe)-important in longterm memory iii. Purkinje layer of the cerebellum-integrates sensory perception with motor control III. ISCHEMIC STROKE A. Regional ischemia to the brain that results in focal neurologic deficits lasting > 24 hours 1. If symptoms last < 24 hours, the event is termed a transient ischemic attack (TIA). B. Subtypes include thrombotic, embolic, and lacunar strokes.

1	B. Subtypes include thrombotic, embolic, and lacunar strokes. 1. Thrombotic stroke is due to rupture of an atherosclerotic plaque. i. Atherosclerosis usually develops at branch points (e.g., bifurcation of internal carotid and middle cerebral artery in the circle of Willis). ii. Results in a pale infarct at the periphery of the cortex (Fig. 17.4) 2. Embolic stroke is due to thromboemboli. 1. Most common source of emboli is the left side of the heart (e.g., atrial fibrillation). ii. Usually involves the middle cerebral artery u1. Results in a hemorrhagic infarct at the periphery of the cortex 3. Lacunar stroke occurs secondary to hyaline arteriolosclerosis, a complication of hypertension. i. Most commonly involves lenticulostriate vessels, resulting in small cystic areas of infarction (Fig. 17.5) 11. Involvement of the internal capsule leads to a pure motor stroke. u1. Involvement of the thalamus leads to a pure sensory stroke.

1	C. Ischemic stroke results in liquefactive necrosis. 1. Eosinophilic change in the cytoplasm of neurons (red neurons, Fig. 17.6A) is an early microscopic finding (12 hours after infarction). 2. Necrosis (24 hours), infiltration by neutrophils (days 1-3) and microglial cells (days 4-7), and gliosis (weeks 2-3) then ensue. 3. Results in formation of a fluid-filled cystic space surrounded by gliosis (Fig. 17.6B) ' ' i w , . •> f Fig. 17.6 Brain infarct. A, Red neurons. B, Cyst formation. (Courtesy of Robert Wollmann, MD) IV. A. Bleeding into brain parenchyma B. Classically due to rupture of Charcot-Bouchard microaneurysms of the lenticulostriate vessels 1. Complication of hypertension; treatment of hypertension reduces incidence by half. 2. Basal ganglia is the most common site (Fig. 17.7). C. Presents as severe headache, nausea, vomiting, and eventual coma V. A. Bleeding into the subarachnoid space (Fig. 17.8)

1	2. Basal ganglia is the most common site (Fig. 17.7). C. Presents as severe headache, nausea, vomiting, and eventual coma V. A. Bleeding into the subarachnoid space (Fig. 17.8) B. Presents as a sudden headache ("worst headache of my life") with nuchal rigidity C. Lumbar puncture shows xanthochromia (yellow hue due to bilirubin). D. Most frequently (85%) due to rupture of a berry aneurysm; other causes include AV malformations and an anticoagulated state. 1. Berry aneurysms are thin-walled saccular outpouchings that lack a media layer (Fig. 17.9), increasing the risk for rupture. 2. Most frequently located in the anterior circle of Willis at branch points of the anterior communicating artery 3. Associated with Marfan syndrome and autosomal dominant polycystic kidney disease I. EPIDURAL HEMATOMA A. Collection of blood between the dura and the skull

1	Associated with Marfan syndrome and autosomal dominant polycystic kidney disease I. EPIDURAL HEMATOMA A. Collection of blood between the dura and the skull B. Classically due to fracture of the temporal bone with rupture of the middle meningeal artery; bleeding separates the dura from the skull. 1. 2. Lucid interval may precede neurologic signs. C. Herniation is a lethal complication. II. SUBDURAL HEMATOMA A. Collection of blood underneath the dura; blood covers the surface of the brain (Fig. 17.10). B. Due to tearing of bridging veins that lie between the dura and arachnoid; usually arises with trauma 1. 2. Presents with progressive neurologic signs Fig. 17.7 lntracerebral hemorrhage, basal Fig. 17.8 Subarachnoid hemorrhage. (Courtesy of ganglia. (Courtesy of Robert Wollmann, MD) Jerome Taxy, MD) 3. Increased rate of occurrence in the elderly due to age-related cerebral atrophy, which stretches the veins C. Herniation is a lethal complication. III. HERNIATION

1	C. Herniation is a lethal complication. III. HERNIATION A. Displacement of brain tissue due to mass effect or increased intracranial pressure B. Tonsillar herniation involves displacement of the cerebellar tonsils into the foramen magnum. 1. Compression of the brain stem leads to cardiopulmonary arrest. C. Subfalcine herniation involves displacement of the cingulate gyrus under the falx cerebri. 1. Compression of the anterior cerebral artery leads to infarction. D. Uncal herniation involves displacement of the temporal lobe uncus under the tentorium cerebelli. 1. Compression of cranial nerve III (oculomotor) leads to the eye moving "down and out" and a dilated pupil. 2. Compression of posterior cerebral artery leads to infarction of occipital lobe (contralateral homonymous hemianopsia). 3. Rupture of the paramedian artery leads to Duret (brainstem) hemorrhage. I. BASIC PRINCIPLES A. Myelin insulates axons, improving the speed and efficiency of conduction. 1.

1	3. Rupture of the paramedian artery leads to Duret (brainstem) hemorrhage. I. BASIC PRINCIPLES A. Myelin insulates axons, improving the speed and efficiency of conduction. 1. Oligodendrocytes myelinate the central nervous system. 2. Schwann cells myelinate the peripheral nervous system. B. Demyelinating disorders are characterized by destruction of myelin or oligodendrocytes; axons are generally preserved. II. LEUKODYSTROPHIES A. Inherited mutations in enzymes necessary for production or maintenance of myelin B. Metachromatic leukodystrophy is due to a deficiency of arylsulfatase (autosomal recessive); most common leukodystrophy 1. Sulfatides cannot be degraded and accumulate in the lysosomes of oligodendrocytes (lysosomal storage disease). C. Krabbe disease is due to a deficiency of galactocerebrosidase (autosomal recessive). 1. Galactocerebroside accumulates in macrophages.

1	C. Krabbe disease is due to a deficiency of galactocerebrosidase (autosomal recessive). 1. Galactocerebroside accumulates in macrophages. D. Adrenoleukodystrophy is due to impaired addition of coenzyme A to long-chain fatty acids (X-linked defect). Fig. 17.9 Berry aneurysm. (Courtesy of Jerome Fig. 17.10 Subdural hematoma. (Courtesy of Fig. 17.11 Gray plaque, multiple sclerosis. Taxy, MD) Robert Wollmann, MD) (Courtesy of Peter Pytel, MD) 1. Accumulation of fatty acids damages adrenal glands and white matter of the brain. III. MULTIPLE SCLEROSIS A. Autoimmune destruction of CNS myelin and oligodendrocytes 1. Most common chronic CNS disease of young adults (20-30 years of age); more commonly seen in women 2. Associated with HLA-DR2 3. More commonly seen in regions away from the equator B. Presents with relapsing neurologic deficits with periods of remission (multiple lesions in time and space). Clinical features include 1. 2. 3. 4.

1	B. Presents with relapsing neurologic deficits with periods of remission (multiple lesions in time and space). Clinical features include 1. 2. 3. 4. Hemiparesis or unilateral loss of sensation (cerebral white matter, usually periventricular) 5. Lower extremity loss of sensation or weakness (spinal cord) 6. Bowel, bladder, and sexual dysfunction (autonomic nervous system) C. Diagnosis is made by MRI and lumbar puncture. 1. MRI reveals plaques (areas of white matter demyelination). 2. Lumbar puncture shows increased lymphocytes, increased immunoglobulins with oligoclonal IgG bands on high resolution electrophoresis, and myelin basic protein. D. Gross examination shows gray-appearing plaques in the white matter (Fig. 17.11). E. Treatment of acute attacks includes high-dose steroids. 1. Long-term treatment with interferon beta slows progression of disease. IV. A. Progressive, debilitating encephalitis leading to death

1	E. Treatment of acute attacks includes high-dose steroids. 1. Long-term treatment with interferon beta slows progression of disease. IV. A. Progressive, debilitating encephalitis leading to death B. Due to slowly progressing, persistent infection of the brain by measles virus. 1. Infection occurs in infancy; neurologic signs arise years later (during childhood). C. Characterized by viral inclusions within neurons (gray matter) and oligodendrocytes (white matter) V. A. JC virus infection of oligodendrocytes (white matter) 1. Immunosuppression (e.g., AIDS or leukemia) leads to reactivation of the latent virus. B. Presents with rapidly progressive neurologic signs (visual loss, weakness, dementia) leading to death VI. CENTRAL PONTINE MYELINOLYSIS A. Focal demyelination of the pons (anterior brain stem) B. Due to rapid intravenous correction of hyponatremia 1. Occurs in severely malnourished patients (e.g., alcoholics and patients with liver disease)

1	B. Due to rapid intravenous correction of hyponatremia 1. Occurs in severely malnourished patients (e.g., alcoholics and patients with liver disease) C. Classically presents as acute bilateral paralysis ("locked in" syndrome) I. BASIC PRINCIPLES A. Characterized by loss of neurons within the gray matter; often due to accumulation of protein which damages neurons B. Degeneration of the cortex leads to dementia. C. Degeneration of the brainstem and basal ganglia leads to movement disorders. II. ALZHEIMER DISEASE (AD) A. Degenerative disease of cortex; most common cause of dementia B. Clinical features 1. Slow-onset memory loss (begins with short-term memory loss and progresses to long-term memory loss) and progressive disorientation 2. 3. 4. Patients become mute and bedridden; infection is a common cause of death. 5. Focal neurologic deficits are not seen in early disease. C. Most cases (95%) are sporadic and seen in the elderly. 1.

1	Patients become mute and bedridden; infection is a common cause of death. 5. Focal neurologic deficits are not seen in early disease. C. Most cases (95%) are sporadic and seen in the elderly. 1. Risk increases with age (doubles every 5 years after the age of60). 2. E4 allele ofapolipoprotein E (APOE) is associated with increased risk, E2 allele with decreased risk. D. Early-onset AD is seen in 1. Familial cases-associated with presenilin 1 and presenilin 2 mutations 2. Down syndrome-commonly occurs by 40 years of age E. Morphologic features include 1. Cerebral atrophy with narrowing of the gyri, widening of the sulci, and dilation of the ventricles (Fig. 17.12A) 2. Neuritic plaques-extracellular core comprised of A~ amyloid with entangled neuritic processes (Fig. 17.12B) i. A~ amyloid is derived from amyloid precursor protein (APP), which is coded on chromosome 21. APP normally undergoes alpha cleavage; beta cleavage results in A~ amyloid.

1	ii. Amyloid may also deposit around vessels, increasing the risk of hemorrhage. 3. Neurofibrillary tangles-intracellular aggregates of fibers composed of hyperphosphorylated tau protein (Fig. 17.12C) i. Tau is a microtubule-associated protein. 4. Loss of cholinergic neurons in the nucleus basalis of Meynert F. Diagnosis is made by clinical and pathological correlation. 1. Presumptive diagnosis is made clinically after excluding other causes. 2. III. VASCULAR DEMENTIA A. Multifocal infarction and injury due to hypertension, atherosclerosis, or vasculitis B. 2nd most common cause of dementia Fig. 17.12 Alzheimer disease. A, Cerebral atrophy. B, Neuritic plaque. C, Neurofibrillary tangle. (A, Courtesy of Jerome Taxy, MD. Band C, Courtesy of Peter Pytel, MD) IV. A. Degenerative disease of the frontal and temporal cortex; spares the parietal and occipital lobes B. Characterized by round aggregates of tau protein (Pick bodies) in neurons of the cortex

1	IV. A. Degenerative disease of the frontal and temporal cortex; spares the parietal and occipital lobes B. Characterized by round aggregates of tau protein (Pick bodies) in neurons of the cortex C. Behavioral and language symptoms arise early; eventually progresses to dementia V. A. Degenerative loss of dopaminergic neurons in the substantia nigra of the basal ganglia (Fig. 17.13A,B) 1. Nigrostriatal pathway of basal ganglia uses dopamine to initiate movement. B. Common disorder related to aging; seen in 2% of older adults C. Unknown etiology; historically, rare cases were related to MPTP exposure (a contaminant in illicit drugs). D. Clinical features ('TRAP') 1. Tremor-pill rolling tremor at rest; disappears with movement 2. Rigidity-cogwheel rigidity in the extremities 3. Akinesia/bradykinesia-slowing of voluntary movement; expressionless face 4.

1	Tremor-pill rolling tremor at rest; disappears with movement 2. Rigidity-cogwheel rigidity in the extremities 3. Akinesia/bradykinesia-slowing of voluntary movement; expressionless face 4. E. Histology reveals loss of pigmented neurons in the substantia nigra and round, eosinophilic inclusions of a-synuclein (Lewy bodies, Fig. 17.13C) in affected neurons. F. Dementia is a common feature oflate disease. 1. Early-onset dementia is suggestive of Lewy body dementia, which is characterized by dementia, hallucinations and parkinsonian features; histology reveals cortical Lewy bodies. VI. HUNTINGTON DISEASE A. Degeneration of GABAergic neurons in the caudate nucleus of the basal ganglia (Fig. 17.14) 1. Autosomal dominant disorder (chromosome 4) characterized by expanded trinucleotide repeats (CAG) in the huntingtin gene 2. Further expansion of repeats during spermatogenesis leads to anticipation.

1	Autosomal dominant disorder (chromosome 4) characterized by expanded trinucleotide repeats (CAG) in the huntingtin gene 2. Further expansion of repeats during spermatogenesis leads to anticipation. B. Presents with chorea that can progress to dementia and depression; average age at presentation is 40 years. C. Suicide is a common cause of death. VII. NORMAL PRESSURE HYDROCEPHALUS A. Increased CSF resulting in dilated ventricles B. Can cause dementia in adults; usually idiopathic Fig. 17.13 Parkinson disease. A, Loss of pigmented neurons in substantia nigra. B, Normal substantia nigra for comparison. C, Lewy body. (A and B, Courtesy of Robert Wollmann, MD) C. Presents as triad of urinary incontinence, gait instability, and dementia ("wet, wobbly, and wacky") D. Lumbar puncture improves symptoms; treatment is ventriculoperitoneal shunting. VIII. SPONGIFORM ENCEPHALOPATHY

1	D. Lumbar puncture improves symptoms; treatment is ventriculoperitoneal shunting. VIII. SPONGIFORM ENCEPHALOPATHY A. Degenerative disease due to prion protein 1. Prion protein is normally expressed in CNS neurons in an a-helical configuration (PrPc). B. Disease arises with conversion to a ~-pleated conformation (PrP'c). 1. Conversion can be sporadic, inherited (familial forms of disease), or transmitted. C. Pathologic protein is not degradable and converts normal protein into the pathologic form, resulting in a vicious cycle. 1. Damage to neurons and glial cells is characterized by intracellular vacuoles (spongy degeneration, Fig. 17.15). D. Creutzfeldt-Jakob disease (CJD) is the most common spongiform encephalopathy. 1. Usually sporadic; rarely can arise due to exposure to prion-infected human tissue (e.g., human growth hormone or corneal transplant) 2.

1	1. Usually sporadic; rarely can arise due to exposure to prion-infected human tissue (e.g., human growth hormone or corneal transplant) 2. Presents as rapidly progressive dementia associated with ataxia (cerebellar involvement) and startle myoclonus i. Periodic sharp waves are seen on EEG. ii. Results in death, usually in < 1 year 3. Variant CJD is a special form of disease that is related to exposure to bovine spongiform encephalopathy ('mad cow'). E. Familial fatal insomnia is an inherited form of prion disease characterized by severe insomnia and an exaggerated startle response. I. BASIC PRINCIPLES A. Can be metastatic (50%) or primary (50%) B. Metastatic tumors characteristically present as multiple, well-circumscribed lesions at the gray-white junction. 1. Lung, breast, and kidney are common sources. C. Primary tumors are classified according to cell type of origin (e.g., astrocytes, meningothelial cells, ependymal cells, oligodendrocytes, or neuroectoderm).

1	C. Primary tumors are classified according to cell type of origin (e.g., astrocytes, meningothelial cells, ependymal cells, oligodendrocytes, or neuroectoderm). D. In adults, primary tumors are usually supratentorial. 1. Most common tumors in adults are glioblastoma multiforme, meningioma, and schwannoma. E. In children, primary tumors are usually infratentorial. Fig. 17.14 Huntington disease. A, Degeneration of caudate nucleus. B, Normal caudate nucleus for Fig. 17.15 Spongiform encephalopath y. (Courtesy comparison. (Courtesy of Peter Pytel, MD) of Peter Pytel, MD) 1. Most common tumors in children are pilocytic astrocytoma, ependymoma, and medulloblastoma. F. Primary malignant CNS tumors are locally destructive, but rarely metastasize. II. GLIOBLASTOMA MULTIFORME (GBM) A. Malignant, high-grade tumor of astrocytes B. Most common primary malignant CNS tumor in adults

1	II. GLIOBLASTOMA MULTIFORME (GBM) A. Malignant, high-grade tumor of astrocytes B. Most common primary malignant CNS tumor in adults C. Usually arises in the cerebral hemisphere; characteristically crosses the corpus callosum ('butterfly' lesion, Fig. 17.16A) D. Characterized by regions of necrosis surrounded by tumor cells (pseudopalisading, Fig. 17.16B) and endothelial cell proliferation; tumor cells are GFAP positive. E. Poor prognosis III. MENINGIOMA A. Benign tumor of arachnoid cells B. Most common benign CNS tumor in adults 1. More commonly seen in women; rare in children C. May present as seizures; tumor compresses, but does not invade, the cortex. D. Imaging reveals a round mass attached to the dura. E. Histology shows a whorled pattern (Fig. 17.17); psammoma bodies may be present. IV. A. Benign tumor of Schwann cells

1	D. Imaging reveals a round mass attached to the dura. E. Histology shows a whorled pattern (Fig. 17.17); psammoma bodies may be present. IV. A. Benign tumor of Schwann cells B. Involves cranial or spinal nerves; within the cranium, most frequently involves cranial nerve VIII at the cerebellopontine angle (presents as loss of hearing and tinnitus) C. Tumor cells are S-100+. D. Bilateral tumors are seen in neurofibromatosis type 2. V. A. Malignant tumor of oligodendrocytes B. Imaging reveals a calcified tumor in the white matter, usually involving the frontal lobe; may present with seizures C. 'Fried-egg' appearance of cells on biopsy (Fig. 17.18) VI. PILOCYTIC ASTROCYTOMA A. Benign tumor of astrocytes B. Most common CNS tumor in children; usually arises in the cerebellum C. Imaging reveals a cystic lesion with a mural nodule (Fig. 17.19A). Fig. 17.16 Glioblastoma multiforme. A, 'Butterfly' lesion. B, Pseudopalisading. (Courtesy of Peter Pytel, MD)

1	C. Imaging reveals a cystic lesion with a mural nodule (Fig. 17.19A). Fig. 17.16 Glioblastoma multiforme. A, 'Butterfly' lesion. B, Pseudopalisading. (Courtesy of Peter Pytel, MD) D. Biopsy shows Rosenthal fibers (thick eosinophilic processes of astrocytes, Fig. 17.19B) and eosinophilic granular bodies; tumor cells are GFAP positive. VII. MEDULLOBLASTOMA A. Malignant tumor derived from the granular cells of the cerebellum (neuroectoderm) B. Usually arises in children C. Histology reveals small, round blue cells; Homer-Wright rosettes may be present. D. Poor prognosis; tumor grows rapidly and spreads via CSF. 1. Metastasis to the cauda equina is termed 'drop metastasis.' VIII. EPENDYMOMA A. Malignant tumor of ependymal cells; usually seen in children B. Most commonly arises in the 4th ventricle; may present with hydrocephalus C. Perivascular pseudorosettes are a characteristic finding on biopsy (Fig. 17.20). IX. CRANIOPHARYNGIOMA

1	B. Most commonly arises in the 4th ventricle; may present with hydrocephalus C. Perivascular pseudorosettes are a characteristic finding on biopsy (Fig. 17.20). IX. CRANIOPHARYNGIOMA A. Tumor that arises from epithelial remnants ofRathke's pouch B. Presents as a supratentorial mass in a child or young adult; may compress the optic chiasm leading to bitemporal hemianopsia C. Calcifications are commonly seen on imaging (derived from "tooth-like" tissue). D. Benign, but tends to recur after resection Fig. 17.17 Meningioma. Fig. 17.18 Oligodendroglioma. (Courtesy of Peter Pytel, MD) .: ~i'~'. , f, . ,. l Fig. 17.19 Pi locytic astrocytoma. A, Cystic lesion with mural nodule. B, Rosentha l fibers. (A, Courtesy Fig. 17.20 Ependymoma. of Peter Pytel, MD) I. ACHONDROPLASIA A. Impaired cartilage proliferation in the growth plate; common cause of dwarfism B. Due to an activating mutation in fibroblast growth factor receptor 3 (FGFR3); autosomal dominant 1.

1	A. Impaired cartilage proliferation in the growth plate; common cause of dwarfism B. Due to an activating mutation in fibroblast growth factor receptor 3 (FGFR3); autosomal dominant 1. Overexpression of FGFR3 inhibits growth. 2. Most mutations are sporadic and related to increased paternal age. C. Clinical features 1. Short extremities with normal-sized head and chest-due to poor endochondral bone formation; intramembranous bone formation is not affected. i. Endochondral bone formation is characterized by formation of a cartilage matrix, which is then replaced by bone; it is the mechanism by which long bones grow. ii. Intramembranous bone formation is characterized by formation of bone without a preexisting cartilage matrix; it is the mechanism by which flat bones (e.g., skull and rib cage) develop. D. Mental function, life span, and fertility are not affected. II. OSTEOGENESIS IMPERFECTA A. Congenital defect of bone formation resulting in structurally weak bone

1	D. Mental function, life span, and fertility are not affected. II. OSTEOGENESIS IMPERFECTA A. Congenital defect of bone formation resulting in structurally weak bone B. Most commonly due to an autosomal dominant defect in collagen type I synthesis C. Clinical features 1. Multiple fractures of bone (can mimic child abuse, but bruising is absent) 2. Blue sclera-Thinning ofscleral collagen reveals underlying choroidal veins. 3. Hearing loss-Bones of the middle ear easily fracture. III. OSTEOPETROSIS A. Inherited defect of bone resorption resulting in abnormally thick, heavy bone that fractures easily B. Due to poor osteoclast function C. Multiple genetic variants exist; carbonic anhydrase II mutation leads to loss of the acidic microenvironment required for bone resorption. D. Clinical features include 1. 2. Anemia, thrombocytopenia, and leukopenia with extramedullary hematopoiesis-due to bony replacement of the marrow (myelophthisic process, Fig. 18.1) 3.

1	D. Clinical features include 1. 2. Anemia, thrombocytopenia, and leukopenia with extramedullary hematopoiesis-due to bony replacement of the marrow (myelophthisic process, Fig. 18.1) 3. Vision and hearing impairment-due to impingement on cranial nerves 4. Hydrocephalus-due to narrowing of the fora men magnum 5. Renal tubular acidosis-seen with carbonic anhydrase II mutation i. Lack ofcarbonic anhydrase results in decreased tubular reabsorption of HCO -, leading to metabolic acidosis. E. Treatment is bone marrow transplant; osteoclasts are derived from monocytes. pathoma.com IV. A. Defective mineralization of osteoid 1. Osteoblasts normally produce osteoid, which is then mineralized with calcium and phosphate to form bone. B. Due to low levels ofvitamin D, which results in low serum calcium and phosphate 1. Vitamin Dis normally derived from the skin upon exposure to sunlight (85%) and from the diet (15%). 2.

1	B. Due to low levels ofvitamin D, which results in low serum calcium and phosphate 1. Vitamin Dis normally derived from the skin upon exposure to sunlight (85%) and from the diet (15%). 2. Activation requires 25 -hydroxylation by the liver followed by 1-alphahydroxylation by the proximal tubule cells of the kidney. 3. i. Intestine-increases absorption of calcium and phosphate ii. Kidney-increases reabsorption of calcium and phosphate iii. Bone-increases resorption of calcium and phosphate 4. Vitamin D deficiency is seen with decreased sun exposure (e.g., northern latitudes), poor diet, malabsorption, liver failure, and renal failure. C. Rickets is due to low vitamin D in children, resulting in abnormal bone mineralization.

1	C. Rickets is due to low vitamin D in children, resulting in abnormal bone mineralization. 1. Most commonly arises in children < 1 year of age; presents with i. Pigeon-breast deformity-inward bending of the ribs with anterior protrusion of the sternum ii. Frontal bossing (enlarged forehead) -due to osteoid deposition on the skull iii. Rachi tic rosary-due to osteoid deposition at the costochondral junction iv. Bowing of the legs may be seen in ambulating children. D. Osteomalacia is due to low vitamin D in adults. 1. Inadequate mineralization results in weak bone with an increased risk for fracture. 2. Laboratory findings include --1.-serum calcium, --1.-serum phosphate, t PTH, and t alkaline phosphatase. V. A. Reduction in trabecular bone mass B. Results in porous bone with an increased risk for fracture C. Risk of osteoporosis is based on peak bone mass (attained in early adulthood) and rate of bone loss that follows thereafter. 1.

1	B. Results in porous bone with an increased risk for fracture C. Risk of osteoporosis is based on peak bone mass (attained in early adulthood) and rate of bone loss that follows thereafter. 1. Peak bone mass is achieved by 30 years of age and is based on (1) genetics (e.g., vitamin D receptor variants), (2) diet, and (3) exercise. 2. Thereafter, slightly less than 1% of bone mass is lost each year; bone mass is lost more quickly with lack of weight-bearing exercise (e.g., space travel), poor diet, or decreased estrogen (e.g., menopause). D. Most common forms of osteoporosis are senile and postmenopausal. Fig. 18.1 Osteopetrosis. (Published with Fig. 18.2 Paget disease of bone. permission from LearningRadiology.com) E. Clinical features 1. Bone pain and fractures in weight-bearing areas such as the vertebrae (leads to loss of height and kyphosis), hip, and distal radius 2. Bone density is measured using a DEXA scan. 3.

1	Bone pain and fractures in weight-bearing areas such as the vertebrae (leads to loss of height and kyphosis), hip, and distal radius 2. Bone density is measured using a DEXA scan. 3. Serum calcium, phosphate, PTH, and alkaline phosphatase are normal; labs help to exclude osteomalacia (which has a similar clinical presentation). F. Treatment includes 1. Exercise, vitamin D, and calcium-limit bone loss 2. Bisphosphonates-induce apoptosis of osteoclasts 3. Estrogen replacement therapy is debated (currently not recommended). 4. Glucocorticoids are contraindicated (worsen osteoporosis). VI. PAGET DISEASE OF BONE A. Imbalance between osteoclast and osteoblast function 1. Usually seen in late adulthood (average age > 60 years) B. Etiology is unknown; possibly viral C. Localized process involving one or more bones; does not involve the entire skeleton D. Three distinct stages are (1) osteoclastic, (2) mixed osteoblastic-osteoclastic, and (3) osteoblastic. 1.

1	C. Localized process involving one or more bones; does not involve the entire skeleton D. Three distinct stages are (1) osteoclastic, (2) mixed osteoblastic-osteoclastic, and (3) osteoblastic. 1. End result is thick, sclerotic bone that fractures easily. 2. Biopsy reveals a mosaic pattern of lamellar bone (Fig. 18.2). E. Clinical features 1. Bone pain-due to microfractures 2. Increasing hat size-Skull is commonly affected. 3. 4. Lion-like facies-involvement of craniofacial bones 5. Isolated elevated alkaline phosphatase-most common cause of isolated elevated alkaline phosphatase in patients > 40 years old F. Treatment includes 1. 2. Bisphosphonates-induces apoptosis of osteoclasts G. Complications include 1. High-output cardiac failure-due to formation of AV shunts in bone 2. VII. OSTEOMYELITIS A. Infection of marrow and bone 1. Usually occurs in children B. Most commonly bacterial; arises via hematogenous spread 1.

1	VII. OSTEOMYELITIS A. Infection of marrow and bone 1. Usually occurs in children B. Most commonly bacterial; arises via hematogenous spread 1. Transient bacteremia (children) seeds metaphysis. 2. Open-wound bacteremia (adults) seeds epiphysis. C. Causes include 1. Staphylococcus aureus-most common cause (90% of cases) 2. 3. 4. 5. Pasteurella-associated with cat or dog bite/scratches 6. D. Clinical features 1. Bone pain with systemic signs of infection (e.g., fever and leukocytosis) 2. Lytic focus (abscess) surrounded by sclerosis of bone on x-ray; lytic focus is called sequestrum, and sclerosis is called involucrum. E. Diagnosis is made by blood culture. _ _____________F_U_N_D_AMENTALS OF PATHOLOGY________. VIII. AVASCULAR (ASEPTIC) NECROSIS A. Ischemic necrosis of bone and bone marrow B. Causes include trauma or fracture (most common), steroids, sickle cell anemia, and caisson disease. C. Osteoarthritis and fracture are major complications. I. OSTEOMA

1	B. Causes include trauma or fracture (most common), steroids, sickle cell anemia, and caisson disease. C. Osteoarthritis and fracture are major complications. I. OSTEOMA A. Benign tumor of bone B. Most commonly arises on the surface of facial bones C. Associated with Gardner syndrome II. OSTEOID OSTEOMA A. Benign tumor of osteoblasts (that produce osteoid) surrounded by a rim of reactive bone B. Occurs in young adults < 25 years ofage (more common in males) C. Arises in cortex of long bones (e.g., femur) D. Presents as bone pain that resolves with aspirin E. Imaging reveals a bony mass(< 2 cm) with a radiolucent core (osteoid). F. Osteoblastoma is similar to osteoid osteoma but is larger(> 2 cm), arises in vertebrae, and presents as bone pain that does not respond to aspirin. III. OSTEOCHONDROMA A. Tumor of bone with an overlying cartilage cap (Fig. 18.3); most common benign tumor ofbone

1	III. OSTEOCHONDROMA A. Tumor of bone with an overlying cartilage cap (Fig. 18.3); most common benign tumor ofbone B. Arises from a lateral projection of the growth plate (metaphysis); bone is continuous with the marrow space. C. Overlying cartilage can transform (rarely) to chondrosarcoma. IV. OSTEOSARCOMA A. Malignant proliferation of osteoblasts B. Peak incidence is seen in teenagers; less commonly seen in the elderly 1. Risk factors include familial retinoblastoma, Paget disease, and radiation exposure. 2. Arises in the metaphysis of long bones, usually the distal femur or proximal tibia (region of the knee) C. Presents as a pathologic fracture or bone pain with swelling D. Imaging reveals a destructive mass with a 'sunburst' appearance and lifting of the periosteum (Codman triangle, Fig. 18.4A). Fig. 18.3 Osteochondroma. (Courtesy of Fig. 18.4 Osteosarcoma. A, X-ray. B, Microscopic appearance. (A, Courtesy of Bu lent Celasun, MD) humpath.com)

1	Fig. 18.3 Osteochondroma. (Courtesy of Fig. 18.4 Osteosarcoma. A, X-ray. B, Microscopic appearance. (A, Courtesy of Bu lent Celasun, MD) humpath.com) E. Biopsy reveals pleomorphic cells that produce osteoid (Fig. 18.4B). V. GIANT CELL TUMOR A. Tumor comprised of multinucleated giant cells and stromal cells B. Occurs in young adults C. Arises in the epiphysis of long bones, usually the distal femur or proximal tibia (region of the knee) D. 'Soap-bubble' appearance on x-ray E. Locally aggressive tumor; may recur VI. EWING SARCOMA A. Malignant proliferation of poorly-differentiated cells derived from neuroectoderm B. Arises in the diaphysis oflong bones; usually in male children(< 15 years ofage) C. 'Onion-skin' appearance on x-ray D. Biopsy reveals small, round blue cells that resemble lymphocytes (Fig. 18.5). 1. Can be confused with lymphoma or chronic osteomyelitis 2. (11;22) translocation is characteristic.

1	D. Biopsy reveals small, round blue cells that resemble lymphocytes (Fig. 18.5). 1. Can be confused with lymphoma or chronic osteomyelitis 2. (11;22) translocation is characteristic. E. Often presents with metastasis; responsive to chemotherapy Vil. CHONDROMA A. Benign tumor of cartilage B. Usually arises in the medulla of small bones of the hands and feet (Fig. 18.6) VIII. CHONDROSARCOMA A. Malignant cartilage-forming tumor B. Arises in medulla of the pelvis or central skeleton IX. METASTATIC TUMORS A. More common than primary tumors B. Usually result in osteolytic (punched-out) lesions 1. Prostatic carcinoma classically produces osteoblastic lesions. I. BASIC PRINCIPLES A. Connection between two bones B. Solid joints are tightly connected to provide structural strength (e.g., cranial sutures). C. Synovial joints have a joint space to allow for motion.

1	A. Connection between two bones B. Solid joints are tightly connected to provide structural strength (e.g., cranial sutures). C. Synovial joints have a joint space to allow for motion. 1. Articular surface of adjoining bones is made ofhyaline cartilage (type II collagen) that is surrounded by a joint capsule. Fig. 18.S Ewing sarcoma. Fig. 18.6 Chondroma. (Published with permi ssion from LearningRadiology.com) 2. Synovium lining the joint capsule secretes fluid rich in hyaluronic acid to lubricate the joint and facilitate smooth motion. II. DEGENERATIVE JOINT DISEASE (OSTEOARTHRITIS) A. Progressive degeneration of articular cartilage; most common type of arthritis B. Most often due to 'wear and tear' C. Major risk factor is age (common after 60 years); additional risk factors include obesity and trauma.

1	B. Most often due to 'wear and tear' C. Major risk factor is age (common after 60 years); additional risk factors include obesity and trauma. D. Affects a limited number of joints (oligoarticular); hips, lower lumbar spine, knees, and the distal interphalangeal joints (DIP) and proximal interphalangeal joints (PIP) of fingers are common sites. E. Classic presentation is joint stiffness in the morning that worsens during the day. F. Pathologic features include 1. Disruption of the cartilage that lines the articular surface (Fig. 18.7); fragments of cartilage floating in the joint space are called 'joint mice.' 2. Eburnation of the subchondral bone 3. Osteophyte formation (reactive bony outgrowths); classically arises in the DIP (Heberden nodes) and PIP (Bouchard nodes) joints of the fingers III. RHEUMATOID ARTHRITIS A. Chronic, systemic autoimmune disease 1. Classically arises in women of late childbearing age 2. Associated with HLA-DR4

1	III. RHEUMATOID ARTHRITIS A. Chronic, systemic autoimmune disease 1. Classically arises in women of late childbearing age 2. Associated with HLA-DR4 B. Characterized by involvement ofjoints 1. Hallmark is synovitis leading to formation of a pannus (inflamed granulation tissue). 2. Leads to destruction of cartilage and ankylosis (fusion) of the joint C. Clinical features 1. Arthritis with morning stiffness that improves with activity. i. Symmetric involvement of PIP joints of the fingers (swan-neck deformity), wrists (radial deviation), elbows, ankles, and knees is characteristic (Fig. 18.8); DIP is usually spared (unlike osteoarthritis). ii. Joint-space narrowing, loss of cartilage, and osteopenia are seen on x-ray. 2. Fever, malaise, weight loss, and myalgias 3. Rheumatoid nodules-central zone of necrosis surrounded by epithelioid histiocytes; arise in skin and visceral organs 4. Vasculitis-Multiple organs may be involved. 5.

1	Rheumatoid nodules-central zone of necrosis surrounded by epithelioid histiocytes; arise in skin and visceral organs 4. Vasculitis-Multiple organs may be involved. 5. Baker cyst-swelling of bursa behind the knee 6. Pleural effusions, lymphadenopathy, and interstitial lung fibrosis Fig. 18.7 Degenerative joint disease. Fig. 18.8 Rheumatoid arthritis. (Courtesy of James Heilman, MD, Wikipedia) D. Laboratory findings 1. IgM autoantibody against Fe portion ofigG (rheumatoid factor); marker of tissue damage and disease activity 2. E. Complications include anemia of chronic disease and secondary amyloidosis. IV. A. Group ofjoint disorders characterized by 1. Lack of rheumatoid factor 2. Axial skeleton involvement 3. HLA-B27 association B. Ankylosing spondyloarthritis involves the sacroiliac joints and spine. 1. Arises in young adults, most often male 2.

1	Axial skeleton involvement 3. HLA-B27 association B. Ankylosing spondyloarthritis involves the sacroiliac joints and spine. 1. Arises in young adults, most often male 2. Presents with low back pain; involvement ofvertebral bodies eventually arises, leading to fusion of the vertebrae ('bamboo spine'). 3. Extra-articular manifestations include uveitis and aortitis (leading to aortic regurgitation). C. Reactive arthritis is characterized by the triad of arthritis, urethritis, and conjunctivitis. 1. Arises in young adults (usually males) weeks after a GI or Chlamydia trachomatis infection D. Psoriatic arthritis is seen in 10% of cases of psoriasis. 1. Involves axial and peripheral joints; DIP joints of the hands and feet are most commonly affected, leading to "sausage" fingers or toes. V. A. Arthritis due to an infectious agent, usually bacterial B. Causes include 1. N gonorrhoeae-young adults; most common cause 2. S aureus-older children and adults; 2nd most common cause

1	V. A. Arthritis due to an infectious agent, usually bacterial B. Causes include 1. N gonorrhoeae-young adults; most common cause 2. S aureus-older children and adults; 2nd most common cause C. Classically involves a single joint, usually the knee D. Presents as a warm joint with limited range of motion; fever, increased white count, and elevated ESR are often present. VI. GOUT A. Deposition of monosodium urate (MSU) crystals in tissues, especially the joints B. Due to hyperuricemia; related to overproduction or decreased excretion of uric acid 1. Uric acid is derived from purine metabolism and is excreted by the kidney. C. Primary gout is the most common form; etiology ofhyperuricemia is unknown. D. Secondary gout is seen with 1. Leukemia and myeloproliferative disorders-Increased cell turnover leads to hyperuricemia. 2. Lesch-Nyhan syndrome-X-linked deficiency ofhypoxanthine-guanine phosphoribosyltransferase (HGPRT); presents with mental retardation and selfmutilation 3.

1	2. Lesch-Nyhan syndrome-X-linked deficiency ofhypoxanthine-guanine phosphoribosyltransferase (HGPRT); presents with mental retardation and selfmutilation 3. Renal insufficiency-decreased renal excretion of uric acid E. Acute gout presents as exquisitely painful arthritis of the great toe (podagra) 1. MSU crystals deposit in the joint, triggering an acute inflammatory reaction. 2. Alcohol or consumption of meat may precipitate arthritis. F. Chronic gout leads to 1. Development of tophi-white, chalky aggregates of uric acid crystals with fibrosis and giant cell reaction in the soft tissue and joints (Fig. 18.9A) 2. Renal failure-Urate crystals may deposit in kidney tubules (urate nephropathy). G. Laboratory findings include hyperuricemia; synovial fluid shows needle-shaped crystals with negative birefringence under polarized light (Fig. 18.9B).

1	G. Laboratory findings include hyperuricemia; synovial fluid shows needle-shaped crystals with negative birefringence under polarized light (Fig. 18.9B). H. Pseudogout resembles gout clinically, but is due to deposition of calcium pyrophosphate dihydrate (CPPD); synovial fluid shows rhomboid-shaped crystals with weakly positive birefringence under polarized light. I. DERMATOMYOSITIS A. Inflammatory disorder of the skin and skeletal muscle B. Unknown etiology; some cases are associated with carcinoma (e.g., gastric carcinoma). C. Clinical features 1. Bilateral proximal muscle weakness; distal involvement can develop late in disease. 2. Rash of the upper eyelids (heliotrope rash); malar rash may also be seen. 3. Red papules on the elbows, knuckles, and knees (Gottron papules) D. Laboratory findings 1. 2. 3. Perimysial inflammation (CD4+ T cells) with perifascicular atrophy on biopsy (Fig. 18.10) E. Treatment is corticosteroids. II. POLYMYOSITIS

1	D. Laboratory findings 1. 2. 3. Perimysial inflammation (CD4+ T cells) with perifascicular atrophy on biopsy (Fig. 18.10) E. Treatment is corticosteroids. II. POLYMYOSITIS A. Inflammatory disorder of skeletal muscle B. Resembles dermatomyositis clinically, but skin is not involved; endomysial inflammation (CDS+ T cells) with necrotic muscle fibers is seen on biopsy. III. X-LINKED MUSCULAR DYSTROPHY A. Degenerative disorder characterized by muscle wasting and replacement of skeletal muscle by adipose tissue B. Due to defects of dystrophin gene 1. Dystrophin is important for anchoring the muscle cytoskeleton to the extracellular matrix. 2. Mutations are often spontaneous; large gene size predisposes to high rate of mutation. C. Duchenne muscular dystrophy is due to deletion of dystrophin. 1. Presents as proximal muscle weakness at 1 year of age; progresses to involve distal muscles i. Calf pseudohypertrophy is a characteristic finding.

1	1. Presents as proximal muscle weakness at 1 year of age; progresses to involve distal muscles i. Calf pseudohypertrophy is a characteristic finding. ii. Serum creatine kinase is elevated. 2. Death results from cardiac or respiratory failure; myocardium is commonly involved. D. Becker muscular dystrophy is due to mutated dystrophin; clinically results in milder disease I. MYASTHENIA GRAVIS A. Autoantibodies against the postsynaptic acetylcholine receptor at the neuromuscular junction B. More commonly seen in women C. Clinical features 1. Muscle weakness that worsens with use and improves with rest; classically involves the eyes, leading to ptosis and diplopia 2. Symptoms improve with anticholinesterase agents. 3. Associated with thymic hyperplasia or thymoma; thymectomy improves symptoms. II. LAMBERT-EATON SYNDROME A. Antibodies against presynaptic calcium channels of the neuromuscular junction

1	3. Associated with thymic hyperplasia or thymoma; thymectomy improves symptoms. II. LAMBERT-EATON SYNDROME A. Antibodies against presynaptic calcium channels of the neuromuscular junction B. Arises as a paraneoplastic syndrome, most commonly due to small cell carcinoma of the lung C. Leads to impaired acetylcholine release 1. Firing of presynaptic calcium channels is required for acetylcholine release. D. Clinical features 1. Proximal muscle weakness that improves with use; eyes are usually spared. 2. Anticholinesterase agents do not improve symptoms. 3. Resolves with resection of the cancer I. LIPOMA A. Benign tumor of adipose tissue B. Most common benign soft tissue tumor in adults II. LIPOSARCOMA A. Malignant tumor of adipose tissue B. Most common malignant soft tissue tumor in adults C. Lipoblast is the characteristic cell. III. RHABDOMYOMA A. Benign tumor of skeletal muscle B. Cardiac rhabdomyoma is associated with tuberous sclerosis. IV. RHABDOMYOSARCOMA

1	C. Lipoblast is the characteristic cell. III. RHABDOMYOMA A. Benign tumor of skeletal muscle B. Cardiac rhabdomyoma is associated with tuberous sclerosis. IV. RHABDOMYOSARCOMA A. Malignant tumor of skeletal muscle B. Most common malignant soft tissue tumor in children C. Rhabdomyoblast is the characteristic cell; desmin positive D. Most common site is the head and neck; vagina is the classic site in young girls. Fig. 18.9 Gout. A, Tophi. B, Negative birefringence. (B, Courtesy of Ed Uthman, MD) Fig. 18.10 Dermatomyositis. (Courtesy of Peter Pytel, MD) I. SKIN A. Functions as a barrier against environmental insults and fluid loss B. Composed of an epidermis and dermis C. Epidermis is comprised of keratinocytes and has four layers (Fig. 19.1). 1. 2. 3. 4. D. Dermis consists of connective tissue, nerve endings, blood and lymphatic vessels, and adnexal structures (e.g., hair shafts, sweat glands, and sebaceous glands). I. ATOPIC (ECZEMATOUS) DERMATITIS

1	I. ATOPIC (ECZEMATOUS) DERMATITIS A. Pruritic, erythematous, oozing rash with vesicles and edema; often involves the face and flexor surfaces B. Type I hypersensitivity reaction; associated with asthma and allergic rhinitis II. CONTACT DERMATITIS A. Pruritic, erythematous, oozing rash with vesicles and edema B. Arises upon exposure to allergens such as 1. 2. Irritant chemicals (e.g., detergents) 3. Drugs (e.g., penicillin) C. Treatment involves removal of the offending agent and topical glucocorticoids, if needed. III. ACNE VULGARIS A. Comedones (whiteheads and blackheads), pustules (pimples), and nodules; extremely common, especially in adolescents B. Due to chronic inflammation of hair follicles and associated sebaceous glands 1. Hormone-associated increase in sebum production (sebaceous glands have androgen receptors) and excess keratin production block follicles, forming comedones. 2.

1	Hormone-associated increase in sebum production (sebaceous glands have androgen receptors) and excess keratin production block follicles, forming comedones. 2. Propionibacterium acnes infection produces lipases that break down sebum, releasing proinflammatory fatty acids; results in pustule or nodule formation C. Treatment includes benzoyl peroxide (antimicrobial) and vitamin A derivatives (e.g., isotretinoin), which reduce keratin production. IV. PSORIASIS A. Well-circumscribed, salmon-colored plaques with silvery scale, usually on extensor surfaces and the scalp (Fig. 19.2A); pitting of nails may also be present. B. Due to excessive keratinocyte proliferation pat homa.com C. Possible autoimmune etiology 1. Associated with HLA-C 2. Lesions often arise in areas of trauma (environmental trigger). D. Histology (Fig. 19.2B) shows 1. 2. Parakeratosis (hyperkeratosis with retention ofkeratinocyte nuclei in the stratum corneum) 3.

1	Lesions often arise in areas of trauma (environmental trigger). D. Histology (Fig. 19.2B) shows 1. 2. Parakeratosis (hyperkeratosis with retention ofkeratinocyte nuclei in the stratum corneum) 3. Collections of neutrophils in the stratum corneum (Munro microabscesses) 4. Thinning of the epidermis above elongated dermal papillae; results in bleeding when scale is picked off (Auspitz sign) E. Treatment involves corticosteroids, UV light with psoralen, or immune-modulating therapy. V. LICHEN PLANUS A. Pruritic, planar, polygonal, purple papules (Fig. 19.3A), often with reticular white lines on their surface (Wickham striae); commonly involves wrists, elbows, and oral mucosa 1. Oral involvement manifests as Wickham striae. B. Histology shows inflammation of the dermal-epidermal junction with a 'saw-tooth' appearance (Fig. 19.3B). C. Etiology is unknown; associated with chronic hepatitis C virus infection I. PEMPHIGUS VULGARIS

1	C. Etiology is unknown; associated with chronic hepatitis C virus infection I. PEMPHIGUS VULGARIS A. Autoimmune destruction of desmosomes between keratinocytes Fig. 19.1 Skin histology, normal. Fig. 19.2 Psoriasis. A, Clinical appearance. B, Microscopic appearance. (A, Courtesy ofVesna PetronicRosic, MD) Fig. 19.3 Lichen planus. A, Clinical appearance. B, Microscopic appearance. (A, Courtesy ofVesna Petronic-Rosic, MD) B. Due to IgG antibody against desmoglein (type II hypersensitivity) C. Presents as skin and oral mucosa bullae (Fig. 19.4A). 1. Acantholysis (separation) of stratum spinosum keratinocytes (normally connected by desmosomes) results in suprabasal blisters. 2. Basal layer cells remain attached to basement membrane via hemidesmosomes ('tombstone' appearance, Fig. 19.4B). 3. Thin-walled bullae rupture easily (Nikolsky sign), leading to shallow erosions with dried crust. 4. Immunofluorescence highlights IgG surrounding keratinocytes in a 'fish net' pattern.

1	3. Thin-walled bullae rupture easily (Nikolsky sign), leading to shallow erosions with dried crust. 4. Immunofluorescence highlights IgG surrounding keratinocytes in a 'fish net' pattern. II. BULLOUS PEMPHIGOID A. Autoimmune destruction ofhemidesmosomes between basal cells and the underlying basement membrane B. Due to IgG antibody against hemidesmosome components (BP180) of the basement membrane C. Presents as blisters of the skin (Fig. 19.SA), usually in the elderly; oral mucosa is spared. 1. Basal cell layer is detached from the basement membrane (Fig. 19.SB). 2. D. Immunofluorescence highlights IgG along basement membrane (linear pattern). III. DERMATITIS HERPETIFORMIS A. Autoimmune deposition of IgA at the tips of dermal papillae B. Presents as pruritic vesicles and bullae that are grouped (herpetiform, Fig. 19.6) C. Strong association with celiac disease; resolves with gluten-free diet

1	B. Presents as pruritic vesicles and bullae that are grouped (herpetiform, Fig. 19.6) C. Strong association with celiac disease; resolves with gluten-free diet Fig. 19.4 Pemphigus vulgaris. A, Clinical appearance. B, Microscopic appearance. (A, Courtesy of Vesna Petronic-Rosic, MD) Fig. 19.5 Bullous pemphigoid. A, Clinical appearance. B, Microscopic appearance. (A, Courtesy of Vesna Petronic-Rosic, MD) IV. ERYTHEMA MULTIFORME (EM) A. Hypersensitivity reaction characterized by targetoid rash and bullae (Fig. 19.7) 1. Targetoid appearance is due to central epidermal necrosis surrounded by erythema. B. Most commonly associated with HSV infection; other associations include Mycoplasma infection, drugs (penicillin and sulfonamides), autoimmune disease (e.g., SLE), and malignancy. C. EM with oral mucosa/lip involvement and fever is termed Stevens-Johnson syndrome (SJS).

1	C. EM with oral mucosa/lip involvement and fever is termed Stevens-Johnson syndrome (SJS). 1. Toxic epidermal necrolysis is a severe form of SJS characterized by diffuse sloughing of skin, resembling a large burn; most often due to an adverse drug reaction I. SEBORRHEIC KERATOSIS A. Benign squamous proliferation; common tumor in the elderly B. Presents as raised, discolored plaques on the extremities or face; often has a coinlike, waxy, 'stuck-on' appearance (Fig. 19.8A) 1. Characterized by keratin pseudocysts on histology (Fig. 19.8B) C. Leser-Trelat sign is the sudden onset of multiple seborrheic keratoses and suggests underlying carcinoma of the GI tract (Fig. 19.SC). II. ACANTHOSIS NIGRICANS A. Epidermal hyperplasia with darkening of the skin ('velvet-like' skin, Fig. 19.9); often involves the axilla or groin Fig. 19.6 Dermatitis herpetiformis. (Courtesy of Fig. 19.7 Erythema multiforme. (Courtesy of Vesna Petronic-Rosic, MD) James Heilman, MD, Wikipedia)

1	Fig. 19.6 Dermatitis herpetiformis. (Courtesy of Fig. 19.7 Erythema multiforme. (Courtesy of Vesna Petronic-Rosic, MD) James Heilman, MD, Wikipedia) Fig. 19.8 Seborrheic keratosis. A, Clinical appearance. B, Microscopic appearance. C, Sign of Leser-Trelat. (A and C, Courtesy of Vesna Petronic-Rosic, MD) B. Associated with insulin resistance (e.g., non-insulin-dependent diabetes) or malignancy (especially gastric carcinoma) III. BASAL CELL CARCINOMA A. Malignant proliferation of the basal cells of the epidermis 1. Most common cutaneous malignancy B. Risk factors stem from DVB-induced DNA damage and include prolonged exposure to sunlight, albinism, and xeroderma pigmentosum. C. Presents as an elevated nodule with a central, ulcerated crater surrounded by dilated (telangiectatic) vessels (Fig. 19.lOA); 'pink, pearl-like papule' 1. Classic location is the upper lip. D. Histology shows nodules of basal cells with peripheral palisading (Fig. 19.lOB).

1	D. Histology shows nodules of basal cells with peripheral palisading (Fig. 19.lOB). E. Treatment is surgical excision; metastasis is rare. IV. SQUAMOUS CELL CARCINOMA A. Malignant proliferation of squamous cells characterized by formation of keratin pearls. B. Risk factors stem from DVB-induced DNA damage and include prolonged exposure to sunlight, albinism, and xeroderma pigmentosum. 1. Additional risk factors include immunosuppressive therapy, arsenic exposure, and chronic inflammation (e.g., scar from burn or draining sinus tract). C. Presents as an ulcerated, nodular mass, usually on the face (classically involving the lower lip) D. Treatment is excision; metastasis is uncommon. E. Actinic keratosis is a precursor lesion of squamous cell carcinoma and presents as a hyperkeratotic, scaly plaque, often on the face, back, or neck.

1	E. Actinic keratosis is a precursor lesion of squamous cell carcinoma and presents as a hyperkeratotic, scaly plaque, often on the face, back, or neck. F. Keratoacanthoma is well-differentiated squamous cell carcinoma that develops rapidly and regresses spontaneously; presents as a cup-shaped tumor filled with keratin debris I. BASIC PRINCIPLES A. Melanocytes are responsible for skin pigmentation and are present in the basal layer of the epidermis. 1. Derived from the neural crest 2. Synthesize melanin in melanosomes using tyrosine as a precursor molecule 3. Pass melanosomes to keratinocytes Fig. 19.9 Acanthosis nigricans. (Courtesy ofVesna Fig. 19.10 Basal cell carcinoma. A, Clinical appearance. B, Microscopic appearance. (A, Courtesy of Petronic-Rosic, MD) Vesna Petronic-Rosic, MD) II. VITILIGO A. Localized loss of skin pigmentation (Fig. 19.11) B. Due to autoimmune destruction of melanocytes III. ALBINISM A. Congenital lack of pigmentation

1	II. VITILIGO A. Localized loss of skin pigmentation (Fig. 19.11) B. Due to autoimmune destruction of melanocytes III. ALBINISM A. Congenital lack of pigmentation B. Due to an enzyme defect (usually tyrosinase) that impairs melanin production C. May involve the eyes (ocular form) or both the eyes and skin (oculocutaneous form) D. Increased risk of squamous cell carcinoma, basal cell carcinoma, and melanoma due to reduced protection against UVB IV. A. Small, tan to brown macule; darkens when exposed to sunlight B. Due to increased number of melanosomes (melanocytes are not increased) V. A. Mask-like hyperpigmentation of the cheeks B. Associated with pregnancy and oral contraceptives VI. NEVUS (MOLE) A. Benign neoplasm of melanocytes B. Congenital nevus is present at birth; often associated with hair (Fig. 19.12) C. Acquired nevus arises later in life. 1.

1	VI. NEVUS (MOLE) A. Benign neoplasm of melanocytes B. Congenital nevus is present at birth; often associated with hair (Fig. 19.12) C. Acquired nevus arises later in life. 1. Begins as nests of melanocytes at the dermal-epidermal junction (junctional nevus); most common mole in children 2. Grows by extension into the dermis (compound nevus) 3. Junctional component is eventually lost resulting in an intradermal nevus, which is the most common mole in adults. D. Characterized by a flat macule or raised papule with symmetry, sharp borders, evenly distributed color, and small diameter(< 6 mm) E. Dysplasia may arise (dysplastic nevus), which is a precursor to melanoma. VII. MELANOMA A. Malignant neoplasm of melanocytes; most common cause of death from skin cancer

1	E. Dysplasia may arise (dysplastic nevus), which is a precursor to melanoma. VII. MELANOMA A. Malignant neoplasm of melanocytes; most common cause of death from skin cancer B. Risk factors are based on UVB-induced DNA damage and include prolonged exposure to sunlight, albinism, and xeroderma pigmentosum; an additional risk factor is dysplastic nevus syndrome (autosomal dominant disorder characterized by formation of dysplastic nevi that may progress to melanoma). C. Presents as a mole-like growth with "ABCD" (Fig. 19.13) Fig, 19,11 Vitiligo. (Courtesy of James Heilman, Fig. 19.12 Congenital nevus. (Courtesy ofVesna Flg. 19,13 Melanoma. (Courtesy ofVesna MD, Wikipedia) Petronic-Rosic, MD) Petronic-Rosic, MD) 1. 2. Borders are irregular. 3. Color is not uniform. 4. D. Characterized by two growth phases 1. Radial growth horizontally along the epidermis and superficial dermis; low risk of metastasis 2.

1	2. Borders are irregular. 3. Color is not uniform. 4. D. Characterized by two growth phases 1. Radial growth horizontally along the epidermis and superficial dermis; low risk of metastasis 2. Vertical growth into the deep dermis 1. Increased risk of metastasis; depth of extension (Breslow thickness) is the most important prognostic factor in predicting metastasis. E. Variants include 1. Superficial spreading-most common subtype; dominant early radial growth results in good prognosis. 2. 3. 4. Acral lentiginous-arises on the palms or soles, often in dark-skinned individuals; not related to UV light exposure I. IMPETIGO A. Superficial bacterial skin infection, most often due to S aureus or S pyogenes B. Commonly affects children C. Presents as erythematous macules that progress to pustules, usually on the face; rupture of pustules results in erosions and dry, crusted, honey-colored serum. II. CELLULITIS

1	C. Presents as erythematous macules that progress to pustules, usually on the face; rupture of pustules results in erosions and dry, crusted, honey-colored serum. II. CELLULITIS A. Deeper (dermal and subcutaneous) infection, usually due to S aureus or Spyogenes B. Presents as a red, tender, swollen rash with fever C. Risk factors include recent surgery, trauma, or insect bite. D. Can progress to necrotizing fasciitis with necrosis ofsubcutaneous tissues due to infection with anaerobic 'flesh-eating' bacteria 1. Production of CO2 leads to crepitus. 2. III. STAPHYLOCOCCAL SCALDED SKIN SYNDROME A. Sloughing of skin with erythematous rash and fever; leads to significant skin loss B. Due to S aureus infection; exfoliative A and B toxins result in epidermolysis of the stratum granulosum. Fig. 19.14 Verruca. (Courtesy ofVesna Petronic-Fig. 19.15 Molluscum bodies. Rosie, MD)

1	B. Due to S aureus infection; exfoliative A and B toxins result in epidermolysis of the stratum granulosum. Fig. 19.14 Verruca. (Courtesy ofVesna Petronic-Fig. 19.15 Molluscum bodies. Rosie, MD) C. Distinguished histologically from toxic epidermal necrolysis by level of skin separation; separation in TEN occurs at the dermal-epidermal junction. IV. A. Flesh-colored papules with a rough surface {Fig. 19.14) B. Due to HPV infection of keratinocytes; characterized by koilocytic change C. Hands and feet are common locations. V. A. Firm, pink, umbilicated papules due to poxvirus; affected keratinocytes show cytoplasmic inclusions (molluscum bodies, Fig. 19.15) B. Most often arise in children; also occur in sexually active adults and immunocompromised individuals

1	What is anatomy? Anatomy includes those structures that can be seen grossly (without the aid of magnification) and microscopically (with the aid of magnification). Typically, when used by itself, the term anatomy tends to mean gross or macroscopic anatomy—that is, the study of structures that can be seen without using a microscopic. Microscopic anatomy, also called histology, is the study of cells and tissues using a microscope. Anatomy forms the basis for the practice of medicine. Anatomy leads the physician toward an understanding of a patient’s disease, whether he or she is carrying out a physical examination or using the most advanced imaging techniques. Anatomy is also important for dentists, chiropractors, physical therapists, and all others involved in any aspect of patient treatment that begins with an analysis of clinical signs. The ability to interpret a clinical observation correctly is therefore the endpoint of a sound anatomical understanding.

1	Observation and visualization are the primary techniques a student should use to learn anatomy. Anatomy is much more than just memorization of lists of names. Although the language of anatomy is important, the network of information needed to visualize the position of physical structures in a patient goes far beyond simple memorization. Knowing the names of the various branches of the external carotid artery is not the same as being able to visualize the course of the lingual artery from its origin in the neck to its termination in the tongue. Similarly, understanding the organization of the soft palate, how it is related to the oral and nasal cavities, and how it moves during swallowing is very different from being able to recite the names of its individual muscles and nerves. An understanding of anatomy requires an understanding of the context in which the terminology can be remembered. How can gross anatomy be studied?

1	How can gross anatomy be studied? The term anatomy is derived from the Greek word temnein, meaning “to cut.” Clearly, therefore, the study of anatomy is linked, at its root, to dissection, although dissection of cadavers by students is now augmented, or even in some cases replaced, by viewing prosected (previously dissected) material and plastic models, or using computer teaching modules and other learning aids. Anatomy can be studied following either a regional or a systemic approach. With a regional approach, each region of the body is studied separately and all aspects of that region are studied at the same time. For example, if the thorax is to be studied, all of its structures are examined.

1	This includes the vasculature, the nerves, the bones, the muscles, and all other structures and organs located in the region of the body defined as the thorax. After studying this region, the other regions of the body (i.e., the abdomen, pelvis, lower limb, upper limb, back, head, and neck) are studied in a similar fashion. In contrast, in a systemic approach, each system of the body is studied and followed throughout the entire body. For example, a study of the cardiovascular system looks at the heart and all of the blood vessels in the body. When this is completed, the nervous system (brain, spinal cord, and all the nerves) might be examined in detail. This approach continues for the whole body until every system, including the nervous, skeletal, muscular, gastrointestinal, respiratory, lymphatic, and reproductive systems, has been studied.

1	Each of these approaches has benefits and deficiencies. The regional approach works very well if the anatomy course involves cadaver dissection but falls short when it comes to understanding the continuity of an entire system throughout the body. Similarly, the systemic approach fosters an understanding of an entire system throughout the body, but it is very difficult to coordinate this directly with a cadaver dissection or to acquire sufficient detail. The anatomical position

1	The anatomical position The anatomical position is the standard reference position of the body used to describe the location of structures (Fig. 1.1). The body is in the anatomical position when standing upright with feet together, hands by the side and face looking forward. The mouth is closed and the facial expression is neutral. The rim of bone under the eyes is in the same horizontal plane as the top of the opening to the ear, and the eyes are open and focused on something in the distance. The palms of the hands face forward with the fingers straight and together and with the pad of the thumb turned 90° to the pads of the fingers. The toes point forward. Three major groups of planes pass through the body in the anatomical position (Fig. 1.1). Coronal planes are oriented vertically and divide the body into anterior and posterior parts.

1	Three major groups of planes pass through the body in the anatomical position (Fig. 1.1). Coronal planes are oriented vertically and divide the body into anterior and posterior parts. Sagittal planes also are oriented vertically but are at right angles to the coronal planes and divide the body into right and left parts. The plane that passes through the center of the body dividing it into equal right and left halves is termed the median sagittal plane. Transverse, horizontal, or axial planes divide the body into superior and inferior parts. Terms to describe location Anterior (ventral) and posterior (dorsal), medial and lateral, superior and inferior Three major pairs of terms are used to describe the location of structures relative to the body as a whole or to other structures (Fig. 1.1).

1	Three major pairs of terms are used to describe the location of structures relative to the body as a whole or to other structures (Fig. 1.1). Anterior (or ventral) and posterior (or dorsal) describe the position of structures relative to the “front” and “back” of the body. For example, the nose is an anterior (ventral) structure, whereas the vertebral column is a posterior (dorsal) structure. Also, the nose is anterior to the ears and the vertebral column is posterior to the sternum. Medial and lateral describe the position of structures relative to the median sagittal plane and the sides of the body. For example, the thumb is lateral to the little finger. The nose is in the median sagittal plane and is medial to the eyes, which are in turn medial to the external ears. Superior and inferior describe structures in reference to the vertical axis of the body. For example, the head is superior to the shoulders and the knee joint is inferior to the hip joint.

1	Superior and inferior describe structures in reference to the vertical axis of the body. For example, the head is superior to the shoulders and the knee joint is inferior to the hip joint. Proximal and distal, cranial and caudal, Other terms used to describe positions include proximal and distal, cranial and caudal, and rostral. Proximal and distal are used with reference to being closer to or farther from a structure’s origin, particularly in the limbs. For example, the hand is distal to the elbow joint. The glenohumeral joint is proximal to the elbow joint. These terms are also used to describe the relative positions of branches along the course of linear structures, such as airways, vessels, and nerves. For example, distal branches occur farther away toward the ends of the system, whereas proximal branches occur closer to and toward the origin of the system.

1	Cranial (toward the head) and caudal (toward the tail) are sometimes used instead of superior and inferior, respectively. Rostral is used, particularly in the head, to describe the position of a structure with reference to the nose. For example, the forebrain is rostral to the hindbrain. Two other terms used to describe the position of structures in the body are superficial and deep. These terms are used to describe the relative positions of two structures with respect to the surface of the body. For example, the sternum is superficial to the heart, and the stomach is deep to the abdominal wall.

1	Superficial and deep can also be used in a more absolute fashion to define two major regions of the body. The superficial region of the body is external to the outer layer of deep fascia. Deep structures are enclosed by this layer. Structures in the superficial region of the body include the skin, superficial fascia, and mammary glands. Deep structures include most skeletal muscles and viscera. Superficial wounds are external to the outer layer of deep fascia, whereas deep wounds penetrate through it. In 1895 Wilhelm Roentgen used the X-rays from a cathode ray tube to expose a photographic plate and produce the first radiographic exposure of his wife’s hand. Over the past 35 years there has been a revolution in body imaging, which has been paralleled by developments in computer technology.

1	X-rays are photons (a type of electromagnetic radiation) and are generated from a complex X-ray tube, which is a type of cathode ray tube (Fig. 1.2). The X-rays are then collimated (i.e., directed through lead-lined shutters to stop them from fanning out) to the appropriate area of the body. As the X-rays pass through the body they are attenuated (reduced in energy) by the tissues. Those X-rays that pass through the tissues interact with the photographic film. In the body: air attenuates X-rays a little; fat attenuates X-rays more than air but less than bone attenuates X-rays the most. These differences in attenuation result in differences in the level of exposure of the film. When the photographic film is developed, bone appears white on the film because this region of the film has been exposed to the least amount of X-rays. Air appears dark on the film because these regions were exposed to the greatest number of X-rays.

1	Modifications to this X-ray technique allow a continuous stream of X-rays to be produced from the X-ray tube and collected on an input screen to allow real-time visualization of moving anatomical structures, barium studies, angiography, and fluoroscopy (Fig. 1.3).

1	To demonstrate specific structures, such as bowel loops or arteries, it may be necessary to fill these structures with a substance that attenuates X-rays more than bowel loops or arteries do normally. It is, however, extremely important that these substances are nontoxic. Barium sulfate, an insoluble salt, is a nontoxic, relatively high-density agent that is extremely useful in the examination of the gastrointestinal tract. When a barium sulfate suspension is ingested it attenuates X-rays and can therefore be used to demonstrate the bowel lumen (Fig. 1.4). It is common to add air to the barium sulfate suspension, by either ingesting “fizzy” granules or directly instilling air into the body cavity, as in a barium enema. This is known as a double-contrast (air/barium) study.

1	For some patients it is necessary to inject contrast agents directly into arteries or veins. In this case, iodine-based molecules are suitable contrast agents. Iodine is chosen because it has a relatively high atomic mass and so markedly attenuates X-rays, but also, importantly, it is naturally excreted via the urinary system. Intra-arterial and intravenous contrast agents are extremely safe and are well tolerated by most patients. Rarely, some patients have an anaphylactic reaction to intra-arterial or intravenous injections, so the necessary precautions must be taken. Intra-arterial and intravenous contrast agents not only help in visualizing the arteries and veins but because they are excreted by the urinary system, can also be used to visualize the kidneys, ureter, and bladder in a process known as intravenous urography.

1	During angiography it is often difficult to appreciate the contrast agent in the vessels through the overlying bony structures. To circumvent this, the technique of subtraction angiography has been developed. Simply, one or two images are obtained before the injection of contrast media. These images are inverted (such that a negative is created from the positive image). After injection of the contrast media into the vessels, a further series of images are obtained, demonstrating the passage of the contrast through the arteries into the veins and around the circulation. By adding the “negative precontrast image” to the positive postcontrast images, the bones and soft tissues are subtracted to produce a solitary image of contrast only. Before the advent of digital imaging this was a challenge, but now the use of computers has made this technique relatively straightforward and instantaneous (Fig. 1.5). Ultrasonography of the body is widely used for all aspects of medicine.

1	Ultrasonography of the body is widely used for all aspects of medicine. Ultrasound is a very high frequency sound wave (not electromagnetic radiation) generated by piezoelectric materials, such that a series of sound waves is produced. Importantly, the piezoelectric material can also receive the sound waves that bounce back from the internal organs. The sound waves are then interpreted by a powerful computer, and a real-time image is produced on the display panel. Developments in ultrasound technology, including the size of the probes and the frequency range, mean that a broad range of areas can now be scanned.

1	Developments in ultrasound technology, including the size of the probes and the frequency range, mean that a broad range of areas can now be scanned. Traditionally ultrasound is used for assessing the abdomen (Fig. 1.6) and the fetus in pregnant women. Ultrasound is also widely used to assess the eyes, neck, soft tissues, and peripheral musculoskeletal system. Probes have been placed on endoscopes, and endoluminal ultrasound of the esophagus, stomach, and duodenum is now routine. Endocavity ultrasound is carried out most commonly to assess the genital tract in women using a transvaginal or transrectal route. In men, transrectal ultrasound is the imaging method of choice to assess the prostate in those with suspected prostate hypertrophy or malignancy.

1	Doppler ultrasound enables determination of flow, its direction, and its velocity within a vessel using simple ultrasound techniques. Sound waves bounce off moving structures and are returned. The degree of frequency shift determines whether the object is moving away from or toward the probe and the speed at which it is traveling. Precise measurements of blood flow and blood velocity can therefore be obtained, which in turn can indicate sites of blockage in blood vessels. Computed tomography (CT) was invented in the 1970s by Sir Godfrey Hounsfield, who was awarded the Nobel Prize in Medicine in 1979. Since this inspired invention there have been many generations of CT scanners.

1	A CT scanner obtains a series of images of the body (slices) in the axial plane. The patient lies on a bed, an X-ray tube passes around the body (Fig. 1.7), and a series of images are obtained. A computer carries out a complex mathematical transformation on the multitude of images to produce the final image (Fig. 1.8).

1	Nuclear magnetic resonance imaging was first described in 1946 and used to determine the structure of complex molecules. The process of magnetic resonance imaging (MRI) is dependent on the free protons in the hydrogen nuclei in molecules of water (H2O). Because water is present in almost all biological tissues, the hydrogen proton is ideal. The protons within a patient’s hydrogen nuclei can be regarded as small bar magnets, which are randomly oriented in space. The patient is placed in a strong magnetic field, which aligns the bar magnets. When a pulse of radio waves is passed through the patient the magnets are deflected, and as they return to their aligned position they emit small radio pulses. The strength and frequency of the emitted pulses and the time it takes for the protons to return to their pre-excited state produce a signal. These signals are analyzed by a powerful computer, and an image is created (Fig. 1.9).

1	By altering the sequence of pulses to which the protons are subjected, different properties of the protons can be assessed. These properties are referred to as the “weighting” of the scan. By altering the pulse sequence and the scanning parameters, T1-weighted images (Fig. 1.10A) and T2-weighted images (Fig. 1.10B) can be obtained. These two types of imaging sequences provide differences in image contrast, which accentuate and optimize different tissue characteristics. From the clinical point of view: Most T1-weighted images show dark fluid and bright fat—for example, within the brain the cerebrospinal fluid (CSF) is dark. T2-weighted images demonstrate a bright signal from fluid and an intermediate signal from fat—for example, in the brain the CSF appears white. MRI can also be used to assess flow within vessels and to produce complex angiograms of the peripheral and cerebral circulation.

1	MRI can also be used to assess flow within vessels and to produce complex angiograms of the peripheral and cerebral circulation. Diffusion-weighted imaging provides information on the degree of Brownian motion of water molecules in various tissues. There is relatively free diffusion in extracellular spaces and more restricted diffusion in intracellular spaces. In tumors and infarcted tissue, there is an increase in intracellular fluid water molecules compared with the extracellular fluid environment resulting in overall increased restricted diffusion, and therefore identification of abnormal from normal tissue. Nuclear medicine involves imaging using gamma rays, which are another type of electromagnetic radiation. The important difference between gamma rays and X-rays is that gamma rays are produced from within the nucleus of an atom when an unstable nucleus decays, whereas X-rays are produced by bombarding an atom with electrons.

1	X-rays is that gamma rays are produced from within the nucleus of an atom when an unstable nucleus decays, whereas X-rays are produced by bombarding an atom with electrons. For an area to be visualized, the patient must receive a gamma ray emitter, which must have a number of properties to be useful, including: a reasonable half-life (e.g., 6 to 24 hours), an easily measurable gamma ray, and energy deposition in as low a dose as possible in the patient’s tissues.

1	The most commonly used radionuclide (radioisotope) is technetium-99m. This may be injected as a technetium salt or combined with other complex molecules. For example, by combining technetium-99m with methylene diphosphonate (MDP), a radiopharmaceutical is produced. When injected into the body this radiopharmaceutical specifically binds to bone, allowing assessment of the skeleton. Similarly, combining technetium-99m with other compounds permits assessment of other parts of the body, for example the urinary tract and cerebral blood flow. Depending on how the radiopharmaceutical is absorbed, distributed, metabolized, and excreted by the body after injection, images are obtained using a gamma camera (Fig. 1.11).

1	Depending on how the radiopharmaceutical is absorbed, distributed, metabolized, and excreted by the body after injection, images are obtained using a gamma camera (Fig. 1.11). Positron emission tomography (PET) is an imaging modality for detecting positron-emitting radionuclides. A positron is an anti-electron, which is a positively charged particle of antimatter. Positrons are emitted from the decay of proton-rich radionuclides. Most of these radionuclides are made in a cyclotron and have extremely short half-lives. The most commonly used PET radionuclide is fluorodeoxyglucose (FDG) labeled with fluorine-18 (a positron emitter). Tissues that are actively metabolizing glucose take up this compound, and the resulting localized high concentration of this molecule compared to background emission is detected as a “hot spot.” PET has become an important imaging modality in the detection of cancer and the assessment of its treatment and recurrence.

1	PET has become an important imaging modality in the detection of cancer and the assessment of its treatment and recurrence. Single photon emission computed tomography (SPECT) is an imaging modality for detecting gamma rays emitted from the decay of injected radionuclides such as technetium-99m, iodine-123, or iodine-131. The rays are detected by a 360-degree rotating camera, which allows the construction of 3D images. SPECT can be used to diagnose a wide range of disease conditions such as coronary artery disease and bone fractures. Imaging is necessary in most clinical specialties to diagnose pathological changes to tissues. It is paramount to appreciate what is normal and what is abnormal. An appreciation of how the image is obtained, what the normal variations are, and what technical considerations are necessary to obtain a radiological diagnosis. Without understanding the anatomy of the region imaged, it is impossible to comment on the abnormal.

1	Plain radiographs are undoubtedly the most common form of image obtained in a hospital or local practice. Before interpretation, it is important to know about the imaging technique and the views obtained as standard. In most instances (apart from chest radiography) the X-ray tube is 1 m away from the X-ray film. The object in question, for example a hand or a foot, is placed upon the film. When describing subject placement for radiography, the part closest to the X-ray tube is referred to first and that closest to the film is referred to second. For example, when positioning a patient for an anteroposterior (AP) radiograph, the more anterior part of the body is closest to the tube and the posterior part is closest to the film. When X-rays are viewed on a viewing box, the right side of the patient is placed to the observer’s left; therefore, the observer views the radiograph as though looking at a patient in the anatomical position.

1	The chest radiograph is one of the most commonly requested plain radiographs. An image is taken with the patient erect and placed posteroanteriorly (PA chest radiograph; that is, with the patient’s back closest to the X-ray tube.). Occasionally, when patients are too unwell to stand erect, films are obtained on the bed in an anteroposterior (AP) position. These films are less standardized than PA films, and caution should always be taken when interpreting AP radiographs. The plain chest radiograph should always be checked for quality. Film markers should be placed on the appropriate side. (Occasionally patients have dextrocardia, which may be misinterpreted if the film marker is placed inappropriately.) A good-quality chest radiograph will demonstrate the lungs, cardiomediastinal contour, diaphragm, ribs, and peripheral soft tissues.

1	Plain abdominal radiographs are obtained in the AP supine position. From time to time an erect plain abdominal radiograph is obtained when small bowel obstruction is suspected.

1	High-density contrast medium is ingested to opacify the esophagus, stomach, small bowel, and large bowel. As described previously (p. 6), the bowel is insufflated with air (or carbon dioxide) to provide a double-contrast study. In many countries, endoscopy has superseded upper gastrointestinal imaging, but the mainstay of imaging the large bowel is the double-contrast barium enema. Typically the patient needs to undergo bowel preparation, in which powerful cathartics are used to empty the bowel. At the time of the examination a small tube is placed into the rectum and a barium suspension is run into the large bowel. The patient undergoes a series of twists and turns so that the contrast passes through the entire large bowel. The contrast is emptied and air is passed through the same tube to insufflate the large bowel. A thin layer of barium coats the normal mucosa, allowing mucosal detail to be visualized (see Fig. 1.4).

1	Intravenous urography is the standard investigation for assessing the urinary tract. Intravenous contrast medium is injected, and images are obtained as the medium is excreted through the kidneys. A series of films are obtained during this period from immediately after the injection up to approximately 20 minutes later, when the bladder is full of contrast medium. This series of radiographs demonstrates the kidneys, ureters, and bladder and enables assessment of the retroperitoneum and other structures that may press on the urinary tract. Computed tomography is the preferred terminology rather than computerized tomography, though both terms are used interchangeably by physicians.

1	Computed tomography is the preferred terminology rather than computerized tomography, though both terms are used interchangeably by physicians. It is important for the student to understand the presentation of images. Most images are acquired in the axial plane and viewed such that the observer looks from below and upward toward the head (from the foot of the bed). By implication: the right side of the patient is on the left side of the image, and the uppermost border of the image is anterior. Many patients are given oral and intravenous contrast media to differentiate bowel loops from other abdominal organs and to assess the vascularity of normal anatomical structures. When intravenous contrast is given, the earlier the images are obtained, the greater the likelihood of arterial enhancement. As the time is delayed between injection and image acquisition, a venous phase and an equilibrium phase are also obtained.

1	The great advantage of CT scanning is the ability to extend and compress the gray scale to visualize the bones, soft tissues, and visceral organs. Altering the window settings and window centering provides the physician with specific information about these structures. There is no doubt that MRI has revolutionized the understanding and interpretation of the brain and its coverings. Furthermore, it has significantly altered the practice of musculoskeletal medicine and surgery. Images can be obtained in any plane and in most sequences. Typically the images are viewed using the same principles as CT. Intravenous contrast agents are also used to further enhance tissue contrast. Typically, MRI contrast agents contain paramagnetic substances (e.g., gadolinium and manganese). Most nuclear medicine images are functional studies. Images are usually interpreted directly from a computer, and a series of representative films are obtained for clinical use.

1	Most nuclear medicine images are functional studies. Images are usually interpreted directly from a computer, and a series of representative films are obtained for clinical use. Whenever a patient undergoes an X-ray or nuclear medicine investigation, a dose of radiation is given (Table 1.1). As a general principle it is expected that the dose given is as low as reasonably possible for a diagnostic image to be obtained. Numerous laws govern the amount of radiation exposure that a patient can undergo for a variety of procedures, and these are monitored to prevent any excess or additional dosage. Whenever a radiograph is booked, the clinician ordering the procedure must appreciate its necessity and understand the dose given to the patient to ensure that the benefits significantly outweigh the risks.

1	Imaging modalities such as ultrasound and MRI are ideal because they do not impart significant risk to the patient. Moreover, ultrasound imaging is the modality of choice for assessing the fetus. Any imaging device is expensive, and consequently the more complex the imaging technique (e.g., MRI) the more expensive the investigation. Investigations must be carried out judiciously, based on a sound clinical history and examination, for which an understanding of anatomy is vital. The skeleton can be divided into two subgroups, the axial skeleton and the appendicular skeleton. The axial skeleton consists of the bones of the skull (cranium), vertebral column, ribs, and sternum, whereas the appendicular skeleton consists of the bones of the upper and lower limbs (Fig. 1.12). The skeletal system consists of cartilage and bone.

1	The skeletal system consists of cartilage and bone. Cartilage is an avascular form of connective tissue consisting of extracellular fibers embedded in a matrix that contains cells localized in small cavities. The amount and kind of extracellular fibers in the matrix varies depending on the type of cartilage. In heavy weightbearing areas or areas prone to pulling forces, the amount of collagen is greatly increased and the cartilage is almost inextensible. In contrast, in areas where weightbearing demands and stress are less, cartilage containing elastic fibers and fewer collagen fibers is common. The functions of cartilage are to: support soft tissues, provide a smooth, gliding surface for bone articulations at joints, and enable the development and growth of long bones.

1	There are three types of cartilage: hyaline—most common; matrix contains a moderate amount of collagen fibers (e.g., articular surfaces of bones); elastic—matrix contains collagen fibers along with a large number of elastic fibers (e.g., external ear); fibrocartilage—matrix contains a limited number of cells and ground substance amidst a substantial amount of collagen fibers (e.g., intervertebral discs). Cartilage is nourished by diffusion and has no blood vessels, lymphatics, or nerves. Bone is a calcified, living, connective tissue that forms the majority of the skeleton. It consists of an intercellular calcified matrix, which also contains collagen fibers, and several types of cells within the matrix. Bones function as: supportive structures for the body, protectors of vital organs, reservoirs of calcium and phosphorus, levers on which muscles act to produce movement, and containers for blood-producing cells.

1	There are two types of bone, compact and spongy (trabecular or cancellous). Compact bone is dense bone that forms the outer shell of all bones and surrounds spongy bone. Spongy bone consists of spicules of bone enclosing cavities containing blood-forming cells (marrow). Classification of bones is by shape. Long bones are tubular (e.g., humerus in upper limb; femur in lower limb). Short bones are cuboidal (e.g., bones of the wrist and ankle). Flat bones consist of two compact bone plates separated by spongy bone (e.g., skull). Irregular bones are bones with various shapes (e.g., bones of the face). Sesamoid bones are round or oval bones that develop in tendons.

1	Bones are vascular and are innervated. Generally, an adjacent artery gives off a nutrient artery, usually one per bone, that directly enters the internal cavity of the bone and supplies the marrow, spongy bone, and inner layers of compact bone. In addition, all bones are covered externally, except in the area of a joint where articular cartilage is present, by a fibrous connective tissue membrane called the periosteum, which has the unique capability of forming new bone. This membrane receives blood vessels whose branches supply the outer layers of compact bone. A bone stripped of its periosteum will not survive. Nerves accompany the vessels that supply the bone and the periosteum. Most of the nerves passing into the internal cavity with the nutrient artery are vasomotor fibers that regulate blood flow. Bone itself has few sensory nerve fibers. On the other hand, the periosteum is supplied with numerous sensory nerve fibers and is very sensitive to any type of injury.

1	Developmentally, all bones come from mesenchyme by either intramembranous ossification, in which mesenchymal models of bones undergo ossification, or endochondral ossification, in which cartilaginous models of bones form from mesenchyme and undergo ossification. The sites where two skeletal elements come together are termed joints. The two general categories of joints (Fig. 1.18) are those in which: the skeletal elements are separated by a cavity (i.e., synovial joints), and there is no cavity and the components are held together by connective tissue (i.e., solid joints). Blood vessels that cross over a joint and nerves that innervate muscles acting on a joint usually contribute articular branches to that joint. Synovial joints are connections between skeletal components where the elements involved are separated by a narrow articular cavity (Fig. 1.19). In addition to containing an articular cavity, these joints have a number of characteristic features.

1	First, a layer of cartilage, usually hyaline cartilage, covers the articulating surfaces of the skeletal elements. In other words, bony surfaces do not normally contact one another directly. As a consequence, when these joints are viewed in normal radiographs, a wide gap seems to separate the adjacent bones because the cartilage that covers the articulating surfaces is more transparent to X-rays than bone. A second characteristic feature of synovial joints is the presence of a joint capsule consisting of an inner synovial membrane and an outer fibrous membrane.

1	A second characteristic feature of synovial joints is the presence of a joint capsule consisting of an inner synovial membrane and an outer fibrous membrane. The synovial membrane attaches to the margins of the joint surfaces at the interface between the cartilage and bone and encloses the articular cavity. The synovial membrane is highly vascular and produces synovial fluid, which percolates into the articular cavity and lubricates the articulating surfaces. Closed sacs of synovial membrane also occur outside joints, where they form synovial bursae or tendon sheaths. Bursae often intervene between structures, such as tendons and bone, tendons and joints, or skin and bone, and reduce the friction of one structure moving over the other. Tendon sheaths surround tendons and also reduce friction.

1	The fibrous membrane is formed by dense connective tissue and surrounds and stabilizes the joint. Parts of the fibrous membrane may thicken to form ligaments, which further stabilize the joint. Ligaments outside the capsule usually provide additional reinforcement. Another common but not universal feature of synovial joints is the presence of additional structures within the area enclosed by the capsule or synovial membrane, such as articular discs (usually composed of fibrocartilage), fat pads, and tendons. Articular discs absorb compression forces, adjust to changes in the contours of joint surfaces during movements, and increase the range of movements that can occur at joints. Fat pads usually occur between the synovial membrane and the capsule and move into and out of regions as joint contours change during movement. Redundant regions of the synovial membrane and fibrous membrane allow for large movements at joints. Descriptions of synovial joints based on shape and movement

1	Descriptions of synovial joints based on shape and movement Synovial joints are described based on shape and movement: based on the shape of their articular surfaces, synovial joints are described as plane (flat), hinge, pivot, bicondylar (two sets of contact points), condylar (ellipsoid), saddle, and ball and socket; based on movement, synovial joints are described as uniaxial (movement in one plane), biaxial (movement in two planes), and multiaxial (movement in three planes). Hinge joints are uniaxial, whereas ball and socket joints are multiaxial. Specific types of synovial joints (Fig. 1.20) Plane joints—allow sliding or gliding movements when one bone moves across the surface of another (e.g., acromioclavicular joint) Hinge joints—allow movement around one axis that passes transversely through the joint; permit flexion and extension (e.g., elbow [humero-ulnar] joint)

1	Hinge joints—allow movement around one axis that passes transversely through the joint; permit flexion and extension (e.g., elbow [humero-ulnar] joint) Pivot joints—allow movement around one axis that passes longitudinally along the shaft of the bone; permit rotation (e.g., atlanto-axial joint) Bicondylar joints—allow movement mostly in one axis with limited rotation around a second axis; formed by two convex condyles that articulate with concave or flat surfaces (e.g., knee joint) Condylar (ellipsoid) joints—allow movement around two axes that are at right angles to each other; permit flexion, extension, abduction, adduction, and circumduction (limited) (e.g., wrist joint) Saddle joints—allow movement around two axes that are at right angles to each other; the articular surfaces are saddle shaped; permit flexion, extension, abduction, adduction, and circumduction (e.g., carpometacarpal joint of the thumb)

1	Ball and socket joints—allow movement around multiple axes; permit flexion, extension, abduction, adduction, circumduction, and rotation (e.g., hip Solid joints are connections between skeletal elements where the adjacent surfaces are linked together either by fibrous connective tissue or by cartilage, usually fibrocartilage (Fig. 1.21). Movements at these joints are more restricted than at synovial joints. Fibrous joints include sutures, gomphoses, and syndesmoses. Sutures occur only in the skull where adjacent bones are linked by a thin layer of connective tissue termed a sutural ligament. Gomphoses occur only between the teeth and adjacent bone. In these joints, short collagen tissue fibers in the periodontal ligament run between the root of the tooth and the bony socket.

1	Gomphoses occur only between the teeth and adjacent bone. In these joints, short collagen tissue fibers in the periodontal ligament run between the root of the tooth and the bony socket. Syndesmoses are joints in which two adjacent bones are linked by a ligament. Examples are the ligamentum flavum, which connects adjacent vertebral laminae, and an interosseous membrane, which links, for example, the radius and ulna in the forearm. Cartilaginous joints include synchondroses and symphyses. Synchondroses occur where two ossification centers in a developing bone remain separated by a layer of cartilage, for example, the growth plate that occurs between the head and shaft of developing long bones. These joints allow bone growth and eventually become completely ossified.

1	Symphyses occur where two separate bones are interconnected by cartilage. Most of these types of joints occur in the midline and include the pubic symphysis between the two pelvic bones, and intervertebral discs between adjacent vertebrae. The skin is the largest organ of the body. It consists of the epidermis and the dermis. The epidermis is the outer cellular layer of stratified squamous epithelium, which is avascular and varies in thickness. The dermis is a dense bed of vascular connective tissue. The skin functions as a mechanical and permeability barrier, and as a sensory and thermoregulatory organ. It also can initiate primary immune responses. Fascia is connective tissue containing varying amounts of fat that separate, support, and interconnect organs and structures, enable movement of one structure relative to another, and allow the transit of vessels and nerves from one area to another. There are two general categories of fascia: superficial and deep.

1	Superficial (subcutaneous) fascia lies just deep to and is attached to the dermis of the skin. It is made up of loose connective tissue usually containing a large amount of fat. The thickness of the superficial fascia (subcutaneous tissue) varies considerably, both from one area of the body to another and from one individual to another. The superficial fascia allows movement of the skin over deeper areas of the body, acts as a conduit for vessels and nerves coursing to and from the skin, and serves as an energy (fat) reservoir.

1	Deep fascia usually consists of dense, organized connective tissue. The outer layer of deep fascia is attached to the deep surface of the superficial fascia and forms a thin fibrous covering over most of the deeper region of the body. Inward extensions of this fascial layer form intermuscular septa that compartmentalize groups of muscles with similar functions and innervations. Other extensions surround individual muscles and groups of vessels and nerves, forming an investing fascia. Near some joints the deep fascia thickens, forming retinacula. These fascial retinacula hold tendons in place and prevent them from bowing during movements at the joints. Finally, there is a layer of deep fascia separating the membrane lining the abdominal cavity (the parietal peritoneum) from the fascia covering the deep surface of the muscles of the abdominal wall (the transversalis fascia). This layer is referred to as extraperitoneal fascia. A similar layer of fascia in the thorax is termed the

1	covering the deep surface of the muscles of the abdominal wall (the transversalis fascia). This layer is referred to as extraperitoneal fascia. A similar layer of fascia in the thorax is termed the endothoracic fascia.

1	The muscular system is generally regarded as consisting of one type of muscle found in the body—skeletal muscle. However, there are two other types of muscle tissue found in the body, smooth muscle and cardiac muscle, that are important components of other systems. These three types of muscle can be characterized by whether they are controlled voluntarily or involuntarily, whether they appear striated (striped) or smooth, and whether they are associated with the body wall (somatic) or with organs and blood vessels (visceral).

1	Skeletal muscle forms the majority of the muscle tissue in the body. It consists of parallel bundles of long, multinucleated fibers with transverse stripes, is capable of powerful contractions, and is innervated by somatic and branchial motor nerves. This muscle is used to move bones and other structures, and provides support and gives form to the body. Individual skeletal muscles are often named on the basis of shape (e.g., rhomboid major muscle), attachments (e.g., sternohyoid muscle), function (e.g., flexor pollicis longus muscle), position (e.g., palmar interosseous muscle), or fiber orientation (e.g., external oblique muscle).

1	Cardiac muscle is striated muscle found only in the walls of the heart (myocardium) and in some of the large vessels close to where they join the heart. It consists of a branching network of individual cells linked electrically and mechanically to work as a unit. Its contractions are less powerful than those of skeletal muscle and it is resistant to fatigue. Cardiac muscle is innervated by visceral motor nerves. Smooth muscle (absence of stripes) consists of elongated or spindle-shaped fibers capable of slow and sustained contractions. It is found in the walls of blood vessels (tunica media), associated with hair follicles in the skin, located in the eyeball, and found in the walls of various structures associated with the gastrointestinal, respiratory, genitourinary, and urogenital systems. Smooth muscle is innervated by visceral motor nerves.

1	The cardiovascular system consists of the heart, which pumps blood throughout the body, and the blood vessels, which are a closed network of tubes that transport the blood. There are three types of blood vessels: arteries, which transport blood away from the heart; veins, which transport blood toward the heart; capillaries, which connect the arteries and veins, are the smallest of the blood vessels and are where oxygen, nutrients, and wastes are exchanged within the tissues. The walls of the blood vessels of the cardiovascular system usually consist of three layers or tunics: tunica externa (adventitia)—the outer connective tissue layer, tunica media—the middle smooth muscle layer (may also contain varying amounts of elastic fibers in medium and large arteries), and tunica intima—the inner endothelial lining of the blood vessels.

1	Arteries are usually further subdivided into three classes, according to the variable amounts of smooth muscle and elastic fibers contributing to the thickness of the tunica media, the overall size of the vessel, and its function. Large elastic arteries contain substantial amounts of elastic fibers in the tunica media, allowing expansion and recoil during the normal cardiac cycle. This helps maintain a constant flow of blood during diastole. Examples of large elastic arteries are the aorta, the brachiocephalic trunk, the left common carotid artery, the left subclavian artery, and the pulmonary trunk. Medium muscular arteries are composed of a tunica media that contains mostly smooth muscle fibers. This characteristic allows these vessels to regulate their diameter and control the flow of blood to different parts of the body. Examples of medium muscular arteries are most of the named arteries, including the femoral, axillary, and radial arteries.

1	Small arteries and arterioles control the filling of the capillaries and directly contribute to the arterial pressure in the vascular system. Veins also are subdivided into three classes. Large veins contain some smooth muscle in the tunica media, but the thickest layer is the tunica externa. Examples of large veins are the superior vena cava, the inferior vena cava, and the portal vein. Small and medium veins contain small amounts of smooth muscle, and the thickest layer is the tunica externa. Examples of small and medium veins are superficial veins in the upper and lower limbs and deeper veins of the leg and forearm. Venules are the smallest veins and drain the capillaries. Although veins are similar in general structure to arteries, they have a number of distinguishing features. The walls of veins, specifically the tunica media, are thin. The luminal diameters of veins are large.

1	The walls of veins, specifically the tunica media, are thin. The luminal diameters of veins are large. There often are multiple veins (venae comitantes) closely associated with arteries in peripheral regions. Valves often are present in veins, particularly in peripheral vessels inferior to the level of the heart. These are usually paired cusps that facilitate blood flow toward the heart. More specific information about the cardiovascular system and how it relates to the circulation of blood throughout the body will be discussed, where appropriate, in each of the succeeding chapters of the text. Lymphatic vessels form an extensive and complex interconnected network of channels, which begin as “porous” blind-ended lymphatic capillaries in tissues of the body and converge to form a number of larger vessels, which ultimately connect with large veins in the root of the neck.

1	Lymphatic vessels mainly collect fluid lost from vascular capillary beds during nutrient exchange processes and deliver it back to the venous side of the vascular system (Fig. 1.28). Also included in this interstitial fluid that drains into the lymphatic capillaries are pathogens, cells of the lymphocytic system, cell products (such as hormones), and cell debris. In the small intestine, certain fats absorbed and processed by the intestinal epithelium are packaged into protein-coated lipid droplets (chylomicrons), which are released from the epithelial cells and enter the interstitial compartment. Together with other components of the interstitial fluid, the chylomicrons drain into lymphatic capillaries (known as lacteals in the small intestine) and are ultimately delivered to the venous system in the neck. The lymphatic system is therefore also a major route of transport for fat absorbed by the gut.

1	The fluid in most lymphatic vessels is clear and colorless and is known as lymph. That carried by lymphatic vessels from the small intestine is opaque and milky because of the presence of chylomicrons and is termed chyle. There are lymphatic vessels in most areas of the body, including those associated with the central nervous system (Louveau A et al., Nature 2015; 523:337-41; Aspelund A et al., J Exp Med 2015; 212:991-9). Exceptions include bone marrow and avascular tissues such as epithelia and cartilage. The movement of lymph through the lymphatic vessels is generated mainly by the indirect action of adjacent structures, particularly by contraction of skeletal muscles and pulses in arteries. Unidirectional flow is maintained by the presence of valves in the vessels.

1	Lymph nodes are small (0.1–2.5 cm long) encapsulated structures that interrupt the course of lymphatic vessels and contain elements of the body’s defense system, such as clusters of lymphocytes and macrophages. They act as elaborate filters that trap and phagocytose particulate matter in the lymph that percolates through them. In addition, they detect and defend against foreign antigens that are also carried in the lymph (Fig. 1.28). Because lymph nodes are efficient filters and flow through them is slow, cells that metastasize from (migrate away from) primary tumors and enter lymphatic vessels often lodge and grow as secondary tumors in lymph nodes. Lymph nodes that drain regions that are infected or contain other forms of disease can enlarge or undergo certain physical changes, such as becoming “hard” or “tender.” These changes can be used by clinicians to detect pathologic changes or to track spread of disease.

1	A number of regions in the body are associated with clusters or a particular abundance of lymph nodes (Fig. 1.29). Not surprisingly, nodes in many of these regions drain the body’s surface, the digestive system, or the respiratory system. All three of these areas are high-risk sites for the entry of foreign pathogens. Lymph nodes are abundant and accessible to palpation in the axilla, the groin and femoral region, and the neck. Deep sites that are not palpable include those associated with the trachea and bronchi in the thorax, and with the aorta and its branches in the abdomen. All lymphatic vessels coalesce to form larger trunks or ducts, which drain into the venous system at sites in the neck where the internal jugular veins join the subclavian veins to form the brachiocephalic veins (Fig. 1.30):

1	Lymph from the right side of the head and neck, the right upper limb, and the right side of the thorax is carried by lymphatic vessels that connect with veins on the right side of the neck. Lymph from all other regions of the body is carried by lymphatic vessels that drain into veins on the left side of the neck. Specific information about the organization of the lymphatic system in each region of the body is discussed in the appropriate chapter. The nervous system can be separated into parts based on structure and on function: structurally, it can be divided into the central nervous system (CNS) and the peripheral nervous system (PNS) (Fig. 1.32); functionally, it can be divided into somatic and visceral parts. The CNS is composed of the brain and spinal cord, both of which develop from the neural tube in the embryo.

1	The CNS is composed of the brain and spinal cord, both of which develop from the neural tube in the embryo. The PNS is composed of all nervous structures outside the CNS that connect the CNS to the body. Elements of this system develop from neural crest cells and as outgrowths of the CNS. The PNS consists of the spinal and cranial nerves, visceral nerves and plexuses, and the enteric system. The detailed anatomy of a typical spinal nerve is described in Chapter 2, as is the way spinal nerves are numbered. Cranial nerves are described in Chapter 8. The details of nerve plexuses are described in chapters dealing with the specific regions in which the plexuses are located.

1	The details of nerve plexuses are described in chapters dealing with the specific regions in which the plexuses are located. The parts of the brain are the cerebral hemispheres, the cerebellum, and the brainstem. The cerebral hemispheres consist of an outer portion, or the gray matter, containing cell bodies; an inner portion, or the white matter, made up of axons forming tracts or pathways; and the ventricles, which are spaces filled with CSF. The cerebellum has two lateral lobes and a midline portion. The components of the brainstem are classically defined as the diencephalon, midbrain, pons, and medulla. However, in common usage today, the term “brainstem” usually refers to the midbrain, pons, and medulla. A further discussion of the brain can be found in Chapter 8.

1	A further discussion of the brain can be found in Chapter 8. The spinal cord is the part of the CNS in the superior two thirds of the vertebral canal. It is roughly cylindrical in shape, and is circular to oval in cross section with a central canal. A further discussion of the spinal cord can be found in Chapter 2. The meninges (Fig. 1.33) are three connective tissue coverings that surround, protect, and suspend the brain and spinal cord within the cranial cavity and vertebral canal, respectively: The dura mater is the thickest and most external of the coverings. The arachnoid mater is against the internal surface of the dura mater. The pia mater is adherent to the brain and spinal cord. Between the arachnoid and pia mater is the subarachnoid space, which contains CSF. A further discussion of the cranial meninges can be found in Chapter 8 and of the spinal meninges in Chapter 2. Functional subdivisions of the CNS

1	A further discussion of the cranial meninges can be found in Chapter 8 and of the spinal meninges in Chapter 2. Functional subdivisions of the CNS Functionally, the nervous system can be divided into somatic and visceral parts. The somatic part (soma, from the Greek for “body”) innervates structures (skin and most skeletal muscle) derived from somites in the embryo, and is mainly involved with receiving and responding to information from the external environment. The visceral part (viscera, from the Greek for “guts”) innervates organ systems in the body and other visceral elements, such as smooth muscle and glands, in peripheral regions of the body. It is concerned mainly with detecting and responding to information from the internal environment. Somatic part of the nervous system The somatic part of the nervous system consists of: nerves that carry conscious sensations from peripheral regions back to the CNS, and nerves that innervate voluntary muscles.

1	The somatic part of the nervous system consists of: nerves that carry conscious sensations from peripheral regions back to the CNS, and nerves that innervate voluntary muscles. Somatic nerves arise segmentally along the developing CNS in association with somites, which are themselves arranged segmentally along each side of the neural tube (Fig. 1.34). Part of each somite (the dermatomyotome) gives rise to skeletal muscle and the dermis of the skin. As cells of the dermatomyotome differentiate, they migrate into posterior (dorsal) and anterior (ventral) areas of the developing body: Cells that migrate anteriorly give rise to muscles of the limbs and trunk (hypaxial muscles) and to the associated dermis. Cells that migrate posteriorly give rise to the intrinsic muscles of the back (epaxial muscles) and the associated dermis.

1	Cells that migrate posteriorly give rise to the intrinsic muscles of the back (epaxial muscles) and the associated dermis. Developing nerve cells within anterior regions of the neural tube extend processes peripherally into posterior and anterior regions of the differentiating dermatomyotome of each somite. Simultaneously, derivatives of neural crest cells (cells derived from neural folds during formation of the neural tube) differentiate into neurons on each side of the neural tube and extend processes both medially and laterally (Fig. 1.35): Medial processes pass into the posterior aspect of the neural tube. Lateral processes pass into the differentiating regions of the adjacent dermatomyotome. Neurons that develop from cells within the spinal cord are motor neurons and those that develop from neural crest cells are sensory neurons.

1	Neurons that develop from cells within the spinal cord are motor neurons and those that develop from neural crest cells are sensory neurons. Somatic sensory and somatic motor fibers that are organized segmentally along the neural tube become parts of all spinal nerves and some cranial nerves. The clusters of sensory nerve cell bodies derived from neural crest cells and located outside the CNS form sensory ganglia. Generally, all sensory information passes into the posterior aspect of the spinal cord, and all motor fibers leave anteriorly. Somatic sensory neurons carry information from the periphery into the CNS and are also called somatic sensory afferents or general somatic afferents (GSAs). The modalities carried by these nerves include temperature, pain, touch, and proprioception. Proprioception is the sense of determining the position and movement of the musculoskeletal system detected by special receptors in muscles and tendons.

1	Somatic motor fibers carry information away from the CNS to skeletal muscles and are also called somatic motor efferents or general somatic efferents (GSEs). Like somatic sensory fibers that come from the periphery, somatic motor fibers can be very long. They extend from cell bodies in the spinal cord to the muscle cells they innervate. Because cells from a specific somite develop into the dermis of the skin in a precise location, somatic sensory fibers originally associated with that somite enter the posterior region of the spinal cord at a specific level and become part of one specific spinal nerve (Fig. 1.36). Each spinal nerve therefore carries somatic sensory information from a specific area of skin on the surface of the body. A dermatome is that area of skin supplied by a single spinal cord level, or on one side, by a single spinal nerve.

1	There is overlap in the distribution of dermatomes, but usually a specific region within each dermatome can be identified as an area supplied by a single spinal cord level. Testing touch in these autonomous zones in a conscious patient can be used to localize lesions to a specific spinal nerve or to a specific level in the spinal cord. Somatic motor nerves that were originally associated with a specific somite emerge from the anterior region of the spinal cord and, together with sensory nerves from the same level, become part of one spinal nerve. Therefore each spinal nerve carries somatic motor fibers to muscles that originally developed from the related somite. A myotome is that portion of a skeletal muscle innervated by a single spinal cord level or, on one side, by a single spinal nerve.

1	Myotomes are generally more difficult to test than dermatomes because each skeletal muscle in the body often develops from more than one somite and is therefore innervated by nerves derived from more than one spinal cord level (Fig. 1.37). Testing movements at successive joints can help in localizing lesions to specific nerves or to a specific spinal cord level. For example: Muscles that move the shoulder joint are innervated mainly by spinal nerves from spinal cord levels C5 and C6. Muscles that move the elbow are innervated mainly by spinal nerves from spinal cord levels C6 and C7. Muscles in the hand are innervated mainly by spinal nerves from spinal cord levels C8 and T1. Visceral part of the nervous system The visceral part of the nervous system, as in the somatic part, consists of motor and sensory components: Sensory nerves monitor changes in the viscera. Motor nerves mainly innervate smooth muscle, cardiac muscle, and glands.

1	Sensory nerves monitor changes in the viscera. Motor nerves mainly innervate smooth muscle, cardiac muscle, and glands. The visceral motor component is commonly referred to as the autonomic division of the PNS and is subdivided into sympathetic and parasympathetic parts. Like the somatic part of the nervous system, the visceral part is segmentally arranged and develops in a parallel fashion (Fig. 1.39). Visceral sensory neurons that arise from neural crest cells send processes medially into the adjacent neural tube and laterally into regions associated with the developing body. These sensory neurons and their processes, referred to as general visceral afferent fibers (GVAs), are associated primarily with chemoreception, mechanoreception, and stretch reception.

1	Visceral motor neurons that arise from cells in lateral regions of the neural tube send processes out of the anterior aspect of the tube. Unlike in the somatic part, these processes, containing general visceral efferent fibers (GVEs), synapse with other cells, usually other visceral motor neurons, that develop outside the CNS from neural crest cells that migrate away from their original positions close to the developing neural tube. The visceral motor neurons located in the spinal cord are referred to as preganglionic motor neurons and their axons are called preganglionic fibers; the visceral motor neurons located outside the CNS are referred to as postganglionic motor neurons and their axons are called postganglionic fibers. The cell bodies of the visceral motor neurons outside the CNS often associate with each other in a discrete mass called a ganglion.

1	The cell bodies of the visceral motor neurons outside the CNS often associate with each other in a discrete mass called a ganglion. Visceral sensory and motor fibers enter and leave the CNS with their somatic equivalents (Fig. 1.40). Visceral sensory fibers enter the spinal cord together with somatic sensory fibers through posterior roots of spinal nerves. Preganglionic fibers of visceral motor neurons exit the spinal cord in the anterior roots of spinal nerves, along with fibers from somatic motor neurons. Postganglionic fibers traveling to visceral elements in the periphery are found in the posterior and anterior rami (branches) of spinal nerves. Visceral motor and sensory fibers that travel to and from viscera form named visceral branches that are separate from the somatic branches. These nerves generally form plexuses from which arise branches to the viscera. Visceral motor and sensory fibers do not enter and leave the CNS at all levels (Fig. 1.41):

1	Visceral motor and sensory fibers do not enter and leave the CNS at all levels (Fig. 1.41): In the cranial region, visceral components are associated with four of the twelve cranial nerves (CN III, VII, IX, and X). In the spinal cord, visceral components are associated mainly with spinal cord levels T1 to L2 and S2 to S4. Visceral motor components associated with spinal levels T1 to L2 are termed sympathetic. Those visceral motor components in cranial and sacral regions, on either side of the sympathetic region, are termed parasympathetic: The sympathetic system innervates structures in peripheral regions of the body and viscera. The parasympathetic system is more restricted to innervation of the viscera only.

1	The parasympathetic system is more restricted to innervation of the viscera only. Spinal sympathetic and spinal parasympathetic neurons share certain developmental and phenotypic features that are different from those of cranial parasympathetic neurons. Based on this, some researchers have suggested reclassifying all spinal visceral motor neurons as sympathetic (Espinosa-Medina I et al. Science 2016;354:893-897). Others are against reclassification, arguing that the results only indicate that the neurons are spinal in origin (Neuhuber W et al. Anat Rec 2017;300:1369-1370). In addition, sacral nerves do not enter the sympathetic trunk, nor do they have postganglionic fibers that travel to the periphery on spinal nerves, as do T1-L2 visceral motor fibers. We have chosen to retain the classification of S2,3,4 visceral motor neurons as parasympathetic. “Parasympathetic” simply means on either side of the “sympathetic,” which correctly describes their anatomy.

1	The sympathetic part of the autonomic division of the PNS leaves thoracolumbar regions of the spinal cord with the somatic components of spinal nerves T1 to L2 (Fig. 1.42). On each side, a paravertebral sympathetic trunk extends from the base of the skull to the inferior end of the vertebral column where the two trunks converge anteriorly to the coccyx at the ganglion impar. Each trunk is attached to the anterior rami of spinal nerves and becomes the route by which sympathetics are distributed to the periphery and all viscera.

1	Visceral motor preganglionic fibers leave the T1 to L2 part of the spinal cord in anterior roots. The fibers then enter the spinal nerves, pass through the anterior rami and into the sympathetic trunks. One trunk is located on each side of the vertebral column (paravertebral) and positioned anterior to the anterior rami. Along the trunk is a series of segmentally arranged ganglia formed from collections of postganglionic neuronal cell bodies where the preganglionic neurons synapse with postganglionic neurons. Anterior rami of T1 to L2 are connected to the sympathetic trunk or to a ganglion by a white ramus communicans, which carries preganglionic sympathetic fibers and appears white because the fibers it contains are myelinated.

1	Preganglionic sympathetic fibers that enter a paravertebral ganglion or the sympathetic trunk through a white ramus communicans may take the following four pathways to target tissues: 1. Peripheral sympathetic innervation at the level of origin of the preganglionic fiber Preganglionic sympathetic fibers may synapse with postganglionic motor neurons in ganglia associated with the sympathetic trunk, after which postganglionic fibers enter the same anterior ramus and are distributed with peripheral branches of the posterior and anterior rami of that spinal nerve (Fig. 1.43). The fibers innervate structures at the periphery of the body in regions supplied by the spinal nerve. The gray ramus communicans connects the sympathetic trunk or a ganglion to the anterior ramus and contains the postganglionic sympathetic fibers. It appears gray because postganglionic fibers are nonmyelinated. The gray ramus communicans is positioned medial to the white ramus communicans.

1	2. Peripheral sympathetic innervation above or below the level of origin of the preganglionic fiber Preganglionic sympathetic fibers may ascend or descend to other vertebral levels where they synapse in ganglia associated with spinal nerves that may or may not have visceral motor input directly from the spinal cord (i.e., those nerves other than T1 to L2) (Fig. 1.44). The postganglionic fibers leave the distant ganglia via gray rami communicantes and are distributed along the posterior and anterior rami of the spinal nerves. The ascending and descending fibers, together with all the ganglia, form the paravertebral sympathetic trunk, which extends the entire length of the vertebral column. The formation of this trunk, on each side, enables visceral motor fibers of the sympathetic part of the autonomic division of the PNS, which ultimately emerge from only a small region of the spinal cord (T1 to L2), to be distributed to peripheral regions innervated by all spinal nerves.

1	White rami communicantes only occur in association with spinal nerves T1 to L2, whereas gray rami communicantes are associated with all spinal nerves. Fibers from spinal cord levels T1 to T5 pass predominantly superiorly, whereas fibers from T5 to L2 pass inferiorly. All sympathetics passing into the head have preganglionic fibers that emerge from spinal cord level T1 and ascend in the sympathetic trunks to the highest ganglion in the neck (the superior cervical ganglion), where they synapse. Postganglionic fibers then travel along blood vessels to target tissues in the head, including blood vessels, sweat glands, small smooth muscles associated with the upper eyelids, and the dilator of the pupil. 3. Sympathetic innervation of thoracic

1	3. Sympathetic innervation of thoracic Preganglionic sympathetic fibers may synapse with postganglionic motor neurons in ganglia and then leave the ganglia medially to innervate thoracic or cervical viscera (Fig. 1.45). They may ascend in the trunk before synapsing, and after synapsing the postganglionic fibers may combine with those from other levels to form named visceral nerves, such as cardiac nerves. Often, these nerves join branches from the parasympathetic system to form plexuses on or near the surface of the target organ, for example, the cardiac and pulmonary plexuses. Branches of the plexus innervate the organ. Spinal cord levels T1 to T5 mainly innervate cranial, cervical, and thoracic viscera. 4. Sympathetic innervation of the abdomen and pelvic regions and the adrenals

1	4. Sympathetic innervation of the abdomen and pelvic regions and the adrenals Preganglionic sympathetic fibers may pass through the sympathetic trunk and paravertebral ganglia without synapsing and, together with similar fibers from other levels, form splanchnic nerves (greater, lesser, least, lumbar, and sacral), which pass into the abdomen and pelvic regions (Fig. 1.46). The preganglionic fibers in these nerves are derived from spinal cord levels T5 to L2. The splanchnic nerves generally connect with sympathetic ganglia around the roots of major arteries that branch from the abdominal aorta. These ganglia are part of a large prevertebral plexus that also has input from the parasympathetic part of the autonomic division of the PNS. Postganglionic sympathetic fibers are distributed in extensions of this plexus, predominantly along arteries, to viscera in the abdomen and pelvis.

1	Some of the preganglionic fibers in the prevertebral plexus do not synapse in the sympathetic ganglia of the plexus but pass through the system to the adrenal gland, where they synapse directly with cells of the adrenal medulla. These cells are homologues of sympathetic postganglionic neurons and secrete adrenaline and noradrenaline into the vascular system. The parasympathetic part of the autonomic division of the PNS (Fig. 1.47) leaves cranial and sacral regions of the CNS in association with: cranial nerves III, VII, IX, and X: III, VII, and IX carry parasympathetic fibers to structures within the head and neck only, whereas X (the vagus spinal nerves S2 to S4: sacral parasympathetic fibers innervate inferior abdominal viscera, pelvic viscera, and the arteries associated with erectile tissues of the perineum.

1	Like the visceral motor nerves of the sympathetic part, the visceral motor nerves of the parasympathetic part generally have two neurons in the pathway. The preganglionic neurons are in the CNS, and fibers leave in the cranial nerves. In the sacral region, the preganglionic parasympathetic fibers form special visceral nerves (the pelvic splanchnic nerves), which originate from the anterior rami of S2 to S4 and enter pelvic extensions of the large prevertebral plexus formed around the abdominal aorta. These fibers are distributed to pelvic and abdominal viscera mainly along blood vessels. The postganglionic motor neurons are in the walls of the viscera. In organs of the gastrointestinal system, preganglionic fibers do not have a postganglionic parasympathetic motor neuron in the pathway; instead, preganglionic fibers synapse directly on neurons in the ganglia of the enteric system. The preganglionic parasympathetic motor fibers in CN

1	The preganglionic parasympathetic motor fibers in CN III, VII, and IX separate from the nerves and connect with one of four distinct ganglia, which house postganglionic motor neurons. These four ganglia are near major branches of CN V. Postganglionic fibers leave the ganglia, join the branches of CN V, and are carried to target tissues (salivary, mucous, and lacrimal glands; constrictor muscle of the pupil; and ciliary muscle in the eye) with these branches. The vagus nerve [X] gives rise to visceral branches along its course. These branches contribute to plexuses associated with thoracic viscera or to the large prevertebral plexus in the abdomen and pelvis. Many of these plexuses also contain sympathetic fibers. When present, postganglionic parasympathetic neurons are in the walls of the target viscera. motor fibers.

1	When present, postganglionic parasympathetic neurons are in the walls of the target viscera. motor fibers. Visceral sensory fibers follow the course of sympathetic fibers entering the spinal cord at similar spinal cord levels. However, visceral sensory fibers may also enter the spinal cord at levels other than those associated with motor output. For example, visceral sensory fibers from the heart may enter at levels higher than spinal cord level T1. Visceral sensory fibers that accompany sympathetic fibers are mainly concerned with detecting pain. Visceral sensory fibers accompanying parasympathetic fibers are carried mainly in IX and X and in spinal nerves S2 to S4. Visceral sensory fibers in IX carry information from chemoreceptors and baroreceptors associated with the walls of major arteries in the neck, and from receptors in the pharynx. Visceral sensory fibers in X include those from cervical viscera, and major vessels and viscera in the thorax and abdomen.

1	Visceral sensory fibers in X include those from cervical viscera, and major vessels and viscera in the thorax and abdomen. Visceral sensory fibers from pelvic viscera and the distal parts of the colon are carried in S2 to S4. Visceral sensory fibers associated with parasympathetic fibers primarily relay information to the CNS about the status of normal physiological processes and reflex activities. The enteric system The enteric nervous system consists of motor and sensory neurons and their support cells, which form two interconnected plexuses, the myenteric and submucous nerve plexuses, within the walls of the gastrointestinal tract (Fig. 1.48). Each of these plexuses is formed by: ganglia, which house the nerve cell bodies and associated cells, and bundles of nerve fibers, which pass between ganglia and from the ganglia into surrounding tissues.

1	Neurons in the enteric system are derived from neural crest cells originally associated with occipitocervical and sacral regions. Interestingly, more neurons are reported to be in the enteric system than in the spinal cord itself. Sensory and motor neurons within the enteric system control reflex activity within and between parts of the gastrointestinal system. These reflexes regulate peristalsis, secretomotor activity, and vascular tone. These activities can occur independently of the brain and spinal cord, but can also be modified by input from preganglionic parasympathetic and postganglionic sympathetic fibers. Sensory information from the enteric system is carried back to the CNS by visceral sensory fibers. Nerve plexuses are either somatic or visceral and combine fibers from different sources or levels to form new nerves with specific targets or destinations (Fig. 1.49). Plexuses of the enteric system also generate reflex activity independent of the CNS.

1	Major somatic plexuses formed from the anterior rami of spinal nerves are the cervical (C1 to C4), brachial (C5 to T1), lumbar (L1 to L4), sacral (L4 to S4), and coccygeal (S5 to Co) plexuses. Except for spinal nerve T1, the anterior rami of thoracic spinal nerves remain independent and do not participate in plexuses. Visceral nerve plexuses are formed in association with viscera and generally contain efferent (sympathetic and parasympathetic) and afferent components (Fig. 1.49). These plexuses include cardiac and pulmonary plexuses in the thorax and a large prevertebral plexus in the abdomen anterior to the aorta, which extends inferiorly onto the lateral walls of the pelvis. The massive prevertebral plexus supplies input to and receives output from all abdominal and pelvic viscera. Specific information about the organization and components of the respiratory, gastrointestinal, and urogenital systems will be discussed in each of the succeeding chapters of this text.

1	Specific information about the organization and components of the respiratory, gastrointestinal, and urogenital systems will be discussed in each of the succeeding chapters of this text. Fig. 1.1 The anatomical position, planes, and terms of location and orientation. Feet togethertoes forwardHands by sidespalms forwardFace looking forwardInferior margin of orbit level withtop of external auditory meatusSagittal planeCoronal planeSuperiorAnteriorPosteriorMedialLateralInferiorTransverse, horizontal,or axial plane Fig. 1.2 Cathode ray tube for the production of X-rays. Fig. 1.3 Fluoroscopy unit. Fig. 1.4 Barium sulfate follow-through. Fig. 1.5 Digital subtraction angiogram. Fig. 1.6 Ultrasound examination of the abdomen. Fig. 1.7 Computed tomography scanner. Fig. 1.8 Computed tomography scan of the abdomen at vertebral level L2. Fig. 1.9 A T2-weighted MR image in the sagittal plane of the pelvic viscera in a woman.

1	Fig. 1.8 Computed tomography scan of the abdomen at vertebral level L2. Fig. 1.9 A T2-weighted MR image in the sagittal plane of the pelvic viscera in a woman. Fig. 1.10 T1-weighted (A) and T2-weighted (B) MR images of the brain in the coronal plane. Fig. 1.11 A gamma camera. Fig. 1.12 The axial skeleton and the appendicular skeleton. Fig. 1.13 Accessory and sesamoid bones. A. Radiograph of the ankle region showing an accessory bone (os trigonum). B. Radiograph of the feet showing numerous sesamoid bones and an accessory bone (os naviculare). Fig. 1.14 A developmental series of radiographs showing the progressive ossification of carpal (wrist) bones from 3 (A) to 10 (D) years of age.

1	Fig. 1.14 A developmental series of radiographs showing the progressive ossification of carpal (wrist) bones from 3 (A) to 10 (D) years of age. Fig. 1.15 T1-weighted image in the coronal plane, demonstrating the relatively high signal intensity returned from the femoral heads and proximal femoral necks, consistent with yellow marrow. In this young patient, the vertebral bodies return an intermediate darker signal that represents red marrow. There is relatively little fat in these vertebrae; hence the lower signal return. Fig. 1.16 Radiograph, lateral view, showing fracture of the ulna at the elbow joint (A) and repair of this fracture (B) using internal fixation with a plate and multiple screws.

1	Fig. 1.16 Radiograph, lateral view, showing fracture of the ulna at the elbow joint (A) and repair of this fracture (B) using internal fixation with a plate and multiple screws. Fig. 1.17 Image of the hip joints demonstrating loss of height of the right femoral head with juxta-articular bony sclerosis and subchondral cyst formation secondary to avascular necrosis. There is also significant wasting of the muscles supporting the hip, which is secondary to disuse and pain. Normal left hipBladderAvascular necrosisWasting of gluteal muscle Fig. 1.18 Joints. A. Synovial joint. B. Solid joint. Fig. 1.19 Synovial joints. A. Major features of a synovial joint. B. Accessory structures associated with synovial joints. Fig. 1.20 Various types of synovial joints. A. Condylar (wrist). B. Gliding (radio-ulnar). C. Hinge (elbow). D. Ball and socket (hip). E. Saddle (carpometacarpal of thumb). F. Pivot (atlanto-axial). Fig. 1.21 Solid joints.

1	Fig. 1.21 Solid joints. Fig. 1.22 This operative photograph demonstrates the focal areas of cartilage loss in the patella and femoral condyles throughout the knee joint. Fig. 1.23 This radiograph demonstrates the loss of joint space in the medial compartment and presence of small spiky osteophytic regions at the medial lateral aspect of the joint. OsteophytesLoss of joint space Fig. 1.24 After knee replacement. This radiograph shows the position of the prosthesis. Fig. 1.25 This is a radiograph, anteroposterior view, of the pelvis after a right total hip replacement. There are additional significant degenerative changes in the left hip joint, which will also need to be replaced. Fig. 1.26 Axial inversion recovery MR imaging series, which suppresses fat and soft tissue and leaves high signal intensity where fluid is seen. A muscle tear in the right adductor longus with edema in and around the muscle is shown. Fig. 1.27 Photograph demonstrating varicose veins.

1	Fig. 1.27 Photograph demonstrating varicose veins. Fig. 1.28 Lymphatic vessels mainly collect fluid lost from vascular capillary beds during nutrient exchange processes and deliver it back to the venous side of the vascular system. Fig. 1.29 Regions associated with clusters or a particular abundance of lymph nodes. Cervical nodes(along courseof internaljugular vein)Axillary nodes(in axilla)Deep nodes(related to aortaand celiac trunkand superior andinferior mesentericarteries)Pericranial ring(base of head)Tracheal nodes(nodes related totrachea and bronchi)Inguinal nodes(along course ofinguinal ligament)Femoral nodes(along femoral vein) Fig. 1.30 Major lymphatic vessels that drain into large veins in the neck.

1	Fig. 1.30 Major lymphatic vessels that drain into large veins in the neck. Fig. 1.31 A. This computed tomogram with contrast, in the axial plane, demonstrates the normal common carotid arteries and internal jugular veins with numerous other nonenhancing nodules that represent lymph nodes in a patient with lymphoma. B. This computed tomogram with contrast, in the axial plane, demonstrates a large anterior soft tissue mediastinal mass that represents a lymphoma. Fig. 1.32 CNS and PNS. Fig. 1.33 Arrangement of meninges in the cranial cavity. Fig. 1.34 Differentiation of somites in a “tubular” embryo. Fig. 1.35 Somatic sensory and motor neurons. Blue lines indicate motor nerves and red lines indicate sensory nerves. Somatic sensory neurondeveloping from neural crest cellsEpaxial (back) musclesHypaxial musclesAxon of motor neuronprojects to muscle developingfrom dermatomyotomeSomatic motor neuroncell body in anterior regionof neural tube Fig. 1.36 Dermatomes.

1	Fig. 1.36 Dermatomes. C6 segment of spinal cordSpinal ganglionDermatomyotomeAutonomous region(where overlap ofdermatomes isleast likely)of C6 dermatome(pad of thumb)Skin on the lateral side of the forearm and on thethumb is innervated by C6 spinal level (spinal nerve).The dermis of the skin in this region develops from the somiteinitially associated with the C6 level of the developing spinal cordCaudalCranialSomite Fig. 1.37 Myotomes. C6 segment of spinal cordMuscles that abduct the arm are innervated by C5 and C6 spinal levels (spinal nerves) and develop from somites initially associated with C5 and C6 regions of developing spinal cordC5 segment of spinal cordDermatomyotomeSomite Fig. 1.38 Dermatomes. A. Anterior view. B. Posterior view. Fig. 1.39 Development of the visceral part of the nervous system.

1	Fig. 1.38 Dermatomes. A. Anterior view. B. Posterior view. Fig. 1.39 Development of the visceral part of the nervous system. Motor nerve endingassociated withblood vessels,sweat glands,arrector pili musclesat peripheryPart of neural crest developinginto spinal gangliaVisceral motor ganglionMotor nerve ending associated with visceraDeveloping gastrointestinal tractSensory nerve endingBody cavity(coelom)Visceral sensory neuron developsfrom neural crest and becomespart of spinal ganglionVisceral motorpreganglionicneuron in lateralregion of CNS(spinal cord)Postganglionic motor neuron is outside CNS.An aggregation of postganglionic neuronal cellbodies forms a peripheral visceral motor ganglion. Fig. 1.40 Basic anatomy of a thoracic spinal nerve. Fig. 1.41 Parts of the CNS associated with visceral motor components. SympatheticT1 to L2spinal segmentsBrainstemcranial nervesIII, VII, IX, XS2 to S4spinal segmentsParasympathetic

1	Fig. 1.41 Parts of the CNS associated with visceral motor components. SympatheticT1 to L2spinal segmentsBrainstemcranial nervesIII, VII, IX, XS2 to S4spinal segmentsParasympathetic Fig. 1.42 Sympathetic part of the autonomic division of the PNS. Abdominal visceraHeartOrgansPeripheralSympathetic nerves followsomatic nerves to periphery(glands, smooth muscle)Pelvic visceraGanglion imparEsophageal plexusPrevertebral plexus Fig. 1.43 Course of sympathetic fibers that travel to the periphery in the same spinal nerves in which they travel out of the spinal cord. Gray ramus communicansT10 spinal nervePosteriorramusAnteriorramusPeripheral distribution of sympatheticscarried peripherally by terminal cutaneousbranches of spinal nerve T1 to L2Motor nerve to sweat glands,smooth muscle of bloodvessels, and arrector pilimuscles in the part of T10dermatome supplied by theanterior ramusT10 spinal segmentWhite ramus communicans

1	Fig. 1.44 Course of sympathetic nerves that travel to the periphery in spinal nerves that are not the ones through which they left the spinal cord. Sympathetic paravertebral trunksPeripheral distribution ofascending sympatheticsPeripheral distribution ofdescending sympathetics(C1) C2 to C8T1 to L2L3 to CoWhite ramus communicansGray ramus communicansPosterior rootGray ramus communicansGray ramus communicansAnterior root Fig. 1.45 Course of sympathetic nerves traveling to the heart. Sympathetic cardiac nervesSympathetic cardiac nervesSympathetic trunkCardiac plexusT1 to T4CervicalWhite ramuscommunicansGray ramuscommunicans Fig. 1.46 Course of sympathetic nerves traveling to abdominal and pelvic viscera.

1	Fig. 1.46 Course of sympathetic nerves traveling to abdominal and pelvic viscera. White ramus communicansGray ramus communicansSacral splanchnic nervesLumbar splanchnic nervesLeast splanchnic nervesLesser splanchnic nervesGreater splanchnic nervesPrevertebral plexusand gangliaParavertebralsympathetic trunkAbdominalandpelvic visceraAortaT5 to T9T12T9 to T10(T10 to T11)L1 to L2 Fig. 1.47 Parasympathetic part of the autonomic division of the PNS. Thoracic visceral plexusPrevertebral plexusAbdominal visceraSynapse with nerve cellsof enteric systemErectile tissues of penisand clitorisS2 to S4Sacral parasympatheticoutflow via pelvicsplanchnic nervesCranial parasympatheticoutflow via cranial nervesHeartSubmandibularganglionPterygopalatineganglionOtic ganglionCiliary ganglion[III][VII][IX][X]Pelvic visceraPupillary constrictionTransition from supply by [X]to pelvic splanchnic nervesSalivary glandsLacrimal glandParotid gland Fig. 1.48 Enteric part of the nervous system.

1	Fig. 1.48 Enteric part of the nervous system. Fig. 1.49 Nerve plexuses. C7C6C5C4C3C2C1T1T2T3T4T5T6T7T8T9T10T11T12L1S1S2S3S4S5L2L3L4L5C8GreaterLeastLesserSOMATIC PLEXUSESVISCERAL PLEXUSESCervical plexusanterior rami C1 to C4Brachial plexusanterior rami C5 to T1Lumbar plexusanterior rami L1 to L4Sacral plexusanterior ramiL4 to S4Parasympathetic [X]S2 to S4 pelvic splanchnic nerves(parasympathetic)Pulmonary branchPulmonary branchesCardiac branchesCardiac plexusThoracic aortic plexusEsophageal plexusPrevertebral plexusVagal trunkGanglion imparSacral splanchnic nervesSplanchnicnervesLumbar splanchnicnerves Fig. 1.50 Mechanism for referred pain from an inflamed appendix to the T10 dermatome. Table 1.1 The approximate dosage of radiation exposure as an order of magnitude In the clinic

1	Fig. 1.50 Mechanism for referred pain from an inflamed appendix to the T10 dermatome. Table 1.1 The approximate dosage of radiation exposure as an order of magnitude In the clinic These are extra bones that are not usually found as part of the normal skeleton, but can exist as a normal variant in many people. They are typically found in multiple locations in the wrist and hands, ankles and feet (Fig. 1.13). These should not be mistaken for fractures on imaging. Sesamoid bones are embedded within tendons, the largest of which is the patella. There are many other sesamoids in the body particularly in tendons of the hands and feet, and most frequently in flexor tendons of the thumb and big toe. Degenerative and inflammatory changes of, as well as mechanical stresses on, the accessory bones and sesamoids can cause pain, which can be treated with physiotherapy and targeted steroid injections, but in some severe cases it may be necessary to surgically remove the bone. In the clinic

1	In the clinic Determination of skeletal age Throughout life the bones develop in a predictable way to form the skeletally mature adult at the end of puberty. In western countries skeletal maturity tends to occur between the ages of 20 and 25 years. However, this may well vary according to geography and socioeconomic conditions. Skeletal maturity will also be determined by genetic factors and disease states. Up until the age of skeletal maturity, bony growth and development follows a typically predictable ordered state, which can be measured through either ultrasound, plain radiographs, or MRI scanning. Typically, the nondominant (left) hand is radiographed, and the radiograph is compared to a series of standard radiographs. From these images the bone age can be determined (Fig. 1.14).

1	In certain disease states, such as malnutrition and hypothyroidism, bony maturity may be slow. If the skeletal bone age is significantly reduced from the patient’s true age, treatment may be required. In the healthy individual the bone age accurately represents the true age of the patient. This is important in determining the true age of the subject. This may also have medicolegal importance. In the clinic The bone marrow serves an important function. There are two types of bone marrow, red marrow (otherwise known as myeloid tissue) and yellow marrow. Red blood cells, platelets, and most white blood cells arise from within the red marrow. In the yellow marrow a few white cells are made; however, this marrow is dominated by large fat globules (producing its yellow appearance) (Fig. 1.15). From birth most of the body’s marrow is red; however, as the subject ages, more red marrow is converted into yellow marrow within the medulla of the long and flat bones.

1	From birth most of the body’s marrow is red; however, as the subject ages, more red marrow is converted into yellow marrow within the medulla of the long and flat bones. Bone marrow contains two types of stem cells. Hemopoietic stem cells give rise to the white blood cells, red blood cells, and platelets. Mesenchymal stem cells differentiate into structures that form bone, cartilage, and muscle. There are a number of diseases that may involve the bone marrow, including infection and malignancy. In patients who develop a bone marrow malignancy (e.g., leukemia) it may be possible to harvest nonmalignant cells from the patient’s bone marrow or cells from another person’s bone marrow. The patient’s own marrow can be destroyed with chemotherapy or radiation and the new cells infused. This treatment is bone marrow transplantation. In the clinic

1	In the clinic Fractures occur in normal bone because of abnormal load or stress, in which the bone gives way (Fig. 1.16A). Fractures may also occur in bone that is of poor quality (osteoporosis); in such cases a normal stress is placed upon a bone that is not of sufficient quality to withstand this force and subsequently fractures. In children whose bones are still developing, fractures may occur across the growth plate or across the shaft. These shaft fractures typically involve partial cortical disruption, similar to breaking a branch of a young tree; hence they are termed “greenstick” fractures.

1	After a fracture has occurred, the natural response is to heal the fracture. Between the fracture margins a blood clot is formed into which new vessels grow. A jelly-like matrix is formed, and further migration of collagen-producing cells occurs. On this soft tissue framework, calcium hydroxyapatite is produced by osteoblasts and forms insoluble crystals, and then bone matrix is laid down. As more bone is produced, a callus can be demonstrated forming across the fracture site. Treatment of fractures requires a fracture line reduction. If this cannot be maintained in a plaster of Paris cast, it may require internal or external fixation with screws and metal rods (Fig. 1.16B). In the clinic

1	In the clinic Avascular necrosis is cellular death of bone resulting from a temporary or permanent loss of blood supply to that bone. Avascular necrosis may occur in a variety of medical conditions, some of which have an etiology that is less than clear. A typical site for avascular necrosis is a fracture across the femoral neck in an elderly patient. In these patients there is loss of continuity of the cortical medullary blood flow with loss of blood flow deep to the retinacular fibers. This essentially renders the femoral head bloodless; it subsequently undergoes necrosis and collapses (Fig. 1.17). In these patients it is necessary to replace the femoral head with a prosthesis. In the clinic

1	In the clinic As the skeleton develops, there are stages of intense growth typically around the ages of 7 to 10 years and later in puberty. These growth spurts are associated with increased cellular activity around the growth plate between the head and shaft of a bone. This increase in activity renders the growth plates more vulnerable to injuries, which may occur from dislocation across a growth plate or fracture through a growth plate. Occasionally an injury may result in growth plate compression, destroying that region of the growth plate, which may result in asymmetrical growth across that joint region. All fractures across the growth plate must be treated with care and expediency, requiring fracture reduction. In the clinic

1	In the clinic Degenerative joint disease is commonly known as osteoarthritis or osteoarthrosis. The disorder is related to aging but not caused by aging. Typically there are decreases in water and proteoglycan content within the cartilage. The cartilage becomes more fragile and more susceptible to mechanical disruption (Fig. 1.22). As the cartilage wears, the underlying bone becomes fissured and also thickens. Synovial fluid may be forced into small cracks that appear in the bone’s surface, which produces large cysts. Furthermore, reactive juxta-articular bony nodules are formed (osteophytes) (Fig. 1.23). As these processes occur, there is slight deformation, which alters the biomechanical forces through the joint. This in turn creates abnormal stresses, which further disrupt the joint. In the United States, osteoarthritis accounts for up to one-quarter of primary health care visits and is regarded as a significant problem.

1	In the United States, osteoarthritis accounts for up to one-quarter of primary health care visits and is regarded as a significant problem. The etiology of osteoarthritis is not clear; however, osteoarthritis can occur secondary to other joint diseases, such as rheumatoid arthritis and infection. Overuse of joints and abnormal strains, such as those experienced by people who play sports, often cause one to be more susceptible to chronic joint osteoarthritis. Various treatments are available, including weight reduction, proper exercise, anti-inflammatory drug treatment, and joint replacement (Fig. 1.24). Arthroscopy is a technique of visualizing the inside of a joint using a small telescope placed through a tiny incision in the skin. Arthroscopy can be performed in most joints. However, it is most commonly performed in the knee, shoulder, ankle, and hip joints.

1	Arthroscopy allows the surgeon to view the inside of the joint and its contents. Notably, in the knee, the menisci and the ligaments are easily seen, and it is possible using separate puncture sites and specific instruments to remove the menisci and replace the cruciate ligaments. The advantages of arthroscopy are that it is performed through small incisions, it enables patients to quickly recover and return to normal activity, and it only requires either a light anesthetic or regional anesthesia during the procedure. In the clinic Joint replacement is undertaken for a variety of reasons. These predominantly include degenerative joint disease and joint destruction. Joints that have severely degenerated or lack their normal function are painful. In some patients, the pain may be so severe that it prevents them from leaving the house and undertaking even the smallest of activities without discomfort.

1	Large joints are commonly affected, including the hip, knee, and shoulder. However, with ongoing developments in joint replacement materials and surgical techniques, even small joints of the fingers can be replaced. Typically, both sides of the joint are replaced; in the hip joint the acetabulum will be reamed, and a plastic or metal cup will be introduced. The femoral component will be fitted precisely to the femur and cemented in place (Fig. 1.25). Most patients derive significant benefit from joint replacement and continue to lead an active life afterward. In a minority of patients who have been fitted with a metal acetabular cup and metal femoral component, an aseptic lymphocyte-dominated vasculitis-associated lesion (ALVAL) may develop, possibly caused by a hypersensitivity response to the release of metal ions in adjacent tissues. These patients often have chronic pain and might need additional surgery to replace these joint replacements with safer models. In the clinic

1	In the clinic The importance of fascias A fascia is a thin band of tissue that surrounds muscles, bones, organs, nerves, and blood vessels and often remains uninterrupted as a 3D structure between tissues. It provides important support for tissues and can provide a boundary between structures. Clinically, fascias are extremely important because they often limit the spread of infection and malignant disease. When infections or malignant diseases cross a fascial plain, a primary surgical clearance may require a far more extensive dissection to render the area free of tumor or infection. A typical example of the clinical importance of a fascial layer would be of that covering the psoas muscle. Infection within an intervertebral body secondary to tuberculosis can pass laterally into the psoas muscle. Pus fills the psoas muscle but is limited from further spread by the psoas fascia, which surrounds the muscle and extends inferiorly into the groin pointing below the inguinal ligament.

1	In the clinic Placement of skin incisions and scarring Surgical skin incisions are ideally placed along or parallel to Langer’s lines, which are lines of skin tension that correspond to the orientation of the dermal collagen fibers. They tend to run in the same direction as the underlying muscle fibers and incisions that are made along these lines tend to heal better with less scarring. In contrast, incisions made perpendicular to Langer’s lines are more likely to heal with a prominent scar and in some severe cases can lead to raised, firm, hypertrophic, or keloid, scars. In the clinic Muscle paralysis is the inability to move a specific muscle or muscle group and may be associated with other neurological abnormalities, including loss of sensation. Major causes include stroke, trauma, poliomyelitis, and iatrogenic factors. Paralysis may be due to abnormalities in the brain, the spinal cord, and the nerves supplying the muscles.

1	In the long term, muscle paralysis will produce secondary muscle wasting and overall atrophy of the region due to disuse. In the clinic Muscle atrophy is a wasting disorder of muscle. It can be produced by a variety of causes, which include nerve damage to the muscle and disuse. Muscle atrophy is an important problem in patients who have undergone long-term rest or disuse, requiring extensive rehabilitation and muscle building exercises to maintain normal activities of daily living. In the clinic Muscle injuries and strains tend to occur in specific muscle groups and usually are related to a sudden exertion and muscle disruption. They typically occur in athletes.

1	In the clinic Muscle injuries and strains tend to occur in specific muscle groups and usually are related to a sudden exertion and muscle disruption. They typically occur in athletes. Muscle tears may involve a small interstitial injury up to a complete muscle disruption (Fig. 1.26). It is important to identify which muscle groups are affected and the extent of the tear to facilitate treatment and obtain a prognosis, which will determine the length of rehabilitation necessary to return to normal activity. In the clinic

1	In the clinic Atherosclerosis is a disease that affects arteries. There is a chronic inflammatory reaction in the walls of the arteries, with deposition of cholesterol and fatty proteins. This may in turn lead to secondary calcification, with reduction in the diameter of the vessels impeding distal flow. The plaque itself may be a site for attraction of platelets that may “fall off” (embolize) distally. Plaque fissuring may occur, which allows fresh clots to form and occlude the vessel. The importance of atherosclerosis and its effects depend upon which vessel is affected. If atherosclerosis occurs in the carotid artery, small emboli may form and produce a stroke. In the heart, plaque fissuring may produce an acute vessel thrombosis, producing a myocardial infarction (heart attack). In the legs, chronic narrowing of vessels may limit the ability of the patient to walk and ultimately cause distal ischemia and gangrene of the toes. In the clinic

1	In the clinic Varicose veins are tortuous dilated veins that typically occur in the legs, although they may occur in the superficial veins of the arm and in other organs. In normal individuals the movement of adjacent leg muscles pumps the blood in the veins to the heart. Blood is also pumped from the superficial veins through the investing layer of fascia of the leg into the deep veins. Valves in these perforating veins may become damaged, allowing blood to pass in the opposite direction. This increased volume and pressure produces dilatation and tortuosity of the superficial veins (Fig. 1.27). Apart from the unsightliness of larger veins, the skin may become pigmented and atrophic with a poor response to tissue trauma. In some patients even small trauma may produce skin ulceration, which requires elevation of the limb and application of pressure bandages to heal.

1	Treatment of varicose veins depends on their location, size, and severity. Typically the superficial varicose veins can be excised and stripped, allowing blood only to drain into the deep system. In the clinic All organs require a blood supply from the arteries and drainage by veins. Within most organs there are multiple ways of perfusing the tissue such that if the main vessel feeding the organ or vein draining the organ is blocked, a series of smaller vessels (collateral vessels) continue to supply and drain the organ. In certain circumstances, organs have more than one vessel perfusing them, such as the hand, which is supplied by the radial and ulnar arteries. Loss of either the radial or the ulnar artery may not produce any symptoms of reduced perfusion to the hand.

1	There are circumstances in which loss of a vein produces significant venous collateralization. Some of these venous collaterals become susceptible to bleeding. This is a considerable problem in patients who have undergone portal vein thrombosis or occlusion, where venous drainage from the gut bypasses the liver through collateral veins to return to the systemic circulation. Normal vascular anastomoses associated with an organ are important. Some organs, such as the duodenum, have a dual blood supply arising from the branches of the celiac trunk and also from the branches of the superior mesenteric artery. Should either of these vessels be damaged, blood supply will be maintained to the organ. The brain has multiple vessels supplying it, dominated by the carotid arteries and the vertebral arteries. Vessels within the brain are end arteries and have a poor collateral circulation; hence any occlusion will produce long-term cerebral damage. In the clinic

1	In the clinic Lymph nodes are efficient filters and have an internal honeycomb of reticular connective tissue filled with lymphocytes. These lymphocytes act on bacteria, viruses, and other bodily cells to destroy them. Lymph nodes tend to drain specific areas, and if infection occurs within a drainage area, the lymph node will become active. The rapid cell turnover and production of local inflammatory mediators may cause the node to enlarge and become tender. Similarly, in patients with malignancy the lymphatics may drain metastasizing cells to the lymph nodes. These can become enlarged and inflamed and will need to be removed if clinically symptomatic. Lymph nodes may become diffusely enlarged in certain systemic illnesses (e.g., viral infection), or local groups may become enlarged with primary lymph node malignancies, such as lymphoma (Fig. 1.31). In the clinic

1	In the clinic A knowledge of dermatomes and myotomes is absolutely fundamental to carrying out a neurological examination. A typical dermatome map is shown in Fig. 1.38. Clinically, a dermatome is that area of skin supplied by a single spinal nerve or spinal cord level. A myotome is that region of skeletal muscle innervated by a single spinal nerve or spinal cord level. Most individual muscles of the body are innervated by more than one spinal cord level, so the evaluation of myotomes is usually accomplished by testing movements of joints or muscle groups. In the clinic

1	In the clinic Referred pain occurs when sensory information comes to the spinal cord from one location but is interpreted by the CNS as coming from another location innervated by the same spinal cord level. Usually, this happens when the pain information comes from a region, such as the gut, which has a low amount of sensory output. These afferents converge on neurons at the same spinal cord level that receive information from the skin, which is an area with a high amount of sensory output. As a result, pain from the normally low output region is interpreted as coming from the normally high output region. Pain is most often referred from a region innervated by the visceral part of the nervous system to a region innervated, at the same spinal cord level, by the somatic side of the nervous system.

1	Pain is most often referred from a region innervated by the visceral part of the nervous system to a region innervated, at the same spinal cord level, by the somatic side of the nervous system. to another. For example, irritation of the peritoneum on the inferior surface of the diaphragm, which is innervated by the phrenic nerve, can be referred to the skin on the top of the shoulder, which is innervated by other somatic nerves arising at the same spinal cord level. A young man sought medical care because of central abdominal pain that was diffuse and colicky. After some hours, the pain began to localize in the right iliac fossa and became constant. He was referred to an abdominal surgeon, who removed a grossly inflamed appendix. The patient made an uneventful recovery.

1	When the appendix becomes inflamed, the visceral sensory fibers are stimulated. These fibers enter the spinal cord with the sympathetic fibers at spinal cord level T10. The pain is referred to the dermatome of T10, which is in the umbilical region (Fig. 1.50). The pain is diffuse, not focal; every time a peristaltic wave passes through the ileocecal region, the pain recurs. This intermittent type of pain is referred to as colic. In the later stages of the disease, the appendix contacts and irritates the parietal peritoneum in the right iliac fossa, which is innervated by somatic sensory nerves. This produces a constant focal pain, which predominates over the colicky pain that the patient felt some hours previously. The patient no longer interprets the referred pain from the T10 dermatome.

1	Although this is a typical history for appendicitis, it should always be borne in mind that the patient’s symptoms and signs may vary. The appendix is situated in a retrocecal position in approximately 70% of patients; therefore it may never contact the parietal peritoneum anteriorly in the right iliac fossa. It is also possible that the appendix is long and may directly contact other structures. As a consequence, the patient may have other symptoms (e.g., the appendix may contact the ureter, and the patient may then develop urological symptoms). Although appendicitis is common, other disorders, for example of the bowel and pelvis, may produce similar symptoms. The Body In the clinic—cont’d

1	Although appendicitis is common, other disorders, for example of the bowel and pelvis, may produce similar symptoms. The Body In the clinic—cont’d The back consists of the posterior aspect of the body and provides the musculoskeletal axis of support for the trunk. Bony elements consist mainly of the vertebrae, although proximal elements of the ribs, superior aspects of the pelvic bones, and posterior basal regions of the skull contribute to the back’s skeletal framework (Fig. 2.1). Associated muscles interconnect the vertebrae and ribs with each other and with the pelvis and skull. The back contains the spinal cord and proximal parts of the spinal nerves, which send and receive information to and from most of the body.

1	The skeletal and muscular elements of the back support the body’s weight, transmit forces through the pelvis to the lower limbs, carry and position the head, and brace and help maneuver the upper limbs. The vertebral column is positioned posteriorly in the body at the midline. When viewed laterally, it has a number of curvatures (Fig. 2.2): The primary curvature of the vertebral column is concave anteriorly, reflecting the original shape of the embryo, and is retained in the thoracic and sacral regions in adults. Secondary curvatures, which are concave posteriorly, form in the cervical and lumbar regions and bring the center of gravity into a vertical line, which allows the body’s weight to be balanced on the vertebral column in a way that expends the least amount of muscular energy to maintain an upright bipedal stance. As stresses on the back increase from the cervical to lumbar regions, lower back problems are common.

1	As stresses on the back increase from the cervical to lumbar regions, lower back problems are common. Muscles of the back consist of extrinsic and intrinsic groups: The extrinsic muscles of the back move the upper limbs and the ribs. The intrinsic muscles of the back maintain posture and move the vertebral column; these movements include flexion (anterior bending), extension, lateral flexion, and rotation (Fig. 2.3). Although the amount of movement between any two vertebrae is limited, the effects between vertebrae are additive along the length of the vertebral column. Also, freedom of movement and extension are limited in the thoracic region relative to the lumbar part of the vertebral column. Muscles in more anterior regions flex the vertebral column.

1	In the cervical region, the first two vertebrae and associated muscles are specifically modified to support and position the head. The head flexes and extends, in the nodding motion, on vertebra CI, and rotation of the head occurs as vertebra CI moves on vertebra CII (Fig. 2.3). Protection of the nervous system The vertebral column and associated soft tissues of the back contain the spinal cord and proximal parts of the spinal nerves (Fig. 2.4). The more distal parts of the spinal nerves pass into all other regions of the body, including certain regions of the head.

1	The major bones of the back are the 33 vertebrae (Fig. 2.5). The number and specific characteristics of the vertebrae vary depending on the body region with which they are associated. There are seven cervical, twelve thoracic, five lumbar, five sacral, and three to four coccygeal vertebrae. The sacral vertebrae fuse into a single bony element, the sacrum. The coccygeal vertebrae are rudimentary in structure, vary in number from three to four, and often fuse into a single coccyx. A typical vertebra consists of a vertebral body and a vertebral arch (Fig. 2.6). The vertebral body is anterior and is the major weightbearing component of the bone. It increases in size from vertebra CII to vertebra LV. Fibrocartilaginous intervertebral discs separate the vertebral bodies of adjacent vertebrae.

1	The vertebral arch is firmly anchored to the posterior surface of the vertebral body by two pedicles, which form the lateral pillars of the vertebral arch. The roof of the vertebral arch is formed by right and left laminae, which fuse at the midline. The vertebral arches of the vertebrae are aligned to form the lateral and posterior walls of the vertebral canal, which extends from the first cervical vertebra (CI) to the last sacral vertebra (vertebra SV). This bony canal contains the spinal cord and its protective membranes, together with blood vessels, connective tissue, fat, and proximal parts of spinal nerves. The vertebral arch of a typical vertebra has a number of characteristic projections, which serve as: attachments for muscles and ligaments, levers for the action of muscles, and sites of articulation with adjacent vertebrae. A spinous process projects posteriorly and generally inferiorly from the roof of the vertebral arch.

1	A spinous process projects posteriorly and generally inferiorly from the roof of the vertebral arch. On each side of the vertebral arch, a transverse process extends laterally from the region where a lamina meets a pedicle. From the same region, a superior articular process and an inferior articular process articulate with similar processes on adjacent vertebrae. Each vertebra also contains rib elements. In the thorax, these costal elements are large and form ribs, which articulate with the vertebral bodies and transverse processes. In all other regions, these rib elements are small and are incorporated into the transverse processes. Occasionally, they develop into ribs in regions other than the thorax, usually in the lower cervical and upper lumbar regions. Muscles in the back can be classified as extrinsic or intrinsic based on their embryological origin and type of innervation (Fig. 2.7).

1	Muscles in the back can be classified as extrinsic or intrinsic based on their embryological origin and type of innervation (Fig. 2.7). The extrinsic muscles are involved with movements of the upper limbs and thoracic wall and, in general, are innervated by anterior rami of spinal nerves. The superficial group of these muscles is related to the upper limbs, while the intermediate layer of muscles is associated with the thoracic wall. All of the intrinsic muscles of the back are deep in position and are innervated by the posterior rami of spinal nerves. They support and move the vertebral column and participate in moving the head. One group of intrinsic muscles also moves the ribs relative to the vertebral column. The spinal cord lies within a bony canal formed by adjacent vertebrae and soft tissue elements (the vertebral canal) (Fig. 2.8): The anterior wall is formed by the vertebral bodies of the vertebrae, intervertebral discs, and associated ligaments.

1	The anterior wall is formed by the vertebral bodies of the vertebrae, intervertebral discs, and associated ligaments. The lateral walls and roof are formed by the vertebral arches and ligaments. Within the vertebral canal, the spinal cord is surrounded by a series of three connective tissue membranes (the meninges): The pia mater is the innermost membrane and is intimately associated with the surface of the spinal cord. The second membrane, the arachnoid mater, is separated from the pia by the subarachnoid space, which contains cerebrospinal fluid. The thickest and most external of the membranes, the dura mater, lies directly against, but is not attached to, the arachnoid mater. In the vertebral canal, the dura mater is separated from surrounding bone by an extradural (epidural) space containing loose connective tissue, fat, and a venous plexus.

1	In the vertebral canal, the dura mater is separated from surrounding bone by an extradural (epidural) space containing loose connective tissue, fat, and a venous plexus. The 31 pairs of spinal nerves are segmental in distribution and emerge from the vertebral canal between the pedicles of adjacent vertebrae. There are eight pairs of cervical nerves (C1 to C8), twelve thoracic (T1 to T12), five lumbar (L1 to L5), five sacral (S1 to S5), and one coccygeal (Co). Each nerve is attached to the spinal cord by a posterior root and an anterior root (Fig. 2.9). After exiting the vertebral canal, each spinal nerve branches into: a posterior ramus—collectively, the small posterior rami innervate the back; and an anterior ramus—the much larger anterior rami innervate most other regions of the body except the head, which is innervated predominantly, but not exclusively, by cranial nerves.

1	The anterior rami form the major somatic plexuses (cervical, brachial, lumbar, and sacral) of the body. Major visceral components of the PNS (sympathetic trunk and prevertebral plexus) of the body are also associated mainly with the anterior rami of spinal nerves. Cervical regions of the back constitute the skeletal and much of the muscular framework of the neck, which in turn supports and moves the head (Fig. 2.10). The brain and cranial meninges are continuous with the spinal cord meninges at the foramen magnum of the skull. The paired vertebral arteries ascend, one on each side, through foramina in the transverse processes of cervical vertebrae and pass through the foramen magnum to participate, with the internal carotid arteries, in supplying blood to the brain. Thorax, abdomen, and pelvis

1	Thorax, abdomen, and pelvis The different regions of the vertebral column contribute to the skeletal framework of the thorax, abdomen, and pelvis (Fig. 2.10). In addition to providing support for each of these parts of the body, the vertebrae provide attachments for muscles and fascia, and articulation sites for other bones. The anterior rami of spinal nerves associated with the thorax, abdomen, and pelvis pass into these parts of the body from the back. The bones of the back provide extensive attachments for muscles associated with anchoring and moving the upper limbs on the trunk. This is less true of the lower limbs, which are firmly anchored to the vertebral column through articulation of the pelvic bones with the sacrum. The upper and lower limbs are innervated by anterior rami of spinal nerves that emerge from cervical and lumbosacral levels, respectively, of the vertebral column.

1	During development, the vertebral column grows much faster than the spinal cord. As a result, the spinal cord does not extend the entire length of the vertebral canal (Fig. 2.11). In the adult, the spinal cord typically ends between vertebrae LI and LII, although it can end as high as vertebra TXII and as low as the disc between vertebrae LII and LIII. Spinal nerves originate from the spinal cord at increasingly oblique angles from vertebrae CI to Co, and the nerve roots pass in the vertebral canal for increasingly longer distances. Their spinal cord level of origin therefore becomes increasingly dissociated from their vertebral column level of exit. This is particularly evident for lumbar and sacral spinal nerves. Each spinal nerve exits the vertebral canal laterally through an intervertebral foramen (Fig. 2.12). The foramen is formed between adjacent vertebral arches and is closely related to intervertebral joints:

1	The superior and inferior margins are formed by notches in adjacent pedicles. The posterior margin is formed by the articular processes of the vertebral arches and the associated joint. The anterior border is formed by the intervertebral disc between the vertebral bodies of the adjacent vertebrae. Any pathology that occludes or reduces the size of an intervertebral foramen, such as bone loss, herniation of the intervertebral disc, or dislocation of the zygapophysial joint (the joint between the articular processes), can affect the function of the associated spinal nerve. Innervation of the back Posterior branches of spinal nerves innervate the intrinsic muscles of the back and adjacent skin. The cutaneous distribution of these posterior rami extends into the gluteal region of the lower limb and the posterior aspect of the head. Parts of dermatomes innervated by the posterior rami of spinal nerves are shown in Fig. 2.13.

1	Skeletal components of the back consist mainly of the vertebrae and associated intervertebral discs. The skull, scapulae, pelvic bones, and ribs also contribute to the bony framework of the back and provide sites for muscle attachment. There are approximately 33 vertebrae, which are subdivided into five groups based on morphology and location (Fig. 2.14): The seven cervical vertebrae between the thorax and skull are characterized mainly by their small size and the presence of a foramen in each transverse process (Figs. 2.14 and 2.15). The 12 thoracic vertebrae are characterized by their articulated ribs (Figs. 2.14 and 2.16); although all vertebrae have rib elements, these elements are small and are incorporated into the transverse processes in regions other than the thorax; but in the thorax, the ribs are separate bones and articulate via synovial joints with the vertebral bodies and transverse processes of the associated vertebrae.

1	Inferior to the thoracic vertebrae are five lumbar vertebrae, which form the skeletal support for the posterior abdominal wall and are characterized by their large size (Figs. 2.14 and 2.17). Next are five sacral vertebrae fused into one single bone called the sacrum, which articulates on each side with a pelvic bone and is a component of the pelvic wall. Inferior to the sacrum is a variable number, usually four, of coccygeal vertebrae, which fuse into a single small triangular bone called the coccyx. In the embryo, the vertebrae are formed intersegmentally from cells called sclerotomes, which originate from adjacent somites (Fig. 2.18). Each vertebra is derived from the cranial parts of the two somites below, one on each side, and the caudal parts of the two somites above. The spinal nerves develop segmentally and pass between the forming vertebrae.

1	A typical vertebra consists of a vertebral body and a posterior vertebral arch (Fig. 2.19). Extending from the vertebral arch are a number of processes for muscle attachment and articulation with adjacent bone. The vertebral body is the weight-bearing part of the vertebra and is linked to adjacent vertebral bodies by intervertebral discs and ligaments. The size of vertebral bodies increases inferiorly as the amount of weight supported increases. The vertebral arch forms the lateral and posterior parts of the vertebral foramen. The vertebral foramina of all the vertebrae together form the vertebral canal, which contains and protects the spinal cord. Superiorly, the vertebral canal is continuous, through the foramen magnum of the skull, with the cranial cavity of the head. The vertebral arch of each vertebra consists of pedicles and laminae (Fig. 2.19): The two pedicles are bony pillars that attach the vertebral arch to the vertebral body.

1	The vertebral arch of each vertebra consists of pedicles and laminae (Fig. 2.19): The two pedicles are bony pillars that attach the vertebral arch to the vertebral body. The two laminae are flat sheets of bone that extend from each pedicle to meet in the midline and form the roof of the vertebral arch. A spinous process projects posteriorly and inferiorly from the junction of the two laminae and is a site for muscle and ligament attachment. A transverse process extends posterolaterally from the junction of the pedicle and lamina on each side and is a site for muscle and ligament attachment, and for articulation with ribs in the thoracic region. Also projecting from the region where the pedicles join the laminae are superior and inferior articular processes (Fig. 2.19), which articulate with the inferior and superior articular processes, respectively, of adjacent vertebrae.

1	Between the vertebral body and the origin of the articular processes, each pedicle is notched on its superior and inferior surfaces. These superior and inferior vertebral notches participate in forming intervertebral foramina. The seven cervical vertebrae are characterized by their small size and by the presence of a foramen in each transverse process. A typical cervical vertebra has the following features (Fig. 2.20A): The vertebral body is short in height and square shaped when viewed from above and has a concave superior surface and a convex inferior surface. Each transverse process is trough shaped and perforated by a round foramen transversarium. The spinous process is short and bifid. The vertebral foramen is triangular. The first and second cervical vertebrae—the atlas and axis—are specialized to accommodate movement of the head.

1	The spinous process is short and bifid. The vertebral foramen is triangular. The first and second cervical vertebrae—the atlas and axis—are specialized to accommodate movement of the head. Vertebra CI (the atlas) articulates with the head (Fig. 2.21). Its major distinguishing feature is that it lacks a vertebral body (Fig. 2.20B). In fact, the vertebral body of CI fuses onto the body of CII during development to become the dens of CII. As a result, there is no intervertebral disc between CI and CII. When viewed from above, the atlas is ring shaped and composed of two lateral masses interconnected by an anterior arch and a posterior arch. Each lateral mass articulates above with an occipital condyle of the skull and below with the superior articular process of vertebra CII (the axis). The superior articular surfaces are bean shaped and concave, whereas the inferior articular surfaces are almost circular and flat.

1	The atlanto-occipital joint allows the head to nod up and down on the vertebral column. The posterior surface of the anterior arch has an articular facet for the dens, which projects superiorly from the vertebral body of the axis. The dens is held in position by a strong transverse ligament of atlas posterior to it and spanning the distance between the oval attachment facets on the medial surfaces of the lateral masses of the atlas. The dens acts as a pivot that allows the atlas and attached head to rotate on the axis, side to side. The transverse processes of the atlas are large and protrude further laterally than those of the other cervical vertebrae and act as levers for muscle action, particularly for muscles that move the head at the atlanto-axial joints.

1	The axis is characterized by the large tooth-like dens, which extends superiorly from the vertebral body (Figs. 2.20B and 2.21). The anterior surface of the dens has an oval facet for articulation with the anterior arch of the atlas. The two superolateral surfaces of the dens possess circular impressions that serve as attachment sites for strong alar ligaments, one on each side, which connect the dens to the medial surfaces of the occipital condyles. These alar ligaments check excessive rotation of the head and atlas relative to the axis. The twelve thoracic vertebrae are all characterized by their articulation with ribs. A typical thoracic vertebra has two partial facets (superior and inferior costal facets) on each side of the vertebral body for articulation with the head of its own rib and the head of the rib below (Fig. 2.20C). The superior costal facet is much larger than the inferior costal facet.

1	Each transverse process also has a facet (transverse costal facet) for articulation with the tubercle of its own rib. The vertebral body of the vertebra is somewhat heart shaped when viewed from above, and the vertebral foramen is circular. The five lumbar vertebrae are distinguished from vertebrae in other regions by their large size (Fig. 2.20D). Also, they lack facets for articulation with ribs. The transverse processes are generally thin and long, with the exception of those on vertebra LV, which are massive and somewhat cone shaped for the attachment of iliolumbar ligaments to connect the transverse processes to the pelvic bones. The vertebral body of a typical lumbar vertebra is cylindrical and the vertebral foramen is triangular in shape and larger than in the thoracic vertebrae.

1	The vertebral body of a typical lumbar vertebra is cylindrical and the vertebral foramen is triangular in shape and larger than in the thoracic vertebrae. The sacrum is a single bone that represents the five fused sacral vertebrae (Fig. 2.20E). It is triangular in shape with the apex pointed inferiorly, and is curved so that it has a concave anterior surface and a correspondingly convex posterior surface. It articulates above with vertebra LV and below with the coccyx. It has two large L-shaped facets, one on each lateral surface, for articulation with the pelvic bones. The posterior surface of the sacrum has four pairs of posterior sacral foramina, and the anterior surface has four pairs of anterior sacral foramina for the passage of the posterior and anterior rami, respectively, of S1 to S4 spinal nerves. The posterior wall of the vertebral canal may be incomplete near the inferior end of the sacrum.

1	The posterior wall of the vertebral canal may be incomplete near the inferior end of the sacrum. The coccyx is a small triangular bone that articulates with the inferior end of the sacrum and represents three to four fused coccygeal vertebrae (Fig. 2.20F). It is characterized by its small size and by the absence of vertebral arches and therefore a vertebral canal. Intervertebral foramina are formed on each side between adjacent parts of vertebrae and associated intervertebral discs (Fig. 2.22). The foramina allow structures, such as spinal nerves and blood vessels, to pass in and out of the vertebral canal. An intervertebral foramen is formed by the inferior vertebral notch on the pedicle of the vertebra above and the superior vertebral notch on the pedicle of the vertebra below. The foramen is bordered: posteriorly by the zygapophysial joint between the articular processes of the two vertebrae, and anteriorly by the intervertebral disc and adjacent vertebral bodies.

1	Each intervertebral foramen is a confined space surrounded by bone and ligament, and by joints. Pathology in any of these structures, and in the surrounding muscles, can affect structures within the foramen. In most regions of the vertebral column, the laminae and spinous processes of adjacent vertebrae overlap to form a reasonably complete bony dorsal wall for the vertebral canal. However, in the lumbar region, large gaps exist between the posterior components of adjacent vertebral arches (Fig. 2.23). These gaps between adjacent laminae and spinous processes become increasingly wide from vertebra LI to vertebra LV. The spaces can be widened further by flexion of the vertebral column. These gaps allow relatively easy access to the vertebral canal for clinical procedures. Joints between vertebrae in the back The two major types of joints between vertebrae are: symphyses between vertebral bodies (Fig. 2.31), and synovial joints between articular processes (Fig. 2.32).

1	The two major types of joints between vertebrae are: symphyses between vertebral bodies (Fig. 2.31), and synovial joints between articular processes (Fig. 2.32). A typical vertebra has a total of six joints with adjacent vertebrae: four synovial joints (two above and two below) and two symphyses (one above and one below). Each symphysis includes an intervertebral disc. Although the movement between any two vertebrae is limited, the summation of movement among all vertebrae results in a large range of movement by the vertebral column. Movements by the vertebral column include flexion, extension, lateral flexion, rotation, and circumduction. Movements by vertebrae in a specific region (cervical, thoracic, and lumbar) are determined by the shape and orientation of joint surfaces on the articular processes and on the vertebral bodies.

1	Movements by vertebrae in a specific region (cervical, thoracic, and lumbar) are determined by the shape and orientation of joint surfaces on the articular processes and on the vertebral bodies. The symphysis between adjacent vertebral bodies is formed by a layer of hyaline cartilage on each vertebral body and an intervertebral disc, which lies between the layers. The intervertebral disc consists of an outer anulus fibrosus, which surrounds a central nucleus pulposus (Fig. 2.31). The anulus fibrosus consists of an outer ring of collagen surrounding a wider zone of fibrocartilage arranged in a lamellar configuration. This arrangement of fibers limits rotation between vertebrae. The nucleus pulposus fills the center of the intervertebral disc, is gelatinous, and absorbs compression forces between vertebrae.

1	The nucleus pulposus fills the center of the intervertebral disc, is gelatinous, and absorbs compression forces between vertebrae. Degenerative changes in the anulus fibrosus can lead to herniation of the nucleus pulposus. Posterolateral herniation can impinge on the roots of a spinal nerve in the intervertebral foramen. The synovial joints between superior and inferior articular processes on adjacent vertebrae are the zygapophysial joints (Fig. 2.32). A thin articular capsule attached to the margins of the articular facets encloses each joint.

1	In cervical regions, the zygapophysial joints slope inferiorly from anterior to posterior and their shape facilitates flexion and extension. In thoracic regions, the joints are oriented vertically and their shape limits flexion and extension, but facilitates rotation. In lumbar regions, the joint surfaces are curved and adjacent processes interlock, thereby limiting range of movement, though flexion and extension are still major movements in the lumbar region. The lateral margins of the upper surfaces of typical cervical vertebrae are elevated into crests or lips termed uncinate processes. These may articulate with the body of the vertebra above to form small “uncovertebral” synovial joints (Fig. 2.33). Joints between vertebrae are reinforced and supported by numerous ligaments, which pass between vertebral bodies and interconnect components of the vertebral arches.

1	Joints between vertebrae are reinforced and supported by numerous ligaments, which pass between vertebral bodies and interconnect components of the vertebral arches. The anterior and posterior longitudinal ligaments are on the anterior and posterior surfaces of the vertebral bodies and extend along most of the vertebral column (Fig. 2.35). The anterior longitudinal ligament is attached superiorly to the base of the skull and extends inferiorly to attach to the anterior surface of the sacrum. Along its length it is attached to the vertebral bodies and intervertebral discs.

1	The posterior longitudinal ligament is on the posterior surfaces of the vertebral bodies and lines the anterior surface of the vertebral canal. Like the anterior longitudinal ligament, it is attached along its length to the vertebral bodies and intervertebral discs. The upper part of the posterior longitudinal ligament that connects CII to the intracranial aspect of the base of the skull is termed the tectorial membrane (see Fig. 2.20B). The ligamenta flava, on each side, pass between the laminae of adjacent vertebrae (Fig. 2.36). These thin, broad ligaments consist predominantly of elastic tissue and form part of the posterior surface of the vertebral canal. Each ligamentum flavum runs between the posterior surface of the lamina on the vertebra below to the anterior surface of the lamina of the vertebra above. The ligamenta flava resist separation of the laminae in flexion and assist in extension back to the anatomical position.

1	The supraspinous ligament connects and passes along the tips of the vertebral spinous processes from vertebra CVII to the sacrum (Fig. 2.37). From vertebra CVII to the skull, the ligament becomes structurally distinct from more caudal parts of the ligament and is called the ligamentum nuchae. The ligamentum nuchae is a triangular, sheet-like structure in the median sagittal plane: The base of the triangle is attached to the skull, from the external occipital protuberance to the foramen magnum. The apex is attached to the tip of the spinous process of vertebra CVII. The deep side of the triangle is attached to the posterior tubercle of vertebra CI and the spinous processes of the other cervical vertebrae. The ligamentum nuchae supports the head. It resists flexion and facilitates returning the head to the anatomical position. The broad lateral surfaces and the posterior edge of the ligament provide attachment for adjacent muscles.

1	Interspinous ligaments pass between adjacent vertebral spinous processes (Fig. 2.38). They attach from the base to the apex of each spinous process and blend with the supraspinous ligament posteriorly and the ligamenta flava anteriorly on each side. Muscles of the back are organized into superficial, intermediate, and deep groups. Muscles in the superficial and intermediate groups are extrinsic muscles because they originate embryologically from locations other than the back. They are innervated by anterior rami of spinal nerves: The superficial group consists of muscles related to and involved in movements of the upper limb. The intermediate group consists of muscles attached to the ribs and may serve a respiratory function. Muscles of the deep group are intrinsic muscles because they develop in the back. They are innervated by posterior rami of spinal nerves and are directly related to movements of the vertebral column and head. Superficial group of back muscles

1	Superficial group of back muscles The muscles in the superficial group are immediately deep to the skin and superficial fascia (Figs. 2.42 to 2.45). They attach the superior part of the appendicular skeleton (clavicle, scapula, and humerus) to the axial skeleton (skull, ribs, and vertebral column). Because these muscles are primarily involved with movements of this part of the appendicular skeleton, they are sometimes referred to as the appendicular group. Muscles in the superficial group include the trapezius, latissimus dorsi, rhomboid major, rhomboid minor, and levator scapulae. The rhomboid major, rhomboid minor, and levator scapulae muscles are located deep to the trapezius muscle in the superior part of the back.

1	Each trapezius muscle is flat and triangular, with the base of the triangle situated along the vertebral column (the muscle’s origin) and the apex pointing toward the tip of the shoulder (the muscle’s insertion) (Fig. 2.43 and Table 2.1). The muscles on both sides together form a trapezoid. The superior fibers of the trapezius, from the skull and upper portion of the vertebral column, descend to attach to the lateral third of the clavicle and to the acromion of the scapula. Contraction of these fibers elevates the scapula. In addition, the superior and inferior fibers work together to rotate the lateral aspect of the scapula upward, which needs to occur when raising the upper limb above the head. Motor innervation of the trapezius is by the accessory nerve [XI], which descends from the neck onto the deep surface of the muscle (Fig. 2.44). Proprioceptive fibers from the trapezius pass in the branches of the cervical plexus and enter the spinal cord at spinal cord levels C3 and C4.

1	The blood supply to the trapezius is from the superficial branch of the transverse cervical artery, the acromial branch of the suprascapular artery, and the dorsal branches of posterior intercostal arteries. Latissimus dorsi is a large, flat triangular muscle that begins in the lower portion of the back and tapers as it ascends to a narrow tendon that attaches to the humerus anteriorly (Figs. 2.42 to 2.45 and Table 2.1). As a result, movements associated with this muscle include extension, adduction, and medial rotation of the upper limb. The latissimus dorsi can also depress the shoulder, preventing its upward movement. The thoracodorsal nerve of the brachial plexus innervates the latissimus dorsi muscle. Associated with this nerve is the thoracodorsal artery, which is the primary blood supply of the muscle. Additional small arteries come from dorsal branches of posterior intercostal and lumbar arteries.

1	Levator scapulae is a slender muscle that descends from the transverse processes of the upper cervical vertebrae to the upper portion of the scapula on its medial border at the superior angle (Figs. 2.43 and 2.45 and Table 2.1). It elevates the scapula and may assist other muscles in rotating the lateral aspect of the scapula inferiorly. The levator scapulae is innervated by branches from the anterior rami of spinal nerves C3 and C4 and the dorsal scapular nerve, and its arterial supply consists of branches primarily from the transverse and ascending cervical arteries. The two rhomboid muscles are inferior to levator scapulae (Fig. 2.45 and Table 2.1). Rhomboid minor is superior to rhomboid major, and is a small, cylindrical muscle that arises from the ligamentum nuchae of the neck and the spinous processes of vertebrae CVII and TI and attaches to the medial scapular border opposite the root of the spine of the scapula.

1	The larger rhomboid major originates from the spinous processes of the upper thoracic vertebrae and attaches to the medial scapular border inferior to rhomboid minor. The two rhomboid muscles work together to retract or pull the scapula toward the vertebral column. With other muscles they may also rotate the lateral aspect of the scapula inferiorly. The dorsal scapular nerve, a branch of the brachial plexus, innervates both rhomboid muscles (Fig. 2.46). Intermediate group of back muscles

1	The dorsal scapular nerve, a branch of the brachial plexus, innervates both rhomboid muscles (Fig. 2.46). Intermediate group of back muscles The muscles in the intermediate group of back muscles consist of two thin muscular sheets in the superior and inferior regions of the back, immediately deep to the muscles in the superficial group (Fig. 2.47 and Table 2.2). Fibers from these two serratus posterior muscles (serratus posterior superior and serratus posterior inferior) pass obliquely outward from the vertebral column to attach to the ribs. This positioning suggests a respiratory function, and at times, these muscles have been referred to as the respiratory group.

1	Serratus posterior superior is deep to the rhomboid muscles, whereas serratus posterior inferior is deep to the latissimus dorsi. Both serratus posterior muscles are attached to the vertebral column and associated structures medially, and either descend (the fibers of the serratus posterior superior) or ascend (the fibers of the serratus posterior inferior) to attach to the ribs. These two muscles therefore elevate and depress the ribs. The serratus posterior muscles are innervated by segmental branches of anterior rami of intercostal nerves. Their vascular supply is provided by a similar segmental pattern through the intercostal arteries. Deep group of back muscles

1	Deep group of back muscles The deep or intrinsic muscles of the back extend from the pelvis to the skull and are innervated by segmental branches of the posterior rami of spinal nerves. They include: the extensors and rotators of the head and neck— the splenius capitis and cervicis (spinotransversales muscles), the extensors and rotators of the vertebral column—the erector spinae and transversospinales, and the short segmental muscles—the interspinales and intertransversarii. The vascular supply to this deep group of muscles is through branches of the vertebral, deep cervical, occipital, transverse cervical, posterior intercostal, subcostal, lumbar, and lateral sacral arteries. The thoracolumbar fascia covers the deep muscles of the back and trunk (Fig. 2.48). This fascial layer is critical to the overall organization and integrity of the region: Superiorly, it passes anteriorly to the serratus posterior muscle and is continuous with deep fascia in the neck.

1	Superiorly, it passes anteriorly to the serratus posterior muscle and is continuous with deep fascia in the neck. In the thoracic region, it covers the deep muscles and separates them from the muscles in the superficial and intermediate groups. Medially, it attaches to the spinous processes of the thoracic vertebrae and, laterally, to the angles of the ribs. The medial attachments of the latissimus dorsi and serratus posterior inferior muscles blend into the thoracolumbar fascia. In the lumbar region, the thoracolumbar fascia consists of three layers: The posterior layer is thick and is attached to the spinous processes of the lumbar vertebrae and sacral vertebrae and to the supraspinous ligament—from these attachments, it extends laterally to cover the erector spinae.

1	The middle layer is attached medially to the tips of the transverse processes of the lumbar vertebrae and intertransverse ligaments—inferiorly, it is attached to the iliac crest and, superiorly, to the lower border of rib XII. The anterior layer covers the anterior surface of the quadratus lumborum muscle (a muscle of the posterior abdominal wall) and is attached medially to the transverse processes of the lumbar vertebrae—inferiorly, it is attached to the iliac crest and, superiorly, it forms the lateral arcuate ligament for attachment of the diaphragm. The posterior and middle layers of the thoracolumbar fascia come together at the lateral margin of the erector spinae (Fig. 2.48). At the lateral border of the quadratus lumborum, the anterior layer joins them and forms the aponeurotic origin for the transversus abdominis muscle of the abdominal wall.

1	The two spinotransversales muscles run from the spinous processes and ligamentum nuchae upward and laterally (Fig. 2.49 and Table 2.3): The splenius capitis is a broad muscle attached to the occipital bone and mastoid process of the temporal bone. The splenius cervicis is a narrow muscle attached to the transverse processes of the upper cervical vertebrae. Together the spinotransversales muscles draw the head backward, extending the neck. Individually, each muscle rotates the head to one side—the same side as the contracting muscle.

1	Together the spinotransversales muscles draw the head backward, extending the neck. Individually, each muscle rotates the head to one side—the same side as the contracting muscle. The erector spinae is the largest group of intrinsic back muscles. The muscles lie posterolaterally to the vertebral column between the spinous processes medially and the angles of the ribs laterally. They are covered in the thoracic and lumbar regions by thoracolumbar fascia and the serratus posterior inferior, rhomboid, and splenius muscles. The mass arises from a broad, thick tendon attached to the sacrum, the spinous processes of the lumbar and lower thoracic vertebrae, and the iliac crest (Fig. 2.50 and Table 2.4). It divides in the upper lumbar region into three vertical columns of muscle, each of which is further subdivided regionally (lumborum, thoracis, cervicis, and capitis), depending on where the muscles attach superiorly.

1	The outer or most laterally placed column of the erector spinae muscles is the iliocostalis, which is associated with the costal elements and passes from the common tendon of origin to multiple insertions into the angles of the ribs and the transverse processes of the lower cervical vertebrae. The middle or intermediate column is the longissimus, which is the largest of the erector spinae subdivision extending from the common tendon of origin to the base of the skull. Throughout this vast expanse, the lateral positioning of the longissimus muscle is in the area of the transverse processes of the various vertebrae. The most medial muscle column is the spinalis, which is the smallest of the subdivisions and interconnects the spinous processes of adjacent vertebrae. The spinalis is most constant in the thoracic region and is generally absent in the cervical region. It is associated with a deeper muscle (the semispinalis capitis) as the erector spinae group approaches the skull.

1	The muscles in the erector spinae group are the primary extensors of the vertebral column and head. Acting bilaterally, they straighten the back, returning it to the upright position from a flexed position, and pull the head posteriorly. They also participate in controlling vertebral column flexion by contracting and relaxing in a coordinated fashion. Acting unilaterally, they bend the vertebral column laterally. In addition, unilateral contractions of muscles attached to the head turn the head to the actively contracting side. The transversospinales muscles run obliquely upward and medially from transverse processes to spinous processes, filling the groove between these two vertebral projections (Fig. 2.51 and Table 2.5). They are deep to the erector spinae and consist of three major subgroups—the semispinalis, multifidus, and rotatores muscles.

1	The semispinalis muscles are the most superficial collection of muscle fibers in the transversospinales group. These muscles begin in the lower thoracic region and end by attaching to the skull, crossing between four and six vertebrae from their point of origin to point of attachment. Semispinalis muscles are found in the thoracic and cervical regions, and attach to the occipital bone at the base of the skull. Deep to the semispinalis is the second group of muscles, the multifidus. Muscles in this group span the length of the vertebral column, passing from a lateral point of origin upward and medially to attach to spinous processes and spanning between two and four vertebrae. The multifidus muscles are present throughout the length of the vertebral column but are best developed in the lumbar region.

1	The small rotatores muscles are the deepest of the transversospinales group. They are present throughout the length of the vertebral column but are best developed in the thoracic region. Their fibers pass upward and medially from transverse processes to spinous processes crossing two vertebrae (long rotators) or attaching to an adjacent vertebra (short rotators). When muscles in the transversospinales group contract bilaterally, they extend the vertebral column, an action similar to that of the erector spinae group. However, when muscles on only one side contract, they pull the spinous processes toward the transverse processes on that side, causing the trunk to turn or rotate in the opposite direction.

1	One muscle in the transversospinales group, the semispinalis capitis, has a unique action because it attaches to the skull. Contracting bilaterally, this muscle pulls the head posteriorly, whereas unilateral contraction pulls the head posteriorly and turns it, causing the chin to move superiorly and turn toward the side of the contracting muscle. These actions are similar to those of the upper erector spinae. The two groups of segmental muscles (Fig. 2.51 and Table 2.6) are deeply placed in the back and innervated by posterior rami of spinal nerves. The first group of segmental muscles are the levatores costarum muscles, which arise from the transverse processes of vertebrae CVII and TI to TXI. They have an oblique lateral and downward direction and insert into the rib below the vertebra of origin in the area of the tubercle. Contraction elevates the ribs.

1	The second group of segmental muscles are the true segmental muscles of the back—the interspinales, which pass between adjacent spinous processes, and the intertransversarii, which pass between adjacent transverse processes. These postural muscles stabilize adjoining vertebrae during movements of the vertebral column to allow more effective action of the large muscle groups. A small group of deep muscles in the upper cervical region at the base of the occipital bone move the head. They connect vertebra CI (the atlas) to vertebra CII (the axis) and connect both vertebrae to the base of the skull. Because of their location they are sometimes referred to as suboccipital muscles (Figs. 2.51 and 2.52 and Table 2.7). They include, on each side: rectus capitis posterior major, rectus capitis posterior minor, obliquus capitis inferior, and obliquus capitis superior.

1	Contraction of the suboccipital muscles extends and rotates the head at the atlanto-occipital and atlanto-axial joints, respectively. The suboccipital muscles are innervated by the posterior ramus of the first cervical nerve, which enters the area between the vertebral artery and the posterior arch of the atlas (Fig. 2.52). The vascular supply to the muscles in this area is from branches of the vertebral and occipital arteries. The suboccipital muscles form the boundaries of the suboccipital triangle, an area that contains several important structures (Fig. 2.52): The rectus capitis posterior major muscle forms the medial border of the triangle. The obliquus capitis superior muscle forms the lateral border. The obliquus capitis inferior muscle forms the inferior border. The contents of the suboccipital triangle include: posterior ramus of CI, vertebral artery, and veins

1	The obliquus capitis inferior muscle forms the inferior border. The contents of the suboccipital triangle include: posterior ramus of CI, vertebral artery, and veins The spinal cord extends from the foramen magnum to approximately the level of the disc between vertebrae LI and LII in adults, although it can end as high as vertebra TXII or as low as the disc between vertebrae LII and LIII (Fig. 2.53). In neonates, the spinal cord extends approximately to vertebra LIII but can reach as low as vertebra LIV. The distal end of the cord (the conus medullaris) is cone shaped. A fine filament of connective tissue (the pial part of the filum terminale) continues inferiorly from the apex of the conus medullaris.

1	The spinal cord is not uniform in diameter along its length. It has two major swellings or enlargements in regions associated with the origin of spinal nerves that innervate the upper and lower limbs. A cervical enlargement occurs in the region associated with the origins of spinal nerves C5 to T1, which innervate the upper limbs. A lumbosacral enlargement occurs in the region associated with the origins of spinal nerves L1 to S3, which innervate the lower limbs. The external surface of the spinal cord is marked by a number of fissures and sulci (Fig. 2.54): The anterior median fissure extends the length of the anterior surface. The posterior median sulcus extends along the posterior surface. The posterolateral sulcus on each side of the posterior surface marks where the posterior rootlets of spinal nerves enter the cord. Internally, the cord has a small central canal surrounded by gray and white matter:

1	Internally, the cord has a small central canal surrounded by gray and white matter: The gray matter is rich in nerve cell bodies, which form longitudinal columns along the cord, and in cross section these columns form a characteristic H-shaped appearance in the central regions of the cord. The white matter surrounds the gray matter and is rich in nerve cell processes, which form large bundles or tracts that ascend and descend in the cord to other spinal cord levels or carry information to and from the brain.

1	The arterial supply to the spinal cord comes from two sources (Fig. 2.55). It consists of: longitudinally oriented vessels, arising superior to the cervical portion of the cord, which descend on the surface of the cord; and feeder arteries that enter the vertebral canal through the intervertebral foramina at every level; these feeder vessels, or segmental spinal arteries, arise predominantly from the vertebral and deep cervical arteries in the neck, the posterior intercostal arteries in the thorax, and the lumbar arteries in the abdomen.

1	After entering an intervertebral foramen, the segmental spinal arteries give rise to anterior and posterior radicular arteries (Fig. 2.55). This occurs at every vertebral level. The radicular arteries follow, and supply, the anterior and posterior roots. At various vertebral levels, the segmental spinal arteries also give off segmental medullary arteries (Fig. 2.55). These vessels pass directly to the longitudinally oriented vessels, reinforcing these.

1	The longitudinal vessels consist of: a single anterior spinal artery, which originates within the cranial cavity as the union of two vessels that arise from the vertebral arteries—the resulting single anterior spinal artery passes inferiorly, approximately parallel to the anterior median fissure, along the surface of the spinal cord; and two posterior spinal arteries, which also originate in the cranial cavity, usually arising directly from a terminal branch of each vertebral artery (the posterior inferior cerebellar artery)—the right and left posterior spinal arteries descend along the spinal cord, each as two branches that bracket the posterolateral sulcus and the connection of posterior roots with the spinal cord.

1	The anterior and posterior spinal arteries are reinforced along their length by eight to ten segmental medullary arteries (Fig. 2.55). The largest of these is the arteria radicularis magna or the artery of Adamkiewicz (Fig. 2.55). This vessel arises in the lower thoracic or upper lumbar region, usually on the left side, and reinforces the arterial supply to the lower portion of the spinal cord, including the lumbar enlargement. Veins that drain the spinal cord form a number of longitudinal channels (Fig. 2.56): Two pairs of veins on each side bracket the connections of the posterior and anterior roots to the cord. One midline channel parallels the anterior median fissure. One midline channel passes along the posterior median sulcus.

1	One midline channel parallels the anterior median fissure. One midline channel passes along the posterior median sulcus. These longitudinal channels drain into an extensive internal vertebral plexus in the extradural (epidural) space of the vertebral canal, which then drains into segmentally arranged vessels that connect with major systemic veins, such as the azygos system in the thorax. The internal vertebral plexus also communicates with intracranial veins.

1	The spinal dura mater is the outermost meningeal membrane and is separated from the bones forming the vertebral canal by an extradural space (Fig. 2.59). Superiorly, it is continuous with the inner meningeal layer of cranial dura mater at the foramen magnum of the skull. Inferiorly, the dural sac dramatically narrows at the level of the lower border of vertebra SII and forms an investing sheath for the pial part of the filum terminale of the spinal cord. This terminal cord-like extension of dura mater (the dural part of the filum terminale) attaches to the posterior surface of the vertebral bodies of the coccyx. As spinal nerves and their roots pass laterally, they are surrounded by tubular sleeves of dura mater, which merge with and become part of the outer covering (epineurium) of the nerves.

1	As spinal nerves and their roots pass laterally, they are surrounded by tubular sleeves of dura mater, which merge with and become part of the outer covering (epineurium) of the nerves. The arachnoid mater is a thin delicate membrane against, but not adherent to, the deep surface of the dura mater (Fig. 2.59). It is separated from the pia mater by the subarachnoid space. The arachnoid mater ends at the level of vertebra SII (see Fig. 2.53). The subarachnoid space between the arachnoid and pia mater contains CSF (Fig. 2.59). The subarachnoid space around the spinal cord is continuous at the foramen magnum with the subarachnoid space surrounding the brain. Inferiorly, the subarachnoid space terminates at approximately the level of the lower border of vertebra SII (see Fig. 2.53).

1	Delicate strands of tissue (arachnoid trabeculae) are continuous with the arachnoid mater on one side and the pia mater on the other; they span the subarachnoid space and interconnect the two adjacent membranes. Large blood vessels are suspended in the subarachnoid space by similar strands of material, which expand over the vessels to form a continuous external coat. The subarachnoid space extends farther inferiorly than the spinal cord. The spinal cord ends at approximately the disc between vertebrae LI and LII, whereas the subarachnoid space extends to approximately the lower border of vertebra SII (see Fig. 2.53). The subarachnoid space is largest in the region inferior to the terminal end of the spinal cord, where it surrounds the cauda equina. As a consequence, CSF can be withdrawn from the subarachnoid space in the lower lumbar region without endangering the spinal cord.

1	The spinal pia mater is a vascular membrane that firmly adheres to the surface of the spinal cord (Fig. 2.59). It extends into the anterior median fissure and reflects as sleeve-like coatings onto posterior and anterior rootlets and roots as they cross the subarachnoid space. As the roots exit the space, the sleeve-like coatings reflect onto the arachnoid mater. On each side of the spinal cord, a longitudinally oriented sheet of pia mater (the denticulate ligament) extends laterally from the cord toward the arachnoid and dura mater (Fig. 2.59). Medially, each denticulate ligament is attached to the spinal cord in a plane that lies between the origins of the posterior and anterior rootlets. Laterally, each denticulate ligament forms a series of triangular extensions along its free border, with the apex of each extension being anchored through the arachnoid mater to the dura mater.

1	Laterally, each denticulate ligament forms a series of triangular extensions along its free border, with the apex of each extension being anchored through the arachnoid mater to the dura mater. The lateral attachments of the denticulate ligaments generally occur between the exit points of adjacent posterior and anterior rootlets. The ligaments function to position the spinal cord in the center of the subarachnoid space. Arrangement of structures in the vertebral canal The vertebral canal is bordered: anteriorly by the bodies of the vertebrae, intervertebral discs, and posterior longitudinal ligament (Fig. 2.60); laterally, on each side by the pedicles and intervertebral foramina; and posteriorly by the laminae and ligamenta flava, and in the median plane the roots of the interspinous ligaments and vertebral spinous processes.

1	Between the walls of the vertebral canal and the dural sac is an extradural space containing a vertebral plexus of veins embedded in fatty connective tissue. The vertebral spinous processes can be palpated through the skin in the midline in thoracic and lumbar regions of the back. Between the skin and spinous processes is a layer of superficial fascia. In lumbar regions, the adjacent spinous processes and the associated laminae on either side of the midline do not overlap, resulting in gaps between adjacent vertebral arches. When carrying out a lumbar puncture (spinal tap), the needle passes between adjacent vertebral spinous processes, through the supraspinous and interspinous ligaments, and enters the extradural space. The needle continues through the dura and arachnoid mater and enters the subarachnoid space, which contains CSF. Each spinal nerve is connected to the spinal cord by posterior and anterior roots (Fig. 2.61):

1	Each spinal nerve is connected to the spinal cord by posterior and anterior roots (Fig. 2.61): The posterior root contains the processes of sensory neurons carrying information to the CNS—the cell bodies of the sensory neurons, which are derived embryologically from neural crest cells, are clustered in a spinal ganglion at the distal end of the posterior root, usually in the intervertebral foramen. The anterior root contains motor nerve fibers, which carry signals away from the CNS—the cell bodies of the primary motor neurons are in anterior regions of the spinal cord. Medially, the posterior and anterior roots divide into rootlets, which attach to the spinal cord. A spinal segment is the area of the spinal cord that gives rise to the posterior and anterior rootlets, which will form a single pair of spinal nerves. Laterally, the posterior and anterior roots on each side join to form a spinal nerve.

1	Each spinal nerve divides, as it emerges from an intervertebral foramen, into two major branches: a small posterior ramus and a much larger anterior ramus (Fig. 2.61): The posterior rami innervate only intrinsic back muscles (the epaxial muscles) and an associated narrow strip of skin on the back. The anterior rami innervate most other skeletal muscles (the hypaxial muscles) of the body, including those of the limbs and trunk, and most remaining areas of the skin, except for certain regions of the head. Near the point of division into anterior and posterior rami, each spinal nerve gives rise to two to four small recurrent meningeal (sinuvertebral) nerves (see Fig. 2.59). These nerves reenter the intervertebral foramen to supply dura, ligaments, intervertebral discs, and blood vessels. All major somatic plexuses (cervical, brachial, lumbar, and sacral) are formed by anterior rami.

1	All major somatic plexuses (cervical, brachial, lumbar, and sacral) are formed by anterior rami. Because the spinal cord is much shorter than the vertebral column, the roots of spinal nerves become longer and pass more obliquely from the cervical to coccygeal regions of the vertebral canal (Fig. 2.62). In adults, the spinal cord terminates at a level approximately between vertebrae LI and LII, but this can range between vertebra TXII and the disc between vertebrae LII and LIII. Consequently, posterior and anterior roots forming spinal nerves emerging between vertebrae in the lower regions of the vertebral column are connected to the spinal cord at higher vertebral levels. Below the end of the spinal cord, the posterior and anterior roots of lumbar, sacral, and coccygeal nerves pass inferiorly to reach their exit points from the vertebral canal. This terminal cluster of roots is the cauda equina. Nomenclature of spinal nerves

1	Nomenclature of spinal nerves There are approximately 31 pairs of spinal nerves (Fig. 2.62), named according to their position with respect to associated vertebrae: eight cervical nerves—C1 to C8, twelve thoracic nerves—T1 to T12, five lumbar nerves—L1 to L5, five sacral nerves—S1 to S5, one coccygeal nerve—Co. The first cervical nerve (C1) emerges from the vertebral canal between the skull and vertebra CI (Fig. 2.63). Therefore cervical nerves C2 to C7 also emerge from the vertebral canal above their respective vertebrae. Because there are only seven cervical vertebrae, C8 emerges between vertebrae CVII and TI. As a consequence, all remaining spinal nerves, beginning with T1, emerge from the vertebral canal below their respective vertebrae.

1	Surface features of the back are used to locate muscle groups for testing peripheral nerves, to determine regions of the vertebral column, and to estimate the approximate position of the inferior end of the spinal cord. They are also used to locate organs that occur posteriorly in the thorax and abdomen. Absence of lateral curvatures When viewed from behind, the normal vertebral column has no lateral curvatures. The vertical skin furrow between muscle masses on either side of the midline is straight (Fig. 2.64). in the sagittal plane When viewed from the side, the normal vertebral column has primary curvatures in the thoracic and sacral/coccygeal regions and secondary curvatures in the cervical and lumbar regions (Fig. 2.65). The primary curvatures are concave anteriorly. The secondary curvatures are concave posteriorly.

1	A number of readily palpable bony features provide useful landmarks for defining muscles and for locating structures associated with the vertebral column. Among these features are the external occipital protuberance, the scapula, and the iliac crest (Fig. 2.66). The external occipital protuberance is palpable in the midline at the back of the head just superior to the hairline. The spine, medial border, and inferior angle of the scapula are often visible and are easily palpable. The iliac crest is palpable along its entire length, from the anterior superior iliac spine at the lower lateral margin of the anterior abdominal wall to the posterior superior iliac spine near the base of the back. The position of the posterior superior iliac spine is often visible as a “sacral dimple” just lateral to the midline. How to identify specific vertebral

1	How to identify specific vertebral Identification of vertebral spinous processes (Fig. 2.67A) can be used to differentiate between regions of the vertebral column and facilitate visualizing the position of deeper structures, such as the inferior ends of the spinal cord and subarachnoid space. The spinous process of vertebra CII can be identified through deep palpation as the most superior bony protuberance in the midline inferior to the skull. Most of the other spinous processes, except for that of vertebra CVII, are not readily palpable because they are obscured by soft tissue. The spinous process of CVII is usually visible as a prominent eminence in the midline at the base of the neck (Fig. 2.67B), particularly when the neck is flexed. Extending between CVII and the external occipital protuberance of the skull is the ligamentum nuchae, which is readily apparent as a longitudinal ridge when the neck is flexed (Fig. 2.67C).

1	Extending between CVII and the external occipital protuberance of the skull is the ligamentum nuchae, which is readily apparent as a longitudinal ridge when the neck is flexed (Fig. 2.67C). Inferior to the spinous process of CVII is the spinous process of TI, which is also usually visible as a midline protuberance. Often it is more prominent than the spinous process of CVII (Fig. 2.67A,B). The root of the spine of the scapula is at the same level as the spinous process of vertebra TIII, and the inferior angle of the scapula is level with the spinous process of vertebra TVII (Fig. 2.67A). The spinous process of vertebra TXII is level with the midpoint of a vertical line between the inferior angle of the scapula and the iliac crest (Fig. 2.67A).

1	The spinous process of vertebra TXII is level with the midpoint of a vertical line between the inferior angle of the scapula and the iliac crest (Fig. 2.67A). A horizontal line between the highest point of the iliac crest on each side crosses through the spinous process of vertebra LIV. The LIII and LV vertebral spinous processes can be palpated above and below the LIV spinous process, respectively (Fig. 2.67A). The sacral dimples that mark the position of the posterior superior iliac spine are level with the SII vertebral spinous process (Fig. 2.67A). The tip of the coccyx is palpable at the base of the vertebral column between the gluteal masses (Fig. 2.67A).

1	The tip of the coccyx is palpable at the base of the vertebral column between the gluteal masses (Fig. 2.67A). The tips of the vertebral spinous processes do not always lie in the same horizontal plane as their corresponding vertebral bodies. In thoracic regions, the spinous processes are long and sharply sloped downward so that their tips lie at the level of the vertebral body below. In other words, the tip of the TIII vertebral spinous process lies at vertebral level TIV. In lumbar and sacral regions, the spinous processes are generally shorter and less sloped than in thoracic regions, and their palpable tips more closely reflect the position of their corresponding vertebral bodies. As a consequence, the palpable end of the spinous process of vertebra LIV lies at approximately the LIV vertebral level. Visualizing the inferior ends of the spinal cord and subarachnoid space

1	Visualizing the inferior ends of the spinal cord and subarachnoid space The spinal cord does not occupy the entire length of the vertebral canal. Normally in adults, it terminates at the level of the disc between vertebrae LI and LII; however, it may end as high as TXII or as low as the disc between vertebrae LII and LIII. The subarachnoid space ends at approximately the level of vertebra SII (Fig. 2.68A).

1	Because the subarachnoid space can be accessed in the lower lumbar region without endangering the spinal cord, it is important to be able to identify the position of the lumbar vertebral spinous processes. The LIV vertebral spinous process is level with a horizontal line between the highest points on the iliac crests. In the lumbar region, the palpable ends of the vertebral spinous processes lie opposite their corresponding vertebral bodies. The subarachnoid space can be accessed between vertebral levels LIII and LIV and between LIV and LV without endangering the spinal cord (Fig. 2.68B). The subarachnoid space ends at vertebral level SII, which is level with the sacral dimples marking the posterior superior iliac spines.

1	A number of intrinsic and extrinsic muscles of the back can readily be observed and palpated. The largest of these are the trapezius and latissimus dorsi muscles (Fig. 2.69A and 2.69B). Retracting the scapulae toward the midline can accentuate the rhomboid muscles (Fig. 2.69C), which lie deep to the trapezius muscle. The erector spinae muscles are visible as two longitudinal columns separated by a furrow in the midline (Fig. 2.69A). Fig. 2.1 Skeletal framework of the back. Fig. 2.2 Curvatures of the vertebral column. Cervical curvature(secondary curvature)Thoracic curvature(primary curvature)Lumbar curvature(secondary curvature)Sacral/coccygeal curvature(primary curvature)Gravity lineConcave primarycurvature of backEarly embryoAdultSomites Fig. 2.3 Back movements. Fig. 2.4 Nervous system. Fig. 2.5 Vertebrae. Fig. 2.6 A typical vertebra. A. Superior view. B. Lateral view. Fig. 2.7 Back muscles. A. Extrinsic muscles. B. Intrinsic muscles.

1	Fig. 2.4 Nervous system. Fig. 2.5 Vertebrae. Fig. 2.6 A typical vertebra. A. Superior view. B. Lateral view. Fig. 2.7 Back muscles. A. Extrinsic muscles. B. Intrinsic muscles. Deep groupSerratus posteriorinferiorSerratus posteriorsuperiorSuboccipitalLevator scapulaeSpleniusRhomboid minorSuperficial groupABIntermediate groupIntrinsic musclesTrue back muscles innervated by posterior rami of spinal nervesRhomboid majorSpinalisIliocostalisErector spinaeLongissimusLatissimusdorsiTrapeziusExtrinsic musclesInnervated by anterior rami of spinal nerves or cranial nerve XI (trapezius) Fig. 2.8 Vertebral canal. Spinal cordPia materSubarachnoid spaceDura materArachnoid materAnterior ramusPosterior ramusPosition of spinal ganglionTransverseprocessSpinousprocessPosterior longitudinalligamentAnterior internal vertebralvenous plexusIntervertebral discExtradural spaceExtradural fatVertebral body Fig. 2.9 Spinal nerves (transverse section). Fig. 2.10 Relationships of the back to other regions.

1	Fig. 2.9 Spinal nerves (transverse section). Fig. 2.10 Relationships of the back to other regions. Cervical region• supports and moves head• transmits spinal cord and vertebral arteries between head and neck Thoracic region• support for thoraxLumbar region• support for abdomenSacral region• transmits weight to lower limbs through pelvic bones• framework for posterior aspect of pelvisVertebral arteries travelin transverse processes ofC6-C1, then pass throughforamen magnum Fig. 2.11 Vertebral canal, spinal cord, and spinal nerves. 1121110112233445595678412345678123C8T1T2T3T4T5T6T7T8T9T10T11T12L1L2L3L4L5S1S2S3S4S5CoC7C6C5C4Cervicalenlargement(of spinal cord)C2C3C1SubarachnoidspaceLumbosacralenlargement(of spinal cord)Arachnoid materEnd of spinalcord at LI–LIIvertebraeEnd ofsubarachnoidspace–sacralvertebra IIDura materPedicles ofvertebraeSpinalganglion Fig. 2.12 Intervertebral foramina. Fig. 2.13 Dermatomes innervated by posterior rami of spinal nerves.

1	Fig. 2.12 Intervertebral foramina. Fig. 2.13 Dermatomes innervated by posterior rami of spinal nerves. C2C3C4T2T3T4T5T6T7T8T9L5S1S2S4S3S5, Co*The dorsal rami of L4 and L5 may not have cutaneousbranches and may therefore not be represented asdermatomes on the backL4L3L2L1T11T12T10 Fig. 2.14 Vertebrae. Fig. 2.15 Radiograph of cervical region of vertebral column. A. Anteroposterior view. B. Lateral view. ARib IICIISpinous process of CVII Vertebralbody of CIIILocation ofintervertebral discVertebra prominens(spinous process of CVII)Posterior tubercleof CI (atlas)B Fig. 2.16 Radiograph of thoracic region of vertebral column. A. Anteroposterior view. B. Lateral view. RibPedicleLocation of intervertebral discSpinous processTransverse processVertebral bodyA BIntervertebral foramenVertebral bodyLocation of intervertebral disc Fig. 2.17 Radiograph of lumbar region of vertebral column. A. Anteroposterior view. B. Lateral view. RibTransverse processPedicleSpinous process of LIVA

1	Fig. 2.17 Radiograph of lumbar region of vertebral column. A. Anteroposterior view. B. Lateral view. RibTransverse processPedicleSpinous process of LIVA Location ofintervertebral discVertebral body of LIIIIntervertebral foramenB Fig. 2.18 Development of the vertebrae. Fig. 2.19 Typical vertebra. Fig. 2.20 Regional vertebrae. A. Typical cervical vertebra. B. Atlas and axis. C. Typical thoracic vertebra. D. Typical lumbar vertebra. E. Sacrum. F. Coccyx.

1	E. Sacrum. F. Coccyx. Transverse processDensDensForamen transversariumSuperior viewSuperior viewSuperior viewPosterior viewPosterosuperior viewBAnterior tuberclePosterior tubercleAnterior archLateral massPosterior archFacet for densFacet for occipital condyleImpressionsfor alarligamentsAlarligamentsTectorial membrane (upper partof posterior longitudinal ligament)PosteriorlongitudinalligamentFacets forattachment ofalar ligamentsAtlas (CI vertebra)Atlas (CI vertebra) and Axis (CII vertebra)Atlas (CIvertebra)and Axis(CII vertebra)and baseof skullAxis (CII vertebra)Transverse ligament of atlasTransverse ligament of atlasVertebral bodyTransverse processTransverseprocessSpinousprocessMammillaryprocessSpinousprocessSuperior viewLateral viewSuperior viewFacet for articulationwith tubercle ofits own ribDemifacet for articulationwith head of rib belowDemifacet for articulationwith head of its own ribCDApical ligamentof densInferior longitudinalband of cruciformligament

1	Anterior viewDorsolateral viewPosterior viewFacet for articulation with pelvic boneEFAnterior sacral foraminaPosterior sacral foraminaCoccygeal cornuIncomplete sacral canal Fig. 2.21 Radiograph showing CI (atlas) and CII (axis) vertebrae. Open mouth, anteroposterior (odontoid peg) view. Superior articularfacet of CIIDensInferior articular faceton lateral mass of CI Fig. 2.22 Intervertebral foramen. Fig. 2.23 Spaces between adjacent vertebral arches in the lumbar region. Fig. 2.24 T1-weighted MR image in the sagittal plane demonstrating a lumbosacral myelomeningocele. There is an absence of laminae and spinous processes in the lumbosacral region. Fig. 2.25 Radiograph of the lumbar region of the vertebral column demonstrating a wedge fracture of the L1 vertebra. This condition is typically seen in patients with osteoporosis.

1	Fig. 2.25 Radiograph of the lumbar region of the vertebral column demonstrating a wedge fracture of the L1 vertebra. This condition is typically seen in patients with osteoporosis. Fig. 2.26 Radiograph of the lumbar region of the vertebral column demonstrating three intrapedicular needles, all of which have been placed into the middle of the vertebral bodies. The high-density material is radiopaque bone cement, which has been injected as a liquid that will harden. Fig. 2.27 Severe scoliosis. A. Radiograph, anteroposterior view. B. Volume-rendered CT, anterior view. Fig. 2.28 Sagittal CT showing kyphosis. Fig. 2.29 Variations in vertebral number. A. Fused vertebral bodies of cervical vertebrae. B. Hemivertebra. C. Axial slice MRI through the LV vertebra. The iliolumbar ligament runs from the tip of the LV vertebra transverse process to the iliac crest. Fused bodies of cervical vertebraeA HemivertebraPartial lumbarization of first sacral vertebraB

1	Fused bodies of cervical vertebraeA HemivertebraPartial lumbarization of first sacral vertebraB Fig. 2.30 A. MRI of a spine with multiple collapsed vertebrae due to diffuse metastatic myeloma infiltration. B1, B2. Positron emission tomography CT (PETCT) study detecting cancer cells in the spine that have high glucose metabolism. Fig. 2.31 Intervertebral joints. Anulus fibrosusNucleus pulposusLayer of hyalinecartilage Fig. 2.32 Zygapophysial joints. Fig. 2.33 Uncovertebral joint. Fig. 2.34 Disc protrusion. T2-weighted magnetic resonance images of the lumbar region of the vertebral column. A. Sagittal plane. B. Axial plane. Fig. 2.35 Anterior and posterior longitudinal ligaments of vertebral column. Fig. 2.36 Ligamenta flava. Fig. 2.37 Supraspinous ligament and ligamentum nuchae. Fig. 2.38 Interspinous ligaments. Fig. 2.39 Axial slice MRI through the lumbar spine demonstrating bilateral hypertrophy of the ligamentum flavum.

1	Fig. 2.38 Interspinous ligaments. Fig. 2.39 Axial slice MRI through the lumbar spine demonstrating bilateral hypertrophy of the ligamentum flavum. Fig. 2.40 Radiograph of lumbar region of vertebral column, oblique view (“Scottie dog”). A. Normal radiograph of lumbar region of vertebral column, oblique view. In this view, the transverse process (nose), pedicle (eye), superior articular process (ear), inferior articular process (front leg), and pars interarticularis (neck) resemble a dog. A fracture of the pars interarticularis is visible as a break in the neck of the dog, or the appearance of a collar. B. Fracture of pars interarticularis. C. CT of lumbar spine shows fracture of the LV pars interarticularis. Fig. 2.41 A. Anterior lumbar interbody fusion (ALIF). B. Posterior lumbar interbody fusion (PLIF). Fig. 2.42 Superficial group of back muscles—trapezius and latissimus dorsi.

1	Fig. 2.41 A. Anterior lumbar interbody fusion (ALIF). B. Posterior lumbar interbody fusion (PLIF). Fig. 2.42 Superficial group of back muscles—trapezius and latissimus dorsi. Spinous process of CVIIAcromionSpine of scapulaIliac crestGreater occipital nerve(posterior ramus of C2)Third occipital nerve(posterior ramus of C3)Medial branches of posterior ramiLateral branches of posterior ramiTrapeziusLatissimus dorsiThoracolumbar fascia Fig. 2.43 Superficial group of back muscles—trapezius and latissimus dorsi, with rhomboid major, rhomboid minor, and levator scapulae located deep to trapezius in the superior part of the back. Fig. 2.44 Innervation and blood supply of trapezius. TrapeziusLatissimus dorsiRhomboid minorRhomboid majorLevator scapulaeAccessory nerve [XI]Superficial branch of transverse cervical artery Fig. 2.45 Rhomboid muscles and levator scapulae. Fig. 2.46 Innervation and blood supply of the rhomboid muscles.

1	Fig. 2.45 Rhomboid muscles and levator scapulae. Fig. 2.46 Innervation and blood supply of the rhomboid muscles. Dorsal scapular nerveTrapeziusLatissimus dorsiRhomboid minorRhomboid majorLevator scapulaeSuperficial branch of transverse cervical arteryDeep branch of transverse cervical artery Fig. 2.47 Intermediate group of back muscles—serratus posterior muscles. Fig. 2.48 Thoracolumbar fascia and the deep back muscles (transverse section). Fig. 2.49 Deep group of back muscles—spinotransversales muscles (splenius capitis and splenius cervicis). Fig. 2.50 Deep group of back muscles—erector spinae muscles. Spinous process of CVIIIliac crestSplenius capitisLongissimus capitis Ligamentum nuchaeLongissimus thoracisLongissimus cervicisSpinalis thoracisSpinalisIliocostalis lumborum Iliocostalis thoracisIliocostalis cervicisIliocostalisLongissimus Fig. 2.51 Deep group of back muscles—transversospinales and segmental muscles.

1	Fig. 2.51 Deep group of back muscles—transversospinales and segmental muscles. Spinous process of CVIIObliquus capitis inferiorObliquus capitis superiorRectus capitis posterior minorRectus capitis posterior majorSemispinalis thoracisIntertransversariusErector spinaeRotatores thoracis(short, long)Levatores costarum(short, long)Semispinalis capitisMultifidus Fig. 2.52 Deep group of back muscles—suboccipital muscles. This also shows the borders of the suboccipital triangle. Spinous process of CIIPosterior ramus of C1Obliquus capitis superior Rectus capitis posterior minorObliquus capitis inferiorRectus capitis posterior majorSplenius capitisSplenius capitisLongissimus capitisSemispinalis cervicisSemispinalis capitisSemispinalis capitisVertebral artery Fig. 2.53 Spinal cord.

1	Fig. 2.53 Spinal cord. End of spinalcord LI–LIIConus medullarisInferior part ofarachnoid materEnd of subarachnoidspace SIICervicalenlargement(of spinal cord)Lumbosacralenlargement(of spinal cord)FilumterminalePial partDural partPedicles ofvertebrae Fig. 2.54 Features of the spinal cord. Fig. 2.55 Arteries that supply the spinal cord. A. Anterior view of spinal cord (not all segmental spinal arteries are shown). B. Segmental supply of spinal cord. Posterior spinal arteryADeep cervical arteryCostocervical trunkThyrocervical trunkSubclavian arteryPosterior intercostalarterySegmentalspinal arteryArtery of Adamkiewicz(branch fromsegmentalspinal artery)Ascending cervicalarteryVertebral arterySegmental medullaryarteriesAnterior spinal arterySegmental medullaryarteries (branch fromsegmental spinalartery)Lateral sacral arterySegmentalspinal artery Fig. 2.56 Veins that drain the spinal cord.

1	Fig. 2.56 Veins that drain the spinal cord. Fig. 2.57 MRI of the spine. There is discitis of the T10-T11 intervertebral disc with destruction of the adjacent endplates. There is also a prevertebral abscess and an epidural abscess, which impinges the cord. Fig. 2.58 CT at the level of CI demonstrates two breaks in the closed ring of the atlas following an axial-loading injury. Fig. 2.59 Meninges. Fig. 2.60 Arrangement of structures in the vertebral canal and the back (lumbar region). Crura of diaphragmAortaPsoasDuraQuadratus lumborumInternal vertebral plexus of veinsin extradural spaceErector spinae musclesLigamenta flavaSupraspinous ligamentInterspinous ligamentLumbar arteryVeinCauda equinaSkinVertebraIntervertebral discIntervertebral foramenLaminaPediclePosterior longitudinal ligament Fig. 2.61 Basic organization of a spinal nerve. Fig. 2.62 Course of spinal nerves in the vertebral canal.

1	Fig. 2.61 Basic organization of a spinal nerve. Fig. 2.62 Course of spinal nerves in the vertebral canal. 1121110112233445595678412345678123C8T1T2T3T4T5T6T7T8T9T10T11T12L1L2L3L4L5S1S2S3S4S5CoC7C6C5C4Cervical enlargement(of spinal cord)C2C3C1Lumbosacral enlargement(of spinal cord)Cauda equinaPedicles of vertebraeSpinal ganglion Fig. 2.63 Nomenclature of the spinal nerves. Nerve C1 emerges betweenskull and CI vertebraNerve C8 emerges inferior topedicle of CVII vertebraNerves C2 to C7 emergesuperior to pediclesNerves T1 to Co emergeinferior to pedicles oftheir respective vertebraeC2C1C3C4C5C6C7C8T1CICVIITIPedicleTransition innomenclatureof nervesT2 Fig. 2.64 Normal appearance of the back. A. In women. B. In men. Fig. 2.65 Normal curvatures of the vertebral column. Fig. 2.66 Back of a woman with major palpable bony landmarks indicated.

1	Fig. 2.64 Normal appearance of the back. A. In women. B. In men. Fig. 2.65 Normal curvatures of the vertebral column. Fig. 2.66 Back of a woman with major palpable bony landmarks indicated. Spine of scapulaInferior angle of scapulaMedial border of scapulaPosition of externaloccipital protuberancePosterior superior iliac spineIliac crest Fig. 2.67 The back with the positions of vertebral spinous processes and associated structures indicated. A. In a man. B. In a woman with neck flexed. The prominent CVII and TI vertebral spinous processes are labeled. C. In a woman with neck flexed to accentuate the ligamentum nuchae.

1	Tip of coccyxSII vertebral spinous processTXII vertebral spinous processTVII vertebral spinous processTIII vertebral spinous processTI vertebral spinous processRoot of spine of scapulaInferior angle of scapulaHighest point of iliac crestIliac crestSacral dimpleCVII vertebral spinous processCII vertebral spinous processPosition of externaloccipital protuberanceLIV vertebral spinous processA Fig. 2.68 Back with the ends of the spinal cord and subarachnoid space indicated. A. In a man. Back with the ends of the spinal cord and subarachnoid space indicated. B. In a woman lying on her side in a fetal position, which accentuates the lumbar vertebral spinous processes and opens the spaces between adjacent vertebral arches. Cerebrospinal fluid can be withdrawn from the subarachnoid space in lower lumbar regions without endangering the spinal cord.

1	Tip of coccyxSII vertebral spinous processTXII vertebral spinous processInferior end of spinal cord(normally betweenLI and LII vertebra)Inferior end ofsubarachnoid spaceALIV vertebral spinous process LIV vertebral spinous processNeedleLV vertebral spinous processTip of coccyxB Fig. 2.69 Back muscles. A. In a man with latissimus dorsi, trapezius, and erector spinae muscles outlined. Back muscles. B. In a man with arms abducted to accentuate the lateral margins of the latissimus dorsi muscles. C. In a woman with scapulae externally rotated and forcibly retracted to accentuate the rhomboid muscles. Fig. 2.70 MRI of the lumbar spine reveals posterior herniation of the L2-3 disc resulting in compression of the cauda equina filaments. Table 2.1 Superficial (appendicular) group of back muscles Table 2.2 Intermediate (respiratory) group of back muscles Table 2.3 Spinotransversales muscles Table 2.4 Erector spinae group of back muscles

1	Table 2.2 Intermediate (respiratory) group of back muscles Table 2.3 Spinotransversales muscles Table 2.4 Erector spinae group of back muscles Table 2.5 Transversospinales group of back muscles Table 2.6 Segmental back muscles Table 2.7 Suboccipital group of back muscles In the clinic Spina bifida is a disorder in which the two sides of vertebral arches, usually in lower vertebrae, fail to fuse during development, resulting in an “open” vertebral canal (Fig. 2.24). There are two types of spina bifida. The commonest type is spina bifida occulta, in which there is a defect in the vertebral arch of LV or SI. This defect occurs in as many as 10% of individuals and results in failure of the posterior arch to fuse in the midline. Clinically, the patient is asymptomatic, although physical examination may reveal a tuft of hair over the spinous processes.

1	The more severe form of spina bifida involves complete failure of fusion of the posterior arch at the lumbosacral junction, with a large outpouching of the meninges. This may contain cerebrospinal fluid (a meningocele) or a portion of the spinal cord (a myelomeningocele). These abnormalities may result in a variety of neurological deficits, including problems with walking and bladder function. In the clinic Vertebroplasty is a relatively new technique in which the body of a vertebra can be filled with bone cement (typically methyl methacrylate). The indications for the technique include vertebral body collapse and pain from the vertebral body, which may be secondary to tumor infiltration. The procedure is most commonly performed for osteoporotic wedge fractures, which are a considerable cause of morbidity and pain in older patients.

1	Osteoporotic wedge fractures (Fig. 2.25) typically occur in the thoracolumbar region, and the approach to performing vertebroplasty is novel and relatively straightforward. The procedure is performed under sedation or light general anesthetic. Using X-ray guidance the pedicle is identified on the anteroposterior image. A metal cannula is placed through the pedicle into the vertebral body. Liquid bone cement is injected via the cannula into the vertebral body (Fig. 2.26). The function of the bone cement is two-fold. First, it increases the strength of the vertebral body and prevents further loss of height. Furthermore, as the bone cement sets, there is a degree of heat generated that is believed to disrupt pain nerve endings. Kyphoplasty is a similar technique that aims to restore some or all of the lost vertebral body height from the wedge fracture by injecting liquid bone cement into the vertebral body. In the clinic

1	In the clinic Scoliosis is an abnormal lateral curvature of the vertebral column (Fig. 2.27). A true scoliosis involves not only the curvature (rightor left-sided) but also a rotational element of one vertebra upon another. The commonest types of scoliosis are those for which we have little understanding about how or why they occur and are termed idiopathic scoliosis. It is thought that there is some initial axial rotation of the vertebrae, which then alters the locations of the mechanical compressive and distractive forces applied through the vertebral growth plates, leading to changes in speed of bone growth and ultimately changes to spinal curvature. These are never present at birth and tend to occur in either the infantile, juvenile, or adolescent age groups. The vertebral bodies and posterior elements (pedicles and laminae) are normal in these patients.

1	When a scoliosis is present from birth (congenital scoliosis) it is usually associated with other developmental abnormalities. In these patients, there is a strong association with other abnormalities of the chest wall, genitourinary tract, and heart disease. This group of patients needs careful evaluation by many specialists. A rare but important group of scoliosis is that in which the muscle is abnormal. Muscular dystrophy is the commonest example. The abnormal muscle does not retain the normal alignment of the vertebral column, and curvature develops as a result. A muscle biopsy is needed to make the diagnosis. Other disorders that can produce scoliosis include bone tumors, spinal cord tumors, and localized disc protrusions. In the clinic

1	Other disorders that can produce scoliosis include bone tumors, spinal cord tumors, and localized disc protrusions. In the clinic Kyphosis is abnormal curvature of the vertebral column in the thoracic region, producing a “hunchback” deformity. This condition occurs in certain disease states, the most dramatic of which is usually secondary to tuberculosis infection of a thoracic vertebral body, where the kyphosis becomes angulated at the site of the lesion. This produces the gibbus deformity, a deformity that was prevalent before the use of antituberculous medication (Fig. 2.28). In the clinic Lordosis is abnormal curvature of the vertebral column in the lumbar region, producing a swayback deformity. In the clinic

1	In the clinic Lordosis is abnormal curvature of the vertebral column in the lumbar region, producing a swayback deformity. In the clinic There are usually seven cervical vertebrae, although in certain diseases these may be fused. Fusion of cervical vertebrae (Fig. 2.29A) can be associated with other abnormalities, for example Klippel-Feil syndrome, in which there is fusion of vertebrae CI and CII or CV and CVI, and may be associated with a high-riding abnormalities. Variations in the number of thoracic vertebrae also are well described.

1	Variations in the number of thoracic vertebrae also are well described. One of the commonest abnormalities in the lumbar vertebrae is a partial fusion of vertebra LV with the sacrum (sacralization of the lumbar vertebra). Partial separation of vertebra SI from the sacrum (lumbarization of first sacral vertebra) may also occur (Fig. 2.29B). The LV vertebra can usually be identified by the iliolumbar ligament, which is a band of connective tissue that runs from the tip of the transverse process of LV to the iliac crest bilaterally (Fig. 2.29C). A hemivertebra occurs when a vertebra develops only on one side (Fig. 2.29B). In the clinic The vertebrae and cancer

1	A hemivertebra occurs when a vertebra develops only on one side (Fig. 2.29B). In the clinic The vertebrae and cancer The vertebrae are common sites for metastatic disease (secondary spread of cancer cells). When cancer cells grow within the vertebral bodies and the posterior elements, they interrupt normal bone cell turnover, leading to either bone destruction or formation and destroying the mechanical properties of the bone. A minor injury may therefore lead to vertebral collapse (Fig. 2.30A). Cancer cells have a much higher glucose metabolism compared with normal adjacent bone cells. These metastatic cancer cells can therefore be detected by administering radioisotope-labeled glucose to a patient and then tracing where the labeled glucose has been metabolized (Fig. 2.30B). Importantly, vertebrae that contain extensive metastatic disease may extrude fragments of tumor into the vertebral canal, compressing nerves and the spinal cord. In the clinic

1	In the clinic Osteoporosis is a pathophysiologic condition in which bone quality is normal but the quantity of bone is deficient. It is a metabolic bone disorder that commonly occurs in women in their 50s and 60s and in men in their 70s. Many factors influence the development of osteoporosis, including genetic predetermination, level of activity and nutritional status, and, in particular, estrogen levels in women. Typical complications of osteoporosis include “crush” vertebral body fractures, distal fractures of the radius, and hip fractures. With increasing age and poor-quality bone, patients are more susceptible to fracture. Healing tends to be impaired in these elderly patients, who consequently require long hospital stays and prolonged rehabilitation.

1	Patients likely to develop osteoporosis can be identified by dual-photon X-ray absorptiometry (DXA) scanning. Low-dose X-rays are passed through the bone, and by counting the number of photons detected and knowing the dose given, the number of X-rays absorbed by the bone can be calculated. The amount of X-ray absorption can be directly correlated with the bone mass, and this can be used to predict whether a patient is at risk for osteoporotic fractures. In the clinic Back pain is an extremely common disorder. It can be related to mechanical problems or to disc protrusion impinging on a nerve. In cases involving discs, it may be necessary to operate and remove the disc that is pressing on the nerve.

1	Not infrequently, patients complain of pain and no immediate cause is found; the pain is therefore attributed to mechanical discomfort, which may be caused by degenerative disease. One of the treatments is to pass a needle into the facet joint and inject it with local anesthetic and corticosteroid. In the clinic Herniation of intervertebral discs

1	In the clinic Herniation of intervertebral discs The discs between the vertebrae are made up of a central portion (the nucleus pulposus) and a complex series of fibrous rings (anulus fibrosus). A tear can occur within the anulus fibrosus through which the material of the nucleus pulposus can track. After a period of time, this material may track into the vertebral canal or into the intervertebral foramen to impinge on neural structures (Fig. 2.34). This is a common cause of back pain. A disc may protrude posteriorly to directly impinge on the cord or the roots of the lumbar nerves, depending on the level, or may protrude posterolaterally adjacent to the pedicle and impinge on the descending root. In cervical regions of the vertebral column, cervical disc protrusions often become ossified and are termed disc osteophyte bars. In the clinic

1	In cervical regions of the vertebral column, cervical disc protrusions often become ossified and are termed disc osteophyte bars. In the clinic Some diseases have a predilection for synovial joints rather than symphyses. A typical example is rheumatoid arthritis, which primarily affects synovial joints and synovial bursae, resulting in destruction of the joint and its lining. Symphyses are usually preserved. In the clinic The ligamenta flava are important structures associated with the vertebral canal (Fig. 2.39). In degenerative conditions of the vertebral column, the ligamenta flava may hypertrophy. This is often associated with hypertrophy and arthritic change of the zygapophysial joints. In combination, zygapophysial joint hypertrophy, ligamenta flava hypertrophy, and a mild disc protrusion can reduce the dimensions of the vertebral canal, producing the syndrome of spinal stenosis. In the clinic

1	In the clinic Vertebral fractures can occur anywhere along the vertebral column. In most instances, the fracture will heal under appropriate circumstances. At the time of injury, it is not the fracture itself but related damage to the contents of the vertebral canal and the surrounding tissues that determines the severity of the patient’s condition. Vertebral column stability is divided into three arbitrary clinical “columns”: the anterior column consists of the vertebral bodies and the anterior longitudinal ligament; the middle column comprises the vertebral body and the posterior longitudinal ligament; and the posterior column is made up of the ligamenta flava, interspinous ligaments, supraspinous ligaments, and the ligamentum nuchae in the cervical vertebral column.

1	Destruction of one of the clinical columns is usually a stable injury requiring little more than rest and appropriate analgesia. Disruption of two columns is highly likely to be unstable and requires fixation and immobilization. A three-column spinal injury usually results in a significant neurological event and requires fixation to prevent further extension of the neurological defect and to create vertebral column stability. At the craniocervical junction, a complex series of ligaments create stability. If the traumatic incident disrupts craniocervical stability, the chances of a significant spinal cord injury are extremely high. The consequences are quadriplegia. In addition, respiratory function may be compromised by paralysis of the phrenic nerve (which arises from spinal nerves C3 to C5), and severe hypotension (low blood pressure) may result from central disruption of the sympathetic part of the autonomic division of the nervous system.

1	Mid and lower cervical vertebral column disruption may produce a range of complex neurological problems involving the upper and lower limbs, although below the level of C5, respiratory function is unlikely to be compromised. Lumbar vertebral column injuries are rare. When they occur, they usually involve significant force. Knowing that a significant force is required to fracture a vertebra, one must assess the abdominal organs and the rest of the axial skeleton for further fractures and visceral rupture. Vertebral injuries may also involve the soft tissues and supporting structures between the vertebrae. Typical examples of this are the unifacetal and bifacetal cervical vertebral dislocations that occur in hyperflexion injuries. The pars interarticularis is a clinical term to describe the specific region of a vertebra between the superior and inferior facet (zygapophysial) joints (Fig. 2.40A). This region is susceptible to trauma, especially in athletes.

1	If a fracture occurs around the pars interarticularis, the vertebral body may slip anteriorly and compress the vertebral canal. The most common sites for pars interarticularis fractures are the LIV and LV levels (Fig. 2.40B,C). (Clinicians often refer to parts of the back in shorthand terms that are not strictly anatomical; for example, facet joints and apophyseal joints are terms used instead of zygapophysial joints, and spinal column is used instead of vertebral column.) It is possible for a vertebra to slip anteriorly upon its inferior counterpart without a pars interarticularis fracture. Usually this is related to abnormal anatomy of the facet joints, facet joint degenerative change. This disorder is termed spondylolisthesis. In the clinic Surgical procedures on the back

1	In the clinic Surgical procedures on the back A prolapsed intervertebral disc may impinge upon the meningeal (thecal) sac, cord, and most commonly the nerve root, producing symptoms attributable to that level. In some instances the disc protrusion will undergo a degree of involution that may allow symptoms to resolve without intervention. In some instances pain, loss of function, and failure to resolve may require surgery to remove the disc protrusion.

1	It is of the utmost importance that the level of the disc protrusion is identified before surgery. This may require MRI scanning and on-table fluoroscopy to prevent operating on the wrong level. A midline approach to the right or to the left of the spinous processes will depend upon the most prominent site of the disc bulge. In some instances removal of the lamina will increase the potential space and may relieve symptoms. Some surgeons perform a small fenestration (windowing) within the ligamentum flavum. This provides access to the canal. The meningeal sac and its contents are gently retracted, exposing the nerve root and the offending disc. The disc is dissected free, removing its effect on the nerve root and the canal.

1	Spinal fusion is performed when it is necessary to fuse one vertebra with the corresponding superior or inferior vertebra, and in some instances multilevel fusion may be necessary. Indications are varied, though they include stabilization after fracture, stabilization related to tumor infiltration, and stabilization when mechanical pain is produced either from the disc or from the posterior elements. There are a number of surgical methods in which a fusion can be performed, through either a posterior approach and fusing the posterior elements, an anterior approach by removal of the disc and either disc replacement or anterior fusion, or in some instances a 360° fusion where the posterior elements and the vertebral bodies are fused (Fig. 2.41A,B). In the clinic

1	In the clinic Weakness in the trapezius, caused by an interruption of the accessory nerve [XI], may appear as drooping of the shoulder, inability to raise the arm above the head because of impaired rotation of the scapula, or weakness in attempting to raise the shoulder (i.e., shrug the shoulder against resistance). A weakness in, or an inability to use, the latissimus dorsi, resulting from an injury to the thoracodorsal nerve, diminishes the capacity to pull the body upward while climbing or doing a pull-up. An injury to the dorsal scapular nerve, which innervates the rhomboids, may result in a lateral shift in the position of the scapula on the affected side (i.e., the normal position of the scapula is lost because of the affected muscle’s inability to prevent antagonistic muscles from pulling the scapula laterally). In the clinic

1	In the clinic The intervertebral discs are poorly vascularized; however, infection within the bloodstream can spread to the discs from the terminal branches of the spinal arteries within the vertebral body endplates, which lie immediately adjacent to the discs (Fig. 2.57). Common sources of infection include the lungs and urinary tract. In the clinic Fractures of the atlas and axis

1	In the clinic Fractures of the atlas and axis Fractures of vertebra CI (the atlas) and vertebra CII (the axis) can potentially lead to the worst types of spinal cord injury including death and paralysis due to injury of the brainstem, which contains the cardiac and respiratory centers. The atlas is a closed ring with no vertebral body. Axial-loading injuries, such as hitting the head while diving into shallow water or hitting the head on the roof of a car in a motor vehicle accident, can cause a “burst” type of fracture, where the ring breaks at more than one site (Fig. 2.58). The British neurosurgeon, Geoffrey Jefferson, first described this fracture pattern in 1920, so these types of fractures are often called Jefferson fractures.

1	Fractures of the axis usually occur due to severe hyperextension and flexion, which can result in fracture of the tip of the dens, base of the dens, or through the body of the atlas. In judicial hangings, there is hyperextension and distraction injury causing fracture through the atlas pedicles and spondylolisthesis of C2 on C3. This type of fracture is often called a hangman’s fracture. In many cases of upper neck injuries, even in the absence of fractures to the atlas or axis, there may be injury to the atlanto-axial ligaments, which can render the neck unstable and pose severe risk to the brainstem and upper spinal cord. In the clinic

1	In the clinic An injury to the spinal cord in the cervical portion of the vertebral column can lead to varying degrees of impairment of sensory and motor function (paralysis) in all 4 limbs, termed quadriplegia or tetraplegia. An injury in upper levels of the cervical vertebral column can result in death because of loss of innervation to the diaphragm. An injury to the spinal cord below the level of TI can lead to varying degrees of impairment in motor and sensory function (paralysis) in the lower limbs, termed paraplegia. In the clinic A lumbar tap (puncture) is carried out to obtain a sample of CSF for examination. In addition, passage of a needle or conduit into the subarachnoid space (CSF space) is used to inject antibiotics, chemotherapeutic agents, and anesthetics.

1	The lumbar region is an ideal site to access the subarachnoid space because the spinal cord terminates around the level of the disc between vertebrae LI and LII in the adult. The subarachnoid space extends to the region of the lower border of the SII vertebra. There is therefore a large CSF-filled space containing lumbar and sacral nerve roots but no spinal cord. Depending on the clinician’s preference, the patient is placed in the lateral or prone position. A needle is passed in the midline in between the spinous processes into the extradural space. Further advancement punctures the dura and arachnoid mater to enter the subarachnoid space. Most needles push the roots away from the tip without causing the patient any symptoms. Once the needle is in the subarachnoid space, fluid can be aspirated. In some situations, it is important to measure CSF pressure.

1	Local anesthetics can be injected into the extradural space or the subarachnoid space to anesthetize the sacral and lumbar nerve roots. Such anesthesia is useful for operations on the pelvis and the legs, which can then be carried out without the need for general anesthesia. When procedures are carried out, the patient must be in the erect position and not lying on his or her side or in the head-down position. If a patient lies on his or her side, the anesthesia is likely to be unilateral. If the patient is placed in the head-down position, the anesthetic can pass cranially and potentially depress respiration.

1	In some instances, anesthesiologists choose to carry out extradural anesthesia. A needle is placed through the skin, supraspinous ligament, interspinous ligament, and ligamenta flava into the areolar tissue and fat around the dura mater. Anesthetic agent is introduced and diffuses around the vertebral canal to anesthetize the exiting nerve roots and diffuse into the subarachnoid space. In the clinic Herpes zoster is the virus that produces chickenpox in children. In some patients the virus remains dormant in the cells of the spinal ganglia. Under certain circumstances, the virus becomes activated and travels along the neuronal bundles to the areas supplied by that nerve (the dermatome). A rash ensues, which is characteristically exquisitely painful. Importantly, this typical dermatomal distribution is characteristic of this disorder. In the clinic

1	In the clinic Back pain is an extremely common condition affecting almost all individuals at some stage during their life. It is of key clinical importance to identify whether the back pain relates to the vertebral column and its attachments or relates to other structures.

1	The failure to consider other potential structures that may produce back pain can lead to significant mortality and morbidity. Pain may refer to the back from a number of organs situated in the retroperitoneum. Pancreatic pain in particular refers to the back and may be associated with pancreatic cancer and pancreatitis. Renal pain, which may be produced by stones in the renal collecting system or renal tumors, also typically refers to the back. More often than not this is usually unilateral; however, it can produce central posterior back pain. Enlarged lymph nodes in the preand para-aortic region may produce central posterior back pain and may be a sign of solid tumor malignancy, infection, or Hodgkin’s lymphoma. An enlarging abdominal aorta (abdominal aortic aneurysm) may cause back pain as it enlarges without rupture. Therefore it is critical to think of this structure as a potential cause of back pain, because treatment will be lifesaving. Moreover, a ruptured abdominal aortic

1	pain as it enlarges without rupture. Therefore it is critical to think of this structure as a potential cause of back pain, because treatment will be lifesaving. Moreover, a ruptured abdominal aortic aneurysm may also cause acute back pain in the first instance.

1	In all patients back pain requires careful assessment not only of the vertebral column but also of the chest and abdomen in order not to miss other important anatomical structures that may produce signs and symptoms radiating to the back. A 50-year-old man was brought to the emergency department with severe lower back pain that had started several days ago. In the past 24 hours he has had two episodes of fecal incontinence and inability to pass urine and now reports numbness and weakness in both his legs. The attending physician performed a physical examination and found that the man had reduced strength during knee extension and when dorsiflexing his feet and toes. He also had reduced reflexes in his knees and ankles, numbness in the perineal (saddle) region, as well as reduced anal sphincter tone.

1	The patient’s symptoms and physical examination findings raised serious concern for compression of multiple lumbar and sacral nerve roots in the spine, affecting both motor and sensory pathways. His reduced power in extending his knees and reduced knee reflexes was suggestive of compression of the L4 nerve roots. His reduced ability to dorsiflex his feet and toes was suggestive of compression of the L5 nerve roots. His reduced ankle reflexes was suggestive of compression of the S1 and S2 nerve roots, and his perineal numbness was suggestive of compression of the S3, S4, and S5 nerve roots. A diagnosis of cauda equina syndrome was made, and the patient was transferred for an urgent MRI scan, which confirmed the presence of a severely herniating L2-3 disc compressing the cauda equina, giving rise to the cauda equina syndrome (Fig. 2.70). The patient underwent surgical decompression of the cauda equina and made a full recovery.

1	The collection of lumbar and sacral nerve roots beyond the conus medullaris has a horsetail-like appearance, from which it derives its name “cauda equina.” Compression of the cauda equina may be caused by a herniating disc (as in this case), fracture fragments following traumatic injury, tumor, abscess, or severe degenerative stenosis of the central canal. Cauda equina syndrome is classed as a surgical emergency to prevent permanent and irreversible damage to the compressed nerve roots. A 45-year-old man was involved in a serious car accident. On examination he had a severe injury to the cervical region of his vertebral column with damage to the spinal cord. In fact, his breathing became erratic and stopped.

1	If the cervical spinal cord injury is above the level of C5, breathing is likely to stop. The phrenic nerve takes origin from C3, C4, and C5 and supplies the diaphragm. Breathing may not cease immediately if the lesion is just below C5, but does so as the cord becomes edematous and damage progresses superiorly. In addition, some respiratory and ventilatory exchange may occur by using neck muscles plus the sternocleidomastoid and trapezius muscles, which are innervated by the accessory nerve [XI]. The patient was unable to sense or move his upper and lower limbs. The patient has paralysis of the upper and lower limbs and is therefore quadriplegic. If breathing is unaffected, the lesion is below the level of C5 or at the level of C5. The nerve supply to the upper limbs is via the brachial plexus, which begins at the C5 level. The site of the spinal cord injury is at or above the C5 level.

1	It is important to remember that although the cord has been transected in the cervical region, the cord below this level is intact. Reflex activity may therefore occur below the injury, but communication with the brain is lost. A 25-year-old woman complained of increasing lumbar back pain. Over the ensuing weeks she was noted to have an enlarging lump in the right groin, which was mildly tender to touch. On direct questioning, the patient also complained of a productive cough with sputum containing mucus and blood, and she had a mild temperature. The chest radiograph revealed a cavitating apical lung mass, which explains the pulmonary history.

1	The chest radiograph revealed a cavitating apical lung mass, which explains the pulmonary history. Given the age of the patient a primary lung cancer is unlikely. The hemoptysis (coughing up blood in the sputum) and the rest of the history suggest the patient has a lung infection. Given the chest radiographic findings of a cavity in the apex of the lung, a diagnosis of tuberculosis (TB) was made. This was confirmed by bronchoscopy and aspiration of pus, which was cultured.

1	During the patient’s pulmonary infection, the tuberculous bacillus had spread via the blood to vertebra LI. The bone destruction began in the cancellous bone of the vertebral body close to the intervertebral discs. This disease progressed and eroded into the intervertebral disc, which became infected. The disc was destroyed, and the infected disc material extruded around the disc anteriorly and passed into the psoas muscle sheath. This is not an uncommon finding for a tuberculous infection of the lumbar portion of the vertebral column. As the infection progressed, the pus spread within the psoas muscle sheath beneath the inguinal ligament to produce a hard mass in the groin. This is a typical finding for a psoas abscess. Fortunately for the patient, there was no evidence of any damage within the vertebral canal.

1	Fortunately for the patient, there was no evidence of any damage within the vertebral canal. The patient underwent a radiologically guided drainage of the psoas abscess and was treated for over 6 months with a long-term antibiotic regimen. She made an excellent recovery with no further symptoms, although the cavities within the lungs remain. It healed with sclerosis. A 72-year-old fit and healthy man was brought to the emergency department with severe back pain beginning at the level of the shoulder blades and extending to the midlumbar region. The pain was of relatively acute onset and was continuous. The patient was able to walk to the gurney as he entered the ambulance; however, at the emergency department the patient complained of inability to use both legs.

1	The attending physician examined the back thoroughly and found no significant abnormality. He noted that there was reduced sensation in both legs, and there was virtually no power in extensor or flexor groups. The patient was tachycardic, which was believed to be due to pain, and the blood pressure obtained in the ambulance measured 120/80 mm Hg. It was noted that the patient’s current blood pressure was 80/40 mm Hg; however, the patient did not complain of typical clinical symptoms of hypotension. On first inspection, it is difficult to “add up” these clinical symptoms and signs. In essence we have a progressive paraplegia associated with severe back pain and an anomaly in blood pressure measurements, which are not compatible with the clinical state of the patient. It was deduced that the blood pressure measurements were obtained in different arms, and both were reassessed.

1	It was deduced that the blood pressure measurements were obtained in different arms, and both were reassessed. The blood pressure measurements were true. In the right arm the blood pressure measured 120/80 mm Hg and in the left arm the blood pressure measured 80/40 mm Hg. This would imply a deficiency of blood to the left arm. The patient was transferred from the emergency department to the CT scanner, and a scan was performed that included the chest, abdomen, and pelvis.

1	The CT scan demonstrated a dissecting thoracic aortic aneurysm. Aortic dissection occurs when the tunica intima and part of the tunica media of the wall of the aorta become separated from the remainder of the tunica media and the tunica adventitia of the aorta wall. This produces a false lumen. Blood passes not only in the true aortic lumen but also through a small hole into the wall of the aorta and into the false lumen. It often reenters the true aortic lumen inferiorly. This produces two channels through which blood may flow. The process of the aortic dissection produces considerable pain for the patient and is usually of rapid onset. Typically the pain is felt between the shoulder blades and radiating into the back, and although the pain is not from the back musculature or the vertebral column, careful consideration of structures other than the back should always be sought.

1	The difference in the blood pressure between the two arms indicates the level at which the dissection has begun. The “point of entry” is proximal to the left subclavian artery. At this level a small flap has been created, which limits the blood flow to the left upper limb, giving the low blood pressure recording. The brachiocephalic trunk has not been affected by the aortic dissection, and hence blood flow remains appropriate to the right upper limb. The paraplegia was caused by ischemia to the spinal cord.

1	The paraplegia was caused by ischemia to the spinal cord. The blood supply to the spinal cord is from a single anterior spinal artery and two posterior spinal arteries. These arteries are fed via segmental spinal arteries at every vertebral level. There are a number of reinforcing arteries (segmental medullary arteries) along the length of the spinal cord—the largest of which is the artery of Adamkiewicz. This artery of Adamkiewicz, a segmental medullary artery, typically arises from the lower thoracic or upper lumbar region, and unfortunately during this patient’s aortic dissection, the origin of this vessel was disrupted. This produces acute spinal cord ischemia and has produced the paraplegia in the patient. Unfortunately, the dissection extended, the aorta ruptured, and the patient succumbed.

1	Unfortunately, the dissection extended, the aorta ruptured, and the patient succumbed. A 55-year-old woman came to her physician with sensory alteration in the right gluteal (buttock) region and in the intergluteal (natal) cleft. Examination also demonstrated low-grade weakness of the muscles of the foot and subtle weakness of the extensor hallucis longus, extensor digitorum longus, and fibularis tertius on the right. The patient also complained of some mild pain symptoms posteriorly in the right gluteal region. A lesion was postulated in the left sacrum.

1	A lesion was postulated in the left sacrum. Pain in the right sacro-iliac region could easily be attributed to the sacro-iliac joint, which is often very sensitive to pain. The weakness of the intrinsic muscles of the foot and the extensor hallucis longus, extensor digitorum longus, and fibularis tertius muscles raises the possibility of an abnormality affecting the nerves exiting the sacrum and possibly the lumbosacral junction. The altered sensation around the gluteal region toward the anus would also support these anatomical localizing features. An X-ray was obtained of the pelvis.

1	An X-ray was obtained of the pelvis. The X-ray appeared on first inspection unremarkable. However, the patient underwent further investigation, including CT and MRI, which demonstrated a large destructive lesion involving the whole of the left sacrum extending into the anterior sacral foramina at the S1, S2, and S3 levels. Interestingly, plain radiographs of the sacrum may often appear normal on first inspection, and further imaging should always be sought in patients with a suspected sacral abnormality. The lesion was expansile and lytic. Most bony metastases are typically nonexpansile. They may well erode the bone, producing lytic type of lesions, or may become very sclerotic (prostate metastases and breast metastases). From time to time we see a mixed pattern of lytic and sclerotic.

1	There are a number of uncommon instances in which certain metastases are expansile and lytic. These typically occur in renal metastases and may be seen in multiple myeloma. The anatomical importance of these specific tumors is that they often expand and impinge upon other structures. The expansile nature of this patient’s tumor within the sacrum was the cause for compression of the sacral nerve roots, producing her symptoms. The patient underwent a course of radiotherapy, had the renal tumor excised, and is currently undergoing a course of chemoimmunotherapy. 122.e1 122.e2 Conceptual Overview • Relationship to Other Regions Fig. 2.20, cont’d Fig. 2.20, cont’d In the clinic—cont’d Fig. 2.55, cont’d Fig. 2.68, cont’d Fig. 2.69, cont’d

1	122.e1 122.e2 Conceptual Overview • Relationship to Other Regions Fig. 2.20, cont’d Fig. 2.20, cont’d In the clinic—cont’d Fig. 2.55, cont’d Fig. 2.68, cont’d Fig. 2.69, cont’d The thorax is an irregularly shaped cylinder with a narrow opening (superior thoracic aperture) superiorly and a relatively large opening (inferior thoracic aperture) inferiorly (Fig. 3.1). The superior thoracic aperture is open, allowing continuity with the neck; the inferior thoracic aperture is closed by the diaphragm. The musculoskeletal wall of the thorax is flexible and consists of segmentally arranged vertebrae, ribs, and muscles and the sternum. The thoracic cavity enclosed by the thoracic wall and the diaphragm is subdivided into three major compartments: a left and a right pleural cavity, each surrounding a lung, and the mediastinum.

1	The thoracic cavity enclosed by the thoracic wall and the diaphragm is subdivided into three major compartments: a left and a right pleural cavity, each surrounding a lung, and the mediastinum. The mediastinum is a thick, flexible soft tissue partition oriented longitudinally in a median sagittal position. It contains the heart, esophagus, trachea, major nerves, and major systemic blood vessels. The pleural cavities are completely separated from each other by the mediastinum. Therefore abnormal events in one pleural cavity do not necessarily affect the other cavity. This also means that the mediastinum can be entered surgically without opening the pleural cavities.

1	Another important feature of the pleural cavities is that they extend above the level of rib I. The apex of each lung actually extends into the root of the neck. As a consequence, abnormal events in the root of the neck can involve the adjacent pleura and lung, and events in the adjacent pleura and lung can involve the root of the neck. One of the most important functions of the thorax is breathing. The thorax not only contains the lungs but also provides the machinery necessary—the diaphragm, thoracic wall, and ribs—for effectively moving air into and out of the lungs. Up and down movements of the diaphragm and changes in the lateral and anterior dimensions of the thoracic wall, caused by movements of the ribs, alter the volume of the thoracic cavity and are key elements in breathing. Protection of vital organs

1	Protection of vital organs The thorax houses and protects the heart, lungs, and great vessels. Because of the upward domed shape of the diaphragm, the thoracic wall also offers protection to some important abdominal viscera. Much of the liver lies under the right dome of the diaphragm, and the stomach and spleen lie under the left. The posterior aspects of the superior poles of the kidneys lie on the diaphragm and are anterior to rib XII, on the right, and to ribs XI and XII, on the left. The mediastinum acts as a conduit for structures that pass completely through the thorax from one body region to another and for structures that connect organs in the thorax to other body regions. The esophagus, vagus nerves, and thoracic duct pass through the mediastinum as they course between the abdomen and neck. The phrenic nerves, which originate in the neck, also pass through the mediastinum to penetrate and supply the diaphragm.

1	The phrenic nerves, which originate in the neck, also pass through the mediastinum to penetrate and supply the diaphragm. Other structures such as the trachea, thoracic aorta, and superior vena cava course within the mediastinum en route to and from major visceral organs in the thorax. The thoracic wall consists of skeletal elements and muscles (Fig. 3.1): Posteriorly, it is made up of twelve thoracic vertebrae and their intervening intervertebral discs; Laterally, the wall is formed by ribs (twelve on each side) and three layers of flat muscles, which span the intercostal spaces between adjacent ribs, move the ribs, and provide support for the intercostal spaces; Anteriorly, the wall is made up of the sternum, which consists of the manubrium of sternum, body of sternum, and xiphoid process.

1	Anteriorly, the wall is made up of the sternum, which consists of the manubrium of sternum, body of sternum, and xiphoid process. The manubrium of sternum, angled posteriorly on the body of sternum at the manubriosternal joint, forms the sternal angle, which is a major surface landmark used by clinicians in performing physical examinations of the thorax. The anterior (distal) end of each rib is composed of costal cartilage, which contributes to the mobility and elasticity of the wall. All ribs articulate with thoracic vertebrae posteriorly. Most ribs (from rib II to IX) have three articulations with the vertebral column. The head of each rib articulates with the body of its own vertebra and with the body of the vertebra above (Fig. 3.2). As these ribs curve posteriorly, each also articulates with the transverse process of its vertebra. Anteriorly, the costal cartilages of ribs I to VII articulate with the sternum.

1	Anteriorly, the costal cartilages of ribs I to VII articulate with the sternum. The costal cartilages of ribs VIII to X articulate with the inferior margins of the costal cartilages above them. Ribs XI and XII are called floating ribs because they do not articulate with other ribs, costal cartilages, or the sternum. Their costal cartilages are small, only covering their tips. The skeletal framework of the thoracic wall provides extensive attachment sites for muscles of the neck, abdomen, back, and upper limbs. A number of these muscles attach to ribs and function as accessory respiratory muscles; some of them also stabilize the position of the first and last ribs. Completely surrounded by skeletal elements, the superior thoracic aperture consists of the body of vertebra TI posteriorly, the medial margin of rib I on each side, and the manubrium anteriorly.

1	Completely surrounded by skeletal elements, the superior thoracic aperture consists of the body of vertebra TI posteriorly, the medial margin of rib I on each side, and the manubrium anteriorly. The superior margin of the manubrium is in approximately the same horizontal plane as the intervertebral disc between vertebrae TII and TIII. The first ribs slope inferiorly from their posterior articulation with vertebra TI to their anterior attachment to the manubrium. Consequently, the plane of the superior thoracic aperture is at an oblique angle, facing somewhat anteriorly. At the superior thoracic aperture, the superior aspects of the pleural cavities, which surround the lungs, lie on either side of the entrance to the mediastinum (Fig. 3.3).

1	At the superior thoracic aperture, the superior aspects of the pleural cavities, which surround the lungs, lie on either side of the entrance to the mediastinum (Fig. 3.3). Structures that pass between the upper limb and thorax pass over rib I and the superior part of the pleural cavity as they enter and leave the mediastinum. Structures that pass between the neck and head and the thorax pass more vertically through the superior thoracic aperture. The inferior thoracic aperture is large and expandable. Bone, cartilage, and ligaments form its margin (Fig. 3.4A). The inferior thoracic aperture is closed by the diaphragm, and structures passing between the abdomen and thorax pierce or pass posteriorly to the diaphragm.

1	The inferior thoracic aperture is closed by the diaphragm, and structures passing between the abdomen and thorax pierce or pass posteriorly to the diaphragm. Skeletal elements of the inferior thoracic aperture are: the body of vertebra TXII posteriorly, rib XII and the distal end of rib XI posterolaterally, the distal cartilaginous ends of ribs VII to X, which unite to form the costal margin anterolaterally, and the xiphoid process anteriorly. The joint between the costal margin and sternum lies roughly in the same horizontal plane as the intervertebral disc between vertebrae TIX and TX. In other words, the posterior margin of the inferior thoracic aperture is inferior to the anterior margin. When viewed anteriorly, the inferior thoracic aperture is tilted superiorly. The musculotendinous diaphragm seals the inferior thoracic aperture (Fig. 3.4B).

1	When viewed anteriorly, the inferior thoracic aperture is tilted superiorly. The musculotendinous diaphragm seals the inferior thoracic aperture (Fig. 3.4B). Generally, muscle fibers of the diaphragm arise radially, from the margins of the inferior thoracic aperture, and converge into a large central tendon. Because of the oblique angle of the inferior thoracic aperture, the posterior attachment of the diaphragm is inferior to the anterior attachment. The diaphragm is not flat; rather, it “balloons” superiorly, on both the right and left sides, to form domes. The right dome is higher than the left, reaching as far as rib V. As the diaphragm contracts, the height of the domes decreases and the volume of the thorax increases. The esophagus and inferior vena cava penetrate the diaphragm; the aorta passes posterior to the diaphragm.

1	The esophagus and inferior vena cava penetrate the diaphragm; the aorta passes posterior to the diaphragm. The mediastinum is a thick midline partition that extends from the sternum anteriorly to the thoracic vertebrae posteriorly, and from the superior thoracic aperture to the inferior thoracic aperture. A horizontal plane passing through the sternal angle and the intervertebral disc between vertebrae TIV and TV separates the mediastinum into superior and inferior parts (Fig. 3.5). The inferior part is further subdivided by the pericardium, which encloses the pericardial cavity surrounding the heart. The pericardium and heart constitute the middle mediastinum. The anterior mediastinum lies between the sternum and the pericardium; the posterior mediastinum lies between the pericardium and thoracic vertebrae. The two pleural cavities are situated on either side of the mediastinum (Fig. 3.6). Each pleural cavity is completely lined by a mesothelial membrane called the pleura.

1	The two pleural cavities are situated on either side of the mediastinum (Fig. 3.6). Each pleural cavity is completely lined by a mesothelial membrane called the pleura. During development, the lungs grow out of the mediastinum, becoming surrounded by the pleural cavities. As a result, the outer surface of each organ is covered by pleura. Each lung remains attached to the mediastinum by a root formed by the airway, pulmonary blood vessels, lymphatic tissues, and nerves. The pleura lining the walls of the cavity is the parietal pleura, whereas that reflected from the mediastinum at the roots and onto the surfaces of the lungs is the visceral pleura. Only a potential space normally exists between the visceral pleura covering lung and the parietal pleura lining the wall of the thoracic cavity.

1	The lung does not completely fill the potential space of the pleural cavity, resulting in recesses, which do not contain lung and are important for accommodating changes in lung volume during breathing. The costodiaphragmatic recess, which is the largest and clinically most important recess, lies inferiorly between the thoracic wall and diaphragm. The superior thoracic aperture opens directly into the root of the neck (Fig. 3.7). The superior aspect of each pleural cavity extends approximately 2 to 3 cm above rib I and the costal cartilage into the neck. Between these pleural extensions, major visceral structures pass between the neck and superior mediastinum. In the midline, the trachea lies immediately anterior to the esophagus. Major blood vessels and nerves pass in and out of the thorax at the superior thoracic aperture anteriorly and laterally to these structures.

1	An axillary inlet, or gateway to the upper limb, lies on each side of the superior thoracic aperture. These two axillary inlets and the superior thoracic aperture communicate superiorly with the root of the neck (Fig. 3.7). Each axillary inlet is formed by: the superior margin of the scapula posteriorly, the clavicle anteriorly, and the lateral margin of rib I medially. The apex of each triangular inlet is directed laterally and is formed by the medial margin of the coracoid process, which extends anteriorly from the superior margin of the scapula. The base of the axillary inlet’s triangular opening is the lateral margin of rib I. Large blood vessels passing between the axillary inlet and superior thoracic aperture do so by passing over rib I. Proximal parts of the brachial plexus also pass between the neck and upper limb by passing through the axillary inlet.

1	Proximal parts of the brachial plexus also pass between the neck and upper limb by passing through the axillary inlet. The diaphragm separates the thorax from the abdomen. Structures that pass between the thorax and abdomen either penetrate the diaphragm or pass posteriorly to it (Fig. 3.8): The inferior vena cava pierces the central tendon of the diaphragm to enter the right side of the mediastinum near vertebral level TVIII. The esophagus penetrates the muscular part of the diaphragm to leave the mediastinum and enter the abdomen just to the left of the midline at vertebral level TX. The aorta passes posteriorly to the diaphragm at the midline at vertebral level TXII. Numerous other structures that pass between the thorax and abdomen pass through or posterior to the diaphragm. The breasts, consisting of mammary glands, superficial fascia, and overlying skin, are in the pectoral region on each side of the anterior thoracic wall (Fig. 3.9).

1	The breasts, consisting of mammary glands, superficial fascia, and overlying skin, are in the pectoral region on each side of the anterior thoracic wall (Fig. 3.9). Vessels, lymphatics, and nerves associated with the breast are as follows: Branches from the internal thoracic arteries and veins perforate the anterior chest wall on each side of the sternum to supply anterior aspects of the thoracic wall. Those branches associated mainly with the second to fourth intercostal spaces also supply the anteromedial parts of each breast. Lymphatic vessels from the medial part of the breast accompany the perforating arteries and drain into the parasternal nodes on the deep surface of the thoracic wall. Vessels and lymphatics associated with lateral parts of the breast emerge from or drain into the axillary region of the upper limb. Lateral and anterior branches of the fourth to sixth intercostal nerves carry general sensation from the skin of the breast.

1	Lateral and anterior branches of the fourth to sixth intercostal nerves carry general sensation from the skin of the breast. When working with patients, physicians use vertebral levels to determine the position of important anatomical structures within body regions.

1	The horizontal plane passing through the disc that separates thoracic vertebrae TIV and TV is one of the most significant planes in the body (Fig. 3.10) because it: passes through the sternal angle anteriorly, marking the position of the anterior articulation of the costal cartilage of rib II with the sternum. The sternal angle is used to find the position of rib II as a reference for counting ribs (because of the overlying clavicle, rib I is not palpable); separates the superior mediastinum from the inferior mediastinum and marks the position of the superior limit of the pericardium; marks where the arch of the aorta begins and ends; passes through the site where the superior vena cava penetrates the pericardium to enter the heart; is the level at which the trachea bifurcates into right and left main bronchi; and marks the superior limit of the pulmonary trunk. Venous shunts from left to right

1	Venous shunts from left to right The right atrium is the chamber of the heart that receives deoxygenated blood returning from the body. It lies on the right side of the midline, and the two major veins, the superior and inferior venae cavae, that drain into it are also located on the right side of the body. This means that, to get to the right side of the body, all blood coming from the left side has to cross the midline. This left-to-right shunting is carried out by a number of important and, in some cases, very large veins, several of which are in the thorax (Fig. 3.11). In adults, the left brachiocephalic vein crosses the midline immediately posterior to the manubrium and delivers blood from the left side of the head and neck, the left upper limb, and part of the left thoracic wall into the superior vena cava.

1	The hemiazygos and accessory hemiazygos veins drain posterior and lateral parts of the left thoracic wall, pass immediately anterior to the bodies of thoracic vertebrae, and flow into the azygos vein on the right side, which ultimately connects with the superior vena cava. The arrangement of vessels and nerves that supply the thoracic wall reflects the segmental organization of the wall. Arteries to the wall arise from two sources: the thoracic aorta, which is in the posterior mediastinum, and a pair of vessels, the internal thoracic arteries, which run along the deep aspect of the anterior thoracic wall on either side of the sternum.

1	the wall, mainly along the inferior margin of each rib (Fig. 3.12A). Running with these vessels are intercostal nerves (the anterior rami of thoracic spinal nerves), which innervate the wall, related parietal pleura, and associated skin. The position of these nerves and vessels relative to the ribs must be considered when passing objects, such as chest tubes, through the thoracic wall. Dermatomes of the thorax generally reflect the segmental organization of the thoracic spinal nerves (Fig. 3.12B). The exception occurs, anteriorly and superiorly, with the first thoracic dermatome, which is located mostly in the upper limb, and not on the trunk. The anterosuperior region of the trunk receives branches from the anterior ramus of C4 via supraclavicular branches of the cervical plexus. The highest thoracic dermatome on the anterior chest wall is T2, which also extends into the upper limb. In the midline, skin over the xiphoid process is innervated by T6.

1	The highest thoracic dermatome on the anterior chest wall is T2, which also extends into the upper limb. In the midline, skin over the xiphoid process is innervated by T6. Dermatomes of T7 to T12 follow the contour of the ribs onto the anterior abdominal wall (Fig. 3.12C). All preganglionic nerve fibers of the sympathetic system are carried out of the spinal cord in spinal nerves T1 to L2 (Fig. 3.13). This means that sympathetic fibers found anywhere in the body ultimately emerge from the spinal cord as components of these spinal nerves. Preganglionic sympathetic fibers destined for the head are carried out of the spinal cord in spinal nerve T1. The thoracic wall is expandable because most ribs articulate with other components of the wall by true joints that allow movement, and because of the shape and orientation of the ribs (Fig. 3.14).

1	The thoracic wall is expandable because most ribs articulate with other components of the wall by true joints that allow movement, and because of the shape and orientation of the ribs (Fig. 3.14). A rib’s posterior attachment is superior to its anterior attachment. Therefore, when a rib is elevated, it moves the anterior thoracic wall forward relative to the posterior wall, which is fixed. In addition, the middle part of each rib is inferior to its two ends, so that when this region of the rib is elevated, it expands the thoracic wall laterally. Finally, because the diaphragm is muscular, it changes the volume of the thorax in the vertical direction. Changes in the anterior, lateral, and vertical dimensions of the thoracic cavity are important for breathing. Innervation of the diaphragm

1	Changes in the anterior, lateral, and vertical dimensions of the thoracic cavity are important for breathing. Innervation of the diaphragm The diaphragm is innervated by two phrenic nerves that originate, one on each side, as branches of the cervical plexus in the neck (Fig. 3.15). They arise from the anterior rami of cervical nerves C3, C4, and C5, with the major contribution coming from C4. The phrenic nerves pass vertically through the neck, the superior thoracic aperture, and the mediastinum to supply motor innervation to the entire diaphragm, including the crura (muscular extensions that attach the diaphragm to the upper lumbar vertebrae). In the mediastinum, the phrenic nerves pass anteriorly to the roots of the lungs.

1	The tissues that initially give rise to the diaphragm are in an anterior position on the embryological disc before the head fold develops, which explains the cervical origin of the nerves that innervate the diaphragm. In other words, the tissue that gives rise to the diaphragm originates superior to the ultimate location of the diaphragm. Spinal cord injuries below the level of the origin of the phrenic nerve do not affect movement of the diaphragm. The cylindrical thorax consists of: a wall, two pleural cavities, the lungs, and the mediastinum.

1	The cylindrical thorax consists of: a wall, two pleural cavities, the lungs, and the mediastinum. The thorax houses the heart and lungs, acts as a conduit for structures passing between the neck and the abdomen, and plays a principal role in breathing. In addition, the thoracic wall protects the heart and lungs and provides support for the upper limbs. Muscles anchored to the anterior thoracic wall provide some of this support, and together with their associated connective tissues, nerves, and vessels, and the overlying skin and superficial fascia, define the pectoral region. The pectoral region is external to the anterior thoracic wall and helps anchor the upper limb to the trunk. It consists of: a superficial compartment containing skin, superficial fascia, and breasts; and a deep compartment containing muscles and associated structures. Nerves, vessels, and lymphatics in the superficial compartment emerge from the thoracic wall, the axilla, and the neck.

1	Nerves, vessels, and lymphatics in the superficial compartment emerge from the thoracic wall, the axilla, and the neck. The breasts consist of mammary glands and associated skin and connective tissues. The mammary glands are modified sweat glands in the superficial fascia anterior to the pectoral muscles and the anterior thoracic wall (Fig. 3.16). The mammary glands consist of a series of ducts and associated secretory lobules. These converge to form 15 to 20 lactiferous ducts, which open independently onto the nipple. The nipple is surrounded by a circular pigmented area of skin termed the areola. A well-developed, connective tissue stroma surrounds the ducts and lobules of the mammary gland. In certain regions, this condenses to form well-defined ligaments, the suspensory ligaments of breast, which are continuous with the dermis of the skin and support the breast. Carcinoma of the breast creates tension on these ligaments, causing pitting of the skin.

1	In nonlactating women, the predominant component of the breasts is fat, while glandular tissue is more abundant in lactating women. The breast lies on deep fascia related to the pectoralis major muscle and other surrounding muscles. A layer of loose connective tissue (the retromammary space) separates the breast from the deep fascia and provides some degree of movement over underlying structures. The base, or attached surface, of each breast extends vertically from ribs II to VI, and transversely from the sternum to as far laterally as the midaxillary line.

1	The base, or attached surface, of each breast extends vertically from ribs II to VI, and transversely from the sternum to as far laterally as the midaxillary line. The breast is related to the thoracic wall and to structures associated with the upper limb; therefore, vascular supply and drainage can occur by multiple routes (Fig. 3.16): laterally, vessels from the axillary artery—superior thoracic, thoraco-acromial, lateral thoracic, and subscapular arteries; medially, branches from the internal thoracic artery; the second to fourth intercostal arteries via branches that perforate the thoracic wall and overlying muscle. Veins draining the breast parallel the arteries and ultimately drain into the axillary, internal thoracic, and intercostal veins. Innervation of the breast is via anterior and lateral cutaneous branches of the second to sixth intercostal nerves. The nipple is innervated by the fourth intercostal nerve. Lymphatic drainage of the breast is as follows:

1	Lymphatic drainage of the breast is as follows: Approximately 75% is via lymphatic vessels that drain laterally and superiorly into axillary nodes (Fig. 3.16). Most of the remaining drainage is into parasternal nodes deep to the anterior thoracic wall and associated with the internal thoracic artery. Some drainage may occur via lymphatic vessels that follow the lateral branches of posterior intercostal arteries and connect with intercostal nodes situated near the heads and necks of ribs. Axillary nodes drain into the subclavian trunks, parasternal nodes drain into the bronchomediastinal trunks, and intercostal nodes drain either into the thoracic duct or into the bronchomediastinal trunks. The breast in men is rudimentary and consists only of small ducts, often composed of cords of cells, that normally do not extend beyond the areola. Breast cancer can occur in men. Muscles of the pectoral region

1	Muscles of the pectoral region Each pectoral region contains the pectoralis major, pectoralis minor, and subclavius muscles (Fig. 3.17 and Table 3.1). All originate from the anterior thoracic wall and insert into bones of the upper limb. The pectoralis major muscle is the largest and most superficial of the pectoral region muscles. It directly underlies the breast and is separated from it by deep fascia and the loose connective tissue of the retromammary space. The pectoralis major has a broad origin that includes the anterior surfaces of the medial half of the clavicle, the sternum, and related costal cartilages. The muscle fibers converge to form a flat tendon, which inserts into the lateral lip of the intertubercular sulcus of the humerus. The pectoralis major adducts, flexes, and medially rotates the arm. The subclavius and pectoralis minor muscles underlie the pectoralis major:

1	The pectoralis major adducts, flexes, and medially rotates the arm. The subclavius and pectoralis minor muscles underlie the pectoralis major: The subclavius is small and passes laterally from the anterior and medial part of rib I to the inferior surface of the clavicle. The pectoralis minor passes from the anterior surfaces of ribs III to V to the coracoid process of the scapula. Both the subclavius and pectoralis minor pull the tip of the shoulder inferiorly. A continuous layer of deep fascia, the clavipectoral fascia, encloses the subclavius and pectoralis minor and attaches to the clavicle above and to the floor of the axilla below.

1	A continuous layer of deep fascia, the clavipectoral fascia, encloses the subclavius and pectoralis minor and attaches to the clavicle above and to the floor of the axilla below. The muscles of the pectoral region form the anterior wall of the axilla, a region between the upper limb and the neck through which all major structures pass. Nerves, vessels, and lymphatics that pass between the pectoral region and the axilla pass through the clavipectoral fascia between the subclavius and pectoralis minor or pass under the inferior margins of the pectoralis major and minor. The thoracic wall is segmental in design and composed of skeletal elements and muscles. It extends between: the superior thoracic aperture, bordered by vertebra TI, rib I, and the manubrium of the sternum; and the inferior thoracic aperture, bordered by vertebra TXII, rib XII, the end of rib XI, the costal margin, and the xiphoid process of the sternum.

1	The skeletal elements of the thoracic wall consist of the thoracic vertebrae, intervertebral discs, ribs, and sternum. There are twelve thoracic vertebrae, each of which is characterized by articulations with ribs. A typical thoracic vertebra has a heart-shaped vertebral body, with roughly equal dimensions in the transverse and anteroposterior directions, and a long spinous process (Fig. 3.18). The vertebral foramen is generally circular and the laminae are broad and overlap with those of the vertebra below. The superior articular processes are flat, with their articular surfaces facing almost directly posteriorly, while the inferior articular processes project from the laminae and their articular facets face anteriorly. The transverse processes are club shaped and project posterolaterally. Articulation with ribs A typical thoracic vertebra has three sites on each side for articulation with ribs.

1	Articulation with ribs A typical thoracic vertebra has three sites on each side for articulation with ribs. Two demifacets (i.e., partial facets) are located on the superior and inferior aspects of the body for articulation with corresponding sites on the heads of adjacent ribs. The superior costal facet articulates with part of the head of its own rib, and the inferior costal facet articulates with part of the head of the rib below. An oval facet (transverse costal facet) at the end of the transverse process articulates with the tubercle of its own rib. Not all vertebrae articulate with ribs in the same fashion (Fig. 3.19): The superior costal facets on the body of vertebra TI are complete and articulate with a single facet on the head of its own rib—in other words, the head of rib I does not articulate with vertebra CVII. Similarly, vertebra TX (and often TIX) articulates only with its own ribs and therefore lacks inferior demifacets on the body.

1	Similarly, vertebra TX (and often TIX) articulates only with its own ribs and therefore lacks inferior demifacets on the body. Vertebrae TXI and TXII articulate only with the heads of their own ribs—they lack transverse costal facets and have only a single complete facet on each side of their bodies. There are twelve pairs of ribs, each terminating anteriorly in a costal cartilage (Fig. 3.20). Although all ribs articulate with the vertebral column, only the costal cartilages of the upper seven ribs, known as true ribs, articulate directly with the sternum. The remaining five pairs of ribs are false ribs: The costal cartilages of ribs VIII to X articulate anteriorly with the costal cartilages of the ribs above. Ribs XI and XII have no anterior connection with other ribs or with the sternum and are often called floating ribs.

1	Ribs XI and XII have no anterior connection with other ribs or with the sternum and are often called floating ribs. A typical rib consists of a curved shaft with anterior and posterior ends (Fig. 3.21). The anterior end is continuous with its costal cartilage. The posterior end articulates with the vertebral column and is characterized by a head, neck, and tubercle. The head is somewhat expanded and typically presents two articular surfaces separated by a crest. The smaller superior surface articulates with the inferior costal facet on the body of the vertebra above, whereas the larger inferior facet articulates with the superior costal facet of its own vertebra. The neck is a short flat region of bone that separates the head from the tubercle. The tubercle projects posteriorly from the junction of the neck with the shaft and consists of two regions, an articular part and a nonarticular part:

1	The tubercle projects posteriorly from the junction of the neck with the shaft and consists of two regions, an articular part and a nonarticular part: The articular part is medial and has an oval facet for articulation with a corresponding facet on the transverse process of the associated vertebra. The raised nonarticular part is roughened by ligament attachments. The shaft is generally thin and flat with internal and external surfaces. The superior margin is smooth and rounded, whereas the inferior margin is sharp. The shaft bends forward just laterally to the tubercle at a site termed the angle. It also has a gentle twist around its longitudinal axis so that the external surface of the anterior part of the shaft faces somewhat superiorly relative to the posterior part. The inferior margin of the internal surface is marked by a distinct costal groove. Distinct features of upper and lower ribs The upper and lower ribs have distinct features (Fig. 3.22).

1	Distinct features of upper and lower ribs The upper and lower ribs have distinct features (Fig. 3.22). Rib I is flat in the horizontal plane and has broad superior and inferior surfaces. From its articulation with vertebra TI, it slopes inferiorly to its attachment to the manubrium of the sternum. The head articulates only with the body of vertebra TI and therefore has only one articular surface. Like other ribs, the tubercle has a facet for articulation with the transverse process. The superior surface of the rib is characterized by a distinct tubercle, the scalene tubercle, which separates two smooth grooves that cross the rib approximately midway along the shaft. The anterior groove is caused by the subclavian vein, and the posterior groove is caused by the subclavian artery. Anterior and posterior to these grooves, the shaft is roughened by muscle and ligament attachments.

1	Rib II, like rib I, is flat but twice as long. It articulates with the vertebral column in a way typical of most ribs. The head of rib X has a single facet for articulation with its own vertebra. Ribs XI and XII articulate only with the bodies of their own vertebrae and have no tubercles or necks. Both ribs are short, have little curve, and are pointed anteriorly. The adult sternum consists of three major elements: the broad and superiorly positioned manubrium of the sternum, the narrow and longitudinally oriented body of the sternum, and the small and inferiorly positioned xiphoid process (Fig. 3.23). Manubrium of the sternum The manubrium of the sternum forms part of the bony framework of the neck and the thorax. The superior surface of the manubrium is expanded laterally and bears a distinct and palpable notch, the jugular notch (suprasternal notch), in the midline.

1	The superior surface of the manubrium is expanded laterally and bears a distinct and palpable notch, the jugular notch (suprasternal notch), in the midline. On either side of this notch is a large oval fossa for articulation with the clavicle. Immediately inferior to this fossa, on each lateral surface of the manubrium, is a facet for the attachment of the first costal cartilage. At the lower end of the lateral border is a demifacet for articulation with the upper half of the anterior end of the second costal cartilage. Body of the sternum The body of the sternum is flat. The anterior surface of the body of the sternum is often marked by transverse ridges that represent lines of fusion between the segmental elements called sternebrae, from which this part of the sternum arises embryologically.

1	The lateral margins of the body of the sternum have articular facets for costal cartilages. Superiorly, each lateral margin has a demifacet for articulation with the inferior aspect of the second costal cartilage. Inferior to this demifacet are four facets for articulation with the costal cartilages of ribs III to VI. At the inferior end of the body of the sternum is a demifacet for articulation with the upper demifacet on the seventh costal cartilage. The inferior end of the body of the sternum is attached to the xiphoid process. The xiphoid process is the smallest part of the sternum. Its shape is variable: it may be wide, thin, pointed, bifid, curved, or perforated. It begins as a cartilaginous structure, which becomes ossified in the adult. On each side of its upper lateral margin is a demifacet for articulation with the inferior end of the seventh costal cartilage.

1	A typical rib articulates with: the bodies of adjacent vertebrae, forming a joint with the head of the rib; and the transverse process of its related vertebra, forming a costotransverse joint (Fig. 3.24). Together, the costovertebral joints and related ligaments allow the necks of the ribs either to rotate around their longitudinal axes, which occurs mainly in the upper ribs, or to ascend and descend relative to the vertebral column, which occurs mainly in the lower ribs. The combined movements of all of the ribs on the vertebral column are essential for altering the volume of the thoracic cavity during breathing. Joint with head of rib

1	Joint with head of rib The two facets on the head of the rib articulate with the superior facet on the body of its own vertebra and with the inferior facet on the body of the vertebra above. This joint is divided into two synovial compartments by an intra-articular ligament, which attaches the crest to the adjacent intervertebral disc and separates the two articular surfaces on the head of the rib. The two synovial compartments and the intervening ligament are surrounded by a single joint capsule attached to the outer margins of the combined articular surfaces of the head and vertebral column. Costotransverse joints are synovial joints between the tubercle of a rib and the transverse process of the related vertebra (Fig. 3.24). The capsule surrounding each joint is thin. The joint is stabilized by two strong extracapsular ligaments that span the space between the transverse process and the rib on the medial and lateral sides of the joint:

1	The costotransverse ligament is medial to the joint and attaches the neck of the rib to the transverse process. The lateral costotransverse ligament is lateral to the joint and attaches the tip of the transverse process to the roughened nonarticular part of the tubercle of the rib. A third ligament, the superior costotransverse ligament, attaches the superior surface of the neck of the rib to the transverse process of the vertebra above. Slight gliding movements occur at the costotransverse joints. The sternocostal joints are joints between the upper seven costal cartilages and the sternum (Fig. 3.25). The joint between rib I and the manubrium is not synovial and consists of a fibrocartilaginous connection between the manubrium and the costal cartilage. The second to seventh joints are synovial and have thin capsules reinforced by surrounding sternocostal ligaments.

1	The joint between the second costal cartilage and the sternum is divided into two compartments by an intraarticular ligament. This ligament attaches the second costal cartilage to the junction of the manubrium and the body of the sternum. Interchondral joints occur between the costal cartilages of adjacent ribs (Fig. 3.25), mainly between the costal cartilages of ribs VII to X, but may also involve the costal cartilages of ribs V and VI. Interchondral joints provide indirect anchorage to the sternum and contribute to the formation of a smooth inferior costal margin. They are usually synovial, and the thin fibrous capsules are reinforced by interchondral ligaments.

1	The joints between the manubrium and the body of the sternum and between the body of the sternum and the xiphoid process are usually symphyses (Fig. 3.25). Only slight angular movements occur between the manubrium and the body of the sternum during respiration. The joint between the body of the sternum and the xiphoid process often becomes ossified with age. A clinically useful feature of the manubriosternal joint is that it can be palpated easily. This is because the manubrium normally angles posteriorly on the body of the sternum, forming a raised feature referred to as the sternal angle. This elevation marks the site of articulation of rib II with the sternum. Rib I is not palpable, because it lies inferior to the clavicle and is embedded in tissues at the base of the neck. Therefore, rib II is used as a reference for counting ribs and can be felt immediately lateral to the sternal angle.

1	In addition, the sternal angle lies on a horizontal plane that passes through the intervertebral disc between vertebrae TIV and TV (see Fig. 3.10). This plane separates the superior mediastinum from the inferior mediastinum and marks the superior border of the pericardium. The plane also passes through the end of the ascending aorta and the beginning of the arch of the aorta, the end of the arch of the aorta and the beginning of the thoracic aorta, and the bifurcation of the trachea, and just superior to the pulmonary trunk (see Fig. 3.79 and 3.86). Intercostal spaces lie between adjacent ribs and are filled by intercostal muscles (Fig. 3.26). Intercostal nerves and associated major arteries and veins lie in the costal groove along the inferior margin of the superior rib and pass in the plane between the inner two layers of muscles.

1	Intercostal nerves and associated major arteries and veins lie in the costal groove along the inferior margin of the superior rib and pass in the plane between the inner two layers of muscles. In each space, the vein is the most superior structure and is therefore highest in the costal groove. The artery is inferior to the vein, and the nerve is inferior to the artery and often not protected by the groove. Therefore, the nerve is the structure most at risk when objects perforate the upper aspect of an intercostal space. Small collateral branches of the major intercostal nerves and vessels are often present superior to the inferior rib below. Deep to the intercostal spaces and ribs, and separating these structures from the underlying pleura, is a layer of loose connective tissue, called endothoracic fascia, which contains variable amounts of fat.

1	Superficial to the spaces are deep fascia, superficial fascia, and skin. Muscles associated with the upper limbs and back overlie the spaces. Muscles of the thoracic wall include those that fill and support the intercostal spaces, those that pass between the sternum and the ribs, and those that cross several ribs between costal attachments (Table 3.2). The muscles of the thoracic wall, together with muscles between the vertebrae and ribs posteriorly (i.e., the levatores costarum and serratus posterior superior and serratus posterior inferior muscles) alter the position of the ribs and sternum and so change the thoracic volume during breathing. They also reinforce the thoracic wall. The intercostal muscles are three flat muscles found in each intercostal space that pass between adjacent ribs (Fig. 3.27). Individual muscles in this group are named according to their positions: The external intercostal muscles are the most superficial.

1	The external intercostal muscles are the most superficial. The internal intercostal muscles are sandwiched between the external and innermost muscles. The innermost intercostal muscles are the deepest of the three muscles. The intercostal muscles are innervated by the related intercostal nerves. As a group, the intercostal muscles provide structural support for the intercostal spaces during breathing. They can also move the ribs.

1	The eleven pairs of external intercostal muscles extend from the inferior margins (lateral edges of costal grooves) of the ribs above to the superior margins of the ribs below. When the thoracic wall is viewed from a lateral position, the muscle fibers pass obliquely anteroinferiorly (Fig. 3.27). The muscles extend around the thoracic wall from the regions of the tubercles of the ribs to the costal cartilages, where each layer continues as a thin connective tissue aponeurosis termed the external intercostal membrane. The external intercostal muscles are most active in inspiration.

1	The eleven pairs of internal intercostal muscles pass between the most inferior lateral edge of the costal grooves of the ribs above, to the superior margins of the ribs below. They extend from parasternal regions, where the muscles course between adjacent costal cartilages, to the angle of the ribs posteriorly (Fig. 3.27). This layer continues medially toward the vertebral column, in each intercostal space, as the internal intercostal membrane. The muscle fibers pass in the opposite direction to those of the external intercostal muscles. When the thoracic wall is viewed from a lateral position, the muscle fibers pass obliquely posteroinferiorly. The internal intercostal muscles are most active during expiration.

1	The innermost intercostal muscles are the least distinct of the intercostal muscles, and the fibers have the same orientation as the internal intercostals (Fig. 3.27). These muscles are most evident in the lateral thoracic wall. They extend between the inner surfaces of adjacent ribs from the medial edge of the costal groove to the deep surface of the rib below. Importantly, the neurovascular bundles associated with the intercostal spaces pass around the thoracic wall in the costal grooves in a plane between the innermost and internal intercostal muscles.

1	The subcostales are in the same plane as the innermost intercostals, span multiple ribs, and are more numerous in lower regions of the posterior thoracic wall (Fig. 3.28A). They extend from the internal surfaces of one rib to the internal surface of the second (next) or third rib below. Their fibers parallel the course of the internal intercostal muscles and extend from the angle of the ribs to more medial positions on the ribs below. The transversus thoracis muscles are found on the deep surface of the anterior thoracic wall (Fig. 3.28B) and in the same plane as the innermost intercostals. The transversus thoracis muscles originate from the posterior aspect of the xiphoid process, the inferior part of the body of the sternum, and the adjacent costal cartilages of the lower true ribs. They pass superiorly and laterally to insert into the lower borders of the costal cartilages of ribs III to VI. They most likely pull these latter elements inferiorly.

1	The transversus thoracis muscles lie deep to the internal thoracic vessels and secure these vessels to the wall. Vessels that supply the thoracic wall consist mainly of posterior and anterior intercostal arteries, which pass around the wall between adjacent ribs in intercostal spaces (Fig. 3.29). These arteries originate from the aorta and internal thoracic arteries, which in turn arise from the subclavian arteries in the root of the neck. Together, the intercostal arteries form a basket-like pattern of vascular supply around the thoracic wall. Posterior intercostal arteries originate from vessels associated with the posterior thoracic wall. The upper two posterior intercostal arteries on each side are derived from the supreme intercostal artery, which descends into the thorax as a branch of the costocervical trunk in the neck. The costocervical trunk is a posterior branch of the subclavian artery (Fig. 3.29).

1	The remaining nine pairs of posterior intercostal arteries arise from the posterior surface of the thoracic aorta. Because the aorta is on the left side of the vertebral column, those posterior intercostal vessels passing to the right side of the thoracic wall cross the midline anterior to the bodies of the vertebrae and therefore are longer than the corresponding vessels on the left. In addition to having numerous branches that supply various components of the wall, the posterior intercostal arteries have branches that accompany lateral cutaneous branches of the intercostal nerves to superficial regions. The anterior intercostal arteries originate directly or indirectly as lateral branches from the internal thoracic arteries (Fig. 3.29).

1	The anterior intercostal arteries originate directly or indirectly as lateral branches from the internal thoracic arteries (Fig. 3.29). Each internal thoracic artery arises as a major branch of the subclavian artery in the neck. It passes anteriorly over the cervical dome of the pleura and descends vertically through the superior thoracic aperture and along the deep aspect of the anterior thoracic wall. On each side, the internal thoracic artery lies posterior to the costal cartilages of the upper six ribs and about 1 cm lateral to the sternum. At approximately the level of the sixth intercostal space, it divides into two terminal branches: the superior epigastric artery, which continues inferiorly into the anterior abdominal wall (Fig. 3.29); and the musculophrenic artery, which passes along the costal margin, goes through the diaphragm, and ends near the last intercostal space.

1	Anterior intercostal arteries that supply the upper six intercostal spaces arise as lateral branches from the internal thoracic artery, whereas those supplying the lower spaces arise from the musculophrenic artery. In each intercostal space, the anterior intercostal arteries usually have two branches: One passes below the margin of the upper rib. The other passes above the margin of the lower rib and meets a collateral branch of the posterior intercostal artery. The distributions of the anterior and posterior intercostal vessels overlap and can develop anastomotic connections. The anterior intercostal arteries are generally smaller than the posterior vessels.

1	In addition to anterior intercostal arteries and a number of other branches, the internal thoracic arteries give rise to perforating branches that pass directly forward between the costal cartilages to supply structures external to the thoracic wall. These vessels travel with the anterior cutaneous branches of the intercostal nerves. Venous drainage from the thoracic wall generally parallels the pattern of arterial supply (Fig. 3.30). Centrally, the intercostal veins ultimately drain into the azygos system of veins or into internal thoracic veins, which connect with the brachiocephalic veins in the neck. Often the upper posterior intercostal veins on the left side come together and form the left superior intercostal vein, which empties into the left brachiocephalic vein. Similarly, the upper posterior intercostal veins on the right side may come together and form the right superior intercostal vein, which empties into the azygos vein.

1	Similarly, the upper posterior intercostal veins on the right side may come together and form the right superior intercostal vein, which empties into the azygos vein. Lymphatic vessels of the thoracic wall drain mainly into lymph nodes associated with the internal thoracic arteries (parasternal nodes), with the heads and necks of ribs (intercostal nodes), and with the diaphragm (diaphragmatic nodes) (Fig. 3.31). Diaphragmatic nodes are posterior to the xiphoid and at sites where the phrenic nerves penetrate the diaphragm. They also occur in regions where the diaphragm is attached to the vertebral column. Parasternal nodes drain into bronchomediastinal trunks. Intercostal nodes in the upper thorax also drain into bronchomediastinal trunks, whereas intercostal nodes in the lower thorax drain into the thoracic duct.

1	Nodes associated with the diaphragm interconnect with parasternal, prevertebral, and juxta-esophageal nodes, brachiocephalic nodes (anterior to the brachiocephalic veins in the superior mediastinum), and lateral aortic/lumbar nodes (in the abdomen). Superficial regions of the thoracic wall drain mainly into axillary lymph nodes in the axilla or parasternal nodes. Innervation of the thoracic wall is mainly by the intercostal nerves, which are the anterior rami of spinal nerves T1 to T11 and lie in the intercostal spaces between adjacent ribs. The anterior ramus of spinal nerve T12 (the subcostal nerve) is inferior to rib XII (Fig. 3.32). A typical intercostal nerve passes laterally around the thoracic wall in an intercostal space. The largest of the branches is the lateral cutaneous branch, which pierces the lateral thoracic wall and divides into an anterior branch and a posterior branch that innervate the overlying skin.

1	The intercostal nerves end as anterior cutaneous branches, which emerge either parasternally, between adjacent costal cartilages, or laterally to the midline, on the anterior abdominal wall, to supply the skin. In addition to these major branches, small collateral branches can be found in the intercostal space running along the superior border of the lower rib. In the thorax, the intercostal nerves carry: somatic motor innervation to the muscles of the thoracic wall (intercostal, subcostal, and transversus thoracis muscles), somatic sensory innervation from the skin and parietal pleura, and postganglionic sympathetic fibers to the periphery. Sensory innervation of the skin overlying the upper thoracic wall is supplied by cutaneous branches (supraclavicular nerves), which descend from the cervical plexus in the neck. In addition to innervating the thoracic wall, intercostal nerves innervate other regions: The anterior ramus of T1 contributes to the brachial plexus.

1	In addition to innervating the thoracic wall, intercostal nerves innervate other regions: The anterior ramus of T1 contributes to the brachial plexus. The lateral cutaneous branch of the second intercostal nerve (the intercostobrachial nerve) contributes to cutaneous innervation of the medial surface of the upper arm. The lower intercostal nerves supply the muscles, skin, and peritoneum of the abdominal wall. The diaphragm is a thin musculotendinous structure that fills the inferior thoracic aperture and separates the thoracic cavity from the abdominal cavity (Fig. 3.34 and see Chapter 4). It is attached peripherally to the: xiphoid process of the sternum, costal margin of the thoracic wall, ends of ribs XI and XII, ligaments that span across structures of the posterior abdominal wall, and vertebrae of the lumbar region. From these peripheral attachments, muscle fibers converge to join the central tendon. The pericardium is attached to the middle part of the central tendon.

1	From these peripheral attachments, muscle fibers converge to join the central tendon. The pericardium is attached to the middle part of the central tendon. In the median sagittal plane, the diaphragm slopes inferiorly from its anterior attachment to the xiphoid, approximately at vertebral level TVIII/IX, to its posterior attachment to the median arcuate ligament, crossing anteriorly to the aorta at approximately vertebral level TXII. Structures traveling between the thorax and abdomen pass through the diaphragm or between the diaphragm and its peripheral attachments: The inferior vena cava passes through the central tendon at approximately vertebral level TVIII. The esophagus passes through the muscular part of the diaphragm, just to the left of midline, approximately at vertebral level TX. The vagus nerves pass through the diaphragm with the esophagus. The aorta passes behind the posterior attachment of the diaphragm at vertebral level TXII.

1	The vagus nerves pass through the diaphragm with the esophagus. The aorta passes behind the posterior attachment of the diaphragm at vertebral level TXII. The thoracic duct passes behind the diaphragm with the aorta. The azygos and hemiazygos veins may also pass through the aortic hiatus or through the crura of the diaphragm. Other structures outside the posterior attachments of the diaphragm lateral to the aortic hiatus include the sympathetic trunks. The greater, lesser, and least splanchnic nerves penetrate the crura.

1	Other structures outside the posterior attachments of the diaphragm lateral to the aortic hiatus include the sympathetic trunks. The greater, lesser, and least splanchnic nerves penetrate the crura. The arterial supply to the diaphragm is from vessels that arise superiorly and inferiorly to it (see Fig. 3.34). From above, pericardiacophrenic and musculophrenic arteries supply the diaphragm. These vessels are branches of the internal thoracic arteries. Superior phrenic arteries, which arise directly from lower parts of the thoracic aorta, and small branches from intercostal arteries contribute to the supply. The largest arteries supplying the diaphragm arise from below it. These arteries are the inferior phrenic arteries, which branch directly from the abdominal aorta.

1	Venous drainage of the diaphragm is by veins that generally parallel the arteries. The veins drain into: the brachiocephalic veins in the neck, the azygos system of veins, or abdominal veins (left suprarenal vein and inferior vena cava). The diaphragm is innervated by the phrenic nerves (C3, C4, and C5), which penetrate the diaphragm and innervate it from its abdominal surface. Contraction of the domes of the diaphragm flattens the diaphragm, thereby increasing thoracic volume. Movements of the diaphragm are essential for normal breathing. One of the principal functions of the thoracic wall and the diaphragm is to alter the volume of the thorax and thereby move air in and out of the lungs. During breathing, the dimensions of the thorax change in the vertical, lateral, and anteroposterior directions.

1	During breathing, the dimensions of the thorax change in the vertical, lateral, and anteroposterior directions. Elevation and depression of the diaphragm significantly alter the vertical dimensions of the thorax. Depression results when the muscle fibers of the diaphragm contract. Elevation occurs when the diaphragm relaxes. Changes in the anteroposterior and lateral dimensions result from elevation and depression of the ribs (Fig. 3.35). The posterior ends of the ribs articulate with the vertebral column, whereas the anterior ends of most ribs articulate with the sternum or adjacent ribs.

1	Because the anterior ends of the ribs are inferior to the posterior ends, when the ribs are elevated, they move the sternum upward and forward. Also, the angle between the body of the sternum and the manubrium may become slightly less acute. When the ribs are depressed, the sternum moves downward and backward. This “pump handle” movement changes the dimensions of the thorax in the anteroposterior direction (Fig. 3.35A). As well as the anterior ends of the ribs being lower than the posterior ends, the middles of the shafts tend to be lower than the two ends. When the shafts are elevated, the middles of the shafts move laterally. This “bucket handle” movement increases the lateral dimensions of the thorax (Fig. 3.35B). Any muscles attaching to the ribs can potentially move one rib relative to another and therefore act as accessory respiratory muscles. Muscles in the neck and the abdomen can fix or alter the positions of upper and lower ribs.

1	Two pleural cavities, one on either side of the mediastinum, surround the lungs (Fig. 3.37): Superiorly, they extend above rib I into the root of the neck. Inferiorly, they extend to a level just above the costal margin. The medial wall of each pleural cavity is the mediastinum. Each pleural cavity is lined by a single layer of flat cells, mesothelium, and an associated layer of supporting connective tissue; together, they form the pleura. The pleura is divided into two major types, based on location: Pleura associated with the walls of a pleural cavity is parietal pleura (Fig. 3.37). Pleura that reflects from the medial wall and onto the surface of the lung is visceral pleura (Fig. 3.37), which adheres to and covers the lung.

1	Pleura that reflects from the medial wall and onto the surface of the lung is visceral pleura (Fig. 3.37), which adheres to and covers the lung. Each pleural cavity is the potential space enclosed between the visceral and parietal pleurae. They normally contain only a very thin layer of serous fluid. As a result, the surface of the lung, which is covered by visceral pleura, directly opposes and freely slides over the parietal pleura attached to the wall. The names given to the parietal pleura correspond to the parts of the wall with which they are associated (Fig. 3.38): Pleura related to the ribs and intercostal spaces is termed the costal part. Pleura covering the diaphragm is the diaphragmatic part. Pleura covering the mediastinum is the mediastinal part. The dome-shaped layer of parietal pleura lining the cervical extension of the pleural cavity is cervical pleura (dome of pleura or pleural cupola).

1	The dome-shaped layer of parietal pleura lining the cervical extension of the pleural cavity is cervical pleura (dome of pleura or pleural cupola). Covering the superior surface of the cervical pleura is a distinct dome-like layer of fascia, the suprapleural membrane (Fig. 3.38). This connective tissue membrane is attached laterally to the medial margin of the first rib and behind to the transverse process of vertebra CVII. Superiorly, the membrane receives muscle fibers from some of the deep muscles in the neck (scalene muscles) that function to keep the membrane taut. The suprapleural membrane provides apical support for the pleural cavity in the root of the neck.

1	In the region of vertebrae TV to TVII, the mediastinal pleura reflects off the mediastinum as a tubular, sleeve-like covering for structures (i.e., airway, vessels, nerves, lymphatics) that pass between the lung and mediastinum. This sleeve-like covering and the structures it contains forms the root of the lung. The root joins the medial surface of the lung at an area referred to as the hilum of the lung. Here, the mediastinal pleura is continuous with the visceral pleura. The parietal pleural is innervated by somatic afferent fibers. The costal pleura is innervated by branches from the intercostal nerves, and pain would be felt in relation to the thoracic wall. The diaphragmatic pleura and the mediastinal pleura are innervated mainly by the phrenic nerves (originating at spinal cord levels C3, C4, and C5). Pain from these areas would refer to the C3, C4, and C5 dermatomes (lateral neck and the supraclavicular region of the shoulder).

1	The peripheral reflections of parietal pleura mark the extent of the pleural cavities (Fig. 3.39). Superiorly, the pleural cavity can project as much as 3 to 4 cm above the first costal cartilage but does not extend above the neck of rib I. This limitation is caused by the inferior slope of rib I to its articulation with the manubrium. Anteriorly, the pleural cavities approach each other posterior to the upper part of the sternum. However, posterior to the lower part of the sternum, the parietal pleura does not come as close to the midline on the left side as it does on the right because the middle mediastinum, containing the pericardium and heart, bulges to the left.

1	Inferiorly, the costal pleura reflects onto the diaphragm above the costal margin. In the midclavicular line, the pleural cavity extends inferiorly to approximately rib VIII. In the midaxillary line, it extends to rib X. From this point, the inferior margin courses somewhat horizontally, crossing ribs XI and XII to reach vertebra TXII. From the midclavicular line to the vertebral column, the inferior boundary of the pleura can be approximated by a line that runs between rib VIII, rib X, and vertebra TXII. The visceral pleura is continuous with the parietal pleura at the hilum of each lung, where structures enter and leave the organ. The visceral pleura is firmly attached to the surface of the lung, including both opposed surfaces of the fissures that divide the lungs into lobes. Although the visceral pleura is innervated by visceral afferent nerves that accompany bronchial vessels, pain is generally not elicited from this tissue.

1	Although the visceral pleura is innervated by visceral afferent nerves that accompany bronchial vessels, pain is generally not elicited from this tissue. The lungs do not completely fill the anterior or posterior inferior regions of the pleural cavities (Fig. 3.40). This results in recesses in which two layers of parietal pleura become opposed. Expansion of the lungs into these spaces usually occurs only during forced inspiration; the recesses also provide potential spaces in which fluids can collect and from which fluids can be aspirated. Anteriorly, a costomediastinal recess occurs on each side where costal pleura is opposed to mediastinal pleura. The largest is on the left side in the region overlying the heart (Fig. 3.40).

1	Anteriorly, a costomediastinal recess occurs on each side where costal pleura is opposed to mediastinal pleura. The largest is on the left side in the region overlying the heart (Fig. 3.40). The largest and clinically most important recesses are the costodiaphragmatic recesses, which occur in each pleural cavity between the costal pleura and diaphragmatic pleura (Fig. 3.40). The costodiaphragmatic recesses are the regions between the inferior margin of the lungs and inferior margin of the pleural cavities. They are deepest after forced expiration and shallowest after forced inspiration.

1	During quiet respiration, the inferior margin of the lung crosses rib VI in the midclavicular line and rib VIII in the midaxillary line, and then courses somewhat horizontally to reach the vertebral column at vertebral level TX. Thus, from the midclavicular line and around the thoracic wall to the vertebral column, the inferior margin of the lung can be approximated by a line running between rib VI, rib VIII, and vertebra TX. The inferior margin of the pleural cavity at the same points is rib VIII, rib X, and vertebra TXII. The costodiaphragmatic recess is the region between the two margins. During expiration, the inferior margin of the lung rises and the costodiaphragmatic recess becomes larger. The two lungs are organs of respiration and lie on either side of the mediastinum surrounded by the right and left pleural cavities. Air enters and leaves the lungs via main bronchi, which are branches of the trachea.

1	The pulmonary arteries deliver deoxygenated blood to the lungs from the right ventricle of the heart. Oxygenated blood returns to the left atrium via the pulmonary veins. The right lung is normally a little larger than the left lung because the middle mediastinum, containing the heart, bulges more to the left than to the right. Each lung has a half-cone shape, with a base, apex, two surfaces, and three borders (Fig. 3.43). The base sits on the diaphragm. The apex projects above rib I and into the root of the neck. The two surfaces—the costal surface lies immediately adjacent to the ribs and intercostal spaces of the thoracic wall. The mediastinal surface lies against the mediastinum anteriorly and the vertebral column posteriorly and contains the comma-shaped hilum of the lung, through which structures enter and leave.

1	The three borders—the inferior border of the lung is sharp and separates the base from the costal surface. The anterior and posterior borders separate the costal surface from the medial surface. Unlike the anterior and inferior borders, which are sharp, the posterior border is smooth and rounded. The lungs lie directly adjacent to, and are indented by, structures contained in the overlying area. The heart and major vessels form bulges in the mediastinum that indent the medial surfaces of the lung; the ribs indent the costal surfaces. Pathology, such as tumors, or abnormalities in one structure can affect the related structure.

1	The root of each lung is a short tubular collection of structures that together attach the lung to structures in the mediastinum (Fig. 3.44). It is covered by a sleeve of mediastinal pleura that reflects onto the surface of the lung as visceral pleura. The region outlined by this pleural reflection on the medial surface of the lung is the hilum, where structures enter and leave. A thin blade-like fold of pleura projects inferiorly from the root of the lung and extends from the hilum to the mediastinum. This structure is the pulmonary ligament. It may stabilize the position of the inferior lobe and may also accommodate the down-and-up translocation of structures in the root during breathing. In the mediastinum, the vagus nerves pass immediately posterior to the roots of the lungs, while the phrenic nerves pass immediately anterior to them.

1	In the mediastinum, the vagus nerves pass immediately posterior to the roots of the lungs, while the phrenic nerves pass immediately anterior to them. Within each root and located in the hilum are: a pulmonary artery, two pulmonary veins, a main bronchus, bronchial vessels, nerves, and lymphatics. Generally, the pulmonary artery is superior at the hilum, the pulmonary veins are inferior, and the bronchi are somewhat posterior in position. On the right side, the lobar bronchus to the superior lobe branches from the main bronchus in the root, unlike on the left where it branches within the lung itself, and is superior to the pulmonary artery. The right lung has three lobes and two fissures (Fig. 3.45A). Normally, the lobes are freely movable against each other because they are separated, almost to the hilum, by invaginations of visceral pleura. These invaginations form the fissures:

1	The oblique fissure separates the inferior lobe (lower lobe) from the superior lobe and the middle lobe of the right lung. The horizontal fissure separates the superior lobe (upper lobe) from the middle lobe. The approximate position of the oblique fissure on a patient, in quiet respiration, can be marked by a curved line on the thoracic wall that begins roughly at the spinous process of the vertebra TIV level of the spine, crosses the fifth interspace laterally, and then follows the contour of rib VI anteriorly (see pp. 241–242). The horizontal fissure follows the fourth intercostal space from the sternum until it meets the oblique fissure as it crosses rib V. The orientations of the oblique and horizontal fissures determine where clinicians should listen for lung sounds from each lobe.

1	The orientations of the oblique and horizontal fissures determine where clinicians should listen for lung sounds from each lobe. The largest surface of the superior lobe is in contact with the upper part of the anterolateral wall and the apex of this lobe projects into the root of the neck. The surface of the middle lobe lies mainly adjacent to the lower anterior and lateral wall. The costal surface of the inferior lobe is in contact with the posterior and inferior walls. When listening to lung sounds from each of the lobes, it is important to position the stethoscope on those areas of the thoracic wall related to the underlying positions of the lobes (see p. 243). The medial surface of the right lung lies adjacent to a number of important structures in the mediastinum and the root of the neck (Fig. 3.45B). These include the: heart, inferior vena cava, superior vena cava, azygos vein, and esophagus.

1	The right subclavian artery and vein arch over and are related to the superior lobe of the right lung as they pass over the dome of the cervical pleura and into the axilla. The left lung is smaller than the right lung and has two lobes separated by an oblique fissure (Fig. 3.46A). The oblique fissure of the left lung is slightly more oblique than the corresponding fissure of the right lung. During quiet respiration, the approximate position of the left oblique fissure can be marked by a curved line on the thoracic wall that begins between the spinous processes of vertebrae TIII and TIV, crosses the fifth interspace laterally, and follows the contour of rib VI anteriorly (see pp. 241–242). As with the right lung, the orientation of the oblique fissure determines where to listen for lung sounds from each lobe.

1	As with the right lung, the orientation of the oblique fissure determines where to listen for lung sounds from each lobe. The largest surface of the superior lobe is in contact with the upper part of the anterolateral wall, and the apex of this lobe projects into the root of the neck. The costal surface of the inferior lobe is in contact with the posterior and inferior walls. When listening to lung sounds from each of the lobes, the stethoscope should be placed on those areas of the thoracic wall related to the underlying positions of the lobes (see p. 243). The inferior portion of the medial surface of the left lung, unlike the right lung, is notched because of the heart’s projection into the left pleural cavity from the middle mediastinum. From the anterior border of the lower part of the superior lobe a tongue-like extension (the lingula of the left lung) projects over the heart bulge.

1	From the anterior border of the lower part of the superior lobe a tongue-like extension (the lingula of the left lung) projects over the heart bulge. The medial surface of the left lung lies adjacent to a number of important structures in the mediastinum and root of the neck (Fig. 3.46B). These include the: heart, aortic arch, thoracic aorta, and esophagus. The left subclavian artery and vein arch over and are related to the superior lobe of the left lung as they pass over the dome of the cervical pleura and into the axilla.

1	The left subclavian artery and vein arch over and are related to the superior lobe of the left lung as they pass over the dome of the cervical pleura and into the axilla. The trachea is a flexible tube that extends from vertebral level CVI in the lower neck to vertebral level TIV/V in the mediastinum where it bifurcates into a right and a left main bronchus (Fig. 3.47). The trachea is held open by C-shaped transverse cartilage rings embedded in its wall—the open part of the C facing posteriorly. The lowest tracheal ring has a hook-shaped structure, the carina, that projects backward in the midline between the origins of the two main bronchi. The posterior wall of the trachea is composed mainly of smooth muscle.

1	Each main bronchus enters the root of a lung and passes through the hilum into the lung itself. The right main bronchus is wider and takes a more vertical course through the root and hilum than the left main bronchus (Fig. 3.47A). Therefore, inhaled foreign bodies tend to lodge more frequently on the right side than on the left. The main bronchus divides within the lung into lobar bronchi (secondary bronchi), each of which supplies a lobe. On the right side, the lobar bronchus to the superior lobe originates within the root of the lung. The lobar bronchi further divide into segmental bronchi (tertiary bronchi), which supply bronchopulmonary segments (Fig. 3.47B).

1	The lobar bronchi further divide into segmental bronchi (tertiary bronchi), which supply bronchopulmonary segments (Fig. 3.47B). Within each bronchopulmonary segment, the segmental bronchi give rise to multiple generations of divisions and, ultimately, to bronchioles, which further subdivide and supply the respiratory surfaces. The walls of the bronchi are held open by discontinuous elongated plates of cartilage, but these are not present in bronchioles. A bronchopulmonary segment is the area of lung supplied by a segmental bronchus and its accompanying pulmonary artery branch. Tributaries of the pulmonary vein tend to pass intersegmentally between and around the margins of segments. Each bronchopulmonary segment is shaped like an irregular cone, with the apex at the origin of the segmental bronchus and the base projected peripherally onto the surface of the lung.

1	Each bronchopulmonary segment is shaped like an irregular cone, with the apex at the origin of the segmental bronchus and the base projected peripherally onto the surface of the lung. A bronchopulmonary segment is the smallest functionally independent region of a lung and the smallest area of lung that can be isolated and removed without affecting adjacent regions. There are ten bronchopulmonary segments in each lung (Fig. 3.48); some of them fuse in the left lung. The right and left pulmonary arteries originate from the pulmonary trunk and carry deoxygenated blood to the lungs from the right ventricle of the heart (Fig. 3.49). The bifurcation of the pulmonary trunk occurs to the left of the midline just inferior to vertebral level TIV/V, and anteroinferiorly to the left of the bifurcation of the trachea. The right pulmonary artery is longer than the left and passes horizontally across the mediastinum (Fig. 3.49).

1	The right pulmonary artery is longer than the left and passes horizontally across the mediastinum (Fig. 3.49). It passes: anteriorly and slightly inferiorly to the tracheal bifurcation and anteriorly to the right main bronchus, and posteriorly to the ascending aorta, superior vena cava, and upper right pulmonary vein. The right pulmonary artery enters the root of the lung and gives off a large branch to the superior lobe of the lung. The main vessel continues through the hilum of the lung, gives off a second (recurrent) branch to the superior lobe, and then divides to supply the middle and inferior lobes. The left pulmonary artery is shorter than the right and lies anterior to the descending aorta and posterior to the superior pulmonary vein (Fig. 3.49). It passes through the root and hilum and branches within the lung.

1	On each side a superior pulmonary vein and an inferior pulmonary vein carry oxygenated blood from the lungs back to the heart (Fig. 3.49). The veins begin at the hilum of the lung, pass through the root of the lung, and immediately drain into the left atrium. The bronchial arteries (Fig. 3.49) and veins constitute the “nutritive” vascular system of the pulmonary tissues (bronchial walls and glands, walls of large vessels, and visceral pleura). They interconnect within the lung with branches of the pulmonary arteries and veins. The bronchial arteries originate from the thoracic aorta or one of its branches: A single right bronchial artery normally arises from the third posterior intercostal artery (but occasionally, it originates from the upper left bronchial artery). Two left bronchial arteries arise directly from the anterior surface of the thoracic aorta—the superior left bronchial artery arises at vertebral level TV, and the inferior one inferior to the left bronchus.

1	The bronchial arteries run on the posterior surfaces of the bronchi and ramify in the lungs to supply pulmonary tissues. The bronchial veins drain into: either the pulmonary veins or the left atrium, and into the azygos vein on the right or into the superior intercostal vein or hemiazygos vein on the left. Structures of the lung and the visceral pleura are supplied by visceral afferents and efferents distributed through the anterior pulmonary plexus and posterior pulmonary plexus (Fig. 3.50). These interconnected plexuses lie anteriorly and posteriorly to the tracheal bifurcation and main bronchi. The anterior plexus is much smaller than the posterior plexus. Branches of these plexuses, which ultimately originate from the sympathetic trunks and vagus nerves, are distributed along branches of the airway and vessels. Visceral efferents from: the vagus nerves constrict the bronchioles; the sympathetic system dilates the bronchioles.

1	Visceral efferents from: the vagus nerves constrict the bronchioles; the sympathetic system dilates the bronchioles. Superficial, or subpleural, and deep lymphatics of the lung drain into lymph nodes called tracheobronchial nodes around the roots of lobar and main bronchi and along the sides of the trachea (Fig. 3.51). As a group, these lymph nodes extend from within the lung, through the hilum and root, and into the posterior mediastinum. Efferent vessels from these nodes pass superiorly along the trachea to unite with similar vessels from parasternal nodes and brachiocephalic nodes, which are anterior to brachiocephalic veins in the superior mediastinum, to form the right and left bronchomediastinal trunks. These trunks drain directly into deep veins at the base of the neck, or may drain into the right lymphatic trunk or thoracic duct.

1	The mediastinum is a broad central partition that separates the two laterally placed pleural cavities (Fig. 3.55). It extends: from the sternum to the bodies of the vertebrae, and from the superior thoracic aperture to the diaphragm (Fig. 3.56). The mediastinum contains the thymus gland, the pericardial sac, the heart, the trachea, and the major arteries and veins. Additionally, the mediastinum serves as a passageway for structures such as the esophagus, thoracic duct, and various components of the nervous system as they traverse the thorax on their way to the abdomen.

1	For organizational purposes, the mediastinum is subdivided into several smaller regions. A transverse plane extending from the sternal angle (the junction between the manubrium and the body of the sternum) to the intervertebral disc between vertebrae TIV and TV separates the mediastinum into the: superior mediastinum, and inferior mediastinum, which is further partitioned into the anterior, middle, and posterior mediastinum by the pericardial sac. The area anterior to the pericardial sac and posterior to the body of the sternum is the anterior mediastinum. The region posterior to the pericardial sac and the diaphragm and anterior to the bodies of the vertebrae is the posterior mediastinum. The area in the middle, which includes the pericardial sac and its contents, is the middle mediastinum (Fig. 3.57). The anterior mediastinum is posterior to the body of the sternum and anterior to the pericardial sac (see Fig. 3.57).

1	The anterior mediastinum is posterior to the body of the sternum and anterior to the pericardial sac (see Fig. 3.57). Its superior boundary is a transverse plane passing from the sternal angle to the intervertebral disc between vertebra TIV and TV, separating it from the superior mediastinum. Its inferior boundary is the diaphragm. Laterally, it is bordered by the mediastinal part of parietal pleura on either side. The major structure in the anterior mediastinum is an inferior extension of the thymus gland (Fig. 3.58). Also present are fat, connective tissue, lymph nodes, mediastinal branches of the internal thoracic vessels, and sternopericardial ligaments, which pass from the posterior surface of the body of the sternum to the fibrous pericardium. The middle mediastinum is centrally located in the thoracic cavity. It contains the pericardium, heart, origins of the great vessels, various nerves, and smaller vessels.

1	The middle mediastinum is centrally located in the thoracic cavity. It contains the pericardium, heart, origins of the great vessels, various nerves, and smaller vessels. The pericardium is a fibroserous sac surrounding the heart and the roots of the great vessels. It consists of two components, the fibrous pericardium and the serous pericardium (Fig. 3.59). The fibrous pericardium is a tough connective tissue outer layer that defines the boundaries of the middle mediastinum. The serous pericardium is thin and consists of two parts: The parietal layer of serous pericardium lines the inner surface of the fibrous pericardium. The visceral layer (epicardium) of serous pericardium adheres to the heart and forms its outer covering.

1	The parietal layer of serous pericardium lines the inner surface of the fibrous pericardium. The visceral layer (epicardium) of serous pericardium adheres to the heart and forms its outer covering. The parietal and visceral layers of serous pericardium are continuous at the roots of the great vessels. The narrow space created between the two layers of serous pericardium, containing a small amount of fluid, is the pericardial cavity. This potential space allows for the relatively uninhibited movement of the heart.

1	The fibrous pericardium is a cone-shaped bag with its base on the diaphragm and its apex continuous with the adventitia of the great vessels (Fig. 3.59). The base is attached to the central tendon of the diaphragm and to a small muscular area of the diaphragm on the left side. Anteriorly, it is attached to the posterior surface of the sternum by sternopericardial ligaments. These attachments help to retain the heart in its position in the thoracic cavity. The sac also limits cardiac distention.

1	The phrenic nerves, which innervate the diaphragm and originate from spinal cord levels C3 to C5, pass through the fibrous pericardium and innervate the fibrous pericardium as they travel from their point of origin to their final destination (Fig. 3.60). Their location, within the fibrous pericardium, is directly related to the embryological origin of the diaphragm and the changes that occur during the formation of the pericardial cavity. Similarly, the pericardiacophrenic vessels are also located within and supply the fibrous pericardium as they pass through the thoracic cavity. The parietal layer of serous pericardium is continuous with the visceral layer of serous pericardium around the roots of the great vessels. These reflections of serous pericardium (Fig. 3.61) occur in two locations: one superiorly, surrounding the arteries—the aorta and the pulmonary trunk; the second more posteriorly, surrounding the veins—the superior and inferior vena cava and the pulmonary veins.

1	The zone of reflection surrounding the veins is J-shaped, and the cul-de-sac formed within the J, posterior to the left atrium, is the oblique pericardial sinus. A passage between the two sites of reflected serous pericardium is the transverse pericardial sinus. This sinus lies posterior to the ascending aorta and the pulmonary trunk, anterior to the superior vena cava, and superior to the left atrium. When the pericardium is opened anteriorly during surgery, a finger placed in the transverse sinus separates arteries from veins. A hand placed under the apex of the heart and moved superiorly slips into the oblique sinus. The pericardium is supplied by branches from the internal thoracic, pericardiacophrenic, musculophrenic, and inferior phrenic arteries, and the thoracic aorta. Veins from the pericardium enter the azygos system of veins and the internal thoracic and superior phrenic veins.

1	Veins from the pericardium enter the azygos system of veins and the internal thoracic and superior phrenic veins. Nerves supplying the pericardium arise from the vagus nerve [X], the sympathetic trunks, and the phrenic nerves. It is important to note that the source of somatic sensation (pain) from the parietal pericardium is carried by somatic afferent fibers in the phrenic nerves. For this reason, “pain” related to a pericardial problem may be referred to the supraclavicular region of the shoulder or lateral neck area dermatomes for spinal cord segments C3, C4, and C5.

1	C3, C4, and C5. The general shape and orientation of the heart are that of a pyramid that has fallen over and is resting on one of its sides. Placed in the thoracic cavity, the apex of this pyramid projects forward, downward, and to the left, whereas the base is opposite the apex and faces in a posterior direction (Fig. 3.63). The sides of the pyramid consist of: a diaphragmatic (inferior) surface on which the pyramid rests, an anterior (sternocostal) surface oriented anteriorly, a right pulmonary surface, and a left pulmonary surface. The base of the heart is quadrilateral and directed posteriorly. It consists of: the left atrium, a small portion of the right atrium, and the proximal parts of the great veins (superior and inferior venae cavae and the pulmonary veins) (Fig. 3.64).

1	Because the great veins enter the base of the heart, with the pulmonary veins entering the right and left sides of the left atrium and the superior and inferior venae cavae at the upper and lower ends of the right atrium, the base of the heart is fixed posteriorly to the pericardial wall, opposite the bodies of vertebrae TV to TVIII (TVI to TIX when standing). The esophagus lies immediately posterior to the base. From the base the heart projects forward, downward, and to the left, ending in the apex. The apex of the heart is formed by the inferolateral part of the left ventricle (Fig. 3.65) and is positioned deep to the left fifth intercostal space, 8 to 9 cm from the midsternal line. Surfaces of the heart The anterior surface faces anteriorly and consists mostly of the right ventricle, with some of the right atrium on the right and some of the left ventricle on the left (Fig. 3.65).

1	The anterior surface faces anteriorly and consists mostly of the right ventricle, with some of the right atrium on the right and some of the left ventricle on the left (Fig. 3.65). The heart in the anatomical position rests on the diaphragmatic surface, which consists of the left ventricle and a small portion of the right ventricle separated by the posterior interventricular groove (Fig. 3.66). This surface faces inferiorly, rests on the diaphragm, is separated from the base of the heart by the coronary sinus, and extends from the base to the apex of the heart. The left pulmonary surface faces the left lung, is broad and convex, and consists of the left ventricle and a portion of the left atrium (Fig. 3.66). The right pulmonary surface faces the right lung, is broad and convex, and consists of the right atrium (Fig. 3.66). Some general descriptions of cardiac orientation refer to right, left, inferior (acute), and obtuse margins:

1	Some general descriptions of cardiac orientation refer to right, left, inferior (acute), and obtuse margins: The right and left margins are the same as the right and left pulmonary surfaces of the heart. The inferior margin is defined as the sharp edge between the anterior and diaphragmatic surfaces of the heart (Figs 3.63 and 3.65)—it is formed mostly by the right ventricle and a small portion of the left ventricle near the apex. The obtuse margin separates the anterior and left pulmonary surfaces (Fig. 3.63)—it is round and extends from the left auricle to the cardiac apex (Fig. 3.65), and is formed mostly by the left ventricle and superiorly by a small portion of the left auricle.

1	For radiological evaluations, a thorough understanding of the structures defining the cardiac borders is critical. The right border in a standard posteroanterior view consists of the superior vena cava, the right atrium, and the inferior vena cava (Fig. 3.67A). The left border in a similar view consists of the arch of the aorta, the pulmonary trunk, left auricle, and the left ventricle. The inferior border in this radiological study consists of the right ventricle and the left ventricle at the apex. In lateral views, the right ventricle is seen anteriorly, and the left atrium is visualized posteriorly (Fig. 3.67B). Internal partitions divide the heart into four chambers (i.e., two atria and two ventricles) and produce surface or external grooves referred to as sulci.

1	Internal partitions divide the heart into four chambers (i.e., two atria and two ventricles) and produce surface or external grooves referred to as sulci. The coronary sulcus circles the heart, separating the atria from the ventricles (Fig. 3.68). As it circles the heart, it contains the right coronary artery, the small cardiac vein, the coronary sinus, and the circumflex branch of the left coronary artery. The anterior and posterior interventricular sulci separate the two ventricles—the anterior interventricular sulcus is on the anterior surface of the heart and contains the anterior interventricular artery and the great cardiac vein, and the posterior interventricular sulcus is on the diaphragmatic surface of the heart and contains the posterior interventricular artery and the middle cardiac vein. These sulci are continuous inferiorly, just to the right of the apex of the heart.

1	These sulci are continuous inferiorly, just to the right of the apex of the heart. The heart functionally consists of two pumps separated by a partition (Fig. 3.69A). The right pump receives deoxygenated blood from the body and sends it to the lungs. The left pump receives oxygenated blood from the lungs and sends it to the body. Each pump consists of an atrium and a ventricle separated by a valve. The thin-walled atria receive blood coming into the heart, whereas the relatively thick-walled ventricles pump blood out of the heart. More force is required to pump blood through the body than through the lungs, so the muscular wall of the left ventricle is thicker than the right. Interatrial, interventricular, and atrioventricular septa separate the four chambers of the heart (Fig. 3.69B). The internal anatomy of each chamber is critical to its function.

1	Interatrial, interventricular, and atrioventricular septa separate the four chambers of the heart (Fig. 3.69B). The internal anatomy of each chamber is critical to its function. In the anatomical position, the right border of the heart is formed by the right atrium. This chamber also contributes to the right portion of the heart’s anterior surface. Blood returning to the right atrium enters through one of three vessels. These are: the superior and inferior venae cavae, which together deliver blood to the heart from the body; and the coronary sinus, which returns blood from the walls of the heart itself. The superior vena cava enters the upper posterior portion of the right atrium, and the inferior vena cava and coronary sinus enter the lower posterior portion of the right atrium.

1	The superior vena cava enters the upper posterior portion of the right atrium, and the inferior vena cava and coronary sinus enter the lower posterior portion of the right atrium. From the right atrium, blood passes into the right ventricle through the right atrioventricular orifice. This opening faces forward and medially and is closed during ventricular contraction by the tricuspid valve. The interior of the right atrium is divided into two continuous spaces. Externally, this separation is indicated by a shallow, vertical groove (the sulcus terminalis cordis), which extends from the right side of the opening of the superior vena cava to the right side of the opening of the inferior vena cava. Internally, this division is indicated by the crista terminalis (Fig. 3.70), which is a smooth, muscular ridge that begins on the roof of the atrium just in front of the opening of the superior vena cava and extends down the lateral wall to the anterior lip of the inferior vena cava.

1	The space posterior to the crista is the sinus of venae cavae and is derived embryologically from the right horn of the sinus venosus. This component of the right atrium has smooth, thin walls, and both venae cavae empty into this space. The space anterior to the crista, including the right auricle, is sometimes referred to as the atrium proper. This terminology is based on its origin from the embryonic primitive atrium. Its walls are covered by ridges called the musculi pectinati (pectinate muscles), which fan out from the crista like the “teeth of a comb.” These ridges are also found in the right auricle, which is an ear-like, conical, muscular pouch that externally overlaps the ascending aorta.

1	An additional structure in the right atrium is the opening of the coronary sinus, which receives blood from most of the cardiac veins and opens medially to the opening of the inferior vena cava. Associated with these openings are small folds of tissue derived from the valve of the embryonic sinus venosus (the valve of the coronary sinus and the valve of inferior vena cava, respectively). During development, the valve of the inferior vena cava helps direct incoming oxygenated blood through the foramen ovale and into the left atrium. Separating the right atrium from the left atrium is the interatrial septum, which faces forward and to the right because the left atrium lies posteriorly and to the left of the right atrium. A depression is clearly visible in the septum just above the orifice of the inferior vena cava. This is the fossa ovalis (oval fossa), with its prominent margin, the limbus fossa ovalis (border of the oval fossa).

1	The fossa ovalis marks the location of the embryonic foramen ovale, which is an important part of fetal circulation. The foramen ovale allows oxygenated blood entering the right atrium through the inferior vena cava to pass directly to the left atrium and so bypass the lungs, which are nonfunctional before birth. Finally, numerous small openings—the openings of the smallest cardiac veins (the foramina of the venae cordis minimae)—are scattered along the walls of the right atrium. These are small veins that drain the myocardium directly into the right atrium. In the anatomical position, the right ventricle forms most of the anterior surface of the heart and a portion of the diaphragmatic surface. The right atrium is to the right of the right ventricle and the right ventricle is located in front of and to the left of the right atrioventricular orifice. Blood entering the right ventricle from the right atrium therefore moves in a horizontal and forward direction.

1	The outflow tract of the right ventricle, which leads to the pulmonary trunk, is the conus arteriosus (infundibulum). This area has smooth walls and derives from the embryonic bulbus cordis. The walls of the inflow portion of the right ventricle have numerous muscular, irregular structures called trabeculae carneae (Fig. 3.71). Most of these are either attached to the ventricular walls throughout their length, forming ridges, or attached at both ends, forming bridges. A few trabeculae carneae (papillary muscles) have only one end attached to the ventricular surface, while the other end serves as the point of attachment for tendon-like fibrous cords (the chordae tendineae), which connect to the free edges of the cusps of the tricuspid valve. There are three papillary muscles in the right ventricle. Named relative to their point of origin on the ventricular surface, they are the anterior, posterior, and septal papillary muscles:

1	There are three papillary muscles in the right ventricle. Named relative to their point of origin on the ventricular surface, they are the anterior, posterior, and septal papillary muscles: The anterior papillary muscle is the largest and most constant papillary muscle, and arises from the anterior wall of the ventricle. The posterior papillary muscle may consist of one, two, or three structures, with some chordae tendineae arising directly from the ventricular wall. The septal papillary muscle is the most inconsistent papillary muscle, being either small or absent, with chordae tendineae emerging directly from the septal wall.

1	The septal papillary muscle is the most inconsistent papillary muscle, being either small or absent, with chordae tendineae emerging directly from the septal wall. A single specialized trabeculum, the septomarginal trabecula (moderator band), forms a bridge between the lower portion of the interventricular septum and the base of the anterior papillary muscle. The septomarginal trabecula carries a portion of the cardiac conduction system, the right bundle of the atrioventricular bundle, to the anterior wall of the right ventricle. The right atrioventricular orifice is closed during ventricular contraction by the tricuspid valve (right atrioventricular valve), so named because it usually consists of three cusps or leaflets (Fig. 3.71). The base of each cusp is secured to the fibrous ring that surrounds the atrioventricular orifice. This fibrous ring helps to maintain the shape of the opening. The cusps are continuous with each other near their bases at sites termed commissures.

1	The naming of the three cusps, the anterior, septal, and posterior cusps, is based on their relative position in the right ventricle. The free margins of the cusps are attached to the chordae tendineae, which arise from the tips of the papillary muscles. During filling of the right ventricle, the tricuspid valve is open, and the three cusps project into the right ventricle. Without the presence of a compensating mechanism, when the ventricular musculature contracts, the valve cusps would be forced upward with the flow of blood and blood would move back into the right atrium. However, contraction of the papillary muscles attached to the cusps by chordae tendineae prevents the cusps from being everted into the right atrium. Simply put, the papillary muscles and associated chordae tendineae keep the valves closed during the dramatic changes in ventricular size that occur during contraction.

1	Simply put, the papillary muscles and associated chordae tendineae keep the valves closed during the dramatic changes in ventricular size that occur during contraction. In addition, chordae tendineae from two papillary muscles attach to each cusp. This helps prevent separation of the cusps during ventricular contraction. Proper closing of the tricuspid valve causes blood to exit the right ventricle and move into the pulmonary trunk. Necrosis of a papillary muscle following a myocardial infarction (heart attack) may result in prolapse of the related valve.

1	Necrosis of a papillary muscle following a myocardial infarction (heart attack) may result in prolapse of the related valve. At the apex of the infundibulum, the outflow tract of the right ventricle, the opening into the pulmonary trunk is closed by the pulmonary valve (Fig. 3.71), which consists of three semilunar cusps with free edges projecting upward into the lumen of the pulmonary trunk. The free superior edge of each cusp has a middle, thickened portion, the nodule of the semilunar cusp, and a thin lateral portion, the lunula of the semilunar cusp (Fig. 3.72).

1	The cusps are named the left, right, and anterior semilunar cusps, relative to their fetal position before rotation of the outflow tracts from the ventricles is complete. Each cusp forms a pocket-like sinus (Fig. 3.72)—a dilation in the wall of the initial portion of the pulmonary trunk. After ventricular contraction, the recoil of blood fills these pulmonary sinuses and forces the cusps closed. This prevents blood in the pulmonary trunk from refilling the right ventricle. The left atrium forms most of the base or posterior surface of the heart. As with the right atrium, the left atrium is derived embryologically from two structures. The posterior half, or inflow portion, receives the four pulmonary veins (Fig. 3.73). It has smooth walls and derives from the proximal parts of the pulmonary veins that are incorporated into the left atrium during development.

1	The anterior half is continuous with the left auricle. It contains musculi pectinati and derives from the embryonic primitive atrium. Unlike the crista terminalis in the right atrium, no distinct structure separates the two components of the left atrium. The interatrial septum is part of the anterior wall of the left atrium. The thin area or depression in the septum is the valve of the foramen ovale and is opposite the floor of the fossa ovalis in the right atrium. During development, the valve of the foramen ovale prevents blood from passing from the left atrium to the right atrium. This valve may not be completely fused in some adults, leaving a “probe patent” passage between the right atrium and the left atrium. The left ventricle lies anterior to the left atrium. It contributes to the anterior, diaphragmatic, and left pulmonary surfaces of the heart, and forms the apex.

1	The left ventricle lies anterior to the left atrium. It contributes to the anterior, diaphragmatic, and left pulmonary surfaces of the heart, and forms the apex. Blood enters the ventricle through the left atrioventricular orifice and flows in a forward direction to the apex. The chamber itself is conical, is longer than the right ventricle, and has the thickest layer of myocardium. The outflow tract (the aortic vestibule) is posterior to the infundibulum of the right ventricle, has smooth walls, and is derived from the embryonic bulbus cordis. The trabeculae carneae in the left ventricle are fine and delicate in contrast to those in the right ventricle. The general appearance of the trabeculae with muscular ridges and bridges is similar to that of the right ventricle (Fig. 3.74).

1	The general appearance of the trabeculae with muscular ridges and bridges is similar to that of the right ventricle (Fig. 3.74). Papillary muscles, together with chordae tendineae, are also observed and their structure is as described above for the right ventricle. Two papillary muscles, the anterior and posterior papillary muscles, are usually found in the left ventricle and are larger than those of the right ventricle. In the anatomical position, the left ventricle is somewhat posterior to the right ventricle. The interventricular septum therefore forms the anterior wall and some of the wall on the right side of the left ventricle. The septum is described as having two parts: a muscular part, and a membranous part.

1	The muscular part is thick and forms the major part of the septum, whereas the membranous part is the thin, upper part of the septum. A third part of the septum may be considered an atrioventricular part because of its position above the septal cusp of the tricuspid valve. This superior location places this part of the septum between the left ventricle and right atrium. The left atrioventricular orifice opens into the posterior right side of the superior part of the left ventricle. It is closed during ventricular contraction by the mitral valve (left atrioventricular valve), which is also referred to as the bicuspid valve because it has two cusps, the anterior and posterior cusps (Fig. 3.74). The bases of the cusps are secured to a fibrous ring surrounding the opening, and the cusps are continuous with each other at the commissures. The coordinated action of the papillary muscles and chordae tendineae is as described for the right ventricle.

1	The aortic vestibule, or outflow tract of the left ventricle, is continuous superiorly with the ascending aorta. The opening from the left ventricle into the aorta is closed by the aortic valve. This valve is similar in structure to the pulmonary valve. It consists of three semilunar cusps with the free edge of each projecting upward into the lumen of the ascending aorta (Fig. 3.75). Between the semilunar cusps and the wall of the ascending aorta are pocket-like sinuses—the right, left, and posterior aortic sinuses. The right and left coronary arteries originate from the right and left aortic sinuses. Because of this, the posterior aortic sinus and cusp are sometimes referred to as the noncoronary sinus and cusp.

1	The functioning of the aortic valve is similar to that of the pulmonary valve with one important additional process: as blood recoils after ventricular contraction and fills the aortic sinuses, it is automatically forced into the coronary arteries because these vessels originate from the right and left aortic sinuses. The cardiac skeleton is a collection of dense, fibrous connective tissue in the form of four rings with interconnecting areas in a plane between the atria and the ventricles. The four rings of the cardiac skeleton surround the two atrioventricular orifices, the aortic orifice and opening of the pulmonary trunks. They are the anulus fibrosus. The interconnecting areas include: the right fibrous trigone, which is a thickened area of connective tissue between the aortic ring and right atrioventricular ring; and the left fibrous trigone, which is a thickened area of connective tissue between the aortic ring and the left atrioventricular ring (Fig. 3.76).

1	The cardiac skeleton helps maintain the integrity of the openings it surrounds and provides points of attachment for the cusps. It also separates the atrial musculature from the ventricular musculature. The atrial myocardium originates from the upper border of the rings, whereas the ventricular myocardium originates from the lower border of the rings. The cardiac skeleton also serves as a dense connective tissue partition that electrically isolates the atria from the ventricles. The atrioventricular bundle, which passes through the anulus, is the single connection between these two groups of myocardium. Two coronary arteries arise from the aortic sinuses in the initial portion of the ascending aorta and supply the muscle and other tissues of the heart. They circle the heart in the coronary sulcus, with marginal and interventricular branches, in the interventricular sulci, converging toward the apex of the heart (Fig. 3.77).

1	The returning venous blood passes through cardiac veins, most of which empty into the coronary sinus. This large venous structure is located in the coronary sulcus on the posterior surface of the heart between the left atrium and left ventricle. The coronary sinus empties into the right atrium between the opening of the inferior vena cava and the right atrioventricular orifice. Right coronary artery. The right coronary artery originates from the right aortic sinus of the ascending aorta. It passes anteriorly and then descends vertically in the coronary sulcus, between the right atrium and right ventricle (Fig. 3.78A). On reaching the inferior margin of the heart, it turns posteriorly and continues in the sulcus onto the diaphragmatic surface and base of the heart. During this course, several branches arise from the main stem of the vessel:

1	An early atrial branch passes in the groove between the right auricle and ascending aorta, and gives off the sinu-atrial nodal branch (Fig. 3.78A), which passes posteriorly around the superior vena cava to supply the sinu-atrial node. A right marginal branch is given off as the right coronary artery approaches the inferior (acute) margin of the heart (Fig. 3.78A,B) and continues along this border toward the apex of the heart. As the right coronary artery continues on the base/ diaphragmatic surface of the heart, it supplies a small branch to the atrioventricular node before giving off its final major branch, the posterior interventricular branch (Fig. 3.78A), which lies in the posterior interventricular sulcus.

1	The right coronary artery supplies the right atrium and right ventricle, the sinu-atrial and atrioventricular nodes, the interatrial septum, a portion of the left atrium, the posteroinferior one third of the interventricular septum, and a portion of the posterior part of the left ventricle. Left coronary artery. The left coronary artery originates from the left aortic sinus of the ascending aorta. It passes between the pulmonary trunk and the left auricle before entering the coronary sulcus. Emerging from behind the pulmonary trunk, the artery divides into its two terminal branches, the anterior interventricular and the circumflex (Fig. 3.78A).

1	The anterior interventricular branch (left anterior descending artery—LAD) (Fig. 3.78A,C) continues around the left side of the pulmonary trunk and descends obliquely toward the apex of the heart in the anterior interventricular sulcus (Fig. 3.78A,C). During its course, one or two large diagonal branches may arise and descend diagonally across the anterior surface of the left ventricle. The circumflex branch (Fig. 3.78A,C) courses toward the left, in the coronary sulcus and onto the base/diaphragmatic surface of the heart, and usually ends before reaching the posterior interventricular sulcus. A large branch, the left marginal artery (Fig. 3.78A,C), usually arises from it and continues across the rounded obtuse margin of the heart. The distribution pattern of the left coronary artery enables it to supply most of the left atrium and left ventricle, and most of the interventricular septum, including the atrioventricular bundle and its branches.

1	Variations in the distribution patterns of coronary arteries. Several major variations in the basic distribution patterns of the coronary arteries occur. The distribution pattern described above for both right and left coronary arteries is the most common and consists of a right dominant coronary artery. This means that the posterior interventricular branch arises from the right coronary artery. The right coronary artery therefore supplies a large portion of the posterior wall of the left ventricle and the circumflex branch of the left coronary artery is relatively small. In contrast, in hearts with a left dominant coronary artery, the posterior interventricular branch arises from an enlarged circumflex branch and supplies most of the posterior wall of the left ventricle (Fig. 3.79).

1	Another point of variation relates to the arterial supply to the sinu-atrial and atrioventricular nodes. In most cases, these two structures are supplied by the right coronary artery. However, vessels from the circumflex branch of the left coronary artery occasionally supply these structures. The coronary sinus receives four major tributaries: the great, middle, small, and posterior cardiac veins.

1	The coronary sinus receives four major tributaries: the great, middle, small, and posterior cardiac veins. Great cardiac vein. The great cardiac vein begins at the apex of the heart (Fig. 3.82A). It ascends in the anterior interventricular sulcus, where it is related to the anterior interventricular artery and is often termed the anterior interventricular vein. Reaching the coronary sulcus, the great cardiac vein turns to the left and continues onto the base/diaphragmatic surface of the heart. At this point, it is associated with the circumflex branch of the left coronary artery. Continuing along its path in the coronary sulcus, the great cardiac vein gradually enlarges to form the coronary sinus, which enters the right atrium (Fig. 3.82B).

1	Middle cardiac vein. The middle cardiac vein (posterior interventricular vein) begins near the apex of the heart and ascends in the posterior interventricular sulcus toward the coronary sinus (Fig. 3.82B). It is associated with the posterior interventricular branch of the right or left coronary artery throughout its course. Small cardiac vein. The small cardiac vein begins in the lower anterior section of the coronary sulcus between the right atrium and right ventricle (Fig. 3.82A). It continues in this groove onto the base/diaphragmatic surface of the heart where it enters the coronary sinus at its atrial end. It is a companion of the right coronary artery throughout its course and may receive the right marginal vein (Fig. 3.82A). This small vein accompanies the marginal branch of the right coronary artery along the acute margin of the heart. If the right marginal vein does not join the small cardiac vein, it enters the right atrium directly.

1	Posterior cardiac vein. The posterior cardiac vein lies on the posterior surface of the left ventricle just to the left of the middle cardiac vein (Fig. 3.82B). It either enters the coronary sinus directly or joins the great cardiac vein. Other cardiac veins. Two additional groups of cardiac veins are also involved in the venous drainage of the heart. The anterior veins of the right ventricle (anterior cardiac veins) are small veins that arise on the anterior surface of the right ventricle (Fig. 3.82A). They cross the coronary sulcus and enter the anterior wall of the right atrium. They drain the anterior portion of the right ventricle. The right marginal vein may be part of this group if it does not enter the small cardiac vein.

1	A group of smallest cardiac veins (venae cordis minimae or veins of Thebesius) have also been described. Draining directly into the cardiac chambers, they are numerous in the right atrium and right ventricle, are occasionally associated with the left atrium, and are rarely associated with the left ventricle. The lymphatic vessels of the heart follow the coronary arteries and drain mainly into: brachiocephalic nodes, anterior to the brachiocephalic veins; and tracheobronchial nodes, at the inferior end of the trachea. The musculature of the atria and ventricles is capable of contracting spontaneously. The cardiac conduction system initiates and coordinates contraction. The conduction system consists of nodes and networks of specialized cardiac muscle cells organized into four basic components: the sinu-atrial node, the atrioventricular node, the atrioventricular bundle with its right and left bundle branches, and the subendocardial plexus of conduction cells (the Purkinje fibers).

1	The unique distribution pattern of the cardiac conduction system establishes an important unidirectional pathway of excitation/contraction. Throughout its course, large branches of the conduction system are insulated from the surrounding myocardium by connective tissue. This tends to decrease inappropriate stimulation and contraction of cardiac muscle fibers. The number of functional contacts between the conduction pathway and cardiac musculature greatly increases in the subendocardial network. Thus, a unidirectional wave of excitation and contraction is established, which moves from the papillary muscles and apex of the ventricles to the arterial outflow tracts. Impulses begin at the sinu-atrial node, the cardiac pacemaker. This collection of cells is located at the superior end of the crista terminalis at the junction of the superior vena cava and the right atrium (Fig. 3.83A).

1	This is also the junction between the parts of the right atrium derived from the embryonic sinus venosus and the atrium proper. The excitation signals generated by the sinu-atrial node spread across the atria, causing the muscle to contract. Concurrently, the wave of excitation in the atria stimulates the atrioventricular node, which is located near the opening of the coronary sinus, close to the attachment of the septal cusp of the tricuspid valve, and within the atrioventricular septum (Fig. 3.83A). The atrioventricular node is a collection of specialized cells that forms the beginning of an elaborate system of conducting tissue, the atrioventricular bundle, which extends the excitatory impulse to all ventricular musculature. The atrioventricular bundle is a direct continuation of the atrioventricular node (Fig. 3.83A). It follows along the lower border of the membranous part of the interventricular septum before splitting into right and left bundles.

1	The right bundle branch continues on the right side of the interventricular septum toward the apex of the right ventricle. From the septum it enters the septomarginal trabecula to reach the base of the anterior papillary muscle. At this point, it divides and is continuous with the final component of the cardiac conduction system, the subendocardial plexus of ventricular conduction cells or Purkinje fibers. This network of specialized cells spreads throughout the ventricle to supply the ventricular musculature, including the papillary muscles. The left bundle branch passes to the left side of the muscular interventricular septum and descends to the apex of the left ventricle (Fig. 3.83B). Along its course it gives off branches that eventually become continuous with the subendocardial plexus of conduction cells (Purkinje fibers). As with the right side, this network of specialized cells spreads the excitation impulses throughout the left ventricle.

1	The autonomic division of the peripheral nervous system is directly responsible for regulating: heart rate, force of each contraction, and cardiac output. Branches from both the parasympathetic and sympathetic systems contribute to the formation of the cardiac plexus. This plexus consists of a superficial part, inferior to the aortic arch and between it and the pulmonary trunk (Fig. 3.84A), and a deep part, between the aortic arch and the tracheal bifurcation (Fig. 3.84B). From the cardiac plexus, small branches that are mixed nerves containing both sympathetic and parasympathetic fibers supply the heart. These branches affect nodal tissue and other components of the conduction system, coronary blood vessels, and atrial and ventricular musculature. Stimulation of the parasympathetic system: decreases heart rate, reduces force of contraction, and constricts the coronary arteries.

1	Stimulation of the parasympathetic system: decreases heart rate, reduces force of contraction, and constricts the coronary arteries. The preganglionic parasympathetic fibers reach the heart as cardiac branches from the right and left vagus nerves. They enter the cardiac plexus and synapse in ganglia located either within the plexus or in the walls of the atria. Stimulation of the sympathetic system: increases heart rate, and increases the force of contraction. Sympathetic fibers reach the cardiac plexus through the cardiac nerves from the sympathetic trunk. Preganglionic sympathetic fibers from the upper four or five segments of the thoracic spinal cord enter and move through the sympathetic trunk. They synapse in cervical and upper thoracic sympathetic ganglia, and postganglionic fibers proceed as bilateral branches from the sympathetic trunk to the cardiac plexus.

1	Visceral afferents from the heart are also a component of the cardiac plexus. These fibers pass through the cardiac plexus and return to the central nervous system in the cardiac nerves from the sympathetic trunk and in the vagal cardiac branches. The afferents associated with the vagal cardiac nerves return to the vagus nerve [X]. They sense alterations in blood pressure and blood chemistry and are therefore primarily concerned with cardiac reflexes.

1	The afferents associated with the cardiac nerves from the sympathetic trunks return to either the cervical or the thoracic portions of the sympathetic trunk. If they are in the cervical portion of the trunk, they normally descend to the thoracic region, where they reenter the upper four or five thoracic spinal cord segments, along with the afferents from the thoracic region of the sympathetic trunk. Visceral afferents associated with the sympathetic system conduct pain sensation from the heart, which is detected at the cellular level as tissue-damaging events (i.e., cardiac ischemia). This pain is often “referred” to cutaneous regions supplied by the same spinal cord levels (see “In the clinic: Referred pain.” p. 46, and “Case 1,” pp. 244–246).

1	The pulmonary trunk is contained within the pericardial sac (Fig. 3.85), is covered by the visceral layer of serous pericardium, and is associated with the ascending aorta in a common sheath. It arises from the conus arteriosus of the right ventricle at the opening of the pulmonary trunk slightly anterior to the aortic orifice and ascends, moving posteriorly and to the left, lying initially anterior and then to the left of the ascending aorta. At approximately the level of the intervertebral disc between vertebrae TV and TVI, opposite the left border of the sternum and posterior to the third left costal cartilage, the pulmonary trunk divides into: the right pulmonary artery, which passes to the right, posterior to the ascending aorta and the superior vena cava, to enter the right lung; and the left pulmonary artery, which passes inferiorly to the arch of the aorta and anteriorly to the descending aorta to enter the left lung.

1	The ascending aorta is contained within the pericardial sac and is covered by a visceral layer of serous pericardium, which also surrounds the pulmonary trunk in a common sheath (Fig. 3.85A). The origin of the ascending aorta is the aortic orifice at the base of the left ventricle, which is level with the lower edge of the third left costal cartilage, posterior to the left half of the sternum. Moving superiorly, slightly forward and to the right, the ascending aorta continues to the level of the second right costal cartilage. At this point, it enters the superior mediastinum and is then referred to as the arch of the aorta. Immediately superior to the point where the ascending aorta arises from the left ventricle are three small outward bulges opposite the semilunar cusps of the aortic valve. These are the posterior, right, and left aortic sinuses. The right and left coronary arteries originate from the right and left aortic sinuses, respectively.

1	The inferior half of the superior vena cava is located within the pericardial sac (Fig. 3.85B). It passes through the fibrous pericardium at approximately the level of the second costal cartilage and enters the right atrium at the lower level of the third costal cartilage. The portion within the pericardial sac is covered with serous pericardium except for a small area on its posterior surface. After passing through the diaphragm, at approximately the level of vertebra TVIII, the inferior vena cava enters the fibrous pericardium. A short portion of this vessel is within the pericardial sac before entering the right atrium. While within the pericardial sac, it is covered by serous pericardium except for a small portion of its posterior surface (Fig. 3.85B).

1	A very short segment of each of the pulmonary veins is also within the pericardial sac. These veins, usually two from each lung, pass through the fibrous pericardium and enter the superior region of the left atrium on its posterior surface. In the pericardial sac, all but a portion of the posterior surface of these veins is covered by serous pericardium. In addition, the oblique pericardial sinus is between the right and left pulmonary veins, within the pericardial sac (Fig. 3.85B). The superior mediastinum is posterior to the manubrium of the sternum and anterior to the bodies of the first four thoracic vertebrae (see Fig. 3.57). Its superior boundary is an oblique plane passing from the jugular notch upward and posteriorly to the superior border of vertebra TI. Inferiorly, a transverse plane passing from the sternal angle to the intervertebral disc between vertebra TIV/V separates it from the inferior mediastinum.

1	Inferiorly, a transverse plane passing from the sternal angle to the intervertebral disc between vertebra TIV/V separates it from the inferior mediastinum. Laterally, it is bordered by the mediastinal part of the parietal pleura on either side. The superior mediastinum is continuous with the neck above and with the inferior mediastinum below. The major structures found in the superior mediastinum (Figs. 3.86 and 3.87) include the: thymus, right and left brachiocephalic veins, left superior intercostal vein, superior vena cava, arch of the aorta with its three large branches, trachea, esophagus, phrenic nerves, vagus nerves, left recurrent laryngeal branch of the left vagus nerve, thoracic duct, and other small nerves, blood vessels, and lymphatics. The thymus is the most anterior component of the superior mediastinum, lying immediately posterior to the manubrium of the sternum. It is an asymmetrical, bilobed structure (see Fig. 3.58).

1	The thymus is the most anterior component of the superior mediastinum, lying immediately posterior to the manubrium of the sternum. It is an asymmetrical, bilobed structure (see Fig. 3.58). The upper extent of the thymus can reach into the neck as high as the thyroid gland; a lower portion typically extends into the anterior mediastinum over the pericardial sac. Involved in the early development of the immune system, the thymus is a large structure in the child, begins to atrophy after puberty, and shows considerable size variation in the adult. In the elderly adult, it is barely identifiable as an organ, consisting mostly of fatty tissue that is sometimes arranged as two lobulated fatty structures. Arteries to the thymus consist of small branches originating from the internal thoracic arteries. Venous drainage is usually into the left brachiocephalic vein and possibly into the internal thoracic veins.

1	Lymphatic drainage returns to multiple groups of nodes at one or more of the following locations: along the internal thoracic arteries (parasternal); at the tracheal bifurcation (tracheobronchial); and in the root of the neck. The left and right brachiocephalic veins are located immediately posterior to the thymus. They form on each side at the junction between the internal jugular and subclavian veins (see Fig. 3.86). The left brachiocephalic vein crosses the midline and joins with the right brachiocephalic vein to form the superior vena cava (Fig. 3.88). The right brachiocephalic vein begins posterior to the medial end of the right clavicle and passes vertically downward, forming the superior vena cava when it is joined by the left brachiocephalic vein. Venous tributaries include the vertebral, first posterior intercostal, and internal thoracic veins. The inferior thyroid and thymic veins may also drain into it.

1	The left brachiocephalic vein begins posterior to the medial end of the left clavicle. It crosses to the right, moving in a slightly inferior direction, and joins with the right brachiocephalic vein to form the superior vena cava posterior to the lower edge of the right first costal cartilage close to the right sternal border. Venous tributaries include the vertebral, first posterior intercostal, left superior intercostal, inferior thyroid, and internal thoracic veins. It may also receive thymic and pericardial veins. The left brachiocephalic vein crosses the midline posterior to the manubrium in the adult. In infants and children the left brachiocephalic vein rises above the superior border of the manubrium and therefore is less protected.

1	The left superior intercostal vein receives the second, third, and sometimes the fourth posterior intercostal veins, usually the left bronchial veins, and sometimes the left pericardiacophrenic vein. It passes over the left side of the aortic arch, lateral to the left vagus nerve and medial to the left phrenic nerve, before entering the left brachiocephalic vein (Fig. 3.89). Inferiorly, it may connect with the accessory hemiazygos vein (superior hemiazygos vein). The vertically oriented superior vena cava begins posterior to the lower edge of the right first costal cartilage, where the right and left brachiocephalic veins join, and terminates at the lower edge of the right third costal cartilage, where it joins the right atrium (see Fig. 3.86). The lower half of the superior vena cava is within the pericardial sac and is therefore contained in the middle mediastinum.

1	The lower half of the superior vena cava is within the pericardial sac and is therefore contained in the middle mediastinum. The superior vena cava receives the azygos vein immediately before entering the pericardial sac and may also receive pericardial and mediastinal veins. The superior vena cava can be easily visualized forming part of the right superolateral border of the mediastinum on a chest radiograph (see Fig. 3.67A). Arch of aorta and its branches

1	The superior vena cava can be easily visualized forming part of the right superolateral border of the mediastinum on a chest radiograph (see Fig. 3.67A). Arch of aorta and its branches The thoracic portion of the aorta can be divided into ascending aorta, arch of the aorta, and thoracic (descending) aorta. Only the arch of the aorta is in the superior mediastinum. It begins when the ascending aorta emerges from the pericardial sac and courses upward, backward, and to the left as it passes through the superior mediastinum, ending on the left side at vertebral level TIV/V (see Fig. 3.86). Extending as high as the midlevel of the manubrium of the sternum, the arch is initially anterior and finally lateral to the trachea. Three branches arise from the superior border of the arch of the aorta; at their origins, all three are crossed anteriorly by the left brachiocephalic vein. The first branch

1	Three branches arise from the superior border of the arch of the aorta; at their origins, all three are crossed anteriorly by the left brachiocephalic vein. The first branch Beginning on the right, the first branch of the arch of the aorta is the brachiocephalic trunk (Fig. 3.90). It is the largest of the three branches and, at its point of origin behind the manubrium of the sternum, is slightly anterior to the other two branches. It ascends slightly posteriorly and to the right. At the level of the upper edge of the right sternoclavicular joint, the brachiocephalic trunk divides into: the right common carotid artery, and the right subclavian artery (see Fig. 3.86). The arteries mainly supply the right side of the head and neck and the right upper limb, respectively. Occasionally, the brachiocephalic trunk has a small branch, the thyroid ima artery, which contributes to the vascular supply of the thyroid gland. The second branch

1	Occasionally, the brachiocephalic trunk has a small branch, the thyroid ima artery, which contributes to the vascular supply of the thyroid gland. The second branch The second branch of the arch of the aorta is the left common carotid artery (Fig. 3.90). It arises from the arch immediately to the left and slightly posterior to the brachiocephalic trunk and ascends through the superior mediastinum along the left side of the trachea. The left common carotid artery supplies the left side of the head and neck. The third branch The third branch of the arch of the aorta is the left subclavian artery (Fig. 3.90). It arises from the arch of the aorta immediately to the left of, and slightly posterior to, the left common carotid artery and ascends through the superior mediastinum along the left side of the trachea. The left subclavian artery is the major blood supply to the left upper limb.

1	The left subclavian artery is the major blood supply to the left upper limb. The ligamentum arteriosum is also in the superior mediastinum and is important in embryonic circulation, when it is a patent vessel (the ductus arteriosus). It connects the pulmonary trunk with the arch of the aorta and allows blood to bypass the lungs during development (Fig. 3.90). The vessel closes soon after birth and forms the ligamentous connection observed in the adult. The trachea is a midline structure that is palpable in the jugular notch as it enters the superior mediastinum. Posterior to it is the esophagus, which is immediately anterior to the vertebral column (Fig. 3.92, and see Figs. 3.86 and 3.87). Significant mobility exists in the vertical positioning of these structures as they pass through the superior mediastinum. Swallowing and breathing cause positional shifts, as may disease and the use of specialized instrumentation.

1	As the trachea and esophagus pass through the superior mediastinum, they are crossed laterally by the azygos vein on the right side and the arch of the aorta on the left side. The trachea divides into the right and left main bronchi at, or just inferior to, the transverse plane between the sternal angle and vertebral level TIV/V (Fig. 3.93), whereas the esophagus continues into the posterior mediastinum. Nerves of the superior mediastinum The vagus nerves [X] pass through the superior and posterior divisions of the mediastinum on their way to the abdominal cavity. As they pass through the thorax, they provide parasympathetic innervation to the thoracic viscera and carry visceral afferents from the thoracic viscera. Visceral afferents in the vagus nerves relay information to the central nervous system about normal physiological processes and reflex activities. They do not transmit pain sensation.

1	Visceral afferents in the vagus nerves relay information to the central nervous system about normal physiological processes and reflex activities. They do not transmit pain sensation. The right vagus nerve enters the superior mediastinum and lies between the right brachiocephalic vein and the brachiocephalic trunk. It descends in a posterior direction toward the trachea (Fig. 3.94), crosses the lateral surface of the trachea, and passes posteriorly to the root of the right lung to reach the esophagus. Just before the esophagus, it is crossed by the arch of the azygos vein. As the right vagus nerve passes through the superior mediastinum, it gives branches to the esophagus, cardiac plexus, and pulmonary plexus.

1	As the right vagus nerve passes through the superior mediastinum, it gives branches to the esophagus, cardiac plexus, and pulmonary plexus. The left vagus nerve enters the superior mediastinum posterior to the left brachiocephalic vein and between the left common carotid and left subclavian arteries (Fig. 3.95). As it passes into the superior mediastinum, it lies just deep to the mediastinal part of the parietal pleura and crosses the left side of the arch of the aorta. It continues to descend in a posterior direction and passes posterior to the root of the left lung to reach the esophagus in the posterior mediastinum. As the left vagus nerve passes through the superior mediastinum, it gives branches to the esophagus, the cardiac plexus, and the pulmonary plexus.

1	As the left vagus nerve passes through the superior mediastinum, it gives branches to the esophagus, the cardiac plexus, and the pulmonary plexus. The left vagus nerve also gives rise to the left recurrent laryngeal nerve, which arises from it at the inferior margin of the arch of the aorta just lateral to the ligamentum arteriosum. The left recurrent laryngeal nerve passes inferior to the arch of the aorta before ascending on its medial surface. Entering a groove between the trachea and esophagus, the left recurrent laryngeal nerve continues superiorly to enter the neck and terminate in the larynx (Fig. 3.96). The phrenic nerves arise in the cervical region mainly from the fourth, but also from the third and fifth, cervical spinal cord segments.

1	The phrenic nerves arise in the cervical region mainly from the fourth, but also from the third and fifth, cervical spinal cord segments. The phrenic nerves descend through the thorax to supply motor and sensory innervation to the diaphragm and its associated membranes. As they pass through the thorax, they provide innervation through somatic afferent fibers to the mediastinal pleura, fibrous pericardium, and parietal layer of serous pericardium. The right phrenic nerve enters the superior mediastinum lateral to the right vagus nerve and lateral and slightly posterior to the beginning of the right brachiocephalic vein (see Fig. 3.94). It continues inferiorly along the right side of this vein and the right side of the superior vena cava.

1	On entering the middle mediastinum, the right phrenic nerve descends along the right side of the pericardial sac, within the fibrous pericardium, anterior to the root of the right lung. The pericardiacophrenic vessels accompany it through most of its course in the thorax (see Fig. 3.60). It leaves the thorax by passing through the diaphragm with the inferior vena cava. The left phrenic nerve enters the superior mediastinum in a position similar to the path taken by the right phrenic nerve. It lies lateral to the left vagus nerve and lateral and slightly posterior to the beginning of the left brachiocephalic vein (see Fig. 3.89), and continues to descend across the left lateral surface of the arch of the aorta, passing superficially to the left vagus nerve and the left superior intercostal vein.

1	On entering the middle mediastinum, the left phrenic nerve follows the left side of the pericardial sac, within the fibrous pericardium, anterior to the root of the left lung, and is accompanied by the pericardiacophrenic vessels (see Fig. 3.60). It leaves the thorax by piercing the diaphragm near the apex of the heart. Thoracic duct in the superior mediastinum The thoracic duct, which is the major lymphatic vessel in the body, passes through the posterior portion of the superior mediastinum (see Figs. 3.87 and 3.92). It: enters the superior mediastinum inferiorly, slightly to the left of the midline, having moved to this position just before leaving the posterior mediastinum opposite vertebral level TIV/V; and continues through the superior mediastinum, posterior to the arch of the aorta, and the initial portion of the left subclavian artery, between the esophagus and the left mediastinal part of the parietal pleura.

1	The posterior mediastinum is posterior to the pericardial sac and diaphragm and anterior to the bodies of the mid and lower thoracic vertebrae (see Fig. 3.57). Its superior boundary is a transverse plane passing from the sternal angle to the intervertebral disc between vertebrae TIV and TV. Its inferior boundary is the diaphragm. Laterally, it is bordered by the mediastinal part of parietal pleura on either side. Superiorly, it is continuous with the superior mediastinum. Major structures in the posterior mediastinum include the: esophagus and its associated nerve plexus, thoracic aorta and its branches, azygos system of veins, thoracic duct and associated lymph nodes, sympathetic trunks, and thoracic splanchnic nerves. The esophagus is a muscular tube passing between the pharynx in the neck and the stomach in the abdomen. It begins at the inferior border of the cricoid cartilage, opposite vertebra CVI, and ends at the cardiac opening of the stomach, opposite vertebra TXI.

1	The esophagus descends on the anterior aspect of the bodies of the vertebrae, generally in a midline position as it moves through the thorax (Fig. 3.97). As it approaches the diaphragm, it moves anteriorly and to the left, crossing from the right side of the thoracic aorta to eventually assume a position anterior to it. It then passes through the esophageal hiatus, an opening in the muscular part of the diaphragm, at vertebral level TX. The esophagus has a slight anterior-to-posterior curvature that parallels the thoracic portion of the vertebral column, and is secured superiorly by its attachment to the pharynx and inferiorly by its attachment to the diaphragm. Relationships to important structures in the posterior mediastinum In the posterior mediastinum, the esophagus is related to a number of important structures. The right side is covered by the mediastinal part of the parietal pleura.

1	In the posterior mediastinum, the esophagus is related to a number of important structures. The right side is covered by the mediastinal part of the parietal pleura. Posterior to the esophagus, the thoracic duct is on the right side inferiorly, but crosses to the left more superiorly. Also on the left side of the esophagus is the thoracic aorta. Anterior to the esophagus, below the level of the tracheal bifurcation, are the right pulmonary artery and the left main bronchus. The esophagus then passes immediately posteriorly to the left atrium, separated from it only by pericardium. Inferior to the left atrium, the esophagus is related to the diaphragm. Structures other than the thoracic duct posterior to the esophagus include portions of the hemiazygos veins, the right posterior intercostal vessels, and, near the diaphragm, the thoracic aorta.

1	Structures other than the thoracic duct posterior to the esophagus include portions of the hemiazygos veins, the right posterior intercostal vessels, and, near the diaphragm, the thoracic aorta. The esophagus is a flexible, muscular tube that can be compressed or narrowed by surrounding structures at four locations (Fig. 3.98): the junction of the esophagus with the pharynx in the neck; in the superior mediastinum where the esophagus is crossed by the arch of the aorta; in the posterior mediastinum where the esophagus is compressed by the left main bronchus; in the posterior mediastinum at the esophageal hiatus in the diaphragm.

1	These constrictions have important clinical consequences. For example, a swallowed object is most likely to lodge at a constricted area. An ingested corrosive substance would move more slowly through a narrowed region, causing more damage at this site than elsewhere along the esophagus. Also, constrictions present problems during the passage of medical instruments. The arterial supply and venous drainage of the esophagus in the posterior mediastinum involve many vessels. Esophageal arteries arise from the thoracic aorta, bronchial arteries, and ascending branches of the left gastric artery in the abdomen. Venous drainage involves small vessels returning to the azygos vein, hemiazygos vein, and esophageal branches to the left gastric vein in the abdomen. Lymphatic drainage of the esophagus in the posterior mediastinum returns to posterior mediastinal and left gastric nodes.

1	Lymphatic drainage of the esophagus in the posterior mediastinum returns to posterior mediastinal and left gastric nodes. Innervation of the esophagus, in general, is complex. Esophageal branches arise from the vagus nerves and sympathetic trunks. Striated muscle fibers in the superior portion of the esophagus originate from the branchial arches and are innervated by branchial efferents from the vagus nerves. Smooth muscle fibers are innervated by cranial components of the parasympathetic part of the autonomic division of the peripheral nervous system, visceral efferents from the vagus nerves. These are preganglionic fibers that synapse in the myenteric and submucosal plexuses of the enteric nervous system in the esophageal wall. Sensory innervation of the esophagus involves visceral afferent fibers originating in the vagus nerves, sympathetic trunks, and splanchnic nerves.

1	Sensory innervation of the esophagus involves visceral afferent fibers originating in the vagus nerves, sympathetic trunks, and splanchnic nerves. The visceral afferents from the vagus nerves are involved in relaying information back to the central nervous system about normal physiological processes and reflex activities. They are not involved in the relay of pain recognition. The visceral afferents that pass through the sympathetic trunks and the splanchnic nerves are the primary participants in detection of esophageal pain and transmission of this information to various levels of the central nervous system.

1	After passing posteriorly to the root of the lungs, the right and left vagus nerves approach the esophagus. As they reach the esophagus, each nerve divides into several branches that spread over this structure, forming the esophageal plexus (Fig. 3.99). There is some mixing of fibers from the two vagus nerves as the plexus continues inferiorly on the esophagus toward the diaphragm. Just above the diaphragm, fibers of the plexus converge to form two trunks: the anterior vagal trunk on the anterior surface of the esophagus, mainly from fibers originally in the left vagus nerve; the posterior vagal trunk on the posterior surface of the esophagus, mainly from fibers originally in the right vagus nerve. The vagal trunks continue on the surface of the esophagus as it passes through the diaphragm into the abdomen.

1	The vagal trunks continue on the surface of the esophagus as it passes through the diaphragm into the abdomen. The thoracic portion of the descending aorta (thoracic aorta) begins at the lower edge of vertebra TIV, where it is continuous with the arch of the aorta. It ends anterior to the lower edge of vertebra TXII, where it passes through the aortic hiatus posterior to the diaphragm. Situated to the left of the vertebral column superiorly, it approaches the midline inferiorly, lying directly anterior to the lower thoracic vertebral bodies (Fig. 3.101). Throughout its course, it gives off a number of branches, which are summarized in Table 3.3. Azygos system of veins

1	Azygos system of veins The azygos system of veins consists of a series of longitudinal vessels on each side of the body that drain blood from the body wall and move it superiorly to empty into the superior vena cava. Blood from some of the thoracic viscera may also enter the system, and there are anastomotic connections with abdominal veins. The longitudinal vessels may or may not be continuous and are connected to each other from side to side at various points throughout their course (Fig. 3.102). The azygos system of veins serves as an important anastomotic pathway capable of returning venous blood from the lower part of the body to the heart if the inferior vena cava is blocked. The major veins in the system are: the azygos vein, on the right; and the hemiazygos vein and the accessory hemiazygos vein, on the left. There is significant variation in the origin, course, tributaries, anastomoses, and termination of these vessels.

1	There is significant variation in the origin, course, tributaries, anastomoses, and termination of these vessels. The azygos vein arises opposite vertebra LI or LII at the junction between the right ascending lumbar vein and the right subcostal vein (Fig. 3.102). It may also arise as a direct branch of the inferior vena cava, which is joined by a common trunk from the junction of the right ascending lumbar vein and the right subcostal vein. The azygos vein enters the thorax through the aortic hiatus of the diaphragm, or it enters through or posterior to the right crus of the diaphragm. It ascends through the posterior mediastinum, usually to the right of the thoracic duct. At approximately vertebral level TIV, it arches anteriorly, over the root of the right lung, to join the superior vena cava before the superior vena cava enters the pericardial sac.

1	Tributaries of the azygos vein include: the right superior intercostal vein (a single vessel formed by the junction of the second, third, and fourth intercostal veins), fifth to eleventh right posterior intercostal veins, the hemiazygos vein, the accessory hemiazygos vein, esophageal veins, mediastinal veins, pericardial veins, and right bronchial veins. The hemiazygos vein (inferior hemiazygos vein) usually arises at the junction between the left ascending lumbar vein and the left subcostal vein (Fig. 3.102). It may also arise from either of these veins alone and often has a connection to the left renal vein. The hemiazygos vein usually enters the thorax through the left crus of the diaphragm, but may enter through the aortic hiatus. It ascends through the posterior mediastinum, on the left side, to approximately vertebral level TIX. At this point, it crosses the vertebral column, posterior to the thoracic aorta, esophagus, and thoracic duct, to enter the azygos vein.

1	Tributaries joining the hemiazygos vein include: the lowest four or five left posterior intercostal veins, esophageal veins, and mediastinal veins. The accessory hemiazygos vein (superior hemiazygos vein) descends on the left side from the superior portion of the posterior mediastinum to approximately vertebral level TVIII (Fig. 3.102). At this point, it crosses the vertebral column to join the azygos vein, or ends in the hemiazygos vein, or has a connection to both veins. Usually, it also has a connection superiorly to the left superior intercostal vein. Vessels that drain into the accessory hemiazygos vein include: the fourth to eighth left posterior intercostal veins, and sometimes, the left bronchial veins. Thoracic duct in the posterior mediastinum

1	Thoracic duct in the posterior mediastinum The thoracic duct is the principal channel through which lymph from most of the body is returned to the venous system. It begins as a confluence of lymph trunks in the abdomen, sometimes forming a saccular dilation referred to as the cisterna chyli (chyle cistern), which drains the abdominal viscera and walls, pelvis, perineum, and lower limbs. The thoracic duct extends from vertebra LII to the root of the neck. Entering the thorax, posterior to the aorta, through the aortic hiatus of the diaphragm, the thoracic duct ascends through the posterior mediastinum to the right of midline between the thoracic aorta on the left and the azygos vein on the right (Fig. 3.103). It lies posterior to the diaphragm and the esophagus and anterior to the bodies of the vertebrae. At vertebral level TV, the thoracic duct moves to the left of midline and enters the superior mediastinum. It continues through the superior mediastinum and into the neck.

1	At vertebral level TV, the thoracic duct moves to the left of midline and enters the superior mediastinum. It continues through the superior mediastinum and into the neck. After being joined, in most cases, by the left jugular trunk, which drains the left side of the head and neck, and the left subclavian trunk, which drains the left upper limb, the thoracic duct empties into the junction of the left subclavian and left internal jugular veins. The thoracic duct usually receives the contents from: the confluence of lymph trunks in the abdomen, descending thoracic lymph trunks draining the lower six or seven intercostal spaces on both sides, upper intercostal lymph trunks draining the upper left five or six intercostal spaces, ducts from posterior mediastinal nodes, and ducts from posterior diaphragmatic nodes.

1	The sympathetic trunks are an important component of the sympathetic part of the autonomic division of the peripheral nervous system and are usually considered a component of the posterior mediastinum as they pass through the thorax. This portion of the sympathetic trunks consists of two parallel cords punctuated by 11 or 12 ganglia (Fig. 3.104). The ganglia are connected to adjacent thoracic spinal nerves by white and gray rami communicantes and are numbered according to the thoracic spinal nerve with which they are associated. In the superior portion of the posterior mediastinum, the trunks are anterior to the neck of the ribs. Inferiorly, they become more medial in position until they lie on the lateral aspect of the vertebral bodies. The sympathetic trunks leave the thorax by passing posterior to the diaphragm under the medial arcuate ligament or through the crura of the diaphragm. Throughout their course the trunks are covered by parietal pleura. Branches from the ganglia

1	Branches from the ganglia Two types of medial branches are given off by the ganglia: The first type includes branches from the upper five ganglia. The second type includes branches from the lower seven ganglia. The first type, which includes branches from the upper five ganglia, consists mainly of postganglionic sympathetic fibers, which supply the various thoracic viscera. These branches are relatively small, and also contain visceral afferent fibers. The second type, which includes branches from the lower seven ganglia, consists mainly of preganglionic sympathetic fibers, which supply the various abdominal and pelvic viscera. These branches are large, also carry visceral afferent fibers, and form the three thoracic splanchnic nerves referred to as the greater, lesser, and least splanchnic nerves (Fig. 3.104).

1	The greater splanchnic nerve on each side usually arises from the fifth to ninth or tenth thoracic ganglia. It descends across the vertebral bodies moving in a medial direction, passes into the abdomen through the crus of the diaphragm, and ends in the celiac ganglion. The lesser splanchnic nerve usually arises from the ninth and tenth, or tenth and eleventh thoracic ganglia. It descends across the vertebral bodies moving in a medial direction, and passes into the abdomen through the crus of the diaphragm to end in the aorticorenal ganglion. The least splanchnic nerve (lowest splanchnic nerve) usually arises from the twelfth thoracic ganglion. It descends and passes into the abdomen through the crus of the diaphragm to end in the renal plexus.

1	The ability to visualize how anatomical structures in the thorax are related to surface features is fundamental to a physical examination. Landmarks on the body’s surface can be used to locate deep structures and to assess function by auscultation and percussion. How to count ribs Knowing how to count ribs is important because different ribs provide palpable landmarks for the positions of deeper structures. To determine the location of specific ribs, palpate the jugular notch at the superior extent of the manubrium of the sternum. Move down the sternum until a ridge is felt. This ridge is the sternal angle, which identifies the articulation between the manubrium of the sternum and the body of the sternum. The costal cartilage of rib II articulates with the sternum at this location. Identify rib II. Then continue counting the ribs, moving in a downward and lateral direction (Fig. 3.105). Surface anatomy of the breast in women

1	Surface anatomy of the breast in women Although breasts vary in size, they are normally positioned on the thoracic wall between ribs II and VI and overlie the pectoralis major muscles. Each mammary gland extends superolaterally around the lower margin of the pectoralis major muscle and enters the axilla (Fig. 3.106). This portion of the gland is the axillary tail or axillary process. The positions of the nipple and areola vary relative to the chest wall depending on breast size. Visualizing structures at the TIV/V The TIV/V vertebral level is a transverse plane that passes through the sternal angle on the anterior chest wall and the intervertebral disc between TIV and TV vertebrae posteriorly. This plane can easily be located, because the joint between the manubrium of the sternum and the body of the sternum forms a distinct bony protuberance that can be palpated. At the TIV/V level (Fig. 3.107): The costal cartilage of rib II articulates with the sternum.

1	The costal cartilage of rib II articulates with the sternum. The superior mediastinum is separated from the inferior mediastinum. The ascending aorta ends and the arch of the aorta begins. The arch of the aorta ends and the thoracic aorta begins. The trachea bifurcates. Visualizing structures in the superior mediastinum A number of structures in the superior mediastinum in adults can be visualized based on their positions relative to skeletal landmarks that can be palpated through the skin (Fig. 3.108). On each side, the internal jugular and subclavian veins join to form the brachiocephalic veins behind the sternal ends of the clavicles near the sternoclavicular joints. The left brachiocephalic vein crosses from left to right behind the manubrium of the sternum. The brachiocephalic veins unite to form the superior vena cava behind the lower border of the costal cartilage of the right first rib.

1	The brachiocephalic veins unite to form the superior vena cava behind the lower border of the costal cartilage of the right first rib. The arch of the aorta begins and ends at the transverse plane between the sternal angle anteriorly and vertebral level TIV/V posteriorly. The arch may reach as high as the midlevel of the manubrium of the sternum. Visualizing the margins of the heart Surface landmarks can be palpated to visualize the outline of the heart (Fig. 3.109). The upper limit of the heart reaches as high as the third costal cartilage on the right side of the sternum and the second intercostal space on the left side of the sternum. The right margin of the heart extends from the right third costal cartilage to near the right sixth costal cartilage. The left margin of the heart descends laterally from the second intercostal space to the apex located near the midclavicular line in the fifth intercostal space.

1	The left margin of the heart descends laterally from the second intercostal space to the apex located near the midclavicular line in the fifth intercostal space. The lower margin of the heart extends from the sternal end of the right sixth costal cartilage to the apex in the fifth intercostal space near the midclavicular line. Where to listen for heart sounds To listen for valve sounds, position the stethoscope downstream from the flow of blood through the valves (Fig. 3.110). The tricuspid valve is heard just to the left of the lower part of the sternum near the fifth intercostal space. The mitral valve is heard over the apex of the heart in the left fifth intercostal space at the midclavicular line. The pulmonary valve is heard over the medial end of the left second intercostal space. The aortic valve is heard over the medial end of the right second intercostal space. Visualizing the pleural cavities and lungs, pleural recesses, and lung lobes and fissures

1	The aortic valve is heard over the medial end of the right second intercostal space. Visualizing the pleural cavities and lungs, pleural recesses, and lung lobes and fissures Palpable surface landmarks can be used to visualize the normal outlines of the pleural cavities and the lungs and to determine the positions of the pulmonary lobes and fissures. Superiorly, the parietal pleura projects above the first costal cartilage. Anteriorly, the costal pleura approaches the midline posterior to the upper portion of the sternum. Posterior to the lower portion of the sternum, the left parietal pleura does not come as close to the midline as it does on the right side. This is because the heart bulges onto the left side (Fig. 3.111A). Inferiorly, the pleura reflects onto the diaphragm above the costal margin and courses around the thoracic wall following an VIII, X, XII contour (i.e., rib VIII in the midclavicular line, rib X in the midaxillary line, and vertebra TXII posteriorly).

1	The lungs do not completely fill the area surrounded by the pleural cavities, particularly anteriorly and inferiorly. Costomediastinal recesses occur anteriorly, particularly on the left side in relationship to the heart bulge. Costodiaphragmatic recesses occur inferiorly between the lower lung margin and the lower margin of the pleural cavity. In quiet respiration, the inferior margin of the lungs travels around the thoracic wall following a VI, VIII, X contour (i.e., rib VI in the midclavicular line, rib VIII in the midaxillary line, and vertebra TX posteriorly). In the posterior view, the oblique fissure on both sides is located in the midline near the spine of vertebra TIV (Figs. 3.111B and 3.112A). It moves laterally in a downward direction, crossing the fourth and fifth intercostal spaces and reaches rib VI laterally.

1	In the anterior view, the horizontal fissure on the right side follows the contour of rib IV and its costal cartilage and the oblique fissures on both sides follow the contour of rib VI and its costal cartilage (Fig. 3.112B). Where to listen for lung sounds The stethoscope placements for listening for lung sounds are shown in Fig. 3.113. Fig. 3.1 Thoracic wall and cavity. Left pleural cavitySternal angleManubrium of sternumXiphoid processInferior thoracic apertureSuperior thoracic apertureBody of sternumRight pleural cavityDiaphragmVertebral columnRib IRibsMediastinum Fig. 3.2 Joints between ribs and vertebrae. Fig. 3.3 Superior thoracic aperture. Manubriumof sternumEsophagusCommon carotid arteryInternal jugular veinRib ISuperior thoracicapertureApex of right lungSubclavianarteryand veinTracheaVertebra TIRib IITracheaPleauralcavity (lung)VeinsNervesArteriesEsophagus Fig. 3.4 A. Inferior thoracic aperture. B. Diaphragm.

1	Fig. 3.4 A. Inferior thoracic aperture. B. Diaphragm. Rib XIDistal cartilaginousends of ribs VII to X;costal marginsCentraltendonLeft domeRight domeAortichiatusEsophagealhiatusInferior thoracicapertureXiphoid processRib XIIVertebra TXIIAB Fig. 3.5 Subdivisions of the mediastinum. Fig. 3.6 Pleural cavities. TracheaLeft pleural cavitysurrounding left lungApex of right lungMediastinumRight main bronchusRight pleural cavityParietal pleuraVisceral pleuraCostodiaphragmaticrecessDiaphragm Fig. 3.7 Superior thoracic aperture and axillary inlet. Fig. 3.8 Major structures passing between abdomen and thorax. Fig. 3.9 Right breast. Fourth thoracicintercostal nerveMammary glandsLactiferousductsLactiferoussinusesPectoralis majorDeep (pectoral) fasciaAxillary processAxillarylymph nodesSecond, third, and fourth anteriorperforating branches of internalthoracic arteryInternalthoracic arteryParasternallymph nodesLymphatic vessel Fig. 3.10 Vertebral level TIV/V.

1	Fig. 3.10 Vertebral level TIV/V. Fig. 3.11 Left-to-right venous shunts. Fig. 3.12 A. Segmental neurovascular supply of thoracic wall. B. Anterior view of thoracic dermatomes associated with thoracic spinal nerves. C. Lateral view of dermatomes associated with thoracic spinal nerves. Left common carotid arteryInternal thoracic arteriesRight subclavian arteryArch of aortaAnteriorcutaneous branchAnteriorintercostal arteryAPosteriorintercostal arteryLateralcutaneous branchIntercostal nerve Fig. 3.13 Sympathetic trunks. Fig. 3.14 Flexible thoracic wall and inferior thoracic aperture. Sternum moves forwardin inspiration because ofrib elevationElevation of lateral aspectof ribs in inspirationDiaphragm descends toincrease thoracic capacityin inspiration Fig. 3.15 Innervation of the diaphragm. Fig. 3.16 Breasts.

1	Fig. 3.15 Innervation of the diaphragm. Fig. 3.16 Breasts. Parasternal nodesSuspensory ligamentsLactiferous sinusesLactiferous ductsRetromammary spaceSecretory lobulesPectoralis major muscleMammary branches ofinternal thoracic arteryAxillary processLateral thoracic arteryLateral axillary nodesCentral axillary nodesApical axillary nodesPectoral branch ofthoracoacromial arteryInternal thoracic arteryAreolaSecretorylobulesLymphatic and venous drainagepasses from lateral and superiorpart of the breast into axillaLymphatic and venousdrainage passes from medial partof the breast parasternallySome lymphatic and venous drainagemay pass from inferior part of thebreast into the abdomenPectoral axillary nodes Fig. 3.17 Muscles and fascia of the pectoral region. Fig. 3.18 Typical thoracic vertebra.

1	Fig. 3.17 Muscles and fascia of the pectoral region. Fig. 3.18 Typical thoracic vertebra. LaminaSpinous processTransverse processPedicleVertebral bodyPosteriorAnteriorVertebral foramenSuperior demifacetFacet for articulation with tubercle of ribSuperiorPosteriorInferiorAnteriorFacet for articulationwith tubercle of ribDemifacets for articulation with head of ribsSuperior articular processInferior articular processSuperior viewSuperolateral view Fig. 3.19 Atypical thoracic vertebrae. Vertebra TIVertebra TXVertebra TXISuperior costal facet for head of rib ISingle complete costal facet for head of rib XNo costal facet on transverse process Fig. 3.20 Ribs. Fig. 3.21 A typical rib. A. Anterior view. B. Posterior view of proximal end of rib. Fig. 3.22 Atypical ribs. Fig. 3.23 Sternum. Fig. 3.24 Costovertebral joints.

1	Fig. 3.20 Ribs. Fig. 3.21 A typical rib. A. Anterior view. B. Posterior view of proximal end of rib. Fig. 3.22 Atypical ribs. Fig. 3.23 Sternum. Fig. 3.24 Costovertebral joints. Joint capsuleVertebraVertebraDiscRibJoint cavitiesCostotransverse jointCostotransverse ligamentSuperior costotransverse ligamentLateral costotransverseligamentJoint with vertebral bodyIntra-articular ligamentSuperolateral viewSuperior view Fig. 3.25 Sternocostal joints. Fig. 3.26 Intercostal space. A. Anterolateral view. B. Details of an intercostal space and relationships. Intercostal space. C. Transverse section.

1	Fig. 3.25 Sternocostal joints. Fig. 3.26 Intercostal space. A. Anterolateral view. B. Details of an intercostal space and relationships. Intercostal space. C. Transverse section. Posterior ramus of spinal nerveLateral branches ofintercostal nerveand vesselsAnterior cutaneousbranch ofintercostal nerve Anterior perforatingbranches ofintercostal vessels Intercostal nerveCostal grooveAortaInternal thoracic arteryand veinPosterior intercostal artery and veinAnterior intercostal artery and veinCollateral branches ofintercostal nerve and vesselsIntercostal veinIntercostal arteryVisceral pleuraParietal pleuraEndothoracic fasciaPleural cavityLungIntercostal nerveCollateral branchesInternal intercostal muscleExternal intercostal muscleSerratus anterior muscleInnermost intercostal muscleSuperficial fasciaSkinAB Fig. 3.27 Intercostal muscles. Fig. 3.28 A. Subcostal muscles. B. Transversus thoracis muscles. Fig. 3.29 Arteries of the thoracic wall.

1	Fig. 3.27 Intercostal muscles. Fig. 3.28 A. Subcostal muscles. B. Transversus thoracis muscles. Fig. 3.29 Arteries of the thoracic wall. Superior epigastric arteryInternal thoracic arteryAortaAnterior perforatingbranchesAnterior intercostal arteryCollateral branch of posteriorintercostal arterySupreme intercostal arteryPosterior intercostal arteryMusculophrenic arterySubclavian arteryCostocervical trunk Fig. 3.30 Veins of the thoracic wall. Fig. 3.31 Major lymphatic vessels and nodes of the thoracic wall. Fig. 3.32 Intercostal nerves. Fig. 3.33 Right thoracotomy for esophageal cancer with intrathoracic large-bore drain. In this case, a neo-esophagus has been fashioned from the stomach. Fig. 3.34 Diaphragm. Fig. 3.35 Movement of thoracic wall during breathing. A. Pump handle movement of ribs and sternum. B. Bucket handle movement of ribs. BuckethandlemovementElevation of lateralshaft of ribSuperior and anteriormovement of sternumBAPump handle

1	BuckethandlemovementElevation of lateralshaft of ribSuperior and anteriormovement of sternumBAPump handle Fig. 3.36 Chest radiograph showing an elevated right hemidiaphragm in a patient with right-sided diaphragmatic paralysis. Fig. 3.37 Pleural cavities. Fig. 3.38 Parietal pleura. Costal partPulmonary ligamentMediastinal partPleura surroundingstructures in root of lungCervical pleuraSuprapleural membraneDiaphragmatic part Fig. 3.39 Pleural reflections. Fig. 3.40 Parietal pleural reflections and recesses. Fig. 3.41 CT image of left pleural effusion. AortaLeft lungRight lungLeft empyema with air-fluid level Fig. 3.42 Pneumothorax in a patient with extensive subcutaneous emphysema. Fig. 3.43 Lungs. Fig. 3.44 Roots and hila of the lungs. Fig. 3.45 A. Right lung. B. Major structures related to the right lung.

1	Fig. 3.43 Lungs. Fig. 3.44 Roots and hila of the lungs. Fig. 3.45 A. Right lung. B. Major structures related to the right lung. DiaphragmBronchusBronchus to superior lobePulmonary arteryPulmonary veinsRib ISubclavian arterySubclavian veinEsophagusPosteriorAnteriorInferior vena cavaAzygos veinLeft brachiocephalic veinRight brachiocephalic veinHeartInferior lobeSuperior lobeMiddle lobeSuperior vena cavaOblique fissureHorizontal fissureAB Fig. 3.46 A. Left lung. B. Major structures related to the left lung. Fig. 3.47 A. Bronchial tree. B. Bronchopulmonary segments. TracheaLeft main bronchusCarinaRight main bronchusLobar bronchiLobar bronchiSegmental bronchiof middle lobeBranch of pulmonary arteryMedial bronchopulmonary segmentof middle lobe of right lungLateral bronchopulmonary segmentof middle lobe of right lungAB Fig. 3.48 Bronchopulmonary segments. A. Right lung. B. Left lung. (Bronchopulmonary segments are numbered and named.)

1	Fig. 3.48 Bronchopulmonary segments. A. Right lung. B. Left lung. (Bronchopulmonary segments are numbered and named.) Fig. 3.49 Pulmonary vessels. A. Diagram of an anterior view. B. Axial computed tomography image showing the left pulmonary artery branching from the pulmonary trunk. C. Axial computed tomography image (just inferior to the image in B) showing the right pulmonary artery branching from the pulmonary trunk. Fig. 3.50 Pulmonary innervation. Fig. 3.51 Lymphatic drainage of lungs. Fig. 3.52 HRCT of patient with emphysema. Fig. 3.53 Bronchoscopic evaluation. A. Of the lower end of the trachea and its main branches. B. Of tracheal bifurcation showing a tumor at the carina.

1	Fig. 3.52 HRCT of patient with emphysema. Fig. 3.53 Bronchoscopic evaluation. A. Of the lower end of the trachea and its main branches. B. Of tracheal bifurcation showing a tumor at the carina. Fig. 3.54 Imaging of the lungs. A. Standard posteroanterior view of the chest showing tumor in upper right lung. B. Axial CT image of lungs showing tumor in right lung. C. Coronal CT image of lungs showing tumor in left lung extending into mediastinum. D. Radionuclide study using FDG PET showing a tumor in the right lung. Fig. 3.55 Cross-section of the thorax showing the position of the mediastinum. Fig. 3.56 Lateral view of the mediastinum. Fig. 3.57 Subdivisions of the mediastinum. Fig. 3.58 Thymus. Fig. 3.59 Sagittal section of the pericardium. FibrouspericardiumParietal layerof serouspericardiumVisceral layerof serouspericardium(epicardium)PericardialcavityJunction between fibrous pericardium and adventitia of great vessels Fig. 3.60 Phrenic nerves and pericardiacophrenic vessels.

1	Fig. 3.60 Phrenic nerves and pericardiacophrenic vessels. Fig. 3.61 Posterior portion of pericardial sac showing reflections of serous pericardium. Arch of aortaSuperior vena cavaInferior vena cavaBranch of rightpulmonary arteryAscending aortaThoracic aortaCut edge of pericardiumRight pulmonary veinsLeft pulmonary arteryLeft pulmonary veinsOblique pericardial sinus(formed by reflection onto thepulmonary veins of heart)Transverse pericardial sinus(separates arteries from veins) Fig. 3.62 Coronal CT showing pericardial effusion. Fig. 3.63 Schematic illustration of the heart showing orientation, surfaces, and margins. Fig. 3.64 Base of the heart. Arch of aortaSuperior vena cavaInferior vena cavaRight pulmonary arterySulcus terminalisRight pulmonary veinsRight atriumRight ventricleApexLeft atriumLeft inferior pulmonary veinLeft superior pulmonary veinLeft ventricleCoronary sinusLeft pulmonary artery Fig. 3.65 Anterior surface of the heart.

1	Fig. 3.65 Anterior surface of the heart. RALABicuspidvalveTricuspid valveAortic valvePulmonary trunkPulmonary valveAortaRVLVArch of aortaSuperior vena cavaInferior vena cavaRight coronaryarteryRight atriumRight ventricleAscending aortaInferior marginApexSmall cardiac veinPulmonary trunkLeft auricleLeft ventricleObtuse marginAnterior interventricular grooveGreat cardiac veinAnterior interventricular branch of left coronary artery Fig. 3.66 Diaphragmatic surface of the heart. Arch of aortaSuperior vena cavaInferior vena cavaRight pulmonary arteryRight pulmonary veinsRight atriumRight ventricleMiddle cardiac veinMarginal branch ofright coronary arteryApexLeft atriumLeft pulmonary veinsLeft ventricleCoronary sinusLeft pulmonary arteryPosterior interventricular groovePosterior interventricularbranch of right coronaryartery Fig. 3.67 Chest radiographs. A. Standard posteroanterior view of the chest. B. Standard lateral view of the heart.

1	Fig. 3.67 Chest radiographs. A. Standard posteroanterior view of the chest. B. Standard lateral view of the heart. Right ventricleLeft atrium Right atriumArch of aortaInferior vena cavaABPulmonary trunkLeft auricleLeft ventricle Superior vena cava Apex of heart Fig. 3.68 Sulci of the heart. A. Anterior surface of the heart. B. Diaphragmatic surface and base of the heart. Fig. 3.69 A. The heart has two pumps. B. Magnetic resonance image of midthorax showing all four chambers and septa. Fig. 3.70 Internal view of right atrium. Arch of aortaSuperior vena cavaInferior vena cavaLimbus of fossa ovalisCrista terminalisMusculi pectinatiRight ventricleFossa ovalisValve of coronary sinusOpening of coronary sinusRight auricleValve of inferior vena cava Fig. 3.71 Internal view of the right ventricle.

1	Fig. 3.71 Internal view of the right ventricle. Arch of aortaSuperior vena cavaTricuspidvalveRight auricleRight atriumAnterior cuspSeptal cuspPosterior cuspPosterior papillary muscleTrabeculae carneaeAnterior papillary muscleChordae tendineaePulmonary trunkPulmonaryvalveLeft auricleSeptal papillary muscleSeptomarginal trabeculaAnterior semilunar cuspRight semilunar cuspLeft semilunar cuspConus arteriosus Fig. 3.72 Posterior view of the pulmonary valve. Fig. 3.73 Left atrium. A. Internal view. B. Axial computed tomography image showing the pulmonary veins entering the left atrium. Arch of aortaMitral valveLeft auricleABPulmonary arteriesPulmonary veinsValve of foramen ovaleLeft ventricleLeft atriumAscending aortaRight ventricleRight pulmonary veinLeft atriumEsophagusThoracic aortaLeft pulmonary vein Fig. 3.74 Internal view of the left ventricle.

1	Fig. 3.74 Internal view of the left ventricle. Arch of aortaCoronary sinusMitral valve posterior cuspPulmonary arteriesPulmonary veinsAnterior papillarymuscleMitral valve anterior cuspPosterior papillarymuscleChordae tendineaeTrabeculae carneaeLeft atrium Fig. 3.75 Anterior view of the aortic valve. Fig. 3.76 Cardiac skeleton (atria removed). Right fibrous trigoneLeft fibrous trigoneLeft atrioventricular ringFibrous ring of pulmonary valveAtrioventricular bundleRight atrioventricular ringFibrous ring of aortic valveAntAntAntRtRtPostPostPostPosteriorAnteriorLeftRightSeptalLtLt Fig. 3.77 Cardiac vasculature. A. Anterior view. B. Superior view (atria removed). Fig. 3.78 A. Anterior view of coronary arterial system. Right dominant coronary artery. B. Left anterior oblique view of right coronary artery. C. Right anterior oblique view of left coronary artery.

1	C. Right anterior oblique view of left coronary artery. Right marginal branch Posterior interventricular branchRight coronary artery Left marginal branch Circumflex branchAnterior interventricular branchRight marginal branchof right coronary artery Right coronary arteryRight atriumRight ventricleSinu-atrial nodal branchof right coronary arteryABCPosterior interventricularbranch of right coronary artery Anterior interventricularbranch of leftcoronary artery Left coronary arteryCircumflex branchof left coronary arteryLeft marginal branchof circumflex branchDiagonal branch ofanterior interventricular branchLeft auricleLeft ventricle Fig. 3.79 Left dominant coronary artery.

1	Fig. 3.79 Left dominant coronary artery. Right marginal branchof right coronary arteryRight coronary arteryPosterior interventricular branch ofcircumflex branch of left coronary arterySinu-atrial nodal branchof left coronary arteryAnterior interventricularbranch of left coronary artery Left coronary arteryCircumflex branchof left coronary arteryLeft marginal branchof circumflex branchDiagonal branch ofanterior interventricular branch Fig. 3.80 A and B. Axial maximum intensity projection (MIP) CT image through the heart. A. Normal anterior interventricular (left anterior descending) artery. B. Stenotic (calcified) anterior interventricular (left anterior descending) artery. C and D. Vertical long axis multiplanar reformation (MRP) CT image through the heart. C. Normal anterior interventricular (left anterior descending) artery. D. Stenotic (calcified) anterior interventricular (left anterior descending) artery.

1	Fig. 3.81 Heart sounds and how they relate to valve closure, the electrocardiogram (ECG), and ventricular pressure. RPQST1st2nd1stSYSTOLESYSTOLEDIASTOLEVentricularpressureECGHeartsoundsAtrial contractionClosure of mitraland tricuspid valvesClosure of aortic andpulmonary valves"lub""lub""dub" Fig. 3.82 Major cardiac veins. A. Anterior view of major cardiac veins. B. Posteroinferior view of major cardiac veins. Fig. 3.83 Conduction system of the heart. A. Right chambers. B. Left chambers. Fig. 3.84 Cardiac plexus. A. Superficial. B. Deep. Left vagus nerveRight vagus nerveVagal cardiac branchesVagal cardiac branchesCardiac nerves fromsympathetic trunkSuperior vena cavaArch of aortaSuperficial cardiac plexusPulmonary trunkLeft recurrent laryngeal nerveRight recurrent laryngeal nerveLeft vagus nerveRight vagus nerveCardiac nerves from sympathetic trunkDeep cardiac plexusVagal cardiac branchesVagal cardiac branchesAB

1	Fig. 3.85 Major vessels within the middle mediastinum. A. Anterior view. B. Posterior view. Ascending aortaPulmonary trunkSuperiorvena cavaSuperior vena cavaInferior vena cavaOblique pericardial sinusRight pulmonaryarteryRight pulmonaryveinsRight atriumLeft pulmonaryveinsLeft pulmonaryarteryArch of aortaAB Fig. 3.86 Structures in the superior mediastinum. Right internal jugular veinRight common carotid arteryLeft common carotid arteryLeft subclavian arteryRight subclavian arteryRight pulmonary arteryLeft pulmonary arteryPulmonary trunkLeft subclavian veinLeft brachiocephalic veinRight brachiocephalic veinRight subclavian veinLeft internal jugular veinTracheaEsophagusEsophagusArch of aortaAscending aortaThoracic aortaLeft main bronchusRight main bronchusSuperior vena cava Fig. 3.87 Cross section through the superior mediastinum at the level of vertebra TIII. A. Diagram. B. Axial computed tomography image.

1	Fig. 3.87 Cross section through the superior mediastinum at the level of vertebra TIII. A. Diagram. B. Axial computed tomography image. ThymusManubrium of sternumLeft brachiocephalic veinRight brachiocephalic veinBrachiocephalic trunkLeft phrenic nerveRight phrenic nerveLeft vagus nerveLeft recurrent laryngeal nerveRight vagus nerveLeft common carotid arteryLeft subclavian arteryThoracic ductEsophagusTIIITracheaABLeft common carotid arteryEsophagusLeft subclavian arteryTracheaLeft brachiocephalic veinBrachiocephalic trunkRight brachiocephalic vein Fig. 3.88 Superior mediastinum with thymus removed. Fig. 3.89 Left superior intercostal vein. Fig. 3.90 Superior mediastinum with thymus and venous channels removed. Fig. 3.91 Axial CT showing aortic dissection. Fig. 3.92 Cross section through the superior mediastinum at the level of vertebra TIV. A. Diagram. B. Axial computed tomography image.

1	Fig. 3.91 Axial CT showing aortic dissection. Fig. 3.92 Cross section through the superior mediastinum at the level of vertebra TIV. A. Diagram. B. Axial computed tomography image. Manubrium of sternumThymusLeft phrenic nerveRight phrenicnerveArch of aortaLeft vagus nerveRight vagusnerveLeft recurrent laryngeal nerveThoracic ductTIVSuperior vena cavaTracheaArch ofazygos veinArch ofazygos veinEsophagusBAEsophagusTracheaArch of aortaSuperior vena cava Fig. 3.93 Trachea in the superior mediastinum. TracheaLeft brachiocephalicveinBrachiocephalictrunkLeft mainbronchusRight main bronchusPulmonary trunkSuperior venacavaArch of aortaTIV/V vertebrallevel Fig. 3.94 Right vagus nerve passing through the superior mediastinum. Fig. 3.95 Left vagus nerve passing through the superior mediastinum. Fig. 3.96 Left recurrent laryngeal nerve passing through the superior mediastinum.

1	Fig. 3.95 Left vagus nerve passing through the superior mediastinum. Fig. 3.96 Left recurrent laryngeal nerve passing through the superior mediastinum. Left recurrent laryngeal nerveLeft vagus nerveRight mainbronchusTIV/VvertebrallevelLeft main bronchusLigamentum arteriosumLeft pulmonary arteryLeft subclavian arteryPulmonary trunkEsophagusEsophagusTracheaThoracic aortaArch of aorta Fig. 3.97 Esophagus. Right main bronchusLeft main bronchusLeft subclavian arteryLeft common carotid arteryEsophagusEsophagusTracheaThoracic aortaArch of aortaBrachiocephalic trunkDiaphragm Fig. 3.98 Sites of normal esophageal constrictions. EsophagusTracheaPharynxDiaphragmJunction of esophagus with pharynxWhere esophagus iscrossed by arch ofaortaWhere esophagus is compressed by left main bronchusAt the esophageal hiatusPosition ofesophagusposterior toleft atrium Fig. 3.99 Esophageal plexus. Fig. 3.100 Axial CT showing esophageal cancer. Fig. 3.101 Thoracic aorta and branches.

1	Fig. 3.99 Esophageal plexus. Fig. 3.100 Axial CT showing esophageal cancer. Fig. 3.101 Thoracic aorta and branches. Left subclavian arterySupremeintercostal arterySuperior leftbronchialarteryRightbronchialarteryEsophagusEsophagusTracheaArch of aortaPosteriorintercostalarteriesMediastinalbranchesEsophageal branches Fig. 3.102 Azygos system of veins. Left superior intercostal veinRight superior intercostal veinAccessory hemiazygos veinHemiazygos veinAzygos veinOpening of azygos veininto superior vena cavaPosterior intercostal veinRight subcostal veinAscending lumbar veinRight ascending lumbar veinInferior vena cava Fig. 3.103 Thoracic duct. Fig. 3.104 Thoracic portion of sympathetic trunks.

1	Fig. 3.103 Thoracic duct. Fig. 3.104 Thoracic portion of sympathetic trunks. Fig. 3.105 Anterior view of chest wall with the locations of skeletal structures shown. A. In women. The location of the nipple relative to a specific intercostal space varies depending on the size of the breasts, which may not be symmetrical. B. In men. Note the location of the nipple in the fourth intercostal space. ClavicleCostal cartilageCoracoid processCostal marginJugular notchSternoclavicular jointManubrium of sternumRib IRib XXiphoid processASternal angleIIIIIIVVVIVIIVIIIIXBody of sternum ClavicleCostal cartilageCoracoid processSternal angleCostal marginJugular notchSternoclavicular jointManubrium of sternumRib IRib XBody of sternumXiphoid processBIIIIIIVVVIVIIVIIIIX Fig. 3.106 A. Close-up view of nipple and surrounding areola of the breast. B. Lateral view of the chest wall of a woman showing the axillary process of the breast.

1	Fig. 3.106 A. Close-up view of nipple and surrounding areola of the breast. B. Lateral view of the chest wall of a woman showing the axillary process of the breast. Fig. 3.107 Anterior view of the chest wall of a man showing the locations of various structures related to the TIV/V level. Fig. 3.108 Anterior view of the chest wall of a man showing the locations of different structures in the superior mediastinum as they relate to the skeleton. Right internal jugular veinRight common carotid arteryLeft common carotid arteryLeft subclavian arteryRight subclavian arteryRight pulmonaryarteryLeft pulmonaryarteryPulmonary trunkLeft subclavian veinLeft brachiocephalicveinRight brachiocephalic veinRight subclavian veinLeft internal jugular veinTracheaEsophagusEsophagusArch of aortaAscending aortaThoracic aortaLeft main bronchusRight mainbronchusSuperiorvena cava Fig. 3.109 Anterior view of the chest wall of a man showing skeletal structures and the surface projection of the heart.

1	Fig. 3.109 Anterior view of the chest wall of a man showing skeletal structures and the surface projection of the heart. Fig. 3.110 Anterior view of the chest wall of a man showing skeletal structures, heart, location of the heart valves, and auscultation points. Fig. 3.111 Views of the chest wall showing the surface projections of the lobes and the fissures of the lungs. A. Anterior view in a woman. On the right side, the superior, middle, and inferior lobes are illustrated. On the left side, the superior and inferior lobes are illustrated. B. Posterior view in a woman. On both sides, the superior and inferior lobes are illustrated. The middle lobe on the right side is not visible in this view.

1	Fig. 3.112 Views of the chest wall. A. Posterior view in a woman with arms abducted and hands positioned behind her head. On both sides, the superior and inferior lobes of the lungs are illustrated. When the scapula is rotated into this position, the medial border of the scapula parallels the position of the oblique fissure and can be used as a guide for determining the surface projection of the superior and inferior lobes of the lungs. B. Lateral view in a man with his right arm abducted. The superior, middle, and inferior lobes of the right lung are illustrated. The oblique fissure begins posteriorly at the level of the spine of vertebra TIV, passes inferiorly crossing rib IV, the fourth intercostal space, and rib V. It crosses the fifth intercostal space at the midaxillary line and continues anteriorly along the contour of rib VI. The horizontal fissure crosses rib V in the midaxillary space and continues anteriorly, crossing the fourth intercostal space and following the contour

1	anteriorly along the contour of rib VI. The horizontal fissure crosses rib V in the midaxillary space and continues anteriorly, crossing the fourth intercostal space and following the contour of rib IV and its costal cartilage to the sternum.

1	Fig. 3.113 Views of the chest wall of a man with stethoscope placements for listening to the lobes of the lungs. A. Anterior views. B. Posterior views. Apex of right lungApex of left lungSuperior lobe of right lungSuperior lobe of left lungMiddle lobe of right lungInferior lobe of right lungInferior lobe of left lungABIIIIIIIVVVIVIIVIIIIXXXIXIIIIIIIIIVVVIVIIVIIIIXX Fig. 3.114 A. Normal left coronary artery angiogram. B. Left coronary artery angiogram showing decreased flow due to blockages. C. Mechanism for perceiving heart pain in T1–4 dermatomes. Fig. 3.115 Axial maximum intensity projection (MIP) CT image through the heart. A. Normal anterior interventricular (left anterior descending) artery. B. Stenotic (calcified) anterior interventricular (left anterior descending) artery. eFig. 3.116 Cervical ribs. A. Neck radiograph demonstrating bilateral cervical ribs. B. Coronal computed tomography image showing cervical ribs.

1	eFig. 3.116 Cervical ribs. A. Neck radiograph demonstrating bilateral cervical ribs. B. Coronal computed tomography image showing cervical ribs. eFig. 3.117 Chest radiograph demonstrating an air/fluid level in the pleural cavity. eFig. 3.118 Chest radiograph of an individual with a pacemaker. The pacemaker wires (2) can be seen traveling through the venous system to the heart where one ends in the right atrium and the other ends in the right ventricle. eFig. 3.119 Chest radiograph demonstrating translucent notches along the inferior border of ribs III to VI. eFig. 3.120 A. CT image of aortic dissection. B. Normal aorta (left) and an aortic dissection (right). The line in the right figure indicates the plane of the CT scan shown in A. The true lumen surroundedby the collapsed intima and mediaCollapsed intima and mediaABThe false lumenThe false lumenAscendingaortaThoracic aortaThe true lumenEntrypointReturnpoint eFig. 3.121 Chest radiograph showing left upper lobe infection.

1	Table 3.1 Muscles of the pectoral region Table 3.2 Muscles of the thoracic wall Table 3.3 Branches of the thoracic aorta In the clinic Axillary tail of breast It is important for clinicians to remember when evaluating the breast for pathology that the upper lateral region of the breast can project around the lateral margin of the pectoralis major muscle and into the axilla. This axillary process (axillary tail) may perforate deep fascia and extend as far superiorly as the apex of the axilla. In the clinic Breast cancer is one of the most common malignancies in women. It develops in the cells of the acini, lactiferous ducts, and lobules of the breast. Tumor growth and spread depends on the exact cellular site of origin of the cancer. These factors affect the response to surgery, chemotherapy, and radiotherapy. Breast tumors spread via the lymphatics and veins, or by direct invasion.

1	When a patient has a lump in the breast, a diagnosis of breast cancer is confirmed by a biopsy and histological evaluation. Once confirmed, the clinician must attempt to stage the tumor. Staging the tumor means defining the: size of the primary tumor, exact site of the primary tumor, number and sites of lymph node spread, and organs to which the tumor may have spread. Computed tomography (CT) scanning of the body may be carried out to look for any spread to the lungs (pulmonary metastases), liver (hepatic metastases), or bone (bony metastases). Further imaging may include bone scanning using radioactive isotopes, which are avidly taken up by the tumor metastases in bone, and PET-CT, which can visualize active foci of the metastatic disease in the body.

1	Lymph drainage of the breast is complex. Lymph vessels pass to axillary, supraclavicular, and parasternal nodes and may even pass to abdominal lymph nodes, as well as to the opposite breast. Containment of nodal metastatic breast cancer is therefore potentially difficult because it can spread through many lymph node groups. Subcutaneous lymphatic obstruction and tumor growth pull on connective tissue ligaments in the breast, resulting in the appearance of an orange peel texture (peau d’orange) on the surface of the breast. Further subcutaneous spread can induce a rare manifestation of breast cancer that produces a hard, woody texture to the skin (cancer en cuirasse).

1	A mastectomy (surgical removal of the breast) involves excision of breast tissue. Within the axilla the breast tissue must be removed from the medial axillary wall. Closely applied to the medial axillary wall is the long thoracic nerve. Damage to this nerve can result in paralysis of the serratus anterior muscle, producing a characteristic “winged” scapula. It is also possible to damage the nerve to the latissimus dorsi muscle, and this may affect extension, medial rotation, and adduction of the humerus. In the clinic Cervical ribs are present in approximately 1% of the population. A cervical rib is an accessory rib articulating with vertebra CVII; the anterior end attaches to the superior border of the anterior aspect of rib I. Plain radiographs may demonstrate cervical ribs as small horn-like structures (see Fig. 3.106).

1	Plain radiographs may demonstrate cervical ribs as small horn-like structures (see Fig. 3.106). It is often not appreciated by clinicians that a fibrous band commonly extends from the anterior tip of the small cervical ribs to rib I, producing a “cervical band” that is not visualized on radiography. In patients with cervical ribs and cervical bands, structures that normally pass over rib I (see Fig. 3.7) are elevated by, and pass over, the cervical rib and band.

1	Clinically, “thoracic outlet syndrome” is used to describe symptoms resulting from abnormal compression of the brachial plexus of nerves as it passes over the first rib and through the axillary inlet into the upper limb. The anterior ramus of T1 passes superiorly out of the superior thoracic aperture to join and become part of the brachial plexus. The cervical band from a cervical rib is one cause of thoracic outlet syndrome by putting upward stresses on the lower parts of the brachial plexus as they pass over the cervical band and related cervical rib. In the clinic Collection of sternal bone marrow The subcutaneous position of the sternum makes it possible to place a needle through the hard outer cortex into the internal (or medullary) cavity containing bone marrow. Once the needle is in this position, bone marrow can be aspirated. Evaluation of this material under the microscope helps clinicians diagnose certain blood diseases such as leukemia. In the clinic

1	In the clinic Single rib fractures are of little consequence, though extremely painful. After severe trauma, ribs may be broken in two or more places. If enough ribs are broken, a loose segment of chest wall, a flail segment (flail chest), is produced. When the patient takes a deep inspiration, the flail segment moves in the opposite direction to the chest wall, preventing full lung expansion and creating a paradoxically moving segment. If a large enough segment of chest wall is affected, ventilation may be impaired and assisted ventilation may be required until the ribs have healed. In the clinic Surgical access to the chest A surgical access is potentially more challenging in the chest given the rigid nature of the thoracic cage. Moreover, access is also dependent upon the organ that is operated upon and its relationships to subdiaphragmatic structures and structures in the neck. The most common approaches are a median sternotomy and a lateral thoracotomy.

1	A median sternotomy involves making a vertical incision in the sternum from just below the sternal notch to the distal end of the xiphoid process. Care must be taken not to cause injury to the vessels, in particular to the brachiocephalic veins. Bleeding from the branches of the internal thoracic artery can occur and needs to be controlled. Opening the sternum causes traction on the upper ribs and may lead to rib fractures. Sometimes partial sternotomy is performed with the incision involving only the upper part of the sternum and ending at the level of manubriosternal junction or just below. A median sternotomy allows access to the heart, including coronary arteries and valves, pericardium, great vessels, anterior mediastinum, and thymus, as well as to the lower trachea. It can also be used for removal of retrosternal goiter or during esophagectomy. The incision can be extended laterally into the supraclavicular region, giving access to the subclavian and carotid arteries.

1	A lateral thoracotomy gives access to the ipsilateral hemithorax and its contents including the lung, mediastinum, esophagus, and heart (left lateral thoracotomy) (Fig. 3.33). However, it involves division of muscles of the thoracic wall which leads to significant postoperative pain that needs to be well controlled to avoid restricted lung function. The incision starts at the anterior axillary line and then passes below the tip of the scapula and is extended superiorly between the posterior midline and medial border of the scapula. The pleural cavity is entered through an intercostal space. In older patients and those with osteoporosis, a short segment of rib is often resected to minimize the risk of a rib fracture.

1	Minimally invasive thoracic surgery (video-assisted thoracic surgery [VATS]) involves making small (1-cm) incisions in the intercostal spaces, placing a small camera on a telescope, and manipulating other instruments through additional small incisions. A number of procedures can be performed in this manner, including lobectomy, lung biopsy, and esophagectomy. In the clinic Insertion of a chest tube is a commonly performed procedure and is indicated to relieve air or fluid trapped in the thorax between the lung and the chest wall (pleural cavity). This procedure is done for pneumothorax, hemothorax, hemopneumothorax, malignant pleural effusion empyema, hydrothorax, and chylothorax, and also after thoracic surgery.

1	The position of the thoracostomy tube is usually between the anterior axillary and midaxillary anatomical lines from anterior to posterior and in either the fourth or fifth intercostal space. The position of the ribs in this region should be clearly marked. Anesthetic should be applied to the superior border of the rib and the inferior aspect of the intercostal space, including one rib and space above and one rib and space below. The neurovascular bundle runs in the neurovascular plane, which lies in the superior aspect of the intercostal space (just below the rib); hence, the reason for positioning the tube on the superior border of a rib (i.e., at the lowest position in the intercostal space).

1	Chest tube insertion is now commonly done with direct ultrasound guidance. This approach allows the physician both to assess whether the pleural effusion is simple or complex and loculated, and to select the safest site for entering the pleural space. In some cases of pneumothorax, a chest drain can be inserted under computed tomography-guidance, especially in patients with underlying lung disease where it is difficult to differentiate a large bulla from free air in the pleural space. In the clinic Local anesthesia of intercostal nerves produces excellent analgesia in patients with chest trauma and in those patients requiring anesthesia for a thoracotomy, mastectomy, or upper abdominal surgical procedures. The intercostal nerves are situated inferior to the rib borders in the neurovascular bundle. Each neurovascular bundle is situated deep to the external and internal intercostal muscle groups.

1	The intercostal nerves are situated inferior to the rib borders in the neurovascular bundle. Each neurovascular bundle is situated deep to the external and internal intercostal muscle groups. The nerve block may be undertaken using a “blind” technique or under direct imaging guidance. The patient is placed in the appropriate position to access the rib. Typically, under ultrasound guidance, a needle may be advanced into the region of the subcostal groove, followed by an injection with a local anesthetic. Depending on the type of anesthetic used, analgesia may be shortor long-acting. Given the position of the neurovascular bundle and the subcostal groove, complications may include puncture of the parietal pleura and an ensuing pneumothorax. Bleeding may also occur if the artery or vein is damaged during the procedure. In the clinic

1	In the clinic In cases of phrenic nerve palsy, diaphragmatic paralysis ensues, which is manifested by the elevation of the diaphragm muscle on the affected side (Fig. 3.36). The most important cause of the phrenic nerve palsy that should never be overlooked is malignant infiltration of the nerve by lung cancer. Other causes include postviral neuropathy (in particular, related to varicella zoster virus), trauma, iatrogenic injury during thoracic surgery, and degenerative changes in the cervical spine with compression of the C3–C5 nerve roots. Most patients with unilateral diaphragmatic paralysis are asymptomatic and require no treatment. Some may report shortness of breath, particularly on exertion. Bilateral paralysis of the diaphragm is rare but can cause significant respiratory distress.

1	Surgical plication of the diaphragm can be performed in cases with respiratory compromise and is often done laparoscopically. The surgeon creates folds in the paralyzed diaphragm and sutures them in place, reducing the mobility of the diaphragmatic muscle. There is usually good improvement in lung function, exercise tolerance, and shortness of breath after the procedure. In the clinic A pleural effusion occurs when excess fluid accumulates within the pleural space. As the fluid accumulates within the pleural space the underlying lung is compromised and may collapse as the volume of fluid increases. Once a pleural effusion has been diagnosed, fluid often will be aspirated to determine the cause, which can include infection, malignancy, cardiac failure, hepatic disease, and pulmonary embolism. A large pleural effusion needs to be drained to allow the collapsed part of the lung to reexpand and improve breathing (Fig. 3.41). In the clinic

1	In the clinic A pneumothorax is a collection of gas or air within the pleural cavity (Fig. 3.42). When air enters the pleural cavity the tissue elasticity of the parenchyma causes the lung to collapse within the chest, impairing the lung function. Occasionally, the gas within the pleural cavity may accumulate to such an extent that the mediastinum is “pushed” to the opposite side, compromising the other lung. This is termed a tension pneumothorax and requires urgent treatment. Most pneumothoraces are spontaneous (i.e., they occur in the absence of no known pathology and no known lung disease). In addition, pneumothoraces may occur as a result of trauma, inflammation, smoking, and other underlying pulmonary diseases. Certain pulmonary metastases, such as in patients with osteosarcoma, may cause spontaneous pneumothorax especially after chemotherapy. The occurrence of pneumothorax interferes with cancer treatment and increases mortality.

1	The symptoms of pneumothorax are often determined by the degree of air leak and the rate at which the accumulation of gas occurs and the ensuing lung collapses. They include pain, shortness of breath, and cardiorespiratory collapse, if severe. In the clinic Imaging the lungs Medical imaging of the lungs is important because they are one of the commonest sites for disease in the body. While the body is at rest, the lungs exchange up to 5 L of air per minute, and this may contain pathogens and other potentially harmful elements (e.g., allergens). Techniques to visualize the lung range from plain chest radiographs to high-resolution computed tomography (CT), which enables precise localization of a lesion within the lung. In the clinic

1	In the clinic High-resolution computed tomography (HRCT) is a diagnostic method for assessing the lungs but more specifically the interstitium of the lungs. The technique involves obtaining narrow cross-sectional slices of 1 to 2 mm. These scans enable the physician and radiologist to view the patterns of disease and their distribution. Diseases that may be easily demonstrated using this procedure include emphysema (Fig. 3.52), pneumoconiosis (coal worker’s pneumoconiosis), and asbestosis. HRCT is also useful in regular follow-ups of patients with interstitial disease to monitor disease progression. In the clinic

1	In the clinic Patients who have an endobronchial lesion (i.e., a lesion within a bronchus) may undergo bronchoscopic evaluation of the trachea and its main branches (Fig. 3.53). The bronchoscope is passed through the nose into the oropharynx and is then directed by a control system past the vocal cords into the trachea. The bronchi are inspected and, if necessary, small biopsies are obtained. Bronchoscopy can also be used in combination with ultrasound (a technique known as EBUS, endobronchial ultrasound). An ultrasound probe is inserted through a working channel of the bronchoscope to visualize the airway walls and adjacent structures. EBUS allows an accurate localization of the lesion and therefore provides a higher diagnostic yield. It can be used for sampling of mediastinal and hilar lymph nodes or to assist in transbronchial biopsy of pulmonary nodules. In the clinic It is important to stage lung cancer because the treatment depends on its stage.

1	In the clinic It is important to stage lung cancer because the treatment depends on its stage. If a small malignant nodule is found within the lung, it can sometimes be excised and the prognosis is excellent. Unfortunately, many patients present with a tumor mass that has invaded structures in the mediastinum or the pleurae or has metastasized. The tumor may then be inoperable and is treated with radiotherapy and chemotherapy. Spread of the tumor is by lymphatics to lymph nodes within the hila, mediastinum, and root of the neck. A key factor affecting the prognosis and ability to cure the disease is the distant spread of metastases. Imaging methods to assess spread include plain radiography (Fig. 3.54A), computed tomography (CT; Fig. 3.54B,C), and magnetic resonance imaging (MRI). Increasingly, radionuclide studies using fluorodeoxyglucose positron emission tomography (FDG PET; Fig. 3.54D) are being used.

1	In FDG PET a gamma radiation emitter is attached to a glucose molecule. In areas of high metabolic activity (i.e., the tumor), excessive uptake occurs and is recorded by a gamma camera. In the clinic Pericarditis is an inflammatory condition of the pericardium. Common causes are viral and bacterial infections, systemic illnesses (e.g., chronic renal failure), and after myocardial infarction.

1	Pericarditis is an inflammatory condition of the pericardium. Common causes are viral and bacterial infections, systemic illnesses (e.g., chronic renal failure), and after myocardial infarction. Pericarditis must be distinguished from myocardial infarction because the treatment and prognosis are quite different. As in patients with myocardial infarction, patients with pericarditis complain of continuous central chest pain that may radiate to one or both arms. Unlike myocardial infarction, however, the pain from pericarditis may be relieved by sitting forward. An electrocardiogram (ECG) is used to help differentiate between the two conditions. It usually shows diffuse ST elevation. Echocardiography can also be performed if there is clinical or radiographic suspicion of pericardial effusion. In the clinic

1	In the clinic Normally, only a tiny amount of fluid is present between the visceral and parietal layers of the serous pericardium. In certain situations, this space can be filled with excess fluid (pericardial effusion) (Fig. 3.62). Because the fibrous pericardium is a “relatively fixed” structure that cannot expand easily, a rapid accumulation of excess fluid within the pericardial sac compresses the heart (cardiac tamponade), resulting in biventricular failure. Removing the fluid with a needle inserted into the pericardial sac can relieve the symptoms. In the clinic

1	In the clinic Abnormal thickening of the pericardial sac (constrictive pericarditis), which usually involves only the parietal pericardium, but can also less frequently involve the visceral layer, can compress the heart, impairing heart function and resulting in heart failure. It can present acutely but often results in a chronic condition when thickened pericardium with fibrin deposits causes pericardial inflammation, leading to chronic scarring and pericardial calcification. As a result, normal filling during the diastolic phase of the cardiac cycle is severely restricted. The diagnosis is made by inspecting the jugular venous pulse in the neck. In normal individuals, the jugular venous pulse drops on inspiration. In patients with constrictive pericarditis, the reverse happens and this is called Kussmaul’s sign. Treatment often involves surgical opening of the pericardial sac. In the clinic

1	In the clinic Valve problems consist of two basic types: incompetence (insufficiency), which results from poorly functioning valves; and stenosis, a narrowing of the orifice, caused by the valve’s inability to open fully. Mitral valve disease is usually a mixed pattern of stenosis and incompetence, one of which usually predominates. Both stenosis and incompetence lead to a poorly functioning valve and subsequent heart changes, which include: left ventricular hypertrophy (this is appreciably less marked in patients with mitral stenosis); increased pulmonary venous pressure; pulmonary edema; and enlargement (dilation) and hypertrophy of the left atrium. Mitral valve stenosis can be congenital or acquired; in the latter, the most common cause is rheumatic fever. Stenosis usually occurs decades after an acute episode of rheumatic endocarditis.

1	Mitral valve stenosis can be congenital or acquired; in the latter, the most common cause is rheumatic fever. Stenosis usually occurs decades after an acute episode of rheumatic endocarditis. Aortic valve disease, both aortic stenosis and aortic regurgitation (backflow), can produce marked heart failure. Aortic valve stenosis is the most common type of cardiac valve disease and results from atherosclerosis causing calcification of the valve leaflets. It can also be caused by postinflammatory or postrheumatic conditions. These may lead to aortic regurgitation such as infective endocarditis, degenerative valve disease, rheumatic fever, or trauma.

1	Valve disease in the right side of the heart (affecting the tricuspid or pulmonary valve) is most likely caused by infection. Intravenous drug use, alcoholism, indwelling catheters, and extensive burns predispose to infection of the valves, particularly the tricuspid valve. The resulting valve dysfunction produces abnormal pressure changes in the right atrium and right ventricle, and these can induce cardiac failure. In the clinic In practice, physicians use alternative names for the coronary vessels. The short left coronary artery is referred to as the left main stem vessel. One of its primary branches, the anterior interventricular artery, is termed the left anterior descending artery (LAD). Similarly, the terminal branch of the right coronary artery, the posterior interventricular artery, is termed the posterior descending artery (PDA). In the clinic

1	In the clinic A heart attack occurs when the perfusion to the myocardium is insufficient to meet the metabolic needs of the tissue, leading to irreversible tissue damage. The most common cause is a total occlusion of a major coronary artery. Occlusion of a major coronary artery, usually due to atherosclerosis, leads to inadequate oxygenation of an area of myocardium and cell death (Fig. 3.80). The severity of the problem will be related to the size and location of the artery involved, whether or not the blockage is complete, and whether there are collateral vessels to provide perfusion to the territory from other vessels. Depending on the severity, patients can develop pain (angina) or a myocardial infarction (MI).

1	This is a technique in which a long fine tube (a catheter) is inserted into the femoral artery in the thigh and passed through the external and common iliac arteries and into the abdominal aorta. It continues to be moved upward through the thoracic aorta to the origins of the coronary arteries. The coronaries may also be approached via the radial or brachial arteries. A fine wire is then passed into the coronary artery and is used to cross the stenosis. A fine balloon is then passed over the wire and may be inflated at the level of the obstruction, thus widening it; this is termed angioplasty. More commonly, this is augmented by placement of a fine wire mesh (a stent) inside the obstruction to hold it open. Other percutaneous interventions are suction extraction of a coronary thrombus and rotary ablation of a plaque.

1	If coronary artery disease is too extensive to be treated by percutaneous intervention, surgical coronary artery bypass grafting may be necessary. The great saphenous vein, in the lower limb, is harvested and used as a graft. It is divided into several pieces, each of which is used to bypass blocked sections of the coronary arteries. The internal thoracic and radial arteries can also be used. In the clinic The most common abnormalities that occur during development are those produced by a defect in the atrial and ventricular septa.

1	A defect in the interatrial septum allows blood to pass from one side of the heart to the other from the chamber with the higher pressure to the chamber with the lower pressure; this is clinically referred to as a shunt. An atrial septal defect (ASD) allows oxygenated blood to flow from the left atrium (higher pressure) across the ASD into the right atrium (lower pressure), resulting in a left to right shunt and volume overload in the right-sided circulation. Many patients with ASD are asymptomatic, but in some cases the ASD may cause symptoms and needs to be closed surgically or by endovascular devices. Occasionally, increased blood flow into the right atrium over many years leads to right atrial and right ventricular hypertrophy and enlargement of the pulmonary trunk, resulting in pulmonary arterial hypertension. In such cases, the patients can present with shortness of breath, increasing tiredness, palpitations, fainting episodes and heart failure. In ASD, the left ventricle is not

1	arterial hypertension. In such cases, the patients can present with shortness of breath, increasing tiredness, palpitations, fainting episodes and heart failure. In ASD, the left ventricle is not enlarged as it is not affected by increased returning blood volume.

1	The most common of all congenital heart defects are those that occur in the ventricular septum—ventriculoseptal defect (VSD). These lesions are most frequent in the membranous portion of the septum and they allow blood to flow from the left ventricle (higher pressure) to the right ventricle (lower pressure), leading to an abnormal communication between the systemic and pulmonary circulation. This leads to right ventricular hypertrophy, increased pulmonary blood flow, elevated arterial pulmonary pressure, and increased blood volume returning to the left ventricle, causing its dilation. Increased pulmonary pressure in most severe cases may cause pulmonary edema. If large enough and left untreated, VSDs can produce marked clinical problems that might require surgery. VSD may be an isolated abnormality or part of a syndromic constellation, such as the tetralogy of Fallot.

1	The tetralogy of Fallot, the most common cyanotic congenital heart disorder diagnosed soon after birth, classically consists of four abnormalities: pulmonary stenosis, VSD, overriding aorta (originating to a varying degree from the right ventricle), and right ventricular hypertrophy. The underdevelopment of the right ventricle and pulmonary stenosis reduce blood flow to the lungs, leading to reduced volume of oxygenated blood returning to the heart. The defect in the interventricular septum causes mixing of oxygenated and nonoxygenated blood. The mixed blood is then delivered by the aorta to the major organs, resulting in poor oxygenation and cyanosis. Infants can present with cyanosis at birth or develop episodes of cyanosis while feeding or crying (tet spells). Most affected infants require surgical intervention. The advent of cardiopulmonary bypass was crucial in delivering highly satisfactory surgical results.

1	Occasionally, the ductus arteriosus, which connects the left branch of the pulmonary artery to the inferior aspect of the aortic arch, fails to close at birth. This is termed a patent or persistent ductus arteriosus (PDA). When this occurs, the oxygenated blood in the aortic arch (higher pressure) passes into the left branch of the pulmonary artery (lower pressure) and produces pulmonary hypertension and left atrial and ventricular enlargement. The prognosis in patients with isolated PDA is extremely good, as most do not have any major sequelae after surgical closure. All of these defects produce a left-to-right shunt, indicating that oxygenated blood from the left side of the heart is being mixed with deoxygenated blood from the right side of the heart before being recirculated into the pulmonary circulation. These shunts are normally compatible with life, but surgery or endovascular treatment may be necessary.

1	Rarely, a shunt is right-to-left. In isolation, this is fatal; however, this type of shunt is often associated with other anomalies, so some deoxygenated blood is returned to the lungs and the systemic circulation. In the clinic Auscultation of the heart reveals the normal audible cardiac cycle, which allows the clinician to assess heart rate, rhythm, and regularity. Furthermore, cardiac murmurs that have characteristic sounds within the phases of the cardiac cycle can be demonstrated (Fig. 3.81). In the clinic Classic symptoms of heart attack

1	In the clinic Classic symptoms of heart attack The typical symptoms are chest heaviness or pressure, which can be severe, lasting more than 20 minutes, and often associated with sweating. The pain in the chest (which may be described as an “elephant sitting on my chest” or by using a clenched fist to describe the pain [Levine sign]) often radiates to the arms (left more common than the right), and can be associated with nausea. The severity of ischemia and infarction depends on the rate at which the occlusion or stenosis has occurred and whether or not collateral channels have had a chance to develop. In the clinic Are heart attack symptoms the same in men and women?

1	In the clinic Are heart attack symptoms the same in men and women? Although men and women can experience the typical symptoms of severe chest pain, cold sweats, and pain in the left arm, women are more likely than men to have subtler, less recognizable symptoms. These may include abdominal pain, achiness in the jaw or back, nausea, shortness of breath, or simply fatigue. The mechanism of this difference is not understood, but it is important to consider cardiac ischemia for a wide range of symptoms. In the clinic The cardiac conduction system can be affected by coronary artery disease. The normal rhythm may be disturbed if the blood supply to the coronary conduction system is disrupted. If a dysrhythmia affects the heart rate or the order in which the chambers contract, heart failure and death may ensue. In the clinic Ectopic parathyroid glands in the thymus

1	In the clinic Ectopic parathyroid glands in the thymus The parathyroid glands develop from the third pharyngeal pouch, which also forms the thymus. The thymus is therefore a common site for ectopic parathyroid glands and, potentially, ectopic parathyroid hormone production. In the clinic Large systemic veins are used to establish central venous access for administering large amounts of fluid, drugs, and blood. Most of these lines (small-bore tubes) are introduced through venous puncture into the axillary, subclavian, or internal jugular veins. The lines are then passed through the main veins of the superior mediastinum, with the tips of the lines usually residing in the distal portion of the superior vena cava or in the right atrium. Similar devices, such as dialysis lines, are inserted into patients who have renal failure, so that a large volume of blood can be aspirated through one channel and reinfused through a second channel. In the clinic

1	In the clinic Using the superior vena cava to access the inferior vena cava Because the superior and inferior venae cavae are oriented along the same vertical axis, a guidewire, catheter, or line can be passed from the superior vena cava through the right atrium and into the inferior vena cava. This is a common route of access for such procedures as: transjugular liver biopsy, transjugular intrahepatic portosystemic shunts (TIPS), and insertion of an inferior vena cava filter to catch emboli dislodged from veins in the lower limb and pelvis (i.e., patients with deep vein thrombosis [DVT]). In the clinic Coarctation of the aorta

1	In the clinic Coarctation of the aorta Coarctation of the aorta is a congenital abnormality in which the aortic lumen is constricted just distal to the origin of the left subclavian artery. At this point, the aorta becomes significantly narrowed and the blood supply to the lower limbs and abdomen is diminished. Over time, collateral vessels develop around the chest wall and abdomen to supply the lower body. Dilated and tortuous intercostal vessels, which form a bypass to supply the descending thoracic aorta, may lead to erosions of the inferior margins of the ribs. This can be appreciated on chest radiographs as inferior rib notching and is usually seen in long standing cases. The coarctation also affects the heart, which has to pump the blood at higher pressure to maintain peripheral perfusion. This in turn may produce cardiac failure. In the clinic

1	In the clinic Diffuse atherosclerosis of the thoracic aorta may occur in patients with vascular disease, but this rarely produces symptoms. There are, however, two clinical situations in which aortic pathology can produce life-threatening situations. The aorta has three fixed points of attachment: the aortic valve, the ligamentum arteriosum, and the point of passing behind the median arcuate ligament of the diaphragm to enter the abdomen. The rest of the aorta is relatively free from attachment to other structures of the mediastinum. A serious deceleration injury (e.g., in a road traffic accident) is most likely to cause aortic trauma at these fixed points.

1	In certain conditions, such as in severe arteriovascular disease, the wall of the aorta can split longitudinally, creating a false channel, which may or may not rejoin into the true lumen distally (Fig. 3.91). This aortic dissection occurs between the intima and media anywhere along its length. If it occurs in the ascending aorta or arch of the aorta, blood flow in the coronary and cerebral arteries may be disrupted, resulting in myocardial infarction or stroke. In the abdomen the visceral vessels may be disrupted, producing ischemia to the gut or kidneys. In the clinic

1	In the clinic The normal aortic arch courses to the left of the trachea and passes over the left main bronchus. A right-sided aortic arch occurs when the vessel courses to the right of the trachea and passes over the right main bronchus. A right-sided arch of aorta is rare and may be asymptomatic. It can be associated with dextrocardia (right-sided heart) and, in some instances, with complete situs inversus (left-to-right inversion of the body’s organs). It can also be associated with abnormal branching of the great vessels, particularly with an aberrant left subclavian artery. In the clinic Abnormal origin of great vessels

1	In the clinic Abnormal origin of great vessels Great vessels occasionally have an abnormal origin, including: a common origin of the brachiocephalic trunk and the left common carotid artery, the left vertebral artery originating from the aortic arch, and the right subclavian artery originating from the distal portion of the aortic arch and passing behind the esophagus to supply the right arm—as a result, the great vessels form a vascular ring around the trachea and the esophagus, which can potentially produce difficulty swallowing. This configuration is one of the most common aortic arch abnormalities. In the clinic The vagus nerves, recurrent laryngeal nerves,

1	In the clinic The vagus nerves, recurrent laryngeal nerves, The left recurrent laryngeal nerve is a branch of the left vagus nerve. It passes between the pulmonary artery and the aorta, a region known clinically as the aortopulmonary window, and may be compressed in any patient with a pathological mass in this region. This compression results in left vocal cord paralysis and hoarseness of the voice. Lymph node enlargement, often associated with the spread of lung cancer, is a common condition that may produce compression. Chest radiography is therefore usually carried out for all patients whose symptoms include a hoarse voice.

1	More superiorly, in the root of the neck, the right vagus nerve gives off the right recurrent laryngeal nerve, which “hooks” around the right subclavian artery as it passes over the cervical pleura. If a patient has a hoarse voice and a right vocal cord palsy is demonstrated at laryngoscopy, chest radiography with an apical lordotic view should be obtained to assess for cancer in the right lung apex (Pancoast’s tumor). In the clinic When patients present with esophageal cancer, it is important to note which portion of the esophagus contains the tumor because tumor location determines the sites to which the disease will spread (Fig. 3.100). Esophageal cancer spreads quickly to lymphatics, draining to lymph nodes in the neck and around the celiac artery. Endoscopy or barium swallow is used to assess the site. CT and MRI may be necessary to stage the disease. Once the extent of the disease has been assessed, treatment can be planned. In the clinic

1	Once the extent of the disease has been assessed, treatment can be planned. In the clinic The first case of esophageal rupture was described by Herman Boerhaave in 1724. This case was fatal, but early diagnosis has increased the survival rate up to 65%. If the disease is left untreated, mortality is 100%. Typically, the rupture occurs in the lower third of the esophagus with a sudden rise in intraluminal esophageal pressure produced by vomiting secondary to an uncoordination and failure of the cricopharyngeus muscle to relax. Because the tears typically occur on the left, they are often associated with a large left pleural effusion that contains the gastric contents. In some patients, subcutaneous emphysema may be demonstrated. Treatment is optimal with urgent surgical repair. A 65-year-old man was admitted to the emergency room with severe central chest pain that radiated to the neck and predominantly to the left arm. He was overweight and a known heavy smoker.

1	A 65-year-old man was admitted to the emergency room with severe central chest pain that radiated to the neck and predominantly to the left arm. He was overweight and a known heavy smoker. On examination he appeared gray and sweaty. His blood pressure was 74/40 mm Hg (normal range 120/80 mm Hg). An electrocardiogram (ECG) was performed and demonstrated anterior myocardial infarction. An urgent echocardiograph demonstrated poor left ventricular function. The cardiac angiogram revealed an occluded vessel (Fig. 3.114A,B). Another approach to evaluating coronary arteries in patients is to perform maximum intensity projection (MIP) CT studies (Fig. 3.115A,B). This patient underwent an emergency coronary artery bypass graft and made an excellent recovery. He has now lost weight, stopped smoking, and exercises regularly.

1	When cardiac cells die during a myocardial infarction, pain fibers (visceral afferents) are stimulated. These visceral sensory fibers follow the course of sympathetic fibers that innervate the heart and enter the spinal cord between the TI and TIV levels. At this level, somatic afferent nerves from spinal nerves T1 to T4 also enter the spinal cord via the posterior roots. Both types of afferents (visceral and somatic) synapse with interneurons, which then synapse with second neurons whose fibers pass across the cord and then ascend to the somatosensory areas of the brain that represent the T1 to T4 levels. The brain is unable to distinguish clearly between the visceral sensory distribution and the somatic sensory distribution and therefore the pain is interpreted as arising from the somatic regions rather than the visceral organ (i.e., the heart; Fig. 3.114C). The patient was breathless because his left ventricular function was poor.

1	The patient was breathless because his left ventricular function was poor. When the left ventricle fails, it produces two effects. First, the contractile force is reduced. This reduces the pressure of the ejected blood and lowers the blood pressure. The left atrium has to work harder to fill the failing left ventricle. This extra work increases left atrial pressure, which is reflected in an increased pressure in the pulmonary veins, and this subsequently creates a higher pulmonary venular pressure. This rise in pressure will cause fluid to leak from the capillaries into the pulmonary interstitium and then into the alveoli. Such fluid is called pulmonary edema and it markedly restricts gas exchange. This results in shortness of breath. This man had a blocked left coronary artery, as shown in Fig. 3.114B. It is important to know which coronary artery is blocked.

1	This man had a blocked left coronary artery, as shown in Fig. 3.114B. It is important to know which coronary artery is blocked. The left coronary artery supplies the majority of the left side of the heart. The left main stem vessel is approximately 2 cm long and divides into the circumflex artery, which lies between the atrium and the ventricle in the coronary sulcus, and the anterior interventricular artery, which is often referred to as the left anterior descending artery (LAD). When the right coronary artery is involved with arterial disease and occludes, associated disorders of cardiac rhythm often result because the sinu-atrial and the atrioventricular nodes derive their blood supplies predominantly from the right coronary artery. When this patient sought medical care, his myocardial function was assessed using ECG, echocardiography, and angiography. During a patient’s initial examination, the physician will usually assess myocardial function.

1	During a patient’s initial examination, the physician will usually assess myocardial function. After obtaining a clinical history and carrying out a physical examination, a differential diagnosis for the cause of the malfunctioning heart is made. Objective assessment of myocardial and valve function is obtained in the following ways:

1	ECG/EKG (electrocardiography)—a series of electrical traces taken around the long and short axes of the heart that reveal heart rate and rhythm and conduction defects. In addition, it demonstrates the overall function of the right and left sides of the heart and points of dysfunction. Specific changes in the ECG relate to the areas of the heart that have been involved in a myocardial infarction. For example, a right coronary artery occlusion produces infarction in the area of myocardium it supplies, which is predominantly the inferior aspect; the infarct is therefore called an inferior myocardial infarction. The ECG changes are demonstrated in the leads that visualize the inferior aspect of the myocardium (namely, leads II, III, and aVF).

1	Chest radiography—reveals the size of the heart and chamber enlargement. Careful observation of the lungs will demonstrate excess fluid (pulmonary edema), which builds up when the left ventricle fails and can produce marked respiratory compromise and death unless promptly treated. Blood tests—the heart releases enzymes during myocardial infarction, namely lactate dehydrogenase (LDH), creatine kinase (CK), and aspartate transaminase (AST). These plasma enzymes are easily measured in the hospital laboratory and used to determine the diagnosis at an early stage. Further specific enzymes termed isoenzymes can also be determined (creatine kinase MB isoenzyme [CKMB]). Newer tests include an assessment for troponin (a specific component of the myocardium), which is released when cardiac cells die during myocardial infarction.

1	Exercise testing—patients are connected to an ECG monitor and exercised on a treadmill. Areas of ischemia, or poor blood flow, can be demonstrated, so localizing the vascular abnormality. Nuclear medicine—thallium (a radioactive X-ray emitter) and its derivatives are potassium analogs. They are used to determine areas of coronary ischemia. If no areas of myocardial uptake are demonstrated when these substances are administered to a patient the myocardium is dead. Coronary angiography—small arterial catheters are maneuvered from a femoral artery puncture site through the femoral artery and aorta and up to the origins of the coronary vessels. X-ray contrast medium is then injected to demonstrate the coronary vessels and their important branches. If there is any narrowing (stenosis), angioplasty may be carried out. In angioplasty tiny balloons are passed across the narrowed areas and inflated to refashion the vessel and so prevent further coronary ischemia and myocardial infarction.

1	A 53-year-old man presented to the emergency department with a 5-hour history of sharp pleuritic chest pain and shortness of breath. The day before he was on a long haul flight, returning from his holidays. He was usually fit and well and was a keen mountain climber. He had no previous significant medical history. On physical examination his lungs were clear, he was tachypneic at 24/min, and his saturation was reduced to 92% on room air. Pulmonary embolism was suspected and the patient was referred for a CT pulmonary angiogram. The study demonstrated clots within the right and left main pulmonary arteries. There was no pleural effusion, lung collapse, or consolidation. He was immediately started on subcutaneous enoxaparin and converted to oral anticoagulation over the course of a couple of days. The whole treatment lasted 6 months as no other risk factors (except immobilization during a long haul flight) were identified. There were no permanent sequelae.

1	The embolic material usually originates in the peripheral deep veins of the lower limbs and less commonly in the pelvic, renal, or upper limb deep veins. The material gets detached from the main thrombus in the deep veins and travels into the pulmonary circulation, where it can lodge either in the pulmonary trunk and main pulmonary arteries, giving rise to central pulmonary embolism or in the lobar, segmental, or subsegmental branches, giving rise to peripheral embolism. The gravity of symptoms is partly dependent on the thrombus load and on which part of the pulmonary arterial tree is affected. Large pulmonary embolisms can lead to severe hemodynamic and respiratory compromise and death (e.g., a saddle thrombus lodged in the pulmonary trunk and in both main pulmonary arteries). Common risk factors include immobilization, surgery, trauma, malignancy, pregnancy, oral contraceptives, and hereditary factors.

1	Common risk factors include immobilization, surgery, trauma, malignancy, pregnancy, oral contraceptives, and hereditary factors. A young man has black areas of skin on the tips of his fingers of his left hand. A clinical diagnosis of platelet emboli was made and a source of the emboli sought. Emboli can arise from many sources. They are clots and plugs of tissue, usually platelets, that are carried from a source to eventually reside in small vessels which they may occlude. Arterial emboli may arise in the heart or in the arteries that supply the region affected. In cases of infected emboli, bacteria grow on the valve and are showered off into the peripheral circulation. A neck radiograph and coronal CT image of the neck demonstrates a cervical rib (eFig. 3.116). Cervical ribs may produce three distinct disease entities:

1	A neck radiograph and coronal CT image of the neck demonstrates a cervical rib (eFig. 3.116). Cervical ribs may produce three distinct disease entities: Arterial compression and embolization—the cervical rib (or band) on the undersurface of the distal portion of the subclavian artery reduces the diameter of the vessel and allows eddy currents to form. Platelets aggregate and atheroma may develop in this region. This debris can be dislodged and flow distally within the upper limb vessels to block off blood flow to the fingers and the hand, a condition called distal embolization. Tension on the T1 nerve—the T1 nerve, which normally passes over rib I, is also elevated by the presence of a cervical rib; thus the patient may experience a sensory disturbance over the medial aspect of the forearm, and develop wasting of the intrinsic muscles of the hand. Compression of the subclavian vein—this may induce axillary vein thrombosis.

1	Compression of the subclavian vein—this may induce axillary vein thrombosis. A Doppler ultrasound scan revealed marked stenosis of the subclavian artery at the outer border of the rib with abnormal flow distal to the narrowing. Within this region of abnormal flow there was evidence of thrombus adherent to the vessel wall. This patient underwent surgical excision of the cervical rib and had no further symptoms. A 52-year-old man presented with headaches and shortness of breath. He also complained of coughing up small volumes of blood. Clinical examination revealed multiple dilated veins around the neck. A chest radiograph demonstrated an elevated diaphragm on the right and a tumor mass, which was believed to be a primary bronchogenic carcinoma. By observing the clinical findings and applying anatomical knowledge, the site of the tumor can be inferred.

1	By observing the clinical findings and applying anatomical knowledge, the site of the tumor can be inferred. The multiple dilated veins around the neck are indicative of venous obstruction. The veins are dilated on both sides of the neck, implying that the obstruction must be within a common vessel, the superior vena cava. Anterior to the superior vena cava in the right side of the chest is the phrenic nerve, which supplies the diaphragm. Because the diaphragm is elevated, suggesting paralysis, it is clear that the phrenic nerve has been involved with the tumor. A 35-year-old man was shot during an armed robbery. The bullet entry wound was in the right fourth intercostal space, above the nipple. A chest radiograph obtained on admission to the emergency room demonstrated complete collapse of the lung. A further chest radiograph performed 20 minutes later demonstrated an air/fluid level in the pleural cavity (eFig. 3.117).

1	A further chest radiograph performed 20 minutes later demonstrated an air/fluid level in the pleural cavity (eFig. 3.117). Three common pathological processes may occur in the pleural cavity. If air is introduced into the pleural cavity, a pneumothorax develops and the lung collapses because of its own elastic recoil. The pleural space fills with air, which may further compress the lung. Most patients with a collapsed lung are unlikely to have respiratory impairment. Under certain conditions, air may enter the pleural cavity at such a rate that it shifts and pushes the mediastinum to the opposite side of the chest. This is called tension pneumothorax and is potentially lethal, requiring urgent treatment by insertion of an intercostal tube to remove the air. The commonest causes of pneumothorax are rib fractures and positive pressure ventilation lung damage.

1	The pleural cavity may fill with fluid (a pleural effusion) and this can be associated with many diseases (e.g., lung infection, cancer, abdominal sepsis). It is important to aspirate fluid from these patients to relieve any respiratory impairment and to carry out laboratory tests on the fluid to determine its nature. Severe chest trauma can lead to development of hemopneumothorax. A tube must be inserted to remove the blood and air that has entered the pleural space and prevent respiratory impairment. This man needs treatment to drain either the air or fluid or both. The pleural space can be accessed by passing a needle between the ribs into the pleural cavity. In a normal healthy adult, the pleural space is virtually nonexistent; therefore, any attempt to introduce a needle into this space is unlikely to succeed and the procedure may damage the underlying lung.

1	Before any form of chest tube is inserted, the rib must be well anesthetized by infiltration because its periosteum is extremely sensitive. The intercostal drain should pass directly on top of the rib. Insertion adjacent to the lower part of the rib may damage the artery, vein, and nerve, which lie within the neurovascular bundle. Appropriate sites for insertion of a chest drain are either in the fourth or fifth intercostal space between the anterior axillary and midaxillary anatomical lines. This position is determined by palpating the sternal angle, which is the point of articulation of rib II. Counting inferiorly will determine the rib number and simple observation will determine the positions of the anterior axillary and midaxillary lines. Insertion of any tube or needle below the fifth interspace runs an appreciable risk of crossing the pleural recesses and placing the needle or the drain into either the liver or the spleen, depending upon which side the needle is inserted.

1	An elderly woman was admitted to the emergency room with severe cardiac failure. She had a left-sided pacemaker box, which had been inserted for a cardiac rhythm disorder (fast atrial fibrillation) many years previously. An ECG demonstrated fast atrial fibrillation. A chest radiograph showed that the wire from the pacemaker had broken under the clavicle. Anatomical knowledge of this region of the chest explains why the wire broke.

1	Anatomical knowledge of this region of the chest explains why the wire broke. Many patients have cardiac pacemakers. A wire arises from the pacemaker, which lies within the subcutaneous tissue over the pectoralis major muscle and travels from the pacemaker under the skin to pierce the axillary vein just beneath the clavicle, lateral to the subclavius muscle. The wire then passes through the subclavian vein, the brachiocephalic vein, the superior vena cava, and the right atrium, and lies on the wall of the right ventricle (where it can stimulate the heart to contract) (eFig. 3.118). If the wire pierces the axillary vein directly adjacent to the subclavius muscle, it is possible that after many years of shoulder movement the subclavius muscle stresses and breaks the wire, causing the pacemaker to fail. Every effort is made to place the insertion point of the wire as far laterally as feasible within the first part of the axillary vein.

1	A 20-year-old man visited his family doctor because he had a cough. A chest radiograph demonstrated translucent notches along the inferior border of ribs III to VI (eFig. 3.119). He was referred to a cardiologist and a diagnosis of coarctation of the aorta was made. The rib notching was caused by dilated collateral intercostal arteries. Coarctation of the aorta is a narrowing of the aorta distal to the left subclavian artery. This narrowing can markedly reduce blood flow to the lower body. Many of the vessels above the narrowing therefore enlarge due to the increased pressure so that blood can reach the aorta below the level of the narrowing. Commonly, the internal thoracic, superior epigastric, and musculophrenic arteries enlarge anteriorly. These arteries supply the anterior intercostal arteries, which anastomose with the posterior intercostal arteries that allow blood to flow retrogradely into the aorta. Enlargement of the intertcostal vessels results in notching of the ribs.

1	The first and second posterior intercostal vessels are supplied from the costocervical trunk, which arises from the subclavian artery proximal to the coarctation, so do not enlarge and do not induce rib notching. A 62-year-old man was admitted to the emergency room with severe interscapular pain. His past medical history indicated that he was otherwise fit and well; however, it was noted he was 6’ 9” and had undergone previous eye surgery for dislocating lenses. On examination the man was pale, clammy, and hypotensive. The pulse in his right groin was weak. An ECG demonstrated an inferior myocardial infarction. Serum blood tests revealed poor kidney function and marked acidosis. The patient was transferred to the CT scanner and a diagnosis of aortic dissection was made.

1	The patient was transferred to the CT scanner and a diagnosis of aortic dissection was made. Aortic dissection is an uncommon disorder in which a small tear occurs within the aortic wall (eFig. 3.120). The aortic wall contains three layers, an intima, a media, and an adventitia. A tear in the intima extends into the media and peels it away, forming a channel within the wall of the vessel. Usually the blood reenters the main vessel wall distal to its point of entry. The myocardial infarction Aortic dissection may extend retrogradely to involve the coronary sinus of the right coronary artery. Unfortunately, in this patient’s case the right coronary artery became occluded as the dissection passed into the origin. In normal individuals the right coronary artery supplies the anterior inferior aspect of the myocardium, and this is evident as an anterior myocardial infarct on an ECG. The ischemic left leg

1	The ischemic left leg The two channels within the aorta have extended throughout the length of the aorta into the right iliac system and to the level of the right femoral artery. Although blood flows through these structures it often causes reduced blood flow. Hence the reduced blood flow into the left lower limb renders it ischemic. The patient became acidotic.

1	The patient became acidotic. All cells in the body produce acid, which is excreted in the urine or converted into water with the production of carbon dioxide, which is removed with ventilation. Unfortunately, when organs become extremely ischemic they release significant amounts of hydrogen ions. Typically, this occurs when the gut becomes ischemic. With the pattern of dissection, (1) the celiac trunk, superior mesenteric artery, and inferior mesenteric artery can be effectively removed from the circulation or (2) the blood flow within these vessels can be significantly impeded, rendering the gut ischemic and hence accounting for the relatively high hydrogen ion levels. Similarly the dissection can impair blood flow to the kidneys, which decreases their ability to function.

1	Similarly the dissection can impair blood flow to the kidneys, which decreases their ability to function. The patient underwent emergency surgery and survived. Interestingly, the height of the patient and the previous lens surgery would suggest a diagnosis of Marfan syndrome, and a series of blood tests and review of the family history revealed this was so. A 35-year-old male patient presented to his family practitioner because of recent weight loss (14 lb over the previous 2 months). He also complained of a cough with streaks of blood in the sputum (hemoptysis) and left-sided chest pain. Recently, he noticed significant sweating, especially at night, which necessitated changing his sheets.

1	On examination the patient had a low-grade temperature and was tachypneic (breathing fast). There was reduced expansion of the left side of the chest. When the chest was percussed it was noted that the anterior aspect of the left chest was dull, compared to the resonant percussion note of the remainder of the chest. Auscultation (listening with a stethoscope) revealed decreased breath sounds, which were hoarse in nature (bronchial breathing). A diagnosis of chest infection was made. Chest infection is a common disease. In most patients the infection affects the large airways and bronchi. If the infection continues, exudates and transudates are produced, filling the alveoli and the secondary pulmonary lobules. The diffuse patchy nature of this type of infection is termed bronchial pneumonia. Given the patient’s specific clinical findings, bronchial pneumonia was unlikely.

1	Given the patient’s specific clinical findings, bronchial pneumonia was unlikely. From the clinical findings it was clear that the patient was likely to have a pneumonia confined to a lobe. Because there are only two lobes in the left lung, the likely diagnosis was a left upper lobe pneumonia. A chest radiograph was obtained (eFig. 3.121). The posteroanterior view of the chest demonstrated an area of veil-like opacification throughout the whole of the left lung. Knowing the position of the oblique fissure, any consolidation within the left upper lobe will produce this veil-like shadowing. Lateral radiographs are usually not necessary but would demonstrate opacification anteriorly and superiorly that ends abruptly at the oblique fissure. Upper lobe pneumonias are unusual because most patients develop gravity-dependent infection. Certain infections, however, are typical within the middle and upper lobes, commonly, tuberculosis (TB) and histoplasmosis.

1	A review of the patient’s history suggested a serious and chronic illness and the patient was admitted to hospital. After admission a bronchoscopy was carried out and sputum was aspirated from the left upper lobe bronchus. This was cultured in the laboratory and also viewed under the microscope and tuberculous bacilli (TB) were identified. A 68-year-old man came to his family physician complaining of discomfort when swallowing (dysphagia). The physician examined the patient and noted since his last visit he had lost approximately 18 lb over 6 months. Routine blood tests revealed the patient was anemic and he was referred to the gastroenterology unit. A diagnosis of esophageal cancer was made and the patient underwent a resection, which involved a chest and abdominal incision. After 4 years the patient remains well though still subject to follow-up.

1	The patient underwent a flexible endoscopic examination of the esophagus in which a tube is placed through the mouth and into the esophagus and a camera is placed on the end of the tube. It is also possible to use biopsy forceps to obtain small portions of tissue for adequate diagnosis. The diagnosis of esophageal carcinoma was made (squamous cell type) and the patient underwent a staging procedure. Staging of any malignancy is important because it determines the extent of treatment and allows the physician to determine the patient’s prognosis. In this case our patient underwent a CT scan of the chest and abdomen, which revealed no significant lymph nodes around the lower third esophageal tumor. The abdominal scan revealed no evidence of spread to the nodes around the celiac trunk and no evidence of spread to the liver. Bleeding was the cause of the anemia.

1	The abdominal scan revealed no evidence of spread to the nodes around the celiac trunk and no evidence of spread to the liver. Bleeding was the cause of the anemia. Many tumors of the gastrointestinal system are remarkably friable, and with the passage of digested material across the tumor, low-grade chronic bleeding occurs. Over a period of time the patient is rendered anemic, which in the first instance is asymptomatic; however, it can be diagnosed on routine blood tests. Complex surgery is planned.

1	Complex surgery is planned. The length of the esophagus is approximately 22 cm. Tumor spread can occur through the submucosal route and also through locoregional lymph nodes. The lymph nodes drain along the arterial supply to the esophagus, which is predominantly supplied by the inferior thyroid artery, esophageal branches from the thoracic aorta, and branches from the left gastric artery. The transthoracic esophagectomy procedure involves placing the patient supine. A laparotomy is performed to assess for any evidence of disease in the abdominal cavity. The stomach is mobilized, with preservation of the right gastric and right gastro-omental arteries. The short gastric vessels and left gastric vessels are divided, and a pyloromyotomy is also performed.

1	The abdominal wound is then closed and the patient is placed in the left lateral position. A right posterolateral thoracotomy is performed through the fifth intercostal space, and the azygos vein is divided to provide full access to the whole length of the esophagus. The stomach is delivered through the diaphragmatic hiatus. The esophagus is resected and the stomach is anastomosed to the cervical esophagus. The patient made an uneventful recovery. Most esophageal cancers are diagnosed relatively late and often have lymph node metastatic spread. A number of patients will also have a spread of tumor to the liver. The overall prognosis for esophageal cancer is poor, with approximately a 25%, 5-year survival rate. Diagnosing esophageal cancer in its early stages before lymph node spread is ideal and can produce a curative procedure. Our patient went on to have chemotherapy and enjoys a good quality of life 4 years after his operation.

1	Our patient went on to have chemotherapy and enjoys a good quality of life 4 years after his operation. A 45-year-old woman, with a history of breast cancer in the left breast, returned to her physician. Unfortunately the disease had spread to the axillary lymph nodes and bones (bony metastatic disease). A surgeon duly resected the primary breast tumor with a wide local excision and then performed an axillary nodal clearance. The patient was then referred to an oncologist for chemotherapy. Chemotherapy was delivered through a portacath, which is a subcutaneous reservoir from which a small catheter passes under the skin into the internal jugular vein. The patient duly underwent a portacath insertion without complication, completed her course of chemotherapy, and is currently doing well 5 years later.

1	The portacath was placed on the patient’s right anterior chest wall and the line was placed into the right internal jugular vein. The left internal jugular vein and subcutaneous tissues were not used. The reason for not using this site was that the patient had previously undergone an axillary dissection on the left, and the lymph nodes and lymphatics were removed. Placement of a portacath in this region may produce an inflammatory response and may even get infected. Unfortunately, because there are no lymphatics to drain away infected material and to remove bacteria, severe sepsis and life-threatening infection may ensue. How was it placed?

1	How was it placed? The ultrasound shows an axial image across the root of the neck on the right demonstrating the right common carotid artery and the right internal jugular vein. The internal jugular vein is the larger of the two structures and generally demonstrates normal respiratory variation, compressibility, and a size dependence upon the patient’s position (when the patient is placed in the head-down position, the vein fills and makes puncture easy). The risks of the procedure As with all procedures and operations there is always a small risk of complication. These risks are always balanced against the potential benefits of the procedure. Placing the needle into the internal jugular vein can be performed under ultrasound guidance, which reduces the risk of puncturing the common carotid artery. Furthermore, by puncturing under direct vision it is less likely that the operator will hit the lung apex and pierce the superior pleural fascia, which may produce a pneumothorax.

1	The position of the indwelling catheter The catheter is placed through the right internal jugular vein and into the right brachiocephalic vein. The tip of the catheter is then placed more inferiorly at the junction of the right atrium and the superior vena cava. The reason for placing the catheter in such a position relates to the agents that are infused. Most chemotherapeutic agents are severely cytotoxic (kill cells), and enabling good mixing with the blood prevents thrombosis and vein wall irritation. A 15-year-old girl presented to the emergency department with a 1-week history of productive cough with copious purulent sputum, increasing shortness of breath, fatigue, fever around 38.5° C, and no response to oral amoxicillin prescribed to her by a family physician. The patient was diagnosed with cystic fibrosis shortly after birth and had multiple admissions to the hospital for pulmonary and gastrointestinal manifestations of the disease.

1	Physical examination on the current admission to the ER revealed widespread inspiratory crackles, mild tachycardia of 105/min, and fever of 38.2° C. Diagnosis of infective exacerbation of bronchiectasis was made. Sputum was sent for microbiology, which later came back positive for Pseudomonas aeruginosa, a common pathogen isolated in such patients.

1	Cystic fibrosis is an autosomal recessive disorder affecting the function of exocrine glands due to a gene mutation, leading to an abnormally low concentration of chloride in exocrine secretions, rendering them thick and sticky. Thick secretions cause blockage and subsequent damage to the airways, bowel, pancreas, liver, and reproductive tract. In the lungs, thick nonclearing secretions lead to recurrent infections and persistent inflammation, resulting in permanent distortion and dilation of the distal bronchi, a condition known as bronchiectasis. Bronchiectasis can be seen on plain chest radiographs as tubular (tram track like) structures, particularly affecting the upper lobes. Computed tomography can easily demonstrate the extent of airway damage and identify potential pulmonary complications of cystic fibrosis such as lobar collapse, pneumothorax, or enlargement of the pulmonary trunk due to pulmonary hypertension.

1	The patient was admitted for a course of broad-spectrum intravenous antibiotics and intensive chest physiotherapy and made satisfactory recovery from the acute episode. She was discharged home on oral prophylactic antibiotics with an ongoing physiotherapy program. 247.e1 247.e2 Conceptual Overview • Relationship to Other Regions Fig. 3.12, cont’d Fig. 3.26, cont’d In the clinic—cont’d Regional Anatomy • Movements of the Thoracic Wall and Diaphragm During Breathing In the clinic—cont’d Surface Anatomy • Visualizing Structures at the TIV/V Vertebral Level Surface Anatomy • Visualizing the Margins of the Heart Surface Anatomy • Visualizing the Pleural Cavities and Lungs, Pleural Recesses, and Lung Lobes and Fissures Surface Anatomy • Where to Listen for Lung Sounds Fig. 3.114, cont’d The abdomen is a roughly cylindrical chamber extending from the inferior margin of the thorax to the superior margin of the pelvis and the lower limb (Fig. 4.1A).

1	Fig. 3.114, cont’d The abdomen is a roughly cylindrical chamber extending from the inferior margin of the thorax to the superior margin of the pelvis and the lower limb (Fig. 4.1A). The inferior thoracic aperture forms the superior opening to the abdomen and is closed by the diaphragm. Inferiorly, the deep abdominal wall is continuous with the pelvic wall at the pelvic inlet. Superficially, the inferior limit of the abdominal wall is the superior margin of the lower limb. The chamber enclosed by the abdominal wall contains a single large peritoneal cavity, which freely communicates with the pelvic cavity. Abdominal viscera are either suspended in the peritoneal cavity by mesenteries or positioned between the cavity and the musculoskeletal wall (Fig. 4.1B).

1	Abdominal viscera are either suspended in the peritoneal cavity by mesenteries or positioned between the cavity and the musculoskeletal wall (Fig. 4.1B). Abdominal viscera include: major elements of the gastrointestinal system—the caudal end of the esophagus, stomach, small and large intestines, liver, pancreas, and gallbladder; the spleen; components of the urinary system—kidneys and ureters; the suprarenal glands; and major neurovascular structures. The abdomen houses major elements of the gastrointestinal system (Fig. 4.2), the spleen, and parts of the urinary system. Much of the liver, gallbladder, stomach, and spleen and parts of the colon are under the domes of the diaphragm, which project superiorly above the costal margin of the thoracic wall, and as a result these abdominal viscera are protected by the thoracic wall. The superior poles of the kidneys are deep to the lower ribs.

1	Viscera not under the domes of the diaphragm are supported and protected predominantly by the muscular walls of the abdomen. One of the most important roles of the abdominal wall is to assist in breathing: It relaxes during inspiration to accommodate expansion of the thoracic cavity and the inferior displacement of abdominal viscera during contraction of the diaphragm (Fig. 4.3). During expiration, it contracts to assist in elevating the domes of the diaphragm, thus reducing thoracic volume. Material can be expelled from the airway by forced expiration using the abdominal muscles, as in coughing or sneezing. Contraction of abdominal wall muscles can dramatically increase intraabdominal pressure when the diaphragm is in a fixed position (Fig. 4.4). Air is retained in the lungs by closing valves in the larynx in the neck. Increased intra-abdominal pressure assists in voiding the contents of the bladder and rectum and in giving birth.

1	The abdominal wall consists partly of bone but mainly of muscle (Fig. 4.5). The skeletal elements of the wall (Fig. 4.5A) are: the five lumbar vertebrae and their intervening intervertebral discs, the superior expanded parts of the pelvic bones, and bony components of the inferior thoracic wall, including the costal margin, rib XII, the end of rib XI, and the xiphoid process. Muscles make up the rest of the abdominal wall (Fig. 4.5B): Lateral to the vertebral column, the quadratus lumborum, psoas major, and iliacus muscles reinforce the posterior aspect of the wall. The distal ends of the psoas major and iliacus muscles pass into the thigh and are major flexors of the hip joint. Lateral parts of the abdominal wall are predominantly formed by three layers of muscles, which are similar in orientation to the intercostal muscles of the thorax—transversus abdominis, internal oblique, and external oblique.

1	Anteriorly, a segmented muscle (the rectus abdominis) on each side spans the distance between the inferior thoracic wall and the pelvis. Structural continuity between posterior, lateral, and anterior parts of the abdominal wall is provided by (aponeuroses) derived from muscles of the lateral wall. A fascial layer of varying thickness separates the abdominal wall from the peritoneum, which lines the abdominal cavity. The general organization of the abdominal cavity is one in which a central gut tube (gastrointestinal system) is suspended from the posterior abdominal wall and partly from the anterior abdominal wall by thin sheets of tissue (mesenteries; Fig. 4.6): a ventral (anterior) mesentery for proximal regions of the gut tube; a dorsal (posterior) mesentery along the entire length of the system. Different parts of these two mesenteries are named according to the organs they suspend or with which they are associated.

1	Different parts of these two mesenteries are named according to the organs they suspend or with which they are associated. Major viscera, such as the kidneys, that are not suspended in the abdominal cavity by mesenteries are associated with the abdominal wall. The abdominal cavity is lined by peritoneum, which consists of an epithelial-like single layer of cells (the mesothelium) together with a supportive layer of connective tissue. Peritoneum is similar to the pleura and serous pericardium in the thorax. The peritoneum reflects off the abdominal wall to become a component of the mesenteries that suspend the viscera. Parietal peritoneum lines the abdominal wall. Visceral peritoneum covers suspended organs. Normally, elements of the gastrointestinal tract and its derivatives completely fill the abdominal cavity, making the peritoneal cavity a potential space, and on the adjacent abdominal wall slide freely against one another.

1	Abdominal viscera are either intraperitoneal or retroperitoneal: Intraperitoneal structures, such as elements of the gastrointestinal system, are suspended from the abdominal wall by mesenteries; Structures that are not suspended in the abdominal cavity by a mesentery and that lie between the parietal peritoneum and abdominal wall are retroperitoneal in position. Retroperitoneal structures include the kidneys and ureters, which develop in the region between the peritoneum and the abdominal wall and remain in this position in the adult. During development, some organs, such as parts of the small and large intestines, are suspended initially in the abdominal cavity by a mesentery, and later become retroperitoneal secondarily by fusing with the abdominal wall (Fig. 4.7).

1	Large vessels, nerves, and lymphatics are associated with the posterior abdominal wall along the median axis of the body in the region where, during development, the peritoneum reflects off the wall as the dorsal mesentery, which supports the developing gut tube. As a consequence, branches of the neurovascular structures that pass to parts of the gastrointestinal system are unpaired, originate from the anterior aspects of their parent structures, and travel in mesenteries or pass retroperitoneally in areas where the mesenteries secondarily fuse to the wall. Generally, vessels, nerves, and lymphatics to the abdominal wall and to organs that originate as retroperitoneal structures branch laterally from the central neurovascular structures and are usually paired, one on each side.

1	The superior aperture of the abdomen is the inferior thoracic aperture, which is closed by the diaphragm (see pp. 126-127). The margin of the inferior thoracic aperture consists of vertebra TXII, rib XII, the distal end of rib XI, the costal margin, and the xiphoid process of the sternum. The musculotendinous diaphragm separates the abdomen from the thorax. The diaphragm attaches to the margin of the inferior thoracic aperture, but the attachment is complex posteriorly and extends into the lumbar area of the vertebral column (Fig. 4.8). On each side, a muscular extension (crus) firmly anchors the diaphragm to the anterolateral surface of the vertebral column as far down as vertebra LIII on the right and vertebra LII on the left. Because the costal margin is not complete posteriorly, the diaphragm is anchored to arch-shaped (arcuate) ligaments, which span the distance between available bony points and the intervening soft tissues:

1	Medial and lateral arcuate ligaments cross muscles of the posterior abdominal wall and attach to vertebrae, the transverse processes of vertebra LI and rib XII, respectively. A median arcuate ligament crosses the aorta and is continuous with the crus on each side. The posterior attachment of the diaphragm extends much farther inferiorly than the anterior attachment. Consequently, the diaphragm is an important component of the posterior abdominal wall, to which a number of viscera are related. The abdominal wall is continuous with the pelvic wall at the pelvic inlet, and the abdominal cavity is continuous with the pelvic cavity. The circular margin of the pelvic inlet is formed entirely by bone: posteriorly by the sacrum, anteriorly by the pubic symphysis, and laterally, on each side, by a distinct bony rim on the pelvic bone (Fig. 4.9).

1	Because of the way in which the sacrum and attached pelvic bones are angled posteriorly on the vertebral column, the pelvic cavity is not oriented in the same vertical plane as the abdominal cavity. Instead, the pelvic cavity projects posteriorly, and the inlet opens anteriorly and somewhat superiorly (Fig. 4.10). The abdomen is separated from the thorax by the diaphragm. Structures pass between the two regions through or posterior to the diaphragm (see Fig. 4.8). The pelvic inlet opens directly into the abdomen and structures pass between the abdomen and pelvis through it. The peritoneum lining the abdominal cavity is continuous with the peritoneum in the pelvis. Consequently, the abdominal cavity is entirely continuous with the pelvic cavity (Fig. 4.11). Infections in one region can therefore freely spread into the other.

1	The bladder expands superiorly from the pelvic cavity into the abdominal cavity and, during pregnancy, the uterus expands freely superiorly out of the pelvic cavity into the abdominal cavity. The abdomen communicates directly with the thigh through an aperture formed anteriorly between the inferior margin of the abdominal wall (marked by the inguinal ligament) and the pelvic bone (Fig. 4.12). Structures that pass through this aperture are: the major artery and vein of the lower limb; the femoral nerve, which innervates the quadriceps femoris muscle, which extends the knee; lymphatics; and the distal ends of psoas major and iliacus muscles, which flex the thigh at the hip joint. As vessels pass inferior to the inguinal ligament, their names change—the external iliac artery and vein of the abdomen become the femoral artery and vein of the thigh. Arrangement of abdominal viscera in the adult

1	Arrangement of abdominal viscera in the adult A basic knowledge of the development of the gastrointestinal tract is needed to understand the arrangement of viscera and mesenteries in the abdomen (Fig. 4.13). The early gastrointestinal tract is oriented longitudinally in the body cavity and is suspended from surrounding walls by a large dorsal mesentery and a much smaller ventral mesentery. Superiorly, the dorsal and ventral mesenteries are anchored to the diaphragm. The primitive gut tube consists of the foregut, the midgut, and the hindgut. Massive longitudinal growth of the gut tube, rotation of selected parts of the tube, and secondary fusion of some viscera and their associated mesenteries to the body wall participate in generating the adult arrangement of abdominal organs. Development of the foregut

1	Development of the foregut In abdominal regions, the foregut gives rise to the distal end of the esophagus, the stomach, and the proximal part of the duodenum. The foregut is the only part of the gut tube suspended from the wall by both the ventral and dorsal mesenteries. A diverticulum from the anterior aspect of the foregut grows into the ventral mesentery, giving rise to the liver and gallbladder, and, ultimately, to the ventral part of the pancreas. The dorsal part of the pancreas develops from an outgrowth of the foregut into the dorsal mesentery. The spleen develops in the dorsal mesentery in the region between the body wall and presumptive stomach. In the foregut, the developing stomach rotates clockwise and the associated dorsal mesentery, containing the spleen, moves to the left and greatly expands. During this process, part of the mesentery becomes associated with, and secondarily fuses with, the left side of the body wall.

1	At the same time, the duodenum, together with its dorsal mesentery and an appreciable part of the pancreas, swings to the right and fuses to the body wall. Secondary fusion of the duodenum to the body wall, massive growth of the liver in the ventral mesentery, and fusion of the superior surface of the liver to the diaphragm restrict the opening to the space enclosed by the ballooned dorsal mesentery associated with the stomach. This restricted opening is the omental foramen (epiploic foramen). The part of the abdominal cavity enclosed by the expanded dorsal mesentery, and posterior to the stomach, is the omental bursa (lesser sac). Access, through the omental foramen, to this space from the rest of the peritoneal cavity (greater sac) is inferior to the free edge of the ventral mesentery.

1	Part of the dorsal mesentery that initially forms part of the lesser sac greatly enlarges in an inferior direction, and the two opposing surfaces of the mesentery fuse to form an apron-like structure (the greater omentum). The greater omentum is suspended from the greater curvature of the stomach, lies over other viscera in the abdominal cavity, and is the first structure observed when the abdominal cavity is opened anteriorly. Development of the midgut The midgut develops into the distal part of the duodenum and the jejunum, ileum, ascending colon, and proximal two-thirds of the transverse colon. A small yolk sac projects anteriorly from the developing midgut into the umbilicus.

1	Rapid growth of the gastrointestinal system results in a loop of the midgut herniating out of the abdominal cavity and into the umbilical cord. As the body grows in size and the connection with the yolk sac is lost, the midgut returns to the abdominal cavity. While this process is occurring, the two limbs of the midgut loop rotate counterclockwise around their combined central axis, and the part of the loop that becomes the cecum descends into the inferior right aspect of the cavity. The superior mesenteric artery, which supplies the midgut, is at the center of the axis of rotation. The cecum remains intraperitoneal, the ascending colon fuses with the body wall becoming secondarily retroperitoneal, and the transverse colon remains suspended by its dorsal mesentery (transverse mesocolon). The greater omentum hangs over the transverse colon and the mesocolon and usually fuses with these structures. Development of the hindgut

1	Development of the hindgut The distal one-third of the transverse colon, descending colon, sigmoid colon, and superior part of the rectum develop from the hindgut. Proximal parts of the hindgut swing to the left and become the descending colon and sigmoid colon. The descending colon and its dorsal mesentery fuse to the body wall, while the sigmoid colon remains intraperitoneal. The sigmoid colon passes through the pelvic inlet and is continuous with the rectum at the level of vertebra SIII. Skin and muscles of the anterior The anterior rami of thoracic spinal nerves T7 to T12 follow the inferior slope of the lateral parts of the ribs and cross the costal margin to enter the abdominal wall (Fig. 4.14). Intercostal nerves T7 to T11 supply skin and muscle of the abdominal wall, as does the subcostal nerve T12. In addition, T5 and T6 supply upper parts of the external oblique muscle of the abdominal wall; T6 also supplies cutaneous innervation to skin over the xiphoid.

1	T6 also supplies cutaneous innervation to skin over the xiphoid. Skin and muscle in the inguinal and suprapubic regions of the abdominal wall are innervated by L1 and not by thoracic nerves. Dermatomes of the anterior abdominal wall are indicated in Figure 4.14. In the midline, skin over the infrasternal angle is T6 and that around the umbilicus is T10. L1 innervates skin in the inguinal and suprapubic regions. Muscles of the abdominal wall are innervated segmentally in patterns that generally reflect the patterns of the overlying dermatomes. The groin is a weak area in the anterior abdominal wall During development, the gonads in both sexes descend from their sites of origin on the posterior abdominal wall into the pelvic cavity in women and the developing scrotum in men (Fig. 4.15).

1	During development, the gonads in both sexes descend from their sites of origin on the posterior abdominal wall into the pelvic cavity in women and the developing scrotum in men (Fig. 4.15). Before descent, a cord of tissue (the gubernaculum) passes through the anterior abdominal wall and connects the inferior pole of each gonad with primordia of the scrotum in men and the labia majora in women (labioscrotal swellings). A tubular extension (the processus vaginalis) of the peritoneal cavity and the accompanying muscular layers of the anterior abdominal wall project along the gubernaculum on each side into the labioscrotal swellings.

1	In men, the testis, together with its neurovascular structures and its efferent duct (the ductus deferens) descends into the scrotum along a path, initially defined by the gubernaculum, between the processus vaginalis and the accompanying coverings derived from the abdominal wall. All that remains of the gubernaculum is a connective tissue remnant that attaches the caudal pole of the testis to the scrotum. The inguinal canal is the passage through the anterior abdominal wall created by the processus vaginalis. The spermatic cord is the tubular extension of the layers of the abdominal wall into the scrotum that contains all structures passing between the testis and the abdomen. The distal sac-like terminal end of the spermatic cord on each side contains the testis, associated structures, and the now isolated part of the peritoneal cavity (the cavity of the tunica vaginalis).

1	In women, the gonads descend to a position just inside the pelvic cavity and never pass through the anterior abdominal wall. As a result, the only major structure passing through the inguinal canal is a derivative of the gubernaculum (the round ligament of the uterus). In both men and women, the groin (inguinal region) is a weak area in the abdominal wall (Fig. 4.15) and is the site of inguinal hernias.

1	The transpyloric plane is a horizontal plane that transects the body through the lower aspect of vertebra LI (Fig. 4.16). It: is about midway between the jugular notch and the pubic symphysis, and crosses the costal margin on each side at roughly the ninth costal cartilage; crosses through the opening of the stomach into the duodenum (the pyloric orifice), which is just to the right of the body of LI; the duodenum then makes a characteristic C-shaped loop on the posterior abdominal wall and crosses the midline to open into the jejunum just to the left of the body of vertebra LII, whereas the head of the pancreas is enclosed by the loop of the duodenum, and the body of the pancreas extends across the midline to the left; crosses through the body of the pancreas; and approximates the position of the hila of the kidneys; though because the left kidney is slightly higher than the right, the transpyloric plane crosses through the inferior aspect of the left hilum and the superior part of

1	of the hila of the kidneys; though because the left kidney is slightly higher than the right, the transpyloric plane crosses through the inferior aspect of the left hilum and the superior part of the right hilum.

1	The gastrointestinal system and its derivatives are supplied by three major arteries

1	Three large unpaired arteries branch from the anterior surface of the abdominal aorta to supply the abdominal part of the gastrointestinal tract and all of the structures (liver, pancreas, and gallbladder) to which this part of the gut gives rise during development (Fig. 4.17). These arteries pass through derivatives of the dorsal and ventral mesenteries to reach the target viscera. These vessels therefore also supply structures such as the spleen and lymph nodes that develop in the mesenteries. These three arteries are: the celiac artery, which branches from the abdominal aorta at the upper border of vertebra LI and supplies the foregut; the superior mesenteric artery, which arises from the abdominal aorta at the lower border of vertebra LI and supplies the midgut; and the inferior mesenteric artery, which branches from the abdominal aorta at approximately vertebral level LIII and supplies the hindgut. Venous shunts from left to right

1	Venous shunts from left to right All blood returning to the heart from regions of the body other than the lungs flows into the right atrium of the heart. The inferior vena cava is the major systemic vein in the abdomen and drains this region together with the pelvis, perineum, and both lower limbs (Fig. 4.18). The inferior vena cava lies to the right of the vertebral column and penetrates the central tendon of the diaphragm at approximately vertebral level TVIII. A number of large vessels cross the midline to deliver blood from the left side of the body to the inferior vena cava. One of the most significant is the left renal vein, which drains the kidney, suprarenal gland, and gonad on the same side. Another is the left common iliac vein, which crosses the midline at approximately vertebral level LV to join with its partner on the right to form the inferior vena cava. These veins drain the lower limbs, the pelvis, the perineum, and parts of the abdominal wall.

1	Other vessels crossing the midline include the left lumbar veins, which drain the back and posterior abdominal wall on the left side. All venous drainage from the through the liver Blood from abdominal parts of the gastrointestinal system and the spleen passes through a second vascular bed, in the liver, before ultimately returning to the heart (Fig. 4.19). Venous blood from the digestive tract, pancreas, gallbladder, and spleen enters the inferior surface of the liver through the large hepatic portal vein. This vein then ramifies like an artery to distribute blood to small endothelial-lined hepatic sinusoids, which form the vascular exchange network of the liver. After passing through the sinusoids, the blood collects in a number of short hepatic veins, which drain into the inferior vena cava just before the inferior vena cava penetrates the diaphragm and enters the right atrium of the heart.

1	Normally, vascular beds drained by the hepatic portal system interconnect, through small veins, with beds drained by systemic vessels, which ultimately connect directly with either the superior or inferior vena cava. Among the clinically most important regions of overlap between the portal and caval systems are those at each end of the abdominal part of the gastrointestinal system: around the inferior end of the esophagus; around the inferior part of the rectum. Small veins that accompany the degenerate umbilical vein (round ligament of the liver) establish another important portacaval anastomosis. The round ligament of the liver connects the umbilicus of the anterior abdominal wall with the left branch of the portal vein as it enters the liver. The small veins that accompany this ligament form a connection between the portal system and para-umbilical regions of the abdominal wall, which drain into systemic veins.

1	Other regions where portal and caval systems interconnect include: where the liver is in direct contact with the diaphragm (the bare area of the liver); where the wall of the gastrointestinal tract is in direct contact with the posterior abdominal wall (retroperitoneal areas of the large and small intestine); and the posterior surface of the pancreas (much of the pancreas is secondarily retroperitoneal). Blockage of the hepatic portal vein or of vascular channels in the liver

1	Blockage of the hepatic portal vein or of vascular channels in the liver Blockage of the hepatic portal vein or of vascular channels in the liver can affect the pattern of venous return from abdominal parts of the gastrointestinal system. Vessels that interconnect the portal and caval systems can become greatly enlarged and tortuous, allowing blood in tributaries of the portal system to bypass the liver, enter the caval system, and thereby return to the heart. Portal hypertension can result in esophageal and rectal varices and in caput medusae in which systemic vessels that radiate from para-umbilical veins enlarge and become visible on the abdominal wall. Abdominal viscera are supplied by a large prevertebral plexus Innervation of the abdominal viscera is derived from a large prevertebral plexus associated mainly with the anterior and lateral surfaces of the aorta (Fig. 4.20). Branches are distributed to target tissues along vessels that originate from the abdominal aorta.

1	The prevertebral plexus contains sympathetic, parasympathetic, and visceral sensory components: Sympathetic components originate from spinal cord levels T5 to L2. Parasympathetic components are from the vagus nerve [X] and spinal cord levels S2 to S4. Visceral sensory fibers generally parallel the motor pathways. The abdomen is the part of the trunk inferior to the thorax (Fig. 4.21). Its musculomembranous walls surround a large cavity (the abdominal cavity), which is bounded superiorly by the diaphragm and inferiorly by the pelvic inlet. The abdominal cavity may extend superiorly as high as the fourth intercostal space, and is continuous inferiorly with the pelvic cavity. It contains the peritoneal cavity and the abdominal viscera. Topographical divisions of the abdomen are used to describe the location of abdominal organs and the pain associated with abdominal problems. The two schemes most often used are: a four-quadrant pattern and a nine-region pattern.

1	A horizontal transumbilical plane passing through the umbilicus and the intervertebral disc between vertebrae LIII and LIV and intersecting with the vertical median plane divides the abdomen into four quadrants—the right upper, left upper, right lower, and left lower quadrants (Fig. 4.22). The nine-region pattern is based on two horizontal and two vertical planes (Fig. 4.23). The superior horizontal plane (the subcostal plane) is immediately inferior to the costal margins, which places it at the lower border of the costal cartilage of rib X and passing posteriorly through the body of vertebra LIII. (Note, however, that sometimes the transpyloric plane, halfway between the jugular notch and the symphysis pubis or halfway between the umbilicus and the inferior end of the body of the sternum, passing posteriorly through the lower border of vertebra LI and intersecting with the costal margin at the ends of the ninth costal cartilages, is used instead.)

1	The inferior horizontal plane (the intertubercular plane) connects the tubercles of the iliac crests, which are palpable structures 5 cm posterior to the anterior superior iliac spines, and passes through the upper part of the body of vertebra LV. The vertical planes pass from the midpoint of the clavicles inferiorly to a point midway between the anterior superior iliac spine and pubic symphysis. These four planes establish the topographical divisions in the nine-region organization. The following designations are used for each region: superiorly the right hypochondrium, the epigastric region, and the left hypochondrium; inferiorly the right groin (inguinal region), pubic region, and left groin (inguinal region); and in the middle the right flank (lateral region), the umbilical region, and the left flank (lateral region) (Fig. 4.23).

1	The abdominal wall covers a large area. It is bounded superiorly by the xiphoid process and costal margins, posteriorly by the vertebral column, and inferiorly by the upper parts of the pelvic bones. Its layers consist of skin, superficial fascia (subcutaneous tissue), muscles and their associated deep fascias, extraperitoneal fascia, and parietal peritoneum (Fig. 4.24). The superficial fascia of the abdominal wall (subcutaneous tissue of abdomen) is a layer of fatty connective tissue. It is usually a single layer similar to, and continuous with, the superficial fascia throughout other regions of the body. However, in the lower region of the anterior part of the abdominal wall, below the umbilicus, it forms two layers: a superficial fatty layer and a deeper membranous layer.

1	The superficial fatty layer of superficial fascia (Camper’s fascia) contains fat and varies in thickness (Figs. 4.25 and 4.26). It is continuous over the inguinal ligament with the superficial fascia of the thigh and with a similar layer in the perineum. In men, this superficial layer continues over the penis and, after losing its fat and fusing with the deeper layer of superficial fascia, continues into the scrotum where it forms a specialized fascial layer containing smooth muscle fibers (the dartos fascia). In women, this superficial layer retains some fat and is a component of the labia majora.

1	The deeper membranous layer of superficial fascia (Scarpa’s fascia) is thin and membranous, and contains little or no fat (Fig. 4.25). Inferiorly, it continues into the thigh, but just below the inguinal ligament, it fuses with the deep fascia of the thigh (the fascia lata; Fig. 4.26). In the midline, it is firmly attached to the linea alba and the symphysis pubis. It continues into the anterior part of the perineum where it is firmly attached to the ischiopubic rami and to the posterior margin of the perineal membrane. Here, it is referred to as the superficial perineal fascia (Colles’ fascia).

1	In men, the deeper membranous layer of superficial fascia blends with the superficial layer as they both pass over the penis, forming the superficial fascia of the penis, before they continue into the scrotum where they form the dartos fascia (Fig. 4.25). Also in men, extensions of the deeper membranous layer of superficial fascia attached to the pubic symphysis pass inferiorly onto the dorsum and sides of the penis to form the fundiform ligament of penis. In women, the membranous layer of the superficial fascia continues into the labia majora and the anterior part of the perineum.

1	There are five muscles in the anterolateral group of abdominal wall muscles: three flat muscles whose fibers begin posterolaterally, pass anteriorly, and are replaced by an aponeurosis as the muscle continues toward the midline—the external oblique, internal oblique, and transversus abdominis muscles; two vertical muscles, near the midline, which are enclosed within a tendinous sheath formed by the aponeuroses of the flat muscles—the rectus abdominis and pyramidalis muscles. Each of these five muscles has specific actions, but together the muscles are critical for the maintenance of many normal physiological functions. By their positioning, they form a firm, but flexible, wall that keeps the abdominal viscera within the abdominal cavity, protects the viscera from injury, and helps maintain the position of the viscera in the erect posture against the action of gravity.

1	In addition, contraction of these muscles assists in both quiet and forced expiration by pushing the viscera upward (which helps push the relaxed diaphragm further into the thoracic cavity) and in coughing and vomiting. All these muscles are also involved in any action that increases intraabdominal pressure, including parturition (childbirth), micturition (urination), and defecation (expulsion of feces from the rectum). The most superficial of the three flat muscles in the anterolateral group of abdominal wall muscles is the external oblique, which is immediately deep to the superficial fascia (Fig. 4.27, Table 4.1). Its laterally placed muscle fibers pass in an inferomedial direction, while its large aponeurotic component covers the anterior part of the abdominal wall to the midline. Approaching the midline, the aponeuroses are entwined, forming the linea alba, which extends from the xiphoid process to the pubic symphysis.

1	The lower border of the external oblique aponeurosis forms the inguinal ligament on each side (Fig. 4.27). This thickened reinforced free edge of the external oblique aponeurosis passes between the anterior superior iliac spine laterally and the pubic tubercle medially (Fig. 4.28). It folds under itself forming a trough, which plays an important role in the formation of the inguinal canal. Several other ligaments are also formed from extensions of the fibers at the medial end of the inguinal ligament: The lacunar ligament is a crescent-shaped extension of fibers at the medial end of the inguinal ligament that pass backward to attach to the pecten pubis on the superior ramus of the pubic bone (Figs. 4.28 and 4.29). Additional fibers extend from the lacunar ligament along the pecten pubis of the pelvic brim to form the pectineal (Cooper’s) ligament.

1	Additional fibers extend from the lacunar ligament along the pecten pubis of the pelvic brim to form the pectineal (Cooper’s) ligament. Deep to the external oblique muscle is the internal oblique muscle, which is the second of the three flat muscles (Fig. 4.30, Table 4.1). This muscle is smaller and thinner than the external oblique, with most of its muscle fibers passing in a superomedial direction. Its lateral muscular components end anteriorly as an aponeurosis that blends into the linea alba at the midline. Deep to the internal oblique muscle is the transversus abdominis muscle (Fig. 4.31, Table 4.1), so named because of the direction of most of its muscle fibers. It ends in an anterior aponeurosis, which blends with the linea alba at the midline.

1	Each of the three flat muscles is covered on its anterior and posterior surfaces by a layer of deep (or investing) fascia. In general, these layers are unremarkable except for the layer deep to the transversus abdominis muscle (the transversalis fascia), which is better developed. The transversalis fascia is a continuous layer of deep fascia that lines the abdominal cavity and continues into the pelvic cavity. It crosses the midline anteriorly, associating with the transversalis fascia of the opposite side, and is continuous with the fascia on the inferior surface of the diaphragm. It is continuous posteriorly with the deep fascia covering the muscles of the posterior abdominal wall and attaches to the thoracolumbar fascia.

1	After attaching to the crest of the ilium, the transversalis fascia blends with the fascia covering the muscles associated with the upper regions of the pelvic bones and with similar fascia covering the muscles of the pelvic cavity. At this point, it is referred to as the parietal pelvic (or endopelvic) fascia. There is therefore a continuous layer of deep fascia surrounding the abdominal cavity that is thick in some areas, thin in others, attached or free, and participates in the formation of specialized structures. The two vertical muscles in the anterolateral group of abdominal wall muscles are the large rectus abdominis and the small pyramidalis (Fig. 4.32, Table 4.1).

1	The two vertical muscles in the anterolateral group of abdominal wall muscles are the large rectus abdominis and the small pyramidalis (Fig. 4.32, Table 4.1). The rectus abdominis is a long, flat muscle and extends the length of the anterior abdominal wall. It is a paired muscle, separated in the midline by the linea alba, and it widens and thins as it ascends from the pubic symphysis to the costal margin. Along its course, it is intersected by three or four transverse fibrous bands or tendinous intersections (Fig. 4.32). These are easily visible on individuals with well-developed rectus abdominis muscles. The second vertical muscle is the pyramidalis. This small, triangular muscle, which may be absent, is anterior to the rectus abdominis and has its base on the pubis, and its apex is attached superiorly and medially to the linea alba (Fig. 4.32).

1	The rectus abdominis and pyramidalis muscles are enclosed in an aponeurotic tendinous sheath (the rectus sheath) formed by a unique layering of the aponeuroses of the external and internal oblique, and transversus abdominis muscles (Fig. 4.33). The rectus sheath completely encloses the upper three-quarters of the rectus abdominis and covers the anterior surface of the lower one-quarter of the muscle. As no sheath covers the posterior surface of the lower quarter of the rectus abdominis muscle, the muscle at this point is in direct contact with the transversalis fascia. The formation of the rectus sheath surrounding the upper three-quarters of the rectus abdominis muscle has the following pattern: The anterior wall consists of the aponeurosis of the external oblique and half of the aponeurosis of the internal oblique, which splits at the lateral margin of the rectus abdominis.

1	The anterior wall consists of the aponeurosis of the external oblique and half of the aponeurosis of the internal oblique, which splits at the lateral margin of the rectus abdominis. The posterior wall of the rectus sheath consists of the other half of the aponeurosis of the internal oblique and the aponeurosis of the transversus abdominis. At a point midway between the umbilicus and the pubic symphysis, corresponding to the beginning of the lower one-quarter of the rectus abdominis muscle, all of the aponeuroses move anterior to the rectus muscle. There is no posterior wall of the rectus sheath and the anterior wall of the sheath consists of the aponeuroses of the external oblique, the internal oblique, and the transversus abdominis muscles. From this point inferiorly, the rectus abdominis muscle is in direct contact with the transversalis fascia. Marking this point of transition is an arch of fibers (the arcuate line; see Fig. 4.32).

1	Deep to the transversalis fascia is a layer of connective tissue, the extraperitoneal fascia, which separates the transversalis fascia from the peritoneum (Fig. 4.34). Containing varying amounts of fat, this layer not only lines the abdominal cavity but is also continuous with a similar layer lining the pelvic cavity. It is abundant on the posterior abdominal wall, especially around the kidneys, continues over organs covered by peritoneal reflections, and, as the vasculature is located in this layer, extends into mesenteries with the blood vessels. Viscera in the extraperitoneal fascia are referred to as retroperitoneal.

1	In the description of specific surgical procedures, the terminology used to describe the extraperitoneal fascia is further modified. The fascia toward the anterior side of the body is described as preperitoneal (or, less commonly, properitoneal) and the fascia toward the posterior side of the body has been described as retroperitoneal (Fig. 4.35). Examples of the use of these terms would be the continuity of fat in the inguinal canal with the preperitoneal fat and a transabdominal preperitoneal laparoscopic repair of an inguinal hernia. Deep to the extraperitoneal fascia is the peritoneum (see Figs. 4.6 and 4.7 on pp. 260-261). This thin serous membrane lines the walls of the abdominal cavity and, at various points, reflects onto the abdominal viscera, providing either a complete or a partial covering. The peritoneum lining the walls is the parietal peritoneum; the peritoneum covering the viscera is the visceral peritoneum.

1	The continuous lining of the abdominal walls by the parietal peritoneum forms a sac. This sac is closed in men but has two openings in women where the uterine tubes provide a passage to the outside. The closed sac in men and the semiclosed sac in women is called the peritoneal cavity. The skin, muscles, and parietal peritoneum of the anterolateral abdominal wall are supplied by T7 to T12 and L1 spinal nerves. The anterior rami of these spinal nerves pass around the body, from posterior to anterior, in an inferomedial direction (Fig. 4.36). As they proceed, they give off a lateral cutaneous branch and end as an anterior cutaneous branch.

1	The intercostal nerves (T7 to T11) leave their intercostal spaces, passing deep to the costal cartilages, and continue onto the anterolateral abdominal wall between the internal oblique and transversus abdominis muscles (Fig. 4.37). Reaching the lateral edge of the rectus sheath, they enter the rectus sheath and pass posterior to the lateral aspect of the rectus abdominis muscle. Approaching the midline, an anterior cutaneous branch passes through the rectus abdominis muscle and the anterior wall of the rectus sheath to supply the skin. Spinal nerve T12 (the subcostal nerve) follows a similar course as the intercostals. Branches of L1 (the iliohypogastric nerve and ilio-inguinal nerve), which originate from the lumbar plexus, follow similar courses initially, but deviate from this pattern near their final destination.

1	Along their course, nerves T7 to T12 and L1 supply branches to the anterolateral abdominal wall muscles and the underlying parietal peritoneum. All terminate by supplying skin: Nerves T7 to T9 supply the skin from the xiphoid process to just above the umbilicus. T10 supplies the skin around the umbilicus. T11, T12, and L1 supply the skin from just below the umbilicus to, and including, the pubic region (Fig. 4.38). Additionally, the ilio-inguinal nerve (a branch of L1) supplies the anterior surface of the scrotum or labia majora, and sends a small cutaneous branch to the thigh.

1	Additionally, the ilio-inguinal nerve (a branch of L1) supplies the anterior surface of the scrotum or labia majora, and sends a small cutaneous branch to the thigh. Numerous blood vessels supply the anterolateral abdominal wall. Superficially: the superior part of the wall is supplied by branches from the musculophrenic artery, a terminal branch of the internal thoracic artery, and the inferior part of the wall is supplied by the medially placed superficial epigastric artery and the laterally placed superficial circumflex iliac artery, both branches of the femoral artery (Fig. 4.39).

1	At a deeper level: the superior part of the wall is supplied by the superior epigastric artery, a terminal branch of the internal thoracic artery; the lateral part of the wall is supplied by branches of the tenth and eleventh intercostal arteries and the subcostal artery; and the inferior part of the wall is supplied by the medially placed inferior epigastric artery and the laterally placed deep circumflex iliac artery, both branches of the external iliac artery. The superior and inferior epigastric arteries both enter the rectus sheath. They are posterior to the rectus abdominis muscle throughout their course, and anastomose with each other (Fig. 4.40). Veins of similar names follow the arteries and are responsible for venous drainage. Lymphatic drainage of the anterolateral abdominal wall follows the basic principles of lymphatic drainage:

1	Veins of similar names follow the arteries and are responsible for venous drainage. Lymphatic drainage of the anterolateral abdominal wall follows the basic principles of lymphatic drainage: Superficial lymphatics above the umbilicus pass in a superior direction to the axillary nodes, while drainage below the umbilicus passes in an inferior direction to the superficial inguinal nodes. Deep lymphatic drainage follows the deep arteries back to parasternal nodes along the internal thoracic artery, lumbar nodes along the abdominal aorta, and external iliac nodes along the external iliac artery.

1	The groin (inguinal region) is the area of junction between the anterior abdominal wall and the thigh. In this area, the abdominal wall is weakened from changes that occur during development and a peritoneal sac or diverticulum, with or without abdominal contents, can therefore protrude through it, creating an inguinal hernia. This type of hernia can occur in both sexes, but it is most common in males. The inherent weakness in the anterior abdominal wall in the groin is caused by changes that occur during the development of the gonads. Before the descent of the testes and ovaries from their initial position high in the posterior abdominal wall, a peritoneal outpouching (the processus vaginalis) forms (Fig. 4.41), protruding through the various layers of the anterior abdominal wall and acquiring coverings from each: The transversalis fascia forms its deepest covering.

1	The transversalis fascia forms its deepest covering. The second covering is formed by the musculature of the internal oblique (a covering from the transversus abdominis muscle is not acquired because the processus vaginalis passes under the arching fibers of this abdominal wall muscle). Its most superficial covering is the aponeurosis of the external oblique. As a result the processus vaginalis is transformed into a tubular structure with multiple coverings from the layers of the anterior abdominal wall. This forms the basic structure of the inguinal canal. The final event in this development is the descent of the testes into the scrotum or of the ovaries into the pelvic cavity. This process depends on the development of the gubernaculum, which extends from the inferior border of the developing gonad to the labioscrotal swellings (Fig. 4.41). The processus vaginalis is immediately anterior to the gubernaculum within the inguinal canal.

1	The processus vaginalis is immediately anterior to the gubernaculum within the inguinal canal. In men, as the testes descend, the testes and their accompanying vessels, ducts, and nerves pass through the inguinal canal and are therefore surrounded by the same fascial layers of the abdominal wall. Testicular descent completes the formation of the spermatic cord in men. In women, the ovaries descend into the pelvic cavity and become associated with the developing uterus. Therefore, the only remaining structure passing through the inguinal canal is the round ligament of the uterus, which is a remnant of the gubernaculum.

1	The development sequence is concluded in both sexes when the processus vaginalis obliterates. If this does not occur or is incomplete, a potential weakness exists in the anterior abdominal wall and an inguinal hernia may develop. In males, only proximal regions of the processus vaginalis obliterate. The distal end expands to enclose most of the testis in the scrotum. In other words, the cavity of the tunica vaginalis in men forms as an extension of the developing peritoneal cavity that becomes separated off during development.

1	The inguinal canal is a slit-like passage that extends in a downward and medial direction, just above and parallel to the lower half of the inguinal ligament. It begins at the deep inguinal ring and continues for approximately 4 cm, ending at the superficial inguinal ring (Fig. 4.42). The contents of the canal are the genital branch of the genitofemoral nerve, the spermatic cord in men, and the round ligament of the uterus in women. Additionally, in both sexes, the ilio-inguinal nerve passes through part of the canal, exiting through the superficial inguinal ring with the other contents.

1	The deep (internal) inguinal ring is the beginning of the inguinal canal and is at a point midway between the anterior superior iliac spine and the pubic symphysis (Fig. 4.43). It is just above the inguinal ligament and immediately lateral to the inferior epigastric vessels. Although sometimes referred to as a defect or opening in the transversalis fascia, it is actually the beginning of the tubular evagination of transversalis fascia that forms one of the coverings (the internal spermatic fascia) of the spermatic cord in men or the round ligament of the uterus in women.

1	The superficial (external) inguinal ring is the end of the inguinal canal and is superior to the pubic tubercle (Fig. 4.44). It is a triangular opening in the aponeurosis of the external oblique, with its apex pointing superolaterally and its base formed by the pubic crest. The two remaining sides of the triangle (the medial crus and the lateral crus) are attached to the pubic symphysis and the pubic tubercle, respectively. At the apex of the triangle the two crura are held together by crossing (intercrural) fibers, which prevent further widening of the superficial ring. As with the deep inguinal ring, the superficial inguinal ring is actually the beginning of the tubular evagination of the aponeurosis of the external oblique onto the structures traversing the inguinal canal and emerging from the superficial inguinal ring. This continuation of tissue over the spermatic cord is the external spermatic fascia.

1	The anterior wall of the inguinal canal is formed along its entire length by the aponeurosis of the external oblique muscle (Fig. 4.44). It is also reinforced laterally by the lower fibers of the internal oblique that originate from the lateral two-thirds of the inguinal ligament (Fig. 4.45). This adds an additional covering over the deep inguinal ring, which is a potential point of weakness in the anterior abdominal wall. Furthermore, as the internal oblique muscle covers the deep inguinal ring, it also contributes a layer (the cremasteric fascia containing the cremasteric muscle) to the coverings of the structures traversing the inguinal canal.

1	The posterior wall of the inguinal canal is formed along its entire length by the transversalis fascia (see Fig. 4.43). It is reinforced along its medial one-third by the conjoint tendon (inguinal falx; Fig. 4.45). This tendon is the combined insertion of the transversus abdominis and internal oblique muscles into the pubic crest and pectineal line. As with the internal oblique muscle’s reinforcement of the area of the deep inguinal ring, the position of the conjoint tendon posterior to the superficial inguinal ring provides additional support to a potential point of weakness in the anterior abdominal wall. The roof (superior wall) of the inguinal canal is formed by the arching fibers of the transversus abdominis and internal oblique muscles (Figs. 4.45 and 4.46). They pass from their lateral points of origin from the inguinal ligament to their common medial attachment as the conjoint tendon.

1	The floor (inferior wall) of the inguinal canal is formed by the medial one-half of the inguinal ligament. This rolled-under, free margin of the lowest part of the aponeurosis of the external oblique forms a gutter or trough on which the contents of the inguinal canal are positioned. The lacunar ligament reinforces most of the medial part of the gutter. The contents of the inguinal canal are: the spermatic cord in men, and the round ligament of the uterus and genital branch of the genitofemoral nerve in women. These structures enter the inguinal canal through the deep inguinal ring and exit it through the superficial inguinal ring.

1	These structures enter the inguinal canal through the deep inguinal ring and exit it through the superficial inguinal ring. Additionally, the ilio-inguinal nerve (L1) passes through part of the inguinal canal. This nerve is a branch of the lumbar plexus, enters the abdominal wall posteriorly by piercing the internal surface of the transversus abdominis muscle, and continues through the layers of the anterior abdominal wall by piercing the internal oblique muscle. As it continues to pass inferomedially, it enters the inguinal canal. It continues down the canal to exit through the superficial inguinal ring. The spermatic cord begins to form proximally at the deep inguinal ring and consists of structures passing between the abdominopelvic cavities and the testis, and the three fascial coverings that enclose these structures (Fig. 4.47).

1	The structures in the spermatic cord include: the ductus deferens, the artery to the ductus deferens (from the inferior vesical artery), the testicular artery (from the abdominal aorta), the pampiniform plexus of veins (testicular veins), the cremasteric artery and vein (small vessels associated with the cremasteric fascia), the genital branch of the genitofemoral nerve (innervation to the cremasteric muscle), sympathetic and visceral afferent nerve fibers, lymphatics, and remnants of the processus vaginalis. These structures enter the deep inguinal ring, proceed down the inguinal canal, and exit from the superficial inguinal ring, having acquired the three fascial coverings during their journey. This collection of structures and fascias continues into the scrotum where the structures connect with the testes and the fascias surround the testes. Three fascias enclose the contents of the spermatic cord:

1	Three fascias enclose the contents of the spermatic cord: The internal spermatic fascia, which is the deepest layer, arises from the transversalis fascia and is attached to the margins of the deep inguinal ring. The cremasteric fascia with the associated cremasteric muscle, which is the middle fascial layer, arises from the internal oblique muscle. The external spermatic fascia, which is the most superficial covering of the spermatic cord, arises from the aponeurosis of the external oblique muscle and is attached to the margins of the superficial inguinal ring (Fig. 4.47A). Round ligament of the uterus

1	Round ligament of the uterus The round ligament of the uterus is a cord-like structure that passes from the uterus to the deep inguinal ring where it enters the inguinal canal (Fig. 4.47B). It passes down the inguinal canal and exits through the superficial inguinal ring. At this point, it has changed from a cord-like structure to a few strands of tissue, which attach to the connective tissue associated with the labia majora. As it traverses the inguinal canal, it acquires the same coverings as the spermatic cord in men. As the round ligament exits the superficial inguinal ring, the coverings are indistinguishable from the tissue strands of the ligament itself.

1	The round ligament of the uterus is the long distal part of the original gubernaculum in the fetus that extends from the ovary to the labioscrotal swellings. From its attachment to the uterus, the round ligament of the uterus continues to the ovary as the ligament of the ovary that develops from the short proximal end of the gubernaculum. An inguinal hernia is the protrusion or passage of a peritoneal sac, with or without abdominal contents, through a weakened part of the abdominal wall in the groin. It occurs because the peritoneal sac enters the inguinal canal either: indirectly, through the deep inguinal ring, or directly, through the posterior wall of the inguinal canal. Inguinal hernias are therefore classified as either indirect or direct.

1	Inguinal hernias are therefore classified as either indirect or direct. The indirect inguinal hernia is the most common of the two types of inguinal hernia and is much more common in men than in women (Fig. 4.48). It occurs because some part, or all, of the embryonic processus vaginalis remains open or patent. It is therefore referred to as being congenital in origin.

1	The protruding peritoneal sac enters the inguinal canal by passing through the deep inguinal ring, just lateral to the inferior epigastric vessels. The extent of its excursion down the inguinal canal depends on the amount of processus vaginalis that remains patent. If the entire processus vaginalis remains patent, the peritoneal sac may traverse the length of the canal, exit the superficial inguinal ring, and continue into the scrotum in men or the labia majus in women. In this case, the protruding peritoneal sac acquires the same three coverings as those associated with the spermatic cord in men or the round ligament of the uterus in women.

1	A peritoneal sac that enters the medial end of the inguinal canal directly through a weakened posterior wall is a direct inguinal hernia (Fig. 4.49). It is usually described as acquired because it develops when abdominal musculature has been weakened, and is commonly seen in mature men. The bulging occurs medial to the inferior epigastric vessels in the inguinal triangle (Hesselbach’s triangle), which is bounded: laterally by the inferior epigastric artery, medially by the rectus abdominis muscle, and inferiorly by the inguinal ligament (Fig. 4.50). Internally, a thickening of the transversalis fascia (the iliopubic tract) follows the course of the inguinal ligament (Fig. 4.50). A direct inguinal hernia does not traverse the entire length of the inguinal canal but may exit through the superficial inguinal ring. When this occurs, the peritoneal sac acquires a layer of external spermatic fascia and can extend, like an indirect hernia, into the scrotum.

1	A thin membrane (the peritoneum) lines the walls of the abdominal cavity and covers much of the viscera. The parietal peritoneum lines the walls of the cavity and the visceral peritoneum covers the viscera. Between the parietal and visceral layers of peritoneum is a potential space (the peritoneal cavity). Abdominal viscera either are suspended in the peritoneal cavity by folds of peritoneum (mesenteries) or are outside the peritoneal cavity. Organs suspended in the cavity are referred to as intraperitoneal (Fig. 4.53); organs outside the peritoneal cavity, with only one surface or part of one surface covered by peritoneum, are retroperitoneal. Innervation of the peritoneum

1	Innervation of the peritoneum The parietal peritoneum associated with the abdominal wall is innervated by somatic afferents carried in branches of the associated spinal nerves and is therefore sensitive to well-localized pain. The visceral peritoneum is innervated by visceral afferents that accompany autonomic nerves (sympathetic and parasympathetic) back to the central nervous system. Activation of these fibers can lead to referred and poorly localized sensations of discomfort, and to reflex visceral motor activity. The peritoneal cavity is subdivided into the greater sac and the omental bursa (lesser sac; Fig. 4.54). The greater sac accounts for most of the space in the peritoneal cavity, beginning superiorly at the diaphragm and continuing inferiorly into the pelvic cavity. It is entered once the parietal peritoneum has been penetrated.

1	The omental bursa is a smaller subdivision of the peritoneal cavity posterior to the stomach and liver and is continuous with the greater sac through an opening, the omental (epiploic) foramen (Fig. 4.55). Surrounding the omental (epiploic) foramen are numerous structures covered with peritoneum. They include the portal vein, hepatic artery proper, and bile duct anteriorly; the inferior vena cava posteriorly; the caudate lobe of the liver superiorly; and the first part of the duodenum inferiorly. Omenta, mesenteries, and ligaments Throughout the peritoneal cavity numerous peritoneal folds connect organs to each other or to the abdominal wall. These folds (omenta, mesenteries, and ligaments) develop from the original dorsal and ventral mesenteries, which suspend the developing gastrointestinal tract in the embryonic coelomic cavity. Some contain vessels and nerves supplying the viscera, while others help maintain the proper positioning of the viscera.

1	The omenta consist of two layers of peritoneum, which pass from the stomach and the first part of the duodenum to other viscera. There are two: the greater omentum, derived from the dorsal mesentery, and the lesser omentum, derived from the ventral mesentery. The greater omentum is a large, apron-like, peritoneal fold that attaches to the greater curvature of the stomach and the first part of the duodenum (Fig. 4.59). It drapes inferiorly over the transverse colon and the coils of the jejunum and ileum (see Fig. 4.54). Turning posteriorly, it ascends to associate with, and become adherent to, the peritoneum on the superior surface of the transverse colon and the anterior layer of the transverse mesocolon before arriving at the posterior abdominal wall.

1	Usually a thin membrane, the greater omentum always contains an accumulation of fat, which may become substantial in some individuals. Additionally, there are two arteries and accompanying veins, the right and left gastro-omental vessels, between this double-layered peritoneal apron just inferior to the greater curvature of the stomach. The other two-layered peritoneal omentum is the lesser omentum (Fig. 4.60). It extends from the lesser curvature of the stomach and the first part of the duodenum to the inferior surface of the liver (Figs. 4.54 and 4.60). A thin membrane continuous with the peritoneal coverings of the anterior and posterior surfaces of the stomach and the first part of the duodenum, the lesser omentum is divided into: a medial hepatogastric ligament, which passes between the stomach and liver, and a lateral hepatoduodenal ligament, which passes between the duodenum and liver.

1	The hepatoduodenal ligament ends laterally as a free margin and serves as the anterior border of the omental foramen (Fig. 4.55). Enclosed in this free edge are the hepatic artery proper, the bile duct, and the portal vein. Additionally, the right and left gastric vessels are between the layers of the lesser omentum near the lesser curvature of the stomach. Mesenteries are peritoneal folds that attach viscera to the posterior abdominal wall. They allow some movement and provide a conduit for vessels, nerves, and lymphatics to reach the viscera and include: the mesentery—associated with parts of the small intestine, the transverse mesocolon—associated with the transverse colon, and the sigmoid mesocolon—associated with the sigmoid colon. All of these are derivatives of the dorsal mesentery.

1	All of these are derivatives of the dorsal mesentery. The mesentery is a large, fan-shaped, double-layered fold of peritoneum that connects the jejunum and ileum to the posterior abdominal wall (Fig. 4.61). Its superior attachment is at the duodenojejunal junction, just to the left of the upper lumbar part of the vertebral column. It passes obliquely downward and to the right, ending at the ileocecal junction near the upper border of the right sacro-iliac joint. In the fat between the two peritoneal layers of the mesentery are the arteries, veins, nerves, and lymphatics that supply the jejunum and ileum.

1	The transverse mesocolon is a fold of peritoneum that connects the transverse colon to the posterior abdominal wall (Fig. 4.61). Its two layers of peritoneum leave the posterior abdominal wall across the anterior surface of the head and body of the pancreas and pass outward to surround the transverse colon. Between its layers are the arteries, veins, nerves, and lymphatics related to the transverse colon. The anterior layer of the transverse mesocolon is adherent to the posterior layer of the greater omentum.

1	The sigmoid mesocolon is an inverted, V-shaped peritoneal fold that attaches the sigmoid colon to the abdominal wall (Fig. 4.61). The apex of the V is near the division of the left common iliac artery into its internal and external branches, with the left limb of the descending V along the medial border of the left psoas major muscle and the right limb descending into the pelvis to end at the level of vertebra SIII. The sigmoid and superior rectal vessels, along with the nerves and lymphatics associated with the sigmoid colon, pass through this peritoneal fold. Peritoneal ligaments consist of two layers of peritoneum that connect two organs to each other or attach an organ to the body wall, and may form part of an omentum. They are usually named after the structures being connected. For example, the splenorenal ligament connects the left kidney to the spleen and the gastrophrenic ligament connects the stomach to the diaphragm.

1	The abdominal esophagus represents the short distal part of the esophagus located in the abdominal cavity. Emerging through the right crus of the diaphragm, usually at the level of vertebra TX, it passes from the esophageal hiatus to the cardial orifice of the stomach just left of the midline (Fig. 4.62). Associated with the esophagus, as it enters the abdominal cavity, are the anterior and posterior vagal trunks: The anterior vagal trunk consists of several smaller trunks whose fibers mostly come from the left vagus nerve; rotation of the gut during development moves these trunks to the anterior surface of the esophagus. Similarly, the posterior vagal trunk consists of a single trunk whose fibers mostly come from the right vagus nerve, and rotational changes during development move this trunk to the posterior surface of the esophagus.

1	The arterial supply to the abdominal esophagus (Fig. 4.63) includes: esophageal branches from the left gastric artery (from the celiac trunk), and esophageal branches from the left inferior phrenic artery (from the abdominal aorta). The stomach is the most dilated part of the gastrointestinal tract and has a J-like shape (Figs. 4.64 and 4.65). Positioned between the abdominal esophagus and the small intestine, the stomach is in the epigastric, umbilical, and left hypochondrium regions of the abdomen. The stomach is divided into four regions: the cardia, which surrounds the opening of the esophagus into the stomach; the fundus of the stomach, which is the area above the level of the cardial orifice; the body of the stomach, which is the largest region of the stomach; and the pyloric part, which is divided into the pyloric antrum and pyloric canal and is the distal end of the stomach.

1	The most distal portion of the pyloric part of the stomach is the pylorus (Fig. 4.64). It is marked on the surface of the organ by the pyloric constriction and contains a thickened ring of gastric circular muscle, the pyloric sphincter, that surrounds the distal opening of the stomach, the pyloric orifice (Figs. 4.64 and 4.65B). The pyloric orifice is just to the right of midline in a plane that passes through the lower border of vertebra LI (the transpyloric plane). Other features of the stomach include: the greater curvature, which is a point of attachment for the gastrosplenic ligament and the greater omentum; the lesser curvature, which is a point of attachment for the lesser omentum; the cardial notch, which is the superior angle created when the esophagus enters the stomach; and the angular incisure, which is a bend on the lesser curvature.

1	The arterial supply to the stomach (Fig. 4.63) includes: the left gastric artery from the celiac trunk, the right gastric artery, often from the hepatic artery proper, the right gastro-omental artery from the gastroduodenal artery, the left gastro-omental artery from the splenic artery, and the posterior gastric artery from the splenic artery (variant and not always present). The small intestine is the longest part of the gastrointestinal tract and extends from the pyloric orifice of the stomach to the ileocecal fold. This hollow tube, which is approximately 6 to 7 m long with a narrowing diameter from beginning to end, consists of the duodenum, the jejunum, and the ileum.

1	The first part of the small intestine is the duodenum. This C-shaped structure, adjacent to the head of the pancreas, is 20 to 25 cm long and is above the level of the umbilicus; its lumen is the widest of the small intestine (Fig. 4.66). It is retroperitoneal except for its beginning, which is connected to the liver by the hepatoduodenal ligament, a part of the lesser omentum. The duodenum is divided into four parts (Fig. 4.66). The superior part (first part) extends from the pyloric orifice of the stomach to the neck of the gallbladder, is just to the right of the body of vertebra LI, and passes anteriorly to the bile duct, gastroduodenal artery, portal vein, and inferior vena cava. Clinically, the beginning of this part of the duodenum is referred to as the ampulla or duodenal cap, and most duodenal ulcers occur in this part of the duodenum.

1	The descending part (second part) of the duodenum is just to the right of midline and extends from the neck of the gallbladder to the lower border of vertebra LIII. Its anterior surface is crossed by the transverse colon, posterior to it is the right kidney, and medial to it is the head of the pancreas. This part of the duodenum contains the major duodenal papilla, which is the common entrance for the bile and pancreatic ducts, and the minor duodenal papilla, which is the entrance for the accessory pancreatic duct. The junction of the foregut and the midgut occurs just below the major duodenal papilla. The inferior part (third part) of the duodenum is the longest section, crossing the inferior vena cava, the aorta, and the vertebral column (Figs. 4.65B and 4.66). It is crossed anteriorly by the superior mesenteric artery and vein.

1	The ascending part (fourth part) of the duodenum passes upward on, or to the left of, the aorta to approximately the upper border of vertebra LII and terminates at the duodenojejunal flexure. This duodenojejunal flexure is surrounded by a fold of peritoneum containing muscle fibers called the suspensory muscle (ligament) of duodenum (ligament of Treitz).

1	This duodenojejunal flexure is surrounded by a fold of peritoneum containing muscle fibers called the suspensory muscle (ligament) of duodenum (ligament of Treitz). The arterial supply to the duodenum (Fig. 4.67) includes: branches from the gastroduodenal artery, the supraduodenal artery from the gastroduodenal artery, duodenal branches from the anterior superior pancreaticoduodenal artery (from the gastroduodenal artery), duodenal branches from the posterior superior pancreaticoduodenal artery (from the gastroduodenal artery), duodenal branches from the anterior inferior pancreaticoduodenal artery (from the inferior pancreaticoduodenal artery—a branch of the superior mesenteric artery), duodenal branches from the posterior inferior pancreaticoduodenal artery (from the inferior pancreaticoduodenal artery—a branch of the superior mesenteric artery), and the first jejunal branch from the superior mesenteric artery.

1	The jejunum and ileum make up the last two sections of the small intestine (Fig. 4.68). The jejunum represents the proximal two-fifths. It is mostly in the left upper quadrant of the abdomen and is larger in diameter and has a thicker wall than the ileum. Additionally, the inner mucosal lining of the jejunum is characterized by numerous prominent folds that circle the lumen (plicae circulares). The less prominent arterial arcades and longer vasa recta (straight arteries) compared to those of the ileum are a unique characteristic of the jejunum (Fig. 4.69). The arterial supply to the jejunum includes jejunal arteries from the superior mesenteric artery. The ileum makes up the distal three-fifths of the small intestine and is mostly in the right lower quadrant. Compared to the jejunum, the ileum has thinner walls, fewer and less prominent mucosal folds (plicae circulares), shorter vasa recta, more mesenteric fat, and more arterial arcades (Fig. 4.69).

1	The ileum opens into the large intestine, where the cecum and ascending colon join together. Two flaps projecting into the lumen of the large intestine (the ileocecal fold) surround the opening (Fig. 4.70). The flaps of the ileocecal fold come together at their end, forming ridges. Musculature from the ileum continues into each flap, forming a sphincter. Possible functions of the ileocecal fold include preventing reflux from the cecum to the ileum, and regulating the passage of contents from the ileum to the cecum. The arterial supply to the ileum (Fig. 4.71) includes: ileal arteries from the superior mesenteric artery, and an ileal branch from the ileocolic artery (from the superior mesenteric artery). The large intestine extends from the distal end of the ileum to the anus, a distance of approximately 1.5 m in adults. It absorbs fluids and salts from the gut contents, thus forming feces, and consists of the cecum, appendix, colon, rectum, and anal canal (Figs. 4.79 and 4.80).

1	Beginning in the right groin as the cecum, with its associated appendix, the large intestine continues upward as the ascending colon through the right flank and into the right hypochondrium (Fig. 4.81). Just below the liver, it bends to the left, forming the right colic flexure (hepatic flexure), and crosses the abdomen as the transverse colon to the left hypochondrium. At this position, just below the spleen, the large intestine bends downward, forming the left colic flexure (splenic flexure), and continues as the descending colon through the left flank and into the left groin. It enters the upper part of the pelvic cavity as the sigmoid colon, continues on the posterior wall of the pelvic cavity as the rectum, and terminates as the anal canal.

1	It enters the upper part of the pelvic cavity as the sigmoid colon, continues on the posterior wall of the pelvic cavity as the rectum, and terminates as the anal canal. The general characteristics of most of the large intestine (Fig. 4.79) are: its large internal diameter compared to that of the small intestine; peritoneal-covered accumulations of fat (the omental appendices) are associated with the colon; the segregation of longitudinal muscle in its walls into three narrow bands (the taeniae coli), which are primarily observed in the cecum and colon and less visible in the rectum; and the sacculations of the colon (the haustra of the colon). The cecum is the first part of the large intestine (Fig. 4.82). It is inferior to the ileocecal opening and in the right iliac fossa. It is generally considered to be an intraperitoneal structure because of its mobility, even though it normally is not suspended in the peritoneal cavity by a mesentery.

1	The cecum is continuous with the ascending colon at the entrance of the ileum and is usually in contact with the anterior abdominal wall. It may cross the pelvic brim to lie in the true pelvis. The appendix is attached to the posteromedial wall of the cecum, just inferior to the end of the ileum (Fig. 4.82).

1	The appendix is a narrow, hollow, blind-ended tube connected to the cecum. It has large aggregations of lymphoid tissue in its walls and is suspended from the terminal ileum by the mesoappendix (Fig. 4.83), which contains the appendicular vessels. Its point of attachment to the cecum is consistent with the highly visible free taeniae leading directly to the base of the appendix, but the location of the rest of the appendix varies considerably (Fig. 4.84). It may be: posterior to the cecum or the lower ascending colon, or both, in a retrocecal or retrocolic position; suspended over the pelvic brim in a pelvic or descending position; below the cecum in a subcecal location; or anterior to the terminal ileum, possibly contacting the body wall, in a pre-ileal position or posterior to the terminal ileum in a postileal position.

1	The surface projection of the base of the appendix is at the junction of the lateral and middle one-third of a line from the anterior superior iliac spine to the umbilicus (McBurney’s point). People with appendicular problems may describe pain near this location. The arterial supply to the cecum and appendix (Fig. 4.85) includes: the anterior cecal artery from the ileocolic artery (from the superior mesenteric artery), the posterior cecal artery from the ileocolic artery (from the superior mesenteric artery), and the appendicular artery from the ileocolic artery (from the superior mesenteric artery). The colon extends superiorly from the cecum and consists of the ascending, transverse, descending, and sigmoid colon (Fig. 4.88). Its ascending and descending segments are (secondarily) retroperitoneal and its transverse and sigmoid segments are intraperitoneal.

1	At the junction of the ascending and transverse colon is the right colic flexure, which is just inferior to the right lobe of the liver (Fig. 4.89). A similar, but more acute bend (the left colic flexure) occurs at the junction of the transverse and descending colon. This bend is just inferior to the spleen, is higher and more posterior than the right colic flexure, and is attached to the diaphragm by the phrenicocolic ligament.

1	Immediately lateral to the ascending and descending colon are the right and left paracolic gutters (Fig. 4.88). These depressions are formed between the lateral margins of the ascending and descending colon and the posterolateral abdominal wall and are gutters through which material can pass from one region of the peritoneal cavity to another. Because major vessels and lymphatics are on the medial or posteromedial sides of the ascending and descending colon, a relatively blood-free mobilization of the ascending and descending colon is possible by cutting the peritoneum along these lateral paracolic gutters.

1	The final segment of the colon (the sigmoid colon) begins above the pelvic inlet and extends to the level of vertebra SIII, where it is continuous with the rectum (Fig. 4.88). This S-shaped structure is quite mobile except at its beginning, where it continues from the descending colon, and at its end, where it continues as the rectum. Between these points, it is suspended by the sigmoid mesocolon. The arterial supply to the ascending colon (Fig. 4.90) includes: the colic branch from the ileocolic artery (from the superior mesenteric artery), the anterior cecal artery from the ileocolic artery (from the superior mesenteric artery), the posterior cecal artery from the ileocolic artery (from the superior mesenteric artery), and the right colic artery from the superior mesenteric artery.

1	The arterial supply to the transverse colon (Fig. 4.90) includes: the right colic artery from the superior mesenteric artery, the middle colic artery from the superior mesenteric artery, and the left colic artery from the inferior mesenteric artery. The arterial supply to the descending colon (Fig. 4.90) includes the left colic artery from the inferior mesenteric artery. The arterial supply to the sigmoid colon (Fig. 4.90) includes sigmoidal arteries from the inferior mesenteric artery. Anastomotic connections between arteries supplying the colon can result in a marginal artery that courses along the ascending, transverse, and descending parts of the large bowel (Fig. 4.90). Extending from the sigmoid colon is the rectum (Fig. 4.91). The rectosigmoid junction is usually described as being at the level of vertebra SIII or at the end of the sigmoid mesocolon because the rectum is a retroperitoneal structure.

1	The anal canal is the continuation of the large intestine inferior to the rectum. The arterial supply to the rectum and anal canal (Fig. 4.92) includes: the superior rectal artery from the inferior mesenteric artery, the middle rectal artery from the internal iliac artery, and the inferior rectal artery from the internal pudendal artery (from the internal iliac artery). The liver is the largest visceral organ in the body and is primarily in the right hypochondrium and epigastric region, extending into the left hypochondrium (or in the right upper quadrant, extending into the left upper quadrant) (Fig. 4.101). Surfaces of the liver include: a diaphragmatic surface in the anterior, superior, and posterior directions; and a visceral surface in the inferior direction (Fig. 4.102).

1	Surfaces of the liver include: a diaphragmatic surface in the anterior, superior, and posterior directions; and a visceral surface in the inferior direction (Fig. 4.102). The diaphragmatic surface of the liver, which is smooth and domed, lies against the inferior surface of the diaphragm (Fig. 4.103). Associated with it are the subphrenic and hepatorenal recesses (Fig. 4.102): The subphrenic recess separates the diaphragmatic surface of the liver from the diaphragm and is divided into right and left areas by the falciform ligament, a structure derived from the ventral mesentery in the embryo. The hepatorenal recess is a part of the peritoneal cavity on the right side between the liver and the right kidney and right suprarenal gland. The subphrenic and hepatorenal recesses are continuous anteriorly.

1	The subphrenic and hepatorenal recesses are continuous anteriorly. The visceral surface of the liver is covered with visceral peritoneum except in the fossa for the gallbladder and at the porta hepatis (gateway to the liver; Fig. 4.104), and structures related to it include the following (Fig. 4.105): esophagus, right anterior part of the stomach, superior part of the duodenum, lesser omentum, gallbladder, right colic flexure, right transverse colon, right kidney, and right suprarenal gland. The porta hepatis serves as the point of entry into the liver for the hepatic arteries and the portal vein, and the exit point for the hepatic ducts (Fig. 4.104).

1	The porta hepatis serves as the point of entry into the liver for the hepatic arteries and the portal vein, and the exit point for the hepatic ducts (Fig. 4.104). The liver is attached to the anterior abdominal wall by the falciform ligament and, except for a small area of the liver against the diaphragm (the bare area), the liver is almost completely surrounded by visceral peritoneum (Fig. 4.105). Additional folds of peritoneum connect the liver to the stomach (hepatogastric ligament), the duodenum (hepatoduodenal ligament), and the diaphragm (right and left triangular ligaments and anterior and posterior coronary ligaments). The bare area of the liver is a part of the liver on the diaphragmatic surface where there is no intervening peritoneum between the liver and the diaphragm (Fig. 4.105): The anterior boundary of the bare area is indicated by a reflection of peritoneum—the anterior coronary ligament.

1	The anterior boundary of the bare area is indicated by a reflection of peritoneum—the anterior coronary ligament. The posterior boundary of the bare area is indicated by a reflection of peritoneum—the posterior coronary ligament. Where the coronary ligaments come together laterally, they form the right and left triangular ligaments. The liver is divided into right and left lobes by the falciform ligament anterosuperiorly and the fissure for the ligamentum venosum and ligamentum teres on the visceral surface. (Fig. 4.104). The right lobe of the liver is the largest lobe, whereas the left lobe of the liver is smaller. The quadrate and caudate lobes are described as arising from the right lobe of the liver but functionally are distinct.

1	The quadrate lobe is visible on the anterior part of the visceral surface of the liver and is bounded on the left by the fissure for the ligamentum teres and on the right by the fossa for the gallbladder. Functionally, it is related to the left lobe of the liver. The caudate lobe is visible on the posterior part of the visceral surface of the liver. It is bounded on the left by the fissure for the ligamentum venosum and on the right by the groove for the inferior vena cava. Functionally, it is separate from the right and the left lobes of the liver. The arterial supply to the liver includes: the right hepatic artery from the hepatic artery proper (a branch of the common hepatic artery from the celiac trunk), and the left hepatic artery from the hepatic artery proper (a branch of the common hepatic artery from the celiac trunk).

1	The gallbladder is a pear-shaped sac lying on the visceral surface of the right lobe of the liver in a fossa between the right and quadrate lobes (Fig. 4.104). It has: a rounded end (fundus of the gallbladder), which may project from the inferior border of the liver; a major part in the fossa (body of the gallbladder), which may be against the transverse colon and the superior part of the duodenum; and a narrow part (neck of the gallbladder) with mucosal folds forming the spiral fold. The arterial supply to the gallbladder (Fig. 4.106) is the cystic artery from the right hepatic artery (a branch of the hepatic artery proper). The gallbladder receives, concentrates, and stores bile from the liver. The pancreas lies mostly posterior to the stomach (Figs. 4.107 and 4.108). It extends across the posterior abdominal wall from the duodenum, on the right, to the spleen, on the left.

1	The pancreas lies mostly posterior to the stomach (Figs. 4.107 and 4.108). It extends across the posterior abdominal wall from the duodenum, on the right, to the spleen, on the left. The pancreas is (secondarily) retroperitoneal except for a small part of its tail and consists of a head, uncinate process, neck, body, and tail. The head of the pancreas lies within the C-shaped concavity of the duodenum. Projecting from the lower part of the head is the uncinate process, which passes posterior to the superior mesenteric vessels. The neck of the pancreas is anterior to the superior mesenteric vessels. Posterior to the neck of the pancreas, the superior mesenteric and splenic veins join to form the portal vein. The body of the pancreas is elongate and extends from the neck to the tail of the pancreas. The tail of the pancreas passes between layers of the splenorenal ligament.

1	The body of the pancreas is elongate and extends from the neck to the tail of the pancreas. The tail of the pancreas passes between layers of the splenorenal ligament. The pancreatic duct begins in the tail of the pancreas (Fig. 4.109). It passes to the right through the body of the pancreas and, after entering the head of the pancreas, turns inferiorly. In the lower part of the head of the pancreas, the pancreatic duct joins the bile duct. The joining of these two structures forms the hepatopancreatic ampulla (ampulla of Vater), which enters the descending (second) part of the duodenum at the major duodenal papilla. Surrounding the ampulla is the sphincter of ampulla (sphincter of Oddi), which is a collection of smooth muscles. The accessory pancreatic duct empties into the duodenum just above the major duodenal papilla at the minor duodenal papilla (Fig. 4.109). If the accessory duct is followed from the minor papilla into the head of the pancreas, a branch point is discovered:

1	One branch continues to the left, through the head of the pancreas, and may connect with the pancreatic duct at the point where it turns inferiorly. A second branch descends into the lower part of the head of the pancreas, anterior to the pancreatic duct, and ends in the uncinate process. The main and accessory pancreatic ducts usually communicate with each other. The presence of these two ducts reflects the embryological origin of the pancreas from dorsal and ventral buds from the foregut.

1	The arterial supply to the pancreas (Fig. 4.110) includes the: gastroduodenal artery from the common hepatic artery (a branch of the celiac trunk), anterior superior pancreaticoduodenal artery from the gastroduodenal artery, posterior superior pancreaticoduodenal artery from the gastroduodenal artery, dorsal pancreatic artery from the inferior pancreatic artery (a branch of the splenic artery), great pancreatic artery from the inferior pancreatic artery (a branch of the splenic artery), anterior inferior pancreaticoduodenal artery from the inferior pancreaticoduodenal artery (a branch of the superior mesenteric artery), and posterior inferior pancreaticoduodenal artery from the inferior pancreaticoduodenal artery (a branch of the superior mesenteric artery).

1	The duct system for the passage of bile extends from the liver, connects with the gallbladder, and empties into the descending part of the duodenum (Fig. 4.111). The coalescence of ducts begins in the liver parenchyma and continues until the right and left hepatic ducts are formed. These drain the respective lobes of the liver. The two hepatic ducts combine to form the common hepatic duct, which runs near the liver, with the hepatic artery proper and portal vein in the free margin of the lesser omentum. As the common hepatic duct continues to descend, it is joined by the cystic duct from the gallbladder. This completes the formation of the bile duct. At this point, the bile duct lies to the right of the hepatic artery proper and usually to the right of, and anterior to, the portal vein in the free margin of the lesser omentum. The omental foramen is posterior to these structures at this point.

1	The omental foramen is posterior to these structures at this point. The bile duct continues to descend, passing posteriorly to the superior part of the duodenum before joining with the pancreatic duct to enter the descending part of the duodenum at the major duodenal papilla (Fig. 4.111). The spleen develops as part of the vascular system in the part of the dorsal mesentery that suspends the developing stomach from the body wall. In the adult, the spleen lies against the diaphragm, in the area of rib IX to rib X (Fig. 4.112). It is therefore in the left upper quadrant, or left hypochondrium, of the abdomen. The spleen is connected to the: greater curvature of the stomach by the gastrosplenic ligament, which contains the short gastric and gastro-omental vessels; and left kidney by the splenorenal ligament (Fig. 4.113), which contains the splenic vessels. Both these ligaments are parts of the greater omentum.

1	Both these ligaments are parts of the greater omentum. The spleen is surrounded by visceral peritoneum except in the area of the hilum on the medial surface of the spleen (Fig. 4.114). The splenic hilum is the entry point for the splenic vessels, and occasionally the tail of the pancreas reaches this area. The arterial supply to the spleen (Fig. 4.115) is the splenic artery from the celiac trunk. The abdominal aorta begins at the aortic hiatus of the diaphragm, anterior to the lower border of vertebra TXII (Fig. 4.121). It descends through the abdomen, anterior to the vertebral bodies, and by the time it ends at the level of vertebra LIV it is slightly to the left of midline. The terminal branches of the abdominal aorta are the two common iliac arteries. Anterior branches of the abdominal aorta The abdominal aorta has anterior, lateral, and posterior branches as it passes through the abdominal cavity.

1	Anterior branches of the abdominal aorta The abdominal aorta has anterior, lateral, and posterior branches as it passes through the abdominal cavity. The three anterior branches supply the gastrointestinal viscera: the celiac trunk and the superior mesenteric and inferior mesenteric arteries (Fig. 4.121). The primitive gut tube can be divided into foregut, midgut, and hindgut regions. The boundaries of these regions are directly related to the areas of distribution of the three anterior branches of the abdominal aorta (Fig. 4.122). The foregut begins with the abdominal esophagus and ends just inferior to the major duodenal papilla, midway along the descending part of the duodenum. It includes the abdominal esophagus, stomach, duodenum (superior to the major papilla), liver, pancreas, and gallbladder. The spleen also develops in relation to the foregut region. The foregut is supplied by the celiac trunk.

1	The midgut begins just inferior to the major duodenal papilla, in the descending part of the duodenum, and ends at the junction between the proximal two-thirds and distal one-third of the transverse colon. It includes the duodenum (inferior to the major duodenal papilla), jejunum, ileum, cecum, appendix, ascending colon, and right two-thirds of the transverse colon. The midgut is supplied by the superior mesenteric artery (Fig. 4.122). The hindgut begins just before the left colic flexure (the junction between the proximal two-thirds and distal one-third of the transverse colon) and ends midway through the anal canal. It includes the left one-third of the transverse colon, descending colon, sigmoid colon, rectum, and upper part of the anal canal. The hindgut is supplied by the inferior mesenteric artery (Fig. 4.122).

1	The celiac trunk is the anterior branch of the abdominal aorta supplying the foregut. It arises from the abdominal aorta immediately below the aortic hiatus of the diaphragm (Fig. 4.123), anterior to the upper part of vertebra LI. It immediately divides into the left gastric, splenic, and common hepatic arteries. The left gastric artery is the smallest branch of the celiac trunk. It ascends to the cardioesophageal junction and sends esophageal branches upward to the abdominal part of the esophagus (Fig. 4.123). Some of these branches continue through the esophageal hiatus of the diaphragm and anastomose with esophageal branches from the thoracic aorta. The left gastric artery itself turns to the right and descends along the lesser curvature of the stomach in the lesser omentum. It supplies both surfaces of the stomach in this area and anastomoses with the right gastric artery.

1	The splenic artery, the largest branch of the celiac trunk, takes a tortuous course to the left along the superior border of the pancreas (Fig. 4.123). It travels in the splenorenal ligament and divides into numerous branches, which enter the hilum of the spleen. As the splenic artery passes along the superior border of the pancreas, it gives off numerous small branches to supply the neck, body, and tail of the pancreas (Fig. 4.124). Approaching the spleen, the splenic artery gives off short gastric arteries, which pass through the gastrosplenic ligament to supply the fundus of the stomach. It also gives off the left gastro-omental artery, which runs to the right along the greater curvature of the stomach, and anastomoses with the right gastro-omental artery. The common hepatic artery is a medium-sized branch of the celiac trunk that runs to the right and divides into its two terminal branches, the hepatic artery proper and the gastroduodenal artery (Figs. 4.123 and 4.124).

1	The hepatic artery proper ascends toward the liver in the free edge of the lesser omentum. It runs to the left of the bile duct and anterior to the portal vein, and divides into the right and left hepatic arteries near the porta hepatis (Fig. 4.125). As the right hepatic artery nears the liver, it gives off the cystic artery to the gallbladder. The right gastric artery often originates from the hepatic artery proper but it can also arise from the common hepatic artery or from the left hepatic, gastroduodenal, or supraduodenal arteries. It courses to the left and ascends along the lesser curvature of the stomach in the lesser omentum, supplies adjacent areas of the stomach, and anastomoses with the left gastric artery.

1	The gastroduodenal artery may give off the supraduodenal artery and does give off the posterior superior pancreaticoduodenal artery near the upper border of the superior part of the duodenum. After these branch the gastroduodenal artery continues descending posterior to the superior part of the duodenum. Reaching the lower border of the superior part of the duodenum, the gastroduodenal artery divides into its terminal branches, the right gastro-omental artery and the anterior superior pancreaticoduodenal artery (Fig. 4.124). The right gastro-omental artery passes to the left, along the greater curvature of the stomach, eventually anastomosing with the left gastro-omental artery from the splenic artery. The right gastro-omental artery sends branches to both surfaces of the stomach and additional branches descend into the greater omentum.

1	The anterior superior pancreaticoduodenal artery descends and, along with the posterior superior pancreaticoduodenal artery, supplies the head of the pancreas and the duodenum (Fig. 4.124). These vessels eventually anastomose with the anterior and posterior branches of the inferior pancreaticoduodenal artery. The superior mesenteric artery is the anterior branch of the abdominal aorta supplying the midgut. It arises from the abdominal aorta immediately below the celiac artery (Fig. 4.126), anterior to the lower part of vertebra LI.

1	The superior mesenteric artery is crossed anteriorly by the splenic vein and the neck of the pancreas. Posterior to the artery are the left renal vein, the uncinate process of the pancreas, and the inferior part of the duodenum. After giving off its first branch (the inferior pancreaticoduodenal artery), the superior mesenteric artery gives off jejunal and ileal arteries on its left (Fig. 4.126). Branching from the right side of the main trunk of the superior mesenteric artery are three vessels—the middle colic, right colic, and ileocolic arteries—which supply the terminal ileum, cecum, ascending colon, and two-thirds of the transverse colon.

1	The inferior pancreaticoduodenal artery is the first branch of the superior mesenteric artery. It divides immediately into anterior and posterior branches, which ascend on the corresponding sides of the head of the pancreas. Superiorly, these arteries anastomose with anterior and posterior superior pancreaticoduodenal arteries (see Figs. 4.125 and 4.126). This arterial network supplies the head and uncinate process of the pancreas and the duodenum. Distal to the inferior pancreaticoduodenal artery, the superior mesenteric artery gives off numerous branches. Arising on the left is a large number of jejunal and ileal arteries supplying the jejunum and most of the ileum (Fig. 4.127). These branches leave the main trunk of the artery, pass between two layers of the mesentery, and form anastomosing arches or arcades as they pass outward to supply the small intestine. The number of arterial arcades increases distally along the gut.

1	There may be single and then double arcades in the area of the jejunum, with a continued increase in the number of arcades moving into and through the area of the ileum. Extending from the terminal arcade are vasa recta (straight arteries), which provide the final direct vascular supply to the walls of the small intestine. The vasa recta supplying the jejunum are usually long and close together, forming narrow windows visible in the mesentery. The vasa recta supplying the ileum are generally short and far apart, forming low broad windows.

1	The middle colic artery is the first of the three branches from the right side of the main trunk of the superior mesenteric artery (Fig. 4.127). Arising as the superior mesenteric artery emerges from beneath the pancreas, the middle colic artery enters the transverse mesocolon and divides into right and left branches. The right branch anastomoses with the right colic artery while the left branch anastomoses with the left colic artery, which is a branch of the inferior mesenteric artery.

1	Continuing distally along the main trunk of the superior mesenteric artery, the right colic artery is the second of the three branches from the right side of the main trunk of the superior mesenteric artery (Fig. 4.126). It is an inconsistent branch, and passes to the right in a retroperitoneal position to supply the ascending colon. Nearing the colon, it divides into a descending branch, which anastomoses with the ileocolic artery, and an ascending branch, which anastomoses with the middle colic artery. The final branch arising from the right side of the superior mesenteric artery is the ileocolic artery (Fig. 4.127). This passes downward and to the right toward the right iliac fossa where it divides into superior and inferior branches: The superior branch passes upward along the ascending colon to anastomose with the right colic artery. The inferior branch continues toward the ileocolic junction, dividing into colic, cecal, appendicular, and ileal branches (Fig. 4.127).

1	The inferior branch continues toward the ileocolic junction, dividing into colic, cecal, appendicular, and ileal branches (Fig. 4.127). The specific pattern of distribution and origin of these branches is variable: The colic branch crosses to the ascending colon and passes upward to supply the first part of the ascending colon. Anterior and posterior cecal branches, arising either as a common trunk or as separate branches, supply corresponding sides of the cecum. The appendicular branch enters the free margin of and supplies the mesoappendix and the appendix. The ileal branch passes to the left and ascends to supply the final part of the ileum before anastomosing with the superior mesenteric artery.

1	The ileal branch passes to the left and ascends to supply the final part of the ileum before anastomosing with the superior mesenteric artery. The inferior mesenteric artery is the anterior branch of the abdominal aorta that supplies the hindgut. It is the smallest of the three anterior branches of the abdominal aorta and arises anterior to the body of vertebra LIII. Initially, the inferior mesenteric artery descends anteriorly to the aorta and then passes to the left as it continues inferiorly (Fig. 4.128). Its branches include the left colic artery, several sigmoid arteries, and the superior rectal artery. The left colic artery is the first branch of the inferior mesenteric artery (Fig. 4.128). It ascends retroperitoneally, dividing into ascending and descending branches:

1	The left colic artery is the first branch of the inferior mesenteric artery (Fig. 4.128). It ascends retroperitoneally, dividing into ascending and descending branches: The ascending branch passes anteriorly to the left kidney, then enters the transverse mesocolon, and passes superiorly to supply the upper part of the descending colon and the distal part of the transverse colon; it anastomoses with branches of the middle colic artery. The descending branch passes inferiorly, supplying the lower part of the descending colon, and anastomoses with the first sigmoid artery. The sigmoid arteries consist of two to four branches, which descend to the left, in the sigmoid mesocolon, to supply the lowest part of the descending colon and the sigmoid colon (Fig. 4.128). These branches anastomose superiorly with branches from the left colic artery and inferiorly with branches from the superior rectal artery.

1	The terminal branch of the inferior mesenteric artery is the superior rectal artery (Fig. 4.128). This vessel descends into the pelvic cavity in the sigmoid mesocolon, crossing the left common iliac vessels. Opposite vertebra SIII, the superior rectal artery divides. The two terminal branches descend on each side of the rectum, dividing into smaller branches in the wall of the rectum. These smaller branches continue inferiorly to the level of the internal anal sphincter, anastomosing along the way with branches from the middle rectal arteries (from the internal iliac artery) and the inferior rectal arteries (from the internal pudendal artery).

1	Venous drainage of the spleen, pancreas, gallbladder, and abdominal part of the gastrointestinal tract, except for the inferior part of the rectum, is through the portal system of veins, which deliver blood from these structures to the liver. Once blood passes through the hepatic sinusoids, it passes through progressively larger veins until it enters the hepatic veins, which return the venous blood to the inferior vena cava just inferior to the diaphragm. The portal vein is the final common pathway for the transport of venous blood from the spleen, pancreas, gallbladder, and abdominal part of the gastrointestinal tract. It is formed by the union of the splenic vein and the superior mesenteric vein posterior to the neck of the pancreas at the level of vertebra LII (Fig. 4.131).

1	Ascending toward the liver, the portal vein passes posterior to the superior part of the duodenum and enters the right margin of the lesser omentum. As it passes through this part of the lesser omentum, it is anterior to the omental foramen and posterior to both the bile duct, which is slightly to its right, and the hepatic artery proper, which is slightly to its left (see Fig. 4.125, p. 347). On approaching the liver, the portal vein divides into right and left branches, which enter the liver parenchyma. Tributaries to the portal vein include: right and left gastric veins draining the lesser curvature of the stomach and abdominal esophagus, cystic veins from the gallbladder, and the para-umbilical veins, which are associated with the obliterated umbilical vein and connect to veins on the anterior abdominal wall (Fig. 4.133 on p. 357).

1	The splenic vein forms from numerous smaller vessels leaving the hilum of the spleen (Fig. 4.132). It passes to the right, passing through the splenorenal ligament with the splenic artery and the tail of the pancreas. Continuing to the right, the large, straight splenic vein is in contact with the body of the pancreas as it crosses the posterior abdominal wall. Posterior to the neck of the pancreas, the splenic vein joins the superior mesenteric vein to form the portal vein. Tributaries to the splenic vein include: short gastric veins from the fundus and left part of the greater curvature of the stomach, the left gastro-omental vein from the greater curvature of the stomach, pancreatic veins draining the body and tail of the pancreas, and usually the inferior mesenteric vein.

1	The superior mesenteric vein drains blood from the small intestine, cecum, ascending colon, and transverse colon (Fig. 4.132). It begins in the right iliac fossa as veins draining the terminal ileum, cecum, and appendix join, and ascends in the mesentery to the right of the superior mesenteric artery. Posterior to the neck of the pancreas, the superior mesenteric vein joins the splenic vein to form the portal vein.

1	Posterior to the neck of the pancreas, the superior mesenteric vein joins the splenic vein to form the portal vein. As a corresponding vein accompanies each branch of the superior mesenteric artery, tributaries to the superior mesenteric vein include jejunal, ileal, ileocolic, right colic, and middle colic veins. Additional tributaries include: the right gastro-omental vein, draining the right part of the greater curvature of the stomach, and the anterior and posterior inferior pancreaticoduodenal veins, which pass alongside the arteries of the same name; the anterior superior pancreaticoduodenal vein usually empties into the right gastro-omental vein, and the posterior superior pancreaticoduodenal vein usually empties directly into the portal vein.

1	The inferior mesenteric vein drains blood from the rectum, sigmoid colon, descending colon, and splenic flexure (Fig. 4.132). It begins as the superior rectal vein and ascends, receiving tributaries from the sigmoid veins and the left colic vein. All these veins accompany arteries of the same name. Continuing to ascend, the inferior mesenteric vein passes posterior to the body of the pancreas and usually joins the splenic vein. Occasionally, it ends at the junction of the splenic and superior mesenteric veins or joins the superior mesenteric vein.

1	Lymphatic drainage of the abdominal part of the gastrointestinal tract, as low as the inferior part of the rectum, as well as the spleen, pancreas, gallbladder, and liver, is through vessels and nodes that eventually end in large collections of pre-aortic lymph nodes at the origins of the three anterior branches of the abdominal aorta, which supply these structures. These collections are therefore referred to as the celiac, superior mesenteric, and inferior mesenteric groups of pre-aortic lymph nodes. Lymph from viscera is supplied by three routes: The celiac trunk (i.e., structures that are part of the abdominal foregut) drains to pre-aortic nodes near the origin of the celiac trunk (Fig. 4.134)—these celiac nodes also receive lymph from the superior mesenteric and inferior mesenteric groups of pre-aortic nodes, and lymph from the celiac nodes enters the cisterna chyli.

1	The superior mesenteric artery (i.e., structures that are part of the abdominal midgut) drains to pre-aortic nodes near the origin of the superior mesenteric artery (Fig. 4.134)—these superior mesenteric nodes also receive lymph from the inferior mesenteric groups of pre-aortic nodes, and lymph from the superior mesenteric nodes drains to the celiac nodes. The inferior mesenteric artery (i.e., structures that are part of the abdominal hindgut) drains to pre-aortic nodes near the origin of the inferior mesenteric artery (Fig. 4.134), and lymph from the inferior mesenteric nodes drains to the superior mesenteric nodes. Abdominal viscera are innervated by both extrinsic and intrinsic components of the nervous system: Extrinsic innervation involves receiving motor impulses from, and sending sensory information to, the central nervous system.

1	Extrinsic innervation involves receiving motor impulses from, and sending sensory information to, the central nervous system. Intrinsic innervation involves the regulation of digestive tract activities by a generally self-sufficient network of sensory and motor neurons (the enteric nervous system). Abdominal viscera receiving extrinsic innervation include the abdominal part of the gastrointestinal tract, the spleen, the pancreas, the gallbladder, and the liver. These viscera send sensory information back to the central nervous system through visceral afferent fibers and receive motor impulses from the central nervous system through visceral efferent fibers. The visceral efferent fibers are part of the sympathetic and parasympathetic parts of the autonomic division of the peripheral nervous system.

1	The visceral efferent fibers are part of the sympathetic and parasympathetic parts of the autonomic division of the peripheral nervous system. Structural components serving as conduits for these afferent and efferent fibers include posterior and anterior roots of the spinal cord, respectively, spinal nerves, anterior rami, white and gray rami communicantes, the sympathetic trunks, splanchnic nerves carrying sympathetic fibers (thoracic, lumbar, and sacral), parasympathetic fibers (pelvic), the prevertebral plexus and related ganglia, and the vagus nerves [X]. The enteric nervous system consists of motor and sensory neurons in two interconnected plexuses in the walls of the gastrointestinal tract. These neurons control the coordinated contraction and relaxation of intestinal smooth muscle and regulate gastric secretion and blood flow.

1	The sympathetic trunks are two parallel nerve cords extending on either side of the vertebral column from the base of the skull to the coccyx (Fig. 4.135). As they pass through the neck, they lie posterior to the carotid sheath. In the upper thorax, they are anterior to the necks of the ribs, while in the lower thorax they are on the lateral aspect of the vertebral bodies. In the abdomen, they are anterolateral to the lumbar vertebral bodies and, continuing into the pelvis, they are anterior to the sacrum. The two sympathetic trunks come together anterior to the coccyx to form the ganglion impar.

1	Throughout the extent of the sympathetic trunks, small raised areas are visible. These collections of neuronal cell bodies outside the CNS are the paravertebral sympathetic ganglia. There are usually: three ganglia in the cervical region, eleven or twelve ganglia in the thoracic region, four ganglia in the lumbar region, four or five ganglia in the sacral region, and the ganglion impar anterior to the coccyx (Fig. 4.135). The ganglia and trunks are connected to adjacent spinal nerves by gray rami communicantes throughout the length of the sympathetic trunk and by white rami communicantes in the thoracic and upper lumbar parts of the trunk (T1 to L2). Neuronal fibers found in the sympathetic trunks include preganglionic and postganglionic sympathetic fibers and visceral afferent fibers.

1	The splanchnic nerves are important components in the innervation of the abdominal viscera. They pass from the sympathetic trunk or sympathetic ganglia associated with the trunk, to the prevertebral plexus and ganglia anterior to the abdominal aorta. There are two different types of splanchnic nerves, depending on the type of visceral efferent fiber they are carrying: The thoracic, lumbar, and sacral splanchnic nerves carry preganglionic sympathetic fibers from the sympathetic trunk to ganglia in the prevertebral plexus, and also visceral afferent fibers. The pelvic splanchnic nerves carry preganglionic parasympathetic fibers from anterior rami of S2, S3, and S4 spinal nerves to an extension of the prevertebral plexus in the pelvis (the inferior hypogastric plexus or pelvic plexus).

1	Three thoracic splanchnic nerves pass from sympathetic ganglia along the sympathetic trunk in the thorax to the prevertebral plexus and ganglia associated with the abdominal aorta in the abdomen (Fig. 4.136): The greater splanchnic nerve arises from the fifth to the ninth (or tenth) thoracic ganglia and travels to the celiac ganglion in the abdomen (a prevertebral ganglion associated with the celiac trunk). The lesser splanchnic nerve arises from the ninth and tenth (or tenth and eleventh) thoracic ganglia and travels to the aorticorenal ganglion. The least splanchnic nerve, when present, arises from the twelfth thoracic ganglion and travels to the renal plexus. There are usually two to four lumbar splanchnic nerves, which pass from the lumbar part of the sympathetic trunk or associated ganglia and enter the prevertebral plexus (Fig. 4.136).

1	There are usually two to four lumbar splanchnic nerves, which pass from the lumbar part of the sympathetic trunk or associated ganglia and enter the prevertebral plexus (Fig. 4.136). Similarly, the sacral splanchnic nerves pass from the sacral part of the sympathetic trunk or associated ganglia and enter the inferior hypogastric plexus, which is an extension of the prevertebral plexus into the pelvis.

1	The pelvic splanchnic nerves (parasympathetic root) are unique. They are the only splanchnic nerves that carry parasympathetic fibers. In other words, they do not originate from the sympathetic trunks. Rather, they originate directly from the anterior rami of S2 to S4. Preganglionic parasympathetic fibers originating in the sacral spinal cord pass from the S2 to S4 spinal nerves to the inferior hypogastric plexus (Fig. 4.136). Once in this plexus, some of these fibers pass upward, enter the abdominal prevertebral plexus, and distribute with the arteries supplying the hindgut. This provides the pathway for innervation of the distal one-third of the transverse colon, the descending colon, and the sigmoid colon by preganglionic parasympathetic fibers.

1	The abdominal prevertebral plexus is a collection of nerve fibers that surrounds the abdominal aorta and is continuous onto its major branches. Scattered throughout the length of the abdominal prevertebral plexus are cell bodies of postganglionic sympathetic fibers. Some of these cell bodies are organized into distinct ganglia, while others are more random in their distribution. The ganglia are usually associated with specific branches of the abdominal aorta and named after these branches. The three major divisions of the abdominal prevertebral plexus and associated ganglia are the celiac, aortic, and superior hypogastric plexuses (Fig. 4.137). The celiac plexus is the large accumulation of nerve fibers and ganglia associated with the roots of the celiac trunk and superior mesenteric artery immediately below the aortic hiatus of the diaphragm. Ganglia associated with the celiac plexus include two celiac ganglia, a single superior mesenteric ganglion, and two aorticorenal ganglia.

1	The aortic plexus consists of nerve fibers and associated ganglia on the anterior and lateral surfaces of the abdominal aorta extending from just below the origin of the superior mesenteric artery to the bifurcation of the aorta into the two common iliac arteries. The major ganglion in this plexus is the inferior mesenteric ganglion at the root of the inferior mesenteric artery. The superior hypogastric plexus contains numerous small ganglia and is the final part of the abdominal prevertebral plexus before the prevertebral plexus continues into the pelvic cavity.

1	The superior hypogastric plexus contains numerous small ganglia and is the final part of the abdominal prevertebral plexus before the prevertebral plexus continues into the pelvic cavity. Each of these major plexuses gives origin to a number of secondary plexuses, which may also contain small ganglia. These plexuses are usually named after the vessels with which they are associated. For example, the celiac plexus is usually described as giving origin to the superior mesenteric plexus and the renal plexus, as well as other plexuses that extend out along the various branches of the celiac trunk. Similarly, the aortic plexus has secondary plexuses consisting of the inferior mesenteric plexus, the spermatic plexus, and the external iliac plexus. Inferiorly, the superior hypogastric plexus divides into the hypogastric nerves, which descend into the pelvis and contribute to the formation of the inferior hypogastric or pelvic plexus (Fig. 4.137).

1	The abdominal prevertebral plexus receives: preganglionic parasympathetic and visceral afferent fibers from the vagus nerves [X], preganglionic sympathetic and visceral afferent fibers from the thoracic and lumbar splanchnic nerves, and preganglionic parasympathetic fibers from the pelvic splanchnic nerves. Parasympathetic innervation of the abdominal part of the gastrointestinal tract and of the spleen, pancreas, gallbladder, and liver is from two sources—the vagus nerves [X] and the pelvic splanchnic nerves. The vagus nerves [X] enter the abdomen associated with the esophagus as the esophagus passes through the diaphragm (Fig. 4.138) and provide parasympathetic innervation to the foregut and midgut.

1	The vagus nerves [X] enter the abdomen associated with the esophagus as the esophagus passes through the diaphragm (Fig. 4.138) and provide parasympathetic innervation to the foregut and midgut. After entering the abdomen as the anterior and posterior vagal trunks, they send branches to the abdominal prevertebral plexus. These branches contain preganglionic parasympathetic fibers and visceral afferent fibers, which are distributed with the other components of the prevertebral plexus along the branches of the abdominal aorta. The pelvic splanchnic nerves, carrying preganglionic parasympathetic fibers from S2 to S4 spinal cord levels, enter the inferior hypogastric plexus in the pelvis. Some of these fibers move upward into the inferior mesenteric part of the prevertebral plexus in the abdomen (Fig. 4.138). Once there, these fibers are distributed with branches of the inferior mesenteric artery and provide parasympathetic innervation to the hindgut.

1	The enteric system is a division of the visceral part of the nervous system and is a local neuronal circuit in the wall of the gastrointestinal tract. It consists of motor and sensory neurons organized into two interconnected plexuses (the myenteric and submucosal plexuses) between the layers of the gastrointestinal wall, and the associated nerve fibers that pass between the plexuses and from the plexuses to the adjacent tissue (Fig. 4.139). The enteric system regulates and coordinates numerous gastrointestinal tract activities, including gastric secretory activity, gastrointestinal blood flow, and the contraction and relaxation cycles of smooth muscle (peristalsis). Although the enteric system is generally independent of the central nervous system, it does receive input from postganglionic sympathetic and preganglionic parasympathetic neurons that modifies its activities. Sympathetic innervation of the stomach The pathway of sympathetic innervation of the stomach is as follows:

1	Sympathetic innervation of the stomach The pathway of sympathetic innervation of the stomach is as follows: A preganglionic sympathetic fiber originating at the T6 level of the spinal cord enters an anterior root to leave the spinal cord. At the level of the intervertebral foramen, the anterior root (which contains the preganglionic fiber) and a posterior root join to form a spinal nerve. Outside the vertebral column, the preganglionic fiber leaves the anterior ramus of the spinal nerve through the white ramus communicans. The white ramus communicans, containing the preganglionic fiber, connects to the sympathetic trunk. Entering the sympathetic trunk, the preganglionic fiber does not synapse but passes through the trunk and enters the greater splanchnic nerve. The greater splanchnic nerve passes through the crura of the diaphragm and enters the celiac ganglion. In the celiac ganglion, the preganglionic fiber synapses with a postganglionic neuron.

1	The greater splanchnic nerve passes through the crura of the diaphragm and enters the celiac ganglion. In the celiac ganglion, the preganglionic fiber synapses with a postganglionic neuron. The postganglionic fiber joins the plexus of nerve fibers surrounding the celiac trunk and continues along its branches. The postganglionic fiber travels through the plexus of nerves accompanying the branches of the celiac trunk supplying the stomach and eventually reaches its point of distribution. This input from the sympathetic system may modify the activities of the gastrointestinal tract controlled by the enteric nervous system.

1	This input from the sympathetic system may modify the activities of the gastrointestinal tract controlled by the enteric nervous system. The posterior abdominal region is posterior to the abdominal part of the gastrointestinal tract, the spleen, and the pancreas (Fig. 4.140). This area, bounded by bones and muscles making up the posterior abdominal wall, contains numerous structures that not only are directly involved in the activities of the abdominal contents but also use this area as a conduit between body regions. Examples include the abdominal aorta and its associated nerve plexuses, the inferior vena cava, the sympathetic trunks, and lymphatics. There are also structures originating in this area that are critical to the normal function of other regions of the body (i.e., the lumbar plexus of nerves), and there are organs that associate with this area during development and remain in it in the adult (i.e., the kidneys and suprarenal glands). Lumbar vertebrae and the sacrum

1	Lumbar vertebrae and the sacrum Projecting into the midline of the posterior abdominal area are the bodies of the five lumbar vertebrae (Fig. 4.141). The prominence of these structures in this region is due to the secondary curvature (a forward convexity) of the lumbar part of the vertebral column. The lumbar vertebrae can be distinguished from cervical and thoracic vertebrae because of their size. They are much larger than any other vertebrae in any other region. The vertebral bodies are massive and progressively increase in size from vertebra LI to LV. The pedicles are short and stocky, the transverse processes are long and slender, and the spinous processes are large and stubby. The articular processes are large and oriented medially and laterally, which promotes flexion and extension in this part of the vertebral column. Between each lumbar vertebra is an intervertebral disc, which completes this part of the midline boundary of the posterior abdominal wall.

1	Between each lumbar vertebra is an intervertebral disc, which completes this part of the midline boundary of the posterior abdominal wall. The midline boundary of the posterior abdominal wall, inferior to the lumbar vertebrae, consists of the upper margin of the sacrum (Fig. 4.141). The sacrum is formed by the fusion of the five sacral vertebrae into a single, wedge-shaped bony structure that is broad superiorly and narrows inferiorly. Its concave anterior surface and its convex posterior surface contain anterior and posterior sacral foramina for the anterior and posterior rami of spinal nerves to pass through. The ilia, which are components of each pelvic bone, attach laterally to the sacrum at the sacro-iliac joints (Fig. 4.141). The upper part of each ilium expands outward into a thin wing-like area (the iliac fossa). The medial side of this region of each iliac bone, and the related muscles, are components of the posterior abdominal wall.

1	Superiorly, ribs XI and XII complete the bony framework of the posterior abdominal wall (Fig. 4.141). These ribs are unique in that they do not articulate with the sternum or other ribs, they have a single articular facet on their heads, and they do not have necks or tubercles. Rib XI is posterior to the superior part of the left kidney, and rib XII is posterior to the superior part of both kidneys. Also, rib XII serves as a point of attachment for numerous muscles and ligaments. Muscles forming the medial, lateral, inferior, and superior boundaries of the posterior abdominal region fill in the bony framework of the posterior abdominal wall (Table 4.2). Medially are the psoas major and minor muscles, laterally is the quadratus lumborum muscle, inferiorly is the iliacus muscle, and superiorly is the diaphragm (Figs. 4.142 and 4.143).

1	Medially, the psoas major muscles cover the anterolateral surface of the bodies of the lumbar vertebrae, filling in the space between the vertebral bodies and the transverse processes (Fig. 4.142). Each of these muscles arises from the bodies of vertebra TXII and all five lumbar vertebrae, from the intervertebral discs between each vertebra, and from the transverse processes of the lumbar vertebrae. Passing inferiorly along the pelvic brim, each muscle continues into the anterior thigh, under the inguinal ligament, to attach to the lesser trochanter of the femur. The psoas major muscle flexes the thigh at the hip joint when the trunk is stabilized and flexes the trunk against gravity when the body is supine. It is innervated by anterior rami of nerves L1 to L3.

1	Associated with the psoas major muscle is the psoas minor muscle, which is sometimes absent. Lying on the surface of the psoas major when present, this slender muscle arises from vertebrae TXII and LI and the intervening intervertebral disc; its long tendon inserts into the pectineal line of the pelvic brim and the iliopubic eminence. The psoas minor is a weak flexor of the lumbar vertebral column and is innervated by the anterior ramus of nerve L1. Laterally, the quadratus lumborum muscles fill the space between rib XII and the iliac crest on both sides of the vertebral column (Fig. 4.142). They are overlapped medially by the psoas major muscles; along their lateral borders are the transversus abdominis muscles.

1	Each quadratus lumborum muscle arises from the transverse process of vertebra LV, the iliolumbar ligament, and the adjoining part of the iliac crest. The muscle attaches superiorly to the transverse process of the first four lumbar vertebrae and the inferior border of rib XII. The quadratus lumborum muscles depress and stabilize the twelfth ribs and contribute to lateral bending of the trunk. Acting together, the muscles may extend the lumbar part of the vertebral column. They are innervated by anterior rami of T12 and L1 to L4 spinal nerves. Inferiorly, an iliacus muscle fills the iliac fossa on each side (Fig. 4.142). From this expansive origin covering the iliac fossa, the muscle passes inferiorly, joins with the psoas major muscle, and attaches to the lesser trochanter of the femur. As they pass into the thigh, these combined muscles are referred to as the iliopsoas muscle.

1	Like the psoas major muscle, the iliacus flexes the thigh at the hip joint when the trunk is stabilized and flexes the trunk against gravity when the body is supine. It is innervated by branches of the femoral nerve. Superiorly, the diaphragm forms the boundary of the posterior abdominal region. This musculotendinous sheet also separates the abdominal cavity from the thoracic cavity. Structurally, the diaphragm consists of a central tendinous part into which the circumferentially arranged muscle fibers attach (Fig. 4.143). The diaphragm is anchored to the lumbar vertebrae by musculotendinous crura, which blend with the anterior longitudinal ligament of the vertebral column: The right crus is the longest and broadest of the crura and is attached to the bodies of vertebrae LI to LIII and the intervening intervertebral discs (Fig. 4.144). Similarly, the left crus is attached to vertebrae LI and LII and the associated intervertebral disc.

1	Similarly, the left crus is attached to vertebrae LI and LII and the associated intervertebral disc. The crura are connected across the midline by a tendinous arch (the median arcuate ligament), which passes anterior to the aorta (Fig. 4.144). Lateral to the crura, a second tendinous arch is formed by the fascia covering the upper part of the psoas major muscle. This is the medial arcuate ligament, which is attached medially to the sides of vertebrae LI and LII and laterally to the transverse process of vertebra LI (Fig. 4.144). A third tendinous arch, the lateral arcuate ligament, is formed by a thickening in the fascia that covers the quadratus lumborum. It is attached medially to the transverse process of vertebra LI and laterally to rib XII (Fig. 4.144). The medial and lateral arcuate ligaments serve as points of origin for some of the muscular components of the diaphragm. Structures passing through or around the diaphragm

1	The medial and lateral arcuate ligaments serve as points of origin for some of the muscular components of the diaphragm. Structures passing through or around the diaphragm Numerous structures pass through or around the diaphragm (Fig. 4.143): The aorta passes posterior to the diaphragm and anterior to the vertebral bodies at the lower level of vertebra TXII; it is between the two crura of the diaphragm and posterior to the median arcuate ligament, just to the left of midline. Accompanying the aorta through the aortic hiatus is the thoracic duct and, sometimes, the azygos vein. The esophagus passes through the musculature of the right crus of the diaphragm at the level of vertebra TX, just to the left of the aortic hiatus. Passing through the esophageal hiatus with the esophagus are the anterior and posterior vagal trunks, the esophageal branches of the left gastric artery and vein, and a few lymphatic vessels.

1	Passing through the esophageal hiatus with the esophagus are the anterior and posterior vagal trunks, the esophageal branches of the left gastric artery and vein, and a few lymphatic vessels. The third large opening in the diaphragm is the caval opening, through which the inferior vena cava passes from the abdominal cavity to the thoracic cavity (Fig. 4.143) at approximately vertebra TVIII in the central tendinous part of the diaphragm. Accompanying the inferior vena cava through the caval opening is the right phrenic nerve. The left phrenic nerve passes through the muscular part of the diaphragm just anterior to the central tendon on the left side. Additional structures pass through small openings either in or just outside the diaphragm as they pass from the thoracic cavity to the abdominal cavity (Fig. 4.143): The greater, lesser, and least (when present) splanchnic nerves pass through the crura, on either side. The hemi-azygos vein passes through the left crus.

1	The greater, lesser, and least (when present) splanchnic nerves pass through the crura, on either side. The hemi-azygos vein passes through the left crus. Passing posterior to the medial arcuate ligament, on either side, are the sympathetic trunks. Passing anterior to the diaphragm, just deep to the ribs, are the superior epigastric vessels. Other vessels and nerves (i.e., the musculophrenic vessels and intercostal nerves) also pass through the diaphragm at various points. The classic appearance of the right and left domes of the diaphragm is caused by the underlying abdominal contents pushing these lateral areas upward, and by the fibrous pericardium, which is attached centrally, causing a flattening of the diaphragm in this area (Fig. 4.145).

1	The domes are produced by: the liver on the right, with some contribution from the right kidney and the right suprarenal gland, and the fundus of the stomach and spleen on the left, with contributions from the left kidney and the left suprarenal gland. Although the height of these domes varies during breathing, a reasonable estimate in normal expiration places the left dome at the fifth intercostal space and the right dome at rib V. This is important to remember when percussing the thorax. During inspiration, the muscular part of the diaphragm contracts, causing the central tendon of the diaphragm to be drawn inferiorly. This results in some flattening of the domes, enlargement of the thoracic cavity, and a reduction in intrathoracic pressure. The physiological effect of these changes is that air enters the lungs and venous return to the heart is enhanced. There is blood supply to the diaphragm on its superior and inferior surfaces:

1	There is blood supply to the diaphragm on its superior and inferior surfaces: Superiorly, the musculophrenic and pericardiacophrenic arteries, both branches of the internal thoracic artery, and the superior phrenic artery, a branch of the thoracic aorta, supply the diaphragm. Inferiorly, the inferior phrenic arteries, branches of the abdominal aorta, supply the diaphragm (see Fig. 4.143). Venous drainage is through companion veins to these arteries. Innervation of the diaphragm is primarily by the phrenic nerves. These nerves, from the C3 to C5 spinal cord levels, provide all motor innervation to the diaphragm and sensory fibers to the central part. They pass through the thoracic cavity, between the mediastinal pleura and the pericardium, to the superior surface of the diaphragm. At this point, the right phrenic nerve accompanies the inferior vena cava through the diaphragm and the left phrenic nerve passes through the diaphragm by itself (see

1	Fig. 4.143). Additional sensory fibers are supplied to the peripheral areas of the diaphragm by intercostal nerves. The bean-shaped kidneys are retroperitoneal in the posterior abdominal region (Fig. 4.149). They lie in the extraperitoneal connective tissue immediately lateral to the vertebral column. In the supine position, the kidneys extend from approximately vertebra TXII superiorly to vertebra LIII inferiorly, with the right kidney somewhat lower than the left because of its relationship with the liver. Although they are similar in size and shape, the left kidney is a longer and more slender organ than the right kidney, and nearer to the midline. Relationships to other structures The anterior surface of the right kidney is related to numerous structures, some of which are separated from the kidney by a layer of peritoneum and some of which are directly against the kidney (Fig. 4.150): A small part of the superior pole is covered by the right suprarenal gland.

1	A small part of the superior pole is covered by the right suprarenal gland. Moving inferiorly, a large part of the rest of the upper part of the anterior surface is against the liver and is separated from it by a layer of peritoneum. Medially, the descending part of the duodenum is retroperitoneal and contacts the kidney. The inferior pole of the kidney, on its lateral side, is directly associated with the right colic flexure and, on its medial side, is covered by a segment of the intraperitoneal small intestine. The anterior surface of the left kidney is also related to numerous structures, some with an intervening layer of peritoneum and some directly against the kidney (Fig. 4.150): A small part of the superior pole, on its medial side, is covered by the left suprarenal gland. The rest of the superior pole is covered by the intraperitoneal stomach and spleen. Moving inferiorly, the retroperitoneal pancreas covers the middle part of the kidney.

1	The rest of the superior pole is covered by the intraperitoneal stomach and spleen. Moving inferiorly, the retroperitoneal pancreas covers the middle part of the kidney. On its lateral side, the lower half of the kidney is covered by the left colic flexure and the beginning of the descending colon, and, on its medial side, by the parts of the intraperitoneal jejunum. Posteriorly, the right and left kidneys are related to similar structures (Fig. 4.151). Superiorly is the diaphragm and inferior to this, moving in a medial to lateral direction, are the psoas major, quadratus lumborum, and transversus abdominis muscles. The superior pole of the right kidney is anterior to rib XII, while the same region of the left kidney is anterior to ribs XI and XII. The pleural sacs and specifically the costodiaphragmatic recesses therefore extend posterior to the kidneys. Also passing posterior to the kidneys are the subcostal vessels and nerves and the iliohypogastric and ilio-inguinal nerves.

1	Also passing posterior to the kidneys are the subcostal vessels and nerves and the iliohypogastric and ilio-inguinal nerves. The kidneys are enclosed in and associated with a unique arrangement of fascia and fat. Immediately outside the renal capsule, there is an accumulation of extraperitoneal fat—the perinephric fat (perirenal fat), which completely surrounds the kidney (Fig. 4.152). Enclosing the perinephric fat is a membranous condensation of the extraperitoneal fascia (the renal fascia). The suprarenal glands are also enclosed in this fascial compartment, usually separated from the kidneys by a thin septum. The renal fascia must be incised in any surgical approach to this organ. At the lateral margins of each kidney, the anterior and posterior layers of the renal fascia fuse (Fig. 4.152). This fused layer may connect with the transversalis fascia on the lateral abdominal wall.

1	Above each suprarenal gland, the anterior and posterior layers of the renal fascia fuse and blend with the fascia that covers the diaphragm. Medially, the anterior layer of the renal fascia continues over the vessels in the hilum and fuses with the connective tissue associated with the abdominal aorta and the inferior vena cava (Fig. 4.152). In some cases, the anterior layer may cross the midline to the opposite side and blend with its companion layer. The posterior layer of the renal fascia passes medially between the kidney and the fascia covering the quadratus lumborum muscle to fuse with the fascia covering the psoas major muscle. Inferiorly, the anterior and posterior layers of the renal fascia enclose the ureters. In addition to perinephric fat and the renal fascia, a final layer of paranephric fat (pararenal fat) completes the fat and fascias associated with the kidney (Fig. 4.152). This fat accumulates posterior and posterolateral to each kidney.

1	Each kidney has a smooth anterior and posterior surface covered by a fibrous capsule, which is easily removable except during disease. On the medial margin of each kidney is the hilum of the kidney, which is a deep vertical slit through which renal vessels, lymphatics, and nerves enter and leave the substance of the kidney (Fig. 4.153). Internally, the hilum is continuous with the renal sinus. Perinephric fat continues into the hilum and sinus and surrounds all structures. Each kidney consists of an outer renal cortex and an inner renal medulla. The renal cortex is a continuous band of pale tissue that completely surrounds the renal medulla. Extensions of the renal cortex (the renal columns) project into the inner aspect of the kidney, dividing the renal medulla into discontinuous aggregations of triangular-shaped tissue (the renal pyramids).

1	The bases of the renal pyramids are directed outward, toward the renal cortex, while the apex of each renal pyramid projects inward, toward the renal sinus. The apical projection (renal papilla) contains the openings of the papillary ducts draining the renal tubules and is surrounded by a minor calyx. The minor calices receive urine from the papillary ducts and represent the proximal parts of the tube that will eventually form the ureter (Fig. 4.153). In the renal sinus, several minor calices unite to form a major calyx, and two or three major calices unite to form the renal pelvis, which is the funnel-shaped superior end of the ureters.

1	A single large renal artery, a lateral branch of the abdominal aorta, supplies each kidney. These vessels usually arise just inferior to the origin of the superior mesenteric artery between vertebrae LI and LII (Fig. 4.154). The left renal artery usually arises a little higher than the right, and the right renal artery is longer and passes posterior to the inferior vena cava. As each renal artery approaches the renal hilum, it divides into anterior and posterior branches, which supply the renal parenchyma. Accessory renal arteries are common. They originate from the lateral aspect of the abdominal aorta, either above or below the primary renal arteries, enter the hilum with the primary arteries or pass directly into the kidney at some other level, and are commonly called extrahilar arteries.

1	Multiple renal veins contribute to the formation of the left and right renal veins, both of which are anterior to the renal arteries (Fig. 4.154A). Importantly, the longer left renal vein crosses the midline anterior to the abdominal aorta and posterior to the superior mesenteric artery and can be compressed by an aneurysm in either of these two vessels (Fig. 4.154B). The lymphatic drainage of each kidney is to the lateral aortic (lumbar) nodes around the origin of the renal artery. The ureters are muscular tubes that transport urine from the kidneys to the bladder. They are continuous superiorly with the renal pelvis, which is a funnel-shaped structure in the renal sinus. The renal pelvis is formed from a condensation of two or three major calices, which in turn are formed by the condensation of several minor calices (see Fig. 4.153). The minor calices surround a renal papilla.

1	The renal pelvis narrows as it passes inferiorly through the hilum of the kidney and becomes continuous with the ureter at the ureteropelvic junction (Fig. 4.155). Inferior to this junction, the ureters descend retroperitoneally on the medial aspect of the psoas major muscle. At the pelvic brim, the ureters cross either the end of the common iliac artery or the beginning of the external iliac artery, enter the pelvic cavity, and continue their journey to the bladder. At three points along their course the ureters are constricted (Fig. 4.155): The first point is at the ureteropelvic junction. The second point is where the ureters cross the common iliac vessels at the pelvic brim. The third point is where the ureters enter the wall of the bladder. Kidney stones can become lodged at these constrictions. The ureters receive arterial branches from adjacent vessels as they pass toward the bladder (Fig. 4.155): The renal arteries supply the upper end.

1	The ureters receive arterial branches from adjacent vessels as they pass toward the bladder (Fig. 4.155): The renal arteries supply the upper end. The middle part may receive branches from the abdominal aorta, the testicular or ovarian arteries, and the common iliac arteries. In the pelvic cavity, the ureters are supplied by one or more arteries from branches of the internal iliac arteries. In all cases, arteries reaching the ureters divide into ascending and descending branches, which form longitudinal anastomoses. Lymphatic drainage of the ureters follows a pattern similar to that of the arterial supply. Lymph from: the upper part of each ureter drains to the lateral aortic (lumbar) nodes, the middle part of each ureter drains to lymph nodes associated with the common iliac vessels, and the inferior part of each ureter drains to lymph nodes associated with the external and internal iliac vessels.

1	Ureteric innervation is from the renal, aortic, superior hypogastric, and inferior hypogastric plexuses through nerves that follow the blood vessels. Visceral efferent fibers come from both sympathetic and parasympathetic sources, whereas visceral afferent fibers return to T11 to L2 spinal cord levels. Ureteric pain, which is usually related to distention of the ureter, is therefore referred to cutaneous areas supplied by T11 to L2 spinal cord levels. These areas would most likely include the posterior and lateral abdominal wall below the ribs and above the iliac crest, the pubic region, the scrotum in males, the labia majora in females, and the proximal anterior aspect of the thigh. The suprarenal glands are associated with the superior pole of each kidney (Fig. 4.163). They consist of an outer cortex and an inner medulla. The right gland is shaped like a pyramid, whereas the left gland is semilunar in shape and the larger of the two.

1	Anterior to the right suprarenal gland is part of the right lobe of the liver and the inferior vena cava, whereas anterior to the left suprarenal gland is part of the stomach, pancreas, and, on occasion, the spleen. Parts of the diaphragm are posterior to both glands. The suprarenal glands are surrounded by the perinephric fat and enclosed in the renal fascia, though a thin septum separates each gland from its associated kidney. The arterial supply to the suprarenal glands is extensive and arises from three primary sources (Fig. 4.163): As the bilateral inferior phrenic arteries pass upward from the abdominal aorta to the diaphragm, they give off multiple branches (superior suprarenal arteries) to the suprarenal glands. A middle branch (middle suprarenal artery) to the suprarenal glands usually arises directly from the abdominal aorta. Inferior branches (inferior suprarenal arteries) from the renal arteries pass upward to the suprarenal glands.

1	Inferior branches (inferior suprarenal arteries) from the renal arteries pass upward to the suprarenal glands. In contrast to this multiple arterial supply is the venous drainage, which usually consists of a single vein leaving the hilum of each gland. On the right side, the right suprarenal vein is short and almost immediately enters the inferior vena cava, while on the left side, the left suprarenal vein passes inferiorly to enter the left renal vein. The suprarenal gland is mainly innervated by preganglionic sympathetic fibers from spinal levels T8-L1 that pass through both the sympathetic trunk and the prevertebral plexus without synapsing. These preganglionic fibers directly innervate cells of the adrenal medulla.

1	The abdominal aorta begins at the aortic hiatus of the diaphragm as a midline structure at approximately the lower level of vertebra TXII (Fig. 4.164). It passes downward on the anterior surface of the bodies of vertebrae LI to LIV, ending just to the left of midline at the lower level of vertebra LIV. At this point, it divides into the right and left common iliac arteries. This bifurcation can be visualized on the anterior abdominal wall as a point approximately 2.5 cm below the umbilicus or even with a line extending between the highest points of the iliac crest. As the abdominal aorta passes through the posterior abdominal region, the prevertebral plexus of nerves and ganglia covers its anterior surface. It is also related to numerous other structures: Anterior to the abdominal aorta, as it descends, are the pancreas and splenic vein, the left renal vein, and the inferior part of the duodenum.

1	Anterior to the abdominal aorta, as it descends, are the pancreas and splenic vein, the left renal vein, and the inferior part of the duodenum. Several left lumbar veins cross it posteriorly as they pass to the inferior vena cava. On its right side are the cisterna chyli, thoracic duct, azygos vein, right crus of the diaphragm, and the inferior vena cava. On its left side is the left crus of the diaphragm. Branches of the abdominal aorta (Table 4.3) can be classified as: visceral branches supplying organs, posterior branches supplying the diaphragm or body wall, or terminal branches. The visceral branches are either unpaired or paired vessels. The three unpaired visceral branches that arise from the anterior surface of the abdominal aorta (Fig. 4.164) are: the celiac trunk, which supplies the abdominal foregut, the superior mesenteric artery, which supplies the abdominal midgut, and the inferior mesenteric artery, which supplies the abdominal hindgut.

1	The paired visceral branches of the abdominal aorta (Fig. 4.164) include: the middle suprarenal arteries—small, lateral branches of the abdominal aorta arising just above the renal arteries that are part of the multiple vascular supply to the suprarenal gland; the renal arteries—lateral branches of the abdominal aorta that arise just inferior to the origin of the superior mesenteric artery between vertebrae LI and LII, and supply the kidneys; and the testicular or ovarian arteries—anterior branches of the abdominal aorta that arise below the origin of the renal arteries, and pass downward and laterally on the anterior surface of the psoas major muscle. The posterior branches of the abdominal aorta are vessels supplying the diaphragm or body wall. They consist of the inferior phrenic arteries, the lumbar arteries, and the median sacral artery (Fig. 4.164).

1	The inferior phrenic arteries arise immediately inferior to the aortic hiatus of the diaphragm either directly from the abdominal aorta, as a common trunk from the abdominal aorta, or from the base of the celiac trunk (Fig. 4.164). Whatever their origin, they pass upward, provide some arterial supply to the suprarenal gland, and continue onto the inferior surface of the diaphragm. There are usually four pairs of lumbar arteries arising from the posterior surface of the abdominal aorta (Fig. 4.164). They run laterally and posteriorly over the bodies of the lumbar vertebrae, continue laterally, passing posterior to the sympathetic trunks and between the transverse processes of adjacent lumbar vertebrae, and reach the abdominal wall. From this point onward, they demonstrate a branching pattern similar to a posterior intercostal artery, which includes providing segmental branches that supply the spinal cord.

1	The final posterior branch is the median sacral artery (Fig. 4.164). This vessel arises from the posterior surface of the abdominal aorta just superior to the bifurcation and passes in an inferior direction, first over the anterior surface of the lower lumbar vertebrae and then over the anterior surface of the sacrum and coccyx. The inferior vena cava returns blood from all structures below the diaphragm to the right atrium of the heart. It is formed when the two common iliac veins come together at the level of vertebra LV, just to the right of midline. It ascends through the posterior abdominal region anterior to the vertebral column immediately to the right of the abdominal aorta (Fig. 4.166), continues in a superior direction, and leaves the abdomen by piercing the central tendon of the diaphragm at the level of vertebra TVIII.

1	During its course, the anterior surface of the inferior vena cava is crossed by the right common iliac artery, the root of the mesentery, the right testicular or ovarian artery, the inferior part of the duodenum, the head of the pancreas, the superior part of the duodenum, the bile duct, the portal vein, and the liver, which overlaps and on occasion completely surrounds the vena cava (Fig. 4.166). Tributaries to the inferior vena cava include the: common iliac veins, lumbar veins, right testicular or ovarian vein, renal veins, right suprarenal vein, inferior phrenic veins, and hepatic veins. There are no tributaries from the abdominal part of the gastrointestinal tract, the spleen, the pancreas, or the gallbladder, because veins from these structures are components of the portal venous system, which first passes through the liver.

1	Of the venous tributaries mentioned above, the lumbar veins are unique in their connections and deserve special attention. Not all of the lumbar veins drain directly into the inferior vena cava (Fig. 4.167): The fifth lumbar vein generally drains into the iliolumbar vein, a tributary of the common iliac vein. The third and fourth lumbar veins usually drain into the inferior vena cava. The first and second lumbar veins may empty into the ascending lumbar veins. The ascending lumbar veins are long, anastomosing venous channels that connect the common iliac, iliolumbar, and lumbar veins with the azygos and hemi-azygos veins of the thorax (Fig. 4.167). If the inferior vena cava becomes blocked, the ascending lumbar veins become important collateral channels between the lower and upper parts of the body.

1	If the inferior vena cava becomes blocked, the ascending lumbar veins become important collateral channels between the lower and upper parts of the body. Lymphatic drainage from most deep structures and regions of the body below the diaphragm converges mainly on collections of lymph nodes and vessels associated with the major blood vessels of the posterior abdominal region (Fig. 4.168). The lymph then predominantly drains into the thoracic duct. Major lymphatic channels that drain different regions of the body as a whole are summarized in Table 4.4 (also see Chapter 1, pp. 27–28, for discussion of lymphatics in general).

1	Table 4.4 (also see Chapter 1, pp. 27–28, for discussion of lymphatics in general). Approaching the aortic bifurcation, the collections of lymphatics associated with the two common iliac arteries and veins merge, and multiple groups of lymphatic vessels and nodes associated with the abdominal aorta and inferior vena cava pass superiorly. These collections may be subdivided into pre-aortic nodes, which are anterior to the abdominal aorta, and right and left lateral aortic or lumbar nodes (para-aortic nodes), which are positioned on either side of the abdominal aorta (Fig. 4.168). As these collections of lymphatics pass through the posterior abdominal region, they continue to collect lymph from a variety of structures. The lateral aortic or lumbar lymph nodes (para-aortic nodes) receive lymphatics from the body wall, the kidneys, the suprarenal glands, and the testes or ovaries.

1	The pre-aortic nodes are organized around the three anterior branches of the abdominal aorta that supply the abdominal part of the gastrointestinal tract, as well as the spleen, pancreas, gallbladder, and liver. They are divided into celiac, superior mesenteric, and inferior mesenteric nodes, and receive lymph from the organs supplied by the similarly named arteries. Finally, the lateral aortic or lumbar nodes form the right and left lumbar trunks, whereas the pre-aortic nodes form the intestinal trunk (Fig. 4.168). These trunks come together and form a confluence that, at times, appears as a saccular dilation (the cisterna chyli). This confluence of lymph trunks is posterior to the right side of the abdominal aorta and anterior to the bodies of vertebrae LI and LII. It marks the beginning of the thoracic duct. Nervous system in the posterior

1	Nervous system in the posterior Several important components of the nervous system are in the posterior abdominal region. These include the sympathetic trunks and associated splanchnic nerves, the plexus of nerves and ganglia associated with the abdominal aorta, and the lumbar plexus of nerves. The sympathetic trunks pass through the posterior abdominal region anterolateral to the lumbar vertebral bodies, before continuing across the sacral promontory and into the pelvic cavity (Fig. 4.169). Along their course, small raised areas are visible. These represent collections of neuronal cell bodies—primarily postganglionic neuronal cell bodies—which are located outside the central nervous system. They are sympathetic paravertebral ganglia. There are usually four ganglia along the sympathetic trunks in the posterior abdominal region.

1	Also associated with the sympathetic trunks in the posterior abdominal region are the lumbar splanchnic nerves (Fig. 4.169). These components of the nervous system pass from the sympathetic trunks to the plexus of nerves and ganglia associated with the abdominal aorta. Usually two to four lumbar splanchnic nerves carry preganglionic sympathetic fibers and visceral afferent fibers. The abdominal prevertebral plexus is a network of nerve fibers surrounding the abdominal aorta. It extends from the aortic hiatus of the diaphragm to the bifurcation of the aorta into the right and left common iliac arteries. Along its route, it is subdivided into smaller, named plexuses (Fig. 4.170): Beginning at the diaphragm and moving inferiorly, the initial accumulation of nerve fibers is referred to as the celiac plexus—this subdivision includes nerve fibers associated with the roots of the celiac trunk and superior mesenteric artery.

1	Continuing inferiorly, the plexus of nerve fibers extending from just below the superior mesenteric artery to the aortic bifurcation is the abdominal aortic plexus (Fig. 4.170). At the bifurcation of the abdominal aorta, the abdominal prevertebral plexus continues inferiorly as the superior hypogastric plexus. Throughout its length, the abdominal prevertebral plexus is a conduit for: preganglionic sympathetic and visceral afferent fibers from the thoracic and lumbar splanchnic nerves, preganglionic parasympathetic and visceral afferent fibers from the vagus nerves [X], and preganglionic parasympathetic fibers from the pelvic splanchnic nerves (Fig. 4.171).

1	Associated with the abdominal prevertebral plexus are clumps of nervous tissue (the prevertebral ganglia), which are collections of postganglionic sympathetic neuronal cell bodies in recognizable aggregations along the abdominal prevertebral plexus; they are usually named after the nearest branch of the abdominal aorta. They are therefore referred to as celiac, superior mesenteric, aorticorenal, and inferior mesenteric ganglia (Fig. 4.172). These structures, along with the abdominal prevertebral plexus, play a critical role in the innervation of the abdominal viscera. Common sites for pain referred from the abdominal viscera and from the heart are given in Table 4.5. The lumbar plexus is formed by the anterior rami of nerves L1 to L3 and most of the anterior ramus of L4 (Fig. 4.173 and Table 4.6). It also receives a contribution from the T12 (subcostal) nerve.

1	The lumbar plexus is formed by the anterior rami of nerves L1 to L3 and most of the anterior ramus of L4 (Fig. 4.173 and Table 4.6). It also receives a contribution from the T12 (subcostal) nerve. Branches of the lumbar plexus include the iliohypogastric, ilio-inguinal, and genitofemoral nerves, the lateral cutaneous nerve of the thigh (lateral femoral cutaneous), and femoral and obturator nerves. The lumbar plexus forms in the substance of the psoas major muscle anterior to its attachment to the transverse processes of the lumbar vertebrae (Fig. 4.174). Therefore, relative to the psoas major muscle, the various branches emerge either: anterior—genitofemoral nerve, medial—obturator nerve, or lateral—iliohypogastric, ilio-inguinal, and femoral nerves and the lateral cutaneous nerve of the thigh.

1	The iliohypogastric and ilio-inguinal nerves arise as a single trunk from the anterior ramus of nerve L1 (Fig. 4.173). Either before or soon after emerging from the lateral border of the psoas major muscle, this single trunk divides into the iliohypogastric and the ilio-inguinal nerves. The iliohypogastric nerve passes across the anterior surface of the quadratus lumborum muscle, posterior to the kidney. It pierces the transversus abdominis muscle and continues anteriorly around the body between the transversus abdominis and internal oblique muscles. Above the iliac crest, a lateral cutaneous branch pierces the internal and external oblique muscles to supply the posterolateral gluteal skin (Fig. 4.175).

1	The remaining part of the iliohypogastric nerve (the anterior cutaneous branch) continues in an anterior direction, piercing the internal oblique just medial to the anterior superior iliac spine as it continues in an obliquely downward and medial direction. Becoming cutaneous, just above the superficial inguinal ring, after piercing the aponeurosis of the external oblique, it distributes to the skin in the pubic region (Fig. 4.175). Throughout its course, it also supplies branches to the abdominal musculature. The ilio-inguinal nerve is smaller than, and inferior to, the iliohypogastric nerve as it crosses the quadratus lumborum muscle. Its course is more oblique than that of the iliohypogastric nerve, and it usually crosses part of the iliacus muscle on its way to the iliac crest. Near the anterior end of the iliac crest, it pierces the transversus abdominis muscle, and then pierces the internal oblique muscle and enters the inguinal canal.

1	The ilio-inguinal nerve emerges through the superficial inguinal ring, along with the spermatic cord, and provides cutaneous innervation to the upper medial thigh, the root of the penis, and the anterior surface of the scrotum in men, or the mons pubis and labium majus in women (Fig. 4.175). Throughout its course, it also supplies branches to the abdominal musculature. The genitofemoral nerve arises from the anterior rami of nerves L1 and L2 (Fig. 4.173). It passes downward in the substance of the psoas major muscle until it emerges on the anterior surface of the psoas major. It then descends on the surface of the muscle, in a retroperitoneal position, passing posterior to the ureter. It eventually divides into genital and femoral branches.

1	The genital branch continues downward and enters the inguinal canal through the deep inguinal ring. It continues through the canal and: in men, innervates the cremasteric muscle and terminates on the skin in the upper anterior part of the scrotum, and in women, accompanies the round ligament of the uterus and terminates on the skin of the mons pubis and labium majus. The femoral branch descends on the lateral side of the external iliac artery and passes posterior to the inguinal ligament, entering the femoral sheath lateral to the femoral artery. It pierces the anterior layer of the femoral sheath and the fascia lata to supply the skin of the upper anterior thigh (Fig. 4.175). Lateral cutaneous nerve of thigh (L2 and L3)

1	Lateral cutaneous nerve of thigh (L2 and L3) The lateral cutaneous nerve of the thigh arises from the anterior rami of nerves L2 and L3 (Fig. 4.173). It emerges from the lateral border of the psoas major muscle, passing obliquely downward across the iliacus muscle toward the anterior superior iliac spine (Fig. 4.175). It passes posterior to the inguinal ligament and enters the thigh. The lateral cutaneous nerve of the thigh supplies the skin on the anterior and lateral thigh to the level of the knee (Fig. 4.175). Obturator nerve (L2 to L4) The obturator nerve arises from the anterior rami of nerves L2 to L4 (Fig. 4.173). It descends in the psoas major muscle, emerging from its medial side near the pelvic brim (Fig. 4.174). The obturator nerve continues posterior to the common iliac vessels, passes across the lateral wall of the pelvic cavity, and enters the obturator canal, through which the obturator nerve gains access to the medial compartment of the thigh.

1	In the area of the obturator canal, the obturator nerve divides into anterior and posterior branches. On entering the medial compartment of the thigh, the two branches are separated by the obturator externus and adductor brevis muscles. Throughout their course through the medial compartment, these two branches supply: articular branches to the hip joint, muscular branches to the obturator externus, pectineus, adductor longus, gracilis, adductor brevis, and adductor magnus muscles, cutaneous branches to the medial aspect of the thigh, and in association with the saphenous nerve, cutaneous branches to the medial aspect of the upper part of the leg and articular branches to the knee joint (Fig. 4.175). Femoral nerve (L2 to L4)

1	Femoral nerve (L2 to L4) The femoral nerve arises from the anterior rami of nerves L2 to L4 (Fig. 4.173). It descends through the substance of the psoas major muscle, emerging from the lower lateral border of the psoas major (Fig. 4.174). Continuing its descent, the femoral nerve lies between the lateral border of the psoas major and the anterior surface of the iliacus muscle. It is deep to the iliacus fascia and lateral to the femoral artery as it passes posterior to the inguinal ligament and enters the anterior compartment of the thigh. Upon entering the thigh, it immediately divides into multiple branches. Cutaneous branches of the femoral nerve include: medial and intermediate cutaneous nerves supplying the skin on the anterior surface of the thigh, and the saphenous nerve supplying the skin on the medial surface of the leg (Fig. 4.175).

1	Muscular branches innervate the iliacus, pectineus, sartorius, rectus femoris, vastus medialis, vastus intermedius, and vastus lateralis muscles. Articular branches supply the hip and knee joints. Visualization of the position of abdominal viscera is fundamental to a physical examination. Some of these viscera or their parts can be felt by palpating through the abdominal wall. Surface features can be used to establish the positions of deep structures. Defining the surface projection of the abdomen Palpable landmarks can be used to delineate the extent of the abdomen on the surface of the body. These landmarks are: the costal margin above and the pubic tubercle, anterior superior iliac spine, and iliac crest below (Fig. 4.176). The costal margin is readily palpable and separates the abdominal wall from the thoracic wall.

1	The costal margin is readily palpable and separates the abdominal wall from the thoracic wall. A line between the anterior superior iliac spine and the pubic tubercle marks the position of the inguinal ligament, which separates the anterior abdominal wall above from the thigh of the lower limb below. The iliac crest separates the posterolateral abdominal wall from the gluteal region of the lower limb. The upper part of the abdominal cavity projects above the costal margin to the diaphragm, and therefore abdominal viscera in this region of the abdomen are protected by the thoracic wall. The level of the diaphragm varies during the breathing cycle. The dome of the diaphragm on the right can reach as high as the fourth costal cartilage during forced expiration. How to find the superficial inguinal ring

1	How to find the superficial inguinal ring The superficial inguinal ring is an elongate triangular defect in the aponeurosis of the external oblique (Fig. 4.177). It lies in the lower medial aspect of the anterior abdominal wall and is the external opening of the inguinal canal. The inguinal canal and superficial ring are larger in men than in women: In men, structures that pass between the abdomen and the testis pass through the inguinal canal and superficial inguinal ring. In women, the round ligament of the uterus passes through the inguinal canal and superficial inguinal ring to merge with connective tissue of the labium majus. The superficial inguinal ring is superior to the pubic crest and tubercle and to the medial end of the inguinal ligament:

1	The superficial inguinal ring is superior to the pubic crest and tubercle and to the medial end of the inguinal ligament: In men, the superficial inguinal ring can be easily located by following the spermatic cord superiorly to the lower abdominal wall—the external spermatic fascia of the spermatic cord is continuous with the margins of the superficial inguinal ring. In women, the pubic tubercle can be palpated and the ring is superior and lateral to it. The deep inguinal ring, which is the internal opening to the inguinal canal, lies superior to the inguinal ligament, midway between the anterior superior iliac spine and pubic symphysis. The pulse of the femoral artery can be felt in the same position but below the inguinal ligament. Because the superficial inguinal ring is the site where inguinal hernias appear, particularly in men, the ring and related parts of the inguinal canal are often evaluated during physical examination. How to determine lumbar vertebral levels

1	How to determine lumbar vertebral levels Lumbar vertebral levels are useful for visualizing the positions of viscera and major blood vessels. The approximate positions of the lumbar vertebrae can be established using palpable or visible landmarks (Fig. 4.178): A horizontal plane passes through the medial ends of the ninth costal cartilages and the body of the LI vertebra—this transpyloric plane cuts through the body midway between the suprasternal (jugular) notch and the pubic symphysis. A horizontal plane passes through the lower edge of the costal margin (tenth costal cartilage) and the body of the LIII vertebra—the umbilicus is normally on a horizontal plane that passes through the disc between the LIII and LIV vertebrae. A horizontal plane (supracristal plane) through the highest point on the iliac crest passes through the spine and body of the LIV vertebra; A plane through the tubercles of the crest of the ilium passes through the body of the LV vertebra.

1	A plane through the tubercles of the crest of the ilium passes through the body of the LV vertebra. Visualizing structures at the LI The LI vertebral level is marked by the transpyloric plane, which cuts transversely through the body midway between the jugular notch and pubic symphysis, and through the ends of the ninth costal cartilages (Fig. 4.179). At this level are: the beginning and upper limit of the end of the duodenum, the hila of the kidneys, the neck of the pancreas, and the origin of the superior mesenteric artery from the aorta. The left and right colic flexures also are close to this level. Visualizing the position of major Each of the vertebral levels in the abdomen is related to the origin of major blood vessels (Fig. 4.180): The celiac trunk originates from the aorta at the upper border of the LI vertebra. The superior mesenteric artery originates at the lower border of the LI vertebra. The renal arteries originate at approximately the LII vertebra.

1	The superior mesenteric artery originates at the lower border of the LI vertebra. The renal arteries originate at approximately the LII vertebra. The inferior mesenteric artery originates at the LIII vertebra. The aorta bifurcates into the right and left common iliac arteries at the level of the LIV vertebra. The left and right common iliac veins join to form the inferior vena cava at the LV vertebral level. Using abdominal quadrants to locate The abdomen can be divided into quadrants by a vertical median plane and a horizontal transumbilical plane, which passes through the umbilicus (Fig. 4.181): The liver and gallbladder are in the right upper quadrant. The stomach and spleen are in the left upper quadrant. The cecum and appendix are in the right lower quadrant. The end of the descending colon and sigmoid colon are in the left lower quadrant.

1	The stomach and spleen are in the left upper quadrant. The cecum and appendix are in the right lower quadrant. The end of the descending colon and sigmoid colon are in the left lower quadrant. Most of the liver is under the right dome of the diaphragm and is deep to the lower thoracic wall. The inferior margin of the liver can be palpated descending below the right costal margin when a patient is asked to inhale deeply. On deep inspiration, the edge of the liver can be felt “slipping” under the palpating fingers placed under the costal margin. A common surface projection of the appendix is McBurney’s point, which is one-third of the way up along a line from the right anterior superior iliac spine to the umbilicus. Defining surface regions to which pain from the gut is referred

1	McBurney’s point, which is one-third of the way up along a line from the right anterior superior iliac spine to the umbilicus. Defining surface regions to which pain from the gut is referred The abdomen can be divided into nine regions by a midclavicular sagittal plane on each side and by the subcostal and intertubercular planes, which pass through the body transversely (Fig. 4.182). These planes separate the abdomen into: three central regions (epigastric, umbilical, pubic), and three regions on each side (hypochondrium, flank, groin). Pain from the abdominal part of the foregut is referred to the epigastric region, pain from the midgut is referred to the umbilical region, and pain from the hindgut is referred to the pubic region. Where to find the kidneys The kidneys project onto the back on either side of the midline and are related to the lower ribs (Fig. 4.183): The left kidney is a little higher than the right and reaches as high as rib XI.

1	The kidneys project onto the back on either side of the midline and are related to the lower ribs (Fig. 4.183): The left kidney is a little higher than the right and reaches as high as rib XI. The superior pole of the right kidney reaches only as high as rib XII. The lower poles of the kidneys occur around the level of the disc between the LIII and LIV vertebrae. The hila of the kidneys and the beginnings of the ureters are at approximately the LI vertebra. The ureters descend vertically anterior to the tips of the transverse processes of the lower lumbar vertebrae and enter the pelvis. Where to find the spleen The spleen projects onto the left side and back in the area of ribs IX to XI (Fig. 4.184). The spleen follows the contour of rib X and extends from the superior pole of the left kidney to just posterior to the midaxillary line. Fig. 4.1 Abdomen. A. Boundaries. B. Arrangement of abdominal contents. Inferior view.

1	Fig. 4.1 Abdomen. A. Boundaries. B. Arrangement of abdominal contents. Inferior view. Fig. 4.2 The abdomen contains and protects the abdominal viscera. Fig. 4.3 The abdomen assists in breathing. InspirationExpirationDiaphragmRelaxation of diaphragmContraction ofdiaphragmContraction of abdominal musclesRelaxation ofabdominalmuscles Fig. 4.4 Increasing intraabdominal pressure to assist in micturition, defecation, and childbirth. Laryngeal cavity closedAir retained in thoraxFixed diaphragmContraction of abdominal wallIncrease inintraabdominalpressureMicturitionChild birthDefecation Fig. 4.5 Abdominal wall. A. Skeletal elements. B. Muscles. Fig. 4.6 The gut tube is suspended by mesenteries. Branch of aortaGastrointestinal tractAortaDorsal mesenteryVentral mesenteryKidney—posterior toperitoneumVisceral peritoneumParietal peritoneum Fig. 4.7 A series showing the progression (A to C) from an intraperitoneal organ to a secondarily retroperitoneal organ.

1	Fig. 4.7 A series showing the progression (A to C) from an intraperitoneal organ to a secondarily retroperitoneal organ. MesenteryVisceral peritoneumGastrointestinal tractGastrointestinal tractGastrointestinal tractParietal peritoneumArtery to gastrointestinal tractRetroperitoneal structuresMesentery before fusion with wallIntraperitoneal part of gastrointestinal tractSecondary retroperitoneal part of gastrointestinal tract ABC Fig. 4.8 Inferior thoracic aperture and the diaphragm. Fig. 4.9 Pelvic inlet. Ala of sacrumS IL VPelvic inletInguinal ligamentPelvic bone Fig. 4.10 Orientation of abdominal and pelvic cavities. Thoracic wallAxis of abdominal cavityAbdominal cavityPelvic cavityPelvic inletAxis of pelvic cavity Fig. 4.11 The abdominal cavity is continuous with the pelvic cavity. RectumPeritoneumPelvic inletBladderUterusShadow of ureterShadow of internal iliac vessels Fig. 4.12 Structures passing between the abdomen and thigh.

1	RectumPeritoneumPelvic inletBladderUterusShadow of ureterShadow of internal iliac vessels Fig. 4.12 Structures passing between the abdomen and thigh. Fig. 4.13 A series (A to H) showing the development of the gut and mesenteries. Fig. 4.14 Innervation of the anterior abdominal wall. Fig. 4.15 Inguinal region. A. Development. B. In men. C. In women. Inferior vena cavaRight testicular arteryLeft testicular arteryRight testicular veinLeft testicular veinAortaPelvic brimLeft ductus deferensDuctus deferensInguinal canalSuperficial inguinal ringDeep inguinal ringSpermatic cordRemnant ofgubernaculumEpididymisTestisTunica vaginalisTesticularartery and veinBInferior vena cavaAortaLeft renal arteryLeft ovarian arteryLeft renal veinLeft ovarian veinPelvic inletUterine tubeRound ligament of uterus (remnantsof gubernaculum)UterusSuperficialinguinal ringC Fig. 4.16 Vertebral level LI.

1	Fig. 4.16 Vertebral level LI. Jugular notchRightkidneyLI (transpyloric)planePubic symphysisCostal marginPyloric orifice betweenstomach and duodenumPosition of umbilicus Fig. 4.17 Blood supply of the gut. A. Relationship of vessels to the gut and mesenteries. B. Anterior view. Fig. 4.18 Left-to-right venous shunts. Fig. 4.19 Hepatic portal system. Fig. 4.20 Prevertebral plexus. Sympathetic inputParasympathetic inputPrevertebral plexusLumbar splanchnicnerves (L1, L2)Pelvic splanchnic nerves (S2 to S4)Anterior and posterior vagus trunks (cranial)Greater, lesser, and least splanchnic nerves(T5 to T12) Fig. 4.21 Boundaries of the abdominal cavity. Fig. 4.22 Four-quadrant topographical pattern. Fig. 4.23 Nine-region organizational pattern. Fig. 4.24 Layers of the abdominal wall. Fig. 4.25 Superficial fascia.

1	Fig. 4.22 Four-quadrant topographical pattern. Fig. 4.23 Nine-region organizational pattern. Fig. 4.24 Layers of the abdominal wall. Fig. 4.25 Superficial fascia. Superficial fasciaFatty layer(Camper's fascia)Membranous layer(Scarpa's fascia)SkinPubic symphysisPenisDartos fasciaScrotumInguinal ligamentAponeurosis of external obliqueFascia lata of thigh Fig. 4.26 Continuity of membranous layer of superficial fascia into other areas. Continuity with superficialpenile fasciaContinuity withdartos fasciaExternal oblique muscleand aponeurosisMembranous layer ofsuperficial fascia (Scarpa's fascia)Attachment to fascia lataSuperficial perinealfascia (Colles' fascia)Attachment toischiopubic rami Fig. 4.27 External oblique muscle and its aponeurosis. Linea albaLatissimus dorsi muscleExternal oblique muscleAbdominal part ofpectoralis major muscleAponeurosis of external obliqueInguinal ligamentAnterior superior iliac spine Fig. 4.28 Ligaments formed from the external oblique aponeurosis.

1	Fig. 4.28 Ligaments formed from the external oblique aponeurosis. Fig. 4.29 Ligaments of the inguinal region. Fig. 4.30 Internal oblique muscle and its aponeurosis. External oblique muscleRib XInternal oblique muscleand aponeurosisLinea albaExternal oblique muscleAponeurosis of external obliqueAnterior superior iliac spine Fig. 4.31 Transversus abdominis muscle and its aponeurosis. External oblique muscleRib XTransversus abdominismuscle and aponeurosisLinea albaExternal oblique muscleAponeurosis of internal obliqueAponeurosis of external obliqueAnterior superior iliac spine Fig. 4.32 Rectus abdominis and pyramidalis muscles. External oblique musclePosterior wall of rectus sheathRectus abdominis muscleTendinous intersectionInternal oblique muscle Arcuate lineTransversalis fasciaLinea albaPyramidalis muscle

1	External oblique musclePosterior wall of rectus sheathRectus abdominis muscleTendinous intersectionInternal oblique muscle Arcuate lineTransversalis fasciaLinea albaPyramidalis muscle Fig. 4.33 Organization of the rectus sheath. A. Transverse section through the upper three-quarters of the rectus sheath. B. Transverse section through the lower one-quarter of the rectus sheath. Fig. 4.34 Transverse section showing the layers of the abdominal wall. Fig. 4.35 Subdivisions of the extraperitoneal fascia. Fig. 4.36 Innervation of the anterolateral abdominal wall. External oblique muscleand aponeurosisIliohypogastric nerve (L1)Xiphoid processIlio-inguinal nerve (L1)Iliac crestAnterior cutaneousbranches T7 to T12Lateral cutaneousbranches T7 to T12 Fig. 4.37 Path taken by the nerves innervating the anterolateral abdominal wall. Fig. 4.38 Dermatomes of the anterolateral abdominal wall. Fig. 4.39 Arterial supply to the anterolateral abdominal wall.

1	Fig. 4.38 Dermatomes of the anterolateral abdominal wall. Fig. 4.39 Arterial supply to the anterolateral abdominal wall. Fig. 4.40 Superior and inferior epigastric arteries. Fig. 4.41 Descent of the testis from week 7 (postfertilization) to birth. Fig. 4.42 Inguinal canal. Linea albaSuperficialinguinal ringDeep inguinal ringExternal oblique muscleAponeurosis of external obliqueInguinal ligamentSpermatic cordAnterior superioriliac spine Fig. 4.43 Deep inguinal ring and the transversalis fascia. Fig. 4.44 Superficial inguinal ring and the aponeurosis of the external oblique. Inguinal ligamentFemoral artery and veinExternal oblique muscleAponeurosis of external oblique Spermatic cordSuperficial inguinal ringAnterior superior iliac spine Fig. 4.45 Internal oblique muscle and the inguinal canal. Inguinal ligamentFemoral artery and veinInternal oblique muscleAponeurosis of internal oblique Spermatic cordConjoint tendonAnterior superior iliac spine

1	Inguinal ligamentFemoral artery and veinInternal oblique muscleAponeurosis of internal oblique Spermatic cordConjoint tendonAnterior superior iliac spine Fig. 4.46 Transversus abdominis muscle and the inguinal canal. Fig. 4.47 A. Spermatic cord (men). B. Round ligament of uterus (women). Internal spermatic fasciaParietal layer of the tunica vaginalisAVisceral layer of the tunica vaginalisCavity of the tunica vaginalisExternal spermatic fasciaCremasteric fasciaDeep inguinal ringInternaloblique muscleSuperficial inguinal ringTesticular artery andpampiniform plexus of veinsParietal peritoneumArtery to ductus deferensCremasteric vesselsGenital branch of genitofemoral nerveInferior epigastric vesselsDuctus deferensExternal obliqueaponeurosisTransversusabdominis muscleConjoint tendonExternal obliqueaponeurosisTransversalisfasciaIlioinguinalnerveIlioinguinal nerveExtraperitoneal fascia

1	Internaloblique muscleParietal peritoneumGenital branch ofgenitofemoral nerveGenital branch ofgenitofemoral nerveInferior epigastric vesselsRound ligament of uterusExternal obliqueaponeurosisTransversusabdominis muscleMembranous layerof superficial fasciaSuperficial fascia(fatty layers) Fine connectivetissue strandsBConjoint tendonExternal obliqueaponeurosisSkin of mons pubisIlioinguinalnerveIlioinguinal nerveExtraperitoneal fascia Fig. 4.48 Indirect inguinal hernia. Fig. 4.49 Direct inguinal hernia. Fig. 4.50 Right inguinal triangle. A. Internal view. B. Laparoscopic view showing the parietal peritoneum still covering the area. Inferior epigastricvesselsDirect herniaTesticular vesselsPosition of deepinguinal ringBLateralMedialExternal iliac vesselsDuctus deferens Fig. 4.51 Coronal CT shows a large inguinal hernia containing loops of large and small bowel (arrow) on the left side of a male patient.

1	Fig. 4.51 Coronal CT shows a large inguinal hernia containing loops of large and small bowel (arrow) on the left side of a male patient. Fig. 4.52 Right indirect inguinal hernia. T2, fat saturated, weighted magnetic resonance image in the coronal plane of a male groin. Fig. 4.53 A. Intraperitoneal. B. Retroperitoneal. Fig. 4.54 Greater and lesser sacs of the peritoneal cavity. Fig. 4.55 Transverse section illustrating the continuity between the greater and lesser sacs through the omental (epiploic) foramen. Fig. 4.56 Coronal CT shows ascites fluid in abdominal cavity. Fig. 4.57 Peritoneal metastasis on the surface of the liver. Computed tomogram in the axial plane of the upper abdomen. Peritoneal metastasison surface of liverAortaInferior vena cavaLiverSpleenLeft kidney Fig. 4.58 Radiograph of subdiaphragmatic gas. Fig. 4.59 Greater omentum. Fig. 4.60 Lesser omentum.

1	Peritoneal metastasison surface of liverAortaInferior vena cavaLiverSpleenLeft kidney Fig. 4.58 Radiograph of subdiaphragmatic gas. Fig. 4.59 Greater omentum. Fig. 4.60 Lesser omentum. Liver (retracted )GallbladderStomachLesser omentumDuodenumDescending colonAscending colonOmental foramenLesser curvature of the stomachHepatogastric ligamentHepatoduodenal ligament Fig. 4.61 Peritoneal reflections, forming mesenteries, outlined on the posterior abdominal wall. Root of the transverse mesocolonRoot of the sigmoid mesocolonRoot of the mesentery Fig. 4.62 Abdominal esophagus. RightvagusnerveTracheaArch of aortaAortaThoracicesophagusAbdominal esophagusRight crus ofdiaphragmLeft vagus nerve Fig. 4.63 Arterial supply to the abdominal esophagus and stomach. Fig. 4.64 Stomach. Fig. 4.65 Radiograph, using barium, showing the stomach and duodenum. A. Double-contrast radiograph of the stomach. B. Double-contrast radiograph showing the duodenal cap.

1	Fig. 4.64 Stomach. Fig. 4.65 Radiograph, using barium, showing the stomach and duodenum. A. Double-contrast radiograph of the stomach. B. Double-contrast radiograph showing the duodenal cap. Fundus of stomachPyloric orificeSuperior part of duodenumABPyloric antrumEsophagusNormal duodenal capPyloric antrum of stomachInferior duodenumDuodenal jejunal flexureBody of stomachDescending part of duodenumPyloric canalPyloric sphincter Fig. 4.66 Duodenum. PancreasDuodenum—superior partDuodenum—descending partDuodenum —ascending partDuodenum—inferior partDescending colonAscending colonSpleenAbdominal aortaEsophagusL1L2L3Bile ductRight suprarenal glandRight kidneyGallbladderPosition of majorduodenal papillaPosition of minorduodenal papillaLeft kidneySuperior mesenteric vein and arteryPortal veinInferior vena cava Fig. 4.67 Arterial supply to the duodenum. Fig. 4.68 Radiograph, using barium, showing the jejunum and ileum.

1	Fig. 4.67 Arterial supply to the duodenum. Fig. 4.68 Radiograph, using barium, showing the jejunum and ileum. Fig. 4.69 Differences in the arterial supply to the small intestine. A. Jejunum. B. Ileum. Fig. 4.70 Ileocecal junction. A. Radiograph showing ileocecal junction. B. Illustration showing ileocecal junction and the ileocecal fold. C. Endoscopic image of the ileocecal fold. Fig. 4.71 Arterial supply to the ileum. Fig. 4.72 The endoscope is a flexible plastic tube that can be controlled from the proximal end. Through a side portal various devices can be inserted, which run through the endoscope and can be used to obtain biopsies and to perform minor endoluminal surgical procedures (e.g., excision of polyps). Fig. 4.73 Endoscopic images of the gastroesophageal junction. A. Normal. B. Esophageal cancer at esophageal junction. Fig. 4.74 Endoscopic image of the pyloric antrum of the stomach looking toward the pylorus.

1	Fig. 4.74 Endoscopic image of the pyloric antrum of the stomach looking toward the pylorus. Fig. 4.75 Endoscopic image showing normal appearance of the second part of the duodenum. Fig. 4.76 Small bowel visualization using MRI in coronal plane. Fig. 4.77 Axial CT shows sigmoid colon wall thickening caused by tumor. Fig. 4.78 Vasculature associated with a Meckel’s diverticulum. A. Surgical image of Meckel’s diverticulum. B. Digital subtraction angiography. Fig. 4.79 Large intestine. Transverse colonSigmoid colonAscending colonRight colic flexureAppendixCecumHaustra of colonOmental appendicesLeft colic flexureAnal canalRectumIleumTaeniae coli Fig. 4.80 Radiograph, using barium, showing the large intestine. Fig. 4.81 Position of the large intestine in the nine-region organizational pattern. Fig. 4.82 Cecum and appendix. Fig. 4.83 Mesoappendix and appendicular vessels. Fig. 4.84 Positions of the appendix. Fig. 4.85 Arterial supply to the cecum and appendix.

1	Fig. 4.82 Cecum and appendix. Fig. 4.83 Mesoappendix and appendicular vessels. Fig. 4.84 Positions of the appendix. Fig. 4.85 Arterial supply to the cecum and appendix. Fig. 4.86 Inflamed appendix. Ultrasound scan. Fig. 4.87 Axial CT shows inflamed appendix. Fig. 4.88 Colon. Fig. 4.89 Right and left colic flexures. Fig. 4.90 Arterial supply to the colon. Fig. 4.91 Rectum and anal canal. Fig. 4.92 Arterial supply to the rectum and anal canal. Posterior view. Fig. 4.93 Small bowel malrotation and volvulus. Radiograph of stomach, duodenum, and upper jejunum using barium. Fig. 4.94 Small bowel malrotation. Radiograph of stomach, duodenum, and jejunum using barium.

1	Fig. 4.94 Small bowel malrotation. Radiograph of stomach, duodenum, and jejunum using barium. Fig. 4.95 This radiograph of the abdomen, anteroposterior view, demonstrates a number of dilated loops of small bowel. Small bowel can be identified by the plicae circulares that pass from wall to wall as indicated. The large bowel is not dilated. The cause of the small bowel dilatation is an adhesion after pelvic surgery. Dilation of small bowelPlicae circulares Fig. 4.96 Coronal CT demonstrates dilated and fluid-filled loops of small bowel in patient with small bowel obstruction. Dilated and fluid-filledloops of small bowel Fig. 4.97 Coronal CT of abdomen shows fluid-filled and dilated ascending and transverse colon in patient with large bowel obstruction. Fig. 4.98 This oblique radiograph demonstrates contrast passing through a colonic stent that has been placed to relieve bowel obstruction prior to surgery.

1	Fig. 4.98 This oblique radiograph demonstrates contrast passing through a colonic stent that has been placed to relieve bowel obstruction prior to surgery. Fig. 4.99 This double-contrast barium enema demonstrates numerous small outpouchings throughout the distal large bowel predominantly within the descending colon and the sigmoid colon. These small outpouchings are diverticula and in most instances remain quiescent. Fig. 4.100 Axial CT of inflamed sigmoid colon in patient with diverticulitis. Fig. 4.101 Position of the liver in the abdomen. Fig. 4.102 Surfaces of the liver and recesses associated with the liver. Fig. 4.103 Diaphragmatic surface of the liver. Fig. 4.104 Visceral surface of the liver. A. Illustration. B. Abdominal computed tomogram, with contrast, in the axial plane.

1	Fig. 4.103 Diaphragmatic surface of the liver. Fig. 4.104 Visceral surface of the liver. A. Illustration. B. Abdominal computed tomogram, with contrast, in the axial plane. Left lobe of liverLeft lobe of liverRight lobeof liverCaudate lobeRight lobe of liverQuadrate lobeQuadrate lobeGallbladderAnteriorPosteriorFundusBodyNeckCystic ductPorta hepatisPortal veinFissure for ligamentum teresFissure for ligamentumvenosumHepatic artery properHepatic ductsBile ductABGallbladderPortal veinInferior vena cavaStomachNeck of pancreasSpleenLeft kidneyAortaRight crusLeft crus Fig. 4.105 Posterior view of the bare area of the liver and associated ligaments. Inferior vena cavaSuprarenal impressionLeft lobe of liverRight lobe of liver Renal impressionCaudate lobeGastric impressionEsophageal impressionColic impressionQuadrate lobeFalciform ligamentGallbladderPorta hepatisAnterior coronary ligamentPosterior coronary ligamentRight triangularligamentLeft triangular ligamentBare areaFundusBodyNeck

1	Fig. 4.106 Arterial supply to the liver and gallbladder. A. Schematic. B. Laparoscopic surgical view of cystic duct and cystic artery. Fig. 4.107 Pancreas. Fig. 4.108 Abdominal images. A. Abdominal computed tomogram, with contrast, in the axial plane. B. Abdominal ultrasound scan. GallbladderPancreasPortal veinSplenic veinStomachLeft colonic flexureSpleenLeft kidneyAortaInferior vena cavaRight lobe of liverRight crusALeft crus Left lobe of liverBSplenic arterySuperior mesenteric arteryPancreasLeft renal veinAortaInferior vena cavaVertebra Fig. 4.109 Pancreatic duct system. Fig. 4.110 Arterial supply to the pancreas. Posterior view. Fig. 4.111 Bile drainage. A. Duct system for passage of bile. B. Percutaneous transhepatic cholangiogram demonstrating the bile duct system. GallbladderCystic ductBile ductBile ductRight hepatic ductLeft hepatic ductCommon hepatic ductCystic ductNeedleCommonhepatic ductMain pancreatic ductDescending part of duodenumDescending part of duodenumAB

1	Fig. 4.112 Spleen. Fig. 4.113 Splenic ligaments and related vasculature. Fig. 4.114 Surfaces and hilum of the spleen. Fig. 4.115 Arterial supply to the spleen. Fig. 4.116 Division of the liver into segments based upon the distributions of the bile ducts and hepatic vessels (Couinaud’s segments). Fig. 4.117 Gallbladder containing multiple stones. Ultrasound scan. Fig. 4.118 Magnetic resonance cholangiopancreatography (MRCP) in the coronal plane. Fig. 4.119 Endoscopic retrograde cholangiopancreatography (ERCP) of biliary system. Endoscope with side-viewing optic mechanismStent in common bile duct Fig. 4.120 Coronal CT of the abdomen containing a massively enlarged spleen (splenomegaly). Fig. 4.121 Anterior branches of the abdominal aorta. Fig. 4.122 Divisions of the gastrointestinal tract into foregut, midgut, and hindgut, summarizing the primary arterial supply to each segment.

1	Fig. 4.121 Anterior branches of the abdominal aorta. Fig. 4.122 Divisions of the gastrointestinal tract into foregut, midgut, and hindgut, summarizing the primary arterial supply to each segment. Fig. 4.123 Celiac trunk. A. Distribution of the celiac trunk. B. Digital subtraction angiography of the celiac trunk and its branches. Fig. 4.124 Arterial supply to the pancreas. Fig. 4.125 Distribution of the common hepatic artery. Fig. 4.126 Initial branching and relationships of the superior mesenteric artery. Fig. 4.127 Superior mesenteric artery. A. Distribution of the superior mesenteric artery. B. Digital subtraction angiography of the superior mesenteric artery and its branches. Fig. 4.128 Inferior mesenteric artery. A. Distribution of the inferior mesenteric artery. B. Digital subtraction angiography of the inferior mesenteric artery and its branches. Fig. 4.129 Arterial supply to the abdominal parts of the gastrointestinal system and to the spleen.

1	Fig. 4.129 Arterial supply to the abdominal parts of the gastrointestinal system and to the spleen. Fig. 4.130 Enlarged marginal artery connecting the superior and inferior mesenteric arteries. Digital subtraction angiogram. Fig. 4.131 Portal vein. Fig. 4.132 Venous drainage of the abdominal portion of the gastrointestinal tract. Fig. 4.133 Portosystemic anastomoses. LiverSpleenSplenic veinSuperior mesenteric veinSuperior rectal veinInferior rectal veinsInferior vena cavaInferior mesenteric veinLeft gastric veinPortal veinRectumStomachTributaries to azygos veinSuperficial veinson abdominal wallExternal iliac veinInternal iliac veinCommon iliac veinPara-umbilical veinsthat accompany theligamentum teres Fig. 4.134 Lymphatic drainage of the abdominal portion of the gastrointestinal tract. Fig. 4.135 Sympathetic trunks. Fig. 4.136 Splanchnic nerves. Fig. 4.137 Abdominal prevertebral plexus and ganglia.

1	Fig. 4.135 Sympathetic trunks. Fig. 4.136 Splanchnic nerves. Fig. 4.137 Abdominal prevertebral plexus and ganglia. Fig. 4.138 Parasympathetic innervation of the abdominal portion of the gastrointestinal tract. Fig. 4.139 The enteric system. Fig. 4.140 Posterior abdominal region. Fig. 4.141 Osteology of the posterior abdominal wall. Fig. 4.142 Muscles of the posterior abdominal wall. Fig. 4.143 Diaphragm. Left phrenic nerveRight phrenic nerveInferior phrenicarteryAortaThoracic ductEsophagus with anteriorand posterior vagal trunksLIVLIIILIILISuperior epigastric arteryCentral tendonInferior vena cavaHemi-azygos veinGreater splanchnic nerveLesser splanchnic nerveLeast splanchnic nerveLeft crusSympathetic trunkRight crus Fig. 4.144 Crura of the diaphragm. Fig. 4.145 Right and left domes of the diaphragm. Chest radiograph. Fig. 4.146 Fetal diaphragmatic hernia in utero. T2-weighted MR image. Fetus in coronal plane, mother in sagittal plane.

1	Fig. 4.145 Right and left domes of the diaphragm. Chest radiograph. Fig. 4.146 Fetal diaphragmatic hernia in utero. T2-weighted MR image. Fetus in coronal plane, mother in sagittal plane. Fetal vertebralcolumnFetal abdominalcontents (fluid-filledloops of intestine)in left side ofthoracic cavityFetal headNormal fetal lung development on rightside of thoracic cavityFetal diaphragmdeveloped onright sideMaternal lumbarvertebra Fig. 4.147 Lower esophagus and upper stomach showing a hiatal hernia. Radiograph using barium. Fig. 4.148 Coronal CT of hiatal hernia. Fig. 4.149 Retroperitoneal position of the kidneys in the posterior abdominal region. Inferior vena cavaDiaphragmRight suprarenal glandLeft suprarenal glandRight kidneyLeft kidneyCut edges of peritoneumAbdominal aortaEsophagus Fig. 4.150 Structures related to the anterior surface of each kidney.

1	Fig. 4.150 Structures related to the anterior surface of each kidney. StomachLiverPancreasDescending colonSmall intestineSpleenJejunumRight colic flexureLeft colic flexureDescending part of duodenumRight suprarenal glandLeft suprarenal gland Fig. 4.151 Structures related to the posterior surface of each kidney. Fig. 4.152 Organization of fat and fascia surrounding the kidney. Fig. 4.153 Internal structure of the kidney. Renal arteryRenal veinPyramid in renal medullaRenal sinusMinor calyxRenal cortexRenal papillaRenal columnRenal pelvisMajor calyxUreterHilum of kidney Fig. 4.154 A. Renal vasculature. B. CT image showing long left renal vein crossing the midline. Fig. 4.155 Ureters. Internal iliac arteryExternal iliac arteryCommon iliac arteryGonadal arteriesLeft kidneyLeft renal arteryRight renal arteryRight kidneyAbdominal aortaBladderUreterThird constriction—entrance to bladderSecond constriction—pelvic inletFirst constriction—ureteropelvic junctionUreter

1	Fig. 4.156 Low-dose axial CT of urinary tract (CT KUB) displays stone in left renal pelvis. Fig. 4.157 Tumor in the right kidney growing toward, and possibly invading, the duodenum. Computed tomogram in the axial plane. Fig. 4.158 Tumor in the right kidney spreading into the right renal vein. Computed tomogram in the axial plane. Fig. 4.159 Transitional cell carcinoma in the pelvis of the right kidney. Coronal computed tomogram reconstruction. Fig. 4.160 This radiograph demonstrates a double-J stent (anteroposterior view). The superior aspect of the double-J stent is situated within the renal pelvis. The stent passes through the ureter, describing the path of the ureter, and the tip of the double-J stent is projected over the bladder, which appears as a slightly dense area on the radiograph.

1	Fig. 4.161 Kidney transplant. A. This image demonstrates an MR angiogram of the bifurcation of the aorta. Attaching to the left external iliac artery is the donor artery for a kidney that has been transplanted into the left iliac fossa. B. Abdominal computed tomogram, in the axial plane, showing the transplanted kidney in the left iliac fossa. AAbdominal aortaCommon iliac arteryExternal iliacarteryInternal iliacarteryThe left external iliac arteryhas been used to connectto the donor kidneyTransplant kidneyin the left iliacfossa Fig. 4.162 Coronal view of 3-D urogram using multidetector computed tomography. Fig. 4.163 Arterial supply to the suprarenal glands. Fig. 4.164 Abdominal aorta.

1	Fig. 4.162 Coronal view of 3-D urogram using multidetector computed tomography. Fig. 4.163 Arterial supply to the suprarenal glands. Fig. 4.164 Abdominal aorta. Fig. 4.165 Volume-rendered reconstruction using multidetector computed tomography of patient with an infrarenal abdominal aortic aneurysm before (A) and after (B) endovascular aneurysm repair. Note the image only demonstrates the intraluminal contrast and not the entire vessel. White patches in the aorta represent intramural calcium. Fig. 4.166 Inferior vena cava. Fig. 4.167 Lumbar veins. Fig. 4.168 Abdominal lymphatics. Inferior vena cavaIntestinal trunkRight lumbar trunk with lateral aortic (lumbar) nodesLeft lumbar trunk withlateral aortic (lumbar) nodesExternal iliac nodesExternal iliac nodesInternal iliac nodesCommon iliac nodesCeliac nodesSuperior mesenteric nodesInferior mesenteric nodesCisterna chyliPre-aortic nodes Fig. 4.169 Sympathetic trunks passing through the posterior abdominal region.

1	Fig. 4.169 Sympathetic trunks passing through the posterior abdominal region. Fig. 4.170 Prevertebral plexus and ganglia in the posterior abdominal region. Fig. 4.171 Nerve fibers passing through the abdominal prevertebral plexus and ganglia. Fig. 4.172 Prevertebral ganglia associated with the prevertebral plexus. Fig. 4.173 Lumbar plexus. T12L1L2L3L4To lumbosacral trunkObturator nerveFemoralnerveTo iliacusmuscleLateral cutaneousnerve of thighGenitofemoralnerveIlio-inguinal nerveIliohypogastricnerve Fig. 4.174 Lumbar plexus in the posterior abdominal region. Subcostal nerveIliohypogastric nerveIlio-inguinal nerveLateral cutaneous nerve of thighFemoral nerveGenitofemoral nerveObturator nerveSubcostal nerve (T12)Iliohypogastric nerve (L1)Psoas major muscleIlio-inguinal nerve (L1)Lateral cutaneous nerve of thigh (L2,L3)Femoral nerve (L2 to L4)Genitofemoral nerve (L1,L2)Iliacus muscleObturator nerve (L2 to L4)Lumbosacral trunks(L4,L5)

1	Fig. 4.175 Cutaneous distribution of the nerves from the lumbar plexus. L1T12T12T12T11T11T10T10Ilio-inguinal nerve (L1)Ilio-inguinal nerve (L1)Genitofemoral nerve (L1,L2)Lateral cutaneous nerve of thigh (L2,L3)Obturator nerve (L2 to L4)Cutaneous branch of obturator nerve (L2 to L4)Femoral nerve (L2 to L4)Lateral cutaneous branchof iliohypogastric nerve (L1)Anterior cutaneous branchof iliohypogastric nerve (L1)Femoral branch of genitofemoral nerve (L1,L2)Lateral cutaneous nerve of thigh (L2,L3)Intermediate cutaneous from femoral nerveMedial cutaneous fromfemoral nerveSaphenous nerve from femoral nerve Fig. 4.176 Interior view of the abdominal region of a man. Palpable bony landmarks, the inguinal ligament, and the position of the diaphragm are indicated. Fig. 4.177 Groin. A. In a man. B. In a woman. C. Examination of the superficial inguinal ring and related regions of the inguinal canal in a man.

1	Fig. 4.177 Groin. A. In a man. B. In a woman. C. Examination of the superficial inguinal ring and related regions of the inguinal canal in a man. Deep inguinal ringAponeurosis of external obliqueAponeurosis of external obliqueSuperficial inguinal ringPosition of pubic symphysisABCAnterior superior iliac spineSpermatic cordInguinal ligamentFemoral arteryDeep inguinal ringSuperficial inguinal ringPosition of pubic symphysisAnterior superior iliac spineRound ligament of uterusInguinal ligamentFemoral artery Fig. 4.178 Landmarks used for establishing the positions of lumbar vertebrae are indicated. Anterior view of the abdominal region of a man. End of ninth costal cartilageLower edge of tenthcostal cartilageHighest point on iliac crestLILIILIIILIVLVPubic symphysisTubercle of crest of iliumJugular notchTranspyloric planeSubcostal planeSupracristal planeIntertubercular planeUmbilicus910

1	Fig. 4.179 LI vertebral level and the important viscera associated with this level. Anterior view of the abdominal region of a man. KidneyEnd of ninth costal cartilageLI9Pubic symphysisJugular notchDuodenumTranspyloric planeNeck of pancreasSuperior mesenteric artery Fig. 4.180 Major vessels projected onto the body’s surface. Anterior view of the abdominal region of a man. Upper border of LICeliac trunkAortaLower border of LISuperior mesenteric arteryLIII Inferior mesenteric arteryLIV Bifurcation of aortaLV Joining of common iliacveins to form the inferiorvena cavaLII Approximate originof renal arteryLITXIILIILIIILIVLVPubic symphysisJugular notchTranspyloric planeSubcostal planeSupracristal planeIntertubercular planeUmbilicusInferior vena cava910 Fig. 4.181 Abdominal quadrants and the positions of major viscera. Anterior view of a man. Fig. 4.182 The nine regions of the abdomen. Anterior view of a woman.

1	Fig. 4.181 Abdominal quadrants and the positions of major viscera. Anterior view of a man. Fig. 4.182 The nine regions of the abdomen. Anterior view of a woman. Fig. 4.183 Surface projection of the kidneys and ureters. Posterior view of the abdominal region of a woman. Fig. 4.184 Surface projection of the spleen. Posterior view of a man. eFig. 4.187 Transjugular liver biopsy needle in the right hepatic vein. Radiograph. eFig. 4.188 Subphrenic collection of pus and gas. Computed tomogram in the axial plane. Subphrenic collection of pus and gas eFig. 4.189 Position of a transjugular intrahepatic portosystemic shunt stent. Radiograph. eFig. 4.190 Functioning transjugular intrahepatic portosystemic shunt. Venogram. Fig. 4.185 Tumor in the head of the pancreas. Computed tomogram in the axial plane. eFig. 4.191 A computed tomogram, in the axial plane, of the pelvis demonstrates a loop of sigmoid colon with numerous diverticula and a large abscess in the pelvic cavity.

1	eFig. 4.191 A computed tomogram, in the axial plane, of the pelvis demonstrates a loop of sigmoid colon with numerous diverticula and a large abscess in the pelvic cavity. Fig. 4.186 This postcontrast computed tomogram, in the axial plane, demonstrates two metastases situated within the right lobe of the liver. The left lobe of the liver is clear. The larger of the two metastases is situated to the right of the middle hepatic vein, which lies in the principal plane of the liver dividing the left and right sides of the liver. Table 4.1 Abdominal wall muscles Table 4.2 Posterior abdominal wall muscles Table 4.3 Branches of the abdominal aorta Table 4.4 Lymphatic drainage Table 4.5 Referred pain pathways (visceral afferents) Table 4.6 Branches of the lumbar plexus In the clinic

1	Table 4.3 Branches of the abdominal aorta Table 4.4 Lymphatic drainage Table 4.5 Referred pain pathways (visceral afferents) Table 4.6 Branches of the lumbar plexus In the clinic Access to the abdomen and its contents is usually obtained through incisions in the anterior abdominal wall. Traditionally, incisions have been placed at and around the region of surgical interest. The size of these incisions was usually large to allow good access and optimal visualization of the abdominal cavity. As anesthesia has developed and muscle-relaxing drugs have become widely used, the abdominal incisions have become smaller. Currently, the most commonly used large abdominal incision is a central craniocaudad incision from the xiphoid process to the symphysis pubis, which provides wide access to the whole of the abdominal contents and allows an exploratory procedure to be performed (laparotomy). In the clinic

1	In the clinic Laparoscopic surgery, also known as minimally invasive or keyhole surgery, is performed by operating through a series of small incisions no more than 1 to 2 cm in length. As the incisions are much smaller than those used in traditional abdominal surgery, patients experience less postoperative pain and have shorter recovery times. There is also a favorable cosmetic outcome with smaller scars. Several surgical procedures such as appendectomy, cholecystectomy, and hernia repair, as well as numerous orthopaedic, urological, and gynecological procedures, are now commonly performed laparoscopically.

1	During the operation, a camera known as a laparoscope is used to transmit live, magnified images of the surgical field to a monitor viewed by the surgeon. The camera is inserted into the abdominal cavity through a small incision, called a port-site, usually at the umbilicus. In order to create enough space to operate, the abdominal wall is elevated by inflating the cavity with gas, typically carbon dioxide. Other long, thin surgical instruments are then introduced through additional port-sites, which can be used by the surgeon to operate. The placement of these port-sites is carefully planned to allow optimal access to the surgical field.

1	Laparoscopic surgery has been further enhanced with the use of surgical robots. Using these systems the surgeon moves the surgical instruments indirectly by controlling robotic arms, which are inserted into the operating field through small incisions. Robot-assisted surgery is now routinely used worldwide and has helped overcome some of the limitations of laparoscopy by enhancing the surgeon’s dexterity. The robotic system is precise, provides the surgeon with a 3D view of the surgical field, and allows improved degree of rotation and manipulation of the surgical instruments. Several procedures such as prostatectomy and cholecystectomy can now be performed with this method.

1	Laparoendoscopic single-site surgery, also known as single-port laparoscopy, is the most recent advance in laparoscopic surgery. This method uses a single incision, usually umbilical, to introduce a port with several operating channels and can be performed with or without robotic assistance. Benefits include less postoperative pain, a faster recovery time, and an even better cosmetic result than traditional laparoscopic surgery. In the clinic

1	In the clinic In men, the cremaster muscle and cremasteric fascia form the middle or second covering of the spermatic cord. This muscle and its associated fascia are supplied by the genital branch of the genitofemoral nerve (L1/L2). Contraction of this muscle and the resulting elevation of the testis can be stimulated by a reflex arc. Gently touching the skin at and around the anterior aspect of the superior part of the thigh stimulates the sensory fibers in the ilio-inguinal nerve. These sensory fibers enter the spinal cord at level L1. At this level, the sensory fibers stimulate the motor fibers carried in the genital branch of the genitofemoral nerve, which results in contraction of the cremaster muscle and elevation of the testis.

1	The cremasteric reflex is more active in children, tending to diminish with age. As with many reflexes, it may be absent in certain neurological disorders. Although it can be used for testing spinal cord function at level L1 in men, its clinical use is limited. In the clinic Masses around the groin Around the groin there is a complex confluence of anatomical structures. Careful examination and good anatomical knowledge allow determination of the correct anatomical structure from which the mass arises and therefore the diagnosis. The most common masses in the groin are hernias. The key to groin examination is determining the position of the inguinal ligament. The inguinal ligament passes between the anterior superior iliac spine laterally and the pubic tubercle medially. Inguinal hernias are above the inguinal ligament and are usually more apparent on standing. A visual assessment of the lump is necessary, bearing in mind the anatomical landmarks of the inguinal ligament.

1	In men, it is wise to examine the scrotum to check for a lump. If an abnormal mass is present, an inability to feel its upper edge suggests that it may originate from the inguinal canal and might be a hernia. By placing the hand over the lump and asking the patient to cough, the lump bulges outward. An attempt should be made to reduce the swelling by applying gentle, firm pressure over the lump. If the lump is reducible, the hand should be withdrawn and careful observation will reveal recurrence of the mass. The position of an abnormal mass in the groin relative to the pubic tubercle is very important, as are the presence of increased temperature and pain, which may represent early signs of strangulation or infection. As a general rule: An inguinal hernia appears through the superficial inguinal ring above the pubic tubercle and crest. A femoral hernia (see below) appears through the femoral canal below and lateral to the pubic tubercle.

1	A femoral hernia (see below) appears through the femoral canal below and lateral to the pubic tubercle. A hernia is the protrusion of a viscus, in part or in whole, through a normal or abnormal opening. The viscus usually carries a covering of parietal peritoneum, which forms the lining of the hernial sac. Hernias occur in a variety of regions. The commonest site is the groin of the lower anterior abdominal wall. In some patients, inguinal hernias are present from birth (congenital) and are caused by the persistence of the processus vaginalis and the passage of viscera through the inguinal canal. Acquired hernias occur in older patients and causes include raised intraabdominal pressure (e.g., from repeated coughing associated with lung disease), damage to nerves of the anterior abdominal wall (e.g., from surgical abdominal incisions), and weakening of the walls of the inguinal canal.

1	One of the potential problems with hernias is that bowel and fat may become stuck within the hernial sac. This can cause appreciable pain and bowel obstruction, necessitating urgent surgery. Another potential risk is strangulation of the hernia, in which the blood supply to the bowel is cut off at the neck of the hernial sac, rendering the bowel ischemic and susceptible to perforation (Fig. 4.51). The hernial sac of an indirect inguinal hernia enters the deep inguinal ring and passes through the inguinal canal. If the hernia is large enough, the hernial sac may emerge through the superficial inguinal ring. In men, such a hernia may extend into the scrotum (Fig. 4.52). The hernial sac of a direct inguinal hernia pushes forward through the posterior wall of the inguinal canal immediately posterior to the superficial inguinal ring. The hernia protrudes directly forward medial to the inferior epigastric vessels and through the superficial inguinal ring.

1	The differentiation between an indirect and a direct inguinal hernia is made during surgery when the inferior epigastric vessels are identified at the medial edge of the deep internal ring: An indirect hernial sac passes lateral to the inferior epigastric vessels. A direct hernia is medial to the inferior epigastric vessels. Inguinal hernias occur more commonly in men than in women possibly because men have a much larger inguinal canal than women.

1	A direct hernia is medial to the inferior epigastric vessels. Inguinal hernias occur more commonly in men than in women possibly because men have a much larger inguinal canal than women. A femoral hernia passes through the femoral canal and into the medial aspect of the anterior thigh. The femoral canal lies at the medial edge of the femoral sheath, which contains the femoral artery, femoral vein, and lymphatics. The neck of the femoral canal is extremely narrow and is prone to trapping bowel within the sac, so making this type of hernia irreducible and susceptible to bowel strangulation. Femoral hernias are usually acquired, are not congenital, and most commonly occur in middle-aged and elderly populations. In addition, because women generally have wider pelvises than men, they tend to occur more commonly in women.

1	The groin can loosely be defined as the area where the leg meets the trunk near the midline. Here the abdominal muscles of the trunk blend in with the adductor muscles of the thigh, the medial end of the inguinal ligament attaches to the pubic tubercle, the pubic symphysis attaches the two pubic bones together, and the superficial (external) inguinal ring occurs. It also is in and around this region where there is considerable translation of force during most athletic and sporting activities. Pain in the groin or pubic region can be due to numerous causes, which include inflammatory changes at the pubic symphysis, insertional problems of the rectus abdominis/adductor longus, and hernias.

1	Umbilical hernias are rare. Occasionally, they are congenital and result from failure of the small bowel to return to the abdominal cavity from the umbilical cord during development. After birth, umbilical hernias may result from incomplete closure of the umbilicus (navel). Overall, most of these hernias close in the first year of life, and surgical repair is not generally attempted until later. Para-umbilical hernias may occur in adults at and around the umbilicus and often have small necks, so requiring surgical treatment. Incisional hernias occur through a defect in a scar of a previous abdominal operation. Usually, the necks of these hernias are wide and do not therefore strangulate the viscera they contain. A spigelian hernia passes upward through the arcuate line into the lateral border at the lower part of the posterior rectus sheath. It may appear as a tender mass on one side of the lower anterior abdominal wall.

1	Abdominopelvic cavity hernias can also develop in association with the pelvic walls, and sites include the obturator canal, the greater sciatic foramen and above and below the piriformis muscle. In the clinic A small volume of peritoneal fluid within the peritoneal cavity lubricates movement of the viscera suspended in the abdominal cavity. It is not detectable on any available imaging such as ultrasound or computed tomography. In various pathological conditions (e.g., in liver cirrhosis, acute pancreatitis, or heart failure) the volume of peritoneal fluid can increase; this is known as ascites. In cases of high volume of free intraperitoneal fluid, marked abdominal distention can be observed (Fig. 4.56). The peritoneal space has a large surface area, which facilitates the spread of disease through the peritoneal cavity and over the bowel and visceral surfaces. Conversely, this large surface area can be used for administering certain types of treatment and a number of procedures.

1	Patients with obstructive hydrocephalus (an excessive accumulation of cerebrospinal fluid within the cerebral ventricular system) require continuous drainage of this fluid. This is achieved by placing a fine-bore catheter through the skull into the cerebral ventricles and placing the extracranial part of the tube beneath the scalp and skin of the neck and chest wall, and then through the abdominal wall into the peritoneal cavity. Cerebrospinal fluid drains through the tube into the peritoneal cavity, where it is absorbed. People who develop renal failure require dialysis to live. There are two methods.

1	People who develop renal failure require dialysis to live. There are two methods. In the first method (hemodialysis), blood is taken from the circulation, dialyzed through a complex artificial membrane, and returned to the body. A high rate of blood flow is required to remove excess body fluid, exchange electrolytes, and remove noxious metabolites. To accomplish this, either an arteriovenous fistula is established surgically (by connecting an artery to a vein, usually in the upper limb, and requiring approximately six weeks to “mature”) and is cannulated each time the patient returns for dialysis, or a large-bore cannula is placed into the right atrium, through which blood can be aspirated and returned.

1	In the second method (peritoneal dialysis), the peritoneum is used as the dialysis membrane. The large surface area of the peritoneal cavity is an ideal dialysis membrane for fluid and electrolyte exchange. To accomplish dialysis, a small tube is inserted through the abdominal wall and dialysis fluid is injected into the peritoneal cavity. Electrolytes and molecules are exchanged across the peritoneum between the fluid and blood. Once dialysis is completed, the fluid is drained. Peritoneal spread of disease

1	Peritoneal spread of disease The large surface area of the peritoneal cavity allows infection and malignant disease to spread easily throughout the abdomen (Fig. 4.57). If malignant cells enter the peritoneal cavity by direct invasion (e.g., from colon or ovarian cancer), spread may be rapid. Similarly, a surgeon excising a malignant tumor and releasing malignant cells into the peritoneal cavity may cause an appreciable worsening of the patient’s prognosis. Infection can also spread across the large surface area. The peritoneal cavity can also act as a barrier to, and container of, disease. Intraabdominal infection therefore tends to remain below the diaphragm rather than spread into other body cavities.

1	The peritoneal cavity can also act as a barrier to, and container of, disease. Intraabdominal infection therefore tends to remain below the diaphragm rather than spread into other body cavities. A perforated bowel (e.g., caused by a perforated duodenal ulcer) often leads to the release of gas into the peritoneal cavity. This peritoneal gas can be easily visualized on an erect chest radiograph—gas can be demonstrated in extremely small amounts beneath the diaphragm. A patient with severe abdominal pain and subdiaphragmatic gas needs a laparotomy (Fig. 4.58). In the clinic The greater omentum

1	In the clinic The greater omentum When a laparotomy is performed and the peritoneal cavity is opened, the first structure usually encountered is the greater omentum. This fatty double-layered vascular membrane hangs like an apron from the greater curvature of the stomach, drapes over the transverse colon, and lies freely suspended within the abdominal cavity. It is often referred to as the “policeman of the abdomen” because of its apparent ability to migrate to any inflamed area and wrap itself around the organ to wall off inflammation. When a part of bowel becomes inflamed, it ceases peristalsis. This aperistaltic area is referred to as a local paralytic ileus. The remaining noninflamed part of the bowel continues to move and “massages” the greater omentum to the region where there is no peristalsis. The localized inflammatory reaction spreads to the greater omentum, which then adheres to the diseased area of bowel.

1	The greater omentum is also an important site for metastatic tumor spread. Direct omental spread by a transcoelomic route is common for carcinoma of the ovary. As the metastases develop within the greater omentum, it becomes significantly thickened. In computed tomography imaging and during laparotomy, the thickened omentum is referred to as an “omental cake.” In the clinic Epithelial transition between the abdominal esophagus and stomach

1	In the clinic Epithelial transition between the abdominal esophagus and stomach The gastroesophageal junction is demarcated by a transition from one epithelial type (nonkeratinized stratified squamous epithelium) to another epithelial type (columnar epithelium). In some people, the histological junction does not lie at the anatomical gastroesophageal junction but occurs more proximally in the lower third of the esophagus. This may predispose these people to esophageal ulceration and is also associated with an increased risk of adenocarcinoma. In certain conditions, like gastroesophageal reflux, the stratified squamous epithelium in the esophagus can undergo metaplasia and the epithelium in the lower esophagus is replaced by columnar epithelium, a condition called Barrett’s esophagus. The presence of Barrett’s esophagus predisposes these people to the development of esophageal malignancy (adenocarcinoma). In the clinic

1	Duodenal ulcers usually occur in the superior part of the duodenum and are much less common than they were 50 years ago. At first, there was no treatment and patients died from hemorrhage or peritonitis. As surgical techniques developed, patients with duodenal ulcers were subjected to extensive upper gastrointestinal surgery to prevent ulcer recurrence and for some patients the treatment was dangerous. As knowledge and understanding of the mechanisms for acid secretion in the stomach increased, drugs were developed to block acid stimulation and secretion indirectly (histamine H2-receptor antagonists) and these have significantly reduced the morbidity and mortality rates of this disease. Pharmacological therapy can now directly inhibit the cells of the stomach that produce acid with, for example, proton pump inhibitors. Patients are also screened for the bacteria Helicobacter pylori, which when eradicated (by antibiotic treatment) significantly reduces duodenal ulcer formation.

1	Anatomically, duodenal ulcers tend to occur either anteriorly or posteriorly. Posterior duodenal ulcers erode either directly onto the gastroduodenal artery or, more commonly, onto the posterior superior pancreaticoduodenal artery, which can produce torrential hemorrhage, which may be fatal in some patients. Treatment may involve extensive upper abdominal surgery with ligation of the vessels or by endovascular means whereby the radiologist may place a very fine catheter retrogradely from the femoral artery into the celiac artery. The common hepatic artery and the gastroduodenal artery are cannulated and the bleeding area may be blocked using small coils, which stem the flow of blood.

1	Anterior duodenal ulcers erode into the peritoneal cavity, causing peritonitis. This intense inflammatory reaction and the local ileus promote adhesion of the greater omentum, which attempts to seal off the perforation. The stomach and duodenum usually contain considerable amounts of gas, which enters the peritoneal cavity and can be observed on a chest radiograph of an erect patient as subdiaphragmatic gas. In most instances, treatment for the ulcer perforation is surgical. In the clinic Examination of the upper and lower It is often necessary to examine the esophagus, stomach, duodenum, proximal jejunum, and colon for disease. After taking an appropriate history and examining the patient, most physicians arrange a series of simple blood tests to look for bleeding, inflammation, and tumors. The next steps in the investigation assess the three components of any loop of bowel, namely, the lumen, the wall, and masses extrinsic to the bowel, which may compress or erode into it.

1	Examination of the bowel lumen Barium sulfate solutions may be swallowed by the patient and can be visualized using an X-ray fluoroscopy unit. The lumen can be examined for masses (e.g., polyps and tumors) and peristaltic waves can be assessed. Patients may also be given carbon dioxide–releasing granules to fill the stomach so that the barium thinly coats the mucosa, resulting in images displaying fine mucosal detail. These tests are relatively simple and can be used to image the esophagus, stomach, duodenum, and small bowel. For imaging the large bowel, a barium enema can be used to introduce barium sulfate into the colon. Colonoscopy and CT colonography are also used. Examination of the bowel wall and extrinsic masses

1	Examination of the bowel wall and extrinsic masses Endoscopy is a minimally invasive diagnostic medical procedure that can be used to assess the interior surfaces of an organ by inserting a tube into the body. The instrument is typically made of a flexible plastic material through which a light source and eyepiece are attached at one end. The images are then projected to a monitor. Some systems allow passage of small instruments through the main bore of the endoscope to obtain biopsies and to also undertake small procedures (e.g., the removal of polyps). In gastrointestinal and abdominal medicine an endoscope is used to assess the esophagus, stomach, duodenum, and proximal small bowel (Figs. 4.72 to 4.75). The tube is swallowed by the patient under light sedation and is extremely well tolerated.

1	Assessment of the colon (colonoscopy) is performed by passage of the long flexible tube through the anus and into the rectum. The endoscope is then advanced into the colon to the cecum and sometimes to the terminal ileum. The patient undergoes bowel preparation before the examination to allow good visualization of the entire large bowel. Specially designed solutions are taken orally to help clear the bowel of fecal material. Air, water, and suction may be used during the examination to improve visualization. Biopsies, polyp removal, cauterization of bleeding, and stent placement can also be performed using additional instruments that can be passed through special openings in the colonoscope.

1	Cross sectional imaging using computed tomography or magnetic resonance is another way to assess the bowel lumen and wall. Magnetic resonance is particularly useful in assessment of the small bowel because it allows dynamic assessment of bowel distention and motility and provides good visualization of segmental or continuous bowel wall thickening and mural or mucosal ulcerations and also can demonstrate increased vascularity of the small bowel mesentery (Fig. 4.76). It is usually performed in patients with inflammatory bowel diseases, such as Crohn’s disease.

1	CT colonography (also called virtual colonoscopy or CT pneumocolon) is an alternative way to visualize and assess the colon for abnormal lesions such as polyps or strictures with the use spiral CT to produce high-resolution 3D views of the large bowel. It is less invasive than traditional colonoscopy, but to achieve good-quality images the patient needs to take bowel preparations to ensure bowel cleansing, and the colon needs to be insufflated with CO2. If a tumor is present (Fig. 4.77), both CT and MRI are used to assess regional disease (MRI), abnormal lymph nodes (MRI, CT), and distant metastases (CT). In the clinic

1	In the clinic A Meckel’s diverticulum (Fig. 4.78) is the remnant of the proximal part of the yolk stalk (vitelline duct) that extends into the umbilical cord in the embryo and lies on the antimesenteric border of the ileum. It appears as a blind-ended tubular outgrowth of bowel. Although it is an uncommon finding (occurring in approximately 2% of the population), it is always important to consider the diagnosis of Meckel’s diverticulum because it does produce symptoms in a small number of patients. It may contain gastric mucosa and therefore lead to ulceration and hemorrhage. Other typical complications include intussusception, diverticulitis, and obstruction. In the clinic

1	In the clinic These imaging techniques can provide important information about the wall of the bowel that may not be obtained from barium or endoscopic studies. Thickening of the wall may indicate inflammatory change or tumor and is always regarded with suspicion. If a tumor is demonstrated, the locoregional spread can be assessed, along with lymphadenopathy and metastatic spread. Endoscopic ultrasound (EUS) uses a small ultrasound device placed on the end of the endoscope to assess the upper gastrointestinal tract. It can produce extremely high-powered views of the mucosa and submucosa and therefore show whether a tumor is resectable. It also provides guidance to the clinician when taking a biopsy. In the clinic Carcinoma of the stomach

1	In the clinic Carcinoma of the stomach Carcinoma of the stomach is a common gastrointestinal malignancy. Chronic gastric inflammation (gastritis), pernicious anemia, and polyps predispose to the development of this aggressive cancer, which is usually not diagnosed until late in the course of the disease. Symptoms include vague epigastric pain, early fullness with eating, bleeding leading to chronic anemia, and obstruction.

1	The diagnosis may be made using barium and conventional radiology or endoscopy, which allows a biopsy to be obtained at the same time. Ultrasound scanning is used to check the liver for metastatic spread, and, if negative, computed tomography is carried out to assess for surgical resectability. If carcinoma of the stomach is diagnosed early, a curative surgical resection is possible. However, because most patients do not seek treatment until late in the disease, the overall 5-year survival rate is between 5% and 20%, with a mean survival time of between 5 and 8 months. In the clinic

1	In the clinic Acute appendicitis is an abdominal emergency. It usually occurs when the appendix is obstructed by either a fecalith or enlargement of the lymphoid nodules. Within the obstructed appendix, bacteria proliferate and invade the appendix wall, which becomes damaged by pressure necrosis. In some instances, this may resolve spontaneously; in other cases, inflammatory change (Figs. 4.86 and 4.87) continues and perforation ensues, which may lead to localized or generalized peritonitis. Most patients with acute appendicitis have localized tenderness in the right groin. Initially, the pain begins as a central, periumbilical, colicky type of pain, which tends to come and go. After 6 to 10 hours, the pain tends to localize in the right iliac fossa and becomes constant. Patients may develop a fever, nausea, and vomiting. The etiology of the pain for appendicitis is described in Case 1 of Chapter 1 on p. 48. The treatment for appendicitis is appendectomy. In the clinic

1	The treatment for appendicitis is appendectomy. In the clinic Congenital disorders of the gastrointestinal tract The normal positions of the abdominal viscera result from a complex series of rotations that the gut tube undergoes and from the growth of the abdominal cavity to accommodate changes in the size of the developing organs (see pp. 265-268). A number of developmental anomalies can occur during gut development, many of which appear in the neonate or infant, and some of which are surgical emergencies. Occasionally, such disorders are diagnosed only in adults.

1	Malrotation is incomplete rotation and fixation of the midgut after it has passed from the umbilical sac and returned to the abdominal coelom (Figs. 4.93 and 4.94). The proximal attachment of the small bowel mesentery begins at the suspensory muscle of duodenum (ligament of Treitz), which determines the position of the duodenojejunal junction. The mesentery of the small bowel ends at the level of the ileocecal junction in the right lower quadrant. This long line of fixation of the mesentery prevents accidental twists of the gut. If the duodenojejunal flexure or the cecum does not end up in its usual site, the origin of the small bowel mesentery shortens, which permits twisting of the small bowel around the axis of the superior mesenteric artery. Twisting of the bowel, in general, is termed volvulus. Volvulus of the small bowel may lead to a reduction of blood flow and infarction.

1	In some patients, the cecum ends up in the midabdomen. From the cecum and the right side of the colon a series of peritoneal folds (Ladd’s bands) develop that extend to the right undersurface of the liver and compress the duodenum. A small bowel volvulus may then occur as well as duodenal obstruction. Emergency surgery may be necessary to divide the bands. In the clinic A bowel obstruction can be either functional or due to a true obstruction. Mechanical obstruction is caused by an intraluminal, mural, or extrinsic mass which can be secondary to a foreign body, obstructing tumor in the wall, or extrinsic compression from an adhesion, or embryological band (Fig. 4.95).

1	A functional obstruction is usually due to an inability of the bowel to peristalse, which again has a number of causes, and most frequently is a postsurgical state due to excessive intraoperative bowel handling. Other causes may well include abnormality of electrolytes (e.g., sodium and potassium) rendering the bowel paralyzed until correction has occurred. The signs and symptoms of obstruction depend on the level at which the obstruction has occurred. The primary symptom is central abdominal, intermittent, colicky pain as the peristaltic waves try to overcome the obstruction. Abdominal distention will occur if it is a low obstruction (distal), allowing more proximal loops of bowel to fill with fluid. A high obstruction (in the proximal small bowel) may not produce abdominal distention. Vomiting and absolute constipation, including the inability to pass flatus, will ensue.

1	Vomiting and absolute constipation, including the inability to pass flatus, will ensue. Early diagnosis is important because considerable fluid and electrolytes enter the bowel lumen and fail to be reabsorbed, which produces dehydration and electrolyte abnormalities. Furthermore, the bowel continues to distend, compromising the blood supply within the bowel wall, which may lead to ischemia and perforation. The symptoms and signs are variable and depend on the level of obstruction. Small bowel obstruction is typically caused by adhesions following previous surgery, and history should always be sought for any operations or abdominal interventions (e.g., previous appendectomy). Other causes include bowel passing into hernias (e.g., inguinal) and bowel twisting on its own mesentery (volvulus). Examination of hernial orifices is mandatory in patients with bowel obstruction (Fig. 4.96).

1	Large bowel obstruction is commonly caused by a tumor. Other potential causes include hernias and inflammatory diverticular disease of the sigmoid colon (Fig. 4.97). The treatment is intravenous replacement of fluid and electrolytes, analgesia, and relief of obstruction. The passage of a nasogastric tube allows aspiration of fluid from the stomach. In many instances, small bowel obstruction, typically secondary to adhesions, will settle with nonoperative management. Large bowel obstruction may require an urgent operation to remove the obstructing lesion, or a temporary bypass procedure (e.g., defunctioning colostomy) (Fig. 4.98). In the clinic

1	In the clinic Diverticular disease is the development of multiple colonic diverticula, predominantly throughout the sigmoid colon, though the whole colon may be affected (Fig. 4.99). The sigmoid colon has the smallest diameter of any portion of the colon and is therefore the site where intraluminal pressure is potentially the highest. Poor dietary fiber intake and obesity are also linked to diverticular disease. The presence of multiple diverticula does not necessarily mean the patient requires any treatment. Moreover, many patients have no other symptoms or signs. Patients tend to develop symptoms and signs when the neck of the diverticulum becomes obstructed by feces and becomes infected. Inflammation may spread along the wall, causing abdominal pain. When the sigmoid colon becomes inflamed (diverticulitis), abdominal pain and fever ensue (Fig. 4.100).

1	Because of the anatomical position of the sigmoid colon there are a number of complications that may occur. The diverticula can perforate to form an abscess in the pelvis. The inflammation may produce an inflammatory mass, obstructing the left ureter. Inflammation may also spread to the bladder, producing a fistula between the sigmoid colon and the bladder. In these circumstances patients may develop a urinary tract infection and rarely have fecal material and gas passing per urethra. The diagnosis is based upon clinical examination and often CT scanning. In the first instance, patients will be treated with antibiotic therapy; however, a surgical resection may be necessary if symptoms persist. In the clinic It is occasionally necessary to surgically externalize bowel to the anterior abdominal wall. Externalization of bowel plays an important role in patient management. These extraanatomical bypass procedures use our anatomical knowledge and in many instances are life saving.

1	Gastrostomy is performed when the stomach is attached to the anterior abdominal wall and a tube is placed through the skin into the stomach. Typically this is performed to feed the patient when it is impossible to take food and fluid orally (e.g., complex head and neck cancer). The procedure can be performed either surgically or through a direct needlestick puncture under sedation in the anterior abdominal wall. Similarly the jejunum is brought to the anterior abdominal wall and fixed. The jejunostomy is used as a site where a feeding tube is placed through the anterior abdominal wall into the proximal efferent small bowel. An ileostomy is performed when small bowel contents need to be diverted from the distal bowel. An ileostomy is often performed to protect a distal surgical anastomosis, such as in the colon to allow healing after surgery.

1	There are a number of instances when a colostomy may be necessary. In many circumstances it is performed to protect the distal large bowel after surgery. A further indication would include large bowel obstruction with imminent perforation wherein a colostomy allows decompression of the bowel and its contents. This is a safe and temporizing procedure performed when the patient is too unwell for extensive bowel surgery. It is relatively straightforward and carries reduced risk, preventing significant morbidity and mortality. An end colostomy is necessary when the patient has undergone a surgical resection of the rectum and anus (typically for cancer).

1	An end colostomy is necessary when the patient has undergone a surgical resection of the rectum and anus (typically for cancer). An ileal conduit is an extraanatomical procedure and is performed after resection of the bladder for tumor. In this situation a short segment of small bowel is identified. The bowel is divided twice to produce a 20-cm segment of small bowel on its own mesentery. This isolated segment of bowel is used as a conduit. The remaining bowel is joined together. The proximal end is anastomosed to the ureters, and the distal end is anastomosed to the anterior abdominal wall. Hence, urine passes from the kidneys into the ureters and through the short segment of small bowel to the anterior abdominal wall.

1	When patients have either an ileostomy, colostomy, or ileal conduit it is necessary for them to fix a collecting bag onto the anterior abdominal wall. Contrary to one’s initial thoughts these bags are tolerated extremely well by most patients and allow patients to live a nearly normal and healthy life. In the clinic The pancreas develops from ventral and dorsal buds from the foregut. The dorsal bud forms most of the head, neck, and body of the pancreas. The ventral bud rotates around the bile duct to form part of the head and the uncinate process. If the ventral bud splits (becomes bifid), the two segments may encircle the duodenum. The duodenum is therefore constricted and may even undergo atresia, and be absent at birth because of developmental problems. After birth, the child may fail to thrive and may vomit due to poor gastric emptying.

1	Sometimes an annular pancreas is diagnosed in utero by ultrasound scanning. The obstruction of the duodenum may prevent the fetus from swallowing enough amniotic fluid, which may increase the overall volume of amniotic fluid in the amniotic sac surrounding the fetus (polyhydramnios). In the clinic

1	In the clinic Pancreatic cancer accounts for a significant number of deaths and is often referred to as the “silent killer.” Malignant tumors of the pancreas may occur anywhere within the pancreas but are most frequent within the head and the neck. There are a number of nonspecific findings in patients with pancreatic cancer, including upper abdominal pain, loss of appetite, and weight loss. Depending on the exact site of the cancer, obstruction of the bile duct may occur, which can produce obstructive jaundice. Although surgery is indicated in patients where there is a possibility of cure, most detected cancers have typically spread locally, invading the portal vein and superior mesenteric vessels, and may extend into the porta hepatis. Lymph node spread also is common and these factors would preclude curative surgery.

1	Given the position of the pancreas, a surgical resection is a complex procedure involving resection of the region of pancreatic tumor usually with part of the duodenum, necessitating a complex bypass procedure. In the clinic Segmental anatomy of the liver For many years the segmental anatomy of the liver was of little importance. However, since the development of liver resection surgery, the size, shape, and segmental anatomy of the liver have become clinically important, especially with regard to liver resection for metastatic disease. Indeed, with detailed knowledge of the segments, curative surgery can be performed in patients with tumor metastases.

1	Indeed, with detailed knowledge of the segments, curative surgery can be performed in patients with tumor metastases. The liver is divided by the principal plane, which divides the organ into halves of approximately equal size. This imaginary line is defined by a parasagittal line that passes through the gallbladder fossa to the inferior vena cava. It is in this plane that the middle hepatic vein is found. Importantly, the principal plane divides the left half of the liver from the right half. The lobes of the liver are unequal in size and bear only little relevance to operative anatomy. The traditional eight-segment anatomy of the liver relates to the hepatic arterial, portal, and biliary drainage of these segments (Fig. 4.116). The caudate lobe is defined as segment I, and the remaining segments are numbered in a clockwise fashion up to segment VIII. The features are extremely consistent between individuals.

1	The caudate lobe is defined as segment I, and the remaining segments are numbered in a clockwise fashion up to segment VIII. The features are extremely consistent between individuals. From a surgical perspective, a right hepatectomy would involve division of the liver in the principal plane in which segments V, VI, VII, and VIII would be removed, leaving segments I, II, III, and IV. In the clinic Gallstones are present in approximately 10% of people over the age of 40 and are more common in women. They consist of a variety of components but are predominantly a mixture of cholesterol and bile pigment. They may undergo calcification, which can be demonstrated on plain radiographs. Gallstones may be visualized incidentally as part of a routine abdominal ultrasound scan (Fig. 4.117) or on a plain radiograph.

1	The easiest way to confirm the presence of gallstones is by performing a fasting ultrasound examination of the gallbladder. The patient refrains from eating for 6 hours to ensure the gallbladder is well distended and there is little shadowing from overlying bowel gas. The examination may also identify bile duct dilation and the presence of cholecystitis. Magnetic resonance cholangiopancreatography (MRCP) is another way to image the gallbladder and biliary tree. MRCP uses fluid present in the bile ducts and in the pancreatic duct as a contrast agent to show stones as well as filling defects within the gallbladder and intrahepatic or extrahepatic bile ducts. It can demonstrate strictures in the biliary tree and can also be used to visualize liver and pancreatic anatomy (Fig. 4.118).

1	From time to time, gallstones impact in the region of Hartmann’s pouch, which is a bulbous region of the neck of the gallbladder. When the gallstone lodges in this area, the gallbladder cannot empty normally and contractions of the gallbladder wall produce severe pain. If this persists, a cholecystectomy (removal of the gallbladder) may be necessary. Sometimes the gallbladder may become inflamed (cholecystitis). If the inflammation involves the related parietal peritoneum of the diaphragm, pain may not only occur in the right upper quadrant of the abdomen but may also be referred to the shoulder on the right side. This referred pain is due to the innervation of the visceral peritoneum of the diaphragm by spinal cord levels (C3 to C5) that also innervate skin over the shoulder. In this case, one somatic sensory region of low sensory output (diaphragm) is referred to another somatic sensory region of high sensory output (dermatomes).

1	From time to time, small gallstones pass into the bile duct and are trapped in the region of the sphincter of the ampulla, which obstructs the flow of bile into the duodenum. This, in turn, produces jaundice.

1	Endoscopic retrograde cholangiopancreatography (ERCP) can be undertaken to remove obstructing gallstones within the biliary tree. This procedure combines endoluminal endoscopy with fluoroscopy to diagnose and treat problems in the biliary and pancreatic ducts. An endoscope with a side-viewing optical system is advanced through the esophagus and stomach and placed in the second part of the duodenum where the major papilla (the ampulla of Vater) is identified. This is where the pancreatic duct converges with the common bile duct. The papilla is initially examined for possible abnormalities (stuck stone or malignant growth) and a biopsy may be taken if necessary. Then either the bile duct or pancreatic duct is cannulated and a small amount of radiopaque contrast medium is injected to visualize either the bile duct (cholangiogram) or pancreatic duct (pancreatogram) (Fig. 4.119). If a stone is present, it can be removed with a stone basket or an extraction balloon. Usually, a

1	to visualize either the bile duct (cholangiogram) or pancreatic duct (pancreatogram) (Fig. 4.119). If a stone is present, it can be removed with a stone basket or an extraction balloon. Usually, a sphincterotomy is performed before stone removal to ease its passage through the distal bile duct.

1	In cases of biliary tree obstruction caused by benign or malignant strictures, a stent can be placed into the common bile duct or into one of the main hepatic ducts to allow opening of the narrowed segment. The patency of the newly inserted stent is confirmed by instillation of more contrast medium to demonstrate free flow of contrast through the stent. In the clinic Jaundice is a yellow discoloration of the skin caused by excess bile pigment (bilirubin) within the plasma. The yellow color is best appreciated by looking at the normally white sclerae of the eyes, which turn yellow. The extent of the elevation of the bile pigments and the duration for which they have been elevated account for the severity of jaundice. Simplified explanation to understanding the types of jaundice and their anatomical causes

1	Simplified explanation to understanding the types of jaundice and their anatomical causes When red blood cells are destroyed by the reticuloendothelial system, the iron from the hemoglobin molecule is recycled, whereas the porphyrin ring (globin) compounds are broken down to form fat-soluble bilirubin. On reaching the liver via the bloodstream, the fat-soluble bilirubin is converted to a water-soluble form of bilirubin. This water-soluble bilirubin is secreted into the biliary tree and then in turn into the bowel, where it forms the dark color of the stool. This type of jaundice is usually produced by conditions where there is an excessive breakdown of red blood cells (e.g., in incompatible blood transfusion and hemolytic anemia).

1	This type of jaundice is usually produced by conditions where there is an excessive breakdown of red blood cells (e.g., in incompatible blood transfusion and hemolytic anemia). The complex biochemical reactions for converting fat-soluble into water-soluble bilirubin may be affected by inflammatory change within the liver (e.g., from hepatitis or chronic liver disease, such as liver cirrhosis) and poisons (e.g., paracetamol overdose). Any obstruction of the biliary tree can produce jaundice, but the two most common causes are gallstones within the bile duct and an obstructing tumor at the head of the pancreas. In the clinic From a clinical point of view, there are two main categories of spleen disorders: rupture and enlargement.

1	In the clinic From a clinical point of view, there are two main categories of spleen disorders: rupture and enlargement. This tends to occur when there is localized trauma to the left upper quadrant. It may be associated with left lower rib fractures. Because the spleen has such an extremely thin capsule, it is susceptible to injury even when there is no damage to surrounding structures, and because the spleen is highly vascular, when ruptured, it bleeds profusely into the peritoneal cavity. Splenic rupture should always be suspected with blunt abdominal injury. Current treatments preserve as much of the spleen as possible, but some patients require splenectomy.

1	The spleen is an organ of the reticuloendothelial system involved in hematopoiesis and immunological surveillance. Diseases that affect the reticuloendothelial system (e.g., leukemia or lymphoma) may produce generalized lymphadenopathy and enlargement of the spleen (splenomegaly) (Fig. 4.120). The spleen often enlarges when performing its normal physiological functions, such as when clearing microorganisms and particulates from the circulation, producing increased antibodies in the course of sepsis, or removing deficient or destroyed erythrocytes (e.g., in thalassemia and spherocytosis). Splenomegaly may also be a result of increased venous pressure caused by congestive heart failure, splenic vein thrombosis, or portal hypertension. An enlarged spleen is prone to rupture. In the clinic Vascular supply to the gastrointestinal system

1	In the clinic Vascular supply to the gastrointestinal system The abdominal parts of the gastrointestinal system are supplied mainly by the celiac trunk and the superior mesenteric and inferior mesenteric arteries (Fig. 4.129): The celiac trunk supplies the lower esophagus, stomach, superior part of the duodenum, and proximal half of the descending part of the duodenum. The superior mesenteric artery supplies the rest of the duodenum, the jejunum, the ileum, the ascending colon, and the proximal two-thirds of the transverse colon. The inferior mesenteric artery supplies the rest of the transverse colon, the descending colon, the sigmoid colon, and most of the rectum.

1	The inferior mesenteric artery supplies the rest of the transverse colon, the descending colon, the sigmoid colon, and most of the rectum. Along the descending part of the duodenum there is a potential watershed area between the celiac trunk blood supply and the superior mesenteric arterial blood supply. It is unusual for this area to become ischemic, whereas the watershed area between the superior mesenteric artery and the inferior mesenteric artery, at the splenic flexure, is extremely vulnerable to ischemia. In certain disease states, the region of the splenic flexure of the colon can become ischemic. When this occurs, the mucosa sloughs off, rendering the patient susceptible to infection and perforation of the large bowel, which then requires urgent surgical attention.

1	Arteriosclerosis may occur throughout the abdominal aorta and at the openings of the celiac trunk and the superior mesenteric and inferior mesenteric arteries. Not infrequently, the inferior mesenteric artery becomes occluded. Interestingly, many of these patients do not suffer any complications, because anastomoses between the right, middle, and left colic arteries gradually enlarge, forming a continuous marginal artery. The distal large bowel therefore becomes supplied by this enlarged marginal artery (marginal artery of Drummond), which replaces the blood supply of the inferior mesenteric artery (Fig. 4.130).

1	If the openings of the celiac trunk and superior mesenteric artery become narrowed, the blood supply to the gut is diminished. After a heavy meal, the oxygen demand of the bowel therefore outstrips the limited supply of blood through the stenosed vessels, resulting in severe pain and discomfort (mesenteric angina). Patients with this condition tend not to eat because of the pain and rapidly lose weight. The diagnosis is determined by aortic angiography, and the stenoses of the celiac trunk and superior mesenteric artery are best appreciated in the lateral view. In the clinic Cirrhosis is a complex disorder of the liver, the diagnosis of which is confirmed histologically. When a diagnosis is suspected, a liver biopsy is necessary. Cirrhosis is characterized by widespread hepatic fibrosis interspersed with areas of nodular regeneration and abnormal reconstruction of preexisting lobular architecture. The presence of cirrhosis implies previous or continuing liver cell damage.

1	The etiology of cirrhosis is complex and includes toxins (alcohol), viral inflammation, biliary obstruction, vascular outlet obstruction, nutritional (malnutrition) causes, and inherited anatomical and metabolic disorders. As the cirrhosis progresses, the intrahepatic vasculature is distorted, which in turn leads to increased pressure in the portal vein and its draining tributaries (portal hypertension). Portal hypertension produces increased pressure in the splenic venules, leading to splenic enlargement. At the sites of portosystemic anastomosis (see below), large dilated veins (varices) develop. These veins are susceptible to bleeding and may produce marked blood loss, which in some instances can be fatal.

1	The liver is responsible for the production of numerous proteins, including those of the clotting cascade. Any disorder of the liver (including infection and cirrhosis) may decrease the production of these proteins and so prevent adequate blood clotting. Patients with severe cirrhosis of the liver have a significant risk of serious bleeding, even from small cuts; in addition, when varices rupture, there is a danger of rapid exsanguination. As the liver progressively fails, the patient develops salt and water retention, which produces skin and subcutaneous edema. Fluid (ascites) is also retained in the peritoneal cavity, which can hold many liters. The poorly functioning liver cells (hepatocytes) are unable to break down blood and blood products, leading to an increase in the serum bilirubin level, which manifests as jaundice.

1	The poorly functioning liver cells (hepatocytes) are unable to break down blood and blood products, leading to an increase in the serum bilirubin level, which manifests as jaundice. With the failure of normal liver metabolism, toxic metabolic by-products do not convert to nontoxic metabolites. This buildup of noxious compounds is made worse by the numerous portosystemic shunts, which allow the toxic metabolites to bypass the liver. Patients may develop severe neurological features, called hepatic encephalopathy, that can manifest as acute confusion, epileptic fits, or psychotic state. Hepatic encephalopathy is one of the urgent criteria for liver transplantation; if the condition is not reversed, it leads to irreversible neurological damage and death.

1	The hepatic portal system drains blood from the visceral organs of the abdomen to the liver. In normal individuals, 100% of the portal venous blood flow can be recovered from the hepatic veins, whereas in patients with elevated portal vein pressure (e.g., from cirrhosis), there is significantly less blood flow to the liver. The rest of the blood enters collateral channels, which drain into the systemic circulation at specific points (Fig. 4.133). The largest of these collaterals occur at: the gastroesophageal junction around the cardia of the stomach—where the left gastric vein and its tributaries form a portosystemic anastomosis with tributaries to the azygos system of veins of the caval system; the anus—the superior rectal vein of the portal system anastomoses with the middle and inferior rectal veins of the systemic venous system; and the anterior abdominal wall around the umbilicus—the para-umbilical veins anastomose with veins on the anterior abdominal wall.

1	When the pressure in the portal vein is elevated, venous enlargement (varices) tend to occur at and around the sites of portosystemic anastomoses and these enlarged veins are called: varices at the anorectal junction, esophageal varices at the gastroesophageal junction, and caput medusae at the umbilicus. Esophageal varices are susceptible to trauma and, once damaged, may bleed profusely, requiring urgent surgical intervention. In the clinic

1	Esophageal varices are susceptible to trauma and, once damaged, may bleed profusely, requiring urgent surgical intervention. In the clinic Surgery for obesity is also known as weight loss surgery and bariatric surgery. This type of surgery has become increasingly popular over the last few years for patients who are unable to achieve significant weight loss through appropriate diet modification and exercise programs. It is often regarded as a last resort. Importantly, we have to recognize the increasing medical impact that overweight patients pose. With obesity the patient is more likely to develop diabetes and cardiovascular problems and may suffer from increased general health disorders. All of these have a significant impact on health care budgeting and are regarded as serious conditions for the “health of a nation.”

1	There are a number of surgical options to treat obesity. Surgery for patients who are morbidly obese can be categorized into two main groups: malabsorptive procedures and restrictive procedures. There are a variety of bypass procedures that produce a malabsorption state, preventing further weight gain and also producing weight loss. There are complications, which may include anemia, osteoporosis, and diarrhea (e.g., jejunoileal bypass). Restrictive procedures involve placing a band or stapling in or around the stomach to decrease the size of the organ. This reduction produces an earlier feeling of satiety and prevents the patient from overeating. Probably the most popular procedure currently in the United States is gastric bypass surgery. This procedure involves stapling the proximal stomach and joining a loop of small bowel to the small gastric remnant. The procedure is usually performed by fashioning a Roux-en-Y loop with alimentary and pancreaticobiliary limbs.

1	The other type of the procedure, sleeve gastrectomy, is increasing in popularity because it can be used in patients deemed to be at high risk for gastric bypass surgery. It involves reduction of the gastric lumen by removing a large portion of the stomach along the greater curvature. Any overweight patient undergoing surgery faces significant risk and increased morbidity, with mortality rates from 1% to 5%. In the clinic

1	Any overweight patient undergoing surgery faces significant risk and increased morbidity, with mortality rates from 1% to 5%. In the clinic At first glance, it is difficult to appreciate why the psoas muscle sheath is of greater importance than any other muscle sheath. The psoas muscle and its sheath arise not only from the lumbar vertebrae but also from the intervertebral discs between each vertebra. This disc origin is of critical importance. In certain types of infection, the intervertebral disc is preferentially affected (e.g., tuberculosis and salmonella discitis). As the infection of the disc develops, the infection spreads anteriorly and anterolaterally. In the anterolateral position, the infection passes into the psoas muscle sheath, and spreads within the muscle and sheath, and may appear below the inguinal ligament as a mass. In the clinic To understand why a hernia occurs through the diaphragm, it is necessary to consider the embryology of the diaphragm.

1	In the clinic To understand why a hernia occurs through the diaphragm, it is necessary to consider the embryology of the diaphragm. The diaphragm is formed from four structures— the septum transversum, the posterior esophageal mesentery, the pleuroperitoneal membrane, and the peripheral rim—which eventually fuse together, separating the abdominal cavity from the thoracic cavity. The septum transversum forms the central tendon, which develops from a mesodermal origin superior to the embryo’s head and then moves to its more adult position during folding of the cephalic portion of the embryo. Fusion of the various components of the diaphragm may fail, and hernias may occur through the failed points of fusion (Fig. 4.146). The commonest sites are: between the xiphoid process and the costal margins on the right (Morgagni’s hernia), and through an opening on the left when the pleuroperitoneal membrane fails to close the pericardioperitoneal canal (Bochdalek’s hernia).

1	Hernias may also occur through the central tendon and through a congenitally large esophageal hiatus. Morgagni’s and Bochdalek’s hernias tend to appear at or around the time of birth or in early infancy. They allow abdominal bowel to enter the thoracic cavity, which may compress the lungs and reduce respiratory function. Most of these hernias require surgical closure of the diaphragmatic defect. However, large hernias can lead to pulmonary hypoplasia and the long-term outcome depends more on the degree of the hypoplasia rather than on the surgical repair itself. Occasionally, small defects within the diaphragm fail to permit bowel through, but do allow free movement of fluid. Patients with ascites may develop pleural effusions, while patients with pleural effusions may develop ascites when these defects are present. In the clinic

1	In the clinic At the level of the esophageal hiatus, the diaphragm may be lax, allowing the fundus of the stomach to herniate into the posterior mediastinum (Figs. 4.147 and 4.148). This typically causes symptoms of acid reflux. Ulceration may occur and may produce bleeding and anemia. The diagnosis is usually made by barium studies or endoscopy. Hiatal hernia is often asymptomatic and is frequently found incidentally on CT imaging performed for unrelated complaints. Treatment in the first instance is by medical management, although surgery may be necessary. In the clinic

1	In the clinic Urinary tract stones (calculi) occur more frequently in men than in women, are most common in people aged between 20 and 60 years, and are usually associated with sedentary lifestyles. The stones are polycrystalline aggregates of calcium, phosphate, oxalate, urate, and other soluble salts within an organic matrix. The urine becomes saturated with these salts, and small variations in the pH cause the salts to precipitate. Typically the patient has pain that radiates from the infrascapular region (loin) into the groin, and even into the scrotum or labia majora. Blood in the urine (hematuria) may also be noticed. Infection must be excluded because certain species of bacteria are commonly associated with urinary tract stones. The complications of urinary tract stones include infection, urinary obstruction, and renal failure. Stones may also develop within the bladder and produce marked irritation, causing pain and discomfort.

1	The diagnosis of urinary tract stones is based upon history and examination. Stones are often visible on abdominal radiographs. Special investigations include: ultrasound scanning, which may demonstrate the dilated renal pelvis and calices when the urinary system is obstructed. This is the preferred way of imaging in pregnant women or when clinical suspicion is low. low-dose CT of the urinary tract (CT KUB), which allows the detection of even smaller stones, shows the exact level of obstruction and, based on the size, density, and location of the stone, can help the urologist plan a procedure to remove the stone if necessary (extracorporeal shock wave lithotripsy versus ureteroscopy, percutaneous nephrolithotomy, or, extremely rare these days, open surgery) (Fig. 4.156). an intravenous urogram, which will demonstrate the obstruction, pinpoint the exact level of the stone is currently less often used because access to low-dose CT KUB has increased. In the clinic

1	an intravenous urogram, which will demonstrate the obstruction, pinpoint the exact level of the stone is currently less often used because access to low-dose CT KUB has increased. In the clinic Most tumors that arise in the kidney are renal cell carcinomas. These tumors develop from the proximal tubular epithelium. Approximately 5% of tumors within the kidney are transitional cell tumors, which arise from the urothelium of the renal pelvis. Most patients typically have blood in the urine (hematuria), pain in the infrascapular region (loin), and a mass.

1	Renal cell tumors (Figs. 4.157 and 4.158) are unusual because not only do they grow outward from the kidney, invading the fat and fascia, but they also spread into the renal vein. This venous extension is rare for any other type of tumor, so, when seen, renal cell carcinoma should be suspected. In addition, the tumor may spread along the renal vein and into the inferior vena cava, and in rare cases can grow into the right atrium across the tricuspid valve and into the pulmonary artery. Treatment for most renal cancers is surgical removal, even when metastatic spread is present, because some patients show regression of metastases.

1	Treatment for most renal cancers is surgical removal, even when metastatic spread is present, because some patients show regression of metastases. Transitional cell carcinoma arises from the urothelium. The urothelium is present from the calices to the urethra and behaves as a “single unit.” Therefore, when patients develop transitional carcinomas within the bladder, similar tumors may also be present within upper parts of the urinary tract. In patients with bladder cancer, the whole of the urinary tract must always be investigated to exclude the possibility of other tumors (Fig. 4.159). This is currently achieved by performing a dual-phase CT urogram that allows visualization of the renal parenchyma and the collecting system at the same time. In the clinic

1	In the clinic A nephrostomy is a procedure where a tube is placed through the lateral or posterior abdominal wall into the renal cortex to lie within the renal pelvis. The function of this tube is to allow drainage of urine from the renal pelvis through the tube externally (Fig. 4.160). The kidneys are situated on the posterior abdominal wall, and in thin healthy subjects may be only up to 2 to 3 cm from the skin. Access to the kidney is relatively straightforward, because the kidney can be easily visualized under ultrasound guidance. Using local anesthetic, a needle can be placed, under ultrasound direction, through the skin into the renal cortex and into the renal pelvis. A series of wires and tubes can be passed through the needle to position the drainage catheter.

1	The indications for such a procedure are many. In patients with distal ureteric obstruction the back pressure of urine within the ureters and the kidney significantly impairs the function of the kidney. This will produce renal failure and ultimately death. Furthermore, a dilated obstructed system is also susceptible to infection. In many cases, there is not only obstruction producing renal failure but also infected urine within the system. In the clinic Renal transplantation is now a common procedure undertaken in patients with end-stage renal failure. Transplant kidneys are obtained from either living or deceased donors. The living donors are carefully assessed, because harvesting a kidney from a normal healthy individual, even with modern-day medicine, carries a small risk. Deceased kidney donors are brain dead or have suffered cardiac death. The donor kidney is harvested with a small cuff of aortic and venous tissue. The ureter is also harvested.

1	An ideal place to situate the transplant kidney is in the left or the right iliac fossa (Fig. 4.161). A curvilinear incision is made paralleling the iliac crest and pubic symphysis. The external oblique muscle, internal oblique muscle, transversus abdominis muscle, and transversalis fascia are divided. The surgeon identifies the parietal peritoneum but does not enter the peritoneal cavity. The parietal peritoneum is medially retracted to reveal the external iliac artery, external iliac vein, and bladder. In some instances the internal iliac artery of the recipient is mobilized and anastomosed directly as an end-to-end procedure onto the renal artery of the donor kidney. Similarly the internal iliac vein is anastomosed to the donor vein. In the presence of a small aortic cuff of tissue the donor artery is anastomosed to the recipient external iliac artery and similarly for the venous anastomosis. The ureter is easily tunneled obliquely through the bladder wall with a straightforward

1	donor artery is anastomosed to the recipient external iliac artery and similarly for the venous anastomosis. The ureter is easily tunneled obliquely through the bladder wall with a straightforward anastomosis.

1	The left and right iliac fossae are ideal locations for the transplant kidney because a new space can be created without compromise to other structures. The great advantage of this procedure is the proximity to the anterior abdominal wall, which permits easy ultrasound visualization of the kidney and Doppler vascular assessment. Furthermore, in this position biopsies are easily obtained. The extraperitoneal approach enables patients to make a swift recovery. In the clinic Investigation of the urinary tract After an appropriate history and examination of the patient, including a digital rectal examination to assess the prostate in men, special investigations are required.

1	After an appropriate history and examination of the patient, including a digital rectal examination to assess the prostate in men, special investigations are required. Cystoscopy is a technique that allows visualization of the urinary bladder and urethra using an optical system attached to a flexible or rigid tube (cystoscope). Images are displayed on a monitor, as done in other endoscopic studies. Biopsies, bladder stone removal, removal of foreign bodies from the bladder, and bleeding cauterization can be performed during cystoscopy. Cystoscopy is helpful in establishing the causes of macroscopic and microscopic hematuria, assessing bladder and urethral diverticula and fistulas, as well as serving as a tool to investigate patients with voiding problems.

1	An IVU is one of the most important and commonly carried out radiological investigations (Fig. 4.162). The patient is injected with iodinated contrast medium. Most contrast media contain three iodine atoms spaced around a benzene ring. The relatively high atomic number of iodine compared to the atomic number of carbon, hydrogen, and oxygen attenuates the radiation beam. After intravenous injection, contrast media are excreted predominantly by glomerular filtration, although some are secreted by the renal tubules. This allows visualization of the collecting system as well as the ureters and bladder. Ultrasound can be used to assess kidney size and the size of the calices, which may be dilated when obstructed. Although the ureters are poorly visualized using ultrasound, the bladder can be easily seen when full. Ultrasound measurements of bladder volume can be obtained before and after micturition.

1	Nuclear medicine is an extremely useful tool for investigating the urinary tract because radioisotope compounds can be used to estimate renal cell mass and function and assess the parenchyma for renal scarring. These tests are often very useful in children when renal scarring and reflux disease is suspected. In the clinic An abdominal aortic aneurysm is a dilation of the aorta and generally tends to occur in the infrarenal region (the region at or below the renal arteries). As the aorta expands, the risk of rupture increases, and it is now generally accepted that when an aneurysm reaches 5.5 cm or greater an operation will significantly benefit the patient. With the aging population, the number of abdominal aortic aneurysms is increasing. Moreover, with the increasing use of imaging techniques, a number of abdominal aortic aneurysms are identified in asymptomatic patients.

1	For many years the standard treatment for repair was an open operative technique, which involved a large incision from the xiphoid process of the sternum to the symphysis pubis and dissection of the aneurysm. The aneurysm was excised and a tubular woven graft was sewn into place. Recovery may take a number of days, even weeks, and most patients would be placed in the intensive care unit after the operation. Further developments and techniques have led to a new type of procedure being performed to treat abdominal aortic aneurysms—the endovascular graft (Fig. 4.165).

1	Further developments and techniques have led to a new type of procedure being performed to treat abdominal aortic aneurysms—the endovascular graft (Fig. 4.165). The technique involves surgically dissecting the femoral artery below the inguinal ligament. A small incision is made in the femoral artery and the preloaded compressed graft with metal support struts is passed on a large catheter into the abdominal aorta through the femoral artery. Using X-ray for guidance the graft is opened, lining the inside of the aorta. Limb attachments are made to the graft that extend into the common iliac vessels. This bifurcated tube device effectively excludes the abdominal aortic aneurysm. This type of device is not suitable for all patients. Patients who receive this device do not need to go to the intensive care unit. Many patients leave the hospital within 24 to 48 hours. Importantly, this device can be used for patients who were deemed unfit for open surgical repair. In the clinic

1	In the clinic Deep vein thrombosis is a potentially fatal condition where a clot (thrombus) is formed in the deep venous system of the legs and the veins of the pelvis. Virchow described the reasons for thrombus formation as decreased blood flow, abnormality of the constituents of blood, and abnormalities of the vessel wall. Common predisposing factors include hospitalization and surgery, the oral contraceptive pill, smoking, and air travel. Other factors include clotting abnormalities (e.g., protein S and protein C deficiency). The diagnosis of deep vein thrombosis may be difficult to establish, with symptoms including leg swelling and pain and discomfort in the calf. It may also be an incidental finding. In practice, patients with suspected deep vein thrombosis undergo a D-dimer blood test, which measures levels of a fibrin degradation product. If this is positive there is a high association with deep vein thrombosis.

1	The consequences of deep vein thrombosis are twofold. Occasionally the clot may dislodge and pass into the venous system through the right side of the heart and into the main pulmonary arteries. If the clots are of significant size they obstruct blood flow to the lung and may produce instantaneous death. Secondary complications include destruction of the normal valvular system in the legs, which may lead to venous incompetency and chronic leg swelling with ulceration. The treatment for deep vein thrombosis is prevention. In order to prevent deep vein thrombosis, patients are optimized by removing all potential risk factors. Subcutaneous heparin may be injected and the patient wears compression stockings to prevent venous stasis while in the hospital.

1	In certain situations it is not possible to optimize the patient with prophylactic treatment, and it may be necessary to insert a filter into the inferior vena cava that traps any large clots. It may be removed after the risk period has ended. In the clinic From a clinical perspective, retroperitoneal lymph nodes are arranged in two groups. The pre-aortic lymph node group drains lymph from the embryological midline structures, such as the liver, bowel, and pancreas. The para-aortic lymph node group (the lateral aortic or lumbar nodes), on either side of the aorta, drain lymph from bilateral structures, such as the kidneys and adrenal glands. Organs embryologically derived from the posterior abdominal wall also drain lymph to these nodes. These organs include the ovaries and the testes (importantly, the testes do not drain lymph to the inguinal regions).

1	In general, lymphatic drainage follows standard predictable routes; however, in the presence of disease, alternate routes of lymphatic drainage will occur. There are a number of causes for enlarged retroperitoneal lymph nodes. In the adult, massively enlarged lymph nodes are a feature of lymphoma, and smaller lymph node enlargement is observed in the presence of infection and metastatic malignant spread of disease (e.g., colon cancer). The treatment for malignant lymph node disease is based upon a number of factors, including the site of the primary tumor (e.g., bowel) and its histological cell type. Normally, the primary tumor is surgically removed and the lymph node spread and metastatic organ spread (e.g., to the liver and the lungs) are often treated with chemotherapy and radiotherapy. In certain instances it may be considered appropriate to resect the lymph nodes in the retroperitoneum (e.g., for testicular cancer).

1	The surgical approach to retroperitoneal lymph node resection involves a lateral paramedian incision in the midclavicular line. The three layers of the anterolateral abdominal wall (external oblique, internal oblique, and transversus abdominis) are opened and the transversalis fascia is divided. The next structure the surgeon sees is the parietal peritoneum. Instead of entering the parietal peritoneum, which is standard procedure for most intraabdominal operations, the surgeon gently pushes the parietal peritoneum toward the midline, which moves the intraabdominal contents and allows a clear view of the retroperitoneal structures. On the left, the para-aortic lymph node group is easily demonstrated, with a clear view of the abdominal aorta and kidney. On the right the inferior vena cava is demonstrated and has to be retracted to access the right para-aortic lymph node chain.

1	The procedure of retroperitoneal lymph node dissection is extremely well tolerated and lacks the problems of entering the peritoneal cavity (e.g., paralytic ileus). Unfortunately, a complication of a vertical incision in the midclavicular line is division of the segmental nerve supply to the rectus abdominis muscle. This produces muscle atrophy and asymmetrical proportions of the anterior abdominal wall. A 45-year-old man had mild epigastric pain, and a diagnosis of esophageal reflux was made. He was given appropriate medication, which worked well. However, at the time of the initial consultation, the family practitioner requested a chest radiograph, which demonstrated a prominent hump on the left side of the diaphragm and old rib fractures. The patient was recalled for further questioning.

1	The patient was recalled for further questioning. He was extremely pleased with the treatment he had been given for his gastroesophageal reflux, but was concerned about being recalled for further history and examination. During the interview, he revealed that he had previously been involved in a motorcycle accident and had undergone a laparotomy for a “rupture.” The patient did not recall what operation was performed, but was assured at the time that the operation was a great success. The patient is likely to have undergone a splenectomy. In any patient who has had severe blunt abdominal trauma (such as that caused by a motorcycle accident), lower left-sided rib fractures are an extremely important sign of appreciable trauma.

1	In any patient who has had severe blunt abdominal trauma (such as that caused by a motorcycle accident), lower left-sided rib fractures are an extremely important sign of appreciable trauma. A review of the patient’s old notes revealed that at the time of the injury the spleen was removed surgically, but it was not appreciated that there was a small rupture of the dome of the left hemidiaphragm. The patient gradually developed a hernia through which bowel could enter, producing the “hump” on the diaphragm seen on the chest radiograph. Because this injury occurred many years ago and the patient has been asymptomatic, it is unlikely that the patient will come to any harm and was discharged.

1	Because this injury occurred many years ago and the patient has been asymptomatic, it is unlikely that the patient will come to any harm and was discharged. A medical student was asked to inspect the abdomen of two patients. On the first patient he noted irregular veins radiating from the umbilicus. On the second patient he noted irregular veins, coursing in a caudal to cranial direction, over the anterior abdominal wall from the groin to the chest. He was asked to explain his findings and determine the significance of these features.

1	In the first patient the veins were draining radially away from the periumbilical region. In normal individuals, enlarged veins do not radiate from the umbilicus. In patients with portal hypertension the portal venous pressure is increased as a result of hepatic disease. Small collateral veins develop at and around the obliterated umbilical vein. These veins pass through the umbilicus and drain onto the anterior abdominal wall, forming a portosystemic anastomosis. The eventual diagnosis for this patient was cirrhosis of the liver.

1	The finding of veins draining in a caudocranial direction on the anterior abdominal wall in the second patient is not typical for veins on the anterior abdominal wall. When veins are so prominent, it usually implies that there is an obstruction to the normal route of venous drainage and an alternative route has been taken. Typically, blood from the lower limbs and the retroperitoneal organs drains into the inferior vena cava and from here to the right atrium of the heart. This patient had a chronic thrombosis of the inferior vena cava, preventing blood from returning to the heart by the “usual” route.

1	Blood from the lower limbs and the pelvis may drain via a series of collateral vessels, some of which include the superficial inferior epigastric veins, which run in the superficial fascia. These anastomose with the superior, superficial, and deep epigastric venous systems to drain into the internal thoracic veins, which in turn drain into the brachiocephalic veins and the superior vena cava. After the initial inferior vena cava thrombosis, the veins of the anterior abdominal wall and other collateral pathways hypertrophy to accommodate the increase in blood flow. A 52-year-old woman visited her family physician with complaints of increasing lethargy and vomiting. The physician examined her and noted that compared to previous visits she had lost significant weight. She was also jaundiced, and on examination of the abdomen a well-defined 10-cm rounded mass was palpable below the liver edge in the right upper quadrant (Fig. 4.185).

1	The clinical diagnosis was carcinoma of the head of the pancreas. It is difficult to appreciate how such a precise diagnosis can be made clinically when only three clinical signs have been described. The patient’s obstruction was in the distal bile duct. When a patient has jaundice, the causes are excessive breakdown of red blood cells (prehepatic), hepatic failure (hepatic jaundice), and posthepatic causes, which include obstruction along the length of the biliary tree. The patient had a mass in her right upper quadrant that was palpable below the liver; this was the gallbladder. In healthy individuals, the gallbladder is not palpable. An expanded gallbladder indicates obstruction either within the cystic duct or below the level of the cystic duct insertion (i.e., the bile duct). The patient’s vomiting was related to the position of the tumor.

1	The patient’s vomiting was related to the position of the tumor. It is not uncommon for vomiting and weight loss (cachexia) to occur in patients with a malignant disease. The head of the pancreas lies within the curve of the duodenum, primarily adjacent to the descending part of the duodenum. Any tumor mass in the region of the head of the pancreas is likely to expand and may encase and invade the duodenum. Unfortunately, in this patient’s case, this happened, producing almost complete obstruction. Further discussion with the patient revealed that she was vomiting relatively undigested food soon after each meal. A CT scan demonstrated further complications.

1	A CT scan demonstrated further complications. In the region of the head and neck of the pancreas are complex anatomical structures, which may be involved with a malignant process. The CT scan confirmed a mass in the region of the head of the pancreas, which invaded the descending part of the duodenum. The mass extended into the neck of the pancreas and had blocked the distal part of the bile duct and the pancreatic duct. Posteriorly the mass had directly invaded the portal venous confluence of the splenic and superior mesenteric veins, producing a series of gastric, splenic, and small bowel varices. This patient underwent palliative chemotherapy, but died 7 months later. A 44-year-old woman had been recently diagnosed with melanoma on the toe and underwent a series of investigations.

1	This patient underwent palliative chemotherapy, but died 7 months later. A 44-year-old woman had been recently diagnosed with melanoma on the toe and underwent a series of investigations. Melanoma (properly called malignant melanoma) can be an aggressive form of skin cancer that spreads to lymph nodes and multiple other organs throughout the body. The malignant potential is dependent upon its cellular configuration and also the depth of its penetration through the skin. The patient developed malignant melanoma in the foot, which spread to the lymph nodes of the groin. The inguinal lymph nodes were resected; however, it was noted on follow-up imaging that the patient had developed two metastatic lesions within the right lobe of the liver. Surgeons and physicians considered the possibility of removing these lesions. A CT scan was performed that demonstrated the lesions within segments V and VI of the liver (Fig. 4.186).

1	Surgeons and physicians considered the possibility of removing these lesions. A CT scan was performed that demonstrated the lesions within segments V and VI of the liver (Fig. 4.186). The segmental anatomy of the liver is important because it enables the surgical planning for resection. The surgery was undertaken and involved identifying the portal vein and the confluence of the right and left hepatic ducts. The liver was divided in the imaginary principal plane of the middle hepatic vein. The main hepatic duct and biliary radicals were ligated and the right liver was successfully resected. The segments remaining included the left lobe of the liver.

1	The segments remaining included the left lobe of the liver. The patient underwent a surgical resection of segments V, VI, VII, and VIII. The remaining segments included IVa, IVb, I, II, and III. It is important to remember that the lobes of the liver do not correlate with the hepatic volume. The left lobe of the liver contains only segments II and III. The right lobe of the liver contains segments IV, V, VI, VII, and VIII. Hence, cross-sectional imaging is important when planning surgical segmental resection. A 55-year-old man developed severe jaundice and a massively distended abdomen. A diagnosis of cirrhosis of the liver was made, and further confirmatory tests demonstrated that the patient had significant ascites (free fluid within the peritoneal cavity). A liver biopsy was necessary to confirm the cirrhosis, but there was some debate about how this biopsy should be obtained (eFig. 4.187).

1	In patients with cirrhosis it is important to determine the extent of the cirrhosis and the etiology. History, examination, and blood tests are useful and are supported by complex radiological investigations. To begin treatment and determine the prognosis, a sample of liver tissue must be obtained. However, there are important issues to consider when taking a liver biopsy from a patient with suspected cirrhosis. One issue is liver function. The liver function of patients with suspected liver disease is poor, as demonstrated by the patient’s jaundice—an inability to conjugate bilirubin. Importantly, because some liver products are blood-clotting factors involved in the clotting cascade, the blood-clotting ability of patients with severe liver disease is significantly impaired. These patients therefore have a high risk of bleeding. Another issue is the presence of ascites.

1	Another issue is the presence of ascites. Normally the liver rests against the lateral and anterior abdominal walls. This direct contact can be useful for care after a liver biopsy has been obtained. After the procedure, the patient lies over the region where the biopsy has been obtained and the weight of the liver stems any localized bleeding. When patients have significant ascites, the liver cannot be compressed against the walls of the abdomen and blood may pour freely into the ascitic fluid. The patient has ascites, so another approach for a liver biopsy must be considered. The patient was referred to the radiology department for a transjugular liver biopsy.

1	The patient has ascites, so another approach for a liver biopsy must be considered. The patient was referred to the radiology department for a transjugular liver biopsy. The skin around the jugular vein in the neck was anesthetized. Access was obtained through insertion of a needle and a guidewire. The guidewire was advanced through the right internal jugular vein and into the right brachiocephalic vein. It entered the superior vena cava, was passed along the posterior wall of the atrium, and entered the superior aspect of the inferior vena cava. A catheter was inserted over the wire and directed into the right hepatic vein. Using a series of dilators, the hole was enlarged and a biopsy needle was placed over the wire and into the right hepatic vein. The liver was biopsied through the right hepatic vein and the biopsy sample was removed. A simple suture was used to close the internal jugular vein in the neck, and minor compression stemmed any blood flow.

1	Assuming that the biopsy needle does not penetrate the liver capsule, it is not important how much the patient bleeds from the liver, because this bleeding will enter the hepatic vein and is immediately returned to the circulation. A 30-year-old man had a diffuse and poorly defined epigastric mass. Further examination revealed asymmetrical scrotal enlargement. As part of her differential diagnosis, the resident considered the possibility that the man had testicular cancer with regional abdominal para-aortic nodal involvement (the lateral aortic, or lumbar, nodes). A primary testicular neoplasm is the most common tumor in men between the ages of 25 and 34 and accounts for between 1% and 2% of all malignancies in men. A family history of testicular cancer and maldescent of the testis are strong predisposing factors. Spread of the tumor is typically to the lymph node chains that drain the testes.

1	Spread of the tumor is typically to the lymph node chains that drain the testes. The testes develop from structures adjacent to the renal vessels in the upper abdomen, between the transversalis fascia and the peritoneum. They normally migrate through the inguinal canals into the scrotum just before birth. The testes take with them their arterial supply, their venous drainage, their nerve supply, and their lymphatics. A computed tomography scan revealed a para-aortic lymph node mass in the upper abdomen and enlarged lymph nodes throughout the internal and common iliac lymph node chains. Assuming the scrotal mass was a carcinoma of the testes, which would normally drain into the lateral aortic (lumbar) nodes in the upper abdomen, it would be very unusual for iliac lymphadenopathy to be present. Further examination of the scrotal mass was required.

1	Further examination of the scrotal mass was required. A transillumination test of the scrotum on the affected side was positive. An ultrasound scan revealed normal right and left testes and a large fluid collection around the right testis. A diagnosis of a right-sided hydrocele was made. Scrotal masses are common in young males, and determining the exact anatomical site of the scrotal mass is of utmost clinical importance. Any mass that arises from the testis should be investigated to exclude testicular cancer. Masses that arise from the epididymis and scrotal lesions, such as fluid (hydrocele) or hernias, are also clinically important but are not malignant. The ultrasound scan revealed fluid surrounding the testis, which is diagnostic of a hydrocele. Simple cysts arising from and around the epididymis (epididymal cysts) can be easily defined. A diagnosis of lymphoma was suspected.

1	A diagnosis of lymphoma was suspected. Lymphoma is a malignant disease of lymph nodes. Most lymphomas are divided into two specific types, namely Hodgkin’s lymphoma and non-Hodgkin’s lymphoma. If caught early the prognosis following radical chemotherapy is excellent.

1	The patient underwent a biopsy, which was performed from the posterior approach. He was placed in the prone position in the computed tomography (CT) scanner. A fine needle with a special cutting device was used to obtain a lymph node sample. A left-sided approach was used because the inferior vena cava is on the right side and the nodes were in the para-aortic regions (i.e., the biopsy needle would have to pass between the inferior vena cava and the aorta from a posterior approach, which is difficult). The skin was anesthetized using local anesthetic at the lateral border of the quadratus lumborum muscle. The needle was angled at approximately 45° within the quadratus lumborum muscle and entered the retroperitoneum to lie beside the left-sided para-aortic lymph nodes. Because this procedure is performed using CT guidance, the operator can advance the needle slowly, taking care not to “hit” other retroperitoneal structures.

1	A good biopsy was obtained and the diagnosis was Hodgkin’s lymphoma. The patient underwent chemotherapy and 2 years later is in full remission and leads an active life. A 35-year-old man had a soft mass approximately 3 cm in diameter in the right scrotum. The diagnosis was a right indirect inguinal hernia. What were the examination findings? The mass was not tender and the physician was not able to “get above it.” The testes were felt separate from the mass, and a transillumination test (in which a bright light is placed behind the scrotum and the scrotal sac is viewed from the front) was negative. (A positive test occurs when the light penetrates through the scrotum.) When the patient stood up, a positive cough “impulse” was felt within the mass. After careful and delicate maneuvering, the mass could be massaged into the inguinal canal, so emptying from the scrotum. When the massaging hand was removed, the mass recurred in the scrotum.

1	After careful and delicate maneuvering, the mass could be massaged into the inguinal canal, so emptying from the scrotum. When the massaging hand was removed, the mass recurred in the scrotum. An indirect inguinal hernia enters the inguinal canal through the deep inguinal ring. It passes through the inguinal canal to exit through the superficial inguinal ring in the aponeurosis of the external oblique muscle. The hernia sac lies superior and medial to the pubic tubercle and enters into the scrotum within the spermatic cord. A direct inguinal hernia passes directly through the posterior wall of the inguinal canal. It does not pass down the inguinal canal. If large enough, it may pass through the superficial inguinal ring and into the scrotum.

1	A 25-year-old man developed severe pain in the left lower quadrant of his abdomen. The pain was diffuse and relatively constant but did ease for short periods of time. On direct questioning the patient indicated that the pain was in the inguinal region and radiated posteriorly into his left infrascapular region (loin). A urine dipstick was positive for blood (hematuria). A diagnosis of a ureteric stone (calculus) was made. The patient’s initial infrascapular pain, which later radiated to the left groin, relates to passage of the ureteric stone along the ureter. The origin of the pain relates to ureteral distention. A series of peristaltic waves along the ureter transport urine along the length of the ureter from the kidney to the bladder. As the ureteric stone obstructs the kidney, the ureter becomes distended, resulting in an exacerbation of the pain. The peristaltic waves are superimposed upon the distention, resulting in periods of exacerbation and periods of relief.

1	The pain is referred. The visceral afferent (sensory) nerve fibers from the ureter pass into the spinal cord, entering the first and second lumbar segments of the spinal cord. Pain is thus referred to cutaneous regions innervated by somatic sensory nerves from the same spinal cord levels. The patient was investigated by a CT scan. Traditionally, a plain radiograph was used to look for the radiopaque stone (90% of renal stones are radiopaque) and this often proceeded to intravenous urography to determine precise location of renal stones. Currently, low-dose CT of the collecting system is used to visualize the renal stones and pinpoint the level of obstruction, using low radiation dose and no intravenous contrast.

1	An ultrasound scan may also be useful to assess for pelvicaliceal dilation and may reveal stones at the pelviureteral junction or the vesicoureteric junction. Ultrasound is also valuable for assessing other causes of obstruction (e.g., tumors at and around the ureteric orifices in the bladder) and is particularly useful in pregnant women when imaging with the use of radiation is a concern. Usually an intravenous urogram would be carried out to enable assessment of the upper urinary tracts and precise location of the stone. Not infrequently, CT scans of the abdomen are also obtained. These scans not only give information about the kidneys, ureters, and bladder but also show the position of the stone and other associated pathology. If this patient’s infrascapular pain was on the right and predominantly within the right lower abdomen, appendicitis would also have to be excluded. A CT scan would enable differentiation of appendicitis and urinary colic.

1	A 27-year-old woman was admitted to the surgical ward with appendicitis. She underwent an appendectomy. It was noted at operation that the appendix had perforated and there was pus within the abdominal cavity. The appendix was removed and the stump tied. The abdomen was washed out with warm saline solution. The patient initially made an uneventful recovery, but by day 7 she had become unwell, with pain over her right shoulder and spiking temperatures. This patient had developed an intraabdominal abscess. Any operation on the bowel may involve peritoneal contamination with fecal contents and fecal flora. This may not be appreciated at the time of the operation. Over the postoperative period an inflammatory reaction ensued and an abscess cavity developed, filling with pus. Typically, the observation chart revealed a “swinging” pyrexia (fever). The most common sites for abscess to develop are the pelvis and the hepatorenal recess.

1	The most common sites for abscess to develop are the pelvis and the hepatorenal recess. When a patient is in the supine position, the lowest points in the abdominal and pelvic cavities are the posterior superior aspect of the peritoneal cavity (the hepatorenal recess) and, in women, the recto-uterine pouch (pouch of Douglas). The shoulder pain suggested that the abscess was in the hepatorenal recess and that the pain was referred from the diaphragm. The motor and sensory innervation of the diaphragm is from nerves C3 to C5. The somatic pain sensation from the parietal peritoneum covering the undersurface of the diaphragm is carried into the spinal cord by the phrenic nerve (C3 to C5) and is interpreted by the brain as coming from skin over the shoulder—a region supplied by other somatic sensory nerves entering the same levels of the spinal cord as those from the diaphragm. A chest radiograph demonstrated elevation of the right hemidiaphragm.

1	A chest radiograph demonstrated elevation of the right hemidiaphragm. This elevation of the right hemidiaphragm was due to the pus tracking from the hepatorenal space around the lateral and anterior aspect of the liver to sit on top of the liver in a subphrenic position. An ultrasound scan demonstrated this collection of fluid. The abscess cavity could be clearly seen by placing the ultrasound probe between ribs XI and XII. The inferior border of the right lower lobe lies at rib X in the midaxillary line. When the probe is placed between ribs XI and XII the ultrasound waves pass between the intercostal muscles and the parietal pleura laterally on the chest wall, and continue through the parietal pleura overlying the diaphragm into the cavity of the abscess, which lies below the diaphragm.

1	Drainage was not done by an intercostal route. Instead, using CT guidance and local anesthesia, a subcostal drain was established and 1 liter of pus was removed (eFig. 4.188). It is important to bear in mind that placing a drain through the pleural cavity into the abdominal cavity effectively allows intraabdominal pus to pass into the thoracic cavity, and that this may produce an empyema (pus in the pleural space). The patient made a slow and uneventful recovery.

1	The patient made a slow and uneventful recovery. A 45-year-old man developed a low-grade rectal carcinoma just above the anorectal margin. He underwent an abdominoperineal resection of the tumor and was left with a left lower abdominal colostomy (see below). Unfortunately, the man’s wife left him for a number of reasons, including lack of sexual desire. He “turned to drink” and over the ensuing years developed cirrhosis. He was brought into the emergency room with severe bleeding from enlarged veins around his colostomy. An emergency transjugular intrahepatic portosystemic shunt was created, which stopped all bleeding (eFigs. 4.189 and 4.190). He is now doing well in a rehabilitation program. A colostomy was necessary because of the low site of the tumor.

1	A colostomy was necessary because of the low site of the tumor. Carcinoma of the colon and rectum usually develops in older patients, but some people do get tumors early in life. Most tumors develop from benign polyps, which undergo malignant change. As the malignancy develops it invades through the wall of the bowel and then metastasizes to local lymphatics. The tumor extends within the wall for a few centimeters above and below its origin. Lymphatic spread is to local and regional lymph nodes and then to the pre-aortic lymph node chain. These drain eventually into the thoracic duct.

1	When this man was assessed for surgery, the tumor was so close to the anal margin that resection of the sphincters was necessary to be certain that the tumor margins were clear. The bowel cannot be joined to the anus without sphincters because the patient would be fecally incontinent. At surgery the tumor was excised, including the locoregional lymph node chains and the peritumoral fat around the rectum. The free end of the sigmoid colon was brought through a hole in the anterior abdominal wall. The bowel was then carefully sutured to the anterior abdominal wall to allow placement of a bag to collect the feces. This is a colostomy. Contrary to their usual immediate negative reaction to having a bag on the anterior abdominal wall, most patients cope extremely well, especially if they have been cured of cancer.

1	Contrary to their usual immediate negative reaction to having a bag on the anterior abdominal wall, most patients cope extremely well, especially if they have been cured of cancer. This patient’s pelvic nerves were damaged. The radical pelvic surgical dissection damaged the pelvic parasympathetic nerve supply necessary for erection of the penis. Unfortunately, this was not well explained to the patient, which in some part led to the failure of his relationship. With any radical surgery in the pelvis, the nerves that supply the penis or clitoris may be damaged, so interfering with sexual function. This patient was bleeding from stomal varices. As he developed a serious drinking problem, his liver became cirrhotic and this damaged the normal liver architecture. This in turn increased the blood pressure in the portal vein (portal hypertension).

1	In patients with portal hypertension small anastomoses develop between the veins of the portal system and the veins of the systemic circulation. These portosystemic anastomoses are usually of little consequence; however, at the gastroesophageal junction, they lie in a submucosal and mucosal position and are subject to trauma. Torrential hemorrhage may occur from even minor trauma, and death may ensue following blood loss. These varices require urgent treatment, which includes injecting sclerosant substances, banding, and even surgical ligation.

1	Fortunately, most of the other portosystemic anastomoses are of relatively little consequence. In patients with colostomies, small veins may develop between the veins of the large bowel (portal system drainage) and cutaneous veins on the anterior abdominal wall (systemic veins). If these veins become enlarged because of portal hypertension, they are subject to trauma as feces are passed through the colostomy. Torrential hemorrhage may ensue if they are damaged. A procedure was carried out to lower the portal pressure. To reduce the pressure in the portal vein in this patient, several surgical procedures were considered. These included sewing the side of the portal vein onto the inferior vena cava (portacaval shunt) and sewing the splenic vein onto the renal vein (a splenorenal shunt). These procedures, however, require a large abdominal incision and are extremely complex. As an alternative, it was decided to create a transjugular intrahepatic portosystemic shunt.

1	Creating a transjugular intrahepatic portosystemic shunt is a relatively new technique that may be carried out under local anesthesia. Using a right internal jugular approach, a long needle is placed through the internal jugular vein, the superior vena cava, and the right atrium, into the inferior vena cava. The right hepatic vein is cannulated and, with special steering wires, a needle is passed through the hepatic substance directly into the right branch of the portal vein. A small balloon is passed over the wire and through the hepatic substance and is inflated. After the balloon has been removed, a metallic stent (a flexible wire tube) is placed across this tract in the liver to keep it open. Blood now freely flows from the portal vein into the right hepatic vein, creating a portosystemic shunt.

1	As a result of this procedure, the pressure in this patient’s portal system is lower and similar to that of the systemic venous system, so reducing the potential for bleeding at the portosystemic anastomoses (i.e., the colostomy). A 62-year-old man came to the emergency department with swelling of both legs and a large left varicocele (enlarged and engorged varicose veins around the left testis and within the left pampiniform plexus of veins). The patient was known to have a left renal cell carcinoma and was due to have this operated on the following week. Anatomically it is possible to link all of the findings with the renal cell carcinoma by knowing the biology of the tumor. Renal cell carcinoma tends to grow steadily and predictably. Typically, when the tumor is less than 3 to 4 cm, it remains confined to the kidney. Large tumors have the propensity to grow into the renal vein, the inferior vena cava and the right atrium and through the heart into the pulmonary artery.

1	The tumor grew into the renal vein. As the tumor grew into the renal vein it blocked off all tributaries draining into the vein, the largest of which is the left testicular vein. This blockage of the left testicular vein caused a dilation of the veins around the left testis (a varicocele occurred). The swollen legs were accounted for by caval obstruction. The tumor grew along the renal vein and into the inferior vena cava toward the heart. Renal tumors can grow rapidly; in this case the tumor grew rapidly into the inferior vena cava, occluding it. This increased the pressure in the leg veins, resulting in swelling and pitting edema of the ankles. The patient unfortunately died on the operating table.

1	The patient unfortunately died on the operating table. In this patient’s case, a “tongue” of tumor grew into the inferior vena cava. At the time of surgery, the initial dissection mobilized the kidney on its vascular pedicle; however, a large portion of tumor became detached in the inferior vena cava. The tumor embolus passed through the right atrium and right ventricle and occluded the pulmonary artery. This could not be cleared at the time of surgery, and the patient succumbed. A 65-year-old businessman came to the emergency department with severe lower abdominal pain that was predominantly central and left sided. He had pain radiating into the left loin, and he also noticed he was passing gas and fecal debris as he urinated. A CT scan of his abdomen and pelvis was performed (eFig. 4.191).

1	A CT scan of his abdomen and pelvis was performed (eFig. 4.191). The CT scan demonstrated a collection of fluid (likely a pelvic abscess) in the left iliac fossa. Associated with this collection of fluid was significant bowel wall thickening of the sigmoid colon and multiple small diverticula arising throughout the sigmoid colon. Gas was present in the bladder. An obstruction was noted in the left ureter and the left pelvicalyceal system. The patient underwent an urgent operation.

1	The patient underwent an urgent operation. As the surgeons entered into the abdominal cavity through a midline incision, the tissues in the left iliac fossa were significantly inflamed. The surgeon used his hand to mobilize the sigmoid colon and entered a cavity from which there was a “whoosh” of pus as indicated on the CT scan. The pus was washed out and drained. The sigmoid colon was remarkably thickened and inflamed and stuck to the dome of the bladder. Careful finger dissection revealed a small perforation in the dome of the bladder, allowing the passage of fecal material and gas into the bladder and producing the patient’s symptoms of pneumaturia and fecaluria. The sigmoid colon was resected. The rectal stump was oversewn and the descending colon was passed through the anterior abdominal wall to form a colostomy. The bladder was catheterized and the small hole in the dome of the bladder was oversewn.

1	The patient had a difficult postoperative period in the intensive care unit where he remained pyrexial and septic. The colostomy began to function well. An ultrasound was performed and demonstrated the continued dilation in the left kidney, and the patient underwent a nephrostomy. Under ultrasound guidance a drainage catheter was placed into the renal pelvis through the renal cortex on the left. A significant amount of pus was drained from the renal tract initially; however, after 24 hours urine passed freely. The likely cause for the obstruction was the inflammation around the distal ureter on the left. It is also possible that a small ureteric perforation occurred, allowing bacteria to enter the urinary tract. The patient made a further uneventful recovery with resumption of normal renal function and left the hospital.

1	The patient made a further uneventful recovery with resumption of normal renal function and left the hospital. On return to the surgeon in the outpatient clinic some weeks later, the patient did not wish to continue with his colostomy and bag. Further to discussion, surgery was planned to “rejoin” the patient. At operation the colostomy was “taken down” and the rectal stump was identified. There was, however, a significant gap between the bowel ends. To enable the bowel to be sutured, the descending colon was mobilized from the posterior abdominal wall. An anastomosis was performed and the patient left the hospital 1 week later and currently remains well. A 72-year-old man was brought to the emergency department with an abdominal aortic aneurysm (an expansion of the infrarenal abdominal aorta). The aneurysm measured 10 cm, and after discussion with the patient it was scheduled for repair. The surgical and endovascular treatment options were explained to the patient.

1	The surgical and endovascular treatment options were explained to the patient. Treatment of abdominal aortic aneurysms has been, for many years, an operative procedure where the dilation (ballooning) of the aorta is resected and a graft is sewn into position. A modern option is to place a graft to line the aneurysm from within the artery (endovascular aneurysm repair). In this technique the surgeon dissects the femoral artery and makes a small hole in it. The graft is compressed within a catheter and the catheter is passed through the femoral artery and the iliac arterial system into the distal abdominal aorta. The graft can then be released inside the aorta, effectively relining it to prevent further expansion of the aneurysm. Occasionally the relined aneurysm may continue to enlarge after the endovascular graft has been placed and a cause needs to be identified.

1	Occasionally the relined aneurysm may continue to enlarge after the endovascular graft has been placed and a cause needs to be identified. A Doppler ultrasound investigation of the abdomen and a CT scan revealed there was flow between the endovascular lining and the wall of the aneurysm. The likely sources for this bleeding were assessed. The graft usually begins below the level of the renal arteries and divides into two limbs that end in the common iliac arteries. The aneurysm may continue to be fed from any vessels between the graft and the aneurysm wall. These vessels can include the lumbar arteries and the inferior mesenteric artery. Interestingly, blood usually flows from the abdominal aorta into the inferior mesenteric artery and the lumbar arteries; however, with the changes in flow dynamics with the graft in place, blood may flow in the opposite direction through these branches, thereby leading to enlargement of the aneurysm.

1	Blood flow was from the superior mesenteric artery into the aneurysm sac. Above the level of the graft the superior mesenteric artery arises normally. From the right colic and middle colic branches a marginal branch around the colon anastomoses, in the region of the splenic flexure, with marginal branches from the inferior mesenteric artery (this can become a hypertrophied vessel known as the marginal artery of Drummond). In this situation, blood passed retrogradely into the inferior mesenteric artery, filling the aneurysm and allowing it to remain pressurized and expand. The inferior mesenteric artery was ligated laparoscopically and the aneurysm failed to expand further. Over the ensuing 6 months the aneurysm contracted. The patient remains fit and healthy, with two small scars in the groin. 412.e1 412.e2 Fig. 4.1, cont’d Conceptual Overview • Relationship to Other Regions Fig. 4.13, cont’d Fig. 4.15, cont’d Fig. 4.47, cont’d. Fig. 4.50, cont’d In the clinic—cont’d

1	412.e1 412.e2 Fig. 4.1, cont’d Conceptual Overview • Relationship to Other Regions Fig. 4.13, cont’d Fig. 4.15, cont’d Fig. 4.47, cont’d. Fig. 4.50, cont’d In the clinic—cont’d In the clinic—cont’d In the clinic—cont’d In the clinic—cont’d In the clinic—cont’d In the clinic—cont’d In the clinic—cont’d In the clinic—cont’d In the clinic—cont’d In the clinic—cont’d Surface Anatomy • How to Find the Superficial Inguinal Ring Surface Anatomy • Visualizing Structures at the LI Vertebral Level Surface Anatomy • Using Abdominal Quadrants to Locate Major Viscera Surface Anatomy • Where to Find the Spleen The pelvis and perineum are interrelated regions associated with the pelvic bones and terminal parts of the vertebral column. The pelvis is divided into two regions: The superior region related to upper parts of the pelvic bones and lower lumbar vertebrae is the false pelvis (greater pelvis) and is generally considered part of the abdominal cavity (Fig. 5.1).

1	The superior region related to upper parts of the pelvic bones and lower lumbar vertebrae is the false pelvis (greater pelvis) and is generally considered part of the abdominal cavity (Fig. 5.1). The true pelvis (lesser pelvis) is related to the inferior parts of the pelvic bones, sacrum, and coccyx, and has an inlet and an outlet. The bowl-shaped pelvic cavity enclosed by the true pelvis consists of the pelvic inlet, walls, and floor. This cavity is continuous superiorly with the abdominal cavity and contains elements of the urinary, gastrointestinal, and reproductive systems. The perineum (Fig. 5.1) is inferior to the floor of the pelvic cavity; its boundaries form the pelvic outlet. The perineum contains the external genitalia and external openings of the genitourinary and gastrointestinal systems. Contains and supports the bladder, rectum, anal canal, and reproductive tracts

1	Contains and supports the bladder, rectum, anal canal, and reproductive tracts Within the pelvic cavity, the bladder is positioned anteriorly and the rectum posteriorly in the midline. As it fills, the bladder expands superiorly into the abdomen. It is supported by adjacent elements of the pelvic bone and by the pelvic floor. The urethra passes through the pelvic floor to the perineum, where, in women, it opens externally (Fig. 5.2A) and in men it enters the base of the penis (Fig. 5.2B). Continuous with the sigmoid colon at the level of vertebra SIII, the rectum terminates at the anal canal, which penetrates the pelvic floor to open into the perineum. The anal canal is angled posteriorly on the rectum. This flexure is maintained by muscles of the pelvic floor and is relaxed during defecation. A skeletal muscle sphincter is associated with the anal canal and the urethra as each passes through the pelvic floor.

1	The pelvic cavity contains most of the reproductive tract in women and part of the reproductive tract in men. In women, the vagina penetrates the pelvic floor and connects with the uterus in the pelvic cavity. The uterus is positioned between the rectum and the bladder. A uterine (fallopian) tube extends laterally on each side toward the pelvic wall to open near the ovary. In men, the pelvic cavity contains the site of connection between the urinary and reproductive tracts. It also contains major glands associated with the reproductive system—the prostate and two seminal vesicles. Anchors the roots of the external genitalia In both genders, the roots of the external genitalia, the clitoris and the penis, are firmly anchored to: the bony margin of the anterior half of the pelvic outlet, and a thick, fibrous, perineal membrane, which fills the area (Fig. 5.3). The roots of the external genitalia consist of erectile (vascular) tissues and associated skeletal muscles.

1	The roots of the external genitalia consist of erectile (vascular) tissues and associated skeletal muscles. The pelvic inlet is somewhat heart shaped and completely ringed by bone (Fig. 5.4). Posteriorly, the inlet is bordered by the body of vertebra SI, which projects into the inlet as the sacral promontory. On each side of this vertebra, wing-like transverse processes called the alae (wings) contribute to the margin of the pelvic inlet. Laterally, a prominent rim on the pelvic bone continues the boundary of the inlet forward to the pubic symphysis, where the two pelvic bones are joined in the midline. Structures pass between the pelvic cavity and the abdomen through the pelvic inlet. During childbirth, the fetus passes through the pelvic inlet from the abdomen, into which the uterus has expanded during pregnancy, and then passes through the pelvic outlet.

1	During childbirth, the fetus passes through the pelvic inlet from the abdomen, into which the uterus has expanded during pregnancy, and then passes through the pelvic outlet. The walls of the true pelvis consist predominantly of bone, muscle, and ligaments, with the sacrum, coccyx, and inferior half of the pelvic bones forming much of them. Two ligaments—the sacrospinous and the sacrotuberous ligaments—are important architectural elements of the walls because they link each pelvic bone to the sacrum and coccyx (Fig. 5.5A). These ligaments also convert two notches on the pelvic bones—the greater and lesser sciatic notches—into foramina on the lateral pelvic walls. Completing the walls are the obturator internus and piriformis muscles (Fig. 5.5B), which arise in the pelvis and exit through the sciatic foramina to act on the hip joint. The diamond-shaped pelvic outlet is formed by both bone and ligaments (Fig. 5.6). It is limited anteriorly in the midline by the pubic symphysis.

1	The diamond-shaped pelvic outlet is formed by both bone and ligaments (Fig. 5.6). It is limited anteriorly in the midline by the pubic symphysis. On each side, the inferior margin of the pelvic bone projects posteriorly and laterally from the pubic symphysis to end in a prominent tuberosity, the ischial tuberosity. Together, these elements construct the pubic arch, which forms the margin of the anterior half of the pelvic outlet. The sacrotuberous ligament continues this margin posteriorly from the ischial tuberosity to the coccyx and sacrum. The pubic symphysis, ischial tuberosities, and coccyx can all be palpated. The pelvic floor, which separates the pelvic cavity from the perineum, is formed by muscles and fascia (Fig. 5.7).

1	The pelvic floor, which separates the pelvic cavity from the perineum, is formed by muscles and fascia (Fig. 5.7). Two levator ani muscles attach peripherally to the pelvic walls and join each other at the midline by a connective tissue raphe. Together they are the largest components of the bowlor funnel-shaped structure known as the pelvic diaphragm, which is completed posteriorly by the coccygeus muscles. These latter muscles overlie the sacrospinous ligaments and pass between the margins of the sacrum and the coccyx and a prominent spine on the pelvic bone, the ischial spine. The pelvic diaphragm forms most of the pelvic floor and in its anterior regions contains a U-shaped defect, which is associated with elements of the urogenital system. The anal canal passes from the pelvis to the perineum through a posterior circular orifice in the pelvic diaphragm. The pelvic floor is supported anteriorly by: the perineal membrane, and muscles in the deep perineal pouch.

1	The pelvic floor is supported anteriorly by: the perineal membrane, and muscles in the deep perineal pouch. The perineal membrane is a thick, triangular fascial sheet that fills the space between the arms of the pubic arch, and has a free posterior border (Fig. 5.7). The deep perineal pouch is a narrow region superior to the perineal membrane. The margins of the U-shaped defect in the pelvic diaphragm merge into the walls of the associated viscera and with muscles in the deep perineal pouch below. The vagina and the urethra penetrate the pelvic floor to pass from the pelvic cavity to the perineum. The pelvic cavity is lined by peritoneum continuous with the peritoneum of the abdominal cavity that drapes over the superior aspects of the pelvic viscera, but in most regions, does not reach the pelvic floor (Fig. 5.8A).

1	The pelvic viscera are located in the midline of the pelvic cavity. The bladder is anterior and the rectum is posterior. In women, the uterus lies between the bladder and rectum (Fig. 5.8B). Other structures, such as vessels and nerves, lie deep to the peritoneum in association with the pelvic walls and on either side of the pelvic viscera. The perineum lies inferior to the pelvic floor between the lower limbs (Fig. 5.9). Its margin is formed by the pelvic outlet. An imaginary line between the ischial tuberosities divides the perineum into two triangular regions. Anteriorly, the urogenital triangle contains the roots of the external genitalia and, in women, the openings of the urethra and the vagina (Fig. 5.9A). In men, the distal part of the urethra is enclosed by erectile tissues and opens at the end of the penis (Fig. 5.9B). Posteriorly, the anal triangle contains the anal aperture.

1	Posteriorly, the anal triangle contains the anal aperture. The cavity of the true pelvis is continuous with the abdominal cavity at the pelvic inlet (Fig. 5.10A). All structures passing between the pelvic cavity and abdomen, including major vessels, nerves, and lymphatics, as well as the sigmoid colon and ureters, pass via the inlet. In men, the ductus deferens on each side passes through the anterior abdominal wall and over the inlet to enter the pelvic cavity. In women, ovarian vessels, nerves, and lymphatics pass through the inlet to reach the ovaries, which lie on each side just inferior to the pelvic inlet. Three apertures in the pelvic wall communicate with the lower limb (Fig. 5.10A): the obturator canal, the greater sciatic foramen, and the lesser sciatic foramen.

1	Three apertures in the pelvic wall communicate with the lower limb (Fig. 5.10A): the obturator canal, the greater sciatic foramen, and the lesser sciatic foramen. The obturator canal forms a passageway between the pelvic cavity and the adductor region of the thigh, and is formed in the superior aspect of the obturator foramen, between bone, a connective tissue membrane, and muscles that fill the foramen. The lesser sciatic foramen, which lies inferior to the pelvic floor, provides communication between the gluteal region and the perineum (Fig. 5.10B). The pelvic cavity also communicates directly with the perineum through a small gap between the pubic symphysis and the perineal membrane (Fig. 5.10B). The pelvic cavity projects posteriorly

1	The pelvic cavity also communicates directly with the perineum through a small gap between the pubic symphysis and the perineal membrane (Fig. 5.10B). The pelvic cavity projects posteriorly In the anatomical position, the anterior superior iliac spines and the superior edge of the pubic symphysis lie in the same vertical plane (Fig. 5.11). Consequently, the pelvic inlet is angled 50°–60° forward relative to the horizontal plane, and the pelvic cavity projects posteriorly from the abdominal cavity. Meanwhile, the urogenital part of the pelvic outlet (the pubic arch) is oriented in a nearly horizontal plane, whereas the posterior part of the outlet is positioned more vertically. The urogenital triangle of the perineum therefore faces inferiorly, while the anal triangle faces more posteriorly. Important structures cross the ureters in the pelvic cavity

1	Important structures cross the ureters in the pelvic cavity The ureters drain the kidneys, course down the posterior abdominal wall, and cross the pelvic inlet to enter the pelvic cavity. They continue inferiorly along the lateral pelvic wall and ultimately connect with the base of the bladder. An important structure crosses the ureters in the pelvic cavity in both men and women—in women, the uterine artery crosses the ureter lateral to the cervix of the uterus (Fig. 5.12A), and in men, the ductus deferens crosses over the ureter just posterior to the bladder (Fig. 5.12B). The prostate in men and the uterus in women are anterior to the rectum In men, the prostate gland is situated immediately anterior to the rectum, just above the pelvic floor (Fig. 5.13). It can be felt by digital palpation during a rectal examination.

1	In men, the prostate gland is situated immediately anterior to the rectum, just above the pelvic floor (Fig. 5.13). It can be felt by digital palpation during a rectal examination. In both sexes, the anal canal and the lower rectum also can be evaluated during a rectal examination by a clinician. In women, the cervix and lower part of the body of the uterus also are palpable. However, these structures can more easily be palpated with a bimanual examination where the index and middle fingers of a clinician’s hand are placed in the vagina and the other hand is placed on the lower anterior abdominal wall. The organs are felt between the two hands. This bimanual technique can also be used to examine the ovaries and uterine tubes. The perineum is innervated by sacral spinal

1	The perineum is innervated by sacral spinal Dermatomes of the perineum in both men and women are from spinal cord levels S3 to S5, except for the anterior regions, which tend to be innervated by spinal cord level L1 by nerves associated with the abdominal wall (Fig. 5.14). Dermatomes of L2 to S2 are predominantly in the lower limb. Most of the skeletal muscles contained in the perineum and the pelvic floor, including the external anal sphincter and external urethral sphincter, are innervated by spinal cord levels S2 to S4. Much of the somatic motor and sensory innervation of the perineum is provided by the pudendal nerve from spinal cord levels S2 to S4. Nerves are related to bone

1	Much of the somatic motor and sensory innervation of the perineum is provided by the pudendal nerve from spinal cord levels S2 to S4. Nerves are related to bone The pudendal nerve is the major nerve of the perineum and is directly associated with the ischial spine of the pelvis (Fig. 5.15). On each side of the body, these spines and the attached sacrospinous ligaments separate the greater sciatic foramina from the lesser sciatic foramina on the lateral pelvic wall. The pudendal nerve leaves the pelvic cavity through the greater sciatic foramen and then immediately enters the perineum inferiorly to the pelvic floor by passing around the ischial spine and through the lesser sciatic foramen (Fig. 5.15). The ischial spine can be palpated transvaginally in women and is the landmark that can be used for administering a pudendal nerve block. Parasympathetic innervation from spinal cord levels S2 to S4 controls erection

1	Parasympathetic innervation from spinal cord levels S2 to S4 controls erection The parasympathetic innervation from spinal cord levels S2 to S4 controls genital erection in both women and men (Fig. 5.16). On each side, preganglionic parasympathetic nerves leave the anterior rami of the sacral spinal nerves and enter the inferior hypogastric plexus (pelvic plexus) on the lateral pelvic wall. The two inferior hypogastric plexuses are inferior extensions of the abdominal prevertebral plexus that forms on the posterior abdominal wall in association with the abdominal aorta. Nerves derived from these plexuses penetrate the pelvic floor to innervate the erectile tissues of the clitoris in women and the penis in men. Muscles and fascia of the pelvic floor and perineum intersect at the perineal body

1	Muscles and fascia of the pelvic floor and perineum intersect at the perineal body Structures of the pelvic floor intersect with structures in the perineum at the perineal body (Fig. 5.17). This poorly defined fibromuscular node lies at the center of the perineum, approximately midway between the two ischial tuberosities. Converging at the perineal body are: the levator ani muscles of the pelvic diaphragm, and muscles in the urogenital and anal triangles of the perineum, including the skeletal muscle sphincters associated with the urethra, vagina, and anus. The course of the urethra is different in men In women, the urethra is short and passes inferiorly from the bladder through the pelvic floor and opens directly into the perineum (Fig. 5.18A).

1	The course of the urethra is different in men In women, the urethra is short and passes inferiorly from the bladder through the pelvic floor and opens directly into the perineum (Fig. 5.18A). In men the urethra passes through the prostate before coursing through the deep perineal pouch and perineal membrane and then becomes enclosed within the erectile tissues of the penis before opening at the end of the penis (Fig. 5.18B). The penile part of the male urethra has two angles: The more important of these is a fixed angle where the urethra bends anteriorly in the root of the penis after passing through the perineal membrane. Another angle occurs distally where the unattached part of the penis curves inferiorly—when the penis is erect, this second angle disappears. It is important to consider the different courses of the urethra in men and women when catheterizing patients and when evaluating perineal injuries and pelvic pathology.

1	It is important to consider the different courses of the urethra in men and women when catheterizing patients and when evaluating perineal injuries and pelvic pathology. The pelvis is the region of the body surrounded by the pelvic bones and the inferior elements of the vertebral column. It is divided into two major regions: the superior region is the false (greater) pelvis and is part of the abdominal cavity; the inferior region is the true (lesser) pelvis, which encloses the pelvic cavity. The bowl-shaped pelvic cavity is continuous above with the abdominal cavity. The rim of the pelvic cavity (the pelvic inlet) is completely encircled by bone. The pelvic floor is a fibromuscular structure separating the pelvic cavity above from the perineum below.

1	The perineum is inferior to the pelvic floor and its margin is formed by the pelvic outlet. The perineum contains: the terminal openings of the gastrointestinal and urinary systems, the external opening of the reproductive tract, and the roots of the external genitalia. The bones of the pelvis consist of the right and left pelvic (hip) bones, the sacrum, and the coccyx. The sacrum articulates superiorly with vertebra LV at the lumbosacral joint. The pelvic bones articulate posteriorly with the sacrum at the sacro-iliac joints and with each other anteriorly at the pubic symphysis. The pelvic bone is irregular in shape and has two major parts separated by an oblique line on the medial surface of the bone (Fig. 5.19A): The pelvic bone above this line represents the lateral wall of the false pelvis, which is part of the abdominal cavity. The pelvic bone below this line represents the lateral wall of the true pelvis, which contains the pelvic cavity.

1	The pelvic bone below this line represents the lateral wall of the true pelvis, which contains the pelvic cavity. The linea terminalis is the lower two-thirds of this line and contributes to the margin of the pelvic inlet. The lateral surface of the pelvic bone has a large articular socket, the acetabulum, which, together with the head of the femur, forms the hip joint (Fig. 5.19B). Inferior to the acetabulum is the large obturator foramen, most of which is closed by a flat connective tissue membrane, the obturator membrane. A small obturator canal remains open superiorly between the membrane and adjacent bone, providing a route of communication between the lower limb and the pelvic cavity. The posterior margin of the bone is marked by two notches separated by the ischial spine: the greater sciatic notch, and the lesser sciatic notch. The posterior margin terminates inferiorly as the large ischial tuberosity.

1	The posterior margin terminates inferiorly as the large ischial tuberosity. The irregular anterior margin of the pelvic bone is marked by the anterior superior iliac spine, the anterior inferior iliac spine, and the pubic tubercle. Components of the pelvic bone Each pelvic bone is formed by three elements: the ilium, pubis, and ischium. At birth, these bones are connected by cartilage in the area of the acetabulum; later, at between 16 and 18 years of age, they fuse into a single bone (Fig. 5.20). Of the three components of the pelvic bone, the ilium is the most superior in position. The ilium is separated into upper and lower parts by a ridge on the medial surface (Fig. 5.21A).

1	Of the three components of the pelvic bone, the ilium is the most superior in position. The ilium is separated into upper and lower parts by a ridge on the medial surface (Fig. 5.21A). Posteriorly, the ridge is sharp and lies immediately superior to the surface of the bone that articulates with the sacrum. This sacral surface has a large L-shaped facet for articulating with the sacrum and an expanded, posterior roughened area for the attachment of the strong ligaments that support the sacro-iliac joint (Fig. 5.21). Anteriorly, the ridge separating the upper and lower parts of the ilium is rounded and termed the arcuate line. The arcuate line forms part of the linea terminalis and the pelvic brim. The portion of the ilium lying inferiorly to the arcuate line is the pelvic part of the ilium and contributes to the wall of the lesser or true pelvis.

1	The portion of the ilium lying inferiorly to the arcuate line is the pelvic part of the ilium and contributes to the wall of the lesser or true pelvis. The upper part of the ilium expands to form a flat, fan-shaped “wing,” which provides bony support for the lower abdomen, or false pelvis. This part of the ilium provides attachment for muscles functionally associated with the lower limb. The anteromedial surface of the wing is concave and forms the iliac fossa. The external (gluteal) surface of the wing is marked by lines and roughenings and is related to the gluteal region of the lower limb (Fig. 5.21B). The entire superior margin of the ilium is thickened to form a prominent crest (the iliac crest), which is the site of attachment for muscles and fascia of the abdomen, back, and lower limb and terminates anteriorly as the anterior superior iliac spine and posteriorly as the posterior superior iliac spine.

1	A prominent tubercle, the tuberculum of the iliac crest, projects laterally near the anterior end of the crest; the posterior end of the crest thickens to form the iliac tuberosity. Inferior to the anterior superior iliac spine of the crest, on the anterior margin of the ilium, is a rounded protuberance called the anterior inferior iliac spine. This structure serves as the point of attachment for the rectus femoris muscle of the anterior compartment of the thigh and the iliofemoral ligament associated with the hip joint. A less prominent posterior inferior iliac spine occurs along the posterior border of the sacral surface of the ilium, where the bone angles forward to form the superior margin of the greater sciatic notch. The anterior and inferior part of the pelvic bone is the pubis (Fig. 5.21). It has a body and two arms (rami).

1	The anterior and inferior part of the pelvic bone is the pubis (Fig. 5.21). It has a body and two arms (rami). The body is flattened dorsoventrally and articulates with the body of the pubic bone on the other side at the pubic symphysis. The body has a rounded pubic crest on its superior surface that ends laterally as the prominent pubic tubercle. The superior pubic ramus projects posterolaterally from the body and joins with the ilium and ischium at its base, which is positioned toward the acetabulum. The sharp superior margin of this triangular surface is termed the pecten pubis (pectineal line), which forms part of the linea terminalis of the pelvic bone and the pelvic inlet. Anteriorly, this line is continuous with the pubic crest, which also is part of the linea terminalis and pelvic inlet. The superior pubic ramus is marked on its inferior surface by the obturator groove, which forms the upper margin of the obturator canal.

1	The inferior ramus projects laterally and inferiorly to join with the ramus of the ischium. The ischium is the posterior and inferior part of the pelvic bone (Fig. 5.21). It has: a large body that projects superiorly to join with the ilium and the superior ramus of the pubis, and a ramus that projects anteriorly to join with the inferior ramus of the pubis. The posterior margin of the bone is marked by a prominent ischial spine that separates the lesser sciatic notch, below, from the greater sciatic notch, above. The most prominent feature of the ischium is a large tuberosity (the ischial tuberosity) on the posteroinferior aspect of the bone. This tuberosity is an important site for the attachment of lower limb muscles and for supporting the body when sitting.

1	The sacrum, which has the appearance of an inverted triangle, is formed by the fusion of the five sacral vertebrae (Fig. 5.22). The base of the sacrum articulates with vertebra LV, and its apex articulates with the coccyx. Each of the lateral surfaces of the bone bears a large L-shaped facet for articulation with the ilium of the pelvic bone. Posterior to the facet is a large roughened area for the attachment of ligaments that support the sacro-iliac joint. The superior surface of the sacrum is characterized by the superior aspect of the body of vertebra SI and is flanked on each side by an expanded wing-like transverse process termed the ala. The anterior edge of the vertebral body projects forward as the promontory. The anterior surface of the sacrum is concave; the posterior surface is convex. Because the transverse processes of adjacent sacral vertebrae fuse lateral to the position of the intervertebral foramina and lateral to the bifurcation of spinal nerves into posterior and

1	is convex. Because the transverse processes of adjacent sacral vertebrae fuse lateral to the position of the intervertebral foramina and lateral to the bifurcation of spinal nerves into posterior and anterior rami, the posterior and anterior rami of spinal nerves S1 to S4 emerge from the sacrum through separate foramina. There are four pairs of anterior sacral foramina on the anterior surface of the sacrum for anterior rami, and four pairs of posterior sacral foramina on the posterior surface for the posterior rami. The sacral canal is a continuation of the vertebral canal that terminates as the sacral hiatus.

1	The small terminal part of the vertebral column is the coccyx, which consists of four fused coccygeal vertebrae (Fig. 5.22) and, like the sacrum, has the shape of an inverted triangle. The base of the coccyx is directed superiorly. The superior surface bears a facet for articulation with the sacrum and two horns, or cornua, one on each side, that project upward to articulate or fuse with similar downward-projecting cornua from the sacrum. These processes are modified superior and inferior articular processes that are present on other vertebrae. Each lateral surface of the coccyx has a small rudimentary transverse process, extending from the first coccygeal vertebra. Vertebral arches are absent from coccygeal vertebrae; therefore no bony vertebral canal is present in the coccyx.

1	The sacrum articulates superiorly with the lumbar part of the vertebral column. The lumbosacral joints are formed between vertebra LV and the sacrum and consist of: the two zygapophysial joints, which occur between adjacent inferior and superior articular processes, and an intervertebral disc that joins the bodies of vertebrae LV and SI (Fig. 5.23A). These joints are similar to those between other vertebrae, with the exception that the sacrum is angled posteriorly on vertebra LV. As a result, the anterior part of the intervertebral disc between the two bones is thicker than the posterior part. The lumbosacral joints are reinforced by strong iliolumbar and lumbosacral ligaments that extend from the expanded transverse processes of vertebra LV to the ilium and the sacrum, respectively (Fig. 5.23B).

1	The sacro-iliac joints transmit forces from the lower limbs to the vertebral column. They are synovial joints between the L-shaped articular facets on the lateral surfaces of the sacrum and similar facets on the iliac parts of the pelvic bones (Fig. 5.24A). The joint surfaces have an irregular contour and interlock to resist movement. The joints often become fibrous with age and may become completely ossified.

1	Each sacro-iliac joint is stabilized by three ligaments: the anterior sacro-iliac ligament, which is a thickening of the fibrous membrane of the joint capsule and runs anteriorly and inferiorly to the joint (Fig. 5.24B); the interosseous sacro-iliac ligament, which is the largest, strongest ligament of the three, and is positioned immediately posterosuperior to the joint and attaches to adjacent expansive roughened areas on the ilium and sacrum, thereby filling the gap between the two bones (Fig. 5.24A,C); and the posterior sacro-iliac ligament, which covers the interosseous sacro-iliac ligament (Fig. 5.24C).

1	The pubic symphysis lies anteriorly between the adjacent surfaces of the pubic bones (Fig. 5.25). Each of the joint’s surfaces is covered by hyaline cartilage and is linked across the midline to adjacent surfaces by fibrocartilage. The joint is surrounded by interwoven layers of collagen fibers and the two major ligaments associated with it are: the superior pubic ligament, located above the joint, and the inferior pubic ligament, located below it. In the anatomical position, the pelvis is oriented so that the front edge of the top of the pubic symphysis and the anterior superior iliac spines lie in the same vertical plane (Fig. 5.26). As a consequence, the pelvic inlet, which marks the entrance to the pelvic cavity, is tilted to face anteriorly, and the bodies of the pubic bones and the pubic arch are positioned in a nearly horizontal plane facing the ground.

1	The pelvises of women and men differ in a number of ways, many of which have to do with the passing of a baby through a woman’s pelvic cavity during childbirth. The pelvic inlet in women is circular (Fig. 5.27A) compared with the heart-shaped pelvic inlet (Fig. 5.27B) in men. The more circular shape is partly caused by the less distinct promontory and broader alae in women. The angle formed by the two arms of the pubic arch is larger in women (80°–85°) than it is in men (50°–60°). The ischial spines generally do not project as far medially into the pelvic cavity in women as they do in men. The true pelvis is cylindrical and has an inlet, a wall, and an outlet. The inlet is open, whereas the pelvic floor closes the outlet and separates the pelvic cavity, above, from the perineum, below.

1	The pelvic inlet is the circular opening between the abdominal cavity and the pelvic cavity through which structures traverse between the abdomen and pelvic cavity. It is completely surrounded by bones and joints (Fig. 5.28). The promontory of the sacrum protrudes into the inlet, forming its posterior margin in the midline. On either side of the promontory, the margin is formed by the alae of the sacrum. The margin of the pelvic inlet then crosses the sacro-iliac joint and continues along the linea terminalis (i.e., the arcuate line, the pecten pubis or pectineal line, and the pubic crest) to the pubic symphysis. The walls of the pelvic cavity consist of the sacrum, the coccyx, the pelvic bones inferior to the linea terminalis, two ligaments, and two muscles. Ligaments of the pelvic wall

1	The walls of the pelvic cavity consist of the sacrum, the coccyx, the pelvic bones inferior to the linea terminalis, two ligaments, and two muscles. Ligaments of the pelvic wall The sacrospinous and sacrotuberous ligaments (Fig. 5.29A) are major components of the lateral pelvic walls that help define the apertures between the pelvic cavity and adjacent regions through which structures pass. The smaller of the two, the sacrospinous ligament, is triangular, with its apex attached to the ischial spine and its base attached to the related margins of the sacrum and the coccyx. The sacrotuberous ligament is also triangular and is superficial to the sacrospinous ligament. Its base has a broad attachment that extends from the posterior superior iliac spine of the pelvic bone, along the dorsal aspect and the lateral margin of the sacrum, and onto the dorsolateral surface of the coccyx. Laterally, the apex of the ligament is attached to the medial margin of the ischial tuberosity.

1	These ligaments stabilize the sacrum on the pelvic bones by resisting the upward tilting of the inferior aspect of the sacrum (Fig. 5.29B). They also convert the greater and lesser sciatic notches of the pelvic bone into foramina (Fig. 5.29A,B). The greater sciatic foramen lies superior to the sacrospinous ligament and the ischial spine. The lesser sciatic foramen lies inferior to the ischial spine and sacrospinous ligament between the sacrospinous and sacrotuberous ligaments. Muscles of the pelvic wall Two muscles, the obturator internus and the piriformis, contribute to the lateral walls of the pelvic cavity. These muscles originate in the pelvic cavity but attach peripherally to the femur. The obturator internus is a flat, fan-shaped muscle that originates from the deep surface of the obturator membrane and from associated regions of the pelvic bone that surround the obturator foramen (Fig. 5.30 and Table 5.1).

1	The muscle fibers of the obturator internus converge to form a tendon that leaves the pelvic cavity through the lesser sciatic foramen, makes a 90° bend around the ischium between the ischial spine and ischial tuberosity, and then passes posterior to the hip joint to insert on the greater trochanter of the femur. The obturator internus forms a large part of the anterolateral wall of the pelvic cavity. The piriformis is triangular and originates in the bridges of bone between the four anterior sacral foramina. It passes laterally through the greater sciatic foramen, crosses the posterosuperior aspect of the hip joint, and inserts on the greater trochanter of the femur above the insertion of the obturator internus muscle (Fig. 5.30 and Table 5.1).

1	A large part of the posterolateral wall of the pelvic cavity is formed by the piriformis. In addition, this muscle separates the greater sciatic foramen into two regions, one above the muscle and one below. Vessels and nerves coursing between the pelvic cavity and the gluteal region pass through these two regions. Apertures in the pelvic wall Each lateral pelvic wall has three major apertures through which structures pass between the pelvic cavity and other regions: the obturator canal, the greater sciatic foramen, and the lesser sciatic foramen. At the top of the obturator foramen is the obturator canal, which is bordered by the obturator membrane, the associated obturator muscles, and the superior pubic ramus (Fig. 5.31). The obturator nerve and vessels pass from the pelvic cavity to the thigh through this canal.

1	The greater sciatic foramen is a major route of communication between the pelvic cavity and the lower limb (Fig. 5.31). It is formed by the greater sciatic notch in the pelvic bone, the sacrotuberous and the sacrospinous ligaments, and the spine of the ischium. The piriformis muscle passes through the greater sciatic foramen, dividing it into two parts. The superior gluteal nerves and vessels pass through the foramen above the piriformis. Passing through the foramen below the piriformis are the inferior gluteal nerves and vessels, the sciatic nerve, the pudendal nerve, the internal pudendal vessels, the posterior femoral cutaneous nerves, and the nerves to the obturator internus and quadratus femoris muscles. The lesser sciatic foramen is formed by the lesser sciatic notch of the pelvic bone, the ischial spine, the sacrospinous ligament, and the sacrotuberous ligament (Fig. 5.31).

1	The lesser sciatic foramen is formed by the lesser sciatic notch of the pelvic bone, the ischial spine, the sacrospinous ligament, and the sacrotuberous ligament (Fig. 5.31). The tendon of the obturator internus muscle passes through this foramen to enter the gluteal region of the lower limb. Because the lesser sciatic foramen is positioned below the attachment of the pelvic floor, it acts as a route of communication between the perineum and the gluteal region. The pudendal nerve and internal pudendal vessels pass between the pelvic cavity (above the pelvic floor) and the perineum (below the pelvic floor), by first passing out of the pelvic cavity through the greater sciatic foramen and then looping around the ischial spine and sacrospinous ligament to pass through the lesser sciatic foramen to enter the perineum. The nerve to obturator internus follows a similar course.

1	The pelvic outlet is diamond shaped, with the anterior part of the diamond defined predominantly by bone and the posterior part mainly by ligaments (Fig. 5.32). In the midline anteriorly, the boundary of the pelvic outlet is the pubic symphysis. Extending laterally and posteriorly, the boundary on each side is the inferior border of the body of the pubis, the inferior ramus of the pubis, the ramus of the ischium, and the ischial tuberosity. Together, the elements on both sides form the pubic arch. From the ischial tuberosities, the boundaries continue posteriorly and medially along the sacrotuberous ligament on both sides to the coccyx. Terminal parts of the urinary and gastrointestinal tracts and the vagina pass through the pelvic outlet. The area enclosed by the boundaries of the pelvic outlet and below the pelvic floor is the perineum.

1	The area enclosed by the boundaries of the pelvic outlet and below the pelvic floor is the perineum. The pelvic floor is formed by the pelvic diaphragm and, in the anterior midline, the perineal membrane and the muscles in the deep perineal pouch. The pelvic diaphragm is formed by the levator ani and the coccygeus muscles from both sides. The pelvic floor separates the pelvic cavity, above, from the perineum, below. The pelvic diaphragm The pelvic diaphragm is the muscular part of the pelvic floor. Shaped like a bowl or funnel and attached superiorly to the pelvic walls, it consists of the levator ani and the coccygeus muscles (Fig. 5.34 and Table 5.2).

1	The pelvic diaphragm’s circular line of attachment to the cylindrical pelvic wall passes, on each side, between the greater sciatic foramen and the lesser sciatic foramen. Thus: the greater sciatic foramen is situated above the level of the pelvic floor and is a route of communication between the pelvic cavity and the gluteal region of the lower limb; and the lesser sciatic foramen is situated below the pelvic floor, providing a route of communication between the gluteal region of the lower limb and the perineum. The two levator ani muscles originate from each side of the pelvic wall, course medially and inferiorly, and join together in the midline. The attachment to the pelvic wall follows the circular contour of the wall and includes: the posterior aspect of the body of the pubic bone, a linear thickening called the tendinous arch, in the fascia covering the obturator internus muscle, and the spine of the ischium.

1	At the midline, the muscles blend together posterior to the vagina in women and around the anal aperture in both sexes. Posterior to the anal aperture, the muscles come together as a ligament or raphe called the anococcygeal ligament (anococcygeal body) and attaches to the coccyx. Anteriorly, the muscles are separated by a U-shaped defect or gap termed the urogenital hiatus. The margins of this hiatus merge with the walls of the associated viscera and with muscles in the deep perineal pouch below. The hiatus allows the urethra (in both men and women), and the vagina (in women), to pass through the pelvic diaphragm (Fig. 5.34). The levator ani muscles are divided into at least three collections of muscle fibers, based on site of origin and relationship to viscera in the midline: the pubococcygeus, the puborectalis, and the iliococcygeus muscles.

1	The pubococcygeus originates from the body of the pubis and courses posteriorly to attach along the midline as far back as the coccyx. This part of the muscle is further subdivided on the basis of association with structures in the midline into the puboprostaticus (levator prostatae), the pubovaginalis, and the puboanalis muscles. A second major collection of muscle fibers, the puborectalis portion of the levator ani muscles, originates, in association with the pubococcygeus muscle, from the pubis and passes inferiorly on each side to form a sling around the terminal part of the gastrointestinal tract. This muscular sling maintains an angle or flexure, called the perineal flexure, at the anorectal junction. This angle functions as part of the mechanism that keeps the end of the gastrointestinal system closed.

1	The final part of the levator ani muscle is the iliococcygeus. This part of the muscle originates from the fascia that covers the obturator internus muscle. It joins the same muscle on the other side in the midline to form a ligament or raphe that extends from the anal aperture to the coccyx. The levator ani muscles help support the pelvic viscera and maintain closure of the rectum and vagina. They are innervated directly by branches from the anterior ramus of S4 and by branches of the pudendal nerve (S2 to S4). The two coccygeus muscles, one on each side, are triangular and overlie the sacrospinous ligaments; together they complete the posterior part of the pelvic diaphragm (Fig. 5.34 and Table 5.2). They are attached, by their apices, to the tips of the ischial spines and, by their bases, to the lateral margins of the coccyx and adjacent margins of the sacrum.

1	The coccygeus muscles are innervated by branches from the anterior rami of S3 and S4 and participate in supporting the posterior aspect of the pelvic floor. The perineal membrane and deep The perineal membrane is a thick fascial, triangular structure attached to the bony framework of the pubic arch (Fig. 5.36A). It is oriented in the horizontal plane and has a free posterior margin. Anteriorly, there is a small gap (blue arrow in Fig. 5.36A) between the membrane and the inferior pubic ligament (a ligament associated with the pubic symphysis). The perineal membrane is related above to a thin space called the deep perineal pouch (deep perineal space) (Fig. 5.36B), which contains a layer of skeletal muscle and various neurovascular elements.

1	The deep perineal pouch is open above and is not separated from more superior structures by a distinct layer of fascia. The parts of the perineal membrane and structures in the deep perineal pouch, enclosed by the urogenital hiatus above, therefore contribute to the pelvic floor and support elements of the urogenital system in the pelvic cavity, even though the perineal membrane and deep perineal pouch are usually considered parts of the perineum. The perineal membrane and adjacent pubic arch provide attachment for the roots of the external genitalia and the muscles associated with them (Fig. 5.36C). The urethra penetrates vertically through a circular hiatus in the perineal membrane as it passes from the pelvic cavity, above, to the perineum, below. In women, the vagina also passes through a hiatus in the perineal membrane just posterior to the urethral hiatus.

1	Within the deep perineal pouch, a sheet of skeletal muscle functions as a sphincter, mainly for the urethra, and as a stabilizer of the posterior edge of the perineal membrane (Fig. 5.37 and Table 5.3). Anteriorly, a group of muscle fibers surround the urethra and collectively form the external urethral sphincter. Two additional groups of muscle fibers are associated with the urethra and vagina in women. One group forms the sphincter urethrovaginalis, which surrounds the urethra and vagina as a unit. The second group forms the compressor urethrae, on each side, which originate from the ischiopubic rami and meet anterior to the urethra. Together with the external urethral sphincter, the sphincter urethrovaginalis and compressor urethrae facilitate closing of the urethra.

1	In both men and women, a deep transverse perineal muscle on each side parallels the free margin of the perineal membrane and joins with its partner at the midline. These muscles are thought to stabilize the position of the perineal body, which is a midline structure along the posterior edge of the perineal membrane. The perineal body is an ill-defined but important connective tissue structure into which muscles of the pelvic floor and the perineum attach (Fig. 5.38). It is positioned in the midline along the posterior border of the perineal membrane, to which it attaches. The posterior end of the urogenital hiatus in the levator ani muscles is also connected to it.

1	The deep transverse perineal muscles intersect at the perineal body; in women, the sphincter urethrovaginalis also attaches to the perineal body. Other muscles that connect to the perineal body include the external anal sphincter, the superficial transverse perineal muscles, and the bulbospongiosus muscles of the perineum. The pelvic viscera include parts of the gastrointestinal system, the urinary system, and the reproductive system. The viscera are arranged in the midline, from front to back; the neurovascular supply is through branches that pass medially from vessels and nerves associated with the pelvic walls. Pelvic parts of the gastrointestinal system consist mainly of the rectum and the anal canal, although the terminal part of the sigmoid colon is also in the pelvic cavity (Fig. 5.39).

1	Pelvic parts of the gastrointestinal system consist mainly of the rectum and the anal canal, although the terminal part of the sigmoid colon is also in the pelvic cavity (Fig. 5.39). The rectum is continuous: above, with the sigmoid colon at about the level of vertebra SIII, and below, with the anal canal as this structure penetrates the pelvic floor and passes through the perineum to end as the anus. The rectum, the most posterior element of the pelvic viscera, is immediately anterior to and follows the concave contour of the sacrum. The anorectal junction is pulled forward (perineal flexure) by the action of the puborectalis part of the levator ani muscle, so the anal canal moves in a posterior direction as it passes inferiorly through the pelvic floor.

1	In addition to conforming to the general curvature of the sacrum in the anteroposterior plane, the rectum has three lateral curvatures; the upper and lower curvatures to the right and the middle curvature to the left. The lower part of the rectum is expanded to form the rectal ampulla. Finally, unlike the colon, the rectum lacks distinct taeniae coli muscles, omental appendices, and sacculations (haustra of the colon). The anal canal begins at the terminal end of the rectal ampulla where it narrows at the pelvic floor. It terminates as the anus after passing through the perineum. As it passes through the pelvic floor, the anal canal is surrounded along its entire length by the internal and external anal sphincters, which normally keep it closed.

1	The lining of the anal canal bears a number of characteristic structural features that reflect the approximate position of the anococcygeal membrane in the fetus (which closes the terminal end of the developing gastrointestinal system in the fetus) and the transition from gastrointestinal mucosa to skin in the adult (Fig. 5.39B). The upper part of the anal canal is lined by mucosa similar to that lining the rectum and is distinguished by a number of longitudinally oriented folds known as anal columns, which are united inferiorly by crescentic folds termed anal valves. Superior to each valve is a depression termed an anal sinus. The anal valves together form a circle around the anal canal at a location known as the pectinate line, which marks the approximate position of the anal membrane in the fetus.

1	Inferior to the pectinate line is a transition zone known as the anal pecten, which is lined by nonkeratinized stratified squamous epithelium. The anal pecten ends inferiorly at the anocutaneous line (“white line”), or where the lining of the anal canal becomes true skin. Given the position of the colon and rectum in the abdominopelvic cavity and its proximity to other organs, it is extremely important to accurately stage colorectal tumors: a tumor in the pelvis, for example, could invade the uterus or bladder. Assessing whether spread has occurred may involve ultrasound scanning, computed tomography, and magnetic resonance imaging. The pelvic parts of the urinary system consist of the terminal parts of the ureters, the bladder, and the proximal part of the urethra (Fig. 5.40).

1	The pelvic parts of the urinary system consist of the terminal parts of the ureters, the bladder, and the proximal part of the urethra (Fig. 5.40). The ureters enter the pelvic cavity from the abdomen by passing through the pelvic inlet. On each side, the ureter crosses the pelvic inlet and enters the pelvic cavity in the area anterior to the bifurcation of the common iliac artery. From this point, it continues along the pelvic wall and floor to join the base of the bladder. In the pelvis, the ureter is crossed by: the ductus deferens in men, and the uterine artery in women. The bladder is the most anterior element of the pelvic viscera. Although it is entirely situated in the pelvic cavity when empty, it expands superiorly into the abdominal cavity when full (Fig. 5.40). The empty bladder is shaped like a three-sided pyramid that has tipped over to lie on one of its margins (Fig. 5.41A). It has an apex, a base, a superior surface, and two inferolateral surfaces.

1	The empty bladder is shaped like a three-sided pyramid that has tipped over to lie on one of its margins (Fig. 5.41A). It has an apex, a base, a superior surface, and two inferolateral surfaces. The apex of the bladder is directed toward the top of the pubic symphysis; a structure known as the median umbilical ligament (a remnant of the embryological urachus that contributes to the formation of the bladder) continues from it superiorly up the anterior abdominal wall to the umbilicus.

1	The base of the bladder is shaped like an inverted triangle and faces posteroinferiorly. The two ureters enter the bladder at each of the upper corners of the base, and the urethra drains inferiorly from the lower corner of the base. Inside, the mucosal lining on the base of the bladder is smooth and firmly attached to the underlying smooth muscle coat of the wall—unlike elsewhere in the bladder where the mucosa is folded and loosely attached to the wall. The smooth triangular area between the openings of the ureters and urethra on the inside of the bladder is known as the trigone (Fig. 5.41B). The inferolateral surfaces of the bladder are cradled between the levator ani muscles of the pelvic diaphragm and the adjacent obturator internus muscles above the attachment of the pelvic diaphragm. The superior surface is slightly domed when the bladder is empty; it balloons upward as the bladder fills. Neck of bladder

1	Neck of bladder The neck of the bladder surrounds the origin of the urethra at the point where the two inferolateral surfaces and the base intersect. The neck is the most inferior part of the bladder and also the most “fixed” part. It is anchored into position by a pair of tough fibromuscular bands, which connect the neck and pelvic part of the urethra to the posteroinferior aspect of each pubic bone. In women, these fibromuscular bands are termed pubovesical ligaments (Fig. 5.42A). Together with the perineal membrane and associated muscles, the levator ani muscles, and the pubic bones, these ligaments help support the bladder. In men, the paired fibromuscular bands are known as puboprostatic ligaments because they blend with the fibrous capsule of the prostate, which surrounds the neck of the bladder and adjacent part of the urethra (Fig. 5.42B).

1	Although the bladder is considered to be pelvic in the adult, it has a higher position in children. At birth, the bladder is almost entirely abdominal; the urethra begins approximately at the upper margin of the pubic symphysis. With age, the bladder descends until after puberty when it assumes the adult position. The urethra begins at the base of the bladder and ends with an external opening in the perineum. The paths taken by the urethra differ significantly in women and men. In women, the urethra is short, being about 4 cm long. It travels a slightly curved course as it passes inferiorly through the pelvic floor into the perineum, where it passes through the deep perineal pouch and perineal membrane before opening in the vestibule that lies between the labia minora (Fig. 5.45A).

1	The urethral opening is anterior to the vaginal opening in the vestibule. The inferior aspect of the urethra is bound to the anterior surface of the vagina. Two small para- urethral mucous glands (Skene’s glands) are associated with the lower end of the urethra. Each drains via a duct that opens onto the lateral margin of the external urethral orifice. In men, the urethra is long, about 20 cm, and bends twice along its course (Fig. 5.45B). Beginning at the base of the bladder and passing inferiorly through the prostate, it passes through the deep perineal pouch and perineal membrane and immediately enters the root of the penis. As the urethra exits the deep perineal pouch, it bends forward to course anteriorly in the root of the penis. When the penis is flaccid, the urethra makes another bend, this time inferiorly, when passing from the root to the body of the penis. During erection, the bend between the root and body of the penis disappears.

1	The urethra in men is divided into preprostatic, prostatic, membranous, and spongy parts. Preprostatic part. The preprostatic part of the urethra is about 1 cm long, extends from the base of the bladder to the prostate, and is associated with a circular cuff of smooth muscle fibers (the internal urethral sphincter). Contraction of this sphincter prevents retrograde movement of semen into the bladder during ejaculation. Prostatic part. The prostatic part of the urethra (Fig. 5.45C) is 3 to 4 cm long and is surrounded by the prostate. In this region, the lumen of the urethra is marked by a longitudinal midline fold of mucosa (the urethral crest). The depression on each side of the crest is the prostatic sinus; the ducts of the prostate empty into these two sinuses.

1	Midway along its length, the urethral crest is enlarged to form a somewhat circular elevation (the seminal colliculus). In men, the seminal colliculus is used to determine the position of the prostate gland during transurethral transection of the prostate. A small blind-ended pouch—the prostatic utricle (thought to be the homologue of the uterus in women)—opens onto the center of the seminal colliculus. On each side of the prostatic utricle is the opening of the ejaculatory duct of the male reproductive system. Therefore the connection between the urinary and reproductive tracts in men occurs in the prostatic part of the urethra. Membranous part. The membranous part of the urethra is narrow and passes through the deep perineal pouch (Fig. 5.45B). During its transit through this pouch, the urethra, in both men and women, is surrounded by skeletal muscle of the external urethral sphincter.

1	Spongy urethra. The spongy urethra is surrounded by erectile tissue (the corpus spongiosum) of the penis. It is enlarged to form a bulb at the base of the penis and again at the end of the penis to form the navicular fossa (Fig. 5.45B). The two bulbo-urethral glands in the deep perineal pouch are part of the male reproductive system and open into the bulb of the spongy urethra. The external urethral orifice is the sagittal slit at the end of the penis. The reproductive system in men has components in the abdomen, pelvis, and perineum (Fig. 5.47A). The major components are a testis, epididymis, ductus deferens, and ejaculatory duct on each side, and the urethra and penis in the midline. In addition, three types of accessory glands are associated with the system: a single prostate, a pair of seminal vesicles, and a pair of bulbo-urethral glands.

1	The design of the reproductive system in men is basically a series of ducts and tubules. The arrangement of parts and linkage to the urinary tract reflects its embryological development. The testes originally develop high on the posterior abdominal wall and then descend, normally before birth, through the inguinal canal in the anterior abdominal wall and into the scrotum of the perineum. During descent, the testes carry their vessels, lymphatics, and nerves, as well as their principal drainage ducts, the ductus deferens (vas deferens) with them. The lymph drainage of the testes is therefore to the lateral aortic or lumbar nodes and pre-aortic nodes in the abdomen, and not to the inguinal or pelvic lymph nodes.

1	Each ellipsoid-shaped testis is enclosed within the end of an elongated musculofascial pouch, which is continuous with the anterior abdominal wall and projects into the scrotum. The spermatic cord is the tube-shaped connection between the pouch in the scrotum and the abdominal wall. The sides and anterior aspect of the testis are covered by a closed sac of peritoneum (the tunica vaginalis), which originally connected to the abdominal cavity. Normally after testicular descent, the connection closes, leaving a fibrous remnant.

1	Each testis (Fig. 5.47B) is composed of seminiferous tubules and interstitial tissue surrounded by a thick connective tissue capsule (the tunica albuginea). Spermatozoa are produced by the seminiferous tubules. The 400 to 600 highly coiled seminiferous tubules are modified at each end to become straight tubules, which connect to a collecting chamber (the rete testis) in a thick, vertically oriented linear wedge of connective tissue (the mediastinum testis), projecting from the capsule into the posterior aspect of the gonad. Approximately 12 to 20 efferent ductules originate from the upper end of the rete testis, penetrate the capsule, and connect with the epididymis.

1	The epididymis courses along the posterolateral side of the testis (Fig. 5.47B). It has two distinct components: the efferent ductules, which form an enlarged coiled mass that sits on the posterior superior pole of the testis and forms the head of the epididymis; and the true epididymis, which is a single, long coiled duct into which the efferent ductules all drain, and which continues inferiorly along the posterolateral margin of the testis as the body of the epididymis and enlarges to form the tail of the epididymis at the inferior pole of the testis. During passage through the epididymis, spermatozoa acquire the ability to move and fertilize an egg. The epididymis also stores spermatozoa until ejaculation. The end of the epididymis is continuous with the ductus deferens.

1	The ductus deferens is a long muscular duct that transports spermatozoa from the tail of the epididymis in the scrotum to the ejaculatory duct in the pelvic cavity (Fig. 5.47A). It ascends in the scrotum as a component of the spermatic cord and passes through the inguinal canal in the anterior abdominal wall. After passing through the deep inguinal ring, the ductus deferens bends medially around the lateral side of the inferior epigastric artery and crosses the external iliac artery and the external iliac vein at the pelvic inlet to enter the pelvic cavity. The duct descends medially on the pelvic wall, deep to the peritoneum, and crosses the ureter posterior to the bladder. It continues inferomedially along the base of the bladder, anterior to the rectum, almost to the midline, where it is joined by the duct of the seminal vesicle to form the ejaculatory duct.

1	Between the ureter and ejaculatory duct, the ductus deferens expands to form the ampulla of the ductus deferens. The ejaculatory duct penetrates through the prostate gland to connect with the prostatic urethra. Each seminal vesicle is an accessory gland of the male reproductive system that develops as a blind-ended tubular outgrowth from the ductus deferens (Fig. 5.47A). The tube is coiled with numerous pocket-like outgrowths and is encapsulated by connective tissue to form an elongate structure situated between the bladder and rectum. The seminal vesicle is immediately lateral to and follows the course of the ductus deferens at the base of the bladder. The duct of the seminal vesicle joins the ductus deferens to form the ejaculatory duct (Fig. 5.48). Secretions from the seminal vesicle contribute significantly to the volume of the ejaculate (semen).

1	The prostate is an unpaired accessory structure of the male reproductive system that surrounds the urethra in the pelvic cavity (Figs. 5.47A and 5.48). It lies immediately inferior to the bladder, posterior to the pubic symphysis, and anterior to the rectum. The prostate is shaped like an inverted rounded cone with a larger base, which is continuous above with the neck of the bladder, and a narrower apex, which rests below on the pelvic floor. The inferolateral surfaces of the prostate are in contact with the levator ani muscles that together cradle the prostate between them. The prostate develops as 30 to 40 individual complex glands, which grow from the urethral epithelium into the surrounding wall of the urethra. Collectively, these glands enlarge the wall of the urethra into what is known as the prostate; however, the individual glands retain their own ducts, which empty independently into the prostatic sinuses on the posterior aspect of the urethral lumen (see Fig. 5.45C).

1	Secretions from the prostate, together with secretions from the seminal vesicles, contribute to the formation of semen during ejaculation. The ejaculatory ducts pass almost vertically in an anteroinferior direction through the posterior aspect of the prostate to open into the prostatic urethra. The bulbo-urethral glands (see Fig. 5.47A), one on each side, are small, pea-shaped mucous glands situated within the deep perineal pouch. They are lateral to the membranous part of the urethra. The duct from each gland passes inferomedially through the perineal membrane, to open into the bulb of the spongy urethra at the root of the penis. Together with small glands positioned along the length of the spongy urethra, the bulbo-urethral glands contribute to lubrication of the urethra and the pre-ejaculatory emission from the penis.

1	Together with small glands positioned along the length of the spongy urethra, the bulbo-urethral glands contribute to lubrication of the urethra and the pre-ejaculatory emission from the penis. The reproductive tract in women is contained mainly in the pelvic cavity and perineum, although during pregnancy, the uterus expands into the abdominal cavity. Major components of the system consist of: an ovary on each side, and a uterus, vagina, and clitoris in the midline (Fig. 5.50). In addition, a pair of accessory glands (the greater vestibular glands) are associated with the tract. Like the testes in men, the ovaries develop high on the posterior abdominal wall and then descend before birth, bringing with them their vessels, lymphatics, and nerves. Unlike the testes, the ovaries do not migrate through the inguinal canal into the perineum, but stop short and assume a position on the lateral wall of the pelvic cavity (Fig. 5.51).

1	The ovaries are the sites of egg production (oogenesis). Mature eggs are ovulated into the peritoneal cavity and normally directed into the adjacent openings of the uterine tubes by cilia on the ends of the uterine tubes. The ovaries lie adjacent to the lateral pelvic wall just inferior to the pelvic inlet. Each of the two almond-shaped ovaries is about 3 cm long and is suspended by a mesentery (the mesovarium) that is a posterior extension of the broad ligament. The uterus is a thick-walled muscular organ in the midline between the bladder and rectum (see Fig. 5.51). It consists of a body and a cervix, and inferiorly it joins the vagina (Fig. 5.53). Superiorly, uterine tubes project laterally from the uterus and open into the peritoneal cavity immediately adjacent to the ovaries.

1	The body of the uterus is flattened anteroposteriorly and, above the level of origin of the uterine tubes (Fig. 5.53), has a rounded superior end (fundus of the uterus). The cavity of the body of the uterus is a narrow slit, when viewed laterally, and is shaped like an inverted triangle, when viewed anteriorly. Each of the superior corners of the cavity is continuous with the lumen of a uterine tube; the inferior corner is continuous with the central canal of the cervix. Implantation of the blastocyst normally occurs in the body of the uterus. During pregnancy, the uterus dramatically expands superiorly into the abdominal cavity.

1	Implantation of the blastocyst normally occurs in the body of the uterus. During pregnancy, the uterus dramatically expands superiorly into the abdominal cavity. The uterine tubes extend from each side of the superior end of the body of the uterus to the lateral pelvic wall and are enclosed within the upper margins of the mesosalpinx portions of the broad ligaments (see p. 477). Because the ovaries are suspended from the posterior aspect of the broad ligaments, the uterine tubes pass superiorly over, and terminate laterally to, the ovaries.

1	Each uterine tube has an expanded trumpet-shaped end (the infundibulum), which curves around the superolateral pole of the related ovary (Fig. 5.54). The margin of the infundibulum is rimmed with small finger-like projections termed fimbriae. The lumen of the uterine tube opens into the peritoneal cavity at the narrowed end of the infundibulum. Medial to the infundibulum, the tube expands to form the ampulla and then narrows to form the isthmus, before joining with the body of the uterus. The fimbriated infundibulum facilitates the collection of ovulated eggs from the ovary. Fertilization normally occurs in the ampulla.

1	The fimbriated infundibulum facilitates the collection of ovulated eggs from the ovary. Fertilization normally occurs in the ampulla. The cervix forms the inferior part of the uterus and is shaped like a short, broad cylinder with a narrow central channel. The body of the uterus normally arches forward (anteflexed on the cervix) over the superior surface of the emptied bladder (Fig. 5.55A). In addition, the cervix is angled forward (anteverted) on the vagina so that the inferior end of the cervix projects into the upper anterior aspect of the vagina. Because the end of the cervix is dome shaped, it bulges into the vagina, and a gutter, or fornix, is formed around the margin of the cervix where it joins the vaginal wall (Fig. 5.55B). The tubular central canal of the cervix opens, below, as the external os, into the vaginal cavity and, above, as the internal os, into the uterine cavity.

1	The vagina is the copulatory organ in women. It is a distensible fibromuscular tube that extends from the perineum through the pelvic floor and into the pelvic cavity (Fig. 5.57A). The internal end of the canal is enlarged to form a region called the vaginal vault. The anterior wall of the vagina is related to the base of the bladder and to the urethra; in fact, the urethra is embedded in, or fused to, the anterior vaginal wall. Posteriorly, the vagina is related principally to the rectum. Inferiorly, the vagina opens into the vestibule of the perineum immediately posterior to the external opening of the urethra. From its external opening (the introitus), the vagina courses posterosuperiorly through the perineal membrane and into the pelvic cavity, where it is attached by its anterior wall to the circular margin of the cervix.

1	The vaginal fornix is the recess formed between the margin of the cervix and the vaginal wall. Based on position, the fornix is subdivided into a posterior fornix, an anterior fornix, and two lateral fornices (Fig. 5.57A and see Fig. 5.55). The vaginal canal is normally collapsed so that the anterior wall is in contact with the posterior wall. By using a speculum to open the vaginal canal, a physician can see the domed inferior end of the cervix, the vaginal fornices, and the external os of the cervical canal in a patient (Fig. 5.57B). During intercourse, semen is deposited in the vaginal vault. Spermatozoa make their way into the external os of the cervical canal, pass through the cervical canal into the uterine cavity, and then continue through the uterine cavity into the uterine tubes where fertilization normally occurs in the ampulla.

1	Fascia in the pelvic cavity lines the pelvic walls, surrounds the bases of the pelvic viscera, and forms sheaths around blood vessels and nerves that course medially from the pelvic walls to reach the viscera in the midline. This pelvic fascia is a continuation of the extraperitoneal connective tissue layer found in the abdomen.

1	In women, a rectovaginal septum separates the posterior surface of the vagina from the rectum (Fig. 5.58A). Condensations of fascia form ligaments that extend from the cervix to the anterior (pubocervical ligament), lateral (transverse cervical or cardinal ligament), and posterior (uterosacral ligament) pelvic walls (Fig. 5.58A). These ligaments, together with the perineal membrane, the levator ani muscles, and the perineal body, are thought to stabilize the uterus in the pelvic cavity. The most important of these ligaments are the transverse cervical or cardinal ligaments, which extend laterally from each side of the cervix and vaginal vault to the related pelvic wall.

1	In men, a condensation of fascia around the anterior and lateral region of the prostate (prostatic fascia) contains and surrounds the prostatic plexus of veins and is continuous posteriorly with the rectovesical septum, which separates the posterior surface of the prostate and base of the bladder from the rectum (Fig. 5.58B). The peritoneum of the pelvis is continuous at the pelvic inlet with the peritoneum of the abdomen. In the pelvis, the peritoneum drapes over the pelvic viscera in the midline, forming: pouches between adjacent viscera, and folds and ligaments between viscera and pelvic walls.

1	Anteriorly, median and medial umbilical folds of peritoneum cover the embryological remnants of the urachus and umbilical arteries, respectively (Fig. 5.59). These folds ascend out of the pelvis and onto the anterior abdominal wall. Posteriorly, peritoneum drapes over the anterior and lateral aspects of the upper third of the rectum, but only the anterior surface of the middle third of the rectum is covered by peritoneum; the lower third of the rectum is not covered at all.

1	In women, the uterus lies between the bladder and rectum, and the uterine tubes extend from the superior aspect of the uterus to the lateral pelvic walls (Fig. 5.59A). As a consequence, a shallow vesico-uterine pouch occurs anteriorly, between the bladder and uterus, and a deep recto-uterine pouch (pouch of Douglas) occurs posteriorly, between the uterus and rectum. In addition, a large fold of peritoneum (the broad ligament), with a uterine tube enclosed in its superior margin and an ovary attached posteriorly, is located on each side of the uterus and extends to the lateral pelvic walls.

1	In the midline, the peritoneum descends over the posterior surface of the uterus and cervix and onto the vaginal wall adjacent to the posterior vaginal fornix. It then reflects onto the anterior and lateral walls of the rectum. The deep pouch of peritoneum formed between the anterior surface of the rectum and posterior surfaces of the uterus, cervix, and vagina is the recto-uterine pouch. A sharp sickle-shaped ridge of peritoneum (recto-uterine fold) occurs on each side near the base of the recto-uterine pouch. The recto-uterine folds overlie the uterosacral ligaments, which are condensations of pelvic fascia that extend from the cervix to the posterolateral pelvic walls.

1	The broad ligament is a sheet-like fold of peritoneum, oriented in the coronal plane that runs from the lateral pelvic wall to the uterus, and encloses the uterine tube in its superior margin and suspends the ovary from its posterior aspect (Fig. 5.59A). The uterine arteries cross the ureters at the base of the broad ligaments, and the ligament of the ovary and round ligament of the uterus are enclosed within the parts of the broad ligament related to the ovary and uterus, respectively. The broad ligament has three parts: the mesometrium, the largest part of the broad ligament, which extends from the lateral pelvic walls to the body of the uterus; the mesosalpinx, the most superior part of the broad ligament, which suspends the uterine tube in the pelvic cavity; and the mesovarium, a posterior extension of the broad ligament, which attaches to the ovary.

1	The peritoneum of the mesovarium is continuous with the ovarian surface (germinal) epithelium (see Fig. 5.59A insert). The ovaries are positioned with their long axis in the vertical plane. The ovarian vessels, nerves, and lymphatics enter the superior pole of the ovary from a lateral position and are covered by another raised fold of peritoneum, which with the structures it contains forms the suspensory ligament of the ovary (infundibulopelvic ligament).

1	The inferior pole of the ovary is attached to a fibromuscular band of tissue (the ligament of the ovary), which courses medially in the margin of the mesovarium to the uterus and then continues anterolaterally as the round ligament of the uterus (Fig. 5.59A). The round ligament of the uterus passes over the pelvic inlet to reach the deep inguinal ring and then courses through the inguinal canal to end in connective tissue related to the labium majus in the perineum. Both the ligament of the ovary and the round ligament of the uterus are remnants of the gubernaculum, which attaches the gonad to the labioscrotal swellings in the embryo. In men, the visceral peritoneum drapes over the top of the bladder onto the superior poles of the seminal vesicles and then reflects onto the anterior and lateral surfaces of the rectum (Fig. 5.59B). A rectovesical pouch occurs between the bladder and rectum.

1	The sacral and coccygeal plexuses are situated on the posterolateral wall of the pelvic cavity and generally occur in the plane between the muscles and blood vessels. They are formed by the ventral rami of S1 to Co, with a significant contribution from L4 and L5, which enter the pelvis from the lumbar plexus (Fig. 5.60). Nerves from these mainly somatic plexuses contribute to the innervation of the lower limb and muscles of the pelvis and perineum. Cutaneous branches supply skin over the medial side of the foot, the posterior aspect of the lower limb, and most of the perineum.

1	The sacral plexus on each side is formed by the anterior rami of S1 to S4, and the lumbosacral trunk (L4 and L5) (Fig. 5.61). The plexus is formed in relation to the anterior surface of the piriformis muscle, which is part of the posterolateral pelvic wall. Sacral contributions to the plexus pass out of the anterior sacral foramina and course laterally and inferiorly on the pelvic wall. The lumbosacral trunk, consisting of part of the anterior ramus of L4 and all of the anterior ramus of L5, courses vertically into the pelvic cavity from the abdomen by passing immediately anterior to the sacro-iliac joint.

1	Gray rami communicantes from ganglia of the sympathetic trunk connect with each of the anterior rami and carry postganglionic sympathetic fibers destined for the periphery to the somatic nerves (Fig. 5.62). In addition, special visceral nerves (pelvic splanchnic nerves) originating from S2 to S4 deliver preganglionic parasympathetic fibers to the pelvic part of the prevertebral plexus (Figs. 5.60 and 5.61). Each anterior ramus has ventral and dorsal divisions that combine with similar divisions from other levels to form terminal nerves (Fig. 5.61). The anterior ramus of S4 has only a ventral division. Branches of the sacral plexus include the sciatic nerve and gluteal nerves, which are major nerves of the lower limb, and the pudendal nerve, which is the nerve of the perineum (Table 5.4). Numerous smaller branches supply the pelvic wall, floor, and lower limb.

1	Most nerves originating from the sacral plexus leave the pelvic cavity by passing through the greater sciatic foramen inferior to the piriformis muscle, and enter the gluteal region of the lower limb. Other nerves leave the pelvic cavity using different routes; a few nerves do not leave the pelvic cavity and course directly into the muscles in the pelvic cavity. Finally, two nerves that leave the pelvic cavity through the greater sciatic foramen loop around the ischial spine and sacrospinous ligament and pass medially through the lesser sciatic foramen to supply structures in the perineum and lateral pelvic wall.

1	Sciatic nerve. The sciatic nerve is the largest nerve of the body and carries contributions from L4 to S3 (Figs. 5.60 and 5.61). It: forms on the anterior surface of the piriformis muscle and leaves the pelvic cavity through the greater sciatic foramen inferior to the piriformis; passes through the gluteal region into the thigh, where it divides into its two major branches, the common fibular nerve (common peroneal nerve) and the tibial nerve—dorsal divisions of L4, L5, S1, and S2 are carried in the common fibular part of the nerve and the ventral divisions of L4, L5, S1, S2, and S3 are carried in the tibial part; innervates muscles in the posterior compartment of the thigh and muscles in the leg and foot; and carries sensory fibers from the skin of the foot and lateral leg.

1	Pudendal nerve. The pudendal nerve forms anteriorly to the lower part of the piriformis muscle from ventral divisions of S2 to S4 (Figs. 5.60 and 5.61). It: leaves the pelvic cavity through the greater sciatic foramen, inferior to the piriformis muscle, and enters the gluteal region; courses into the perineum by immediately passing around the sacrospinous ligament, where the ligament joins the ischial spine, and through the lesser sciatic foramen (this course takes the nerve out of the pelvic cavity, around the peripheral attachment of the pelvic floor, and into the perineum); is accompanied throughout its course by the internal pudendal vessels; and innervates skin and skeletal muscles of the perineum, including the external anal and external urethral sphincters.

1	Other branches of the sacral plexus. Other branches of the sacral plexus include: motor branches to muscles of the gluteal region, pelvic wall, and pelvic floor (superior and inferior gluteal nerves, nerve to obturator internus and superior gemellus, nerve to quadratus femoris and inferior gemellus, nerve to piriformis, nerves to levator ani); and sensory nerves to skin over the inferior gluteal region and posterior aspects of the thigh and upper leg (perforating cutaneous nerve and posterior cutaneous nerve of the thigh) (Figs. 5.60 and 5.61). The superior gluteal nerve, formed by branches from the dorsal divisions of L4 to S1, leaves the pelvic cavity through the greater sciatic foramen superior to the piriformis muscle and supplies muscles in the gluteal region—gluteus medius, gluteus minimus, and tensor fasciae latae (tensor of fascia lata) muscles.

1	The inferior gluteal nerve, formed by branches from the dorsal divisions of L5 to S2, leaves the pelvic cavity through the greater sciatic foramen inferior to the piriformis muscle and supplies the gluteus maximus, the largest muscle in the gluteal region. Both superior and inferior gluteal nerves are accompanied by corresponding arteries. The nerve to the obturator internus and the associated superior gemellus muscle originates from the ventral divisions of L5 to S2 and leaves the pelvic cavity through the greater sciatic foramen inferior to the piriformis muscle. Like the pudendal nerve, it passes around the ischial spine and through the lesser sciatic foramen to enter the perineum and supply the obturator internus muscle from the medial side of the muscle, inferior to the attachment of the levator ani muscle.

1	The nerve to the quadratus femoris muscle and the inferior gemellus muscle, and the posterior cutaneous nerve of the thigh (posterior femoral cutaneous nerve) also leave the pelvic cavity through the greater sciatic foramen inferior to the piriformis muscle and course to muscles and skin, respectively, in the lower limb. Unlike most of the other nerves originating from the sacral plexus, which leave the pelvic cavity through the greater sciatic foramen either above or below the piriformis muscle, the perforating cutaneous nerve leaves the pelvic cavity by penetrating directly through the sacrotuberous ligament and then courses to skin over the inferior aspect of the buttocks. The nerve to the piriformis and a number of small nerves to the levator ani and coccygeus muscles originate from the sacral plexus and pass directly into their target muscles without leaving the pelvic cavity.

1	The obturator nerve (L2 to L4) is a branch of the lumbar plexus. It passes inferiorly along the posterior abdominal wall within the psoas muscle, emerges from the medial surface of the psoas, passes posteriorly to the common iliac artery and medially to the internal iliac artery at the pelvic inlet, and then courses along the lateral pelvic wall. It leaves the pelvic cavity by traveling through the obturator canal and supplies the adductor region of the thigh. The small coccygeal plexus has a minor contribution from S4 and is formed mainly by the anterior rami of S5 and Co, which originate inferiorly to the pelvic floor. They penetrate the coccygeus muscle to enter the pelvic cavity and join with the anterior ramus of S4 to form a single trunk, from which small anococcygeal nerves originate (Table 5.4). These nerves penetrate the muscle and the overlying sacrospinous and sacrotuberous ligaments and pass superficially to innervate skin in the anal triangle of the perineum.

1	The paravertebral part of the visceral nervous system is represented in the pelvis by the inferior ends of the sympathetic trunks (Fig. 5.63A). Each trunk enters the pelvic cavity from the abdomen by passing over the ala of the sacrum medially to the lumbosacral trunks and posteriorly to the iliac vessels. The trunks course inferiorly along the anterior surface of the sacrum, where they are positioned medially to the anterior sacral foramina. Four ganglia occur along each trunk. Anteriorly to the coccyx, the two trunks join to form a single small terminal ganglion (the ganglion impar). The principal function of the sympathetic trunks in the pelvis is to deliver postganglionic sympathetic fibers to the anterior rami of sacral nerves for distribution to the periphery, mainly to parts of the lower limb and perineum. This is accomplished by gray rami communicantes, which connect the trunks to the sacral anterior rami.

1	In addition to gray rami communicantes, other branches (the sacral splanchnic nerves) join and contribute to the pelvic part of the prevertebral plexus associated with innervating pelvic viscera (Fig. 5.63A). Pelvic extensions of the prevertebral plexus The pelvic parts of the prevertebral plexus carry sympathetic, parasympathetic, and visceral afferent fibers (Fig. 5.63A). Pelvic parts of the plexus are associated with innervating pelvic viscera and erectile tissues of the perineum. The prevertebral plexus enters the pelvis as two hypogastric nerves, one on each side, that cross the pelvic inlet medially to the internal iliac vessels (Fig. 5.63A). The hypogastric nerves are formed by the separation of the fibers in the superior hypogastric plexus, into right and left bundles. The superior hypogastric plexus is situated anterior to vertebra LV between the promontory of the sacrum and the bifurcation of the aorta.

1	When the hypogastric nerves are joined by pelvic splanchnic nerves carrying preganglionic parasympathetic fibers from S2 to S4, the pelvic plexuses (inferior hypogastric plexuses) are formed (Fig. 5.63). The inferior hypogastric plexuses, one on each side, course in an inferior direction around the pelvic walls, medially to major vessels and somatic nerves. They give origin to the following subsidiary plexuses, which innervate the pelvic viscera: the rectal plexus, the uterovaginal plexus, the prostatic plexus, and the vesical plexus. Terminal branches of the inferior hypogastric plexuses penetrate and pass through the deep perineal pouch and innervate erectile tissues of the penis and the clitoris in the perineum (Fig. 5.63B). In men, these nerves, called cavernous nerves, are extensions of the prostatic plexus. The pattern of distribution of similar nerves in women is not entirely clear, but they are likely extensions of the uterovaginal plexus.

1	Sympathetic fibers enter the inferior hypogastric plexuses from the hypogastric nerves and from branches (sacral splanchnic nerves) of the upper sacral parts of the sympathetic trunks (Fig. 5.63A). Ultimately, these nerves are derived from preganglionic fibers that leave the spinal cord in the anterior roots, mainly of T10 to L2. These fibers: innervate blood vessels, cause contraction of smooth muscle in the internal urethral sphincter in men and the internal anal sphincters in both men and women, cause smooth muscle contraction associated with the reproductive tract and with the accessory glands of the reproductive system, and are important in moving secretions from the epididymis and associated glands into the urethra to form semen during ejaculation.

1	Parasympathetic fibers enter the pelvic plexus in pelvic splanchnic nerves that originate from spinal cord levels S2 to S4 (Fig. 5.63A). They: are generally vasodilatory, stimulate bladder contraction, stimulate erection, and modulate activity of the enteric nervous system of the colon distal to the left colic flexure (in addition to pelvic viscera, some of the fibers from the pelvic plexus course superiorly in the prevertebral plexus, or as separate nerves, and pass into the inferior mesenteric plexus of the abdomen). Visceral afferent fibers follow the course of the sympathetic and parasympathetic fibers to the spinal cord. Afferent fibers that enter the cord in lower thoracic levels and lumbar levels with sympathetic fibers generally carry pain; however, pain fibers from the cervix and some pain fibers from the bladder and urethra may accompany parasympathetic nerves to sacral levels of the spinal cord.

1	The major artery of the pelvis and perineum is the internal iliac artery on each side (Fig. 5.64). In addition to providing a blood supply to most of the pelvic viscera, pelvic walls and floor, and structures in the perineum, including erectile tissues of the clitoris and the penis, this artery gives rise to branches that follow nerves into the gluteal region of the lower limb. Other vessels that originate in the abdomen and contribute to the supply of pelvic structures include the median sacral artery and, in women, the ovarian arteries.

1	The internal iliac artery originates from the common iliac artery on each side, approximately at the level of the intervertebral disc between LV and SI, and lies anteromedial to the sacro-iliac joint (Fig. 5.64). The vessel courses inferiorly over the pelvic inlet and then divides into anterior and posterior trunks at the level of the superior border of the greater sciatic foramen. Branches from the posterior trunk contribute to the supply of the lower posterior abdominal wall, the posterior pelvic wall, and the gluteal region. Branches from the anterior trunk supply the pelvic viscera, the perineum, the gluteal region, the adductor region of the thigh, and, in the fetus, the placenta. Branches of the posterior trunk of the internal iliac artery are the iliolumbar artery, the lateral sacral artery, and the superior gluteal artery (Fig. 5.64).

1	Branches of the posterior trunk of the internal iliac artery are the iliolumbar artery, the lateral sacral artery, and the superior gluteal artery (Fig. 5.64). The iliolumbar artery ascends laterally back out of the pelvic inlet and divides into a lumbar branch and an iliac branch. The lumbar branch contributes to the supply of the posterior abdominal wall, psoas and quadratus lumborum muscles, and cauda equina, via a small spinal branch that passes through the intervertebral foramen between LV and SI. The iliac branch passes laterally into the iliac fossa to supply muscle and bone. The lateral sacral arteries, usually two, originate from the posterior division of the internal iliac artery and course medially and inferiorly along the posterior pelvic wall. They give rise to branches that pass into the anterior sacral foramina to supply related bone and soft tissues, structures in the vertebral (sacral) canal, and skin and muscle posterior to the sacrum.

1	The superior gluteal artery is the largest branch of the internal iliac artery and is the terminal continuation of the posterior trunk. It courses posteriorly, usually passing between the lumbosacral trunk and anterior ramus of S1, to leave the pelvic cavity through the greater sciatic foramen above the piriformis muscle and enter the gluteal region of the lower limb. This vessel makes a substantial contribution to the blood supply of muscles and skin in the gluteal region and also supplies branches to adjacent muscles and bones of the pelvic walls. Branches of the anterior trunk of the internal iliac artery include the superior vesical artery, the umbilical artery, the inferior vesical artery, the middle rectal artery, the uterine artery, the vaginal artery, the obturator artery, the internal pudendal artery, and the inferior gluteal artery (Fig. 5.65).

1	The first branch of the anterior trunk is the umbilical artery, which gives origin to the superior vesical artery and then travels forward just inferior to the margin of the pelvic inlet. Anteriorly, the vessel leaves the pelvic cavity and ascends on the internal aspect of the anterior abdominal wall to reach the umbilicus. In the fetus, the umbilical artery is large and carries blood from the fetus to the placenta. After birth, the vessel closes distally to the origin of the superior vesical artery and eventually becomes a solid fibrous cord. On the anterior abdominal wall, the cord raises a fold of peritoneum termed the medial umbilical fold. The fibrous remnant of the umbilical artery itself is the medial umbilical ligament.

1	The superior vesical artery normally originates from the root of the umbilical artery and courses medially and inferiorly to supply the superior aspect of the bladder and distal parts of the ureter. In men, it also may give rise to an artery that supplies the ductus deferens. The inferior vesical artery occurs in men and supplies branches to the bladder, ureter, seminal vesicle, and prostate. The vaginal artery in women is the equivalent of the inferior vesical artery in men and, descending to the vagina, supplies branches to the vagina and to adjacent parts of the bladder and rectum. The vaginal artery and uterine artery may originate together as a common branch from the anterior trunk, or the vaginal artery may arise independently.

1	The middle rectal artery courses medially to supply the rectum. The vessel anastomoses with the superior rectal artery, which originates from the inferior mesenteric artery in the abdomen, and the inferior rectal artery, which originates from the internal pudendal artery in the perineum. The obturator artery courses anteriorly along the pelvic wall and leaves the pelvic cavity via the obturator canal. Together with the obturator nerve, above, and obturator vein, below, it enters and supplies the adductor region of the thigh.

1	The internal pudendal artery courses inferiorly from its origin in the anterior trunk and leaves the pelvic cavity through the greater sciatic foramen inferior to the piriformis muscle. In association with the pudendal nerve on its medial side, the vessel passes laterally to the ischial spine and then through the lesser sciatic foramen to enter the perineum. The internal pudendal artery is the main artery of the perineum. Among the structures it supplies are the erectile tissues of the clitoris and the penis. The inferior gluteal artery is a large terminal branch of the anterior trunk of the internal iliac artery. It passes between the anterior rami S1 and S2 or S2 and S3 of the sacral plexus and leaves the pelvic cavity through the greater sciatic foramen inferior to the piriformis muscle. It enters and contributes to the blood supply of the gluteal region and anastomoses with a network of vessels around the hip joint.

1	The uterine artery in women courses medially and anteriorly in the base of the broad ligament to reach the cervix (Figs. 5.65B and 5.66). Along its course, the vessel crosses the ureter and passes superiorly to the lateral vaginal fornix. Once the vessel reaches the cervix, it ascends along the lateral margin of the uterus to reach the uterine tube, where it curves laterally and anastomoses with the ovarian artery. The uterine artery is the major blood supply to the uterus and enlarges significantly during pregnancy. Through anastomoses with other arteries, the vessel contributes to the blood supply of the ovary and vagina as well.

1	In women, the gonadal (ovarian) vessels originate from the abdominal aorta and then descend to cross the pelvic inlet and supply the ovaries. They anastomose with terminal parts of the uterine arteries (Fig. 5.66). On each side, the vessels travel in the suspensory ligament of the ovary (the infundibulopelvic ligament) as they cross the pelvic inlet to the ovary. Branches pass through the mesovarium to reach the ovary and through the mesometrium of the broad ligament to anastomose with the uterine artery. The ovarian arteries enlarge significantly during pregnancy to augment the uterine blood supply.

1	The median sacral artery (Figs. 5.65A and 5.66) originates from the posterior surface of the aorta just superior to the aortic bifurcation at vertebral level LIV in the abdomen. It descends in the midline, crosses the pelvic inlet, and then courses along the anterior surface of the sacrum and coccyx. It gives rise to the last pair of lumbar arteries and to branches that anastomose with the iliolumbar and lateral sacral arteries. Pelvic veins follow the course of all branches of the internal iliac artery except for the umbilical artery and the iliolumbar artery (Fig. 5.67A). On each side, the veins drain into internal iliac veins, which leave the pelvic cavity to join common iliac veins situated just superior and lateral to the pelvic inlet.

1	Within the pelvic cavity, extensive interconnected venous plexuses are associated with the surfaces of the viscera (bladder, rectum, prostate, uterus, and vagina). Together, these plexuses form the pelvic plexus of veins. The part of the venous plexus surrounding the rectum and anal canal drains via superior rectal veins (tributaries of inferior mesenteric veins) into the hepatic portal system, and via middle and inferior rectal veins into the caval system. This pelvic plexus is an important portacaval shunt when the hepatic portal system is blocked (Fig. 5.67B).

1	The inferior part of the rectal plexus around the anal canal has two parts, an internal and an external. The internal rectal plexus is in connective tissue between the internal anal sphincter and the epithelium lining the canal. This plexus connects superiorly with longitudinally arranged branches of the superior rectal vein that lie one in each anal column. When enlarged, these branches form varices or internal hemorrhoids, which originate above the pectinate line and are covered by colonic mucosa. The external rectal plexus circles the external anal sphincter and is subcutaneous. Enlargement of vessels in the external rectal plexus results in external hemorrhoids.

1	The single deep dorsal vein that drains erectile tissues of the clitoris and the penis does not follow branches of the internal pudendal artery into the pelvic cavity. Instead, this vein passes directly into the pelvic cavity through a gap formed between the arcuate pubic ligament and the anterior margin of the perineal membrane. The vein joins the prostatic plexus of veins in men and the vesical (bladder) plexus of veins in women. (Superficial veins that drain the skin of the penis and corresponding regions of the clitoris drain into the external pudendal veins, which are tributaries of the great saphenous vein in the thigh.) In addition to tributaries of the internal iliac vein, median sacral veins and ovarian veins parallel the courses of the median sacral artery and ovarian artery, respectively, and leave the pelvic cavity to join veins in the abdomen:

1	The median sacral veins coalesce to form a single vein that joins either the left common iliac vein or the junction of the two common iliac veins to form the inferior vena cava. The ovarian veins follow the course of the corresponding arteries; on the left, they join the left renal vein and, on the right, they join the inferior vena cava in the abdomen. Lymphatics from most pelvic viscera drain mainly into lymph nodes distributed along the internal iliac and external iliac arteries and their associated branches (Fig. 5.68), which drain into nodes associated with the common iliac arteries and then into the lateral aortic or lumbar nodes associated with the lateral surfaces of the abdominal aorta. In turn, these lateral aortic or lumbar nodes drain into the lumbar trunks, which continue to the origin of the thoracic duct at approximately vertebral level TXII.

1	Lymphatics from the ovaries and related parts of the uterus and uterine tubes leave the pelvic cavity superiorly and drain, via vessels that accompany the ovarian arteries, directly into lateral aortic or lumbar nodes and, in some cases, into the pre-aortic nodes on the anterior surface of the aorta. In addition to draining pelvic viscera, nodes along the internal iliac artery also receive drainage from the gluteal region of the lower limb and from deep areas of the perineum. The perineum is a diamond-shaped region positioned inferiorly to the pelvic floor between the thighs. Its peripheral boundary is the pelvic outlet; its ceiling is the pelvic diaphragm (the levator ani and coccygeus muscles); and its narrow lateral walls are formed by the walls of the pelvic cavity below the attachment of the levator ani muscle (Fig. 5.69A). The perineum is divided into an anterior urogenital triangle and a posterior anal triangle.

1	The perineum is divided into an anterior urogenital triangle and a posterior anal triangle. The urogenital triangle is associated with the openings of the urinary systems and the reproductive systems and functions to anchor the external genitalia. The anal triangle contains the anus and the external anal sphincter. The pudendal nerve (S2 to S4) and the internal pudendal artery are the major nerve and artery of the region. The margin of the perineum is marked by the inferior border of the pubic symphysis at its anterior point, the tip of the coccyx at its posterior point, and the ischial tuberosities at each of the lateral points (Fig. 5.69A). The lateral margins are formed by the ischiopubic rami anteriorly and by the sacrotuberous ligaments posteriorly. The pubic symphysis, the ischial tuberosities, and the coccyx can be palpated on the patient.

1	The perineum is divided into two triangles by an imaginary line between the two ischial tuberosities (Fig. 5.69A). Anterior to the line is the urogenital triangle and posterior to the line is the anal triangle. Significantly, the two triangles are not in the same plane. In the anatomical position, the urogenital triangle is oriented in the horizontal plane, whereas the anal triangle is tilted upward at the transtubercular line so that it faces more posteriorly. The roof of the perineum is formed mainly by the levator ani muscles that separate the pelvic cavity, above, from the perineum, below. These muscles, one on each side, form a coneor funnel-shaped pelvic diaphragm, with the anal aperture at its inferior apex in the anal triangle. Anteriorly, in the urogenital triangle, a U-shaped defect in the muscles, the urogenital hiatus, allows the passage of the urethra and vagina.

1	Anteriorly, in the urogenital triangle, a U-shaped defect in the muscles, the urogenital hiatus, allows the passage of the urethra and vagina. The perineal membrane (see pp. 449–451) is a thick fibrous sheet that fills the urogenital triangle (Fig. 5.69B). It has a free posterior border, which is anchored in the midline to the perineal body and is attached laterally to the pubic arch. Immediately superior to the perineal membrane is a thin region termed the deep perineal pouch, containing a layer of skeletal muscle and neurovascular tissues. Among the skeletal muscles in the pouch (see p. 451, Fig. 5.37) is the external urethral sphincter. The perineal membrane and deep perineal pouch provide support for the external genitalia, which are attached to its inferior surface. Also, the parts of the perineal membrane and deep perineal pouch inferior to the urogenital hiatus in the levator ani provide support for the pelvic viscera, above.

1	The urethra leaves the pelvic cavity and enters the perineum by passing through the deep perineal pouch and perineal membrane. In women, the vagina also passes through these structures posterior to the urethra.

1	Because the levator ani muscles course medially from their origin on the lateral pelvic walls, above, to the anal aperture and urogenital hiatus, below, inverted wedge-shaped gutters occur between the levator ani muscles and adjacent pelvic walls as the two structures diverge inferiorly (Fig. 5.70). In the anal triangle, these gutters, one on each side of the anal aperture, are termed ischio-anal fossae. The lateral wall of each fossa is formed mainly by the ischium, obturator internus muscle, and sacrotuberous ligament. The medial wall is the levator ani muscle. The medial and lateral walls converge superiorly where the levator ani muscle attaches to the fascia overlying the obturator internus muscle. The ischio-anal fossae allow movement of the pelvic diaphragm and expansion of the anal canal during defecation.

1	The ischio-anal fossae of the anal triangle are continuous anteriorly with recesses that project into the urogenital triangle superior to the deep perineal pouch. These anterior recesses of the ischio-anal fossae are shaped like three-sided pyramids that have been tipped onto one of their sides (Fig. 5.70C). The apex of each pyramid is closed and points anteriorly toward the pubis. The base is open and continuous posteriorly with its related ischio-anal fossa. The inferior wall of each pyramid is the deep perineal pouch. The superomedial wall is the levator ani muscle, and the superolateral wall is formed mainly by the obturator internus muscle. The ischio-anal fossae and their anterior recesses are normally filled with fat.

1	The anal triangle of the perineum faces posteroinferiorly and is defined laterally by the medial margins of the sacrotuberous ligaments, anteriorly by a horizontal line between the two ischial tuberosities, and posteriorly by the coccyx. The ceiling of the anal triangle is the pelvic diaphragm, which is formed by the levator ani and coccygeus muscles. The anal aperture occurs centrally in the anal triangle and is related on either side to an ischio-anal fossa. The major muscle in the anal triangle is the external anal sphincter.

1	The external anal sphincter, which surrounds the anal canal, is formed by skeletal muscle and consists of three parts—deep, superficial, and subcutaneous—arranged sequentially along the canal from superior to inferior (Fig. 5.69B, Table 5.5). The deep part is a thick ring-shaped muscle that circles the upper part of the anal canal and blends with the fibers of the levator ani muscle. The superficial part also surrounds the anal canal, but is anchored anteriorly to the perineal body and posteriorly to the coccyx and anococcygeal ligament. The subcutaneous part is a horizontally flattened disc of muscle that surrounds the anal aperture just beneath the skin. The external anal sphincter is innervated by inferior rectal branches of the pudendal nerve and by branches directly from the anterior ramus of S4.

1	The urogenital triangle of the perineum is the anterior half of the perineum and is oriented in the horizontal plane. It contains the roots of the external genitalia (Fig. 5.71) and the openings of the urogenital system. The urogenital triangle is defined: laterally by the ischiopubic rami, posteriorly by an imaginary line between the ischial tuberosities, and anteriorly by the inferior margin of the pubic symphysis. As with the anal triangle, the roof or ceiling of the urogenital triangle is the levator ani muscle. Unlike the anal triangle, the urogenital triangle contains a strong fibromuscular support platform, the perineal membrane and deep perineal pouch (see pp. 449–451), which is attached to the pubic arch. Anterior extensions of the ischio-anal fossae occur between the deep perineal pouch and the levator ani muscle on each side.

1	Anterior extensions of the ischio-anal fossae occur between the deep perineal pouch and the levator ani muscle on each side. Between the perineal membrane and the membranous layer of superficial fascia is the superficial perineal pouch. The principal structures in this pouch are the erectile tissues of the penis and clitoris and associated skeletal muscles. Structures in the superficial perineal pouch The superficial perineal pouch contains: erectile structures that join together to form the penis in men and the clitoris in women, and skeletal muscles that are associated mainly with parts of the erectile structures attached to the perineal membrane and adjacent bone. Each erectile structure consists of a central core of expandable vascular tissue and its surrounding connective tissue capsule. Two sets of erectile structures join to form the penis and the clitoris.

1	Two sets of erectile structures join to form the penis and the clitoris. A pair of cylindrically shaped corpora cavernosa, one on each side of the urogenital triangle, are anchored by their proximal ends to the pubic arch. These attached parts are often termed the crura (from the Latin for “legs”) of the clitoris or the penis. The distal ends of the corpora, which are not attached to bone, form the body of the clitoris in women and the dorsal parts of the body of the penis in men. The second set of erectile tissues surrounds the openings of the urogenital system.

1	The second set of erectile tissues surrounds the openings of the urogenital system. In women, a pair of erectile structures, termed the bulbs of the vestibule, are situated, one on each side, at the vaginal opening and are firmly anchored to the perineal membrane (Fig. 5.71A). Small bands of erectile tissues connect the anterior ends of these bulbs to a single, small, pea-shaped erectile mass, the glans clitoris, which is positioned in the midline at the end of the body of the clitoris and anterior to the opening of the urethra.

1	In men, a single large erectile mass, the corpus spongiosum, is the structural equivalent to the bulbs of the vestibule, the glans clitoris, and the interconnecting bands of erectile tissues in women (Fig. 5.71B). The corpus spongiosum is anchored at its base to the perineal membrane. Its proximal end, which is not attached, forms the ventral part of the body of the penis and expands over the end of the body of the penis to form the glans penis. This pattern in men results from the absence of a vaginal opening and from the fusion of structures across the midline during embryological development. As the originally paired erectile structures fuse, they enclose the urethral opening and form an additional channel that ultimately becomes most of the penile part of the urethra. As a consequence of this fusion and growth in men, the urethra is enclosed by the corpus spongiosum and opens at the end of the penis. This is unlike the situation in women, where the urethra is not enclosed by

1	of this fusion and growth in men, the urethra is enclosed by the corpus spongiosum and opens at the end of the penis. This is unlike the situation in women, where the urethra is not enclosed by erectile tissue of the clitoris and opens directly into the vestibule of the perineum.

1	The clitoris is composed of two corpora cavernosa and the glans clitoris (Fig. 5.71A). As in the penis, it has an attached part (root) and a free part (body). Unlike the root of the penis, the root of the clitoris technically consists only of the two crura. (Although the bulbs of the vestibule are attached to the glans clitoris by thin bands of erectile tissue, they are not included in the attached part of the clitoris.) The body of the clitoris, which is formed only by the unattached parts of the two corpora cavernosa, angles posteriorly and is embedded in the connective tissues of the perineum. The body of the clitoris is supported by a suspensory ligament that attaches superiorly to the pubic symphysis. The glans clitoris is attached to the distal end of the body and is connected to the bulbs of the vestibule by small bands of erectile tissue. The glans clitoris is exposed in the perineum and the body of the clitoris can be palpated through skin.

1	The penis is composed mainly of the two corpora cavernosa and the single corpus spongiosum, which contains the urethra (Fig. 5.71B.) As in the clitoris, it has an attached part (root) and a free part (body): The root of the penis consists of the two crura, which are proximal parts of the corpora cavernosa attached to the pubic arch, and the bulb of the penis, which is the proximal part of the corpus spongiosum anchored to the perineal membrane. The body of the penis, which is covered entirely by skin, is formed by the tethering of the two proximal free parts of the corpora cavernosa and the related free part of the corpus spongiosum.

1	The body of the penis, which is covered entirely by skin, is formed by the tethering of the two proximal free parts of the corpora cavernosa and the related free part of the corpus spongiosum. The base of the body of the penis is supported by two ligaments: the suspensory ligament of the penis (attached superiorly to the pubic symphysis), and the more superficially positioned fundiform ligament of the penis (attached above to the linea alba of the anterior abdominal wall and split below into two bands that pass on each side of the penis and unite inferiorly). Because the anatomical position of the penis is erect, the paired corpora are defined as dorsal in the body of the penis and the single corpus spongiosum as ventral, even though the positions are reversed in the nonerect (flaccid) penis. The corpus spongiosum expands to form the head of the penis (glans penis) over the distal ends of the corpora cavernosa (Fig. 5.71B).

1	The corpus spongiosum expands to form the head of the penis (glans penis) over the distal ends of the corpora cavernosa (Fig. 5.71B). Erection of the penis and clitoris is a vascular event generated by parasympathetic fibers carried in pelvic splanchnic nerves from the anterior rami of S2 to S4, which enter the inferior hypogastric part of the prevertebral plexus and ultimately pass through the deep perineal pouch and perineal membrane to innervate the erectile tissues. Stimulation of these nerves causes specific arteries in the erectile tissues to relax. This allows blood to fill the tissues, causing the penis and clitoris to become erect. Arteries supplying the penis and clitoris are branches of the internal pudendal artery; branches of the pudendal nerve (S2 to S4) carry general sensory nerves from the penis and clitoris.

1	Arteries supplying the penis and clitoris are branches of the internal pudendal artery; branches of the pudendal nerve (S2 to S4) carry general sensory nerves from the penis and clitoris. The greater vestibular glands (Bartholin’s glands) are seen in women. They are small, pea-shaped mucous glands that lie posterior to the bulbs of the vestibule on each side of the vaginal opening and are the female homologues of the bulbo-urethral glands in men (Fig. 5.71). However, the bulbo-urethral glands are located within the deep perineal pouch, whereas the greater vestibular glands are in the superficial perineal pouch. The duct of each greater vestibular gland opens into the vestibule of the perineum along the posterolateral margin of the vaginal opening. Like the bulbo-urethral glands in men, the greater vestibular glands produce secretion during sexual arousal.

1	Like the bulbo-urethral glands in men, the greater vestibular glands produce secretion during sexual arousal. The superficial perineal pouch contains three pairs of muscles: the ischiocavernosus, bulbospongiosus, and superficial transverse perineal muscles (Fig. 5.72 and Table 5.6). Two of these three pairs of muscles are associated with the roots of the penis and clitoris; the other pair is associated with the perineal body. The two ischiocavernosus muscles cover the crura of the penis and clitoris (Fig. 5.72). Each muscle is anchored to the medial margin of the ischial tuberosity and related ischial ramus and passes forward to attach to the sides and inferior surface of the related crus, and forces blood from the crus into the body of the erect penis and clitoris. The two bulbospongiosus muscles are associated mainly with the bulbs of the vestibule in women and with the attached part of the corpus spongiosum in men (Fig. 5.72).

1	The two bulbospongiosus muscles are associated mainly with the bulbs of the vestibule in women and with the attached part of the corpus spongiosum in men (Fig. 5.72). In women, each bulbospongiosus muscle is anchored posteriorly to the perineal body and courses anterolaterally over the inferior surface of the related greater vestibular gland and the bulb of the vestibule to attach to the surface of the bulb and to the perineal membrane (Fig. 5.72A). Other fibers course anterolaterally to blend with the fibers of the ischiocavernosus muscle, and still others travel anteriorly and arch over the body of the clitoris.

1	In men, the bulbospongiosus muscles are joined in the midline to a raphe on the inferior surface of the bulb of the penis. The raphe is anchored posteriorly to the perineal body. Muscle fibers course anterolaterally, on each side, from the raphe and perineal body to cover each side of the bulb of the penis and attach to the perineal membrane and connective tissue of the bulb. Others extend anterolaterally to associate with the crura and attach anteriorly to the ischiocavernosus muscles. In both men and women, the bulbospongiosus muscles compress attached parts of the erect corpus spongiosum and bulbs of the vestibule and force blood into more distal regions, mainly the glans. In men, the bulbospongiosus muscles have two additional functions: They facilitate emptying of the bulbous part of the penile urethra following urination (micturition). Their reflex contraction during ejaculation is responsible for the pulsatile emission of semen from the penis.

1	Their reflex contraction during ejaculation is responsible for the pulsatile emission of semen from the penis. The paired superficial transverse perineal muscles follow a course parallel to the posterior margin of the inferior surface of the perineal membrane (Fig. 5.72). These flat band-shaped muscles, which are attached to ischial tuberosities and rami, extend medially to the perineal body in the midline and stabilize the perineal body. Superficial features of the external genitalia

1	Superficial features of the external genitalia In women, the clitoris and vestibular apparatus, together with a number of skin and tissue folds, form the vulva (Fig. 5.73). On either side of the midline are two thin folds of skin termed the labia minora. The region enclosed between them, and into which the urethra and vagina open, is the vestibule. Anteriorly, the labia minora each bifurcate, forming a medial and a lateral fold. The medial folds unite to form the frenulum of the clitoris, that joins the glans clitoris. The lateral folds unite ventrally over the glans clitoris and the body of the clitoris to form the prepuce of the clitoris (hood). The body of the clitoris extends anteriorly from the glans clitoris and is palpable deep to the prepuce and related skin. Posterior to the vestibule, the labia minora unite, forming a small transverse fold, the frenulum of the labia minora (the fourchette).

1	Within the vestibule, the vaginal orifice is surrounded to varying degrees by a ring-like fold of membrane, the hymen, which may have a small central perforation or may completely close the vaginal opening. Following rupture of the hymen (resulting from first sexual intercourse or injury), irregular remnants of the hymen fringe the vaginal opening. The orifices of the urethra and the vagina are associated with the openings of glands. The ducts of the para-urethral glands (Skene’s glands) open into the vestibule, one on each side of the lateral margin of the urethra. The ducts of the greater vestibular glands (Bartholin’s glands) open adjacent to the posterolateral margin of the vaginal opening in the crease between the vaginal orifice and remnants of the hymen.

1	Lateral to the labia minora are two broad folds, the labia majora, which unite anteriorly to form the mons pubis. The mons pubis overlies the inferior aspect of the pubic symphysis and is anterior to the vestibule and the clitoris. Posteriorly, the labia majora do not unite and are separated by a depression termed the posterior commissure, which overlies the position of the perineal body.

1	Superficial components of the genital organs in men consist of the scrotum and the penis (Fig. 5.74). The scrotum is the male homologue of the labia majora in women. In the fetus, labioscrotal swellings fuse across the midline, resulting in a single scrotum into which the testes and their associated musculofascial coverings, blood vessels, nerves, lymphatics, and drainage ducts descend from the abdomen. The remnant of the line of fusion between the labioscrotal swellings in the fetus is visible on the skin of the scrotum as a longitudinal midline raphe that extends from the anus, over the scrotal sac, and onto the inferior aspect of the body of the penis. The penis consists of a root and body. The attached root of the penis is palpable posterior to the scrotum in the urogenital triangle of the perineum. The pendulous part of the penis (body of penis) is entirely covered by skin; the tip of the body is covered by the glans penis.

1	The external urethral orifice is a sagittal slit, normally positioned at the tip of the glans. The inferior margin of the urethral orifice is continuous with a midline raphe of the penis, which represents a line of fusion formed in the glans as the urethra develops in the fetus. The base of this raphe is continuous with the frenulum of the glans, which is a median fold of skin that attaches the glans to more loosely attached skin proximal to the glans. The base of the glans is expanded to form a raised circular margin (the corona of the glans); the two lateral ends of the corona join inferiorly at the midline raphe of the glans. The depression posterior to the corona is the neck of the glans. Normally, a fold of skin at the neck of the glans is continuous anteriorly with thin skin that tightly adheres to the glans and posteriorly with thicker skin loosely attached to the body. This fold, known as the prepuce, extends forward to cover the glans. The prepuce is removed during male

1	tightly adheres to the glans and posteriorly with thicker skin loosely attached to the body. This fold, known as the prepuce, extends forward to cover the glans. The prepuce is removed during male circumcision, leaving the glans exposed.

1	Superficial fascia of the urogenital triangle The superficial fascia of the urogenital triangle is continuous with similar fascia on the anterior abdominal wall. As with the superficial fascia of the abdominal wall, the perineal fascia has a membranous layer on its deep surface. This membranous layer (Colles’ fascia), is attached: posteriorly to the perineal membrane and therefore does not extend into the anal triangle (Fig. 5.75), and to the ischiopubic rami that form the lateral borders of the urogenital triangle and therefore does not extend into the thigh (Fig. 5.75). It defines the external limits of the superficial perineal pouch, lines the scrotum or labia, and extends around the body of the penis and clitoris.

1	It defines the external limits of the superficial perineal pouch, lines the scrotum or labia, and extends around the body of the penis and clitoris. Anteriorly, the membranous layer of fascia is continuous over the pubic symphysis and pubic bones with the membranous layer of fascia on the anterior abdominal wall. In the lower lateral abdominal wall, the membranous layer of abdominal fascia is attached to the deep fascia of the thigh just inferior to the inguinal ligament. Because the membranous layer of fascia encloses the superficial perineal pouch and continues up the anterior abdominal wall, fluids or infectious materials that accumulate in the pouch can track out of the perineum and onto the lower abdominal wall. This material will not track into the anal triangle or the thigh because the fascia fuses with deep tissues at the borders of these regions.

1	The major somatic nerve of the perineum is the pudendal nerve. This nerve originates from the sacral plexus and carries fibers from spinal cord levels S2 to S4. It leaves the pelvic cavity through the greater sciatic foramen inferior to the piriformis muscle, passes around the sacrospinous ligament, and then enters the anal triangle of the perineum by passing medially through the lesser sciatic foramen. As it enters and courses through the perineum, it travels along the lateral wall of the ischio-anal fossa in the pudendal canal, which is a tubular compartment formed in the fascia that covers the obturator internus muscle. This pudendal canal also contains the internal pudendal artery and accompanying veins. The pudendal nerve (Fig. 5.76) has three major terminal branches—the inferior rectal and perineal nerves and the dorsal nerve of the penis or clitoris—which are accompanied by branches of the internal pudendal artery (Fig. 5.77).

1	The inferior rectal nerve is often multiple, penetrates through the fascia of the pudendal canal, and courses medially across the ischio-anal fossa to innervate the external anal sphincter and related regions of the levator ani muscles. The nerve is also general sensory for the skin of the anal triangle. The perineal nerve passes into the urogenital triangle and gives rise to motor and cutaneous branches. The motor branches supply skeletal muscles in the superficial and deep perineal pouches. The largest of the sensory branches is the posterior scrotal nerve in men and the posterior labial nerve in women.

1	The dorsal nerve of the penis and clitoris enters the deep perineal pouch (Fig. 5.76). It passes along the lateral margin of the pouch and then exits by passing inferiorly through the perineal membrane in a position just inferior to the pubic symphysis where it meets the body of the clitoris or the penis. It courses along the dorsal surface of the body to reach the glans. The dorsal nerve is sensory to the penis and clitoris, particularly to the glans. Other somatic nerves that enter the perineum are mainly sensory and include branches of the ilio-inguinal, genitofemoral, posterior femoral cutaneous, and anococcygeal nerves. Visceral nerves enter the perineum by two routes:

1	Visceral nerves enter the perineum by two routes: Those to the skin, which consist mainly of postganglionic sympathetics, are delivered into the region along the pudendal nerve. These fibers join the pudendal nerve from gray rami communicantes that connect pelvic parts of the sympathetic trunks to the anterior rami of the sacral spinal nerves (see p. 481 and Fig. 5.62). Those to erectile tissues enter the region mainly by passing through the deep perineal pouch from the inferior hypogastric plexus in the pelvic cavity (see p. 488 and Fig. 5.63B). The fibers that stimulate erection are parasympathetic fibers, which enter the inferior hypogastric plexus via pelvic splanchnic nerves from spinal cord levels of S2 to S4 (see Fig. 5.63A,B). The most significant artery of the perineum is the internal pudendal artery (Fig. 5.77). Other arteries entering the area include the external pudendal, the testicular, and the cremasteric arteries.

1	The most significant artery of the perineum is the internal pudendal artery (Fig. 5.77). Other arteries entering the area include the external pudendal, the testicular, and the cremasteric arteries. The internal pudendal artery originates as a branch of the anterior trunk of the internal iliac artery in the pelvis (Fig. 5.77). Along with the pudendal nerve, it leaves the pelvis through the greater sciatic foramen inferior to the piriformis muscle. It passes around the ischial spine, where the artery lies lateral to the nerve, enters the perineum by coursing through the lesser sciatic foramen, and accompanies the pudendal nerve in the pudendal canal on the lateral wall of the ischio-anal fossa. The branches of the internal pudendal artery are similar to those of the pudendal nerve in the perineum and include the inferior rectal and perineal arteries, and branches to the erectile tissues of the penis and clitoris (Fig. 5.77).

1	One or more inferior rectal arteries originate from the internal pudendal artery in the anal triangle and cross the ischio-anal fossa medially to branch and supply muscle and related skin (Fig. 5.77). They anastomose with middle and superior rectal arteries from the internal iliac artery and the inferior mesenteric artery, respectively, to form a network of vessels that supply the rectum and anal canal. The perineal artery originates near the anterior end of the pudendal canal and gives off a transverse perineal branch, and a posterior scrotal or labial artery to surrounding tissues and skin (Fig. 5.77). Terminal part of the internal pudendal artery The terminal part of the internal pudendal artery accompanies the dorsal nerve of the penis or clitoris into the deep perineal pouch and supplies branches to the tissues in the deep perineal pouch and erectile tissues.

1	Branches that supply the erectile tissues in men include the artery to the bulb of the penis, the urethral artery, the deep artery of the penis, and the dorsal artery of the penis (Fig. 5.77). The artery of the bulb of the penis has a branch that supplies the bulbo-urethral gland and then penetrates the perineal membrane to supply the corpus spongiosum. A urethral artery also penetrates the perineal membrane and supplies the penile urethra and surrounding erectile tissue to the glans.

1	A urethral artery also penetrates the perineal membrane and supplies the penile urethra and surrounding erectile tissue to the glans. Near the anterior margin of the deep perineal pouch, the internal pudendal artery bifurcates into two terminal branches. A deep artery of the penis penetrates the perineal membrane to enter the crus and supply the crus and corpus cavernosum of the body. The dorsal artery of the penis penetrates the anterior margin of the perineal membrane to meet the dorsal surface of the body of the penis. The vessel courses along the dorsal surface of the penis, medial to the dorsal nerve, and supplies the glans penis and superficial tissues of the penis; it also anastomoses with branches of the deep artery of the penis and the urethral artery. Branches that supply the erectile tissues in women are similar to those in men. Arteries of the bulb of the vestibule supply the bulb of the vestibule and related vagina.

1	Branches that supply the erectile tissues in women are similar to those in men. Arteries of the bulb of the vestibule supply the bulb of the vestibule and related vagina. Deep arteries of the clitoris supply the crura and corpus cavernosum of the body. Dorsal arteries of the clitoris supply surrounding tissues and the glans. The external pudendal arteries consist of a superficial vessel and a deep vessel, which originate from the femoral artery in the thigh. They course medially to enter the perineum anteriorly and supply related skin of the penis and scrotum or the clitoris and labia majora. In men, the testicular arteries originate from the abdominal aorta and descend into the scrotum through the inguinal canal to supply the testes. Also, cremasteric arteries, which originate from the inferior epigastric branch of the external iliac artery, accompany the spermatic cord into the scrotum.

1	In women, small cremasteric arteries follow the round ligament of the uterus through the inguinal canal. Veins in the perineum generally accompany the arteries and join the internal pudendal veins that connect with the internal iliac vein in the pelvis (Fig. 5.78). The exception is the deep dorsal vein of the penis or clitoris that drains mainly the glans and the corpora cavernosa. The deep dorsal vein courses along the midline between the dorsal arteries on each side of the body of the penis or clitoris, passes though the gap between the inferior pubic ligament and the deep perineal pouch, and connects with the plexus of veins surrounding the prostate in men or bladder in women.

1	External pudendal veins, which drain anterior parts of the labia majora or the scrotum and overlap with the area of drainage of the internal pudendal veins, connect with the femoral vein in the thigh. Superficial dorsal veins of the penis or clitoris that drain skin are tributaries of the external pudendal veins. Lymphatic vessels from deep parts of the perineum accompany the internal pudendal blood vessels and drain mainly into internal iliac nodes in the pelvis. Lymphatic channels from superficial tissues of the penis or the clitoris accompany the superficial external pudendal blood vessels and drain mainly into superficial inguinal nodes, as do lymphatic channels from the scrotum or labia majora (Fig. 5.79). The glans penis, glans clitoris, labia minora, and terminal inferior end of the vagina drain into deep inguinal nodes and external iliac nodes.

1	Lymphatics from the testes drain via channels that ascend in the spermatic cord, pass through the inguinal canal, and course up the posterior abdominal wall to connect directly with lateral aortic or lumbar nodes and pre-aortic nodes around the aorta, at approximately vertebral levels LI and LII. Therefore disease from the testes tracks superiorly to nodes high in the posterior abdominal wall and not to inguinal or iliac nodes. Surface anatomy of the pelvis and perineum Palpable bony features of the pelvis are used as landmarks for: locating soft tissue structures, visualizing the orientation of the pelvic inlet, and defining the margins of the perineum. The ability to recognize the normal appearance of structures in the perineum is an essential part of a physical examination. In women, the cervix can be visualized directly by opening the vaginal canal using a speculum.

1	In women, the cervix can be visualized directly by opening the vaginal canal using a speculum. In men, the size and texture of the prostate in the pelvic cavity can be assessed by digital palpation through the anal aperture. Orientation of the pelvis and perineum in the anatomical position In the anatomical position, the anterior superior iliac spines and the anterior superior edge of the pubic symphysis lie in the same vertical plane. The pelvic inlet faces anterosuperiorly. The urogenital triangle of the perineum is oriented in an almost horizontal plane and faces inferiorly, whereas the anal triangle is more vertical and faces posteriorly (Figs. 5.80 and 5.81). How to define the margins of the perineum The pubic symphysis, ischial tuberosities, and tip of the sacrum are palpable on patients and can be used to define the boundaries of the perineum. This is best done with patients lying on their backs with their thighs flexed and abducted in the lithotomy position (Fig. 5.82).

1	The ischial tuberosities are palpable on each side as large bony masses near the crease of skin (gluteal fold) between the thigh and gluteal region. They mark the lateral corners of the diamond-shaped perineum. The tip of the coccyx is palpable in the midline posterior to the anal aperture and marks the most posterior limit of the perineum. The anterior limit of the perineum is the pubic symphysis. In women, this is palpable in the midline deep to the mons pubis. In men, the pubic symphysis is palpable immediately superior to where the body of the penis joins the lower abdominal wall.

1	Imaginary lines that join the ischial tuberosities with the pubic symphysis in front, and with the tip of the coccyx behind, outline the diamond-shaped perineum. An additional line between the ischial tuberosities divides the perineum into two triangles, the urogenital triangle anteriorly and anal triangle posteriorly. This line also approximates the position of the posterior margin of the perineal membrane. The midpoint of this line marks the location of the perineal body or central tendon of the perineum. Identification of structures in the anal triangle

1	Identification of structures in the anal triangle The anal triangle is the posterior half of the perineum. The base of the triangle faces anteriorly and is an imaginary line joining the two ischial tuberosities. The apex of the triangle is the tip of the coccyx; the lateral margins can be approximated by lines joining the coccyx to the ischial tuberosities. In both women and men, the major feature of the anal triangle is the anal aperture in the center of the triangle. Fat fills the ischio-anal fossa on each side of the anal aperture (Fig. 5.83). Identification of structures in the urogenital triangle of women

1	Identification of structures in the urogenital triangle of women The urogenital triangle is the anterior half of the perineum. The base of the triangle faces posteriorly and is an imaginary line joining the two ischial tuberosities. The apex of the triangle is the pubic symphysis. The lateral margins can be approximated by lines joining the pubic symphysis to the ischial tuberosities. These lines overlie the ischiopubic rami, which can be felt on deep palpation. In women, the major contents of the urogenital triangle are the clitoris, the vestibule, and skin folds that together form the vulva (Fig. 5.84A,B).

1	In women, the major contents of the urogenital triangle are the clitoris, the vestibule, and skin folds that together form the vulva (Fig. 5.84A,B). Two thin skin folds, the labia minora, enclose between them a space termed the vestibule into which the vagina and the urethra open (Fig. 5.84C). Gentle lateral traction on the labia minora opens the vestibule and reveals a soft tissue mound on which the urethra opens. The para-urethral (Skene’s) glands, one on each side, open into the skin crease between the urethra and the labia minora (Fig. 5.84D). Posterior to the urethra is the vaginal opening. The vaginal opening (introitus) is ringed by remnants of the hymen that originally closes the vaginal orifice and is usually ruptured during the first sexual intercourse. The ducts of the greater vestibular (Bartholin’s) glands, one on each side, open into the skin crease between the hymen and the adjacent labium minus (Fig. 5.84D).

1	The labia minora each bifurcate anteriorly into medial and lateral folds. The medial folds unite at the midline to form the frenulum of the clitoris. The larger lateral folds also unite across the midline to form the clitoral hood or prepuce that covers the glans clitoris and distal parts of the body of the clitoris. Posterior to the vaginal orifice, the labia minora join, forming a transverse skin fold (the fourchette). The labia majora are broad folds positioned lateral to the labia minora. They come together in front to form the mons pubis, which overlies the inferior aspect of the pubic symphysis. The posterior ends of the labia majora are separated by a depression termed the posterior commissure, which overlies the position of the perineal body.

1	The cervix is visible when the vaginal canal is opened with a speculum (Fig. 5.84E). The external cervical os opens onto the surface of the dome-shaped cervix. A recess or gutter, termed the fornix, occurs between the cervix and the vaginal wall and is further subdivided, based on location, into anterior, posterior, and lateral fornices. The roots of the clitoris occur deep to surface features of the perineum and are attached to the ischiopubic rami and the perineal membrane. The bulbs of the vestibule (Fig. 5.84F), composed of erectile tissues, lie deep to the labia minora on either side of the vestibule. These erectile masses are continuous, via thin bands of erectile tissues, with the glans clitoris, which is visible under the clitoral hood. The greater vestibular glands occur posterior to the bulbs of the vestibule on either side of the vaginal orifice.

1	The crura of the clitoris are attached, one on each side, to the ischiopubic rami. Each crus is formed by the attached part of the corpus cavernosum. Anteriorly, these erectile corpora detach from bone, curve posteroinferiorly, and unite to form the body of the clitoris. The body of the clitoris underlies the ridge of skin immediately anterior to the clitoral hood (prepuce). The glans clitoris is positioned at the end of the body of the clitoris. Identification of structures in the urogenital triangle of men In men, the urogenital triangle contains the root of the penis. The testes and associated structures, although they migrate into the scrotum from the abdomen, are generally evaluated with the penis during a physical examination.

1	The scrotum in men is homologous to the labia majora in women. Each oval testis is readily palpable through the skin of the scrotum (Fig. 5.85A). Posterolateral to the testis is an elongated mass of tissue, often visible as a raised ridge that contains lymphatics and blood vessels of the testis, and the epididymis and ductus deferens. A midline raphe (Fig. 5.85B) is visible on the skin separating left and right sides of the scrotum. In some individuals, this raphe is prominent and extends from the anal aperture, over the scrotum and along the ventral surface of the body of the penis, to the frenulum of the glans. The root of the penis is formed by the attached parts of the corpus spongiosum and the corpora cavernosa. The corpus spongiosum is attached to the perineal membrane and can be easily palpated as a large mass anterior to the perineal body. This mass, which is covered by the bulbospongiosus muscles, is the bulb of penis.

1	The corpus spongiosum detaches from the perineal membrane anteriorly, becomes the ventral part of the body of the penis (shaft of penis), and eventually terminates as the expanded glans penis (Fig. 5.85C,D). The crura of the penis, one crus on each side, are the attached parts of the corpora cavernosa and are anchored to the ischiopubic rami (Fig. 5.85E). The corpora cavernosa are unattached anteriorly and become the paired erectile masses that form the dorsal part of the body of the penis. The glans penis caps the anterior ends of the corpora cavernosa. Fig. 5.1 Pelvis and perineum. Fig. 5.2 The pelvis and perineum contain and support terminal parts of the gastrointestinal, urinary, and reproductive systems. A. In women. B. In men. Fig. 5.3 The perineum contains and anchors the roots of the external genitalia. A. In women. B. In men.

1	Fig. 5.3 The perineum contains and anchors the roots of the external genitalia. A. In women. B. In men. Obturator foramenIschialtuberosityPerineal membraneRoots of external genitaliaBody of clitorisUrethral orificeVaginal orificeGlans of clitorisObturator foramenIschial tuberosityRoot of penisBody of penisOpening of urethraGlans of penisPerineal membraneAB Fig. 5.4 Pelvic inlet. Pelvic inletAla of sacrumSacro-iliac jointCoccyxIschial spineAnterior superior iliac spineObturator foramenIschial tuberosityPubic symphysisIschiopubic ramusPubic tubercleSI body Fig. 5.5 Pelvic walls. A. Bones and ligaments of the pelvic walls. B. Muscles of the pelvic walls. Greater sciatic foramenLesser sciatic foramenObturator foramenSacrotuberous ligamentSacrospinous ligamentIschial tuberosityIschiopubic ramusPubic tubercleAnterior superioriliac spineMargin of pelvic inletA Margin of pelvic inletPiriformis muscleObturator internus muscleB Fig. 5.6 Pelvic outlet.

1	Margin of pelvic inletPiriformis muscleObturator internus muscleB Fig. 5.6 Pelvic outlet. Ischiopubic ramusPubic symphysisIschial tuberosityCoccyxSacrotuberous ligamentPubic tubercleObturator foramenAnterior superior iliac spineAcetabulumMargin of pelvic outletSacrum Fig. 5.7 Pelvic floor. Fig. 5.8 Pelvic cavity and peritoneum. A. In men (sagittal section). B. In women (anterior view). Pelvic cavity linedby peritoneumAPerineal membraneand deep perineal pouchLevator aniRectumAortaPeritoneumPelvic inletExternal iliac arteryInternal iliac artery(artery of pelvis)BladderUterusB Fig. 5.9 Perineum. A. In women. B. In men. Roots of external genitaliaPerineal membraneLevator aniSacrotuberous ligamentAnal triangleAnal apertureUrethral orificeUrogenital triangleVaginal orificeRoots of external genitaliaLevator aniAnal apertureAnal triangleUrogenital trianglePerineal membraneABUrethral orifice

1	Fig. 5.10 Areas of communication between the true pelvis and other regions. A. Between the true pelvis, abdomen, and lower limb. B. Between the perineum and other regions. Gap between pubic symphysis and perineal membrane• Dorsal vein of penis and clitorisSacrotuberous ligamentSacrospinous ligamentOrifices in floor• Urethra• Vagina• AnusLesser sciatic foramen• Obturator internus muscle• Pudendal nerve• Internal pudendal vein and arteryB Fig. 5.11 Orientation of the pelvis and pelvic cavity in the anatomical position. Coronal planeAnterior superioriliac spinePubic tuberclePubic symphysisSacrumSacrospinous ligamentSacrotuberous ligamentAnal triangle of perineumUrogenital triangle of perineumPelvic inlet Fig. 5.12 Structures that cross the ureters in the pelvic cavity. A. In women. B. In men. Fig. 5.13 Position of the prostate gland. Fig. 5.14 Dermatomes of the perineum. A. In women. B. In men. Fig. 5.15 Pudendal nerve.

1	Fig. 5.13 Position of the prostate gland. Fig. 5.14 Dermatomes of the perineum. A. In women. B. In men. Fig. 5.15 Pudendal nerve. S1S2S3S4Sacrospinous ligamentPudendal nerveIschial spineAttachment of levatorani and coccygeus(pelvic floor) Fig. 5.16 Pelvic splanchnic nerves from spinal levels S2 to S4 control erection. Hypogastric nerveInferior hypogastric plexusAttachment of pelvic floor(levator ani and coccygeus)Pelvic splanchnic nerves(from S2 to S4)Urogenital triangleAnal triangleProstateNerves to erectile tissue Fig. 5.17 Perineal body. Fig. 5.18 Course of the urethra. A. In women. B. In men. Fig. 5.19 Right pelvic bone. A. Medial view. B. Lateral view. Fig. 5.20 Ilium, ischium, and pubis. Fig. 5.21 Components of the pelvic bone. A. Medial surface. B. Lateral surface. Fig. 5.22 Sacrum and coccyx. A. Anterior view. B. Posterior view. C. Lateral view. Fig. 5.23 Lumbosacral joints and associated ligaments. A. Lateral view. B. Anterior view.

1	Fig. 5.22 Sacrum and coccyx. A. Anterior view. B. Posterior view. C. Lateral view. Fig. 5.23 Lumbosacral joints and associated ligaments. A. Lateral view. B. Anterior view. Fig. 5.24 Sacro-iliac joints and associated ligaments. A. Lateral view. B. Anterior view. C. Posterior view. Fig. 5.25 Pubic symphysis and associated ligaments. Fig. 5.26 Orientation of the pelvis (anatomical position). Fig. 5.27 Structure of the bony pelvis. A. In women. B. In men. The angle formed by the pubic arch can be approximated by the angle between the thumb and index finger for women and the angle between the index finger and middle finger for men as shown in the insets. Fig. 5.28 Pelvic inlet. PromontoryMargin of alaSacro-iliac jointArcuatelinePectenpubisPubic crestLinea terminalisPubic symphysisPubictubercle Fig. 5.29 Sacrospinous and sacrotuberous ligaments. A. Medial view of right side of pelvis. B. Function of the ligaments.

1	Fig. 5.29 Sacrospinous and sacrotuberous ligaments. A. Medial view of right side of pelvis. B. Function of the ligaments. Fig. 5.30 Obturator internus and piriformis muscles (medial view of right side of pelvis). Fig. 5.31 Apertures in the pelvic wall. Obturator canal – obturator nerve and vesselsObturator internus muscleGreater sciatic foramen,above and belowpiriformis muscleLesser sciatic foramenSuperior gluteal nerveand vesselsSciatic nerve, inferior gluteal, posterior femoral cutaneous,and quadratus femoris nerves,and the inferior gluteal and internal pudendal vesselsPudendal nerve and internalpudendal vessels and nerveto obturator internus Fig. 5.32 Pelvic outlet. Pubic archPubic symphysisIschial tuberosityBody of pubisCoccyxSacrotuberous ligament Fig. 5.33 Sagittal T2-weighted magnetic resonance image of the lower abdomen and pelvis of a pregnant woman. Fig. 5.34 Pelvic diaphragm. Fig. 5.35 MRI defecating proctogram in sagittal plane showing active defecation.

1	Fig. 5.34 Pelvic diaphragm. Fig. 5.35 MRI defecating proctogram in sagittal plane showing active defecation. Fig. 5.36 Perineal membrane and deep perineal pouch. A. Inferior view. B. Superolateral view. Perineal membrane and deep perineal pouch. C. Medial view. Obturator foramenIschiopubic ramusIschial tuberosityPubic symphysisOpening for urethraLine of attachment for margin ofurogenital hiatus of levator aniDeep perineal pouchBAInferior pubic ligamentPerineal membranePerineal membrane Obturator internus muscleCoccygeus muscleAnococcygeal ligamentLevator ani muscleSacrospinous ligamentRoot of penisCDeep perineal pouchPerineal membrane Fig. 5.37 Muscles in the deep perineal pouch. A. In women. B. In men. Fig. 5.38 Perineal body. Fig. 5.39 Rectum and anal canal. A. Left pelvic bone removed. B. Longitudinal section. Fig. 5.40 Pelvic parts of the urinary system.

1	B. In men. Fig. 5.38 Perineal body. Fig. 5.39 Rectum and anal canal. A. Left pelvic bone removed. B. Longitudinal section. Fig. 5.40 Pelvic parts of the urinary system. External iliac arteryInternal iliac arteryCommon iliac arteryUreterUrethraNeck of bladderBladderFull bladderEmpty bladder Fig. 5.41 Bladder. A. Superolateral view. B. The trigone. Anterior view with the anterior part of the bladder cut away. Internal urethralorificeOpening of uretersUretersUretersUrethraUrethraInferolateralsurfacesSuperior surfaceApexTrigoneTrigoneMedianumbilicalligamentABBase Fig. 5.42 Ligaments that anchor the neck of the bladder and pelvic part of the urethra to the pelvic bones. A. In women. B. In men. Fig. 5.43 Intravenous urogram demonstrating a stone in the lower portion of the ureter. A. Control radiograph. B. Intravenous urogram, postmicturition. Fig. 5.44 Intravenous urogram demonstrating a small tumor in the wall of the bladder. Fig. 5.45 Urethra. A. In women. B. In men.

1	Fig. 5.44 Intravenous urogram demonstrating a small tumor in the wall of the bladder. Fig. 5.45 Urethra. A. In women. B. In men. C. Prostatic part of the urethra in men. 4. Spongy part of urethraBladderBladderProstate2nd bend when penis is flaccidABBulbo-urethral gland and duct1st bendPenisInternal urethral sphincter(smooth muscle)External urethral sphincter(skeletal muscle)Deep perineal pouchPerineal membraneGlans clitorisExternal urethral sphincterNavicular fossaDeep perineal pouchPerineal membraneExternal urethral orificeDuct of Skene's glandPara-urethral gland (Skene's gland)Greater vestibular gland Vaginal opening in deep perineal pouchand perineal membraneExternal urethral orifice3. Membranous part of urethra2. Prostatic part of urethraUrethra1. Preprostatic part of urethra

1	Prostatic utricleExternal urethralsphincter (skeletal muscle)COpenings ofejaculatory ductsOpenings ofducts of glandularelements of prostateProstateInternal urethral sphincter(smooth muscle)Urethral crestSeminal colliculusGlandular elementsof prostateFibromuscularstroma(smooth muscle andfibrous connective tissue)Deep perineal pouchPerineal membraneProstatic sinuses Fig. 5.46 Ultrasound demonstrating the bladder. A. Full bladder. B. Postmicturition bladder. Fig. 5.47 Reproductive system in men. A. Overview. B. Testis and surrounding structures. External iliac arteryUreterProstateInferiorepigastricarteryAmpulla of ductusdeferensDeep inguinalringSuperficial inguinal ringInguinal canalSpermatic cordTestisTail of epididymisHead of epididymisBody of epididymisTunica vaginalisMusculofascial pouchDuctus deferensDuctus deferensBulbo-urethral gland in deep perineal pouchSeminal vesicleEjaculatory ductsScrotumA

1	Capsule(tunica albuginea)Seminiferous tubuleLigamentous remnantof processus vaginalisBParietal layerCavityTunicavaginalisVisceral layerEfferent ductulesHead of epididymisDuctus deferensBody of epididymisRete testis in mediastinum testisTail of epididymisStraight tubule Fig. 5.48 The prostate gland. Zonal anatomy. Seminal vesicleArea of seminalcolliculusExternal urethralsphincterEjaculatory ductTransitionalzoneUrethraPeripheral zoneCentral zoneAnterior region(nonglandular)Penile urethraAmpulla of ductusdeferens Fig. 5.49 Axial T2-weighted magnetic resonance images of prostate problems. A. A small prostatic cancer in the peripheral zone of a normal-sized prostate. B. Benign prostatic hypertrophy. Fig. 5.50 Reproductive system in women. Glans clitorisBladderOvaryVaginaUterine tubeLigament of ovaryRound ligamentof uterusDeep inguinal ringSuperficial inguinal ringGreater vestibularglandBulb of vestibuleUterus Fig. 5.51 Ovaries and broad ligament.

1	Fig. 5.51 Ovaries and broad ligament. Ovarian vesselsMesovariumSuspensory ligamentof ovaryRound ligamentof the uterusLigament of ovaryInguinal canalLabium majorusDeep inguinal ringSuperficial inguinal ring Fig. 5.52 Sagittal magnetic resonance image demonstrating ovarian cancer. Fig. 5.53 Uterus. Anterior view. The anterior halves of the uterus and vagina have been cut away. BodyFundusVaginaOpening of uterine tubeUterine tubeCervix Fig. 5.54 Uterine tubes. AmpullaIsthmusOvaryAnteriorPosteriorMedialLateralFundus of uterusInfundibulumFimbriaeOpening of theuterine tubeRound ligamentof uterusLigament of ovary Fig. 5.55 Uterus and vagina. A. Angles of anteflexion and anteversion. B. The cervix protrudes into the vagina. Anterior fornixVaginal canalAngle of anteversionAngle ofanteflexionAxis ofuterinebodyABPosteriorfornixInternal osExternal osAxis of cervixAxis of vagina

1	Anterior fornixVaginal canalAngle of anteversionAngle ofanteflexionAxis ofuterinebodyABPosteriorfornixInternal osExternal osAxis of cervixAxis of vagina Fig. 5.56 Picture taken through a speculum inserted into the vagina demonstrating cervical cancer. See Fig. 5.84E on p. 519 for a view of the normal cervix. Fig. 5.57 Vagina. A. Left half of pelvis cut away. B. Vaginal fornices and cervix as viewed through a speculum. Lateral fornixBladderABPosterior fornixLateral fornixCervixAnterior fornixBlade of speculumBlade of speculumVaginal vaultVaginaExternal vaginal openingUterusRectumRound ligamentof uterus Fig. 5.58 Pelvic fascia. A. In women. B. In men. Anal canalProstateProstatic plexus of veinsPuboprostatic ligamentRectumProstatic fasciaRectovesicalseptumB A Transverse cervical ligamentPubocervical ligamentUterosacral ligamentRectovaginal septum Fig. 5.59 Peritoneum in the pelvis. A. In women. B. In men.

1	Fig. 5.59 Peritoneum in the pelvis. A. In women. B. In men. Inferior epigastricarteryBroad ligamentVesico-uterine pouchLateral umbilical foldMedial umbilical foldLigament of ovaryMedian umbilical foldRecto-uterine pouchRecto-uterine foldUreterRound ligamentof uterusSuspensoryligamentof ovaryAUterine tubeMesosalpinxBroad ligamentOvarian surface(germinal)epitheliumRound ligamentof uterusOvaryUreterUterine arterySagittal section of broad ligamentMesovariumMesometrium Fig. 5.60 Sacral and coccygeal plexuses.

1	Fig. 5.60 Sacral and coccygeal plexuses. Lumbosacral trunkSuperior gluteal nerveInferior gluteal nerveNerve to piriformis muscleObturator nerve(from lumbar plexus)Sciatic nervePosterior femoralcutaneous nervePudendal nervePerforatingcutaneous nerveNerves to coccygeus,levator ani, and externalanal sphincter musclesAnococcygeal nervesCoccygeal plexusL4L5S1S2S3S4Pelvic splanchnic nerves (parasympathetics from S2 to S4)Nerve to obturatorinternus and superiorgemellus musclesNerve to quadratusfemoris and inferiorgemellus muscles Fig. 5.61 Components and branches of the sacral and coccygeal plexuses.

1	Fig. 5.61 Components and branches of the sacral and coccygeal plexuses. L4L5S1Sciatic nerveTo obturator internus muscleTo quadratus femoris andinferior gemellus musclesPudendal nervePelvic splanchnic nervesPelvic splanchnicnervePosterior femoral cutaneous nervePerforating cutaneous nerveInferior glutealSuperior glutealLumbosacral trunkCommon fibular partTibial partS2S3S4S5CoTo piriformis muscleAnococcygeal nervesVentral divisionsDorsal divisionsAnterior ramiCoccygeal plexusTo levator ani, coccygeus, and external anal sphincter musclesSacral plexus Fig. 5.62 Sympathetic trunks in the pelvis. Gray ramus communicansSympathetic trunksGanglion imparSacral splanchnicnerves to inferiorhypogastric plexus Fig. 5.63 Pelvic extensions of the prevertebral plexus. A. Anterior view. B. Anteromedial view of right side of plexus.

1	Fig. 5.63 Pelvic extensions of the prevertebral plexus. A. Anterior view. B. Anteromedial view of right side of plexus. L5S1S2S3S4Pelvic splanchnic nerves(parasympatheticsfrom S2 to S4)Sacral splanchnic nervesGray ramicommunicantesSympathetictrunkHypogastric nerveGanglionimparSuperior hypogastric plexusInferior hypogastricplexusPelvic parasympathetics ascendingSympathetics descendingA Fig. 5.64 Branches of the posterior trunk of the internal iliac artery. Fig. 5.65 Branches of the anterior trunk of the internal iliac artery. A. Male. B. Female. S1S2S3S4Median sacral artery (from aorta in abdomen)Internal iliac arteryAnterior trunk of internal iliac arteryDorsal artery of penisObturator arterySuperior vesical arteryInferior vesical arteryInferior gluteal arteryInternal pudendal arteryMiddle rectal artery Umbilical arteryA Fig. 5.66 Uterine and vaginal arteries.

1	Fig. 5.66 Uterine and vaginal arteries. Fig. 5.67 Pelvic veins. A. In a man with the left side of the pelvis and most of the viscera removed. B. Veins associated with the rectum and anal canal. Internal iliac veinMedian sacral veinMedian sacral veinObturator veinInternal pudendal veinMiddle rectal veinVesical plexusProstatic plexus of veinsAInternal pudendal veinMiddle rectal veinInferior rectal veinSuperior rectal veinTo hepatic portal systemTo caval systemTo caval systemAnal canalPelvic floorRectumBExternal venous plexusInternal rectalplexus Fig. 5.68 Pelvic lymphatics. Fig. 5.69 Borders and ceiling of the perineum. A. Boundaries of the perineum. B. Perineal membrane. Fig. 5.70 Ischio-anal fossae and their anterior recesses. A. Anterolateral view with left pelvic wall removed. B. Inferior view. C. Anterolateral view with pelvic walls and diaphragm removed.

1	Fig. 5.70 Ischio-anal fossae and their anterior recesses. A. Anterolateral view with left pelvic wall removed. B. Inferior view. C. Anterolateral view with pelvic walls and diaphragm removed. Obturator internus muscleIschio-anal fossaeAnterior recesses of ischio-anal fossaeSacrotuberous ligamentSacrospinous ligamentCoccygeus muscleAObturator internus muscleTendon of obturatorinternus muscleIschio-anal fossaeAnterior recesses of ischio-anal fossaeBObturator internus muscleAnterior recesses of ischio-anal fossaeLevator aniCDeep perineal pouchDeep perineal pouchPerineal membranePerineal membrane Fig. 5.71 Erectile tissues of clitoris and penis. A. Clitoris. B. Penis.

1	Fig. 5.71 Erectile tissues of clitoris and penis. A. Clitoris. B. Penis. Corpora cavernosaGlans penisExternal urethralorificeNavicular fossaof urethraCrus of penis(attached part ofcorpus cavernosum)Crus of clitoris(attached part ofcorpus cavernosum)Corpora cavernosaCorpus spongiosumcontaining urethraBody of clitoris(cross section)Body of penis(cross section)Bulb of vestibuleBulb of penis(attached part ofcorpus spongiosum)Glans clitorisGreater vestibular glandin superficial pouchBulbo-urethral glandwithin deep pouchSkinAB Fig. 5.72 Muscles in the superficial perineal pouch. A. In women. B. In men. Midline rapheIschiocavernosus muscleBulbospongiosusmusclePerineal bodySuperficial transverse perineal muscleFundiform ligament of penisSuspensory ligament of penisIschiocavernosus muscleBulbospongiosusmusclePerineal bodySuperficial transverse perineal muscleSuspensory ligament of clitorisAB

1	Fig. 5.73 Superficial features of the perineum in women. A. Overview. B. Close-up of external genitalia. Ischial tuberosity(palpable)Urogenital triangleABMons pubisAnal triangleUrethral openingVestibule(between labia minora)Vaginal openingPubic symphysis(palpable)Coccyx(palpable)Anal aperturePosterior commissureOpening of duct ofparaurethral glandFrenulumPrepuce of clitorisGlans clitorisLateral foldMedial foldLabium minusHymenFourchetteOpening of duct ofgreater vestibular gland Fig. 5.74 Superficial features of the perineum in men. A. Overview. B. Close-up of external genitalia. Ischial tuberosity(palpable)Urogenital triangleSkin overlying spermatic cordSkin overlyingbulb of penisAnal trianglePubic symphysis(palpable)Coccyx(palpable)Anal apertureRapheScrotumFrenulum of glansExternal urethral orificeGlans penisPrepuceNeck of glansCorona of glansAB Fig. 5.75 Superficial fascia. A. Lateral view. B. Anterior view.

1	Fig. 5.75 Superficial fascia. A. Lateral view. B. Anterior view. Membranous layerof superficial fasciaAnterior superior iliac spineAttachment of membranouslayer of superficial fasciato fascia lata of thigh Posterior margin ofperineal membrane Fused to posterior margin ofperineal membraneMuscles of abdominal wallInguinal ligamentFascia lata of thighPubic tubercleAB Fig. 5.76 Pudendal nerve. A. In men. B. In women. S2S3S4Obturator internus muscleInferior rectal nerveDorsal nerve of penisSacrospinous ligamentCoccygeus muscleLevator animusclePerineal nervePosterior scrotal nervePudendal nervePudendal canal infascia of obturator internusMotor branches to skeletal muscle in urogenital triangleA Dorsal nerve of clitorisPosterior labial nerveMotor branchesInferior rectal nervePudendal nervePerineal nerveB Fig. 5.77 Arteries in the perineum.

1	Dorsal nerve of clitorisPosterior labial nerveMotor branchesInferior rectal nervePudendal nervePerineal nerveB Fig. 5.77 Arteries in the perineum. Internal iliac arteryInternal pudendal arteryInferior rectal arteryInternal pudendal arteryin fascia of obturator internusDorsal artery of penis(dorsal artery of clitoris in women)Perineal arteryPosterior scrotal artery(posterior labial artery in women)Artery of bulb of penis (artery of vestibular bulb in women)Artery to bulbUrethral arteryDeep artery of penis(deep artery of clitoris in women) Fig. 5.78 Perineal veins. Internal pudendal veinInferior rectal veinProstatic plexus of veins(vesical plexus in women)Posterior scrotal vein (or posterior labial vein in women)Deep dorsal vein (or deep dorsal vein of clitoris in women) Fig. 5.79 Lymphatic drainage of the perineum.

1	Fig. 5.79 Lymphatic drainage of the perineum. Thoracic ductLymph from testisLILateral aortic(lumbar) nodesExternal iliac nodesSuperficial inguinal nodesLymph fromsuperficial tissues ofpenis and scrotum(clitoris and labia majorain women)Deep inguinal nodesInguinal ligamentTestisLymph from glans penis(glans clitoris, labia minora,and lower part of vaginain women)Pre-aortic nodes Fig. 5.80 Lateral view of the pelvic area with the position of the skeletal features indicated. The orientation of the pelvic inlet, urogenital triangle, and anal triangle is also shown. A. In a woman. B. In a man. Plane of pelvic inletPlane of urogenital trianglePlane of anal triangleTuberculum of iliac crestHigh point of iliac crestPosterior superior iliac spineAnterior superior iliac spinePubic tubercleAB

1	Plane of pelvic inletPlane of urogenital trianglePlane of anal triangleTuberculum of iliac crestHigh point of iliac crestPosterior superior iliac spineAnterior superior iliac spinePubic tubercleAB Fig. 5.81 Anterior view of the pelvic area. A. In a woman showing the position of the pubic symphysis. B. In a man showing the position of the pubic tubercle, pubic symphysis, and anterior superior iliac spine. Fig. 5.82 Inferior view of the perineum in the lithotomy position. Boundaries, subdivisions, and palpable landmarks are indicated. A. In a man. B. In a woman. Fig. 5.83 Anal triangle with the anal aperture and position of the ischio-anal fossae indicated. A. In a man. B. In a woman. Anal triangleBIschial tuberosityIschial tuberosityPosition of perineal bodyPosition of ischio-anal fossaPosition of ischio-anal fossaCoccyxAnal apertureAnal triangleAIschial tuberosityPosition of perineal bodyPosition of ischio-anal fossaPosition of ischio-anal fossaCoccyxLabium minusAnal aperture

1	Fig. 5.84 Structures in the urogenital triangle of a woman. A. Inferior view of the urogenital triangle of a woman with major features indicated. B. Inferior view of the vestibule. The labia minora have been pulled apart to open the vestibule. Also indicated are the glans clitoris, the clitoral hood, and the frenulum of the clitoris. C. Inferior view of the vestibule showing the urethral and vaginal orifices and the hymen. The labia minora have been pulled further apart than in Figure 5.84B. D. Inferior view of the vestibule with the left labium minus pulled to the side to show the regions of the vestibule into which the greater vestibular and para-urethral glands open. E. View through the vaginal canal of the cervix. F. Inferior view of the urogenital triangle of a woman with the erectile tissues of the clitoris and vestibule and the greater vestibular glands indicated with overlays.

1	ABCDGlans clitorisGlans clitorisGlans clitorisFrenulumof clitorisVestibuleSkin overlyingbody of clitorisLabiumminusPosteriorcommissure(overliesperineal body)Labium minusLabium majusPrepuce (hood) of clitorisVaginal opening(introitus)Vaginal opening External urethral orificeExternal urethral orificeRemnants of hymenFourchetteArea of opening of the duct of the para-urethralglandArea of opening of the duct of the greater vestibular gland EFSkin overlying body of clitorisCervixExternalcervical osPosteriorfornixAnteriorfornixGlans clitorisBody of clitoris (unattached parts of corpora cavernosa)Bulb of vestibuleGreater vestibular glandMons pubisCrus clitoris (attached part of corpus cavernosum) Fig. 5.85 Structures in the urogenital triangle of a man. A. Inferior view. B. Ventral surface of the body of the penis. C. Anterior view of the glans penis showing the urethral opening. D. Lateral view of the body of the penis and glans.

1	C. Anterior view of the glans penis showing the urethral opening. D. Lateral view of the body of the penis and glans. E. Inferior view of the urogenital triangle of a man with the erectile tissues of the penis indicated with overlays. Position of perineal bodyABBody of penisVentral surface of body of penisGlans penisGlans penisTestisTestisIschial tuberosityFrenulumRapheEpididymis, vas deferens,vessels, nerves, and lymphatics CDorsal surface ofbody of penisUrethral orificeNeck of glansGlans penisPrepuceCorona of glansPosition of perineal bodyEBody of penis (unattached parts of corpus spongiosum and corpora cavernosa)Bulb of penis (attached part of corpus spongiosum)Crus of penis (attached part of corpus cavernosum)Glans penis D Fig. 5.86 Left testicular venogram demonstrating the pampiniform plexus of veins. Fig. 5.87 Sagittal computed tomogram demonstrating a pelvic kidney.

1	Fig. 5.86 Left testicular venogram demonstrating the pampiniform plexus of veins. Fig. 5.87 Sagittal computed tomogram demonstrating a pelvic kidney. eFig. 5.88 Digital subtraction aorto-iliac angiogram. A. Normal circulation pattern. B. Occluded left common iliac artery. eFig. 5.89 Sagittal MRI of the pelvic cavity. A. Measurement of a fibroid before the uterine artery embolization. B. Measurement of a fibroid 6 months after the embolization. The size of the fibroid has decreased. BA56.3 mm46.1 mm Table 5.1 Muscles of the pelvic walls Table 5.2 Muscles of the pelvic diaphragm Table 5.3 Muscles within the deep perineal pouch Table 5.4 Branches of the sacral and coccygeal plexuses (spinal segments in parentheses do not consistently participate) Table 5.5 Muscles of the anal triangle Table 5.6 Muscles of the superficial perineal pouch In the clinic

1	Table 5.5 Muscles of the anal triangle Table 5.6 Muscles of the superficial perineal pouch In the clinic In certain diseases (e.g., leukemia), a sample of bone marrow must be obtained to assess the stage and severity of the problem. The iliac crest is often used for such bone marrow biopsies. The iliac crest lies close to the surface and is easily palpated. A bone marrow biopsy is performed by injecting anesthetic in the skin and passing a cutting needle through the cortical bone of the iliac crest. The bone marrow is aspirated and viewed under a microscope. Samples of cortical bone can also be obtained in this way to provide information about bone metabolism. In the clinic

1	In the clinic The pelvis can be viewed as a series of anatomical rings. There are three bony rings and four fibro-osseous rings. The major bony pelvic ring consists of parts of the sacrum, ilium, and pubis, which forms the pelvic inlet. Two smaller subsidiary rings are the obturator foramina. The greater and lesser sciatic foramina formed by the greater and lesser sciatic notches and the sacrospinous and sacrotuberous ligaments form the four fibro-osseous rings. The rings, which are predominantly bony (i.e., the pelvic inlet and the obturator foramina), are brittle rings. It is not possible to break one side of the ring without breaking the other side of the ring, which in clinical terms means that if a fracture is demonstrated on one side, a second fracture should always be suspected. Fractures of the pelvis may occur in isolation; however, they usually occur in trauma patients and warrant special mention.

1	Fractures of the pelvis may occur in isolation; however, they usually occur in trauma patients and warrant special mention. Owing to the large bony surfaces of the pelvis, a fracture produces an area of bone that can bleed significantly. A large hematoma may be produced, which can compress organs such as the bladder and the ureters. This blood loss may occur rapidly, reducing the circulating blood volume and, unless this is replaced, the patient will become hypovolemic and shock will develop. Pelvic fractures may also disrupt the contents of the pelvis, leading to urethral disruption, potential bowel rupture, and nerve damage. In the clinic Common problems with the sacro-iliac joints

1	Pelvic fractures may also disrupt the contents of the pelvis, leading to urethral disruption, potential bowel rupture, and nerve damage. In the clinic Common problems with the sacro-iliac joints As with many weight-bearing joints, degenerative changes may occur with the sacro-iliac joints and cause pain and discomfort in the region. In addition, disorders associated with the major histocompatibility complex antigen HLA-B27, such as ankylosing spondylitis, psoriatic arthritis, inflammatory arthritis associated with inflammatory bowel disease, and reactive arthritis (the group referred to as seronegative spondyloarthropathies), can produce specific inflammatory changes within these joints. In the clinic

1	In the clinic Transverse and sagittal measurements of a woman’s pelvic inlet and outlet can help in predicting the likelihood of a successful vaginal delivery. These measurements include: the sagittal inlet (between the promontory and the top of the pubic symphysis), the maximum transverse diameter of the inlet, the bispinous outlet (the distance between ischial spines), and the sagittal outlet (the distance between the tip of the coccyx and the inferior margin of the pubic symphysis). These measurements can be obtained using magnetic resonance imaging, which carries no radiation risk for the fetus or mother (Fig. 5.33). In the clinic

1	These measurements can be obtained using magnetic resonance imaging, which carries no radiation risk for the fetus or mother (Fig. 5.33). In the clinic At the beginning of defecation, closure of the larynx stabilizes the diaphragm and intraabdominal pressure is increased by contraction of abdominal wall muscles. As defecation proceeds, the puborectalis muscle surrounding the anorectal junction relaxes, which straightens the anorectal angle. Both the internal and the external anal sphincters also relax to allow feces to move through the anal canal. Normally, the puborectal sling maintains an angle of about 90° between the rectum and the anal canal and acts as a “pinch valve” to prevent defecation. When the puborectalis muscle relaxes, the anorectal angle increases to about 130° to 140°.

1	The fatty tissue of the ischio-anal fossa allows for changes in the position and size of the anal canal and anus during defecation. During evacuation, the anorectal junction moves down and back and the pelvic floor usually descends slightly. During defecation, the circular muscles of the rectal wall undergo a wave of contraction to push feces toward the anus. As feces emerge from the anus, the longitudinal muscles of the rectum and levator ani bring the anal canal back up, the feces are expelled, and the anus and rectum return to their normal positions. A magnetic resonance defecating proctogram is a fairly new imaging technique that allows assessment of different phases of defecation, including rectal function and behavior of the pelvic floor musculature during this process. It is useful in detecting pelvic organ abnormal descent/prolapse during dynamic scanning and potential formation of cystocele or rectocele (Fig. 5.35). In the clinic

1	In the clinic During childbirth the perineal body may be stretched and torn. Traditionally it was felt that if a perineal tear is likely, the obstetrician may proceed with an episiotomy. This is a procedure in which an incision is made in the perineal body to allow the head of the fetus to pass through the vagina. There are two types of episiotomies: a median episiotomy cuts through the perineal body, while a mediolateral episiotomy is an incision 45° from the midline. The maternal benefits of this procedure have been thought to be less traumatic to the perineum and to result in decreased pelvic floor dysfunction after childbirth. However, more recent evidence suggests that an episiotomy should not be performed routinely. Review of data has failed to show a decrease in pelvic floor damage with routine use of episiotomies. In the clinic

1	In the clinic A digital rectal examination (DRE) is performed by placing the gloved and lubricated index finger into the rectum through the anus. The anal mucosa can be palpated for abnormal masses, and in women, the posterior wall of the vagina and the cervix can be palpated. In men, the prostate can be evaluated for any extraneous nodules or masses. In many instances the digital rectal examination may be followed by proctoscopy or colonoscopy. An ultrasound probe may be placed into the rectum to assess the gynecological structures in females and the prostate in the male before performing a prostatic biopsy. A digital rectal examination also allows detection of fresh or altered blood in the rectum in patients with acute gastrointestinal bleeding or chronic anemia. In the clinic Carcinoma of the colon and rectum

1	In the clinic Carcinoma of the colon and rectum Carcinoma of the colon and rectum (colorectum) is a common and often lethal disease. Recent advances in surgery, radiotherapy, and chemotherapy have only slightly improved 5-year survival rates. The biological behavior of tumors of the colon and rectum is relatively predictable. Most of the tumors develop from benign polyps, some of which undergo malignant change. The overall prognosis is related to: the degree of tumor penetration through the bowel wall, the presence or absence of lymphatic dissemination, and the presence or absence of systemic metastases.

1	Given the position of the colon and rectum in the abdominopelvic cavity and its proximity to other organs, it is extremely important to accurately stage colorectal tumors; a tumor in the pelvis, for example, could invade the uterus or bladder. Assessing whether or not spread has occurred usually involves computed tomography (assessment for distal metastases) and magnetic resonance imaging (local staging). Endoscopic ultrasound (EUS) is also used in some instances for local staging of rectal cancer. In the clinic Iatrogenic injury of the ureters

1	Ureters can be injured during various surgeries within the abdomen and pelvis as they lie close to the dissection planes. The most common surgeries that can result in ureteric injury are total abdominal hysterectomy and bilateral salpingo-oophorectomy (removal of the uterus, fallopian tubes and ovaries), laparoscopic vaginal hysterectomy, laparoscopic anterior resection of the rectum, and open left hemicolectomy. At increased risk of ureteric injury are patients with a bulky tumor (uterine, colonic, rectal) and those with a history of previous operations or pelvic irradiation, all of which make dissection of tissues more difficult. During surgery, the ureter can be crushed, cut open, devascularized, or avulsed. It can also be injured during cryoablation or electric cauterization to control intraoperative bleeding. Ureters can also undergo trauma during the course of ureteroscopy, a procedure where a small endoscope is introduced through the urethra and urinary bladder into one of the

1	intraoperative bleeding. Ureters can also undergo trauma during the course of ureteroscopy, a procedure where a small endoscope is introduced through the urethra and urinary bladder into one of the ureters to treat stones or tumors of the ureter (usually due to a tear or electrocauterization).

1	Ureteric injury leads to high morbidity due to infection and in most severe cases to renal impairment. The prognosis is improved when the diagnosis is made intraoperatively and the ureter is repaired immediately. Delayed diagnosis leads to urine leakage and contamination of the abdominal and pelvic cavity, development of sepsis, and in the case of injury near the vagina, a uretero-vaginal fistula can develop. When the diagnosis is made postoperatively, sometimes diversion of urine flow is required and percutaneous nephrostomy is performed. In the clinic

1	In the clinic In some patients, small calculi (stones) form in the kidneys. These may pass down the ureter, causing ureteric obstruction, and into the bladder (Fig. 5.43), where insoluble salts further precipitate on these small calculi to form larger calculi. Often, these patients develop (or may already have) problems with bladder emptying, which leaves residual urine in the bladder. This urine may become infected, which alters the pH of the urine, permitting further precipitation of insoluble salts. If small enough, the stones may be removed via a transurethral route using specialized instruments. If the stones are too big, it may be necessary to make a suprapubic incision and enter the bladder retroperitoneally to remove them. In the clinic

1	If the stones are too big, it may be necessary to make a suprapubic incision and enter the bladder retroperitoneally to remove them. In the clinic In certain instances it is necessary to catheterize the bladder through the anterior abdominal wall. For example, when the prostate is markedly enlarged and it is impossible to pass a urethral catheter, a suprapubic catheter may be placed. The bladder is a retroperitoneal structure and when full lies adjacent to the anterior abdominal wall. Ultrasound visualization of the bladder may be useful in assessing the size of this structure and, importantly, differentiating this structure from other potential abdominal masses. The procedure of suprapubic catheterization is straightforward and involves the passage of a small catheter on a needle in the midline approximately 2 cm above the pubic symphysis. The catheter passes easily into the bladder without compromise of other structures and permits free drainage. In the clinic

1	In the clinic Bladder cancer (Fig. 5.44) is the most common tumor of the urinary tract and is usually a disease of the sixth and seventh decades, although there is an increasing trend for younger patients to develop this disease. Approximately one-third of bladder tumors are multifocal; fortunately, two-thirds are superficial tumors and amenable to local treatment. Bladder tumors may spread through the bladder wall and invade local structures, including the rectum, uterus (in women), and lateral walls of the pelvic cavity. Prostatic involvement is not uncommon in male patients. The disease spreads via the internal iliac lymph nodes. Spread to distant metastatic sites rarely includes the lung. Large bladder tumors may produce complications, including invasion and obstruction of the ureters. Ureteric obstruction can then obstruct the kidneys and induce kidney failure. Moreover, bladder tumors can invade other structures of the pelvic cavity.

1	Treatment for early-stage tumors includes local resection with preservation of the bladder. Diffuse tumors may be treated with local chemotherapy; more extensive tumors may require radical surgical removal of the bladder (cystectomy) and, in men, the prostate (prostatectomy). Bladder reconstruction (formation of so-called neobladder) is performed in patients after cystectomy using part of a bowel, most commonly the ileum. In the clinic The relatively short length of the urethra in women makes them more susceptible than men to bladder infection. The primary symptom of urinary tract infection in women is usually inflammation of the bladder (cystitis). The infection can be controlled in most instances by oral antibiotics and resolves without complication. In children under 1 year of age, infection from the bladder may spread via the ureters to the kidneys, where it can produce renal damage and ultimately lead to renal failure. Early diagnosis and treatment are necessary.

1	In the clinic Urethral catheterization is often performed to drain urine from a patient’s bladder when the patient is unable to micturate. When inserting urinary catheters, it is important to appreciate the gender anatomy of the patient. In men: The spongy urethra is surrounded by the erectile tissue of the bulb of the penis immediately inferior to the deep perineal pouch. The wall of this short segment of urethra is relatively thin and angles superiorly to pass through the deep perineal pouch; at this position the urethra is vulnerable to damage, notably during cystoscopy. The membranous part of the urethra runs superiorly as it passes through the deep perineal pouch. The prostatic part of the urethra takes a slight concave curve anteriorly as it passes through the prostate gland.

1	The prostatic part of the urethra takes a slight concave curve anteriorly as it passes through the prostate gland. In women, it is much simpler to pass catheters and cystoscopes because the urethra is short and straight. Urine may therefore be readily drained from a distended bladder without significant concern for urethral rupture. Occasionally, it is impossible to pass any form of instrumentation through the urethra to drain the bladder, usually because there is a urethral stricture or prostatic enlargement. In such cases, an ultrasound of the lower abdomen will demonstrate a full bladder (Fig. 5.46) behind the anterior abdominal wall. A suprapubic catheter may be inserted into the bladder with minimal trauma through a small incision under local anesthetic. In the clinic

1	In the clinic Tumors of the testis account for a small percentage of malignancies in men. However, they generally occur in younger patients (between 20 and 40 years of age). When diagnosed at an early stage, most of these tumors are curable by surgery and chemotherapy. Early diagnosis of testicular tumors is extremely important. Abnormal lumps can be detected by palpation, and diagnosis can be made using ultrasound. Simple ultrasound scanning can reveal the extent of the local tumor, usually at an early stage. Surgical removal of the malignant testis is often carried out using an inguinal approach. The testis is not usually removed through a scrotal incision, because it is possible to spread tumor cells into the subcutaneous tissues of the scrotum, which has a different lymphatic drainage than the testis. In the clinic

1	Interrupted descent of testis leads to an empty scrotal sac and abnormal location of the testis, which can lie anywhere along the usual route of descent. Most commonly the testis is present in the inguinal canal, where it can be palpated. This condition is usually diagnosed at birth or within the first year of life. A higher incidence of ectopic (undescended) testis occurs in premature births (30%) than in term births (3–5%). Normally, the ectopic testis can complete its descent within the first 3 months after a child is born; therefore watchful waiting is recommended for the first couple of months. A specialist referral is usually made at 6 months if the testis is still absent from the scrotal sac. It is crucial to make the diagnosis early so that an appropriate management plan can be initiated to avoid or reduce the risk of complications such as testicular malignancy, subfertility or infertility, testicular torsion, and inguinal hernia (due to patent processus vaginalis). If

1	be initiated to avoid or reduce the risk of complications such as testicular malignancy, subfertility or infertility, testicular torsion, and inguinal hernia (due to patent processus vaginalis). If surgical correction is required, the ectopic testis is moved from the inguinal canal into the scrotum (orchiopexy). During mobilization of the testis, dissection of tissues must be performed carefully to avoid injuring the ilioinguinal nerve adjacent to the spermatic cord. At the time of orchiopexy, the patent processus vaginalis is closed and any inguinal hernia, if present, is repaired.

1	In the clinic The ductus deferens transports spermatozoa from the tail of the epididymis in the scrotum to the ejaculatory duct in the pelvic cavity. Because it has a thick smooth muscle wall, it can be easily palpated in the spermatic cord between the testes and the superficial inguinal ring. Also, because it can be accessed through skin and superficial fascia, it is amenable to surgical dissection and surgical division. When this is carried out bilaterally (vasectomy), the patient is rendered sterile—this is a useful method for male contraception. In the clinic

1	In the clinic Prostate cancer is one of the most commonly diagnosed malignancies in men, and often the disease is advanced at diagnosis. Prostate cancer typically occurs in the peripheral zone of the prostate (see Fig. 5.48) and is relatively asymptomatic. In many cases, it is diagnosed by a digital rectal examination (DRE) (Fig. 5.49A) and by blood tests, which include serum acid phosphatase and serum prostate-specific antigen (PSA). In rectal exams, the tumorous prostate feels “rock” hard. The diagnosis is usually made by obtaining a number of biopsies of the prostate. Ultrasound is used during the biopsy procedure to image the prostate for the purpose of taking measurements and for needle placement. Ultrasound can also be used to aid planning radiotherapy by placing special metal markers, called fiducials, under direct ultrasound guidance, through the rectal wall into or near the tumor. This allows maximization of the radiation dose to the tumor while protecting healthy tissue.

1	Benign prostatic hypertrophy is a disease of the prostate that occurs with increasing age in most men (Fig. 5.49B). It generally involves the more central regions of the prostate (see Fig. 5.48), which gradually enlarge. The prostate feels “bulky” on DRE. Owing to the more central hypertrophic change of the prostate, the urethra is compressed, and a urinary outflow obstruction develops in a number of patients. With time, the bladder may become hypertrophied in response to the urinary outflow obstruction. In some male patients, the obstruction becomes so severe that urine cannot be passed and transurethral or suprapubic catheterization is necessary. Despite being a benign disease, benign prostatic hypertrophy can therefore have a marked effect on the daily lives of many patients. In the clinic

1	In the clinic Ovarian cancer remains one of the major challenges in oncology. The ovaries contain numerous cell types, all of which can undergo malignant change and require different imaging and treatment protocols and ultimately have different prognoses. Ovarian tumors most commonly originate from the ovarian surface (germinal) epithelium that is continuous at a sharp transition zone with the peritoneum of the mesovarium. Many factors have been linked with the development of ovarian tumors, including a strong family history. Ovarian cancer may occur at any age, but more typically it occurs in older women.

1	Many factors have been linked with the development of ovarian tumors, including a strong family history. Ovarian cancer may occur at any age, but more typically it occurs in older women. Cancer of the ovaries may spread via the blood and lymphatics, and frequently metastasizes directly into the peritoneal cavity. Such direct peritoneal cavity spread allows the passage of tumor cells along the paracolic gutters and over the liver from where this disease may disseminate easily. Unfortunately, many patients already have metastatic and diffuse disease (Fig. 5.52) at the time of diagnosis. In the clinic Imaging the ovary

1	In the clinic Imaging the ovary The ovaries can be visualized using ultrasound. If the patient drinks enough water, the bladder becomes enlarged and full. This fluid-filled cavity provides an excellent acoustic window, behind which the uterus and ovaries may be identified by transabdominal scanning with ultrasound. This technique also allows obstetricians and technicians to view a fetus and record its growth throughout pregnancy. Some patients are not suitable for transabdominal scanning, in which case a probe may be passed into the vagina, permitting close visualization of the uterus, the contents of the recto-uterine pouch (pouch of Douglas), and the ovaries. The ovaries can also be visualized laparoscopically. Many countries have introduced screening programs for cervical cancer where women are regularly called for smear tests. In the clinic

1	In the clinic A hysterectomy is the surgical removal of the uterus. This is usually complete excision of the body, fundus, and cervix of the uterus, though occasionally the cervix may be left in situ. In some instances the uterine (fallopian) tubes and ovaries are removed as well. This procedure is called a total abdominal hysterectomy and bilateral salpingo-oophorectomy. Hysterectomy, oophorectomy, and salpingo-oophorectomy may be performed in patients who have reproductive malignancy, such as uterine, cervical, and ovarian cancers. Other indications include a strong family history of reproductive disorders, endometriosis, and excessive bleeding. Occasionally the uterus may need to be removed postpartum because of excessive postpartum bleeding.

1	A hysterectomy is performed through a transverse suprapubic incision (Pfannenstiel’s incision). During the procedure tremendous care is taken to identify the distal ureters and to ligate the nearby uterine arteries without damage to the ureters. In the clinic After ovulation, the unfertilized egg is gathered by the fimbriae of the uterine tube. The egg passes into the uterine tube where it is normally fertilized in the ampulla. The zygote then begins development and passes into the uterine cavity where it implants in the uterine wall. A simple and effective method of birth control is to surgically ligate (clip) the uterine tubes, preventing spermatozoa from reaching the ovum. This simple short procedure is performed under general anesthetic. A small laparoscope is passed into the peritoneal cavity and special equipment is used to identify the tubes. In the clinic Carcinoma of the cervix and uterus

1	In the clinic Carcinoma of the cervix and uterus Carcinoma of the cervix (Fig. 5.56) and uterus is a common disease. Diagnosis is by inspection, cytology (examination of the cervical cells), imaging, biopsy, and dilation and curettage (D&C) of the uterus. Carcinoma of the cervix and uterus may be treated by local resection, removal of the uterus (hysterectomy), and adjuvant chemotherapy. The tumor spreads via lymphatics to the internal and common iliac lymph nodes. Many countries have introduced screening programs for cervical cancer where women are regularly called for smear tests. The age of women included in the screening population varies depending on the country. In the clinic The recto-uterine pouch

1	In the clinic The recto-uterine pouch The recto-uterine pouch (pouch of Douglas) is an extremely important clinical region situated between the rectum and uterus. When the patient is in the supine position, the recto-uterine pouch is the lowest portion of the abdominopelvic cavity and is a site where infection and fluids typically collect. It is impossible to palpate this region transabdominally, but it can be examined by transvaginal and transrectal digital palpation. If an abscess is suspected, it may be drained through the vagina or the rectum without necessitating transabdominal surgery. In the clinic

1	Pudendal block anesthesia is performed to relieve the pain associated with childbirth. Although the use of this procedure is less common since the widespread adoption of epidural anesthesia, it provides an excellent option for women who have a contraindication to neuraxial anesthesia (e.g., spinal anatomy, low platelets, too close to delivery). Pudendal blocks are also used for certain types of chronic pelvic pain and in some rectal or urological procedures. The injection is usually given where the pudendal nerve crosses the lateral aspect of the sacrospinous ligament near its attachment to the ischial spine. During childbirth, a finger inserted into the vagina can palpate the ischial spine. The needle is passed transcutaneously to the medial aspect of the ischial spine and around the sacrospinous ligament. Infiltration is performed and the perineum is anesthetized. Pudendal nerve blocks can also be performed with imaging guidance (using fluoroscopy, computed tomography, or

1	the sacrospinous ligament. Infiltration is performed and the perineum is anesthetized. Pudendal nerve blocks can also be performed with imaging guidance (using fluoroscopy, computed tomography, or ultrasound) to localize the nerve rather than relying purely on anatomical landmarks.

1	In the clinic It may be necessary to perform radical surgery to cure cancer of the prostate. To do this, the prostate and its attachments around the base of the bladder, including the seminal vesicles, must be removed en masse. Parts of the inferior hypogastric plexus in this region give rise to nerves that innervate the erectile tissues of the penis. Impotence may occur if these nerves cannot be or are not preserved during removal of the prostate. For the same reasons, women may experience sexual dysfunction if similar nerves are damaged during pelvic surgery, for example, during a total hysterectomy. In the clinic

1	In the clinic This is a new and innovative way of performing radical prostatectomy in patients with prostate cancer. The patient is placed on an operating table near a so-called patient unit consisting of a high-resolution camera and three arms containing microsurgical instruments. The surgeon operates the robot from a computer console and views the surgical field on a monitor as magnified 3D images. The operator usually makes a number of incisions between 1 cm to 2 cm wide through which the camera and surgical instruments are inserted into the pelvis. The surgeon’s hand movements are filtered and translated by the robot into very fine and precise movements of the microtools. This markedly increases the precision of prostate removal and reduces the risk of nerve damage and potential development of postsurgical erectile dysfunction. In the clinic Abscesses in the ischio-anal fossae

1	In the clinic Abscesses in the ischio-anal fossae The anal mucosa is particularly vulnerable to injury and may be easily torn by hard feces. Occasionally, patients develop inflammation and infection of the anal canal (sinuses or crypts). This infection can spread between the sphincters, producing intersphincteric fistulas. The infection can tract superiorly into the pelvic cavity or laterally into the ischio-anal fossae. In the clinic

1	In the clinic A hemorrhoid is an engorgement of the venous plexus at or inside the anal sphincter. It is a common complaint and has prevalence of approximately 4% in the United States. Hemorrhoids have a slight genetic predisposition; however, straining during bowel movements, obesity, and sedentary lifestyle can also produce hemorrhoids. The symptoms include irritation, pain, and swelling. Hemorrhoids occurring at the anal verge (distal boundary of the anal canal) are typically called external hemorrhoids. Internal hemorrhoids occur inside the rectum and have a tendency to bleed. Prolapsed hemorrhoids are internal hemorrhoids that pass outside the anal canal and form lumps, which may undergo thrombosis and become painful.

1	There are many treatments for hemorrhoids, which include ligation above the pectinate (dentate) line using simple rubber bands or surgical excision. Surgery to this region is not without complications and care must be taken to preserve the internal anal sphincter. In the back of every physician’s mind is the concern that the rectal bleeding or symptoms may not be attributable to hemorrhoids. Therefore, excluding a tumor within the bowel is as important as treating the hemorrhoids. In the clinic Emission and ejaculation of semen In men, emission is the formation of semen, and ejaculation is the expulsion of semen from the penis.

1	In the clinic Emission and ejaculation of semen In men, emission is the formation of semen, and ejaculation is the expulsion of semen from the penis. Although erection of the penis is a vascular event generated by parasympathetic nerves from spinal levels S2–S4, the formation of semen in the urethra is caused by the contraction of smooth muscle of the ducts and glands of the reproductive system that is innervated by the sympathetic part of the visceral nervous system. Ejaculation of semen from the penis is through the action of skeletal muscles innervated by somatic motor nerves.

1	Smooth muscle in the duct system of the male reproductive tract and in the accessory glands is innervated by sympathetic fibers from the lower thoracic and upper lumbar spinal levels (T12, L1,2). The fibers pass into the prevertebral plexus and are then distributed to target tissues. Semen is formed as luminal contents from the ducts (epididymis, ductus deferens, ampulla of the ductus deferens) and glands (prostate, seminal vesicles) are moved into the urethra at the base of the penis by the contraction of smooth muscle in the walls of the structures.

1	Pulsatile emission of semen from the penis is generated by the reflex contraction of the bulbospongiosus muscle that forces semen from the base of the penis and out of the external urethral meatus. Bulbospongiosus muscle is innervated by somatic motor fibers carried in the pudendal nerve (S2–S4). Contraction of the internal urethral sphincter and periurethral smooth muscle, innervated by the sympathetic part of the visceral nervous system, prevents retrograde ejaculation into the bladder. In the clinic

1	Erectile dysfunction (ED) is a complex condition in which men are unable to initiate or maintain penile erection. When this affects erections during sleep, and with self-stimulation as well as with a partner, vascular and/or nerve impairment is present. This generalized type of ED increases with age and is recognized as a risk factor for coronary artery disease. It is frequently associated with cardiovascular disease, diabetes, and neurological conditions including Parkinson’s disease, spinal cord injuries, multiple sclerosis, and as nerve damage from pelvic surgeries or radiation for pelvic malignancies. Low testosterone states can impair erections and consistently prevent sleep-induced erections. Medications including serotonin reuptake inhibitors (SSRIs), thiazides and anti-androgens can also underlie ED. When only partnered erections are problematic, psychological factors underlie the dysfunction—the normal erections from sleep confirming healthy vascular and neurological

1	can also underlie ED. When only partnered erections are problematic, psychological factors underlie the dysfunction—the normal erections from sleep confirming healthy vascular and neurological function. Most cases of ED are multifactorial in etiology, and all markedly lessen quality of life and a person’s well-being and can lead to depression and low self-esteem as well as emotional and social isolation.

1	Delayed (or absent) ejaculation can result from nerve damage in conditions such as diabetes, Parkinson’s disease, spinal cord injuries, multiple sclerosis, complications after major pelvic surgeries and pelvic irradiation. Ejaculation is absent after radical prostatectomy for prostate cancer (which also removes the seminal vesicles), but orgasm is still possible as the pudendal nerve is spared. SSRIs, neuroleptics, alcohol, and recreational drugs (marijuana, cocaine, and heroin) often delay orgasm and therefore ejaculation, as in health the two coincide (even though the nerves involved are different).

1	Because erectile tissues in the clitoris have similar innervation and blood supply to the penis, vulvar swelling is likely compromised by the same conditions that cause ED in men. However, it appears that this is a rare cause of female sexual dysfunction. Reduced clitoral swelling is rarely symptomatic. Note that phosphodiesterase type 5 (PDE5) inhibitors (sildenafil) do not improve female sexual dysfunction even in conditions such as diabetes. Research confirms that otherwise healthy women with complaints of low sexual arousal have a physiologically normal increase in genital congestion in response to visual sexual stimuli, even though they do not find the stimuli mentally sexually arousing. Loss of genital sexual sensitivity from somatic nerve damage from multiple sclerosis or diabetes can be highly symptomatic and preclude orgasm. Medications preventing orgasm in men can also affect women. In the clinic Urethral rupture may occur at a series of well-defined anatomical points.

1	In the clinic Urethral rupture may occur at a series of well-defined anatomical points. The commonest injury is a rupture of the proximal spongy urethra below the perineal membrane. The urethra is usually torn when structures of the perineum are caught between a hard object (e.g., a steel beam or crossbar of a bicycle) and the inferior pubic arch. Urine escapes through the rupture into the superficial perineal pouch and descends into the scrotum and up onto the anterior abdominal wall deep to the superficial fascia. In association with severe pelvic fractures, urethral rupture may occur at the prostatomembranous junction above the deep perineal pouch. The urine will extravasate into the true pelvis.

1	In association with severe pelvic fractures, urethral rupture may occur at the prostatomembranous junction above the deep perineal pouch. The urine will extravasate into the true pelvis. The worst and most serious urethral rupture is related to serious pelvic injuries where there is complete disruption of the puboprostatic ligaments. The prostate is dislocated superiorly not only by the ligamentous disruption but also by the extensive hematoma formed within the true pelvis. The diagnosis can be made by palpating the elevated prostate during a digital rectal examination.

1	A 25-year-old man visited his family physician because he had a “dragging feeling” in the left side of his scrotum. He was otherwise healthy and had no other symptoms. During examination, the physician palpated the left testis, which was normal, although he noted soft nodular swelling around the superior aspect of the testes and the epididymis. In his clinical notes, he described these findings as a “bag of worms” (Fig. 5.86). The bag of worms was a varicocele. The venous drainage of the testis is via the pampiniform plexus of veins that runs within the spermatic cord. A varicocele is a collection of dilated veins that arise from the pampiniform plexus. In many ways, they are similar to varicose veins that develop in the legs. Typically, the patient complains of a dragging feeling in the scrotum and around the testis, which is usually worse toward the end of the day. The family physician recommended surgical treatment, with a recommendation for surgery through an inguinal incision.

1	The family physician recommended surgical treatment, with a recommendation for surgery through an inguinal incision. A simple surgical technique divides the skin around the inguinal ligament. The aponeurosis of the external oblique muscle is divided in the anterior abdominal wall to display the spermatic cord. Careful inspection of the spermatic cord reveals the veins, which are surgically ligated. Another option is to embolize the varicocele. In this technique, a small catheter is placed via the right femoral vein. The catheter is advanced along the external iliac vein and the common iliac vein and into the inferior vena cava. The catheter is then positioned in the left renal vein, and a venogram is performed to demonstrate the origin of the left testicular vein. The catheter is advanced down the left testicular vein into the veins of the inguinal canal and the pampiniform plexus. Metal coils to occlude the vessels are injected, and the catheter is withdrawn.

1	The patient asked how blood would drain from the testis after the operation. Although the major veins of the testis had been occluded, small collateral veins running within the scrotum and around the outer aspect of the spermatic cord permitted drainage without recurrence of the varicocele. A young woman visited her family practitioner because she had mild upper abdominal pain. An ultrasound demonstrated gallstones within the gallbladder, which explained the patient’s pain. However, when the technician assessed the pelvis, she noted a mass behind the bladder, which had sonographic findings similar to a kidney (Fig. 5.87). What did the sonographer do next? Having demonstrated this pelvic mass behind the bladder, the sonographer assessed both kidneys. The patient had a normal right kidney. However, the left kidney could not be found in its usual place. The technician diagnosed a pelvic kidney.

1	A pelvic kidney can be explained by the embryology. The kidneys develop from a complex series of structures that originate adjacent to the bladder within the fetal pelvis. As development proceeds and the functions of the various parts of the developing kidneys change, they attain a superior position in the upper abdomen adjacent to the abdominal aorta and inferior vena cava, on the posterior abdominal wall. A developmental arrest or complication may prevent the kidney from obtaining its usual position. Fortunately, it is unusual for patients to have any symptoms relating to a pelvic kidney. This patient had no symptoms attributable to the pelvic kidney and she was discharged.

1	This patient had no symptoms attributable to the pelvic kidney and she was discharged. A 19-year-old woman presented to the emergency department with a 36-hour history of lower abdominal pain that was sharp and initially intermittent, later becoming constant and severe. The patient also reported feeling nauseated and vomited once in the ER. She did not have diarrhea and had opened her bowels normally 8 hours before admission. She had no symptoms of dysuria. She was afebrile, slightly tachycardic at 95/min, and had a normal blood pressure. Blood results showed mild leukocytosis of 11.6 x 109/L and normal renal and liver function tests. She reported being sexually active with a long-term partner. She was never pregnant, and the urine pregnancy test on admission was negative.

1	On physical examination there was tenderness in the right iliac fossa with guarding. On vaginal examination a tender mass in the right adnexal region was felt. The patient subsequently underwent a transvaginal ultrasound examination for evaluation of adnexal pathology. The scan showed a markedly enlarged right ovary measuring up to 8 cm in long axis with echogenic stroma and peripherally distributed follicles. There was no internal vascularity when color Doppler was applied. A small amount of free fluid was seen in the pouch of Douglas. The diagnosis of ovarian torsion was made.

1	Ovarian torsion is the twisting of an ovary on its suspensory ligament, which contains arterial, venous, and lymphatic vessels (forming so-called vascular pedicle), leading to a compromised blood supply. Initially, the venous and lymphatic circulation is compromised, resulting in ovarian edema and enlargement. The arterial flow is maintained longer due to thicker and less compressible arterial walls. Prolonged torsion leads to increased internal ovarian pressure that eventually results in arterial thrombosis, ischemia of the ovarian tissue, and necrosis. If the correct diagnosis and treatment are delayed, the patient may develop generalized sepsis. The symptoms are nonspecific, making the diagnosis of ovarian torsion challenging. There is often no significant past medical history.

1	The symptoms are nonspecific, making the diagnosis of ovarian torsion challenging. There is often no significant past medical history. At surgery, the right ovary was hemorrhagic and necrotic with the pedicle twisted 360 degrees. The left ovary was normal in appearance. Right-sided salpingo-oophorectomy was performed, and histopathological examination confirmed completely necrotic ovary without any residual normal ovarian tissue. The patient made a quick recovery after surgical intervention. Ovarian torsion is encountered in women of all ages, but those of reproductive age have much higher prevalence. Torsion of a normal ovary is uncommon and is seen more frequently in adolescent population, with elongated pelvic ligaments, fallopian tube spasm, or more mobile fallopian tubes or mesosalpinx cited as contributing factors.

1	A young man developed pain in his right gluteal region, in the posterior aspect of the thigh and around the posterior and lateral aspects of the leg. On further questioning, he reported that the pain also radiated over the lateral part of the foot, particularly around the lateral malleolus. The areas of pain correspond to dermatomes L4 to S3 nerves. Over the following weeks, the patient began to develop muscular weakness, predominantly footdrop. These findings are consistent with loss of the motor function and sensory change in the common fibular nerve, which is a branch of the sciatic nerve in the lower limb. A computed tomography (CT) scan of the abdomen and pelvis revealed a mass in the posterior aspect of the right side of the pelvis. The mass was anterior to the piriformis muscle and adjacent to the rectum.

1	On the anterior belly of the piriformis muscle, the sciatic nerve is formed from the roots of L4 to S3 nerves. The mass in the patient’s pelvis compressed this nerve, producing his sensory and motor dysfunction. During surgery, the mass was found to be a benign nerve tumor and was excised. This patient had no long-standing neurological deficit. A 65-year-old man was examined by a surgical intern because he had a history of buttock pain and impotence. On examination he had a reduced peripheral pulse on the left foot compared to the right. On direct questioning, the patient revealed that he experienced severe left-sided buttock pain after walking 100 yards. After a short period of rest, he could walk another 100 yards before the same symptoms recurred. He also noticed that over the past year he was unable to obtain an erection. He smoked heavily and was on no other drugs or treatment.

1	He smoked heavily and was on no other drugs or treatment. The pain in the left buttock is ischemic in nature. He gives a typical history relating to lack of blood flow to the muscles. A similar finding is present when muscular branches of the femoral artery are occluded or stenosed. Such patients develop similar (ischemic) pain in the calf muscles called intermittent claudication. How does the blood get to the gluteal muscles? Blood arrives at the aortic bifurcation and then passes into the common iliac arteries, which divide into the internal and external iliac vessels. The internal iliac artery then divides into anterior and posterior divisions, which in turn give rise to vessels that leave the pelvis by passing through the greater sciatic foramen and supply the gluteal muscles. The internal pudendal artery also arises from the anterior division of the internal iliac artery and supplies the penis.

1	The patient’s symptoms occur on the left side, suggesting that an obstruction exists on that side only. Because the patient’s symptoms occur on the left side only, the lesion is likely in the left common iliac artery (eFig. 5.88) and is preventing blood flow into the external and internal iliac arteries on the left side. “How will I be treated?” asked the patient. The patient was asked to stop smoking and begin regular exercise. Other treatment options include unblocking the lesion by ballooning the blockage to reopen the vessels or by a surgical bypass graft. Stopping smoking and regular exercise improved the patient’s walking distance. The patient underwent the less invasive procedure of ballooning the vessel (angioplasty) and as a result he was able to walk unhindered and to have an erection.

1	A 50-year-old woman was admitted to hospital for surgical resection of the uterus (hysterectomy) for cancer. The surgeon was also going to remove all the pelvic lymph nodes and carry out a bilateral salpingo-oophorectomy (removal of uterine tubes and ovaries). The patient was prepared for this procedure and underwent routine surgery. Twenty-five hours after surgery, it was noted that the patient had passed no urine and her abdomen was expanding. An ultrasound scan demonstrated a considerable amount of fluid within the abdomen. Fluid withdrawn from the abdomen was tested and found to be urine. It was postulated that this patient’s ureters had been damaged during the surgery.

1	It was postulated that this patient’s ureters had been damaged during the surgery. The pelvic part of the ureter courses posteroinferiorly and external to the parietal peritoneum on the lateral wall of the pelvis anterior to the internal iliac artery. It continues in its course to a point approximately 2 cm superior to the ischial spine and then passes anteromedially and superior to the levator ani muscles. Importantly, the ureter closely adheres to the peritoneum. The only structure that passes between the ureter and the peritoneum in men is the ductus deferens. In women, however, as the ureter descends on the pelvic wall, it passes under the uterine artery. The ureter continues close to the lateral fornix of the vagina, especially on the left, and enters the posterosuperior angle of the bladder. It was at this point that the ureter was inadvertently damaged.

1	It was at this point that the ureter was inadvertently damaged. Knowing the anatomy and recognizing the possibility of ureteric damage enabled the surgeons to reestablish continuity of the ureter surgically. The patient was hospitalized a few days longer than expected and made an uneventful recovery. A 25-year-old woman was admitted to the emergency department with a complaint of pain in her right iliac fossa. The pain had developed rapidly over approximately 40 minutes and was associated with cramps and vomiting. The surgical intern made an initial diagnosis of appendicitis.

1	The typical history for appendicitis is a central abdominal, colicky (intermittent waxing and waning) pain, which over a period of hours localizes to become a constant pain in the right iliac fossa. The central colicky pain is typical for a poorly localized visceral type of pain. As the parietal peritoneum becomes inflamed, the pain becomes localized. Although this patient does have right iliac fossa pain, the history is not typical for appendicitis (although it must be remembered that patients may not always have a classical history for appendicitis). The surgical intern asked a more senior colleague for an opinion.

1	The surgical intern asked a more senior colleague for an opinion. The senior colleague considered other anatomical structures that lie within the right iliac fossa as a potential cause of pain. These include the appendix, the cecum, and the small bowel. Musculoskeletal pain and referred pain could also be potential causes. In women, pain may also arise from the ovary, fallopian tube, and uterus. In a young patient, diseases of these organs are rare. Infection and pelvic inflammatory disease may occur in the younger patient and need to be considered. The patient gave no history of these disorders.

1	The patient gave no history of these disorders. Upon further questioning, however, the patient revealed that her last menstrual period was 6 weeks before this examination. The senior physician realized that a potential cause of the abdominal pain was a pregnancy outside the uterus (ectopic pregnancy). The patient was rushed for an abdominal ultrasound, which revealed no fetus or sac in the uterus. She was also noted to have a positive pregnancy test. The patient underwent surgery and was found to have a ruptured fallopian tube caused by an ectopic pregnancy. Whenever a patient has apparent pelvic pain, it is important to consider the gender-related anatomical differences. Ectopic pregnancy should always be considered in women of childbearing age.

1	Whenever a patient has apparent pelvic pain, it is important to consider the gender-related anatomical differences. Ectopic pregnancy should always be considered in women of childbearing age. A 35-year-old woman visited her family practitioner because she had a “bloating” feeling and an increase in abdominal girth. The family practitioner examined the lower abdomen, which revealed a mass that extended from the superior pubic rami to the level of the umbilicus. The superior margin of the mass was easily palpated, but the inferior margin appeared to be less well defined. This patient has a pelvic mass. When examining a patient in the supine position, the observer should uncover the whole of the abdomen.

1	This patient has a pelvic mass. When examining a patient in the supine position, the observer should uncover the whole of the abdomen. Inspection revealed a bulge in the lower abdomen to the level of the umbilicus. Palpation revealed a hard and slightly irregular mass with well-defined superior and lateral borders and a less well-defined inferior border, giving the impression that the mass continued into the pelvis. The lesion was dull to percussion. Auscultation did not reveal any abnormal sounds. The doctor pondered which structures this mass may be arising from. When examining the pelvis, it is important to remember the sex differences. Common to both men and women are the rectum, bowel, bladder, and musculature. Certain pathological states are also common to both sexes, including the development of pelvic abscesses and fluid collections.

1	In men, the prostate cannot be palpated transabdominally, and it is extremely rare for it to enlarge to such an extent in benign diseases. Aggressive prostate cancer can spread throughout the whole of the pelvis, although this is often associated with bowel obstruction and severe bladder symptoms. In women, a number of organs can develop large masses, including the ovaries (solid and cystic tumors), the embryological remnants within the broad ligaments, and the uterus (pregnancy and fibroids). The physician asked further questions. It is always important to establish whether the patient is pregnant (occasionally, pregnancy may come as a surprise to the patient).

1	The physician asked further questions. It is always important to establish whether the patient is pregnant (occasionally, pregnancy may come as a surprise to the patient). This patient’s pregnancy test was negative. After the patient emptied her bladder, there was no change in the mass. The physician thought the mass might be a common benign tumor of the uterus (fibroid). To establish the diagnosis, he obtained an ultrasound scan of the pelvis, which confirmed that the mass stemmed from the uterus. The patient was referred to a gynecologist, and after a long discussion regarding her symptomatology, fertility, and risks, the surgeon and the patient agreed that a hysterectomy (surgical removal of the uterus) would be an appropriate course of therapy. The patient sought a series of opinions from other gynecologists, all of whom agreed that surgery was the appropriate option. The fibroid was removed with no complications.

1	The patient sought a series of opinions from other gynecologists, all of whom agreed that surgery was the appropriate option. The fibroid was removed with no complications. A 52-year-old woman was referred to a gynecologist. Magnetic resonance imaging (MRI) indicated the presence of uterine fibroids. After a long discussion regarding her symptomatology, fertility, and risks, she was offered the choice between a hysterectomy (surgical removal of the uterus) or uterine artery embolization. A uterine artery embolization is a procedure where an interventional radiologist uses a catheter to inject small particles into the uterine arteries. This reduces the blood supply to the fibroids and causes them to shrink. The patient opted for the uterine artery embolization. An MRI performed 6 months after the embolization procedure showed a favorable reduction in the size of the uterine fibroids (eFig. 5.89). 524.e2 524.e1 Conceptual Overview • Relationship to Other Regions

1	524.e2 524.e1 Conceptual Overview • Relationship to Other Regions Fig. 5.10, cont’d Fig. 5.36, cont’d Fig. 5.45, cont’d Fig. 5.47, cont’d Fig. 5.59, cont’d Table 5.4 Branches of the sacral and coccygeal plexuses (spinal segments in parentheses do not consistently participate)—cont’d Fig. 5.63, cont’d Fig. 5.65, cont’d Fig. 5.76, cont’d Surface Anatomy • How to Define the Margins of the Perineum Surface Anatomy • Identification of Structures in the Anal Triangle Surface Anatomy • Identification of Structures in the Urogenital Triangle of Men Fig. 5.84, cont’d Surface Anatomy • Identification of Structures in the Urogenital Triangle of Men Fig. 5.85, cont’d Posterior compartment of leg 615 Lateral compartment of leg 622 Anterior compartment of leg 624 Tarsal tunnel, retinacula, and arrangement of major structures at the ankle 642 Arches of the foot 644 Fibrous sheaths of toes 645 Avoiding the sciatic nerve 659 Finding the femoral artery in the femoral

1	Arches of the foot 644 Fibrous sheaths of toes 645 Avoiding the sciatic nerve 659 Finding the femoral artery in the femoral Identifying structures around the knee 660 Visualizing the contents of the popliteal fossa 662 Finding the tarsal tunnel—the gateway to the foot 663 Identifying tendons around the ankle and in the foot 664 Finding the dorsalis pedis artery 665 Approximating the position of the plantar arterial arch 665

1	Finding the dorsalis pedis artery 665 Approximating the position of the plantar arterial arch 665 The lower limb is directly anchored to the axial skeleton by a sacroiliac joint and by strong ligaments, which link the pelvic bone to the sacrum. It is separated from the abdomen, back, and perineum by a continuous line (Fig. 6.1), which: joins the pubic tubercle with the anterior superior iliac spine (position of the inguinal ligament) and then continues along the iliac crest to the posterior superior iliac spine to separate the lower limb from the anterior and lateral abdominal walls; passes between the posterior superior iliac spine and along the dorsolateral surface of the sacrum to the coccyx to separate the lower limb from the muscles of the back; and joins the medial margin of the sacrotuberous ligament, the ischial tuberosity, the ischiopubic ramus, and the pubic symphysis to separate the lower limb from the perineum.

1	The lower limb is divided into the gluteal region, thigh, leg, and foot on the basis of major joints, component bones, and superficial landmarks (Fig. 6.2): The gluteal region is posterolateral and between the iliac crest and the fold of skin (gluteal fold) that defines the lower limit of the buttocks. Anteriorly, the thigh is between the inguinal ligament and the knee joint—the hip joint is just inferior to the middle third of the inguinal ligament, and the posterior thigh is between the gluteal fold and the knee. The leg is between the knee and ankle joint. The foot is distal to the ankle joint. The femoral triangle and popliteal fossa, as well as the posteromedial side of the ankle, are important areas of transition through which structures pass between regions (Fig. 6.3).

1	The femoral triangle and popliteal fossa, as well as the posteromedial side of the ankle, are important areas of transition through which structures pass between regions (Fig. 6.3). The femoral triangle is a pyramid-shaped depression formed by muscles in the proximal regions of the thigh and by the inguinal ligament, which forms the base of the triangle. The major blood supply and one of the nerves of the limb (femoral nerve) enter into the thigh from the abdomen by passing under the inguinal ligament and into the femoral triangle. The popliteal fossa is posterior to the knee joint and is a diamond-shaped region formed by muscles of the thigh and leg. Major vessels and nerves pass between the thigh and leg through the popliteal fossa.

1	Most nerves, vessels, and flexor tendons that pass between the leg and foot pass through a series of canals (collectively termed the tarsal tunnel) on the posteromedial side of the ankle. The canals are formed by adjacent bones and a flexor retinaculum, which holds the tendons in position. Support the body weight A major function of the lower limb is to support the weight of the body with minimal expenditure of energy. When standing erect, the center of gravity is anterior to the edge of the SII vertebra in the pelvis (Fig. 6.4). The vertical line through the center of gravity is slightly posterior to the hip joints, anterior to the knee and ankle joints, and directly over the almost circular support base formed by the feet on the ground and holds the knee and hip joints in extension.

1	The organization of ligaments at the hip and knee joints, together with the shape of the articular surfaces, particularly at the knee, facilitates “locking” of these joints into position when standing, thereby reducing the muscular energy required to maintain a standing position. A second major function of the lower limbs is to move the body through space. This involves the integration of movements at all joints in the lower limb to position the foot on the ground and to move the body over the foot. Movements at the hip joint are flexion, extension, abduction, adduction, medial and lateral rotation, and circumduction (Fig. 6.5). The knee and ankle joints are primarily hinge joints. Movements at the knee are mainly flexion and extension (Fig. 6.6A). Movements at the ankle are dorsiflexion (movement of the dorsal side of the foot toward the leg) and plantarflexion (Fig. 6.6B).

1	During walking, many anatomical features of the lower limbs contribute to minimizing fluctuations in the body’s center of gravity and thereby reduce the amount of energy needed to maintain locomotion and produce a smooth, efficient gait (Fig. 6.7). They include pelvic tilt in the coronal plane, pelvic rotation in the transverse plane, movement of the knees toward the midline, flexion of the knees, and complex interactions between the hip, knee, and ankle. As a result, during walking, the body’s center of gravity normally fluctuates only 5 cm in both vertical and lateral directions. The bones of the gluteal region and the thigh are the pelvic bone and the femur (Fig. 6.8). The large ball and socket joint between these two bones is the hip joint.

1	The bones of the gluteal region and the thigh are the pelvic bone and the femur (Fig. 6.8). The large ball and socket joint between these two bones is the hip joint. The femur is the bone of the thigh. At its distal end, its major weight-bearing articulation is with the tibia, but it also articulates anteriorly with the patella (knee cap). The patella is the largest sesamoid bone in the body and is embedded in the quadriceps femoris tendon. The joint between the femur and tibia is the principal articulation of the knee joint, but the joint between the patella and femur shares the same articular cavity. Although the main movements at the knee are flexion and extension, the knee joint also allows the femur to rotate on the tibia. This rotation contributes to “locking” of the knee when fully extended, particularly when standing. The leg contains two bones: The tibia is medial in position, is larger than the laterally positioned fibula, and is the weight-bearing bone.

1	The leg contains two bones: The tibia is medial in position, is larger than the laterally positioned fibula, and is the weight-bearing bone. The fibula does not take part in the knee joint and forms only the most lateral part of the ankle joint—proximally, it forms a small synovial joint (superior tibiofibular joint) with the inferolateral surface of the head of the tibia. The tibia and fibula are linked along their lengths by an interosseous membrane, and at their distal ends by a fibrous inferior tibiofibular joint, and little movement occurs between them. The distal surfaces of the tibia and fibula together form a deep recess. The ankle joint is formed by this recess and part of one of the tarsal bones of the foot (talus), which projects into the recess. The ankle is most stable when dorsiflexed.

1	The bones of the foot consist of the tarsal bones, the metatarsals, and the phalanges (Fig. 6.9). There are seven tarsal bones, which are organized in two rows with an intermediate bone between the two rows on the medial side. Inversion and eversion of the foot, or turning the sole of the foot inward and outward, respectively, occur at joints between the tarsal bones. The tarsal bones articulate with the metatarsals at tarsometatarsal joints, which allow only limited sliding movements. Independent movements of the metatarsals are restricted by deep transverse metatarsal ligaments, which effectively link together the distal heads of the bones at the metatarsophalangeal joints. There is a metatarsal for each of the five digits, and each digit has three phalanges except for the great toe (digit I), which has only two. The metatarsophalangeal joints allow flexion, extension, abduction, and adduction of the digits, but the range of movement is more restricted than in the hand.

1	The metatarsophalangeal joints allow flexion, extension, abduction, and adduction of the digits, but the range of movement is more restricted than in the hand. The interphalangeal joints are hinge joints and allow flexion and extension. The bones of the foot are not organized in a single plane so that they lie flat on the ground. Rather, the metatarsals and tarsals form longitudinal and transverse arches (Fig. 6.10). The longitudinal arch is highest on the medial side of the foot. The arches are flexible in nature and are supported by muscles and ligaments. They absorb and transmit forces during walking and standing. Muscles of the gluteal region consist predominantly of extensors, rotators, and abductors of the hip joint (Fig. 6.11). In addition to moving the thigh on a fixed pelvis, these muscles also control the movement of the pelvis relative to the limb bearing the body’s weight (weight-bearing or stance limb) while the other limb swings forward (swing limb) during walking.

1	Major flexor muscles of the hip (iliopsoas—psoas major and iliacus) do not originate in the gluteal region or the thigh. Instead, they are attached to the posterior abdominal wall and descend through the gap between the inguinal ligament and pelvic bone to attach to the proximal end of the femur (Fig. 6.12). Muscles in the thigh and leg are separated into three compartments by layers of fascia, bones, and ligaments (Fig. 6.13). In the thigh, there are medial (adductor), anterior (extensor), and posterior (flexor) compartments: Most muscles in the medial compartment act mainly on the hip joint. The large muscles (hamstrings) in the posterior compartment act on the hip (extension) and knee (flexion) because they attach to both the pelvis and bones of the leg. Muscles in the anterior compartment (quadriceps femoris) predominantly extend the knee. Muscles in the leg are divided into lateral (fibular), anterior, and posterior compartments:

1	Muscles in the anterior compartment (quadriceps femoris) predominantly extend the knee. Muscles in the leg are divided into lateral (fibular), anterior, and posterior compartments: Muscles in the lateral compartment predominantly evert the foot. Muscles in the anterior compartment dorsiflex the foot and extend the digits. Muscles in the posterior compartment plantarflex the foot and flex the digits; one of the muscles can also flex the knee because it attaches superiorly to the femur. Specific muscles in each of the three compartments in the leg also provide dynamic support for the arches of the foot. Muscles found entirely in the foot (intrinsic muscles) modify the forces produced by tendons entering the toes from the leg and provide dynamic support for the longitudinal arches of the foot when walking, particularly when levering the body forward on the stance limb just before toe-off.

1	Unlike in the upper limb where most structures pass between the neck and limb through a single axillary inlet, in the lower limb, there are four major entry and exit points between the lower limb and the abdomen, pelvis, and perineum (Fig. 6.14). These are: the gap between the inguinal ligament and pelvic bone, the greater sciatic foramen, the obturator canal (at the top of the obturator foramen), and the lesser sciatic foramen. The lower limb communicates directly with the abdomen through a gap between the pelvic bone and the inguinal ligament (Fig. 6.14). Structures passing though this gap include: muscles—psoas major, iliacus, and pectineus; nerves—femoral and femoral branch of the genitofemoral nerves, and the lateral cutaneous nerve of the thigh; vessels—femoral artery and vein; and lymphatics.

1	This gap between the pelvic bone and the inguinal ligament is a weak area in the abdominal wall and often associated with abnormal protrusion of the abdominal cavity and contents into the thigh (femoral hernia). This type of hernia usually occurs where the lymphatic vessels pass through the gap (the femoral canal). Structures within the pelvis communicate with the lower limb through two major apertures (Fig. 6.14). Posteriorly, structures communicate with the gluteal region through the greater sciatic foramen and include: a muscle—piriformis; nerves—sciatic, superior and inferior gluteal, and pudendal nerves; and vessels—superior and inferior gluteal arteries and veins, and the internal pudendal artery. The sciatic nerve is the largest peripheral nerve of the body and is the major nerve of the lower limb.

1	The sciatic nerve is the largest peripheral nerve of the body and is the major nerve of the lower limb. Anteriorly, the obturator nerve and vessels pass between the pelvis and thigh through the obturator canal. This canal is formed between bone at the top of the obturator foramen and the obturator membrane, which closes most of the foramen during life. Structures pass between the perineum and gluteal region through the lesser sciatic foramen (Fig. 6.14). The most important with respect to the lower limb is the tendon of the obturator internus muscle. The nerve and artery of the perineum (the internal pudendal artery and pudendal nerve) pass out of the pelvis through the greater sciatic foramen into the gluteal region and then immediately pass around the ischial spine and sacrospinous ligament and through the lesser sciatic foramen to enter the perineum. Innervation is by lumbar and sacral

1	Innervation is by lumbar and sacral Somatic motor and general sensory innervation of the lower limb is by peripheral nerves emanating from the lumbar and sacral plexuses on the posterior abdominal and pelvic walls. These plexuses are formed by the anterior rami of L1 to L3 and most of L4 (lumbar plexus) and L4 to S5 (sacral plexus).

1	Nerves originating from the lumbar and sacral plexuses and entering the lower limb carry fibers from spinal cord levels L1 to S3 (Fig. 6.15). Nerves from lower sacral segments innervate the perineum. Terminal nerves exit the abdomen and pelvis through a number of apertures and foramina and enter the limb. As a consequence of this innervation, lumbar and upper sacral nerves are tested clinically by examining the lower limb. In addition, clinical signs (such as pain, pins-and-needles sensations, paresthesia, and fascicular muscle twitching) resulting from any disorder affecting these spinal nerves (e.g., herniated intervertebral disc in the lumbar region) appear in the lower limb.

1	Dermatomes in the lower limb are shown in Fig. 6.16. Regions that can be tested for sensation and are reasonably autonomous (have minimal overlap) are: over the inguinal ligament—L1, lateral side of the thigh—L2, lower medial side of the thigh—L3, medial side of the great toe (digit I)—L4, medial side of digit II—L5, little toe (digit V)—S1, back of the thigh—S2, and skin over the gluteal fold—S3. The dermatomes of S4 and S5 are tested in the perineum. Selected joint movements are used to test myotomes (Fig. 6.17). For example: Flexion of the hip is controlled primarily by L1 and L2. Extension of the knee is controlled mainly by L3 and L4. Knee flexion is controlled mainly by L5 to S2. Plantarflexion of the foot is controlled predominantly by S1 and S2. Adduction of the digits is controlled by S2 and S3. In an unconscious patient, both somatic sensory and somatic motor functions of spinal cord levels can be tested using tendon reflexes:

1	Adduction of the digits is controlled by S2 and S3. In an unconscious patient, both somatic sensory and somatic motor functions of spinal cord levels can be tested using tendon reflexes: A tap on the patellar ligament at the knee tests predominantly L3 and L4. A tendon tap on the calcaneal tendon posterior to the ankle (tendon of gastrocnemius and soleus) tests S1 and S2. Each of the major muscle groups or compartments in the lower limb is innervated primarily by one or more of the major nerves that originate from the lumbar and sacral plexuses (Fig. 6.18): Large muscles in the gluteal region are innervated by the superior and inferior gluteal nerves. Most muscles in the anterior compartment of the thigh are innervated by the femoral nerve (except the tensor fasciae latae, which are innervated by the superior gluteal nerve).

1	Most muscles in the anterior compartment of the thigh are innervated by the femoral nerve (except the tensor fasciae latae, which are innervated by the superior gluteal nerve). Most muscles in the medial compartment are innervated mainly by the obturator nerve (except the pectineus, which is innervated by the femoral nerve, and part of the adductor magnus, which is innervated by the tibial division of the sciatic nerve). Most muscles in the posterior compartment of the thigh and the leg and in the sole of the foot are innervated by the tibial part of the sciatic nerve (except the short head of the biceps femoris in the posterior thigh, which is innervated by the common fibular division of the sciatic nerve). The anterior and lateral compartments of the leg and muscles associated with the dorsal surface of the foot are innervated by the common fibular part of the sciatic nerve.

1	The anterior and lateral compartments of the leg and muscles associated with the dorsal surface of the foot are innervated by the common fibular part of the sciatic nerve. In addition to innervating major muscle groups, each of the major peripheral nerves originating from the lumbar and sacral plexuses carries general sensory information from patches of skin (Fig. 6.19). Sensation from these areas can be used to test for peripheral nerve lesions: The femoral nerve innervates skin on the anterior thigh, medial side of the leg, and medial side of the ankle. The obturator nerve innervates the medial side of the thigh. The tibial part of the sciatic nerve innervates the lateral side of the ankle and foot. The common fibular nerve innervates the lateral side of the leg and the dorsum of the foot. Nerves related to bone

1	The common fibular nerve innervates the lateral side of the leg and the dorsum of the foot. Nerves related to bone The common fibular branch of the sciatic nerve curves laterally around the neck of the fibula when passing from the popliteal fossa into the leg (Fig. 6.20). The nerve can be rolled against bone just distal to the attachment of biceps femoris to the head of the fibula. In this location, the nerve can be damaged by impact injuries, fractures to the bone, or leg casts that are placed too high. Large veins embedded in the subcutaneous (superficial) fascia of the lower limb (Fig. 6.21) often become distended (varicose). These vessels can also be used for vascular transplantation. The most important superficial veins are the great and small saphenous veins, which originate from the medial and lateral sides, respectively, of a dorsal venous arch in the foot.

1	The most important superficial veins are the great and small saphenous veins, which originate from the medial and lateral sides, respectively, of a dorsal venous arch in the foot. The great saphenous vein passes up the medial side of the leg, knee, and thigh to pass through an opening in deep fascia covering the femoral triangle and join with the femoral vein. The small saphenous vein passes behind the distal end of the fibula (lateral malleolus) and up the back of the leg to penetrate deep fascia and join the popliteal vein posterior to the knee. The external surfaces of the pelvic bones, sacrum, and coccyx are predominantly the regions of the pelvis associated with the lower limb, although some muscles do originate from the deep or internal surfaces of these bones and from the deep surfaces of the lumbar vertebrae, above (Fig. 6.22).

1	Each pelvic bone is formed by three bones (ilium, ischium, and pubis), which fuse during childhood. The ilium is superior and the pubis and ischium are anteroinferior and posteroinferior, respectively. The ilium articulates with the sacrum. The pelvic bone is further anchored to the end of the vertebral column (sacrum and coccyx) by the sacrotuberous and sacrospinous ligaments, which attach to a tuberosity and spine on the ischium. The outer surface of the ilium, and the adjacent surfaces of the sacrum, coccyx, and sacrotuberous ligament are associated with the gluteal region of the lower limb and provide extensive muscle attachment. The ischial tuberosity provides attachment for many of the muscles in the posterior compartment of the thigh, and the ischiopubic ramus and body of the pubis are associated mainly with muscles in the medial compartment of the thigh. The head of the femur articulates with the acetabulum on the lateral surface of the pelvic bone.

1	The upper fan-shaped part of the ilium is associated on its inner side with the abdomen and on its outer side with the lower limb. The top of this region is the iliac crest, which ends anteriorly as the anterior superior iliac spine and posteriorly as the posterior superior iliac spine. A prominent lateral expansion of the crest just posterior to the anterior superior iliac spine is the tuberculum of the iliac crest. The anterior inferior iliac spine is on the anterior margin of the ilium, and below this, where the ilium fuses with the pubis, is a raised area of bone (the iliopubic eminence). The gluteal surface of the ilium faces posterolaterally and lies below the iliac crest. It is marked by three curved lines (inferior, anterior, and posterior gluteal lines), which divide the surface into four regions:

1	The inferior gluteal line originates just superior to the anterior inferior iliac spine and curves inferiorly across the bone to end near the posterior margin of the acetabulum—the rectus femoris muscle attaches to the anterior inferior iliac spine and to a roughened patch of bone between the superior margin of the acetabulum and the inferior gluteal line. The anterior gluteal line originates from the lateral margin of the iliac crest between the anterior superior iliac spine and the tuberculum of the iliac crest, and arches inferiorly across the ilium to disappear just superior to the upper margin of the greater sciatic foramen—the gluteus minimus muscle originates from between the inferior and anterior gluteal lines.

1	The posterior gluteal line descends almost vertically from the iliac crest to a position near the posterior inferior iliac spine—the gluteus medius muscle attaches to bone between the anterior and posterior gluteal lines, and the gluteus maximus muscle attaches posterior to the posterior gluteal line. The ischial tuberosity is posteroinferior to the acetabulum and is associated mainly with the hamstring muscles of the posterior thigh (Fig. 6.23). It is divided into upper and lower areas by a transverse line. The upper area of the ischial tuberosity is oriented vertically and is further subdivided into two parts by an oblique line, which descends, from medial to lateral, across the surface: The more medial part of the upper area is for the attachment of the combined origin of the semitendinosus muscle and the long head of the biceps femoris muscle. The lateral part is for the attachment of the semimembranosus muscle.

1	The lateral part is for the attachment of the semimembranosus muscle. The lower area of the ischial tuberosity is oriented horizontally and is divided into medial and lateral regions by a ridge of bone: The lateral region provides attachment for part of the adductor magnus muscle. The medial part faces inferiorly and is covered by connective tissue and by a bursa. When sitting, this medial part supports the body weight. The sacrotuberous ligament is attached to a sharp ridge on the medial margin of the ischial tuberosity. The external surfaces of the ischiopubic ramus anterior to the ischial tuberosity and the body of the pubis provide attachment for muscles of the medial compartment of the thigh (Fig. 6.23). These muscles include the adductor longus, adductor brevis, adductor magnus, pectineus, and gracilis.

1	The large cup-shaped acetabulum for articulation with the head of the femur is on the lateral surface of the pelvic bone in the region where the ilium, pubis, and ischium fuse (Fig. 6.24). The margin of the acetabulum is marked inferiorly by a prominent notch (acetabular notch). The wall of the acetabulum consists of nonarticular and articular parts: The nonarticular part is rough and forms a shallow circular depression (the acetabular fossa) in central and inferior parts of the acetabular floor—the acetabular notch is continuous with the acetabular fossa. The articular surface is broad and surrounds the anterior, superior, and posterior margins of the acetabular fossa. The smooth crescent-shaped articular surface (the lunate surface) is broadest superiorly where most of the body’s weight is transmitted through the pelvis to the femur. The lunate surface is deficient inferiorly at the acetabular notch.

1	The acetabular fossa provides attachment for the ligament of the head of the femur, whereas blood vessels and nerves pass through the acetabular notch. The femur is the bone of the thigh and the longest bone in the body. Its proximal end is characterized by a head and neck, and two large projections (the greater and lesser trochanters) on the upper part of the shaft (Fig. 6.26). The head of the femur is spherical and articulates with the acetabulum of the pelvic bone. It is characterized by a nonarticular pit (fovea) on its medial surface for the attachment of the ligament of the head. The neck of the femur is a cylindrical strut of bone that connects the head to the shaft of the femur. It projects superomedially from the shaft at an angle of approximately 125°, and projects slightly forward. The orientation of the neck relative to the shaft increases the range of movement of the hip joint.

1	The upper part of the shaft of the femur bears a greater and lesser trochanter, which are attachment sites for muscles that move the hip joint. The greater trochanter extends superiorly from the shaft of the femur just lateral to the region where the shaft joins the neck of the femur (Fig. 6.26). It continues posteriorly where its medial surface is deeply grooved to form the trochanteric fossa. The lateral wall of this fossa bears a distinct oval depression for attachment of the obturator externus muscle. The greater trochanter has an elongate ridge on its anterolateral surface for attachment of the gluteus minimus and a similar ridge more posteriorly on its lateral surface for attachment of the gluteus medius. Between these two points, the greater trochanter is palpable.

1	On the medial side of the superior aspect of the greater trochanter and just above the trochanteric fossa is a small impression for attachment of the obturator internus and its associated gemelli muscles, and immediately above and behind this feature is an impression on the margin of the trochanter for attachment of the piriformis muscle. The lesser trochanter is smaller than the greater trochanter and has a blunt conical shape. It projects posteromedially from the shaft of the femur just inferior to the junction with the neck (Fig. 6.26). It is the attachment site for the combined tendons of psoas major and iliacus muscles. Extending between the two trochanters and separating the shaft from the neck of the femur are the intertrochanteric line and intertrochanteric crest.

1	Extending between the two trochanters and separating the shaft from the neck of the femur are the intertrochanteric line and intertrochanteric crest. The intertrochanteric line is a ridge of bone on the anterior surface of the upper margin of the shaft that descends medially from a tubercle on the anterior surface of the base of the greater trochanter to a position just anterior to the base of the lesser trochanter (Fig. 6.26). It is continuous with the pectineal line (spiral line), which curves medially under the lesser trochanter and around the shaft of the femur to merge with the medial margin of the linea aspera on the posterior aspect of the femur.

1	The intertrochanteric crest is on the posterior surface of the femur and descends medially across the bone from the posterior margin of the greater trochanter to the base of the lesser trochanter (Fig. 6.26). It is a broad smooth ridge of bone with a prominent tubercle (the quadrate tubercle) on its upper half, which provides attachment for the quadratus femoris muscle. Shaft of the femur The shaft of the femur descends from lateral to medial in the coronal plane at an angle of 7° from the vertical axis (Fig. 6.27). The distal end of the femur is therefore closer to the midline than the upper end of the shaft. The middle third of the shaft of the femur is triangular in shape with smooth lateral and medial margins between anterior, lateral (posterolateral), and medial (posteromedial) surfaces. The posterior margin is broad and forms a prominent raised crest (the linea aspera).

1	The linea aspera is a major site of muscle attachment in the thigh. In the proximal third of the femur, the medial and lateral margins of the linea aspera diverge and continue superiorly as the pectineal line and gluteal tuberosity, respectively (Fig. 6.27): The pectineal line curves anteriorly under the lesser trochanter and joins the intertrochanteric line. The gluteal tuberosity is a broad linear roughening that curves laterally to the base of the greater trochanter. The gluteus maximus muscle is attached to the gluteal tuberosity. The triangular area enclosed by the pectineal line, the gluteal tuberosity, and the intertrochanteric crest is the posterior surface of the proximal end of the femur.

1	The triangular area enclosed by the pectineal line, the gluteal tuberosity, and the intertrochanteric crest is the posterior surface of the proximal end of the femur. The hip joint is a synovial articulation between the head of the femur and the acetabulum of the pelvic bone (Fig. 6.30A). The joint is a multiaxial ball and socket joint designed for stability and weight-bearing at the expense of mobility. Movements at the joint include flexion, extension, abduction, adduction, medial and lateral rotation, and circumduction. When considering the effects of muscle action on the hip joint, the long neck of the femur and the angulation of the neck on the shaft of the femur must be borne in mind. For example, medial and lateral rotation of the femur involves muscles that move the greater trochanter forward and backward, respectively, relative to the acetabulum (Fig. 6.30B).

1	The articular surfaces of the hip joint are: the spherical head of the femur, and the lunate surface of the acetabulum of the pelvic bone. The acetabulum almost entirely encompasses the hemispherical head of the femur and contributes substantially to joint stability. The nonarticular acetabular fossa contains loose connective tissue. The lunate surface is covered by hyaline cartilage and is broadest superiorly. Except for the fovea, the head of the femur is also covered by hyaline cartilage. The rim of the acetabulum is raised slightly by a fibrocartilaginous collar (the acetabular labrum). Inferiorly, the labrum bridges across the acetabular notch as the transverse acetabular ligament and converts the notch into a foramen (Fig. 6.31A).

1	The ligament of the head of the femur is a flat band of delicate connective tissue that attaches at one end to the fovea on the head of the femur and at the other end to the acetabular fossa, transverse acetabular ligament, and margins of the acetabular notch (Fig. 6.31B). It carries a small branch of the obturator artery, which contributes to the blood supply of the head of the femur. The synovial membrane attaches to the margins of the articular surfaces of the femur and acetabulum, forms a tubular covering around the ligament of the head of the femur, and lines the fibrous membrane of the joint (Figs. 6.31B and 6.32). From its attachment to the margin of the head of the femur, the synovial membrane covers the neck of the femur before reflecting onto the fibrous membrane (Fig. 6.32).

1	The fibrous membrane that encloses the hip joint is strong and generally thick. Medially, it is attached to the margin of the acetabulum, the transverse acetabular ligament, and the adjacent margin of the obturator foramen (Fig. 6.33A). Laterally, it is attached to the intertrochanteric line on the anterior aspect of the femur and to the neck of the femur just proximal to the intertrochanteric crest on the posterior surface. Three ligaments reinforce the external surface of the fibrous membrane and stabilize the joint: the iliofemoral, pubofemoral, and ischiofemoral ligaments.

1	Three ligaments reinforce the external surface of the fibrous membrane and stabilize the joint: the iliofemoral, pubofemoral, and ischiofemoral ligaments. The iliofemoral ligament is anterior to the hip joint and is triangular shaped (Fig. 6.33B). Its apex is attached to the ilium between the anterior inferior iliac spine and the margin of the acetabulum and its base is attached along the intertrochanteric line of the femur. Parts of the ligament attached above and below the intertrochanteric line are thicker than the part attached to the central part of the line. This results in the ligament having a Y appearance. The pubofemoral ligament is anteroinferior to the hip joint (Fig. 6.33B). It is also triangular in shape, with its base attached medially to the iliopubic eminence, adjacent bone, and obturator membrane. Laterally, it blends with the fibrous membrane and with the deep surface of the iliofemoral ligament.

1	The ischiofemoral ligament reinforces the posterior aspect of the fibrous membrane (Fig. 6.33C). It is attached medially to the ischium, just posteroinferior to the acetabulum, and laterally to the greater trochanter deep to the iliofemoral ligament. The fibers of all three ligaments are oriented in a spiral fashion around the hip joint so that they become taut when the joint is extended. This stabilizes the joint and reduces the amount of muscle energy required to maintain a standing position. Vascular supply to the hip joint is predominantly through branches of the obturator artery, medial and lateral circumflex femoral arteries, superior and inferior gluteal arteries, and the first perforating branch of the deep artery of the thigh. The articular branches of these vessels form a network around the joint (Fig. 6.34). The hip joint is innervated by articular branches from the femoral, obturator, and superior gluteal nerves, and the nerve to the quadratus femoris.

1	The hip joint is innervated by articular branches from the femoral, obturator, and superior gluteal nerves, and the nerve to the quadratus femoris. Gateways to the lower limb There are four major routes by which structures pass from the abdomen and pelvis into and out of the lower limb. These are the obturator canal, the greater sciatic foramen, the lesser sciatic foramen, and the gap between the inguinal ligament and the anterosuperior margin of the pelvis (Fig. 6.35). The obturator canal is an almost vertically oriented passageway at the anterosuperior edge of the obturator foramen (Fig. 6.35). It is bordered: above by a groove (obturator groove) on the inferior surface of the superior ramus of the pubic bone, and below by the upper margin of the obturator membrane, which fills most of the obturator foramen, and by muscles (obturator internus and externus) attached to the inner and outer surfaces of the obturator membrane and surrounding bone.

1	The obturator canal connects the abdominopelvic region with the medial compartment of the thigh. The obturator nerve and vessels pass through the canal. The greater sciatic foramen is formed on the posterolateral pelvic wall and is the major route for structures to pass between the pelvis and the gluteal region of the lower limb (Fig. 6.35). The margins of the foramen are formed by: the greater sciatic notch, parts of the upper borders of the sacrospinous and sacrotuberous ligaments, and the lateral border of the sacrum. The piriformis muscle passes out of the pelvis into the gluteal region through the greater sciatic foramen and separates the foramen into two parts, a part above the muscle and a part below: The superior gluteal nerve and vessels pass through the greater sciatic foramen above the piriformis.

1	The superior gluteal nerve and vessels pass through the greater sciatic foramen above the piriformis. The sciatic nerve, inferior gluteal nerve and vessels, pudendal nerve and internal pudendal vessels, posterior cutaneous nerve of the thigh, nerve to the obturator internus and gemellus superior, and nerve to the quadratus femoris and gemellus inferior pass through the greater sciatic foramen below the muscle. The lesser sciatic foramen is inferior to the greater sciatic foramen on the posterolateral pelvic wall (Fig. 6.35). It is also inferior to the lateral attachment of the pelvic floor (levator ani and coccygeus muscles) to the pelvic wall and therefore connects the gluteal region with the perineum: The tendon of the obturator internus passes from the lateral pelvic wall through the lesser sciatic foramen into the gluteal region to insert on the femur.

1	The tendon of the obturator internus passes from the lateral pelvic wall through the lesser sciatic foramen into the gluteal region to insert on the femur. The pudendal nerve and internal pudendal vessels, which first exit the pelvis by passing through the greater sciatic foramen below the piriformis muscle, enter the perineum below the pelvic floor by passing around the ischial spine and sacrospinous ligament and medially through the lesser sciatic foramen. Gap between the inguinal ligament

1	Gap between the inguinal ligament The large crescent-shaped gap between the inguinal ligament above and the anterosuperior margin of the pelvic bone below is the major route of communication between the abdomen and the anteromedial aspect of the thigh (Fig. 6.35). The psoas major, iliacus, and pectineus muscles pass through this gap to insert onto the femur. The major blood vessels (femoral artery and vein) and lymphatics of the lower limb also pass through it, as does the femoral nerve, to enter the femoral triangle of the thigh. Nerves that enter the lower limb from the abdomen and pelvis are terminal branches of the lumbosacral plexus on the posterior wall of the abdomen and the posterolateral walls of the pelvis (Fig. 6.36 and Table 6.1).

1	The lumbar plexus is formed by the anterior rami of spinal nerves L1 to L3 and part of L4 (see Chapter 4, pp. 398–401). The rest of the anterior ramus of L4 and the anterior ramus of L5 combine to form the lumbosacral trunk, which enters the pelvic cavity and joins with the anterior rami of S1 to S3 and part of S4 to form the sacral plexus (see Chapter 5, pp. 480–486). Major nerves that originate from the lumbosacral plexus and leave the abdomen and pelvis to enter the lower limb include the femoral nerve, obturator nerve, sciatic nerve, superior gluteal nerve, and inferior gluteal nerve. Other nerves that also originate from the plexus and enter the lower limb to supply skin or muscle include the lateral cutaneous nerve of the thigh, nerve to the obturator internus, nerve to the quadratus femoris, posterior cutaneous nerve of the thigh, perforating cutaneous nerve, and branches of the ilio-inguinal and genitofemoral nerves.

1	The femoral nerve carries contributions from the anterior rami of L2 to L4 and leaves the abdomen by passing through the gap between the inguinal ligament and superior margin of the pelvis to enter the femoral triangle on the anteromedial aspect of the thigh (Fig. 6.35 and Table 6.1). In the femoral triangle it is lateral to the femoral artery. The femoral nerve: innervates all muscles in the anterior compartment of the thigh, in the abdomen, gives rise to branches that innervate the iliacus and pectineus muscles, and innervates skin over the anterior aspect of the thigh, the anteromedial side of the knee, the medial side of the leg, and the medial side of the foot.

1	The obturator nerve, like the femoral nerve, originates from L2 to L4. It descends along the posterior abdominal wall, passes through the pelvic cavity and enters the thigh by passing through the obturator canal (Fig. 6.36 and Table 6.1). The obturator nerve innervates: all muscles in the medial compartment of the thigh, except the part of the adductor magnus muscle that originates from the ischium and the pectineus muscle, which are innervated by the sciatic and the femoral nerves, respectively; the obturator externus muscle; and skin on the medial side of the upper thigh. The sciatic nerve is the largest nerve of the body and carries contributions from L4 to S3. It leaves the pelvis through the greater sciatic foramen inferior to the piriformis muscle, enters and passes through the gluteal region (Fig. 6.36 and Table 6.1), and then enters the posterior compartment of the thigh where it divides into its two major branches: the common fibular nerve, and the tibial nerve.

1	Posterior divisions of L4 to S2 are carried in the common fibular part of the nerve and the anterior divisions of L4 to S3 are carried in the tibial part. The sciatic nerve innervates: all muscles in the posterior compartment of the thigh, the part of the adductor magnus originating from the ischium, all muscles in the leg and foot, and skin on the lateral side of the leg and the lateral side and sole of the foot. The gluteal nerves are major motor nerves of the gluteal region. The superior gluteal nerve (Fig. 6.36 and Table 6.1) carries contributions from the anterior rami of L4 to S1, leaves the pelvis through the greater sciatic foramen above the piriformis muscle, and innervates: the gluteus medius and minimus muscles, and the tensor fasciae latae muscle.

1	The inferior gluteal nerve (Fig. 6.36 and Table 6.1) is formed by contributions from L5 to S2, leaves the pelvis through the greater sciatic foramen inferior to the piriformis muscle, and enters the gluteal region to supply the gluteus maximus. Terminal sensory branches of the ilio-inguinal nerve (L1) and the genitofemoral nerve (L1, L2) descend into the upper thigh from the lumbar plexus. The ilio-inguinal nerve originates from the superior part of the lumbar plexus, descends around the abdominal wall in the plane between the transversus abdominis and internal oblique muscles, and then passes through the inguinal canal to leave the abdominal wall through the superficial inguinal ring (Fig. 6.36 and Table 6.1). Its terminal branches innervate skin on the medial side of the upper thigh and adjacent parts of the perineum.

1	The genitofemoral nerve passes anteroinferiorly through the psoas major muscle on the posterior abdominal wall and descends on the anterior surface of the psoas major (Fig. 6.36 and Table 6.1). Its genital branch innervates anterior aspects of the perineum. Its femoral branch passes into the thigh by crossing under the inguinal ligament where it is lateral to the femoral artery. It passes superficially to innervate skin over the upper central part of the anterior thigh. Lateral cutaneous nerve of thigh The lateral cutaneous nerve of the thigh originates from L2 and L3. It leaves the abdomen either by passing through the gap between the inguinal ligament and the pelvic bone just medial to the anterior superior iliac spine or by passing directly through the inguinal ligament (Fig. 6.36 and Table 6.1). It supplies skin on the lateral side of the thigh. Nerve to quadratus femoris and nerve

1	Nerve to quadratus femoris and nerve The nerve to the quadratus femoris (L4 to S1) and the nerve to the obturator internus (L5 to S2) are small motor nerves that originate from the sacral plexus. Both nerves pass through the greater sciatic foramen inferior to the piriformis muscle and enter the gluteal region (Fig. 6.36 and Table 6.1): The nerve to the obturator internus supplies the gemellus superior muscle in the gluteal region and then loops around the ischial spine and enters the perineum through the lesser sciatic foramen to penetrate the perineal surface of the obturator internus muscle. The nerve to the quadratus femoris supplies the gemellus inferior and quadratus femoris muscles. Posterior cutaneous nerve of thigh

1	The nerve to the quadratus femoris supplies the gemellus inferior and quadratus femoris muscles. Posterior cutaneous nerve of thigh The posterior cutaneous nerve of the thigh is formed by contributions from S1 to S3 and leaves the pelvic cavity through the greater sciatic foramen inferior to the piriformis muscle (Fig. 6.36 and Table 6.1). It passes vertically through the gluteal region deep to the gluteus maximus and enters the posterior thigh and innervates: a longitudinal band of skin over the posterior aspect of the thigh that continues into the upper leg, and skin over the gluteal fold, over the upper medial part of the thigh and in the adjacent regions of the perineum.

1	The perforating cutaneous nerve is a small sensory nerve formed by contributions from S2 and S3. It leaves the pelvic cavity by penetrating directly through the sacrotuberous ligament (Fig. 6.36 and Table 6.1) and passes inferiorly around the lower border of the gluteus maximus where it overlaps with the posterior cutaneous nerve of the thigh in innervating skin over the medial aspect of the gluteal fold. The major artery supplying the lower limb is the femoral artery (Fig. 6.37), which is the continuation of the external iliac artery in the abdomen. The external iliac artery becomes the femoral artery as the vessel passes under the inguinal ligament to enter the femoral triangle in the anterior aspect of the thigh. Branches supply most of the thigh and all of the leg and foot. the obturator artery Other vessels supplying parts of the lower limb include the superior and inferior gluteal arteries and the obturator artery (Fig. 6.37).

1	the obturator artery Other vessels supplying parts of the lower limb include the superior and inferior gluteal arteries and the obturator artery (Fig. 6.37). The superior and inferior gluteal arteries originate in the pelvic cavity as branches of the internal iliac artery (see Chapter 5, pp. 489–492) and supply the gluteal region. The superior gluteal artery leaves the pelvis through the greater sciatic foramen above the piriformis muscle, and the inferior gluteal artery leaves through the same foramen but below the piriformis muscle. The obturator artery is also a branch of the internal iliac artery in the pelvic cavity (see Chapter 5, pp. 490–491) and passes through the obturator canal to enter and supply the medial compartment of the thigh.

1	Branches of the femoral, inferior gluteal, superior gluteal, and obturator arteries, together with branches from the internal pudendal artery of the perineum, interconnect to form an anastomotic network in the upper thigh and gluteal region. The presence of these anastomotic channels may provide collateral circulation when one of the vessels is interrupted. Veins draining the lower limb form superficial and deep groups. The deep veins generally follow the arteries (femoral, superior gluteal, inferior gluteal, and obturator). The major deep vein draining the limb is the femoral vein (Fig. 6.38). It becomes the external iliac vein when it passes under the inguinal ligament to enter the abdomen.

1	The superficial veins are in the subcutaneous connective tissue and are interconnected with and ultimately drain into the deep veins. The superficial veins form two major channels—the great saphenous vein and the small saphenous vein. Both veins originate from a dorsal venous arch in the foot: The great saphenous vein originates from the medial side of the dorsal venous arch and then ascends up the medial side of the leg, knee, and thigh to connect with the femoral vein just inferior to the inguinal ligament. The small saphenous vein originates from the lateral side of the dorsal venous arch, ascends up the posterior surface of the leg, and then penetrates deep fascia to join the popliteal vein posterior to the knee; proximal to the knee, the popliteal vein becomes the femoral vein. Most lymphatic vessels in the lower limb drain into superficial and deep inguinal nodes located in the fascia just inferior to the inguinal ligament (Fig. 6.39).

1	Most lymphatic vessels in the lower limb drain into superficial and deep inguinal nodes located in the fascia just inferior to the inguinal ligament (Fig. 6.39). The superficial inguinal nodes, approximately ten in number, are in the superficial fascia and parallel the course of the inguinal ligament in the upper thigh. Medially, they extend inferiorly along the terminal part of the great saphenous vein. Superficial inguinal nodes receive lymph from the gluteal region, lower abdominal wall, perineum, and superficial regions of the lower limb. They drain, via vessels that accompany the femoral vessels, into external iliac nodes associated with the external iliac artery in the abdomen. The deep inguinal nodes, up to three in number, are medial to the femoral vein (Fig. 6.39).

1	The deep inguinal nodes, up to three in number, are medial to the femoral vein (Fig. 6.39). The deep inguinal nodes receive lymph from deep lymphatics associated with the femoral vessels and from the glans penis (or clitoris) in the perineum. They interconnect with the superficial inguinal nodes and drain into the external iliac nodes via vessels that pass along the medial side of the femoral vein as it passes under the inguinal ligament. The space through which the lymphatic vessels pass under the inguinal ligament is the femoral canal. In addition to the inguinal nodes, there is a small collection of deep nodes posterior to the knee close to the popliteal vessels (Fig. 6.39). These popliteal nodes receive lymph from superficial vessels, which accompany the small saphenous vein, and from deep areas of the leg and foot. They ultimately drain into the deep and superficial inguinal nodes. Deep fascia and the saphenous opening

1	Deep fascia and the saphenous opening The outer layer of deep fascia in the lower limb forms a thick “stocking-like” membrane, which covers the limb and lies beneath the superficial fascia (Fig. 6.40A). This deep fascia is particularly thick in the thigh and gluteal region and is termed the fascia lata. The fascia lata is anchored superiorly to bone and soft tissues along a line of attachment that defines the upper margin of the lower limb. Beginning anteriorly and circling laterally around the limb, this line of attachment includes the inguinal ligament, iliac crest, sacrum, coccyx, sacrotuberous ligament, inferior ramus of the pubic bone, body of the pubic bone, and superior ramus of the pubic bone. Inferiorly, the fascia lata is continuous with the deep fascia of the leg.

1	Inferiorly, the fascia lata is continuous with the deep fascia of the leg. The fascia lata is thickened laterally into a longitudinal band (the iliotibial tract), which descends along the lateral margin of the limb from the tuberculum of the iliac crest to a bony attachment just below the knee (Fig. 6.40B). The superior aspect of the fascia lata in the gluteal region splits anteriorly to enclose the tensor fasciae latae muscle and posteriorly to enclose the gluteus maximus muscle: The tensor fasciae latae muscle is partially enclosed by and inserts into the superior and anterior aspects of the iliotibial tract. Most of the gluteus maximus muscle inserts into the posterior aspect of the iliotibial tract.

1	Most of the gluteus maximus muscle inserts into the posterior aspect of the iliotibial tract. The tensor fasciae latae and gluteus maximus muscles, working through their attachments to the iliotibial tract, hold the leg in extension once other muscles have extended the leg at the knee joint. The iliotibial tract and its two associated muscles also stabilize the hip joint by preventing lateral displacement of the proximal end of the femur away from the acetabulum. The fascia lata has one prominent aperture on the anterior aspect of the thigh just inferior to the medial end of the inguinal ligament (the saphenous opening), which allows the great saphenous vein to pass from superficial fascia through the deep fascia to connect with the femoral vein (Fig. 6.41).

1	The margin of the saphenous opening is formed by the free medial edge of the fascia lata as it descends from the inguinal ligament and spirals around the lateral side of the great saphenous vein and medially under the femoral vein to attach to the pectineal line (pecten pubis) of the pelvic bone. The femoral triangle is a wedge-shaped depression formed by muscles in the upper thigh at the junction between the anterior abdominal wall and the lower limb (Fig. 6.42): The base of the triangle is the inguinal ligament. The medial border is the medial margin of the adductor longus muscle in the medial compartment of the thigh. The lateral margin is the medial margin of the sartorius muscle in the anterior compartment of the thigh. The floor of the triangle is formed medially by the pectineus and adductor longus muscles in the medial compartment of the thigh and laterally by the iliopsoas muscle descending from the abdomen.

1	The floor of the triangle is formed medially by the pectineus and adductor longus muscles in the medial compartment of the thigh and laterally by the iliopsoas muscle descending from the abdomen. The apex of the femoral triangle points inferiorly and is continuous with a fascial canal (adductor canal), which descends medially down the thigh and posteriorly through an aperture in the lower end of one of the largest of the adductor muscles in the thigh (the adductor magnus muscle) to open into the popliteal fossa behind the knee. The femoral nerve, artery, and vein and lymphatics pass between the abdomen and lower limb under the inguinal ligament and in the femoral triangle (Fig. 6.43). The femoral artery and vein pass inferiorly through the adductor canal and become the popliteal vessels behind the knee where they meet and are distributed with branches of the sciatic nerve, which descends through the posterior thigh from the gluteal region.

1	From lateral to medial, major structures in the femoral triangle are the femoral nerve, the femoral artery, the femoral vein, and lymphatic vessels. The femoral artery can be palpated in the femoral triangle just inferior to the inguinal ligament and midway between the anterior superior iliac spine and the pubic symphysis.

1	In the femoral triangle, the femoral artery and vein and the associated lymphatic vessels are surrounded by a funnel-shaped sleeve of fascia (the femoral sheath). The sheath is continuous superiorly with the transversalis fascia and iliac fascia of the abdomen and merges inferiorly with connective tissue associated with the vessels. Each of the three structures surrounded by the sheath is contained within a separate fascial compartment within the sheath. The most medial compartment (the femoral canal) contains the lymphatic vessels and is conical in shape. The opening of this canal superiorly is potentially a weak point in the lower abdomen and is the site for femoral hernias. The femoral nerve is lateral to and not contained within the femoral sheath. The gluteal region lies posterolateral to the bony pelvis and proximal end of the femur (Fig. 6.44). Muscles in the region mainly abduct, extend, and laterally rotate the femur relative to the pelvic bone.

1	The gluteal region communicates anteromedially with the pelvic cavity and perineum through the greater sciatic foramen and lesser sciatic foramen, respectively. Inferiorly, it is continuous with the posterior thigh. The sciatic nerve enters the lower limb from the pelvic cavity by passing through the greater sciatic foramen and descending through the gluteal region into the posterior thigh and then into the leg and foot. The pudendal nerve and internal pudendal vessels pass between the pelvic cavity and perineum by passing first through the greater sciatic foramen to enter the gluteal region and then immediately passing through the lesser sciatic foramen to enter the perineum. The nerve to the obturator internus and gemellus superior follows a similar course. Other nerves and vessels that pass through the greater sciatic foramen from the pelvic cavity supply structures in the gluteal region itself.

1	Muscles of the gluteal region (Table 6.2) are composed mainly of two groups: a deep group of small muscles, which are mainly lateral rotators of the femur at the hip joint and include the piriformis, obturator internus, gemellus superior, gemellus inferior, and quadratus femoris; a more superficial group of larger muscles, which mainly abduct and extend the hip and include the gluteus minimus, gluteus medius, and gluteus maximus; an additional muscle in this group, the tensor fasciae latae, stabilizes the knee in extension by acting on a specialized longitudinal band of deep fascia (the iliotibial tract) that passes down the lateral side of the thigh to attach to the proximal end of the tibia in the leg. Many of the important nerves in the gluteal region are in the plane between the superficial and deep groups of muscles.

1	Many of the important nerves in the gluteal region are in the plane between the superficial and deep groups of muscles. The piriformis muscle is the most superior of the deep group of muscles (Fig. 6.45) and is a muscle of the pelvic wall and of the gluteal region (see Chapter 5, p. 443). It originates from between the anterior sacral foramina on the anterolateral surface of the sacrum and passes laterally and inferiorly through the greater sciatic foramen. In the gluteal region, the piriformis passes posterior to the hip joint and attaches to a facet on the upper margin of the greater trochanter of the femur. The piriformis externally rotates and abducts the femur at the hip joint and is innervated in the pelvic cavity by the nerve to the piriformis, which originates as branches from S1 and S2 of the sacral plexus (see Chapter 5, p. 485).

1	In addition to its action on the hip joint, the piriformis is an important landmark because it divides the greater sciatic foramen into two regions, one above and one below the piriformis. Vessels and nerves pass between the pelvis and gluteal region by passing through the greater sciatic foramen either above or below the piriformis. The obturator internus muscle, like the piriformis muscle, is a muscle of the pelvic wall and of the gluteal region (Fig. 6.45). It is a flat fan-shaped muscle originating from the medial surface of the obturator membrane and adjacent bone of the obturator foramen (see Chapter 5, pp. 442–443). Because the pelvic floor attaches to a thickened band of fascia across the medial surface of the obturator internus, the obturator internus forms: the anterolateral wall of the pelvic cavity above the pelvic floor, and the lateral wall of the ischio-anal fossa in the perineum below the pelvic floor.

1	The muscle fibers of the obturator internus converge to form a tendon, which bends 90° around the ischium between the ischial spine and ischial tuberosity and passes through the lesser sciatic foramen to enter the gluteal region. The tendon then passes posteroinferiorly to the hip joint and attaches to the medial surface of the superior margin of the greater trochanter of the femur just inferior to the attachment of the piriformis muscle. The obturator internus laterally rotates and abducts the femur at the hip joint and is innervated by the nerve to the obturator internus. The gemellus superior and inferior (gemelli is Latin for “twins”) are a pair of triangular muscles associated with the upper and lower margins of the obturator internus tendon (Fig. 6.45): The base of the gemellus superior originates from the gluteal surface of the ischial spine. The base of the gemellus inferior originates from the upper gluteal and pelvic surfaces of the ischial tuberosity.

1	The base of the gemellus inferior originates from the upper gluteal and pelvic surfaces of the ischial tuberosity. Fibers of the gemellus muscles attach along the length of the obturator internus tendon, and the apices of the two muscles insert with the tendon of the obturator internus on the greater trochanter of the femur. The gemellus superior is innervated by the nerve to the obturator internus, and the gemellus inferior is innervated by the nerve to the quadratus femoris. The gemellus muscles act with the obturator internus muscle to laterally rotate and abduct the femur at the hip joint. The quadratus femoris muscle is the most inferior of the deep group of muscles in the gluteal region (Fig. 6.45). It is a flat rectangular muscle below the obturator internus muscle and its associated gemellus muscles.

1	The quadratus femoris is attached at one end to a linear roughening on the lateral aspect of the ischium just anterior to the ischial tuberosity and at the other end to the quadrate tubercle on the intertrochanteric crest of the proximal femur. The quadratus femoris laterally rotates the femur at the hip joint and is innervated by the nerve to the quadratus femoris. The gluteus minimus and medius muscles are two muscles of the more superficial group in the gluteal region (Fig. 6.45). The gluteus minimus is a fan-shaped muscle that originates from the external surface of the expanded upper part of the ilium, between the inferior gluteal line and the anterior gluteal line. The muscle fibers converge inferiorly and laterally to form a tendon, which inserts into a broad linear facet on the anterolateral aspect of the greater trochanter.

1	The gluteus medius overlies the gluteus minimus and is also fan shaped. It has a broad origin from the external surface of the ilium between the anterior gluteal line and posterior gluteal line and inserts on an elongate facet on the lateral surface of the greater trochanter. The gluteus medius and minimus muscles abduct the lower limb at the hip joint and reduce pelvic drop over the opposite swing limb during walking by securing the position of the pelvis on the stance limb (Fig. 6.45B). Both muscles are innervated by the superior gluteal nerve. The gluteus maximus is the largest muscle in the gluteal region and overlies most of the other gluteal muscles (Fig. 6.46).

1	The gluteus maximus is the largest muscle in the gluteal region and overlies most of the other gluteal muscles (Fig. 6.46). The gluteus maximus is quadrangular in shape and has a broad origin extending from a roughened area of the ilium behind the posterior gluteal line and along the dorsal surface of the lower sacrum and the lateral surface of the coccyx to the external surface of the sacrotuberous ligament. It is also attached to fascia overlying the gluteus medius muscle and, between the ilium and sacrum, to fascia covering the erector spinae muscle, and is often described as being enclosed within two layers of the fascia lata, which covers the thigh and gluteal region.

1	Laterally, the upper and superficial lower parts of the gluteus maximus insert into the posterior aspect of a tendinous thickening of the fascia lata (the iliotibial tract), which passes over the lateral surface of the greater trochanter and descends down the thigh and into the upper leg. Deep distal parts of the muscle attach to the elongate gluteal tuberosity of the proximal femur. The gluteus maximus mainly extends the flexed thigh at the hip joint. Through its insertion into the iliotibial tract, it also stabilizes the knee and hip joints. It is innervated by the inferior gluteal nerve. The tensor fasciae latae muscle is the most anterior of the superficial group of muscles in the gluteal region and overlies the gluteus minimus and the anterior part of the gluteus medius (Fig. 6.47).

1	The tensor fasciae latae originates from the outer margin of the iliac crest from the anterior superior iliac spine to approximately the tuberculum of the iliac crest. The muscle fibers descend to insert into the anterior aspect of the iliotibial tract of deep fascia, which runs down the lateral side of the thigh and attaches to the upper tibia. Like the gluteus maximus muscle, the tensor fasciae latae is enclosed within a compartment of the fascia lata. The tensor fasciae latae stabilizes the knee in extension and, working with the gluteus maximus muscle on the iliotibial tract lateral to the greater trochanter, stabilizes the hip joint by holding the head of the femur in the acetabulum (Fig. 6.47). It is innervated by the superior gluteal nerve.

1	Seven nerves enter the gluteal region from the pelvis through the greater sciatic foramen (Fig. 6.48): the superior gluteal nerve, sciatic nerve, nerve to the quadratus femoris, nerve to the obturator internus, posterior cutaneous nerve of the thigh, pudendal nerve, and inferior gluteal nerve. An additional nerve, the perforating cutaneous nerve, enters the gluteal region by passing directly through the sacrotuberous ligament. Some of these nerves, such as the sciatic and pudendal nerves, pass through the gluteal region en route to other areas. Nerves such as the superior and inferior gluteal nerves innervate structures in the gluteal region. Many of the nerves in the gluteal region are in the plane between the superficial and deep groups of muscles.

1	Of all the nerves that pass through the greater sciatic foramen, the superior gluteal nerve is the only one that passes above the piriformis muscle (Fig. 6.48). After entering the gluteal region, the nerve loops up over the inferior margin of the gluteus minimus and travels anteriorly and laterally in the plane between the gluteus minimus and medius muscles. The superior gluteal nerve supplies branches to the gluteus minimus and medius muscles and terminates by innervating the tensor fasciae latae muscle.

1	The superior gluteal nerve supplies branches to the gluteus minimus and medius muscles and terminates by innervating the tensor fasciae latae muscle. The sciatic nerve enters the gluteal region through the greater sciatic foramen inferior to the piriformis muscle (Fig. 6.48). It descends in the plane between the superficial and deep group of gluteal region muscles, crossing the posterior surfaces of first the obturator internus and associated gemellus muscles and then the quadratus femoris muscle. It lies just deep to the gluteus maximus at the midpoint between the ischial tuberosity and the greater trochanter. At the lower margin of the quadratus femoris muscle, the sciatic nerve enters the posterior thigh. The sciatic nerve is the largest nerve in the body and innervates all muscles in the posterior compartment of the thigh that flex the knee and all muscles that work the ankle and foot. It also innervates a large area of skin in the lower limb. Nerve to quadratus femoris

1	Nerve to quadratus femoris The nerve to the quadratus femoris enters the gluteal region through the greater sciatic foramen inferior to the piriformis muscle and deep to the sciatic nerve (Fig. 6.48). Unlike other nerves in the gluteal region, the nerve to the quadratus femoris lies anterior to the plane of the deep muscles. The nerve to the quadratus femoris descends along the ischium deep to the tendon of the obturator internus muscle and associated gemellus muscles to penetrate and innervate the quadratus femoris. It supplies a small branch to the gemellus inferior. Nerve to obturator internus

1	Nerve to obturator internus The nerve to the obturator internus enters the gluteal region through the greater sciatic foramen inferior to the piriformis muscle and between the posterior cutaneous nerve of the thigh and the pudendal nerve (Fig. 6.48). It supplies a small branch to the gemellus superior and then passes over the ischial spine and through the lesser sciatic foramen to innervate the obturator internus muscle from the medial surface of the muscle in the perineum. Posterior cutaneous nerve of the thigh The posterior cutaneous nerve of the thigh enters the gluteal region through the greater sciatic foramen inferior to the piriformis muscle and immediately medial to the sciatic nerve (Fig. 6.48). It descends through the gluteal region just deep to the gluteus maximus and enters the posterior thigh.

1	The posterior cutaneous nerve of the thigh has a number of gluteal branches, which loop around the lower margin of the gluteus maximus muscle to innervate skin over the gluteal fold. A small perineal branch passes medially to contribute to the innervation of the skin of the scrotum or labia majora in the perineum. The main trunk of the posterior cutaneous nerve of the thigh passes inferiorly, giving rise to branches that innervate the skin on the posterior thigh and leg. The pudendal nerve enters the gluteal region through the greater sciatic foramen inferior to the piriformis muscle and medial to the sciatic nerve (Fig. 6.48). It passes over the sacrospinous ligament and immediately passes through the lesser sciatic foramen to enter the perineum. The course of the pudendal nerve in the gluteal region is short and the nerve is often hidden by the overlying upper margin of the sacrotuberous ligament.

1	The pudendal nerve is the major somatic nerve of the perineum and has no branches in the gluteal region. The inferior gluteal nerve enters the gluteal region through the greater sciatic foramen inferior to the piriformis muscle and along the posterior surface of the sciatic nerve (Fig. 6.48). It penetrates and supplies the gluteus maximus muscle. The perforating cutaneous nerve is the only nerve in the gluteal region that does not enter the area through the greater sciatic foramen. It is a small nerve that leaves the sacral plexus in the pelvic cavity by piercing the sacrotuberous ligament. It then loops around the lower border of the gluteus maximus to supply the skin over the medial aspect of the gluteus maximus (Fig. 6.48).

1	Two arteries enter the gluteal region from the pelvic cavity through the greater sciatic foramen, the inferior gluteal artery and the superior gluteal artery (Fig. 6.50). They supply structures in the gluteal region and posterior thigh and have important collateral anastomoses with branches of the femoral artery. The inferior gluteal artery originates from the anterior trunk of the internal iliac artery in the pelvic cavity. It leaves the pelvic cavity with the inferior gluteal nerve through the greater sciatic foramen inferior to the piriformis muscle (Fig. 6.50). The inferior gluteal artery supplies adjacent muscles and descends through the gluteal region and into the posterior thigh where it supplies adjacent structures and anastomoses with perforating branches of the femoral artery. It also supplies a branch to the sciatic nerve.

1	The superior gluteal artery originates from the posterior trunk of the internal iliac artery in the pelvic cavity. It leaves the pelvic cavity with the superior gluteal nerve through the greater sciatic foramen above the piriformis muscle (Fig. 6.50). In the gluteal region, it divides into a superficial branch and a deep branch: The superficial branch passes onto the deep surface of the gluteus maximus muscle. The deep branch passes between the gluteus medius and minimus muscles. In addition to adjacent muscles, the superior gluteal artery contributes to the supply of the hip joint. Branches of the artery also anastomose with the lateral and medial femoral circumflex arteries from the deep femoral artery in the thigh, and with the inferior gluteal artery (Fig. 6.51).

1	Inferior and superior gluteal veins follow the inferior and superior gluteal arteries into the pelvis where they join the pelvic plexus of veins. Peripherally, the veins anastomose with superficial gluteal veins, which ultimately drain anteriorly into the femoral vein. Deep lymphatic vessels of the gluteal region accompany the blood vessels into the pelvic cavity and connect with internal iliac nodes. Superficial lymphatics drain into the superficial inguinal nodes on the anterior aspect of the thigh. The thigh is the region of the lower limb that is approximately between the hip and knee joints (Fig. 6.52): Anteriorly, it is separated from the abdominal wall by the inguinal ligament. Posteriorly, it is separated from the gluteal region by the gluteal fold superficially, and by the inferior margins of the gluteus maximus and quadratus femoris on deeper planes. Structures enter and leave the top of the thigh by three routes:

1	Structures enter and leave the top of the thigh by three routes: Posteriorly, the thigh is continuous with the gluteal region and the major structure passing between the two regions is the sciatic nerve. Anteriorly, the thigh communicates with the abdominal cavity through the aperture between the inguinal ligament and pelvic bone, and major structures passing through this aperture are the iliopsoas and pectineus muscles; the femoral nerve, artery, and vein; and lymphatic vessels. Medially, structures (including the obturator nerve and associated vessels) pass between the thigh and pelvic cavity through the obturator canal. The thigh is divided into three compartments by intermuscular septa between the posterior aspect of the femur and the fascia lata (the thick layer of deep fascia that completely surrounds or invests the thigh; Fig. 6.52C): The anterior compartment of the thigh contains muscles that mainly extend the leg at the knee joint.

1	The anterior compartment of the thigh contains muscles that mainly extend the leg at the knee joint. The posterior compartment of the thigh contains muscles that mainly extend the thigh at the hip joint and flex the leg at the knee joint. The medial compartment of the thigh consists of muscles that mainly adduct the thigh at the hip joint. The sciatic nerve innervates muscles in the posterior compartment of the thigh, the femoral nerve innervates muscles in the anterior compartment of the thigh, and the obturator nerve innervates most muscles in the medial compartment of the thigh. The major artery, vein, and lymphatic channels enter the thigh anterior to the pelvic bone and pass through the femoral triangle inferior to the inguinal ligament. Vessels and nerves passing between the thigh and leg pass through the popliteal fossa posterior to the knee joint.

1	The skeletal support for the thigh is the femur. Most of the large muscles in the thigh insert into the proximal ends of the two bones of the leg (tibia and fibula) and flex and extend the leg at the knee joint. The distal end of the femur provides origin for the gastrocnemius muscles, which are predominantly in the posterior compartment of the leg and plantarflex the foot. Shaft and distal end of femur The shaft of the femur is bowed forward and has an oblique course from the neck of the femur to the distal end (Fig. 6.53). As a consequence of this oblique orientation, the knee is close to the midline under the body’s center of gravity.

1	The middle part of the shaft of the femur is triangular in cross section (Fig. 6.53D). In the middle part of the shaft, the femur has smooth medial (posteromedial), lateral (posterolateral), and anterior surfaces and medial, lateral, and posterior borders. The medial and lateral borders are rounded, whereas the posterior border forms a broad roughened crest—the linea aspera. In proximal and distal regions of the femur, the linea aspera widens to form an additional posterior surface. At the distal end of the femur, this posterior surface forms the floor of the popliteal fossa, and its margins form the medial and lateral supracondylar lines. The medial supracondylar line terminates at a prominent tubercle (the adductor tubercle) on the superior aspect of the medial condyle of the distal end. Just lateral to the lower end of the medial supracondylar line is an elongate roughened area of bone for the proximal attachment of the medial head of the gastrocnemius muscle (Fig. 6.52).

1	The distal end of the femur is characterized by two large condyles, which articulate with the proximal head of the tibia. The condyles are separated posteriorly by an intercondylar fossa and are joined anteriorly where they articulate with the patella. The surfaces of the condyles that articulate with the tibia are rounded posteriorly and become flatter inferiorly. On each condyle, a shallow oblique groove separates the surface that articulates with the tibia from the more anterior surface that articulates with the patella. The surfaces of the medial and lateral condyles that articulate with the patella form a V-shaped trench, which faces anteriorly. The lateral surface of the trench is larger and steeper than the medial surface. The walls of the intercondylar fossa bear two facets for the superior attachment of the cruciate ligaments, which stabilize the knee joint (Fig. 6.53):

1	The walls of the intercondylar fossa bear two facets for the superior attachment of the cruciate ligaments, which stabilize the knee joint (Fig. 6.53): The wall formed by the lateral surface of the medial condyle has a large oval facet, which covers most of the inferior half of the wall, for attachment of the proximal end of the posterior cruciate ligament. The wall formed by the medial surface of the lateral condyle has a posterosuperior smaller oval facet for attachment of the proximal end of the anterior cruciate ligament. Epicondyles, for the attachment of collateral ligaments of the knee joint, are bony elevations on the nonarticular outer surfaces of the condyles (Fig. 6.53). Two facets separated by a groove are just posterior to the lateral epicondyle: The upper facet is for attachment of the lateral head of the gastrocnemius muscle. The inferior facet is for attachment of the popliteus muscle.

1	The upper facet is for attachment of the lateral head of the gastrocnemius muscle. The inferior facet is for attachment of the popliteus muscle. The tendon of the popliteus muscle lies in the groove separating the two facets. The medial epicondyle is a rounded eminence on the medial surface of the medial condyle. Just posterosuperior to the medial epicondyle is the adductor tubercle. The patella (knee cap) is the largest sesamoid bone (a bone formed within the tendon of a muscle) in the body and is formed within the tendon of the quadriceps femoris muscle as it crosses anterior to the knee joint to insert on the tibia. The patella is triangular: Its apex is pointed inferiorly for attachment to the patellar ligament, which connects the patella to the tibia (Fig. 6.54). Its base is broad and thick for the attachment of the quadriceps tendon from above.

1	Its base is broad and thick for the attachment of the quadriceps tendon from above. Its posterior surface articulates with the femur and has medial and lateral facets, which slope away from a raised smooth ridge—the lateral facet is larger than the medial facet for articulation with the larger corresponding surface on the lateral condyle of the femur. Proximal end of tibia The tibia is the medial and larger of the two bones in the leg, and is the only one that articulates with the femur at the knee joint. The proximal end of the tibia is expanded in the transverse plane for weight-bearing and consists of a medial condyle and a lateral condyle, which are both flattened in the horizontal plane and overhang the shaft (Fig. 6.55). The superior surfaces of the medial and lateral condyles are articular and separated by an intercondylar region, which contains sites of attachment for strong ligaments (cruciate ligaments) and interarticular cartilages (menisci) of the knee joint.

1	The articular surfaces of the medial and lateral condyles and the intercondylar region together form a “tibial plateau,” which articulates with and is anchored to the distal end of the femur. Inferior to the condyles on the proximal part of the shaft is a large tibial tuberosity and roughenings for muscle and ligament attachments. The tibial condyles are thick horizontal discs of bone attached to the top of the tibial shaft (Fig. 6.55). The medial condyle is larger than the lateral condyle and is better supported over the shaft of the tibia. Its superior surface is oval for articulation with the medial condyle of the femur. The articular surface extends laterally onto the side of the raised medial intercondylar tubercle. The superior surface of the lateral condyle is circular and articulates above with the lateral condyle of the femur. The medial edge of this surface extends onto the side of the lateral intercondylar tubercle.

1	The superior articular surfaces of both the lateral and medial condyles are concave, particularly centrally. The outer margins of the surfaces are flatter and are the regions in contact with the interarticular discs (menisci) of fibrocartilage in the knee joint. The nonarticular posterior surface of the medial condyle bears a distinct horizontal groove for part of the attachment of the semimembranosus muscle, and the undersurface of the lateral condyle bears a distinct circular facet for articulation with the proximal head of the fibula. The intercondylar region of the tibial plateau lies between the articular surfaces of the medial and lateral condyles (Fig. 6.55). It is narrow centrally where it is raised to form the intercondylar eminence, the sides of which are elevated further to form medial and lateral intercondylar tubercles.

1	The intercondylar region bears six distinct facets for the attachment of menisci and cruciate ligaments. The anterior intercondylar area widens anteriorly and bears three facets: The most anterior facet is for attachment of the anterior end (horn) of the medial meniscus. Immediately posterior to the most anterior facet is a facet for the attachment of the anterior cruciate ligament. A small facet for the attachment of the anterior end (horn) of the lateral meniscus is just lateral to the site of attachment of the anterior cruciate ligament. The posterior intercondylar area also bears three attachment facets: The most anterior is for attachment of the posterior horn of the lateral meniscus. Posteromedial to the most anterior facet is the site of attachment for the posterior horn of the medial meniscus. Behind the site of attachment for the posterior horn of the medial meniscus is a large facet for the attachment of the posterior cruciate ligament.

1	Behind the site of attachment for the posterior horn of the medial meniscus is a large facet for the attachment of the posterior cruciate ligament. In addition to these six sites of attachment for menisci and cruciate ligaments, a large anterolateral region of the anterior intercondylar area is roughened and perforated by numerous small nutrient foramina for blood vessels. This region is continuous with a similar surface on the front of the tibia above the tuberosity and lies against infrapatellar connective tissue. The tibial tuberosity is a palpable inverted triangular area on the anterior aspect of the tibia below the site of junction between the two condyles (Fig. 6.55). It is the site of attachment for the patellar ligament, which is a continuation of the quadriceps femoris tendon below the patella. Shaft of tibia

1	Shaft of tibia The shaft of the tibia is triangular in cross section and has three surfaces (posterior, medial, and lateral) and three borders (anterior, interosseous, and medial) (Fig. 6.55D): The anterior border is sharp and descends from the tibial tuberosity where it is continuous superiorly with a ridge that passes along the lateral margin of the tuberosity and onto the lateral condyle. The interosseous border is a subtle vertical ridge that descends along the lateral aspect of the tibia from the region of bone anterior and inferior to the articular facet for the head of the fibula. The medial border is indistinct superiorly where it begins at the anterior end of the groove on the posterior surface of the medial tibial condyle, but is sharp in midshaft.

1	The medial border is indistinct superiorly where it begins at the anterior end of the groove on the posterior surface of the medial tibial condyle, but is sharp in midshaft. The large medial surface of the shaft of the tibia, between the anterior and medial borders, is smooth and subcutaneous, and is palpable along almost its entire extent. Medial and somewhat inferior to the tibial tuberosity, this medial surface bears a subtle, slightly roughened elongate elevation. This elevation is the site of the combined attachment of three muscles (sartorius, gracilis, and semitendinosus), which descend from the thigh. The posterior surface of the shaft of the tibia, between the interosseous and medial borders, is widest superiorly where it is crossed by a roughened oblique line (the soleal line). The lateral surface, between the anterior and interosseous borders, is smooth and unremarkable. Proximal end of fibula

1	The lateral surface, between the anterior and interosseous borders, is smooth and unremarkable. Proximal end of fibula The fibula is the lateral bone of the leg and does not take part in formation of the knee joint or in weight-bearing. It is much smaller than the tibia and has a small proximal head, a narrow neck, and a delicate shaft, which ends as the lateral malleolus at the ankle. The head of the fibula is a globe-shaped expansion at the proximal end of the fibula (Fig. 6.56). A circular facet on the superomedial surface is for articulation above with a similar facet on the inferior aspect of the lateral condyle of the tibia. Just posterolateral to this facet, the bone projects superiorly as a blunt apex (styloid process). The lateral surface of the head of the fibula bears a large impression for the attachment of the biceps femoris muscle. A depression near the upper margin of this impression is for attachment of the fibular collateral ligament of the knee joint.

1	The neck of the fibula separates the expanded head from the shaft. The common fibular nerve lies against the posterolateral aspect of the neck. Like the tibia, the shaft of the fibula has three borders (anterior, posterior, and interosseous) and three surfaces (lateral, posterior, and medial), which lie between the borders (Fig. 6.56): The anterior border is sharp midshaft and begins superiorly from the anterior aspect of the head. The posterior border is rounded and descends from the region of the styloid process of the head. The interosseous border is medial in position. The three surfaces of the fibula are associated with the three muscular compartments (lateral, posterior, and anterior) of the leg. Muscles of the thigh are arranged in three compartments separated by intermuscular septa (Fig. 6.57).

1	Muscles of the thigh are arranged in three compartments separated by intermuscular septa (Fig. 6.57). The anterior compartment of the thigh contains the sartorius and the four large quadriceps femoris muscles (rectus femoris, vastus lateralis, vastus medialis, and vastus intermedius). All are innervated by the femoral nerve. In addition, the terminal ends of the psoas major and iliacus muscles pass into the upper part of the anterior compartment from sites of origin on the posterior abdominal wall. These muscles are innervated by branches directly from the anterior rami of L1 to L3 (psoas major) or from the femoral nerve (iliacus) as it passes down the abdominal wall.

1	The medial compartment of the thigh contains six muscles (gracilis, pectineus, adductor longus, adductor brevis, adductor magnus, and obturator externus). All except the pectineus, which is innervated by the femoral nerve, and part of the adductor magnus, which is innervated by the sciatic nerve, are innervated by the obturator nerve. The posterior compartment of the thigh contains three large muscles termed the “hamstrings.” All are innervated by the sciatic nerve. Muscles in the anterior compartment (Table 6.3) act on the hip and knee joints: the psoas major and iliacus act on the hip joint, the sartorius and rectus femoris act on both the hip and knee joints, and the vastus muscles act on the knee joint. The psoas major and iliacus muscles originate on the posterior abdominal wall and descend into the upper part of the anterior compartment of the thigh through the lateral half of the gap between the inguinal ligament and the pelvic bone (Fig. 6.58).

1	Although the iliacus and psoas major originate as separate muscles in the abdomen, both insert by a common tendon onto the lesser trochanter of the femur and together are usually referred to as the iliopsoas muscle. The iliopsoas is a powerful flexor of the thigh at the hip joint and can also contribute to lateral rotation of the thigh. The psoas major is innervated by branches from the anterior rami of L1 to L3 and the iliacus is innervated by branches from the femoral nerve in the abdomen. Quadriceps femoris—vastus medialis, intermedius, and lateralis and rectus femoris The large quadriceps femoris muscle consists of three vastus muscles (vastus medialis, vastus intermedius, and vastus lateralis) and the rectus femoris muscle (Fig. 6.59).

1	The large quadriceps femoris muscle consists of three vastus muscles (vastus medialis, vastus intermedius, and vastus lateralis) and the rectus femoris muscle (Fig. 6.59). The quadriceps femoris muscle mainly extends the leg at the knee joint, but the rectus femoris component also assists flexion of the thigh at the hip joint. Because the vastus muscles insert into the margins of the patella as well as into the quadriceps femoris tendon, they stabilize the position of the patella during knee joint movement. The quadriceps femoris is innervated by the femoral nerve with contributions mainly from spinal segments L3 and L4. A tap with a tendon hammer on the patellar ligament therefore tests reflex activity mainly at spinal cord levels L3 and L4. The vastus muscles originate from the femur, whereas the rectus femoris muscle originates from the pelvic bone. All attach first to the patella by the quadriceps femoris tendon and then to the tibia by the patellar ligament.

1	The vastus medialis originates from a continuous line of attachment on the femur, which begins anteromedially on the intertrochanteric line and continues posteroinferiorly along the pectineal line and then descends along the medial lip of the linea aspera and onto the medial supracondylar line. The fibers converge onto the medial aspect of the quadriceps femoris tendon and the medial border of the patella (Fig. 6.59). The vastus intermedius originates mainly from the upper two-thirds of the anterior and lateral surfaces of the femur and the adjacent intermuscular septum (Fig. 6.59). It merges into the deep aspect of the quadriceps femoris tendon and also attaches to the lateral margin of the patella and lateral condyle of the tibia.

1	A tiny muscle (articularis genus) originates from the femur just inferior to the origin of the vastus intermedius and inserts into the suprapatellar bursa associated with the knee joint (Fig. 6.59). This articular muscle, which is often part of the vastus intermedius muscle, pulls the bursa away from the knee joint during extension. The vastus lateralis is the largest of the vastus muscles (Fig. 6.59). It originates from a continuous line of attachment, which begins anterolaterally from the superior part of the intertrochanteric line of the femur and then circles laterally around the bone to attach to the lateral margin of the gluteal tuberosity and continues down the upper part of the lateral lip of the linea aspera. Muscle fibers converge mainly onto the quadriceps femoris tendon and the lateral margin of the patella. Unlike the vastus muscles, which cross only the knee joint, the rectus femoris muscle crosses both the hip and the knee joints (Fig. 6.59).

1	Unlike the vastus muscles, which cross only the knee joint, the rectus femoris muscle crosses both the hip and the knee joints (Fig. 6.59). The rectus femoris has two tendinous heads of origin from the pelvic bone: one from the anterior inferior iliac spine (straight head), and the other from a roughened area of the ilium immediately superior to the acetabulum (reflected head) (Fig. 6.59). The two heads of the rectus femoris unite to form an elongate muscle belly, which lies anterior to the vastus intermedius muscle and between the vastus lateralis and vastus medialis muscles, to which it is attached on either side. At the distal end, the rectus femoris muscle converges on the quadriceps femoris tendon and inserts on the base of the patella.

1	The patellar ligament is functionally the continuation of the quadriceps femoris tendon below the patella and is attached above to the apex and margins of the patella and below to the tibial tuberosity (Fig. 6.59). The more superficial fibers of the quadriceps femoris tendon and the patellar ligament are continuous over the anterior surface of the patella, and lateral and medial fibers are continuous with the ligament beside the margins of the patella. The sartorius muscle is the most superficial muscle in the anterior compartment of the thigh and is a long strap-like muscle that descends obliquely through the thigh from the anterior superior iliac spine to the medial surface of the proximal shaft of the tibia (Fig. 6.59). Its flat aponeurotic insertion into the tibia is immediately anterior to the insertion of the gracilis and semitendinosus muscles.

1	The sartorius, gracilis, and semitendinosus muscles attach to the tibia in a three-pronged pattern on the tibia, so their combined tendons of insertion are often termed the pes anserinus (Latin for “goose foot”). In the upper one-third of the thigh, the medial margin of the sartorius forms the lateral margin of the femoral triangle. In the middle one-third of the thigh, the sartorius forms the anterior wall of the adductor canal. The sartorius muscle assists in flexing the thigh at the hip joint and the leg at the knee joint. It also abducts the thigh and rotates it laterally, as when resting the foot on the opposite knee when sitting. The sartorius is innervated by the femoral nerve.

1	The sartorius is innervated by the femoral nerve. There are six muscles in the medial compartment of the thigh (Table 6.4): gracilis, pectineus, adductor longus, adductor brevis, adductor magnus, and obturator externus (Fig. 6.60). Collectively, all these muscles except the obturator externus mainly adduct the thigh at the hip joint; the adductor muscles may also medially rotate the thigh. Obturator externus is a lateral rotator of the thigh at the hip joint. The gracilis is the most superficial of the muscles in the medial compartment of thigh and descends almost vertically down the medial side of the thigh (Fig. 6.60). It is attached above to the outer surface of the ischiopubic ramus of the pelvic bone and below to the medial surface of the proximal shaft of the tibia, where it lies sandwiched between the tendon of sartorius in front and the tendon of the semitendinosus behind.

1	The pectineus is a flat quadrangular muscle (Fig. 6.61). It is attached above to the pectineal line of the pelvic bone and adjacent bone, and descends laterally to attach to an oblique line extending from the base of the lesser trochanter to the linea aspera on the posterior surface of the proximal femur. From its origin on the pelvic bone, the pectineus passes into the thigh below the inguinal ligament and forms part of the floor of the medial half of the femoral triangle. The pectineus adducts and flexes the thigh at the hip joint and is innervated by the femoral nerve. The adductor longus is a flat fan-shaped muscle that originates from a small rough triangular area on the external surface of the body of the pubis just inferior to the pubic crest and lateral to the pubic symphysis (Fig. 6.61). It expands as it descends posterolaterally to insert via an aponeurosis into the middle third of the linea aspera.

1	The adductor longus contributes to the floor of the femoral triangle, and its medial margin forms the medial border of the femoral triangle. The muscle also forms the proximal posterior wall of the adductor canal. The adductor longus adducts and medially rotates the thigh at the hip joint and is innervated by the anterior division of the obturator nerve. The adductor brevis lies posterior to the pectineus and adductor longus. It is a triangular muscle attached at its apex to the body of the pubis and inferior pubic ramus just superior to the origin of the gracilis muscle (Fig. 6.61). The muscle is attached by its expanded base via an aponeurosis to a vertical line extending from lateral to the insertion of the pectineus into the upper aspect of the linea aspera lateral to the attachment of the adductor longus. The adductor brevis adducts and medially rotates the thigh at the hip joint and is innervated by the obturator nerve.

1	The adductor brevis adducts and medially rotates the thigh at the hip joint and is innervated by the obturator nerve. The adductor magnus is the largest and deepest of the muscles in the medial compartment of the thigh (Fig. 6.62). The muscle forms the distal posterior wall of the adductor canal. Like the adductor longus and brevis muscles, the adductor magnus is a triangular or fan-shaped muscle anchored by its apex to the pelvis and attached by its expanded base to the femur.

1	On the pelvis, the adductor magnus is attached along a line that extends from the inferior pubic ramus, above the attachments of the adductor longus and brevis muscles, and along the ramus of the ischium to the ischial tuberosity. The part of the muscle that originates from the ischiopubic ramus expands laterally and inferiorly to insert on the femur along a vertical line of attachment that extends from just inferior to the quadrate tubercle and medial to the gluteal tuberosity, along the linea aspera and onto the medial supracondylar line. This lateral part of the muscle is often termed the “adductor part” of the adductor magnus.

1	The medial part of the adductor magnus, often called the “hamstring part,” originates from the ischial tuberosity of the pelvic bone and descends almost vertically along the thigh to insert via a rounded tendon into the adductor tubercle on the medial condyle of the distal head of the femur. It also inserts via an aponeurosis up onto the medial supracondylar line. A large circular gap inferiorly between the hamstring and adductor parts of the muscle is the adductor hiatus (Fig. 6.62), which allows the femoral artery and associated veins to pass between the adductor canal on the anteromedial aspect of the thigh and the popliteal fossa posterior to the knee. The adductor magnus adducts and medially rotates the thigh at the hip joint. The adductor part of the muscle is innervated by the obturator nerve and the hamstring part is innervated by the tibial division of the sciatic nerve.

1	The obturator externus is a flat fan-shaped muscle. Its expansive body is attached to the external aspect of the obturator membrane and adjacent bone (Fig. 6.62). The muscle fibers converge posterolaterally to form a tendon, which passes posterior to the hip joint and neck of the femur to insert on an oval depression on the lateral wall of the trochanteric fossa. The obturator externus externally rotates the thigh at the hip joint and is innervated by the posterior branch of the obturator nerve. There are three long muscles in the posterior compartment of the thigh: biceps femoris, semitendinosus, and semimembranosus (Table 6.5)—and they are collectively known as the hamstrings (Fig. 6.63). All except the short head of the biceps femoris cross both the hip and knee joints. As a group, the hamstrings flex the leg at the knee joint and extend the thigh at the hip joint. They are also rotators at both joints.

1	The biceps femoris muscle is lateral in the posterior compartment of the thigh and has two heads (Fig. 6.63): The long head originates with the semitendinosus muscle from the inferomedial part of the upper area of the ischial tuberosity. The short head arises from the lateral lip of the linea aspera on the shaft of the femur. The muscle belly of the long head crosses the posterior thigh obliquely from medial to lateral and is joined by the short head distally. Together, fibers from the two heads form a tendon, which is palpable on the lateral side of the distal thigh. The main part of the tendon inserts into the lateral surface of the head of the fibula. Extensions from the tendon blend with the fibular collateral ligament and with ligaments associated with the lateral side of the knee joint.

1	The biceps femoris flexes the leg at the knee joint. The long head also extends and laterally rotates the hip. When the knee is partly flexed, the biceps femoris can laterally rotate the leg at the knee joint. The long head is innervated by the tibial division of the sciatic nerve and the short head is innervated by the common fibular division of the sciatic nerve. The semitendinosus muscle is medial to the biceps femoris muscle in the posterior compartment of the thigh (Fig. 6.63). It originates with the long head of the biceps femoris muscle from the inferomedial part of the upper area of the ischial tuberosity. The spindle-shaped muscle belly ends in the lower half of the thigh and forms a long cord-like tendon, which lies on the semimembranosus muscle and descends to the knee. The tendon curves around the medial condyle of the tibia and inserts into the medial surface of the tibia just posterior to the tendons of the gracilis and sartorius muscles as part of the pes anserinus.

1	The semitendinosus flexes the leg at the knee joint and extends the thigh at the hip joint. Working with the semimembranosus, it also medially rotates the thigh at the hip joint and medially rotates the leg at the knee joint. The semitendinosus muscle is innervated by the tibial division of the sciatic nerve. The semimembranosus muscle lies deep to the semitendinosus muscle in the posterior compartment of the thigh (Fig. 6.63). It is attached above to the superolateral impression on the ischial tuberosity and below mainly to the groove and adjacent bone on the medial and posterior surfaces of the medial tibial condyle. Expansions from the tendon also insert into and contribute to the formation of ligaments and fascia around the knee joint. The semimembranosus flexes the leg at the knee joint and extends the thigh at the hip joint. Working with the semitendinosus muscle, it medially rotates the thigh at the hip joint and the leg at the knee joint.

1	The semimembranosus muscle is innervated by the tibial division of the sciatic nerve. Three arteries enter the thigh: the femoral artery, the obturator artery, and the inferior gluteal artery. Of these, the femoral artery is the largest and supplies most of the lower limb. The three arteries contribute to an anastomotic network of vessels around the hip joint. The femoral artery is the continuation of the external iliac artery and begins as the external iliac artery passes under the inguinal ligament to enter the femoral triangle on the anterior aspect of the upper thigh (Fig. 6.65). The femoral artery is palpable in the femoral triangle just inferior to the inguinal ligament midway between the anterior superior iliac spine and the pubic symphysis.

1	The femoral artery passes vertically through the femoral triangle and then continues down the thigh in the adductor canal. It leaves the canal by passing through the adductor hiatus in the adductor magnus muscle and becomes the popliteal artery behind the knee. A cluster of four small branches—superficial epigastric artery, superficial circumflex iliac artery, superficial external pudendal artery, and deep external pudendal artery—originate from the femoral artery in the femoral triangle and supply cutaneous regions of the upper thigh, lower abdomen, and perineum. Deep artery of thigh

1	Deep artery of thigh The largest branch of the femoral artery in the thigh is the deep artery of the thigh (profunda femoris artery), which originates from the lateral side of the femoral artery in the femoral triangle and is the major source of blood supply to the thigh (Fig. 6.65). The deep artery of the thigh immediately passes: posteriorly between the pectineus and adductor longus muscles and then between the adductor longus and adductor brevis muscles, and then travels inferiorly between the adductor longus and adductor magnus, eventually penetrating through the adductor magnus to connect with branches of the popliteal artery behind the knee. The deep artery of the thigh has lateral and medial circumflex femoral branches and three perforating branches.

1	The deep artery of the thigh has lateral and medial circumflex femoral branches and three perforating branches. The lateral circumflex femoral artery normally originates proximally from the lateral side of the deep artery of the thigh, but may arise directly from the femoral artery (Fig. 6.66). It passes deep to the sartorius and rectus femoris and divides into three terminal branches: One vessel (ascending branch) ascends laterally deep to the tensor fasciae latae muscle and connects with a branch of the medial circumflex femoral artery to form a channel, which circles the neck of the femur and supplies the neck and head of the femur. One vessel (descending branch) descends deep to the rectus femoris, penetrates the vastus lateralis muscle, and connects with a branch of the popliteal artery near the knee.

1	One vessel (descending branch) descends deep to the rectus femoris, penetrates the vastus lateralis muscle, and connects with a branch of the popliteal artery near the knee. One vessel (transverse branch) passes laterally to pierce the vastus lateralis and then circles around the proximal shaft of the femur to anastomose with branches from the medial femoral circumflex artery, the inferior gluteal artery, and the first perforating artery to form the cruciate anastomosis around the hip.

1	The medial circumflex femoral artery normally originates proximally from the posteromedial aspect of the deep artery of the thigh, but may originate from the femoral artery (Fig. 6.66). It passes medially around the shaft of the femur, first between the pectineus and iliopsoas and then between the obturator externus and adductor brevis muscles. Near the margin of the adductor brevis the vessel gives off a small branch, which enters the hip joint through the acetabular notch and anastomoses with the acetabular branch of the obturator artery. The main trunk of the medial circumflex femoral artery passes over the superior margin of the adductor magnus and divides into two major branches deep to the quadratus femoris muscle: One branch ascends to the trochanteric fossa and connects with branches of the gluteal and lateral circumflex femoral arteries.

1	One branch ascends to the trochanteric fossa and connects with branches of the gluteal and lateral circumflex femoral arteries. The other branch passes laterally to participate with branches from the lateral circumflex femoral artery, the inferior gluteal artery, and the first perforating artery in forming an anastomotic network of vessels around the hip.

1	The three perforating arteries branch from the deep artery of the thigh (Fig. 6.66) as it descends anterior to the adductor brevis muscle—the first originates above the muscle, the second originates anterior to the muscle, and the third originates below the muscle. All three penetrate through the adductor magnus near its attachment to the linea aspera to enter and supply the posterior compartment of the thigh. Here, the vessels have ascending and descending branches, which interconnect to form a longitudinal channel, which participates above in forming an anastomotic network of vessels around the hip and inferiorly anastomoses with branches of the popliteal artery behind the knee.

1	The obturator artery originates as a branch of the internal iliac artery in the pelvic cavity and enters the medial compartment of the thigh through the obturator canal (Fig. 6.67). As it passes through the canal, it bifurcates into an anterior branch and a posterior branch, which together form a channel that circles the margin of the obturator membrane and lies within the attachment of the obturator externus muscle. Vessels arising from the anterior and posterior branches supply adjacent muscles and anastomose with the inferior gluteal and medial circumflex femoral arteries. In addition, an acetabular vessel originates from the posterior branch, enters the hip joint through the acetabular notch, and contributes to the supply of the head of the femur.

1	Veins in the thigh consist of superficial and deep veins. Deep veins generally follow the arteries and have similar names. Superficial veins are in the superficial fascia, interconnect with deep veins, and do not generally accompany arteries. The largest of the superficial veins in the thigh is the great saphenous vein. The great saphenous vein originates from a venous arch on the dorsal aspect of the foot and ascends along the medial side of the lower limb to the proximal thigh (see p. 560). Here it passes through the saphenous ring in deep fascia covering the anterior thigh to connect with the femoral vein in the femoral triangle (see p. 566). There are three major nerves in the thigh, each associated with one of the three compartments. The femoral nerve is associated with the anterior compartment of the thigh, the obturator nerve is associated with the medial compartment of the thigh, and the sciatic nerve is associated with the posterior compartment of the thigh.

1	The femoral nerve originates from the lumbar plexus (spinal cord segments L2–L4) on the posterior abdominal wall and enters the femoral triangle of the thigh by passing under the inguinal ligament (Fig. 6.68). In the femoral triangle the femoral nerve lies on the lateral side of the femoral artery and is outside the femoral sheath, which surrounds the vessels. Before entering the thigh, the femoral nerve supplies branches to the iliacus and pectineus muscles. Immediately after passing under the inguinal ligament, the femoral nerve divides into anterior and posterior branches, which supply muscles of the anterior compartment of the thigh and skin on the anterior and medial aspects of the thigh and on the medial sides of the leg and foot.

1	Branches of the femoral nerve (Fig. 6.68) include: anterior cutaneous branches, which penetrate deep fascia to supply skin on the front of the thigh and knee; numerous motor nerves, which supply the quadriceps femoris muscles (rectus femoris, vastus lateralis, vastus intermedius, and vastus medialis muscles) and the sartorius muscle; and one long cutaneous nerve, the saphenous nerve, which supplies skin as far distally as the medial side of the foot. The saphenous nerve accompanies the femoral artery through the adductor canal, but does not pass through the adductor hiatus with the femoral artery. Rather, the saphenous nerve penetrates directly through connective tissues near the end of the canal to appear between the sartorius and gracilis muscles on the medial side of the knee. Here the saphenous nerve penetrates deep fascia and continues down the medial side of the leg to the foot, and supplies skin on the medial side of the knee, leg, and foot.

1	The obturator nerve is a branch of the lumbar plexus (spinal cord segments L2–L4) on the posterior abdominal wall. It descends in the psoas muscle, and then passes out of the medial margin of the psoas muscle to enter the pelvis (Fig. 6.69). The obturator nerve continues along the lateral pelvic wall and then enters the medial compartment of the thigh by passing through the obturator canal. It supplies most of the adductor muscles and skin on the medial aspect of the thigh. As the obturator nerve enters the thigh, it divides into two branches, an anterior branch and a posterior branch, which are separated by the adductor brevis muscle: The posterior branch descends behind the adductor brevis muscle and on the anterior surface of the adductor magnus muscle, and supplies the obturator externus and adductor brevis muscles and the part of the adductor magnus that attaches to the linea aspera.

1	The anterior branch descends on the anterior surface of the adductor brevis muscle and is behind the pectineus and adductor longus muscles—it supplies branches to the adductor longus, gracilis, and adductor brevis muscles, and often contributes to the supply of the pectineus muscle, and cutaneous branches innervate the skin on the medial side of the thigh. The sciatic nerve is a branch of the lumbosacral plexus (spinal cord segments L4–S3) and descends into the posterior compartment of the thigh from the gluteal region (Fig. 6.70). It innervates all muscles in the posterior compartment of the thigh and then its branches continue into the leg and foot. In the posterior compartment of the thigh, the sciatic nerve lies on the adductor magnus muscle and is crossed by the long head of the biceps femoris muscle.

1	In the posterior compartment of the thigh, the sciatic nerve lies on the adductor magnus muscle and is crossed by the long head of the biceps femoris muscle. Proximal to the knee, and sometimes within the pelvis, the sciatic nerve divides into its two terminal branches: the tibial nerve and the common fibular nerve. These nerves travel vertically down the thigh and enter the popliteal fossa posterior to the knee. Here, they meet the popliteal artery and vein. The tibial part of the sciatic nerve, either before or after its separation from the common fibular nerve, supplies branches to all muscles in the posterior compartment of the thigh (long head of biceps femoris, semimembranosus, semitendinosus) except the short head of the biceps femoris, which is innervated by the common fibular part (Fig. 6.70). The tibial nerve descends through the popliteal fossa, enters the posterior compartment of the leg, and continues into the sole of the foot.

1	The tibial nerve descends through the popliteal fossa, enters the posterior compartment of the leg, and continues into the sole of the foot. The tibial nerve innervates: all muscles in the posterior compartment of the leg, all intrinsic muscles in the sole of the foot including the first two dorsal interossei muscles, which also may receive innervation from the deep fibular nerve, and skin on the posterolateral side of the lower half of the leg and lateral side of the ankle, foot, and little toe, and skin on the sole of the foot and toes. The common fibular part of the sciatic nerve innervates the short head of the biceps femoris in the posterior compartment of the thigh and then continues into the lateral and anterior compartments of the leg and onto the foot (Fig. 6.70).

1	The common fibular nerve innervates: all muscles in the anterior and lateral compartments of the leg, one muscle (extensor digitorum brevis) on the dorsal aspect of the foot, the first two dorsal interossei muscles in the sole of the foot, and skin over the lateral aspect of the leg, and ankle, and over the dorsal aspect of the foot and toes. The knee joint is the largest synovial joint in the body. It consists of: the articulation between the femur and tibia, which is weight-bearing, and the articulation between the patella and the femur, which allows the pull of the quadriceps femoris muscle to be directed anteriorly over the knee to the tibia without tendon wear (Fig. 6.71). Two fibrocartilaginous menisci, one on each side, between the femoral condyles and tibia accommodate changes in the shape of the articular surfaces during joint movements.

1	Two fibrocartilaginous menisci, one on each side, between the femoral condyles and tibia accommodate changes in the shape of the articular surfaces during joint movements. The detailed movements of the knee joint are complex, but basically the joint is a hinge joint that allows mainly flexion and extension. Like all hinge joints, the knee joint is reinforced by collateral ligaments, one on each side of the joint. In addition, two very strong ligaments (the cruciate ligaments) interconnect the adjacent ends of the femur and tibia and maintain their opposed positions during movement. Because the knee joint is involved in weight-bearing, it has an efficient “locking” mechanism to reduce the amount of muscle energy required to keep the joint extended when standing.

1	Because the knee joint is involved in weight-bearing, it has an efficient “locking” mechanism to reduce the amount of muscle energy required to keep the joint extended when standing. The articular surfaces of the bones that contribute to the knee joint are covered by hyaline cartilage. The major surfaces involved include: the two femoral condyles, and the adjacent surfaces of the superior aspect of the tibial condyles. The surfaces of the femoral condyles that articulate with the tibia in flexion of the knee are curved or round, whereas the surfaces that articulate in full extension are flat (Fig. 6.72). The articular surfaces between the femur and patella are the V-shaped trench on the anterior surface of the distal end of the femur where the two condyles join and the adjacent surfaces on the posterior aspect of the patella. The joint surfaces are all enclosed within a single articular cavity, as are the intraarticular menisci between the femoral and tibial condyles.

1	There are two menisci, which are fibrocartilaginous C-shaped cartilages, in the knee joint, one medial (medial meniscus) and the other lateral (lateral meniscus) (Fig. 6.73). Both are attached at each end to facets in the intercondylar region of the tibial plateau. The medial meniscus is attached around its margin to the capsule of the joint and to the tibial collateral ligament, whereas the lateral meniscus is unattached to the capsule. Therefore, the lateral meniscus is more mobile than the medial meniscus. The menisci are interconnected anteriorly by a transverse ligament of the knee. The lateral meniscus is also connected to the tendon of the popliteus muscle, which passes superolaterally between this meniscus and the capsule to insert on the femur.

1	The menisci improve congruency between the femoral and tibial condyles during joint movements where the surfaces of the femoral condyles articulating with the tibial plateau change from small curved surfaces in flexion to large flat surfaces in extension. The synovial membrane of the knee joint attaches to the margins of the articular surfaces and to the superior and inferior outer margins of the menisci (Fig. 6.75A). The two cruciate ligaments, which attach in the intercondylar region of the tibia below and the intercondylar fossa of the femur above, are outside the articular cavity, but enclosed within the fibrous membrane of the knee joint. Posteriorly, the synovial membrane reflects off the fibrous membrane of the joint capsule on either side of the posterior cruciate ligament and loops forward around both ligaments thereby excluding them from the articular cavity.

1	Anteriorly, the synovial membrane is separated from the patellar ligament by an infrapatellar fat pad. On each side of the pad, the synovial membrane forms a fringed margin (an alar fold), which projects into the articular cavity. In addition, the synovial membrane covering the lower part of the infrapatellar fat pad is raised into a sharp midline fold directed posteriorly (the infrapatellar synovial fold), which attaches to the margin of the intercondylar fossa of the femur. The synovial membrane of the knee joint forms pouches in two locations to provide low-friction surfaces for the movement of tendons associated with the joint: The smallest of these expansions is the subpopliteal recess (Fig. 6.75A), which extends posterolaterally from the articular cavity and lies between the lateral meniscus and the tendon of the popliteus muscle, which passes through the joint capsule.

1	The second expansion is the suprapatellar bursa (Fig. 6.75B), a large bursa that is a continuation of the articular cavity superiorly between the distal end of the shaft of the femur and the quadriceps femoris muscle and tendon—the apex of this bursa is attached to the small articularis genus muscle, which pulls the bursa away from the joint during extension of the knee. Other bursae associated with the knee but not normally communicating with the articular cavity include the subcutaneous prepatellar bursa, deep and subcutaneous infrapatellar bursae, and numerous other bursae associated with tendons and ligaments around the joint (Fig. 6.75B). The prepatellar bursa is subcutaneous and anterior to the patella. The deep and subcutaneous infrapatellar bursae are on the deep and subcutaneous sides of the patellar ligament, respectively.

1	The prepatellar bursa is subcutaneous and anterior to the patella. The deep and subcutaneous infrapatellar bursae are on the deep and subcutaneous sides of the patellar ligament, respectively. The fibrous membrane of the knee joint is extensive and is partly formed and reinforced by extensions from tendons of the surrounding muscles (Fig. 6.76). In general, the fibrous membrane encloses the articular cavity and the intercondylar region: On the medial side of the knee joint, the fibrous membrane blends with the tibial collateral ligament and is attached on its internal surface to the medial meniscus. Laterally, the external surface of the fibrous membrane is separated by a space from the fibular collateral ligament and the internal surface of the fibrous membrane is not attached to the lateral meniscus.

1	Anteriorly, the fibrous membrane is attached to the margins of the patella where it is reinforced with tendinous expansions from the vastus lateralis and vastus medialis muscles, which also merge above with the quadriceps femoris tendon and below with the patellar ligament. The fibrous membrane is reinforced anterolaterally by a fibrous extension from the iliotibial tract and posteromedially by an extension from the tendon of the semimembranosus (the oblique popliteal ligament), which reflects superiorly across the back of the fibrous membrane from medial to lateral. The upper end of the popliteus muscle passes through an aperture in the posterolateral aspect of the fibrous membrane of the knee and is enclosed by the fibrous membrane as its tendon travels around the joint to insert on the lateral aspect of the lateral femoral condyle.

1	The major ligaments associated with the knee joint are the patellar ligament, the tibial (medial) and fibular (lateral) collateral ligaments, and the anterior and posterior cruciate ligaments. The patellar ligament is basically the continuation of the quadriceps femoris tendon inferior to the patella (Fig. 6.76). It is attached above to the margins and apex of the patella and below to the tibial tuberosity. The collateral ligaments, one on each side of the joint, stabilize the hinge-like motion of the knee (Fig. 6.77). The cord-like fibular collateral ligament is attached superiorly to the lateral femoral epicondyle just above the groove for the popliteus tendon. Inferiorly, it is attached to a depression on the lateral surface of the fibular head. It is separated from the fibrous membrane by a bursa.

1	The broad and flat tibial collateral ligament is attached by much of its deep surface to the underlying fibrous membrane. It is anchored superiorly to the medial femoral epicondyle just inferior to the adductor tubercle and descends anteriorly to attach to the medial margin and medial surface of the tibia above and behind the attachment of the sartorius, gracilis, and semitendinosus tendons. The two cruciate ligaments are in the intercondylar region of the knee and interconnect the femur and tibia (Figs. 6.77D and 6.78). They are termed “cruciate” (Latin for “shaped like a cross”) because they cross each other in the sagittal plane between their femoral and tibial attachments: The anterior cruciate ligament attaches to a facet on the anterior part of the intercondylar area of the tibia and ascends posteriorly to attach to a facet at the back of the lateral wall of the intercondylar fossa of the femur.

1	The posterior cruciate ligament attaches to the posterior aspect of the intercondylar area of the tibia and ascends anteriorly to attach to the medial wall of the intercondylar fossa of the femur. The anterior cruciate ligament crosses lateral to the posterior cruciate ligament as they pass through the intercondylar region. The anterior cruciate ligament prevents anterior displacement of the tibia relative to the femur and the posterior cruciate ligament restricts posterior displacement (Fig. 6.78). When standing, the knee joint is locked into position, thereby reducing the amount of muscle work needed to maintain the standing position (Fig. 6.79). One component of the locking mechanism is a change in the shape and size of the femoral surfaces that articulate with the tibia: In flexion, the surfaces are the curved and rounded areas on the posterior aspects of the femoral condyles.

1	In flexion, the surfaces are the curved and rounded areas on the posterior aspects of the femoral condyles. As the knee is extended, the surfaces move to the broad and flat areas on the inferior aspects of the femoral condyles. Consequently the joint surfaces become larger and more stable in extension. Another component of the locking mechanism is medial rotation of the femur on the tibia during extension. Medial rotation and full extension tightens all the associated ligaments. Another feature that keeps the knee extended when standing is that the body’s center of gravity is positioned along a vertical line that passes anterior to the knee joint. The popliteus muscle unlocks the knee by initiating lateral rotation of the femur on the tibia.

1	The popliteus muscle unlocks the knee by initiating lateral rotation of the femur on the tibia. Vascular supply to the knee joint is predominantly through descending and genicular branches from the femoral, popliteal, and lateral circumflex femoral arteries in the thigh and the circumflex fibular artery and recurrent branches from the anterior tibial artery in the leg. These vessels form an anastomotic network around the joint (Fig. 6.80). The knee joint is innervated by branches from the obturator, femoral, tibial, and common fibular nerves. The small proximal tibiofibular joint is synovial in type and allows very little movement (Fig. 6.82). The opposing joint surfaces, on the undersurface of the lateral condyle of the tibia and on the superomedial surface of the head of the fibula, are flat and circular. The capsule is reinforced by anterior and posterior ligaments.

1	The popliteal fossa is an important area of transition between the thigh and leg and is the major route by which structures pass from one region to the other. The popliteal fossa is a diamond-shaped space behind the knee joint formed between muscles in the posterior compartments of the thigh and leg (Fig. 6.83A): The margins of the upper part of the diamond are formed medially by the distal ends of the semitendinosus and semimembranosus muscles and laterally by the distal end of the biceps femoris muscle. The margins of the smaller lower part of the space are formed medially by the medial head of the gastrocnemius muscle and laterally by the plantaris muscle and the lateral head of the gastrocnemius muscle. The floor of the fossa is formed by the capsule of the knee joint and adjacent surfaces of the femur and tibia, and, more inferiorly, by the popliteus muscle.

1	The floor of the fossa is formed by the capsule of the knee joint and adjacent surfaces of the femur and tibia, and, more inferiorly, by the popliteus muscle. The roof is formed by deep fascia, which is continuous above with the fascia lata of the thigh and below with deep fascia of the leg. The major contents of the popliteal fossa are the popliteal artery, the popliteal vein, and the tibial and common fibular nerves (Fig. 6.83B). The tibial and common fibular nerves originate proximal to the popliteal fossa as the two major branches of the sciatic nerve. They are the most superficial of the neurovascular structures in the popliteal fossa and enter the region directly from above under the margin of the biceps femoris muscle: The tibial nerve descends vertically through the popliteal fossa and exits deep to the margin of the plantaris muscle to enter the posterior compartment of the leg.

1	The tibial nerve descends vertically through the popliteal fossa and exits deep to the margin of the plantaris muscle to enter the posterior compartment of the leg. The common fibular nerve exits by following the biceps femoris tendon over the lower lateral margin of the popliteal fossa, and continues to the lateral side of the leg where it swings around the neck of the fibula and enters the lateral compartment of the leg. The popliteal artery is the continuation of the femoral artery in the anterior compartment of the thigh, and begins as the femoral artery passes posteriorly through the adductor hiatus in the adductor magnus muscle. The popliteal artery appears in the popliteal fossa on the upper medial side under the margin of the semimembranosus muscle. It descends obliquely through the fossa with the tibial nerve and enters the posterior compartment of the leg where it ends just lateral to the midline of the leg by dividing into the anterior and posterior tibial arteries.

1	The popliteal artery is the deepest of the neurovascular structures in the popliteal fossa and is therefore difficult to palpate; however, a pulse can usually be detected by deep palpation near the midline. In the popliteal fossa, the popliteal artery gives rise to branches, which supply adjacent muscles, and to a series of geniculate arteries, which contribute to vascular anastomoses around the knee. The popliteal vein is superficial to and travels with the popliteal artery. It exits the popliteal fossa superiorly to become the femoral vein by passing through the adductor hiatus. Roof of popliteal fossa

1	Roof of popliteal fossa The roof of the popliteal fossa is covered by superficial fascia and skin (Fig. 6.83C). The most important structure in the superficial fascia is the small saphenous vein. This vessel ascends vertically in the superficial fascia on the back of the leg from the lateral side of the dorsal venous arch in the foot. It ascends to the back of the knee where it penetrates deep fascia, which forms the roof of the popliteal fossa, and joins with the popliteal vein. One other structure that passes through the roof of the fossa is the posterior cutaneous nerve of the thigh, which descends through the thigh superficial to the hamstring muscles, passes through the roof of the popliteal fossa, and then continues inferiorly with the small saphenous vein to innervate skin on the upper half of the back of the leg. The leg is that part of the lower limb between the knee joint and ankle joint (Fig. 6.84):

1	The leg is that part of the lower limb between the knee joint and ankle joint (Fig. 6.84): Proximally, most major structures pass between the thigh and leg through or in relation to the popliteal fossa behind the knee. Distally, structures pass between the leg and foot mainly through the tarsal tunnel on the posteromedial side of the ankle, the exceptions being the anterior tibial artery and the ends of the deep and superficial fibular nerves, which enter the foot anterior to the ankle. The bony framework of the leg consists of two bones, the tibia and fibula, arranged in parallel. The fibula is much smaller than the tibia and is on the lateral side of the leg. It articulates superiorly with the inferior aspect of the lateral condyle of the proximal tibia, but does not take part in formation of the knee joint. The distal end of the fibula is firmly anchored to the tibia by a fibrous joint and forms the lateral malleolus of the ankle joint.

1	The tibia is the weight-bearing bone of the leg and is therefore much larger than the fibula. Above, it takes part in the formation of the knee joint and below it forms the medial malleolus and most of the bony surface for articulation of the leg with the foot at the ankle joint. The leg is divided into anterior (extensor), posterior (flexor), and lateral (fibular) compartments by: an interosseous membrane, which links adjacent borders of the tibia and fibula along most of their length; two intermuscular septa, which pass between the fibula and deep fascia surrounding the limb; and direct attachment of the deep fascia to the periosteum of the anterior and medial borders of the tibia (Fig. 6.84).

1	Muscles in the anterior compartment of the leg dorsiflex the ankle, extend the toes, and invert the foot. Muscles in the posterior compartment plantarflex the ankle, flex the toes, and invert the foot. Muscles in the lateral compartment evert the foot. Major nerves and vessels supply or pass through each compartment. Shaft and distal end of tibia The shaft of the tibia is triangular in cross section and has anterior, interosseous, and medial borders and medial, lateral, and posterior surfaces (Fig. 6.85): The anterior and medial borders and the entire medial surface are subcutaneous and easily palpable. The interosseous border of the tibia is connected, by the interosseous membrane, along its length to the interosseous border of the fibula. The posterior surface is marked by an oblique line (the soleal line).

1	The posterior surface is marked by an oblique line (the soleal line). The soleal line descends across the bone from the lateral side to the medial side where it merges with the medial border. In addition, a vertical line descends down the upper part of the posterior surface from the midpoint of the soleal line. It disappears in the lower one-third of the tibia. The shaft of the tibia expands at both the upper and lower ends to support the body’s weight at the knee and ankle joints. The distal end of the tibia is shaped like a rectangular box with a bony protuberance on the medial side (the medial malleolus; Fig. 6.81). The upper part of the box is continuous with the shaft of the tibia while the lower surface and the medial malleolus articulate with one of the tarsal bones (talus) to form a large part of the ankle joint.

1	The posterior surface of the box-like distal end of the tibia is marked by a vertical groove, which continues inferiorly and medially onto the posterior surface of the medial malleolus. The groove is for the tendon of the tibialis posterior muscle. The lateral surface of the distal end of the tibia is occupied by a deep triangular notch (the fibular notch), to which the distal head of the fibula is anchored by a thickened part of the interosseous membrane. Shaft and distal end of fibula The fibula is not involved in weight-bearing. The fibular shaft is therefore much narrower than the shaft of the tibia. Also, and except for the ends, the fibula is enclosed by muscles.

1	The fibula is not involved in weight-bearing. The fibular shaft is therefore much narrower than the shaft of the tibia. Also, and except for the ends, the fibula is enclosed by muscles. Like the tibia, the shaft of the fibula is triangular in cross section and has three borders and three surfaces for the attachment of muscles, intermuscular septa, and ligaments (Fig. 6.85). The interosseous border of the fibula faces and is attached to the interosseous border of the tibia by the interosseous membrane. Intermuscular septa attach to the anterior and posterior borders. Muscles attach to the three surfaces. The narrow medial surface faces the anterior compartment of the leg, the lateral surface faces the lateral compartment of the leg, and the posterior surface faces the posterior compartment of the leg. The posterior surface is marked by a vertical crest (medial crest), which divides the posterior surface into two parts each attached to a different deep flexor muscle.

1	The posterior surface is marked by a vertical crest (medial crest), which divides the posterior surface into two parts each attached to a different deep flexor muscle. The distal end of the fibula expands to form the spade-shaped lateral malleolus (Fig. 6.85). The medial surface of the lateral malleolus bears a facet for articulation with the lateral surface of the talus, thereby forming the lateral part of the ankle joint. Just superior to this articular facet is a triangular area, which fits into the fibular notch on the distal end of the tibia. Here the tibia and fibula are joined together by the distal end of the interosseous membrane. Posteroinferior to the facet for articulation with the talus is a pit or fossa (the malleolar fossa) for the attachment of the posterior talofibular ligament associated with the ankle joint. The posterior surface of the lateral malleolus is marked by a shallow groove for the tendons of the fibularis longus and fibularis brevis muscles.

1	The posterior surface of the lateral malleolus is marked by a shallow groove for the tendons of the fibularis longus and fibularis brevis muscles. Interosseous membrane of leg The interosseous membrane of the leg is a tough fibrous sheet of connective tissue that spans the distance between facing interosseous borders of the tibial and fibular shafts (Fig. 6.86). The collagen fibers descend obliquely from the interosseous border of the tibia to the interosseous border of the fibula, except superiorly where there is a ligamentous band, which ascends from the tibia to fibula. There are two apertures in the interosseous membrane, one at the top and the other at the bottom, for vessels to pass between the anterior and posterior compartments of the leg. The interosseous membrane not only links the tibia and fibula together, but also provides an increased surface area for muscle attachment.

1	The interosseous membrane not only links the tibia and fibula together, but also provides an increased surface area for muscle attachment. The distal ends of the fibula and tibia are held together by the inferior aspect of the interosseous membrane, which spans the narrow space between the fibular notch on the lateral surface of the distal end of the tibia and the corresponding surface on the distal end of the fibula. This expanded end of the interosseous membrane is reinforced by anterior and posterior tibiofibular ligaments. This firm linking together of the distal ends of the tibia and fibula is essential to produce the skeletal framework for articulation with the foot at the ankle joint. Posterior compartment of leg

1	Posterior compartment of leg Muscles in the posterior (flexor) compartment of the leg are organized into two groups, superficial and deep, separated by a layer of deep fascia. Generally, the muscles mainly plantarflex and invert the foot and flex the toes. All are innervated by the tibial nerve. The superficial group of muscles in the posterior compartment of the leg comprises three muscles—the gastrocnemius, plantaris, and soleus (Table 6.6)—all of which insert onto the heel (calcaneus) of the foot and plantarflex the foot at the ankle joint (Fig. 6.87). As a unit, these muscles are large and powerful because they propel the body forward off the planted foot during walking and can elevate the body upward onto the toes when standing. Two of the muscles (gastrocnemius and plantaris) originate on the distal end of the femur and can also flex the knee.

1	The gastrocnemius muscle is the most superficial of the muscles in the posterior compartment and is one of the largest muscles in the leg (Fig. 6.87). It originates from two heads, one lateral and one medial: The medial head is attached to an elongate roughening on the posterior aspect of the distal femur just behind the adductor tubercle and above the articular surface of the medial condyle. The lateral head originates from a distinct facet on the upper lateral surface of the lateral femoral condyle where it joins the lateral supracondylar line. At the knee, the facing margins of the two heads of the gastrocnemius form the lateral and medial borders of the lower end of the popliteal fossa. In the upper leg, the heads of the gastrocnemius combine to form a single elongate muscle belly, which forms much of the soft tissue bulge identified as the calf.

1	In the upper leg, the heads of the gastrocnemius combine to form a single elongate muscle belly, which forms much of the soft tissue bulge identified as the calf. In the lower leg, the muscle fibers of the gastrocnemius converge with those of the deeper soleus muscle to form the calcaneal tendon, which attaches to the calcaneus (heel) of the foot. The gastrocnemius plantarflexes the foot at the ankle joint and can also flex the leg at the knee joint. It is innervated by the tibial nerve. The plantaris has a small muscle belly proximally and a long thin tendon, which descends through the leg and joins the calcaneal tendon (Fig. 6.87). The muscle takes origin superiorly from the lower part of the lateral supracondylar ridge of the femur and from the oblique popliteal ligament associated with the knee joint.

1	The short spindle-shaped muscle body of the plantaris descends medially, deep to the lateral head of the gastrocnemius, and forms a thin tendon, which passes between the gastrocnemius and soleus muscles and eventually fuses with the medial side of the calcaneal tendon near its attachment to the calcaneus. The plantaris contributes to plantarflexion of the foot at the ankle joint and flexion of the leg at the knee joint, and is innervated by the tibial nerve. The soleus is a large flat muscle under the gastrocnemius muscle (Fig. 6.87). It is attached to the proximal ends of the fibula and tibia, and to a tendinous ligament, which spans the distance between the two heads of attachment to the fibula and tibia: On the proximal end of the fibula, the soleus originates from the posterior aspect of the head and adjacent surface of the neck and upper shaft of the fibula. On the tibia, the soleus originates from the soleal line and adjacent medial border.

1	On the tibia, the soleus originates from the soleal line and adjacent medial border. The ligament, which spans the distance between the attachments to the tibia and fibula, arches over the popliteal vessels and tibial nerve as they pass from the popliteal fossa into the deep region of the posterior compartment of the leg. In the lower leg, the soleus muscle narrows to join the calcaneal tendon that attaches to the calcaneus. The soleus muscle, together with the gastrocnemius and plantaris, plantarflexes the foot at the ankle joint. It is innervated by the tibial nerve. There are four muscles in the deep posterior compartment of the leg (Fig. 6.88)—the popliteus, flexor hallucis longus, flexor digitorum longus, and tibialis posterior (Table 6.7). The popliteus muscle acts on the knee, whereas the other three muscles act mainly on the foot.

1	The popliteus is the smallest and most superior of the deep muscles in the posterior compartment of the leg. It unlocks the extended knee at the initiation of flexion and stabilizes the knee by resisting lateral (external) rotation of the tibia on the femur. It is flat and triangular in shape, forms part of the floor of the popliteal fossa (Fig. 6.88), and is inserted into a broad triangular region above the soleal line on the posterior surface of the tibia. The popliteus muscle ascends laterally across the lower aspect of the knee and originates from a tendon, which penetrates the fibrous membrane of the joint capsule of the knee. The tendon ascends laterally around the joint where it passes between the lateral meniscus and the fibrous membrane and then into a groove on the inferolateral aspect of the lateral femoral condyle. The tendon attaches to and originates from a depression at the anterior end of the groove.

1	When initiating gait from a standing position, contraction of the popliteus laterally rotates the femur on the fixed tibia, unlocking the knee joint. The popliteus muscle is innervated by the tibial nerve. The flexor hallucis longus muscle originates on the lateral side of the posterior compartment of the leg and inserts into the plantar surface of the great toe on the medial side of the foot (Fig. 6.88). It arises mainly from the lower two-thirds of the posterior surface of the fibula and adjacent interosseous membrane.

1	The muscle fibers of the flexor hallucis longus converge inferiorly to form a large cord-like tendon, which passes behind the distal head of the tibia and then slips into a distinct groove on the posterior surface of the adjacent tarsal bone (talus) of the foot. The tendon curves anteriorly first under the talus and then under a shelf of bone (the sustentaculum tali), which projects medially from the calcaneus, and then continues anteriorly through the sole of the foot to insert on the inferior surface of the base of the distal phalanx of the great toe. The flexor hallucis longus flexes the great toe. It is particularly active during the toe-off phase of walking when the body is propelled forward off the stance leg and the great toe is the last part of the foot to leave the ground. It can also contribute to plantarflexion of the foot at the ankle joint and is innervated by the tibial nerve.

1	The flexor digitorum longus muscle originates on the medial side of the posterior compartment of the leg and inserts into the lateral four digits of the foot (Fig. 6.88). It arises mainly from the medial side of the posterior surface of the tibia inferior to the soleal line. The flexor digitorum longus descends in the leg and forms a tendon, which crosses posterior to the tendon of the tibialis posterior muscle near the ankle joint. The tendon continues inferiorly in a shallow groove behind the medial malleolus and then swings forward to enter the sole of the foot. It crosses inferior to the tendon of the flexor hallucis longus muscle to reach the medial side of the foot and then divides into four tendons, which insert on the plantar surfaces of the bases of the distal phalanges of digits II to V.

1	The flexor digitorum longus flexes the lateral four toes. It is involved with gripping the ground during walking and propelling the body forward off the toes at the end of the stance phase of gait. It is innervated by the tibial nerve. The tibialis posterior muscle originates from the interosseous membrane and the adjacent posterior surfaces of the tibia and fibula (Fig. 6.88). It lies between and is overlapped by the flexor digitorum longus and the flexor hallucis longus muscles.

1	Near the ankle, the tendon of the tibialis posterior is crossed superficially by the tendon of the flexor digitorum longus muscle and lies medial to this tendon in the groove on the posterior surface of the medial malleolus. The tendon curves forward under the medial malleolus and enters the medial side of the foot. It wraps around the medial margin of the foot to attach to the plantar surfaces of the medial tarsal bones, mainly to the tuberosity of the navicular and to the adjacent region of the medial cuneiform. The tibialis posterior inverts and plantarflexes the foot, and supports the medial arch of the foot during walking. It is innervated by the tibial nerve. The popliteal artery is the major blood supply to the leg and foot and enters the posterior compartment of the leg from the popliteal fossa behind the knee (Fig. 6.89).

1	The popliteal artery is the major blood supply to the leg and foot and enters the posterior compartment of the leg from the popliteal fossa behind the knee (Fig. 6.89). The popliteal artery passes into the posterior compartment of the leg between the gastrocnemius and popliteus muscles. As it continues inferiorly it passes under the tendinous arch formed between the fibular and tibial heads of the soleus muscle and enters the deep region of the posterior compartment of the leg where it immediately divides into an anterior tibial artery and a posterior tibial artery. Two large sural arteries, one on each side, branch from the popliteal artery to supply the gastrocnemius, soleus, and plantaris muscles (Fig. 6.89). In addition, the popliteal artery gives rise to branches that contribute to a collateral network of vessels around the knee joint (see Fig. 6.80).

1	The anterior tibial artery passes forward through the aperture in the upper part of the interosseous membrane and enters and supplies the anterior compartment of the leg. It continues inferiorly onto the dorsal aspect of the foot. The posterior tibial artery supplies the posterior and lateral compartments of the leg and continues into the sole of the foot (Fig. 6.89). The posterior tibial artery descends through the deep region of the posterior compartment of the leg on the superficial surfaces of the tibialis posterior and flexor digitorum longus muscles. It passes through the tarsal tunnel behind the medial malleolus and into the sole of the foot. In the leg, the posterior tibial artery supplies adjacent muscles and bone and has two major branches, the circumflex fibular artery and the fibular artery:

1	In the leg, the posterior tibial artery supplies adjacent muscles and bone and has two major branches, the circumflex fibular artery and the fibular artery: The circumflex fibular artery passes laterally through the soleus muscle and around the neck of the fibula to connect with the anastomotic network of vessels surrounding the knee (Fig. 6.89; see also Fig. 6.80). The fibular artery parallels the course of the tibial artery, but descends along the lateral side of the posterior compartment adjacent to the medial crest on the posterior surface of the fibula, which separates the attachments of the tibialis posterior and flexor hallucis longus muscles. The fibular artery supplies adjacent muscles and bone in the posterior compartment of the leg and also has branches that pass laterally through the intermuscular septum to supply the fibularis muscles in the lateral compartment of the leg.

1	A perforating branch that originates from the fibular artery distally in the leg passes anteriorly through the inferior aperture in the interosseous membrane to anastomose with a branch of the anterior tibial artery. The fibular artery passes behind the attachment between the distal ends of the tibia and fibula and terminates in a network of vessels over the lateral surface of the calcaneus. Deep veins in the posterior compartment generally follow the arteries. The nerve associated with the posterior compartment of the leg is the tibial nerve (Fig. 6.90), a major branch of the sciatic nerve that descends into the posterior compartment from the popliteal fossa. The tibial nerve passes under the tendinous arch formed between the fibular and tibial heads of the soleus muscle and passes vertically through the deep region of the posterior compartment of the leg on the surface of the tibialis posterior muscle with the posterior tibial vessels.

1	The tibial nerve leaves the posterior compartment of the leg at the ankle by passing through the tarsal tunnel behind the medial malleolus. It enters the foot to supply most intrinsic muscles and skin. In the leg, the tibial nerve gives rise to: branches that supply all the muscles in the posterior compartment of the leg, and two cutaneous branches, the sural nerve and medial calcaneal nerve. Branches of the tibial nerve that innervate the superficial group of muscles of the posterior compartment and popliteus muscle of the deep group originate high in the leg between the two heads of the gastrocnemius muscle in the distal region of the popliteal fossa (Fig. 6.91). Branches innervate the gastrocnemius, plantaris, and soleus muscles, and pass more deeply into the popliteus muscle.

1	Branches to the deep muscles of the posterior compartment originate from the tibial nerve deep to the soleus muscle in the upper half of the leg and innervate the tibialis posterior, flexor hallucis longus, and flexor digitorum longus muscles. The sural nerve originates high in the leg between the two heads of the gastrocnemius muscle (Fig. 6.90). It descends superficial to the belly of the gastrocnemius muscle and penetrates through the deep fascia approximately in the middle of the leg where it is joined by a sural communicating branch from the common fibular nerve. It passes down the leg, around the lateral malleolus, and into the foot. The sural nerve supplies skin on the lower posterolateral surface of the leg and the lateral side of the foot and little toe. The medial calcaneal nerve is often multiple and originates from the tibial nerve low in the leg near the ankle and descends onto the medial side of the heel.

1	The medial calcaneal nerve is often multiple and originates from the tibial nerve low in the leg near the ankle and descends onto the medial side of the heel. The medial calcaneal nerve innervates skin on the medial surface and sole of the heel (Fig. 6.90). Lateral compartment of leg There are two muscles in the lateral compartment of the leg—the fibularis longus and fibularis brevis (Fig. 6.91 and Table 6.8). Both evert the foot (turn the sole outward) and are innervated by the superficial fibular nerve, which is a branch of the common fibular nerve. The fibularis longus muscle arises in the lateral compartment of the leg, but its tendon crosses under the foot to attach to bones on the medial side (Fig. 6.91). It originates from both the upper lateral surface of the fibula and from the anterior aspect of the fibular head and occasionally up onto the adjacent region of the lateral tibial condyle.

1	The common fibular nerve passes anteriorly around the fibular neck between the attachments of the fibularis longus to the fibular head and shaft. Distally, the fibularis longus descends in the leg to form a tendon, which, in order: passes posterior to the lateral malleolus in a shallow bony groove, swings forward to enter the lateral side of the foot, descends obliquely down the lateral side of the foot where it curves forward under a bony tubercle (fibular trochlea) of the calcaneus, enters a deep groove on the inferior surface of one of the other tarsal bones (the cuboid), and swings under the foot to cross the sole and attach to the inferior surfaces of bones on the medial side of the foot (lateral sides of the base of metatarsal I and the distal end of the medial cuneiform). The fibularis longus everts and plantarflexes the foot.

1	The fibularis longus everts and plantarflexes the foot. In addition, the fibularis longus, tibialis anterior, and tibialis posterior muscles, which all insert on the undersurfaces of bones on the medial side of the foot, together act as a stirrup to support the arches of the foot. The fibularis longus supports mainly the lateral and transverse arches. The fibularis longus is innervated by the superficial fibular nerve. The fibularis brevis muscle is deep to the fibularis longus muscle in the leg and originates from the lower two-thirds of the lateral surface of the shaft of the fibula (Fig. 6.91). The tendon of the fibularis brevis passes behind the lateral malleolus with the tendon of the fibularis longus muscle and then curves forward across the lateral surface of the calcaneus to attach to a tubercle on the lateral surface of the base of metatarsal V (the metatarsal associated with the little toe).

1	The fibularis brevis assists in eversion of the foot and is innervated by the superficial fibular nerve. No major artery passes vertically through the lateral compartment of the leg. It is supplied by branches (mainly from the fibular artery in the posterior compartment of the leg) that penetrate into the lateral compartment (Fig. 6.92). Deep veins generally follow the arteries. The nerve associated with the lateral compartment of the leg is the superficial fibular nerve. This nerve originates as one of the two major branches of the common fibular nerve, which enters the lateral compartment of the leg from the popliteal fossa (Fig. 6.92B).

1	The common fibular nerve originates from the sciatic nerve in the posterior compartment of the thigh or in the popliteal fossa (Fig. 6.92A), and follows the medial margin of the biceps femoris tendon over the lateral head of the gastrocnemius muscle and toward the fibula. Here it gives origin to two cutaneous branches, which descend in the leg: the sural communicating nerve, which joins the sural branch of the tibial nerve and contributes to innervation of skin over the lower posterolateral side of the leg; and the lateral sural cutaneous nerve, which innervates skin over the upper lateral leg. The common fibular nerve continues around the neck of the fibula and enters the lateral compartment by passing between the attachments of the fibularis longus muscle to the head and shaft of the fibula. Here the common fibular nerve divides into its two terminal branches: the superficial fibular nerve, and the deep fibular nerve.

1	The superficial fibular nerve descends in the lateral compartment deep to the fibularis longus and innervates the fibularis longus and fibularis brevis (Fig. 6.91B). It then penetrates deep fascia in the lower leg and enters the foot where it divides into medial and lateral branches, which supply dorsal areas of the foot and toes except for: the web space between the great and second toes, which is supplied by the deep fibular nerve; and the lateral side of the little toe, which is supplied by the sural branch of the tibial nerve. The deep fibular nerve passes anteromedially through the intermuscular septum into the anterior compartment of the leg, which it supplies. Anterior compartment of leg

1	The deep fibular nerve passes anteromedially through the intermuscular septum into the anterior compartment of the leg, which it supplies. Anterior compartment of leg There are four muscles in the anterior compartment of the leg—the tibialis anterior, extensor hallucis longus, extensor digitorum longus, and fibularis tertius (Fig. 6.93 and Table 6.9). Collectively they dorsiflex the foot at the ankle joint, extend the toes, and invert the foot. All are innervated by the deep fibular nerve, which is a branch of the common fibular nerve. The tibialis anterior muscle is the most anterior and medial of the muscles in the anterior compartment of the leg (Fig. 6.93). It originates mainly from the upper two-thirds of the lateral surface of the shaft of the tibia and adjacent surface of the interosseous membrane. It also originates from deep fascia.

1	The muscle fibers of the tibialis anterior converge in the lower one-third of the leg to form a tendon, which descends into the medial side of the foot, where it attaches to the medial and inferior surfaces of one of the tarsal bones (medial cuneiform) and adjacent parts of metatarsal I associated with the great toe. The tibialis anterior dorsiflexes the foot at the ankle joint and inverts the foot at the intertarsal joints. During walking, it provides dynamic support for the medial arch of the foot. The tibialis anterior is innervated by the deep fibular nerve. The extensor hallucis longus muscle lies next to and is partly overlapped by the tibialis anterior muscle (Fig. 6.93). It originates from the middle one-half of the medial surface of the fibula and adjacent interosseous membrane.

1	The tendon of the extensor hallucis longus appears between the tendons of the tibialis anterior and extensor digitorum longus in the lower one-half of the leg and descends into the foot. It continues anteriorly on the medial side of the dorsal surface of the foot to near the end of the great toe where it inserts on the upper surface of the base of the distal phalanx. The extensor hallucis longus extends the great toe. Because it crosses anterior to the ankle joint, it also dorsiflexes the foot at the ankle joint. Like all muscles in the anterior compartment of the leg, the extensor hallucis longus muscle is innervated by the deep fibular nerve.

1	The extensor digitorum longus muscle is the most posterior and lateral of the muscles in the anterior compartment of the leg (Fig. 6.93). It originates mainly from the upper one-half of the medial surface of the fibula lateral to and above the origin of the extensor hallucis longus muscle, and extends superiorly onto the lateral condyle of the tibia. Like the tibialis anterior muscle, it also originates from deep fascia. The extensor digitorum longus muscle descends to form a tendon, which continues into the dorsal aspect of the foot, where it divides into four tendons, which insert, via dorsal digital expansions, into the dorsal surfaces of the bases of the middle and distal phalanges of the lateral four toes. The extensor digitorum longus extends the toes and dorsiflexes the foot at the ankle joint, and is innervated by the deep fibular nerve.

1	The extensor digitorum longus extends the toes and dorsiflexes the foot at the ankle joint, and is innervated by the deep fibular nerve. The fibularis tertius muscle is normally considered part of the extensor digitorum longus (Fig. 6.93). The fibularis tertius originates from the medial surface of the fibula immediately below the origin of the extensor digitorum longus muscle and the two muscles are normally connected. The tendon of the fibularis tertius descends into the foot with the tendon of the extensor digitorum longus. On the dorsal aspect of the foot, it deviates laterally to insert into the dorsomedial surface of the base of metatarsal V (the metatarsal associated with the little toe). The fibularis tertius assists in dorsiflexion and possibly eversion of the foot, and is innervated by the deep fibular nerve.

1	The fibularis tertius assists in dorsiflexion and possibly eversion of the foot, and is innervated by the deep fibular nerve. The artery associated with the anterior compartment of the leg is the anterior tibial artery, which originates from the popliteal artery in the posterior compartment of the leg and passes forward into the anterior compartment of the leg through an aperture in the interosseous membrane. The anterior tibial artery descends through the anterior compartment on the interosseous membrane (Fig. 6.94). In the distal leg, it lies between the tendons of the tibialis anterior and extensor hallucis longus muscles. It leaves the leg by passing anterior to the distal end of the tibia and ankle joint and continues onto the dorsal aspect of the foot as the dorsalis pedis artery. In the proximal leg, the anterior tibial artery has a recurrent branch, which connects with the anastomotic network of vessels around the knee joint.

1	In the proximal leg, the anterior tibial artery has a recurrent branch, which connects with the anastomotic network of vessels around the knee joint. Along its course, the anterior tibial artery supplies numerous branches to adjacent muscles and is joined by the perforating branch of the fibular artery, which passes forward through the lower aspect of the interosseous membrane from the posterior compartment of the leg. Distally, the anterior tibial artery gives rise to an anterior medial malleolar artery and an anterior lateral malleolar artery, which pass posteriorly around the distal ends of the tibia and fibula, respectively, and connect with vessels from the posterior tibial and fibular arteries to form an anastomotic network around the ankle. Deep veins follow the arteries and have similar names.

1	Deep veins follow the arteries and have similar names. The nerve associated with the anterior compartment of the leg is the deep fibular nerve (Fig. 6.94). This nerve originates in the lateral compartment of the leg as one of the two divisions of the common fibular nerve. The deep fibular nerve passes anteromedially through the intermuscular septum that separates the lateral from the anterior compartments of the leg and then passes deep to the extensor digitorum longus. It reaches the anterior interosseous membrane where it meets and descends with the anterior tibial artery. The deep fibular nerve: innervates all muscles in the anterior compartment; then continues into the dorsal aspect of the foot where it innervates the extensor digitorum brevis, contributes to the innervation of the first two dorsal interossei muscles, and supplies the skin between the great and second toes.

1	The foot is the region of the lower limb distal to the ankle joint. It is subdivided into the ankle, the metatarsus, and the digits. There are five digits consisting of the medially placed digits, ending laterally with the little toe (digit V) (Fig. 6.95). The foot has a superior surface (dorsum of foot) and an inferior surface (sole; Fig. 6.95). Abduction and adduction of the toes are defined with respect to the long axis of the second digit. Unlike in the hand, where the thumb is oriented 90° to the other fingers, the great toe is oriented in the same position as the other toes. The foot is the body’s point of contact with the ground and provides a stable platform for upright stance. It also levers the body forward during walking.

1	There are three groups of bones in the foot (Fig. 6.96): the seven tarsal bones, which form the skeletal framework for the ankle; metatarsals (I to V), which are the bones of the metatarsus; and the phalanges, which are the bones of the toes—each toe has three phalanges, except for the great toe, which has two. The tarsal bones are arranged in a proximal group and a distal group with an intermediate bone between the two groups on the medial side of the foot (Fig. 6.96A). The proximal group consists of two large bones, the talus (Latin for “ankle”) and the calcaneus (Latin for “heel”): The talus is the most superior bone of the foot and sits on top of and is supported by the calcaneus (Fig. 6.96B)—it articulates above with the tibia and fibula to form the ankle joint and also projects forward to articulate with the intermediate tarsal bone (navicular) on the medial side of the foot.

1	The calcaneus is the largest of the tarsal bones—posteriorly it forms the bony framework of the heel and anteriorly it projects forward to articulate with one of the distal group of tarsal bones (cuboid) on the lateral side of the foot. The talus, when viewed from the medial or lateral sides, is snail-shaped (Fig. 6.97A,B). It has a rounded head, which is projected forward and medially at the end of a short broad neck, which is connected posteriorly to an expanded body. Anteriorly, the head of the talus is domed for articulation with a corresponding circular depression on the posterior surface of the navicular bone. Inferiorly, this domed articular surface is continuous with an additional three articular facets separated by smooth ridges: The anterior and middle facets articulate with adjacent surfaces on the calcaneus bone.

1	The anterior and middle facets articulate with adjacent surfaces on the calcaneus bone. The other facet, medial to the facets for articulation with the calcaneus, articulates with a ligament—the plantar calcaneonavicular ligament (spring ligament)—which connects the calcaneus to the navicular under the head of the talus. The neck of the talus is marked by a deep groove (the sulcus tali), which passes obliquely forward across the inferior surface from medial to lateral, and expands dramatically on the lateral side. Posterior to the sulcus tali is a large facet (posterior calcaneal surface) for articulation with the calcaneus. The superior aspect of the body of the talus is elevated to fit into the socket formed by the distal ends of the tibia and fibula to form the ankle joint: The upper (trochlear) surface of this elevated region articulates with the inferior end of the tibia. The medial surface articulates with the medial malleolus of the tibia.

1	The upper (trochlear) surface of this elevated region articulates with the inferior end of the tibia. The medial surface articulates with the medial malleolus of the tibia. The lateral surface articulates with the lateral malleolus of the fibula. Because the lateral malleolus is larger and projects more inferiorly than the medial malleolus at the ankle joint, the corresponding lateral articular surface on the talus is larger and projects more inferiorly than the medial surface. The lower part of the lateral surface of the body of the talus, which supports the lower part of the facet for articulation with the fibula, forms a bony projection (the lateral process). The inferior surface of the body of the talus has a large oval concave facet (the posterior calcaneal articular facet) for articulation with the calcaneus.

1	The inferior surface of the body of the talus has a large oval concave facet (the posterior calcaneal articular facet) for articulation with the calcaneus. The posterior aspect of the body of the talus consists of a backward and medially facing projection (the posterior process). The posterior process is marked on its surface by a lateral tubercle and a medial tubercle, which bracket between them the groove for the tendon of the flexor hallucis longus as it passes from the leg into the foot. The calcaneus sits under and supports the talus. It is an elongate, irregular, box-shaped bone with its long axis generally oriented along the midline of the foot, but deviating lateral to the midline anteriorly (Fig. 6.98). The calcaneus projects behind the ankle joint to form the skeletal framework of the heel. The posterior surface of this heel region is circular and divided into upper, middle, and lower parts. The calcaneal tendon (Achilles tendon) attaches to the middle part:

1	The upper part is separated from the calcaneal tendon by a bursa. The lower part curves forward, is covered by subcutaneous tissue, is the weight-bearing region of the heel, and is continuous onto the plantar surface of the bone as the calcaneal tuberosity. The calcaneal tuberosity projects forward on the plantar surface as a large medial process and a small lateral process separated from each other by a V-shaped notch (Fig. 6.98B). At the anterior end of the plantar surface is a tubercle (the calcaneal tubercle) for the posterior attachment of the short plantar ligament of the sole of the foot.

1	The lateral surface of the calcaneus has a smooth contour except for two slightly raised regions (Fig. 6.98C). One of these raised areas—the fibular trochlea (peroneal tubercle)—is anterior to the middle of the surface and often has two shallow grooves, which pass, one above the other, obliquely across its surface. The tendons of the fibularis brevis and longus muscles are bound to the trochlea as they pass over the lateral side of the calcaneus. Superior and posterior to the fibular trochlea is a second raised area or tubercle for attachment of the calcaneofibular part of the lateral collateral ligament of the ankle joint. The medial surface of the calcaneus is concave and has one prominent feature associated with its upper margin (the sustentaculum tali; Fig. 6.98A), which is a shelf of bone projecting medially and supporting the more posterior part of the head of the talus.

1	The underside of the sustentaculum tali has a distinct groove running from posterior to anterior and along which the tendon of the flexor hallucis longus muscle travels into the sole of the foot. The superior surface of the sustentaculum tali has a facet (middle talar articular surface) for articulation with the corresponding middle facet on the head of the talus. Anterior and posterior talar articular surfaces are on the superior surface of the calcaneus itself (Fig. 6.98A): The anterior talar articular surface is small and articulates with the corresponding anterior facet on the head of the talus. The posterior talar articular surface is large and is approximately near the middle of the superior surface of the calcaneus. Between the posterior talar articular surface, which articulates with the body of the talus, and the other two articular surfaces, which articulate with the head of the talus, is a deep groove (the calcaneal sulcus; Fig. 6.98A,C).

1	The calcaneal sulcus on the superior surface of the calcaneus and the sulcus tali on the inferior surface of the talus together form the tarsal sinus, which is a large gap between the anterior ends of the calcaneus and talus that is visible when the skeleton of the foot is viewed from its lateral aspect (Fig. 6.99). The intermediate tarsal bone on the medial side of the foot is the navicular (boat shaped) (Fig. 6.96). This bone articulates behind with the talus and articulates in front and on the lateral side with the distal group of tarsal bones. One distinctive feature of the navicular is a prominent rounded tuberosity for the attachment of the tibialis posterior tendon, which projects inferiorly on the medial side of the plantar surface of the bone. From lateral to medial, the distal group of tarsal bones consists of (Fig. 6.96):

1	From lateral to medial, the distal group of tarsal bones consists of (Fig. 6.96): The cuboid (Greek for “cube”), which articulates posteriorly with the calcaneus, medially with the lateral cuneiform, and anteriorly with the bases of the lateral two metatarsals—the tendon of the fibularis longus muscle lies in a prominent groove on the anterior plantar surface, which passes obliquely forward across the bone from lateral to medial. Three cuneiforms (Latin for “wedge”)—the lateral, intermediate, and medial cuneiform bones, in addition to articulating with each other, articulate posteriorly with the navicular bone and anteriorly with the bases of the medial three metatarsals. There are five metatarsals in the foot, numbered I to V from medial to lateral (Fig. 6.100). Metatarsal I, associated with the great toe, is shortest and thickest. The second is the longest. Each metatarsal has a head at the distal end, an elongate shaft in the middle, and a proximal base.

1	Each metatarsal has a head at the distal end, an elongate shaft in the middle, and a proximal base. The head of each metatarsal articulates with the proximal phalanx of a toe and the base articulates with one or more of the distal group of tarsal bones. The plantar surface of the head of metatarsal I also articulates with two sesamoid bones. The sides of the bases of metatarsals II to V also articulate with each other. The lateral side of the base of metatarsal V has a prominent tuberosity, which projects posteriorly and is the attachment site for the tendon of the fibularis brevis muscle. The phalanges are the bones of the toes (Fig. 6.100). Each toe has three phalanges (proximal, middle, and distal), except for the great toe, which has only two (proximal and distal). Each phalanx consists of a base, a shaft, and a distal head: The base of each proximal phalanx articulates with the head of the related metatarsal.

1	Each phalanx consists of a base, a shaft, and a distal head: The base of each proximal phalanx articulates with the head of the related metatarsal. The head of each distal phalanx is nonarticular and flattened into a crescent-shaped plantar tuberosity under the plantar pad at the end of the digit. In each toe, the total length of the phalanges combined is much shorter than the length of the associated metatarsal. The ankle joint is synovial in type and involves the talus of the foot and the tibia and fibula of the leg (Fig. 6.102). The ankle joint mainly allows hinge-like dorsiflexion and plantarflexion of the foot on the leg. The distal end of the fibula is firmly anchored to the larger distal end of the tibia by strong ligaments. Together, the fibula and tibia create a deep bracket-shaped socket for the upper expanded part of the body of the talus: The roof of the socket is formed by the inferior surface of the distal end of the tibia.

1	The roof of the socket is formed by the inferior surface of the distal end of the tibia. The medial side of the socket is formed by the medial malleolus of the tibia. The longer lateral side of the socket is formed by the lateral malleolus of the fibula. The articular surfaces are covered by hyaline cartilage. The articular part of the talus is shaped like a short half-cylinder tipped onto its flat side with one end facing lateral and the other end facing medial. The curved upper surface of the half-cylinder and the two ends are covered by hyaline cartilage and fit into the bracket-shaped socket formed by the distal ends of the tibia and fibula.

1	When viewed from above, the articular surface of the talus is much wider anteriorly than it is posteriorly. As a result, the bone fits tighter into its socket when the foot is dorsiflexed and the wider surface of the talus moves into the ankle joint than when the foot is plantarflexed and the narrower part of the talus is in the joint. The joint is therefore most stable when the foot is dorsiflexed. The articular cavity is enclosed by a synovial membrane, which attaches around the margins of the articular surfaces, and by a fibrous membrane, which covers the synovial membrane and is also attached to the adjacent bones. The ankle joint is stabilized by medial (deltoid) and lateral ligaments. The medial (deltoid) ligament is large, strong (Fig. 6.103), and triangular in shape. Its apex is attached above to the medial malleolus and its broad base is attached below to a line that extends from the tuberosity of the navicular bone in front to the medial tubercle of the talus behind.

1	The medial ligament is subdivided into four parts based on the inferior points of attachment: The part that attaches in front to the tuberosity of the navicular and the associated margin of the plantar calcaneonavicular ligament (spring ligament), which connects the navicular bone to the sustentaculum tali of the calcaneus bone behind, is the tibionavicular part of the medial ligament. The tibiocalcaneal part, which is more central, attaches to the sustentaculum tali of the calcaneus bone. The posterior tibiotalar part attaches to the medial side and medial tubercle of the talus. The fourth part (the anterior tibiotalar part) is deep to the tibionavicular and tibiocalcaneal parts of the medial ligament and attaches to the medial surface of the talus. The lateral ligament of the ankle is composed of three separate ligaments, the anterior talofibular ligament, the posterior talofibular ligament, and the calcaneofibular ligament (Fig. 6.104):

1	The lateral ligament of the ankle is composed of three separate ligaments, the anterior talofibular ligament, the posterior talofibular ligament, and the calcaneofibular ligament (Fig. 6.104): The anterior talofibular ligament is a short ligament, and attaches the anterior margin of the lateral malleolus to the adjacent region of the talus. The posterior talofibular ligament runs horizontally backward and medially from the malleolar fossa on the medial side of the lateral malleolus to the posterior process of the talus. The calcaneofibular ligament is attached above to the malleolar fossa on the posteromedial side of the lateral malleolus and passes posteroinferiorly to attach below to a tubercle on the lateral surface of the calcaneus. The numerous synovial joints between the individual tarsal bones mainly invert, evert, supinate, and pronate the foot: Inversion and eversion is turning the whole sole of the foot inward and outward, respectively.

1	Inversion and eversion is turning the whole sole of the foot inward and outward, respectively. Pronation is rotating the front of the foot laterally relative to the back of the foot, and supination is the reverse movement. Pronation and supination allow the foot to maintain normal contact with the ground when in different stances or when standing on irregular surfaces. The major joints at which movements occur include the subtalar, talocalcaneonavicular, and calcaneocuboid joints (Fig. 6.105). The talocalcaneonavicular and calcaneocuboid joints together form what is often referred to as the transverse tarsal joint. Intertarsal joints between the cuneiforms and between the cuneiforms and the navicular allow only limited movement. The joint between the cuboid and navicular is normally fibrous.

1	Intertarsal joints between the cuneiforms and between the cuneiforms and the navicular allow only limited movement. The joint between the cuboid and navicular is normally fibrous. The subtalar joint is between: the large posterior calcaneal facet on the inferior surface of the talus, and the corresponding posterior talar facet on the superior surface of the calcaneus. The articular cavity is enclosed by synovial membrane, which is covered by a fibrous membrane. The subtalar joint allows gliding and rotation, which are involved in inversion and eversion of the foot. Lateral, medial, posterior, and interosseous talocalcaneal ligaments stabilize the joint. The interosseous talocalcaneal ligament lies in the tarsal sinus (Fig. 6.106). The talocalcaneonavicular joint is a complex joint in which the head of the talus articulates with the calcaneus and plantar calcaneonavicular ligament (spring ligament) below and the navicular in front (Fig. 6.107A).

1	The talocalcaneonavicular joint allows gliding and rotation movements, which together with similar movements of the subtalar joint are involved with inversion and eversion of the foot. It also participates in pronation and supination. The parts of the talocalcaneonavicular joint between the talus and calcaneus are: the anterior and middle calcaneal facets on the inferior surface of the talar head, and the corresponding anterior and middle talar facets on the superior surface and sustentaculum tali, respectively, of the calcaneus (Fig. 6.107B). The part of the joint between the talus and the plantar calcaneonavicular ligament (spring ligament) is between the ligament and the medial facet on the inferior surface of the talar head. The joint between the navicular and talus is the largest part of the talocalcaneonavicular joint and is between the ovoid anterior end of the talar head and the corresponding concave posterior surface of the navicular.

1	The capsule of the talocalcaneonavicular joint, which is a synovial joint, is reinforced: posteriorly by the interosseous talocalcaneal ligament, superiorly by the talonavicular ligament, which passes between the neck of the talus and adjacent regions of the navicular, and inferiorly by the plantar calcaneonavicular ligament (spring ligament) (Fig. 6.107C,D). The lateral part of the talocalcaneonavicular joint is reinforced by the calcaneonavicular part of the bifurcate ligament, which is a Y-shaped ligament superior to the joint. The base of the bifurcate ligament is attached to the anterior aspect of the superior surface of the calcaneus and its arms are attached to: the dorsomedial surface of the cuboid (calcaneocuboid ligament), and the dorsolateral part of the navicular (calcaneonavicular ligament).

1	The plantar calcaneonavicular ligament (spring ligament) is a broad thick ligament that spans the space between the sustentaculum tali behind and the navicular bone in front (Fig. 6.107B,C). It supports the head of the talus, takes part in the talocalcaneonavicular joint, and resists depression of the medial arch of the foot. The calcaneocuboid joint is a synovial joint between: the facet on the anterior surface of the calcaneus, and the corresponding facet on the posterior surface of the cuboid. The calcaneocuboid joint allows sliding and rotating movements involved with inversion and eversion of the foot, and also contributes to pronation and supination of the forefoot on the hindfoot. The calcaneocuboid joint is reinforced by the bifurcate ligament (see above) and by the long plantar ligament and the plantar calcaneocuboid ligament (short plantar ligament).

1	The calcaneocuboid joint is reinforced by the bifurcate ligament (see above) and by the long plantar ligament and the plantar calcaneocuboid ligament (short plantar ligament). The plantar calcaneocuboid ligament (short plantar ligament) is short, wide, and very strong, and connects the calcaneal tubercle to the inferior surface of the cuboid (Fig. 6.108A). It not only supports the calcaneocuboid joint, but also assists the long plantar ligament in resisting depression of the lateral arch of the foot. The long plantar ligament is the longest ligament in the sole of the foot and lies inferior to the plantar calcaneocuboid ligament (Fig. 6.108B): Posteriorly, it attaches to the inferior surface of the calcaneus between the tuberosity and the calcaneal tubercle. Anteriorly, it attaches to a broad ridge and a tubercle on the inferior surface of the cuboid bone behind the groove for the fibularis longus tendon.

1	Anteriorly, it attaches to a broad ridge and a tubercle on the inferior surface of the cuboid bone behind the groove for the fibularis longus tendon. More superficial fibers of the long plantar ligament extend to the bases of the metatarsal bones. The long plantar ligament supports the calcaneocuboid joint and is the strongest ligament, resisting depression of the lateral arch of the foot. The tarsometatarsal joints between the metatarsal bones and adjacent tarsal bones are plane joints and allow limited sliding movements (Fig. 6.109). The range of movement of the tarsometatarsal joint between the metatarsal of the great toe and the medial cuneiform is greater than that of the other tarsometatarsal joints and allows flexion, extension, and rotation. The tarsometatarsal joints, with the transverse tarsal joint, take part in pronation and supination of the foot.

1	The metatarsophalangeal joints are ellipsoid synovial joints between the sphere-shaped heads of the metatarsals and the corresponding bases of the proximal phalanges of the digits. The metatarsophalangeal joints allow extension and flexion, and limited abduction, adduction, rotation, and circumduction. The joint capsules are reinforced by medial and lateral collateral ligaments, and by plantar ligaments, which have grooves on their plantar surfaces for the long tendons of the digits (Fig. 6.109). Four deep transverse metatarsal ligaments link the heads of the metatarsals together and enable the metatarsals to act as a single unified structure (Fig. 6.109). The ligaments blend with the plantar ligaments of the adjacent metatarsophalangeal joints.

1	The metatarsal of the great toe is oriented in the same plane as the metatarsals of the other toes and is linked to the metatarsal of the second toe by a deep transverse metatarsal ligament. In addition, the joint between the metatarsal of the great toe and medial cuneiform has a limited range of motion. The great toe therefore has a very restricted independent function—unlike the thumb in the hand, where the metacarpal is oriented 90° to the metacarpals of the fingers, there is no deep transverse metacarpal ligament between the metacarpals of the thumb and index finger, and the joint between the metacarpal and carpal bone allows a wide range of motion. The interphalangeal joints are hinge joints that allow mainly flexion and extension. They are reinforced by medial and lateral collateral ligaments and by plantar ligaments (Fig. 6.109). Tarsal tunnel, retinacula, and arrangement of major structures at the ankle

1	Tarsal tunnel, retinacula, and arrangement of major structures at the ankle The tarsal tunnel is formed on the posteromedial side of the ankle by: a depression formed by the medial malleolus of the tibia, the medial and posterior surfaces of the talus, the medial surface of the calcaneus, and the inferior surface of the sustentaculum tali of the calcaneus; and an overlying flexor retinaculum (Fig. 6.110). The flexor retinaculum is a strap-like layer of connective tissue that spans the bony depression formed by the medial malleolus, the medial and posterior surfaces of the talus, the medial surface of the calcaneus, and the inferior surface of the sustentaculum tali (Fig. 6.110). It attaches above to the medial malleolus and below and behind to the inferomedial margin of the calcaneus. The retinaculum is continuous above with the deep fascia of the leg and below with the deep fascia (plantar aponeurosis) of the foot.

1	The retinaculum is continuous above with the deep fascia of the leg and below with the deep fascia (plantar aponeurosis) of the foot. Septa from the flexor retinaculum convert grooves on the bones into tubular connective tissue channels for the tendons of the flexor muscles as they pass into the sole of the foot from the posterior compartment of the leg (Fig. 6.110). Free movement of the tendons in the channels is facilitated by synovial sheaths, which surround the tendons. Two compartments on the posterior surface of the medial malleolus are for the tendons of the tibialis posterior and flexor digitorum longus muscles. The tendon of the tibialis posterior is medial to the tendon of the flexor digitorum longus.

1	Immediately lateral to the tendons of the tibialis posterior and flexor digitorum longus, the posterior tibial artery with its associated veins and the tibial nerve pass through the tarsal tunnel into the sole of the foot. The pulse of the posterior tibial artery can be felt through the flexor retinaculum midway between the medial malleolus and the calcaneus. Lateral to the tibial nerve is the compartment on the posterior surface of the talus and the undersurface of the sustentaculum tali for the tendon of the flexor hallucis longus muscle. Two extensor retinacula strap the tendons of the extensor muscles to the ankle region and prevent tendon bowing during extension of the foot and toes (Fig. 6.111): A superior extensor retinaculum is a thickening of deep fascia in the distal leg just superior to the ankle joint and attached to the anterior borders of the fibula and tibia.

1	A superior extensor retinaculum is a thickening of deep fascia in the distal leg just superior to the ankle joint and attached to the anterior borders of the fibula and tibia. An inferior retinaculum is Y-shaped, attached by its base to the lateral side of the upper surface of the calcaneus, and crosses medially over the foot to attach by one of its arms to the medial malleolus, whereas the other arm wraps medially around the foot and attaches to the medial side of the plantar aponeurosis. The tendons of the extensor digitorum longus and fibularis tertius pass through a compartment on the lateral side of the proximal foot. Medial to these tendons, the dorsalis pedis artery (terminal branch of the anterior tibial artery), the tendon of the extensor hallucis longus muscle, and finally the tendon of the tibialis anterior muscle pass under the extensor retinacula.

1	Fibular (peroneal) retinacula bind the tendons of the fibularis longus and fibularis brevis muscles to the lateral side of the foot (Fig. 6.112): A superior fibular retinaculum extends between the lateral malleolus and the calcaneus. An inferior fibular retinaculum attaches to the lateral surface of the calcaneus around the fibular trochlea and blends above with the fibers of the inferior extensor retinaculum. At the fibular trochlea, a septum separates the compartment for the tendon of the fibularis brevis muscle above from that for the fibularis longus below. Arches of the foot The bones of the foot do not lie in a horizontal plane. Instead, they form longitudinal and transverse arches relative to the ground (Fig. 6.113), which absorb and distribute downward forces from the body during standing and moving on different surfaces.

1	The longitudinal arch of the foot is formed between the posterior end of the calcaneus and the heads of the metatarsals (Fig. 6.113A). It is highest on the medial side, where it forms the medial part of the longitudinal arch, and lowest on the lateral side, where it forms the lateral part. The transverse arch of the foot is highest in a coronal plane that cuts through the head of the talus and disappears near the heads of the metatarsals, where these bones are held together by the deep transverse metatarsal ligaments (Fig. 6.113B). Ligaments and muscles support the arches of the foot (Fig. 6.114): Ligaments that support the arches include the plantar calcaneonavicular (spring ligament), plantar calcaneocuboid (short plantar ligament), and long plantar ligaments, and the plantar aponeurosis. Muscles that provide dynamic support for the arches during walking include the tibialis anterior and posterior and the fibularis longus.

1	Muscles that provide dynamic support for the arches during walking include the tibialis anterior and posterior and the fibularis longus. The plantar aponeurosis is a thickening of deep fascia in the sole of the foot (Fig. 6.115). It is firmly anchored to the medial process of the calcaneal tuberosity and extends forward as a thick band of longitudinally arranged connective tissue fibers. The fibers diverge as they pass anteriorly and form digital bands, which enter the toes and connect with bones, ligaments, and dermis of the skin. Distal to the metatarsophalangeal joints, the digital bands of the plantar aponeurosis are interconnected by transverse fibers, which form superficial transverse metatarsal ligaments. The plantar aponeurosis supports the longitudinal arch of the foot and protects deeper structures in the sole. Fibrous sheaths of toes

1	The plantar aponeurosis supports the longitudinal arch of the foot and protects deeper structures in the sole. Fibrous sheaths of toes The tendons of the flexor digitorum longus, flexor digitorum brevis, and flexor hallucis longus muscles enter fibrous digital sheaths or tunnels on the plantar aspect of the digits (Fig. 6.116). These fibrous sheaths begin anterior to the metatarsophalangeal joints and extend to the distal phalanges. They are formed by fibrous arches and cruciate (cross-shaped) ligaments attached posteriorly to the margins of the phalanges and to the plantar ligaments associated with the metatarsophalangeal and interphalangeal joints. These fibrous tunnels hold the tendons to the bony plane and prevent tendon bowing when the toes are flexed. Within each tunnel, the tendons are surrounded by a synovial sheath.

1	These fibrous tunnels hold the tendons to the bony plane and prevent tendon bowing when the toes are flexed. Within each tunnel, the tendons are surrounded by a synovial sheath. The tendons of the extensor digitorum longus, extensor digitorum brevis, and extensor hallucis longus pass into the dorsal aspect of the digits and expand over the proximal phalanges to form complex dorsal digital expansions (“extensor hoods”) (Fig. 6.117). Each extensor hood is triangular in shape with the apex attached to the distal phalanx, the central region attached to the middle (toes II to V) or proximal (toe I) phalanx, and each corner of the base wrapped around the sides of the metatarsophalangeal joint. The corners of the hoods attach mainly to the deep transverse metatarsal ligaments.

1	Many of the intrinsic muscles of the foot insert into the free margin of the hood on each side. The attachment of these muscles into the extensor hoods allows the forces from these muscles to be distributed over the toes to cause flexion of the metatarsophalangeal joints while at the same time extending the interphalangeal joints (Fig. 6.117). The function of these movements in the foot is uncertain, but they may prevent overextension of the metatarsophalangeal joints and flexion of the interphalangeal joints when the heel is elevated off the ground and the toes grip the ground during walking.

1	Intrinsic muscles of the foot originate and insert in the foot: the extensor digitorum brevis and extensor hallucis brevis on the dorsal aspect of the foot; all other intrinsic muscles—the dorsal and plantar interossei, flexor digiti minimi brevis, flexor hallucis brevis, flexor digitorum brevis, quadratus plantae (flexor accessorius), abductor digiti minimi, abductor hallucis, and lumbricals—are on the plantar side of the foot in the sole where they are organized into four layers. Intrinsic muscles mainly modify the actions of the long tendons and generate fine movements of the toes. All intrinsic muscles of the foot are innervated by the medial and lateral plantar branches of the tibial nerve except for the extensor digitorum brevis, which is innervated by the deep fibular nerve. The first two dorsal interossei also may receive part of their innervation from the deep fibular nerve. On the dorsal aspect

1	On the dorsal aspect The extensor digitorum brevis is attached to a roughened area on the superolateral surface of the calcaneus lateral to the tarsal sinus (Fig. 6.118 and Table 6.10). The flat muscle belly passes anteromedially over the foot, deep to the tendons of the extensor digitorum longus, and forms three tendons, which enter digits II, III, and IV. The tendons join the lateral sides of the tendons of the extensor digitorum longus. The extensor digitorum brevis extends the middle three toes through attachments to the long extensor tendons and extensor hoods. It is innervated by the deep fibular nerve. The extensor hallucis brevis originates in conjunction with the extensor digitorum brevis. Its tendon attaches to the base of the proximal phalanx of the great toes. The muscle extends the metatarsophalangeal joint of the great toe and is innervated by the deep fibular nerve. In the sole

1	In the sole The muscles in the sole of the foot are organized into four layers. From superficial to deep, or plantar to dorsal, these layers are the first, second, third, and fourth layers. There are three components in the first layer of muscles, which is the most superficial of the four layers and is immediately deep to the plantar aponeurosis (Fig. 6.119 and Table 6.11). From medial to lateral, these muscles are the abductor hallucis, flexor digitorum brevis, and abductor digiti minimi. The abductor hallucis muscle forms the medial margin of the foot and contributes to a soft tissue bulge on the medial side of the sole (Fig. 6.119). It originates from the medial process of the calcaneal tuberosity and adjacent margins of the flexor retinaculum and plantar aponeurosis. It forms a tendon that inserts on the medial side of the base of the proximal phalanx of the great toe and on the medial sesamoid bone associated with the tendon of the flexor hallucis brevis muscle.

1	The abductor hallucis abducts and flexes the great toe at the metatarsophalangeal joint and is innervated by the medial plantar branch of the tibial nerve. The flexor digitorum brevis muscle lies immediately superior to the plantar aponeurosis and inferior to the tendons of the flexor digitorum longus in the sole of the foot (Fig. 6.119). The flat spindle-shaped muscle belly originates as a tendon from the medial process of the calcaneal tuberosity and from the adjacent plantar aponeurosis. The muscle fibers of the flexor digitorum brevis converge anteriorly to form four tendons, which each enter one of the lateral four toes. Near the base of the proximal phalanx of the toe, each tendon splits to pass dorsally around each side of the tendon of the flexor digitorum longus and attach to the margins of the middle phalanx. The flexor digitorum brevis flexes the lateral four toes at the proximal interphalangeal joints and is innervated by the medial plantar branch of the tibial nerve.

1	The flexor digitorum brevis flexes the lateral four toes at the proximal interphalangeal joints and is innervated by the medial plantar branch of the tibial nerve. The abductor digiti minimi muscle is on the lateral side of the foot and contributes to the large lateral plantar eminence on the sole (Fig. 6.119). It has a broad base of origin, mainly from the lateral and medial processes of the calcaneal tuberosity and from a fibrous band of connective tissue, which connects the calcaneus with the base of metatarsal V. The abductor digiti minimi forms a tendon, which travels in a shallow groove on the plantar surface of the base of metatarsal V and continues forward to attach to the lateral side of the base of the proximal phalanx of the little toe. The abductor digiti minimi abducts the little toe at the metatarsophalangeal joint and is innervated by the lateral plantar branch of the tibial nerve.

1	The abductor digiti minimi abducts the little toe at the metatarsophalangeal joint and is innervated by the lateral plantar branch of the tibial nerve. The second muscle layer in the sole of the foot is associated with the tendons of the flexor digitorum longus muscle, which pass through this layer, and consists of the quadratus plantae and four lumbrical muscles (Fig. 6.120 and Table 6.12). The quadratus plantae muscle is a flat quadrangular muscle with two heads of origin (Fig. 6.120): One of the heads originates from the medial surface of the calcaneus inferior to the sustentaculum tali. The other head originates from the inferior surface of the calcaneus anterior to the lateral process of the calcaneal tuberosity and the attachment of the long plantar ligament. The quadratus plantae muscle inserts into the lateral side of the tendon of the flexor digitorum longus in the proximal half of the sole of the foot near where the tendon divides.

1	The quadratus plantae muscle inserts into the lateral side of the tendon of the flexor digitorum longus in the proximal half of the sole of the foot near where the tendon divides. The quadratus plantae assists the flexor digitorum longus tendon in flexing the toes and may also adjust the “line of pull” of this tendon as it enters the sole of the foot from the medial side. The muscle is innervated by the lateral plantar nerve. The lumbrical muscles are four worm-like muscles that originate from the tendons of the flexor digitorum longus and pass dorsally to insert into the free medial margins of the extensor hoods of the four lateral toes (Fig. 6.120). The first lumbrical originates from the medial side of the tendon of the flexor digitorum longus that is associated with the second toe. The remaining three muscles are bipennate and originate from the sides of adjacent tendons.

1	The lumbrical muscles act through the extensor hoods to resist excessive extension of the metatarsophalangeal joints and flexion of the interphalangeal joints when the heel leaves the ground during walking. The first lumbrical is innervated by the medial plantar nerve, while the other three are innervated by the lateral plantar nerve. There are three muscles in the third layer in the sole of the foot (Fig. 6.122 and Table 6.13): Two (the flexor hallucis brevis and adductor hallucis) are associated with the great toe. The third (the flexor digiti minimi brevis) is associated with the little toe. The flexor hallucis brevis muscle has two tendinous heads of origin (Fig. 6.122): The lateral head originates from the plantar surfaces of the cuboid, behind the groove for the fibularis longus, and adjacent surface of the lateral cuneiform. The medial head originates from the tendon of the tibialis posterior muscle as it passes into the sole of the foot.

1	The medial head originates from the tendon of the tibialis posterior muscle as it passes into the sole of the foot. The medial and lateral heads unite and give rise to a muscle belly, which itself is separated into medial and lateral parts adjacent to the plantar surface of metatarsal I. Each part of the muscle gives rise to a tendon that inserts on either the lateral or medial side of the base of the proximal phalanx of the great toe. A sesamoid bone occurs in each tendon of the flexor hallucis brevis as it crosses the plantar surface of the head of metatarsal I. The tendon of the flexor hallucis longus passes between the sesamoid bones. The flexor hallucis brevis flexes the metatarsophalangeal joint of the great toe and is innervated by the medial plantar nerve. The adductor hallucis muscle originates by two muscular heads, transverse and oblique, which join near their ends to insert into the lateral side of the base of the proximal phalanx of the great toe (Fig. 6.122):

1	The transverse head originates from the plantar ligaments associated with the metatarsophalangeal joints of the lateral three toes and from the associated deep transverse metatarsal ligaments—the muscle crosses the sole of the foot transversely from lateral to medial and joins the oblique head near the base of the great toe. The oblique head is larger than the transverse head and originates from the plantar surfaces of the bases of metatarsals II to IV and from the sheath covering the fibularis longus muscle—this head passes anterolaterally through the sole of the foot and joins the transverse head. The tendon of insertion of the adductor hallucis attaches to the lateral sesamoid bone associated with the tendon of the flexor hallucis brevis muscle in addition to attaching to the proximal phalanx. The adductor hallucis adducts the great toe at the metatarsophalangeal joint and is innervated by the lateral plantar nerve.

1	The adductor hallucis adducts the great toe at the metatarsophalangeal joint and is innervated by the lateral plantar nerve. The flexor digiti minimi brevis muscle originates from the plantar surface of the base of metatarsal V and adjacent sheath of the fibularis longus tendon (Fig. 6.122). It inserts on the lateral side of the base of the proximal phalanx of the little toe. The flexor digiti minimi brevis flexes the little toe at the metatarsophalangeal joint and is innervated by the lateral plantar nerve. There are two muscle groups in the deepest muscle layer in the sole of the foot, the dorsal and plantar interossei (Fig. 6.123 and Table 6.14). The four dorsal interossei are the most superior muscles in the sole of the foot and abduct the second to fourth toes relative to the long axis through the second toe (Fig. 6.123). All four muscles are bipennate and originate from the sides of adjacent metatarsals.

1	The tendons of the dorsal interossei insert into the free margin of the extensor hoods and base of the proximal phalanges of the toes. The second toe can be abducted to either side of its long axis, so it has two dorsal interossei associated with it, one on each side. The third and fourth toes have a dorsal interosseous muscle on their lateral sides only. The great and little toes have their own abductors (the abductor hallucis and abductor digiti minimi) in the first layer of muscles in the sole of the foot. In addition to abduction, the dorsal interossei act through the extensor hoods to resist extension of the metatarsophalangeal joints and flexion of the interphalangeal joints. The dorsal interossei are innervated by the lateral plantar nerve. The first and second dorsal interossei also receive branches on their superior surfaces from the deep fibular nerve.

1	The dorsal interossei are innervated by the lateral plantar nerve. The first and second dorsal interossei also receive branches on their superior surfaces from the deep fibular nerve. The three plantar interossei adduct the third, fourth, and little toes toward the long axis through the second toe (Fig. 6.123). Each plantar interosseous muscle originates from the medial side of its associated metatarsal and inserts into the medial free margin of the extensor hood and base of the proximal phalanx. The great toe has its own adductor (the adductor hallucis) in the third layer of muscles in the sole of the foot and the second toe is adducted back to its longitudinal axis by using one of its dorsal interossei. In addition to adduction, the plantar interossei act through the extensor hoods to resist extension of the metatarsophalangeal joints and flexion of the interphalangeal joints. All are innervated by the lateral plantar nerve.

1	Blood supply to the foot is by branches of the posterior tibial and dorsalis pedis (dorsal artery of the foot) arteries. The posterior tibial artery enters the sole and bifurcates into lateral and medial plantar arteries. The lateral plantar artery joins with the terminal end of the dorsalis pedis artery (the deep plantar artery) to form the deep plantar arch. Branches from this arch supply the toes. The dorsalis pedis artery is the continuation of the anterior tibial artery, passes onto the dorsal aspect of the foot and then inferiorly, as the deep plantar artery, between metatarsals I and II to enter the sole of the foot.

1	The posterior tibial artery enters the foot through the tarsal tunnel on the medial side of the ankle and posterior to the medial malleolus. Midway between the medial malleolus and the heel, the pulse of the posterior tibial artery is palpable because here the artery is covered only by a thin layer of retinaculum, by superficial connective tissue, and by skin. Near this location, the posterior tibial artery bifurcates into a small medial plantar artery and a much larger lateral plantar artery.

1	The lateral plantar artery passes anterolaterally into the sole of the foot, first deep to the proximal end of the abductor hallucis muscle and then between the quadratus plantae and flexor digitorum brevis muscles (Fig. 6.124). It reaches the base of metatarsal V where it lies in the groove between the flexor digitorum brevis and abductor digiti minimi muscles. From here, the lateral plantar artery curves medially to form the deep plantar arch, which crosses the deep plane of the sole on the metatarsal bases and the interossei muscles. Between the bases of metatarsals I and II, the deep plantar arch joins with the terminal branch (deep plantar artery) of the dorsalis pedis artery, which enters the sole from the dorsal side of the foot.

1	Major branches of the deep plantar arch include: a digital branch to the lateral side of the little toe; four plantar metatarsal arteries, which supply digital branches to adjacent sides of toes I to V and the medial side of the great toe; and three perforating arteries, which pass between the bases of metatarsals II to V to anastomose with vessels on the dorsal aspect of the foot. The medial plantar artery passes into the sole of the foot by passing deep to the proximal end of the abductor hallucis muscle (Fig. 6.124). It supplies a deep branch to adjacent muscles and then passes forward in the groove between the abductor hallucis and the flexor digitorum brevis muscles. It ends by joining the digital branch of the deep plantar arch, which supplies the medial side of the great toe.

1	Near the base of metatarsal I, the medial plantar artery gives rise to a superficial branch, which divides into three vessels that pass superficial to the flexor digitorum brevis muscle to join the plantar metatarsal arteries from the deep plantar arch. The dorsalis pedis artery is the continuation of the anterior tibial artery and begins as the anterior tibial artery crosses the ankle joint (Fig. 6.125). It passes anteriorly over the dorsal aspect of the talus, navicular, and intermediate cuneiform bones, and then passes inferiorly, as the deep plantar artery, between the two heads of the first dorsal interosseous muscle to join the deep plantar arch in the sole of the foot. The pulse of the dorsalis pedis artery on the dorsal surface of the foot can be felt by gently palpating the vessel against the underlying tarsal bones between the tendons of the extensor hallucis longus and the extensor digitorum longus to the second toe.

1	Branches of the dorsalis pedis artery include lateral and medial tarsal branches, an arcuate artery, and a first dorsal metatarsal artery: The tarsal arteries pass medially and laterally over the tarsal bones, supplying adjacent structures and anastomosing with a network of vessels formed around the ankle. The arcuate artery passes laterally over the dorsal aspect of the metatarsals near their bases and gives rise to three dorsal metatarsal arteries, which supply dorsal digital arteries to adjacent sides of digits II to V, and to a dorsal digital artery that supplies the lateral side of digit V. The first dorsal metatarsal artery (the last branch of the dorsalis pedis artery before the dorsalis pedis artery continues as the deep plantar artery into the sole of the foot) supplies dorsal digital branches to adjacent sides of the great and second toes.

1	The dorsal metatarsal arteries connect with perforating branches from the deep plantar arch and similar branches from the plantar metatarsal arteries. There are interconnected networks of deep and superficial veins in the foot. The deep veins follow the arteries. Superficial veins drain into a dorsal venous arch on the dorsal surface of the foot over the metatarsals (Fig. 6.126): The great saphenous vein originates from the medial side of the arch and passes anterior to the medial malleolus and onto the medial side of the leg. The small saphenous vein originates from the lateral side of the arch and passes posterior to the lateral malleolus and onto the back of the leg. The foot is supplied by the tibial, deep fibular, superficial fibular, sural, and saphenous nerves: All five nerves contribute to cutaneous or general sensory innervation.

1	The foot is supplied by the tibial, deep fibular, superficial fibular, sural, and saphenous nerves: All five nerves contribute to cutaneous or general sensory innervation. The tibial nerve innervates all intrinsic muscles of the foot except for the extensor digitorum brevis, which is innervated by the deep fibular nerve. The deep fibular nerve often also contributes to the innervation of the first and second dorsal interossei. The tibial nerve enters the foot through the tarsal tunnel posterior to the medial malleolus. In the tunnel, the nerve is lateral to the posterior tibial artery, and gives origin to medial calcaneal branches, which penetrate the flexor retinaculum to supply the heel. Midway between the medial malleolus and the heel, the tibial nerve bifurcates with the posterior tibial artery into: a large medial plantar nerve, and a smaller lateral plantar nerve (Fig. 6.127). The medial and lateral plantar nerves lie together between their corresponding arteries.

1	The medial and lateral plantar nerves lie together between their corresponding arteries. The medial plantar nerve is the major sensory nerve in the sole of the foot (Fig. 6.127). It innervates skin on most of the anterior two-thirds of the sole and adjacent surfaces of the medial three and one-half toes, which includes the great toe. In addition to this large area of plantar skin, the nerve also innervates four intrinsic muscles—the abductor hallucis, flexor digitorum brevis, flexor hallucis brevis, and first lumbrical. The medial plantar nerve passes into the sole of the foot deep to the abductor hallucis muscle and forward in the groove between the abductor hallucis and flexor digitorum brevis, supplying branches to both these muscles.

1	The medial plantar nerve supplies a digital branch (proper plantar digital nerve) to the medial side of the great toe and then divides into three nerves (common plantar digital nerves) on the plantar surface of the flexor digitorum brevis, which continue forward to supply proper plantar digital branches to adjacent surfaces of toes I to IV. The nerve to the first lumbrical originates from the first common plantar digital nerve. The lateral plantar nerve is an important motor nerve in the foot because it innervates all intrinsic muscles in the sole, except for the muscles supplied by the medial plantar nerve (the abductor hallucis, flexor digitorum brevis, flexor hallucis brevis, and first lumbrical) (Fig. 6.127). It also innervates a strip of skin on the lateral side of the anterior two-thirds of the sole and the adjacent plantar surfaces of the lateral one and one-half digits.

1	The lateral plantar nerve enters the sole of the foot by passing deep to the proximal attachment of the abductor hallucis muscle. It continues laterally and anteriorly across the sole between the flexor digitorum brevis and quadratus plantae muscles, supplying branches to both these muscles, and then divides near the head of metatarsal V into deep and superficial branches. The superficial branch of the lateral plantar nerve gives rise to a proper plantar digital nerve, which supplies skin on the lateral side of the little toe, and to a common plantar digital nerve, which divides to supply proper plantar digital nerves to skin on the adjacent sides of toes IV and V. The proper plantar digital nerve to the lateral side of the little toe also innervates the flexor digiti minimi brevis and the dorsal and plantar interossei muscles between metatarsals IV and V.

1	The proper plantar digital nerve to the lateral side of the little toe also innervates the flexor digiti minimi brevis and the dorsal and plantar interossei muscles between metatarsals IV and V. The deep branch of the lateral plantar nerve is motor and accompanies the lateral plantar artery deep to the long flexor tendons and the adductor hallucis muscle. It supplies branches to the second to fourth lumbrical muscles, the adductor hallucis muscle, and all interossei except those between metatarsals IV and V, which are innervated by the superficial branch. The deep fibular nerve innervates the extensor digitorum brevis, contributes to the innervation of the first two dorsal interossei muscles, and supplies general sensory branches to the skin on the adjacent dorsal sides of the first and second toes and to the web space between them (Fig. 6.128).

1	The deep fibular nerve enters the dorsal aspect of the foot on the lateral side of the dorsalis pedis artery, and is parallel with and lateral to the tendon of the extensor hallucis longus muscle. Just distal to the ankle joint, the nerve gives origin to a lateral branch, which innervates the extensor digitorum brevis from its deep surface. The deep fibular nerve continues forward on the dorsal surface of the foot, penetrates deep fascia between metatarsals I and II near the metatarsophalangeal joints, and then divides into two dorsal digital nerves, which supply skin over adjacent surfaces of toes I and II down to the beginning of the nail beds. Small motor branches, which contribute to the supply of the first two dorsal interossei muscles, originate from the deep fibular nerve before it penetrates deep fascia.

1	Small motor branches, which contribute to the supply of the first two dorsal interossei muscles, originate from the deep fibular nerve before it penetrates deep fascia. The superficial fibular nerve is sensory to most skin on the dorsal aspect of the foot and toes except for skin on adjacent sides of toes I and II (which is innervated by the deep fibular nerve) and skin on the lateral side of the foot and little toe (which is innervated by the sural nerve; Fig. 6.128). The superficial fibular nerve penetrates deep fascia on the anterolateral side of the lower leg and enters the dorsal aspect of the foot in superficial fascia. It gives rise to cutaneous branches and dorsal digital nerves along its course.

1	The sural nerve is a cutaneous branch of the tibial nerve that originates high in the leg. It enters the foot in superficial fascia posterior to the lateral malleolus close to the short saphenous vein. Terminal branches innervate skin on the lateral side of the foot and dorsolateral surface of the little toe (Fig. 6.128B). The saphenous nerve is a cutaneous branch of the femoral nerve that originates in the thigh. Terminal branches enter the foot in superficial fascia on the medial side of the ankle and supply skin on the medial side of the proximal foot (Fig. 6.128B). Tendons, muscles, and bony landmarks in the lower limb are used to locate major arteries, veins, and nerves.

1	Tendons, muscles, and bony landmarks in the lower limb are used to locate major arteries, veins, and nerves. Because vessels are large, they can be used as entry points to the vascular system. In addition, vessels in the lower limb are farthest from the heart and the most inferior in the body. Therefore, the nature of peripheral pulses in the lower limb can give important information about the status of the circulatory system in general. Sensation and muscle action in the lower limb are tested to assess lumbar and sacral regions of the spinal cord. Avoiding the sciatic nerve

1	Sensation and muscle action in the lower limb are tested to assess lumbar and sacral regions of the spinal cord. Avoiding the sciatic nerve The sciatic nerve innervates muscles in the posterior compartment of the thigh, muscles in the leg and foot, and an appreciable area of skin. It enters the lower limb in the gluteal region (Fig. 6.129) and passes inferiorly midway between two major palpable bony landmarks, the greater trochanter and the ischial tuberosity. The greater trochanter can be easily felt as a hard bony protuberance about one hand’s width inferior to the midpoint of the iliac crest. The ischial tuberosity is palpable just above the gluteal fold. The gluteal region can be divided into quadrants by two lines positioned using palpable bony landmarks. One line descends vertically from the highest point of the iliac crest.

1	The gluteal region can be divided into quadrants by two lines positioned using palpable bony landmarks. One line descends vertically from the highest point of the iliac crest. The other line passes horizontally through the first line midway between the highest point of the iliac crest and the horizontal plane through the ischial tuberosity. The sciatic nerve curves through the upper lateral corner of the lower medial quadrant and descends along the lateral margin of the lower medial quadrant. Injections can be carried out in the anterior corner of the upper lateral quadrant to avoid injury to the sciatic nerve and major vessels in the region (Fig. 6.129B). Finding the femoral artery in the femoral triangle The femoral artery passes into the femoral triangle (Fig. 6.130) of the lower limb from the abdomen.

1	Finding the femoral artery in the femoral triangle The femoral artery passes into the femoral triangle (Fig. 6.130) of the lower limb from the abdomen. The femoral triangle is the depression formed in the anterior thigh between the medial margin of the adductor longus muscle, the medial margin of the sartorius muscle, and the inguinal ligament. The tendon of the adductor longus muscle can be palpated as a cord-like structure that attaches to bone immediately inferior to the pubic tubercle. The sartorius muscle originates from the anterior superior iliac spine and crosses anteriorly over the thigh to attach to the medial aspect of the tibia below the knee joint. The inguinal ligament attaches to the anterior superior iliac spine laterally and the pubic tubercle medially.

1	The inguinal ligament attaches to the anterior superior iliac spine laterally and the pubic tubercle medially. The femoral artery descends into the thigh from the abdomen by passing under the inguinal ligament and into the femoral triangle. In the femoral triangle, its pulse is easily felt just inferior to the inguinal ligament midway between the pubic symphysis and the anterior superior iliac spine. Medial to the artery is the femoral vein and medial to the vein is the femoral canal, which contains lymphatics and lies immediately lateral to the pubic tubercle. The femoral nerve lies lateral to the femoral artery. Identifying structures around the knee

1	Identifying structures around the knee The patella is a prominent palpable feature at the knee. The quadriceps femoris tendon attaches superiorly to it and the patellar ligament connects the inferior surface of the patella to the tibial tuberosity (Fig. 6.131). The patellar ligament and the tibial tuberosity are easily palpable. A tap on the patellar ligament (tendon) tests reflex activity mainly at spinal cord levels L3 and L4. The head of the fibula is palpable as a protuberance on the lateral surface of the knee just inferior to the lateral condyle of the tibia. It can also be located by following the tendon of the biceps femoris inferiorly. The common fibular nerve passes around the lateral surface of the neck of the fibula just inferior to the head and can often be felt as a cord-like structure in this position.

1	The common fibular nerve passes around the lateral surface of the neck of the fibula just inferior to the head and can often be felt as a cord-like structure in this position. Another structure that can usually be located on the lateral side of the knee is the iliotibial tract. This flat tendinous structure, which attaches to the lateral tibial condyle, is most prominent when the knee is fully extended. In this position, the anterior edge of the tract raises a sharp vertical fold of skin posterior to the lateral edge of the patella. Visualizing the contents of the popliteal fossa

1	Visualizing the contents of the popliteal fossa The popliteal fossa is a diamond-shaped depression formed between the hamstrings and gastrocnemius muscle posterior to the knee. The inferior margins of the diamond are formed by the medial and lateral heads of the gastrocnemius muscle. The superior margins are formed laterally by the biceps femoris muscle and medially by the semimembranosus and semitendinosus muscles. The tendons of the biceps femoris muscle and the semitendinosus muscle are palpable and often visible. The head of the fibula is palpable on the lateral side of the knee and can be used as a landmark for identifying the biceps femoris tendon and the common fibular nerve, which curves laterally out of the popliteal fossa and crosses the neck of the fibula just inferior to the head.

1	The popliteal fossa contains the popliteal artery, the popliteal vein, the tibial nerve, and the common fibular nerve (Fig. 6.132). The popliteal artery is the deepest of the structures in the fossa and descends through the region from the upper medial side. As a consequence of its position, the popliteal artery pulse is difficult to find, but usually can be detected on deep palpation just medial to the midline of the fossa. The small saphenous vein penetrates deep fascia in the upper part of the posterior leg and joins the popliteal vein. Finding the tarsal tunnel—the gateway to the foot The tarsal tunnel (Fig. 6.133) is formed on the medial side of the foot in the groove between the medial malleolus and the heel (calcaneal tuberosity) and by the overlying flexor retinaculum.

1	The tarsal tunnel (Fig. 6.133) is formed on the medial side of the foot in the groove between the medial malleolus and the heel (calcaneal tuberosity) and by the overlying flexor retinaculum. The posterior tibial artery and tibial nerve enter the foot through the tarsal tunnel. The tendons of the tibialis posterior, flexor digitorum longus, and flexor hallucis longus also pass through the tarsal tunnel in compartments formed by septa of the flexor retinaculum. The order of structures passing through the tunnel from the anteromedial to posterolateral are the tendon of the tibialis posterior, the tendon of the flexor digitorum longus, the posterior tibial artery and associated veins, the tibial nerve, and the tendon of the flexor hallucis longus (“Tom, Dick, and a very nervous Harry”). The tibial artery is palpable just posteroinferior to the medial malleolus on the anterior face of the visible groove between the heel and medial malleolus.

1	The tibial artery is palpable just posteroinferior to the medial malleolus on the anterior face of the visible groove between the heel and medial malleolus. Identifying tendons around the ankle and in the foot Numerous tendons can be identified around the ankle and in the foot (Fig. 6.134) and can be used as useful landmarks for locating vessels or testing spinal reflexes. The tibialis anterior tendon is visible on the medial side of the ankle anterior to the medial malleolus. The calcaneal tendon is the largest tendon entering the foot and is prominent on the posterior aspect of the foot as it descends from the leg to the heel. A tap with a tendon hammer on this tendon tests reflex activity of spinal cord levels S1 and S2. When the foot is everted, the tendons of the fibularis longus and fibularis brevis raise a linear fold of skin, which descends from the lower leg to the posterior edge of the lateral malleolus.

1	When the foot is everted, the tendons of the fibularis longus and fibularis brevis raise a linear fold of skin, which descends from the lower leg to the posterior edge of the lateral malleolus. The tendon of the fibularis brevis is often evident on the lateral surface of the foot descending obliquely to the base of metatarsal V. The tendons of the fibularis tertius, extensor digitorum longus, and extensor hallucis longus are visible on the dorsal aspect of the foot from lateral to medial. Finding the dorsalis pedis artery The nature of the dorsalis pedis pulse (Fig. 6.135) is important for assessing peripheral circulation because the dorsalis pedis artery is the farthest palpable vessel from the heart. Also, it is the lowest palpable artery in the body when a person is standing.

1	The dorsalis pedis artery passes onto the dorsal aspect of the foot and anteriorly over the tarsal bones where it lies between and is parallel to the tendon of the extensor hallucis longus and the tendon of the extensor digitorum longus to the second toe. It is palpable in this position. The terminal branch of the dorsalis pedis artery passes into the plantar surface of the foot between the two heads of the first dorsal interosseous muscle. Approximating the position of the plantar arterial arch The blood supply of the foot is provided by branches of the posterior tibial and dorsalis pedis arteries. The posterior tibial artery enters the plantar surface of the foot through the tarsal tunnel and divides into a lateral and a medial plantar artery.

1	The posterior tibial artery enters the plantar surface of the foot through the tarsal tunnel and divides into a lateral and a medial plantar artery. The lateral plantar artery curves laterally across the posterior half of the sole and then curves medially as the plantar arch (Fig. 6.136) through the anterior sole. Between the bases of metatarsals I and II, the plantar arch joins the terminal branch (deep plantar artery) of the dorsalis pedis artery. Most of the foot is supplied by the plantar arch. The medial plantar artery passes anteriorly through the sole, connects with branches of the plantar arch, and supplies the medial side of the great toe. Superficial veins in the lower limb often become enlarged. Also, because the veins are long, they can be removed and used elsewhere in the body as vascular grafts.

1	Superficial veins in the lower limb often become enlarged. Also, because the veins are long, they can be removed and used elsewhere in the body as vascular grafts. Superficial veins (Fig. 6.137) in the lower limb begin as a dorsal venous arch in the foot. The medial side of the arch curves superiorly anterior to the medial malleolus and passes up the leg and thigh as the great saphenous vein. This vein passes through an aperture in the fascia lata (saphenous ring) to join with the femoral vein in the femoral triangle. The lateral side of the dorsal venous arch in the foot passes posterior to the lateral malleolus and up the posterior surface of the leg as the small saphenous vein. This vessel passes through the deep fascia in the upper one-third of the leg and connects with the popliteal vein in the popliteal fossa posterior to the knee.

1	Peripheral pulses can be felt at four locations in the lower limb (Fig. 6.138): femoral pulse in the femoral triangle—femoral artery inferior to the inguinal ligament and midway between the anterior superior iliac spine and the pubic symphysis; popliteal pulse in the popliteal fossa—popliteal artery deep in the popliteal fossa near the midline; posterior tibial pulse in the tarsal tunnel—posterior tibial artery posteroinferior to the medial malleolus in the groove between the medial malleolus and the heel (calcaneal tuberosity); dorsalis pedis pulse on the dorsal aspect of the foot—dorsalis pedis artery as it passes distally over the tarsal bones between the tendon of the extensor hallucis longus and the tendon of the extensor digitorum longus to the second toe. Fig. 6.1 Upper margin of the lower limb. Fig. 6.2 Regions of the lower limb. Fig. 6.3 Areas of transition. Inguinal ligamentFemoral trianglePopliteal fossa(posterior to knee)Tarsal tunnel

1	Fig. 6.1 Upper margin of the lower limb. Fig. 6.2 Regions of the lower limb. Fig. 6.3 Areas of transition. Inguinal ligamentFemoral trianglePopliteal fossa(posterior to knee)Tarsal tunnel Fig. 6.4 Center and line of gravity. Center ofgravityCenterof gravityposterior tohip jointLine of gravityAnterior to kneeAnterior to ankle Fig. 6.5 Movements of the hip joint. A. Flexion and extension. B. Abduction and adduction. C. External and internal rotation. D. Circumduction. Fig. 6.6 Movements of the knee and ankle. A. Knee flexion and extension. B. Ankle dorsiflexion and plantarflexion. Fig. 6.7 Some of the determinants of gait.

1	D. Circumduction. Fig. 6.6 Movements of the knee and ankle. A. Knee flexion and extension. B. Ankle dorsiflexion and plantarflexion. Fig. 6.7 Some of the determinants of gait. Pelvic rotation in transverse planeminimizes drop in center of gravity byeffectively lengthening the limbsKnee flexion on full stance. Limb minimizes rise incenter of gravity by effectively shortening the limbPelvic tilt (drop) on swing side minimizes rise incenter of gravityMovement of knees toward midline(adduction of hip) minimizes lateralshift in center of gravityAbduction on stanceside controls andlimits the dropLateral shift incenter of gravityWith adductionof hip (kneesmove towardmidline)No adductionof hip (knees donot move towardmidline)Vertical shift incenter of gravityInternal rotationof hip jointExternal rotation of hip jointCenter of gravity with knee not flexedCenter of gravity with knee flexedFlexion Fig. 6.8 Bones and joints of the lower limb. Fig. 6.9 Bones of the foot.

1	Fig. 6.8 Bones and joints of the lower limb. Fig. 6.9 Bones of the foot. Fig. 6.10 Longitudinal and transverse arches of the foot. Fig. 6.11 Muscles of the gluteal region. Extensor(gluteus maximus)Abductors(gluteus mediusand gluteus minimus)Rotators(piriformis, obturatorinternus, gemelli,quadratus femoris) Fig. 6.12 Major flexors of the hip. Fig. 6.13 Muscle compartments in the thigh and leg. Fig. 6.14 Apertures of communication between the lower limb and other regions. Fig. 6.15 Innervation of the lower limb. LumbarIliohypogastric (L1)Genitofemoral (L1, L2)Lateral cutaneous nerveof thigh (L2, L3)Obturator nerve (L2 to L4)Femoral nerve (L2 to L4)SacralSuperior gluteal nerve (L4 to S1)Sciatic nerve (L4 to S3)Inferior gluteal nerve (L5 to S2)Pudendal nerve (S2 to S4)Tibial nerve (branch of sciatic)(L4 to S3)Common fibular nerve (branch of sciatic)(L4 to S2)Sacrospinous ligamentIlio-inguinal (L1)LVLIVLIIILIILI

1	Fig. 6.16 Dermatomes of the lower limb. Dots indicate autonomous zones (i.e., with minimal overlap). Fig. 6.17 Movements generated by myotomes. L1, L2L3, L4L5 to S2S1, S2Adduction of toes S2, S3 Fig. 6.18 Major nerves of the lower limb (colors indicate regions of motor innervation). Femoral nerve(anterior compartmentof thigh)Obturator(medial compartmentof thigh)Commonfibular nerveSuperficial branch(lateral compartment of leg)Deep branch(anterior compartment of leg)Sciatic nerve(posterior compartmentof thigh, leg, andsole of foot)Superior and inferiorgluteal nerves Fig. 6.19 Regions of skin innervated by peripheral nerves.

1	Fig. 6.19 Regions of skin innervated by peripheral nerves. Obturator nerveFemoral nerve (anteriorcutaneous nerves of thigh)Femoral nerve (saphenous nerve)Common fibular nerve(lateral cutaneous of calf)Common fibular nerve(superficial branch)Common fibular nerve(deep branch)Medial plantar nervePosterior cutaneous nerve of thigh(from sacral plexus)Posterior rami (L1 to L3)Posterior rami (S1 to S3)Obturator nerveFemoral nerve (saphenous nerve)Tibial nerve (sural nerve)Tibial nerve (sural nerve)Lateral plantar nerveTibial nerve (medial calcanealbranches)Lateral cutaneousnerve of thigh(from lumbar plexus) Fig. 6.20 Nerves related to bone. Deep branchCommon fibularnerve (neck of fibula)Superficial branch Fig. 6.21 Superficial veins. Fig. 6.22 External surface of the bony pelvis. Lateral view.

1	Fig. 6.20 Nerves related to bone. Deep branchCommon fibularnerve (neck of fibula)Superficial branch Fig. 6.21 Superficial veins. Fig. 6.22 External surface of the bony pelvis. Lateral view. LIV spineHorizontal plane through top of iliac crestAnterior gluteal linePosterior gluteal linePosterior superior iliac spineSacrumSacrospinous ligamentIschial spineSacrotuberous ligamentPosterior inferior iliac spineAcetabulumIschial tuberosityIliumInferior gluteal lineIschiumPubisPubic tubercleIliopubic eminenceAnterior inferior iliac spineInguinal ligamentAnterior superior iliac spineAnterior abdominal wallIliac crestTuberculum of iliac crest Fig. 6.23 Ischial tuberosity. Posterolateral view.

1	Fig. 6.23 Ischial tuberosity. Posterolateral view. AcetabulumIschial spineFor attachment of sacrotuberous ligamentFor attachment of semitendinosus andlong head of biceps femoris muscleCovered by connective tissue and bursaFor attachment of adductor magnus muscleFor attachment of semimembranosus muscleIschiopubic ramusObturator foramenBody of pubic bone Fig. 6.24 Acetabulum. Fig. 6.25 Multiple fractures of the pelvis. Radiograph with contrast in the bladder. A large accumulation of blood is deforming the bladder. Fig. 6.26 Proximal end of the femur (right). A. Anterior view. B. Medial view. C. Posterior view. D. Lateral view.

1	Fig. 6.26 Proximal end of the femur (right). A. Anterior view. B. Medial view. C. Posterior view. D. Lateral view. ABTrochanteric fossaHeadNeckNeckAttachment site for piriformis muscleGreater trochanterAttachment site for gluteus minimusShaftLesser trochanterIntertrochanteric lineTubercleGreater trochanterPiriformisFoveaObturator internusTrochanteric fossaEnd of intertrochanteric linePectineal line (spiral line)Lesser trochanterOval depression forobturator externusAttachment ofgluteus mediusQuadrate tubercleGreater trochanterFoveaNeckQuadrate tubercleLesser trochanterPectineal line (spiral line)Medial margin of linea asperaLinea asperaLateral margin of linea asperaGluteal tuberosityAttachment site for gluteus mediusIntertrochanteric crestGluteus minimusGluteus mediusLesser trochanterCD Fig. 6.27 Shaft of the femur. On the right is a posterior view of proximal shaft of right femur.

1	Fig. 6.27 Shaft of the femur. On the right is a posterior view of proximal shaft of right femur. Fig. 6.28 This radiograph of the pelvis, anteroposterior view, demonstrates a fracture of the neck of the femur. Fractured neck of femur Fig. 6.29 Anteroposterior radiograph showing an intertrochanteric fracture of proximal end of femur. Fig. 6.30 Hip joint. A. Articular surfaces. Anterior view. B. Movement of the neck of the femur during medial and lateral rotation. Superior view. Acetabulum of pelvic boneAcetabular labrumHead of femurSuperior viewLateral rotationMedial rotationAB Fig. 6.31 Hip joint. A. Transverse acetabular ligament. B. Ligament of the head of the femur. The head of the femur has been laterally rotated out of the acetabulum to show the ligament.

1	Fig. 6.31 Hip joint. A. Transverse acetabular ligament. B. Ligament of the head of the femur. The head of the femur has been laterally rotated out of the acetabulum to show the ligament. ABAcetabular labrum Lunate surfaceTransverseacetabular ligamentAcetabularforamenObturator foramenAcetabular fossaArtery of ligament of headSynovial sleeve around ligamentLigament of head of femurIschial tuberosityObturator membranePubisPubic tubercleObturator arteryAcetabular branchof obturator arteryCut synovial membrane Fig. 6.32 Synovial membrane of the hip joint. Synovial membraneLine of attachment around head of femurMembrane reflects back to attach to margin ofacetabulum Fig. 6.33 Fibrous membrane and ligaments of the hip joint. A. Fibrous membrane of the joint capsule. Anterior view. B. Iliofemoral and pubofemoral ligaments. Anterior view. C. Ischiofemoral ligament. Posterior view. Fig. 6.34 Blood supply of the hip joint.

1	Fig. 6.34 Blood supply of the hip joint. Internal iliac arteryCommon iliac arteryExternal iliac arteryLateral circumflex femoral artery1st perforating arteryDeep artery of thighMedial circumflex femoral arteryFemoral arteryObturator arteryInferior gluteal arterySuperior gluteal artery Fig. 6.35 Gateways to the lower limb.

1	Fig. 6.35 Gateways to the lower limb. Sacrospinous ligamentSacrotuberous ligamentPelvic cavityIlio-inguinal nerveAbdominal cavityGap between inguinal ligament and pelvic bone:• Psoas major, iliacus, pectineus muscles• Femoral artery• Femoral vein• Lymphatics• Femoral branch of genitofemoral nerve• Lateral cutaneous nerve of thigh• Femoral nerveObturator canal:• obturator nerve• obturator vesselsLesser sciatic foramen:• Obturator internus muscle tendon• Pudendal nerve and internal pudendal vessels pass into perineum from gluteal regionGreater sciatic foramen below piriformis muscle:• Sciatic nerve• Inferior gluteal nerve, artery, vein• Pudendal nerve• Internal pudendal artery and vein• Posterior femoral cutaneous nerve• Nerve to obturator internus and gemellus superior muscles• Nerve to quadratus femoris and gemellus inferior musclesGreater sciatic foramen abovepiriformis muscle:• Superior gluteal nerve, artery, veinPiriformis muscle

1	Fig. 6.36 Branches of the lumbosacral plexus. L3 anterior ramusPerforating cutaneous nerveLumbosacral trunkSuperior gluteal nerveNerves to quadratus femoris and obturator internusInguinal ligamentInferior gluteal nervePosterior cutaneous nerve of thighSciatic nerveS1S2Sacrotuberous ligamentSacrospinous ligamentObturator nerveFemoral branch of genitofemoral nerveFemoral nerveLateral cutaneous nerve of thighIlio-inguinal nerveL1 anterior ramusL2 anterior ramus Fig. 6.37 Arteries of the lower limb. Fig. 6.38 Veins of the lower limb. Fig. 6.39 Lymphatic drainage of the lower limb. Fig. 6.40 Fascia lata. A. Right limb. Anterior view. B. Lateral view. ABInguinal ligamentInguinal ligamentDeep fascia of legFascia lataSaphenous openingAnterior superior iliac spineAnterior superior iliac spineTuberculum of iliac crestFascia lataGluteus maximusTensor fascia lataIliotibial tractPubic tuberclePubic tubercle Fig. 6.41 Saphenous ring. Anterior view.

1	Fig. 6.41 Saphenous ring. Anterior view. Fig. 6.42 Boundaries of the femoral triangle. Fig. 6.43 Contents of the femoral triangle. Fig. 6.44 Gluteal region. Posterior view. Fig. 6.45 Deep muscles in the gluteal region. A. Posterior view. B. Function. ABGemellus superior Gemellus inferiorObturator internusQuadratus femorisGluteus mediusGluteus minimusGreater sciatic foramen abovepiriformisPiriformis muscleGreater sciatic foramen belowpiriformisContraction of gluteus minimus and mediuson stance side prevents excessive pelvic tiltduring swing phase on opposite side Fig. 6.46 Gluteus maximus muscle. Posterior view. Gluteus mediusGluteus maximusAttachment of gluteus maximus to iliotibial tractIliotibial tractAttachment of deep fibers to gluteal tuberosity Fig. 6.47 Tensor fasciae latae. Left gluteal region, lateral view. Tensor fasciae lataeTubercle of crest of iliumGluteus mediusIliotibial tractGluteus minimusGluteus maximusFascia lataDeep fascia of legTibiaAttachment to tibia

1	Tensor fasciae lataeTubercle of crest of iliumGluteus mediusIliotibial tractGluteus minimusGluteus maximusFascia lataDeep fascia of legTibiaAttachment to tibia Fig. 6.48 Nerves of the gluteal region. Posterior view. Superior gluteal nerveTensor fasciae latae muscleInferior gluteal nerveIliotibial tractPosterior cutaneous nerve of thighNerve to quadratus femorismuscle (deep to gemelli, obturator internus, and quadratus femoris)Gluteus maximusPerforating cutaneous nerveNerve to obturator internusPudendal nervePiriformis muscleSciatic nerve Fig. 6.49 Site for intramuscular injections in the gluteal region. Fig. 6.50 Arteries of the gluteal region. Deep branchLateral femoral circumflex arteryMedial femoral circumflex arteryFirst perforating artery fromdeep artery of thighInferior gluteal artery and veinSuperficial branchSuperior gluteal artery and veinPiriformis muscle

1	Fig. 6.51 Anastomoses between gluteal arteries and vessels originating from the femoral artery in the thigh. Posterior view. Superior gluteal arteryFirst perforating arterySecond perforating arteryThird perforating arteryFemoral arteryDeep artery of thighMedial femoral circumflex arteryLateral femoralcircumflex arteryInferior gluteal artery Fig. 6.52 Thigh. A. Posterior view. B. Anterior view. C. Cross section through the midthigh. ABCInguinal ligamentAbdominal wallGap between inguinal ligament and pelvic boneObturator canalAnterior superior iliac spinePubic tubercleMedialAnteriorLateral Posterior Anterior compartmentPosterior compartmentMedial compartmentPopliteal fossa(posterior to knee)Popliteal fossaSciatic nerveQuadratus femorisInferior margin of gluteus maximusGluteal fold Fig. 6.53 Shaft and distal end of femur. A. Lateral view. B. Anterior view. C. Posterior view. D. Cross section through shaft of femur.

1	Fig. 6.53 Shaft and distal end of femur. A. Lateral view. B. Anterior view. C. Posterior view. D. Cross section through shaft of femur. Lateral epicondyleMedial epicondyleMedial condyleLateral borderLateral(posterolateral)surfaceMedial(posteromedial)surfaceMedialborderLateral condyleLateral epicondyleFacet for attachment of lateral head of gastrocnemiusFacet for attachment of the tendon of popliteus muscleFacet for attachmentof the tendon ofpopliteus muscleAdductor tubercleAdductor tuberclePatellar surfacePosterior surfaceMedial supracondylar lineLateral supracondylar lineIntercondylar fossaFacet for attachment of posterior cruciate ligamentFacet for attachment of anterior cruciate ligamentRoughened area for attachment of medial head of gastrocnemius muscleFacet for attachmentof lateral head ofgastrocnemius muscleLinea asperaLinea asperaAnterior surfaceABCD Fig. 6.54 Patella. A. Anterior view. B. Posterior view. C. Superior view.

1	Fig. 6.54 Patella. A. Anterior view. B. Posterior view. C. Superior view. Fig. 6.55 Proximal end of the tibia. A. Superior view, tibial plateau. B. Anterior view. C. Posterior view. D. Cross section through the shaft of tibia. AIntercondylar regionAttachment of posterior cruciate ligamentPosterior attachment of lateral meniscusArea of articulation with lateral meniscusAnterior attachment of lateral meniscusAttachment of anterior cruciate ligamentTuberosityTubercles of intercondylar eminenceRoughened and perforated areaAnterior attachment of medial meniscusArea of articulation with medial meniscusPosterior attachment of medial meniscus

1	BCLateral condyleRoughened and perforated areaSite of attachment of sartorius, gracilis, and semitendinosus musclesShaft of tibiaGrooveTibial tuberosityMedial condyleAnterior attachment ofmedial meniscusTubercles of intercondylar eminenceAnterior Anterior borderLateral surfaceInterosseous borderPosterior surfacePosteriorMedial borderMedial surfaceAttachment of medial meniscusAttachment of posterior cruciate ligamentSoleal lineArticular facet for proximal head of fibulaD Fig. 6.56 Proximal end of the fibula. A. Anterior view. B. Cross section through the shaft of fibula. ABApexAttachment site for fibular collateral ligament of kneeAttachment site for tendon of biceps femoris muscleCommon fibular nerveLateral surfaceShaftMedial surfaceMedial part of posterior surfaceNeckFacet for articulationwith inferior surfaceof lateral condyle of tibiaHeadInterosseousborderPosterior borderLateral surfaceAnterior borderMedial surfacePosterior surfaceMedial crest onposterior surface

1	Fig. 6.57 Transverse section through the midthigh. Fig. 6.58 Psoas major and iliacus muscles. Fig. 6.59 Muscles of the anterior compartment of thigh. SartoriusSartoriusStraight head of rectus femorisReflected head of rectus femorisVastus lateralisRectus femorisVastus medialisQuadriceps femoris tendonPatellaPatellar ligamentPes anserinusSartoriusGracilisSemitendinosusAttachment of pes anserinusTibial tuberosityPatellar ligamentQuadriceps femoris tendonSuprapatellar bursaArticularis genusVastus medialisRectus femorisVastus intermediusVastus lateralisSartoriusPosterior compartment of thighVastus lateralisVastus medialisVastus intermediusMedial compartment of thighAdductor canal Fig. 6.60 Muscles of the medial compartment of thigh. Anterior view.

1	Fig. 6.60 Muscles of the medial compartment of thigh. Anterior view. Obturator externusAdductor magnusPectineusAdductor longusAdductor brevisAnterior compartment of thighSartorius attachmentGracilisGracilisSemitendinosus attachmentAdductor hiatusAdductor canalAdductor longusAdductor magnusPosterior compartment of thighPes anserinus Fig. 6.61 Pectineus, adductor longus, and adductor brevis muscles. Anterior view. Fig. 6.62 Adductor magnus and obturator externus muscles. Anterior view. Obturator externusAdductor magnus (adductor part)Adductor magnus (hamstring part)Adductor tubercleAdductor hiatusPerforations for branches of deep artery of thigh• Terminal end of deep artery of thigh Fig. 6.63 Muscles of the posterior compartment of thigh. Posterior view.

1	Fig. 6.63 Muscles of the posterior compartment of thigh. Posterior view. Ischial tuberosityQuadratus femorisAdductor magnusHamstring part of adductor magnusLong head of biceps femorisShort head of biceps femorisPart of semimembranosus thatinserts into capsule around knee jointOn anterior aspect of tibia attaches to pes anserinusSemimembranosusSemitendinosus Fig. 6.64 Coronal MRI of the posterior pelvis and thigh showing a hamstring avulsion injury. Fig. 6.65 Femoral artery.

1	Fig. 6.64 Coronal MRI of the posterior pelvis and thigh showing a hamstring avulsion injury. Fig. 6.65 Femoral artery. Superficial epigastric arterySartorius muscleDeep artery of thighRectus femoris muscleVastus medialis muscleGracilis muscleDeep externalpudendal arterySuperficial externalpudendal arteryVastus lateralis muscleVastus medialis muscleSartorius muscleArtery passes posteriorlythrough adductor hiatus andbecomes popliteal arteryArtery in adductor canalFemoral artery • Midway between anterior superior iliac spine and pubic symphysis inferior to inguinal ligamentExternal iliac arterySuperficial externaliliac arteryPubic symphysis Fig. 6.66 Deep artery of thigh. A. Anterior view. B. Posterior view.

1	Fig. 6.66 Deep artery of thigh. A. Anterior view. B. Posterior view. BSuperior gluteal arteryLateral femoral circumflex arteryCruciate anastomosesMedial circumflex femoral arteryFirst perforating arterySecond perforating arteryThird perforating arteryTerminal end of deepartery of thighPopliteal arteryAdductor magnus muscleAdductor hiatusInferior gluteal arteryPiriformis muscleAPsoas and iliacus musclesSartorius muscleRectus femoris muscleLateral circumflex femoral arteryAscending branchDescending branchVastus lateralis muscleVastus intermedius muscleCut vastus medialis muscleQuadriceps femoris tendonTerminal end of deepartery of thighGracilis muscleSartorius muscleAdductor magnus muscleFirst, second, and third perforating arteriesAdductor brevis muscleAdductor longus musclePectineus muscleMedial circumflex femoral arteryDeep artery of thigh Fig. 6.67 Obturator artery.

1	Fig. 6.67 Obturator artery. Ligament ofhead offemurObturatorarteryAnterior branchAcetabular branchPosterior branchObturatorexternus muscleArtery of ligament of head of femur Fig. 6.68 Femoral nerve. Nerve to pectineusAnterior branchNerves to iliacusNerve to sartoriusRectus femoris muscleVastus lateralis muscleVastus medialis muscleSaphenous nerveSaphenous nervePes anserinusSartorius muscleGracilis muscleAnterior cutaneous branchesAdductor magnus musclePectineus muscleAdductor longus musclePosterior branchFemoral nerve Fig. 6.69 Obturator nerve. Posterior branchObturator externus musclePsoas and iliacus musclesPectineus muscleAdductor brevis muscleAdductor longus muscleBranch to adductor magnus from posterior branchAdductor magnus muscleGracilis muscleCutaneous branchAnterior branchObturator nerve Fig. 6.70 Sciatic nerve.

1	Fig. 6.70 Sciatic nerve. Piriformis muscleQuadratus femoris muscleSciatic nerveAdductor magnus muscleLong head of biceps femoris muscleLong head of biceps femoris muscleShort head of biceps femoris muscleCommon fibular nerveTibial nervePopliteal artery and veinSemimembranosus muscleSemitendinosus muscleBranch to part of adductor magnus originatingfrom ischial tuberosity Fig. 6.71 Knee joint. Joint capsule is not shown. Fig. 6.72 Articular surfaces of the knee joint. A. Extended. B. Flexed. C. Anterior view (flexed). Fig. 6.73 Menisci of the knee joint. A. Superior view. Menisci of the knee joint. B. Normal knee joint showing the medial meniscus. T2-weighted magnetic resonance image in the sagittal plane. C. Normal knee joint showing the lateral meniscus. T2-weighted magnetic resonance image in the sagittal plane. APatellar ligamentJoint capsuleMedial meniscusPopliteus tendonLateral meniscusTransverse ligament of the kneeInfrapatellar fat

1	APatellar ligamentJoint capsuleMedial meniscusPopliteus tendonLateral meniscusTransverse ligament of the kneeInfrapatellar fat Fig. 6.74 Meniscal injury and repair. A. Sagittal MRI of a knee joint showing tear of the medial meniscus. B. Coronal MRI of a knee showing a truncated lateral meniscus after partial meniscectomy to treat a tear. Fig. 6.75 Synovial membrane of the knee joint and associated bursae. A. Superolateral view; patella and femur not shown. B. Paramedial sagittal section through the knee. Fig. 6.76 Fibrous membrane of the knee joint capsule. A. Anterior view. B. Posterior view.

1	Fig. 6.76 Fibrous membrane of the knee joint capsule. A. Anterior view. B. Posterior view. Fig. 6.77 Collateral ligaments of the knee joint. A. Lateral view. B. Medial view. C. Normal knee joint showing the patellar ligament and the fibular collateral ligament. T1-weighted magnetic resonance image in the sagittal plane. D. Normal knee joint showing the tibial collateral ligament, the medial and lateral menisci, and the anterior and posterior cruciate ligaments. T1-weighted magnetic resonance image in the coronal plane. Fig. 6.78 Cruciate ligaments of the knee joint. Superolateral view. Fig. 6.79 Knee “locking” mechanism. Medial rotation of femur on tibia tightens ligamentsFlat surface offemoral condylesis in contact withtibia and stabilizesjointLine of center of gravity isanterior to knee joint andmaintains extension Fig. 6.80 Anastomoses of arteries around the knee. Anterior view.

1	Fig. 6.80 Anastomoses of arteries around the knee. Anterior view. Femoral arteryAdductorhiatusDescendinggenicular arteryAdductormagnusSaphenousbranchSuperior medialgenicular arteryPoplitealarteryInferior medialgenicular arteryPosteriortibial arteryAnteriortibial arteryInterosseousmembraneRecurrent branchof anterior tibialCircumflexfibular arteryInferior lateralgenicular arterySuperior lateralgenicular arteryDescending branch of lateral circumflex femoral artery Fig. 6.81 Sagittal MRI of knee joint showing rupture of the anterior cruciate ligament. Fig. 6.82 Tibiofibular joint. Fig. 6.83 Popliteal fossa. A. Boundaries. B. Nerves and vessels. C. Superficial structures.

1	Fig. 6.82 Tibiofibular joint. Fig. 6.83 Popliteal fossa. A. Boundaries. B. Nerves and vessels. C. Superficial structures. ABCLinea asperaBiceps femoris muscle(short head)Biceps femorismuscle(long head)PoplitealfossaPlantarismuscleLateral head ofgastrocnemiusmusclePopliteusmuscleMedial head ofgastrocnemiusmuscleSemitendinosusmuscleSemimembranosus muscleAdductorhiatusAdductor magnus muscleSciatic nerveTibial nerveSmallsaphenousveinSmallsaphenousveinCommonfibular nervePopliteal arteryPopliteal veinFemoral veinFemoral arteryPosteriorcutaneousnerve of thighPosteriorcutaneousnerve of thighLateralNerveVeinArteryMedial Fig. 6.84 Posterior view of leg; cross section through the left leg (inset). Fig. 6.85 Tibia and fibula. A. Anterior view. B. Posterior view. C. Cross section through shafts. D. Posteromedial view of distal ends.

1	Fig. 6.85 Tibia and fibula. A. Anterior view. B. Posterior view. C. Cross section through shafts. D. Posteromedial view of distal ends. Lateral surfaceLateralsurfaceInterosseousmembraneInterosseousborderMedialsurfaceAnteriorborderMedialborderPosteriorborderPosterior surfacePosterior surfaceMedial crestInterosseous borderInterosseousborderMedialsurfaceAnteriorborderAnteriorborderGroove for fibularis longus and brevis musclesRoughened triangulararea that fits intofibular groove of tibiaABCDFibulaTibiaFibular grooveon tibiaSoleal lineArticular surfaces for talusMedial malleolusGroove for tendon oftibialis posterior muscleMalleolar fossaLateralmalleolusLateral malleolus Fig. 6.86 Interosseous membrane. A. Anterior view. B. Posteromedial view. Aperture for anterior tibial vesselsABAperture for perforating branch of fibular arteryAnterior tibiofibular ligamentInterosseous membraneInterosseous membranePosterior tibiofibular ligament

1	Aperture for anterior tibial vesselsABAperture for perforating branch of fibular arteryAnterior tibiofibular ligamentInterosseous membraneInterosseous membranePosterior tibiofibular ligament Fig. 6.87 Superficial group of muscles in the posterior compartment of leg. A. Posterior view. B. Lateral view. APlantarisMedial head ofgastrocnemiusPopliteal vessels and tibial nerveLigament spanning distance betweenfibular and tibial origins of soleusTendon of plantarisCalcaneal (Achilles) tendonCalcaneusGastrocnemiusSoleusLateral head of gastrocnemiusMedialLateralBSoleusCalcaneal tendonGastrocnemiusCalcaneus Fig. 6.88 Deep group of muscles in the posterior compartment of leg.

1	Fig. 6.88 Deep group of muscles in the posterior compartment of leg. PopliteusSoleal lineTibialisposteriorOrigin oftibialisposteriorVerticallineFlexordigitorumlongusOrigin of flexordigitorum longusOrigin of flexorhallucis longusGroove onmedialmalleolusMedialTuberosity ofnavicularMedialcuneiformGroove on inferiorsurface of sustentaculumtali of calcaneus boneGroove on posteriorsurface of talusFlexor hallucislongusLateral Fig. 6.89 Arteries in the posterior compartment of leg. Adductor hiatusSuperior medialgenicular arterySuperior lateralgenicular arteryAdductormagnus musclePopliteal arterySural arteriesMedial head ofgastrocnemiusmuscle Popliteus musclePosterior tibial arteryPosterior tibial arteryPerforating terminalbranch of fibular arteryBranches thatperforate intermuscularseptum to enter lateralcompartmentFibular arteryCircumflexfibular arteryAnterior tibial artery(passes throughaperture ininterosseousmembrane)Popliteal vein

1	Fig. 6.90 Tibial nerve. A. Posterior view. B. Sural nerve. Fig. 6.91 Muscles in the lateral compartment of leg. A. Lateral view. B. Inferior view of the right foot, with the foot plantarflexed at the ankle. ABCommon fibular nerveFibularislongusFibularisbrevisFibular trochleaof calcaneus boneAnteriorborder of fibulaInterosseousmembraneMedialcuneiformMetatarsal IGroove oninferior aspectof cuboidFibularisbrevistendonFibularislongustendon Fig. 6.92 Common fibular nerve, and nerves and arteries of the lateral compartment of leg. A. Posterior view, right leg. B. Lateral view, right leg. ABSural communicating nervePenetrates deep fasciaSural nervePenetrates deep fasciaLateral sural nerveCommon fibular nerveSuperficial fibular nervePerforating branches of fibular arteryin posterior compartment(vessels in and around fibula)Deep fibularnerve Fig. 6.93 Muscles of the anterior compartment of leg.

1	Fig. 6.93 Muscles of the anterior compartment of leg. Anterior surfaceof fibulaExtensordigitorumlongusOrigin of extensordigitorum longusLateral surfaceof fibulaExtensor hallucis longusOrigin of extensorhallucis longusFibularistertiusAttachment to inferior surface of medial cuneiform and metatarsal ISubcutaneoussurface of tibiaTibialisanteriorOrigin of tibialisanterior Fig. 6.94 Anterior tibial artery and deep fibular nerve. Fig. 6.95 Foot. A. Dorsal aspect, right foot. B. Plantar aspect, right foot, showing the surface in contact with the ground when standing. AbductionAdductionGreat toeDigit IDigit IIDigit IIIDigit IVDigit VPhalangesLittle toeMetatarsals (I–V)Tarsal bonesABCut surface of medialmalleolus (tibia)Cut surface of lateralmalleolus (fibula)Calcaneal tendonMedial malleolusLateral malleolusHeelSesamoid bones Fig. 6.96 Bones of the foot. A. Dorsal view, right foot. B. Lateral view, right foot.

1	Fig. 6.96 Bones of the foot. A. Dorsal view, right foot. B. Lateral view, right foot. TalusPosteriorprocessof the talusBCalcaneusFibular trochleaGrooveCuboidMedial tubercleCuneiformsNavicularPhalangesMetatarsalsNavicularIntermediatetarsal boneTuberosity (on undersurface)CalcaneusTalusLateral processLateraltubercleGroove for tendon offlexor hallucis longusProximal groupof tarsal bonesMedialIntermediateLateralCuneiformsDistal groupof tarsal bonesCuboidDistalMiddleProximalA Fig. 6.97 Talus. A. Medial view. B. Inferior view. ABPosterior AnteriorArticular surfacefor navicularArticular surfacefor navicularNeckHeadBodyAnteriorPosteriorArticular surface withmedial malleolusMedialtubercleLateraltubercleGroove for flexorhallucis longusGroove for flexorhallucis longusArticular surface withdistal end of tibiaAnterior calcanealsurfaceMiddle calcanealsurfaceSulcus taliPosterior processof talusPosteriorcalcaneal surfaceArticular surface forcalcaneonavicularligament

1	Fig. 6.98 Calcaneus. A. Superior view. B. Inferior view. C. Lateral view. ABCAnterior talar articular surfacePosterior talararticular surfacePosterior talararticular surfaceAnterior talararticular surfaceMiddle talararticular surfaceMiddle talararticular surfaceSustentaculum taliMedialMedialMiddle part of posterior surface(insertion of calcaneal tendon)Upper part ofposterior surfaceLateralCalcanealsulcusArticular surface with cuboid boneCalcanealtubercleLateralprocessNotchCalcaneal tuberosity(lower part of posterior surface)MedialprocessGroove for tendon offlexor hallucis longusAttachment of calcaneofibularpart of lateral collateral ligamentof ankle jointFibular trochleaCalcaneal sulcus Fig. 6.99 Tarsal sinus. Lateral view, right foot. Fig. 6.100 Metatarsals and phalanges. Dorsal view, right foot. Fig. 6.101 Radiograph of ankle showing talar beak.

1	Fig. 6.99 Tarsal sinus. Lateral view, right foot. Fig. 6.100 Metatarsals and phalanges. Dorsal view, right foot. Fig. 6.101 Radiograph of ankle showing talar beak. Fig. 6.102 Ankle joint. A. Anterior view with right foot plantarflexed. B. Schematic of joint, posterior view. C. Superior view of the talus to show the shape of the articular surface. ABCInterosseousligamentTibiaMedialmalleolusLateralmalleolusLateral malleolusTalusFibulaFibulaTibiaArticular surface of talusArticular surfacenarrow posteriorlyPosteriorMedial malleolusAnteriorArticular surface wide anteriorly Fig. 6.103 Medial ligament of the ankle joint, right foot. Medialtubercleof talusSustentaculum taliof calcaneus bonePlantar calcaneonavicular ligamentTuberosity ofnavicular bonePosteriortibiotalar partTibiocalcaneal partTibionavicular partAnterior tibiotalar partMedial ligament of the ankle joint Fig. 6.104 Lateral ligament of the ankle joint. A. Lateral view, right foot. B. Posterior view, right foot.

1	Fig. 6.104 Lateral ligament of the ankle joint. A. Lateral view, right foot. B. Posterior view, right foot. Malleolar fossaAnterior talofibular ligamentCalcaneofibular ligamentPosteriortalofibularligamentPosteriortalofibular ligamentPosteriorprocess of talusMalleolar fossaFibulaTibiaTalusAB Fig. 6.105 Intertarsal joints, right foot. Fig. 6.106 Interosseous talocalcaneal ligament. Lateral view, right foot. Fig. 6.107 Talocalcaneonavicular joint. A. Medial view, right foot. B. Superior view, right foot, talus removed. C. Ligaments, medial view, right foot. D. Ligaments, lateral view, right foot. Fig. 6.108 Plantar ligaments, right foot. A. Plantar calcaneocuboid ligament (short plantar ligament). B. Long plantar ligament. Fig. 6.109 Tarsometatarsal, metatarsophalangeal, and interphalangeal joints, and the deep transverse metatarsal ligaments, right foot.

1	Fig. 6.109 Tarsometatarsal, metatarsophalangeal, and interphalangeal joints, and the deep transverse metatarsal ligaments, right foot. Fig. 6.110 Tarsal tunnel and flexor retinaculum. Posteromedial view, right foot. A. Bones. B. Tarsal tunnel and flexor retinaculum. ABTibiaTalusCalcaneusFlexor retinaculumTarsaltunnelTendon of flexordigitorum longusTendon oftibialis posteriorPulse of post-tibial arterymidway between heeland medial malleolusTendon offlexorhallucislongusTibial nervePosteriortibial arteryTibialis posteriorFlexor digitorumlongusTibial arteryTibial veinTibial nerveFlexor hallucislongus Fig. 6.111 Extensor retinacula, right foot. Anterior tibial arteryTendon of extensorhallucis longusTendon oftibialis anteriorDorsalispedis arteryFirst dorsalinterosseousmuscleInferior extensorretinaculumSuperior extensorretinaculumExtensordigitorum longusFibularis tertius Fig. 6.112 Fibular retinacula. Lateral view, right foot.

1	Fig. 6.112 Fibular retinacula. Lateral view, right foot. Tendons of fibularis longus andbrevis musclesSuperior fibular retinaculumInferior fibular retinaculum(at fibular trochlea on calcaneus) Fig. 6.113 Arches of the foot. A. Longitudinal arches, right foot. B. Transverse arch, left foot. Fig. 6.114 Support for arches of the foot. A. Ligaments. Medial view, right foot. B. Cross section through the foot to show tendons of muscles supporting the arches, left foot. Fig. 6.115 Plantar aponeurosis, right foot. Superficial transversemetatarsal ligamentsMedial process ofcalcaneal tuberosityPlantar aponeurosisAnterior arm of inferiorextensor retinaculum Fig. 6.116 Fibrous digital sheaths, right foot. Fig. 6.117 Extensor hoods. 1st dorsal interosseous muscleExtensor tendonsExtensor hoodDeep transverse metatarsal ligamentLumbricalFlexor digitorum longusExtension of PIP joints prevents overflexionFlexion of MTP jointprevents overextension

1	Fig. 6.118 Extensor digitorum brevis muscle, right foot. Fig. 6.119 First layer of muscles in the sole of the right foot. Fig. 6.120 Second layer of muscles in the sole of the right foot. Fig. 6.121 MRI of posterior foot in a patient with plantar fasciitis shows thickening of the plantar aponeurosis at the calcaneal insertion and a calcaneal spur. Fig. 6.122 Third layer of muscles in the sole of the right foot. FlexorhallucisbrevisTransverse headOblique headAdductor hallucisFlexor digitiminimi brevisTendon of fibularislongus muscleTendon of tibialisposterior muscleTendon of flexorhallucis longus Fig. 6.123 Fourth layer of muscles in the sole of the right foot. Fig. 6.124 Arteries in the sole of the right foot. Digital branchesDeep plantar artery: terminal branchof dorsalis pedis arteryPlantar metatarsal arteriesPerforatingvesselsDeepplantar archLateral plantararteryPosterior tibial arteryMedial plantarartery Fig. 6.125 Dorsalis pedis artery right foot.

1	Fig. 6.125 Dorsalis pedis artery right foot. ExtensorhoodDorsaldigitalarteriesAnterior tibial arteryDorsalis pedisarteryAnterior medialmalleolar arteryAnterior lateralmalleolar arteryMedial and lateraltarsal branchesExtensorhallucislongusDorsalis pedisarteryDeep plantararteryFirst dorsalmetatarsalarteryFirst dorsalinterosseousmuscleArcuate arteryTendon of extensordigitorum longusto toe II Fig. 6.126 Superficial veins of the right foot. Fig. 6.127 Lateral and medial plantar nerves. A. Sole of the right foot. B. Cutaneous distribution of right foot. Fig. 6.128 A.Terminal branches of superficial and deep fibular nerves in the right foot. B. Cutaneous distribution right foot. Deep fibularnerveABBranches tofirst and seconddorsal interosseiExtensordigitorumbrevisBranch of deepfibular to extensordigitorum brevisSuperficialfibular nerveSaphenous nerveSuperficialfibular nerveDeep fibular nerveSural nerve

1	Fig. 6.129 Avoiding the sciatic nerve. A. Posterior view of the gluteal region of a man with the position of the sciatic nerve indicated. B. Posterolateral view of the left gluteal region with gluteal quadrants and the position of the sciatic nerve indicated. Fig. 6.130 Position of the femoral artery in the femoral triangle. Anterior thigh. Inguinal ligamentAnterior superior iliac spineFemoral nerveFemoral arteryFemoral veinLymphatics passing through femoral canalMedial margin ofsartorius muscleMedial margin of adductor longus musclePubic tuberclePubic symphysis Fig. 6.131 Identifying structures around the knee. A. Anterior view of the right knee. B. Lateral view of the partially flexed right knee. C. Lateral view of the extended right knee, thigh, and gluteal region.

1	C. Lateral view of the extended right knee, thigh, and gluteal region. ABCVastus lateralisVastus medialisQuadriceps tendonQuadriceps femoris musclePatellaPatellaPatellarligamentPatellar ligamentTibial tuberosityTendon ofbiceps femorisHead of fibulaCommon fibular nerveTensor fasciae lataeIliotibial tractHamstring musclesGluteus maximus Fig. 6.132 Visualizing the contents of the popliteal fossa. Posterior view of the left knee. Popliteal fossaPopliteal arteryPopliteal veinCommon fibular nerveBiceps femoris muscle and tendonHead of fibulaPenetrates deep fasciaLateral head of gastrocnemius muscleMedial head of gastrocnemius muscleTibial nerveSemimembranosus muscleSemitendinosus tendonSmall saphenous vein Fig. 6.133 Finding the tarsal tunnel—the gateway to the foot. Fig. 6.134 Identifying tendons around the ankle and in the foot. A. Medial side of the right foot. B. Posterior aspect of the right foot. C. Lateral side of the right foot. D. Dorsal aspect of the right foot.

1	Fig. 6.135 Finding the dorsalis pedis artery. Dorsalis pedis arteryExtensor digitorumlongus tendon to second toeExtensor hallucislongus tendon Fig. 6.136 Position of the plantar arch. Fig. 6.137 Major superficial veins. A. Dorsal aspect of the right foot. B. Anterior view of right lower limb. C. Posterior aspect of the left thigh, leg, and foot. Fig. 6.138 Where to feel peripheral arterial pulses in the lower limb. Fig. 6.139 A. Normal knee joint showing the tibial collateral ligament and the medial and lateral menisci. Proton density (PD)-weighted magnetic resonance image in the coronal plane. B. Knee joint showing a torn tibial collateral ligament. PD-weighted magnetic resonance image in the coronal plane. Fig. 6.140 A. Knee joint showing an intact anterior cruciate ligament. T2-weighted magnetic resonance image in the sagittal plane.

1	Fig. 6.140 A. Knee joint showing an intact anterior cruciate ligament. T2-weighted magnetic resonance image in the sagittal plane. B. Knee joint showing a torn anterior cruciate ligament. T2-weighted magnetic resonance image in the sagittal plane. C. Knee joint showing a torn medial meniscus (the broken off portion of the posterior horn has moved into the anterior aspect of the joint giving the impression of a ‘double meniscus’ in this location). Proton density-weighted magnetic resonance image in the sagittal plane. AFemurPatellaAnterior cruciate ligamentTibiaBFemurTorn anteriorcruciate ligamentTibiaAnterior cruciateligament fragmentPatellaCMedial femoral condyleTorn posterior “horn”of medial meniscusTibiaAnterior “horn” of medial meniscusDisplaced fragment of posterior hornof medial meniscus

1	Fig. 6.141 Radiograph (A) and MRI (B) of soft tissue ulceration and erosion in the adjacent calcaneus. After debridement and placement of antibiotics beads in the wound there is progressive healing (C). eFig. 6.142 Popliteal fossa showing position of the popliteal artery and vein and sciatic nerve. T1-weighted magnetic resonance image in the axial plane. eFig. 6.143 Pulmonary embolus. Axial computed tomogram. Left atriumAortaRight inferior pulmonary artery with embolusEmbolus eFig. 6.144 Ankle showing a ruptured calcaneal tendon. T2-weighted magnetic resonance image in the sagittal plane. eFig. 6.145 A. Normal ankle joint showing an intact anterior talofibular ligament. T1-weighted magnetic resonance image in the axial plane. B. Ankle joint showing a torn anterior talofibular ligament. T2-weighted magnetic resonance image in the axial plane. Table 6.1 Branches of the lumbosacral plexus associated with the lower limb

1	Table 6.1 Branches of the lumbosacral plexus associated with the lower limb Table 6.2 Muscles of the gluteal region (spinal segments in bold are the major segments innervating the muscle) Table 6.3 Muscles of the anterior compartment of thigh (spinal segments in bold are the major segments innervating the muscle) Table 6.4 Muscles of the medial compartment of thigh (spinal segments in bold are the major segments innervating the muscle) Table 6.5 Muscles of the posterior compartment of thigh (spinal segments in bold are the major segments innervating the muscle) Table 6.6 Superficial group of muscles in the posterior compartment of leg (spinal segments in bold are the major segments innervating the muscle) Table 6.7 Deep group of muscles in the posterior compartment of leg (spinal segments in bold are the major segments innervating the muscle) Table 6.8 Muscles of the lateral compartment of leg (spinal segments in bold are the major segments innervating the muscle)

1	Table 6.8 Muscles of the lateral compartment of leg (spinal segments in bold are the major segments innervating the muscle) Table 6.9 Muscles of the anterior compartment of leg (spinal segments in bold are the major segments innervating the muscle) Table 6.10 Muscles of the dorsal aspect of the foot (spinal segments in bold are the major segments innervating the muscle) Table 6.11 First layer of muscles in the sole of the foot (spinal segments in bold are the major segments innervating the muscle) Table 6.12 Second layer of muscles in the sole of the foot (spinal segments in bold are the major segments innervating the muscle) Table 6.13 Third layer of muscles in the sole of the foot (spinal segments in bold are the major segments innervating the muscle) Table 6.14 Fourth layer of muscles in the sole of the foot (spinal segments in bold are the major segments innervating the muscle) In the clinic

1	Table 6.14 Fourth layer of muscles in the sole of the foot (spinal segments in bold are the major segments innervating the muscle) In the clinic The pelvic bones, sacrum, and associated joints form a bony ring surrounding the pelvic cavity. Soft tissue and visceral organ damage must be suspected when the pelvis is fractured. Patients with multiple injuries and evidence of chest, abdominal, and lower limb trauma should also be investigated for pelvic trauma. Pelvic fractures can be associated with appreciable blood loss (concealed exsanguination) and blood transfusion is often required. In addition, this bleeding tends to form a significant pelvic hematoma, which can compress nerves, press on organs, and inhibit pelvic visceral function (Fig. 6.25). There are many ways of classifying pelvic fractures, which enable the surgeon to determine the appropriate treatment and the patient’s prognosis. Pelvic fractures are generally of four types.

1	There are many ways of classifying pelvic fractures, which enable the surgeon to determine the appropriate treatment and the patient’s prognosis. Pelvic fractures are generally of four types. Type 1 injuries occur without disruption of the bony pelvic ring (e.g., a fracture of the iliac crest). These types of injuries are unlikely to represent significant trauma, though in the case of a fracture of the iliac crest, blood loss needs to be assessed. Type 2 injuries occur with a single break in the bony pelvic ring. An example of this would be a single fracture with diastasis (separation) of the symphysis pubis. Again, these injuries are relatively benign in nature, but it may be appropriate to assess for blood loss. Type 3 injuries occur with double breaks in the bony pelvic ring. These include bilateral fractures of the pubic rami, which may produce urethral damage. Type 4 injuries occur at and around the acetabulum.

1	Type 4 injuries occur at and around the acetabulum. Other types of pelvic ring injuries include fractures of the pubic rami and disruption of the sacro-iliac joint with or without dislocation. This may involve significant visceral pelvic trauma and hemorrhage. Other general pelvic injuries include stress fractures and insufficiency fractures, as seen in athletes and elderly patients with osteoporosis, respectively. In the clinic

1	Femoral neck fractures (Fig. 6.28) can interrupt the blood supply to the femoral head. The blood supply to the head and neck is primarily from an arterial ring formed by the branches of the medial and lateral circumflex femoral arteries around the base of the femoral neck. From here, vessels course along the neck, penetrate the capsule, and supply the femoral head. The blood supply to the femoral head and femoral neck is further enhanced by the artery of the ligamentum teres, a branch of the obturator artery, which is generally small and variable. Femoral neck fractures may disrupt associated vessels and lead to necrosis of the femoral head. Femoral neck fractures can be divided into three categories depending on the location of the fracture line: subcapital (fracture line passes across the femoral head-neck junction), transcervical (fracture line passes through the midportion of the femoral neck), and basicervical (fracture line passes across the base of the neck). Subcapital

1	the femoral head-neck junction), transcervical (fracture line passes through the midportion of the femoral neck), and basicervical (fracture line passes across the base of the neck). Subcapital fractures have the highest risk of developing necrosis of the femoral head, and basicervical fractures have the lowest risk. Elderly patients with osteoporosis tend to have transverse subcapital fractures following low-energy trauma such as a fall from a standing height. Conversely, younger patients usually sustain more vertical fractures of the distal femoral neck (basicervical) after high-energy trauma such as a fall from a great height or due to axial load applied to an abducted knee, such as during a motor vehicle accident.

1	In the clinic In these fractures, the break usually runs from the greater trochanter through to the lesser trochanter and does not involve the femoral neck. Intertrochanteric fractures preserve the femoral neck blood supply and do not render the femoral head ischemic. They are most commonly seen in the elderly and result from low-energy impact (Fig. 6.29). Sometimes isolated fractures of the greater or the lesser trochanter can occur. An isolated fracture of the lesser trochanter in adults is most commonly pathological and due to an underlying malignant deposit. In the clinic An appreciable amount of energy is needed to fracture the femoral shaft. This type of injury is therefore accompanied by damage to the surrounding soft tissues, which include the muscle compartments and the structures they contain. In the clinic

1	In the clinic The normal flow of blood in the lower limbs is from the skin and subcutaneous tissues to the superficial veins, which drain via perforating veins to the deep veins, which in turn drain into the iliac veins and inferior vena cava. The normal flow of blood in the venous system depends upon the presence of competent valves, which prevent reflux. Venous return is supplemented with contraction of the muscles in the lower limb, which pump the blood toward the heart. When venous valves become incompetent they tend to place extra pressure on more distal valves, which may also become incompetent. This condition produces dilated tortuous superficial veins (varicose veins) in the distribution of the great (long) and small (short) saphenous venous systems.

1	Varicose veins occur more commonly in women than in men, and symptoms are often aggravated by pregnancy. Some individuals have a genetic predisposition to developing varicose veins. Valves may also be destroyed when a deep vein thrombosis occurs if the clot incorporates the valve into its interstices; during the process of healing and recanalization the valve is destroyed, rendering it incompetent. Typical sites for valvular incompetence include the junction between the great (long) saphenous vein and the femoral vein, perforating veins in the midthigh, and the junction between the small (short) saphenous vein and the popliteal vein.

1	Varicose veins may be unsightly, and soft tissue changes may occur with chronic venous incompetence. As the venous pressure rises, increased venular and capillary pressure damages the cells, and blood and blood products extrude into the soft tissue. This may produce a brown pigmentation in the skin, and venous eczema may develop. Furthermore, if the pressure remains high the skin may break down and ulcerate, and many weeks of hospitalization may be needed for this to heal. Treatments for varicose veins include tying off the valve, “stripping” (removing) the great (long) and small (short) saphenous systems, and in some cases valvular reconstruction. In the clinic Thrombosis may occur in the deep veins of the lower limb and within the pelvic veins. Its etiology was eloquently described by Virchow, who described the classic triad (venous stasis, injury to the vessel wall, and hypercoagulable states) that precipitates thrombosis.

1	In some patients a deep vein thrombosis (DVT) in the calf veins may propagate into the femoral veins. This clot may break off and pass through the heart to enter the pulmonary circulation, resulting in occlusion of the pulmonary artery, cardiopulmonary arrest, and death. A significant number of patients undergoing surgery are likely to develop a DVT, so most surgical patients are given specific prophylactic treatment to prevent thrombosis. A typical DVT prophylactic regimen includes anticoagulant injections and graduated stockings (to prevent deep venous stasis and facilitate emptying of the deep veins). Although physicians aim to prevent the formation of DVT, it is not always possible to detect it because there may be no clinical signs. Calf muscle tenderness, postoperative pyrexia, and limb swelling can be helpful clues. The diagnosis is predominantly made by duplex Doppler sonography or rarely by ascending venography.

1	If DVT is confirmed, intravenous and oral anticoagulation are started to prevent extension of the thrombus. In the clinic Vascular access to the lower limb Deep and inferior to the inguinal ligament are the femoral artery and femoral vein. The femoral artery is palpable as it passes over the femoral head and may be easily demonstrated using ultrasound. If arterial or venous access is needed rapidly, a physician can use the femoral approach to these vessels. Many radiological procedures involve catheterization of the femoral artery or the femoral vein to obtain access to the contralateral lower limb, the ipsilateral lower limb, the vessels of the thorax and abdomen, and the cerebral vessels. Cardiologists also use the femoral artery to place catheters in vessels around the arch of the aorta and into the coronary arteries to perform coronary angiography and angioplasty.

1	Cardiologists also use the femoral artery to place catheters in vessels around the arch of the aorta and into the coronary arteries to perform coronary angiography and angioplasty. Access to the femoral vein permits catheters to be maneuvered into the renal veins, the gonadal veins, the right atrium, and the right side of the heart, including the pulmonary artery and distal vessels of the pulmonary tree. Access to the superior vena cava and the great veins of the neck is also possible. In the clinic Trendelenburg’s sign occurs in people with weak or paralyzed abductor muscles (gluteus medius and gluteus minimus) of the hip. The sign is demonstrated by asking the patient to stand on one limb. When the patient stands on the affected limb, the pelvis severely drops over the swing limb.

1	Positive signs are typically found in patients with damage to the superior gluteal nerve. Damage to this nerve may occur with associated pelvic fractures, with space-occupying lesions within the pelvis extending into the greater sciatic foramen, and in some cases relating to hip surgery during which there has been disruption of and subsequent atrophy of the insertion of the gluteus medius and gluteus minimus tendons on the greater trochanter. In patients with a positive Trendelenburg’s sign, gait also is abnormal. Typically during the stance phase of the affected limb, the weakened abductor muscles allow the pelvis to tilt inferiorly over the swing limb. The patient compensates for the pelvic drop by lurching the trunk to the affected side to maintain the level of the pelvis throughout the gait cycle. In the clinic

1	In the clinic From time to time it is necessary to administer drugs intramuscularly, that is, by direct injection into muscles. This procedure must be carried out without injuring neurovascular structures. A typical site for an intramuscular injection is the gluteal region. The sciatic nerve passes through this region and needs to be avoided. The safest place to inject is the upper outer quadrant of either gluteal region. The gluteal region can be divided into quadrants by two imaginary lines positioned using palpable bony landmarks (Fig. 6.49). One line descends vertically from the highest point of the iliac crest. Another line is horizontal and passes through the first line midway between the highest point of the iliac crest and the horizontal plane through the ischial tuberosity.

1	It is important to remember that the gluteal region extends as far forward as the anterior superior iliac spine. The sciatic nerve curves through the upper lateral corner of the lower medial quadrant and descends along the medial margin of the lower lateral quadrant. Occasionally, the sciatic nerve bifurcates into its tibial and common fibular branches in the pelvis, in which case the common fibular nerve passes into the gluteal region through, or even above, the piriformis muscle. The superior gluteal nerve and vessels normally enter the gluteal region above the piriformis and pass superiorly and forward. The anterior corner of the upper lateral quadrant is normally used for injections to avoid injuring any part of the sciatic nerve or other nerves and vessels in the gluteal region. A needle placed in this region enters the gluteus medius anterosuperior to the margin of the gluteus maximus. In the clinic

1	In the clinic Compartment syndrome occurs when there is swelling within a fascial enclosed muscle compartment in the limbs. Typical causes include limb trauma, intracompartment hemorrhage, and limb compression. As pressure within the compartment elevates, capillary blood flow and tissue perfusion is compromised, which can ultimately lead to neuromuscular damage if not treated. In the clinic Muscle injuries to the lower limb Muscle injuries may occur as a result of direct trauma or as part of an overuse syndrome. Muscle injuries may occur as a minor muscle tear, which may be demonstrated as a focal area of fluid within the muscle. With increasingly severe injuries, more muscle fibers are torn and this may eventually result in a complete muscle tear. The usual muscles in the thigh that tear are the hamstring muscles. Tears in the muscles below the knee typically occur within the soleus muscle, though other muscles may be affected.

1	Injury to the hamstring muscles is a common source of pain in athletes, particularly in those competing in sports requiring a high degree of power and speed (such as sprinting, track and field, football) where the hamstring muscles are very susceptible to injury from excessive stretching.

1	The injury can range from a mild muscle strain to a complete tear of a muscle or a tendon. It usually occurs during sudden accelerations and decelerations or rapid change in direction. In adults, the most commonly injured is the muscle-tendon junction, which is a wide transition zone between the muscle and the tendon. An avulsion of the ischial tuberosity with proximal hamstring origin attachment is common in the adolescent population, particularly during sudden hip flexion because the ischial apophysis is the weakest element of the proximal hamstring unit in this age group (Fig. 6.64). Both ultrasound and MRI can be used to assess the hamstring injury with the MRI providing not only the information about the extent of the injury but also give some indication about the prognosis (future risk of re-tear, loss of function, etc). In the clinic

1	In the clinic Peripheral vascular disease is often characterized by reduced blood flow to the legs. This disorder may be caused by stenoses (narrowing) and/or occlusions (blockages) in the lower aorta and the iliac, femoral, tibial, and fibular vessels. Patients typically have chronic leg ischemia and “acute on chronic” leg ischemia. Chronic leg ischemia is a disorder in which vessels have undergone atheromatous change, and often there is significant luminal narrowing (usually over 50%). Most patients with peripheral arterial disease have widespread arterial disease (including cardiovascular and cerebrovascular disease), which may be clinically asymptomatic. Some of these patients develop such severe ischemia that the viability of the limb is threatened (critical limb ischemia).

1	The commonest symptom of chronic leg ischemia is intermittent claudication. Patients typically have a history of pain that develops in the calf muscles (usually associated with occlusions or narrowing in the femoral artery) or the buttocks (usually associated with occlusion or narrowing in the aorto-iliac segments). The pain experienced in these muscles is often cramplike and occurs with walking. The patient rests and is able to continue walking up to the same distance until the pain recurs and stops walking as before. In some patients with chronic limb ischemia, an acute event blocks the vessels or reduces the blood supply to such a degree that the viability of the limb is threatened. Occasionally a leg may become acutely ischemic with no evidence of underlying atheromatous disease. In these instances a blood clot is likely to have embolized from the heart. Patients with mitral valve disease and atrial fibrillation are prone to embolic disease.

1	Critical limb ischemia occurs when the blood supply to the limb is so poor that the viability of the limb is severely threatened, and in this case many patients develop gangrene, ulceration, and severe rest pain in the foot. These patients require urgent treatment, which may be in the form of surgical reconstruction, radiological angioplasty, or even amputation. In the clinic Menisci can get torn during forceful rotation or twisting of the knee, but significant trauma is not always necessary for a tear to occur. There are various patterns of meniscal tearing depending on the cleavage plane such as vertical tears (perpendicular to the tibial plateau), horizontal tears (parallel to the long axis of the meniscus and perpendicular to the tibial plateau), or bucket handle tears (longitudinal tear where the torn portion of the meniscus forms a handle shaped fragment which gets displaced into the intercondylar notch).

1	The patient usually complains of pain localized to the medial or lateral side of the knee, knee locking or clicking, sensation of knee giving way, and swelling, which can be intermittent and usually delayed. MRI is the modality of choice to assess meniscal tears and detect other associated injuries, such as ligamentous tears and articular cartilage damage (Fig. 6.74A). Arthroscopy is usually performed to repair a tear, debride the damaged meniscal material, or rarely remove the entire torn meniscus (Fig. 6.74B). In the clinic The collateral ligaments are responsible for stabilizing the knee joint, controlling its sideway movements, and protecting the knee from excessive motion.

1	In the clinic The collateral ligaments are responsible for stabilizing the knee joint, controlling its sideway movements, and protecting the knee from excessive motion. Injury to the fibular collateral ligament occurs when excessive outward force is applied to the medial side of the knee (varus force), and is less common than an injury to the tibial collateral ligament that is damaged when excessive force is applied inward to the lateral side of the joint (valgus force). Injuries to the tibial collateral ligament can be part of a so called “unhappy triad” that also involves tears of the medial meniscus and the anterior cruciate ligament. The spectrum of injuries to collateral ligaments of the knee range from minor sprains where the ligaments are slightly stretched, but still able to stabilize the knee joint, to full thickness tears where all fibers are torn and the ligaments lose their stabilizing function. In the clinic

1	The anterior cruciate ligament (ACL) is most frequently injured during non-contact activities when there is a sudden change in the direction of movement (cutting or pivoting) (Fig. 6.81). Contact sports may also result in ACL injury due to sudden twisting, hyperextension, and valgus force related to direct collision. The injury usually affects the mid-portion of the ligament and manifests itself as a complete or partial discontinuity of the fibers or abnormal orientation and contour of the ligament. With an acute ACL tear, a sudden click or pop can be heard and the knee becomes rapidly swollen. Several tests are used to clinically assess the injury, and the diagnosis is usually confirmed by MRI. A full thickness ACL tear causes instability of the knee joint. The treatment depends on the desired level of activity of the patient. In those with high activity levels, surgical reconstruction of the ligament is required. Those with low activity levels may opt for knee bracing and

1	on the desired level of activity of the patient. In those with high activity levels, surgical reconstruction of the ligament is required. Those with low activity levels may opt for knee bracing and physiotherapy; however, in the long term the internal damage to the knee leads to the development of early osteoarthritis.

1	A tear to the posterior cruciate ligament (PCL) requires significant force, so it rarely occurs in isolation. It usually occurs during hyperextension of the knee or as a result of a direct blow to a bent knee such as when striking the knee against the dashboard in a motor vehicle accident. Typically, the injury presents as posterior displacement of the tibia on physical examination (the so called tibial sag sign). Patients complain of knee pain and swelling, inability to bear weight, and instability. The diagnosis is confirmed on MRI. The management, as in ACL injury, depends on the degree of the injury (sprain, partial thickness, full thickness) and the level of desired activity. In the clinic Degenerative joint disease occurs throughout many joints within the body. Articular degeneration may result from an abnormal force across the joint with a normal cartilage or a normal force with abnormal cartilage.

1	Typically degenerative joint disease occurs in synovial joints and the process is called osteoarthritis. In the joints where osteoarthritis occurs the cartilage and bony tissues are usually involved, with limited change within the synovial membrane. The typical findings include reduction in the joint space, eburnation (joint sclerosis), osteophytosis (small bony outgrowths), and bony cyst formation. As the disease progresses the joint may become malaligned, its movement may become severely limited, and there may be significant pain. The commonest sites for osteoarthritis include the small joints of the hands and wrist, and in the lower limb, the hip and knee are typically affected, though the tarsometatarsal and metatarsophalangeal articulations may undergo similar changes.

1	The etiology of degenerative joint disease is unclear, but there are some associations, including genetic predisposition, increasing age (males tend to be affected younger than females), overuse or underuse of joints, and nutritional and metabolic abnormalities. Further factors include joint trauma and pre-existing articular disease or deformity. The histological findings of osteoarthritis consist of degenerative changes within the cartilage and the subchondral bone. Further articular damage worsens these changes, which promote further abnormal stresses upon the joint. As the disease progresses the typical finding is pain, which is usually worse on rising from bed and at the end of a day’s activity. Commonly it is aggravated by the extremes of movement or unaccustomed exertion. Stiffness and limitation of movement may ensue.

1	Treatment in the first instance includes alteration of lifestyle to prevent pain and simple analgesia. As symptoms progress a joint replacement may be necessary, but although joint replacement appears to be the panacea for degenerative joint disease, it is not without risks and complications, which include infection and failure in the short and long term. In the clinic Examination of the knee joint It is important to establish the nature of the patient’s complaint before any examination. The history should include information about the complaint, the signs and symptoms, and the patient’s lifestyle (level of activity). This history may give a significant clue to the type of injury and the likely findings on clinical examination, for example, if the patient was kicked around the medial aspect of the knee, a valgus deformity injury to the tibial collateral ligament might be suspected.

1	The examination should include assessment in the erect position, while walking, and on the couch. The affected side must be compared with the unaffected side. There are many tests and techniques for examining the knee joint, including the following. Lachman’s test—the patient lies on the couch. The examiner places one hand around the distal femur and the other around the proximal tibia and then elevates the knee, producing 20° of flexion. The patient’s heel rests on the couch. The examiner’s thumb must be on the tibial tuberosity. The hand on the tibia applies a brisk anteriorly directed force. If the movement of the tibia on the femur comes to a sudden stop, it is a firm endpoint. If it does not come to a sudden stop, the endpoint is described as soft and is associated with a tear of the anterior cruciate ligament.

1	Anterior drawer test—a positive anterior drawer test is when the proximal head of a patient’s tibia can be pulled anteriorly on the femur. The patient lies supine on the couch. The knee is flexed to 90° and the heel and sole of the foot are placed on the couch. The examiner sits gently on the patient’s foot, which has been placed in a neutral position. The index fingers are used to check that the hamstrings are relaxed while the other fingers encircle the upper end of the tibia and pull the tibia. If the tibia moves forward, the anterior cruciate ligament is torn. Other peripheral structures, such as the medial meniscus or meniscotibial ligaments, must also be damaged to elicit this sign.

1	Pivot shift test—there are many variations of this test. The patient’s foot is wedged between the examiner’s body and elbow. The examiner places one hand flat under the tibia pushing it forward with the knee in extension. The other hand is placed against the patient’s thigh pushing it the other way. The lower limb is taken into slight abduction by the examiner’s elbow with the examiner’s body acting as a fulcrum to produce the valgus. The examiner maintains the anterior tibial translation and the valgus and initiates flexion of the patient’s knee. At about 20°–30° the pivot shift will occur as the lateral tibial plateau reduces. This test demonstrates damage to the posterolateral corner of the knee joint and the anterior cruciate ligament.

1	Posterior drawer test—a positive posterior drawer test occurs when the proximal head of a patient’s tibia can be pushed posteriorly on the femur. The patient is placed in a supine position and the knee is flexed to approximately 90° with the foot in the neutral position. The examiner sits gently on the patient’s foot placing both thumbs on the tibial tuberosity and pushing the tibia backward. If the tibial plateau moves, the posterior cruciate ligament is torn. Assessment of other structures of the knee Assessment of the tibial collateral ligament can be performed by placing a valgus stress on the knee. Assessment of lateral and posterolateral knee structures requires more complex clinical testing. The knee will also be assessed for: joint line tenderness, patellofemoral movement and instability, presence of an effusion, muscle injury, and popliteal fossa masses.

1	The knee will also be assessed for: joint line tenderness, patellofemoral movement and instability, presence of an effusion, muscle injury, and popliteal fossa masses. After the clinical examination has been carried out, further investigations usually include plain radiography and possibly magnetic resonance imaging, which allows the radiologist to assess the menisci, cruciate ligaments, collateral ligaments, bony and cartilaginous surfaces, and soft tissues. Arthroscopy may be carried out and damage to any internal structures repaired or trimmed. An arthroscope is a small camera that is placed into the knee joint through the anterolateral or anteromedial aspect of the knee joint. The joint is filled with a saline solution and the telescope is manipulated around the knee joint to assess the cruciate ligaments, menisci, and cartilaginous surfaces. In the clinic Anterolateral ligament of the knee

1	In the clinic Anterolateral ligament of the knee A ligament associated at its origin with the fibular collateral ligament of the knee has been described. This ligament (anterolateral ligament of the knee) courses from the lateral femoral epicondyle to the anterolateral region of the proximal end of the tibia and may control internal rotation of the tibia. (J Anat 2013;223:321–328) In the clinic The popliteal artery can become abnormally dilated, forming an aneurysm. The artery is considered aneurysmal when its diameter exceeds 7 mm. Although popliteal artery aneurysms can occur in isolation, they are most commonly associated with aneurysms in other large vessels such as the femoral artery or the thoracic or abdominal aorta. Therefore, once a popliteal aneurysm has been detected, the entire arterial tree needs to be investigated for the presence of coexisting aneurysms elsewhere in the body.

1	Popliteal artery aneurysms tend to undergo thrombosis and are less likely to rupture than other aneurysms. Therefore the complications are mainly related to distal embolization of the arterial tree and lower limb ischemia, which in the most severe cases can lead to leg amputation. Ultrasound with duplex Doppler is the most helpful way of diagnosing a popliteal artery aneurysm because it can demonstrate abnormal dilation of the artery, confirm or rule out thrombus within the aneurysm, and help distinguish it from other masses of the popliteal fossa such as a synovial cyst (Baker’s cyst). Popliteal artery aneurysms are usually repaired surgically in view of high risk of thromboembolic complications. In the clinic

1	In the clinic Rupture of the calcaneal tendon is often related to sudden or direct trauma. This type of injury frequently occurs in a normal healthy tendon. In addition, there are certain conditions that may predispose the tendon to rupture. Among these conditions are tendinopathy (due to overuse, or to age-related degenerative changes) and previous calcaneal tendon interventions such as injections of pharmaceuticals and the use of certain antibiotics (quinolone group). The diagnosis of calcaneal tendon rupture is relatively straightforward. The patient typically complains of “being kicked” or “shot” behind the ankle, and clinical examination often reveals a gap in the tendon. In the clinic Neurological examination of the legs

1	In the clinic Neurological examination of the legs Some of the commonest conditions that affect the legs are peripheral neuropathy (particularly associated with diabetes mellitus), lumbar nerve root lesions (associated with pathology of the intervertebral discs), fibular nerve palsy, and spastic paraparesis. Look for muscle wasting—loss of muscle mass may indicate loss of or reduced innervation.

1	Look for muscle wasting—loss of muscle mass may indicate loss of or reduced innervation. Test the power in muscle groups—hip flexion (L1, L2—iliopsoas—straight leg raise); knee flexion (L5 to S2—hamstrings—the patient tries to bend the knee while the examiner applies force to the leg to hold the knee in extension); knee extension (L3, L4—quadriceps femoris—the patient attempts to keep the leg straight while the examiner applies a force to the leg to flex the knee joint); ankle plantarflexion (S1, S2—the patient pushes the foot down while the examiner applies a force to the plantar surface of the foot to dorsiflex the ankle joint); ankle dorsiflexion (L4, L5—the patient pulls the foot upward while the examiner applies a force to the dorsal aspect of the foot to plantarflex the ankle joint).

1	Examine knee and ankle reflexes—a tap with a tendon hammer on the patellar ligament (tendon) tests reflexes at the L3–L4 spinal levels, and tapping the calcaneal tendon tests reflexes at the S1–S2 spinal levels. Assess status of general sensory input to lumbar and upper sacral spinal cord levels—test light touch, pin prick, and vibration sense at dermatomes in the lower limb. In the clinic

1	Assess status of general sensory input to lumbar and upper sacral spinal cord levels—test light touch, pin prick, and vibration sense at dermatomes in the lower limb. In the clinic Footdrop is an inability to dorsiflex the foot. Patients with footdrop have a characteristic “steppage” gait. As the patient walks, the knee of the affected limb is elevated to an abnormal height during the swing phase to prevent the foot from dragging. At the end of the swing phase, the foot “slaps” the ground. Also, the unaffected limb often acquires a characteristic tiptoe pattern of gait during the stance phase. A typical cause of footdrop is damage to the common fibular nerve, which may occur with fractures of the fibular neck. Other causes include disc protrusion compressing the L5 nerve root, disorders of the sciatic nerve and the lumbosacral plexus, and pathologies of the spinal cord and brain. In the clinic

1	In the clinic The common fibular nerve is susceptible to injury as it passes around the lateral aspect of the neck of the fibula. It can be injured as a result of a direct trauma (blow or laceration), secondary to knee injury (knee dislocation), or as a consequence of a proximal fibular fracture. Sometimes damage to the nerve can be iatrogenic, that is, damaged during arthroscopy or knee surgery. Symptoms of common fibular nerve injury are often observed in bed-bound patients, particularly in those with decreased levels of consciousness, due to prolonged external pressure to the knee leading to nerve compression and neuropathy. Similarly, application of a tight cast or a brace to the leg can compress the nerve, producing symptoms of fibular muscle palsy. Apart from a foot drop, other symptoms of common fibular nerve injury include loss of sensation over the lateral aspect of the leg and dorsum of the foot, and wasting of fibular and anterior tibial muscles. In the clinic

1	In the clinic Occasionally there is a superior projection of the distal aspect of the talus, which is beak-shaped (Fig. 6.101). It is often associated with the presence of a bony or fibrous joint between the talus and calcaneus. In the clinic Fracture of the talus The talus is an unusual bone because it ossifies from a single primary ossification center, which initially appears in the neck. The posterior aspect of the talus appears to ossify last, normally after puberty. In up to 50% of people there is a small accessory ossicle (the os trigonum) posterior to the lateral tubercle of the posterior process. Articular cartilage covers approximately 60% of the talar surface and there are no direct tendon or muscle attachments to the bone.

1	One of the problems with fractures of the talus is that the blood supply to the bone is vulnerable to damage. The main blood supply to the bone enters the talus through the tarsal sinus from a branch of the posterior tibial artery. This vessel supplies most of the neck and the body of the talus. Branches of the dorsalis pedis artery enter the superior aspect of the talar neck and supply the dorsal portion of the head and neck, and branches from the fibular artery supply a small portion of the lateral talus. Fractures of the neck of the talus often interrupt the blood supply to the talus, so making the body and posterior aspect of the talus susceptible to osteonecrosis, which may in turn lead to premature osteoarthritis and require extensive surgery. In the clinic An appreciation of ankle anatomy is essential to understand the wide variety of fractures that may occur at and around the ankle joint.

1	In the clinic An appreciation of ankle anatomy is essential to understand the wide variety of fractures that may occur at and around the ankle joint. The ankle joint and related structures can be regarded as a fibro-osseous ring oriented in the coronal plane. The upper part of the ring is formed by the joint between the distal ends of the fibula and tibia and by the ankle joint itself. The sides of the ring are formed by the ligaments that connect the medial malleolus and lateral malleolus to the adjacent tarsal bones. The bottom of the ring is not part of the ankle joint, but consists of the subtalar joint and the associated ligaments. Visualizing the ankle joint and surrounding structures as a fibro-osseous ring allows the physician to predict the type of damage likely to result from a particular type of injury. For example, an inversion injury may fracture the medial malleolus and tear ligaments anchoring the lateral malleolus to the tarsal bones.

1	The ring may be disrupted not only by damage to the bones (which produces fractures), but also by damage to the ligaments. Unlike bone fractures, damage to ligaments is unlikely to be appreciated on plain radiographs. When a fracture is noted on a plain radiograph, the physician must always be aware that there may also be appreciable ligamentous disruption. The Ottawa ankle rules were developed to assist clinicians in deciding whether patients with acute ankle injuries require investigation with radiographs in order to avoid unnecessary studies. Named after the hospital where they were developed, the rules are highly sensitive and have reduced the utilization of unwarranted ankle radiographs since their implementation. An ankle x-ray series is required if there is ankle pain and any of the following: Bone tenderness along the distal 6 cm of the posterior tibia or tip of the medial malleolus

1	An ankle x-ray series is required if there is ankle pain and any of the following: Bone tenderness along the distal 6 cm of the posterior tibia or tip of the medial malleolus Bone tenderness along the distal 6 cm of the posterior fibula or tip of the lateral malleolus Inability to bear weight for four steps both immediately after the injury and in the emergency department A foot x-ray series is required if there is midfoot pain and any of the following: Bone tenderness at the base of the fifth metatarsal Bone tenderness at the navicular bone Inability to bear weight for four steps both immediately after the injury and in the emergency department In the clinic

1	Bone tenderness at the navicular bone Inability to bear weight for four steps both immediately after the injury and in the emergency department In the clinic A bunion occurs on the medial aspect of the first metatarsophalangeal joint. This is an extremely important area of the foot because it is crossed by tendons and ligaments, which transmit and distribute the body’s weight during movement. It is postulated that abnormal stresses in this region of the joint may produce the bunion deformity. Clinically, a bunion is a significant protuberance of bone that may include soft tissue around the medial aspect of the first metatarsophalangeal joint. As it progresses, the toe appears to move toward the smaller toes, producing crowding of the digits. This deformity tends to occur among people who wear high-heeled or pointed shoes, but osteoporosis and a hereditary predisposition are also risk factors.

1	This deformity tends to occur among people who wear high-heeled or pointed shoes, but osteoporosis and a hereditary predisposition are also risk factors. Typically the patient’s symptoms are pain, swelling, and inflammation. The bunion tends to enlarge and may cause problems in obtaining appropriate footwear. Initial treatment is by adding padding to shoes, changing the type of footwear used, and taking anti-inflammatory drugs. Some patients may need surgery to correct the deformity and realign the toe. In the clinic

1	In the clinic The plantar aponeurosis is a flat band of connective tissue that supports the arch of the sole of the foot. It runs from the calcaneal tuberosity to the base of the toes. Overuse and increased strain on the plantar aponeurosis, such as excessive running and standing, and increased body weight, can lead to micro-tears and degeneration within the aponeurosis at the heel with disorganization of the collagen fibers. Patients typically present with mild to severe heel pain, which appears thickened on imaging (Fig. 6.121). It is usually successfully treated with intense physiotherapy, but may require image-guided injection therapies. In severe cases, the diseased section of the fascia needs to be surgically removed. In the clinic

1	In the clinic A Morton’s neuroma is an enlarged common plantar nerve, usually in the third interspace between the third and fourth toes. In this region of the foot the lateral plantar nerve often unites with the medial plantar nerve. As the two nerves join, the resulting nerve is typically larger in diameter than those of the other toes. Also, it is in a relatively subcutaneous position, just above the fat pad of the foot close to the artery and the vein. Above the nerve is the deep transverse metatarsal ligament, which is a broad strong structure holding the metatarsals together. Typically, as the patient enters the “push-off” phase of walking the interdigital nerve is sandwiched between the ground and the deep transverse metatarsal ligament. The forces tend to compress the common plantar nerve, which can be irritated, in which case there is usually some associated inflammatory change and thickening.

1	Typically, patients experience pain in the third interspace, which may be sharp or dull and is usually worsened by wearing shoes and walking. Treatment may include injection of anti-inflammatory drugs, or it may be necessary to surgically remove the lesion. In the clinic Clubfoot is a congenital deformity in which babies are born with one or both feet pointing inward and downward. It is treated with gentle manipulation of the affected foot and with plaster casts to straighten the foot, which is usually followed by a minor surgical procedure where the calcaneal tendon is cut to release the foot into a better position.

1	A young man was enjoying a long weekend skiing at a European ski resort. While racing a friend he caught an inner edge of his right ski. He lost his balance and fell. During his tumble he heard an audible “click.” After recovering from his spill, he developed tremendous pain in his right knee. He was unable to carry on skiing for that day, and by the time he returned to his chalet, his knee was significantly swollen. He went immediately to see an orthopedic surgeon. The orthopedic surgeon carefully reviewed the mechanism of injury.

1	The orthopedic surgeon carefully reviewed the mechanism of injury. The man was skiing down the slope with both skis in parallel. The ankles were held rigid in the boots and the knees were slightly flexed. A momentary loss of concentration led to the skier catching the inner edge of his right ski. This effect was to force the boot and calf into external rotation. Furthermore, the knee was forced into a valgus position (bowed laterally away from the midline) and the skier tumbled. Both skis were detached from the boots as the bindings released them. A series of structures within the knee joint were damaged sequentially.

1	A series of structures within the knee joint were damaged sequentially. As the knee went into external rotation and valgus, the anterior cruciate ligament became taut, acting as a fulcrum. The tibial collateral ligament was stressed and the lateral compartment of the knee compressed. As the force increased, the tibial collateral ligament was torn (Fig. 6.139A,B), as was the medial meniscus (Fig. 6.140C). Finally, the anterior cruciate ligament, which was taut, gave way (Fig. 6.140A,B). The joint became swollen some hours afterward. Disruption of the anterior cruciate ligament characteristically produces marked joint swelling. The ligament is extrasynovial and intracapsular and has a rich blood supply. As the ligament was torn it ruptured into the joint. Blood from the tear irritates the synovial membrane and also enters the joint. These factors produce gradual swelling of the joint over the ensuing hours with significant fluid accumulation in the joint cavity.

1	The patient had a surgical reconstruction of the anterior cruciate ligament. It is difficult to find a man-made substance that can act in the same way as the anterior cruciate ligament and demonstrate the same physical properties. Surgeons have devised ingenious ways of reconstructing the anterior cruciate ligament. Two of the commonest methods use the patellar ligament (tendon) and hamstrings to reconstruct the ligament. The patient had further surgical procedures. The tibial collateral ligament was explored and resutured. Using arthroscopic techniques, the tear in the medial meniscus was débrided to prevent further complications. A 45-year-old man with diabetes mellitus visited his nurse because he had an ulcer on his foot that was not healing despite daily dressings.

1	A 45-year-old man with diabetes mellitus visited his nurse because he had an ulcer on his foot that was not healing despite daily dressings. Diabetes can lead to vascular disease of large and medium arteries, narrowing the lumen and reducing blood supply to the extremities, thereby impairing healing. In addition, diabetes can also affect blood supply to nerves, which leads to peripheral neuropathy. Peripheral neuropathy results in reduced sensation, and therefore minor injuries can often go unnoticed. This patient has developed an ulcer on his heel, which is a pressure point and likely to be under repeated strain. The nurse examined the ulcer and found that the ulcer was looking infected with pus at the base of the ulcer and asked for a specialist orthopedic opinion, who requested an x-ray and an MRI. The MRI and x-ray both demonstrated infection invading into the calcaneus with destruction of the bone (Fig. 6.141A,B).

1	The patient required surgical washout with removal of the dead and infected bone (debridement) and was given long-term antibiotic treatment (Fig. 6.141C). A young woman came to a vascular surgeon with a series of large dilated tortuous veins in her right leg. The rest of her leg was otherwise unremarkable. A diagnosis of varicose veins was made and the surgeon needed to determine the site of valvular incompetence.

1	A diagnosis of varicose veins was made and the surgeon needed to determine the site of valvular incompetence. There are typical points where incompetent valves occur between the superficial and the deep veins. In these regions the varicosities tend to become marked. The typical sites are: at the saphena varix—the saphenofemoral junction where the femoral vein is joined by the great saphenous vein; in the midthigh perforating vein between the great saphenous vein and the femoral vein; in the calf the three sites where perforators occur, 5, 10, and 15 cm above the medial malleolus between the great saphenous vein and the deep veins of the calf; and at the junction of the small saphenous vein and the popliteal vein.

1	The surgeon asked the patient to lie supine on the bed and elevated the leg. A tourniquet was placed around the upper thigh below the saphenofemoral junction and the patient was asked to stand up. No veins were demonstrated filling on the medial aspect of the thigh and lower limb. The effect of the tourniquet is to compress the great saphenous vein while permitting blood to flow in the deep venous system of the femoral vein and the deep femoral vein. Because there was no filling of the medial varicose veins below the level of the tourniquet, the surgeon assumed that the valve at the saphenofemoral junction was incompetent and would require surgical treatment. However, during the tourniquet maneuver the surgeon also noted some veins around the posterior and posterolateral aspect of the calf.

1	However, during the tourniquet maneuver the surgeon also noted some veins around the posterior and posterolateral aspect of the calf. A similar technique was performed by application of a tourniquet just below the level of the knee joint while the leg was elevated. The patient stood up and no veins were demonstrated filling in the posterior and posterolateral aspect of the calf. These findings suggested to the surgeon that there was also incompetence of the valve for the small saphenous system where it anastomoses with the popliteal vein. Surgery was planned.

1	Surgery was planned. A small transverse incision was made below the level of the inguinal ligament where the great saphenous vein passes through the saphenous ring in the deep fascia. This can be easily palpated as a small circular defect in the fascia. The saphenofemoral junction was identified and the great saphenous vein was ligated, at its anastomosis with the femoral vein. The great saphenous vein was stripped using special surgical techniques and removed. The patient was placed prone for the second part of the operation. A small incision was made transversely at the level of the skin crease in the popliteal fossa. However, the surgeon had difficulty identifying the junction between the small saphenous vein and the popliteal vein. After considerable time the surgeon located what he thought was the small saphenous vein and the structure was ligated and the wound closed.

1	The following day the patient was sent home, but returned to the clinic after 2 weeks complaining of problems walking. On examination there was absence of dorsiflexion, a sensory disturbance over the lateral aspect of the leg and foot, and obvious wasting of the fibular muscles. As the patient walked, the foot was dragged between steps. A clinical diagnosis of footdrop was made and a common fibular nerve injury was diagnosed. The injury occurred at the time of surgery.

1	Within the popliteal fossa are the popliteal artery, popliteal vein, and sciatic nerve (and its divisions). The popliteal artery is the deepest structure. The popliteal vein is superficial to the artery and the sciatic nerve is superficial to the vein (eFig. 6.142). Importantly, the sciatic nerve divides at the apex of the popliteal fossa. The tibial nerve continues into the lower popliteal fossa. The common fibular nerve passes laterally adjacent to the biceps femoris muscle to become superficial and wrap around the fibula neck. It was concluded that the surgeon had accidentally ligated the common fibular nerve rather than the small saphenous vein, thus producing this patient’s symptoms. A 72-year-old woman was admitted to the emergency room after falling at home. She complained of a severe pain in her right hip and had noticeable bruising on the right side of the face. On admission it was noted that the patient’s right leg was shorter than her left leg and externally rotated.

1	On admission it was noted that the patient’s right leg was shorter than her left leg and externally rotated. An initial series of investigations was carried out, including a plain radiograph of the pelvis. The plain radiograph of the pelvis demonstrated a displaced fracture through the right neck of the femur.

1	The plain radiograph of the pelvis demonstrated a displaced fracture through the right neck of the femur. The apparent shortening and external rotation of the leg on clinical examination were accounted for by spasm of the muscles connecting the pelvis to the trochanters and proximal femur. Of the muscles that surround the hip joint the largest group is the adductor group (adductor longus, brevis, and magnus) and the psoas major. The psoas major inserts onto the lesser trochanter and its action is to externally rotate and flex the hip. The fulcrum of action of the psoas major is the femoral head in the acetabulum. However, when the femoral neck is detached its overriding action pulls the femur proximally and into external rotation. The external rotation is exacerbated by the spasm in the adductor muscles. Extensive medical testing was necessary before surgery. It is important to remember that elderly patients may have a number of coexisting diseases.

1	Extensive medical testing was necessary before surgery. It is important to remember that elderly patients may have a number of coexisting diseases. The patient then underwent a hemiarthroplasty. Hemiarthroplasty is a surgical procedure in which the femoral head is removed from the acetabulum. The femoral neck is trimmed close to the trochanters and the medullary cavity of the femoral shaft is reamed. A metal hip prosthesis is inserted into the medullary cavity of the femur and the head of the prosthesis is placed into the acetabulum, in which it articulates. Importantly, the acetabulum is not replaced in straightforward cases, though a prosthetic acetabulum may be inserted if clinically appropriate. An arthroplasty was the only procedure that could be performed.

1	An arthroplasty was the only procedure that could be performed. The blood supply to the femoral head is from three sources—the artery within the ligament of the head of the femur, vessels in the medullary cavity, and vessels deep to the synovium running in the retinacula of the fibrous capsule of the hip joint. With increasing age, the medullary cavity undergoes fatty replacement of the normal red marrow, thus attenuating the medullary blood supply. The artery within the ligament of the head of the femur also becomes attenuated and this is often associated with atherosclerotic arterial disease.

1	Unfortunately for this patient, the sole blood supply to the head of the femur was via the vessels in the retinacula fibers, which were transected at the time of the fracture. If the patient had an intertrochanteric fracture instead, the vessels of the retinacula fibers would not have been damaged and a different approach to surgical fixation could be undertaken without the need for a hemiarthroplasty. The patient has osteoporosis. Osteoporosis is a common condition affecting older people, but is significantly more frequent in postmenopausal women. Many fractures of the femoral neck in elderly patients occur because the strength of the bone is significantly reduced when it is osteoporotic. Other common sites for osteoporotic fractures include the distal radius and the thoracic and lumbar vertebrae.

1	A 28-year-old woman was seen by her family practitioner for a routine pregnancy checkup at 36 weeks’ gestational age. Neither the patient nor the family physician had any concerns about the pregnancy. However, the patient did complain of unilateral swelling of her left leg, which had gradually increased over the previous 2 days. Furthermore, the evening before her visit she developed some sharp chest pain, which was exacerbated by deep breaths. The family physician ordered a duplex ultrasound scan of the left leg venous system. Ultrasound scanning of the vascular tree can demonstrate flow and occlusion of arteries and veins.

1	The family physician ordered a duplex ultrasound scan of the left leg venous system. Ultrasound scanning of the vascular tree can demonstrate flow and occlusion of arteries and veins. The probe was placed over the left femoral vein and no flow was demonstrated. Furthermore, the vein could not be compressed, and no alteration of flow with breathing could be demonstrated. Some flow was demonstrated in the deep femoral vein and in the great saphenous vein. No flow was demonstrated throughout the length of the left femoral vein, the popliteal vein, and the tibial veins. The technician scanned the opposite side, where excellent flow was demonstrated within the right femoral venous system. In addition, when the calf was gently massaged an augmentation to the flow was noted. It was possible to demonstrate alteration of flow with respiratory excursion and venous compression was satisfactory. A diagnosis of extensive left-sided deep vein thrombosis was made.

1	Certain patients are more prone to deep vein thrombosis. Three major factors predispose a patient to thrombosis: reduced or stagnant blood flow in the veins—significant stasis of blood (which may be due to lack of movement), reduced muscular calf pump effect, and obstruction to flow may occur; injury to the vein wall—venous trauma may damage the vessel walls, promoting thrombus formation; hypercoagulability of the blood—hypercoagulable states are often associated with abnormal levels of certain clotting factors, such as antithrombin III, protein C, and protein S. In this patient, compression of the left external iliac veins by the gravid uterus was the likely cause of stasis, which led to the deep vein thrombosis. The chest pain was due to pulmonary emboli.

1	In this patient, compression of the left external iliac veins by the gravid uterus was the likely cause of stasis, which led to the deep vein thrombosis. The chest pain was due to pulmonary emboli. Small emboli were thrown off from the leading edge of the thrombus through the heart to lodge in the lungs. Small emboli cause typical pleuritic chest pain, which is exacerbated by breathing. In isolation these small pulmonary emboli may affect respiratory function, but they may be the precursor to a large and potentially fatal pulmonary embolus (eFig. 6.143). Anticoagulation was instituted and the patient had an uneventful delivery. A 45-year-old man had recently taken up squash. During a game he attempted a forehand shot and noticed severe sudden pain in his heel. He thought his opponent had hit him with his racket. When he turned, though, he realized his opponent was too far away to have hit him.

1	Within minutes there was marked swelling of the ankle. The patient was unable to plantarflex his foot and had to stop the game. Afterward an appreciable subcutaneous hematoma developed in the ankle. The diagnostic possibilities include a bone or soft tissue injury. A bony injury was excluded because there was no bone tenderness. The patient had a significant soft tissue injury. On examination he had significant swelling of the ankle with a subcutaneous hematoma. He was unable to stand on tiptoe on the right leg, and in the prone position a palpable defect was demonstrated within the calcaneal tendon. A diagnosis of calcaneal tendon rupture was made. This patient has a typical history of ruptured calcaneal tendon and the clinical findings support this. Magnetic resonance imaging was carried out and confirmed the diagnosis (eFig. 6.144). The patient underwent an operative repair. The tendon healed well, though the patient has not gone back to playing squash.

1	The patient underwent an operative repair. The tendon healed well, though the patient has not gone back to playing squash. A 67-year-old man was noted to have a mass at the back of his knee. The mass measured approximately 4 cm in transverse diameter. The patient was otherwise fit and well and had no other history of note. The mass was arising from one of the structures in the popliteal fossa. Within the popliteal fossa there is a neurovascular bundle that contains the sciatic nerve (and its two divisions), the popliteal artery and the popliteal vein. There are also numerous small bursae associated with the posterior aspect of the knee joint and the muscles and tendons in this region. It is possible that this mass is arising from the posterior structures of the knee joint, which include synovial outpouchings, the menisci, and the muscles and tendons of this region.

1	The commonest masses demonstrated within the popliteal fossa are a popliteal cyst, a popliteal aneurysm, and an arterial adventitial cyst. Further clinical examination revealed that this mass was pulsatile and demonstrated a bruit (an audible rumbling made by turbulent blood flow) on auscultation. A diagnosis of popliteal artery aneurysm was made. A popliteal artery aneurysm is an abnormal dilation of the popliteal artery. It is unusual for it to be greater than 5 cm because symptoms usually develop before it reaches this size. Unlike aneurysms elsewhere in the body, the natural history of popliteal aneurysms is to embolize, with the mural thrombus producing ischemia distal to the lesion, rather than rupture. It is mandatory to examine the rest of the arterial tree in patients with a popliteal aneurysm because aneurysms may be bilateral and there is an association with abdominal aortic aneurysms.

1	The other diagnostic possibilities include a popliteal cyst and an adventitial cyst. A popliteal cyst (Baker’s cyst) is a synovial outpouching that arises from the posteromedial aspect of the knee joint. The synovial membrane of the knee joint outpouches between the medial head of the gastrocnemius and the semimembranosus tendon to lie medially within the popliteal fossa. Occasionally it tracks inferiorly to lie in and around the tendons that form the pes anserinus (sartorius, gracilis, and semitendinosus). An arterial adventitial cyst is an uncommon cystic structure that arises from the wall of the artery. An ultrasound investigation was carried out. Using real-time ultrasound the dimensions of the popliteal aneurysm were characterized and the flow in the vessels was demonstrated. Furthermore, a popliteal cyst and adventitial cyst were completely excluded. The patient underwent a surgical excision and graft interposition and has made an uneventful recovery.

1	The patient underwent a surgical excision and graft interposition and has made an uneventful recovery. A young long-distance runner came to her physician with acute swelling around the lateral aspect of her ankle. This injury occurred directly after accidentally running into a pothole in the pavement. A fractured ankle was suspected. Plain anteroposterior and lateral radiographs of the ankle revealed no evidence of any bone injury to account for the patient’s soft tissue swelling. The patient was given a pair of crutches and analgesics and told to rest. A diagnosis of a simple sprain was made. Over the ensuing weeks the swelling and edema within the soft tissue of the ankle decreased and the patient began to run, but noted that the ankle kept “giving way.” She went to an orthopedic surgeon for further assessment. There was a positive anterior drawer test of the ankle joint.

1	There was a positive anterior drawer test of the ankle joint. At this stage it is important to review the mechanism of injury. Typically when running on a hard surface the final phase of push-off involves supination of the foot. If the foot is caught in a pothole or divot, this supinating maneuver continues and inverts the ankle joint in plantarflexion. This puts significant strain on the lateral ligament complex and, given the appropriate circumstances, disruption (in order) of the ligament structures occurs from anterior to posterior. First, the anterior talofibular ligament is disrupted, followed by the calcaneofibular ligament, and then the posterior talofibular ligament. As each of these ligaments is disrupted, the severity of the soft tissue injury is significantly enhanced and the chance of permanent ankle instability is increased.

1	On examination any positive anterior drawer test of the ankle (4–5 mm compared to the opposite side) suggests an injury to the anterior talofibular ligament. The anterior talofibular ligament can be assessed by placing the foot in marked plantarflexion. If there is over 10° of difference between the affected foot and nonaffected foot, an anterior talofibular ligament disruption is suspected. It is extremely rare for all three ligaments to be disrupted, and if so there are usually other significant ankle injuries. Magnetic resonance imaging (MRI) was carried out to assess ligament damage. MRI is excellent for demonstrating the medial and lateral ligament complexes of the ankle as well as the soft tissues that support the bones of the posterior foot. Unfortunately for this patient there was a tear of the anterior talofibular ligament (eFig. 6.145), which had to be repaired surgically. 670.e2 670.e1 In the clinic—cont’d

1	Unfortunately for this patient there was a tear of the anterior talofibular ligament (eFig. 6.145), which had to be repaired surgically. 670.e2 670.e1 In the clinic—cont’d Table 6.1 Branches of the lumbosacral plexus associated with the lower limb—cont’d Regional Anatomy • Deep Fascia and the Saphenous Opening Fig. 6.73, cont’d Surface Anatomy • Avoiding the Sciatic Nerve Surface Anatomy • Identifying Structures Around the Knee Surface Anatomy • Finding the Tarsal Tunnel—the Gateway to the Foot Surface Anatomy • Approximating the Position of the Plantar Arterial Arch Anterior compartment of the forearm 766 Posterior compartment of the forearm 775 Carpal tunnel and structures at the wrist 788 Bony landmarks and muscles of the posterior scapular region 810 Visualizing the axilla and locating contents and related structures 811 Locating the brachial artery in the arm 812 The triceps brachii tendon and position of the radial nerve 813

1	Visualizing the axilla and locating contents and related structures 811 Locating the brachial artery in the arm 812 The triceps brachii tendon and position of the radial nerve 813 Identifying tendons and locating major vessels and nerves in the distal forearm 815 Normal appearance of the hand 816 Position of the flexor retinaculum and the recurrent branch of the median nerve 817 Motor function of the median and ulnar nerves in the hand 817 Visualizing the positions of the superficial and deep palmar arches 818 The upper limb is associated with the lateral aspect of the lower portion of the neck and with the thoracic wall. It is suspended from the trunk by muscles and a small skeletal articulation between the clavicle and the sternum—the sternoclavicular joint. Based on the position of its major joints and component bones, the upper limb is divided into shoulder, arm, forearm, and hand (Fig. 7.1A).

1	The shoulder is the area of upper limb attachment to the trunk (Fig. 7.1B). The arm is the part of the upper limb between the shoulder and the elbow joint; the forearm is between the elbow joint and the wrist joint; and the hand is distal to the wrist joint. The axilla, cubital fossa, and carpal tunnel are significant areas of transition between the different parts of the limb (Fig. 7.2). Important structures pass through, or are related to, each of these areas. The axilla is an irregularly shaped pyramidal area formed by muscles and bones of the shoulder and the lateral surface of the thoracic wall. The apex or inlet opens directly into the lower portion of the neck. The skin of the armpit forms the floor. All major structures that pass between the neck and arm pass through the axilla.

1	The cubital fossa is a triangularly shaped depression formed by muscles anterior to the elbow joint. The major artery, the brachial artery, passing from the arm to the forearm passes through this fossa, as does one of the major nerves of the upper limb, the median nerve. The carpal tunnel is the gateway to the palm of the hand. Its posterior, lateral, and medial walls form an arch, which is made up of small carpal bones in the proximal region of the hand. A thick band of connective tissue, the flexor retinaculum, spans the distance between each side of the arch and forms the anterior wall of the tunnel. The median nerve and all the long flexor tendons passing from the forearm to the digits of the hand pass through the carpal tunnel. Positioning the hand Unlike the lower limb, which is used for support, stability, and locomotion, the upper limb is highly mobile for positioning the hand in space.

1	Positioning the hand Unlike the lower limb, which is used for support, stability, and locomotion, the upper limb is highly mobile for positioning the hand in space. The shoulder is suspended from the trunk predominantly by muscles and can therefore be moved relative to the body. Sliding (protraction and retraction) and rotating the scapula on the thoracic wall changes the position of the glenohumeral joint (shoulder joint) and extends the reach of the hand (Fig. 7.3). The glenohumeral joint allows the arm to move around three axes with a wide range of motion. Movements of the arm at this joint are flexion, extension, abduction, adduction, medial rotation (internal rotation), lateral rotation (external rotation), and circumduction (Fig. 7.4).

1	The major movements at the elbow joint are flexion and extension of the forearm (Fig. 7.5A). At the other end of the forearm, the distal end of the lateral bone, the radius, can be flipped over the adjacent head of the medial bone, the ulna. Because the hand is articulated with the radius, it can be efficiently moved from a palm-anterior position to a palm-posterior position simply by crossing the distal end of the radius over the ulna (Fig. 7.5B). This movement, termed pronation, occurs solely in the forearm. Supination returns the hand to the anatomical position. At the wrist joint, the hand can be abducted, adducted, flexed, extended, and circumducted (Fig. 7.6). These movements, combined with those of the shoulder, arm, and forearm, enable the hand to be placed in a wide range of positions relative to the body. The hand as a mechanical tool

1	The hand as a mechanical tool One of the major functions of the hand is to grip and manipulate objects. Gripping objects generally involves flexing the fingers against the thumb. Depending on the type of grip, muscles in the hand act to: modify the actions of long tendons that emerge from the forearm and insert into the digits of the hand, and produce combinations of joint movements within each digit that cannot be generated by the long flexor and extensor tendons alone coming from the forearm. The hand as a sensory tool The hand is used to discriminate between objects on the basis of touch. The pads on the palmar aspect of the fingers contain a high density of somatic sensory receptors. Also, the sensory cortex of the brain devoted to interpreting information from the hand, particularly from the thumb, is disproportionately large relative to that for many other regions of skin.

1	The bones of the shoulder consist of the scapula, clavicle, and proximal end of the humerus (Fig. 7.7). The clavicle articulates medially with the manubrium of the sternum and laterally with the acromion of the scapula, which arches over the joint between the glenoid cavity of the scapula and the head of the humerus (the glenohumeral joint). The humerus is the bone of the arm (Fig. 7.7). The distal end of the humerus articulates with the bones of the forearm at the elbow joint, which is a hinge joint that allows flexion and extension of the forearm. The forearm contains two bones: The lateral bone is the radius. The medial bone is the ulna (Fig. 7.7). At the elbow joint, the proximal ends of the radius and ulna articulate with each other as well as with the humerus. In addition to flexing and extending the forearm, the elbow joint allows the radius to spin on the humerus while sliding against the head of the ulna during pronation and supination of the hand.

1	In addition to flexing and extending the forearm, the elbow joint allows the radius to spin on the humerus while sliding against the head of the ulna during pronation and supination of the hand. The distal portions of the radius and the ulna also articulate with each other. This joint allows the end of the radius to flip from the lateral side to the medial side of the ulna during pronation of the hand. The wrist joint is formed between the radius and carpal bones of the hand and between an articular disc, distal to the ulna, and carpal bones. The bones of the hand consist of the carpal bones, the metacarpals, and the phalanges (Fig. 7.7). The five digits in the hand are the thumb and the index, middle, ring, and little fingers. Joints between the eight small carpal bones allow only limited amounts of movement; as a result, the bones work together as a unit. The five metacarpals, one for each digit, are the primary skeletal foundation of the palm (Fig. 7.7).

1	The five metacarpals, one for each digit, are the primary skeletal foundation of the palm (Fig. 7.7). The joint between the metacarpal of the thumb (metacarpal I) and one of the carpal bones allows greater mobility than the limited sliding movement that occurs at the carpometacarpal joints of the fingers. Distally, the heads of metacarpals II to V (i.e., except that of the thumb) are interconnected by strong ligaments. Lack of this ligamentous connection between the metacarpal bones of the thumb and index finger together with the biaxial saddle joint between the metacarpal bone of the thumb and the carpus provide the thumb with greater freedom of movement than the other digits of the hand. The bones of the digits are the phalanges (Fig. 7.7). The thumb has two phalanges, while each of the other digits has three.

1	The bones of the digits are the phalanges (Fig. 7.7). The thumb has two phalanges, while each of the other digits has three. The metacarpophalangeal joints are biaxial condylar joints (ellipsoid joints) that allow abduction, adduction, flexion, extension, and circumduction (Fig. 7.8). Abduction and adduction of the fingers is defined in reference to an axis passing through the center of the middle finger in the anatomical position. The middle finger can therefore abduct both medially and laterally and adduct back to the central axis from either side. The interphalangeal joints are primarily hinge joints that allow only flexion and extension.

1	Some muscles of the shoulder, such as the trapezius, levator scapulae, and rhomboids, connect the scapula and clavicle to the trunk. Other muscles connect the clavicle, scapula, and body wall to the proximal end of the humerus. These include the pectoralis major, pectoralis minor, latissimus dorsi, teres major, and deltoid (Fig. 7.9A,B). The most important of these muscles are the four rotator cuff muscles—the subscapularis, supraspinatus, infraspinatus, and teres minor muscles—which connect the scapula to the humerus and provide support for the glenohumeral joint (Fig. 7.9C). Muscles in the arm and forearm are separated into anterior (flexor) and posterior (extensor) compartments by layers of fascia, bones, and ligaments (Fig. 7.10).

1	Muscles in the arm and forearm are separated into anterior (flexor) and posterior (extensor) compartments by layers of fascia, bones, and ligaments (Fig. 7.10). The anterior compartment of the arm lies anteriorly in position and is separated from muscles of the posterior compartment by the humerus and by medial and lateral intermuscular septa. These intermuscular septa are continuous with the deep fascia enclosing the arm and attach to the sides of the humerus. In the forearm, the anterior and posterior compartments are separated by a lateral intermuscular septum, the radius, the ulna, and an interosseous membrane, which joins adjacent sides of the radius and ulna (Fig. 7.10). Muscles in the arm act mainly to move the forearm at the elbow joint, while those in the forearm function predominantly to move the hand at the wrist joint and the fingers and thumb.

1	Muscles in the arm act mainly to move the forearm at the elbow joint, while those in the forearm function predominantly to move the hand at the wrist joint and the fingers and thumb. Muscles found entirely in the hand, the intrinsic muscles, generate delicate movements of the digits of the hand and modify the forces produced by tendons coming into the fingers and thumb from the forearm. Included among the intrinsic muscles of the hand are three small thenar muscles, which form a soft tissue mound, called the thenar eminence, over the palmar aspect of metacarpal I. The thenar muscles allow the thumb to move freely relative to the other fingers.

1	The upper limb is directly related to the neck. Lying on each side of the superior thoracic aperture at the base of the neck is an axillary inlet, which is formed by: the lateral margin of rib I, the posterior surface of the clavicle, the superior margin of the scapula, and the medial surface of the coracoid process of the scapula (Fig. 7.11). The major artery and vein of the upper limb pass between the thorax and the limb by passing over rib I and through the axillary inlet. Nerves, predominantly derived from the cervical portion of the spinal cord, also pass through the axillary inlet and the axilla to supply the upper limb. Muscles that attach the bones of the shoulder to the trunk are associated with the back and the thoracic wall and include the trapezius, levator scapulae, rhomboid major, rhomboid minor, and latissimus dorsi (Fig. 7.12).

1	The breast on the anterior thoracic wall has a number of significant relationships with the axilla and upper limb. It overlies the pectoralis major muscle, which forms most of the anterior wall of the axilla and attaches the humerus to the chest wall (Fig. 7.13). Often, part of the breast known as the axillary process extends around the lateral margin of the pectoralis major into the axilla. of the breast is predominantly into lymph nodes in the axilla. Several arteries and veins that supply or drain the gland also originate from, or drain into, major axillary vessels. Innervation of the upper limb is by the brachial plexus, which is formed by the anterior rami of cervical spinal nerves C5 to C8, and T1 (Fig. 7.14). This plexus is initially formed in the neck and then continues through the axillary inlet into the axilla. Major nerves that ultimately innervate the arm, forearm, and hand originate from the brachial plexus in the axilla.

1	As a consequence of this innervation pattern, clinical testing of lower cervical and T1 nerves is carried out by examining dermatomes, myotomes, and tendon reflexes in the upper limb. Another consequence is that the clinical signs of problems related to lower cervical nerves—pain; pins-and-needles sensations, or paresthesia; and muscle twitching—appear in the upper limb. Dermatomes of the upper limb (Fig. 7.15A) are often tested for sensation. Areas where overlap of dermatomes is minimal include the: upper lateral region of the arm for spinal cord level C5, palmar pad of the thumb for spinal cord level C6, pad of the index finger for spinal cord level C7, pad of the little finger for spinal cord level C8, and skin on the medial aspect of the elbow for spinal cord level T1. Selected joint movements are used to test myotomes (Fig. 7.15B): Abduction of the arm at the glenohumeral joint is controlled predominantly by C5.

1	Selected joint movements are used to test myotomes (Fig. 7.15B): Abduction of the arm at the glenohumeral joint is controlled predominantly by C5. Flexion of the forearm at the elbow joint is controlled primarily by C6. Extension of the forearm at the elbow joint is controlled mainly by C7. Flexion of the fingers is controlled mainly by C8. Abduction and adduction of the index, middle, and ring fingers is controlled predominantly by T1. In an unconscious patient, both somatic sensory and motor functions of spinal cord levels can be tested using tendon reflexes: A tap on the tendon of the biceps in the cubital fossa tests mainly for spinal cord level C6. A tap on the tendon of the triceps posterior to the elbow tests mainly for C7. The major spinal cord level associated with innervation of the diaphragm, C4, is immediately above the spinal cord levels associated with the upper limb.

1	The major spinal cord level associated with innervation of the diaphragm, C4, is immediately above the spinal cord levels associated with the upper limb. Evaluation of dermatomes and myotomes in the upper limb can provide important information about potential breathing problems that might develop as complications of damage to the spinal cord in regions just below the C4 spinal level. Each of the major muscle compartments in the arm and forearm and each of the intrinsic muscles of the hand is innervated predominantly by one of the major nerves that originate from the brachial plexus in the axilla (Fig. 7.16A): All muscles in the anterior compartment of the arm are innervated by the musculocutaneous nerve.

1	All muscles in the anterior compartment of the arm are innervated by the musculocutaneous nerve. The median nerve innervates the muscles in the anterior compartment of the forearm, with two exceptions—one flexor of the wrist (the flexor carpi ulnaris muscle) and part of one flexor of the fingers (the medial half of the flexor digitorum profundus muscle) are innervated by the ulnar nerve. Most intrinsic muscles of the hand are innervated by the ulnar nerve, except for the thenar muscles and two lateral lumbrical muscles, which are innervated by the median nerve. All muscles in the posterior compartments of the arm and forearm are innervated by the radial nerve. In addition to innervating major muscle groups, each of the major peripheral nerves originating from the from patches of skin quite different from dermatomes (Fig. 7.16B). Sensation in these areas can be used to test for peripheral nerve lesions:

1	The musculocutaneous nerve innervates skin on the anterolateral side of the forearm. The median nerve innervates the palmar surface of the lateral three and one-half digits, and the ulnar nerve innervates the medial one and one-half digits. The radial nerve supplies skin on the posterior surface of the forearm and the dorsolateral surface of the hand. Nerves related to bone Three important nerves are directly related to parts of the humerus (Fig. 7.17): The axillary nerve, which supplies the deltoid muscle, a major abductor of the humerus at the glenohumeral joint, passes around the posterior aspect of the upper part of the humerus (the surgical neck). The radial nerve, which supplies all of the extensor muscles of the upper limb, passes diagonally around the posterior surface of the middle of the humerus in the radial groove.

1	The radial nerve, which supplies all of the extensor muscles of the upper limb, passes diagonally around the posterior surface of the middle of the humerus in the radial groove. The ulnar nerve, which is ultimately destined for the hand, passes posteriorly to a bony protrusion, the medial epicondyle, on the medial side of the distal end of the humerus. Fractures of the humerus in any one of these three regions can endanger the related nerve. Large veins embedded in the superficial fascia of the upper limb are often used to access a patient’s vascular system and to withdraw blood. The most significant of these veins are the cephalic, basilic, and median cubital veins (Fig. 7.18). The cephalic and basilic veins originate from the dorsal venous network on the back of the hand.

1	The cephalic and basilic veins originate from the dorsal venous network on the back of the hand. The cephalic vein originates over the anatomical snuffbox at the base of the thumb, passes laterally around the distal forearm to reach the anterolateral surface of the limb, and then continues proximally. It crosses the elbow, then passes up the arm into a triangular depression—the clavipectoral triangle (deltopectoral triangle)—between the pectoralis major muscle, deltoid muscle, and clavicle. In this depression, the vein passes into the axilla by penetrating deep fascia just inferior to the clavicle. The basilic vein originates from the medial side of the dorsal venous network of the hand and passes proximally up the posteromedial surface of the forearm. It passes onto the anterior surface of the limb just inferior to the elbow and then continues proximally to penetrate deep fascia about midway up the arm.

1	At the elbow, the cephalic and basilic veins are connected by the median cubital vein, which crosses the roof of the cubital fossa. Orientation of the thumb The thumb is positioned at right angles to the orientation of the index, middle, ring, and little fingers (Fig. 7.19). As a result, movements of the thumb occur at right angles to those of the other digits. For example, flexion brings the thumb across the palm, whereas abduction moves it away from the fingers at right angles to the palm. Importantly, with the thumb positioned at right angles to the palm, only a slight rotation of metacarpal I on the wrist brings the pad of the thumb into a position directly facing the pads of the other fingers. This opposition of the thumb is essential for normal hand function. The shoulder is the region of upper limb attachment to the trunk.

1	The shoulder is the region of upper limb attachment to the trunk. The bone framework of the shoulder consists of: the clavicle and scapula, which form the pectoral girdle (shoulder girdle), and the proximal end of the humerus. The superficial muscles of the shoulder consist of the trapezius and deltoid muscles, which together form the smooth muscular contour over the lateral part of the shoulder. These muscles connect the scapula and clavicle to the trunk and to the arm, respectively. The clavicle is the only bony attachment between the trunk and the upper limb. It is palpable along its entire length and has a gentle S-shaped contour, with the forward-facing convex part medial and the forward-facing concave part lateral. The acromial (lateral) end of the clavicle is flat, whereas the sternal (medial) end is more robust and somewhat quadrangular in shape (Fig. 7.20).

1	The acromial end of the clavicle has a small oval facet on its surface for articulation with a similar facet on the medial surface of the acromion of the scapula. The sternal end has a much larger facet for articulation mainly with the manubrium of the sternum, and to a lesser extent, with the first costal cartilage. The inferior surface of the lateral third of the clavicle possesses a distinct tuberosity consisting of a tubercle (the conoid tubercle) and lateral roughening (the trapezoid line), for attachment of the important coracoclavicular ligament. In addition, the surfaces and margins of the clavicle are roughened by the attachment of muscles that connect the clavicle to the thorax, neck, and upper limb. The superior surface is smoother than the inferior surface.

1	The scapula is a large, flat triangular bone with: three angles (lateral, superior, and inferior), three borders (superior, lateral, and medial), two surfaces (costal and posterior), and three processes (acromion, spine, and coracoid process) (Fig. 7.21). The lateral angle of the scapula is marked by a shallow, somewhat comma-shaped glenoid cavity, which articulates with the head of the humerus to form the glenohumeral joint (Fig. 7.21B,C). A large triangular-shaped roughening (the infraglenoid tubercle) inferior to the glenoid cavity is the site of attachment for the long head of the triceps brachii muscle. A less distinct supraglenoid tubercle is located superior to the glenoid cavity and is the site of attachment for the long head of the biceps brachii muscle. A prominent spine subdivides the posterior surface of the scapula into a small, superior supraspinous fossa and a much larger, inferior infraspinous fossa (Fig. 7.21A).

1	A prominent spine subdivides the posterior surface of the scapula into a small, superior supraspinous fossa and a much larger, inferior infraspinous fossa (Fig. 7.21A). The acromion, which is an anterolateral projection of the spine, arches over the glenohumeral joint and articulates, via a small oval facet on its distal end, with the clavicle. The region between the lateral angle of the scapula and the attachment of the spine to the posterior surface of the scapula is the greater scapular notch (spinoglenoid notch). Unlike the posterior surface, the costal surface of the scapula is unremarkable, being characterized by a shallow concave subscapular fossa over much of its extent (Fig. 7.21B). The costal surface and margins provide for muscle attachment, and the costal surface, together with its related muscle (subscapularis), moves freely over the underlying thoracic wall.

1	The lateral border of the scapula is strong and thick for muscle attachment, whereas the medial border and much of the superior border is thin and sharp. The superior border is marked on its lateral end by: the coracoid process, a hook-like structure that projects anterolaterally and is positioned directly inferior to the lateral part of the clavicle; and the small but distinct suprascapular notch, which lies immediately medial to the root of the coracoid process. The spine and acromion can be readily palpated on a patient, as can the tip of the coracoid process, the inferior angle, and much of the medial border of the scapula. The proximal end of the humerus consists of the head, the anatomical neck, the greater and lesser tubercles, the surgical neck, and the superior half of the shaft of the humerus (Fig. 7.22). The head is half-spherical in shape and projects medially and somewhat superiorly to articulate with the much smaller glenoid cavity of the scapula.

1	The head is half-spherical in shape and projects medially and somewhat superiorly to articulate with the much smaller glenoid cavity of the scapula. The anatomical neck is very short and is formed by a narrow constriction immediately distal to the head. It lies between the head and the greater and lesser tubercles laterally, and between the head and the shaft more medially. The greater and lesser tubercles are prominent landmarks on the proximal end of the humerus and serve as attachment sites for the four rotator cuff muscles of the glenohumeral joint. The greater tubercle is lateral in position. Its superior surface and posterior surface are marked by three large smooth facets for muscle tendon attachments: The superior facet is for attachment of the supraspinatus muscle. The middle facet is for attachment of the infraspinatus. The inferior facet is for attachment of the teres minor.

1	The superior facet is for attachment of the supraspinatus muscle. The middle facet is for attachment of the infraspinatus. The inferior facet is for attachment of the teres minor. The lesser tubercle is anterior in position and its surface is marked by a large smooth impression for attachment of the subscapularis muscle. A deep intertubercular sulcus (bicipital groove) separates the lesser and greater tubercles and continues inferiorly onto the proximal shaft of the humerus (Fig. 7.22). The tendon of the long head of the biceps brachii passes through this sulcus. Roughenings on the lateral and medial lips and on the floor of the intertubercular sulcus mark sites for the attachment of the pectoralis major, teres major, and latissimus dorsi muscles, respectively.

1	The lateral lip of the intertubercular sulcus is continuous inferiorly with a large V-shaped deltoid tuberosity on the lateral surface of the humerus midway along its length (Fig. 7.22), which is where the deltoid muscle inserts onto the humerus. In approximately the same position, but on the medial surface of the bone, there is a thin vertical roughening for attachment of the coracobrachialis muscle.

1	In approximately the same position, but on the medial surface of the bone, there is a thin vertical roughening for attachment of the coracobrachialis muscle. One of the most important features of the proximal end of the humerus is the surgical neck (Fig. 7.22). This region is oriented in the horizontal plane between the expanded proximal part of the humerus (head, anatomical neck, and tubercles) and the narrower shaft. The axillary nerve and the posterior circumflex humeral artery, which pass into the deltoid region from the axilla, do so immediately posterior to the surgical neck. Because the surgical neck is weaker than more proximal regions of the bone, it is one of the sites where the humerus commonly fractures. The associated nerve (axillary) and artery (posterior circumflex humeral) can be damaged by fractures in this region. The three joints in the shoulder complex are the sternoclavicular, acromioclavicular, and glenohumeral joints.

1	The three joints in the shoulder complex are the sternoclavicular, acromioclavicular, and glenohumeral joints. The sternoclavicular joint and the acromioclavicular joint link the two bones of the pectoral girdle to each other and to the trunk. The combined movements at these two joints enable the scapula to be positioned over a wide range on the thoracic wall, substantially increasing “reach” by the upper limb. The glenohumeral joint (shoulder joint) is the articulation between the humerus of the arm and the scapula.

1	The glenohumeral joint (shoulder joint) is the articulation between the humerus of the arm and the scapula. The sternoclavicular joint occurs between the proximal end of the clavicle and the clavicular notch of the manubrium of the sternum together with a small part of the first costal cartilage (Fig. 7.23). It is synovial and saddle shaped. The articular cavity is completely separated into two compartments by an articular disc. The sternoclavicular joint allows movement of the clavicle, predominantly in the anteroposterior and vertical planes, although some rotation also occurs. The sternoclavicular joint is surrounded by a joint capsule and is reinforced by four ligaments: The anterior and posterior sternoclavicular ligaments are anterior and posterior, respectively, to the joint. An interclavicular ligament links the ends of the two clavicles to each other and to the superior surface of the manubrium of the sternum.

1	An interclavicular ligament links the ends of the two clavicles to each other and to the superior surface of the manubrium of the sternum. The costoclavicular ligament is positioned laterally to the joint and links the proximal end of the clavicle to the first rib and related costal cartilage. The acromioclavicular joint is a small synovial joint between an oval facet on the medial surface of the acromion and a similar facet on the acromial end of the clavicle (Fig. 7.24, also see Fig. 7.31). It allows movement in the anteroposterior and vertical planes together with some axial rotation.

1	The acromioclavicular joint is surrounded by a joint capsule and is reinforced by: a small acromioclavicular ligament superior to the joint and passing between adjacent regions of the clavicle and acromion, and a much larger coracoclavicular ligament, which is not directly related to the joint, but is an important strong accessory ligament, providing much of the weight-bearing support for the upper limb on the clavicle and maintaining the position of the clavicle on the acromion—it spans the distance between the coracoid process of the scapula and the inferior surface of the acromial end of the clavicle and comprises an anterior trapezoid ligament (which attaches to the trapezoid line on the clavicle) and a posterior conoid ligament (which attaches to the related conoid tubercle).

1	The glenohumeral joint is a synovial ball and socket articulation between the head of the humerus and the glenoid cavity of the scapula (Fig. 7.25). It is multiaxial with a wide range of movements provided at the cost of skeletal stability. Joint stability is provided, instead, by the rotator cuff muscles, the long head of the biceps brachii muscle, related bony processes, and extracapsular ligaments. Movements at the joint include flexion, extension, abduction, adduction, medial rotation, lateral rotation, and circumduction. The articular surfaces of the glenohumeral joint are the large spherical head of the humerus and the small glenoid cavity of the scapula (Fig. 7.25). Each of the surfaces is covered by hyaline cartilage.

1	The glenoid cavity is deepened and expanded peripherally by a fibrocartilaginous collar (the glenoid labrum), which attaches to the margin of the fossa. Superiorly, this labrum is continuous with the tendon of the long head of the biceps brachii muscle, which attaches to the supraglenoid tubercle and passes through the articular cavity superior to the head of the humerus. The synovial membrane attaches to the margins of the articular surfaces and lines the fibrous membrane of the joint capsule (Fig. 7.26). The synovial membrane is loose inferiorly. This redundant region of synovial membrane and related fibrous membrane accommodates abduction of the arm.

1	The synovial membrane protrudes through apertures in the fibrous membrane to form bursae, which lie between the tendons of surrounding muscles and the fibrous membrane. The most consistent of these is the subtendinous bursa of the subscapularis, which lies between the subscapularis muscle and the fibrous membrane. The synovial membrane also folds around the tendon of the long head of the biceps brachii muscle in the joint and extends along the tendon as it passes into the intertubercular sulcus. All these synovial structures reduce friction between the tendons and adjacent joint capsule and bone.

1	In addition to bursae that communicate with the articular cavity through apertures in the fibrous membrane, other bursae are associated with the joint but are not connected to it. These occur: between the acromion (or deltoid muscle) and supraspinatus muscle (or joint capsule) (the subacromial or subdeltoid bursa), between the acromion and skin, between the coracoid process and the joint capsule, and in relationship to tendons of muscles around the joint (coracobrachialis, teres major, long head of triceps brachii, and latissimus dorsi muscles). The fibrous membrane of the joint capsule attaches to the margin of the glenoid cavity, outside the attachment of the glenoid labrum and the long head of the biceps brachii muscle, and to the anatomical neck of the humerus (Fig. 7.27).

1	On the humerus, the medial attachment occurs more inferiorly than the neck and extends onto the shaft. In this region, the fibrous membrane is also loose or folded in the anatomical position. This redundant area of the fibrous membrane accommodates abduction of the arm. Openings in the fibrous membrane provide continuity of the articular cavity with bursae that occur between the joint capsule and surrounding muscles and around the tendon of the long head of the biceps brachii muscle in the intertubercular sulcus.

1	The fibrous membrane of the joint capsule is thickened: anterosuperiorly in three locations to form superior, middle, and inferior glenohumeral ligaments, which pass from the superomedial margin of the glenoid cavity to the lesser tubercle and inferiorly related anatomical neck of the humerus (Fig. 7.27); superiorly between the base of the coracoid process and the greater tubercle of the humerus (the coracohumeral ligament); and between the greater and lesser tubercles of the humerus (transverse humeral ligament)—this holds the tendon of the long head of the biceps brachii muscle in the intertubercular sulcus (Fig. 7.27). Joint stability is provided by surrounding muscle tendons and a skeletal arch formed superiorly by the coracoid process and acromion and the coraco-acromial ligament (Fig. 7.28).

1	Joint stability is provided by surrounding muscle tendons and a skeletal arch formed superiorly by the coracoid process and acromion and the coraco-acromial ligament (Fig. 7.28). Tendons of the rotator cuff muscles (the supraspinatus, infraspinatus, teres minor, and subscapularis muscles) blend with the joint capsule and form a musculotendinous collar that surrounds the posterior, superior, and anterior aspects of the glenohumeral joint (Figs. 7.28 and 7.29). This cuff of muscles stabilizes and holds the head of the humerus in the glenoid cavity of the scapula without compromising the arm’s flexibility and range of motion. The tendon of the long head of the biceps brachii muscle passes superiorly through the joint and restricts upward movement of the humeral head on the glenoid cavity. Vascular supply to the glenohumeral joint is predominantly through branches of the anterior and posterior circumflex humeral and suprascapular arteries.

1	Vascular supply to the glenohumeral joint is predominantly through branches of the anterior and posterior circumflex humeral and suprascapular arteries. The glenohumeral joint is innervated by branches from the posterior cord of the brachial plexus, and from the suprascapular, axillary, and lateral pectoral nerves. The two most superficial muscles of the shoulder are the trapezius and deltoid muscles (Fig. 7.35 and Table 7.1). Together, they provide the characteristic contour of the shoulder: The trapezius attaches the scapula and clavicle to the trunk. The deltoid attaches the scapula and clavicle to the humerus. Both the trapezius and deltoid are attached to opposing surfaces and margins of the spine of the scapula, acromion, and clavicle. The scapula, acromion, and clavicle can be palpated between the attachments of the trapezius and deltoid.

1	Deep to the trapezius the scapula is attached to the vertebral column by three muscles—the levator scapulae, rhomboid minor, and rhomboid major. These three muscles work with the trapezius (and with muscles found anteriorly) to position the scapula on the trunk. The trapezius muscle has an extensive origin from the axial skeleton, which includes sites on the skull and the vertebrae, from CI to TXII (Fig. 7.36). From CI to CVII, the muscle attaches to the vertebrae through the ligamentum nuchae. The muscle inserts onto the skeletal framework of the shoulder along the inner margins of a continuous U-shaped line of attachment oriented in the horizontal plane, with the bottom of the U directed laterally. Together, the left and right trapezius muscles form a diamond or trapezoid shape, from which the name is derived. The trapezius muscle is a powerful elevator of the shoulder and also rotates the scapula to extend the reach superiorly.

1	The trapezius muscle is a powerful elevator of the shoulder and also rotates the scapula to extend the reach superiorly. Innervation of the trapezius muscle is by the accessory nerve [XI] and the anterior rami of cervical nerves C3 and C4 (Fig. 7.36). These nerves pass vertically along the deep surface of the muscle. The accessory nerve can be evaluated by testing the function of the trapezius muscle. This is most easily done by asking patients to shrug their shoulders against resistance. The deltoid muscle is large and triangular in shape, with its base attached to the scapula and clavicle and its apex attached to the humerus (Fig. 7.36). It originates along a continuous U-shaped line of attachment to the clavicle and the scapula, mirroring the adjacent insertion sites of the trapezius muscle. It inserts into the deltoid tuberosity on the lateral surface of the shaft of the humerus. The major function of the deltoid muscle is abduction of the arm.

1	The major function of the deltoid muscle is abduction of the arm. The deltoid muscle is innervated by the axillary nerve, which is a branch of the posterior cord of the brachial plexus. The axillary nerve and associated blood vessels (the posterior circumflex humeral artery and vein) enter the deltoid by passing posteriorly around the surgical neck of the humerus. The levator scapulae originates from the transverse processes of CI to CIV vertebrae (Fig. 7.36). It descends laterally to attach to the posterior surface of the medial border of the scapula from the superior angle to the smooth triangular area of bone at the root of the spine. The levator scapulae muscle is innervated by the dorsal scapular nerve and directly from C3 and C4 spinal nerves. The levator scapulae elevates the scapula.

1	The levator scapulae muscle is innervated by the dorsal scapular nerve and directly from C3 and C4 spinal nerves. The levator scapulae elevates the scapula. The rhomboid minor and major muscles attach medially to the vertebral column and descend laterally to attach to the medial border of the scapula inferior to the levator scapulae muscle (Fig. 7.36). The rhomboid minor originates from the lower end of the ligamentum nuchae and the spines of CVII and TI vertebrae. It inserts laterally into the smooth triangular area of bone at the root of the spine of the scapula on the posterior surface. The rhomboid major originates from the spines of vertebrae TII to TV and from the intervening supraspinous ligaments. It descends laterally to insert along the posterior surface of the medial border of the scapula from the insertion of the rhomboid minor to the inferior angle. The rhomboid muscles are innervated by the dorsal scapular nerve, which is a branch of the brachial plexus.

1	The rhomboid muscles are innervated by the dorsal scapular nerve, which is a branch of the brachial plexus. The rhomboid minor and major retract and elevate the scapula. The posterior scapular region occupies the posterior aspect of the scapula and is located deep to the trapezius and deltoid muscles (Fig. 7.37 and Table 7.2). It contains four muscles, which pass between the scapula and proximal end of the humerus: the supraspinatus, infraspinatus, teres minor, and teres major muscles. The posterior scapular region also contains part of one additional muscle, the long head of the triceps brachii, which passes between the scapula and the proximal end of the forearm. This muscle, along with other muscles of the region and the humerus, participates in forming a number of spaces through which nerves and vessels enter and leave the region. The supraspinatus, infraspinatus, and teres minor muscles are components of the rotator cuff, which stabilizes the glenohumeral joint.

1	The supraspinatus, infraspinatus, and teres minor muscles are components of the rotator cuff, which stabilizes the glenohumeral joint. The supraspinatus and infraspinatus muscles originate from two large fossae, one above and one below the spine, on the posterior surface of the scapula (Fig. 7.37). They form tendons that insert on the greater tubercle of the humerus. The tendon of the supraspinatus passes under the acromion, where it is separated from the bone by a subacromial bursa, passes over the glenohumeral joint, and inserts on the superior facet of the greater tubercle. The tendon of the infraspinatus passes posteriorly to the glenohumeral joint and inserts on the middle facet of the greater tubercle. The supraspinatus participates in abduction of the arm. The infraspinatus laterally rotates the humerus.

1	The supraspinatus participates in abduction of the arm. The infraspinatus laterally rotates the humerus. The teres minor muscle is a cord-like muscle that originates from a flattened area of the scapula immediately adjacent to its lateral border below the infraglenoid tubercle (Fig. 7.37). Its tendon inserts on the inferior facet of the greater tubercle of the humerus. The teres minor laterally rotates the humerus and is a component of the rotator cuff. The teres major muscle originates from a large oval region on the posterior surface of the inferior angle of the scapula (Fig. 7.37). This broad cord-like muscle passes superiorly and laterally and ends as a flat tendon that attaches to the medial lip of the intertubercular sulcus on the anterior surface of the humerus. The teres major medially rotates and extends the humerus. Long head of triceps brachii

1	Long head of triceps brachii The long head of the triceps brachii muscle originates from the infraglenoid tubercle and passes somewhat vertically down the arm to insert, with the medial and lateral heads of this muscle, on the olecranon of the ulna (Fig. 7.37). The triceps brachii is the primary extensor of the forearm at the elbow joint. Because the long head crosses the glenohumeral joint, it can also extend and adduct the humerus. The importance of the triceps brachii in the posterior scapular region is that its vertical course between the teres minor and teres major, together with these muscles and the humerus, forms spaces through which nerves and vessels pass between regions. Gateways to the posterior scapular region

1	Gateways to the posterior scapular region The suprascapular foramen is the route through which structures pass between the base of the neck and the posterior scapular region (Fig. 7.37). It is formed by the suprascapular notch of the scapula and the superior transverse scapular (suprascapular) ligament, which converts the notch into a foramen. The suprascapular nerve passes through the suprascapular foramen; the suprascapular artery and the suprascapular vein follow the same course as the nerve, but normally pass immediately superior to the superior transverse scapular ligament and not through the foramen (Fig. 7.38).

1	The quadrangular space provides a passageway for nerves and vessels passing between more anterior regions (the axilla) and the posterior scapular region (Fig. 7.37). In the posterior scapular region, its boundaries are formed by: the inferior margin of the teres minor, the surgical neck of the humerus, the superior margin of the teres major, and the lateral margin of the long head of the triceps brachii. The axillary nerve and the posterior circumflex humeral artery and vein pass through this space (Fig. 7.38). The triangular space is an area of communication between the axilla and the posterior scapular region (Fig. 7.37). When viewed from the posterior scapular region, the triangular space is formed by: the medial margin of the long head of the triceps brachii, the superior margin of the teres major, and the inferior margin of the teres minor. The circumflex scapular artery and vein pass through this gap (Fig. 7.38).

1	The circumflex scapular artery and vein pass through this gap (Fig. 7.38). The triangular interval is formed by: the lateral margin of the long head of the triceps brachii, the shaft of the humerus, and the inferior margin of the teres major (Fig. 7.37). Because this space is below the inferior margin of the teres major, which defines the inferior boundary of the axilla, the triangular interval serves as a passageway between the anterior and posterior compartments of the arm and between the posterior compartment of the arm and the axilla. The radial nerve, the profunda brachii artery (deep artery of arm), and associated veins pass through it (Fig. 7.38). The two major nerves of the posterior scapular region are the suprascapular and axillary nerves, both of which originate from the brachial plexus in the axilla (Fig. 7.38).

1	The two major nerves of the posterior scapular region are the suprascapular and axillary nerves, both of which originate from the brachial plexus in the axilla (Fig. 7.38). The suprascapular nerve originates in the base of the neck from the superior trunk of the brachial plexus. It passes posterolaterally from its origin, through the suprascapular foramen to reach the posterior scapular region, where it lies in the plane between bone and muscle (Fig. 7.38). It innervates the supraspinatus muscle and then passes through the greater scapular (spinoglenoid) notch, between the root of the spine of the scapula and the glenoid cavity, to terminate in and innervate the infraspinatus muscle. Generally, the suprascapular nerve has no cutaneous branches.

1	Generally, the suprascapular nerve has no cutaneous branches. The axillary nerve originates from the posterior cord of the brachial plexus. It exits the axilla by passing through the quadrangular space in the posterior wall of the axilla, and enters the posterior scapular region (Fig. 7.38). Together with the posterior circumflex humeral artery and vein, it is directly related to the posterior surface of the surgical neck of the humerus. The axillary nerve innervates the deltoid and teres minor muscles. In addition, it has a cutaneous branch, the superior lateral cutaneous nerve of the arm, which carries general sensation from the skin over the inferior part of the deltoid muscle. Three major arteries are found in the posterior scapular region: the suprascapular, posterior circumflex humeral, and circumflex scapular arteries. These arteries contribute to an interconnected vascular network around the scapula (Fig. 7.39).

1	The suprascapular artery originates in the base of the neck as a branch of the thyrocervical trunk, which, in turn, is a major branch of the subclavian artery (Figs. 7.38 and 7.39). The vessel may also originate directly from the third part of the subclavian artery. The suprascapular artery normally enters the posterior scapular region superior to the suprascapular foramen, whereas the nerve passes through the foramen. In the posterior scapular region, the vessel runs with the suprascapular nerve. In addition to supplying the supraspinatus and infraspinatus muscles, the suprascapular artery contributes branches to numerous structures along its course. The posterior circumflex humeral artery originates from the third part of the axillary artery in the axilla (Figs. 7.38 and 7.39).

1	The posterior circumflex humeral artery originates from the third part of the axillary artery in the axilla (Figs. 7.38 and 7.39). The posterior circumflex humeral artery and axillary nerve leave the axilla through the quadrangular space in the posterior wall and enter the posterior scapular region. The vessel supplies the related muscles and the glenohumeral joint. The circumflex scapular artery is a branch of the subscapular artery that also originates from the third part of the axillary artery in the axilla (Figs. 7.38 and 7.39). The circumflex scapular artery leaves the axilla through the triangular space and enters the posterior scapular region, passes through the origin of the teres minor muscle, and forms anastomotic connections with other arteries in the region. Veins in the posterior scapular region generally follow the arteries and connect with vessels in the neck, back, arm, and axilla.

1	Veins in the posterior scapular region generally follow the arteries and connect with vessels in the neck, back, arm, and axilla. The axilla is the gateway to the upper limb, providing an area of transition between the neck and the arm (Fig. 7.40A). Formed by the clavicle, the scapula, the upper thoracic wall, the humerus, and related muscles, the axilla is an irregularly shaped pyramidal space with: four sides, an inlet, and a floor (base) (Fig. 7.40A,B). The axillary inlet is continuous superiorly with the neck, and the lateral part of the floor opens into the arm. All major structures passing into and out of the upper limb pass through the axilla (Fig. 7.40C). Apertures formed between muscles in the anterior and posterior walls enable structures to pass between the axilla and immediately adjacent regions (the posterior scapular, pectoral, and deltoid regions).

1	The axillary inlet is oriented in the horizontal plane and is somewhat triangular in shape, with its apex directed laterally (Fig. 7.40A,B). The margins of the inlet are completely formed by bone: The medial margin is the lateral border of rib I. The anterior margin is the posterior surface of the clavicle. The posterior margin is the superior border of the scapula up to the coracoid process. The apex of the triangularly shaped axillary inlet is lateral in position and is formed by the medial aspect of the coracoid process. Major vessels and nerves pass between the neck and the axilla by crossing over the lateral border of rib I and through the axillary inlet (Fig. 7.40A). The subclavian artery, the major blood vessel supplying the upper limb, becomes the axillary artery as it crosses the lateral margin of rib I and enters the axilla. Similarly, the axillary vein becomes the subclavian vein as it passes over the lateral margin of rib I and leaves the axilla to enter the neck.

1	At the axillary inlet, the axillary vein is anterior to the axillary artery, which, in turn, is anterior to the trunks of the brachial plexus. The inferior trunk (lower trunk) of the brachial plexus lies directly on rib I in the neck, as does the subclavian artery and vein. As they pass over rib I, the vein and artery are separated by the insertion of the anterior scalene muscle (Fig. 7.40A). The anterior wall of the axilla is formed by the lateral part of the pectoralis major muscle, the underlying pectoralis minor and subclavius muscles, and the clavipectoral fascia (Table 7.3). The pectoralis major muscle is the largest and most superficial muscle of the anterior wall (Fig. 7.41). Its inferior margin underlies the anterior axillary fold, which marks the anteroinferior border of the axilla. The muscle has two heads: The clavicular head originates from the medial half of the clavicle.

1	The clavicular head originates from the medial half of the clavicle. The sternocostal head originates from the medial part of the anterior thoracic wall—often, fibers from this head continue inferiorly and medially to attach to the anterior abdominal wall, forming an additional abdominal part of the muscle. The muscle inserts into the lateral lip of the intertubercular sulcus of the humerus. The parts of the muscle that have a superior origin on the trunk insert lower and more anteriorly on the lateral lip of the intertubercular sulcus than the parts of the muscle that originate inferiorly. Acting together, the two heads of the pectoralis major flex, adduct, and medially rotate the arm at the glenohumeral joint. The clavicular head flexes the arm from an extended position, whereas the sternocostal head extends the arm from a flexed position, particularly against resistance.

1	The pectoralis major is innervated by the lateral and medial pectoral nerves, which originate from the brachial plexus in the axilla. The subclavius muscle is a small muscle that lies deep to the pectoralis major muscle and passes between the clavicle and rib I (Fig. 7.42). It originates medially, as a tendon, from rib I at the junction between the rib and its costal cartilage. It passes laterally and superiorly to insert via a muscular attachment into an elongate shallow groove on the inferior surface of the middle third of the clavicle. The function of the subclavius is not entirely clear, but it may act to pull the shoulder down by depressing the clavicle and may also stabilize the sternoclavicular joint by pulling the clavicle medially. The subclavius muscle is innervated by a small branch from the superior trunk of the brachial plexus.

1	The subclavius muscle is innervated by a small branch from the superior trunk of the brachial plexus. The pectoralis minor muscle is a small triangular-shaped muscle that lies deep to the pectoralis major muscle and passes from the thoracic wall to the coracoid process of the scapula (Fig. 7.42). It originates as three muscular slips from the anterior surfaces and upper margins of ribs III to V and from the fascia overlying muscles of the related intercostal spaces. The muscle fibers pass superiorly and laterally to insert into the medial and upper aspects of the coracoid process. The pectoralis minor muscle protracts the scapula (by pulling the scapula anteriorly on the thoracic wall) and depresses the lateral angle of the scapula. The pectoralis minor is innervated by the medial pectoral nerve, which originates from the brachial plexus in the axilla.

1	The pectoralis minor is innervated by the medial pectoral nerve, which originates from the brachial plexus in the axilla. The clavipectoral fascia is a thick sheet of connective tissue that connects the clavicle to the floor of the axilla (Fig. 7.42). It encloses the subclavius and pectoralis minor muscles and spans the gap between them. Structures travel between the axilla and the anterior wall of the axilla by passing through the clavipectoral fascia either between the pectoralis minor and subclavius muscles or inferior to the pectoralis minor muscle. Important structures that pass between the subclavius and pectoralis minor muscles include the cephalic vein, the thoraco-acromial artery, and the lateral pectoral nerve. The lateral thoracic artery leaves the axilla by passing through the fascia inferior to the pectoralis minor muscle.

1	The lateral thoracic artery leaves the axilla by passing through the fascia inferior to the pectoralis minor muscle. The medial pectoral nerve leaves the axilla by penetrating directly through the pectoralis minor muscle to supply this muscle and to reach the pectoralis major muscle. Occasionally, branches of the medial pectoral nerve pass around the lower margin of the pectoralis minor to reach and innervate the overlying pectoralis major muscle. The medial wall of the axilla consists of the upper thoracic wall (the ribs and related intercostal tissues) and the serratus anterior muscle (Fig. 7.43 and Table 7.4, and see Fig. 7.40).

1	The medial wall of the axilla consists of the upper thoracic wall (the ribs and related intercostal tissues) and the serratus anterior muscle (Fig. 7.43 and Table 7.4, and see Fig. 7.40). The serratus anterior muscle originates as a number of muscular slips from the lateral surfaces of ribs I to IX and the intervening deep fascia overlying the related intercostal spaces (Fig. 7.43). The muscle forms a flattened sheet, which passes posteriorly around the thoracic wall to insert primarily on the costal surface of the medial border of the scapula. The serratus anterior pulls the scapula forward over the thoracic wall and facilitates scapular rotation. It also keeps the costal surface of the scapula closely opposed to the thoracic wall.

1	The serratus anterior pulls the scapula forward over the thoracic wall and facilitates scapular rotation. It also keeps the costal surface of the scapula closely opposed to the thoracic wall. The serratus anterior is innervated by the long thoracic nerve, which is derived from the roots of the brachial plexus, passes through the axilla along the medial wall, and passes vertically down the serratus anterior muscle on its external surface, just deep to skin and superficial fascia. The only major structure that passes directly through the medial wall and into the axilla is the intercostobrachial nerve (Fig. 7.43). This nerve is the lateral cutaneous branch of the second intercostal nerve (anterior ramus of T2). It communicates with a branch of the brachial plexus (the medial cutaneous nerve of the arm) in the axilla and supplies skin on the upper posteromedial side of the arm, which is part of the T2 dermatome.

1	The lateral wall of the axilla is narrow and formed entirely by the intertubercular sulcus of the humerus (Fig. 7.44). The pectoralis major muscle of the anterior wall attaches to the lateral lip of the intertubercular sulcus. The latissimus dorsi and teres major muscles of the posterior wall attach to the floor and medial lip of the intertubercular sulcus, respectively (Table 7.5). The posterior wall of the axilla is complex (Fig. 7.45 and see Fig. 7.50). Its bone framework is formed by the costal surface of the scapula. Muscles of the wall are: the subscapularis muscle (associated with the costal surface of the scapula), the distal parts of the latissimus dorsi and teres major muscles (which pass into the wall from the back and posterior scapular region), and the proximal part of the long head of the triceps brachii muscle (which passes vertically down the wall and into the arm).

1	Gaps between the muscles of the posterior wall form apertures through which structures pass between the axilla, posterior scapular region, and posterior compartment of the arm. The subscapularis muscle forms the largest component of the posterior wall of the axilla. It originates from, and fills, the subscapular fossa and inserts on the lesser tubercle of the humerus (Figs. 7.45 and 7.46). The tendon crosses immediately anterior to the joint capsule of the glenohumeral joint. Together with three muscles of the posterior scapular region (the supraspinatus, infraspinatus, and teres minor muscles), the subscapularis is a member of the rotator cuff muscle group, which stabilizes the glenohumeral joint. The subscapularis is innervated by branches of the brachial plexus (the superior and inferior subscapular nerves), which originate in the axilla.

1	The subscapularis is innervated by branches of the brachial plexus (the superior and inferior subscapular nerves), which originate in the axilla. The inferolateral aspect of the posterior wall of the axilla is formed by the terminal part of the teres major muscle and the tendon of the latissimus dorsi muscle (Fig. 7.45). These two structures lie under the posterior axillary fold, which marks the posteroinferior border of the axilla. The flat tendon of the latissimus dorsi muscle curves around the inferior margin of the teres major muscle on the posterior wall to insert into the floor of the intertubercular sulcus of the humerus, anterior to and slightly above the most distal attachment of the teres major muscle to the medial lip of the intertubercular sulcus. As a consequence, the inferior margin of the teres major muscle defines the inferior limit of the axilla laterally.

1	The axillary artery becomes the brachial artery of the arm as it crosses the inferior margin of the teres major muscle. Long head of the triceps brachii The long head of the triceps brachii muscle passes vertically through the posterior wall of the axilla, and, together with surrounding muscles and adjacent bones, results in the formation of three apertures through which major structures pass through the posterior wall: the quadrangular space, the triangular space, and the triangular interval (Fig. 7.45). Gateways in the posterior wall (See also “Gateways to the posterior scapular region,” pp. 706–710, and Figs. 7.37 and 7.38.)

1	Gateways in the posterior wall (See also “Gateways to the posterior scapular region,” pp. 706–710, and Figs. 7.37 and 7.38.) The quadrangular space provides a passageway for nerves and vessels passing between the axilla and the more posterior scapular and deltoid regions (Fig. 7.45). When viewed from anteriorly, its boundaries are formed by: the inferior margin of the subscapularis muscle, the surgical neck of the humerus, the superior margin of the teres major muscle, and the lateral margin of the long head of the triceps brachii muscle. Passing through the quadrangular space are the axillary nerve and the posterior circumflex humeral artery and vein. The triangular space is an area of communication between the axilla and the posterior scapular region (Fig. 7.45). When viewed from anteriorly, it is formed by: the medial margin of the long head of the triceps brachii muscle, the superior margin of the teres major muscle, and the inferior margin of the subscapularis muscle.

1	The circumflex scapular artery and vein pass into this space. This triangular interval is formed by: the lateral margin of the long head of the triceps brachii muscle, the shaft of the humerus, and the inferior margin of the teres major muscle (Fig. 7.45). The radial nerve passes out of the axilla traveling through this interval to reach the posterior compartment of the arm. The floor of the axilla is formed by fascia and a dome of skin that spans the distance between the inferior margins of the walls (Fig. 7.47 and see Fig. 7.40B). It is supported by the clavipectoral fascia. On a patient, the anterior axillary fold is more superior in position than is the posterior axillary fold. Inferiorly, structures pass into and out of the axilla immediately lateral to the floor where the anterior and posterior walls of the axilla converge and where the axilla is continuous with the anterior compartment of the arm. Contents of the axilla

1	Contents of the axilla Passing through the axilla are the major vessels, nerves, and lymphatics of the upper limb. The space also contains the proximal parts of two muscles of the arm, the axillary process of the breast, and collections of lymph nodes, which drain the upper limb, chest wall, and breast. The proximal parts of the biceps brachii and coracobrachialis muscles pass through the axilla (Table 7.6). The biceps brachii muscle originates as two heads (Fig. 7.48): The short head originates from the apex of the coracoid process of the scapula and passes vertically through the axilla and into the arm where it joins the long head.

1	The short head originates from the apex of the coracoid process of the scapula and passes vertically through the axilla and into the arm where it joins the long head. The long head originates as a tendon from the supraglenoid tubercle of the scapula, passes over the head of the humerus deep to the joint capsule of the glenohumeral joint, and enters the intertubercular sulcus where it is held in position by a ligament, the transverse humeral ligament, which spans the distance between the greater and lesser tubercles; the tendon passes through the axilla in the intertubercular sulcus and forms a muscle belly in the proximal part of the arm. The long and short heads of the muscle join in distal regions of the arm and primarily insert as a single tendon into the radial tuberosity in the forearm.

1	The long and short heads of the muscle join in distal regions of the arm and primarily insert as a single tendon into the radial tuberosity in the forearm. The biceps brachii muscle is primarily a powerful flexor of the forearm at the elbow joint and a powerful supinator in the forearm. Because both heads originate from the scapula, the muscle also acts as an accessory flexor of the arm at the glenohumeral joint. In addition, the long head prevents superior movement of the humerus on the glenoid cavity. The biceps brachii muscle is innervated by the musculocutaneous nerve. The coracobrachialis muscle, together with the short head of the biceps brachii muscle, originates from the apex of the coracoid process (Fig. 7.48). It passes vertically through the axilla to insert on a small linear roughening on the medial aspect of the humerus, approximately midshaft. The coracobrachialis muscle flexes the arm at the glenohumeral joint.

1	The coracobrachialis muscle flexes the arm at the glenohumeral joint. In the axilla, the medial surface of the coracobrachialis muscle is pierced by the musculocutaneous nerve, which innervates and then passes through the muscle to enter the arm. The axillary artery supplies the walls of the axilla and related regions, and continues as the major blood supply to the more distal parts of the upper limb (Fig. 7.49). The subclavian artery in the neck becomes the axillary artery at the lateral margin of rib I and passes through the axilla, becoming the brachial artery at the inferior margin of the teres major muscle. The axillary artery is separated into three parts by the pectoralis minor muscle, which crosses anteriorly to the vessel (Fig. 7.49): The first part is proximal to the pectoralis minor. The second part is posterior to the pectoralis minor. The third part is distal to the pectoralis minor. Generally, six branches arise from the axillary artery:

1	The second part is posterior to the pectoralis minor. The third part is distal to the pectoralis minor. Generally, six branches arise from the axillary artery: One branch, the superior thoracic artery, originates from the first part. Two branches, the thoraco-acromial artery and the lateral thoracic artery, originate from the second part. Three branches, the subscapular artery, the anterior circumflex humeral artery, and the posterior circumflex humeral artery, originate from the third part (Fig. 7.50). The superior thoracic artery is small and originates from the anterior surface of the first part of the axillary artery (Fig. 7.50). It supplies upper regions of the medial and anterior axillary walls.

1	The thoraco-acromial artery is short and originates from the anterior surface of the second part of the axillary artery just posterior to the medial (superior) margin of the pectoralis minor muscle (Fig. 7.50). It curves around the superior margin of the muscle, penetrates the clavipectoral fascia, and immediately divides into four branches—the pectoral, deltoid, clavicular, and acromial branches, which supply the anterior axillary wall and related regions. Additionally, the pectoral branch contributes vascular supply to the breast, and the deltoid branch passes into the clavipectoral triangle where it accompanies the cephalic vein and supplies adjacent structures (see Fig. 7.41).

1	The lateral thoracic artery arises from the anterior surface of the second part of the axillary artery posterior to the lateral (inferior) margin of the pectoralis minor (Fig. 7.50). It follows the margin of the muscle to the thoracic wall and supplies the medial and anterior walls of the axilla. In women, branches emerge from around the inferior margin of the pectoralis major muscle and contribute to the vascular supply of the breast. The subscapular artery is the largest branch of the axillary artery and is the major blood supply to the posterior wall of the axilla (Fig. 7.50). It also contributes to the blood supply of the posterior scapular region. The subscapular artery originates from the posterior surface of the third part of the axillary artery, follows the inferior margin of the subscapularis muscle for a short distance, and then divides into its two terminal branches, the circumflex scapular artery and the thoracodorsal artery.

1	The circumflex scapular artery passes through the triangular space between the subscapularis, teres major, and long head of the triceps muscle. Posteriorly, it passes inferior to, or pierces, the origin of the teres minor muscle to enter the infraspinous fossa. It anastomoses with the suprascapular artery and the deep branch (dorsal scapular artery) of the transverse cervical artery, thereby contributing to an anastomotic network of vessels around the scapula. The thoracodorsal artery approximately follows the lateral border of the scapula to the inferior angle. It contributes to the vascular supply of the posterior and medial walls of the axilla. The anterior circumflex humeral artery is small compared to the posterior circumflex humeral artery, and originates from the lateral side of the third part of the axillary artery (Fig. 7.50). It passes anterior to the surgical neck of the humerus and anastomoses with the posterior circumflex humeral artery.

1	This anterior circumflex humeral artery supplies branches to surrounding tissues, which include the glenohumeral joint and the head of the humerus. The posterior circumflex humeral artery originates from the lateral surface of the third part of the axillary artery immediately posterior to the origin of the anterior circumflex humeral artery (Fig. 7.50). With the axillary nerve, it leaves the axilla by passing through the quadrangular space between the teres major, teres minor, and long head of the triceps brachii muscle and the surgical neck of the humerus. The posterior circumflex humeral artery curves around the surgical neck of the humerus and supplies the surrounding muscles and the glenohumeral joint. It anastomoses with the anterior circumflex humeral artery and with branches from the profunda brachii, suprascapular, and thoraco-acromial arteries.

1	The axillary vein begins at the lower margin of the teres major muscle and is the continuation of the basilic vein (Fig. 7.51), which is a superficial vein that drains the posteromedial surface of the hand and forearm and penetrates the deep fascia in the middle of the arm. The axillary vein passes through the axilla medial and anterior to the axillary artery and becomes the subclavian vein as the vessel crosses the lateral border of rib I at the axillary inlet. Tributaries of the axillary vein generally follow the branches of the axillary artery. Other tributaries include brachial veins that follow the brachial artery and the cephalic vein.

1	The cephalic vein is a superficial vein that drains the lateral and posterior parts of the hand, the forearm, and the arm. In the area of the shoulder, it passes into an inverted triangular cleft (the clavipectoral triangle) between the deltoid muscle, pectoralis major muscle, and clavicle. In the superior part of the clavipectoral triangle, the cephalic vein passes deep to the clavicular head of the pectoralis major muscle and pierces the clavipectoral fascia to join the axillary vein. Many patients who are critically ill have lost blood or fluid, which requires replacement. Access to a peripheral vein is necessary to replace the fluid. The typical sites for venous access are the cephalic vein in the hand or veins that lie within the superficial tissues of the cubital fossa.

1	The brachial plexus is a somatic nerve plexus formed by the anterior rami of C5 to C8, and most of the anterior ramus of T1 (Fig. 7.52). The plexus originates in the neck, passes laterally and inferiorly over rib I, and enters the axilla. The parts of the brachial plexus, from medial to lateral, are roots, trunks, divisions, and cords. All major nerves that innervate the upper limb originate from the brachial plexus, mostly from the cords. Proximal parts of the brachial plexus are posterior to the subclavian artery in the neck, while more distal regions of the plexus surround the axillary artery.

1	The roots of the brachial plexus are the anterior rami of C5 to C8, and most of T1. Close to their origin, the roots receive gray rami communicantes from the sympathetic trunk (Fig. 7.52). These carry postganglionic sympathetic fibers onto the roots for distribution to the periphery. The roots and trunks enter the posterior triangle of the neck by passing between the anterior scalene and middle scalene muscles and lie superior and posterior to the subclavian artery. The three trunks of the brachial plexus originate from the roots, pass laterally over rib I, and enter the axilla (Fig. 7.52): The superior trunk is formed by the union of C5 and C6 roots. The middle trunk is a continuation of the C7 root. The inferior trunk is formed by the union of the C8 and T1 roots. The inferior trunk lies on rib I posterior to the subclavian artery; the middle and superior trunks are more superior in position.

1	The inferior trunk is formed by the union of the C8 and T1 roots. The inferior trunk lies on rib I posterior to the subclavian artery; the middle and superior trunks are more superior in position. Each of the three trunks of the brachial plexus divides into an anterior and a posterior division (Fig. 7.52): The three anterior divisions form parts of the brachial plexus that ultimately give rise to peripheral nerves associated with the anterior compartments of the arm and forearm. The three posterior divisions combine to form parts of the brachial plexus that give rise to nerves associated with the posterior compartments. No peripheral nerves originate directly from the divisions of the brachial plexus. The three cords of the brachial plexus originate from the divisions and are related to the second part of the axillary artery (Fig. 7.52):

1	The three cords of the brachial plexus originate from the divisions and are related to the second part of the axillary artery (Fig. 7.52): The lateral cord results from the union of the anterior divisions of the upper and middle trunks and therefore has contributions from C5 to C7—it is positioned lateral to the second part of the axillary artery. The medial cord is medial to the second part of the axillary artery and is the continuation of the anterior division of the inferior trunk—it contains contributions from C8 and T1. The posterior cord occurs posterior to the second part of the axillary artery and originates as the union of all three posterior divisions—it contains contributions from all roots of the brachial plexus (C5 to T1).

1	Most of the major peripheral nerves of the upper limb originate from the cords of the brachial plexus. Generally, nerves associated with the anterior compartments of the upper limb arise from the medial and lateral cords and nerves associated with the posterior compartments originate from the posterior cord. Branches (Table 7.7) Branches of the roots In addition to small segmental branches from C5 to C8 to muscles of the neck and a contribution of C5 to the phrenic nerve, the roots of the brachial plexus give rise to the dorsal scapular and long thoracic nerves (Fig. 7.53). The dorsal scapular nerve: originates from the C5 root of the brachial plexus, passes posteriorly, often piercing the middle scalene muscle in the neck, to reach and travel along the medial border of the scapula (Fig. 7.54), and innervates the rhomboid major and minor muscles from their deep surfaces.

1	The long thoracic nerve: originates from the anterior rami of C5 to C7, passes vertically down the neck, through the axillary inlet, and down the medial wall of the axilla to supply the serratus anterior muscle (Fig. 7.54), and lies on the superficial aspect of the serratus anterior muscle. Branches of the trunks The only branches from the trunks of the brachial plexus are two nerves that originate from the superior trunk (upper trunk): the suprascapular nerve and the nerve to the subclavius muscle (Fig. 7.53). The suprascapular nerve (C5 and C6): originates from the superior trunk of the brachial plexus, passes laterally through the posterior triangle of the neck (Fig. 7.54) and through the suprascapular foramen to enter the posterior scapular region, innervates the supraspinatus and infraspinatus muscles, and is accompanied in the lateral parts of the neck and in the posterior scapular region by the suprascapular artery.

1	The nerve to the subclavius muscle (C5 and C6) is a small nerve that: originates from the superior trunk of the brachial plexus, passes anteroinferiorly over the subclavian artery and vein, and innervates the subclavius muscle. Branches of the lateral cord Three nerves originate entirely or partly from the lateral cord (Fig. 7.53). The lateral pectoral nerve is the most proximal of the branches from the lateral cord. It passes anteriorly, together with the thoraco-acromial artery, to penetrate the clavipectoral fascia that spans the gap between the subclavius and pectoralis minor muscles (Fig. 7.55), and innervates the pectoralis major muscle.

1	The musculocutaneous nerve is a large terminal branch of the lateral cord. It passes laterally to penetrate the coracobrachialis muscle and pass between the biceps brachii and brachialis muscles in the arm, and innervates all three flexor muscles in the anterior compartment of the arm, terminating as the lateral cutaneous nerve of the forearm. The lateral root of the median nerve is the largest terminal branch of the lateral cord and passes medially to join a similar branch from the medial cord to form the median nerve (Fig. 7.55). Branches of the medial cord The medial cord has five branches (Fig. 7.55).

1	Branches of the medial cord The medial cord has five branches (Fig. 7.55). The medial pectoral nerve is the most proximal branch. It receives a communicating branch from the lateral pectoral nerve and then passes anteriorly between the axillary artery and axillary vein. Branches of the nerve penetrate and supply the pectoralis minor muscle. Some of these branches pass through the muscle to reach and supply the pectoralis major muscle. Other branches occasionally pass around the inferior or lateral margin of the pectoralis minor muscle to reach the pectoralis major muscle.

1	The medial cutaneous nerve of the arm (medial brachial cutaneous nerve) passes through the axilla and into the arm where it penetrates deep fascia and supplies skin over the medial side of the distal third of the arm. In the axilla, the nerve communicates with the intercostobrachial nerve of T2. Fibers of the medial cutaneous nerve of the arm innervate the upper part of the medial surface of the arm and floor of the axilla. The medial cutaneous nerve of the forearm (medial antebrachial cutaneous nerve) originates just distal to the origin of the medial cutaneous nerve of the arm. It passes out of the axilla and into the arm where it gives off a branch to the skin over the biceps brachii muscle, and then continues down the arm to penetrate the deep fascia with the basilic vein, continuing inferiorly to supply the skin over the anterior surface of the forearm. It innervates skin over the medial surface of the forearm down to the wrist.

1	The medial root of the median nerve passes laterally to join with a similar root from the lateral cord to form the median nerve anterior to the third part of the axillary artery.

1	The ulnar nerve is a large terminal branch of the medial cord (Fig. 7.55). However, near its origin, it often receives a communicating branch from the lateral root of the median nerve originating from the lateral cord and carrying fibers from C7 (see Fig. 5.73B). The ulnar nerve passes through the arm and forearm into the hand where it innervates all intrinsic muscles of the hand (except for the three thenar muscles and the two lateral lumbrical muscles). On passing through the forearm, branches of the ulnar nerve innervate the flexor carpi ulnaris muscle and the medial half of the flexor digitorum profundus muscle. The ulnar nerve innervates skin over the palmar surface of the little finger, medial half of the ring finger, and associated palm and wrist, and the skin over the dorsal surface of the medial part of the hand.

1	Median nerve. The median nerve is formed anterior to the third part of the axillary artery by the union of lateral and medial roots originating from the lateral and medial cords of the brachial plexus (Fig. 7.55). It passes into the arm anterior to the brachial artery and through the arm into the forearm, where branches innervate most of the muscles in the anterior compartment of the forearm (except for the flexor carpi ulnaris muscle and the medial half of the flexor digitorum profundus muscle, which are innervated by the ulnar nerve). The median nerve continues into the hand to innervate: the three thenar muscles associated with the thumb, the two lateral lumbrical muscles associated with movement of the index and middle fingers, and the skin over the palmar surface of the lateral three and one-half digits and over the lateral side of the palm and middle of the wrist.

1	The musculocutaneous nerve, the lateral root of the median nerve, the median nerve, the medial root of the median nerve, and the ulnar nerve form an M over the third part of the axillary artery (Fig. 7.55). This feature, together with penetration of the coracobrachialis muscle by the musculocutaneous nerve, can be used to identify components of the brachial plexus in the axilla. Branches of the posterior cord Five nerves originate from the posterior cord of the brachial plexus: the superior subscapular nerve, the thoracodorsal nerve, the inferior subscapular nerve, the axillary nerve, and the radial nerve (Fig. 7.53). All these nerves except the radial nerve innervate muscles associated with the shoulder region or the posterior wall of the axilla; the radial nerve passes into the arm and forearm.

1	All these nerves except the radial nerve innervate muscles associated with the shoulder region or the posterior wall of the axilla; the radial nerve passes into the arm and forearm. The superior subscapular, thoracodorsal, and inferior subscapular nerves originate sequentially from the posterior cord and pass directly into muscles associated with the posterior axillary wall (Fig. 7.56). The superior subscapular nerve is short and passes into and supplies the subscapularis muscle. The thoracodorsal nerve is the longest of these three nerves and passes vertically along the posterior axillary wall. It penetrates and innervates the latissimus dorsi muscle. The inferior subscapular nerve also passes inferiorly along the posterior axillary wall and innervates the subscapularis and teres major muscles.

1	The axillary nerve originates from the posterior cord and passes inferiorly and laterally along the posterior wall to exit the axilla through the quadrangular space (Fig. 7.56). It passes posteriorly around the surgical neck of the humerus and innervates both the deltoid and teres minor muscles. A superior lateral cutaneous nerve of the arm originates from the axillary nerve after passing through the quadrangular space and loops around the posterior margin of the deltoid muscle to innervate skin in that region. The axillary nerve is accompanied by the posterior circumflex humeral artery.

1	The radial nerve is the largest terminal branch of the posterior cord (Fig. 7.56). It passes out of the axilla and into the posterior compartment of the arm by passing through the triangular interval between the inferior border of the teres major muscle, the long head of the triceps brachii muscle, and the shaft of the humerus. It is accompanied through the triangular interval by the profunda brachii artery, which originates from the brachial artery in the anterior compartment of the arm. The radial nerve and its branches innervate: all muscles in the posterior compartments of the arm and forearm, and the skin on the posterior aspect of the arm and forearm, the lower lateral surface of the arm, and the dorsal lateral surface of the hand. The posterior cutaneous nerve of the arm (posterior brachial cutaneous nerve) originates from the radial nerve in the axilla and innervates skin on the posterior surface of the arm.

1	The posterior cutaneous nerve of the arm (posterior brachial cutaneous nerve) originates from the radial nerve in the axilla and innervates skin on the posterior surface of the arm. All lymphatics from the upper limb drain into lymph nodes in the axilla (Fig. 7.57). In addition, axillary nodes receive drainage from an extensive area on the adjacent trunk, which includes regions of the upper back and shoulder, the lower neck, the chest, and the upper anterolateral abdominal wall. Axillary nodes also receive drainage from approximately 75% of the mammary gland. The 20–30 axillary nodes are generally divided into five groups on the basis of location. Humeral (lateral) nodes posteromedial to the axillary vein receive most of the lymphatic drainage from the upper limb. Pectoral (anterior) nodes occur along the inferior margin of the pectoralis minor muscle along the course of the lateral thoracic vessels and receive drainage from the abdominal wall, the chest, and the mammary gland.

1	Subscapular (posterior) nodes on the posterior axillary wall in association with the subscapular vessels drain the posterior axillary wall and receive lymphatics from the back, the shoulder, and the neck. Central nodes are embedded in axillary fat and receive tributaries from humeral, subscapular, and pectoral groups of nodes. Apical nodes are the most superior group of nodes in the axilla and drain all other groups of nodes in the region. In addition, they receive lymphatic vessels that accompany the cephalic vein as well as vessels that drain the superior region of the mammary gland. Efferent vessels from the apical group converge to form the subclavian trunk, which usually joins the venous system at the junction between the right subclavian vein and the right internal jugular vein in the neck. On the left, the subclavian trunk usually joins the thoracic duct in the base of the neck. Axillary process of the mammary gland

1	Axillary process of the mammary gland Although the mammary gland is in superficial fascia overlying the thoracic wall, its superolateral region extends along the inferior margin of the pectoralis major muscle toward the axilla. In some cases, this may pass around the margin of the muscle to penetrate deep fascia and enter the axilla (Fig. 7.58). This axillary process rarely reaches as high as the apex of the axilla. The arm is the region of the upper limb between the shoulder and the elbow (Fig. 7.59). The superior aspect of the arm communicates medially with the axilla. Inferiorly, a number of important structures pass between the arm and the forearm through the cubital fossa, which is positioned anterior to the elbow joint. The arm is divided into two compartments by medial and lateral intermuscular septa, which pass from each side of the humerus to the outer sleeve of deep fascia that surrounds the limb (Fig. 7.59).

1	The arm is divided into two compartments by medial and lateral intermuscular septa, which pass from each side of the humerus to the outer sleeve of deep fascia that surrounds the limb (Fig. 7.59). The anterior compartment of the arm contains muscles that predominantly flex the elbow joint; the posterior compartment contains muscles that extend the joint. Major nerves and vessels supply and pass through each compartment. The skeletal support for the arm is the humerus (Fig. 7.60). Most of the large muscles of the arm insert into the proximal ends of the two bones of the forearm, the radius and the ulna, and flex and extend the forearm at the elbow joint. In addition, the muscles predominantly situated in the forearm that move the hand originate at the distal end of the humerus. Shaft and distal end of the humerus

1	Shaft and distal end of the humerus In cross section, the shaft of the humerus is somewhat triangular with: anterior, lateral, and medial borders, and anterolateral, anteromedial, and posterior surfaces (Fig. 7.60). The posterior surface of the humerus is marked on its superior aspect by a linear roughening for the attachment of the lateral head of the triceps brachii muscle, beginning just inferior to the surgical neck and passing diagonally across the bone to the deltoid tuberosity. The middle part of the posterior surface and adjacent part of the anterolateral surface are marked by the shallow radial groove, which passes diagonally down the bone and parallel to the sloping posterior margin of the deltoid tuberosity. The radial nerve and the profunda brachii artery lie in this groove. Approximately in the middle of the shaft, the medial border is marked by thin elongate roughening for the attachment of the coracobrachialis muscle.

1	Approximately in the middle of the shaft, the medial border is marked by thin elongate roughening for the attachment of the coracobrachialis muscle. Intermuscular septa, which separate the anterior compartment from the posterior compartment, attach to the medial and lateral borders (Fig. 7.61). Distally, the bone becomes flattened, and these borders expand as the lateral supraepicondylar ridge (lateral supracondylar ridge) and the medial supraepicondylar ridge (medial supracondylar ridge). The lateral supraepicondylar ridge is more pronounced than the medial ridge and is roughened for the attachment of muscles found in the posterior compartment of the forearm. The distal end of the humerus, which is flattened in the anteroposterior plane, bears a condyle, two epicondyles, and three fossae, as follows (Fig. 7.61). The condyle The two articular parts of the condyle, the capitulum and the trochlea, articulate with the two bones of the forearm.

1	The condyle The two articular parts of the condyle, the capitulum and the trochlea, articulate with the two bones of the forearm. The capitulum articulates with the radius of the forearm. Lateral in position and hemispherical in shape, it projects anteriorly and somewhat inferiorly and is not visible when the humerus is viewed from the posterior aspect. The trochlea articulates with the ulna of the forearm. It is pulley shaped and lies medial to the capitulum. Its medial edge is more pronounced than its lateral edge and, unlike the capitulum, it extends onto the posterior surface of the bone. The two epicondyles The two epicondyles lie adjacent, and somewhat superior, to the trochlea and capitulum (Fig. 7.61).

1	The two epicondyles The two epicondyles lie adjacent, and somewhat superior, to the trochlea and capitulum (Fig. 7.61). The medial epicondyle, a large bony protuberance, is the major palpable landmark on the medial side of the elbow, and projects medially from the distal end of the humerus. On its surface, it bears a large oval impression for the attachment of muscles in the anterior compartment of the forearm. The ulnar nerve passes from the arm into the forearm around the posterior surface of the medial epicondyle and can be palpated against the bone in this location. The lateral epicondyle is much less pronounced than the medial epicondyle. It is lateral to the capitulum and has a large irregular impression for the attachment of muscles in the posterior compartment of the forearm. The three fossae Three fossae occur superior to the trochlea and capitulum on the distal end of the humerus (Fig. 7.61).

1	The three fossae Three fossae occur superior to the trochlea and capitulum on the distal end of the humerus (Fig. 7.61). The radial fossa is the least distinct of the fossae and occurs immediately superior to the capitulum on the anterior surface of the humerus. The coronoid fossa is adjacent to the radial fossa and is superior to the trochlea. The largest of the fossae, the olecranon fossa, occurs immediately superior to the trochlea on the posterior surface of the distal end of the humerus. These three fossae accommodate projections from the bones in the forearm during movements of the elbow joint. Proximal end of the radius The proximal end of the radius consists of a head, a neck, and the radial tuberosity (Fig. 7.62A,B).

1	Proximal end of the radius The proximal end of the radius consists of a head, a neck, and the radial tuberosity (Fig. 7.62A,B). The head of the radius is a thick disc-shaped structure oriented in the horizontal plane. The circular superior surface is concave for articulation with the capitulum of the humerus. The thick margin of the disc is broad medially where it articulates with the radial notch on the proximal end of the ulna. The neck of the radius is a short and narrow cylinder of bone between the expanded head and the radial tuberosity on the shaft. The radial tuberosity is a large blunt projection on the medial surface of the radius immediately inferior to the neck. Much of its surface is roughened for the attachment of the biceps brachii tendon. The oblique line of the radius continues diagonally across the shaft of the bone from the inferior margin of the radial tuberosity. Proximal end of the ulna

1	Proximal end of the ulna The proximal end of the ulna is much larger than the proximal end of the radius and consists of the olecranon, the coronoid process, the trochlear notch, the radial notch, and the tuberosity of the ulna (Fig. 7.63A,B). The olecranon is a large projection of bone that extends proximally from the ulna. Its anterolateral surface is articular and contributes to the formation of the trochlear notch, which articulates with the trochlea of the humerus. The superior surface is marked by a large roughened impression for the attachment of the triceps brachii muscle. The posterior surface is smooth, shaped somewhat triangularly, and can be palpated as the “tip of the elbow.” The coronoid process projects anteriorly from the proximal end of the ulna (Fig. 7.63). Its superolateral surface is articular and participates, with the olecranon, in forming the trochlear notch. The lateral surface is marked by the radial notch for articulation with the head of the radius.

1	Just inferior to the radial notch is a fossa that allows the radial tuberosity to change position during pronation and supination. The posterior margin of this fossa is broadened to form the supinator crest. The anterior surface of the coronoid process is triangular, with the apex directed distally, and has a number of roughenings for muscle attachment. The largest of these roughenings, the tuberosity of the ulna, is at the apex of the anterior surface and is the attachment site for the brachialis muscle. The anterior compartment of the arm contains three muscles—the coracobrachialis, brachialis, and biceps brachii muscles—which are innervated predominantly by the musculocutaneous nerve. The posterior compartment contains one muscle—the triceps brachii muscle—which is innervated by the radial nerve.

1	The posterior compartment contains one muscle—the triceps brachii muscle—which is innervated by the radial nerve. The coracobrachialis muscle extends from the tip of the coracoid process of the scapula to the medial side of the midshaft of the humerus (Fig. 7.64 and Table 7.8). It passes through the axilla and is penetrated and innervated by the musculocutaneous nerve. The coracobrachialis muscle flexes the arm. The biceps brachii muscle has two heads: The short head of the muscle originates from the coracoid process in conjunction with the coracobrachialis. The long head originates as a tendon from the supraglenoid tubercle of the scapula (Fig. 7.64 and Table 7.8). The tendon of the long head passes through the glenohumeral joint superior to the head of the humerus and then passes through the intertubercular sulcus and enters the arm. In the arm, the tendon joins with its muscle belly and, together with the muscle belly of the short head, overlies the brachialis muscle.

1	The long and short heads converge to form a single tendon, which inserts onto the radial tuberosity. As the tendon enters the forearm, a flat sheet of connective tissue (the bicipital aponeurosis) fans out from the medial side of the tendon to blend with deep fascia covering the anterior compartment of the forearm. The biceps brachii muscle is a powerful flexor of the forearm at the elbow joint; it is also the most powerful supinator of the forearm when the elbow joint is flexed. Because the two heads of the biceps brachii muscle cross the glenohumeral joint, the muscle can also flex the glenohumeral joint. The biceps brachii muscle is innervated by the musculocutaneous nerve. A tap on the tendon of the biceps brachii at the elbow is used to test predominantly spinal cord segment C6.

1	The biceps brachii muscle is innervated by the musculocutaneous nerve. A tap on the tendon of the biceps brachii at the elbow is used to test predominantly spinal cord segment C6. The brachialis muscle originates from the distal half of the anterior aspect of the humerus and from adjacent parts of the intermuscular septa, particularly on the medial side (Fig. 7.64 and Table 7.8). It lies beneath the biceps brachii muscle, is flattened dorsoventrally, and converges to form a tendon, which attaches to the tuberosity of the ulna. The brachialis muscle flexes the forearm at the elbow joint. Innervation of the brachialis muscle is predominantly by the musculocutaneous nerve. A small component of the lateral part is innervated by the radial nerve. The only muscle of the posterior compartment of the arm is the triceps brachii muscle (Fig. 7.65 and Table 7.9). The triceps brachii muscle has three heads: The long head originates from the infraglenoid tubercle of the scapula.

1	The long head originates from the infraglenoid tubercle of the scapula. The medial head originates from the extensive area on the posterior surface of the shaft of the humerus inferior to the radial groove. The lateral head originates from a linear roughening superior to the radial groove of the humerus. The three heads converge to form a large tendon, which inserts on the superior surface of the olecranon of the ulna. The triceps brachii muscle extends the forearm at the elbow joint. Innervation of the triceps brachii is by branches of the radial nerve. A tap on the tendon of the triceps brachii tests predominantly spinal cord segment C7. The major artery of the arm, the brachial artery, is found in the anterior compartment (Fig. 7.66A). Beginning as a continuation of the axillary artery at the lower border of the teres major muscle, it terminates just distal to the elbow joint where it divides into the radial and ulnar arteries.

1	In the proximal arm, the brachial artery lies on the medial side. In the distal arm, it moves laterally to assume a position midway between the lateral epicondyle and the medial epicondyle of the humerus. It crosses anteriorly to the elbow joint where it lies immediately medial to the tendon of the biceps brachii muscle. The brachial artery is palpable along its length. In proximal regions, the brachial artery can be compressed against the medial side of the humerus. Branches of the brachial artery in the arm include those to adjacent muscles and two ulnar collateral vessels, which contribute to a network of arteries around the elbow joint (Fig. 7.66B). Additional branches are the profunda brachii artery and nutrient arteries to the humerus, which pass through a foramen in the anteromedial surface of the humeral shaft.

1	The profunda brachii artery, the largest branch of the brachial artery, passes into and supplies the posterior compartment of the arm (Fig. 7.66A,B). It enters the posterior compartment with the radial nerve and together they pass through the triangular interval, which is formed by the shaft of the humerus, the inferior margin of the teres major muscle, and the lateral margin of the long head of the triceps muscle. They then pass along the radial groove on the posterior surface of the humerus deep to the lateral head of the triceps brachii muscle. Branches of the profunda brachii artery supply adjacent muscles and anastomose with the posterior circumflex humeral artery. The artery terminates as two collateral vessels, which contribute to an anastomotic network of arteries around the elbow joint (Fig. 7.66B). Paired brachial veins pass along the medial and lateral sides of the brachial artery, receiving tributaries that accompany branches of the artery (Fig. 7.67).

1	Paired brachial veins pass along the medial and lateral sides of the brachial artery, receiving tributaries that accompany branches of the artery (Fig. 7.67). In addition to these deep veins, two large subcutaneous veins, the basilic vein and the cephalic vein, are located in the arm. The basilic vein passes vertically in the distal half of the arm, penetrates deep fascia to assume a position medial to the brachial artery, and then becomes the axillary vein at the lower border of the teres major muscle. The brachial veins join the basilic, or axillary, vein. The cephalic vein passes superiorly on the anterolateral aspect of the arm and through the anterior wall of the axilla to reach the axillary vein.

1	The cephalic vein passes superiorly on the anterolateral aspect of the arm and through the anterior wall of the axilla to reach the axillary vein. The musculocutaneous nerve leaves the axilla and enters the arm by passing through the coracobrachialis muscle (Fig. 7.68). It passes diagonally down the arm in the plane between the biceps brachii and brachialis muscles. After giving rise to motor branches in the arm, it emerges laterally to the tendon of the biceps brachii muscle at the elbow, penetrates deep fascia, and continues as the lateral cutaneous nerve of the forearm. The musculocutaneous nerve provides: motor innervation to all muscles in the anterior compartment of the arm, and sensory innervation to skin on the lateral surface of the forearm.

1	The musculocutaneous nerve provides: motor innervation to all muscles in the anterior compartment of the arm, and sensory innervation to skin on the lateral surface of the forearm. The median nerve enters the arm from the axilla at the inferior margin of the teres major muscle (Fig. 7.68). It passes vertically down the medial side of the arm in the anterior compartment and is related to the brachial artery throughout its course: In proximal regions, the median nerve is immediately lateral to the brachial artery. In more distal regions, the median nerve crosses to the medial side of the brachial artery and lies anterior to the elbow joint. The median nerve has no major branches in the arm, but a branch to one of the muscles of the forearm, the pronator teres muscle, may originate from the nerve immediately proximal to the elbow joint.

1	The median nerve has no major branches in the arm, but a branch to one of the muscles of the forearm, the pronator teres muscle, may originate from the nerve immediately proximal to the elbow joint. The ulnar nerve enters the arm with the median nerve and axillary artery (Fig. 7.68). It passes through proximal regions medial to the axillary artery. In the middle of the arm, the ulnar nerve penetrates the medial intermuscular septum and enters the posterior compartment where it lies anterior to the medial head of the triceps brachii muscle. It passes posterior to the medial epicondyle of the humerus and then into the anterior compartment of the forearm. The ulnar nerve has no major branches in the arm.

1	The ulnar nerve has no major branches in the arm. The radial nerve originates from the posterior cord of the brachial plexus and enters the arm by crossing the inferior margin of the teres major muscle (Fig. 7.69). As it enters the arm, it lies posterior to the brachial artery. Accompanied by the profunda brachii artery, the radial nerve enters the posterior compartment of the arm by passing through the triangular interval.

1	As the radial nerve passes diagonally, from medial to lateral, through the posterior compartment, it lies in the radial groove directly on bone. On the lateral side of the arm, it passes anteriorly through the lateral intermuscular septum and enters the anterior compartment where it lies between the brachialis muscle and a muscle of the posterior compartment of the forearm—the brachioradialis muscle, which attaches to the lateral supraepicondylar ridge of the humerus. The radial nerve enters the forearm anterior to the lateral epicondyle of the humerus, just deep to the brachioradialis muscle. In the arm, the radial nerve has muscular and cutaneous branches (Fig. 7.69).

1	In the arm, the radial nerve has muscular and cutaneous branches (Fig. 7.69). Muscular branches include those to the triceps brachii, brachioradialis, and extensor carpi radialis longus muscles. In addition, the radial nerve contributes to the innervation of the lateral part of the brachialis muscle. One of the branches to the medial head of the triceps brachii muscle arises before the radial nerve’s entrance into the posterior compartment and passes vertically down the arm in association with the ulnar nerve. Cutaneous branches of the radial nerve that originate in the posterior compartment of the arm are the inferior lateral cutaneous nerve of the arm and the posterior cutaneous nerve of the forearm, both of which penetrate through the lateral head of the triceps brachii muscle and the overlying deep fascia to become subcutaneous. The elbow joint is a complex joint involving three separate articulations, which share a common synovial cavity (Fig. 7.71).

1	The elbow joint is a complex joint involving three separate articulations, which share a common synovial cavity (Fig. 7.71). The joints between the trochlear notch of the ulna and the trochlea of the humerus and between the head of the radius and the capitulum of the humerus are primarily involved with hinge-like flexion and extension of the forearm on the arm and, together, are the principal articulations of the elbow joint. The joint between the head of the radius and the radial notch of the ulna, the proximal radio-ulnar joint, is involved with pronation and supination of the forearm. The articular surfaces of the bones are covered with hyaline cartilage. The synovial membrane originates from the edges of the articular cartilage and lines the radial fossa, the coronoid fossa, the olecranon fossa, the deep surface of the joint capsule, and the medial surface of the trochlea (Fig. 7.72).

1	The synovial membrane is separated from the fibrous membrane of the joint capsule by pads of fat in regions overlying the coronoid fossa, the olecranon fossa, and the radial fossa. These fat pads accommodate the related bony processes during extension and flexion of the elbow. Attachments of the brachialis and triceps brachii muscles to the joint capsule overlying these regions pull the attached fat pads out of the way when the adjacent bony processes are moved into the fossae. The fibrous membrane of the joint capsule overlies the synovial membrane, encloses the joint, and attaches to the medial epicondyle and the margins of the olecranon, coronoid, and radial fossae of the humerus (Fig. 7.73). It also attaches to the coronoid process and olecranon of the ulna. On the lateral side, the free inferior margin of the joint capsule passes around the neck of the radius from an anterior attachment to the coronoid process of the ulna to a posterior attachment to the base of the olecranon.

1	The fibrous membrane of the joint capsule is thickened medially and laterally to form collateral ligaments, which support the flexion and extension movements of the elbow joint (Fig. 7.73). In addition, the external surface of the joint capsule is reinforced laterally where it cuffs the head of the radius with a strong anular ligament of the radius. Although this ligament blends with the fibrous membrane of the joint capsule in most regions, they are separate posteriorly. The anular ligament of the radius also blends with the radial collateral ligament. The anular ligament of the radius and related joint capsule allow the radial head to slide against the radial notch of the ulna and pivot on the capitulum during pronation and supination of the forearm.

1	The deep surface of the fibrous membrane of the joint capsule and the related anular ligament of the radius that articulate with the sides of the radial head are lined by cartilage. A pocket of synovial membrane (sacciform recess) protrudes from the inferior free margin of the joint capsule and facilitates rotation of the radial head during pronation and supination. Vascular supply to the elbow joint is through an anastomotic network of vessels derived from collateral and recurrent branches of the brachial, profunda brachii, radial, and ulnar arteries. The elbow joint is innervated predominantly by branches of the radial and musculocutaneous nerves, but there may be some innervation by branches of the ulnar and median nerves.

1	The elbow joint is innervated predominantly by branches of the radial and musculocutaneous nerves, but there may be some innervation by branches of the ulnar and median nerves. The cubital fossa is an important area of transition between the arm and the forearm. It is located anterior to the elbow joint and is a triangular depression formed between two forearm muscles: the brachioradialis muscle originating from the lateral supra-epicondylar ridge of the humerus, and the pronator teres muscle originating from the medial epicondyle of the humerus (Fig. 7.77A). The base of the triangle is an imaginary horizontal line between the medial and lateral epicondyles. The bed or floor of the fossa is formed mainly by the brachialis muscle. The major contents of the cubital fossa, from lateral to medial, are: the tendon of the biceps brachii muscle, the brachial artery, and the median nerve (Fig. 7.77B).

1	The major contents of the cubital fossa, from lateral to medial, are: the tendon of the biceps brachii muscle, the brachial artery, and the median nerve (Fig. 7.77B). The brachial artery normally bifurcates into the radial and ulnar arteries in the apex of the fossa (Fig. 7.77B), although this bifurcation may occur much higher in the arm, even in the axilla. When taking a blood pressure reading from a patient, the clinician places the stethoscope over the brachial artery in the cubital fossa. The median nerve lies immediately medial to the brachial artery and leaves the fossa by passing between the ulnar and humeral heads of the pronator teres muscle (Fig. 7.77C).

1	The median nerve lies immediately medial to the brachial artery and leaves the fossa by passing between the ulnar and humeral heads of the pronator teres muscle (Fig. 7.77C). The brachial artery and the median nerve are covered and protected anteriorly in the distal part of the cubital fossa by the bicipital aponeurosis (Fig. 7.77B). This flat connective tissue membrane passes between the medial side of the tendon of the biceps brachii muscle and deep fascia of the forearm. The sharp medial margin of the bicipital aponeurosis can often be felt. The radial nerve lies just under the lip of the brachioradialis muscle, which forms the lateral margin of the fossa (Fig. 7.77C). In this position, the radial nerve divides into superficial and deep branches: The superficial branch continues into the forearm just deep to the brachioradialis muscle.

1	The superficial branch continues into the forearm just deep to the brachioradialis muscle. The deep branch passes between the two heads of the supinator muscle (see pp. 778–780 and Fig. 7.92) to access the posterior compartment of the forearm. The ulnar nerve does not pass through the cubital fossa. Instead, it passes posterior to the medial epicondyle. The roof of the cubital fossa is formed by superficial fascia and skin. The most important structure within the roof is the median cubital vein (Fig. 7.77D), which passes diagonally across the roof and connects the cephalic vein on the lateral side of the upper limb with the basilic vein on the medial side. The bicipital aponeurosis separates the median cubital vein from the brachial artery and median nerve. Other structures within the roof are cutaneous nerves—the medial cutaneous and lateral cutaneous nerves of the forearm.

1	The forearm is the part of the upper limb that extends between the elbow joint and the wrist joint. Proximally, most major structures pass between the arm and forearm through, or in relation to, the cubital fossa, which is anterior to the elbow joint (Fig. 7.79). The exception is the ulnar nerve, which passes posterior to the medial epicondyle of the humerus. Distally, structures pass between the forearm and the hand through, or anterior to, the carpal tunnel (Fig. 7.79). The major exception is the radial artery, which passes dorsally around the wrist to enter the hand posteriorly. The bone framework of the forearm consists of two parallel bones, the radius and the ulna (Figs. 7.79 and 7.80B). The radius is lateral in position and is small proximally, where it articulates with the humerus, and large distally, where it forms the wrist joint with the carpal bones of the hand.

1	The ulna is medial in the forearm, and its proximal and distal dimensions are the reverse of those for the radius: the ulna is large proximally and small distally. Proximal and distal joints between the radius and the ulna allow the distal end of the radius to swing over the adjacent end of the ulna, resulting in pronation and supination of the hand. As in the arm, the forearm is divided into anterior and posterior compartments (Fig. 7.79). In the forearm, these compartments are separated by: a lateral intermuscular septum, which passes from the anterior border of the radius to deep fascia surrounding the limb; an interosseous membrane, which links adjacent borders of the radius and ulna along most of their length; and the attachment of deep fascia along the posterior border of the ulna.

1	Muscles in the anterior compartment of the forearm flex the wrist and digits and pronate the hand. Muscles in the posterior compartment extend the wrist and digits and supinate the hand. Major nerves and vessels supply or pass through each compartment. Shaft and distal end of radius The shaft of the radius is narrow proximally, where it is continuous with the radial tuberosity and neck, and much broader distally, where it expands to form the distal end (Fig. 7.80). Throughout most of its length, the shaft of the radius is triangular in cross section, with: three borders (anterior, posterior, and interosseous), and three surfaces (anterior, posterior, and lateral).

1	The anterior border begins on the medial side of the bone as a continuation of the radial tuberosity. In the superior third of the bone, it crosses the shaft diagonally, from medial to lateral, as the oblique line of the radius. The posterior border is distinct only in the middle third of the bone. The interosseous border is sharp and is the attachment site for the interosseous membrane, which links the radius to the ulna. The anterior and posterior surfaces of the radius are generally smooth, whereas an oval roughening for the attachment of the pronator teres marks approximately the middle of the lateral surface of the radius. Viewed anteriorly, the distal end of the radius is broad and somewhat flattened anteroposteriorly (Fig. 7.80). Consequently, the radius has expansive anterior and posterior surfaces and narrow medial and lateral surfaces. Its anterior surface is smooth and unremarkable, except for the prominent sharp ridge that forms its lateral margin.

1	The posterior surface of the radius is characterized by the presence of a large dorsal tubercle, which acts as a pulley for the tendon of one of the extensor muscles of the thumb (extensor pollicis longus). The medial surface is marked by a prominent facet for articulation with the distal end of the ulna (Fig. 7.80). The lateral surface of the radius is diamond shaped and extends distally as a radial styloid process. The distal end of the bone is marked by two facets for articulation with two carpal bones (the scaphoid and lunate). Shaft and distal end of ulna The shaft of the ulna is broad superiorly where it is continuous with the large proximal end and narrow distally to form a small distal head (Fig. 7.81). Like the radius, the shaft of the ulna is triangular in cross section and has: three borders (anterior, posterior, and interosseous), and three surfaces (anterior, posterior, and medial).

1	The anterior border is smooth and rounded. The posterior border is sharp and palpable along its entire length. The interosseous border is also sharp and is the attachment site for the interosseous membrane, which joins the ulna to the radius. The anterior surface of the ulna is smooth, except distally where there is a prominent linear roughening for the attachment of the pronator quadratus muscle. The medial surface is smooth and unremarkable. The posterior surface is marked by lines, which separate different regions of muscle attachments to bone. The distal end of the ulna is small and characterized by a rounded head and the ulnar styloid process (Fig. 7.81). The anterolateral and distal part of the head is covered by articular cartilage. The ulnar styloid process originates from the posteromedial aspect of the ulna and projects distally.

1	The distal radio-ulnar joint occurs between the articular surface of the head of the ulna, with the ulnar notch on the end of the radius, and with a fibrous articular disc, which separates the radio-ulnar joint from the wrist joint (Fig. 7.82). The triangular-shaped articular disc is attached by its apex to a roughened depression on the ulna between the styloid process and the articular surface of the head, and by its base to the angular margin of the radius between the ulnar notch and the articular surface for the carpal bones. The synovial membrane is attached to the margins of the distal radio-ulnar joint and is covered on its external surface by a fibrous joint capsule. The distal radio-ulnar joint allows the distal end of the radius to move anteromedially over the ulna.

1	The distal radio-ulnar joint allows the distal end of the radius to move anteromedially over the ulna. The interosseous membrane is a thin fibrous sheet that connects the medial and lateral borders of the radius and ulna, respectively (Fig. 7.82). Collagen fibers within the sheet pass predominantly inferiorly from the radius to the ulna. The interosseous membrane has a free upper margin, which is situated just inferior to the radial tuberosity, and a small circular aperture in its distal third. Vessels pass between the anterior and posterior compartments superior to the upper margin and through the inferior aperture. The interosseous membrane connects the radius and ulna without restricting pronation and supination and provides attachment for muscles in the anterior and posterior compartments. The orientation of fibers in the membrane is also consistent with its role in transferring forces from the radius to the ulna and ultimately, therefore, from the hand to the humerus.

1	Pronation and supination of the hand occur entirely in the forearm and involve rotation of the radius at the elbow and movement of the distal end of the radius over the ulna (Fig. 7.83). At the elbow, the superior articular surface of the radial head spins on the capitulum while, at the same time, the articular surface on the side of the head slides against the radial notch of the ulna and adjacent areas of the joint capsule and anular ligament of the radius. At the distal radio-ulnar joint, the ulnar notch of the radius slides anteriorly over the convex surface of the head of the ulna. During these movements, the bones are held together by: the anular ligament of the radius at the proximal radio-ulnar joint, the interosseous membrane along the lengths of the radius and ulna, and the articular disc at the distal radio-ulnar joint (Fig. 7.83).

1	Because the hand articulates predominantly with the radius, the translocation of the distal end of the radius medially over the ulna moves the hand from the palm-anterior (supinated) position to the palm-posterior (pronated) position. Two muscles supinate and two muscles pronate the hand (Fig. 7.83). Biceps brachii. The biceps brachii muscle, the largest of the four muscles that supinate and pronate the hand, is a powerful supinator as well as a flexor of the elbow joint. It is most effective as a supinator when the forearm is flexed. Supinator. The second of the muscles involved with supination is the supinator muscle. Located in the posterior compartment of the forearm, it has a broad origin, from the supinator crest of the ulna and the lateral epicondyle of the humerus and from ligaments associated with the elbow joint.

1	The supinator muscle curves around the posterior surface and the lateral surface of the upper third of the radius to attach to the shaft of the radius superior to the oblique line. The tendon of the biceps brachii muscle and the supinator muscle both become wrapped around the proximal end of the radius when the hand is pronated (Fig. 7.83). When they contract, they unwrap from the bone, producing supination of the hand. Pronator teres and pronator quadratus. Pronation results from the action of the pronator teres and pronator quadratus muscles (Fig. 7.83). Both these muscles are in the anterior compartment of the forearm: The pronator teres runs from the medial epicondyle of the humerus to the lateral surface of the radius, approximately midway along the shaft. The pronator quadratus extends between the anterior surfaces of the distal ends of the radius and ulna.

1	The pronator quadratus extends between the anterior surfaces of the distal ends of the radius and ulna. When these muscles contract, they pull the distal end of the radius over the ulna, resulting in pronation of the hand (Fig. 7.83). Anconeus. In addition to hinge-like flexion and extension at the elbow joint, some abduction of the distal end of the ulna also occurs and maintains the position of the palm of the hand over a central axis during pronation (Fig. 7.84). The muscle involved in this movement is the anconeus muscle, which is a triangular muscle in the posterior compartment of the forearm that runs from the lateral epicondyle to the lateral surface of the proximal end of the ulna. Muscles in the anterior (flexor) compartment of the forearm occur in three layers: superficial, intermediate, and deep. Generally, these muscles are associated with: movements of the wrist joint, flexion of the fingers including the thumb, and pronation.

1	All muscles in the anterior compartment of the forearm are innervated by the median nerve, except for the flexor carpi ulnaris muscle and the medial half of the flexor digitorum profundus muscle, which are innervated by the ulnar nerve. All four muscles in the superficial layer—the flexor carpi ulnaris, palmaris longus, flexor carpi radialis, and pronator teres—have a common origin from the medial epicondyle of the humerus, and, except for the pronator teres, extend distally from the forearm into the hand (Fig. 7.85 and Table 7.10). The flexor carpi ulnaris muscle is the most medial of the muscles in the superficial layer of flexors, having a long linear origin from the olecranon and posterior border of the ulna, in addition to an origin from the medial epicondyle of the humerus (Fig. 7.85A,B).

1	The ulnar nerve enters the anterior compartment of the forearm by passing through the triangular gap between the humeral and ulnar heads of the flexor carpi ulnaris (Fig. 7.85B). The muscle fibers converge on a tendon that passes distally and attaches to the pisiform bone of the wrist. From this point, force is transferred to the hamate bone of the wrist and to the base of metacarpal V by the pisohamate and pisometacarpal ligaments. The flexor carpi ulnaris muscle is a powerful flexor and adductor of the wrist and is innervated by the ulnar nerve (Table 7.10). The palmaris longus muscle, which is absent in about 15% of the population, lies between the flexor carpi ulnaris and the flexor carpi radialis muscles (Fig. 7.85A). It is a spindle-shaped muscle with a long tendon, which passes into the hand and attaches to the flexor retinaculum and to a thick layer of deep fascia, the palmar aponeurosis, which underlies and is attached to the skin of the palm and fingers.

1	In addition to its role as an accessory flexor of the wrist joint, the palmaris longus muscle also opposes shearing forces on the skin of the palm during gripping (Table 7.10). The flexor carpi radialis muscle is lateral to the palmaris longus and has a large and prominent tendon in the distal half of the forearm (Fig. 7.85A and Table 7.10). Unlike the tendon of the flexor carpi ulnaris, which forms the medial margin of the distal forearm, the tendon of the flexor carpi radialis muscle is positioned just lateral to the midline. In this position, the tendon can be easily palpated, making it an important landmark for finding the pulse in the radial artery, which lies immediately lateral to it. The tendon of the flexor carpi radialis passes through a compartment formed by bone and fascia on the lateral side of the anterior surface of the wrist and attaches to the anterior surfaces of the bases of metacarpals II and III.

1	The flexor carpi radialis is a powerful flexor of the wrist and can also abduct the wrist. The pronator teres muscle originates from the medial epicondyle and supraepicondylar ridge of the humerus and from a small linear region on the medial edge of the coronoid process of the ulna (Fig. 7.85A). The median nerve often exits the cubital fossa by passing between the humeral and ulnar heads of this muscle. The pronator teres crosses the forearm and attaches to an oval roughened area on the lateral surface of the radius approximately midway along the bone. The pronator teres forms the medial border of the cubital fossa and rotates the radius over the ulna during pronation (Table 7.10).

1	The pronator teres forms the medial border of the cubital fossa and rotates the radius over the ulna during pronation (Table 7.10). The muscle in the intermediate layer of the anterior compartment of the forearm is the flexor digitorum superficialis muscle (Fig. 7.86). This large muscle has two heads: the humero-ulnar head, which originates mainly from the medial epicondyle of the humerus and from the adjacent medial edge of the coronoid process of the ulna; and the radial head, which originates from the anterior oblique line of the radius. The median nerve and ulnar artery pass deep to the flexor digitorum superficialis between the two heads. In the distal forearm, the flexor digitorum superficialis forms four tendons, which pass through the carpal tunnel of the wrist and into the four fingers. The tendons for the ring and middle fingers are superficial to the tendons for the index and little fingers.

1	In the forearm, carpal tunnel, and proximal regions of the four fingers, the tendons of the flexor digitorum superficialis are anterior to the tendons of the flexor digitorum profundus muscle. Near the base of the proximal phalanx of each finger, the tendon of the flexor digitorum superficialis splits into two parts to pass posteriorly around each side of the tendon of the flexor digitorum profundus and ultimately attach to the margins of the middle phalanx (Fig. 7.86). The flexor digitorum superficialis flexes the metacarpophalangeal joint and proximal interphalangeal joint of each finger; it also flexes the wrist joint (Table 7.11). There are three deep muscles in the anterior compartment of the forearm: the flexor digitorum profundus, flexor pollicis longus, and pronator quadratus (Fig. 7.87).

1	There are three deep muscles in the anterior compartment of the forearm: the flexor digitorum profundus, flexor pollicis longus, and pronator quadratus (Fig. 7.87). The flexor digitorum profundus muscle originates from the anterior and medial surfaces of the ulna and from the adjacent half of the anterior surface of the interosseous membrane (Fig. 7.87). It gives rise to four tendons, which pass through the carpal tunnel into the four medial fingers. Throughout most of their course, the tendons are deep to the tendons of the flexor digitorum superficialis muscle. Opposite the proximal phalanx of each finger, each tendon of the flexor digitorum profundus passes through a split formed in the overlying tendon of the flexor digitorum superficialis muscle and passes distally to insert into the anterior surface of the base of the distal phalanx. In the palm, the lumbrical muscles originate from the sides of the tendons of the flexor digitorum profundus (see Fig. 7.108).

1	In the palm, the lumbrical muscles originate from the sides of the tendons of the flexor digitorum profundus (see Fig. 7.108). Innervation of the medial and lateral halves of the flexor digitorum profundus varies as follows: The lateral half (associated with the index and middle fingers) is innervated by the anterior interosseous nerve (branch of the median nerve). The medial half (the part associated with the ring and little fingers) is innervated by the ulnar nerve. The flexor digitorum profundus flexes the metacarpophalangeal joints and the proximal and distal interphalangeal joints of the four fingers. Because the tendons cross the wrist, it can flex the wrist joint as well (Table 7.12).

1	The flexor pollicis longus muscle originates from the anterior surface of the radius and the adjacent half of the anterior surface of the interosseous membrane (Fig. 7.87). It is a powerful muscle and forms a single large tendon, which passes through the carpal tunnel, lateral to the tendons of the flexor digitorum superficialis and flexor digitorum profundus muscles, and into the thumb where it attaches to the base of the distal phalanx. The flexor pollicis longus flexes the thumb and is innervated by the anterior interosseous nerve (branch of the median nerve) (Table 7.12). The pronator quadratus muscle is a flat square-shaped muscle in the distal forearm (Fig. 7.87). It originates from a linear ridge on the anterior surface of the lower end of the ulna and passes laterally to insert onto the flat anterior surface of the radius. It lies deep to, and is crossed by, the tendons of the flexor digitorum profundus and flexor pollicis longus muscles.

1	The pronator quadratus muscle pulls the distal end of the radius anteriorly over the ulna during pronation and is innervated by the anterior interosseous nerve (branch of the median nerve) (Table 7.12). The largest arteries in the forearm are in the anterior compartment, pass distally to supply the hand, and give rise to vessels that supply the posterior compartment (Fig. 7.88). The brachial artery enters the forearm from the arm by passing through the cubital fossa. At the apex of the cubital fossa, it divides into its two major branches, the radial and ulnar arteries.

1	The brachial artery enters the forearm from the arm by passing through the cubital fossa. At the apex of the cubital fossa, it divides into its two major branches, the radial and ulnar arteries. The radial artery originates from the brachial artery at approximately the neck of the radius and passes along the lateral aspect of the forearm (Fig. 7.88). It is: just deep to the brachioradialis muscle in the proximal half of the forearm, related on its lateral side to the superficial branch of the radial nerve in the middle third of the forearm, and medial to the tendon of the brachioradialis muscle and covered only by deep fascia, superficial fascia, and skin in the distal forearm.

1	In the distal forearm, the radial artery lies immediately lateral to the large tendon of the flexor carpi radialis muscle and directly anterior to the pronator quadratus muscle and the distal end of the radius (Fig. 7.88). In the distal forearm, the radial artery can be located using the flexor carpi radialis muscle as a landmark. The radial pulse can be felt by gently palpating the radial artery against the underlying muscle and bone. The radial artery leaves the forearm, passes around the lateral side of the wrist, and penetrates the posterolateral aspect of the hand between the bases of metacarpals I and II (Fig. 7.88). Branches of the radial artery in the hand often provide the major blood supply to the thumb and lateral side of the index finger.

1	Branches of the radial artery originating in the forearm include: a radial recurrent artery, which contributes to an anastomotic network around the elbow joint and to numerous vessels that supply muscles on the lateral side of the forearm (see Fig. 7.66B); a small palmar carpal branch, which contributes to an anastomotic network of vessels that supply the carpal bones and joints; a somewhat larger branch, the superficial palmar branch, which enters the hand by passing through, or superficial to, the thenar muscles at the base of the thumb (Fig. 7.88) and anastomoses with the superficial palmar arch formed by the ulnar artery. The ulnar artery is larger than the radial artery and passes down the medial side of the forearm (Fig. 7.88). It leaves the cubital fossa by passing deep to the pronator teres muscle, and then passes through the forearm in the fascial plane between the flexor carpi ulnaris and flexor digitorum profundus muscles.

1	In the distal forearm, the ulnar artery often remains tucked under the anterolateral lip of the flexor carpi ulnaris tendon, and is therefore not easily palpable. In distal regions of the forearm, the ulnar nerve is immediately medial to the ulnar artery. The ulnar artery leaves the forearm, enters the hand by passing lateral to the pisiform bone and superficial to the flexor retinaculum of the wrist, and arches over the palm (Fig. 7.88). It is often the major blood supply to the medial three and one-half digits.

1	Branches of the ulnar artery that arise in the forearm include: the ulnar recurrent artery with anterior and posterior branches, which contribute to an anastomotic network of vessels around the elbow joint (see Fig. 7.66B); numerous muscular arteries, which supply surrounding muscles; the common interosseous artery, which divides into anterior and posterior interosseous arteries (Fig. 7.88); and two small carpal arteries (dorsal carpal branch and palmar carpal branch), which supply the wrist. The posterior interosseous artery passes dorsally over the proximal margin of the interosseous membrane into the posterior compartment of the forearm.

1	The posterior interosseous artery passes dorsally over the proximal margin of the interosseous membrane into the posterior compartment of the forearm. The anterior interosseous artery passes distally along the anterior aspect of the interosseous membrane and supplies muscles of the deep compartment of the forearm and the radius and ulna. It has numerous branches, which perforate the interosseous membrane to supply deep muscles of the posterior compartment; it also has a small branch, which contributes to the vascular network around the carpal bones and joints. Perforating the interosseous membrane in the distal forearm, the anterior interosseous artery terminates by joining the posterior interosseous artery. Deep veins of the anterior compartment generally accompany the arteries and ultimately drain into brachial veins associated with the brachial artery in the cubital fossa.

1	Deep veins of the anterior compartment generally accompany the arteries and ultimately drain into brachial veins associated with the brachial artery in the cubital fossa. Nerves in the anterior compartment of the forearm are the median and ulnar nerves and the superficial branch of the radial nerve (Fig. 7.89). The median nerve innervates the muscles in the anterior compartment of the forearm except for the flexor carpi ulnaris and the medial part of the flexor digitorum profundus (ring and little fingers). It leaves the cubital fossa by passing between the two heads of the pronator teres muscle and passing between the humero-ulnar and radial heads of the flexor digitorum superficialis muscle (Fig. 7.89).

1	The median nerve continues a straight linear course distally down the forearm in the fascia on the deep surface of the flexor digitorum superficialis muscle. Just proximal to the wrist, it moves around the lateral side of the muscle and becomes more superficial in position, lying between the tendons of the palmaris longus and flexor carpi radialis muscles. It leaves the forearm and enters the palm of the hand by passing through the carpal tunnel deep to the flexor retinaculum. Most branches to the muscles in the superficial and intermediate layers of the forearm originate medially from the nerve just distal to the elbow joint.

1	Most branches to the muscles in the superficial and intermediate layers of the forearm originate medially from the nerve just distal to the elbow joint. The largest branch of the median nerve in the forearm is the anterior interosseous nerve, which originates between the two heads of the pronator teres, passes distally down the forearm with the anterior interosseous artery, innervates the muscles in the deep layer (the flexor pollicis longus, the lateral half of the flexor digitorum profundus, and the pronator quadratus) and terminates as articular branches to joints of the distal forearm and wrist. A small palmar branch originates from the median nerve in the distal forearm immediately proximal to the flexor retinaculum (Fig. 7.89), passes superficially into the hand, and innervates the skin over the base and central palm. This palmar branch is spared in carpal tunnel syndrome because it passes into the hand superficial to the flexor retinaculum of the wrist.

1	The ulnar nerve passes through the forearm and into the hand, where most of its major branches occur. In the forearm, the ulnar nerve innervates only the flexor carpi ulnaris muscle and the medial part (ring and little fingers) of the flexor digitorum profundus muscle (Fig. 7.89). The ulnar nerve enters the anterior compartment of the forearm by passing posteriorly around the medial epicondyle of the humerus and between the humeral and ulnar heads of the flexor carpi ulnaris muscle. After passing down the medial side of the forearm in the plane between the flexor carpi ulnaris and the flexor digitorum profundus muscles, it lies under the lateral lip of the tendon of the flexor carpi ulnaris proximal to the wrist. The ulnar artery is lateral to the ulnar nerve in the distal two-thirds of the forearm, and both the ulnar artery and nerve enter the hand by passing superficial to the flexor retinaculum and immediately lateral to the pisiform bone (Fig. 7.89).

1	In the forearm the ulnar nerve gives rise to: muscular branches to the flexor carpi ulnaris and to the medial half of the flexor digitorum profundus that arise soon after the ulnar nerve enters the forearm; and two small cutaneous branches—the palmar branch originates in the middle of the forearm and passes into the hand to supply skin on the medial side of the palm; the larger dorsal branch originates from the ulnar nerve in the distal forearm and passes posteriorly deep to the tendon of the flexor carpi ulnaris and innervates skin on the posteromedial side of the back of the hand and most skin on the posterior surfaces of the medial one and one-half digits. The radial nerve bifurcates into deep and superficial branches under the margin of the brachioradialis muscle in the lateral border of the cubital fossa (Fig. 7.89).

1	The radial nerve bifurcates into deep and superficial branches under the margin of the brachioradialis muscle in the lateral border of the cubital fossa (Fig. 7.89). The deep branch is predominantly motor and passes between the superficial and deep layers of the supinator muscle to access and supply muscles in the posterior compartment of the forearm. The superficial branch of the radial nerve is sensory. It passes down the anterolateral aspect of the forearm deep to the brachioradialis muscle and in association with the radial artery. Approximately two-thirds of the way down the forearm, the superficial branch of the radial nerve passes laterally and posteriorly around the radial side of the forearm deep to the tendon of the brachioradialis. The nerve continues into the hand where it innervates skin on the posterolateral surface.

1	Muscles in the posterior compartment of the forearm occur in two layers: a superficial and a deep layer. The muscles are associated with: movement of the wrist joint, extension of the fingers and thumb, and supination. All muscles in the posterior compartment of the forearm are innervated by the radial nerve. The seven muscles in the superficial layer are the brachioradialis, extensor carpi radialis longus, extensor carpi radialis brevis, extensor digitorum, extensor digiti minimi, extensor carpi ulnaris, and anconeus (Fig. 7.90). All have a common origin from the supraepicondylar ridge and lateral epicondyle of the humerus and, except for the brachioradialis and anconeus, extend as tendons into the hand. The brachioradialis muscle originates from the proximal part of the supraepicondylar ridge of the humerus and passes through the forearm to insert on the lateral side of the distal end of the radius just proximal to the radial styloid process (Fig. 7.90).

1	In the anatomical position, the brachioradialis is part of the muscle mass overlying the anterolateral surface of the forearm and forms the lateral boundary of the cubital fossa. Because the brachioradialis is anterior to the elbow joint, it acts as an accessory flexor of this joint even though it is in the posterior compartment of the forearm. Its action is most efficient when the forearm is midpronated and it forms a prominent bulge as it acts against resistance. The radial nerve emerges from the posterior compartment of the arm just deep to the brachioradialis in the distal arm and innervates the brachioradialis. Lateral to the cubital fossa, the brachioradialis lies over the radial nerve and its bifurcation into deep and superficial branches. In more distal regions, the brachioradialis lies over the superficial branch of the radial nerve and radial artery (Table 7.13).

1	The extensor carpi radialis longus muscle originates from the distal part of the supraepicondylar ridge and the lateral epicondyle of the humerus; its tendon inserts on the dorsal surface of the base of metacarpal II (Fig. 7.90). In proximal regions, it is deep to the brachioradialis muscle. The extensor carpi radialis longus muscle extends and abducts the wrist, and is innervated by the radial nerve before the nerve divides into superficial and deep branches (Table 7.13). The extensor carpi radialis brevis muscle originates from the lateral epicondyle of the humerus, and the tendon inserts onto adjacent dorsal surfaces of the bases of metacarpals II and III (Fig. 7.90). Along much of its course, the extensor carpi radialis brevis lies deep to the extensor carpi radialis longus.

1	The extensor carpi radialis brevis muscle extends and abducts the wrist, and is innervated by the deep branch of the radial nerve before the nerve passes between the two heads of the supinator muscle (Table 7.13). The extensor digitorum muscle is the major extensor of the four fingers (index, middle, ring, and little fingers). It originates from the lateral epicondyle of the humerus and forms four tendons, each of which passes into a finger (Fig. 7.90). On the dorsal surface of the hand, adjacent tendons of the extensor digitorum are interconnected. In the fingers, each tendon inserts, via a triangular-shaped connective tissue aponeurosis (the extensor hood), into the base of the dorsal surfaces of the middle and distal phalanges. The extensor digitorum muscle is innervated by the posterior interosseous nerve, which is the continuation of the deep branch of the radial nerve after it emerges from the supinator muscle (Table 7.13).

1	The extensor digiti minimi muscle is an accessory extensor of the little finger and is medial to the extensor digitorum in the forearm (Fig. 7.90). It originates from the lateral epicondyle of the humerus and inserts, together with the tendon of the extensor digitorum, into the extensor hood of the little finger. The extensor digiti minimi is innervated by the posterior interosseous nerve (Table 7.13). The extensor carpi ulnaris muscle is medial to the extensor digiti minimi (Fig. 7.90). It originates from the lateral epicondyle, and its tendon inserts into the medial side of the base of metacarpal V. The extensor carpi ulnaris extends and adducts the wrist, and is innervated by the posterior interosseous nerve (Table 7.13).

1	The extensor carpi ulnaris extends and adducts the wrist, and is innervated by the posterior interosseous nerve (Table 7.13). The anconeus muscle is the most medial of the superficial extensors and has a triangular shape. It originates from the lateral epicondyle of the humerus and has a broad insertion into the posterolateral surface of the olecranon and related posterior surface of the ulna (see Fig. 7.84). The anconeus abducts the ulna during pronation to maintain the center of the palm over the same point when the hand is flipped. It is also considered to be an accessory extensor of the elbow joint. The anconeus is innervated by the branch of the radial nerve that innervates the medial head of the triceps brachii muscle (Table 7.13). The deep layer of the posterior compartment of the forearm consists of five muscles: supinator, abductor pollicis longus, extensor pollicis brevis, extensor pollicis longus, and extensor indicis (Fig. 7.91).

1	Except for the supinator muscle, all these deep layer muscles originate from the posterior surfaces of the radius, ulna, and interosseous membrane and pass into the thumb and fingers. Three of these muscles—the abductor pollicis longus, extensor pollicis brevis, and extensor pollicis longus—emerge from between the extensor digitorum and the extensor carpi radialis brevis tendons of the superficial layer and pass into the thumb. Two of the three “outcropping” muscles (the abductor pollicis longus and extensor pollicis brevis) form a distinct muscular bulge in the distal posterolateral surface of the forearm. All muscles of the deep layer are innervated by the posterior interosseous nerve, the continuation of the deep branch of the radial nerve. The supinator muscle has two layers, which insert together on the proximal aspect of the radius (Fig. 7.91):

1	The supinator muscle has two layers, which insert together on the proximal aspect of the radius (Fig. 7.91): The more superficial (humeral) layer originates mainly from the lateral epicondyle of the humerus and the related anular ligament and the radial collateral ligament of the elbow joint. The deep (ulnar) layer originates mainly from the supinator crest on the posterolateral surface of the ulna. From their sites of origin, the two layers wrap around the posterior and lateral aspect of the head, neck, and proximal shaft of the radius to insert on the lateral surface of the radius superior to the anterior oblique line and to the insertion of the pronator teres muscle. The supinator muscle supinates the forearm and hand. The deep branch of the radial nerve innervates the supinator muscle and passes to the posterior compartment of the forearm by passing between the two heads of this muscle (Table 7.14).

1	The deep branch of the radial nerve innervates the supinator muscle and passes to the posterior compartment of the forearm by passing between the two heads of this muscle (Table 7.14). The abductor pollicis longus muscle originates from the proximal posterior surfaces of the radius and the ulna and from the related interosseous membrane (Fig. 7.91). In the distal forearm, it emerges between the extensor digitorum and extensor carpi radialis brevis muscles to form a tendon that passes into the thumb and inserts on the lateral side of the base of metacarpal I. The tendon contributes to the lateral border of the anatomical snuffbox at the wrist. The major function of the abductor pollicis longus is to abduct the thumb at the joint between the metacarpal I and trapezium bones (Table 7.14).

1	The major function of the abductor pollicis longus is to abduct the thumb at the joint between the metacarpal I and trapezium bones (Table 7.14). The extensor pollicis brevis muscle arises distal to the origin of the abductor pollicis longus from the posterior surface of the radius and interosseous membrane (Fig. 7.91). Together with the abductor pollicis longus, it emerges between the extensor digitorum and extensor carpi radialis brevis muscles to form a bulge on the posterolateral surface of the distal forearm. The tendon of the extensor pollicis brevis passes into the thumb and inserts on the dorsal surface of the base of the proximal phalanx. At the wrist, the tendon contributes to the lateral border of the anatomical snuffbox. The extensor pollicis brevis extends the metacarpophalangeal and carpometacarpal joints of the thumb (Table 7.14).

1	The extensor pollicis brevis extends the metacarpophalangeal and carpometacarpal joints of the thumb (Table 7.14). The extensor pollicis longus muscle originates from the posterior surface of the ulna and adjacent interosseous membrane and inserts via a long tendon into the dorsal surface of the distal phalanx of the thumb (Fig. 7.91). Like the abductor pollicis longus and extensor pollicis brevis, the tendon of this muscle emerges between the extensor digitorum and the extensor carpi radialis brevis muscles. However, it is held away from the other two deep muscles of the thumb by passing medially around the dorsal tubercle on the distal end of the radius. The tendon forms the medial margin of the anatomical snuffbox at the wrist. The extensor pollicis longus extends all joints of the thumb (Table 7.14).

1	The extensor pollicis longus extends all joints of the thumb (Table 7.14). The extensor indicis muscle is an accessory extensor of the index finger. It originates distal to the extensor pollicis longus from the posterior surface of the ulna and adjacent interosseous membrane (Fig. 7.91). The tendon passes into the hand and inserts into the extensor hood of the index finger with the tendon of the extensor digitorum (Table 7.14). The blood supply to the posterior compartment of the forearm occurs predominantly through branches of the radial, posterior interosseous, and anterior interosseous arteries (Fig. 7.92).

1	The blood supply to the posterior compartment of the forearm occurs predominantly through branches of the radial, posterior interosseous, and anterior interosseous arteries (Fig. 7.92). The posterior interosseous artery originates in the anterior compartment from the common interosseous branch of the ulnar artery and passes posteriorly over the proximal margin of the interosseous membrane and into the posterior compartment of the forearm. It contributes a branch, the recurrent interosseous artery (see Fig. 7.66B), to the vascular network around the elbow joint and then passes between the supinator and abductor pollicis longus muscles to supply the superficial extensors. After receiving the terminal end of the anterior interosseous artery, the posterior interosseous artery terminates by joining the dorsal carpal arch of the wrist.

1	The anterior interosseous artery, also a branch of the common interosseous branch of the ulnar artery, is situated in the anterior compartment of the forearm on the interosseous membrane. It has numerous perforating branches, which pass directly through the interosseous membrane to supply deep muscles of the posterior compartment. The terminal end of the anterior interosseous artery passes posteriorly through an aperture in the interosseous membrane in distal regions of the forearm to join the posterior interosseous artery. The radial artery has muscular branches, which contribute to the supply of the extensor muscles on the radial side of the forearm. Deep veins of the posterior compartment generally accompany the arteries. They ultimately drain into brachial veins associated with the brachial artery in the cubital fossa.

1	Deep veins of the posterior compartment generally accompany the arteries. They ultimately drain into brachial veins associated with the brachial artery in the cubital fossa. The nerve of the posterior compartment of the forearm is the radial nerve (Fig. 7.92). Most of the muscles are innervated by the deep branch, which originates from the radial nerve in the lateral wall of the cubital fossa deep to the brachioradialis muscle and becomes the posterior interosseous nerve after emerging from between the superficial and deep layers of the supinator muscle in the posterior compartment of the forearm. In the lateral wall of the cubital fossa, and before dividing into superficial and deep branches, the radial nerve innervates the brachioradialis and extensor carpi radialis longus muscles.

1	In the lateral wall of the cubital fossa, and before dividing into superficial and deep branches, the radial nerve innervates the brachioradialis and extensor carpi radialis longus muscles. The deep branch innervates the extensor carpi radialis brevis, then passes between the two layers of the supinator muscle and follows the plane of separation between the two layers dorsally and laterally around the proximal shaft of the radius to the posterior aspect of the forearm. It supplies the supinator muscle and then emerges, as the posterior interosseous nerve, from the muscle to lie between the superficial and deep layers of muscles. The posterior interosseous nerve supplies the remaining muscles in the posterior compartment and terminates as articular branches, which pass deep to the extensor pollicis longus muscle to reach the wrist.

1	The hand (Fig. 7.93) is the region of the upper limb distal to the wrist joint. It is subdivided into three parts: the wrist (carpus), the metacarpus, and the digits (five fingers including the thumb). The five digits consist of the laterally positioned thumb and, medial to the thumb, the four fingers—the index, middle, ring, and little fingers. In the normal resting position, the fingers form a flexed arcade, with the little finger flexed most and the index finger flexed least. In the anatomical position, the fingers are extended. The hand has an anterior surface (palm) and a dorsal surface (dorsum of hand). Abduction and adduction of the fingers are defined with respect to the long axis of the middle finger (Fig. 7.93). In the anatomical position, the long axis of the thumb is rotated 90° to the rest of the digits so that the pad of the thumb points medially; consequently, movements of the thumb are defined at right angles to the movements of the other digits of the hand.

1	The hand is a mechanical and sensory tool. Many of the features of the upper limb are designed to facilitate positioning the hand in space. There are three groups of bones in the hand: The eight carpal bones are the bones of the wrist. The five metacarpals (I to V) are the bones of the metacarpus. The phalanges are the bones of the digits—the thumb has only two; the rest of the digits have three (Fig. 7.94). The carpal bones and metacarpals of the index, middle, ring, and little fingers (metacarpals II to V) tend to function as a unit and form much of the bony framework of the palm. The metacarpal of the thumb functions independently and has increased flexibility at the carpometacarpal joint to provide opposition of the thumb to the fingers. The small carpal bones of the wrist are arranged in two rows, a proximal and a distal row, each consisting of four bones (Fig. 7.94).

1	The small carpal bones of the wrist are arranged in two rows, a proximal and a distal row, each consisting of four bones (Fig. 7.94). From lateral to medial and when viewed from anteriorly, the proximal row of bones consists of: the boat-shaped scaphoid, the lunate, which has a crescent shape, the three-sided triquetrum bone, and the pea-shaped pisiform (Fig. 7.94). The pisiform is a sesamoid bone in the tendon of the flexor carpi ulnaris and articulates with the anterior surface of the triquetrum. The scaphoid has a prominent tubercle on its lateral palmar surface that is directed anteriorly. From lateral to medial and when viewed from anteriorly, the distal row of carpal bones consists of: the irregular four-sided trapezium bone, the four-sided trapezoid, the capitate, which has a head, and the hamate, which has a hook (Fig. 7.94). The trapezium articulates with the metacarpal bone of the thumb and has a distinct tubercle on its palmar surface that projects anteriorly.

1	The trapezium articulates with the metacarpal bone of the thumb and has a distinct tubercle on its palmar surface that projects anteriorly. The largest of the carpal bones, the capitate, articulates with the base of metacarpal III. The hamate, which is positioned just lateral and distal to the pisiform, has a prominent hook (hook of hamate) on its palmar surface that projects anteriorly. The carpal bones have numerous articular surfaces (Fig. 7.94). All of them articulate with each other, and the carpal bones in the distal row articulate with the metacarpals of the digits. With the exception of the metacarpal of the thumb, all movements of the metacarpal bones on the carpal bones are limited. The expansive proximal surfaces of the scaphoid and lunate articulate with the radius to form the wrist joint.

1	The expansive proximal surfaces of the scaphoid and lunate articulate with the radius to form the wrist joint. The carpal bones do not lie in a flat plane; rather, they form an arch, whose base is directed anteriorly (Fig. 7.94). The lateral side of this base is formed by the tubercles of the scaphoid and trapezium. The medial side is formed by the pisiform and the hook of the hamate. The flexor retinaculum attaches to, and spans the distance between, the medial and lateral sides of the base to form the anterior wall of the so-called carpal tunnel. The sides and roof of the carpal tunnel are formed by the arch of the carpal bones. Each of the five metacarpals is related to one digit: Metacarpal I is related to the thumb. Metacarpals II to V are related to the index, middle, ring, and little fingers, respectively (Fig. 7.94). Each metacarpal consists of a base, a shaft (body), and distally, a head.

1	Metacarpals II to V are related to the index, middle, ring, and little fingers, respectively (Fig. 7.94). Each metacarpal consists of a base, a shaft (body), and distally, a head. All of the bases of the metacarpals articulate with the carpal bones; in addition, the bases of the metacarpal bones of the fingers articulate with each other. All of the heads of the metacarpals articulate with the proximal phalanges of the digits. The heads form the knuckles on the dorsal surface of the hand when the fingers are flexed. The phalanges are the bones of the digits (Fig. 7.94): The thumb has two—a proximal and a distal phalanx. The rest of the digits have three—a proximal, a middle, and a distal phalanx. Each phalanx has a base, a shaft (body), and distally, a head. The base of each proximal phalanx articulates with the head of the related metacarpal bone.

1	Each phalanx has a base, a shaft (body), and distally, a head. The base of each proximal phalanx articulates with the head of the related metacarpal bone. The head of each distal phalanx is nonarticular and flattened into a crescent-shaped palmar tuberosity, which lies under the palmar pad at the end of the digit. The wrist joint is a synovial joint between the distal end of the radius and the articular disc overlying the distal end of the ulna, and the scaphoid, lunate, and triquetrum (Fig. 7.94). Together, the articular surfaces of the carpals form an oval shape with a convex contour, which articulates with the corresponding concave surface of the radius and articular disc. The wrist joint allows movement around two axes. The hand can be abducted, adducted, flexed, and extended at the wrist joint. Because the radial styloid process extends further distally than does the ulnar styloid process, the hand can be adducted to a greater degree than it can be abducted.

1	Because the radial styloid process extends further distally than does the ulnar styloid process, the hand can be adducted to a greater degree than it can be abducted. The capsule of the wrist joint is reinforced by palmar radiocarpal, palmar ulnocarpal, and dorsal radiocarpal ligaments. In addition, radial and ulnar collateral ligaments of the wrist joint span the distance between the styloid processes of the radius and ulna and the adjacent carpal bones. These ligaments reinforce the medial and lateral sides of the wrist joint and support them during flexion and extension. The synovial joints between the carpal bones share a common articular cavity. The joint capsule of the joints is reinforced by numerous ligaments. Although movement at the carpal joints (intercarpal joints) is limited, the joints do contribute to the positioning of the hand in abduction, adduction, flexion, and, particularly, extension.

1	Although movement at the carpal joints (intercarpal joints) is limited, the joints do contribute to the positioning of the hand in abduction, adduction, flexion, and, particularly, extension. There are five carpometacarpal joints between the metacarpals and the related distal row of carpal bones (Fig. 7.94). The saddle joint, between metacarpal I and the trapezium, imparts a wide range of mobility to the thumb that is not a feature of the rest of the digits. Movements at this carpometacarpal joint are flexion, extension, abduction, adduction, rotation, and circumduction. The carpometacarpal joints between metacarpals II to V and the carpal bones are much less mobile than the carpometacarpal joint of the thumb, allowing only limited gliding movements. Movement of the joints increases medially, so metacarpal V slides to the greatest degree. This can be best observed on the dorsal surface of the hand as it makes a fist.

1	The joints between the distal heads of the metacarpals and the proximal phalanges of the digits are condylar joints, which allow flexion, extension, abduction, adduction, circumduction, and limited rotation (Fig. 7.94). The capsule of each joint is reinforced by the palmar ligament and by medial and lateral collateral ligaments. The three deep transverse metacarpal ligaments (Fig. 7.95) are thick bands of connective tissue connecting the palmar ligaments of the metacarpophalangeal joints of the fingers to each other. They are important because, by linking the heads of the metacarpal bones together, they restrict the movement of these bones relative to each other. As a result, they help form a unified skeletal framework for the palm of the hand.

1	Significantly, a deep transverse metacarpal ligament does not occur between the palmar ligament of the metacarpophalangeal joint of the thumb and the palmar ligament of the index finger. The absence of this ligament, and the presence of a saddle joint between metacarpal I and the trapezium, are responsible for the increased mobility of the thumb relative to the rest of the digits of the hand. Interphalangeal joints of hand The interphalangeal joints of the hand are hinge joints that allow mainly flexion and extension. They are reinforced by medial and lateral collateral ligaments and palmar ligaments. Carpal tunnel and structures at the wrist The carpal tunnel is formed anteriorly at the wrist by a deep arch formed by the carpal bones and the flexor retinaculum (see Fig. 7.94). The base of the carpal arch is formed medially by the pisiform and the hook of the hamate and laterally by the tubercles of the scaphoid and trapezium.

1	The base of the carpal arch is formed medially by the pisiform and the hook of the hamate and laterally by the tubercles of the scaphoid and trapezium. The flexor retinaculum is a thick connective tissue ligament that bridges the space between the medial and lateral sides of the base of the arch and converts the carpal arch into the carpal tunnel. The four tendons of the flexor digitorum profundus, the four tendons of the flexor digitorum superficialis, and the tendon of the flexor pollicis longus pass through the carpal tunnel, as does the median nerve (Fig. 7.98). The flexor retinaculum holds the tendons to the bony plane at the wrist and prevents them from “bowing.”

1	The flexor retinaculum holds the tendons to the bony plane at the wrist and prevents them from “bowing.” Free movement of the tendons in the carpal tunnel is facilitated by synovial sheaths, which surround the tendons. All the tendons of the flexor digitorum profundus and flexor digitorum superficialis are surrounded by a single synovial sheath; a separate sheath surrounds the tendon of the flexor pollicis longus. The median nerve is anterior to the tendons in the carpal tunnel. The tendon of the flexor carpi radialis is surrounded by a synovial sheath and passes through a tubular compartment formed by the attachment of the lateral aspect of the flexor retinaculum to the margins of a groove on the medial side of the tubercle of the trapezium.

1	The ulnar artery, ulnar nerve, and tendon of the palmaris longus pass into the hand anterior to the flexor retinaculum and therefore do not pass through the carpal tunnel (Fig. 7.98). The tendon of the palmaris longus is not surrounded by a synovial sheath. The radial artery passes dorsally around the lateral side of the wrist and lies adjacent to the external surface of the scaphoid. The extensor tendons pass into the hand on the medial, lateral, and posterior surfaces of the wrist in six compartments defined by an extensor retinaculum and lined by synovial sheaths (Fig. 7.98): The tendons of the extensor digitorum and extensor indicis share a compartment and synovial sheath on the posterior surface of the wrist. The tendons of the extensor carpi ulnaris and extensor digiti minimi have separate compartments and sheaths on the medial side of the wrist.

1	The tendons of the extensor carpi ulnaris and extensor digiti minimi have separate compartments and sheaths on the medial side of the wrist. The tendons of the abductor pollicis longus and extensor pollicis brevis muscles, the extensor carpi radialis longus and extensor carpi radialis brevis muscles, and the extensor pollicis longus muscle pass through three compartments on the lateral surface of the wrist. The palmar aponeurosis is a triangular condensation of deep fascia that covers the palm and is anchored to the skin in distal regions (Fig. 7.99). The apex of the triangle is continuous with the palmaris longus tendon, when present; otherwise, it is anchored to the flexor retinaculum. From this point, fibers radiate to extensions at the bases of the digits that project into each of the index, middle, ring, and little fingers and, to a lesser extent, the thumb. Transverse fibers interconnect the more longitudinally arranged bundles that continue into the digits.

1	Transverse fibers interconnect the more longitudinally arranged bundles that continue into the digits. Vessels, nerves, and long flexor tendons lie deep to the palmar aponeurosis in the palm. The palmaris brevis, a small intrinsic muscle of the hand, is a quadrangular-shaped subcutaneous muscle that overlies the hypothenar muscles, ulnar artery, and superficial branch of the ulnar nerve at the medial side of the palm (Fig. 7.99). It originates from the palmar aponeurosis and flexor retinaculum and inserts into the dermis of the skin on the medial margin of the hand. The palmaris brevis deepens the cup of the palm by pulling on skin over the hypothenar eminence and forming a distinct ridge. This may improve grip. The palmaris brevis is innervated by the superficial branch of the ulnar nerve.

1	The palmaris brevis is innervated by the superficial branch of the ulnar nerve. The “anatomical snuffbox” is a term given to the triangular depression formed on the posterolateral side of the wrist and metacarpal I by the extensor tendons passing into the thumb (Fig. 7.100). Historically, ground tobacco (snuff) was placed in this depression before being inhaled into the nose. The base of the triangle is at the wrist and the apex is directed into the thumb. The impression is most apparent when the thumb is extended: The lateral border is formed by the tendons of the abductor pollicis longus and extensor pollicis brevis. The medial border is formed by the tendon of the extensor pollicis longus. The floor of the impression is formed by the scaphoid and trapezium, and the distal ends of the tendons of the extensor carpi radialis longus and extensor carpi radialis brevis.

1	The floor of the impression is formed by the scaphoid and trapezium, and the distal ends of the tendons of the extensor carpi radialis longus and extensor carpi radialis brevis. The radial artery passes obliquely through the anatomical snuffbox, deep to the extensor tendons of the thumb and lies adjacent to the scaphoid and trapezium. Terminal parts of the superficial branch of the radial nerve pass subcutaneously over the snuffbox as does the origin of the cephalic vein from the dorsal venous arch of the hand.

1	Terminal parts of the superficial branch of the radial nerve pass subcutaneously over the snuffbox as does the origin of the cephalic vein from the dorsal venous arch of the hand. After exiting the carpal tunnel, the tendons of the flexor digitorum superficialis and profundus muscles cross the palm and enter fibrous sheaths on the palmar aspect of the digits (Fig. 7.101). These fibrous sheaths: begin proximally, anterior to the metacarpophalangeal joints, and extend to the distal phalanges; are formed by fibrous arches and cruciate (cross-shaped) ligaments, which are attached posteriorly to the margins of the phalanges and to the palmar ligaments associated with the metacarpophalangeal and interphalangeal joints; and hold the tendons to the bony plane and prevent the tendons from bowing when the digits are flexed.

1	Within each tunnel, the tendons are surrounded by a synovial sheath. The synovial sheaths of the thumb and little finger are continuous with the sheaths associated with the tendons in the carpal tunnel (Fig. 7.101). The tendons of the extensor digitorum and extensor pollicis longus muscles pass onto the dorsal aspect of the digits and expand over the proximal phalanges to form complex “extensor hoods” or “dorsal digital expansions” (Fig. 7.103A). The tendons of the extensor digiti minimi, extensor indicis, and extensor pollicis brevis muscles join these hoods.

1	Each extensor hood is triangular, with: the apex attached to the distal phalanx, the central region attached to the middle phalanx (index, middle, ring, and little fingers) or proximal phalanx (thumb), and each corner of the base wrapped around the sides of the metacarpophalangeal joint—in the index, middle, ring, and little fingers, the corners of the hoods attach mainly to the deep transverse metacarpal ligaments; in the thumb, the hood is attached on each side to muscles. In addition to other attachments, many of the intrinsic muscles of the hand insert into the free margin of the hood on each side. By inserting into the extensor hood, these intrinsic muscles are responsible for complex delicate movements of the digits that could not be accomplished with the long flexor and extensor tendons alone.

1	In the index, middle, ring, and little fingers, the lumbrical, interossei, and abductor digiti minimi muscles attach to the extensor hoods. In the thumb, the adductor pollicis and abductor pollicis brevis muscles insert into and anchor the extensor hood. Because force from the small intrinsic muscles of the hand is applied to the extensor hood distal to the fulcrum of the metacarpophalangeal joints, the muscles flex these joints (Fig. 7.103B). Simultaneously, the force is transferred dorsally through the hood to extend the interphalangeal joints. This ability to flex the metacarpophalangeal joints, while at the same time extending the interphalangeal joints, is entirely due to the intrinsic muscles of the hand working through the extensor hoods. This type of precision movement is used in the upstroke when writing a t (Fig. 7.103C).

1	The intrinsic muscles of the hand are the palmaris brevis (described on p. 791; see Fig. 7.99), interossei, adductor pollicis, thenar, hypothenar, and lumbrical muscles (Figs. 7.104 to 7.108). Unlike the extrinsic muscles that originate in the forearm, insert in the hand, and function in forcefully gripping (“power grip”) with the hand, the intrinsic muscles occur entirely in the hand and mainly execute precision movements (“precision grip”) with the fingers and thumb. All of the intrinsic muscles of the hand are innervated by the deep branch of the ulnar nerve except for the three thenar and two lateral lumbrical muscles, which are innervated by the median nerve. The intrinsic muscles are predominantly innervated by spinal cord segment T1 with a contribution from C8.

1	The interossei are muscles between and attached to the metacarpals (Figs. 7.104 and 7.105). They insert into the proximal phalanx of each digit and into the extensor hood and are divided into two groups, the dorsal interossei and the palmar interossei. All of the interossei are innervated by the deep branch of the ulnar nerve. Collectively, the interossei abduct and adduct the digits and contribute to the complex flexion and extension movements generated by the extensor hoods. Dorsal interossei are the most dorsally situated of all of the intrinsic muscles and can be palpated through the skin on the dorsal aspect of the hand (Fig. 7.104). There are four bipennate dorsal interosseous muscles between, and attached to, the shafts of adjacent metacarpal bones (Fig. 7.104). Each muscle inserts both into the base of the proximal phalanx and into the extensor hood of its related digit. The tendons of the dorsal interossei pass dorsal to the deep transverse metacarpal ligaments:

1	The tendons of the dorsal interossei pass dorsal to the deep transverse metacarpal ligaments: The first dorsal interosseous muscle is the largest and inserts into the lateral side of the index finger. The second and third dorsal interossei insert into the lateral and medial sides, respectively, of the middle finger. The fourth dorsal interosseous muscle inserts into the medial side of the ring finger. In addition to generating flexion and extension movements of the fingers through their attachments to the extensor hoods, the dorsal interossei are the major abductors of the index, middle, and ring fingers, at the metacarpophalangeal joints (Table 7.15). The middle finger can abduct medially and laterally with respect to the long axis of the middle finger and consequently has a dorsal interosseous muscle on each side. The thumb and little finger have their own abductors in the thenar and hypothenar muscle groups, respectively, and therefore do not have dorsal interossei.

1	The radial artery passes between the two heads of the first dorsal interosseous muscle as it passes from the anatomical snuffbox on the posterolateral side of the wrist into the deep aspect of the palm. The three (or four) palmar interossei are anterior to the dorsal interossei, and are unipennate muscles originating from the metacarpals of the digits with which each is associated (Fig. 7.105). The first palmar interosseous muscle is rudimentary and often considered part of either the adductor pollicis or the flexor pollicis brevis. When present, it originates from the medial side of the palmar surface of metacarpal I and inserts into both the base of the proximal phalanx of the thumb and into the extensor hood. A sesamoid bone often occurs in the tendon attached to the base of the phalanx. The second palmar interosseous muscle originates from the medial surface of metacarpal II and inserts into the medial side of the extensor hood of the index finger.

1	The second palmar interosseous muscle originates from the medial surface of metacarpal II and inserts into the medial side of the extensor hood of the index finger. The third and fourth palmar interossei originate from the lateral surfaces of metacarpals IV and V and insert into the lateral sides of the respective extensor hoods. Like the tendons of the dorsal interossei, the tendons of the palmar interossei pass dorsal to the deep transverse metacarpal ligaments. The palmar interossei adduct the thumb, index, ring, and little fingers with respect to a long axis through the middle finger. The movements occur at the metacarpophalangeal joints. Because the muscles insert into the extensor hoods, they also produce complex flexion and extension movements of the digits (Table 7.15).

1	The adductor pollicis is a large triangular muscle anterior to the plane of the interossei that crosses the palm (Fig. 7.106). It originates as two heads: a transverse head from the anterior aspect of the shaft of metacarpal III, and an oblique head, from the capitate and adjacent bases of metacarpals II and III. The two heads converge laterally to form a tendon, which often contains a sesamoid bone, that inserts into both the medial side of the base of the proximal phalanx of the thumb and into the extensor hood. The radial artery passes anteriorly and medially between the two heads of the muscle to enter the deep plane of the palm and form the deep palmar arch. The adductor pollicis is a powerful adductor of the thumb and opposes the thumb to the rest of the digits in gripping (Table 7.15).

1	The adductor pollicis is a powerful adductor of the thumb and opposes the thumb to the rest of the digits in gripping (Table 7.15). The three thenar muscles (the opponens pollicis, flexor pollicis brevis, and abductor pollicis brevis muscles) are associated with opposition of the thumb to the fingers and with delicate movements of the thumb (Fig. 7.107) and are responsible for the prominent swelling (thenar eminence) on the lateral side of the palm at the base of the thumb. The thenar muscles are innervated by the recurrent branch of the median nerve. The opponens pollicis muscle is the largest of the thenar muscles and lies deep to the other two (Fig. 7.107). Originating from the tubercle of the trapezium and the adjacent flexor retinaculum, it inserts along the entire length of the lateral margin and adjacent lateral palmar surface of metacarpal I. The opponens pollicis rotates and flexes metacarpal

1	The opponens pollicis rotates and flexes metacarpal I on the trapezium, so bringing the pad of the thumb into a position facing the pads of the fingers (Table 7.15). The abductor pollicis brevis muscle overlies the opponens pollicis and is proximal to the flexor pollicis brevis muscle (Fig. 7.107). It originates from the tubercles of the scaphoid and trapezium and from the adjacent flexor retinaculum, and inserts into the lateral side of the base of the proximal phalanx of the thumb and into the extensor hood. The abductor pollicis brevis abducts the thumb, principally at the metacarpophalangeal joint. Its action is most apparent when the thumb is maximally abducted and the proximal phalanx is moved out of line with the long axis of the metacarpal bone (Table 7.15).

1	The flexor pollicis brevis muscle is distal to the abductor pollicis brevis (Fig. 7.107). It originates mainly from the tubercle of the trapezium and adjacent flexor retinaculum, but it may also have deeper attachments to other carpal bones and associated ligaments. It inserts into the lateral side of the base of the proximal phalanx of the thumb. The tendon often contains a sesamoid bone. The flexor pollicis brevis flexes the metacarpophalangeal joint of the thumb (Table 7.15). The hypothenar muscles (the opponens digiti minimi, abductor digiti minimi, and flexor digiti minimi brevis) contribute to the swelling (hypothenar eminence) on the medial side of the palm at the base of the little finger (Fig. 7.107). The hypothenar muscles are similar to the thenar muscles in name and in organization. Unlike the thenar muscles, the hypothenar muscles are innervated by the deep branch of the ulnar nerve and not by the recurrent branch of the median nerve.

1	Unlike the thenar muscles, the hypothenar muscles are innervated by the deep branch of the ulnar nerve and not by the recurrent branch of the median nerve. The opponens digiti minimi muscle lies deep to the other two hypothenar muscles (Fig. 7.107). It originates from the hook of the hamate and from the adjacent flexor retinaculum and it inserts into the medial margin and palmar surface of metacarpal V. Its base is penetrated by the deep branches of the ulnar nerve and ulnar artery. The opponens digiti minimi rotates metacarpal V toward the palm; however, because of the simple shape of the carpometacarpal joint and the presence of a deep transverse metacarpal ligament, which attaches the head of metacarpal V to that of the ring finger, the movement is much less dramatic than that of the thumb (Table 7.15).

1	The abductor digiti minimi muscle overlies the opponens digiti minimi (Fig. 7.107). It originates from the pisiform bone, the pisohamate ligament, and the tendon of the flexor carpi ulnaris, and inserts into the medial side of the base of the proximal phalanx of the little finger and into the extensor hood. The abductor digiti minimi is the principal abductor of the little finger (Table 7.15). The flexor digiti minimi brevis muscle is lateral to the abductor digiti minimi (Fig. 7.107). It originates from the hook of the hamate bone and the adjacent flexor retinaculum and inserts with the abductor digiti minimi muscle into the medial side of the base of the proximal phalanx of the little finger. The flexor digiti minimi brevis flexes the metacarpophalangeal joint. There are four lumbrical (worm-like) muscles, each of which is associated with one of the fingers. The muscles originate from the tendons of the flexor digitorum profundus in the palm:

1	There are four lumbrical (worm-like) muscles, each of which is associated with one of the fingers. The muscles originate from the tendons of the flexor digitorum profundus in the palm: The medial two lumbricals are bipennate and originate from the flexor digitorum profundus tendons associated with the middle and ring fingers and the ring and little fingers, respectively. The lateral two lumbricals are unipennate muscles, originating from the flexor digitorum profundus tendons associated with the index and middle fingers, respectively. The lumbricals pass dorsally around the lateral side of each finger, and insert into the extensor hood (Fig. 7.108). The tendons of the muscles are anterior to the deep transverse metacarpal ligaments. The lumbricals are unique because they link flexor tendons with extensor tendons. Through their insertion into the extensor hoods, they participate in flexing the metacarpophalangeal joints and extending the interphalangeal joints.

1	The medial two lumbricals are innervated by the deep branch of the ulnar nerve; the lateral two lumbricals are innervated by digital branches of the median nerve (Table 7.15). The blood supply to the hand is by the radial and ulnar arteries, which form two interconnected vascular arches (superficial and deep) in the palm (Fig. 7.109). Vessels to the digits, muscles, and joints originate from the two arches and the parent arteries: The radial artery contributes substantially to the supply of the thumb and the lateral side of the index finger. The remaining digits and the medial side of the index finger are supplied mainly by the ulnar artery.

1	The remaining digits and the medial side of the index finger are supplied mainly by the ulnar artery. The ulnar artery and ulnar nerve enter the hand on the medial side of the wrist (Fig. 7.110). The vessel lies between the palmaris brevis and the flexor retinaculum and is lateral to the ulnar nerve and the pisiform bone. Distally, the ulnar artery is medial to the hook of the hamate bone and then swings laterally across the palm, forming the superficial palmar arch, which is superficial to the long flexor tendons of the digits and just deep to the palmar aponeurosis. On the lateral side of the palm, the arch communicates with a palmar branch of the radial artery.

1	One branch of the ulnar artery in the hand is the deep palmar branch (Figs. 7.109 and 7.110), which arises from the medial aspect of the ulnar artery, just distal to the pisiform, and penetrates the origin of the hypothenar muscles. It curves medially around the hook of the hamate to access the deep plane of the palm and to anastomose with the deep palmar arch derived from the radial artery. Branches from the superficial palmar arch include: a palmar digital artery to the medial side of the little finger, and three large, common palmar digital arteries, which ultimately provide the principal blood supply to the lateral side of the little finger, both sides of the ring and middle fingers, and the medial side of the index finger (Fig. 7.110); they are joined by palmar metacarpal arteries from the deep palmar arch before bifurcating into the proper palmar digital arteries, which enter the fingers.

1	The radial artery curves around the lateral side of the wrist and passes over the floor of the anatomical snuffbox and into the deep plane of the palm by penetrating anteriorly through the back of the hand (Figs. 7.109 and 7.111). It passes between the two heads of the first dorsal interosseous muscle and then between the two heads of the adductor pollicis to access the deep plane of the palm and form the deep palmar arch. The deep palmar arch passes medially through the palm between the metacarpal bones and the long flexor tendons of the digits. On the medial side of the palm, it communicates with the deep palmar branch of the ulnar artery (Figs. 7.109 and 7.111).

1	Before penetrating the back of the hand, the radial artery gives rise to two vessels: a dorsal carpal branch, which passes medially as the dorsal carpal arch, across the wrist and gives rise to three dorsal metacarpal arteries, which subsequently divide to become small dorsal digital arteries, which enter the fingers; and the first dorsal metacarpal artery, which supplies adjacent sides of the index finger and thumb. Two vessels, the princeps pollicis artery and the radialis indicis artery, arise from the radial artery in the plane between the first dorsal interosseous and adductor pollicis. The princeps pollicis artery is the major blood supply to the thumb, and the radialis indicis artery supplies the lateral side of the index finger.

1	The deep palmar arch gives rise to: three palmar metacarpal arteries, which join the common palmar digital arteries from the superficial palmar arch; and three perforating branches, which pass posteriorly between the heads of origin of the dorsal interossei to anastomose with the dorsal metacarpal arteries from the dorsal carpal arch. As generally found in the upper limb, the hand contains interconnected networks of deep and superficial veins. The deep veins follow the arteries; the superficial veins drain into a dorsal venous network on the back of the hand over the metacarpal bones (Fig. 7.112). The cephalic vein originates from the lateral side of the dorsal venous network and passes over the anatomical snuffbox into the forearm. The basilic vein originates from the medial side of the dorsal venous network and passes into the dorsomedial aspect of the forearm.

1	The basilic vein originates from the medial side of the dorsal venous network and passes into the dorsomedial aspect of the forearm. The hand is supplied by the ulnar, median, and radial nerves (Figs. 7.113 to 7.115). All three nerves contribute to cutaneous or general sensory innervation. The ulnar nerve innervates all intrinsic muscles of the hand except for the three thenar muscles and the two lateral lumbricals, which are innervated by the median nerve. The radial nerve only innervates skin on the dorsolateral side of the hand. The ulnar nerve enters the hand lateral to the pisiform and posteromedially to the ulnar artery (Fig. 7.113). Immediately distal to the pisiform, it divides into a deep branch, which is mainly motor, and a superficial branch, which is mainly sensory.

1	The deep branch of the ulnar nerve passes with the deep branch of the ulnar artery (Fig. 7.113). It penetrates and supplies the hypothenar muscles to reach the deep aspect of the palm, arches laterally across the palm, deep to the long flexors of the digits, and supplies the interossei, the adductor pollicis, and the two medial lumbricals. In addition, the deep branch of the ulnar nerve contributes small articular branches to the wrist joint. As the deep branch of the ulnar nerve passes across the palm, it lies in a fibro-osseous tunnel (Guyon’s canal) between the hook of the hamate and the flexor tendons. Occasionally, small outpouchings of synovial membrane (ganglia) from the joints of the carpus compress the nerve within this canal, producing sensory and motor symptoms.

1	The superficial branch of the ulnar nerve innervates the palmaris brevis muscle and continues across the palm to supply skin on the palmar surface of the little finger and the medial half of the ring finger (Fig. 7.113). The median nerve is the most important sensory nerve in the hand because it innervates skin on the thumb, index and middle fingers, and lateral side of the ring finger (Fig. 7.115). The nervous system, using touch, gathers information about the environment from this area, particularly from the skin on the thumb and index finger. In addition, sensory information from the lateral three and one-half digits enables the fingers to be positioned with the appropriate amount of force when using precision grip. The median nerve also innervates the thenar muscles that are responsible for opposition of the thumb to the other digits.

1	The median nerve also innervates the thenar muscles that are responsible for opposition of the thumb to the other digits. The median nerve enters the hand by passing through the carpal tunnel and divides into a recurrent branch and palmar digital branches (Fig. 7.115). The recurrent branch of the median nerve innervates the three thenar muscles. Originating from the lateral side of the median nerve near the distal margin of the flexor retinaculum, it curves around the margin of the retinaculum and passes proximally over the flexor pollicis brevis muscle. The recurrent branch then passes between the flexor pollicis brevis and abductor pollicis brevis to end in the opponens pollicis.

1	The palmar digital nerves cross the palm deep to the palmar aponeurosis and the superficial palmar arch and enter the digits. They innervate skin on the palmar surfaces of the lateral three and one-half digits and cutaneous regions over the dorsal aspects of the distal phalanges (nail beds) of the same digits. In addition to skin, the digital nerves supply the lateral two lumbrical muscles. Superficial branch of the radial nerve The only part of the radial nerve that enters the hand is the superficial branch (Fig. 7.116). It enters the hand by passing over the anatomical snuffbox on the dorsolateral side of the wrist. Terminal branches of the nerve can be palpated or “rolled” against the tendon of the extensor pollicis longus as they cross the anatomical snuffbox.

1	The superficial branch of the radial nerve innervates skin over the dorsolateral aspect of the palm and the dorsal aspects of the lateral three and one-half digits distally to approximately the terminal interphalangeal joints. Tendons, muscles, and bony landmarks in the upper limb are used to locate major arteries, veins, and nerves. Asking patients to maneuver their upper limbs in specific ways is essential for performing neurological examinations. Tendons are used to test reflexes associated with specific spinal cord segments. Vessels are used clinically as points of entry into the vascular system (for collecting blood and administering pharmaceuticals and nutrients), and for taking blood pressure and pulses. Nerves can become entrapped or be damaged in regions where they are related to bone or pass through confined spaces. Bony landmarks and muscles of the posterior scapular region

1	Nerves can become entrapped or be damaged in regions where they are related to bone or pass through confined spaces. Bony landmarks and muscles of the posterior scapular region The medial border, inferior angle, and part of the lateral border of the scapula can be palpated on a patient, as can the spine and acromion. The superior border and angle of the scapula are deep to soft tissue and are not readily palpable. The supraspinatus and infraspinatus muscles can be palpated above and below the spine, respectively (Fig. 7.117). The trapezius muscle is responsible for the smooth contour of the lateral side of the neck and over the superior aspect of the shoulder. The deltoid muscles form the muscular eminence inferior to the acromion and around the glenohumeral joint. The axillary nerve passes posteriorly around the surgical neck of the humerus deep to the deltoid muscle.

1	The latissimus dorsi muscle forms much of the muscle mass underlying the posterior axillary skin fold extending obliquely upward from the trunk to the arm. The teres major muscle passes from the inferior angle of the scapula to the upper humerus and contributes to this posterior axillary skin fold laterally. Visualizing the axilla and locating contents and related structures The axillary inlet and outlet and walls of the axilla can be established using skin folds and palpable bony landmarks (Fig. 7.118). The anterior margin of the axillary inlet is the clavicle, which can be palpated along its entire length. The lateral limit of the axillary inlet is approximated by the tip of the coracoid process, which is palpable immediately below the lateral third of the clavicle and deep to the medial margin of the deltoid muscle. The inferior margin of the anterior axillary wall is the anterior axillary skin fold, which overlies the lower margin of the pectoralis major muscle.

1	The inferior margin of the anterior axillary wall is the anterior axillary skin fold, which overlies the lower margin of the pectoralis major muscle. The inferior margin of the posterior axillary wall is the posterior axillary skin fold, which overlies the margins of the teres major muscle laterally and latissimus dorsi muscle medially. The medial wall of the axilla is the upper part of the serratus anterior muscle overlying the thoracic wall. The long thoracic nerve passes vertically out of the axilla and down the lateral surface of the serratus anterior muscle in a position just anterior to the posterior axillary skin fold. The lateral boundary of the axilla is the humerus. The floor of the axilla is the dome of skin between the posterior and anterior axillary skin folds. Major vessels, nerves, and lymphatics travel between the upper limb and the trunk by passing through the axilla.

1	Major vessels, nerves, and lymphatics travel between the upper limb and the trunk by passing through the axilla. The axillary artery, axillary vein, and components of the brachial plexus pass through the axilla and into the arm by traveling lateral to the dome of skin that forms the floor. This neurovascular bundle can be palpated by placing a hand into this dome of skin and pressing laterally against the humerus. The cephalic vein travels in superficial fascia in the cleft between the deltoid muscle and the pectoralis major muscle and penetrates deep fascia in the clavipectoral triangle to join with the axillary vein. Locating the brachial artery in the arm The brachial artery is on the medial side of the arm in the cleft between the biceps brachii and triceps brachii muscles (Fig. 7.119). The median nerve courses with the brachial artery, whereas the ulnar nerve deviates posteriorly from the vessel in distal regions. The triceps brachii tendon and position of the radial nerve

1	The triceps brachii tendon and position of the radial nerve The triceps brachii muscle forms the soft tissue mass posterior to the humerus, and the tendon inserts onto the olecranon of the ulna, which is readily palpable and forms the bony protuberance at the “tip” of the elbow (Fig. 7.120). The brachioradialis muscle is also visible as a muscular bulge on the lateral aspect of the arm. It is particularly prominent when the forearm is half pronated, flexed at the elbow against resistance, and viewed anteriorly. The radial nerve in the distal arm emerges from behind the humerus to lie deep to the brachioradialis muscle. The cubital fossa lies anterior to the elbow joint and contains the biceps brachii tendon, the brachial artery, and the median nerve (Fig. 7.121).

1	The cubital fossa lies anterior to the elbow joint and contains the biceps brachii tendon, the brachial artery, and the median nerve (Fig. 7.121). The base of the cubital fossa is an imaginary line between the readily palpable medial and lateral epicondyles of the humerus. The lateral and medial borders are formed by the brachioradialis and pronator teres muscles, respectively. The margin of the brachioradialis can be found by asking a subject to flex the semipronated forearm against resistance. The margin of the pronator teres can be estimated by an oblique line extending between the medial epicondyle and the midpoint along the length of the lateral surface of the forearm. The approximate apex of the cubital fossa is where this line meets the margin of the brachioradialis muscle.

1	Contents of the cubital fossa, from lateral to medial, are the tendon of the biceps brachii, the brachial artery, and the median nerve. The tendon of the biceps brachii is easily palpable. Often the cephalic, basilic, and median cubital veins are visible in the subcutaneous fascia overlying the cubital fossa. The ulnar nerve passes behind the medial epicondyle of the humerus and can be “rolled” here against the bone. The radial nerve travels into the forearm deep to the margin of the brachioradialis muscle anterior to the elbow joint. Identifying tendons and locating major vessels and nerves in the distal forearm Tendons that pass from the forearm into the hand are readily visible in the distal forearm and can be used as landmarks to locate major vessels and nerves.

1	Tendons that pass from the forearm into the hand are readily visible in the distal forearm and can be used as landmarks to locate major vessels and nerves. In the anterior aspect of the distal forearm, the tendons of the flexor carpi radialis, flexor carpi ulnaris, and palmaris longus muscles can be easily located either by palpating or by asking a patient to flex the wrist against resistance. The tendon of flexor carpi radialis is located approximately at the junction between the lateral and middle thirds of an imaginary line drawn transversely across the distal forearm. The radial artery is immediately lateral to this tendon and this site is used for taking a radial pulse (Fig. 7.122A).

1	The tendon of the flexor carpi ulnaris is easily palpated along the medial margin of the forearm and inserts on the pisiform, which can also be palpated by following the tendon to the base of the hypothenar eminence of the hand. The ulnar artery and ulnar nerve travel through the distal forearm and into the hand under the lateral lip of the flexor carpi ulnaris tendon and lateral to the pisiform. The palmaris longus tendon may be absent, but when present, lies medial to the flexor carpi radialis tendon and is particularly prominent when the wrist is flexed against resistance. The median nerve is also medial to the flexor carpi radialis tendon and lies under the palmaris longus tendon. The long tendons of the digits of the hand are deep to the median nerve and between the long flexors of the wrist. Their position can be visualized by rapidly and repeatedly flexing and extending the fingers from medial to lateral.

1	In the posterior distal forearm and wrist, the tendons of the extensor digitorum (Fig. 7.122B) are in the midline and radiate into the index, middle, ring, and little fingers from the wrist. The distal ends of the tendons of the extensor carpi radialis longus and brevis muscles are on the lateral side of the wrist (Fig. 7.122C) and can be accentuated by making a tight fist and extending the wrist against resistance. The tendon of the extensor carpi ulnaris can be felt on the far medial side of the wrist between the distal end of the ulna and the wrist.

1	Hyperextension and abduction of the thumb reveals the anatomical snuffbox (Fig. 7.122D). The medial margin of this triangular area is the tendon of the extensor pollicis longus, which swings around the dorsal tubercle of the radius and then travels into the thumb. The lateral margin is formed by the tendons of the extensor pollicis brevis and abductor pollicis longus. The radial artery passes through the anatomical snuffbox when traveling laterally around the wrist to reach the back of the hand and penetrate the base of the first dorsal interosseous muscle to access the deep aspect of the palm of the hand. The pulse of the radial artery can be felt in the floor of the anatomical snuffbox in the relaxed wrist. The cephalic vein crosses the roof of the anatomical snuffbox, and cutaneous branches of the radial nerve can be felt by moving a finger back and forth along the tendon of the extensor pollicis longus muscle. Normal appearance of the hand

1	Normal appearance of the hand In the resting position, the palm and digits of the hand have a characteristic appearance. The fingers form a flexed arcade, with the little finger flexed the most and the index finger flexed the least (Fig. 7.123A). The pad of the thumb is positioned at a 90° angle to the pads of the fingers. A thenar eminence occurs at the base of the thumb and is formed by the underlying thenar muscles. A similar hypothenar eminence occurs along the medial margin of the palm at the base of the little finger. The appearance of the thenar and hypothenar eminences, and the positions of the fingers change when the ulnar and median nerves are compromised. Major superficial veins of the upper limb begin in the hand from a dorsal venous network (Fig. 7.123B), which overlies the metacarpals. The basilic vein originates from the medial side of the network and the cephalic vein originates from the lateral side.

1	Position of the flexor retinaculum and the recurrent branch of the median nerve The proximal margin of the flexor retinaculum can be determined using two bony landmarks. The pisiform bone is readily palpable at the distal end of the flexor carpi ulnaris tendon. The tubercle of the scaphoid can be palpated at the distal end of the flexor carpi radialis tendon as it enters the wrist (Fig. 7.124). An imaginary line between these two points marks the proximal margin of the flexor retinaculum. The distal margin of the flexor retinaculum is approximately deep to the point where the anterior margin of the thenar eminence meets the hypothenar eminence near the base of the palm. The recurrent branch of the median nerve lies deep to the skin and deep fascia overlying the anterior margin of the thenar eminence near the midline of the palm. Motor function of the median and ulnar nerves in the hand

1	Motor function of the median and ulnar nerves in the hand The ability to flex the metacarpophalangeal joints while at the same time extending the interphalangeal joints of the fingers is entirely dependent on the intrinsic muscles of the hand (Fig. 7.125A). These muscles are mainly innervated by the deep branch of the ulnar nerve, which carries fibers from spinal cord level (C8)T1. Adducting the fingers to grasp an object placed between them is caused by the palmar interossei muscles, which are innervated by the deep branch of the ulnar nerve carrying fibers from spinal cord level (C8)T1. The ability to grasp an object between the pad of the thumb and the pad of one of the fingers depends on normal functioning of the thenar muscles, which are innervated by the recurrent branch of the median nerve carrying fibers from spinal cord level C8(T1). Visualizing the positions of the superficial and deep palmar arches

1	Visualizing the positions of the superficial and deep palmar arches The positions of the superficial and deep palmar arches in the hand can be visualized using bony landmarks, muscle eminences, and skin creases (Fig. 7.126). The superficial palmar arch begins as a continuation of the ulnar artery, which lies lateral to the pisiform bone at the wrist. The arch curves laterally across the palm anterior to the long flexor tendons in the hand. The arch reaches as high as the proximal transverse skin crease of the palm and terminates laterally by joining a vessel of variable size, which crosses the thenar eminence from the radial artery in the distal forearm.

1	The deep palmar arch originates on the lateral side of the palm deep to the long flexor tendons and between the proximal ends of metacarpals I and II. It arches medially across the palm and terminates by joining the deep branch of the ulnar artery, which passes through the base of the hypothenar muscles and between the pisiform and hook of the hamate. The deep palmar arch is more proximal in the hand than the superficial palmar arch and lies approximately one-half of the distance between the distal wrist crease and the proximal transverse skin crease of the palm. Peripheral pulses can be felt at six locations in the upper limb (Fig. 7.127). Axillary pulse: axillary artery in the axilla lateral to the apex of the dome of skin covering the floor of the axilla. Brachial pulse in midarm: brachial artery on the medial side of the arm in the cleft between the biceps brachii and triceps brachii muscles. This is the position where a blood pressure cuff is placed.

1	Brachial pulse in the cubital fossa: brachial artery medial to the tendon of the biceps brachii muscle. This is the position where a stethoscope is placed to hear the pulse of the vessel when taking a blood pressure reading. Radial pulse in the distal forearm: radial artery immediately lateral to the tendon of the flexor carpi radialis muscle. This is the most common site for “taking a pulse.” Ulnar pulse in the distal forearm: ulnar artery immediately under the lateral margin of the flexor carpi ulnaris tendon and proximal to the pisiform. Radial pulse in the anatomical snuffbox: radial artery as it crosses the lateral side of the wrist between the tendon of the extensor pollicis longus muscle and the tendons of the extensor pollicis brevis and abductor pollicis longus muscles. Fig. 7.1 Upper limb. A. Anterior view of the upper limb. B. Superior view of the shoulder.

1	Fig. 7.1 Upper limb. A. Anterior view of the upper limb. B. Superior view of the shoulder. Spinous process of vertebra TINeckShoulderGlenohumeral jointArmElbow jointForearmWrist jointHandBAThoracic wallRib IRib IAxillaScapulaManubriumof sternumClavicle Fig. 7.2 Areas of transition in the upper limb. Fig. 7.3 Movements of the scapula. A. Rotation. B. Protraction and retraction. Fig. 7.4 Movements of the arm at the glenohumeral joint. Fig. 7.5 Movements of the forearm. A. Flexion and extension at the elbow joint. B. Pronation and supination. Fig. 7.6 Movements of the hand at the wrist joint. Fig. 7.7 Bones of the upper limb. Fig. 7.8 Movements of the metacarpophalangeal (A) and interphalangeal (B) joints. Fig. 7.9 Muscles of the shoulder. A. Posterior shoulder. B. Anterior shoulder. C. Rotator cuff muscles.

1	Fig. 7.8 Movements of the metacarpophalangeal (A) and interphalangeal (B) joints. Fig. 7.9 Muscles of the shoulder. A. Posterior shoulder. B. Anterior shoulder. C. Rotator cuff muscles. SupraspinatusSubscapularisHumerusAcromionSpine of scapulaInfraspinatusTeres minorCoracoid processTrapeziusDeltoidPectoralis majorTeres majorLatissimus dorsiABCTrapeziusLatissimus dorsiLevator scapulaeRhomboid minorRhomboid major Fig. 7.10 Muscle components in the arm and forearm. Fig. 7.11 Relationship of the upper limb to the neck. Axillary arteryAxillary veinAxillaMedial margin ofcoracoid processHumerusAxillary inletSuperior margin of scapulaNerves to upper limbLateral margin of rib I Fig. 7.12 Muscles of the back and thoracic wall. Fig. 7.13 Breast. Fig. 7.14 Innervation of the upper limb. Anterior ramiNervesMusculocutaneous nerve(C5 to C7)Radial nerve(C5 to C8,T1)Median nerve(C6 to C8,T1)Ulnar nerve(C[7], C8, T1)C5C6C7C8T1Brachial plexus

1	Fig. 7.14 Innervation of the upper limb. Anterior ramiNervesMusculocutaneous nerve(C5 to C7)Radial nerve(C5 to C8,T1)Median nerve(C6 to C8,T1)Ulnar nerve(C[7], C8, T1)C5C6C7C8T1Brachial plexus Fig. 7.15 Dermatomes and myotomes in the upper limb. A. Dermatomes. B. Movements produced by myotomes. T1T1T1T2T2C6C6C6C5C5C5C4C4C3C3C8C8C8C7C7C7AAbduction of armC5Flexion of elbowC(5)6Adduction and abduction of digitsT1Flexion of digitsC8BC(6)7(8) Fig. 7.16 Nerves of upper limb. A. Major nerves in the arm and forearm. B. Anterior and posterior areas of skin innervated by major peripheral nerves in the arm and forearm.

1	Radial nerve • All muscles in posterior compartment of arm and forearmUlnar nerve• Most intrinsic muscles in hand• Flexor carpi ulnaris and medial half of flexor digitorum profundus in forearmMusculocutaneous nerve• All muscles in anterior compartment of armMedian nerve• Most flexors in forearm• Thenar muscles in handABUlnar nerveMedian nerveRadial nerve• Inferior lateral cutaneous nerve of arm• Posterior cutaneous nerve of arm• Posterior cutaneous nerve of forearmMusculocutaneous nerve• Lateral cutaneous nerve of forearmRadial nerve• Superficial branchPosteriorT1T2Ulnar nerveMedian nerveRadial nerve• Inferior lateral cutaneous nerve of armAxillary nerve• Superior lateral cutaneous nerve of armAxillary nerve• Superior lateral cutaneous nerve of armMusculocutaneous nerve• Lateral cutaneous nerve of forearmRadial nerve• Superficial branchAnteriorT1T2 Fig. 7.17 Nerves related to the humerus.

1	Fig. 7.17 Nerves related to the humerus. Radial nerveUlnar nerveAxillary nerveRadial groove of humerusSurgical neck of humerusMedial epicondyle Fig. 7.18 Veins in the superficial fascia of upper limb. The area of the cubital fossa is shown in yellow. Clavipectoral triangleClavicleCephalic veinBiceps brachiiAxillary veinCephalic veinPectoralis majorBasilic veinMedian cubital veinDorsal venous network of handBasilic veinCubital fossaDeltoid Fig. 7.19 A to C. Movements of the thumb. Fig. 7.20 Right clavicle. LateralMedialSuperior viewSurface for articulationwith acromionAnterior viewSurface for articulation with manubrium of sternum andfirst costal cartilageTrapezoid lineInferior viewConoid tubercleConoid tubercle Fig. 7.21 Scapula. A. Posterior view of right scapula. B. Anterior view of costal surface. C. Lateral view.

1	Fig. 7.21 Scapula. A. Posterior view of right scapula. B. Anterior view of costal surface. C. Lateral view. Articular surface for clavicleCoracoidprocessSuperior borderAnterior view of scapulaSuperior angleMedial borderSubscapular fossaAcromionGlenoid cavityLateral borderInfraglenoid tubercleInferior angleAAcromionSupraglenoid tubercleSuperior angleCoracoid processGlenoid cavityInfraglenoid tubercleInferior angleLateral borderSpinous processLateral viewCMedial borderLateral borderSuperior borderSuperior angleInferior angleArticular surface for clavicleCoracoid processPosterior viewAcromionGlenoid cavityInfraglenoid tubercleSupraspinousfossaSuprascapular notchGreater scapular notch (or spinoglenoid notch)InfraspinousfossaSpine of scapulaB Fig. 7.22 Proximal end of right humerus.

1	Fig. 7.22 Proximal end of right humerus. HeadSuperior facet on greater tubercle(supraspinatus)GreatertubercleIntertubercularsulcusIntertubercularsulcusLesser tubercle(subscapularis)Lateral lip, floor,and medial lip ofintertubercularsulcus (pectoralismajor, latissimusdorsi, and teresmajor, respectively)Deltoid tuberosity(deltoid)Deltoid tuberosity(deltoid)Anatomical neckSurgical neckLateral viewAnterior viewAttachmentfor pectoralis majorAttachment forcoracobrachialisGreatertubercleSuperior facet(supraspinatus)Middle facet(infraspinatus)Inferior facet(teres minor)Surgical neckAnatomicalneckPosterior view Fig. 7.23 Sternoclavicular joint. A. Bones and ligaments. B. Volume-rendered reconstruction using multidetector computed tomography.

1	Fig. 7.23 Sternoclavicular joint. A. Bones and ligaments. B. Volume-rendered reconstruction using multidetector computed tomography. CostoclavicularligamentInterclavicularligamentClavicular notchAnteriorsternoclavicularligamentManubrium ofsternumSternal angleArticular disc(capsule and ligamentsremoved anteriorlyto expose joint)Attachment sitefor rib IIFirst costalcartilageRib IVertebral body of TIILeft clavicleSternalangleRib IRib IIManubriumof sternumAB Fig. 7.24 Right acromioclavicular joint. Fig. 7.25 Glenohumeral joint. A. Articular surfaces of right glenohumeral joint. B. Radiograph of a normal glenohumeral joint. Tendon of longhead of bicepsbrachii muscleGlenoid cavityABGlenoid labrumTransverse humeral ligamentClavicleAcromionGlenoid cavityHead of humerusHead ofhumerus Fig. 7.26 Synovial membrane and joint capsule of right glenohumeral joint.

1	Fig. 7.26 Synovial membrane and joint capsule of right glenohumeral joint. Synovial sheathSynovial membraneRedundant synovial membrane in adductionSubtendinous bursa of subscapularisLong head of biceps brachii tendonCoracohumeralligamentLong head of biceps brachiitendonFibrous membrane of joint capsule Fig. 7.27 Capsule of right glenohumeral joint. Superior glenohumeral ligamentMiddle glenohumeral ligamentInferiorglenohumeralligamentCoracohumeral ligamentTransversehumeral ligamentSynovialsheathTendon oflong headof bicepsbrachiiRedundant capsuleAperture for subtendinousbursa of subscapularis Fig. 7.28 Lateral view of right glenohumeral joint and surrounding muscles with proximal end of humerus removed.

1	Fig. 7.28 Lateral view of right glenohumeral joint and surrounding muscles with proximal end of humerus removed. SupraspinatusInfraspinatusTeres minorTeres majorSynovial membraneSubtendinous bursaof subscapularisLong head of biceps brachii tendonLong head of tricepsbrachiiShort head of biceps brachiiand coracobrachialisLatissimus dorsiPectoralis majorGlenoid labrum Fibrous membraneSubscapularisDeltoidAcromionCoracoid processCoraco-acromial ligamentGlenoid cavitySubacromial bursa (subdeltoid) Fig. 7.29 Magnetic resonance image (T1-weighted) of a normal glenohumeral joint in the sagittal plane. SupraspinatusInfraspinatusPosteriorAnteriorClavicleAcromionTeres minorHead of humerusCoracoid processSubscapularis Fig. 7.30 There is an oblique fracture of the middle third of the right clavicle. Fracture of clavicleAcromioclavicular joint

1	Fig. 7.30 There is an oblique fracture of the middle third of the right clavicle. Fracture of clavicleAcromioclavicular joint Fig. 7.31 Radiographs of acromioclavicular joints. A. Normal right acromioclavicular joint. B. Dislocated right acromioclavicular joint (shoulder separation). Acromioclavicular jointAcromionClavicle Head of humerusClavicleAcromionHumerusAB Fig. 7.32 Radiograph showing an anteroinferior dislocation of the shoulder joint. AcromionGlenoid cavityClavicleHead of humerus Fig. 7.33 Magnetic resonance image of a full-thickness tear of the supraspinatus tendon as it inserts onto the greater tubercle of the humerus. Fig. 7.34 Ultrasound of shoulder showing needle placement into the subdeltoid/subacromial bursa. DeltoidHead of humerusNeedleSubacromial-subdeltoid bursa Fig. 7.35 Lateral view of trapezius and deltoid muscles. AcromionSpine of thescapulaTrapeziusDeltoidDeltoid tuberosity of humerusClavicle

1	DeltoidHead of humerusNeedleSubacromial-subdeltoid bursa Fig. 7.35 Lateral view of trapezius and deltoid muscles. AcromionSpine of thescapulaTrapeziusDeltoidDeltoid tuberosity of humerusClavicle Fig. 7.36 Attachments and neurovascular supply of the trapezius and deltoid muscles. AcromionSpine of scapulaTrapeziusDeltoidDeltoid tuberosity of humerusRhomboid minorRhomboid majorClavicleSuperior nuchal lineExternal occipitalprotuberanceMastoid processLigamentum nuchaeSpinous processes and interspinous ligaments to TXIIAccessory nerve (XI)Levator scapulaeAxillary nervePosterior circumflex humeral arteryLine of attachment of trapeziusLine of attachment of deltoid Fig. 7.37 Right posterior scapular region.

1	Fig. 7.37 Right posterior scapular region. Suprascapular notch (foramen) SupraspinatusInfraspinatusCut edge of trapeziusCut edge of deltoidSurgical neck of humerusMedial lip of intertubercular sulcusTriangular intervalTriangular spaceOlecranonLong head of triceps brachiiTeres majorTeres minorQuadrangular spaceCut edge of lateral head of triceps brachii Fig. 7.38 Arteries and nerves associated with gateways in the posterior scapular region. Posterior circumflex humeral arteryCircumflex scapular arterySuperior transverse scapular ligamentSuprascapular nerveSuprascapular arteryAxillary nerveProfunda brachii arteryRadial nerveTo deltoidTo skin on lateral part of deltoidCut edge of lateral head of triceps brachii Fig. 7.39 Arterial anastomoses around the shoulder.

1	Fig. 7.39 Arterial anastomoses around the shoulder. ClaviclePosterior circumflexhumeral arteryAnterior circumflexhumeral arterySubscapular arteryCircumflex scapular arterySuprascapular arteryAxillary arteryProfunda brachii arteryBrachial arteryRight subclavian arteryDeep branch of transversecervical arteryTransverse cervical arteryRib IRight common carotid arteryThyrocervical trunk Fig. 7.40 Axilla. A. Walls and transition between neck and arm. Axilla. B. Boundaries. C. Continuity with the arm. Lateral wallAnterior wallPosterior wallSkinMedial wallCoracoid processAnterior scalene muscleMiddle scalene muscleClavicleLateral margin of rib IA

1	Axilla. B. Boundaries. C. Continuity with the arm. Lateral wallAnterior wallPosterior wallSkinMedial wallCoracoid processAnterior scalene muscleMiddle scalene muscleClavicleLateral margin of rib IA Axillary sheathsurroundingarteries, veins, nerves, and lymphaticsApex of inletInletAxillaSkin of armSkin on floor of axillaPosterior wall• Subscapularis, teres major, and latissimus dorsi muscles, and long head of triceps brachii muscleLateral wall• Intertubercular sulcusInlet• Lateral margin of rib I• Clavicle• Superior margin of scapula to coracoid processAnterior wall• Pectoralis major and minor muscles• Subclavius muscle• Clavipectoral fasciaMedial wall• Upper thoracic wall • Serratus anterior muscleFloor • Skin of armpit• Opens laterally into armBC Fig. 7.41 Pectoralis major muscle. Fig. 7.42 Pectoralis minor and subclavius muscles and clavipectoral fascia.

1	Fig. 7.41 Pectoralis major muscle. Fig. 7.42 Pectoralis minor and subclavius muscles and clavipectoral fascia. Pectoralis majorPectoralis majorPectoralis minorClavipectoral fasciaCephalic veinLateral pectoral nerveMedial pectoralnervesPectoral branch of thoraco-acromial arterySubclaviusAttachment of fasciato floor of axilla Fig. 7.43 Medial wall of the axilla. A. Lateral view. B. Lateral view with lateral angle of scapula retracted posteriorly. C. Anterior view. IIIIIIIVVVIVIILong thoracic nerveLong thoracic nerveLateral angle ofscapula pulledposterolaterallyaway from thethoracic wallIntercostobrachial nerve (lateral cutaneousbranch of T2)Serratus anteriorSerratus anteriorSerratus anteriorABC Fig. 7.44 Lateral wall of the axilla. Fig. 7.45 Posterior wall of the axilla.

1	Fig. 7.44 Lateral wall of the axilla. Fig. 7.45 Posterior wall of the axilla. Teres majorLatissimus dorsiLong head of triceps brachiiSubscapularisSuprascapular foramen• Suprascapular nerveTriangular interval • Radial nerve • Profunda brachii arteryTriangular space • Circumflex scapular arteryQuadrangular space • Axillary nerve • Posterior circumflex humeral artery and vein Fig. 7.46 Magnetic resonance image of the glenohumeral joint in the transverse or horizontal plane. Glenoid cavityBiceps tendon in intertubercular sulcusSubscapularisGlenoid labrumTeres minor and infraspinatus musclesHead of humerusAnteriorPosterior Fig. 7.47 Floor of the axilla. Axillary sheathArmDome of skin onfloor of axillaAnterior axillary skin foldPosterior axillaryskin fold Fig. 7.48 Contents of the axilla: muscles. Long head of biceps brachiiShort head of biceps brachiiTendon of biceps brachiiCoracobrachialisBicipital aponeurosisTransverse humeral ligament

1	Fig. 7.48 Contents of the axilla: muscles. Long head of biceps brachiiShort head of biceps brachiiTendon of biceps brachiiCoracobrachialisBicipital aponeurosisTransverse humeral ligament Fig. 7.49 Contents of the axilla: the axillary artery. Lower border of teres majorPectoralis minorLateral margin of rib ISubclaviusSubclavian artery1st part2nd part3rd partBrachial arteryAxillary artery Fig. 7.50 Branches of the axillary artery. Posterior circumflex humeral artery(quadrangular space)Anterior circumflex humeral arteryProfunda brachii artery(triangular interval)Long head of triceps brachiiThoraco-acromial arterySubclaviusPectoralis minorSubscapular arterySubscapularisSuperior thoracic arteryLateral thoracic arteryCircumflex scapular branch(triangular space)Thoracodorsal arteryLatissimus dorsiTeres major Fig. 7.51 Axillary vein. Fig. 7.52 Brachial plexus. A. Major components in the neck and axilla. B. Schematic showing parts of the brachial plexus.

1	Fig. 7.51 Axillary vein. Fig. 7.52 Brachial plexus. A. Major components in the neck and axilla. B. Schematic showing parts of the brachial plexus. TerminalnervesCordsDivisionsTrunksRoots(anterior rami)C5C6C7C8T1SuperiorMiddleInferiorLateralPosteriorPosteriorPosteriorPosteriorMedialAnteriorAnteriorAnterior Arrangedaround 2nd part of axillary arteryBSuperior cervical sympathetic ganglionInferior cervical sympathetic ganglionMiddle cervical sympathetic ganglionGray ramuscommunicansRoots (anterior rami of C5 to T1)Trunks (superior, middle, inferior)Divisions (anterior, posterior)Cords (medial, lateral, posterior)C8C7C6C5T1Middle scalene muscleAnterior scalene tendonA Fig. 7.53 Brachial plexus. A. Schematic showing branches of the brachial plexus. B. Relationships to the axillary artery.

1	Terminal nervesCordsDivisionsTrunksRoots (anterior rami)C5C6C7C8T1SuperiorMiddleInferiorLateralPosteriorPosteriorPosteriorPosteriorMedialAnteriorAnteriorAnterior ALateral pectoral nerveMedial pectoral nerveMedial cutaneous nerve of armMedial cutaneous nerve of forearmMusculocutaneousMedianRadialUlnarAxillaryLong thoracicnerveSuprascapular nerveDorsal scapular nerveContribution to phrenic nerveNerve to subclaviusSuperiorsubscapular nerveThoracodorsal nerveInferior subscapular nerveDorsal scapular nerveC5C6C7C8T1T2Suprascapular nerveLateral pectoral nerveMedial cordPosterior cordLateral cordMedial pectoral nerveSecond part of axillary arteryMusculocutaneous nerveAxillary nerveMedian nerveC7 fibersRadial nerveUlnar nerveSuperior subscapular nerveThoracodorsal nerveInferior subscapular nerveMedial cutaneous nerve of armMedial cutaneous nerve of forearmLong thoracic nerveIntercostobrachial nerve (lateral cutaneous branch of T2)Nerve to subclaviusB

1	Fig. 7.54 Branches of the roots and trunks of the brachial plexus. Dorsal scapular nerveMiddle scalene muscleAnterior scalene tendonT1 intercostal nerveC5 branch to phrenic nervePhrenic nerveSubclavian veinNerve to subclaviusAxillary arteryLong thoracic nerveSerratus anteriorSuprascapular foramenSuprascapular nerve Fig. 7.55 Branches of the lateral and medial cords of the brachial plexus. Lateral pectoral nerveMedial pectoral nerveAxillary arteryMusculocutaneous nerveMedian nerveUlnar nerveLateral cutaneousnerve of armMedial cutaneous nerve of forearmMedial cutaneous nerve of armLateral cordMedial cordPectoralis minorT1 intercostal nerve Fig. 7.56 Branches of the posterior cord of the brachial plexus. Axillary nerveSuperior subscapular nerveThoracodorsal nerveInferior subscapular nerveRadial nervePosterior cutaneousnerve of arm Fig. 7.57 Lymph nodes and vessels in the axilla.

1	Axillary nerveSuperior subscapular nerveThoracodorsal nerveInferior subscapular nerveRadial nervePosterior cutaneousnerve of arm Fig. 7.57 Lymph nodes and vessels in the axilla. Humeral nodesCentral nodesPectoral nodesApical nodesInfraclavicular nodesSubscapular nodesAnterior scaleneMost of upper limbSome of upper limbAnterolateral body wall and centrolateral part of mammary glandRight subclavian trunkSuperior part of mammary gland Fig. 7.58 Axillary process of the breast. Fig. 7.59 Arm. A. Proximal and distal relationships. B. Transverse section through the middle of the arm. AxillaCubitalfossaLateral intermuscular septumMedial intermuscular septumHumerusAnterior (flexor) compartmentPosterior (extensor) compartmentDeep fasciaForearmABArmLine of section Fig. 7.60 Humerus. Posterior view. Fig. 7.61 Distal end of the humerus. Fig. 7.62 A. Anterior view of the proximal end of the radius. B. Radiograph of the elbow joint (anteroposterior view).

1	Fig. 7.60 Humerus. Posterior view. Fig. 7.61 Distal end of the humerus. Fig. 7.62 A. Anterior view of the proximal end of the radius. B. Radiograph of the elbow joint (anteroposterior view). BTrochleaCapitulumLateral epicondyleMedial epicondyleRadiusHead of radiusHumerusUlnaOblique lineHeadRadial tuberosityNeckLateralMedialA Fig. 7.63 A. Lateral, anterior, medial, and posterior views of the proximal end of the ulna. B. Radiograph of the elbow joint (lateral view). Fig. 7.64 Coracobrachialis, biceps brachii, and brachialis muscles. Long head of bicepsbrachii muscleShort head of bicepsbrachii muscleCoracobrachialis muscleTransverse humeral ligamentBrachialis muscleTuberosity of ulnaBicipital aponeurosis (cut )Radial tuberosity Fig. 7.65 Triceps muscle. OlecranonLong head of triceps brachiiLateral head of triceps brachiiMedial head of triceps brachiiLateral head of triceps brachiiRadial groove of humerus Fig. 7.66 Brachial artery. A. In context. Brachial artery. B. Branches.

1	Fig. 7.66 Brachial artery. A. In context. Brachial artery. B. Branches. Fig. 7.67 Veins of the arm. Axillary veinCoracobrachialisInferior margin of teres majorPaired brachial veinsBasilic veinBrachialisBasilic vein (subcutaneous superficial vein)Basilic vein penetratesdeep fasciaDeep veins accompanying arteriesCephalic veinBiceps brachii Fig. 7.68 Musculocutaneous, median, and ulnar nerves in the arm. Median nerveUlnar nerveMedial epicondyleLateral cutaneous nerve of forearmMusculocutaneous nerveMusculocutaneous nerveMedial cordLateral cordRadial nerveMedial intermuscular septum Fig. 7.69 Radial nerve in the arm. Ulnar nerveProfunda brachii arteryTriangular intervalInferior lateral cutaneous nerve of armPosterior cutaneous nerve of forearmRadial nerve (in radial groove)Medial epicondyleBranch to medial head of triceps brachii Fig. 7.70 Radiograph of the humerus demonstrating a midshaft fracture, which may disrupt the radial nerve.

1	Fig. 7.70 Radiograph of the humerus demonstrating a midshaft fracture, which may disrupt the radial nerve. Fig. 7.71 Components and movements of the elbow joint. A. Bones and joint surfaces. B. Flexion and extension. C. Pronation and supination. D. Radiograph of a normal elbow joint (anteroposterior view). Fig. 7.72 Synovial membrane of elbow joint (anterior view). Fig. 7.73 Elbow joint. A. Joint capsule and ligaments of the right elbow joint. B. Magnetic resonance image of the elbow joint in the coronal plane. RadialcollateralligamentAUlnarcollateralligamentAnular ligamentof radiusSacciform recessof synovialmembraneBUlnar collateralligamentRadial collateral ligamentHead of radiusHumerusMedial epicondyleUlna Fig. 7.74 Radiograph of an elbow showing a fracture of the olecranon and involving the insertion of the triceps brachii muscle. Fig. 7.75 Radiographs of elbow joint development. A. At age 2 years. B. At age 5 years. C. At age 5–6 years. D. At age 12 years.

1	Fig. 7.75 Radiographs of elbow joint development. A. At age 2 years. B. At age 5 years. C. At age 5–6 years. D. At age 12 years. Fig. 7.76 MRI of right elbow showing swelling of the ulnar nerve in the cubital tunnel posterior to the medial epicondyle, consistent with nerve compression. Fig. 7.77 Cubital fossa. A. Margins. B. Contents. C. Position of the radial nerve. D. Superficial structures.

1	Fig. 7.77 Cubital fossa. A. Margins. B. Contents. C. Position of the radial nerve. D. Superficial structures. Ulnar nerveRadial arteryUlnar arteryPronator teres (humeral head)Pronator teres(ulnar head)Median nerveMedian cubitalveinUlnar nerveMedian nerveBasilic veinBasilic veinMedial cutaneousnerve of forearmLateral cutaneousnerve of forearmForearm extensorsLine betweenlateral and medialepicondylesForearm flexorsBrachioradialisBrachialisRadial nerveRadial nerveCephalic veinMusculocutaneous nerveBrachioradialis(pulled back )Deep branchof radial nerveSupinatorSuperficial branch of radial nerveTendon (biceps brachii)Biceps brachiiRadial arteryUlnar arteryTriceps brachiiABCDPronator teres Artery(brachial)Nerve(median)BicipitalaponeurosisMedial intermuscularseptumCubital fossaMedialepicondyleLateralepicondyleTendon (biceps)Artery (brachial)Nerve (median)Ulnar nerveRadial nerve Fig. 7.78 Digital subtraction angiograms of forearm demonstrating a surgically created radiocephalic fistula.

1	Fig. 7.78 Digital subtraction angiograms of forearm demonstrating a surgically created radiocephalic fistula. A. Anteroposterior view. B. Lateral view. Fig. 7.79 Forearm. A. Proximal and distal relationships of the forearm. B. Transverse section through the middle of the forearm. Cubital fossaCarpal tunnelUlnaMedian nerveLong flexortendons of digitsBiceps tendonBrachial arteryMedian nerveUlnaRadiusRadiusPosteriorcompartmentDeep fasciaBALateral intermuscular septumAnterior compartmentInterosseous membraneArmElbow jointForearmWrist jointHand Fig. 7.80 Radius. A. Shaft and distal end of the right radius. B. Radiograph of the forearm (anteroposterior view). Fig. 7.81 Shaft and distal end of right ulna.

1	Fig. 7.80 Radius. A. Shaft and distal end of the right radius. B. Radiograph of the forearm (anteroposterior view). Fig. 7.81 Shaft and distal end of right ulna. AnteriorsurfaceAnterior border(rounded)Posterior border(sharp)Anterior surfaceInterosseousborderMedialsurfaceAnterior borderPosterior surfaceUlnar styloid processRougheningfor attachmentof pronator quadratusAttachment ofarticular discInterosseousborderTrochlearnotchTuberosity of ulnaCoronoidprocessRadial notchOlecranonAnterior viewDistal view Fig. 7.82 Distal radio-ulnar joint and the interosseous membrane. Fig. 7.83 Pronation and supination. PronatedSupinatedSupinatorBiceps brachiiPronator teresPronator teresand pronatorquadratuscontractSupinator andbiceps brachiicontractPronator quadratusSupinatedAxis of movement Fig. 7.84 Abduction of the distal end of the ulna by the anconeus during pronation. Abduction ofulna by anconeusduring pronationAxis of movementwith abduction of ulnaAnconeus

1	Fig. 7.84 Abduction of the distal end of the ulna by the anconeus during pronation. Abduction ofulna by anconeusduring pronationAxis of movementwith abduction of ulnaAnconeus Fig. 7.85 Superficial layer of forearm muscles. A. Superficial muscles (flexor retinaculum not shown). B. Flexor carpi ulnaris muscle. Median nerveFlexor carpi radialisPalmaris longusFlexor carpi ulnaris Palmar aponeurosisPisometacarpal ligamentPisiformUlnar head offlexor carpi ulnarisABHumeral head ofpronator teresUlnar head ofpronator teresHumeral head offlexor carpi ulnarisUlnar nerveUlnar nerveBrachial arteryUlnar arteryRadial arteryPronator teres (cut )Pisohamate ligamentHook of hamate Fig. 7.86 Intermediate layer of forearm muscles. Median nerveFlexor digitorumsuperficialisUlnar nerve Ulnar arteryFlexor retinaculumHumero-ulnarhead of flexordigitorumsuperficialisUlnar arteryRadial head of flexordigitorumsuperficialisMedian nerve Fig. 7.87 Deep layer of forearm muscles.

1	Fig. 7.87 Deep layer of forearm muscles. Fig. 7.88 Arteries of the anterior compartment of the forearm. Fig. 7.89 Nerves of anterior forearm. Fig. 7.90 Superficial layer of muscles in the posterior compartment of the forearm. A. Brachioradialis muscle (anterior view). B. Superficial muscles (posterior view). Fig. 7.91 Deep layer of muscles in the posterior compartment of the forearm. Fig. 7.92 Posterior interosseous artery and radial nerve in posterior compartment of forearm. Posteriorinterosseous nerve(continuation ofdeep branch ofradial nerve)Commoninterosseous arteryAnteriorinterosseous arteryUlnar arteryPosteriorinterosseous arteryInterosseous membraneSuperficial branchDeep branchRadial nerveBranch tobrachioradialisBranch to extensorcarpi radialis brevisBranch to extensorcarpi radialis longusAnteriorinterosseous arteryPosteriorinterosseous arteryAnterior viewPosterior view

1	Fig. 7.93 Right hand. The fingers are shown in a normal resting arcade in which they are flexed. In the anatomical position, the digits are straight and adducted. Carpal bonesThumbLittleRingIndexFingersRadiusWrist jointUlnaProximal skin creaseDistal skin creaseMetacarpalsDigits ofthe handAdductionAdductionAbductionAbductionMiddle Fig. 7.94 Right hand and wrist joint. A. Bones. Right hand and wrist joint. B. Radiograph of a normal hand and wrist joint (anteroposterior view). C. Magnetic resonance image of a normal wrist joint in the coronal plane. RadiusWrist jointIIIIIIIVVScaphoidTrapezoidLunateUlnaTriquetrumTriquetrumPisiformPisiformHook ofhamateHamateHamatePhalangesAMetacarpalsCarpal bonesCapitateCapitateTrapeziumTrapeziumProximalMiddleDistalProximalCarpal bonesCarpal archCarpal archDistalTubercle of trapeziumTrapezoidTubercle of scaphoidTubercle Fig. 7.95 Deep transverse metacarpal ligaments, right hand.

1	Fig. 7.95 Deep transverse metacarpal ligaments, right hand. Fig. 7.96 Wrist radiographs (posteroanterior view). A. Normal. B. Scaphoid fracture. Fig. 7.97 Radiograph of wrist showing sclerosis in the lunate consistent with avascular necrosis (Kienbock’s disease). Fig. 7.98 Carpal tunnel. A. Structure and relations. B. Magnetic resonance image of a normal wrist in the axial plane. C. Magnetic resonance image of a normal wrist in the coronal plane. Fig. 7.99 Palmar aponeurosis, right hand. Fig. 7.100 Anatomical snuffbox, left hand. Fig. 7.101 Fibrous digital sheaths and synovial sheaths of the right hand. Fig. 7.102 MRI of the wrist showing fluid and inflammation associated with the first extensor compartment, consistent with De Quervain’s tenosynovitis. Fig. 7.103 Extensor hood. A and B. Middle finger, left hand. C. Function of extensor hoods and intrinsic muscles.

1	Fig. 7.103 Extensor hood. A and B. Middle finger, left hand. C. Function of extensor hoods and intrinsic muscles. Extensor hoodExtensordigitorum tendonDorsal interosseousmuscleFlexor digitorumprofundus tendonFulcrum ofmetacarpophalangeal jointABCContraction of intrinsicmuscles (lumbricals andinterossei muscles)Deep transversemetacarpal ligamentMiddle fingerFulcrums ofinterphalangeal jointsExtension ofinterphalangeal jointsFlexion ofmetacarpophalangeal jointExtendedFlexedUpstrokePalmar ligamentLumbrical muscle Fig. 7.104 Dorsal interossei (palmar view), right hand. Fig. 7.105 Palmar interossei (palmar view), right hand. AdductionAdductionFirst palmarinterosseous(rudimentary: whenpresent is oftenconsidered part ofeither adductorpollicis or flexorpollicis brevis)Insertion intodorsal expansion Fig. 7.106 Adductor pollicis, right hand. Oblique head of adductor pollicisTransverse head of adductor pollicisRadial artery(deep palmar arch)Sesamoid bone

1	Fig. 7.106 Adductor pollicis, right hand. Oblique head of adductor pollicisTransverse head of adductor pollicisRadial artery(deep palmar arch)Sesamoid bone Fig. 7.107 Thenar and hypothenar muscles, right hand. Flexor retinaculumMedian nerveFlexor carpi ulnarisOpponens digiti minimiAbductor digiti minimiFlexor digiti minimi brevisDeep branch ofulnar artery and nerveRecurrent branch of median nerveAdductor pollicis and first palmarinterosseous insert into medialside of extensor hoodFlexor pollicis brevis and abductorpollicis brevis insert into lateral side of extensor hoodExtensor hoodFlexor pollicis brevisThree thenar musclesThree hypothenar musclesOpponens pollicisAbductor pollicis brevis Fig. 7.108 Lumbrical muscles, right hand.

1	Fig. 7.108 Lumbrical muscles, right hand. Flexor retinaculumFlexor digitorumsuperficialis tendon (cut )Flexor digitorumprofundus tendonAttached to dorsal hoodFirst and second lumbricals(unipennate)Third and fourth lumbricals(bipennate)Flexor pollicislongus tendonDeep transversemetacarpal ligament Fig. 7.109 Arterial supply of the right hand. Fig. 7.110 Superficial palmar arch, right hand. Fig. 7.111 Deep palmar arch, right hand.

1	Fig. 7.109 Arterial supply of the right hand. Fig. 7.110 Superficial palmar arch, right hand. Fig. 7.111 Deep palmar arch, right hand. Radial arteryUlnar arteryUlnar nerveDeep branchof ulnar arteryPerforatingarteryPalmarmetacarpalarteriesDeep palmar archAdductorpollicismuscleRadialis indicisarteryPrincepspollicis arteryFirst dorsalinterosseous muscleFirst dorsalmetacarpalarteryMainly radial arteryDorsalmetacarpalarteriesDorsal carpalnetworkDorsalcarpal archDorsaldigitalarteriesDorsalcarpal branchof ulnar arteryPosteriorinterosseousarteryDorsal viewarcharteryExtensordigitorumtendons(cut)DorsalmetacarpalarteriesFirst dorsalmetacarpalarteryDorsal carpalDorsal carpalbranch of ulnarExtensor pollicis brevisAbductor pollicis longusExtensor pollicis longusExtensor retinaculumExtensor carpiradialis longusRadial artery inanatomical snuffboxExtensor carpiradialis brevisDorsal interosseiDorsal digitalarteriesDorsal branches of properpalmar digital arteries

1	Fig. 7.112 Dorsal venous arch of the right hand. Fig. 7.113 Ulnar nerve in the right hand. Palmar viewDorsal viewMedial twolumbricalmusclesPalmar branch of ulnarnerve from forearmArea of distribution ofsuperficial branch of ulnarnerve in handDorsal branch of ulnarnerve from forearmUlnar nerveDeep branch(of ulnar nerve)Superficial branch(of ulnar nerve)Ulnar artery Fig. 7.114 Typical appearance of a “clawed hand” due to a lesion of the ulnar nerve. Fig. 7.115 Median nerve in the right hand. Recurrent branch(of median nerve)Lateral twolumbrical musclesPalmar branch of mediannerve from forearmDigital nervesMedian nervePalmar branch(of median nerve)Abductor pollicis brevisFlexor pollicis brevisPalmar viewDorsal view Fig. 7.116 Radial nerve in the right hand. Fig. 7.117 Bony landmarks and muscles of the posterior scapular region. Posterior view of shoulder and back.

1	Fig. 7.116 Radial nerve in the right hand. Fig. 7.117 Bony landmarks and muscles of the posterior scapular region. Posterior view of shoulder and back. Teres major muscleTrapezius muscleDeltoid muscleAxillary nerveLatissimus dorsi muscleSpine of scapulaAcromionSupraspinatus muscleInfraspinatus muscleTeres minor musclePosterior axillary skin fold Fig. 7.118 Visualizing the axilla and locating its contents and related structures. A. Anterior shoulder showing folds and walls of the axilla. B. Anterior shoulder showing outlet and floor of the axilla. C. Anterior view showing the axillary neurovascular bundle and long thoracic nerve. D. Anterior view of the shoulder showing the clavipectoral triangle with the cephalic vein.

1	ClavicleCoracoid processHumerusAnterior axillaryskin foldAnterior wallPosterior wallMedial wallLateral wallNeurovascular bundleSerratus anteriormuscleLong thoracic nerveAxillaDeltoid musclePectoralis major muscleCephalic veinClavipectoral triangleFloor of axillaOpening ofaxilla into armAnterior axillary skin foldPosterioraxillary skin foldABCD Fig. 7.119 Locating the brachial artery in the right arm (medial view of arm with brachial artery, median nerve, and ulnar nerve). Fig. 7.120 Triceps brachii tendon and position of the radial nerve (posterior view of right arm). Fig. 7.121 Cubital fossa (anterior view, right arm). A. Anterior view. B. Boundaries and contents. C. Showing radial and ulnar nerves, and veins. Fig. 7.122 Identifying tendons and locating major vessels and nerves in the distal right forearm. A. Anterior distal forearm and wrist. B. Posterior distal forearm and wrist. C. Lateral view of posterior wrist and forearm. D. Anatomical snuffbox.

1	Fig. 7.123 Normal appearances of the right hand. A. Palmar view with the thenar and hypothenar eminences and finger arcade. B. Dorsal view with dorsal venous network. Fig. 7.124 Anterior view of left hand to show the position of the flexor retinaculum and recurrent branch of the median nerve. Recurrent branch of the median nervePisiform Tubercle ofthe scaphoidFlexor carpiradialis tendonMedian nerveFlexor carpiulnaris tendonFlexor retinaculumHypothenar eminenceThenar eminence Fig. 7.125 Motor function of the ulnar and median nerves in the hand. A. Flexing the metacarpophalangeal joints and extending the interphalangeal joints: the “ta-ta” position. B. Grasping an object between the fingers. C. Grasping an object between the pad of the thumb and pad of the index finger.

1	Fig. 7.126 Visualizing the positions of the superficial and deep palmar arches, left hand. The proximal transverse skin crease of the palm and distal wrist crease are labeled and the superficial and deep palmar arches shown in overlay. This also shows the position of the pisiform and the hook of the hamate. Distal transverseskin crease of palmPisiform Distal wrist creaseProximal transverseskin crease of palmProximalwrist creaseUlnar arteryRadial arteryHook of hamateDeep palmar archSuperficial palmar arch Fig. 7.127 Where to take peripheral artery pulses in the upper limb. A. Pulse points. B. Placement of blood pressure cuff and stethoscope. ABBrachial pulse in midarmBrachial pulse in the cubital fossaUlnar pulse in distal forearmRadial pulse in the anatomical snuffboxRadial pulse in distal forearmAxillary pulse eFig. 7.128 Ultrasound showing a completely torn supraspinatus tendon with fluid in the subacromial subdeltoid bursa.

1	Deltoid muscleTear in supraspinatus tendonNormal supraspinatus tendonHead of humerusHead of humerus eFig. 7.129 The radiograph, anteroposterior view, demonstrates an anteroinferior dislocation of the humeral head at the glenohumeral joint. Table 7.1 Muscles of the shoulder (spinal segments in bold are the major segments innervating the muscle) Table 7.2 Muscles of the posterior scapular region (spinal segments in bold are the major segments innervating the muscle) Table 7.3 Muscles of the anterior wall of the axilla (spinal segments in bold are the major segments innervating the muscle) Table 7.4 Muscle of the medial wall of the axilla (spinal segment in bold is the major segment innervating the muscle) Table 7.5 Muscles of the lateral and posterior wall of the axilla (spinal segments in bold are the major segments innervating the muscle; spinal segments in parentheses do not consistently innervate the muscle)

1	Table 7.6 Muscles having parts that pass through the axilla (spinal segments in bold are the major segments innervating the muscle) Table 7.7 Branches of brachial plexus (parentheses indicate that a spinal segment is a minor component of the nerve or is inconsistently present in the nerve) Table 7.8 Muscles of the anterior compartment of the arm (spinal segments in bold are the major segments innervating the muscle) Table 7.9 Muscle of the posterior compartment of the arm (spinal segment indicated in bold is the major segment innervating the muscle) Table 7.10 Superficial layer of muscles in the anterior compartment of the forearm (spinal segments indicated in bold are the major segments innervating the muscle) Table 7.11 Intermediate layer of muscles in the anterior compartment of the forearm (spinal segment indicated in bold is the major segment innervating the muscle)

1	Table 7.11 Intermediate layer of muscles in the anterior compartment of the forearm (spinal segment indicated in bold is the major segment innervating the muscle) Table 7.12 Deep layer of muscles in the anterior compartment of the forearm (spinal segments indicated in bold are the major segments innervating the muscle) Table 7.13 Superficial layer of muscles in the posterior compartment of the forearm (spinal segments indicated in bold are the major segments innervating the muscle) Table 7.14 Deep layer of muscles in the posterior compartment of the forearm (spinal segments indicated in bold are the major segments innervating the muscle) Table 7.15 Intrinsic muscles of the hand (spinal segments indicated in bold are the major segments innervating the muscle) In the clinic Fracture of the proximal humerus

1	Table 7.15 Intrinsic muscles of the hand (spinal segments indicated in bold are the major segments innervating the muscle) In the clinic Fracture of the proximal humerus It is extremely rare for fractures to occur across the anatomical neck of the humerus because the obliquity of such a fracture would have to traverse the thickest region of bone. Typically fractures occur around the surgical neck of the humerus. Although the axillary nerve and posterior circumflex humeral artery may be damaged with this type of fracture, this rarely happens. It is important that the axillary nerve is tested before relocation to be sure that the injury has not damaged the nerve and that the treatment itself does not cause a neurological deficit. In the clinic Fractures of the clavicle and dislocations of the acromioclavicular and sternoclavicular joints

1	In the clinic Fractures of the clavicle and dislocations of the acromioclavicular and sternoclavicular joints The clavicle provides the only bony connection between the upper limb and trunk. Given its relative size and the potential forces that it transmits from the upper limb to the trunk, it is not surprising that it is often fractured. The typical site of fracture is the middle third (Fig. 7.30). The medial and lateral thirds are rarely fractured. The acromial end of the clavicle tends to dislocate at the acromioclavicular joint with trauma (Fig. 7.31). The outer third of the clavicle is joined to the scapula by the conoid and trapezoid ligaments of the coracoclavicular ligament.

1	A minor injury tends to tear the fibrous joint capsule and ligaments of the acromioclavicular joint, resulting in acromioclavicular separation on a plain radiograph. More severe trauma will disrupt the conoid and trapezoid ligaments of the coracoclavicular ligament, which results in elevation and upward subluxation of the clavicle. The typical injury at the medial end of the clavicle is an anterior or posterior dislocation of the sternoclavicular joint. Importantly, a posterior dislocation of the clavicle may impinge on the great vessels in the root of the neck and compress or disrupt them. In the clinic Dislocations of the glenohumeral joint The glenohumeral joint is extremely mobile, providing a wide range of movement at the expense of stability. The relatively small bony glenoid cavity, supplemented by the less robust fibrocartilaginous glenoid labrum and the ligamentous support, make it susceptible to dislocation.

1	Anterior dislocation (Fig. 7.32) occurs most frequently and is usually associated with an isolated traumatic incident (clinically, all anterior dislocations are anteroinferior). In some cases, the anteroinferior glenoid labrum is torn with or without a small bony fragment. Once the joint capsule and cartilage are disrupted, the joint is susceptible to further (recurrent) dislocations. When an anteroinferior dislocation occurs, the axillary nerve may be injured by direct compression of the humeral head on the nerve inferiorly as it passes through the quadrangular space. Furthermore, the “lengthening” effect of the humerus may stretch the radial nerve, which is tightly bound within the radial groove, and produce a radial nerve paralysis. Occasionally, an anteroinferior dislocation is associated with a fracture, which may require surgical reduction.

1	Posterior dislocation is extremely rare; when seen, the clinician should focus on its cause, the most common being extremely vigorous muscle contractions, which may be associated with an epileptic seizure caused by electrocution. Treatment of recurrent instability can be challenging. The aims of treatment are to maintain function and range of movement while preventing instability (subluxation, dislocation, and the “feeling” of dislocation). This can be achieved through physical therapy and shoulder “re-education.” If this fails, capsular tightening and stabilization of the labrum can be achieved arthroscopically. If the problem persists, the coracoid process can be divided at the base, maintaining continuity of the muscular attachments. The process is transferred and a screw fixed to the anterior inferior border of the glenoid to form a buttress to prevent future dislocations. In the clinic

1	In the clinic The two main disorders of the rotator cuff are impingement and tendinopathy. The muscle most commonly involved is supraspinatus as it passes beneath the acromion and the acromioclavicular ligament. This space, beneath which the supraspinatus tendon passes, is of fixed dimensions. Swelling of the supraspinatus muscle, excessive fluid within the subacromial/subdeltoid bursa, or subacromial bony spurs may produce significant impingement when the arm is abducted. The blood supply to the supraspinatus tendon is relatively poor. Repeated trauma, in certain circumstances, makes the tendon susceptible to degenerative change, which may result in calcium deposition, producing extreme pain. The calcium deposits can be extracted through a needle under image guidance and often have the consistency of toothpaste.

1	When the supraspinatus tendon has undergone significant degenerative change, it is more susceptible to trauma, and partialor full-thickness tears may develop (Fig. 7.33). These tears are most common in older patients and may result in considerable difficulty in carrying out normal activities of daily living such as combing hair. However, complete tears may be entirely asymptomatic. In the clinic Inflammation of the subacromial (subdeltoid) bursa Between the supraspinatus and deltoid muscles laterally and the acromion medially, there is a bursa referred to clinically as the subacromial or subdeltoid bursa. In patients who have injured the shoulder or who have supraspinatus tendinopathy, this bursa may become inflamed, making movements of the glenohumeral joint painful. These inflammatory changes may be treated by injection of a corticosteroid and local anesthetic agent (Fig. 7.34). In the clinic

1	In the clinic Hypertrophy of the quadrangular space muscles or fibrosis of the muscle edges may impinge on the axillary nerve. Uncommonly, this produces weakness of the deltoid muscle. Typically it produces atrophy of the teres minor muscle, which may affect the control that the rotator cuff muscles exert on shoulder movement. In the clinic ”Winging” of the scapula Because the long thoracic nerve passes down the lateral thoracic wall on the external surface of the serratus anterior muscle, just deep to skin and subcutaneous fascia, it is vulnerable to damage. Loss of function of this muscle causes the medial border, and particularly the inferior angle, of the scapula to elevate away from the thoracic wall, resulting in characteristic “winging” of the scapula, on pushing forward with the arm. Furthermore, normal elevation at the arm is no longer possible. In the clinic Imaging the blood supply to the upper limb

1	In the clinic Imaging the blood supply to the upper limb When there is clinical evidence of vascular compromise to the upper limb, or vessels are needed to form an arteriovenous fistula (which is necessary for renal dialysis), imaging is required to assess the vessels. Ultrasound is a useful tool for carrying out a noninvasive assessment of the vessels of the upper limb from the third part of the subclavian artery to the deep and superficial palmar arteries. Blood flow can be quantified and anatomical variants can be noted.

1	Angiography is carried out in certain cases. The femoral artery is punctured below the inguinal ligament and a long catheter is placed through the iliac arteries and around the arch of the aorta to enter either the left subclavian artery or the brachiocephalic trunk and then the right subclavian artery. Radiopaque contrast agents are injected into the vessel and radiographs are obtained as the contrast agents pass first through the arteries, then the capillaries, and finally the veins. In the clinic Trauma to the arteries of the upper limb The arterial supply to the upper limb is particularly susceptible to trauma in places where it is relatively fixed or in a subcutaneous position. Fracture of rib I

1	The arterial supply to the upper limb is particularly susceptible to trauma in places where it is relatively fixed or in a subcutaneous position. Fracture of rib I As the subclavian artery passes out of the neck and into the axilla, it is fixed in position by the surrounding muscles to the superior surface of rib I. A rapid deceleration injury involving upper thoracic trauma may cause a first rib fracture, which may significantly compromise the distal part of the subclavian artery or the first part of the axillary artery. Fortunately, there are anastomotic connections between branches of the subclavian artery and the axillary artery, which form a network around the scapula and proximal end of the humerus; therefore, even with complete vessel transection, the arm is rarely rendered completely ischemic (ischemia is poor blood supply to an organ or a limb). Anterior dislocation of the humeral head

1	Anterior dislocation of the humeral head Anterior dislocation of the humeral head may compress the axillary artery, resulting in vessel occlusion. This is unlikely to render the upper limb completely ischemic, but it may be necessary to surgically reconstruct the axillary artery to obtain pain-free function. Importantly, the axillary artery is intimately related to the brachial plexus, which may be damaged at the time of anterior dislocation. In the clinic There are a number of routes through which central venous access may be obtained. The “subclavian route” and the jugular routes are commonly used by clinicians. The subclavian route is a misnomer that remains the preferred term in clinical practice. In fact, most clinicians enter the first part of the axillary vein.

1	There are a number of patients that undergo catheterization of the subclavian vein/axillary vein. Entering the subclavian vein/axillary vein is a relatively straightforward technique. The clavicle is identified and a sharp needle is placed in the infraclavicular region, aiming superomedially. When venous blood is aspirated, access has been obtained. This route is popular for long-term venous access, such as Hickman lines, and for shorter-term access where multiple-lumen catheters are inserted (e.g., intensive care unit).

1	The subclavian vein/axillary vein is also the preferred site for insertion of pacemaker wires. There is, however, a preferred point of entry into the vein to prevent complications. The vein should be punctured in the midclavicular line or lateral to this line. The reason for this puncture site is the course of the vein and its relationship to other structures. The vein passes anterior to the artery, superior to the first rib, and inferior to the clavicle as it courses toward the thoracic inlet. Beneath the clavicle is situated the subclavius muscle. Should the puncture of the vein enter where the subclavius muscle is related to the axillary vein, the catheter or wire may become kinked at this point. Moreover, the constant contraction and relaxation of this muscle will induce fatigue in the line and wire, which may ultimately lead to fracture. A fractured pacemaker wire or a rupture in a chemotherapy catheter can have severe consequences for the patient. In the clinic

1	In the clinic Injuries to the brachial plexus The brachial plexus is an extremely complex structure. When damaged, it requires meticulous clinical history taking and examination. Assessment of the individual nerve functions can be obtained by nerve conduction studies and electromyography, which assess the latency of muscle contraction when the nerve is artificially stimulated. Brachial plexus injuries are usually the result of blunt trauma producing nerve avulsions and disruption. These injuries are usually devastating for the function of the upper limb and require many months of dedicated rehabilitation for even a small amount of function to return. Spinal cord injuries in the cervical region and direct pulling injuries tend to affect the roots of the brachial plexus. Severe trauma to the first rib usually affects the trunks. The divisions and cords of the brachial plexus can be injured by dislocation of the glenohumeral joint. In the clinic

1	In the clinic Lymphatic drainage from the lateral part of the breast passes through nodes in the axilla. Significant disruption to the normal lymphatic drainage of the upper limb may occur if a mastectomy or a surgical axillary nodal clearance has been carried out for breast cancer. Furthermore, some patients have radiotherapy to the axilla to prevent the spread of metastatic disease, but a side effect of this is the destruction of the tiny lymphatics as well as the cancer cells. If the lymphatic drainage of the upper limb is damaged, the arm may swell and pitting edema (lymphedema) may develop. In the clinic Rupture of biceps tendon

1	If the lymphatic drainage of the upper limb is damaged, the arm may swell and pitting edema (lymphedema) may develop. In the clinic Rupture of biceps tendon It is relatively unusual for muscles and their tendons to rupture in the upper limb; however, the tendon that most commonly ruptures is the tendon of the long head of the biceps brachii muscle. In isolation, this has relatively little effect on the upper limb, but it does produce a characteristic deformity—on flexing the elbow, there is an extremely prominent bulge of the muscle belly as its unrestrained fibers contract—the “Popeye” sign. Distal biceps tendon rupture also occurs. It is important to determine the site of the rupture, whether it’s at the musculotendinous junction, midtendon, or at the insertion because this will determine the surgical approach for repair. In the clinic

1	In the clinic Blood pressure measurement is an extremely important physiological parameter. High blood pressure (hypertension) requires treatment to prevent long-term complications such as stroke. Low blood pressure may be caused by extreme blood loss, widespread infection, or poor cardiac output (e.g., after myocardial infarction). Accurate measurement of blood pressure is essential.

1	Most clinicians use a sphygmomanometer and a stethoscope. The sphygmomanometer is a device that inflates a cuff around the midportion of the arm to compress the brachial artery against the humerus. The cuff is inflated so it exceeds the systolic blood pressure (greater than 120 mm Hg). The clinician places a stethoscope over the brachial artery in the cubital fossa and listens (auscultates) for the pulse. As the pressure in the arm cuff of the sphygmomanometer is reduced just below the level of the systolic blood pressure, the pulse becomes audible as a regular thumping sound. As the pressure in the sphygmomanometer continues to drop, the regular thumping sound becomes clearer. When the pressure in the sphygmomanometer is less than that of the diastolic blood pressure, the audible thumping sound becomes inaudible. Using the simple scale on the sphygmomanometer, the patient’s blood pressure can be determined. The normal range is 90–120/60–80 mm Hg (systolic blood pressure/diastolic

1	sound becomes inaudible. Using the simple scale on the sphygmomanometer, the patient’s blood pressure can be determined. The normal range is 90–120/60–80 mm Hg (systolic blood pressure/diastolic blood pressure).

1	In the clinic Radial nerve injury in the arm The radial nerve is tightly bound with the profunda brachii artery between the medial and lateral heads of the triceps brachii muscle in the radial groove. If the humerus is fractured, the radial nerve may become stretched or transected in this region, leading to permanent damage and loss of function. This injury is typical (Fig. 7.70) and the nerve should always be tested when a fracture of the midshaft of the humerus is suspected. The patient’s symptoms usually include wrist drop (due to denervation of the extensor muscles) and sensory changes over the dorsum of the hand. In the clinic Median nerve injury in the arm In the arm and forearm the median nerve is usually not injured by trauma because of its relatively deep position. The commonest neurological problem associated with the median nerve is compression beneath the flexor retinaculum at the wrist (carpal tunnel syndrome).

1	On very rare occasions, a fibrous band may arise from the anterior aspect of the humerus beneath which the median nerve passes. This is an embryological remnant of the coracobrachialis muscle and is sometimes called the ligament of Struthers; occasionally, it may calcify. This band can compress the median nerve, resulting in weakness of the flexor muscles in the forearm and the thenar muscles. Nerve conduction studies will demonstrate the site of nerve compression. In the clinic Supracondylar fracture of the humerus

1	In the clinic Supracondylar fracture of the humerus Elbow injuries in children may result in a transverse fracture of the distal end of the humerus, above the level of the epicondyles. This fracture is termed a supracondylar fracture. The distal fragment and its soft tissues are pulled posteriorly by the triceps muscle. This posterior displacement effectively “bowstrings” the brachial artery over the irregular proximal fracture fragment. In children, this is a relatively devastating injury: the muscles of the anterior compartment of the forearm are rendered ischemic and form severe contractions, significantly reducing the function of the anterior compartment and flexor muscles (Volkmann’s ischemic contracture). In the clinic

1	In the clinic Pulled elbow is a disorder that typically occurs in children under 5 years of age. It is commonly caused by a sharp pull of the child’s hand, usually when the child is pulled up a curb. The not-yet-developed head of the radius and the laxity of the anular ligament of the radius allow the head to sublux from this cuff of tissue. Pulled elbow is extremely painful, but can be treated easily by simple supination and compression of the elbow joint by the clinician. When the radial head is relocated the pain subsides immediately and the child can continue with normal activity. In the clinic Fracture of the olecranon Fractures of the olecranon can result from a direct blow to the olecranon or from a fall onto an outstretched hand (Fig. 7.74). The triceps inserts into the olecranon and injuries can cause avulsion of the muscle. In the clinic Developmental changes in the elbow joint

1	In the clinic Developmental changes in the elbow joint The elbow joint can be injured in many ways; the types of injuries are age dependent. When a fracture or soft tissue trauma is suspected, a plain lateral and an anteroposterior radiograph are obtained. In an adult it is usually not difficult to interpret the radiograph, but in children additional factors require interpretation.

1	As the elbow develops in children, numerous secondary ossification centers appear before and around puberty. It is easy to mistakenly interpret these as fractures. In addition, it is also possible for the epiphyses and apophyses to be “pulled off” or disrupted. Therefore, when interpreting a child’s radiograph of the elbow, the physician must know the child’s age (Fig. 7.75). Fusion occurs at around the time of puberty. An understanding of the normal epiphyses and apophyses and their normal relationship to the bones will secure a correct diagnosis. The approximate ages of appearance of the secondary ossification centers around the elbow joint are: capitulum—1 year, head (of radius)—5 years, medial epicondyle—5 years, trochlea—11 years, olecranon—12 years, and lateral epicondyle—13 years. In the clinic Fracture of the head of the radius

1	In the clinic Fracture of the head of the radius A fracture of the head of the radius is a common injury and can cause appreciable morbidity. It is one of the typical injuries that occur with a fall on the outstretched hand. On falling, the force is transmitted to the radial head, which fractures. These fractures typically result in loss of full extension, and potential surgical reconstruction may require long periods of physiotherapy to obtain a full range of movement at the elbow joint.

1	A lateral radiograph of a fracture of the head of the radius typically demonstrates the secondary phenomenon of this injury. When the bone is fractured, fluid fills the synovial cavity, elevating the small pad of fat within the coronoid and olecranon fossae. These fat pads appear as areas of lucency on the lateral radiograph—the “fat pad” sign. This radiological finding is useful because fracture of the head of the radius is not always clearly visible. If there is an appropriate clinical history, tenderness around the head of the radius, and a positive fat pad sign, a fracture can be inferred clinically even if no fracture can be identified on the radiograph, and appropriate treatment can be instituted. In the clinic

1	In the clinic It is not uncommon for people who are involved in sports such as golf and tennis to develop an overuse strain of the origins of the flexor and extensor muscles of the forearm. The pain is typically around the epicondyles and usually resolves after rest and physical therapy. It may also be treated with injection of the patient’s own plasma, rich in platelets, into the tendon to promote tendon healing and repair. If pain and inflammation persist, surgical division of the extensor or flexor origin from the bone may be necessary. Typically, in tennis players this pain occurs on the lateral epicondyle and common extensor origin (tennis elbow), whereas in golfers it occurs on the medial epicondyle and common flexor origin. In the clinic

1	In the clinic Osteoarthritis is extremely common and is usually most severe in the dominant limb. From time to time an arthritic elbow may undergo such degenerative change that small bone fragments appear in the articular cavity. Given the relatively small joint space, these fragments can result in an appreciable reduction in flexion and extension, and typically lodge within the olecranon and coronoid fossae. In the clinic Ulnar nerve injury at the elbow Posterior to the medial epicondyle of the humerus the ulnar nerve is bound in a fibro-osseous tunnel (the cubital tunnel) by a retinaculum. Older patients may develop degenerative changes within this tunnel, which compresses the ulnar nerve when flexed. The repeated action of flexion and extension of the elbow may cause local nerve damage, resulting in impaired function of the ulnar nerve. Accessory muscles and localized neuritis in this region secondary to direct trauma may also produce ulnar nerve damage (Fig. 7.76).

1	In the clinic Construction of a dialysis fistula Many patients throughout the world require renal dialysis for kidney failure. The patient’s blood is filtered and cleaned by the dialysis machine. Blood therefore has to be taken from patients into the filtering device and then returned to them. This process of dialysis occurs over many hours and requires considerable flow rates of 250–500 mL per minute. To enable such large volumes of blood to be removed from and returned to the body, the blood is taken from vessels that have a high flow. As no veins in the peripheral limbs have such high flow, a surgical procedure is necessary to create such a system. In most patients, the radial artery is anastomosed (joined) to the cephalic vein (Fig. 7.78) at the wrist, or the brachial artery is anastomosed to the cephalic vein at the elbow. Some surgeons place an arterial graft between these vessels.

1	After six weeks, the veins increase in size in response to their arterial blood flow and are amenable to direct cannulation or dialysis. In the clinic Fractures of the radius and ulna The radius and ulna are attached to the humerus proximally and the carpal bones distally by a complex series of ligaments. Although the bones are separate, they behave as one. When a severe injury occurs to the forearm it usually involves both bones, resulting in either fracture of both bones or more commonly a fracture of one bone and a dislocation of the other. Commonly, the mechanism of injury and the age of the patient determine which of these are likely to occur. There are three classic injuries to the radius and ulna: Monteggia’s fracture is a fracture of the proximal third of the ulna and an anterior dislocation of the head of the radius at the elbow.

1	There are three classic injuries to the radius and ulna: Monteggia’s fracture is a fracture of the proximal third of the ulna and an anterior dislocation of the head of the radius at the elbow. Galeazzi’s fracture is a fracture of the distal third of the radius associated with subluxation (partial dislocation) of the head of the ulna at the wrist joint. Colles’ fracture is a fracture, and posterior displacement, of the distal end of the radius. Whenever a fracture of the radius or ulna is demonstrated radiographically, further images of the elbow and wrist should be obtained to exclude dislocations. In the clinic Transection of the radial or ulnar artery

1	In the clinic Transection of the radial or ulnar artery Adult patients may transect the radial or ulnar artery because these vessels are relatively subcutaneous. A typical method of injury is when the hand is forced through a plate glass window. Fortunately, the dual supply to the hand usually enables the surgeon to tie off either the ulnar or the radial artery, without significant consequence. In the clinic Fracture of the scaphoid and avascular necrosis of the proximal scaphoid The commonest carpal injury is a fracture across the waist of the scaphoid bone (Fig. 7.96). It is uncommon to see other injuries. In approximately 10% of individuals, the scaphoid bone has a sole blood supply from the radial artery, which enters through the distal portion of the bone to supply the proximal portion. When a fracture occurs across the waist of the scaphoid, the proximal portion therefore undergoes avascular necrosis. It is impossible to predict which patients have this blood supply.

1	In the clinic Interruption of the blood supply to the lunate can lead to avascular necrosis of the lunate, known as Kienbock’s disease (Fig. 7.97). This can cause pain and stiffness and arthritis in the longer term. In the clinic A large median artery is an anatomical variant found in some individuals, where a persistent artery runs alongside the median nerve in one or both forearms and through the carpal tunnel. Individuals are at risk from heavy bleeding from deep cuts to the wrist. In the clinic

1	In the clinic Carpal tunnel syndrome is an entrapment syndrome caused by pressure on the median nerve within the carpal tunnel. The etiology of this condition is often obscure, though in some instances the nerve injury may be a direct effect of increased pressure on the median nerve caused by overuse, swelling of the tendons and tendon sheaths (e.g., rheumatoid arthritis), and cysts arising from the carpal joints. Increased pressure in the carpal tunnel is thought to cause venous congestion that produces nerve edema and anoxic damage to the capillary endothelium of the median nerve itself. Patients typically report pain and pins-and-needles sensations in the distribution of the median nerve. Weakness and loss of muscle bulk of the thenar muscles may also occur. Gently tapping over the median nerve (in the region of the flexor retinaculum) readily produces these symptoms (Tinel’s sign).

1	Initial treatment is aimed at reducing the inflammation and removing any repetitive insults that produce the symptoms. If this does not lead to improvement, nerve conduction studies will be necessary to confirm nerve entrapment, which may require surgical decompression of the flexor retinaculum. In the clinic The palmar fascia can become abnormally thickened in certain individuals, causing the fingers to progressively develop a fixed flexion position. This results in loss of dexterity and function, and in severe cases requires surgical removal of the abnormal tissue. In the clinic The anatomical snuffbox is an important clinical region. When the hand is in ulnar deviation, the scaphoid becomes palpable within the snuffbox. This position enables the physician to palpate the bone to assess for a fracture. The pulse of the radial artery can also be felt in the snuffbox. In the clinic

1	In the clinic De Quervain’s syndrome is an inflammatory disorder that occurs within the first dorsal extensor compartment and involves the extensor pollicis brevis tendon and abductor pollicis longus tendon and their common tendon sheath (Fig. 7.102). Patients typically present with significant wrist pain preventing appropriate flexion/extension and abduction of the thumb. The cause of this disorder is often overuse. For example, the syndrome is common in young mothers who are constantly lifting young children. Other causes include inflammatory disorders such as rheumatoid arthritis. In the clinic

1	In the clinic Tenosynovitis is inflammation of a tendon and its sheath. The condition may be caused by overuse; however, it can also be associated with other disorders such as rheumatoid arthritis and connective tissue pathologies. If the inflammation becomes severe and ensuing fibrosis occurs, the tendon will not run smoothly within the tendon sheath, and typically within the fingers the tendon may stick or require excess force to fully extend and flex, producing a “triggering” phenomenon. In the clinic Trigger finger is a common disorder of late childhood and adulthood and is typically characterized by catching or snapping and occasionally locking of the flexor tendon(s) in the hand. Trigger finger can be associated with significant dysfunction and pain. The triggering is usually related to fibrosis and tightening of the flexor tendon sheath at the level of the metacarpophalangeal joint. In the clinic

1	In the clinic To test for adequate anastomoses between the radial and ulnar arteries, compress both the radial and ulnar arteries at the wrist, then release pressure from one or the other, and determine the filling pattern of the hand. If there is little connection between the deep and superficial palmar arteries, only the thumb and lateral side of the index finger will fill with blood (become red) when pressure on the radial artery alone is released. In the clinic

1	In the clinic In many patients, venous access is necessary for obtaining blood for laboratory testing and administering fluid and intravenous drugs. The ideal sites for venous access are typically in the cubital fossa and in the cephalic vein adjacent to the anatomical snuffbox. The veins are simply distended by use of a tourniquet. A tourniquet should be applied enough to allow the veins to become prominent. For straightforward blood tests the antecubital vein is usually the preferred site, and although it may not always be visible, it is easily palpated. The cephalic vein is generally the preferred site for a short-term intravenous cannula. In the clinic The ulnar nerve is most commonly injured at two sites: the elbow and the wrist. At the elbow, the nerve lies posterior to the medial epicondyle. At the wrist, the ulnar nerve passes superficial to the flexor retinaculum and lies lateral to the pisiform bone.

1	At the elbow, the nerve lies posterior to the medial epicondyle. At the wrist, the ulnar nerve passes superficial to the flexor retinaculum and lies lateral to the pisiform bone. Ulnar nerve lesions are characterized by “clawing” of the hand, in which the metacarpophalangeal joints of the fingers are hyperextended and the interphalangeal joints are flexed because the function of most of the intrinsic muscles of the hand is lost (Fig. 7.114). Clawing is most pronounced in the medial fingers because the function of all intrinsic muscles of these digits is lost while in the lateral two digits, the lumbricals are innervated by the median nerve. Function of the adductor pollicis muscle is also lost.

1	In lesions of the ulnar nerve at the elbow, function of the flexor carpi ulnaris muscle and flexor digitorum profundus to the medial two digits is lost as well. Clawing of the hand, particularly of the little and ring fingers, is worse with lesions of the ulnar nerve at the wrist than at the elbow because interruption of the nerve at the elbow paralyzes the ulnar half of the flexor digitorum profundus, which leads to lack of flexion at the distal interphalangeal joints in these fingers. Ulnar nerve lesions at the elbow and wrist result in impaired sensory innervation on the palmar aspect of the medial one and one-half digits. Damage to the ulnar nerve at the wrist or at a site proximal to the wrist can be distinguished by evaluating the status of function of the dorsal branch (cutaneous) of the ulnar nerve, which originates in distal regions of the forearm. This branch innervates skin over the dorsal surface of the hand on the medial side. In the clinic

1	In the clinic Around the elbow joint the radial nerve divides into its two terminal branches—the superficial branch and the deep branch. The most common radial nerve injury is damage to the nerve in the radial groove of the humerus, which produces a global paralysis of the muscles of the posterior compartment, resulting in wrist drop. Radial nerve damage can result from fracture of the shaft of the humerus as the radial nerve spirals around in the radial groove. The typical injury produces reduction of sensation in the cutaneous distribution, predominantly over the posterior aspect of the hand. Severing the posterior interosseous nerve (continuation of deep branch of radial nerve) may paralyze the muscles of the posterior compartment of the forearm, but the nerve supply is variable. Typically, the patient may not be able to extend the fingers.

1	The distal branches of the superficial branch of the radial nerve can be readily palpated as “cords” passing over the tendon of the extensor pollicis longus in the anatomical snuffbox. Damage to these branches is of little consequence because they supply only a small area of skin. A 57-year-old woman underwent a right mastectomy for a breast cancer. The surgical note reported that all of the breast tissue had been removed, including the axillary process. In addition, the surgeon had dissected all lymph nodes within the axilla with their surrounding fat. The patient made an uneventful recovery. At the first follow-up appointment, the patient’s husband told the surgeon that she had now developed a bony “spike” on her back. The surgeon was intrigued and asked the patient to reveal this spike. At examination, the spike was the inferior angle of the scapula, which appeared to be sticking out posteriorly (“winged”). Raising the arms accentuated this structure.

1	The medial border of the scapula was accentuated and it was noted that there was some loss of bulk of the serratus anterior muscle, which attaches to the tip of the scapula. The nerve to this muscle was damaged. During the surgery on the axilla, the long thoracic nerve was damaged as it passed down the lateral thoracic wall on the external surface of the serratus anterior, just deep to the skin and subcutaneous fascia. Because the nerve was transected, it is unlikely that the patient will improve, but she was happy that she had an adequate explanation for the spike. A 25-year-old woman was involved in a motor vehicle accident and thrown from her motorcycle. When she was admitted to the emergency room, she was unconscious. A series of tests and investigations were performed, one of which included chest radiography. The attending physician noted a complex fracture of the first rib on the left. Many important structures that supply the upper limb pass over rib I.

1	Many important structures that supply the upper limb pass over rib I. It is important to test the nerves that supply the arm and hand, although this is extremely difficult to do in an unconscious patient. However, some muscle reflexes can be determined using a tendon hammer. Also, it may be possible to test for pain reflexes in patients with altered consciousness levels. Palpation of the axillary artery, brachial artery, radial artery, and ulnar artery pulses is necessary because a fracture of the first rib can sever and denude the subclavian artery, which passes over it. A chest drain was immediately inserted because the lung had collapsed. The fractured first rib had damaged the visceral and parietal pleurae, allowing air from a torn lung to escape into the pleural cavity. The lung collapsed, and the pleural cavity filled with air, which impaired lung function. A tube was inserted between the ribs, and the air was sucked out to re-inflate the lung.

1	A tube was inserted between the ribs, and the air was sucked out to re-inflate the lung. The first rib is a deep structure at the base of the neck. It is not uncommon for ribs to be broken after minor injuries, including sports injuries. However, rib I, which lies at the base of the neck, is surrounded by muscles and soft tissues that provide it with considerable protection. Therefore a patient with a fracture of the first rib has undoubtedly been subjected to a considerable force, which usually occurs in a deceleration injury. Other injuries should always be sought and the patient should be managed with a high level of concern for deep neck and mediastinal injuries. A resident was asked to carry out a clinical assessment of a patient’s hand. He examined the following:

1	A resident was asked to carry out a clinical assessment of a patient’s hand. He examined the following: The musculoskeletal system includes the bones, joints, muscles, and tendons. The resident looked for abnormalities and muscle wasting. Knowing which areas are wasted identifies the nerve that supplies them. She palpated the individual bones and palpated the scaphoid with the wrist in ulnar deviation. She examined the movement of joints because they may be restricted by joint disease or inability of muscular contraction. Palpation of both radial and ulnar pulses is necessary. The resident looked for capillary return to assess how well the hand was perfused. Examination of the nerves The three main nerves to the hand should be tested.

1	Examination of the nerves The three main nerves to the hand should be tested. The median nerve innervates the skin on the palmar aspect of the lateral three and one-half digits, the dorsal aspect of the distal phalanx, half of the middle phalanges of the same fingers, and a variable amount on the radial side of the palm of the hand. Median nerve damage results in wasting of the thenar eminence, absence of abduction of the thumb, and absence of opposition of the thumb. The ulnar nerve innervates the skin of the anterior and posterior surfaces of the little finger and the ulnar side of the ring finger, the skin over the hypothenar eminence, and a similar strip of skin posteriorly. Sometimes the ulnar nerve innervates all the skin of the ring finger and the ulnar side of the middle finger.

1	An ulnar nerve palsy results in wasting of the hypothenar eminence, absent flexion of the distal interphalangeal joints of the little and ring fingers, and absent abduction and adduction of the fingers. Adduction of the thumb also is affected. The radial nerve innervates a small area of skin over the lateral aspect of metacarpal I and the back of the first web space. The radial nerve also produces extension of the wrist and extension of the metacarpophalangeal and interphalangeal joints and of the digits. A very simple examination would include tests for the median nerve by opposition of the thumb, for the ulnar nerve by abduction and adduction of the digits, and for the radial nerve by extension of the wrist and fingers and feeling on the back of the first web space.

1	A 45-year-old man came to his physician complaining of pain and weakness in his right shoulder. The pain began after a fall on his outstretched hand approximately 6 months previously. The patient recalled having some minor shoulder tenderness but no other specific symptoms. He was otherwise fit and well. On examination of the shoulder, there was marked wasting of the muscles in the supraspinous and infraspinous fossae. The patient found initiation of abduction difficult and there was a weakness of lateral rotation of the humerus. The wasted muscles were the supraspinatus and infraspinatus. The cause of the muscle wasting was disuse. Muscle atrophy (wasting) occurs through a variety of disorders. Disuse atrophy is one of the most common causes. Examples of disuse atrophy include the loss of muscle bulk after fracture immobilization in a plaster cast. The opposite effect can also be demonstrated—when muscles are overused they become bulkier (hypertrophy).

1	The supraspinatus and infraspinatus muscles are supplied by the suprascapular nerve (C5, C6), which originates from the superior trunk of the brachial plexus. Given that only these muscles were involved, it is highly likely that the muscle atrophy is caused by denervation. Denervation may result from a direct nerve transection, nerve compression, or a pharmacological effect on the nerve. The typical site for compression of the suprascapular nerve is the suprascapular notch (foramen) on the superior margin of the scapula. The patient’s apparently minor injury damaged the fibrocartilaginous glenoid labrum, which allowed a cyst to form and pass along the superior border of the scapula to the suprascapular notch (foramen), where the cyst compressed the suprascapular nerve. Surgical excision of the damaged glenoid labrum and removal of the cyst improved the patient’s symptoms.

1	Surgical excision of the damaged glenoid labrum and removal of the cyst improved the patient’s symptoms. A surgeon wished to carry out a complex procedure on a patient’s wrist, and asked the anesthesiologist whether the whole arm could be numbed while the patient was awake. Within 20 minutes the anesthesiologist had carried out the procedure after injecting 10 mL of local anesthetic into the axilla. The surgeon went ahead with the operation and the patient did not feel a thing. The anesthetic was injected into the axillary sheath. It would be almost impossible to anesthetize the wrist in the forearm because local anesthetic would have to be placed accurately around the ulnar, median, and radial nerves. Furthermore, all of the cutaneous branches of the forearm would also have to be anesthetized individually, which would take a considerable amount of time and probably produce subtotal anesthesia.

1	The nerves of the upper limb originate from the brachial plexus, which surrounds the axillary artery within the axilla. Importantly, the axillary artery, axillary vein, and brachial plexus lie within the sleeve-like covering of fascia termed the axillary sheath. By injecting the anesthetic into the space enclosed by the axillary sheath, all of the nerves of the brachial plexus were paralyzed. It is possible with a patient’s arm abducted and externally rotated (palm behind the head) to easily palpate the axillary artery and therefore locate the position of the axillary sheath. Once the axillary artery has been identified, a small needle can be placed beside the vessel and local anesthetic can be injected on both sides of it. The local anesthetic tracks along the axillary sheath in this region. The brachial plexus surrounding the axillary artery is therefore completely anesthetized and an effective local anesthetic “block” is achieved.

1	“Could there be any complications?” asks the patient. Potential complications are a direct needle spike of the branches of the brachial plexus, damage to the axillary artery, and inadvertent arterial injection of the local anesthetic. Fortunately, these are rare in skilled hands. A 35-year-old woman comes to her physician complaining of tingling and numbness in the fingertips of the first, second, and third digits (thumb, index, and middle fingers). The symptoms were provoked by arm extension. Local anesthesia was also present around the base of the thenar eminence. The problem was diagnosed as median nerve compression.

1	The problem was diagnosed as median nerve compression. The median nerve is formed from the lateral and medial cords of the brachial plexus anterior to the axillary artery and passes into the arm anterior to the brachial artery. At the level of the elbow joint it sits medial to the brachial artery, both of which are medial to the tendon of the biceps. In the forearm the nerve courses through the anterior compartment and passes deep to the flexor retinaculum. It innervates most of the muscles of the forearm, the thenar muscles, the two lateral lumbricals, and the skin over the palmar surface of the lateral three and one-half digits and over the lateral side of the palm and the middle of the wrist. In this patient, the median nerve initially was believed to be trapped below the flexor retinaculum (carpal tunnel syndrome).

1	In this patient, the median nerve initially was believed to be trapped below the flexor retinaculum (carpal tunnel syndrome). Carpal tunnel syndrome is a common problem in young to middle-aged patients. Typically the nerve becomes compressed within the carpal tunnel. This syndrome may be associated with a number of medical conditions, such as thyroid disease and pregnancy. Occasionally a small ganglion or a tumor situated within the carpal tunnel can also compress the nerve. Other possibilities include tenosynovitis in patients with rheumatoid arthritis.

1	Nerve conduction studies were performed to confirm the clinical findings. Nerve conduction studies are a series of tests that send small electrical impulses along the length of a variety of nerves in order to measure the speed at which the nerve conducts these pulses. The speed of the nerve pulse can be measured and is referred to as the latency. In our patient it was noted that the nerve had normal latency to the elbow joint; however, below the elbow joint there was increased latency. The nerve conduction studies indicated that the compression site was at the elbow joint.

1	The nerve conduction studies indicated that the compression site was at the elbow joint. The clinical findings are not consistent with carpal tunnel syndrome. The clinician should have been alerted to this problem given that the patient experienced numbness over the thenar eminence of the hand. This clue indicates an understanding of the anatomy. Compression of the nerve within the carpal tunnel does not produce this numbness, because the small cutaneous branch that supplies this region is proximal to the flexor retinaculum. The nerve compromise was caused by the ligament of Struthers, which is an embryological remnant of the coracobrachialis muscle. It is an extremely rare finding. Occasionally it may ossify and cross the nerve, artery, and vein to produce compression in arm extension. Although this is very rare and unusual, it illustrates the complex course of the median nerve.

1	After a hard day’s studying, two medical students decided to meet for coffee. The more senior student said to the freshman that he would bet him $50 that he could not lift a matchbook with a finger. The freshman placed $50 on the table and the bet was on. The senior medical student told the freshman to make a fist and place it in a palm-downward position, so that the middle phalanges of the fingers were in direct contact with the bar counter. He was then told to extend his middle finger so that it stuck forward while maintaining the middle phalanges of the index finger, the ring finger, and the little finger on the bar surface. A matchbook was placed on top of the freshman’s middle fingernail and he was told to flip it. He couldn’t. He lost the $50. Extension of the index, middle, ring, and little fingers is performed by the extensor digitorum muscle.

1	Extension of the index, middle, ring, and little fingers is performed by the extensor digitorum muscle. Placing the fist in a palm-down position on the table and pressing the middle phalanges onto the table effectively immobilizes the action of the extensor digitorum. The freshman was therefore unable to elevate his middle finger (which was sticking out). It is important to remember that if this same procedure is carried out leaving the index or little finger free to move, they do. This is because these two digits are extended not only by the extensor digitorum muscle but also by the extensor indicis muscle (index finger) and extensor digiti minimi muscle (little finger). A 70-year-old woman came to an orthopedic surgeon with right shoulder pain and failure to initiate abduction of the shoulder. Further examination revealed loss of muscle bulk in the supraspinous fossa. The supraspinatus muscle was damaged.

1	Abduction of the humerus at the glenohumeral joint is initiated by the supraspinatus muscle. After the shoulder has been abducted to 10°–15°, the deltoid muscle continues the movement. The patient was able to abduct her arm by lowering and tilting the glenohumeral joint inferiorly to enable the deltoid to obtain its mechanical advantage. The loss of muscle bulk in the supraspinous fossa suggested muscle atrophy. Muscle atrophy occurs when a muscle is not used. The orthopedic surgeon thought that there was a tear of the supraspinatus tendon beneath the acromion. If this was so, the muscle would atrophy. The diagnosis was confirmed by ultrasound scan.

1	The diagnosis was confirmed by ultrasound scan. The patient was seated on a stool and her right shoulder was uncovered. The patient’s hand was placed over her right buttock, a position that acts to externally rotate and extend the shoulder, exposing the supraspinatus tendon for ultrasound scan examination. The ultrasound revealed a completely torn tendon with fluid in the subacromial subdeltoid bursa (eFig. 7.128). The patient underwent a surgical repair and made a good recovery. A 35-year-old baseball pitcher came to the clinic with a history of a recurrent dislocation of the shoulder (eFig. 7.129). An MRI scan was performed to assess the shoulder joint prior to any treatment. The MRI demonstrates the anatomical structures in multiple planes, allowing the physician to obtain an overview of the shoulder and to assess any intraarticular or extraarticular structures that may have been damaged and require surgical repair.

1	The MRI demonstrated a divot in the posterosuperior aspect of the humeral head and a small fragment of bone and glenoid labrum that had become separated in the anteroinferior aspect of the glenoid cavity. Shoulder dislocation is not an uncommon problem and may occur as a “once-off” or with repetitive injury may be recurrent. Recurrent dislocations may be bilateral and symmetrical (a memory aid is “torn loose or born loose”).

1	The MRI findings are typical for an anteroinferior dislocation, which is the most common type; moreover the MRI demonstrates the injuries that occur within the joint at the time of dislocation. These injuries include the abutment of the posterosuperior aspect of the humeral head on the anteroinferior aspect of the glenoid cavity. This type of injury, when recurrent, may avulse a small fragment of the glenoid labrum, and in some cases this may attach to a small fragment of bone (the Bankart lesion). When the shoulder is relocated, the integrity of the capsular attachment anteroinferiorly has been disrupted, potentially making the shoulder somewhat prone to further dislocation. An arthroscopic repair was performed.

1	An arthroscopic repair was performed. Arthroscopy of the shoulder is an established method for assessing the shoulder joint. Portals of entry are anterior and posterior and small holes in the capsule are made percutaneously. The shoulder joint is filled with saline, which distends it, allowing the arthroscope to move around the joint and inspect the joint surfaces, including the labrum. The labrum and its bony fragment were reattached and sutured using anchor sutures (somewhat similar to staples). The anterior aspect of the capsule was also tightened. The patient made an uneventful recovery. After the procedure the arm was held in internal rotation and remained adducted. Gentle exercise and physiotherapy were performed and the patient returned to playing baseball. 821.e1 821.e2 Conceptual Overview • Relationship to Other Regions Fig. 7.40, cont’d

1	821.e1 821.e2 Conceptual Overview • Relationship to Other Regions Fig. 7.40, cont’d Table 7.7 Branches of brachial plexus (parentheses indicate that a spinal segment is a minor component of the nerve or is inconsistently present in the nerve)—cont’d Fig. 7.63, cont’d Fig. 7.66, cont’d Regional Anatomy • Anterior Compartment of the Forearm Regional Anatomy • Anterior Compartment of the Forearm Regional Anatomy • Anterior Compartment of the Forearm Regional Anatomy • Anterior Compartment of the Forearm Regional Anatomy • Posterior Compartment of the Forearm Regional Anatomy • Posterior Compartment of the Forearm Regional Anatomy • Posterior Compartment of the Forearm Regional Anatomy • Posterior Compartment of the Forearm Fig. 7.94, cont’d Surface Anatomy • Visualizing the Axilla and Locating Contents and Related Structures Surface Anatomy • Identifying Tendons and Locating Major Vessels and Nerves in the Distal Forearm

1	Surface Anatomy • Visualizing the Axilla and Locating Contents and Related Structures Surface Anatomy • Identifying Tendons and Locating Major Vessels and Nerves in the Distal Forearm Surface Anatomy • Motor Function of the Median and Ulnar Nerves in the Hand Arrangement of meninges and spaces 865 Anterior triangle of the neck 995 Posterior triangle of the neck 1012 Root of the neck 1019 Gaps in the pharyngeal wall and structures passing through them 1035 Cavity of the larynx 1048 Function of the larynx 1053 Walls, floor, and roof 1065 Multiple nerves innervate the oral cavity 1077 Walls: the cheeks 1080 Anatomical position of the head and major landmarks 1110 Visualizing structures at the CIII/CIV and CVI vertebral levels 1111 How to outline the anterior and posterior triangles of the neck 1112 How to locate the cricothyroid ligament 1113 How to find the thyroid gland 1114 Estimating the position of the middle meningeal artery 1114

1	How to locate the cricothyroid ligament 1113 How to find the thyroid gland 1114 Estimating the position of the middle meningeal artery 1114 Major features of the face 1115 The eye and lacrimal apparatus 1116 The head and neck are anatomically complex areas of the body. The head is composed of a series of compartments, which are formed by bone and soft tissues. They are: the cranial cavity, two ears, two orbits, two nasal cavities, and an oral cavity (Fig. 8.1). The cranial cavity is the largest compartment and contains the brain and associated membranes (meninges). Most of the ear apparatus on each side is contained within one of the bones forming the floor of the cranial cavity. The external parts of the ears extend laterally from these regions.

1	Most of the ear apparatus on each side is contained within one of the bones forming the floor of the cranial cavity. The external parts of the ears extend laterally from these regions. The two orbits contain the eyes. They are cone-shaped chambers immediately inferior to the anterior aspect of the cranial cavity, and the apex of each cone is directed posteromedially. The walls of the orbits are bone, whereas the base of each conical chamber can be opened and closed by the eyelids. The nasal cavities are the upper parts of the respiratory tract and are between the orbits. They have walls, floors, and ceilings, which are predominantly composed of bone and cartilage. The anterior openings to the nasal cavities are nares (nostrils), and the posterior openings are choanae (posterior nasal apertures). Continuous with the nasal cavities are air-filled extensions (paranasal sinuses), which project laterally, superiorly, and posteriorly into surrounding bones.

1	Continuous with the nasal cavities are air-filled extensions (paranasal sinuses), which project laterally, superiorly, and posteriorly into surrounding bones. The largest, the maxillary sinuses, are inferior to the orbits. The oral cavity is inferior to the nasal cavities, and separated from them by the hard and soft palates. The floor of the oral cavity is formed entirely of soft tissues. The anterior opening to the oral cavity is the oral fissure (mouth), and the posterior opening is the oropharyngeal isthmus. Unlike the nares and choanae, which are continuously open, both the oral fissure and oropharyngeal isthmus can be opened and closed by surrounding soft tissues.

1	In addition to the major compartments of the head, two other anatomically defined regions (infratemporal fossa and pterygopalatine fossa) of the head on each side are areas of transition from one compartment of the head to another (Fig. 8.2). The face and scalp also are anatomically defined areas of the head and are related to external surfaces. The infratemporal fossa is an area between the posterior aspect (ramus) of the mandible and a flat region of bone (lateral plate of the pterygoid process) just posterior to the upper jaw (maxilla). This fossa, bounded by bone and soft tissues, is a conduit for one of the major cranial nerves—the mandibular nerve (the mandibular division of the trigeminal nerve [V3]), which passes between the cranial and oral cavities.

1	The pterygopalatine fossa on each side is just posterior to the upper jaw. This small fossa communicates with the cranial cavity, the infratemporal fossa, the orbit, the nasal cavity, and the oral cavity. A major structure passing through the pterygopalatine fossa is the maxillary nerve (the maxillary division of the trigeminal nerve [V2]). The face is the anterior aspect of the head and contains a unique group of muscles that move the skin relative to underlying bone and control the anterior openings to the orbits and oral cavity (Fig. 8.3). The scalp covers the superior, posterior, and lateral regions of the head (Fig. 8.3). The neck extends from the head above to the shoulders and thorax below (Fig. 8.4). Its superior boundary is along the inferior margins of the mandible and bone features on the posterior aspect of the skull. The posterior neck is higher than the anterior neck to connect cervical viscera with the posterior openings of the nasal and oral cavities.

1	The inferior boundary of the neck extends from the top of the sternum, along the clavicle, and onto the adjacent acromion, a bony projection of the scapula. Posteriorly, the inferior limit of the neck is less well defined, but can be approximated by a line between the acromion and the spinous process of vertebra CVII, which is prominent and easily palpable. The inferior border of the neck encloses the base of the neck. The neck has four major compartments (Fig. 8.5), which are enclosed by an outer musculofascial collar: The vertebral compartment contains the cervical vertebrae and associated postural muscles. The visceral compartment contains important glands (thyroid, parathyroid, and thymus), and parts of the respiratory and digestive tracts that pass between the head and thorax. The two vascular compartments, one on each side, contain the major blood vessels and the vagus nerve.

1	The two vascular compartments, one on each side, contain the major blood vessels and the vagus nerve. The neck contains two specialized structures associated with the digestive and respiratory tracts—the larynx and pharynx. The larynx (Fig. 8.6) is the upper part of the lower airway and is attached below to the top of the trachea and above, by a flexible membrane, to the hyoid bone, which in turn is attached to the floor of the oral cavity. A number of cartilages form a supportive framework for the larynx, which has a hollow central channel. The dimensions of this central channel can be adjusted by soft tissue structures associated with the laryngeal wall. The most important of these are two lateral vocal folds, which project toward each other from adjacent sides of the laryngeal cavity. The upper opening of the larynx (laryngeal inlet) is tilted posteriorly, and is continuous with the pharynx.

1	The pharynx (Fig. 8.6) is a chamber in the shape of a half-cylinder with walls formed by muscles and fascia. Above, the walls are attached to the base of the skull, and below to the margins of the esophagus. On each side, the walls are attached to the lateral margins of the nasal cavities, the oral cavity, and the larynx. The two nasal cavities, the oral cavity, and the larynx therefore open into the anterior aspect of the pharynx, and the esophagus opens inferiorly. The part of the pharynx posterior to the nasal cavities is the nasopharynx. Those parts posterior to the oral cavity and larynx are the oropharynx and laryngopharynx, respectively. The head houses and protects the brain and all the receptor systems associated with the special senses—the nasal cavities associated with smell, the orbits with vision, the ears with hearing and balance, and the oral cavity with taste. Contains upper parts of respiratory

1	Contains upper parts of respiratory The head contains the upper parts of the respiratory and digestive systems—the nasal and oral cavities—which have structural features for modifying the air or food passing into each system. The head and neck are involved in communication. Sounds produced by the larynx are modified in the pharynx and oral cavity to produce speech. In addition, the muscles of facial expression adjust the contours of the face to relay nonverbal signals. Positioning the head The neck supports and positions the head. Importantly, it enables an individual to position sensory systems in the head relative to environmental cues without moving the entire body. Connects the upper and lower respiratory and digestive tracts

1	Connects the upper and lower respiratory and digestive tracts The neck contains specialized structures (pharynx and larynx) that connect the upper parts of the digestive and respiratory tracts (nasal and oral cavities) in the head, with the esophagus and trachea, which begin relatively low in the neck and pass into the thorax. The many bones of the head collectively form the skull (Fig. 8.7A). Most of these bones are interconnected by sutures, which are immovable fibrous joints (Fig. 8.7B). In the fetus and newborn, large membranous and unossified gaps (fontanelles) between the bones of the skull, particularly between the large flat bones that cover the top of the cranial cavity (Fig. 8.7C), allow: the head to deform during its passage through the birth canal, and postnatal growth.

1	Most of the fontanelles close during the first year of life. Full ossification of the thin connective tissue ligaments separating the bones at the suture lines begins in the late twenties, and is normally completed in the fifth decade of life. There are only three pairs of synovial joints on each side in the head. The largest are the temporomandibular joints between the lower jaw (mandible) and the temporal bone. The other two synovial joints are between the three tiny bones in the middle ear, the malleus, incus, and stapes. The seven cervical vertebrae form the bony framework of the neck. Cervical vertebrae (Fig. 8.8A) are characterized by: small bodies, bifid spinous processes, and transverse processes that contain a foramen (foramen transversarium). Together the foramina transversaria form a longitudinal passage on each side of the cervical vertebral column for blood vessels (vertebral artery and veins) passing between the base of the neck and the cranial cavity.

1	The typical transverse process of a cervical vertebra also has anterior and posterior tubercles for muscle attachment. The anterior tubercles are derived from the same embryological elements that give rise to ribs in the thoracic region. Occasionally, cervical ribs develop from these elements, particularly in association with the lower cervical vertebrae. The upper two cervical vertebrae (CI and CII) are modified for moving the head (Fig. 8.8B–E; see also Chapter 2). The hyoid bone is a small U-shaped bone (Fig. 8.9A) oriented in the horizontal plane just superior to the larynx, where it can be palpated and moved from side to side. The body of the hyoid bone is anterior and forms the base of the U. The two arms of the U (greater horns) project posteriorly from the lateral ends of the body. The hyoid bone does not articulate directly with any other skeletal elements in the head and neck.

1	The two arms of the U (greater horns) project posteriorly from the lateral ends of the body. The hyoid bone does not articulate directly with any other skeletal elements in the head and neck. The hyoid bone is a highly movable and strong bony anchor for a number of muscles and soft tissue structures in the head and neck. Significantly, it is at the interface between three dynamic compartments: Superiorly, it is attached to the floor of the oral cavity. Inferiorly, it is attached to the larynx. Posteriorly, it is attached to the pharynx (Fig. 8.9B). The soft palate is a soft tissue flap-like structure “hinged” to the back of the hard palate (Fig. 8.10A) with a free posterior margin. It can be elevated and depressed by muscles (Fig. 8.10B). The soft palate and associated structures can be clearly seen through an open mouth. The skeletal muscles of the head and neck can be grouped on the basis of function, innervation, and embryological derivation. In the head

1	The skeletal muscles of the head and neck can be grouped on the basis of function, innervation, and embryological derivation. In the head The muscle groups in the head include: the extra-ocular muscles (move the eyeball and open the upper eyelid), muscles of the middle ear (adjust the movement of the middle ear bones), muscles of facial expression (move the face), muscles of mastication (move the jaw—temporo- mandibular joint), muscles of the soft palate (elevate and depress the palate), and muscles of the tongue (move and change the contour of the tongue). In the neck In the neck, major muscle groups include: muscles of the pharynx (constrict and elevate the pharynx), muscles of the larynx (adjust the dimensions of the air pathway), strap muscles (position the larynx and hyoid bone in the neck), muscles of the outer cervical collar (move the head and upper limb), and postural muscles in the muscular compartment of the neck (position the neck and head).

1	The superior thoracic aperture (thoracic inlet) opens directly into the base of the neck (Fig. 8.11). Structures passing between the head and thorax pass up and down through the superior thoracic aperture and the visceral compartment of the neck. At the base of the neck, the trachea is immediately anterior to the esophagus, which is directly anterior to the vertebral column. There are major veins, arteries, and nerves anterior and lateral to the trachea. There is an axillary inlet (gateway to the upper limb) on each side of the superior thoracic aperture at the base of the neck (Fig. 8.11): Structures such as blood vessels pass over rib I when passing between the axillary inlet and thorax. Cervical components of the brachial plexus pass directly from the neck through the axillary inlets to enter the upper limb. In the neck, the two important vertebral levels (Fig.

1	Cervical components of the brachial plexus pass directly from the neck through the axillary inlets to enter the upper limb. In the neck, the two important vertebral levels (Fig. 8.12) are: between CIII and CIV, at approximately the superior border of the thyroid cartilage of the larynx (which can be palpated) and where the major artery on each side of the neck (the common carotid artery) bifurcates into internal and external carotid arteries; and between CV and CVI, which marks the lower limit of the pharynx and larynx, and the superior limit of the trachea and esophagus—the indentation between the cricoid cartilage of the larynx and the first tracheal ring can be palpated. The internal carotid artery has no branches in the neck and ascends into the skull to supply much of the brain. It also supplies the eye and orbit. Other regions of the head and neck are supplied by branches of the external carotid artery. Airway in the neck

1	Airway in the neck The larynx (Fig. 8.13) and the trachea are anterior to the digestive tract in the neck, and can be accessed directly when upper parts of the system are blocked. A cricothyrotomy makes use of the easiest route of access through the cricothyroid ligament (cricovocal membrane, cricothyroid membrane) between the cricoid and thyroid cartilages of the larynx. The ligament can be palpated in the midline, and usually there are only small blood vessels, connective tissue, and skin (though occasionally, a small lobe of the thyroid gland—pyramidal lobe) overlying it. At a lower level, the airway can be accessed surgically through the anterior wall of the trachea by tracheostomy. This route of entry is complicated because large veins and part of the thyroid gland overlie this region. There are twelve pairs of cranial nerves and their defining feature is that they exit the cranial cavity through foramina or fissures.

1	There are twelve pairs of cranial nerves and their defining feature is that they exit the cranial cavity through foramina or fissures. All cranial nerves innervate structures in the head or neck. In addition, the vagus nerve [X] descends through the neck and into the thorax and abdomen where it innervates viscera. Parasympathetic fibers in the head are carried out of the brain as part of four cranial nerves—the oculomotor nerve [III], the facial nerve [VII], the glossopharyngeal nerve [IX], and the vagus nerve [X] (Fig. 8.14). Parasympathetic fibers in the oculomotor nerve [III], the facial nerve [VII], and the glossopharyngeal nerve [IX] destined for target tissues in the head leave these nerves, and are distributed with branches of the trigeminal nerve [V]. The vagus nerve [X] leaves the head and neck to deliver parasympathetic fibers to the thoracic and abdominal viscera. There are eight cervical nerves (C1 to C8):

1	The vagus nerve [X] leaves the head and neck to deliver parasympathetic fibers to the thoracic and abdominal viscera. There are eight cervical nerves (C1 to C8): C1 to C7 emerge from the vertebral canal above their respective vertebrae. C8 emerges between vertebrae CVII and TI (Fig. 8.15A). The anterior rami of C1 to C4 form the cervical plexus. The major branches from this plexus supply the strap muscles, the diaphragm (phrenic nerve), skin on the anterior and lateral parts of the neck, skin on the upper anterior thoracic wall, and skin on the inferior parts of the head (Fig. 8.15B). The anterior rami of C5 to C8, together with a large component of the anterior ramus of T1, form the brachial plexus, which innervates the upper limb. Functional separation of the digestive

1	The anterior rami of C5 to C8, together with a large component of the anterior ramus of T1, form the brachial plexus, which innervates the upper limb. Functional separation of the digestive The pharynx is a common chamber for the digestive and respiratory tracts. Consequently, breathing can take place through the mouth as well as through the nose, and material from the oral cavity can potentially enter either the esophagus or the larynx. Importantly: The lower airway can be accessed through the oral cavity by intubation. The digestive tract (esophagus) can be accessed through the nasal cavity by feeding tubes. Normally, the soft palate, epiglottis, and soft tissue structures within the larynx act as valves to prevent food and liquid from entering lower parts of the respiratory tract (Fig. 8.16A).

1	Normally, the soft palate, epiglottis, and soft tissue structures within the larynx act as valves to prevent food and liquid from entering lower parts of the respiratory tract (Fig. 8.16A). During normal breathing, the airway is open and air passes freely through the nasal cavities (or oral cavity), pharynx, larynx, and trachea (Fig. 8.16A). The lumen of the esophagus is normally closed because, unlike the airway, it has no skeletal support structures to hold it open. When the oral cavity is full of liquid or food, the soft palate is swung down (depressed) to close the oropharyngeal isthmus, thereby allowing manipulation of food and fluid in the oral cavity while breathing (Fig. 8.16C). When swallowing, the soft palate and parts of the larynx act as valves to ensure proper movement of food from the oral cavity into the esophagus (Fig. 8.16D).

1	When swallowing, the soft palate and parts of the larynx act as valves to ensure proper movement of food from the oral cavity into the esophagus (Fig. 8.16D). The soft palate elevates to open the oropharyngeal isthmus while at the same time sealing off the nasal part of the pharynx from the oral part. This prevents food and fluid from moving upward into the nasopharynx and nasal cavities. The epiglottis of the larynx closes the laryngeal inlet and much of the laryngeal cavity becomes occluded by opposition of the vocal folds and soft tissue folds superior to them. In addition, the larynx is pulled up and forward to facilitate the moving of food and fluid over and around the closed larynx and into the esophagus.

1	In newborns, the larynx is high in the neck and the epiglottis is above the level of the soft palate (Fig. 8.16E). Babies can therefore suckle and breathe at the same time. Liquid flows around the larynx without any danger of entering the airway. During the second year of life, the larynx descends into the low cervical position characteristic of adults. Triangles of the neck The two muscles (trapezius and sternocleidomastoid) that form part of the outer cervical collar divide the neck into anterior and posterior triangles on each side (Fig. 8.17). The boundaries of each anterior triangle are: the median vertical line of the neck, the inferior margin of the mandible, and the anterior margin of the sternocleidomastoid muscle. The posterior triangle is bounded by: the middle one-third of the clavicle, the anterior margin of the trapezius, and the posterior margin of the sternocleidomastoid.

1	The posterior triangle is bounded by: the middle one-third of the clavicle, the anterior margin of the trapezius, and the posterior margin of the sternocleidomastoid. Major structures that pass between the head and thorax can be accessed through the anterior triangle. The posterior triangle in part lies over the axillary inlet, and is associated with structures (nerves and vessels) that pass into and out of the upper limb. The skull has 22 bones, excluding the ossicles of the ear. Except for the mandible, which forms the lower jaw, the bones of the skull are attached to each other by sutures, are immobile, and form the cranium. The cranium can be subdivided into: an upper domed part (the calvaria), which covers the cranial cavity containing the brain, a base that consists of the floor of the cranial cavity, and a lower anterior part—the facial skeleton (viscerocranium).

1	The bones forming the calvaria are mainly the paired temporal and parietal bones, and parts of the unpaired frontal, sphenoid, and occipital bones. The bones forming the base of the cranium are mainly parts of the sphenoid, temporal, and occipital bones. The bones forming the facial skeleton are the paired nasal bones, palatine bones, lacrimal bones, zygomatic bones, maxillae and inferior nasal conchae and the unpaired vomer. The mandible is not part of the cranium nor part of the facial skeleton. The anterior view of the skull includes the forehead superiorly, and, inferiorly, the orbits, the nasal region, the part of the face between the orbit and the upper jaw, the upper jaw, and the lower jaw (Fig. 8.18). The forehead consists of the frontal bone, which also forms the superior part of the rim of each orbit (Fig. 8.18).

1	The forehead consists of the frontal bone, which also forms the superior part of the rim of each orbit (Fig. 8.18). Just superior to the rim of the orbit on each side are the raised superciliary arches. These are more pronounced in men than in women. Between these arches is a small depression (the glabella). Clearly visible in the medial part of the superior rim of each orbit is the supra-orbital foramen (supra-orbital notch; Table 8.1). Medially, the frontal bone projects inferiorly forming a part of the medial rim of the orbit. Laterally, the zygomatic process of the frontal bone projects inferiorly forming the upper lateral rim of the orbit. This process articulates with the frontal process of the zygomatic bone. The lower lateral rim of the orbit, as well as the lateral part of the inferior rim of the orbit is formed by the zygomatic bone (the cheekbone).

1	The lower lateral rim of the orbit, as well as the lateral part of the inferior rim of the orbit is formed by the zygomatic bone (the cheekbone). Superiorly, in the nasal region the paired nasal bones articulate with each other in the midline, and with the frontal bone superiorly. The center of the frontonasal suture formed by the articulation of the nasal bones and the frontal bone is the nasion. Laterally, each nasal bone articulates with the frontal process of each maxilla. Inferiorly, the piriform aperture is the large opening in the nasal region and the anterior opening of the nasal cavity. It is bounded superiorly by the nasal bones and laterally and inferiorly by each maxilla. Visible through the piriform aperture are the fused nasal crests, forming the lower part of the bony nasal septum and ending anteriorly as the anterior nasal spine, and the paired inferior nasal conchae.

1	The part of the face between the orbit and the upper teeth and each upper jaw is formed by the paired maxillae. Superiorly, each maxilla contributes to the inferior and medial rims of the orbit. Laterally, the zygomatic process of each maxilla articulates with the zygomatic bone and medially, the frontal process of each maxilla articulates with the frontal bone. Inferiorly, the part of each maxilla, lateral to the opening of the nasal cavity, is the body of the maxilla. On the anterior surface of the body of the maxilla, just below the inferior rim of the orbit, is the infra-orbital foramen (Table 8.1). Inferiorly, each maxilla ends as the alveolar process, which contains the teeth and forms the upper jaw.

1	Inferiorly, each maxilla ends as the alveolar process, which contains the teeth and forms the upper jaw. The lower jaw (mandible) is the most inferior structure in the anterior view of the skull. It consists of the body of the mandible anteriorly and the ramus of the mandible posteriorly. These meet posteriorly at the angle of the mandible. All these parts of the mandible are visible, to some extent, in the anterior view. The body of the mandible is arbitrarily divided into two parts: The lower part is the base of the mandible. The upper part is the alveolar part of the mandible. The alveolar part of the mandible contains the teeth and is resorbed when the teeth are removed. The base of the mandible has a midline swelling (the mental protuberance) on its anterior surface where the two sides of the mandible come together. Just lateral to the mental protuberance, on either side, are slightly more pronounced bumps (mental tubercles).

1	Laterally, a mental foramen (Table 8.1) is visible halfway between the upper border of the alveolar part of the mandible and the lower border of the base of the mandible. Continuing past this foramen is a ridge (the oblique line) passing from the front of the ramus onto the body of the mandible. The oblique line is a point of attachment for muscles that depress the lower lip. The lateral view of the skull consists of the lateral wall of the cranium, which includes lateral portions of the calvaria and the facial skeleton, and half of the lower jaw (Fig. 8.19): Bones forming the lateral portion of the calvaria include the frontal, parietal, occipital, sphenoid, and temporal bones. Bones forming the visible part of the facial skeleton include the nasal, maxilla, and zygomatic bones. The mandible forms the visible part of the lower jaw. Lateral portion of the calvaria

1	Bones forming the visible part of the facial skeleton include the nasal, maxilla, and zygomatic bones. The mandible forms the visible part of the lower jaw. Lateral portion of the calvaria The lateral portion of the calvaria begins anteriorly with the frontal bone. In upper regions, the frontal bone articulates with the parietal bone at the coronal suture. The parietal bone then articulates with the occipital bone at the lambdoid suture. In lower parts of the lateral portion of the calvaria, the frontal bone articulates with the greater wing of the sphenoid bone (Fig. 8.19), which then articulates with the parietal bone at the sphenoparietal suture, and with the anterior edge of the temporal bone at the sphenosquamous suture.

1	The junction where the frontal, parietal, sphenoid, and temporal bones are in close proximity is the pterion. The clinical consequences of a skull fracture in this area can be very serious. The bone in this area is particularly thin and overlies the anterior division of the middle meningeal artery, which can be torn by a skull fracture in this area, resulting in an extradural hematoma. The final articulation across the lower part of the lateral portion of the calvaria is between the temporal bone and the occipital bone at the occipitomastoid suture. A major contributor to the lower portion of the lateral wall of the cranium is the temporal bone (Fig. 8.19), which consists of several parts:

1	A major contributor to the lower portion of the lateral wall of the cranium is the temporal bone (Fig. 8.19), which consists of several parts: The squamous part has the appearance of a large flat plate, forms the anterior and superior parts of the temporal bone, contributes to the lateral wall of the cranium, and articulates anteriorly with the greater wing of the sphenoid bone at the sphenosquamous suture, and with the parietal bone superiorly at the squamous suture. The zygomatic process is an anterior bony projection from the lower surface of the squamous part of the temporal bone that initially projects laterally and then curves anteriorly to articulate with the temporal process of the zygomatic bone to form the zygomatic arch.

1	Immediately below the origin of the zygomatic process from the squamous part of the temporal bone is the tympanic part of the temporal bone, and clearly visible on the surface of this part is the external acoustic opening leading to the external acoustic meatus (ear canal). The petromastoid part, which is usually separated into a petrous part and a mastoid part for descriptive purposes. The mastoid part is the most posterior part of the temporal bone, and is the only part of the petromastoid part of the temporal bone seen on a lateral view of the skull. It is continuous with the squamous part of the temporal bone anteriorly, and articulates with the parietal bone superiorly at the parietomastoid suture, and with the occipital bone posteriorly at the occipitomastoid suture. These two sutures are continuous with each other, and the parietomastoid suture is continuous with the squamous suture.

1	Inferiorly, a large bony prominence (the mastoid process) projects from the inferior border of the mastoid part of the temporal bone. This is a point of attachment for several muscles. Medial to the mastoid process, the styloid process projects from the lower border of the temporal bone. Visible part of the facial skeleton The bones of the viscerocranium visible in a lateral view of the skull include the nasal, maxilla, and zygomatic bones (Fig. 8.19) as follows: A nasal bone anteriorly. The maxilla with its alveolar process containing teeth forming the upper jaw; anteriorly, it articulates with the nasal bone; superiorly, it contributes to the formation of the inferior and medial borders of the orbit; medially, its frontal process articulates with the frontal bone; laterally, its zygomatic process articulates with the zygomatic bone.

1	The zygomatic bone, an irregularly shaped bone with a rounded lateral surface that forms the prominence of the cheek, is a visual centerpiece in this view— medially, it assists in the formation of the inferior rim of the orbit through its articulation with the zygomatic process of the maxilla; superiorly, its frontal process articulates with the zygomatic process of the frontal bone assisting in the formation of the lateral rim of the orbit; laterally, seen prominently in this view of the skull, the horizontal temporal process of the zygomatic bone projects backward to articulate with the zygomatic process of the temporal bone and so form the zygomatic arch. Usually a small foramen (the zygomaticofacial foramen; Table 8.1) is visible on the lateral surface of the zygomatic bone. A zygomaticotemporal foramen is present on the medial deep surface of the bone.

1	The final bony structure visible in a lateral view of the skull is the mandible. Inferiorly in the anterior part of this view, it consists of the anterior body of the mandible, a posterior ramus of the mandible, and the angle of the mandible where the inferior margin of the mandible meets the posterior margin of the ramus (Fig. 8.19). The teeth are in the alveolar part of the body of the mandible and the mental protuberance is visible in this view. The mental foramen is on the lateral surface of the body, and on the superior part of the ramus condylar and coronoid processes extend upward. The condylar process is involved in articulation of the mandible with the temporal bone, and the coronoid process is the point of attachment for the temporalis muscle. The occipital, parietal, and temporal bones are seen in the posterior view of the skull.

1	The occipital, parietal, and temporal bones are seen in the posterior view of the skull. Centrally the flat or squamous part of the occipital bone is the main structure in this view of the skull (Fig. 8.20). It articulates superiorly with the paired parietal bones at the lambdoid suture and laterally with each temporal bone at the occipitomastoid sutures. Along the lambdoid suture small islands of bone (sutural bones or wormian bones) may be observed. Several bony landmarks are visible on the occipital bone. There is a midline projection (the external occipital protuberance) with curved lines extending laterally from it (superior nuchal lines). The most prominent point of the external occipital protuberance is the inion. About 1 inch (2.5 cm) below the superior nuchal lines two additional lines (the inferior nuchal lines) curve laterally. Extending downward from the external occipital protuberance is the external occipital crest.

1	Laterally, the temporal bones are visible in the posterior view of the skull, with the mastoid processes being the prominent feature (Fig. 8.20). On the inferomedial border of each mastoid process is a notch (the mastoid notch), which is a point of attachment for the posterior belly of the digastric muscle. The frontal bone, parietal bones, and occipital bone are seen in a superior view of the skull (Fig. 8.21). These bones make up the superior part of the calvaria or the calva (skullcap). In an anterior to posterior direction: The unpaired frontal bone articulates with the paired parietal bones at the coronal suture. The two parietal bones articulate with each other in the midline at the sagittal suture. The parietal bones articulate with the unpaired occipital bone at the lambdoid suture. The junction of the sagittal and coronal sutures is the bregma, and the junction of the sagittal and lambdoid sutures is the lambda.

1	The junction of the sagittal and coronal sutures is the bregma, and the junction of the sagittal and lambdoid sutures is the lambda. The only foramina visible in this view of the skull may be the paired parietal foramina, posteriorly, one on each parietal bone just lateral to the sagittal suture (Fig. 8.21). The bones making up the calvaria (Fig. 8.22) are unique in their structure, consisting of dense internal and external tables of compact bone separated by a layer of spongy bone (the diploë). The base of the skull is seen in the inferior view and extends anteriorly from the middle incisor teeth posteriorly to the superior nuchal lines and laterally to the mastoid processes and zygomatic arches (Fig. 8.23).

1	For descriptive purposes the base of the skull is often divided into: an anterior part, which includes the teeth and the hard palate, a middle part, which extends from behind the hard palate to the anterior margin of the foramen magnum, and a posterior part, which extends from the anterior edge of the foramen magnum to the superior nuchal lines. The main features of the anterior part of the base of the skull are the teeth and the hard palate. The teeth project from the alveolar processes of the two maxillae. These processes are together arranged in a U-shaped alveolar arch that borders the hard palate on three sides (Fig. 8.23). The hard palate is composed of the palatine processes of each maxilla anteriorly and the horizontal plates of each palatine bone posteriorly.

1	The hard palate is composed of the palatine processes of each maxilla anteriorly and the horizontal plates of each palatine bone posteriorly. The paired palatine processes of each maxilla meet in the midline at the intermaxillary suture, the paired maxillae and the paired palatine bones meet at the palatomaxillary suture, and the paired horizontal plates of each palatine bone meet in the midline at the interpalatine suture.

1	Several additional features are also visible when the hard palate is examined: the incisive fossa in the anterior midline immediately posterior to the teeth, the walls of which contain incisive foramina (the openings of the incisive canals, which are passageways between the hard palate and nasal cavity); the greater palatine foramina near the posterolateral border of the hard palate on each side, which lead to greater palatine canals; just posterior to the greater palatine foramina, the lesser palatine foramina in the pyramidal process of each palatine bone, which lead to lesser palatine canals; a midline pointed projection (the posterior nasal spine) in the free posterior border of the hard palate. The middle part of the base of the skull is complex: Forming the anterior half are the vomer and sphenoid bones. Forming the posterior half are the occipital and paired temporal bones.

1	The middle part of the base of the skull is complex: Forming the anterior half are the vomer and sphenoid bones. Forming the posterior half are the occipital and paired temporal bones. Anteriorly, the small vomer is in the midline, resting on the sphenoid bone (Fig. 8.23). It contributes to the formation of the bony nasal septum separating the two choanae. Most of the anterior part of the middle part of the base of the skull consists of the sphenoid bone. The sphenoid bone is made up of a centrally placed body, paired greater and lesser wings projecting laterally from the body, and two downward projecting pterygoid processes immediately lateral to each choana. Three parts of the sphenoid bone, the body, greater wings, and pterygoid processes, are seen in the inferior view of the skull (Fig. 8.23). The lesser wing of the sphenoid is not seen in the inferior view. The body of the sphenoid is a centrally placed cube of bone containing two large air sinuses separated by a septum.

1	The body of the sphenoid is a centrally placed cube of bone containing two large air sinuses separated by a septum. It articulates anteriorly with the vomer, ethmoid, and palatine bones, posterolaterally with the temporal bones, and posteriorly with the occipital bone. Extending downward from the junction of the body and the greater wings are the pterygoid processes (Fig. 8.23). Each of these processes consists of a narrow medial plate and broader lateral plate separated by the pterygoid fossa. Each medial plate of the pterygoid process ends inferiorly with a hook-like projection, the pterygoid hamulus, and divides superiorly to form the small, shallow scaphoid fossa. Just superior to the scaphoid fossa, at the root of the medial plate of the pterygoid process is the opening of the pterygoid canal, which passes forward from near the anterior margin of the foramen lacerum.

1	Lateral to the lateral plate of the pterygoid process is the greater wing of the sphenoid (Fig. 8.23), which not only forms a part of the base of the skull but also continues laterally to form part of the lateral wall of the skull. It articulates laterally and posteriorly with parts of the temporal bone. Important features visible on the surface of the greater wing in an inferior view of the skull are the foramen ovale and the foramen spinosum on the posterolateral border extending outward from the upper end of the lateral plate of the pterygoid process. In the posterior half of the middle part of the base of the skull are the occipital bone and the paired temporal bones (Fig. 8.23). The occipital bone, or more specifically its basilar part, is in the midline immediately posterior to the body of the sphenoid. It extends posteriorly to the foramen magnum and is bounded laterally by the temporal bones.

1	Prominent on the basilar part of the occipital bone is the pharyngeal tubercle, a bony protuberance for the attachment of parts of the pharynx to the base of the skull (Fig. 8.23). Immediately lateral to the basilar part of the occipital bone is the petrous part of the petromastoid part of each temporal bone. Wedge-shaped in its appearance, with its apex anteromedial, the petrous part of the temporal bone is between the greater wing of the sphenoid anteriorly and the basilar part of the occipital bone posteriorly. The apex forms one of the boundaries of the foramen lacerum, an irregular opening filled in life with cartilage (Fig. 8.23). The other boundaries of the foramen lacerum are the basilar part of the occipital bone medially and the body of the sphenoid anteriorly. Posterolateral from the foramen lacerum along the petrous part of the temporal bone is the large circular opening for the carotid canal.

1	Posterolateral from the foramen lacerum along the petrous part of the temporal bone is the large circular opening for the carotid canal. Between the petrous part of the temporal bone and the greater wing of the sphenoid is a groove for the cartilaginous part of the pharyngotympanic tube (auditory tube). This groove continues posterolaterally into a bony canal in the petrous part of the temporal bone for the pharyngotympanic tube. Just lateral to the greater wing of the sphenoid is the squamous part of the temporal bone, which participates in the temporomandibular joint. It contains the mandibular fossa, which is a concavity where the head of the mandible articulates with the base of the skull. An important feature of this articulation is the prominent articular tubercle, which is the downward projection of the anterior border of the mandibular fossa (Fig. 8.23).

1	The posterior part of the base of the skull extends from the anterior edge of the foramen magnum posteriorly to the superior nuchal lines (Fig. 8.23). It consists of parts of the occipital bone centrally and the temporal bones laterally. The occipital bone is the major bony element of this part of the base of the skull (Fig. 8.23). It has four parts organized around the foramen magnum, which is a prominent feature of this part of the base of the skull and through which the brain and spinal cord are continuous. The parts of the occipital bone are the squamous part, which is posterior to the foramen magnum, the lateral parts, which are lateral to the foramen magnum, and the basilar part, which is anterior to the foramen magnum (Fig. 8.23). The squamous and lateral parts are components of the posterior part of the base of the skull.

1	The squamous and lateral parts are components of the posterior part of the base of the skull. The most visible feature of the squamous part of the occipital bone when examining the inferior view of the skull is a ridge of bone (the external occipital crest), which extends downward from the external occipital protuberance toward the foramen magnum. The inferior nuchal lines arc laterally from the midpoint of the crest. Immediately lateral to the foramen magnum are the lateral parts of the occipital bones, which contain numerous important structural features.

1	Immediately lateral to the foramen magnum are the lateral parts of the occipital bones, which contain numerous important structural features. On each anterolateral border of the foramen magnum are the rounded occipital condyles (Fig. 8.23). These paired structures articulate with the atlas (vertebra CI). Posterior to each condyle is a depression (the condylar fossa) containing a condylar canal, and anterior and superior to each condyle is the large hypoglossal canal. Lateral to each hypoglossal canal is a large, irregular jugular foramen formed by opposition of the jugular notch of the occipital bone and jugular notch of the temporal bone. Laterally in the posterior part of the base of the skull is the temporal bone. The parts of the temporal bone seen in this location are the mastoid part of the petromastoid part and the styloid process (Fig. 8.23).

1	The lateral edge of the mastoid part is identified by the large cone-shaped mastoid process projecting from its inferior surface. This prominent bony structure is the point of attachment for several muscles. On the medial aspect of the mastoid process is the deep mastoid notch, which is also an attachment point for a muscle. Anteromedial to the mastoid process is the needle-shaped styloid process projecting from the lower border of the temporal bone. The styloid process is also a point of attachment for numerous muscles and ligaments. Finally, between the styloid process and the mastoid process is the stylomastoid foramen. The cranial cavity is the space within the cranium that contains the brain, meninges, proximal parts of the cranial nerves, blood vessels, and cranial venous sinuses.

1	The cranial cavity is the space within the cranium that contains the brain, meninges, proximal parts of the cranial nerves, blood vessels, and cranial venous sinuses. The calvaria is the dome-shaped roof that protects the superior aspect of the brain. It consists mainly of the frontal bone anteriorly, the paired parietal bones in the middle, and the occipital bone posteriorly (Fig. 8.24). Sutures visible internally include: the coronal suture, between the frontal and parietal bones, the sagittal suture, between the paired parietal bones, and the lambdoid suture, between the parietal and occipital bones. Visible junctions of these sutures are the bregma, where the coronal and sagittal sutures meet, and the lambda, where the lambdoid and sagittal sutures meet. Other markings on the internal surface of the calva include bony ridges and numerous grooves and pits.

1	From anterior to posterior, features seen on the bony roof of the cranial cavity are: a midline ridge of bone extending from the surface of the frontal bone (the frontal crest), which is a point of attachment for the falx cerebri (a specialization of the dura mater that partially separates the two cerebral hemispheres); at the superior point of the termination of the frontal crest the beginning of the groove for the superior sagittal sinus, which widens and deepens posteriorly and marks the position of the superior sagittal sinus (an intradural venous structure); on either side of the groove for the superior sagittal sinus throughout its course, a small number of depressions and pits (the granular foveolae), which mark the location of arachnoid granulations (prominent structures readily identifiable when a brain with its meningeal coverings is examined; the arachnoid granulations are involved in the reabsorption of cerebrospinal fluid); and on the lateral aspects of the roof of the

1	identifiable when a brain with its meningeal coverings is examined; the arachnoid granulations are involved in the reabsorption of cerebrospinal fluid); and on the lateral aspects of the roof of the cranial cavity, smaller grooves created by various meningeal vessels.

1	The floor of the cranial cavity is divided into anterior, middle, and posterior cranial fossae. Parts of the frontal, ethmoid, and sphenoid bones form the anterior cranial fossa (Fig. 8.25). Its floor is composed of: frontal bone in the anterior and lateral direction, ethmoid bone in the midline, and two parts of the sphenoid bone posteriorly, the body (midline) and the lesser wings (laterally). The anterior cranial fossa is above the nasal cavity and the orbits, and it is filled by the frontal lobes of the cerebral hemispheres. Anteriorly, a small wedge-shaped midline crest of bone (the frontal crest) projects from the frontal bone. This is a point of attachment for the falx cerebri. Immediately posterior to the frontal crest is the foramen cecum (Table 8.2). This foramen between the frontal and ethmoid bones may transmit emissary veins connecting the nasal cavity with the superior sagittal sinus.

1	Posterior to the frontal crest is a prominent wedge of bone projecting superiorly from the ethmoid (the crista galli). This is another point of attachment for the falx cerebri, which is the vertical extension of dura mater partially separating the two cerebral hemispheres. Lateral to the crista galli is the cribriform plate of the ethmoid bone (Fig. 8.25). This is a sieve-like structure, which allows small olfactory nerve fibers to pass through its foramina from the nasal mucosa to the olfactory bulb. The olfactory nerves are commonly referred to collectively as the olfactory nerve [I].

1	On each side of the ethmoid, the floor of the anterior cranial fossa is formed by relatively thin plates of frontal bone (the orbital part of the frontal bone), which also forms the roof of the orbit below. Posterior to both the frontal and ethmoid bones, the rest of the floor of the anterior cranial fossa is formed by the body and lesser wings of the sphenoid. In the midline, the body extends anteriorly between the orbital parts of the frontal bone to reach the ethmoid bone and posteriorly it extends into the middle cranial fossa. The boundary between the anterior and middle cranial fossae in the midline is the anterior edge of the prechiasmatic sulcus, a smooth groove stretching between the optic canals across the body of the sphenoid. Lesser wings of the sphenoid The two lesser wings of the sphenoid project laterally from the body of the sphenoid and form a distinct boundary between the lateral parts of the anterior and middle cranial fossae.

1	The two lesser wings of the sphenoid project laterally from the body of the sphenoid and form a distinct boundary between the lateral parts of the anterior and middle cranial fossae. Overhanging the anterior part of the middle cranial fossae, each lesser wing ends laterally as a sharp point at the junction of the frontal bone and the greater wing of the sphenoid near the upper lateral edge of the superior orbital fissure that is formed between the greater and lesser wings.

1	Medially each lesser wing widens, curves posteriorly, and ends as a rounded anterior clinoid process (Fig. 8.25). These processes serve as the anterior point of attachment for the tentorium cerebelli, which is a sheet of dura that separates the posterior part of the cerebral hemispheres from the cerebellum. Just anterior to each anterior clinoid process is a circular opening in the lesser wing of the sphenoid (the optic canal), through which the ophthalmic artery and optic nerve [II] pass as they exit the cranial cavity to enter the orbit. The optic canals are usually included in the middle cranial fossa. The middle cranial fossa consists of parts of the sphenoid and temporal bones (Fig. 8.26). The boundary between the anterior and middle cranial fossae in the midline is the anterior edge of the prechiasmatic sulcus, which is a smooth groove stretching between the optic canals across the body of the sphenoid.

1	The posterior boundaries of the middle cranial fossa are formed by the anterior surface, as high as the superior border, of the petrous part of the petromastoid part of the temporal bone. The floor in the midline of the middle cranial fossa is elevated and formed by the body of the sphenoid. Lateral to this are large depressions formed on either side by the greater wing of the sphenoid and the squamous part of the temporal bone. These depressions contain the temporal lobes of the brain. Just posterior to the chiasmatic sulcus is the uniquely modified remainder of the body of the sphenoid (the sella turcica), which consists of a deep central area (the hypophyseal fossa) containing the pituitary gland with anterior and posterior vertical walls of bone (Fig. 8.26). The anterior wall of the sella is vertical in position with its superior extent visible as a slight elevation (the tuberculum sellae) at the posterior edge of the chiasmatic sulcus.

1	The anterior wall of the sella is vertical in position with its superior extent visible as a slight elevation (the tuberculum sellae) at the posterior edge of the chiasmatic sulcus. Lateral projections from the corners of the tuberculum sellae (the middle clinoid processes) are sometimes evident. The posterior wall of the sella turcica is the dorsum sellae, a large ridge of bone projecting upward and forward. At the top of this bony ridge the lateral edges contain rounded projections (the posterior clinoid processes), which are points of attachment, like the anterior clinoid processes, for the tentorium cerebelli. Lateral to each side of the body of the sphenoid, the floor of the middle cranial fossa is formed on either side by the greater wing of the sphenoid (Fig. 8.26).

1	Lateral to each side of the body of the sphenoid, the floor of the middle cranial fossa is formed on either side by the greater wing of the sphenoid (Fig. 8.26). A diagonal gap, the superior orbital fissure, separates the greater wing of the sphenoid from the lesser wing and is a major passageway between the middle cranial fossa and the orbit. Passing through the fissure are the oculomotor nerve [III], the trochlear nerve [IV], the ophthalmic nerve [V1], the abducent nerve [VI], and ophthalmic veins. Posterior to the medial end of the superior orbital fissure on the floor of the middle cranial fossa is a rounded foramen projecting in an anterior direction (the foramen rotundum), through which the maxillary nerve [V2] passes from the middle cranial fossa to the pterygopalatine fossa.

1	Posterolateral to the foramen rotundum is a large oval opening (the foramen ovale), which allows structures to pass between the extracranial infratemporal fossa and the middle cranial fossa. The mandibular nerve [V3], lesser petrosal nerve (carrying fibers from the tympanic plexus that originally came from the glossopharyngeal nerve [IX]) and, occasionally, a small vessel (the accessory middle meningeal artery), pass through this foramen. Posterolateral from the foramen ovale is the small foramen spinosum (Fig. 8.26). This opening also connects the infratemporal fossa with the middle cranial fossa. The middle meningeal artery and its associated veins pass through this foramen and, once inside, the groove for the middle meningeal artery across the floor and lateral wall of the middle cranial fossa clearly marks their path.

1	Posteromedial to the foramen ovale is the rounded intracranial opening of the carotid canal. Directly inferior to this opening is an irregular foramen (the foramen lacerum) (Fig. 8.26). Clearly observed in the inferior view of the skull, the foramen lacerum is closed in life by a cartilaginous plug, and no structures pass through it completely. The posterior boundary of the middle cranial fossa is formed by the anterior surface of the petrous part of the petromastoid part of the temporal bone. Medially, there is a slight depression (trigeminal impression) in the anterior surface of the petrous part of the temporal bone (Fig. 8.26), which marks the location of the sensory ganglion for the trigeminal nerve [V].

1	Lateral to the trigeminal impression and on the anterior surface of the petrous part of the temporal bone is a small linear groove that passes in a superolateral direction and ends in a foramen (the groove and hiatus for the greater petrosal nerve). The greater petrosal nerve is a branch of the facial nerve [VII]. Anterolateral to the groove for the greater petrosal nerve is a second, smaller groove and hiatus for the lesser petrosal nerve, a branch from the tympanic plexus carrying fibers that originally came from the glossopharyngeal nerve [IX] (Fig. 8.26). Above and lateral to the small openings for the greater and lesser petrosal nerves, near the superior ridge of the petrous part of the temporal bone, is a rounded protrusion of bone (the arcuate eminence) produced by the underlying anterior semicircular canal of the inner ear.

1	Just anterior and lateral to the arcuate eminence the anterior surface of the petrous part of the temporal bone is slightly depressed. This region is the tegmen tympani, and marks the thin bony roof of the middle ear cavity. The posterior cranial fossa consists mostly of parts of the temporal and occipital bones, with small contributions from the sphenoid and parietal bones (Fig. 8.27). It is the largest and deepest of the three cranial fossae and contains the brainstem (midbrain, pons, and medulla) and the cerebellum. The anterior boundaries of the posterior cranial fossa in the midline are the dorsum sellae and the clivus (Fig. 8.27). The clivus is a slope of bone that extends upward from the foramen magnum. It is formed by contributions from the body of the sphenoid and from the basilar part of the occipital bone. Laterally the anterior boundaries of the posterior cranial fossa are the superior border of the petrous part of the petromastoid part of the temporal bone.

1	Laterally the anterior boundaries of the posterior cranial fossa are the superior border of the petrous part of the petromastoid part of the temporal bone. Posteriorly the squamous part of the occipital bone to the level of the transverse groove is the major boundary, while laterally the petromastoid part of the temporal bone and small parts of the occipital and parietal bones border the fossa. Centrally, in the deepest part of the posterior cranial fossa, is the largest foramen in the skull, the foramen magnum. It is surrounded by the basilar part of the occipital bone anteriorly, the lateral parts of the occipital bone on either side, and the squamous part of the occipital bone posteriorly. The spinal cord passes superiorly through the foramen magnum to continue as the brainstem. Also passing through the foramen magnum are the vertebral arteries, the meninges, and the spinal roots of the accessory nerve [XI].

1	Also passing through the foramen magnum are the vertebral arteries, the meninges, and the spinal roots of the accessory nerve [XI]. The clivus slopes upward from the foramen magnum. Lateral to the clivus is a groove for the inferior petrosal sinus between the basilar part of the occipital bone and the petrous part of the petromastoid part of the temporal bone (Fig. 8.27). Laterally, across the upper half of the posterior surface of the petrous part of the temporal bone, is an oval foramen (the internal acoustic meatus). The facial [VII] and vestibulocochlear [VIII] nerves, and the labyrinthine artery pass through it. Inferior to the internal acoustic meatus the temporal bone is separated from the occipital bone by the large jugular foramen (Fig. 8.27). Leading to this foramen from the medial side is the groove for the inferior petrosal sinus, and from the lateral side the groove for the sigmoid sinus.

1	The sigmoid sinus passes into the jugular foramen, and is continuous with the internal jugular vein, while the inferior petrosal sinus empties into the internal jugular vein in the area of the jugular foramen. Also passing through the jugular foramen are the glossopharyngeal nerve [IX], the vagus nerve [X], and the accessory nerve [XI]. Medial to the jugular foramen is a large rounded mound of the occipital bone (the jugular tubercle). Just inferior to this, and superior to the foramen magnum, is the hypoglossal canal, through which the hypoglossal nerve [XII] leaves the posterior cranial fossa, and a meningeal branch of the ascending pharyngeal artery enters the posterior cranial fossa. Just posterolateral to the hypoglossal canal is the small condylar canal that, when present, transmits an emissary vein. Squamous part of the occipital bone The squamous part of the occipital bone has several prominent features (Fig. 8.27):

1	Squamous part of the occipital bone The squamous part of the occipital bone has several prominent features (Fig. 8.27): Running upward in the midline from the foramen magnum is the internal occipital crest. On either side of the internal occipital crest, the floor of the posterior cranial fossa is concave to accommodate the cerebellar hemispheres. The internal occipital crest ends superiorly in a bony prominence (the internal occipital protuberance). Extending laterally from the internal occipital protuberance are grooves produced by the transverse sinuses, which continue laterally, eventually joining a groove for each sigmoid sinus—each of these grooves then turns inferiorly toward the jugular foramina. The transverse and sigmoid sinuses are intradural venous sinuses. Foramina and fissures through which major structures enter and leave the cranial cavity

1	The transverse and sigmoid sinuses are intradural venous sinuses. Foramina and fissures through which major structures enter and leave the cranial cavity Foramina and fissures through which major structures pass between the cranial cavity and other regions are summarized in Fig. 8.28. The brain, as well as the spinal cord, is surrounded by three layers of membranes (the meninges, Fig. 8.31A)—a tough, outer layer (the dura mater), a delicate, middle layer (the arachnoid mater), and an inner layer firmly attached to the surface of the brain (the pia mater). The cranial meninges are continuous with, and similar to, the spinal meninges through the foramen magnum, with one important distinction—the cranial dura mater consists of two layers, and only one of these is continuous through the foramen magnum (Fig. 8.31B). The cranial dura mater is a thick, tough, outer covering of the brain. It consists of an outer periosteal layer and an inner meningeal layer (Fig. 8.31A):

1	The cranial dura mater is a thick, tough, outer covering of the brain. It consists of an outer periosteal layer and an inner meningeal layer (Fig. 8.31A): The outer periosteal layer is firmly attached to the skull, is the periosteum of the cranial cavity, contains the meningeal arteries, and is continuous with the periosteum on the outer surface of the skull at the foramen magnum and other intracranial foramina (Fig. 8.31B). The inner meningeal layer is in close contact with the arachnoid mater and is continuous with the spinal dura mater through the foramen magnum. The two layers of dura separate from each other at numerous locations to form two unique types of structures (Fig. 8.31A): dural partitions, which project inward and incompletely separate parts of the brain, and intracranial venous structures.

1	The dural partitions project into the cranial cavity and partially subdivide the cranial cavity. They include the falx cerebri, tentorium cerebelli, falx cerebelli, and diaphragma sellae. The falx cerebri (Fig. 8.32) is a crescent-shaped downward projection of meningeal dura mater from the dura lining the calva that passes between the two cerebral hemispheres. It is attached anteriorly to the crista galli of the ethmoid bone and frontal crest of the frontal bone. Posteriorly it is attached to and blends with the tentorium cerebelli.

1	The tentorium cerebelli (Fig. 8.32) is a horizontal projection of the meningeal dura mater that covers and separates the cerebellum in the posterior cranial fossa from the posterior parts of the cerebral hemispheres. It is attached posteriorly to the occipital bone along the grooves for the transverse sinuses. Laterally, it is attached to the superior border of the petrous part of the temporal bone, ending anteriorly at the anterior and posterior clinoid processes. The anterior and medial borders of the tentorium cerebelli are free, forming an oval opening in the midline (the tentorial notch), through which the midbrain passes. The falx cerebelli (Fig. 8.32) is a small midline projection of meningeal dura mater in the posterior cranial fossa. It is attached posteriorly to the internal occipital crest of the occipital bone and superiorly to the tentorium cerebelli. Its anterior edge is free and is between the two cerebellar hemispheres.

1	The final dural projection is the diaphragma sellae (Fig. 8.32). This small horizontal shelf of meningeal dura mater covers the hypophyseal fossa in the sella turcica of the sphenoid bone. There is an opening in the center of the diaphragma sellae through which passes the infundibulum, connecting the pituitary gland with the base of the brain, and any accompanying blood vessels. The arterial supply to the dura mater (Fig. 8.33) travels in the outer periosteal layer of the dura and consists of: anterior meningeal arteries in the anterior cranial fossa, the middle and accessory meningeal arteries in the middle cranial fossa, and the posterior meningeal artery and other meningeal branches in the posterior cranial fossa. All are small arteries except for the middle meningeal artery, which is much larger and supplies the greatest part of the dura. The anterior meningeal arteries are branches of the ethmoidal arteries.

1	The anterior meningeal arteries are branches of the ethmoidal arteries. The middle meningeal artery is a branch of the maxillary artery. It enters the middle cranial fossa through the foramen spinosum and divides into anterior and posterior branches: The anterior branch passes in an almost vertical direction to reach the vertex of the skull, crossing the pterion during its course. The posterior branch passes in a posterosuperior direction, supplying this region of the middle cranial fossa. The accessory meningeal artery is usually a small branch of the maxillary artery that enters the middle cranial fossa through the foramen ovale and supplies areas medial to this foramen. The posterior meningeal artery and other meningeal branches supplying the dura mater in the posterior cranial fossa come from several sources (Fig. 8.33): The posterior meningeal artery, the terminal branch of the ascending pharyngeal artery, enters the posterior cranial fossa through the jugular foramen.

1	The posterior meningeal artery, the terminal branch of the ascending pharyngeal artery, enters the posterior cranial fossa through the jugular foramen. A meningeal branch from the ascending pharyngeal artery enters the posterior cranial fossa through the hypoglossal canal. Meningeal branches from the occipital artery enter the posterior cranial fossa through the jugular foramen and the mastoid foramen. A meningeal branch from the vertebral artery arises as the vertebral artery enters the posterior cranial fossa through the foramen magnum. Innervation of the dura mater (Fig. 8.34) is by small meningeal branches of all three divisions of the trigeminal nerve [V1, V2, and V3], the vagus nerve [X], and the first, second, and, sometimes, third cervical nerves. (Possible involvement of the glossopharyngeal [IX] and hypoglossal nerves [XII] in the posterior cranial fossa has also been reported.)

1	In the anterior cranial fossa meningeal branches from the ethmoidal nerves, which are branches of the ophthalmic nerve [V1], supply the floor and the anterior part of the falx cerebri. Additionally, a meningeal branch of the ophthalmic nerve [V1] turns and runs posteriorly, supplying the tentorium cerebelli and the posterior part of the falx cerebri. The middle cranial fossa is supplied medially by meningeal branches from the maxillary nerve [V2] and laterally, along the distribution of the middle meningeal artery, by meningeal branches from the mandibular nerve [V3]. The posterior cranial fossa is supplied by meningeal branches from the first, second, and, sometimes, third cervical nerves, which enter the fossa through the foramen magnum, the hypoglossal canal, and the jugular foramen. Meningeal branches of the vagus nerve [X] have also been described. (Possible contributions from the glossopharyngeal [IX] and hypoglossal [XII] nerves have also been reported.)

1	The arachnoid mater is a thin, avascular membrane that lines, but is not adherent to, the inner surface of the dura mater (Fig. 8.35). From its inner surface thin processes or trabeculae extend downward, cross the subarachnoid space, and become continuous with the pia mater. Unlike the pia, the arachnoid does not enter the grooves or fissures of the brain, except for the longitudinal fissure between the two cerebral hemispheres. The pia mater is a thin, delicate membrane that closely invests the surface of the brain (Fig. 8.35). It follows the contours of the brain, entering the grooves and fissures on its surface, and is closely applied to the roots of the cranial nerves at their origins. Arrangement of meninges and spaces There is a unique arrangement of meninges coupled with real and potential spaces within the cranial cavity (Fig. 8.35). A potential space is related to the dura mater, while a real space exists between the arachnoid mater and the pia mater.

1	A potential space is related to the dura mater, while a real space exists between the arachnoid mater and the pia mater. The potential space between dura mater and bone is the extradural space (Fig. 8.35). Normally, the outer or periosteal layer of dura mater is firmly attached to the bones surrounding the cranial cavity. This potential space between dura and bone can become a fluid-filled actual space when a traumatic event results in a vascular hemorrhage. Bleeding into the extradural space primarily due to rupture of a meningeal artery or less often from a torn dural venous sinus results in an extradural hematoma.

1	Anatomically, a true subdural space does not exist. Blood collecting in this region (subdural hematoma) due to injury represents a dissection of the dural border cell layer, which is the innermost lining of the meningeal dura. Dural border cells are flattened cells surrounded by extracellular spaces filled with amorphous material. While very infrequent, an occasional cell junction may be seen between these cells and the underlying arachnoid layer. Bleeding due to the tearing of a cerebral vein as it crosses through the dura to enter a dural venous sinus can result in a subdural hematoma.

1	Deep to the arachnoid mater is the only normally occurring fluid-filled space associated with the meninges, the subarachnoid space (Fig. 8.35). It occurs because the arachnoid mater clings to the inner surface of the dura mater and does not follow the contour of the brain, while the pia mater, being against the surface of the brain, closely follows the grooves and fissures on the surface of the brain. The narrow subarachnoid space is therefore created between these two membranes (Fig. 8.35). The subarachnoid space surrounds the brain and spinal cord and in certain locations it enlarges into expanded areas (subarachnoid cisterns). It contains cerebrospinal fluid (CSF) and blood vessels. Cerebrospinal fluid is produced by the choroid plexus, primarily in the ventricles of the brain. It is a clear, colorless, cell-free fluid that circulates through the subarachnoid space surrounding the brain and spinal cord.

1	The CSF returns to the venous system through arachnoid villi. These project as clumps (arachnoid granulations) into the superior sagittal sinus, which is a dural venous sinus, and its lateral extensions, the lateral lacunae (Fig. 8.35). The brain is a component of the central nervous system. During development the brain can be divided into five continuous parts (Figs. 8.36 and 8.37). From rostral (or cranial) to caudal they are: The telencephalon (cerebrum) becomes the large cerebral hemispheres. The surface of these hemispheres consists of elevations (gyri) and depressions (sulci), and the hemispheres are partially separated by a deep longitudinal fissure. The cerebrum fills the area of the cranial cavity above the tentorium cerebelli and is subdivided into lobes based on position.

1	The diencephalon, which is hidden from view in the adult brain by the cerebral hemispheres, consists of the thalamus, hypothalamus, and other related structures, and classically is considered to be the most rostral part of the brainstem. (However, in common usage today, the term brainstem usually refers to the midbrain, pons, and medulla.) The mesencephalon (midbrain), which is the first part of the brainstem seen when an intact adult brain is examined, spans the junction between the middle and posterior cranial fossae. The metencephalon, which gives rise to the cerebellum (consisting of two lateral hemispheres and a midline part in the posterior cranial fossa below the tentorium cerebelli) and the pons (anterior to the cerebellum, and is a bulging part of the brainstem in the most anterior part of the posterior cranial fossa against the clivus and dorsum sellae).

1	The myelencephalon (medulla oblongata), the caudalmost part of the brainstem, ends at the foramen magnum or the uppermost rootlets of the first cervical nerve and to which cranial nerves VI to XII are attached. The brain receives its arterial supply from two pairs of vessels, the vertebral and internal carotid arteries (Fig. 8.38), which are interconnected in the cranial cavity to produce a cerebral arterial circle (of Willis). The two vertebral arteries enter the cranial cavity through the foramen magnum and just inferior to the pons fuse to form the basilar artery. The two internal carotid arteries enter the cranial cavity through the carotid canals on either side.

1	The two internal carotid arteries enter the cranial cavity through the carotid canals on either side. Each vertebral artery arises from the first part of each subclavian artery (Fig. 8.38) in the lower part of the neck, and passes superiorly through the foramen transversarium of the upper six cervical vertebrae. On entering the cranial cavity through the foramen magnum each vertebral artery gives off a small meningeal branch. Continuing forward, the vertebral artery gives rise to three additional branches before joining with its companion vessel to form the basilar artery (Figs. 8.38 and 8.39): The first is a posterior inferior cerebellar artery.

1	The first is a posterior inferior cerebellar artery. A second branch is the posterior spinal artery, which passes posteriorly around the medulla and then descends on the posterior surface of the spinal cord in the area of the attachment of the posterior roots—there are two posterior spinal arteries, one on each side (although the posterior spinal arteries can originate directly from the vertebral arteries, they more commonly branch from the posterior inferior cerebellar arteries). A third branch joins with its companion from the other side to form the single anterior spinal artery, which then descends in the anterior median fissure of the spinal cord.

1	A third branch joins with its companion from the other side to form the single anterior spinal artery, which then descends in the anterior median fissure of the spinal cord. The basilar artery travels in a rostral direction along the anterior aspect of the pons (Fig. 8.39). Its branches in a caudal to rostral direction include the anterior inferior cerebellar arteries, several small pontine arteries, and the superior cerebellar arteries. The basilar artery ends as a bifurcation, giving rise to two posterior cerebral arteries. The two internal carotid arteries arise as one of the two terminal branches of the common carotid arteries (Fig. 8.38). They proceed superiorly to the base of the skull where they enter the carotid canal. Entering the cranial cavity each internal carotid artery gives off the ophthalmic artery, the posterior communicating artery, the middle cerebral artery, and the anterior cerebral artery (Fig. 8.39).

1	The cerebral arterial circle (of Willis) is formed at the base of the brain by the interconnecting vertebrobasilar and internal carotid systems of vessels (Fig. 8.38). This anastomotic interconnection is accomplished by: an anterior communicating artery connecting the left and right anterior cerebral arteries to each other, and two posterior communicating arteries, one on each side, connecting the internal carotid artery with the posterior cerebral artery (Figs. 8.38 and 8.39). Venous drainage of the brain begins internally as networks of small venous channels lead to larger cerebral veins, cerebellar veins, and veins draining the brainstem, which eventually empty into dural venous sinuses. The dural venous sinuses are endothelial-lined spaces between the outer periosteal and the inner meningeal layers of the dura mater, and eventually lead to the internal jugular veins.

1	Also emptying into the dural venous sinuses are diploic veins, which run between the internal and external tables of compact bone in the roof of the cranial cavity, and emissary veins, which pass from outside the cranial cavity to the dural venous sinuses (Fig. 8.43). The emissary veins are important clinically because they can be a conduit through which infections can enter the cranial cavity because they have no valves. The dural venous sinuses include the superior sagittal, inferior sagittal, straight, transverse, sigmoid, and occipital sinuses, the confluence of sinuses, and the cavernous, sphenoparietal, superior petrosal, inferior petrosal, and basilar sinuses (Fig. 8.44, Table 8.3).

1	The superior sagittal sinus is in the superior border of the falx cerebri (Fig. 8.44). It begins anteriorly at the foramen cecum, where it may receive a small emissary vein from the nasal cavity, and ends posteriorly in the confluence of sinuses, usually bending to the right to empty into the right transverse sinus. The superior sagittal sinus communicates with lateral extensions (lateral lacunae) of the sinus containing numerous arachnoid granulations. The superior sagittal sinus usually receives cerebral veins from the superior surface of the cerebral hemispheres, diploic and emissary veins, and veins from the falx cerebri. The inferior sagittal sinus is in the inferior margin of the falx cerebri (Fig. 8.44). It receives a few cerebral veins and veins from the falx cerebri, and ends posteriorly at the anterior edge of the tentorium cerebelli, where it is joined by the great cerebral vein and together with the great cerebral vein forms the straight sinus (Fig. 8.44).

1	The straight sinus continues posteriorly along the junction of the falx cerebri and the tentorium cerebelli and ends in the confluence of sinuses, usually bending to the left to empty into the left transverse sinus. The straight sinus usually receives blood from the inferior sagittal sinus, cerebral veins (from the posterior part of the cerebral hemispheres), the great cerebral vein (draining deep areas of the cerebral hemispheres), superior cerebellar veins, and veins from the falx cerebri. Confluence of sinuses, transverse and The superior sagittal and straight sinuses, and the occipital sinus (in the falx cerebelli) empty into the confluence of sinuses, which is a dilated space at the internal occipital protuberance (Fig. 8.44) and is drained by the right and left transverse sinuses. The paired transverse sinuses extend in horizontal directions from the confluence of sinuses where the tentorium cerebelli joins the lateral and posterior walls of the cranial cavity.

1	The paired transverse sinuses extend in horizontal directions from the confluence of sinuses where the tentorium cerebelli joins the lateral and posterior walls of the cranial cavity. The right transverse sinus usually receives blood from the superior sagittal sinus and the left transverse sinus usually receives blood from the straight sinus. The transverse sinuses also receive blood from the superior petrosal sinus, veins from the inferior parts of the cerebral hemispheres and the cerebellum, and diploic and emissary veins. As the transverse sinuses leave the surface of the occipital bone, they become the sigmoid sinuses (Fig. 8.44), which turn inferiorly, grooving the parietal, temporal, and occipital bones, before ending at the beginning of the internal jugular veins. The sigmoid sinuses also receive blood from cerebral, cerebellar, diploic, and emissary veins.

1	The paired cavernous sinuses are against the lateral aspect of the body of the sphenoid bone on either side of the sella turcica (Figs. 8.45 and 8.46). They are of great clinical importance because of their connections and the structures that pass through them. The cavernous sinuses receive blood not only from cerebral veins but also from the ophthalmic veins (from the orbit) and emissary veins (from the pterygoid plexus of veins in the infratemporal fossa). These connections provide pathways for infections to pass from extracranial sites into intracranial locations. In addition, because structures pass through the cavernous sinuses and are located in the walls of these sinuses they are vulnerable to injury due to inflammation. Structures passing through each cavernous sinus are: the internal carotid artery, and the abducent nerve [VI].

1	Structures passing through each cavernous sinus are: the internal carotid artery, and the abducent nerve [VI]. Structures in the lateral wall of each cavernous sinus are, from superior to inferior: the oculomotor nerve [III], the trochlear nerve [IV], the ophthalmic nerve [V1], and the maxillary nerve [V2]. Connecting the right and left cavernous sinuses are the intercavernous sinuses on the anterior and posterior sides of the pituitary stalk (Fig. 8.44). Sphenoparietal sinuses drain into the anterior ends of each cavernous sinus. These small sinuses are along the inferior surface of the lesser wings of the sphenoid and receive blood from the diploic and meningeal veins.

1	The superior petrosal sinuses drain the cavernous sinuses into the transverse sinuses. Each superior petrosal sinus begins at the posterior end of the cavernous sinus, passes posterolaterally along the superior margin of the petrous part of each temporal bone, and connects to the transverse sinus (Fig. 8.44). The superior petrosal sinuses also receive cerebral and cerebellar veins. The inferior petrosal sinuses also begin at the posterior ends of the cavernous sinuses. These bilateral sinuses pass posteroinferiorly in a groove between the petrous part of the temporal bone and the basal part of the occipital bone, ending in the internal jugular veins. They assist in draining the cavernous sinuses and also receive blood from cerebellar veins and veins from the internal ear and brainstem. Basilar sinuses connect the inferior petrosal sinuses to each other and to the vertebral plexus of veins. They are on the clivus, just posterior to the sella turcica of the sphenoid bone (Fig. 8.44).

1	The 12 pairs of cranial nerves are part of the peripheral nervous system (PNS) and pass through foramina or fissures in the cranial cavity. All nerves except one, the accessory nerve [XI], originate from the brain. In addition to having somatic and visceral components similar to those of spinal nerves, some cranial nerves also contain special sensory and motor components (Tables 8.4 and 8.5). The special sensory components are associated with hearing, seeing, smelling, balancing, and tasting. Special motor components include those that innervate skeletal muscles derived embryologically from the pharyngeal arches and not from somites. In human embryology, six pharyngeal arches are designated, but the fifth pharyngeal arch never develops. Each of the pharyngeal arches that does develop is associated with a developing cranial nerve or one of its branches. These cranial nerves carry efferent fibers that innervate the musculature derived from the pharyngeal arch.

1	Innervation of the musculature derived from the five pharyngeal arches that do develop is as follows: first arch—trigeminal nerve [V3], second arch—facial nerve [VII], third arch—glossopharyngeal nerve [IX], fourth arch—superior laryngeal branch of the vagus nerve [X], sixth arch—recurrent laryngeal branch of the vagus nerve [X], posterior arches—accessory nerve [XI]. The olfactory nerve [I] carries special afferent (SA) fibers for the sense of smell. Its sensory neurons have: peripheral processes that act as receptors in the nasal mucosa, and central processes that return information to the brain. The receptors are in the roof and upper parts of the nasal cavity, and the central processes, after joining into small bundles, enter the cranial cavity by passing through the cribriform plate of the ethmoid bone (Fig. 8.53). They terminate by synapsing with secondary neurons in the olfactory bulbs (Fig. 8.54).

1	The optic nerve [II] carries SA fibers for vision. These fibers return information to the brain from photoreceptors in the retina. Neuronal processes leave the retinal receptors, join into small bundles, and are carried by the optic nerves to other components of the visual system in the brain. The optic nerves enter the cranial cavity through the optic canals (Fig. 8.53). The oculomotor nerve [III] carries two types of fibers: General somatic efferent (GSE) fibers innervate most of the extra-ocular muscles. General visceral efferent (GVE) fibers are part of the parasympathetic part of the autonomic division of the PNS. The oculomotor nerve [III] leaves the anterior surface of the brainstem between the midbrain and the pons (Fig. 8.54). It enters the anterior edge of the tentorium cerebelli, continues in an anterior direction in the lateral wall of the cavernous sinus (Figs. 8.53 and 8.54; see Fig. 8.45), and leaves the cranial cavity through the superior orbital fissure.

1	In the orbit, the GSE fibers in the oculomotor nerve innervate levator palpebrae superioris, superior rectus, inferior rectus, medial rectus, and inferior oblique muscles. The GVE fibers are preganglionic parasympathetic fibers that synapse in the ciliary ganglion and ultimately innervate the sphincter pupillae muscle, responsible for pupillary constriction, and the ciliary muscles, responsible for accommodation of the lens for near vision.

1	The trochlear nerve [IV] is a cranial nerve that carries GSE fibers to innervate the superior oblique muscle, an extra-ocular muscle in the orbit. It arises in the midbrain and is the only cranial nerve to exit from the posterior surface of the brainstem (Fig. 8.54). After curving around the midbrain, it enters the inferior surface of the free edge of the tentorium cerebelli, continues in an anterior direction in the lateral wall of the cavernous sinus (Figs. 8.53 and 8.54; see Fig. 8.45), and enters the orbit through the superior orbital fissure. The trigeminal nerve [V] is the major general sensory nerve of the head and also innervates muscles that move the lower jaw. It carries general somatic afferent (GSA) and branchial efferent (BE) fibers:

1	The GSA fibers provide sensory input from the face, anterior one-half of the scalp, mucous membranes of the oral and nasal cavities and the paranasal sinuses, the nasopharynx, part of the ear and external acoustic meatus, part of the tympanic membrane, the orbital contents and conjunctiva, and the dura mater in the anterior and middle cranial fossae. The BE fibers innervate the muscles of mastication; the tensor tympani, tensor veli palatini, and mylohyoid muscles; and the anterior belly of the digastric muscle. The trigeminal nerve exits from the anterolateral surface of the pons as a large sensory root and a small motor root (Fig. 8.54). These roots continue forward out of the posterior cranial fossa and into the middle cranial fossa by passing over the medial tip of the petrous part of the temporal bone (Fig. 8.53).

1	In the middle cranial fossa the sensory root expands into the trigeminal ganglion (Fig. 8.53), which contains cell bodies for the sensory neurons in the trigeminal nerve and is comparable to a spinal ganglion. The ganglion is in a depression (the trigeminal depression) on the anterior surface of the petrous part of the temporal bone, in a dural cave (the trigeminal cave). The motor root is below and completely separate from the sensory root at this point. Arising from the anterior border of the trigeminal ganglion are the three terminal divisions of the trigeminal nerve, which in descending order are: the ophthalmic nerve (ophthalmic division [V1]). the maxillary nerve (maxillary division [V2]), and the mandibular nerve (mandibular division [V3]). The ophthalmic nerve [V1] passes forward in the dura of the lateral wall of the cavernous sinus (see Fig. 8.45), leaves the cranial cavity, and enters the orbit through the superior orbital fissure (Fig. 8.53).

1	The ophthalmic nerve [V1] carries sensory branches from the eyes, conjunctiva, and orbital contents, including the lacrimal gland. It also receives sensory branches from the nasal cavity, frontal sinus, ethmoidal cells, falx cerebri, dura in the anterior cranial fossa and superior parts of the tentorium cerebelli, upper eyelid, dorsum of the nose, and the anterior part of the scalp. The maxillary nerve [V2] passes forward in the dura mater of the lateral wall of the cavernous sinus just inferior to the ophthalmic nerve [V1] (see Fig. 8.45), leaves the cranial cavity through the foramen rotundum (Fig. 8.53), and enters the pterygopalatine fossa. The maxillary nerve [V2] receives sensory branches from the dura in the middle cranial fossa, the nasopharynx, the palate, the nasal cavity, teeth of the upper jaw, maxillary sinus, and skin covering the side of the nose, the lower eyelid, the cheek, and the upper lip.

1	The mandibular nerve [V3] leaves the inferior margin of the trigeminal ganglion and leaves the skull through the foramen ovale (Fig. 8.53), and enters the infratemporal fossa. The motor root of the trigeminal nerve also passes through the foramen ovale and unites with the sensory component of the mandibular nerve [V3] outside the skull. Thus the mandibular nerve [V3] is the only division of the trigeminal nerve that contains a motor component. Outside the skull the motor fibers innervate the four muscles of mastication (temporalis, masseter, and medial and lateral pterygoids), as well as the tensor tympani muscle, the tensor veli palatini muscle, the anterior belly of the digastric muscle, and the mylohyoid muscle.

1	The mandibular nerve [V3] also receives sensory branches from the skin of the lower face, cheek, lower lip, anterior part of the external ear, part of the external acoustic meatus and the temporal region, the anterior two-thirds of the tongue, the teeth of the lower jaw, the mastoid air cells, the mucous membranes of the cheek, the mandible, and dura in the middle cranial fossa. The abducent nerve [VI] carries GSE fibers to innervate the lateral rectus muscle in the orbit. It arises from the brainstem between the pons and medulla and passes forward, piercing the dura covering the clivus (Figs. 8.53 and 8.54). Continuing upward in a dural canal, it crosses the superior edge of the petrous part of the temporal bone, enters and crosses the cavernous sinus (see Fig. 8.45) just inferolateral to the internal carotid artery, and enters the orbit through the superior orbital fissure. The facial nerve [VII] carries GSA, SA, GVE, and BE fibers:

1	The facial nerve [VII] carries GSA, SA, GVE, and BE fibers: The GSA fibers provide sensory input from part of the external acoustic meatus and deeper parts of the auricle. The SA fibers are for taste from the anterior two-thirds of the tongue. The GVE fibers are part of the parasympathetic part of the autonomic division of the PNS and stimulate secretomotor activity in the lacrimal gland, submandibular and sublingual salivary glands, and glands in the mucous membranes of the nasal cavity, and hard and soft palates. The BE fibers innervate the muscles of the face (muscles of facial expression) and scalp derived from the second pharyngeal arch, and the stapedius muscle, the posterior belly of the digastric muscle, and the stylohyoid muscle. The facial nerve [VII] attaches to the lateral surface of the brainstem, between the pons and medulla oblongata (Fig. 8.54). It consists of a large motor root and a smaller sensory root (the intermediate nerve):

1	The intermediate nerve contains the SA fibers for taste, the parasympathetic GVE fibers, and the GSA fibers. The larger motor root contains the BE fibers. The motor and sensory roots cross the posterior cranial fossa and leave the cranial cavity through the internal acoustic meatus (Fig. 8.53). After entering the facial canal in the petrous part of the temporal bone, the two roots fuse and form the facial nerve [VII]. Near this point the nerve enlarges as the geniculate ganglion, which is similar to a spinal ganglion containing cell bodies for sensory neurons. At the geniculate ganglion the facial nerve [VII] turns and gives off the greater petrosal nerve, which carries mainly preganglionic parasympathetic (GVE) fibers (Table 8.6). The facial nerve [VII] continues along the bony canal, giving off the nerve to the stapedius and the chorda tympani, before exiting the skull through the stylomastoid foramen.

1	The facial nerve [VII] continues along the bony canal, giving off the nerve to the stapedius and the chorda tympani, before exiting the skull through the stylomastoid foramen. The chorda tympani carries taste (SA) fibers from the anterior two-thirds of the tongue and preganglionic parasympathetic (GVE) fibers destined for the submandibular ganglion (Table 8.6). The vestibulocochlear nerve [VIII] carries SA fibers for hearing and balance, and consists of two divisions: a vestibular component for balance, and a cochlear component for hearing. The vestibulocochlear nerve [VIII] attaches to the lateral surface of the brainstem, between the pons and medulla, after emerging from the internal acoustic meatus and crossing the posterior cranial fossa (Figs. 8.53 and 8.54). The two divisions combine into the single nerve seen in the posterior cranial fossa within the substance of the petrous part of the temporal bone.

1	The glossopharyngeal nerve [IX] carries GVA, GSA, SA, GVE, and BE fibers: The GVA fibers provide sensory input from the carotid body and sinus. The GSA fibers provide sensory input from the posterior one-third of the tongue, palatine tonsils, oropharynx, and mucosa of the middle ear, pharyngotympanic tube, and mastoid air cells. The SA fibers are for taste from the posterior one-third of the tongue. The GVE fibers are part of the parasympathetic part of the autonomic division of the PNS and stimulate secretomotor activity in the parotid salivary gland. The BE fibers innervate the muscle derived from the third pharyngeal arch (the stylopharyngeus muscle).

1	The BE fibers innervate the muscle derived from the third pharyngeal arch (the stylopharyngeus muscle). The glossopharyngeal nerve [IX] arises as several rootlets on the anterolateral surface of the upper medulla oblongata (Fig. 8.54). The rootlets cross the posterior cranial fossa and enter the jugular foramen (Fig. 8.53). Within the jugular foramen, and before exiting from it, the rootlets merge to form the glossopharyngeal nerve. Within or immediately outside the jugular foramen are two ganglia (the superior and inferior ganglia), which contain the cell bodies of the sensory neurons in the glossopharyngeal nerve [IX].

1	Within or immediately outside the jugular foramen are two ganglia (the superior and inferior ganglia), which contain the cell bodies of the sensory neurons in the glossopharyngeal nerve [IX]. Branching from the glossopharyngeal nerve [IX] either within or immediately outside the jugular foramen is the tympanic nerve. This branch reenters the temporal bone, enters the middle ear cavity, and participates in the formation of the tympanic plexus. Within the middle ear cavity it provides sensory innervation to the mucosa of the cavity, pharyngotympanic tube, and mastoid air cells. The tympanic nerve also contributes GVE fibers, which leave the tympanic plexus in the lesser petrosal nerve—a small nerve that exits the temporal bone, enters the middle cranial fossa, and descends through the foramen ovale to exit the cranial cavity carrying preganglionic parasympathetic fibers to the otic ganglion (Table 8.6). The vagus nerve [X] carries GSA, GVA, SA, GVE, and BE fibers:

1	The vagus nerve [X] carries GSA, GVA, SA, GVE, and BE fibers: The GSA fibers provide sensory input from the larynx, laryngopharynx, deeper parts of the auricle, part of the external acoustic meatus, and the dura mater in the posterior cranial fossa. The GVA fibers provide sensory input from the aortic body chemoreceptors and aortic arch baroreceptors, and the esophagus, bronchi, lungs, heart, and abdominal viscera in the foregut and midgut. The SA fibers are for taste around the epiglottis and pharynx. The GVE fibers are part of the parasympathetic part of the autonomic division of the PNS and stimulate smooth muscle and glands in the pharynx, larynx, thoracic viscera, and abdominal viscera of the foregut and midgut. The BE fibers innervate one muscle of the tongue (palatoglossus), the muscles of the soft palate (except the tensor veli palatini), pharynx (except the stylopharyngeus), and larynx.

1	The BE fibers innervate one muscle of the tongue (palatoglossus), the muscles of the soft palate (except the tensor veli palatini), pharynx (except the stylopharyngeus), and larynx. The vagus nerve arises as a group of rootlets on the anterolateral surface of the medulla oblongata just inferior to the rootlets arising to form the glossopharyngeal nerve [IX] (Fig. 8.54). The rootlets cross the posterior cranial fossa and enter the jugular foramen (Fig. 8.53). Within this foramen, and before exiting from it, the rootlets merge to form the vagus nerve [X]. Within or immediately outside the jugular foramen are two ganglia, the superior (jugular) and inferior (nodose) ganglia, which contain the cell bodies of the sensory neurons in the vagus nerve [X].

1	The accessory nerve [XI] is a cranial nerve that carries BE fibers to innervate the sternocleidomastoid and trapezius muscles (see Diogo R et al. Nature 2015;520:466–473). It is a unique cranial nerve because its roots arise from motor neurons in the upper five segments of the cervical spinal cord. These fibers leave the lateral surface of the spinal cord and, joining together as they ascend, enter the cranial cavity through the foramen magnum (Fig. 8.54). The accessory nerve [XI] continues through the posterior cranial fossa and exits through the jugular foramen (Fig. 8.53). It then descends in the neck to innervate the sternocleidomastoid and trapezius muscles from their deep surfaces. Cranial root of the accessory nerve

1	Cranial root of the accessory nerve Some descriptions of the accessory nerve [XI] refer to a few rootlets arising from the caudal part of the medulla oblongata on the anterolateral surface just inferior to the rootlets arising to form the vagus nerve [X] as the “cranial” root of the accessory nerve (Fig. 8.54). Leaving the medulla, the cranial roots course with the “spinal” roots of the accessory nerve [XI] into the jugular foramen, at which point the cranial roots join the vagus nerve [X]. As part of the vagus nerve [X], they are distributed to the pharyngeal musculature innervated by the vagus nerve [X] and are therefore described as being part of the vagus nerve [X].

1	The hypoglossal nerve [XII] carries GSE fibers to innervate all intrinsic muscles and most of the extrinsic muscles of the tongue. It arises as several rootlets from the anterior surface of the medulla (Fig. 8.54), passes laterally across the posterior cranial fossa, and exits through the hypoglossal canal (Fig. 8.53). This nerve innervates the hyoglossus, styloglossus, and genioglossus muscles and all intrinsic muscles of the tongue. A face-to-face meeting is an important initial contact between individuals. Part of this exchange is the use of facial expressions to convey emotions. In fact, a physician can gain important information about an individual’s general health by observing a patient’s face.

1	Thus an understanding of the unique organization of the various structures between the superciliary arches superiorly, the lower edge of the mandible inferiorly, and as far back as the ears on either side, the area defined as the face, is particularly useful in the practice of medicine. The muscles of the face (Fig. 8.56) develop from the second pharyngeal arch and are innervated by branches of the facial nerve [VII]. They are in the superficial fascia, with origins from either bone or fascia, and insertions into the skin. Because these muscles control expressions of the face, they are sometimes referred to as muscles of “facial expression.” They also act as sphincters and dilators of the orifices of the face (i.e., the orbits, nose, and mouth). This organizational arrangement into functional groups provides a logical approach to understanding these muscles (Table 8.7). Two muscles are associated with the orbital group—the orbicularis oculi and the corrugator supercilii.

1	Two muscles are associated with the orbital group—the orbicularis oculi and the corrugator supercilii. The orbicularis oculi is a large muscle that completely surrounds each orbital orifice and extends into each eyelid (Fig. 8.57). It closes the eyelids. It has two major parts: The outer orbital part is a broad ring that encircles the orbital orifice and extends outward beyond the orbital rim. The inner palpebral part is in the eyelids and consists of muscle fibers originating in the medial corner of the eye that arch across each lid to attach laterally. The orbital and palpebral parts have specific roles to play during eyelid closure. The palpebral part closes the eye gently, whereas the orbital part closes the eye more forcefully and produces some wrinkling on the forehead. An additional small lacrimal part of the orbicularis oculi muscle is deep, medial in position, and attaches to bone posterior to the lacrimal sac of the lacrimal apparatus in the orbit.

1	An additional small lacrimal part of the orbicularis oculi muscle is deep, medial in position, and attaches to bone posterior to the lacrimal sac of the lacrimal apparatus in the orbit. The second muscle in the orbital group is the much smaller corrugator supercilii (Fig. 8.57), which is deep to the eyebrows and the orbicularis oculi muscle and is active when frowning. It arises from the medial end of the superciliary arch, passing upward and laterally to insert into the skin of the medial half of the eyebrow. It draws the eyebrows toward the midline, causing vertical wrinkles above the nose. Three muscles are associated with the nasal group—the nasalis, the procerus, and the depressor septi nasi (Fig. 8.58). The largest and best developed of the muscles of the nasal group is the nasalis, which is active when the nares are flared (Fig. 8.58). It consists of a transverse part (the compressor naris) and an alar part (the dilator naris):

1	The transverse part of the nasalis compresses the nares—it originates from the maxilla and its fibers pass upward and medially to insert, along with fibers from the same muscle on the opposite side, into an aponeurosis across the dorsum of the nose. The alar part of the nasalis draws the alar cartilages downward and laterally, so opening the nares—it originates from the maxilla, below and medial to the transverse part, and inserts into the alar cartilage. The procerus is a small muscle superficial to the nasal bone and is active when an individual frowns (Fig. 8.58). It arises from the nasal bone and the upper part of the lateral nasal cartilage and inserts into the skin over the lower part of the forehead between the eyebrows. It may be continuous with the frontal belly of the occipitofrontalis muscle of the scalp. The procerus draws the medial border of the eyebrows downward to produce transverse wrinkles over the bridge of the nose.

1	The procerus draws the medial border of the eyebrows downward to produce transverse wrinkles over the bridge of the nose. The final muscle in the nasal group is the depressor septi nasi, another muscle that assists in widening the nares (Fig. 8.58). Its fibers arise from the maxilla above the central incisor tooth and ascend to insert into the lower part of the nasal septum. The depressor septi nasi pulls the nose inferiorly, so assisting the alar part of the nasalis in opening the nares. The muscles in the oral group move the lips and cheek. They include the orbicularis oris and buccinator muscles, and a lower and upper group of muscles (Fig. 8.59). Many of these muscles intersect just lateral to the corner of the mouth on each side at a structure termed the modiolus.

1	The orbicularis oris is a complex muscle consisting of fibers that completely encircle the mouth (Fig. 8.59). Its function is apparent when pursing the lips, as occurs during whistling. Some of its fibers originate near the midline from the maxilla superiorly and the mandible inferiorly, whereas other fibers are derived from both the buccinator, in the cheek, and the numerous other muscles acting on the lips. It inserts into the skin and mucous membrane of the lips, and into itself. Contraction of the orbicularis oris narrows the mouth and closes the lips. The buccinator forms the muscular component of the cheek and is used every time air expanding the cheeks is forcefully expelled (Figs. 8.59 and 8.60). It is in the space between the mandible and the maxilla, deep to the other facial muscles in the area.

1	The buccinator arises from the posterior part of the maxilla and mandible opposite the molar teeth and the pterygomandibular raphe, which is a tendinous band between the pterygoid hamulus superiorly and the mandible inferiorly and is a point of attachment for the buccinator and superior pharyngeal constrictor muscles. The fibers of the buccinator pass toward the corner of the mouth to insert into the lips, blending with fibers from the orbicularis oris in a unique fashion. Central fibers of the buccinator cross so that lower fibers enter the upper lip and upper fibers enter the lower lip (Fig. 8.60). The highest and lowest fibers of the buccinator do not cross and enter the upper and lower lips, respectively. Contraction of the buccinator presses the cheek against the teeth. This keeps the cheek taut and aids in mastication by preventing food from accumulating between the teeth and the cheek. The muscle also assists in the forceful expulsion of air from the cheeks.

1	Lower group of oral muscles The muscles in the lower group consist of the depressor anguli oris, depressor labii inferioris. and mentalis (Fig. 8.59). The depressor anguli oris is active during frowning. It arises along the side of the mandible below the canine, premolar, and first molar teeth and inserts into skin and the upper part of the orbicularis oris near the corner of the mouth. It depresses the corner of the mouth. The depressor labii inferioris arises from the front of the mandible, deep to the depressor anguli oris. Its fibers move superiorly and medially, some merging with fibers from the same muscle on the opposite side and fibers from the orbicularis oris before inserting into the lower lip. It depresses the lower lip and moves it laterally.

1	The mentalis helps position the lip when drinking from a cup or when pouting. It is the deepest muscle of the lower group arising from the mandible just inferior to the incisor teeth, with its fibers passing downward and medially to insert into the skin of the chin. It raises and protrudes the lower lip as it wrinkles the skin of the chin. Upper group of oral muscles The muscles of the upper group of oral muscles consist of the risorius, zygomaticus major, zygomaticus minor, levator labii superioris, levator labii superioris alaeque nasi, and levator anguli oris (Fig. 8.59). The risorius helps produce a grin (Fig. 8.59). It is a thin, superficial muscle that extends laterally from the corner of the mouth in a slightly upward direction. Contraction of its fibers pulls the corner of the mouth laterally and upward.

1	The zygomaticus major and zygomaticus minor help produce a smile (Fig. 8.59). The zygomaticus major is a superficial muscle that arises deep to the orbicularis oculi along the posterior part of the lateral surface of the zygomatic bone, and passes downward and forward, blending with the orbicularis oris and inserting into skin at the corner of the mouth. The zygomaticus minor arises from the zygomatic bone anterior to the origin of the zygomaticus major, parallels the path of the zygomaticus major, and inserts into the upper lip medial to the corner of the mouth. Both zygomaticus muscles raise the corner of the mouth and move it laterally. The levator labii superioris deepens the furrow between the nose and the corner of the mouth during sadness (Fig. 8.59). It arises from the maxilla just superior to the infra-orbital foramen, and its fibers pass downward and medially to blend with the orbicularis oris and insert into the skin of the upper lip.

1	The levator labii superioris alaeque nasi is medial to the levator labii superioris, arises from the maxilla next to the nose, and inserts into both the alar cartilage of the nose and skin of the upper lip (Fig. 8.59). It may assist in flaring the nares. The levator anguli oris is more deeply placed and covered by the other two levators and the zygomaticus muscles (Fig. 8.59). It arises from the maxilla, just inferior to the infra-orbital foramen and inserts into the skin at the corner of the mouth. It elevates the corner of the mouth and may help deepen the furrow between the nose and the corner of the mouth during sadness. Several additional muscles or groups of muscles not in the area defined as the face, but derived from the second pharyngeal arch and innervated by the facial nerve [VII], are considered muscles of facial expression. They include the platysma, auricular, and occipitofrontalis muscles (see Fig. 8.56).

1	The platysma is a large, thin sheet of muscle in the superficial fascia of the neck. It arises below the clavicle in the upper part of the thorax and ascends through the neck to the mandible. At this point, the more medial fibers insert on the mandible, whereas the lateral fibers join with muscles around the mouth. The platysma tenses the skin of the neck and can move the lower lip and corners of the mouth down. Three of these muscles, “other muscles of facial expression,” are associated with the ear—the anterior, superior, and posterior auricular muscles (Fig. 8.61): The anterior muscle is anterolateral and pulls the ear upward and forward. The superior muscle is superior and elevates the ear. The posterior muscle is posterior and retracts and elevates the ear.

1	The superior muscle is superior and elevates the ear. The posterior muscle is posterior and retracts and elevates the ear. The occipitofrontalis is the final muscle in this category of “other muscles of facial expression” and is associated with the scalp (see Fig. 8.56). It consists of a frontal belly anteriorly and an occipital belly posteriorly. An aponeurotic tendon connects the two: The frontal belly covers the forehead and is attached to the skin of the eyebrows. The occipital belly arises from the posterior aspect of the skull and is smaller than the frontal belly. The occipitofrontalis muscles move the scalp and wrinkle the forehead.

1	The occipital belly arises from the posterior aspect of the skull and is smaller than the frontal belly. The occipitofrontalis muscles move the scalp and wrinkle the forehead. The parotid glands are the largest of the three pairs of main salivary glands in the head and numerous structures pass through them. They are anterior to and below the lower half of the ear, superficial, posterior, and deep to the ramus of the mandible (Fig. 8.62). They extend down to the lower border of the mandible and up to the zygomatic arch. Posteriorly they cover the anterior part of the sternocleidomastoid muscle and continue anteriorly to halfway across the masseter muscle.

1	The parotid duct leaves the anterior edge of the parotid gland midway between the zygomatic arch and the corner of the mouth (Fig. 8.62). It crosses the face in a transverse direction and, after crossing the medial border of the masseter muscle, turns deeply into the buccal fat pad and pierces the buccinator muscle. It opens into the oral cavity near the second upper molar tooth. pass just deep to the parotid gland. These include the facial nerve [VII], the external carotid artery and its branches, and the retromandibular vein and its tributaries (Fig. 8.62). The facial nerve [VII] exits the skull through the stylomastoid foramen and then passes into the parotid gland, where it usually divides into upper and lower trunks. These pass through the substance of the parotid gland, where there may be further branching and anastomosing of the nerves.

1	Five terminal groups of branches of the facial nerve [VII]—the temporal, zygomatic, buccal, marginal mandibular, and cervical branches—emerge from the upper, anterior, and lower borders of the parotid gland (Fig. 8.62). The intimate relationships between the facial nerve [VII] and the parotid gland mean that surgical removal of the parotid gland is a difficult dissection if all branches of the facial nerve [VII] are to be spared. The external carotid artery enters into or passes deep to the inferior border of the parotid gland (Fig. 8.62). As it continues in a superior direction, it gives off the posterior auricular artery before dividing into its two terminal branches (the maxillary and superficial temporal arteries) near the lower border of the ear: The maxillary artery passes horizontally, deep to the mandible. The superficial temporal artery continues in a superior direction and emerges from the upper border of the gland after giving off the transverse facial artery.

1	The superficial temporal artery continues in a superior direction and emerges from the upper border of the gland after giving off the transverse facial artery. The retromandibular vein is formed in the substance of the parotid gland when the superficial temporal and maxillary veins join together (Fig. 8.62), and passes inferiorly in the substance of the parotid gland. It usually divides into anterior and posterior branches just below the inferior border of the gland. The parotid gland receives its arterial supply from the numerous arteries that pass through its substance. Sensory innervation of the parotid gland is provided by the auriculotemporal nerve, which is a branch of the mandibular nerve [V3]. This division of the trigeminal nerve exits the skull through the foramen ovale.

1	The auriculotemporal nerve also carries secretomotor fibers to the parotid gland. These postganglionic parasympathetic fibers have their origin in the otic ganglion associated with the mandibular nerve [V3] and are just inferior to the foramen ovale. Preganglionic parasympathetic fibers to the otic ganglion come from the glossopharyngeal nerve [IX]. During development a cranial nerve becomes associated with each of the pharyngeal arches. Because the face is primarily derived from the first and second pharyngeal arches, innervation of neighboring facial structures is as follows: The trigeminal nerve [V] innervates facial structures derived from the first arch. The facial nerve [VII] innervates facial structures derived from the second arch. Because the face is derived developmentally from a number of structures originating from the first pharyngeal arch, cutaneous innervation of the face is by branches of the trigeminal nerve [V].

1	Because the face is derived developmentally from a number of structures originating from the first pharyngeal arch, cutaneous innervation of the face is by branches of the trigeminal nerve [V]. The trigeminal nerve [V] divides into three major divisions— the ophthalmic [V1], maxillary [V2], and mandibular [V3]—before leaving the middle cranial fossa (Fig. 8.64). Each of these divisions passes out of the cranial cavity to innervate a part of the face, so most of the skin covering the face is innervated solely by branches of the trigeminal nerve [V]. The exception is a small area covering the angle and lower border of the ramus of the mandible and parts of the ear, where the facial [VII], vagus [X], and cervical nerves contribute to the innervation.

1	The ophthalmic nerve [V1] exits the skull through the superior orbital fissure and enters the orbit. Its branches (Fig. 8.64) that innervate the face include: the supra-orbital and supratrochlear nerves, which leave the orbit superiorly and innervate the upper eyelid, forehead, and scalp; the infratrochlear nerve, which exits the orbit in the medial angle to innervate the medial half of the upper eyelid, the skin in the area of the medial angle, and the side of the nose; the lacrimal nerve, which exits the orbit in the lateral angle to innervate the lateral half of the upper eyelid and the skin in the area of the lateral angle; and the external nasal nerve, which supplies the anterior part of the nose (Fig. 8.65).

1	The maxillary nerve [V2] exits the skull through the foramen rotundum. Branches (Fig. 8.64) that innervate the face include: a small zygomaticotemporal branch, which exits the zygomatic bone and supplies a small area of the anterior temple above the zygomatic arch; a small zygomaticofacial branch, which exits the zygomatic bone and supplies a small area of skin over the zygomatic bone; and the large infra-orbital nerve, which exits the maxilla through the infra-orbital foramen and immediately divides into multiple branches to supply the lower eyelid, cheek, side of the nose, and upper lip (Fig. 8.65).

1	The mandibular nerve [V3] exits the skull through the foramen ovale. Branches (Fig. 8.65) innervating the face include: the auriculotemporal nerve, which enters the face just posterior to the temporomandibular joint, passes through the parotid gland, and ascends just anterior to the ear to supply the external acoustic meatus, the surface of the tympanic membrane (eardrum), and a large area of the temple; the buccal nerve, which is on the surface of the buccinator muscle supplying the cheek; and the mental nerve, which exits the mandible through the mental foramen and immediately divides into multiple branches to supply the skin and mucous membrane of the lower lip and skin of the chin (Fig. 8.65). The muscles of the face, as well as those associated with the external ear and the scalp, are derived from the second pharyngeal arch. The cranial nerve associated with this arch is the facial nerve [VII] and therefore branches of the facial nerve [VII] innervate all these muscles.

1	The facial nerve [VII] exits the posterior cranial fossa through the internal acoustic meatus. It passes through the temporal bone, giving off several branches, and emerges from the base of the skull through the stylomastoid foramen (Fig. 8.66). At this point it gives off the posterior auricular nerve. This branch passes upward, behind the ear, to supply the occipital belly of the occipitofrontalis muscle of the scalp and the posterior auricular muscle of the ear. The main stem of the facial nerve [VII] then gives off another branch, which innervates the posterior belly of the digastric muscle and the stylohyoid muscle. At this point, the facial nerve [VII] enters the deep surface of the parotid gland (Fig. 8.66B).

1	Once in the parotid gland, the main stem of the facial nerve [VII] usually divides into upper (temporofacial) and lower (cervicofacial) branches. As these branches pass through the substance of the parotid gland they may branch further or take part in an anastomotic network (the parotid plexus). Whatever types of interconnections occur, five terminal groups of branches of the facial nerve [VII]—the temporal, zygomatic, buccal, marginal mandibular, and cervical branches—emerge from the parotid gland (Fig. 8.66A). Although there are variations in the pattern of distribution of the five terminal groups of branches, the basic pattern is as follows: Temporal branches exit from the superior border of the parotid gland to supply muscles in the area of the temple, forehead, and supra-orbital area. Zygomatic branches emerge from the anterosuperior border of the parotid gland to supply muscles in the infra-orbital area, the lateral nasal area, and the upper lip.

1	Zygomatic branches emerge from the anterosuperior border of the parotid gland to supply muscles in the infra-orbital area, the lateral nasal area, and the upper lip. Buccal branches emerge from the anterior border of the parotid gland to supply muscles in the cheek, the upper lip, and the corner of the mouth. Marginal mandibular branches emerge from the anteroinferior border of the parotid gland to supply muscles of the lower lip and chin. Cervical branches emerge from the inferior border of the parotid gland to supply the platysma. The arterial supply to the face is primarily from branches of the external carotid artery, though there is some limited supply from a branch of the internal carotid artery. Similarly, most of the venous return is back to the internal jugular vein, though some important connections from the face result in venous return through a clinically relevant intracranial pathway involving the cavernous sinus.

1	The facial artery is the major vessel supplying the face (Fig. 8.67). It branches from the anterior surface of the external carotid artery, passes up through the deep structures of the neck, and appears at the lower border of the mandible after passing posterior to the submandibular gland. Curving around the inferior border of the mandible just anterior to the masseter, where its pulse can be felt, the facial artery then enters the face. From this point the facial artery runs upward and medially in a tortuous course. It passes along the side of the nose and terminates as the angular artery at the medial corner of the eye. Along its path the facial artery is deep to the platysma, risorius, and zygomaticus major and minor, superficial to the buccinator and levator anguli oris, and may pass superficially to or through the levator labii superioris. Branches of the facial artery include the superior and inferior labial branches and the lateral nasal branch (Fig. 8.67).

1	Branches of the facial artery include the superior and inferior labial branches and the lateral nasal branch (Fig. 8.67). The labial branches arise near the corner of the mouth: The inferior labial branch supplies the lower lip. The superior labial branch supplies the upper lip, and also provides a branch to the nasal septum. Near the midline, the superior and inferior labial branches anastomose with their companion arteries from the opposite side of the face. This provides an important connection between the facial arteries and the external carotid arteries of opposite sides. The lateral nasal branch is a small branch arising from the facial artery as it passes along the side of the nose. It supplies the lateral surface and dorsum of the nose. Another contributor to the vascular supply of the face is the transverse facial artery (Fig. 8.67), which is a branch of the superficial temporal artery (the smaller of the two terminal branches of the external carotid artery).

1	The transverse facial artery arises from the superficial temporal artery within the substance of the parotid gland, passes through the gland, and crosses the face in a transverse direction. Lying on the superficial surface of the masseter muscle, it is between the zygomatic arch and the parotid duct. Branches of the maxillary artery The maxillary artery, the larger of the two terminal branches of the external carotid artery, gives off several small branches which contribute to the arterial supply to the face: The infra-orbital artery enters the face through the infra-orbital foramen and supplies the lower eyelid, upper lip, and the area between these structures. The buccal artery enters the face on the superficial surface of the buccinator muscle and supplies structures in this area. The mental artery enters the face through the mental foramen and supplies the chin. Branches of the ophthalmic artery

1	The mental artery enters the face through the mental foramen and supplies the chin. Branches of the ophthalmic artery Three small arteries from the internal carotid artery also contribute to the arterial supply of the face. These vessels arise from the ophthalmic artery, a branch of the internal carotid artery, after the ophthalmic artery enters the orbit: The zygomaticofacial and zygomaticotemporal arteries come from the lacrimal branch of the ophthalmic artery (Fig. 8.67), enter the face through the zygomaticofacial and zygomaticotemporal foramina, and supply the area of the face over the zygomatic bone. The dorsal nasal artery, a terminal branch of the ophthalmic artery, exits the orbit in the medial corner, and supplies the dorsum of the nose. The supraorbital and supratrochlear arteries supply the anterior scalp.

1	The supraorbital and supratrochlear arteries supply the anterior scalp. The facial vein is the major vein draining the face (Fig. 8.67). Its point of origin is near the medial corner of the orbit as the supratrochlear and supra-orbital veins come together to form the angular vein. This vein becomes the facial vein as it proceeds inferiorly and assumes a position just posterior to the facial artery. The facial vein descends across the face with the facial artery until it reaches the inferior border of the mandible. Here the artery and vein part company and the facial vein passes superficial to the submandibular gland to enter the internal jugular vein. Throughout its course the facial vein receives tributaries from veins draining the eyelids, external nose, lips, cheek, and chin that accompany the various branches of the facial artery.

1	Throughout its course the facial vein receives tributaries from veins draining the eyelids, external nose, lips, cheek, and chin that accompany the various branches of the facial artery. The transverse facial vein is a small vein that accompanies the transverse facial artery in its journey across the face (Fig. 8.67). It empties into the superficial temporal vein within the substance of the parotid gland. As it crosses the face, the facial vein has numerous connections with venous channels passing into deeper regions of the head (Fig. 8.68): near the medial corner of the orbit, it communicates with ophthalmic veins; in the area of the cheek it communicates with veins passing into the infra-orbital foramen; it also communicates with veins passing into deeper regions of the face (i.e., the deep facial vein connecting with the pterygoid plexus of veins).

1	All these venous channels have interconnections with the intracranial cavernous sinus through emissary veins that connect intracranial with extracranial veins. There are no valves in the facial vein or any other venous channels in the head, so blood can move in any direction. Because of the interconnections between the veins, infections of the face, primarily above the mouth (i.e., the “danger area”) should be handled with great care to prevent the dissemination of infectious material in an intracranial direction.

1	Lymphatic drainage from the face primarily moves toward three groups of lymph nodes (Fig. 8.69): submental nodes inferior and posterior to the chin, which drain lymphatics from the medial part of the lower lip and chin bilaterally; submandibular nodes superficial to the submandibular gland and inferior to the body of the mandible, which drain the lymphatics from the medial corner of the orbit, most of the external nose the medial part of the cheek, the upper lip, and the lateral part of the lower lip that follows the course of the facial artery; pre-auricular and parotid nodes anterior to the ear, which drain lymphatics from most of the eyelids, a part of the external nose, and the lateral part of the cheek. The scalp is the part of the head that extends from the superciliary arches anteriorly to the external occipital protuberance and superior nuchal lines posteriorly. Laterally it continues inferiorly to the zygomatic arch.

1	The scalp is a multilayered structure with layers that can be defined by the word itself: S—skin, C—connective tissue (dense), A—aponeurotic layer, L—loose connective tissue, and P—pericranium (Fig. 8.70). Examining the layers of the scalp reveals that the first three layers are tightly held together, forming a single unit. This unit is sometimes referred to as the scalp proper and is the tissue torn away during serious “scalping” injuries. The skin is the outer layer of the scalp (Figs. 8.70 and 8.71). It is similar structurally to skin throughout the body with the exception that hair is present on a large amount of it. Deep to the skin is dense connective tissue. This layer anchors the skin to the third layer and contains the arteries, veins, and nerves supplying the scalp. When the scalp is cut, the dense connective tissue surrounding the vessels tends to hold cut vessels open. This results in profuse bleeding.

1	The deepest layer of the first three layers is the aponeurotic layer. Firmly attached to the skin by the dense connective tissue of the second layer, this layer consists of the occipitofrontalis muscle, which has a frontal belly anteriorly, an occipital belly posteriorly, and an aponeurotic tendon— the epicranial aponeurosis (galea aponeurotica)—connecting the two (Fig. 8.72). The frontal belly of the occipitofrontalis begins anteriorly where it is attached to the skin of the eyebrows. It passes upward, across the forehead, to become continuous with the aponeurotic tendon. Posteriorly, each occipital belly of the occipitofrontalis arises from the lateral part of the superior nuchal line of the occipital bone and the mastoid process of the temporal bone. It also passes superiorly to attach to the aponeurotic tendon.

1	The occipitofrontalis muscles move the scalp, wrinkle the forehead, and raise the eyebrows. The frontal belly is innervated by temporal branches of the facial nerve [VII] and the posterior belly by the posterior auricular branch. A layer of loose connective tissue separates the aponeurotic layer from the pericranium and facilitates movement of the scalp proper over the calvaria (Figs. 8.70 and 8.72). Because of its consistency, infections tend to localize and spread through the loose connective tissue (also see “In the clinic” on p. 878). The pericranium is the deepest layer of the scalp and is the periosteum on the outer surface of the calvaria. It is attached to the bones of the calvaria but is removable, except in the area of the sutures.

1	Sensory innervation of the scalp is from two major sources, cranial nerves or cervical nerves, depending on whether it is anterior or posterior to the ears and the vertex of the head (Fig. 8.73), The occipitofrontalis muscle is innervated by branches of the facial nerve [VII]. Anterior to the ears and the vertex Branches of the trigeminal nerve [V] supply the scalp anterior to the ears and the vertex of the head (Fig. 8.73). These branches are the supratrochlear, supra-orbital, zygomaticotemporal, and auriculotemporal nerves: The supratrochlear nerve exits the orbit, passes through the frontalis muscle, continues superiorly across the front of the forehead, and supplies the front of the forehead near the midline. The supra-orbital nerve exits the orbit through the supra-orbital notch or foramen, passes through the frontalis muscle, and continues superiorly across the scalp as far back as the vertex of the head.

1	The zygomaticotemporal nerve exits the skull through a foramen in the zygomatic bone and supplies the scalp over a small anterior area of the temple. The auriculotemporal nerve exits from the skull, deep to the parotid gland, passes just anterior to the ear, continues superiorly anterior to the ear until nearly reaching the vertex of the head, and supplies the scalp over the temporal region and anterior to the ear to near the vertex. Posterior to the ears and the vertex Posterior to the ears and vertex, sensory innervation of the scalp is by cervical nerves, specifically branches from spinal cord levels C2 and C3 (Fig. 8.73). These branches are the great auricular, the lesser occipital, the greater occipital, and the third occipital nerves:

1	The great auricular nerve is a branch of the cervical plexus, arises from the anterior rami of the C2 and C3 spinal nerves, ascends on the surface of the sternocleidomastoid muscle, and innervates a small area of the scalp just posterior to the ear. The lesser occipital nerve is also a branch of the cervical plexus, arises from the anterior ramus of the C2 spinal nerve, ascends on the posterior border of the sternocleidomastoid muscle, and supplies an area of the scalp posterior and superior to the ear. The greater occipital nerve is a branch of the posterior ramus of the C2 spinal nerve, emerges just inferior to the obliquus capitis inferior muscle, ascends superficial to the suboccipital triangle, pierces the semispinalis capitis and trapezius muscles, and then spreads out to supply a large part of the posterior scalp as far superiorly as the vertex.

1	The third occipital nerve is a branch of the posterior ramus of the C3 spinal nerve, pierces the semispinalis capitis and trapezius muscles, and supplies a small area of the lower part of the scalp. Arteries supplying the scalp (Fig. 8.74) are branches of either the external carotid artery or the ophthalmic artery, which is a branch of the internal carotid artery. Branches from the ophthalmic artery The supratrochlear and supra-orbital arteries supply the anterior and superior aspects of the scalp. They branch from the ophthalmic artery while it is in the orbit, continue through the orbit, and exit onto the forehead in association with the supratrochlear and supra-orbital nerves. Like the nerves, the arteries ascend across the forehead to supply the scalp as far posteriorly as the vertex of the head. Branches from the external carotid artery

1	Branches from the external carotid artery Three branches of the external carotid artery supply the largest part of the scalp—the superficial temporal, posterior auricular, and occipital arteries supply the lateral and posterior aspects of the scalp (Fig. 8.74): The smallest branch (the posterior auricular artery) leaves the posterior aspect of the external carotid artery, passes through deeper structures, and emerges to supply an area of the scalp posterior to the ear. Also arising from the posterior aspect of the external carotid artery is the occipital artery, which ascends in a posterior direction, passes through several layers of back musculature, and emerges to supply a large part of the posterior aspect of the scalp.

1	The third arterial branch supplying the scalp is the superficial temporal artery, a terminal branch of the external carotid artery that passes superiorly, just anterior to the ear, divides into anterior and posterior branches, and supplies almost the entire lateral aspect of the scalp. Veins draining the scalp follow a pattern similar to the arteries: The supratrochlear and supra-orbital veins drain the anterior part of the scalp from the superciliary arches to the vertex of the head (Fig. 8.74), pass inferior to the superciliary arches, communicate with the ophthalmic veins in the orbit, and continue inferiorly to participate in the formation of the angular vein, which is the upper tributary to the facial vein. The superficial temporal vein drains the entire lateral area of the scalp before passing inferiorly to join in the formation of the retromandibular vein.

1	The superficial temporal vein drains the entire lateral area of the scalp before passing inferiorly to join in the formation of the retromandibular vein. The posterior auricular vein drains the area of the scalp posterior to the ear and eventually empties into a tributary of the retromandibular vein. The occipital vein drains the posterior aspect of the scalp from the external occipital protuberance and superior nuchal lines to the vertex of the head; deeper, it passes through the musculature in the posterior neck to join in the formation of the plexus of veins in the suboccipital triangle. Lymphatic drainage of the scalp generally follows the pattern of arterial distribution.

1	Lymphatic drainage of the scalp generally follows the pattern of arterial distribution. The lymphatics in the occipital region initially drain to occipital nodes near the attachment of the trapezius muscle at the base of the skull (Fig. 8.75). Further along the pathway occipital nodes drain into upper deep cervical nodes. There is also some direct drainage to upper deep cervical nodes from this part of the scalp. Lymphatics from the upper part of the scalp drain in two directions: Posterior to the vertex of the head they drain to mastoid nodes (retro-auricular/posterior auricular nodes) posterior to the ear near the mastoid process of the temporal bone, and efferent vessels from these nodes drain into upper deep cervical nodes. Anterior to the vertex of the head they drain to pre-auricular and parotid nodes anterior to the ear on the surface of the parotid gland.

1	Anterior to the vertex of the head they drain to pre-auricular and parotid nodes anterior to the ear on the surface of the parotid gland. Finally, there may be some lymphatic drainage from the forehead to the submandibular nodes through efferent vessels that follow the facial artery. The orbits are bilateral structures in the upper half of the face below the anterior cranial fossa and anterior to the middle cranial fossa that contain the eyeball, the optic nerve, the extra-ocular muscles, the lacrimal apparatus, adipose tissue, fascia, and the nerves and vessels that supply these structures.

1	Seven bones contribute to the framework of each orbit (Fig. 8.76). They are the maxilla, zygomatic, frontal, ethmoid, lacrimal, sphenoid, and palatine bones. Together they give the bony orbit the shape of a pyramid, with its wide base opening anteriorly onto the face and its apex extending in a posteromedial direction. Completing the pyramid configuration are medial, lateral, superior, and inferior walls. The apex of the pyramid-shaped bony orbit is the optic foramen, whereas the base (the orbital rim) is formed: superiorly by the frontal bone, medially by the frontal process of the maxilla, inferiorly by the zygomatic process of the maxilla and the zygomatic bone, and laterally by the zygomatic bone, the frontal process of the zygomatic bone, and the zygomatic process of the frontal bone.

1	The roof (superior wall) of the bony orbit is made up of the orbital part of the frontal bone with a small contribution from the sphenoid bone (Fig. 8.76). This thin plate of bone separates the contents of the orbit from the brain in the anterior cranial fossa. Unique features of the superior wall include: anteromedially, the trochlear fovea, for the attachment of a pulley through which the superior oblique muscle passes, and the possible intrusion of part of the frontal sinus; anterolaterally, a depression (the lacrimal fossa) for the orbital part of the lacrimal gland. Posteriorly, the lesser wing of the sphenoid bone completes the roof. The medial walls of the paired bony orbits are parallel to each other and each consists of four bones—the maxilla, lacrimal, ethmoid, and sphenoid bones (Fig. 8.76).

1	The medial walls of the paired bony orbits are parallel to each other and each consists of four bones—the maxilla, lacrimal, ethmoid, and sphenoid bones (Fig. 8.76). The largest contributor to the medial wall is the orbital plate of the ethmoid bone. This part of the ethmoid bone contains collections of ethmoidal cells, which are clearly visible in a dried skull. Also visible, at the junction between the roof and the medial wall, usually associated with the frontoethmoidal suture, are the anterior and posterior ethmoidal foramina. The anterior and posterior ethmoidal nerves and vessels leave the orbit through these openings.

1	Anterior to the ethmoid bone is the small lacrimal bone, and completing the anterior part of the medial wall is the frontal process of the maxilla. These two bones participate in the formation of the lacrimal groove, which contains the lacrimal sac and is bound by the posterior lacrimal crest (part of the lacrimal bone) and the anterior lacrimal crest (part of the maxilla). Posterior to the ethmoid bone the medial wall is completed by a small part of the sphenoid bone, which forms a part of the medial wall of the optic canal. The floor (inferior wall) of the bony orbit, which is also the roof of the maxillary sinus, consists primarily of the orbital surface of the maxilla (Fig. 8.76), with small contributions from the zygomatic and palatine bones. Beginning posteriorly and continuing along the lateral boundary of the floor of the bony orbit is the inferior orbital fissure. Beyond the anterior end of the fissure the zygomatic bone completes the floor of the bony orbit.

1	Posteriorly, the orbital process of the palatine bone makes a small contribution to the floor of the bony orbit near the junction of the maxilla, ethmoid, and sphenoid bones. The lateral wall of the bony orbit consists of contributions from two bones—anteriorly, the zygomatic bone and posteriorly, the greater wing of the sphenoid bone (Fig. 8.76). The superior orbital fissure is between the greater wing of the sphenoid and the lesser wing of the sphenoid that forms part of the roof. The upper and lower eyelids are anterior structures that, when closed, protect the surface of the eyeball. The space between the eyelids, when they are open, is the palpebral fissure. The layers of the eyelids, from anterior to posterior, consist of skin, subcutaneous tissue, voluntary muscle, the orbital septum, the tarsus, and conjunctiva (Fig. 8.77). The upper and lower eyelids are basically similar in structure except for the addition of two muscles in the upper eyelid.

1	The upper and lower eyelids are basically similar in structure except for the addition of two muscles in the upper eyelid. The skin of the eyelids is not particularly substantial, and only a thin layer of connective tissue separates the skin from the underlying voluntary muscle layer (Fig. 8.77). The thin layer of connective tissue and its loose arrangement account for the accumulation of fluid (blood) when an injury occurs. The muscle fibers encountered next in an anteroposterior direction through the eyelid belong to the palpebral part of the orbicularis oculi (Fig. 8.77). This muscle is part of the larger orbicularis oculi muscle, which consists primarily of two parts—an orbital part, which surrounds the orbit, and the palpebral part, which is in the eyelids. The orbicularis oculi is innervated by the facial nerve [VII] and closes the eyelids.

1	The palpebral part is thin and anchored medially by the medial palpebral ligament (Fig. 8.78), which attaches to the anterior lacrimal crest and laterally blends with fibers from the muscle in the lower eyelid at the lateral palpebral ligament (Fig. 8.78). A third part of the orbicularis oculi muscle that can be identified consists of fibers on the medial border, which pass deeply to attach to the posterior lacrimal crest. These fibers form the lacrimal part of the orbicularis oculi, which may be involved in the drainage of tears.

1	Deep to the palpebral part of the orbicularis oculi is an extension of periosteum into both the upper and lower eyelids from the margin of the orbit (Fig. 8.79). This is the orbital septum, which extends downward into the upper eyelid and upward into the lower eyelid and is continuous with the periosteum outside and inside the orbit (Fig. 8.79). The orbital septum attaches to the tendon of the levator palpebrae superioris muscle in the upper eyelid and attaches to the tarsus in the lower eyelid. Providing major support for each eyelid is the tarsus (Fig. 8.80). There is a large superior tarsus in the upper eyelid and a smaller inferior tarsus in the lower eyelid (Fig. 8.80). These plates of dense connective tissue are attached medially to the anterior lacrimal crest of the maxilla by the medial palpebral ligament and laterally to the orbital tubercle on the zygomatic bone by the lateral palpebral ligament.

1	Although the tarsal plates in the upper and lower eyelids are generally similar in structure and function, there is one unique difference. Associated with the tarsus in the upper eyelid is the levator palpebrae superioris muscle (Fig. 8.80), which raises the eyelid. Its origin is from the posterior part of the roof of the orbit, just superior to the optic foramen, and it inserts into the anterior surface of the superior tarsus, with the possibility of a few fibers attaching to the skin of the upper eyelid. It is innervated by the oculomotor nerve [III]. In companion with the levator palpebrae superioris muscle is a collection of smooth muscle fibers passing from the inferior surface of the levator to the upper edge of the superior tarsus (see Fig. 8.77). Innervated by postganglionic sympathetic fibers from the superior cervical ganglion, this muscle is the superior tarsal muscle.

1	Loss of function of either the levator palpebrae superioris muscle or the superior tarsal muscle results in a ptosis or drooping of the upper eyelid. The structure of the eyelid is completed by a thin membrane (the conjunctiva), which covers the posterior surface of each eyelid (see Fig. 8.77). This membrane covers the full extent of the posterior surface of each eyelid before reflecting onto the outer surface (sclera) of the eyeball. It attaches to the eyeball at the junction between the sclera and the cornea. With this membrane in place, a conjunctival sac is formed when the eyelids are closed, and the upper and lower extensions of this sac are the superior and inferior conjunctival fornices (Fig. 8.77).

1	Embedded in the tarsal plates are tarsal glands (see Fig. 8.77), which empty onto the free margin of each eyelid. These glands are modified sebaceous glands and secrete an oily substance that increases the viscosity of the tears and decreases the rate of evaporation of tears from the surface of the eyeball. Blockage and inflammation of a tarsal gland is a chalazion and is on the inner surface of the eyelid. The tarsal glands are not the only glands associated with the eyelids. Associated with the eyelash follicles are sebaceous and sweat glands (see Fig. 8.77). Blockage and inflammation of either of these is a stye and is on the edge of the eyelid.

1	The arterial supply to the eyelids is from the numerous vessels in the area (Fig. 8.81). They include: the supratrochlear, supra-orbital, lacrimal, and dorsal nasal arteries from the ophthalmic artery; the angular artery from the facial artery; the transverse facial artery from the superficial temporal artery; and branches from the superficial temporal artery itself. Venous drainage follows an external pattern through veins associated with the various arteries and an internal pattern moving into the orbit through connections with the ophthalmic veins. Lymphatic drainage is primarily to the parotid nodes, with some drainage from the medial corner of the eye along lymphatic vessels associated with the angular and facial arteries to the submandibular nodes. Innervation of the eyelids includes both sensory and motor components.

1	Innervation of the eyelids includes both sensory and motor components. The sensory nerves are all branches of the trigeminal nerve [V] (Fig. 8.82). Palpebral branches arise from: the supra-orbital, supratrochlear, infratrochlear, and lacrimal branches of the ophthalmic nerve [V1]; and the infra-orbital branch of the maxillary nerve [V2]. Motor innervation is from: the facial nerve [VII], which innervates the palpebral part of the orbicularis oculi; the oculomotor nerve [III], which innervates the levator palpebrae superioris; and sympathetic fibers, which innervate the superior tarsal muscle. Loss of innervation of the orbicularis oculi by the facial nerve [VII] causes an inability to close the eyelids tightly and the lower eyelid droops away, resulting in a spillage of tears. Loss of innervation of the levator palpebrae superioris by the oculomotor nerve causes an inability to open the superior eyelid voluntarily, producing a complete ptosis.

1	Loss of innervation of the levator palpebrae superioris by the oculomotor nerve causes an inability to open the superior eyelid voluntarily, producing a complete ptosis. Loss of innervation of the superior tarsal muscle by sympathetic fibers causes a constant partial ptosis. The lacrimal apparatus is involved in the production, movement, and drainage of fluid from the surface of the eyeball. It is made up of the lacrimal gland and its ducts, the lacrimal canaliculi, the lacrimal sac, and the nasolacrimal duct. The lacrimal gland is anterior in the superolateral region of the orbit (Fig. 8.83) and is divided into two parts by the levator palpebrae superioris (Fig. 8.84): The larger orbital part is in a depression, the lacrimal fossa, in the frontal bone. The smaller palpebral part is inferior to the levator palpebrae superioris in the superolateral part of the eyelid. Numerous ducts empty the glandular secretions into the lateral part of the superior fornix of the conjunctiva.

1	Numerous ducts empty the glandular secretions into the lateral part of the superior fornix of the conjunctiva. Fluid is continually being secreted by the lacrimal gland and moved across the surface of the eyeball from lateral to medial as the eyelids blink. The fluid accumulates medially in the lacrimal lake and is drained from the lake by the lacrimal canaliculi, one canaliculus associated with each eyelid (Fig. 8.83). The lacrimal punctum is the opening through which fluid enters each canaliculus. Passing medially, the lacrimal canaliculi eventually join the lacrimal sac between the anterior and posterior lacrimal crests, posterior to the medial palpebral ligament and anterior to the lacrimal part of the orbicularis oculi muscle (Figs. 8.85 and 8.86). When the orbicularis oculi muscle contracts during blinking, the small lacrimal part of the muscle may dilate the lacrimal sac and draw tears into it through the canaliculi from the conjunctival sac.

1	The innervation of the lacrimal gland involves three different components (Fig. 8.87). Sensory neurons from the lacrimal gland return to the CNS through the lacrimal branch of the ophthalmic nerve [V1]. Secretomotor fibers from the parasympathetic part of the autonomic division of the PNS stimulate fluid secretion from the lacrimal gland. These preganglionic parasympathetic neurons leave the CNS in the facial nerve [VII], enter the greater petrosal nerve (a branch of the facial nerve [VII]), and continue with this nerve until it becomes the nerve of the pterygoid canal (Fig. 8.87).

1	The nerve of the pterygoid canal eventually joins the pterygopalatine ganglion where the preganglionic parasympathetic neurons synapse on postganglionic parasympathetic neurons. The postganglionic neurons join the maxillary nerve [V2] and continue with it until the zygomatic nerve branches from it, and travel with the zygomatic nerve until it gives off the zygomaticotemporal nerve, which eventually distributes postganglionic parasympathetic fibers in a small branch that joins the lacrimal nerve. The lacrimal nerve passes to the lacrimal gland.

1	Sympathetic innervation of the lacrimal gland follows a similar path as parasympathetic innervation. Postganglionic sympathetic fibers originating in the superior cervical ganglion travel along the plexus surrounding the internal carotid artery (Fig. 8.87). They leave this plexus as the deep petrosal nerve and join the parasympathetic fibers in the nerve of the pterygoid canal. Passing through the pterygopalatine ganglion, the sympathetic fibers from this point onward follow the same path as the parasympathetic fibers to the lacrimal gland. The arterial supply to the lacrimal gland is by branches from the ophthalmic artery and venous drainage is through the ophthalmic veins. Numerous structures enter and leave the orbit through a variety of openings (Fig. 8.88).

1	Numerous structures enter and leave the orbit through a variety of openings (Fig. 8.88). When the bony orbit is viewed from an anterolateral position, the round opening at the apex of the pyramidal-shaped orbit is the optic canal, which opens into the middle cranial fossa and is bounded medially by the body of the sphenoid and laterally by the lesser wing of the sphenoid. Passing through the optic canal are the optic nerve and the ophthalmic artery (Fig. 8.89). Just lateral to the optic canal is a triangular-shaped gap between the roof and lateral wall of the bony orbit. This is the superior orbital fissure and allows structures to pass between the orbit and the middle cranial fossa (Fig. 8.88). Passing through the superior orbital fissure are the superior and inferior branches of the oculomotor nerve [III], the trochlear nerve [IV], the abducent nerve [VI], the lacrimal, frontal, and nasociliary branches of the ophthalmic nerve [V1], and the superior ophthalmic vein (Fig. 8.89).

1	Separating the lateral wall of the orbit from the floor of the orbit is a longitudinal opening, the inferior orbital fissure (Fig. 8.88). Its borders are the greater wing of the sphenoid and the maxilla, palatine, and zygomatic bones. This long fissure allows communication between: the orbit and the pterygopalatine fossa posteriorly, the orbit and the infratemporal fossa in the middle, and the orbit and the temporal fossa posterolaterally. Passing through the inferior orbital fissure are the maxillary nerve [V2] and its zygomatic branch, the infra-orbital vessels, and a vein communicating with the pterygoid plexus of veins. Beginning posteriorly and crossing about two-thirds of the inferior orbital fissure, a groove (the infra-orbital groove) is encountered, which continues anteriorly across the floor of the orbit (Fig. 8.88). This groove connects with the infra-orbital canal that opens onto the face at the infra-orbital foramen.

1	The infra-orbital nerve, part of the maxillary nerve [V2], and vessels pass through this structure as they exit onto the face. Associated with the medial wall of the bony orbit are several smaller openings (Fig. 8.88). The anterior and posterior ethmoidal foramina are at the junction between the superior and medial walls. These openings provide exits from the orbit into the ethmoid bone for the anterior and posterior ethmoidal nerves and vessels. Completing the openings on the medial wall is a canal in the lower part of the wall anteriorly. Clearly visible is the depression for the lacrimal sac formed by the lacrimal bone and the frontal process of the maxilla. This depression is continuous with the nasolacrimal canal, which leads to the inferior nasal meatus. Contained within the nasolacrimal canal is the nasolacrimal duct, a part of the lacrimal apparatus.

1	The periosteum lining the bones that form the orbit is the periorbita (Fig. 8.90A). It is continuous at the margins of the orbit with the periosteum on the outer surface of the skull and sends extensions into the upper and lower eyelids (the orbital septa). At the various openings where the orbit communicates with the cranial cavity the periorbita is continuous with the periosteal layer of dura mater. In the posterior part of the orbit, the periorbita thickens around the optic canal and the central part of the superior orbital fissure. This is the point of origin of the four rectus muscles and is the common tendinous ring. Fascial sheath of the eyeball The fascial sheath of the eyeball (bulbar sheath) is a layer of fascia that encloses a major part of the eyeball (Figs. 8.91 and 8.92): Posteriorly, it is firmly attached to the sclera (the white part of the eyeball) around the point of entrance of the optic nerve into the eyeball.

1	Posteriorly, it is firmly attached to the sclera (the white part of the eyeball) around the point of entrance of the optic nerve into the eyeball. Anteriorly, it is firmly attached to the sclera near the edge of the cornea (the clear part of the eyeball). Additionally, as the muscles approach the eyeball, the investing fascia surrounding each muscle blends with the fascial sheath of the eyeball as the muscles pass through and continue to their point of attachment. A specialized lower part of the fascial sheath of the eyeball is the suspensory ligament (Figs. 8.91 and 8.92), which supports the eyeball. This “sling-like” structure is made up of the fascial sheath of the eyeball and contributions from the two inferior ocular muscles and the medial and lateral ocular muscles. Check ligaments of the medial and lateral

1	Check ligaments of the medial and lateral Other fascial specializations in the orbit are the check ligaments (Fig. 8.92). These are expansions of the investing fascia covering the medial and lateral rectus muscles, which attach to the medial and lateral walls of the bony orbit: The medial check ligament is an extension from the fascia covering the medial rectus muscle and attaches immediately posterior to the posterior lacrimal crest of the lacrimal bone. The lateral check ligament is an extension from the fascia covering the lateral rectus muscle and is attached to the orbital tubercle of the zygomatic bone. Functionally, the positioning of these ligaments seems to restrict the medial and lateral rectus muscles, thus the names of the fascial specializations.

1	Functionally, the positioning of these ligaments seems to restrict the medial and lateral rectus muscles, thus the names of the fascial specializations. There are two groups of muscles within the orbit: extrinsic muscles of eyeball (extra-ocular muscles) involved in movements of the eyeball or raising upper eyelids, and intrinsic muscles within the eyeball, which control the shape of the lens and size of the pupil. The extrinsic muscles include the levator palpebrae superioris, superior rectus, inferior rectus, medial rectus, lateral rectus, superior oblique, and inferior oblique. The intrinsic muscles include the ciliary muscle, the sphincter pupillae, and the dilator pupillae. Of the seven muscles in the extrinsic group of muscles, one raises the eyelids, whereas the other six move the eyeball itself (Table 8.8).

1	Of the seven muscles in the extrinsic group of muscles, one raises the eyelids, whereas the other six move the eyeball itself (Table 8.8). The movements of the eyeball, in three dimensions, (Fig. 8.93) are: elevation—moving the pupil superiorly, depression—moving the pupil inferiorly, abduction—moving the pupil laterally, adduction—moving the pupil medially, internal rotation (intorsion)—rotating the upper part of the pupil medially (or toward the nose), and external rotation (extorsion)—rotating the upper part of the pupil laterally (or toward the temple). The axis of each orbit is directed slightly laterally from back to front, but each eyeball is directed anteriorly (Fig. 8.94). Therefore the pull of some muscles has multiple effects on the movement of the eyeball, whereas that of others has a single effect.

1	Levator palpebrae superioris raises the upper eyelid (Table 8.8). It is the most superior muscle in the orbit, originating from the roof, just anterior to the optic canal on the inferior surface of the lesser wing of the sphenoid (Fig. 8.95B). Its primary point of insertion is into the anterior surface of the superior tarsus, but a few fibers also attach to the skin of the upper eyelid and the superior conjunctival fornix. Innervation is by the superior branch of the oculomotor nerve [III]. Contraction of the levator palpebrae superioris raises the upper eyelid. A unique feature of the levator palpebrae superioris is that a collection of smooth muscle fibers passes from its inferior surface to the upper edge of the superior tarsus (see Fig. 8.77). This group of smooth muscle fibers (the superior tarsal muscle) help maintain eyelid elevation and are innervated by postganglionic sympathetic fibers from the superior cervical ganglion.

1	Loss of oculomotor nerve [III] function results in complete ptosis or drooping of the superior eyelid, whereas loss of sympathetic innervation to the superior tarsal muscle results in partial ptosis. Four rectus muscles occupy medial, lateral, inferior, and superior positions as they pass from their origins posteriorly to their points of attachment on the anterior half of the eyeball (Fig. 8.95 and Table 8.8). They originate as a group from a common tendinous ring at the apex of the orbit and form a cone of muscles as they pass forward to their attachment on the eyeball. The superior and inferior rectus muscles have complicated actions because the apex of the orbit, where the muscles originate, is medial to the central axis of the eyeball when looking directly forward: The superior rectus originates from the superior part of the common tendinous ring above the optic canal.

1	The superior rectus originates from the superior part of the common tendinous ring above the optic canal. The inferior rectus originates from the inferior part of the common tendinous ring below the optic canal (Fig. 8.96). As these muscles pass forward in the orbit to attach to the anterior half of the eyeball, they are also directed laterally (Fig. 8.95). Because of these orientations: Contraction of the superior rectus elevates, adducts, and internally rotates the eyeball (Fig. 8.97A). Contraction of the inferior rectus depresses, adducts, and externally rotates the eyeball (Fig. 8.97A). The superior branch of the oculomotor nerve [III] innervates the superior rectus, and the inferior branch of the oculomotor nerve [III] innervates the inferior rectus.

1	The superior branch of the oculomotor nerve [III] innervates the superior rectus, and the inferior branch of the oculomotor nerve [III] innervates the inferior rectus. To isolate the function of and to test the superior and inferior rectus muscles, a patient is asked to track a physician’s finger laterally and then either upward or downward (Fig. 8.97B). The first movement brings the axis of the eyeball into alignment with the long axis of the superior and inferior rectus muscles. Moving the finger upward tests the superior rectus muscle and moving it downward tests the inferior rectus muscle (Fig. 8.97B). The orientation and actions of the medial and lateral rectus muscles are more straightforward than those of the superior and inferior rectus muscles.

1	The orientation and actions of the medial and lateral rectus muscles are more straightforward than those of the superior and inferior rectus muscles. The medial rectus originates from the medial part of the common tendinous ring medial to and below the optic canal, whereas the lateral rectus originates from the lateral part of the common tendinous ring as the common tendinous ring bridges the superior orbital fissure (Fig. 8.96). The medial and lateral rectus muscles pass forward and attach to the anterior half of the eyeball (Fig. 8.95). Contraction of medial rectus adducts the eyeball, whereas contraction of lateral rectus abducts the eyeball (Fig. 8.97A). The inferior branch of the oculomotor nerve [III] innervates the medial rectus, and the abducent nerve [VI] innervates the lateral rectus.

1	The inferior branch of the oculomotor nerve [III] innervates the medial rectus, and the abducent nerve [VI] innervates the lateral rectus. To isolate the function of and test the medial and lateral rectus muscles, a patient is asked to track a physician’s finger medially and laterally, respectively, in the horizontal plane (Fig. 8.97B). The oblique muscles are in the superior and inferior parts of the orbit, do not originate from the common tendinous ring, are angular in their approaches to the eyeball, and, unlike the rectus muscles, attach to the posterior half of the eyeball (Table 8.8). The superior oblique arises from the body of the sphenoid, superior and medial to the optic canal and medial to the origin of the levator palpebrae superioris (Figs. 8.95 and 8.96). It passes forward, along the medial border of the roof of the orbit, until it reaches a fibrocartilaginous pulley (the trochlea), which is attached to the trochlear fovea of the frontal bone.

1	The tendon of the superior oblique passes through the trochlea and turns laterally to cross the eyeball in a posterolateral direction. It continues deep to the superior rectus muscle and inserts into the outer posterior quadrant of the eyeball. Contraction of the superior oblique therefore directs the pupil down and out (Fig. 8.97A). The trochlear nerve [IV] innervates the superior oblique along its superior surface. To isolate the function of and to test the superior oblique muscle, a patient is asked to track a physician’s finger medially to bring the axis of the tendon of the muscle into alignment with the axis of the eyeball, and then to look down, which tests the muscle (Fig. 8.97B).

1	The inferior oblique is the only extrinsic muscle that does not take origin from the posterior part of the orbit. It arises from the medial side of the floor of the orbit, just posterior to the orbital rim, and is attached to the orbital surface of the maxilla just lateral to the nasolacrimal groove (Fig. 8.95). The inferior oblique crosses the floor of the orbit in a posterolateral direction between the inferior rectus and the floor of the orbit, before inserting into the outer posterior quadrant just under the lateral rectus. Contraction of the inferior oblique directs the pupil up and out (Fig. 8.97A). The inferior branch of the oculomotor nerve innervates the inferior oblique. To isolate the function of and to test the inferior oblique muscle, a patient is asked to track a physician’s finger medially to bring the axis of the eyeball into alignment with the axis of the muscle and then to look up, which tests the muscle (Fig. 8.97B).

1	Six of the seven extrinsic muscles of the orbit are directly involved in movements of the eyeball. For each of the rectus muscles, the medial, lateral, inferior, and superior, and the superior and inferior obliques, a specific action or group of actions can be described (Table 8.8). However, these muscles do not act in isolation. They work as teams of muscles in the coordinated movement of the eyeball to position the pupil as needed. For example, although the lateral rectus is the muscle primarily responsible for moving the eyeball laterally, it is assisted in this action by the superior and inferior oblique muscles. The arterial supply to the structures in the orbit, including the eyeball, is by the ophthalmic artery (Fig. 8.99). This vessel is a branch of the internal carotid artery, given off immediately after the internal carotid artery leaves the cavernous sinus. The ophthalmic artery passes into the orbit through the optic canal with the optic nerve.

1	In the orbit the ophthalmic artery initially lies inferior and lateral to the optic nerve (Fig. 8.99). As it passes forward in the orbit, it crosses superior to the optic nerve and proceeds anteriorly on the medial side of the orbit.

1	In the orbit the ophthalmic artery gives off numerous branches as follows: the lacrimal artery, which arises from the ophthalmic artery on the lateral side of the optic nerve, and passes anteriorly on the lateral side of the orbit, supplying the lacrimal gland, muscles, the anterior ciliary branch to the eyeball, and the lateral sides of the eyelid; the central retinal artery, which enters the optic nerve, proceeds down the center of the nerve to the retina, and is clearly seen when viewing the retina with an ophthalmoscope—occlusion of this vessel or of the parent artery leads to blindness; the long and short posterior ciliary arteries, which are branches that enter the eyeball posteriorly, piercing the sclera, and supplying structures inside the eyeball; the muscular arteries, which are branches supplying the intrinsic muscles of the eyeball; the supra-orbital artery, which usually arises from the ophthalmic artery immediately after it has crossed the optic nerve, proceeds

1	which are branches supplying the intrinsic muscles of the eyeball; the supra-orbital artery, which usually arises from the ophthalmic artery immediately after it has crossed the optic nerve, proceeds anteriorly, and exits the orbit through the supra-orbital foramen with the supra-orbital nerve—it supplies the forehead and scalp as it passes across these areas to the vertex of the skull; the posterior ethmoidal artery, which exits the orbit through the posterior ethmoidal foramen to supply the ethmoidal cells and nasal cavity; the anterior ethmoidal artery, which exits the orbit through the anterior ethmoidal foramen, enters the cranial cavity giving off the anterior meningeal branch, and continues into the nasal cavity supplying the septum and lateral wall, and ending as the dorsal nasal artery; the medial palpebral arteries, which are small branches supplying the medial area of the upper and lower eyelids; the dorsal nasal artery, which is one of the two terminal branches of the

1	artery; the medial palpebral arteries, which are small branches supplying the medial area of the upper and lower eyelids; the dorsal nasal artery, which is one of the two terminal branches of the ophthalmic artery, leaves the orbit to supply the upper surface of the nose; and the supratrochlear artery, which is the other terminal branch of the ophthalmic artery and leaves the orbit with the supratrochlear nerve, supplying the forehead as it passes across it in a superior direction.

1	There are two venous channels in the orbit, the superior and inferior ophthalmic veins (Fig. 8.100). The superior ophthalmic vein begins in the anterior area of the orbit as connecting veins from the supra-orbital vein and the angular vein join together. It passes across the superior part of the orbit, receiving tributaries from the companion veins to the branches of the ophthalmic artery and veins draining the posterior part of the eyeball. Posteriorly, it leaves the orbit through the superior orbital fissure and enters the cavernous sinus. The inferior ophthalmic vein is smaller than the superior ophthalmic vein, begins anteriorly, and passes across the inferior part of the orbit. It receives various tributaries from muscles and the posterior part of the eyeball as it crosses the orbit.

1	The inferior ophthalmic vein leaves the orbit posteriorly by: joining with the superior ophthalmic vein, passing through the superior orbital fissure on its own to join the cavernous sinus, or passing through the inferior orbital fissure to join with the pterygoid plexus of veins in the infratemporal fossa. Because the ophthalmic veins communicate with the cavernous sinus, they act as a route by which infections can spread from outside to inside the cranial cavity. Numerous nerves pass into the orbit and innervate structures within its bony walls. They include the optic nerve [II], the oculomotor nerve [III], the trochlear nerve [IV], the abducent nerve [VI], and autonomic nerves. Other nerves such as the ophthalmic nerve [V1] innervate orbital structures and then travel out of the orbit to innervate other regions.

1	The optic nerve [II] is not a true cranial nerve, but rather an extension of the brain carrying afferent fibers from the retina of the eyeball to the visual centers of the brain. The optic nerve is surrounded by the cranial meninges, including the subarachnoid space, which extends as far forward as the eyeball. Any increase in intracranial pressure therefore results in increased pressure in the subarachnoid space surrounding the optic nerve. This may impede venous return along the retinal veins, causing edema of the optic disc (papilledema), which can be seen when the retina is examined using an ophthalmoscope. The optic nerve leaves the orbit through the optic canal (Fig. 8.101). It is accompanied in the optic canal by the ophthalmic artery. The oculomotor nerve [III] leaves the anterior surface of the brainstem between the midbrain and the pons. It passes forward in the lateral wall of the cavernous sinus.

1	The oculomotor nerve [III] leaves the anterior surface of the brainstem between the midbrain and the pons. It passes forward in the lateral wall of the cavernous sinus. Just before entering the orbit the oculomotor nerve [III] divides into superior and inferior branches (Fig. 8.102). These branches enter the orbit through the superior orbital fissure, lying within the common tendinous ring (Fig. 8.101). Inside the orbit the small superior branch passes upward over the lateral side of the optic nerve to innervate the superior rectus and levator palpebrae superioris muscles (Fig. 8.102). The large inferior branch divides into three branches: one passing below the optic nerve as it passes to the medial side of the orbit to innervate the medial rectus muscle, a second descending to innervate the inferior rectus muscle, and the third descending as it runs forward along the floor of the orbit to innervate the inferior oblique muscle (Fig. 8.102).

1	As the third branch descends, it gives off the branch to the ciliary ganglion. This is the parasympathetic root to the ciliary ganglion and carries preganglionic parasympathetic fibers that will synapse in the ciliary ganglion with postganglionic parasympathetic fibers. The postganglionic fibers are distributed to the eyeball through short ciliary nerves and innervate the sphincter pupillae and ciliary muscles. The trochlear nerve [IV] arises from the posterior surface of the midbrain, and passes around the midbrain to enter the edge of the tentorium cerebelli. It continues on an intradural path arriving in and passing through the lateral wall of the cavernous sinus just below the oculomotor nerve [III].

1	Just before entering the orbit, the trochlear nerve ascends, passing across the oculomotor nerve [III] and entering the orbit through the superior orbital fissure above the common tendinous ring (Fig. 8.101). In the orbit the trochlear nerve [IV] ascends and turns medially, crossing above the levator palpebrae superioris muscle to enter the upper border of the superior oblique muscle (Fig. 8.103). The abducent nerve [VI] arises from the brainstem between the pons and medulla. It enters the dura covering the clivus and continues in a dural canal until it reaches the cavernous sinus. The abducent nerve enters the cavernous sinus and runs through the sinus lateral to the internal carotid artery. It passes out of the sinus and enters the orbit through the superior orbital fissure within the common tendinous ring (Fig. 8.101). Once in the orbit it courses laterally to supply the lateral rectus muscle.

1	Preganglionic sympathetic fibers arise from the upper segments of the thoracic spinal cord, mainly T1. They enter the sympathetic chain through white rami communicantes, and ascend to the superior cervical ganglion where they synapse with postganglionic sympathetic fibers. The postganglionic fibers are distributed along the internal carotid artery and its branches. The postganglionic sympathetic fibers destined for the orbit travel with the ophthalmic artery. Once in the orbit the fibers are distributed to the eyeball either by: passing through the ciliary ganglion, without synapsing, and joining the short ciliary nerves, which pass from the ganglion to the eyeball; or passing through long ciliary nerves to reach the eyeball. In the eyeball postganglionic sympathetic fibers innervate the dilator pupillae muscle.

1	In the eyeball postganglionic sympathetic fibers innervate the dilator pupillae muscle. The ophthalmic nerve [V1] is the smallest and most superior of the three divisions of the trigeminal nerve. This purely sensory nerve receives input from structures in the orbit and from additional branches on the face and scalp. Leaving the trigeminal ganglion, the ophthalmic nerve [V1] passes forward in the lateral wall of the cavernous sinus inferior to the trochlear [IV] and oculomotor [III] nerves. Just before it enters the orbit it divides into three branches—the nasociliary, lacrimal, and frontal nerves (Fig. 8.104). These branches enter the orbit through the superior orbital fissure with the frontal and lacrimal nerves outside the common tendinous ring, and the nasociliary nerve within the common tendinous ring (Fig. 8.101).

1	The lacrimal nerve is the smallest of the three branches of the ophthalmic nerve [V1]. Once in the orbit it passes forward along the upper border of the lateral rectus muscle (Fig. 8.105). It receives a branch from the zygomaticotemporal nerve, which carries parasympathetic and sympathetic postganglionic fibers for distribution to the lacrimal gland. Reaching the anterolateral aspect of the orbit, the lacrimal nerve supplies the lacrimal gland, conjunctiva, and lateral part of the upper eyelid. The frontal nerve is the largest branch of the ophthalmic nerve [V1] and receives sensory input from areas outside the orbit. Exiting the superior orbital fissure, this branch passes forward between the levator palpebrae superioris and the periorbita on the roof of the orbit (Fig. 8.101). About midway across the orbit it divides into its two terminal branches—the supra-orbital and supratrochlear nerves (Figs. 8.104 and 8.105):

1	The supratrochlear nerve continues forward in an anteromedial direction, passing above the trochlea, exits the orbit medial to the supra-orbital foramen, and supplies the conjunctiva and skin of the upper eyelid and the skin on the lower medial part of the forehead. The supra-orbital nerve is the larger of the two branches, continues forward, passing between the levator palpebrae superioris muscle and the periorbita covering the roof of the orbit (Fig. 8.105), exits the orbit through the supra-orbital notch and ascends across the forehead and scalp, supplying the upper eyelid and conjunctiva, the forehead, and as far posteriorly as the middle of the scalp.

1	The nasociliary nerve is intermediate in size between the frontal and lacrimal nerves and is usually the first branch from the ophthalmic nerve (Fig. 8.104). It is most deeply placed in the orbit, entering the area within the common tendinous ring between the superior and inferior branches of the oculomotor nerve [III] (see Fig. 8.101). Once in the orbit, the nasociliary nerve crosses the superior surface of the optic nerve as it passes in a medial direction below the superior rectus muscle (Figs. 8.104 and 8.106). Its first branch, the communicating branch with the ciliary ganglion (sensory root to the ciliary ganglion), is given off early in its path through the orbit.

1	The nasociliary nerve continues forward along the medial wall of the orbit, between the superior oblique and the medial rectus muscles, giving off several branches (Fig. 8.106). These include: the long ciliary nerves, which are sensory to the eyeball but may also contain sympathetic fibers for pupillary dilation; the posterior ethmoidal nerve, which exits the orbit through the posterior ethmoidal foramen to supply posterior ethmoidal cells and the sphenoidal sinus; the infratrochlear nerve, which distributes to the medial part of the upper and lower eyelids, the lacrimal sac, and skin of the upper half of the nose; and the anterior ethmoidal nerve, which exits the orbit through the anterior ethmoidal foramen to supply the anterior cranial fossa, nasal cavity, and skin of the lower half of the nose (Fig. 8.106).

1	The ciliary ganglion is a parasympathetic ganglion of the oculomotor nerve [III]. It is associated with the nasociliary branch of the ophthalmic nerve [V1] and is the site where preganglionic and postganglionic parasympathetic neurons synapse as fibers from this part of the autonomic division of the PNS make their way to the eyeball. The ciliary ganglion is also traversed by postganglionic sympathetic fibers and sensory fibers as they travel to the eyeball. The ciliary ganglion is a very small ganglion, in the posterior part of the orbit immediately lateral to the optic nerve and between the optic nerve and the lateral rectus muscle (Fig. 8.106). It is usually described as receiving at least two, and possibly three, branches or roots from other nerves in the orbit.

1	As the inferior branch of the oculomotor nerve [III] passes the area of the ciliary ganglion, it sends a branch to the ganglion (the parasympathetic root). The parasympathetic branch carries preganglionic parasympathetic fibers, which enter the ganglion and synapse with postganglionic parasympathetic fibers within the ganglion (Fig. 8.107). The postganglionic parasympathetic fibers leave the ganglion through short ciliary nerves, which enter the posterior aspect of the eyeball around the optic nerve. In the eyeball the parasympathetic fibers innervate: the sphincter pupillae muscle, responsible for pupillary constriction, and the ciliary muscle, responsible for accommodation of the lens of the eye for near vision.

1	A second branch (the sensory root), passes from the nasociliary nerve to the ganglion (Fig. 8.107). This branch enters the posterosuperior aspect of the ganglion, and carries sensory fibers, which pass through the ganglion and continue along the short ciliary nerves to the eyeball. These fibers are responsible for sensory innervation to all parts of the eyeball; however, the sympathetic fibers also may take alternative routes to the eyeball.

1	The third branch to the ciliary ganglion is the most variable. This branch, when present, is the sympathetic root and contains postganglionic sympathetic fibers from the superior cervical ganglion (Fig. 8.107). These fibers travel up the internal carotid artery, leave the plexus surrounding the artery in the cavernous sinus, and enter the orbit through the common tendinous ring. In the orbit they enter the posterior aspect of the ciliary ganglion, cross the ganglion, and continue along the short ciliary nerves to the eyeball; however, the sympathetic fibers also may take alternative routes to the eyeball.

1	Sympathetic fibers to the eyeball may not enter the ganglion as a separate sympathetic root. Rather, the postganglionic sympathetic fibers may leave the plexus associated with the internal carotid artery in the cavernous sinus, join the ophthalmic nerve [V1], and course into the ciliary ganglion in the sensory root from the nasociliary nerve. In addition, the sympathetic fibers carried in the nasociliary nerve may not enter the ganglion at all and may course directly into the eyeball in the long ciliary nerves (Fig. 8.107). Whatever their path, postganglionic sympathetic fibers reach the eyeball and innervate the dilator pupillae muscle. The globe-shaped eyeball occupies the anterior part of the orbit. Its rounded shape is disrupted anteriorly, where it bulges outward. This outward projection represents about one-sixth of the total area of the eyeball and is the transparent cornea (Fig. 8.108).

1	Posterior to the cornea and in order from front to back are the anterior chamber, the iris and pupil, the posterior chamber, the lens, the postremal (vitreous) chamber, and the retina. The anterior chamber is the area directly posterior to the cornea and anterior to the colored part of the eye (iris). The central opening in the iris is the pupil. Posterior to the iris and anterior to the lens is the smaller posterior chamber. The anterior and posterior chambers are continuous with each other through the pupillary opening. They are filled with a fluid (aqueous humor), which is secreted into the posterior chamber, flows into the anterior chamber through the pupil, and is absorbed into the scleral venous sinus (the canal of Schlemm), which is a circular venous channel at the junction between the cornea and the iris (Fig. 8.108).

1	The aqueous humor supplies nutrients to the avascular cornea and lens and maintains the intra-ocular pressure. If the normal cycle of its production and absorption is disturbed so that the amount of fluid increases, intra-ocular pressure will increase. This condition (glaucoma) can lead to a variety of visual problems. The lens separates the anterior one-fifth of the eyeball from the posterior four-fifths (Fig. 8.108). It is a transparent, biconvex elastic disc attached circumferentially to muscles associated with the outer wall of the eyeball. This lateral attachment provides the lens with the ability to change its refractive ability to maintain visual acuity. The clinical term for opacity of the lens is a cataract.

1	The posterior four-fifths of the eyeball, from the lens to the retina, is occupied by the postremal (vitreous) chamber (Fig. 8.108). This segment is filled with a transparent, gelatinous substance—the vitreous body (vitreous humor). This substance, unlike aqueous humor, cannot be replaced. Walls of the eyeball Surrounding the internal components of the eyeball are the walls of the eyeball. They consist of three layers: an outer fibrous layer, a middle vascular layer, and an inner retinal layer (Fig. 8.108). The outer fibrous layer consists of the sclera posteriorly and the cornea anteriorly. The middle vascular layer consists of the choroid posteriorly and is continuous with the ciliary body and iris anteriorly. The inner layer consists of the optic part of the retina posteriorly and the nonvisual retina that covers the internal surface of the ciliary body and iris anteriorly. The arterial supply to the eyeball is from several sources:

1	The arterial supply to the eyeball is from several sources: The short posterior ciliary arteries are branches from the ophthalmic artery that pierce the sclera around the optic nerve and enter the choroid layer (Fig. 8.108). The long posterior ciliary arteries, usually two, enter the sclera on the medial and lateral sides of the optic nerve and proceed anteriorly in the choroid layer to anastomose with the anterior ciliary arteries. The anterior ciliary arteries are branches of the arteries supplying the muscles (Fig. 8.108)—as the muscles attach to the sclera, these arteries pierce the sclera to anastomose with the long posterior ciliary arteries in the choroid layer. The central retinal artery that has traversed the optic nerve and enters the area of the retina at the optic disc.

1	The central retinal artery that has traversed the optic nerve and enters the area of the retina at the optic disc. Venous drainage of the eyeball is primarily related to drainage of the choroid layer. Four large veins (the vorticose veins) are involved in this process. They exit through the sclera from each of the posterior quadrants of the eyeball and enter the superior and inferior ophthalmic veins. There is also a central retinal vein accompanying the central retinal artery. Fibrous layer of the eyeball The fibrous layer of the eyeball consists of two components—the sclera covers the posterior and lateral parts of the eyeball, about five-sixths of the surface, and the cornea covers the anterior part (Fig. 8.108).

1	The sclera is an opaque layer of dense connective tissue that can be seen anteriorly through its conjunctival covering as the “white of the eye.” It is pierced by numerous vessels and nerves, including the optic nerve posteriorly and provides attachment for the various muscles involved in eyeball movements. The fascial sheath of the eyeball covers the surface of the sclera externally from the entrance of the optic nerve to the corneoscleral junction while internally the surface of the sclera is loosely attached to the choroid of the vascular layer. Continuous with the sclera anteriorly is the transparent cornea. It covers the anterior one-sixth of the surface of the eyeball and, being transparent, allows light to enter the eyeball. Vascular layer of the eyeball The vascular layer of the eyeball consists of three continuous parts—the choroid, the ciliary body, and the iris from posterior to anterior (Fig. 8.108).

1	Vascular layer of the eyeball The vascular layer of the eyeball consists of three continuous parts—the choroid, the ciliary body, and the iris from posterior to anterior (Fig. 8.108). The choroid is posterior and represents approximately two-thirds of the vascular layer. It is a thin, highly vascular, pigmented layer consisting of smaller vessels adjacent to the retina and larger vessels more peripherally. It is firmly attached to the retina internally and loosely attached to the sclera externally. Extending from the anterior border of the choroid is the ciliary body (Fig. 8.108). This triangular-shaped structure, between the choroid and the iris, forms a complete ring around the eyeball. Its components include the ciliary muscle and the ciliary processes (Fig. 8.110).

1	The ciliary muscle consists of smooth muscle fibers arranged longitudinally, circularly, and radially. Controlled by parasympathetics traveling to the orbit in the oculomotor nerve [III], these muscle fibers, on contraction, decrease the size of the ring formed by the ciliary body. The ciliary processes are longitudinal ridges projecting from the inner surface of the ciliary body (Fig. 8.110). Extending from them are zonular fibers attached to the lens of the eyeball, which suspend the lens in its proper position and collectively form the suspensory ligament of the lens. Contraction of the ciliary muscle decreases the size of the ring formed by the ciliary body. This reduces tension on the suspensory ligament of the lens. The lens therefore becomes more rounded (relaxed) resulting in accommodation of the lens for near vision. Ciliary processes also contribute to the formation of aqueous humor.

1	Ciliary processes also contribute to the formation of aqueous humor. Completing the vascular layer of the eyeball anteriorly is the iris (Fig. 8.108). This circular structure, projecting outward from the ciliary body, is the colored part of the eye with a central opening (the pupil). Controlling the size of the pupil are smooth muscle fibers (sphincter pupillae) and myoepithelial cells (dilator pupillae) within the iris (Fig. 8.110): Fibers arranged in a circular pattern make up the sphincter pupillae muscle (Table 8.9), which is innervated by parasympathetics—contraction of its fibers decreases or constricts the pupillary opening. Contractile fibers arranged in a radial pattern make up the dilator pupillae muscle, which is innervated by sympathetics—contraction of its fibers increases or dilates the pupillary opening. Inner layer of the eyeball

1	Inner layer of the eyeball The inner layer of the eyeball is the retina (Fig. 8.108). It consists of two parts. Posteriorly and laterally is the optic part of the retina, which is sensitive to light, and anteriorly is the nonvisual part, which covers the internal surface of the ciliary body and the iris. The junction between these parts is an irregular line (the ora serrata). Optic part of the retina The optic part of the retina consists of two layers, an outer pigmented layer and an inner neural layer: The pigmented layer is firmly attached to the choroid and continues anteriorly over the internal surface of the ciliary body and iris. The neural layer, which can be further subdivided into its various neural components, is only attached to the pigmented layer around the optic nerve and at the ora serrata. It is the neural layer that separates in the case of a detached retina. Several obvious features are visible on the posterior surface of the optic part of the retina.

1	It is the neural layer that separates in the case of a detached retina. Several obvious features are visible on the posterior surface of the optic part of the retina. The optic disc is where the optic nerve leaves the retina (Fig. 8.109). It is lighter than the surrounding retina and branches of the central retinal artery spread from this point outward to supply the retina. As there are no lightsensitive receptor cells in the optic disc, it is referred to as a blind spot in the retina. Lateral to the optic disc a small area with a hint of yellowish coloration is the macula lutea with its central depression, the fovea centralis (Fig. 8.109). This is the thinnest area of the retina and visual sensitivity here is higher than elsewhere in the retina because it has fewer rods (light-sensitive receptor cells that function in dim light and are insensitive to color) and more cones (light-sensitive receptor cells that respond to bright light and are sensitive to color).

1	The ear is the organ of hearing and balance. It has three parts (Fig. 8.113): The first part is the external ear consisting of the part attached to the lateral aspect of the head and the canal leading inward. The second part is the middle ear—a cavity in the petrous part of the temporal bone bounded laterally, and separated from the external canal, by a membrane and connected internally to the pharynx by a narrow tube. The third part is the internal ear consisting of a series of cavities within the petrous part of the temporal bone between the middle ear laterally and the internal acoustic meatus medially. The internal ear converts the mechanical signals received from the middle ear, which start as sound captured by the external ear, into electrical signals to transfer information to the brain. The internal ear also contains receptors that detect motion and position.

1	The external ear consists of two parts. The part projecting from the side of the head is the auricle (pinna) and the canal leading inward is the external acoustic meatus. The auricle is on the side of the head and assists in capturing sound. It consists of cartilage covered with skin and arranged in a pattern of various elevations and depressions (Fig. 8.114). The large outside rim of the auricle is the helix. It ends inferiorly at the fleshy lobule, the only part of the auricle not supported by cartilage. The hollow center of the auricle is the concha of the auricle. The external acoustic meatus leaves from the depths of this area. Just anterior to the opening of the external acoustic meatus, in front of the concha, is an elevation (the tragus). Opposite the tragus, and above the fleshy lobule, is another elevation (the antitragus). A smaller curved rim, parallel and anterior to the helix, is the antihelix.

1	Numerous intrinsic and extrinsic muscles are associated with the auricle: The intrinsic muscles pass between the cartilaginous parts of the auricle and may change the shape of the auricle. The extrinsic muscles, the anterior, superior, and posterior auricular muscles, pass from the scalp or skull to the auricle and may also play a role in positioning of the auricle (see Fig. 8.56). Both groups of muscles are innervated by the facial nerve [VII]. Sensory innervation of the auricle is from many sources (Fig. 8.115): The outer more superficial surfaces of the auricle are supplied by the great auricular nerve (anterior and posterior inferior portions) and the lesser occipital nerve (posterosuperior portion) from the cervical plexus and the auriculotemporal branch of the mandibular nerve [V3] (anterosuperior portion).

1	The deeper parts of the auricle are supplied by the vagus nerve [X] (the auricular branch) and the facial nerve [VII] (which sends a branch to the auricular branch of the vagus nerve [X]). The arterial supply to the auricle is from numerous sources. The external carotid artery supplies the posterior auricular artery, the superficial temporal artery supplies anterior auricular branches, and the occipital artery supplies a branch. Venous drainage is through vessels following the arteries. Lymphatic drainage of the auricle passes anteriorly into parotid nodes and posteriorly into mastoid nodes, and possibly into the upper deep cervical nodes.

1	Lymphatic drainage of the auricle passes anteriorly into parotid nodes and posteriorly into mastoid nodes, and possibly into the upper deep cervical nodes. The external acoustic meatus extends from the deepest part of the concha to the tympanic membrane (eardrum), a distance of approximately 1 inch (2.5 cm) (Fig. 8.116). Its walls consist of cartilage and bone. The lateral one-third is formed from cartilaginous extensions from some of the auricular cartilages and the medial two-thirds is a bony tunnel in the temporal bone. Throughout its length the external acoustic meatus is covered with skin, some of which contains hair and modified sweat glands producing cerumen (earwax). Its diameter varies, being wider laterally and narrow medially.

1	The external acoustic meatus does not follow a straight course. From the external opening it passes upward in an anterior direction, then turns slightly posteriorly still passing upward, and finally, turns again in an anterior direction with a slight descent. For examination purposes, observation of the external acoustic meatus and tympanic membrane can be improved by pulling the ear superiorly, posteriorly, and slightly laterally. Sensory innervation of the external acoustic meatus is from several of the cranial nerves. The major sensory input travels through branches of the auriculotemporal nerve, a branch of the mandibular nerve [V3] (anterior and superior walls), and in the auricular branch of the vagus nerve [X] (posterior and inferior walls). A minor sensory input may also come from a branch of the facial nerve [VII] to the auricular branch of the vagus nerve [X].

1	The tympanic membrane separates the external acoustic meatus from the middle ear (Figs. 8.117 and 8.118). It is at an angle, sloping medially from top to bottom and posteriorly to anteriorly. Its lateral surface therefore faces inferiorly and anteriorly. It consists of a connective tissue core lined with skin on the outside and mucous membrane on the inside. Around the periphery of the tympanic membrane a fibrocartilaginous ring attaches it to the tympanic part of the temporal bone. At its center, a concavity is produced by the attachment on its internal surface of the lower end of the handle of the malleus, part of the malleus bone in the middle ear. This point of attachment is the umbo of the tympanic membrane. Anteroinferior to the umbo of the tympanic membrane a bright reflection of light, referred to as the cone of light, is usually visible when examining the tympanic membrane with an otoscope.

1	Anteroinferior to the umbo of the tympanic membrane a bright reflection of light, referred to as the cone of light, is usually visible when examining the tympanic membrane with an otoscope. Superior to the umbo in an anterior direction is the attachment of the rest of the handle of the malleus (Fig. 8.118). At the most superior extent of this line of attachment a small bulge in the membrane marks the position of the lateral process of the malleus as it projects against the internal surface of the tympanic membrane. Extending away from this elevation, on the internal surface of the membrane, are the anterior and posterior malleolar folds. Superior to these folds the tympanic membrane is thin and slack (the pars flaccida), whereas the rest of the membrane is thick and taut (the pars tensa). Innervation of the external and internal surfaces of the tympanic membrane is by several cranial nerves:

1	Innervation of the external and internal surfaces of the tympanic membrane is by several cranial nerves: Sensory innervation of the skin on the outer surface of the tympanic membrane is primarily by the auriculotemporal nerve, a branch of the mandibular nerve [V3] with additional participation of the auricular branch of the vagus nerve [X], a small contribution by a branch of the facial nerve [VII] to the auricular branch of the vagus nerve [X], and possibly a contribution from the glossopharyngeal nerve [IX]. Sensory innervation of the mucous membrane on the inner surface of the tympanic membrane is carried entirely by the glossopharyngeal [IX] nerve.

1	Sensory innervation of the mucous membrane on the inner surface of the tympanic membrane is carried entirely by the glossopharyngeal [IX] nerve. The middle ear is an air-filled, mucous membrane–lined space in the temporal bone between the tympanic membrane laterally and the lateral wall of the internal ear medially. It is described as consisting of two parts (Fig. 8.119): the tympanic cavity immediately adjacent to the tympanic membrane, and the epitympanic recess superiorly.

1	The middle ear communicates with the mastoid area posteriorly and the nasopharynx (via the pharyngotympanic tube) anteriorly. Its basic function is to transmit vibrations of the tympanic membrane across the cavity of the middle ear to the internal ear. It accomplishes this through three interconnected but movable bones that bridge the space between the tympanic membrane and the internal ear. These bones are the malleus (connected to the tympanic membrane), the incus (connected to the malleus by a synovial joint), and the stapes (connected to the incus by a synovial joint, and attached to the lateral wall of the internal ear at the oval window). The middle ear has a roof and a floor, and anterior, posterior, medial, and lateral walls (Fig. 8.120).

1	The middle ear has a roof and a floor, and anterior, posterior, medial, and lateral walls (Fig. 8.120). The tegmental wall (roof) of the middle ear consists of a thin layer of bone, which separates the middle ear from the middle cranial fossa. This layer of bone is the tegmen tympani on the anterior surface of the petrous part of the temporal bone. The jugular wall (floor) of the middle ear consists of a thin layer of bone that separates it from the internal jugular vein. Occasionally, the floor is thickened by the presence of mastoid air cells. Near the medial border of the floor is a small aperture, through which the tympanic branch from the glossopharyngeal nerve [IX] enters the middle ear.

1	Near the medial border of the floor is a small aperture, through which the tympanic branch from the glossopharyngeal nerve [IX] enters the middle ear. The membranous (lateral) wall of the middle ear consists almost entirely of the tympanic membrane, but because the tympanic membrane does not extend superiorly into the epitympanic recess, the upper part of the membranous wall of the middle ear is the bony lateral wall of the epitympanic recess. The mastoid (posterior) wall of the middle ear is only partially complete. The lower part of this wall consists of a bony partition between the tympanic cavity and mastoid air cells. Superiorly, the epitympanic recess is continuous with the aditus to the mastoid antrum (Figs. 8.120 and 8.121).

1	Associated with the mastoid wall are: the pyramidal eminence, a small elevation through which the tendon of the stapedius muscle enters the middle ear; and the opening through which the chorda tympani nerve, a branch of the facial nerve [VII], enters the middle ear. The anterior wall of the middle ear is only partially complete. The lower part consists of a thin layer of bone that separates the tympanic cavity from the internal carotid artery. Superiorly, the wall is deficient because of the presence of: a large opening for the entrance of the pharyngotympanic tube into the middle ear, and a smaller opening for the canal containing the tensor tympani muscle. The foramen for the exit of the chorda tympani nerve from the middle ear is also associated with this wall (Fig. 8.120).

1	The foramen for the exit of the chorda tympani nerve from the middle ear is also associated with this wall (Fig. 8.120). The labyrinthine (medial) wall of the middle ear is also the lateral wall of the internal ear. A prominent structure on this wall is a rounded bulge (the promontory) produced by the basal coil of the cochlea, which is an internal ear structure involved with hearing (Fig. 8.120). Associated with the mucous membrane covering the promontory is a plexus of nerves (the tympanic plexus), which consists primarily of contributions from the tympanic branch of the glossopharyngeal nerve [IX] and branches from the internal carotid plexus. It supplies the mucous membrane of the middle ear, the mastoid area, and the pharyngotympanic tube.

1	Additionally, a branch of the tympanic plexus (the lesser petrosal nerve) leaves the promontory and the middle ear, travels across the anterior surface of the petrous part of the temporal bone, and leaves the middle cranial fossa through the foramen ovale to enter the otic ganglion. Other structures associated with the labyrinthine wall are two openings, the oval and round windows, and two prominent elevations (Fig. 8.120): The oval window is posterosuperior to the promontory, is the point of attachment for the base of the stapes (footplate), and ends the chain of bones that transfer vibrations initiated by the tympanic membrane to the cochlea of the internal ear. The round window is posteroinferior to the promontory. Posterior and superior to the oval window on the medial wall is the prominence of the facial canal, which is a ridge of bone produced by the facial nerve [VII] in its canal as it passes through the temporal bone.

1	Just above and posterior to the prominence of the facial canal is a broader ridge of bone (prominence of the lateral semicircular canal) produced by the lateral semicircular canal, which is a structure involved in detecting motion. Posterior to the epitympanic recess of the middle ear is the aditus to the mastoid antrum, which is the opening to the mastoid antrum (Fig. 8.121). The mastoid antrum is a cavity continuous with collections of air-filled spaces (the mastoid cells), throughout the mastoid part of the temporal bone, including the mastoid process. The mastoid antrum is separated from the middle cranial fossa above by only the thin tegmen tympani. The mucous membrane lining the mastoid air cells is continuous with the mucous membrane throughout the middle ear. Therefore infections in the middle ear can easily spread into the mastoid area.

1	The mucous membrane lining the mastoid air cells is continuous with the mucous membrane throughout the middle ear. Therefore infections in the middle ear can easily spread into the mastoid area. The pharyngotympanic tube connects the middle ear with the nasopharynx (Fig. 8.122) and equalizes pressure on both sides of the tympanic membrane. Its opening in the middle ear is on the anterior wall, and from here it extends forward, medially, and downward to enter the nasopharynx just posterior to the inferior meatus of the nasal cavity. It consists of: a bony part (the one-third nearest the middle ear); and a cartilaginous part (the remaining two-thirds). The opening of the bony part is clearly visible on the inferior surface of the skull at the junction of the squamous and petrous parts of the temporal bone immediately posterior to the foramen ovale and foramen spinosum.

1	The arterial supply to the pharyngotympanic tube is from several sources. Branches arise from the ascending pharyngeal artery (a branch of the external carotid artery) and from two branches of the maxillary artery (the middle meningeal artery and the artery of the pterygoid canal). Venous drainage of the pharyngotympanic tube is to the pterygoid plexus of veins in the infratemporal fossa. Innervation of the mucous membrane lining the pharyngotympanic tube is primarily from the tympanic plexus because it is continuous with the mucous membrane lining the tympanic cavity, the internal surface of the tympanic membrane, and the mastoid antrum and mastoid cells. This plexus receives its major contribution from the tympanic nerve, a branch of the glossopharyngeal nerve [IX]. The bones of the middle ear consist of the malleus, incus, and stapes. They form an osseous chain across the middle ear from the tympanic membrane to the oval window of the internal ear (Fig. 8.123).

1	Muscles associated with the auditory ossicles modulate movement during the transmission of vibrations. The malleus is the largest of the auditory ossicles and is attached to the tympanic membrane. Identifiable parts include the head of the malleus, neck of the malleus, anterior and lateral processes, and handle of the malleus (Fig. 8.123). The head of the malleus is the rounded upper part of the malleus in the epitympanic recess. Its posterior surface articulates with the incus. Inferior to the head of the malleus is the constricted neck of the malleus, and below this are the anterior and lateral processes: The anterior process is attached to the anterior wall of the middle ear by a ligament. The lateral process is attached to the anterior and posterior malleolar folds of the tympanic membrane. The downward extension of the malleus, below the anterior and lateral processes, is the handle of the malleus, which is attached to the tympanic membrane.

1	The downward extension of the malleus, below the anterior and lateral processes, is the handle of the malleus, which is attached to the tympanic membrane. The second bone in the series of auditory ossicles is the incus. It consists of the body of the incus and long and short limbs (Fig. 8.123): The enlarged body of the incus articulates with the head of the malleus and is in the epitympanic recess. The long limb extends downward from the body, paralleling the handle of the malleus, and ends by bending medially to articulate with the stapes. The short limb extends posteriorly and is attached by a ligament to the upper posterior wall of the middle ear. The stapes is the most medial bone in the osseous chain and is attached to the oval window. It consists of the head of the stapes, anterior and posterior limbs, and the base of the stapes (Fig. 8.123): The head of the stapes is directed laterally and articulates with the long process of the incus.

1	The head of the stapes is directed laterally and articulates with the long process of the incus. The two limbs separate from each other and attach to the oval base. The base of the stapes fits into the oval window on the labyrinthine wall of the middle ear. Muscles associated with the ossicles Two muscles are associated with the bony ossicles of the middle ear—the tensor tympani and stapedius (Fig. 8.124 and Table 8.10). The tensor tympani muscle lies in a bony canal above the pharyngotympanic tube. It originates from the cartilaginous part of the pharyngotympanic tube, the greater wing of the sphenoid, and its own bony canal, and passes through its canal in a posterior direction, ending in a rounded tendon that inserts into the upper part of the handle of the malleus. Innervation of the tensor tympani is by a branch from the mandibular nerve [V3].

1	Innervation of the tensor tympani is by a branch from the mandibular nerve [V3]. Contraction of the tensor tympani pulls the handle of the malleus medially. This tenses the tympanic membrane, reducing the force of vibrations in response to loud noises. The stapedius muscle is a very small muscle that originates from inside the pyramidal eminence, which is a small projection on the mastoid wall of the middle ear (Fig. 8.124). Its tendon emerges from the apex of the pyramidal eminence and passes forward to attach to the posterior surface of the neck of the stapes. The stapedius is innervated by a branch from the facial nerve [VII]. Contraction of the stapedius muscle, usually in response to loud noises, pulls the stapes posteriorly and prevents excessive oscillation.

1	Contraction of the stapedius muscle, usually in response to loud noises, pulls the stapes posteriorly and prevents excessive oscillation. Numerous arteries supply the structures in the middle ear: the two largest branches are the tympanic branch of the maxillary artery and the mastoid branch of the occipital or posterior auricular arteries; smaller branches come from the middle meningeal artery, the ascending pharyngeal artery, the artery of the pterygoid canal, and tympanic branches from the internal carotid artery. Venous drainage of the middle ear returns to the pterygoid plexus of veins and the superior petrosal sinus.

1	Venous drainage of the middle ear returns to the pterygoid plexus of veins and the superior petrosal sinus. The tympanic plexus innervates the mucous membrane lining the walls and contents of the middle ear, which includes the mastoid area and the pharyngotympanic tube. It is formed by the tympanic nerve, a branch of the glossopharyngeal nerve [IX], and from branches of the internal carotid plexus. The tympanic plexus occurs in the mucous membrane covering the promontory, which is the rounded bulge on the labyrinthine wall of the middle ear (Fig. 8.125). As the glossopharyngeal nerve [IX] exits the skull through the jugular foramen, it gives off the tympanic nerve. This branch reenters the skull through a small foramen and passes through the bone to the middle ear.

1	Once in the middle ear, the tympanic nerve forms the tympanic plexus, along with branches from the plexus of nerves surrounding the internal carotid artery (caroticotympanic nerves). Branches from the tympanic plexus supply the mucous membranes of the middle ear, including the pharyngotympanic tube and the mastoid area. The tympanic plexus also gives off a major branch (the lesser petrosal nerve), which supplies preganglionic parasympathetic fibers to the otic ganglion (Fig. 8.125). The lesser petrosal nerve leaves the area of the promontory, exits the middle ear, travels through the petrous part of the temporal bone, and exits onto the anterior surface of the petrous part of the temporal bone through a hiatus just below the hiatus for the greater petrosal nerve (Fig. 8.126). It continues diagonally across the anterior surface of the temporal bone before exiting the middle cranial fossa through the foramen ovale. Once outside the skull it enters the otic ganglion.

1	The internal ear consists of a series of bony cavities (the bony labyrinth) and membranous ducts and sacs (the membranous labyrinth) within these cavities. All these structures are in the petrous part of the temporal bone between the middle ear laterally and the internal acoustic meatus medially (Figs. 8.127 and 8.128). The bony labyrinth consists of the vestibule, three semicircular canals, and the cochlea (Fig. 8.128). These bony cavities are lined with periosteum and contain a clear fluid (the perilymph). Suspended within the perilymph but not filling all spaces of the bony labyrinth is the membranous labyrinth, which consists of the semicircular ducts, the cochlear duct, and two sacs (the utricle and the saccule). These membranous spaces are filled with endolymph. The structures in the internal ear convey information to the brain about balance and hearing: The cochlear duct is the organ of hearing. The semicircular ducts, utricle, and saccule are the organs of balance.

1	The cochlear duct is the organ of hearing. The semicircular ducts, utricle, and saccule are the organs of balance. The nerve responsible for these functions is the vestibulocochlear nerve [VIII], which divides into vestibular (balance) and cochlear (hearing) parts after entering the internal acoustic meatus (Fig. 8.128). The vestibule, which contains the oval window in its lateral wall, is the central part of the bony labyrinth (Fig. 8.129). It communicates anteriorly with the cochlea and posterosuperiorly with the semicircular canals. A narrow canal (the vestibular aqueduct) leaves the vestibule, and passes through the temporal bone to open on the posterior surface of the petrous part of the temporal bone.

1	A narrow canal (the vestibular aqueduct) leaves the vestibule, and passes through the temporal bone to open on the posterior surface of the petrous part of the temporal bone. Projecting in a posterosuperior direction from the vestibule are the anterior, posterior, and lateral semicircular canals (Fig. 8.129). Each of these canals forms two-thirds of a circle connected at both ends to the vestibule and with one end dilated to form the ampulla. The canals are oriented so that each canal is at right angles to the other two.

1	Projecting in an anterior direction from the vestibule is the cochlea, which is a bony structure that twists on itself two and one-half to two and three-quarter times around a central column of bone (the modiolus). This arrangement produces a cone-shaped structure with a base of the cochlea that faces posteromedially and an apex that faces anterolaterally (Fig. 8.130). This positions the wide base of the modiolus near the internal acoustic meatus, where it is entered by branches of the cochlear part of the vestibulocochlear nerve [VIII]. Extending laterally throughout the length of the modiolus is a thin lamina of bone (the lamina of the modiolus, or spiral lamina). Circling around the modiolus, and held in a central position by its attachment to the lamina of the modiolus, is the cochlear duct, which is a component of the membranous labyrinth.

1	Attached peripherally to the outer wall of the cochlea, the cochlear duct creates two canals (the scala vestibuli and the scala tympani), which extend throughout the cochlea and are continuous with each other at the apex through a narrow slit (the helicotrema): The scala vestibuli is continuous with the vestibule. The scala tympani is separated from the middle ear by the secondary tympanic membrane covering the round window (Fig. 8.131). Finally, near the round window is a small channel (the cochlear canaliculus), which passes through the temporal bone and opens on its inferior surface into the posterior cranial fossa. This provides a connection between the perilymph-containing cochlea and the subarachnoid space (Fig. 8.131). The membranous labyrinth is a continuous system of ducts and sacs within the bony labyrinth. It is filled with endolymph and separated from the periosteum that covers the walls of the bony labyrinth by perilymph.

1	Consisting of two sacs (the utricle and the saccule) and four ducts (the three semicircular ducts and the cochlear duct), the membranous labyrinth has unique functions related to balance and hearing: The utricle, saccule, and three semicircular ducts are part of the vestibular apparatus (i.e., organs of balance). The cochlear duct is the organ of hearing. The general organization of the parts of the membranous labyrinth (Fig. 8.131) places: the cochlear duct within the cochlea of the bony labyrinth, anteriorly, the three semicircular ducts within the three semicircular canals of the bony labyrinth, posteriorly, and the saccule and utricle within the vestibule of the bony labyrinth, in the middle. Organs of balance Five of the six components of the membranous labyrinth are concerned with balance. These are the two sacs (the utricle and the saccule) and three ducts (the anterior, posterior, and lateral semicircular ducts). Utricle, saccule, and endolymphatic duct

1	Utricle, saccule, and endolymphatic duct The utricle is the larger of the two sacs. It is oval, elongated and irregular in shape and is in the posterosuperior part of the vestibule of the bony labyrinth. The three semicircular ducts empty into the utricle. Each semicircular duct is similar in shape, including a dilated end forming the ampulla, to its complementary bony semicircular canal, only much smaller. The saccule is a smaller, rounded sac lying in the anteroinferior part of the vestibule of the bony labyrinth (Fig. 8.131). The cochlear duct empties into it.

1	The saccule is a smaller, rounded sac lying in the anteroinferior part of the vestibule of the bony labyrinth (Fig. 8.131). The cochlear duct empties into it. The utriculosaccular duct establishes continuity between all components of the membranous labyrinth and connects the utricle and saccule. Branching from this small duct is the endolymphatic duct, which enters the vestibular aqueduct (a channel through the temporal bone) to emerge onto the posterior surface of the petrous part of the temporal bone in the posterior cranial fossa. Here the endolymphatic duct expands into the endolymphatic sac, which is an extradural pouch that functions in resorption of endolymph.

1	Functionally, sensory receptors for balance are organized into unique structures that are located in each of the components of the vestibular apparatus. In the utricle and saccule the sense organ is the macula of the utricle and the macula of the saccule, respectively, and in the ampulla of each of the three semicircular ducts it is the crista. The utricle responds to linear acceleration in the horizontal plane and sideways head tilts, while the saccule responds to linear acceleration in the vertical plane, such as forward-backward and upward-downward movements. In contrast, the receptors in the three semicircular ducts respond to rotational movement in any direction. Organ of hearing

1	Organ of hearing The cochlear duct has a central position in the cochlea of the bony labyrinth dividing it into two canals (the scala vestibuli and the scala tympani). It is maintained in this position by being attached centrally to the lamina of the modiolus, which is a thin lamina of bone extending from the modiolus (the central bony core of the cochlea) and peripherally to the outer wall of the cochlea (Fig. 8.132).

1	Thus, the triangular-shaped cochlear duct has: an outer wall against the bony cochlea consisting of thickened, epithelial-lined periosteum (the spiral ligament), a roof (the vestibular membrane), which separates the endolymph in the cochlear duct from the perilymph in the scala vestibuli and consists of a membrane with a connective tissue core lined on either side with epithelium, and a floor, which separates the endolymph in the cochlear duct from the perilymph in the scala tympani and consists of the free edge of the lamina of the modiolus, and a membrane (the basilar membrane) extending from this free edge of the lamina of the modiolus to an extension of the spiral ligament covering the outer wall of the cochlea. The spiral organ is the organ of hearing, rests on the basilar membrane, and projects into the enclosed, endolymph-filled cochlear duct (Fig. 8.132).

1	The spiral organ is the organ of hearing, rests on the basilar membrane, and projects into the enclosed, endolymph-filled cochlear duct (Fig. 8.132). The arterial supply to the internal ear is divided between vessels supplying the bony labyrinth and the membranous labyrinth. The bony labyrinth is supplied by the same arteries that supply the surrounding temporal bone—these include an anterior tympanic branch from the maxillary artery, a stylomastoid branch from the posterior auricular artery, and a petrosal branch from the middle meningeal artery.

1	The membranous labyrinth is supplied by the labyrinthine artery, which either arises from the anteroinferior cerebellar artery or is a direct branch of the basilar artery—whatever its origin, it enters the internal acoustic meatus with the facial [VII] and vestibulocochlear [VIII] nerves and eventually divides into: a cochlear branch, which passes through the modiolus and supplies the cochlear duct; and one or two vestibular branches, which supply the vestibular apparatus. Venous drainage of the membranous labyrinth is through vestibular veins and cochlear veins, which follow the arteries. These come together to form a labyrinthine vein, which eventually empties into either the inferior petrosal sinus or the sigmoid sinus.

1	The vestibulocochlear nerve [VIII] carries special afferent fibers for hearing (the cochlear component) and balance (the vestibular component). It enters the lateral surface of the brainstem, between the pons and medulla, after exiting the temporal bone through the internal acoustic meatus and crossing the posterior cranial fossa. Inside the temporal bone, at the distal end of the internal acoustic meatus, the vestibulocochlear nerve divides to form: the cochlear nerve, and the vestibular nerve. The vestibular nerve enlarges to form the vestibular ganglion, before dividing into superior and inferior parts, which distribute to the three semicircular ducts and the utricle and saccule (see Fig. 8.128).

1	The cochlear nerve enters the base of the cochlea and passes upward through the modiolus. The ganglion cells of the cochlear nerve are in the spiral ganglion at the base of the lamina of the modiolus as it winds around the modiolus (Fig. 8.130). Branches of the cochlear nerve pass through the lamina of the modiolus to innervate the receptors in the spiral organ. Facial nerve [VII] in the temporal bone The facial nerve [VII] is closely associated with the vestibulocochlear nerve [VIII] as it enters the internal acoustic meatus of the temporal bone. Traveling through the temporal bone, its path and several of its branches are directly related to the internal and middle ears. The facial nerve [VII] enters the internal acoustic meatus in the petrous part of the temporal bone (Fig. 8.133A). The vestibulocochlear nerve and the labyrinthine artery accompany it.

1	The facial nerve [VII] enters the internal acoustic meatus in the petrous part of the temporal bone (Fig. 8.133A). The vestibulocochlear nerve and the labyrinthine artery accompany it. At the distal end of the internal acoustic meatus, the facial nerve [VII] enters the facial canal and continues laterally between the internal and middle ears. At this point the facial nerve [VII] enlarges and bends posteriorly and laterally. The enlargement is the sensory geniculate ganglion. As the facial canal continues, the facial nerve [VII] turns sharply downward, and running in an almost vertical direction, it exits the skull through the stylomastoid foramen (Fig. 8.133A).

1	Greater petrosal nerve. At the geniculate ganglion, the facial nerve [VII] gives off the greater petrosal nerve (Fig. 8.133A). This is the first branch of the facial nerve [VII]. The greater petrosal nerve leaves the geniculate ganglion, travels anteromedially through the temporal bone, and emerges through the hiatus for the greater petrosal nerve on the anterior surface of the petrous part of the temporal bone (see Fig. 8.126). The greater petrosal nerve carries preganglionic parasympathetic fibers to the pterygopalatine ganglion. Continuing beyond the bend, the position of the facial nerve [VII] is indicated on the medial wall of the middle ear by a bulge (see Fig. 8.125). Nerve to stapedius and chorda tympani. Near the beginning of its vertical descent, the facial nerve [VII] gives off a small branch, the nerve to the stapedius (Fig. 8.133), which innervates the stapedius muscle, and just before it exits the skull the facial nerve [VII] gives off the chorda tympani nerve.

1	The chorda tympani does not immediately exit the temporal bone, but ascends to enter the middle ear through its posterior wall, passing near the upper aspect of the tympanic membrane between the malleus and incus (Fig. 8.133B). It then exits the middle ear through a canal leading to the petrotympanic fissure and exits the skull through this fissure to join the lingual nerve in the infratemporal fossa. Transmission of sound

1	Transmission of sound A sound wave enters the external acoustic meatus and strikes the tympanic membrane moving it medially (Fig. 8.134). As the handle of the malleus is attached to this membrane, it also moves medially. This moves the head of the malleus laterally. Because the heads of the malleus and incus articulate with each other, the head of the incus is also moved laterally. This pushes the long process of the incus medially. The long process articulates with the stapes, so its movement causes the stapes to move medially. In turn, because the base of the stapes is attached to the oval window, the oval window is also moved medially. This action completes the transfer of a large-amplitude, low-force, airborne wave that vibrates the tympanic membrane into a small-amplitude, high-force vibration of the oval window, which generates a wave in the fluid-filled scala vestibuli of the cochlea.

1	The wave established in the perilymph of the scala vestibuli moves through the cochlea and causes an outward bulging of the secondary tympanic membrane covering the round window at the lower end of the scala tympani (Fig. 8.134). This causes the basilar membrane to vibrate, which in turn leads to stimulation of receptor cells in the spiral organ. The receptor cells send impulses back to the brain through the cochlear part of the vestibulocochlear nerve [VIII] where they are interpreted as sound. If the sounds are too loud, causing excessive movement of the tympanic membrane, contraction of the tensor tympani muscle (attached to the malleus) and/or the stapedius muscle (attached to the stapes) dampens the vibrations of the ossicles and decreases the force of the vibrations reaching the oval window. The temporal and infratemporal fossae are interconnected spaces on the lateral side of the head (Fig. 8.135). Their boundaries are formed by bone and soft tissues.

1	The temporal and infratemporal fossae are interconnected spaces on the lateral side of the head (Fig. 8.135). Their boundaries are formed by bone and soft tissues. The temporal fossa is superior to the infratemporal fossa, above the zygomatic arch, and communicates with the infratemporal fossa below through the gap between the zygomatic arch and the more medial surface of the skull. The infratemporal fossa is a wedge-shaped space deep to the masseter muscle and the underlying ramus of the mandible. Structures that travel between the cranial cavity, neck, pterygopalatine fossa, floor of the oral cavity, floor of the orbit, temporal fossa, and superficial regions of the head pass through it.

1	Of the four muscles of mastication (masseter, temporalis, medial pterygoid, and lateral pterygoid) that move the lower jaw at the temporomandibular joint, one (masseter) is lateral to the infratemporal fossa, two (medial and lateral pterygoid) are in the infratemporal fossa, and one fills the temporal fossa. Bones that contribute significantly to the boundaries of the temporal and infratemporal fossae include the temporal, zygomatic, and sphenoid bones, and the maxilla and mandible (Figs. 8.136 and 8.137). Parts of the frontal and parietal bones are also involved. The squamous part of the temporal bone forms part of the bony framework of the temporal and infratemporal fossae. The tympanic part of the temporal bone forms the posteromedial corner of the roof of the infratemporal fossa, and also articulates with the head of the mandible to form the temporomandibular joint.

1	The tympanic part of the temporal bone forms the posteromedial corner of the roof of the infratemporal fossa, and also articulates with the head of the mandible to form the temporomandibular joint. The lateral surface of the squamous part of the temporal bone is marked by two surface features on the medial wall of the temporal fossa: a transversely oriented supramastoid crest, which extends posteriorly from the base of the zygomatic process and marks the posteroinferior border of the temporal fossa; and a vertically oriented groove for the middle temporal artery, a branch of the superficial temporal artery.

1	Two features that participate in forming the temporomandibular joint on the inferior aspect of the root of the zygomatic process are the articular tubercle and the mandibular fossa. Both are elongate from medial to lateral. Posterior to the mandibular fossa is the external acoustic meatus. The tympanic part of the temporal bone is a flat concave plate of bone that curves inferiorly from the back of the mandibular fossa and forms part of the wall of the external auditory meatus. When viewed from inferiorly, there is a distinct tympanosquamous fissure between the tympanic and squamous parts of the temporal bone. Medially, a small slip of bone from the petrous part of the temporal bone insinuates itself into the fissure and forms a petrotympanic fissure between it and the tympanic part (Fig. 8.136). The chorda tympani nerve exits the skull and enters the infratemporal fossa through the medial end of the petrotympanic fissure.

1	The chorda tympani nerve exits the skull and enters the infratemporal fossa through the medial end of the petrotympanic fissure. The parts of the sphenoid bone that form part of the bony framework of the infratemporal fossa are the lateral plate of the pterygoid process and the greater wing (Fig. 8.136). The greater wing also forms part of the medial wall of the temporal fossa. The greater wings extend one on each side from the body of the sphenoid. They project laterally from the body and curve superiorly. The inferior and lateral surfaces form the roof of the infratemporal fossa and the medial wall of the temporal fossa, respectively.

1	The sharply angled boundary between the lateral and inferior surfaces of the greater wing is the infratemporal crest (Fig. 8.136). Two apertures (the foramen ovale and the foramen spinosum) pass through the base of the greater wing and allow the mandibular nerve [V3] and the middle meningeal artery, respectively, to pass between the middle cranial fossa and infratemporal fossa. In addition, one or more small sphenoidal emissary foramina penetrate the base of the greater wing anteromedial to the foramen ovale and allow emissary veins to pass between the pterygoid plexus of veins in the infratemporal fossa and the cavernous sinus in the middle cranial fossa. Projecting vertically downward from the greater wing immediately medial to the foramen spinosum is the irregularly shaped spine of the sphenoid, which is the attachment site for the cranial end of the sphenomandibular ligament.

1	The lateral plate of the pterygoid process is a vertically oriented sheet of bone that projects posterolaterally from the pterygoid process (Fig. 8.136). Its lateral and medial surfaces provide attachment for the lateral and medial pterygoid muscles, respectively. The posterior surface of the maxilla contributes to the anterior wall of the infratemporal fossa (Fig. 8.136). This surface is marked by a foramen for the posterosuperior alveolar nerve and vessels. The superior margin forms the inferior border of the inferior orbital fissure. The zygomatic bone is a quadrangular-shaped bone that forms the palpable bony prominence of the cheek: A maxillary process extends anteromedially to articulate with the zygomatic process of the maxilla. A frontal process extends superiorly to articulate with the zygomatic process of the frontal bone. A temporal process extends posteriorly to articulate with the zygomatic process of the temporal bone to complete the zygomatic arch.

1	A temporal process extends posteriorly to articulate with the zygomatic process of the temporal bone to complete the zygomatic arch. A small zygomaticofacial foramen on the lateral surface of the zygomatic bone transmits the zygomaticofacial nerve and vessels onto the cheek. A thin plate of bone extends posteromedially from the frontal process and contributes to the lateral wall of the orbit on one side and the anterior wall of the temporal fossa on the other. A zygomaticotemporal foramen on the temporal fossa surface of the plate where it attaches to the frontal process is for the zygomaticotemporal nerve. Ramus of mandible The ramus of the mandible is quadrangular in shape and has medial and lateral surfaces and condylar and coronoid processes (Fig. 8.137). The lateral surface of the ramus of the mandible is generally smooth except for the presence of a few obliquely oriented ridges. Most of the lateral surface provides attachment for the masseter muscle.

1	The posterior and inferior borders of the ramus intersect to form the angle of the mandible, while the superior border is notched to form the mandibular notch. The anterior border is sharp and is continuous below with the oblique line on the body of the mandible. The coronoid process extends superiorly from the junction of the anterior and superior borders of the ramus. It is a flat, triangular process that provides attachment for the temporalis muscle. The condylar process extends superiorly from the posterior and superior borders of the ramus. It consists of: the head of the mandible, which is expanded medially and participates in forming the temporomandibular joint; and the neck of the mandible, which bears a shallow depression (the pterygoid fovea) on its anterior surface for attachment of the lateral pterygoid muscle.

1	The medial surface of the ramus of the mandible is the lateral wall of the infratemporal fossa (Fig. 8.137B). Its most distinctive feature is the mandibular foramen, which is the superior opening of the mandibular canal. The inferior alveolar nerve and vessels pass through this foramen. Immediately anterosuperior to the mandibular foramen is a triangular elevation (the lingula) for attachment of the mandibular end of the sphenomandibular ligament. An elongate groove (the mylohyoid groove) extends anteroinferiorly from the mandibular foramen. The nerve to the mylohyoid is in this groove. Posteroinferior to the mylohyoid groove and mandibular foramen, the medial surface of the ramus of the mandible is roughened for attachment of the medial pterygoid muscle. The temporomandibular joints, one on each side, allow opening and closing of the mouth and complex chewing or side-to-side movements of the lower jaw.

1	The temporomandibular joints, one on each side, allow opening and closing of the mouth and complex chewing or side-to-side movements of the lower jaw. Each joint is synovial and is formed between the head of the mandible and the articular fossa and articular tubercle of the temporal bone (Fig. 8.138A). Unlike most other synovial joints where the articular surfaces of the bones are covered by a layer of hyaline cartilage, those of the temporomandibular joint are covered by fibrocartilage. In addition, the joint is completely divided by a fibrous articular disc into two parts: The lower part of the joint allows mainly the hinge-like depression and elevation of the mandible. The upper part of the joint allows the head of the mandible to translocate forward (protrusion) onto the articular tubercle and backward (retraction) into the mandibular fossa. Opening the mouth involves both depression and protrusion (Fig. 8.138B).

1	Opening the mouth involves both depression and protrusion (Fig. 8.138B). The forward or protrusive movement allows greater depression of the mandible by preventing backward movement of the angle of the mandible into structures in the neck. The synovial membrane of the joint capsule lines all nonarticular surfaces of the upper and lower compartments of the joint and is attached to the margins of the articular disc. The fibrous membrane of the joint capsule encloses the temporomandibular joint complex and is attached: above along the anterior margin of the articular tubercle, laterally and medially along the margins of the articular fossa, posteriorly to the region of the tympanosquamous suture, and below around the upper part of the neck of the mandible. The articular disc attaches around its periphery to the inner aspect of the fibrous membrane.

1	The articular disc attaches around its periphery to the inner aspect of the fibrous membrane. Three extracapsular ligaments are associated with the temporomandibular joint—the lateral, sphenomandibular, and the stylomandibular ligaments (Fig. 8.139): The lateral ligament is closest to the joint, just lateral to the capsule, and runs diagonally backward from the margin of the articular tubercle to the neck of the mandible. The sphenomandibular ligament is medial to the temporomandibular joint, runs from the spine of the sphenoid bone at the base of the skull to the lingula on the medial side of the ramus of the mandible. The stylomandibular ligament passes from the styloid process of the temporal bone to the posterior margin and angle of the mandible. Movements of the mandible

1	The stylomandibular ligament passes from the styloid process of the temporal bone to the posterior margin and angle of the mandible. Movements of the mandible A chewing or grinding motion occurs when the movements at the temporomandibular joint on one side are coordinated with a reciprocal set of movements at the joint on the other side. Movements of the mandible include depression, elevation, protrusion, and retraction (Fig. 8.140): Depression is generated by the digastric, geniohyoid, and mylohyoid muscles on both sides, is normally assisted by gravity, and, because it involves forward movement of the head of the mandible onto the articular tubercle, the lateral pterygoid muscles are also involved. Elevation is a very powerful movement generated by the temporalis, masseter, and medial pterygoid muscles and also involves movement of the head of the mandible into the mandibular fossa.

1	Elevation is a very powerful movement generated by the temporalis, masseter, and medial pterygoid muscles and also involves movement of the head of the mandible into the mandibular fossa. Protraction is mainly achieved by the lateral pterygoid muscle, with some assistance by the medial pterygoid. Retraction is carried out by the geniohyoid and digastric muscles, and by the posterior and deep fibers of the temporalis and masseter muscles, respectively. Except for the geniohyoid muscle, which is innervated by the C1 spinal nerve, all muscles that move the temporomandibular joints are innervated by the mandibular nerve [V3] by branches that originate in the infratemporal fossa. The masseter muscle is a powerful muscle of mastication that elevates the mandible (Fig. 8.141 and Table 8.11). It overlies the lateral surface of the ramus of the mandible.

1	The masseter muscle is a powerful muscle of mastication that elevates the mandible (Fig. 8.141 and Table 8.11). It overlies the lateral surface of the ramus of the mandible. The masseter muscle is quadrangular in shape and is anchored above to the zygomatic arch and below to most of the lateral surface of the ramus of the mandible. The more superficial part of the masseter originates from the maxillary process of the zygomatic bone and the anterior two-thirds of the zygomatic process of the maxilla. It inserts into the angle of the mandible and related posterior part of the lateral surface of the ramus of the mandible. The deep part of the masseter originates from the medial aspect of the zygomatic arch and the posterior part of its inferior margin and inserts into the central and upper part of the ramus of the mandible as high as the coronoid process.

1	The masseter is innervated by the masseteric nerve from the mandibular nerve [V3] and supplied with blood by the masseteric artery from the maxillary artery. The masseteric nerve and artery originate in the infratemporal fossa and pass laterally over the margin of the mandibular notch to enter the deep surface of the masseter muscle. The temporal fossa is a narrow fan-shaped space that covers the lateral surface of the skull (Fig. 8.142A): Its upper margin is defined by a pair of temporal lines that arch across the skull from the zygomatic process of the frontal bone to the supramastoid crest of the temporal bone. It is limited laterally by the temporal fascia, which is a tough, fan-shaped aponeurosis overlying the temporalis muscle and attached by its outer margin to the superior temporal line and by its inferior margin to the zygomatic arch.

1	Anteriorly, it is limited by the posterior surface of the frontal process of the zygomatic bone and the posterior surface of the zygomatic process of the frontal bone, which separate the temporal fossa behind from the orbit in front. Its inferior margin is marked by the zygomatic arch laterally and by the infratemporal crest of the greater wing of the sphenoid medially (Fig. 8.142B)—between these two features, the floor of the temporal fossa is open medially to the infratemporal fossa and laterally to the region containing the masseter muscle. The major structure in the temporal fossa is the temporalis muscle. Also passing through the fossa is the zygomaticotemporal branch of the maxillary nerve [V2], which enters the region through the zygomaticotemporal foramen on the temporal fossa surface of the zygomatic bone.

1	The temporalis muscle is a large, fan-shaped muscle that fills much of the temporal fossa (Fig. 8.143). It originates from the bony surfaces of the fossa superiorly to the inferior temporal line and is attached laterally to the surface of the temporal fascia. The more anterior fibers are oriented vertically while the more posterior fibers are oriented horizontally. The fibers converge inferiorly to form a tendon, which passes between the zygomatic arch and the infratemporal crest of the greater wing of the sphenoid to insert on the coronoid process of the mandible. The temporalis muscle attaches down the anterior surface of the coronoid process and along the related margin of the ramus of the mandible, almost to the last molar tooth.

1	The temporalis muscle attaches down the anterior surface of the coronoid process and along the related margin of the ramus of the mandible, almost to the last molar tooth. The temporalis is a powerful elevator of the mandible. Because this movement involves posterior translocation of the head of the mandible from the articular tubercle of the temporal bone and back into the mandibular fossa, the temporalis also retracts the mandible or pulls it posteriorly. In addition, the temporalis participates in side-to-side movements of the mandible. The temporalis is innervated by deep temporal nerves that originate from the mandibular nerve [V3] in the infratemporal fossa and then pass into the temporal fossa. Blood supply of the temporalis is by deep temporal arteries, which travel with the nerves, and the middle temporal artery, which penetrates the temporal fascia at the posterior end of the zygomatic arch.

1	The deep temporal nerves, usually two in number, originate from the anterior trunk of the mandibular nerve [V3] in the infratemporal fossa (Fig. 8.144). They pass superiorly and around the infratemporal crest of the greater wing of the sphenoid to enter the temporal fossa deep to the temporalis muscle, and supply the temporalis muscle. The zygomaticotemporal nerve is a branch of the zygomatic nerve (see Fig. 8.87, p. 922). The zygomatic nerve is a branch of the maxillary nerve [V2], which originates in the pterygopalatine fossa and passes into the orbit. The zygomaticotemporal nerve enters the temporal fossa through one or more small foramina on the temporal fossa surface of the zygomatic bone. Branches of the zygomaticotemporal nerve pass superiorly between the bone and the temporalis muscle to penetrate the temporal fascia and supply the skin of the temple (Fig. 8.144).

1	Branches of the zygomaticotemporal nerve pass superiorly between the bone and the temporalis muscle to penetrate the temporal fascia and supply the skin of the temple (Fig. 8.144). Normally two in number, these vessels originate from the maxillary artery in the infratemporal fossa and travel with the deep temporal nerves around the infratemporal crest of the greater wing of the sphenoid to supply the temporalis muscle (Fig. 8.144). They anastomose with branches of the middle temporal artery. The middle temporal artery originates from the superficial temporal artery just superior to the root of the zygomatic arch between this structure and the external ear (Fig. 8.144). It penetrates the temporalis fascia, passes under the margin of the temporalis muscle, and travels superiorly on the deep surface of the temporalis muscle. The middle temporal artery supplies the temporalis and anastomoses with branches of the deep temporal arteries.

1	The middle temporal artery supplies the temporalis and anastomoses with branches of the deep temporal arteries. The wedge-shaped infratemporal fossa is inferior to the temporal fossa and between the ramus of the mandible laterally and the wall of the pharynx medially. It has a roof, a lateral wall, and a medial wall, and is open to the neck posteroinferiorly (Fig. 8.145): The roof is formed by the inferior surfaces of the greater wing of the sphenoid and the temporal bone, contains the foramen spinosum, foramen ovale, and the petrotympanic fissure, and lateral to the infratemporal crest of the greater wing of the sphenoid, is open superiorly to the temporal fossa. The lateral wall is the medial surface of the ramus of the mandible, which contains the opening to the mandibular canal.

1	The lateral wall is the medial surface of the ramus of the mandible, which contains the opening to the mandibular canal. The medial wall is formed anteriorly by the lateral plate of the pterygoid process and more posteriorly by the pharynx and by two muscles of the soft palate (tensor and levator veli palatini muscles), and contains the pterygomaxillary fissure anteriorly, which allows structures to pass between the infratemporal and pterygopalatine fossae. The anterior wall is formed by part of the posterior surface of the maxilla and contains the alveolar foramen, and the upper part opens as the inferior orbital fissure into the orbit. Major contents of the infratemporal fossa include the sphenomandibular ligament, medial and lateral pterygoid muscles (Table 8.11), the maxillary artery, the mandibular nerve [V3], branches of the facial nerve [VII] and the glossopharyngeal nerve [IX], and the pterygoid plexus of veins.

1	The sphenomandibular ligament is an extracapsular ligament of the temporomandibular joint. It is attached superiorly to the spine of the sphenoid bone and expands inferiorly to attach to the lingula of the mandible and the posterior margin of the mandibular foramen (Fig. 8.146). The medial pterygoid muscle is quadrangular in shape and has deep and superficial heads (Fig. 8.146): The deep head is attached above to the medial surface of the lateral plate of the pterygoid process and the associated surface of the pyramidal process of the palatine bone, and descends obliquely downward, medial to the sphenomandibular ligament, to attach to the roughened medial surface of the ramus of the mandible near the angle of the mandible. The superficial head originates from the tuberosity of the maxilla and adjacent pyramidal process of the palatine bone and joins with the deep head to insert on the mandible.

1	The superficial head originates from the tuberosity of the maxilla and adjacent pyramidal process of the palatine bone and joins with the deep head to insert on the mandible. The medial pterygoid mainly elevates the mandible. Because it passes obliquely backward to insert into the mandible, it also assists the lateral pterygoid muscle in protruding the lower jaw. The medial pterygoid is innervated by the nerve to the medial pterygoid from the mandibular nerve [V3]. The lateral pterygoid is a thick triangular muscle and like the medial pterygoid muscle has two heads (Fig. 8.147): The upper head originates from the roof of the infratemporal fossa (inferior surface of the greater wing of the sphenoid and the infratemporal crest) lateral to the foramen ovale and foramen spinosum.

1	The lower head is larger than the upper head and originates from the lateral surface of the lateral plate of the pterygoid process, and the inferior part insinuates itself between the cranial attachments of the two heads of the medial pterygoid. The fibers from both heads of the lateral pterygoid muscle converge to insert into the pterygoid fovea of the neck of the mandible and into the capsule of the temporomandibular joint in the region where the capsule is attached internally to the articular disc. Unlike the medial pterygoid muscle whose fibers tend to be oriented vertically, those of the lateral pterygoid are oriented almost horizontally. As a result, when the lateral pterygoid contracts it pulls the articular disc and head of the mandible forward onto the articular tubercle and is therefore the major protruder of the lower jaw. The lateral pterygoid is innervated by the nerve to the lateral pterygoid from the mandibular nerve [V3].

1	The lateral pterygoid is innervated by the nerve to the lateral pterygoid from the mandibular nerve [V3]. When the lateral and medial pterygoids contract on only one side, the chin moves to the opposite side. When opposite movements at the two temporomandibular joints are coordinated, a chewing movement results. The mandibular nerve [V3] is the largest of the three divisions of the trigeminal nerve [V]. Unlike the ophthalmic [V1] and maxillary [V2] nerves, which are purely sensory, the mandibular nerve [V3] is both motor and sensory.

1	Unlike the ophthalmic [V1] and maxillary [V2] nerves, which are purely sensory, the mandibular nerve [V3] is both motor and sensory. In addition to carrying general sensation from the teeth and gingivae of the mandible, the anterior two-thirds of the tongue, mucosa on the floor of the oral cavity, the lower lip, skin over the temple and lower face, and part of the cranial dura mater, the mandibular nerve [V3] also carries motor innervation to most of the muscles that move the mandible, one of the muscles (tensor tympani) in the middle ear, and one of the muscles of the soft palate (tensor veli palatini). All branches of the mandibular nerve [V3] originate in the infratemporal fossa. Like the ophthalmic [V1] and maxillary [V2] nerves, the sensory part of the mandibular nerve [V3] originates from the trigeminal ganglion in the middle cranial fossa (Fig. 8.148):

1	Like the ophthalmic [V1] and maxillary [V2] nerves, the sensory part of the mandibular nerve [V3] originates from the trigeminal ganglion in the middle cranial fossa (Fig. 8.148): The sensory part of the mandibular nerve [V3] drops vertically through the foramen ovale and enters the infratemporal fossa between the tensor veli palatini muscle and the upper head of the lateral pterygoid muscle. The small motor root of the trigeminal nerve [V] passes medial to the trigeminal ganglion in the cranial cavity, then passes through the foramen ovale and immediately joins the sensory part of the mandibular nerve [V3]. Soon after the sensory and motor roots join, the mandibular nerve [V3] gives rise to a small meningeal branch and to the nerve to the medial pterygoid, and then divides into anterior and posterior trunks (Fig. 8.148):

1	Branches from the anterior trunk are the buccal, masseteric, and deep temporal nerves, and the nerve to the lateral pterygoid, all of which, except the buccal nerve (which is predominantly sensory) are motor nerves. Branches from the posterior trunk are the auriculotemporal, lingual, and inferior alveolar nerves, all of which, except a small nerve (nerve to the mylohyoid) that branches from the inferior alveolar nerve, are sensory nerves. The meningeal branch originates from the medial side of the mandibular nerve [V3] and ascends to leave the infratemporal fossa with the middle meningeal artery and reenter the cranial cavity through the foramen spinosum (Fig. 8.148). It is sensory for the dura mater, mainly of the middle cranial fossa, and also supplies the mastoid cells that communicate with the middle ear. Nerve to medial pterygoid

1	Nerve to medial pterygoid The nerve to the medial pterygoid also originates medially from the mandibular nerve [V3] (Fig. 8.148). It descends to enter and supply the deep surface of the medial pterygoid muscle. Near its origin from the mandibular nerve [V3], it has two small branches: One of these supplies the tensor veli palatini. The other ascends to supply the tensor tympani muscle, which occupies a small bony canal above and parallel to the pharyngotympanic tube in the temporal bone. The buccal nerve is a branch of the anterior trunk of the mandibular nerve [V3] (Fig. 8.148). It is predominantly a sensory nerve, but may also carry the motor innervation to the lateral pterygoid muscle and to part of the temporalis muscle.

1	The buccal nerve passes laterally between the upper and lower heads of the lateral pterygoid and then descends around the anterior margin of the insertion of the temporalis muscle to the anterior margin of the ramus of the mandible, often slipping through the tendon of the temporalis. It continues into the cheek lateral to the buccinator muscle to supply general sensory nerves to the adjacent skin and oral mucosa and the buccal gingivae of the lower molars. The masseteric nerve is a branch of the anterior trunk of the mandibular nerve [V3] (Fig. 8.148; also see Fig. 8.141). It passes laterally over the lateral pterygoid muscle and through the mandibular notch to penetrate and supply the masseter muscle.

1	The deep temporal nerves, usually two in number, originate from the anterior trunk of the mandibular nerve [V3] (Fig. 8.148; also see Fig. 8.144). They pass laterally above the lateral pterygoid muscle and curve around the infratemporal crest to ascend in the temporal fossa and supply the temporalis muscle from its deep surface. Nerve to lateral pterygoid The nerve to the lateral pterygoid may originate directly as a branch from the anterior trunk of the mandibular nerve [V3] or from its buccal branch (Fig. 8.148). From its origin, it passes directly into the deep surface of the lateral pterygoid muscle. The auriculotemporal nerve is the first branch of the posterior trunk of the mandibular nerve [V3] and originates as two roots, which pass posteriorly around the middle meningeal artery ascending from the maxillary artery to the foramen spinosum (Fig. 8.149).

1	The auriculotemporal nerve passes first between the tensor veli palatini muscle and the upper head of the lateral pterygoid muscle, and then between the sphenomandibular ligament and the neck of the mandible. It curves laterally around the neck of the mandible and then ascends deep to the parotid gland between the temporomandibular joint and ear. The terminal branches of the auriculotemporal nerve carry general sensation from skin over a large area of the temple. In addition, the auriculotemporal nerve contributes to sensory innervation of the external ear, the external auditory meatus, tympanic membrane, and temporomandibular joint. It also delivers postganglionic parasympathetic nerves from the glossopharyngeal nerve [IX] to the parotid gland.

1	The lingual nerve is a major sensory branch of the posterior trunk of the mandibular nerve [V3] (Fig. 8.149A,B). It carries general sensation from the anterior two-thirds of the tongue, oral mucosa on the floor of the oral cavity, and lingual gingivae associated with the lower teeth. The lingual nerve is joined high in the infratemporal fossa by the chorda tympani branch of the facial nerve [VII] (Fig. 8.149C), which carries: taste from the anterior two-thirds of the tongue, and parasympathetic fibers to all salivary glands below the level of the oral fissure. The lingual nerve first descends between the tensor veli palatini muscle and the lateral pterygoid muscle, where it is joined by the chorda tympani nerve, and then descends across the lateral surface of the medial pterygoid muscle to enter the oral cavity.

1	The lingual nerve enters the oral cavity between the posterior attachment of the mylohyoid muscle to the mylohyoid line and the attachment of the superior constrictor of the pharynx to the pterygomandibular raphe. As the lingual nerve enters the floor of the oral cavity, it is in a shallow groove on the medial surface of the mandible immediately inferior to the last molar tooth. In this position, it is palpable through the oral mucosa and in danger when one is operating on the molar teeth and gingivae (Fig. 8.149C). The lingual nerve passes into the tongue on the lateral surface of the hyoglossus muscle where it is attached to the submandibular ganglion. This ganglion is where the preganglionic parasympathetic fibers carried from the infratemporal fossa into the floor of the oral cavity on the lingual nerve synapse with postganglionic parasympathetic fibers (see Fig. 8.150).

1	The inferior alveolar nerve, like the lingual nerve, is a major sensory branch of the posterior trunk of the mandibular nerve [V3] (Fig. 8.149A–C). In addition to innervating all lower teeth and much of the associated gingivae, it also supplies the mucosa and skin of the lower lip and skin of the chin. It has one motor branch, which innervates the mylohyoid muscle and the anterior belly of the digastric muscle.

1	The inferior alveolar nerve originates deep to the lateral pterygoid muscle from the posterior trunk of the mandibular nerve [V3] in association with the lingual nerve. It descends on the lateral surface of the medial pterygoid muscle, passes between the sphenomandibular ligament and the ramus of the mandible, and then enters the mandibular canal through the mandibular foramen. Just before entering the mandibular foramen, it gives origin to the nerve to the mylohyoid (Fig. 8.149C), which lies in the mylohyoid groove inferior to the foramen and continues anteriorly below the floor of the oral cavity to innervate the mylohyoid muscle and the anterior belly of the digastric muscle. The inferior alveolar nerve passes anteriorly within the mandibular canal of the lower jaw. The mandibular canal and its contents are inferior to the roots of the molar teeth, and the roots can sometimes curve around the canal making extraction of these teeth difficult.

1	The inferior alveolar nerve supplies branches to the three molar teeth and the second premolar tooth and associated labial gingivae, and then divides into its two terminal branches: the incisive nerve, which continues in the mandibular canal to supply the first premolar, incisor, and canine teeth, and related gingivae; and the mental nerve, which exits the mandible through the mental foramen and supplies the lower lip and chin (Fig. 8.149A,B). The mental nerve is palpable and sometimes visible through the oral mucosa adjacent to the roots of the premolar teeth. Chorda tympani and the lesser petrosal nerve Branches of two cranial nerves join branches of the mandibular nerve [V3] in the infratemporal fossa (Fig. 8.150). These are the chorda tympani branch of the facial nerve [VII] and the lesser petrosal nerve, a branch of the tympanic plexus in the middle ear, which had its origin from a branch of the glossopharyngeal nerve [IX] (see Fig. 8.125, p. 953).

1	The chorda tympani (Fig. 8.150) carries taste from the anterior two-thirds of the tongue and parasympathetic innervation to all salivary glands below the level of the oral fissure. The chorda tympani originates from the facial nerve [VII] within the temporal bone and in association with the mastoid wall of the middle ear, passes anteriorly through a small canal, and enters the lateral aspect of the middle ear. As it continues anterosuperiorly across the middle ear, it is separated from the tympanic membrane by the handle of the malleus. It leaves the middle ear through the medial end of the petrotympanic fissure, enters the infratemporal fossa, descends medial to the spine of the sphenoid and then to the lateral pterygoid muscle, and joins the lingual nerve.

1	Preganglionic parasympathetic fibers carried in the chorda tympani synapse with postganglionic parasympathetic fibers in the submandibular ganglion, which “hangs off” the lingual nerve in the floor of the oral cavity (Fig. 8.150). Postganglionic parasympathetic fibers leave the submandibular ganglion and either: reenter the lingual nerve to travel with its terminal branches to reach target tissues, or pass directly from the submandibular ganglion into glands (Fig. 8.150). The taste (SA) fibers do not pass through the ganglion and are distributed with terminal branches of the lingual nerve. The lesser petrosal nerve carries mainly parasympathetic fibers destined for the parotid gland (Fig. 8.150). The preganglionic parasympathetic fibers are located in the glossopharyngeal nerve [IX] as it exits the jugular foramen at the base of the skull. Branching from the glossopharyngeal nerve [IX] either within or immediately outside the jugular foramen is the tympanic nerve (Fig. 8.150B).

1	The tympanic nerve reenters the temporal bone through a small foramen on the ridge of bone separating the jugular foramen from the carotid canal and ascends through a small bony canal (inferior tympanic canaliculus) to the promontory located on the labyrinthine (medial) wall of the middle ear. Here it participates in the formation of the tympanic plexus. The lesser petrosal nerve is a branch of this plexus (Fig. 8.150B). The lesser petrosal nerve contains mainly preganglionic parasympathetic fibers. It leaves the middle ear and enters the middle cranial fossa through a small opening on the anterior surface of the petrous part of the temporal bone just lateral and inferior to the opening for the greater petrosal nerve, a branch of the facial nerve [VII]. The lesser petrosal nerve then passes medially and descends through the foramen ovale with the mandibular nerve [V3].

1	In the infratemporal fossa, the preganglionic parasympathetic fibers synapse with cell bodies of postganglionic parasympathetic fibers in the otic ganglion located on the medial side of the mandibular nerve [V3] around the origin of the nerve to the medial pterygoid. Postganglionic parasympathetic fibers leave the otic ganglion and join the auriculotemporal nerve, which carries them to the parotid gland. The maxillary artery is the largest branch of the external carotid artery in the neck and is a major source of blood supply for the nasal cavity, the lateral wall and roof of the oral cavity, all teeth, and the dura mater in the cranial cavity. It passes through and supplies the infratemporal fossa and then enters the pterygopalatine fossa, where it gives origin to terminal branches (Fig. 8.151).

1	The maxillary artery originates within the substance of the parotid gland and then passes forward, between the neck of the mandible and sphenomandibular ligament, into the infratemporal fossa. It ascends obliquely through the infratemporal fossa to enter the pterygopalatine fossa by passing through the pterygomaxillary fissure. This part of the vessel may pass either lateral or medial to the lower head of the lateral pterygoid. If it passes medial to the lower head, the maxillary artery then loops laterally between the upper and lower heads of the lateral pterygoid to access the pterygomaxillary fissure. Branches of the maxillary artery are as follows (Fig. 8.151): The first part of the maxillary artery (the part between the neck of the mandible and the sphenomandibular ligament) gives origin to two major branches (the middle meningeal and inferior alveolar arteries) and a number of smaller branches (deep auricular, anterior tympanic, and accessory meningeal).

1	The second part of the maxillary artery (the part related to the lateral pterygoid muscle) gives origin to deep temporal, masseteric, buccal, and pterygoid branches, which course with branches of the mandibular nerve [V3]. The third part of the maxillary artery is in the pterygopalatine fossa (see Fig. 8.158) The middle meningeal artery ascends vertically from the maxillary artery and passes through the foramen spinosum to enter the cranial cavity (Fig. 8.151). In the infratemporal fossa, it passes superiorly between the sphenomandibular ligament on the medial side and the lateral pterygoid muscle on the lateral side. Just inferior to the foramen spinosum, it passes between the two roots of the auriculotemporal nerve at their origin from the mandibular nerve [V3] (Fig. 8.151). The middle meningeal artery is the largest of the meningeal vessels and supplies much of the dura mater, bone, and related bone marrow of the cranial cavity walls.

1	The middle meningeal artery is the largest of the meningeal vessels and supplies much of the dura mater, bone, and related bone marrow of the cranial cavity walls. Within the cranial cavity, the middle meningeal artery and its branches travel in the periosteal (outer) layer of dura mater, which is tightly adherent to the bony walls. As major branches of the middle meningeal artery pass superiorly up the walls of the cranial cavity, they can be damaged by lateral blows to the head. When the vessels are torn, the leaking blood, which is under arterial pressure, slowly separates the dura mater from its attachment to the bone, resulting in an extradural hematoma. The inferior alveolar artery descends from the maxillary artery to enter the mandibular foramen and canal with the inferior alveolar nerve (Fig. 8.151). It is distributed with the inferior alveolar nerve and supplies all lower teeth, and contributes to the supply of the buccal gingivae, chin, and lower lip.

1	Before entering the mandible, the inferior alveolar artery gives origin to a small mylohyoid branch, which accompanies the nerve to the mylohyoid. Deep auricular, anterior tympanic, and accessory meningeal arteries The deep auricular, anterior tympanic, and accessory meningeal arteries are small branches from the first part of the maxillary artery and contribute to the blood supply of the external acoustic meatus, deep surface of the tympanic membrane, and cranial dura mater, respectively. The accessory meningeal branch also contributes small branches to surrounding muscles in the infratemporal fossa before ascending through the foramen ovale into the cranial cavity to supply the dura mater. Branches from the second part Deep temporal arteries, usually two in number, originate from the second part of the maxillary artery and travel with the deep temporal nerves to supply the temporalis muscle in the temporal fossa (Fig. 8.151).

1	Numerous pterygoid arteries also originate from the second part of the maxillary artery and supply the pterygoid muscles. The masseteric artery, also from the second part of the maxillary artery, accompanies the masseteric nerve laterally through the mandibular notch to supply the masseter muscle. The buccal artery is distributed with the buccal nerve and supplies skin, muscle, and oral mucosa of the cheek. The pterygoid plexus is a network of veins between the medial and lateral pterygoid muscles, and between the lateral pterygoid and temporalis muscles (Fig. 8.152).

1	The pterygoid plexus is a network of veins between the medial and lateral pterygoid muscles, and between the lateral pterygoid and temporalis muscles (Fig. 8.152). Veins that drain regions supplied by arteries branching from the maxillary artery in the infratemporal fossa and pterygopalatine fossa connect with the pterygoid plexus. These tributary veins include those that drain the nasal cavity, roof and lateral wall of the oral cavity, all teeth, muscles of the infratemporal fossa, paranasal sinuses, and nasopharynx. In addition, the inferior ophthalmic vein from the orbit can drain through the inferior orbital fissure into the pterygoid plexus.

1	Significantly, small emissary veins often connect the pterygoid plexus in the infratemporal fossa to the cavernous sinus in the cranial cavity. These emissary veins, which pass through the foramen ovale, through the cartilage that fills the foramen lacerum, and through a small sphenoidal foramen on the medial side of the lateral plate of the pterygoid process at the base of the skull, are a route by which infections can spread into the cranial cavity from structures, such as the teeth, that are drained by the pterygoid plexus. Also, because there are no valves in veins of the head and neck, anesthetic inadvertently injected under pressure into veins of the pterygoid plexus can backflow into tissues or into the cranial cavity. The pterygoid plexus connects: posteriorly, via a short maxillary vein, with the retromandibular vein in the neck; and anteriorly, via a deep facial vein, with the facial vein on the face.

1	The pterygoid plexus connects: posteriorly, via a short maxillary vein, with the retromandibular vein in the neck; and anteriorly, via a deep facial vein, with the facial vein on the face. The pterygopalatine fossa is an inverted teardrop-shaped space between bones on the lateral side of the skull immediately posterior to the maxilla (Fig. 8.153). Although small in size, the pterygopalatine fossa communicates via fissures and foramina in its walls with the: middle cranial fossa, infratemporal fossa, floor of the orbit, lateral wall of the nasal cavity, oropharynx, and roof of the oral cavity.

1	Because of its strategic location, the pterygopalatine fossa is a major site of distribution for the maxillary nerve [V2] and for the terminal part of the maxillary artery. It also contains the pterygopalatine ganglion where preganglionic parasympathetic fibers originating in the facial nerve [VII] synapse with postganglionic parasympathetic fibers and these fibers, along with sympathetic fibers originating from the T1 spinal cord level join branches of the maxillary nerve [V2]. All the upper teeth receive their innervation and blood supply from the maxillary nerve [V2] and the terminal part of the maxillary artery, respectively, that pass through the pterygopalatine fossa. The walls of the pterygopalatine fossa are formed by parts of the palatine, maxilla, and sphenoid bones (Fig. 8.153): The anterior wall is formed by the posterior surface of the maxilla. The medial wall is formed by the lateral surface of the palatine bone.

1	The anterior wall is formed by the posterior surface of the maxilla. The medial wall is formed by the lateral surface of the palatine bone. The posterior wall and roof are formed by parts of the sphenoid bone. The part of the sphenoid bone that contributes to the formation of the pterygopalatine fossa is the anterosuperior surface of the pterygoid process (Fig. 8.154). Opening onto this surface are two large foramina: The maxillary nerve [V2] passes through the most lateral and superior of these—the foramen rotundum—which communicates posteriorly with the middle cranial fossa (Fig. 8.154B). The greater petrosal nerve from the facial nerve [VII] and sympathetic fibers from the internal carotid plexus join to form the nerve of the pterygoid canal that passes forward into the pterygopalatine fossa through the more medial and inferior foramen—the anterior opening of the pterygoid canal.

1	The pterygoid canal (Fig. 8.154A) is a bony canal running horizontally through the root of the pterygoid process of the sphenoid bone. It opens anteriorly into the pterygopalatine fossa. Posteriorly it continues through the cartilage filling the foramen lacerum and opens into the middle cranial fossa just anteroinferior to the internal carotid artery as the vessel enters the cranial cavity through the carotid canal (Fig. 8.154B). Seven foramina and fissures provide apertures through which structures enter and leave the pterygopalatine fossa (Fig. 8.155): The foramen rotundum and pterygoid canal communicate with the middle cranial fossa and open onto the posterior wall. A small palatovaginal canal opens onto the posterior wall and leads to the nasopharynx. The palatine canal leads to the roof of the oral cavity (hard palate) and opens inferiorly. The sphenopalatine foramen opens onto the lateral wall of the nasal cavity and is in the medial wall.

1	The palatine canal leads to the roof of the oral cavity (hard palate) and opens inferiorly. The sphenopalatine foramen opens onto the lateral wall of the nasal cavity and is in the medial wall. The lateral aspect of the pterygopalatine fossa is continuous with the infratemporal fossa via a large gap (the pterygomaxillary fissure) between the posterior surface of the maxilla and pterygoid process of the sphenoid bone. The superior aspect of the anterior wall of the fossa opens into the floor of the orbit via the inferior orbital fissure. The maxillary nerve [V2] and terminal part of the maxillary artery enter and branch within the pterygopalatine fossa. In addition, the nerve of the pterygoid canal enters the fossa carrying: preganglionic parasympathetic fibers from the greater petrosal branch of the facial nerve [VII], and postganglionic sympathetic fibers from the deep petrosal branch of the carotid plexus.

1	The preganglionic parasympathetic fibers synapse in the pterygopalatine ganglion and both the sympathetic and postganglionic parasympathetic fibers pass with branches of the maxillary nerve [V2] out of the fossa and into adjacent regions. In addition to nerves and arteries, veins and lymphatics also pass through the pterygopalatine fossa. The maxillary nerve [V2] is purely sensory. It originates from the trigeminal ganglion in the cranial cavity, exits the middle cranial fossa, and enters the pterygopalatine fossa through the foramen rotundum (Fig. 8.156). It passes anteriorly through the fossa and exits as the infra-orbital nerve through the inferior orbital fissure. While passing through the pterygopalatine fossa, the maxillary nerve [V2] gives rise to the zygomatic nerve, the posterior superior alveolar nerve, and two ganglionic branches (Fig. 8.156). The two ganglionic branches originate from its inferior surface and pass through the pterygopalatine ganglion.

1	Postganglionic parasympathetic fibers, arising in the pterygopalatine ganglion, join the general sensory branches of the maxillary nerve [V2] in the pterygopalatine ganglion, as do postganglionic sympathetic fibers from the carotid plexus. The three types of fibers leave the ganglion as orbital, palatine, nasal, and pharyngeal branches. Orbital branches. The orbital branches are small and pass through the inferior orbital fissure to contribute to the supply of the orbital wall and of the sphenoidal and ethmoidal sinuses. Greater and lesser palatine nerves. The greater and lesser palatine nerves (Fig. 8.156) pass inferiorly from the pterygopalatine ganglion, enter and pass through the palatine canal, and enter the oral surface of the palate through the greater and lesser palatine foramina. The greater palatine nerve passes forward on the roof of the oral cavity to innervate mucosa and glands of the hard palate and the adjacent gingiva, almost as far forward as the incisor teeth.

1	The greater palatine nerve passes forward on the roof of the oral cavity to innervate mucosa and glands of the hard palate and the adjacent gingiva, almost as far forward as the incisor teeth. In the palatine canal, the greater palatine nerve gives origin to posterior inferior nasal nerves, which pass medially through small foramina in the perpendicular plate of the palatine bone and contribute to the innervation of the lateral nasal wall. After passing through the lesser palatine foramen, the lesser palatine nerve passes posteriorly to supply the soft palate. Nasal nerves. The nasal nerves (Fig. 8.156), approximately seven in number, pass medially through the sphenopalatine foramen to enter the nasal cavity. Most pass anteriorly to supply the lateral wall of the nasal cavity, while others pass across the roof to supply the medial wall.

1	One of the nerves passing across the roof to supply the medial wall of the nasal cavity (the nasopalatine nerve) is the largest of the nasal nerves and passes anteriorly down the nasal septum, through the incisive canal and fossa in the hard palate to enter the roof of the oral cavity and supply mucosa, gingiva, and glands adjacent to the incisor teeth. Pharyngeal nerve. The pharyngeal nerve (Fig. 8.156) passes posteriorly from the pterygopalatine ganglion, and leaves the fossa through the palatovaginal canal, which it then exits to supply the mucosa and glands of the nasopharynx. Zygomatic nerve. The zygomatic nerve (Fig. 8.156) originates directly from the maxillary nerve [V2] in the pterygopalatine fossa, which it leaves to enter the orbit through the inferior orbital fissure. It passes forward on the lateral orbital wall and divides into zygomaticotemporal and zygomaticofacial branches:

1	The zygomaticotemporal branch continues forward at the base of the lateral orbital wall, passes through a small bony canal in the zygomatic bone to enter the temporal fossa through a small foramen in the lateral orbital margin on the posterior surface of the frontal process of the zygomatic bone, and passes superficially to supply skin over the temple. The zygomaticofacial branch also passes forward at the base of the lateral orbital wall and leaves through a small bony canal, in the orbital margin, which opens via multiple small foramina on the anterolateral surface of the zygomatic bone, and its branches supply the adjacent skin.

1	Posterior superior alveolar nerve. The posterior superior alveolar nerve (Fig. 8.156) originates from the maxillary nerve [V2] in the pterygopalatine fossa and passes laterally out of the fossa through the pterygomaxillary fissure to enter the infratemporal fossa. It continues laterally and inferiorly to enter the posterior surface of the maxilla through a small alveolar foramen approximately midway between the last molar tooth and the inferior orbital fissure. It then passes inferiorly just deep to the mucosa of the maxillary sinus to join the superior dental plexus. The posterior superior alveolar nerve supplies the molar teeth and adjacent buccal gingivae, and contributes to the supply of the maxillary sinus.

1	The posterior superior alveolar nerve supplies the molar teeth and adjacent buccal gingivae, and contributes to the supply of the maxillary sinus. Infra-orbital nerve. The infra-orbital nerve (Fig. 8.156) is the anterior continuation of the maxillary nerve [V2] that leaves the pterygopalatine fossa through the inferior orbital fissure. It lies first in the infra-orbital groove in the floor of the orbit and then continues forward in the infra-orbital canal. While in the infra-orbital groove and canal, the infra-orbital nerve gives origin to middle and anterior superior alveolar nerves, respectively, which ultimately join the superior alveolar plexus to supply the upper teeth: The middle superior alveolar nerve also supplies the maxillary sinus. The anterior superior alveolar nerve also gives origin to a small nasal branch, which passes medially through the lateral wall of the nasal cavity to supply parts of the areas of the nasal floor and walls.

1	The infra-orbital nerve exits the infra-orbital canal through the infra-orbital foramen inferior to the orbital margin and divides into nasal, palpebral, and superior labial branches: Nasal branches supply skin over the lateral aspect of the external nose and part of the nasal septum. Palpebral branches supply skin of the lower eyelid. Superior labial branches supply skin over the cheek and upper lip, and the related oral mucosa. Nerve of the pterygoid canal and the pterygopalatine ganglion The nerve of the pterygoid canal (Fig. 8.157) is formed in the middle cranial fossa by the union of: the greater petrosal nerve (a branch of the facial nerve [VII]), and the deep petrosal nerve (a branch of the internal carotid plexus). The nerve of the pterygoid canal passes into the pterygopalatine fossa and joins the pterygopalatine ganglion. It carries mainly preganglionic parasympathetic and postganglionic sympathetic fibers.

1	The nerve of the pterygoid canal passes into the pterygopalatine fossa and joins the pterygopalatine ganglion. It carries mainly preganglionic parasympathetic and postganglionic sympathetic fibers. The greater petrosal nerve, which originates from the geniculate ganglion of the facial nerve [VII] in the temporal bone, exits the temporal bone through a small canal that opens via a fissure onto the anterior surface of the petrous part of the temporal bone. It passes anteromedially along the posterior margin of the middle cranial fossa and then under the internal carotid artery to reach the superior surface of the cartilage filling the foramen lacerum. As the greater petrosal nerve passes under the internal carotid artery, it is joined by the deep petrosal nerve to form the nerve of the pterygoid canal.

1	As the greater petrosal nerve passes under the internal carotid artery, it is joined by the deep petrosal nerve to form the nerve of the pterygoid canal. The greater petrosal nerve carries parasympathetic innervation to all glands above the oral fissure, including: mucous glands in the nasal cavity, salivary glands in the upper half of the oral cavity, and the lacrimal gland in the orbit. The greater petrosal nerve also carries some taste (SA) fibers from the soft palate in the lesser palatine nerve. The deep petrosal nerve is formed by postganglionic sympathetic fibers that originate in the superior cervical sympathetic ganglion in the neck and leave the ganglion as the internal carotid nerve. Preganglionic fibers that synapse in the ganglion are from the T1 spinal nerve.

1	Preganglionic fibers that synapse in the ganglion are from the T1 spinal nerve. The internal carotid nerve forms the internal carotid plexus around the internal carotid artery as the internal carotid artery passes through the skull and into the cranial cavity. Some of the fibers from the internal carotid plexus converge to form the deep petrosal nerve, which leaves the internal carotid plexus in the middle cranial fossa and joins the greater petrosal branch of the facial nerve [VII]. The deep petrosal nerve carries postganglionic sympathetic fibers destined mainly for blood vessels.

1	The deep petrosal nerve carries postganglionic sympathetic fibers destined mainly for blood vessels. The nerve of the pterygoid canal enters the superior surface of the cartilage that fills the foramen lacerum and passes anteriorly through the cartilage to enter the pterygoid canal in the root of the pterygoid process of the sphenoid bone. It passes through the canal and into the pterygopalatine fossa where it joins the pterygopalatine ganglion formed around the branches of the maxillary nerve [V2] (Fig. 8.157). The pterygopalatine ganglion is the largest of the four parasympathetic ganglia in the head and is formed by the cell bodies of the postganglionic neurons associated with preganglionic parasympathetic fibers of the facial nerve [VII] carried by the greater petrosal nerve and the nerve of the pterygoid canal.

1	The postganglionic parasympathetic fibers that originate in the pterygopalatine ganglion, together with postganglionic sympathetic fibers passing through the ganglion, join fibers from the ganglionic branches of the maxillary nerve [V2] to form orbital, palatine, nasal, and pharyngeal branches, which leave the ganglion. Other postganglionic parasympathetic and sympathetic fibers pass superiorly through the ganglionic branches of the maxillary nerve [V2] to enter the main trunk of the maxillary nerve and be distributed with the zygomatic, posterior superior alveolar, and infra-orbital nerves. Of these, the postganglionic parasympathetic and sympathetic fibers that pass into the orbit with the zygomatic nerve are particularly important because they ultimately innervate the lacrimal gland. Innervation of the lacrimal gland

1	Innervation of the lacrimal gland Approximately midway along the orbital wall, the postganglionic parasympathetic and sympathetic fibers leave the zygomaticotemporal branch of the zygomatic nerve and form a special autonomic nerve, which travels up the lateral orbital wall to join the lacrimal nerve (Fig. 8.157; also see Fig. 8.87). The lacrimal nerve is a major general sensory branch of the ophthalmic nerve [V1], which passes forward in the orbit at the margin between the lateral wall and roof. The postganglionic parasympathetic and sympathetic fibers pass with the lacrimal nerve to the lacrimal gland. A lesion anywhere along the course of parasympathetic fibers that leave the brain as part of the facial nerve [VII] and are ultimately carried to the lacrimal gland along branches of the ophthalmic nerve [V1] results in “dry eye” and can eventually lead to loss of vision in the affected eye.

1	The maxillary artery is a major branch of the external carotid artery in the neck. It originates adjacent to the neck of the mandible, passes forward through the infratemporal fossa, and then enters the pterygopalatine fossa through the pterygomaxillary fissure (Fig. 8.158). The part of the maxillary artery in the pterygopalatine fossa (the third part) is anterior to the pterygopalatine ganglion and gives origin to branches that accompany branches of the maxillary nerve [V2] and the pterygopalatine ganglion. Branches of the maxillary artery include the posterior superior alveolar, infra-orbital, greater palatine, pharyngeal, and sphenopalatine arteries, and the artery of the pterygoid canal (Fig. 8.158). Collectively, these branches supply much of the nasal cavity, the roof of the oral cavity, and all upper teeth. In addition, they contribute to the blood supply of the sinuses, oropharynx, and floor of the orbit.

1	Posterior superior alveolar artery. The posterior superior alveolar artery (Fig. 8.158) originates from the maxillary artery as it passes through the pterygomaxillary fissure. It meets the posterior superior alveolar nerve, accompanies it through the alveolar foramen on the infratemporal surface of the maxilla, and supplies the molar and premolar teeth, adjacent gingiva, and the maxillary sinus. Infra-orbital artery. The infra-orbital artery (Fig. 8.158) passes forward with the infra-orbital nerve and leaves the pterygopalatine fossa through the inferior orbital fissure. With the infra-orbital nerve, it lies in the infra-orbital groove and infra-orbital canal, and emerges through the infra-orbital foramen to supply parts of the face.

1	Within the infra-orbital canal, the infra-orbital artery gives origin to: branches that contribute to the blood supply of structures near the floor of the orbit—the inferior rectus and inferior oblique muscles, and the lacrimal sac; and anterior superior alveolar arteries (Fig. 8.158), which supply the incisor and canine teeth and the maxillary sinus. Greater palatine artery. The greater palatine artery (Fig. 8.158) passes inferiorly with the palatine nerves into the palatine canal. It gives origin to a lesser palatine branch (Fig. 8.158), which passes through the lesser palatine foramen to supply the soft palate, and then continues through the greater palatine foramen to supply the hard palate. The latter vessel passes forward on the inferior surface of the palate to enter the incisive fossa and pass superiorly through the incisive canal to supply the anterior aspect of the septal wall of the nasal cavity.

1	Pharyngeal branch. The pharyngeal branch (Fig. 8.158) of the maxillary artery travels posteriorly and leaves the pterygopalatine fossa through the palatovaginal canal with the pharyngeal nerve. It supplies the posterior aspect of the roof of the nasal cavity, the sphenoidal sinus, and the pharyngotympanic tube. Sphenopalatine artery. The sphenopalatine artery (Fig. 8.158) is the terminal branch of the maxillary artery. It leaves the pterygopalatine fossa medially through the sphenopalatine foramen and accompanies the nasal nerves, giving off: posterior lateral nasal arteries, which supply the lateral wall of the nasal cavity and contribute to the supply of the paranasal sinuses; and posterior septal branches, which travel medially across the roof to supply the nasal septum—the largest of these branches passes anteriorly down the septum to anastomose with the end of the greater palatine artery.

1	Artery of pterygoid canal. The artery of the pterygoid canal passes posteriorly into the pterygoid canal. It supplies surrounding tissues and terminates, after passing inferiorly through cartilage filling the foramen lacerum, in the mucosa of the nasopharynx. Veins that drain areas supplied by branches of the terminal part of the maxillary artery generally travel with these branches back into the pterygopalatine fossa. The veins coalesce in the pterygopalatine fossa and then pass laterally through the pterygomaxillary fissure to join the pterygoid plexus of veins in the infratemporal fossa (Fig. 8.159). The infra-orbital vein, which drains the inferior aspect of the orbit, may pass directly into the infratemporal fossa through the lateral aspect of the inferior orbital fissure, so bypassing the pterygopalatine fossa.

1	The neck is a tube providing continuity from the head to the trunk. It extends anteriorly from the lower border of the mandible to the upper surface of the manubrium of the sternum, and posteriorly from the superior nuchal line on the occipital bone of the skull to the intervertebral disc between the CVII and TI vertebrae. Within the tube, four compartments provide longitudinal organization (Fig. 8.160): The visceral compartment is anterior and contains parts of the digestive and respiratory systems, and several endocrine glands. The vertebral compartment is posterior and contains the cervical vertebrae, spinal cord, cervical nerves, and muscles associated with the vertebral column. The two vascular compartments, one on each side, are lateral and contain the major blood vessels and the vagus nerve [X]. All these compartments are contained within unique layers of cervical fascia. For descriptive purposes the neck is divided into anterior and posterior triangles (Fig. 8.161):

1	All these compartments are contained within unique layers of cervical fascia. For descriptive purposes the neck is divided into anterior and posterior triangles (Fig. 8.161): The boundaries of the anterior triangle are the anterior border of the sternocleidomastoid muscle, the inferior border of the mandible, and the midline of the neck. The boundaries of the posterior triangle are the posterior border of the sternocleidomastoid muscle, the anterior border of the trapezius muscle, and the middle one-third of the clavicle. The fascia of the neck has a number of unique features. The superficial fascia in the neck contains a thin sheet of muscle (the platysma), which begins in the superficial fascia of the thorax, runs upward to attach to the mandible and blend with the muscles on the face, is innervated by the cervical branch of the facial nerve [VII], and is only found in this location.

1	Deep to the superficial fascia, the deep cervical fascia is organized into several distinct layers (Fig. 8.160). These include: an investing layer, which surrounds all structures in the neck; the prevertebral layer, which surrounds the vertebral column and the deep muscles associated with the back; the pretracheal layer, which encloses the viscera of the neck; and the carotid sheaths, which receive a contribution from the other three fascial layers and surround the two major neurovascular bundles on either side of the neck. The investing layer completely surrounds the neck and encloses the trapezius and sternocleidomastoid muscles (Fig. 8.162).

1	The investing layer completely surrounds the neck and encloses the trapezius and sternocleidomastoid muscles (Fig. 8.162). Attaching posteriorly to the ligamentum nuchae and the spinous process of the CVII vertebra, this fascial layer splits as it passes forward to enclose the trapezius muscle, reunites into a single layer as it forms the roof of the posterior triangle, splits again to surround the sternocleidomastoid muscle, and reunites again to join its twin from the other side. Anteriorly, the investing fascia merges with fascia surrounding the infrahyoid muscles. The investing fascia is attached: superiorly to the external occipital protuberance and the superior nuchal line, laterally to the mastoid process and zygomatic arch, and inferiorly to the spine of the scapula, the acromion, the clavicle, and the manubrium of the sternum.

1	The external and anterior jugular veins, and the lesser occipital, great auricular, transverse cervical, and supraclavicular nerves, all branches of the cervical plexus, pierce the investing fascia. The prevertebral layer is a cylindrical layer of fascia that surrounds the vertebral column and the muscles associated with it (Fig. 8.162). Muscles in this group include the prevertebral muscles, the anterior, middle, and posterior scalene muscles, and the deep muscles of the back.

1	The prevertebral fascia is attached posteriorly along the length of the ligamentum nuchae, and superiorly forms a continuous circular line attaching to the base of the skull. This circle begins: anteriorly as the fascia attaches to the basilar part of the occipital bone, the area of the jugular foramen, and the carotid canal; continues laterally, attaching to the mastoid process; and continues posteriorly along the superior nuchal line ending at the external occipital protuberance, where it associates with its partner from the opposite side. Anteriorly, the prevertebral fascia is attached to the anterior surfaces of the transverse processes and bodies of vertebrae CI to CVII. The prevertebral fascia passing between the attachment points on the transverse processes is unique. In this location, it splits into two layers, creating a longitudinal fascial space containing loose connective tissue that extends from the base of the skull through the thorax (Figs. 8.162 and 8.163).

1	There is one additional specialization of the prevertebral fascia in the lower region of the neck. The prevertebral fascia in an anterolateral position extends from the anterior and middle scalene muscles to surround the brachial plexus and subclavian artery as these structures pass into the axilla. This fascial extension is the axillary sheath. The pretracheal layer consists of a collection of fascias that surround the trachea, esophagus, and thyroid gland (Fig. 8.162). Anteriorly, it consists of a pretracheal fascia that crosses the neck and encloses the infrahyoid muscles, and covers the trachea and the thyroid gland. The pretracheal fascia begins superiorly at the hyoid bone and ends inferiorly in the upper thoracic cavity. Laterally, this fascia encloses the thyroid gland and more posteriorly is continuous with fascia that surrounds the esophagus.

1	Posterior to the pharynx, the pretracheal layer is referred to as the buccopharyngeal fascia and separates the pharynx from the prevertebral layer (Fig. 8.163). The buccopharyngeal fascia begins superiorly at the base of the skull and merges with fascia covering the esophagus that then continues inferiorly into the thoracic cavity. Each carotid sheath is a column of fascia that surrounds the common carotid artery, the internal carotid artery, the internal jugular vein, and the vagus nerve as these structures pass through the neck (Fig. 8.162). It receives contributions from the investing, prevertebral, and pretracheal layers, though the extent of each component’s contribution varies. The arrangement of the various layers of cervical fascia organizes the neck into four longitudinal compartments (Fig. 8.160): The first compartment is the largest, includes the other three, and consists of the area surrounded by the investing layer.

1	The first compartment is the largest, includes the other three, and consists of the area surrounded by the investing layer. The second compartment consists of the vertebral column and the deep muscles associated with this structure, and is the area contained within the prevertebral layer. The third compartment (the visceral compartment) contains the pharynx, the trachea, the esophagus, and the thyroid gland, which are surrounded by the pretracheal layer. Finally, there is a compartment (the carotid sheath) consisting of the neurovascular structures that pass from the base of the skull to the thoracic cavity, and the sheath enclosing these structures receives contributions from the other cervical fascias. Between the fascial layers in the neck are spaces that may provide a conduit for the spread of infections from the neck to the mediastinum. (Fig. 8.163):

1	Between the fascial layers in the neck are spaces that may provide a conduit for the spread of infections from the neck to the mediastinum. (Fig. 8.163): The first is the pretracheal space between the investing layer of cervical fascia covering the posterior surface of the infrahyoid muscles and the pretracheal fascia (covering the anterior surface of the trachea and the thyroid gland), which passes between the neck and the anterior part of the superior mediastinum. The second is the retropharyngeal space between the buccopharyngeal fascia (on the posterior surface of the pharynx and esophagus) and the prevertebral fascia (on the anterior surface of the transverse processes and bodies of the cervical vertebrae), which extends from the base of the skull to the upper part of the posterior mediastinum.

1	The third space is within the prevertebral layer covering the anterior surface of the transverse processes and bodies of the cervical vertebrae. This layer splits into two laminae to create a fascial space that begins at the base of the skull and extends through the posterior mediastinum to the diaphragm. The external jugular and anterior jugular veins are the primary venous channels for superficial venous drainage of the neck (Fig. 8.164). The external jugular vein is formed posterior to the angle of the mandible as the posterior auricular vein and the retromandibular vein join: The posterior auricular vein drains the scalp behind and above the ear.

1	The posterior auricular vein drains the scalp behind and above the ear. The retromandibular vein is formed when the superficial temporal and maxillary veins join in the substance of the parotid gland and it descends to the angle of mandible, where it divides into an anterior and a posterior division (Fig. 8.164)—the posterior division joins the posterior auricular vein to form the external jugular vein, and the anterior division joins the facial vein to form the common facial vein, which passes deep and becomes a tributary to the internal jugular vein. Once formed, the external jugular vein passes straight down the neck in the superficial fascia and is superficial to the sternocleidomastoid muscle throughout its course, crossing it diagonally as it descends.

1	Reaching the lower part of the neck, just superior to the clavicle and immediately posterior to the sternocleidomastoid muscle, the external jugular vein pierces the investing layer of cervical fascia, passes deep to the clavicle, and enters the subclavian vein. Tributaries received by the external jugular vein along its course include the posterior external jugular vein (draining superficial areas of the back of the neck) and the transverse cervical and suprascapular veins (draining the posterior scapular region). The anterior jugular veins, although variable and inconsistent, are usually described as draining the anterior aspect of the neck (Fig. 8.164). These paired venous channels, which begin as small veins, come together at or just superior to the hyoid bone. Once formed, each anterior jugular vein descends on either side of the midline of the neck.

1	Inferiorly, near the medial attachment of the sternocleidomastoid muscle, each anterior jugular vein pierces the investing layer of cervical fascia to enter the subclavian vein. Occasionally, the anterior jugular vein may enter the external jugular vein immediately before the external jugular vein enters the subclavian vein. Often, the right and left anterior jugular veins communicate with each other, being connected by a jugular venous arch in the area of the suprasternal notch. Anterior triangle of the neck The anterior triangle of the neck is outlined by the anterior border of the sternocleidomastoid muscle laterally, the inferior border of the mandible superiorly, and the midline of the neck medially (Fig. 8.166). It is further subdivided into several smaller triangles as follows: The submandibular triangle is outlined by the inferior border of the mandible superiorly and the anterior and posterior bellies of the digastric muscle inferiorly.

1	The submandibular triangle is outlined by the inferior border of the mandible superiorly and the anterior and posterior bellies of the digastric muscle inferiorly. The submental triangle is outlined by the hyoid bone inferiorly, the anterior belly of the digastric muscle laterally, and the midline. The muscular triangle is outlined by the hyoid bone superiorly, the superior belly of the omohyoid muscle, and the anterior border of the sternocleidomastoid muscle laterally, and the midline. The carotid triangle is outlined by the superior belly of the omohyoid muscle anteroinferiorly, the stylohyoid muscle and posterior belly of the digastric superiorly, and the anterior border of the sternocleidomastoid muscle posteriorly.

1	Each of these triangles contains numerous structures that can be identified as being within a specific triangle, passing into a specific triangle from outside the area, originating in one triangle and passing to another triangle, or passing through several triangles while passing through the region. A discussion of the anterior triangle of the neck must therefore combine a systemic approach, describing the muscles, vessels, and nerves in the area, with a regional approach, describing the contents of each triangle. The muscles in the anterior triangle of the neck (Table 8.12) can be grouped according to their location relative to the hyoid bone: Muscles superior to the hyoid are classified as suprahyoid muscles and include the stylohyoid, digastric, mylohyoid, and geniohyoid. Muscles inferior to the hyoid are infrahyoid muscles and include the omohyoid, sternohyoid, thyrohyoid, and sternothyroid.

1	Muscles inferior to the hyoid are infrahyoid muscles and include the omohyoid, sternohyoid, thyrohyoid, and sternothyroid. The four pairs of suprahyoid muscles are related to the submental and submandibular triangles (Fig. 8.166). They pass in a superior direction from the hyoid bone to the skull or mandible and raise the hyoid, as occurs during swallowing. The stylohyoid muscle arises from the base of the styloid process and passes anteroinferiorly to attach to the lateral area of the body of the hyoid bone (Fig. 8.167). During swallowing it pulls the hyoid bone posterosuperiorly and it is innervated by the facial nerve [VII]. The digastric muscle has two bellies connected by a tendon, which attaches to the body of the hyoid bone (Fig. 8.167): The posterior belly arises from the mastoid notch on the medial side of the mastoid process of the temporal bone. The anterior belly arises from the digastric fossa on the lower inside of the mandible.

1	The anterior belly arises from the digastric fossa on the lower inside of the mandible. The tendon between the two bellies, which is attached to the body of the hyoid bone, is the point of insertion of both bellies. Because of this arrangement, the muscle has multiple actions depending on which bone is fixed: When the mandible is fixed, the digastric muscle raises the hyoid bone. When the hyoid bone is fixed, the digastric muscle opens the mouth by lowering the mandible. Innervation of the digastric muscle is from two different cranial nerves. The innervation of the posterior belly of the digastric muscle is by the facial nerve [VII], whereas the anterior belly of the muscle is innervated by the mandibular division [V3] of the trigeminal nerve [V].

1	The mylohyoid muscle is superior to the anterior belly of the digastric and, with its partner from the opposite side, forms the floor of the mouth (Fig. 8.167). It originates from the mylohyoid line on the medial surface of the body of the mandible and inserts into the hyoid bone and also blends with the mylohyoid muscle from the opposite side. This mylohyoid muscle supports and elevates the floor of the mouth and elevates the hyoid bone. It is innervated by the mandibular division [V3] of the trigeminal nerve [V]. The geniohyoid muscle is superior to the floor of the oral cavity and is not generally considered a muscle of the anterior triangle of the neck; however, it can be regarded as a suprahyoid muscle. It is the final muscle in the suprahyoid group (Fig. 8.167). A narrow muscle, it is superior to the medial part of each mylohyoid muscle. The muscles from each side are next to each other in the midline.

1	The geniohyoid arises from the inferior mental spine of the mandible and passes backward and downward to insert on the body of the hyoid bone. It has two functions depending on which bone is fixed: Fixation of the mandible elevates and pulls the hyoid bone forward. Fixation of the hyoid bone pulls the mandible downward and inward. The geniohyoid is innervated by a branch from the anterior ramus of C1 carried along the hypoglossal nerve [XII]. The four infrahyoid muscles are related to the muscular triangle (Fig. 8.166). They attach the hyoid bone to inferior structures and depress the hyoid bone. They also provide a stable point of attachment for the suprahyoid muscles. Because of their appearance, they are sometimes referred to as the “strap muscles.”

1	The sternohyoid muscle is a long, thin muscle originating from the posterior aspect of the sternoclavicular joint and adjacent manubrium of the sternum (Fig. 8.168). It ascends to insert onto the body of the hyoid bone. It depresses the hyoid bone and is innervated by the anterior rami of C1 to C3 through the ansa cervicalis. Lateral to the sternohyoid muscle is the omohyoid muscle (Fig. 8.168). This muscle consists of two bellies with an intermediate tendon in both the posterior and anterior triangles of the neck: The inferior belly begins on the superior border of the scapula, medial to the suprascapular notch, and passes forward and upward across the posterior triangle ending at the intermediate tendon. The superior belly begins at the intermediate tendon and ascends to attach to the body of the hyoid bone just lateral to the attachment of the sternohyoid. The intermediate tendon is attached to the clavicle, near its medial end, by a fascial sling.

1	The intermediate tendon is attached to the clavicle, near its medial end, by a fascial sling. The omohyoid depresses and fixes the hyoid bone. It is innervated by the anterior rami of C1 to C3 through the ansa cervicalis. The thyrohyoid muscle is deep to the superior parts of the omohyoid and sternohyoid (Fig. 8.168). Originating at the oblique line on the lamina of the thyroid cartilage it passes upward to insert into the greater horn and adjacent aspect of the body of the hyoid bone. The thyrohyoid muscle has variable functions depending on which bone is fixed. Generally, it depresses the hyoid, but when the hyoid is fixed it raises the larynx (e.g., when high notes are sung). It is innervated by fibers from the anterior ramus of C1 that travel with the hypoglossal nerve [XII].

1	Lying beneath the sternohyoid and in continuity with the thyrohyoid, the sternothyroid is the last muscle in the infrahyoid group (Fig. 8.168). It arises from the posterior surface of the manubrium of the sternum and passes upward to attach to the oblique line on the lamina of the thyroid cartilage. The sternothyroid muscle draws the larynx (thyroid cartilage) downward and is innervated by the anterior rami of C1 to C3 through the ansa cervicalis. Passing through the anterior triangle of the neck are the common carotid arteries and their branches, the external and internal carotid arteries. These vessels supply all structures of the head and neck. Associated with this arterial system are the internal jugular vein and its tributaries. These vessels receive blood from all structures of the head and neck. The common carotid arteries are the beginning of the carotid system (Fig. 8.169):

1	The common carotid arteries are the beginning of the carotid system (Fig. 8.169): The right common carotid artery originates from the brachiocephalic trunk immediately posterior to the right sternoclavicular joint and is entirely in the neck throughout its course. The left common carotid artery begins in the thorax as a direct branch of the arch of the aorta and passes superiorly to enter the neck near the left sternoclavicular joint. Both right and left common carotid arteries ascend through the neck, just lateral to the trachea and esophagus, within a fascial compartment (the carotid sheath). They give off no branches as they pass through the neck. Near the superior edge of the thyroid cartilage each common carotid artery divides into its two terminal branches—the external and internal carotid arteries (Fig. 8.170).

1	Near the superior edge of the thyroid cartilage each common carotid artery divides into its two terminal branches—the external and internal carotid arteries (Fig. 8.170). The superior part of each common carotid artery and its division into external and internal carotid arteries occurs in the carotid triangle (Fig. 8.170), which is a subdivision of the anterior triangle of the neck (see Fig. 8.166). At the bifurcation, the common carotid artery and the beginning of the internal carotid artery are dilated. This dilation is the carotid sinus (Fig. 8.171) and contains receptors that monitor changes in blood pressure and are innervated by a branch of the glossopharyngeal nerve [IX]. Another accumulation of receptors in the area of the bifurcation is responsible for detecting changes in blood chemistry, primarily oxygen content. This is the carotid body and is innervated by branches from both the glossopharyngeal [IX] and vagus [X] nerves.

1	After its origin, the internal carotid artery ascends toward the base of the skull (Fig. 8.171). It gives off no branches in the neck and enters the cranial cavity through the carotid canal in the petrous part of the temporal bone. The internal carotid arteries supply the cerebral hemispheres, the eyes and the contents of the orbits, and the forehead. The external carotid arteries begin giving off branches immediately after the bifurcation of the common carotid arteries (Fig. 8.171 and Table 8.13) as follows: The superior thyroid artery is the first branch—it arises from the anterior surface near or at the bifurcation and passes in a downward and forward direction to reach the superior pole of the thyroid gland. The ascending pharyngeal artery is the second and smallest branch—it arises from the posterior aspect of the external carotid artery and ascends between the internal carotid artery and the pharynx.

1	The ascending pharyngeal artery is the second and smallest branch—it arises from the posterior aspect of the external carotid artery and ascends between the internal carotid artery and the pharynx. The lingual artery arises from the anterior surface of the external carotid artery just above the superior thyroid artery at the level of the hyoid bone, passes deep to the hypoglossal nerve [XII], and passes between the middle constrictor of the pharynx and hyoglossus muscles. The facial artery is the third anterior branch of the external carotid artery—it arises just above the lingual artery, passes deep to the stylohyoid and posterior belly of the digastric muscles, continues deep between the submandibular gland and mandible, and emerges over the edge of the mandible just anterior to the masseter muscle, to enter the face.

1	The occipital artery arises from the posterior surface of the external carotid artery, near the level of origin of the facial artery, passes upward and posteriorly deep to the posterior belly of the digastric muscle, and emerges on the posterior aspect of the scalp. The posterior auricular artery is a small branch arising from the posterior surface of the external carotid artery and passes upward and posteriorly. The superficial temporal artery is one of the terminal branches and appears as an upward continuation of the external carotid artery—beginning posterior to the neck of the mandible, it passes anterior to the ear, crosses the zygomatic process of the temporal bone, and above this point divides into anterior and posterior branches.

1	The maxillary artery is the larger of the two terminal branches of the external carotid artery—arising posterior to the neck of the mandible, it passes through the parotid gland, continues medial to the neck of the mandible and into the infratemporal fossa, and continues through this area into the pterygopalatine fossa. Collecting blood from the skull, brain, superficial face, and parts of the neck, the internal jugular vein begins as a dilated continuation of the sigmoid sinus, which is a dural venous sinus. This initial dilated part is referred to as the superior bulb of jugular vein and receives another dural venous sinus (the inferior petrosal sinus) soon after it is formed. It exits the skull through the jugular foramen associated with the glossopharyngeal [IX], vagus [X], and accessory [XI] nerves, and enters the carotid sheath.

1	The internal jugular vein traverses the neck within the carotid sheath, initially posterior to the internal carotid artery, but passes to a more lateral position farther down. It remains lateral to the common carotid artery through the rest of the neck with the vagus nerve [X] posterior and partially between the two vessels. The paired internal jugular veins join with the subclavian veins posterior to the sternal end of the clavicle to form the right and left brachiocephalic veins (Fig. 8.169). Tributaries to each internal jugular vein include the inferior petrosal sinus, and the facial, lingual, pharyngeal, occipital, superior thyroid, and middle thyroid veins. Numerous cranial and peripheral nerves: pass through the anterior triangle of the neck as they continue to their final destination, send branches to structures in or forming boundaries of the anterior triangle of the neck, and while in the anterior triangle of the neck, send branches to nearby structures.

1	The cranial nerves in these categories include the facial [VII], glossopharyngeal [IX], vagus [X], accessory [XI], and hypoglossal [XII]. Branches of spinal nerves in these categories include the transverse cervical nerve from the cervical plexus and the upper and lower roots of the ansa cervicalis. After emerging from the stylomastoid foramen, the facial nerve [VII] gives off branches that innervate two muscles associated with the anterior triangle of the neck: the posterior belly of the digastric, and the stylohyoid. The facial nerve [VII] also innervates the platysma muscle that overlies the anterior triangle and part of the posterior triangle of the neck.

1	The facial nerve [VII] also innervates the platysma muscle that overlies the anterior triangle and part of the posterior triangle of the neck. The glossopharyngeal nerve [IX] leaves the cranial cavity through the jugular foramen. It begins its descent between the internal carotid artery and the internal jugular vein, lying deep to the styloid process and the muscles associated with the styloid process. As the glossopharyngeal nerve [IX] completes its descent, it passes forward between the internal and external carotid arteries, and curves around the lateral border of the stylopharyngeus muscle (Fig. 8.172). At this point, it continues in an anterior direction, deep to the hyoglossus muscle, to reach the base of the tongue and the area of the palatine tonsil. As the glossopharyngeal nerve [IX] passes through the area of the anterior triangle of the neck it innervates the stylopharyngeus muscle, sends a branch to the carotid sinus, and supplies sensory branches to the pharynx.

1	The vagus nerve [X] exits the cranial cavity through the jugular foramen between the glossopharyngeal [IX] and accessory [XI] nerves. Outside the skull the vagus nerve [X] enters the carotid sheath and descends through the neck enclosed in this structure medial to the internal jugular vein and posterior to the internal carotid and common carotid arteries (Fig. 8.173). Branches of the vagus nerve [X] as it passes through the anterior triangle of the neck include a motor branch to the pharynx, a branch to the carotid body, the superior laryngeal nerve (which divides into external and internal laryngeal branches), and possibly a cardiac branch.

1	The accessory nerve [XI] is the most posterior of the three cranial nerves exiting the cranial cavity through the jugular foramen. It begins its descent medial to the internal jugular vein, emerging from between the internal jugular vein and internal carotid artery to cross the lateral surface of the internal jugular vein as it passes downward and backward to disappear either into or beneath the anterior border of the sternocleidomastoid muscle (Fig. 8.174). The accessory nerve gives off no branches as it passes through the anterior triangle of the neck.

1	The accessory nerve gives off no branches as it passes through the anterior triangle of the neck. The hypoglossal nerve [XII] leaves the cranial cavity through the hypoglossal canal and is medial to the internal jugular vein and internal carotid artery immediately outside the skull. As it descends, it passes outward between the internal jugular vein and internal carotid artery (Fig. 8.175). At this point it passes forward, hooking around the occipital artery, across the lateral surfaces of the internal and external carotid arteries and the lingual artery, and then continues deep to the posterior belly of the digastric and stylohyoid muscles. It passes over the surface of the hyoglossus muscle and disappears deep to the mylohyoid muscle. The hypoglossal nerve [XII], which supplies the tongue, does not give off any branches as it passes through the anterior triangle of the neck.

1	The hypoglossal nerve [XII], which supplies the tongue, does not give off any branches as it passes through the anterior triangle of the neck. The transverse cervical nerve is a branch of the cervical plexus arising from the anterior rami of cervical nerves C2 and C3. It emerges from beneath the posterior border of the sternocleidomastoid muscle, near the middle of the muscle, and loops around the sternocleidomastoid to cross its anterior surface in a transverse direction (Fig. 8.176). It continues across the neck and provides cutaneous innervation to this area. The ansa cervicalis is a loop of nerve fibers from cervical nerves C1 to C3 that innervate the “strap muscles” in the anterior triangle of the neck (Fig. 8.177). It begins as branches from the cervical nerve C1 join the hypoglossal nerve [XII] soon after it leaves the skull.

1	As the hypoglossal nerve [XII] completes its descent and begins to pass forward across the internal and external carotid arteries, some of the cervical nerve fibers leave it and descend between the internal jugular vein and the internal, and then common, carotid arteries. These nerve fibers are the superior root of the ansa cervicalis and innervate the superior belly of the omohyoid muscle, and the upper parts of the sternohyoid and sternothyroid muscles. Completing the loop is a direct branch from the cervical plexus containing nerve fibers from the second and third cervical nerves C2 and C3 (Fig. 8.177). This is the inferior root of the ansa cervicalis. It descends either medial or lateral to the internal jugular vein before turning medially to join the superior root. At this location, the ansa cervicalis gives off branches that innervate the inferior belly of the omohyoid, and the lower parts of the sternohyoid and sternothyroid muscles. Elements of the gastrointestinal

1	Elements of the gastrointestinal The esophagus, trachea, pharynx, and larynx lie in the neck and are related to the anterior triangles. The esophagus is part of the gastrointestinal system and has only a short course in the lower neck. It begins at vertebral level CVI, where it is continuous with the pharynx above and courses inferiorly to pass through the thoracic inlet. It lies directly anterior to the vertebral column (Fig. 8.178B). The trachea is part of the lower airway and, like the esophagus, begins at vertebral level CVI, where it is continuous with the larynx above (Fig. 8.178B). The trachea lies directly anterior to the esophagus and passes inferiorly in the midline to enter the thorax. The pharynx is a common pathway for air and food, and the head with similar compartments in the lower neck (see pp. 1029–1041). The larynx is the upper end of the lower airway. It is continuous with the trachea below and the pharynx posterosuperiorly (see pp. 1041–1058).

1	The larynx is the upper end of the lower airway. It is continuous with the trachea below and the pharynx posterosuperiorly (see pp. 1041–1058). The thyroid and parathyroid glands are endocrine glands positioned anteriorly in the neck. Both glands begin as pharyngeal outgrowths that migrate caudally to their final positions as development continues. The thyroid gland is a large, unpaired gland, while the parathyroid glands, usually four in number, are small and are on the posterior surface of the thyroid gland. The thyroid gland is anterior in the neck below and lateral to the thyroid cartilage (Fig. 8.178). It consists of two lateral lobes (which cover the anterolateral surfaces of the trachea, the cricoid cartilage, and the lower part of the thyroid cartilage) with an isthmus that connects the lateral lobes and crosses the anterior surfaces of the second and third tracheal cartilages.

1	Lying deep to the sternohyoid, sternothyroid, and omohyoid muscles, the thyroid gland is in the visceral compartment of the neck. This compartment also includes the pharynx, trachea, and esophagus and is surrounded by the pretracheal layers of fascia. The thyroid gland arises as a median outgrowth from the floor of the pharynx near the base of the tongue. The foramen cecum of the tongue indicates the site of origin and the thyroglossal duct marks the path of migration of the thyroid gland to its final adult location. The thyroglossal duct usually disappears early in development, but remnants may persist as a cyst or as a connection to the foramen cecum (i.e., a fistula). There may also be functional thyroid gland: associated with the tongue (a lingual thyroid), anywhere along the path of migration of the thyroid gland, or extending upward from the gland along the path of the thyroglossal duct (a pyramidal lobe). Two major arteries supply the thyroid gland.

1	Two major arteries supply the thyroid gland. Superior thyroid artery. The superior thyroid artery is the first branch of the external carotid artery (Fig. 8.179). It descends, passing along the lateral margin of the thyrohyoid muscle, to reach the superior pole of the lateral lobe of the gland where it divides into anterior and posterior glandular branches: The anterior glandular branch passes along the superior border of the thyroid gland and anastomoses with its twin from the opposite side across the isthmus (Fig. 8.179). The posterior glandular branch passes to the posterior side of the gland and may anastomose with the inferior thyroid artery (Fig. 8.180).

1	The posterior glandular branch passes to the posterior side of the gland and may anastomose with the inferior thyroid artery (Fig. 8.180). Inferior thyroid artery. The inferior thyroid artery is a branch of the thyrocervical trunk, which arises from the first part of the subclavian artery (Figs. 8.179 and 8.180). It ascends along the medial edge of the anterior scalene muscle, passes posteriorly to the carotid sheath, and reaches the inferior pole of the lateral lobe of the thyroid gland. At the thyroid gland the inferior thyroid artery divides into an: inferior branch, which supplies the lower part of the thyroid gland and anastomoses with the posterior branch of the superior thyroid artery, and an ascending branch, which supplies the parathyroid glands. Occasionally, a small thyroid ima artery arises from the brachiocephalic trunk or the arch of the aorta and ascends on the anterior surface of the trachea to supply the thyroid gland.

1	Occasionally, a small thyroid ima artery arises from the brachiocephalic trunk or the arch of the aorta and ascends on the anterior surface of the trachea to supply the thyroid gland. Three veins drain the thyroid gland (Fig. 8.179): The superior thyroid vein primarily drains the area supplied by the superior thyroid artery. The middle and inferior thyroid veins drain the rest of the thyroid gland. The superior and middle thyroid veins drain into the internal jugular vein, and the inferior thyroid veins empty into the right and left brachiocephalic veins, respectively. Lymphatic drainage of the thyroid gland is to nodes beside the trachea (paratracheal nodes) and to deep cervical nodes inferior to the omohyoid muscle along the internal jugular vein.

1	Lymphatic drainage of the thyroid gland is to nodes beside the trachea (paratracheal nodes) and to deep cervical nodes inferior to the omohyoid muscle along the internal jugular vein. The thyroid gland is closely related to the recurrent laryngeal nerves. After branching from the vagus nerve [X] and looping around the subclavian artery on the right and the arch of the aorta on the left, the recurrent laryngeal nerves ascend in a groove between the trachea and esophagus (Fig. 8.180). They pass deep to the posteromedial surface of the lateral lobes of the thyroid gland and enter the larynx by passing deep to the lower margin of the inferior constrictor of the pharynx.

1	Together with branches of the inferior thyroid arteries, the recurrent laryngeal nerves are clearly related to, and may pass through ligaments, one on each side, that bind the thyroid gland to the trachea and to the cricoid cartilage of the larynx. These relationships need to be considered when surgically removing or manipulating the thyroid gland. The parathyroid glands are two pairs of small, ovoid, yellowish structures on the deep surface of the lateral lobes of the thyroid gland. They are designated as the superior and inferior parathyroid glands (Fig. 8.180). However, their position is quite variable and they may be anywhere from the carotid bifurcation superiorly to the mediastinum inferiorly. Derived from the third (the inferior parathyroid glands) and fourth (the superior parathyroid glands) pharyngeal pouches, these paired structures migrate to their final adult positions and are named accordingly.

1	The arteries supplying the parathyroid glands are the inferior thyroid arteries, and venous and lymphatic drainage follows that described for the thyroid gland. Location of structures in different regions of the anterior triangle of the neck The regional location of major structures in the anterior triangle of the neck is summarized in Table 8.14. Structures can be identified as being within a specific subdivision, passing into a specific subdivision from outside the area, originating in one subdivision and passing to another subdivision, or passing through several subdivisions while traversing the region. Posterior triangle of the neck

1	Posterior triangle of the neck The posterior triangle of the neck is on the lateral aspect of the neck in direct continuity with the upper limb (Fig. 8.183). It is bordered: anteriorly by the posterior edge of the sternocleidomastoid muscle, posteriorly by the anterior edge of the trapezius muscle, basally by the middle one-third of the clavicle, and apically by the occipital bone just posterior to the mastoid process where the attachments of the trapezius and sternocleidomastoid come together. The roof of the posterior triangle consists of an investing layer of cervical fascia that surrounds the sternocleidomastoid and trapezius muscles as it passes through the region. The muscular floor of the posterior triangle is covered by the prevertebral layer of cervical fascia; and from superior to inferior consists of the splenius capitis, levator scapulae, and the posterior, middle, and anterior scalene muscles.

1	Numerous muscles participate in forming the borders and floor of the posterior triangle of the neck (Table 8.15). In addition, the omohyoid muscle passes across the inferior part of the posterior triangle before disappearing under the sternocleidomastoid muscle and emerging in the anterior triangle (Fig. 8.184). It is enclosed in the investing layer of cervical fascia and crosses the posterior triangle from lateral to medial as it continues in a superior direction. It originates on the superior border of the scapula, just medial to the scapular notch and eventually inserts into the inferior border of the body of the hyoid bone. It has two bellies connected by a tendon, which is anchored by a fascial sling to the clavicle: The superior belly is in the anterior triangle. The inferior belly crosses the posterior triangle, subdividing it into a small, omoclavicular or subclavian triangle inferiorly and a much larger occipital triangle superiorly.

1	The inferior belly crosses the posterior triangle, subdividing it into a small, omoclavicular or subclavian triangle inferiorly and a much larger occipital triangle superiorly. The omohyoid is innervated by branches of the ansa cervicalis (anterior rami from C1 to C3) and it depresses the hyoid bone. One of the most superficial structures passing through the posterior triangle of the neck is the external jugular vein (Fig. 8.185). This large vein forms near the angle of the mandible, when the posterior branch of the retromandibular and posterior auricular veins join, and descends through the neck in the superficial fascia. After crossing the sternocleidomastoid muscle, the external jugular vein enters the posterior triangle and continues its vertical descent. In the lower part of the posterior triangle, the external jugular vein pierces the investing layer of cervical fascia and ends in the subclavian vein.

1	In the lower part of the posterior triangle, the external jugular vein pierces the investing layer of cervical fascia and ends in the subclavian vein. Tributaries to the external jugular vein while it traverses the posterior triangle of the neck include the transverse cervical, suprascapular, and anterior jugular veins. Several arteries are found within the boundaries of the posterior triangle of the neck. The largest is the third part of the subclavian artery as it crosses the base of the posterior triangle (Fig. 8.186). The first part of the subclavian artery ascends to the medial border of the anterior scalene muscle from either the brachiocephalic trunk on the right side or directly from the arch of the aorta on the left side. It has numerous branches. The second part of the subclavian artery passes laterally between the anterior and middle scalene muscles, and one branch may arise from it.

1	The second part of the subclavian artery passes laterally between the anterior and middle scalene muscles, and one branch may arise from it. The third part of the subclavian artery emerges from between the anterior and middle scalene muscles to cross the base of the posterior triangle (Fig. 8.186). It extends from the lateral border of the anterior scalene muscle to the lateral border of rib I where it becomes the axillary artery and continues into the upper limb. A single branch (the dorsal scapular artery) may arise from the third part of the subclavian artery. This branch passes posterolaterally to reach the superior angle of the scapula where it descends along the medial border of the scapula posterior to the rhomboid muscles.

1	Two other small arteries also cross the base of the posterior triangle. These are the transverse cervical and the suprascapular arteries (Fig. 8.186). They are both branches of the thyrocervical trunk, which arises from the first part of the subclavian artery. After branching from the thyrocervical trunk, the transverse cervical artery passes laterally and slightly posteriorly across the base of the posterior triangle anterior to the anterior scalene muscle and the brachial plexus. Reaching the deep surface of the trapezius muscle, it divides into superficial and deep branches: The superficial branch continues on the deep surface of the trapezius muscle. The deep branch continues on the deep surface of the rhomboid muscles near the medial border of the scapula.

1	The superficial branch continues on the deep surface of the trapezius muscle. The deep branch continues on the deep surface of the rhomboid muscles near the medial border of the scapula. The suprascapular artery, also a branch of the thyrocervical trunk, passes laterally, in a slightly downward direction across the lowest part of the posterior triangle, and ends up posterior to the clavicle (Fig. 8.186). Approaching the scapula, it passes over the superior transverse scapular ligament and distributes branches to muscles on the posterior surface of the scapula. Veins accompany all the arteries described previously.

1	Veins accompany all the arteries described previously. The subclavian vein is a continuation of the axillary vein and begins at the lateral border of rib I. As it crosses the base of the posterior triangle, the external jugular, and, possibly, the suprascapular and transverse cervical veins enter it (Fig. 8.185). It ends by joining with the internal jugular vein to form the brachiocephalic vein near the sternoclavicular joint. In the posterior triangle it is anterior to, and slightly lower than, the subclavian artery and passes anterior to the anterior scalene muscle. Transverse cervical and suprascapular veins travel with each of the similarly named arteries. These veins become tributaries to either the external jugular vein or the initial part of the subclavian vein.

1	A variety of nerves pass through or are within the posterior triangle. These include the accessory nerve [XI], branches of the cervical plexus, components forming the brachial plexus, and branches of the brachial plexus.

1	The accessory nerve [XI] exits the cranial cavity through the jugular foramen. It descends through the neck in a posterior direction, to reach the anterior border of the sternocleidomastoid muscle. Passing either deep to or through and innervating the sternocleidomastoid muscle, the accessory nerve [XI] continues to descend and enters the posterior triangle (Fig. 8.187). It crosses the posterior triangle, still in an obliquely downward direction, within the investing layer of cervical fascia as this fascia crosses between the sternocleidomastoid and trapezius muscles. When the accessory nerve [XI] reaches the anterior border of the trapezius muscle, it continues on the deep surface of the trapezius and innervates it. The superficial location of the accessory nerve as it crosses the posterior triangle makes it susceptible to injury. The cervical plexus is formed by the anterior rami of cervical nerves C1 to C4 (Fig. 8.188).

1	The cervical plexus is formed by the anterior rami of cervical nerves C1 to C4 (Fig. 8.188). The cervical plexus forms in the substance of the muscles making up the floor of the posterior triangle within the prevertebral layer of cervical fascia, and consists of: muscular (or deep) branches, and cutaneous (or superficial) branches. The cutaneous branches are visible in the posterior triangle emerging from beneath the posterior border of the sternocleidomastoid muscle (Fig. 8.187).

1	The cutaneous branches are visible in the posterior triangle emerging from beneath the posterior border of the sternocleidomastoid muscle (Fig. 8.187). Muscular (deep) branches of the cervical plexus distribute to several groups of muscles. A major branch is the phrenic nerve, which supplies the diaphragm with both sensory and motor innervation (Fig. 8.188). It arises from the anterior rami of cervical nerves C3 to C5. Hooking around the upper lateral border of the anterior scalene muscle, the nerve continues inferiorly across the anterior surface of the anterior scalene within the prevertebral fascia to enter the thorax (Fig. 8.189). As the nerve descends in the neck, it is “pinned” to the anterior scalene muscle by the transverse cervical and suprascapular arteries.

1	Several muscular branches of the cervical plexus supply prevertebral and lateral vertebral muscles, including the rectus capitis anterior, rectus capitis lateralis, longus colli, and longus capitis (Fig. 8.189 and Table 8.16). The cervical plexus also contributes to the formation of the superior and inferior roots of the ansa cervicalis (Fig. 8.188). This loop of nerves receives contributions from the anterior rami of the cervical nerves C1 to C3 and innervates the infrahyoid muscles. Cutaneous (superficial) branches of the cervical plexus are visible in the posterior triangle as they pass outward from the posterior border of the sternocleidomastoid muscle (Figs. 8.187 and 8.188): The lesser occipital nerve consists of contributions from cervical nerve C2 (Fig. 8.188), ascends along the posterior border of the sternocleidomastoid muscle, and distributes to the skin of the neck and scalp posterior to the ear.

1	The great auricular nerve consists of branches from cervical nerves C2 and C3, emerges from the posterior border of the sternocleidomastoid muscle, and ascends across the muscle to the base of the ear, supplying the skin of the parotid region, the ear, and the mastoid area. The transverse cervical nerve consists of branches from the cervical nerves C2 and C3, passes around the midpart of the sternocleidomastoid muscle, and continues horizontally across the muscle to supply the lateral and anterior parts of the neck. The supraclavicular nerves are a group of cutaneous nerves from cervical nerves C3 and C4 that, after emerging from beneath the posterior border of the sternocleidomastoid muscle, descend and supply the skin over the clavicle and shoulder as far inferiorly as rib II.

1	The brachial plexus forms from the anterior rami of cervical nerves C5 to C8 and thoracic nerve T1. The contributions of each of these nerves, which are between the anterior and middle scalene muscles, are the roots of the brachial plexus. As the roots emerge from between these muscles, they form the next component of the brachial plexus (the trunks) as follows: the anterior rami of C5 and C6 form the upper trunk, the anterior ramus of C7 forms the middle trunk, the anterior rami of C8 and T1 form the lower trunk. The trunks cross the base of the posterior triangle (see Fig. 8.186). Several branches of the brachial plexus may be visible in the posterior triangle (see Fig. 7.54 on pg. 730). These include the: dorsal scapular nerve to the rhomboid muscles, long thoracic nerve to the serratus anterior muscle, nerve to the subclavius muscle, and suprascapular nerve to the supraspinatus and infraspinatus muscles. Root of the neck

1	Root of the neck The root of the neck (Fig. 8.190) is the area immediately superior to the superior thoracic aperture and axillary inlets. It is bounded by: the top of the manubrium of the sternum and superior margin of the clavicle anteriorly, and the top of the thoracic vertebra TI and the superior margin of the scapula to the coracoid process posteriorly. It contains structures passing between the neck, thorax, and upper limb. There is also an extension of the thoracic cavity projecting into the root of the neck (Fig. 8.190). This consists of an upward projection of the pleural cavity, on both sides, and includes the cervical part of the parietal pleura (cupula), and the apical part of the superior lobe of each lung. Anteriorly, the pleural cavity extends above the top of the manubrium of the sternum and superior border of rib I, while posteriorly, due to the downward slope of the superior thoracic aperture, the pleural cavity remains below the top of vertebra TI.

1	The subclavian arteries on both sides arch upward out of the thorax to enter the root of the neck (Fig. 8.191). The right subclavian artery begins posterior to the sternoclavicular joint as one of two terminal branches of the brachiocephalic trunk. It arches superiorly and laterally to pass anterior to the extension of the pleural cavity in the root of the neck and posterior to the anterior scalene muscle. Continuing laterally across rib I, it becomes the axillary artery as it crosses its lateral border. The left subclavian artery begins lower in the thorax than the right subclavian artery as a direct branch of the arch of the aorta. Lying posterior to the left common carotid artery and lateral to the trachea, it ascends and arches laterally, passing anterior to the extension of the pleural cavity and posterior to the anterior scalene muscle. It continues laterally over rib I, and becomes the axillary artery as it crosses the lateral border of rib I.

1	Both subclavian arteries are divided into three parts by the anterior scalene muscle (Fig. 8.191): The first part extends from the origin of the artery to the anterior scalene muscle. The second part is the part of the artery posterior to the anterior scalene muscle. The third part is the part lateral to the anterior scalene muscle before the artery reaches the lateral border of rib I. All branches from the right and left subclavian arteries arise from the first part of the artery, except in the case of one branch (the costocervical trunk) on the right side (Fig. 8.191). The branches include the vertebral artery, the thyrocervical trunk, the internal thoracic artery, and the costocervical trunk.

1	The vertebral artery is the first branch of the subclavian artery as it enters the root of the neck (Fig. 8.191). A large branch, arising from the first part of the subclavian artery medial to the anterior scalene muscle, it ascends and enters the foramen in the transverse process of vertebra CVI. Continuing to pass superiorly, the vertebral artery passes through the foramina of vertebrae CV to CI. At the superior border of vertebra CI, the artery turns medially and crosses the posterior arch of vertebra CI. From here it passes through the foramen magnum to enter the posterior cranial fossa. The second branch of the subclavian artery is the thyrocervical trunk (Fig. 8.191). It arises from the first part of the subclavian artery medial to the anterior scalene muscle, and divides into three branches—the inferior thyroid, the transverse cervical, and the suprascapular arteries.

1	Inferior thyroid artery. The inferior thyroid artery (Fig. 8.191) is the superior continuation of the thyrocervical trunk. It ascends, anterior to the anterior scalene muscle, and eventually turns medially, crossing posterior to the carotid sheath and its contents and anterior to the vertebral artery. Reaching the posterior surface of the thyroid gland it supplies the thyroid gland. When the inferior thyroid artery turns medially, it gives off an important branch (the ascending cervical artery), which continues to ascend on the anterior surface of the prevertebral muscles, supplying these muscles and sending branches to the spinal cord.

1	Transverse cervical artery. The middle branch of the thyrocervical trunk is the transverse cervical artery (Fig. 8.191). This branch passes laterally, across the anterior surface of the anterior scalene muscle and the phrenic nerve, and enters and crosses the base of the posterior triangle of the neck. It continues to the deep surface of the trapezius muscle, where it divides into superficial and deep branches: The superficial branch continues on the deep surface of the trapezius muscle. The deep branch continues on the deep surface of the rhomboid muscles near the medial border of the scapula.

1	The superficial branch continues on the deep surface of the trapezius muscle. The deep branch continues on the deep surface of the rhomboid muscles near the medial border of the scapula. Suprascapular artery. The lowest branch of the thyrocervical trunk is the suprascapular artery (Fig. 8.191). This branch passes laterally, crossing anterior to the anterior scalene muscle, the phrenic nerve, the third part of the subclavian artery, and the trunks of the brachial plexus. At the superior border of the scapula, it crosses over the superior transverse scapular ligament and enters the supraspinatus fossa. The third branch of the subclavian artery is the internal thoracic artery (Fig. 8.191). This artery branches from the inferior edge of the subclavian artery and descends.

1	The third branch of the subclavian artery is the internal thoracic artery (Fig. 8.191). This artery branches from the inferior edge of the subclavian artery and descends. It passes posterior to the clavicle and the large veins in the region and anterior to the pleural cavity. It enters the thoracic cavity posterior to the ribs and anterior to the transversus thoracis muscle and continues to descend giving off numerous branches. The final branch of the subclavian artery in the root of the neck is the costocervical trunk (Fig. 8.191). It arises in a slightly different position, depending on the side: On the left, it arises from the first part of the subclavian artery, just medial to the anterior scalene muscle. On the right, it arises from the second part of the subclavian artery.

1	On the left, it arises from the first part of the subclavian artery, just medial to the anterior scalene muscle. On the right, it arises from the second part of the subclavian artery. On both sides, the costocervical trunk ascends and passes posteriorly over the dome of the pleural cavity and continues in a posterior direction behind the anterior scalene muscle. Eventually it divides into two branches—the deep cervical and the supreme intercostal arteries: The deep cervical artery ascends in the back of the neck and anastomoses with the descending branch of the occipital artery. The supreme intercostal artery descends anterior to rib I and divides to form the posterior intercostal arteries for the first two intercostal spaces. Numerous veins pass through the root of the neck. Small veins accompany each of the arteries described above, and large veins form major drainage channels.

1	Numerous veins pass through the root of the neck. Small veins accompany each of the arteries described above, and large veins form major drainage channels. The subclavian veins begin at the lateral margin of rib I as continuations of the axillary veins. Passing medially on each side, just anterior to the anterior scalene muscles, each subclavian vein is joined by the internal jugular vein to form the brachiocephalic veins. The only tributary to each subclavian vein is an external jugular vein. The veins accompanying the numerous arteries in this region empty into other veins. Several nerves and components of the nervous system pass through the root of the neck.

1	The veins accompanying the numerous arteries in this region empty into other veins. Several nerves and components of the nervous system pass through the root of the neck. The phrenic nerves are branches of the cervical plexus and arise on each side as contributions from the anterior rami of cervical nerves C3 to C5 come together. Passing around the upper lateral border of each anterior scalene muscle, the phrenic nerves continue inferiorly across the anterior surface of each anterior scalene muscle within the prevertebral layer of cervical fascia (Fig. 8.192). Leaving the lower edge of the anterior scalene muscle each phrenic nerve passes between the subclavian vein and artery to enter the thorax and continue to the diaphragm. The vagus nerves [X] descend through the neck within the carotid sheath, posterior to and just between the common carotid artery and the internal jugular vein.

1	The vagus nerves [X] descend through the neck within the carotid sheath, posterior to and just between the common carotid artery and the internal jugular vein. In the lower part of the neck, the vagus nerves [X] give off cardiac branches, which continue downward and medially, passing posterior to the subclavian arteries to disappear into the thorax. In the root of the neck, each vagus nerve [X] passes anterior to the subclavian artery and posterior to the subclavian vein as it enters the thorax (Fig. 8.192). The right and left recurrent laryngeal nerves are visible as they originate in (the right recurrent laryngeal nerve), or pass through (the left recurrent laryngeal nerve), the root of the neck.

1	The right and left recurrent laryngeal nerves are visible as they originate in (the right recurrent laryngeal nerve), or pass through (the left recurrent laryngeal nerve), the root of the neck. The right recurrent laryngeal nerve is a branch of the right vagus nerve [X] as it reaches the lower edge of the first part of the subclavian artery in the root of the neck. It passes around the subclavian artery and upward and medially in a groove between the trachea and the esophagus as it heads to the larynx. The left recurrent laryngeal nerve is a branch of the left vagus nerve [X] as it crosses the arch of the aorta in the superior mediastinum. It passes below and behind the arch of the aorta and ascends beside the trachea to the larynx (Fig. 8.192).

1	Various components of the sympathetic nervous system are visible as they pass through the root of the neck (Fig. 8.193). These include: the cervical part of the sympathetic trunk, the ganglia associated with the cervical part of the sympathetic trunk, and cardiac nerves branching from the cervical part of the sympathetic trunk. The sympathetic trunks are two parallel cords that run from the base of the skull to the coccyx. Along the way they are punctuated by ganglia, which are collections of neuronal cell bodies outside the CNS. Cervical part of the sympathetic trunk The cervical part of the sympathetic trunk is anterior to the longus colli and longus capitis muscles, and posterior to the common carotid artery in the carotid sheath and the internal carotid artery. It is connected to each cervical spinal nerve by a gray ramus communicans (Fig. 8.194). There are no white rami communicantes in the cervical region.

1	Three ganglia are usually described along the course of the sympathetic trunk in the cervical region, and in these ganglia ascending preganglionic sympathetic fibers from upper thoracic spinal cord levels synapse with postganglionic sympathetic fibers. The postganglionic sympathetic fibers are distributed in branches from these ganglia. Superior cervical ganglion. A very large superior cervical ganglion in the area of cervical vertebrae CI and CII marks the superior extent of the sympathetic trunk (Figs. 8.193 and 8.194). Its branches pass to: the internal carotid and external carotid arteries, forming plexuses around these vessels, cervical spinal nerves C1 to C4 through gray rami communicantes, the pharynx, and the heart as superior cardiac nerves.

1	Middle cervical ganglion. A second ganglion inferior to the superior cervical ganglion along the course of the sympathetic trunk (the middle cervical ganglion) is encountered at about the level of cervical vertebra CVI (Figs. 8.193 and 8.194). Branches from this ganglion pass to: cervical spinal nerves C5 and C6 through gray rami communicantes, and the heart as middle cardiac nerves. Inferior cervical ganglion. At the lower end of the cervical part of the sympathetic trunk is another ganglion (the inferior cervical ganglion), which becomes very large when it combines with the first thoracic ganglion and forms the cervicothoracic ganglion (stellate ganglion). The inferior cervical ganglion (Figs. 8.193 and 8.194) is anterior to the neck of rib I and the transverse process of cervical vertebra CVII, and posterior to the first part of the subclavian artery and the origin of the vertebral artery.

1	Branches from this ganglion pass to: spinal nerves C7 to T1 through gray rami communicantes, the vertebral artery, forming a plexus associated with this vessel, and the heart as inferior cardiac nerves. This ganglion may also receive white rami communicantes from thoracic spinal nerve T1, and occasionally, from T2. The thoracic duct is a major lymphatic channel that begins in the abdomen, passes superiorly through the thorax, and ends in the venous channels in the neck. It passes through the lower thoracic cavity in the midline with: the thoracic aorta on the left, the azygos vein on the right, and the esophagus anteriorly.

1	It passes through the lower thoracic cavity in the midline with: the thoracic aorta on the left, the azygos vein on the right, and the esophagus anteriorly. At about the level of thoracic vertebra TV the thoracic duct passes to the left and continues to ascend just to the left of the esophagus. It passes through the superior mediastinum and enters the root of the neck to the left of the esophagus (Fig. 8.195). Arching laterally, it passes posterior to the carotid sheath and turns inferiorly in front of the thyrocervical trunk, the phrenic nerve, and the vertebral artery.

1	The thoracic duct terminates in the junction between the left internal jugular and the left subclavian veins (Fig. 8.195). Near its junction with the venous system it is joined by: the left jugular trunk, which drains lymph from the left side of the head and neck, the left subclavian trunk, which drains lymph from the left upper limb, and occasionally, the left bronchomediastinal trunk, which drains lymph from the left half of the thoracic structures (Fig. 8.196). A similar confluence of three lymphatic trunks occurs on the right side of the body. Emptying into the junction between the right internal jugular and right subclavian veins are: the right jugular trunk from the head and neck, the right subclavian trunk from the right upper limb, and occasionally, the right bronchomediastinal trunk carrying lymph from the structures in the right half of the thoracic cavity and the right upper intercostal spaces (Fig. 8.196).

1	There is variability in how these trunks enter the veins. They may combine into a single right lymphatic duct to enter the venous system or enter as three separate trunks. Lymphatics of the neck A description of the organization of the lymphatic system in the neck becomes a summary of the lymphatic system in the head and neck. It is impossible to separate the two regions. The components of this system include superficial nodes around the head, superficial cervical nodes along the external jugular vein, and deep cervical nodes forming a chain along the internal jugular vein (Fig. 8.197). The basic pattern of drainage is for superficial lymphatic vessels to drain to the superficial nodes. Some of these drain to the superficial cervical nodes on their way to the deep cervical nodes and others drain directly to the deep cervical nodes.

1	Five groups of superficial lymph nodes form a ring around the head and are primarily responsible for the lymphatic drainage of the face and scalp. Their pattern of drainage is very similar to the area of distribution of the arteries near their location.

1	Beginning posteriorly these groups (Fig. 8.197) are: occipital nodes near the attachment of the trapezius muscle to the skull and associated with the occipital artery—lymphatic drainage is from the posterior scalp and neck; mastoid nodes (retro-auricular/posterior auricular nodes) posterior to the ear near the attachment of the sternocleidomastoid muscle and associated with the posterior auricular artery—lymphatic drainage is from the posterolateral half of the scalp; pre-auricular and parotid nodes anterior to the ear and associated with the superficial temporal and transverse facial arteries—lymphatic drainage is from the anterior surface of the auricle, the anterolateral scalp, the upper half of the face, the eyelids, and the cheeks; submandibular nodes inferior to the body of the mandible and associated with the facial artery—lymphatic drainage is from structures along the path of the facial artery as high as the forehead, as well as the gingivae, the teeth, and the tongue;

1	mandible and associated with the facial artery—lymphatic drainage is from structures along the path of the facial artery as high as the forehead, as well as the gingivae, the teeth, and the tongue; submental nodes inferior and posterior to the chin—lymphatic drainage is from the center part of the lower lip, the chin, the floor of the mouth, the tip of the tongue, and the lower incisor teeth.

1	Lymphatic flow from these superficial lymph nodes passes in several directions: Drainage from the occipital and mastoid nodes passes to the superficial cervical nodes along the external jugular vein. Drainage from the pre-auricular and parotid nodes, the submandibular nodes, and the submental nodes passes to the deep cervical nodes. The superficial cervical nodes are a collection of lymph nodes along the external jugular vein on the superficial surface of the sternocleidomastoid muscle (Fig. 8.197). They primarily receive lymphatic drainage from the posterior and posterolateral regions of the scalp through the occipital and mastoid nodes, and send lymphatic vessels in the direction of the deep cervical nodes.

1	The deep cervical nodes are a collection of lymph nodes that form a chain along the internal jugular vein (Fig. 8.197). They are divided into upper and lower groups where the intermediate tendon of the omohyoid muscle crosses the common carotid artery and the internal jugular vein. The most superior node in the upper deep cervical group is the jugulodigastric node (Fig. 8.197). This large node is where the posterior belly of the digastric muscle crosses the internal jugular vein and receives lymphatic drainage from the tonsils and tonsillar region. Another large node, usually associated with the lower deep cervical group because it is at or just inferior to the intermediate tendon of the omohyoid muscle, is the jugulo-omohyoid node (Fig. 8.197). This node receives lymphatic drainage from the tongue. The deep cervical nodes eventually receive all lymphatic drainage from the head and neck either directly or through regional groups of nodes.

1	The deep cervical nodes eventually receive all lymphatic drainage from the head and neck either directly or through regional groups of nodes. From the deep cervical nodes, lymphatic vessels form the right and left jugular trunks, which empty into the right lymphatic duct on the right side or the thoracic duct on the left side. The pharynx is a musculofascial half-cylinder that links the oral and nasal cavities in the head to the larynx and esophagus in the neck (Fig. 8.198). The pharyngeal cavity is a common pathway for air and food. The pharynx is attached above to the base of the skull and is continuous below, approximately at the level of vertebra CVI, with the top of the esophagus. The walls of the pharynx are attached anteriorly to the margins of the nasal cavities, oral cavity, and larynx. Based on these anterior relationships the pharynx is subdivided into three regions, the nasopharynx, oropharynx, and laryngopharynx:

1	The posterior apertures (choanae) of the nasal cavities open into the nasopharynx. The posterior opening of the oral cavity (oropharyngeal isthmus) opens into the oropharynx. The superior aperture of the larynx (laryngeal inlet) opens into the laryngopharynx. In addition to these openings, the pharyngeal cavity is related anteriorly to the posterior one-third of the tongue and to the posterior aspect of the larynx. The pharyngotympanic tubes open into the lateral walls of the nasopharynx. Lingual, pharyngeal, and palatine tonsils are on the deep surface of the pharyngeal walls. The pharynx is separated from the posteriorly positioned vertebral column by a thin retropharyngeal space containing loose connective tissue.

1	The pharynx is separated from the posteriorly positioned vertebral column by a thin retropharyngeal space containing loose connective tissue. Although the soft palate is generally considered as part of the roof of the oral cavity, it is also related to the pharynx. The soft palate is attached to the posterior margin of the hard palate and is a type of “flutter valve” that can: swing up (elevate) to close the pharyngeal isthmus, and seal off the nasopharynx from the oropharynx, and swing down (depress) to close the oropharyngeal isthmus and seal off the oral cavity from the oropharynx.

1	The superior and anterior margins of the pharyngeal wall are attached to bone and cartilage, and to ligaments. The two sides of the pharyngeal wall are welded together posteriorly in the midline by a vertically oriented cord-like ligament (the pharyngeal raphe). This connective tissue structure descends from the pharyngeal tubercle on the base of the skull to the level of cervical vertebra CVI where the raphe blends with connective tissue in the posterior wall of the esophagus.

1	There is an irregular C-shaped line of pharyngeal wall attachment on the base of the skull (Fig. 8.200). The open part of the C faces the nasal cavities. Each arm of the C begins at the posterior margin of the medial plate of the pterygoid process of the sphenoid bone, just inferior to the cartilaginous part of the pharyngotympanic tube. The line crosses inferior to the pharyngotympanic tube and then passes onto the petrous part of the temporal bone where it is just medial to the roughening for the attachment of one of the muscles (levator veli palatini) of the soft palate. From here, the line swings medially onto the occipital bone and joins the line from the other side at a prominent elevation of bone in the midline (the pharyngeal tubercle). Anterior vertical line of attachment for the lateral pharyngeal walls

1	Anterior vertical line of attachment for the lateral pharyngeal walls The vertical line of attachment for the lateral pharyngeal walls to structures related to the nasal and oral cavities and larynx is discontinuous and in three parts (Fig. 8.201). On each side, the anterior line of attachment of the lateral pharyngeal wall begins superiorly on the posterior edge of the medial pterygoid plate of the sphenoid bone just inferior to where the pharyngotympanic tube lies against this plate. It continues inferiorly along the edge of the medial plate of the pterygoid process and onto the pterygoid hamulus. From this point, the line descends along the pterygomandibular raphe to the mandible where this part of the line terminates.

1	The pterygomandibular raphe is a linear cord-like connective tissue ligament that spans the distance between the tip of the pterygoid hamulus and a triangular roughening immediately posterior to the third molar on the mandible. It joins a muscle of the lateral pharyngeal wall (superior constrictor) with a muscle of the lateral wall of the oral cavity (buccinator). The second part of the line of attachment of the lateral pharyngeal wall is related to the hyoid bone. It begins on the lower aspect of the stylohyoid ligament, which connects the tip of the styloid process of the temporal bone to the lesser horn of the hyoid bone. The line continues onto the lesser horn and then turns and runs posteriorly along the entire upper surface of the greater horn of the hyoid where it terminates.

1	The most inferior and third part of the line of attachment of the lateral pharyngeal wall begins superiorly on the superior tubercle of the thyroid cartilage, and descends along the oblique line to the inferior tubercle. From the inferior tubercle, the line of attachment continues over the cricothyroid muscle along a tendinous thickening of fascia to the cricoid cartilage where it terminates. The pharyngeal wall is formed by skeletal muscles and by fascia. Gaps between the muscles are reinforced by the fascia and provide routes for structures to pass through the wall. The muscles of the pharynx are organized into two groups based on the orientation of muscle fibers. The constrictor muscles have fibers oriented in a circular direction relative to the pharyngeal wall, whereas the longitudinal muscles have fibers oriented vertically.

1	The constrictor muscles have fibers oriented in a circular direction relative to the pharyngeal wall, whereas the longitudinal muscles have fibers oriented vertically. The three constrictor muscles on each side are major contributors to the structure of the pharyngeal wall (Fig. 8.202 and Table 8.17) and their names indicate their position—superior, middle, and inferior constrictor muscles. Posteriorly, the muscles from each side are joined together by the pharyngeal raphe. Anteriorly, these muscles attach to bones, cartilages, and ligaments related to the lateral margins of the nasal and oral cavities and the larynx. The constrictor muscles overlap each other in a fashion resembling the walls of three flower pots stacked one on the other. The inferior constrictors overlap the lower margins of the middle constrictors and, in the same way, the middle constrictors overlap the superior constrictors. Collectively, the muscles constrict or narrow the pharyngeal cavity.

1	Collectively, the muscles constrict or narrow the pharyngeal cavity. When the constrictor muscles contract sequentially from top to bottom, as in swallowing, they move a bolus of food through the pharynx and into the esophagus. All of the constrictors are innervated by the pharyngeal branch of the vagus nerve [X]. The superior constrictor muscles together bracket the upper part of the pharyngeal cavity (Fig. 8.202). Each muscle is attached anteriorly to the pterygoid hamulus, pterygomandibular raphe, and adjacent bone of the mandible. From these attachments, the muscle fans out posteriorly and joins with its partner muscle from the other side at the pharyngeal raphe. A special band of muscle (the palatopharyngeal sphincter) originates from the anterolateral surface of the soft palate and circles the inner aspect of the pharyngeal wall, blending with the inner aspect of the superior constrictor.

1	When the superior constrictor constricts during swallowing, it forms a prominent ridge on the deep aspect of the pharyngeal wall that catches the margin of the elevated soft palate, which then seals closed the pharyngeal isthmus between the nasopharynx and oropharynx. The middle constrictor muscles are attached to the lower aspect of the stylohyoid ligament, the lesser horn of the hyoid bone, and the entire upper surface of the greater horn of the hyoid (Fig. 8.202). Like the superior constrictors, the middle constrictor muscles fan out posteriorly and attach to the pharyngeal raphe. The posterior part of the middle constrictors overlaps the superior constrictors. The inferior constrictor muscles attach anteriorly to the oblique line of the thyroid cartilage, the cricoid cartilage, and a ligament that spans between these two attachments to cartilage and crosses the cricothyroid muscle (Fig. 8.202).

1	Like the other constrictor muscles, the inferior constrictor muscles spread out posteriorly and attach to the pharyngeal raphe. The posterior part of the inferior constrictors overlaps the middle constrictors. Inferiorly, the muscle fibers blend with and attach into the wall of the esophagus. The parts of the inferior constrictors attached to the cricoid cartilage bracket the narrowest part of the pharyngeal cavity. The three longitudinal muscles of the pharyngeal wall (Fig. 8.203 and Table 8.18) are named according to their origins—stylopharyngeus from the styloid process of the temporal bone, salpingopharyngeus from the cartilaginous part of the pharyngotympanic tube (salpinx is Greek for “tube”), and palatopharyngeus from the soft palate. From their sites of origin, these muscles descend and attach into the pharyngeal wall.

1	The longitudinal muscles elevate the pharyngeal wall, or during swallowing, pull the pharyngeal wall up and over a bolus of food being moved through the pharynx and into the esophagus. The cylindrical stylopharyngeus muscle (Fig. 8.203A) originates from the base of the medial surface of the styloid process of the temporal bone and descends between the superior and middle constrictor muscles to fan out on, and blend with, the deep surface of the pharyngeal wall. It is innervated by the glossopharyngeal nerve [IX]. The salpingopharyngeus (Fig. 8.203B) is a small muscle originating from the inferior aspect of the pharyngotympanic tube, descending on, and blending into, the deep surface of the pharyngeal wall. It is innervated by the vagus nerve [X].

1	The palatopharyngeus (Fig. 8.203B), in addition to being a muscle of the pharynx, is also a muscle of the soft palate (see pp. 1098–1099). It is attached to the upper surface of the palatine aponeurosis, and passes posteriorly and inferiorly to blend with the deep surface of the pharyngeal wall. The palatopharyngeus forms an important fold in the overlying mucosa (the palatopharyngeal arch). This arch is visible through the oral cavity and is a landmark for finding the palatine tonsil, which is immediately anterior to it on the oropharyngeal wall. In addition to elevating the pharynx, the palatopharyngeus participates in closing the oropharyngeal isthmus by depressing the palate and moving the palatopharyngeal fold toward the midline. The palatopharyngeus is innervated by the vagus nerve [X]. The pharyngeal fascia is separated into two layers, which sandwich the pharyngeal muscles between them:

1	The palatopharyngeus is innervated by the vagus nerve [X]. The pharyngeal fascia is separated into two layers, which sandwich the pharyngeal muscles between them: A thin layer (buccopharyngeal fascia) coats the outside of the muscular part of the wall and is a component of the pretracheal layer of cervical fascia (see p. 991). A much thicker layer (pharyngobasilar fascia) lines the inner surface. The fascia reinforces the pharyngeal wall where muscle is deficient. This is particularly evident above the level of the superior constrictor where the pharyngeal wall is formed almost entirely of fascia (Fig. 8.203). This part of the wall is reinforced externally by muscles of the soft palate (tensor and levator veli palatini). Gaps in the pharyngeal wall and structures passing through them Gaps between muscles of the pharyngeal wall provide important routes for muscles and neurovascular tissues (Fig. 8.204).

1	Gaps in the pharyngeal wall and structures passing through them Gaps between muscles of the pharyngeal wall provide important routes for muscles and neurovascular tissues (Fig. 8.204). Above the margin of the superior constrictor, the pharyngeal wall is deficient in muscle and completed by pharyngeal fascia. The tensor and levator veli palatini muscles of the soft palate initially descend from the base of the skull and are lateral to the pharyngeal fascia. In this position, they reinforce the pharyngeal wall: The levator veli palatini passes through the pharyngeal fascia inferior to the pharyngotympanic tube and enters the soft palate. The tendon of the tensor veli palatini turns medially around the pterygoid hamulus and passes through the origin of the buccinator muscle to enter the soft palate.

1	The tendon of the tensor veli palatini turns medially around the pterygoid hamulus and passes through the origin of the buccinator muscle to enter the soft palate. One of the largest and most important apertures in the pharyngeal wall is between the superior and middle constrictor muscles of the pharynx and the posterior border of the mylohyoid muscle, which forms the floor of the mouth (Fig. 8.204). This triangular-shaped gap (oropharyngeal triangle) not only enables the stylopharyngeus to slip into the pharyngeal wall, but also allows muscles, nerves, and vessels to pass between regions lateral to the pharyngeal wall and the oral cavity, particularly to the tongue. The gap between the middle and inferior constrictor muscles allows the internal laryngeal vessels and nerve access to the aperture in the thyrohyoid membrane to enter the larynx.

1	The gap between the middle and inferior constrictor muscles allows the internal laryngeal vessels and nerve access to the aperture in the thyrohyoid membrane to enter the larynx. The recurrent laryngeal nerves and accompanying inferior laryngeal vessels enter the larynx posterior to the inferior horn of the thyroid cartilage deep to the inferior margin of the inferior constrictor muscle. The nasopharynx is behind the posterior apertures (choanae) of the nasal cavities and above the level of the soft palate (Fig. 8.205). Its ceiling is formed by the sloping base of the skull and consists of the posterior part of the body of the sphenoid bone and the basal part of the occipital bone. The ceiling and lateral walls of the nasopharynx form a domed vault at the top of the pharyngeal cavity that is always open.

1	The cavity of the nasopharynx is continuous below with the cavity of the oropharynx at the pharyngeal isthmus. The position of the pharyngeal isthmus is marked on the pharyngeal wall by a mucosal fold caused by the underlying palatopharyngeal sphincter, which is part of the superior constrictor muscle. Elevation of the soft palate and constriction of the palatopharyngeal sphincter closes the pharyngeal isthmus during swallowing and separates the nasopharynx from the oropharynx. There is a large collection of lymphoid tissue (the pharyngeal tonsil) in the mucosa covering the roof of the nasopharynx. Enlargement of this tonsil, known then as adenoids, can occlude the nasopharynx so that breathing is only possible through the oral cavity (Fig. 8.205A). The most prominent features on each lateral wall of the nasopharynx are: the pharyngeal opening of the pharyngotympanic tube, and mucosal elevations and folds covering the end of the pharyngotympanic tube and the adjacent muscles.

1	The opening of the pharyngotympanic tube is posterior to and slightly above the level of the hard palate, and lateral to the top of the soft palate (Fig. 8.205A). Because the pharyngotympanic tube projects into the nasopharynx from a posterolateral direction, its posterior rim forms an elevation or bulge on the pharyngeal wall. Posterior to this tubal elevation (torus tubarius) is a deep recess (pharyngeal recess) (Fig. 8.205A). Mucosal folds related to the pharyngotympanic tube include: the small vertical salpingopharyngeal fold, which descends from the tubal elevation and overlies the salpingopharyngeus muscle, and a broad fold or elevation (torus levatorius) that appears to emerge from just under the opening of the pharyngotympanic tube, continues medially onto the upper surface of the soft palate, and overlies the levator veli palatini muscle.

1	The oropharynx is posterior to the oral cavity, inferior to the level of the soft palate, and superior to the upper margin of the epiglottis (Fig. 8.205). The palatoglossal folds (arches), one on each side, that cover the palatoglossal muscles, mark the boundary between the oral cavity and the oropharynx. The arched opening between the two folds is the oropharyngeal isthmus. Just posterior and medial to these folds are another pair of folds (arches), the palatopharyngeal folds, one on each side, that overlie the palatopharyngeus muscles.

1	The anterior wall of the oropharynx inferior to the oropharyngeal isthmus is formed by the upper part of the posterior one-third or pharyngeal part of the tongue. Large collections of lymphoid tissue (the lingual tonsils) are in the mucosa covering this part of the tongue. A pair of mucosal pouches (valleculae), one on each side of the midline, between the base of the tongue and epiglottis, are depressions formed between a midline mucosal fold and two lateral folds that connect the tongue to the epiglottis. The palatine tonsils are on the lateral walls of the oropharynx. On each side, there is a large ovoid collection of lymphoid tissue in the mucosa lining the superior constrictor muscle and between the palatoglossal and palatopharyngeal arches. The palatine tonsils are visible through the oral cavity just posterior to the palatoglossal folds.

1	When holding liquid or solids in the oral cavity, the oropharyngeal isthmus is closed by depression of the soft palate, elevation of the back of the tongue, and movement toward the midline of the palatoglossal and palatopharyngeal folds. This allows a person to breathe while chewing or manipulating material in the oral cavity. On swallowing, the oropharyngeal isthmus is opened, the palate is elevated, the laryngeal cavity is closed, and the food or liquid is directed into the esophagus. A person cannot breathe and swallow at the same time because the airway is closed at two sites, the pharyngeal isthmus and the larynx. The laryngopharynx extends from the superior margin of the epiglottis to the top of the esophagus at the level of vertebra CVI (Fig. 8.205). The laryngeal inlet opens into the anterior wall of the laryngopharynx. Inferior to the laryngeal inlet, the anterior wall consists of the posterior aspect of the larynx.

1	The laryngeal inlet opens into the anterior wall of the laryngopharynx. Inferior to the laryngeal inlet, the anterior wall consists of the posterior aspect of the larynx. There is another pair of mucosal recesses (piriform fossae) between the central part of the larynx and the more lateral lamina of the thyroid cartilage. The piriform fossae form channels that direct solids and liquids from the oral cavity around the raised laryngeal inlet and into the esophagus. Collections of lymphoid tissue in the mucosa of the pharynx surrounding the openings of the nasal and oral cavities (Waldeyer’s tonsillar ring) are part of the body’s defense system. The largest of these collections form distinct masses (tonsils). Tonsils occur mainly in three areas (Fig. 8.205): The pharyngeal tonsil, known as adenoids when enlarged, is in the midline on the roof of the nasopharynx.

1	The pharyngeal tonsil, known as adenoids when enlarged, is in the midline on the roof of the nasopharynx. The palatine tonsils are on each side of the oropharynx between the palatoglossal and palatopharyngeal arches just posterior to the oropharyngeal isthmus. (The palatine tonsils are visible through the open mouth of a patient when the tongue is depressed.) The lingual tonsils refer collectively to numerous lymphoid nodules on the posterior one-third of the tongue. Small lymphoid nodules also occur in the pharyngotympanic tube near its opening into the nasopharynx, and on the upper surface of the soft palate. Numerous vessels supply the pharyngeal wall (Fig. 8.206). Arteries that supply upper parts of the pharynx include: the ascending pharyngeal artery, the ascending palatine and tonsillar branches of the facial artery, and numerous branches of the maxillary and the lingual arteries. All these vessels are from the external carotid artery.

1	All these vessels are from the external carotid artery. Arteries that supply the lower parts of the pharynx include pharyngeal branches from the inferior thyroid artery, which originates from the thyrocervical trunk of the subclavian artery. The major blood supply to the palatine tonsil is from the tonsillar branch of the facial artery, which penetrates the superior constrictor muscle. Veins of the pharynx form a plexus, which drains superiorly into the pterygoid plexus in the infratemporal fossa, and inferiorly into the facial and internal jugular veins (Fig. 8.207). Lymphatic vessels from the pharynx drain into the deep cervical nodes and include retropharyngeal (between the nasopharynx and vertebral column), paratracheal, and infrahyoid nodes (Fig. 8.207). The palatine tonsils drain through the pharyngeal wall into the jugulodigastric nodes in the region where the facial vein drains into the internal jugular vein (and inferior to the posterior belly of the digastric muscle).

1	Motor and most sensory innervation (except for the nasal region) of the pharynx is mainly through branches of the vagus [X] and glossopharyngeal [IX] nerves, which form a plexus in the outer fascia of the pharyngeal wall (Fig. 8.208A). The pharyngeal plexus is formed by: the pharyngeal branch of the vagus nerve [X], branches from the external laryngeal nerve from the superior laryngeal branch of the vagus nerve [X], and pharyngeal branches of the glossopharyngeal nerve [IX]. The pharyngeal branch of the vagus nerve [X] originates from the upper part of its inferior ganglion above the origin of the superior laryngeal nerve and is the major motor nerve of the pharynx. All muscles of the pharynx are innervated by the vagus nerve [X] mainly through the pharyngeal plexus, except for the stylopharyngeus, which is innervated directly by a branch of the glossopharyngeal nerve [IX] (Fig. 8.208B). Each subdivision of the pharynx has a different sensory innervation:

1	Each subdivision of the pharynx has a different sensory innervation: The nasopharynx is innervated by a pharyngeal branch of the maxillary nerve [V2] that originates in the pterygopalatine fossa and passes through the palatovaginal canal in the sphenoid bone to reach the roof of the pharynx. The oropharynx is innervated by the glossopharyngeal nerve [IX] via the pharyngeal plexus. The laryngopharynx is innervated by the vagus nerve [X] via the internal branch of the superior laryngeal nerve. The glossopharyngeal nerve [IX] is related to the pharynx throughout most of its course outside the cranial cavity.

1	The glossopharyngeal nerve [IX] is related to the pharynx throughout most of its course outside the cranial cavity. After exiting the skull through the jugular foramen, the glossopharyngeal nerve [IX] descends on the posterior surface of the stylopharyngeus muscle (Fig. 8.208B), passes onto the lateral surface of the stylopharyngeus, and then passes anteriorly through the gap (oropharyngeal triangle) between the superior constrictor, middle constrictor, and mylohyoid muscles to eventually reach the posterior aspect of the tongue. As the glossopharyngeal nerve [IX] passes under the free edge of the superior constrictor, it is just inferior to the palatine tonsil lying on the deep surface of the superior constrictor.

1	As the glossopharyngeal nerve [IX] passes under the free edge of the superior constrictor, it is just inferior to the palatine tonsil lying on the deep surface of the superior constrictor. Pharyngeal branches to the pharyngeal plexus and a motor branch to the stylopharyngeus muscle are among branches that originate from the glossopharyngeal nerve [IX] in the neck. Because sensory innervation of the oropharynx is by the glossopharyngeal nerve [IX], this nerve carries sensory innervation from the palatine tonsil and is also the afferent limb of the gag reflex (see “In the clinic” on p. 889). The larynx is a hollow musculoligamentous structure with a cartilaginous framework that caps the lower respiratory tract. The cavity of the larynx is continuous below with the trachea, and above opens into the pharynx immediately posterior and slightly inferior to the tongue and the posterior opening (oropharyngeal isthmus) of the oral cavity (Fig. 8.209A,B).

1	The larynx is both a valve (or sphincter) to close the lower respiratory tract, and an instrument to produce sound. It is composed of: three large unpaired cartilages (cricoid, thyroid, and epiglottis), three pairs of smaller cartilages (arytenoid, corniculate, and cuneiform), and a fibro-elastic membrane and numerous intrinsic muscles. The larynx is suspended from the hyoid bone above and attached to the trachea below by membranes and ligaments. It is highly mobile in the neck and can be moved up and down and forward and backward by the action of extrinsic muscles that attach either to the larynx itself or to the hyoid bone. During swallowing, the dramatic upward and forward movements of the larynx facilitate closing the laryngeal inlet and opening the esophagus. Motor and sensory innervation of the larynx is provided by the vagus nerve [X].

1	Motor and sensory innervation of the larynx is provided by the vagus nerve [X]. The cricoid cartilage is the most inferior of the laryngeal cartilages and completely encircles the airway (Fig. 8.210). It is shaped like a signet ring with a broad lamina of cricoid cartilage posterior to the airway and a much narrower arch of cricoid cartilage circling anteriorly. The posterior surface of the lamina is characterized by two shallow oval depressions separated by a vertical ridge. The esophagus is attached to the ridge and the depressions are for attachment of the posterior crico-arytenoid muscles. The cricoid cartilage has two articular facets on each side for articulation with other laryngeal cartilages: One facet is on the sloping superolateral surface of the lamina and articulates with the base of an arytenoid cartilage.

1	One facet is on the sloping superolateral surface of the lamina and articulates with the base of an arytenoid cartilage. The other facet is on the lateral surface of the lamina near its base and is for articulation with the medial surface of the inferior horn of the thyroid cartilage. The thyroid cartilage (Fig. 8.211) is the largest of the laryngeal cartilages. It is formed by a right and a left lamina, which are widely separated posteriorly, but converge and join anteriorly. The most superior point of the site of fusion between the two broad flat laminae projects forward as the laryngeal prominence (Adam’s apple). The angle between the two laminae is more acute in men (90°) than in women (120°) so the laryngeal prominence is more apparent in men than women.

1	Just superior to the laryngeal prominence, the superior thyroid notch separates the two laminae as they diverge laterally. Both the superior thyroid notch and the laryngeal prominence are palpable landmarks in the neck. There is a less distinct inferior thyroid notch in the midline along the base of the thyroid cartilage. The posterior margin of each lamina of the thyroid cartilage is elongated to form a superior horn and an inferior horn: The medial surface of the inferior horn has a facet for articulation with the cricoid cartilage. The superior horn is connected by a lateral thyrohyoid ligament to the posterior end of the greater horn of the hyoid bone. The lateral surface of each thyroid lamina is marked by a ridge (the oblique line), which curves anteriorly from the base of the superior horn to a little short of midway along the inferior margin of the lamina.

1	The ends of the oblique line are expanded to form superior and inferior thyroid tubercles. The oblique line is a site of attachment for the extrinsic muscles of the larynx (sternothyroid, thyrohyoid, and inferior constrictor). The epiglottis is a leaf-shaped cartilage attached by its stem to the posterior aspect of the thyroid cartilage at the angle (Fig. 8.212) and projects posterosuperiorly from its attachment to the thyroid cartilage. The attachment is via the thyro-epiglottic ligament in the midline approximately midway between the laryngeal prominence and the inferior thyroid notch. The upper margin of the epiglottis is behind the pharyngeal part of the tongue. The inferior half of the posterior surface of the epiglottis is raised slightly to form an epiglottic tubercle. The two arytenoid cartilages are pyramid-shaped cartilages with three surfaces, a base of arytenoid cartilage and an apex of arytenoid cartilage (Fig. 8.213):

1	The two arytenoid cartilages are pyramid-shaped cartilages with three surfaces, a base of arytenoid cartilage and an apex of arytenoid cartilage (Fig. 8.213): The base is concave and articulates with the sloping articular facet on the superolateral surface of the lamina of cricoid cartilage. The apex articulates with a corniculate cartilage. The medial surface of each cartilage faces the other. The anterolateral surface has two depressions, separated by a ridge, for muscle (vocalis) and ligament (vestibular ligament) attachment. The posterior surface is covered by the transverse arytenoid muscle (see Fig. 8.223). The anterior angle of the base is elongated into a vocal process to which the vocal ligament is attached. The lateral angle is similarly elongated into a muscular process for attachment of the posterior and lateral crico-arytenoid muscles.

1	The corniculate cartilages (Fig. 8.214) are two small conical cartilages whose bases articulate with the apices of the arytenoid cartilages. Their apices project posteromedially toward each other. These two small club-shaped cartilages (Fig. 8.214) lie anterior to the corniculate cartilages and are suspended in the part of the fibro-elastic membrane of the larynx that attaches the arytenoid cartilages to the lateral margin of the epiglottis. The thyrohyoid membrane is a tough fibro-elastic ligament that spans between the superior margin of the thyroid cartilage below and the hyoid bone above (Fig. 8.215). It is attached to the superior margin of the thyroid laminae and adjacent anterior margins of the superior horns, and ascends medial to the greater horns and posterior to the body of the hyoid bone to attach to the superior margins of these structures.

1	An aperture in the lateral part of the thyrohyoid membrane on each side is for the superior laryngeal artery, the internal branch of the superior laryngeal nerve, and lymphatics. The posterior borders of the thyrohyoid membrane are thickened to form the lateral thyrohyoid ligaments. The membrane is also thickened anteriorly in the midline to form the median thyrohyoid ligament. Occasionally, there is a small cartilage (triticeal cartilage) in each lateral thyrohyoid ligament. The hyo-epiglottic ligament (Fig. 8.215) extends from the midline of the epiglottis, anterosuperiorly to the body of the hyoid bone. The cricotracheal ligament (Fig. 8.215) runs from the lower border of the cricoid cartilage to the adjacent upper border of the first tracheal cartilage. Fibro-elastic membrane of the larynx

1	The cricotracheal ligament (Fig. 8.215) runs from the lower border of the cricoid cartilage to the adjacent upper border of the first tracheal cartilage. Fibro-elastic membrane of the larynx The fibro-elastic membrane of the larynx links together the laryngeal cartilages and completes the architectural framework of the laryngeal cavity. It is composed of two parts—a lower conus elasticus and an upper quadrangular membrane. The conus elasticus (Fig. 8.216) is attached to the arch of cricoid cartilage and extends superiorly to end in a free upper margin within the space enclosed by the thyroid cartilage. On each side, this upper free margin attaches: anteriorly to the thyroid cartilage, and posteriorly to the vocal processes of the arytenoid cartilages. The free margin between these two points of attachment is thickened to form the vocal ligament, which is under the vocal fold (true vocal cord) of the larynx.

1	The free margin between these two points of attachment is thickened to form the vocal ligament, which is under the vocal fold (true vocal cord) of the larynx. The conus elasticus is also thickened anteriorly in the midline to form a distinct median cricothyroid ligament, which spans the distance between the arch of cricoid cartilage and the inferior thyroid notch and adjacent deep surface of the thyroid cartilage up to the attachment of the vocal ligaments. In emergency situations, when the airway is blocked above the level of the vocal folds, the median cricothyroid ligament can be perforated to establish an airway. Except for small vessels and the occasional presence of a pyramidal lobe of the thyroid gland, normally there are few structures between the median cricothyroid ligament and skin.

1	The quadrangular membrane on each side runs between the lateral margin of the epiglottis and the anterolateral surface of the arytenoid cartilage on the same side (Fig. 8.217). It is also attached to the corniculate cartilage, which articulates with the apex of arytenoid cartilage. Each quadrangular membrane has a free upper margin, between the top of the epiglottis and the corniculate cartilage, and a free lower margin. The free lower margin is thickened to form the vestibular ligament under the vestibular fold (false vocal cord) of the larynx. The vestibular ligament is attached posteriorly to the superior depression on the anterolateral surface of the arytenoid cartilage and anteriorly to the thyroid angle just superior to the attachment of the vocal ligament.

1	On each side, the vestibular ligament of the quadrangular membrane is separated from the vocal ligament of the cricothyroid ligament below by a gap. Because the vestibular ligament attaches to the anterolateral surface of the arytenoid cartilage and the vocal ligament attaches to the vocal process of the same cartilage, the vestibular ligament is lateral to the vocal ligament when viewed from above (Fig. 8.218). The joints between the inferior horns of the thyroid cartilage and the cricoid cartilage, and between the cricoid cartilage and arytenoid cartilages are synovial. Each is surrounded by a capsule and is reinforced by associated ligaments. The cricothyroid joints enable the thyroid cartilage to move forward and tilt downward on the cricoid cartilage (Fig. 8.219).

1	Because the vocal ligaments pass between the posterior aspect of the thyroid angle and the arytenoid cartilages that sit on the lamina of cricoid cartilage, forward movement and downward rotation of the thyroid cartilage on the cricoid cartilage effectively lengthens and puts tension on the vocal ligaments. The crico-arytenoid joints between articular facets on the superolateral surfaces of the cricoid cartilage and the bases of the arytenoid cartilages enable the arytenoid cartilages to slide away or toward each other and to rotate so that the vocal processes pivot either toward or away from the midline. These movements abduct and adduct the vocal ligaments (Fig. 8.220). Cavity of the larynx The central cavity of the larynx (Fig. 8.221) is tubular and lined by mucosa. Its architectural support is provided by the fibro-elastic membrane of the larynx and by the laryngeal cartilages to which it is attached.

1	The superior aperture of the cavity (laryngeal inlet) opens into the anterior aspect of the pharynx just below and posterior to the tongue (Fig. 8.221A): Its anterior border is formed by mucosa covering the superior margin of the epiglottis. Its lateral borders are formed by mucosal folds (ary-epiglottic folds), which enclose the superior margins of the quadrangular membranes and adjacent soft tissues, and two tubercles on the more posterolateral margin of the laryngeal inlet on each side mark the positions of the underlying cuneiform and corniculate cartilages. Its posterior border in the midline is formed by a mucosal fold that forms a depression (interarytenoid notch) between the two corniculate tubercles.

1	Its posterior border in the midline is formed by a mucosal fold that forms a depression (interarytenoid notch) between the two corniculate tubercles. The inferior opening of the laryngeal cavity is continuous with the lumen of the trachea, is completely encircled by the cricoid cartilage, and is horizontal in position unlike the laryngeal inlet, which is oblique and points posterosuperiorly into the pharynx. In addition, the inferior opening is continuously open, whereas the laryngeal inlet can be closed by downward movement of the epiglottis. Two pairs of mucosal folds, the vestibular and vocal folds, which project medially from the lateral walls of the laryngeal cavity, constrict it and divide it into three major regions—the vestibule, a middle chamber, and the infraglottic cavity (Fig. 8.221B): The vestibule is the upper chamber of the laryngeal cavity between the laryngeal inlet and the vestibular folds, which encloses the vestibular ligaments and associated soft tissues.

1	The vestibule is the upper chamber of the laryngeal cavity between the laryngeal inlet and the vestibular folds, which encloses the vestibular ligaments and associated soft tissues. The middle part of the laryngeal cavity is very thin and is between the vestibular folds above and the vocal folds below. The infraglottic space is the most inferior chamber of the laryngeal cavity and is between the vocal folds (which encloses the vocal ligaments and related soft tissues) and the inferior opening of the larynx.

1	On each side, the mucosa of the middle cavity bulges laterally through the gap between the vestibular and vocal ligaments to produce an expanded trough-shaped space (a laryngeal ventricle) (Fig. 8.221A). An elongate tubular extension of each ventricle (laryngeal saccule) projects anterosuperiorly between the vestibular fold and thyroid cartilage and may reach as high as the top of the thyroid cartilage. Within the walls of these laryngeal saccules are numerous mucous glands. Mucus secreted into the saccules lubricates the vocal folds. When viewed from above (Fig. 8.221C,D), there is a triangular opening (the rima vestibuli) between the two adjacent vestibular folds at the entrance to the middle chamber of the laryngeal cavity. The apex of the opening is anterior and its base is formed by the posterior wall of the laryngeal cavity.

1	Inferior to the vestibular folds, the vocal folds (true vocal cords) and adjacent mucosa-covered parts of the arytenoid cartilages form the lateral walls of a similar, but narrower, triangular opening (the rima glottidis between the two adjacent vocal folds). This opening separates the middle chamber above from the infraglottic cavity below. The base of this triangular opening is formed by the fold of mucosa (interarytenoid fold) at the bottom of the interarytenoid notch. Both the rima glottidis and the rima vestibuli can be opened and closed by movement of the arytenoid cartilages and associated fibro-elastic membranes.

1	Both the rima glottidis and the rima vestibuli can be opened and closed by movement of the arytenoid cartilages and associated fibro-elastic membranes. The intrinsic muscles of the larynx (Table 8.19) adjust tension in the vocal ligaments, open and close the rima glottidis, control the inner dimensions of the vestibule, close the rima vestibuli, and facilitate closing of the laryngeal inlet. They do this mainly by: acting on the cricothyroid and crico-arytenoid joints, adjusting the distance between the epiglottis and arytenoid cartilages, pulling directly on the vocal ligaments, and forcing soft tissues associated with the quadrangular membranes and vestibular ligaments toward the midline. The fan-shaped cricothyroid muscles are attached to the anterolateral surfaces of the arch of the cricoid cartilage and expand superiorly and posteriorly to attach to the thyroid cartilage (Fig. 8.222). Each muscle has an oblique part and a straight part:

1	Each muscle has an oblique part and a straight part: The oblique part runs in a posterior direction from the arch of the cricoid cartilage to the inferior horn of the thyroid cartilage. The straight part runs more vertically from the arch of the cricoid cartilage to the posteroinferior margin of the thyroid lamina. The cricothyroid muscles move the cricothyroid joints. They pull the thyroid cartilage forward and rotate it down relative to the cricoid cartilage. These actions lengthen the vocal folds. The cricothyroid muscles are the only intrinsic muscles of the larynx innervated by the superior laryngeal branches of the vagus nerves [X]. All other intrinsic muscles are innervated by the recurrent laryngeal branches of the vagus nerves [X].

1	There is a right and a left posterior crico-arytenoid muscle (Fig. 8.223). The fibers of each muscle originate from a large shallow depression on the posterior surface of the lamina of the cricoid cartilage, and run superiorly and laterally to converge on the muscular processes of the arytenoid cartilage. The posterior crico-arytenoid muscles abduct and externally (laterally) rotate the arytenoid cartilages, thereby opening the rima glottidis. These muscles are the primary abductors of the vocal folds. They are innervated by the recurrent laryngeal branches of the vagus nerves [X]. The lateral crico-arytenoid muscle on each side originates from the upper surface of the arch of the cricoid cartilage, and runs posteriorly and superiorly to insert on the muscular process of the arytenoid cartilage (Fig. 8.223). The lateral crico-arytenoid muscles internally rotate the arytenoid cartilages. These movements result in adducted (closed) vocal folds.

1	The lateral crico-arytenoid muscles internally rotate the arytenoid cartilages. These movements result in adducted (closed) vocal folds. The lateral crico-arytenoids are innervated by the recurrent laryngeal branches of the vagus nerves [X]. The single transverse arytenoid muscle spans the distance between adjacent lateral margins of the arytenoid cartilages and covers the posterior surfaces of these cartilages (Fig. 8.223). It adducts the arytenoid cartilages and is innervated by the recurrent laryngeal branches of the vagus nerves [X]. Each of the two oblique arytenoid muscles runs from the posterior surface of the muscular process of one arytenoid cartilage to the apex of the arytenoid cartilage on the other side (Fig. 8.223). Some fibers of the muscle continue laterally around the margin of the arytenoid cartilage and into the ary-epiglottic fold where they continue as the ary-epiglottic part of the muscle (Fig. 8.224).

1	The oblique arytenoids can narrow the laryngeal inlet by constricting the distance between the arytenoid cartilages and the epiglottis. They are innervated by the recurrent laryngeal branches of the vagus nerves [X]. The vocalis muscles are elongate muscles lateral to and running parallel with each vocal ligament (Fig. 8.223). The fibers in each muscle are attached posteriorly to the lateral surface of the vocal process and adjacent depression on the anterolateral surface of the arytenoid cartilage, and anteriorly insert along the length of the vocal ligament to the thyroid angle. The vocalis muscles adjust tension in the vocal folds and are innervated by the recurrent laryngeal branches of the vagus nerves [X].

1	The vocalis muscles adjust tension in the vocal folds and are innervated by the recurrent laryngeal branches of the vagus nerves [X]. The two thyro-arytenoid muscles are broad flat muscles lateral to the fibro-elastic membrane of the larynx and the laryngeal ventricles and saccules (Fig. 8.224). Each muscle runs from a vertical line of origin on the lower half of the thyroid angle and adjacent external surface of the cricothyroid ligament to the anterolateral surface of the arytenoid cartilage. Some of the fibers may continue into the ary-epiglottic fold and reach the margin of the epiglottis. These fibers are the thyro-epiglottic part of the muscle.

1	Because the thyro-arytenoid muscles are broad and lateral to the quadrangular membrane, they act as a sphincter of the vestibule by pushing soft tissues medial to the muscles toward the midline. The muscles also narrow the laryngeal inlet by pulling the arytenoid cartilages forward while simultaneously pulling the epiglottis toward the arytenoid cartilages. The thyro-arytenoid muscles are innervated by the recurrent laryngeal branches of the vagus nerves [X]. Function of the larynx The larynx is an elaborate sphincter for the lower respiratory tract and provides a mechanism for producing sounds. Adjustments of the size of the central cavity of the larynx result from changes in the dimensions of the rima glottidis, the rima vestibuli, the vestibule, and the laryngeal inlet (Fig. 8.225). These changes result from muscle actions and laryngeal mechanics.

1	During quiet respiration, the laryngeal inlet, vestibule, rima vestibuli, and rima glottidis are open. The arytenoid cartilages are abducted and the rima glottidis is triangular shaped (Fig. 8.225A). During forced inspiration (Fig. 8.225B), the arytenoid cartilages are rotated laterally, mainly by the action of the posterior crico-arytenoid muscles. As a result, the vocal folds are abducted and the rima glottidis widens into a rhomboid shape, which effectively increases the diameter of the laryngeal airway. When phonating, the arytenoid cartilages and vocal folds are adducted and air is forced through the closed rima glottidis (Fig. 8.225C). This action causes the vocal folds to vibrate against each other and produce sounds, which can then be modified by the upper parts of the airway and oral cavity. Tension in the vocal folds can be adjusted by the vocalis and cricothyroid muscles.

1	Effort closure of the larynx (Fig. 8.225D) occurs when air is retained in the thoracic cavity to stabilize the trunk, for example during heavy lifting, or as part of the mechanism for increasing intra-abdominal pressure. During effort closure, the rima glottidis is completely closed, as is the rima vestibuli and lower parts of the vestibule. The result is to completely and forcefully shut the airway.

1	During swallowing, the rima glottidis, rima vestibuli, and vestibule are closed and the laryngeal inlet is narrowed. In addition, the larynx moves up and forward. This action causes the epiglottis to swing downward toward the arytenoid cartilages and to effectively narrow or close the laryngeal inlet (Fig. 8.225E). The up and forward movement of the larynx also opens the esophagus, which is attached to the posterior aspect of the lamina of the cricoid cartilage. All these actions together prevent solids and liquids from entry into the airway and facilitate their movement through the piriform fossae into the esophagus. The major blood supply to the larynx is by the superior and inferior laryngeal arteries (Fig. 8.226):

1	The major blood supply to the larynx is by the superior and inferior laryngeal arteries (Fig. 8.226): The superior laryngeal artery originates near the upper margin of the thyroid cartilage from the superior thyroid branch of the external carotid artery, and accompanies the internal branch of the superior laryngeal nerve through the thyrohyoid membrane to reach the larynx. The inferior laryngeal artery originates from the inferior thyroid branch of the thyrocervical trunk of the subclavian artery low in the neck and, together with the recurrent laryngeal nerve, ascends in the groove between the esophagus and trachea—it enters the larynx by passing deep to the margin of the inferior constrictor muscle of the pharynx. Veins draining the larynx accompany the arteries: Superior laryngeal veins drain into superior thyroid veins, which in turn drain into the internal jugular veins (Fig. 8.227).

1	Veins draining the larynx accompany the arteries: Superior laryngeal veins drain into superior thyroid veins, which in turn drain into the internal jugular veins (Fig. 8.227). Inferior laryngeal veins drain into inferior thyroid veins, which drain into the left brachiocephalic vein. Lymphatics drain regions above and below the vocal folds: Those above the vocal folds follow the superior laryngeal artery and terminate in deep cervical nodes associated with the bifurcation of the common carotid artery. Those below the vocal folds drain into deep nodes associated with the inferior thyroid artery or with nodes associated with the front of the cricothyroid ligament or upper trachea. Sensory and motor innervation of the larynx is by two branches of the vagus nerves [X]—the superior laryngeal nerves and the recurrent laryngeal nerves (Fig. 8.228).

1	Sensory and motor innervation of the larynx is by two branches of the vagus nerves [X]—the superior laryngeal nerves and the recurrent laryngeal nerves (Fig. 8.228). The superior laryngeal nerves originate from the inferior vagal ganglia high in the neck (Fig. 8.228). On each side, the nerve descends medial to the internal carotid artery and divides into internal and external branches just above the level of the superior horn of the hyoid bone: The external branch (external laryngeal nerve) descends along the lateral wall of the pharynx to supply and penetrate the inferior constrictor of the pharynx and ends by supplying the cricothyroid muscle. The internal branch (internal laryngeal nerve) passes anteroinferiorly to penetrate the thyrohyoid membrane—it is mainly sensory and supplies the laryngeal cavity down to the level of the vocal folds.

1	The recurrent laryngeal nerves are (Fig. 8.228): sensory to the laryngeal cavity below the level of the vocal folds, and motor to all intrinsic muscles of the larynx except for the cricothyroid. The left recurrent laryngeal nerve originates in the thorax, whereas the right recurrent laryngeal nerve originates in the root of the neck. Both nerves generally ascend in the neck in the groove between the esophagus and trachea and enter the larynx deep to the margin of the inferior constrictor. They may pass medial to, lateral to, or through the lateral ligament of the thyroid gland, which attaches the thyroid gland to the trachea and lower part of the cricoid cartilage on each side. The two nasal cavities are the uppermost parts of the respiratory tract and contain the olfactory receptors. They are elongated wedge-shaped spaces with a large inferior base and a narrow superior apex (Figs. 8.229 and 8.230) and are held open by a skeletal framework consisting mainly of bone and cartilage.

1	The smaller anterior regions of the cavities are enclosed by the external nose, whereas the larger posterior regions are more central within the skull. The anterior apertures of the nasal cavities are the nares, which open onto the inferior surface of the nose. The posterior apertures are the choanae, which open into the nasopharynx. The nasal cavities are separated: from each other by a midline nasal septum, from the oral cavity below by the hard palate, and from the cranial cavity above by parts of the frontal, ethmoid, and sphenoid bones. Lateral to the nasal cavities are the orbits. Each nasal cavity has a floor, roof, medial wall, and lateral wall (Fig. 8.230A). The lateral wall is characterized by three curved shelves of bone (conchae), which are one above the other and project medially and inferiorly across the nasal cavity (Fig. 8.230B). The medial, anterior, and posterior margins of the conchae are free.

1	The conchae divide each nasal cavity into four air channels (Fig. 8.230C,D): an inferior nasal meatus between the inferior concha and the nasal floor, a middle nasal meatus between the inferior and middle concha, a superior nasal meatus between the middle and superior concha, and a spheno-ethmoidal recess between the superior concha and the nasal roof. These conchae increase the surface area of contact between tissues of the lateral wall and the respired air. The openings of the paranasal sinuses, which are extensions of the nasal cavity that erode into the surrounding bones during childhood and early adulthood, are on the lateral wall and roof of the nasal cavities (Fig. 8.231). In addition, the lateral wall also contains the opening of the nasolacrimal duct, which drains tears from the eye into the nasal cavity. Each nasal cavity consists of three general regions—the nasal vestibule, the respiratory region, and the olfactory region (Fig. 8.232):

1	Each nasal cavity consists of three general regions—the nasal vestibule, the respiratory region, and the olfactory region (Fig. 8.232): The nasal vestibule is a small dilated space just internal to the naris that is lined by skin and contains hair follicles. The respiratory region is the largest part of the nasal cavity, has a rich neurovascular supply, and is lined by respiratory epithelium composed mainly of ciliated and mucous cells. The olfactory region is small, is at the apex of each nasal cavity, is lined by olfactory epithelium, and contains the olfactory receptors.

1	The olfactory region is small, is at the apex of each nasal cavity, is lined by olfactory epithelium, and contains the olfactory receptors. In addition to housing receptors for the sense of smell (olfaction), the nasal cavities adjust the temperature and humidity of respired air by the action of a rich blood supply, and trap and remove particulate matter from the airway by filtering the air through hair in the vestibule and by capturing foreign material in abundant mucus. The mucus normally is moved posteriorly by cilia on epithelial cells in the nasal cavities and is swallowed. Innervation of the nasal cavities is by three cranial nerves: Olfaction is carried by the olfactory nerve [I]. General sensation is carried by the trigeminal nerve [V], the anterior region by the ophthalmic nerve [V1], and the posterior region by the maxillary nerve [V2].

1	General sensation is carried by the trigeminal nerve [V], the anterior region by the ophthalmic nerve [V1], and the posterior region by the maxillary nerve [V2]. All glands are innervated by parasympathetic fibers in the facial nerve [VII] (greater petrosal nerve), which join branches of the maxillary nerve [V2] in the pterygopalatine fossa. Sympathetic fibers are ultimately derived from the T1 spinal cord level. They synapse mainly in the superior cervical sympathetic ganglion, and postganglionic fibers reach the nasal cavities along blood vessels, or by joining branches of the maxillary nerve [V2] in the pterygopalatine fossa. Blood supply to the nasal cavities is by: terminal branches of the maxillary and facial arteries, which originate from the external carotid artery, and ethmoidal branches of the ophthalmic artery, which originates from the internal carotid artery.

1	Bones that contribute to the skeletal framework of the nasal cavities include: the unpaired ethmoid, sphenoid, frontal, and vomer bones, and the paired nasal, maxillary, palatine, and lacrimal bones and inferior conchae. Of all the bones associated with the nasal cavities, the ethmoid is a key element. The single ethmoid bone is one of the most complex bones in the skull. It contributes to the roof, lateral wall, and medial wall of both nasal cavities, and contains the ethmoidal cells (ethmoidal sinuses). The ethmoid bone is cuboidal in overall shape (Fig. 8.233A) and is composed of two rectangular box- shaped ethmoidal labyrinths, one on each side, united superiorly across the midline by a perforated sheet of bone (the cribriform plate). A second sheet of bone (the perpendicular plate) descends vertically in the median sagittal plane from the cribriform plate to form part of the nasal septum.

1	Each ethmoidal labyrinth is composed of two delicate sheets of bone, which sandwich between them the ethmoidal cells. The lateral sheet of bone (the orbital plate) is flat and forms part of the medial wall of the orbit. The medial sheet of bone forms the upper part of the lateral wall of the nasal cavity and is characterized by two processes and a swelling (Fig. 8.233B)—the two processes are curved shelves of bone (the superior and middle conchae), which project across the nasal cavity and curve downward ending in free medial margins, while inferior to the origin of the middle concha, the middle ethmoidal cells form a prominent bulge (the ethmoidal bulla), on the medial wall of the labyrinth.

1	Extending anterosuperiorly from just under the bulla is a groove (the ethmoidal infundibulum), which continues upward, and narrows to form a channel that penetrates the ethmoidal labyrinth and opens into the frontal sinus. This channel is for the frontonasal duct, which drains the frontal sinus. The superior surface of the ethmoidal labyrinth articulates with the frontal bone, which usually completes the roof of the ethmoidal cells, while the anterior surface articulates with the frontal process of the maxilla and with the lacrimal bone. The inferior surface articulates with the upper medial margin of the maxilla. A delicate irregularly shaped projection (the uncinate process) on the anterior aspect of the inferior surface of the ethmoidal labyrinth extends posteroinferiorly across a large defect (maxillary hiatus) in the medial wall of the maxilla to articulate with the inferior concha.

1	The cribriform plate is at the apex of the nasal cavities and fills the ethmoidal notch in the frontal bone (Fig. 8.233) and separates the nasal cavities below from the cranial cavity above. Small perforations in the bone allow the fibers of the olfactory nerve [I] to pass between the two regions. A large triangular process (the crista galli) at the midline on the superior surface of the cribriform plate anchors a fold (falx cerebri) of dura mater in the cranial cavity. The perpendicular plate of the ethmoid bone is quadrangular in shape, descends in the midline from the cribriform plate, and forms the upper part of the median nasal septum (Fig. 8.233). It articulates: posteriorly with the sphenoidal crest on the body of the sphenoid bone, anteriorly with the nasal spine on the frontal bone and with the site of articulation at the midline between the two nasal bones, and inferiorly and anteriorly with the septal cartilage and posteriorly with the vomer.

1	The external nose extends the nasal cavities onto the front of the face and positions the nares so that they point downward (Fig. 8.234). It is pyramidal in shape with its apex anterior in position. The upper angle of the nose between the openings of the orbits is continuous with the forehead. Like posterior regions, the anterior parts of the nasal cavities found within the nose are held open by a skeletal framework, which is composed partly of bone and mainly of cartilage: The bony parts are where the nose is continuous with the skull—here the nasal bones and parts of the maxillae and frontal bones provide support. Anteriorly, and on each side, support is provided by lateral processes of the septal cartilage, major alar and three or four minor alar cartilages, and a single septal cartilage in the midline that forms the anterior part of the nasal septum.

1	There are four paranasal air sinuses—the ethmoidal cells, and the sphenoidal, maxillary, and frontal sinuses (Fig. 8.235A,B). Each is named according to the bone in which it is found. The paranasal sinuses develop as outgrowths from the nasal cavities and erode into the surrounding bones. All of the paranasal sinuses: are lined by respiratory mucosa, which is ciliated and mucus secreting, open into the nasal cavities, and are innervated by branches of the trigeminal nerve [V]. The frontal sinuses, one on each side, are variable in size and are the most superior of the sinuses (Fig. 8.235A–C). Each is triangular in shape and is in the part of the frontal bone under the forehead. The base of each triangular sinus is oriented vertically in the bone at the midline above the bridge of the nose and the apex is laterally approximately one-third of the way along the upper margin of the orbit.

1	Each frontal sinus drains onto the lateral wall of the middle meatus via the frontonasal duct, which penetrates the ethmoidal labyrinth and continues as the ethmoidal infundibulum at the front end of the semilunar hiatus. The frontal sinuses are innervated by branches of the supra-orbital nerve from the ophthalmic nerve [V1]. Their blood supply is from branches of the anterior ethmoidal arteries. The ethmoidal cells on each side fill the ethmoidal labyrinth (Fig. 8.235A,B). Each cluster of cells is separated from the orbit by the thin orbital plate of the ethmoidal labyrinth, and from the nasal cavity by the medial wall of the ethmoidal labyrinth. The ethmoidal cells are formed by a variable number of individual air chambers, which are divided into anterior, middle, and posterior ethmoidal cells based on the location of their apertures on the lateral wall of the nasal cavity: The anterior ethmoidal cells open into the ethmoidal infundibulum or the frontonasal duct.

1	The anterior ethmoidal cells open into the ethmoidal infundibulum or the frontonasal duct. The middle ethmoidal cells open onto the ethmoidal bulla, or onto the lateral wall just above this structure. The posterior ethmoidal cells open onto the lateral wall of the superior nasal meatus. Because the ethmoidal cells often erode into bones beyond the boundaries of the ethmoidal labyrinth, their walls may be completed by the frontal, maxillary, lacrimal, sphenoid, and palatine bones. The ethmoidal cells are innervated by: the anterior and posterior ethmoidal branches of the nasociliary nerve from the ophthalmic nerve [V1], and the maxillary nerve [V2] via orbital branches from the pterygopalatine ganglion. The ethmoidal cells receive their blood supply through branches of the anterior and posterior ethmoidal arteries.

1	The ethmoidal cells receive their blood supply through branches of the anterior and posterior ethmoidal arteries. The maxillary sinuses, one on each side, are the largest of the paranasal sinuses and completely fill the bodies of the maxillae (Fig. 8.235A,B). Each is pyramidal in shape with the apex directed laterally and the base deep to the lateral wall of the adjacent nasal cavity. The medial wall or base of the maxillary sinus is formed by the maxilla, and by parts of the inferior concha and palatine bone that overlie the maxillary hiatus. The opening of the maxillary sinus is near the top of the base, in the center of the semilunar hiatus, which grooves the lateral wall of the middle nasal meatus. Relationships of the maxillary sinus are as follows: The superolateral surface (roof) is related above to the orbit. The anterolateral surface is related below to the roots of the upper molar and premolar teeth and in front to the face.

1	The superolateral surface (roof) is related above to the orbit. The anterolateral surface is related below to the roots of the upper molar and premolar teeth and in front to the face. The posterior wall is related behind to the infratemporal fossa. The maxillary sinuses are innervated by infra-orbital and alveolar branches of the maxillary nerve [V2], and receive their blood through branches from the infra-orbital and superior alveolar branches of the maxillary arteries. The sphenoidal sinuses, one on either side within the body of the sphenoid, open into the roof of the nasal cavity via apertures on the posterior wall of the spheno-ethmoidal recess (Fig. 8.235C,D). The apertures are high on the anterior walls of the sphenoid sinuses. The sphenoidal sinuses are related: above to the cranial cavity, particularly to the pituitary gland and to the optic chiasm, laterally, to the cranial cavity, particularly to the cavernous sinuses, and below and in front, to the nasal cavities.

1	Because only thin shelves of bone separate the sphenoidal sinuses from the nasal cavities below and hypophyseal fossa above, the pituitary gland can be surgically approached through the roof of the nasal cavities by passing first through the anteroinferior aspect of the sphenoid bone and into the sphenoidal sinuses and then through the top of the sphenoid bone into the hypophyseal fossa. Innervation of the sphenoidal sinuses is provided by: the posterior ethmoidal branch of the ophthalmic nerve [V1], and the maxillary nerve [V2] via orbital branches from the pterygopalatine ganglion. The sphenoidal sinuses are supplied by branches of the pharyngeal arteries from the maxillary arteries. Walls, floor, and roof The medial wall of each nasal cavity is the mucosa-covered surface of the thin nasal septum, which is oriented vertically in the median sagittal plane and separates the right and left nasal cavities from each other.

1	The nasal septum (Fig. 8.236) consists of: the septal nasal cartilage anteriorly, posteriorly, mainly the vomer and the perpendicular plate of the ethmoid bone, small contributions by the nasal bones where they meet in the midline, and the nasal spine of the frontal bone, and contributions by the nasal crests of the maxillary and palatine bones, rostrum of the sphenoid bone, and the incisor crest of the maxilla. The floor of each nasal cavity (Fig. 8.237) is smooth, concave, and much wider than the roof. It consists of: soft tissues of the external nose, and the upper surface of the palatine process of the maxilla and the horizontal plate of the palatine bone, which together form the hard palate. The naris opens anteriorly into the floor, and the superior aperture of the incisive canal is deep to the mucosa immediately lateral to the nasal septum near the front of the hard palate.

1	The naris opens anteriorly into the floor, and the superior aperture of the incisive canal is deep to the mucosa immediately lateral to the nasal septum near the front of the hard palate. The roof of the nasal cavity is narrow and is highest in central regions where it is formed by the cribriform plate of the ethmoid bone (Fig. 8.238). Anterior to the cribriform plate the roof slopes inferiorly to the nares and is formed by: the nasal spine of the frontal bone and the nasal bones, and the lateral processes of the septal cartilage and major alar cartilages of the external nose. Posteriorly, the roof of each cavity slopes inferiorly to the choana and is formed by: the anterior surface of the sphenoid bone, the ala of the vomer and adjacent sphenoidal process of the palatine bone, and the vaginal process of the medial plate of the pterygoid process.

1	Underlying the mucosa, the roof is perforated superiorly by openings in the cribriform plate, and anterior to these openings by a separate foramen for the anterior ethmoidal nerve and vessels. The opening between the sphenoidal sinus and the spheno-ethmoidal recess is on the posterior slope of the roof. The lateral wall of each nasal cavity is complex and is formed by bone, cartilage, and soft tissues. Bony support for the lateral wall (Fig. 8.239A) is provided by: the ethmoidal labyrinth, superior concha, middle concha and uncinate process, the perpendicular plate of the palatine bone, the medial pterygoid plate of the sphenoid bone, the medial surfaces of the lacrimal bones and maxillae, and the inferior concha.

1	In the external nose, the lateral wall of the cavity is supported by cartilage (lateral process of the septal cartilage and major and minor alar cartilages) and by soft tissues. The surface of the lateral wall is irregular in contour and is interrupted by the three nasal conchae. The inferior, middle, and superior conchae (Fig. 8.239B) extend medially across the nasal cavity, separating it into four air channels, an inferior, middle, and superior meatus and a spheno-ethmoidal recess. The conchae do not extend forward into the external nose. The anterior end of each concha curves inferiorly to form a lip that overlies the end of the related meatus. Immediately inferior to the attachment of the middle concha and just anterior to the midpoint of the concha, the lateral wall of the middle meatus elevates to form the dome-shaped ethmoidal bulla (Fig. 8.239C). This is formed by the underlying middle ethmoidal cells, which expand the medial wall of the ethmoidal labyrinth.

1	Inferior to the ethmoidal bulla is a curved gutter (the semilunar hiatus), which is formed by the mucosa covering the lateral wall as it spans a defect in the bony wall between the ethmoidal bulla above and the uncinate process below. The anterior end of the semilunar hiatus forms a channel (the ethmoidal infundibulum), which curves upward and continues as the frontonasal duct through the anterior part of the ethmoidal labyrinth to open into the frontal sinus. The nasolacrimal duct and most of the paranasal sinuses open onto the lateral wall of the nasal cavity (Fig. 8.239C): The nasolacrimal duct opens onto the lateral wall of the inferior nasal meatus under the anterior lip of the inferior concha—it drains tears from the conjunctival sac of the eye into the nasal cavity and originates at the inferior end of the lacrimal sac on the anteromedial wall of the orbit.

1	The frontal sinus drains via the frontonasal duct and ethmoidal infundibulum into the anterior end of the semilunar hiatus on the lateral wall of the middle nasal meatus—the anterior ethmoidal cells drain into the frontonasal duct or ethmoidal infundibulum (in some cases, the frontal sinus drains directly into the anterior end of the middle nasal meatus and the frontonasal duct ends blindly in the anterior ethmoidal cells). The middle ethmoidal cells open onto or just above the ethmoidal bulla. The posterior ethmoidal cells usually open onto the lateral wall of the superior nasal meatus. The large maxillary sinus opens into the semilunar hiatus, usually just inferior to the center of the ethmoidal bulla—this opening is near the roof of the maxillary sinus. The only paranasal sinus that does not drain onto the lateral wall of the nasal cavity is the sphenoidal sinus, which usually opens onto the sloping posterior roof of the nasal cavity.

1	The only paranasal sinus that does not drain onto the lateral wall of the nasal cavity is the sphenoidal sinus, which usually opens onto the sloping posterior roof of the nasal cavity. The nares are oval apertures on the inferior aspect of the external nose and are the anterior openings of the nasal cavities (Fig. 8.240A). They are held open by the surrounding alar cartilages and septal cartilage, and by the inferior nasal spine and adjacent margins of the maxillae. Although the nares are continuously open, they can be widened further by the action of the related muscles of facial expression (nasalis, depressor septi nasi, and levator labii superioris alaeque nasi muscles; Fig. 8.240B).

1	The choanae are the oval-shaped openings between the nasal cavities and the nasopharynx (Fig. 8.241). Unlike the nares, which have flexible borders of cartilage and soft tissues, the choanae are rigid openings completely surrounded by bone, and their margins are formed: inferiorly by the posterior border of the horizontal plate of the palatine bone, laterally by the posterior margin of the medial plate of the pterygoid process, and medially by the posterior border of the vomer. The roof of the choanae is formed: anteriorly by the ala of the vomer and the vaginal process of the medial plate of the pterygoid process, and posteriorly by the body of the sphenoid bone. There are a number of routes by which nerves and vessels enter and leave the soft tissues lining each nasal cavity (Fig. 8.242), and these include the cribriform plate, sphenopalatine foramen, incisive canal, and small foramina in the lateral wall, and around the margin of the nares.

1	The fibers of the olfactory nerve [I] exit the nasal cavity and enter the cranial cavity through perforations in the cribriform plate. In addition, small foramina between the cribriform plate and surrounding bone allow the anterior ethmoidal nerve, a branch of the ophthalmic nerve [V1], and accompanying vessels to pass from the orbit into the cranial cavity and then down into the nasal cavity. In addition, there is a connection in some individuals between nasal veins and the superior sagittal sinus of the cranial cavity through a prominent foramen (the foramen cecum) in the midline between the crista galli and frontal bone.

1	One of the most important routes by which nerves and vessels enter and leave the nasal cavity is the sphenopalatine foramen in the posterolateral wall of the superior nasal meatus. This foramen is just superior to the attachment of the posterior end of the middle nasal concha and is formed by the sphenopalatine notch in the palatine bone and the body of the sphenoid bone. The sphenopalatine foramen is a route of communication between the nasal cavity and the pterygopalatine fossa. Major structures passing through the foramen are: the sphenopalatine branch of the maxillary artery, the nasopalatine branch of the maxillary nerve [V2], and superior nasal branches of the maxillary nerve [V2].

1	Another route by which structures enter and leave the nasal cavities is through the incisive canal in the floor of each nasal cavity. This canal is immediately lateral to the nasal septum and just posterosuperior to the root of the central incisor in the maxilla. The two incisive canals, one on each side, both open into the single unpaired incisive fossa in the roof of the oral cavity and transmit: the nasopalatine nerve from the nasal cavity into the oral cavity, and the terminal end of the greater palatine artery from the oral cavity into the nasal cavity. Small foramina in the lateral wall Other routes by which vessels and nerves get into and out of the nasal cavity include the nares and small foramina in the lateral wall: Internal nasal branches of the infra-orbital nerve of the maxillary nerve [V2] and alar branches of the nasal artery from the facial artery loop around the margin of the naris to gain entry to the lateral wall of the nasal cavity from the face.

1	Inferior nasal branches from the greater palatine branch of the maxillary nerve [V2] enter the lateral wall of the nasal cavity from the palatine canal by passing through small foramina on the lateral wall. The nasal cavities have a rich vascular supply for altering the humidity and temperature of respired air. In fact, the submucosa of the respiratory region, particularly that related to the conchae and septum, is often described as “erectile” or “cavernous” because the tissue enlarges or shrinks depending on the amount of blood flowing into the system. Arteries that supply the nasal cavity include vessels that originate from both the internal and external carotid arteries (Fig. 8.243): Vessels that originate from branches of the external carotid artery include the sphenopalatine, greater palatine, superior labial, and lateral nasal arteries. Vessels that originate from branches of the internal carotid artery are the anterior and posterior ethmoidal arteries.

1	Vessels that originate from branches of the internal carotid artery are the anterior and posterior ethmoidal arteries. The largest vessel supplying the nasal cavity is the sphenopalatine artery (Fig. 8.243), which is the terminal branch of the maxillary artery in the pterygopalatine fossa. It leaves the pterygopalatine fossa and enters the nasal cavity by passing medially through the sphenopalatine foramen and onto the lateral wall of the nasal cavity. Posterior lateral nasal branches supply a large part of the lateral wall and anastomose anteriorly with branches from the anterior and posterior ethmoidal arteries, and with lateral nasal branches of the facial artery.

1	Posterior septal branches of the sphenopalatine artery pass over the roof of the cavity and onto the nasal septum where they contribute to the blood supply of the medial wall. One of these latter branches continues forward down the nasal septum to anastomose with the terminal end of the greater palatine artery and septal branches of the superior labial artery. The terminal end of the greater palatine artery enters the anterior aspect of the floor of the nasal cavity by passing up through the incisive canal from the roof of the oral cavity (Fig. 8.243).

1	The terminal end of the greater palatine artery enters the anterior aspect of the floor of the nasal cavity by passing up through the incisive canal from the roof of the oral cavity (Fig. 8.243). Like the sphenopalatine artery, the greater palatine artery arises in the pterygopalatine fossa as a branch of the maxillary artery. It passes first onto the roof of the oral cavity by passing down through the palatine canal and greater palatine foramen to the posterior aspect of the palate, then passes forward on the undersurface of the palate, and up through the incisive fossa and canal to reach the floor of the nasal cavity. The greater palatine artery supplies anterior regions of the medial wall and adjacent floor of the nasal cavity, and anastomoses with the septal branch of the sphenopalatine artery. The superior labial artery and the lateral nasal artery originate from the facial artery on the front of the face.

1	The superior labial artery and the lateral nasal artery originate from the facial artery on the front of the face. The superior labial artery originates from the facial artery near the lateral end of the oral fissure and passes medially in the lip, supplying the lip and giving rise to branches that supply the nose and nasal cavity. An alar branch supplies the region around the lateral aspect of the naris and a septal branch passes into the nasal cavity and supplies anterior regions of the nasal septum. The lateral nasal artery originates from the facial artery in association with the margin of the external nose and contributes to the blood supply of the external nose. Alar branches pass around the lateral margin of the naris and supply the nasal vestibule.

1	The anterior and posterior ethmoidal arteries (Fig. 8.243) originate in the orbit from the ophthalmic artery, which originates in the cranial cavity as a major branch of the internal carotid artery. They pass through canals in the medial wall of the orbit between the ethmoidal labyrinth and frontal bone, supply the adjacent paranasal sinuses, and then enter the cranial cavity immediately lateral and superior to the cribriform plate. The posterior ethmoidal artery descends into the nasal cavity through the cribriform plate and has branches to the upper parts of the medial and lateral walls.

1	The posterior ethmoidal artery descends into the nasal cavity through the cribriform plate and has branches to the upper parts of the medial and lateral walls. The anterior ethmoidal artery passes forward, with the accompanying anterior ethmoidal nerve, in a groove on the cribriform plate and enters the nasal cavity by descending through a slit-like foramen immediately lateral to the crista galli. It gives rise to branches that supply the medial (septal) and lateral wall of the nasal cavity and then continues forward on the deep surface of the nasal bone, and terminates by passing between the nasal bone and lateral nasal cartilage to emerge on the external nose as the external nasal branch to supply skin and adjacent tissues.

1	Vessels that supply the nasal cavities form extensive anastomoses with each other. This is particularly evident in the anterior region of the medial wall where there are anastomoses between branches of the greater palatine, sphenopalatine, superior labial, and anterior ethmoidal arteries, and where the vessels are relatively close to the surface (Fig. 8.243B). This area is the major site of nosebleeds, or epistaxis. Veins draining the nasal cavities generally follow the arteries (Fig. 8.244): Veins that pass with branches that ultimately originate from the maxillary artery drain into the pterygoid plexus of veins in the infratemporal fossa. Veins from anterior regions of the nasal cavities join the facial vein.

1	Veins from anterior regions of the nasal cavities join the facial vein. In some individuals, an additional nasal vein passes superiorly through a midline aperture (the foramen cecum), in the frontal bone anterior to the crista galli, and joins with the anterior end of the superior sagittal sinus. Because this nasal vein connects an intracranial venous sinus with extracranial veins, it is classified as an emissary vein. Emissary veins in general are routes by which infections can track from peripheral regions into the cranial cavity. Veins that accompany the anterior and posterior ethmoidal arteries are tributaries of the superior ophthalmic vein, which is one of the largest emissary veins and drains into the cavernous sinus on either side of the hypophyseal fossa. Nerves that innervate the nasal cavities (Fig. 8.245) are: the olfactory nerve [I] for olfaction, and branches of the ophthalmic [V1] and maxillary [V2] nerves for general sensation.

1	Nerves that innervate the nasal cavities (Fig. 8.245) are: the olfactory nerve [I] for olfaction, and branches of the ophthalmic [V1] and maxillary [V2] nerves for general sensation. Secretomotor innervation of mucous glands in the nasal cavities and paranasal sinuses is by parasympathetic fibers from the facial nerve [VII], which mainly join branches of the maxillary nerve [V2] in the pterygopalatine fossa. The olfactory nerve [I] is composed of axons from receptors in the olfactory epithelium at the top of each nasal cavity. Bundles of these axons pass superiorly through perforations in the cribriform plate to synapse with neurons in the olfactory bulb of the brain. Branches from the ophthalmic nerve [V1] Branches from the ophthalmic nerve [V1] that innervate the nasal cavity are the anterior and posterior ethmoidal nerves, which originate from the nasociliary nerve in the orbit.

1	The anterior ethmoidal nerve (Fig. 8.245) travels with the anterior ethmoidal artery and leaves the orbit through a canal between the ethmoidal labyrinth and the frontal bone. It passes through and supplies the adjacent ethmoidal cells and frontal sinus, and then enters the cranial cavity immediately lateral and superior to the cribriform plate. It then travels forward in a groove on the cribriform plate and enters the nasal cavity by descending through a slit-like foramen immediately lateral to the crista galli. It has branches to the medial and lateral wall of the nasal cavity and then continues forward on the undersurface of the nasal bone. It passes onto the external surface of the nose by traveling between the nasal bone and lateral nasal cartilage, and then terminates as the external nasal nerve, which supplies skin around the naris, in the nasal vestibule, and on the tip of the nose.

1	Like the anterior ethmoidal nerve, the posterior ethmoidal nerve leaves the orbit through a similar canal in the medial wall of the orbit. It terminates by supplying the mucosa of the ethmoidal cells and sphenoidal sinus and normally does not extend into the nasal cavity itself. Branches from the maxillary nerve [V2] A number of nasal branches from the maxillary nerve [V2] innervate the nasal cavity. Many of these nasal branches (Fig. 8.245) originate in the pterygopalatine fossa, which is just lateral to the lateral wall of the nasal cavity, and leave the fossa to enter the nasal cavity by passing medially through the sphenopalatine foramen or through smaller foramina in the lateral wall: A number of these nerves (posterior superior lateral nasal nerves) pass forward on and supply the lateral wall of the nasal cavity. Others (posterior superior medial nasal nerves) cross the roof to the nasal septum and supply both these regions.

1	Others (posterior superior medial nasal nerves) cross the roof to the nasal septum and supply both these regions. The largest of these nerves is the nasopalatine nerve, which passes forward and down the medial wall of the nasal cavity to pass through the incisive canal onto the roof of the oral cavity, and terminates by supplying the oral mucosa posterior to the incisor teeth. Other nasal nerves (posterior inferior nasal nerves) originate from the greater palatine nerve, descending from the pterygopalatine fossa in the palatine canal just lateral to the nasal cavity, and pass through small bony foramina to innervate the lateral wall of the nasal cavity. A small nasal nerve also originates from the anterior superior alveolar branch of the infra-orbital nerve and passes medially through the maxilla to supply the lateral wall near the anterior end of the inferior concha.

1	Secretomotor innervation of glands in the mucosa of the nasal cavity and paranasal sinuses is by preganglionic parasympathetic fibers carried in the greater petrosal branch of the facial nerve [VII]. These fibers enter the pterygopalatine fossa and synapse in the pterygopalatine ganglion (see Fig. 8.157 and pp. 986–987). Postganglionic parasympathetic fibers then join branches of the maxillary nerve [V2] to leave the fossa and ultimately reach target glands.

1	Sympathetic innervation, mainly involved with regulating blood flow in the nasal mucosa, is from spinal cord level T1. Preganglionic sympathetic fibers enter the sympathetic trunk and ascend to synapse in the superior cervical sympathetic ganglion. Postganglionic sympathetic fibers pass onto the internal carotid artery, enter the cranial cavity, and then leave the internal carotid artery to form the deep petrosal nerve, which joins the greater petrosal nerve of the facial nerve [VII] and enters the pterygopalatine fossa (see Figs. 8.156 and 8.157 and pp. 984–986). Like the parasympathetic fibers, the sympathetic fibers follow branches of the maxillary nerve [V2] into the nasal cavity. Lymph from anterior regions of the nasal cavities drains forward onto the face by passing around the margins of the nares (Fig. 8.246). These lymphatics ultimately connect with the submandibular nodes.

1	Lymph from posterior regions of the nasal cavity and the paranasal sinuses drains into upper deep cervical nodes. Some of this lymph passes first through the retropharyngeal nodes. The oral cavity is inferior to the nasal cavities (Fig. 8.247A). It has a roof and floor and lateral walls, opens onto the face through the oral fissure, and is continuous with the cavity of the pharynx at the oropharyngeal isthmus. The roof of the oral cavity consists of the hard and soft palates. The floor is formed mainly of soft tissues, which include a muscular diaphragm and the tongue. The lateral walls (cheeks) are muscular and merge anteriorly with the lips surrounding the oral fissure (the anterior opening of the oral cavity). The posterior aperture of the oral cavity is the oropharyngeal isthmus, which opens into the oral part of the pharynx.

1	The posterior aperture of the oral cavity is the oropharyngeal isthmus, which opens into the oral part of the pharynx. The oral cavity is separated into two regions by the upper and lower dental arches consisting of the teeth and alveolar bone that supports them (Fig. 8.247B): The outer oral vestibule, which is horseshoe shaped, is between the dental arches and the deep surfaces of the cheeks and lips—the oral fissure opens into it and can be opened and closed by muscles of facial expression, and by movements of the lower jaw. The inner oral cavity proper is enclosed by the dental arches. The degree of separation between the upper and lower arches is established by elevating or depressing the lower jaw (mandible) at the temporomandibular joint. The oropharyngeal isthmus at the back of the oral cavity proper can be opened and closed by surrounding soft tissues, which include the soft palate and tongue. The oral cavity has multiple functions:

1	The oral cavity has multiple functions: It is the inlet for the digestive system involved with the initial processing of food, which is aided by secretions from salivary glands. It manipulates sounds produced by the larynx and one outcome of this is speech. It can be used for breathing because it opens into the pharynx, which is a common pathway for food and air. For this reason, the oral cavity can be used by physicians to access the lower airway, and dentists use “rubber dams” to prevent debris such as tooth fragments from passing through the oropharyngeal isthmus and pharynx into either the esophagus or the lower airway. Multiple nerves innervate the oral cavity General sensory innervation is carried predominantly by branches of the trigeminal nerve [V]: The upper parts of the cavity, including the palate and the upper teeth, are innervated by branches of the maxillary nerve [V2].

1	The upper parts of the cavity, including the palate and the upper teeth, are innervated by branches of the maxillary nerve [V2]. The lower parts, including the teeth and oral part of the tongue, are innervated by branches of the mandibular nerve [V3]. Taste (special afferent [SA]) from the oral part or anterior two-thirds of the tongue is carried by branches of the facial nerve [VII], which join and are distributed with branches of the trigeminal nerve [V]. Parasympathetic fibers to the glands within the oral cavity are also carried by branches of the facial nerve [VII], which are distributed with branches of the trigeminal nerve [V]. Sympathetic fibers in the oral cavity ultimately come from spinal cord level T1, synapse in the superior cervical sympathetic ganglion, and are eventually distributed to the oral cavity along branches of the trigeminal nerve [V] or directly along blood vessels.

1	All muscles of the tongue are innervated by the hypoglossal nerve [XII], except the palatoglossus, which is innervated by the vagus nerve [X]. All muscles of the soft palate are innervated by the vagus nerve [X], except for the tensor veli palatini, which is innervated by a branch from the mandibular nerve [V3]. The muscle (mylohyoid) that forms the floor of the oral cavity is also innervated by the mandibular nerve [V3]. Bones that contribute to the skeletal framework of the oral cavity or are related to the anatomy of structures in the oral cavity include: the paired maxillae, palatine, and temporal bones, and the unpaired mandible, sphenoid, and hyoid bones. In addition, the cartilaginous parts of the pharyngotympanic tubes on the inferior aspect of the base of the skull are related to the attachment of muscles of the soft palate.

1	In addition, the cartilaginous parts of the pharyngotympanic tubes on the inferior aspect of the base of the skull are related to the attachment of muscles of the soft palate. The two maxillae contribute substantially to the architecture of the roof of the oral cavity. The parts involved are the alveolar and palatine processes (Fig. 8.248A). The palatine process is a horizontal shelf that projects from the medial surface of each maxilla. It originates just superior to the medial aspect of the alveolar process and extends to the midline where it is joined, at a suture, with the palatine process from the other side. Together, the two palatine processes form the anterior two-thirds of the hard palate.

1	In the midline on the inferior surface of the hard palate and at the anterior end of the intermaxillary suture is a single small fossa (incisive fossa) just behind the incisor teeth. Two incisive canals, one on each side, extend posterosuperiorly from the roof of this fossa to open onto the floor of the nasal cavity. The canals and fossae allow passage of the greater palatine vessels and the nasopalatine nerves. The parts of each L-shaped palatine bone that contribute to the roof of the oral cavity are the horizontal plate and the pyramidal process (Fig. 8.248A). The horizontal plate projects medially from the inferior aspect of the palatine bone and is joined by sutures to its partner in the midline and, on the same side, with the palatine process of the maxilla anteriorly.

1	A single posterior nasal spine is formed at the midline where the two horizontal plates join and projects backward from the margin of the hard palate. The posterior margin of the horizontal plates and the posterior nasal spine are associated with attachment of the soft palate. The greater palatine foramen, formed mainly by the horizontal plate of the palatine bone and completed laterally by the adjacent part of the maxilla, opens onto the posterolateral aspect of the horizontal plate. This foramen is the inferior opening of the palatine canal, which continues superiorly into the pterygopalatine fossa and transmits the greater palatine nerve and vessels to the palate. Also opening onto the palatine bone is the lesser palatine foramen. This foramen is the inferior opening of the short lesser palatine canal, which branches from the greater palatine canal and transmits the lesser palatine nerve and vessels to the soft palate.

1	The pyramidal process projects posteriorly and fills the space between the inferior ends of the medial and lateral plates of the pterygoid process of the sphenoid bone. The pterygoid processes and spines of the sphenoid bone are associated with structures related to the soft palate, which forms part of the roof of the oral cavity (Fig. 8.248A). The pterygoid processes descend, one on each side, from the lateral aspect of the body of the sphenoid bone. Each process has a medial and a lateral plate. These two vertically oriented plates project from the posterior aspect of the process. The V-shaped gap that occurs inferiorly between the two plates is filled by the pyramidal process of the palatine bone.

1	Projecting posterolaterally from the inferior margin of the medial plate of the pterygoid process is an elongate hook-shaped structure (the pterygoid hamulus). This hamulus is immediately behind the alveolar arch and inferior to the posterior margin of the hard palate. It is: a “pulley” for one of the muscles (tensor veli palatini) of the soft palate, and the attachment site for the upper end of the pterygomandibular raphe, which is attached below to the mandible and joins together the superior constrictor of the pharynx and the buccinator muscle of the cheek. At the root of the medial plate of the pterygoid process on the base of the skull is a small canoe-shaped fossa (scaphoid fossa), which begins just medial to the foramen ovale and descends anteriorly and medially to the root of the medial plate of the pterygoid process (Fig. 8.248A). This fossa is for the attachment of one of the muscles of the soft palate (tensor veli palatini).

1	The spines of the sphenoid, one on each side, are vertical projections from the inferior surfaces of the greater wings of the sphenoid bone (Fig. 8.248A). Each spine is immediately posteromedial to the foramen spinosum. The medial aspect of the spine provides attachment for the most lateral part of the tensor veli palatini muscle of the soft palate. The styloid process and inferior aspect of the petrous part of the temporal bone provide attachment for muscles associated with the tongue and soft palate, respectively. The styloid process projects anteroinferiorly from the underside of the temporal bone. It can be as long as 1 inch (2.5 cm) and points toward the lesser horn of the hyoid bone to which it is attached by the stylohyoid ligament (Fig. 8.248B). The root of the styloid process is immediately anterior to the stylomastoid foramen and lateral to the jugular foramen. The styloglossus muscle of the tongue attaches to the anterolateral surface of the styloid process.

1	The inferior aspect of the temporal bone has a triangular roughened area immediately anteromedial to the opening of the carotid canal (Fig. 8.248A). The levator veli palatini muscle of the soft palate is attached here. Cartilaginous part of the pharyngotympanic tube The trumpet-shaped cartilaginous part of the pharyngotympanic tube is in a groove between the anterior margin of the petrous part of the temporal bone and the posterior margin of the greater wing of the sphenoid (Fig. 8.248A). The medial and lateral walls of the cartilaginous part of the pharyngotympanic tube are formed mainly of cartilage, whereas the more inferolateral wall is more fibrous and is known as the membranous lamina. The apex of the cartilaginous part of the pharyngotympanic tube connects laterally to the opening of the bony part in the temporal bone.

1	The apex of the cartilaginous part of the pharyngotympanic tube connects laterally to the opening of the bony part in the temporal bone. The expanded medial end of the cartilaginous part of the pharyngotympanic tube is immediately posterior to the upper margin of the medial plate of the pterygoid process and opens into the nasopharynx. The cartilaginous part of the pharyngotympanic tube is lateral to the attachment of the levator veli palatini muscle and medial to the spine of the sphenoid. The tensor veli palatini muscle is attached, in part, to the membranous lamina. The mandible is the bone of the lower jaw (Fig. 8.249). It consists of a body of right and left parts, which are fused anteriorly in the midline (mandibular symphysis), and two rami. The site of fusion is particularly visible on the external surface of the bone as a small vertical ridge in the midline.

1	The upper surface of the body of the mandible bears the alveolar arch (Fig. 8.249B), which anchors the lower teeth, and on its external surface on each side is a small mental foramen (Fig. 8.249B). Posterior to the mandibular symphysis on the internal surface of the mandible are two pairs of small spines, one pair immediately above the other pair. These are the superior and inferior mental spines (superior and inferior genial spines) (Fig. 8.249A,C), and are attachment sites for a pair of muscles that pass into the tongue and a pair of muscles that connect the mandible to the hyoid bone. Extending from the midline and originating inferior to the mental spines is a raised line or ridge (the mylohyoid line) (Fig. 8.249C), which runs posteriorly and superiorly along the internal surface of each side of the body of the mandible to end just below the level of the last molar tooth.

1	Above the anterior one-third of the mylohyoid line is a shallow depression (the sublingual fossa) (Fig. 8.249C), and below the posterior two-thirds of the mylohyoid line is another depression (the submandibular fossa) (Fig. 8.249C). Between the last molar tooth and the mylohyoid line is a shallow groove for the lingual nerve. Immediately posterior to the last molar tooth on the medial upper surface of the body of the mandible is a small triangular depression (retromolar triangle) (Fig. 8.249A,C). The pterygomandibular raphe attaches just medial to the apex of this triangle and extends from here to the tip of the pterygoid hamulus above. The ramus of the mandible, one on each side, is quadrangular shaped and oriented in the sagittal plane. On the medial surface of the ramus is a large mandibular foramen for transmission of the inferior alveolar nerve and vessels (Fig. 8.249C).

1	The hyoid bone is a small U-shaped bone in the neck between the larynx and the mandible. It has an anterior body of hyoid bone and two large greater horns, one on each side, which project posteriorly and superiorly from the body (Fig. 8.250). There are two small conical lesser horns on the superior surface where the greater horns join with the body. The stylohyoid ligaments attach to the apices of the lesser horns. The hyoid bone is a key bone in the neck because it connects the floor of the oral cavity in front with the pharynx behind and the larynx below. Walls: the cheeks The walls of the oral cavity are formed by the cheeks. Each cheek consists of fascia and a layer of skeletal muscle sandwiched between skin externally and oral mucosa internally. The thin layer of skeletal muscle within the cheeks is principally the buccinator muscle.

1	The buccinator muscle is one of the muscles of facial expression (Fig. 8.251). It is in the same plane as the superior constrictor muscle of the pharynx. In fact, the posterior margin of the buccinator muscle is joined to the anterior margin of the superior constrictor muscle by the pterygomandibular raphe, which runs between the tip of the pterygoid hamulus of the sphenoid bone above and a roughened area of bone immediately behind the last molar tooth on the mandible below. The buccinator and superior constrictor muscles therefore provide continuity between the walls of the oral and pharyngeal cavities. The buccinator muscle, in addition to originating from the pterygomandibular raphe, also originates directly from the alveolar part of the mandible and alveolar process of the maxilla.

1	The buccinator muscle, in addition to originating from the pterygomandibular raphe, also originates directly from the alveolar part of the mandible and alveolar process of the maxilla. From its three sites of origin, the muscle fibers of the buccinator run forward to blend with those of the orbicularis oris muscle and to insert into the modiolus, which is a small button-shaped nodule of connective tissue at the interface between the muscles of the lips and cheeks on each side. The buccinator muscle holds the cheeks against the alveolar arches and keeps food between the teeth when chewing. The buccinator is innervated by the buccal branch of the facial nerve [VII]. General sensation from the skin and oral mucosa of the cheeks is carried by the buccal branch of the mandibular nerve [V3].

1	The floor of the oral cavity proper is formed mainly by three structures: a muscular diaphragm, which fills the U-shaped gap between the left and right sides of the body of the mandible and is composed of the paired mylohyoid muscles; two cord-like geniohyoid muscles above the diaphragm, which run from the mandible in front to the hyoid bone behind; and the tongue, which is superior to the geniohyoid muscles. Also present in the floor of the oral cavity proper are salivary glands and their ducts. The largest of these glands, on each side, are the sublingual gland and the oral part of the submandibular gland. The two thin mylohyoid muscles (Table 8.20), one on each side, together form a muscular diaphragm that defines the inferior limit of the floor of the oral cavity (Fig. 8.252A). Each muscle is triangular in shape with its apex pointed forward.

1	The lateral margin of each triangular muscle is attached to the mylohyoid line on the medial side of the body of the mandible. From here, the muscle fibers run slightly downward to the medial margin at the midline where the fibers are joined together with those of their partner muscle on the other side by a raphe. The raphe extends from the posterior aspect of the mandibular symphysis in front to the body of the hyoid bone behind. The posterior margin of each mylohyoid muscle is free except for a small medial attachment to the hyoid bone. The mylohyoid muscles: contribute structural support to the floor of the oral cavity, participate in elevating and pulling forward the hyoid bone, and therefore the attached larynx, during the initial stages of swallowing, and when the hyoid bone is fixed in position, depress the mandible and open the mouth.

1	Like the muscles of mastication, the mylohyoid muscles are innervated by the mandibular nerve [V3]. The specific branch that innervates the mylohyoid muscles is the nerve to the mylohyoid from the inferior alveolar nerve. The geniohyoid muscles (Table 8.20) are paired cord-like muscles that run, one on either side of the midline, from the inferior mental spines on the posterior surface of the mandibular symphysis to the anterior surface of the body of the hyoid bone (Fig. 8.252B,C). They are immediately superior to the mylohyoid muscles in the floor of the mouth and inferior to the genioglossus muscles that form part of the root of the tongue. The geniohyoid muscles: mainly pull the hyoid bone, and therefore the attached larynx, up and forward during swallowing; and because they pass posteroinferiorly from the mandible to the hyoid bone, when the hyoid bone is fixed, they can act with the mylohyoid muscles to depress the mandible and open the mouth.

1	Unlike other muscles that move the mandible at the temporomandibular joint, the geniohyoid muscles are innervated by a branch of cervical nerve C1, which “hitchhikes” from the neck along the hypoglossal nerve [XII] into the floor of the oral cavity. Gateway into the floor of the oral cavity In addition to defining the lower limit of the floor of the oral cavity, the free posterior border of the mylohyoid muscle on each side forms one of the three margins of a large triangular aperture (oropharyngeal triangle), which is a major route by which structures in the upper neck and infratemporal fossa of the head pass to and from structures in the floor of the oral cavity (Fig. 8.253). The other two muscles that complete the margins of the aperture are the superior and middle constrictor muscles of the pharynx.

1	Most structures that pass through the aperture are associated with the tongue and include muscles (hyoglossus, styloglossus), vessels (lingual artery and vein), nerves (lingual, hypoglossal [XII], glossopharyngeal [IX]), and lymphatics. A large salivary gland (the submandibular gland) is “hooked” around the free posterior margin of the mylohyoid muscle and therefore also passes through the opening. The tongue is a muscular structure that forms part of the floor of the oral cavity and part of the anterior wall of the oropharynx (Fig. 8.254A). Its anterior part is in the oral cavity and is somewhat triangular in shape with a blunt apex of the tongue. The apex is directed anteriorly and sits immediately behind the incisor teeth. The root of the tongue is attached to the mandible and the hyoid bone. The superior surface of the oral or anterior two-thirds of the tongue is oriented in the horizontal plane.

1	The superior surface of the oral or anterior two-thirds of the tongue is oriented in the horizontal plane. The pharyngeal surface or posterior one-third of the tongue curves inferiorly and becomes oriented more in the vertical plane. The oral and pharyngeal surfaces are separated by a V-shaped terminal sulcus of the tongue. This terminal sulcus forms the inferior margin of the oropharyngeal isthmus between the oral and pharyngeal cavities. At the apex of the V-shaped sulcus is a small depression (the foramen cecum of the tongue), which marks the site in the embryo where the epithelium invaginated to form the thyroid gland. In some people a thyroglossal duct persists and connects the foramen cecum on the tongue with the thyroid gland in the neck. The superior surface of the oral part of the tongue is covered by hundreds of papillae (Fig. 8.254B): Filiform papillae are small cone-shaped projections of the mucosa that end in one or more points.

1	Filiform papillae are small cone-shaped projections of the mucosa that end in one or more points. Fungiform papillae are rounder in shape and larger than the filiform papillae, and tend to be concentrated along the margins of the tongue. The largest of the papillae are the vallate papillae, which are blunt-ended cylindrical papillae invaginations in the tongue’s surface—there are only about 8 to 12 vallate papillae in a single V-shaped line immediately anterior to the terminal sulcus of the tongue. Foliate papillae are linear folds of mucosa on the sides of the tongue near the terminal sulcus of tongue. The papillae in general increase the area of contact between the surface of the tongue and the contents of the oral cavity. All except the filiform papillae have taste buds on their surfaces. Inferior surface of tongue

1	Inferior surface of tongue The undersurface of the oral part of the tongue lacks papillae, but does have a number of linear mucosal folds (see Fig. 8.265). A single median fold (the frenulum of the tongue) is continuous with the mucosa covering the floor of the oral cavity, and overlies the lower margin of a midline sagittal septum, which internally separates the right and left sides of the tongue. On each side of the frenulum is a lingual vein, and lateral to each vein is a rough fimbriated fold. The mucosa covering the pharyngeal surface of the tongue is irregular in contour because of the many small nodules of lymphoid tissue in the submucosa. These nodules are collectively the lingual tonsil. There are no papillae on the pharyngeal surface. The bulk of the tongue is composed of muscle (Fig. 8.254 and Table 8.21).

1	There are no papillae on the pharyngeal surface. The bulk of the tongue is composed of muscle (Fig. 8.254 and Table 8.21). The tongue is completely divided into left and right halves by a median sagittal septum composed of connective tissue. This means that all muscles of the tongue are paired. There are intrinsic and extrinsic lingual muscles. Except for the palatoglossus, which is innervated by the vagus nerve [X], all muscles of the tongue are innervated by the hypoglossal nerve [XII]. The intrinsic muscles of the tongue (Fig. 8.255) originate and insert within the substance of the tongue. They are divided into superior longitudinal, inferior longitudinal, transverse, and vertical muscles, and they alter the shape of the tongue by: lengthening and shortening it, curling and uncurling its apex and edges, and flattening and rounding its surface.

1	Working in pairs or one side at a time the intrinsic muscles of the tongue contribute to precision movements of the tongue required for speech, eating, and swallowing. Extrinsic muscles of the tongue (Fig. 8.255 and Table 8.21) originate from structures outside the tongue and insert into the tongue. There are four major extrinsic muscles on each side, the genioglossus, hyoglossus, styloglossus, and palatoglossus. These muscles protrude, retract, depress, and elevate the tongue. The thick fan-shaped genioglossus muscles make a substantial contribution to the structure of the tongue. They occur on either side of the midline septum that separates left and right halves of the tongue.

1	The genioglossus muscles originate from the superior mental spines on the posterior surface of the mandibular symphysis immediately superior to the origin of the geniohyoid muscles from the inferior mental spines (Fig. 8.256). From this small site of origin, each muscle expands posteriorly and superiorly. The most inferior fibers attach to the hyoid bone. The remaining fibers spread out superiorly to blend with the intrinsic muscles along virtually the entire length of the tongue. The genioglossus muscles: depress the central part of the tongue, and protrude the anterior part of the tongue out of the oral fissure (i.e., stick the tongue out). Like most muscles of the tongue, the genioglossus muscles are innervated by the hypoglossal nerves [XII].

1	Like most muscles of the tongue, the genioglossus muscles are innervated by the hypoglossal nerves [XII]. Asking a patient to “stick your tongue out” can be used as a test for the hypoglossal nerves [XII]. If the nerves are functioning normally, the tongue should protrude evenly in the midline. If the nerve on one side is not fully functional, the tip of the tongue will point to that side. The hyoglossus muscles are thin quadrangular muscles lateral to the genioglossus muscles (Fig. 8.257).

1	The hyoglossus muscles are thin quadrangular muscles lateral to the genioglossus muscles (Fig. 8.257). Each hyoglossus muscle originates from the entire length of the greater horn and the adjacent part of the body of the hyoid bone. At its origin from the hyoid bone, the hyoglossus muscle is lateral to the attachment of the middle constrictor muscle of the pharynx. The muscle passes superiorly and anteriorly through the gap (oropharyngeal triangle) between the superior constrictor, middle constrictor, and mylohyoid to insert into the tongue lateral to the genioglossus and medial to the styloglossus. The hyoglossus muscle depresses the tongue and is innervated by the hypoglossal nerve [XII]. An important landmark. The hyoglossus muscle is an important landmark in the floor of the oral cavity: The lingual artery from the external carotid artery in the neck enters the tongue deep to the hyoglossus, between the hyoglossus and genioglossus.

1	The lingual artery from the external carotid artery in the neck enters the tongue deep to the hyoglossus, between the hyoglossus and genioglossus. The hypoglossal nerve [XII] and lingual nerve (branch of the mandibular nerve [V3]), from the neck and infratemporal fossa of the head, respectively, enter the tongue on the external surface of the hyoglossus. The styloglossus muscles originate from the anterior surface of the styloid processes of the temporal bones. From here, each muscle passes inferiorly and medially through the gap (oropharyngeal triangle) between the middle constrictor, superior constrictor, and mylohyoid muscles to enter the lateral surface of the tongue where they blend with the superior margin of the hyoglossus and with the intrinsic muscles (Fig. 8.258). The styloglossus muscles retract the tongue and pull the back of the tongue superiorly. They are innervated by the hypoglossal nerves [XII].

1	The styloglossus muscles retract the tongue and pull the back of the tongue superiorly. They are innervated by the hypoglossal nerves [XII]. The palatoglossus muscles are muscles of the soft palate and the tongue. Each originates from the undersurface of the palatine aponeurosis and passes anteroinferiorly to the lateral side of the tongue (Fig. 8.259). The palatoglossus muscles: elevate the back of the tongue, move the palatoglossal arches of mucosa toward the midline, and depress the soft palate. These movements facilitate closing of the oropharyngeal isthmus and as a result separate the oral cavity from the oropharynx. Unlike other muscles of the tongue, but similar to most other muscles of the soft palate, the palatoglossus muscles are innervated by the vagus nerves [X]. The major artery of the tongue is the lingual artery (Fig. 8.260).

1	The major artery of the tongue is the lingual artery (Fig. 8.260). On each side, the lingual artery originates from the external carotid artery in the neck adjacent to the tip of the greater horn of the hyoid bone. It forms an upward bend and then loops downward and forward to pass deep to the hyoglossus muscle, and accompanies the muscle through the aperture (oropharyngeal triangle) formed by the margins of the mylohyoid, superior constrictor, and middle constrictor muscles, and enters the floor of the oral cavity. The lingual artery then travels forward in the plane between the hyoglossus and genioglossus muscles to the apex of the tongue. In addition to the tongue, the lingual artery supplies the sublingual gland, gingiva, and oral mucosa in the floor of the oral cavity. The tongue is drained by dorsal lingual and deep lingual veins (Fig. 8.260).

1	The tongue is drained by dorsal lingual and deep lingual veins (Fig. 8.260). The deep lingual veins are visible through the mucosa on the undersurface of the tongue. Although they accompany the lingual arteries in anterior parts of the tongue, they become separated from the arteries posteriorly by the hyoglossus muscles. On each side, the deep lingual vein travels with the hypoglossal nerve [XII] on the external surface of the hyoglossus muscle and passes out of the floor of the oral cavity through the aperture (oropharyngeal triangle) formed by the margins of the mylohyoid, superior constrictor, and middle constrictor muscles. It joins the internal jugular vein in the neck. The dorsal lingual vein follows the lingual artery between the hyoglossus and genioglossus muscles and, like the deep lingual vein, drains into the internal jugular vein in the neck. Innervation of the tongue is complex and involves a number of nerves (Figs. 8.260 and 8.261).

1	Innervation of the tongue is complex and involves a number of nerves (Figs. 8.260 and 8.261). Taste (SA) and general sensation from the pharyngeal part of the tongue are carried by the glossopharyngeal nerve [IX]. The glossopharyngeal nerve [IX] leaves the skull through the jugular foramen and descends along the posterior surface of the stylopharyngeus muscle. It passes around the lateral surface of the stylopharyngeus and then slips through the posterior aspect of the gap (oropharyngeal triangle) between the superior constrictor, middle constrictor, and mylohyoid muscles. The nerve then passes forward on the oropharyngeal wall just below the inferior pole of the palatine tonsil and enters the pharyngeal part of the tongue deep to the styloglossus and hyoglossus muscles. In addition to taste and general sensation on the posterior one-third of the tongue, branches creep anterior to the terminal sulcus of the tongue to carry taste (SA) and general sensation from the vallate papillae.

1	General sensory innervation from the anterior two-thirds or oral part of the tongue is carried by the lingual nerve, which is a major branch of the mandibular nerve [V3]. It originates in the infratemporal fossa and passes anteriorly into the floor of the oral cavity by passing through the gap (oropharyngeal triangle) between the mylohyoid, superior constrictor, and middle constrictor muscles (Fig. 8.262). As it travels through the gap, it passes immediately inferior to the attachment of the superior constrictor to the mandible and continues forward on the medial surface of the mandible adjacent to the last molar tooth and deep to the gingiva. In this position, the nerve can be palpated against the bone by placing a finger into the oral cavity. The lingual nerve then continues anteromedially across the floor of the oral cavity, loops under the submandibular duct, and ascends into the tongue on the external and superior surface of the hyoglossus muscle.

1	In addition to general sensation from the oral part of the tongue, the lingual nerve also carries general sensation from the mucosa on the floor of the oral cavity and gingiva associated with the lower teeth. The lingual nerve also carries parasympathetic and taste fibers from the oral part of the tongue that are part of the facial nerve [VII]. Taste (SA) from the oral part of the tongue is carried into the central nervous system by the facial nerve [VII]. Special sensory (SA) fibers of the facial nerve [VII] leave the tongue and oral cavity as part of the lingual nerve. The fibers then enter the chorda tympani nerve, which is a branch of the facial nerve [VII] that joins the lingual nerve in the infratemporal fossa (Fig. 8.262; also see p. 976). All muscles of the tongue are innervated by the hypoglossal nerve [XII] except for the palatoglossus muscle, which is innervated by the vagus nerve [X].

1	All muscles of the tongue are innervated by the hypoglossal nerve [XII] except for the palatoglossus muscle, which is innervated by the vagus nerve [X]. The hypoglossal nerve [XII] leaves the skull through the hypoglossal canal and descends almost vertically in the neck to a level just below the angle of the mandible (Fig. 8.263). Here it angles sharply forward around the sternocleidomastoid branch of the occipital artery, crosses the external carotid artery, and continues forward, crossing the loop of the lingual artery, to reach the external surface of the lower one-third of the hyoglossus muscle. The hypoglossal nerve [XII] follows the hyoglossus muscle through the gap (oropharyngeal triangle) between the superior constrictor, middle constrictor, and mylohyoid muscles to reach the tongue.

1	The hypoglossal nerve [XII] follows the hyoglossus muscle through the gap (oropharyngeal triangle) between the superior constrictor, middle constrictor, and mylohyoid muscles to reach the tongue. In the upper neck, a branch from the anterior ramus of C1 joins the hypoglossal nerve [XII]. Most of these C1 fibers leave the hypoglossal nerve [XII] as the superior root of the ansa cervicalis (Fig. 8.263). Near the posterior border of the hyoglossus muscle, the remaining fibers leave the hypoglossal nerve [XII] and form two nerves: the thyrohyoid branch, which remains in the neck to innervate the thyrohyoid muscle, and the branch to the geniohyoid, which passes into the floor of the oral cavity to innervate the geniohyoid. All lymphatic vessels from the tongue ultimately drain into the deep cervical chain of nodes along the internal jugular vein:

1	All lymphatic vessels from the tongue ultimately drain into the deep cervical chain of nodes along the internal jugular vein: The pharyngeal part of the tongue drains through the pharyngeal wall directly into mainly the jugulodigastric node of the deep cervical chain. The oral part of the tongue drains both directly into the deep cervical nodes, and indirectly into these nodes by passing first through the mylohyoid muscle and into submental and submandibular nodes. The submental nodes are inferior to the mylohyoid muscles and between the digastric muscles, while the submandibular nodes are below the floor of the oral cavity along the inner aspect of the inferior margins of the mandible. The tip of the tongue drains through the mylohyoid muscle into the submental nodes and then into mainly the jugulo-omohyoid node of the deep cervical chain.

1	The tip of the tongue drains through the mylohyoid muscle into the submental nodes and then into mainly the jugulo-omohyoid node of the deep cervical chain. Salivary glands are glands that open or secrete into the oral cavity. Most are small glands in the submucosa or mucosa of the oral epithelium lining the tongue, palate, cheeks, and lips, and open into the oral cavity directly or via small ducts. In addition to these small glands are much larger glands, which include the paired parotid, submandibular, and sublingual glands. The parotid gland (see pp. 900–901) on each side is entirely outside the boundaries of the oral cavity in a shallow triangular-shaped trench (Fig. 8.264) formed by: the sternocleidomastoid muscle behind, the ramus of the mandible in front, and superiorly, the base of the trench is formed by the external acoustic meatus and the posterior aspect of the zygomatic arch.

1	The gland normally extends anteriorly over the masseter muscle, and inferiorly over the posterior belly of the digastric muscle. The parotid duct passes anteriorly across the external surface of the masseter muscle and then turns medially to penetrate the buccinator muscle of the cheek and open into the oral cavity adjacent to the crown of the second upper molar tooth. The parotid gland encloses the external carotid artery, the retromandibular vein, and the origin of the extracranial part of the facial nerve [VII]. The elongate submandibular glands are smaller than the parotid glands but larger than the sublingual glands. Each is hook shaped (Fig. 8.265A,B):

1	The elongate submandibular glands are smaller than the parotid glands but larger than the sublingual glands. Each is hook shaped (Fig. 8.265A,B): The larger arm of the hook is directed forward in the horizontal plane below the mylohyoid muscle and is therefore outside the boundaries of the oral cavity—this larger superficial part of the gland is directly against a shallow impression on the medial side of the mandible (submandibular fossa) inferior to the mylohyoid line. The smaller arm of the hook (or deep part) of the gland loops around the posterior margin of the mylohyoid muscle to enter and lie within the floor of the oral cavity where it is lateral to the root of the tongue on the lateral surface of the hyoglossus muscle. The submandibular duct emerges from the medial side of the deep part of the gland in the oral cavity and passes forward to open on the summit of a small sublingual caruncle (papilla) beside the base of the frenulum of the tongue (Fig. 8.265C,D).

1	The lingual nerve loops under the submandibular duct, crossing first the lateral side and then the medial side of the duct, as the nerve descends anteromedially through the floor of the oral cavity and then ascends into the tongue. The sublingual glands are the smallest of the three major paired salivary glands. Each is almond shaped and is immediately lateral to the submandibular duct and associated lingual nerve in the floor of the oral cavity (Fig. 8.265). Each sublingual gland lies directly against the medial surface of the mandible where it forms a shallow groove (sublingual fossa) superior to the anterior one-third of the mylohyoid line. The superior margin of the sublingual gland raises an elongate fold of mucosa (sublingual fold), which extends from the posterolateral aspect of the floor of the oral cavity to the sublingual papilla beside the base of the frenulum of the tongue at the midline anteriorly (Fig. 8.265D).

1	The sublingual gland drains into the oral cavity via numerous small ducts (minor sublingual ducts), which open onto the crest of the sublingual fold. Occasionally, the more anterior part of the gland is drained by a duct (major sublingual duct) that opens together with the submandibular duct on the sublingual caruncle. Vessels that supply the parotid gland originate from the external carotid artery and from its branches that are adjacent to the gland. The submandibular and sublingual glands are supplied by branches of the facial and lingual arteries. Veins from the parotid gland drain into the external jugular vein, and those from the submandibular and sublingual glands drain into lingual and facial veins. Lymphatic vessels from the parotid gland drain into nodes that are on or in the gland. These parotid nodes then drain into superficial and deep cervical nodes.

1	Lymphatic vessels from the parotid gland drain into nodes that are on or in the gland. These parotid nodes then drain into superficial and deep cervical nodes. Lymphatics from the submandibular and sublingual glands drain mainly into submandibular nodes and then into deep cervical nodes, particularly the jugulo-omohyoid node. Parasympathetic innervation to all salivary glands in the oral cavity is by branches of the facial nerve [VII], which join branches of the maxillary [V2] and mandibular [V3] nerves to reach their target destinations. The parotid gland, which is entirely outside the oral cavity, receives its parasympathetic innervation from fibers that initially traveled in the glossopharyngeal nerve [IX], which eventually join a branch of the mandibular nerve [V3] in the infratemporal fossa (Fig. 8.266).

1	All salivary glands above the level of the oral fissure, as well as all mucus glands in the nose and the lacrimal gland in the orbit, are innervated by parasympathetic fibers carried in the greater petrosal branch of the facial nerve [VII] (Fig. 8.266). Preganglionic parasympathetic fibers carried in this nerve enter the pterygopalatine fossa and synapse with postganglionic parasympathetic fibers in the pterygopalatine ganglion formed around branches of the maxillary nerve [V2]. Postganglionic parasympathetic fibers join general sensory branches of the maxillary nerve, such as the palatine nerves, destined for the roof of the oral cavity, to reach their target glands. All glands below the level of the oral fissure, which include those small glands in the floor of the oral cavity, in the lower lip, and in the tongue, and the larger submandibular and sublingual glands, are innervated by parasympathetic fibers carried in the chorda tympani branch of the facial nerve [VII] (Fig. 8.266).

1	The chorda tympani joins the lingual branch of the mandibular nerve [V3] in the infratemporal fossa and passes with it into the oral cavity. On the external surface of the hyoglossus muscle, preganglionic parasympathetic fibers leave the inferior aspect of the lingual nerve to synapse with postganglionic parasympathetic fibers in the submandibular ganglion, which appears to hang off the lingual nerve (Fig. 8.267). Postganglionic parasympathetic fibers leave the ganglion and pass directly to the submandibular and sublingual glands while others hop back onto the lingual nerve and travel with branches of the lingual nerve to target glands. Sympathetic innervation to the salivary glands is from spinal cord level T1. Preganglionic sympathetic fibers enter the sympathetic trunk and ascend to synapse in the superior cervical sympathetic ganglion (Fig. 8.268). Postganglionic fibers hop onto adjacent blood vessels and nerves to reach the glands.

1	The roof of the oral cavity consists of the palate, which has two parts—an anterior hard palate and a posterior soft palate (Fig. 8.269). The hard palate separates the oral cavity from the nasal cavities. It consists of a bony plate covered above and below by mucosa: Above, it is covered by respiratory mucosa and forms the floor of the nasal cavities. Below, it is covered by a tightly bound layer of oral mucosa and forms much of the roof of the oral cavity (Fig. 8.269). The palatine processes of the maxillae form the anterior three-quarters of the hard palate. The horizontal plates of the palatine bones form the posterior one-quarter. In the oral cavity, the upper alveolar arch borders the hard palate anteriorly and laterally. Posteriorly, the hard palate is continuous with the soft palate.

1	The mucosa of the hard palate in the oral cavity possesses numerous transverse palatine folds (palatine rugae) and a median longitudinal ridge (palatine raphe), which ends anteriorly in a small oval elevation (incisive papilla). The incisive papilla (Fig. 8.269) overlies the incisive fossa formed between the horizontal plates of the maxillae immediately behind the incisor teeth. The soft palate (Fig. 8.269) continues posteriorly from the hard palate and acts as a valve that can be: depressed to help close the oropharyngeal isthmus, and elevated to separate the nasopharynx from the oropharynx. The soft palate is formed and moved by four muscles and is covered by mucosa that is continuous with the mucosa lining the pharynx and oral and nasal cavities. The small tear-shaped muscular projection that hangs from the posterior free margin of the soft palate is the uvula. Muscles of the soft palate

1	The small tear-shaped muscular projection that hangs from the posterior free margin of the soft palate is the uvula. Muscles of the soft palate Five muscles (Table 8.22) on each side contribute to the formation and movement of the soft palate. Two of these, the tensor veli palatini and levator veli palatini, descend into the palate from the base of the skull. Two others, the palatoglossus and palatopharyngeus, ascend into the palate from the tongue and pharynx, respectively. The last muscle, the musculus uvulae, is associated with the uvula. All muscles of the palate are innervated by the vagus nerve [X], except for the tensor veli palatini, which is innervated by the mandibular nerve [V3] (via the nerve to the medial pterygoid). Tensor veli palatini and the palatine aponeurosis The tensor veli palatini muscle is composed of two parts—a vertical muscular part and a more horizontal fibrous part, which forms the palatine aponeurosis (Fig. 8.270A).

1	The tensor veli palatini muscle is composed of two parts—a vertical muscular part and a more horizontal fibrous part, which forms the palatine aponeurosis (Fig. 8.270A). The vertical part of the tensor veli palatini is thin and triangular in shape with its base attached to the skull and its apex pointed inferiorly. The base is attached along an oblique line that begins medially at the scaphoid fossa near the root of the pterygoid process of the sphenoid bone and continues laterally along the membranous part of the pharyngotympanic tube to the spine of the sphenoid bone. The tensor veli palatini descends vertically along the lateral surface of the medial plate of the pterygoid process and pharyngeal wall to the pterygoid hamulus where the fibers converge to form a small tendon (Fig. 8.270A).

1	The tendon loops 90° medially around the pterygoid hamulus, penetrating the origin of the buccinator muscle as it does, and expands like a fan to form the fibrous horizontal part of the muscle. This fibrous part is continuous across the midline with its partner on the other side to form the palatine aponeurosis. The palatine aponeurosis is attached anteriorly to the margin of the hard palate, but is unattached posteriorly where it ends in a free margin. This expansive aponeurosis is the major structural element of the soft palate to which the other muscles of the palate attach. The tensor veli palatini: tenses (makes firm) the soft palate so that the other muscles attached to the palate can work more effectively, and opens the pharyngotympanic tube when the palate moves during yawning and swallowing as a result of its attachment superiorly to the membranous part of the pharyngotympanic tube.

1	The tensor veli palatini is innervated by the nerve to the medial pterygoid from the mandibular nerve [V3]. The levator veli palatini muscle originates from the base of the skull and descends to the upper surface of the palatine aponeurosis (Fig. 8.270B). On the skull, it originates from a roughened area on the petrous part of the temporal bone immediately anterior to the opening of the carotid canal. Some fibers also originate from adjacent parts of the pharyngotympanic tube. The levator veli palatini passes anteroinferiorly through fascia of the pharyngeal wall, passes medial to the pharyngotympanic tube, and inserts onto the palatine aponeurosis (Fig. 8.270B). Its fibers interlace at the midline with those of the levator veli palatini on the other side.

1	Unlike the tensor veli palatini muscles, the levator veli palatini muscles do not pass around each pterygoid hamulus, but course directly from the base of the skull to the upper surface of the palatine aponeurosis. Therefore, they are the only muscles that can elevate the palate above the neutral position and close the pharyngeal isthmus between the nasopharynx and oropharynx. The levator veli palatini is innervated by the vagus nerve [X] through the pharyngeal branch to the pharyngeal plexus. Clinically, the levator veli palatini can be tested by asking a patient to say “ah.” If the muscle on each side is functioning normally, the palate elevates evenly in the midline. If one side is not functioning, the palate deviates away from the abnormal side.

1	The palatopharyngeus muscle originates from the superior surface of the palatine aponeurosis and passes posterolaterally over its margin to descend and become one of the longitudinal muscles of the pharyngeal wall (Fig. 8.270C). It is attached to the palatine aponeurosis by two flat lamellae separated by the levator veli palatini muscle. The more anterior and lateral of these two lamellae is attached to the posterior margin of the hard palate as well as to the palatine aponeurosis. The two palatopharyngeus muscles, one on each side, underlie the palatopharyngeal arches on the oropharyngeal wall. The palatopharyngeal arches lie posterior and medial to the palatoglossal arches when viewed anteriorly through the oral cavity (Fig. 8.271). On each side, the palatine tonsil is between the palatopharyngeal and palatoglossal arches on the lateral oropharyngeal wall (Fig. 8.271A).

1	On each side, the palatine tonsil is between the palatopharyngeal and palatoglossal arches on the lateral oropharyngeal wall (Fig. 8.271A). The palatopharyngeus muscles: depress the palate and move the palatopharyngeal arches toward the midline like curtains—both these actions help close the oropharyngeal isthmus; and elevate the pharynx during swallowing. The palatopharyngeus is innervated by the vagus nerve [X] through the pharyngeal branch to the pharyngeal plexus. The palatoglossus muscle attaches to the inferior (oral) surface of the palatine aponeurosis and passes inferiorly and anteriorly into the lateral surface of the tongue (Fig. 8.272). The palatoglossus muscle underlies a fold of mucosa that arches from the soft palate to the tongue. These palatoglossal arches, one on each side, are lateral and anterior to the palatopharyngeal arches and define the lateral margins of the oropharyngeal isthmus (Fig. 8.271A).

1	The palatine tonsil is between the palatoglossal and palatopharyngeal arches on the lateral oropharyngeal wall (Figs. 8.271 and 8.272). The palatoglossus muscles depress the palate, move the palatoglossal arches toward the midline like curtains, and elevate the back of the tongue. These actions help close the oropharyngeal isthmus. The palatoglossus is innervated by the vagus nerve [X] through the pharyngeal branch to the pharyngeal plexus. The musculus uvulae originates from the posterior nasal spine on the posterior margin of the hard palate and passes directly posteriorly over the dorsal aspect of the palatine aponeurosis to insert into connective tissue underlying the mucosa of the uvula (Fig. 8.272). It passes between the two lamellae of the palatopharyngeus superior to the attachment of the levator veli palatini. Along the midline, the musculus uvulae blends with its partner on the other side.

1	The musculus uvulae elevates and retracts the uvula. This action thickens the central part of the soft palate and helps the levator veli palatini muscles close the pharyngeal isthmus between the nasopharynx and oropharynx. The musculus uvulae is innervated by the vagus nerve [X] through the pharyngeal branch to the pharyngeal plexus. Arteries of the palate include the greater palatine branch of the maxillary artery, the ascending palatine branch of the facial artery, and the palatine branch of the ascending pharyngeal artery. The maxillary, facial, and ascending pharyngeal arteries are all branches that arise in the neck from the external carotid artery (Fig. 8.273).

1	The ascending palatine artery of the facial artery ascends along the external surface of the pharynx. The palatine branch loops medially over the top of the superior constrictor muscle of the pharynx to penetrate the pharyngeal fascia with the levator veli palatini muscle and follow the levator veli palatini to the soft palate. The palatine branch of the ascending pharyngeal artery follows the same course as the palatine branch of the ascending palatine artery from the facial artery and may replace the vessel.

1	The palatine branch of the ascending pharyngeal artery follows the same course as the palatine branch of the ascending palatine artery from the facial artery and may replace the vessel. The greater palatine artery originates from the maxillary artery in the pterygopalatine fossa. It descends into the palatine canal where it gives origin to a small lesser palatine branch, and then continues through the greater palatine foramen onto the inferior surface of the hard palate (Fig. 8.274). The greater palatine artery passes forward on the hard palate and then leaves the palate superiorly through the incisive canal to enter the medial wall of the nasal cavity where it terminates. The greater palatine artery is the major artery of the hard palate. It also supplies palatal gingiva. The lesser palatine branch passes through the lesser palatine foramen just posterior to the greater palatine foramen, and contributes to the vascular supply of the soft palate.

1	Veins from the palate generally follow the arteries and ultimately drain into the pterygoid plexus of veins in the infratemporal fossa (Fig. 8.275; also see pp. 980–981), or into a network of veins associated with the palatine tonsil, which drain into the pharyngeal plexus of veins or directly into the facial vein. Lymphatic vessels from the palate drain into deep cervical nodes (Fig. 8.275). The palate is supplied by the greater and lesser palatine nerves and the nasopalatine nerve (Figs. 8.274 and 8.276). originate in the pterygopalatine fossa from the maxillary nerve [V2]. palate) fibers from a branch of the facial nerve [VII] join the nerves in the pterygopalatine fossa, as do the sympathetics (mainly to blood vessels) ultimately derived from the T1 spinal cord level. The greater and lesser palatine nerves descend through the pterygopalatine fossa and palatine canal to reach the palate (Fig. 8.276):

1	The greater and lesser palatine nerves descend through the pterygopalatine fossa and palatine canal to reach the palate (Fig. 8.276): The greater palatine nerve travels through the greater palatine foramen and turns anteriorly to supply the hard palate and gingiva as far as the first premolar. The lesser palatine nerve passes posteromedially to supply the soft palate. The nasopalatine nerve also originates in the pterygopalatine fossa, but passes medially into the nasal cavity. It continues medially over the roof of the nasal cavity to reach the medial wall, then anteriorly and obliquely down the wall to reach the incisive canal in the anterior floor, and descends through the incisive canal and fossa to reach the inferior surface of the hard palate (Fig. 8.276). The nasopalatine nerve supplies gingiva and mucosa adjacent to the incisors and canine.

1	The nasopalatine nerve supplies gingiva and mucosa adjacent to the incisors and canine. The oral fissure is the slit-like opening between the lips that connects the oral vestibule to the outside (Fig. 8.277). It can be opened and closed, and altered in shape by the movements of the muscles of facial expression associated with the lips and surrounding regions, and by movements of the lower jaw (mandible). The lips are entirely composed of soft tissues (Fig. 8.277B). They are lined internally by oral mucosa and covered externally by skin. Externally, there is an area of transition from the thicker skin that covers the face to the thinner skin that overlies the margins of the lips and continues as oral mucosa onto the deep surfaces of the lips. Blood vessels are closer to the surface in areas where the skin is thin and as a consequence there is a vermilion border that covers the margins of the lips.

1	Blood vessels are closer to the surface in areas where the skin is thin and as a consequence there is a vermilion border that covers the margins of the lips. The upper lip has a shallow vertical groove on its external surface (the philtrum) sandwiched between two elevated ridges of skin (Fig. 8.277A). The philtrum and ridges are formed embryologically by fusion of the medial nasal processes. On the inner surface of both lips, a fold of mucosa (the median labial frenulum) connects the lip to the adjacent gum. The lips enclose the orbicularis oris muscle, neurovascular tissues, and labial glands (Fig. 8.277B). The small pea-shaped labial glands are between the muscle tissue and the oral mucosa and open into the oral vestibule.

1	A number of muscles of facial expression control the shape and size of the oral fissure. The most important of these is the orbicularis oris muscle, which encircles the orifice and acts as a sphincter. A number of other muscles of facial expression blend into the orbicularis oris or other tissues of the lips and open or adjust the contours of the oral fissure. These include the buccinator, levator labii superioris, zygomaticus major and minor, levator anguli oris, depressor labii inferioris, depressor anguli oris, and platysma (see pp. 897–899). The oropharyngeal isthmus is the opening between the oral cavity and the oropharynx (see Fig. 8.271). It is formed: laterally by the palatoglossal arches; superiorly by the soft palate; and inferiorly by the sulcus terminalis of the tongue that divides the oral surface of the tongue (anterior two-thirds) from the pharyngeal surface (posterior one-third).

1	The oropharyngeal isthmus can be closed by elevation of the posterior aspect of the tongue, depression of the palate, and medial movement of the palatoglossal arches toward the midline. Medial movement of the palatopharyngeal arches medial and posterior to the palatoglossal arches is also involved in closing the oropharyngeal isthmus. By closing the oropharyngeal isthmus, food or liquid can be held in the oral cavity while breathing. The teeth are attached to sockets (alveoli) in two elevated arches of bone on the mandible below and the maxillae above (alveolar arches). If the teeth are removed, the alveolar bone is resorbed and the arches disappear. The gingivae (gums) are specialized regions of the oral mucosa that surround the teeth and cover adjacent regions of the alveolar bone. The different types of teeth are distinguished on the basis of morphology, position, and function (Fig. 8.278A).

1	The different types of teeth are distinguished on the basis of morphology, position, and function (Fig. 8.278A). In adults, there are 32 teeth, 16 in the upper jaw and 16 in the lower jaw. On each side in both maxillary and mandibular arches are two incisor, one canine, two premolar, and three molar teeth. The incisor teeth are the “front teeth” and have one root and a chisel-shaped crown, which “cuts.” The canine teeth are posterior to the incisors, are the longest teeth, have a crown with a single pointed cusp, and “grasp.” The premolar teeth (bicuspids) have a crown with two pointed cusps, one on the buccal (cheek) side of the tooth and the other on the lingual (tongue) or palatal (palate) side, generally have one root (but the upper first premolar next to the canine may have two), and “grind.” The molar teeth are behind the premolar teeth, have three roots and crowns with three to five cusps, and “grind.”

1	The molar teeth are behind the premolar teeth, have three roots and crowns with three to five cusps, and “grind.” Two successive sets of teeth develop in humans, deciduous teeth (“baby” teeth) (Fig. 8.278B) and permanent teeth (“adult” teeth). The deciduous teeth emerge from the gingivae at between six months and two years of age. Permanent teeth begin to emerge and replace the deciduous teeth at around age six years, and can continue to emerge into adulthood. The 20 deciduous teeth consist of two incisor, one canine, and two molar teeth on each side of the upper and lower jaws. These teeth are replaced by the incisor, canine, and premolar teeth of the permanent teeth. The permanent molar teeth erupt posterior to the deciduous molars and require the jaws to elongate forward to accommodate them. All teeth are supplied by vessels that branch either directly or indirectly from the maxillary artery (Fig. 8.279).

1	All teeth are supplied by vessels that branch either directly or indirectly from the maxillary artery (Fig. 8.279). All lower teeth are supplied by the inferior alveolar artery, which originates from the maxillary artery in the infratemporal fossa. The vessel enters the mandibular canal of the mandible, passes anteriorly in bone supplying vessels to the more posterior teeth, and divides opposite the first premolar into incisor and mental branches. The mental branch leaves the mental foramen to supply the chin, while the incisor branch continues in bone to supply the anterior teeth and adjacent structures. All upper teeth are supplied by anterior and posterior superior alveolar arteries.

1	All upper teeth are supplied by anterior and posterior superior alveolar arteries. The posterior superior alveolar artery originates from the maxillary artery just after the maxillary artery enters the pterygopalatine fossa and it leaves the fossa through the pterygomaxillary fissure. It descends on the posterolateral surface of the maxilla, branches, and enters small canals in the bone to supply the molar and premolar teeth. The anterior superior alveolar artery originates from the infra-orbital artery, which arises from the maxillary artery in the pterygopalatine fossa. The infra-orbital artery leaves the pterygopalatine fossa through the inferior orbital fissure and enters the inferior orbital groove and canal in the floor of the orbit. The anterior superior alveolar artery originates from the infra-orbital artery in the infra-orbital canal. It passes through bone and branches to supply the incisor and canine teeth.

1	The gingivae are supplied by multiple vessels and the source depends on which side of each tooth the gingiva is—the side facing the oral vestibule or cheek (vestibular or buccal side), or the side facing the tongue or palate (lingual or palatal side): Buccal gingiva of the lower teeth is supplied by branches from the inferior alveolar artery, whereas the lingual side is supplied by branches from the lingual artery of the tongue. Buccal gingiva of the upper teeth is supplied by branches of the anterior and posterior superior alveolar arteries. Palatal gingiva is supplied by branches from the nasopalatine (incisor and canine teeth) and greater palatine (premolar and molar teeth) arteries. Veins from the upper and lower teeth generally follow the arteries (Fig. 8.279).

1	Veins from the upper and lower teeth generally follow the arteries (Fig. 8.279). Inferior alveolar veins from the lower teeth, and superior alveolar veins from the upper teeth drain mainly into the pterygoid plexus of veins in the infratemporal fossa, although some drainage from the anterior teeth may be via tributaries of the facial vein. The pterygoid plexus drains mainly into the maxillary vein and ultimately into the retromandibular vein and jugular system of veins. In addition, small communicating vessels pass superiorly, from the plexus, and pass through small emissary foramina in the base of the skull to connect with the cavernous sinus in the cranial cavity. Infection originating in the teeth can track into the cranial cavity through these small emissary veins. Venous drainage from the teeth can also be via vessels that pass through the mental foramen to connect with the facial vein.

1	Venous drainage from the teeth can also be via vessels that pass through the mental foramen to connect with the facial vein. Veins from the gingivae also follow the arteries and ultimately drain into the facial vein or into the pterygoid plexus of veins. Lymphatic vessels from the teeth and gingivae drain mainly into submandibular, submental, and deep cervical nodes (Fig. 8.280). All nerves that innervate the teeth and gingivae are branches of the trigeminal nerve [V] (Figs. 8.281 and 8.282). The lower teeth are all innervated by branches from the inferior alveolar nerve, which originates in the infratemporal fossa from the mandibular nerve [V3] (Figs. 8.281 and 8.282). The inferior alveolar nerve and its accompanying vessels enter the mandibular foramen on the medial surface of the ramus of the mandible and travel anteriorly through the bone in the mandibular canal. Branches to the back teeth originate directly from the inferior alveolar nerve.

1	Adjacent to the first premolar tooth, the inferior alveolar nerve divides into incisive and mental branches: The incisive branch innervates the first premolar, the canine, and the incisor teeth, together with the associated vestibular (buccal) gingiva. The mental nerve exits the mandible through the mental foramen and innervates the chin and lower lip. Anterior, middle, and posterior superior All upper teeth are innervated by the anterior, middle, and posterior superior alveolar nerves, which originate directly or indirectly from the maxillary nerve [V2] (Figs. 8.281 and 8.282).

1	All upper teeth are innervated by the anterior, middle, and posterior superior alveolar nerves, which originate directly or indirectly from the maxillary nerve [V2] (Figs. 8.281 and 8.282). The posterior superior alveolar nerve originates directly from the maxillary nerve [V2] in the pterygopalatine fossa, exits the pterygopalatine fossa through the pterygomaxillary fissure, and descends on the posterolateral surface of the maxilla. It enters the maxilla through a small foramen approximately midway between the pterygomaxillary fissure and the last molar tooth, and passes through the bone in the wall of the maxillary sinus. The posterior superior alveolar nerve then innervates the molar teeth through the superior alveolar plexus formed by the posterior, middle, and anterior alveolar nerves. The middle and anterior superior alveolar nerves originate from the infra-orbital branch of the maxillary nerve [V2] in the floor of the orbit:

1	The middle and anterior superior alveolar nerves originate from the infra-orbital branch of the maxillary nerve [V2] in the floor of the orbit: The middle superior alveolar nerve arises from the infra-orbital nerve in the infra-orbital groove, passes through the bone in the lateral wall of the maxillary sinus, and innervates the premolar teeth via the superior alveolar plexus. The anterior superior alveolar nerve originates from the infra-orbital nerve in the infra-orbital canal, passes through the maxilla in the anterior wall of the maxillary sinus, and via the superior alveolar plexus, supplies the canine and incisor teeth. Innervation of gingivae Like the teeth, the gingivae are innervated by nerves that ultimately originate from the trigeminal nerve [V] (Fig. 8.282): Gingiva associated with the upper teeth is innervated by branches derived from the maxillary nerve [V2]. Gingiva associated with the lower teeth is innervated by branches of the mandibular nerve [V3].

1	Gingiva associated with the lower teeth is innervated by branches of the mandibular nerve [V3]. The gingiva on the buccal side of the upper teeth is innervated by the anterior, middle, and superior alveolar nerves, which also innervate the adjacent teeth. Gingiva on the palatal (lingual) side of the same teeth is innervated by the nasopalatine and the greater palatine nerves: The nasopalatine nerve innervates gingiva associated with the incisor and canine teeth. The greater palatine nerve supplies gingiva associated with the remaining teeth. The gingiva associated with the (buccal) side of the mandibular incisor, canine, and premolar teeth is innervated by the mental branch of the inferior alveolar nerve. Gingiva on the buccal side of the mandibular molar teeth is innervated by the buccal nerve, which originates in the infratemporal fossa from the mandibular nerve [V3]. Gingiva adjacent to the lingual surface of all lower teeth is innervated by the lingual nerve.

1	Skeletal landmarks in the head and neck are used for locating major blood vessels, glands, and muscles, and for locating points of access to the airway. Neurological examination of the cranial and upper cervical nerves is carried out by assessing function in the head and neck. In addition, information about the general status of body health can often be obtained by evaluating surface features, the eye and the oral cavity, and the characteristics of speech. Anatomical position of the head The head is in the anatomical position when the inferior margins of the bony orbits and the superior margins of the external acoustic meatuses are in the same horizontal plane (Frankfort plane). In addition to the external acoustic meatus and the bony margin of the orbit, other features that are palpable include the head of the mandible, zygomatic arch, zygomatic bone, mastoid process, and external occipital protuberance (Fig. 8.283).

1	The head of the mandible is anterior to the external ear and behind and inferior to the posterior end of the zygomatic arch. It is best found by opening and closing the jaw and palpating the head of the mandible as it moves forward onto the articular tubercle and then back into the mandibular fossa, respectively. The zygomatic arch extends forward from the region of the temporomandibular joint to the zygomatic bone, which forms a bony prominence lateral to the inferior margin of the anterior opening of the orbit. The mastoid process is a large bony protuberance that is easily palpable posterior to the inferior aspect of the external acoustic meatus. The superior end of the sternocleidomastoid muscle attaches to the mastoid process. The external occipital protuberance is palpable in the midline posteriorly where the contour of the skull curves sharply forward. This landmark marks the point superficially where the back of the neck joins the head.

1	Another clinically useful feature of the head is the vertex. This is the highest point of the head in the anatomical position and marks the approximate point on the scalp where there is a transition from cervical to cranial innervation of the scalp. Anterior to the vertex, the scalp and face are innervated by the trigeminal nerve [V]. Posterior to the vertex, the scalp is innervated by branches from cervical spinal nerves. Visualizing structures at the CIII/CIV and CVI vertebral levels Two vertebral levels in the neck are associated with important anatomical features (Fig. 8.284). The intervertebral disc between the CIII and CIV vertebrae is in the same horizontal plane as the bifurcation of the common carotid artery into the internal and external carotid arteries. This level is approximately at the upper margin of the thyroid cartilage.

1	Vertebral level CVI marks the transition from pharynx to esophagus and larynx to trachea. The CVI vertebral level therefore marks the superior ends of the esophagus and trachea and is approximately at the level of the inferior margin of the cricoid cartilage. How to outline the anterior and posterior triangles of the neck The boundaries of the anterior and posterior triangles on each side of the neck are easily established using readily visible bony and muscular landmarks (Fig. 8.285). The base of each anterior triangle is the inferior margin of the mandible, the anterior margin is the midline of the neck, and the posterior margin is the anterior border of the sternocleidomastoid muscle. The apex of each anterior triangle points inferiorly and is at the suprasternal notch.

1	The anterior triangles are associated with structures such as the airway and digestive tract, and nerves and vessels that pass between the thorax and head. They are also associated with the thyroid and parathyroid glands. The base of each posterior triangle is the middle one-third of the clavicle. The medial margin is the posterior border of the sternocleidomastoid muscle, and the lateral margin is the anterior border of the trapezius muscle. The apex points superiorly and is immediately posteroinferior to the mastoid process. The posterior triangles are associated with nerves and vessels that pass into and out of the upper limbs. How to locate the cricothyroid ligament An important structure to locate in the neck is the median cricothyroid ligament (Fig. 8.286) because artificial penetration of this membrane in emergency situations can provide access to the lower airway when the upper airway above the level of the vocal folds is blocked.

1	The ligament can be easily found using palpable features of the larynx as landmarks. Using a finger to gently feel laryngeal structures in the midline, first find the thyroid notch in the superior margin of the thyroid cartilage and then move the finger inferiorly over the laryngeal prominence and down the anterior surface of the thyroid angle. As the finger crosses the inferior margin of the thyroid cartilage in the midline, a soft depression is felt before the finger slides onto the arch of the cricoid cartilage, which is hard. The soft depression between the lower margin of the thyroid cartilage and the arch of the cricoid is the position of the median cricothyroid ligament. A tube passed through the median cricothyroid ligament enters the airway just inferior to the position of the vocal folds of the larynx.

1	A tube passed through the median cricothyroid ligament enters the airway just inferior to the position of the vocal folds of the larynx. Structures that may occur in or cross the midline between the skin and the median cricothyroid ligament include the pyramidal lobe of the thyroid gland and small vessels, respectively. Inferior to the cricoid cartilage, the upper cartilage of the larynx can sometimes be palpated above the level of the isthmus of the thyroid gland that crosses the trachea anteriorly. The landmarks used for finding the cricothyroid ligament are similar in men and women; however, because the laminae of the thyroid cartilage meet at a more acute angle in men, the structures are more prominent in men than in women. How to find the thyroid gland

1	How to find the thyroid gland The left and right lobes of the thyroid gland are in the anterior triangles in the lower neck on either side of the airway and digestive tract inferior to the position of the oblique line of the thyroid cartilage (Fig. 8.287). In fact, the sternothyroid muscles, which attach superiorly to the oblique lines, lie anterior to the lobes of the thyroid gland and prevent the lobes from moving upward in the neck. The lobes of the thyroid gland can be most easily palpated by finding the thyroid prominence and arch of the cricoid cartilage and then feeling posterolateral to the larynx. The isthmus of the thyroid gland crosses anterior to the upper end of the trachea and can be easily palpated in the midline inferior to the arch of the cricoid.

1	The isthmus of the thyroid gland crosses anterior to the upper end of the trachea and can be easily palpated in the midline inferior to the arch of the cricoid. The presence of the isthmus of the thyroid gland makes palpating the tracheal cartilages difficult in the neck. Also, the presence of the isthmus of the thyroid gland and the associated vessels found in and crossing the midline makes it difficult to artificially enter the airway anteriorly through the trachea. This procedure, a tracheostomy, is a surgical procedure. Estimating the position of the middle The middle meningeal artery (Fig. 8.288) is a branch of the maxillary artery in the infratemporal fossa. It enters the skull through the foramen spinosum and is within the dura mater lining the cranial cavity. In lateral blows to the head the middle meningeal artery can be ruptured, leading to extradural hemorrhage and eventual death if not treated.

1	In lateral blows to the head the middle meningeal artery can be ruptured, leading to extradural hemorrhage and eventual death if not treated. The anterior branch of the middle meningeal artery is the part of the vessel most often torn. This branch is in the temple region of the head, approximately midway between the superior margin of the orbit and the upper part of the external ear in the pterion region. The pterion is a small circular area enclosing the region where the sphenoid, frontal, parietal, and temporal bones of the skull come together. Lateral blows to the head can fracture the internal table of bone of the skull and tear the middle meningeal artery in the outer layer of dura mater that is fused to the cranium. Blood under pulsatile arterial pressure leaks out of the vessel and gradually separates the dura from the bone, forming a progressively larger extradural hematoma. Major features of the face

1	Major features of the face The major features of the face are those related to the anterior openings of the orbit, the nasal cavities, and the oral cavity (Fig. 8.289). The palpebral fissures are between the upper and lower eyelids and can be opened and closed. The oral fissure is the gap between the upper and lower lips and can also be opened and closed. The sphincter muscles of the oral and palpebral fissures are the orbicularis oris and orbicularis oculi muscles, respectively. These muscles are innervated by the facial nerve [VII]. The nares are the anterior apertures of the nasal cavities and are continuously open. The vertical groove in the midline between the external nose and the upper lip is the philtrum.

1	The nares are the anterior apertures of the nasal cavities and are continuously open. The vertical groove in the midline between the external nose and the upper lip is the philtrum. Sensory innervation of the face is carried by the trigeminal nerve [V]. The three divisions of this nerve are represented on the face and can be tested by touching the forehead (the ophthalmic nerve [V1]), the anterior cheek (the maxillary nerve [V2]), and skin over the anterior body of the mandible (the mandibular nerve [V3]). The eye and lacrimal apparatus Major features of the eye include the sclera, cornea, iris, and pupil (Fig. 8.290). The cornea is continuous with the sclera and is the clear circular region of the external covering of the eye through which the pupil and iris are visible. The sclera is not transparent and is normally white.

1	The upper and lower eyelids of each eye enclose between them the palpebral fissure. The eyelids come together at the medial and lateral palpebral commissures on either side of each eye. At the medial side of the palpebral fissure and lateral to the medial palpebral commissure is a small triangular soft tissue structure (the lacrimal lake). The elevated mound of tissue on the medial side of the lacrimal lake is the lacrimal caruncle, and the lateral margin overlying the sclera is the lacrimal fold. The lacrimal apparatus consists of the lacrimal gland and the system of ducts and channels that collects the tears and drain them into the nasal cavity. Tears hydrate and maintain the transparency of the cornea.

1	The lacrimal gland is associated with the upper eyelid and is in a small depression in the lateral roof of the orbit just posterior to the orbital margin. The multiple small ducts of the gland open into the upper margin of the conjunctival sac, which is the thin gap between the deep surface of the eyelid and the cornea. Tears are swept medially over the eye by blinking and are collected in small openings (lacrimal puncta), one on each of the upper and lower eyelids near the lacrimal lake. Each punctum is on a small raised mound of tissue (a lacrimal papilla), and is the opening of a small canal (lacrimal canaliculus) that connects with the lacrimal sac. The lacrimal sac is in the lacrimal fossa on the medial side of the orbit. From the lacrimal sac, tears drain via the nasolacrimal duct into the nasal cavity.

1	The lacrimal sac is in the lacrimal fossa on the medial side of the orbit. From the lacrimal sac, tears drain via the nasolacrimal duct into the nasal cavity. The external ear (Fig. 8.291) consists of the auricle and the external acoustic meatus. The auricle is supported by cartilage and is covered by skin. The external acoustic meatus is near the anterior margin of the auricle. The auricle is characterized by a number of depressions, eminences, and folds. The folded outer margin of the auricle is the helix, which ends inferiorly as the lobule. A smaller fold (the antihelix) parallels the contour of the helix and is separated from it by a depression (the scaphoid fossa). The tragus is a small eminence anteroinferior to the external acoustic meatus. Opposite the tragus and at the end of the antihelix is another eminence (the antitragus). The depression between the tragus and antitragus is the intertragic incisure.

1	The deepest depression (the concha) is bracketed by the antihelix and leads into the external acoustic meatus. Other depressions include the triangular fossa and the cymba conchae. Arterial pulses can be felt at four locations in the head and neck (Fig. 8.292). Carotid pulse—the common or external carotid artery can be palpated in the anterior triangle of the neck. This is one of the strongest pulses in the body. The pulse can be obtained by palpating either the common carotid artery posterolateral to the larynx or the external carotid artery immediately lateral to the pharynx midway between the superior margin of the thyroid cartilage below and the greater horn of the hyoid bone above. Facial pulse—the facial artery can be palpated as it crosses the inferior border of the mandible immediately adjacent to the anterior margin of the masseter muscle.

1	Facial pulse—the facial artery can be palpated as it crosses the inferior border of the mandible immediately adjacent to the anterior margin of the masseter muscle. Temporal pulse—the superficial temporal artery can be palpated anterior to the ear and immediately posterosuperior to the position of the temporomandibular joint. Temporal pulse—the anterior branch of the superficial temporal artery can be palpated posterior to the zygomatic process of the frontal bone as it passes lateral to the temporal fascia and into anterolateral regions of the scalp. In some individuals pulsations of the superficial temporal artery can be seen through the skin. Fig. 8.1 Major compartments of the head and neck. Fig. 8.2 Areas of transition from one compartment of the head to another. Infratemporal fossaLateral plate ofpterygoid processMandibular nerve [V3]Pterygopalatine fossaMaxillary nerve [V2]Ramus of mandible Fig. 8.3 Muscles of the face. Fig. 8.4 Boundaries of the neck.

1	Infratemporal fossaLateral plate ofpterygoid processMandibular nerve [V3]Pterygopalatine fossaMaxillary nerve [V2]Ramus of mandible Fig. 8.3 Muscles of the face. Fig. 8.4 Boundaries of the neck. Vertebra CVIIMandibleMastoid processSuperior nuchal lineAcromionManubrium of sternumClavicle Fig. 8.5 Major compartments of the neck. Fig. 8.6 Specialized structures of the neck. A. Conceptual view. B. Anatomical view. Fig. 8.7 Skull. A. Bones. B. Sutures. C. Fontanelles and lambdoid suture. Fig. 8.8 Cervical vertebrae. A. Typical features. B. Atlas—vertebra CI (superior view). C. Axis—vertebra CII (anterior view). D. Atlas and axis (anterolateral view). E. Atlanto-occipital joint (posterior view).

1	Foramen transversariumAnterior tuberclePosterior tubercleArchSuperior articular facetBodyTransverse processSpinous processDensAnterior archArticular facetfor densLateral massSuperior articular surface (for occipital condyle)Posterior archBodyAtlas (CI)Axis (CII)Articular facetfor densABCDForamenmagnumOccipital condyleOccipital boneApical ligamentof densSuperior longitudinal bandof cruciform ligamentInferior longitudinal bandof cruciform ligamentTransverseligament of atlasAlar ligamentsETectorial membrane (upper partof posterior longitudinal ligament)Posteriorlongitudinalligament Fig. 8.9 Hyoid. A. Bone. B. Attachments. ABLesser hornGreater hornBody of hyoid boneFloor of mouth (mylohyoid muscle)Thyrohyoid membraneStylohyoid ligamentMiddle pharyngeal constrictor muscleInferior pharyngeal constrictor muscleEpiglottis Fig. 8.10 Soft palate. A. Position. B. Muscles. Fig. 8.11 Superior thoracic aperture and axillary inlets. Fig. 8.12 Important vertebral levels—CIII/CIV and CV/CVI.

1	Fig. 8.10 Soft palate. A. Position. B. Muscles. Fig. 8.11 Superior thoracic aperture and axillary inlets. Fig. 8.12 Important vertebral levels—CIII/CIV and CV/CVI. Fig. 8.13 Larynx and associated structures in the neck. Fig. 8.14 Cranial nerves and parasympathetic innervation. Fig. 8.15 Cervical nerves. A. Structure. B. Dermatomes. Phrenic nerveBrachial plexus(C5 to T1)Cervical plexus(C1 to C4)Cutaneous nervesAnsa cervicalis tostrap musclesAC3C4C2C2C3C4Ophthalmic nerve [V1]Trigeminal nerve [V]Maxillary nerve [V2]Mandibular nerve [V3]Anterior rami (C2 to C4) ClavicleAcromionPosterior rami (C2 to C4)External occipital protuberanceB Fig. 8.16 Larynx, soft palate, epiglottis, and oropharyngeal isthmus. A. Overall design. B. Normal breathing. C. Breathing with food or liquid in the oral cavity. D. Swallowing. E. In a newborn child.

1	B. Normal breathing. C. Breathing with food or liquid in the oral cavity. D. Swallowing. E. In a newborn child. Soft palate(opens and closesoropharyngeal isthmus)Cranial cavityEarsChoanaeNasopharynxCVI vertebral levelPharynxEsophagusScapulaAxillary inletClavicleSuperior thoracic aperture(thoracic inlet)Manubrium of sternumTracheaVocal folds(together with other soft tissuestructures open and closecavity of larynx)Epiglottis(opens and closes laryngeal inlet)Oropharyngeal isthmusOral cavityNasal cavitiesOrbitsARib IVertebra TIOropharynxLaryngopharynxLaryngeal inletLarynx Laryngeal inletand laryngealcavity openBack oftongue elevated,palate depressedOropharyngealisthmus closedLarynx andhyoid pulledup and forwardresulting in openingthe esophagusEpiglottisclosed overlaryngeal inletOropharyngealisthmus openOpening between nasal andoral parts of pharynx closedby soft palateSoft palatein neutral positionMilk pathwayTracheaNasal cavityEEsophagusSoft palateLaryngealinletBCD

1	Fig. 8.17 Anterior and posterior triangles of neck. Fig. 8.18 Anterior view of the skull. GlabellaNasal boneFrontal boneSuperciliary archSupra-orbital notch(foramen)Zygomatic process(of frontal bone)Zygomatic boneFrontal process (of maxilla)Infra-orbital foramenInferior nasal conchaMaxillaOblique lineMandibleMental foramenMental tubercleMental protuberanceAngle of mandibleAlveolar part of mandibleZygomatic process (of maxilla)NasionNasal crestPiriform apertureAlveolar processRamus of mandibleBody of mandibleAnterior nasal spine Fig. 8.19 Lateral view of the skull.

1	Fig. 8.19 Lateral view of the skull. Sphenoparietal sutureCoronal sutureFrontal bonePterionSphenosquamous sutureGreater wing(of sphenoid bone)ZygomaticofacialforamenZygomatic boneMaxillaMental foramenBody of mandibleTemporal process (of zygomatic bone)Alveolar part(of mandible)Condylar processAngleZygomatic process (of temporal bone)Coronoid process Ramus of mandibleStyloid processMastoid processTympanic part (of temporal bone)Mastoid part of temporal boneOccipitomastoidsutureOccipital boneAsterionLambdoidsutureParietomastoidsutureParietal boneSquamous sutureSquamous part (of temporal bone)Nasal boneLacrimal boneZygomaticotemporalforamen(on deep surface ofzygomatic bone) Fig. 8.20 Posterior view of the skull. Fig. 8.21 Superior view of the skull. Fig. 8.22 Calvaria. Fig. 8.23 Inferior view of the skull.

1	Fig. 8.20 Posterior view of the skull. Fig. 8.21 Superior view of the skull. Fig. 8.22 Calvaria. Fig. 8.23 Inferior view of the skull. Incisive fossaHard palate (maxilla)Hard palate (palatine bone)Greater palatine foramenHamulusLesser palatine foramenLateral plate of pterygoidprocessMedial plate of pterygoidprocessVomerBody of sphenoidArticular tubercleMandibular fossaForamen ovaleForamen spinosumPetrous part oftemporal boneSquamous part oftemporal boneStyloid processStylomastoid foramenJugular foramenCarotid canalInferior nuchal lineOccipital condyleExternal occipital protuberanceSuperior nuchal lineExternal occipital crestForamen magnumPharyngeal tubercleHypoglossal canalMastoid processMastoid notchBasilar part of occipital boneForamen lacerumGroove for auditory tubeOpening of pterygoid canalPterygoid processScaphoid fossaPterygoid fossaGreater wing (of sphenoid bone)Posterior nasal aperture (choana)Pyramidal process of palatine boneAlveolar archPosterior nasal spine

1	Fig. 8.24 Roof of the cranial cavity. Frontal boneFrontal crestGroove for superiorsagittal sinusBregmaGranular foveolaeSagittal sutureLambdoid sutureOccipital boneLambdaParietal boneGrooves for middlemeningeal arteryCoronal sutureGroove for anterior branch of middle meningeal artery Fig. 8.25 Anterior cranial fossa. Foramen cecumForamina of cribriform plateBody of (sphenoid)Frontal crestOrbital part (of frontal bone)Crista galliCribriform plate (of ethmoid bone)Lesser wing (of sphenoid)Anterior clinoid process Fig. 8.26 Middle cranial fossa. Optic canalSuperior orbital fissureGreater wing (of sphenoid)Foramen rotundumGroove for middlemeningeal arteryForamen ovaleForamen spinosumForamen lacerumTegmen tympaniDorsum sellaeMiddle clinoid processPrechiasmatic sulcusTuberculum sellaeHypophyseal fossaPosterior clinoid processGroove and hiatus for lesser petrosal nerveGroove and hiatus for greater petrosal nerveArcuate eminenceTrigeminal impressionOpening of carotid canal

1	Fig. 8.27 Posterior cranial fossa. Superior border of petrous part of temporal boneInternal acoustic meatusJugular foramenHypoglossal canalForamen magnumClivusJugular tubercleGroove for sigmoid sinusGroove for inferior petrosal sinusGroove for transverse sinusInternal occipital crestInternal occipital protuberance Fig. 8.28 Summary of foramina and fissures through which major structures enter and leave the cranial cavity. A. Floor of cranial cavity. Also indicated are the regions between which each foramen or fissure communicates. B. Inferior aspect of cranium.

1	Foramen ovale:• [V3] Mandibular division of [V] (trigeminal nerve)Jugular foramen:• [IX] Glossopharyngeal nerve• [X] Vagus nerve• [XI] Accessory nerve• Internal jugular veinForamen magnum:• Spinal cord• Vertebral arteries Roots of accessory nerve [XI] pass from upper region of spinal cord through the foramen magnum into the cranial cavity and then leave the cranial cavity through the jugular foramenForamen spinosum:• Middle meningeal arteryStylomastoid foramen:• [VII] Facial nerveCarotid canal:• Internal carotid arteryHypoglossal canal:• [XII] Hypoglossal nerveCribriform plate: (anterior cranial fossa/nasal cavity)• [I] Olfactory nerves Optic canal:(middle cranial fossa/orbit)• [II] Optic nerve• Ophthalmic arterySuperior orbital fissure:(middle cranial fossa/orbit)• [V1] Ophthalmic division of [V] (trigeminal nerve)• [III] Oculomotor nerve• [IV] Trochlear nerve• [VI] Abducent nerve• Superior ophthalmic veinForamen rotundum:(middle cranial fossa/pterygopalatine

1	[V1] Ophthalmic division of [V] (trigeminal nerve)• [III] Oculomotor nerve• [IV] Trochlear nerve• [VI] Abducent nerve• Superior ophthalmic veinForamen rotundum:(middle cranial fossa/pterygopalatine fossa)• [V2] Maxillary division of [V] (trigeminal nerve)Foramen ovale:(middle cranial fossa/infratemporal fossa)• [V3] Mandibular division of [V] (trigeminal nerve)Foramen lacerum(filled with cartilage in life)Jugular foramen:(posterior cranial fossa/neck)• [IX] Glossopharyngeal nerve• [X] Vagus nerve• [XI] Accessory nerve• Internal jugular veinABForamen magnum:(posterior cranial fossa/neck)• Spinal cord• Vertebral arteries Roots of accessory nerve [XI] pass from upper region of spinal cord through the foramen magnum into the cranial cavity and then leave the cranial cavity through the jugular foramen Foramen spinosum:(middle cranial fossa/infratemporal fossa)• Middle meningeal arteryCarotid canal:(middle cranial fossa/neck)• Internal carotid arteryHypoglossal

1	cavity through the jugular foramen Foramen spinosum:(middle cranial fossa/infratemporal fossa)• Middle meningeal arteryCarotid canal:(middle cranial fossa/neck)• Internal carotid arteryHypoglossal canal:(posterior cranial fossa/neck)• [XII] Hypoglossal nerveInternal acoustic meatus:(posterior cranial fossa/ear, and neckvia stylomastoid foramen)• [VII] Facial nerve• [VIII] Vestibulocochlear nerve Labyrnthine artery and vein

1	Fig. 8.29 Skull fracture seen on a skull radiograph (patient in supine position). Fig. 8.30 Ultrasound scans. A. Normal carotid bifurcation. B. Internal carotid artery stenosis. Fig. 8.31 Cranial meninges. A. Superior coronal view. B. Continuity with the spinal meninges. Intracranial venous structure(superior sagittal sinus)Outer periosteal layer of dura materInner meningeal layer of dura materArachnoid materPia materDural partition (falx cerebri)SkullDura materSubarachnoid spaceABMeningeal layer of dura materForamen magnumPeriosteal layer of dura materSkullPeriosteumSpinal dura materSpinal extradural spaceVertebra CI Fig. 8.32 Dural partitions. A. Diagram. B. Dissection. Fig. 8.33 Dural arterial supply.

1	Fig. 8.32 Dural partitions. A. Diagram. B. Dissection. Fig. 8.33 Dural arterial supply. Middle meningeal arteryPosition of pterionMeningeal branch(from occipital artery)Meningeal branch(from vertebral artery)Posterior meningeal artery(from ascendingpharyngeal artery)Meningeal branch(from ascendingpharyngeal artery)Middlemeningeal arteryOccipital arteryAscending pharyngeal arteryExternal carotid arteryMaxillary arteryAnterior meningeal arteries(from ethmoidal arteries) Fig. 8.34 Dural innervation. Ophthalmic divisionof trigeminal nerve[V1]Ophthalmic division of trigeminal nerve [V1](tentorium cerebelli)Maxillary division of trigeminal nerve [V2]Mandibular division of trigeminal nerve [V3]Cervical nervesOphthalmic division oftrigeminal nerve [V1](falx cerebri) Fig. 8.35 Arrangement of the meninges and spaces. Fig. 8.36 Lateral view of the brain. Fig. 8.37 Sagittal section of the brain.

1	Fig. 8.35 Arrangement of the meninges and spaces. Fig. 8.36 Lateral view of the brain. Fig. 8.37 Sagittal section of the brain. Fig. 8.38 Arterial supply to the brain. A. Diagram. B. Magnetic resonance angiogram showing normal carotid and vertebral arteries. C. Enhanced CT scan of carotid vessels. Fig. 8.39 Arteries on the base of the brain. Fig. 8.40 Different imaging modalities used to evaluate a stroke (arrows). A. CT scan. B. T2weighted CT. C. Diffusion-weighted image (DWI). D. Apparent diffusion coefficient image (ADC). Fig. 8.41 Basilar tip aneurysm. A. Three-dimensional cranial cutaway CT scan. B. Magnified view of aneurysm. Fig. 8.42 Anterior communicating aneurysm. A. Left carotid angiogram. B. Left carotid angiogram after embolization. Fig. 8.43 Dural venous sinuses. Fig. 8.44 Veins, meninges, and dural venous sinuses.

1	Fig. 8.43 Dural venous sinuses. Fig. 8.44 Veins, meninges, and dural venous sinuses. Sigmoid sinusInferior sagittal sinusSuperior petrosal sinusBasilar sinusSphenoparietal sinusIntercavernous sinusCavernous sinusOphthalmic veinPterygoid plexus of veinsSuperior petrosal sinusSigmoid sinusInferior petrosal sinusRight transverse sinusGreat cerebral veinConfluence of sinusesStraight sinusSuperior sagittal sinus Fig. 8.45 Cavernous sinuses. Pituitary glandInternal carotid arteryDura materAbducent nerve [VI]Maxillary division of trigeminal nerve [V2]Cavernous (venous) sinusesSphenoidal (paranasal) sinusesOphthalmic division of trigeminal nerve [V1]Trochlear nerve [IV]Oculomotor nerve [III]Diaphragma sellae Fig. 8.46 Lateral view of right cavernous sinus with meningeal layer of dura removed to show contents.

1	Fig. 8.46 Lateral view of right cavernous sinus with meningeal layer of dura removed to show contents. Trochlea nerve [IV]Abducent nerve [VI]Abducent nerve [VI]Oculomotor nerve [III]Oculomotor nerve [III]Infundibulum (stalk of pituitary gland)Anterior clinoid processPosterior clinoid processTentorium cerebelliCut edge of dura materMaxillary nerve [V2]Ophthalmic nerve [V1]Trigeminal ganglionMandibular nerve [V3]Internal carotid arteryOptic nerve [III]Trochlea nerve [IV]Trigeminal nerve [V] Fig. 8.47 Scalp and meninges.

1	Neurovascular bundleV1V2Connected togetheras a structural unitSkinAponeurosisPeriosteum1234BoneOutertableInnertableDiploëAnterior cerebral arteryInternal carotidarteryDiploic veinSuperior sagittal sinusCavernous sinusOptic tractsDuraPeriostial layerMeningeal layerArachnoidSubarachnoid spacePiaEmissary vein: can spread infection fromthe scalp into the cranial cavityConnective tissue:contains majornerves and vesselsof the scalpFractureLoose connective tissue (danger area)• In scalping injuries, this is the layer in which separation occurs.• Infection can easily spread in this layer.• Blunt trauma can result in hemorrhage in this layer (blood can spread forward into the face, resulting in “black eyes”).Rupture of the middle meningeal artery (branches) by fracture of the inner table of boneresults in extradural hematoma. Under pressure, the blood progressively separates dura from the bone.Aneurysm• Ruptured aneurysms of vessels of the cerebral arterial circle hemorrhage directly into

1	in extradural hematoma. Under pressure, the blood progressively separates dura from the bone.Aneurysm• Ruptured aneurysms of vessels of the cerebral arterial circle hemorrhage directly into the subarachnoid space and CSF.Tear to cerebral vein where it crosses dura to enter cranial venous sinus can result in subdural hematoma. The tearseparates a thin layer of meningeal dura from that which remains attached to the periosteal layer. As a result, thehematoma is covered by an inner limiting membrane derived from part of the meningeal dura. ExtraduralhematomaSubdural hematomaVIIVIII1234

1	Fig. 8.48 Extradural hematoma. Axial CT scan of brain. Shift of the falx cerebriExtradural hematoma Fig. 8.49 Chronic (low-density) subdural hematoma. Axial CT scan of brain. Fig. 8.50 Subarachnoid hemorrhage. Axial CT scan of brain. Fig. 8.51 MRI of the brain shows peripherally enhancing tuberculosis lesions in the left temporal lobe and cerebral peduncle. Fig. 8.52 MRI of the brain reveals an incidental Chiari I malformation with herniation of the the cerebellar tonsils through the foramen magnum, giving rise to a cone shape. Fig. 8.53 Cranial nerves exiting the cranial cavity. Fig. 8.54 Cranial nerves on the base of the brain. Fig. 8.56 Facial muscles. Fig. 8.57 Orbital group of facial muscles. Fig. 8.58 Nasal group of facial muscles. Fig. 8.59 Oral group of facial muscles. Fig. 8.60 Buccinator muscle. Fig. 8.61 Auricular muscles. Fig. 8.62 Parotid gland. A. Lateral view. B. Cross section. Fig. 8.63 Tumor in parotid gland. Axial CT scan.

1	Fig. 8.60 Buccinator muscle. Fig. 8.61 Auricular muscles. Fig. 8.62 Parotid gland. A. Lateral view. B. Cross section. Fig. 8.63 Tumor in parotid gland. Axial CT scan. Fig. 8.64 Trigeminal nerve [V] leaving the skull. Fig. 8.65 Cutaneous distribution of the trigeminal nerve [V]. Third occipital(from posterior ramus of C3)Lesser occipitaland great auricular(from cervical plexus)Great auricular nerve(from anterior ramusof C2 and C3)Lesser occipital nerveZygomaticotemporalnervesSupra-orbital nerveAuriculotemporalnerveGreater occipital(from posteriorramus of C2)Supratrochlear nerveOphthalmic nerve [V1]Maxillary nerve [V2]Mandibular nerve [V3]External nasalnerveInfratrochlear nerveZygomaticofacial nerveInfra-orbital nerveBuccal nerveMental nerveTransverse cervical(from anterior ramus of C2 and C3)Transverse cervical Fig. 8.66 Facial nerve [VII] on the face. A. Terminal branches. B. Branches before entering the parotid gland.

1	Fig. 8.66 Facial nerve [VII] on the face. A. Terminal branches. B. Branches before entering the parotid gland. Fig. 8.67 Vasculature of the face. A. Lateral view. B. Branches of the maxillary artery. Fig. 8.68 Intracranial venous connections. Fig. 8.69 Lymphatic drainage of the face. Fig. 8.70 SCALP. Fig. 8.71 Layers of the scalp. Fig. 8.72 Occipitofrontalis muscle. A. Frontal belly. B. Occipital belly. Fig. 8.73 Innervation of the scalp. Fig. 8.74 Vasculature of the scalp. Fig. 8.75 Lymphatic drainage of the scalp. Fig. 8.76 Bones of the orbit. Optic canalFrontal boneEthmoidal foraminaEthmoid boneLacrimalgrooveLacrimal bonePalatine boneMaxillaInferior orbital fissureZygomatic boneGreater wing of sphenoidSuperior orbital fissureLesser wing of sphenoid Fig. 8.77 Eyelids.

1	Fig. 8.77 Eyelids. PeriosteumLevator palpebraesuperioris muscleSuperior conjunctival fornixConjunctivaTarsusSebaceous glandof eyelashTarsal glandOrbital septumSuperior tarsal muscle(smooth muscle)Tendon of levator palpebrae superioris muscleOrbicularis oculimuscle Fig. 8.78 Orbicularis oculi muscle. Fig. 8.79 Orbital septum. PeriosteumOrbital septumOrbital septumPeriosteumTendon of levator palpebrae superioris muscle Fig. 8.80 Tarsal plates. Tendon of levatorpalpebrae superioris muscleSuperior tarsusAnterior lacrimal crestMedial palpebral ligamentInferior tarsusOrbital septumLateral palpebral ligamentOrbital septum Fig. 8.81 Vasculature of the eyelids. Fig. 8.82 Innervation of the eyelids. Fig. 8.83 Lacrimal gland, anterior view. MedialPunctaLacrimal sacNasolacrimal ductLacrimalcanaliculiLacrimal glandTendon of levator palpebrae superioris muscle Fig. 8.84 Lacrimal gland and levator palpebrae superioris.

1	MedialPunctaLacrimal sacNasolacrimal ductLacrimalcanaliculiLacrimal glandTendon of levator palpebrae superioris muscle Fig. 8.84 Lacrimal gland and levator palpebrae superioris. Orbital part of lacrimal glandLacrimal vessels and nervePalpebral part oflacrimal glandTendon oflevator palpebrae superiorisOrbital septum Fig. 8.85 The lacrimal sac. Fig. 8.86 Position of lacrimal sac. Medial palpebral ligamentLacrimal part of orbicularis oculi muscleOrbital septum Posterior lacrimal crestLacrimal sacAnterior lacrimal crestPeriosteumAnteriorPosteriorLateral Fig. 8.87 Innervation of the lacrimal gland.

1	Fig. 8.87 Innervation of the lacrimal gland. Lacrimal nerveLacrimal glandForamen rotundumMaxillary nerve [V2]Pterygoid canalGreater petrosal nerveDeep petrosal nerveNerve ofpterygoid canalSympathetic plexusInternal carotid arteryBranch of zygomaticotemporal nerveZygomatic nervePterygopalatine ganglionSensory fibersSympathetic postganglionic fibersParasympathetic preganglionic fibersParasympathetic postganglionic fibersZygomaticotemporal nerveZygomaticofacial nerve Fig. 8.88 Openings into the bony orbit. Nasolacrimal canalInferior orbital fissure Optic canalFrontal boneEthmoidal foraminaEthmoid boneLacrimal bonePalatine boneMaxillaInfra-orbital grooveZygomatic boneGreater wing of sphenoidSuperior orbital fissureLesser wing of sphenoid Fig. 8.89 Optic canal and superior orbital fissure.

1	Fig. 8.89 Optic canal and superior orbital fissure. Lacrimal branch of the ophthalmic nerve [V1]Nasociliary branch of ophthalmic nerve [V1]Frontal branch of the ophthalmic nerve [V1]Optic nerveOptic canalInferior ophthalmic veinSuperior ophthalmic veinOphthalmic arteryInferior branch of oculomotor nerve [III]Abducent nerve [VI]Trochlear nerve [IV]Superior branch of oculomotor nerve [III]Superior orbital fissureInferior orbital fissureLateralMedial Fig. 8.90 Periorbita. A. Lateral view. B. Common tendinous ring. Fig. 8.91 Fascial sheath of the eyeball. Fig. 8.92 Check ligaments. A. Anterior view. B. Superior view. Medial rectus muscleMedial rectus muscleLateral rectus muscleLateral rectus muscleInferior rectus muscleInferior oblique muscleMedial check ligamentCheck ligament of medial rectusmuscleCheck ligament of lateral rectus muscleLateral check ligamentSuspensory ligamentSuspensory ligamentSuspensoryligamentFascial sheathFascial sheathPeriosteumPeriorbitaLacrimal sacAB

1	Fig. 8.93 Movements of the eyeball. Fig. 8.94 Axes of the eyeball and orbit. Axis of eyeballAxis of orbitMedial Fig. 8.95 Muscles of the eyeball. A. Superior view. B. Lateral view. C. Coronal magnetic resonance image through the eye. Fig. 8.96 Origins of muscles of the eyeball, coronal view. Superior orbital fissureInferior orbital fissureLacrimal nerveFrontal nerveNasociliary nerveOptic nerveLateral rectusMedial rectusSuperior rectusSuperior obliqueLevator palpebrae superiorisInferior rectusInferior ophthalmic veinOphthalmic arteryInferior division of oculomotor nerve [III]Superior division of oculomotor nerve [III]Abducent nerve [VI]Trochlear nerve [IV]LateralMedial Fig. 8.97 Actions of muscles of the eyeball. A. Action of individual muscles (anatomical action). B. Movement of eye when testing specific muscle (clinical testing).

1	Fig. 8.97 Actions of muscles of the eyeball. A. Action of individual muscles (anatomical action). B. Movement of eye when testing specific muscle (clinical testing). AbductionAdductionElevationDepressionInferiorobliqueSuperiorobliqueSuperiorrectusInferiorrectusLateralrectusMedialrectusMuscle testedDirection to moveeye when testingmuscleSuperior rectusInferior rectusLateral rectusMedial rectusInferior obliqueSuperior obliqueLook laterally and upwardLook laterally and downwardLook laterally Look mediallyLook medially and upwardLook medially and downwardABMedialLateral Fig. 8.98 The “H-test.” Right eyeLateral rectus [VI]Superior rectus [III]Inferior rectus [III]Medial rectus [III]Superior oblique [IV]Inferior oblique [III]1.2.3.4.5.6.Medial rectus [III]Inferior oblique [III]Superior oblique [IV]Lateral rectus [VI]Inferior rectus [III]Superior rectus [III]Left eye142635 Fig. 8.99 Arterial supply to the orbit and eyeball. Fig. 8.100 Venous drainage of the orbit and eyeball.

1	Fig. 8.99 Arterial supply to the orbit and eyeball. Fig. 8.100 Venous drainage of the orbit and eyeball. Fig. 8.101 Innervation of the orbit and eyeball. Lacrimal branch of ophthalmic nerve [V1]Nasociliary branchof ophthalmic nerve [V1]Frontal branch of ophthalmic nerve [V1]Optic nerveOptic canalInferior ophthalmic veinSuperior ophthalmic veinOphthalmic arteryInferior branch of oculomotor nerve [III]Superior branch of oculomotor nerve [III]Abducent nerve [VI]Trochlear nerve [IV]Common tendinous ringLateralMedial Fig. 8.102 Oculomotor nerve [III] and its divisions. Fig. 8.103 Trochlear nerve [IV] in the orbit. Fig. 8.104 Ophthalmic nerve [V1] and its divisions. Fig. 8.105 Relationship of the ophthalmic nerve [V1] and its divisions to the muscles of the eyeball. Fig. 8.106 Course of the nasociliary nerve (from [V1]) in the orbit.

1	Fig. 8.105 Relationship of the ophthalmic nerve [V1] and its divisions to the muscles of the eyeball. Fig. 8.106 Course of the nasociliary nerve (from [V1]) in the orbit. Long ciliary nervesShort ciliary nervesLacrimal glandLacrimal nerve (from [V1])Lateral rectus Ciliary ganglionAbducent nerve[VI]Inferior branch of the oculomotor nerve [III]Superior branch of the oculomotor nerve [III]Medial rectus muscleNasociliary nerve (from [V1])Posterior ethmoidal nerveAnterior ethmoidal nerveInfratrochlear nerveLateral Fig. 8.107 Ciliary ganglion. Fig. 8.108 Eyeball. Fig. 8.109 Ophthalmoscopic view of posterior chamber of the right eye. Fig. 8.110 Ciliary body. Fig. 8.111 Layers of the retina in a healthy eye. A. HD-OCT scan of a healthy eye. B. Schematic indicating the layers of the retina on an HD-OCT scan of a healthy eye. C. Diagram illustrating the layers of the retina. Fig. 8.112 High-definition optical coherence tomography (HD-OCT). A. Diseased eye. B. Healthy eye.

1	Fig. 8.112 High-definition optical coherence tomography (HD-OCT). A. Diseased eye. B. Healthy eye. Fig. 8.113 Right ear. Fig. 8.114 Auricle. Fig. 8.115 Sensory innervation of the auricle. Auriculotemporalbranch of themandibular nerve [V3]Great auricular nerve(C2,C3)Facial nerve [VII]Vagus nerve [X]Lesser occipitalnerve (C2) Fig. 8.116 External acoustic meatus. Fig. 8.117 Middle ear. Fig. 8.118 Tympanic membrane (right ear). A. Diagram. B. Otoscopic view. ABPars flaccidaPosteriormalleolar foldHandle ofmalleusUmboCone of lightAnteriormalleolar foldLateral process(of malleus) Fig. 8.119 Parts of the middle ear. Fig. 8.120 Boundaries of the right middle ear.

1	Fig. 8.119 Parts of the middle ear. Fig. 8.120 Boundaries of the right middle ear. Tegmen tympaniTensor tympani musclePharyngotympanic tubeLesser petrosal nerveBranch from internalcarotid plexusSympathetic plexusInternal carotid arteryChorda tympani nerveTympanic branch of the glossopharyngeal nerve [IX]Internal jugular veinPromontoryRound windowFacial nerve [VII]Chorda tympani nervePyramidal eminenceAditus tomastoid antrumProminence of facial canalProminence of lateral semicircular canalOval window Fig. 8.121 Mastoid antrum and surrounding bone. A. Diagram. B. High-resolution CT scan of left ear (petrous temporal bone). BAditus to mastoid antrumTegmen tympaniEpitympanic recessPharyngotympanic tubeMiddle earMastoid processCochleaMiddle earExternal auditorymeatusMastoid air cellsMastoid air cellsMastoid antrumA Fig. 8.122 Pharyngotympanic tube. Fig. 8.123 Auditory ossicles. A. Malleus. B. Incus. C. Stapes.

1	Fig. 8.122 Pharyngotympanic tube. Fig. 8.123 Auditory ossicles. A. Malleus. B. Incus. C. Stapes. Head of malleusNeck ofmalleusABCAnterior processHandle of malleusLateralprocessIncus articulationShort limbMalleus articulationLong limbBody ofincusBase of stapesAnterior limbHead of stapesPosterior limb Fig. 8.124 Muscles associated with the auditory ossicles (right ear). MalleusIncusPyramidal eminenceTendon ofstapedius muscleFootplate of stapesTympanic membranePharyngotympanic tubeTensor tympani muscle Fig. 8.125 Innervation of the middle ear. Prominence of lateralsemicircular canalTensor tympani musclePromontoryLesser petrosal nervePharyngotympanic tubeBranch from internal carotid plexus(caroticotympanic nerve)Tympanic nerve(from glossopharyngeal nerve [IX])Tympanic plexusRound windowStapesProminence of facial canal Fig. 8.126 Grooves and hiatuses for the greater and lesser petrosal nerves. Fig. 8.127 Location of the internal ear in temporal bone. Fig. 8.128 Internal ear.

1	Fig. 8.126 Grooves and hiatuses for the greater and lesser petrosal nerves. Fig. 8.127 Location of the internal ear in temporal bone. Fig. 8.128 Internal ear. Fig. 8.129 Bony labyrinth. SacculeHelicotremaDura materCochleaCochlear ductScala tympaniScala vestibuliPharyngotympanic tubeRound windowOpening of cochlear canaliculusTympanic membraneVestibuleUtricleAmpullaAnterior semicircular canal and ductVestibular aqueductLateral semicircular canal and ductPosterior semicircular canal and ductStapes in oval window Fig. 8.130 Cochlea. ModiolusScala vestibuliScalatympaniCochlear ductLamina of modiolusCochlear nerveSpiral ganglionHelicotrema Fig. 8.131 Membranous labyrinth. Fig. 8.132 Membranous labyrinth, cross section. Vestibular membraneScala vestibuliModiolusLamina of modiolusScala tympaniBasilar membraneSpiral ligamentSpiral organ Fig. 8.133 A. Facial nerve in the temporal bone. B. Chorda tympani in the temporal bone. Fig. 8.134 Transmission of sound.

1	Fig. 8.133 A. Facial nerve in the temporal bone. B. Chorda tympani in the temporal bone. Fig. 8.134 Transmission of sound. Fig. 8.135 Temporal and infratemporal fossae. Zygomatic archArticular tubercleMandibular fossaGroove for middletemporal arteryRamus of mandibleExternal acousticmeatusSupramastoid crestTemporal fossaInfratemporal fossaMasseter muscle Fig. 8.136 Bony features related to the temporal and infratemporal fossae.

1	Fig. 8.136 Bony features related to the temporal and infratemporal fossae. Greater wingof sphenoid boneFrontal boneFrontal process ofzygomatic boneZygomaticofacialforamenZygomaticotemporalforamen(on deep surface ofzygomatic bone)Posterior surfaceof maxillaZygomatic boneMaxillary process ofzygomatic boneLateral plate of pterygoidprocess of sphenoid bonePalatine bonePterygopalatinefossaTympanomastoidfissureMastoid processTympanic plateSpine of sphenoidStyloid processPterygomaxillary fissure(leading into pterygopalatine fossa)PterygoidhamulusExternal acousticmeatusSupramastoid crestGroove for middletemporal arteryMandibular fossaArticular tubercleForamen spinosumInfratemporal crestForamen ovaleInfratemporalsurface of maxillaAlveolar foramenPetrotympanic fissureSquamous part oftemporal bone Fig. 8.137 Mandible. A. Lateral view of left side. B. Medial view of left side. Fig. 8.138 Temporomandibular joint. A. Mouth closed. B. Mouth open.

1	Fig. 8.137 Mandible. A. Lateral view of left side. B. Medial view of left side. Fig. 8.138 Temporomandibular joint. A. Mouth closed. B. Mouth open. ABUpper joint cavityArticular discArticular tubercleFibrocartilage onarticular surfaceCapsuleLower joint cavitySynovialmembraneMandibular fossaForward movement of discand mandible at upper jointLateral pterygoidmuscleProtrusionDepressionHinge movementat lower joint Fig. 8.139 Ligaments associated with the temporomandibular joint. Fig. 8.140 Movements of the temporomandibular joint. Protrusion• Lateral pterygoid assisted by medial pterygoidRetraction• Posterior fibers of temporalis, deep part of masseter, and geniohyoid and digastricDepression• Gravity• Digastric, geniohyoid, and mylohyoid musclesElevation• Temporalis, masseter, medial pterygoid Fig. 8.141 Masseter muscle. Fig. 8.142 Temporal fossa. A. Lateral view. B. Lateral view showing the infratemporal fossa. Fig. 8.143 Temporalis muscle. Lateral view.

1	Fig. 8.141 Masseter muscle. Fig. 8.142 Temporal fossa. A. Lateral view. B. Lateral view showing the infratemporal fossa. Fig. 8.143 Temporalis muscle. Lateral view. Fig. 8.144 Nerves and arteries of the temporal fossa. Temporal fasciaZygomaticotemporal nerve(branch of maxillary nerve [V2])Deep temporal arteriesMandibular nerve [V3]Deep temporal nervesZygomaticofacial nerveInfratemporal crestMaxillary artery in infratemporal fossaExternal carotid arterySuperficial temporal arteryMiddle temporal arteryTemporalis muscle Fig. 8.145 Borders of the infratemporal fossa.

1	Fig. 8.145 Borders of the infratemporal fossa. Foramen spinosumInfratemporal crestForamen ovaleTensor veli palatiniLevator veli palatiniAlveolar foramenLateral plate of pterygoid processPterygomaxillary fissure(leading into pterygopalatine fossa)Posterior surface of maxillaPterygopalatine fossaGreater wing of sphenoid boneMasseterMylohyoidHyoglossusMiddle constrictorSuperior constrictorPharynxSpine of sphenoidHead and neck of mandiblePetrotympanic fissurePterygomandibular raphe Fig. 8.146 Medial pterygoid muscle. Foramen spinosumInfratemporal crestForamen ovaleTensor veli palatiniLevator veli palatiniDeep headmedial pterygoidSuperficial headmedial pterygoidSpine of sphenoidPetrotympanic fissureLingulaMandibular canalSphenomandibular ligament Fig. 8.147 Lateral pterygoid muscle. Infratemporal crestUpper head of lateral pterygoidDeep head medial pterygoidSuperficial headmedial pterygoidLower headlateral pterygoidCapsuleArticular discSphenomandibular ligament

1	Infratemporal crestUpper head of lateral pterygoidDeep head medial pterygoidSuperficial headmedial pterygoidLower headlateral pterygoidCapsuleArticular discSphenomandibular ligament Fig. 8.148 Mandibular nerve [V3]—anterior trunk. Meningeal branch and nerve to medial pterygoid. Deep headmedial pterygoidTrigeminal ganglionUpper head lateral pterygoid (cut )Lower head lateral pterygoid (cut )Nerve to medial pterygoidBranch to tensor veli palatiniMeningeal branchPosterior trunkBranch to tensor tympaniAnterior trunkDeep temporal nervesBuccal nerveNerve to lateral pterygoidMasseteric nerve Fig. 8.149 Mandibular nerve [V3]—posterior trunk. A. Lateral view. B. Anterior view. C. Anteromedial view. Petrotympanic fissureChorda tympani nerveLingual nerveAIncisive nerveMental nerveNerve to mylohyoidInferior alveolar nerveAuriculotemporal nerve

1	B. Anterior view. C. Anteromedial view. Petrotympanic fissureChorda tympani nerveLingual nerveAIncisive nerveMental nerveNerve to mylohyoidInferior alveolar nerveAuriculotemporal nerve Medial pterygoidmuscleLingulaLingual nerveMental nerveIncisive nerveInferior alveolar nerveSphenomandibular ligamentChorda tympaniInferior alveolar nerveSphenomandibular ligamentSuperior constrictor muscleSubmandibular ganglionNerve to mylohyoidGreater horns of hyoid boneHyoglossus muscleGenioglossus muscleGeniohyoid muscleLingual nerveTemporalis tendonPterygomandibular raphe (cut )Buccal nerve (branch of anterior trunk)BCFacial nerve [VII]Trigeminal nerve [V]Mandibular nerve [V3] Fig. 8.150 Chorda tympani and lesser petrosal nerves. A. Course after emerging from the skull. B. Course of parasympathetic fibers.

1	Fig. 8.150 Chorda tympani and lesser petrosal nerves. A. Course after emerging from the skull. B. Course of parasympathetic fibers. APetrotympanic fissureChorda tympani nerve from [VII]Lesser petrosal nerve [IX]Lingual nerveOtic ganglion (medial to [V3])TongueLingual nerveSublingual glandMylohyoidSubmandibular glandSubmandibular ganglionPreganglionic parasympathetic fibers from glossopharyngeal nerve [IX]Postganglionic parasympathetic fibers from otic ganglionPreganglionic parasympathetic fibers from facial nerve [VII]Postganglionic parasympathetic fibers from submandibular ganglionAuriculotemporal nerveTop of parotid glandAuriculotemporal nerve

1	Preganglionic parasympathetic fibers from glossopharyngeal nerve [IX]Postganglionic parasympathetic fibers from otic ganglionPreganglionic parasympathetic fibers from facial nerve [VII]Postganglionic parasympathetic fibers from submandibular ganglionTympanic plexusLesser petrosal nerveOtic ganglionChorda tympani (carries taste fromthe anterior 2/3 of the tongue)Sublingual glandSubmandibularglandChorda tympanicarries parasympatheticinnervation to all glandsbelow the oral fissureSubmandibularganglionLingual nerveAuriculotemporal nerveParotid glandTympanic membraneChorda tympaniTympanic nerveInferior ganglionGreater petrosal nerveBGlossopharyngeal nerve [IX]Facial nerve [VII]Mandibular nerve [V3]Maxillary nerve [V2]Ophthalmic nerve [V1]Trigeminal nerve [V] Fig. 8.151 Maxillary artery.

1	Fig. 8.151 Maxillary artery. Branches of middlemeningeal in cranial cavityMiddle meningeal arteryArtery to masseterUpper head oflateral pterygoid (cut )Pterygoid arteryPterygopalatine fossaLower head of lateralpterygoid (cut )Buccal arteryMental arteryInferior alveolar arteryExternal carotidMaxillary arteryAuriculotemporal nerveSuperficial temporal arteryDeep temporal arteries Fig. 8.152 Pterygoid plexus of veins. Maxillary veinEmissary veins(connect with cavernous sinus)Inferior ophthalmic veinDeep facial veinFacial veinInferior alveolar veinSuperficial temporal veinExternal jugular veinInternal jugular veinPosterior auricular veinRetromandibular vein Fig. 8.153 Pterygopalatine fossa. A. Anterolateral view. B. Lateral view. Fig. 8.154 Sphenoid bone. A. Anterior view. B. Posterosuperior view.

1	Fig. 8.153 Pterygopalatine fossa. A. Anterolateral view. B. Lateral view. Fig. 8.154 Sphenoid bone. A. Anterior view. B. Posterosuperior view. AForamen rotundumSurface related topterygopalatine fossaPterygoid canalPalatovaginal grooveBLesser wingGreater wingForamen rotundumPosterior opening of bony part of pterygoid canalPterygoid processPart of pterygoid canal incartilage of foramen lacerumCartilage filling foramen lacerumGreater petrosal nerve of VIIMaxillary nerve [V2]Internal carotid arterySuperior orbital fissure Fig. 8.155 Gateways of the pterygopalatine fossa. Sphenopalatine foramennasal cavityInferior orbital fissurefloor of orbitPterygomaxillary fissureinfratemporal fossaPalatine canalroof of oral cavity (palate)Pterygoid canalcranial cavity(middle cranial fossa)Foramen rotundumcranial cavity(middle cranial fossa)Palatovaginal canalnasopharynx Fig. 8.156 Maxillary nerve [V2]. A. Terminal branches. B. In relationship to the pterygopalatine ganglion.

1	Fig. 8.156 Maxillary nerve [V2]. A. Terminal branches. B. In relationship to the pterygopalatine ganglion. Fig. 8.157 Nerve of the pterygoid canal. A. Overview. B. In relationship to the pterygopalatine ganglion. ABLacrimal nerveLacrimal glandParasympathetic nerves in branch of zygomaticotemporal nerveZygomatic nerveNerve of pterygoid canalCartilage filling foramen lacerumDeep petrosal nerveInternal carotid nerveFacialnerve [VII]Preganglionic sympathetic nerves from T1Superior cervical sympathetic ganglionSympathetic trunkGeniculate ganglionGreater petrosal nerveInternal carotid plexus[V1][V2][V3]Internal carotid arteryPterygopalatine fossaInferior orbital fissureInfra-orbital nervePreganglionic parasympathetic nervesPostganglionic parasympathetic nervesPreganglionic sympathetic nervesPostganglionic sympathetic nervesNerve of pterygoid canal Fig. 8.158 Maxillary artery in the pterygopalatine fossa.

1	Fig. 8.158 Maxillary artery in the pterygopalatine fossa. Pharyngeal arterySphenopalatine arteryInfra-orbital arterySeptal part of greater palatine arteryAnterior superior alveolar arteryPosterior superior alveolar arteryGreater palatine arteryLesser palatine arteryMaxillary artery ininfratemporal fossaNasopharynxArtery of pterygoid canalCartilage filling foramen lacerum Fig. 8.159 Veins of the pterygopalatine fossa. Fig. 8.160 Compartments of the neck. Fig. 8.161 Anterior and posterior triangles of the neck. Inferior border of mandibleAnteriortriangleSternocleidomastoid muscleClavicleTrapezius musclePosteriortriangle Fig. 8.162 Fascia of neck, transverse view. Fig. 8.163 Fascia of the neck, sagittal view. Investing layerInfrahyoid musclesPretracheal fasciaManubrium of sternumPretracheal spaceFascial space within prevertebral layerRetropharyngeal spacePrevertebral layerBuccopharyngeal fascia(posterior portion ofpretracheal layer) Fig. 8.164 Superficial veins of neck.

1	Fig. 8.164 Superficial veins of neck. Fig. 8.165 Placing a central venous catheter in the neck. A. Clinical procedure. B. Chest radiograph showing that the tip of the catheter is in the origin of the right atrium. Fig. 8.166 Borders and subdivisions of the anterior triangle of the neck. Submandibular triangleAnterior belly ofdigastric muscleSubmental triangleHyoid boneSuperior belly of omohyoid muscleMuscular triangleSternocleidomastoid muscleTrapezius musclePosterior triangleCarotid triangleStylohyoid musclePosterior belly of digastric muscle Fig. 8.167 Suprahyoid muscles. A. Lateral view. B. Inferior view. Fig. 8.168 Infrahyoid muscles. Fig. 8.169 Origin of common carotid arteries. TracheaEsophagusLeft common carotid arteryLeft internal jugular veinLeft subclavian arteryLeft subclavian veinClavicleSuperior vena cavaRight subclavian veinRight subclavian arteryRight internal jugular veinRight common carotid arteryArch of aortaLeft brachiocephalic veinRight brachiocephalic vein

1	Fig. 8.170 Carotid triangle. SternocleidomastoidmuscleCarotid triangleSuperior belly ofomohyoid muscleCommon carotid arteryPosterior belly of digastric muscleInternal carotid arteryExternal carotid artery Fig. 8.171 Carotid system. Fig. 8.172 Glossopharyngeal nerve [IX] in the anterior triangle of the neck. Fig. 8.173 Vagus nerve [X] in the anterior triangle of the neck. Fig. 8.174 Accessory nerve [XI] in the posterior triangle of the neck. Fig. 8.175 Hypoglossal nerve [XII]. A. Surgical view of hypoglossal nerve in anterior triangle of the neck. B. Diagram. Hyoglossus muscleExternal carotidarterySuperior root ofansa cervicalisSuperior thyroid arteryInternal jugular veinInternal jugularveinPosterior belly ofdigastric muscle (cut )Stylohyoid muscleSternocleidomastoidbranch of occipital arteryHypoglossal nerveHypoglossal nerveSternocleidomastoid branchof occipital arteryPosterior belly of digastric muscleOccipital arteryAB

1	Fig. 8.176 Transverse cervical nerve in the anterior triangle of the neck. Fig. 8.177 Ansa cervicalis. Hypoglossal nerveC1C3C2Superior root of ansa cervicalisInferior root of ansa cervicalisOmohyoid muscle(superior belly)Sternohyoid muscleSternothyroid muscleThyrohyoid muscleOmohyoid muscle(inferior belly) Fig. 8.178 Thyroid gland in the anterior triangle of neck. A. Anterior view. B. Transverse view. C. Ultrasound scan—compound axial view of the neck. D. Ultrasound scan—axial view of the neck. E. Nuclear medicine scan—normal thyroid uptake of pertechnetate in the neck. Fig. 8.179 Vasculature of the thyroid: anterior view. Fig. 8.180 Superior and inferior thyroid arteries and left and right recurrent laryngeal nerves and thyroid and parathyroid glands. A. Posterior view. B. Surgical (anterolateral) view of parathyroid gland with left lobe of thyroid retracted. BLeft lobe of thyroid glandParathyroid gland

1	BLeft lobe of thyroid glandParathyroid gland Fig. 8.181 Surgical view of left lobe of enlarged thyroid (goiter) retracted to show close association with recurrent laryngeal nerve. Left lobe of thyroid glandLeft recurrent laryngeal nerve Fig. 8.182 Ectopic parathyroid adenoma in superior mediastinum. Noncontrast hybrid single photon emission computed tomography/computed tomography (SPECT/CT). A. Transverse view. B. Sagittal view. C. Coronal view. Fig. 8.183 Borders of the posterior triangle of the neck. Sternocleidomastoid muscleTrapezius muscleOmoclavicular orsubclavian triangleOccipital triangleHyoid bonePosteriortriangleSuperior belly ofomohyoid muscleInferior belly of omohyoid muscle Fig. 8.184 Muscles of the posterior triangle of the neck. Sternocleidomastoid muscleClavicleInferior belly of omohyoid muscleAcromion ofscapulaTrapezius muscleAnterior scalene muscleMiddle scalene musclePosterior scalene muscleLevator scapulae muscleSplenius capitis muscle

1	Fig. 8.185 External jugular vein in the posterior triangle of the neck. Fig. 8.186 Arteries in the posterior triangle of the neck. Trapezius muscleMiddle scalene muscleTransverse cervical arteryBrachial plexusSuprascapular artery3rd part of subclavian arteryPhrenic nerveAnterior scalene muscleSubclavian veinExternal jugular veinInternal jugular veinClavicle1st part of subclavian arteryVagus nerveCommon carotid arteryThyrocervical trunkInferior thyroid arterySternocleidomastoid muscle Fig. 8.187 Accessory nerve and cutaneous branches of the cervical plexus in the posterior triangle of the neck. Fig. 8.188 Cervical plexus. Fig. 8.189 Prevertebral and lateral vertebral muscles supplied by cervical plexus. Fig. 8.190 Root of the neck. TracheaRib IManubrium of sternumCervical pleuraTI vertebraEsophagus Fig. 8.191 Vasculature of the root of the neck. Fig. 8.192 Nerves in the root of the neck. Fig. 8.193 Components of the sympathetic nervous system in the root of the neck.

1	Fig. 8.191 Vasculature of the root of the neck. Fig. 8.192 Nerves in the root of the neck. Fig. 8.193 Components of the sympathetic nervous system in the root of the neck. Fig. 8.194 Cervical part of the sympathetic trunk. Fig. 8.195 Thoracic duct in the root of the neck. Fig. 8.196 Termination of lymphatic trunks in the root of the neck. Fig. 8.197 Lymphatic system in the neck. Fig. 8.198 Pharynx. Fig. 8.199 Neck regions (levels) that are used clinically to evaluate lymph nodes. Fig. 8.200 Line of attachment of the pharynx to the base of the skull.

1	Fig. 8.198 Pharynx. Fig. 8.199 Neck regions (levels) that are used clinically to evaluate lymph nodes. Fig. 8.200 Line of attachment of the pharynx to the base of the skull. Cartilaginous position of pharyngotympanic tubeChoanae (posterior openings of nasal cavities)Pterygoid hamulusLine of attachment of pharynxRoughening on petrous part of temporal bone for attachment of levator veli palatiniExternal acoustic meatusPharyngeal tubercleJugular foramenCarotid canalMedial plate of pterygoid process of sphenoidPetrous part of temporal boneScaphoid fossa on sphenoid bone (for attachment of tensor veli palatini) Fig. 8.201 Attachments of the lateral pharyngeal wall. Fig. 8.202 Constrictor muscles of the pharynx. A. Lateral view. B. Posterior view.

1	Fig. 8.201 Attachments of the lateral pharyngeal wall. Fig. 8.202 Constrictor muscles of the pharynx. A. Lateral view. B. Posterior view. ABPosition of palatopharyngeal sphincteron deep surface of superior constrictorSuperior constrictorMiddle constrictorInferior constrictorEsophagusPharyngeal tuberclePharyngeal fasciaStylohyoid ligamentStylopharyngeus musclePharyngeal rapheStyloid process Fig. 8.203 Longitudinal muscles of the pharynx. A. Stylopharyngeus muscle. B. Medial view. Fig. 8.204 Gaps between muscles in the pharyngeal wall. Pharyngeal fasciaStylopharyngeusSuperior constrictorMiddle constrictorInferior constrictorOropharyngealtriangle:structures (muscles,nerves, vessels)passing into and outof the oral cavityInternal laryngealnerve and vesselsEsophagusRecurrent laryngealnerve and vesselsTracheaMylohyoidBuccinator Fig. 8.205 Mucosal features of the pharynx. A. Lateral view. B. Posterior view with the pharyngeal wall opened. C. Superior view.

1	Fig. 8.205 Mucosal features of the pharynx. A. Lateral view. B. Posterior view with the pharyngeal wall opened. C. Superior view. ABCFold overlyingpalatopharyngealsphincterTorus levatorius(fold overlyinglevator veli palatini)Torus tubariusPharyngeal recessPharyngeal tonsilPharyngeal opening of thepharyngotympanic tubeSalpingopharyngeal foldPalatine tonsilPalatopharyngeal arch(overliespalatopharyngeusmuscle)Laryngeal inletEsophagusTracheaValleculaLingual tonsilsPalatoglossal arch(margin of oropharyngeal isthmus)TongueNasal cavityNasopharynxOropharynxLaryngopharynxOropharyngealisthmusPharyngeal tonsilChoanaeTorus tubariusTorus levatoriusSoft palatePalatopharyngeal archPalatopharyngeal archPalatine tonsilValleculae (anterior to epiglottis)Laryngeal inletEsophagusPiriform fossaLingual tonsilLingual tonsilPalatine tonsilEpiglottisValleculaPiriform fossaSalpingopharyngealfoldPharyngealrecessesNasal cavityOral cavityTracheaLarynxPharynxEsophagus Fig. 8.206 Arterial supply of the pharynx.

1	Fig. 8.206 Arterial supply of the pharynx. Pharyngeal branch(supplies roof of nasopharynx)Superficial temporal arteryAscending palatinearteryAscending pharyngeal arteryInternal carotid arteryCommon carotid arteryPharyngeal branchesInferior thyroid arteryThyrocervical trunkSubclavian arteryLingual arteryExternal carotid arteryFacial arteryTonsillar branchMaxillary artery Fig. 8.207 Venous and lymphatic drainage of the pharynx. Fig. 8.208 Innervation of the pharynx. A. Lateral view. B. Posterior view showing innervation of stylopharyngeus muscle. ABPharyngeal branch of [V2]Nasopharynx–sensory [V2]Oropharynx–sensory [IX]Laryngopharynx–sensory [X]Superiorlaryngeal nerveInferiorganglion of [X]External laryngeal nerve(branch of superior laryngealnerve from [X])Internal laryngeal nerve(branch of superiorlaryngeal nervefrom [X])Pharyngealbranch of [IX]Pharyngealbranch of [X][IX][V2]IXMotor branch to stylopharyngeus Fig. 8.209 Larynx. A. Relationship to other cavities. B. Lateral view.

1	Fig. 8.209 Larynx. A. Relationship to other cavities. B. Lateral view. Fig. 8.210 Cricoid cartilage. A. Anterolateral view. B. Posterior view. Facet for articulation witharytenoid cartilageFacet for articulation withinferior horn of thyroid cartilageTracheaCricoidcartilageLaminaArchAirwayAFacet for articulation witharytenoid cartilageRidgeFacet for articulation with inferior horn of thyroid cartilageDepressionsB Fig. 8.211 Thyroid cartilage. A. Anterolateral view. B. Superior view. Fig. 8.212 Epiglottis. A. Anterolateral view. B. Posterior surface. CricoidTracheaEpiglottic tubercleThyro-epiglottic ligamentRight thyroid laminaAnterior surface of epiglottisPosterior surface of epiglottisAB Fig. 8.213 Arytenoid cartilages.

1	CricoidTracheaEpiglottic tubercleThyro-epiglottic ligamentRight thyroid laminaAnterior surface of epiglottisPosterior surface of epiglottisAB Fig. 8.213 Arytenoid cartilages. ApexPosterior surfaceMedial surfaceRidge on anterolateral surfaceMuscular processArytenoidcartilageDepression for attachment of vocalis musclesDepression for attachment of vestibular ligamentBase (concave – for articulation with cricoid)Vocal processArticular facet forcorniculate cartilageAnterolateral surface Fig. 8.214 Corniculate and cuneiform cartilages. Fig. 8.215 Extrinsic ligaments of the larynx. Lateral thyrohyoid ligamentsAperture for internal branch of superior laryngeal nerve and associated arteryThyrohyoid membraneCricotracheal ligamentMedian thyrohyoid ligamentHyo-epiglottic ligamentHyoid boneTriticeal cartilage Fig. 8.216 Cricothyroid ligament. Fig. 8.217 Quadrangular membrane. Fig. 8.218 Fibro-elastic membrane of the larynx (superior view).

1	Fig. 8.216 Cricothyroid ligament. Fig. 8.217 Quadrangular membrane. Fig. 8.218 Fibro-elastic membrane of the larynx (superior view). EpiglottisVestibular ligamentQuadrangular membraneConus elasticusMuscular process of arytenoidVocal process of arytenoidCorniculate cartilageVocal ligament Fig. 8.219 Movements of the cricothyroid joints. Fig. 8.220 Movements of the crico-arytenoid joints. Fig. 8.221 Laryngeal cavity. A. Posterolateral view. B. Posterior view (cut away). C. Superior view through the laryngeal inlet. D. Labeled photograph of the larynx, superior view.

1	Fig. 8.221 Laryngeal cavity. A. Posterolateral view. B. Posterior view (cut away). C. Superior view through the laryngeal inlet. D. Labeled photograph of the larynx, superior view. EpiglottisEpiglottisEpiglottisAry-epiglottic foldAry-epiglottic foldCut edge of mucosaLaryngeal sacculeVestibuleVestibuleCut edge of right thyroid laminaLaryngeal ventricleInfraglottic spaceInterarytenoid notchCorniculate tubercleCorniculate tubercleCuneiform tubercleCuneiform tubercleCuneiform tubercleLaryngeal inletLaryngeal inletVestibular fold(mucosa overlying vestibular ligament)Vestibular fold (false vocal cord)Vocal fold (mucosa overlying vocal ligament)Vocal fold (true vocal cord)TracheaCricoid archMiddle part of cavityLaryngeal sacculeRima vestibuliRima glottidisRima glottidis (opening between vocal cords)Interarytenoid foldCorniculate tubercleVestibular foldAry-epiglottic foldVocal fold ABCDLaryngopharynx (closed)Piriform recessTongueAnteriorPosterior Fig. 8.222 Cricothyroid muscle.

1	Fig. 8.222 Cricothyroid muscle. Fig. 8.223 Crico-arytenoid, oblique and transverse arytenoid, and vocalis muscles. Fig. 8.224 Thyro-arytenoid muscle. Superior thyroid notchAry-epiglottic part ofoblique arytenoid muscleSacculeThyro-arytenoid muscleThyro-epiglotticpart of thyro-arytenoid muscle Fig. 8.225 Laryngeal function. A. Quiet respiration. B. Forced inspiration. C. Phonation. D. Effort closure. E. Swallowing. Quiet respirationForced inspirationPhonationEffort closureSwallowingVocal foldVocal foldsclosedVestibular foldVestibularfolds closedAry-epiglottic foldLaryngeal inletLaryngealinlet narrowedEpiglottis swingsdown to arytenoidsEpiglottis• Vocal folds abducted and rima glottidis wide open• Vestibule open• Vocal folds adducted and stridulating as air is forced between them• Vestibule open• Vocal folds and vestibular folds adducted• Rima glottidis and vestibule closedABCDE Fig. 8.226 Arterial supply of the larynx, left lateral view.

1	Fig. 8.226 Arterial supply of the larynx, left lateral view. Fig. 8.227 Venous drainage of the larynx, anterior view. Superior laryngeal veinMiddle thyroid veinSuperior thyroid veinThyrohyoid membraneMedian cricothyroid ligamentInferior thyroid veinThyroid glandManubrium of sternumRight subclavian veinInferior laryngeal veinRight internal jugular veinHyoid bone Fig. 8.228 Innervation of the larynx. Inferior vagal ganglionLeft vagus nerveThyrohyoid membranePosition of vocal foldsMedian cricothyroid ligamentLeft recurrent laryngeal nerveTracheaLeft subclavian arteryAortic archLeft pulmonary arteryRight pulmonary arteryLigamentum arteriosumPulmonary trunkEsophagusManubriumRight subclavian arteryRight recurrent laryngeal nerveCricothyroid muscleRight vagus nerveExternal laryngeal nerveSuperior laryngeal nerveInternal laryngeal nerve Fig. 8.229 Nasal cavities (anterolateral view). Relationship to other cavities.

1	Fig. 8.229 Nasal cavities (anterolateral view). Relationship to other cavities. Fig. 8.230 Nasal cavities. A. Floor, roof, and lateral walls. B. Conchae on lateral walls. C. Coronal section. D. Air channels in right nasal cavity. Fig. 8.231 Paranasal sinuses and nasolacrimal duct. Fig. 8.232 Regions of the nasal cavities. Fig. 8.233 Ethmoid bone. A. Overall shape. B. Coronal section through skull.

1	Fig. 8.231 Paranasal sinuses and nasolacrimal duct. Fig. 8.232 Regions of the nasal cavities. Fig. 8.233 Ethmoid bone. A. Overall shape. B. Coronal section through skull. ABAnteriorPosteriorCrista galliLeft ethmoidal labyrinthOrbital plateUncinate processMiddle conchaPerpendicular plateChannel for frontonasal ductopening into frontal sinusRight ethmoidal labyrinthCribriform plateSuperior conchaEthmoidal bullaMiddle conchaUncinate processInfundibulumCranial cavityCribriform platePerpendicular plateNasal cavitiesOrbitOrbitMiddle ethmoidal cellsSuperior conchaEthmoidal bullaMiddle conchaUncinate processInferior concha boneVomerOral cavityPalatine process of maxillary boneMaxillarysinusMaxillarysinusOrbital plate ofethmoidal labyrinthOrbital plate offrontal boneCrista galli Fig. 8.234 External nose.

1	Fig. 8.234 External nose. Nasal boneFrontal process of maxillaLacrimal boneNasolacrimal grooveMinor alar cartilagesSeptal cartilageNarisMajor alarcartilageSuperior margin ofseptal cartilageLateral process ofseptal cartilage Fig. 8.235 Paranasal sinuses. A. Anterior view. B. Posteroanterior skull radiograph. C. Paramedian view of right nasal cavity. D. Lateral skull radiograph. ABEthmoidalcellsFrontal sinusesZygomatic process of frontal boneSuperior orbital fissureFrontalsinusesEthmoidal cellsMaxillarysinusesMaxillary sinusRoots of posteriorupper molarsOrbital plate ofethmoid boneNasal septumForamen rotundum Fig. 8.236 Medial wall of the nasal cavity—the nasal septum. Nasal spine of frontal bonePerpendicular plate of ethmoid bonePituitary fossaSphenoidal sinusVomerNasal crest ofmaxillary andpalatine bonesIncisor crestSeptalcartilageNasal bone Fig. 8.237 Floor of the nasal cavity (superior view).

1	Fig. 8.237 Floor of the nasal cavity (superior view). Septal cartilageNarisAnterior nasal spineIncisive canalPalatine process of maxillaHorizontal plate of palatineSoft palateNasal crestsMaxillary sinus Fig. 8.238 Roof of the nasal cavity. Cribriform plateOpening of sphenoidal sinusAla of vomerVomerSphenoidal rostrum(articulates in themidline with the vomer)Nasal bonesNasal spine of frontal bone Fig. 8.239 Lateral wall of the nasal cavity. A. Bones. B. Covered with mucosa. C. Conchae broken away at attachment to lateral wall. AFrontal process of maxillaSuperior conchaMiddle conchaMedial pterygoid plate ofsphenoid boneUncinate process of ethmoidPerpendicular plateof palatine boneInferior conchaMinor alar cartilageMajor alar cartilageLateral process ofseptal cartilageLacrimal boneNasal bone

1	BCOpening ofpharyngotympanic tubeNasopharynxSoft palateInferior conchaMiddle conchaSuperior conchaOpening of posterior ethmoidalcells into lateral wall of superior meatusOpening of sphenoidal sinusinto spheno-ethmoidal recessOpening of middle ethmoidalcells onto ethmoidal bullaSemilunar hiatusOpening of maxillary sinus infloor of semilunar hiatus Opening of nasolacrimal ductInfundibulum opening of frontonasalduct that drains the frontal sinusand anterior ethmoidal cells Fig. 8.240 Nares. A. Inferior view. B. Associated muscles. NaresAMajor alar cartilageMinor alar cartilagesInferior nasal spine of maxillaConnective tissueSeptal cartilageBOrbitAttachment to frontalprocess of maxillaNasalis muscleAttachment to maxillaDepressor septi nasiNarisLevator labii superioris alaeque nasi Fig. 8.241 Choanae (posterior view). A. Overview. B. Magnified view.

1	Fig. 8.241 Choanae (posterior view). A. Overview. B. Magnified view. Ala of vomerSphenoidal rostrumSphenoidal process of palatine bonePalatine boneMaxillaPalatovaginal canalVomerBAVomerSphenoid boneMedial pterygoidplate of sphenoidHorizontal plate of palatine bonePyramidal process of palatine boneOral cavityChoanaeChoanaeVaginal process of medial pterygoid plate Fig. 8.242 Gateways to the nasal cavities. Fig. 8.243 Arterial supply of the nasal cavities. A. Lateral wall of the right nasal cavity. B. Septum (medial wall of right nasal cavity).

1	Fig. 8.242 Gateways to the nasal cavities. Fig. 8.243 Arterial supply of the nasal cavities. A. Lateral wall of the right nasal cavity. B. Septum (medial wall of right nasal cavity). ABAnterior ethmoidal arteryPosterior ethmoidal arterySuperior conchaMiddle conchaInferior conchaGreater palatine arteryPosterior lateral nasalbranches of sphenopalatine arterySphenopalatine arteryAlar branch oflateral nasal arteryExternal nasalartery from anteriorethmoidal arterySeptal branch ofanterior ethmoidal arterySeptal branch ofposterior ethmoidal arteryArea of significantanastomoses (proneto “nosebleeds”)Posterior septal branch ofsphenopalatine arteryTerminal part ofgreater palatine arterySeptal branch from nasalartery from superior labial artery Fig. 8.244 Venous drainage of the nasal cavities. Nasal vein in foramen cecumDrainage to cavernoussinus in cranial cavityDrainage to pterygoid plexusin infratemporal fossaDrainage to facial vein

1	Fig. 8.244 Venous drainage of the nasal cavities. Nasal vein in foramen cecumDrainage to cavernoussinus in cranial cavityDrainage to pterygoid plexusin infratemporal fossaDrainage to facial vein Fig. 8.245 Innervation of the nasal cavities. A. Lateral wall of right nasal cavity. B. Medial wall of right nasal cavity. ABPosterior inferiorlateral nasal nervesInternal nasal branchesof infra-orbital nerveExternal nasalbranch of anteriorethmoidal nerveSeptal branch ofanterior ethmoidal nerveOlfactory nerve [I](septal branches)Olfactory bulbAnterior ethmoidal nerveOlfactory nerve [I]Sphenopalatine foramenPosterior superiorlateral nasal nervesNasal branch of anteriorsuperior alveolar nerveNasopalatine nerve Fig. 8.246 Lymphatic drainage of the nasal cavities. Fig. 8.247 Oral cavity. A. Relationship to other cavities. B. Oral vestibule and oral cavity proper.

1	Fig. 8.246 Lymphatic drainage of the nasal cavities. Fig. 8.247 Oral cavity. A. Relationship to other cavities. B. Oral vestibule and oral cavity proper. Fig. 8.248 Base and lateral aspects of the skull. A. Features in the base of the skull related to structures associated with the oral cavity. B. Styloid process of the temporal bone.

1	Fig. 8.248 Base and lateral aspects of the skull. A. Features in the base of the skull related to structures associated with the oral cavity. B. Styloid process of the temporal bone. Petrous part of temporal boneScaphoid fossaForamen ovaleSpine of sphenoidOpening to bony partof pharyngotympanic tubeForamen spinosumCarotid canalStyloid process of temporal boneMastoid processStylomastoid foramenJugular foramenRoughening for attachment of levator veli palatiniForamen lacerum (closed by cartilage)Cartilaginous part of pharyngotympanic tubeGreater wing of sphenoidMembranous lamina ofcartilaginous part of pharyngotympanic tubeAIncisive fossaPalatine process of maxillaAlveolar process of maxillaHorizontal plate of palatine boneGreater palatine foramenLesser palatine foramenLateral plate of pterygoid processMedial plate of pterygoid processIntermaxillary suturePosterior nasal spinePyramidal process of palatine bonePterygoid hamulus

1	Fig. 8.249 Mandible. A. Superior view. B. Lateral view. C. Medial view. Fig. 8.250 Hyoid bone. A. Anterior view. B. Lateral view. Fig. 8.251 Buccinator muscle. Attachment to maxillaSuperior constrictorPterygomandibular rapheAttachment to mandibleBuccinatorOrbicularis orisModiolus Fig. 8.252 A. Mylohyoid muscles. B. Geniohyoid muscles. C. Lateral view. Superior mental spinesGeniohyoidGeniohyoidMylohyoidMylohyoidSuperior mental spinesMylohyoid lineInferior mental spinesRapheGreater hornBody of hyoidFree posterior marginSubmandibular fossaSublingual fossa ABC Fig. 8.253 Gateway into the floor of the oral cavity. Superiorconstrictorof pharynxMiddleconstrictorof pharynxMylohyoidTriangular aperture (oropharyngeal triangle) between mylohyoid,superior constrictor, and middle constrictor Fig. 8.254 Tongue. A. Paramedian sagittal section. B. Superior view.

1	Fig. 8.254 Tongue. A. Paramedian sagittal section. B. Superior view. AOral part (anterior two-thirds)Foramen cecumand terminal sulcusPharyngeal part (posterior one-third)Hyoid boneRoot of tongueMylohyoid muscleGeniohyoid muscleMandibleInferior surfaceLower lipOral vestibuleFiliform papillaeTerminal sulcusForamen cecumBOropharynxPharyngeal part of tongueVallate papillaeFoliate papillaeFungiform papillae Fig. 8.255 Muscles of the tongue. Fig. 8.256 Genioglossus muscles. A. Posterior view. B. Lateral (left) view. Fig. 8.257 Hyoglossus muscles. A. Posterior view. B. Lateral (left) view. Fig. 8.258 Styloglossus muscles. Fig. 8.259 Palatoglossus muscles. Hard palatePalatine aponeurosisof soft palatePalatoglossus muscle (underliesthe palatoglossusarch of mucosa)Uvula Fig. 8.260 Arteries, veins, and nerves of the tongue.

1	Hard palatePalatine aponeurosisof soft palatePalatoglossus muscle (underliesthe palatoglossusarch of mucosa)Uvula Fig. 8.260 Arteries, veins, and nerves of the tongue. Lingual nerve(from [V3])Chorda tympani (from [VII])Hypoglossalnerve [XII]OccipitalarterySternocleidomastoid branch of occipital arteryLingual arteryCommon carotid arteryInternal jugular veinDorsal lingual veinDeep lingual veinHyoglossusGlossopharyngealnerve [IX] Fig. 8.261 Innervation of the tongue. Fig. 8.262 Lingual nerve in the floor of the oral cavity (medial view). Fig. 8.263 Hypoglossal nerve and C1 fibers. Hypoglossal nerveC1C1 fibersC3C2ThyrohyoidSuperior root ofansa cervicalisNerve to thyrohyoid(C1)GeniohyoidNerve to geniohyoid (C1) Fig. 8.264 Parotid gland. MasseterBuccinatorParotid duct(penetrates buccinator oppositecrown of 2nd upper molar tooth)SternocleidomastoidParotid glandExternal acoustic meatus

1	Fig. 8.264 Parotid gland. MasseterBuccinatorParotid duct(penetrates buccinator oppositecrown of 2nd upper molar tooth)SternocleidomastoidParotid glandExternal acoustic meatus Fig. 8.265 Submandibular and sublingual glands. A. Medial view. B. Posterior view. C. Anterior view. D. Anterosuperior view. Superior constrictor musclePterygomandibular rapheSmall ducts of sublingual glandSubmandibular ductSublingual glandSublingual glandSubmandibular ductGenioglossus muscleHyoglossus muscleSuperficialDeepSuperficialDeepSubmandibularglandLingual nerveABSubmandibular ductSublingual fold overlyingsublingual glandSublingual caruncleOpening ofsubmandibular ductOpening of leftsubmandibular ductSublingual carunclesLingual veinFrenulum of tongueOpening of ducts from sublingual glandCDDeep lingual veinFimbriated foldFrenulum of tongue Fig. 8.266 Summary of parasympathetic (secretomotor) innervation of glands in the head.

1	Fig. 8.266 Summary of parasympathetic (secretomotor) innervation of glands in the head. Lacrimal glandGlands onpalatePterygopalatine ganglionPalatine nerveLabial glandsLingual glandsSublingual glandSubmandibular glandSubmandibular ganglionOtic ganglionAuriculotemporal nerve (from [V3])Parotid gland innervated by [IX]Chorda tympani[V][VII][IX]Preganglionic parasympathetic fibers from [IX]Greater petrosal nerveAll glands abovelevel of oral fissureinnervated by greater petrosal of [VII]All glands below level of oral fissureinnervated bychorda tympani of [VII] Fig. 8.267 Course of parasympathetic fibers carried in the chorda tympani nerve. Fig. 8.268 Summary of sympathetic innervation of glands in the head. Fig. 8.269 Palate. Fig. 8.270 A. Tensor veli palatini muscles and the palatine aponeurosis. B. Levator veli palatini muscles. C. Palatopharyngeus muscles.

1	Fig. 8.269 Palate. Fig. 8.270 A. Tensor veli palatini muscles and the palatine aponeurosis. B. Levator veli palatini muscles. C. Palatopharyngeus muscles. Muscular part of tensor veli palatiniCartilaginous part ofpharyngotympanic tubeFibrous part of pharyngotympanic tubePalatine aponeurosisPterygoid hamulusPosition of palatopharyngealsphincterPharyngeal rapheSuperior constrictor of pharynxPterygomandibular rapheBuccinator musclePterygopalatine fossaABCNasal cavityMedial pterygoid plateLateral pterygoid plateNasal septumLevator veli palatiniPalatopharyngeus Fig. 8.271 Open mouth with soft palate. A. Oropharyngeal isthmus opened. B. Oropharyngeal isthmus closed.

1	Fig. 8.271 Open mouth with soft palate. A. Oropharyngeal isthmus opened. B. Oropharyngeal isthmus closed. ABPalatoglossal archPalatopharyngeal archPosterior wall of oropharynxSoft palateSoft palateTongueUvulaPalatine tonsilAnterior margin oforopharyngeal isthmus(palatoglossal arch)Closure of oropharyngeal isthmus• Medial and downward movement of palatoglossal arches• Medial and downward movement of palatopharyngeal arches• Upward movement of tongue• Downward and forward movement of soft palate Fig. 8.272 Palatoglossus muscles and musculus uvulae. Musculus uvulaePalatoglossusfrom underside of aponeurosisPalatine tonsil Fig. 8.273 Arteries of the palate. Fig. 8.274 Palatine nerves and arteries. Incisive fossaNasopalatine nerveGreater palatine nerveGreater palatine foramenLesser palatine foramenLesser palatine nerveUvulaBranches from ascending palatine artery of facial artery and palatine branch of ascending pharyngeal arteryLesser palatine arteryGreater palatine artery

1	Fig. 8.275 Venous and lymphatic drainage of the palate. Fig. 8.276 Innervation of the palate. Greater petrosal nerve (preganglionic parasympatheticand special sensory [taste])[VII]Deep petrosal nerve (postganglionic sympathetic)Superior cervical sympathetic ganglionSympathetic trunkPreganglionicsympathetic from T1UvulaInternal carotid arteryNerve of pterygoid canalLesser palatine nerveLesser palatine foramenGreater palatine nerveNasopalatine nerveGreater palatine foramenPalatine canalPterygopalatine ganglionNasopalatine nerveMaxillary nerve[V] Fig. 8.277 Oral fissure and lips. A. Anterior view. B. Sagittal section. PhiltrumVestibuleOrbicularis oris muscleOral fissureVermilion border of lipFacial arterySuperior and inferior labial arteriesABLabial salivary glandsArtery and veinVermilion bordersOrbicularis oris muscleBuccinator muscle Fig. 8.278 Teeth. A. Adult upper and lower permanent teeth. B. Deciduous (“baby”) teeth.

1	Fig. 8.278 Teeth. A. Adult upper and lower permanent teeth. B. Deciduous (“baby”) teeth. AIncisorsCaninesPremolarsMolarsMolarsPremolarsCaninesIncisorsMaxillary sinusRoots related to maxillary sinusPremolars3212132121Roots related tomandibular canalMolarsCanineIncisorsUpperLowerMolarsIncisorsCaninesBUpperLower Fig. 8.279 Arteries and veins of the teeth. Cavernous sinusin cranial cavityEmissary veinsInfra-orbitalartery and veinPosterior superior alveolarartery and veinAnterior superior alveolarartery and veinInferior alveolar artery andvein in mandibular canalFacial veinInternal jugular veinExternal jugular veinExternal carotid arteryPterygoid plexus of veinsRetromandibular veinMaxillary veinMaxillary artery Fig. 8.280 Lymphatic drainage of the teeth and gums. Fig. 8.281 Innervation of the teeth. Fig. 8.282 Innervation of the teeth and gums.

1	Fig. 8.280 Lymphatic drainage of the teeth and gums. Fig. 8.281 Innervation of the teeth. Fig. 8.282 Innervation of the teeth and gums. TeethAnterior superior alveolar nerve (from [V2])Middle superior alveolar nerve (from [V2])Posterior superioralveolar nerve (from [V2])UpperLowerMain trunk of inferioralveolar nerve (from [V3])Incisive branch of inferioralveolar nerve (from [V3])Nasopalatine nerve (from [V2])Greater palatine nerve (from [V2])GingivaeAnterior superior alveolar nerve (from [V2])Middle superior alveolar nerve (from [V2])Posterior superior alveolar nerve (from [V2]) Lingual nerve (from [V3])Buccal nerve (from [V3])Mental nerve from inferior alveolar nerve (from [V3]) Fig. 8.283 Anatomical position of the head and major landmarks. Lateral head and neck of a man.

1	Fig. 8.283 Anatomical position of the head and major landmarks. Lateral head and neck of a man. External occipitalprotuberanceCervical spinal nervesExternal acoustic meatusSternocleidomastoid muscleAngle of mandibleMastoid processVertexZygomatic boneFrankfort lineInferior margin of orbitPosition of head of mandiblePosition of zygomatic arch[V1][V2][V3] Fig. 8.284 Visualizing structures at the CIII/IV and CVI vertebral levels. Lateral head and neck of a man. Vertebral level CVI• Arch of cricoid cartilage• Superior end of esophagus• Superior end of tracheaVertebral level CIII/IV• Upper margin of thyroid cartilage• Bifurcation of common carotid arteryBifurcation of common carotidFrankfort lineEsophagusArch of cricoidPharynx Fig. 8.285 How to outline the anterior and posterior triangles of the neck. A. In a woman, anterolateral view. The left anterior triangle is indicated. B. In a man, anterior view of the posterior triangle.

1	ABMidline of neckAnterior margin ofsternocleidomastoidAnterior margin oftrapeziusClaviclePosterior margin ofsternocleidomastoidPosterior triangleAnterior triangleInferior margin of mandibleStructures coursing betweenhead and thorax are associatedwith the anterior trianglesStructures coursing betweenthorax/neck and upper limb areassociated with the posterior triangles Fig. 8.286 How to locate the median cricothyroid ligament. A. In a man, lateral view of head and neck. B. In a woman, lateral view of head and neck. C. In a man, anterior neck with the chin elevated. D. In a woman, anterior neck with the chin elevated. ABThyroid notchLaryngeal prominencePosition of mediancricothyroid ligamentArch of cricoid cartilage Thyroid notchLaryngeal prominencePosition of median cricothyroid ligamentArch of cricoidcartilageIsthmus of thyroid glandCD Fig. 8.287 How to find the thyroid gland. A. In a woman, anterior view of neck. B. In a man, anterior view of neck.

1	Fig. 8.287 How to find the thyroid gland. A. In a woman, anterior view of neck. B. In a man, anterior view of neck. Position of oblique line on thyroid cartilageHyoid boneThyroid notchLaryngeal prominenceMedian cricothyroidligamentArch of cricoidLeft lobe of thyroid glandRight lobe of thyroid glandIsthmus of thyroid glandAB Fig. 8.288 Estimating the position of the middle meningeal artery. Lateral head and neck of a man. External earExternal acoustic meatusPterionFrankfort lineInferior margin of orbitSuperior margin of orbit Fig. 8.289 Major features of the face. Anterior head and neck of a woman. Region for testing [V1]Palpebral fissureOral fissureNostril[V1][V2][V3]Region fortesting [V2]Region for testingsensory of [V3]Orbicularis orisPhiltrumOrbicularis oculi

1	Region for testing [V1]Palpebral fissureOral fissureNostril[V1][V2][V3]Region fortesting [V2]Region for testingsensory of [V3]Orbicularis orisPhiltrumOrbicularis oculi Fig. 8.290 Eye and lacrimal apparatus. A. Face of a woman. Lacrimal apparatus and the flow of tears are indicated. B. Left eye and surrounding structures. C. Left eye and surrounding structures with lower eyelid pulled down to reveal the lacrimal papilla and lacrimal punctum. ALacrimal glandFlow of tearsNasolacrimal ductInferior canaliculusLacrimal sacUpper eyelidLower eyelidLacrimal lakeScleraPupilPalpebral fissureLacrimal foldLacrimal caruncleIrisMedial commissureLateral commissureLacrimal papillaLacrimal punctumBC Fig. 8.291 External ear. Lateral view of the right ear of a woman. Fig. 8.292 Where to take arterial pulses in the head and neck. Fig. 8.293 Coronal CT scan demonstrating an orbital blowout fracture. eFig. 8.294 Ultrasound scan (axial view) demonstrating a stone in a dilated parotid duct.

1	Fig. 8.293 Coronal CT scan demonstrating an orbital blowout fracture. eFig. 8.294 Ultrasound scan (axial view) demonstrating a stone in a dilated parotid duct. eFig. 8.295 Coronal MRI showing pituitary macroadenoma. Table 8.1 External foramina of the skull Table 8.2 Internal foramina of the skull Table 8.3 Dural venous sinuses Table 8.4 Cranial nerve functional components Other terminology used when describing functional components: *Special sensory, or special visceral afferent (SVA): smell, taste. Special somatic afferent (SSA): vision, hearing, balance. **Special visceral efferent (SVE) or branchial motor. Table 8.5 Cranial nerves (see Table 8.4 for abbreviations) Table 8.6 Parasympathetic ganglia of the head Table 8.7 Muscles of the face Table 8.8 Extrinsic (extra-ocular) muscles Table 8.9 Intrinsic muscles of the eye Table 8.10 Muscles of the middle ear Table 8.11 Muscles of mastication Table 8.12 Anterior triangle of neck (suprahyoid and infrahyoid muscles)

1	Table 8.9 Intrinsic muscles of the eye Table 8.10 Muscles of the middle ear Table 8.11 Muscles of mastication Table 8.12 Anterior triangle of neck (suprahyoid and infrahyoid muscles) Table 8.13 Branches of the external carotid artery Table 8.14 Subdivisions of the anterior triangle of the neck—a regional approach Table 8.15 Muscles associated with the posterior triangle of the neck; parentheses indicate possible involvement Table 8.16 Prevertebral and lateral vertebral muscles Table 8.17 Constrictor muscles of the pharynx Table 8.18 Longitudinal muscles of the pharynx Table 8.19 Intrinsic muscles of the larynx Table 8.20 Muscles in the floor of the oral cavity Table 8.21 Muscles of the tongue Table 8.22 Muscles of the soft palate In the clinic

1	Table 8.19 Intrinsic muscles of the larynx Table 8.20 Muscles in the floor of the oral cavity Table 8.21 Muscles of the tongue Table 8.22 Muscles of the soft palate In the clinic Some babies can be born with ossified fusion (synostosis) of one or more of the cranial sutures. This can result in an irregular head shape because the pattern and direction of skull growth are altered. In the majority of cases the cause is unknown, and in a minority of cases it may be caused by a genetic syndrome. In the clinic Medical imaging of the head

1	In the clinic Medical imaging of the head Until recently, the standard method of imaging the head was plain radiography. The radiographs are taken in three standard projections—the posteroanterior view, the lateral view, and the Towne’s view (anteroposterior [AP] axial—head in anatomical position). Additional views are obtained to assess the foramina at the base of the skull and the facial bones. Currently, skull radiographs are used in cases of trauma, but such use is declining. Skull fractures are relatively easily detected (Fig. 8.29). The patient is assessed and treatment is based upon the underlying neurological or potential neurological complications.

1	Since the development of computed tomography (CT), cerebral CT has become the “workhorse” of neuroradiological examination. It is ideally used for head injury because the brain and its coverings can be easily and quickly examined and blood is easily detected. By altering the mathematical algorithm of the data set the bones can also be demonstrated. With intravenous contrast, CT angiography can be used to demonstrate the position and the size of an intracerebral aneurysm before endovascular treatment. Magnetic resonance imaging (MRI) is unsurpassed by other imaging techniques in its ability for contrast resolution. The brain and its coverings, cerebrospinal fluid (CSF), and vertebral column can be easily and quickly examined. Newer imaging sequences permit CSF suppression to define periventricular lesions.

1	Magnetic resonance angiography has been extremely useful in determining the completeness of the intracranial vasculature (circle of Willis), which is necessary in some surgical conditions. MRI is also a powerful tool in the assessment of carotid stenosis. It is now possible to carry out intracranial Doppler studies, which enable a surgeon to detect whether a patient is experiencing cerebral embolization from a carotid plaque. Extracranial ultrasound is extremely important in tumor staging and in assessing neck masses and the carotid bifurcation (Fig. 8.30). Ultrasound is useful in children because they have an acoustic window through the fontanelles. In the clinic Fractures of the skull vault

1	Ultrasound is useful in children because they have an acoustic window through the fontanelles. In the clinic Fractures of the skull vault The skull vault is a remarkably strong structure because it protects our most vital organ, the brain. The shape of the skull vault is of critical importance and its biomechanics prevent fracture. From a clinical standpoint skull fractures alert clinicians to the nature and force of an injury and potential complications. The fracture itself is usually of little consequence (unlike, say, a fracture of the tibia). Of key importance is the need to minimize the extent of primary brain injury and to treat potential secondary complications, rather than focusing on the skull fracture. Skull fractures that have particular significance include depressed skull fractures, compound fractures, and pterion fractures.

1	In a depressed skull fracture a bony fragment is depressed below the normal skull convexity. This may lead to secondary arterial and venous damage with hematoma formation. A primary brain injury can also result from this type of fracture. In a compound fracture there is a fracture of the bone together with a breach of the skin, which may allow an infection to enter. Typically these fractures are associated with scalp lacerations and can usually be treated with antibiotics. Important complications of compound fractures include meningitis, which may be fatal. A more subtle type of compound fracture involves fractures across the sinuses. These may not be appreciated on first inspection, but are an important potential cause of morbidity and should be considered in patients who develop intracranial infections secondary to trauma.

1	The pterion is an important clinical point on the lateral aspect of the skull. At the pterion the frontal, parietal, greater wing of the sphenoid, and temporal bones come together. Importantly, deep to this structure is the middle meningeal artery. An injury to this point of the skull is extremely serious because damage to this vessel may produce a significant extradural hematoma, which can be fatal. In the clinic Hydrocephalus is a dilation of the cerebral ventricular system, which is due to either an obstruction to the flow of CSF, an overproduction of CSF, or a failure of reabsorption of CSF.

1	Hydrocephalus is a dilation of the cerebral ventricular system, which is due to either an obstruction to the flow of CSF, an overproduction of CSF, or a failure of reabsorption of CSF. Cerebrospinal fluid is secreted by the choroid plexus within the lateral, third, and fourth ventricles of the brain. As it is produced it passes from the lateral ventricles through the interventricular foramina (the foramina of Monro) to enter the third ventricle. From the third ventricle it passes through the cerebral aqueduct (aqueduct of Sylvius) into the fourth ventricle, and from here it passes into the subarachnoid space via the midline foramen or the two lateral foramina (foramen of Magendie and foramina of Luschka). The CSF passes around the spinal cord inferiorly, envelops the brain superiorly, and is absorbed through the arachnoid granulations in the walls of the dural venous sinuses. In adults almost half a liter of CSF is produced per day.

1	In adults the commonest cause of hydrocephalus is an interruption of the normal CSF absorption through the arachnoid granulations. This occurs when blood enters the subarachnoid space after subarachnoid hemorrhage, passes over the brain, and interferes with normal CSF absorption. To prevent severe hydrocephalus it may be necessary to place a small catheter through the brain into the ventricular system to relieve the pressure. Other causes of hydrocephalus include congenital obstruction of the aqueduct of Sylvius and a variety of tumors (e.g., a midbrain tumor), where the mass obstructs the aqueduct. Rare causes include choroid plexus tumors that secrete CSF.

1	In children, hydrocephalus is always dramatic in its later stages. The hydrocephalus increases the size and dimensions of the ventricle, and as a result the brain enlarges. Because the skull sutures are not fused, the head expands. Cranial enlargement in utero may make a vaginal delivery impossible, and delivery then has to be by caesarean section. Both CT and MRI enable a radiologist to determine the site of obstruction and in most cases the cause of the obstruction. A distinction must be made between ventricular enlargement due to hydrocephalus and that due to a variety of other causes (e.g., cerebral atrophy). In the clinic Leakage of CSF from the subarachnoid space may occur after any procedure in and around the brain, spinal cord, and meningeal membranes. These procedures include lumbar spine surgery, epidural injection, and CSF aspiration.

1	In “cerebrospinal fluid leak” syndrome, CSF leaks out of the subarachnoid space and through the dura mater for no apparent reason. The clinical consequences of this include dizziness, nausea, fatigue, and a metallic taste in the mouth. Other effects also include facial nerve weakness and double vision. In the clinic Meningitis is a rare infection of the leptomeninges (the leptomeninges are a combination of the arachnoid mater and the pia mater). Infection of the meninges typically occurs via a blood-borne route, though in some cases it may be by direct spread (e.g., trauma) or from the nasal cavities through the cribriform plate in the ethmoid bone. Certain types of bacterial inflammation of the meninges are so virulent that overwhelming inflammation and sepsis with cerebral irritation can cause the patient to rapidly pass into a coma and die. Meningitis is usually treatable with antibiotics.

1	Meningitis is usually treatable with antibiotics. Certain types of bacteria that produce meningitis produce other effects; for example, subcutaneous hemorrhage (ecchymoses) is a feature of meningococcal meningitis. The typical history of meningitis is nonspecific at first. The patient may have mild headache, fever, drowsiness, and nausea. As the infection progresses, photophobia (light intolerance) and ecchymosis may ensue. Straight leg raising causes marked neck pain and discomfort (Kernig’s sign) and an emergency hospital admission is warranted. Immediate treatment consists of very-high-dose intravenous antibiotics and supportive management. In the clinic Determination of the anatomical structure from which a tumor arises is of the utmost importance, particularly when it arises within the cranial vault. Misinterpretation of the location of a lesion and its site of origin may have devastating consequences for the patient.

1	When assessing any lesion in the brain, it is important to define whether it is intra-axial (within the brain) or extra-axial (outside the brain). Typical extra-axial tumors include meningiomas (tumors of the meninges) and acoustic neuromas. Meningiomas typically arise from the meninges, with preferred sites including regions at and around the falx cerebri, the free edge of the tentorium cerebelli, and the anterior margin of the middle cranial fossa. Acoustic neuromas are typically at and around the vestibulocochlear nerve [VIII] and in the cerebellopontine angle. Intra-axial lesions are either primary or secondary. By far the commonest type are the secondary brain lesions, which in most cases are metastatic tumor deposits. Metastatic tumor lesions are typically found in patients with either breast carcinoma or lung carcinoma, though many other malignancies can give rise to cerebral metastases.

1	Metastatic tumor lesions are typically found in patients with either breast carcinoma or lung carcinoma, though many other malignancies can give rise to cerebral metastases. Primary brain lesions are rare and range from benign tumors to extremely aggressive lesions with a poor prognosis. These tumors arise from the different cell lines and include gliomas, oligodendrocytomas, and choroid plexus tumors. Primary brain tumors may occur at any age, though there is a small peak incidence in the first few years of life followed by a later peak in early to middle age. In the clinic

1	In the clinic A stroke, or cerebrovascular accident (CVA), is defined as the interruption of blood flow to the brain or brainstem resulting in impaired neurological function lasting more than 24 hours. Neurological impairment resolving within 24 hours is known as a transient ischemic attack (TIA) or mini-stroke. Based on their etiology, strokes are broadly classified as either ischemic or hemorrhagic. Ischemic strokes are further divided into those caused by thrombotic or embolic phenomena. The latter is by far the commonest type of stroke and is often caused by emboli that originate from atherosclerotic plaques in the carotid arteries that migrate into and block smaller intracranial vessels. Hemorrhagic strokes are caused by rupture of blood vessels.

1	The risk factors for stroke are those of cardiovascular disease, such as diabetes, hypertension, and smoking. In younger patients underlying clotting disorders, use of oral contraceptives, and illicit substance abuse (such as cocaine) are additional causes. The symptoms and signs of a stroke depend on the distribution of impaired brain perfusion. Common presentations include rapid-onset hemiparesis or hemisensory loss, visual field deficits, dysarthria, ataxia, and a decreased level of consciousness. Stroke is a neurological emergency. It is therefore important to establish the diagnosis as early as possible so that urgent and potentially life-saving treatment can be administered. Potent thrombolytic (blood-thinning) drugs can restore cerebral blood flow and improved patient outcome if administered within 3 to 4.5 hours of onset of the patient’s symptoms.

1	Following initial clinical history taking and neurological examination, all patients with suspected stroke should undergo urgent brain imaging with computed tomography (CT). This is to identify hemorrhagic strokes for which thrombolytic therapy is contraindicated and to exclude an alternative diagnosis such as malignancy. In ischemic stroke, early CT imaging may appear normal or can show a relatively darker area of low density that corresponds to the region of abnormal brain perfusion. Due to subsequent brain edema and swelling, the affected brain also loses its normal sulcal pattern (Fig. 8.40A). If thrombolysis is performed, a 24-hour follow-up CT scan is routinely carried out to evaluate for complications such as intracranial hemorrhage.

1	Additional diagnostic workup of stroke includes hematological and biochemical blood tests to identify causes such as hypoglycemia or underlying clotting disorders. A toxicology screen may be useful to identify substance intoxication, which can mimic stroke.

1	The full extent of neurological injury can be evaluated on subsequent magnetic resonance imaging (MRI) of the brain, which has better soft tissue resolution compared to CT. MRI is also useful for identifying strokes that may be too small to detect on a CT scan. MRI scans are produced by using complicated algorithms that create a series of images, also known as sequences. Various sequences can be obtained to assess different anatomical and physiological properties of the brain. A stroke, whether acute or chronic, will appear as a bright region on a sequence that is sensitive to fluid (T2 weighted) (Fig. 8.40B). To identify whether a stroke is acute, further sequences are obtained, known as diffusion-weighted imaging (DWI) (Fig. 8.40C) and the apparent diffusion coefficient (ADC) (Fig. 8.40D) map. These evaluate the diffusion of water molecules in the brain. If the region of abnormality appears bright on the DWI sequence and dark on the ADC map, this is known as restricted diffusion,

1	map. These evaluate the diffusion of water molecules in the brain. If the region of abnormality appears bright on the DWI sequence and dark on the ADC map, this is known as restricted diffusion, which is compatible with an acute stroke. These changes can persist for up to a week after the initial insult.

1	Imaging of the carotid and vertebral arteries is also performed to assess for any treatable atherosclerotic changes and stenosis. This can be done with ultrasound, CT, or less frequently, MRI. Management of a stroke is multidisciplinary. Supportive treatment to stabilize the patient is a priority. Stroke specialists, speech and language therapists, occupational therapists, and physiotherapists have key roles in patient rehabilitation. Long-term use of antiplatelet drugs such as aspirin and modification of cardiovascular disease risk factors are important in the secondary prevention of stroke. In the clinic Endarterectomy is a surgical procedure to remove atheromatous plaque from arteries.

1	In the clinic Endarterectomy is a surgical procedure to remove atheromatous plaque from arteries. Atheromatous plaques occur in the subendothelial layer of vessels and consist of lipid-laden macrophages and cholesterol debris. The developing plaque eventually accumulates fibrous connective tissue and calcifies. Plaque commonly occurs around vessel bifurcations, limiting blood flow, and may embolize to distal organs. During endarterectomy, plaque is removed and the vessel reopened. In many instances a patch of material is sewn over the hole in the vessel, enabling improved flow and preventing narrowing from the suturing of the vessel. In the clinic

1	In the clinic Cerebral aneurysms arise from the vessels in and around the cerebral arterial circle (of Willis). They typically occur in and around the anterior communicating artery, the posterior communicating artery, the branches of the middle cerebral artery, the distal end of the basilar artery (Fig. 8.41), and the posterior inferior cerebellar artery.

1	As the aneurysms enlarge, they have a significant risk of rupture. Typically patients have no idea that there is anything wrong. As the aneurysm ruptures, the patient complains of a sudden-onset “thunderclap” headache that produces neck stiffness and may induce vomiting. In a number of patients death ensues, but many patients reach the hospital, where the diagnosis is established. An initial CT scan demonstrates blood within the subarachnoid space, and this may be associated with an intracerebral bleed. Further management usually includes cerebral angiography, which enables the radiologist to determine the site, size, and origin of the aneurysm.

1	Usually patients undergo complex surgery to ligate the neck of the aneurysm. More recently radiological intervention has superseded the management of some aneurysms in specific sites. This treatment involves cannulation of the femoral artery, and placement of a long catheter through the aorta into the carotid circulation and thence into the cerebral circulation. The tip of the catheter is placed within the aneurysm and is packed with fine microcoils (Fig. 8.42), which seals the rupture. In the clinic Summary of relationships and clinical significance of the scalp and meninges (Fig. 8.47). In the clinic Head trauma is a common injury and is a significant cause of morbidity and death. Head injury may occur in isolation, but often the patient has other injuries; it should always be suspected in patients with multiple injuries. Among patients with multiple trauma, 50% die from the head injury. At the time of the initial head injury two processes take place.

1	At the time of the initial head injury two processes take place. First the primary brain injury may involve primary axonal and cellular damage, which results from the shearing deceleration forces within the brain. These injuries are generally not repairable. Further primary brain injuries include intracerebral hemorrhage and penetrating injuries, which may directly destroy gray and white matter. The secondary injuries are sequelae of the initial trauma. They include scalp laceration, fracture of the cranial vault, disruption of intracerebral arteries and veins, intracerebral edema, and infection. In most cases these can be treated if diagnosed early, and rapid and effective treatment will significantly improve the patient’s recovery and prognosis. In the clinic Types of intracranial hemorrhage The many causes of a primary brain hemorrhage include aneurysm rupture, hypertension (intracerebral hematoma secondary to high blood pressure), and bleeding after cerebral infarction.

1	The many causes of a primary brain hemorrhage include aneurysm rupture, hypertension (intracerebral hematoma secondary to high blood pressure), and bleeding after cerebral infarction. An extradural hemorrhage (Fig. 8.48) is caused by arterial damage and results from tearing of the branches of the middle meningeal artery, which typically occurs in the region of the pterion. Blood collects between the periosteal layer of the dura and the calvaria and under arterial pressure slowly expands. The typical history is of a blow to the head (often during a sporting activity) that produces a minor loss of consciousness. Following the injury the patient usually regains consciousness and has a lucid interval for a period of hours. After this, rapid drowsiness and unconsciousness ensue, which may lead to death.

1	A subdural hematoma (Fig. 8.49) results from venous bleeding, usually from torn cerebral veins where they enter the superior sagittal sinus. The tear and resulting seepage of blood separates the thin layer of dural border cells from the rest of the dura as the hematoma develops. Patients at most risk of developing a subdural hematoma are the young and elderly. The increased CSF space in patients with cerebral atrophy results in a greater than normal stress on the cerebral veins entering the sagittal sinus. The clinical history usually includes a trivial injury followed by an insidious loss of consciousness or alteration of personality. Subarachnoid hemorrhage (Fig. 8.50) may occur in patients who have undergone significant cerebral trauma, but typically it results from a ruptured intracerebral aneurysm arising from the vessels supplying and around the arterial circle (of Willis). In the clinic Tuberculosis of the central nervous system

1	In the clinic Tuberculosis of the central nervous system Tuberculosis (TB) may invade the central nervous system, including the brain, spinal cord, and meninges (Fig. 8.51). Symptoms of brain TB include headache, neck stiffness, weight loss, and fever. Symptoms of spinal cord TB include leg weakness and fecal and urinary incontinence. Meningitis can cause altered mental status, fever, and seizures. Treatment usually requires a cocktail of drugs for 1 year, but treatment for brain TB can require 2 years. In the clinic Emissary veins connect extracranial veins with intracranial veins and are important clinically because they can be a conduit through which infections can enter the cranial cavity. Emissary veins lack valves, as do the majority of veins in the head and neck. In the clinic

1	In the clinic Concussion (mild traumatic brain injury [MTBI]) is the most common type of traumatic brain injury. The injury typically results from a rapid deceleration of the head or by a rotation of the brain within the cranial cavity. General symptoms of MTBI can include posttraumatic amnesia, confusion, loss of consciousness, headache, dizziness, vomiting, lack of motor coordination, and light sensitivity. The diagnosis of concussion, MTBI, is based on the event, the current neurological status, and the state of consciousness of the patient. In the clinic Clinical assessment of patients with head injury Clinical assessment of patients with head injury always appears relatively straightforward. In reality it is usually far from straightforward. Patients may have a wide spectrum of modes of injury from a simple fall to complex multiple trauma. The age of the patient and ability to communicate about the injuries are important factors.

1	Patients may have a wide spectrum of modes of injury from a simple fall to complex multiple trauma. The age of the patient and ability to communicate about the injuries are important factors. The circumstances in which the injury may have occurred should be documented because some head injuries result from a serious assault, and the physician may be required to give evidence to a court of law. Determining the severity of head injury may be difficult because some injuries occur as a result of or in association with alcohol intoxication. Even when the diagnosis has been made and the correct management has been instigated, the circumstances in which the injury occurred and the environment to which the patient will return after treatment need to be reviewed to prevent further injuries (e.g., an elderly person tripping on loose carpet on a staircase).

1	A thorough clinical examination includes all systems, but with a special focus on the central and peripheral nervous systems. The level of consciousness must also be assessed and accurately documented using the Glasgow Coma Scale, which allows clinicians to place a numerical value upon the level of consciousness so that any deterioration or improvement can be measured and quantified. The Glasgow Coma Scale was proposed in 1974 and is now widely accepted throughout the world. There is a total score of 15 points, such that 15/15 indicates that the patient is alert and fully oriented, whereas 3/15 indicates a severe and deep coma. The points score comprises a best motor response (total of 6 points), best verbal response (total of 5 points), and best eye movement response (total of 4 points). In the clinic Treatment of head injury

1	In the clinic Treatment of head injury Treatment of primary brain injury is extremely limited. Axonal disruption and cellular death are generally irrecoverable. Whenever the brain is injured, like most tissues, it swells. Because the brain is encased within a fixed space (the skull), swelling impairs cerebral function and has two other important effects. First, the swelling compresses the blood supply into the skull, resulting in a physiologically dramatic increase in blood pressure. Second, the cerebral swelling may be diffuse, eventually squeezing the brain and brainstem through the foramen magnum (coning). This compression and disruption of the brainstem may lead to a loss of basic cardiorespiratory function, and death will ensue. Focal cerebral edema may cause one side of the brain to herniate beneath the falx cerebri (falcine herniation).

1	Simple measures to prevent the swelling include hyperventilation (which alters the intracerebral acid–base balance and decreases swelling) and intravenous corticosteroids (though their action is often delayed). Extracerebral hematoma may be removed surgically. The outcome of patients with head injury depends on how the secondary injury is managed. Even with a severe primary injury, patients may recover to lead a normal life. In the clinic

1	The outcome of patients with head injury depends on how the secondary injury is managed. Even with a severe primary injury, patients may recover to lead a normal life. In the clinic The skull is a closed bony compartment, and the brain and cerebrospinal fluid are maintained physiologically within a narrow intracranial pressure range. Any new space-occupying lesion, such as a hematoma, an injury that leads to brain swelling, or a brain tumor, can increase intracranial pressure and compress the brain. In severe cases, the brain may be squeezed down into the foramen magnum, giving it a cone shape, termed cerebral herniation, or “coning.” This may in turn compress the brainstem and upper cervical spinal cord, which can be fatal.

1	Congenital herniation or coning of the cerebellar tonsils through the foramen magnum can also occur if the posterior fossa is too small, a condition known as Chiari I malformation (Fig. 8.52). This often causes no problems in childhood and may only start causing symptoms in adulthood. In the clinic In the clinic Overview of cranial nerves Afferent—Trigeminal nerve (CN V) Efferent—Facial nerve (CN VII) Afferent—Glossopharyngeal nerve (CN IX) Efferent—Vagus nerve (CN X) Afferent—optic nerve (CN II) Efferent—oculomotor nerve (CN III) Fig. 8.55 Overview of cranial nerves.

1	Glossopharyngeal nerve [IX]Special sensory – taste (posterior 1/3 of tongue)Somatic sensory – posterior 1/3 of tongue, oropharynx,palatine tonsil, middle ear, pharyngotympanic tube, andmastoid air cellsBranchial motor – stylopharyngeusVisceral motor – (parasympathetic) – secretomotor to theparotid glandVisceral sensory – from carotid body and sinusFacial nerve [VII] Branchial motor – all muscles of facial expression, andstapedius, stylohyoid, and posterior belly of digastricEfferent (motor) fibersV3V3V2V1Afferent (sensory) fibersFacial nerve [VII] (intermediate nerve)Special sensory – taste (anterior 2/3 of tongue)Somatic sensory – part of external acoustic meatus anddeeper parts of auricleVisceral motor (parasympathetic) – secretomotor to allsalivary glands except for parotid gland; all mucous glandsassociated with the oral and nasal cavities; lacrimal glandVestibulocochlear nerve [VIII]Special sensory – hearing and balanceOlfactory nerve [I]Special sensory – smellOptic nerve

1	all mucous glandsassociated with the oral and nasal cavities; lacrimal glandVestibulocochlear nerve [VIII]Special sensory – hearing and balanceOlfactory nerve [I]Special sensory – smellOptic nerve [II]Special sensory – visionTrigeminal nerve [V] sensory rootSomatic sensory – eyes, orbital contents, face, sinuses, teeth, nasal cavities, oral cavity, anterior 2/3 of tongue, nasopharynx, dura, anterior part of external ear,and part of external acoustic meatusTrigeminal nerve [V] motor rootBranchial motor – the four muscles of mastication(medial pterygoid, lateral pterygoid, masseter, temporalis)and mylohyoid, anterior belly of digastric, tensor tympani,and tensor veli palatiniOculomotor nerve [III]Somatic motor – five extra-ocular muscles (superior rectus, medial rectus,inferior oblique, inferior rectus, and levator palpebrae superioris)Visceral motor – ciliary muscles and sphincter pupillae musclesTrochlear nerve [IV]Somatic motor – one extra-ocularmuscle (superior oblique)Abducent

1	inferior rectus, and levator palpebrae superioris)Visceral motor – ciliary muscles and sphincter pupillae musclesTrochlear nerve [IV]Somatic motor – one extra-ocularmuscle (superior oblique)Abducent nerve [VI]Somatic motor – one extra-ocularmuscle (lateral rectus)Hypoglossal nerve [XII]Somatic motor – all muscles of thetongue except palatoglossusAccessory nerve [XI]Branchial motor – sternocleidomastoidand trapeziusVagus nerve [X]Somatic sensory – larynx, laryngopharynx, deeper parts ofauricle, and part of external acoustic meatusSpecial sensory – taste from epiglottis and pharynxBranchial motor – all muscles of pharynx except for stylopharyngeus; all muscles of the soft palate except for tensor veli palatini, all intrinsic muscles of larynxVisceral motor – (parasympathetic) – thoracic viscera andabdominal viscera to end of midgutVisceral sensory – thoracic viscera and abdominal viscerato end of midgut, chemoand baroreceptors(and in some cases carotid body)

1	In the clinic Facelift surgery (rhytidectomy) aims to lift up and pull back the skin in the lower half of the face and neck to make the face more taught. Careful placement of the incisions is important to ensure there is no skin or facial distortion and to avoid hair loss. The commonest incisions are placed in the temporal region on each side, extending to the helices of the ears, then tracking behind the tragus, around the earlobes, and then to the occiput.

1	Botox is derived from the toxin produced by the bacterium Clostridium botulinum, which blocks neuromuscular junctions resulting in muscle relaxation. It is used in many therapies including strabismus (crossed eyes) where it is injected into extra-ocular muscles. Its injection is also used to treat uncontrolled blinking (blepharospasm), spastic muscle conditions, and overactive bladder disorders, as well as to relax facial muscles to improve the cosmetic appearances of lines and wrinkles and to treat patients with excessive sweating (hyperhidrosis). In the clinic The parotid gland is the largest of the paired salivary glands and is enclosed within the split investing layer of deep cervical fascia. The parotid gland produces a watery saliva and salivary amylase, which are necessary for food bolus formation, oral digestion, and smooth passage of the bolus into the upper gastrointestinal tract. Tumors of the parotid gland

1	Tumors of the parotid gland The commonest tumors of the parotid gland (Fig. 8.63) are benign and typically involve the more superficial part of the gland. These include pleomorphic adenoma and adenolymphoma. Their importance is in relation to their anatomical position. The relationship of any tumor to the branches of the facial nerve [VII] must be defined because resection of the tumor may damage the nerve. It is not uncommon for stones to develop within the parotid gland. They typically occur within the main confluence of the ducts and within the main parotid duct. The patient usually complains of intense pain when salivating and tends to avoid foods that produce this symptom. The pain can be easily reproduced in the clinic by squirting lemon juice into the patient’s mouth.

1	Surgery depends upon where the stone is. If it is within the anterior aspect of the duct, a simple incision in the buccal mucosa with a sphincterotomy may allow removal. If the stone is farther back within the main duct, complete gland excision may be necessary. In the clinic The complexity of the facial nerve [VII] is demonstrated by the different pathological processes and sites at which these processes occur.

1	In the clinic The complexity of the facial nerve [VII] is demonstrated by the different pathological processes and sites at which these processes occur. The facial nerve [VII] is formed from the nuclei within the brainstem emerging at the junction of the pons and the medulla. It enters the internal acoustic meatus, passes to the geniculate ganglion (which gives rise to further branches), and emerges from the skull base after a complex course within the temporal bone, leaving through the stylomastoid foramen. It enters the parotid gland and gives rise to five terminal groups of branches that supply muscles in the face and a number of additional branches that supply deeper or more posterior muscles. A series of lesions may affect the nerve along its course, and it is possible, with good clinical expertise, to determine the exact site of the lesion in relation to the course of the nerve.

1	A primary brainstem lesion affecting the motor nucleus of the facial nerve [VII] would lead to ipsilateral (same side) weakness of the whole face. However, because the upper part of the nucleus receives motor input from the left and right cerebral hemispheres a lesion occurring above the nucleus leads to contralateral lower facial weakness. In this example, motor innervation to the upper face is spared because the upper part of the nucleus receives input from both hemispheres. Preservation and loss of the special functions are determined by the extent of the lesion. Lesions at and around the geniculate ganglion Typically lesions at and around the geniculate ganglion are accompanied by loss of motor function on the whole of the ipsilateral (same) side of the face. Taste to the anterior two-thirds of the tongue, lacrimation, and some salivation also are likely to be affected because the lesion is proximal to the greater petrosal and chorda tympani branches of the nerve.

1	Lesions at and around the stylomastoid foramen Lesions at and around the stylomastoid foramen are the commonest abnormality of the facial nerve [VII] and usually result from a viral inflammation of the nerve within the bony canal before exiting through the stylomastoid foramen. Typically the patient has an ipsilateral loss of motor function of the whole side of the face. Not only does this produce an unusual appearance, but it also complicates chewing of food. Lacrimation and taste may not be affected if the lesion remains distal to the greater petrosal and chorda tympani branches that originate deep in the temporal bone. In the clinic Trigeminal neuralgia (tic douloureux) is a complex sensory disorder of the sensory root of the trigeminal nerve. Typically the pain is in the region of the mandibular [V3] and maxillary [V2] nerves, and is usually of sudden onset, is excruciating in nature, and may be triggered by touching a sensitive region of skin.

1	The etiology of trigeminal neuralgia is unknown, although anomalous blood vessels lying adjacent to the sensory route of the maxillary [V2] and mandibular [V3] nerves may be involved. If symptoms persist and are unresponsive to medical care, surgical exploration of the trigeminal nerve (which is not without risk) may be necessary to remove any aberrant vessels. In the clinic The scalp has an extremely rich blood supply from the external carotid arteries, so lacerations of the scalp tend to bleed profusely. Importantly, scalp bleeding is predominantly arterial, because of two reasons. First, in the erect position the venous pressure is extremely low. Second, the vessels do not retract and close when lacerated because the connective tissue in which they are found holds them open. In the clinic

1	In the clinic Fractures of the orbit are not uncommon and may involve the orbital margins with extension into the maxilla, frontal, and zygomatic bones. These fractures are often part of complex facial fractures. Fractures within the orbit frequently occur within the floor and the medial wall; however, superior and lateral wall fractures also occur. Inferior orbital floor fractures are one of the commonest types of injuries. These fractures may drag the inferior oblique muscle and associated tissues into the fracture line. In these instances, patients may have upward gaze failure (upward gaze diplopia) in the affected eye. Medial wall fractures characteristically show air within the orbit in radiographs. This is due to fracture of the ethmoidal labyrinth, permitting direct continuity between the orbit and the ethmoidal paranasal sinuses. Occasionally, patients feel a full sensation within the orbit when blowing the nose. In the clinic

1	In the clinic Horner’s syndrome is caused by any lesion that leads to a loss of sympathetic function in the head. It is characterized by three typical features: pupillary constriction due to paralysis of the dilator pupillae muscle, partial ptosis (drooping of the upper eyelid) due to paralysis of the superior tarsal muscle, and absence of sweating on the ipsilateral side of the face and the neck due to absence of innervation of the sweat glands. Secondary changes may also include: ipsilateral vasodilation due to loss of the normal sympathetic control of the subcutaneous blood vessels, and enophthalmos (sinking of the eye)—believed to result from paralysis of the orbitalis muscle, although this is an uncommon feature of Horner’s syndrome. The orbitalis muscle spans the inferior orbital fissure and helps maintain the forward position of orbital contents.

1	The orbitalis muscle spans the inferior orbital fissure and helps maintain the forward position of orbital contents. The commonest cause for Horner’s syndrome is a tumor eroding the cervicothoracic ganglion, which is typically an apical lung tumor. A surgically induced Horner’s syndrome may be necessary for patients who suffer severe hyperhidrosis (sweating). This often debilitating condition may be so severe that patients are confined to their home for fear of embarrassment. Treatment is relatively straightforward. The patient is anesthetized and a bifurcate endotracheal tube is placed into the left and right main bronchi. A small incision is made in the intercostal space on the appropriate side, and a surgically induced pneumothorax is created. The patient is ventilated through the contralateral lung.

1	Using an endoscope the apex of the thoracic cavity can be viewed from inside and the cervicothoracic ganglion readily identified. Obliterative techniques include thermocoagulation and surgical excision. After the ganglion has been destroyed, the endoscope is removed, the lung is reinflated, and the small hole is sutured. In the clinic Examination of the eye Examination of the eye includes assessment of the visual capabilities, the extrinsic musculature and its function, and disease processes that may affect the eye in isolation or as part of the systemic process. Examination of the eye includes tests for visual acuity, astigmatism, visual fields, and color interpretation (to exclude color blindness) in a variety of circumstances. The physician also assesses the retina, the optic nerve and its coverings, the lens, and the cornea. The extrinsic muscles are supplied by the abducent nerve [VI], the trochlear nerve [IV], and the oculomotor nerve [III].

1	The extrinsic muscles are supplied by the abducent nerve [VI], the trochlear nerve [IV], and the oculomotor nerve [III]. The extrinsic muscles work synergistically to provide appropriate and conjugate eye movement: lateral rectus—abducent nerve [VI], superior oblique—trochlear nerve [IV], and remainder—oculomotor nerve [III]. The eye may be affected in systemic diseases. Diabetes mellitus typically affects the eye and may cause cataracts, macular disease, and retinal hemorrhage, all impairing vision. Occasionally unilateral paralysis of the extra-ocular muscles occurs and is due to brainstem injury or direct nerve injury, which may be associated with tumor compression or trauma. The paralysis of a muscle is easily demonstrated when the patient attempts to move the eye in the direction associated with normal action of that muscle. Typically the patient complains of double vision (diplopia). Loss of innervation of the muscles around the eye

1	Loss of innervation of the muscles around the eye Loss of innervation of the orbicularis oculi by the facial nerve [VII] causes an inability to close the eyelids tightly, allowing the lower eyelid to droop away causing spillage of tears. This loss of tears allows drying of the conjunctiva, which may ulcerate, so allowing secondary infection. Loss of innervation of the levator palpebrae superioris by oculomotor nerve [III] damage causes an inability of the superior eyelid to elevate, producing a complete ptosis. Usually, oculomotor nerve [III] damage is caused by severe head injury. Loss of innervation of the superior tarsal muscle by sympathetic fibers causes a constant partial ptosis. Any lesion along the sympathetic trunk can induce this. An apical pulmonary malignancy should always be suspected because the ptosis may be part of Horner’s syndrome (see “In the clinic” on p. 920). In the clinic The “H-test”

1	In the clinic The “H-test” A simple “formula” for remembering the nerves that innervate the extraocular muscles is “LR6SO4 and all the rest are 3” (lateral rectus [VI], superior oblique [IV], all the rest including levator palpebrae superioris are [III]). The function of all extrinsic muscles and their nerves [III, IV, VI] that move the eyeball in both orbits can all easily be tested at the same time by having the patient track, without moving his or her head, an object such as the tip of a pen or a finger moved in an “H” pattern—starting from the midline between the two eyes (Fig. 8.98). In the clinic Intraocular pressure will rise if the normal cycle of aqueous humor fluid production and absorption is disturbed so that the amount of fluid increases. This condition is glaucoma and can lead to a variety of visual problems including blindness, which results from compression of the retina and its blood supply. In the clinic

1	In the clinic With increasing age and in certain disease states the lens of the eye becomes opaque. Increasing opacity results in increasing visual impairment. A common operation is excision of the cloudy lens and replacement with a new man-made lens. In the clinic Direct visualization of the postremal (vitreous) chamber of the eye is possible in most clinical settings. It is achieved using an ophthalmoscope, which is a small battery-operated light with a tiny lens that allows direct visualization of the postremal (vitreous) chamber and the posterior wall of the eye through the pupil and the lens. It is sometimes necessary to place a drug directly onto the eye to dilate the pupil for better visualization. The optic nerve, observed as the optic disc, is easily seen. The typical four branches of the central retinal artery and the fovea are also seen.

1	The optic nerve, observed as the optic disc, is easily seen. The typical four branches of the central retinal artery and the fovea are also seen. Using ophthalmoscopy the physician can look for diseases of the optic nerve, vascular abnormalities, and changes within the retina (Fig. 8.109). In the clinic High-definition optical coherence tomography (HD-OCT) (Fig. 8.111) is a procedure used to obtain subsurface images of translucent or opaque materials. It is similar to ultrasound, except that it uses light instead of sound to produce high-resolution cross-sectional images. It is especially useful in the diagnosis and management of optic nerve and retinal diseases. An epiretinal membrane (Fig. 8.112) is a thin sheet of fibrous tissue that develops on the surface of the retina in the area of the macula and can cause visual problems. If the visual problems are significant, surgical removal of the membrane may be necessary. In the clinic

1	In the clinic The eustachian tube links the middle ear and pharynx and balances the pressure between the outer and middle ear. Colds and allergies, particularly in children, can result in swelling of the lining of the eustachian tube, which can then impair normal drainage of fluid from the middle ear. The fluid then builds up behind the tympanic membrane, providing an attractive environment for bacteria and viruses to grow and cause otitis media. Left untreated, otitis media can lead to perforation of the tympanic membrane, hearing loss, meningitis, and brain abscess. In the clinic Examination of the ear The ear comprises three components—the external, middle, and internal ear. Clinical examination is carried out to assess hearing and balance. Further examination involves use of an otoscope or other imaging techniques.

1	Clinical examination is carried out to assess hearing and balance. Further examination involves use of an otoscope or other imaging techniques. The external ear is easily examined. The external acoustic meatus and the tympanic membrane require otoscopic examination (Fig. 8.118B). An otoscope is a device through which light can be shone and the image magnified to inspect the external acoustic meatus and the tympanic membrane. The examination begins by grasping the posterosuperior aspect of the ear and gently retracting it to straighten the external auditory meatus. The normal tympanic membrane is relatively translucent and has a gray–reddish tinge. The handle of the malleus is visible near the center of the membrane. In the 5 o’clock position a cone of light is always demonstrated. The middle ear is investigated by CT and MRI to visualize the malleus, incus, and stapes. The relationship of these bones to the middle ear cavity is determined and any masses identified.

1	The middle ear is investigated by CT and MRI to visualize the malleus, incus, and stapes. The relationship of these bones to the middle ear cavity is determined and any masses identified. The inner ear is also assessed by CT and MRI. In the clinic Swimmer’s ear, often called otitis externa, is a painful condition resulting from an infection in the external acoustic meatus. It frequently occurs in swimmers. In the clinic Surfer’s ear, which is prevalent among individuals who surf or swim in cold water, results from the development of a “bony lump” in the external acoustic meatus. Growth of the lump eventually constricts the meatus and reduces hearing in the affected ear. In the clinic Although perforation of the tympanic membrane (eardrum) has many causes, trauma and infection are the most common. Ruptures of the tympanic membrane tend to heal spontaneously, but surgical intervention may be necessary if the rupture is large.

1	Ruptures of the tympanic membrane tend to heal spontaneously, but surgical intervention may be necessary if the rupture is large. Occasionally, it may be necessary to enter the middle ear through the tympanic membrane. Because the chorda tympani runs in the upper one-third of the tympanic membrane, incisions are always below this level. The richer blood supply to the posterior aspect of the tympanic membrane determines the standard surgical approach in the posteroinferior aspect. Otitis media (infection of the middle ear) is common and can lead to perforation of the tympanic membrane. The infection can usually be treated with antibiotics. If the infection persists, the chronic inflammatory change may damage the ossicular chain and other structures within the middle ear to produce deafness. In the clinic

1	In the clinic Infection within the mastoid antrum and mastoid cells is usually secondary to infection in the middle ear. The mastoid cells provide an excellent culture medium for infection. Infection of the bone (osteomyelitis) may also develop, spreading into the middle cranial fossa. Drainage of the pus within the mastoid air cells is necessary and there are numerous approaches for doing this. When undertaking this type of surgery, it is extremely important that care is taken not to damage the mastoid wall of the middle ear to prevent injury to the facial nerve [VII]. Any breach of the inner table of the cranial vault may allow bacteria to enter the cranial cavity and meningitis will ensue. In the clinic A lingual nerve injury proximal to where the chorda tympani joins it in the infratemporal fossa will produce loss of general sensation from the anterior two-thirds of the tongue, oral mucosa, gingivae, the lower lip, and the chin.

1	If a lingual nerve lesion is distal to the site where it is joined by the chorda tympani, secretion from the salivary glands below the oral fissure and taste from the anterior two-thirds of the tongue will also be lost. In the clinic Anesthesia of the inferior alveolar nerve is widely practiced by most dentists. The inferior alveolar nerve is one of the largest branches of the mandibular nerve [V3], carries the sensory branches from the teeth and mandible, and receives sensory information from the skin over most of the mandible. The inferior alveolar nerve passes into the mandibular canal, courses through the body of the mandible, and eventually emerges through the mental foramen into the chin.

1	The inferior alveolar nerve passes into the mandibular canal, courses through the body of the mandible, and eventually emerges through the mental foramen into the chin. of the inferior alveolar nerve by local anesthetic. To anesthetize this nerve the needle is placed lateral to the anterior arch of the fauces (palatoglossal arch) in the oral cavity and is advanced along the medial border around the inferior third of the ramus of the mandible so that anesthetic can be deposited in this region. It is also possible to anesthetize the infra-orbital and buccal nerves, depending on where the anesthesia is needed. In the clinic

1	It is also possible to anesthetize the infra-orbital and buccal nerves, depending on where the anesthesia is needed. In the clinic In most instances, access to peripheral veins of the arm and the leg will suffice for administering intravenous drugs and fluids and for obtaining blood for analysis; however, in certain circumstances it is necessary to place larger-bore catheters in the central veins, for example, for dialysis, parenteral nutrition, or the administration of drugs that have a tendency to produce phlebitis.

1	“Blind puncture” of the subclavian and jugular veins to obtain central venous access used to be standard practice. However, subclavian vein puncture is not without complications. As the subclavian vein passes inferiorly, posterior to the clavicle, it passes over the apex of the lung. Any misplacement of a needle into or through this structure may puncture the apical pleura, producing a pneumothorax. Inadvertent arterial puncture and vein laceration may also produce a hemopneumothorax. A puncture of the internal jugular vein (Fig. 8.165) carries fewer risks, but local hematoma and damage to the carotid artery are again important complications. Current practice is to identify major vessels using ultrasound and to obtain central venous access under direct vision to avoid any significant complication. In the clinic

1	Current practice is to identify major vessels using ultrasound and to obtain central venous access under direct vision to avoid any significant complication. In the clinic The jugular venous pulse is an important clinical sign that enables the physician to assess the venous pressure and waveform and is a reflection of the functioning of the right side of the heart. In the clinic The thyroid gland develops from a small region of tissue near the base of the tongue. This tissue descends as the thyroglossal duct from the foramen cecum in the posterior aspect of the tongue to pass adjacent to the anterior aspect of the middle of the hyoid bone. The thyroid tissue continues to migrate inferiorly and eventually comes to rest at the anterior aspect of the trachea in the root of the neck.

1	Consequently, the migration of thyroid tissue may be arrested anywhere along the embryological descent of the gland. Ectopic thyroid tissue is relatively rare. More frequently seen is the cystic change that arises from the thyroglossal duct. The usual symptom of a thyroglossal duct cyst is a midline mass. Ultrasound easily demonstrates its nature and position, and treatment is by surgical excision. The whole of the duct as well as a small part of the anterior aspect of the hyoid bone must be excised to prevent recurrence. In the clinic A thyroidectomy is a common surgical procedure. In most cases it involves excision of part or most of the thyroid gland. This surgical procedure is usually carried out for benign diseases, such as multinodular goiter and thyroid cancer.

1	Given the location of the thyroid gland, there is a possibility of damaging other structures when carrying out a thyroidectomy, namely the parathyroid glands and the recurrent laryngeal nerve (Fig. 8.181). Assessment of the vocal folds is necessary before and after thyroid surgery because the recurrent laryngeal nerves are closely related to ligaments that bind the gland to the larynx and can be easily traumatized during surgical procedures. In the clinic Thyroid gland pathology is extremely complex. In essence, thyroid gland pathology should be assessed from two points of view. First, the thyroid gland may be diffusely or focally enlarged, for which there are numerous causes. Second, the thyroid gland may undersecrete or oversecrete the hormone thyroxine.

1	One of the commonest disorders of the thyroid gland is a multinodular goiter, which is a diffuse irregular enlargement of the thyroid gland with areas of thyroid hypertrophy and colloid cyst formation. Most patients are euthyroid (i.e., have normal serum thyroxine levels). The typical symptom is a diffuse mass in the neck, which may be managed medically or may need surgical excision if the mass is large enough to affect the patient’s life or cause respiratory problems. Isolated nodules in the thyroid gland may be a dominant nodule in a multinodular gland or possibly an isolated tumor of the thyroid gland. Isolated tumors may or may not secrete thyroxine depending on their cellular morphology. Treatment is usually by excision.

1	Immunological diseases may affect the thyroid gland and may overstimulate it to produce excessive thyroxine. These diseases may be associated with other extrathyroid manifestations, which include exophthalmos, pretibial myxedema, and nail changes. Other causes of diffuse thyroid stimulation include viral thyroiditis. Some diseases may cause atrophy of the thyroid gland, leading to undersecretion of thyroxine (myxedema). In the clinic The parathyroid glands develop from the third and fourth pharyngeal pouches and translocate to their more adult locations during development. The position of the glands can be highly variable, sometimes being situated high in the neck or in the thorax. Tumors develop in any of these locations (Fig. 8.182). In the clinic

1	In the clinic Damage to either the right or left recurrent laryngeal nerve may lead initially to a hoarse voice and finally to an inability to speak. Recurrent laryngeal nerve palsy can occur from disruption of the nerves anywhere along their course. Furthermore, interruption of the vagus nerves before the division of the recurrent laryngeal nerves can also produce vocal symptoms. Lung cancer in the apex of the right lung can affect the right recurrent laryngeal nerve, whereas cancers that infiltrate into the area between the pulmonary artery and aorta, an area known clinically as the “aortopulmonary window,” can affect the left recurrent laryngeal nerve. Thyroid surgery also can traumatize the recurrent laryngeal nerves. In the clinic Clinical lymphatic drainage of the head and neck

1	In the clinic Clinical lymphatic drainage of the head and neck Enlargement of the neck lymph nodes (cervical lymphadenopathy) is a common manifestation of disease processes that occur in the head and neck. It is also a common manifestation of diffuse diseases of the body, which include lymphoma, sarcoidosis, and certain types of viral infection such as glandular fever and human immunodeficiency virus (HIV) infection. Evaluation of cervical lymph nodes is extremely important in determining the nature and etiology of the primary disease process that has produced nodal enlargement. Clinical evaluation includes a general health assessment, particularly relating to symptoms from the head and neck. Examination of the nodes themselves often gives the clinician a clue as to the nature of the pathological process. Soft, tender, and inflamed lymph nodes suggest an acute inflammatory process, which is most likely to be infective.

1	Soft, tender, and inflamed lymph nodes suggest an acute inflammatory process, which is most likely to be infective. Firm multinodular large-volume rubbery nodes often suggest a diagnosis of lymphoma. Examination should also include careful assessment of other nodal regions, including the supraclavicular fossae, the axillae, the retroperitoneum, and the inguinal regions. Further examination may include digestive tract endoscopy, chest radiography, and body CT scanning. Most cervical lymph nodes are easily palpable and suitable for biopsy to establish a tissue diagnosis. Biopsy can be performed using ultrasound for guidance and good samples of lymph nodes may be obtained.

1	The lymphatic drainage of the neck is somewhat complex, clinically. A relatively simple “level” system of nodal enlargement has been designed that is extremely helpful in evaluating lymph node spread of primary head and neck tumors. Once the number of levels of nodes are determined, and the size of the lymph nodes, the best mode of treatment can be instituted. This may include surgery, radiotherapy, and chemotherapy. The lymph node level also enables a prognosis to be made. The levels are as follows (Fig. 8.199): Level I—from the midline of the submental triangle up to the level of the submandibular gland. Level II—from the skull base to the level of the hyoid bone anteriorly from the posterior border of the sternocleidomastoid muscle. Level III—the inferior aspect of the hyoid bone to the bottom cricoid arch and anterior to the posterior border of the sternocleidomastoid up to the midline.

1	Level III—the inferior aspect of the hyoid bone to the bottom cricoid arch and anterior to the posterior border of the sternocleidomastoid up to the midline. Level IV—from the inferior aspect of the cricoid to the top of the manubrium of the sternum and anterior to the posterior border of the sternocleidomastoid muscle. Level V—posterior to the sternocleidomastoid muscle and anterior to the trapezius muscle above the level of the clavicle. Level VI—below the hyoid bone and above the jugular (sternal) notch in the midline. Level VII—below the level of the jugular (sternal) notch. In the clinic

1	Level VI—below the hyoid bone and above the jugular (sternal) notch in the midline. Level VII—below the level of the jugular (sternal) notch. In the clinic In emergency situations, when the airway is blocked above the level of the vocal folds, the median cricothyroid ligament can be perforated and a small tube inserted through the incision to establish an airway. Except for small vessels and the occasional presence of a pyramidal lobe of the thyroid gland, normally there are few structures between the median cricothyroid ligament and the skin. In the clinic A tracheostomy is a surgical procedure in which a hole is made in the trachea and a tube is inserted to enable ventilation. A tracheostomy is typically performed when there is obstruction to the larynx as a result of inhalation of a foreign body, severe edema secondary to anaphylactic reaction, or severe head and neck trauma.

1	The typical situation in which a tracheostomy is performed is in the calm atmosphere of an operating theater. A small transverse incision is placed in the lower third of the neck anteriorly. The strap muscles are deviated laterally and the trachea can be easily visualized. Occasionally it is necessary to divide the isthmus of the thyroid gland. An incision is made in the second and third tracheal rings and a small tracheostomy tube inserted. After the tracheostomy has been in situ for the required length of time, it is simply removed. The hole through which it was inserted almost inevitably closes without any intervention. Patients with long-term tracheostomies are unable to vocalize because no air is passing through the vocal cords. In the clinic

1	Patients with long-term tracheostomies are unable to vocalize because no air is passing through the vocal cords. In the clinic Laryngoscopy is a medical procedure that is used to inspect the larynx. The functions of laryngoscopy include the evaluation of patients with difficulty swallowing, assessment of the vocal cords, and assessment of the larynx for tumors, masses, and weak voice.

1	The larynx is typically visualized using two methods. Indirect laryngoscopy involves passage of a small rod-mounted mirror (not dissimilar to a dental mirror) into the oropharynx permitting indirect visualization of the larynx. Direct laryngoscopy can be performed using a device with a curved metal tip that holds the tongue and epiglottis forward, allowing direct inspection of the larynx. This procedure can be performed only in the unconscious patient or in a patient in whom the gag reflex is not intact. Other methods of inspection include the passage of fiberoptic endoscopes through either the oral cavity or nasal cavity. In the clinic The nasal septum is typically situated in the midline; however, septal deviation to one side or the other is not uncommon, and in many cases is secondary to direct trauma. Extreme septal deviation can produce nasal occlusion. The deviation can be corrected surgically. In the clinic

1	In the clinic Most cancers of the oral cavity, oropharynx, nasopharynx, larynx, sinuses, and salivary glands arise from the epithelial cells that line them, resulting in squamous cell carcinoma. The majority of these are related to cell damage caused by smoking and alcohol use. Certain viruses are also related to cancers in the head and neck, including human papillomavirus (HPV) and Epstein-Barr virus (EBV). A 50-year-old overweight woman came to the doctor complaining of hoarseness of voice and noisy breathing. She was also concerned at the increase in size of her neck. On examination she had a slow pulse rate (45 beats per minute). She also had an irregular knobby mass in the anterior aspect of the lower neck, which deviated the trachea to the right. A clinical diagnosis of a multinodular goiter and hypothyroidism was made.

1	A clinical diagnosis of a multinodular goiter and hypothyroidism was made. Enlargement of the thyroid gland is due to increased secretion of thyroid-stimulating hormone, which is usually secondary to diminished output of thyroid hormones. The thyroid undergoes periods of activity and regression, which can lead to the formation of nodules, some of which are solid and some of which are partially cystic (colloid cysts). This nodule formation is compounded by areas of fibrosis within the gland. Other causes of multinodular goiter include iodine deficiency and in certain circumstances, drugs that interfere with the metabolism and production of thyroxine. The typical symptom of a goiter is a painless swelling of the thyroid gland. It may be smooth or nodular, and occasionally it may extend into the superior mediastinum as a retrosternal goiter. The trachea was deviated.

1	The trachea was deviated. The enlargement of the thyroid gland due to a multinodular goiter may not be symmetrical. In this case there was significant asymmetrical enlargement of the left lobe of the thyroid deviating the trachea to the right. The patient had a hoarse voice and noisy breathing. If the thyroid gland enlargement is significant it can compress the trachea, narrowing it to such an extent that a “crowing sound” is heard during inspiration (stridor). Other possible causes for hoarseness include paralysis of the vocal cord due to compression of the left recurrent laryngeal nerve from the goiter. Of concern is the possibility of malignant change within the goiter directly invading the recurrent laryngeal nerve. Fortunately, malignant change is rare within the thyroid gland.

1	When patients have a relatively low production of thyroxine such that the basal metabolic rate is reduced they become more susceptible to infection, including throat and upper respiratory tract infections. On examination the thyroid gland moved during swallowing. Characteristically, an enlarged thyroid gland is evident as a neck mass arising on one or both sides of the trachea. The enlarged thyroid gland moves on swallowing because it is attached to the larynx by the pretracheal fascia. The patient was hypothyroid. Hypothyroidism refers to the clinical and biochemical state in which the thyroid gland is underactive (hyperthyroidism refers to an overactive thyroid gland). Some patients have thyroid masses and no clinical or biochemical abnormalities—these patients are euthyroid.

1	The hormone thyroxine controls the basal metabolic rate; therefore, low levels of thyroxine affect the resting pulse rate and may produce other changes, including weight gain, and in some cases depression. The patient was insistent upon surgery. After discussion about the risks and complications, a subtotal thyroidectomy was performed. After the procedure the patient complained of tingling in her hands and feet and around her mouth, and carpopedal spasm. These symptoms are typical of tetany and are caused by low serum calcium levels.

1	The etiology of the low serum calcium level was trauma and bruising of the four parathyroid glands left in situ after the operation. Undoubtedly the trauma of removal of such a large thyroid gland produced a change within the parathyroid gland, which failed to function appropriately. The secretion of parathyroid hormone rapidly decreased over the next 24 hours, resulting in increased excitability of peripheral nerves, manifest by carpopedal spasm and orofacial tingling. Muscle spasms can also be elicited by tapping the facial nerve [VII] as it emerges from the parotid gland to produce twitching of the facial muscles (Chvostek’s sign). The patient recovered from these symptoms due to a low calcium level over the next 24 hours. At her return to the clinic the patient was placed on supplementary oral thyroxine, which is necessary after removal of the thyroid gland. The patient also complained of a hoarse voice.

1	At her return to the clinic the patient was placed on supplementary oral thyroxine, which is necessary after removal of the thyroid gland. The patient also complained of a hoarse voice. The etiology of her hoarse voice was damage to the recurrent laryngeal nerve. The recurrent laryngeal nerve lies close to the thyroid gland. It may be damaged in difficult surgical procedures, and this may produce unilateral spasm of the ipsilateral vocal cord to produce a hoarse voice. Since the thyroidectomy and institution of thyroxine treatment, the patient has lost weight and has no further complaints.

1	Since the thyroidectomy and institution of thyroxine treatment, the patient has lost weight and has no further complaints. A 33-year-old man was playing cricket for his local Sunday team. As the new bowler pitched the ball short, it bounced higher than he anticipated and hit him on the side of his head. He immediately fell to the ground unconscious, but after about 30 seconds he was helped to his feet and felt otherwise well. It was noted he had some bruising around his temple. He decided not to continue playing and went to watch the match from the side. Over the next hour he became extremely sleepy and was eventually unrousable. He was rushed to hospital. When he was admitted to hospital, the patient’s breathing was shallow and irregular and it was necessary to intubate him. A skull radiograph demonstrated a fracture in the region of the pterion. No other abnormality was demonstrated other than minor soft tissue bruising over the left temporal fossa. A CT scan was performed.

1	A CT scan was performed. The CT scan demonstrated a lentiform area of high density within the left cranial fossa. A diagnosis of extradural hemorrhage was made. Fractures in the region of the pterion are extremely dangerous. A division of the middle meningeal artery passes deep to this structure and is subject to laceration and disruption, especially in conjunction with a skull injury in this region. In this case the middle meningeal artery was torn and started to bleed, producing a large extradural clot. The patient’s blood pressure began to increase.

1	The patient’s blood pressure began to increase. Within the skull there is a fixed volume and clearly what goes in must come out (e.g., blood, cerebrospinal fluid). If there is a space-occupying lesion, such as an extradural hematoma, there is no space into which it can decompress. As the lesion expands, the brain becomes compressed and the intracranial pressure increases. This pressure compresses vessels, so lowering the cerebral perfusion pressure. To combat this the homeostatic mechanisms of the body increase the blood pressure to overcome the increase in intracerebral pressure. Unfortunately, the increase in intracranial pressure is compounded by the cerebral edema that occurs at and after the initial insult. An urgent surgical procedure was performed.

1	An urgent surgical procedure was performed. Burr holes were placed around the region of the hematoma and it was evacuated. The small branch of the middle meningeal artery was ligated and the patient spent a few days in the intensive care unit. Fortunately the patient made an uneventful recovery. A 35-year-old man was involved in a fight and sustained a punch to the right orbit. He came to the emergency department with double vision. The double vision was only in one plane. Examination of the orbits revealed that when the patient was asked to look upward the right eye was unable to move superiorly when adducted. There was some limitation in general eye movement. Assessment of the lateral rectus muscle (abducent nerve [VI]), superior oblique muscle (trochlear nerve [IV]), and the rest of the eye muscles (oculomotor nerve [III]) was otherwise unremarkable. The patient underwent a CT scan.

1	The patient underwent a CT scan. A CT scan of the facial bones demonstrated a fracture through the floor of the orbit (Fig. 8.293). A careful review of this CT scan demonstrated that the inferior oblique muscle had been pulled inferiorly with the fragment of bone in the fracture. This produced a tethering effect, so when the patient was asked to gaze in the upward direction, the left eye was able to do so but the right eye was unable to because of the tethered inferior oblique muscle. The patient underwent surgical exploration to elevate the small bony fragment and return the inferior oblique to its appropriate position. On follow-up the patient had no complications.

1	The patient underwent surgical exploration to elevate the small bony fragment and return the inferior oblique to its appropriate position. On follow-up the patient had no complications. A 25-year-old man complained of significant swelling in front of his right ear before and around mealtimes. This swelling was associated with considerable pain, which was provoked by the ingestion of lemon sweets. On examination he had tenderness around the right parotid region and a hard nodule was demonstrated in the buccal mucosa adjacent to the right upper molar teeth. A diagnosis of parotid duct calculus was made.

1	A diagnosis of parotid duct calculus was made. The formation of stones in the salivary glands is not uncommon, but it is more likely in the submandibular gland than in the parotid gland because the saliva is more mucinous and the duct has a long upward course from the floor of the mouth. Nevertheless, stones do form in the parotid gland and the parotid ducts. Notably, most parotid duct calculi and submandibular duct calculi occur in mouths with excellent dental hygiene and mucosa. An ultrasound scan was performed. An initial ultrasound scan demonstrated a stone in the distal end of the right parotid duct with evidence of ductal dilation (eFig. 8.294). Assessment of the gland also demonstrated dilated ducts within the gland and evidence of intraparotid lymphadenopathy. The patient was treated with antibiotics.

1	The patient was treated with antibiotics. A course of antibiotics was given to remove the bacteria that had produced the inflammation. On return to the doctor some days later the gland was normal in size and there was no evidence of inflammation or infection. An operation was necessary. The stone was at the distal end of the parotid duct and it would seem logical and straightforward to make a small incision at the sphincter in the buccal mucosa and deliver the stone, thus permitting the gland to drain normally. Unfortunately, in this patient’s case the gland was significantly destroyed by the chronic obstruction and bacterial infection. Furthermore, smaller calculi were also demonstrated in the gland at ultrasound. On direct questioning it appeared that the patient had had numerous attacks over the previous 4–5 years and it was decided that the parotid gland should be removed surgically.

1	The patient consented for removal of the parotid gland and a discussion of the possibility for loss of facial function and facial paralysis was had with the patient at this time.

1	Within the parotid gland the facial nerve [VII] divides into its five terminal branches. At operation the gland is displayed and extremely careful dissection is necessary to peel away the parotid gland from the branches of the facial nerve [VII]. This procedure was made more difficult by the chronic inflammatory change within the gland. After the procedure the patient made a good recovery, though there was some mild paralysis of the whole of the right side of the face. Importantly, taste to the anterior two-thirds of the tongue was preserved. The taste fibers to the anterior two-thirds of the tongue travel in the chorda tympani nerve, which is a branch of the facial nerve [VII]. This nerve leaves the facial nerve [VII] to join the lingual nerve proximal to the parotid gland; therefore, any damage to the facial nerve [VII] within the parotid gland does not affect special sensation (taste).

1	Over the following week the paralysis improved and was likely due to nerve bruising during the procedure. The patient remained asymptomatic. A 60-year-old woman was brought to the emergency department with acute right-sided weakness, predominantly in the upper limb, which lasted for 24 hours. She made an uneventful recovery, but was extremely concerned about the nature of her illness and went to see her local doctor. A diagnosis of a transient ischemic attack (TIA) was made. A TIA is a neurological deficit resolving within 24 hours. It is a type of stroke. Neurological deficits may be permanent or transient. Most transient events resolve within 21 days; any failure of resolution beyond 21 days is an established stroke. An investigation into the cause of the TIA was undertaken. Eighty-five percent of all strokes result from cerebral infarction, of which most are due to embolization. A duplex Doppler scan of the carotid vessels was performed.

1	Eighty-five percent of all strokes result from cerebral infarction, of which most are due to embolization. A duplex Doppler scan of the carotid vessels was performed. The majority of emboli originate from plaques that develop at and around the carotid bifurcation. Emboli consist of platelet aggregates, cholesterol, and atheromatous debris. Emboli may also arise from the heart secondary to cardiac tumors or myocardial infarction. The lesion in the brain was on the left side. The motor cortex for the whole of the right side of the body is represented in the left motor strip of the brain, which sits on the precentral gyrus. The duplex Doppler ultrasound scan demonstrated a significant narrowing (stenosis) of the left internal carotid artery with evidence of plaque formation and abnormal flow in this region. The narrowing was approximately 90%. Treatment required an operation.

1	Treatment required an operation. A carotid endarterectomy (removal of the stenosis and the atheromatous plaque) was planned. This procedure is indicated in the presence of an ulcerating plaque with stenosis. The procedure was carried out under general anesthetic and a curvilinear incision was placed in the left side of the neck. The common carotid, external carotid, and internal carotid arteries were displayed. All vessels were clamped and a shunt was placed from the common carotid artery into the internal carotid artery to maintain cerebral blood flow during the procedure. The internal carotid artery was opened and the plaque excised.

1	After the procedure the patient did extremely well and suffered no further cerebral events. However, a new medical student examined the patient the following day and demonstrated a number of interesting findings. These included altered skin sensation inferior to the left mandible, altered sensation on the left side of the soft palate, a paralyzed left vocal cord, inability to shrug the left shoulder, and a tongue that deviated to the left. The etiology of these injuries was due to localized nerve trauma.

1	The etiology of these injuries was due to localized nerve trauma. This constellation of neurological deficits can be accounted for by trauma to the nerves that are close to the carotid bifurcation. The changes in skin sensation can be accounted for by a neurapraxia due to damage to cervical nerves. The alteration in sensation in the soft palate is due to neurapraxia of the glossopharyngeal nerve [IX]. The paralyzed left cord results from neurapraxia of the recurrent laryngeal nerve, while the inability to shrug the shoulder is due to neurapraxia of the accessory nerve [XI]. Deviation of the tongue can be accounted for by damage to the hypoglossal nerve [XII]. Most of these changes are transient and are usually due to traction injuries during the surgical procedure.

1	Most of these changes are transient and are usually due to traction injuries during the surgical procedure. A 33-year-old fit and well woman came to the emergency department complaining of double vision and pain behind her right eye. She had no other symptoms. On examination of the right eye the pupil was dilated. There was a mild ptosis. Testing of eye movement revealed that the eye turned down and out and the pupillary reflex was not present. These findings revealed that the patient had an ipsilateral third nerve palsy (palsy of the oculomotor nerve [III]). The oculomotor nerve [III] is the main motor nerve to the ocular and extra-ocular muscles. It arises from the midbrain and pierces the dura mater to run in the lateral wall of the cavernous sinus. The oculomotor nerve [III] leaves the cranial cavity and enters the orbit through the superior orbital fissure. Within this fissure it divides into its superior and inferior divisions.

1	The site of the nerve lesion needs to be assessed. Third nerve palsy may involve the nucleus of the oculomotor nerve [III], which typically spares the pupil and is painless. The pupillary reflexes are supplied from the autonomic fibers of the Edinger–Westphal nucleus, which pass through the ciliary ganglion. The lesion cannot be a primary oculomotor nerve [III] nuclear injury. As both the pupillary reflexes and vision are affected, the lesion is likely to be along the course of the oculomotor nerve [III]. Medical conditions such as diabetes mellitus and vascular disease may produce an isolated oculomotor nerve [III] injury, but they are not associated with pain. The lesion was caused by an aneurysm.

1	The lesion was caused by an aneurysm. One of the commonest causes of a third nerve palsy is pressure on the nerve from a posterior communicating artery aneurysm, which lies parallel to the nerve on the anterior aspect of the brainstem. As the aneurysm abuts the outside of the oculomotor nerve [III], it involves the parasympathetic fibers, which lead to a predominance of the loss of pupillary function over general function. The aneurysm was imaged with an angiogram. The patient initially underwent CT and MRI scanning. Currently, the definitive test for assessment of aneurysms arising from the circle of Willis and its branches is a digital subtraction angiogram. The angiogram demonstrated the posterior communicating artery aneurysm. The patient underwent surgery and made an excellent recovery.

1	A 10-year-old boy was brought to an ENT surgeon (ear, nose, and throat surgeon) with epistaxis (nose bleeding). The bleeding was associated with his nose picking habit. However, the bleeding was profuse and on two occasions required hospital admission and nasal packing. On inspection an indurated area was noted. The typical findings are an indurated area in the anterior inferior aspect of the nasal septum (Kiesselbach’s area). This is a very vascular area that has a considerable number of veins, which are often traumatized during nose picking. The patient underwent treatment. Typical treatment is cauterization of these prominent veins in Kiesselbach’s area, which is usually performed by a simple local analgesia and the application of silver nitrate. Unfortunately, the boy was involved in a fight the next day and again developed severe epistaxis, which again was difficult to control.

1	Unfortunately, the boy was involved in a fight the next day and again developed severe epistaxis, which again was difficult to control. Not only is there a rich venous plexus around Kiesselbach’s area, but there is also a significant arterial supply, which is provided from the nasal septal branches of the posterior and anterior ethmoidal arteries and the branches of the greater palatine artery. These are supplemented from the septal branches of the superior labial artery. In most cases treatment is conservative.

1	In most cases treatment is conservative. Conservative treatment usually involves packing the nasal cavity until bleeding has stopped and correcting any bleeding abnormality. In patients with bleeding refractory to medical treatment a series of maneuvers have been employed, including ligating the anterior and posterior ethmoidal arteries through a medial incision in the canthus orbit, or by ligating other major arteries supplying the nasal cavity. Unfortunately, many of these procedures fail because of the rich and diverse origin of blood supply to the nasal cavity. Determination of the specific site of bleeding can be achieved radiologically. By placing a catheter from the femoral artery through the aorta and into the carotid circulation the sphenopalatine artery can be easily cannulated from the maxillary branch of the external carotid artery. Bleeding can usually be demonstrated and the vessel can be embolized using small particles.

1	Fortunately in this young boy’s case, bleeding stopped after further medical management and he remained asymptomatic. A 30-year-old woman came to her doctor with a history of amenorrhea (absence of menses) and galactorrhea (the production of breast milk). She was not pregnant and appeared otherwise fit and well. Serum prolactin was measured. Prolactin is a hormone produced by the pituitary gland and necessary for the production of breast milk postpartum. This hormone was markedly elevated. Further clinical tests demonstrated visual field defects. The patient went to see an optometrist who performed a visual field assessment and demonstrated a reduction in the lateral aspects of the normal visual fields. This was bilateral and symmetrical—a bilateral temporal hemianopia. The visual pathways have now determined the site of the lesion.

1	The visual pathways have now determined the site of the lesion. Visual information from the temporal fields is projected onto the medial aspect of the retina bilaterally. The visual information from the medial aspects of the retina is carried in fibers that cross the midline through the optic chiasm to the opposite side. The lesion is in the area of the optic chiasm. Any disruption of the optic chiasm produces the field defect of bitemporal hemianopia. Tumors of the optic chiasm are unusual, though gliomas do occur. More frequently, compression of the optic chiasm by tumors in the vicinity is the usual cause for bitemporal hemianopia. A pituitary tumor was diagnosed. The optic chiasm is anterior and extremely close to the pituitary gland. Given that the patient is producing excessive amounts of prolactin (a pituitary tumor) and there is loss of the function of the chiasm, the most likely clinical explanation is an exophytic pituitary tumor compressing the optic chiasm.

1	An MRI scan was performed and demonstrated a large tumor (macroadenoma) of the pituitary gland. Drug treatment was commenced and the tumor shrank (eFig. 8.295). The endocrinological effects of the prolactin secretion also stopped. Follow-up scans were performed. Over the ensuing few years the tumor shrank. Unfortunately, the patient again began to secrete prolactin and surgery was performed. A transsphenoidal approach was undertaken. With meticulous accuracy a series of very fine instruments was passed through the nasal cavity into the sphenoid bone. The bone was drilled and via this approach the pituitary gland was removed. Extreme care must be taken because on both sides of the pituitary gland is the cavernous sinus through which the internal carotid artery, oculomotor nerve [III], trochlear nerve [IV], trigeminal nerve [V], and abducent nerve [VI] pass. 1121.e2 1121.e1 Fig. 8.7, cont’d Skull. Conceptual Overview • Relationship to Other Regions Fig. 8.16, cont’d

1	1121.e2 1121.e1 Fig. 8.7, cont’d Skull. Conceptual Overview • Relationship to Other Regions Fig. 8.16, cont’d In the clinic—cont’d In the clinic—cont’d In the clinic—cont’d In the clinic—cont’d In the clinic—cont’d Table 8.5 Cranial nerves (see Table 8.4 for abbreviations)—cont’d In the clinic—cont’d Table 8.7 Muscles of the face—cont’d In the clinic—cont’d In the clinic—cont’d Fig. 8.149, cont’d In the clinic—cont’d Fig. 8.235, cont’d Fig. 8.239, cont’d Surface Anatomy • Visualizing Structures at the CIII/CIV and CVI Vertebral Levels Surface Anatomy • How to Locate the Cricothyroid Ligament Surface Anatomy • Major Features of the Face

1	Surface Anatomy • Visualizing Structures at the CIII/CIV and CVI Vertebral Levels Surface Anatomy • How to Locate the Cricothyroid Ligament Surface Anatomy • Major Features of the Face Neuroanatomy and neuroscience are fields of science that seek to explain embryonic development, structural organization, and physiological function of the nervous system. Both fields work together to help identify the simple to the most complex questions of human sensory, motor, behavioral, and higher cognitive functions. The focus of this chapter is to introduce the basic structures and functions of the individual and systemic components of the human nervous system. Part I: Nervous system

1	Part I: Nervous system Organization of the human nervous system is structurally divided into the central nervous system (CNS) and peripheral nervous system (PNS) (eFig. 9.1). Components of the CNS are the brain and spinal cord, which are enclosed within the cranial cavity and vertebral column of the axial skeleton. Peripheral nervous system structures include cranial nerves, spinal nerves, autonomic nerves, and the enteric nervous system.

1	During the third week of development the outermost layer of the embryo—the ectoderm—thickens to form a neural plate (eFig. 9.2A). This plate develops a longitudinally running neural groove, which deepens so that it is flanked on either side by neural folds (eFig. 9.2B). These folds further develop and eventually fuse during a process called neurulation to form a long tubelike structure called the neural tube with an inner lumen called the neural canal (eFig. 9.3). Fusion of the tube starts at the midpoint and extends cranially and caudally so that the tube is fully formed by the fourth week. Continued proliferation of the cells at the cephalic end cause the neural tube to dilate and form the three primary brain vesicles (eFig. 9.4): the prosencephalon (forebrain), mesencephalon (midbrain), and rhombencephalon (hindbrain), which later give rise to the structures of the brain. Caudally, the neural tube lengthens and narrows to form the spinal cord. The neural canal forms the cavities of

1	and rhombencephalon (hindbrain), which later give rise to the structures of the brain. Caudally, the neural tube lengthens and narrows to form the spinal cord. The neural canal forms the cavities of the ventricular system in the brain and central canal of the spinal cord (eTable 9.1). The peripheral nervous system consists of cranial nerves, spinal nerves, spinal ganglia, the enteric system, and autonomic ganglia. The peripheral nervous system is formed by nerve fibers that extend out of the central nervous system and by neurons and their fibers that develop from migratory neural crest cells (eFig. 9.4A). Like the neural tube, neural crest cells originate from surface ectoderm and initially lie on each side of the developing CNS.

1	Several terms are used to identify the orientation and location of neural structures. The orientation nomenclature is quite simple in organisms such as fish and reptiles, which have a linear nervous system. For these animals, ventral (Latin for “belly”) is oriented toward the ground, dorsal (Latin for “back”) toward the sky, rostral (Latin for “beak”) toward the snout, and caudal (Latin for “tail) toward the tail (eFig. 9.5). Because humans are bipedal and maintain an erect posture, the nervous system makes an obligatory bend of 80 to 90 degrees at the midbrain–diencephalic junction. Because of this, directional references such as ventral, dorsal, rostral, and caudal have different meanings along different locations of the CNS structures (eFig. 9.6A). An additional set of terms that remain constant in their reference to orientation of nervous system structures are anterior, posterior, superior, and inferior.

1	When studied through imaging or in histopathology, the nervous system is observed in sections cut from one of three different planes: a coronal plane, which divides the nervous system into anterior and posterior parts; the sagittal plane, which is oriented at a right angle to the coronal plane and divides the nervous system into left and right parts; and a horizontal (also referred to as axial or transverse) plane, which divides the nervous system into superior and inferior parts (eFig. 9.6). Note that a sagittal plane passing through the midline may also be referred to as a midsagittal section, whereas a section taken just lateral to the midline is referred to as a parasagittal section.

1	Nerve cells (neurons) and glial cells are the primary cellular components of the nervous system. Neurochemical signaling is predominantly carried out through a complex series of physiological connections between adjoining neurons. Glial cells participate in a constellation of functions that are vital for proper brain function. Their historically appreciated contribution to neuronal function has expanded to include recognition of their role in regulating the content of the extracellular space and regulation of neurotransmitters at the synaptic junction.

1	Neurons consist of a cell body (or soma), which contains the cell nucleus, short processes called dendrites for receiving input from other neurons, and long processes called axons, which conduct signals away from the cell body (eTable 9.2). Depending on their location, neuronal morphology can be quite variable. The majority of mammalian neurons are multipolar, indicating that there are several dendrites from one end and a single axon that branches extensively at its terminus (eFig. 9.7). Some additional neuronal types are bipolar, unipolar, and pseudounipolar (eFig. 9.8).

1	To prevent the loss of linear signal propagation, glial cells form a phospholipid-based layer of insulation called the myelin sheath along the length of the axon (eFig. 9.7). The myelin sheath is formed by oligodendrocytes in the CNS and Schwann cells in the PNS. Interspersed between the segments of myelin are exposed segments of the axon called nodes of Ranvier, which have a large population of voltage-gated ion channels. Presence of the ion channels facilitates rapid conduction of the action potential (a transient voltage change in the axonal membrane) from node to node in a process called saltatory conduction (eFig. 9.7).

1	Functionally, the nervous system is organized into a somatic nervous system and visceral nervous system. The somatic nervous system consists of nerves that carry conscious sensation from peripheral regions back to the CNS and nerves that exit the CNS to innervate voluntary (skeletal) muscles. In contrast, the visceral nervous system consists of nerves that carry sensory information into and motor (autonomic) innervation out of the CNS to regulate homeostatic functions. Further discussion of the somatic and visceral nervous systems will be presented within the context of the subsequent “Spinal Cord” section. Part II: Brain

1	Part II: Brain Externally, the outer surface of the brain, or cerebral cortex, is composed of six layers of cell bodies referred to as gray matter. Internally, the myelinated axonal processes of these cells extend into the cerebral hemispheres. Because of the whitish appearance of these large bundles of myelinated axons, they are referred to as white matter. In the brain, gray matter is predominantly located on the cortical surface and the white matter runs deep inside the cerebral hemispheres; the opposite is true for the spinal cord, where the white matter is superficial to the gray matter. Topographically, the surface of the cerebral hemispheres has a series of elevations called gyri and infoldings referred to as sulci, both of which significantly increase the surface area of the brain.

1	Structurally, each cerebral hemisphere is divided into four major anatomical lobes: frontal, parietal, occipital, and temporal (eFig. 9.9A). The frontal lobes are located anteriorly and are separated from the more posterior parietal lobe by the central sulcus (sulcus of Rolando) (eFig. 9.9A). Laterally, the frontal lobe is separated from the temporal lobe by the lateral sulcus (fissure of Sylvius). Although there is no specific demarcation between the parietal and occipital lobe laterally, along the medial aspect of the hemispheres the two lobes are separated by the parieto-occipital sulcus (eFig. 9.9B). Along the midline, the cerebral hemispheres are separated from one another by the longitudinal fissure (interhemispheric fissure, sagittal fissure). Concealing a small area of cortex called the insula laterally are portions of the frontal, parietal, and temporal lobes collectively referred to as the operculum (Latin for “lid”) (eFig. 9.10). The insula represents fusion of the

1	called the insula laterally are portions of the frontal, parietal, and temporal lobes collectively referred to as the operculum (Latin for “lid”) (eFig. 9.10). The insula represents fusion of the telencephalon and diencephalon and can be seen by gently prying open the lateral sulcus.

1	The path to and from the cerebral cortex is achieved through various white matter pathways coursing through the spinal cord, brainstem, and cerebral hemispheres. Beneath the gray matter of the cortical surface is an expansion of white matter referred to as the corona radiata. This white matter pathway condenses to form the internal capsule, a V-shaped structure when viewed in horizontal sections that contains axons traversing to and from various cortical and deep nuclear structures (eFig. 9.11). The internal capsule is divided into three parts based on connections to different parts of the cortex and underlying structures. The most anterior portion of this white matter pathway is the anterior limb, which is bounded medially by the head of the caudate and laterally by the globus pallidus and putamen. The anterior limb transitions into the genu (Latin for “knee”) at the level of the interventricular foramen (of Monro) and completes its course as the posterior limb, situated lateral to

1	and putamen. The anterior limb transitions into the genu (Latin for “knee”) at the level of the interventricular foramen (of Monro) and completes its course as the posterior limb, situated lateral to the thalamus and medial to the globus pallidus and putamen. In addition to this more vertical stream of axonal connections is the horizontally running corpus callosum. The corpus callosum (eFig. 9.12) is formed by myelinated axons horizontally linking the two cerebral hemispheres to one another, and it is divided into a rostrum, genu, body, and splenium (eFig. 9.12).

1	The ventricular system is derived from the inner lumen of the developing neural tube. As the brain continues to grow, the caverns and canals of the ventricular system adapt to the shape of the cerebral hemispheres, diencephalon, pons, medulla, and cerebellum, which form the surrounding walls (eFig. 9.13). Inferior and lateral to the corpus callosum are two large, fluid-filled cavities that represent the beginning of the ventricular system. These most rostral cavities are the two C-shaped lateral ventricles, located within the cerebral hemispheres (eFig. 9.14). As the lateral ventricles extend through all of the lobes of the cerebral hemispheres, they are divided into five named parts. In the frontal lobe is the anterior (frontal) horn, which transitions into the body within the frontal and parietal lobes (eFig. 9.15). Projecting into the occipital lobe is the posterior (occipital) horn (eFig. 9.15). A final horn extends inferiorly and anteriorly as the inferior (temporal) horn in the

1	parietal lobes (eFig. 9.15). Projecting into the occipital lobe is the posterior (occipital) horn (eFig. 9.15). A final horn extends inferiorly and anteriorly as the inferior (temporal) horn in the temporal lobe (eFig. 9.15). Near the splenium of the corpus callosum, the body, posterior, and inferior horns come together at the atrium/trigone of the lateral ventricles (eFig. 9.15). Lining most of the ventricles is the choroid plexus (eFig. 9.16), a series of modified ependymal cells responsible for producing 0.5 L of cerebrospinal fluid (CSF) a day in adults.

1	From the lateral ventricles, CSF flows through the interventricular foramen (of Monro) to the slitlike third ventricle, which is surrounded by the thalamus and hypothalamus (eFig. 9.15). The third ventricle communicates with the fourth ventricle via the cerebral aqueduct (aqueduct of Sylvius), which courses through the midbrain (eFig. 9.15). Surrounded by the pons and medulla anteriorly and the cerebellum posteriorly, the fourth ventricle sends CSF out of the ventricular system and into the subarachnoid space via the lateral foramina of Luschka and midline foramen of Magendie (eFig. 9.15).

1	Within the bony encasement of the skull and vertebral column, the CNS is surrounded by three concentric, connective tissue coverings called meninges (from Greek word meninx for “membrane”), which act to support and stabilize the brain and spinal cord. The focus of this section will be on the cranial meninges. The spinal meninges, which have a slightly different configuration, will be discussed in the “Spinal Cord” section.

1	The outermost covering is the dura (Latin for “hard”) mater (Latin for “mother”), a tough, fibrous sheet composed of two layers. The outer periosteal layer is adherent to the skull, and the inner meningeal layer lies against the underlying arachnoid mater (eFig. 9.17). Although these two layers are closely adherent to one another, they do separate in some regions to form dural venous sinuses, which receive cerebral venous drainage (eFig. 9.18). The anatomy of the venous sinuses will be discussed in the “Cerebral Vasculature” section. Two potential spaces exist as the epidural (extradural) space, superficial to the periosteal layer, and subdural space, deep to the meningeal dural layer. These spaces can become filled with blood during vascular trauma (eTable 9.3). Within the cranial cavity, the meningeal layer of dura mater folds in on itself in several areas to form dural reflections, or septa. These reflections are known as the falx (Latin for “sickle”) cerebri between the cerebral

1	the meningeal layer of dura mater folds in on itself in several areas to form dural reflections, or septa. These reflections are known as the falx (Latin for “sickle”) cerebri between the cerebral hemispheres, as the tentorium cerebelli between the cerebral hemispheres and cerebellum, and as the falx cerebelli between the cerebellar hemispheres (eFig. 9.17). A smaller reflection, the diaphragm sellae, covers the pituitary fossa and underlying pituitary gland.

1	Deep to the dura mater is the arachnoid (from the Greek word arachne meaning “spider’s web”) mater. The outer layer of the arachnoid mater is composed of several layers of flattened cells that lie adjacent to the meningeal layer of the dura mater (eFig. 9.19). Strands of connective tissue extend from this outer layer to form arachnoid trabeculae, which connect internally to the pia mater (eFig. 9.19). The pia mater forms a thin, veil-like layer that closely follows the gyri and sulci on the surface of the brain. The pia and arachnoid matter are separated by a subarachnoid space that contains CSF and the major blood vessels supplying the brain.

1	Vascular supply to the brain is divided into the anterior circulation arising from the internal carotid arteries and posterior circulation from the vertebral arteries (eFig. 9.20). The internal carotid arteries arise from the branching of the common carotid arteries at the level of the fourth cervical vertebra. Bilaterally the arteries course through the neck to enter the middle cranial fossa through the carotid canal. The arteries then make a series of turns to pass through the petrous portion of the temporal bone and cavernous sinus before entering the subarachnoid space just lateral to the optic chiasm. Upon exiting the cavernous sinus, the internal carotid artery gives rise to the ophthalmic artery and then continues superiorly to give off the posterior communicating artery and anterior choroidal arteries before terminating as the anterior and middle cerebral arteries (eFig. 9.21).

1	The two anterior cerebral arteries anastomose proximally via the anterior communicating artery, anterior to the optic chiasm (eFig. 9.22). Distal to this connection, the anterior cerebral artery (ACA) courses along the medial aspect of the cerebral hemisphere within the longitudinal fissure and follows the superior border of the corpus callosum to the anterior portion of the parietal lobe. Along its course two large branches arise: the callosomarginal artery, which follows the cingulate sulcus, and the pericallosal artery, which is immediately adjacent to the corpus callosum (eFig. 9.23). Given its course and branches, the ACA perfuses most of the medial aspect of the brain from the frontal lobe to the anterior portion of the parietal lobe.

1	Branching laterally from the internal carotid artery, the middle cerebral artery (MCA) penetrates the lateral fissure and gives off the lenticulostriate striate arteries (eFig. 9.24) before bifurcating into superior and inferior divisions, which loop extensively along the insula and frontal operculum before emerging on the lateral convexity of the cerebrum. The superior division perfuses the cortex above the lateral fissure, including the lateral frontal lobe and a small portion of the anterior parietal lobe (eFig. 9.21). The inferior division perfuses the cortex below the lateral fissure, including the temporal lobe and the anterolateral portion of the parietal lobe.

1	The posterior cerebral cortex receives vascular supply from the vertebral-basilar system of arteries. This system begins with the vertebral arteries bilaterally, which arise from the subclavian arteries and ascend through the foramen transversarium of the cervical vertebrae in the neck. After entering the foramen magnum at the level of the pontomedullary junction, the arteries join to form the basilar artery, which courses along the midline of the ventral brainstem (eFig. 9.20). At the level of the midbrain, the basilar artery gives rise to the posterior cerebral artery (PCA), which turns posteriorly and gives rise to branches that perfuse the inferior and medial temporal and occipital lobes. Also at the PCA, a connecting artery, the posterior communicating artery, branches off and connects to the internal carotid artery (eFig. 9.22).

1	Venous drainage of the cerebral hemispheres follows a system of deep veins, superficial veins, and dural venous sinuses before reaching the internal jugular vein. Before reaching the internal jugular veins, the superficial and deep veins connect to the dural sinuses located between the periosteal and meningeal layers of the dura. None of the vessels in this network have valves present in their lumen.

1	Running along the superior edge of the falx cerebri is the superior sagittal sinus. The superior sagittal sinus continues posteriorly to drain into the transverse sinuses bilaterally (eFig. 9.25A). Each transverse sinus turns inferiorly to form the sigmoid sinus, which exits the jugular foramen to become the internal jugular vein. Along the inferior margin of the falx cerebri is the inferior sagittal sinus (eFig. 9.25B). Posteriorly, the inferior sagittal sinus joins the great vein of Galen to form the straight sinus. The point where the straight sinus, superior sagittal sinus, and occipital sinus join is known as the confluence of the sinuses (eFig. 9.25B). The confluence of sinuses is drained by the transverse sinuses. Located on either side of the hypophysial fossa is a plexus of veins referred to as the cavernous sinus (eFig. 9.26). In addition to receiving drainage from the other sinuses, the cavernous sinus also receives the ophthalmic veins. The cavernous sinus is drained by

1	referred to as the cavernous sinus (eFig. 9.26). In addition to receiving drainage from the other sinuses, the cavernous sinus also receives the ophthalmic veins. The cavernous sinus is drained by the superior petrosal sinus into the transverse sinus and inferior petrosal sinuses into the internal jugular vein.

1	Venous drainage from the superficial veins is primarily received by the superior sagittal sinus and cavernous sinus. Although the pattern of superficial veins coursing through the subarachnoid space from the cerebral cortex is quite variable, three veins appear to be fairly constant. Positioned in parallel to the lateral fissure is the superficial middle cerebral vein, which drains into the cavernous sinus from the temporal lobe (eFig. 9.25A). Connecting to the superficial middle cerebral vein perpendicularly is the superior anastomotic vein (of Trolard) (eFig. 9.25A). This vein courses superiorly across the parietal lobe to drain into the superior sagittal sinus. Also connecting to the superficial middle cerebral vein perpendicularly is the inferior anastomotic vein (of Labbé) (eFig. 9.25A). The inferior anastomotic vein passes inferiorly along the temporal lobe to drain into the transverse sinus.

1	In contrast to the superficial veins, deep veins are more constant in their organization. Most of the deep veins eventually drain into the great cerebral vein (of Galen) before entering the dural venous sinuses (eFig. 9.27). Traveling adjacent to the ACA and MCA are the anterior cerebral vein and deep middle cerebral vein. These deep veins join to form the basal vein (of Rosenthal), which continues around the lateral aspect of the midbrain. Formed at the interventricular foramen by the joining of the septal and thalamostriate veins bilaterally are the internal cerebral veins (eFig. 9.27). Posterior to the midbrain, the internal cerebral veins and basal veins join to form the great cerebral vein (of Galen) (eFig. 9.27). From here the great cerebral vein joins the inferior sagittal sinus to form the straight sinus. Part III: Thalamus

1	Part III: Thalamus The thalamus (Greek for “inner chamber”) is a large, egg-shaped mass of gray matter derived from the diencephalon of the developing brain (see eTable 9.1). The most significant role of the thalamus is as a synaptic relay for pathways projecting to the cerebral cortex. However, the thalamus also acts as a gatekeeper to prevent or enhance information transfer, depending on the behavioral state. Sensory, motor, limbic, and modulatory signals from behavioral and arousal circuits all have synaptic relays within the thalamic nuclei.

1	Because of its location deep within the brain, the thalamus is neighbored by several structures and also portions of the ventricular system. Anteriorly, the thalamus extends forward to contact the interventricular foramen, which connects the lateral and third ventricles (eFig. 9.28). Together, the thalamic nuclear masses and the ventrally located hypothalamus comprise the lateral walls of the third ventricle. Immediately lateral to the thalamus is the posterior limb of the internal capsule (eFig. 9.29). Related to the dorsal aspect of the thalamus is the body of the lateral ventricle, and extending caudally is the midbrain portion of the brainstem (eFig. 9.28). Across the midline of the third ventricle, the two thalamic masses are interconnected by the interthalamic adhesion.

1	Based on their relationship to the internal medullary lamina, a Y-shaped band of myelinated axons coursing rostrocaudally through the thalamus, the thalamic nuclei are classified into four groups: (1) anterior, (2) medial, (3) lateral, and (4) intralaminar (eFig. 9.30). In addition to this structural categorization, the nuclei are divided into three major functional classes: (1) relay, (2) intralaminar, and (3) reticular. As mentioned earlier, most of the thalamus is composed of relay nuclei, which have reciprocal excitatory connections with the cortex. Relay nuclei are further subdivided into specific and nonspecific based on their projections to specific areas of the primary sensory and motor cortex or more diffuse cortical projections. The majority of specific relay nuclei reside in the lateral thalamus—in fact, all sensory modalities, with the exception of olfaction, have relays in the lateral thalamus before reaching their primary cortical target. Details of specific and

1	in the lateral thalamus—in fact, all sensory modalities, with the exception of olfaction, have relays in the lateral thalamus before reaching their primary cortical target. Details of specific and nonspecific nuclei and their cortical connections can be reviewed in eTable 9.4.

1	Intralaminar nuclei reside within the internal medullary lamina and have numerous reciprocal projections, primarily with the basal ganglia and reticular formation (eFig. 9.30).There are two major functional regions of the intralaminar nuclei: rostral and caudal. The rostral intralaminar nuclei have reciprocal connections with the basal ganglia and also relay input from the ascending reticular activating system. Caudal intralaminar nuclei, which include the large centromedian nucleus, are predominantly involved in basal ganglia circuitry.

1	The reticular nucleus of the thalamus is a thin, sheet-like structure along the lateral aspect of the thalamus just medial to the posterior limb of the internal capsule (eFig. 9.30). Unlike the rest of the thalamic nuclei, the reticular nucleus does not send projections to the cerebral cortex, but rather receives input from other thalamic nuclei and the cortex, which project back to the thalamus. Functionally, this organization of inputs and outputs, along with the GABAergic neurons of the reticular nucleus, allows it to regulate thalamic activity quite effectively. Vascular supply to the thalamus arises from penetrating branches from the ACA, anterior choroidal artery branching from the internal carotid, lenticulostriate arteries of the middle cerebral artery and thalamoperforator arteries from the posterior cerebral arteries. Part IV: Brainstem

1	Part IV: Brainstem The brainstem is a stalklike structure within the posterior cranial fossa of the skull connecting the forebrain and spinal cord (eFig. 9.31). From rostral to caudal, the brainstem consists of the midbrain, pons, and medulla oblongata. Broadly speaking, the brainstem has three main functions: (1) it is a conduit for tracts ascending and descending through the CNS; (2) it houses cranial nerve nuclei III to XII (note that the CNXI is in the cervical spinal cord); and (3) it is the location for reflex centers related to respiration, cardiovascular function, and regulation of consciousness. Externally, each portion of the brainstem has a distinct appearance and structural features that define its many functional roles.

1	Approximately 2 cm in length, the midbrain connects the forebrain to the pons caudally and the cerebellum posteriorly (eFig. 9.32A). Along the midline of the anterior surface there is a deep depression, the interpeduncular fossa, which has several perforations on its surface where small vessels perforate through the floor of the fossa. On either side of the interpeduncular fossa are the crus cerebri (eFig. 9.32B). Projecting medially from the crus is cranial nerve III, the oculomotor nerve (eFig. 9.33A). The most prominent features on the posterior surface are the superior and inferior colliculi (eFig. 9.32A). Between the inferior colliculi, cranial nerve IV, the trochlear nerve, emerges, crosses the midline, and wraps around the lateral aspect of the midbrain (eFig. 9.32A). Laterally, the superior brachium and inferior brachium can be seen as they project in an anterolateral direction from the superior and inferior colliculi.

1	Caudal to the midbrain, the pons is approximately 2.5 cm in length and connects the midbrain to the medulla (eFig. 9.32B). The midline of the anterior surface has a shallow groove, the basilar groove, where the basilar artery resides along its course (eFig. 9.32B). On either side of the basilar groove, the pons has a prominent convex shape as a result of the vast number of fibers bridging through the pons to the cerebellum through the middle cerebellar peduncle. Along the anterolateral surface of the pons, cranial nerve V, the motor and sensory root of the trigeminal nerve, emerges (eFig. 9.33B). At the junction of the pons and medulla, from medial to lateral, cranial nerves VI (abducent), VII (facial), and VIII (vestibulocochlear) emerge (eFig. 9.33C). The posterior surface of the pons faces the cerebellum and forms the floor of the fourth ventricle (eFig. 9.32A). Along the midline of the posterior surface is a prominent bump, the facial colliculus, which represents the relationship

1	the cerebellum and forms the floor of the fourth ventricle (eFig. 9.32A). Along the midline of the posterior surface is a prominent bump, the facial colliculus, which represents the relationship between the facial nerve fibers as they wind around the nucleus of the abducent nerve (eFig. 9.33C).

1	The medulla is the longest portion of the brainstem, measuring approximately 3 cm in length (eFig. 9.32A). At the rostral connection of the medulla to the pons, it has a broad conical shape that tapers caudally before connecting with the spinal cord at the level of the foramen magnum. Along the midline of the anterior surface is the anterior median fissure, which continues on to the anterior surface of the spinal cord (eFig. 9.33B). Lateral to the fissure are the medullary pyramids composed of motor fibers descending from the cerebral cortex (eFig. 9.33B). Continuing caudally, the pyramids eventually give way to the decussation of the pyramids where the majority of motor fibers decussate to the opposing side. Lateral to the pyramids are the olives, which represent the underlying inferior olivary nuclei. It is at the junction of the pyramid and the olive that the rootlets of cranial nerve XII, the hypoglossal nerve, emerge (eFig. 9.33D). Oriented posterior to the olives are the

1	olivary nuclei. It is at the junction of the pyramid and the olive that the rootlets of cranial nerve XII, the hypoglossal nerve, emerge (eFig. 9.33D). Oriented posterior to the olives are the inferior cerebellar peduncles, which form a connection between the cerebellum and medulla (eFig. 9.33D). At the junction of the olive and inferior cerebellar peduncles from rostral to caudal, rootlets of cranial nerves IX (glossopharyngeal), X (vagus), and XI (spinal accessory) emerge. Like the pons, the posterior surface of the medulla forms the floor of the fourth ventricle (eFig. 9.32A). Coursing down the midline of the posterior medulla is the posterior median sulcus, which continues into the spinal cord. On either side of the sulcus are the gracile and cuneate tubercles, formed by the underlying gracile nucleus and cuneate nuclei (eFig. 9.32A).

1	Internal structures of the brainstem can be identified by their general location in the tectum (Latin for “roof”), tegmentum (Latin for “covering”), or basis (eFig. 9.34). To identify the structures present in each level of the brainstem, it is best to view serial sections stained for myelin. This way, nuclear groups and myelinated axons can be more easily distinguished from one another.

1	The tectum is the most obvious landmark of the midbrain, as it consists of the prominent superior colliculi, inferior colliculi, and underlying cerebral aqueduct. Given the short length of the midbrain, stained serial sections typically include either the superior colliculi, which are located rostrally, or the inferior colliculi, which are caudal. In addition to the superior colliculi, within a rostral section of the midbrain prominent nuclei such as the oculomotor nuclei, Edinger–Westphal nuclei, red nuclei, mesencephalic nuclei of cranial nerve V, and substantia nigra can be seen (eFig. 9.35). In caudal sections of the midbrain the inferior colliculus, trochlear nucleus, mesencephalic nuclei of cranial nerve V, and substantia nigra are present (eFig. 9.36).

1	Sections from the rostral pons are significant for pontine nuclei and multiple transversely oriented pontocerebellar fibers en route to the contralateral cerebellum. Interspersed among these horizontally running axon are longitudinally running corticospinal fibers (eFig. 9.37). Dorsal to this collection of fiber bundles, the medial lemniscus is oriented horizontally, forming a borderlike structure between the basal and tegmental portion of the pons (eFig. 9.37). At the lateral edge of the medial lemniscus, the spinothalamic tract can be seen, because it neighbors the superior cerebellar peduncle (eFig. 9.37). Near the midline, the medial longitudinal fasciculus resides just ventral to the periaqueductal gray matter.

1	At mid-pontine levels the superior cerbellar peduncles form the lateral walls of the expanding fourth ventricle (eFig. 9.38). Also at this level, the prominent middle cerebellar peduncles can also be seen, along with the motor nucleus and principal sensory nucleus of cranial nerve V, the trigeminal nerve. In the caudal aspect of the pons, the abducent nucleus can be appreciated just lateral to the medial longitudinal fasciculus (eFig. 9.39). Also present at this level is the medial vestibular nucleus in addition to the anterior and posterior cochlear nuclei.

1	From rostral to caudal the internal structures of the medulla will be briefly described from three levels: (1) level of the inferior olivary nucleus, (2) decussation of the internal arcuate fibers, and (3) decussation of the pyramids. Nuclei visible in a transverse section at the level of the inferior olivary nucleus include those associated with cranial nerves VIII (vestibulocochlear), IX (glossopharyngeal), X (vagus), XI (spinal accessory), and XII (hypoglossal) (eFig. 9.40). At this level, the medial lemniscus maintains a vertical position immediately adjacent to the midline. Dorsal to the medial lemniscus are the medial longitudinal fasciculus and hypoglossal nucleus. A large distinguishing structure at this rostral level is the laterally oriented inferior cerebellar peduncle, which connects to the cerebellum posteriorly.

1	Continuing caudally at the level of the internal arcuate fibers, the dorsal aspect of the medulla is populated by the gracile nucleus medially, followed by the cuneate and spinal trigeminal nucleus laterally. Ventral to the cuneate and gracile nuclei, their neuronal axons can be seen decussating as internal arcuate fibers to form the medial lemniscus near the midline of the medulla. Lateral to the internal arcuate fibers and ventral to the spinal trigeminal tract, the spinocerebellar and anterolateral tracts can be seen along the perimeter of the medulla (eFig. 9.41). Before transitioning into the spinal cord, the pyramidal decussation can be observed along the midline of the caudal medulla (eFig. 9.36). Dorsal to these decussating fibers, the gracile and cuneate nuclei begin to emerge as their fasciculi continue rostrally. Note that the spinal accessory nucleus (CN XI) is located in the cervical spinal cord and not the medulla.

1	Vascular supply to the brainstem, and other structures in the posterior cranial fossa, is provided by branches off of the vertebrobasilar system of arteries. As mentioned in the “Cerebral Vasculature” section, the vertebrobasilar system begins with the vertebral arteries bilaterally, which arise from the subclavian arteries and ascend through the foramen transversaria of cervical vertebrae C6 to C2 in the neck. At the pontomedullary junction, the vertebral arteries fuse to form the single basilar artery. The basilar artery continues rostrally and terminates as the paired posterior cerebral arteries at the pontomesencephalic junction.

1	Before merging and forming the basilar artery, each vertebral artery gives rise to a posterior inferior cerebellar artery (PICA) and posterior spinal artery and contributes to the formation of the anterior spinal artery (eFig. 9.42). The PICA perfuses the lateral aspect of the medulla and the inferior portion of the cerebellum. The medial and anterior portions of the medulla receive vascular supply from the paramedian branches off of the vertebral and anterior spinal arteries. At the level of the caudal pons, the anterior inferior cerebellar artery (AICA) branches off of the basilar artery and perfuses the lateral portion of the caudal pons (eFig. 9.43A). Rostral levels of the lateral pons are perfused by circumferential branches of the basilar artery (eFig. 9.43A and B). Medial portions of the pons are perfused by paramedian branches off of the basilar artery as it continues rostrally toward the midbrain.

1	Just before the midbrain, the superior cerebellar arteries branch off of the basilar artery and supply the superior cerebellar peduncles and caudal aspect of the dorsal midbrain before reaching the superior portion of the cerebellar hemispheres. Paramedian branches from the basilar artery supply the medial aspect of the midbrain (eFig. 9.43C). The final branches emerging from the top of the basilar artery are the PCAs, which perfuse the lateral aspect of the midbrain before reaching the thalamus, medial occipital, and inferior temporal lobes (eFig. 9.43C and D). Part V: Spinal cord

1	Part V: Spinal cord The spinal cord is continuous with the medulla oblongata near the foramen magnum at the base of the skull. Cylindrical in shape, it occupies the vertebral canal of the vertebral column to the LI and LII vertebral level in an adult (eFig. 9.44). Numerous ascending and descending axonal tracts course through the spinal cord and connect with the brain to convey sensory (afferent) and motor (efferent) information for facilitation of movement, reflexes, sensory input, and feedback mechanisms.

1	Like the brain, the spinal cord is surrounded by three concentric meninges: the dura mater, arachnoid mater, and pia mater. The spinal dura mater is continuous with the inner meningeal layer of the cranial dura mater and extends inferiorly to the posterior surface of the vertebral body of S2 (eFig. 9.45). It is separated from the bony vertebral canal by the epidural/extradural space (eFig. 9.45). In addition, unlike in the cranial cavity, the underlying arachnoid mater is not tightly adherent to the dura mater, and instead has a theoretical plane or potential space called the subdural space.

1	Although the arachnoid mater of the spinal cord has a less adherent relationship with the dura mater than in the cranial cavity, the overall structure of the arachnoid mater is the same (eFig. 9.46A). The subarachnoid space created by the loose relationship of the arachnoid and underlying pia mater extends inferiorly to the level of the SII vertebra (eFig. 9.45). Given that the spinal cord terminates near the LI–LII vertebrae, this lower termination point of the subarachnoid space creates a safe and enlarged space for accessing CSF in the clinical setting (eFig. 9.45).

1	The innermost pia mater is a highly vascular layer that is adherent to the surface of the spinal cord. Midway between the anterior and posterior roots the pia mater forms a flat continuous sheet, the denticulate ligament (eFig. 9.46B). At the posterior and anterior rootlets, sleevelike projections from the denticulate ligament extend out through the arachnoid mater to attach onto the dura mater. These delicate attachments anchor and position the spinal cord within the central area of the subarachnoid space.

1	The anterior and posterior surfaces of the spinal cord have several longitudinally running fissures and sulci. Along the midline on the anterior surface of the spinal cord is a deep separation, the anterior median fissure (eFig. 9.47). Posteriorly the spinal cord has a shallower separation, the posterior median sulcus, which is flanked on either side by a posterolateral sulcus (eFig. 9.47). Emerging from the spinal cord are a series of rootlets, which coalesce to form anterior and posterior roots at the corresponding cord segment (eFig. 9.48). These anterior and posterior roots converge to form 31 pairs of spinal nerves, which extend the length of the spinal cord (eFig. 9.44). Along the length of the spinal cord two regions are enlarged to accommodate the numerous neurons innervating the upper and lower extremities. The cervical enlargement extends from C5 to T1 and innervates the upper extremities, whereas the lumbar enlargement extends from L2 to S3 and innervates the lower

1	the upper and lower extremities. The cervical enlargement extends from C5 to T1 and innervates the upper extremities, whereas the lumbar enlargement extends from L2 to S3 and innervates the lower extremities (eFig. 9.44).

1	A cross-section of the spinal cord reveals an inner H-shaped gray matter consisting of neuronal cell bodies and an outer white matter composed of myelinated neuronal axons. The ventral or anterior horns of gray matter contain cell bodies of motor neurons, whereas the dorsal or posterior horns contain cell bodies receiving sensory information (eFig. 9.47). An enlargement of the lateral portion of the gray matter, termed the intermediolateral cell column, can be seen in the T1 to L2 region of the spinal cord (eFig. 9.49). This region enlarges to accommodate the preganglionic cell bodies of the sympathetic nervous system. To further define the diverse cytoarchitecture of the gray matter, it is divided into 10 zones known as Rexed’s laminae (eFig. 9.50). These will be referred to as they relate to later discussions of the ascending and descending tracts within the spinal cord.

1	The anterior funiculus of the white matter consists of motor axons, whereas the posterior funiculus consists of axons conveying sensory information (eFig. 9.47). The lateral funiculus has a mixture of axons conveying both sensory and motor information.

1	Sensory information entering the CNS from peripheral sensory receptors is conducted through a series of neurons that synapse with targets in the spinal cord, cerebral cortex, and other brain structures. The sensory modalities carried in these pathways include pain, temperature, tactile, and proprioceptive input. Conscious perception of sensory input is transmitted through neuronal pathways, which reach the primary somatosensory region of the cerebral cortex. In addition to conscious sensory input, there is subconscious sensory input, which is transmitted to other structures such as the cerebellum. For simplicity, in this section we will review the sensory pathways that reach conscious perception and discuss the pathways conveying subconscious sensory input in the section on cerebellum.

1	Two somatosensory pathways ascend within the spinal cord to reach the cortex: (1) the anterolateral pathways, which convey sensations of pain, temperature, and crude touch; and (2) the posterior column–medial lemniscal pathway, which conveys sensations of discriminative or fine touch, vibration, and conscious proprioception (eFig. 9.51). Both of these pathways transmit information through a series of three neurons. We will review the anterolateral pathway first.

1	The anterolateral pathways are composed of three tracts: the spinothalamic, spinoreticular, and spinomesencephalic tract. Separate aspects of pain are conveyed through the spinothalamic tract, so we will follow the course of those neurons first. The first-order neuronal cell body of axons forming the spinothalamic tract is located in a spinal ganglion (eFig. 9.52). Axons then enter the spinal cord through the posterior root to reach the posterior horn. From here, axons have two courses: some synapse immediately on second-order neurons in the posterior horn gray matter (lamina I and V), and others have axonal collaterals that ascend or descend one to two spinal cord segments in the posterolateral tract of Lissauer before synapsing with the second-order neurons in the gray matter (eFig. 9.51). Axons of the second-order neurons then cross obliquely over two to three spinal cord segments within the anterior commissure of the spinal cord to join the anterolateral tract on the contralateral

1	Axons of the second-order neurons then cross obliquely over two to three spinal cord segments within the anterior commissure of the spinal cord to join the anterolateral tract on the contralateral side (eFig. 9.52). These second-order axons ascend through the CNS to reach the third-order neuronal cell bodies in the ventral posterior lateral nucleus of the thalamus (eFig. 9.52). Axons from the third-order neurons then project through the posterior limb of the internal capsule to reach the primary somatosensory cortex (eFig. 9.52).

1	The spinoreticular and spinomesencephalic tracts have a similar beginning as the spinothalamic tract. The principal difference is the target structure of the second-order axons. Rather than project to the thalamus as the spinothalamic tract does, the spinoreticular tract projects to the reticular formation in the brainstem to convey the emotional and arousal aspects of pain (eFig. 9.52), and the spinomesencephalic tract projects to the periaqueductal gray matter and superior colliculi in the midbrain for the central modulation of pain (eFig. 9.52).

1	First-order neuronal cells bodies of the posterior column–medial lemniscal pathway are located in a spinal ganglion (eFig. 9.53). Axons then enter the spinal cord through the posterior root to reach either the gracile fasciculus (gracile means “thin”), which carries information from the lower limb and trunk, or the cuneate fasciculus (cuneate means “wedge-shaped”), which carries information from the upper limb and neck. These first-order axons then ascend ipsilaterally to the caudal medulla and synapse with the second-order neuronal cell bodies within the nucleus gracilis and nucleus cuneatus (eFig. 9.53). Axons of these second-order neurons then cross over as the internal arcuate fibers to form the medial lemniscus in the contralateral medulla (eFig. 9.53). These second-order axons ascend through the brainstem to reach the third-order neuronal cell bodies in the ventral posterior lateral nucleus of the thalamus (eFig. 9.53). Axons from the third-order neurons then project through the

1	through the brainstem to reach the third-order neuronal cell bodies in the ventral posterior lateral nucleus of the thalamus (eFig. 9.53). Axons from the third-order neurons then project through the posterior limb of the internal capsule to reach the primary somatosensory cortex (eFig. 9.53).

1	Descending tracts through the spinal cord are involved in voluntary movements; postural movements; and coordination of head, neck, and eye movements. These pathways originate from the cerebral cortex and brainstem and are influenced by sensory input and feedback circuitry from the cerebellum and basal ganglia. Structures that influence regulation of motor planning and voluntary control will be discussed in subsequent sections. In this section we will review the tracts of the medial and lateral motor systems. The tracts in each of these systems are composed of an upper motor neuron with cell bodies located in the cerebral cortex or brainstem and a lower motor neuron with cell bodies located in the spinal cord gray matter. We will begin by exploring the tracts of the lateral motor system first.

1	Tracts of the lateral motor system include the lateral corticospinal tract and rubrospinal tract. Both are located in the lateral column of the spinal cord white matter and synapse on lower motor neuronal cell bodies in the lateral aspect of the anterior horn gray matter.

1	Clinically, the most important tract is the lateral corticospinal tract, because it is responsible for controlling movement of the upper and lower extremities. Cell bodies of upper motor neurons forming this tract are located in the primary motor cortex (eFig. 9.54). Axons of these upper motor neurons converge in the corona radiata and descend through the posterior limb of the internal capsule to reach the crus cerebri of the midbrain. These axons continue through the anterior aspect of the pons as small bundles to accommodate the transverse pontocerebellar fibers, which are also present in this location. Once the fibers reach the medulla, they are again grouped together and form a large swelling known as the pyramid (eFig. 9.54). At the caudal medulla, before transitioning into the spinal cord, most of the axons decussate over to the contralateral side to form the lateral corticospinal tract (eFig. 9.54). The remaining axons will stay ipsilateral and form the anterior corticospinal

1	cord, most of the axons decussate over to the contralateral side to form the lateral corticospinal tract (eFig. 9.54). The remaining axons will stay ipsilateral and form the anterior corticospinal tract, a tract included in the medial motor systems.

1	After decussating and forming the lateral corticospinal tract, the axons descend through the spinal cord to synapse on the cell bodies of lower motor neurons in the lateral portion of the anterior horn gray matter. Axons of these lower motor neurons then exit the spinal cord through the anterior root (eFig. 9.54). The other lateral motor system pathway is the rubrospinal tract (eFig. 9.55). Cell bodies of upper motor neurons in this pathway begin in the red nucleus of the midbrain. After leaving the red nucleus, the axons cross the midline as the ventral tegmental decussation and descend as the rubrospinal tract through the brainstem and lateral column of the spinal cord white matter (eFig. 9.55). These axons only descend to cervical regions of the spinal cord, and axons synapse with interneurons in the anterior horn gray matter to facilitate flexor muscle activity and inhibit extensor muscle activity of the upper limb.

1	Tracts of the medial motor system regulate axial or truncal muscles involved in maintaining posture, balance, automatic gait-related movements, and orientating movements of the head and neck. Unlike the lateral motor system, the tracts in this system primarily project bilaterally on interneurons within the spinal cord. This makes it difficult to test each tract individually in the clinical system. We will briefly review the four tracts of the medial motor system beginning with the anterior corticospinal tract.

1	The anterior corticospinal tract is formed by the remaining descending upper motor neurons that did not decussate in the caudal medulla to form the lateral corticospinal tract. These upper motor neurons, which remain ipsilateral to form the anterior corticospinal tract, descend through the medial aspect of the anterior spinal cord to the level of the upper thoracic region (eFig. 9.54). These axons project bilaterally to synapse on cell bodies of lower motor neurons in the medial portion of the anterior horn gray matter. Axons of these lower motor neurons then exit the spinal cord through the anterior root.

1	Tectospinal tract axons arise from cell bodies located in the superior colliculus of the dorsal midbrain (eFig. 9.56). These axons decussate in the dorsal tegmental decussation shortly after leaving the nucleus to form the tectospinal tract along the midline of the brainstem. The tectospinal tract continues through the brainstem near the medial longitudinal fasciculus and into cervical regions of the spinal cord near the anterior median fissure. Within the cervical spinal cord, axons project bilaterally to synapse on cell bodies of interneurons in the anterior horn gray matter. As the superior colliculus receives visual input, it is believed that the tectospinal tract modulates reflex postural movements in response to visual stimuli.

1	Vestibulospinal tract axons arise from vestibular nuclei located in the pons and medulla. The medial vestibular nucleus gives rise to the medial vestibulospinal tract, which projects bilaterally to thoracic regions of the spinal cord, and the lateral vestibular nucleus gives rise to the lateral vestibulospinal tract, which descends ipsilaterally through the entire length of the spinal cord to synapse on interneurons in the anterior horn gray matter (eFig. 9.57). Given that the vestibular nuclei receive sensory input from the inner ear and cerebellum, this tract facilitates activity of extensor/antigravity muscles and inhibits activity of flexor muscles to maintain balance and an upright posture. As an example, the change in head position induced during tripping initiates extension of the upper limb and/or lower limb to prevent oneself from falling forward.

1	Reticulospinal tract axons arise from the reticular formation in the pons and medulla. The axons of the pontine and medullary reticulospinal tracts descend ipsilaterally through the length of the spinal cord in the anterior white matter and synapse with interneurons in the anterior horn gray matter. They are believed to function in regulating voluntary movements in reflex activity and autonomic outflow (eTables 9.5 and 9.6).

1	Vascular perfusions to the spinal cord are supplied by three longitudinally running vessels and several segmental branches. The longitudinally running vessels are the anterior spinal artery and two posterior spinal arteries. The posterior spinal arteries originate in the cranial cavity as branches of either the vertebral artery or PICA. These arteries descend along the length of the posterior spinal cord on the posterolateral sulcus. The single anterior spinal artery originates within the cranial cavity from the union of two contributing branches from the vertebral arteries. The anterior spinal artery descends along the length of the anterior spinal cord on the anterior median fissure.

1	Reinforcing vascular supply to these longitudinally running vessels is provided by eight to ten segmental medullary arteries. The largest segmental medullary artery is the artery of Adamkiewicz in the lower thoracic or upper lumbar region. This vessel is typically on the left side and contributes significantly to perfusion of the lower portion of the spinal cord. Venous drainage of the spinal cord occurs through a series of longitudinally running channels that connect with the anterior and posterior spinal veins on the surface of the cord. Part VI: Basal nuclei

1	Part VI: Basal nuclei The basal nuclei are a collection of gray-matter structures named for their location deep within the base of the forebrain. Functionally, the basal nuclei have a significant role in controlling posture and voluntary movement through connections to the thalamus, cortex, and neighboring basal nuclei structures. In addition to their role in posture and movement, the basal nuclei have connections to limbic system pathways, which govern the expression of various behaviors and motivational states. For the purposes of this section, we will focus on reviewing the structures of the basal nuclei and pathways involved in controlling posture and voluntary movement.

1	A variety of terminology is used to refer to the structures of the basal nuclei individually and based on their collective morphology. To appreciate the three-dimensional shape of the basal nuclei and their relationship to surrounding structures, it is best to view them in horizontal and coronal brain sections taken at different levels of the brain. The corpus striatum (Latin for “striped body”) includes the caudate nucleus and lentiform nucleus. This collection of structures received their name because of the striated appearance of bands that interconnect the caudate nucleus and putamen of the lentiform nucleus through the anterior limb of the internal capsule (eFig. 9.58).

1	The lentiform nucleus (Latin for “lens-shaped”) includes the globus pallidus and putamen, which appear lens-shaped when viewed laterally. Both of these structures are lateral to the internal capsule, which separates them from the thalamus and caudate nucleus medially (eFig. 9.58). Laterally, the putamen is bordered by the external capsule, a thin layer of white matter adjacent to a thin gray-matter layer called the claustrum. Beyond the claustrum is the external capsule, which borders the white matter of the insula (eFig. 9.58).

1	Medial to the internal capsule is the caudate nucleus. The caudate nucleus is a large C-shaped structure divided into a head, body, and tail, which closely follows the shape of the lateral ventricle (eFig. 9.58). Rostrally, the head of the caudate has a large rounded shape that contributes to the lateral wall of the anterior horn of the lateral ventricle (eFig. 9.58). Also at this level, the head of the caudate is continuous with the putamen (eFig. 9.59). Because of this close relationship the putamen and caudate are referred to collectively as the striatum. At the level of the interventricular foramen, the head of the caudate transitions to the body. The body of the caudate is long and narrows substantially as it transitions from the head to the tail (eFig. 9.59). Along its course the body contributes to the floor of the lateral ventricle. Near the posterior border of the thalamus the body of the caudate transitions into the tail. The tail continues anteriorly within the roof of the

1	contributes to the floor of the lateral ventricle. Near the posterior border of the thalamus the body of the caudate transitions into the tail. The tail continues anteriorly within the roof of the inferior horn of the lateral ventricle to terminate in the amygdaloid nucleus (eFig. 9.59).

1	Input to the basal nuclei is primarily received by the striatum (caudate nucleus and putamen), and output predominantly leaves from the globus pallidus. Many structures send input to the striatum, including all areas of the cerebral cortex, thalamic nuclei, subthalamic nucleus, brainstem, and substantia nigra. To understand how the basal nuclei integrates all of this incoming information to influence motor activity, two simplified neuronal loops are described: the direct pathway and the indirect pathway.

1	The direct pathway has a series of connections through the basal nuclei, which result in an overall increase in motor activity. This pathway begins with input to the striatum, which sends axonal connections to the globus pallidus (eFig. 9.60A). The globus pallidus then has output connections to the thalamus, which completes the circuit with axonal connections back to the cortex. The indirect pathway has a similar course, with the addition of output connections to the subthalamic nucleus, which results in an overall decrease in motor activity (eFig. 9.60B). Part VII: Cerebellum

1	Part VII: Cerebellum The cerebellum is the largest structure of the hindbrain. It resides within the posterior cranial fossa and is composed of two large hemispheres, which are connected by the vermis in the midline (eFig. 9.61). Functionally, the cerebellum plays a role in maintaining balance and influencing posture and is responsible for coordinating movements by synchronizing contraction and relaxation of voluntary muscles. We will first examine the structural organization of the cerebellum and then review how these structures contribute to the circuitry of the cerebellum.

1	Within the posterior cranial fossa, the cerebellum is covered by the tentorium cerebelli of the dura mater (eFig. 9.17) and connects to the posterior surface of the brainstem via the superior, middle, and inferior cerebellar peduncles (eFig. 9.62). Anteriorly, the cerebellum forms the roof of the fourth ventricle (eFig. 9.14). On its surface, the cerebellum has several convoluted folds, or folia, separated by fissures. Two of these fissures serve as landmarks to divide the cerebellum into three lobes. Superiorly, the primary fissure separates the anterior lobe from the posterior lobe (eFig. 9.61). Anteriorly and inferiorly, the posterolateral fissure defines the structures of the flocculonodular lobe, which includes the flocculus from each hemisphere and nodule of the vermis (eFig. 9.63). A third fissure, the horizontal fissure, borders the superior and inferior surfaces of the cerebellum (eFig. 9.64).

1	Each fold or folia of the cerebellar cortex has a central core of white matter covered by a thin layer of gray matter superficially. In sections parallel to the median plane, the branching pattern of the folia can be appreciated; this is often referred to as the arbor vitae (eFig. 9.28). Deep within the white matter of each hemisphere are four masses of cerebellar nuclei. From lateral to medial they are the dentate, emboliform, globose, and fastigial (eFig. 9.65). Note that the emboliform and globose are collectively referred to as the interposed nuclei. Output from the cerebellum originates from one of these four nuclear complexes before leaving through the superior cerebellar peduncle, predominantly. In general, the output from each cerebellar hemisphere coordinates movement on the ipsilateral side of the body.

1	Functionally, the cerebellar cortex can be divided into three areas. The vermis in the midline influences movements along the axis of the body, including the neck, trunk, abdomen, and pelvis (eFig. 9.66). Adjacent to the vermis, the intermediate zone controls muscles of the distal upper and lower limbs. The lateral zone participates in motor planning to coordinate sequential movements of the entire body. Input to these functional areas of cerebellar cortex come from the cerebral cortex, spinal cord, and brainstem by passing predominantly through the middle and inferior cerebellar peduncles. Fibers entering the cerebellum proceed as mossy fibers (from various regions) or climbing fibers (from olivary nucleus). Mossy fibers form excitatory synapses with dendrites of the granule cells, in the granule cell layer (eFig. 9.67). From here, the granule cells send axons to the molecular layer, where they bifurcate into parallel fibers that run longitudinally in the folia. The parallel fibers

1	granule cell layer (eFig. 9.67). From here, the granule cells send axons to the molecular layer, where they bifurcate into parallel fibers that run longitudinally in the folia. The parallel fibers then synapse on Purkinje cells in the outermost Purkinje cell layer (eFig. 9.67). Climbing fibers project to the Purkinje cell layer and form powerful excitatory connections with the Purkinje cells. The Purkinje cells then project to the deep cerebellar nuclei (eFig. 9.67).

1	As mentioned earlier, the cerebellum receives input from the cerebral cortex, brainstem, and spinal cord. Input from the cerebral cortex to the cerebellum is primarily involved in voluntary muscle control and coordination of movement. Axonal projections from the cerebral cortex destined for the cerebellum descend through the internal capsule and terminate on pontine nuclei (eFig. 9.68). The axons from the pontine nuclei then cross over as transverse fibers to enter the contralateral cerebellum through the middle cerebellar peduncle.

1	Input from the spinal cord to the cerebellum conveys information from muscles and joints to influence muscle tone and posture. The primary spinal cord pathways with connections to the cerebellum include the anterior or ventral spinocerebellar and the posterior or dorsal spinocerebellar tracts. These tracts originate from joint and cutaneous mechanoreceptors and ascend through the spinal cord to enter the ipsilateral cerebellum primarily through the inferior cerebellar peduncle (eFig. 9.69). A final source of cerebellar input arises from the vestibular nuclei and reticular formation in the brainstem. The connections are primarily involved in reflexive maintenance of balance. These nuclei send axonal projections to the ipsilateral cerebellum through the inferior cerebellar peduncle.

1	Output from the cerebellum originates from one of the four deep cerebellar nuclei. The largest collection of fibers leaving the cerebellum originates from the dentate nucleus. Axons from this nuclear complex project to the contralateral ventral nucleus of the thalamus after decussating in the superior cerebellar peduncle. From here, axons of the thalamic nuclei project to the motor cortex (eFig. 9.70). This pathway influences posture and movement.

1	The emboliform and globose nuclei, or interposed nuclei, have a similar course as the axons from the dentate, but with the addition of another synaptic target. Axons from the interposed nuclei decussate in the superior cerebellar peduncle to synapse on the contralateral ventral nucleus of the thalamus and the contralateral red nucleus in the midbrain (eFig. 9.70). Axons leaving the red nucleus descend to the inferior olivary nucleus in the medulla. The axonal projections from the interposed nuclei function in monitoring and correcting motor activity of the upper and lower extremities.

1	Axons from the fastigial nucleus project to the vestibular nuclei, reticular formation, contralateral ventral nucleus of the thalamus, and contralateral tectum. Vestibular axons pass through the inferior cerebellar peduncle to reach the ipsilateral vestibular nucleus and uncinate fasciculus to reach the contralateral vestibular nucleus (eFig. 9.70). Also going through the inferior cerebellar peduncle are axons heading to the reticular formation. Ascending in the superior cerebellar peduncles are axons that will synapse with the contralateral tectum and contralateral ventral nucleus of the thalamus.

1	The cerebellum is perfused by the vertebrobasilar system of arteries (eFig. 9.42). Before merging and forming the basilar artery, each vertebral artery gives rise to a PICA and posterior spinal artery and contributes to the formation of the anterior spinal artery (eFig. 9.71). The PICA perfuses the inferior portion of the cerebellum. At the level of the caudal pons, the AICA branches off the basilar artery and perfuses the anterior and lateral portion of the cerebellum, as well as the middle and inferior cerebellar peduncles (eFig. 9.71). Just before the midbrain, the superior cerebellar arteries branch off of the basilar artery and supply the superior cerebellar peduncles and superior portion of the cerebellar hemispheres (eFig. 9.71). Part VIII: Visual System

1	Part VIII: Visual System The visual system is a complex special sensory system that begins in the eyeball and has neuronal connections to the thalamus, brainstem, primary visual cortex, and association cortices. In addition to mediating visual perception, these connections are also involved in higher visual functions, such as determining spatial relationships between objects and structural features of objects. In this section we will explore the primary or geniculate visual pathway from the retina to the primary visual cortex.

1	Although the retinal layer of the eyeball is often considered the beginning of visual perception, the anterior structures of the eye play an important role in visual perception too. The anteriormost structure is the cornea and is the first layer through which light, the visual stimulus, enters the eye (eFig. 9.72). This transparent layer overlies the aqueous humor of the anterior chamber and the pupil, a small central aperture that controls how much light is admitted into the eye. After passing through the pupil light is refracted through the lens, which along with the ciliary body, separates the anterior portion of the eye from the posterior portion (eFig. 9.72). The lens is a clear biconvex structure that rounds in shape as the ciliary muscle contracts and relaxes the suspensory ligaments attached to the borders of the lens, a parasympathetically controlled process referred to as accommodation.

1	After the lens, light passes through the vitreous humor of the posterior chamber and is projected onto the layers of the retina (eFig. 9.73). The retina is composed of a non-neural layer and several layers of neural cells with synaptic connections. To reduce the amount of light reflected in the eye, the choroid layer lining the inner surface of the sclera (eFig. 9.72), along with the pigment epithelium layer (non-neural) of the retina, absorb and refract some of the light stimulus (eFig. 9.73). Interdigitating between the pigment epithelial cells, the photoreceptors transduce the light stimulus into an electrical signal in a process called phototransduction. Images formed on the retina are inverted in both vertical and lateral dimensions (eFig. 9.74). Because of this, the visual field is defined as having four quadrants: left/right and upper/lower.

1	Photoreceptors include rods, which are very sensitive to light and essential for vision in dimly lit conditions, and cones, which are responsible for color vision and high visual acuity. Although rods and cones are both distributed across the retina, rods outnumber cones by twentyfold and are concentrated in the periphery of the retina. Cones predominate near the macula and are the only photoreceptors present at the fovea (eFig. 9.72). The fovea represents the primary visual axis of the eye and is the location of maximal visual acuity. Despite their functional differences, both the rods and cones have an outer light-sensing segment, an inner segment, and a synaptic terminal. These synaptic terminals contact bipolar cells, the first-order neurons in the visual pathway (eFig. 9.73). Bipolar cells then synapse with ganglion cells, the second-order neurons in the visual pathway. Two other cell types present in the retina are horizontal and amacrine cells. These transversely oriented

1	cells then synapse with ganglion cells, the second-order neurons in the visual pathway. Two other cell types present in the retina are horizontal and amacrine cells. These transversely oriented interneurons moderate the excitation level of bipolar and ganglion cells.

1	Axons from the ganglion cells coalesce at the optic disc, which is absent of photoreceptors and thus creates a blind spot in the visual field. As they leave the optic disc they form the optic nerve, acquire a myelin sheath provided by oligodendrocytes, and are invested by the cranial meninges (eFig. 9.72). These morphological features derived during embryonic development, define the optic nerve as a component of the CNS. Anterior to the infundibular stalk, the optic nerves converge at the optic chiasm. Within the chiasm, axons from the nasal portion of the retina decussate and enter into the contralateral optic tract (eFig. 9.75). Conversely, axons from the temporal portion of the retina stay ipsilateral to enter the ipsilateral optic tract (eFig. 9.75).

1	Continuing posteriorly, the optic tracts course around the midbrain to enter the lateral geniculate nucleus of the thalamus (eFig. 9.75). At this level, a small portion of the fibers from the optic tract travel to the pretectal area and superior colliculus to mediate the pupillary light reflex. Axons leaving the lateral geniculate nucleus form the optic radiations, which continue on to the primary visual cortex in the occipital lobe (eFig. 9.76). Fibers traveling in the lower portion of the optic radiations terminate on the lower half of the primary visual cortex, whereas fibers in the upper portion terminate on the upper half of the cortex (eFig. 9.76). A full review of how the visual field is represented throughout the visual pathway can be reviewed in eFig. 9.77.

1	As mentioned previously, images formed on the retina are inverted in both vertical and lateral dimensions. In addition, the fibers from the nasal portion of the hemiretina decussate at the optic chiasm. Because of this, the optic tracts, thalamus, optic radiations, and primary visual cortex receive information relating only to the contralateral half of the visual field. Knowledge of how the visual field is represented throughout the visual pathway is essential for identifying the location of lesions in patients with visual field deficits. Examples of lesions in the five major structures of the visual pathway (optic nerve, optic chiasm, optic tract, optic radiations, primary visual cortex) and their associated visual field deficits are represented in eFig. 9.78. Part IX: Auditory and

1	Part IX: Auditory and Cranial nerve VIII, the vestibulocochlear nerve, conveys sensory information from the vestibular and auditory organs of the inner ear to the pontomedullary junction of the brainstem. Although these sensory modalities are conveyed to the brainstem by a common nerve bundle, each of these sensory functions has different central pathways. In this section we will first review the central auditory pathways and then the vestibular pathways.

1	Sound waves, the auditory stimulus, are directed toward the external acoustic meatus by the pinna of the outer ear and through the ear canal to the tympanic membrane (eFig. 9.79). Vibration of the tympanic membrane, induced by the incoming sound waves, is transmitted to the three ossicle bones (malleus, incus, and stapes) of the middle ear. These bones amplify sound waves so they can be converted to pressure waves at the oval window of the fluid-filled cochlea. To prevent damage from loud or high-decibel sounds, movements of the malleus and stapes are reduced by the tensor tympani and stapedius muscles.

1	In the cochlea, sound waves are converted into an electrical signal at the organ of Corti. The organ of Corti rests upon the basilar membrane in the scala media, which is filled with endolymph (eFig. 9.80). In addition to contributing to the formation of the scala media with the vestibular membrane, the basilar membrane separates the cochlea into the scala vestibuli and scala tympani, which are filled with perilymph and are continuous with one another at the helicotrema (eFig. 9.80).

1	As the sound waves move through the perilymph they displace the basilar membrane, which causes deflection of the hair cells in the organ of Corti (eFig. 9.81A). These sensory receptors synapse with sensory neurons, which have cell bodies in spiral ganglion located within the modiolus of the cochlea (eFig. 9.80). Axons leaving the modiolus form the cochlear nerve near the base of the cochlea. The cochlear nerve then passes through the internal acoustic meatus and subarachnoid space to enter the pontomedullary junction at the cerebellopontine angle.

1	The first-order axons entering the brainstem from the cochlea terminate ipsilaterally on the dorsal and ventral cochlear nuclei. From here, second-order axons have several synaptic targets. Second-order axons forming the ascending auditory pathway ascend to the pons and project bilaterally on the superior olivary nucleus (eFig. 9.82). These bilateral projections are important for auditory acuity and localizing the origin of a sound. From here, fibers continue to ascend as the lateral lemniscus and terminate in the inferior colliculus of the midbrain. Axons leaving the inferior colliculus then have a synaptic relay in the medial geniculate nucleus of the thalamus before reaching the final synaptic target: the primary auditory cortex (superior temporal gyrus) of the temporal lobe (eFig. 9.82). Throughout this pathway, from the basilar membrane to the primary auditory cortex, auditory stimuli are tonotopically represented. This is analogous to the somatotopic map previously discussed in

1	Throughout this pathway, from the basilar membrane to the primary auditory cortex, auditory stimuli are tonotopically represented. This is analogous to the somatotopic map previously discussed in the section on somatosensory systems pathways.

1	In addition to the ascending auditory pathway, the superior olivary nucleus receives descending input from the primary auditory cortex as a form of feedback (eFig. 9.82). The superior olivary nucleus then sends descending olivocochlear fibers to the organ of Corti, which has an inhibitory function on the hair cells to prevent damage from harmfully loud sounds. It is also believed that the superior olivary nucleus has connections with the motor nuclei of the trigeminal and facial nerve to mediate reflexive contraction of the tensor tympani and stapedius muscles in response to loud sounds.

1	The vestibular nerve transmits afferent or sensory information regarding movement and position of the head from the vestibular organs, which include semicircular ducts, utricle, and saccule. Like the cochlear apparatus, each of these vestibular organs is located within a membranous (ductal) portion of the vestibular apparatus, which is surrounded by a bony (canal) portion (eFig. 9.83). The cell bodies for these sensory receptors are located in the vestibular (Scarpa’s) ganglion within the internal acoustic meatus (eFig. 9.83). The vestibular nerve then passes through the internal acoustic meatus and subarachnoid space to enter the pontomedullary junction at the cerebellopontine angle.

1	The central processes of the vestibular axons predominantly terminate in the four vestibular nuclei (superior, inferior, medial, and lateral), which are located in the rostral medulla and caudal pons (eFig. 9.40). Axons leaving the vestibular nuclei have several ascending and descending synaptic targets. These pathways connect with visual motor, descending motor, and cerebellar pathways to coordinate movement and maintain posture and balance (eFig. 9.84).

1	Axons ascending after leaving the vestibular nuclei form the medial longitudinal fasciculus to reach the oculomotor, trochlear, and abducent nuclei (eFig. 9.85). These connections coordinate movement of the head and eyes so that visual fixation on an object can be maintained. Other axons ascending from the vestibular nuclei project to the cerebral cortex after a synaptic relay in the ventral posterior nucleus of the thalamus (eFig. 9.84B). This connection assists in consciously orienting oneself in space. In addition to these connections, axons leaving the vestibular nuclei pass through the inferior cerebellar peduncle to reach the cerebellum and modulate equilibrium (eFig. 9.84B).

1	Axons descending after leaving the vestibular nuclei form the medial and lateral vestibulospinal tracts ipsilaterally (eFig. 9.84B). As mentioned in the previous section on medial motor systems in the spinal cord, the axons in these pathways synapse on interneurons within the anterior horn gray matter to influence spinal motor neuron activity and maintain posture and balance. Part X: Hypothalamus The hypothalamus is a neuroendocrine organ that regulates physiological processes for survival such as consumption of fluid and food, temperature control, the sleep–wake cycle, growth, and reproduction. As a result of this vast array of functions, the nuclei of the hypothalamus make connections with several other neural and endocrine structures in the body (eFig. 9.86). In this section we will explore the nuclei of the hypothalamus and their main connections.

1	Located in the ventralmost aspect of the diencephalon, the hypothalamus is bounded by the lamina terminalis anteriorly, the hypothalamic sulcus superiorly, and the tegmentum of the midbrain posteriorly (eFig. 9.87). Laterally the hypothalamus is bordered by the substantia innominata rostrally and the posterior limb of the internal capsule caudally. It forms the floor in addition to the lower portion of the lateral walls in the third ventricle. When viewing the external surface of the ventral brain, the area containing the hypothalamus is circumscribed by the optic chiasm, optic tract, crus cerebri, and caudal edge of the mammillary bodies (eFig. 9.88).

1	Continuous with the hypothalamus inferiorly is the pituitary gland. These two structures are connected by the infundibulum and hypophysial stalk just caudal to the optic chiasm (eFig. 9.87). The intimate relationship of the hypothalamus to the portal circulation of the pituitary gland allows the hypothalamus to be an efficient regulator of hormone synthesis and release. Releasing and inhibiting factors synthesized by the hypothalamus pass through these portal vessels via the tuberoinfundibular tract (eFig. 9.89) to reach the anterior pituitary (adenohypophysis) and control release of hormones produced by the anterior pituitary such as adrenocorticotropic hormone, luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone, growth hormone, and prolactin (eFig. 9.90).

1	A second connection exists between the hypothalamus and pituitary through nerve fibers, which originate in the supraoptic region and paraventricular nuclei of the medial hypothalamus and extend through the hypothalamohypophyseal tract to the posterior lobe of the pituitary for release into the circulatory system (eFig. 9.89). Internally, the hypothalamus is composed of many small nuclei, which are divided by a parasagittal plane into a medial and lateral zone. Landmarks for this dividing point are the columns of the fornix and the mammillothalamic tract as they reside within this sagittal plane (eFig. 9.91). The hypothalamus is also divided by coronal planes into a preoptic area and periventricular zone. Note that the periventricular zone is not to be confused with the paraventricular nucleus, which is a thin region of cell bodies lying medial to the medial zone.

1	Within the lateral zone of the hypothalamus is a large bundle of axons forming the medial forebrain bundle (MFB) (eFig. 9.90). Theses fibers interconnect the hypothalamus with the septal nuclei rostrally and nuclear complexes within the brainstem. Axons arising from the large lateral nucleus in this zone enter the MFB and function in promoting feeding behavior. The other and smaller nuclear group in this zone is the tuberal group. Axons from the tuberal nuclei make connections with the anterior pituitary through the tuberoinfundibular tract to regulate release of hormones in the hypophysial portal system and to the cerebellum to regulate motor activity (eFig. 9.89).

1	The medial zone is divided into three regions: supraoptic, tuberal, and mammillary. There are four nuclei in the supraoptic region, which play a role in thermoregulation, osmoregulation, and the sleep–wake cycle. The supraoptic and paraventricular nuclei in this region synthesize antidiuretic hormone or vasopressin, which stimulates water uptake and oxytocin for stimulation of uterine contractions and lactation in the mammary glands. Axons from these nuclei are conveyed by the hypothalamohypophyseal tract to the posterior pituitary for release into the circulatory system (eFig. 9.89). A third nuclear group in this region is the suprachiasmatic nucleus. This nucleus receives input directly from the retina to influence circadian rhythms, which contribute to the light–dark cycle. The anterior nucleus is the final group and functions predominantly in regulating body temperature.

1	The tuberal region in the medial zone contains three nuclei: ventromedial, dorsomedial, and arcuate. The largest and best defined is the ventromedial nucleus, which functions as a satiety center to decrease feeding behavior. Posterior to the ventromedial nucleus is the dorsomedial nucleus, which functions in the behavioral expression of rage or aggressive behavior. Finally, the arcuate nucleus serves as a center for releasing hormones, which are transmitted by the tuberoinfundibular tract and hypophysial portal system to the anterior pituitary (eFig. 9.89).

1	The mammillary region or mammillary body is the final group of nuclei in the medial zone. Four nuclei comprise this region: medial, intermediate, lateral mammillary, and posterior hypothalamic. The best defined is the medial mammillary nucleus, as it is the primary site for the termination of axons from the postcommissural fornix. This pathway originates from the subiculum of the hippocampal complex and plays a key role in memory. The medial mammillary nuclei also connects to structures of the limbic system. The periventricular zone resides medial to the medial zone and adjacent to the ependymal cells of the third ventricle. Neurons from this zone predominantly synthesize releasing hormones. Axons from these neuronal cells project through the tuberoinfundibular tract to the hypophysial portal system to influence release of hormones from the anterior pituitary.

1	Through the review of the hypothalamus thus far, it is apparent that this small, 4-gram structure has a significant role in regulating visceral, endocrine, and behavioral system functions through multiple pathways. It is important to remember the majority of the pathways mentioned represent input–output relationships between the hypothalamus and other structures. For a review of neural and non-neural inputs and outputs, refer to the summary figures (eFig. 9.92A and B). Part XI: Olfactory and

1	Part XI: Olfactory and The sense of olfaction has a role in both pleasurable experiences and survival. The same receptors that allow us to enjoy the food we consume or experience odorants in the environment also help us avoid spoiled food or potentially hazardous situations like a fire. Unlike the other special sensory system pathways, the olfactory sensory pathway is unique in that it does not have a thalamic relay before reaching the primary olfactory cortex. In this section we will review the course of the neurons in the olfactory system and their connection to the limbic system.

1	Three types of olfactory receptors make up the olfactory epithelium along the lateral and septal walls of the nasal cavity. These cells allow for regeneration (basal stem cells), support (sustentacular cells), and transmission of information (olfactory receptor neurons). Each olfactory receptor neuron has an olfactory vesicle with cilia that contain receptors for odorant molecules and an unmyelinated axon that passes through the cribriform plate to terminate in the olfactory bulb (eFig. 9.93). As the olfactory receptor neurons originate embryologically from the CNS, they are considered part of the CNS and not the PNS.

1	After synapsing with the mitral cells in the glomeruli of the olfactory bulb, mitral cell axons converge to form the olfactory tract. The olfactory tract then divides into medial and lateral olfactory striae to reach different synaptic targets (eFig. 9.94). Some of the axons in the medial olfactory striae travel through the diagonal band to reach the septal area, whereas others cross the midline in the anterior commissure and inhibit mitral cell activity in the contralateral olfactory bulb to enhance localization of the olfactory stimulant. Axons in the lateral olfactory stria primarily terminate in the piriform cortex/primary olfactory cortex of the uncus and in the amygdala (eFig. 9.94). The medial forebrain bundle, traveling through the lateral hypothalamus, connects the olfactory cortex with both the hypothalamus and the brainstem to regulate autonomic responses such as arousal through the reticular formation, salivation, and gastric contraction.

1	The limbic system is composed of several cortical and subcortical structures that participate in an intricate network of connections to regulate complicated behaviors such as memory, emotions, homeostatic functions, and motivational state. In this section we will review the major structures and pathways that form the limbic system. Grossly, the limbic lobe includes a ring-shaped area of cortical structures that border the brainstem. These cortical areas include the cingulate gyrus, parahippocampal gyrus, and subcallosal area (eFig. 9.12). Laterally, the insular cortex also participates in limbic system function (eFig. 9.10). Nuclear structures of the limbic system include the amygdala, hippocampal formation, anterior and mediodorsal thalamic nuclei, septal nuclei in the forebrain, and nucleus accumbens (eFig. 9.95).

1	The amygdaloid nucleus is an almond-shaped structure located anterior to the inferior horn of the lateral ventricle and tail of the caudate within the temporal lobe (eFig. 9.96). Structurally, the amygdala consists of three nuclear regions: a large basolateral group and a smaller corticomedial group, which includes the central nucleus. Functionally, the amygdala is primarily associated with the emotion of fear, but it also has an important role in autonomic and neuroendocrine pathways. Connections of the amygdala are predominantly bidirectional and follow three different pathways: the uncinate fasciculus, stria terminalis, and ventral amygdalofugal pathway (eFig. 9.97). Connections to cortical areas pass through the uncinate fasciculus, which progresses anterior to the amygdala. Projections to the septal area and hypothalamus follow the stria terminalis (eFig. 9.98A–D). Fibers forming the ventral amygdalofugal pathway project to thalamic nuclei and several brainstem and forebrain

1	to the septal area and hypothalamus follow the stria terminalis (eFig. 9.98A–D). Fibers forming the ventral amygdalofugal pathway project to thalamic nuclei and several brainstem and forebrain structures (eFig. 9.98A–D).

1	The nucleus accumbens resides with the ventral forebrain adjacent to where the putamen and head of the caudate become continuous with one another (eFig. 9.99). Afferent axons to the nucleus accumbens come from the amygdala through the amygdalofugal pathway, hippocampal formation by way of the fornix, basal forebrain area from the stria terminalis, and ventral tegmentum through the medial forebrain bundle (eFig. 9.98A–D). Efferent axons leaving the nucleus accumbens project directly to the hypothalamus and globus pallidus and reach nuclei in the brainstem through the medial forebrain bundle. Its connections to the globus pallidus represent an important connection of the limbic system to the motor system. The overall function of the nucleus accumbens is recognized as a gratification center and has been shown to play a role in behaviors related to addiction.

1	The septal region is located rostral to the anterior commissure along the medial aspect of the cerebral hemispheres (eFig. 9.99). This region appears to play a role in pleasurable behaviors. Conversely, lesion studies indicate that damage to this area evokes behaviors of extreme displeasure or rage. Afferent axons to the septal area arise from the amygdala, hippocampus, olfactory tract, and monoaminergic nuclei in the brainstem (eFigs. 9.100 and 9.101). The septal area also connects to a collection of cholinergic neurons along the wall and roof of the third ventricle known as the habenular nuclei. Axons from the habenular nuclei project to the interpeduncular nucleus of the reticular formation, which is believed to play a role in the sleep–wake cycle (eFig. 9.100).

1	The hippocampal formation is located in the medial ventral temporal lobe (eFig. 9.102). It consists of the hippocampus, dentate gyrus, and subiculum (eFig. 9.103A and B). The hippocampal formation plays a role in memory processes such as episodic memory, short-term memory, working memory, and consolidation of memories. Input to the hippocampal formation is primarily received by the entorhinal cortex from association cortices. Because of this, it is believed that the “storage” of memories is in the association and primary cortices, not in the medial temporal lobe. Neurons from the entorhinal cortex project to the hippocampal formation by two pathways: the perforant pathway and alvear pathway. The perforant courses directly through the hippocampal sulcus to reach the dentate gyrus (eFig. 9.104B). As the hippocampus resembles the appearance of a ram’s horn, early on it was named cornu ammonis (horn of Ammon). Based on its cytoarchitecture, it was subdivided into four regions termed the

1	As the hippocampus resembles the appearance of a ram’s horn, early on it was named cornu ammonis (horn of Ammon). Based on its cytoarchitecture, it was subdivided into four regions termed the cornu ammonis 1 to 4 (eFig. 9.104A). From the dentate gyrus, axons project to CA3 of the hippocampus. Axons from the hippocampus leave via the fornix or as Shaffer collaterals to reach CA1. Axons from CA1 may enter the fornix or project to the subiculum. Finally, axons from the subiculum enter the fornix or go back to the entorhinal cortex.

1	A second afferent pathway from the entorhinal cortex to the hippocampal formation is through the alvear pathway. Axons in the alvear pathway project directly on to CA1 and CA3 of the hippocampus (eFig. 9.104B). Similar to the perforant pathway, axons leaving the alvear pathway primarily originate from CA1 and CA3, which then project to the subiculum.

1	Efferent axons leaving the hippocampal formation primarily exit from the subiculum and form the fornix (Latin for “arch”), a white matter structure that arches over the ventricular system (eFig. 9.95). The fornix begins with axons exiting the hippocampus to form the alveus along the ventricular surface of the hippocampus. As the axons come together medially, they form a bundle referred to as the fimbria of the fornix. The fornix then emerges from the hippocampal formation and curves under the corpus callosum before coursing medially to run adjacent to the midline (eFig. 9.105). At the anterior commissure, the fornix divides into a precommissural fornix and postcommissural fornix to reach the nucleus accumbens, septal nuclei, medial frontal cortex, mammillary nucleus, ventromedial nucleus of the hypothalamus, and anterior nucleus of the dorsal thalamus (eFig. 9.95).

1	Through this section we have described a collection of anatomical structures and defined their connections with other areas of the brain and brainstem without exploring how these individual structures are interconnected with one another. In the 1930s James Papez, an American neurologist, described a circuit that links these structures and cortical areas together in a way that was thought to be involved in the experience and expression of emotion. This is referred to as the Papez circuit (eFig. 9.106). The circuit begins with fibers from the subiculum, which then enter the fornix to reach the mammillary nuclei. These axons then project through the mammillothalamic tract to the anterior nucleus of the thalamus. Next, the axons from the anterior nucleus of the thalamus project through the internal capsule to the cingulate gyrus. Lastly, the cingulum fibers from the cingulate cortex project to the parahippocampal gyrus and then the entorhinal cortex and hippocampal formation (eFig.

1	the internal capsule to the cingulate gyrus. Lastly, the cingulum fibers from the cingulate cortex project to the parahippocampal gyrus and then the entorhinal cortex and hippocampal formation (eFig. 9.106). Papez’s description of this circuit is useful for reviewing the major limbic system pathways; however, the role of some of the structures in the pathway has been shown to play little or no role in the expression of emotion. In addition, many of the structures that do play a role in the expression of emotion also have a role in other functions.

1	In the olowingpages I have attempted to set orth, as brily as seemed to be consistent with thoroughness, the scientic basis or and the practical application of the obstetrical art. At the same time, I have endeavored to present the more practical aspects of obstetrics in such a manner as to be of direct service to the obstetrician at the bedside. -J. Whitridge Williams (1903) So reads the introduction to Williams' irst edition of this textbook, Obstetrics-A Text-Book or the Use of Students and Practitioners. In this 25th edition, we strive to follow the tenets described by Williams. And, each chapter begins with a quote from his original textbook.

1	he science and clinical practice of obstetrics is concerned with human reproduction. hrough quality perinatal care, the specialty promotes the health and well-being of the pregnant woman and her fetus. Such care entails appropriate recognition and treatment of complications, supervision of labor and delivery, initial care of the newborn, and management of the puerperium. Postpartum care promotes health and provides family planning options. he importance of obstetrics is relected by the use of maternal and neonatal outcomes as an index of the quality of health and life among nations. Intuitively, indices that reflect poor obstetrical and perinatal outcomes would lead to the assumption that medical care for the entire population is lacking. With those thoughts, we now provide a synopsis of the current state of maternal and newborn health in the United States as it relates to obstetrics.

1	The National Vital Statistics System of the United States is the oldest and most successful example of intergovernmental data sharing in public health. This agency collects statistics through vital registration systems that operate in various jurisdictions. These systems are legally responsible for registration of births, fetal deaths, deaths, marriages, and divorces. Legal authority resides individually with the 50 states; two regions-the District of Columbia and New York City; and ive territoriesAmerican Samoa, Guam, the Northern Mariana Islands, Puerto Rico, and the Virgin Islands. The standard birth certificate was revised in 1989 to include more information on medical and lifestyle risk factors and obstetrical practices. In 2003, an extensively revised Standard Certificate of Live Birth was implemented in the United States. The enhanced data categories and speciic examples of each are summarized in Table

1	By 2013,i35 states had implemented the revised birth certificate representing 76 percent of all births (MacDorman, 2015). Importantly, the 2003 version of the population death certificate contains a pregnancy checkbox to eventually be implemented by all states a oseph, 2017). The uniform use of standard deinitions is encouraged by the World Health Organization as well as the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017). Such uniformity allows data comparison not only between states or regions of the country but also between countries. Still, not all deinitions are uniformly Overview of Obstetrics TABLE 1-1. General Categories of New Information Added to the 2003 Revision of the Birth Certificate Risk factors in pregnancy-Examples: prior preterm birth, prior eclampsia Obstetrical procedures-Examples: tocolysis, cerclage, external cephalic version

1	Labor-Examples: noncephalic presentation, glucocorticoids for fetal lung maturation, antibiotics during labor Delivery-Examples: unsuccessful operative vaginal delivery, trial of labor with prior cesarean delivery Newborn-Examples: assisted ventilation, surfactant therapy, congenital anomalies applied. For example, the American College of Obstetricians and Gynecologists recommends that reporting include all fetuses and neonates born weighing at minimum 500 g, whether alive or dead. But, not all states follow this recommendation. Speciically, 28 states stipulate that fetal deaths beginning at 20 weeks' gestation should be recorded as such; eight states report all products of conception as fetal deaths; and still others use a minimum birthweight of 350 g, 400 g, or 500 g to deine fetal death. To further the confusion, the National Vital Statistics Reports tabulates fetal deaths from gestations that are 20 weeks or older (Centers for Disease Control and Prevention, 2016). his is

1	death. To further the confusion, the National Vital Statistics Reports tabulates fetal deaths from gestations that are 20 weeks or older (Centers for Disease Control and Prevention, 2016). his is problematic because the 50th percentile for fetal weight at 20 weeks approximates 325 to 350 g-considerably less than the 500-g deinition. Indeed, a birthweight of 500 g corresponds closely with the 50th percentile for 22 weeks' gestation.

1	Deinitions recommended by the National Center for Health Statistics and the Centers for Disease Control and Prevention are as follows: Perinatal period. The interval between the birth of a neonate born after 20 weeks' gestation and the 28 completed days after that birth. When perinatal rates are based on birthweight, rather than gestational age, it is recommended that the perinatal period be deined as commencing at the birth of a 500-g neonate. Birth. he complete expulsion or extraction from the mother of a fetus after 20 weeks' gestation. As described above, in the absence of accurate dating criteria, fetuses weighing < 500 g are usually not considered as births but rather are termed abortuses for purposes of vital statistics. Birthweight. The weight of a neonate determined immediately after delivery or as soon thereafter as feasible. It should be expressed to the nearest gram.

1	Birthweight. The weight of a neonate determined immediately after delivery or as soon thereafter as feasible. It should be expressed to the nearest gram. Birth rate. The number of live births per 1000 population. Fertility rate. The number of live births per 1000 females aged 15 through 44 years. Live birth. he term used to record a birth whenever the newborn at or sometime after birth breathes spontaneously or shows any other sign of life such as a heartbeat or deinite spontaneous movement of voluntary muscles. Heartbeats are distinguished from transient cardiac contractions, and respirations are diferentiated from fleeting respiratory efforts or gasps. Stillbirth or fetal death. The absence of signs of life at or after birth. Early neonatal death. Death of a liveborn neonate during the irst 7 days after birth. Late neonatal death. Death after 7 days but before 29 days.

1	Early neonatal death. Death of a liveborn neonate during the irst 7 days after birth. Late neonatal death. Death after 7 days but before 29 days. Stillbirth rate or fetal death rate. The number of stillborn neonates per 1000 neonates born, including live births and stillbirths. Neonatal mortality rate. The number of neonatal deaths per 1000 live births. Perinatal mortality rate. he number of stillbirths plus neonatal deaths per 1000 total births. Infant death. All deaths of liveborn infants from birth through 12 months of age. Infant mortality rate. The number of infant deaths per 1000 live births. Low birthweight. A newborn whose weight is <2500 g. Very low birthweight. A newborn whose weight is < 1500 g. Extremely low birthweight. A newborn whose weight is < 1000 g.

1	Low birthweight. A newborn whose weight is <2500 g. Very low birthweight. A newborn whose weight is < 1500 g. Extremely low birthweight. A newborn whose weight is < 1000 g. Term neonate. A neonate born any time ater 37 completed weeks of gestation and up until 42 completed weeks of gestation (260 to 294 days). The American College of Obstetricians and Gynecologists (2016b) and Society for MaternalFetal Medicine endorse and encourage specific gestational age designations. Eary term refers to neonates born at 37 completed weeks up to 386/7 weeks. Full term denotes those born at 39 completed weeks up to 406r weeks. Last, late term describes neonates born at 41 completed weeks up to weeks. Preterm neonate. A neonate born before 37 completed weeks (the 259th day). A neonate born before 34 completed weeks is early preterm, whereas a neonate born between 34 and 36 completed weeks is late preterm.

1	Postterm neonate. A neonate born anytime after completion of the 42nd week, beginning with day 295. Abortus. A fetus or embryo removed or expelled from the uterus during the first half of gestation-20 weeks or less, or in the absence of accurate dating criteria, born weighing <500 g. Induced termination of pregnancy. The purposeful interruption of an intrauterine pregnancy that has the intention other than to produce a liveborn neonate and that does not result in a live birth. This deinition excludes retention of products of conception following fetal death. Direct maternal death. he death of the mother that results from obstetrical complications of pregnancy, labor, or the puerperium and from interventions, omissions, incorrect treatment, or a chain of events resulting from any of these factors. An example is maternal death from exsanguination after uterine rupture.

1	Indirect maternal death. A maternal death that is not directly due to an obstetrical cause. Death results from previously existing disease or a disease developing during pregnancy, labor, or the puerperium that was aggravated by maternal physiological adaptation to pregnancy. An example is maternal death from complications of mitral valve stenosis. Nonmaternal death. Death of the mother that results from accidental or incidental causes not related to pregnancy. An example is death from an automobile accident or concurrent malignancy.

1	Nonmaternal death. Death of the mother that results from accidental or incidental causes not related to pregnancy. An example is death from an automobile accident or concurrent malignancy. Maternal mortality ratio. The number of maternal deaths that result from the reproductive process per 100,000 live births. Used more commonly, but less accurately, are the terms maternal mortaliy rate or maternal death rate. The term ratio is more accurate because it includes in the numerator the number of deaths regardless of pregnancy outcome-for example, live births, stillbirths, and ectopic pregnancieswhereas the denominator includes the number of live births. Pregnancy-associated death. The death of a woman, from any cause, while pregnant or within 1 calendar year of termination of pregnancy, regardless of the duration and the site of pregnancy.

1	Pregnancy-associated death. The death of a woman, from any cause, while pregnant or within 1 calendar year of termination of pregnancy, regardless of the duration and the site of pregnancy. Pregnancy-related death. A pregnancy-associated death that results from: (1) complications of pregnancy itself, (2) the chain of events initiated by pregnancy that led to death, or (3) aggravation of an unrelated condition by the physiologicalior pharmacological efects of pregnancy and that subsequently caused death.

1	According to the Centers for Disease Control and Prevention (CDC), the fertility rate of women aged 15 to 44 years in the United States in 2015 was 62.5 live births per 1000 women (Martin, 2017). This rate began slowly trending downward in 1990 and has now dropped below that for replacement births. This indicates a population decline (Hamilton, 2012). There were 3.98 million births in 2015, and this constituted the lowest birth rate ever recorded for the United States-12.3 per 1000 population. The birth rate decreased for all major ethnic and racial groups, for adolescents and unmarried women, and for those aged 20 to 24 years. For women older than 30 years, the birth rate rose slightly. Almost half of newborns in 2010 in the United States were minorities: Hispanic-25 percent, AfricanAmerican-14 percent, and Asian-4 percent (Frey, 2011).

1	he total number of pregnancies and their outcomes in 2015 are shown in Table 1-2. According to the Guttmacher Institute (2016b), 45 percent of births in the United States are unintended at the time of conception. Importantly, the overall proportion of aExciudes spontaneous abortions and ectopic pregnancies. Data from Martin, 201l7. unintended births has declined only slightly since 2001. Unmarried women, black women, and women with less education or income are more likely to have unplanned pregnancies. In Table 1-2, induced abortion information derives from CDC abortion surveillance data from 45 states combined with Guttmacher Institute data on induced abortion. hese data have been collected beginning in 1976. Since Roe v. Wade legalization of abortion, more than 46 million American women have chosen legalized abortions. As discussed later, this provides a compelling argument for easily accessible family planning.

1	Several indices are used to assess obstetrical and perinatal outcomes as measures of medical care quality. As noted, the perinatal mortality rate includes the numbers of stillbirths and neonatal deaths per 1000 total births. In 2013, the perinatal mortality rate was 9.98 per 1000 births (Fig. 1-1) (MacDorman, 2015). There were 25,972 fetal deaths at gestational ages of 20 weeks or older. Fetal deaths at 28 weeks or more have been declining since 1990, whereas rates for those between 20 and 27 weeks are static (Fig. 1-2). By way of comparison, there were a total of 19,041 neonatal deaths in 2006-meaning that nearly 60 percent of the perinatal deaths in the United States were fetal.

1	here were 6.1 infant deaths per 1000 live births in 2013 compared with 6.8 in 2001 (MacDorman, 2015). The three leading causes of infant death-congenital malformations, low birthweight, and sudden infant death syndrome-accounted for almost half of all deaths (Heron, 2015). Infants born at the lowest gestational ages and birthweights add substantively to these mortality rates. For example, more than half of all infant deaths in 2005 were in the 2 percent of infants born before FIGURE ,A-, Perinatal mortality rates: United States, 2000-2013. (Reproduced with permission from MacDorman MF, Gregory EC: Fetal and perinatal mortality: United States, 2013. Natl Vital Stat Rep. 2015 Jul 23;64(8):1-24.) 0..C ::l� 0 Con , .�A.� 8.s

1	(Reproduced with permission from MacDorman MF, Gregory EC: Fetal and perinatal mortality: United States, 2013. Natl Vital Stat Rep. 2015 Jul 23;64(8):1-24.) 0..C ::l� 0 Con , .�A.� 8.s FIGURE 1-2 Fetal and neonatal deaths: United States, 2000-2013. (Modified with permission from MacDorman MF, Gregory EC: Fetal and perinatal mortality: United States, 2013. Natl Vital Stat Rep. 2015 Jul 23;64(8):1-24.) 32 weeks' gestation. Indeed, the percentage of infant deaths related to preterm birth increased from 34.6 percent in 2000 to 36.5 percent in 2005. When analyzed by birthweight, two thirds of infant deaths were in low-birthweight neonates. Of particular interest are infants with birthweights <500 g, for whom neonatal intensive care can now be ofered.

1	As shown in Figure 1-3, maternal mortality rates dropped precipitously in the United States during the 20th century. Pregnancy-related deaths are so uncommon as to be measured per 100,000 births. The CDC (2017a) has maintained data on pregnancy-related deaths since 1986 in its Pregnancy Mortality Surveillance System. In the latest report, Creanga and coworkers (2017) described 2009 pregnancy-related deaths during the period from 2011 to 2013. Approximately 5 percent were early-pregnancy deaths due to ectopic gestation or abortive outcomes. he deadly obstetrical triad ofhemorrhage, preeclampsia, and infection has accounted for a third of all deaths (Fig. 1-4). hromboembolism, cardiomyopathy, and other cardiovascular �CD ""..D FIGURE 1-3 Maternal mortality rates for the United States, 19502003. (Data from Berg, 2010; Hoyert, 2007.) 11.4 �Yuf 11.8 �� � .��t ,��o Cause of pregnancy-related deaths

1	FIGURE 1-3 Maternal mortality rates for the United States, 19502003. (Data from Berg, 2010; Hoyert, 2007.) 11.4 �Yuf 11.8 �� � .��t ,��o Cause of pregnancy-related deaths FIGURE 1-4 Six common causes of pregnancy-related deaths for the United States, 2006-2010. (Data from Creanga, 2015.) disease together accounted for another third. Other signii-. cant contributors were amnionic luid embolism (5.3 percent) and cerebrovascular accidents (6.2 percent). Anesthesia-related deaths were at an all-time low-only 0.7 percent. Similar causes were reported for selected cohorts for years 2008 to 2009 and 2013 to 2014 (MacDorman, 2017).

1	Shown in Figure 1-5, the pregnancy-related mortality ratio of 23.8 per 100,000 live births in 2014 is the highest during the previous 40 years. And, according to the Institute ofHealth Metrics, it was 28 per 100,000 in 2013 (Tavernise, 2016). This rise simply may be that more women are dying, however, other factors explain this doubling of the rate from 1990 to 2013 aoseph, 2017). The irst is an artiicial elevation caused by the International Statistical Classiication of Diseases, 10th Revision (ICD-I0), implemented in 1999. Second, improved ..C-) ) .=19 FIGURE 1-5 Estimated maternal mortality rates in 48 states and the District ofColumbia. (Data from MacDorman, 2016.) Overview of Obstetrics 14.6 13.6

1	FIGURE 1-5 Estimated maternal mortality rates in 48 states and the District ofColumbia. (Data from MacDorman, 2016.) Overview of Obstetrics 14.6 13.6 FIGURE 1-6 Trends in maternal mortality ratio (per 100,000 live births) by race: United States, 2005-2014. (Data from Moaddab, 2016.) reporting definitely contributes to the rise (MacDorman, 2016b, 2017). In the past, maternal deaths were notoriously underreported (Koonin, 1997). Third, and related to the second explanation, the rate of rise is at least partially due to the revised death certificate and its pregnancy checkbox described earlier (Main, 2015). Fourth, the number of pregnant women with severe chronic health conditions, which place women at higher risk, is greater (Centers for Disease Control and Prevention, 2017a). Finally, the increased proportion of births to women older than 40 years contribute to higher mortality rates (MacDorman, 2017).

1	Whatever the cause, the apparent sharp rise of the maternal mortality rates has galvanized the obstetrical community to action (Chescheir, 2015). According to Barbieri (2015), the Joint Commission has recommended that birthing centers establish standardized protocols and implement simulation eforts. D' Alton and colleagues (2016) described eforts of a working group to lower morbidity and mortality rates. Another consideration is the obvious disparity of higher mortality rates among black, Hispanic, and white women as shown in Figure 1-6. Racial disparities translate to health care availability, access, or utilization (Howell, 2016; Moaddab, 2016). And, maternal mortality is disparately high in rural compared with metropolitan areas (Maron, 2017).

1	Importantly, many of the reported maternal deaths are considered preventable. Berg and colleagues (2005) estimated that this may be up to a third of pregnancy-related deaths in white women and up to half of those in black women. In one evaluation of an insured cohort, 28 percent of 98 maternal deaths were judged preventable (Clark, 2008). hus, although significant progress has been made, further eforts are imperative for obstetrics in the 21 st century.

1	his serves as another measure to guide prevention eforts. Lowering medical error rates serves to diminish risks for maternal mortality or severe maternal morbidity. The terms near misses or close calls were introduced and defined as unplanned events caused by error that do not result in patient injury but have the potential to do so (Institute for Safe Medication Practices, 2009). hese are much more common than injury events, but for obvious reasons, they are more diicult to identiy and quantiy. Systems designed to encourage reporting have been installed in various institutions and allow focused safety eforts (Clark, 20i12; Main, 2017; Shields, 2017). The American College of Obstetricians and Gynecologists and the Society for Maternal-Fetal Medicine (20 16f) have provided lists of suggested screening topics for this purpose.

1	Several data systems now measure indicators of unplanned events caused by errors that have injurious potential. This evolution followed inadequacies in the ability of hospitalization coding to reflect the severity of maternal complications. Thus, coding indicators or modifiers are used to allow analysis of serious adverse clinical events (Clark, 2012; King, 2012). Such a system was implemented by the World Health Organization. It has been validated in Brazil and accurately reflects maternal death rates (Souza, 2012). Similar systems are in use in Britain as the UK Obstetric Surveilance System-UKOSS (Knight, 2005,i2008). In the United States, one example is the National Partnership for Maternal Safety (D'Alton, 2016; Main, 2015).

1	To study severe morbidity, the CDC analyzed more than 50 million maternity records from the Nationwide Inpatient Sample from 1998 to 2009 (Callaghan, 2012). They used ICD9-CM codes and reported that 129 per 10,000 of these gravidas had at least one indicator for severe morbidity (Table 1--).hus, for every maternal death, approximately 200 women experience severe morbidity. he CDC (20 17b) estimates that 65,000 women per year have such maternal morbidity. These numbers are greatest in smaller hospitals with < 1000 deliveries annually (Hehir, 2017). Finally, as with mortality rates, there are serious racial and ethnic disparities for severe maternal morbidity, and black women are disproportionately afected (Creanga, 2014). Various topics have been in the forefront for obstetrical providers in the 4 years since the last edition of this textbook. In the following, we discuss several of these topics. • U.S. Health Care in Crisis

1	• U.S. Health Care in Crisis In a 2016 issue of the Jounal of the American Medical Association fAA), then-President Barack Obama presented a summary of the Afordable Care Act (ACA), so-called Obamacare. He described the successes, the challenges ahead, and the policy implications of the policy (Bauchner, 2016). He summarized three lessons from his experiences with the ACA. First, change is especially diicult in the face of hyperpartisanship. Second, special interests pose a continued obstacle to change. Third, he stressed the importance of pragmatism. Here, he was referring to the pragmatism necessary when the ACA did not work efectively on day 1 of implementation. TABLE 1-3. Severe Maternal Morbidity Indicators Injuries of thorax, abdomen, and pelvis Intracranial injuries Conversion of cardiac rhythm Hysterectomy Summarized from the Centers for Disease Control and Prevention,l2017b.

1	Injuries of thorax, abdomen, and pelvis Intracranial injuries Conversion of cardiac rhythm Hysterectomy Summarized from the Centers for Disease Control and Prevention,l2017b. At this same time, draconian cuts to Medicaid were being proposed, and President Obama ended his JM1A report with a quotation from John Kasich, the Republican governor of Ohio. "For those that live in the shadows of life, those who are the least among us, I will not accept the fact that the most vulnerable in our state should be ignored. We can help them." hese potential efects to Medicaid ripple into the specialty of obstetrics. In 2010, it was estimated that Medicaid insured 48 percent of the births in the United States (lvlarkus, 2013). Importantly, Medicaid covered a disproportionate number of complicated births. Speciically, Medicaid insured more than half of all hospital stays for preterm and low-birthweight infants and approximately 45 percent of infant hospital stays due to birth defects.

1	he young, healthy Americans who were expected to financially bolster the ACA ultimately enrolled in insuicient numbers to ensure long-term ACA sustainability. hus, long-term options included repair or repeal of the ACA. hroughout Donald Trump's campaign for the presidency of the United States, he made repeal of the ACA a focus of his candidacy. As of this writing, both the United States House of Representatives and the Senate have grappled with "repeal and replace" for 6 months. According to the Congressional Budget Oice, this action would result in 23 million Americans losing health care insurance and cuts in Medicaid dollars (Fiedler, 2017). he latter was to be accomplished by transferring funding of Medicaid from the Federal government to the states.

1	Overview of Obstetrics 7 hese potential outcomes have prompted considerable debate among voters, and "repeal and replace" has become politically charged. Currently, the Senate has been unable to recruit suficient Republican votes for Senate passage of such a bill. We suggest that the health care crisis should be reframed and redirected instead to a critical analysis of health care costs and resource utilization. that spending on health care in the United States in 2015 accounted for 17.8 percent of the gross domestic product

1	that spending on health care in the United States in 2015 accounted for 17.8 percent of the gross domestic product GDP (Voelker, 2010). he total amount of health-care spend ing-$3.2 trillion-equated to an estimated $10,000 per person. Moreover, compared with 12 other high-income coun tries, health-care spending in the United States as a proportion of GDP was approximately 50 percent more than the next highest country. Yet, health-care outcomes, which included infant mortality rates, were worse in the United States. And, approximately two thirds of U.S. infant deaths result from complications stemming from preterm births (Matthews, 2015). Indeed, in its 2010 annual global Premature Birth Report Card, the United States garnered a grade of "D" from the March of Dimes for its recognition and prevention of pre term labor in the more than 540,000 neonates born annually before 37 weeks' gestation.

1	Causes for the excessive health care costs in the United States are attributed, in part, to greater use of medical technology and excessive prices (Squires, 2017). Two recent studies demonstrate the detrimental efect of obstetrics on health care costs. The first report by Nelson and coworkers (2017) described the inefectiveness of 17 -alpha hydroxyprogesterone caproate (17OHP-C) to prevent recurrent preterm birth. Methodology for this trial is presented in Chapter 42 (p. 817). Several lessons can be learned from this investigation. First, use of 17 -OHP-C was legitimized in the United States by a national consensus committee using expert opinion. hese opinions were promulgated, despite FDA reservations that the evidence was lacking in several important respects. However, once approved, 17 -OHP-C was sold by one pharmaceutical company for $1500 for a single, 250-mg injectable dose. Remarkably, this same dose could be compounded and purchased for $25 from local pharmacies. In the

1	17 -OHP-C was sold by one pharmaceutical company for $1500 for a single, 250-mg injectable dose. Remarkably, this same dose could be compounded and purchased for $25 from local pharmacies. In the subsequent price-gouging controversy, members of the United States Congress intervened to permit continued use of the less expensive 17-0 HP -C.

1	he second study is a multisite prospective trial of the efectiveness of transvaginal sonography to screen for cervical-length shortening to predict preterm birth (Esplin, 2017). A total of 9410 nulliparous women were studied. he Society for Maternal-Fetal Medicine and the American College of Obstetricians and Gynecologists (2016d) both legitimized universal cervicallength screening in their joint Committee Opinion (Bloom, 2017). And, by 2015, one survey of78 Maternal-Fetal Medicine fellowship programs showed that 68 percent were using universal cervical-length screening to predict preterm birth (Khalifeh, 2017). It was estimated that a modest Medicaid rate of $237 per cervical-length ultrasound would result in approximately $350 million in added health care costs. But, Esplin and associates (2017) found that routine screening for a short cervix was not beneficial. hat is, a widely used intervention was actually inefective. his is a clear example of how unproven technology can seep

1	(2017) found that routine screening for a short cervix was not beneficial. hat is, a widely used intervention was actually inefective. his is a clear example of how unproven technology can seep into widespread practice.

1	These two reports highlight a substantial problem in U.S. health care, namely, inefective yet expensive interventions introduced into broad use without robust evidence. These two reports also speak to a demand for robust scientific evidence. Scrutiny of other ingredients in the health-care paradigm such as prices for hospitalization, prices for surgical procedures, and prices charged by health insurance companies may illuminate similar contributions to the health care fiscal crisis. In past editions of this textbook, the rising cesarean delivery rate was considered problematic. his rate has leveled, but there are still imperatives in progress to help lower this rate. One collateral source of cesarean delivery morbidity is from the growing incidence of morbidly adherent placentas encountered in women with a prior hysterotomy incision, discussed in Chapters 31 and 41.

1	Breakthroughs in fetal testing and diagnosis continue to stun. By 2012, prenatal gene micro array techniques were used for clinical management (Dugof, 2012). he advantages of these techniques are outlined in Chapters 13 and 14. Wapner and coworkers (2012) compared chromosomal microarray analysis of maternal blood with karyotyping for chromosomal anomalies. Reddy and associates (2012) applied this technology to stillbirth evaluation and reported it to be superior to karyotyping. Another report by Talkowski and colleagues (2012) described whole-genome sequencing of a fetus using maternal blood.

1	Screening for fetal aneuploidy using cell-free DNA (cDNA) was first introduced in 2011. The technique is described in Chapter 14 (p. 284), and it is based on isolation of free fetal (placental) DNA in maternal blood. In a landmark study, Norton and associates (2015) found that cDNA had a higher sensitivity and speciicity compared with standard prenatal screening for trisomy 21 fetuses. Still, invasive testing is currently necessary to confirm a positive cDNA test result (Chitty, 2015; Snyder, 2015).

1	• The Ob/Gyn Hospitalist he term "hospitalist" was coined in the 1990s and referred to physicians whose primary professional focus was generalized care of hospitalized patients. From this concept came the obstetrical and gynecological hospitalist whose primary role was to care for hospitalized obstetrical patients and to help manage their emergencies. These physicians could also provide urgent gynecological care and emergency department consultation. Alternative terms include "obstetrical hospitalist" or "laborist," but the preferred standardized term by the American College of Obstetricians and Gynecologists (2016e) is "Ob/Gyn hospitalist." lthough not a recognized subspecialty of obstetrics and gynecology, the Ob/Gyn hospitalist movement has gained momentum. he Society of Ob-Gyn Hospitalists had 528 members in 2017 (Burkard, 2017). Various practice models are described to fit the needs of a wide spectrum of obstetrical volumes (McCue, 2016). In addition to providing lifestyle

1	had 528 members in 2017 (Burkard, 2017). Various practice models are described to fit the needs of a wide spectrum of obstetrical volumes (McCue, 2016). In addition to providing lifestyle modifications, Ob/Gyn hospitalists are used by some hospitals to improve the quality and safety of their women's services and to reduce adverse events. Aside from a possible lowering of the labor induction rate, studies are needed to demonstrate improved outcomes with these providers (American College of Obstetricians and Gynecologists, 2016e; Srinivas, 2016).

1	he American College of Obstetricians and Gynecologists periodically surveys its fellows concerning the efect of liability on their practice. he 2015 Survey on Professional Liability is the 12th such report since 1983 (Carpentieri, 2015). From this survey, it appears that there is still a "liability crisis," and the reasons for it are complex. Because it is largely driven by money and politics, a consensus seems unlikely. Although some interests are diametrically opposite, other factors contribute to the problem's complexity. For example, each state has its own laws and opinions on tort reform. In some states, annual premiums for obstetricians approach $300,000-expenses that at least partially are borne by the patient and certainly by the entire health-care system. In 2011, all tort costs in the United States totaled nearly $265 billion. his is an astounding 1.8 percent of the gross domestic product and averages to a cost of $838 per citizen (Towers Watson, 2015).

1	he American College of Obstetricians and Gynecologists (2016a,c) has taken a lead in adopting a fair system for malpractice litigation-or maloccurrence itigation. And nationally, there is the possibility of federl tort reform under the Trump administration (Lockwood, 2017; Mello, 2017). Following a slight decline from 1990 through 2004, the percentage of out-of-hospital births in the United States increased from 0.86 to 1.5 percent-almost 75 percent-through 2014 (MacDorman, 2016a). Of these home births, only a third are attended by nurse midwives certiied by the American Midwife Certiication Board (Grtinebaum, 2015; Snowden, 2015).

1	Proponents of home births cite successes derived from laudatory observational data from England and he Netherlands (de Jonge, 2015; Van der Kooy, 2011). Data from the United States, however, are less convincing and indicate a higher incidence of perinatal morbidity and mortality (Grtinebaum, 2014, 2015; Snowden, 2015; Wasden, 2014; Wax, 2010). hese latter findings have led Chervenak and coworkers (2013, 2015) to question the ethics of participation in planned home births. Greene and Ecker (2015) take a broader view. Given data from these more recently cited studies, they are of the view that these data empower women to make a rational decision regarding home delivery. he American College of Obstetricians and Gynecologists (20 17b) believes that hospitals and accredited birth centers ofer the safest settings, but that each woman has the right to make a medically informed decision regarding delivery.

1	Politics and religion over the years have led to various govern mental interferences with the reproductive rights of women. adolescents. This is despite all reports of the overwhelming suc cess of such programs. One example is the exclusion of Planned Parenthood ailiates from the Texas Medicaid fee-for-service family planning program. In some groups of women served, there was discontinuation of contraception and an increased rate of Medicaid binhs (Stevenson, 2016),

1	According to the Gurrmacher Institute (2016a), publicly funded family planning services are needed by 20 million American women. In 2014, such services prevenred nearly 2 million unintended pregnancies and 700,000 abonions in the United States. The fate of family planning services is not fully determined, while waiting for decisions regarding provisions within the 2017 American Healrh Care Ace (AHCA). or "Trumpcare." In his response ro news that the AHCA may dismantle cOlHraceptive coverage, American College of Obstetricians and Gynecologises President Dt. Haywood Brown (2017) called this a deep disregard for women's health.

1	According to the CDC (2014). there were 259 million prescriptions written in 2012 for opioid medications. In 2013, more than a rhird of American adults reported prescrip tion opioid use (Han, 2017). These fteely available-albeit requiring a prescription-addictive drugs are associated with opioid use disorders. It remains uncercain if opioid use is terarogenic (Lind, 2017). Still, their abuse by pregnam women has caused an unprecedented rise in the 1eonata/ abstinence syndrome. described furrher in Chapters 12 (p. 248) and 33 (p. 625). Treatment of opioid abuse in pregnancy and its sequelae result in $1.5 billion annually in hospital charges.

1	For obstetrical providers ro bener deal with opioid-addicted pregnant women and their fetus-newborns, the Eu.nice Kennedy Shriver National Institute of Child Helth and Human Development convened a workshop in 2016 co study many aspecrs of the problem (Reddy. 2017). he Workshop was cosponsored by the American College of Obstetricians and Gynecologisrs, the American Academy of Pediatrics, the Society for Maternal-Fetal Medicine, the CDC, and the March of Dimes. Several copies were addressed, and hopefully implementation of these indings will help improve maternal treatment and neonatal outcomes (American College of Obstetricians and Gynecologists. 2017a).

1	The bold new concept of in-vitro fercilization (IVF) produced the irst IVF baby in Britain in 1978. This was soon followed in 1981 with an American success. After four decades. the Society for Assisted Reproductive Technology (SART) reports that more than 1 million babies have been born in the United States using assisted reproductive technologies (ART) ofered by 440 clinics (Fox. 2017). ter IS years of experimental preparation. the promise of a successful human uterine transplant was finally realized with n Overview of Obstetrics 9 IVF-conceived livebon neonate in Sweden (Brinnstrom, 2015).

1	Overview of Obstetrics 9 IVF-conceived livebon neonate in Sweden (Brinnstrom, 2015). During pregnancy, the mother was treated with t:lcrolimus, azathioprine, and corticosteroids and underwent cesarean delivery at 32 weeks for preeclampsia and abnormal feral heart rate testing. This was followed by uterine trmsplamation programs at the C1cvebnd Clinic and Baylor Medical Center in Dallas (F1yckt. 2016. 2017, Testa. 2017). In 2017. the Swedish team had completed a nine-patient trial, in which seven women had become pregnant and ive had sllccessful deliveries (Kuehn, 2017). Also, in Dallas, the irst such newbon in the Unired Stares was born (Rice. 2017).

1	Meanwhile, researchers at Children's Hospital of Philadelphia pursued a 20-year goal in search of an artiicial womb (Yuko, 2017). Using incubatOr technology, [he team devised an artiicial amnionic sac. hrough this, (he umbilical vessels were perfused and drained, and the blood was retuned to systems [hat performed exrracorporeal membrane oxygenation and dialysis. To date, lamb fetuses have been kept alive for as long as 1 month. Adverse efects of cerebrovascular hypotension and hypoxemia are conjectural but highly worrisome. 1he ethical and legal challenges of these new technologies are daunting. Of those that arose from IVF, mOSt are setded. For the Q[her twO endeavors, there are likely many years of ethical and legal milestones ahead. American Academy of Pediatrics, American College of Obstetricians and Gynecologisrs: Guidelines for perinatal care, 8th ed. Elk Grove Village, AAP,e2017

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1	MacDorman MF, Declercq E: Trends and charactcristics of United States outof-hospital births 2004-20n14: new information on risk staws and access to carc. Birth 43(2): 1 16, 2016a MacDonnan "-IF, Declercq E, Cabral H, et ;\1: Recent increases in the U.S. maternal mortality rate: disentangling trends from measuremem issues. Obstet GynecoIn128(3):447, 2016b �lacDorman MF, Declercq E, 1110ma ME: Trends in matenal mortality by sociodcmographic characteristics and cause of death in 27 states and the Dimi" of Columbia. Obstet Gynccol 129(5):811,n2017 MacDorman MF, Gregory EC: Fe!'al and perinatal mortality: United States, 2013. Nat! Vital Star R:p 64(8): 1, 2015 Main EK, Cape V, Abreo A, et al: Reduction of severe matcnal morbidity from hemorrhage using a state perinatal qualiry collaborative. Am J Obstet Gynccol 216(3):298.el, 2017

1	Main EK, Cape V, Abreo A, et al: Reduction of severe matcnal morbidity from hemorrhage using a state perinatal qualiry collaborative. Am J Obstet Gynccol 216(3):298.el, 2017 Main EK, Coffman D, Scavone B� I,et al: National partnership for materna! safety: consensus bundle on obstetric hemorrhage. Obstet Gynreol 126( I); 155, 2015 �Iarkus AR, Andres E, West D, et Il: Medicaid covered births, 2008 through 2010, in rhe concexr of the implementation of health reform. Womens Health Issues 23(5):e273. 2013 �1aron DF: 1.laternal health care is disappearing in rural America. Scientiic American, February 15,n2017 Martin JA, Hamilton BE, Osterman MJK, et al: Births: inal data for 2015. National Vital Statistics Report 66: 1, 2017

1	Martin JA, Hamilton BE, Osterman MJK, et al: Births: inal data for 2015. National Vital Statistics Report 66: 1, 2017 Matthews RJ, MaeDorman MF, Thoma ME: In.1l1t mortality s:uistis from the 2013 period linked birth/in..lJl( death data set. Nat! Vital Stat Rep 64(9): 1,2015 t\IcCue B, Fagnant R, Townsend A, et al: Deinitions of obstetric and ynecologic hospitalists. Qbstel GynecoIn127(2):393, 2016 �Iello MM, Kachalia A, Sruddert D\I: Medicalnliabiliry�prospeclS for federal reform. N Engl J �Ied r6(19):1806, 2017 �loaddab A, Dildy GA, Brown H L, et al: Health care disparity and state-speciic pregnancy-related mortality in the United States, 2005-2014. Obstet Gynccol 128:869,n2016 Nelson DB, Mcintire DO, McDonald J, et al: 17-Alpha hydroxyprogestetone caprOate did not reduce the ratc of recurrent prcterm birth in a prospective cohort study. Am J Obstet Gynecol 216:600.e 1, 2017

1	NortOn ME. Jacobsson B, Swamy GK, et al: Cell-free DNA analysis for noninvasive examination of trisomy. N Engl J Moo 373(26):2582, 2015 Reddy UM, Davis )M, Ren Z, e1al: Opioid cin pregnancy, neonatal abstinence syndrome, and childhood Outcomes. Obsret Gynecol 130(1): 10. 201 Reddy UM, Page GP, Saade GR, et al: Karyotype versus microarra), testing for generic abnormalities after stillbirth. N Engl) Med 367(23):2185, 2012 Rice S: Baylor doctors deliver irst baby born in U.S. after uterine transplant. Dallas i\lorning News. December 2, 2017 Shields LE, Wiesner S, Klein C, et al: Early standardized treatment of critical blood pressure elevations is associated with a reduction in eclampsia and severe maternal morbidity. Am J Obstet GynecoI216(4):415.el, 2017 Snowden JM, Tilden EL, Snyder J, et al: Planned out-of-hospital birth and birth outcomes. N Engl J Med 373:2642,r2015

1	Snowden JM, Tilden EL, Snyder J, et al: Planned out-of-hospital birth and birth outcomes. N Engl J Med 373:2642,r2015 Snyder M, Simmons LE, Kitzman JO, et al: Copy-number variation and false positive prenatal aneuploidy screening results. N Engl J Med 372(1 ): 1639, 2015 Souza JP, Cecatti JG, Haddad SM, et al: he WHO maternal near-miss approach and the maternal severity index model (MSI): tools for assessing the management of severe maternal morbidity. PLoS One 7(8):e44129, 2012 Squires 0, Anderson C: US health care from a global perspective, spending, use of services, prices, and health in ] 3 countries. Issue Brief (Commonw Fund) 15:1,r2015 Srinivas SK, Small OS, Macheras M, et al: Evaluating the impact of the laborist model of obstetric care on maternal and neonatal outcomes. Am J Obstet GynecoIr215:770.el,r2016 Stevenson AJ, Flores-Vazquez 1M, Allgeyer RL, et al: Efect of removal of Planned Parenthood from the Texas women's health program. N Engl J Med 374(9):853, 2016

1	Stevenson AJ, Flores-Vazquez 1M, Allgeyer RL, et al: Efect of removal of Planned Parenthood from the Texas women's health program. N Engl J Med 374(9):853, 2016 Overview of Obstetrics 11 Talkowski ME, Ordulu Z, Pillalamarri V, et al: Clinical diagnosis by wholegenome sequencing of a prenatal sample. N EnglJ Med 367(23):2226, 2012 Tavenise S: Maternal mortality rate in U.S. rises, deying global trend, study finds. New York Times, September 21,r2016 Testa G, Koon EC, Johannesson L, et al: Living donor uterus transplantation: a single center's observations and lessons leaned from early setbacks to technical success. Am J Transplant, April 22, 2017 [Epub ahead of print] Towers Watson: Update of U.S. tort cost trends. Available at: towerswatson. com. Accessed July 30,r201

1	Towers Watson: Update of U.S. tort cost trends. Available at: towerswatson. com. Accessed July 30,r201 Yan der Kooy J, Poeran J, de Graaf JP, et l: Planned home compared with planned hospital births in the Netherlands. Obstet Gynecol 118(5): 1037, 2011 Yoelker R: US preterm births: "0" is for dismal. JAMA 303(2):116,r2010 Wapner RJ, Martin CL, Levy B, et al: Chromosomal microarray versus karyo typing for prenatal diagnosing. N Engl J Med 367(23):2175, 2012 Wasden S, Perlman J, Chasen S, et al: Home birth and risk of neonatal hypoxic ischemic encephalopathy. Am J Obstet Gynecol 21 0:S251, 2014 Wax JR, Lucas FJ, Lamont M, et al: Maternal and newbon oLltcomes in planned home birth vs planned hospital births: a metaanalysis. Am J Obstet GynecoIr203(3):243,r2010 Yuko E: Weighing the ethics of artiicial wombs. New York Times, May 8,r2017 ANTERIOR ABDOMINAL WALL . . . . . . . . . . . . . . . . . . . . . 14 EXTERNAL GENERATIVE ORGANS ... .... .. 16

1	Yuko E: Weighing the ethics of artiicial wombs. New York Times, May 8,r2017 ANTERIOR ABDOMINAL WALL . . . . . . . . . . . . . . . . . . . . . 14 EXTERNAL GENERATIVE ORGANS ... .... .. 16 INTERNAL GENERATIVE ORGANS .......... ..... . 23 LOWER URINARY TRACT STRUCTURES ... . 28 MUSCULOSKELETAL PELVIC ANATOMY . .. 29 As the mechanism of labour is essentialy a process of accommodation between the oetus and the passage through which it must pass, it is apparent that obstetrics lacked a scientiic oundation until the anatomy of the bony pelvis and of the sot parts connected with it was cleary understood -J. Whitridge Williams (1903) • Skin, Subcutaneous Layer, and Fascia he anterior abdominal wall confines abdominal viscera, stretches to accommodate the expanding uterus, and provides surgical access to the internal reproductive organs. hus, a comprehensive knowledge of its layered structure is required to surgically enter the peritoneal cavity.

1	Langer lines describe the orientation of dermal fibers within the skin. In the anterior abdominal wall, they are arranged transversely. As a result, vertical skin incisions sustain greater lateral tension and thus, in general, develop wider scars. In contrast, low transverse incisions, such as the Pfannenstiel, follow Langer lines and lead to superior cosmetic results. The subcutaneous layer can be separated into a superficial, predominantly fatty layer-Camper fascia, and a deeper membranous layer-Scarpa fascia. Camper fascia continues onto the perineum to provide fatty substance to the mons pubis and labia majora and then to blend with the fat of the ischioanal fossa. Scarpa fascia continues inferiorly onto the perineum as Colles fascia, described on page 19.

1	Beneath the subcutaneous layer, the anterior abdominal wall muscles consist of the midline rectus abdominis and pyramidalis muscles as well as the external oblique, internal oblique, and transversus abdominis muscles, which extend across the entire wall (Fig. The ibrous aponeuroses of these three latter muscles form the primary fascia of the anterior abdominal wall. These fuse in the midline at the linea alba, which normally measures 10 to 15 mm wide below the umbilicus (Beer, 2009). n abnormally wide separation may reflect diastasis recti or hernia.

1	These three aponeuroses also invest the rectus abdominis muscle as the rectus sheath. The construction of this sheath varies above and below a boundary, termed the arcuate line (see Fig. 2-1). Cephalad to this border, the aponeuroses invest the rectus abdominis bellies on both dorsal and ventral surfaces. Caudal to this line, all aponeuroses lie ventral or supericial to the rectus abdominis muscle, and only the thin transversalis fascia and peritoneum lie beneath the rectus (Loukas, 2008). This transition of rectus sheath composition can be seen best in the upper third of a midline vertical abdominal incision. he paired small triangular pyramidalis muscles originate from the pubic crest and insert into the linea alba. These muscles lie atop the rectus abdominis muscle but beneath the anterior rectus sheath.

1	he supericial epigastric, supericial circumlex iliac, and supericial external pudendal arteries arise from the femoral artery just below the inguinal ligament within the femoral triangle Rectus abdominis m. External oblique m. Internal oblique m. Transversus abdominis m. Lateral cutaneous n. Genital branch of genitofemoral n. Internal thoracic a. Superior epigastric a. Inferior epigastric a. Superficial circumflex iliac a. External pudendal a. with emerging round ligament, ilioinguinal & genitofemoral nn.

1	Internal thoracic a. Superior epigastric a. Inferior epigastric a. Superficial circumflex iliac a. External pudendal a. with emerging round ligament, ilioinguinal & genitofemoral nn. FIGURE 2-1 Anterior abdominal wall anatomy. (Modified with permission from Corton MM: Anatomy. In Hoffman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016) (see Fig. 2-0. These vessels supply the skin and subcutaneous layers of the anterior abdominal wall and mons pubis. Of these three, the superficial epigastric vessels are surgically important to the obstetrician and course diagonally from their origin toward the umbilicus. With a low transverse skin incision, these vessels can usually be identified at a depth halway between the skin and the anterior rectus sheath. They lie above Scarpa fascia and several centimeters from the midline. Ideally, these vessels are identiied and surgically occluded.

1	In contrast, the inferior "deep" epigastric vessels are branches of the external iliac vessels and supply anterior abdominal wall muscles and fascia. Of surgical relevance, the inferior epigastric vessels initially course lateral to, then posterior to the rectus abdominis muscles, which they supply. Above the arcuate line, these vessels course ventral to the posterior rectus sheath and lie between this sheath and the posterior surface of the rectus muscles. Near the umbilicus, the inferior epigastric vessels anastomose with the superior epigastric artery and vein, which are branches of the internal thoracic vessels. Clinically, when a Maylard incision is used for cesarean delivery, the inferior epigastric vessels may be lacerated lateral to the rectus belly during muscle transection. Preventively, identiication and surgical occlusion are preferable. These vessels rarely may rupture following abdominal trauma and create a rectus sheath hematoma (Toicher, 2010; Wai, 2015).

1	On each side of the lower anterior abdominal wall, Hesselbach triangle is the region bounded laterally by the inferior epigastric vessels, inferiorly by the inguinal ligament, and medially by the lateral border of the rectus abdominis muscle. Hernias that protrude through the abdominal wall in Hesselbach triangle are termed direct inguinal hernias. In contrast, indirect inguinal hernias do so through the deep inguinal ring, which lies lateral to this triangle, and then may exit out the supericial inguinal ring. he entire anterior abdominal wall is innervated by intercostal nerves (T 7-"i )ithe subcostal nerve (T'2)•' and the ilio hypogastric and the ilioinguinal nerves (L,). Of these, the Intecostal n. Internaloblique m. External __.oblique m. Lateral Anterior ramus

1	FIGURE 2-2 Intercostal and subcostal nerves are the anterior rami of spinal nerves. In this figure, an intercostal nerve extends ventrally between the transversus abdominis and internal oblique muscles. During this path, the nerve gives rise to lateral and anterior cutaneous branches, which innervate the anterior abdominal wall. As shown by the inserted needle, the transversus abdominis plane (TAP) block takes advantage of this anatomy. (Modified with permission from Hawkins JL: Anesthesia for the pregnant woman. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al: Cunningham and Gilstraps's Operative Obstetrics, 3rd ed. New York, McGraw Hill Education, 201o7.) intercostal and subcostal nerves are anterior rami of the thoracic spinal nerves and run along the lateral and then anterior abdominal wall between the transversus abdominis and internal oblique muscles (Fig. 2-2). his space, termed the transversus abdominis plane, can be used for postcesarean analgesia blockade (Chap. 25, p.

1	wall between the transversus abdominis and internal oblique muscles (Fig. 2-2). his space, termed the transversus abdominis plane, can be used for postcesarean analgesia blockade (Chap. 25, p. 500) (Fusco, 2015; Tawfik, 2017). Others report rectus sheath or ilioinguinaliliohypogastric nerve blocks to decrease postoperative pain (Mei, 2011; Wolfson, 2012).

1	Near the rectus abdominis lateral borders, anterior branches of the intercostal and subcostal nerves pierce the posterior sheath, rectus muscle, and then anterior sheath to reach the skin. hus, these nerve branches may be severed during a Pfannenstiel incision creation during the step in which the overlying anterior rectus sheath is separated from the rectus abdominis muscle.

1	In contrast, the iliohypogastric and ilioinguinal nerves originate from the anterior ramus of the first lumbar spinal nerve. They emerge lateral to the psoas muscle and travel retroperitoneally across the quadratus lumborum inferomedially toward the iliac crest. Near this crest, both nerves pierce the transversus abdominis muscle and course ventromedially. At a site 2 to 3 cm medial to the anterior superior iliac spine, the nerves then pierce the internal oblique muscle and course supericial to it toward the midline (Whiteside, 2003). The iliohypogastric nerve perforates the external oblique aponeurosis near the lateral rectus border to provide sensation to the skin over the suprapubic area (see Fig. 2-1). The ilioinguinal nerve in its course medially travels through the inguinal canal and exits through the supericial inguinal ring, which forms by splitting of external abdominal oblique aponeurosis fibers. This nerve supplies the skin of the mons pubis, upper labia majora, and medial

1	exits through the supericial inguinal ring, which forms by splitting of external abdominal oblique aponeurosis fibers. This nerve supplies the skin of the mons pubis, upper labia majora, and medial upper thigh.

1	The ilioinguinal and iliohypogastric nerves can be severed during a low transverse incision or entrapped during closure, especially if incisions extend beyond the lateral borders of the rectus abdominis muscle (Rahn, 2010). These nerves carry sensory information only, and injury leads to loss of sensation within the areas supplied. Rarely, chronic pain may develop (Whiteside, 2005). he T 10 dermatome approximates the level of the umbilicus. Analgesia to this level is suitable for labor and vaginal birth. Regional analgesia for cesarean delivery or for puerperal sterilization ideally extends to T4' Mons Pubis, Labia, and Clitoris

1	Mons Pubis, Labia, and Clitoris The pudenda-commonly designated the vulva-includes all structures visible externally from the symphysis pubis to the perineal body. This includes the mons pubis, labia majora and minora, clitoris, hymen, vestibule, urethral opening, greater vestibular or Bartholin glands, minor vestibular glands, and paraurethral glands (Fig. 2-3). he vulva receives innervations and vascular support from the pudendal nerve (p. 22). he mons pubis is a fat-illed cushion overlying the symphysis pubis. After puberty, the mons pubis skin is covered by curly hair that forms the triangular escutcheon, whose base aligns with the upper margin of the symphysis pubis. In men and some hirsute women, the escutcheon extends farther onto the anterior abdominal wall toward the umbilicus.

1	Labia majora usually are 7 to 8 cm long, 2 to 3 cm wide, and 1 to 1.5 cm thick. They are continuous directly with the mons pubis superiorly, and the round ligaments terminate at their upper borders. Hair covers the labia majora, and apocrine, eccrine, and sebaceous glands are abundant. Beneath the skin, a dense connective tissue layer is nearly void of muscular elements but is rich in elastic fibers and fat. his fat mass provides bulk to the labia majora and is supplied with a rich venous plexus. During pregnancy, this vasculature may develop varicosities, especially in multiparas, from increased venous pressure created by the enlarging uterus. hey appear as engorged tortuous veins or as small grapelike clusters, but they are typically asymptomatic and require no treatment.

1	Each labium minus is a thin tissue fold that lies medial to each labium majus. he labia minora extend superiorly, where each divides into two lamellae. From each side, the lower lamellae fuse to form the frenulum of the clitoris, and the upper lamellae merge to form the prepuce (see Fig. 2-3). Inferiorly, the labia minora extend to approach the midline as low ridges of tissue that join to form the fourchette. The labia minora dimensions vary greatly among individuals, with lengths from 2 to 10 cm and widths from 1 to 5 cm (Lloyd, 2005). Structurally, the labia minora are composed of connective tissue with numerous vessels, elastin ibers, and very few smooth muscle ibers. hey are supplied with many nerve endings and are extremely sensitive (Ginger, 20i1i1a; Schober, 2015). The epithelia of the labia minora difer with location. Thinly keratinized stratiied squamous epithelium covers the Glans of clitoris Inferior fascia of levator ani mm./ External anal sphincter m.

1	Glans of clitoris Inferior fascia of levator ani mm./ External anal sphincter m. FIGURE 2-3 Vulvar structures and subcutaneous layer of the anterior perineal triangle. Note the continuity of Colles and Scarpa fasciae. Inset: Vestibule boundaries and openings onto vestibule. (Reproduced with permission from Corton MM: Anatomy. In Hofman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.) outer surface of each labium. On their inner surface, the lateral portion is covered by this same epithelium up to a demarcating line, termed Hart line. Medial to this line, each labium is covered by squamous epithelium that is nonkeratinized. he labia minora lack hair follicles, eccrine glands, and apocrine glands. However, sebaceous glands are numerous (Wilkinson, 2011).

1	he clitoris is the principal female erogenous organ. It is located beneath the prepuce, above the frenulum and urethra, and projects downward and inward toward the vaginal opening. The clitoris rarely exceeds 2 cm in length and is composed of a glans, a corpus or body, and two crura (Verkauf, 1992). he glans is usually less than 0.5 cm in diameter, is covered by stratified squamous epithelium, and is richly innervated. he clitoral body contains two corpora cavernosa. Extending from the clitoral body, each corpus cavernosum diverges laterally to form a long, narrow crus. Each crus lies along the inferior surface of its respective ischiopubic ramus and deep to the ischiocavernosus muscle. The clitoral blood supply stems from branches of the internal pudendal artery. Specifically, the deep artery of the clitoris supplies the clitoral body, whereas the dorsal artery of the clitoris supplies the glans and prepuce.

1	In adult women, the vestibule is an almond-shaped area that is enclosed by Hart line laterally, the external surface of the hymen medially, the clitoral frenulum anteriorly, and the fourchette posteriorly (see Fig. 2-3). The vestibule is usually perforated by six openings: the urethra, the vagina, two Bartholin gland ducts, and two ducts of the largest paraurethral glands-the Skene glands. he posterior portion of the vestibule between the fourchette and the vaginal opening is called the fossa navic ularis. It is usually observed only in nulliparas.

1	Skene glands. he posterior portion of the vestibule between the fourchette and the vaginal opening is called the fossa navic ularis. It is usually observed only in nulliparas. The bilateral Bartholin glands, also termed greater vestibular glands, measure 0.5 to 1 cm in diameter. On their respective side, each lies inferior to the vestibular bulb and deep to the inferior end of the bulbospongiosus muscle (former bulbocavernosus muscle). A duct extends medially from each gland, measures 1.5 to 2 cm long, and opens distal to the hymeneal ring-one at 5 and the other at 7 o'clock on the vestibule. Following trauma or infection, either duct may swell and obstruct to form a cyst or, if infected, an abscess. In contrast, the minor vestibular glands are shallow glands lined by simple mucinsecreting epithelium and open along Hart line.

1	he paraurethral glands are a collective arborization of glands whose numerous small ducts open predominantly along the entire inferior aspect of the urethra. he two largest are called Skene glands, and their ducts typically lie distally and near the urethral meatus. Clinically, inlammation and duct obstruction of any of the paraurethral glands can lead to urethral diverticulum formation. The urethral opening or meatus is in the midline of the vestibule, 1 to 1.5 cm below the pubic arch, and a short distance above the vaginal opening. In adult women, the hymen is a membrane of varying thickness that surrounds the vaginal opening more or less completely. Perineal body Fuseddistal segment of urethra & vagina FIGURE 2-4 Vagina and surrounding anatomy. (Reproduced with permission from Corton MM: Anatomy. In Hofman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 201o6.)

1	Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 201o6.) It is composed mainly of elastic and collagenous connective tissue, and both outer and inner surfaces are covered by nonkeratinized stratified squamous epithelium. he aperture of the intact hymen ranges in diameter from pinpoint to one that admits one or even two ingertips. As a rule, the hymen is torn at several sites during irst coitus. However, identical tears may form by other penetration, for example, by tampons used during menstruation. The edges of the torn tissue soon reepithelialize. In pregnant women, the hymeneal epithelium is thick and rich in glycogen. Changes produced in the hymen by childbirth are usually readily recognizable. For example, over time, the hymen transforms into several nodules of various sizes, termed hymeneal or myrtiform caruncles.

1	Proximal to the hymen, the vagina is a musculomembranous tube that extends to the uterus and is interposed lengthwise between the bladder and the rectum (Fig. 2-4). Anteriorly, the vagina is separated from the bladder and urethra by connective tissue-the vesicovaginal septum. Posteriorly, between the lower portion of the vagina and the rectum, similar tissues together form the rectovaginal septum. The upper fourth of the vagina is separated from the rectum by the rectouterine pouch, also called the cul-de-sac or pouch of Douglas. Normally, the anterior and posterior walls of the vaginal lumen lie in contact, with only a slight space intervening at the lateral margins. Vaginal length varies considerably, but commonly, the anterior wall measures 6 to 8 cm, whereas the posterior vaginal wall is 7 to 10 cm. The upper end of the vaginal Posteriorcul-de-sac of Douglas sphincter m. External anal sphincter m.

1	Posteriorcul-de-sac of Douglas sphincter m. External anal sphincter m. vault is subdivided by the cervix into anterior, posterior, and two lateral fornices. Clinically, the internal pelvic organs usually can be palpated through the thin walls of these fornices. The vaginal lining is composed of nonkeratinized stratified squamous epithelium and underlying lamina propria. In premenopausal women, this lining is thrown into numerous thin transverse ridges, known as rugae, which line the anterior and posterior vaginal walls along their length. Deep to this, a muscular layer contains smooth muscle, collagen, and elastin. Beneath this muscularis lies an adventitial layer consisting of collagen and elastin (Weber, 1997).

1	The vagina lacks glands. Instead, it is lubricated by a transudate that originates from the vaginal subepithelial capillary plexus and crosses the permeable epithelium (Kim, 201l). Due to increased vascularity during pregnancy, vaginal secretions are notably increased. At times, this may be confused with amnionic fluid leakage, and clinical diferentiation of these two is described in Chapter 22 (p. 435). Mter birth-related epithelial trauma and healing, fragments of stratified epithelium occasionally are embedded beneath the vaginal surface. Similar to its native tissue, this buried epithelium continues to shed degenerated cells and keratin. As a result, epidermal inclusion cysts, which are filled with keratin debris, may form. hese are a common vaginal cyst. The vagina has an abundant vascular supply. The proximal portion is supplied by the cervical branch of the uterine artery and by the vaginal artery. he latter may variably arise from the

1	The vagina has an abundant vascular supply. The proximal portion is supplied by the cervical branch of the uterine artery and by the vaginal artery. he latter may variably arise from the Vesicovaginal space (filled with loose connective tissue) uterine or inferior vesical artery or directly from the internal iliac artery. The middle rectal artery contributes supply to the posterior vaginal wall, whereas the distal walls receive contributions from the internal pudendal artery. At each level, vessels supplying each side of the vagina course medially across the anterior or posterior vaginal wall and form midline anastomoses. An extensive venous plexus also surrounds the vagina and follows the course of the arteries. Lymphatics from the lower third, along with those of the vulva, drain primarily into the inguinal lymph nodes. Those from the middle third drain into the internal iliac nodes, and those from the upper third drain into the external, internal, and common iliac nodes.

1	his diamond-shaped area between the thighs has boundaries that mirror those of the bony pelvic outlet: the pubic symphysis anteriorly, ischiopubic rami and ischial tuberosities anterolaterally, sacrotuberous ligaments posterolaterally, and coccyx posteriorly. An arbitrary line joining the ischial tuberosities divides the perineum into an anterior triangle, also called the urogenital triangle, and a posterior triangle, termed the anal triangle.

1	he perineal body is a fibromuscular pyramidal mass found in the midline at the junction between these anterior and posterior triangles (Fig. 2-5). Also called the central tendon of the perineum, the perineal body sonographically measures 8 mm tall and 14 mm wide and thick (Santoro, 2016). It serves as the junction for several structures and provides significant perineal support (Shafik, 2007). Superficially, the bulbospongiosus, superficial transverse perineal, and external anal sphincter muscles converge on the perineal body. More deeply, the perineal membrane, portions of the pubococcygeus muscle, and internal anal sphincter contribute (Larson, 2010). The perineal body Ischiocavernosus m. Cut edge of Colles fascia Bulbospongiosus m. Superficial transverse perineal m. External anal sphincter m. is incised by an episiotomy incision and is torn with second-, third-, and fourth-degree lacerations. Superficial Space of the Anterior Triangle

1	External anal sphincter m. is incised by an episiotomy incision and is torn with second-, third-, and fourth-degree lacerations. Superficial Space of the Anterior Triangle This triangle is bounded by the pubic rami superiorly, the ischial tuberosities laterally, and the supericial transverse perineal muscles posteriorly. It is divided into supericial and deep spaces by the perineal membrane. This membranous partition is a dense ibrous sheet that was previously known as the inferior fascia of the urogenital diaphragm. The perineal membrane attaches laterally to the ischiopubic rami, medially to the distal third of the urethra and vagina, posteriorly to the perineal body, and anteriorly to the arcuate ligament of the pubis (see Fig. 2-5) .

1	The supericial space of the anterior triangle is bounded deeply by the perineal membrane and supericially by Co lIes fascia. As noted earlier, Colles fascia is the continuation of Scarpa fascia onto the perineum. On the perineum, Co lIes fascia securely attaches laterally to the pubic rami and fascia lata of the thigh, inferiorly to the supericial transverse perineal muscle and inferior border of the perineal membrane, and medially to the urethra, clitoris, and vagina. As such, the superficial space of the anterior triangle is a relatively closed compartment.

1	his supericial pouch contains several important structures, which include the Bartholin glands, vestibular bulbs, clitoral body and crura, branches of the pudendal vessels and nerve, and the ischiocavernosus, bulbospongiosus, and supericial transverse perineal muscles. Of these muscles, the ischiocavernosus muscles each attach on their respective side to the medial aspect of the ischial tuberosity inferiorly and the ischiopubic ramus laterally. Anteriorly, each attaches to a clitoral crus and may help maintain clitoral erection by compressing the crus to obstruct venous drainage. he bilateral bulbospongiosus muscles overlie the vestibular bulbs and Crus of clitoriS Cut edge of ischiocavernous m. Levator ani m. �Gluteus maximus m.

1	Crus of clitoriS Cut edge of ischiocavernous m. Levator ani m. �Gluteus maximus m. FIGURE 2-5 Superficial space of the anterior perineal triangle and posterior perineal triangle. Structures on the left side of the image can be seen after removal of Colles fascia. Those on the right side are noted after removal of the superficial muscles of the anterior triangle. (Modified with permission from Corton MM: Anatomy. In Hoffman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.)

1	Bartholin glands. They attach to the body of the clitoris anteriorly and the perineal body posteriorly. The muscles constrict the vaginal lumen and aid release of secretions from the Bartholin glands. They also may contribute to clitoral erection by compressing the deep dorsal vein of the clitoris. The bulbospongiosus and ischiocavernosus muscles also pull the clitoris downward. Last, the superficial transverse perineal muscles are narrow strips that attach to the ischial tuberosities laterally and the perineal body medially. They may be attenuated or even absent, but when present, they contribute to the perineal body (Corton, 2016).

1	The vestibular bulbs are almond-shaped aggregations of veins that lie beneath the bulbospongiosus muscle on either side of the vestibule. They measure 3 to 4 cm long, 1 to 2 cm wide, and 0.5 to 1 cm thick. The bulbs terminate inferiorly at approximately the middle of the vaginal opening and extend upward toward the clitoris. Their anterior extensions merge in the midline, below the clitoral body. During childbirth, veins in the vestibular bulbs may be lacerated or even rupture to create a vulvar hematoma enclosed within the supericial space of the anterior triangle (Fig. 41-11, p. 765).

1	Deep Space of the Anterior Triangle his space lies deep to the perineal membrane and extends up into the pelvis (Mirilas, 2004). In contrast to the supericial perineal space, the deep space is continuous superiorly with the pelvic cavity (Corton, 2005). It contains portions of urethra and vagina, certain portions of internal pudendal artery branches, and muscles of the striated urogenital sphincter complex (Fig. 2-6).

1	Urethra. The female urethra measures 3 to 4 cm and originates within the bladder trigone (p. 28). he distal two thirds of the urethra are fused with the anterior vaginal wall. The epithelialilining of the urethra changes from transitional epithelium proximally to nonkeratinized stratiied squamous epithelium distally. The walls of the urethra consist of two layers of smooth muscle, an inner longitudinal and an outer circular. This is in turn surrounded by a circular layer of skeletal muscle referred to as the sphincter urethrae or rhabdosphincter (see Fig. 2-6). Approximately at the junction of the middle and lower third of the urethra, and just above or deep to the perineal membrane, two strap skeletal muscles called the urethrovaginal sphincter and compressor urethrae are found. Together with the sphincter urethrae, these constitute the striated urogenital sphincter complex. This complex supplies constant tonus and provides emergency relex contraction to sustain continence.

1	Distal to the level of the perineal membrane, the walls of the urethra consist of ibrous tissue, serving as the nozzle that directs the urine stream. Here, the urethra has a prominent submucosal layer that is lined by hormonally sensitive stratiied squamous epithelium. Within the submucosal layer on the dorsal (vaginal) surface of the urethra lie the paraurethral glands, described earlier (p. 17). he urethra receives its blood supply from branches of the inferior vesical, vaginal, or internal pudendal arteries. Although still controversial, the pudendal nerve is believed to innervate the most distal part of the striated urogenital sphincter complex. Somatic eferent branches from 52-54 that course along the inferior hypogastric plexus variably innervate the sphincter urethrae.

1	Dorsal v. of clitoris Labium minus (cut) Hymeneal ring Sphincter urethrae m.}Striated Compressor urethrae m. urogenital sphincter Urethrovaginal sphincter m. complex Ischiopubic ramus (cut) Perineal membrane Ischiopubic ramus Cut edge of perineal membrane Bulbospongiosus m. (cut) Ischiocavernous m. (cut) Superficial transverse perineal m. External anal sphincter m. Gluteus maximus m. Compressor urethrae m. Urethrovaginal sphincter m. Pubococcygeus }(pubovisceral) m . Pub"ecto'" m. Levator ani mm. . lliococcygeus m."S FIGURE 2-6 Deep space of anterior triangle of the perineum. Structures on the right side of the image can be seen after remova l of the perineal membrane. Also shown are structures that attach to the perineal body: bulbospongiosus, superficial transverse perineal, external anal sphincter, and puboperinealis muscles as well as perineal membrane. (Reproduced with permission from Corton MM: Anatomy. In Hoffman BL, Schorge JO, Bradshaw KD, et al (eds): Gynecology, 3rd ed. New

1	sphincter, and puboperinealis muscles as well as perineal membrane. (Reproduced with permission from Corton MM: Anatomy. In Hoffman BL, Schorge JO, Bradshaw KD, et al (eds): Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.) ·m. External anal sphincter m. Longitudinal smooth m. layer Circular smooth m. layer Plicae transversalis recti

1	Internal anal sphincter m . FIGURE 2-7 Anal canal and ischioanal fossa. (Reproduced with permission from Corton MM: Anatomy. In Hoffman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.) Found deep to the anterior and posterior triangles, this broad muscular sling provides substantial support to the pelvic viscera. The pelvic diaphragm is composed of the levator ani and the coccygeus muscles. The levator ani, in turn, contains the pubococcygeus, puborectalis, and iliococcygeus muscles. The pubococcygeus muscle is also termed the pubovisceral muscle and is subdivided based on points of insertion and function. These include the pubovaginalis, puboperinealis, and puboanalis muscles, which insert into the vagina, perineal body, and anus, respectively (Kearney, 2004).

1	Vaginal birth conveys signiicant risk for damage to the levator ani or to its innervation (DeLancey, 2003; Weidner, 2006). Evidence supports that levator ani avulsion may predispose women to greater risk of pelvic organ prolapse (Dietz, 2008; Schwertner-Tiepelmann, 2012). For this reason, current research eforts are aimed at minimizing these injuries. his triangle contains the ischioanal fossae, anal canal, and anal sphincter complex, which consists of the internal anal sphincter, external anal sphincter, and puborectalis muscle. Branches of the pudendal nerve and internal pudendal vessels are also found within this triangle.

1	Ischioanal Fossae. Also known as ischiorectal fossae, these two fat-filled wedge-shaped spaces are found on either side of the anal canal and comprise the bulk of the posterior triangle (Fig. 2-7). Each fossa has skin as its supericial base, whereas its deep apex is formed by the junction of the levator ani and obturator internus muscles. Other borders include: laterally, the obturator internus muscle fascia and ischial tuberosity; inferomedially, the anal canal and sphincter complex; superomedially, the inferior fascia of the downwardly sloping levator ani; posteriorly, the gluteus maxim us muscle and sacrotuberous ligament; and anteriorly, the inferior border of the anterior tri angle.

1	The fat found within each fossa provides support to surrounding organs yet allows rectal distention during defecation and vaginal stretching during delivery. Clinically, injury to vessels in the posterior triangle can lead to hematoma formation in the ischioanal fossa, and the potential for large accumulation in these easily distensible spaces. Moreover, the two fossae communicate dorsally, behind the anal canal. This can be especially important because an episiotomy infection or hematoma may extend from one fossa into the other. Anal Canal. his distal continuation of the rectum begins at the level of levator ani attachment to the rectum and ends at the anal skin. Along this 4-to 5-cm length, the mucosa consists of columnar epithelium in the uppermost portion. However, at the pectinate line, also termed dentate line, simple stratified squamous epithelium begins and continues to the anal verge. At the verge, keratin and skin adnexa join the squamous epithelium.

1	The anal canal has several tissue layers (see Fig. 2-7). Inner layers include the anal mucosa, the internal anal sphincter, and an intersphincteric space that contains continuation of the rectum's longitudinal smooth muscle layer. An outer layer contains the puborectalis muscle as its cephalad component and the external anal sphincter caudally. Within the anal canal, three highly vascularized submucosal arteriovenous plexuses, termed anal cushions, aid complete closure of the canal and fecal continence when apposed. Increasing uterine size, excessive straining, and hard stool create increased pressure that ultimately leads to degeneration and subsequent laxity of the cushion's supportive connective tissue base. These cushions then protrude into and downward through the anal canal. This leads to venous engorgement within the cushionsnow termed hemorrhoids. Venous stasis results in inflammation, erosion of the cushion's epithelium, and then bleeding. internus m. Pudendal n.

1	internus m. Pudendal n. External hemorrhoids are those that arise distal to the pectinate line. They are covered by stratiied squamous epithelium and receive sensory innevation from the inferior rectal nerve. Accordingly, pain and a palpable mass are typical complaints. Following resolution, a hemorrhoidal tag may remain and is composed of redundant anal skin and ibrotic tissue. In contrast, internal hemorrhoids are those that form above the pectinate line and are covered by insensitive anorectal mucosa. hese may prolapse or bleed but rarely become painful unless they undergo thrombosis or necrosis.

1	Anal Sphincter Complex. Two sphincters surround the anal canal to provide fecal continence-the external and internal anal sphincters. Both lie near the vagina and may be torn during vaginal delivery. he internal anal sphincter (lAS) is a distal continuation of the rectal circular smooth muscle layer. It receives predominantly parasympathetic ibers, which pass through the pelvic splanchnic nerves. long its length, this sphincter is supplied by the superior, middle, and inferior rectal arteries. he lAS contributes the bulk of anal canal resting pressure for fecal continence and relaxes prior to defecation. The lAS measures 3 to 4 cm in length, and at its distal margin, it overlaps the external sphincter for 1 to 2 cm (DeLancey, 1997). he distal site at which this overlap ends, called the intersphincteric groove, is palpable on digital examination.

1	In contrast, the external anal sphincter (EAS) is a striated muscle ring that anteriorly attaches to the perineal body and posteriorly connects to the coccyx via the anococygealiligament. The EAS maintains a constant resting contraction to aid continence, provides additional squeeze pressure when continence is threatened, yet relaxes for defecation. he external sphincter receives Posterior labial n. Ischiocavernosus m. Bulbospongiosus m. perineal m. Perineal n. Pudendal n. sphincter m. Levator ani mm. blood supply from the inferior rectal artery, which is a branch of the internal pudendal artery. Somatic motor ibers from the inferior rectal branch of the pudendal nerve supply innervation. Clinically, the AS and EAS may be involved in third-and fourthdegree lacerations during vaginal delivery, and reunion of these rings is integral to defect repair (Chap. 27, p. 532).

1	This is formed from the anterior rami of S24 spinl nerves. It courses between the piriformis and cocygeus muscles and exits through the greater sciatic foramen at a location posterior to the sacrospinous ligament and just medil to the ischial spine (Barber, 2002; Maldonado, 2015). Thus, when injecting local anesthetic for a pudendal nerve block, the ischial spine serves an identifiable landmark (Chap. 25, p. 489). The pudendal nerve then runs beneath the sacrospinous ligament and above the sacrotuberous ligament as it reenters the lesser sciatic foramen to course along the obturator intern us muscle. Atop this muscle, the nerve lies within the pudendal canal, also known as Alcock canal, which is formed by splitting of the obturator internus investing fascia (Shafik, 1999). In general, the pudendal nerve is relatively fixed as it courses behind the sacrospinous ligament and within the pudendal canal. Accordingly, it may be at risk of stretch injury during downward displacement of the

1	pudendal nerve is relatively fixed as it courses behind the sacrospinous ligament and within the pudendal canal. Accordingly, it may be at risk of stretch injury during downward displacement of the pelvic floor during childbirth (Lien, 2005).

1	he pudendal nerve leaves this canal to enter the perineum and divides into three terminal branches (Fig. 2-8). The first of these, the dorsal nerve of the clitoris, runs between the ischiocavernosus muscle and perineal membrane to supply the clitoral glans (Ginger, 2011 b). Second, the perineal nerve runs supericial to the perineal membrane (Montoya, 201l). It divides into Ilioinguinal & genitofemoral nn. branches Glans & crus of clitoriS sphincter mm. /' Perineal membrane with window that exposes striated urogenital sphincter mm. Dorsal n. & a. of Perineal n. & a. Perineal branch of cutaneous n. Inferior rectal n. & a. Gluteus maximus m.

1	/' Perineal membrane with window that exposes striated urogenital sphincter mm. Dorsal n. & a. of Perineal n. & a. Perineal branch of cutaneous n. Inferior rectal n. & a. Gluteus maximus m. FIGURE 2-8 Pudendal nerve and vessels. (Reproduced with permission from Corton MM: Anatomy. In Hoffman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.) posterior labial branches and muscular branches, which serve the labial skin and the anterior perineal triangle muscles, respectively. Last, the inferior rectal branch runs through the ischioanal fossa to supply the external anal sphincter, the anal mucosa, and the perianal skin (vlahakkanukrauh, 2005). The major blood supply to the perineum is via the internal pudendal artery, and its branches mirror the divisions of the pudendal nerve.

1	The nonpregnant uterus lies in the pelvic cavity between the bladder anteriorly and the rectum posteriorly. Almost the entire posterior wall of the uterus is covered by serosa, that is, visceral peritoneum (Fig. 2-9). The lower portion of this peritoneum forms the anterior boundary of the rectouterine cul-de-sac, or pouch of Douglas. Only the upper portion of the anterior uterine wall is covered by visceral peritoneum. At the caudal border of this portion, the peritoneum relects forward onto the bladder dome to create the vesicouterine pouch. As a result, the lower portion of the anterior uterine wall is separated from the posterior wall of the bladder only by a well-defined loose connective tissue layer-the vesicouterine space. Clinically, during cesarean delivery, the peritoneum of the vesicouterine pouch is sharply incised, and the vesicouterine space is entered. Dissection caudally within this space lifts the bladder safely of the lower uterine segment for hysterotomy and delivery

1	pouch is sharply incised, and the vesicouterine space is entered. Dissection caudally within this space lifts the bladder safely of the lower uterine segment for hysterotomy and delivery (Chap. 30, p. 573).

1	The uterus is pear shaped and consists of two major but unequal parts. The upper, larger portion is the body or corpus, whereas the lower smaller cervix projects into the vagina. The isthmus is the union site of these two. It is of special obstetrical significance because it forms the lower uterine segment during pregnancy. At each superolateral margin of the body is a uterine cornu, from which a fallopian tube emerges. This area also contains the origins of the round and ovarian ligaments. Between the points of fallopian tube insertion is the convex upper uterine segment termed the fundus. The bulk of the uterine body, but not the cervix, is muscle. The inner surfaces of the anterior and posterior walls lie almost in contact, and the cavity between these walls forms a mere slit. The nulligravid uterus measures 6 to 8 cm in length compared with 9 to 10 cm in multiparas. The uterus averages 60 g and typically weighs more in parous women (Langlois, 1970; Sheikhazadi,i2010).

1	Pregnancy stimulates remarkable uterine growth due to muscle iber hypertrophy. The uterine fundus, a previously flattened convexity between tubal insertions, now becomes dome shaped. Moreover, the round ligaments appear to insert at the junction of the middle and upper thirds of the organ. The fallopian tubes elongate, but the ovaries grossly appear unchanged. This portion of the uterus is cylindrical and has small apertures at each end-the internal and external cervical ora. The endocervical canal runs through the cervix and connects these ora. The cervix is divided into upper and lower portions by the vagina's attachment to its outer surface. he upper portionthe portio supravaginalis-begins at the internal os, which corresponds to the level at which the peritoneum is reflected up onto the bladder (Fig. 2-10). The lower cervical portion protrudes into the vagina as the portio vaginalis.

1	Before childbirth, the external cervical os is a small, regular, oval opening. After labor, especially vaginal childbirth, the orifice FIGURE 2-9 Anterior (A), right lateral (8), and posterior (C)views of the uterus of an adult woman. a = oviduct; b = round ligament; = ovarian ligament; Ur ureter. Tubal & ovarian branches of uterine & ovarian aa. External iliac a. Uterine a. Internal iliac a. Inferior epigastric a. & n. vaginalis of cervix FIGURE 2-10 Uterus, adnexa, and associated anatomy. (Reproduced with permission from Corton MM: Anatomy. In Hoffman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.) is converted into a transverse slit that is divided such that there are the so-called anterior and posterior cervical lips. If torn deeply during labor or delivery, the cervix may heal in such a manner that it appears irregular, nodular, or stellate (Fig. 36-1, p. 653).

1	The cervical surface that radially surrounds the external os is called the ectocervix and is lined predominantly by nonkeratinized stratified squamous epithelium. In contrast, the endocervical canal is covered by a single layer of mucin-secreting columnar epithelium, which creates deep cleftlike infoldings or "glands." Commonly during pregnancy, the endocervical epithelium moves out and onto the ectocervix in a physiological process termed eversion (Chap. 4, p. 51). The cervical stroma is composed mainly of collagen, elastin, and proteoglycans, but very little smooth muscle. As described in Chapter 21 (p. 409), changes in the amount, composition, and orientation of these components lead to cervical ripening prior to labor onset. In early pregnancy, increased vascularity within the cervix stroma beneath the epithelium creates an ectocervical blue tint that is characteristic of Chadwick sign. Cervical edema leads to softening-Goodell sign, whereas isthmic softening is Hegar sign.

1	Most of the uterus is composed of myometrium, which contains smooth muscle bundles united by connective tissue with many elastic fibers. Interlacing myometrial ibers surround myometrial vessels and contract to compress these. This anatomy allows hemostasis at the placental site during the third stage of labor. he number of myometrial muscle ibers varies by location (Schwalm, 1966). Levels progressively diminish caudally such that, in the cervix, muscle makes up only 10 percent of the tissue mass. The uterine body's inner wall has relatively more muscle than its outer layers. And, in the anterior and posterior walls, the muscle content is greater than in the lateral walls. During pregnancy, the upper myometrium undergoes marked hypertrophy, but cervical muscle content does not change significantly.

1	The uterine cavity is lined with endometrium, which is composed of an overlying epithelium, invaginating glands, and a supportive, vascular stroma. As discussed in Chapter 5 (p. 83), the endometrium varies greatly throughout the menstrual cycle. This layer is divided into a functionalis layer, which is sloughed with menses, and a basalis layer, which serves to regenerate the functionalis layer following each menses. During pregnancy, the endometrium is termed decidua and undergoes dramatic hormonally driven alterations. Several ligaments extend from the uterine surface toward the pelvic sidewalls and include the round, broad, cardinal, and uterosacral ligaments (Figs. 2-10 and 2-11) . Despite their appellation, the round and broad ligaments provide no substantial uterine support, which contrasts with the cardinal and uterosacral ligaments.

1	he round ligament originates somewhat below and anterior to the origin of the fallopian tubes. Clinically, this orientation can aid fallopian tube identification during puerperal sterilization. External iliac a. & v. Lateral vaginal sulcus Adventitial layer of anterior vaginal wall Rectum

1	Lateral vaginal sulcus Adventitial layer of anterior vaginal wall Rectum FIGURE 2-1 1 Pelvic viscera and their connective tissue support. (Reproduced with permission from Corton MM: Anatomy. In Hoffman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.) his is important if pelvic adhesions limit tubal mobility and thus, hinder fimbria visualization and tubal confirmation prior to ligation. Each round ligament extends laterally and down into the inguinal canal, through which it passes, to terminate in the upper portion of the ipsilateral labium majus. Sampson artery, a branch of the uterine artery, runs within this ligament. In nonpregnant women, the round ligament varies from 3 to 5 mm in diameter and is composed of smooth muscle bundles separated by ibrous tissue septa (Mahran, 1965). During pregnancy, these ligaments undergo considerable hypertrophy and increase appreciably in both length and diameter.

1	The broad ligaments are two winglike structures that extend from the lateral uterine margins to the pelvic sidewalls. Each broad ligament consists of a double-layer drape of peritoneum. The anterior and posterior layers of this drape are termed the anterior and posterior leaves, respectively. In forming the broad ligament, this peritoneum folds over structures extending from each cornu. Peritoneum that folds over the fallopian tube is termed the mesosalpinx, that around the round ligament is the mesoteres, and that over the ovarian ligament is the mesovarium. Peritoneum that extends beneath the imbriated end of the fallopian tube toward the pelvic wall forms the suspensory ligament or the infundibulopelvic ligament of the ovary. This contains nerves and the ovarian vessels, and during pregnancy, these vessels, especially the venous plexuses, are dramatically enlarged. Speciically, the diameter of the ovarian vascular pedicle increases from 0.9 cm to reach 2.6 cm at term (Hodgkinson,

1	these vessels, especially the venous plexuses, are dramatically enlarged. Speciically, the diameter of the ovarian vascular pedicle increases from 0.9 cm to reach 2.6 cm at term (Hodgkinson, 1953).

1	The cardinal ligament-also called the transverse cervicaliligament or Mackenrodt ligament-anchors medially to the uterus and upper vagina. The cardinal ligament is the thick base of the broad ligament. As such, during cesarean hysterectomy, sturdy clamps and suture are required for its transection and ligation. Each uterosacral ligament originates with a posterolateral attachment to the supravaginal portion of the cervix and inserts into the fascia over the sacrum, with some variations (Ramanah, 2012; Umek, 2004). These ligaments are composed of connective tissue, small bundles of vessels and nerves, and some smooth muscle. Covered by peritoneum, these ligaments form the lateral boundaries of the pouch of Douglas. The term parametrium is used to describe the connective tissues adjacent and lateral to the uterus within the broad ligament. Paracervical tissues are those adjacent to the cervix, whereas paracolpium is that tissue lateral to the vaginal walls.

1	During pregnancy, there is marked hypertrophy of the uterine vasculature, which is supplied principally from the uterine and ovarian arteries (see Fig. 2-10). The uterine artery, a main branch of the internal iliac artery-previously called the hypogastric artery-enters the base of the broad ligament. he uterine artery courses medially to the lateral side of the uterus. Approximately 2 cm lateral to the cervix, the uterine artery crosses over the ureter. This proximity is of great surgical signiicance, as the ureter may be injured or ligated during hysterectomy when the uterine vessels are clamped and ligated.

1	Once the uterine artery has reached the supravaginal portion of the cervix, it divides. The smaller cervicovaginal artery supplies blood to the lower cervix and upper vagina. The main uterine artery branch rurns abruptly upward and travels cephalad along the lateral margin of the uterus. Along its path, this main artery provides a branch of considerable size to the upper cervix and then numerous other medial branches serially penetrate the body of the uterus to form the arcuate arteries. As indicated by the name, each branch arches across the organ by coursing within the myometrium just beneath the serosal surface. Arcuate vessels from each side anastomose at the uterine midline. Radial artery branches originate at right angles from the arcuate arteries and travel inward through the myometrium, enter the endometrium/decidua, and branch there to become either basal arteries or coiled spiral arteries. he spiral arteries supply the funcrionalis layer. Also called the straight arteries,

1	enter the endometrium/decidua, and branch there to become either basal arteries or coiled spiral arteries. he spiral arteries supply the funcrionalis layer. Also called the straight arteries, the basal arteries extend only into the basalis layer.

1	As the uterine artery courses cephalad, it gives rise to Sampson artery of the round ligament. Just before the main uterine artery vessel reaches the fallopian rube, it divides into three terminal branches. The ovarian branch of the uterine artery forms an anastomosis with the terminal branch of the ovarian artery; Internal iliac a. the rubal branch makes its way through the mesosalpinx and supplies part of the fallopian tube; and the fundal branch penetrates the uppermost uterus.

1	Internal iliac a. the rubal branch makes its way through the mesosalpinx and supplies part of the fallopian tube; and the fundal branch penetrates the uppermost uterus. In addition to the uterine artery, the uterus receives blood supply from the ovarian artery (see Fig. 2-10). This artery is a direct branch of the aorta and enters the broad ligament through the infundibulopelvic ligament. At the ovarian hilum, it divides into smaller branches that enter the ovary. As the ovarian artery runs along the hilum, it also sends several branches through the mesosalpinx to supply the fallopian rubes. Its main stem, however, traverses the entire length of the broad ligament toward the uterine cornu. Here, it forms an anastomosis with the ovarian branch of the uterine artery. This dual uterine blood supply creates a vascular reserve to prevent uterine ischemia if ligation of the uterine or internal iliac artery is performed to control postpartum hemorrhage.

1	Uterine veins accompany their respective arteries. As such, the arcuate veins unite to form the uterine vein, which empties into the internal iliac vein and then the common iliac vein. Some of the blood from the upper uterus, the ovary, and the upper part of the broad ligament is collected by several veins. Within the broad ligament, these veins form the large pampiniform plexus that terminates in the ovarian vein. From here, the right ovarian vein empties into the vena cava, whereas the left ovarian vein empties into the left renal vein. Ureter Inferior mesenteric a. Ovarian a. External iliaca. Iliolumbar a. Obturatora. Accessory obturator a. Superior rectal a. Inferior epigastric a. Round ligament (cut) Lateralsacral a. Medial umbilical ligament Superior gluteal a. Superior vesical aa. Inferior gluteal a. Inferior vesical a. Vaginal a. Internal pudendala. Uterine a. (ascending branch) Sampson a.toround ligament Uterus

1	Superior vesical aa. Inferior gluteal a. Inferior vesical a. Vaginal a. Internal pudendala. Uterine a. (ascending branch) Sampson a.toround ligament Uterus FIGURE 2-12 Pelvic arteries. (Reproduced with permission from Corton MM: Anatomy. In Hoffman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.)

1	Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.) Blood supply to the pelvis is predominantly provided by branches of the internal iliac artery (Fig. 2-12). These branches are organized into anterior and posterior divisions, and subsequent branches are highly variable between individuals. The anterior division provides blood supply to the pelvic organs and perineum and includes the inferior gluteal, internal pudendal, middle rectal, vaginal, uterine, and obturator arteries, as well as the umbilical artery and its continuation as the superior vesical artery. The posterior division branches extend to the buttock and thigh and include the superior gluteal, lateral sacral, and iliolumbar arteries. For this reason, during internal iliac artery ligation, many advocate ligation distal to the posterior division to avoid compromised blood low to the areas supplied by this division (Bleich, 2007).

1	The lymphatics from the uterine corpus are distributed to two groups of nodes. One set of vessels drains into the internal iliac nodes. The other set, after joining lymphatics from the ovarian region, terminates in the paraaortic lymph nodes. Lymphatics from the cervix terminate mainly in the internal iliac nodes, which are situated near the bifurcation of the common iliac vessels. As a brief review, the peripheral nevous system is divided into a somatic division, which innervates skeletal muscle, and an autonomic division, which innervates smooth muscle, cardiac muscle, and glands. Pelvic visceral innervation is predominantly autonomic, which is further divided into sympathetic and parasympathetic components.

1	Sympathetic innervation to pelvic viscera begins with the superior hypogastric plexus, also termed the presacral nerve (Fig. 2-13). Beginning below the aortic bifurcation and extending downward retroperitoneally, this plexus is formed by sympathetic fibers arising from spinal levels TID through L2• At the level of the sacral promontory, this superior hypogastric plexus divides into a right and a left hypogastric nerve, which run downward along the pelvis sidewalls (Ripperda, 20i1i5). Fibers to vestibular bulbs FIGURE 2-13 Pelvic innervation. (Reproduced with permission from Corton MM: Anatomy. In Hofman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.)

1	Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.) In contrast, parasympathetic innervation to the pelvic viscera derives from neurons at spinal levels 52 through 54' heir axons exit as part of the anterior rami of the spinal nerves for those levels. These combine on each side to form the pelvic splanchnic nerves, also termed nervi erigentes.

1	Blending of the two hypogastric nerves (sympathetic) and the two pelvic splanchnic nerves (parasympathetic) gives rise to the inferior hypogastric plexus, also termed the pelvic plexus. his retroperitoneal plaque of nerves lies at the 54 and 55 level (Spackman, 2007). From here, fibers of this plexus accompany internal iliac artery branches to their respective pelvic viscera. Thus, the inferior hypogastric plexus divides into three plexuses. he vesical plexus innervates the bladder, and the middle rectal plexus travels to the rectum. he uterovaginal plexus, also termed F rankenhauser plexus, reaches the proximal fallopian tubes, uterus, and upper vagina. Extensions of the inferior hypogastric plexus also reach the perineum along the vagina and urethra to innervate the clitoris and vestibular bulbs (Montoya, 2011). Of these, the uterovaginal plexus is composed of variably sized ganglia, but particularly of a large ganglionic plate that is situated on either side of the cervix,

1	vestibular bulbs (Montoya, 2011). Of these, the uterovaginal plexus is composed of variably sized ganglia, but particularly of a large ganglionic plate that is situated on either side of the cervix, proximate to the uterosacral and cardinal ligaments (Ramanah, 2012).

1	For the uterus, most of its aferent sensory fibers ascend through the inferior hypogastric plexus and enter the spinal cord via T 10 through T 12 and L] spinal nerves. These transmit the painful stimuli of contractions to the central nervous system. For the cervix and upper part of the birth canal, sensory nerves pass through the pelvic splanchnic nerves to the second, third, and fourth sacral nerves. Last, those from the lower portion of the birth canal pass primarily through the pudendal nerve. Anesthetic blocks used during delivery target these levels of innervation. long the pelvic sidewall, each ovary usually rests in the ovarian fossa ofWaldeyer, which is a slight depression between the external and internal iliac vessels. During childbearing years, ovaries variably measure 2.5 to 5 cm in length, 1.5 to 3 cm in width, and 0.6 to 1.5 cm in thickness.

1	he ovarian ligament, also called the uteroovarian ligament, originates from the upper posterolateral portion of the uterus, just beneath the tubal insertion level, and extends to the uterine pole of the ovary (see Fig. 2-10). Measuring a few centimeters long and 3 to 4 mm in diameter, this ligament is made up of muscle and connective tissue and is covered by peritoneum-the mesovarium. Blood supply reaches the ovary through this double-layered mesovarium to enter the ovarian hilum. The ovary consists of an outer cortex and inner medulla. In young women, the cortex is smooth, has a dull white surface, and is lined by single layer of cuboidal epithelium, the germinal epithelium ofWaldeyer. his epithelium is supported by a connective tissue condensation, the tunica albuginea. Beneath this, the ovarian cortex contains oocytes and developing follicles. The medulla is composed of loose connective tissue, numerous arteries and veins, and a small amount of smooth muscle ibers.

1	The ovaries are supplied with both sympathetic and parasympathetic nerves. he sympathetic nerves are derived primarily from the ovarian plexus that accompanies the ovarian vessels and originates in the renal plexus. Others are derived from the plexus that surrounds the ovarian branch of the uterine artery. Parasympathetic input is from the vagus nerve. Sensory aferents follow the ovarian artery and enter at T]o spinal cord level.

1	Also called oviducts, these serpentine tubes extend laterlly 8 to 14 cm from the uterine cornua. hey are anatomically classified along their length s an interstitial portion, isthmus, ampulla, and inundibulum (Fig. 2-14). vIost proximal, the interstitial portion is embodied within the uterine muscular wall. Next, the narrow 2-to 3-mm wide isthmus widens gradually into the 5-to 8-mm wide ampulla. Last, the inundibulum is the unnel-shaped fimbriated distal extremity of the tube, which opens into the abdominal cavity. These latter three extrauterine portions are covered by the mesosalpinx at the superior margin of the broad ligament.

1	In cross section, the extrauterine fallopian tube contains a mesosalpinx, myosalpinx, and endosalpinx. he outer of these, the mesosalpinx, is a single-cell mesothelial layer unctioning as visceral peritoneum. In the myosalpinx, smooth muscle is arranged in an inner circular and an outer longitudinal layer. The tubal musculature undergoes rhythmic contractions constantly, the rate of which varies with cyclical ovarian hormonal changes.

1	The tubal mucosa or endosalpinx is a single layer of columnar epithelium composed of ciliated, secretory, and intercalary cells resting on a sparse lamina propria. Clinically, its close proximity to the underlying myosalpinx contributes to easy invasion by ectopic trophoblast. The tubal mucosa is arranged in longitudinal folds that become progressively more complex toward the fimbria. In the ampulla, the lumen is occupied almost completely by the arborescent mucosa. The current produced by the tubal cilia is such that the direction of flow is toward the uterine cavity. Tubal peristalsis created by cilia and muscular layer contraction is believed to be an important factor in ovum transport (Croxatto, 2002).

1	he tubes are supplied richly with elastic tissue, blood vessels, and lymphatics. Their sympathetic innervation is extensive, in contrast to their parasympathetic innervation. This nerve supply derives partly from the ovarian plexus and partly from the uterovaginal plexus. Sensory aferent ibers ascend to T]o spinal cord levels. Anteriorly, the bladder rests against the inner surface of the pubic bones and then, as it fills, also against the anterior abdominal wall. Posteriorly, it rests against the vagina and cer vix. he bladder is divided into a dome and a base approximately at the level of the ureteral oriices. The dome is thin walled and distensible, whereas the base is thicker and undergoes less distention during filling. The vesical trigone lies in the bladder base and contains both ureteral oriices and the internal

1	A B FIGURE 2-14 The fallopian tube of an adult woman with cross-sectioned illustrations of the gross structure in several portions: (A) isthmus, (B) ampUlla, and (C) infundibulum. Below these are photographs of corresponding histological sections. (Used with permission from Dr. Kelley S. Carrick.) urinary meatus (see Fig. 2-11). he urethral lumen begins at this meatus and then courses through the bladder base for less than 1 cm. This region where the urethral lumen traverses the bladder base is called the bladder neck. The bladder wall consists of coarse bundles of smooth muscle known as the detrusor muscle, which extends into the proximal part of the urethra. A submucosal layer intervenes between this detrusor muscle and the mucosa. he bladder mucosa consists of transitional epithelium and underlying lamina propria.

1	The blood supply to the bladder arises from the superior vesical arteries, which are branches of the patent portion of the umbilical artery, and from the middle and inferior vesical arteries, which, when present, often arise from either the internal pudendal or the vaginal arteries (see Fig. 2-12). The nerve supply to the bladder arises from the vesical plexus, a component of the inferior hypogastric plexus (see Fig. 2-13).

1	As the ureter enters the pelvis, it crosses over the bifurcation of the common iliac artery and passes just medial to the ovarian vessels (see Fig. 2-10). As the ureter descends into the pelvis, it lies medial to the internal iliac branches and anterolateral to the uterosacral ligaments. The ureter then traverses through the cardinal ligament approximately 1 to 2 cm lateral to the cervix. Near the level of the uterine isthmus, it courses below the uterine artery and travels anteromedially toward the bladder base. In this path, it runs close to the upper third of the anterior vaginal wall (Rahn, 2007). Finally, the ureter enters the bladder and travels obliquely for approximately 1.5 cm before opening at the ureteral oriices.

1	The pelvic ureter receives blood supply from the vessels it passes: the common iliac, internal iliac, uterine, and superior vesical vessels. The ureter's course runs medial to these vessels, and thus its blood supply reaches the ureter from lateral sources. This is important during ure teral isolation. Vascular anastomoses on the connective tissue sheath enveloping the ureter form a longitudinal nenvork of vessels. The pelvis is composed of four bonesthe sacrum, coccyx, and two innominate bones. Each innominate bone is formed by the fusion of three bones-the ilium, ischium, and pubis (Fig. 2-15). Both innominate bones are joined to the sacrum at the sacroiliac synchondroses and to one another at the symphysis pubis.

1	Anteriorly, the pelvic bones are joined together by the symphysis pubis. This structure consists of fibrocartilage and the superior and inferior pubic ligaments. The latter ligament is frequently designated the arcuate ligament of the pubis. Posteriorly, the pelvic bones are joined by articulations between the sacrum and the iliac portion of the innominate bones to form the sacroiliac joints.

1	The pelvic joints in general have a limited degree of mobility. However, during pregnancy, these joints relax remarkably at term. As one result, upward gliding of the sacroiliac joint, which is greatest in the dorsal lithotomy position, may increase the diameter of the outlet by 1.5 to 2.0 cm for delivery (Borell, 1957). Sacroiliac joint mobility also likely aids the McRoberts maneuver to release an obstructed shoulder in cases of shoulder dystocia (Chap. 27, p. 521). These changes may also contribute to the success of the modified squatting position to hasten second-stage labor (Cardosi, 1989). The squatting position may increase the interspinous diameter and the pelvic outlet diameter (Russell, 1969, 1982).

1	FIGURE 2-15 The innominate bone is composed of the pubis (brown), ischium (red), and ilium (blue). Of the three anteroposterior diameters of the pelvic inlet, only the diagonal conjugate can be measured clinically. The important obstetrical conjugate is derived by subtracting 15 cm from the diagonal conjugate. • Planes and Diameters of the Pelvis The pelvis is conceptually divided into false and true components. The false pelvis lies above the linea terminalis, and the true pelvis is below this boundary (Fig. 2-16). he false pelvis is bounded posteriorly by the lumbar vertebra and laterally by the iliac fossa. In front, the boundary is formed by the lower portion of the anterior abdominal wall. he pelvis is described as having four imaginary planes: 1. The plane of the pelvic inlet-the superior strait. 2. The plane of the pelvic outlet-the inferior strait. 3. he plane of the midpelvis-the least pelvic dimensions. 4.

1	The plane of the pelvic inlet-the superior strait. 2. The plane of the pelvic outlet-the inferior strait. 3. he plane of the midpelvis-the least pelvic dimensions. 4. he plane of greatest pelvic dimension-of no obstetrical significance. he pelvic inlet, also called the superior strait, is the superior plane of the true pelvis. It is bounded posteriorly by the promontory and alae of the sacrum, laterally by the linea terminalis, and anteriorly by the horizontal pubic rami and the symphysis pubis. During labor, fetal head engagement is deined by the fetal head's biparietal diameter passing through this plane.

1	Four diameters of the pelvic inlet are usually described: anteroposterior, transverse, and two oblique diameters. Of these, distinct anteroposterior diameters have been described using specific landmarks. Most cephalad, the anteroposterior diameter, termed the true conjugate, extends from the uppermost margin of the symphysis pubis to the sacral promontory (see Fig. 2-15). he clinically important obstetrical conjugate is the shortest distance between the sacral promontory and the symphysis pubis. Normally, this measures 10 cm or more, but unfortunately, it cannot be measured directly with examining fingers. hus, the obstetrical conjugate is estimated indirectly by subtracting 1.5 to 2 cm from the diagonal conjugate. To measure the diagonal conjugate, a hand with the palm oriented laterally extends its index finger to the promontory. The distance from the fingertip to the point at which the lowest margin of the symphysis strikes the same inger's base is the diagonal conjugate.

1	he transverse diameter is constructed at right angles to the obstetrical conjugate and represents the greatest distance between the linea terminalis on either side (see Fig. 2-16). It usually intersects the obstetrical conjugate at a point approximately 5 cm in front of the promontory and measures approximately 13 cm. The midpelvis is measured at the level of the ischial spines, also called the midplane or plane of least pelvic dimensions (see Fig. 2-16). During labor, the degree of fetal head descent into the true pelvis may be described by station, and the midpelvis and ischial spines serve to mark zero station. The interspinous diameter is 10 cm or slightly greater and is usually the smallest pelvic diameter. The anteroposterior diameter through the level of the ischial spines normally measures at least 11.5 cm.

1	The pelvic outlet consists of two approximately triangular areas whose boundaries mirror those of the perineal triangle described earlier (p. 19). They have a common base, which is a line drawn between the two ischial tuberosities. The apex of the posterior triangle is the tip of the sacrum, and the (, .Iium FIGURE 2-16 Axial view of a normal female pelvis. The clinically important obstetrical conjugate and transverse diameter of the pelvic inlet are illustrated. The interspinous diameter of the mid pelvis is also marked. Anthropotd ,.,• Intermediates • .. 4 FIGURE 2-17 The four parent pelvic types of the Caldwell-Moloy classification. A line passing through the widest transverse diameter divides the inlets into posterior (P) and anterior (A) segments.

1	FIGURE 2-17 The four parent pelvic types of the Caldwell-Moloy classification. A line passing through the widest transverse diameter divides the inlets into posterior (P) and anterior (A) segments. lateral boundaries are the sacrotuberous ligaments and the ischial tuberosities. 'he anrerior triangle is formed by the descending inferior rami of rhe pubic bones. These rami unite at an angle of 90 [0 100 degrees ro form a rounded arch under which the fetal head must pass. Unless there is Signiicant pelvic bony disease, rhe pelvic ourler seldom obstructs vaginal delivery.

1	The Caldwell-Moloy (I933, 1934) anaromical classiication of rhe pelvis is based on shape, and its concepts aid an understanding of labor mechanisms. Speciically, the grearesr transverse diameter of rhe inler and its division into alHerior and posterior segmelHs are used to classiy rhe pelvis as gynecoid, anthropoid, android, or platypelloid. The posterior segmelH determines the type of pelvis, whereas the anterior segment determines the tendency. These are both determined because many pelves are nor pure but are mixed types. For example, a gynecoid pelvis with an android tendency means that rhe posterior pelvis is gynecoid and the anterior pelvis is android shaped. From viewing the four basic types in 2-17I, the coniguration of the gynecoid pelvis would intuitively seem suited for delivery of most fetuses. Indeed, Caldwell (I 939) reported [hac rhe gynecoid pelvis was found in almost half of women.

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1	Ginger VA, Cold j, Yang CC: Surgical anatomy of the dorsal nerve of the clitoris. Neuroural Urodyn 30(3):412, 2011 b Hawkins JL: Anesthesia for the pregnant woman. In Yeomans ER, Hofman BL, Gilstrap LC Ill, et al: Cunningham and Gilsuaps's Operative Obstetrics, 3rd ed. New York, McGraw-Hill Education, 2017 Hodgkinson CP: Physiology of the ovarian veins during pregnancy. Obstet Gynecol 1 (1):26, 19S3 Kearney R, Sawhney R, Delancey )0: Levator ani muscle anatomy evaluated by origin-insertion pairs. Obstet Gynecol 104:168,e2004 Kim SO, Oh K), Lee HS, et al: Expression of aquaporin water channels in the vagina in premenopausal women.e) Sex Med 8(7):I92S, 2011 Langlois PL: The si'Le of the normal uterus.e) Reprod Med 4:220, 1970

1	Langlois PL: The si'Le of the normal uterus.e) Reprod Med 4:220, 1970 Larson KA, Yousuf A, Lewicky-Gaupp C, et al: Perineal body anatomy in living women: 3-dimensional analysis using thin-slice magnetic resonance imaging. Am J Obstet GynecoI203(S):494.elS, 2010 ien KC, Morgan OM, Delancey )0, et al: Pudendal nerve stretch during vaginal birth: a 3D computer simulation. m J Obstet Gynecol 192(S): 1669, 200S Lloyde). Crouch NS, Minto CL, et al: Female genital appearance: "normality" unfold" BjOG 112(5),643,e2005 Loukas M, r,1yers C, Shah R, et al: Arcuate line of the rectus sheath: clinical approach. Anat Sci Int 83(3):140, 2008 Mahakkanukrauh p, Surin p, Vaidhayakarn P: Anatomical study of the pudendal nerve adjacent to the sacrospinous ligament. Clin Anat 18:200, 200S Mahran '1: The microscopic anatomy of the round ligament. J Obstet Gynae col Br Commomv 72:614, 1965

1	Maldonado rA, Chin K, Garcia A, et 1: Anatomic variations of pudendal nerve within pelvis and pudendal canal: clinical applications. m ) Obstet GynecoIe213(S):727,e20IS Mei W, lin C, Feng L, et al: Bilateral ultrasound-guided transversus abdominis plane block combined with iliOinguinal-iliohypogastric nerve block for cesarean delivey anesthesia. Anesth Analg 113(1):134,2011 Mirilas p, Skandalakis )E: Urogenital diaphragm: an erroneous concept casting its shadow over the sphincter urethrae and deep perineal space. J m Coli Surg 198:279,e2004 Montoya TI, Calver L, Carrick S, et al: Anatomic relationships of the pudendal nerve branches. Am J Obstet Gynecol 20S(S):S04.el, 2011 Rahn DO, Bleich AT, Wai CY, et al: Anatomic relationships of the distal third of the pelvic ureter, trigone, and urethra in unembalmed female cadavers. Am J Obstet Gynecol 197(6):668.e 1, 2007

1	Rahn DO, Bleich AT, Wai CY, et al: Anatomic relationships of the distal third of the pelvic ureter, trigone, and urethra in unembalmed female cadavers. Am J Obstet Gynecol 197(6):668.e 1, 2007 Rahn DD, Phelan IN, Roshanravan SM, et al: Anterior abdominal wall nerve and vessel anatomy: clinical implications for gynecologic surgery. m ) Obstet Gyneol 202(3):234.el, 2010 Ramanah R, Berger MB, Parrarre BM, et al: Anatomy and histology of apical support: a literature review concerning cardinal and uterosacral ligaments. Int Urogynecole) 23(11):e1483, 2012 Ripperda CM, Jackson A, Phelan )N, et al: Anatomic relationships of the pelvic autonomic nervous system in female cadavers: clinical applications to pelvic surgery. Oral presentation at AUGS Annual Scientiic Meeting, 13-17 October, 20lS Russell )G: iVloulding of the pelvic outlet. J Obstet Gynaecol Br Commonw 76:817.e1969 Russell JG: The rationale of primitive delivery positions. B)OG 89:712, 1982

1	Russell )G: iVloulding of the pelvic outlet. J Obstet Gynaecol Br Commonw 76:817.e1969 Russell JG: The rationale of primitive delivery positions. B)OG 89:712, 1982 Santoro GA, Shobeiri SA, Petros PP, et al: Perineal body anatomy seen by three-dimensional endovaginal ultrasound of asymptomatic nulliparae. Colorectal Dis 18(4):400, 2016 Schober J, A.lrdsma N, lvlayoglou L, et al: Terminal innervation of female genitalia, cutaneous sensory receptors of the epithelium of the labia minora. Clin Anat 28(3):392, 20lS Schwalm H, Dubrauszky V: The structure of the musculature of the human uterus-muscles and connective tissue. m) Obstet Cynecol 94:391, 1966 Schwertner-Tiepelmann N, Thakar R, Sultan AH, et al: Obstetric levator ani muscle injuries: current status. Ultrasound Obstet GynecoI39(4):372, 2012 Shaik A, Doss SH: Pudendal canal: surgical anatomy and clinical implications. m Surg 6S: 176, 1999

1	m Surg 6S: 176, 1999 Shaik A, Sibai OE, Shaik A, et al: A novel concept for the surgical anatomy of the perineal body. Dis Colon Rectum SO(l2):2120, 2007 Sheikhazadi A, Sadr SS, Ghadyani MH, et al: Study of the normal internal organ weights in Tehran's population.) Forensic Leg Med 17(2):78, 2010 Spackman R, Wrigley B, RobertS A, et al: The inferior hypogastriC plexus: a diferent view.) Obstet Gynaecoi 27(2):130, 2007 Tawik MM, Mohamed YM, Elbadrawi E, et al: Transversus abdominis plane block versus wound iniltration for analgeSia after cesarean delivery: a randomized controlled trial. Anesth Analg 124(4):1291, 2017 Tolcher :lC, Nitsche)F, Arendt f, et al: Spontaneous rectus sheath hematoma pregnancy: case report and revie' of the literature. Obstet Cynecol Su� 65(8),517, 2010 Umek H, Morgan DM, Ashton-Miller )A, et al: Quantitative analysis of uterosacral ligament origin and insertion poims by magnetic resonance imaging. Obstet Gynecol 103:447,e2004

1	Umek H, Morgan DM, Ashton-Miller )A, et al: Quantitative analysis of uterosacral ligament origin and insertion poims by magnetic resonance imaging. Obstet Gynecol 103:447,e2004 VerkaufBS, Von Thron J, O'Brien WF: Clitoral size in normal women. Obstet Gynecol 80(1):41, 1992 Wai C, Bhatia K, Clegg I: RecTUS sheath haematoma: a rare cause of abdominal pain in pregnancy. 1m) Obstet Anesth 24(2): 194, 2015 Weber M, Walters MD: Anterior vaginal prolapse: review of anatomy and techniques of surgical repair. Obstet Gynecol 89:311, 1997 (leidner AC, Jamison MG, Branham V, et 1: Neuropathic injury to the levator ani occurs in I in 4 primiparous women. m J Obstet Gynecole19S: 18SI, 2006 Whiteside JL, Barber MD: IIlioinguinalliliohypogastric neurectomy for management of inrractable right lower quadrant pain ater cesarean section: a case report. J Reprod IvIed SO(l 1):8S7, 200S

1	Whiteside JL, Barber MD: IIlioinguinalliliohypogastric neurectomy for management of inrractable right lower quadrant pain ater cesarean section: a case report. J Reprod IvIed SO(l 1):8S7, 200S Whiteside JL, Barber MD, Walters MD, et 1: Anatomy of ilioinguinal and iliohypogastric nerves in relation to trocar placement and low transverse incisions. m) Obsret Gynecol 189: 1574, 2003 Wilkinson E), Massoll NA: Benign diseases of the vulva. In Kurman R), Ellenson LH, Ronnen BM (eds): Blaustein's Pathology of the Female Genital Tract, 6th ed. New York, Springer, 2011, p 3 Wolfson A, Lee A), Wong P, et al: Bilateral multi-injection iliohypogastricilioinguinal nerve block in conjunction with neuraxial morphine is superior to neuraxial morphine alone for postcesarean analgeSia. J Clin Anesth 24(4),298,e2012 GENITOURINARY TRACT DEVELOPMENT ..... . .. 33 SEXUAL DIFFERENTIATION .. . ..... ............. 38..... DISORDERS OF SEX .... . ... . 38..........

1	GENITOURINARY TRACT DEVELOPMENT ..... . .. 33 SEXUAL DIFFERENTIATION .. . ..... ............. 38..... DISORDERS OF SEX .... . ... . 38.......... BLADDER AND PERINEAL ABNORMALITIES ...... , ... 41 MULLERIAN ABNORMALITIES .... .. ..... ....... 41...... UTERINE FLEXION ......... , .............. , .. , ... 46 Abnormalities in the development or fusion of one or both MUllerian ducts may result in maormations which sometimes possess an obstetrical signicance. Pregnancy may be associated with anyione of these maormations, provided an ovum be cast of rom the ovaries and no serious obstacle be opposed to the upward passage of the spermatozoa and their subsequent union with it. -]. Whitridge Williams (1903)

1	-]. Whitridge Williams (1903) In females, the external genitalia, gonads, and miillerian ducts each derive from diferent primordia and in close association with the urinary tract and hindgut. Abnormal embryogenesis during this process is thought to be multifactorial and can create sporadic anomalies. Several of these can lead to infertility, subfertility, miscarriage, or preterm delivery. Thus, knowledge of genitourinary system development is essential. • Embryology of the Urinary System Between the 3rd and 5th gestational weeks, an elevation ofintermediate mesoderm on each side of the fetus-the urogenital ridge-begins development into the urogenital tract. Subsequently, the urogenital ridge divides into the genital ridge, destined to become the ovary, and into the nephrogenic ridge (Fig. he nephrogenic ridges develop into the mesonephros (mesonephric kidney) and paired mesonephric ducts, also termed wolian ducts, which connect to the cloaca.

1	he nephrogenic ridges develop into the mesonephros (mesonephric kidney) and paired mesonephric ducts, also termed wolian ducts, which connect to the cloaca. The early urinary tract develops from the mesonephros and its mesonephric ducts (Fig. 3-2A). Recall that evolution of the renal system passes sequentially through the pronephric and mesonephric stages to reach the permanent metanephric system. Between the 4th and 5th weeks, each mesonephric duct gives rise to a ureteric bud, which grows cephalad toward its respective mesonephros (Fig. 3-2B), As each bud lengthens, it induces diferentiation of the metanephros, which will become the inal kidney (Fig. 3-2C). Each mesonephros degenerates near the end of the first trimester, and without testosterone, the mesonephric ducts regress as well.

1	he cloaca begins as a common opening for the embryonic urinary, genital, and alimentary tracts. By the 7th week it becomes divided by the urorectal septum to create the rectum and the urogenital sinus (Fig. 3-2D). The urogenital sinus is considered in three parts: (1) the cephalad or vesicle portion, which forms the urinary bladder; (2) the middle or pelvic portion, which creates the female urethra; and (3) the caudal or phallic part, which gives rise to the distal vagina and to the greater vestibular (Bartholin) and paraurethral glands.

1	• Embryology of the Genital Tract he fallopian tubes, uterus, and upper vagina derive from the miillerian ducts, also termed paramesonephric ducts, which form adjacent to each mesonephros (see Fig. 3-2B). These ducts extend downward and then turn medially to meet and fuse together in the midline. he uterus is formed by this union of the two miillerian ducts at approximately the 10th week (Fig. 3-2E). Fusion to create the uterus begins in the middle and then extends both caudally and cephalad. With cellular FIGURE 3-2 Embryonic development of the female genitourinary tract (A-F). (Reproduced with permission from Shatzkes DR, Haller JO, Velcek FT: Imaging of uterovaginal anomalies in the pediatric patient, Urol Radiol 1991;13(1):58-66.)

1	FIGURE 3-1 A. Cross-section of an embryo at 4 to 6 weeks. B. Large ameboid primordial germ cells migrate (arrows) from the yolk sac to the area of germinal epithelium, within the genital ridge. C. Migration of sympathetic cells from the spinal ganglia to a region above the developing kidney. Remnant of mesonephric duct Bladder-Urethra/ F Ureter -...--Uterus ---Rectum proliferation at the upper portion, a thick wedge of tissue cre ates the characteristic piriform uterine shape. At the same time, dissolution of cells at the lower pole forms the irst uterine cav ity (Fig. 3-2F). As the upper wedge-shaped septum is slowly reabsorbed, the inal uterine cavity is usually formed by the 20th week. If the two mullerian ducts fail to fuse, then two separate uterine horns remain. In contrast, resorption failure of the common tissue between them results in various degrees of persistent uterine septum.

1	As the distal end of the fused mullerian ducts contacts the uro genital sinus, this induces endodermal outgrowths from the sinus termed the sinovaginal bulbs. hese bulbs proliferate and fuse to form the vaginal plate, which later resorbs to form the vaginal lumen. his vaginal canalization is generally completed by the 20th week. However, the lumen remains separated from the urogenital sinus by the hymeneal membrane. This membrane further degenerates to leave only the hymeneal ring.

1	The close association of the mesonephric (wolian) and paramesonephric (mullerian) ducts explains the simultaneous abnormalities in their end organs. Kenney and colleagues (1i984) showed that up to half of females with urerovaginal malformations have associated urinary tract defects. Anomalies most frequently associated with renal defects are unicornuate uterus, uterine didelphys, and agenesis syndromes, whereas arcuate and bicornuate are less commonly linked (Reichman, 2010). When mullerian anomalies are identiied, the urinary system can be evaluated with magnetic resonance (MR) imaging, sonography, or intravenous pyelography (Hall-Craggs, 2013). With mullerian anomalies, ovaries are functionally normal but have a higher incidence of anatomical maldescent into the pelvis (Allen, 2012; Dabirashrafi, 1994).

1	As discussed, the mesonephric ducts usually degenerate, however, persistent remnants may become clinically apparent. Mesonephric or wolian vestiges can persist as Gartner duct cysts. These are typically located in the proximal anterolateral vaginal wall but may be found at other sites along the vaginal length. They can be further characterized by MR imaging, which provides excellent image resolution at soft tissue interfaces. Most cysts are asymptomatic and benign and usually do not require surgical excision. Intraabdominal wolian remnants in the female include a few blind tubules in the mesovarium-the epoophoronand similar ones adjacent to the uterus-paroophoron (see Fig. 3-2F) (Moore, 2013). The epoophoron or paroophoron may develop into clinically identiiable cysts in the adult. • Embryology of the Gonads

1	• Embryology of the Gonads At approximately 4 weeks, gonads derive from coelomic epithelium covering the medial and ventral surface of the nephrogenic cord at a site between the eighth thoracic and fourth lumbar segments. Because of this separate gonadal and mullerian derivation, women with mullerian defects typically have functionally normal ovaries and are phenotypic females. he coelomic epithelium thickens to form the genital ridge, also known as the gonadal ridge. Strands of these epithelial cells extend into the underlying mesenchyme as the primary sex cords. By the sixth week, primordial germ cells have migrated from the yolk sac to enter the genital ridge mesenchyme (Fig. 3-3). The primordial germ cells are then incorporated into the primary sex cords.

1	In the seventh week, the sexes can be distinguished, and tes tes are recognized during microscopic sectioning by their well deined radiating testis cords. These cords are separated from the coelomic epithelium by mesenchyme that is to become the tunica albuginea. The testis cords develop into the seminiferous tubules and rete testis. he rete testis establishes connection with small tubes arising of the mesonephric duct. These small tubes become the eferent ducts that drain into the epididymis and then into the vas deferens, which are main mesonephric duct derivatives.

1	In the female embryo, the primary sex cords give rise to the medullary cords, which persist only for a short time. The coelomic epithelium again proliferates into the underlying mesenchyme, and these strands are the cortical cords. By the fourth month, the cortical cords begin to form isolated cell clusters called primordial follicles. These follicles contain the oogonia, which derive from primordial germ cells and are surrounded by a single layer of lattened follicular cells derived from the cortical cords. Follicular cells serve as supporting nutrient cells. By 8 months, the ovary has become a long, narrow, lobulated structure that is attached to the body wall by the mesovarium. The coelomic epithelium has been separated by a band of connective tissue-tunica albuginea-from the cortex. At this stage, the cortex contains follicles and is well deined from the inner medulla, which is composed of abundant blood vessels, lymphatic vessels, and nerve ibers.

1	• Embryology of the External Genitalia Early development of the external genitalia is similar in both sexes. By 6 weeks' gestation, three external protuberances have developed surrounding the cloacal membrane. These are the left and right cloacal folds, which meet ventrally to form the genital tubercle (Fig. 3-4). With division of the cloacal membrane into anal and urogenital membranes, the cloacal folds become the anal and urethral folds, respectively. Lateral to the urethral folds, genital swellings arise, and these become the labioscrotal folds. Between the urethral folds, the urogenital sinus extends onto the surface of the enlarging genital tubercle to form the urethral groove. By week 7, the urogenital membrane ruptures, exposing the cavity of the urogenital sinus to amnionic fluid.

1	The genital tubercle elongates to form the phallus in males and the clitoris in females. Still, it is not possible to visually diferentiate between male and female external genitalia until week 12. In the male fetus, dihydrotestosterone (DHT) forms locally by the 5-a reduction of testosterone. DHT prompts the anogenital distance to lengthen, the phallus to enlarge, and the labioscrotal folds to fuse and form the scrotum. In the female fetus, without DHT, the anogenital distance does not lengthen, and the labioscrotal and urethral folds do not fuse (Fig. 3-4C). The genital tubercle bends caudally to become the clitoris, and the urogenital sinus forms the vestibule of the vagina. The labioscrotal folds create the labia majora, whereas the urethral folds persist as the labia minora. Female external genital diferentiation is complete by 11 weeks, whereas male external genital diferentiation is complete by 14 weeks.

1	Degenerating paramesonephric duct Primordial germ cell Uterine tube -FIGURE 3-3 Embryonic gonad diferentiation. TDF Persistent paramesonephric duct Coelomic epithelium Degenerating medullary cords = testis-determining factor. Labioscrotal/ , FIGURE 3-4 Development ofthe external genitalia. A. Indifferent stage. B. Virilization ofexternal genitalia. C. Feminization. (Reproduced with permission from Bradshaw KD: Anatomical disorders. In Hoffman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw Hill Education, 201o6.) Defining gender incorporates genetic gender, gonadal gender, and phenotypic gender. Genetic gender-X or Y -is established at fertilization. However, for the irst 6 weeks, development of male and female embf) os is morphologically indistinguishable.

1	Gonadal gender is heralded by the diferentiation of the primordial gonad into a testis or an ovary. If a Y chromosome is present, the gonad begins developing into a testis. Testis development is directed by a protein called the testis-determiningactor (TDF), which modulates the transcription of several genes involved in gonadal diferentiation. TDF is encoded by the sexdetermining region (SR) gene, located on the short arm of the Y chromosome. But testis development is much more complex and requires other autosomal genes (Nistal, 2015a). he importance of the SRYgene is demonstrated in severl paradoxical conditions. First, 46,X phenotypic males can result from translocation of the Y chromosome fragment containing SRY to the X chromosome during meiosis of male germ cells Wu, 2014). Similarly, 46,Y individuals can appear phenotypically female if they carry a mutation in the SRYgene (Helszer, 2013).

1	Last, phenoypic gender begins at 8 weeks' gestation. Before this, urogenital tract development in both sexes is indistinguishable. Thereafter, diferentiation of the internal and external genitalia to the male phenotype is dependent on testicular function. In the absence of a testis, female diferentiation ensues irrespective of genetic gender (Table 3-1).

1	In males, the fetal testis secretes a protein called mullerianinhibiting substance (MIS), also called antimullerian hormone (AMH) . It acts locally as a paracrine factor to cause mullerian duct regression. Thus, it prevents the development of uterus, fallopian tube, and upper vagina. AMH is produced by the Sertoli cells of the seminiferous tubules. Importantly, these tubules appear in fetal gonads and secrete AMH before diferentiation of Leydig cells, which are the cellular site of testosterone synthesis. AMH is secreted as early as 7 weeks, and mullerian duct regression is completed by 9 to 10 weeks. Because AMH acts locally near its site of formation, if a testis were absent on one side, the mullerian duct on that side would persist, and the uterus and fallopian tube would develop on that side.

1	Apparently through stimulation initially by human chorionic gonadotropin (hCG), and later by fetal pituitary luteinizing hormone (LH), the fetal testes secrete testosterone. his hormone acts directly on the wolian duct to efect the development of the vas deferens, epididymis, and seminal vesicles. Testosterone also enters fetal blood and acts on the external genitalia anlage. In these tissues, testosterone is converted to 5a-DHT to cause virilization of the external genitalia. As evident from the prior discussion, abnormal sex development may involve the gonads, internal duct system, or external genitalia. Rates vary and approximate 1 in every 1000 to 4500 births (Murphy, 2011; Ocal, 2011). The nomenclature used to describe disorders of sex development (DSDs) has evolved. Current classiication of these disorders include: (1) sex chromosome DSDs, (2) 46,Y DSDs, and (3) 46,X DSDs (Table 3-2) (Hughes, 2006).

1	Other important terms describe the abnormal phenotypic findings that can be found. First, some disorders of sexual development are associated with abnormal, underdeveloped gonads, that is, gonadal dysgenesis. With this, if a testis is poorly formed, it is called a dysgenetic testis, and if an ovary is poorly formed, it is called a streak gona. In afected patients, the underdeveloped gonad ultimately fails, which is indicated by elevated gonadotropin levels. Another important clinical sequela is that patients bearing a Y chromosome are at high risk of developing a germ cell tumor in the dysgenetic gonad. A second term, ambiguous genitalia, describes genitalia that do not appear clearly male or female. Abnormalities may include TABLE 3-1. Embryonic Urogenital Structures and Their Adult Homologues Uteroovarian and round ligaments Epoophoron, paroophoron Gartner duct Uterus, fallopian tubes, upper vagina Bladder, urethra Seminiferous tubules, Sertoli cells Gubernaculum testis

1	Uteroovarian and round ligaments Epoophoron, paroophoron Gartner duct Uterus, fallopian tubes, upper vagina Bladder, urethra Seminiferous tubules, Sertoli cells Gubernaculum testis Efferent ductules, paradidymis Epididymis, ductus deferens, Prostatic utricle, appendix of testis Bladder, urethra Floor of penile urethra TABLE 3-2. Disorders of Sex Development (DSD) Classification 45,X Turnerd 47,xXY Klinefelterd 45,X/46,XY Mixed gonadal dysgenesis 46,XX/46,XY Ovotesticular DSD 46,XY DSD 46,XX DSD aAnd syndrome variants. AMH = antimullerian hormone; hCG = human chorionic gonadotropin; LH = luteinizing hormone. Adapted with permission from Hughes lA, Houk C, Ahmed SF, et al: Consensus statement on management of intersex disorders. j Pediatr Urol 2:148,l2006. hypospadias, undescended testes, micropenis or enlarged clitoris, labial fusion, and labial mass.

1	hypospadias, undescended testes, micropenis or enlarged clitoris, labial fusion, and labial mass. Last, ovotesticular deines states characterized by ovarian and testicular tissue in the same individual. It was formerly termed true hermaphroditism. In these cases, diferent types of gonads can be paired. he types of gonads that may be paired include a normal testis, a normal ovary, a streak gonad, dysgenetic testis, or an ovotestis. In the latter, both ovarian and testicular elements are combined within the same gonad. With ovotesticular DSDs, the internal ductal system structure depends on the ipsilateral gonad and its degree of determination. Speciically, the amount of AMH and testosterone determines the degree to which the internal ductal system is masculinized or feminized. Extenal genitalia are usually ambiguous and undermasculinized due to inadequate testosterone. • Sex Chromosome Disorders of Sex

1	• Sex Chromosome Disorders of Sex Sex chromosome disorders of sexual development typically arise from an abnormal number of sex chromosomes. Of these, Turner and linefelter syndromes are most frequently encoun tered (Nielsen, 1990). Turner syndrome is caused by de novo loss or severe struc tural abnormality of one X chromosome in a phenotypic female. Most afected fetuses are spontaneously aborted. How ever, in girls with Turner syndrome who survive, phenotype varies widely, bur nearly all afected patients have short stat ure. Associated problems include cardiac anomalies (especially coarctation of the aorta), renal anomalies, hearing impairment, otitis media and mastoiditis, and an increased incidence of hypertension, achlorhydria, diabetes mellirus, and Hashimoto thyroiditis. It is the most common form of gonadal dysgenesis that leads to primary ovarian failure. In these cases, the uterus and vagina are normal and capable of responding to exogenous hormones (Matthews, 2017).

1	Another sex chromosome disorder is Klineelter syndrome (47,Y). hese individuals tend to be tall, undervirilized males with gynecomastia and small, firm testes. They have sig nificantly reduced fertility from hypogonadism due to gradual testicular cell failure. These men are at increased risk for germ cell rumors, osteoporosis, hypothyroidism, diabetes mellirus, breast cancer, cardiovascular abnormalities, and cognitive and psychosocial problems (Aksglaede, 2013; Calogero, 2017). Several karyotypes can create a coexistent ovary and testis, and thus ovotesticular DSD is found in all three DSD categories (see Table 3-2). In the sex chromosome group, ovotesticular DSD may arise from a 46,XX/46,XY karyotype. Here, an ovary, testis, or ovotestis may be paired. he phenotype in general mirrors that for ovotesticular disorders, which are described earlier on this page.

1	For others in the sex chromosome DSD group, ovotesticular disorder arises from a chromosomal mosaic such as 45,Xl46,XY. With this karyotype, a picture ofmixed gonadal dysgenesis shows a streak gonad on one side and a dysgenetic or normal testis on the other. The phenotypical appearance ranges from undervirilized male to ambiguous genitalia to Turner stigmata. • 46,XY Disorders of Sex Development Insuicient androgen exposure of a ferus destined to be a male leads to 46,XY DSD-formerly called male pseudohermaphroditism. he karyotype is 46,XY and testes are frequently present. he uterus is generally absent as a result of normal embryonic AMH production by Sertoli cells. hese subjects are most often sterile from abnormal spermatogenesis and have a small phallus that is inadequate for sexual function. As seen in Table 3-2, etiology of 46,XY DSD may stem from abnormal testis development or from abnormal androgen production or action.

1	46,XY Gonadal Dysgenesis his spectrum of abnormal gonad underdevelopment includes pure or complete, partial, or mixed 46,XY gonadal dysgenesis. These are deined by the amount of normal testicular tissue and by karyotype. Because of the potential for germ cell rumors in dysgeneric testes and intraabdominal testes, afected patients routinely have been advised to undergo gonadectomy Giang, 2016). Of these, pure gonadal dysgenesis results from a mutation in the SRY gene or in other genes with testis-determining efects (Hutson, 2014). his leads to underdeveloped dysgenetic gonads that fail to produce androgens or AMH. Formerly named Swyer syndrome, the condition creates a normal prepubertal female phenotype and a normal miillerian system due to absentiAMH.

1	Partial gonaal dysgenesis deines those with gonad development intermediate between normal and dysgenetic testes. Depending on the percentage of underdeveloped testis, wolian and miille rian structures and genital ambiguity are variably expressed. Mixed gonadal dysgenesis is one type of ovotesticular disorder of sexual diferentiation. As discussed on page 39, one gonad is streak, and the other is a normal or a dysgenetic testis. Of afected individuals, 15 percent have a 46,Y karyotype (Nistal, 20 15 b). The phenotype is wide ranging as with partial gonadal dysgenesis. Last, testicular regression can follow initial testis development. A broad phenotypic spectrum is possible and depends on the timing of testis failure.

1	Last, testicular regression can follow initial testis development. A broad phenotypic spectrum is possible and depends on the timing of testis failure. In some cases, 46,Y disorders of sexual diferentiation stem from abnormalities in: (1) testosterone biosynthesis, (2) LH receptor function, (3) AMH function, or (4) androgen receptor action. First, the sex steroid biosynthesis pathway can sufer enzymatic defects that block testosterone production. Depending on the timing and degree of blockade, undervirilized males or phenotypic females may result. In contrast to these central enzymatic defects, peripheral defects may be causative. Namely, abnormal 5-. reductase type 2 enzyme action leads to impaired conversion of testosterone to DHT and thus to undervirilization.

1	Second, hCG/LH receptor abnormalities within the testes can lead to Leydig cell aplasia/hypoplasia and impaired testosterone production. In contrast, disorders of MH and AMH receptors result in persistent miillerian duct syndrome (PMDS). Afected patients appear as males but have a persistent uterus and fallopian tubes due to failed AMH action. Last, the androgen receptor may be defective and result in androgen-insensitivity syndrome (AIS). Resistance to androgens may be incomplete and associated with varying degrees of virilization and genital ambiguity. Milder forms have been described in men with severe male factor infertility and poor virilization.

1	Females with complete androgen-insensitivity syndrome (CAIS) appear as phenotypically normal females at birth. They often present at puberty with primary amenorrhea. External genitalia appear normal; scant or absent pubic and axillary hair are noted; the vagina is shortened or blind ending; and the uterus and fallopian tubes are absent. However, these individuals develop breasts during pubertal maturation due to abundant androgen to estrogen conversion. Testes may be palpable in the labia or inguinal area or may be found intraabdominally. Surgical excision of the testes after puberty is recommended to decrease the associated risk of germ cell tumors, which may be as high as 20 to 30 percent. • 46,XX Disorders of Sex Development As seen in Table 3-2, etiology of 46,X disorders of sexual differentiation may stem from abnormal ovarian development or from excess androgen exposure.

1	• 46,XX Disorders of Sex Development As seen in Table 3-2, etiology of 46,X disorders of sexual differentiation may stem from abnormal ovarian development or from excess androgen exposure. Disorders of ovarian development of those with a 46,X complement include: (1) gonadal dysgenesis, (2) testicular DSD, and (3) ovotesticular DSD. With 4, X gonadal dysgenesis, similar to Turner syndrome, streak gonads develop. These lead to hypogonadism, prepubertal normal female genitalia, and normal miillerian structures, but other Turner stigmata are absent.

1	With 4,X testicuar DSD, several possible genetic mutations lead to testis-like formation within the ovary-streak gonad, dysgenetic testis, or ovotestis. Defects may stem from SRY translocation onto one X chromosome. In individuals without SRY translocation, other genes with testis-determining efects are most likely activated. Regardless, production of MH prompts miillerian system regression, and androgens promote wolian system development and external genitalia masculinization. Spermatogenesis, however, is absent due to a lack of needed genes on the long arm of the Y chromosome. hese individuals are not usually diagnosed until puberty or during infertility evaluation. With 4,X ovotesticular DSD, individuals possess a unilateral ovotestis with a contralateral ovary or testis, or bilateral ovotestes. Phenotypic indings depend on the degree of androgen exposures and mirror those for other ovotesticular DSDs discussed on page 39.

1	Discordance between gonadal sex (46,X) and the phenotypic appearance of external genitalia (masculinized) may also result from excessive fetal androgen exposure. his was previously termed female pseudohermaphroditism. In afected individuals, the ovaries and female internal ductal structures such as the uterus, cervix, and upper vagina are present. hus, patients are potentially fertile. he external genitalia, however, are virilized to a varying degree depending on the amount and timing of androgen exposure. he three embryonic structures that are commonly afected by elevated androgen levels or ovarian development disorders are the clitoris, labioscrotal folds, and urogenital sinus. As a result, virilization may range from modest clitoromegaly to posterior labial fusion and development of a phallus with a penile urethra. Degrees of virilization can be described by the Prader score, which ranges from 0 for a normal-appearing female to 5 for a normal, virilized male.

1	Fetal, placental, or maternal sources can provide the excessive androgen levels. Maternally derived androgen excess may come from virilizing ovarian tumors such as luteoma and Sertoli-Leydig cell tumor or from virilizing adrenal tumors. Fortunately, these neoplasms infrequently cause fetal efects because of the tremendous ability of placental syncytiotrophoblast to convert C19 steroids-androstenedione and testosterone-to estradiol via the enzyme aromatase (Chap. 5, p. 103). As another source, drugs such as testosterone, danazol, and other androgen derivatives may cause fetal virilization. Of fetal sources, exposure can arise from fetal congenital adrenal hyperplasia (CAH). his stems from a fetal enzyme dei ciency in the steroidogenic pathway that leads to androgen accu mulation. The most common defect is 21-hydroxylase deiciency. CAH is a frequent cause of virilization and has an incidence approximating 1 in 10,000 to 20,000 live births (Speiser, 2010).

1	With CAH, phenotypes depend on the location of the enzyme defect in the steroidogenic pathway and on the severity of the resulting enzymatic deiciency (Miller, 201i1). With severe enzymatic deiciency, afected newborns have life-threatening salt wasting and virilization. Other mutations may prompt virilization alone (Auchus, 2015). he mildest abnormalities present later and are described as "nonclassic," "late-onset," or "adult-onset" CAH. In these patients, activation of the adrenal axis at puberty increases steroidogenesis and unmasks a mild enzymatic deficiency. Excess androgen provides negative feedback to gonadotropin-releasing hormone (GnRH) receptors in the hypothalamus. These patients oten present with hirsutism, acne, and anovulation. Thus, lateonset CAH may mimic polycystic ovarian syndrome (McCannCrosby, 2014). In some instances, CAH can be diagnosed antenatally. Early maternal dexamethasone therapy can dampen androgen excess to minimize virilization (Chap. 16, p. 317).

1	Of rare placental sources, placental aromatase deiciency from a fetal CP 19 gene mutation causes an accumulation of placental androgen and underproduction of placental estrogens (Chap. 5, p. 105) Gones, 2007). Consequently, both the mother and the 46,X fetus are virilized. Delivery of a newborn with a disorder of sexual diferentiation is a potential medical emergency and can create possible longlasting psychosexual and social ramiications for the individual and family. Ideally, as soon as the afected neonate is stable, parents are encouraged to hold the child. he newborn is referred to as "your baby," and suggested terms include "phallus," "gonads," "folds," and "urogenital sinus" to reference underdeveloped structures. The obstetrician explains that the genitalia are incompletely formed and emphasizes the seriousness of the situation and the need for rapid consultation and laboratory testing.

1	Because similar or identical phenotypes may have several etiologies, identiication of a speciic DSD may require several diagnostic tools (McCann-Crosby, 2015). Relevant neonatal physicl examination evaluates: (1) ability to palpate gonads in the labioscrotal or inguinal regions, (2) ability to palpate uterus during rectl examination, (3) phallus size, (4) genitalia pigmentation, and (5) presence of other syndromic features. The newborn metabolic condition is assessed, as hyperkalemia, hyponatremia, and hypoglycemia may indicate CAH. he mother is examined for signs of hyperandrogenism. Other neonatal tests include genetic studies, hormone measurements, imaging, and in some cases endoscopic, laparoscopic, and gonadal biopsy. Sonography shows the presence or absence of miillerian/wolian structures, n locate the gonads, and can identiy associated mlformations such as renal anomalies.

1	Very early during embryo formation, a bilaminar cloacal membrane lies at the caudal end of the germinal disc and forms the infraumbilical abdominal wall. Normally, an ingrowth of mesoderm between the ectodermal and endodermal layers of the cloacal membrane leads to formation of the lower abdomi nal musculature and pelvic bones. Without this reinforcement, the cloacal membrane may prematurely rupture, and depend ing on the extent of the infraumbilical defect, cloacal exstrophy, bladder exstrophy, or epispadias may result. Of these, cloacal exstrophy is rare. It includes the triad of omphalocele, bladder exstrophy, and imperforate anus.

1	Of these, cloacal exstrophy is rare. It includes the triad of omphalocele, bladder exstrophy, and imperforate anus. Bladder exstrophy is uncommon and is characterized by an exposed bladder lying outside the abdomen. Associated ind symphysis pubis. At the same time, however, the uterus, fal lopian tubes, and ovaries are typically normal except for occa sional miillerian duct fusion defects. Pregnancy with bladder exstrophy is associated with greater risk for antepartum pyelonephritis, urinary retention, ureteral obstruction, pelvic organ prolapse, preterm birth, and breech presentation. The guidelines for pregnancy (Eswara, 2016). Due to the exten encountered, some recommend planned cesarean delivery at a tertiary center (Deans, 2012; Dy, 2015; Greenwell, 2003). Epispadias without bladder exstrophy is rare and develops in association with other anomalies such as a widened, patulous tened mons pubis. Vertebral abnormalities and pubic symphy sis diathesis are also common .

1	Clitoral anomalies are unusual. One is clitoral duplication or biid clitoris, which is rare and usually develops in association with bladder exstrophy or epispadias. With female phallic urethra, the urethra opens at the clitoral tip. Last, clitoromegaly noted at birth suggests fetal exposure to excessive androgens (p. 40). In other cases, congenital clitoromegaly in females born extremely premature is a rare but well-recognized finding thought to be due to transient androgen levels in these neonates (Greaves, 2008).

1	As noted, the hymen marks the embryological boundary between structures derived from the miillerian and urogenital sinus. Hymeneal anomalies include imperforate, microperforate, cribriform (sievelike), navicular (boat-shaped), and septate hymens. They result from failure of the inferior end of the vaginal plate-the hymeneal membrane-to canalize. Their incidences approximate 1 in 1000 to 2000 females (American College of Obstetricians and Gynecologists, 2016). During the neonatal period, signiicant amounts of mucus can be secreted due to maternal estrogen stimulation. With an imperforate hymen, secretions collect to form a bulging, translucent yellow-gray mass, termed hydro-or mucocolpos, at the vaginal introitus. Most are asymptomatic and resolve as mucus is reabsorbed and estrogen levels decrease, but rarely can cause perinatal urinary retention from their mass efects Gohal, 2009).

1	Four principal deformities arise from defective miillerian duct embryological steps: (1) agenesis of both ducts, either focally or along the entire duct length; (2) unilateral maturation of one mullerian duct with incomplete or absent development of the opposite side; (3) absent or faulty midline fusion of the ducts; or (4) defective canalization. Various classifications have been proposed, and Table 3-3 shows the one from the American Fertility Society (1988). It separates anomalies into groups with similar clinical characteristics, prognosis for pregnancy, and treatment. It also includes one for abnormalities associated with fetal exposure to diethylstilbestrol (DES). Several other classiication systems have been crafted, but this one is the most widely used (Acien, 2011; Di Spiezio Sardo, 2015; Oppelt, 2005).

1	Mullerian anomalies may be suspected by symptoms or physical findings such as vaginal septa, blind-ending vagina, or duplicated cevix. Amenorrhea may be an initial complaint for those with agenesis of a mullerian component. In those with outlet obstruction, pelvic pain from occult blood that accumulates and distends the vagina, uterus, or fallopian tubes may arise from functioning endometrium. Endometriosis and its associated dysmenorrhea, dyspareunia, and chronic pain are also frequent with outlet obstruction. Class I segmental defects are caused by mullerian hypoplasia or agenesis as shown in Figure 3-5. These developmental defects can afect the vagina, cervix, uterus, or fallopian tubes and may be isolated or may coexist with other mullerian defects. TABLE 3-3. Classification of MU"erian Anomalies I. Segmental mullerian hypoplasia or agenesis ab. Vaginal . Cervical c. d. e. II. Unicornuate uterus a. b. c. d. III. Uterine didelphys IV. Bicornuate uterus a.

1	I. Segmental mullerian hypoplasia or agenesis ab. Vaginal . Cervical c. d. e. II. Unicornuate uterus a. b. c. d. III. Uterine didelphys IV. Bicornuate uterus a. Complete-division to internal os b. V. Septate uterus a. Complete-septum to internal os b. VI. Arcuate VII. Diethylstilbestrol related Data from American Fertility Society: The American Fertility Society classifications of adnexal adhesions, distal tubal occlUSion, tubal occlusion secondary to tubal ligation, tubal pregnancies, Mullerian anomalies and intrauterine adhesions, Fertil Steril 1988 Jun;49(6):944-955.

1	Of all vaginal anomalies, vaginal agenesis is the most profound and may be isolated or associated with other mullerian anomalies. One example is the Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome, in which upper vaginal agenesis is typically associated with uterine hypoplasia or agenesis. Less often, this syndrome also displays abnormalities of the renal, skeletal, and auditory systems. his triad is known by the acronym MURCS, which reflects mullerian duct aplasia, renal aplasia, and �ervicothoracic �omite dysplasia (Rall, 2015).

1	he obstetrical significance of vaginal anomalies depends greatly on the degree of obstruction. Complete vaginal agenesis, unless corrected operatively, precludes pregnancy by vaginal intercourse. With MRKH syndrome, a functional vagina can be created, but uterine agenesis proscribes childbearing. In these women, however, ova can be retrieved for in vitro fertilization (IVF) in a surrogate mother (F riedler, 2016). U terine transplantation is currently experimental but holds future promise for these women Qohannesson, 2016). Of other vaginal anomalies, congenital septa may form longitudinally or transversely, and each can arise from a fusion or resorption defect. Longitudinal septa divide the vagina into right and left portions. hey may be complete and extend the entire vaginal length. Partial septa usually form high in the vagina but may develop at lower levels. Septa are typically associated with other mullerian anomalies (Haddad, 1997).

1	During labor, a complete longitudinal vaginal septum usually does not cause dystocia because the vaginal side through which the fetus descends dilates satisfactorily. An incomplete or partially obstructed longitudinal septum, however, may interfere with descent. Occasionally, a woman with a distal longitudinal septum presents in labor. During second-stage labor, this septum usually becomes attenuated by pressure from the fetal head. After ensuring adequate analgesia, the inferior attachment of the septum is isolated, clamped, transected, and ligated. Following placenta delivery, the superior attachment can be transected while carefully avoiding urethral injury.

1	A transverse septum poses an obstruction of variable thickness. It may develop at any depth within the vagina, but most are in the lower third (Williams, 2014). These mayior may not be perforate, and thus obstruction or infertility is variably present. In labor, perforate strictures may be mistaken for the upper limit of the vaginal vault, and the septal opening is misidentiied as an undilated cervical os (Kumar, 2014). If encountered during labor, and ater the external os has dilated completely, the head impinges on the septum and causes it to bulge downward. If the septum does not yield, slight stretching pressure on its opening usually leads to further dilatation, but occasionally cruciate incisions are required to permit delivery (Blanton, 2003). If there is a thick transverse septum, however, cesarean delivery may be necessary. Developmental abnormalities of the cervix include partial or complete agenesis, duplication, and longitudinal septa.

1	Developmental abnormalities of the cervix include partial or complete agenesis, duplication, and longitudinal septa. I. Hypoplasia/agenesis II. Unicornuate A. Vaginal B. Cervical A. Communicating B. Noncommunicating C. Fundal D. Tubal E. Combined C. No cavity D. No horn III. Didelphys IV. Bicornuate A. Complete B. Partial V. Septate VI. Arcuate VII. DES related A. Complete B. Partial FIGURE 3-5 Classification of mOllerian anomalies. DES = diethylstilbestrol.(Modified with permission from American Fertility Society: The American Fertility Society classifications of adnexal adhesions, distal tubal occlusion,tubalocclusion secondary to tubal ligation, tubal pregnancies, MOlierian anomalies and intrauterine adhesions, Ferril Steril 1988 Jun;49(6):944-55.l

1	Uncorrected complete agenesis is incompatible with pregnancy, and in vitro ferrilization with gestational surrogacy is an option. Surgical correction by uterovaginal anastomosis has resulted in successful pregnancy (Kriplani, 201e2). Significant complications accompany this correcrive surgery, and rhe need for clear preoperative anatomy delineation has been emphasized by Rock (2010) and Roberrs (2011) and their colleagues. For this reason, they recommend hysterecromy for complete cervi� cal agenesis and reserve reconsrruction attemprs for carefully selected patients with cervical dysgenesis.

1	From a large variery, a few of the more common congenital urerine malformations are shown in Table 3-3. An accurate population prevalence of these is diicult to assess because the best diagnostiC techniques are invasive. That said, the prevalence found with imaging ranges from 0.4 to 10 percent, and rates in women with recurrent miscarriage are signiicantly higher (Byrne. 2000; Dreisler. 2014; Saravclos. 2008). In a general population, the mOSt common inding is arcuare urerus, followed in descending order by septate, bicornuate, didelphic, and unicornuate classes (Chan, 2011b).

1	ination, cesarean delivery, rubal sterilization, or infertility evaluation. Depending on clinical presenrarion, diagnostic tools may include sonography, hysterosalpingography, magnetic� resonance imaging, laparoscopy, and hysteroscopy. Each has limitations, and rhese may be used in combination ro completely deine anatomy. In women undergoing fertiliry evaluation, hysterosalpingography (HSG) is commonly selected for uterine cavity and tubal patency assessment. It is contraindi� cared during pregnancy. HSG poorly deines the external urerine contour and can delineate only patent cavities. Regarding patency, remember that some uniconuate rudimentary horns lack a caviry. Also, ourlet Obstructions will preclude dye illing.

1	In most clinical senings, rwo-dimensional transvaginal sonography (2-D VS) is initially performed. For this indication, the pooled accuracy for VS is 90 to 92 percent (Pellerito. 1992). Saline infusion sonography (SIS) improves delineation of the endome[fium and intenal merine morphology, bur only with a patent endometrial cavity. It also is contraindicated in pregnancy. Three-dimensional (3-D) sonography is more accurate than 2·D sonography because it provides uterine images from virtually any angle. Thus, coronal images can be constructed as shown in Figure 3-6, and these are essential in evaluating borh

1	FIGURE 3-6 Three-dimensional transvaginal sonographic images. A. Bicornuate uterus with an 8-week gestation. The external fundal contour (red doted ine) dips centrally below the intercornual line, and the endometrial cavities communicate. B. Septate uterus with a S-week gestation. The external fundal contour is normal and convex 'elow dotted ine), and the long septum (asterisk) extends caudad in the midline. C. Arcuate uterus with an 8-week gestation. The external fundal contour is normal and convex (red dotted line), but the fundal endometrial cavity is slightly indented (arrow). internal and exrernaJ uterine contours (Grimbizis, 2016). Both 2-D and 3-D sonography arc suirable for use in pregnancy.

1	internal and exrernaJ uterine contours (Grimbizis, 2016). Both 2-D and 3-D sonography arc suirable for use in pregnancy. Several studies have reported very good concordance between 3-D TVS and MR imaging of muUerian anomalies (Deutch, 2008; Graupera, 2015). MR imaging is often preferred for complex anatomy, especially cases for which corrective surgery is planned. MR imaging provides clear delineation of both the internal and external uterine anatomy and has a reported accuracy of up to 100 percent for mullerian anomaly evaluation (Bermejo, 20 I0; Pellerito, 1992). In addition, com plex anomalies and commonly associatcd secondary diagnoses such as renal or skeletal anomalies can be concurrendy evaluated. Precautions wirh MR imaging in pregnancy are discussed in Chapter 46 (p. 910).

1	In some women undergoing an inferrility evaluation. hysteroscopy and laparoscopy may be selected co assess for mullerian anomalies; screen for endomctriosis, which is often coexistent; and exclude other tubal or uterine cavity pathologies (Puscheck, 2008; Saravelo" 2008). In pregnancy, these approaches are rarely used co diagnose mullerian anomalies. and hysteroscopy is contraindicated.

1	Wirh this abnormality, the underdeveloped or rudimentary horn may be absent. If present, it may or may nor communicatewith the domin�nr horn �nd may or may not contain an endomerrium-Iined cavity (see Fig. 3-5). General population estimates cite an incidence of I in 4000 women (Reichman, 2009). This anomaly may be detected during ferrility evaluation by HSG. Bur as nored, noncommunicating or noncavirary rudimentary horns may not ill with dye. If this anomaly is suspected. 3-D sonography increases diagnostic accuracy, but again MR imaging may be preferred. Importantly, 40 percent of afected women will have renal anomalies (Fedele, 1996).

1	This mullerian anomaly carries signiicant obstetrical risks, including first-and second-trimester miscarriage. malpresentation, fetal-growth restricrion. fetal demise. prematurely ruprured membranes, and preterm delivery (Chan, 2011 a; Hua, 2011; Reichman, 2009). Abnormal uterine blood Row, cervicl incompetence, and diminished cavity size and muscle mass of the hemiurerus are postulated ro underlie these risks (Donderwinkel, 1992).

1	Rudimentary horns also increase the risk for an ectopic pregnancy within rhe remnant, which may be disastrous. This risk includes noncommunicating cavitary rudiments, for which transperironeal sperm migration permirs ovum fertilization and pregnancy (Nahum, 2004). In a report of 70 sllch pregnancies, Rolen and associares (1966) found that the rudimentary uterine horn ruptured prior to 20 weeks in most. Nahum (2002) reviewed the literature from 1900 to 1999 and identiied 588 rudimentary horn pregnancies. Half had uterine rupture, and 80 percent did so before the third rrimesrer. Of the rotal 588, the neonatal survival race was only 6 percent. Imaging allows an earlier diagnOSiS of rudimentary horn pregnancy so that it can be treated either medically with methotrexare or surgically before rupture (Dove, 2017; Edelman, 2003; Khati, 2012; Worley, 2008). lthough not emphasized in Figure 3-5, the anachmem sire between rhe rudimentary horn at times can be broad and vascular.

1	If diagnosed in a nonpregnant woman, mOSt recommend prophylactic excision of a horn thac has a cavity (Fedele, 2005; Rackow, 2007). Data regarding subsequent pregnancy after excision are scarce. In one series of eight women, all had a preterm cesarean delivery (pados, 2014). This mullerian anomaly arises from a complere lack of fusion rhat results in twO enrirely separate hemiureri, cervices, and usually (yo vaginas (see Fig. 3-5). It is common among marsupials, for example, the American possum-Didelphys virginiana. Most women have a double vagina or a longitudinal vaginal septum. Urerine didelphys may be isolated. Or, it may compose a triad with an Qbstructed hemiyagina and with ipsilateral renal Igenesis (OHVlRA), also known as HerlynWerner-Wunderlich syndrome (Tong, 2013).

1	These anomalies are suspected on pelvic examinarion by identificarion of a longitudinal vaginal septum and two cervices. During HSG for fertility evaluation, contrast shows twO separate endocervical canals. These open into separare noncommunicating fusiform endometrial cavities char each ends wirh a solitary fallopian tube. In women without fertility issues, 2-or 3-D TVS is a logical initial imaging tool, and separate diver gent uterine horns with a large intervening fundal cleft are seen. Endometrial cavities are uniformly separate. MR imaging may be valuable in cases without classic indings. Adverse obstetrical outcomes associated with uterine didel phys are similar but less frequent than those seen with unicornu ate uterus. Increased risks include miscarriage, pre term birth, and malpresentation (Chan, 2011a; Grimbizis, 2001; Hua, 2011).

1	Metroplasty for either uterine didelphys or bicornuate uterus involves resection of intervening myometrium and fundal recombination (Alborzi, 2015). hese rarely performed surgeries are chosen for highly selected patients with otherwise unexplained miscarriages. Moreover, no evidence-based data conirm the eicacy of such surgical repair. his fusion anomaly results in two hemiuteri. As shown in Figure 3-5, the central myometrium runs either partially or completely to the cervix. A complete bicornuate uterus may extend to the internal cervical os and have a single cervix (bicornuate unicollis) or reach the external os (bicornuate bicollis). As with uterine didelphys, a coexistent longitudinal vaginal septum is not uncommon.

1	Radiological discrimination of a bicornuate uterus from a septate uterus can be challenging. This distinction, however, is important because septate uterus can be treated with hysteroscopic septal resection. HSG or 2-D TVS may initially suggest an anomaly, but further distinction is provided by 3-D TVS or MR imaging (see Fig. 3-6). With these, an intercornual angle greater than 105 degrees typiies a bicornuate uterus, whereas one less than 75 degrees indicates a septate uterus. Fundal contour also assists, and a straight line drawn between the imaged tubal ostia serves as the defining threshold. Referent to this, an intrafundal downward cleft measuring �1 em or more is indicative of bicornuate uterus. A septate uterus shows a cleft depth < 1 em, or it may have a normal fundal contour.

1	Bicornuate uterus carries increased risks for adverse obstetrical outcomes that include miscarriage, preterm birth, and malpresentation. As discussed in the prior section, rare surgical correction by metroplasty is reserved for highly selected patients. With this anomaly, a resorption defect leads to a persistent complete or partial longitudinal uterine septum (see Fig. 3-5). Less often, a complete vaginocervicouterine septum is found (Ludwin, 2013). Many septate uteri are identiied during evaluation of infertility or recurrent pregnancy loss. Although an abnormality may be identiied with HSG or 2-D TVS, typically 3-D TVS or MR imaging is required to diferentiate this from a bicornuate uterus (see Fig. 3-6).

1	Septate anomalies are associated with diminished fertility and increased risks for adverse pregnancy outcomes that include miscarriage, preterm delivery, and malpresentation (Chan, 201i1a; Ghi, 2012). Hysteroscopic septal resection has been shown to improve pregnancy rates and outcomes (Mollo, 2009; Pabu:cu, 2004). From their metaanalysis, Valle and colleagues (20l3) reported a 63-percent pregnancy rate and 50-percent live birth rate following resection. his malformation is a mild deviation from the normally developed uterus. Although some studies report no increased adverse associated outcomes, others have found excessive secondtrimester losses, preterm labor, and malpresentation (Chan, 2011a; Mucowski, 2010; Woelfer, 2001). Treatment with Cerclage

1	Treatment with Cerclage Some women with uterine anomalies and repetitive pregnancy losses may beneit from transvaginal or transabdominal cervical cerclage (Golan, 1992; Groom, 2004). Others with partial cervical atresia or hypoplasia may also benefit (Hampton, 1990; Ludmir, 1991). Candidacy for cerclage is determined by the same criteria used for women without such defects, which is discussed in Chapter 18 (p. 354).

1	During the 1960s, a synthetic nonsteroidal estrogen-diethylstilbestrol (DES)-was used to treat pregnant women for threatened abortion, preterm labor, preeclampsia, and diabetes. he treatment was remarkably inefective. Later, it was also discovered that women exposed as fetuses had increased risks of developing several specific reproductive-tract abnormalities. hese included vaginal clear cell adenocarcinoma, cervical intraepithelial neoplasia, small-cell cervical carcinoma, and vaginal adenosis. Afected women had identiiable structural variations in the cervix and vagina that include transverse septa, circumferential ridges, and cervical collars. Uteri potentially had smaller cavities, shortened upper uterine segments, or T-shaped and other irregular cavities (see Fig. 3-5) (Kaufman, 1984).

1	These women sufer impaired conception rates and higher rates of miscarriage, ectopic pregnancy, and preterm delivery, especially in those with structural abnormalities (Kaufman, 2000; Palmer, 2001). Now, more than 50 years after DES use was proscribed, most afected women are past childbearing age, but higher rates of earlier menopause, cervical intraepithelial neoplasia, and breast cancer are reported in exposed women (Hatch, 2006; Hoover, 2011; Troisi, 2016).

1	he fallopian tubes develop from the unpaired distal ends of the mlillerian ducts. Congenital anomalies include accessory ostia, complete or segmental tubal agenesis, and several embryonic cystic remnants. The most common is a small, benign cyst attached by a pedicle to the distal end of the fallopian tube-the hydatid of Morgagni. In other cases, benign paratubal cysts may be of mesonephric or mesothelial origin. Last, in utero exposure to DES is associated with various tubal abnormalities. Of these, short, tortuous tubes or ones with shriveled fimbria and small ostia are linked to infertility (DeCherney, 1981). The pregnant uterus may infrequently show exaggerated flexion. Mild or moderate lexion is typically inconsequential, but congenital or acquired extremes may lead to pregnancy complications.

1	The pregnant uterus may infrequently show exaggerated flexion. Mild or moderate lexion is typically inconsequential, but congenital or acquired extremes may lead to pregnancy complications. Antlexion describes forward angling of the uterine fundus in the sagittal plane relative to the cervix. Exaggerated degrees usually pose no problem in early pregnancy. Later, however, particularly when the abdominal wall is lax such as with diastasis recti or ventral hernia, the uterus may fall forward. In extreme cases, the fundus lies below the lower margin of the symphysis. Sometimes, this abnormal uterine position prevents proper transmission of labor contractions, but this is usually overcome by repositioning and application of an abdominal binder.

1	Retrolexion describes posterior uterine fundal angling in the sagittal plane. A growing retrolexed uterus will occasionally become incarcerated in the hollow of the sacrum. Symptoms include abdominal discomfort, pelvic pressure, and voiding dysfunction or retention. During bimanual pelvic examination, the cervix will be anterior and behind the symphysis pubis, whereas the uterus is appreciated as a mass wedged in the pelvis. Sonography or MR imaging can aid the clinical diagnosis (Gardner, 2013; Grossenburg, 2011; van Beekhuizen, 2003).

1	With continued uterine growth, the incarcerated uterus can spontaneously resolve over 1 to 2 weeks. An indwelling urinary catheter or intermittent self-catheterization may be needed in the interim to empty the bladder. Persistent cases require manual repositioning. For this, after bladder catheterization, the uterus can usually be pushed out of the pelvis when the woman is placed in a knee-chest position. Often, this is best accomplished by digital pressure applied through the rectum. FIGURE 3-7 Anterior sacculation of a pregnant uterus. Note the markedly attenuated anterior uterine wall and atypical location of the true uterine fundus. Rood, 2014). Conscious sedation, spinal analgesia, or general anesthesia may be necessary. Following repositioning, the catheter is left in place until bladder tone returns. Insertion of a soft pessary for a few weeks usually prevents recurrent incarceration.

1	Lettieri and colleagues (1994) described seven cases of uterine incarceration not amenable to these simple procedures. In two women, laparoscopy was used at 14 weeks' gestation to reposition the uterus using the round ligaments for traction. Alternatively, in case series, advancing a colonoscope or colonoscopic insulation was used to dislodge an incarcerated uterus (Dierickx, 2011; Newell, 2014; Seubert, 1999).

1	Rarely, sacculation may form as extensive lower uterine segment dilatation due to persistent entrapment of the pregnant uterus in the pelvis (Fig. 3-7). In these extreme cases, sonography and MR imaging are typically required to deine anatomy (Gottschalk, 2008; Lee, 2008). Cesarean delivery is necessary when sacculation is marked, and Spearing (1978) stressed the importance of clariying the distorted anatomy. An elongated vagina passing above the level of a fetal head that is deeply placed into the pelvis suggests a sacculation or an abdominal pregnancy. The Foley catheter is frequently palpated above the level of the umbilicus! Spearing (1978) recommended extending the abdominal incision above the umbilicus and delivering the entire uterus from the abdomen before hysterotomy. This will restore correct anatomical relationships and prevent inadvertent incisions into and through the vagina and bladder. Unfortunately, this may not always be possible (Singh, 2007). As a inal caveat, a

1	restore correct anatomical relationships and prevent inadvertent incisions into and through the vagina and bladder. Unfortunately, this may not always be possible (Singh, 2007). As a inal caveat, a true uterine diverticulum has been mistaken for uterine sacculation (Rajiah, 2009).

1	he uterus commonly rotates to the maternal right during pregnancy. Rarely, uterine rotation exceeds 180 degrees to cause torsion. Most cases of torsion result from uterine leiomyomas, miillerian anomalies, fetal malpresentation, pelvic adhesions, or laxity of the abdominal wall or uterine ligaments. Jensen (1992) reviewed 212 cases and reported that associated symptoms may include obstructed labor, intestinal or urinary Anterior wall complaints, abdominal pain, uterine sacculation hypertonus, vaginal bleeding, and hypotension.

1	Anterior wall complaints, abdominal pain, uterine sacculation hypertonus, vaginal bleeding, and hypotension. Most cases of uterine torsion are found at the time of cesarean delivery. In some women, torsion can be conirmed preoperatively with MR imaging, which shows a twisted vagina that appears X-shaped rather than its normal H-shape (Nicholson, 1995). As with uterine incarceration, during cesarean delivery, a severely displaced uterus should be repositioned anatomically before hysterotomy. In some cases, an inability to reposition or a failure to recognize the torsion may lead to a posterior hysterotomy inci sion (Albayrak, 2011; Picone, 2006; Acitll P, Aden MI; lhe hisrory of female genital (rael malformation classiica rions and proposal of an updau'd s,slcm. Hum Reprod Update 17:693, 2011 AksgJacdc , JUlll A: Testicular �unction and lertiliry in men and Klinefelter syndrome: a review. Ellr J Endocrinol 168(4):R67, 2013

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1	Obstet Gynecol Suv 49:642, 1994 Ludmir J, Jackson GM, Samuels P: Transvaginal cerclage under ultrasound guidance in cases of severe cervical hypoplasia. Obstet Gynecol 78: 1067, 1991 Ludwin A, Ludwin I, Pitynski K, et al: Diferentiating between a double cervix or cervical duplication and a complete septate uterus with longitudinal vaginal septum. Taiwan J Obstet Gynecol 52(2):308, 2013 Matthews 0, Bath L, Hagler W, et l: Hormone supplementation for pubertal induction in girls. Arch Dis Child 102(10):975,o2017 McCann-Croshy B, Chen MJ, et al: Non-classical congenital adrenal hyperplasia: targets of treatment and transition. Pediatr Endocrinol Rev 12(2):224, 2014 McCann-Crosby B, Sutton VR: Disorders of sexual development. Clin PerinawIo42(2):395,o2015 Miller L, Auchus RJ: The molecular biology, biochemistry, and physiology of human sterOidogenesis and its disorders. Endocr Rev 32(1):81, 2011

1	Mollo A, De Franciscis P, Colacurci N, et al: Hysteroscopic resection of the septum improves the pregnancy rate of women with unexplained infertility: a prospective controlled trial. Ferri! SteriIo91(6):2628, 2009 Moore L, Persaud V, Torchia MG: he urogenital system. In The Developing Human. Philadelphia, Saunders, 2013, P 272 Mucowski SJ, Herndon CN, Rosen MP: The arcuate uterine anomaly: a critical appraisal of its diagnostiC and clinical relevance. Obstet Gynecol Surv 65(7),449,o2010 Murphy C, Allen L, Jamieson A: Ambiguous genitalia in the newborn: an overview and teaching tool. J Pediatr Adolesc GynecoIo24:236, 2011 Nahum G, Stanislaw H, McMahon C: Preventing ecropic pregnancies: how often does transperiwneal transmigration of sperm occur in efecting human pregnancy? BJOG 111:706,o2004 Nahum GG: Rudimentary uterine horn pregnancy: the 20th-century worldwide experience of 588 cases. J Reprod Med 47: 151, 2002

1	Nahum GG: Rudimentary uterine horn pregnancy: the 20th-century worldwide experience of 588 cases. J Reprod Med 47: 151, 2002 Newell SO, Crofts JF, Gram SR: The incarcerated gravid uterus: complications and lessons learned. Obstet Gynecol 123(2 Pt 2 Suppl (2 Pt 2 Suppl 2))A23,o2014 Nicholson K, Coulson Cc, McCoy MC, er al: Pelvic magnetic resonance imaging in the evaluation of uterine torsion. Obstet Gynecol 85(5 Pt 2),888, 1995 Nielsen J, Wohlert M: Sex chromosome abnormalities found among 34,910 newborn children: results from a 13-year incidence study in rhus, Denmark. Birth Defects Orig Artic Ser 26:209, 1990 Nistal IvI, Paniagua R, Gonzalez-Peramato p, et al: Perspectives in pediatric pathology, chapter I. normal development of testicular structures; from the bipotemial gonad to the fetal testis. Pediarr De\' PathoIo18(2}:88, 2015a

1	Nistal M, Paniagua R, Gonlez-Peramato P, et al: Perspectives in pediatric pathology, chapter 5. gonadal dysgeneSiS. Pediatr Dev PathoIo18(4):259, 2015b Ocal G: Current concepts in disorders of sexual development. J Clin Res Pediatr EndocrinoI3(3): \05,o2011 Oppelt P, Renner sr, Brucker S, et al. The VCUAM (Vagina Cervix Uterus Adnex-associated Malformation) Classiication: a new classiication for genital malformations. Ferril Sterilo84: 1493,o2005 Pabw:cu R, Gomel V: Reproductive outcome after hysteroscopic metroplasfY in women with septate uterus and othervrise unexplained infertility. Ferril Sterilo81:o1675,o2004 Pados G, Tsolakidis 0, Athanatos 0, et aI: Reproductive and obstetric outcome after laparoscopic excision of functional, non-communicating broadly attached rudimentary hon: a case series. Eur J Obstet Gynecol Reprod Bioi 182,33,o2014 Palmer JR, Hatch EE, Rao RS, et al: Infertility among womcn exposed prenatally to diethylstilbestrol. Am J Epidemiol 154:31G, 2001

1	Palmer JR, Hatch EE, Rao RS, et al: Infertility among womcn exposed prenatally to diethylstilbestrol. Am J Epidemiol 154:31G, 2001 Pellerito JS, lvlcCarthy S�'I, Doyle MB, et al: Diagnosis of uterine anomalies: relative accuracy of MR imaging, endovaginal sonography, and hysterosalpingography. Radiology 183:795, 1992 Picone 0, Fubini A, Ooumerc S, et al: Cesarean delivery by posterior hysterotomy due to torsion of the pregnant uterus. Obstet Gynecol 107(2 Pt 2),533, 2006 Puscheck EE, Cohen L: Congenital malformations of the uterus: the role of ultrasound. Semin Reprod Med 26(3):223, 2008 Rackow BW, Arici A: Reproductive performance of womcn with miillerian anomalies. Curr Opin Obstet Gynecol 19(3):229, 2007 Rajiah P, Eastwood KL, Gunn ML, ct aI: Uterinc diverticulum. Obstct Gynecol 113(2 Pt 2):525, 2009

1	Rajiah P, Eastwood KL, Gunn ML, ct aI: Uterinc diverticulum. Obstct Gynecol 113(2 Pt 2):525, 2009 Ral K, Eisenbeis S, Henninger V: Typical and atypical associated indings in a group of 346 patients with Mayer-Rokitansky-Kuester-Hauser syndrome. J Pediatr Adolesc Gynecol 28(5):362, 2015 Reichman 0, Laufer MR: Congenital uterine anomalies afecting reproduction. Best Pract Res Clin Obstet GvnaecoIo24(2):193, 2010 Reichman 0, Laufer MR, Robinson 3K: Pregnancy outcomes in unicornuate uteri: a review. Fertil Sterilo91(5): 1886,o2009 Roberts CP, Rock JA: Surgical methods in the treatment of congenital anomalies of the uterine cervix. Curr Opin Obstet Gynecol 23(4):251, 2011 Rock JA, Roberts CP, Jones HW Jr: Congenital anomalies of the uterine cervix: lessons from 30 cases managed clinically by a common protocol. Fertil Steril 94(5),1858,o2010 Rolen AC, Choquette AJ, Semmens JP: Rudimentary uterine horn: obstetric and gynecologic implications. Obstet Gynecol 27:806, 1966

1	Rolen AC, Choquette AJ, Semmens JP: Rudimentary uterine horn: obstetric and gynecologic implications. Obstet Gynecol 27:806, 1966 Rood K, Markham KB: Torsion of a term gravid uterus: a possible cause of intrauterine growth restriction and abnormal umbilical artery Doppler indings. J Ultrasound Med 33(10):1873, 2014 Saravelos SH, Cocksedge A, Li TC: Prevalence and diagnosis of congenital uterine anomalies in women with reproductive failure: a critical appraisal. Hum Reprod Update 14(5):415,o2008 Seubert DE, Puder KS, Goldmeier P, et al: Colonoscopic release of the incarcerated gravid uterus. Obstet Gynecol 94:792, 1999 Singh MN, Payappagoudar J, Lo J: Incarcerated retroverted uterus in the third trimester complicated by postpartum pulmonary embolism. Obstet Gynecol 109A98, 2007 Spearing GJ: Uterine sacculation. Obstet GynecoIo51:IIS, 1978

1	Spearing GJ: Uterine sacculation. Obstet GynecoIo51:IIS, 1978 Speiser PW, Azziz R, Baskin LS, ct al: Congenital adrenal hyperplasia due to steroid 2 I-hydroxylase deiciency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 95(9):4133, 2010 Tongo], Zhu L, Lang J: Clinical characteristics of 70 patients with HerlynWerner-Wunderlich syndrome. Int J Gynaecol Obstet 121 (2): 173, 2013 Troisi R, Hatch EE, Palmer JR, et al: Prenatal diethylstilbestrol exposure and high-grade squamous cell neoplasia of the lower genital traCt. Am J Obstet Gynecol 215(3):322.el, 2016 Valle RF, Ekpo GE: Hysteroscopic metroplasty for the septate uterus: review and meta-analysis. J Minim Invasive GynecoIo20(1):22, 2013 Van Beekhuizen HJ, Bodewes HW, Tepe EM, et al: Role of magnetic reso nance imaging in the diagnosis of incarceration of the gravid uterus. Obstct Gynecol 102:1o134,o2003

1	Van Beekhuizen HJ, Bodewes HW, Tepe EM, et al: Role of magnetic reso nance imaging in the diagnosis of incarceration of the gravid uterus. Obstct Gynecol 102:1o134,o2003 Williams CE, Nakhal RS, Hall-Craggs A, et al: Transverse vaginal septae: management and long-term outcomes. BJOG 121(13):1653,o2014 Woelfer B, Salim R, Banerjee S, et al: Reproductive outcomes in women with congenital uterinc anomalies detected by three-dimensional ultrasound screening. Obstet GynecoIo98:1099, 2001 Worley KC, Hnat i'vID, Cunningham FG: Advanced xtrauterine pregnancy: diagnostic and therapeutic challenges. Am J Obstet Gynecol 198:287.e 1, 2008 WU QY, U N, Li W, et al: Clinical, molecular and cytogenetic analysis of 46,X testicular disorder of scx developmcnt with SRY-positive. BMC Urol 14:70,o2014 REPRODUCTIVE TRACT ...........................49 BREASTS....................................... 53 SKIN .......................................... 53

1	REPRODUCTIVE TRACT ...........................49 BREASTS....................................... 53 SKIN .......................................... 53 METABOLIC CHANGES. . . . . . . . . . . . . . . . . . . . . . . . . . . 54 HEMATOLOGICAL CHANGES ...................... 57 CARDIOVASCULAR SYSTEM ....................... 60 RESPIRATORY ............................64 URINARY SYSTEM ............................... 65 GASTROINTESTINAL TRACT .......................68 ENDOCRINE SYSTEM ................... ...... .. 68 MUSCULOSKELETAL SYSTEM ...................... 72 CENTRAL NERVOUS SYSTEM . . . . . . . . . . . . . . . . . . . . . . 72 The matenal organism reacts to a greater or lesser extent under the inluence of pregnancy, but naturaly the most characteristic changes are observed in the generative tract, and especialy the uterus, which undergoes a very marked increase in size. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) In the irst edition of this textbook, Williams devoted only 10 pages to the physiology of pregnancy, and half were focused on uterine growth. Many gestational changes begin soon after fertilization and continue throughout pregnancy. Equally astounding is that the woman is returned almost completely to her prep regnancy state after delivery and lactation. Most pregnancy-related changes are prompted by stimuli provided by the fetus and placenta. Virtually every organ system undergoes alterations, and these can appreciably modiy criteria for disease diagnosis and treatment. hus, an understanding of pregnancy adaptations is essential to avoid misinterpretation. Moreover, some physiological changes can unmask or worsen preexisting disease.

1	In the nonpregnant woman, the uterus weighs approximately 70 g and is almost solid, except for a cavity of 10 mL or less. During pregnancy, the uterus is transformed into a thin-walled muscular organ of suicient capacity to accommodate the fetus, placenta, and amnionic luid. he total volume of the contents at term averages 5 L but may be 20 L or more! hus, by the end of pregnancy, the uterus has achieved a capacity that is 500 to 1000 times greater than the nonpregnant state. The corresponding increase in uterine weight is such that, by term, the organ weighs nearly 1100 g.

1	During pregnancy, uterine enlargement involves stretching and marked hypertrophy of muscle cells, whereas the production of new myocytes is limited. Fibrous tissue also accumulates, particularly in the external muscle layer, together with a considerable rise in elastic tissue content. The walls of the corpus considerably thicken and strengthen during the first few months of pregnancy but then gradually thin. By term, the myometrium is only 1 to 2 cm thick, and the fetus usually can be palpated through the soft, readily indentable uterine walls. Uterine hypertrophy early in pregnancy probably is stimulated by the action of estrogen and perhaps progesterone. Thus, similar uterine changes can be observed with ectopic pregnancy. But after approximately 12 weeks' gestation, uterine growth is related predominantly to pressure exerted by the expanding products of conception.

1	Within the uterus, enlargement is most marked in the fundus. The extent of uterine hypertrophy is also influenced by the position of the placenta. Namely, the myometrium surrounding the placental site grows more rapidly than does the rest. The uterine musculature during pregnancy is arranged in three strata. The irst is an outer hoodlike layer, which arches over the fundus and extends into the various ligaments. The middle layer is a dense network of muscle fibers perforated in all directions by blood vessels. Last is an internal layer, with sphincter-like ibers around the fallopian tube orifices and internal cervical os. Most of the uterine wall is formed by the middle layer. Here, each myocyte has a double curve so that the interlacing of any two cells forms a figure eight. This arrangement is crucial and permits myocytes to contract after delivery and constrict penetrating blood vessels to halt bleeding.

1	For the first few weeks, the uterus maintains its original piriform or pear shape. But, as pregnancy advances, the corpus and fundus become globular and almost spherical by 12 weeks' gestation. Subsequently, the organ grows more rapidly in length than in width and becomes ovoid. By the end of 12 weeks, the enlarged uterus extends out of the pelvis. With this, it contacts the anterior abdominal wall, displaces the intestines laterally and superiorly, and ultimately reaches almost to the liver. With uterine ascent, it usually rotates to the right, and this dextrorotation likely is caused by the rectosigmoid on the left side of the pelvis. As the uterus rises, tension is exerted on the broad and round ligaments.

1	With the pregnant woman standing, the longitudinal axis of the uterus corresponds to an extension of the pelvic inlet axis. The abdominal wall supports the uterus and maintains this axis, unless the wall is lax. When the pregnant woman lies supine, the uterus falls back to rest on the vertebral column and the adjacent great vessels.

1	Beginning in early pregnancy, the uterus contracts irregularly, and these may be perceived as mild cramps. During the second trimester, these contractions can be detected by bimanual examination. In 1872, J. Braxton Hicks first brought attention to these contractions, which now bear his name. hese appear unpredictably and sporadically and are usually nonrhythmic. Their intensity varies between 5 and 25 mm Hg (Alvarez, 1950). Until near term, these Braxton Hicks contractions are infrequent, but their number rises during the last week or two. At this time, the uterus may contract as often as every 10 to 20 minutes and with some degree of rhythmicity. Correspondingly, uterine electrical activity is low and uncoordinated early in gestation, but becomes progressively more intense and synchronized by term (Garield, 2005; Rabotti, 2015). This synchrony develops twice as fast in multiparas compared with nulliparas (Govindan, 2015). Late in pregnancy, these contractions may cause some

1	by term (Garield, 2005; Rabotti, 2015). This synchrony develops twice as fast in multiparas compared with nulliparas (Govindan, 2015). Late in pregnancy, these contractions may cause some discomfort and account for so-called false labor.

1	The delivery of most substances essential for fetal and placental growth, metabolism, and waste removal requires the placental intervillous space to be adequately perfused (Chap. 5, p. 94). Placental perfusion depends on total uterine blood low, but simultaneous measurement of uterine, ovarian, and collateral vessels is not yet possible, even using magnetic resonance (MR) angiography (Pates, 2010). Using ultrasound to study the uterine arteries, uteroplacental blood low has been measured to increase progressively during pregnancy-from approximately 450 mUmin in the mid trimester to nearly 500 to 750 mU min at 36 weeks (Flo, 2014; Wilson, 2007). hese measures are similar to uterine artery blood flow estimates ascertained indirectly using clearance rates of androstenedione and xenon-133 (Edman, 1981; Kauppila, 1980). These values also mirror older ones-500 to 750 mUmin-obtained with invasive methods (Assali, 1953; Browne, 1953; Metcalfe, 1955). Logically, such massively increased

1	(Edman, 1981; Kauppila, 1980). These values also mirror older ones-500 to 750 mUmin-obtained with invasive methods (Assali, 1953; Browne, 1953; Metcalfe, 1955). Logically, such massively increased uteroplacental blood flow requires adaptation of the uterine veins as well. The resultant increased venous caliber and distensibility can result in uterine vein varices that in rare instances may rupture (Lim, 2014).

1	As noted irst from animal studies, uterine contractions, either spontaneous or induced, lower uterine blood flow proportionally to contraction intensity (Assali, 1968). A tetanic contraction yields a precipitous fall in uterine blood flow. In humans, three-dimensional power Doppler angiography has also demonstrated reduced uterine blood low during contractions Gones, 2009). Using a similar technique, resistance to blood low in both maternal and fetal vessels was found to be greater during the second stage of labor compared with the first (Baron, 2015). Given that baseline uterine blood low is diminished in pregnancies complicated by fetal-growth restriction, these fetuses may tolerate spontaneous labor less efectively (Ferrazzi, 2011; Simeone, 2017).

1	The vessels that supply the uterine corpus widen and elongate yet preserve their contractile function (Mandala, 2012). In contrast, the spiral arteries, which directly supply the placenta, vasodilate but completely lose contractility. This presumably results from endovascular trophoblast invasion that destroys the intramural muscular elements (Chap. 5, p. 91). It is this vasodilation that allows maternal-placental blood low to progressively rise during gestation. Given that blood flow increases proportionally to the fourth power of the radius of the vessel, small increases in vessel diameter result in tremendous augmentation of uterine artery blood low. For example, in one study, the uterine artery diameter grew from only 3.3 mm to 3.7 mm between 22 and 29 weeks' gestation, but mean velocity increased 50 percent, from 29 to 43 cm/sec (Flo, 2010).

1	The downstream fall in vascular resistance is another key factor that accelerates low velocity and shear stress in upstream vessels. In turn, shear stress leads to circumferential vessel growth. Nitric oxide-a potent vasodilator-appears to play a central role in regulating this process and is discussed later (p. 63). Indeed, endothelial shear stress and several hormones and growth facrors all augment endorhelial nitric oxide synthase (eNOS) and nitric oxide producrion (Crummer, 2009; Lim, 2015; Mandala, 2012; Pang, 2015). Facrors include estrogen, progesterone, activin, placental growth facror (PIG F), and vas cular endorhelial growrh factor (VEGF), which is a promoter of angiogenesis. As an impo((am aside, VEGF and PIGF signaling is attenuated in response to excess placenral secretion of their soluble recepror-soluble FMS-like yrosille kinase I (sFlt-I). An ing PIGF and VEGF concentrations and is importanr in pre eclampsia parhogenesis (Chap. 40, p. 716).

1	Normal pregnancy is also characterized by vascular refrac mriness CO the pressor efects of infused angiotensin II, and rhis raises ureropiacenral blood Aow (Rosenfeld, 1981,e2012). relaxin and certain adipocyrokines (Vodsrrcil, 201e2). Chemeri1 is an adipocycokine secreted by several rissues, including the placenra (Garces, 2013; Kasher-Meron, 2014). Irs concentra tion rises as gestadon advances and serves ro increase human umbilical eNOS activity, which mediates greater blood Aow (Wang, 2015). Anorher adipocyrokine-viotil-raises VEGF cells derived from the placemal amnion (Astern, 2013). Orher adipocyrokines include lepfin, resistin, and 1diponectin, which (Pole', 2014).

1	Last, cenain micro RNA species mediate vascular remodeling and uterine blood low early in placentation (Sama, 2015). In particular, members of the miR-1e7-92 clusrer and miR-34 are important in spiral artery remodeling and invasion. Abnormalities of micro-RNA function have been reported in preeclampsia, fetal-growth rescriction, and gestational diaberes.

1	As early as 1 momh after conception, the cervix begins to soften and gain bluish mnes. These result from increased vascularity and edema of the entire cervix, from changes in the collagen necwork, and from hypertrophy and hyperplasia of the cervical glands (Peralra, 2015; Srraach, 2005). Alrhough rhe cervix contains a small amollnt of smooth muscle, its major componenr is connective (issue. Rearrangemem of rhis collagen-rich tissue aids rhe cervix in retention of the pregnancy until term, in dilatation to aid delivery, and in posrpartum repair and reconstitution ro permir a subsequenr successful pregnancy (Myers, 2015). As derailed in Chapter 21 (p. 409), cervical ripening involves connective tissue remodeling (har lowers collagen and proreoglycan concentrations and raises warer comenr compared wirh rhe nonpregnanr cervix.

1	Cervical glands undergo marked proliferation, and by the end of pregnancy, they occupy up to one half of the entire cervical mass. This normal pregnancy-induced change promprs an extension, or eversion, of (he proliferating columnar endocervical glands onto the ecrocervical portio (rig. 4-1). This tissue appears red and velvety and bleeds even with minor trauma, such as with Pap tesring. FIGURE 4-1 Cervical eversion of pregnancy as viewed through a colposcope. The eversion represents columnar epithelium on the portio of the cervix. (Used with permission from Dr. Claudia Werner.)

1	FIGURE 4-1 Cervical eversion of pregnancy as viewed through a colposcope. The eversion represents columnar epithelium on the portio of the cervix. (Used with permission from Dr. Claudia Werner.) The endocervical mucosal cells produce copious amounts of tenacious mucus thar obstruct the cervical canal soon after conception (Bastholm, 2017). This mucus is rich in immunoglobulins and cytokines and may acr as an immunological barrier to prorec( the uterine conrents against infection (Hansen, 2014; Wang, 2014). Ar labor onser, if not before, this mucus plug is expelled, resulting in a bloody show. Moreover, the cervical mucus consistency changes during pregnancy. Speciically, in most pregnant women, as a result of progesterone, when cervical mucus is spread and dried on a glass slide, it shows poor crysrallization, termed beading. In some gravidas, as a resulr of amnionic Auid leakage. an arborization of ice-like crystals, calledening, is seen microscopically.

1	HistOlogically, basal cells near rhe squamocolumnar junction can be prominenr in size, shape, and staining quality in pregnancy. 111ese changes are considered ro be estrogen induced. In addition, pregnancy is associated with bO[h endoccrvical gland hyperplasia and hypersecretory appearance-the Arias-Stela raction-which can make diferenriating these from truly atypical glandular cells during Pap test evaluarion particularly diiculr (Rosai, 2015). Ovularion ceases during pregnancy, and maturation of new follicles is suspended. The single corpus luteum found in graVidas functions maximally during the irst 6 to 7 weeks of pregnancy--4 ro 5 weeks postovulation. Thereafter, ir contributes relarively linie to progesrcrone production. Surgical removal of the corpus luteum before 7 weeks prompts a rapid fall in matenal serum progesrerone levels and spontaneous abortion (Csapo, 1973). Afrer this time, however, corpus lureum excision ordinarily does nO[ cause abortion.

1	An extrauterine decidual reaction on and jusr beneath the ovarian surface is common in pregnancy and is usually observed ar cesarean delivery. 1l1ese slighdy elevated clear or red patches bleed easily and may, on irst glance, resemble freshly torn adhesions. Similar decidual reactions arc seen on the uterine serosa and other pelvic, or even extrapelvic, abdominal organs (Bloom. 2010). These areas arise from subcoelomic mesenchyme or eniomcrcioric lesions that have been stimulated by progesterone. They histologically appear similar to progestin-stimulated intrauterine endometrial stroma (Kim. 2015). The enormous caliber of the ovarian veins viewed at cesarean delivery is starding. Hodgkinson (1953) found that the diametcr of the ovarian vascular pedicle increased during pregnancy from 0.9 em to approximately 2.6 em at term. Again, recall that Aow in a tubular structure increases exponentially as the diameter enlarges.

1	This protein hormone is secreted by the corpus lureum, the decidua. and the placenta in a pattern similar to that of human chorionic gonadotropin (hCG) (Chap. 5. p. 102). Relaxin is also expressed in brain, hean, and kidney. Ie is mentioned here because its secretion by the corpus lureum appears to aid many maternal physiological adaptations, such as remodeling of reproductive-tract connective tissue to accommodate labor (Conrad. 2013; Vrachnis. 2015). Relaxin also appears importam in initiating augmented renal hemodynamics, lowering serum osmolality, and increasing arterial compliance, which are all associated with normal pregnancy (Conrad. 2014a). Despite its name, serum relaxin levels do nOt contribute to greater peripheral joint laxity or pelvic girdle pain during pregnancy (Aldabe. 2012; Marnach. 2003; V0l1estad. 2012).

1	These benign ovarian lesions reAect exaggerated physiological follicle stimulation, which is termed hyperreactio luteinalis. These usually bilateral cystic ovaries are moderately to massively enlarged. The reaction is usually linked to markedly elevated serum heG levels. Logically, theca-Iurein CYStS are found frequenrlywith gestational trophoblastic disease (Fig. 20-3. p. 391). They also can develop with the placentomegaly that can accompany diabetes, anti-D alloimmunization, and multi fetal gestation (Malinowski, 2015). Hyperreactio luteinalis is associated with preeclampsia and hyperthyroidism, which may contribure to elevated risks for fetal-growth restriction and preterm birth (Cavorerro. 2014; Lynn. 2013; Mlinowski. 2015). hese cystS also are encountered in women with otherwise uncomplicated pregnancies. In these cases, an exaggerated response of the ovaries to normal levels of circulating heG is suspected (Sarmento Gon,alves. 2015).

1	Although usually asymptomatic, hemorrhage into the CYStS can cause acme abdominal pain (Amoah. 2011). Maternal virilization may be seen in up to 30 percent of women, however, virilization of the fetus has only rarely been reported (Malinowski. 2015). Maternal indings that include temporal balding, hirsutism, and clitoromegaly are associated with massively elevated levels of androstenedione and testosterone. The diagnosis typically is based on sonographic findings of bilateral enlarged ovaries containing multiple cysts in the appropriate clinical settings. The condition is self-limited and resolves following delivery. Its management is reviewed by Malinowski (2015) and discussed further in Chapter 63 (p. 1199). The fallopian rube musculature, rhat is, the myosalpinx, undergoes little hypertrophy during pregnancy. The epithelium of the mdosnpinxsomwhat Ranens. Decidual cells may develop in rhe stroma of the endosalpinx, but a continuous decidual membrane is not formed.

1	Rarely, a fallopian tube may twisr during uterine enlarge� mem (Macedo, 2017). This torsion is more common with comorbid pararubal or ovarian cysts (Lee, 2015). During pregnancy, greater vascularity and hyperemia develop in the skin and muscles of the perineum and vulva, and the underlying abundant connective tissue softens. This augmented vascularity prominently afects the vagina and cervix and results in the violet color characreristic of Chadwick sign.

1	Within the vagina, the considerably elevated volume of cervical secretions during pregnancy forms a somewhat thick, white discharge. The pH is acidic, varying from 3.5 to 6. This pH results from increased production of lactic acid by lactobacillus acidophi/us during metabolism of glycogen energy stores in the vaginal epithelium. Pregnancy is associated with an elevated risk of vulvovaginal candidiasis. particularly during the second and third trimesters. Higher infection rates may stem from immunological and hormonal changes and from greater vaginl glycogen stores (Aguin. 2015). The vaginal walls undergo striking changes in preparation for the distention that accompanies labor and delivery. These alterations include considerable epithelial thickening, connective tissue loosening, and smooth muscle cell hypertrophy.

1	Pelvic Organ Prolapse Quantiication (POP-Q) and threedimensional sonography studies show that vaginal suppOrt changes across pregnancy. In particular, vaginal lengthening, posterior vaginal wall and hiatal relaxation, increased levaror hiaral area, and greater irst-trimester vaginal elastase activity are all associated with uncomplicated spontaneous vaginal delivery (Oliphant. 2014). The larger hiatal area persists in women who deliver vaginally compared with women delivering by prelabor or early-labor cesarean delivery. However. all women show greater hiatal distensibility after delivery, which is potentially a facror in later pelvic floor dysfunction (van Veelen, 2015).

1	In women with apical vaginal prolapse. the cervix, and occasionally a portion of the uterine body, can protrude variably from the vulva during early pregnancy. With further growth. the uterus usually rises above the pelvis and can draw the cervix up with it. If the uterus persists in its prolapsed position, symptoms of incarceration may develop at 10 to 14 weeks' gestation (Chap. 3, p. 46). s a preventive measure. [he urerus can be replaced early in pregnancy and held in position with a suitable pessary.

1	Attenuation of anterior vaginal wall suppOrt can lead to prolapse of the bladder, that is, a cystocele. Urinary stasis with a cystocele predisposes to infection. Pregnancy may also worsen coexistent stress urinary incontinmce (SUi), likely because urerhral closing pressures do nor risc suiciently to compensate for altered bladder neck support. Urinary incontinence afects nearly 20 percent of women during the first trimester and nearly 40 percent during the third trimester. Most cases stem from SUI rather than urgency urinary incontinence (Abdullah, 2016a; Franco, 2014; Iosif, 1980). In primigravidas, maternal age greater than 30 years, obesity, smoking, constipation, and gestational diabetes mellitus are all risk factors associated with SUI development during pregnancy (Sangsawang, 2014).

1	Attenuation of posterior vaginal wall support can result in a rectocele. A large defect may fill with feces that occasionally can be evacuated only digitally. During labor, a cystocele or rectocele can block fetal descent unless they are emptied and pushed out of the way. Rarely, an enterocele of considerable size may bulge into the vagina. If the mass interferes with delivery, the hernia sac and its abdominal contents are gently reduced to permit fetal descent.

1	In early pregnancy, women often experience breast tenderness and paresthesias. Mter the second month, the breasts grow in size, and delicate veins are visible just beneath the skin. he nipples become considerably larger, more deeply pigmented, and more erectile. Mter the first few months, a thick, yellowish luid-colosrum-can often be expressed from the nipples by gentle massage. During the same months, the areolae become broader and more deeply pigmented. Scattered through each areola are several small elevations, the glands of Montgomey, which are hypertrophic sebaceous glands. If breasts gain extensive size, skin striae similar to those observed in the abdomen may develop. Rarely, breasts can become pathologically enlarged-referred to as gigantomastia-which may require postpartum surgical reduction (Fig. 4-2) (Eler Dos Reis, 2014; Rezai,i2015).

1	For most normal pregnancies, prepregnancy breast size and ultimate volume of breast milk do not correlate, as multiple factors influence milk production (Hartmann, 2007). These FIGURE 4-2 Gigantomastia in a woman near term. (Used with permission from Dr. Patricia Santiago-Munoz.) factors and gestation breast changes are further discussed in Chapter 36 (p. 656). Skin changes are common, and Fernandes and Amaral (2015) described dermatological changes in more than 900 pregnant women. They found at least one physiological cutaneous change in 89 percent of the women examined. Dermatologic pathologies during pregnancy are found in Chapter 62.

1	Beginning after midpregnancy, reddish, slightly depressed streaks commonly develop in the abdominal skin and some times in the skin over the breasts and thighs. These are called striae gravidarum or stretch marks. In multiparas, glistening, silvery lines that represent the cicatrices of previous striae fre quently coexist. In one study of 800 primiparas, 70 percent their breasts; and 41 percent on their hips and thighs (Picard, 2015). The strongest associated risk factors included younger maternal age, family history, and prepregnancy weight and weight gain during pregnancy. The etiology of striae gravi darum is unknown, and there are no preventive steps or defini tive treatments (Korgavkar, 2015).

1	Occasionally, the muscles of the abdominal walls do not withstand the tension of the expanding pregnancy. As a result, rectus muscles separate in the midline, creating diastasis recti of varying extent. If severe, a considerable portion of the anterior uterine wall is covered by only a layer of skin, attenuated fascia, and peritoneum to form a ventral hernia.

1	This develops in up to 90 percent of women and is usually more accentuated in those with darker complexion (Ikino, 2015). Of speciic sites, the pigmented skin line in the midline of the anterior abdominal wall-the linea alba-takes on dark brownblack pigmentation to form the linea nigra. Occasionally, irregular brownish patches of varying size appear on the face and neck, giving rise to chloasma or melasma gravidarum-the mask of pregnancy. Pigmentation of the areolae and genital skin may also be accentuated. Mter delivery, these pigmentary changes usually disappear or at least regress considerably. Oral contraceptives may cause similar alterations (Handel, 2014).

1	he etiology of these pigmentary changes is incompletely understood, however, hormonal and genetic factors play a role. For example, levels of melanocyte-stimulating hormone, a polypeptide similar to corticotropin, are elevated remarkably throughout pregnancy, and estrogen and progesterone also are reported to have melanocyte-stimulating efects . Angiomas, called vascular spiders, are particularly common on the face, neck, upper chest, and arms. These are minute, red skin papules with radicles branching out from a central lesion. he condition is often designated as nevus, angioma, TABLE 4-1. Additional Energy Demands During Normal Pregnancya Rates of Tissue Deposition Energy Cost of Pregnancy Estimated from Basal Metabolic Rate and Energy Deposition aAssumes an average gestational weight gain of 12 kg. bEfficiency of food energy utilization for protein and fat deposition estimated as 0.90. Adapted from the World Health Organization, 2004.

1	bEfficiency of food energy utilization for protein and fat deposition estimated as 0.90. Adapted from the World Health Organization, 2004. or telangiectasis. Palmar eythema is encountered during pregnancy. Both conditions lack clinical significance and disappear in most gravidas shortly after pregnancy. hey are likely the consequence ofhyperestrogenemia. In addition to these discrete lesions, increased cutaneous blood flow in pregnancy serves to dissipate excess heat generated by the augmented metabolism. hroughout life, the human hair follicle undergoes a pattern of cyclic activity that includes periods of hair growth (anagen phase), apoptosis-driven involution (catagen phase), and a resting period (telogen phase). Based on a study of 116 healthy pregnant women, the anagen phase lengthens during pregnancy and the telogen rate increases postpartum (Gizlenti, 2014). Neither is exaggerated in most gravidas, but excessive hair loss in the puerperium is termed telogen fluvium.

1	In response to the greater demands of the rapidly growing fetus and placenta, the pregnant woman undergoes metabolic changes that are numerous and intense. By the third trimester, maternal basal metabolic rate rises by 20 percent compared with that of the nonpregnant state (Berggren, 2015). This rate grows by an additional 10 percent in women with a twin gestation (Shinagawa, 2005). Viewed another way, the additional total pregnancy energy demand associated with normal pregnancy approximates kcal (World Health Organization, 2004). This is stratified as 85, 285, and 475 kcal/d during the irst, second, and third trimester, respectively (Table 4-1). Of note, Abeysekera and coworkers (2016) reported that women accrue fat mass during pregnancy despite the increased total energy expenditure and without signiicant change in energy intake. This suggests more eicient energy storage.

1	Most of the normal weight gain in pregnancy is attributable to the uterus and its contents, the breasts, and expanded blood and extravascular extracellular fluid volumes. A smaller fraction results from metabolic alterations that promote accumulation of cellular water, fat, and protein, which are so-called maternal reserves. The average weight gain during pregnancy approximates 12.5 kg or 27.5 Ib, and this value has remained consistent across studies and over time (Hytten, 1991; Jebeile, 2016). Weight gain is considered in urther detail in Table 4-2 and in Chapter 9 (p. 165). In pregnancy, greater water retention is normal and mediated in part by a drop in plasma osmolality of 10 mOsm/kg. This decline develops in early pregnancy and is induced by a reset of osmotic thresholds for thirst and vasopressin secretion (Fig. 4-3) TABLE 4-2. Weight Gain Based on Pregnancy-Related Components Total 650 4000 8500 12,500 Modified from Hytten, 1991l. Weeks of pregnancy

1	TABLE 4-2. Weight Gain Based on Pregnancy-Related Components Total 650 4000 8500 12,500 Modified from Hytten, 1991l. Weeks of pregnancy FIGURE 4-3 Mean values (black line) ± standard deviations (blue lines) for plasma osmolality (Posm) measured at weekly intervals in nine women from preconception to 16 weeks. LMP = last menstrual period; MP = menstrual period. (Redrawn with permission from Davison JM, Dunlop W: Renal hemodynamics and tubular function in normal human pregnancy. Kidney Int 18:1o52, 1980.) (Davison, 1981; Lindheimer, 2001). Relaxin and other hormones are thought to playia role (Conrad, 2013). At term, the water content of the fetus, placenta, and amnionic luid approximates 3.5 L. Another 3.0 L accumulates from expanded maternal blood volume and from uterus and breast growth. Thus, the minimum amount of extra water that the average woman accrues during normal pregnancy approximates 6.5 L. This corresponds to 14.3 lb.

1	Clearly demonstrable pitting edema of the ankles and legs is seen in most pregnant women, especially at the end of the day. This fluid accumulation, which may amount to a liter or so, results from greater venous pressure below the level of the uterus as a consequence of partial vena cava occlusion. A decline in interstitial colloid osmotic pressure induced by normal pregnancy also favors edema late in pregnancy (0ian, 1985). Longitudinal studies of body composition show a progressive accumulation of total body water and fat mass during pregnancy. These two components as well as initial maternal weight and weight gained during pregnancy are highly associated with neonatal birthweight (Lederman, 1999; MardonesSantander, 1998). "Over-nourished" women are more likely to deliver oversized neonates, even when glucose tolerant (Di Benedetto, 2012).

1	The products of conception, the uterus, and maternal blood are relatively rich in protein rather than fat or carbohydrate. At term, the normally grown fetus and placenta together weigh about 4 kg and contain approximately 500 g of protein, or about half of the total pregnancy increase. The remaining 500 g is added to the uterus as contractile protein, to the breasts primarily in the glands, and to maternal blood as hemoglobin and plasma proteins.

1	Amino acid concentrations are higher in the fetal than in the maternal compartment and generally result from facilitated transport across the placenta (Cleal, 2011; Panitchob, 2015). This greater concentration is largely regulated by the placenta through an incompletely understood process. In particular, placental transport is variable for individuals and for diferent amino acids. For example, tyrosine is a conditionally essential amino acid in the preterm neonate but not in the fetus (Van den Akker, 2010, 2011). The placenta concentrates amino acids into the fetal circulation and is also involved in protein synthesis, oxidation, and transamination of some nonessential amino acids (Galan, 2009).

1	Maternal protein intake does not appear to be a critical determinant for birthweight among well-nourished women (Chong, 2015). Still, recent data suggest that current recommendations for protein intake may be too low. These guidelines are extrapolated from nonpregnant adults and may underestimate actual needs. Stephens and colleagues (2015) prospectively analyzed maternal protein intake and metabolism. They estimated average requirements of 1.22 g/kg/d of protein for early pregnancy and 1.52 g/kg/d for late pregnancy. These levels are higher than the current recommendation of 0.88 g/kg/d. The daily requirements for dietary protein intake during pregnancy are discussed in Chapter 9 (p. 167). Normal pregnancy is characterized by mild fasting hypoglycemia, postprandial hyperglycemia, and hyperinsulinemia (Fig. 4-4). his elevated basal level of plasma insulin in normal 1 PM 6 PM 12 M SAM MEALS: SAM t t t

1	FIGURE 4-4 Diurnal changes in plasma glucose and insulin in normal late pregnancy. (Redrawn from Phelps, 1981o.) pregnancy is associated with several unique responses to glucose ingestion. Speciically, after an oral glucose meal, gravidas demonstrate prolonged hyperglycemia and hyperinsulinemia and a greater suppression of glucagon (Phelps, 1981). This cannot be explained by an increased metabolism of insulin because its half-life during pregnancy is not changed appreciably (Lind, 1977). Instead, this response relects a pregnancy-induced state of peripheral insulin resistance, which ensures a sustained postprandial supply of glucose to the fetus. Indeed, insulin sensitivity in late normal pregnancy is 30 to 70 percent lower than that of nonpregnant women (Lowe, 2014).

1	The mechanisms responsible for this reduced insulin sensitivity include numerous endocrine and inlammatory factors (Angueira, 2015). In particular, pregnancy-related hormones such as progesterone, placentally derived growth hormone, prolactin, and cortisol; cytokines such as tumor necrosis factor; and hormones derived from central adiposity, particularly leptin and its interplay with prolactin, all have a role in the insulin resistance of pregnancy. Even so, insulin resistance is not the only factor to elevate postprandial glucose values. Hepatic gluconeogenesis is augmented during both diabetic and nondiabetic pregnancies, particularly in the third trimester (Angueira, 2015).

1	Overnight, the pregnant woman changes from a postprandial state characterized by elevated and sustained glucose levels to a fasting state characterized by decreased plasma glucose and some amino acids. Plasma concentrations of free fatty acids, triglycerides, and cholesterol are also higher in the fasting state. his pregnancy-induced switch in fuels from glucose to lipids has been called accelerated starvation. Certainly, when fasting is prolonged in the pregnant woman, these alterations are exaggerated and ketonemia rapidly appears.

1	he concentrations of lipids, lipoproteins, and apolipoproteins in plasma rise appreciably during pregnancy (Appendix, p. 1259). Increased insulin resistance and estrogen stimulation during pregnancy are responsible for the maternal hyperlipidemia. Augmented lipid synthesis and food intake contribute to maternal fat accumulation during the first two trimesters (Herrera, 2014). In the third trimester, however, fat storage declines or ceases. his is a consequence of enhanced lipolytic activity, and decreased lipoprotein lipase activity reduces circulating triglyceride uptake into adipose tissue. his transition to a catabolic state favors maternal use oflipids as an energy source and spares glucose and amino acids for the fetus.

1	Maternal hyperlipidemia is one of the most consistent and striking changes of lipid metabolism during late pregnancy. Triacylglycerol and cholesterol levels in very-low-density lipoproteins (VLDLs), low-density lipoproteins (LDLs), and highdensity lipoproteins (HDLs) are increased during the third trimester compared with those in nonpregnant women. During the third trimester, the average level of total serum cholesterol is 267 ± 30 mg/dL, ofLDL-C is 136 ± 33 mg/dL, ofHDL-C is 81 ± 17 mg/dL, and of triglycerides is 245 ± 73 mg/dL (Lippi, 2007). After delivery, the concentrations of these lipids, lipoproteins, and apolipoproteins decline. Breastfeeding drops maternal triglyceride levels but increases those of HDL-C. The efects of breastfeeding on total cholesterol and LDL-C levels are unclear (Gunderson, 2014).

1	Hyperlipidemia is theoretically a concern because it is associated with endothelial dysfunction. From studies, however, endothelium-dependent vasodilation responses actually improve across pregnancy (Saarelainen, 2006). Ihis is partly because increased HDL-C concentrations likely inhibit LDL oxidation and thus protect the endothelium. hese indings suggest that the increased cardiovascular disease risk in multiparas may be related to factors other than maternal hypercholesterolemia. This peptide hormone is primarily secreted by adipose tissue in nonpregnant humans. It plays a key role in body fat and energy expenditure regulation and in reproduction. For example, leptin is important for implantation, cell proliferation, and angiogenesis (Vazquez, 2015). Leptin deiciency is associated with anovulation and infertility, whereas certain leptin mutations cause extreme obesity (Tsai, 2015).

1	Among normal-weight pregnant women, serum leptin levels rise and peak during the second trimester and plateau until term in concentrations two to four times higher than those in nonpregnant women. Among obese women, leptin levels correlate with adiposity (Ozias, 2015; Tsai, 2015). In all cases, leptin levels fall after delivery, relecting the significant amounts produced by the placenta (Vazquez, 2015). Leptin participates in regulating energy metabolism during pregnancy. Interestingly, despite the rise in leptin concentrations during pregnancy, reduced leptin sensitivity to food intake during pregnancy has been described (Chehab, 2014; Vazquez, 2015). This "leptin resistance" may serve to promote energy storage during pregnancy and for later lactation.

1	Higher leptin levels during pregnancy may be disadvantageous under certain situations, such as in maternal obesity. Leptin functions as a proinflammatory cytokine in white adipose tissue, which may dysregulate the inlammatory cascade and lead to placental dysfunction in obese women (Vazquez, 2015). In addition, abnormally elevated leptin levels have been associated with preeclampsia and gestational diabetes (Bao, 2015; Taylor, 2015). Fetal leptin is important for the development of several organs that include the pancreas, kidney, heart, and brain. Fetal levels correlate with maternal body mass index (BMI) and birthweight. Lower levels are linked to fetal-growth restriction (Brifa, 2015; Tsai, 2015).

1	Dozens of hormones with metabolic and/or inflammatory functions are produced by adipose tissue. Adiponectin is a peptide produced primarily in maternal fat but not in the placenta (Haghiac, 2014). Adiponectin levels inversely correlate with adiposity, and it acts as a potent insulin sensitizer. Despite reduced adiponectin levels in women with gestational diabetes, directed assays are not useful for predicting diabetes development (Hauguel-de Mouzon, 2013). Ghrelin is a peptide secreted principally by the stomach in response to hunger. It cooperates with other neuroendocrine factors, such as leptin, in energy homeostasis modulation. Ghrelin is also expressed in the placenta and likely has a role in fetal growth and cell proliferation (Gonzalez-Dominguez, 2016). Angelidis and associates (2012) have reviewed the many functions of ghrelin in the regulation of reproductive function.

1	Viatin is a peptide that was first identified as a growth fac tor for B lymphocytes, but it is mainly produced within adipose tissue. Mumtaz and colleagues (2015) propose that elevated levels of visfatin and leptin impair uterine contractility. Such findings may provide a physiological basis for the observation that maternal obesity raises the risk for dysfunctional labor.

1	During normal pregnancy, nearly 1000 mEq of sodium and 300 mEq of potassium are retained (Lindheimer, 1987). Although the glomerular filtration rate of sodium and potassium is increased, the excretion of these electrolytes is unchanged during pregnancy as a result of enhanced tubular resorption (Brown, 1986, 1988). Although total accumulations of sodium and potassium are elevated, their serum concentrations are diminished slightly (Appendix, p. 1257). Several mechanisms may explain these lower levels (Odutayo, 2012). In the case of potassium, it possibly involves the expanded plasma volume of pregnancy. With respect to sodium, osmoregulation is altered and the threshold for arginine vasopressin release is lowered. his promotes free water retention and diminished sodium levels.

1	Total serum calcium levels, which include both ionized and nonionized calcium, decrease during pregnancy. his reduction consequent decline in the amount of circulating protein-bound nonionized calcium. Serum ionized calcium levels, however, remain unchanged (Olausson, 2012).

1	he developing fetus imposes a significant demand on maternal calcium homeostasis. For example, the fetal skeleton accretes approximately 30 g of calcium by term, 80 percent of which is deposited during the third trimester. his demand is largely met by a doubling of maternal intestinal calcium absorption mediated partly by 1,25-dihydroxyvitamin D3• hese higher levels of vitamin D are possibly stimulated by a twofold rise in PTH-related peptide levels produced by several tissues including the placenta (Kovacs, 2006; Olausson, 2012). To help compensate, dietary intake of suicient calcium is necessary to prevent excess depletion from the mother. A list of all recommended daily allowances is found in Table 9-5 (p. 167). his is especially important for pregnant adolescents, in whom bones are still developing. Unfortunately, a lack of robust data prevents drawing firm conclusions regarding the utility of calcium and vitamin D supplements during pregnancy (De-Regil, 2016).

1	Serum manesium levels also decline during pregnancy. Bardicef and colleagues (1995) concluded that pregnancy is actually a state of extracellular magnesium depletion. Compared with nonpregnant women, both total and ionized magnesium concentrations are significantly lower during normal pregnancy (Rylander, 2014). Serum phosphate levels lie within the nonpregnant range (Larsson, 2008). Although calcitonin is an important regulator of serum calcium and phosphate, the importance of calcitonin as it relates to pregnancy is poorly understood (Olausson, 2012).

1	Iodine requirements increase during normal pregnancy for several reasons (Moleti, 2014; Zimmermann, 2012). First, maternal thyroxine production rises to maintain maternal euthyroidism and to transfer thyroid hormone to the fetus prior to fetal thyroid functioning. Second, fetal thyroid hormone production increases during the second half of pregnancy. This contributes to greater maternal iodine requirements because iodide readily crosses the placenta. hird, the primary route of iodine excretion is through the kidney. Beginning in early pregnancy, the iodide glomerular iltration rate increases by 30 to 50 percent. In sum, because of greater thyroid hormone production, fetal iodine requirements, and augmented renal clearance, dietary iodine needs are higher during normal gestation. lthough the placenta has the ability to store iodine, whether this organ functions to protect the fetus from inadequate maternal dietary iodine is currently unknown (Burns, 2011). Iodine deiciency is discussed

1	placenta has the ability to store iodine, whether this organ functions to protect the fetus from inadequate maternal dietary iodine is currently unknown (Burns, 2011). Iodine deiciency is discussed later in this chapter (p. 71) and in Chapter 58 (p. 1126). At the other extreme, maternal supplements containing excessive iodine have been associated with congenital hypothyroidism. his stems from autoregulation in the thyroid gland-known as the Wof-Chaikof fect-to curb thyroxine production in response to iodide overconsumption (Connelly, 2012).

1	With respect to most other minerals, pregnancy induces little change in their metabolism other than their retention in amounts equivalent to those needed for growth. An important exception is the considerably greater requirement for iron, which is discussed subsequently. he well-known hypervolemia associated with normal pregnancy averages 40 to 45 percent above the nonpregnant blood volume after 32 to 34 weeks' gestation (Pritchard, 1965; Zeeman, 2009). In individual women, expansion varies considerably. In some, accumulated volume rises only modestly, whereas in others blood volume nearly doubles. A fetus is not essential, as augmented blood volume develops in some with hydatidiform mole. Pregnancy-induced hypervolemia serves several functions.

1	Pregnancy-induced hypervolemia serves several functions. First, it meets the metabolic demands of the enlarged uterus and its greatly hypertrophied vascular system. Second, it provides abundant nutrients and elements to support the rapidly growing placenta and fetus. Third, the expanded intravascular volume protects the mother, and in turn the fetus, against the deleterious efects of impaired venous return in the supine and erect positions. Last, it safeguards the mother against the adverse efects of parturition-associated blood loss.

1	Maternal blood volume begins to accrue during the irst trimester. By 12 menstrual weeks, plasma volume expands by approximately 15 percent compared with that prior to pregnancy (Bernstein, 2001). Maternal blood volume grows most rapidly during the midtrimester, rises at a much slower rate during the third trimester, and reaches a plateau during the last 6.5 � 6.0 ! 5.5 5.0 4.0 3.5 • Twin pregnancy• Singleton pregnancy 3.0 7 -6 -ls 5E 4)E) 3�'5r� 2:� Menses First Second Third Postpartum

1	FIGURE 4-6 Estimated daily iron requirements during pregnancy in a 55-kg woman. (Modified from Koenig, 2014.) (n 10) and singletons (n 40). Data shown as medians. (Data from Thomsen, 1994.) several weeks of pregnancy (Fig. 4-5). Blood volume accrues even more dramatically in twin gestations. During blood volume expansion, plasma volume and erythrocyte number rise. Although more plasma than erythrocytes is usually added to the maternal circulation, the increase in erythrocyte volume is considerable and averages 450 mL (Pritchard, 1960). Moderate erythroid hyperplasia develops in the bone marrow, and the reticulocyte count is elevated slightly during normal pregnancy. These changes are almost certainly related to an elevated maternal plasma erythropoietin level.

1	Because ofgreat plasma augmentation, both hemoglobin concentration and hematocrit decline slightly during pregnancy (Appendix, p. 1255).s aresult, whole bloodviscositydecreases (Huisman, 1987). Hemoglobin concentration at term averages 12.5 g/dL, and in approximately 5 percent ofwomen it is below 11.0 g/dL. Thus, a hemoglobin concentration below 11.0 g/dL, especially late in pregnancy, is considered abnormal and usually due to irondeficiency anemia rather than pregnancy hypervolemia.

1	The total iron content of normal adult women ranges from 2.0 to 2.5 g, or approximately half that found normally in men. Most of this is incorporated in hemoglobin or myoglobin, and thus, iron stores of normal young women only approximate 300 mg (Pritchard, 1964). Although the lower iron levels in women may be partly due to menstrual blood loss, other factors have a role, particularly hepcidin-a peptide hormone that functions as a homeostatic regulator of systemic iron metabolism. Hepcidin levels rise with inlammation, but drop with iron deficiency and several hormones, including testosterone, estrogen, vitamin D, and possibly prolactin (Uu, 2016; Wang, 2015). Lower hepcidin levels are associated with greater absorption ofiron via ferroportin in enterocytes (Camaschella, 2015).

1	Of the approximate 1000 mg ofiron required for normal pregnancy, about 300 mg is actively transferred to the fetus and placenta, and another 200 mg is lost through various normal excretion routes, primarily the gastrointestinal tract. These are obligatory losses and accrue even when the mother is iron deficient. The average increase in the total circulating erythrocyte volume-about 450 mL-requires another 500 mg. Recall that each 1 mL oferythrocytes contains 1.1 mg of iron.

1	As shown in Figure 4-6, because most iron is used during the latter halfofpregnancy, the iron requirement becomes large after midpregnancy and averages 6 to 7 mg/d (Pritchard, 1970). In most women, this amount is usually not available from iron stores or diet. Thus, without supplemental iron, the optimal rise in maternal erythrocyte volume will not develop, and the hemoglobin concentration and hematocrit will fall appreciably as plasma volume rises. At the same time, fetal red cell production is not impaired because the placenta transfers iron even if the mother has severe iron-deficiency anemia. In severe cases, we have documented maternal hemoglobin values of 3 gldL, and at the same time, fetuses had hemoglobin concentrations of 16 gldL. The mechanisms of placental iron transport and regulation are complex (Koenig, 2014; McArdle, 2014).

1	Ifthe nonanemic pregnant woman is not given supplemental iron, then serum iron andferritin concentrations decline atermidpregnancy. Importantly, hepcidin levels drop early in pregnancy (Hedengran, 2016; Koenig, 2014). s noted, lower hepcidin levels aid iron transfer into the maternal circulation via ferroportin in enterocytes. Lower hepcidin levels also augment iron transport into the fetus via ferroportin in syncytiotrophoblast. With normal vaginal delivery, 500 to 600 mL of blood is typically lost, and thus not all the maternal iron added in the form of hemoglobin is spent (Pritchard, 1965). he excess hemoglobin iron becomes stored iron.

1	Pregnancy is associated with suppression of various humoral and cell-mediated immunological functions (Chap. 5, p. 95). his permits accommodation of the "foreign" semiallogeneic fetal graft that contains antigens of both maternal and paternal origin (Redman, 2014). he tolerance that exists at the maternal-fetal interface remains a great unsolved medical mystery. This tolerance is complex and involves certain immune system adaptations and crosstalk among the maternal microbiome, uterine decidua, and trophoblast. In particular, areas of the uterus that were previously considered sterile are colonized with bacteria. In most cases, these microbes are believed to be commensal and play a tolerizing and protective role. Indeed, commensal organisms may inhibit the proliferation of certain pathogens. Several reviewers have described these relationships (Mor, 2015; Racicot, 2014; Sisti, 2016).

1	tection at the maternal-fetal interface involves the expression of the trophoblast. Recall that all cells of the body express a "badge" immune responses. For most cells of the body, this "badge" is known as MHC Class Ia. However, it is uncommon for two unrelated individuals to share compatible MHC class Ia. This creates a potential problem for reproduction because half of the fetus is composed of paternally derived antigens. To circumvent this problem, trophoblast cells express a form of MHC that does not vary between individuals. This "nonclassic" MHC is known as human leukocyte antigen class Ib and includes HLA-E, HLA F, and HLA-G. Recognition of these HLA class Ib proteins by natural killer cells residing within the decidua inhibits their activ ity and promotes immune quiescence (Djurisic, 2014).

1	Another immune adaptation that promotes tolerances stems from important changes in CD4 T lymphocyte subpopulations in pregnancy. First, Th1-mediated immunity shifts to h2-mediated immunity. Indeed, an important antiinlammatory component of pregnancy involves suppression ofT-helper (Th) 1 and T-cytotoxic (Tc) 1 cells, which lower secretion of interleukin-2 (IL-2), interferon-a, and tumor necrosis factor (TNF). Moreover, suppressed Th1 response is thought to be a requisite for pregnancy continuation. It also may explain pregnancy-related remission of some autoimmune disorders such as rheumatoid arthritis, multiple sclerosis, and Hashimoto thyroiditis-which are cell-mediated immune diseases stimulated by h1 cytokines (Kumru, 2005). With suppression of Th1 cells, there is upregulation of Th2 cells to increase secretion of IL-4, IL-10, and IL-13 (Michimata, 2003). hese h2 cytokines promote humoral, or antibody-based, immunity. hus, autoimmune diseases mediated mainly by autoantibodies,

1	cells to increase secretion of IL-4, IL-10, and IL-13 (Michimata, 2003). hese h2 cytokines promote humoral, or antibody-based, immunity. hus, autoimmune diseases mediated mainly by autoantibodies, such as systemic lupus erythematosus, may lare if the disease is already active in early pregnancy. But, the transition to an antibody-mediated immunity is an important defense during pregnancy and early puerperium. In cervical mucus, peak levels of immunoglobulins A and G (IgA and IgG) are significantly higher during pregnancy, and the immunoglobulin-rich cervical mucus plug creates a barrier to ascending infection (Hansen, 2014; Wang, 2014). Similarly, IgG is transferred to the developing fetus in the third trimester as a form of passive immunity, ostensibly in anticipation of birth. Further, immunoglobulins secreted into breast milk during lactation augment neonatal defenses against infection.

1	Other subpopulations of CD4 T lymphocytes serve mucosal and barrier immunity. These specific CD4-positive cells are known as h17 cells and Treg cells. h17 cells are proinflammatory and express the cytokine IL-17 and the retinoic acid receptor-related orphan receptors (RORs). Treg cells express the transcription factor forkhead box protein-3 (FOXP3) and confer tolerizing activity. here is a shift toward Treg CD4 cells in the first trimester, which peaks during the second trimester and falls toward delivery (Figueiredo, 2016). his shift may promote tolerance at the maternal-fetal interface (La Rocca, 2014). In particular, failure of these CD4 T lymphocyte subpopulation alterations may be related to preeclampsia development (Vargas-Rojas, 2016).

1	nonpregnant values, and upper values approach 15,000/jLL (Appendix, p. 1255). During labor and the early puerpe rium, values may become markedly elevated, attaining levels of 25,000/LL or greater. The cause is unknown, but the same response occurs during and after strenuous exercise. The leuko cytosis possibly represents the reappearance of leukocytes previ ously shunted out of active circulation. The distribution of lymphocyte cell types is also altered during pregnancy. Speciically, B lymphocytes numbers are unchanged, but the absolute numbers of T lymphocytes rise and create a relative increase. Concurrently, the ratio of CD4 to CD8 T lymphocytes does not change (Kuhnert, 1998).

1	Many tests performed to diagnose inlammation cannot be used reliably during pregnancy. For example, leukocyte alkaline phosphatase levels-used to evaluate myeloproliferative disorders-are elevated beginning early in pregnancy. he concentration of C-reactive protein, an acute-phase serum reactant, rises rapidly in response to tissue trauma or inflammation. Median C-reactive protein levels in pregnancy and labor are higher than for nonpregnant women (Anderson, 2013; Watts, 1991). Of nonlaboring gravidas, 95 percent had levels of 1.5 mg/ dL or less, and gestational age did not afect serum levels. Another marker of inlammation, the erythrocyte sedimentation rate (ESR), is increased in normal pregnancy because of elevated plasma globulins and ibrinogen levels. Complement actors C3 and C4 levels also significantly rise during the second and third trimesters (Gallery, 1981; Richani, 2005). Last, concentrations ofprocalcitonin, a normal precursor of calcitonin, increase at the end of the

1	also significantly rise during the second and third trimesters (Gallery, 1981; Richani, 2005). Last, concentrations ofprocalcitonin, a normal precursor of calcitonin, increase at the end of the third trimester and through the irst few postpartum days. Procalcitonin levels rise with severe bacterial infections but remain low in viral infections and nonspecific inlammatory disease. However, measured levels poorly predict development of overt or subclinical chorioamnionitis after premature rupture of membranes (Thornburg, 2016).

1	During normal pregnancy, both coagulation and fibrinolysis are augmented but remain balanced to maintain hemostasis (Kenny, 2014). Evidence of activation includes increased concentrations of all clotting factors except factors I and III (Table 4-3). Of procoagulants, the level and rate of thrombin generation throughout gestation progressively increase (McLean, 2012). In normal nonpregnant women, plasma ibrinogen (factor 1) averages 300 mg/dL and ranges from 200 to 400 mg/dL. During normal pregnancy, the fibrinogen concentration rises approximately 50 percent. In late pregnancy, it averages 450 mg/dL, with a range from 300 to 600 mg/dL. his contributes greatly to ap <.05. Data shown as mean ± standard deviation. PlT = partial thromboplastin time; tPA = tissue activator. Data from Uchikova, 2005. the striking increase in the ESR. Also, levels of factor XIIIibrin stabilizing oetor-signiicantly drop as normal pregnancy advances (Sharief, 2014).

1	Data from Uchikova, 2005. the striking increase in the ESR. Also, levels of factor XIIIibrin stabilizing oetor-signiicantly drop as normal pregnancy advances (Sharief, 2014). The end product of the coagulation cascade is ibrin formation, and the main function of the fibrinolytic system is to remove excess ibrin (Fig. 41-29, p. 784). Tissue plasminogen activator (tP A) converts plasminogen into plasmin, which causes ibrinolysis and produces ibrin-degradation products such as D-dimers. Although somewhat conlicting, most evidence suggests that fibrinolytic activity is reduced in normal pregnancy (Kenny, 2014). As reviewed by Cunningham and Nelson (2015), these changes favor ibrin formation. Although this is countered by increased levels of plasminogen, the net result is that pregnancy is a procoagulant state. Such changes serve to ensure hemostatic control during normal pregnancy, particularly during delivery when a certain amount of blood loss is expected.

1	Several proteins are natural inhibitors of coagulation, including proteins C and S and antithrombin (Fig. 52-I, p. 1005). Inherited or acquired deficiencies of these and other natural regulatory proteins-collectively referred to as thrombophiliasaccount for many thromboembolic episodes during pregnancy. hey are discussed in Chapter 52 (p. 1006).

1	Activated protein , along with the cofactors protein Siand factor V, functions as an anticoagulant by neutralizing the procoagulants factor Va and factor VIlla. During pregnancy, resistance to activated protein C grows progressively and is related to a concomitant drop in free protein S levels and greater factor VIII concentrations. Between the irst and third trimesters, activated protein C levels decline from 2.4 to 1.9 V/mL, and free protein S concentrations diminish from 0.4 to 0.16 V/ mL (Cunningham, 2015; Walker, 1997). Antithrombin levels decrease by 13 percent between midpregnancy and term and fall 30 percent from this baseline until 12 hours after delivery. By 72 hours after delivery, there is a return to baseline Games, 2014).

1	Normal pregnancy promotes platelet changes. In one study, the average platelet count declined slightly during pregnancy to 213,OOO/1L compared with 250,000/1L in nonpregnant controls (Boehlen, 2000). hrombocytopenia deined as below the 2.5th percentile corresponded to a platelet count of 116,OOO/1L. Lower platelet concentrations are partially due to hemodilution. Also, platelet consumption is likely augmented and creates a greater proportion of younger and therefore larger platelets (Han, 2014; Valera, 2010). Further, levels of several markers of platelet activation rise with gestational age but drop postpartum (Robb, 2010). Because of splenic enlargement, there may be an element of "hypersplenism," in which platelets are prematurely destroyed (Kenny, 2014).

1	By the end of normal pregnancy, the spleen enlarges by up to 50 percent compared with that in the irst trimester (Maymon, 2007). Moreover, Gayer and coworkers (2012) found that splenic size was 68-percent greater compared with that of nonpregnant controls. he cause of this splenomegaly is unknown, but it might follow the increased blood volume and/or the hemodynamic changes of pregnancy.

1	Changes in cardiac function become apparent during the irst 8 weeks of pregnancy (Hibbard, 2014). Cardiac output is increased as early as the ifth week and reflects a reduced systemic vascular resistance and an increased heart rate. Compared with prepregnancy measurements, brachial systolic blood pressure, diastolic blood pressure, and central systolic blood pressure are all signiicantly lower 6 to 7 weeks from the last menstrual period (Mahendru, 2012). he resting pulse rate rises approximately 10 beats/min during pregnancy. Nelson and associates (2015) found that for both normal and overweight women, heart rate increased signiicantly between 12 and 16 weeks' and between 32 and 36 weeks' gestation. Between weeks 10 and 20, plasma volume expansion begins, and preload rises. his augmented preload results in signiicantly larger left atrial volumes and ejection fractions (Cong, 2015).

1	Ventricular performance during pregnancy is inluenced by both the decrease in systemic vascular resistance and changes in pulsatile arterial low. Multiple factors contribute to this overall altered hemodynamic function, which allows the physiological demands of the fetus to be met while maintaining maternal cardiovascular integrity (Hibbard, 2014). hese changes during the last half of pregnancy and efects of maternal posture are summarized in Figure 4-7. As the diaphragm becomes progressively elevated, the heart is displaced to the left and upward and is rotated on its long axis. As a result, the apex is moved somewhat laterally from its usual position and produces a larger cardiac silhouette in chest radiographs. Furthermore, gravidas normally have some degree of benign pericardial efusion, which may enlarge the

1	FIGURE 4-7 Left ventricular stroke volume across pregnancy compared with 12-week postpartum (nonpregnant) values for normal-weight women in the supine and lateral positions. (Data from Nelson, 201o5.) cardiac silhouette (Enein, 1987). These factors make it dii cult to precisely identiy moderate degrees of cardiomegaly by simple radiographic studies. graphic changes, and the most common is slight left-axis devia tion due to the altered heart position. Q waves in leads II, III and avF and lat or inverted T-waves in leads III, V1-V3 may also occur (Sunitha, 2014). During pregnancy, many of the normal cardiac souns are modiied. hese include: (1) an exaggerated splitting ofthe irst heart sound and increased loudness ofboth components, (2) no deinite changes in the aortic and pulmonary elements of the second sound, and (3) a loud, easily heard third sound (Cut forth, 1966). In 90 percent of gravidas, they also heard a sys expiration in others and that disappeared shortly after delivery.

1	A soft diastolic murmur was noted transiently in 20 percent, and continuous murmurs arising from the breast vasculature in 10 percent (Fig. 49-1, p. 950). Structurally, the expanding plasma volume seen during nor mal pregnancy is relected by enlarging cardiac end-systolic and end-diastolic dimensions. Concurrently, however, septal thick ness or ejection fraction does not change. This is because the dimensional changes are accompanied by substantive ventricu lar remodeling, which is characterized by left-ventricular mass expansion of 30 to 35 percent near term. In the nonpregnant state, the heart is capable of remodeling in response to stimuli such as hypertension and exercise. Such cardiacplasticiy likely as that in exercise, and pathological hypertrophy-such as with hypertension (Hill, 2008). Stewart and colleagues (2016) used cardiac MR imaging to prospectively evaluate cardiac remodeling during pregnancy.

1	Stewart and colleagues (2016) used cardiac MR imaging to prospectively evaluate cardiac remodeling during pregnancy. Compared with the first trimester, left ventricular mass increased signiicantly beginning at 26 to 30 weeks' gestation, and this continued until delivery (Fig. 4-8). This remodeling is concen tric and proportional to maternal size for both normal and over weight women and resolved within 3 months ofdelivery. Certainly for clinical purposes, ventricular unction during pregnancy is normal, as estimated by the Braunwad ventricuar unction graph (Fig. 4-9). For the given illing pressures, cardiac ..I3 .g 90 ; ..c)> .J=80

1	FIGURE 4-8 Let ventricular mass of normal-weight and overweight women across pregnancy compared with 12-week postpartum (nonpregnant) values. (Data from Stewart, 2016.) output is appropriate andthus cardiac unction during pregnancy is eudynamic. Of the metabolic changes that occur in the heart during pregnancy, the eiciency of cardiac work-which is the product ofcardiac output X mean arterial pressure-is estimated to rise by approximately 25 percent. The associated increase in oxygen consumption is primarily accomplished via increased coronary blood low rather than increased extraction (Liu, 2014).

1	When measured in the lateral recumbent position at rest, cardiac output increases signiicantly beginning in earlypregnancy. It continues to rise and remains elevated during the remainder ofpregnancy. In a supine woman, a large uterus rather consistently compresses veins and diminishes venous return from the lower body. It also may compress the aorta (Bieniarz, 1968). In response, cardiac illing may be reduced and cardiac output ..J= FIGURE 4-9 Relationship between let ventricular stroke work index (LVSWI), cardiac output, and pulmonary capillary wedge pressure (PCWP) in 10 normal pregnant women in the third trimester. (Data from Clark, 1989.) TABLE 4-4. Central Hemodynamic Changes in 10 Normal Nulliparous Women Near Term and 6.2 ± 1.0 4.3 ± 0.9 +43% 18.0 ± 1.5 20.8 ± 1.0 -14% 10.5 ± 2.7 14.5 ± 2.5 -28% 48 ± 6 41 ± 8 NSC aMeasured in lateral recumbent position. bChanges significant unless NSC = no significant change.

1	bChanges significant unless NSC = no significant change. COP = colloid osmotic pressure; PCWP = pulmonary capillary wedge pressure. Data from Clark, 1989. lessened. Speciically, cardiac MR imaging shows that when a woman rolls from her back onto her left side, cardiac output at 26 to 30 weeks' gestation rises by approximately 20 percent and at 32 to 34 weeks by 10 percent (Nelson, 2015). Consistent with this, Simpson and James (2005) found that fetal oxygen saturation is approximately 10 percent higher ifa laboring woman lies in a lateral recumbent position compared with supine. Upon standing, cardiac output falls to the same degree as in the nonpregnant woman (Easterling, 1988).

1	In multifetal pregnancies, compared with singletons, maternal cardiac output is augmented further by almost another 20 percent. Ghi and coworkers (2015) used transthoracic echocardiography to show that irst-trimester cardiac output with twins (mean 5.50 Umin) was more than 20 percent greater than postpartum values. Cardiac output values in the second (6.31 L/min) and third (6.29 Umin) trimesters were increased an additional 15 percent compared with irst-trimester output. Left atrial and left ventricular end-diastolic diameters are also longer with twins due to augmented preload (Kametas, 2003). The greater heart rate and inotropic contractility imply that cardiovascular reserve is reduced in multifetal gestations. During first-stage labor, cardiac output rises moderately. During the second stage, with vigorous expulsive eforts, it is appreciably greater. The pregnancy-induced increase is lost ater delivery, at times dependent on blood loss.

1	During the second stage, with vigorous expulsive eforts, it is appreciably greater. The pregnancy-induced increase is lost ater delivery, at times dependent on blood loss. Clark and associates (1989) conducted invasive studies to measure hemodynamic function late in pregnancy (Table 4-4). Right heart catheterization was performed in 10 healthy nulliparas at 35 to 38 weeks' gestation, and again at 11 to 13 weeks postpartum. Late pregnancy was associated with the expected increases in heart rate, stroke volume, and cardiac output. Systemic vascular and pulmonary vascular resistance both dropped signiicantly, as did colloid osmotic pressure. Pulmonary capillary wedge pressure and central venous pressure did not change appreciably. Thus, although cardiac output rises, left ventricular function as measured by stroke work index remains similar to the nonpregnant normal range (see Fig. 4-9). Put another way, normal pregnancy is not a continuous "high-output" state.

1	Changes in posture afect arterial blood pressure (Fig. 4-10). Brachial artery pressure when sitting is lower than that when in the lateral recumbent supine position (Bamber, 2003). Additionally, systolic blood pressure is lower in the lateral positions compared with either the flexed sitting or supine positions (Armstrong, 2011). Arterial pressure usually declines to a nadir at 24 to 26 weeks' gestation and rises thereafter. Diastolic pressure decreases more than systolic. FIGURE 4-10 Sequential changes (±SEM) in blood pressure throughout pregnancy in 69 women in supine (blue lines) and left lateral recumbent positions (red lines). PP = postpartum. (Adapted from Wilson, 1980.) Morris and associates (2015) studied measures of vas cular compliance before pregnancy, during pregnancy, and postpartum. Compared with healthy nonpregnant controls, stifness, measured using pulse wave velocity, were observed between the prepregnant and the postpartum time periods.

1	sibly help explain why the risk of preeclampsia is reduced in subsequent pregnancies. Antecubital venous pressure remains unchanged during pregnancy. In the supine position, however, femoral venous pressure rises steadily, from approximately 8 mm Hg early in pregnancy to 24 mm Hg at term. Venous blood flow in the legs is retarded during pregnancy except when the lateral recumbent position is assumed (Wright, 1950). his tendency toward blood stagnation in the lower extremities during later pregnancy is attriburable to occlusion of the pelvic veins and inferior vena cava by the enlarged uterus. he elevated venous pressure returns to normal when the pregnant woman lies on her side and immediately after delivery (McLennan, 1943). These alterations contribute to the dependent edema frequently experienced and to the development of varicose veins in the legs and vulva, as well as hemorrhoids. These changes also predispose to deep-vein thrombosis.

1	In approximately 10 percent of women, supine compression of the great vessels by the uterus causes signiicant arterial hypotension, sometimes referred to as the supine hypotensive syndrome (Kinsella, 1994). Also when supine, uterine arterial pressure-and thus uterine blood flow-is signiicantly lower than that in the brachial artery. Evidence to support whether this directly afects fetal heart rate patterns in uncomplicated low-risk pregnancies is conlicting (Armstrong, 2011; Ibrahim, 2015; Tamas, 2007). Similar changes can also be seen with hemorrhage or with spinal analgesia. • Renin, Angiotensin II, and Plasma Volume

1	• Renin, Angiotensin II, and Plasma Volume The renin-angiotensin-aldosterone axis is intimately involved in blood pressure control via sodium and water balance. All components of this system show increased levels in normal pregnancy. Renin is produced by both the maternal kidney and the placenta, and greater amounts of renin substrate (angiotensinogen) are produced by both maternal and fetal liver. Elevated angiotensinogen levels result, in part, from augmented estrogen production during normal pregnancy and are important in irst-trimester blood pressure maintenance (Lumbers, 2014).

1	Gant and associates (1973) reported that nulliparas who remained normotensive became and stayed refractory to the pressor efects of infused angiotensin II. Conversely, those who ultimately became hypertensive developed, but then lost, this refractoriness. The diminished vascular responsiveness to angiotensin II may be progesterone related. Normally, pregnant women lose their acquired vascular refractoriness to angiotensin II within 15 to 30 minutes after the placenta is delivered. Large amounts of intramuscular progesterone given during late labor delay this diminishing refractoriness.

1	Large amounts of intramuscular progesterone given during late labor delay this diminishing refractoriness. At least two species of these-atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)-are secreted by cardiomyocytes in response to chamber-wall stretching. These peptides regulate blood volume by provoking natriuresis, diuresis, and vascular smooth-muscle relaxation. In nonpregnant and pregnant patients, levels of BNP and of amino-terminal pro-brain natriuretic peptide (Nt pro-BNP), as well as newer analytes such as suppressor of tumorigenicity 2 (ST2), may be useful in screening for depressed left ventricular systolic function and determining chronic heart failure prognosis (Ghashghaei, 2016).

1	During normal pregnancy, plasma ANP and BNP levels are maintained in the nonpregnant range despite greater plasma volume (Yurteri-Kaplan, 2012). In one study, median BNP levels were stable across pregnancy with values <20 pg/mL (Resnik, 2005). BNP levels are increased in severe preeclamp sia, and this may be caused by cardiac strain from increased afterload (Afshani, 2013). It would appear that ANP-induced ume expansion and in the elevated plasma aldosterone concen trations characteristic of normal pregnancy.

1	Elevated prostaglandin production during pregnancy is thought to have a central role in control of vascular tone, blood pressure, and sodium balance. Renal medullary prostaglandin E2 synthesis is markedly elevated during late pregnancy and is presumed to be natriuretic. Levels of prostacyclin (PGI2), the principal prostaglandin of endothelium, also rise during late pregnancy. PGI2 regulates blood pressure and platelet function. It helps maintain vasodilation during pregnancy, and its deiciency is associated with pathological vasoconstriction (Shah, 2015). Thus, the ratio of PGI2 to thromboxane in maternal urine and blood is considered important in preeclampsia pathogenesis (Majed, 2012).

1	Several endothelins are generated in pregnancy. Endothelin-1 is a potent vasoconstrictor produced in endothelial and vascular smooth muscle cells and regulates local vasomotor tone (George, 2011; Lankhorst, 2016). Its production is stimulated by angiotensin II, arginine vasopressin, and thrombin. Endothelins, in turn, stimulate secretion of ANP, aldosterone, and catecholamines. Vascular sensitivity to endothelin-1 is not altered during normal pregnancy. Pathologically elevated levels may playia role in preeclampsia (Saleh, 2016). This potent vasodilator is released by endothelial cells and may modiy vascular resistance during pregnancy. Moreover, nitric oxide is an important mediator of placental vascular tone and development (Krause, 2011; Kulandavelu, 2013). Abnormal nitric oxide synthesis has been linked to preeclampsia development (Laskowska, 2015; Vignini, 2016).

1	Of anatomic changes, the diaphragm rises approximately 4 cm during pregnancy (Fig. 4-11). he subcostal angle widens appreciably as the transverse diameter of the thoracic cage lengthens approximately 2 cm. The thoracic circumference increases about 6 cm, but not suiciently to prevent reduced residual lung volumes created by the elevated diaphragm. Even so, diaphragmatic excursion is greater in pregnant than in nonpregnant women.

1	Of physiological lung changes, unctional residual capaciy (PRe) decreases by approximately 20 to 30 percent or 400 to 700 mL during pregnancy (Fig. 4-12). his capacity is composed of expiratory reserve volume-which drops 15 to 20 percent or 200 to 300 mL-and residual volume-which decreases 20 to 25 percent or 200 to 400 mL. FRe and residual volume decline progressively across pregnancy due to diaphragm elevation. Signiicant reductions are observed by the sixth month. Inspiratory capaciy, the maximum volume that can be inhaled from FRe, rises by 5 to 10 percent or 200 to 350 mL during pregnancy. Total lung capaci-the combination of FRe and inspiratory capacity-is unchanged or decreases by less than 5 percent at term Decreased plasma osmolality also results in less respiratory depression (Moen, 2014). his provides an additional mechanism for the increased minute ventilation seen in pregnancy, and one that is not dependent on progesterone.

1	Regarding pulmonary function, peak expiratory low rates rise progressively as gestation advances (Grindheim, 2012). Lung compliance is unafected by pregnancy. Airway conductance is increased and total pulmonary resistance reduced, possibly as a result of progesterone. The maximum breathing capaciy andorced or timed vital capaciy are not altered appreciably. It is unclear whether the critical closing volume-the lung volume at which airways in the dependent parts of the lung begin to close during expiration-is higher in pregnancy (Hegewald, 2011). Pulmonary function with a singleton pregnancy does not signiicantly difer from that with twins (MAulife, 2002; Siddiqui, 2014). Importantly, the greater oxygen requirements and perhaps the increased critical closing volume imposed by pregnancy make respiratory diseases more serious.

1	Demir and colleagues (2015) studied nasal physiology in 85 pregnant women. Although the minimal cross-sectional area decreased between the irst and third trimesters, subjective reports ,/ 103.5° "" II , " \\ . (37 weeks) : (Hegewald, 2011). The respiratory rate is essentially unchanged, but tidal volume and resting minute ven as pregnancy advances. Kolar zyk and coworkers (2005) .. -� is-7 em .--_ - reported significantly greater mean tidal volumes-0.66 to 0.8 LI min-and resting minute ventilations-10.7 to 14.1 Llmin-compared with those of nonpregnant women. The elevated minute ventilation is caused by several factors. These include enhanced respira ,;, . ,----------.---.A..,, .I \IIII,\\ ..<: ::: --------------- toy drive primarily due to the stimulatory action of proges terone, low expiratory reserve

1	Chest wall measurements in nonpregnant (left) and pregnant women (right). The subcostal angle increases, as does the anteroposterior and transverse diameters of the chest wall and chest wall circumference. These changes compensate for the 4-cm elevation of the diaphragm volume, and compensated respiso that total lung capacity is not significantly reduced. (Redrawn from Hegewald MJ, Crapo RO: ratory alkalosis (Heenan, 2003). Respiratory physiology in pregnancy. Clin Chest Med 32(1 ):1,2011.) 5 4 TLC 2 FVC FAC RV Not pregnant Pregnant (7-9 mos.) 4 FVC 3 2 RV

1	FIGURE 4-1 2 Changes in lung volumes with pregnancy. The most significant changes are reduction in functional residual capacity (FRC) and its subcomponents, expiratory reserve volume (ERV) and residual volume (RV), as well as increases in inspiratory capacity (IC) and tidal volume (). (Redrawn from Hegewald MJ, Crapo RO: Respiratory physiology in pregnancy. Clin Chest Med 32(1):1,2011.) of nasal congestion or total nasal resistance did not signiicantly difer among trimesters or compared with nonpregnant controls.

1	he amount ofoxygen delivered into the lungs by the increased tidal volume clearly exceeds oxygen requirements imposed by pregnancy. Moreover, the total hemoglobin mass and, in turn, total oxygen-carrying capacity rise appreciably during normal pregnancy, as does cardiac output. Consequently, the maternal arteriovenous oxygen diference is diminished. Oxygen consumption grows approximately 20 percent during pregnancy, and it is approximately 10 percent higher in multifetal gestations (Ajjimaporn, 2014). During labor, oxygen consumption increases 40 to 60 percent (Bobrowski, 2010).

1	A greater awareness of a desire to breathe is common even early in pregnancy (Milne, 1978). his may be interpreted as dyspnea, which may suggest pulmonary or cardiac abnormalities when none exist. This physiological dyspnea, which should not interfere with normal physical activity, is thought to result from greater tidal volume that lowers the blood Peo2 slightly and paradoxically causes dyspnea. he increased respiratory efort during pregnancy, and in turn the reduction in the partial pressure ofcarbon dioxide in blood (Peo2), is likely induced in large part by progesterone and to a lesser degree by estrogen. Progesterone acts centrally, where it lowers the threshold and raises the sensitivity ofthe chemoreflex response to carbon dioxide (C02) 0ensen, 2005).

1	To compensate for the resulting respiratory alkalosis, plasma bicarbonate levels normally drop from 26 to 22 mmollL. Although blood pH is increased only minimally, it does shift the oxygen dissociation curve to the left. his shift increases the ainity of maternal hemoglobin for oxygen-the Bohr fectthereby lowering the oxygen-releasing capacity of maternal blood. This is ofset because the slight pH rise also stimulates an increase in 2,3-diphosphoglycerate in maternal erythrocytes. ): �:3 This shifts the curve back to the right (Tsai, 1982). hus, reduced Peo2 from maternal hyperventilation aids CO2 (waste) transfer from the fetus to the mother while also aiding oxygen release to the fetus.

1	he urinary system undergoes several remarkable changes in pregnancy (Table 4-5). Kidney size grows approximately 1.0 cm (Cietak, 1985). Both theglomerulariltration rate (CPR) and renal plasma low increase early in pregnancy. he GFR rises as much as 25 percent by the second week after conception and 50 percent by the beginning of the second trimester. his hyperiltration results from two principal factors. First, hypervolemia-induced hemodilution lowers the protein concentration and oncotic pressure ofplasma entering the glomerular microcirculation. Second, renal plasma flow increases by approximately 80 percent before the end of the irst trimester (Conrad, 2014b; Odutayo, 2012). As shown in Figure 4-13, , 25 FIGURE 4-13 Percentage increment in glomerular filtration rate (GFR) and renal plasma flow (RPF) across gestation and in the puerperium. (Data from Odutayo, 201o2.) TABLE 4-5. Renal Changes in Normal Pregnancy

1	TABLE 4-5. Renal Changes in Normal Pregnancy Renal function Glomerular filtration rate and renal plasma flow increase ...50% Maintenance of Decreased bicarbonate threshold; acid-base progesterone stimulates respiratory center osmotic thresholds for AVP release and thirst decrease; hormonal disposal rates increase Size returns to normal postpartum Can be confused with obstructive uropathy; retained urine leads to collection errors; renal infections are more virulent; may be responsible for "distention syndrome"; elective pyelography should be deferred to at least 12 weeks postpartum Serum creatinine decreases during normal gestation; >0.8 mg/dL (> 72 �mol/L) creatinine already borderline; protein, amino acid, and glucose excretion all increase

1	Serum creatinine decreases during normal gestation; >0.8 mg/dL (> 72 �mol/L) creatinine already borderline; protein, amino acid, and glucose excretion all increase Serum bicarbonate decreased by 4-5 mEq/L; Pc02 decreased 10 mm Hg; a Pc02 of 40 mm Hg already represents CO2 retention 5 mEq/L) during normal gestation; increased placental metabolism of AVP may cause transient diabetes insipidus during pregnancy AVP = vasopressin; IVP = intravenous pyelography; Pc02= partial pressure carbon dioxide. Modified from Lindheimer, 2000. elevated GFR persists until term, even though renal plasma flow declines during late pregnancy. Primarily as a consequence of this elevated GFR, approximately 60 percent of nulliparas during the third trimester experience urinary frequency, and 80 percent experience nocturia (F rederice, 2013).

1	During the puerperium, a marked GFR persists during the first postpartum day, principally from the reduced glomerular capillary oncotic pressure. A reversal of the gestational hypervolemia and hemodilution, still evident on the first postpartum day, eventuates by the second week postpartum (Odutayo, 2012). Studies suggest that relaxin, discussed earlier (p. 52), may mediate both increased GFR and renal blood flow during pregnancy (Conrad, 2014a; Helal, 2012). Relaxin boosts renal nitric oxide production, which leads to renal vasodilation and lowered renal aferent and eferent arteriolar resistance. This augments renal blood flow and GFR (Bramham, 2016). Relaxin may also increase vascular gelatinase activity during pregnancy, which leads to renal vasodilation, glomerular hyperiltration, and reduced myogenic reactivity of small renal arteries (Odutayo, 2012).

1	As with blood pressure, maternal posture may considerably inluence several aspects of renal function. Late in pregnancy, the sodium excretion rate in the supine position averages less than half that in the lateral recumbent position. The efects of posture on GFR and renal plasma low vary. One unusual feature of the pregnancy-induced changes in renal excretion is the remarkably increased amounts of some nutrients lost in the urine. Amino acids and water-soluble vitamins are excreted in much greater amounts (Shibata, 2013).

1	Of renal function tests, serum creatinine levels decline during normal pregnancy from a mean of 0.7 to 0.5 mg/dL. Values of 0.9 mg/dL or reater sugest underying renal disease and prompt further evaluation. Creatinine clearance in pregnancy averages 30 percent higher than the 100 to 115 mLi min in nonpregnant women. his is a useful test to estimate renal function, provided that complete urine collection is made during an accurately timed period. If this is not done precisely, results are misleading (Lindheimer, 2000, 2010). During the day, pregnant women tend to accumulate water as dependent edema, and at night, while recumbent, they mobilize this fluid with diuresis. This reversal of the usual nonpregnant diurnal pattern of urinary flow causes nocturia, and urine is more dilute than in nonpregnant women. Failure of a pregnant woman to excrete concentrated urine after withholding fluids for approximately 18 hours does not necessarily signiy renal damage. In fact, the kidneys in these

1	women. Failure of a pregnant woman to excrete concentrated urine after withholding fluids for approximately 18 hours does not necessarily signiy renal damage. In fact, the kidneys in these circumstances function perfectly normally by excreting mobilized extracellular fluid of relatively low osmolality.

1	Glucosuria during pregnancy may not be abnormal. The appreciably increased GFR, together with impaired tubular reabsorptive capacity for filtered glucose, accounts for most cases of glucosuria. Chesley (1963) calculated that about a sixth of pregnant women will spill glucose in the urine. hat said, although common during pregnancy, when glucosuria is identiied, a search for diabetes mellitus is pursued. Hematuria frequently results from contamination during collection. If not, it most often suggests urinary tract disease or infection. Hematuria is common after diicult labor and delivery because of trauma to the bladder and urethra.

1	Proteinuria is typically deined in nonpregnant subjects as a protein excretion rate of more than 150 mg/ d. Because of the aforementioned hyperiltration and possible reduction of tubular reabsorption, proteinuria during pregnancy is usually considered signiicant once a protein excretion threshold total protein excretion by gestational age. Mean and 95-percent confidence limits are outlined. (Redrawn from Higby K, Suiter CR,

1	Phelps JY, et al: Normal values of urinary albumin and total protein excretion during pregnancy. Am J Obstet Gynecol 171 :984, 1994.) of at least 300 mg/d is reached (Odutayo, 2012). Higby and coworkers (1994) measured protein excretion in 270 normal women throughout pregnancy (Fig. 4-14). Mean 24-hour excretion for all three trimesters was 11i5 mg, and the upper 95-percent conidence limit was 260 mg/d without significant diferences by trimester. They showed that albumin excretion is minimal and ranges from 5 to 30 mg/d. Proteinuria increases with gestational age, which corresponds with the peak in CFR (see Fig. 4-13)(Odutayo, 2012).

1	Measuring Urine Protein. The three most commonly employed approaches for assessing proteinuria are the qualitative classic dipstick, the quantitative 24-hour collection, and the albumin/ creatinine or protein/creatinine ratio of a single voided urine specimen. he pitfalls of each approach have been reviewed by Conrad (20 14b) and Bramham (2016) and their colleagues. he principal problem with dipstick assessment is that it fails to account for renal concentration or dilution of urine. For example, with polyuria and extremely dilute urine, a negative or trace dipstick could actually be associated with excessive protein excretion.

1	he 24-hour urine collection is afected by urinaty tract dilatation, which is discussed in the next section. he dilated tract may lead to errors related both to retention-hundreds of milliliters of urine remaining in the dilated tract-and to timing-the remaining urine may have formed hours before the collection. To minimize these pitfalls, the patient is irst hydrated and positioned in lateral recumbency-the deinitive nonobstructive posture-for 45 to 60 minutes. After this, she is asked to void, and this specimen is discarded. Immediately following this void, her 24-hour collection begins. During the final hour of collection, the patient is again placed in the lateral recumbent position. But, at the end of this hour, the inal collected urine is incorporated into the total collected volume (Lindheimer, 2010).

1	Last, the protein/creatinine ratio is a promising approach because data can be obtained quickly and collection errors are ,S 200 avoided. Disadvantageously, the amount of protein per unit of creatinine excreted during a 24-hour period is not constant, and the thresholds to deine abnormal vary. Nomograms for cated pregnancies have been developed (Waugh, 2003).

1	After the uterus completely rises out of the pelvis, it rests on the ureters. This laterally displaces and compresses them at the pelvic brim. Above this level, elevated intraureteral tonus results, and ureteral dilatation is impressive (Rubi, 1968). It is right sided in 86 percent of women (Fig. 4-15) (Schulman, 1975). This unequal dilatation may result from cushioning provided the left ureter by the sigmoid colon and perhaps from greater right ureteral compression exerted by the dextrorotated uterus. The right ovarian vein complex, which is remarkably dilated during pregnancy, lies obliquely over the right ureter and may also contribute to right ureteral dilatation. Progesterone likely has some additional efect. Van Wagenen and Jenkins (1939) described continued ureteral dilatation after removal of the monkey fetus but with the placenta left in situ. The relatively abrupt onset of dilatation in women at midpregnancy, however, seems more consistent with ureteral compression.

1	Ureteral elongation accompanies distention, and the ureter is frequently thrown into curves of varying size, the smaller of which may be sharply angulated. hese so-called kinks are FIGURE 4-15 Hydronephrosis. Plain film from the 15-minute image of an intravenous pyelogram (IVP). Moderate hydronephrosis on the right (arrows) and mild hydronephrosis on the left (arrowheads) are both normal for this 35-week gestation. poorly named, because the term connotes obstruction. hey are usually single or double curves that, when viewed in a radiograph taken in the same plane as the curve, may appear as acute angulations. Another exposure at right angles nearly always identiies them to be gentle curves. Despite these anatomical changes, complication rates associated with ureteroscopy in pregnant and nonpregnant patients do not difer signiicantly (Semins, 2014).

1	he bladder shows few significant anatomical changes before 12 weeks' gestation. Subsequently, however, increased uterine size, the hyperemia that afects all pelvic organs, and hyperplasia of bladder muscle and connective tissues elevate the trigone and thicken its intraureteric margin. Continuation of this process to term produces marked deepening and widening of the trigone. The bladder mucosa is unchanged other than an increase in the size and tortuosity of its blood vessels.

1	Bladder pressure in primigravidas increases from 8 cm H20 early in pregnancy to 20 cm H20 at term (Iosif, 1980). To compensate for reduced bladder capacity, absolute and functional urethral lengths increased by 6.7 and 4.8 mm, respectively. Concurrently, maximal intraurethral pressure rises from 70 to 93 cm H20, and thus continence is maintained. Still, at least half of women experience some degree of urinary incontinence by the third trimester (Abdullah, 20 16a). Indeed, this is always considered in the diferential diagnosis of ruptured membranes. Near term-particularly in nulliparas, in whom the presenting part often engages before labor-the entire base of the bladder is pushed ventral and cephalad. his converts the normally convex surface into a concavity. As a result, diiculties in diagnostic and therapeutic procedures are greatly accentuated. Moreover, pressure from the presenting part impairs blood and lymph drainage from the bladder base, often rendering the area edematous,

1	diagnostic and therapeutic procedures are greatly accentuated. Moreover, pressure from the presenting part impairs blood and lymph drainage from the bladder base, often rendering the area edematous, easily traumatized, and possibly more susceptible to infection.

1	As pregnancy progresses, the stomach and intestines are displaced cephalad by the enlarging uterus. Consequently, the physical findings in certain diseases are altered. he appendix, for instance, is usually displaced upward and somewhat laterally. At times, it may reach the right lank. Pyrosis (heartburn) is common during pregnancy and is most likely caused by reflux of acidic secretions into the lower esophagus. Although the altered stomach position probably contributes to its frequency, lower esophageal sphincter tone also is decreased. In addition, intraesophageal pressures are lower and intragastric pressures higher in pregnant women. Concurrently, esophageal peristalsis has lower wave speed and lower amplitude (Ulmsten, 1978).

1	Gastric empying time is unchanged during each trimester and compared with nonpregnant women (Macfie, 1991; Wong, 2002, 2007). During labor, however, and especially after administration of analgesics, gastric emptying time may be appreciably prolonged. As a result, one danger of general anesthesia for delivery is regurgitation and aspiration of either food-laden or highly acidic gastric contents. Hemorrhoids are common during pregnancy (Shin, 2015). hey are caused in large measure by constipation and elevated pressure in rectal veins below the level of the enlarged uterus. Liver size does not enlarge during human pregnancy. Hepatic arterial and portal venous blood flow, however, increase substantively (Clapp, 2000).

1	Liver size does not enlarge during human pregnancy. Hepatic arterial and portal venous blood flow, however, increase substantively (Clapp, 2000). Some laboratory test results of hepatic function are altered in normal pregnancy (Appendix, p. 1257). Total alkaline phosphatase activity almost doubles, but much of the rise is attributable to heat-stable placental alkaline phosphatase isozymes. Serum aspartate transaminase (AST) , alanine transaminase (AL T), 1-glutamyl transpeptidase (GGT), and bilirubin levels are slightly lower compared with nonpregnant values (Cattozzo, 2013; Ruiz-Extremera, 2005). The serum albumin concentration declines during pregnancy. By late pregnancy, albumin levels may be near 3.0 g/dL compared with approximately 4.3 g/dL in nonpregnant women (Mendenhall, 1970). Total body albumin levels rise, however, because of pregnancy-associated increased plasma volume. Serum globulin levels are also slightly higher.

1	Leucine aminopeptidase is a proteolytic liver enzyme whose serum levels may be increased with liver disease. Its activity is markedly elevated in pregnant women. he rise, however, results from a pregnancy-speciic enzyme(s) with distinct substrate speciicities (Song, 1968). Pregnancy-induced aminopeptidase has oxytocinase and vasopressinase activity that occasionally causes transient diabetes insipidus.

1	During normal pregnancy, gallbladder contractility is reduced and leads to greater residual volume (Braverman, 1980). Progesterone potentially impairs gallbladder contraction by inhibiting cholecystokinin-mediated smooth muscle stimulation, which is the primary regulator of gallbladder contraction. Impaired emptying, subsequent stasis, and the increased cholesterol saturation of bile in pregnancy contribute to the increased prevalence of cholesterol gallstones in multiparas. In one study, approximately 8 percent of women had gallbladder sludge or stones when imaged at 18 and/or 36 weeks' gestation (Ko, 2014). he pregnancy efects on maternal serum bile acid concentrations are still incompletely characterized. his is despite the long-acknowledged propensity for pregnancy to cause intrahepatic cholestasis and pruritus gravidarum from retained bile salts. Cholestasis of pregnancy is described in Chapter 55 (p. 1059).

1	During normal pregnancy, the pUUltary gland enlarges by approximately 135 percent (Gonzalez, 1988). This increase may suiciently compress the optic chiasma to reduce visual fields. Impaired vision from this is rare and usually due to macro ad enomas (Lee, 2014). Pituitary enlargement is primarily caused by estrogen-stimulated hypertrophy and hyperplasia of the lactotrophs (Feldt-Rasmussen, 2011). And, as discussed subse quently, maternal serum prolactin levels parallel the increasing size. Gonadotrophs decline in number, and corticotrophs and thyrotrophs remain constant. Somatotrophs are generally sup pressed due to negative feedback by the placental production of growth hormone.

1	Peak pituitary size may reach 12 mm in MR images in the irst days postpartum. he gland then involutes rapidly and sen, 2011). he incidence of pituitary prolactinomas is not increased during pregnancy (Scheithauer, 1990). When these tumors are large before pregnancy-a macroadenoma measur ing : 1 0 mm-then growth during pregnancy is more likely (Chap. 58, p. 1132). he maternal pituitary gland is not essential for pregnancy maintenance. Many women have undergone hypophysectomy, completed pregnancy successfully, and entered spontaneous labor while receiving compensatory glucocorticoids, thyroid hormone, and vasopressin.

1	During the first trimester, growth hormone is secreted predominantly from the maternal pituitary gland, and concentrations in serum and amnionic luid lie within the nonpregnant range of 0.5 to 7.5 ng/mL (Kletzky, 1985). As early as 6 weeks' gestation, growth hormone secreted from the placenta becomes detectable, and by approximately 20 weeks, the placenta is the principal source of growth hormone secretion (Perez-Ibave, 2014). Maternal serum values rise slowly from approximately 3.5 ng/mL at 10 weeks to plateau at about 14 ng/mL after 28 weeks. Growth hormone in amnionic fluid peaks at 14 to 15 weeks and slowly declines thereafter to reach baseline values after 36 weeks.

1	Placental growth hormone-which difers from pituitary growth hormone by 13 amino acid residues-is secreted by syncytiotrophoblast in a nonpulsatile fashion (Newbern, 2011). Its regulation and physiological efects are incompletely understood, but it inluences fetal growth via up regulation of insulin-like growth factor 1 (IGF-1). Higher levels have been linked with development of preeclampsia (MinaI, 2007; PerezIbave, 2014). Further, placental expression correlates positively with birthweight but negatively with fetal-growth restriction (Koutsaki, 201l). Maternal serum levels are associated with uterine artery resistance changes (Schiessl, 2007). hat said, fetal growth still progresses in the complete absence of this hormone. Although not absolutely essential, the hormone may act in concert with placental lactogen to regulate fetal growth (Newbern, 2011).

1	Maternal plasma prolactin levels increase markedly during normal pregnancy. Concentrations are usually tenfold greater at term-about 150 ng/mL-compared with those of nonpregnant women. Paradoxically, plasma concentrations drop after delivery even in women who are breastfeeding. During early lactation, pulsatile bursts of prolactin secretion are a response to suckling. The principal function of maternal prolactin is to ensure lac tation. Early in pregnancy, prolactin acts to initiate DNA syn thesis and mitosis of glandular epithelial cells and presecretory alveolar cells of the breast. Prolactin also augments the number of estrogen and prolactin receptors in these cells. Finally, pro lactin promotes mammary alveolar cell RNA synthesis, galac topoiesis, and production of casein, lactalbumin, lactose, and lipids (Andersen, 1982). A woman with isolated prolactin dei ciency failed to lactate after two pregnancies (Kauppila, 1987).

1	This establishes prolactin as a requisite for lactation but not for pregnancy. Grattan (2015) has reviewed the numerous physi ological roles of prolactin for facilitating maternal adaptations to pregnancy. A possible role is proposed for a prolactin frag ment in the genesis of peripartum cardiomyopathy (Chap. 49, p. 963) (Cunningham, 2012). Prolactin is present in amnionic fluid in high concen trations. Levels of up to 10,000 ng/mL are found at 20 to 26 weeks' gestation. Thereafter, levels decline and reach a nadir after 34 weeks. Uterine decidua is the synthesis site of prolactin found in amnionic luid. Although the exact function of amni onic luid prolactin is unknown, impaired water transfer from the fetus into the maternal compartment to thereby prevent fetal dehydration is one suggestion.

1	These two hormones are secreted from the posterior pituitary gland. The roles of oxytocin in parturition and lactation are discussed in Chapters 21 (p. 416) and 36 (p. 657), respectively. Brown and colleagues (2013) have reviewed the complex mechanisms that promote quiescence of oxytocin systems during pregnancy. Levels of antidiuretic hormone, also called vasopressin, do not change during pregnancy. hyrotropin-releasing hormone (TRH) is secreted by the hypothalamus and stimulates thyrotrope cells of the anterior pituitary to release thyroid-stimulating hormone (TSH), also called thyrotropin. TRH levels do not rise during normal pregnancy. However, TRH does cross the placenta and may serve to stimulate the fetal pituitary to secrete TSH (horpe-Beeston, 1991).

1	Serum TSH and hCG levels vary with gestational age (Fig. 4-16). As discussed in Chapter 5 (p. 98), the a-subunits of the two glycoproteins are identical, whereas the 3-subunits, although similar, difer in their amino acid sequence. As a result of this structural similarity, hCG has intrinsic thyrotropic activity, and thus, high serum hCG levels cause thyroid stimulation. Indeed, TSH levels in the irst trimester decline in more than 80 percent of pregnant women, however, they still remain in the normal range for nonpregnant women The thyroid gland boosts production of thyroid hormones by 40 to 100 percent to meet maternal and fetal needs (Moleti, 2014). To accomplish this, the thyroid gland undergoes moderate enlargement during pregnancy caused by glandular hyperplasia and greater vascularity. Mean thyroid volume increases from 12 mL in the first trimester to 15 mL at term (Glinoer, Week of pregnancy

1	Week of pregnancy FIGURE 4-16 Relative changes in maternal and fetal thyroid function across pregnancy. Maternal changes include a marked and early increase in hepatic production of thyroxine-binding globulin (TBG) and placental production of human chorionic gonadotropin (hCG). Increased TBG increases serum thyroxine (T4) concentrations. hCG has thyrotropin-like activity and stimulates maternal free T4 secretion. This transient hCG-induced increase in serum T4 levels inhibits maternal secretion of thyrotropin. Except for minimally increased free T4 levels when hCG peaks, these levels are essentially unchanged. Fetal levels of all serum thyroid analytes increase incrementally across pregnancy. Fetal triiodothyronine (T3) does not increase until late pregnancy. (Modified from Burrow, 1994.) 1990). That said, normal pregnancy does not typically cause significant thyromegaly, and thus any goiter warrants evaluation.

1	Early in the first trimester, levels of the principal carrier protein-thyroid-binding globulin (TBG)-rise, reach their zenith at about 20 weeks, and stabilize at approximately double baseline values for the remainder of pregnancy (see Fig. 4-16). he greater TBG concentrations result from both higher hepatic synthesis rates-due to estrogen stimulation-and lower metabolism rates due to greater TBG sialylation and glycosylation. These elevated TBG levels increase total serum thyroxine (T 4) and triiodothyronine (T 3) concentrations, but do not afect the physiologically important serum ree T4 and ree T3 levels. Specifically, total serum T4 levels rise sharply beginning between 6 and 9 weeks' gestation and reach a plateau at 18 weeks. Serum free T 4 levels rise only slightly and peak along with hCG levels, and then they return to normal.

1	Interestingly, T 4 and T 3 secretion is not similar for all pregnant women (G linoer, 1990). Approximately a third of women experience relative hypothyroxinemia, preferential T3 secretion, and higher, albeit normal, serum TSH levels. hus, thyroidal adjustments during normal pregnancy may vary considerably. The fetus relies on maternal T 4, which crosses the placenta in small quantities to maintain normal fetal thyroid function (Chap. 58, p. 1118). Recall that the fetal thyroid does not begin to concentrate iodine until 10 to 12 weeks' gestation. he synthesis and secretion of thyroid hormone by fetal pituitary TSH ensues at approximately 20 weeks. At birth, approximately 30 percent of the T 4 in umbilical cord blood is of maternal origin (Leung,i2012).

1	Normal suppression of TSH during pregnancy may lead to a misdiagnosis of subclinical hyperthyroidism. Of greater concern is the potential failure to identiy women with early hypothyroidism because of suppressed TSH concentrations. To mitigate the likelihood of such misdiagnoses, Dashe and coworkers (2005) conducted a population-based study at Parkland Hospital to develop gestational-age-specific TSH normal curves for both singleton and twin pregnancies (Fig. 4-17). Similarly, Ashoor and associates (2010) established normal ranges for maternal TSH, free T4, and free T3 at 11 to 13 weeks' gestation. hese complex alterations of thyroid regulation do not appear to alter maternal thyroid status as measured by metabolic studies. lthough basal metabolic rate increases progressively by as much as 25 percent during normal pregnancy, most 4.0 0.4

1	FIGURE 4-17 Gestational age-specific thyroid-stimulating hormone (TSH) nomogram derived from 13,599 singleton pregnancies. The nonpregnant reference values of 4.0 and 0.4 mUll are represented as solid black lines. Upper shaded area represents the 28 percent of singleton pregnancies with TSH values above the 97.5th percentile threshold that would not have been identified as abnormal based on the assay reference value of 4.0 mUll. lower shaded area represents singleton pregnancies that would have been (falsely) identified as having TSH suppression based on the assay reference value of 0.4 mUll. (Data from Dashe, 2005.) of this greater oxygen consumption can be attributed to fetal metabolic activity. If fetal body surface area is considered along with that of the mother, the predicted and observed basal metabolic rates are similar to those in nonpregnant women.

1	Iodine requirements increase during normal pregnancy (Chap. 58, p. 1127). In women with low or marginal intake, deiciency may manifest as low T4 and higher TSH levels. Importantly, more than a third of the world population lives in areas where iodine intake is marginal. For the fetus, early exposure to thyroid hormone is essential for the nervous system, and despite public health programs to supplement iodine, severe iodine deiciency resulting in cretinism afects more than 2 million people globally (Syed, 2015).

1	In one longitudinal investigation of 20 women, all markers of bone turnover rose during normal pregnancy and failed to reach baseline levels by 12 months postpartum (More, 2003). Investigators concluded that the calcium needed for fetal growth and lactation may be drawn at least in part from the maternal skeleton. he factors afecting bone turnover yield a net result favoring fetal skeletal formation at the expense of the mother, such that pregnancy is a vulnerable period for osteoporosis (Sanz-Salvador, 2015). hat said, prevention is diicult due to a paucity of identiiable risk factors. Acute or chronic declines in plasma calcium or acute drops in magnesium levels stimulate parathyroid hormone (PTH) release. Conversely, greater calcium and magnesium levels suppress PTH levels. he action of this hormone on bone resorption, intestinal absorption, and kidney reabsorption is to raise extracellular luid calcium concentrations and lower phosphate levels.

1	Fetal skeleton mineralization requires approximately 30 g of calcium, primarily during the third trimester (Sanz-Salvador, 2015). Although this amounts to only 3 percent of the total calcium held within the maternal skeleton, the provision of calcium still challenges the mother. In most circumstances, augmented maternal calcium absorption provides the additional calcium. During pregnancy, the amount of calcium absorbed rises gradually and reaches approximately 400 mg/ d in the third trimester. Greater calcium absorption appears to be mediated by elevated maternal 1,25-dihydroxyvitamin D concentrations. This occurs despite decreased PTH levels during early pregnancy, which is the normal stimulus for active vitamin D production within the kidney. Indeed, PTH plasma concentrations decline during the irst trimester and then rise progressively throughout the remainder of pregnancy (Pitkin, 1979).

1	he increased production of active vitamin D is likely due to placental production of either PTH or a PTH-related protein (PTH-rP). Outside pregnancy and lactation, PTH-rP is usually detectable only in serum of women with hypercalcemia due to malignancy. During pregnancy, however, PTH-rP concentrations increase signiicantly. his protein is synthesized in both fetal tissues and maternal breasts. he C cells that secrete calcitonin are located predominantly in the perifollicular areas of the thyroid gland. Calcitonin opposes actions of PTH and vitamin D and protects the matenal skeleton during times of calcium stress. Pregnancy and lactation cause profound maternal calcium stress, ostensibly for the sake of the fetus. Indeed, fetal calcitonin levels are at least twofold higher than maternal levels (Ohata, 2016). And although maternal levels fall during pregnancy, they generally rise postpartum (M011er, 2013).

1	Calcium and magnesium promote the biosynthesis and secretion of calcitonin. Various gastric hormones-gastrin, pentagastrin, glucagon, and pancreozymin-and food ingestion also increase calcitonin plasma levels. In normal pregnancy, unlike their fetal counterparts, the maternal adrenal glands undergo little, if any, morphological change. The serum concentration of circulating cortisol rises, but much of it is bound by transcortin, the cortisol-binding globulin. The adrenal secretion rate of this principal glucocorticoid is not elevated, and probably it is lower than in the nonpregnant state. he metabolic clearance rate of cortisol, however, is diminished during pregnancy because its half-life is nearly doubled compared with that for nonpregnant women (Migeon, 1957). Administration of estrogen, including most oral contraceptives, causes changes in serum cortisol levels and transcortin similar to those of pregnancy Qung, 201i1).

1	During early pregnancy, the levels of circulating adrenocorticotropic hormone (A CTH) , also known as corticotropin, are dramatically reduced. As pregnancy progresses, ACTH and free cortisol levels rise equally and strikingly (Fig. 4-18). This ,50

1	FIGURE 4-18 Serial increases in serum cortisol (blue line) and adrenocorticotropic hormone (ACTH) (red line) across normal pregnancy. (Data from Carr, 1981o.) apparent paradox is not understood completely. Some suggest that greater free cortisol levels in pregnancy result from a "resetting" of the maternal feedback mechanism to higher thresholds (Nolten, 1981). his might result from tissue reractoriness to cortisol. Others assert that these incongruities stem from an antagonistic action of progesterone on mineralocorticoids (Keller-Wood, 2001). Thus, in response to elevated progesterone levels during pregnancy, an elevated free cortisol is needed to maintain homeostasis. Other theories include possible roles for higher free cortisol in preparation for the stress of pregnancy, delivery, and lactation. This pattern might also influence postpartum behavior and parenting roles (Conde, 2014).

1	As early as 15 weeks' gestation, the maternal adrenal glands secrete considerably increased amounts of aldosterone, the principal mineralocorticoid. By the third trimester, approximately 1 mg/d is released. If sodium intake is restricted, aldosterone secretion is even further elevated (Watanabe, 1963). Concurrently, levels of renin and angiotensin II substrate normally rise, especially during the latter half of pregnancy. his scenario promotes greater plasma levels of angiotensin II, which acts on the zona glomerulosa of the maternal adrenal glands and accounts for the markedly elevated aldosterone secretion. Some suggest the increased aldosterone secretion during normal pregnancy afords protection against the natriuretic efect of progesterone and atrial natriuretic peptide. Gennari-Moser and colleagues (2011) provide evidence that aldosterone, as well as cortisol, may modulate trophoblast growth and placental size.

1	Maternal plasma levels of this potent mineralocorticosteroid progressively increase during pregnancy. Indeed, plasma levels of deoxycorticosterone rise to near 1500 pg/mL by term, a more than 15-fold increase (Parker, 1980). his marked elevation does not derive from adrenal secretion but instead represents augmented kidney production resulting from estrogen stimulation. The levels of deoxycorticosterone and its sulfate in fetal blood are appreciably higher than those in maternal blood, which suggests transfer of fetal deoxycorticosterone into the maternal compartment.

1	In balance, androgenic aCtiVlty rises during pregnancy, and both maternal plasma levels of androstenedione and testosterone are increased. This inding is not totally explained by alterations in their metabolic clearance. Both androgens are converted to estradiol in the placenta, which increases their clearance rates. Conversely, greater plasma sex hormone-binding globulin levels in gravidas retard testosterone clearance. Thus, the production rates of maternal testosterone and androstenedione during human pregnancy are increased. he source of this higher C19-steroid production is unknown, but it likely originates in the ovary. Interestingly, little or no testosterone in maternal plasma enters the fetal circulation as testosterone. Even when massive testosterone levels are found in the circulation of pregnant women, as with androgen-secreting tumors, testosterone concentrations in umbilical cord blood are likely to be undetectable. his results from the near complete trophoblastic

1	circulation of pregnant women, as with androgen-secreting tumors, testosterone concentrations in umbilical cord blood are likely to be undetectable. his results from the near complete trophoblastic conversion of testosterone to 173-estradiol.

1	Maternal serum and urine levels of dehydroepiandrosterone suate are lower during normal pregnancy. his stems from a greater metabolic clearance through extensive maternal hepatic 16.-hydroxylation and placental conversion to estrogen (Chap. 5, p. 103).

1	Progressive lordosis is a characteristic feature of normal pregnancy. Compensating for the anterior position of the enlarging uterus, lordosis shifts the center of gravity back over the lower extremities. The sacroiliac, sacrococcygeal, and pubic joints have increased mobility during pregnancy. However, as discussed earlier (p. 52), increased joint laxity and associated discomfort during pregnancy do not correlate with increased maternal serum levels of estradiol, progesterone, or relaxin (Aldabe, 2012; Marnach, 2003; V0llestad, 2012). Most relaxation takes place in the first half of pregnancy. It may contribute to maternal posture alterations and in turn create lower back discomfort. s discussed in Chapter 36 (p. 661), although some symphyseal separation likely accompanies many deliveries, those greater than 1 cm may cause significant pain (Shnaekel, 2015).

1	Aching, numbness, and weakness also occasionally are experienced in the upper extremities. his may result from the marked lordosis and associated anterior neck flexion and shoulder girdle slumping, which produce traction on the ulnar and median nerves (Crisp, 1964). he latter may give rise to symptoms mistaken for the carpal tunnel syndrome (Chap. 60, p. 1167). Joint strengthening begins immediately following delivery and is usually complete within 3 to 5 months. Pelvic dimensions measured by MR imaging up to 3 months after delivery are not significantly diferent from prep regnancy measurements (Huerta-Enochian, 2006).

1	Central nervous system changes are relatively few and mostly subtle. Women often report problems with attention, concentration, and memory throughout pregnancy and the early puerperium. Systematic studies of memory in pregnancy, however, are limited and often anecdotal. Keenan and associates (1998) longitudinally investigated memory in pregnant women and a matched control group. hey found pregnancy-related memory decline that was limited to the third trimester. his decline was not attributable to depression, anxiety, sleep deprivation, or other physical changes associated with pregnancy. It was transient and quickly resolved following delivery. Others have found poorer verbal recall and processing speed and worse spatial recognition memory in pregnancy (Farrar, 2014; Henry, 2012).

1	Zeeman and coworkers (2003) used MR imaging to measure cerebral blood low across pregnancy. They found that mean blood flow in the middle and posterior cerebral arteries declined progressively from 147 and 56 mLimin when nonpregnant to 118 and 44 mLlmin late in pregnancy, respectively. Mechanisms and signiicance of the decline are unknown. Pregnancy does notiafect cerebrovascular autoregulation (Bergersen, 2006; Cipolla, 2014).

1	Intraocular pressure drops during pregnancy and is attributed partly to greater vitreous outlow. Corneal sensitivity is decreased, and the greatest changes are late in gestation. Most pregnant women demonstrate a measurable but slight increase in corneal thickness, thought to be due to edema. Consequently, they may have diiculty with previously comfortable contact lenses. Brownish-red opacities on the posterior surface of the cornea-Krukenberg spindles-are observed with a higher than expected frequency during pregnancy. Hormonal efects similar to those observed for skin lesions are believed to cause this increased pigmentation. Other than transient loss of accommodation reported with both pregnancy and lactation, visual function is unafected by pregnancy. These changes during pregnancy and pathological eye aberrations were reviewed by Grant and Chung (2013).

1	Beginning as early as 12 weeks' gestation and extending through the first 2 months postpartum, women have diiculty with falling asleep, frequent awakenings, fewer hours of night sleep, and reduced sleep eiciency (Pavlova, 2011). Abdullah and colleagues (2016b) concluded that sleep apnea is more common in pregnancy, especially in obese patients. he greatest disruption of sleep is encountered postpartum and may contribute to postpartum blues or to frank depression auulia Paavonen, 2017). Abdullah B, Ayub SH, Mohd Zahid AZ, et al: Urinary incontinence in primigravida: the neglected pregnancy predicament. Eur ] Obstet Gynecol Reprod Bioi 198:110, 2016a Abdullah HR, Nagappa M, Siddiqui N, Chung F: Diagnosis and treatment of obstructive sleep apnea during pregnancy. Curr Opin Anaesthesiol 29:317, 2016b

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1	Wilson MJ, Lopez M, Vargas M, et al: Greater uterine artery blood low during pregnancy in multigenerational (Andean) than shorter-term (European) highaltitude residents. AmJ Physiol Regul Integr Comp Physio1r293:R1313, 200 Wong CA, Lofredi M, Ganchif IN, et al: Gastric emptying of water in term pregnancy. Anesthesiology 96: 1395, 2002 Wong CA, McCarthy RJ, Fitzgerald PC, et al: Gastric emptying of water in obese pregnant women at term. Anesth Analg 105:751,r2007 World Health Organization: Human energy requirements. Food and nutrition technical report series 1. Rome, Food and Agriculture Organization of the United Nations, 2004, p 53 Wright HP, Osborn SB, Edmonds DG: Changes in rate of low of venous blood in the leg during pregnancy, measured with radioactive sodium. Surg Gynecol Obstet 90:481, 1950

1	Wright HP, Osborn SB, Edmonds DG: Changes in rate of low of venous blood in the leg during pregnancy, measured with radioactive sodium. Surg Gynecol Obstet 90:481, 1950 Yurteri-Kaplan L, Saber S, Zamudio S: Brain natriuretic peptide in term pregnancy. Reprod Sci 19(5):520,r2012 Zeeman GG, Cunningham FG, Pritchard JA: The magnitude of hemoconcentration with eclampsia. Hypertens Pregnancy 28(2): 127, 2009 Zeeman GG, Hatab M, Twickler OM: Maternal cerebral blood low changes in pregnancies. Am J Obstet Gynecol 189:968,2003 Zimmermann MB: The efects of iodine deiciency in pregnancy and infancy. Paediatr Perinat Epidemiolr26(Supp 1):108,r2012 OMETRIAL CYCLE. . .... 80 DECIDUA. . .... 85 FORMATION.. ............ . . .... 87 PLACENTA AND CHORION . .90 AMNION . . .... 95 UMBILICAL CORD ................... . . .... ..... 97 PLACENTAL HORMONES. .98 INTERACTIONS. . . . . .... . . . . . . ...... . . . . . .. . ... 104

1	PLACENTA AND CHORION . .90 AMNION . . .... 95 UMBILICAL CORD ................... . . .... ..... 97 PLACENTAL HORMONES. .98 INTERACTIONS. . . . . .... . . . . . . ...... . . . . . .. . ... 104 Almost immediately ater the impamltio1 of the ovum, its trophoblast begins to prolerate and lJ1d, the s.rrounding decidual tissue. As it does so, it breaks through the walls of the moumal capilaries, from which the blood escapes and orms cavities, which are bounded party by trophobast and party by decidua. ?he maternal blood spaces established in this manner represent the earliest stages of the intervillous blood spaces of the inure placenta. -). Whitridge Williams (1903)

1	-). Whitridge Williams (1903) In 1903, the histopathological and embryological descrip tions of ovum implanrarion and placental development had been extensively studied and described. However, the origins and functions of pregnancy hormones were largely unknown. Indeed, it was another 25 to 30 years before estrogen and progesterone were discovered. In the past 50 years, remarkable strides have followed to uncover the steps of implantation and placental structure and function.

1	All obstetricians should understand the basic biological steps required for women to successully achieve pregnancy. Several abnormalities can fect each of these and lead to infertili' or pregnancy loss. In most women, spontaneous, cyclical ovulation continues during almost 40 years between menarche and menopause. Without contraception, there are approximately 400 opportunities for pregnancy, namely, the day of ovulation and its few preceding days. his narrow window for fertilization is controlled by tightly regulated production of ovarian steroids. Moreover, these hormones promote optimal endometrial regeneration after menstruation in preparation for the next implantation window.

1	If fertilization occurs, events thac begin ater blastocyst implantation persist until parturition. hese derive from a unique interaction between fetal trophoblasts and the maternal endometrium. which has been transformed into the decidua. The ability of a mother and her fetus to coexist as two distinct immunological systems results from endocrine. paracrine. and immunological modiication of fetal and maternal tissues in a manner not seen elsewhere. In addition. the placenta mediates a unique fetal-maternal communication system. which creates a hormonal environment that initially maintains pregnancy and eventually initiates events leading to parturition.

1	Predictable. regular. cyclical. and spontaneous ovulatory menstrual cycles are regulated by complex interactions of the hypothalamic-pituitay-ovarian axis. Concurrently, cyclical changes in endometrial histology are faithfully reproduced ;-1). Essentials players in this process include follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which are pituitary-derived gonadotropins, and the ovarian sex steroid hormones estrogen and progesterone.

1	FIGURE 5-1 Gonadotropin control of the ovarian and endometrial cycles. The ovarian-endometrial cycle has been structured as a 28-day cycle. The follicular phase (days 1 to 14) is characterized by rising estrogen levels, endometrial thickening, and selection of the dominant "ovulatory" follicle. During the luteal phase (days 14 to 21 I, the corpus luteum (ell produces estrogen and progesterone, which prepare the endometrium for implantation. If implantation occurs, the developing blastocyst begins to produce human chorionic gonadotropin (hCG) and rescues the corpus luteum, thus maintaining progesterone production. FSH = follicle-stimulating hormone; LH = luteinizing hormone. The average cycle duration approximates 28 days but ranges from 25 to 32 days, even for a given woman. The follicular or proliferative phase shows considerable phase-length variation. This contrasts with the luteal or secretory POStovulatory phase of the cycle, which is remarkably constant at 12 to 14 days.

1	The human ovary contains 2 million oocytes at birth, and approximately 400,000 follicles are present at puberty onset (Baker, 1963). These are depleted at a rate of approximately 1000 follicles per month until age 35, when this rate accelerates (Faddy, 1992). Only 400 follicles are normally released during female reproductive life. Therefore, more than 99.9 percent of these undergo atresia through a process of cell death termed apoptosis (Gougeon, 1996; Kaipia, 1997).

1	Follicular development consists of several stages. Primordial follicles undergo gonadotropin-independent recruitment from the resting pool and then progress from primary and secondary follicles to the antral stage. This appears to be controlled by locally produced growth factors. Two members of the transforming growth factor-3 family include growth diferentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP-15), which regulate granulosa cell proliferation and diferentiation as primary follicles grow (Trombly, 2009; Yan, 2001). hey also stabilize and expand the cumulus oocyte complex in the oviduct (Hreinsson, 2002). These factors are produced by oocytes, suggesting that the early steps in follicular development are, in part, oocyte controlled. As antral follicles develop, surrounding stromal cells are recruited, by a yet-to-be-deined mechanism, to become thecal cells.

1	Although not required for early follicular maturation, FSH is required for further development of large antral follicles (Hillier, 2001). During each ovarian FOLLICULAR PHASE LUTEAL PHASE/PREGNANCY cycle. a group of antral follicles, known as a cohon, begins a phase of semisynchro ________ •! CAMP--------.! phase of rhe previous cycle. This FSH increase leading to further follicular developmenr is called the seection winow of C',,""'.o me ovarian cycle (Macklon, 2001). Only follicles progressing to this stage develop Basement membrane the capacity [Q produce estrogen. During rhe follicular phase, estro gen levels rise in proportion to growth � of a dominant follicle and {Q rhe , increase in its number of granulosa cells , , l.

1	During rhe follicular phase, estro gen levels rise in proportion to growth � of a dominant follicle and {Q rhe , increase in its number of granulosa cells , , l. (see Fig. 5-1). These cells are rhe exclucAMP sive site of FSH receptor expression. Cholesterol lle elevation of Circulating FSH levels during the late luteal phase of the previous cycle stimulates an increase in FSH FSH receptors and subsequently, the ability

1	FIGURE 5-2 The two-cell, two-gonadotropin principle of ovarian steroid hormone produc tion. During the follicular phase (left panen, luteinizing hormone (LH) controls theca cell granulosa cells to conven androstenedi production of androstenedione, which diffuses into the adjacent granulosa cells and acts as one into estradiol. The requirement for precursor for estradiol biosynthesis. The granulosa cell capacity to convert androstenedione thecal cells, which respond ro LH, and to estradiol is controlled by follicle-stimulating hormone (FSH), After ovulation (right panen, granulosa cells, which respond to FSH, the corpus luteum forms and both theca-lutein and granulosa-lutein cells respond to LH. represents the two-gonadotropin, twoThe theca-lutein cells continue to produce androstenedione, whereas granulosa-lutein cells ceH hypothesis for estrogen biosynthesis greatly increase their capacity to produce progesterone and to convert androstenedione to estradiol. LH and hCG bind to the

1	whereas granulosa-lutein cells ceH hypothesis for estrogen biosynthesis greatly increase their capacity to produce progesterone and to convert androstenedione to estradiol. LH and hCG bind to the same LH-hCG receptor. If pregnancy occurs (right pane�, (Short, 1962). As shown in 5-2, human chorionic gonadotropin (hCG) rescues the corpus luteum through their shared LH hCG receptor. Low-density lipoproteins (LOL) are an important source of cholesterol for sion of the antrum of growing follicles.

1	steroidogenesis, cAMP =cyclic adenosine monophosphate. The follicle within the cohorr that is most responsive to FSH is likely to be the irst to produce estradiol and initiate expression of LH precise predictor of ovulation. It occurs 34 to 36 hours before receptors. ovum release from the follicle (see Fig. 5-1). LH secretion peaks

1	Mrer the appearance of LH receptors, the preovulatory 10 to 12 hours before ovulation and stimulatcs resumption of granulosa cells begin to secrete small quantities of progesterone. meiosis in the ovum and release of the irst polar body. Studies The preovulatory progesterone secrecion, although somewhat suggest that in response to LH, greater progesterone and proslimited, is believed to exert positive feedback on the estrogentaglandin production by the cumulus cells, as well as GDF9 primed pituitary to either calise or augment LH release. In addiand BMP-15 by the oocyte, activates expression of genes critition, during the late follicular phase, LH stimulates thecal cell cal to formarion of a hyaluronan-rich extracellular matrix by production of androgens, particularly androstenedione, which the cumulus complex (Richards, 2007). As seen in are then transferred to the adjacent follicles where they are aroduring synthesis of this matrix, cumulus cells lose contact with matized to

1	which the cumulus complex (Richards, 2007). As seen in are then transferred to the adjacent follicles where they are aroduring synthesis of this matrix, cumulus cells lose contact with matized to esnadiol (see Fig. 5-2). During the early follicular one another and move outward from the oocyte along the hyalphase, granulosa cells also produce in hi bin 3, which can feed uronan polymer-this process is called expansion. This results back on the pituitary to inhibit FSH release (Groome, 1996). in a 20-fold augmentation of the cumulus complex volume and As the dominant foHicle begins to grow, estradiol and inhibin coincides with an LH-induced remodeling of the ovarian extraproduction rises and results in a decline of follicular-phase cellular matrix. hese allow release of the mature oocyte and FSH. This drop in FSH levels is responsible for the failure of its surrounding cumulus cells through the surface epirhelium. other follicles to reach preovulatory status-the Graaian folActivation of

1	FSH. This drop in FSH levels is responsible for the failure of its surrounding cumulus cells through the surface epirhelium. other follicles to reach preovulatory status-the Graaian folActivation of proteases likely plays a pivotal role in weakening licle stage-during any one cycle. Thus, 95 percent of plasma the follicular basement membrane and ovulation (Curry, 2006; estradiol produced at this time is secreted by the dominant Ny, 2002). follicle-thc one destined to ovulate. Concurrently, the con tralateral ovary is relatively inactive.

1	Following ovulation, rhe corpus luteum develops from the remains of the Graaian follicle in a process referred to as The onset of rhe gonadotropin surge resulting from increasluteiniation. The basement membrane separating the granuing estrogen secretion by preovulatory follicles is a relatively losa-lutein and theca-lutein cells breaks down, and by day 2 FIGURE 5-3 An ovulated cumulus-oocyte complex. An oocyte is at the center of the complex. Cumulus cells are widely separated from each other by the hyaluronan-rich extracellular matrix. (Used with permission from Dr. Kevin J. Doody.) postovulation, blood vessels and capillaries invade the granulosa cell layer. The rapid neo�ascularization of the once-avascular granulosa may be due to angiogenic factors that include vas by theca-lutein and granulosa-lutein cells in response to LH (Albrecht, 2003; Fraser, 2001). During luteinization, these cells undergo hypertrophy and increase their capacity to synthesize hormones.

1	LH is the primary luteotropic factor responsible for corpus luteum maintenance (Vande Wiele, 1970). Indeed, LH injec tions can extend the corpus luteum life span in normal women by 2 weeks (Segalof, 1951). The hormone secretion pattern of the corpus luteum difers from that of the follicle (see Fig. 5-1). As depicted in Figure 5-2, the greater capacity of granulosa-lutein cells to produce progesterone results from enhanced access to considerably more steroidogenic precursors through blood-borne, low-densiry lipoprotein (LDL)-derived cholesterol (Carr, 1981a). Ovarian progesterone production peaks at 25 to 50 mg/d during the midluteal phase. With pregnancy, the corpus luteum continues progesterone production in response to placental human chorionic gonadotropin (hCG), which binds to the same receptor as LH. Estrogen levels follow a more complex pattern of secretion.

1	Estrogen levels follow a more complex pattern of secretion. Specifically, just after ovulation, estrogen levels decline, but then exhibit a secondary rise that reaches a peak production of 0.25 mg/d of 173-estradiol in the midluteal phase. Toward the end of the luteal phase, estradiol production again drops. The human corpus luteum is a transient endocrine organ that, in the absence of pregnancy, will rapidly regress 9 to 11 days after ovulation via apoptosis (Vaskivuo, 2002). The mechanisms that control luteolysis, that is, the regression of the corpus luteum, remain unclear. However, it results in part from dropping levels of circulating LH in the late luteal phase and rising LH insensitivity by luteal cells (Duncan, 1996; Filicori, 1986). he role of other factors is less established. The dramatic drop in circulating estradiol and progesterone levels initiate molecular events that lead to menstruation.

1	Estrogen is the essential hormonal signal on which most events in the normal menstrual cycle depend. They function in many cell types to regulate follicular development, uterine receptivity, and blood flow. The most biologically potent naturally occur ring estrogen is 173-estradiol, which is secreted by granulosa cells of the dominant follicle and luteinized granulosa cells of the corpus luteum. Estradiol action is complex and appears estrogen receptor a (ERa) and 3 (ER3) (Katzen ellenbogen, 2001). These isoforms are the products of separate genes and can exhibit distinct tissue expression. Both estradiol-receptor complexes act as transcriptional factors that become associ ated with the estrogen-response element of speciic genes. They share a robust activation by estradiol. However, diferences in their binding ainities to other estrogens and their cell-speciic may have both distinct and overlapping function (Saunders, 2005).

1	Most progesterone actions on the female reproductive tract are mediated through the nuclear hormone receptors, progesterone-receptor type A (PR-A) and B (PR-B). Progesterone enters cells by difusion, and in responsive tissues it becomes associated with its receptors (Conneely, 2002). Progesteronereceptor isoforms arise from a single gene and regulate transcription of target genes. hese receptors have unique actions. When PR-A and PR-B receptors are coexpressed, it appears that PR-A can inhibit PR-B gene regulation. The endometrial glands and stroma appear to have diferent expression patterns for progesterone receptors that vary during the menstrual cycle (Mote, 1999). Progesterone can also evoke rapid responses, such as changes in intracellular free calcium levels, which cannot be explained by genomic mechanisms. G-protein-coupled membrane receptors for progesterone have been identiied, but their role in the ovarian-endometrial cycle remains to be elucidated (Peluso, 2007).

1	In the endometrium, epithelial cells line the endometrial glands and are supported by stromal cells. These cells and supplying blood vessels replicate rapidly and cyclically in reproductiveaged women and are regenerated each ovarian-endometrial cycle. The superficial endometrium, termed the functionalis layer, is shed and reconstructed from the deeper basalis layer (Fig. 5-4). There is no other example in humans of such cyclical shedding and regrowth of an entire tissue. Fluctuations in estrogen and progesterone levels produce striking efects on the endometrium. Follicular-phase estradiol production is the most important factor in endometrial recovery following menstruation, and both ERa and ER3 receptors are expressed here. Although up to rwo thirds of the functionalis endometrium is fragmented and shed with menses, reepithelialization begins even before menstrual bleeding has ceased. By the fifth day of the endometrial cycle-ifth day of menses-the

1	FIGURE 5-4 The endometrium consists of two layers, the functionalis layer and basalis layer. These are supplied by the spiral and basal arteries, respectively. Numerous glands also span these layers. As the menstrual cycle progresses, greater coiling of the spiral arteries and increased gland folding can be seen. Near the end of the menstrual cycle (day 27), the coiled arteries constrict, deprive blood supply to the functionalis layer, and lead to necrosis and sloughing of this layer.

1	epithelial surface of the endometrium has been restored, and revascularization has begun. he preovulatory endometrium is characterized by proliferation of glandular, stromal, and vascular endothelial cells. During the early part of the proliferative phase, the endometrium is usually less than 2 mm thick. he glands are narrow, tubular structures that pursue almost a straight and parallel course from the basalis layer toward the endometrial cavity. Mitotic igures, especially in the glandular epithelium, are identiied by the fifth cycle day. Mitotic activity in both epithelium and stroma persists until day 16 to 17, that is, 2 to 3 days after ovulation. Blood vessels are numerous and prominent.

1	Clearly, reepithelialization and angiogenesis are important to cessation of endometrial bleeding (Chennazhi, 2009; Rogers, 2009). hese are dependent on tissue regrowth, which is estrogen regulated. Epithelial cell growth also is regulated in part by epidermal growth factor and transforming growth factor a (TGPa). Stromal cells proliferate through paracrine and autocrine actions of estrogen and greater local levels of fibroblast growth factor-9 (Tsai, 2002). Estrogens also raise local production ofVEGF, which causes angiogenesis through vessel elongation in the basalis (Gargett, 2001; Sugino, 2002). By the late proliferative phase, the endometrium thickens from both glandular hyperplasia and augmented stromal ground substance, which is edema and proteinaceous material. he loose stroma is especially prominent, and the glands in the functionalis layer are widely separated. his is compared with those of the basalis layer, in which the glands are more crowded and the stroma is denser.

1	At midcycle, as ovulation nears, glandular epithelium becomes taller and pseudostratiied. he surface epithelial cells acquire numerous microvilli, which increase epithelial surface area, and develop cilia, which move endometrial secretions during the secretory phase (Perenczy, 1976).

1	After ovulation, the estrogen-primed endometrium responds to rising progesterone levels in a highly predictable manner. By day 17, glycogen accumulates in the basal portion of glandular epithelium, creating subnuclear vacuoles and pseudostratification. hese changes likely result from direct progesterone action through receptors expressed in glandular cells (Mote, 2000). On day 18, vacuoles move to the apical portion of the secretory non ciliated cells. By day 19, these cells begin to secrete glycoprotein and mucopolysaccharide contents into the gland lumen (Hafez, 1975). Glandular cell mitosis ceases with secretory activity due to rising progesterone levels, which antagonize the mitotic efects of estrogen. Estradiol action also diminishes because of glandular expression of the type 2 isoform of 173-hydroxysteroid dehydrogenase. his converts estradiol to the less active estrone (Casey, 1996). On cycle days 21 to 24, the stroma becomes edematous. Next, on days 22 to 25, stromal cells

1	of 173-hydroxysteroid dehydrogenase. his converts estradiol to the less active estrone (Casey, 1996). On cycle days 21 to 24, the stroma becomes edematous. Next, on days 22 to 25, stromal cells surrounding the spiral arterioles begin to enlarge, and stromal mitosis becomes apparent. Days 23 to 28 are characterized by pre decidual cells, which surround spiral arterioles.

1	Between days 22 and 25, the secretory-phase endometrium undergoes striking changes associated with predecidual transformation of the upper two thirds of the functionalis layer. The glands exhibit extensive coiling, and luminal secretions become visible. Changes within the endometrium also can mark the so-called window of implantation seen on days 20 to 24. Epithelial surface cells show fewer microvilli and cilia, but luminal protrusions appear on the apical cell surface (Nikas, 2003). hese pinopodes help prepare for blastocyst implantation. hey also coincide with changes in the surface glycocalyx that allow acceptance of a blastocyst (Aplin, 2003). Another highlight of the secretory phase is the continuing growth and development of the spiral arteries. hese vessels arise from the radial arteries, which are myometrial branches of the arcuate and, ultimately, uterine vessels (see Fig. 5-4).

1	he morphological and functional properties of the spiral arter ies are unique and essential to blood flow changes seen during menstruation or implantation. During endometrial growth, spiral arteries lengthen at a rate appreciably greater than the rate of endometrial tissue thickening. his growth discordance obliges even greater coiling. Spiral artery development relects a marked induction of angiogenesis, with widespread vessel sprouting and extension. Such rapid angiogenesis is regulated, in part, through estrogen-and progesterone-regulated synthesis ofVEGF (Ancelin, 2002; Chennazhi, 2009). The midluteal-secretory phase of the endometrial cycle is a tiation. With corpus luteum rescue and continued progesterone secretion, the endometrium is transformed into the decidua. With luteolysis and declining luteal progesterone production, events leading to menstruation are initiated (Critchley, 2006; Thiruchelvam, 2013).

1	With luteolysis and declining luteal progesterone production, events leading to menstruation are initiated (Critchley, 2006; Thiruchelvam, 2013). In the late premenstrual-phase endometrium, the stroma is invaded by neutrophils to create a pseudo inflammatory appearance. These cells iniltrate primarily on the day or two immediately preceding menses onset. he endometrial stromal and epithelial cells produce interleukin-8 (IL-8), a chemotactic-activating factor for neutrophils (Arici, 1993). Similarly, monocyte chemotactic protein-1 (MCP-1) is synthesized by endometrium and promotes monocyte recruitment (Arici, 1995).

1	Leukocyte iniltration is considered key to both endometrial extracellular matrix breakdown and repair of the functionalis layer. The term "inflammatory tightrope" refers to the ability of macrophages to assume phenotypes that vary from proinflammatory and phagocytic to immunosuppressive and reparative. These are likely relevant to menstruation, in which tissue breakdown and restoration occur simultaneously (Evans, 2012; Maybin, 2015). Invading leukocytes secrete enzymes that are members of the matrix metalloprotease (MMP) family. These add to the proteases already produced by endometrial stromal cells and efectively initiate matrix degradation. During menses as tissue shedding is completed, microenvironment-regulated changes in macrophage phenotype then promote repair and resolution (Evans, 2012; Thiruchelvam, 2013).

1	The classic study by Markee (1940) described tissue and vascular alterations in endometrium before menstruation. With endometrial regression, spiral artery coiling becomes suiciently severe that resistance to blood flow rises to cause endometrial hypoxia. Resultant stasis is the primary cause of endometrial ischemia and tissue degeneration. Intense spiral artery vasoconstriction precedes menstruation and also serves to limit menstrual blood loss. Prostaglandins playia key role in the events leading to menstruation that include vasoconstriction, myometrial contractions, and upregulation of proinflammatory responses (Abel, 2002).

1	Prostaglandins playia key role in the events leading to menstruation that include vasoconstriction, myometrial contractions, and upregulation of proinflammatory responses (Abel, 2002). Large amounts of prostaglandins are present in menstrual blood. Painful menstruation is common and likely caused by myometrial contractions and uterine ischemia. This response is believed to be mediated by prostaglandin F2a (PGF2a)-induced spiral artery vasoconstriction, which render the uppermost endometrial zones hypoxic. The hypoxic environment is a potent inducer of angiogenesis and vascular permeability factors such as VEGF.

1	Progesterone withdrawal increases expression of cyclooxygenase 2 (COX-2), also called prostaglandin synthase 2, to synthesize prostaglandins. Withdrawal also lowers expression of 15-hydroxyprostaglandin dehydrogenase (PGDH), which degrades prostaglandins (Casey, 1980, 1989). The net result is higher prostaglandin production by endometrial stromal cells and greater prostaglandin-receptor density on blood vessels and surrounding cells. Actual menstrual bleeding follows rupture of spiral arterioles and consequent hematoma formation. With a hematoma, the superficial endometrium is distended and ruptures. Subsequently, fissures develop in the adjacent functionalis layer, and blood and tissue fragments are sloughed. Hemorrhage stops with arteriolar constriction. Changes that accompany partial tissue necrosis also serve to seal vessel tips.

1	he endometrial surface is restored by growth of langes, or collars, that form the everted free ends of the endometrial glands (Markee, 1940). These flanges rapidly grow in diameter, and epithelial continuity is reestablished by fusion of the edges of these sheets of migrating cells.

1	his is a specialized, highly modiied endometrium of pregnancy. It is essential for hemochorial placentation, that is, one in which maternal blood contacts trophoblasts. This relationship requires trophoblast invasion, and considerable research has focused on the interaction between decidual cells and invading trophoblasts. Decidualization, that is, transformation oO proliferating endometrial stromal cells into specialized secretory cells, is dependent on estrogen, progesterone, androgens, and factors secreted by the implanting blastocyst (Gibson, 2016). The decidua produces factors that regulate endometrial receptivity and modulate immune and vascular cell functions within the maternal-fetal microenvironment. The special relationship existing between the decidua and the invading trophoblasts ensures success of the pregnancy semiallograft yet seemingly deies the laws of transplantation immunology.

1	The decidua is classiied into three parts based on anatomical location. Decidua directly beneath blastocyst implantation is modiied by trophoblast invasion and becomes the decidua basalis. The decidua capsularis overlies the enlarging blastocyst and initially separates the conceptus from the rest of the uterine cavity (Fig. 5-5). This portion is most prominent during the second month of pregnancy and consists of stromal decidual cells covered by a single layer of lattened epithelial cells. Internally, it contacts the avascular, extraembryonic fetal membrane-the chorion laeve. he remainder of the uterus is lined by decidua parietalis. During early pregnancy, there is a space between the decidua capsularis and parietalis because the gestational sac does not fill the entire uterine cavity. The gestation sac is the extraembryonic coelom and also called the chorionic cavity. By 14 to 16 weeks' gestation, the expanding sac has enlarged to completely ill the uterine cavity. The resulting

1	The gestation sac is the extraembryonic coelom and also called the chorionic cavity. By 14 to 16 weeks' gestation, the expanding sac has enlarged to completely ill the uterine cavity. The resulting apposition of the decidua capsularis and parietalis creates the decidua vera, and the uterine cavity is functionally obliterated.

1	Decidua basalis Cervical canal parietal is Chorionic villi cavity FIGURE 5-5 Three portions of the decidua-the basalis, capsularis, and parietalis-are illustrated. In early pregnancy, the decidua begins to thicken, eventually attaining a depth of 5 to 10 mm. With magnification, furrows and numerous small openings, representing the mouths of uterine glands, can be detected. Later in pregnancy, the decidua becomes thinner, presumably because of pressure exerted by the expanding uterine contents. he decidua parietalis and basalis are composed of three layers. here is a surface or compact zone-zona compacta; a middle portion or spongy zone-zona spongiosa-with remnants of glands and numerous small blood vessels; and a basal zone-zona basalis. The zona compacta and spongiosa together form the zona functionalis. The basal zone remains after delivery and gives rise to new endometrium.

1	In human pregnancy, the decidual reaction is completed only with blastocyst implantation. Predecidual changes, however, commence irst during the midluteal phase in endometrial stromal cells adjacent to the spiral arteries and arterioles. hereafter, they spread in waves throughout the uterine endometrium and then from the implantation site. The endometrial stromal cells enlarge to form polygonal or round decidual cells. he nuclei become vesicular, and the cytoplasm becomes clear, slightly basophilic, and surrounded by a translucent membrane. As a consequence of implantation, the blood supply to the decidua capsularis is lost as the embryo-fetus grows. Blood supply to the decidua parietalis through spiral arteries persists. These arteries retain a smooth-muscle wall and endothelium and thereby remain responsive to vasoactive agents.

1	In contrast, the spiral arterial system that supplies the decidua basalis and ultimately the placental intervillous space is altered remarkably. These spiral arterioles and arteries are invaded by trophoblasts, and during this process, the vessel walls in the basalis are destroyed. Only a shell without smooth muscle or endothelial cells remains. Importantly, as a result, these vascular conduits of maternal blood-which become the utero placental vessels-are unresponsive to vasoactive agents. Conversely, the fetal chorionic vessels, which transport blood between the placenta and the fetus, contain smooth muscle and thus do respond to vasoactive agents.

1	Early in pregnancy, the zona spongiosa of the decidua consists of large distended glands, often exhibiting marked hyperplasia and separated by minimal stroma. At first, the glands are lined by typical cylindrical uterine epithelium with abundant secretory activity that contributes to blastocyst nourishment. With advanced pregnancy, the glandular elements largely disappear. he decidua basalis contributes to formation of the placental basal plate (Fig. 5-6). he spongy zone of the decidua basalis consists mainly of arteries and widely dilated veins, and by term, glands have virtually disappeared. Also, the decidua basalis is invaded by many interstitial trophoblasts and trophoblastic giant cells. Although most abundant in the decidua, the giant cells commonly penetrate the upper myometrium. Their number and invasiveness can be so extensive as to resemble choriocarcinoma.

1	The Nitabuch layer is a zone of fibrinoid degeneration in which invading trophoblasts meet the decidua basalis. If the decidua is defective, as in placenta accreta, the Nitabuch layer is usually absent (Chap. 41, p. 778). here is also a more supericial, but inconsistent, deposition of ibrin-Rohr stria-at the bottom of the intervillous space and surrounding the anchoring FIGURE 5-6 Section through a junction of chorion, villi, and decidua basalis in early first-trimester pregnancy. (Used with permission from Dr. Kurt Benirschke.) villi. Decidual necrosis is a normal phenomenon in the irst and probably second trimesters (McCombs, 1964). hus, necrotic decidua obtained through curettage after spontaneous abortion in the irst trimester should not necessarily be interpreted as either a cause or an efect of the pregnancy loss.

1	Both decidua types contain numerous cell groups whose composition varies with gestational stage (Loke, 1995). he primary cellular components are the true decidual cells, which diferentiated from the endometrial stromal cells, and numerous maternal bone marrow-derived cells. Accumulation of lymphocytes with unique properties at the maternal-fetal interface is essential to evoke tolerance mechanisms that prevent maternal immune rejection of the fetus. These include regulatolY T cells, decidual macrophages, and decidual natural killer cells. Collectively, these cells not only provide immunotolerance but also play an important role in trophoblast invasion and vasculogenesis (PrabhuDas, 2015).

1	In addition to placental development, the decidua potentially provides other functions. The decidua is the source of prolactin, which is present in enormous amounts in amnionic luid (Colander, 1978; Riddick, 1979). Decidual prolactin is a product of the same gene that encodes for anterior pituitary prolactin, but the exact physiological role of decidual prolactin is unknown. Notably, decidual prolactin is not to be confused with placental lactogen (hPL), which is produced only by syncytiotrophoblast. Prolactin preferentially enters amnionic luid, and little enters maternal blood. Consequently, prolactin levels in amnionic luid are extraordinarily high and may reach 10,000 ng/mL at 20 to 24 weeks' gestation (Tyson, 1972). This compares with fetal serum levels of 350 ng/mL and maternal serum levels of 150 to 200 ngl mL. As a result, decidual prolactin is a classic example of paracrine function between maternal and fetal tissues.

1	The ferus is dependent on the placenta for pulmonary, hepatic, and renal functions. These are accomplished through the anatomical relationship of the placenta and its uterine interface. In overview, maternal blood spurts from uteroplacental vessels into the placental intervillous space and bathes the outer syncytiotrophoblast. his allows exchange of gases, nutrients, and other substances with fetal capillary blood within the core of each villus. hus, fetal and maternal blood does not normally mix in this hemochorial placenta. A paracrine system also links mother and ferus through the anatomical and biochemical juxtaposition of the maternal decidua parietalis and the extraembryonic chorion laeve, which is fetal. This is an extraordinarily important arrangement for communication between fetus and mother and for maternal immunological acceptance of the conceptus (Cuzeloglu-Kayisli, 2009).

1	With ovulation, the secondary oocyte and adhered cells of the cumulus-oocyte complex are freed from the ovary. Although technically this mass of cells is released into the peritoneal cavity, the oocyte is quickly engulfed by the fallopian tube infundibulum. Further transport through the tube is accomplished by directional movement of cilia and tubal peristalsis. Fertilization, which normally occurs in the oviduct, must take place within a few hours, and no more than a day after ovulation. Because of this narrow window, spermatozoa must be present in the fallopian tube at the time of oocyte arrival. Almost all pregnancies result when intercourse occurs during the 2 days preceding or on the day of ovulation.

1	Fertilization is highly complex. Molecular mechanisms allow spermatozoa to pass between follicular cells; through the zona pellucida, which is a thick glycoprotein layer surrounding the oocyte cell membrane; and into the oocyte cytoplasm. Fusion of the two nuclei and intermingling of maternal and paternal chromosomes creates the zygote. Early human development is described by days or weeks postfertilization, that is, postconceptional. By contrast, in most chapters of this book, clinical pregnancy dating is calculated from the irst day of the last menstrual period (LMP). hus, 1 week postfertilization corresponds to approximately 3 weeks from the LMP in women with regular 28-day cycles. As an example, 8 weeks' gestation refers to 8 completed weeks following the LMP. After fertilization, the zygote-a diploid cell with 46 chromosomes-undergoes cleavage, and zygote cells produced by this division are called blastomeres (Fig. 5-7). In the two-cell

1	After fertilization, the zygote-a diploid cell with 46 chromosomes-undergoes cleavage, and zygote cells produced by this division are called blastomeres (Fig. 5-7). In the two-cell FIGURE 5-7 Zygote cleavage and blastocyst formation. The morula period begins at the 12-to 16-cell stage and ends when the blastocyst forms, which occurs when there are 50 to 60 blastomeres present. The polar bodies, shown in the 2-cell stage, are small nonfunctional cells that soon degenerate. zygote, the blastomeres and polar body continue to be surrounded by the zona pellucida. he zygote undergoes slow cleavage for 3 days while still remaining in the fallopian tube. As the blastomeres continue to divide, a solid mulberry-like ball of cells-the morula-is produced. The morula enters the uterine cavity approximately 3 days after fertilization. Gradual accumulation of luid beween the morula cells leads to formation of the early blastocyst.

1	As early as 4 to 5 days after fertilization, the 58-cell blastula diferentiates into ive embryo-producing cells-the inner cel mass (see Fig. 5-7). he remaining 53 outer cells, called the trophectoderm, are destined to form trophoblasts (Hertig, 1962). Interestingly, the 107 -cell blastocyst is found to be no larger than the earlier cleavage stages, despite the accumulated fluid within the blastocyst cavity. At this stage, the eight formative, embryo-producing cells are surrounded by 99 trophoblastic cells. And, the blastocyst is released from the zona pellucida secondary to secretion of specific proteases from the secretoryphase endometrial glands (O'Sullivan, 2002).

1	Release from the zona pellucida allows blastocyst-produced cytokines and hormones to directly influence endometrial receptivity (Lindhard, 2002). IL-la and IL-lj are secreted by the blastocyst, and these cytokines likely directly influence the endometrium. Embryos also have been shown to secrete hCG, which may influence endometrial receptivity (Licht, 2001; Lobo, 2001). The receptive endometrium is thought to respond by producing leukemia inhibitoY factor (LIF), follistatin, and colony-stimulating factor-l (CSF-l). LIF and follistatin activate signaling pathways that collectively inhibit proliferation and promote diferentiation of the endometrial epithelia and stroma to enable uterine receptivity (Rosario, 20 16b). At the maternal-fetal interface, CSF-1 has proposed immunomodulatory actions and pro angiogenic actions that are required for implantation (Rahmati, 2015).

1	Six or 7 days after fertilization, the blastocyst implants into the uterine wall. This process can be divided into three phases: (1) apposition-initial contact of the blastocyst to the uterine wall; (2) adhesion-increased physical contact between the blastocyst and decidua; and (3) invasion-penetration and invasion of syncytiotrophoblast and cytotrophoblasts into the decidua, inner third of the myometrium, and uterine vasculature. Successful implantation requires a receptive endometrium appropriately primed with estrogen and progesterone by the corpus luteum. Such uterine receptivity is limited to days 20 to 24 of the cycle. Adherence is mediated by cell-surface receptors at the implantation site that interact with blastocyst receptors (Carson, 2002; Lessey, 2002; Lindhard, 2002). If the blastocyst approaches the endometrium after cycle day 24, the potential for adhesion is diminished because antiadhesive glycoprotein synthesis prevents receptor interactions (N avot, 1991).

1	At the time of its interaction with the endometrium, the blastocyst is composed of 100 to 250 cells. The blastocyst loosely adheres to the decidua by apposition. This most commonly occurs on the upper posterior uterine wall. Attachment of the blastocyst trophectoderm to the decidual surface by apposition and adherence appears to be closely regulated by paracrine interactions between these two tissues. Successful endometrial blastocyst adhesion involves modiication in expression of cellular adhesion molecules (CMs). The integrins-one of four families of CAMs-are cell-surface receptors that mediate cell adhesion to extracellular matrix proteins (Lessey, 2002). Endometrial integrins are hormonally regulated, and a specific set of integrins is expressed at implantation (Lessey, 1995). Recognition-site blockade of integrins needed for binding will prevent blastocyst attachment (Kaneko, 2013).

1	Human placental formation begins with the trophectoderm, which gives rise to a trophoblast cell layer encircling the blastocyst. From then until term, trophoblasts playia critical part at the fetal-maternal interface. Trophoblasts exhibit the most variable structure, function, and developmental pattern of all placental components. Their invasiveness promotes implantation, their nutritional role for the conceptus is relected in their name, and their endocrine organ function is essential to maternal physiological adaptations and to pregnancy maintenance.

1	By the eighth day postfertilization, after initial implantation, trophoblasts have diferentiated into an outer multinucleated syncytium-primitive syncytiotrophoblast, and an inner layer of primitive mononuclear cells-cytotrophoblasts. The latter are germinal cells for the syncytium. As cytotrophoblasts proliferate, their cell walls disappear, and the cells fuse to add to the expanding outer layer of syncytiotrophoblast. Each cytotrophoblast has a well-demarcated cell border, a single nucleus, and ability to undergo DNA synthesis and mitosis (Arnholdt, 1991). These are lacking in the syncytiotrophoblast, which provides transport functions of the placenta. It is so named because instead of individual cells, it has an amorphous cytoplasm without cell borders, nuclei that are multiple and diverse in size and shape, and a continuous syncytial lining. This coniguration aids transport.

1	After implantation is complete, trophoblasts further differentiate along two main pathways, giving rise to villous and extravillous trophoblasts. As shown in Figure 5-8, both have distinct functions (Loke, 1995). Villous trophoblasts generate chorionic villi, which primarily transport oxygen, nutrients, and other compounds between the fetus and mother. Extravillous trophoblasts migrate into the decidua and myometrium and also penetrate maternal vasculature, thus coming into contact with various maternal cell types (Pijnenborg, 1994). Extravillous trophoblasts are further classiied as interstitial trophoblasts and endovascular trophoblasts. he interstitial trophoblasts invade the decidua and eventually penetrate the myometrium to form placental-bed giant cells. These trophoblasts also surround spiral arteries. The endovascular trophoblasts penetrate the spiral artery lumens (Pijnenborg, 1983). hese are both discussed in greater detail in subsequent sections.

1	Early End of 1st pregnancy trimester FIGURE 5-8 Extravillous trophoblasts are found outside the villus and can be subdivided into endovascular and interstitial categories. Endovascular trophoblasts invade and transform spiral arteries during pregnancy to create low-resistance blood flow that is characteristic of the placenta. Interstitial trophoblasts invade the decidua and surround spiral arteries.

1	After gentle erosion between epithelial cells of the surface endometrium, invading trophoblasts burrow deeper. At 9 days of development, the blastocyst wall facing the uterine lumen is a single layer of lattened cells. By the 10th day, the blastocyst becomes totally encased within the endometrium (Fig. 5-9). he blastocyst wall opposite the uterine lumen is thicker and comprises two zones-the trophoblasts and the embryo-forming inner cell mass. As early as 7Y2 days postfertilization, the inner cell mass or embryonic disc diferentiates into a thick plate of primitive ectoderm and an underlying layer of endoderm. Some small cells appear between the embryonic disc and the trophoblasts and enclose a space that will become the amnionic cavity.

1	FIGURE 5-9 Drawing of sections through implanted blastocysts. A. At 10 days. B. At 12 days ater fertilization. This stage is characterized by the intercommunication of the lacunae filled with maternal blood. Note in (B) that large cavities have appeared in the extraembryonic mesoderm, forming the beginning of the extraembryonic coelom. Also note that extraembryonic endodermal cells have begun to form on the inside of the primitive yolk sac. (Redrawn from Moore KL, Persaud, W, Torchia, MG (eds): The Developing Human. Clinically Oriented Embryology, 9th edition, Philadelphia, Saunders, 201o3.)

1	Extraembryonic mesenchyme first appears as groups of isolated cells within the blastocyst cavity, and later this mesoderm completely lines the cavity. Spaces form and then fuse within the extraembryonic mesoderm to form the chorionic cavity (extraembryonic coelom). The chorion is composed of trophoblasts and mesenchyme. Some mesenchymal cells eventually will condense to form the body stalk. his stalk joins the embryo to the nutrient chorion and later develops into the umbilical cord. The body stalk can be recognized at an early stage at the caudal end of the embryonic disc (Fig. 7-3, p. 126). As the embryo enlarges, more maternal decidua basalis is invaded by syncytiotrophoblast. Beginning approximately 12 days after conception, the syncytiotrophoblast is permeated

1	As the embryo enlarges, more maternal decidua basalis is invaded by syncytiotrophoblast. Beginning approximately 12 days after conception, the syncytiotrophoblast is permeated FIGURE 5-10 Electron micrograph of term human placenta villus. A villus capillary filled with fetal red blood cells (asterisks) is seen in close proximity to the microvilli border. (Reproduced with permission from Boyd JD, Hamilton WJ: The Human Placenta. Cambridge, Heffer, 1970.) 90 Placentation, Embryogenesis, and Fetal Development by a system of intercommunicating channels called trophoblastic lacunae. After invasion of supericial decidual capillary walls, lacunae become filled with maternal blood. At the same time, the decidual reaction intensifies in the surrounding stroma. This is characterized by decidual stromal cell enlargement and glycogen storage.

1	With deeper blastocyst invasion into the decidua, solid primary villi arise from buds of cytotrophoblasts that protrude into the primitive syncytium before 12 days postfertilization. Primary villi are composed of a cytotrophoblast core covered by syncytiotrophoblast. s the lacunae join, a complicated labyrinth is formed that is partitioned by these solid cytotrophoblastic columns. The trophoblast-lined channels form the intervillous space, and the solid cellular columns form theprimay villous stalks.

1	Beginning on approximately the 12th day after fertilization, mesenchymal cords derived from extraembryonic mesoderm invade the solid trophoblast columns. These form seconday villi. Once angiogenesis begins in the mesenchymal cords, tertiay villi are formed. Although maternal venous sinuses are tapped early in implantation, maternal arterial blood does not enter the intervillous space until around day 15. By approximately the 17th day, however, fetal blood vessels are functional, and a placental circulation is established. The fetal-placental circulation is completed when the blood vessels of the embryo are connected with chorionic vessels. In some villi, angiogenesis fails from lack of circulation. They can be seen normally, but the most striking exaggeration of this process is seen with hydatidiform mole (Fig. 20-1, p. 389).

1	Villi are covered by an outer layer of syncytiotrophoblast and an inner layer of cytotrophoblasts, which are also known as Langhans cels. Cytotrophoblast proliferation at the villous tips produces the trophoblastic cell columns that form anchoring villi. hey are not invaded by fetal mesenchyme, and they are anchored to the decidua at the basal plate. Thus, the base of the intervillous space faces the maternal side and consists of cytotrophoblasts from cell columns, the covering shell of syncytiotrophoblast, and maternal decidua of the basal plate. The base of the chorionic plate forms the roof of the intervillous space. It consists of two layers of trophoblasts externally and fibrous mesoderm internally. The "deinitive" chorionic plate is formed by 8 to 10 weeks as the amnionic and primary chorionic plate mesenchyme fuse together. This formation is accomplished by expansion of the amnionic sac, which also surrounds the connective stalk and the allantois and joins these structures to

1	chorionic plate mesenchyme fuse together. This formation is accomplished by expansion of the amnionic sac, which also surrounds the connective stalk and the allantois and joins these structures to form the umbilical cord (Kaufmann, 1992).

1	Interpretation of the ine structure of the placenta came from electron microscopic studies of Wislocki and Dempsey (1955). There are prominent microvilli on the syncytial surface that correspond to the so-called brush border described by light microscopy. Associated pinocytotic vacuoles and vesicles are related to absorptive and secretory placental functions. Microvilli act to increase surface area in direct contact with maternal blood. This contact between the trophoblast and maternal blood is the deining characteristic of a hemochorial placenta (Fig. 5-10).

1	In early pregnancy, the villi are distributed over the entire periphery of the chorionic membrane (Fig. 5-11). As the blastocyst with its surrounding trophoblasts grows and expands into the decidua, one pole faces the endometrial cavity. he opposite pole will form the placenta. Here, chorionic villi in contact with the decidua basalis proliferate to form the chorion rondosumor leay chorion. As growth of embryonic and extraembryonic tissues continues, the blood supply to the chorion facing the endometrial cavity is restricted. Because of this, villi in contact with the decidua capsularis cease to grow and then degenerate. his portion of the chorion becomes the avascular fetal membrane that abuts the decidua parietalis and is called the chorion laeve-or smooth chorion. This smooth chorion is composed of cytotrophoblasts and fetal mesodermal mesenchyme.

1	Until near the end of the third month, the chorion laeve is separated from the amnion by the exocoelomic cavity. hereater, they are in intimate contact to form an avascular amniochorion. These two structures are important sites of molecular transfer and metabolic activity. Moreover, they constitute an important paracrine arm of the fetal-maternal communication system. • Regulators of Trophoblast Invasion

1	Implantation and endometrial decidualization activate a unique population of maternal immune cells that iniltrate the uterus and play critical functions in trophoblast invasion, angiogenesis, spiral artery remodeling, and maternal tolerance to fetal alloantigens. Decidual natural killer cells (dNK) make up 70 percent of decidual leukocytes in the first trimester and are found in direct contact with trophoblasts. In contrast to natural killer cells in peripheral blood, these cells lack cytotoxic functions. They produce specific cytokines and angiogenic factors to regulate invasion of fetal trophoblasts and spiral artery remodeling (Hanna, 2006). hese and other unique properties distinguish dNK cells from circulating natural killer cells and from natural killer cells in the endometrium before pregnancy (Fu, 2013; Winger, 2013). dNK cells express both IL-8 and interferoninducible protein-1 0, which bind to receptors on invasive trophoblastic cells to promote their decidual invasion

1	before pregnancy (Fu, 2013; Winger, 2013). dNK cells express both IL-8 and interferoninducible protein-1 0, which bind to receptors on invasive trophoblastic cells to promote their decidual invasion toward the spiral arteries. dNK cells also produce proangiogenic factors, including VEGF and placental growth factor (PIGF), which both promote vascular growth in the decidua.

1	FIGURE 5-1 1 Complete abortion specimens. A. Initially, the entire chorionic sac is covered with villi, and the embryo within is not visible B. With further growth, stretch and pressure prompt partial regression of the villi. Remaining villi form the future placenta, whereas the smooth portion is the chorion.

1	Trophoblasts also secrete speciic chemokines that attract the dNK cells to the maternl-fetal interface. hus, both cell types simultaneously attract each other. Decidual macro phages account for approximately 20 percent of leukocytes in the first trimester and elicit an M2-immunomodulatory phenotype (Williams, 2009). Remember, M1 macrophages are pro inflammatory, and M2 macrophages counter proinflammatory responses and promote tissue repair. In addition to a role in angiogenesis and spiral artery remodeling, dNK cells promote phagocytosis of cell debris (Faas, 2017). Concurrent with the critical role of maternal dNK cells and macro phages, T cell subsets aid tolerance toward the allogenic fetus. Regulatory T cells (Tregs) are essential for promoting immune tolerance. Other T cell subsets are present, such s Th 1, Th2 and Th 17, although their unctions are tightly regulated (Ruocco, 2014) .

1	Extravillous trophoblasts of the first-trimester placenta are highly invasive. his process occurs under low-oxygen conditions, and regulatory factors that are induced under hypoxic conditions are contributory (Soares, 2012). Invasive trophoblasts secrete numer vate proteinases already present in the decidua. Trophoblasts produce urokinase-type plasminogen activator, which converts plasminogen into the broadly acting serine protease, plasmin. This in turn both degrades matrix proteins and activates M.1Ps. One member of the MMP family, MMP-9, appears to be criti cal. The timing and extent of trophoblast invasion is regulated by a balanced interplay between pro-and anti invasive factors.

1	One member of the MMP family, MMP-9, appears to be criti cal. The timing and extent of trophoblast invasion is regulated by a balanced interplay between pro-and anti invasive factors. he relative ability to invade maternal tissue in early preg nancy compared with limited invasiveness in late pregnancy is ual factors. Trophoblasts secrete insulin-like growth factor II that promotes invasion into the decidua. Decidual cells secrete insulin-like growth factor binding-protein type 4, which blocks this autocrine loop.

1	Low estradiol levels in the irst trimester are critical for trophoblast invasion and remodeling of the spiral arteries. nimal studies suggest that the rise in second-trimester estradiol levels suppresses and limits vessel remodeling by reducing trophoblast expression of VEGF and speciic integrin receptors (Bonagura, 2012). Namely, extravillous trophoblasts express integrin receptors that recognize the extracellular matrix proteins collagen IV, laminin, and ibronectin. Binding of these matrix proteins and integrin receptors initiates signls to promote trophoblast cell migration and differentiation. However, as pregnancy advances, rising estradiol levels downregulate VEGF and integrin receptor expression. This represses and controls the extent of uterine vessel transformation.

1	One of the most remarkable features of human placental development is the extensive modification of maternal vasculature by trophoblasts, which are by definition of fetal origin. These events occur in the first half of pregnancy and are considered in detail because of their importance to uteroplacental blood flow. They are also integral to some pathological conditions such as preeclampsia, fetal-growth restriction, and preterm birth. Spiral artery modiications are carried out by two populations of extravillous trophoblasts-endovascular trophoblasts, which penetrate the spiral-artery lumen, and interstitial trophoblasts, which surround the arteries (see Fig. 5-8). Interstitial trophoblasts constitute a major portion of the placental bed. They penetrate the decidua and adjacent myometrium and aggregate around spiral arteries. Although less defined, their functions may include vessel preparation for endovascular trophoblast invasion.

1	Endovascular trophoblasts irst enter the spiral artery lumens and initially form cellular plugs. They then destroy vascular endothelium via an apoptosis mechanism and invade and modiy the vascular media. hus, fibrinoid material replaces smooth muscle and connective tissue of the vessel media. Spiral arteries later regenerate endothelium. Invading endovascular trophoblasts can extend several centimeters along the vessel lumen, and they must migrate against arterial flow. Of note, invasion by trophoblasts involves only the decidual spiral arteries and not decidual veins.

1	Uteroplacental vessel development proceeds in two waves or stages (Ramsey, 1980). The irst occurs before 12 weeks' postfertilization, and spiral arteries are invaded and modiied up to the border between the decidua and myometrium. he second wave, between 12 and 16 weeks, involves some invasion of the intramyometrial segments of spiral arteries. Remodeling converts narrow-lumen, muscular spiral arteries into dilated, low-resistance uteroplacental vessels. Molecular mechanisms of these crucial events, their regulation by cytokines, signaling pathways, and their significance in the pathogenesis of preeclampsia and fetal-growth restriction has been reviewed by several authors (Pereira de Sousa, 2017; ie, 2016; Zhang, 2016). Approximately 1 month after conception, maternal blood enters the intervillous space in fountain-like bursts from the spiral arteries. Blood is propelled outside of the maternal vessels and sweeps over and directly bathes the syncytiotrophoblast.

1	Although certain villi of the chorion frondosum extend from the chorionic plate to the decidua to serve as anchoring villi, most villi arborize and end freely within the intervillous space. s gestation proceeds, the short, thick, early stem villi branch to form progressively iner subdivisions and greater numbers of increasingly smaller villi (Fig. 5-12). Each of the truncal or main stem villi and their ramiications constitutes a placental lobule, or cotyledon. Each lobule is supplied with a single chorionic artery. And each lobule has a single vein, so that lobules constitute the functional units of placental architecture.

1	FIGURE 5-12 Electron micrographs (A, C) and photomicrographs (B, D) of early and late human placentas. A and B. Limited branching of villi is seen in this early placenta. C and D. With placental maturation, increasing villous arborization is seen, and villous capillaries lie closer to the surface of each villus. (Photomicrographs used with permission from Dr. Kurt Benirschke. Electron micrographs reproduced with permission from King BF, Menton DN: Scanning electron microscopy of human placental villi from early and late in gestation. Am J Obstet Gynecol 122:824, 1975.) In the irst trimester, placental growth is more rapid than that of the fetus. But by approximately 17 weeks' gestation, placen tal and fetal weights are approximately equal. By term, placen tal weight is approximately one sixth of fetal weight.

1	he mature placenta and its variant forms are discussed in detail in Chapter 6 (p. 112). Briely, viewed from the mater nal surface, the number of slightly elevated convex areas, called lobes, varies from 10 to 38. Lobes are incompletely separated by grooves of variable depth that overlie placental septa, which arise as upward projections of decidua. he total number of placental lobes remains the same throughout gestation, and individual lobes continue to grow-although less actively in the inal weeks (Crawford, 1959). Although grossly visible lobes are commonly referred to as cotyledons, this is not accurate. Correctly used, lobules or cotyledons are the functional units supplied by each main stem villus.

1	Correctly used, lobules or cotyledons are the functional units supplied by each main stem villus. As villi continue to branch and the terminal ramiications become more numerous and smaller, the volume and promi nence of cytotrophoblasts decrease. As the syncytium thins, the fetal vessels become more prominent and lie closer to the sur face (see Fig. 5-10). he villous stroma also exhibits changes as gestation progresses. In early pregnancy, the branching connec tive-tissue cells are separated by an abundant loose intercellular matrix. Later, the villous stroma becomes denser, and the cells are more spindly and closely packed.

1	Another change in the stroma involves the iniltration of Hobauer cels, which are fetal macrophages. hese are nearly round with vesicular, often eccentric nuclei and very granular or vacuolated cytoplasm. hey grow in number and maturational state throughout pregnancy and appear to be important mediators of protection at the maternal-fetal interface Qohnson, 2012). These macrophages are phagocytic, have an immunosuppressive phenotype, can produce various cytokines, and are capable of paracrine regulation of trophoblastic functions (Cervar, 1999; Reyes, 2017). As discussed further in Chapter 64 (p. 1219), recent studies suggest that Zika virus can infect Hofbauer cells to allow fetal transmission (Simoni, 2017).

1	Some of the histological changes that accompany placental growth and maturation improve transport and exchange to meet advancing fetal metabolic requirements. Among these changes are a thinner syncytiotrophoblst, signiicantly reduced cytotrophoblast number, decreased stroma, and increased number of capillaries with close approximation to the syncytial surface. By 16 weeks' gestation, the apparent continuity of the cytotrophoblasts is lost. At term, villi may be focally reduced to a thin layer of syncytium covering minimal villous connective tissue in which thinwalled fetal capillaries abut the trophoblast and dominate the villi. There are some changes in placental architecture that can cause decreased placental exchange eiciency if they are substantive. These include thickening of the basal lamina of trophoblast or capillaries, obliteration of certain fetal vessels, greater villous stroma, and ibrin deposition on the villous surface.

1	FIGURE 5-1 3 Uterus showing a normal placenta and its membranes in situ. Because the placenta is functionally an intimate approximation of the fetal capillary bed to maternal blood, its gross anatomy primarily concerns vascular relations. he fetal surface is covered by the transparent amnion, beneath which chorionic vessels course. A section through the placenta includes amnion, chorion, chorionic villi and intervillous space, decidual (basal) plate, and myometrium (Figs. 5-13 and 5-14). FIGURE 5-14 Photomicrograph of early implanted blastocyst. Trophoblasts are seen invading the decidua basalis. eNS central nervous system. (Used with permission from Dr. Kurt Benirschke.)

1	FIGURE 5-14 Photomicrograph of early implanted blastocyst. Trophoblasts are seen invading the decidua basalis. eNS central nervous system. (Used with permission from Dr. Kurt Benirschke.) Deoxygenated venous-like fetal blood flows to the placenta through the two umbilical arteries. As the cord joins the placenta, these umbilical vessels branch repeatedly beneath the amnion as they run across the chorionic plate. Branching continues within the villi to ultimately form capillary networks in the terminal villous branches. Blood with signiicantly higher oxygen content returns from the placenta via a single umbilical vein to the fetus.

1	The branches of the umbilical vessels that traverse along the fetal surface of the placenta in the chorionic plate are referred to as the placental surface or chorionic vessels. These vessels are responsive to vasoactive substances, but anatomically, morphologically, histologically, and functionally, they are unique. Chorionic arteries always cross over chorionic veins. Vessels are most readily recognized by this interesting relationship, but they are diicult to distinguish by histological criteria. Truncal arteries are perforating branches of the surface arteries that pass through the chorionic plate. Each truncal artery supplies one main stem villus and thus one cotyledon. As the artery penetrates the chorionic plate, its wall loses smooth muscle, and its caliber increases. The loss of muscle continues as the truncal arteries and veins branch into their smaller rami.

1	Before 10 weeks' gestation, there is no end-diastolic low pattern within the umbilical artery at the end of the fetal cardiac cycle (Fisk, 1988; Loquet, 1988). After 10 weeks, however, end-diastolic low appears and is maintained throughout normal pregnancy. Clinically, these flow patterns are studied with Doppler sonography to assess fetal well-being (Chap. 10, p. 213).

1	Mechanisms of placental blood low must allow blood to leave syncytiotrophoblast, rather than remaining in contact with villi long enough for adequate exchange. For this, maternal blood enters through the basal plate and is driven high up toward the chorionic plate by arterial pressure before laterally dispersing (Fig. 5-15). After bathing the external microvillous surface of chorionic villi, maternal blood drains back through venous oriices in the basal plate and enters uterine veins. Thus, maternal blood traverses the placenta randomly without preformed channels. The previously described trophoblast invasion of the spiral arteries creates low-resistance vessels that can accommodate massive increase in uterine perfusion during gestation. Generally, spiral arteries are perpendicular to, but veins are parallel to, the uterine wall. his arrangement aids closure of veins during a uterine contraction and prevents the exit of maternal blood from the intervillous space. The number of

1	to, but veins are parallel to, the uterine wall. his arrangement aids closure of veins during a uterine contraction and prevents the exit of maternal blood from the intervillous space. The number of arterial openings into the intervillous space is gradually reduced by cytotrophoblastic invasion. here are about 120 spiral arterial entries into the intervillous space at term (Brosens, 1963). hese discharge blood in spurts that bathes the adjacent villi (Borell, 1958). After the 30th week, a prominent venous plexus lies between the decidua basalis and myometrium and helps develop the cleavage plane needed for placental separation after delivery.

1	Both inlow and outlow are curtailed during uterine contractions. BIeker and associates (1975) used serial sonography during normal labor and found that placental length, thickness, and surface area grew during contractions. They attributed this to distention of the intervillous space by impairment of venous outlow compared with arterial inlow. During contractions, therefore, a somewhat larger volume of blood is available for exchange even though the rate of low is decreased. Similarly, Doppler velocimetry has shown that diastolic low veloci ty in spiral arteries is diminished during uterine contractions. Thus, principal factors regulating intervillous space blood low are arterial blood pressure, intrauterine pressure, uterine contraction pattern, and factors that act specifically on arterial walls. • Breas in the Placental"Barrier" he placenta does not maintain absolute integrity of the fetal and maternal circulations. here are numerous examples of

1	• Breas in the Placental"Barrier" he placenta does not maintain absolute integrity of the fetal and maternal circulations. here are numerous examples of FIGURE 5-15 Schematic drawing of a section through a full-term placenta. Maternal blood flows into the intervillous spaces in funnel-shaped spurts. Exchanges occur with fetal blood as maternal blood flows around the villi. Infiowing arterial blood pushes venous blood into the endometrial veins, which are scattered over the entire surface of the decidua basalis. Note also that the umbilical arteries carry deoxygenated fetal blood to the placenta and that the umbilical vein carries oxygenated blood to the fetus. Placental lobes are separated from each other by placental (decidual) septa. traicking cells between mother and fetus in both directions. This situation is best exempliied clinically by erythrocyte

1	traicking cells between mother and fetus in both directions. This situation is best exempliied clinically by erythrocyte D-antigen alloimmunization (Chap. 15, p. 301). Fetal cell admixtures likely are small in most cases, although rarely the fetus exsanguinates into the maternal circulation. Fetal cells can also engraft in the mother during pregnancy and can be identiied decades later. Fetal lymphocytes, CD34+ mesenchymal stem cells, and endothelial colony-forming cells all reside in maternal blood, bone marrow, or uterine vascula ture (Nguyen, 2006; Piper, 2007; Sipos, 2013). Termed micro chimerism, such residual stem cells have been implicated in the disparate female:male ratio of autoimmune disorders (Greer, 2011; Stevens, 2006). As discussed in Chapter 59 (p. 1139), they are associated with the pathogenesis of lymphocytic thy roiditis, scleroderma, and systemic lupus erythematosus.

1	Survival of the semi allogenic fetal graft requires complex interactions between fetal trophoblasts and maternal decidual immune cells. The fetal-maternal interface is not immunologically inert. Rather, it is an active hub of interactions that allows implantation and appropriate placental development and ensures immuno tolerance of the fetus. Despite this, a functional immune system must be maintained to protect the mother. Immunogenicity of the Trophoblasts Trophoblastic cells are the only fetus-derived cells in direct contact with maternal tissues and blood. Fetal syncytiotrophoblast synthesizes and secretes numerous factors that regulate the immune responses of maternal cells both at the implantation site and systemically.

1	Human leukocyte antigens (HLAs) are the human analogue of the major histocompatibility complex (MHC) (Hunt, 1992). There are 17 HLA class I genes, including three classic genes, HLA-A, -B, and -C, that encode the major class I (class Ia) transplantation antigens. Three other class I genes, designated HLA-E, -F, and -G, encode class Ib HLA antigens. MHC class I and II antigens are absent from villous trophoblasts, which appear to be immunologically inert at all gestational stages (Weetman, 1999). Invasive extravillous cytotrophoblasts do express MHC class I molecules. Thus, the ability of these cells to bypass transplantation rejection is the focus of considerable study.

1	Mofett-King (2002) reasoned that normal implantation depends on controlled trophoblastic invasion of maternal decidua and spiral arteries. Such invasion must proceed far enough to provide for normal fetal growth and development, but a mechanism must regulate invasion depth. She suggests that dNK cells combined with unique expression of three speciic HLA class I genes in extravillous cytotrophoblasts act in concert to permit and subsequently limit trophoblast invasion.

1	Class I antigens in extravillous cytotrophoblasts are accounted for by the expression of classic HLA-C and nonclassic class Ib molecules of HLA-E and HLA-G. HLA-G antigen is expressed only in humans, with expression restricted to extravillous cytotrophoblasts contiguous with maternal tissues. Embryos used for in vitro fertilization do not implant if they do not express a soluble HLA-G isoform (Fuzzi, 2002). hus, HLA-G may be immunologically permissive of the maternalfetal antigen mismatch (LeBouteiller, 1999). HLA-G has a proposed role in protecting extravillous trophoblasts from immune rejection via modulation of dNK functions (Apps, 2011; Rajagopalan, 2012). Last, Goldman-Wohl and associates (2000) have provided evidence for abnormal HA-G expression in extravillous trophoblasts from women with preeclampsia.

1	Natural killer cells are the predominant population of leukocytes present in midluteal phase endometrium and in decidua throughout the first trimester Oohnson, 1999). By term, however, relatively few dNK cells are present in decidua. In first-trimester decidua, dNK cells lie close to extravillous trophoblasts, and there they purportedly serve to regulate invasion. These dNKs have a distinct phenotype characterized by a high surface density of CD56 or neural cell adhesion molecules (Manaster, 2008; Mofett-King, 2002). Their infiltration is increased by progesterone and by stromal cell production of IL-15 and decidual prolactin (Dunn, 2002; Gubbay, 2002). Although dNK cells have the capacity for cytotoxicity, they are not cytotoxic toward fetal trophoblasts. Their cytotoxic potential is prevented by molecular cues from decidual macrophages. In addition, the expression of specific HLA molecules protects against dNK killing. Also, dNK cells function to restrict trophoblast invasiveness to

1	by molecular cues from decidual macrophages. In addition, the expression of specific HLA molecules protects against dNK killing. Also, dNK cells function to restrict trophoblast invasiveness to protect the mother.

1	Of other cell types, decidual macro phages are distinct from proinflammatory M 1 or antiinlammatory M2 macro phages. Decidual macro phages express the complement receptor COlIc at high or low levels: CD11cHI and CD11cLO. These cells function to regulate adaptive T cell responses; control dNK diferentiation, activation, and cytotoxicity; and produce antiinlammatory cytokines such as IL-10 to ensure fetal tolerance and inhibition of harmful immune responses. Dendritic cells are cells that present antigens to T cells. hey play an important role in the development of a receptive endometrium for implantation.

1	Dendritic cells are cells that present antigens to T cells. hey play an important role in the development of a receptive endometrium for implantation. Maternal T cels, as part of the adaptive immune response, increase in number and function after encounter with a speciic antigen. These cells subsequently retain the ability to respond rapidly in a subsequent encounter with the same antigen. Specific populations of Treg cells persist and can protect against aberrant immune responses. During pregnancy, there is a systemic expansion of maternal Treg cell populations. These cells are FOXP3+ cells with defined fetal speciicity. They are immunosuppressive and playia role in fetal tolerance.

1	At term, the amnion is a tough and tenacious but pliable membrane. his innermost avascular fetal membrane is contiguous with amnionic fluid and plays a role of incredible importance in human pregnancy. he amnion provides almost all tensile strength of the fetal membranes. Thus, its resilience to rupture is vitally important to successful pregnancy outcome. Indeed, preterm rupture of fetal membranes is a major cause of preterm delivery (Chap. 42, p. 819). FIGURE 5-16 Photomicrograph of fetal membranes. From left to right: AE = amnion epithelium; AM = amnion mesenchyme; S zona spongiosa; eM = chorionic mesenchyme; TR = trophoblast; 0= decidua. (Used with permission from Dr. Judith R. Head.)

1	Bourne (1962) described five separate amnion layers. he inner surface, which is bathed by amnionic luid, is an uninterrupted, single layer of cuboidal epithelium (Fig. 5-16). his epithelium is attached irmly to a distinct basement membrane that is connected to an acellular compact layer composed primarily of interstitial collagens. On the outer side of the compact layer, there is a row of ibroblast-like mesenchymal cells, which are widely dispersed at term. here also are a few fetal macrophages in the amnion. he outermost amnion layer is the relatively acellular zona spongiosa, which is contiguous with the second fetal membrane, the chorion laeve. he human amnion lacks smooth muscle cells, nerves, lymphatics, and importantly, blood vessels.

1	Early during implantation, a space develops between the embryonic cell mass and adjacent trophoblastic cells (see Fig. 5-9). Small cells that line this inner surface of trophoblasts have been called amniogenic cells-precursors of amnionic epithelium. The amnion is irst identiiable on the 7th or 8th day of embryo development. It is initially a minute vesicle, which then develops into a small sac that covers the dorsal embryo surface. As the amnion enlarges, it gradually engulfs the growing embryo, which prolapses into its cavity (Benirschke, 2012).

1	Distention of the amnionic sac eventually brings it into contact with the interior surface of the chorion laeve. Apposition of the chorion laeve and amnion near the end of the irst trimester then causes an obliteration of the extraembryonic coelom. he amnion and chorion laeve, although slightly adhered, are never intimately connected and can be separated easily. Placental amnion covers the placental surface and thereby is in contact with the adventitial surface of chorionic vessels. Umbilical amnion covers the umbilical cord. With diamnionic-monochorionic placentas, there is no intervening tissue between the fused amnions. In the conjoined portion of membranes of diamnionic-dichorionic twin placentas, amnions are separated by fused chorion laeve.

1	Amnionic luid ills this amnionic sac. Until about 34 weeks' gestation, the normally clear fluid increases in volume as pregnancy progresses. Mter this, the volume declines. At term, amnionic luid averages 1000 mL, although this may vay widely in normal and especially abnormal conditions. The origin, composition, circulation, and function of amnionic luid are discussed further in Chapter 11 (p. 225). Epithelial cells of the amnion derive from fetal ectoderm of the embryonic disc. hey do not arise by delamination from trophoblasts. his is an important consideration from both embryological and functional perspectives. For example, HLA class I gene expression in amnion is more akin to that in embyonic cells than to that in trophoblasts.

1	he ibroblast-like mesenchymal cell layer of the amnion is likely derived from embryonic mesoderm. Early in human embryogenesis, the amnionic mesenchymal cells lie immediately adjacent to the basal surface of the amnion epithelium. At this time, the amnion surface is a two-cell layer with approximately equal numbers of epithelial and mesenchymal cells. Simultaneously with growth and development, interstitial collagens are deposited between these two cell layers. his marks formation of the amnion compact layer, which separates the two layers of amnion cells.

1	As the amnionic sac expands, the compactness of the mesenchymal cells is progressively reduced, and they become sparsely distributed. Early in pregnancy, amnionic epithelium replicates at a rate appreciably faster than mesenchymal cells. At term, these cells form a continuous uninterrupted epithelium on the fetal amnionic surface. Conversely, mesenchymal cells are widely dispersed, being connected by a ine lattice network of extracellular matrix with the appearance of long slender ibrils.

1	he apical surface of the amnionic epithelium is replete with highly developed microvilli. This structure relects its function as a major site of transfer between amnionic luid and amnion. This epithelium is metabolically active, and its cells synthesize tissue inhibitor of MMP-l, prostaglandin E2 (PGE2), and fetal ibronectin (FN) (Rowe, 1997). Although epithelia produce FN, recent studies suggest that ibronectin functions in the underlying mesenchymal cells. Here, FN promotes synthesis of MMPs that break down the strength-bearing collagens and enhance prostaglandin synthesis to prompt uterine contractions (Mogami, 2013). This pathway is upregulated in premature rupture of membranes induced by thrombin or infection-induced release of FN (Chigusa, 2016; Mogami, 2014).

1	Epithelial cells may respond to signals derived from the fetus or the mother, and they are responsive to various endocrine or paracrine modulators. Examples include oxytocin and vasopressin, both of which increase PGE2 production in vitro (Moore, 1988). They may also produce cytokines such as IL-8 during labor initiation (Elliott, 2001).

1	Amnionic epithelium also synthesizes vasoactive pep tides, including endothelin and parathyroid hormone-related protein (Economos, 1992; Germain, 1992). he tissue produces brain natriuretic peptide (BNP) and corticotropin-releasing hormone (CRH), which are peptides that invoke smooth-muscle relaxation (Riley, 1991; Warren, 1995). BNP production is positively regulated by mechanical stretch in fetal membranes and is proposed to function in uterine quiescence. Epidermal growth factor, a negative regulator ofBNP, is up regulated in the membranes at term and leads to a decline in BNP-regulated uterine quiescence (Carvaj al, 2013). It seems reasonable that vasoactive peptides produced in amnion gain access to the adventitial surface of chorionic vessels. hus, the amnion may be involved in modulating chorionic vessel tone and blood low. Amnionderived vasoactive peptides function in both maternal and fetal tissues in diverse physiological processes. AOter their secretion, these bioactive

1	chorionic vessel tone and blood low. Amnionderived vasoactive peptides function in both maternal and fetal tissues in diverse physiological processes. AOter their secretion, these bioactive agents enter amnionic luid and thereby are available to the fetus by swallowing and inhalation.

1	Mesenchymal cells of the amnionic ibroblast layer are responsible for other major functions. Synthesis of interstitial collagens that compose the compact layer of the amnion-the major source of its tensile strength-takes place in mesenchymal cells (Casey, 1996). At term the generation of cortisol by l1�-hydroxysteroid dehydrogenase may contribute to membrane rupture via reduction of collagen abundance (Mi, 2017). Mesenchymal cells also synthesize cytokines that include IL-6, IL-8, and MCP-I. Cytokine synthesis rises in response to bacterial toxins and IL-1. his functional capacity of amnion mesenchymal cells is an important consideration in amnionic fluid study of labor-associated accumulation of inlammatory mediators (Garcia-Velasco, 1999). Finally, mesenchymal cells may be a greater source of PGE2 than epithelial cells, especially in the case of premature membrane rupture (Mogami, 2013; Whittle, 2000).

1	During tests of tensile strength, the decidua and then the chorion laeve give way long before the amnion ruptures. Indeed, the membranes are elastic and can expand to twice normal size during pregnancy (Benirschke, 2012). The amnion tensile strength resides almost exclusively in the compact layer, which is composed of cross-linked interstitial collagens I and III and lesser amounts of collagens V and VI. Collagens are the primary macromolecules of most connective tissues. Collagen I is the major interstitial collagen in tissues characterized by great tensile strength, such as bone and tendon. In other tissues, collagen III is believed to contribute to tissue integrity and provides both tissue extensibility and tensile strength. For example, the ratio of collagen III to collagen I in the walls of a number of highly extensible tissues-amnionic sac, blood vessels, urinary bladder, bile ducts, intestine, and gravid uterus-is greater than that in nonelastic tissues a efrey, 1991).

1	Amnion tensile strength is regulated in part by fibrillar collagen assembly. his process is influenced by the interaction fibrils with proteoglycans such as decorin and biglycan (Chap. 21, p. 409). Reduction of these proteoglycans is reported to perturb fetal membrane function (Horgan, 2014; Wu, 2014). Fetal membranes overlying the cervix have a regional shift in gene expression and lymphocyte activation that set in motion an inflammatory cascade (Marcellin, 2017). his change may contribute to tissue remodeling and loss of tensile strength in the amnion (Moore, 2009).

1	The amnion is metabolically active, is involved in solute and water transport for amnionic fluid homeostasis, and produces an impressive array of bioactive compounds. The amnion is responsive both acutely and chronically to mechanical stretch, which alters amnionic gene expression (Carvajal, 2013; Nemeth, 2000). This in turn may trigger both autocrine and paracrine responses that include production of MMPs, IL-8, and collagenase (Bryant-Greenwood, 1998; Mogami, 2013). Such factors may modulate changes in membrane properties during labor.

1	he yolk sac and the umbilical vesicle into which it develops are prominent early in pregnancy. At irst, the embryo is a lattened disc interposed between amnion and yolk sac (see Fig. 5-9). Its dorsal surface grows faster than the ventral surface, in association with the elongation of its neural tube. Thus, the embryo bulges into the amnionic sac, and the dorsal part of the yolk sac is incorporated into the embryo body to form the gut. The allantois projects into the base of the body stalk from the caudal wall of the yolk sac and later, from the anterior wall of the hindgut.

1	As pregnancy advances, the yolk sac becomes smaller and its pedicle relatively longer. By the middle of the third month, the expanding amnion obliterates the extraembryonic coelom, fuses with the chorion laeve, and covers the bulging placental disc and the lateral surface of the body stalk. The latter is then called the umbilical cord-or funis. A more detailed description of this cord and potential abnormalities is found in Chapter 6 (p. 117). he cord at term normally has two arteries and one vein (Fig. 5-17). The right umbilical vein usually disappears early during fetal development, leaving only the original left vein.

1	he cord at term normally has two arteries and one vein (Fig. 5-17). The right umbilical vein usually disappears early during fetal development, leaving only the original left vein. The umbilical cord extends from the fetal umbilicus to the fetal surface of the placenta, that is, the chorionic plate. Blood flows from the umbilical vein toward the fetus. Blood then takes a path of least resistance via two routes within the fetus. One is the ductus venosus, which empties directly into the inferior vena cava (Fig. 7-9, p. 130). The other route consists of numerous smaller openings into the hepatic circulation. Blood from the liver flows into the inferior vena cava via the hepatic vein. Resistance in the ductus venosus is controlled by a sphincter that is situated at the origin of the ductus at the umbilical recess and is innervated by a vagus nerve branch.

1	Blood exits the fetus via the two umbilical arteries. These are anterior branches of the internal iliac artery and become obliterated after birth to form the medial umbilical ligaments. FIGURE 5-17 Cross-section of umbilical cord. The large umbilical vein carries oxygenated blood to the fetus (right). To its left are the two smaller umbilical arteries, carrying deoxygenated blood from the fetus to the placenta. (Used with permission from Dr. Mandolin S. Ziadie.) The production of steroid and protein hormones by human trophoblasts is greater in amount and diversity than that of any single endocrine tissue in all of mammalian physiology. A compendium of average production rates for various steroid hormones in nonpregnant and in near-term pregnant women is given in Table

1	It is apparent that alterations in steroid hormone production that accompany normal human pregnancy are incredible. he human placenta also synthesizes an enormous amount of protein and peptide hormones, summarized in Table 5-2. Another remarkable feature is the successful TABLE 5-1 . Steroid Production Rates in Nonpregnant and Near-Term Pregnant Women aEstrogens and progesterone are produced by placenta. Aldosterone is produced by the maternal adrenal in response to the stimulus of angiotenoin II. Deoxycorticosterone is produced in extraglandular tissue sites by way of the 21-hydroxylation of plasma progesterone. Cortisol production during pregnancy is not increased, even though the blood levels are elevated because of decreased clearance caused by increased cortisol-binding globulin. physiological adaptations of pregnant women to the unique endocrine milieu, and this is discussed throughout Chapter 4 (p. 49).

1	physiological adaptations of pregnant women to the unique endocrine milieu, and this is discussed throughout Chapter 4 (p. 49). Chorionic gonadotropin is a glycoprotein with biological activity similar to that of LH, and both act via the same LH-hCG receptor. hCG has with a molecular weight of 36,000 to 40,000 Da and has the highest carbohydrate content of any human hormone-30 percent. The carbohydrate component, and especially the terminal sialic acid, protects the molecule from catabolism. As a result, the 36-hour plasma half-life of intact hCG is much longer than the 2 hours for LH. he hCG molecule is composed of two dissimilar subunits termed . and � subunits. hese are noncovalently linked and are held together by electrostatic and hydrophobic forces. Isolated subunits are unable to bind the LH-hCG receptor and thus lack biological activity.

1	hCG is produced almost exclusively in the placenta, but low levels are synthesized in the fetal kidney. Other fetal tissues produce either the �-subunit or intact hCG molecule (McGregor, 1981,i1983). The hCG hormone is structurally related to three other glycoprotein hormones-LH, FSH, and TSH. All four glycoproteins share a common a-subunit. However, each of their 3-subunits, although sharing certain similarities, is characterized by a distinctly diferent amino acid sequence.

1	Synthesis of the .-and 3-chains of hCG is regulated separately. A single gene located on chromosome 6 encodes the a-subunit. Seven genes on chromosome 19 encode for the �-hCG-3-LH family of subunits. Six genes code for 3-hCG and one for 3-LH (Miller-Lindholm, 1997). Both subunits are synthesized as larger precursors, which are then cleaved by endopeptidases. Intact hCG is then assembled and rapidly released by secretory granule exocytosis (Morrish, 1987). There are multiple forms of hCG in maternal plasma and urine that vary enormously in bioactivity and immunoreactivity. Some result from enzymatic degradation, and others from modifications during molecular synthesis and processing.

1	Before 5 weeks, hCG is expressed both in the syncytiotrophoblast and in cytotrophoblasts (Mamo, 1992). Later, in the irst trimester when maternal serum levels peak, hCG is produced almost solely in the syncytiotrophoblast (Beck, 1986; Kurman, 1984). At this time, mRNA concentrations of both .-and 3-subunits in the syncytiotrophoblast are greater than at term (Hoshina, 1982). This may be an important consideration when hCG is used as a screening procedure to identiy abnormal fetuses.

1	Circulating free levels of 3-subunit are low to undetectable throughout pregnancy. In part, this is the result of its rate-limiting synthesis. Free a-subunits that do not combine with the 3-subunit are found in placental tissue and maternal plasma. Levels of the a-subunit rise gradually and steadily until they plateau at approximately 36 weeks' gestation. At this time, they account for 30 to 50 percent of hormone (Cole, 1997). Thus, .-hCG secretion roughly corresponds to placental mass, whereas secretion of complete hCG molecules is maximal at 8 to 10 weeks. TABLE 5-2. Protein Hormones Produced by the Human Placenta Primary Non-placental Shares Structural or Hormone Site of Expression Function Similarity Functions LH, FSH,lTSH GH, prolactin

1	TABLE 5-2. Protein Hormones Produced by the Human Placenta Primary Non-placental Shares Structural or Hormone Site of Expression Function Similarity Functions LH, FSH,lTSH GH, prolactin The combined hCG molecule is detectable in plasma of pregnant women 7 to 9 days after the midcycle surge of LH that precedes ovulation. hus, hCG likely enters maternal blood at the time of blastocyst implantation. Plasma levels rise rapidly, doubling every 2 days in the first trimester (Fig. 5-18). Appreciable fluctuations in levels for a given patient are observed on the same day.

1	Intact hCG circulates as multiple highly related isoforms with variable cross-reactivity between commercial assays. hus, calculated serum hCG levels can vary considerably among the more than a hundred available assays. his emphasizes the need to use the same assay type when clinically measuring serial hCG levels. Peak maternal plasma levels reach approximately 50,000 to 100,000 mIU/mL between the 60th and 80th days after menses. At 10 to 12 weeks' gestation, plasma levels begin to decline, and a nadir is reached by approximately 16 weeks. Plasma levels are maintained at this lower level for the remainder of pregnancy. The pattern of hCG appearance in fetal blood is similar to that in the mother. Fetal plasma levels, however, are only about 3 percent of those in maternal plasma. Amnionic fluid hCG concentration early in pregnancy is similar to that in maternal plasma. As pregnancy progresses, hCG concentration in Aids maternal adaptation to fetal energy requirements

1	Aids maternal adaptation to fetal energy requirements Relaxes smooth-muscle; initiates parturition? Promotes fetal and maternal glucocorticoid production Regulates trophoblast hCG production Regulates transfer of calcium and other solutes; regulates fetal mineral homeostasis nizing hormone; TSH = thyroid-stimulating hormone. amnionic fluid declines, and near term the levels are approximately 20 percent of those in maternal plasma. Maternal urine contains the same variety of hCG degradation products as maternal plasma. The principal urinary form is the terminal product of hCG degradation, namely, the 500 400 300�I 0E , 200£:) 100 , ..--, , , , I I 4, , I :t > II II ,, 3.1 c 40 I \ , , c I \, " , , 2 FIGURE 5-18 Distinct profiles for the concentrations of human chorionic gonadotropin (hCG), human placental lactogen (hPL), and corticotropin-releasing hormone (CRH) in serum of women throughout normal pregnancy.

1	�-core fragment. Concentrations of this fragment follow the same general pattern as that in maternal plasma, peaking at approximately 10 weeks' gestation. Importantly, the so-called �-subunit antibody used in most pregnancy tests reacts with both intact hCG-the major form in the plasma, and with fragments of hCG-the major forms found in urine. Placental gonadotropin-releasing hormone (GnRH) is likely involved in the regulation of hCG formation. Both GnRH and its receptor are expressed by cytotrophoblasts and by the syncytiotrophoblast (Wolfahrt, 1998). GnRH administration elevates circulating hCG levels, and cultured trophoblasts respond to GnRH treatment and raise hCG secretion (Iwashita, 1993; Siler-Khodr, 1981). Pituitary GnRH production also is regulated by inhibin and activin. In cultured placental cells, activin stimulates and inhibin inhibits GnRH and hCG production (Petraglia, 1989; Steele, 1993).

1	Renal clearance of hCG accounts for 30 percent of its metabolic clearance. he remainder is likely cleared by metabolism in the liver (Wehmann, 1980). Clearances of�-and .-subunits are approximately 10-and 30-fold, respectively, greater than that of intact hCG. In pregnancies complicated by chronic renal disease, hCG clearance can be markedly decreased. Both hCG subunits are required for binding to the LH-hCG receptor in the corpus lute un and the fetal testis. LH-hCG receptors are present in various other tissues, but their roles there are less defined.

1	he best-known biological function of hCG is the so-called rescue and maintenance of corpus luteum function-that is, continued progesterone production. This is only an incomplete explanation for the physiological function of hCG in pregnancy. For example, maximum plasma hCG concentrations are attained well after hCG-stimulated corpus luteum secretion of progesterone has ceased. Speciically, luteal progesterone synthesis begins to decline at about 6 weeks despite continued and increasing hCG production.

1	A second hCG role is stimulation of fetal testicular testosterone secretion. This is maximum approximately when hCG levels peak. hus, at a critical time in male sexual diferentiation, hCG enters fetal plasma from the syncytiotrophoblast. In the fetus, it acts as an LH surrogate to stimulate Leydig cell replication and testosterone synthesis to promote male sexual differentiation (Chap. 3, p. 35). Before approximately 110 days, there is no vascularization of the fetal anterior pituitary from the hypothalamus. Thus, pituitary LH secretion is minimal, and hCG acts as LH before this time. hereafter, as hCG levels fall, pituitary LH maintains modest testicular stimulation.

1	The maternal thyroid gland is also stimulated by large quantities of hCG. In some women with gestational trophoblastic disease, biochemical and clinical evidence of hyperthyroidism sometimes develops (Chap. 20, p. 391). This once was attributed to formation of chorionic thyrotropins by neoplastic trophoblasts. It was subsequently shown, however, that some forms ofhCG bind to TSH receptors on thyrocytes (Hershman, 1999). And, treatment of men with exogenous hCG increases thyroid activity. The thyroid-stimulatory activity in plasma of irst-trimester pregnant women varies appreciably from sample to sample. Modiications of hCG oligosaccharides likely are important in the capacity of hCG to stimulate thyroid function. For example, acidic isoforms stimulate thyroid activity, and some more basic isoforms stimulate iodine uptake (Kraiem, 1994; Tsuruta, 1995; Yoshimura, 1994). Finally, the LH-hCG receptor is expressed by thyrocytes, which suggests that hCG stimulates thyroid activity via the

1	isoforms stimulate iodine uptake (Kraiem, 1994; Tsuruta, 1995; Yoshimura, 1994). Finally, the LH-hCG receptor is expressed by thyrocytes, which suggests that hCG stimulates thyroid activity via the LH-hCG receptor as well as by the TSH receptor (Tomer, 1992).

1	Other hCG functions include promotion of relaxin secretion by the corpus luteum (Duy, 1996). LH-hCG receptors are found in myometrium and in uterine vascular tissue. It has been hypothesized that hCG may promote uterine vascular vasodilatation and myometrial smooth muscle relaxation (Kurtzman, 2001). Chorionic gonadotropin also regulates expansion of dNK cell numbers during early stages of placentation, thus ensuring appropriate establishment of pregnancy (Kane, 2009).

1	here are several clinical circumstances in which substantively higher maternal plasma hCG levels are found. Some examples are multifetal pregnancy, erythroblastosis fetalis associated with fetal hemolytic anemia, and gestational trophoblastic disease. Relatively higher hCG levels may be found in women carrying a fetus with Down syndrome. This observation is used in biochemical screening tests (Chap. 14, p. 281). The reason for the elevation is not clear, but reduced placental maturity has been speculated. Various malignant tumors also produce hCG, sometimes in large amounts-especially trophoblastic neoplasms (Chaps. 9, p. 159 and 20, p. 391).

1	Relatively lower hCG plasma levels are found in women with early pregnancy wastage, including ectopic pregnancy (Chap. 19, p. 373). hCG is produced in very small amounts in normal tissues of men and nonpregnant women, perhaps primarily in the anterior pituitary gland. Nonetheless, the detection of hCG in blood or urine almost always indicates pregnancy (Chap. 9, p. 158). This is a single, nonglycosylated polypeptide chain with a molecular weight of 22,279 Da. The sequences of hPL and of human growth hormone (hGH) are strikingly similar, with 96-percent homology. Also, hPL is structurally similar to human prolactin (hPRL), with a 67-percent amino acid sequence similarity. Because of these similarities, it was called human placental lactogen or chorionic growth hormone. Currently, human placental lactogen is used by most.

1	here are ive genes in the growth hormone-placental lactogen gene cluster that are linked and located on chromosome 17. hPL is concentrated in syncytiotrophoblast, but similar to hCG, hPL is demonstrated in cytotrophoblasts before 6 weeks (Grumbach, 1964; Maruo, 1992). Within 5 to 10 days after conception, hPL is demonstrable in the placenta and can be detected in maternal serum as early as 3 weeks. Levels of mRNA for hPL in syncytiotrophoblast remain relatively con stant throughout pregnancy. his inding supports the idea that the hPL secretion rate is proportional to placental mass. Levels rise steadily until 34 to 36 weeks' gestation. The hPL produc tion rate near term-approximately 1 g/d-is by far the great est of aRY known hormone in humans. The hal-life of hPL in maternal plasma is between 10 and 30 minutes (Walker, 1991). In late pregnancy, maternal serum concentrations reach levels of5 to 15 �g/mL (see Fig. 5-18).

1	In late pregnancy, maternal serum concentrations reach levels of5 to 15 �g/mL (see Fig. 5-18). Very little hPL is detected in fetal blood or in the urine of the mother or newborn. Amnionic fluid levels are somewhat lower than in maternal plasma. hPL is secreted primarily into the maternal circulation, with only very small amounts in cord blood. hus, its role in pregnancy is believed to be mediated through actions in maternal rather than in fetal tissues. None theless, interest continues for the possibility that hPL serves select functions in fetal growth. cesses. First, hPL promotes maternal lipolysis with increased circulating free fatty acid levels. This provides an energy source for maternal metabolism and fetal nutrition. In vitro cytiotrophoblast (Coya, 2005). Prolonged maternal starvation in the irst half of pregnancy leads to higher hPL plasma con centrations.

1	Second, hPL may aid maternal adaptation to fetal energy requirements. For example, increased maternal insulin resistance ensures nutrient flow to the fetus. It also favors protein synthesis and provides a readily available amino acid source to the fetus. To counterbalance the greater insulin resistance and prevent maternal hyperglycemia, maternal insulin levels are increased. Both hPL and prolactin signal through the prolactin receptor to increase maternal beta cell proliferation to augment insulin secretion (Georgia, 2010). In animals, prolactin and hPL upregulate serotonin synthesis, which increases beta cell proliferation (Kim, 2010). Short-term changes in plasma glucose or insulin, however, have relatively little efect on plasma hPL levels. In vitro studies of syncytiotrophoblast suggest that hPL synthesis is stimulated by insulin and insulin-like growth factor-1 and inhibited by PGE2 and PGF2: (Bhaumick, 1987; Genbacev, 1977).

1	Last, hPL is a potent angiogenic hormone. It may serve an important function in fetal vasculature formation (Corbacho, 2002). capacity to synthesize numer ous peptide hormones, including some that are analogous or related to hypothalamic and pituitary hormones. In contrast to their counterparts, some of these placental peptide/protein hormones are not subject to feedback inhibition. he known hypothalamic-releasing or -inhibiting hormones include GnRH, CRH, thyrotropin-releasing hormone (TRH), growth hormone-releasing hormone, and somatostatin. For each, there is an analogous hormone produced in the human placenta (Petraglia, 1992; Siler-Khodr, 1988).

1	GnH in the placenta shows its highest expression in the first trimester (Siler-Khodr, 1978, 1988). Interestingly, it is found in cytotrophoblasts, but not syncytiotrophoblast. Pla centa-derived GnRH functions to regulate trophoblast hCG ofMMP-2 and MMP-9 (Peng, 2016). Placenta-derived GnRH is also the likely cause of elevated maternal GnRH levels in pregnancy (Siler-Khodr, 1984). CH is a member of a larger family of CRH-related pep tides that includes CRH and urocortins (Dautzenberg, 2002). Maternal serum CRH levels increase from 5 to 10 pmollL in the nonpregnant woman to approximately 100 pmollL in the early third trimester of pregnancy and to almost 500 pmollL abruptly during the last 5 to 6 weeks (see Fig. 5-18). Urocortin also is produced by the placenta and secreted into the maternal circulation, but at much lower levels than seen for CRH (Flo rio, 2002). After labor begins, maternal plasma CRH levels rise even further (Petraglia, 1989, 1990).

1	he biological function of CRH synthesized in the placenta, membranes, and decidua has been somewhat deined. CRH receptors are present in many tissues including placenta. Trophoblast, amniochorion, and decidua express both CRH-R1 and CRH-R2 receptors and several variant receptors (Florio, 2000). Both CRH and urocortin enhance trophoblast secretion of adrenocorticotropic hormone (ACTH), supporting an autocrine-paracrine role (Petraglia, 1999). Large amounts of trophoblast CRH enter maternal blood.

1	Other proposed biological roles include induction of smooth-muscle relaxation in vascular and myometrial tissue and immunosuppression. The physiological reverse, however, induction of myometrial contractions, has been proposed for the rising CRH levels seen near term. One hypothesis suggests that CRH may be involved with parturition initiation (Wadhwa, 1998). Some evidence suggests that urocortin 2 expression is induced at term and induces expression of pro inlammatory markers and prostaglandin F receptor expression in the placenta and myometrium (Voltolini, 2015). Prostaglandin formation in the placenta, amnion, chorion laeve, and decidua is increased with CRH treatment Oones, 1989b). hese observations further support a potential action in parturition timing.

1	Glucocorticoids act in the hypothalamus to inhibit CRH release, but in the trophoblast, glucocorticoids stimulate CRH gene expression Oones, 1989a; Robinson, 1988). hus, there may be a novel positive feedback loop in the placenta by which placental CRH stimulates placental ACTH to stimulate fetal and maternal adrenal glucocorticoid production with quent stimulation of placental CRH expression (Nicholson, 2001; Riley, 1991). Growth hormone-releasing hormone has an unknown role (Berry, 1992). Ghrelin is another regulator of hGH secretion that is produced by placental tissue (Horvath, 2001). Trophoblast ghrelin expression peaks at midpregnancy and is a paracrine regulator of diferentiation or is a potential regulator of human growth hormone variant production, described next (Fuglsang, 2005; Gualillo, 2001).

1	ACTH, lipotropin, and �-endorphin, which are all proteolytic products of pro-opiomelanocortin, are recovered from placental extracts (Genazzani, 1975; Odagiri, 1979). he physiological action of placental ACTH is unclear. As discussed, placental CRH stimulates synthesis and release of chorionic ACTH.

1	A human growth hormone variant (hGH-J that is not expressed in the pituitary is expressed in the placenta. The gene encoding hGH-V is located in the hGH-hPL gene cluster on chromosome 17. Sometimes referred to as placental growth hormone, hGH-V is a 191-amino-acid protein that difers in 15 amino acid positions from the sequence for hGH. Although hGH-V retains growth-promoting and antilipogenic functions similar to those of hGH, it has reduced diabetogenic and lactogenic functions relative to hGH (Vickers, 2009). Placental hGH-V presumably is synthesized in the syncytiotrophoblast. It is believed that hGH-V is present in maternal plasma by 21 to 26 weeks' gestation, rises in concentration until approximately 36 weeks, and remains relatively constant thereafter. There is a correlation between the levels of hGH-V in maternal plasma and those of insulin-like growth factor-I. lso, hGH-V secretion by trophoblast in vitro is inhibited by glucose in a dosedependent manner (Patel, 1995).

1	between the levels of hGH-V in maternal plasma and those of insulin-like growth factor-I. lso, hGH-V secretion by trophoblast in vitro is inhibited by glucose in a dosedependent manner (Patel, 1995). Overexpression ofhGH-V in mice causes severe insulin resistance, making it a likely candidate to mediate insulin resistance of pregnancy (Liao, 2016).

1	Expression of relaxin has been demonstrated in human corpus luteum, decidua, and placenta (Bogic, 1995). This peptide is synthesized as a single, 105-amino-acid preprorelaxin molecule that is cleaved to A and B molecules. Relaxin is structurally similar to insulin and insulin-like growth factor. Two of the three relaxin genes-H2 and H3-are transcribed in the corpus luteum (Bathgate, 2002; Hudson, 1983, 1984). Decidua, placenta, and membranes express H 1 and H2 (Hansell, 1991).

1	The rise in maternal circulating relaxin levels seen in early pregnancy is attributed to corpus luteum secretion, and levels parallel those of hCG. Relaxin, along with rising progesterone levels, may act on myometrium to promote relaxation and the quiescence of early pregnancy (Chap. 21, p. 407). In addition, the production of relaxin and relaxin-like factors within the placenta and fetal membranes may play an autocrine-paracrine role in postpartum regulation of extracellular matrix remodeling (Qin, 1997a,b). One important relaxin function is enhancement of the glomerular iltration rate (Chap. 4, p. 66).

1	In pregnancy, circulating parathyroid hormone-related protein (PTH-rP) levels are significantly elevated within maternal but not fetal circulation (Bertelloni, 1994; Saxe, 1997). Many functions of this hormone have been proposed. PTH-rP synthesis is found in several normal adult tissues, especially in reproductive organs that include myometrium, endometrium, corpus luteum, and lactating mammary tissue. PTH-rP is not produced in the parathyroid glands of normal adults. Placentaderived PTH-rP may have an important function to regulate genes involved in transfer of calcium and other solutes. It also contributes to fetal mineral homeostasis in bone, amnionic luid, and the fetal circulation (Simmonds, 2010).

1	This hormone is normally secreted by adipocytes. It functions as an antiobesity hormone that decreases food intake .through its hypothalamic receptor. It also regulates bone growth and immune unction (Cock, 2003; La Cava, 2004). In the placenta, leptin is synthesized by both cytotrophoblasts and syncytiotrophoblast (Henson, 2002). Relative contributions of leptin from maternal adipose tissue versus placenta are currently not well defined, although recent evidence highlights a key regulatory role of placental leptin in placental amino acid transport and fetal growth (Rosario, 20 16a). Maternal serum levels are significantly higher than those in nonpregnant women. Fetalileptin levels correlate positively with birth weight and likely function in fetal development and growth. Studies suggest that reductions in leptin availability contribute to adverse fetal metabolic programing in intrauterine growth-restricted ofspring (Nusken, 2016).

1	This 36-amino-acid peptide is widely distributed in brain. It also is found in sympathetic neurons innervating the cardiovascular, respiratory, gastrointestinal, and genitourinary systems. Neuropeptide Y has been isolated from the placenta and localized in cytotrophoblasts (Petraglia, 1989). Trophoblasts possess neuropeptide Y receptors, and treatment of these with neuropeptide Y causes CRH release (Robidoux, 2000).

1	hese glycoprotein hormones are expressed in male and female reproductive tissues and belong to the transforming growth factor-� family Gones, 2006). Inhibin is a heterodimer made up of one a-subunit and one of two distinct �-subunits, either �A or �B. his yields either inhibin A or inhibin B, respectively. Activin is formed by the combination of the two �-subunits. Activin, inhibin, and their respective receptors are expressed in the placenta. Both activin and inhibin A have proposed functions during cytotrophoblast fusion into the syncytiotrophoblast (Debieve, 2000; Jones, 2006). Activin also stimulates production of placental hormones such as hCG, hPL, progesterone, and estrogen (Luo, 2002; Morrish, 1991; Petraglia, 1989; Song, 1996). Inhibin A opposes activin action in the placenta to inhibit production of hCG and steroidogenesis (Petraglia, 1989). Abnormal levels of inhibin or activin correlate with placental pathologies. For example, elevation in inhibin A levels in the second

1	inhibit production of hCG and steroidogenesis (Petraglia, 1989). Abnormal levels of inhibin or activin correlate with placental pathologies. For example, elevation in inhibin A levels in the second trimester is indicative of fetal Down syndrome. Further, low inhibin levels early in pregnancy may indicate pregnancy failure (Prakash, 2005; Wallace, 1996). Elevations in circulating inhibin and activin levels are reported in women with preeclampsia (Bersinger, 2003).

1	After 6 to 7 weeks' gestation, little progesterone is produced in the ovary (Diczfalusy, 1961). Surgical removal of the corpus luteum or even bilateral oophorectomy during the 7th to 10th week does not decrease excretion rates of urinary pregnanediol, 100.0 50.0 10.0 n ..) 5.0 1.0 ):l u 0.5 0.1 0.05

1	FIGURE 5-19 Plasma levels of progesterone, estradiol, estrone, estetrol, and estriol in women during the course of gestation. (Modified and redrawn with permission from Mesiano S: The endocrinology of human pregnancy and fetoplacental neuroendocrine development. In Strauss JF, Barbieri RL (eds) Yen and Jaffe's Reproductive Endocrinology: Physiology, Pathophysiology, and Clinical Management, 6th ed. Philadephia, Saunders, 2009.) the principal urinary metabolite of progesterone. Before this time, however, corpus luteum removal will result in spontaneous abortion unless an exogenous progestin is given (Chap. 63, p. 1198). After approximately 8 weeks, the placenta assumes progesterone secretion, resulting in a gradual increase in maternal serum levels throughout pregnancy (Fig. 5-19). By term, these levels are 10 to 5000 times those found in nonpregnant women, depending on the stage of the ovarian cycle.

1	The daily production rate of progesterone in late, normal, singleton pregnancies approximates 250 mg. In mulrifetal pregnancies, the daily production rate may exceed 600 mg. Progesterone is synthesized from cholesterol in a two-step enzymatic reauiuI1. r.l�., L-l1UI:�L:lUl ;� C0il v'ci'tC. tv plcg.cidciC ';;ithi� the mitochondria, in a reaction catalyzed by cytochrome P450 cholesterol side-chain cleavage enzyme. Pregnenolone leaves the mitochondria and is converted to progesterone in the endoplasmic reticulum by 33-hydroxysteroid dehydrogenase. Progesterone is released immediately through a process of difusion.

1	Although the placenta produces a prodigious amount of progesterone, the syncytiotrophoblast has a limited capacity for cholesterol biosynthesis. Radiolabeled acetate is incorporated into cholesterol by placental tissue at a slow rate. he rate-limiting enzyme in cholesterol biosynthesis is 3-hydroxy3-methylglutaryl coenzyme A (HMG-CoA) reductase. Because of this, the placenta must rely on an exogenous source, that is, maternal cholesterol, for progesterone formation. he tro biosynthesis (Simpson, 1979, 1980). This mechanism difers from placental production of estrogens, which relies principally on fetal adrenal precursors. Although there is a relationship between fetal well-being and placental estrogen production, this is not the case for placen tal progesterone. Thus, placental endocrine function, including the formation of protein hormones such as hCG and proges terone biosynthesis, may persist for weeks after fetal demise.

1	he metabolic clearance rate of progesterone in pregnant women is similar to that found in men and nonpregnant women. During pregnancy, the plasma concentration of 5a-dihydroprogesterone disproportionately rises due to syn progesterone and fetus-derived precursor (Dombroski, 1997). hus, the concentration ratio of this progesterone metabolite to progesterone is elevated in pregnancy. The mechanisms for this are not deined completely. Progesterone also is converted to the potent mineralocorticoid deoxycorticosterone in preg nant women and in the fetus. The concentration of deoxy corticosterone is strikingly higher in both maternal and fetal compartments (see Table 5-1). The extraadrenal formation of most of its production in pregnancy (Casey, 1982a,b).

1	During the first 2 to 4 weeks of pregnancy, rising hCG levels maintain production of estradiol in the maternal corpus luteum. Production of both progesterone and estrogens in the maternal ovaries drops signiicantly by the 7th week of pregnancy. At this time, there is a luteal-placental transition. By the 7th week, more than half of estrogen entering maternal circulation is produced in the placenta (MacDonald, 1965a; Siiteri, 1963,i1966). Subsequently, the placenta produces a continually increasing magnitude of estrogen. Near term, normal human pregnancy is a hyperestrogenic state, and syncytiotrophoblast is producing estrogen in amounts equivalent to that produced in 1 day by the ovaries of no fewer than 1000 ovulatory women. This hyperestrogenic state terminates abruptly after delivery of the placenta.

1	In human trophoblast, neither cholesterol nor, in turn, proges :e�8!e :�� �e�.'e !s F"�C�!!S0!" For �'tr0g�n hi)"ynth�."is. Thi� is because steroid 17a-hydroxylasel1,20-yase (CP17Al) is not expressed in the human placenta. This essential enzyme converts 17-0H progesterone (a C2I steroid) to androstenedione, which is a C'9 steroid and an estrogen precursor. Consequently, the conversion of C2I steroids to C'9 steroids is not possible. However, dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) are also C'9 steroids and are produced by maternal and fetal adrenal glands. These two steroids can serve as estrogen precursors (Fig. 5-20). Ryan (1959a) found that the placenta had an exceptionally high capacity to convert appropriate C'9 steroids to estrone and estradiol. The conversion of DHEA-S to estradiol requires placental expression of four key enzymes

1	FIGURE 5-20 Schematic presentation of estrogen biosynthesis in the human placenta. Dehydroepiandrosterone sulfate (DHEAS), secreted in prodigious amounts by the fetal adrenal glands, is converted to 16a-hydroxydehydroepiandrosterone sulfate (16aOHDHEAS) in the fetal liver. These steroids, DHEAS and 16aOHDHEAS, are converted in the placenta to estrogens, that is, 17�-estradiol (E) and estriol (E3). Near term, half of E2 is derived from fetal adrenal DHEAS and half from maternal DHEAS. On the other hand, 90 percent of E3 in the placenta arises from fetal 16aOHDHEAS and only 10 percent from all other sources.

1	that are located principally in syncytiotrophoblast (Bonenfant, 2000; Salido, 1990). First, the placenta expresses high levels of steroid sulfatase (STS), which converts the conjugated DHEAS to DHEA. DHEA is then acted upon by 33-hydroxysteroid dehydrogenase type 1 (33HSD) to produce androstenedione. Cytochrome P450 aromatase (CYP19) then converts androstenedione to estrone, which is then converted to estradiol by 173-hydroxysteroid dehydrogenase type 1 (173HSDl). DHEA-S is the major precursor of estrogens in pregnancy (Baulieu, 1963; Siiteri, 1963). However, maternal adrenal glands do not produce suicient amounts of DHA-S to account for more than a fraction oftotal placental estrogen biosynthesis. he fetal adrenal glands are quantitatively the most important source of placental estrogen precursors in human pregnancy. hus, estrogen production duringpregnancy relects the unique interactions among fetal adrenal glands, fetal liver, placenta, and maternal adrenal glands.

1	More than 90 percent of estradiol and estriol formed in syncytiotrophoblast 'enters maternal plasma (Gurpide, 1966). And, 85 percent or more of placental progesterone enters maternal plasma, and little maternal progesterone crosses the placenta to the fetus (Gurpide, 1972). his directional movement ofnewly formed steroid into the maternal circulation stems from basic characteristics of hemochorioendothelial placentation. In this system, steroids secreted from syncytiotrophoblast can enter maternal blood directly. Steroids that leave the syncytium do not enter fetal blood directly. hey must irst traverse the cytotrophoblast layer and then enter the stroma of the villous core and then fetal capillaries. From either ofthese spaces, steroids can reenter the syncytium. he net result of this hemochorial arrangement is that entry of steroids into the maternal circulation is substantially greater than that into fetal blood.

1	Morphologically, functionally, and physiologically, the fetal adrenal glands are remarkable. At term, the fetal adrenal glands weigh the same as those of the adult. More than 85 percent of the fetal gland is composed of a unique fetal zone, which has a great capacity for steroid biosynthesis. Daily steroid production of fetal adrenal glands near term is 100 to 200 mg/d. This compares with resting adult steroid secretion of 30 to 40 mg/d. The fetal zone is lost in the irst year of life and is not present in the adult. In addition to ACTH, fetal adrenal gland growth is influenced by factors secreted by the placenta. This is exempliied by the continued growth of the fetal glands throughout gestation and by rapid involution immediately after birth and placental delivery.

1	Estradiol is the primary placental estrogen product at term. In addition, significant levels of estriol and estetrol are found in the maternal circulation, and levels also rise, particularly late in gestation (see Fig. 5-19). hese hydroxylated forms of estro gen derive from the placenta using substrates formed by the combined eforts of the fetal adrenal gland and fetal liver. For this, high levels of fetal hepatic 16a-hydroxylase act on adre nal-derived steroids. Ryan (1959b) and MacDonald and Siiteri (1965b) found that 16a-hydroxylated C19 steroids, particu larly 16a-hydroxydehydroepiandrosterone (16-0HDHEA), were converted to estriol by placental tissue. Thus, the dispro portionate increase in estriol formation during pregnancy is accounted for by placental synthesis of estriol principally from plasma-borne 16-0HDHEA-sulfate. Near term, the fetus is the source of 90 percent of placental estriol and estetrol precur sors in normal human pregnancy.

1	Maternal estriol and estetrol are produced almost solely by fetal steroid precursors. hus, in the past, levels of these steroids were used as an indicator of fetal well-being. However, the low sensitivity and speciicity of such tests have caused them to be discarded. The precursor for fetal adrenal steroidogenesis is cholesterol. he steroid biosynthesis rate in the fetal gland is so great that its steroidogenesis alone is equivalent to a fourth of the total daily LDL cholesterol turnover in adults. Fetal adrenal glands synthesize cholesterol from acetate. All enzymes involved in cholesterol biosynthesis are elevated compared with those of the adult adrenal gland (Rainey, 2001). Thus, the de novo cholesterol synthesis rate by fetal adrenal tissue is extremely high. Even so, it is insuicient to account for the steroids produced by fetal adrenal glands. Therefore, cholesterol must be assimilated from the fetal circulation and mainly from LDL (Carr, 1980, 1981 b, 1982; Simpson, 1979).

1	Most fetal plasma cholesterol arises by de novo synthesis in the fetal liver (Carr, 1984). The low LDL cholesterol level in fetal plasma is not the consequence of impaired fetal LDL synthesis, but instead results from the rapid use ofLDL by the fetal adrenal glands for steroidogenesis (Parker, 1980, 1983). Several fetal disorders alter the availability of substrate for placental steroid synthesis and thus highlight the interdependence of fetal development and placental function. Fetal demise is followed by a striking reduction in urinary estrogen levels. Similarly, after ligation of the umbilical cord with the fetus and placenta left in situ, placental estrogen production declines markedly (Cassmer, 1959). However, as previously discussed, placental progesterone production is maintained. In sum, an important source of precursors of pla cental estrogen-but not progesterone-biosynthesis is elimi nated with fetal death. Anencephalic etuses have markedly atrophic adrenal glands.

1	Anencephalic etuses have markedly atrophic adrenal glands. his stems from absent hypothalamic-pituitary function, which precludes adrenal stimulation by ACTH. With absence of the adrenal cortex fetal zone, the placental formation of estrogen-especially estriol-is severely limited because of diminished availability of C19 steroid precursors. Indeed, uri nary estrogen levels in women pregnant with an anencephalic fetus are only about 10 percent of those found in normal preg nancy (Frandsen, 1961). With an anencephalic fetus, almost all estrogens produced arise from placental use of maternal plasma DHEA-S. Fetal adrenal cortical hypoplasia occurs in perhaps 1 in 12,500 births (McCabe, 2001). Estrogen production in these pregnan cies is limited, which suggests the absence of C19 precursors.

1	Fetal adrenal cortical hypoplasia occurs in perhaps 1 in 12,500 births (McCabe, 2001). Estrogen production in these pregnan cies is limited, which suggests the absence of C19 precursors. Fetal-placental suatase diciency is associated with very low estrogen levels in otherwise normal pregnancies (France, 1969). Namely, sulfatase deficiency precludes the hydrolysis of C19 steroid sulfates, the first enzymatic step in the placental use of these circulating prehormones for estrogen biosynthesis. This deficiency is an X-linked disorder, and thus all afected fetuses are male. Its estimated frequency is 1 in 2000 to 5000 births and is associated with delayed labor onset. It also is associated with the development of ichthyosis in afected males later in life (Bradshaw, 1986).

1	Fetal-placental aromatase diciency is a rare autosomal recessive disorder in which individuals cannot synthesize endogenous estrogens (Grumbach, 2011; Simpson, 2000). To recall, fetal adrenal DHA-S is converted in the placenta to androstenedione, but in cases of placental aromatase deiciency, androstenedione cannot be converted to estradiol. Rather, androgen metabolites of DHEA produced in the placenta, including androstenedione and some testosterone, are secreted into the maternal or fetal circulation, or both. This can cause virilization of the mother and the female fetus (Belgorosky, 2009; Harada, 1992; Shozu, 1991). Trisomy 21-Down syndrome screening searches for abnormal levels ofhCG, alpha-fetoprotein, and other analytes (Chap. 14, p. 281). It was discovered that serum unconjugated estriol levels low in women with Down syndrome fetuses (Benn, 2002). The likely reason for this is inadequate formation of C19 steroids in the adrenal glands of these trisomic fetuses.

1	Fetal eythroblastosis in some cases of severe fetal D-antigen alloimmunization can lead to elevated maternal plasma estrogen levels. A suspected cause is the greater placental mass from hypertrophy, which can be seen with such fetal hemolytic anemia (Chap. 15, p. 300). Glucocorticoid treatment can cause a striking reduction in placental estrogen formation. Glucocorticoids inhibit ACTH secretion from the maternal and fetal pituitary glands. This diminishes maternal and fetal adrenal secretion of the placental estrogen precursor DHEA-S. With Addison disease, pregnant women show lower estrogen levels, principally estrone and estradiol levels (Baulieu, 1956). The fetal adrenal contribution to estriol synthesis, particularly in later pregnancy, is quantitatively much more important.

1	Maternal androgen-producing tumors can present the placenta with elevated androgen levels. Fortunately, placenta is extraordinary eicient in the aromatization of C19 steroids. For example, Edman and associates (1981) found that virtually all androstenedione entering the intervillous space is taken up by syncytiotrophoblast and converted to estradiol. None of this C19 steroid enters the fetus. Second, a female fetus is rarely virilized if there is a maternal androgen-secreting tumor. The placenta eiciently converts aromatizable C19 steroids, including testosterone, to estrogens, thus precluding transplacental passage. Indeed, virilized female fetuses of women with an androgenproducing tumor may be cases in which a nonaromatizable C19 steroid androgen is produced by the tumor-for example, 5a-dihydrotestosterone. Another explanation is that testosterone is produced very early in pregnancy in amounts that exceed the placental aromatase capacity at that time.

1	Complete hydatidiorm mole and gestational trophoblastic neoplasias lack a fetus and also a fetal adrenal source of C19 steroid precursors for trophoblast estrogen biosynthesis. Consequently, placental estrogen formation is limited to the use of C 19 steroids from the maternal plasma, and therefore estradiol is principally produced (MacDonald, 1964, 1966). Abel MH: Prostanoids and menstruation. In Baird DT, Michie EA (eds): Mechanisms of Menstrual Bleeding. New York, Raven, 2002

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1	Winger EE, Reed JL: The multiple faces of the decidual natural killer cell. Am J Reprod Immunol 70:1, 2013 Wislocki GB, Dempsey EW: Electron microscopy of the human placenta. Anat Rec 123:133, 1955 Wolfahrt S, Kleine B, Rossmanith WG: Detection of gonadotrophin releasing hormone and its receptor mRNA in human placental trophoblasts using in situ reverse transcription-polymerase chain reaction. Mol Hum Reprod 4:999, 1998 Wu Z, Horgan CE, Carr 0, et al: Biglycan and decorin diferentially regulate signaling in the fetal membranes. Matrix Bioi 34:266, 2014 Xie L, Sadovsky Y: he function of miR-519d in cell migration, invasion, and proliferation suggests a role in early placentation. Placenta 48:34, 2016 Yan C, Wang P, DeMayo J, et al: Synergistic roles of bone morphogenetic Protein 15 and growth diferentiation Factor 9 in ovarian function. Mol Endocrinol 15:854, 2001

1	Yan C, Wang P, DeMayo J, et al: Synergistic roles of bone morphogenetic Protein 15 and growth diferentiation Factor 9 in ovarian function. Mol Endocrinol 15:854, 2001 Yoshimura M, Pekary AE, Pang XP, et al: hyrotropic activity of basic isoelectric forms of human chorionic gonadotropin extracted from hydatidiform mole tissues. J Ciin Endocrinol Metab 78:862, 1994 Zhang M, Muralimanoharan S, Wortman AC, et al: Primate-speciic miR-515 family members inhibit key genes in human trophoblast differentiation and are up regulated in preeclampsia. Proc Natl Acad Sci USsA 113(45):E7069, 2016 NORMAL PLACENTA . . . . . . . . . . . . . . . . . . . . . . . . . . .. 111 SHAPE AND SIZE VARIANTS . . . . . . . . . . . . . . . . . . . . .. 112 EXTRACHORIAL PLACENTATION . . . . . . . . . . . . . . . . .. 113 CIRCULATORY DISTURBANCES . . . . . . . . . . . . . . . . . .. 113 PLACENTAL CALCIFICATION. . . . . . . . . . . . . . . . . . . . .. 115 PLACENTAL TUMORS ........................... 115

1	CIRCULATORY DISTURBANCES . . . . . . . . . . . . . . . . . .. 113 PLACENTAL CALCIFICATION. . . . . . . . . . . . . . . . . . . . .. 115 PLACENTAL TUMORS ........................... 115 AMNIOCHORION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 116 UMBILICAL CORD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 117 The placenta, as a rule, presents more or less rounded outlines, but now and again when inserted in the neighbourhood of the internal os it may take on a horseshoe-like appearance, its two branches running partialy around the oriice. -J. Whitridge Williams (1903) he placenta is a fantastic organ in its own right. As discussed in Chapter 5 (p. 88), it provides the indispensable interface between mother and fetus. Indeed, placental anatomy, ogy, and molecular structure remain some of the most intriguing and understudied topics in obstetrics.

1	Although a placental examination by the obstetrician is recommended, by consensus, routine pathological examination is not mandatory. Indeed, speciic conditions that merit submission for detailed inspection are still debated. By way of example, the College of American Pathologists has recommended placental examination for an extensive list of indications (Langston, 1997). Data, however, are insuicient to support all of these. At minimum, the placenta and cord should be inspected in the delivery room. he decision to request pathological examination should be based on clinical and placental findings (Redline, 2008; Roberts, 2008). Listed in Table 6-1 are some of the indications at Parkland Hospital for placental anatomical and histopathological examination.

1	At term, the typical placenta weighs 470 g, is round to oval with a 22-cm diameter, and has a central thickness of 2.5 cm (Benirschke, 2012). It is composed of a placental disc, extraplacental membranes, and three-vessel umbilical cord. The disc surface that lies against the uterine wall is the basal plate, which is divided by clefts into portions-termed cotyledons. he fetal surface is the chorionic plate, into which the umbilical cord inserts, typically in the center. Large fetal vessels that originate from the cord vessels then spread and branch across the chorionic plate before entering stem villi of the placenta parenchyma. In tracing these, fetal arteries almost invariably cross over veins. he chorionic plate and its vessels are covered by thin amnion, which can be easily peeled away from a postdelivery specimen.

1	As recommended by the American Institute of Ultrasound in Medicine (2013), placental location and relationship to the internal cervical os are recorded during prenatal sonographic examinations. As visualized ultrasonically, the normal placenta is homogenous and 2 to 4 cm thick, lies against the myometrium, and indents into the amnionic sac. The retroplacental space is a hypoechoic area that separates the myometrium from the basal plate and measures less than 1 to 2 cm. The umbilical cord is also imaged, its fetal and placental insertion sites examined, and its vessels counted. Many placental lesions can be identiied grossly or sonographically, but other abnormalities require histopathological examination for clariication. A detailed description of these Antepartum infection with fetal risks Anti-CDE alloimmunization Cesarean hysterectomy Oligohydramnios or hydramnios Peripartum fever or infection Preterm delivery Postterm delivery

1	Antepartum infection with fetal risks Anti-CDE alloimmunization Cesarean hysterectomy Oligohydramnios or hydramnios Peripartum fever or infection Preterm delivery Postterm delivery Systemic disorders with known effects Thick or viscid meconium Unexplained late pregnancy bleeding Admission to an acute care nursery Birth weight � 10th or �95th percentile Feta! anemia Vanishing twin beyond the first trimester alndications are organized alphabetically. is beyond the scope of this chapter, and interested readers are referred to textbooks by Benirschke (2012), Fox (2007), and Faye-Petersen (2006) and their colleagues. Moreover, the placenta accrete syndrome and gestational trophoblastic disease are presented in detail in Chapters 20 and 41, respectively.

1	Of variants, placentas may infrequently form as separate, nearly equally sized discs. his bilobate placenta may also be called bipartite placenta or placenta duplex. In these, the cord inserts between the two placental lobes-either into a connecting chorionic bridge or into intervening membranes. A placenta containing three or more equivalently sized lobes is rare and termed multilobate. Unlike this equal distribution, one or more TABLE 6-1. Some Indications for Placental Pathological Examinationa

1	TABLE 6-1. Some Indications for Placental Pathological Examinationa FIGURE 6-1 Succenturiate lobe. A. Vessels extend from the main placental disc to supply the small round succenturiate lobe located beneath it. (Used with permission from Dr. Jaya George.) B. Sonographic imaging with color Doppler shows the main placental disc implanted posteriorly (asterisk). The succenturiate lobe is located on the anterior uterine wall across the amnionic cavity. Vessels are identified as the long red and blue crossing tubular structures that travel within the membranes to connect these two portions of placenta.

1	disparately smaller accessory lobes-succenturiate lobes-may develop in the membranes at a distance from the main placenta (Fig. 6-1). hese lobes have vessels that course through the membranes. Of clinical importance, if these vessels overlie the cervix to create a vasa previa, dangerous fetal hemorrhage can follow vessel laceration (p. 118). n accessory lobe can also be retained in the uterus after delivery to cause postpartum uterine atony and hemorrhage or later endometritis.

1	Rarely, the placental surface area varies from the norm. With placenta membranacea, villi cover all or nearly all the uterine cavity. This may occasionally give rise to serious hemorrhage because of associated placenta previa or accreta (Greenberg, 1991; Pereira, 2013). A ring-shaped placenta may be a variant of placenta membranacea. his placenta is annular, and a partial or complete ring of placental tissue is present. hese abnormalities appear to be associated with a greater likelihood of tion (Faye-Petersen, 2006; Steemers, 1995). With placenta fenestrata, the central portion of a placental disc is missing. In some instances, there is an actual hole in the placenta, but more often, the defect involves only villous tissue, and the chorionic plate remains intact. Clinically, it may erroneously prompt a search for a retained placental cotyledon.

1	During pregnancy, the normal placenta increases its thickness at a rate of approximately 1 mm per week. lthough not measured as a component of routine sonographic evaluation, this thickness typically does not exceed 40 mm (Hoddick, 1985). Placentomegay deines those thicker than 40 mm and commonly results from striking villous enlargement. his may be secondary to maternal diabetes or severe maternal anemia, or to fetal hydrops, anemia, or infection caused by syphilis, toxoplasmosis, parvovirus, or cytomegalovirus. In these conditions, the placenta is homogeneously thickened. Less commonly with placentomegaly, fetal parts are present, but villi are edematous and appear as small placental cysts, such as in cases of partial mole (Chap. 20, p. 391). Cystic vesicles are also seen with placental mesenchymal dysplasia. Vesicles in this rare condition correspond to enlarged stem villi, but unlike molar pregnancy, trophoblast proliferation is not excessive (Woo, 2011).

1	Rather than villous enlargement, placentomegaly often may result from collections of blood or ibrin, which impart heterogeneity to the placenta. Examples of this are discussed on page 114 and include massive perivillous fibrin deposition, intervillous or subchorionic thromboses, and large retroplacental hematomas.

1	he chorionic plate normally extends to the periphery of the placenta and has a diameter similar to that of the basal plate. With extrachorial placentation, however, the chorionic plate fails to extend to this periphery and leads to a chorionic plate that is smaller than the basal plate (Fig. 6-2). Circummarginate and circumvallate placentas are the two types. In a circummarginate placenta, ibrin and old hemorrhage lie between the placenta and the overlying sheer amniochorion. In contrast, with a circumvallate placenta, the chorion periphery is a thickened, opaque, gray-white circular ridge composed of a double fold of chorion and amnion. Sonographically, the double fold can be seen as a thick, linear band of echoes extending from one placental edge to the other. On cross section, however, it appears as two "shelves," with each lying above an opposing placental margin (see Fig. 6-2). his anatomy can help diferentiate this shelf from amnionic bands and amnionic sheets, which are

1	it appears as two "shelves," with each lying above an opposing placental margin (see Fig. 6-2). his anatomy can help diferentiate this shelf from amnionic bands and amnionic sheets, which are described on page 116.

1	In relatively small observational studies of circumvallate placenta diagnosed postpartum, it was associated with increased risk for antepartum bleeding, abruption, fetal demise, and preterm birth (Lademacher, 1981; Suzuki, 2008; Taniguchi, 2014). In a prospective sonographic investigation of 17 cases, however, Shen and associates (2007a) found most circumvallate placentas to be transient. Persistent cases were benign. In general, most otherwise uncomplicated pregnancies with either type of extrachorial placentation have normal outcomes, and no increased surveillance is usually required.

1	Functionally, placental perfusion disorders can be grouped into: (1) those in which maternal blood low to or within the intervillous space is disrupted, and (2) those with disturbed fetal blood flow through the villi. These lesions are frequently identiied in the normal, mature placenta. Although they can limit maximal placental blood flow, functional reserve within the placental prevents harm in most cases. Indeed, some estimate that up to 30 percent of placental villi can be lost without untoward fetal efects (Fox, 2007). If extensive, however, these lesions can profoundly limit fetal growth. Lesions that disrupt perfusion are frequently seen grossly or sonographically, whereas smaller lesions are seen only histologically. With sonography, many of these, such as subchorionic fibrin deposition, perivillous ibrin deposition, and intervillous thrombosis, appear as focal sonolucencies within the placenta. Importantly, in the absence of maternal or fetal

1	FIGURE 6-2 A. In this illustration, circummarginate (left) and circumvallate (right) varieties of extrachorial placentation are shown. Double fold of amnion & chorion A circummarginate placenta is covered by a single layer of amniochorion. B. This transabdominal gray-scale sonographic image shows a circumvallate placenta. The double fold of amnion and chorion creates a broad, opaque white ring and ridge on the fetal surface. complications, isolated placental sonolucencies are considered incidental indings. These collections are caused by slowing of maternal blood low within the intervillous space. In the portion of this space near the chorionic plate, blood stasis is prominent and leads to subsequent ibrin deposition. In viewing the placental fetal surface, subchorionic lesions are commonly seen as white or yellow, firm, round, elevated plaques just beneath the chorionic plate.

1	Stasis of maternal blood flow around an individual villus also results in fibrin deposition and can lead to diminished villous oxygenation and necrosis of syncytiotrophoblast (Fig. 6-3). These small yellow-white placental nodules are grossly visible within the parenchyma of a sectioned placenta. Within limits, these relect normal placental aging.

1	Maternal Floor Infarction. his extreme variant of perivillous fibrin deposition is a dense ibrinoid layer within the placental basal plate and is erroneously termed an infarction. Maternal loor inarction has a thick, yellow or white, irm corrugated surface that impedes normal maternal blood low into the intervillous space. In specific cases that extend beyond the basal plate to entrap villi and obliterate the intervillous space, the term massive perivillous ibrin deposition is used. The etiopathogenesis is unclear, but maternal auto-or alloimmunity appears contributory (Faye-Peterson, 2017; Romero, 2013). Antiphospholipid antibody syndrome and angiogenic factors involved with preeclampsia have also been implicated (Sebire, 2002, 2003; Whitten, 2013).

1	These lesions are not reliably imaged with prenatal sonography, but they may create a thicker basal plate. Afected pregnancies are associated with miscarriage, fetal-growth restriction, preterm delivery, and stillbirth (Andres, 1990; Mandsager, 1994). Importantly, these adverse outcomes can recur in subsequent pregnancies. This is a collection of coagulated maternal blood normally found in the intervillous space mixed with fetal blood from a break in a villus. Grossly, these round or oval collections vary in size up to several centimeters. hey appear red if recent or white-yellow if older, and they develop at any placental depth. Intervillous thrombi are common and typically not associated with adverse fetal sequelae. Because there is potential for a communication between maternal and fetal circulations, large lesions can cause elevated maternal serum alpha-fetoprotein levels (Salaia, 1988).

1	Chorionic villi themselves receive oxygen solely from maternal circulation supplied to the intervillous space. Any uteroplacental disease that diminishes or obstructs this supply can result in infarction of an individual villus. These are common lesions in mature placentas and are benign in limited numbers. If numerous, however, placental insuiciency can develop. When they are thick, centrally located, and randomly distributed, they may be associated with preeclampsia or lupus anticoagulant. As depicted in Figure 6-3, the maternal-placental-fetal unit can develop several hematoma types. These include: (1) retroplacental hematoma-between the placenta and its adjacent decidua; (2) marginal hematoma-between the chorion and

1	FIGURE 6-3 Potential sites of maternally and fetally related placental circulatory disturbances. (Adapted from Faye-Petersen, 2006.) decidua at the placental periphery-known clinically as sub chorionic hemorrhage; (3) subamnionic hematoma-these are of fetal vessel origin and found beneath the amnion but above the chorionic plate, and (4) subchorial thrombus along the roof of the intervillous space and beneath the chorionic plate. With this last type, massive subchorionic hematomas are also known as a Breus mole.

1	Sonographically, hematomas evolve with time and appear hyperechoic to isoechoic in the irst week after hemorrhage, hypoechoic at 1 to 2 weeks, and inally, anechoic after 2 weeks. Most subchorionic hematomas visible sonographically are fairly small and of no clinical consequence. However, extensive retroplacental, marginal, and subchorial collections have been associated with higher rates of miscarriage, stillbirth, placental abruption, and preterm delivery (Ball, 1996; Fung, 2010; vladu, 2006; Tuuli, 2011). In essence, placental abruption is a large, clinically signiicant retroplacental hematoma.

1	Placental lesions that arise from fetal circulatory disturbances are also depicted in Figure 6-3. Deoxygenated fetal blood lows from the two umbilical arteries into arteries within the chorionic plate that divide and send branches out across the placental surface. hese eventually supply individual stem villi, and their thrombosis will obstruct fetal blood low. Distal to the obstruction, afected portions of the villus become nonfunctional. Thrombi in limited numbers are normally found in mature placentas. If many villi are afected, which can be seen with preeclampsia, the fetus may sufer growth restriction, stillbirth, or nonreassuring fetal heart rate patterns (Chisholm, 2015; Lepais, 2014; Saleemuddin, 2010).

1	here is a spectrum of villous capillary lesions. Chorangiosis describes an increased number of capillaries within terminal villi. Its deinition requires :::10 capillaries to be present in :::10 villi in :::10 ields viewed through a lOx microscope lens (Altshuler, 1984). Clinically, long-standing hypoperfusion or hypoxia is thought to be causative (Stanek, 2016). It is often associated with maternal diabetes mellitus (Ogino, 2000). Chorangiomatosis describes increased capillary number in stem villi, but terminal villi are spared. his inding has been linked with fetal-growth restriction and anomalies (Bagby, 2011). Despite these associations, the clinical signiicance of both vascular conditions remains unclear. Chorioangiomas are described subsequently. As indicated earlier, these hematomas lie between the chorionic plate and amnion. They most often are acute events during third-stage labor when cord traction ruptures a vessel near the cord insertion.

1	Large chronic antepartum lesions may cause fetomaternal hemorrhage or fetal-growth restriction (Deans, 1998). They also may be confused with other placental masses such as chorio angioma. In most cases, Doppler interrogation will show absent internal blood low within a hematoma and permit differentiation (Sepulveda, 2000). Calcium salts can be deposited throughout the placenta but are most common on the basal plate. Calciication accrues with advancing gestation, and greater degrees are associated (Bedir Findik, 2015; Klesges, 1998; McKenna, 2005). These hyperechoic deposits can easily be seen sonographically, and a grading scale from 0 to 3 relects increasing calciication with increasing numerical grade (Grannum, 1979). Following this scheme, a grade 0 placenta is homogeneous, lacks calciication, and displays a smooth, lat chorionic plate. A grade 1 placenta lations. Grade 2 shows echogenic stippling at the basal plate.

1	Large, echogenic comma shapes originate from an indented chorionic plate, bur their curve falls short of the basal plate. Last, a grade 3 placenta has echogenic indentations extending from the chorionic plate to the basal plate, which create discrete components that resemble cotyledons. Basal plate densities also increase. As a predictor, this grading scale is not useful for neona tal outcome near term (Hill, 1983; McKenna, 2005; Montan, 1986). However, data from two small studies link grade 3 pla centa prior to 32 weeks with stillbirth and some other adverse pregnancy outcomes (Chen, 2011, 2015).

1	These benign tumors have components similar to blood vessels and stroma of the chorionic villus. lso called chorangiomas, these placental tumors occur with an incidence of approximately 1 percent (Guschmann, 2003). In some cases, fetal-to-maternal hemorrhage across tumor capillaries leads to elevated levels of maternal serum alpha-fetoprotein (MSAFP), prompting sonographic evaluation. heir characteristic sonographic appearance shows a well-circumscribed, rounded, predominantly hypoechoic lesion lying near the chorionic plate and protruding into the amnionic cavity (Fig. 6-4). Documenting increased blood low by color Doppler helps to distinguish these lesions from other placental masses such as hematoma, partial hydatidiform mole, teratoma, metastases, and leiomyoma (Prapas, 2000). Although rare, chorangiocarcinoma tumors clinically mirror chorangiomas (Huang, 2015).

1	Small chorioangiomas are usually asymptomatic. Large tumors, typically those measuring >4 em, can create signiicant arteriovenous shunting within the placenta to cause high-output heart failure, hydrops, and fetal death (l Wattar, 2014). Compression of fetal erythrocytes within tumor vessels can lead to hemolysis and microangiopathic anemia (Bauer, 1978). Hydramnios, pre term delivery, and fetal-growth restriction are other sequelae. Rare cases include tumor vessel rupture, hemorrhage, and fetal death (Batukan, 2001). At the other extreme, rare rumor infarction can lead to symptom reversal (Zalel, 2002). FIGURE 6�4 Placental chorioangioma. A. Color Doppler imaging displays blood flow through a large chorioangioma with its border outlined by white arrows. B. Grossly, the chorioangioma is a round, well-circumcised mass protruding from the fetal surface.

1	Gray-scale and color Doppler interrogation of the placenta and amnionic fluid volume are used to identiy these tumors. Diagnostic tools that can airm associated fetomaternal hemorrhage include MSAFP level and Kleihauer-Betke stain. With fetal concern, echocardiography assesses cardiac function, whereas middle cerebral artery interrogation is used to identiy fetal anemia. Several fetal therapies interfere with the vascular supply to the tumor and reverse fetal heart failure. At specialized perinatal centers, endoscopic laser ablation of feeder vessels to the tumor is most frequently used and is associated with favorable fetal outcomes (Hosseinzadeh, 2015). Of other therapy, fetal transfusion can treat serious anemia, amnioreduction can temporize hydramnios, and digoxin therapy can assist fetal heart failure.

1	Maternal malignant tumors rarely metastasize to the placenta. Of those that do, melanomas, leukemias and lymphomas, and breast cancer are the most common (Al-Adnani, 2007). Tumor cells usually are confined within the intervillous space. As a result, metastasis to the fetus is uncommon but is most often seen with melanoma (lexander, 2003). Similarly, cases in which fetal malignancy metastasizes to the placenta are rare (Reif, 2014). hese are predominantly fetal neuroectodermal tumors, and only one case in the literature describes transplantation of tumor to the maternal uterus (Nath, 1995).

1	Normal genital-tract flora can colonize and infect the membranes, umbilical cord, and eventually the fetus. Bacteria most commonly ascend after prolonged membrane rupture and during labor to cause infection. Organisms initially infect the chorion and adjacent decidua in the area overlying the internal os. Subsequently, progression leads to full-thickness involvement of the membranes-chorioamnionitis. Organisms often then spread along the chorioamnionic surface to colonize and replicate in amnionic luid. Inlammation of the chorionic plate and of the umbilical cord-unisitis-may follow (Kim, 2015; Redline, 2012).

1	Most commonly, there is microscopic or occult chorioamnionitis, which is caused by a wide variety of microorganisms. his is frequently cited as a possible explanation for many otherwise unexplained cases of ruptured membranes, preterm labor, or both as discussed in Chapter 42 (p. 810). In some cases, gross infection is characterized by membrane clouding and is sometimes accompanied by a foul odor that depends on bacterial species. Amnion nodosum is a condition characterized by numerous small, light-tan nodules on the amnion overlying the chorionic plate. hese may be scraped of the fetal surface and contain deposits of fetal squames and fibrin that relect prolonged and severe oligohydramnios (Adeniran, 2007).

1	Two notable bandlike structures can be formed by the fetal membranes. Of these, amnionic band sequence is an anatomical disruption sequence in which amnion bands tether, constrict, or amputate fetal parts. Amnionic bands commonly cause limb-reduction defects, facial clefts, or encephalocele (Barzilay, 2015; Guzman-Huerta, 2013). Umbilical cord compromise is another sequela (Barros, 2014; Heifetz, 1984b). Severe defects of the spine or ventral wall that accompany amnionic bands suggest a limb-body wall complex, described in Chapter 10 (p. 206). Clinically, sonography often irst identiies the sequelae of this sequence rather than the bands themselves. As with any fetal anomaly, targeted sonography is indicated. Identiication of a limb-reduction defect, an encephalocele in an atypical location, or an extremity with edema or positional deformity should prompt careful evaluation for amnionic bands. Management depends on the degree of anatomic deformity.

1	Management depends on the degree of anatomic deformity. Fetoscopic laser interruption of the band may be suitable in highly selected antepartum cases Qavadian, 2013; Mathis, 2015). In contrast, an amnionic sheet is formed by normal amniochorion draped over a preexisting uterine synechia. Generally, these sheets pose little fetal risk, although slightly higher rates of preterm membrane rupture and placental abruption have been described (Korbin, 1998; Nelson, 2010; Tuuli, 2012). Most umbilical cords at delivery are 40 to 70 cm long, and very few measure <30 cm or > 100 cm. Cord length is inluenced positively by both amnionic luid volume and fetal mobility (Miller, 1982). In retrospective studies, short cords have been linked with congenital malformations and intrapartum distress (Baergen, 2001; Krakowiak, 2004; Yamamoto, 2016). Excessively long cords are linked with cord entanglement or prolapse and with fetal anomalies (Olaya-C, 2015; Rayburn, 1981).

1	Because antenatal determination of cord length is technically limited, cord diameter has been evaluated as a predictive marker for fetal outcomes. Some have linked lean cords with poor fetal growth and large-diameter cords with macrosomia (Proctor, 2013). However, the clinical utility of this parameter is still unclear (Barbieri, 2008; Cromi, 2007; Raio, 1999b, 2003).

1	Cord coiling characteristics have been reported but are not currently part of standard sonographic evaluation. Usually the umbilical vessels spiral through the cord in a sinistral, that is, let-twisting direction (Fletcher, 1993; Lacro, 1987). he number of complete coils per centimeter of cord length is termed the umbilical coiling index-Uel (Strong, 1994). A normal, antepartum, sonographically derived UCI is 0.4, and this contrasts with a normal, postpartum, physically measured value of 0.2 (Sebire, 2007). UCls < 10th percentile are considered hypocoile, and those > 90th percentile are hypercoiled. Clinically, the signiicance of coiling extremes is controversial. Some studies evaluating large, unselected cohorts ind no associations between UCI values and poor neonatal outcome Qessop, 2014; Pathak, 2010). In others, extremes are linked with various adverse outcomes but most consistently with intrapartum fetal heart rate abnormalities, preterm labor, or fetal-growth restriction

1	2014; Pathak, 2010). In others, extremes are linked with various adverse outcomes but most consistently with intrapartum fetal heart rate abnormalities, preterm labor, or fetal-growth restriction (Chitra, 2012; de Laat, 2006; Predanic, 2005; Rana, 1995).

1	Counting cord vessel number is a standard component of anatomical evaluation during fetal sonographic examination and immediately after delivery (Fig. 6-5). Embryos initially have two sonographically in the second trimester. They encircle the fetal bladder (asterisk) as extensions of the superior vesical arteries. In this color Doppler sonographic image, a single umbilical artery, shown in red, runs along the bladder wall before joining the umbilical vein (blue) in the cord. Below this, the two vessels of the cord, seen as a larger red and smaller blue circle, are also seen floating in a cross section of a cord segment. umbilical veins. In the irst trimester, the right vein typically atrophies to leave one large vein to accompany the two, thick-walled umbilical arteries. Four-vessel cords are rare and often associated with congenital anomalies (Puvabanditsin, 2011). If it is an isolated inding, however, prognosis can be good (Avnet, 2011).

1	The most common aberration is that of a single umbilical artery (SUA), with a cited incidence of 0.63 percent in liveborn neonates, 1.92 percent with perinatal deaths, and 3 percent in twins (Heifetz, 1984a). Fetuses with major malformations frequently have a single artery. Thus, its identiication often prompts consideration for targeted sonography and possibly fetal echocardiography. The most frequent anomalies are cardiovascular and genitourinary (Hua, 2010; Murphy-Kaulbeck, 2010). In an anomalous fetus, a single artery greatly increases the aneuploidy risk, and amniocentesis is recommended (Dagklis, 2010; Lubusky, 2007).

1	If target sonography inds otherwise normal anatomy, an isolated single artery in an otherwise low-risk pregnancy does not significantly increase the fetal aneuploidy risk. However, as in isolated inding, it has been associated with fetal-growth restriction and perinatal death in some but not all studies (ChettyJohn, 2010; Gutvirtz, 2016; Hua, 2010; Murphy-Kaulbeck, 2010; Voskamp, 2013). hus, while clinical monitoring of growth is reasonable, the value of sonographic surveillance is unclear. A rare anomaly is that of a fused umbilical artery with a shared lumen. It arises from failure of the two arteries to split during embryological development. The common lumen may extend through the entire cord, but, if partial, it is typically found near the placental insertion site (Yamada, 2005). In one report, these were associated with a higher incidence of marginal or velamentous cord insertion, but not congenital fetal anomalies (Fujikura, 2003).

1	Found in most placentas, the Hyrtl anastomosis is a connection between the two umbilical arteries and lies near the cord insertion into the placenta. This anastomosis acts as a pressureequalizing system between the arteries (Gordon, 2007). As a result, redistribution of pressure gradients and blood flow improves placental perfusion, especially during uterine contractions or during compression of one umbilical artery. Fetuses with a single umbilical artery lack this safety valve (Raio, 1999a, 2001). Several structures are housed in the umbilical cord during fetal development, and their remnants may be seen when the mature cord is viewed transversely. Indeed, J auniaux and colleagues (1989) sectioned 1000 cords, and in one fourth of the specimens, they found remnants of vitelline duct, allantoic duct, and embryonic vessels. These were not associated with congenital malformations or perinatal complications. Cysts occasionally are found along the course of the cord.

1	Cysts occasionally are found along the course of the cord. They are designated according to their origin. True cysts are epithelium-lined remnants of the allantoic or vitelline ducts and tend to be located closer to the fetal insertion site. In contrast, the more common pseudocysts form from local degeneration of Wharton jelly and occur anywhere along the cord. Both have a similar sonographic appearance. Single umbilical cord cysts identified in the irst trimester tend to resolve completely, however, multiple cysts may portend miscarriage or aneuploidy (Ghezzi, 2003; Hannaford, 2013). Cysts persisting beyond this time are associated wi th a risk for structural defects and chromosomal anomalies (Bonilla, 2010; Zangen, 2010).

1	The cord normally inserts centrally into the placental disc, but eccentric, marginal, or velamentous insertions are variants. Of these, eccentric insertions in general pose no identifiable fetal risk. Marginal insertion is a common variant-sometimes referred to as a battledore placenta-in which the cord anchors at the placental margin. In one population-based study, the rate was 6 percent in singleton gestations and 11 percent in twins (Ebbing, 20l3). This common insertion variant rarely causes problems, but it and velamentous insertion occasionally result in the cord being pulled of during delivery of the placenta (Ebbing, 2015; Luo, 20l3). In monochorionic twins, this insertion may be associated with weight discordance (Kent, 2011).

1	With velamentous insertion, the umbilical vessels characteristically travel within the membranes before reaching the placental margin (Fig. 6-6) The incidence of velamentous insertion approximates 1 percent but is 6 percent with twins (Ebbing, 2013). It is more commonly seen with placenta previa (Papinniemi, 2007; Raisanen, 2012). Antenatal diagnosis is possible sonographically, and with velamentous insertion, cord vessels are seen traveling along the uterine wall before entering the placental disc. Clinically, vessels are vulnerable to compression, which may lead to fetal hypoperfusion and acidemia. Higher associated rates of low Apgar scores, stillbirth, preterm delivery, and small for gestational age have been noted (Ebbing, 2017; Esakof, 2015; Heinonen, 1996; Vahanian, 2015). Accordingly, monitoring of fetal growth is reasonable either clinically or sonographically (Vintzileos, 2015).

1	Last, with the very uncommon furcate insertion, umbilical vessels lose their protective Wharton jelly shortly before they insert. As a result, they are covered only by an amnion sheath and prone to compression, twisting, and thrombosis. With this condition, vessels travel within the membranes and overlie the cervical os. here, they can be torn with cervical dilatation or membrane rupture, and laceration can lead to rapid fetal exsanguination. Over the cervix, vessels can also be compressed by a presenting fetal part. Fortunately, vasa previa is uncommon and has an incidence of 2 to 6 per 10,000 pregnancies

1	FIGURE 6-6 Velamentous cord insertion. A.The umbilical cord inserts into the membranes. From here, the cord vessels branch and are supported only by membrane until they reach the placental disc. B. When viewed sonographically and using color Doppler, the cord vessels appear to lie against the myometrium as they travel to insert marginally into the placental disc, which lies at the top of this image. (Ruiter, 2016; Sullivan, 2017). Vasa previa is classiied as type 1, in which vessels are part of a velamentous cord insertion, and type 2, in which involved vessels span between portions of a bilobate or a succenturiate placenta (Catanzarite, 2001). Two other risks are conception with in vitro fertilization and second-trimester placenta previa, with or without later migra tion (Baulies, 2007; Schachter, 2003).

1	Compared with intrapartum diagnosis, antepartum diagnosis greatly improves the perinatal survival rate, which ranges from 97 to 100 percent (Oyelese, 2004; Rebarber, 2014; Swank, 2016). Thus, vasa previa is ideally identiied early, although this is not always possible. Clinically, an examiner is occasionally able to palpate or directly see a tubular fetal vessel in the membranes overlying the presenting part. Efective screening for vasa previa begins during scheduled midtrimester sonographic examination. In suspicious cases, transvaginal sonography is added and shows cord vessels inserting into the membranes-rather than directly into the placenta-and vessels running above the cervical internal os (Fig. 6-7). Routine color Doppler interrogation of the placental cord insertion site, particularly in cases of placenta previa or low-lying placenta, may aid its detection. With this, the vessel waveform relects the fetal heart rate. In one systematic review, the median prenatal detection

1	in cases of placenta previa or low-lying placenta, may aid its detection. With this, the vessel waveform relects the fetal heart rate. In one systematic review, the median prenatal detection rate was 93 percent (Ruiter, 2015).

1	Once vasa previa is identiied, subsequent imaging is reasonable because 6 to 17 percent of cases ultimately resolve (Rebarber, 2015; Swank, 2016). Bed rest apparently has no added advantage. Antenatal corticosteroids can be provided as indicated or given prophylactically at 28 to 32 weeks' gestation to cover possible urgent preterm delivery. Antenatal hospitalization may be considered at 30 to 34 weeks to permit surveillance and expedited delivery for labor, bleeding, or rupture of membranes. Data supporting is limited, and admission may best serve women with risk factors that portend early delivery (Society for Maternal-Fetal Medicine, 2015). A few cases FIGURE 6-7 Vasa previa. Using color Doppler, an umbilical vessel (red circle) is seen overlying the internal os. At the bottom, the Doppler waveform seen with this vasa previa has the typical appearance of an umbilical artery, with a pulse rate of 141 beats per minute.

1	of antepartum fetoscopic surgery with vessel laser ablation are described (Hosseinzadeh, 2015; Johnston, 2014). However, current practice is early scheduled cesarean delivery. Robinson mend elective cesarean delivery at 34 to 35 weeks' gestation to balance the risks of perinatal exsanguination versus preterm birth morbidity. he Society for Maternal-Fetal Medicine (2015) considers planned cesarean delivery at 34 to 37 weeks' gestation reasonable. At delivery, the fetus is expeditiously delivered after the hys terotomy incision in case a vessel is lacerated during uterine entry. Delayed cord clamping is not encouraged.

1	At delivery, the fetus is expeditiously delivered after the hys terotomy incision in case a vessel is lacerated during uterine entry. Delayed cord clamping is not encouraged. In all pregnancies, otherwise unexplained vaginal bleeding of vasa previa and a lacerated fetal vessel. In many cases, bleed ing is rapidly fatal, and infant salvage is not possible. With less hemorrhage, however, it may be possible to distinguish fetal ver sus maternal bleeding. Various tests may be used, and each relies on the increased resistance of fetal hemoglobin to denaturing by alkaline or acid reagents (Odunsi, 1996; Oyelese, 1999). • Knots, Strictures, and Loops

1	• Knots, Strictures, and Loops Various mechanical abnormalities in the cord can impede blood low and sometimes cause fetal harm. Of these, true knots are found in approximately 1 percent of births. hese form from fetal movement, and associated risks include hydramnios and diabetes (Hershkovitz, 2001; Raisanen, 2013). Knots are especially common and dangerous in monoamnionic twins, which are discussed in Chapter 45 (p. 874). When true knots are associated with singleton fetuses, the stillbirth risk is increased four-to tenfold (Airas, 2002; S0rnes, 2000).

1	Knots can be found incidentally during antepartum sonography, and a "hanging noose" sign is suggestive (Ramon y Cajal, 2006). hree-dimensional and color Doppler aid diagnostic accuracy (Hasbun, 2007). With these knots, optimal fetal surveillance is unclear but may include umbilical artery Doppler velocimetry, nons tress testing, or subjective fetal movement monitoring (Rodriguez, 2012; Scioscia, 201l). Allowing vaginal delivery is suitable, but abnormal intrapartum fetal heart rate tracings are more often encountered. hat said, cesarean delivery rates are not increased, and cord blood acid-base values are usually normal (Airas, 2002; Maher, 1996). In contrast,alse knots form from focal redundancy and folding of an umbilical cord vessel. hese lack clinical signiicance.

1	In contrast,alse knots form from focal redundancy and folding of an umbilical cord vessel. hese lack clinical signiicance. Cord strictures are focal narrowings of the diameter that usually develop near the fetal cord insertion site (Peng, 2006). Characteristic pathological features include an absence of Wharton jelly and stenosis or obliteration of cord vessels at the narrow segment (Sun, 1995). In most instances, the fetus is stillborn (French, 2005). Even less common is a cord stricture caused by an amnionic band. Cord loops are frequently encountered and are caused by coiling around various fetal parts during movement. A cord around the neck-a nuchal cord-is common, and vaginal delivery is suitable. One loop is reported in 20 to 34 percent of deliveries; two loops in 2.5 to 5 percent; and three loops in 0.2 to 0.5 percent (Kan, 1957; S0rnes, 1995; Spellacy, 1966).

1	During labor, up to 20 percent of fetuses with a nuchal cord have moderate to severe variable heart rate decelerations, and these are associated with a lower umbilical artery pH (Hankins, 1987). Cords wrapped around the body can have similar efects (Kobayashi, 2015). Despite their frequency, nuchal cords are not associated with greater rates of adverse perinatal outcome (Henry, 2013; Sheiner, 2006). Last, a unic presentation describes when the umbilical cord is the presenting part in labor. These are uncommon and most often are associated with fetal malpresentation (Kinugasa, 2007). A funic presentation in some cases is identiied with placental sonography and color low Doppler (Ezra, 2003). Overt or occult cord prolapse can complicate labor. hus, once identiied at term, cesarean delivery is typically recommended.

1	Cord hematomas are rare and generally follow rupture of an umbilical vessel, usually the vein, and bleeding into the Wharton jelly. Hematomas have been associated with abnormal cord length, umbilical vessel aneurysm, trauma, entanglement, umbilical vessel venipuncture, and funisitis (Gualandri, 2008). Most are identiied postpartum, but hematomas are recognized so no graphically as hypoechoic masses that lack blood low (Chou, 2003). Sequelae include stillbirth or intrapartum abnormal fetal heart rate pattern (Abraham, 2015; Barbati, 2009; Sepulveda, 2005; Towers, 2009).

1	Umbilical cord vessel thromboses are rare in utero events and seldom diagnosed antepartum. Approximately 70 percent are venous, 20 percent are venous and arterial, and 10 percent are arterial thromboses (Heifetz, 1988). hese all have high associated rates of stillbirth, fetal-growth restriction, and intrapartum fetal distress (Minakami, 2001; Sato, 2006; Shilling, 2014). If these are identiied antepartum as hypoechoic masses without blood low, data from case reports support consideration of prompt delivery if of viable age (Kanenishi, 2013).

1	n umbilical vein varix can complicate either the intraamnionic or fetal intraabdominal portion of the umbilical vein. So no graphically and complemented by color Doppler, rare intraamnionic varices show cystic dilatation of the umbilical vein that is contiguous with a normal-caliber portion. Of complications, an intraamnionic varix may compress an adjacent umbilical artery or can rupture or thrombose. In cases without these, White and colleagues (1994) recommend fetal surveillance and delivery once fetal maturity is confirmed. However, data are limited and derived from case reports.

1	he rare umbilical artey aneuysm is caused by congenital thinning of the vessel wall with diminished support from Wharton jelly. Indeed, most form at or near the cord placental insertion site, where this support is absent. These are associated with single umbilical artery, trisomy 18, amnionic luid volume extremes, fetal-growth restriction, and stillbirth (Hill, 2010; Vyas, 2016). At least theoretically, these aneurysms could cause fetal compromise and death by compression of the umbilical vein. These aneurysms may appear sonographically as a cyst with a hyperechoic rim. Within the aneurysm, color low and spectral Doppler interrogation demonstrate either lowvelocity or turbulent nonpulsatile flow (Olog, 2011; Sepulveda, 2003; Shen, 2007b). Although not codified, management may include fetal karyotyping, antenatal fetal surveillance, and early delivery to prevent stillbirth (Doehrman, 2014).

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1	Nelson LD, Grobman WA: Obstetric morbidity associated with amniotic sheets. Ultrasound Obstet Gynecol 36(3):324, 2010 Odunsi K, Bullough CH, Henzel], et al: Evaluation of chemical tests for fetal bleeding from vasa previa. Int] Gynaecol Obstet 55(3):20 , 19% Ogino S, Redline RW: Villous capillary lesions of the placenta: distinctions between chorangioma, chorangiomatosis, and chorangiosis. Hum Pathol 31:945,s2000 Olaya-C M, Bernal ]E: Clinical associations to abnormal umbilical cord length in Latin American newbons.s] Neonatal Perinatal Med 8(3):251, 2015 Olog A, homas ]T, Petersen S, et al: Large umbilical artery aneurysm with a live healthy baby delivered at 31 weeks. Fetal Diagn Ther 29(4):331,2011 Oyelese KO, Turner M, Lees e, et al: Vasa previa: an avoidable obstetric tragedy. Obstet Gynecol Surv 54: 138, 1999 Oyelese Y, Catanzarite V, Prefumo F, et al: Vasa previa: the impact of prenatal diagnosis on outcomes. Obstet Gynecol 103:937, 2004

1	Oyelese Y, Catanzarite V, Prefumo F, et al: Vasa previa: the impact of prenatal diagnosis on outcomes. Obstet Gynecol 103:937, 2004 Papinniemi M, Keski-Nisula L, Heinonen S: Placental ratio and risk of velamentous umbilical cord insertion are increased in women with placenta previa. Ams] Perinatol 24:353, 2007 Pathak S, Hook E, Hackett G, et al: Cord coiling, umbilical cord insertion and placental shape in an unselected cohort delivering at term: relationship with common obstetric outcomes. Placenta 31 (11) :963, 2010 Peng HQ, Levitin-Smith M, Rochelson B, et al: Umbilical cord stricture and over coiling are common causes of fetal demise. Pediatr Dev Pathol 9: 14, 2006 Pereira N, Yao R, Guilfoil DS, et al: Placenta membranacea with placenta accreta: radiologic diagnosis and clinical implications. Prenat Diagn 33(13):1293,s2013

1	Pereira N, Yao R, Guilfoil DS, et al: Placenta membranacea with placenta accreta: radiologic diagnosis and clinical implications. Prenat Diagn 33(13):1293,s2013 Prapas N, Liang RI, Hunter D, et al: Color Doppler imaging of placental masses: diferential diagnosis and fetal outcome. Ultrasound Obstet Gynecol 16:559,s2000 Predanic M, Perni SC, Chasen ST, et al: Ultrasound evaluation of abnormal umbilical cord coiling in second trimester of gestation in association with adverse pregnancy outcome. Am] Obstet Gynecol 193:387,2005 Proctor LK, Fitzgerald B, Whittle L, et al: Umbilical cord diameter percentile curves and their correlation to birth weight and placental pathology. Placenta 34(1):62, 2013 Puvabanditsin S, Garrow E, Bhatt M, et al: Four-vessel umbilical cord associated with multiple congenital anomalies: a case report and literature review. Fetal Pediatr Pathol 30(2):98, 2011

1	Puvabanditsin S, Garrow E, Bhatt M, et al: Four-vessel umbilical cord associated with multiple congenital anomalies: a case report and literature review. Fetal Pediatr Pathol 30(2):98, 2011 Raio L, Ghezzi F, Di Naro E, et al: In-utero characterization of the blood flow in the Hyrtl anastomosis. Placenta 22:597,s2001 Raio L, Ghezzi F, Di Naro E, et al: Prenatal assessment of the Hyrtl anastomosis and evaluation of its function: case report. Hum Reprod 14: 1890, 1999a Raio L, Ghezzi F, Di Naro E, et al: Sonographic measurement of the umbilical cord and fetal anthropometric parameters. Eur ] Obstet Gynecol Reprod Bioi 83: 131, 1999b Raio L, Ghezzi F, Di Naro E, et al: Umbilical cord morphologic characteristics and umbilical artery Doppler parameters in intrauterine growth-restricted fetuses. ] Ultrasound Med 22: 1341, 2003

1	Raio L, Ghezzi F, Di Naro E, et al: Umbilical cord morphologic characteristics and umbilical artery Doppler parameters in intrauterine growth-restricted fetuses. ] Ultrasound Med 22: 1341, 2003 Raisanen S, Georgiadis L, Harju M, et al: Risk factors and adverse pregnancy outcomes among births afected by velamentous umbilical cord insertion: a retrospective population-based register study. Eurs] Obstet Gynecol Reprod BioI 165(2):231, 2012 Raisanen S, Georgiadis L, Harju M, et al: True umbilical cord knot and obstetric outcome. Int] Gynaecol Obstet 122(1):18, 2013 Ramon y Cajal CL, Martinez RO: Four-dimensional ultrasonography of a true knot of the umbilical cord. Am] Obstet Gynecol 195:896, 2006 Ranas], Ebert GA, Kappy A: Adverse perinatal outcome in patients with an abnormal umbilical coiling index. Obstet Gynecol 85(4):573, 1995 Rayburn WF, Beynen A, Brinkman DL: Umbilical cord length and intrapartum complications. Obstet Gynecol 57(4):450, 1981

1	Rebarber A, Dolin e, Fox NS, et al: Natural history of vasa previa across gestation using a screening protocol.] Ultrasound Med 33(1):141, 2014 Redline RW: Inlammatory response in acute chorioamnionitis. Semin Fetal Neonatal Med 17(1):20,s2012 Redline RW: Placental pathology: a systematic approach with clinical correlations. Placenta 29(Suppl A):S86, 2008 Reif P, Hofer N, Kolovetsiou-Kreiner V, et al: Metastasis of an undiferentiated fetal soft tissue sarcoma to the maternal compartment of the placenta: maternal aspects, pathology indings and review of the literature on fetal malignancies with placenta metastases. Histopathology 65(6):933,s2014 Roberts D]: Placental pathology, a survival guide. Arch Pathol Lab Med 132(4):641, 2008 Robinson BK, Grobman WA: Efectiveness of timing strategies for delivery of individuals with vasa previa. Obstet Gynecol 117(3):542,s201s1

1	Robinson BK, Grobman WA: Efectiveness of timing strategies for delivery of individuals with vasa previa. Obstet Gynecol 117(3):542,s201s1 Rodriguez N, Angarita M, Casasbuenas A, et al: Three-dimensional highdeinition low imaging in prenatal diagnosis of a true umbilical cord knot. Ultrasound Obstet Gynecol 39(2):245, 2012 Romero R, Whitten A, Korzeniewski S], et al: Maternal Boor infarction/massive perivillous ibrin deposition: a manifestation of maternal antifetal rejection? Am] Reprod Immunol 70(4):285,s2013 Ruiter L, Kok N, Limpens ], et al: Incidence of and risk indicators for vasa praevia: a systematic review. B]OG 123(8):1278,s2016 Ruiter L, Kok N, Limpens J, et al: Systematic review of accuracy of ultrasound in the diagnosis of vasa previa. Ultrasound Obstet Gynecol 45(5):516, 2015

1	Salaia CM, Silberman L, Herrera NE, et al: Placental pathology at term associated with elevated midtrimester maternal serum alpha-fetoprotein concent·ation. Am ] Obstet Gynecol 158(5): 1 064, 1988 Saleemuddin A, Tantbirojn P, Sirois K, et al: Obstetric and perinatal complications in placentas with fetal thrombotic vasculopathy. Pediatr Dev Pathol 13(6):459,s2010 Sato Y, Benirschke K: Umbilical arterial thrombosis with vascular wall necrosis: clinicopathologic indings of 11 cases. Placenta 27: 15, 2006 Schachter M, Tovbin Y, Arieli S, et al: In vitro fertilization is a risk factor for vasa previa. Fertil Steril 78(3):642, 2003

1	Scioscia M, Fornale M, Bruni F, et al: Four-dimensional and Doppler sonography in the diagnosis and surveillance of a true cord knot. ] Clin Ultrasound 39(3):157,s2011 Sebire N]: Pathophysiological signiicance of abnormal umbilical cord coiling index. Ultrasound Obstet Gynecol 30(6):804, 2007 Sebire N], Backos M, El Gaddal S, et al: Placental pathology, antiphospholipid antibodies, and pregnancy outcome in recurrent miscarriage patients. Obstet Gynecol 10 1 :258, 2003 Sebire N], Backos M, Goldin RD, et al: Placental massive perivillous ibrin deposition associated with antiphospholipid antibody syndrome. B]OG 109:570,s2002 Sepulveda W, Aviles G, Carstens E, et al: Prenatal diagnosis of solid placental masses: the value of color Bow imaging. Ultrasound Obstet GynecoIs16:554, 2000 Sepulveda W, Corral E, Kottmann C, et al: Umbilical artery aneUlysm: prenatal identiication in three fetuses with trisomy 18. Ultrasound Obstet Gynecol 21:213, 2003 Sepulveda W, Wong AE, Gonzalez R, et al:

1	W, Corral E, Kottmann C, et al: Umbilical artery aneUlysm: prenatal identiication in three fetuses with trisomy 18. Ultrasound Obstet Gynecol 21:213, 2003 Sepulveda W, Wong AE, Gonzalez R, et al: Fetal death due to umbilical cord hematoma: a rare complication of umbilical cord cyst. ] Matern-Fetal Neonatal Med 18(6):387,s2005 Sheiner E, Abramowicz ]S, Levy A, et al: Nuchal cord is not associated with adverse perinatal outcome. Arch Gynecol Obstet 274:81, 2006 Shen 0, Golomb E, Lavie 0, et al: Placental shelf-a common, typically transient and benign inding on early second-trimester sonography. Ultrasound Obstet Gynecol 29: 192, a Shen 0, Reinus e, Baranov A, et al: Prenatal diagnosis of umbilical artery aneurysm: a potentially lethal anomaly. ] Ultrasound Med 26(2):251, 2007b Shilling e, Walsh e, Downey P, et al: Umbilical artery thrombosis is a rare but clinically important inding: a series of cases with clinical outcomes. Pediatr Dev PathoIs17(2):89, 2014

1	Society for Maternal-Fetal (SMFM) Publications Committee, Sinkey RG, Odibo AO, et al: Diagnosis and management of vasa previa. Am J Obstet GynecoIs213(5):615,s2015 S0nes T: Umbilical cord encirclements and fetal growth restriction. Obstet Gynecol 86:725, 1995 S0nes T: Umbilical cord knots. Acta Obstet Gynecol Scand 79: , 2000 Spellacy erN, Gravem H, Fisch RO: The umbilical cord complications of true knots, nuchal coils and cords around the body. Report from the collaborative study of cerebral palsy. Am J Obstet Gynecol 94: 1136, 1966 Stanek J: Chorangiosis of chorionic villi: what does it really mean? Arch Pathol Lab Med 140(6):58,s2016 5teemers NY, De Rop C, Van Assche A: Zonary placenta. Int J Gynaecol Obstet 51(3):251,s1995 Strong TH Jr, Jarles DL, Vega JS, et al: The umbilical coiling index. Am J ObstetsGynecols1700 Pt 1):29, 1994 Sullivan EA, Javid N, Duncombe G, et al: Vasa previa diagnosis, clinical practice, and outcomes in Australia. Obstet Gynecol 130(3):591, 2017

1	Sullivan EA, Javid N, Duncombe G, et al: Vasa previa diagnosis, clinical practice, and outcomes in Australia. Obstet Gynecol 130(3):591, 2017 Sun Y, Arbuckle S, Hocking G, et al: Umbilical cord stricture and intrauterine fetal death. Pediatr Pathol Lab Med 15:723, 1995 Suzuki S: Clinical signiicance of pregnancies with circumvallate placenta. J Obstet Gynaecol Res 340):51, 2008 Swank ML, Garite TJ, Maurel K, et al: Vasa previa: diagnosis and management. Obstetrix Collaborative Research Network. Am J Obstet Gynecol 215(2):223.e1,s2016 Taniguchi H, Aoki S, Sakamaki K, et al: Circumvallate placenta: associated clinical manifestations and complications-a retrospective study. Obstet Gynecol Int 2014:986230,s2014 Towers CV, Juratsch CE, Garite TJ: The fetal heart monitor tracing in pregnancies complicated by a spontaneous umbilical cord haematoma. J Perinatol 29:517, 2009

1	Towers CV, Juratsch CE, Garite TJ: The fetal heart monitor tracing in pregnancies complicated by a spontaneous umbilical cord haematoma. J Perinatol 29:517, 2009 Tuuli MG, Norman SM, Odibo AO, et al: Perinatal outcomes in women with subchorionic hematoma: a systematic review and meta-analysis. Obstet GynecoIsI17(5):1205,2011 Tuuli MG, Shanks A, Bernhard L, et al: Uterine synechiae and pregnancy complications. Obstet GynecoIs119(4):810, 2012 Vahanian SA, Lavery JA, Ananth CV, et al: Placental implantation abnormalities and risk of preterm delivery: a systematic review and metaanalysis. Am J Obstet Gynecol213(4 Suppl):S78, 2015 Vintzileos AM, Ananth CV, Smulian JC: Using ultrasound in the clinical management of placental implantation abnormalities. Am J Obstet Gynecol 213(4 Suppl):S70, 2015

1	Vintzileos AM, Ananth CV, Smulian JC: Using ultrasound in the clinical management of placental implantation abnormalities. Am J Obstet Gynecol 213(4 Suppl):S70, 2015 Voskamp BJ, Fleurke-Rozema H, Oude-Rengerink K, et al: Relationship of isolated single umbilical artery to fetal growth, aneuploidy and perinatal mortality: systematic review and meta-analysis. Ultrasound Obstet Gynecol 42(6):622,s2013 Vyas NM, Manjeera L, Rai S, et al: Prenatal diagnosis of umbilical artery aneurysm with good fetal outcome and review of literature. J Clin Diagn Res 10(l):QDOl,s2016 White SP, Koinas A: Prenatal diagnosis and management of umbilical vein varix of the intra-amniotic portion of the umbilical vein. J Ultrasound Med 13(2):992, 1994

1	White SP, Koinas A: Prenatal diagnosis and management of umbilical vein varix of the intra-amniotic portion of the umbilical vein. J Ultrasound Med 13(2):992, 1994 Whitten E, Romero R, Korzeniewski SJ, et al: Evidence of an imbalance of angiogeniclantiangiogenic factors in massive perivillous ibrin deposition (maternal Aoor infarction): a placental lesion associated with recurrent miscarriage and fetal death. Am J Obstet GynecoIs208(4):310.el, 2013 Woo GW, Rocha FG, Gaspar-Oishi M, et al: Placental mesenchymal dysplasia. Am J Obstet Gynecol 205(6):e3, 201s1 Yamada S, Hamanishi J, Tanada S, et al: Embryogenesis of fused umbilical arteries in human embryos. Am J Obstet Gynecol 193: 1709, 2005 Yamamoto Y, Aoki S, Oba MS, et al: Relationship between short umbilical cord length and adverse pregnancy outcomes. Fetal Pediatr Pathol 35(2):81, 2016 Zalel Y, Weisz B, Gamzu R, et al: Chorioangiomas of the placenta: sonographic and Doppler low characteristics. Ultrasound Med 21 :909, 2002

1	Zalel Y, Weisz B, Gamzu R, et al: Chorioangiomas of the placenta: sonographic and Doppler low characteristics. Ultrasound Med 21 :909, 2002 Zangen R, Boldes R, Yafe H, et al: Umbilical cord cysts in the second and third trimesters: signiicance and prenatal approach. Ultrasound Obstet Gynecol 36(3):296, 2010 GESTATIONAL AGE ............................. 124 EMBRYONIC DEVELOPMENT . . . . . . . . . . . . . . . . . . . .. 125 FETAL DEVELOPMENT AND PHYSIOLOGY. . . . . . . . . .. 128 ENERGY AND . . . . . . . . . . . . . . . . . . . . . .. 137 PLACENTAL ROLE IN EMBRYOFETAL DEVELOPMENT. .. 139 Our knowledge concening the physioloy of the oetus has been markedy enriched during recent years; nevertheless, when com pared with the adult, it oers many points concening which we are but slighty inormed or prooundy ignorant. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) Since these words were written by Williams in 1903, great strides in the understanding of fetal organogenesis and physiology have been gained. Contemporary obstetrics incorporates physiology and pathophysiology of the fetus, its development, and its environment. An important result is that fetal status has been elevated to that of a patient who, in large measure, can be given the same meticulous care that obstetricians provide for gravidas. In our 2yh edition, the entirety of Section 5 is dedicated to the fetal patient, as are individual chapters in other sections. Indeed, virtually every aspect of obstetrics can afect the developing fetus.

1	Several terms define pregnancy duration and thus fetal age (Fig. 7-1). Gestational age or menstrual age is the time elapsed since the first day of the last menstrual period (LMP), a time that actully precedes conception. This starting time, which is usually approximately 2 weeks before ovulation and fertilization and nearly 3 weeks before blastocyst implantation, has traditionally been used because most women know their approximate last period. Embryologists describe embryofetal development in ovuation age, or the time in days or weeks from ovulation. Another term is postconceptional age, which is nearly identical to ovulation age.

1	Until recently, clinicians customarily calculated menstrual age with term pregnancy averaging approximately 280 days, or 40 weeks between the first day of the LMP and birth. This corresponds to 9 and 113 calendar months. However, menstrual cycle length variability among women renders many of these calculations inaccurate. This realization, combined with the frequent use of first-trimester sonography, has led to more accurate gestational age determination (Duryea, 2015). Much of this change hinges on the accuracy of early sonographic measurement. As a result, the American College of Obstetricians and Gynecologists, the American Institute of Ultrasound in Medicine, and the Society for Maternal-Fetal Medicine (Reddy, 2014) together recommend the following: 1. First-trimester sonography is the most accurate method to establish or reairm gestational age. 2. In conceptions achieved with assisted-reproductive technology, this gestational age is used.

1	First-trimester sonography is the most accurate method to establish or reairm gestational age. 2. In conceptions achieved with assisted-reproductive technology, this gestational age is used. FIGURE 7-1 Terminology used to describe pregnancy duration. 3. If available, the gestational ages calculated from the LMP and from first-trimester sonography are compared, and the estimated date of coninement (EDC) recorded and discussed with the patient. 4. The best obstetrical estimate of gestational age at delivery is recorded on the birth certiicate. he embryofetal crown-rump length in the first trimester is accurate ±5 to 7 days. hus, if sonographic assessment of gestational age difers by more than 5 days prior to 9 weeks' gestation, or by more than 7 days later in the irst trimester, the estimated delivery date is changed.

1	An EDC based on LMP can be quickly estimated as follows: add 7 days to the irst day of the LMP and subtract 3 months. For example, if the irst day of the LMP was October 5, the due date is 10-05 minus 3 (months) plus 7 (days) = 7-12, or July 12 of the following year. This calculation has been termed the Naegele rule. The period of gestation can also be divided into three units of approximately 14 weeks each. These three trimesters are important obstetrical milestones. In addition to estimating the EDC with either Naegele rule or pregnancy "wheels," calculator tools in the electronic medical record and smartphone applications can provide a calculated EDC and gestational age. For example, the American College ofObstetricians and Gynecologists (2016) has developed a calculator application that incorporates sonographic criteria and the LMP or embryo transfer date. This is discussed further in Chapter 10 (p. 183).

1	The complexity of embryofetal development is almost beyond comprehension. Figure 7-2 shows a developmental sequence of various organ systems. New information regarding organ development continues to accrue. For example, imaging techniques help unravel the contributions of gene regulation and tissue interaction to eventual three-dimensional organ morphology (Anderson, 2016; Mohun, 2011). Others have described the sequence of gene activation that underlies cardiac development. During the irst 2 weeks after ovulation and then fertilization, the zygote-or preembryo-develops to the blastocyst stage. The blastocyst implants 6 or 7 days following fertilization. The 58-cell blastocyst diferentiates into ive embryo-producing cells-the inner cel mass-and the remaining 53 cells form placental trophoblast. Details of implantation and early development of the blastocyst and placenta are described in Chapter 5 (p. 87). Ears Canals, Diaphragm Transverse septum, diaphragm

1	Ears Canals, Diaphragm Transverse septum, diaphragm Lungs Tracheoesophageal septum, bronchi, lobes Heart Primitive tube, great vessels, valves. chambers Intestines Foregut, liver, pancreas, midgut gut chlea. inner ears. ossicles --Yolksac . Developing FIGURE 7-3 Early human embryos. Ovulation ages: A. 19 days (presomite). B. 21 days (7 somites). C. 22 days (1o7 somites). (After drawings and models in the Carnegie Institute.) he conceptus is termed an embryo at the beginning of the third week after ovulation and fertilization. Primitive chorionic villi form, and this coincides with the expected day of menses. he embryonic period, during which time organogenesis takes place, lasts 6 weeks. It begins the third week from the LMP through the eighth week. The embryonic disc is well deined, and most pregnancy tests that measure human chorionic

1	FIGURE 7-4 Three-to four-week-old embryos. A, B. Dorsal views of embryos during 22 to 23 days of development showing 8 and 12 somites, gonadotropin (hCG) become positive by this time. As shown in Figure 7-3, the body stalk is now diferentiated. There are villous cores in which angioblastic chorionic mesoderm can be distinguished and a true intervillous space that contains maternal blood. During the third week, fetal blood vessels in the chorionic villi appear. In the fourth week, a cardiovascular system has formed (Fig. 7-4). hereby, a true circulation is established respectively. (-E. Lateral views of embryos during 24 to 28 days, showing 16, 27, and 33 somites, respectively. (Redrawn from Moore KL: The Developing Human: Clinically Oriented Embryology, 4th ed. Philadelphia, Saunders, 1988.)

1	FIGURE 7-5 Embyro photographs. A. Dorsal view of an embryo at 24 to 26 days and corresponding to Figure 7-4C. B. Lateral view of an embryo at 28 days and corresponding to Figure 7-40. C. Lateral view of embryofetus at 56 days, which marks the end of the embryonic period and the beginning of the fetal period. The liver is within the white, halo circle. (From Werth B, Tsiaras A: From Conception to Birth:

1	A Life Unfolds. New York, Doubleday, 2002.) both within the embryo and between the embryo and the chorionic villi. Partitioning of the primitive heart begins. Also in the fourth week, the neural plate forms, and it subsequently folds to form the neural tube. By the end of the ifth menstrual week, the chorionic sac measures approximately 1 cm in diameter. he embryo is 3 mm long and can be measured sonographically. Arm and leg buds have developed, and the amnion is beginning to ensheathe the body stalk, which thereafter becomes the umbilical cord. At the end of the sixth week, the embryo measures approximately 9 mm long, and the neural tube has closed (Fig. 7-5). Cardiac motion is almost always discern able sonographically (Fig. 7-6). The cranial end of the neural tube closes by 38 days from the LMP, and the caudal end closes by 40 days. Thus, the neural tube has closed by the end of the sixth week. And by the end of the eighth week, the crownrump length approximates 22 mm. Fingers and

1	the LMP, and the caudal end closes by 40 days. Thus, the neural tube has closed by the end of the sixth week. And by the end of the eighth week, the crownrump length approximates 22 mm. Fingers and toes are present, and the arms bend at the elbows. The upper lip is complete, and the external ears form definitive elevations on either side of the head. hree-dimensional images and videos of human embryos from the MultiDimensional Human Embryo project are found at: http://embryo.soad. umich.edu/index.html.

1	FIGURE 7-6 A. This image of a 6-week, 4-day embryo depicts measurement of the crown-rump length, which is 7.4 mm at this gestational age. B. Despite the early gestational age, M-mode imaging readily demonstrates embryonic cardiac activity. The heart rate in this image is 124 beats per minute. Transition from the embryonic period to the fetal period occurs at 7 weeks after fertilization, corresponding to 9 weeks after onset of the last menses. At this time, the fetus approximates 24 mm in length, most organ systems have developed, and the fetus enters a period of growth and maturation. These phases are outlined in Figure 7-2.

1	The uterus usually is just palpable above the symphysis pubis. Fetal growth is rapid, and the fetal crown-rump length is 5 to 6 cm (Fig. 7-7). Centers of ossiication have appeared in most fetal bones, and the fingers and toes have become diferentiated. Skin and nails develop, and scattered rudiments of hair appear. he external genitalia are beginning to show deinitive signs of male or female gender. he fetus begins to make spontaneous movements. Fetal growth slows at this time. The crown-rump length is 12 cm, and the fetal weight approximates 150 g (Hadlock, 1991). Practically speaing, the sonographic crown-rump length is not measured beyond 13 weeks, which corresponds to approximately 8.4 cm. Instead, biparietal diameter, head circumference, abdominal circumference, and femur length are measured. Fetal weight in the second and third trimesters is estimated from a combination of these measurements (Chap. 10, p. 184).

1	Eye movements begin at 16 to 18 weeks, coinciding with midbrain maturation. By 18 weeks in the female fetus, the uterus is formed and vaginal canalization begins. By 20 weeks in the male, testicles start to descend. FIGURE 7-7 This image of a 12-week, 3-day embryo depicts measurement of the crown-rump length. The fetal profile, cranium, and a hand and foot are also visible in this image. This is the midpoint of pregnancy as estimated from the LMP. The fetus now weighs somewhat more than 300 g, and weight increases substantially in a linear manner. From this point onward, the fetus moves approximately every minute and is active 10 to 30 percent of the day (DiPietro, 2005). Brown fat forms, and the fetal skin becomes less transparent. Downy lanugo covers its entire body, and some scalp hair can be seen. Cochlear function develops between 22 and 25 weeks, and its maturation continues for 6 months after delivery.

1	The fetus now weighs almost 700 g (Duryea, 2014). he skin is characteristically wrinkled, and fat deposition begins. The head is still comparatively large, and eyebrows and eyelashes are usually recognizable. By 24 weeks, the secretory type II pneumocytes have initiated surfactant secretion (Chap. 32, p. 607). The canalicular period of lung development, during which the bronchi and bronchioles enlarge and alveolar ducts develop, is nearly completed. Despite this, a fetus born at this time will attempt to breathe, but many will die because the terminal sacs, required for gas exchange, have not yet formed. The overall survival rate at 24 weeks is barely above 50 percent, and only approximately 30 percent survive without severe morbidity (Rysavy, 2015). By 26 weeks, the eyes open. Nociceptors are present over all the body, and the neural pain system is developed (Kadic, 2012). The fetal liver and spleen are important sites for hemopoiesis.

1	The crown-rump length approximates 25 cm, and the fetus weighs about 1100 g. The thin skin is red and covered with vernix caseosa. The pupillary membrane has just disappeared from the eyes. Isolated eye blinking peaks at 28 weeks. The bone marrow becomes the major site of hemopoiesis. he otherwise normal neonate born at this age has a 90-percent chance of survival.without physical or neurological impairment. At 32 weeks, the fetus has attained a crown-rump length approximating 28 cm and a weight of about 1800 g. he skin surface is still red and wrinkled. In contrast, by 36 weeks, the fetal crown-rump length averages about 32 cm, and the weight approximates 2800 g (Duryea, 2014). Because of subcutaneous fat deposition, the body has become more rotund, and the previous wrinkled facies is now fuller. Normal fetuses have nearly 100-percent survival rate.

1	This is considered term, and the fetus is now fully developed. The average crown-rump length measures about 36 cm, and the average weight approximates 3500 g.

1	The cranial end of the neural tube closes by 38 days from the LMP, and the caudal end closes by 40 days. Hence, folic acid supplementation to prevent neural-tube defects must be in place before this point to be eicacious (Chap. 9, p. 169). The walls of the neural tube form the brain and spinal cord. The lumen becomes the ventricular system of the brain and the central canal of the spinal cord. During the sixth week, the cranial end of the neural tube forms three primary vesicles. In the seventh week, five secondary vesicles develop: the telencephalon-future cerebral hemispheres; diencephalon-thalami; mesencephalon-midbrain; metencephalon-pons and cerebellum; and myelencephalonmedulla. Meanwhile, lexures develop and fold the brain into its typical configuration. The end of the embryonic period signiies completion of primary and secondary neuralization. At 3 to 4 months' gestation, neuronal proleration peaks.

1	At 3 to 4 months' gestation, neuronal proleration peaks. As expected, disorders in this cerebral development phase profoundly worsen function (Volpe, 2008). Neuronal migration occurs almost simultaneously and peaks at 3 to 5 months. This process is characterized by movement of millions of neuronal cells from their ventricular and subventricular zones to areas of the brain in which they reside for life (Fig. 7-8). U pregulation of gene expression for neuronal migration has been described (Iruretagoyena, 2014). Noninvasive methods to study fetal neurodevelopment have also been reported (Goetzl, 2016).

1	As gestation progresses, the fetal brain appearance steadily changes. Thus, it is possible to identiY fetal age from its external appearance (Volpe, 2008). Neuronal proliferation and migration proceed along with gyral growth and maturation (see Fig. 7-8). Sequential maturation studies by Manganaro (2007) and Dubois (2014) and their colleagues have characterized the developing fetal brain image using magnetic resonance (MR) imaging. Other recent investigations that also used MR imaging have quantiied development of subcortical brain structures from 12 to 22 weeks (Meng, 2012). Myelination of the ventral roots of the cerebrospinal nerves and brainstem begins at approximately 6 months, but most

1	Myelination of the ventral roots of the cerebrospinal nerves and brainstem begins at approximately 6 months, but most A B c �. .�. ''' FIGURE 7-8 Neuronal proliferation and migration are complete at 20 to 24 weeks. During the second half of gestation, organizational events proceed with gyral formation and proliferation, diferentiation, and migration of cellular elements. Approximate gestational ages are shown. A. 20 weeks. B. 35 weeks. C.40 weeks. myelination progresses after birth. This lack of myelin and incomplete skull ossiication permit fetal brain structure to be seen sonographically throughout gestation.

1	myelination progresses after birth. This lack of myelin and incomplete skull ossiication permit fetal brain structure to be seen sonographically throughout gestation. Whereas the superior two thirds of the neural tube give rise to the brain, the inferior third forms the spinal cord. In the embryo, the spinal cord extends along the entire vertebral column length, but after that it lags behind vertebral growth. Ossiication of the entire sacrum is visible sonographically by approximately 21 weeks (Chap. 10, p. 191). By 24 weeks, the spinal cord extends to SIiat birth to L3, and in the adult to LI. Spinal cord myelination begins at midgestation and continues through the irst year of life. Synaptic function is suiciently developed by the eighth week to demonstrate flexion of the neck and trunk (Temiras, 1968). During the third trimester, integration of nervous and muscular function proceeds rapidly.

1	The embryology of the heart is complex. At its earliest stages of formation, the fetal heart undergoes molecular programming, and more than a hundred genes and molecular factors are integral to its morphogenesis. To summarize, the straight cardiac tube is formed by the 23rd day during an intricate morphogenetic sequence, during which each segment arises at a unique time. The tube then undergoes looping, and the chambers then fuse and form septa (Manner, 2009). The valves develop, and the aortic arch forms by vasculogenesis. For a complete description, refer to Chapter 9 in Hursts he Heart (Keller, 2013).

1	his unique circulation is substantially diferent from that of the adult and functions until birth, when it changes dramatically. For example, because fetal blood does not need to enter the pulmonary vasculature to be oxygenated, most of the right ventricular output bypasses the lungs. In addition, the fetal heart chambers work in parallel, not in series, which efectively supplies the brain and heart with more highly oxygenated blood than the rest of the body.

1	Oxygen and nutrient materials required for fetal growth and maturation are delivered from the placenta by the single umbilical vein (Fig. 7-9). The vein then divides into the ductus venosus and the portal sinus. The ductus venosus is the major branch of the umbilical vein and traverses the liver to enter the inferior vena cava directly. Because it does not supply oxygen to the intervening tissues, it carries well-oxygenated blood directly to the heart. In contrast, the portal sinus carries blood to the hepatic veins primarily on the left side of the liver, and oxygen is extracted. The relatively deoxygenated blood from the liver then lows back into the inferior vena cava, which also receives more deoxygenated blood returning from the lower body. Blood flowing to the fetal heart from the inferior vena cava, therefore, consists of an admixture of arterial-like blood that passes directly through the ductus venosus and less well-oxygenated blood that returns from most of the veins below

1	inferior vena cava, therefore, consists of an admixture of arterial-like blood that passes directly through the ductus venosus and less well-oxygenated blood that returns from most of the veins below the level of the diaphragm. The oxygen content of blood delivered to the heart from the inferior vena cava is thus lower than that leaving the placenta.

1	Foramen ovale Umbilical v. Hypogastricaa. Ductus Aorta _ -- FIGURE 7-9 The intricate nature of the fetal circulation is evident. The degree of blood oxygenation in various vessels differs appreciably from that in the postnatal state. aa = arteries; LA = left atrium; LV = left ventricle; RA = right atrium; RV = right ventricle; v = vein.

1	As discussed, the ventricles of the fetal heart work in parallel, not in series. Well-oxygenated blood enters the left ventricle, which supplies the heart and brain, and less oxygenated blood enters the right ventricle, which supplies the rest of the body. These two separate circulations are maintained by the right atrial structure, which efectively directs entering blood to either the left atrium or the right ventricle, depending on its oxygen content. his separation of blood according to its oxygen content is aided by the pattern of blood flow in the inferior vena cava. The well-oxygenated blood tends to course along the medial aspect of the inferior vena cava and the less oxygenated blood flows along the lateral vessel wall. This aids their shunting into opposite sides of the heart. Once this blood enters the right atrium, the configuration of the upper interatrial septum-the crista dividens-preferentially shunts the well-oxygenated blood from the medial side of the inferior vena

1	this blood enters the right atrium, the configuration of the upper interatrial septum-the crista dividens-preferentially shunts the well-oxygenated blood from the medial side of the inferior vena cava and the ductus venosus through the foramen ovale into the left heart and then to the heart and brain (Dawes, 1962). After these tissues have extracted needed oxygen, the resulting less oxygenated blood returns to the right atrium through the superior vena cava.

1	The less oxygenated blood coursing along the lateral wall of the inferior vena cava enters the right atrium and is deflected through the tricuspid valve to the right ventricle. The superior vena cava courses inferiorly and anteriorly as it enters the right atrium, ensuring that less well-oxygenated blood returning from the brain and upper body also will be shunted directly to the right ventricle. Similarly, the ostium of the coronary sinus lies just superior to the tricuspid valve so that less oxygenated blood from the heart also returns to the right ventricle. As a result of this blood flow pattern, blood in the right ventricle is 15 to 20 percent less saturated than blood in the left ventricle.

1	lmost 90 percent of blood exiting the right ventricle is shunted through the ductus arteriosus to the descending aorta. High pulmonary vascular resistance and comparatively lower resistance in the ductus arteriosus and the umbilical-placental vasculature ensure that only about 8 percent of right ventricular output goes to the lungs (Fineman, 2014). Thus, one third of the blood passing through the ductus arteriosus is delivered to the body. The remaining right ventricular output returns to the placenta through the two hypogastric arteries. These two arteries course from the level of the bladder along the abdominal wall to the umbilical ring and into the cord as the umbilical arteries. In the placenta, this blood picks up oxygen and other nutrients and is recirculated through the umbilical vein.

1	After birth, the umbilical vessels, ductus arteriosus, foramen ovale, and ductus venosus normally constrict or collapse. With the functional closure of the ductus arteriosus and the expansion of the lungs, blood leaving the right ventricle preferentially enters the pulmonary vasculature to become oxygenated before it returns to the left heart (Hillman, 2012). Virtually instantaneously, the ventricles, which had worked in parallel in fetal life, now efectively work in series. The more distal portions of the hypogastric arteries undergo atrophy and obliteration within 3 to 4 days after birth. hese become the umbilicaliligaments, whereas the intraabdominal remnants of the umbilical vein form the ligamentum teres. The ductus venosus constricts by 10 to 96 hours after birth and is anatomically closed by 2 to 3 weeks. This ultimately forms the ligamentum venosum (Fineman, 2014).

1	Although precise measurements of human fetoplacental blood volume are lacking, Usher and associates (1963) reported values in term normal newborns to average 78 mLlkg when immediate cord clamping was conducted. Gruenwald (1967) found the fetal blood volume contained in the placenta after prompt cord clamping to average 45 mLlkg of fetal weight. Thus, fetoplacental blood volume at term is approximately 125 mLlkg of fetal weight. This is important when assessing the magnitude of fetomaternal hemorrhage as discussed in Chapter 15 (p. 307).

1	In the early embryo, hemopoiesis is demonstrable first in the yolk sac, followed by the liver, and inally spleen and bone marrow. Both myeloid and erythroid cells are continually pro duced by progenitors that are from hematopoietic stem cells (Golub, 2013; Heinig, 2015). The first erythrocytes released into the fetal circulation are nucleated and macrocytic. The mean cell volume is at least 180 L in the embryo and decreases to 105 to 115 L at term. The erythrocytes of aneuploid fetuses mean cell volumes-130 L on average (Sipes, 1991). As fetal development progresses, more and more of the circulating erythrocytes are smaller and nonnucleated. With fetal growth, both the blood volume in the common fetoplacental circu lation and hemoglobin concentration increase. As shown in Figure 7-10, fetal hemoglobin concentrations rise across preg nancy. The Society for Maternal-Fetal Medicine (2015) rec ommends a cutof hematocrit value of 30 percent to define anemia.

1	Figure 7-10, fetal hemoglobin concentrations rise across preg nancy. The Society for Maternal-Fetal Medicine (2015) rec ommends a cutof hematocrit value of 30 percent to define anemia. Because of their large size, fetal erythrocytes have a short life span, which progressively lengthens to approximately 90 days at term (Pearson, 1966). As a consequence, red blood cell production rises. Reticulocytes are initially present at high levels, but decrease to 4 to 5 percent of the total at term. Fetal erythrocytes difer structurally and metabolically from those in the adult (Baron, 2012). They are more deformable, which serves to ofset their higher viscosity. They also contain several enzymes with appreciably diferent activities.

1	Erythropoiesis is controlled primarily by fetal erythropoietin because maternal erythropoietin does not cross the placenta. Fetal hormone production is influenced by testosterone, estrogen, prostaglandins, thyroid hormone, and lipoproteins (Stockman, 1992). Serum erythropoietin levels rise with fetal maturity. Although the exact production site is disputed, the fetal liver appears to be an important source until renal production begins. There is a close correlation between the erythropoietin concentration in amnionic fluid and that in umbilical venous blood obtained by cordocentesis. After birth, erythropoietin normally may not be detectable for up to 3 months. In contrast, platelet production reaches stable levels by midpregnancy, although there is some variation across gestation (Fig. The fetal and neonatal platelet count is subject to various agents as discussed in Chapter 15 (p. 307).

1	The fetal and neonatal platelet count is subject to various agents as discussed in Chapter 15 (p. 307). This tetrameric protein is composed of two copies of two diferent peptide chains, which determine the type of hemoglobin produced. Normal adult hemoglobin A is made of a and 3 chains. During embryonic and fetal life, various a and 3 chain precursors are produced. This results in the serial production of several diferent embryonic hemoglobins. Genes for 3-type chains are on chromosome 11, and those for a-type chains on chromosome 16. Each of these genes is turned on and then of during fetal life, until a and 3 genes, which direct the production of adult hemoglobin A, are permanentlyiactivated.

1	FIGURE 7-11 Platelet counts by gestational age obtained the first day of life. Mean values and 5th and 95th percentiles are shown. (Data from Christensen RD, Henry E, Antonio DV: Thrombocytosis and thrombocytopenia in the NICU: incidence, mechanisms and treatments, J Matern Fetal Neonatal Med 2012 Oct;25 SuppIo4:15-17) • •• • •••• • • • •• •• •• • Moderateanemia • • ,• •• Mildanemia • Severeanemia•• • • 1.16 :JA12 0.84 ) 0.65 0.55 ) :;)I S FIGURE 7-10 Relationship between fetal hemoglobin across gestational age. Blue dots indicate fetuses with hydrops. (Reproduced with permission from Mari G, Deter RL, Carpenter RL, et al: Noninvasive diagnosis by Doppler ultrasonography offetal anemia due to maternal red-cell alloimmunization. Collaborative Group for Doppler Assessment ofthe Blood Velocity in Anemic Fetuses (Level II-I), N Engl J Med 2000 Jan 6;342(1 ):9-14.)

1	The timing ofproduction ofeach ofthese earlyhemoglobins corresponds to the site of hemoglobin production. Fetal blood is first produced in the yolk sac, where hemoglobins Gower 1, Gower 2, and Portland are made. Erythropoiesis then moves to the liver, where fetal hemoglobin F is produced. When hemopoiesis finally moves to the bone marrow, adult-type hemoglobin A appears in fetal red blood cells and is present in progressively greater amounts as the fetus matures (Pataryas, 1972).

1	he final adult version of the . chain is produced exclusively by 6 weeks. Mter this, there are no functional alternative 400,000 /95th :J:LD 300,000 200,000 ::D)5 ) 100,000 24262830323436384042 versions. Ifan a-gene mutation or deletion occurs, no alternate a-type chain can be substituted to form functional hemoglobin. In contrast, at least two versions of the � chain-6 and ,-remain in production throughout fetal life and beyond. In the case ofa �-gene mutation or deletion, these two other versions of the � chain often continue to be produced, resulting in hemoglobin A2 or hemoglobin F, which substitute for the abnormal or missing hemoglobin.

1	Genes are turned of by methylation of their control region, which is discussed in Chapter 13 (p. 267). In some situations, methylation does not occur. For example, in newborns ofdiabetic women, hemoglobin F may persist due to hypomethylation of thei, gene (Perrine, 1988). With sickle cell anemia, thei, gene remains unmethylated, and large quantities of fetal hemoglobin continue to be produced. s discussed in Chapter 56 (p. 1081), elevated hemoglobin F levels are associated with fewer sickle-cell disease symptoms, and pharmacological modiication ofthese levels by hemoglobin F-inducing drugs is one approach to treatment.

1	As discussed on page 140, there is a functional diference between hemoglobins A and F. At any given oxygen tension and at identical pH, fetal erythrocytes that contain mostly hemoglobin F bind more oxygen than do those that contain nearly all hemoglobin A (Fig. 47-2, p. 920). his is because hemoglobin A binds 2,3-diphosphoglycerate (2,3-DPG) more avidly than does hemoglobin F, thus lowering the ainity of hemoglobin A for oxygen. During pregnancy, maternal 2,3-DPG levels are greater, and because fetal erythrocytes have lower concentrations of 2,3-DPG, the latter has increased oxygen ainity. he amount of hemoglobin F in fetal erythrocytes begins to decrease in the last weeks of pregnancy. At term, approximately three fourths oftotal hemoglobin levels are hemoglobin F. During the irst 6 to 12 months of life, the hemoglobin F proportion continues to decline and eventually reaches the low levels found in adult erythrocytes.

1	With the exception of fibrinogen, there are no embryonic forms of the various hemostatic proteins. he fetus starts producing normal, adult-type procoagulant, ibrinolytic, and anticoagulant proteins by 12 weeks. Because they do not cross the placenta, their concentrations at birth are markedly below the levels that develop within a few weeks of life (Corrigan, 1992). In normal neonates, the levels of factors II, VII, IX, X, XI, and of protein S, protein C, antithrombin, and plasminogen all approximate 50 percent of adult levels. In contrast, levels of factors V, VIII, XIII, and ibrinogen are closer to adult values (Saracco, 2009). Without prophylactic treatment, the levels of vitamin K-dependent coagulation factors usually decrease even further during the irst few days after birth. his decline is ampliied in breastfed infants and may lead to newborn hemorrhage (Chap. 33, p. 626).

1	Fetal ibrinogen, which appears as early as 5 weeks, has the same amino acid composition as adult ibrinogen, however, it has diferent properties (Klagsbrun, 1988). It forms a less compressible clot, and the ibrin monomer has a lower degree of aggregation (Heimark, 1988). Although plasma ibrinogen levels at birth are less than those in nonpregnant adults, the protein is functionally more active than adult ibrinogen (Ignjatovic, 2011). Levels of functional fetal factor XIII-ibrin stabilizing factor-are signiicantly reduced compared with those in adults (Henriksson, 1974). Nielsen (1969) described low levels of plasminogen and elevated ibrinolytic activity in cord plasma compared with that of maternal plasma. Platelet counts in cord blood are in the normal range for nonpregnant adults (see Fig. 7-11).

1	Despite this relative reduction in procoagulants, the fetus appears to be protected from hemorrhage, and fetal bleeding is rare. Even after invasive fetal procedures such as cordocentesis, excessive bleeding is uncommon. Ney and coworkers (1989) have shown that amnionic luid thromboplastins and a factor(s) in Wharton jelly combine to aid coagulation at the umbilical cord puncture site. Various thrombophilias may cause thromboses and pregnancy complications in adults (Chap. 52, p. 1008). If the fetus inherits one of these mutations, thrombosis and infarction can develop in the placenta or fetal organs. his is usually seen with homozygous inheritance. One example is homozygous protein C mutation, which causes purpura fulminans.

1	Liver enzymes and other plasma proteins are produced by the fetus, and these levels do not correlate with maternal levels (Weiner, 1992). Concentrations of plasma proteins, which include albumin, lactic dehydrogenase, aspartate aminotransferase, 1-glutamyl transpeptidase, and alanine transferase, all rise. Conversely, prealbumin levels decline with gestational age (Fryer, 1993). At birth, mean total plasma protein and albumin concentrations in fetal blood are similar to maternal levels. This is important because albumin binds unconjugated bilirubin to prevent kernicterus in the newborn (Chap. 33, p. 626).

1	Lung maturation and biochemical indices of functional fetal lung maturity are important predictors of early neonatal outcome. Morphological or functional immaturity at birth leads to the development of the respiratoy distress syndrome (Chap. 34, p. 636). A suicient amount of surface-active materialscollectively referred to as suactant-in the amnionic fluid is evidence of fetal lung maturity. As Liggins (1994) emphasized, however, the structural and morphological maturation of fetal lung also is extraordinarily important to proper lung function.

1	The limits of viability appear to be determined by the usual process of pulmonary growth. Like the branching of a tree, lung development proceeds along an established timetable that apparently cannot be hastened by antenatal or neonatal therapy. Within this framework, four essential lung development stages are described by Moore (2000). First, the pseudoglandular stage entails growth of the intrasegmental bronchial tree between the 5th and 17th weeks. During this period, the lung looks microscopically like a gland. Second, during the canalicular stage, from 16 to 25 weeks, the bronchial cartilage plates extend peripherally. Each terminal bronchiole gives rise to several respiratory bronchioles, and each of these in turn divides into multiple saccular ducts. Third, the terminal sac stage begins after 25 weeks. During this stage, alveoli give rise to primitive pulmonary alveoli, that is, the terminal sacs. Simultaneously, an extracellular matrix develops from proximal to distal lung

1	begins after 25 weeks. During this stage, alveoli give rise to primitive pulmonary alveoli, that is, the terminal sacs. Simultaneously, an extracellular matrix develops from proximal to distal lung segments until term. Finally, the alveolar stage begins during the late fetal period and continues well into childhood. An extensive capillary network is built, the lymph system forms, and type II pneumocytes begin to produce surfactant. At birth, only approximately 15 percent of the adult number of alveoli is present. hus, the lung continues to grow, adding more alveoli for up to 8 years.

1	Various insults can upset this process, and their timing determines the sequelae. One example is fetal renal agenesis, in which amnionic luid is absent at the beginning oflung growth, and major defects occur in all four developmental stages. In another instance, the fetus with membrane rupture and subsequent oligohydramnios before 20 weeks usually exhibits nearly normal bronchial branching and cartilage development but has immature alveoli. In contrast, membrane rupture after 24 weeks may have minimal long-term efect on pulmonary structure. In another example, various growth factors are expressed abnormally in the fetus with a diaphragmatic hernia (Candilira, 2015). Finally, vitamin D is thought to be important for several aspects oflung development (Hart, 2015; Lykkedegn, 2015).

1	After the irst breath, the terminal sacs must remain expanded despite the pressure imparted by the tissue-to-air interface, and surfactant keeps them from collapsing. Surfactant is formed in type II pneumonocytes that line the alveoli. These cells are characterized by multivesicular bodies that produce the lamellar bodies in which surfactant is assembled. During late fetal life, at a time when the alveolus is characterized by a waterto-tissue interface, the intact lamellar bodies are secreted from the lung and swept into the amnionic fluid during respiratorylike movements that are termed fetal breathing. At birth, with the first breath, an air-to-tissue interface is established in the lung alveolus. Surfactant uncoils from the lamellar bodies and spreads to line the alveolus to prevent alveolar collapse during expiration. hus, the fetal lungs' capacity to produce surfactant establishes lung maturity.

1	Surfactant Composition. Gluck (1972) and Hallman (1976) and their coworkers approximated that 90 percent of surfactant's dry weight is lipid, speciically glycerophospholipids. Proteins account for the other 10 percent. Nearly 80 percent of the glycerophospholipids are phosphatidylcholines (lecithins). The principal active component that constitutes half of surfactant is a speciic lecithin, which is dipalmitoylphosphatidylcholine (DPPC or PC). Phosphatidylglycerol (PG) accounts for another 8 to 15 percent. Its precise role is unclear because newborns without PG usually do well. he other major constituent is phosphatidylinositol (PI). The relative contributions of each component are shown in Figure 7-12.

1	Surfactant Synthesis. Biosynthesis takes place in the type II pneumocytes. he apoproteins are produced in the endoplasmic reticulum, and the glycerophospholipids are synthesized by cooperative interactions of several cellular organelles. Phospholipid is the primary surface tension-lowering component of surfactant, whereas the apoproteins aid the forming and reforming of a surface ilm. The major apoprotein is surfactant A (SP-A), which is a glycoprotein with a molecular weight of 28,000 to 35,000 Da (Whitsett, 1992). It is synthesized in the type II cells, and its content in amnionic fluid increases with gestational age and fetal lung maturity. SP-A gene expression is demonstrable by 29 weeks (Mendelson, 2005). Specifically, SP-Ai and SP-A2 0: : o . 15 -.a are two separate genes on chromosome 10, but their regulation is distinctive and diferent (McCormick, 1994).

1	15 -.a are two separate genes on chromosome 10, but their regulation is distinctive and diferent (McCormick, 1994). Several smaller apoproteins such as SP-B and SP-C are likely important in optimizing the action of surfactant. For example, deletions.in SP-B gene are incompatible with survival despite production of large amounts of surfactant (Hallman, 2013). Corticosteroids and Fetal Lung Maturation. Since Liggins (1969) observed accelerated lung maturation in lamb fetuses given glucocorticosteroids prior to preterm delivery, many suggested that fetal cortisol stimulates lung maturation and surfactant synthesis. It is unlikely that corticosteroids are the only stimulus for augmented surfactant formation. However, when these are administered at certain critical times, they may improve preterm fetal lung maturation. As fetal lung therapy, antenatal betamethasone and dexamethasone use and neonatal replacement surfactant therapy are discussed in Chapter 34 (p. 637).

1	Fetal respiratory muscles develop early, and chest wall movements are detected sonographically as early as 11 weeks (Koos, 2014). From the beginning of the fourth month, the fetus engages in respiratory movement suiciently intense to move amnionic luid in and out of the respiratory tract. Some extrauterine events have efects on fetal breathing, for example, maternal exercise stimulates it (Sussman, 2016). After its embryogenic formation from the yolk sac as the primordial gut, the digestive system forms the intestines and various appendages. The foregut gives rise to the pharynx, lower respiratory system, esophagus, stomach, proximal duodenum, liver, pancreas, and biliary tree. The midgut gives rise to the distal duodenum, jejunum, ileum, cecum, appendix, and the right colon. The hindgut develops into the left colon, rectum, and the superior portion of the anal canal. Numerous malformations develop in these structures from improper rotation, ixation, and partitioning.

1	Swallowing begins at 10 to 12 weeks, coincident with the ability of the small intestine to undergo peristalsis and actively transport glucose (Koldovsky, 1965). As a correlate, neo ..A:.A : 0 of immature gut motility (Singendonk, 2014). Much of the water in swallowed luid is absorbed, and unabsorbed matter o). ::) 0-0.. .-o i:S :.=. 0 is propelled to the lower colon. Gitlin (1974) demonstrated that late in pregnancy, approximately 800 mg of soluble pro tein is ingested daily by the fetus. he stimulus for swallow :.o)B : 0 ing is unclear, but the fetal neural analogue of thirst, gastric o : 0 :.=.=t ..A emptying, and change in the amnionic fluid composition are potential factors (Boyle, 1992). The fetal taste buds may play a role because saccharin injected into amnionic fluid increases ..At 40 swallowing, whereas injection of a noxious chemical inhibits it Gestational age (weeks) (Liley, 1972). Fetal swallowing appears to have little efect on amnionic

1	Gestational age (weeks) (Liley, 1972). Fetal swallowing appears to have little efect on amnionic FIGURE 7-12 Relationship between the levels of lecithindipalmitoyl phosphatidylcholine (PC), phosphatidylinositol (PI), and fluid volume early in pregnancy because the volume swallowed phosphatidylglycerol (PG) in amnionic fluid. is small compared with the total. However, term fetuses swallow between 200 and 760 mL per day-an amount comparable to that of the term neonate (Pritchard, 1966). hus at term, amnionic fluid volume regulation can be substantially altered by fetal swallowing. For example, as discussed in Chapter 11 (p. 227), if swallowing is inhibited, hydramnios is common.

1	Hydrochloric acid and some digestive enzymes are pres ent in the stomach and small intestine in minimal amounts in the early fetus. Intrinsic factor is detectable by 11 weeks, and pepsinogen by 16 weeks. The preterm neonate, depending on its gestational age, may have transient deficiencies of these enzymes (Lebenthal, 1983). Stomach emptying appears to be stimulated primarily by volume. Movement of amnionic luid through the gastroin testinal system may enhance growth and development of the alimentary canal. That said, other regulatory factors likely are involved. For example, anencephalic fetuses, in which swallow ing is limited, often have normal amnionic fluid volume and normal-appearing gastrointestinal tract.

1	Fetal bowel contents consist of various products of secretion, such as glycerophospholipids from the lung, desquamated fetal cells, lanugo, scalp hair, and vernix. It also contains undigested debris from swallowed amnionic fluid. The dark greenish-black color forms from bile pigments, especially biliverdin. Meconium can pass from normal bowel peristalsis in the mature fetus or from vagal stimulation. It can also pass when hypoxia stimulates arginine vasopressin (A VP) release from the fetal pituitary gland. A VP stimulates colonic smooth muscle to contract, resulting in intraamnionic defecation (deVane, 1982; Rosenfeld, 1985). Meconium is toxic to the respiratory system, and its inhalation can result in meconium aspiration syndrome (Chap. 33, p. 620).

1	The hepatic diverticulum is an outgrowth of the endodermal lining of the foregut. Epithelial liver cords and primordial cells diferentiate into hepatic parenchyma. Serum liver enzyme levels increase with gestational age. Still, the fetal liver has a gestational-age-related diminished capacity for converting free unconjugated bilirubin to conjugated bilirubin (Morioka, 2015). Because of hepatic immaturity, the preterm newborn is at particular risk for hyperbilirubinemia (Chap. 33, p. 626). And because the life span of normal fetal macrocytic erythrocytes is shorter than that of the adult, relatively more unconjugated bilirubin is produced. As just noted, the fetal liver conjugates only a small fraction, and this is excreted into the intestine and ultimately oxidized to biliverdin. Most of the unconjugated bilirubin is excreted into the amnionic luid after 12 weeks and transferred across the placenta (Bashore, 1969).

1	Importantly, placental bilirubin transfer is bidirectional. Thus, a woman with severe hemolysis from any cause has excess unconjugated bilirubin that readily passes to the fetus and then into the amnionic fluid. Conversely, conjugated bilirubin is not exchanged to any significant degree between mother and fetus. Most fetal cholesterol derives from hepatic synthesis, which satisies the large demand for low-density lipoprotein (LDL) cholesterol by the fetal adrenal glands. Hepatic glycogen is present in low concentration during the second trimester, but near term, levels rise rapidly and markedly to reach concentrations that are two-to threefold higher than those in the adult liver. After birth, glycogen content falls precipitously.

1	from the endoderm of the foregut. Gene regulation of its development was recently reviewed Qennings, 2015). Insu lin-containing granules can be identiied by 9 to 10 weeks, and insulin is detectable in fetal plasma at 12 weeks (Adam, 1969). The pancreas responds to hyperglycemia by secreting insulin (Obenshain, 1970). Glucagon has been identified in the fetal pancreas at 8 weeks. Although hypoglycemia does not cause an increase in fetal glucagon levels, similar stimuli do so by 12 hours after birth (Chez, 1975). At the same time, however, fetal pancreatic a cells do respond to L-dopa infu sions (Epstein, 1977). Therefore, unresponsiveness to hypo glycemia is likely the consequence of failed glucagon release rather than inadequate production. This is consistent with developmental expression of pancreatic genes in the fetus (Mally, 1994).

1	Most pancreatic enzymes are present by 16 weeks. T rypsin, chymotrypsin, phospholipase A, and lipase are found in the 14-week fetus, and their concentrations increase with gestational age (Wedin, 1992). Amylase has been identiied in amnionic luid at 14 weeks (Davis, 1986). The exocrine function of the fetal pancreas is limited. Physiologically important secretion occurs only after stimulation by a secretagogue such as acetylcholine, which is released locally after vagal stimulation (Werlin, 1992). Cholecystokinin normally is released only after protein ingestion and thus ordinarily would not be found in the fetus.

1	Renal development involves interaction between pluripotential stem cells, undiferentiated mesenchymal cells, and epithelial components (Fanos, 2015). Two primitive urinary systemsthe pronephros and the mesonephros-precede development of the metanephros, which forms the inal kidney (Chap. 3, p. 33). The pronephros involutes by 2 weeks, and the mesonephros produces urine at 5 weeks and degenerates by 11 to 12 weeks. Failure of these two structures either to form or to regress may result in anomalous urinary system development. Between 9 and 12 weeks, the ureteric bud and the nephrogenic blastema interact to produce the metanephros. he kidney and ureter develop from intermediate mesoderm. The bladder and urethra develop from the urogenital sinus. The bladder also develops in part from the allantois.

1	By week 14, the loop of Henle is functional and reabsorption occurs (Smith, 1992). New nephrons continue to be formed until 36 weeks. In preterm neonates, their formation continues after birth. Although the fetal kidneys produce urine, their ability to concentrate and modiy the pH is limited even in the mature fetus. Fetal urine is hypotonic with respect to fetal plasma and has low electrolyte concentrations.

1	Renal vascular resistance is high, and the filtration fraction is low compared with adult values (Smith, 1992). Fetal renal blood low and thus urine production are controlled or inluenced by the renin-angiotensin system, the sympathetic nervous system, prostaglandins, kallikrein, and atrial natriuretic peptide. The glomerular filtration rate increases with gestational age from less than 0.1 mLimin at 12 weeks to 0.3 mLi min at 20 weeks. In later gestation, the rate remains constant when corrected for fetal weight (Smith, 1992). Hemorrhage or hypoxia generally results in a decrease in renal blood flow, glomerular filtration rate, and urine output. Urine usually is found in the bladder even in small fetuses.

1	Urine usually is found in the bladder even in small fetuses. The fetal kidneys start producing urine at 12 weeks. By 18 weeks, they are producing 7 to 14 mLi day, and at term, this increases to 650 mLiday (Wladimirof, 1974). Maternally administered furosemide increases fetal urine formation, whereas uteroplacental insuiciency, fetal growth restriction, and other fetal disorders can lower it. Obstruction of the urethra, bladder, ureters, or renal pelves can damage renal parenchyma and distort fetal anatomy (Muller Brochut, 2014). Kidneys are not essential for survival in utero but inluence control of amnionic luid composition and volume. hus, abnormalities that cause chronic fetal anuria are usually accompanied by oligohydramnios and pulmonary hypoplasia. Pathological correlates and prenatal therapy of urinary tract obstruction are discussed in Chapter 16 (p. 325).

1	The fetal endocrine system is functional for some time before the central nervous system reaches maturity (Mulchahey, 1987). he anterior pituitary gland develops from oral ectodermRathke pouch, whereas the posterior pituitary gland derives from neuroectoderm. As with other organ systems, embryonic development involves a complex and highly spatiotemporally regulated network of signaling molecules and transcription factors (Bancalari, 2012; de Moraes, 2012). Anterior and Intermediate Lobes. The adenohypophysis, or anterior pituitary, diferentiates into five cell types that secrete six protein hormones. Of these types, lactotropes produce prolactin (PRL), somatotropes produce growth hormone (GH), corticotropes produce adrenocorticotropic hormone (ACTH), thyrotropes produce thyroid-stimulating hormone (TSH), and gonadotropes produce luteinizing hormone (LH) and folliclestimulating hormone (FSH).

1	ACTH is irst detected in the fetal pituitary gland at 7 weeks, and GH and LH have been identified by 13 weeks. By the end of the 17th week, the fetal pituitary gland synthesizes and stores all pituitary hormones. Moreover, the fetal pituitary is responsive to tropic hormones and is capable of secreting these early in gestation (Grumbach, 1974). he fetal pituitary secretes �-endorphin, and cord blood levels of �-endorphin and �-lipotrophin rise with fetal PC02 (Browning, 1983). The intermediate lobe in the fetal pituitary gland is well developed. The cells of this structure begin to disappear before term and are absent from the adult pituitary. he principal secretory products of the intermediate lobe cells are .-melanocytestimulating hormone (.-MSH) and �-endorphin.

1	Neurohypophysis. The posterior pituitary gland or neurohypophysis is well developed by 10 to 12 weeks, and oxytocin and arginine vasopressin are demonstrable. Both hormones probably unction in the fetus to conserve water by actions directed largely at the lung and placenta rather than kidney. Vasopressin levels in umbilical cord plasma are increased strikingly compared with maternal levels (Chard, 1971).

1	This gland arises from the endoderm of the second pharyngeal pouch. he thyroid migrates to its inal position and the obliterated thyroglossal duct connects to the foramen cecum of the tongue. he pituitary-thyroid system is functional by the end of the first trimester. The thyroid gland is able to synthesize hormones by 10 to 12 weeks, and thyrotropin, thyroxine, and thyroid-binding globulin (TBG) have been detected in fetal serum as early as 11 weeks (Bernal, 2007). he placenta actively concentrates iodide on the fetal side, and by 12 weeks and throughout pregnancy, the fetal thyroid concentrates iodide more avidly than does the maternal thyroid. hus, maternal administration of either radioiodide or appreciable amounts of ordinary iodide is hazardous after this time (Chap. 58, p. 1121). Normal fetal levels of free thyroxine (T4), free triiodothyronine (T3)' and thyroxin-binding globulin increase steadily throughout gestation (Ballabio, 1989). Compared with adult levels, by 36 weeks,

1	Normal fetal levels of free thyroxine (T4), free triiodothyronine (T3)' and thyroxin-binding globulin increase steadily throughout gestation (Ballabio, 1989). Compared with adult levels, by 36 weeks, fetal serum concentrations of TSH are higher, total and free T3 concentrations are lower, and T4 is similar. This suggests that the fetal pituitary may not become sensitive to feedback until late pregnancy (horpeBeeston, 1991).

1	Fetal thyroid hormone plays a role in the normal development of virtually all fetal tissues, especially the brain (Forhead, 2014; Rovet, 2014). Its inluence is illustrated by congenital hyperthyroidism, which develops when maternal thyroidstimulating antibody crosses the placenta to stimulate the fetal gland to secrete thyroxine (Donnelley, 2015). These fetuses develop large goiters as shown in Figure 58-3 (p. 1121). They also display tachycardia, hepatosplenomegaly, hematological abnormalities, craniosynostosis, and growth restriction. As children, they have perceptual motor diiculties, hyperactivity, and reduced growth (Wenstrom, 1990). Fetal thyroid disease and its treatment are discussed in Chapter 16 (p. 318) . Neonatal efects of fetal thyroid deficiency are discussed in Chapter 58 (p. 1126).

1	The placenta prevents substantial passage of maternal thyroid hormones to the fetus by rapidly deiodinating maternal T 4 and T 3 to form reverse T 3' a relatively inactive thyroid hormone (Vulsma, 1989). Several antithyroid antibodies cross the placenta when present in high concentrations (Pelag, 2002). Those include the long-acting thyroid stimulators (LATS), LATSprotector (LATS-P), and thyroid-stimulating immunoglobulin (TSI). It was previously believed that normal fetal growth and development, which occurred despite fetal hypothyroidism, provided evidence that T 4 was not essential for fetal growth. It is now known, however, that growth proceeds normally because small quantities of maternal T4 prevent antenatal cretinism in fetuses with thyroid agenesis (Forhead, 2014; Vulsma, 1989).

1	The fetus with congenital hypothyroidism typically does not develop stigmata of cretinism until after birth (Abduljabbar, 2012). Because administration of thyroid hormone will prevent this, by state law, all newborns are tested for high serum levels ofTSH (Chap. 32, p. 614). Immediately after birth, thyroid function and metabolism undergo major change. Cooling to room temperature evokes sudden and marked increase in TSH secretion. his in turn causes a progressive increase in serum T 4 levels that are maximal 24 to 36 hours after birth. here are nearly simultaneous eleva tions of serum T levels.

1	These glands develop from two separate tissues. The medulla derives from neural crest ectoderm, whereas the fetal and adult cortex arise from intermediate mesoderm. The gland grows rapidly through cell proliferation and angiogenesis, cellular migration, hypertrophy, and apoptosis (Ishimoto, 2011). Fetal glands are much larger in relation to total body size than in adults. he bulk is made up of the inner or fetal zone of the adrenal cortex and involutes rapidly after birth. his zone is scant to absent in rare instances in which the fetal pituitary gland is congenitally absent. he function of the fetal adrenal glands is discussed in detail in Chapter 5 (p. 104).

1	Infections in utero have provided an opportunity to examine mechanisms of the fetal immune response. Evidence of immunological competence has been reported as early as 13 weeks (Kohler, 1973; Stabile, 1988). In cord blood at or near term, the average level for most components is approximately half that of the adult values (Adinolfi, 1977). B cells diferentiate from pluripotent hemopoietic stem cells that migrate to the liver (Melchers, 2015; Muzzio, 2013). Despite this, in the absence of a direct antigenic stimulus such as infection, fetal plasma immunoglobulins consist almost totally of transferred maternal immunoglobulin G (IgG). hus, antibodies in the newborn are most often relective of maternal immunological experiences (American College of Obstetricians and Gynecologists, 2017). he interaction between maternal and fetal T cells is described in detail in Chapter 5 (p. 95).

1	Maternal IgG transport to the fetus begins at approximately 16 weeks and increases thereafter. he bulk of IgG is acquired during the last 4 weeks of pregnancy (Gitlin, 1971). Accordingly, preterm neonates are poorly endowed with protective maternal antibodies. Newborns begin to slowly produce IgG, and adult values are not attained until age 3 years. In certain situations, the transfer of IgG antibodies from mother to fetus can be harmful rather than protective to the fetus. The classic example is hemolytic disease of the fetus and newborn resulting from Rh-antigen alloimmunization (Chap. 15, p. 301). In the adult, production of immunoglobulin M (IgM) in response to an antigenic stimulus is superseded in a week or so predominantly by IgG production. In contrast, very little

1	In the adult, production of immunoglobulin M (IgM) in response to an antigenic stimulus is superseded in a week or so predominantly by IgG production. In contrast, very little IgM is produced by normal fetuses. With infection, the IgM response is dominant in the fetus and remains so for weeks to months in the newborn. And, because IgM is not trans ported from the mother, any IgM in the fetus or newborn is that which it produced. hus, specific IgM levels in umbilical cord blood may be useful in fetal infection diagnosis. Accord ing to the American College of Obstetricians and Gynecologists (2017), elevated levels of IgM are usually found in newborns with congenital infection such as rubella, cytomegalovirus infection, or toxoplasmosis. In infants, adult levels of IgM are normally attained by age 9 months. mucosal protection against enteric infections. This may explain the small amount of fetal secretory IgA found in amnionic luid (Quan, 1999).

1	mucosal protection against enteric infections. This may explain the small amount of fetal secretory IgA found in amnionic luid (Quan, 1999). he immune system develops early, and B lymphocytes appear in fetal liver by 9 weeks and in blood and spleen by 12 weeks. T lymphocytes begin to leave the thymus at approximately 14 weeks. Despite this, the newborn responds poorly to immunization, and especially poorly to bacterial capsular polysaccharides. his immature response may stem from a deicient response of newborn B cells to polyclonal activators or from a lack of T cells that proliferate in response to specific stimuli (Hayward, 1983). In the newborn, monocytes are able to process and present antigen when tested with maternal antigen-speciic T cells. DNA methylation patterns are developmentally regulated during monocyte-macrophage diferentiation and contribute to the antiinlammatory phenotype in macro phages (Kim, 2012).

1	The origin of most muscles and bones is mesodermal. he skeleton arises from condensed mesenchyme-embryonic connective tissue-which eventually forms hyaline cartilage models of the bones. By the end of the embryonic period, ossification centers have developed, and bones harden by endochondral ossification. The limb buds appear by the fourth week. Most skeletal muscle derives from myogenic precursor cells in the somites. Because of the small amount of yolk in the human ovum, growth of the embryofetus is dependent on maternal nutrients during the irst 2 months. During the first few days after implantation, blastocyst nutrition comes from the interstitial fluid of the endometrium and the surrounding maternal tissue.

1	Maternal adaptations to store and transfer nutrients to the fetus are discussed in Chapter 4 and summarized here. Three major maternal storage depots are the liver, muscle, and adipose tissue. These maternal depots and the storage hormone insulin are intimately involved in the metabolism of the nutrients absorbed from the gut. Maternal insulin secretion is sustained by increased serum levels of glucose and amino acids. he net efect is maternal storage of glucose as glycogen primarily in liver and muscle, retention of some amino acids as protein, and storage of the excess as fat. Storage of maternal fat peaks in the second trimester and then declines as fetal energy demands rise in the third trimester (Pipe, 1979). Interestingly, the placenta appears to act as a nutrient sensor, altering transport based on the maternal supply and environmental stimuli (Fowden, 2006; Jansson, 2006b).

1	During times of fasting, glucose is released from glycogen, but maternal glycogen stores cannot provide an adequate amount of glucose to meet requirements for maternal energy and fetal growth. Augmentation is provided by cleavage of triacylglycerols, stored in adipose tissue, which result in free fatty acids and activation of lipolysis.

1	Although dependent on the mother for nutrition, the fetus also actively participates in providing its own nutrition. At midpregnancy, fetal glucose concentration is independent of maternallevels and may exceed them (Bozzetti, 1988). Glucose is the major nutrient for fetal growth and energy. Logically, mechanisms exist during pregnancy to minimize maternal glucose use so that the limited maternal supply is available to the fetus. Human placental lactogen (hPL), a hormone normally abundant in the mother bur not the fetus, has an insulin antagonist efect. It blocks the peripheral uptake and use of glucose, while promoting mobilization and use of free fatty acids by maternal tissues (Chap. 5, p. 100). his hormone is also diabetogenic as discussed in Chapter 57 (p. 1107).

1	he transfer of D-glucose across cell membranes is accomplished by a carrier-mediated, stereospecific, nonconcentrating process of facilitated difusion. There are 14 glucose transport proteins (GLUTs) encoded by the SLC2A gene family and characterized by tissue-speciic distribution (Leonce, 2006). GLUT-1 and GLUT -3 primarily facilitate glucose uptake by the placenta and are located in the plasma membrane of the syncytiotrophoblast microvilli (Acosta, 2015). DNA methylation regulates expression of placental GLUT genes, with epigenetic modiication across gestation (Novakovic, 2013). It increases as pregnancy advances and is induced by almost all growth factors (Frolova, 2011). GLUT-3 expression is up regulated with fetal growth restriction a anzen, 2013). Lactate is a product of glucose metabolism and transported across the placenta also by facilitated difusion. By way of cotransport with hydrogen ions, lactate is probably transported as lactic acid.

1	Lactate is a product of glucose metabolism and transported across the placenta also by facilitated difusion. By way of cotransport with hydrogen ions, lactate is probably transported as lactic acid. The precise biomolecular events in the pathophysiology of fetal macrosomia are not deined. Nonetheless, fetal hyperinsulinemia is clearly one driving force (Luo, 2012). As discussed in Chapter 44 (p. 845), insulin-like growth factor, fibroblast growth factor, and corticotropin-releasing hormone (CRH) and are important regulators of placental development and function (Gao, 2012; Giudice, 1995). Maternal obesity begets fetal macrosomia (Chap. 44, p. 857). In addition, it is hypothesized that maternal obesity afects fetal cardiomyocyte growth that may result in fetal cardiomyopathy or even congenital heart disease (Roberts, 2015).

1	This polypeptide hormone was originally identified as a product of adipocytes and a regulator of energy homeostasis by curbing appetite. It also contributes to angiogenesis, hemopoiesis, osteogenesis, pulmonary maturation, and neuroendocrine, immune, and reproductive functions (Brifa, 2015; Maymo, 2009). Leptin is produced by the mother, fetus, and placenta. It is expressed in syncytiotrophoblast and fetal vascular endothelial cells. Of placental production, 5 percent enters the fetal circulation, whereas 95 percent is transferred to the mother (Hauguel-de vlouzon, 2006). Leptin concentrations peak in amnionic fluid at midpregnancy (Scott-Finley, 2015).

1	Fetalileptin levels begin rising at approximately 34 weeks and are correlated with fetal weight. This hormone is involved in the development and maturation of the heart, brain, kidneys, and pancreas, and its levels are decreased with fetal growth restriction (Brifa, 2015). Abnormal levels have been associated with fetal growth disorders, gestational diabetes, and preeclampsia (Fasshauer, 2014). Postpartum, leptin levels decline in both the newborn and mother. Perinatal leptin is associated with the development of metabolic syndromes later in life (Brifa, 2015; Granado, 2012).

1	The newborn has a large proportion of fat, which averages 15 percent of body weight (Kimura, 1991). Thus, late in pregnancy, a substantial part of the substrate transferred to the human fetus is stored as fat. Although maternal obesity raises placental fatty acid uptake and fetal fat deposition, it does not appear to afect fetal organ growth (Dube, 2012). Neutral fat in the form of triglycerides does not cross the placenta, but glycerol does. Despite this, evidence supports that abnormal maternal concentrations of triglycerides-both low and high levels-are associated with major congenital anomalies (Nederlof, 2015).

1	There is preferential placental-fetal transfer of long-chain polyunsaturated fatty acids (Gil-Sanchez, 2012). Lipoprotein lipase is present on the maternal but not on the fetal side of the placenta. his arrangement favors hydrolysis of triacylglycerols in the maternal intervillous space yet preserves these neutral lipids in fetal blood. Fatty acids transferred to the fetus can be converted to triglycerides in the fetal liver.

1	The placental uptake and use ofLDL is an alternative mechanism for fetal assimilation of essential fatty acids and amino acids (Chap. 5, p. 103). LDL binds to specific receptors in the coated-pit regions of the syncytiotrophoblast microvilli. The large LDL particle, measuring about 250,000 Da, is taken up by a process of receptor-mediated endocytosis. The apoprotein and cholesterol esters of LD L are hydrolyzed by lysosomal enzymes in the syncytium to yield: (1) cholesterol for progesterone synthesis; (2) free amino acids, including essential amino acids; and (3) essential fatty acids, primarily linoleic acid.

1	he placenta concentrates many amino acids in the syncytiotrophoblast, which are then transferred to the fetal side by difusion. Based on data from cordocentesis blood samples, the amino acid concentration in umbilical cord plasma is greater than in maternal venous or arterial plasma (Morriss, 1994) . Transport system activity is influenced by gestational age and environmental factors. hese include heat stress, hypoxia, under-and overnutrition, and hormones such as glucocorticoids, growth hormone, and leptin (Brifa, 2015; Fowden, 2006). Trophoblastic mammalian target of rapamycin complex 1 (mTORC1) regulates placental amino acid transporters and modulates transfer across the placenta Qansson, 2012). In vivo studies suggest an upregulation of transport for certain amino acids and a greater delivery rate of these to the fetuses of women with gestational diabetes associated with fetal overgrowth Oansson, 2006a).

1	Placental transfer of larger proteins is limited, but there are exceptions. IgG crosses the placenta in large amounts via endocytosis and trophoblast Fc receptors. IgG transfer depends on maternal levels of total IgG, gestational age, placental integrity, IgG subclass, and antigenic potential (Palmeira, 2012). Conversely, the larger immunoglobulins-IgA and IgM-of maternal origin are efectively excluded from the fetus.

1	Calcium and phosphorus are actively transported from mother to fetus. Calcium is transferred for fetal skeletal mineralization (Olausson, 2012). A calcium-binding protein is produced in placenta. Parathyroid hormone-related protein (PTH-rP), as the name implies, acts as a surrogate PTH in many systems (Chap. 5, p. 102). PTH is not found in fetal plasma, but PTHrP is present, suggesting that PTH-rP is the fetal parathormone. The expression of PTH-rP in cytotrophoblasts is modulated by the extracellular concentration of Ca2+ (Hellman, 1992). It seems possible, therefore, that PTH-rP synthesized in decidua, placenta, and other fetal tissues is important in fetal Cai+ transfer and homeostasis.

1	Iodide transport is clearly attributable to a carrier-mediated, energy-requiring active process. And indeed, the placenta concentrates iodide. he concentrations of zinc in the fetal plasma also are greater than those in maternal plasma. Conversely, copper levels in fetal plasma are less than those in maternal plasma. his fact is of particular interest because important copperrequiring enzymes are necessary for fetal development. Placental Sequestration of Heavy Metals he heavy metal-binding protein metallothionein-1 is expressed in human syncytiotrophoblast. his protein binds and sequesters a host of heavy metals, including zinc, copper, lead, and cadmium. Despite this, fetal exposure is variable (Caserta, 2013). For example, lead enters the fetal environment at a level 90 percent of maternal concentrations. In contrast, placental transfer of cadmium is limited (Kopp, 2012). he most common source of environmental cadmium is cigarette smoke.

1	Metallothionein also binds and sequesters copper (Cu2+) in placental tissue. This accounts for the low levels of Cu2+ in cord blood (Iyengar, 2001). It is possible that cadmium provokes metallothionein synthesis in the amnion. his may cause Cu2+ sequestration, a pseudo-copper deficiency, and in turn, diminished tensile strength of the amnion. he concentration of vitamin A (retinol) is greater in fetal than in maternal plasma and is bound to retinol-binding protein and to prealbumin. Retinol-binding protein is transferred from the maternal compartment across the syncytiotrophoblast. The transport of vitamin C-ascorbic acid-from mother to fetus is accomplished by an energy-dependent, carrier-mediated process. Levels of principal vitamin D metabolites, including 1,25-dihydroxycholecalciferol, are greater in maternal plasma than in fetal plasma. The 1 3-hydroxylation of 25-hydroxyvitamin D3 is known to take place in placenta and in decidua.

1	The placenta is the organ of transfer between mother and fetus. Within this maternal-fetal interface, oxygen and nutrients transfer from the mother to the fetus, whereas CO2 and meta bolic wastes are directed from fetus to mother. Fetal blood, which is contained in the fetal capillaries of the chorionic villi, has no direct contact with maternal blood, which remains in the intervillous space. Instead, bidirectional transfer depends on processes that allow or aid the transport through the syncy tiotrophoblast that lines chorionic villi.

1	Over the past few years, it has become apparent that breaks in the chorionic villi permit escape of fetal cells and other bloodborne material into the maternal circulation. This leakage is the mechanism by which some D-negative women become sensitized by the erythrocytes of their D-positive fetus (Chap. 15, p. 301). In fact, after 10 weeks, 10 to 15 percent of cell-free DNA (cDNA) in maternal plasma is placental in origin, that is, trophoblastic DNA (Norton, 2012). he escape of fetal cells can also lead to fetal microchimerism from entrance of allogeneic fetal cells, including trophoblast, into maternal blood and other organs (Rijnik, 2015). Volumes are estimated to range from 1 to 6 cells/ mL at midpregnancy. Some fetal cells become "immortal" in that they persist in the maternal circulation and organs following pregnancy. As discussed in Chapter 59 (p. 1139), the clinical corollary is that some maternal autoimmune diseases may be provoked by such microchimerism.

1	• The Intervillous Space vIaternal blood within the intervillous space is the primary source of maternal-fetal transfer. Blood from the maternal spiral arteries directly bathes the trophoblast layer that surrounds the villi. Substances transferred from mother to fetus irst enter the intervillous space and are then transported to the syncytiotrophoblast. As such, the chorionic villi and intervillous space function together as the fetal lung, gastrointestinal tract, and kidney. Circulation within the intervillous space is described in Chapter 5 (p. 94). Intervillous and uteroplacental blood flow increases throughout the irst trimester of normal pregnancies (Merce, 2009). At term, the residual volume of the intervillous space approximates 140 mL. Moreover, utero placental blood flow near term is estimated to be 700 to 900 mLl min, and most of this blood apparently goes to the intervillous space (Pates, 2010).

1	Active labor contractions reduce blood low into the intervillous space to a degree that depends on contraction intensity. Blood pressure within the intervillous space is significantly less than uterine arterial pressure, but somewhat greater than venous pressure. he latter, in turn, varies depending on several factors, including maternal position (Nelson, 2015). When supine, for example, pressure in the lower part of the inferior vena cava is elevated, and consequently, pressure in the uterine and ovarian veins, and in turn in the intervillous space, is increased.

1	Substances that pass from maternal to fetal blood must irst traverse the syncytiotrophoblast, the attenuated cytotrophoblast layer, the villous stroma, and inally, the fetal capillary wall. Although this histological barrier separates maternal and fetal circulations, it is not a simple physical barrier. First, throughout pregnancy, syncytiotrophoblast actively or passively permits, facilitates, and adjusts the amount and rate of substance transfer to the fetus. The maternal-facing syncytiotrophoblast surface is characterized by a complex microvillous structure. he fetal-facing basal cell membrane is the site of transfer to the intravillous space. Finally, the villous capillaries are an additional site for transport from the intravillous space into fetal blood, or vice versa. In determining the efectiveness of the human placenta as an organ of transfer, several variables are important and shown in Table

1	Zhao and coworkers (2014) have provided a review of the pharmacology of these interactions. Mechanisms of Transfer Most substances with a molecular mass <500 Da pass readily through placental tissue by simple difusion. These include TABLE 7-1. Variables of Maternal-Fetal Substance binding of the substance Maternal blood flow rate through the intervillous space Trophoblast surface area size available for exchange Physical trophoblast properties to permit simple difusion Trophoblast biochemical machinery for active transport Substance metabolism by the placenta during transfer Fetal intervillous capillary surface area size for exchange Fetal blood concentration of the substance

1	Specific binding or carrier proteins in the fetal or maternal ci rcu lation oxygen, CO2, water, most electrolytes, and anesthetic gases (Carter, 2009). Some low-molecular-weight compounds undergo transfer facilitated by syncytiotrophoblast. These are usually those that have low concentrations in maternal plasma but are essential for normal fetal development. Insulin, steroid hormones, and thyroid hormones cross the placenta, but very slowly. he hormones synthesized in situ in the syncytiotrophoblast enter both the maternal and fetal circulations, but not equally (Chap. 5, p. 98). Examples are hCG and hPL concentrations, which are much lower in fetal plasma than in maternal plasma. High-molecularweight substances usually do not traverse the placenta, but there are important exceptions. One is immunoglobulin G-molecular weight 160,000 Da-which is transferred by way of a speciic trophoblast receptor-mediated mechanism (Stach, 2014). Transfer of Oxygen and Carbon Dioxide

1	Transfer of Oxygen and Carbon Dioxide Placental oxygen transfer is blood flow limited. Using estimated uteroplacental blood low, Longo (1991) calculated oxygen delivery to be approximately 8 mL 02/min/kg of fetal weight. Normal values for oxygen and CO2 are presented in Figure 7-13. Because of the continuous passage of oxygen from maternal blood in the intervillous space to the fetus, its oxygen saturation resembles that in maternal capillaries. The average oxygen saturation of intervillous blood is estimated to be 65 to 75 percent, with a partial pressure (P02) of 30 to 35 mm Hg. ;: 50 .N 30 . 2010 n = 58 1 mm Hg approx. = 0.•133 kPa

1	;: 50 .N 30 . 2010 n = 58 1 mm Hg approx. = 0.•133 kPa FIGURE 7-13 Umbilical venous cordocentesis samples obtained in fetuses being evaluated for possible intrauterine infections or hemolysis, but who were found to be healthy. A. Oxygen pressure (PoJ B. Carbon dioxide pressure (Pco2). Shaded areas represent 5th to 95th percentiles. (Modified from Ramsey, MM: Normal Values in Pregnancy. Ramsay MM, James OK, Steer PJ, et al (eds). London, Elsevier, 1996, p 106.) The oxygen saturation of umbilical vein blood is similar but has a somewhat lower oxygen partial pressure. Fetal hemoglobin has a higher oxygen ainity than adult hemoglobin. This is illustrated by the oxyhemoglobin disassociation curve, which is described in Chapter 47 (p. 920).

1	he placenta is highly permeable to CO2, which traverses the chorionic villus by diusion more rapidly than oxygen. Near term, the partial pressure of carbon dioxide (PC02) in the umbilical arteries averages 50 mm Hg, which is approximately 5 mm Hg higher than in the maternal intervillous blood. Fetal blood has less ainity for CO2 than does maternal blood, thereby favoring CO2 transfer from fetus to mother. Also, mild maternal hyperventilation results in a fall in Pc02ilevels, favoring a transfer of CO2 from the fetal compartment to maternal blood.

1	Although simple difusion is an important method of placental transfer, the trophoblast and chorionic villus unit demonstrate enormous selectivity in transfer. his results in diferent metabolite concentrations on the two sides of the villus. Importantly, the levels of many substances that are not synthesized by the fetus are several times higher in fetal than in maternal blood. Ascorbic acid is one example. his relatively low-molecularweight substance might be expected to traverse the placenta by simple difusion. he concentration of ascorbic acid, however, is two to four times higher in fetal plasma than in maternal plasma (Morriss, 1994). Another example is the unidirectional transfer of iron. Typically, maternal plasma iron concentration is much lower than that in her fetus. Even with severe maternal iron-deficiency anemia, the fetal hemoglobin mass is normal. Abduljabbar MS, ii M: Congenital hypothyroidism. ] Pediatr Endocrinol Metab 25(102)13, 2012

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1	SOCIAL HISTORY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 SCREENING TESTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 153 Pregnancy may be associated with certain diseases that existed bore the inception of pregnancy. As a rule, all diseases which subject the organism to a considerable strain are much more serious when occurring in a pregnant woman.

1	-J. Whitridge Williams (1903) he Centers for Disease Control and Prevention (CDC) (2015) defines preconceptional care as "a set of interventions that aim to identiy and modiy biomedical, behavioral, and social risks to a woman's health or pregnancy outcome through prevention and management." To achieve this goal, the CDC has developed an action plan for preconceptional health care in the United States Qohnson, 2006). The American College of Obstetricians and Gynecologists (2017 e) and the Society for Maternal-Fetali1vIedicine (2014) also reairm the importance of preconceptional care, and the following objectives have been established for advancing it: 1. Improve knowledge, attitudes, and behaviors of men and women related to preconceptional health 2. Assure that all childbearing-aged women receive preconceptional care services-including evidence-based risk screening, health promotion, and interventions-that will enable them to enter pregnancy in optimal health 3.

1	Reduce risks indicated by a previous adverse pregnancy outcome through interconceptional interventions to prevent or minimize recurrent adverse outcomes 4. Reduce the disparities in adverse pregnancy outcomes

1	Reduce the disparities in adverse pregnancy outcomes To illustrate potentilly modifiable conditions, data that describe the health status of women who delivered liveborn neonates in the United States in 2004 are reviewed. Table 8-1 demonstrates the high prevalence of many conditions that may be amenable to intervention during the preconceptional and interpregnancy periods. To be successul, however, strategies that mitigate these potential pregnancy risks must be provided before conception. By the time most women realize they are pregnantusually 1 to 2 weeks ater the first missed period-the embryo has already begun to form. hus, many preventive steps-for example, folic acid to avoid neural-tube defects-will be inefective if initiated at this time. Importantly, up to half of all pregnancies in the United States in 2008 were unplanned according to the Guttmacher Institute (2015), and oten these are at greatest risk.

1	Few randomized trials evaluate preconceptional counseling eicacy, in part because withholding such counseling would be unethical. Also, pregnancy outcomes are dependent on the interaction of various maternal, fetal, and environmental factors. hus, ascribing a salutary outcome to a specific intervention is diicult (Moos, 2004; Temel, 2014). However, prospective observational and case-control studies have demonstrated the successes of preconceptional counseling (American College of Obstetrics and Gynecologists, 2016b). \100s and coworkers (1996) assessed the efectiveness of a preconceptional counseling program administered during routine health care provision to reduce unintended pregnancies. he 456 counseled women had a 50-percent greater likelihood of subsequent pregnancies that they considered "intended" compared with 309 uncounseled

1	TABLE 8-1. Prevalence of Prepregnancy Maternal Behaviors, Experiences, Health Conditions, and Previous Poor Birth Outcomesa aln the United States in 2004. bAmong women who were not trying to become pregnant. Data from D'Angelo D, Williams L, Morrow 8, et al: Precon ception and interconception health status of women who recently gave birth to a live-born infant-Pregnancy Risk Assessment Monitoring System (PRAMS), United States, 26 reporting areas, 2004. MMWR 56(1l0):1,l2007. women. Moreover, compared with another group of women who had no health care before pregnancy, the counseled group had a 65-percent higher rate of intended pregnancy. Interesting ethical aspects of paternal lifestyle modification were reviewed by van der Zee and associates (2013).

1	Gynecologists, internists, family practitioners, and pediatricians have the best opportunity to provide preventive counseling during periodic health maintenance examinations. The occasion of a negative pregnancy test is also an excellent time for education. Jack and colleagues (1995) administered a comprehensive preconceptional risk survey to 136 such women, and almost 95 percent reported at least one problem that could afect a uture pregnancy. These included medical or reproductive problems-52 percent; family history of genetic disease-50 percent; increased risk of human immunodeiciency virus infection-30 percent; increased risk of hepatitis B and illegal substance abuse-25 percent; alcohol use-17 percent; and nutritional risks-54 percent. Counselors should be knowledgeable regarding relevant medical diseases, prior surgery, reproductive disorders, or genetic conditions and must be able to interpret data and recommendations provided by other specialists (Simpson, 2014). If the

1	relevant medical diseases, prior surgery, reproductive disorders, or genetic conditions and must be able to interpret data and recommendations provided by other specialists (Simpson, 2014). If the practitioner is uncomfortable providing guidance, the woman or couple should be referred to an appropriate counselor.

1	Women presenting specifically for preconceptional evaluation should be advised that information collection may be time consuming, depending on the number and complexity of factors that require assessment. he intake evaluation includes a thorough review of the medical, obstetrical, social, and family histories. Useul information is more likely to be obtained by asking specific questions regarding each of these histories and each family member than by asking general, open-ended questions. Some important information can be obtained by questionnaires that address these topics. Answers are reviewed with the couple to ensure appropriate follow-up, including obtaining relevant medical records.

1	With specific medical conditions, general points include how pregnancy will afect maternl health and how a high-risk condition might afect the fetus. terward, advice for improving outcome is provided. Some chronic conditions that may ffect pregnancy outcomes include treated or active cancer, prior peripartum cardiomyopathy, and systemic lupus erythematosus (mant, 2015; Buyon, 2015; McNamara, 2015). Importantly, psychological health should be considered (Lassi, 2014). Detailed preconceptional information regarding a few exemplary conditions is found in the next sections and in the other topic-speciic chapters of this text.

1	Because maternal and fetal pathology associated with hyperglycemia is well known, diabetes is the protoype of a condition for which preconceptional counseling is beneficial. Diabetesassociated risks to both mother and fetus are discussed in detail in Chapter 57 (p. 1099). Many of these complications can be avoided if glucose control is optimized before conception. Another important aspect of counseling pertains to the frequent use of teratogenic angiotensin-converting enzyme inhibitors in this population (Podymow, 2015).

1	The American College of Obstetricians and Gynecologists (2016a) has concluded that preconceptional counseling for women with pregestational diabetes is both beneicial and costefective and should be encouraged. The American Diabetes Association has promulgated consensus recommendations for preconceptional care for diabetic women (Kitzmiller, 2008). hese guidelines advise obtaining a thorough inventory of disease duration and related complications and completing a clinical and laboratory examination for end-organ damage. Perhaps most essential, they encourage a preconceptional goal of the lowest hemoglobin Ale level possible without undue hypoglycemic risk to the mother. In addition to assessing diabetic control during the preceding 6 weeks, hemoglobin Ale measurement can also be used to estimate risks for major anomalies as shown in Figure lthough these data are from women with severe overt diabetes, the incidence of fetal anomalies in women who have gestational diabetes with fasting

1	risks for major anomalies as shown in Figure lthough these data are from women with severe overt diabetes, the incidence of fetal anomalies in women who have gestational diabetes with fasting hyperglycemia is increased fourfold compared with that in normal women (Sheield, 2002).

1	Such counseling in diabetic women has been shown to be efective. Leguizam6n and associates (2007) identiied 12 studies that included more than 3200 pregnancies in women with insulin-dependent diabetes. Of the 1618 women without precon o..Ac) 'uE .. 4.6-7.6 7.7-8.6 8.7-9.9 10-10.5 >10.6

1	FIGURE 8-1 Relationship between first-trimester glycosylated hemoglobin values and risk for major congenital malformations in 320 women with insulin-dependent diabetes. (Data from Kitzmiller JL, Gavin LA, Gin GO, et al: Preconception care of diabetics. JAMA 265:731, 1991.) ceptional counseling, 8.3 percent had a fetus with a major congenital anomaly, and this compared with a rate of 2.7 percent in the 1599 women who did have counseling. Tripathi and coworkers (2010) compared outcomes in 588 women with pregestational diabetes in whom approximately half had preconceptional counseling. hose women who received counseling had improved glycemic control before pregnancy and in the first trimester. his group also had higher folate intake rates preconceptionally, and they experienced lower rates of adverse outcomes-deined as a perinatal death or major congenital anomaly. hese cited beneits are accompanied by reduced helth-care costs in diabetic women. From their review, Reece and Homko (2007)

1	adverse outcomes-deined as a perinatal death or major congenital anomaly. hese cited beneits are accompanied by reduced helth-care costs in diabetic women. From their review, Reece and Homko (2007) found that each $1 expended for a preconceptional care program saved between $1.86 and $5.19 in averted medical costs. Despite such beneits, the proportion of diabetic women receiving preconceptional care is suboptimal. In their study of approximately 300 diabetic women in a managed-care plan, Kim and colleagues (2005) found that only approximately one half had preconceptional counseling. Counseling rates are undoubtedly much lower among uninsured and indigent women.

1	Compared with unafected women, those with a seizure disorder carry an undisputed augmented risk of having neonates with structural anomalies (Chap. 12, p. 240). Some early reports indicated that epilepsy conferred an elevated a priori risk for congenital malformations that was independent of anticonvulsant treatment efects. Although more recent publications have largely failed to conirm this increased risk in untreated women, it is diicult to refute entirely because women who are controlled without medication generally have less severe disease (Cassina, 2013; Vajda, 2015). Fried and associates (2004) conducted a metaanalysis of studies comparing epileptic women, both treated and untreated, with controls. In this study, greater malformation rates could only be demonstrated in the ofspring of women who had been exposed to anticonvulsant therapy. Veiby and coworkers (2009) used the Medical Birth Registry of Norway and identiied an increased malformation risk only in women who were

1	of women who had been exposed to anticonvulsant therapy. Veiby and coworkers (2009) used the Medical Birth Registry of Norway and identiied an increased malformation risk only in women who were exposed to valproic acid (5.6 percent) or poly therapy (6.1 percent). Untreated women had anomaly rates that were similar to those of nonepileptic controls. Risks for miscarriage and stillbirths in exposed epileptic women do not appear elevated (Aghajanian, 2015; Bech, 2014).

1	Ideally, seizure control is optimized preconceptionlly. For example, Vajda and colleagues (2008) nalyzed data from the Australian Register of Antiepileptic Drugs in Pregnancy. hey found the seizure risk during pregnancy was 50-to 70-percent lower in women without a seizure in the year preceding pregnancy compared with a group experiencing seizures in this preceding year. No urther advantages accrued if the seizure-free period exceeded a year.

1	Treatment goals attempt to achieve seizure control with monotherapy and with medications considered less teratogenic (Aguglia, 2009; Tomson, 2009). As discussed in detail in Chapter 60 (p. 1159) and shown in Table 8-2, some one-drug regimens are more teratogenic than others. Valproic acid, in particular, is avoided if possible, as this medication has consistently been associated with a greater risk for major congenital malformations than other antiepileptic drugs Qentink, 2010; Vajda, 2015). Trimethadione is contraindicated (Aghajanian, 2015). he American Academy of Neurology recommends consideration of antiseizure medication discontinuation before pregnancy in suitable candidates Qeha, 2005). hese include women who satisy the following criteria: (1) have been seizure-free for 2 to 5 years, (2) display a single seizure type, (3) have a normal neurological examination and normal intelligence, and (4) showeelectroencephalogram results that have normalized with treatment.

1	Epileptic women should be advised to daily take a 4-mg folic acid supplement. Even so, it is not entirely clear that folate supplementation reduces the fetal malformation risk in pregnant women taking anticonvulsant therapy. In one case-control study, Kjer and associates (2008) reported that the congenital abnormality risk was reduced by maternal folate supplementation in fetuses exposed to carbamazepine, phenobarbitl, phenytoin, and primidone. Conversely, from the United Kingdom Epilepsy and TABLE 8-2. First-Trimester .ntiepileptic Monotherapy and the Associated Major Malformation Risk Unexposed controls (442) 1.1 Reference Lamotrigine (1 562) 2.0 1.8 (0.7-4.6) Carbamazepine (1 033) 3.0 2.7 (1 .0-7.0) Phenytoin (4 16) 2.9 2.6 (0.9-7.4) Levetiracetam (450) 2.4 2.2 (0.8-6.4) Topiramate (359) 4.2 3.8 (1 .4-10.6) Valproate (323) 9.3 9.0 (3.4-23.3) Phenobarbital (1 99) 5.5 5.1 (1 .8-1 4.9) Oxcarbazepine (1 82) 2.2 2.0 (0.5-7.4) Gabapentin (1 45) 0.6 (0.07-5.2)

1	Topiramate (359) 4.2 3.8 (1 .4-10.6) Valproate (323) 9.3 9.0 (3.4-23.3) Phenobarbital (1 99) 5.5 5.1 (1 .8-1 4.9) Oxcarbazepine (1 82) 2.2 2.0 (0.5-7.4) Gabapentin (1 45) 0.6 (0.07-5.2) Clonazepam (64) 3.1 2.8 (0.5-14.8) aRisk compared with that of the unexposed reference population of nonepileptic women. n =number of exposed infants. Data from Hernandez-Oraz 5, Smith CR, Shen A., et al: Comparative safety of antiepileptic drugs during pregnancy. Neurology 78:1692, 2012. Pregnancy Register, Morrow and coworkers (2009) compared fetal outcomes of women who received preconceptional folic acid with those who did not receive it until later in pregnancy or not at all. In this study, a paradoxical increse in the number of major congenital malformations was observed in the group who received preconceptional folate. hese investigators concluded that folate metabolism may be only a part of the mechanism by which malformations are induced in women taking these medications.

1	Preconceptional counseling includes assessment of immunity against common pathogens. Also, depending on health status, travel plans, and time of year, other immunizations may be indicated as discussed in Chapter 9 (Table 9-7, p. 172). Vac cines that contain toxoids such as tetanus are suitable before or during gestation. Also, those containing killed bacteria or viruses-such as inluenza, pneumococcus, hepatitis B, menin gococcus, and rabies vaccines-are not associated with adverse fetal outcomes and are not contraindicated preconceptionally or during pregnancy. Conversely, live-virus vaccines are not recommended during pregnancy. Examples are vaccines against varicella-zoster, measles, mumps, rubella, polio, chickenpox, and yellow fever. Moreover, 1 month or longer should ideally pass between vaccination and conception attempts. That said, inadvertent administration of measles, mumps, rubella (MMR) considered indications for pregnancy termination. Most reports indicate that the fetal

1	vaccination and conception attempts. That said, inadvertent administration of measles, mumps, rubella (MMR) considered indications for pregnancy termination. Most reports indicate that the fetal risk is only theoretical. Immunization to smallpox, anthrax, and other bioterrorism diseases should be discussed if clinically appropriate (Chap. 64, p. 1228).

1	With some infections, vaccines are unavailable. One recent example is the Zika virus (Brasil, 2016). For this virus, the CDC has issued travel advisories for pregnant women (Petersen, 2016; Schuler-Faccini, 2016). he CDC (20e16) estimates that 3 percent of neonates born each year in the United States will have at least one birth defect. Importantly, such defects are the leading cause of infant mortality and account for 20 percent of deaths. The benefits of preconceptional counseling usually are measured by comparing the incidence of new cases before and after initiation of a counseling program. Congenital conditions that clearly beneit from patient education include neural-tube defects, phenylketonuria, thalassemias, and other genetic diseases more common in individuals of Eastern European Jewish descent.

1	Pedigree construction using the symbols shown in Figure 8-2 is the most thorough method for obtaining a family history as a part of genetic screening. he health and reproductive status of each "blood relative" should be individually reviewed for medical illnesses, mental retardation, birth defects, infertility, and pregnancy loss. Certain racial, ethnic, or religious backgrounds may indicate elevated risk for specific recessive disorders. Although most women can provide some information regarding their history, their understanding may be limited. For example, several studies have shown that pregnant women often fail to report a birth defect in the family or they report it incorrectly. hus, any disclosed defect or genetic disease should be conirmed by reviewing pertinent medical records or by contacting afected relatives for additional information.

1	he incidence of neural-tube defects (NTDs) is 0.9 per 1000 live births, and they are second only to cardiac anomalies as the most frequent structural fetal malformation (Chap. 13, p. 270). Some NTDs, as well as congenital heart defects, are associated with speciic mutations. One example is the 677C -+=T substitution in the gene that encodes methylene tetrahydrofolate reductase. For this and similar gene defects, the trial conducted by the Medical Research Council Vitamin Study Research Group (1991) showed that preconceptional folic acid therapy significantly reduced the risk for a recurrent NTD by 72 percent. More importantly, because more than 90 percent of neonates with NTDs are born to women at low risk, Czeizel and Dudas (1e992) showed that supplementation reduced the a priori risk of a irst NTD occurrence. It is currently recommended, therefore, that all women who may become pregnant take daily 400 to 800 j1g of folic acid orally before conception and through the irst trimester

1	a irst NTD occurrence. It is currently recommended, therefore, that all women who may become pregnant take daily 400 to 800 j1g of folic acid orally before conception and through the irst trimester (U.S Preventive Services Task Force, 2009). Folate fortiication of cereal grains has been mandatory in the United States since 1998, and this practice has also resulted in decreased neural-tube defect rates (Williams, 2015). Despite the demonstrated benefits of folate supplementation, only half of women have taken folic acid supplementation periconceptionally (de Jong-van den Berg, 2005; Goldberg, 2006). The strongest predictor of use appears to be consultation with a health-care provider before conception.

1	More than 600 mutations have been identified in the phenylalanine hydroxylase gene. The inherited defect in phenylalanine metabolism exempliies diseases in which the fetus may not be at risk to inherit the disorder but may be damaged by maternal disease. Specifically, mothers with phenylketonuria (PKU) who eat an unrestricted diet have abnormally high blood phenylalanine levels. his amino acid readily crosses the placenta and can damage developing fetal organs, especially neural and cardiac tissues (Table 8-3). TABLE 8-3. Frequency of Complications in the Offspring of Women with Untreated Phenylketonuria Data from American Academy of Pediatrics: Matenal phenylketonuria, Pediatrics 2008 \ug;122(2):445-449. No offspring II Proband is II -2 Adopted out of a family FIGURE 8-2 Symbols used for pedigree construction. (Modified with permission from Thompson MW, Mcinnes RR, Huntington FW (eds): Number of children of sex indicated Carrier of X-linked recessive trait

1	Number of children of sex indicated Carrier of X-linked recessive trait Twins of unknown zygosity Genetics in Medicine, 5th ed. Philadelphia, Saunders, 1991o.) With appropriate preconceptional counseling and adherence to a phenylalanine-restricted diet before pregnancy, the incidence of fetal malformations is dramatically reduced (Camp, 2014; Vockley, 2014). Therefore, the phenylalanine concentration is ideally normalized 3 months before conception and then maintained throughout pregnancy (American College of Obstetricians and Gynecologists, 2017b). The target phenylalanine blood concentration is 120 to 360 �mol/L (Camp, 2014).

1	hese disorders of globin-chain synthesis are the most common single-gene disorders worldwide (Forget, 2013; Vichinsky, 2013). As many as 200 million people carry a gene for one of these hemoglobinopathies, and hundreds of mutations are known to cause thalassemia syndromes (Chap. 56, p. 1084). In endemic areas such as Mediterranean and Southeast Asian countries, counseling and other prevention strategies have reduced the incidence of new cases by up to 80 percent (Cao, 2013). The American College of Obstetricians and Gynecologists (2015a) recommends that individuals of high-risk ancestry be ofered carrier screening to allow them informed decision making regarding reproduction and prenatal diagnosis. One method of early prenatal diagnosis is preimplantation genetic diagnosis (PGD), which is coupled with assisted reproductive technologies. Described in Chapter 14 (p. 295), PGD is available for patients at risk for certain thalassemia syndromes (Kuliev, 2011).

1	• Individuals of Eastern European Jewish Descent Most individuals of Jewish ancestry in North America are descended from Ashkenazi Jewish communities and are at increased risk for having ofspring with one of several autosomal recessive disorders. These include Tay-Sachs disease, Gaucher disease, cystic ibrosis, Canavan disease, familial dysautonomia, mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia group C, and Bloom syndrome. The American College ofObstetricians and Gynecologists (20 16c, 2017a) recommends preconceptional counseling and screening for these in this population. Carrier frequency and features of these conditions are discussed in Chapter 14 (p. 290).

1	During preconceptional screening, information is sought regarding infertility; abnormal pregnancy outcomes that may include miscarriage, ectopic pregnancy, and recurrent pregnancy loss; and obstetrical complications such as cesarean delivery, preeclampsia, placental abruption, and preterm delivery (Stubblefield, 2008). As discussed in Chapter 35 (p. 646), details involving a prior stillbirth are especially important. For example, Korteweg and associates (2008) identiied chromosomal abnormalities in 13 percent ofstillborns who underwent karyotyping. Reddy and colleagues (2012) confirmed that chromosomal microarray analysis (CMA) yielded better detection of genetic abnormalities than did standard karyotyping, primarily because nonviable tissue can be used for the analysis. CMA is described and illustrated in Chapter 13 (p. 271). Identiication of a genetic abnormality in a stillborn can help determine the recurrence risk and aid in the preconceptional or prenatal management in subsequent

1	illustrated in Chapter 13 (p. 271). Identiication of a genetic abnormality in a stillborn can help determine the recurrence risk and aid in the preconceptional or prenatal management in subsequent pregnancies.

1	Women at both ends of the reproductive-age spectrum have unique outcomes to be considered. First, according to the CDC, in 2010,i3.4 percent ofbirths in the United Stateswerein women between the ages of 15 and 19 years (Martin, 2012). These adolescents are at increased risk for anemia, preterm delivery, and preeclampsia compared with women aged 20 to 35 years (Usta, 2008). he incidence of sexually transmitted diseases-common in adolescents-is even higher during pregnancy (Niccolai, 2003). Unfortunately, because most of their pregnancies are unplanned, adolescents rarely seek preconceptional counseling.

1	Conceptions after age 35 currently comprise approximately 15 percent ofpregnancies in the United Stares (Martin, 2012). By contrast, these older women are more likely to request preconceptional counseling, either because of postponed pregnancy with a desire to optimize outcomes or because of plans to undergo infertility treatment. Some studies-including data from Parland Hospital presented in Figure 8-3 indicate that ater age 35, the risks for obstetrical complications and for perinatal morbidity and mortality rise (Cunningham, 1995; Waldenstrom, 2015). he older woman who has a chronic illness or who is in poor physical condition usually has readily apparent risks. For rhe physicallyfit woman without medical problems, however, the risks are much lower than previously reported.

1	Overall, the maternal mortality rate is higher in women aged 35 and older. Compared wirh women in rheir 20s, women aged 35 to 39 are 2.5 times more likely and women aged 40 or older are 5.3 times more likely to sufer pregnancy-related mortality (Geller, 2006). Creanga and coworkers (2015) analyzed pregnancy-related deaths in the United States for 2006 through 2010. Although women older than 35 years contributed less than 15 percent of all live births, they constituted 27 percent ofmaternal deaths. For the fetus, maternal age-related risks primarily stem from: (1) indicated preterm delivery for maternal complications such as hypertension and diabetes, (2) spontaneous preterm birth, (3) fetal growth disorders related to chronic maternal disease or multifetal gestation, (4) fetal aneuploidy, and (5) pregnancies resulting from assisted reproductive technology.

1	Recall that older women have subfertility problems. And although the incidence of dizygotic twinning increases with maternal age, the more important cause of multifetal gestation in older women follows the use ofassisted reproductive technology (ART) and ovulation induction. Indeed, according to the CDC, 30 to 40 percent ofall multifetal gestations in the United States in 2012 were conceived with the use ofART (Sunderan, 2015). Morbidity and mortality with multifetal pregnancies stem from preterm delivery. Other obstetrical morbidities, such as placenta previa, abruption, and preeclampsia, are also risks associated with these conceptions (Lukes, 2017; Qin, 2016). Finally, experience has accrued that links ART to higher major congenital malformation rates. Davies and colleagues (2012) reported that of 308,974 births in South Australia, 8.3 percent ofneonates conceived byART had major birth defects.

1	In this analysis, after adjustment for maternal age and other risk factors, intracytoplasmic injection continued to be associated with a signiicantly elevated risk for malformations, but in vitro fertilization did not. Parental history and experiences-paternal and maternal-can exert efects through epigenomic information not contained in the DNA sequence. Examples include variations in sperm and oocyte cytosine methylation and other mechanisms (Cedars, 2015; Lane, 2014). Perhaps one example is the possible link between increasing paternal age and complex neuropsychiatric conditions (Malaspina, 2015). Finally, the incidence of genetic diseases in ofspring caused by new autosomal-dominant mutations in older men is increased. Still, the incidence is low (Chap. 13, p. 265). Accordingly, targeted sonographic examination performed solely for advanced maternal or paternal age is controversial.

1	Fetal risks associated with alcohol, marijuana, cocaine, amphetamines, and heroin are discussed in Chapter 12 (p. 239). he first step in preventing drug-related fetal risk is an honest assessment of use by the patient (American College of Obstetricians and Gynecologists, 2017 c) . Toward this end, questioning should be nonjudgmental. Screening for at-risk drinking can be accomplished using several validated tools. One is the wellstudied T ACE questions (American College of Obstetricians and Gynecologists, 2013). This is a series of four questions concerning .olerance to alcohol, being 4nnoyed by comments about their drinking, attempts to fut down, and a history of drinking early in the morning-the fye opener.

1	In a Canadian study of more than 1000 postpartum patients, Tough and coworkers (2006) found that a high percentage of women reported alcohol use concurrent with conception attempts. Specifically, nearly half of those planning for pregnancy reported a mean of 2.2 drinks daily during early gestation and before they recognized their pregnancy. Of note, Bailey and associates (2008) found that rates of binge drinking and marijuana use by men were unafected by their partner's pregnancy. The frequency and pattern of such behaviors clearly underscore the opportunity for preconceptional counseling.

1	Currently 20 million women in the United States smoke cigarettes (Centers for Disease Control and Prevention, 2014). Smoking in pregnancy has been consistently associated with numerous adverse perinatal outcomes, listed in Chapter 12 (p. 249). These risks are largely mitigated by cessation before pregnancy, highlighting the importance of screening for tobacco use in the preconceptional period and during prenatal care as outlined in Chapter 9 (p. 161). Contact with environmental substances is inescapable. Thus, it is fortunate that only a few agents have been shown to cause adverse pregnancy outcomes (Windham, 2008). Exposures to infectious diseases have myriad deleterious efects, and these are detailed in Chapters 64 and 65. Likewise, contact with some chemicals may impart signiicant maternal and fetal risks. As discussed in Chapters 9 and 12 (pp. 170 and 244), excess exposure to methyl mercury or lead is associated with neurodevelopmental disorders.

1	In the past, some concerns were raised over common everyday exposure to electromagnetic iels such as those emanated by high-voltage power lines, electric blankets, microwave ovens, and cellular phones. Fortunately, no human or animal evidence links these and adverse fetal outcomes (Robert, 1999). he efects of electrical shock are discussed in Chapter 47 (p. 930).

1	Pica is the craving for and consuming of ice, laundry starch, clay, dirt, or other nonfood items. It should be discouraged due to its inherent replacement of healthul food with nutritionally empty products (Chap. 9, p. 174). In some cases, it may represent an unusual physiological response to iron deficiency. Many vegetarian diets are protein deficient but can be corrected by increasing egg and cheese consumption. Anorexia and bulimia increase maternal risks of nutritional deiciencies, electrolte disturbances, cardiac arrhythmias, and gastrointestinal pathology (Becker, 1999). s discussed in Chapter 61 (p. 1180), pregnancy-related complications with these disorders include greater risks of low birthweight, smaller head circumference, microcephaly, and small-for-gestational-age newborns (Kouba, 2005).

1	In contrast to these perinatal morbidities, obesiy is linked with several maternal complications. As discussed in Chapter 48 (p. 939), these include preeclampsia, gestational diabetes, labor abnormalities, cesarean delivery, and operative complications (American College of Obstetricians and Gynecologists, 2015b). Obesity also appears to be associated with a range of structural fetal anomalies (Stothard, 2009).

1	Conditioned pregnant women usually can continue to exercise throughout gestation (American College of Obstetricians and Gynecologists, 2017d). As discussed in Chapter 9 (p. 170), no data suggest that exercise is harmful during pregnancy. One caveat is that as pregnancy progresses, balance problems and joint relaxation may predispose to orthopedic injury. A woman is advised not to exercise to exhaustion, and she should augment heat dissipation and fluid replacement. Further avoidances include prolonged supine position, activities requiring good balance, and extreme weather conditions.

1	Pregnancy can exacerbate interpersonal problems and is a time of elevated risk from an abusive partner. According to the American College of Obstetricians and Gynecologists (2012), approximately 324,000 pregnant women are abused each year. As discussed in Chapter 47 (p. 925), intimate partner violence has been associated with greater risk for several pregnancy-related complications, including hypertension, vaginal bleeding, hyperemesis, preterm delivery, and low-birthweight neonates (Silverman, 2006). Because domestic violence can escalate during pregnancy, even to the point of homicide, the preconceptional period provides an ideal time for screening and if indicated, intervention (Cheng, 2010). s detailed in Chapter 9 (p. 162), the American College of Obstetricians and Gynecologists (2012) provides recommendations and resources for screening both pregnant and nonpregnant women for domestic violence.

1	Certain laboratory tests may help assess the risk for and prevent some pregnancy complications. hese include basic tests that are usually performed during prenatal care and are enumerated in Chapter 9. More speciic tests may assist evaluation of women with certain chronic medical diseases. Examples of some chronic diseases that ideally would be assessed before conception are highlighted in Table 8-4. With several of these, optimizing maternal condition before conception will improve pregnancy outcomes. Cox and coworkers (1e992) reviewed pregnancy outcomes in 1075 high-risk women who received such evaluation. hey reported that the 240 women with hypertension, asthma, or renal, thyroid, or cardiac disease had better outcomes compared with the outcomes from their prior pregnancies. TABLE 8-4. Selected Preconceptional

1	TABLE 8-4. Selected Preconceptional Methyl mercury: Avoid shark, swordfish, king mackerel, and tile fish. Ingest no more than 12 ounces or 2 servings of canned tuna and no more than 6 ounces of albacore per week. Lead: Blood lead testing if a risk factor is identified; treat if indicated according to recommendations. Calculate BMI yearly from Figure 48-1, p. 937 BMI .25 kglm2: Counsel on diet. Test for diabetes and metabolic syndrome if indicated. Consider weight 105s prior to conception. BMI � 78.5 kglm2: Assess for eating disorder. Counsel on cardiac risks during pregnancy; discuss situations in which pregnancy is contraindicated. cardiac function. Discuss medication teratogenicity (warfarin, ACE inh'bitor, ARB) and, if possible, switch to less dangerous agent when conception planned. Ofer genetic counseling to those with congenital cardiac anomalies (Table 49-4, p. 953).

1	Counsel on specific risks during pregnancy. Assess those with long-standing HTN for ventricular hypertrophy, retinopathy, and rena! disease. Optimize blood pressure co trol. If medications indicated, select or switch to an agent appropriate for pregnancy. Counsel on asthma risks during pregnancy. Optimize pulmonary function preconceptionally. Treat women with pharmacologica l step therapy for chronic asthma. Question for personal or family history of thrombotic events or recurrent poor pregnancy outcomes. If a thrombopbilia is found or known, counsel and offer appropriate anticoagulation regimen. Counsel on specific risks during pregnancy. Optimize blood pressure control before conception. Counsel women taking ACE inhibitors and ARBs abolJt teratogenicity and the need to switch agents before pregnancy.

1	Inlammatory bowel disease: Counsel afected women on subfertility risks and risks of adverse pregnancy outcomes. Discuss teratogenicity of methotrexate and the other imnunomodulators. Offer effective contraception during their use and switch agents, if possible, before conception. Hepatitis B: Vaccinate all high-risk women before conception (Table 9-7, p. 1l72). Counsel chronic carriers on transmission prevention to partners and fetus. Treat if indicated. Hepatitis C: Screen high-risk women. Counsel affected ,fomen on risks of disease and transmission. If treatment indicated, discuss ramifications and appropriateness of pregnancy. Iron-deficiency anemia: Iron supplementation. Sickle-cell disease: Screen all black women. Counsel those with trait or disease. Test partner if desired. Thalassemias: Screen women of Southeast Asian or Mediterranean ancestry. Renal d:sease Chap. 9, p. 170 Chap. 12, p. 244 Chap.l48,lp.l936 Chap. 61A, p. 1180 Chap. 49, p. 951 Chap. 12, pp. 241,

1	Renal d:sease Chap. 9, p. 170 Chap. 12, p. 244 Chap.l48,lp.l936 Chap. 61A, p. 1180 Chap. 49, p. 951 Chap. 12, pp. 241, Chap. 50, p. 976 Chap. 51, p. 988 Chap.l52,lp.ll006 Chap. 53, p. 1025 Chap. 12, p. 241 Chap. 54, p. 1050 Chap. 12, pp. 242, Chap. 55, p. 1064 Chap.l56,lp.ll075 TABLE 8-4. Continued Diabetes Chap. 57, p. 1104 Optimize glycemic control to minimize teratogenicity of hyperglycemia. Evaluate for end-organ damage such as retinopathy, nephropathy, hypertension, and others. Discontinue ACE inhibitors. Thyroid disease Chap. 58, p. 1118 Screen those with thyroid disease symptoms. Ensure diet. Treat overt hyper-or hypothyroidism. Counsel on risks to pregnancy outcome. Connective Chap. 59, p. 11l38 RA: Counsel on flare risk after pregnancy. Discuss methotrexate and leflunomide tissue disease Chap. 12, p. 241 teratogenicity, as well as possible effects of other immunomodulators. Switch these agents before conception. Stop NSAIDs by 27 weeks' gestation.

1	Switch these agents before conception. Stop NSAIDs by 27 weeks' gestation. SLE: Counsel on risks during pregnancy. Optimize disease before conception. Discuss mycophenolate mofetil and cyclophosphamide teratogenicity as well as possible effects of newer immunomodulators. Switch these agents before conception. Psych iatric Cha p. 61, p. 11 75 Depression: Screen for symptoms of depression. Counsel on risks of treatment and disorders of untreated illness and the high risk of exacerbation during pregnancy and the puerperium. Neurological Chap. 60, p. 11l59 Seizure disorder: Optimize seizure control using monotherapy if possible. disorders Dermatological Chap. 12, p. 245 Discuss isotretinoin and etretinate teratogenicity and effective contraception disease during their use; switch agents before conception.

1	Cancer Chap. 63, p. 11l92 Counsel on fertility preservation options before cancer therapy and on decreased fertility following certain agents. Discuss appropriateness of pregnancy balanced with need for ongoing cancer therapy and prognosis of the disease state. Infectious Chap. 64, p. 1209 Influenza: Vaccinate all women who will be pregnant during flu season. Vaccinate diseases high-risk women prior to flu season. Malaria: Counsel to avoid travel to endemic areas during conception. If unable, offer effective contraception during travel or provide chemoprophylaxis for those planning pregnancy. Zika virus: See travel restrictions by CDC. Rubella: Screen for rubella immunity. If nonimmune, vaccinate and counsel on the need for effective contraception during the subsequent month. Tdap: tetanus, diphtheria, pertussis: Update vaccination in all reproductive-aged women.

1	Tdap: tetanus, diphtheria, pertussis: Update vaccination in all reproductive-aged women. Varicella: Question regarding immunity. If nonimmune, STDs Chap.l65,lp.l1235 Gonorrhea, syphilis, chlamydial infection: Screen high-risk women and treat as indicated. HIV: Screen at-risk women. Counsel afected women on risks during pregnancy and on perinatal transmission. Discuss initiation of treatment before pregnancy to decrease transmission risk. Offer effective contraception to those not desiring conception. HPV' Provide Pap smear screening per guidelines (Chap. 63, p. 1193). Vaccinate candidate patients. HSV: Provide serological screening to asymptomatic women with affected partners. Counsel affected women on risks of perinatal transmission and on preventative measures during the third trimester and labor.

1	ACE = angiotensin-converting enzyme; ACOG = American College of Obstetricians and Gynecologists; ARB = angiotensinreceptor blocker; BMI = body mass index; CDC = Centers for Disease Control and Prevention; HIV = human immunodeficiency virus; HPV = human papillomavirus; HSV = herpes simplex virus; HTN = hypertension; NSAID = nonsteroidal antiinflammatory drug; RA = rheumatoid arthritis; SLE = systemic lupus erythematosus; STD = sexually transmitted disease. Data from Jack BW, Atrash H, Coonrod DV, et al: The clinical content of preconception care: an and preparation of this supplement, Am J Obstet Gynecol. 2008 Dec;199(6 Suppl 2):5266-S279. Aghajanian P, Gupta M: Helping your epileptic patient. Contemp OB/GYN 60:10,s2015 Aguglia U, Barboni G, Battino 0, et al: Italian consensus conference on epilepsy and pregnancy, labor and puerperium. Epilepsia 50:7, 2009

1	Aguglia U, Barboni G, Battino 0, et al: Italian consensus conference on epilepsy and pregnancy, labor and puerperium. Epilepsia 50:7, 2009 Amant F, Vandenbroucke T, Verheecke M, et a1: Pediatric outcome after maternal cancer diagnosed during pregnancy. N Engl J Med 3(19): 1824, 2015 American Academy of Pediatrics: Maternal phenylketonuria. Pediatrics 122: 445, 2008 American College of Obstetricians and Gynecologists: Intimate partner violence. Committee Opinion No. 518, 2012 American College of Obstetricians and Gynecologists: At-risk drinking and alcohol dependence: obstetric and gynecologic implications. Committee Opinion No. 496, August 2011, Reairmed 2013 American College of Obstetricians and Gynecologists: Hemoglobinopathies in pregnancy. Practice Bulletin No. 78, January 2007, Reairmed 2015a American College of Obstetricians and Gynecologists: Obesiry in pregnancy. Committee Opinion No. 549, January 2013, Reairmed 2015b

1	Committee Opinion No. 549, January 2013, Reairmed 2015b American College of Obstetricians and Gynecologists: Pregestational diabetes mellitus. Practice Bulletin No. 60, March 2005, Reairmed 2016a American College ofsObstetricians and Gynecologists: Reproductive life plan nll1g to reduce unIntended pregnancy. Committee Opinion No. 654, February 2016b American College of Obstetricians and Gynecologists: Screening for fetal aneuploidy. Practice Bulletin No. 163, March 2016c America� Coll�g� of Obstetricians and Gynecologists: Carrier screening for genetIc condltlons. Committee Opinion No. 691, March 2017a Ame:ican College of Obstetricians and Gynecologists: Management of women WIth phenylketonuria. Committee Opinion No. 636, June 2015, Reaffirmed 2017b American College of Obstetricians and Gynecologists: Marijuana use during pregnancy and lactation. Committee Opinion No. 722, July 2015, Reaffirmed 2017c

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1	Kitzmiller JL, Gavin A, Gin GO, et al: Preconception care of diabetics. JAMA 265:731, 1991 Kjer 0, Horvath-Puh6 E, Christensens], et a1: Antiepileptic drug use, folic acid supplementation, and congenital abnormalities: a population-based case-control study. 115(1):98,s2008 Korteweg FJ, Bouman K, Erwich n, et al: Cytogenetic analysis after evaluation of 750 fetal deaths: proposal for diagnostic workup. Obstet Gynecol III (4):865, 2008 Kouba 5, Hallstrom T, Lindholm C, et al: Pregnancy and neonatal outcomes in women with eating disorders. Obstet Gynecol 105:255,s2005 Kuliev A, Pakhalchuk T, Verlinsky 0, et al: Preimplantation genetic for hemoglobinopathies. Hemoglobin 35(5-6):547, 201s1 Lane M, Robker RL, Robertson SA: Parenting from before conception. Science 345(6198):756,s2014 Lassi ZS, Imam AM, Dean SV, et al: Preconception care: screening and management of chronic disease and promoting psychological health. Reprod Health 26:11, 2014

1	Lassi ZS, Imam AM, Dean SV, et al: Preconception care: screening and management of chronic disease and promoting psychological health. Reprod Health 26:11, 2014 Leguizam6n G, Igarzabal ML, Reece EA: Periconceptional care of women with diabetes mellitus. Obstet Gynecol C1in North Am 34:225, 200 Luke B: Pregnancy and birth outcomes in couples with infertility and with and without assisted reproductive technology: with an emphasis on US population-based studies. Am J Obstet Gynecol 217:270, 2017 Maillot F, �ook P, Lilburn M, et al: A practical approach to maternal phenylketonufla management. J Inherit Metab Dis 30: 198, 2007 Malaspina 0, Gilman C, Kranz TM: Paternal age and mental health of ofspring. Ferti! Steril 103(6):1392,s2015 Martin ]A, Hamilton BE, Ventura 5], et al: Births: inal data for 2010. Nat! Vital Stat Rep 61(1):1, 2012

1	Martin ]A, Hamilton BE, Ventura 5], et al: Births: inal data for 2010. Nat! Vital Stat Rep 61(1):1, 2012 McNamara OM, Elkayam U, Alharethi R, et al: Clinical outcomes for peripartum cardiomyopathy in North America: results of the IPAC study (investigations of pregnancy-associated cardiomyopathy). J Am ColI CardioIe66(8): 905,e2015 Medical Research Council Vitamin Study Research Group: Prevention of neural tube defects: results of the Medical Research Council vitamin study. Lancet 338:131, 1991 Moos MK: Preconceptional health promotion: progress in changing a prevention paradigm. J Perinat Neonatal Nurs 18:2,e2004 Moos MK, Bangdiwala SI, Meibohm R, et al: he impact of a preconceptional health promotion program on intendedness of pregnancy. Am J PerinatoIe13:103, 1996

1	Moos MK, Bangdiwala SI, Meibohm R, et al: he impact of a preconceptional health promotion program on intendedness of pregnancy. Am J PerinatoIe13:103, 1996 Morrow JI, Hunt SJ, Russell AJ, et al: Folic acid use and major congenital malformations in ofspring of women with epilepsy: a prospective study from the UK Epilepsy and Pregnancy Register. J Neurol Neurosurg Psychiatry 80(5):506, 2009 Niccolai LM, Ethier A, Kershaw TS, et al: Pregnant adolescents at risk: sexual behaviors and sexually transmitted disease prevalence. Am J Obstet Gynecol 188:63,e2003 Petersen EE, Staples JE, Meaney-Delman 0, et al: Interim guidelines for pregnant women during a Zika virus outbreak-United States, 2016. MMWR 65(2):30,e2016 Podymow T, Joseph G: Preconception and pregnancy management of women with diabetic nephropathy on angiotensin converting enzyme inhibitors. Clin NephroIe83(2):73, 2015

1	Podymow T, Joseph G: Preconception and pregnancy management of women with diabetic nephropathy on angiotensin converting enzyme inhibitors. Clin NephroIe83(2):73, 2015 Qin J, Liu X, Sheng X, et al: Assisted reproductive technology and the risk of pregnancy-related complications and adverse pregnancy outcomes in Singleton pregnancies: a meta-analysis of cohort studies. Fertil Steril 105(1):73, 2016 Reddy UM, Page GP, Saade GR, et al: Karyotype versus microarray testing for genetic abnormalities ater stillbirth. N Engl J Med 367(23):2185,2012 Reece EA, Homko CJ: Prepregnancy care and the prevention of fetal malformations in the pregnancy complicated by diabetes. Clin Obstet Gynecol 50:990, 2007 Robert E: Intrauterine efects of electromagnetiC ields (low frequency, midfrequency F, and microwave): review of epidemiologic studies. Teratology 59:292, 1999

1	Robert E: Intrauterine efects of electromagnetiC ields (low frequency, midfrequency F, and microwave): review of epidemiologic studies. Teratology 59:292, 1999 Schuler-Faccini L, Ribeiro EM, Feitosa 1M, et al: Possible association between Zika virus infection and microcephaly-Brazil, 2015. MMWR 65(3):59, 20e16 Sheield JS, Butler-Koster EL, Casey BM, et al: Maternal diabetes mellitus and infant malformations. Obstet Gynecolel00:925, 2002 Silverman JG, Decker MR, Reed E, et al: Intimate partner violence victimization prior to and during pregnancy among women residing in 26 U.S. States: associations with maternal and neonatal health. Am J Obstet Gynecol 195:140,e2006 Simpson LL: Preconception considerations. Semin Perinatol 38(5):236, 2014

1	Simpson LL: Preconception considerations. Semin Perinatol 38(5):236, 2014 Society for Maternal-Fetal Medicine (SMFM), Sciscione A, Berghella V, et al: Society for Maternal-Fetal Medicine (SMFM) special report: the maternal-fetal medicine subspecialists' role within a health care system. Am J Obstet Gynecole211 (6):607,e2014 Stothard KJ, Tennant PW, Bell R, et al: Maternal oveweight and obesity and the risk of congenital anomalies: a systematic review and meta-analysis. JAMA 301:636,e2009 Stubbleield PG, Coonrod DV, Reddy UM, et al: he clinical content of preconception care: reproductive history. Am J Obstet Gynecol 199(6 Suppl 2):S373, 2008 Sunderan S, Kissin OM, Crawford SB, et al: Assisted reproduction technology suveillance-United States, 20e12. MMWR 64:e1,e20e15

1	Sunderan S, Kissin OM, Crawford SB, et al: Assisted reproduction technology suveillance-United States, 20e12. MMWR 64:e1,e20e15 Temel S, Van Voorst SF, Jack BW, et al: Evidence-based preconceptionalelifestyle interventions. Epidemiol Rev 36:19,e2014 hompson MW, McInnes R, Huntington FW (eds): Genetics in Medicine, 5th ed. Philadelphia, Saunders, 1991 Tomson T, Battino 0: Pregnancy and epilepsy: what should we tell our patients? J Neurol 256(6):856, 2009 Tough S, ToHemire K, Clarke M, et al: Do women change their drinking behaviors while trying to conceive? n opportunity for preconception counseling. Clin Med Res 4:97, 2006 Tripathi A, Rankin J, Aarvold J, et al: Preconception counseling in women with diabetes: a population-based study in the North of England. Diabetes Care 33(3):586,e2010

1	Tripathi A, Rankin J, Aarvold J, et al: Preconception counseling in women with diabetes: a population-based study in the North of England. Diabetes Care 33(3):586,e2010 U.S. Preventive Services Task Force: Final update summary: folic acid to prevent neural tube defects. 2009. Available at: http://ww.uspreventiveservicestaskfo rce. 0rg/Pagel Documen t/U pdateS ummary F inall folic-acidto-prevent-neural-tube-defects-preventive-medication. Accessed April 5, 2016 Usta 1M, Zoorob 0, Abu-Musa A, et al: Obstetric outcome of teenage pregnancies compared with adult pregnancies. Acta Obstet Gynecol 87: 178, 2008 Vajda FJ, Hitchcock A, Graham J, et al: Seizure control in antiepileptic drug treated pregnancy. Epilepsia 49: 172, 2008 Vajda FJ, O'Brien TJ, Graham J, et al: he outcomes of pregnancy in women with untreated epilepsy. Seizure 24: 7, 2015

1	Van der Zee B, de Wert G, Steegers A, et al: Ethical aspects of paternal preconception lifestyle modiication. Am J Obstet Gynecol 209(1): 11, 2013 Veiby G, Daltveit AK, Engelsen BA, et al: Pregnancy, delivery, and outcome for the child in maternal epilepsy. Epilepsia 50(9):2130, 2009 Vichinsky EP: Clinical manifestations of a-thalassemia. Cold Spring Harb Perspect Med 3(5):aOI1742, 2013 Vockley J, Andersson HC, Antshel KM, et al: Phenylalanine hydroxylase deiciency: diagnosis and management guideline. American College of Medical Genetics and Genomics Therapeutics Committee 16:356,2014 Waldenstrom U, Cnattingius S, Norman M, et al: Advanced maternal age and stillbirth risk in nulliparous and parous women. Obstet Gynecol 126(2): 355, 2015 Williams J, Mai CT, Mulinare J, et al: Updated estimates of neural tube defects prevention by mandatory folic acid fortiication-United States, 1995-2011. MMWR 64(1):1, 2015

1	Williams J, Mai CT, Mulinare J, et al: Updated estimates of neural tube defects prevention by mandatory folic acid fortiication-United States, 1995-2011. MMWR 64(1):1, 2015 Windham G, Fenster L: Environmental contaminants and pregnancy outcomes. Fertil Steril 89:e Ill, 2008 DIAGNOSIS OF PREGNANCY. . . . . . . . . . . . . . . . . . . . .. 158 INITIAL PRENATAL EVALUATION .e. .e..... .e.. .e.. ...e. 159 SUBSEQUENT PRENATAL VISITS .... .e. .e......... .e. 164 ..NUTRITIONAL COUNSELING. . . . . . . . . . . . . . . . . . . . .. 165 COMMON CONCERNS . . . . . . . . . . . . . . . . . . . . . . . . . .. 170 The borderline between health and disease is less distincty marked during gestation, and therore, it accordingy becomes necessary to keep pregnant patients under strict supervision, and to be constanty on the alert or the appearance of untoward symptoms.

1	-J. Whitridge Williams (1903) s emphasized above by Williams, prenatal care is important. According to the merican Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) a comprehensive antepartum program is deined as: "a coordinated approach to medical care, continuous risk assessment, and psychological support that optimally begins before conception and extends throughout the postpartum period and interconceptional period." Almost a century after its introduction, prenatal care has become one of the most frequently used health services in the United States. In 2001, there were approximately 50 million prenatal visits. The median was 12.3 visits per pregnancy, and many women had 17 or more visits. Still, as seen from

1	Figure 9-1, 6 to 7 percent of women in this country have late or no prenatal care. In 2014, the percentages of non-Hispanic white, Hispanic, and African-American women who received inadequate or no prenatal care were 4.3, 7.5, and 9.7, respec tively (Child Trends, 2015). he Centers for Disease Control and Prevention (CDC) (2000) analyzed birth certificate data and found that half of women with delayed or no prenatal care wanted to begin care earlier. Barriers to care varied by social and ethnic group, age, and payment method. he most common reason cited was late recognition of pregnancy by the patient. The second most commonly cited obstacle was lack of money or insurance. he third was inability to obtain an appointment.

1	Care designed during the early 1900s focused on lowering the extremely high maternal mortality rate. Prenatal care undoubtedly contributed to the dramatic decline in this mortality rate from 690 deaths per 100,000 births in 1920 to 50 per 100,000 •• Births in all states Births in states using the 1989 birth certificate/revision • Births in states using the 2003 birth certificate/revision FIGURE 9-1 Percentage of births to mothers who received late or no prenatal care-United States, 1990-2014. (Data from Trends, 2015.) by 1955 (Loudon, 1992). And, the low current maternal mortality rate of 10 to 15 per 100,000 is likely associated with the high utilization of this care (Xu, 2010). Indeed, data from 1998 to 2005 from the Pregnancy \10rtality Surveillance System identified a ivefold increased risk for maternal death in women who received no prenatal care (Berg, 2010).

1	Other reports also attest to prenatal care eicacy. In a study of almost 29 million births, the risk for preterm birth, stillbirth, early and late neonatal death, and infant death rose linearly with decreasing prenatal care (Partridge, 2012). Similarly, Leveno and associates (2009) found that a significant decline in preterm births at Parkland Hospital correlated closely with increased use of prenatal care by medically indigent women. Moreover, National Center for Health Statistics data showed that women with prenatal care had an overall stillbirth rate of 2.7 per 1000 compared with 14.1 per 1000 for women without this care (Vintzileos, 2002).

1	Evaluating the format of care, Ickovics and coworkers (2016) compared individual prenatal care and group prenatal care. he latter provided traditional pregnancy surveillance in a group setting with special focus on support, education, and active healthcare participation. Women enrolled in group prenatal care had significantly better pregnancy outcomes. Carter and colleagues (2016) cited similar results. Childbirth education classes are also reported to result in better pregnancy outcomes (Afshar, 2017). Adolescent pregnancies carry special risk, and guidelines have been developed that focus on this subgroup (Fleming, 2015). Few data are available to recommend the practice of ofering tangible incentives to improve prenatal care attendance (Till, 2015).

1	Pregnancy is usually identified when a woman presents with symptoms and possibly a positive home urine pregnancy test result. Typically, these women receive conirmatory testing of urine or blood for human chorionic gonadotropin (hCG). Further, presumptive signs or diagnostic indings of pregnancy may be found during examination. Sonography is often used, particularly if miscarriage or ectopic pregnancy is a concern. 100,000 50,000 E 10,000

1	100,000 50,000 E 10,000 Amenorrhea in a healthy reproductive-aged woman who previously has experienced spontaneous, cyclical, predictable menses is highly suggestive of pregnancy. Menstrual cycles vary appreciably in length among women and even in the same woman (Chap. 5, p. 81). hus, amenorrhea is not a reliable pregnancy indicator until 10 days or more after expected menses have passed. Occasionally, uterine bleeding that mimics menstruation is noted after conception. During the irst month of pregnancy, these episodes are likely the consequence of blastocyst implantation. Still, irst-trimester bleeding should generally prompt evaluation for an abnormal pregnancy.

1	Of other symptoms, maternal perception of fetal movement depends on factors such as parity and habitus. In general, after a irst successful pregnancy, a woman may irst perceive fetal movements between 16 and 18 weeks' gestation. A primigravida may not appreciate fetal movements until approximately 2 weeks later. At about 20 weeks, depending on maternal habitus, an examiner can begin to detect fetal movements. Of pregnancy signs, changes in the lower reproductive tract, uterus, and breasts develop early. These are described in detail in Chapter 4 (p. 49). Detection of hCG in maternal blood and urine is the basis for endocrine assays of pregnancy. Syncytiotrophoblast produces hCG in amounts that increase exponentially during the irst trimester following implantation. A main function of hCG is to prevent involution of the corpus luteum, which is the principal site of progesterone formation during the irst 6 weeks of pregnancy.

1	With a sensitive test, the hormone can be detected in maternal serum or urine by 8 to 9 days after ovulation. he doubling time of serum hCG concentration is 1.4 to 2.0 days. As shown in Figure 9-2, serum levels range widely and increase from the day of implantation. They reach peak levels at 60 to 70 days. Thereafter, the concentration declines slowly until a plateau is reached at approximately 16 weeks' gestation. Measurement of hCG his hormone is a glycoprotein with high carbohydrate content. he general structure of hCG is a heterodimer composed of two dissimilar subunits, designated a and �, which are non covalently linked. The a-subunit is identical to those of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH), but the �-subunit is structurally distinct among these. hus, antibodies were developed with high speciicity for the hCG �-subunit.

1	FIGURE 9-2 Mean concentration (95% CI) of human chorionic gonadotropin (hCG) in serum of This speciicity allows its detecwomen throughout normal pregnancy. tion, and numerous commercial immunoassays are available for measuring serum and urine hCG levels. Although each immunoassay detects a slightly diferent mixture of hCG variants, its free subunits, or its metabolites, all are appropriate for pregnancy testing (Braunstein, 2014). Depending on the assay used, the sensitivity for the laboratory detection limit of hCG in serum is 1.0 mIU/mL or even lower (Wilcox, 2001). False-positive hCG test results are rare (Braunstein, 2002). erroneously with the test antibody directed to hCG in a given assay. The most common factors are heterophilic antibodies.

1	False-positive hCG test results are rare (Braunstein, 2002). erroneously with the test antibody directed to hCG in a given assay. The most common factors are heterophilic antibodies. hese are produced by an individual and bind to the animal derived test antibodies used in a given immunoassay. Thus, women who have worked closely with animals are more likely to develop these antibodies, and alternative laboratory tech niques are available (American College of Obstetricians and Gynecologists, 2017a). Elevated hCG levels may also reflect molar pregnancy and its associated cancers (Chap. 20, p. 391). Other rare causes of positive assays without pregnancy are: (1) exogenous hCG injection used for weight loss, (2) renal fail ure with impaired hCG clearance, (3) physiological pituitary hCG, and (4) hCG-producing tumors that most commonly originate from gastrointestinal sites, ovary, bladder, or lung (Montagnana, 2011).

1	Over-the-counter pregnancy test kits have been available since the early 1970s, and millions are sold annually in the United States. More than 60 such tests are available in this country (Grenache, 2015). Unfortunately, many of these are not as accurate as advertised Gohnson, 2015). For example, Cole and associates (201i1) found that a detection limit of 12.5 mIU/mL would be required to diagnose 95 percent of pregnancies at the time of missed menses, but they reported that only one brand had this degree of sensitivity. Two other brands gave false-positive or invalid results. In fact, with an hCG concentration of 100 mIU/mL, clearly positive results were displayed by only 44 percent of brands. Accordingly, only about 15 percent of pregnancies could be diagnosed at the time of the missed menses. Some manufacturers of even newer home urine assays claim >99-percent accuracy of tests done on the day of-and some up to 4 days before-the expected day of menses. Again, careful analysis

1	menses. Some manufacturers of even newer home urine assays claim >99-percent accuracy of tests done on the day of-and some up to 4 days before-the expected day of menses. Again, careful analysis suggests that these assays are often not as sensitive as advertised Gohnson, 2015).

1	• Sonographic Recognition of Pregnancy Transvaginal sonography has revolutionized early pregnancy imaging and is commonly used to accurately establish gestational age and conirm pregnancy location. A gestational sac-a small anechoic luid collection within the endometrial cavityis the irst sonographic evidence of pregnancy. It may be seen with transvaginal sonography by 4 to 5 weeks' gestation. A luid collection, however, can also be seen within the endometrial cavity with an ectopic pregnancy and is termed a pseudogestational sac or pseudosac (Fig. 19-4, p. 375). Thus, further evaluation may be warranted if this is the only sonographic inding, particularly in a woman with pain or bleeding. A normal

1	FIGURE 9-3 Transvaginal sonogram of a first-trimester intrauterine pregnancy. The double decidual sign is noted surrounding the gestational sac and is defined by the decidua parietalis (white asterisk) and the decidua capsularis (yellow asterisk). The arrow notes the yolk sac, and the crown-rump length of the embryo is marked with measuring calipers. (Used with permission from Dr. Elysia Moschos.) gestational sac implants eccentrically in the endometrium, whereas a pseudosac is seen in the midline of the endometrial cavity. Other potential indicators of early intrauterine pregnancy are an anechoic center surrounded by a single echogenic rim-the intradecidual sign-or two concentric echogenic rings surrounding the gestational sac-the double decidual sin shown in Figure 9-3. If sonography yields equivocal indings, the term pregnancy of unknown location (PUL) is applied. In these cases, serial serum hCG levels and transvaginal sonograms can help diferentiate a normal intrauterine

1	yields equivocal indings, the term pregnancy of unknown location (PUL) is applied. In these cases, serial serum hCG levels and transvaginal sonograms can help diferentiate a normal intrauterine pregnancy from an extrauterine pregnancy or an early miscarriage (Chap. 19, p. 373).

1	If the yolk sac-a brightly echogenic ring with an anechoic center-is seen within the gestational sac, an intrauterine location for the pregnancy is confirmed. The yolk sac can normally be seen by the middle of the ifth week. As shown in Figure 9-3, after 6 weeks, an embryo is seen as a linear structure immediately adjacent to the yolk sac. Cardiac motion is typically noted at this point. Up to 12 weeks' gestation, the crown-rump length is predictive of gestational age within 4 days (Chap. 10, p. 183). Prenatal care is ideally initiated early. Major goals are to: (1) deine the health status of the mother and fetus, (2) estimate the gestational age, and (3) initiate a plan for continued obstetrical care. Typical components of the initial visit are summarized in Table Subsequent care may range from relatively infrequent routine visits to prompt hospitalization because of serious maternal or fetal disease.

1	Subsequent care may range from relatively infrequent routine visits to prompt hospitalization because of serious maternal or fetal disease. Use of a standardized record within a perinatal health-care system greatly aids antepartum and intrapartum management. TABLE 9-1 . Typical Components of Routine Prenatal Care Chap. 9, p. 161 Chap. 9, p. 163 Chap. 40, p. 71l1 Chap. 9, p. 165 Chap. 9, p. 163 Chap. 9, p. 164 Chap. 9, p. 165 Chap. 56, p. 1075 Chap. 15, p. 301 Chap. 15, p. 301 Chap. 63, p. 1193 Chap. 57, p. 1108 Chap. 14, p. 278 Chap. 14, p. 283 Chap. 14, p. 289 Chap. 4, p. 66 Chap. 53, p. 1026 Chap. 64, p. 1215 Chap. 65, p. 1l237 Chap. 65, p. 1239 Chap. 65, p. 1240 Chap. 55, p. 1064 Chap. 65, p. 1247 Chap. 64, p. 1220 Chap. 51, p. 996 aFirst-trimester aneuploidy screening may be ofered between 11 and 14 weeks. A Performed at 28 weeks, if indicated. 8 Test should be ofered. C High-risk women should be retested at the beginning of the third trimester.

1	8 Test should be ofered. C High-risk women should be retested at the beginning of the third trimester. o High-risk women should be screened at the first prenatal visit and again in the third trimester. culture should be between 35 and 37 weeks. HIV human immunodeficiency virus. Standardizing documentation allows communication and care continuity between providers and enables objective measures of care quality to be evaluated over time and across diferent clinical settings (Gregory, 2006). A prototype is provided by the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) in their Guidelines or Perinatal Care, 8th edition. Several deinitions are pertinent to establishment of an accurate prenatal record. 1. Nulligravida-a woman who currently is not pregnant and has never been pregnant. 2.

1	Several deinitions are pertinent to establishment of an accurate prenatal record. 1. Nulligravida-a woman who currently is not pregnant and has never been pregnant. 2. Gravida-a woman who currently is pregnant or has been in the past, irrespective of the pregnancy outcome. With the establishment of the first pregnancy, she becomes a primigravida, and with successive pregnancies, a multigravida. 3. Nullpara-a woman who has never completed a pregnancy beyond 20 weeks' gestation. She may not have been pregnant or may have had a spontaneous or elective abortion(s) or an ectopic pregnancy. 4.

1	3. Nullpara-a woman who has never completed a pregnancy beyond 20 weeks' gestation. She may not have been pregnant or may have had a spontaneous or elective abortion(s) or an ectopic pregnancy. 4. Primipara-a woman who has been delivered only once of a fetus or fetuses born alive or dead with an estimated length of gestation of 20 or more weeks. In the past, a 500-g birthweight threshold was used to define parity. his threshold is now controversial because many states still use this weight to diferentiate a stillborn fetus from an abortus (Chap. 1, p. 3). However, the survival of neonates with birthweights < 500 g is no longer uncommon.

1	5. Multpara-a woman who has completed two or more pregnancies to 20 weeks' gestation or more. Parity is determined by the number of pregnancies reaching 20 weeks. It is not increased to a higher number if multiples are delivered in a given pregnancy. Moreover, stillbirth does not lower this number. In some locales, the obstetrical history is summarized by a series of digits connected by dashes. hese refer to the number of term infants, preterm infants, abortuses younger than 20 weeks, and children currently alive. For example, a woman who is para 2-1-0-3 has had two term deliveries, one preterm delivery, no abortuses, and has three living children. Because these are nonconventional, it is helpful to speciy the outcome of any pregnancy that did not end normally.

1	The normal duration of pregnancy calculated from the irst day of the last normal menstrual period is very close to 280 days or 40 weeks. In a study of 427,581 singleton pregnancies from the Swedish Birth Registry, Bergsj0 and coworkers (1990) found that the mean pregnancy duration was 281 days with a standard deviation of 13 days. However, menstrual cycle length varies among women and renders many of these calculations inaccurate. This, combined with the frequent use of irst-trimester sonography, has changed the method of determining an accurate gestational age (Duryea, 2015).

1	he American College of Obstetricians and Gynecologists (2017e), the American Institute of Ultrasound in Medicine, and the Society for Maternal-Fetal Medicine have concluded that irst-trimester ultrasound is the most accurate method to establish or reairm gestational age. For pregnancies conceived by assisted reproductive technology, embryo age or transfer date is used to assign gestational age. If available, the gestational ages calculated from the last menstrual period and from irst-trimester ultrasound are compared, and this estimated date of delivery is recorded. his is discussed in further detail in Chapter 7 (p. 124) and in Table 10-1 (p. 183).

1	A quick estimate of a pregnancy due date based on menstrual data can be made as follows: add 7 days to the irst day of the last period and subtract 3 months. For example, if the irst day of the last menses was October 5, the due date is 10-05 minus 3 (months) plus 7 (days) = 7-12, or July 12 of the following year. his calculation is the Naegele rule (American College of Obstetricians and Gynecologists, 2017 e).

1	It has become customary to divide pregnancy into three equal epochs or trimesters of approximately 3 calendar months. Historically, the irst trimester extends through completion of 14 weeks, the second through 28 weeks, and the third includes the 29th through 42nd weeks of pregnancy. Thus, there are three periods of 14 weeks each. Certain major obstetrical problems tend to cluster in each of these time periods. For example, most spontaneous abortions take place during the irst trimester, whereas most women with hypertensive disorders due to preg nancy are diagnosed during the third trimester.

1	In modern obstetrics, the clinical use of trimesters to describe a speciic pregnancy is imprecise. For example, it is inappropriate in cases of uterine hemorrhage to categorize the problem temporally as "third-trimester bleeding." Appropriate management for the mother and her fetus will vary remark ably depending on whether bleeding begins early or late in the third trimester (Chap. 41, p. 757). Because precise knowledge of fetal age is imperative for ideal obstetrical management, the clinically appropriate unit is weeks of gestation complete. And more recently, clinicians designate gestational age using com pleted weeks and days, for example, 334/7 weeks or 33 + 4, for 33 completed weeks and 4 days.

1	As elsewhere in medicine, history taking begins with queries concerning medical or surgical disorders. Also, detailed information regarding previous pregnancies is essential as many obstetrical complications tend to recur in subsequent pregnancies. The menstrual and contraceptive histories are also important. Gestational or menstrual age is the number of weeks since the onset of the last menstrual period in women with menstrual cycles lasting 28 to 30 days. For those with irregular menses, sonography in early pregnancy will clariy gestational age. Last, some methods of birth control favor ectopic implantation following method failure (Chap. 38, pp. 683 and 689).

1	Psychosocial Screening. The American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) define psychosocial issues as nonbiomedical factors that afect mental and physical well-being. Women should be screened regardless of social status, education level, race, or ethnicity. Such screening should seek barriers to care, communication obstacles, nutritional status, unstable housing, desire for pregnancy, safety concerns that include intimate-partner violence, depression, stress, and use of substances such as tobacco, alcohol, and illicit drugs. his screening is performed on a regular basis, at least once per trimester, to identiy important issues and reduce adverse pregnancy outcomes. Coker and colleagues (2012) compared pregnancy outcomes in women before and after implementation of a universal psychosocial screening program and found that screened women were less likely to have preterm or low-birthweight newborns, as well as other adverse outcomes.

1	and after implementation of a universal psychosocial screening program and found that screened women were less likely to have preterm or low-birthweight newborns, as well as other adverse outcomes. Speciic screens for depression are presented in Chapter 61 (p. 1174).

1	Cigarette Smoking. Data on this practice have been included on the birth certiicate since 1989. The number of pregnant women who smoke continues to decline. From 2000 to 2010, the prevalences were 12 to 13 percent (Tong, 2013). Based on the Pregnancy Risk Assessment Monitoring System, these women were more likely younger, had less education, and were either Alaska Natives or American Indians (Centers for Disease Control and Prevention, 20 13a). Numerous adverse outcomes have been linked to smoking during pregnancy (U.S. Department of Health and Human Services, 2000). Potential teratogenic efects are reviewed in TABLE 9-2. Five A's of Smoking Cessation ASK about smoking at the first and subsequent prenatal visits.

1	TABLE 9-2. Five A's of Smoking Cessation ASK about smoking at the first and subsequent prenatal visits. ADVISE with clear, strong statements that explain the risks of continued smoking to the woman, fetus, and newborn. ASSESS the patient's willingness to attempt cessation. ASSIST with pregnancy-specific, self-help smoking cessation materials. Ofer a direct referral to the smoker's quit line (1-800-QUIT NOW) to provide ongoing counseling and support. ARRANGE to track smoking progress at subsequent visits. Adapted from Fiore, 2008.

1	Adapted from Fiore, 2008. Chapter 12 (p. 249). Notable among these are greater rates of miscarriage, stillbirth, low birthweight, and preterm delivery (Man, 2006; Tong, 2013). here is also a twofold risk of placenta previa, placental abruption, and premature membrane rupture compared with nonsmokers. hus, the U.S. Preventive Services Task Force recommends that clinicians ofer counseling and efective intervention options to pregnant smokers at the irst and subsequent prenatal visits (Siu, 2015). Although beneits are greatest if smoking ceases early in pregnancy or preferably preconceptionally, quitting at any stage of pregnancy can improve perinatal outcomes (Fiore, 2008).

1	Person-to-person psychosocial interventions are signiicantly more successful in achieving smoking abstinence in pregnancy than is simply advising the woman to quit (Fiore, 2008). One example is a brief counseling session covering the "5As" of smoking cessation (Table 9-2). This approach can be accomplished in 15 minutes or less and is efective when initiated by health-care providers (American College of Obstetricians and Gynecologists, 20 17i).

1	Behavioral interventions and nicotine replacement products are successful in reducing smoking rates (Patnode, 2015). hat said, nicotine replacement has not been suiciently evaluated to determine its efectiveness and safety in pregnancy. Trials evaluating such therapy have yielded conflicting evidence (Coleman, 2015; Pollak, 2007; Spindel, 2016). Two recent randomized trials also produced nonconclusive results. In the Smoking and Nicotine in Pregnancy (SNAP) trial, Cooper and associates (2014) reported a temporary cessation of smoking that may have been associated with improved infant development. In the Study of Nicotine Patch in Pregnancy (SNIPP) trial, Berlin and coworkers (2014) found no diferences in smoking cessation rates or birthweights.

1	Because of limited available evidence to support pharmacotherapy for smoking cessation in pregnancy, the American College of Obstetricians and Gynecologists (2017i) has recommended that if nicotine replacement therapy is used, it should be done with close supervision and after careful consideration of the risks of smoking versus nicotine replacement.

1	Alcohol. Ethyl acohol or ethanol is a potent teratogen that causes a etal syndrome characterized by growth restriction, acial abnormalities, and central nervous system dyunction. s discussed in Chapter 12 (p. 239), women who are pregnant or considering pregnancy should abstain from using any alcoholic beverages. The CDC analyzed data from the Behavioral isk Factor Surveillance System from 2011 to 2013 and estimated that 10 percent of pregnant women used alcohol. It is estimated that 3.3 million women are at risk for such exposure (Green, 2016). he American College of Obstetricians and Gynecologists (2016b) in collaboration with the CDC has developed the Fetal Acohol Spectrum Disorders (FASD) Prevention Program, which provides resources for providers and is available at: http://www. acog.org/ alcohol.

1	Illicit Drugs. It is estimated that 10 percent of fetuses are exposed to one or more illicit drugs. Agents may include heroin and other opiates, cocaine, amphetamines, barbiturates, and marijuana (American Academy of Pediatrics, 2017; merican College of Obstetricians and Gynecologists, 20 15a, 2017 d). As discussed in Chapter 12 (p. 247), chronic use of most of these in large quantities is harmful to the fetus (Metz, 2015). Well-documented sequelae include fetal-growth restriction, low birthweight, and drug withdrawal soon ater birth. Adverse efects of marijuana are less convincing. Women who use such drugs frequently do not seek prenatal care, which in itself is associated with risks for preterm and low-birthweight newborns (EI-Mohandes, 2003; Eriksen, 2016).

1	For women who abuse heroin, methadone maintenance can be initiated within a registered methadone treatment program to reduce complications of illicit opioid use and narcotic withdrawal, to encourage prenatal care, and to avoid drug culture risks (American College of Obstetricians and Gynecologists, 2017£). Available programs can be found through the treatment locator of the Substance Abuse and Mental Health Services Administration at ww.samhsa.gov. Methadone dosages usually are initiated at 10 to 30 mg daily and titrated as needed. In some women, careful methadone taper may be an appropriate option (Stewart, 2013). Although less commonly used, buprenorphine alone or in combination with naloxone may also be ofered and managed by physicians with specific credentialing.

1	Intimate-Partner Violence. his term refers to a pattern of assault and coercive behavior that may include physical injury, psychological abuse, sexual assault, progressive isolation, stalking, deprivation, intimidation, and reproductive coercion (American College of Obstetricians and Gynecologists, 2012). Such violence has been recognized as a major public health problem. Unfortunately, most abused women continue to be victimized during pregnancy. With the possible exception of preeclampsia, domestic violence is more prevalent than any major medical condition detectable through routine prenatal screening (American Academy of Pediatrics and the Ameri can College of Obstetricians and Gynecologists, 2017). The prevalence during pregnancy is estimated to range between 4 and 8 percent. Intimate-partner violence is associated with an increased risk of several adverse perinatal outcomes including preterm delivery, fetal-growth restriction, and perinatal death (Chap. 47, p. 925).

1	The American College of Obstetricians and Gynecologists (2012) has provided methods for domestic violence screening and recommends their use at the irst prenatal visit, then again at least once per trimester, and again at the postpartum visit. Such screening should be done privately and away from family members and friends. Patient self-administered or computerized screenings appear to be as efective for disclosure as clinician-directed interviews (Ahmad, 2009; Chen, 2007). Physicians should be familiar with state laws that may require reporting of intimate-partner violence. Coordination with social services can be invaluable in these cases. The National Domestic Violence Hotline (l-800-799-SAFE [7233]) is a nonprofit telephone referral service that provides individualized information regarding city-speciic shelter locations, counseling resources, and legal advocacy.

1	A thorough, general physical examination should be completed at the initial prenatal encounter. Pelvic examination is performed as part of this evaluation. he cervix is visualized employing a speculum lubricated with warm water or waterbased lubricant gel. Bluish-red passive hyperemia of the cervix is characteristic, but not of itself diagnostic, of pregnancy. Dilated, occluded cervical glands bulging beneath the ectocervical mucosa-nabothian cysts-may be prominent. he cervix is not normally dilated except at the external os. To identiy cytological abnormalities, a Pap test is performed according to current guidelines noted in Chapter 63 (p. 1193). Specimens for identiication of Chlamydia trachomatis and Neisseria gonorrhoeae are also obtained when indicated.

1	Bimanual examination is completed by palpation, with special attention given to the consistency, length, and dilatation of the cervix; to uterine and adnexal size; to the bony pelvic architecture; and to any vaginal or perineal anomalies. Later in pregnancy, fetal presentation often can also be determined. Lesions of the cevix, vagina, or vulva are further evaluated as needed by colposcopy, biopsy, culture, or dark-field examination. The perianal region is visualized, and digital rectal examination performed as required for complaints of rectal pain, bleeding, or mass.

1	Precise knowledge of gestational age is one of the most important aspects of prenatal care because several pregnancy complications may develop for which optimal treatment will depend on fetal age. As discussed earlier and in Chapter 7 (p. 124), irst-trimester sonographic assessment is best correlated with menstrual history. That said, gestational age can also be estimated with considerable precision by carefully performed clinical uterine size examination that is coupled with knowledge of the last menses. Uterine size similar to a small orange roughly correlates with a 6-week gestation; a large orange, with an 8-week pregnancy; and a grapefruit, with one at 12 weeks (Margulies, 2001).

1	Recommended routine tests at the first prenatal encounter are listed in Table 9-1. Initial blood tests include a complete blood count, a determination of blood type with h status, and an antibody screen. The Institute of Medicine recommends universal human immunodeiciency virus (HIV) testing, with patient notiication and right of refusal, as a routine part of prenatal care. The CDC (Branson, 2006) as well as the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (20 16f, 2017) continue to support this practice. If a woman declines testing, this is recorded in the prenatal record. All pregnant women are also screened for hepatitis B virus infection, syphilis, and immunity to rubella at the initial visit. Based on their prospective investigation of 1000 women, Murray and coworkers (2002) concluded that in the absence of hypertension, routine urinalysis beyond the irst prenatal visit was not necessary. A urine culture is recommended by most because

1	women, Murray and coworkers (2002) concluded that in the absence of hypertension, routine urinalysis beyond the irst prenatal visit was not necessary. A urine culture is recommended by most because treating bacteruria signiicantly reduces the likelihood of developing symptomatic urinary tract infections in pregnancy (Chap. 53, p. 1026).

1	Chlamydia trachomatis is isolated from the cervix in 2 to 13 percent of pregnant women. he American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) recommend that all women be screened for chlamydia during the irst prenatal visit, with additional third-trimester testing for those at increased risk. Risk factors include unmarried status, recent change in sexual partner or multiple concurrent partners, age younger than 25 years, inner-city residence, history or presence of other sexually transmitted diseases, and little or no prenatal care. For those testing positive, treatment described in Chapter 65 (p. 1240) is followed by a second testing-a test of cure-3 to 4 weeks after treatment completion.

1	Neisseria gonorrhoeae typically causes lower genital tract infection in pregnancy. It also may cause septic arthritis (Bleich, 2012). Risk factors for gonorrhea are similar to those for chlamydial infection. The American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) recommend that pregnant women with risk factors or those living in an area of high N gonorrhoeae prevalence be screened at the initial prenatal visit and again in the third trimester. Treatment is given for gonorrhea and simultaneously for possible coexisting chlamydial infection (Chap. 65, p. 1240). Test of cure is also recommended following treatment. Many factors can adversely afect maternal and fetal well-being. Some are evident at conception, but many become apparent during the course of pregnancy. The designation of "high-risk pregnancy" is overly vague for an individual woman and probably is best avoided if a more specific diagnosis can be assigned.

1	TABLE 9-3. Conditions for Which Maternal-Fetal Medicine Consultation May Be Beneficial Cardiac disease-moderate to severe disorders Diabetes mellitus with evidence of end-organ damage or uncontroiled hyperglycemia Family or personal history of genetic abnormalities Renal insuficiency if associated with significant proteinuria (::500 mg/24 hour), serum creatininel::1.5 mg/dL, or hypertension Pulmonary disease if severe restrictive or obstructive, including severe asthma Human immunodeficiency virus infection Prior embolus or deep-vein thrombosis Severe systemic disease, including autoimmune conditions Bariatric surgery Epilepsy if poorly controlled or requires more than one anticonvulsant Cancer, especially if treatment is indicated in pregnancy CDE (Rh) or other blood group alloimmunization (excluding ABO, Lewis) Prior or current fetal structural or chromosomal abnormality Periconceptional exposure to known teratogens

1	CDE (Rh) or other blood group alloimmunization (excluding ABO, Lewis) Prior or current fetal structural or chromosomal abnormality Periconceptional exposure to known teratogens Infection with or exposure to organisms that cause congenital infection Higher-order multifetal gestation Severe disorders of amnionic fluid volume Some common risk factors for which consultation is recommended by the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) are shown in Table 9-3. Some conditions may require the involvement of a maternal-fetal medicine subspecialist, geneticist, pediatrician, anesthesiologist, or other medical specialist in the evaluation, counseling, and care of the woman and her fetus.

1	hese are traditionally scheduled at 4-week intervals until 28 weeks, then every 2 weeks until 36 weeks, and weekly thereafter. Women with complicated pregnancies-for example, with twins or diabetes-often require return visits at 1-to 2-week intervals (Luke, 2003; Power, 2013). In 1986, the Department of Health and Human Services convened an expert panel to review the content of prenatal care. his report was subsequently reevaluated and revised in 2005 (Gregory, 2006). he panel recommended, among other things, early and continuing risk assessment that is patient speciic. It also endorsed lexibility in clinical visit spacing; health promotion and education, including preconceptional care; medical and psychosocial interventions; standardized documentation; and expanded prenatal care objectives-to include family health up to 1 year after birth.

1	he World Health Organization conducted a multicenter randomized trial with almost 25,000 women comparing routine prenatal care with an experimental model designed to minimize visits (Villar, 2001). In the new model, women were seen once in the irst trimester and screened for certain risks. hose without anticipated complications-80 percent of those screened-were seen again at 26, 32, and 38 weeks. Compared with routine prenatal care, which required a median of eight visits, the new model required a median of only ive. No disadvantages were attributed to the regimen with fewer visits, and these indings were consistent with other randomized trials (Clement, 1999; McDuie, 1996).

1	At each return visit, the well-being of mother and fetus are assessed (see Table 9-1). Fetal heart rate, growth, and activity and amnionic fluid volume are evaluated. Maternal blood pressure and weight and their extent of change are examined. Symptoms such as headache, altered vision, abdominal pain, nausea and vomiting, bleeding, vaginal luid leakage, and dysuria are sought. Mter 20 weeks' gestation, uterine examination measures size from the symphysis to the fundus. In late pregnancy, vaginal examination often provides valuable information that includes conirmation of the presenting part and its station, clinical estimation of pelvic capacity and configuration, amnionic luid volume adequacy, and cervical consistency, efacement, and dilatation (Chap. 22, p. 435).

1	Between 20 and 34 weeks' gestation, the height of the uterine fundus measured in centimeters correlates closely with gestational age in weeks Gimenez, 1983). his measurement is used to monitor fetal growth and amnionic fluid volume. It is measured along the abdominal wall from the top of the symphysis pubis to the top of the fundus. Importantly, the bladder must be emptied before fundal measurement (Worthen, 1980). Obesity or the presence of uterine masses such as leiomyomas may also limit fundal height accuracy. Moreover, using fundal height alone, fetal-growth restriction may be undiagnosed in up to a third of cases (American College of Obstetricians and Gynecologists, 2015b; Haragan, 2015).

1	Instruments incorporating Doppler ultrasound are often used to easily detect fetal heart action, and in the absence of maternal obesity, heart sounds are almost always detectable by 10 weeks with such instruments (Chap. 10, p. 213). he fetal heart rate ranges from 11e0 to 160 beats per minute and is typically heard as a double sound. Using a standard nonampliied stethoscope, the fetal heart is audible by 20 weeks in 80 percent of women, and by 22 weeks, heart sounds are expected to be heard in all (Herbert, 1987). Because the fetus moves freely in amni onic fluid, the site on the maternal abdomen where fetal heart sounds can be heard best will vary. Additionally, with ultrasonic auscultation, one may hear the unic soule, which is a sharp, whistling sound that is syn chronous with the fetal pulse. It is caused by the rush of blood through the umbilical arteries and may not be heard consistently.

1	In contrast, the uterine soule is a soft, blowing sound that is synchronous with the maternal pulse. It is produced by the pas sage of blood through the dilated uterine vessels and is heard most distinctly near the lower portion of the uterus.

1	Sonography provides invaluable information regarding fetal anatomy, growth, and well-being, and most women in the United States have at least one prenatal sonographic examination during pregnancy (American College of Obstetricians and Gynecologists, 2016h). Continuing trends suggest that the number of these examinations performed per pregnancy is increasing. Siddique and associates (2009) reported that the average number rose from 1.5 in 1995 through 1997 to 2.7 almost 10 years later. his trend was noted in both high-and low-risk pregnancies. he actual clinical utility of this increased use in pregnancy has not been demonstrated, and it is unclear that the cost-benefit ratio is justified (Washington State Health Care Authority, 2010). The American College of Obstetricians and Gynecologists (20e16h) has concluded that sonography should be performed only when there is a valid medical indication and under the lowest possible ultrasound exposure setting. The College further states that a

1	(20e16h) has concluded that sonography should be performed only when there is a valid medical indication and under the lowest possible ultrasound exposure setting. The College further states that a physician is not obligated to perform sonography without a specific indication in a low-risk patient, but that if she requests sonographic screening, it is reasonable to honor her request.

1	If initial results were normal, most tests need not be repeated. Hematocrit or hemoglobin determination, along with serology for syphilis if it is prevalent in the population, is repeated at 28 to 32 weeks (Hollier, 2003; Kiss, 2004). For women at increased risk for HIV acquisition during pregnancy, repeat testing is recommended in the third trimester, preferably before 36 weeks (American College of Obstetricians and Gynecologists, 20 16). Similarly, women who engage in behaviors that place them at high risk for hepatitis B virus infection are retested at the time of hospitalization for delivery. Women who are D (h) negative and are unsensitized should have an antibody screening test repeated at 28 to 29 weeks, and anti-D immunoglobulin is given if they remain unsensitized (Chap. 15, p. 305).

1	he CDC (20e1e0b) recommends that vaginal and rectal group B streptococcal (GBS) cultures be obtained in all women between 35 and 37 weeks' gestation, and the American College of Obstetricians and Gynecologists (20e1e6g) has endorsed this recommendation. Intrapartum antimicrobial prophylaxis is provided to those whose culture results are positive. Women with GBS bacteriuria or a previous infant with invasive disease are given empirical intrapartum prophylaxis. Trials are in progress to test an investigational vaccine (Donders, 2016; Schrag, 2016). hese infections are described further in Chapter 64 (p. 1220).

1	All pregnant women are screened for gestational diabetes mellitus, whether by history, clinical factors, or routine laboratory testing. Although laboratory testing between 24 and 28 weeks' gestation is the most sensitive approach, there may be pregnant women at low risk who are less likely to beneit from testing (American College of Obstetricians and Gynecologists, 2017c). Gestational diabetes is discussed in Chapter 57 (p. 11 07).

1	Serum screening for neural-tube defects is ofered at 15 to 20 weeks. Fetal aneuploidy screening may be performed at 11 to 14 weeks' gestation and/or at 15 to 20 weeks, depending on the protocol selected (Rink, 2016). Additionally, screening for certain genetic abnormalities is ofered to women at increased risk based on family history, ethnic or racial background, or age (American College of Obstetricians and Gynecologists, 20 17h). These are discussed in greater detail in Chapter 14 (p. 277). Some examples include testing for Tay-Sachs disease for persons of Eastern European Jewish or French Canadian ancestry; �-thalassemia for those of Mediterranean, Southeast Asian, Indian, Pakistani, or Mrican ancestry; a-thalassemia for individuals of Southeast Asian or Mrican ancestry; sickle-cell anemia for people of African, Mediterranean, Middle Eastern, Caribbean, Latin American, or Indian descent; and trisomy 21 for those with advanced maternal age.

1	In 2009, the Institute of Medicine and National Research Council revised guidelines for weight gain in pregnancy and continued to stratiy suggested weight gain ranges based on prepregnancy body mass index (BMI) (Table 9-4). The new guidelines included a specific, relatively narrow range of recommended weight gains for obese women. Also, the same recommendations apply to adolescents, short women, and women of TABLE 9-4. Recommendations for Total and Rate of Weight Gain During Pregnancy Underweight 28-40 1 (1l-1l.3) «1l8.5) Normal weight 25-35 1 (0.8-1) (1l8.5-24.9) Overweight 15-25 0.6 (0.5-0.7) (25.0-29.9) Obese (::30.0) 11l-20 0.5 (0.4-0.6) aEmpirical recommendations for weight gain in twin pregnancies include: normal BMI, 37-54 Ib; overweight women, 31-50 Ib; and obese women, 25-42 lb. BMI = body mass index. Modified from the Institute of Medicine and National Research Council, 2009.

1	all racial and ethnic groups. The American College of Obstetricians and Gynecologists (2016i) has endorsed these measures.

1	When the Institute of Medicine guidelines were formulated, concern focused on low-birthweight newborns, however, current emphasis is directed to the obesity epidemic (Catalano, 2007). This explains renewed interest in lower weight gains during pregnancy. Obesity is associated with significantly greater risks for gestational hypertension, preeclampsia, gestational diabetes, macrosomia, cesarean delivery, and other complications (Chap. 48, p. 939). he risk appears "dose related" to prenatal weight gain. In a population-based cohort of more than 120,000 obese pregnant women, those who gained <15 lb had the lowest rates of preeclampsia, large-for-gestational age neonates, and cesarean delivery (Kiel, 2007). Among 100,000 women with normal prepregnancy BMI, De Vader and colleagues (2007) found that those who gained <25 Ib during pregnancy had a lower risk for preeclampsia, failed induction, cephalopelvic disproportion, cesarean delivery, and large-for-gestational age neonates. his cohort,

1	that those who gained <25 Ib during pregnancy had a lower risk for preeclampsia, failed induction, cephalopelvic disproportion, cesarean delivery, and large-for-gestational age neonates. his cohort, however, had an increased risk for smallfor-gestational age newborns. Lifestyle intervention during pregnancy can result in less weight gain (Sagedal, 2017).

1	There is irrefutable evidence that maternal weight gain during pregnancy inluences birthweight. Martin and coworkers (2009) studied this using birth certificate data for 2006. Approximately 60 percent of women gained 26 Ib or more during pregnancy, and maternal weight gain positively correlated with birthweight. Nloreover, women with the greatest risk14 percent-for delivering a newborn weighing <2500 g were those with weight gain < 16 lb. Nearly 20 percent of births to women with such low weight gains were preterm.

1	Meaningful studies of nutrition in human pregnancy are exceedingly diicult to design because experimental dietary deficiency is not ethical. In those instances in which severe nutritional deiciencies have been induced as a consequence of social, economic, or political disaster, coincidental events have oten created many variables, the efects of which are not amenable to quantiication. Some past experiences suggest, however, that in otherwise healthy women, a state of near starvation is required to establish clear diferences in pregnancy outcome.

1	During the severe European winter of 1944 to 1945, nutritional deprivation of known intensity prevailed in a wellcircumscribed area of The Netherlands occupied by the German military (Kyle, 2006). At the lowest point during this Dutch Hunger Winter, rations reached 450 kcal/d, with generalized rather than selective malnutrition. Smith (1947) analyzed the outcomes of pregnancies that were in progress during this 6-month famine. Median neonatal birthweights declined approximately 250 g and rose again ater food became available. This indicated that birthweight can be inluenced significantly by starvation during later pregnancy. he perinatal mortality rate, however, was not altered. Moreover, the incidence of fetal malformations or preeclampsia did not rise signiicantly. Parenthetically, weight loss in obese women during pregnancy is also associated with an increased risk for low-birthweight neonates (Cox Bauer, 2016).

1	Evidence of impaired brain development has been obtained in some animal fetuses whose mothers had been subjected to intense dietary deprivation. Subsequent intellectual development was studied by Stein and associates (1972) in young male adults whose mothers had been starved during pregnancy in the aforementioned Hunger Winter. The comprehensive study was made possible because all males at age 19 underwent compulsory examination for military service. It was concluded that severe dietary deprivation during pregnancy caused no detectable efects on subsequent mental performance. Several studies of the long-term consequences to this cohort of children born to nutritionally deprived women have been performed and have been reviewed by Kyle and Pichard (2006).

1	Several studies of the long-term consequences to this cohort of children born to nutritionally deprived women have been performed and have been reviewed by Kyle and Pichard (2006). Progeny deprived in mid to late pregnancy were lighter, shorter, and thinner at birth, and they had a higher incidences of sub sequent hypertension, reactive airway disease, dyslipidemia, diminished glucose tolerance, and coronary artery disease. Early pregnancy deprivation was associated with greater obesity rates in adult women but not men. Early starvation was also linked to higher rates of central nervous system anomalies, schizophre nia, and schizophrenia-spectrum personality disorders. hese observations and others have led to the concept of etal programming by which adult morbidity and mortality are related to fetal health. Known widely as the Barker hypothesis, as promulgated by Barker and colleagues (1989), this concept is discussed in Chapter 44 (p. 848).

1	Not all the weight gained during pregnancy is lost during and immediately after delivery. Schauberger and coworkers (1992) studied prenatal and postpartum weights in 795 women. Their average weight gain was 28.6 Ib or 12.9 kg. As shown in Figure 9-4, most maternal weight loss was at deliveryapproximately 12 lb or 5.4 kg-and in the ensuing 2 weeksapproximately 9 Ib or 4 kg. An additional 5.5 lb or 2.5 kg was lost between 2 weeks and 6 months postpartum. Thus, average retained pregnancy weight was 2.1 Ib or 1 kg. Excessive weight gain is manifest by accrual of fat and may be partially retained as long-term fat (Berggren, 2016; Widen, 2015). Overall, the more weight that was gained during pregnancy, the more that was lost postpartum. Interestingly, there is no relationship between pre pregnancy BMI or prenatal weight gain and weight retention.

1	Periodically, the Institute of Medicine (2006, 2011) publishes recommended dietary allowances, including those for pregnant aRecommendations measured as adequate intake. Calories are necessary for energy. Whenever caloric intake is inadequate, protein is metabolized rather than being spared for its vital role in fetal growth and development. Total physiologiWeeks of pregnancy cal requirements during pregnancy are not necessarily the sum or lactating women. The latest recommendations are summarized in Table 9-5. Certain prenatal vitamin-mineral supplements may lead to intakes well in excess of the recommended allowances. Moreover, the use of excessive supplements, which often are self-prescribed, has led to concern regarding nutrient toxicities during pregnancy. Those with potentialy toxic fects include iron, zinc, selenium, and vitamins A, B6, C, and D.

1	As shown in Figure 9-5, pregnancy requires an additional 80,000 kcal, mostly during the last 20 weeks. To meet this demand, a caloric increase of 100 to 300 kcal/d is recommended during pregnancy (American Academy of Pediatrics and the American College of Obstetricians and Gynecologists, 2017). This greater intake, however, should not be divided equally during the course of pregnancy. The Institute ofMedicine (2006) recommends adding 0, 340, and 452 kcal/d to the estimated nonpregnantenergy requirements in the irst, second, and third trimesters, respectively. he addition of 1000 kcal/d or more results in fat accrual Qebeile, 2015). From the Institute of Medicine, 2006, 2011. pregnancy may be compensated in whole or in part by reduced physical activity (Hytten, 1991). Protein requirements rise to meet the demands for growth and remodeling of the fetus, placenta, uterus, and breasts, and for 80,000 70,000 60,000 Maintenance ) 50,000 � 40,000 30,000 20,000 10,000

1	Protein requirements rise to meet the demands for growth and remodeling of the fetus, placenta, uterus, and breasts, and for 80,000 70,000 60,000 Maintenance ) 50,000 � 40,000 30,000 20,000 10,000 FIGURE 9-4 Cumulative weight loss from last antepartum visit to 6 months postpartum. *Significantly different from 2-week weight loss; **Significantly different from 6-week weight loss. (Redrawn from Schauberger CW, Rooney BL, Brimer LM: Factors that influence weight loss in the puerperium. Obstet Gynecol 79:424, 1992.)

1	FIGURE 9-5 Cumulative kilocalories required for pregnancy. (Redrawn of ordinary nonpregnant requirements plus those speciic to from Chamberlain G, Broughton-Pipkin F (eds): Clinical Physiology in pregnancy. For example, the additional energy required during Obstetrics, 3rd ed. Oxford, Blackwell Science, 1998.) increased maternal blood volume (Chap. 4, p. 55). During the second half of pregnancy, approximately 1000 g of protein are deposited, amounting to 5 to 6 g/d (Hytten, 1971). To accomplish this, protein intake that approximates 1 g/kg/ d is recommended (see Table 9-5). Data suggest this should be doubled in late gestation (Stephens, 2015). Most amino-acid levels in maternal plasma fall markedly, including ornithine, glycine, taurine, and proline (Hytten, 1991). Exceptions during pregnancy are glutamic acid and alanine, the concentrations of which rise.

1	Preferably, most protein is supplied from animal sources, such as meat, milk, eggs, cheese, poultry, and fish. hese furnish amino acids in optimal combinations. Milk and dairy products are considered nearly ideal sources of nutrients, especially protein and calcium, for pregnant or lactating women. Ingestion of specific ish and potential methylmercury toxicity are discussed on page 170. The intakes recommended by the Institute of Medicine (2006) for various minerals are listed in Table 9-5. With the exception of iron and iodine, practically all diets that supply suicient calories for appropriate weight gain will contain enough minerals to prevent deiciency.

1	Iron requirements are greatly increased during pregnancy, and reasons for this are discussed in Chapter 4 (p. 58). Of the approximately 300 mg of iron transferred to the fetus and placenta and the 500 mg incorporated into the expanding maternal hemoglobin mass, nearly all is used after midpregnancy. During that time, iron requirements imposed by pregnancy and maternal excretion total approximately 7 mg/ d (Pritchard, 1970). Few women have suicient iron stores or dietary intake to supply this amount. Thus, the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (20e17) endorse the recommendation by the National Academy of Sciences that at least 27 mg of elemental iron be supplemented daily to pregnant women. his amount is contained in most prenatal vitamins.

1	Scott and coworkers (1970) established that as little as 30 mg of elemental iron, supplied as ferrous gluconate, sulfate, or fumarate and taken daily throughout the latter half of pregnancy, provides suicient iron to meet pregnancy requirements and protect preexisting iron stores. his amount will also provide for iron requirements of lactation. he pregnant woman may benefit from 60 to 100 mg of elemental iron per day if she is large, has a multifetal gestation, begins supplementation late in pregnancy, takes iron irregularly, or has a somewhat depressed hemoglobin level. he woman who is overtly anemic from iron deficiency responds well to oral supplementation with iron salts. In response, serum ferritin levels rise more than the hemoglobin concentration (Daru, 2016).

1	Iodine is also needed, and the recommended iodine allowance is 220 Lg/d (see Table 9-5). The use of iodized salt and bread products is recommended during pregnancy to ofset the increased fetal requirements and maternal renal losses of iodine. Despite this, iodine intake has declined substantially in the past 15 years, and in some areas it is probably inadequate (Casey, 2017). Severe maternal iodine deficiency predisposes ofspring to endemic cretinism, which is characterized by multiple severe neurological defects. In parts of China and Africa where this condition is common, iodide supplementation very early in pregnancy prevents some cretinism cases (Cao, 1994). To obviate this, many prenatal supplements now contain various quantities of iodine.

1	Cacium is retained by the pregnant woman during gestation and approximates 30 g. Most of this is deposited in the fetus late in pregnancy (Pitkin, 1985). his amount of calcium represents only approximately 2.5 percent of total maternal calcium, most of which is in bone and can readily be mobilized for fetal growth. s another potential use, routine calcium supplementation to prevent preeclampsia has not proved efective (Chap. 40, p. 727). Zinc deficiency if severe may lead to poor appetite, suboptimal growth, and impaired wound healing. During pregnancy, the recommended daily intake approximates 12 mg. But, the safe level of zinc supplementation for pregnant women has not been clearly established. Vegetarians have lower zinc intakes (Foster, 2015). he bulk of studies support zinc supplementation only in zinc-deficient women in poor-resource countries (Nossier, 2015; Ota, 2015).

1	Manesium deficiency as a consequence of pregnancy has not been recognized. Undoubtedly, during prolonged illness with no magnesium intake, the plasma level might become critically low, as it would in the absence of pregnancy. We have observed magnesium deiciency during pregnancies in some with previous intestinal bypass surgery. As a preventive agent, Sibai and coworkers (1989) randomly assigned 400 normotensive primigravid women to 365-mg elemental magnesium supplementation or placebo tablets from 13 to 24 weeks' gestation. Supplementation did not improve any measures of pregnancy outcome.

1	Trace metals include copper, selenium, chromium, and manganese, which all have important roles in certain enzyme functions. In general, most are provided by an average diet. Selenium deficiency is manifested by a frequently fatal cardiomyopathy in young children and reproductive-aged women. Conversely, selenium toxicity resulting from oversupplementation also has been observed. Selenium supplementation is not needed in American women. Potassium concentrations in maternal plasma decline by approximately 0.5 mEq/L by midpregnancy (Brown, 1986). Potassium deficiency develops in the same circumstances as in nonpregnant individuals-a common example is hyperemesis gravidarum.

1	Fluoride metabolism is not altered appreciably during pregnancy (Maheshwari, 1983). Horowitz and Heifetz (1967) concluded that no additional ofspring beneits accrued from maternal ingestion of luoridated water if the newborn ingested such water from birth. Sa Roriz Fonteles and associates (2005) studied microdrill biopsies of deciduous teeth and concluded that antenatal luoride provided no additional fluoride uptake compared with postnatal fluoride alone. Finally, supplemental luoride ingested by lactating women does not raise the luoride concentration in breast milk (Ekstrand, 1981).

1	he increased requirements for most vitamins during pregnancy shown in Table 9-5 usually are supplied by any general diet that provides adequate calories and protein. he excep tion is folic acid during times of unusual requirements, such as pregnancy complicated by protracted vomiting, hemo lytic anemia, or multiple fetuses. hat said, in impoverished countries, routine multivitamin supplementation reduced the incidence of low-birthweight and growth-restricted fetuses, (Fawzi, 2007).

1	neural-tube defect risks (Chap. 13, p. 270). Namely, the CDC (2004) estimated that the number of afected pregnancies had decreased from 4000 pregnancies per year to approximately 3000 per year after mandatory fortiication of cereal products with folic acid in 1998. Perhaps more than half of all neural tube defects can be prevented with daily intake of 400 �g of folic acid throughout the periconceptional period. Evidence development (Ars, 2016). Putting 140 �g of folic acid into each 100 g of grain products may increase the folic acid intake of the average American woman of childbearing age by 1 00 �g/ d. Because nutritional sources alone are insuicient, however, folic acid supplementation is still recommended (American College of Obstetricians and Gynecologists, 20 16e). Likewise, the U.S. Preventive Services Task Force (2009) recommends that all women planning or capable of pregnancy take a daily supplement containing 400 to 800 �g of folic acid.

1	A woman with a prior child with a neural-tube defect can reduce the 2-to 5-percent recurrence risk by more than 70 percent with a daily 4-mg folic acid supplement taken during the month before conception and during the first trimester. As emphasized by the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017), this dose should be consumed as a separate supplement and not as multivitamin tablets. This practice avoids excessive intake of fat-soluble vitamins.

1	Vitamin A, although essential, has been associated with congeni tal malformations when taken in high doses (> 10,000 IU / d) during pregnancy. These malformations are similar to those produced by the vitamin A derivative isotretinoin (Accutane), which is a potent teratogen (Chap. 12, p. 245). Beta-carotene, the precursor of vitamin A found in fruits and vegetables, has not been shown to produce vitamin A toxicity. Most prenatal vitamins contain vitamin A in doses considerably below the teratogenic threshold. Dietary intake of vitamin A in the United States appears to be adequate, and additional supplementation is not routinely recommended. In contrast, vitamin A deiciency is an endemic nutritional problem in the developing world (McCauley, 2015). Vitamin A deficiency, whether overt or subclinical, is associated with night blindness and with an increased risk of maternal anemia and spontaneous preterm birth (West, 2003).

1	Vitamin BJ2 plasma levels drop in normal pregnancy, mostly as a result of reduced plasma levels of their carrier proteinstranscobalamins. Vitamin BJ2 occurs naturally only in foods of animal origin, and strict vegetarians may give birth to neonates whose BI2 stores are low. Likewise, because breast milk of a vegetarian mother contains little vitamin B12, the deiciency may become profound in the breastfed infant (Higginbottom, 1978). Excessive ingestion of vitamin C also can lead to a functional deiciency of vitamin B12. Although its role is still controversial, vitamin BI2 deiciency preconceptionally, similar to folate, may elevate the risk of neural-tube defects (Molloy, 2009).

1	Vitamin B6, which is pyridoxine, does not require supple mentation in most gravidas (Salam, 2015). For women at high risk for inadequate nutrition, a daily 2-mg supplement is rec ommended. As discussed on page 174, vitamin B6, when com bined with the antihistamine doxylamine, is helpful in many cases of nausea and vomiting of pregnancy. Vitamin C allowances during pregnancy are 80 to 85 mg/ d Table 9-5). A reasonable diet should readily provide this amount, and supplementation is not necessary (Rumbold, 2015). Maternal plasma levels decline during pregnancy, whereas cord-blood levels are higher, a phenomenon observed with most water-soluble vitamins.

1	Vitamin D is a fat-soluble vitamin. After being metabolized to its active form, it boosts the eiciency of intestinal calcium absorption and promotes bone mineralization and growth. Unlike most vitamins that are obtained exclusively from dietary intake, vitamin D is also synthesized endogenously with exposure to sunlight. Vitamin D deiciency is common during pregnancy. his is especially true in high-risk groups such as women with limited sun exposure, vegetarians, and ethnic minoritiesparticularly those with darker skin (Bodnar, 2007). Maternal deiciency can cause disordered skeletal homeostasis, congenital rickets, and fractures in the newborn (American College of Obstetricians and Gynecologists, 2017k). Vitamin D supplementation to women with asthma may decrease the likelihood of childhood asthma in their fetuses (Litonjua, 2016). The Food and Nutrition Board of the Institute of Medicine (2011) established that an adequate intake of vitamin D during pregnancy and lactation was 15

1	asthma in their fetuses (Litonjua, 2016). The Food and Nutrition Board of the Institute of Medicine (2011) established that an adequate intake of vitamin D during pregnancy and lactation was 15 �g/d (600 IU/d). In women suspected of having vitamin D deficiency, serum levels of 25-hydroxyvitamin D can be obtained. Even then, the optimal levels in pregnancy have not been established (De-Regil, 2016).

1	Although researchers continue to study the ideal nutritional regimen for the pregnant woman and her fetus, basic tenets for the clinician include: 1. Advise the pregnant woman to eat food types she wants in reasonable amounts and salted to taste. 2. Ensure that food is amply available for socioeconomically deprived women. 3. Monitor weight gain, with a goal of approximately 25 to 35elb in women with a normal BMI. 4. Explore food intake by dietary recall periodically to discover the occasional nutritionally errant diet. 5. Give tablets of simple iron salts that provide at least 27 mg of elemental iron daily. Give folate supplementation before and in the early weeks of pregnancy. Provide iodine supplementation in areas of known dietary insuiciency. 6. Recheck the hematocrit or hemoglobin concentration at 28 to 32 weeks' gestation to detect signiicant anemia. TABLE 9-6. Some Contraindications to Exercise During Pregnancy

1	6. Recheck the hematocrit or hemoglobin concentration at 28 to 32 weeks' gestation to detect signiicant anemia. TABLE 9-6. Some Contraindications to Exercise During Pregnancy Significant cardiovascular or pulmonary disease Significant risk for preterm labor: cerclage, multifetal gestation, significant bleeding, threatened preterm labor, prematurely ruptured membranes Obstetrical complications: preeclampsia, placenta previa, poorly controlled diabetes or epilepsy, morbid obesity, Summarized from American College of Obstetricians and Gynecologists, 2017g.

1	Summarized from American College of Obstetricians and Gynecologists, 2017g. More than half of the children in the United States are born to working mothers. Federal law prohibits employers from excluding women from job categories on the basis that they are or might become pregnant. he Family and Medical Leave Act of 1993 requires that covered employers must grant up to 12 work weeks of unpaid leave to an employee for the birth and care of a newborn child Qackson, 2015). In the absence of complications, most women can continue to work until the onset of labor (American Academy of Pediatrics and the American College of Obstetricians and Gynecologists, 2017).

1	Some types of work, however, may increase pregnancy complication risks. Mozurkewich and colleagues (2000) reviewed 29 studies that involved more than 160,000 pregnancies. With physically demanding work, women had 20-to 60-percent higher rates of preterm birth, fetal-growth restriction, or gestational hypertension. In a prospective study of more than 900 healthy nulliparas, women who worked had a ivefold risk of preeclampsia (Higgins, 2002). Newman and coworkers (2001) reported outcomes in more than 2900 women with singleton pregnancies. Occupational fatigue-estimated by the number of hours standing, intensity of physical and mental demands, and environmental stressors-was associated with an increased risk of preterm premature membrane rupture. For women reporting the highest degrees of fatigue, the risk was 7.4 percent.

1	Thus, any occupation that subjects the gravida to severe physical strain should be avoided. Ideally, no work or play is continued to the extent that undue fatigue develops. Adequate periods of rest should be provided. It seems prudent to advise women with prior pregnancy complications that commonly recur to minimize physical work. In general, pregnant women do not need to limit exercise, provided they do not become excessively fatigued or risk injury (Davenport, 2016). Clapp and associates (2000) reported that both placental size and birthweight were signiicantly greater in women who exercised. Duncombe and coworkers (2006) reported similar findings in 148 women. In contrast, vfagann and colleagues (2002) prospectively analyzed exercise behavior in 750 healthy women and found that working women who exercised had smaller infants and more dysfunctional labors.

1	The American College of Obstetricians and Gynecologists (2017 g) advises a thorough clinical evaluation before recommending an exercise program. In the absence of contraindications listed in Table 9-6, pregnant women are encouraged to engage in regular, moderate-intensity physical activity for at least 150 minutes each week. Each activity should be reviewed individually for its potential risk. Examples of safe activities are walking, running, swimming, stationary cycling, and low-impact aerobics. However, they should refrain from activities with a high risk of falling or abdominal trauma. Similarly, scuba diving is avoided because the fetus is at increased risk for decompression sickness.

1	In the setting of certain pregnancy complications, it is wise to abstain from exercise and even limit physical activity. For example, some women with pregnancy-associated hypertensive disorders, preterm labor, placenta previa, or severe cardiac or pulmonary disease may gain from being sedentary. Also, those with multiple or suspected growth-restricted fetuses may be served by greater rest.

1	Fish are an excellent source of protein, are low in saturated fats, and contain omega-3 fatty acids. The Avon Longitudinal Study of Parents and Children reported beneicial efects on pregnancy outcomes in women who consumed 340 g or more of seafood weekly (Hibbeln, 2007). Because nearly all fish and shellish contain trace amounts of mercury, pregnant and lactating women are advised to avoid specific types of ish with potentially high methylmercury levels. These include shark, swordfish, king mackerel, and tile fish. It is further recommended that pregnant women ingest 8 to 12 ounces of fish weely, but no more than 6 ounces of albacore or "white" tuna (U.S. Environmental Protection Agency, 2014). If the mercury content oflocally caught fish is unknown, then overall fish consumption should be limited to 6 ounces per week (American Academy of Pediatrics and the American College of Obstetricians and Gynecologists, 2017) .

1	Maternal lead exposure has been associated with several adverse maternal and fetal outcomes across a range of maternal blood lead levels (Taylor, 2015). These include gestational hypertension, miscarriage, low birthweight, and neurodevelopmental impairments in exposed pregnancies (American College of Obstetricians and Gynecologists, 2016c). The levels at which these risks rise remains unclear. However, recognizing that such exposure remains a significant health issue for reproductive-aged women, the CDC (201 Oa) has issued guidance for screening and managing exposed pregnant and lactating women. These guidelines, which have been endorsed by the American College of Obstetricians and Gynecologists (2016c), recommend blood lead testing only if a risk factor is identified. If the levels are > 5 /Lg1 dL, then counseling is completed, and the lead source is sought and removed. Subsequent blood levels are obtained. Blood lead levels >45 Lg/dL are consistent with lead poisoning, and women in

1	5 /Lg1 dL, then counseling is completed, and the lead source is sought and removed. Subsequent blood levels are obtained. Blood lead levels >45 Lg/dL are consistent with lead poisoning, and women in this group may be candidates for chelation therapy. Afected pregnancies are best managed in consultation with lead poisoning treatment experts. National and state resources are available at the CDC website: ww.cdc.gov/ncehllead/.

1	Pregnant women are encouraged to wear properly positioned three-point restraints as protection against automobile crash injury (Chap. 47, p. 927). The lap portion of the restraining belt is placed under the abdomen and across her upper thighs. The belt should be comfortably snug. The shoulder belt also is irmly positioned between the breasts. Airbags should not be disabled for the pregnant woman.

1	In general, air travel in a properly pressurized aircraft has no harmful efect on pregnancy (Aerospace Medical Association, 2003). Thus, in the absence of obstetrical or medical complications, the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (20 16a, 2017) have concluded that pregnant women can safely ly up to 36 weeks' gestation. It is recommended that pregnant women observe the same precautions for air travel as the general population. Seatbelts are used while seated. Periodic lower extremity movement and at least hourly ambulation help lower the venous thromboembolism threat. Signiicant risks with travel, especially international travel, are infectious disease acquisition and development of complications remote from adequate healthcare resources (Ryan, 2002).

1	In healthy pregnant women, sexual intercourse usually is not harmul. Whenever miscarriage, placenta previa, or preterm labor threatens, however, coitus is avoided. Nearly 10,000 women enrolled in a prospective investigation by the Vaginal Infection and Prematurity Study Group were interviewed regarding sexual activity (Read, 1993). hey reported a decreased frequency of coitus with advancing gestation. By 36 weeks, 72 percent had intercourse less than once weekly. The decline is attributed to lower desire and fear of harming the pregnancy (Bartellas, 2000; Staruch, 2016).

1	Intercourse speciically late in pregnancy is not harmful. Grudzinskas and coworkers (1979) noted no association between gestational age at delivery and coital frequency during the last 4 weeks of pregnancy. Sayle and colleagues (2001) reported no increased-and actually a decreased-risk of delivery within 2 weeks of intercourse. Tan and associates (2007) studied women scheduled for non urgent labor induction and found that spontaneous labor ensued at equal rates in groups either participating in or abstaining from intercourse. Oral-vaginal intercourse is occasionally hazardous. Aronson and Nelson (1967) described a fatal air embolism late in pregnancy as a result of air blown into the vagina during cunnilingus. Other near-fatal cases have been described (Bernhardt, 1988).

1	Examination of the teeth is included in the prenatal examination, and good dental hygiene is encouraged. Indeed, periodontal disease has been linked to preterm labor. Unfortunately, although its treatment improves dental health, it does not prevent preterm birth (Michalowicz, 2006). Dental caries are not aggravated by pregnancy. Importantly, pregnancy is not a contraindication to dental treatment including dental radiographs (Giglio, 2009) .

1	Current recommendations for immunization during pregnancy are summarized in Table 9-7. Well-publicized concerns regarding a causal link between childhood exposure to the thimerosal preservative in some vaccines and neuropsychological disorders have led to some parents to vaccine prohibition. Although controversy continues, these associations have proven groundless (Sugarman, 2007; Thompson, 2007; Tozzi, 2009). Thus, many vaccines may be used in pregnancy. The American College of Obstetricians and Gynecologists (20 16b) stresses the importance of integrating an efective vaccine strategy into the care of both obstetrical and gynecological patients. The College further emphasizes that information on the safety of vaccines given during pregnancy is subject to change, and recommendations can be found on the CDC website at ww.cdc.gov/vaccines.

1	he frequency of pertussis infection has substantially risen in the United States. Young infants are at increased risk for death from pertussis and are entirely dependent on passive immunization from maternal antibodies until the infant vaccine series is initiated at age 2 months. For this reason, a three-agent tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis (T dap) vaccine is recommended and is safe for pregnant women (Centers for Disease Control and Prevention, 2013b, 2016; Morgan, 2015). However, as demonstrated by Healy and coworkers (2013), maternal antipertussis antibodies are relatively shortlived, and T dap administration before pregnancy-or even in the first half of the current pregnancy-is not likely to provide a high level of newborn antibody protection. Thus, to maximize passive antibody transfer to the fetus, a dose of T dap is ideally given to gravidas between 27 and 36 weeks' gestation (American College of Obstetricians and Gynecologists, 2017j;

1	Thus, to maximize passive antibody transfer to the fetus, a dose of T dap is ideally given to gravidas between 27 and 36 weeks' gestation (American College of Obstetricians and Gynecologists, 2017j; Centers for Disease Control and Prevention, 2013b, 2016).

1	All women who will be pregnant during inluenza season should be ofered vaccination, regardless of gestational age. hose with underlying medical conditions that increase the risk for influenza complications are provided the vaccine before lu season starts. In addition to maternal protection against infection, prenatal maternal vaccination in one study reduced the infant influenza incidence in the irst 6 months of life by 63 percent (Zaman, 2008). Moreover, it reduced all febrile respiratory illnesses in these infants by a third. Women who are susceptible to rubella during pregnancy should receive measles, mumps, rubella (MMR) vaccination postpartum. Although this vaccine is not recommended during TABLE 9-7. Continued Rabies Postexposure prophylaxis Half dose at injury site, half dose in Used in conjunction with rabies deltoid killed-virus vaccine Tetanus Postexposure prophylaxis One dose 1M Used in conjunction with tetanus

1	Tetanus Postexposure prophylaxis One dose 1M Used in conjunction with tetanus Varicella Should be considered for One dose 1M within 96 hours of Indicated also for newborns or exposed pregnant women exposure women who developed varicella to protect against maternal, within 4 days before delivery or not congenital, infection 2 days following delivery Hepatitis A: Postexposure prophylaxis and 0.02 mUkg 1M in one dose Immune globulin should be given Hepatitis A those at high risk as soon as possible and within virus vaccine 2 weeks of exposure; infants should be used born to women who are with hepatitis A incubating the virus or are immune globulin acutely ill at delivery should receive one dose of0.5 mL as soon as possible after birth 'Two doses necessary for students entering institutions of higher education, newly hired medical personnel, and travel abroad. blnactlvated polio vaccine recommended for nonimmunized adults at increased risk.

1	10 = intradermally; 1M = intramuscularly; MMR = measles, mumps, rubella; PO = orally; SC = subcutaneously. From the Centers for Disease Control and Prevention, 201 1; Kim, 201 6. pregnancy, congenital rubella syndrome has never resulted from its inadvertent use. Breastfeeding is compatible with MMR vaccination (Centers for Disease Control and Prevention, 201l). Whether adverse pregnancy outcomes are related to cafeine consumption is somewhat controversial. As summarized from Chapter 18 (p. 348), heavy intake of cofee each day-about five cups or 500 mg of cafeine-slightly raises the miscarriage risk. Studies of "moderate" intake-less than 200 mg dailydid not find a higher risk.

1	It is unclear if cafeine consumption is associated with preterm birth or impaired fetal growth. Clausson and coworkers (2002) found no association between moderate cafeine consumption of less than 500 mg/d and low birthweight, fetalgrowth restriction, or preterm delivery. Bech and associates (2007) randomly assigned more than 1200 pregnant women who drank at least three cups of cofee per day to cafeinated versus decafeinated cofee. They found no diference in birthweight or gestational age at delivery between groups. The CARE Study Group (2008), however, evaluated 2635 lowrisk pregnancies and reported a l.4-fold risk for fetal-growth restriction among those whose daily cafeine consumption was >200 mg/d compared with those who consumed <100 mg/d. he American College of Obstetricians and Gynecologists (20e1e6d) concludes that moderate consumption of caffeine-less than 200 mg/ d-does not appear to be associated with miscarriage or preterm birth, but that the relationship between cafeine

1	(20e1e6d) concludes that moderate consumption of caffeine-less than 200 mg/ d-does not appear to be associated with miscarriage or preterm birth, but that the relationship between cafeine consumption and fetal-growth restriction remains unsettled. The American Dietetic Association (2008) recommends that cafeine intake during pregnancy be limited to less than 300 mg/d, which approximates three 5-oz cups of percolated cofee.

1	Nausea and vomiting are common complaints during the first half of pregnancy. These vary in severity and usually commence between the first and second missed menstrual period and continue until 14 to 16 weeks' gestation. Although nausea and vomiting tend to be worse in the morning-thus erroneously termed morning sickness-both symptoms frequently continue throughout the day. Lacroix and coworkers (2000) found that nausea and vomiting were reported by three fourths of pregnant women and lasted an average of35 days. Halfhad reliefby 14 weeks, and 90 percent by 22 weeks. In 80 percent of these women, nausea lasted all day.

1	Treatment of pregnancy-associated nausea and vomiting seldom provides complete relief, but symptoms can be minimized. Eating small meals at frequent intervals is valuable. One systematic literature search reported that the herbal remedy ginger was likely efective (Borrelli, 2005). Mild symptoms usually respond to vitamin BG given along with doxylamine, but some women require phenothiazine or HI-receptor blocking antiemetics (American College of Obstetricians and Gynecologists, 2015c). In some with hyperemesis gravidarum, vomiting is so severe that dehydration, electrolyte and acid-base disturbances, and starvation ketosis become serious problems.

1	Heartburn is another common complaint of gravidas and is caused by gastric content reflux into the lower esophagus. The greater frequency of regurgitation during pregnancy most likely results from upward displacement and compression of the stomach by the uterus, combined with relaxation of the lower esophageal sphincter. Avoiding bending over or lying flat is preventive. In most pregnant women, symptoms are mild and relieved by a regimen of more frequent but smaller meals. Antacids may provide considerable relief (Phupong, 2015). Specifically, aluminum hydroxide, magnesium trisilicate, or magnesium hydroxide is given alone or in combination. Management of heartburn or nausea that does not respond to simple measures is discussed in Chapter 54 (p. 1045).

1	The craving of pregnant women for strange foods is termed pica. Worldwide, its prevalence is estimated to be 30 percent (Fawcett, 2016). At times, nonfoods such as ice-pagophagia, starch-amylophagia, or clay-geophagia may predominate. This desire is considered by some to be triggered by severe iron deficiency. Although such cravings usually abate after deficiency correction, not all pregnant women with pica are iron deficient. Indeed, if strange "foods" dominate the diet, iron deficiency will be aggravated or will develop eventually.

1	Patel and coworkers (2004) prospectively completed a dietary inventory on more than 3000 women during the second trimester. The prevalence of pica was 4 percent. The most common nonfood items ingested were starch in 64 percent, dirt in 14 percent, sourdough in 9 percent, and ice in 5 percent. he prevalence of anemia was 15 percent in women with pica compared with 6 percent in those without it. Interestingly, the rate of spontaneous preterm birth before 35 weeks was twice as high in women with pica. Women during pregnancy are occasionally distressed by profuse salivation-pyalism. Although usually unexplained, ptyalism sometimes appears to follow salivary gland stimulation by the ingestion of starch. At least 5 percent of pregnancies are estimated to be complicated by new-onset or new-type headache (Spierings, 2016). Common headaches are virtually universal. Acetaminophen is suitable for most of these, and an in-depth discussion is found in Chapter 60 (p. 1057).

1	Low back pain to some extent is reported by nearly 70 percent of gravidas (Liddle, 2015; Wang, 2004). Minor degrees follow excessive strain or significant bending, lifting, or walking. It can be reduced by squatting rather than bending when reaching down, by using a back-support pillow when sitting, and by avoiding high-heeled shoes. Back pain complaints increase with progressing gestation and are more prevalent in obese women and those with a history of low back pain. In some cases, troublesome pain may persist for years after the pregnancy (Noren, 2002).

1	Severe back pain should not be attributed simply to pregnancy until a thorough orthopedic examination has been conducted. Severe pain has other uncommon causes that include pregnancy-associated osteoporosis, disc disease, vertebral osteoarthritis, or septic arthritis (Smith, 2008). More commonly, muscular spasm and tenderness are classiied clinically as acute strain or ibrositis. Although evidence-based clinical research directing care in pregnancy is limited, low back pain usually responds well to analgesics, heat, and rest. Acetaminophen may be used chronically as needed. Nonsteroidal antiinlammatory drugs may also be beneicial but are used only in short courses to avoid fetal efects (Chap. 12, p. 241). Muscle relaxants that include cyclobenzaprine or baclofen may be added when needed. Once acute pain is improved, stabilizing and strengthening exercises provided by physical therapy help improve spine and hip stability, which is essential for the increased load of pregnancy. For

1	Once acute pain is improved, stabilizing and strengthening exercises provided by physical therapy help improve spine and hip stability, which is essential for the increased load of pregnancy. For some, a support belt that stabilizes the sacroiliac joint may be helpful (Gutke, 2015).

1	Venous leg varicosities have a congenital predisposition and accrue with advancing age. They can be aggravated by factors that raise lower extremity venous pressures, such as an enlarging uterus. Femoral venous pressures in the supine gravida rise from 8 mm Hg in early pregnancy to 24 mm Hg at term. Thus, leg varicosities typically worsen as pregnancy advances, especially with prolonged standing. Symptoms vary from cosmetic blemishes and mild discomfort at the end of the day to severe discomfort that requires prolonged rest with feet elevation. Treatment is generally limited to periodic rest with leg elevation, elastic stockings, or both. Surgical correction during pregnancy generally is not advised, although rarely the symptoms may be so severe that injection, ligation, or even stripping of the veins is necessary.

1	Vulvar varicosities frequently coexist with leg varicosities, but they may appear without other venous pathology. Uncommonly, they become massive and almost incapacitating. If these large varicosities rupture, blood loss can be severe. Treatment is with specially itted pantyhose that will also minimize lower extremity varicosities. With particularly bothersome vulvar varicosities, a foam rubber pad suspended across the vulva by a belt can be used to exert pressure on the dilated veins.

1	Hemorrhoids are rectal vein varicosities and may irst appear during pregnancy as pelvic venous pressures rise. Commonly, they are recurrences of previously encountered hemorrhoids. Up to 40 percent of pregnant women develop these (Poskus, 2014). Pain and swelling usually are relieved by topically applied anesthetics, warm soaks, and stool-softening agents. With thrombosis of an external hemorrhoid, pain can be considerable. This may be relieved by incision and removal of the clot following injection of a local anesthetic.

1	Beginning early in pregnancy, many women experience fatigue and need greater amounts of sleep. his likely is due to the soporiic efect of progesterone but may be compounded in the irst trimester by nausea and vomiting. In the latter stages, general discomforts, urinary frequency, and dyspnea can be additive. Sleep duration may be related to obesity and gestational weight gain (Facco, 2016; Lockhart, 2015). Moreover, sleep eiciency appears to progressively diminish as pregnancy advances. Wilson and associates (201i1) performed overnight polysomnography and observed that women in the third trimester had poorer sleep eiciency, more awakenings, and less of both stage 4 (deep) and rapid-eye movement sleep. Women in the irst trimester were also afected, but to a lesser extent. Daytime naps and mild sedatives at bedtime such as diphenhydramine (Benadryl) can be helpful.

1	Since the irst successful cord blood transplantation in 1988, more than 25,000 umbilical cord blood transplantations have been performed to treat hemopoietic cancers and various genetic conditions (Butler, 2011). There are two types of cord blood banks. Public banks promote allogeneic donation, for use by a related or unrelated recipient, similar to blood product donation (Armson, 2015). Private banks were initially developed to store stem cells for future autologous use and charged fees for initial processing and annual storage. The American College of Obstetricians and Gynecologists (20 15d) has concluded that if a woman requests information on umbilical cord banking, information regarding advantages and disadvantages of public versus private banking should be explained. Some states have passed laws that require physicians to inform patients about cord blood banking options. Importantly, few transplants have been performed by using cord blood stored in the absence of a known

1	have passed laws that require physicians to inform patients about cord blood banking options. Importantly, few transplants have been performed by using cord blood stored in the absence of a known indication in the recipient (Screnci, 2016). The likelihood that cord blood would be used for the child or family member of the donor couple is considered remote, and it is recommended that directed donation be considered when an immediate family member carries the diagnosis of a speciic condition known to be treatable by hemopoietic transplantation (Chap. 56, p. 1075).

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1	Wang SM, Dezinno P, Maranets I, et al: Low back pain during pregnancy: prevalence, risk facrors, and outcomes. Obstet Gynecol 104:65, 2004 Washington State Health Care Authority: Ultrasonography (ultrasound) in pregnancy: a health technology assessmenr. 2010. Available at: http://www. hta.hca. wa.gov/ documen ts/i naLreporcul trasound. pdf. Accessed September 19,r2016 West KP: Vitamin A deiciency disorders in children and women. Food Nun Bull 24:578, 2003 Widen EM, Whyatt vl, Hoepner A, et al: Excessive gestational weight gain is associated with long-term body fat and weight retention at 7 y postpartum in African American and Dominican mothers with underweight, normal, and overweight prepregnancy BM!. Am J Clin NLltr 102(6):1460, 2015 Wilcox AJ, Baird DD, Dunson D, et al: Natural limits of pregnancy testing in relation to the expected menstrual period. JAMA 286: 1759, 2001

1	Wilson DL, Barnes M, Ellett L, et al: Decreased sleep eiciency, increased wake after sleep onset and increased cortical arousals in late pregnancy. Aust N Z J Obstet Gynaecol 51(1):38, 2011 Worthen N, Bustillo M: Efect of urinary bladder fullness on fundal height measurements. Am J Obstet Gynecol 138:759, 1980 XU J, Kochanek KD, Murphy SL: Deaths: inal data for 2007. Nat Stat Vit Rep 58(19):1, 2010 Zaman K, Roy E, Arifeen SE, et al: Efectiveness of maternal inluenza immunization in mothers and infants. N Engl J vIed 359(15): 1555, 2008 SONOGRAPHY IN OBSTETRICSe. . . . . . . . . . . . . . . . . . .. 182 TECHNOLOGY AND SAFETYe. . . . . . . . . . . . . . . . . . . . . . 182 GESTATIONAL AGE ASSESSMENT . . . . . . . . . . . . . . . .. 183 FIRST-TRIMESTER SONOGRAPHY ....e.....e...e...... 185 SECOND-AND THIRD-TRIMESTER SONOGRAPHY .... 186 CERVICAL LENGTH ASSESSMENT. . . . . . . . . . . . . . . .. 189 NORMAL AND ABNORMAL FETAL ANATOMY. ....... 191

1	SECOND-AND THIRD-TRIMESTER SONOGRAPHY .... 186 CERVICAL LENGTH ASSESSMENT. . . . . . . . . . . . . . . .. 189 NORMAL AND ABNORMAL FETAL ANATOMY. ....... 191 DOPPLER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 MAGNETIC RESONANCE IMAGING ....e...e.......... 215 After discovey of the Roentgen ray and the demonstration of the various uses to which it might be put, it was thought possible that it might also aord a valuable method ofinvestigating the shape and size of the pelvis. -]. Whitridge Williams (1903)

1	-]. Whitridge Williams (1903) X-ray techniques were just on the horizon when the first edition of this textbook was published. he first application focused on the maternal pelvis without attention to the fetus. hus, congenital abnormalities were routinely not discovered until birth. Subsequent radiographic eforts to evaluate the fetus were later replaced by ultrasonography and more recently by magnetic resonance (MR) imaging, techniques which have become increasingly sophisticated. he subspecialty of fetal medicine has developed only because of these advances, and today's practitioner can hardly imagine obstetrical care without them.

1	Prenatal sonography can be used to accurately assess gestational age, fetal number, viability, and placental location, and it can aid diagnosis of many fetal abnormalities. With improvements in resolution and image display, anomalies are increasingly detected in the irst trimester, and Doppler is used to manage pregnancies complicated by growth impairment or anemia. he American College of Obstetricians and Gynecologists (2016) recommends that prenatal sonography be performed in all pregnancies and considers it an important part of obstetrical care in the United States.

1	The real-time image on the ultrasound screen is produced by sound waves that are relected back from fluid and tissue interfaces of the fetus, amnionic fluid, and placenta. Sector array transducers contain groups of piezoelectric crystals working simultaneously in arrays. These crystals convert electrical energy into sound waves, which are emitted in synchronized pulses. Sound waves pass through tissue layers and are reflected back to the transducer when they encounter an interface between tissues of diferent densities. Dense tissue such as bone produces high-velocity reflected waves, which are displayed as bright echoes on the screen. Conversely, luid generates few relected waves and appears dark. Digital images generated at 50 to more than 100 frames per second undergo postprocessing that yields the appearance of real-time imaging.

1	Ultrasound refers to sound waves traveling at a frequency above 20,000 hertz (cycles per second). Higher-frequency transducers yield better image resolution, whereas lower frequencies penetrate tissue more efectively. Transducers use wide-bandwidth technology to perform within a range of frequencies. In early pregnancy, a 5-to 10-megahertz (MHz) transvaginal transducer usually provides excellent resolution, because the early fetus is close to the transducer. And, in the first and second trimesters, a 4-to 6-MHz transabdominal transducer is similarly close enough to the fetus to yield precise images. By the third trimester, however, a lower frequency 2-to 5-MHz transducer may be needed for tissue penetration-particularly in obese patients-and this can lead to compromised resolution.

1	cation, using the lowest possible exposure setting to gain neces sary information-the AAA principle-as low as reasonably 4chievable. Examinations are performed only by those trained pathology, using techniques to avoid ultrasound exposure beyond what is considered safe for the fetus (American College of Obstetricians and Gynecologists, 2016; American Institute of Ultrasound in Medicine, 2013b). No causal relationship has ognized adverse efect in human pregnancy. The International Society of Ultrasound in Obstetrics and Gynecology (2016) further concludes that there is no scientifically proven associa tion between ultrasound exposure in the first or second trimes ters and autism spectrum disorder or its severity.

1	All sonography machines are required to display two indices: the thermal index and the mechanical index. The thermal index is a measure of the relative probability that the examination may raise the temperature, potentially high enough to induce injury. That said, fetal damage resulting from commercially available ultrasound equipment in routine practice is extremely unlikely. The potential for temperature elevation is higher with longer examination time and is greater near bone than in soft tissue. heoretical risks are higher during organogenesis than later in gestation. The thermal index for soft tissue, Tis, is used before 10 weeks' gestation, and that for bone, ib, is used at or beyond 10 weeks' (American Institute of Ultrasound in Medicine, 2013b). The thermal index is higher with pulsed Doppler applications than with routine B-mode scanning (p. 213). In the first trimester, if pulsed Doppler is clinically indicated, the thermal index should be ;0.7, and the exposure time should

1	Doppler applications than with routine B-mode scanning (p. 213). In the first trimester, if pulsed Doppler is clinically indicated, the thermal index should be ;0.7, and the exposure time should be as brief as possible (American Institute of Ultrasound in Medicine, 2016) . To document the embryonic or fetal heart rate, motion-mode (M-mode) imaging is used instead of pulsed Doppler imaging.

1	The mechanical index is a measure of the likelihood of adverse efects related to rarefactional pressure, such as cavi tationwhich is relevant only in tissues that contain air. Microbubble ultrasound contrast agents are not used in pregnancy for this reason. In mammalian tissues that do not contain gas bodies, no adverse efects have been reported over the range of diagnostically relevant exposures. Because fetuses cannot contain gas bodies, they are not considered at risk. The use of sonography for any nonmedical purpose, such as "keepsake fetal imaging," is considered contray to responsible medical practice and is not condoned by the Food and Drug Administration (2014), the American Institute of Ultrasound in Medicine (2012, 2013b), or the American College ofObstetricians and Gynecologists (2016).

1	The reported prevalence of work-related musculoskeletal discomfort or injury among sonographers approximates 70 percent Ganga, 2012; Roll, 2012). The main risk factors for injury during transabdominal ultrasound examinations are awkward posture, sustained static forces, and various pinch grips used while maneuvering the transducer (Centers for Disease Control and Prevention, 2006). Maternal habitus can be contributory because more force is oten employed when imaging obese patients. The following guidelines may help avert injury: 1. Position the patient close to you on the examination table. As a result, your elbow is close to your body, shoulder abduction is less than 30 degrees, and your thumb is facing up. 2. Adjust the table or chair height so that your forearm is parallel to the floor. 3. If seated, use a chair with back support, support your feet, and keep ankles in neutral position. Do not lean toward the patient or monitor. 4.

1	3. If seated, use a chair with back support, support your feet, and keep ankles in neutral position. Do not lean toward the patient or monitor. 4. Face the monitor squarely and position it so that it is viewed at a neutral angle, such as 15 degrees downward. 5. Avoid reaching, bending, or twisting while scanning. 6. Frequent breaks may prevent muscle strain. Stretching and strengthening exercises can be helpful. The earlier that sonography is performed, the more accurate the gestational age assessment. Specific criteria for "re-dating" a pregnancy, that is, reassigning the gestational age and estimated date of delivery using initial sonogram findings, are shown in Table 1 0-1. The only exception to revising the gestational age based on early sonography is if the pregnancy resulted from assisted reproductive technology, in which case accuracy of gestational age assessment is presumed.

1	Sonographic measurement of the crown-rump length (CRL) is the most accurate method to establish or conirm gestational TABLE 10-1. Sonographic Gestational Age Assessment Gestational Age Parameter(s) to Revisea 9 to <14 wks CRL 14 to <16 wks BPO, HC AC FL 16 to <22 wks BPO, HC AC FL >10 d 22 to <28 wks BPO, HC AC FL >14d :28 weeks BPOt HC AC FL >21 d aSonographic gestational age should be used when the LMP-derived gestational age differs from that obtained with sonography by the threshold value. abdominal circumference; BPO biparietal diameter; CRL = crown-rump length; FL = femur length; HC = head circumference; LMP = last menstrual period. Modified from American College of Obstetricians and Gynecologists,l2017b. FIGURE 10-1 The measured crown-rump length in this 12-week 3-day fetus approximates 6 cm.

1	FIGURE 10-1 The measured crown-rump length in this 12-week 3-day fetus approximates 6 cm. age (Appendix, p. 1263). As noted, transvaginal imaging typi cally yields higher resolution images. The CL is measured in the midsagittaleplane with the embryo or fetus in a neutral, nonlexed position so that its length can be measured in a straight line (Fig. The measurement should include neither the yolk sac nor a limb bud. The mean of three discrete measurements is used. Until 136/7 weeks' gestation, the CL is accurate to within 5 to 7 days (American College of Obstetricians and Gynecologists, 201 7b).

1	Starting at 14°/ weeks, equipment software formulas calculate estimated gestational age and fetal weight from measurements of the biparietal diameter, head and abdominal circumference, and femur length (Fig. 10-2). The estimates are most accurate when multiple parameters are used but may over-or underestimate fetal weight by up to 20 percent (American College of Obstetricians and Gynecologists, 2016). Various nomograms for other fetal structures, including the cerebellar diameter, ear length, ocular distances, thoracic circumference, and lengths of kidney, long bones, and feet, may be used to address speciic questions regarding organ system abnormalities or syndromes (Appendix, p. 1266).

1	The biparietal diameter (BPD) most accurately reflects gestational age, with a variation of 7 to 10 days in the second trimester. The BPD is measured perpendicular to the midline falx in the transthalamic view, at the level of the thalami and cavum septum pellucidum (CSP) (see Fig. 10-2A). Calipers are placed from the outer edge of the skull in the near ield to the inner edge of the skull in the far field. he head circumference (HC) is also measured in the trans thalamic view. An ellipse is

1	FIGURE 10-2 Fetal biometry. A. Transthalamic view. A transverse (axial) image of the head is obtained at the level of the cavum septum pellucidum (arrows) and thalami (asterisks). The biparietal diameter is measured perpendicular to the sagittal midline, from the outer edge of the skull in the near field to the inner edge of the skull in the far field. By convention, the near field is that which is closer to the sonographic transducer. The head circumference is measured circumferentially around the outer border of the skull. B. Femur length. The femur is measured perpendiCUlar to the femoral shaft, from each diaphyseal end, excluding the epiphysis. C. Abdominal circumference. This is a transverse measurement at the level of the stomach (5). The J-shaped structure (arrowheads) indicates the confluence of the umbilical vein and the right portal vein. Ideally, only one rib is visible on each side of the abdomen, indicating that the image was not taken at an oblique angle.

1	placed around the outer edge of the skull or the circumference is calculated using BPD and occipital-frontal diameter (OFD) values. he cephalic index, which is the BPD divided by the OFD, is normally 70 to 86 percent. If the head shape is lattened-dolichocephay, or roundedbrachycephay, the HC is more reliable than the BPD. hese head shape variants may be normal or can be secondary to fetal position or oligohydramnios. But, dolichocephaly can occur with neural-tube defects, and brachycephaly may be seen in fetuses with Down syndrome. lso, with abnormal skull shape, craniosynostosis and other craniofacial abnormalities are a consideration.

1	he femur length (FL) correlates well with both BPD and gestational age. It is measured with the beam perpendicular to the long axis of the shat. Calipers are placed at each end of the calcified diaphysis and exclude the epiphysis. For gestational age estimation, it has a variation of 7 to 11 days in the second trimester (see Fig. 1O-2B). A femur measurement that is <2.5th percentile for gestational age or that is shortened to :;90 percent of that expected based on the measured BPD is a minor marker for Down syndrome (Chap. 14, p.287). he normal range for the FL to abdominal circumference (AC) ratio is generally 20 to 24 percent. A dramatically foreshortened FL or a FL-to-AC ratio below 18 percent prompts evaluation for a skeletal dysplasia (p. 210).

1	Of biometric parameters, AC is most afected by fetal growth. Thus, for gestational age estimation, AC has the greatest variation, which can reach 2 to 3 weeks in the second trimester. To measure the AC, a circle is placed outside the fetal skin in a transverse image that contains the stomach and the confluence of the umbilical vein with the portal sinus (see Fig. 10-2C). The image should appear as round as possible and ideally contains no more than 1 rib on each side of the abdomen. he kidneys should not be visible in the image. Variability of the sonographic gestationl age estimate increases with advancing gestation. Accordingly, pregnancies not imaged prior to 22 weeks to conirm or revise gestational age are considered suboptimaly ated (American College of Obstetricians and Gynecologists, 2017a). Although the estimate is improved by averaging multiple parameters, if one parameter difers TABLE 10-2. Some Indications for First-Trimester Ultrasound Examination

1	TABLE 10-2. Some Indications for First-Trimester Ultrasound Examination Define the cause of vaginal bleeding Assist chorionic villus sampling, embryo transfer, and Assess for certain fetal anomalies, such as anencephaly, in a bnormaIities Measure nuchal translucency /hen part of a screening Modified from the American Institute of Ultrasound in significantly from the others, consideration should be given to excluding it from the calculation. he outlier may result from poor visibility, but it could also indicate a fetal abnormality or growth problem. Reference tables such as the one in the Appendix (p. 1264) may be used to estimate fetal weight percentiles.

1	Indications for sonography before 14 weeks' gestation are listed in Table 10-2. Early pregnancy can be evaluated using transabdominal or transvaginal sonography, or both. he components listed in Table 10-3 should be assessed. First-trimester sonography can reliably diagnose anembryonic gestation, embryonic demise, ectopic pregnancy, and gestational trophoblastic disease. he irst trimester is also the ideal time to evaluate the uterus, adnexa, and cul-de-sac. Determination of TABLE 10-3. Components of Standard Ultrasound Examination by Trimester Gestational sac size, location, and number Embryo and/or yolk sac identification Crown-rump length Fetal number, including amnionicity and chorionicity of the first rimester Evaluation of the maternal uterus, adnexa, and cul-de-sac Evaluat"on of the fetal nuchal region, with consideration of fetal :chal translucenry assessment Fetal number, including amnionicity and chorionicity of multifetal gestations

1	Fetal number, including amnionicity and chorionicity of multifetal gestations Placental location, appearance, and relationship to tie internal cervical os, with documentation of placental cord insertion site when technically possible Fetal anatomical survey, including documentation of technical limitations Evaluation of the maternal uterus, adnexa, and cervix when Modified from the American Institute of Ultrasound in Medicine, 2013a. chorionicity in a multifetal gestation is most accurate in the first trimester (Chap. 45, p. 868).

1	Modified from the American Institute of Ultrasound in Medicine, 2013a. chorionicity in a multifetal gestation is most accurate in the first trimester (Chap. 45, p. 868). n intrauterine gestational sac is reliably visualized with transvaginal sonography by 5 weeks, and an embryo with cardiac activity by 6 weeks (Fig. 10-3). The embryo should be visible transvaginally once the mean sac diameter has reached 25 mm-otherwise the gestation is anembyonic. Cardiac motion is usually visible with transvaginal imaging when the embryo length reaches 5 mm. In embryos <7 mm without cardiac activity, subsequent examination may be needed to determine viability (American College of Obstetricians and Gynecologists, 2016). At Parkland hospital, first-trimester demise is diagnosed if the embryo has reached 10 mm and lacks cardiac motion. Other criteria for this diagnosis are found in Chapter 18 (Table 18-3, p. 350).

1	Nuchal translucency (NT) evaluation is a component of first-trimester aneuploidy screening, discussed in Chapter 14 (p. 281). It represents the maximum thickness of the subcutaneous translucent area between the skin and soft tissue overlying the fetal spine at the back of the neck. NT is measured in the sagittal plane between 11 and 14 weeks' gestation using precise criteria (Table 10-4). When the NT measurement is increased, the risk for fetal aneuploidy and various structural anomalies-in particular heart defects-is signiicantly elevated.

1	Assessment for selected fetal abnormalities in an at-risk pregnancy is done with irst-trimester sonography (see Table 10-2). Research in this area has focused on anatomy visible at 11 to 14 weeks' gestation, to coincide with sonography performed as part of aneuploidy screening. ith current technoloy, it is not realistic to expect that all major abnomalities detectable in the second trimester may be visualized in the irst trimester. Thus, irst-trimester scanning should not replace second-trimester anatomical evaluation (American College of Obstetricians and Gynecologists, 2016). FIGURE 10-3 A. The measured crown-rump length is approximately 7 mm in this 6-week embryo. B. M-mode demonstrates embryonic cardiac activity and a heart rate of 124 beats per minute.

1	As examples, in one study of more than 40,000 pregnancies undergoing sonographic aneuploidy screening between 11 and 14 weeks, basic anatomical evaluation yielded a detection rate of approximately 40 percent for structural abnormalities (Syngelaki, 2011). Bromley and colleagues (2014) similarly found that late irst-trimester sonography identiied major abnormalities in 0.5 percent of pregnancies, representing approximately 40 percent of pregnancies with anomalies detected prenatally. Detection rates are very high for anencephaly, alobar holoprosencephaly, and ventral wall defects. But, in one analysis of more than 60,000 pregnancies with these early scans, only a third of major cardiac anomalies were identiied, and no cases of microcephaly, agenesis of the corpus callosum, cerebellar abnormalities, congenital pulmonary airway malformations, or bowel obstruction were detected (Syngelaki, 2011). In another study of low-risk or unselected pregnancies, 32 percent of anomalies were

1	abnormalities, congenital pulmonary airway malformations, or bowel obstruction were detected (Syngelaki, 2011). In another study of low-risk or unselected pregnancies, 32 percent of anomalies were detected, whereas in pregnancies described as high-risk, anomaly detection exceeded 60 percent (Karim, 2017).

1	It is recommended that sonography be routinely ofered to all pregnant women between 18 and 22 weeks' gestation (American TABLE 10-4. Guidelines for Nuchal Translucency (NT) Measurement The margins of NT edges must be clear enough for proper caliper placement The fetus must be in the midsagittal plane The image must be magnified so that it is filled by the fetal head, neck, and upper thorax The fetal neck must be in a neutral position, not flexed and not hyperextended The amnion must be seen as separate from the NT line Electronic calipers must be used to perform the measurement The + calipers must be placed on the inner borders of the nuchal space with none of the horizontal crossbar itself protruding into the space The calipers must be placed perpendicular to the long axis of the fetus The measurement must be obtained at the widest space of the NT From the American Institute of Ultrasound in Medicine, 20 13a, with permission.

1	From the American Institute of Ultrasound in Medicine, 20 13a, with permission. TABLE 10-5. Some Indications for Second-or ThirdTrimester Ultrasound Examination Preterm premature rupture of membranes and/or preterm labor Adjunct to cervical cerclage Adjunct to amniocentesis or other procedure Adjunct to external cephalic version Assessment for findings that raise the aneuploidy risk Follow-up evaluation of a fetal anomaly History of congenital anomaly in prior pregnancy Adapted from the American Institute of Ultrasound in Medicine,l201l3a.

1	Follow-up evaluation of a fetal anomaly History of congenital anomaly in prior pregnancy Adapted from the American Institute of Ultrasound in Medicine,l201l3a. College of Obstetricians and Gynecologists, 2016). This time interval permits accurate assessment of gestational age, fetal anatomy, placental location, and cervical length. Recognizing that the gestational age at which abnormalities are identified may afect pregnancy management options, providers may opt to perform the examination prior to 20 weeks. The many additional indications for second-and third-trimester sonography are listed in Table 10-5. The three examination types are standard, specialized-which includes targeted sonography, and limited.

1	The standard sonogram includes evaluation of fetal number and presentation, cardiac activity, amnionic fluid volume, placental position, fetal biometry, and fetal anatomy (American Institute of Ultrasound in Medicine, 2013b). When technically feasible, the maternal cervix and adnexa are examined as clinically appropriate. Components are found in Table 10-3, and the fetal anatomical structures that should be evaluated are listed in Table 10-6. With twins or other multiples, documentation also includes the number of chorions and amnions, comparison of fetal sizes, estimation of amnionic luid volume within each sac, and fetal sex determination (Chap. 45, p. 868).

1	he targeted sonogram is a type of specialized examination. It is performed when the risk for a fetal anatomical or genetic abnor mality is elevated because of history, screening test result, or abnor mal inding during standard examination (Table 10-7) . Targeted sonograms include a detailed anatomical survey, the components of which are shown in Table 10-6. Because it carries the CPT code 76811, this sonogram is colloquilly called the "76811 examina tion." It is intended to be indication-driven and should not be repeated later in the absence of an extenuating circumstance.

1	have expertise in fetal imaging, through both training and ongo ing experience (Wax, 2014). For many of the targeted examina tion components, the physician determines on a case-by-case basis whether assessment is needed (American College of Obstetricians and Gynecologists, 2016). Other types of specilized examinations include fetal echocardiography, Doppler evaluation, and the bio physical proile, which is described in Chapter 17 (p. 337). A limited sonogram is performed to address a speciic clini cal question. Examples include evaluation of fetal presentation, viability, amnionic luid volume, or placental location. In the absence of an emergency, a limited examination is only per formed if a standard sonogram has already been completed. Otherwise, provided that the gestational age is at least 18 weeks, a standard sonogram is recommended.

1	Otherwise, provided that the gestational age is at least 18 weeks, a standard sonogram is recommended. With current advances in imaging technology, approximately 50 percent of major fetal abnormalities overall are detected with standard sonography (Rydberg, 2017). he sensitivity of sonography for detecting fetal anomalies varies according to factors such as gestational age, maternal habitus, fetal position, equipment features, examination type, operator skill, and the speciic abnormality in question. For example, maternal obesity has been associated with a 20-percent reduction in the anomaly detection rate (Dashe, 2009). Detection also varies considerably according to the abnormality.

1	Detection also varies considerably according to the abnormality. For example, population-based data from 18 registries comprise the EUROCAT network. Between 2011 and 2015, EUROCAT (2017) prenatal detection rates for selected fetal anomaliesexcluding genetic conditions-were as follows: anencephaly, 99 percent; spina bifida, 89 percent; hydrocephaly, 78 percent; clet lip/palate, 68 percent; hypoplastic let heart, 87 percent; transposition of the great vessels, 64 percent; diaphragmatic hernia, 74 percent; gastroschisis, 94 percent; omphalocele, 92 percent; bilateral renal agenesis, 94 percent; posterior urethral valves, 79 percent; limb-reduction defects, 57 percent; and clubfoot, 57 percent.

1	Importantly, however, the overall anomaly detection rate, excluding aneuploidy, was below 40 percent. his relects inclusion of anomalies with minimal or no sonographic detection in the second trimester, such as microcephaly, choanal atresia, cleft palate, Hirschsprung disease, anal atresia, and congenital skin disorders. hese are mentioned because clinicians tend to focus on abnormalities amenable to sonographic detection, whereas those not readily detectable may be equally devastating to families. Therore, evey sonographic examination should include a frank discussion of examination limitations. Most anomalous neonates are born to women whose pregnancies are otherwise considered low-risk, that is, without an TABLE 10-6. Components of Standard and Targeted Fetal Anatomic Surveys Head, face, and neck Lateral cerebral ventricles Choroid plexus Midline falx Cavum septum pellucidum Cerebellum Cistena magna Upper lip Consideration of nuchal skin fold measurement at 15-20 weeks

1	Head, face, and neck Lateral cerebral ventricles Choroid plexus Midline falx Cavum septum pellucidum Cerebellum Cistena magna Upper lip Consideration of nuchal skin fold measurement at 15-20 weeks Four-chamber view of the heart Left ventricular outflow tract Right ventricular outflow tract Stomach: presence, size, and situs Cervical, thoracic, lumbar, and sacral spine Head, face, and neck Integrity and shape of cranium Third ventriclea Fourth ventriclea Corpus callosuma Cerebellar lobes, vermis Brain parenchyma Profile Coronal nose, lips, lensa Palatea, maxilla, mandible, and tonguea Ear pOSition and sizea Orbitsa Neck Integrity of diaphragm Abdomen Small and large bowela Adrenal glandsa Gallbladdera Liver Renal arteriesa Spleen° Integrity of abdominal wall Integrity of spine and overlying soft tissue Architecture, position, number Hands Digitsa: number, position aWhen medically indicated (determined on case-by-case basis).

1	Integrity of spine and overlying soft tissue Architecture, position, number Hands Digitsa: number, position aWhen medically indicated (determined on case-by-case basis). Modified from the American Institute of Ultrasound in Medicine, 2013a; Wax, 2014.

1	indication for targeted sonography. hus, for standard sonographic examinations, accurate documentation and quality assurance are essential to optimize detection rates. Practice guidelines and standards established by organizations such as the American Institute of Ultrasound in Medicine (2013b) and the Internationl Society of Ultrasound in Obstetrics and Gynecology (Salomon, 2011) have undoubtedly contributed to improvements in anomaly detection rates. Ultrasound practice accreditation is a process ofered by the American Institute of Ultrasound in Medicine and the American College of Radiology that was developed to improve imaging quality and adherence to guidelines. It includes review of images and their storage, ultrasound equipment, report generation, and the qualifications of physicians and sonographers. The Society for Maternal-Fetal Medicine (2013) recommends that whenever possible, obstetrical ultrasound examinations by maternal-fetal medicine sub specialists be performed by

1	and sonographers. The Society for Maternal-Fetal Medicine (2013) recommends that whenever possible, obstetrical ultrasound examinations by maternal-fetal medicine sub specialists be performed by accredited practices.

1	Evaluation of amnionic luid volume is a component of every second-or third-trimester sonogram, and volumes vary with TABLE 10-7. Indications for Targeted Fetal Anatomical Ultrasound Examination Prior fetus or neonate with a structural or genetic/chromosomal abnormality Current pregnancy with known or suspected fetal abnormality or confirmed growth abnormality Maternal diabetes diagnosed before 24 weeks' gestation Assisted repr0ductive technology to achieve conception Matenal prepregnancy body mass index >30 kg/m2 Multifetal gestation (Chap. 45, p. 864) Nuchal translucency measurement ::3.0 mm Parental carriage of genetic/chromosomal abnormality Maternal age ::35 at delivery Nuchal translucency measurement ::3.0 mm Other condition affecting the fetus Congenital infection (Chaps. 64 and 65) Alloimmunization (Chap. 15, p. 301) Amnionic fluid abnormality (Chap. 11l, p. 227) Modified from Jax, 2014, 2015.

1	Other condition affecting the fetus Congenital infection (Chaps. 64 and 65) Alloimmunization (Chap. 15, p. 301) Amnionic fluid abnormality (Chap. 11l, p. 227) Modified from Jax, 2014, 2015. gestational age. Oligohydramnios indicates an amnionic fluid volume below normal range, and subjective crowding of the fetus is often noted. Hydramnios-also calledpoyhydramniosdefines a volume above a given normal threshold. Amnionic luid volume is usually assessed semiquantitatively. Measurements include either the single deepest vertical luid pocket or the sum of the deepest vertical pockets from each of four equal uterine quadrants-the amnionic luid index (Phelan, 1987). Reference ranges have been established for both measurements from 16 weeks' gestation onward. he single deepest vertical pocket is normally between 2 and 8 cm, and the amnionic luid index normally ranges between 8 and 24 cm. A further discussion and images are provided in Chapter 11 (p. 227).

1	Evaluation of the relationship between the placenta and the internal cervical os is an essential component of the standard sonogram. Abnormalities of the placenta and umbilical cord are reviewed in Chapter 6 (p. 111). Although the cervix may be imaged transabdominally (Fig. 10-4), this is often limited FIGURE 10-4 A. Transabdominal image of the cervix depicting the internal os and external as. B. Transvaginal imaging a more accurate evaluation of the cervix and should be used for medical decision-making. In this image, arrowheads mark the endocervical canal. (Used with permission from Dr. Emily Adhikari.) TABLE 10-8. Criteria for Transvaginal Evaluation of the Cervix Imaging the Cervix Maternal bladder should be empty. Transducer is inserted under real-time observation, identiying midsagittal intenal os, and then extenal os, while keeping the internal os in view.

1	Maternal bladder should be empty. Transducer is inserted under real-time observation, identiying midsagittal intenal os, and then extenal os, while keeping the internal os in view. Internal os, external os, and entire endocervical canal should be visible. The internal os may appear as a small triangular indentation at the junction of the amnionic cavity and endocervical canal. Image is enlarged so that the cervix fills approximately 75% of the screen. Anterior and posterior width of the cervix should be approximately equal. Transducer is pulled back slightly until the image begins to blur, ensuring that pressure is not placed on the cervix, then inserted only enough to restore a clear image. and without fundal or suprapubic pressure, to assess for dynamic change-or shortening on real-time imaging. Measuring the Cervix Calipers are placed where anterior and posterior walls of cervix meet. Endocervical canal appears as a faint, linear echodensity.

1	Measuring the Cervix Calipers are placed where anterior and posterior walls of cervix meet. Endocervical canal appears as a faint, linear echodensity. If canal has a curved contour, a straight line between the internal and external os will deviate from the path of the endocervical canal. If midpoint of the line between the internal and external canal deviates by �3 mm from the endocervical canal, measure the cervical length in two linear segments. Funneling, sludge (debris), or dynamic change is noted. At least three separate images are measured during a period of at least 3 minutes to allow for dynamic change. Visualization of cervical shortening on real-time imaging, with or without fundal or suprapubic pressure, raises preterm birth risks. Shortest cervical length image that meets all criteria should be used. Modified from lams, 2013.

1	Shortest cervical length image that meets all criteria should be used. Modified from lams, 2013. by technical factors that include maternal habitus, cervical position, or shadowing by the fetal presenting part. In addition, the maternal bladder or pressure from the transducer may artificially elongate the appearance of the cervix. As a result, values from transabdominal or transvaginal measurement of the cervix can difer significantly.

1	If the cervix appears shortened or if it cannot be adequately visualized during transabdominal evaluation, transvaginal assessment is considered (American Institute of Ultrasound in Medicine, 2013b). Only cervical length measurements obtained transvaginally at or beyond 16 weeks' gestation are considered sufficiently accurate for clinical decision-making (see Fig. 10-4). A foreshortened cervix is associated with an elevated risk for preterm birth, particularly in the setting of prior preterm birth, and the degree of risk rises proportionally with the degree of cervical shortening (Chap. 42, p. 815).

1	To measure the cervix transvaginally, the imaging criteria shown in Table 10-8 are followed. he endocervical canal should be visible in its entirety, and images ideally are obtained over several minutes to allow for dynamic change. During examination, visible funneling or debris is sought. Funneling is a protrusion of amnionic membranes into a portion of the endocervical canal that has dilated (Fig. 10-5). Funneling is not an independent predictor of preterm birth, however, it is associated with cervical shortening, and transvaginal assessment is recommended if a funnel is suspected transabdominally. The cervical length is measured distal to the funnel, because the base of the funnel becomes the functional internal os. If the cervix is dilated, as with cervical insuiciency, the membranes may prolapse through the endocervical canal and into the vagina, producing an

1	FIGURE 10-5 Transvaginal image depicting a foreshortened cervix with funneling. Funneling is a protrusion of amnionic membranes into a portion of the endocervical canal that has dilated. The distal protruding edge of the funnel becomes the functional internal os (left arrow). Thus, the measured cervical length, which lies between the arrows, should not include the funnel. (Used with permission from Dr. Emily Adhikari.) FIGURE 10-7 Transcerebellar view of the posterior fossa, demonstrating measurement of the cerebellum (+), cisterna magna (x), and nuchal fold thickness (backet). Care is taken not to angle obliquely down the spine, which may artificially increase the nuchal fold measurement.

1	FIGURE 10-6 The transventricular view depicts the lateral ventricles, which contain the echogenic choroid plexus (CP). The lateral ventricle is measured at the atrium (arrows), which is the confluence of the temporal and occipital horns. A normal measurement is between 5 and 10 mm throughout the second and third trimesters. The atria measured 6 mm in this 21-week fetus. hourglass appearance. Sludge or debris represents an aggregate of particulate matter within the amnionic sac, close to the internal os. In pregnancies at risk for preterm birth, sludge is associated with a further increased risk.

1	Many fetal anomalies and syndromes may be characterized with targeted sonography, and selected abnormalities are discussed subsequently. This list is not intended to be comprehensive but covers abnormalities commonly detected with standard sonography and those that are potentially amenable to fetal therapy. Sonographic features of chromosomal abnormalities are reviewed in Chapters 13 and 14, and fetal therapy is discussed in Chapter 16.

1	Standard sonographic evaluation of the fetal brain includes three transverse (axial) views. The transthalamic view is used to measure the BPD and HC and includes the midline falx, cavum septum pellucidum (CSP), and thalami (see Fig. 10-IA). The CSP is the space between the two laminae that separate the frontal horns of the lateral ventricles. Inability to visualize a normal CSP may indicate a midline brain abnormality such as agenesis of the corpus callosum, lobar holoprosencephaly, or septo-optic dysplasia (de Morsier syndrome). The transventricular view includes the lateral ventricles, which contain the echogenic choroid plexus (Fig. 10-6). The ventricles are measured at their atrium, which is the confluence of the temporal and occipital horns. The transcerebellar view is obtained by angling the transducer back through the posterior fossa (Fig. 10-7). In this view, the cerebellum and cisterna magna are measured, and between 15 and about 20 weeks, the nuchal skinfold thickness may

1	angling the transducer back through the posterior fossa (Fig. 10-7). In this view, the cerebellum and cisterna magna are measured, and between 15 and about 20 weeks, the nuchal skinfold thickness may also be measured. From 15 until 22 weeks' gestation, the cerebellar diameter in millimeters is roughly equivalent to the gestational age in weeks (Goldstein, 1987). The cisterna magna normally measures between 2 and 10 mm. Efacement of the cisterna magna is present in the Chiari II maormation, discussed later (p. 193).

1	Imaging of the spine includes evaluation of the cervical, thoracic, lumbar, and sacral regions (Fig. 10-8). Representative spinal images for record keeping are often obtained in the sagittal or coronal plane, but real-time imaging of each spinal segment in the transverse plane is more sensitive for anomaly detection. Transverse images demonstrate three ossification centers. The anterior ossiication center is the vertebral body, and the posterior paired ossification centers represent the FIGURE 10-8 Normal fetal spine. In this sagittal image of a 21-week fetus, the cervical (, thoracic (, lumbar (, and sacral spine (5) are depicted. Arrows denote the parallel rows of paired posterior ossification centers-representing the junction of vertebralolamina and pedicles.

1	FIGURE 10-9 Anencephaly/acrania. A. Acrania. This ll-week fetus has absence of the cranium, with protrusion of a disorganized mass of brain tissue that resembles a "shower cap" (arrows) and a characteristic triangular facial appearance. B. Anencephaly. This sagittal image shows the absence of forebrain and cranium above the skull base and orbit. The long white arrow points to the fetal orbit, and the short white arrow indicates the nose. junction of vertebral laminae and pedicles. Ossiication of the spine proceeds in a cranial-caudal fashion, such that ossification of the upper sacrum (51-52) is not generally visible sonographically before 16 weeks' gestation, and ossification of the entire sacrum may not be visible until 21 weeks (De Biasio, 2003). Thus, detection of some spinal abnormalities can be challenging in the early second trimester.

1	If a brain or spinal abnormality is identified, targeted sonography is indicated. he International Society of Ultrasound in Obstetrics and Gynecology (2007) has published guidelines for a "fetal neurosonogram." Fetal MR imaging may also be helpful (p. 217). hese defects include anencephaly, myelomeningocele (also called spina bifida), cephalocele, and other rare spinal fusion (or schisis) abnormalities. hey result from incomplete closure of the neural tube by the embryonic age of 26 to 28 days. heir birth prevalence is 0.9 in 1000 in the United States and most of Europe and 1.3 in 1000 in the United Kingdom (Cragan, 2009; Dolk, 2010). Many neural-tube defects can be prevented with folic acid supplementation. When isolated, neural-tube defect inheritance is multifactorial, and the recurrence risk without periconceptional folic acid supplementation is 3 to 5 percent (Chap. 13, p. 270).

1	Screening for neural-tube defects with maternal serum alpha-fetoprotein (MSAFP) has been ofered routinely as part of prenatal care since the 1980s (Chap. 14, p. 283). Women currently have the option of neural-tube defect screening with MSAFP, sonography, or both (American College of Obstetricians and Gynecologists, 2016). Serum screening is generally performed between 15 and 20 weeks' gestation. And, if using an upper threshold of 2.5 multiples of the median (MoM), the anticipated detection rate is at least 90 percent for fetal anencephaly and 80 percent for myelomeningocele. Targeted sonography is the preferred diagnostic test, and in addition to characterizing the neural-tube defect, it may identiy other abnormalities or conditions that also result in MSAFP elevation (Table 14-6, p. 283).

1	Anencephay is characterized by absence of the cranium and telencephalic structures above the level of the skull base and orbits (Fig. 10-9). Acrania is absence of the cranium with protrusion of disorganized brain tissue. Both are uniformly lethal and are generally considered together, with anencephaly as the final stage of acrania (Bronshtein, 1991). These anomalies are often diagnosed in the late first trimester, and with adequate visualization, virtually all cases may be diagnosed in the second trimester. Inability to image the BPD raises suspicion. The face often appears triangular, and sagittal images readily demonstrate absence of the ossified cranium. Hydramnios from impaired fetal swallowing is common in the third trimester. Cphaocee is the herniation of meninges through a cranial defect, tpically located in the midline occipital region (Fig. 10-10).

1	Cphaocee is the herniation of meninges through a cranial defect, tpically located in the midline occipital region (Fig. 10-10). FIGURE 10-10 Encephalocele. This transverse image depicts a large defect in the occipital region of the cranium (arrows) through which meninges and brain tissue have herniated. FIGURE 10-1 1 Myelomeningocele, In this sagittal image of a lumbosacral myelomeningocele, the arrowheads indicate nerve roots within the anechoic herniated sac. The overlying skin is visible above the level of the spinal defect but abruptly stops at the defect (arrow),

1	When brain tissue herniates through the skull defect, the anomaly is termed an encephaocee. Herniation of the cerebellum and other posterior fossa structures constitutes a Chiari II maomaion. Associated hydrocephalus and microcephaly are common, and survivors have a high incidence of neurological deficits and intellectual disability. Cephalocele is an important feature of the autosomal recessive Meckel-Guber syndrome, which includes cystic renal dysplasia and polydactyly. A cephalocele not located in the occipital midline rises suspicion for amnionic-band sequence (Chap. 6, p. 116).

1	Spina bia is a defect in the vertebrae, typically the dorsal arches, with exposure of the meninges and spinal cord. The birth prevalence approximates 1 in 2000 (Cragan, 2009; Dolk, 2010). Most cases are open spina bia-the defect includes the skin and sot tissues. Herniation of a meningeal sac containing neural elements is termed a myelomeningocele (Fig. 10-1i1). When only a meningeal sac is present, the defect is a meningocele. Although the sac may be easier to image in the sagittal plane, transverse images more readily demonstrate separation or splaying of the laterl processes.

1	Detection of spina bifida is aided by two characteristic cranial findings (Nicolaides, 1986). Scalloping of the frontal bones is termed the lemon sign, and anterior curvature of the cerebellum with efacement of the cisterna magna is the banana sign (Fig. 10-12). These indings are manifestations of the Chiari II malformation, also called the Arnold-Chiari maormation. This develops when downward displacement of the spinal cord pulls a portion of the cerebellum through the foramen magnum and into the upper cervical canal. Ventriculomegay is another frequent associated sonographic finding, particularly after midgestation. More than 80 percent of infants with open spina bifida require ventriculoperitoneal shunt placement. A small BPD is often present as well. Children with spina biida require multidisciplinary care to address problems related to the defect, therapeutic shunting, and deicits in swallowing, bladder and bowel function, and ambulation. Fetal myelomeningocele surgery is

1	multidisciplinary care to address problems related to the defect, therapeutic shunting, and deicits in swallowing, bladder and bowel function, and ambulation. Fetal myelomeningocele surgery is discussed

1	Chapter 16 (p. 319). Characterized by distention of the cerebral ventricles by cerebrospinal luid (CSF), this finding is a nonspecific marker of abnormal brain development (Pilu, 2011). The atrium normally measures between 5 and 10 mm from 15 weeks' gestation until term (see Fig. 10-6). Mild ventriculomegaly is diagnosed when the atrial width measures 10 to 15 mm (Fig. 10-13), and overt or severe ventriculomegaly when it exceeds 15 mm. The larger the atrium, the greater the likelihood of an abnormal outcome (Gaglioti, 2009; J06, 2008). CSF is produced within the ventricles by the choroid plexus, which is composed of loose connective tissue surrounding an epithelium-lined capillary core. he choroid plexus often appears to dangle within the ventricle when severe ventriculomegaly is present.

1	Ventriculomegaly may be caused by various genetic and environmental insults. It may be due to other central nervous system (CNS) abnormalities-such as Dandy-Walker malformation or holoprosencephaly, to an obstructive process-such as aqueductal stenosis, or to a destructive process-such as porencephaly or an intracranial teratoma. Initial evaluation includes a targeted examination of fetal anatomy, testing for congenital infections such as cytomegalovirus and toxoplasmosis, and chromosomal microarray analysis, which is described in Chapter 13 (p. 271). Fetal MR imaging should be considered to assess for associated abnormalities that may not be detectable sonographically.

1	FIGURE 10-12 Cranial findings in myelomeningocele, A. Image of a fetal head at the level of the lateral ventricles demonstrates inward bowing or scalloping of the frontal bones (arrows)-the lemon sign. B. Image of a fetal head at the level of the posterior fossa shows anterior curvature of the cerebellum (arrows) with effacement of the cisterna magna-the banana sign. FIGURE 10-13 Ventriculomegaly. In this transverse view of the cranium, the white line depicts measurement of the atrium of the lateral ventricle, which measured 12 mm, consistent with mild ventriculomegaly.

1	Prognosis is generally determined by etiology, severity, and rate of progression. However, even with mild-appearing and isolated ventriculomegaly, prognosis can vary widely. In a systematic review of nearly 1500 mild-to-moderate cases, 1 to 2 percent were associated with congenital infection, 5 percent with aneuploidy, and 12 percent with neurological abnormality (Devaseelan, 2010). A neurological abnormality was significantly more common if ventriculomegly progressed with advancing gestation. Agenesis of the Corpus Callosum

1	Agenesis of the Corpus Callosum The corpus callosum is the major iber bundle connecting reciprocal regions of the cerebral hemispheres. With complete agenesis of the corpus callosum, a normal cavum septum pellucidum cannot be visualized sonographically. Also, the frontal horns are displaced laterally, and the atria show mild enlargement posteriorly-such that the ventricle has a characteristic "teardrop" appearance (Fig. 10-14). Callosal dysgenesis involves only the caudal portions-the body and splenium-and consequently may be more diicult to detect prenatally.

1	In population-based studies, agenesis of the corpus callosum has a prevalence of 1 in 5000 births (Glass, 2008; Szabo, 2011). In a review of apparently isolated cases, fetal MR imaging identiied additional brain abnormalities in more than 20 percent (Sotiriadis, 2012). If the anomaly was still considered isolated following MR imaging, normal developmental outcome was reported in 75 percent of cases, but severe disability occurred in 12 percent. Agenesis of the corpus callosum is associated with other anomalies, aneuploidy, and more than 200 genetic syndromes. Thus, genetic counseling can be challenging. FIGURE 10-14 Agenesis of the corpus callosum. This image demonstrates a "teardrop" shaped ventricle with mild ventriculomegaly (dotted line) and laterally displaced frontal horns (arrow). A normal cavum septum pellucidum cannot be visualized.

1	In early normal brain development, the prosencephalon or forebrain divides as it becomes the telencephalon and diencephalon. With holoprosencephaly, the prosencephalon fails to divide completely into two separate cerebral hemispheres and underlying paired diencephalic structures. Main forms of holoprosencephaly are a continuum that contains, with decreasing severity, alobar, semilobar, and lobar types. In the most severe form-alobar holoprosencephay-a single monoventricle, with or without a covering mantle of cortex, surrounds fused central thalami (Fig. 10-15). In semilobar holoprosencephay, partial separation of the hemispheres occurs. Lobar holoprosencephay is characterized by a variable degree of fusion of frontal structures and should be considered when a normal CSP cannot be seen.

1	Diferentiation into two cerebral hemispheres is induced by prechordal mesenchyme, which is also responsible for diferentiation of the midline face. Thus, holoprosencephaly may be associated with anomalies of the orbits and eyeshypotelorism, cyclopia, or micro-ophthalmia; lips-median cleft; or nose-ethmocephaly, cebocephaly, or arhinia with proboscis (see Fig. 10-15). The birth prevalence of holoprosencephaly is only 1 in 10,000 to 15,000. However, the abnormality has been identiied in nearly 1 in 250 early abortuses, which attests to the extremely high in-utero lethality (Orioli, 2010; Yamada, 2004). The alobar form accounts for 40 to 75 percent of cases, and 30 to 40 percent have a numerical chromosomal abnormality, particularly trisomy 13 (Orioli, 2010; Solomon, 2010). Conversely, two thirds of trisomy 13 cases are found to have holoprosencephaly. Fetal karyotype or chromosomal microarray analysis should be ofered when this anomaly is identiied.

1	This posterior fossa abnormality is characterized by agenesis of the cerebellar vermis, posterior fossa enlargement, and elevation of the tentorium. Sonographically, luid in the enlarged FIGURE 10-15 Alobar holoprosencephaly. A. Transverse cranial image of a fetus with alobar holoprosencephaly, depicting fused thalami (Th) encircled by a monoventricle () with a covering mantle (M) of cortex. The midline falx is absent. (Reproduced with permission from Rafael Levy, ROMS.) B. In this profile view of the face, a soft tissue mass-a proboscis (arrow), protrudes from the region of the forehead.

1	cisterna magna visibly communicates with the fourth ventricle through the cerebellar vermis defect, with visible separation of the cerebellar hemispheres (Fig. 10-16). The birth prevalence approximates 1 in 12,000 (Long, 2006). Associated anomalies and aneuploidy are common. These include ventriculomegaly in 30 to 40 percent, other anomalies in approximately 50 percent, and aneuploidy in 40 percent (Ecker, 2000; Long, 2006). Dandy-Walker malformation is also associated with numerous genetic and sporadic syndromes, congenital viral infections, and teratogen exposure, all of which greatly afect the prognosis. Thus, the initial evaluation mirrors that for ventriculomegaly (p. 193).

1	Inerior vermian agenesis, also called Dandy-Walker variant, is a term used when only the inferior portion of the vermis is absent. But, even when vermian agenesis appears to be partial and relatively subtle, the prevalence of associated anomalies and aneuploidy is still high, and the prognosis is often poor (Ecker, 2000; Long, 2006). Schizencephaly is a rare brain abnormality characterized by clefts in one or both cerebral hemispheres, typically involving the perisylvian fissure. The cleft is lined by heterotopic gray matter and communicates with the ventricle, extending through the cortex to the pial surface (Fig. 10-17). Schizencephaly is believed to be an abnormality of neuronal migration, which explains its typically delayed recognition until after midpregnancy (Howe, 2012). It is associated with absence of the cavum septum pellucidum, resulting in the frontal horn communication shown in the image below.

1	FIGURE 10-16 Dandy-Walker malformation. This transcerebellar image demonstrates agenesis of the cerebellar vermis. The cerebellar hemispheres (+) are widely separated by a fluid collection that connects the 4th ventricle (asterisk) to the enlarged cisterna magna (eM). FIGURE 10-17 Schizencephaly. This transverse image ofthe fetal head shows a large cleft that extends from the right lateral ventricle through the cortex. Because the borders of the cleft are sepa rate, the defect is termed open-lipped. (Used with permission from Michael Davidson, ROMS.) FIGURE 10-19 Midline face. This view demonstrates the integrity of the upper lip. FIGURE 10-18 Sacrococcygeal teratoma. Sonographically, this tumor appears as a solid and/or cystic mass that arises from the anterior sacrum and tends to extend inferiorly and externally as it grows. In this image, a 7 x 6 cm inhomogeneous solid mass is visible below the normal-appearing sacrum. There is also an internal component to the tumor.

1	In contrast, porencephaly is a cystic space within the brain that is lined by white matter and mayior may not communicate with the ventricular system. It is generally considered to be a destructive lesion and may develop following intracranial hemorrhage in the setting of neonatal alloimmune thrombocytopenia or following death of a monochorionic co-twin (Fig. 45-20, p. 878). Fetal MR imaging should be considered when either of these CNS anomalies is identiied.

1	his germ cell tumor is one of the most common tumors in neonates, with a birth prevalence of approximately 1 in 28,000 (Derikx, 2006; Swamy, 2008). It is thought to arise from the totipotent cells along Hensen node, anterior to the coccyx. Classification of sacrococcygeal teratoma (SCT) includes four types (Altman, 1974). Type 1 is predominantly external with a minimal presacral component; type 2 is predominantly external but with a significant intrapelvic component; type 3 is predominantly internal but with abdominal extension; and type 4 is entirely internal with no external component. he tumor histological type may be mature, immature, or malignant.

1	Sonographiclly, SCT appears as a solid and/or cystic mass that arises rom the anterior sacrum and usually extends inferiorly and externally as it grows (Fig. 10-18). Solid components oten have varying echogenicity, appear disorganized, and may enlarge rapidly with advancing gestation. Internal pelvic components may be more challenging to visualize, and fetal MR imaging should be considered. Hydramnios is frequent, and hydrops may develop from high-output cardiac failure, either as a consequence of tumor vascularity or secondary to bleeding within the tumor and resultant anemia. Mentioned throughout this chapter, hydrops is more ully described in Chapter 15 (p. 309). Fetuses with tumors >5 cm oten require cesarean delivery, and classical hysterotomy may be needed (Gucciardo, 2011). s shown in Figure 16-3 (p. 320), fetal surgery is suitable for some SCT cases.

1	his rare anomaly is characterized by absence of the sacral spine and often portions of the lumbar spine. It is approximately 25 times more common in diabetic pregnancies (Garne, 2012). Sonographic indings include a spine that appears abnormally short, lacks normal lumbosacral curvature, and terminates abruptly above the level of the iliac wings. Because the sacrum does not lie between the iliac wings, they are abnormally close together and may appear "shield-like." here may also be abnormal positioning of the lower extremities and lack of normal local soft tissue development. Caudal regression should be diferentiated from sirenomelia, which is a rare anomaly characterized by a single fused lower extremity that occupies the midline.

1	Normal fetal lips and nose are shown in Figure 10-19. A fetal profile is not a required component of standard examination but may be helpful in identiying cases of micrognathia-an abnormally small jaw (Fig. 10-20). Micrognathia should be considered in the evaluation of hydramnios (Chap. 11, p. 227). Use of the ex-utero intrapartum treatment (EI) procedure for severe micrognathia is discussed in Chapter 16 (p. 327). here are three main types of clefts. he irst type, clt lip and palate, always involves the lip, may also involve the hard palate, can be unilateral or bilateral, and has a birth prevalence that approximates 1 in 1000 (Cragan, 2009; Dolk, 2010). If isolated, the inheritance is multifactorial-with a recurrence risk of 3 to 5 percent for one prior afected child. If a cleft is visible in the upper lip, a transverse image at the level of the alveolar ridge may demonstrate that the defect also involves the primary palate (Fig. 10-21).

1	In one systematic review of low-risk pregnancies, cleft lip was identiied so no graphically in only about half of cases FIGURE 10-20 Fetal profile. A. This image depicts a normal fetal profile. B. This fetus has severe micrognathia, which creates a severely recessed chin. (Maarse, 2010). Approximately 40 percent of those detected in prenatal series are associated with other anomalies or syndromes, and aneuploidy is common (Maarse, 2011; Oferdal, 2008). The rate of associated anomalies is highest for bilateral defects that involve the palate. Using data from the Utah Birth Defect Network, Walker and associates (2001) identified aneuploidy in 1 percent with cleft lip alone, 5 percent with unilateral cleft lip and palate, and 13 percent with bilateral cleft lip and palate. It is reasonable to ofer fetal chromosomal microarray analysis when a cleft is identiied.

1	he second type of cleft is isolated clt palate. I t begins at the uvula, may involve the soft palate, and occasionally involves the hard palate-but does not involve the lip. The birth prevalence approximates 1 in 2000 (Dolk, 2010). Identiication of isolated cleft palate has been described using specialized 2-and 3-dimensional sonography (Ramos, 2010; Wilhelm, 2010). However, it is not expected to be visualized during a standard sonographic examination (Maarse, 2011; Oferdal, 2008). A third type of cleft is median clt lp, which is found in association with several conditions. hese include agenesis of the primary palate, hypotelorism, and holoprosencephaly. FIGURE 10-21 Cleft lip/palate. A. This fetus has a prominent unilateral (left-sided) cleft lip. B. Transverse view of the palate in the same fetus demonstrates a defect in the alveolar ridge (arrow), The tongue ) is also visible.

1	Median clefts may also be associated with hypertelorism and frontonasal hyperplasia, formerly called the median clt ace syndrome. This venolymphatic malformation is characterized by luidilled sacs that extend from the posterior neck (Fig. 10-22). Cystic hygromas may be diagnosed as early as the irst trimester and vary widely in size. They are believed to develop when lymph from the head fails to drain into the jugular vein and accumulates instead in jugular lymphatic sacs. Their birth prevalence approximates 1 in 5000. But, reflecting the high in-utero lethality of the condition, the first-trimester incidence exceeds 1 in 300 (Malone, 2005).

1	Up to 70 percent of cystic hygromas are associated with aneuploidy. When cystic hygromas are diagnosed in the irst trimester, trisomy 21 is the most common aneuploidy, followed by 45,X and trisomy 18 (Kharrat, 2006; Malone, 2005). Firsttrimester fetuses with cystic hygromas are ive times more likely to be aneuploid than fetuses with a thickened nuchal translucency. When cystic hygromas are diagnosed in the second trimester, approximately 75 percent of aneuploid cases are 45,X-Turner syndrome Gohnson, 1993; Shulman, 1992).

1	Even in the absence of aneuploidy, cystic hygromas confer a significantly greater risk for other anomalies, particularly cardiac anomalies that are flow-related. These include hypoplastic let heart and coarctation of the aorta. Cystic hygromas also may be part of a genetic syndrome. One is Noonan yndrome, an autosomal dominant disorder that shares several features with Turner syndrome, including short stature, lymphedema, high-arched palate, and oten pulmonary valve stenosis. Large cystic hygromas are usually associated with hydrops fetalis, rarely resolve, FIGURE 10-22 Cystic hygromas. A. This 9-week fetus with a cystic hygroma (arrow) was later found to have Noonan syndrome. B. Massive multiseptated hygromas (arrowheads) in the setting of hydrops fetalis at 15 weeks' gestation.

1	and carry a poor prognosis. Small hygromas may undergo spontaneous resolution, and provided that fetal karyotype and echocardiography results are normal, the prognosis may be good. The likelihood of a nonanomalous liveborn neonate with normal karyotype following identiication of first-trimester hygroma is approximately 1 in 6 (Kharrat, 2006; Malone, 2005).

1	he lungs appear homogeneous and surround the heart. In the four-chamber view of the heart, they comprise approximately two thirds of the area, with the heart occupying the remaining third. The thoracic circumference is measured at the skin line in a transverse plane at the level of the four-chamber view. In cases of suspected pulmonary hypoplasia secondary to a small thorax, such as with severe skeletal dysplasia, comparison with a reference table may be helpul (Appendix, p. 1266). Various abnormalities may appear so no graphically as cystic or solid space-occupying lesions or as an efusion outlining the heart or lung(s). Fetal therapy for thoracic abnormalities is discussed in Chapter 16 (p. 324).

1	This is a defect in the diaphragm through which abdominal organs herniate into the thorax. It is let-sided in approximately 75 percent of cases, right-sided in 20 percent, and bilateral in 5 percent (Gallot, 2007). he prevalence of congenital diaphragmatic hernia (CDH) is 1 in 3000 to 4000 births (Cragan, 2009; Dolk, 2010). Associated anomalies and aneuploidy are found in 40 percent of cases (Gallot, 2007; Stege, 2003). With suspected CDH, targeted sonography and fetal echo cardiography should be performed, and fetal chromosomal microarray analysis should be ofered. In population-based series, the presence of an associated abnormality reduces the overall survival rate of neonates with CDH from approximately 50 percent to about 20 percent (Colvin, 2005; Gallot, 2007). If there are no associated abnormalities, the major causes of neonatal mortality are pulmonary hypoplasia and pulmonary hypertension.

1	Sonographically, left-sided CDH typically shows dextroposition of the heart toward the right side of the thorax and a cardiac axis pointing toward the midline (Fig. 10-23). Associated findings include the stomach bubble or bowel peristalsis in the chest and a wedge-shaped mass-the liver-located anteriorly in the left hemithorax. Liver herniation complicates at least 50 percent of cases and is associated with a 30-percent reduction in the survival rate (Mullassery, 2010). With large lesions, impaired swallowing and mediastinal shift may result in hydramnios and hydrops, respectively. Eforts to reduce neonatal mortality rates and need for extracorporeal membrane oxygenation (ECMO) have focused on indicators such as the sonographic lung-to-head ratio, MR imaging measurements of lung volume, and the degree of liver herniation Gani, 2012; Oluyomi-Obi, 2016; Worley, 2009). These and fetal therapy for CDH are reviewed in Chapter 16 (p. 323).

1	FIGURE 10-23 Congenital diaphragmatic hernia. In this transverse view of the thorax, the heart is shifted to the far right side of the chest by a let-sided diaphragmatic hernia containing stomach (5), liver (, and bowel (B). This abnormality represents a hamartomatous overgrowth of terminal bronchioles that communicates with the tracheobronchial tree. It is also called congenital pulmonay airway maormation (CPM), based on an understanding that not all histopathological types are cystic or adenomatoid (Azizkhan, 2008; Stocker, 1977, 2002). The estimated prevalence is 1 in 6000 to 8000 births, and this rate is rising because of improved sonographic detection of milder cases (Burge, 2010; Duncombe, 2002).

1	Sonographically, congenital cystic adenomatoid malformation (CCAM) is a well-circumscribed thoracic mass that may appear solid and echogenic or may have one or multiple variably sized cysts (Fig. 10-24). It usually involves one lobe, has blood supply from the pulmonary artery, and drains into the pulmonary veins. Lesions with cysts :5 mm are generally FIGURE 10-24 Transverse (A) and sagittal (8) images of a 26-week fetus with a very large left-sided microcystic congenital cystic adenomatoid malformation (CCAM). The mass () fills the thorax and has shifted the heart to the far right side of the chest, with development of ascites (asterisks). Fortunately, the mass did not continue to grow, the ascites resolved, and the neonate was delivered at term and did well following resection. termed macrocystic, and lesions with cysts <5 mm are termed microcystic (Adzick, 1985).

1	termed macrocystic, and lesions with cysts <5 mm are termed microcystic (Adzick, 1985). In a review of 645 CCM cases, the overall survival rate exceeded 95 percent, and 30 percent of cases demonstrated apparent prenatal resolution. The other 5 percent of cases typically very large lesions with associated mediastinal shift were complicated by hydrops, and the prognosis was poor (Cavoretto, 2008). CCAMs often become less conspicuous with advancing gestation. However, a subset of CCAMs dem onstrates rapid growth between 18 and 26 weeks' gestation. lesions to forestall growth and potentially ameliorate hydrops (Curran, 2010; Peranteau, 2016). If a large dominant cyst is present, thoracoamnionic shunt placement may lead to hydrops resolution. Fetal therapy for CCAM is discussed in Chapter 16 (p.i324). Also called a bronchopulmonary sequestration, this abnormal ity is an accessory lung bud "sequestered" from the tracheo bronchial tree, that is, a mass of nonfunctioning lung tissue.

1	Also called a bronchopulmonary sequestration, this abnormal ity is an accessory lung bud "sequestered" from the tracheo bronchial tree, that is, a mass of nonfunctioning lung tissue. Most cases diagnosed prenatally are extralobar, which means they are enveloped in their own pleura. Overall, however, most sequestrations present in adulthood and are intralobar-within the pleura of another lobe. Extralobar pulmonary sequestra tion is considered significantly less common than CCAM, and no precise prevalence has been reported. Lesions have a left sided predominance and most often involve the left lower lobe. Of cases, 10 to 20 percent are located below the diaphragm, 10 percent of cases (Yildirim, 2008).

1	Of cases, 10 to 20 percent are located below the diaphragm, 10 percent of cases (Yildirim, 2008). Sonographically, pulmonary sequestration presents as a homogeneous, echogenic thoracic mass (Fig. 10-25A). Thus, it may resemble a microcystic CCAM. However, the blood supply is from the systemic circulation-from the aorta rather than the pulmonary artery (see Fig. 10-25B). In 5 to 10 percent with pulmonary sequestration, a large ipsilateral pleural efusion develops, and without treatment, this may result in pulmonary hypoplasia or hydrops (see Fig. 1 0-25C,D). Therapeutic thoracoamnionic shunting of efusions is discussed in Chapter 16 (p. 324). Hydrops may also result from mediastinal shift or high-output cardiac failure due to the left-toright shunt imposed by the mass. In the absence of a pleural efusion, the reported survival rate exceeds 95 percent, and 40 percent of cases demonstrate apparent prenatal resolution (Cavoretto, 2008).

1	This rare anomaly usually results from laryngeal or tracheal atresia. The normal egress of lung fluid is obstructed, and the tracheobronchial tree and lungs become massively distended. Sonographically, the lungs appear brightly echogenic, the bronchi are dilated, the diaphragm is flattened or everted, and the heart is compressed (Fig. 10-26). Venous return is impaired and ascites develops, typically followed by hydrops. In one review of 118 cases, associated anomalies were identiied in more than 50 percent (Sanford, 2012). Congenital high airway obstruction sequence (CHAOS) is a feature of the autosomal recessive Fraser syndrome and has been associated with the 22q 11.2 deletion syndrome. In some cases, the obstructed airway spontaneously perforates, which potentially confers a better prognosis. he EIT procedure has significantly improved outcome in selected cases (Chap. 16, p. 327).

1	FIGURE 10-25 Pulmonary sequestration. A. This transverse image at the level of the 4-chamber view of the heart depicts a pulmonary sequestration involving the left lower lobe in a 25-week fetus. Mass effect leads to dextroposition of the heart to the right side of the chest. B. A sagittal image shows the pulmonary sequestration supplied by a branch of the abdominal aorta. C. Over the next 3 weeks, a large ipsilateral pleural effusion develops (asterisk), resulting in mediastinal shift and dextroposition of the heart to the far right thorax. D. Following placement of a double-pigtail shunt through the chest wall and into the effusion, the effusion drained and the lung significantly reexpanded. Arrows points to coils of the pigtail shunt. (Used with permission from Dr. Elaine Duryea.)

1	FIGURE 10-26 Congenital high airway obstruction sequence (CHAOS). The lungs appear brightly echogenic, and one is marked by an "L." The bronchi, one of which is noted by an arrow, are dilated with fluid. Flattening and eversion of the diaphragm is common, as is ascites (asterisks).

1	Cardiac malformations are the most common class of congenital anomalies, and their overall prevalence is 8 in 1000 births (Cragan, 2009). Almost 90 percent of cardiac defects are multifactorial or polygenic in origin, another 1 to 2 percent result from a single-gene disorder or gene-deletion syndrome, and 1 to 2 percent stem from exposure to a teratogen such as isotretinoin, hydantoin, or maternal diabetes. Based on data from population-based registries, approximately 1 in 8 liveborn and stillborn neonates with a congenital heart defect has a chromosomal abnormality (Dolk, 2010; Hartman, 201l). Of chromosomal abnormalities associated with cardiac anomalies, trisomy 21 accounts for more than 50 percent of cases. Others are trisomy 18, 22q 11.2 deletion, trisomy 13, and monosomy X (Hartman, 2011). Of these aneuploid fetuses, 50 to 70 percent also have extracardiac anomalies. Chromo defects are found.

1	Traditionally, detection of congenital cardiac anomalies is more challenging than for anomalies of other organ systems. 40 percent of those with major cardiac anomalies before 22 weeks' gestation, and specialized sonography may identiy 80 percent (Romosan, 2009; Trivedi, 2012). For selected anoma lies, prenatal detection may improve neonatal survival. his requiring prostaglandin infusion after birth to keep the duc tus arteriosus open (Franklin, 2002; Mahle, 2001; Tworetzky, 2001). Standard cardiac assessment includes a four-chamber view, evaluation of rate and rhythm, and evaluation of the left and right ventricular outlow tracts (Figs. 10-27 and 10-28A-C). Examination of the cardiac outflow tracts aids detection of abnormalities that might not be appreciated in the four-cham ber view. hese include tetralogy of Fallot, transposition of the great vessels, and truncus arteriosus.

1	heour-chamber view is a transverse image of the fetal tho rax at a level immediately above the diaphragm. It allows evalu ation of cardiac size, position in the thorax, cardiac axis, atria and ventricles, foramen ovale, atrial septum primum, interven tricular septum, and atrioventricular valves (see Fig. 10-27). he atria and ventricles should be similar in size, and the apex of the heart should form a 45-degree angle with the left anterior chest wall. Abnormalities of cardiac axis are frequently encoun tered with structural cardiac anomalies and occur in more than a third (Shipp, 1995).

1	The lt ventricular oulow tract view is a transverse image just above the diaphragm and demonstrates that the ascending aorta arises entirely from the left ventricle. The inteventricular septum is shown to be in continuity with the anterior wall of the aorta, and the mitral valve in continuity with the posterior wall of the aorta (see Fig. 10-28B). Ventricular septal defects and outflow tract abnormalities are often visible in this view (Fig. 10-29).

1	The right ventricular oulow tract view shows the right ventricle giving rise to the pulmonary artery (see Fig. 10-28C). Together, the left and right outflow tract views demonstrate the normal perpendicular orientation of the aorta and pulmonary artery, and the comparable size of these great arteries. Structures visible in the right ventricular outlow tract view include the right ventricle and the main pulmonary artery, which subsequently branches into the right and left pulmonary arteries. hese structures are also visible in the short axis view, shown in Figure 10-28E. This is a specialized examination of fetal cardiac structure and function designed to identiy and characterize abnormalities. Guidelines for its performance have been developed collaboratively by the American Institute of Ultrasound in Medicine,

1	FIGURE 10-27 The four-chamber view. A. Diagram demonstrat ing measurement of the cardiac axis from the four-chamber view of the fetal heart. B. Sonogram of the four-chamber view at 22 weeks' gestation shows the normal symmetry of the atria and ventricles, normal position of the mitral and triscuspid valves, pulmonary veins entering the left atrium, and descending aorta (Ao). L = left; LA = left atrium; LV = left ventricle; R = right; RA = right atrium; RV = right ventricle.

1	American College of Obstetricians and Gynecologists, Society for Maternal-Fetal .Medicine, American Society of Echocardiography, and American College of Radiology. Echocardiography indications include suspected fetal cardiac anomaly, extracardiac anomaly, or chromosomal abnormality; fetal arrhythmia; hydrops; thick nuchal translucency; monochorionic twin gestation; irst-degree relative to the fetus with a congenital cardiac defect; in vitro fertilization; maternal antiRo or anti-La antibodies; exposure to a medication associated with cardiac defects; and maternal metabolic disease associated with cardiac defects-such as pregestational diabetes or phenylketonuria (American Institute of Ultrasound in Medicine, 2013a). Components of the examination are listed in TABLE 10-9. Components of Fetal Echocardiography EvaIuation of atria Evaluation of ventricles Evaluation of great vessels Cardiac and visceral situs Atrioventricular junctions Ventriculoarterial junctions

1	EvaIuation of atria Evaluation of ventricles Evaluation of great vessels Cardiac and visceral situs Atrioventricular junctions Ventriculoarterial junctions Scanning planes, gray scale Right ventricular outflow tract Three-vessel and trachea view Short-axis view, low (ventricles) Short-axis view, high (outflow tracts) Aortic arch aPulsed-wave Doppler sonography should be used as an adjunct to evaluate these structures. Cardiac biometry and functional assessment are optional but should be considered for suspected structural/ functional abnormalities. Adapted from the American Institute of Ultrasound in Medicine,l201l3b.

1	Adapted from the American Institute of Ultrasound in Medicine,l201l3b. FIGURE 10-30 Endocardial cushion defect. A. During ventricular systole, the lateral leaflets of the mitral and triscuspid valves come together in the midline. But the atrioventricular valve plane is abnormal, a common atrium (A) is observed, and there is a visible defect (arrow) in the interventricular septum. B. During diastolic filling, opening of the atrioventricular valves more clearly demonstrates the absence of their medial leaflets. Table 10-9, and examples of nine required gray-scale imaging views are shown in Figure 10-28. Examples of selected cardiac anomalies are reviewed below.

1	Ventricular Septal Defect. This is the most common congenital cardiac anomaly and is found in approximately 1 in 300 births (Cragan, 2009; Dolk, 2010). Even with adequate visualization, the prenatal detection rate of ventricular septal defect (VSD) is low. A defect may be appreciated in the membranous or muscular portion of the interventricular septum in the four-chamber view, and color Doppler demonstrates flow through the defect. Imaging of the left ventricular outflow tract may show discontinuity of the interventricular septum as it becomes the wall of the aorta (see Fig. 10-29). Fetal VSD is associated with other abnormalities and aneuploidy, and chromosomal microarray analysis should be ofered. hat said, the prognosis for an isolated defect is good. More than a third of prenatally diagnosed VSDs close in utero, and another third close in the irst year of life (Axt-Fliedner, 2006; Paladini, 2002).

1	Endocardial Cushion Defect. This is also called an atrioventricular (A ) septal dect or A V canal dect. It has a prevalence of approximately 1 in 2500 births and is associated with trisomy 21 in more than half of cases (Christensen, 2013; Cragan, 2009; Dolk, 2010). The endocardial cushions are the crux of the heart, and defects jointly involve the atrial septum primum, interventricular septum, and medial leaflets of the mitral and tricuspid valves (Fig. 10-30). Approximately 6 percent of cases occur with heterotaxy syndromes, that is, those in which the heart and/or abdominal organs are on the incorrect side. Endocardial cushion defects associated with heterotaxy can have comorbid conduction system abnormalities resulting in third-degree AV block, which confers a poor prognosis (Chap. 16, p. 316).

1	Hypoplastic Let Heart Syndrome. This anomaly is found in approximately 1 in 4000 births (Cragan, 2009; Dolk, 2010). Sonographically, the let side of the heart may appear "illed-in" or the let ventricle may be so small and attenuated that a ventricular chamber is diicult to appreciate (Fig. 10-31). There may be no visible let ventricular inlow or outflow, and reversal of low may be documented in the aortic arch. Although this anomaly was once considered a lethal prognosis, 70 percent of afected infants may now survive to adulthood (Feinstein, 2012). Postnatal treatment consists of a three-stage palliative repair or cardiac transplantation. Still, morbidity remains high, and developmental delays are common (Lloyd, 2017; Paladini, 2017). This is a ductal-dependent lesion for which neonatal administration of prostaglandin therapy is essential. Fetal therapy for hypoplastic left heart is discussed in Chapter 16 (p. 326).

1	FIGURE 10-28 Fetal echocardiography gray-scale imaging planes. A. Four-chamber view. B. Left ventricular outflow tract view. The white arrow illustrates the mitral valve becoming the wall of the aorta. The arrow with asterisk marks the interventricular septum becoming the opposing aortic wall. C. Right ventricular outflow tract view. D. Three vessel and trachea view. E. High short-axis view (outflow tracts). F. Low short-axis view (ventricles). G. Aortic arch view. H. Ductal arch view. I. Superior and inferior vena cavae views. Ao = aorta; IVC = inferior vena cava; LA = left atrium; LV = left ventricle; PA = pulmonary artery; RA = right atrium; RV = right ventricle; SVC = superior vena cava. branous) portion of the interventricular septum. B.The left-ventricular outflow tract view of the same fetus demonstrates a break (arrow) in continuity between the interventricular septum and the anterior wall of the aorta.

1	B.The left-ventricular outflow tract view of the same fetus demonstrates a break (arrow) in continuity between the interventricular septum and the anterior wall of the aorta. FIGURE 10-29 Ventricular septal defect. A.oln this four-chamber view of a 22-week fetus, a defect (arrow) is noted in the superior (mem Tetralogy of Fallot. This anomaly occurs in approximately 1 in 3000 births (Cragan, 2009; Dolk, 2010; Nelson, 2016). It includes a ventricular septal defect; an overriding aorta; a pulmonary valve abnormality, typically stenosis; and right ventricular hypertrophy (Fig. 10-32). he last does not present before birth. Due to the location of the ventricular septal defect, the four-chamber view may appear normal.

1	Following postnatal repair, the 20-year survival rates exceed 95 percent (Knott-Craig, 1998). However, cases with pulmonay atresia have a more complicated course. There is also a variant in which the pulmonary valve is absent. These afected fetuses are at risk for hydrops and for tracheomalacia from compression of the trachea by an enlarged pulmonary artery. Cardiac Rhabdomyoma. his is the most common cardiac tumor. Approximately 50 percent of cases are associated with tuberous sclerosis, an autosomal dominant disease with multiorgan system manifestations. Tuberous sclerosis is caused by mutations in the hamartin (TSCl) and tuberin (TSC2) genes.

1	Cardiac rhabdomyomas appear as well-circumscribed echo genic masses, pler depicts flow from the right atrium to the right ventricle only, and left ventricular filling is usually within the ventricles or out not visible. C. The left ventricular outflow tract view demonstrates marked narrowing of the aorta (Ao). RV = right ventricle; LA = low tracts. They may be single or is the hypoplastic aortic root. (Used with permission from Rafael Levy, ROMS.) multiple; may grow in size during gestation; and occasionally, may lead to inlow or outflow obstruction. In cases without obstruction or large tumor size, the prognosis is relatively good from a cardiac standpoint, because the tumors tend to regress after the neonatal period. Because extracardiac findings of tuberous sclerosis may not be apparent with prenatal sonography, MR imaging may be considered to evaluate fetal CNS anatomy (p. 217).

1	Motion-mode or M-mode imaging is a linear display of cardiac cycle events, with time on the x-axis and motion on the y-axis. It is often used to measure embryonic or fetal heart rate (Fig. 10-33). If an abnormality of heart rate or rhythm is identiied, M-mode imaging permits separate evaluation of atrial and ventricular waveforms. Thus, it is particularly useful for characterizing arrhythmias and their response to treatment (Chap. 16, p. 316). M-mode can also be used to assess ventricular function and atrial and ventricular outputs. Premature Atrial Contractions. Also called atrial extrasystoles, these are the most common fetal arrhythmia and a frequent

1	Premature Atrial Contractions. Also called atrial extrasystoles, these are the most common fetal arrhythmia and a frequent FIGURE 10-31 Hypoplastic left heart syndrome. A. In this 4-chamber view at 16 weeks, the let ventricle (L) appears "filled in" and is Significantly smaller than the right ventricle (R). The tricuspid valve () is open, whereas the mitral valve appears closed (asterisk). B. Color Dop let atrium. D. The tiny circle (arrow) in this short axis view FIGURE 10-32 Tetralogy of Fallot. This image shows a ventricular septal defect with an overriding aorta in a fetus with tetralogy of Fallot. The arrow points to the aortic valve. The left ventricle (LV)and right ventricle (R) are labeled. FIGURE 10-33 M-mode, or motion mode, is a linear display of the events of the cardiac cycle, with time on the x-axis and motion on the y-axis. M-mode is used commonly to measure the fetal heart rate, as in this 12-week fetus.

1	finding. They represent cardiac conduction system immaturity and typically resolve later in gestation or in the neonatal period. Premature atrial contractions (PACs) may be conducted and thus sound like an extra beat. However, they are more commonly blocked, and with handheld Doppler they sound like a dropped beat. As shown in Figure 10-34, the dropped beat may be demonstrated with M-mode evaluation as a compensatory FIGURE 10-34 M-mode. In this image, there is normal concordance between atrial (A) and ventricular contractions (). Movement of the tricuspid valve en is also shown. There is also a premature atrial contraction (arrow) and a subsequent early ventricular contraction, followed by a compensatory pause. pause that follows the premature contraction.

1	pause that follows the premature contraction. P ACs sometimes occur with an atrial septal aneurysm but are not associated with major structural cardiac abnormalities. Older case reports describe an association with maternal cafeine consumption and with hydralazine (Lodeiro, 1989; Oei, 1989). In approximately 2 percent of cases, afected fetuses are later identified to have a supraventricular tachycardia (S) that requires urgent treatment (Copel, 2000). Accordingly, pregnancies with fetal PACs are often followed with fetal heart rate assessment as often as every 1 to 2 weeks until ectopy resolves. T reatment of fetal SVT and other arrhythmias is discussed in Chapter 16 (p. 316) . he integrity of the abdominal wall is assessed at the level of the cord insertion during the standard examination (Fig. 10-35). Ventral wall defects include gastroschisis, omphalocele, and body stalk anomaly.

1	Ventral wall defects include gastroschisis, omphalocele, and body stalk anomaly. Gastroschisis is a full-thickness abdominal wall defect located to the right of the umbilical cord insertion. Bowel herniates through the defect into the amnionic cavity (Fig. 10-36). he prevalence is approximately 1 in 2000 births Oones, 2016; Nelson, 2015). Gastroschisis is the one major anomaly more common in fetuses of younger mothers, and the average maternal age is 20 years (Santiago-Munoz, 2007). Coexisting bowel abnormalities such as jjunal atresia are found in approximately 15 percent of cases (Nelson, 2015; Overcash, 2014). Gastroschisis is not associated with aneuploidy, and the survival rate is 90 to 95 percent (Kitchanan, 2000; Nelson, 2015; Nembhard, 2001).

1	Fetal-growth restriction complicates gastroschisis in 15 to 40 percent of cases (Overcash, 2014; Santiago-Munoz, 2007). Growth restriction does not appear to be associated with adverse outcomes such as longer hospitalization or higher mortality rate (Nelson, 2015; Overcash, 2014). However, earlier gestational age at delivery does pose a risk for adverse outcome with gastroschisis, and planned delivery at 36 to 37 weeks does not confer neonatal benefit (Al-Kaf, 2016; Overcash, 2014; South, 2013).

1	Omphalocele complicates 1 in 3000 to 5000 pregnancies (Canield, 2006; Dolk, 2010). It forms when the lateral ectomesodermal folds fail to meet in the midline. This leaves the abdominal contents covered only by a two-layered sac of amnion and peritoneum into which the umbilical cord inserts (Fig. 10-37). More than half of cases are associated with other major anomalies or aneuploidy. Omphalocele also is a component of syndromes such as Beckwith-Wiedemann, cloacal exstrophy, and pentaloy of Cantrell. Smaller defects confer greater risk for aneuploidy (De Veciana, 1994). Chromosomal microarray analysis should be ofered in all cases of omphalocele. FIGURE 10-35 Normal ventral wall. Transverse view of the abdomen in a second-trimester fetus with an intact anterior abdominal wall and normal cord insertion.

1	FIGURE 10-35 Normal ventral wall. Transverse view of the abdomen in a second-trimester fetus with an intact anterior abdominal wall and normal cord insertion. Body stalk anomay, also known as limb-body-wall complex or cyllosoma, is a rare, lethal anomaly characterized by abnormal formation of the body wall. Typically, no abdominal wall is visible, and the abdominal organs extrude into the extraamnionic coelom. here is close approximation or fusion of the body to the placenta, and the umbilical cord is extremely short. Acute-angle scoliosis is another feature. Amnionic bands are often identified. FIGURE 10-36 Gastroschisis. This 18-week fetus has a fullthickness ventral wall defect to the right of the cord insertion (arrowhea), through which multiple small bowel loops (B) have herniated into the amnionic cavity. FIGURE 10-37 Omphalocele. Transverse view of the abdomen showing an omphalocele as a large abdominal wall defect with exteriorized liver covered by a thin membrane.

1	FIGURE 10-37 Omphalocele. Transverse view of the abdomen showing an omphalocele as a large abdominal wall defect with exteriorized liver covered by a thin membrane. he stomach is visible in nearly all fetuses after 14 weeks' gestation. If the stomach is not seen during initial evaluation, the examination is repeated, and targeted sonography should be considered. Nonvisualization of the stomach may be secondary to impaired swallowing in the setting of oligohydramnios or to underlying causes such as esophageal atresia, a craniofacial anomaly, or a CNS or musculoskeletal abnormality. Fetuses with hydrops may also have impaired swallowing.

1	he bowel, liver, gallbladder, and spleen can be identiied in many second-and third-trimester fetuses. Bowel appearance changes with fetal maturation. Occasionally, it may be bright or echogenic, which may indicate small amounts of swallowed intraamnionic blood, especially with comorbid MSAFP elevation. Bowel that appears as bright as fetal bone confers a slightly greater risk for underlying gastrointestinal malformations, for cystic ibrosis, for trisomy 21, and for congenital infection such as cytomegalovirus (Fig. 14-3, p. 287). Bowel atresia is characterized by obstruction and proximal bowel dilation. In general, the more proximal the obstruction, the more likely it is to lead to hydramnios. At times, hydramnios from proximal small-bowel obstruction can be suiciently severe to result in maternal respiratory compromise or preterm labor and may necessitate amnioreduction (Chap. 11, p. 230).

1	Esophageal atresia occurs in approximately 1 in 4000 births (Cragan, 2009; Pedersen, 2012). It may be suspected when the stomach cannot be visualized and hydramnios is present. hat said, in up to 90 percent of cases, a concomitant tracheoesophageal istula allows luid to enter the stomach, such that prenatal detection is problematic. More than half have associated anomalies or genetic syndromes. Multiple malformations are present in 30 percent of cases, and aneuploidy such as trisom) 18 or 21, in 10 percent (Pedersen, 2012). Cardiac, urinary tract, and other gastrointestinal abnormalities are the most frequently associated anomalies. Approximately 10 percent of FIGURE 10-38 Duodenal atresia. The double-bubble sign represents distension of the stomach (5) and the first part of the duodenum (0), as seen on this axial abdominal image. Demonstrating continuity between the stomach and proximal duodenum confirms that the second "bubble" is the proximal duodenum.

1	cases of esophageal atresia occur as part of the V ACTERL asso ciation, which is yertebral defects, !nal atresia, £ardiac defects, !racheo�sophageal istula, renal anomalies, and limb abnormali ties (Pedersen, 2012).

1	Duodenal atresia is found in approximately 1 in 10,000 births (Best, 2012; Dolk, 2010). It is characterized by the sonographic double-bubble sin, which represents distention of the stomach and the irst part of the duodenum (Fig. 10-38). This finding is usually not present before 22 to 24 weeks' gestation. Demonstrating continuity between the stomach and proximal duodenum confirms that the second "bubble" is the proximal duodenum. Approximately 30 percent of afected fetuses have an associated chromosomal abnormality or genetic syndrome, particularly trisomy 21. Of cases without a genetic abnormality, a third have associated anomalies, most commonly cardiac defects and other gastrointestinal abnormalities (Best, 2012). Obstructions in the more distal small bowel usually result in multiple dilated loops that may have enhanced peristaltic activity.

1	Large-bowel obstructions and anal atresia are less readily diagnosed by sonography, because hydramnios is not a typical feature and the bowel may not be signiicantly dilated. A transverse view through the pelvis may show an enlarged rectum as an anechoic structure between the bladder and the sacrum. The fetal kidneys are visible adjacent to the spine, frequently in the first trimester and routinely by 18 weeks' gestation (Fig. 10-39). he length of the kidney approximates 20 mm at 20 weeks and grows by about 1.1 mm each week thereafter (Chitty, 2003). With advancing gestation, the kidneys become relatively less echogenic, and a rim of perinephric fat aids visualization of their margins. he fetal bladder is readily visible in the second trimester as a round, anechoic structure in the anterior midline of the

1	he fetal bladder is readily visible in the second trimester as a round, anechoic structure in the anterior midline of the FIGURE 10-39 Normal fetal kidneys. The kidneys are visible adjacent to the fetal spine in this 29-week fetus. With advancing gestation, a rim of perinephric fat aids visualization of the kidney margins. A physiological amount of urine is visible in the renal pelves and is marked in one kidney by an arrow. pelvis. With application of Doppler, the bladder is outlined by the two superior vesical arteries as they become the umbilical arteries of the umbilical cord (Fig. 10-40 and Chap. 6, p. 1i17). The fetal ureters and urethra are not visible sonographically unless abnormally dilated.

1	The placenta and membranes are the major sources of amnionic fluid early in pregnancy. However, after 18 weeks' gestation, most of the luid is produced by the kidneys (Chap. 11, p. 225). Fetal urine production rises from 5 mLlhr at 20 weeks to approximately 50 mLlhr at term (Rabinowitz, 1989). Normal amnionic luid volume in the second half of pregnancy suggests urinary tract patency with at least one functioning kidney. But, unexplained oligohydramnios suggests a urinary tract defect or placental perfusion abnormality. FIGURE 10-40 Normal fetal bladder. The normal fetal bladder is readily visible as a round, fluid-filled structure in the anterior pelvis, outlined by the two superior vesical arteries as they become the umbilical arteries of the umbilical cord.

1	FIGURE 10-41 Renal pelvis dilatation. This common finding is identified in 1 to 5 percent of pregnancies. A.oln this 34-week fetus with mild renal pelvis dilatation, the anterior-posterior diameter of the renal pelvis measured 7 mm in the transverse plane. B. Sagittal image of the kidney in a 32-week fetus with severe renal pelvis dilatation secondary to ureteropelvic junction obstruction. The arrow points to one of the rounded calyces. This finding is present in 1 to 5 percent of fetuses. It is also called urinary tract dilatation or hydronephrosis. In 40 to 90 percent of cases, it is transient or physiological and does not represent an underlying abnormality (Ismaili, 2003; Nguyen, 2010). In approximately a third of cases, a urinary tract abnormality is conirmed in the neonatal period. Of these, ureteropelvic junction (UP]) obstruction and vesicoureteral relux UR) are the most frequent.

1	he fetal renal pelvis is measured anterior to posterior in a transverse plane, and calipers are placed on the inner border of the fluid collection (Fig. 10-41). Although various thresholds have been deined, the pelvis is typically considered dilated if it exceeds 4 mm in the second trimester or 7 mm at approximately 32 weeks' gestation (Reddy, 2014). Typically, the second-trimester threshold is used to identiy pregnancies that warrant subsequent third-trimester evaluation. he Society for Fetal Urology categorized renal pelvis dilatation based on a metaanalysis of more than 100,000 screened pregnancies (Table 10-10) (Lee, 2006; Nguyen, 2010). he degree of dilatation correlates with the likelihood of an underlying abnormality. Other suggestive indings of pathology TABLE 10-10. Risk for Postnatal Urinary Abnormality According to Degree of Renal Pelvis Dilatationa Second Third Postnatal Dilatation Trimester Abnormality 4 to <7 mm 7 to <9 mm

1	TABLE 10-10. Risk for Postnatal Urinary Abnormality According to Degree of Renal Pelvis Dilatationa Second Third Postnatal Dilatation Trimester Abnormality 4 to <7 mm 7 to <9 mm Moderate 7 to �1 0 mm 9 to �15 mm 45% aSociety for Fetal Urology Classification. Modified from Lee, 2006; Nguyen, 2010. include calyceal dilatation, cortical thinning, or dilatation elsewhere along the urinary tract. Mild pyelectasis in the second trimester is associated with a slightly greater risk for Down syndrome and is considered a soft marker for this (Fig. 14-3, p.i287).

1	Ureteropelvic Junction Obstruction. This condition is the most common abnormality associated with renal pelvis dilatation. The birth prevalence is 1 in 1000 to 2000, and males are afected three times more often than females (Williams, 2007; Woodward, 2002). Obstruction is generally functional rather than anatomical, and it is bilateral in up to a fourth of cases. he likelihood of ureteropelvic junction obstruction rises from 5 percent with mild renal pelvis dilatation to more than 50 percent with severe dilatation (Lee, 2006).

1	Duplicated Renal Collecting System. In this anatomical anomaly, the upper and lower poles of the kidney-called moieties-are each drained b) a separate ureter (Fig. 10-42). Duplication is found in approximately 1 in 4000 pregnancies, is more common in females, and is bilateral in 15 to 20 percent of cases (James, 1998; Vergani, 1998; Whitten, 2001). Sonographically, an intervening tissue band separates two distinct renal pelves. Development of hydronephrosis or ureteral dilatation may occur due to abnormal implantation of one or both ureters within the bladder-a relationship that reflects the anatomical Weigert-Meyer rule. The upper pole ureter may develop obstruction from a ureterocele within the bladder, whereas the lower pole ureter has a shortened intravesical segment that predisposes to vesicoureteral reflux (see Fig. 10-42B). Thus, both moieties may become dilated from diferent etiologies, and both are at risk for loss of function.

1	The prevalence of bilateral renal agenesis is approximately in 8000 births, whereas that of unilateral renal agenesis is 1 in FIGURE 10-42 Duplicated renal collecting system. The upper and lower moieties of the kidney are each drained by a separate ureter. A. Renal pelvis dilatation is visible in both the upper (U) and lower (L) pole moieties, which are separated by an intervening band of renal tissue (arrowhead). B.The bladder, encircled by the highlighted umbilical arteries, contains a ureterocele (arrowhead). 1000 births (Cragan, 2009; Dolk, 2010; Sheih, 1989; Wiesel, 2005). When a kidney is absent, color Doppler imaging of the descending aorta demonstrates absence of the ipsilateral renal artery (Fig. 10-43). In addition, the ipsilateral adrenal gland typically enlarges to ill the renal fossa, termed the ying down adrenal sin (Hofman, 1992). As with other fetal anomalies, amniocentesis for chromosomal microarray analysis should be considered.

1	If renal agenesis is bilateral, no urine is produced, and the resulting anhydramnios leads to pulmonary hypoplasia, limb contractures, and a distinctively compressed face. When this combination results from renal agenesis, it is called Poter syndrome, after Dr. Edith Potter, who described it in 1946. When these abnormalities result from severely decreased amnionic luid volume from another etiology, such as bilateral multicystic dysplastic kidney or autosomal recessive polycystic kidney disease, it is called Poter sequence. he prognosis for these abnormalities is extremely poor.

1	This severe form of renal dysplasia results in a nonfunctioning kidney. The nephrons and collecting ducts do not form normally, such that primitive ducts are surrounded by ibromuscular tissue, and the ureter is atretic (Hains, 2009). Sonographically, the kidney contains numerous smooth-walled cysts of varying size that do not communicate with the renal pelvis and are surrounded by echogenic cortex (Fig. 10-44). Unilateral multicystic dysplastic kidney (MCDK) has a prevalence of 1 in 4000 births. Contralateral renal abnormalities are present in 30 to 40 percent-most frequently vesicoureteral relux or ureteropelvic junction obstruction (Schreuder, 2009). Nonrenal anomalies have been reported in 25 percent of cases, and cystic dysplasia may occur as a component of many genetic syndromes (Lazebnik, 1999; Schreuder, 2009). If MCDK is isolated and unilateral, the prognosis is generally good.

1	FIGURE 10-43 Renal agenesis. A.oln this coronal image of the fetal abdomen, color Doppler shows the course of the abdominal aorta. The ultrasound beam is perpendicular to the aorta, demonstrating absence of the renal arteries bilaterally. B. This coronal image of a fetus with unilateral renal agenesis shows the adrenal gland (arrowheads) filling the renal fossa, termed the "lying-down" adrenal sign. The adrenal gland has a hypoechoic cortex and hyperechoic medulla. FIGURE 10-44 Multicystic dysplastic kidneys. Coronal view of the fetal abdomen demonstrates markedly enlarged kidneys containing multiple cysts of varying sizes that do not communicate with a renal pelvis. FIGURE 10-45 Posterior urethral valve. In this 19-week fetus with severe bladder outlet obstruction, the bladder is dilated and thickwalled, with dilatation of the proximal urethra that resembles a "keyhole." Adjacent to the bladder is an enlarged kidney with evidence of cystic dysplasia, conferring a poor prognosis.

1	Bilateral MCDK is found in approximately 1 in 12,000 births. It is associated with severely decreased amnionic luid volume starting early in gestation. his leads to Potter sequence and a poor prognosis (Lazebnik, 1999).

1	Of the hereditary polycystic diseases, only the infantile form of autosomal recessive poyystic kidney disease (APD) may be reliably diagnosed prenatally. ARPKD is a chronic, progressive disease of the kidneys and liver that results in cystic dilatation of the renal collecting ducts and in congenital hepatic ibrosis (Turkbey, 2009). he carrier frequency of a disease-causing mutation in the PHDI gene approximates 1 in 70, and the disease prevalence is 1 in 20,000 (Zerres, 1998). he phenotypic variability of ARPKD ranges from lethal pulmonary hypoplasia at birth to presentation in late childhood or even adulthood with predominantly hepatic manifestations. Sonographically, infantile ARPKD displays abnormally large kidneys that ill and distend the fetal abdomen and have a solid, ground-glass texture. Severe oligohydramnios confers a poor prognosis.

1	Autosomal dominant poyystic kidney disease ADPD), which is far more common, usually does not manifest until adulthood (Chap. 53, p. 1031). Even so, some fetuses with AD PKD have mild renal enlargement and enhanced renal echogenicity in the setting of normal amnionic luid volume. he diferential diagnosis for these findings includes several genetic syndromes, aneuploidy, or normal variant.

1	Distal obstruction of the urinary tract is more frequent in male fetuses, and the most common etiology is posterior urethral valves. Characteristically, the bladder and proximal urethra are dilated, termed the "keyhole" sign, and the bladder wall is thick (Fig. 10-45). Oligohydramnios, particularly before midpregnancy, portends a poor prognosis because of pulmonary hypoplasia. Unfortunately, the outcome may be poor even with normal amnionic fluid volume. Evaluation includes a careful search for associated anomalies, which may occur in 40 percent of cases, and for aneuploidy, which has been reported in 5 to 8 percent (Hayden, 1988; Hobbins, 1984; Mann, 2010). If neither are present, afected male fetuses with severe oligohydramnios who have fetal urinary electrolytes suggesting a potentially favorable prognosis may be fetal therapy candidates. Evaluation and treatment of fetal bladder outlet obstruction is discussed in Chapter 16 (p. 325).

1	The 2015 revision of the Nosology and Classification of Genetic Skeletal Disorders includes an impressive 436 skeletal anomalies in 42 groups, characterized by genetic abnormalities, phenotypic features, or radiographic criteria (Bonafe, 2015). he two types of skeletal dysplasias are osteochondrodyspasiasthe generalized abnormal development of bone and/or cartilage, and dysostoses-which are abnormalities of individual bones, for example, poydacyy. In addition to these maormations, skeletal abnormalities include dormations, as with some cases of clubfoot, and disruptions such as limb-reduction defects. The prevalence of skeletal dysplasias approximates 3 in 10,000 births. Two groups account for more than half of all cases: the ibroblast growth octor 3 (FGF3) chondrodysplasia group and the osteogenesis impeecta and decreased bone density group. Each occurs in 0.8 in 10,000 births (Stevenson, 2012).

1	Evaluation of a pregnancy with suspected skeletal dysplasia includes a survey of every long bone, as well as the hands and feet, skull size and shape, clavicles, scapulae, thorax, and spine. Reference tables are used to determine which long bones are afected and ascertain the degree of shortening (Appendix, p. 1267). Involvement of all long bones is termed micromelia, whereas predominant involvement of only the proximal, intermediate, or distal long bone segments is termed rhizomelia, meso melia, and acromelia, respectively. he degree of ossiication should be noted, as should presence of bowing or fractures. Each of these may provide clues to narrow the diferential diagnosis and occasionally suggest a specific skeletal dysplasia. Many, if not most, skeletal dysplasias have a genetic component, and knowledge of specific mutations has advanced dramatically (Bonafe, 2015).

1	nosis, it is frequently possible to determine whether a skeletal dysplasia is lethal. Lethal dysplasias show profound long bone shortening, with measurements <5th percentile, and display cent (Nelson, 2014; Rahemtullah, 1997; Ramus, 1998). Gen erally, other sonographic abnormalities are evident. Pulmonary hypoplasia is suggested by a thoracic circumference < 80 per cent of the abdominal circumference value, by a thoracic cir cumference <2.5th percentile, and a cardiac circumference > 50 percent of the thoracic circumference value (Appendix, p. 1266). Afected pregnancies also may develop hydramnios and/or hydrops (Nelson, 2014).

1	he FGFR3 chondrodysplasias include achondroplasia and thanatophoric dysplasia. Achondroplasia, also called heterozygous achondroplasia, is the most common nonlethal skeletal dysplasia. An impressive 98 percent of cases are due to a speciic point mutation in the FGFJ gene. It has an autosomal dominant inheritance, and 80 percent of cases result from a new mutation. Achondroplasia is characterized by long bone shortening that is predominantly rhizomelic, an enlarged head with frontal bossing, depressed nasal bridge, exaggerated lumbar lordosis, and a trident configuration of the hands. Intelligence is typically normal. Sonographically, the femur and humerus measurements may not lie below the 5th percentile until the early third trimester. hus, this condition is usually not diagnosed until late in pregnancy. In homozygotes, which represent 25 percent of the ofspring of heterozygous parents, the condition is characterized by greater long bone shortening and is lethal.

1	he other major class of FGFR3 dysplasias, thanatophoric dysplasia, is the most common lethal skeletal disorder. It is characterized by severe micromelia, and afected fetuses-particularly those with type II-may develop a characteristic cloverleaf skull deformity (Kleeblattschade) due to craniosynostosis. More than 99 percent of cases may be con firmed with genetic testing. Osteogenesis impeecta represents a group of skeletal dysplasias typiied by hypomineralization. here are multiple types, and more than 90 percent of cases are characterized by a mutation in the COLIAI or COLIA2 gene. Type IIa, also called the perinatal form, is lethal. It displays a profound lack of skull ossification, such that gentle pressure on the maternal abdomen from the ultrasound transducer results in visible skull deformation 10-46).

1	FIGURE 10-46 Osteogenesis imperfecta. Type Ila, which is lethal, is characterized by such profound lack of skull ossification that gentle pressure on the maternal abdomen from the ultrasound transducer results in visible deformation (flattening) of the skull (arrowheads). Other features include multiple in-utero fractures and ribs that appear "beaded." Inheritance is autosomal dominant, such that all cases result from either new mutations or gonadal mosaicism (Chap. 13, p. 264). Another skeletal dysplasia that results in severe hypomineralization is hypophosphatasia, which has an autosomal recessive inheritance.

1	his disorder is notable for a deformed talus and shortened Achilles tendon. he afected foot is abnormally fixed and positioned with equinus (downward pointing), varus (inward rotation), and forefoot adduction. Most cases are considered malformations, with a multifactorial genetic component. However, an association with environmental factors and with early amniocentesis suggests that deformation also plays a role (T redwell, 2001). Sonographically, the footprint is visible in the same plane as the tibia and ibula (Fig. 10-47).

1	he prevalence of clubfoot approximates 1 in 1000 births, and the male:female ratio is 2:1 (Carey, 2003; Pavone, 2012). Clubfoot is bilateral in approximately 50 percent of afected individuals, and associated anomalies are present in at least 50 percent of all cases (lvfammen, 2004; Sharma, 201i1). Frequently associated anomalies include neuraltube defects, arthrogryposis, and myotonic dystrophy and other genetic syndromes. In cases with associated anomalies, aneuploidy is found in approximately 30 percent. In contrast, the rate is <4 percent when clubfoot appears isolated (Lauson, 2010; Sharma, 2011). hus, a careful search for associated structural abnormalities is warranted, and chromosomal microarray analysis may be considered. FIGURE 10-47 Foot position. A. Normal fetal lower leg, demonstrating normal position of the foot. B. With talipes equinovarus, the foot "print" is visible in the same plane as the tibia and fibula.

1	FIGURE 10-47 Foot position. A. Normal fetal lower leg, demonstrating normal position of the foot. B. With talipes equinovarus, the foot "print" is visible in the same plane as the tibia and fibula. Documentation of the arms and legs is a component of the standard examination. he absence or hypoplasia of all or part of one or more extremities is a limb-reduction dect. he birth prevalence is 4 to 8 in 10,000 (Kucik, 2012; Stoll, 2010; Vasluian, 2013). Approximately half of these are isolated defects, up to on� third occur as part of a recognized syndrome, and individuals in the remaining cases have other coexisting anomalies (Stoll, 2010; Vasluian, 2013). Upper extremities are afected more frequently than lower ones. Of categories, a terminal transverse limb dect lacks part or all of a distal limb to create a stump (Fig. 10-48). his is more common than a longitudinal dect, which is complete or partial absence of the long bone(s) on only one side of a given extremity.

1	Absence of an entire extremity is termed amelia. Phocomelia, associated with thalidomide exposure, is an absence of one or more long bones with the hands or feet attached to the trunk (Chap. 12, p. 246). Limb-reduction defects are associated with numerous genetic syndromes, such as Roberts syndrome, an autosomal recessive condition characterized by tetraphocomelia. A clubhand dormiy, usually from an absent radius, is associated with trisomy 18 and is also a component of the thrombocytopenia-absent radius syndrome (Fig. 13-5B, p. 258). Limb-reduction defects may occur in the setting of a disruption such as amnionic-band sequence (Chap. 6, p. 116). hey have also been associated with chorionic villus sampling when performed before 10 weeks' gestation (Fig. 14-6, p. 294).

1	During the past two decades, three-dimensional (3-D) sonography has gone from a novelty to a standard feature of most modern ultrasound equipment (Fig. 10-49). 3-D sonography is not routiney used during a standard examination nor considered a required modality. However, it may be a component of specialized evaluations. Most 3-D scanning uses a special transducer developed for this purpose. After a region of interest is identiied, a 3-D volume is acquired that can be rendered to display axial, sagittal, coronal, or oblique images. Sequential "slices" may be generated, similar to computed tomographic (CT) or MR images. Unlike two-dimensional (2-D) scanning, which appears to be in "real time," 3-D imaging is static and obtained by processing a volume of stored images. With our-dimensional (4-DJ sonography, also known as real-time 3-D sonography, rapid reconstruction of the rendered images conveys the impression that the scanning is in real time.

1	FIGURE 10-48 Transverse limb-reduction defect. A. At 18 weeks' gestation, only a rudimentary hand was visible. B. By 24 weeks, the radius and ulna were normal in size and appearance, and small rudimentary digits were evident.

1	For 4-D imaging, one application known as spatiotemporal image co rrelatio n-S TIC improves visualization of cardiac anatomy. During an automated sweep over the heart, the STIC application acquires a volume that includes thousands of 2D images captured at a rate as high as 150 frames per second (Devore, 2003). These individual images are obtained at diferent locations in the heart but at the same point in time. These views are subsequently arranged according to their spatial and temporal domains. his permits display of an ordered sequence of volume sets in a continuous cine loop (or video clip) of the cardiac cycle (Yeo, 2016). For example, after obtaining a volume sweep over the cardiac apex, an application such as Fetal Intelligent Navigation Echocardiography (FINE) can be applied to FIGURE 10-49 Fetal face. Surface rendered three-dimensional image of a normal fetal face and hand at 32 weeks. display videos of each of the diferent cardiac views shown in

1	FIGURE 10-49 Fetal face. Surface rendered three-dimensional image of a normal fetal face and hand at 32 weeks. display videos of each of the diferent cardiac views shown in Fig. 10-28 (Garcia, 2016). It is hoped that such technology may eventually improve detection of fetal cardiac anomalies. For selected anomalies, such as those of the face and skel eton, for tumors, and for some cases of neural-tube defects, 3-D sonography can add useful information (American College of

1	Obstetricians and Gynecologists, 2016; Goncalves, 2005). That said, comparisons of 3-D and conventional 2-D sonography for the diagnosis of most congenital anomalies have not demon strated better overall detection rates (Goncalves, 2006; Reddy, 2008). The American College of Obstetricians and Gynecolo gists (2016) concludes that proof of a clinical advantage of 3-D S sonography for prenatal diagnosis is generally lacking. D hen sound waves strike a moving target, the frequency of the waves reflected back is shifted in proportion to the velocity and direction of that moving target-a phenomenon known as the Doppler sht. Because the magnitude and direction of the frequency shift depend on the relative motion of the moving target, Doppler can help evaluate low within blood vessels. The Doppler equation is shown in Figure 10-50.

1	n important component of the equation is the angle of insonation, abbreviated as theta (8). This is the angle between the sound waves from the transducer and low within the vessel. Measurement error becomes large when 8 is not close to zero, in other words, when blood flow is not coming directy toward or away from the transducer. For this reason, ratios are often used to compare diferent waveform components, allowing cosine 8 to cancel out of the equation. Figure 10-51 is a schematic of the Doppler waveform and describes the three ratios commonly used. he simplest is the systolic-diastolic ratio (SID ratio), which compares the maximal (or peak) systolic low with end-diastolic flow to evaluate downstream impedance to flow. Currently, two types of Doppler modalities are available for clinical use. Continuous-wave Doppler equipment has two separate types of crystals-one transmits high-frequency sound waves, and ,,,I ,

1	Continuous-wave Doppler equipment has two separate types of crystals-one transmits high-frequency sound waves, and ,,,I , FIGURE 10-50 Doppler equation. Ultrasound emanating from the transducer with initial frequency fo strikes blood moving at velocity v. Reflected frequency fd is dependent on angle e between beam of sound and vessel. FIGURE 10-51 Doppler systolic-diastolic waveform indices of blood flow velocity. S represents the peak systolic flow or velocity, and D indicates the end-diastolic flow or velocity. The mean, which is the time-average mean velocity, is calculated from computerdigitized waveforms. another continuously captures signals. In M-mode imaging, continuous-wave Doppler is used to evaluate motion through time, however, it cannot image individual vessels.

1	another continuously captures signals. In M-mode imaging, continuous-wave Doppler is used to evaluate motion through time, however, it cannot image individual vessels. Pulsed-wave Doppler uses only one crystal, which transmits the signal and then waits until the returning signal is received before transmitting another one. It allows precise targeting and visualization of the vessel of interest. Pulsed-wave Doppler can be conigured to allow color-low mapping-such that blood flowing toward the transducer is displayed in red and that flowing away from the transducer appears in blue. Various combinations of pulsed-wave Doppler, color-flow Doppler, and real-time sonography are commercially available.

1	Umbilical artery Doppler has been subjected to more rigorous assessment than any previous test of fetal health. he umbilical artery difers from other vessels in that it normally has forward low throughout the cardiac cycle. Moreover, the amount of low during diastole increases as gestation advances, and this reflects decreasing placental impedance. As a result, the SID ratio normaly declines rom approximatey 4.0 at 20 weeks' gestation, to generaly less than 3.0 ter 30 weeks' and inaly to 2.0 at term. Because of downstream impedance to low, more end-diastolic low is observed at the placental cord insertion than at the fetal ventral wall. hus, abnormalities such as absent or reversed enddiastolic low will appear first at the fetal cord insertion site. The International Society of Ultrasound in Obstetrics and Gynecology recommends that umbilical artery Doppler measurements be made in a free loop of cord (Bhide, 2013). However, assessment close to the ventral wall insertion may optimize

1	in Obstetrics and Gynecology recommends that umbilical artery Doppler measurements be made in a free loop of cord (Bhide, 2013). However, assessment close to the ventral wall insertion may optimize measurement reproducibility when low is diminished (Berkley, 2012).

1	The waveform is considered abnormal if the SID ratio is >95th percentile for gestational age. In extreme cases of growth restriction, end-diastolic flow can become absent or even reversed (Fig. 44-8, p. 854). Such reversal of end-diastolic flow has been associated with greater than 70-percent obliteration of the small muscular arteries in placental tertiary stem villi (Kingdom, 1997; Morrow, 1989). As described in Chapter 44 (p. 854), umbilical artery Doppler aids management of fetal-growth restriction and has been associated with improved outcome in these cases (American College of Obstetricians and Gynecologists, 2015). It is not recommended for complications other than growth restriction. Similarly, its use for growth-restriction screening is not advised (Berkley, 2012). Abnormal umbilical artery Doppler findings should prompt a complete fetal evaluation, if not already done, because abnormal measurements are associated with major fetal anomalies and aneuploidy (Wenstrom, 1991).

1	Doppler evaluation of the ductus arteriosus has been used primarily to monitor fetuses exposed to indomethacin and other nonsteroidal antiinlammatory agents (NSAIDs). Indomethacin, which is used by some for tocolysis, may cause ductal constriction or closure, particularly when used in the third trimester (Huhta, 1987). The resulting increased pulmonary flow can cause reactive hypertrophy of the pulmonary arterioles and eventual development of pulmonary hypertension. In a review of 12 randomized controlled trials involving more than 200 exposed pregnancies, Koren and coworkers (2006) reported that NSAIDs raised the odds of ductal constriction 15-fold. When these agents are indicated, their duration is typically limited to less than 72 hours. And, women taking NSAIDs are closely monitored so that these can be discontinued if ductal constriction is identified. Fortunately, ductal constriction is often reversible after NSAID discontinuation.

1	Uterine blood low is estimated to rise from 50 mLlmin early in gestation to 500 to 750 mLlmin by term. he uterine artery Doppler waveform is characterized by high diastolic low velocities and markedly turbulent flow. Greater resistance to flow and development of a diastolic notch are associated with later development of gestational hypertension, preeclampsia, and fetal-growth restriction. Zeeman and coworkers (2003) also found that women with chronic hypertension who had elevated uterine artery impedance at 16 to 20 weeks' gestation were at greater risk to develop superimposed preeclampsia. However, the technique, best testing interval, and defining criteria for this indication have not been standardized. As the predictive value of uterine artery Doppler testing is considered to be low, its use for screening or for clinical decision-making is not recommended in either high-risk or low-risk pregnancies (Sciscione, 2009).

1	Doppler interrogation of the middle cerebral artery (MCA) has been investigated and applied clinically for fetal anemia detection and fetal-growth restriction evaluation. Anatomically, the path of the MCA is such that flow often approaches the transducer "head-on," allowing for accurate determination of low velocity (Fig. 10-52). he MCA is imaged in an axial view of the head at the base of the skull, ideally within 2 mm of the internal carotid artery origin. Velocity measurement is optimal when the insonating angle is close to zero, and no more than 30 degrees of angle correction should be used. In general, velocity assessment is not performed in other fetal vessels, because a larger insonating angle is needed and confers significant measurement error.

1	When fetal anemia is present, the peak systolic velociy is enhanced due to greater cardiac output and decreased blood viscosity (Segata, 2004). This has permitted the reliable, noninvasive detection of fetal anemia in cases of blood-group alloimmunization. Mari and colleagues (2000) demonstrated that an MCA peak systolic velocity threshold of 1.50 MoM could reliably identiy fetuses with moderate or severe anemia. As discussed in Chapter 15 (p. 303), MCA peak systolic velocity has replaced FIGURE 10-52 Middle cerebral artery (MCA) Doppler. A. Color Doppler of the circle of Willis, demonstrating the correct location to sample the MCA. B. The waveform demonstrates a peak systolic velocity exceeding 70 cm/sec in a 32-week fetus with severe fetal anemia secondary to Rh alloimmunization. invasive testing with amniocentesis as the preferred test for fetal anemia detection (Society for Maternal-Fetal Medicine, 2015).

1	invasive testing with amniocentesis as the preferred test for fetal anemia detection (Society for Maternal-Fetal Medicine, 2015). MCA Doppler has also been studied as an adjunct in evaluation of fetal-growth restriction. Fetal hypoxemia is believed to result in increased blood flow to the brain, heart, and adrenal glands, leading to greater end-diastolic low in the MCA. his phenomenon, "brain-sparing," is actually a misnomer, as it is not protective for the fetus but rather is associated with perinatal morbidity and mortality (Bahado-Singh, 1999; CruzMartinez, 2011). The utility ofMCA Doppler to aid the timing of delivery is uncertain. It has not been evaluated in randomized trials or adopted as standard practice in the management of growth restriction (American College of Obstetricians and Gynecologists, 2015; Berkley, 2012).

1	he ductus venosus is imaged as it branches from the umbilical vein at approximately the level of the diaphragm. Fetal position poses more of a challenge in imaging the ductus venosus than it does with either the umbilical artery or the middle cerebral artery. he waveform is biphasic and normally has forward flow throughout the cardiac cycle. The irst peak reflects ventricular systole, and the second is ventricular diastolic illing. These are followed by a nadir during atrial contraction-termed the a-wave.

1	It is believed that Doppler indings in preterm fetuses with growth restriction show a progression in which umbilical artery Doppler abnormlities are followed by ones in the middle cerebral artery and then in the ductus venosus. However, manifestations of these abnormalities vary widely (Berkley, 2012). With severe fetal-growth restriction, cardiac dysfunction may lead to low in the a-wave that is decreased, absent, and eventually reversed, along with pulsatile low in the umbilical vein (Fig. 10-53).

1	Ductus venosus abnormalities have potential to identiy preterm growth-restricted fetuses that are at greatest risk for adverse outcomes (Baschat, 2003, 2004; Bilardo, 2004; Figueras, 2009). As noted by the Society for Maternal-Fetal Medicine, however, they have not been suiciently evaluated in randomized trials (Berkley, 2012). In sum, Doppler assessment of vessels other than the umbilical artery has not been shown to improve perinatal outcome, and thus their role in clinical practice remains uncertain (American College of Obstetricians and Gynecologists, 2015).

1	Image resolution with MR is oten superior to that with sonography because it is not as hindered by bony interfaces, maternal obesity, oligohydramnios, or an engaged fetal head. Thus, it can serve as an adjunct to sonography in evaluating suspected fetal abnormalities. Examples include complex abnormalities of the fetal CNS, thorax, gastrointestinal system, genitourinary system, and musculoskeletal system. MR has also been used in the evaluation of maternal pelvic masses and placental invasion. MR imaging, however, is not portable, it is time-consuming, and its use is generally limited to referral centers with expertise in fetal imaging. To guide clinical use, the American College of Radiology and Society for Pediatric Radiology (2015) have developed

1	To guide clinical use, the American College of Radiology and Society for Pediatric Radiology (2015) have developed FIGURE 10-53 Venous Doppler abnormalities. A. Reversal of a-wave flow in the ductus venosus. Arrows depict a-waves below the baseline. This finding may be identified with cardiac dysfunction in the setting of severe fetal-growth restriction. B. Pulsatile flow in the umbilical vein. The undulating umbilical venous waveform below the baseline indicates tricuspid regurgitation. Above the baseline is the umbilical artery waveform, in which there is no visible end-diastolic flow. Because the venous waveform is below the baseline in this image, it is not possible to determine whether the umbilical artery end-diastolic flow is reversed.

1	a practice guideline for fetal MR imaging. This document acknowledges primacy of sonography as the preferred screening modality. Moreover, it recommends that fetal MR imaging be used for problem solving to ideally contribute to prenatal diagnosis, counseling, treatment, and delivery planning. Specific indications for fetal MR imaging are listed in Table 10-11 and are discussed subsequently. MR imaging uses no ionizing radiation, but theoretical concerns include the efects of luctuating electromagnetic ields and high sound-intensity levels. The strength of the magnetic TABLE 10-11. Fetal Conditions for Which Magnetic Resonance Imaging May Be Indicateda Agenesis of the corpus callosurn Solid or cyJtic masses Vascular malformations Hydranencephaly Infarction. Hemorrhage Skull, face, and neck Other abnormalities with potential airway obstruction Evaluation of pulmonary hypoplasia secondary to diaphragmatic hernia, oligohydramnios, chest mass, or ske!etal dysplasia

1	Skull, face, and neck Other abnormalities with potential airway obstruction Evaluation of pulmonary hypoplasia secondary to diaphragmatic hernia, oligohydramnios, chest mass, or ske!etal dysplasia Abdomen, pelvis, and retroperitoneum Abdominopelvic cystic mass evaluation Tumor evaluation (sacrococcygeal teratoma, neuroblastoma, hemangioma, suprarenal or renal masses) Complex genitourinary anomalies (bladder outlet obstruction syndromes, bladder exstrophy, cloacal exstrophy) Assess rena! anomalies with oligohydramnios Diagnose bowel anomalies (anorectal malformations, Complications of monochorionic twins Determine vascular anatomy prior to laser treatment Assess morbidity ater death of a monochorionic co-twin

1	Diagnose bowel anomalies (anorectal malformations, Complications of monochorionic twins Determine vascular anatomy prior to laser treatment Assess morbidity ater death of a monochorionic co-twin Fetal brain anatomy before and after surgical intervention Anomalies for which fetal surgery is planned aln some cases, magnetic resonance (MR) imaging is indicated only if the anomaly is ed but cannot be adequately characterized sonographically, vvhich is assessed on a case-by case basis. Data from American College of Radiology, 2015.

1	Data from American College of Radiology, 2015. field is measured in tesLa (), and most imaging studies during pregnancy are performed using 1.5 T. A few preliminary studies advocate the use of 3 T for fetal imaging to potentially improve signal-to-noise ratios and thus image clarity (Victoria, 2016). For safety, all clinical examinations must adhere to the speciic absorption rate, which is regulated by the Food and Drug Administration, and the ALARA principle should be followed. Thus, for routine clinical examinations, the lower ield strength of 1.5 T is recommended (Prayer, 2017).

1	Human and tissue studies support the safety of fetal MR imaging. Repetitive exposure of human lung fibroblasts to a static 1.5-T magnetic ield does not afect cellular proliferation (Wiskirchen, 1999). Fetal heart rate patterns have been evaluated before and during MR imaging, and no signiicant diferences were observed (Vadeyar, 2000). Children exposed to MR as fetuses do not show a greater incidence of disease or disability when tested at age 9 months or 3 years (Baker, 1994; Clements, 2000).

1	Glover and associates (1995) attempted to mimic the sound level experienced by the fetal ear by having an adult volunteer swallow a microphone while the stomach was illed with a liter of luid to represent the amnionic sac. Sound intensity was attenuated at least 30-dB from the body surface to the fluidfilled stomach and reduced the sound pressure from 120 dB to below 90 dB. This level is considerably less than the 135 dB experienced from vibroacoustic stimulation used in antepartum well-being assessments (Chap. 17, p. 337). Cochlear function testing in infants exposed to 1.5-T MR imaging as fetuses showed no hearing impairment (Reeves, 2010).

1	he American College of Radiology (2013) concludes that based on available evidence, MR imaging has no documented deleterious efects on the developing fetus. Therefore, MR imaging can be performed in pregnancy if data are needed to care for the fetus or mother. Health-care providers who are pregnant may work in and around an MR unit, but it is recommended that they not remain in the MR scanner magnet room-known as Zone IV-while an examination is in progress.

1	Gadolinium-based MR contrast agents are gadolinium (Gd3+) chelates. These contrast agents readily enter the fetal circulation and are excreted via fetal urination into amnionic luid, where they may remain for an indeterminate period before being ingested and reabsorbed. he longer the gadolinium-chelate molecule remains in a protected space such as the amnionic sac, the greater the potential for dissociation of the toxic Gd3+ ion. Accordingly, gadolinium contrast should be avoided during pregnancy because of this potential for dissociation. Routine use of gadolinium is not recommended unless there are overwhelming potential benefits (American College of Radiology, 2013). In adults with renal disease, this contrast agent has been associated with development of nephrogenic systemic fibrosis, a potentially severe complication.

1	Before MR examination, all women complete a written safety questionnaire that includes information about metallic implants, pacemakers, or other metal-or iron-containing devices that may alter the study (American College of Radiology, 2013). Iron supplementation may cause artifact in the colon but does not usually afect the resolution of fetal images. In more than nancy during the past 15 years, maternal anxiety secondary to claustrophobia and/or fear of MR equipment has developed in less than 1 percent of our patients. To reduce maternal anxiety in this small group, a single oral dose of diazepam, 5 to 10 mg, or lorazepam, 1 to 2 mg, may be given.

1	To begin an MR examination, women are placed in a supine or left lateral decubitus position. A torso coil is used in most circumstances to send and receive the radiofrequency pulses, but a body coil can be used alone to accommodate large mater nal habitus. A series of three-plane localizers, or scout views, are obtained relative to the maternal coronal, sagittal, and axial planes. he gravid uterus is imaged in the maternal axial plane (7-mm slices, 0 gap) with a T2-weighted fast acquisition. Typically, these may be a single-shot fast spin echo sequence (SSFSE), half-Fourier acquisition single-shot turbo spin echo (HASTE), or rapid acquisition with relaxation enhancement (AE), depending on the brand of machine. Next, a fast T1-weighted acquisition such as spoiled gradient echo (SPGR) is performed (7-mm thickness, 0 gap). hese large-field-of-view acquisitions through the maternal abdomen and pelvis are par ticularly good for identiying fetal and maternal anatomy.

1	Orthogonal images of targeted fetal or maternal structures are then obtained. In these cases, 3-to 5-mm slice thickness, o gap T2-weighted acquisitions are performed in the coronal, sagittal, and axial planes. Depending on the anatomy and underlying suspected abnormality, T1-weighted images can be performed to evaluate for subacute hemorrhage, fat, or location of normal structures that appear bright on these sequences, such as liver and meconium in the colon (Brugger, 2006; Zaretsky, 2003b).

1	Short Tl inversion recovey (STIR) and frequency-selective fat-saturated T2-weighted images may provide diferentiation in cases in which the water content of the abnormality is similar to that of the normal structure. An example is a thoracic mass compared with normal lung. Difusion-weighted imaging may be employed to evaluate for restricted difusion, which can be seen in ischemia, cellular tumors, or clotted blood (Brugger, 2006; Zaretsky, 2003b). Our series also includes an axial brain 3-to 5-mm T2-weighted sequence to obtain head biometry for gestational age estimation using the biparietal diameter and head circumference (Reichel, 2003).

1	Whenever a fetal abnormality is identiied, indings from the afected organ and other organ systems should be thoroughly characterized. Accordingly, a fetal anatomical survey is generally completed during each MR examination. In a recent prospective study, nearly 95 percent of the anatomical components recommended by the Inter- national Society of Ultrasound in Obstetrics and Gynecology were visible at 30 weeks' gestation (Millischer, 2013). he aorta and pulmonary artery were the most diicult to evaluate. Zaretsky and coworkers (2003a) similarly found that with the exclusion of cardiac structures, fetal anatomical evaluation was possible in 99 percent of cases. For intracranial anomalies, very fast T2-weighted images produce excellent tissue contrast, and eSF-containing structures are hyperintense or bright. This allows exquisite detail of the posterior fossa, midline structures, and cerebral cortex. T1-weighted images are used to identiy hemorrhage.

1	eNS biometry obtained with MR imaging is comparable with that obtained using sonography (Twickler, 2002). Nomograms have been published for multiple intracranial structures, including corpus callosum and cerebellar vermis lengths (Garel, 2004; Tilea, 2009). MR imaging provides valuable added information for cerebral abnormalities suspected sonographically (Benacerraf, 2007; Li, 2012). In early studies, MR imaging changed the diagnosis in 40 to 50 percent of cases and afected management in 15 to 50 percent (Levine, 1999b; Simon, 2000; Twickler, 2003). Additional information is more likely to be gained when the examination is performed beyond 24 weeks' gestation. More recently, Griiths and associates (2017) reported that MR evaluation of suspected fetal brain anomalies identified additional indings in nearly 50 percent and changed prognosis in 20 percent.

1	Fetuses with a cerebral abnormality may have a signiicant lag in cortical development. Levine and colleagues (l999a) demonstrated that MR imaging accurately portrays cerebral gyration and sulcation patterns (Fig. 10-54). Sonography permits limited evaluation of subtle migrational abnormalities, and MR imaging provides greater accuracy, particularly later in gestation. For ventriculomegaly, fetal MR imaging is selected to help identiy associated underlying eNS dysmorphology (p. 193). In cases of septo-optic dysplasia, MR imaging may confirm absence of the septum pellucidum and display hypoplastic FIGURE 10-54 Axial images of the fetal brain at 23 weeks' gestation (A) and at 33 weeks (8) demonstrate the normal gyration and sulcation progression during fetal development. These images were obtained using a Half Fourier Acquisition Single Shot Turbo Spin Echo (HASTE) sequence because it is relatively motion insensitive.

1	optic tracts (Fig. 10-55). In other fetuses, MR imaging can also assist with identiying agenesis or dysgenesis of the corpus callosum and characterizing migrational abnormalities (Benacerraf, 2007; Li, 2012; Twickler, 2003). Another fetal MR imaging indication is evaluation of suspected intraventricular hemorrhage (IVH). Fetal IVH risk factors include atypical-appearing ventriculomegaly, neonatal alloimmune thrombocytopenia, and a monochorionic multifetal gestation complicated by demise of one fetus or by severe twin-twin transu FIGURE 10-55 Septo-optic dysplasia. Axial (A) and coronal (B) images at 30 weeks' ges sion syndrome (Hu, 2006). If hemorrhage tation confirm absence of the cavum septum pellucidum (arrowheads) in both. There is is seen, MR imaging characteristics may also associated mild ventriculomegaly (arrow).

1	FIGURE 10-56 Terminal myelocystocele, 36 weeks' gestation. A. In this sagittal T2-weighted image, the spinal cord is tethered, expanding into the terminal cyst (arrow). B. Seen in this Tl-weighted image, the meningocele and terminal cyst are covered by subcutaneous fat (arrows) and skin. indicate which structures are involved and approximately when bleeding occurred. In the setting of congenital fetal infections, ,1R imaging can delineate the variable degrees of neural parenchymal abnormality and subsequent maldevelopment (Soares de Oliveira-Szejnfeld, 2016). Aside from cerebral structure, suspected spinal dysraphisms, including neural-tube defects, can also be further characterized for surgical planning. Figure 10-56 demonstrates a complex skin-covered dysraphism with associated tethering of the spinal cord. his terminal myelocystocele will benefit from early intervention following delivery.

1	Many thoracic abnormalities are readily seen with targeted sonography. MR imaging, however, may help delineate the location and size of space-occupying thoracic lesions and quantiy remaining lung tissue volumes. MR imaging can aid in characterizing the type of congenital cystic adenomatoid malformation and in visualizing the blood supply of pulmonary sequestration (p. 200). With congenital diaphragmatic hernia, MR imaging can help veriy and quantiy the abdominal organs within the thorax. his includes the volume of herniated liver and compressed lung tissue volumes (Fig. 10-57) (Debus, 2013; Lee, 2011; Mehollin-Ray, 2012). Also in fetuses with diaphragmatic hernia, MR imaging can assist in identiying other organ-system abnormalities, which may greatly afect fetal prognosis (Kul, 2012). MR imaging similarly is used for chest evaluation in skeletal dysplasia and to measure lung volumes in pregnancies with prolonged oligohydramnios secondary to renal disease or ruptured membranes

1	MR imaging similarly is used for chest evaluation in skeletal dysplasia and to measure lung volumes in pregnancies with prolonged oligohydramnios secondary to renal disease or ruptured membranes (Messerschmidt, 2011; Zaretsky, 2005).

1	When sonographic viewing of fetal abdominal abnormalities is limited by oligohydramnios or maternal obesity, MR imaging may add value (Caire, 2003). Hawkins and coworkers (2008) found that lack of signal in a contracted fetal bladder on T2-weighted sequences was associated with lethal renal abnormalities (Fig. 10-58). Diferences in signal characteristics between meconium in the fetal colon and urine in the bladder may permit deinition of cystic abdominal abnormalities (Farhataziz, 2005). Given the predictable pattern of meconium accumulation within the gastrointestinal tract and high signal intensity on T1-weighted sequences, MR imaging is a complementary tool in diagnosing gastrointestinal abnormalities and complex cloacal malformations (Furey, 2016). Peritoneal calciications related to meconium peritonitis are more readily apparent

1	FIGURE 10-57 A. Coronal image of normal lungs on a balanced sequence at 29 weeks' gestation. The liver (L) and stomach (5) lie below the diaphragm. B. Left-sided congenital diaphragmatic hernia (CDH) (dotted ellipse) seen on balanced sequence at 33 weeks. C. The T1o-weighted sequence confirms the subdiaphragmatic position of the liver and better delineates the small bowel (arrow) and meconium-containing colon (arrowhead) that have herniated into the chest. D. Another image of a left-sided CDH at 22 weeks demonstrates no normal lung, the heart (H) displaced into the right chest, and an elevated liver (dotted ellipse). sonographically, whereas pseudocysts and resultant abnormalities of meconium migration are better delineated with MR imaging . • Adjunct to Fetal Therapy

1	As indications for fetal therapy have grown, MR imaging is used preoperatively to outline abnormalities. At some centers, before laser ablation of placental anastomoses for twin-twin transfusion syndrome, MR imaging is performed to assess the fetal brain for IVH or periventricular leukomalacia (Chap. 45, p. 878) (Hu, 2006; Kline-Fath, 2007). Because of its precision in visualizing brain and spine indings in cases of myelomeningocele, it is often used preoperatively. For sacrococcygeal teratomas, if fetal surgery is considered, MR imaging may identiY tumor extension into the fetal pelvis (Avni, 2002; Neubert, 2004; Perrone, 2017). With a fetal neck mass for which an EIT is considered, MR imaging may help delineate the lesion extent and its efect on the oral cavity, hypopharynx, and trachea (Hirose, 2003; Lazar, 2012; Ogamo, 2005; Shiraishi, 2000). Finally, vfR imaging can also calculate a jaw index when an EIT procedure may be needed for severe micrognathia (MacArthur, 2012; Morris,

1	(Hirose, 2003; Lazar, 2012; Ogamo, 2005; Shiraishi, 2000). Finally, vfR imaging can also calculate a jaw index when an EIT procedure may be needed for severe micrognathia (MacArthur, 2012; Morris, 2009). Fetal therapy is discussed in Chapter 16 (p. 327).

1	FIGURE 10-58 A. Sagittal short T1 inversion recovery (STIR) image through a fetus with posterior urethral valve at 23 weeks' gestation. Notice the characteristic dilation of the posterior urethra (arrowhead). B. At 31 weeks, a coronal image shows progression of severe hydronephrosis, cystic changes in the parenchyma, hydroureter, and anhydramnios. The lungs (L) show a decreased signal and are small. C. An axial balanced sequence shows a distended bladder (B) with thickened wall (arrows). The clinical importance of identiying women with placenta accreta is discussed in Chapter 41 (p. 777). Sonography is gen erally used to identiy placental invasion into the myometrium, however, MR evaluation is an adjunct for indeterminate cases.

1	bands on T2-weighted images, focal bulging, and placental heterogeneity (Leyendecker, 2012). When used in a comple mentary role, MR imaging sensitivity is high for detection of placental invasion, although the depth of invasion is diicult to predict. Clinical risk factors and sonographic indings should be taken into account when interpreting MR placental images.

1	Of these, MR difusion tensor imaging and tractography may allow urther understanding of neural development and more precise definition of abnormality and pathology (Kasprian, 2008; Mitter, 2015). Automatic and semiautomated extraction of quantitative data from MR imaging volumetric acquisitions of fetal brain and placenta may enable subanalyses of massive datasets not previously possible with laborious manual segmentation (Tourbier, 2017; Wang, 2016). Using multiparametric MR of the placenta in vivo will expand our understanding of function and pathology without risk to mother or fetus. Last, although echocardiography will always be paramount in fetal heart assessment, MR imaging may contribute to volumetric cardiac analysis and will urther evaluation of the aorta, which is oten diicult to completely examine sonographically (Lloyd, 2017).

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1	Wilhelm L, Borgers H: The "equals sign": a novel marker in the diagnosis of fetal isolated sot palate. Ultrasound Obstet Gynecol 36:439, 2010 Williams B, Tareen B, Resnick M: Pathophysiology and treatment of ureteropelvic junction obstruction. Curr Urol Rep 8: Ill, 2007 Wiskirchen J, Groenewaeller EF, Kehlbach R, et al: Long-term efects of repetitive exposure to a static magnetic ield 1.o5 T on proliferation of human fetal lung ibroblasts. Magn Reson Med 41 :464, 1999 Woodward M, Frank 0: Postnatal management of antenatal hydronephrosis. BJU Int 89:o149, 2002 Worley KC, Dashe JS, Barber RG, et al: Fetal magnetic resonance imaging in isolated diaphragmatic hernia: volume of herniated liver and neonatal outcome. Am J Obstet GynecoI200(3):318.e1, 2009 Yamada 5, Uwabe C, Fujii 5, et al: Phenotypic variability in human embryonic holoprosencephaly in the Kyoto collection. Birth Defects Res Part A Clin Mol TeratoIo70:495,o2004

1	Yamada 5, Uwabe C, Fujii 5, et al: Phenotypic variability in human embryonic holoprosencephaly in the Kyoto collection. Birth Defects Res Part A Clin Mol TeratoIo70:495,o2004 Yeo L, Romero R: How to acquire cardiac volumes for sonographic examination of the fetal heart, part I. J Ultrasound Med 35 (5): 1 021, 2016 Yildirim G, Gungorduk K, Asian H, et al: Prenatal diagnosis of extralobar pulmonary sequestration. Arch Gynecol Obstet 278: 181, 2008 Zaretsky M, Ramus R, McIntire 0, et al: MRI calculation of lung volumes to predict outcome in fetuses with genitourinary abnormalities. AJR Am J RoentgenoIo185:1328,o2005 Zaretsky MV, McIntire DO, Twickler DM: Feasibility of the fetal anatomic and maternal pelvic survey by magnetic resonance imaging at term. Am J Obstet Gynecol 189:997, 2003a Zaretsky MY, Twickler OM: Magnetic imaging in obstetrics. Clin Obstet Gynecol 46:868, 2003b

1	Zaretsky MY, Twickler OM: Magnetic imaging in obstetrics. Clin Obstet Gynecol 46:868, 2003b Zeeman GG, McIntire DO, Twickler DM: Maternal and fetal artery Doppler findings in women with chronic hypertension who subsequently develop superimposed pre-eclampsia. J Matern Fetal Neonatal Med 14:318,o2003 Zerres K, Mucher G, Becker J, et al: Prenatal diagnosis of autosomal recessive polycystic kidney disease: molecular genetics, clinical experience, and fetal morphology. Am J Med Genet 6:l37, 1998 NORMAL AMNIONIC FLUID VOLUME .......... .....225 PHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 225 SONOGRAPHIC ASSESSMENT . .......... . .i..226 HYDRAMNIOS ......... ...i...i..i......... .....227 OLIGOHYDRAMNIOS ..i........ .... ....... .....230 BORDERLINE OLIGOHYDRAMNIOS ........... ...i.. 232

1	HYDRAMNIOS ......... ...i...i..i......... .....227 OLIGOHYDRAMNIOS ..i........ .... ....... .....230 BORDERLINE OLIGOHYDRAMNIOS ........... ...i.. 232 It is generaly agreed that amniotic luid represents in great part a transudation from the matenal vesses, but many authorities consider that a portion of it is derived rom urinay secretion of the oetus. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) At the time Williams wrote this, the fetal kidney was thought by many to be nonfunctional. Since that time, however, much has been learned of this complex multifunctional liquor amnii. mnionic fluid serves several roles during pregnancy. Fetal breathing of amnionic fluid is essential for normal lung growth, and fetal swallowing permits gastrointestinal (GI) tract development. Amnionic fluid also creates a physical space for fetal movement, which is necessary for neuromusculoskeletal maturation. It further guards against umbilical cord compression and protects the fetus from trauma. Amnionic luid even has bacteriostatic properties. Abnormalities of volume may result from fetal or placental pathology-indicating a problem with luid production or its circulation. These volume extremes may be associated with increased risks for adverse pregnancy outcome.

1	Amnionic luid volume increases from approximately 30 mL at 10 weeks to 200 mL by 16 weeks and reaches 800 mL by the mid-third trimester (Brace, 1989; Magann, 1997). The luid is approximately 98-percent water. A full-term fetus contains roughly 2800 mL of water and the placenta another 400 mL, such that the term uterus holds nearly 4 liters of water (Modena, 2004). Abnormally decreased luid volume is termed oligohydramnios, whereas abnormally increased luid volume is termed hydramnios or poyhydramnios.

1	Early in pregnancy, the amnionic cavity is filled with luid that is similar in composition to extracellular luid. During the first half of pregnancy, transfer of water and other small molecules takes place across the amnion-transmembranous low; across the fetal vessels on placental surface-intramembranous low; and transcutaneous low-across fetal skin. Fetal urine production begins between 8 and 11 weeks' gestation, but it does not become a major component of amnionic luid until the second trimester, which explains why fetuses with lethal renal abnormalities may not manifest severe oligohydramnios until after 18 weeks. Water transport across the fetal skin continues until keratinization occurs at 22 to 25 weeks. This explains why extremely preterm neonates can experience signiicant luid loss across their skin. With advancing gestation, four pathways playia major role in amnionic luid volume regulation (Table

1	With advancing gestation, four pathways playia major role in amnionic luid volume regulation (Table First, fetal urination is the primary source of amnionic luid in the second half of pregnancy. By term, fetal urine production may exceed 1 liter per day, and the entire amnionic luid volume is recirculated on a daily basis. Fetal urine osmolality is similar to that of amnionic luid and significantly hypotonic to that of maternal and fetal plasma. Speciically, the osmolality of maternal TABLE 11-1. Amnionic Fluid Volume Regulation in Late Pregnancy Data from Magann, 201l1; Modena, 2004; Moore, 201l0.

1	TABLE 11-1. Amnionic Fluid Volume Regulation in Late Pregnancy Data from Magann, 201l1; Modena, 2004; Moore, 201l0. and fetal plasma approximates 280 mOsm/mL, whereas that of amnionic fluid is about 260 mOsm/L. The hypotonicity of amnionic luid accounts for significant intramembranous fluid transfer across and into fetal vessels on the placental surface. This transfer reaches 400 mL per day and is a second regulator of fluid volume (Mann, 1996). In the setting of maternal dehydration, the resultant increase in maternal osmolality favors fluid transfer from the fetus to the mother, and then from the amnionic luid compartment into the fetus (Moore, 2010).

1	n important third source of amnionic fluid regulation is the respiratory tract. Approximately 350 mL of lung fluid is produced daily late in gestation, and half of this is immediately swallowed. Last, fetal swallowing is the primary mechanism for amnionic luid resorption and averages 500 to 1000 mL per day (Mann, 1996). Impaired swallowing, secondary to either a central nervous system abnormality or GI tract obstruction, can result in an impressive degree of hydramnios. The remaining pathways are transmembranous and transcutaneous low, which together account for a far smaller proportion of fluid transport in the second half of pregnancy.

1	From a practical standpoint, the actual volume of amnionic luid is rarely measured outside of the research setting. That said, direct measurement and dye-dilution methods of luid quantification have contributed to our understanding of normal physiology. These measurements have further been used to validate sonographic fluid assessment techniques. Dye dilution involves injecting a small quantity of a dye such as aminohippurate into the amnionic cavity under sonographic guidance and then sampling the amnionic fluid to determine the dye concentration and hence to calculate the volume.

1	Brace and Wolf (1989) reviewed 12 studies done through the 1960s in which amnionic fluid volume was assessed using these measurement techniques. Although fluid volume increased across gestation, they found that the mean value did not change significantly between 22 and 39 weeks-it was approximately 750 mL. There was considerable variation at each week of gestation, especially in the mid-third trimester, when the 5th percentile was 300 mL and the 95th percentile nearly 2000 mL. In contrast, Magann and colleagues (1997), using dye-dilution measurements, found that amnionic fluid volume rose with advancing gestation. Specifically, the average fluid volume was approximately 400 mL between 22 and 30 weeks, doubling thereafter to a mean of 800 mL. he volume remained at this level until 40 weeks and then declined by approximately 8 percent per week. The two reports difered in the regression methodology employed, and despite diferent conclusions, both identiied a wide normal range,

1	40 weeks and then declined by approximately 8 percent per week. The two reports difered in the regression methodology employed, and despite diferent conclusions, both identiied a wide normal range, particularly in the third trimester. This normal variation is similarly observed sonographically.

1	Amnionic fluid volume evaluation is a component of every standard sonogram performed in the second or third trimester (Chap. 10, p. 188). It may be measured using either of two semi-quantitative techniques, the single deepest pocket of fluid or the amnionic fluid index (API), which was described by Phelan and associates (1987). Both measurements are reproducible and, in the setting of a fluid abnormality, can be followed serially over time to assess trends and to aid communication among providers. For these reasons, semiquantitative assessment of amnionic luid is preferred to qualitative or subjective estimation (American College of Obstetricians and Gynecologists, 2016). Using either technique, a fluid pocket must be at least 1 em in width to be considered adequate. Fetal parts or loops of umbilical cord may be visible in the pocket, but they are not included in the measurement. Color Doppler is generally used to veriY that umbilical cord is not within the measurement.

1	his is also called the largest or maximal vertical pocket of amnionic fluid. he ultrasound transducer is held perpendicular to the floor and parallel to the long axis of the woman. Then, while scanning in the sagittal plane, the largest vertical pocket of luid is identified and measured. The single deepest pocket measurement is considered normal f above 2 cm and less than 8 cm, with values below and above this range indicating oligohydramnios and hydramnios, respectivey. hese thresholds are based on data from Chamberlain and associates (1984) and correspond to the 3rd and 97th percentiles. When evaluating twin pregnancies and other multi fetal gestations, a single deepest pocket of amnionic fluid is assessed in each gestational sac, again using a normal range of more than 2 em to less than 8 em (Hernandez, 2012; Society for Maternal-Fetal Medicine, 2013). The fetal biophysical profile similarly uses a single deepest vertical pocket threshold of more than 2 em to indicate normal

1	than 8 em (Hernandez, 2012; Society for Maternal-Fetal Medicine, 2013). The fetal biophysical profile similarly uses a single deepest vertical pocket threshold of more than 2 em to indicate normal amnionic luid volume. This is discussed further in Chapter 17 (p. 337).

1	28 Mild hydramnios 26 24E 22>;'20 ) 180.� 16Q:J=14= > 12·c0 10·cE 8« 6 4 2 0 FIGURE 11-1 Amnionic fluid index (AFI) according to gestational-age-specific and threshold values. The blue curves represent the 25th, 50th, and 975th AFI percentile values, based on the nomogram by Moore (1o990). Red and tan curves represent 50th percentile values for AFI from Machado (2007) and from Hinh and Ladinsky (2005), respectively. The light blue and yellow shaded bars indicate threshold values used to define hydramnios and oligohydramnios, respectively.

1	or a gestational age-specific percentile reference range. he AFI is generally considered normal if greater than 5 cm and below 24 or 25 cm. Values outside these ranges indicate oligohydramnios and hydramnios, respectively. The upper threshold of 24 cm is used in consensus documents (American College of Obstetricians and Gynecologists, 2016; Reddy, 2014). The 25-cm threshold is oten applied in research studies (Khan, 2017; Luo, 2017; Pri-Paz, 2012). Moore and Cayle (1990) have provided normal curves for AFI values based on a cross-sectionl evaluation of nearly 800 uncomplicated pregnancies. he mean AFI was found to be between 12 and 15 cm from 16 weeks until 40 weeks' gestation. Other investigators have published nomograms with similar mean values (Hinh, 2005; Machado, 2007). Figure 11-1 depicts these AFI nomogram reference values in relation to commonly used thresholds for hydramnios and oligohydramnios.

1	This is an abnormally increased amnionic luid volume, and it complicates 1 to 2 percent of singleton pregnancies (Dashe, 2002; Khan, 2017; Pri-Paz, 2012). It is more frequently noted in multifetal gestations (Hernandez, 2012). Hydramnios may be suspected if the uterine size exceeds that expected for gestational age. The uterus may feel tense, and palpating fetal small parts or auscultating fetal heart tones may be diicult. An extreme example is shown in Figure 11-2. Hydramnios may be urther categorized according to degree. Such categorization is primarily used in research studies to stratiy risks. Several groups have termed hydramnios as mid if the AFI is 25 to 29.9 cm; moderate, if 30 to 34.9 cm; and severe, if 35 cm or more (Lazebnik, 1999; Luo, 2016; Odibo, 2016; Pri-Paz, 2012). Mild hydramnios is the most common, comprising

1	As with the single deepest luid pocket measurement, the ultrasound transducer is held perpendicular to the loor and parallel to the long axis of the woman. The uterus is divided into four equal quadrants-the right and left upper and lower quadrants, respectively. The AFI is the sum of the single deepest pocket from each quadrant. The intraobserver variability of the AFI approximates 1 cm, and the interobserver variability is about 2 cm. Variations are larger when luid volumes are above the normal range (Moore, 1990; Rutherford, 1987). A useful guideline is that the AFI approximates three times the single deepest pocket of fluid (Hill, 2003). Determination of whether the AFI is normal may be based on either a static numerical threshold FIGURE 11-2 Severe hydramnios-5500 mL of amnionic fluid was measured at delivery. 97.5th percentile 2.5th percentile

1	FIGURE 11-2 Severe hydramnios-5500 mL of amnionic fluid was measured at delivery. 97.5th percentile 2.5th percentile FIGURE "-3 Sonogram of severe hydramnios at 35 weeks in a pregnancy complicated by fetal aqueductal stenosis. This pocket of amnionic fluid measureso> 15 cm, and the amnionic fluid index measured nearly 50 cm. approximately two thirds of cases; moderate hydramnios accounts for about 20 percent; and severe hydramnios for approximately 15 percent. Using the single deepest pocket of amnionic fluid, mild hydramnios is defined as 8 to 9.9 cm, moderate as 10 to 11.9 cm, and severe hydramnios as 12 cm or more 11-3). In general, severe hydramnios is far more likely to have an underlying etiology and to have consequences for the pregnancy than mild hydramnios, which is frequently idiopathic and benign.

1	Underlying causes ofhydramnios include fetal anomalies-either structural abnormalities or genetic syndromes-in approximately 15 percent, and diabetes in 15 to 20 percent (Table 11-2). Congenital infection, red blood cell alloimmunization, and placental chorioangioma are less frequent etiologies. Infections that may present with hydramnios include cytomegalovirus, toxoplasmosis, syphilis, and parvovirus. Hydramnios is oten a component of hydrops etalis, and several of the above causes-selected anomalies, infections, and alloimmunization-may result in a hydropic fetus and placenta. he underlying pathophysiology in such cases is complex but is frequently related to a high cardiacoutput state. Severe fetal anemia is a classic example. Because the etiologies of hydramnios are so varied, hydramnios treatment also difers and is tailored in most cases to the underlying cause.

1	Selected anomalies and the likely mechanism by which they cause hydramnios are shown in Table 11-3. Many of these abnormalities are depicted and discussed in Chapter 10. Because of this association, targeted sonography is indicated whenever hydramnios is identiied. If a fetal abnormality is encountered concurrent with hydramnios, amniocentesis with chromosomal microarray analysis should be ofered, because the aneuploidy risk is signiicantly elevated (Dashe, 2002; Pri-Paz, 2012).

1	Importantly, the degree of hydramnios correlates with the likelihood of an anomalous infant (Lazebnik, 1999; Pri-Paz, 2012). At Parkland Hospital, the prevalence of an anomalous neonate was approximately 8 percent with mild hydramnios, 12 percent with moderate hydramnios, and more than 30 percent with severe hydramnios (Dashe, 2002). Even if no abnormality was detected with targeted sonography, the likelihood of a major anomaly identified at birth was 1 to 2 percent if hydramnios was mild or moderate and 10 percent if hydramnios was severe. he overall reported risk that an underlying anomaly will be discovered ater delivery has ranged from 9 percent in the neonatal period to 28 percent among infants followed to 1 year of age (Abele, 2012; Dorleijn, 2009). he anomaly risk is particularly high with hydramnios coexistent with fetal-growth restriction (Lazebnik, 1999).

1	Although amnionic luid volume abnormalities are associated with fetal malformations, the converse is not usually the case. In the Spanish Collaborative Study of Congenital Malformations that included more than 27,000 anomalous infants, only 4 percent of pregnancies were complicated by hydramnios, and another 3 percent with oligohydramnios (Martinez-Frias, 1999). he amnionic luid glucose concentration is higher in diabetic women than in those without diabetes, and the AFI may TABLE "e-2. Hydramnios: Prevalence and Associated Etiologies-Values in Percent aA signiicant correlation was identified between severity of hydramnios and likelihood of an anomalous infant. TABLE 11-3. Selected Anomalies and Mechanism for Hydramnios Cystic adenomatoid malformation? Tetralogy of Fallet with absent Thyroto/icosis� Functional cardiac etiology Cardiom 'apathy, myocarditisa Cardiac arrhythmia tnali flutter, atrial fibrillation, supraventricular tachycardia Bradyarrhythmiaa.

1	Functional cardiac etiology Cardiom 'apathy, myocarditisa Cardiac arrhythmia tnali flutter, atrial fibrillation, supraventricular tachycardia Bradyarrhythmiaa. Neurological or muscular etiology Arthrogryposis, akinesia sequence Myotonic dystrophy Neoplastic etiology Sacrococcygeal teratomaa Mesoblastic nephromaa aPoses risk for hydrops. CHAOS = congenital high-airway obstruction sequence; GI = gastrointestinal.

1	CHAOS = congenital high-airway obstruction sequence; GI = gastrointestinal. correlate with the amnionic fluid glucose concentration (Dashe, Idiopathic Hydramnios 2000; Spellacy, 1973; Weiss, 1985). Such findings support the his accounts for up to 70 percent of cases of hydramnios and is hypothesis that maternal hyperglycemia causes fetal hyperglythus identified in as many as 1 percent of pregnanciescemia, with resulting fetal osmotic diuresis into the amnionic (Panting-Kemp, 1999; Pri-Paz, 2012; Wiegand, 2016). Idiopathic luid compartment. hat said, rescreening for gestational diahydramnios is rarely identiied during midtrimester sonography betes in pregnancies with hydramnios does not appear to be and is oten an incidental inding later in gestation. he gestabeneicial, provided that the second-trimester glucose tolerance tional age at sonographic detection usually lies between 32 and 35 test result was normal (F rank Wolf, 2017).

1	weeks (Abele, 2012; Odibo, 2016; Wiegand, 2016). Although it is a diagnosis of exclusion, an underlying fetal abnormality may subsequently become apparent with advancing gestation, particu Hydramnios is generally deined in multifetal gestations as larly if the degree of hydramnios becomes severe. In the absence a single deepest amnionic luid pocket measuring 8 cm or of an etiology, idiopathic hydramnios is mild in approximately 80 more. It may be further characterized as moderate if the single percent of cases, and resolution is reported in more than a third deepest pocket is at least 10 cm and severe if this pocket is of afected pregnancies (Odibo, 2016; Wiegand, 2016). Mild, at least 12 cm. In a review of nearly 2000 twin gestations, idiopathic hydramnios is most commonly a benign finding, and Hernandez and colleagues (20i12) identified hydramnios in associated pregnancy outcomes are usually good.

1	18 percent of both monochorionic and dichorionic pregnancies. As in singletons, severe hydramnios was more strongly associated with fetal abnormalities. In monochorionic gestations, hydramnios of one sac and oligohydramnios of the Unless hydramnios is severe or develops rapidly, maternal other are diagnostic criteria for twin-twin transfusion synsymptoms are infrequent. With chronic hydramnios, luid drome (TTTS), discussed in Chapter 45 (p. 878). Isolated accumulates gradually, and a woman may tolerate excessive hydramnios of one sac also may precede the development abdominal distention with relatively little discomfort. Acute of this syndrome (Chon, 2014). In the absence of TTTS, hydramnios, however, tends to develop earlier in pregnancy. hydramnios does not generally raise pregnancy risks in nonIt may result in preterm labor before 28 weeks or in symptoms anomalous twins (Hernandez, 2012). that become so debilitating as to necessitate intervention.

1	Symptoms may arise from pressure exerted within the overdistended uterus and upon adjacent organs. When distention is excessive, such as that shown in Figure 11-2, the mother may sufer dyspnea and orthopnea to such a degree that she may be able to breathe comfortably only when upright. Edema may develop as a consequence of major venous system compression by the enlarged uterus, and it tends to be most pronounced in the lower extremities, vulva, and abdominal wall. Rarely, oliguria may result from ureteral obstruction by the enlarged uterus (Chap. 53, p. 1037). Maternal complications such as these are typically associated with severe hydramnios from an underlying etiology.

1	Maternal complications associated with hydramnios include placental abruption, uterine dysfunction during labor, and postpartum hemorrhage. Placental abruption is fortunately infrequent. It may result from the rapid decompression of an overdistended uterus that follows fetal-membrane rupture or therapeutic amnioreduction. With prematurely ruptured membranes, a placental abruption occasionally occurs days or weeks after amniorrhexis. Uterine dysfunction consequent to overdistention may lead to postpartum atony and, in turn, postpartum hemorrhage.

1	Some outcomes more common with hydramnios include birthweight >4000 g, cesarean delivery, and importantly, perinatal mortality. Pregnancies with idiopathic hydramnios are associated with birthweights exceeding 4000 g in nearly 25 percent of cases, and the likelihood appears to be greater if the hydramnios is moderate or severe (Luo, 2016; Odibo, 2016; Wiegand, 2016). A rationale for this association is that larger fetuses have higher urine output, by virtue of their increased volume of distribution, and fetal urine is the largest contributor to amnionic luid volume. Cesarean delivery rates are also higher in pregnancies with idiopathic hydramnios, with reported rates of 35 to 55 percent (Dorleijn, 2009; Khan, 2017; Odibo, 2016).

1	An unresolved question is whether hydramnios alone raises the risk for perinatal mortality. Some studies have found no increase in stillbirth or neonatal death rates with idiopathic hydramnios, whereas others show a greater risk (Khan, 2017; Pilliod, 2015; Wiegand, 2016). Using birth certificate data from the state of California, Pilliod and coworkers (2015) identified hydramnios in 0.4 percent of singleton, nonanomalous pregnancies, and afected pregnancies had significantly greater stillbirth rates. At 37 weeks, the stillbirth risk was sevenfold higher in pregnancies with hydramnios. By 40 weeks, this risk was more than tenfold higher-66 per 10,000 births compared with 6 per 10,000 without hydramnios.

1	isks appear to be compounded when a growth-restricted fetus is identiied with hydramnios (Erez, 2005). The combination also has a recognized association with trisomy 18. When an underlying cause is identified, degree of hydramnios has been associated with likelihood of preterm delivery, smallfor-gestational age newborn, and perinatal mortality (Pri-Paz, 2012). However, idiopathic hydramnios is generally not associated with preterm birth (Magann, 2010; Many, 1995; PantingKemp, 1999).

1	s previously noted, treatment is directed to the underlying cause. Occasionally, severe hydramnios may result in early preterm labor or the development of maternal respiratory compromise. In such cases, large-volume amniocentesis-termed amnioreduction-may be needed. he technique is similar to that for genetic amniocentesis, described in Chapter 14 (p. 292). One diference is that it is generally done with a larger needle, 18-or 20-gauge, and uses either an evacuated container bottle or a larger syringe. Approximately 1000 to 2000 mL of luid is slowly withdrawn over 20 to 30 minutes, depending on the severity of hydramnios and gestational age. he goal is to restore amnionic fluid volume to the upper normal range. Hydramnios severe enough to necessitate amnioreduction almost invariably has an underlying cause, and subsequent amnioreduction procedures may be required as oten as weekly or even semiweekly.

1	In a review of 138 singleton pregnancies requiring amnioreduction for hydramnios, a fetal GI malformation was identified in 20 percent, a chromosomal abnormality or genetic condition in almost 30 percent, and a neurological abnormality in 8 percent (Dickinson, 2014). In only 20 percent of cases was the hydramnios idiopathic. he initial amnioreduction procedure in this series was performed at 31 weeks' gestation, and the median gestational age at delivery was 36 weeks. Complications within 48 hours of amnioreduction included deliveY in 4 percent and ruptured membranes in 1 percent. here was no instance of chorioamnionitis, placental abruption, or bradycardia requiring delivery (Dickinson, 2014).

1	his is an abnormally decreased amount of amnionic luid. Oligohydramnios complicates approximately 1 to 2 percent of pregnancies (Casey, 2000; Petrozella, 2011). When no measurable pocket of amnionic fluid is identiied, the term anhydramnios may be used. Unlike hydramnios, which is often mild and often confers a benign prognosis in the absence of an underlying etiolo)i, oligohydramnios is always a cause for concern, as discussed on page 231.

1	The sonographic diagnosis of oligohydramnios is usualy based on an API less than 5 cm or a single deepest pocket of amnionic luid below 2 cm (American College of Obstetricians and Gynecologists, 2016). Using the Moore nomogram, an API threshold of 5 em is below the 2.5th percentile throughout the second and third trimesters (see Fig. 11-1). Either criterion is considered acceptable. However, use of API rather than single deepest pocket will identiy more pregnancies as having oligohydramnios, without evidence of improvement in pregnancy outcomes (Kehl, 2016; Nabhan, 2010). When evaluating multifetal pregnancies for TTTS, a single deepest pocket below 2 em is used to define oligohydramnios (Society for Maternal-Fetal Medicine, 2013).

1	Pregnancies complicated by oligohydramnios include those in which the amnionic luid volume has been severely diminished since the early second trimester and those in which the fluid volume was normal until near-term or even full-term. The prognosis depends heavily on the underlying cause and thus varies. Whenever oligohydramnios is diagnosed, it becomes an important consideration in clinical management. When amnionic fluid volume is abnormally decreased from the early second trimester, it may reflect a fetal abnormality that precludes normal urination, or it may represent a placental abnormality suiciently severe to impair perfusion. In either circumstance, the prognosis is poor. Ruptured membranes should be excluded, and targeted sonography is performed to assess for fetal and placental abnormalities.

1	When amnionic luid volume becomes abnormally decreased in the late second or in the third trimester, it is very often associated with fetal-growth restriction, with a placental abnormality, or with a maternal complication such as preeclampsia or vascular disease (Table 11-4). The underlying cause in such cases is frequently uteroplacental insuiciency, which can impair fetal growth and reduce fetal urine output. Exposure to selected medications has also been linked with oligohydramnios as discussed subsequently. Investigation of third-trimester oligohydramnios generally includes evaluation for ruptured membranes and sonography to assess fetal growth. Umbilical artery Doppler studies are recommended if growth restriction is identiied (Chap. 10, p. 213). Oligohydramnios is commonly encountered in lateterm and postterm pregnancies (Chap. 43, p. 837). Magann and coworkers (1997) found that amnionic luid volume decreased by approximately 8 percent per week beyond 40 weeks.

1	By approximately 18 weeks, the fetal kidneys are the main contributor to amnionic luid volume. Selected renal abnormalities that lead to absent fetal urine production include bilateral renal agenesis, bilateral multicystic dysplastic kidney, unilateral renal agenesis with contralateral multicystic dysplastic kidney, and the infantile form of autosomal recessive poycystic kidney dis ease. Urinary abnormalities may also result in oligohydramnios because of fetal bladder outlet obstruction. Examples of this are posterior urethral valves, urethral atresia or stenosis, or the megacystis microcolon intestinal hypoperistalsis syndrome. Complex fetal genitourinary abnormalities such as persistent cloaca and sirenomelia similarly may result in a lack of amnionic luid. Many of these renal and urinary abnormalities are discussed and depicted in Chapter 10 (p. 208). If no amnionic luid is visible beyond the mid-second trimester due to a genitourinary etiology, the prognosis is extremely poor

1	abnormalities are discussed and depicted in Chapter 10 (p. 208). If no amnionic luid is visible beyond the mid-second trimester due to a genitourinary etiology, the prognosis is extremely poor unless fetal therapy is an option. Fetuses with bladder-outlet obstruction may be candidates for vesicoamnionic shunt placement (Chap. 16, p. 325).

1	Oligohydramnios has been associated with exposure to drugs that block the renin-angiotensin system. These include angiotensin-converting enzyme (ACE) inhibitors, angiotensin-receptor blockers, and nonsteroidal antiinlammatory drugs (NSAIDs). When taken in the second or third trimester, ACE inhibitors and angiotensin-receptor blockers may create fetal hypotension, renal hypoperfusion, and renal ischemia, with subsequent anuric renal failure (Bullo, 2012; Guron, 2000). Fetal skull bone hypoplasia and limb contractures have also been described (Schaefer, 2003). NSAIDs can be associated with fetal ductus arteriosus constriction and with lower fetal urine production. In neonates, their use may result in acute and chronic renal insuiciency (Fanos, 2011). These agents are discussed in Chapter 12 (p. 241). Oligohydramnios is associated with adverse pregnancy outcomes. Casey and colleagues (2000) found that an AFI �5 em

1	Oligohydramnios is associated with adverse pregnancy outcomes. Casey and colleagues (2000) found that an AFI �5 em TABLE 11-4. Pregnancy Outcomes in Women Diagnosed with Oligohydramnios between 24 and 34 Weeks' Gestation AFI �5 em AFI 8 to 24 em Factor (n = 166) (n = 28,1s85) P Value Major malformation 42 (25) 634 (2) <.00l1 1) <.001 Gestational age at deliverya 35.1 ± 3.3 39.2 ± 2.0 <.001 Preterm birth, spontaneousa 49 (42) 1698 (6) <.001 Preterm birth, indicateda 20) 405 (2) <.001 Cesarean delivery for nonreassuring 10l(9) 1083 (4) <.001 Birthweight < 10th percentilea 61 (53) 3388 (12) <.001 <3rd percentilea 43 (37) 1130 (4) <.001 1) <.OOllb Data expressed as No. (%) and mean ± standard deviation. aAnomalous infants excluded. bThis difference was no longer significant after adjustment for gestational age at delivery. Data from 2011.

1	complicated 2 percent of pregnancies undergoing sonography at Parkland Hospital ater 34 weeks' gestation. Fetal malformation rates were elevated in those with oligohydramnios. Even in their absence, rates of stillbirth, growth restriction, nonreassUfing heart rate pattern, and meconium aspiration syndrome were higher than in nonafected pregnancies. Petrozella and associates (2011) similarly reported that an AFI :;5 em identiied between 24 and 34 weeks was associated with increased risks for stillbirth, spontaneous or medically indicated preterm birth, heart rate pattern abnormalities, and growth restriction (see Table 11-4). In one metaanalysis comprising more than 10,000 pregnancies, women with oligohydramnios had a twofold greater risk for cesarean delivery for fetal distress and a ivefold higher risk for an Apgar score <7 at 5 minutes compared with pregnancies with a normal AFI (Chauhan, 1999).

1	As discussed, evidence suggests that if oligohydramnios is defined as an AFI :;5 em rather than a single deepest pocket :;2 em, more pregnancies will be classiied as such. One review of trials encompassing more than 3200 high-risk and low-risk pregnancies compared outcomes according to which deinition was used (Nabhan, 2008). Rates of cesarean delivery, neonatal intensive care unit admission, umbilical artery pH <7.1, or Apgar score <7 at 5 minutes did not difer between groups. Using AFI criteria, however, twice as many pregnancies were diagnosed with oligohydramnios. In this group, there was a doubling of the labor induction rate and a 50-percent increase in the cesarean delivery rate for fetal distress. Kehl and colleagues (2016) performed a prospective trial with more than 1000 term pregnancies in which women with oligohydramnios, deined either by an AFI < 5 em or a single deepest pocket <2 em, were randomized to labor induction or expectant care. Signiicantly more pregnancies were

1	in which women with oligohydramnios, deined either by an AFI < 5 em or a single deepest pocket <2 em, were randomized to labor induction or expectant care. Signiicantly more pregnancies were diagnosed with oligohydramnios using the AFI criterion-10 percent compared with just 2 percent-when single deepest pocket was used. This led to a higher rate of labor induction in the AFI group, but no diference in neonatal outcomes.

1	When diminished amnionic luid is first identiied before the mid-second trimester, particularly before 20 to 22 weeks, pulmonary hypoplasia is a signiicant concern. he underlying etiology is a major factor in the prognosis for such pregnancies. Severe oligohydramnios secondary to a renal abnormality generally has a lethal prognosis. If a placental hematoma or chronic abruption is severe enough to result in oligohydramnios-the chronic abruption-oligohydramnios sequence (CAOS)-it commonly also causes growth restriction (Chap. 41, p. 768). he prognosis for this constellation is similarly poor. Oligohydramnios that results from membrane rupture in the second trimester is reviewed in Chapter 42 (p. 821).

1	Initially, an evaluation for fetal anomalies and growth is essential. In a pregnancy complicated by oligohydramnios and fetal-growth restriction, close fetal surveillance is important because of associated morbidity and mortality (Chap. 44, p. 855). Oligohydramnios detected before 36 weeks' gestation in the presence of normal fetal anatomy and growth is generally managed expectantly in conjunction with enhanced fetal surveillance. However, evidence of fetal or maternal compromise will override potential complications from preterm delivery. Antepartum management of oligohydramnios may include maternal hydration. In a recent review of 16 trials of pregnancies with apparent isolated oligohydramnios, oral or intravenous hydration was associated with significant improvement in the AFI. However, it was not clear whether this translated into better pregnancy outcomes (Gizzo, 2015).

1	Amnioinfusion, discussed in Chapter 24 (p. 475), may be used intrapartum to help resolve variable fetal heart rate decelerations. It is not considered treatment for oligohydramnios per se, although the decelerations are presumed secondary to umbilical cord compression resulting from lack of amnionic luid. Amnioinfusion is not the standard of care for other etiologies of oligohydramnios and is not generally recommended.

1	he term borderline AFI or borderline oligohydramnios is somewhat controversial. It usually refers to an AFI between 5 and 8 em (Magann, 2011; Petrozella, 2011). Through the mid-third trimester, an AFI value of 8 em is below the 5th percentile on the Moore nomogram (see Fig. 11-1). Petrozella and colleagues (2011) found that pregnancies between 24 and 34 weeks with an AFI between 5 and 8 em were not more likely than those with an AFI above 8 em to be complicated by maternal hypertension, stillbirth, or neonatal death. That said, higher rates of preterm delivery, cesarean delivery for a nonreassuring fetal heart rate pattern, and fetal-growth restriction were found. Wood and colleagues (2014) similarly reported a higher rate of fetal-growth restriction in pregnancies with borderline AFI. Thus, results evaluating pregnancy outcomes with borderline AFI have been mixed. Magann and associates (2011) concluded that evidence is insuicient to support fetal testing or delivery in this setting.

1	Abele H, Starz S, Hoopmann M, et al: Idiopathic polyhydramnios and postnatal abnormalities. Fetal Diagn Ther 32(4) :251, 2012 American College of Obstetricians and Gynecologists: Ultrasound in pregnancy. Practice Bulletin No. 175, December 2016 Biggio JR Jr, Wenstrom D, Dubard MB, et al: Hydramnios prediction of adverse perinatal outcome. Obstet Gynecol 94:773, 1999 Brace A, Wolf EJ: Normal amniotic luid volume changes throughout pregnancy. Am J Obstet Gynecol 161 (2):382, 1989 Bulla M, Tschumi S, Bucher BS: Pregnancy outcome following exposure to angiotensin-converting enzyme inhibitors or angiotensin receptor antagonists: a systematic review. Hypertension 60:444, 2012 Casey BM, Mcintire DO, Bloom SL, et al: Pregnancy outcomes after antepartum diagnosis of oligohydramnios at or beyond 34 weeks' gestation. Am J Obstet GynecoIo182:909, 2000

1	Casey BM, Mcintire DO, Bloom SL, et al: Pregnancy outcomes after antepartum diagnosis of oligohydramnios at or beyond 34 weeks' gestation. Am J Obstet GynecoIo182:909, 2000 Chamberlain PF, Manning FA, Morrison I, et al: Ultrasound evaluation of amniotic fluid. he relationship of marginal and decreased amniotic fluid volumes to perinatal outcome. Am J Obstet GynecoIo150:245, 1984 Chauhan Sp, Sanderson M, Hendrix NW, et al: Perinatal outcome and amniotic fluid index in the antepartum and intrapartum periods: a meta-analysis. Am J Obstet Gynecol 181: 1473, 1999 Chon AH, Korst LM, Llanes A, et al: Midtrimester isolated polyhydramnios in monochorionic diamniotic multiple gestations. Am J Obstet Gynecol 211(3):303.e1,o2014 Dashe ]S, McIntire DO, Ramus M, et al: Hydramnios: anomaly prevalence and sonographic detection. Obstet Gynecolo100(l):134, 2002

1	Dashe ]S, McIntire DO, Ramus M, et al: Hydramnios: anomaly prevalence and sonographic detection. Obstet Gynecolo100(l):134, 2002 Dashe ]S, Nathan L, McIntire DO, et al: Correlation between amniotic fluid glucose concentration and amniotic fluid volume in pregnancy complicated by diabetes. Amo] Obstet Gynecol 182(4):901,o2000 Dickinson ]E, Tjioe ¥, Jude E, et al: Amnioreduction in the management of polyhydramnios complicating singleton pregnancies. Amo] Obstet Gynecol 211:434.e.1,o2014 Dorleijn OM, Cohen-Overbeek TE, Groenendaal F, et al: Idiopathic polyhydramnios and postnatal indings.] Matern Fetal Neonatal Med 22(4):315, 2009 Erez 0, Shoham-Vardi I, Sheiner E, et al: Hydramnios and small for gestational age are independent risk factors for neonatal mortality and maternal morbidity. Arch Gynecol Obstet 271 (4):296,o2005

1	Erez 0, Shoham-Vardi I, Sheiner E, et al: Hydramnios and small for gestational age are independent risk factors for neonatal mortality and maternal morbidity. Arch Gynecol Obstet 271 (4):296,o2005 Fanos V, Marcialis MA, Bassareo PP, et al: Renal safety of Non Steroidal Anti Inflammatory Drugs (NSAIDs) in the pharmacologic treatment of patent ductus arteriosus.] Matern Fetal Neonatal Med 24(S1):50, 201o1 Frank Wolf M, Peleg 0, Stahl-Rosenzweig T, et al: Isolated polyhydramnios in the third trimester: is a gestational diabetes evaluation of value? Gynecol EndocrinoIo33(1l):849,o2017 Gizzo S, Noventa M, Vitagliano A, et al: An update on maternal hydration strategies for amniotic luid improvement in isolated oligohydramnios and normohydramnios: evidence from a systematic review of literature and meta-analysis. PLoS One 10(12):e0144334, 2015

1	Golan A, Wolman I, Saller Y, et al: Hydramnios in singleton pregnancy: sonographic prevalence and etiology. Gynecol Obstet Invest 35:91, 1993 Guron G, Friberg P: An intact renin-angiotensin system is a prerequisite for normal renal development.] Hypertens 18(2):123,o2000 Hernandez ]S, Twickler OM, McIntire OM, et al: Hydramnios in twin gestations. Obstet GynecoIo120(4):759, 2012 Hill LM, Sohaey R, Nyberg DA: Abnormalities of amniotic fluid. In Nyberg DA, McGahan ]P, Pretorius DH, et al (eds): Diagnostic Imaging of Fetal Anomalies. Philadelphia, Lippincott Williams & Wilkins, 2003 Hinh NO, Ladinsky ]L: Amniotic fluid index measurements in normal pregnancy after 28 gestational weeks. Int] Gynaecol Obstet 91:132, 2005 Kehl S, Schelkle A, Thomas A, et al: Single deepest vertical pocket or amniotic luid index as evaluation test for predicting adverse pregnancy outcome (SAFE trial): a multicenter open-label, randomized controlled trial. Ultrasound Obstet Gynecol 47:674,o2016

1	Khan S, Donnellyo]: Outcome of pregnancy in women diagnosed with idiopathic polyhydramnios. Aust N Z] Obstet Gynaecol 57(1):57,o2017 Lazebnik N, Many A: The severity of polyhydramnios, estimated fetal weight and preterm delivery are independent risk factors for the presence of congenital anomalies. Gynecol Obstet Invest 48:28, 1999 Luo QQ, Zou L, Gao H, et al: Idiopathic polyhydramnios at term and pregnancy outcomes: a multicenter observational study.o] Matern Fetal Neonatal Med 30(14):1755, 2017 Machado MR, Cecatti ]G, Krupa F, et al: Curve of amniotic fluid index measurements in low risk pregnancy. Acta Obstet Gynecol Scand 86:37, 2007 Magann EF, Bass ]0, Chauhan SP, et al: Amniotic luid volume in normal singleton pregnancies. Obstet Gynecol 90(4):524, 1997 Magann EF, Chauhan CP, Hitt WC, et al: Borderline or marginal amniotic fluid index and peripartum outcomes: a review of the literature. ] Ultrasound Med 30(4):523, 2011

1	Magann EF, Chauhan CP, Hitt WC, et al: Borderline or marginal amniotic fluid index and peripartum outcomes: a review of the literature. ] Ultrasound Med 30(4):523, 2011 Magann EF, Doherty 0, LutegendorfMA, et al: Peripartum outcomes of highrisk pregnancies complicated by oligo-and polyhydramnios: a prospective longitudinal study. ] Obstet Gynaecol Res 36(2):268, 2010 Mann SE, Nijland M], Ross MG: Mathematic modeling of human amniotic fluid dynamics. Amo] Obstet Gynecol 175(4):937, 1996 Many A, Hill LM, Lazebnik N, et al: he association between polyhydramnios and preterm delivery. Obstet Gynecol 86(3):389, 1995 Martinez-Frias ML, Bermejo E, Rodriguez-Pinilla E, et al: Maternal and fetal factors related to abnormal amniotic luid. ] Perinatol 19:514, 1999 Modena AB, Fieni S: Amniotic luid dynamics. Acta Bio Medica Ateneo Parmanese 75(Suppl 1):11,o2004 Moore TR: Amniotic fluid dynamics relect fetal and maternal health and disease. Obstet Gynecol 116(3):759,o2010

1	Moore TR, Cayle ]E: The amniotic luid index in normal human pregnancy. Amo] Obstet GynecoIo162(5):1168, 1990 Nabhan AF, Abdelmoula YA: Amniotic luid index versus single deepest vertical pocket as a screening test for preventing adverse pregnancy outcome. Cochrane Database Syst Rev 3:CD006953, 2008 Odibo IN, Newville TM, Ounpraseuth ST, et al: Idiopathic polyhydramnios: persistence across gestation and impact on pregnancy outcomes. Euro] Obstet Gynecol Reprod Bioi 199: 175, 2016 Panting-Kemp A, Nguyen T, Chang E, et al: Idiopathic polyhydramnios and perinatal outcome. Am ] Obstet Gynecol 181 (5): 1 079, 1999 Petrozella LN, DasheJS, McIntire DO, et al: Clinical signiicance of borderline amniotic luid index and oligohydramnios in preterm pregnancy. Obstet Gynecol 117(2 pt 1):338, 2011 Phelan ]P, Smith CV, Broussard P, et al: Amniotic luid volume assessment with the four-quadrant technique at 36-42 weeks' gestation. ] Reprod Med 32:540, 1987

1	Phelan ]P, Smith CV, Broussard P, et al: Amniotic luid volume assessment with the four-quadrant technique at 36-42 weeks' gestation. ] Reprod Med 32:540, 1987 Pilliod A, Page ]M, Butwick RM, et al: he risk of fetal death in nonanomalous pregnancies afected by polyhydramnios. Am ] Obstet Gynecol 213:410.e.1,o2015 Pri-Paz S, Khalek N, Fuchs vf, et al: Maximal amniotic luid index as a prognostic factor in pregnancies complicated by polyhydramnios. Ultrasound Obstet Gynecol 39(6):648, 2012 Reddy UM, Abuhamad AZ, Levin 0, et al: Fetal imaging: executive summary of a joint Eunice Kennedy Shriver National Institute of Child Health and Human Development, Society for Maternal-Fetal Medicine, American Institute of Ultrasound in Medicine, ArIerican College of Obstetricians and Gynecologists, American College of Radiology, Society for Pediatric Radiology, and Society of Radiologists in Ultrasound Fetal Imaging Workshop. Obstet GynecoIo123(5):1070, 2014

1	Rutherford SE, Smith CV, Phelan ]P, et al: Four-quadrant assessment of amniotic fluid volume. Interobserver and intraobserver variation. ] Reprod Med 32(8):587, 1987 Schaefer C: Angiotensin II-receptor-antagonists: further evidence of fetotoxicity but not teratogenicity. Birth Defects Res A Clin Mol Teratol 67(8):591, 2003 Society for Maternal-Fetal Medicine, Simpson LL: Twin-twin transfusion syndrome. Amo] Obstet GynecoIo208(1):3, 2013 Spellacy N, Buhi WC, Bradley B, et al: Maternal, fetal, amniotic fluid levels of glucose, insulin, and growth hormone. Obstet GynecoIo41:323, 1973 Weiss PA, Hofmann H, Winter R, et al: Amniotic luid glucose values in nor mal and abnormal pregnancies. Obstet Gynecol 65:333, 1985 Wiegand SL, Beamon q, Chescheir NC, et al: Idiopathic polyhydramnios: severity and perinatal morbidity. Amo] Perinatol 33(7):658, 2016

1	Wiegand SL, Beamon q, Chescheir NC, et al: Idiopathic polyhydramnios: severity and perinatal morbidity. Amo] Perinatol 33(7):658, 2016 Wood SL, Newton ]M, Wang L, et al: Borderline amniotic luid index and its relation to fetal intolerance of labor: a 2-center retrospective cohort study. ] Ultrasound Med 33(4):705, 2014 CHAPTER 12 Teratology, Teratogens, and Fetotoxic Agents TERATOLOGY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 CRITERIA FOR DETERMINING TERATOGENICITY .e... 235 COUNSELING FOR MEDICATION EXPOSURE . ..e...e. 238 KNOWN AND SUSPECTED TERATOGENS .e.. .e. 239 Al inectious diseases have a tendency to bring about death of the chid and its subsequent expulsion from the uterus. The atal result is usualy due to the transmission of toxins, and occasionaly the speciic micro-organisms from the mother to the chil. Poisoning with phosphorus, lead, iluminating gas, and other substances may lead to similar results. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) Other than referring to fetal deformities that might impede vaginal delivery, little is written in the first edition of this book regarding teratogens and fetal malformations. his is despite the fact that birth defects are common, and 2 to 3 percent of all newborns have a major congenital abnormality detectable at birth (Cragan, 2009; Dolk, 2010). There are undoubtedly medications that pose significant risk to the developing embryo or fetus (Table 12-1). However, 80 percent of birth defects do not have an obvious etiology, and of those with an identified cause, nearly 95 percent' of cases have chromosomal or genetic origins (Feldkamp, 2017). The Food and Drug Administration (FDA) (2005) estimates that less than 1 percent of all birth defects are caused by medications. Their remarkably small contribution to congenital abnormalities is shown in Figure

1	That said, signiicant concern surrounds medication use in pregnancy. This is because so many pregnant women are prescribed medications and because safety data are often lacking. Investigators from the National Birth Defects Prevention Study found that women take an average of two to three medications per pregnancy and that 70 percent use medication in the first trimester (Mitchell, 2011). And, in one review of medications approved by the FDA between 2000 and 2010, the Teratogen Information System (TERIS) advisory board deemed the TABLE 12-1. Selected Teratogens and Fetotoxic Agents

1	TABLE 12-1. Selected Teratogens and Fetotoxic Agents Teratology, Teratogens, and Fetotoxic Agents 235 teratogen may be deined as any agent that acts during embryonic or fetal development to produce a permanent alteration of form or function. Thus, a teratogen may be a medication or other chemical substance, a physical or environmental factor such as heat or radiation, a maternal metabolite as in diabetes or phenylketonuria, or an infection such as cytomegalovirus. Even obesity is considered a teratogen (Stothard, 2009; Waller, 2007). Strictly defined, a teratogen causes only structural abnormalities. A hadegen-after the god Hades-is an agent that interferes with organ maturation and function, and from a population-based review of 270,878 births. pregnancy risk "undetermined" for more than 95 percent of these agents (Adam, 2011). he study of birth defects and their etiology is termed teratology, derived from the Greek teratos, meaning monster. A

1	he study of birth defects and their etiology is termed teratology, derived from the Greek teratos, meaning monster. A TABLE 12-2. Criteria for Determining Teratogenicity Essential Criteria: 1. Careful delineation of clinical cases, particularly if there is a speciic defect or syndrome 2. Proof that exposure occurred at critical time during development (see Fig. 12-2) 3. Consistent indings by at least two epidemiological studies with: a. exclusion of bias, b. adjustment for confounding variables, c. adequate sample size (power), d. prospective ascertainment if possible, and e. relative risk (RR) of 3.0 or greater, some recommend RR of 6.0 or greater For a rare environmental exposure associated with a rare defect, at least three reported cases. This is easiest if defect is severe Ancillary Criteria: 4. The association is biologically plausible 5. Teratogenicity in experimental animals is important but not essential 6.

1	Ancillary Criteria: 4. The association is biologically plausible 5. Teratogenicity in experimental animals is important but not essential 6. The agent acts in an unaltered form in an experimental model Data from from Shepard 1994, 2002a. term teratogen is used to refer to all three types of agents. The guidelines shown in Table 12-2 were proposed by Shepard (1994) as a framework for discussion and have proven useful for more than 25 years. Although each individual criterion is not required to establish teratogenicity, the following tenets must be considered (Shepard, 2002a): • The defect has been completely characterized. This is preferably done by a geneticist or dysmorphologist because various genetic and environmental factors may produce similar anomalies. It is easiest to prove causation when a rare exposure produces a rare defect, when at least three cases with the same exposure have been identified, and when the defect is severe.

1	• he agent must cross the placenta. Although almost all drugs cross the placenta, transport must be of suicient quantity to directly inluence embryonic or fetal development or to alter maternal or placental metabolism to exert an indirect efect. Placental transfer depends on maternal metabolism; on speciic characteristics of the drug, such as protein binding and storage, molecular size, electrical charge, and lipid solubility; and on placental metabolism, such as by the cytochrome P 450 enzyme system. In early pregnancy, the placenta also has a relatively thick membrane that slows difusion. • Exposure must occur during a critical developmental period.

1	• Exposure must occur during a critical developmental period. o Thepreimplantation period is the 2 weeks between fertilization and implantation and is known as the "all or none" period. As the zygote undergoes cleavage, an insult damaging a large number of cells typically causes embryonic death. However, if only a few cells are injured, compensation may be possible and allow normal development (Clayton-Smith, 1996). From animal data, insults that appreciably diminish the cell number in the inner cell mass may produce a dose-dependent diminution in body length or size (Iahnaccone, 1987). Fertilization to bilaminar disc formation FIGURE 12-2 Timing of organogenesis during the embryonic period. (Reproduced with permission from Salder W: Langman's Medical Embryology, 6th ed. Baltimore, Williams & Wilkins; 1990.)

1	FIGURE 12-2 Timing of organogenesis during the embryonic period. (Reproduced with permission from Salder W: Langman's Medical Embryology, 6th ed. Baltimore, Williams & Wilkins; 1990.) The embyonic period extends from the second through the eighth week postconception. It encompasses organogenesis and is thus the most crucial period with regard to structural malformations. Critical developmental periods for each organ system are illustrated in Figure 12-2. heetal period, which is beyond 8 weeks postconception, is characterized by continued maturation and unctional development. During this time, certain organs remain vulnerable. • A biologically plausible association is supportive. Because birth defects and medication exposures are both common, they may be temporally but not causally related.

1	• A biologically plausible association is supportive. Because birth defects and medication exposures are both common, they may be temporally but not causally related. • Epidemiological indings must be consistent. Because initial evaluation of teratogen exposure is often retrospective, it may be hampered by recall bias, inadequate reporting, and incomplete assessment of the exposed population. Potential confounding factors include varying dosages, concomitant drug therapy, and comorbid maternal disease(s). Familial and environmental variables can also inluence development of birth defects. hus, an important criterion for teratogenicity is that two or more high-quality epidemiological studies report similar findings. Finally, a relative risk of 3.0 or reater is generally considered necessary to support the hypothesis, whereas a lesser risk is interpreted with caution (Khoury, 1992).

1	• he suspected teratogen causes a defect in animal studies. his criterion is not obligatory. In fact, the Teratology Society (2005) states that establishment of causation in teratology-related litigation requires human data.

1	Failure to employ these tenets and criteria has contributed to erroneous conclusions regarding the safety of some widely used drugs. he poster child for this is the medicolegal iasco surrounding Bendectin. This antiemetic was a combination of doxylamine and pyridoxine, with or without dicyclomine, and was safe and efective for nausea and vomiting in early pregnancy. More than 30 million women used this drug worldwide, and the 3-percent congenital anomaly rate among exposed fetuses was not diferent from the background rate (McKeigue, 1994). Despite considerable evidence that this combination of an antihistamine and a B-vitamin is not teratogenic, Bendectin was the target of numerous lawsuits, and the inancial burden of defending these forced its withdrawal from the marketplace. As a consequence, hospitalizations for hyperemesis doubled (Koren, 1998). Ironically, the combination of doxylamine and pyridoxine was subsequently remarketed under the brand name Diclegis and was approved by

1	hospitalizations for hyperemesis doubled (Koren, 1998). Ironically, the combination of doxylamine and pyridoxine was subsequently remarketed under the brand name Diclegis and was approved by the FDA in 2013.

1	he study of medication safetyor teratogenicity-in pregnant women is fraught with complications. First, animal studies are considered necessary but insuicient. For example, thalidomide is harmless in sveral animal species but resulted in phocomelia in thousands of children born across Europe in the late 1950s and early 1960s. Second, medications are rarely approved by the FDA for a pregnancy-related indication. Instead, pregnant women are considered a special population and summarily excluded from medication trials. Last, drug concentration and thus embyo-fetl exposure are afected by pregnancy physiology. hese include changes in volume of distribution, cardiac output, gastrointestinal absorption, hepatic metabolism, and renal clearance. In the absence of research trials, counseling is based on case reports or series, casecontrol studies, cohort studies, and pregnancy registry data.

1	Many, if not most, major teratogens were irst described by clinicians who observed a rare defect occurring after a rare exposure. his has been termed the "astute clinician model" (Carey, 2009). Congenital rubella syndrome was identiied in this way by Gregg (1941), an Australian ophthalmologist whose observations challenged the view that the uterine environment was impervious to noxious agents. Other teratogens identiied through case series include thalidomide and alcohol a ones, 1973; Lenz, 1962). Shepard (2002a) recommended that establishment of teratogenicity in this way requires proven exposure at a critical time in development and probably at least three such cases, each carefully delineated. Unfortunately, teratogens are less likely to be identiied if the exposure is uncommon, if the defects are relatively nonspeciic, or if abnormalities develop in only a small proportion of exposed fetuses. A major limitation of case series is their lack of a control group.

1	hese studies begin with groups of afected infants (cases) and unafected controls and are structured to allow retrospective assessment of prenatal exposure to particular substances. Casecontrol studies are an eicient way to study rare outcomes (Alwan, 2015). These permit investigators to evaluate associations and generate useful hypotheses. However, case-control studies have inherent potential for recal bias. Namely, parents of an afected infant are often more likely to recall exposure than those whose child is not ill. Confounding by indication is another concern, that is, the indication for the medication may be the cause of the birth defect. And importantly, birth defect registries have statistical power to detect small diferences that may not be clinically meaningful. Grimes and Schulz (2012) have cautioned that unless odds ratios in case-control studies are above three-to fourfold, the observed associations may not be correct.

1	The National Birth Defects Prevention Study n excellent example of a population-based case-control study is the National Birth Defects Prevention Study (NBDPS). Funded by Congress and coordinated by the National Center on Birth Defects and Developmental Disabilities, the NBDPS . took place between 1997 and 2013 across ten states with active birth defects surveillance programs. Clinical geneticists reviewed each potential case, and standardized telephone interviews were conducted with mothers whose pregnancies were afected or unafected to obtain information regarding medication exposure and risk factors (Mitchell, 2011i; Reehuis, 2015). Live births, stillbirths, and terminated pregnancies were included and totaled approximately 32,000 cases and nearly 12,000 controls.

1	he NBDPS has yielded more than 200 scientiic manuscripts. It identified novel-although often small-associations between individual birth defects and the following classes of medications: antibiotics, antidepressants, antiemetics, antihypertensives, asthma medications, nonsteroidal antiinlammatory drugs (NSAIDs), and opioids (Ailes, 2016; Broussard, 2011; Fisher, 2017; Hernandez, 2012; Lin, Teratology, Teratogens, and Fetotoxic Agents 237 2012; Munsie, 201i1). he NBDPS also found associations between birth defects and exposures such as secondhand smoke, pesticides, and nitrogen oxide, which is a marker of traic-related air pollution (Hoyt, 2016; Rocheleau, 2015; Stingone,i2017). The NBDPS did have limitations related to study design.

1	First, interviews were conducted 6 weeks to 2 years following delivery, which raised the likelihood of recall bias. For example, 25 percent of women could not remember which antibiotic they had taken (Ailes, 2016). Another weakness was that only two thirds of women agreed to participate, and there were diferences in ethnicity and socioeconomic status between cases and controls. These factors potentially led to selection bias (Reefhuis, 2015). In addition, medical records were not reviewed to veriy dosage, and this precluded assessment of dose-response relationships. And a major limitation was that because the NBDPS included only a small number of cases of each birth defect and analyzed them for multiple maternal exposures, it was not possible to adjust for multiple comparisons. As a result, some of the associations observed were likely due to chance (Alwan, 2015). For example, the study of antibiotics and birth defects included 43 comparisons and identiied four signiicant

1	As a result, some of the associations observed were likely due to chance (Alwan, 2015). For example, the study of antibiotics and birth defects included 43 comparisons and identiied four signiicant associations, but chance alone predicted that two associations would be identiied (Ailes, 2016). Last, the low absolute risk of an abnormality complicates counseling and prenatal management. In many instances, the risk identiied by the NBDPS was as low as 1 case per 1000 exposed pregnancies.

1	These studies begin with cohorts of pregnant women who are exposed or unexposed to a particular medication. The percentage of infants or children afected with birth defects is examined in each cohort. Because individual birth defects are rare, cohort studies require a vey large sample size. Medicaid datasets and private insurance claims databases are commonly used for cohort studies of teratogenicity in the United States (Ehrenstein, 2010). Inability to adjust for confounding variables-such as the indication for which the medication was needed-may be an important limitation of this study design.

1	Potentially harmful agents may be monitored by clinicians who prospectively enroll exposed pregnancies in a registry. he FDA (20 17b) maintains an active list on their webpage titled Pregnancy Registries. As of 2017, this included registries for 100 individual medications and for medication groups used to treat asthma, autoimmune disease, cancer, epilepsy, human immunodeiciency virus (HIV) infection, and transplant rejection. Similar to case series, exposure registries are hampered by lack of a control group. The prevalence of an abnormality identiied through a registry requires knowledge of the baseline prevalence of that anomaly in the population. Investigators typically use a birth defect registry to assess population prevalence. One example is the Metropolitan Atlanta Congenital Defects Program, which is an active surveillance program established in 1967 for fetuses and infants with birth defects. medications in pregnancy. Five categories-A, B, C, D, and

1	medications in pregnancy. Five categories-A, B, C, D, and X-were intended to summarize available evidence from

1	Questions regarding medication and illicit drug use should human or animal studies of embryonic-fetal risk. These letbe part of routine preconceptional and prenatal care. Misinters also conveyed beneits of the given medication balanced formation is common. Individuals tend to underestimate the against its potential risks. The system, shown in Table 12-3, background risk for birth defects in the general population and was intended to simpliY risk-beneit data. exaggerate potential risks associated with medication exposure. Unfortunately, information regarding medication risk was In a recent population-based study of more than 270,000 births very oten incomplete and led to an overreliance on the deinifrom Utah that included 5500 fetuses and infants with major tion of the letter category alone. However, a higher letter grade birth defects, only 4 cases were attributed to medication expodid not necessarily mean greater risk, and drugs in the same catesure (see Fig. 12-1) (Feldkamp, 2017).

1	However, a higher letter grade birth defects, only 4 cases were attributed to medication expodid not necessarily mean greater risk, and drugs in the same catesure (see Fig. 12-1) (Feldkamp, 2017). And yet, Koren and gory oten had very diferent risks. Very few medications-fewer colleagues (1989) reported that a fourth of women exposed to than 1 percent-had demonstrated safety in human pregnancy nonteratogenic drugs thought they had a 25-percent risk for (category A), and most had no safety data in human or animal fetal anomalies. Misinformation may be amplified by inaccurate studies (category C). Another diiculty was that the classiication reports in the lay press. Knowledgeable counseling may allay system did not address inadvertent exposure, a common reason anxiety considerably and may even avert pregnancy termination. for counseling. Ultimately, it is the responsibility of the clinician

1	Several sources are available to assist providers with accurate to interpret letter category information in the context of mediand updated risk information. PubMed is a free tool from the cation dosage and route, timing of exposure during pregnancy, National Center for Biomedical Information that aids rapid other medications used, and underlying medical condition(s). search of published research. Online databases, such as Reprotox, To address these deiciencies, new labeling requirements were TERIS, and Shepard's Online Catalog of Teratogenic Agents, created and went into efect in 2015. Updates to older mediofer reviews of medication risks. hey summarize human and cations will be phased in over time (Food and Drug Adminanimal studies of teratogenicity and fetotoxicity, address the istration, 2014). With the new requirements, the FDA letter quality of the available evidence, and provide magnitude of risk. categories have been (or will be) removed from all prescription Lactmed, a

1	2014). With the new requirements, the FDA letter quality of the available evidence, and provide magnitude of risk. categories have been (or will be) removed from all prescription Lactmed, a database from the National Library of Medicine, drug and biological product labeling. he format for providing speciically deals with medication use by breastfeeding women. information includes a summary of risks, clinical considerations, Its entries on speciic medications describe levels in breast milk and available data. The pregnancy subsection has registry inforand potential efects on the infant. Finally, with recent changes mation, if available, as well as labor and delivery information. to the FDA labeling requirements, discussed next, the manufacFor each medication, a lactation subsection-formerly called turer's prescribing information has become increasingly helpful. "nursing mothers"-is included. There is also a section to address potential risks in females and males with reproductive

1	called turer's prescribing information has become increasingly helpful. "nursing mothers"-is included. There is also a section to address potential risks in females and males with reproductive potential .

1	• The Food and Drug Administration: In 1979, the FDA developed a letter classiication system In addition to potential embryonic and fetal risks from drug in an efort to provide therapeutic guidance for prescribing exposure, counseling should discuss the risks and/or genetic TABLE 12-3. Food and Drug Administration Letter Categories for Drugs and Medications (1•979-2015)a Category A: Studies in pregnant women have not shown an increased risk for fetal abnormalities if administered during the first (second, third, or all) trimester(s) of pregnancy, and the possibility of fetal harm appears remote. Category B: Animal reproduction studies have been performed and have revealed no evidence of impaired fertility or harm to the fetus. Animal studies have shown an adverse effect, but adequate and well-controlled studies in pregnant women have failed to demonstrate a risk to the fetus during the first trimester of pregnancy, and there is no evidence of a risk in later trimesters.

1	Category C: Animal reproduction studies have shown that this medication is teratogenic (or embryocidal or has other adverse effect), and there are no adequate and well-controlled studies in pregnant women. There are no animal reproduction studies and no adequate and well-contolled studies in humans. Category 0: This medication can cause fetal harm when administered to a pregnant woman. If this drug is used during pregnancy or if a woman becomes pregnant while taking this medication, she should be apprised of the potential hazard to the fetus. Category X: This medication is contraindicated in women who are or may become pregnant. It may cause fetal harm. aMedications approved after June 2015 are not assigned a letter category, whereas older medications will have letter categories phased out after this date.

1	implications of the underlying condition for which the drug is given. Risks associated with not treating the condition are also described. Even the manner in which information is presented afects perception. For example, women given negative information-such as a 2-percent chance of a malformed newborn-are more likely to perceive an exaggerated risk than women given positive information-such as a 98-percent chance of an unafected infant Gasper, 2001). Instead of citing a higher odds ratio, it may be helpful to provide the absolute risk for a particular defect or the attributable risk, which is the diference between prevalence in exposed and unexposed individuals (Conover, 2011). The association between oral corticosteroid medications and cleft lip sounds far more concerning when presented as a tripling or 200-percent increase in risk than when described as an increase from 1 per 1000 to 3 per 1000 or as a 99.7 -percent likelihood of no cleft development following exposure.

1	With a few notable exceptions, most commonly prescribed drugs and medications can be used with relative safety during pregnancy. Many drugs discussed in this chapter are lowrisk teratogens, which are medications that produce defects in fewer than 10 per 1000 maternal exposures (Shepard, 2002a). Because risks conferred by low-risk teratogens are so close to the population background rate of fetal anomalies, they may not be a major factor in deciding whether to discontinue treatment for an important condition (Shepard, 2002b). Remember that al women have an approximate 3-percent chance of having a newborn with a birth dect. Although exposure to a confirmed teratogen may elevate this risk, the magnitude of the increase is usually only 1 or 2 percent or at most, doubled or tripled. The concept of risk versus benefit is often central to counseling. Some untreated diseases pose a more serious threat to both mother and fetus than medication exposure risks.

1	Considering the thousands of compounds available, relatively few medications and other substances are considered to be major human teratogens. The most common examples are listed in Table 12-1. With few exceptions, in every clinical situation potentially requiring therapy with a known teratogen, alternative drugs can be given with relative safety. Realizing limitations in available evidence, pregnant women should be advised to take any medication only when it is clearly needed. In general, targeted sonography is indicated if there has been exposure to any major teratogen during the embryonic period. Ethanol is a potent and prevalent teratogen. It is considered the leading cause of preventable developmental disabilities worldwide (Hoyme, 2016). In the United States, 8 percent of pregnant women report drinking alcohol and between 1 and 2 percent admit to binge drinking (Centers for Disease Control and Prevention, 2012).

1	The fetal efects of alcohol abuse have been recognized since the 1800s. Lemoine (1968) and Jones (1973) and their coworkers are credited with describing the spectrum of alcohol-related Teratology, Teratogens, and Fetotoxic Agents 239 fetal defects known asetal acohol syndrome (Table 12-4). For every child with the syndrome, many more are born with neurobehavioral deicits from alcohol exposure (American College of Obstetricians and Gynecologists, 2013). Fetal acohol spectrum disorder is an umbrella term that includes ive conditions attributed to prenatal alcohol damage: (1) fetal alcohol syndrome, partial fetal alcohol syndrome, (3) alcohol-related birth defects, (4) alcohol-related neurodevelopmental disorder, and neurobehavioral disorder associated with prenatal alcohol exposure (Williams, 2015). The birth prevalence of fetal alcohol syndrome is estimated to be as high as 1 percent in the United States (Centers for Disease Control, 2012; Guerri, 2009).

1	TABLE 12-4. Criteria for Prenatal Alcohol Exposure, Fetal Alcohol Syndrome, and Alcohol-Related Birth Defects 1. ::6 drinks per week for ::2 weeks 2. ::3 drinks per occasion for ::2 occasions 3. Risk identiied with a validated screening questionnaire 4. 5. Documentation of an alcohol-related legal or social 1. Dysmorphic facial features (::2 required) a. b. Thin vermilion border of the upper lip c. 2. Prenatal and/or postnatal growth impairment, � 10th percentile 3. Abnormal brain growth, morphogenesis, or physiology (::1 required) a. b. c. 4. Neurobehavioral impairment (deined as > 1.5 SO below mean) a. Child <3 years: developmental delay b. Child ::3 years: global cognitive impairment, r cognitive deicit in at least 1 neurobehavioral domain, or behavioral deicit in at least 1 domain Cardiac: atrial or ventricular septal defect, aberrant great vessels, conotruncal heart defects

1	Cardiac: atrial or ventricular septal defect, aberrant great vessels, conotruncal heart defects Skeletal: radioulnar synostosis, vertebral segmentation defects, joint contractures, scoliosis Renal: aplastic or hypoplastic kidneys, dysplastic kidneys, horseshoe kidney, ureteral duplication Eyes: strabismus, ptosis, retinal vascular abnormalities, optic nerve hypoplasia Ears: conductive or neurosensory hearing loss Data from Hoyme, 2016. FIGURE 12-3 Fetal alcohol syndrome. A.At 2Y2 years. B. At 12 years. Note persistence of short palpebral fissures, epicanthal folds, flat midface, hypoplastic philtrum, and thin upper vermilion border. (Reproduced with permission from Streissguth AP, Clarren, SK, Jones KL. Natural history of fetal alcohol syndrome: a la-year follow-up of eleven patients, Lancet. 1985 Jul 13;2(8446):85-91.) But, studies of school children have identiied fetal alcohol spectrum disorder in 2 to 5 percent (May, 2009, 2014).

1	But, studies of school children have identiied fetal alcohol spectrum disorder in 2 to 5 percent (May, 2009, 2014). Fetal alcohol syndrome has speciic criteria (see Table 12-4). hese include central nervous system (CNS) abnormalities, preor postnatal growth impairment, and a characteristic pattern of minor facial abnormalities (Fig. 12-3). Similar criteria have been established for the other conditions that make up fetal alcohol spectrum disorder (Hoyme, 2016). Prenatal alcohol exposure criteria are also available to assist with assessment.

1	Alcohol-related birth defects include cardiac and renal anomalies, orthopedic problems, and abnormalities of the eyes and ears (see Table 12-4). An association has further been reported between periconceptional alcohol use and omphalocele and gastroschisis (Richardson, 2011). here are no established sonographic criteria for prenatal diagnosis of fetal alcohol syndrome. hat said, in some cases, major abnormalities or growth restriction may be suggestive (Paintner, 2012).

1	Fetal vulnerability to alcohol is modiied by genetic components, nutritional status, environmental factors, coexisting maternal disease, and maternal age (Abel, 1995). The Centers for Disease Control and Prevention (CDC) and the American Academy of Pediatrics have stressed that no amount of alcohol can be considered safe in pregnancy (Williams, 2015). Binge drinking, however, is believed to pose particularly high risk for alcohol-related birth defects and has also been linked to a higher risk for stillbirth (Centers for Disease Control, 2012; Maier, 2001; Strandberg-Larsen, 2008). Traditionally, women with epilepsy requiring treatment with medication were informed that their risk for fetal malformations was increased. More recent data suggest that the risk may not be as great as once thought, particularly for newer agents. The most frequently reported anomalies are orofacial clets, cardiac malformations, and neural-tube defects.

1	Of agents in current use, valproic acid confers the greatest risk (Vajda, 2014). he North American Antiepileptic Drug (NAAED) Pregnancy Registry reported that major malformations developed in 9 percent of fetuses with irst-trimester valproate exposure. his included a 4-percent risk for neural-tube defects (Hernandez-Diaz, 2012). School-aged children with in utero exposure to valproic acid have poorer cognitive development-including signiicantly lower intelligence quotient (I) scores-than children exposed to other antiepileptic drugs (Bromley, 2014; Meador, 2009).

1	Regarding other speciic anticonvulsants, one recent metaanalysis identiied higher malformation rates among exposed children compared with rates among children born to women with untreated epilepsy. Rates were twofold higher among children exposed to carbamazepine or phenytoin, threefold higher among those exposed to phenobarbital, and fourfold higher among those exposed to topiramate as mono therapy (Weston, 2016). The risk for fetal malformations is approximately doubled if multiple agents are required (Vajda, 2016). Several older anticonvulsants also produce a constellation of malformations similar to the fetal hydantoin syndrome, which is described in Figure 1i2-4.

1	Figure 1i2-4. FIGURE 12-4 Fetal hydantoin syndrome. A. Facial features including upturned nose, mild midfacial hypoplasia, and long upper lip with thin vermilion border. B. Distal digital hypoplasia. (Reproduced with permission from Buehler BA 1, Delimont D, van Waes M, et al: Prenatal prediction of risk of the fetal hydantoin syndrome, N Engl J Med. 1990 May 31;322(22):1567-1572.) These risks do not appear to hold for the newer agents leve tiracetam and lamotrigine, although the number of reported pregnancies to date is smaller (M0Igaard-Nielsen, 2011; Weston, 2016). The Motherisk Program reviewed eight stud ies of levetiracetam and concluded that mono therapy was associated with a 2-percent major malformation rate, which is no diferent from that for the general population (Chaudhry, 2014). Providers are encouraged to enroll pregnant women treated with antiepileptic medication in the NAAED Pregnancy Reg istry. Management of epilepsy in pregnancy is discussed in

1	Providers are encouraged to enroll pregnant women treated with antiepileptic medication in the NAAED Pregnancy Reg istry. Management of epilepsy in pregnancy is discussed in Chapter 60 (p. 1159). These medications may result in ACE-inhibitor etopathy. Normal renal development depends on the fetal renin-angiotensin system. ACE-inhibitor medication may cause fetal hypotension and renal hypoperfusion, with subsequent ischemia and anuria (Guron, 2000; Pryde, 1993). Reduced perfusion can result in fetal-growth restriction and calvarium maldevelopment, and oligohydramnios may lead to pulmonary hypoplasia and limb contractures (Barr, 1991). Because angiotensin-receptor blockers have a similar mechanism of action, concerns regarding fetotoxicity have been generalized to include this entire medication class.

1	Concerns were also raised about ACE-inhibitor embryotoxiciy, although these have largely been disproven. In 2006, a review of 29,000 infants from the Tennessee Medicaid database identified a two-to threefold greater risk for neonatal cardiac and CNS abnormalities among the 209 that had prenatal ACE-inhibitor exposure (Cooper, 2006). Subsequent larger studies have not corroborated these observations. First, in a retrospective cohort study of more than 460,000 pregnancies, risks for birth defects were not greater with ACE inhibitors than with other antihypertensive medications (Li, 201i1). Similarly, Bateman and coworkers (2017) reviewed 1.3 million pregnancies from the Medicaid Analytic eXtract and found no higher risk for any malformation with ACE-inhibitor exposure after adjusting for confounding factors such as diabetes. hus, women with inadvertent irst-trimester exposure to these medications can be reassured. However, given the many therapeutic options for treating hypertension

1	confounding factors such as diabetes. hus, women with inadvertent irst-trimester exposure to these medications can be reassured. However, given the many therapeutic options for treating hypertension during pregnancy, discussed in Chapter 50 (p. 980), it is recommended that ACE inhibitors and angiotensin receptor-blocking drugs be avoided in pregnancy.

1	From this class of drugs, fluconazole has been associated with a pattern of congenital malformations resembling the autosomal recessiveAntly-Bixler syndrome. Abnormalities include oral clets, abnormal facies, and cardiac, skull, long-bone, and joint abnormalities. Such indings have been reported only with chronic, irsttrimester, high-dose treatment at doses of 400 to 800 mg daily. Regarding low-dose treatment of vulvovaginal candidiasis, the Motherisk Program recently conducted a systematic review of pregnancies with first-trimester fluconazole exposure of 150

1	Regarding low-dose treatment of vulvovaginal candidiasis, the Motherisk Program recently conducted a systematic review of pregnancies with first-trimester fluconazole exposure of 150 Teratology, Teratogens, and Fetotoxic Agents 241 or 300 mg in total (Alsaad, 2015). The overall risk for birth defects was not greater, although a small increase in rates of cardiac malformations could not be excluded. A populationbased cohort study from Denmark identified a threefold greater risk for tetralogy of FaIlor following exposure to low-dose fluconazole (M0Igaard-Nielsen, 2013). The birth prevalence of tetralogy of Fallot rose from 3 to 10 cases per 10,000. This is a risk so low that we would not endorse specialized sonography for this indication. Notably, investigators did not identiy increased risks for 14 other birth defects previously associated with exposure to high-dose azole antifungal agents (M0IgaardNielsen,i20l3).

1	such as ibuprofen and indomethacin. They exert their efects by inhibiting prostaglandin synthesis. In a report from the NBDPS, at least 20 percent of pregnant women recall first trimester NSAID use, particularly ibuprofen and aspirin, and such exposure is not a major risk factor for birth defects (Her nandez, 2012).

1	NBDPS, at least 20 percent of pregnant women recall first trimester NSAID use, particularly ibuprofen and aspirin, and such exposure is not a major risk factor for birth defects (Her nandez, 2012). However, when taken in late pregnancy, indomethacin may cause constriction of the fetal ductus arteriosus and subsequent pulmonary hypertension. Fetal ductal constriction is more likely when the drug is taken in the third trimester for longer than 72 hours. he risk is IS-fold higher among indomethacin-exposed pregnancies (Koren, 2006). he drug also may decrease fetal urine production and amnionic luid volume (Rasanen, 1995; van der Heijden, 1994; Walker, 1994). In one systematic review, indomethacin tocolysis was associated with neonatal morbidity (Hammers, 2015a,b). Specifically, the risk for bronchopulmonary dysplasia, severe intraventricular hemorrhage, and necrotizing enterocolitis was increased approximately 50 percent (odds ratio 1.5).

1	With aspirin, a low dosage of 100 mg daily or less does not confer a greater risk for constriction of the ductus arteriosus or for adverse infant outcomes (Di Sessa, 1994; Grab, 2000). As with other NSAIDs, however, high-dose aspirin use should be avoided, particularly in the third trimester.

1	his is a pyrimidine-synthesis inhibitor used to treat rheuma toid arthritis but is contraindicated in pregnancy. In several animal species, it results in fetal hydrocephalus, eye anoma lies, skeletal abnormalities, and embryo death when given at or below human-equivalent doses (Sanoi-Aventis, 2016). The active metabolite, terilunomide, is detectable in plasma for up to 2 years following discontinuation of the medication. Women who become pregnant while taking leflunomide, and even those of childbearing potential who have discontinued it, are recommended to undergo an accelerated drug elimination procedure with either cholestyramine or activated charcoal (Sanoi-Aventis, 2016). Reassuringly, in a cohort of 60 women with irst-trimester lelunomide exposure who completed cholestyramine washout, the rate of birth defects was not increased (Chambers, 2010).

1	Medications used to treat infections are among those most frequently administered during pregnancy. Over the years, experience has accrued regarding their general safety. With a few exceptions cited below, most of the commonly used antimicrobial agents are considered safe for the embryo-fetus. Some preterm neonates treated with gentamicin or streptomycin have developed nephrotoxicity and otoroxicity. Despite theoretical concern for potential fetal toxicity, no adverse efects have been demonstrated, and no congenital defects resulting from prenatal exposure have been identified.

1	his antimicrobial is not considered teratogenic and is no lon ger routinely used in the United States. More than 50 years ago, a constellation of findings termed the gray baby syndrome was described in neonates who received the medication. Pre..i term newborns were unable to conjugate and excrete the drug and manifested abdominal distention, respiratory abnormali ties, an ashen-gray color, and vascular collapse (Weiss, 1960). due to theoretical concerns. From NBDPS results, first-trimester nitrofurantoin exposure is linked to a twofold risk for cleft lip (Ailes, 2016; Crider, 2009). Considering that the birth prevalence of clefts approximates 1 case per 1000, the likelihood that a nitrofurantoin-exposed fetus would not have a cleft would thus be 998 per 1000. For other birth defects, initial associations with this antibiotic did not persist in the inal NBDPS cohort (Ailes, 2016).

1	In one systematic review of nitrofurantoin exposure in pregnancy, results of cohort and case-control studies difered (Goldberg, 2015). Five cohort studies included 9275 exposed pregnancies and nearly 1.5 million unexposed pregnancies, and the review found no higher risk for any malformation. However, among three case-control studies that had nearly 40,000 cases matched with 130,000 controls, the rate of hypoplastic left heart syndrome was threefold greater (Goldberg, 2015). For context, this increase in risk would result in a birth prevalence of fewer than 1 case per 1000 exposed infants. The American College of Obstetricians and Gynecologists (2017 e) has concluded that irst-trimester nitrofurantoin use is appropriate if no suitable alternatives are available.

1	These drugs are often combined with trimethoprim and used to treat infections during pregnancy. One indication is treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection. The NBDPS, which included 107 pregnancies with periconceptional trimethoprim-sulfamethoxazole exposure and birth defects, identiied a fivefold greater risk to have ofspring with esophageal atresia or diaphragmatic hernia (Ailes, 2016). Similar to findings with nitrofurantoin exposure, this degree of increase would confer a risk of approximately 1 case per 1000 exposed infants for these selected birth defects. However, these findings have not been corroborated by other reports. One review from the Medication Exposure in Pregnancy Risk Evaluation Program included more than 7500 infants with irst-trimester exposure to trimethoprim-sulfamethoxazole (Hansen, 2016). Compared with either unexposed infants or those exposed to penicillins or cephalosporins, no greater risk for any congenital abnormality was

1	exposure to trimethoprim-sulfamethoxazole (Hansen, 2016). Compared with either unexposed infants or those exposed to penicillins or cephalosporins, no greater risk for any congenital abnormality was identified. The American College of Obstetricians and Gynecologists (2017 e) considers sulfonamides appropriate for irst-trimester use if suitable alternatives are lacking.

1	Sulfonamides displace bilirubin from protein-binding sites. Thus, if given near the time of preterm delivery, these agents theoretically might worsen neonatal hyperbilirubinemia. However, a population-based review of more than 800,000 births from Denmark found no association between exposure to sulfamethoxazole in late pregnancy and neonatal jaundice (Klarskov, 2013). These drugs are no longer commonly used in pregnant women. They are associated with yellowish-brown discoloration of deciduous teeth when used after 25 weeks' gestation. The risk for subsequent dental caries does not appear greater (Billings, 2004; Kutscher, 1966). In contrast, a recent systematic review of doxycycline in pregnancy identified no higher rates of either birth defects or staining of deciduous teeth (Cross, 2016).

1	Cancer management in pregnancy includes many chemotherapeutic agents generally considered to be at least potentially toxic to the embryo, fetus, or both. For the many novel polyclonal antibody therapies designated as antineoplastics, there are few data concerning their safety. Some risks with these and with other antineoplastic agents are discussed in Chapter 63 (p. 1191). A few of the more common agents for which experience in pregnancy has accrued are considered next.

1	This alkylating agent inflicts a chemical insult on developing fetal tissues and leads to cell death and heritable DNA alterations in surviving cells. Pregnancy loss rates are increased, and reported malformations include skeletal abnormalities, limb defects, cleft palate, and eye abnormalities (Enns, 1999; Kirshon, 1988). Surviving infants may have growth abnormalities and developmental delays. Environmental exposure among health-care workers is associated with a higher risk for spontaneous abortion (Chap. 18, p. 348).

1	This folic-acid antagonist is a potent terarogen. It is used for cancer chemotherapy, immunosuppression of autoimmune diseases and psoriasis, nonsurgical treatment of ecropic pregnancy, and medical abortion. It is similar in action to aminopterin, which is no longer used clinically, and can cause defects known collectively as the fetal methotrexate-aminopterin syndrome. This includes craniosynosrosis with a "clover-leaf' skull, wide nasal bridge, low-set ears, micrognathia, and limb abnormalities (Del Campo, 1999). Teratology, Teratogens, and Fetotoxic Agents 243 he embryo is thought to be most vulnerable at 8 to 10 weeks postconception and at dosages of at least 10 mg/week. However, this is not universally accepted (Feldkamp, 1993).

1	he standard 50 mg/m2 dose given to treat ectopic pregnancy or to induce elective abortion exceeds this threshold dose. Some reports have suggested an association with cardiac anomalies, particularly conotruncal defects, in intrauterine pregnancies inadvertently treated with methotrexate for suspected ectopic pregnancy (Dawson, 2014; Hyoun, 2012). Thus, ongoing pregnancies after treatment with methotrexate-especially if used in conjunction with misoprostol-raise serious concerns for fetal malformations (N urmohamed, 2011).

1	This nonsteroidal selective estrogen-receptor modulator (SERM) is used as an adjuvant to treat breast cancer. No pattern of birth defects has been described in limited case reports and series (Braems, 2011). However, tamoxifen has been associated with malformations similar to those caused by diethylstilbestrol (DES) exposure in rodents, including vaginal adenosis. Consequently, women who become pregnant on therapy or within 2 months of its discontinuation should be apprised of the potentialilong-term risks of a DES-like syndrome.

1	his is a recombinant monoclonal antibody directed to the human epidermal growth factor receptor 2 (HER2) protein. Used to treat breast cancers that express HER2 protein, this drug has not been associated with fetal malformations. However, cases of oligohydramnios sequence resulting in pulmonary hypoplasia, renal failure, skeletal abnormalities, and neonatal deaths have been reported (Genentech, 2017). Surveillance for these complications is recommended for exposed pregnancies and for those treated at any time in the 7 months prior to conception. A trastuzumab pregnancy exposure registry and a pregnancy pharmacovigilance program have been established to monitor pregnancy outcomes. hese warnings also apply to those treated with ado-trastuzumab emtansine. The number of drugs used to treat viral infections has increased rapidly during the past 20 years. For most, experience in pregnant women is limited.

1	The number of drugs used to treat viral infections has increased rapidly during the past 20 years. For most, experience in pregnant women is limited. This nucleoside analogue is a component of therapy for hepatitis C infection, discussed in Chapter 55 (p. 1065). Ribavirin causes birth defects in multiple animal species at doses signiicantly lower than those recommended for human use. Reported malformations include skull, palate, eye, skeleton, and gastrointestinal abnormalities. he drug has a half-life of 12 days and persists in extravascular compartments following therapy discontinuation. Treated women must use two forms of contraception and have monthly pregnancy tests while on therapy and for 6 months following drug discontinuation (Genentech, 2015). Ribavirin use is also contraindicated in men whose partners are pregnant.

1	This is a nonnucleoside reverse transcriptase inhibitor used to treat HIV infection (Chap. 65, p. 1249). CNS and ocular abnormalities have been reported in cynomolgus monkeys treated with doses comparable to those used in humans. Several case reports also describe neural-tube defects following human exposure to efavirenz. Reassuringly, the Antiretroviral Pregnancy Registry has identiied no increased birth defect rates in more than 800 pregnancies with irst-trimester exposure (Bristol-Meyers Squibb, 2017b).

1	Bosentan, ambrisentan, and macitentan are three endothelinreceptor antagonists used to treat pulmonary arterial hypertension (Chap. 49, p. 962). he endothelin-receptor signaling pathway is important for neural-crest development. Mice deicient in endothelin receptors develop neural-crest cell defects that include craniofacial and cardiac outflow tract abnormalities (de Raaf, 2015). Each of these three agents has been found to cause similar birth defects in multiple animal species (Actelion, 2017). No human data are available. Endothelin-receptor antagonists may be obtained only through restricted access programs, each of which has stringent requirements that include contraception and monthly pregnancy testing (Actelion, 2016, 2017; Gilead, 2015). Some of the functions and efects of male and female hormones on the developing fetus are discussed in Chapter 3 (p. 38). It is intuitive that exposure of female fetuses to excessive male sex hormones-and vice versa-might be detrimental.

1	Androgen exposure in reproductive-aged women typically stems from anabolic steroid use to accrue lean body mass and muscular strength. Exposure of a female fetus may cause varying degrees of virilization and may result in ambiguous genitalia similar to that encountered in congenital adrenal hyperplasia . Findings can include labioscrotal fusion with irst-trimester exposure and phallic enlargement from later fetal exposure (Grumbach, 1960; Schardein, 1980). This ethinyl testosterone derivative has weak androgenic activity. It is used to treat endometriosis, immune thrombocytopenic purpura, migraine headaches, premenstrual syndrome, and fibrocystic breast disease. In a review of inadvertent exposure during early pregnancy, Brunskill (1992) reported that 40 percent of exposed female fetuses were virilized. There was a doserelated pattern of clitoromegaly, fused labia, and urogenital sinus malformation.

1	This medication is included for historical context. From 1940 until 1971, between 2 and 10 million pregnant women were given this synthetic estrogen for ill-advised indications. It was removed from the market after Herbst and associates (1971) reported a series of eight women exposed to DES in utero who developed an otherwise rare neoplasm, vaginal clear-cell adenocarcinoma. With no relationship to drug dosage, the absolute cancer risk approximates 1 case per 1000 DES-exposed fetuses. Twofold greater rates of vaginal and cervical intraepithelial neoplasia were also described (Vessey, 1989).

1	DES exposure has further been associated with genital tract abnormalities in exposed fetuses of both genders. Women may have a hypoplastic, T-shaped uterine cavity; cervical collars, hoods, septa, and coxcombs; and "withered" fallopian tubes (Goldberg, 1999; Salle, 1996). Some are described and illustrated in Chapter 3 (p. 45). Later in life, women exposed in utero have slightly higher rates of earlier menopause and breast cancer (Hoover, 2011). Men may develop epididymal cysts, microphallus, hypospadias, cryptorchidism, and testicular hypoplasia (Klip, 2002; Stillman, 1982). Some of the immune functions necessary for pregnancy maintenance are discussed in Chapter 5 (p. 95). Given these important interactions, immunosuppressant drugs logically might afect pregnancy.

1	Some of the immune functions necessary for pregnancy maintenance are discussed in Chapter 5 (p. 95). Given these important interactions, immunosuppressant drugs logically might afect pregnancy. hese medications include glucocorticoids and mineralocorticoids, which have antiinflammatory and immunosuppressive actions. They are frequently used to treat serious disorders such as asthma and autoimmune disease. Corticosteroids have been associated with clefts in animal studies. In one metaanalysis of case-control studies by the Motherisk Program, systemic corticosteroid exposure was associated with a threefold increase in the rate of clefts. his is an absolute risk of 3 cases per 1000 exposed fetuses (Park-Wyllie, 2000). A 10-year prospective cohort study by the same group, however, did not identiy higher risks for major malformations. Based on these findings, corticosteroids are not considered to represent a major teratogenic risk.

1	Unlike other corticosteroids, the active metabolite of prednisone, which is prednisolone, is inactivated by the placental enzyme 11�-hydroxysteroid dehydrogenase 2. Thus, it may not efectively reach the fetus. This inosine monophosphate dehydrogenase inhibitor, and a related agent, mycophenolic acid, are immunosuppressants. They are used to prevent rejection in organ-transplant recipients and to treat autoimmune disease (Chap. 59, p. 1142). Mycophenolate is a potent teratogen. From the National Transplantation Pregnancy Registry, of pregnancies in which mycophenolate was not discontinued until after the first trimester, birth defects complicated 30 percent, and another 30 percent spontaneously aborted (ing, 2017). One prospective review by the European Network of Teratology Information Services similarly identified a spontaneous loss rate of nearly 30 percent in exposed pregnancies. More than 20 percent of liveborn infants had major anomalies (Hoeltzenbein, 2012).

1	Many afected infants have a pattern of defects termed mycophenolate embyopathy. his includes microtia, auditory canal atresia, clefts, coloboma and other eye anomalies, short ingers with hypoplastic nails, and cardiac defects (Anderka, 2009; Merlob, 2009). A Risk Evaluation and Mitigation Strategy (REMS) has been developed for mycophenolate prescribers who treat women with reproductive potential. REMS are safety strategies mandated by the FDA to help manage known risks associated with a medicine yet still allow patients to have access to the benefits of a given drug.

1	Radioactive iodine-131 is used for treatment of thyroid cancer and thyrotoxicosis and for diagnostic thyroid scanning. It is also a component of iodine-131 tositumomab therapy, which is employed to treat a type of non-Hodgkin lymphoma. Radioiodine is contraindicated during pregnancy because it readily crosses the placenta and is then concentrated in the fetal thyroid gland by 12 weeks' gestation. It may cause severe or irreversible fetal and neonatal hypothyroidism, which can lead to decreased mental capacity and delayed skeletal maturation Qubilant DraxImage, 2016). Pregnancy testing should be performed before administration of radioiodine-131. Prenatal lead exposure is associated with fetal-growth abnormalities and with childhood developmental delay and behavioral abnormalities. According to the CDC (2010), no level of lead exposure is considered safe in pregnancy. Care and testing for at-risk pregnancies is discussed in Chapter 9 (p. 170).

1	Environmental spills of methyl mercury in Minamata Bay, Japan, and rural Iraq demonstrated that the developing nervous system is particularly susceptible to this heavy metal. Prenatal exposure causes disturbances in neuronal cell division and migration. This leads to a range of defects from developmental delay to microcephaly and severe brain damage (Choi, 1978). he principal concern for prenatal mercury exposure is the consumption of certain species of large fish (Chap. 9, p. 170). The FDA (2017a) advises that pregnant women and breastfeeding mothers avoid consumption of king mackerel, marlin, orange roughy, shark, swordfish, tilefish, and bigeye tuna. Treatment of psychiatric illness in pregnancy, including a discussion of the risks and benefits of various psychiatric medications, is described in Chapter 61 (p. 1173). Selected birth defects and adverse efects associated with specific medications are presented here.

1	This medication has been associated with Ebstein anomaly, a rare cardiac abnormality that otherwise complicates only 1 per 20,000 births. Ebstein anomaly is characterized by apical Teratology, Teratogens, and Fetotoxic Agents 245 displacement of the tricuspid valve, often resulting in severe tri confer signiicant morbidity. A report from the Lithium Baby Registry initially suggested that the risk for Ebstein anomaly was as high as 3 percent. However, subsequent series have iden right-sided cardiac anomalies of only 1 to 4 per 1000 exposed pregnancies (Patorno, 2017; Yacobi, 2008). In a review of fout case-control studies that included more than 200 infants with Ebstein anomaly, no cases were attributed to lithium exposure (Cohen, 1994).

1	Ebstein anomaly, no cases were attributed to lithium exposure (Cohen, 1994). ery. he manufacturer recommends that if possible, the dosage should be decreased or drug discontinued 2 to 3 days prior to delivery to reduce this risk (West-Ward, 2016). Findings typically persist for 1 to 2 weeks and may include neonatal hypothyroidism, diabetes insipidus, cardiomegaly, bradycar dia, electrocardiogram abnormalities, cyanosis, and hypotonia (American College of Obstetricians and Gynecologists, 2016).

1	As a class, these medications are not considered major teratogens (American College of Obstetricians and Gynecologists, 2016). The one exception is paroxetine, which has been associated with a higher risk for cardiac anomalies, particularly atrial and ventricular septal defects. Three large databases-a Swedish national registry, a United States insurance claims database, and the Motherisk Program-each identified a 1.5-to twofold greater risk for cardiac malformations following first-trimester paroxetine exposure (Bar-Oz, 2007; Sebela, 2017). For these reasons, the American College of Obstetricians and Gynecologists (2016) recommends that paroxetine be avoided in women planning pregnancy. Fetal echocardiography should be considered for those with first-trimester paroxetine exposure.

1	Neonatal efects have been associated with prenatal exposure to selective serotonin-reuptake inhibitors (SSRIs) and selective norepinephrine-reuptake inhibitors (SNRIs). Approximately 25 percent of neonates exposed to SSRIs in late pregnancy manifest one or more nonspecific findings considered to represent poor neonatal adaptation (Chambers, 2006; Costei, 2002; Jordan, 2008). Collectively termed the neonatal behavioral syndrome, findings can include jitteriness, irritability, hyper-or hypotonia, feeding abnormalities, vomiting, hypoglycemia, thermoregulatory instability, and respiratory abnormalities. Fortunately, these neonatal behaviors are typically mild and self-limited and last approximately 2 days. Jordan and coworkers (2008) reported that afected newborns were not more likely to require a higher level of care, to experience respiratory abnormalities, or to have prolonged hospitalization. Rarely, neonates exposed to SSRIs in late pregnancy demonstrated more severe adaptation

1	require a higher level of care, to experience respiratory abnormalities, or to have prolonged hospitalization. Rarely, neonates exposed to SSRIs in late pregnancy demonstrated more severe adaptation abnormalities (Ornoy, 2017).

1	Another concern with late-pregnancy exposure is the possible association of SSRI medications with persistent pulmonary hypertension of the newborn (PPH). he baseline incidence approximates 2 cases per 1000 term newborns. PPHN is characterized by elevated pulmonary vascular resistance with right-to-Ieft shunting and resultant hypoxemia. Two recent population-based cohort studies-together involving more than 5 million pregnancies-identiied an attributable risk of only 1 to 2 cases per 1000 births (Huybrechts, 2015; Kieler, 2012). Not only is the risk for this condition quite low, but cases associated with SSRI medication have not been severe (Ornoy,i2017).

1	No antipsychotic medications are considered teratogenic. Exposed neonates can manifest abnormal extrapyramidal muscle movements and withdrawal symptoms that include agitation, abnormally enhanced or diminished muscle tone, tremor, sleepiness, feeding diiculty, and respiratory abnormalities. These indings are nonspeciic and transient, similar to the neonatal behavioral syndrome that can follow SSRI exposure. An FDA (2011) alert cites all medications in this class. These include older drugs such as haloperidol and chlorpromazine, as well as newer medications such as aripiprazole, olanzapine, quetiapine, and risperidone.

1	hese vitamin A derivatives are among the most potent human teratogens. hree retinoids available in the United States are highly teratogenic when orally administered-isotretinoin, acitretin, and bexarotene. By inhibiting neural-crest cell migration during embryogenesis, they create a pattern of cranial neural-crest defects-termed retinoic acid embryopath-that involve the CNS, face, heart, and thymus (Fig. 12-5). Specific anomalies may include ventriculomegaly, maldevelopment of the facial bones or cranium, microtia or anotia, micrognathia, cleft palate, conotruncal heart defects, and thymic aplasia or hypoplasia.

1	13-cis-Retinoic acid is a vitamin A isomer that stimulates epithelial cell diferentiation and is used for dermatological disorders, especially cystic nodular acne. First-trimester exposure is associated with a high rate of pregnancy loss, and up to a third of fetuses have malformations (Lammer, 1985). he iPLEDGE program is an FDA-mandated REMS for isotretinoin and is found at: ww.ipledgeprogram.com.ihis webbased, restricted-distribution program requires participation for all patients, physicians, and pharmacies to help eliminate embryonic-fetal exposure. Although other countries have instituted similar programs, inadvertent exposure remains a global concern (Crijns, 201l).

1	This retinoid is used to treat severe psoriasis and was introduced to replace etretinate. he latter is a lipophilic retinoid with such a long hal-life (120 days) that birth defects resulted more than 2 years after therapy was discontinued. Although acitretin has a short half-life, it is metabolized to etretinate, and thus remains in the body for prolonged periods (Stiefel Laboratories, 2015). To obviate exposure, the manufacturer of acitretin has developed a pregnancy risk management program. Called "Do Your P.A.R.T"-Eregnancy prevention Actively Required during FIGURE 12-5 Isotretinoin embryopathy. A. Bilateral microtia or anotia with stenosis of external (Kaplan, 2015). These results may be reassuring to pregnant women with inadvertent exposure. Notably, the manufacturer of tazarotene cautions that application over a suicient body surface area could be comparable to oral treatment. Accordingly, its use in pregnancy is not recommended (Allergan, 2017).

1	There are wo natural forms of vitamin A. Beta-carotene, which is a precursor of provitamin A, is found in fruits and vegetables and has never been shown to cause birth defects (Oakley, 1995). Retinol is preformed vitamin A, ear canal. B. Flat, depressed nasal bridge and ocular hypertelorism. (Used with permission from Dr. Edward Lammer.) and after Ireatment, this program promotes a delay of conception for at least 3 years following therapy discontinuation.

1	Dr. Edward Lammer.) and after Ireatment, this program promotes a delay of conception for at least 3 years following therapy discontinuation. This retinoid is used to treat cutaneous T-cell lymphoma. When given to rats in doses comparable to those for human therapy, fetuses developed eye and ear abnormalities, cleft palate, and incomplete ossification. For a woman to receive this medication, the manufacturer requires two forms of contraception that are initiated 1 month before therapy and are continued for 1 month after bexarotene discontinuation. This is coupled with monthly pregnancy testing during treatment (Valeant Pharmaceuticals, 2015). Males who have partners who could become pregnant are advised to use condoms during sexual intercourse while taking bexarotene and for 1 month after discontinuing therapy.

1	These compounds, initially used to treat acne, have become so popular for the treatment of sun damage that they are called cosmeceuticals (Panchaud, 2012). The most commonly used topical agents are tretinoin, isotretinoin, and adapalene. Systemic absorption is low, and this argues against plausible teratogenicity.

1	Isolated case reports have described malformations following topical tretinoin, and it is unknown whether this is due to variability in absorption or perhaps potential individual susceptibility (Kaplan, 2015). A prospective study by the European Network of Teratology Information Services found no higher rates of birth defects or spontaneous losses, and no case of retinoid embryopathy (Panchaud, 2012). One systematic review by the Motherisk Program included 635 pregnancies with exposure to topical retinoids. Investigators similarly identified no higher risk for congenital malformations, spontaneous abortion, stillbirth, low birthweight, or preterm delivery cranial neural-crest defects when more than 10,000 IU per day is consumed in the first trimester (Rothman, 1995). It seems reasonable to avoid doses of preformed preparations that exceed the recommended 3000 IU daily allowance (American Academy of Pediatrics, 2017).

1	Possibly the most notorious human teratogen, thalidomide causes malformations in 20 percent of fetuses exposed between 34 and 50 days menstrual age. The characteristic malformation is phocomelia-an absence of one or more long bones. As a result, hands or feet are attached to the trunk, occasionally by a small rudimentary bone. Cardiac malformations, gastrointestinal abnormalities, external ear malformations, eye anomalies, and other limb-reduction defects are also common following thalidomide exposure. The manufacturer reports that up to 40 percent of afected newborns do not survive the neonatal period (Celgene, 2017 a).

1	Thalidomide was marketed outside the United States from 1956 to 1960, before its teratogenicity was appreciated. The ensuing disaster, with thousands of afected children, was instructive of several important teratological principles. First, the placenta is not an efective barrier to the transfer of toxic substances from mother to embryo (Dally, 1998). Second, diferent species show extreme variability in their susceptibility to drugs and chemicals. Namely, thalidomide produced no defects in multiple rodent species and was assumed to be safe for humans. Last, exposure timing and defect type are often closely related (Vargesson, 2015). For example, upper-limb amelia may develop with thalidomide exposure during days 24 to 30 postconception, upper-limb phocomelia with exposure during days 24 to 33, and lower-limb phocomelia with exposure during days 27 to 33.

1	halidomide was irst approved in the United States in 1999 and currently is used to treat erythema leprosum nodosum and multiple myeloma (Celgene, 2017a). The FDA has mandated a web-based, restricted-distribu tion program for thalidomide, called THALOMID REMS, which is required before patients, physicians, and pharmacies can access the medication. Lenalidomide is an analogue of thalidomide that is used to treat some types of myelodys myeloma. It crosses the placenta in multiple animal species, and gene, 2017b). Because of obvi ous teratogenicity concerns, a similar to that used for thalido mide has been developed.

1	his anticoagulant is a vitamin K antagonist with a long halflife. Because of its low molecular weight, it readily crosses the placenta and may cause embryotoxic and fetotoxic efects. Warfarin analogues, such as Coumadin, are considered contraindicated in pregnancy. An exception, as discussed in Chapter 49 (p. 954), is treatment of women with mechanical heart valves who are at high risk for thromboembolism (Bristol-Myers Squibb,i2017a).

1	Waarin embryopathy is characterized by stippled epiphyses and nasal hypoplasia (Fig. 12-6). In one review of 63 cases attributed to warfarin exposure, 80 percent displayed characteristic indings, which include depressed nasal bridge with nasal hypoplasia and choanal atresia, along with stippled epiphyses of the femur, humerus, calcanei, and distal phalanges (Van Driel, 2002). It may result from exposure between the 6th and 9th weeks' gestation (Hall, 1980). he prevalence of the warfarin embryopathy following exposure during this critical period is estimated to be 6 percent (van Driel, 2002). One metaanalysis of cases in which the warfarin dosage was ; 5 mg/ d identiied embryopathy in 1 percent of exposed fetuses. his suggests that risk may be dose dependent (Hassouna, 2014).

1	If used beyond the first trimester, warfarin may lead to hemorrhage into fetal structures, which can cause abnormal growth and deformation from scarring (Warkany, 1976). Nearly 50 percent of reported embryopathy cases also have CNS anomalies (van Driel, 2002). Abnormalities can include agenesis of the corpus callosum; cerebellar vermian agenesis, which is the Dandy-Walker malformation; microphthalmia; and optic atrophy (Hall, 1980). Afected infants are also at risk for blindness, deafness, and developmental delays. With various herbal remedies, associated risks are more challenging to estimate because studies are few and because these compounds are not FDA-regulated. he European Committee of Herbal Medicinal Products provides assessment reports and Teratology, Teratogens, and Fetotoxic Agents 247 FIGURE 12-6 Warfarin embryopathy or fetal warfarin syndrome: nasal hypoplasia and depressed nasal bridge seen in a fetal sonographic image (A) and in the same newborn (8).

1	FIGURE 12-6 Warfarin embryopathy or fetal warfarin syndrome: nasal hypoplasia and depressed nasal bridge seen in a fetal sonographic image (A) and in the same newborn (8). monographs on selected herbal substances and preparations, but safety data are generally limited (Wiesner, 2017). Animal studies have not been conducted, and thus knowledge of complications often derives from reports of acute toxicity (Hepner, 2002; Sheehan, 1998). Further, the identity, quantity, and purity of each ingredient are usually unknown. Given these uncertainties, it seems prudent to counsel pregnant women to avoid these substances. A list of selected herbal compounds and their potential efects is shown in Table 12-5.

1	Not uncommonly, fetuses are exposed to one or more illicit drugs. Assessment of outcomes attributable to these drugs may be confounded by factors such as poor maternal health, malnutrition, infectious disease, and polysubstance abuse. Moreover, illegal substances may contain toxic contaminants such as lead, cyanide, herbicides, and pesticides. Impurities added as diluents may independently have serious adverse perinatal efects. As noted on page 239, alcohol is a significant teratogen. Because it is legally obtained and ubiquitous, its use also confounds the study of illicit drug teratogenicity.

1	hese sympathomimetic amines are not considered to be major teratogens. Methamphetamine enhances dopamine release and blocks its reuptake. It is prescribed to treat attention-deicit/ hyperactivity disorder and narcolepsy. Methamphetamine abuse has been rising in the United States since the late 1980s (American College of Obstetricians and Gynecologists, 2017b). In utero exposure has been consistently associated with higher rates of small-for-gestational age newborns (Gorman, 2014; Smith, 2006). Hypertensive complications, placental abruption, preterm birth, and stillbirth are other associated complications (Gorman, 2014). Behavioral abnormalities have been described in both infants and school-aged children (Eze, 2016). TABLE 12-5. Pharmacological Actions and Adverse Effects of Some Herbal Medicines Echinacea: purple coneflower root Ephedra: ma huang Garlic: ajo Kava: awa, intoxicating pepper, kawa Valerian: all heal, garden heliotrope, vandal root Yohimbe

1	Echinacea: purple coneflower root Ephedra: ma huang Garlic: ajo Kava: awa, intoxicating pepper, kawa Valerian: all heal, garden heliotrope, vandal root Yohimbe Contains linoleic acids, a prostaglandin precursor Cyclooxygenase inhibitor, thromboxane synthetase inhibitor Lowers blood glucose; inhibition of platelet aggregation Sedation, anxiolysis Data from Ang-Lee, 2001; Briggs, 2015; Hall, 2012; Wiesner, 2017.

1	Lowers blood glucose; inhibition of platelet aggregation Sedation, anxiolysis Data from Ang-Lee, 2001; Briggs, 2015; Hall, 2012; Wiesner, 2017. With this CNS stimulant, most adverse outcomes result from its vasoconstrictive and hypertensive efects. Serious potential maternal complications are cerebrovascular hemorrhage, myocardial damage, and placental abruption. Studies of congenital abnormalities and cocaine exposure have yielded conflicting results, but associations with cleft palate, cardiovascular abnormalities, and urinary tract anomalies have been reported (Chasnof, 1988; Lipshultz, 1991; van Gelder, 2009). Cocaine use is also associated with fetal-growth restriction and preterm delivery. Children exposed as fetuses have risks for behavioral abnormalities and cognitive impairments (Bada, 2011; Gouin, 2011).

1	The dramatic rise in narcotic use among non-pregnant and pregnant individuals has been aptly termed an epidemic. Opioids are not considered to be major teratogens. he NBDPS did identiY a slightly greater risk for spina biida, gastroschisis, and cardiac abnormalities with periconceptional opioid exposure (Broussard, 2011). The American College of Obstetricians Allergic reactions; decreases immunosuppressant efectiveness; possible immunosuppression with long-term use arrhythmias, myocardial ischemia, stroke; depletes endogenous catecholamines; lifethreatening interaction with monoamine oxidase inhibitors Possible complications if used for labor induction Risk of bleeding, especially when combined with other platelet aggregation inhibitors Increased risk of bleeding Risk of bleeding; interferes with monoamine oxidase inhibitors Hypoglycemia; hypertension; risk of bleeding

1	Increased risk of bleeding Risk of bleeding; interferes with monoamine oxidase inhibitors Hypoglycemia; hypertension; risk of bleeding Sedation; hepatotoxicity, benzodiazepine-like acute withdrawal Hypertension, arrhythmias and Gynecologists (2017 c) stresses that this potential, small increase in birth defects with maintenance therapy should be weighed against the risks associated with uncontrolled opioid abuse. Heroin addiction is associated with adverse pregnancy outcomes from the efects of repeated narcotic withdrawal on the fetus and placenta (American College of Obstetricians and Gynecologists, 2017 c). These include preterm birth, placental abruption, fetal-growth restriction, and fetal death.

1	Neonatal narcotic withdrawal, called the neonatal abstinence syndrome, may manifest in 40 to 90 percent of exposed newborns (Blinick, 1973; Creanga, 2012; Dashe, 2002; Zelson, 1973). As discussed in Chapter 33 (p. 625), CNS irritability may progress to seizures if untreated and may be accompanied by tachypnea, apneic episodes, poor feeding, and failure to thrive. At-risk neonates are closely monitored using a scoring system, and those severely afected are treated with opioids (Finnegan, 1975). he proportion of exposed newborns developing neonatal abstinence syndrome has risen significantly in recent years (Creanga, 2012; Lind, 2015).

1	he American College of Obstetricians and Gynecologists (2017 c) recommends that pregnant women with opioid-use disorder be maintained on opioid-agonist therapy to reduce the risks associated with illicit opioid abuse and associated behaviors. Treatment includes either methadone, usually through a licensed outpatient opioid treatment program, or buprenorphine, which may be given in an oice-based setting by a licensed buprenorphine prescriber. A multidisciplinary treatment program is recommended to reduce the likelihood of additional opioid abuse while on maintenance therapy. he College (2017 c) discourages withdrawal from methadone during pregnancy because of high relapse rates. At Parkland Hospital, pregnant opioid users who decline maintenance therapy are ofered inpatient hospitalization for controlled methadone taper, with the goal of reducing the likelihood of neonatal abstinence syndrome (Dashe, 2002; Stewart, 2013).

1	This is the illicit drug most commonly used in pregnancy (American College of Obstetricians and Gynecologists, 2017a). Based on data from the National Survey on Drug Use and Health, the prevalence of marijuana use in pregnancy was nearly 4 percent in 2014 (Brown, 2017). Cannabinoids are not considered to be major teratogens, but there is concern because endogenous cannabinoids play key roles in human brain development. In one metaanalysis of nearly 8000 exposed pregnancies, adverse outcomes such as preterm birth and low birthweight were increased only in the presence of concomitant tobacco use (Conner, 2016).

1	Phencyclidine (PCP) or angel dust is not associated with congenital anomalies. More than half of exposed newborns, however, experience withdrawal symptoms characterized by tremors, jitteriness, and irritability. Toluene is a common solvent used in paints and glue. Occupational exposure is reported to have signiicant fetal risks (Wilkins-Haug, 1997). When abused by women in early pregnancy, it is associated with toluene embyopathy, which is phenotypically similar to fetal alcohol syndrome. Abnormalities include pre-and postnatal growth deficiency, microcephaly, midface hypoplasia, short palpebral fissures, and wide nasal bridge (Pearson, 1994) . Up to 40 percent of exposed children have developmental delays (Arnold, 1994).

1	Cigarette smoke contains a complex mixture of nicotine, cotinine, cyanide, thiocyanate, carbon monoxide, cadmium, lead, and various hydrocarbons (Stillerman, 2008). In addition to being fetotoxic, many of these substances have vasoactive efects or reduce oxygen levels. Tobacco is not considered a major teratogen, although selected birth defects have been reported to occur with greater frequency among newborns of women who smoke. It is plausible that the vasoactive properties of tobacco smoke could produce congenital defects related to vascular disturbances. For example, the prevalence of Poland sequence, which is caused by an interruption in the vascular supply to one side of the fetal chest and ipsilateral arm, is twofold greater in smokers (Martinez-Frias, 1999). A small increased risk for cardiac anomalies has also been reported and may be dose related (Alverson, 2011; Malik, 2008; Sullivan,

1	Teratology, Teratogens, and Fetotoxic Agents 249 2015). One study analyzing more than 6 million births found an association between maternal smoking and hydrocephaly, microcephaly, omphalocele, gastroschisis, cleft lip and palate, and hand abnormalities (Honein, 2001). Electronic nicotine delivery systems are not considered safe, as nicotine may have adverse efects on fetal brain and lung development (American College of 0bstetricians and Gynecologists, 2017 d).

1	he best-documented adverse reproductive outcome from smoking is a dose-response reduction in fetal growth. Newborns of mothers who smoke weigh on average 200 g less than newborns of nonsmokers (D'Souza, 1981). Smoking doubles the risk of low birthweight and raises the risk of fetal-growth restriction two-to threefold (Werler, 1997). Even secondhand smoke increases the risk for low birthweight (Hegaard, 2006). Women who stop smoking early in pregnancy may have neonates with normal birthweights (Cliver, 1995). Other adverse outcomes associated with cigarette smoking include preterm birth, placenta previa, placenta abruption, spontaneous abortion, and sudden infant death syndrome (American College of Obstetricians and Gynecologists, 2017d). Risks of childhood asthma and obesity are also increased. Abel EL, Hannigan ]H: Maternal risk factors in fetal alcohol syndrome: provocative and permissive inluences. Neurotoxicol Teratolo17(4):445, 1995

1	Abel EL, Hannigan ]H: Maternal risk factors in fetal alcohol syndrome: provocative and permissive inluences. Neurotoxicol Teratolo17(4):445, 1995 Actelion Pharmaceuticals: Opsumit (Macitentan) prescribing information, 2017. Available at: http://www.opsumit.com/opsumit-prescribing-information. pd. Accessed September 24, 2017 Actelion Pharmaceuticals: T racleer (Bosentan) prescribing information, 2016. Available at: www.tracleer.com/assets/PD Rs/T racleer_Full_Prescribin� Information. pdf. Accessed September 24, 2017 Adam MP, Polifka ]E, Friedman ]M: Evolving knowledge of the teratogenicity of medications in human pregnancy. Am] Med Genet C Semin Med Genet 157(3):175,o2011 Ailes EC, Gilboa SM, Gill SK, et al: Association between antibiotic use among pregnant women and urinary tract infections in the irst trimester and birth defects, National Birth Defects Prevention Study 1997 to 2011. Birth Defects Res A Clin Mol Teratol 106(1o1):940, 2016

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1	May PA, Baete A, Russo J, et al: Prevalence and characteristics of fetal alcohol spectrum disorders. Pediatrics 134(5):855,o2014 May PA, Gossage JP, Kalberg WO, et al: Prevalence and epidemiologic characteristics ofFASD from various research methods with an emphasis on recent in-school studies. Dev Disabil Res Rev 15(3): 176, 2009 McKeigue PM, Lamm SH, Linn S, et al: Bendectin and birth defects: 1. A meta-analysis of the epidemiologic studies. Teratology 50(1):27, 1994 Meador KJ, Baker GA, Browning N, et al: Cognitive function at 3 years of age ater fetal exposure to antiepileptic drugs. N EnglJ Med 360(16):1597,o2009 Merlob P, Stahl B, Klinger G: Tetrada of the possible mycophenolate mofetil embryopathy: a review. Reprod ToxicoIo28(1):105, 2009 Mitchell A, Gilboa SM, Werler MM, et al: Medication use during pregnancy, with particular focus on prescription drugs: 1976-2008. Am J Obstet GynecoIo205(1):51,e1,2011

1	Mitchell A, Gilboa SM, Werler MM, et al: Medication use during pregnancy, with particular focus on prescription drugs: 1976-2008. Am J Obstet GynecoIo205(1):51,e1,2011 M0lgaard-Nielsen D, Hviid A: Newer-generation antiepileptic drugs and the risk of major birth defects. JAMA 305(19):1996,o2011 M01gaard-Nielsen D, Pasternak B, Hviid A: Use of oral fluconazole during pregnancy and the risk of birth defects. N Engl J Med 369(9):830, 2013 Munsie JW, Lin S, Browne ML, et al: Maternal bronchodilator use and the risk of oral clefts. Hum Reprod 26(11):3147,o2011 Nurmohamed L, Moretti ME, Schechter T, et al: Outcome following highdose methotrexate in pregnancies misdiagnosed as ectopic. Am J Obstet GynecoIo205(6):533.e1,o2011 Oakley GP, Erickson JD: Vitamin A and birth defects. N Engl J Med 333(21):1414, 1995 Ornoy A, Koren G: Selective serotonin reuptake inhibitors during pregnancy: do we have now more definite answers related to prenatal exposure? Birth Defects Res 109(12):898:2017

1	Ornoy A, Koren G: Selective serotonin reuptake inhibitors during pregnancy: do we have now more definite answers related to prenatal exposure? Birth Defects Res 109(12):898:2017 Paintner A, Williams AD, Burd L: Fetal alcohol spectrum disorders-implications for child neurology, Part 2: diagnosis and management. J Child NeuroIo27(3):355,o2012 Panchaud A, Csajka C, Merlob P, et al: Pregnancy outcome following exposure to topical retinoids: a multicenter prospective study. J Clin Pharmacol 52(12):1844,o2012 Park-Wyllie L, Mazzota P, Pastuszak A, et al: Birth defects after maternal exposure to corticosteroids: prospective cohort study and meta-analysis of epidemiological studies. Teratology 62(6):385, 2000 Patorno E, Huybrechts KF, Bateman BT, et al: Lithium use in pregnancy and the risk of cardiac malformations. N Engl J Med 376(23):2245, 2017

1	Patorno E, Huybrechts KF, Bateman BT, et al: Lithium use in pregnancy and the risk of cardiac malformations. N Engl J Med 376(23):2245, 2017 Pearson MA, Hoyme HE, Seaver LH, et al: Toluene embryopathy: delineation of the phenotype and comparison with fetal alcohol syndrome. Pediatrics 93(2):211, 1994 Pryde PG, Sedman AB, Nugent CE, et al: Angiotensin converting enzyme inhibitor fetopathy. J Am Soc NephroIo3(9):1575, 1993 Rasanen J, Jouppila P: Fetal cardiac function and ductus arteriosus during indomethacin and sulindac therapy for threatened preterm labor: a randomized study. Am J Obstet Gynecol 173(1):20, 1995 Reefhuis J, Gilboa SM, Anderka M, et al: The National Birth Defects Prevention Study: a review of the methods. Birth Defects Res A Clin Mol Teratol 103(8):656,o2015

1	Reefhuis J, Gilboa SM, Anderka M, et al: The National Birth Defects Prevention Study: a review of the methods. Birth Defects Res A Clin Mol Teratol 103(8):656,o2015 Richardson S, Browne ML, Rasmussen SA, et al: Associations between periconceptional alcohol consumption and craniosynostosis, omphalocele, and gastroschisis. Birth Defects Res A Clin Mol Teratol 91 (7):623,2011 Rocheleau CM, Bertke SJ, Lawson CC, et al: Maternal occupational pesticide exposure and risk of congenital heart defects in the National Birth Defects Prevention Study. Birth Defects Res A Clin Mol Teratolo103(1 0):823,2015 Rothman KJ, Moore LL, Singer MR, et al: Teratogenicity of high vitamin A intake. N Engl J Med 333(21):1369, 1995 Sadler W (ed): Langman's Medical Embryology, 6th ed. Baltimore, Williams & Wilkins, 1990, p 130 Salle B, Sergeant P, Awada A, et al: Transvaginal ultrasound studies of vascular and morphological changes in uteri exposed to diethylstilbestrol in utero. Hum Reprod 11(11):2531, 1996

1	Salle B, Sergeant P, Awada A, et al: Transvaginal ultrasound studies of vascular and morphological changes in uteri exposed to diethylstilbestrol in utero. Hum Reprod 11(11):2531, 1996 Sanofi-Aventis: Arava (Lelunomide) prescribing information, 20o16. Available at: http://products.sanoi.us/arava/arava.htmll. Accessed September 24, 2017 Schardein JL: Congenital abnormalities and hormones during pregnancy: a clinical review. Teratology 22(3):251,o1980 Sebela Pharmaceuticals: Pexeva (Paroxetine) prescribing information, 2017. Available at: ww.pexeva.com/pdf/Pexeva_20140728_ver .pd. Accessed September 24, 2017 Sheehan DM: Herbal medicines, phytoestrogens and toxicity: risk:beneit considerations. Proc Soc Exp BioI Med 21 (3):379, 1998 Shepard TH: Annual commentary on human teratogens. Teratology 66(6):275, 2002a Shepard TH: Letters: "proof' of human teratogenicity. Teratology 50(2):97, 1994

1	Shepard TH: Annual commentary on human teratogens. Teratology 66(6):275, 2002a Shepard TH: Letters: "proof' of human teratogenicity. Teratology 50(2):97, 1994 Shepard TH, Brent L, Friedman JM, et al: Update on new developments in the study of human teratogens. Teratology 65(4): 153, 2002b Smith LM, LaGasse LL, Derauf C, et al: he infant development, environment, and lifestyle study: efects of prenatal methamphetamine exposure, polydrug exposure, and poverty on intrauterine growth. Pediatrics 118(3): 1149,2006 Stewart D, Nelson DB, Adhikari EH, et al: he obstetrical and neonatal impact of maternal opioid detoxification in pregnancy. Am J Obstet Gynecol 209(3):267, 2013 Stiefel Laboratories Soriatane (acitretin) prescribing information. 2015. Available at: https:/ Iwww.gsksource.com/pharma/ con tentl dam I G laxoSmi thKline/USI en/P rescribin�Informationl Soriatanel pdfl SO RIA T ANEPI-MG. PDF. Accessed September 24,o2017

1	Available at: https:/ Iwww.gsksource.com/pharma/ con tentl dam I G laxoSmi thKline/USI en/P rescribin�Informationl Soriatanel pdfl SO RIA T ANEPI-MG. PDF. Accessed September 24,o2017 Stillerman KP, Mattison DR, Giudice LC, et al: Environmental exposures and adverse pregnancy outcomes: a review of the science. Reprod Sci 15(7):631, 2008 Stillman RJ: In utero exposure to diethylstilbestrol: adverse efects on the reproductive tract and reproductive performance in male and female ofspring. Am J Obstet Gynecol 142(7):905, 1982 Stingone JA, Luben TJ, Carmichael SL, et al: Maternal exposure to nitrogen dioxide, intake of methyl nutrients and congenital heart defects in ofspring. AmoJ EpidemioIo186(6):719, 2017 Stothard KJ, Tennant PWG, Bell R, et al: Maternal overweight and obesity and the risk of congenital anomalies: a systematic review and meta-analysis. JAMA 301(6):636,o2009

1	Strandberg-Larsen K, Nielsen NR, Gf0nbaek M, et al: Binge drinking in pregnancy and risk of fetal death. Obstet Gynecol III(3):602, 2008 Streissguth AP, Clarren SK, Jones L: Natural history of fetal alcohol syndrome: a 10-year follow-up of eleven patients. Lancet 2:85, 1985 Sullivan PM, Dervan A, Reiger S, et al: Risk of congenital heart defects in the ofspring of smoking mothers: a population-based study. J Pediatr 166(4):978,o2015 Teratology Society Public Afairs Committee: Causation in teratology-related litigation. Birth Def Res A Clin Mol Teratol 3(6):421,o2005 Vajda FJ, O'Brien TJ, Lander CM, et al: Antiepileptic drug combinations not involving valproate and the risk of fetal malformations. Epilepsia 57(7):1048,o2016

1	Vajda FJ, O'Brien TJ, Lander CM, et al: Antiepileptic drug combinations not involving valproate and the risk of fetal malformations. Epilepsia 57(7):1048,o2016 Valeant Pharmaceuticals: Tagretin (Bexarotene) prescribing information, 2015. Available at: http://ww.valeant.com/Portals/25/PDF/TargretinCapsulesP1.pd?ver=2016-05-1o1-044521-020. Accessed September 24,o2017 van der Heijden BJ, Carlus C, Narcy F, et al: Persistent anuria, neonatal death, and renal microcystic lesions after prenatal exposure to indomethacin. Am J Obstet GynecoIo171(3):617, 1994 Van Driel D, Wesseling J, Sauer PJ, et al: Teratogen update: fetal efects after in utero exposure to coumarins overview of cases, follow-up findings, and pathogenesis. Teratology 66(3):127,o2002 Van Gelder MM, Reehuis J, Caton R, et al: Maternal periconceptional illicit drug use and the risk of congenital malformations. Epidemiology 20:60, 2009 Vargesson N: halidomide-induced teratogenesis: history and mechanisms.

1	Birth Defects Res C Embryo Today 105(2):140, 2015 Vessey MP: Epidemiological studies of the efects of diethylstilbestrol. IARC Sci Pub1o335, 1989 Walker MP, Moore TR, Brace A: Indomethacin and arginine vasopressin interaction in the fetal kidney. A mechanism of oliguria. Am J Obstet Gynecol 171(5):1234, 1994 Waller DK, Shaw GM, Rasmussen SA, et al: Prepregnancy obesity as risk factor for structural birth defects. Arch Pediatr Adolesc Med 161(8):745,2007 Warkany J: Warfarin embryopathy. Teratology 14(2):205, 1976 Weiss CF, Glazko AJ, Weston JK: Chloramphenicol in the newborn infant: a physiologic explanation of its toxicity when given in excessive doses. N Engl J Med 262:787, 1960 Werler MM: Teratogen update: smoking and reproductive outcomes. Teratology 55(6):382, 1997 Weston J, Bromley R, Jackson CF, et al: Monotherapy treatment of epilepsy in pregnancy: congenital malformation outcomes in the child. Cochrane Database Syst Rev 11 :CDO 1 0224, 2016

1	Weston J, Bromley R, Jackson CF, et al: Monotherapy treatment of epilepsy in pregnancy: congenital malformation outcomes in the child. Cochrane Database Syst Rev 11 :CDO 1 0224, 2016 West-Ward Pharmaceuticals: Lithium prescribing information, 20o16. Available at: https:/ Idailymed.nlm.nih.gov/dailymed/fda/fdaDruXsl.cfm?setid =a226a88d-eb5 7 -4c96-afda-93980 1 bcaOa9&type=display. Accessed September 24, 2017 Wiesner J, Knoss W: Herbal medicinal products in pregnancy-which data are available? Reprod Toxicol 72:142, 201 Wilkins-Haug L: Teratogen update: toluene. Teratology 55(2):145, 1997 Williams JF, Smith VC, American Academy of Pediatrics Committee on Substance Abuse: Fetal alcohol spectrum disorders. Pediatrics 136(5):e1395, 2015 Yacobi S, Ornoy A: Is lithium a real teratogen? What can we conclude from the prospective versus retrospective studies? A review. Isr J Psychiatry Relat Sci 45(2):95, 2008

1	Yacobi S, Ornoy A: Is lithium a real teratogen? What can we conclude from the prospective versus retrospective studies? A review. Isr J Psychiatry Relat Sci 45(2):95, 2008 Zelson C, Lee SJ, Casalino M: Neonatal narcotic addiction: comparative efects of maternal intake of heroin and methadone. N Engl J Med 289(23):1216, 1973 GENOMICS IN OBSTETRICSe. . . . . . . . . . . . . . . . . . . . . . . 253 ABNORMALITIES OF CHROMOSOME NUMBER .e.e. .e. 254 ABNORMALITIES OF CHROMOSOME STRUCTURE .... 260 CHROMOSOMAL MOSAICISM .. .e. .e.... .e.. ... . . 263 MODES OF INHERITANCE .. .e... ..e...e. ..e.e. .e. ..e. 264 CYTOGENETIC ANALYSIS . . . . . . . . . . . . . . . . . . . . . . . . 270 CHROMOSOMAL MICROARRAY ANALYSIS . .e.. .e. .e... 271 WHOLE GENOME AND WHOLE EXOME SEQUENCINGe.. 272 FETAL DNA IN THE MATERNAL CIRCULATION ...e. .e. 273

1	CHROMOSOMAL MICROARRAY ANALYSIS . .e.. .e. .e... 271 WHOLE GENOME AND WHOLE EXOME SEQUENCINGe.. 272 FETAL DNA IN THE MATERNAL CIRCULATION ...e. .e. 273 Foetal death may be due to abnormalities in the ovum itsef or due to some disease on the part of the mother, and now and again of the ather. The death of the oetus is frequenty due to abnormalities in the development of the embyo which are inconsistent with oetal le. -J. Whitridge Williams (1903) In Williams' irst edition of Obstetrics, he rarely referenced inherited conditions that Gregor Mendel had described 50 years earlier. Fast-forward to 2017, when the science of genetics is a major obstetrical discipline.

1	Genetics is the study of genes, heredity, and the variation of inherited characteristics. Medical genetics deals with the etiology and pathogenesis of human diseases that are at least partially genetic in origin, along with their prediction and prevention. Thus, it is closely linked to genomics, which is the study of gene function and interaction. In addition to chromosomal, mendelian, and nonmendelian genetic conditions reviewed in this chapter, medical genetics includes prenatal and preimplantation genetic diagnosis, as well as newborn genetic screening, which are discussed in Chapters 14 and 32, respectively.

1	Genetic disease is common. Between 2 and 3 percent of newborns have a recognized structural defect. In another 3 percent of individuals, a defect is diagnosed by age 5, and another 8 to 10 percent of persons are discovered by age 18 to have one or more functional or developmental abnormalities. Advances in genomics are used increasingly to provide information regarding susceptibility to genetic diseases, and every indication suggests that this ield will reshape prenatal diagnosis.

1	Completed in 2003, the Human Genome Project identiied more than 25,000 human genes and led to rapid expansion of genomic research to better understand disease biology (McKusick, 2003). More than 99 percent of our DNA is identical. However, genetic code varies every 200 to 500 base pairs, usually as a single-nucleotide polymorphism. The human genome contains more than 80 million such genetic variants, and understanding their potential role in disease requires not only sophisticated interpretation but also integration of resources (Rehm, 2015).

1	The National Center for Biotechnology Information (NCB!) maintains genetic and genomic databases that are freely accessible to clinicians and researchers. Several of these databases are particularly useful in obstetrics and maternal-fetal medicine practice. he GeneReviews database provides in-depth clinical information for nearly 700 genetic conditions, including diagnostic criteria, management, and genetic counseling considerations (National Center for Biotechnology Information, 2017a). The Genetic Testing Registry (GTR) database contains information regarding the beneits and limitations of available tests for a given disorder. It lists more than 48,000 genetic tests and instructions for specimen collection and transport to individual laboratories throughout the world (National Center for Biotechnology Information, 2017b). Another database, Online Mendelian Inheritance in Man (OMIM), is a comprehensive catalog of human genes and phenotypes that allows clinicians to search for

1	for Biotechnology Information, 2017b). Another database, Online Mendelian Inheritance in Man (OMIM), is a comprehensive catalog of human genes and phenotypes that allows clinicians to search for syndromes based on particular traits or abnormalities. As of early 2017, OMIM included more than 15,000 genes and nearly 5000 mendelian and mitochondrial conditions with a known molecular basis Oohns Hopkins University, 2017). The National Library of Medicine (2017) has also established a database of genetic information intended for patients-one that trainees may find especially helpful-the Genetics Home Reerence (GHR). his database contains data on more than 2400 genetic conditions and genes, including resources for families.

1	Chromosomal abnormalities igure prominently in genetic disease. Aneuploidy accounts for more than 50 percent of irst-trimester miscarriages, approximately 20 percent of second-trimester losses, and 6 to 8 percent of stillbirths and early-childhood deaths (Reddy, 2012; Stevenson, 2004; Wou, 2016). In the European Surveillance of Congenital Anomalies (EUROCAT) network of population-based registries, chromosomal abnormalities were identiied in 0.4 percent of births (Wellesley, 2012). Of recognized pregnancies with aneuploidy, trisomy 21 composes just more than half of all cases. Trisomy 18 accounts for almost 15 percent, and trisomy 13 for 5 percent (Fig. Karyotypes are described using the International System for Human Cytogenomic Nomenclature (McGowan-Jordan, Trisomy 21 (23:10,000) 0 45,X (3:10,000) Trisomy 18 (6: 1 0,000) o 47,XXX; 47,XXY; 47,XYY (2:1e0,000) o Trisomy 13 (2:10,000) o Other (23: 10,000)

1	Trisomy 21 (23:10,000) 0 45,X (3:10,000) Trisomy 18 (6: 1 0,000) o 47,XXX; 47,XXY; 47,XYY (2:1e0,000) o Trisomy 13 (2:10,000) o Other (23: 10,000) FIGURE 13-1 Prevalence and relative proportion of selected chromosomal abnormalities from EUROCAT (European Surveillance of Congenital Anomalies) population-based registries that included > 10,000 aneuploid live births, fetal deaths, and pregnancy terminations, 2000-2006. (Data from Wellesley, 2012.) 2016). Abnormalities fall into two broad categories-those of chromosome number, such as trisom)i, and those of chromosome structure, such as a deletion or translocation. Each chromosome has a short arm, termed the "p" or petit arm, and a long arm, known as the "q" arm, selected because it is the next letter in the alphabet. The two arms are separated by the centromere.

1	When reporting a karyotype, the total number of chromosomes is listed irst, corresponding to the number of centro meres. This is followed by the sex chromosomes, X or Y, and then by a description of any structural variation. Speciic abnormalities are indicated by standard abbreviations, such as del (deletion) and inv (inversion). he afected region or bands of the p or q arms are then designated, so that the reader will know the exact abnormality location and type. Examples are shown in Table

1	Terminology is similar for fluorescence in situ hybridization. Described on page 270, this technique is used to rapidly identiy of a specific chromosome abnormality and veriy suspected microdeletion or microduplication syndromes. The report begins with the designation ish for in situ hybridization performed on metaphase cells and nuc ish for hybridization performed on interphase nuclei. If no abnormality is identified, this is followed by the probe's specific chromosomal region, such as 22q 11i.2, and then the name of the probe and the number of signals visualized-for example, HIRAx2. If a deletion is identified, del is included before the chromosomal region, and the name of the probe is followed by a minus sign (HIRA-), as shown in Table 13-1. The 22q 11.2 microdeletion syndrome is discussed on page 260.

1	A recent addition to the standard nomenclature is terminology to represent copy number variants identiied by chromosomal microarray anaysis, which is discussed on page 271. Copy number variant is another term for a microdeletion or microduplication of DNA too small to be visualized with a standard karyotype. The array designation begins with the abbreviation arr and the version of the genome build to which the nucleotide designations are aligned, such as GRCh38 for Genome Reference Consortium human build 38. his is followed by the number of the chromosome on which the abnormality is identified, by the p or q arm, and by the speciic bands in question. Array reports next include the afected base pair coordinates, thus conveying the exact size and location within the genome for every abnormality identiied-including copy number variants of uncertain signiicance . • Abnormalities of Chromosome Number

1	• Abnormalities of Chromosome Number The most easily recognized chromosomal abnormalities are numerical. Aneuploidy is inheritance of either an extra chromosome-resulting in trisomy, or loss of a chromosomemonosomy. These difer from poyploidy, which is an abnormal number of haploid chromosome sets, such as triploidy. The estimated incidence of various numerical chromosomal abnormalities is shown in Figure 13-1. These account for approximately half of all chromosomal abnormalities. In most cases, trisomy results from nondisjunction,

1	TABLE 13-1. Examples of Karyotype Designations Using the 2016 International System for Human Cytogenetic Nomenclature 46,XX Normal female chromosome constitution 47,XY,+21 Male with trisomy 21 47,XX,+21/46,XX Female who is a mosaic of trisomy 21 cells and cells with normal constitution 46,XY,del(4)(p 14) Male with terminal deletion (del) of the short arm of chromosome 4 at band p 14 46,XX,dup(5)(p 14p 15.3) Female with duplication (dup) of the short arm of chromosome 5 from band p14 to band p15.3 45,XY,derCi 3;14)(q 1 O;q 10) Male with balanced robertsonian translocation (der) of the long arms of chromosomes 13 and 14-the karyotype now has one normal 13, one normal 14, and the translocation chromosome. reducing the normal 46 chromosome complement to 45 46,XX,t(11 ;22)(q23;q 11l.2) Female with a balanced reciprocal translocation (t) between chromosomes 11 and 22, with breakpoints at 11 q23 and 22q 11l.2 46,XY,inv(3)(p21 q 13) Male with inversion (inv) of chromosome 3 that extends from

1	with a balanced reciprocal translocation (t) between chromosomes 11 and 22, with breakpoints at 11 q23 and 22q 11l.2 46,XY,inv(3)(p21 q 13) Male with inversion (inv) of chromosome 3 that extends from p21 to q 13-a pericentric inversion because it includes the centromere 46,X,r(X)(p22.1 q27) Female with one normal X and one ring (r) X chromosome, with the regions distal to p22.1 and q27 deleted from the ring 46,X,i(X)(q 10) Female with one normal X chromosome and an isochromosome (i) of the long arm of the ish 22q11l.2(HIRAx2) FISH of metaphase cells using a probe for the HIRA locus of the 22q 11l.2 region, with 2 signals identified (no evidence of microdeletion) ish del(22)(q 11.2q 11l.2) (HIRA-) FISH of metaphase cells using a probe for the HIRA locus of the 22q11l.2 region, with only one signal identified, consistent with the microdeletion arr[GRCh38] 18p 11.32q23 Microarray analysis (arr), genome build GRCh38, showing a single copy gain on (102328_79093443)x3 chromosome 18 from

1	one signal identified, consistent with the microdeletion arr[GRCh38] 18p 11.32q23 Microarray analysis (arr), genome build GRCh38, showing a single copy gain on (102328_79093443)x3 chromosome 18 from band p11.32 to band q23 (essentially the entire chromosome), consistent with trisomy 18 arr[GRCh38] 4q32.2q35.1 Microarray analysis (arr), genome build GRCh38, showing a copy loss on the long arm of (163146681_183022312)x1 chromosome 4 at bands q32.2 through q35.1 (19.9 Mb) arr[GRCh38] 15q 11l.2q26 SNP microarray analysis (arr), genome build GRCh 38, showing homozygosity for the entire (23123715_101888908)x2 long arm of chromosome 15 cycle regulator; SNP = single nucleotide pleomorphism.

1	Used with permission from Dr. Kathleen S. Wilson. which is failure of normal chromosomal pairing and separation during meiosis. Nondisjunction may occur if the chromosomes: (1) fail to pair up, (2) pair up properly but separate prematurely, or (3) fail to separate.

1	he risk of any autosomal trisomy rises steeply with maternal age, particularly after age 35 (Fig. 13-2). Oocytes are suspended in midprophase of meiosis I from birth until ovulation, in some cases for 50 years. Following completion of meiosis at ovulation, nondisjunction results in one gamete having two copies of the afected chromosome, leading to trisomy if fertilized. he other gamete, receiving no copy of the afected chromosome, will be monosomic if fertilized. It is estimated that 10 to 20 percent of oocytes are aneuploid secondary to meiotic errors, compared with 3 to 4 percent of sperm. lthough each chromosome pair is equally likely to have a segregation error, programs in the United States, 2006-2010, which included live it is rare for trisomies other than 21, 18, or 13 to result in a births, stillbirths, and pregnancy terminations. (Data from Mai, 2013. term pregnancy, and most fetuses with trisomies 18 and 13 die Redrawn with permission from Dashe JS: Aneuploidy screening.

1	in a births, stillbirths, and pregnancy terminations. (Data from Mai, 2013. term pregnancy, and most fetuses with trisomies 18 and 13 die Redrawn with permission from Dashe JS: Aneuploidy screening. before term. Obstet GynecoIo128(1):181, 2016.) 6 10 11'.13 14 16 19

1	FIGURE 13-3 Abnormal male karyotype with trisomy 21, consistent with Down syndrome (47,XY,+21). (Used with permission from Dr. Frederick Elder.) Mter a pregnancy with an autosomal trisomy, the risk for any autosomal trisomy in a future pregnancy approximates 1 percent until the woman's age-related risk exceeds this. Accordingly, prenatal diagnosis with chorionic villus sampling or amniocentesis is ofered in these subsequent pregnancies (Chap. 14, p. 292). Parental chromosomal studies are not indicated unless the afected pregnancy was caused by an unbalanced translocation or other structural rearrangement.

1	Trisomy 21-Down Syndrome. In 1866, ]. L. H. Down described a group of intellectually disabled children with distinctive physical features. Nearly 100 years later, Lejeune (1959) demonstrated that Down syndrome is caused by an autosomal trisomy (Fig. 13-3). Trisomy 21 causes 95 percent of Down syndrome cases, whereas 3 to 4 percent of cases are due to a robertsonian translocation, described later (p. 262). he remaining 1 to 2 percent results from an isochromosome or from mosaicism. he nondisjunction that yields trisomy 21 occurs during meiosis I in approximately 75 percent of cases, and the remaining events are during meiosis II. Down syndrome is the most common nonlethal trisomy.

1	Down syndrome is the most common nonlethal trisomy. Its approximate prevalence is 1 in 500 recognized pregnancies. However, fetal losses and pregnancy terminations yield an estimated prevalence of 13.5 in 10,000 births in the United States-1 per 740 (Mai, 2013; Parker, 2010). he fetal death rate beyond 20 weeks' gestation approximates 5 percent (Loane, 2013). Coinciding with the older maternal age distribution during the past four decades, the prevalence of Down syndrome has risen approximately 33 percent (Loane, 2013; Parker, 2010; Shin, 2009). Notably, adult women with Down syndrome are fertile, and a third of their ofspring will have Down syndrome (Scharrer, 1975). Males with Down syndrome are almost always sterile because of markedly reduced spermatogenesis.

1	Approximately 30 percent of second-trimester fetuses with Down syndrome have a major malformation that can be identified sonographically (Hussamy, 2017; Vintzileos, 1995). As discussed in Chapter 14 (p. 286), when both major anomalies and minor aneuploidy markers are considered, an estimated 50 to 60 percent of Down syndrome pregnancies can be detected sonographically (American College of Obstetricians and Gynecologists, 2016d). Approximately half of liveborn neonates with Down syndrome are found to have cardiac defects, particularly ventricular septal defects and endocardial cushion defects (Figs. 10-29 and 10-30, p. 202) (Bergstrom, 2016; Freeman, 2008). Gastrointestinal abnormalities are identiied in 12 percent and include esophageal atresia, Hirschsprung disease, and duodenal atresia (Fig. 10-38, p. 207) (Bull, 2011). Characteristic features of Down syndrome are shown in

1	Characteristic features of Down syndrome are shown in Figure 13-4. Typical indings include brachycephaly; epicanthaI folds and up-slanting palpebral issures; Brushield spots, which are grayish spots on the periphery of the iris; a lat nasal bridge; and hypotonia. Infants often have loose skin at the nape of the neck, short ingers, a single palmar crease, hypoplasia of the middle phalanx of the fifth finger, and a prominent space or "sandal-toe gap" between the irst and second toes. Some of these indings are prenatal sonographic markers for Down syndrome, reviewed in Chapter 14 (p. 286). Health problems common in children with Down syndrome include hearing loss in 75 percent, severe optical refractive

1	Health problems common in children with Down syndrome include hearing loss in 75 percent, severe optical refractive FIGURE 13-4 Trisomy 21-Down syndrome. A. Characteristic facial appearance. B. Redundant nuchal tissue. C. Single transverse palmar crease. (Used with permission from Dr. Charles P. Read and Dr. Lewis Waber.) errors in 50 percent, cataracts in 15 percent, obstructive sleep apnea in 60 percent, thyroid disease in 15 percent, and a higher incidence of leukemia (Bull, 2011). The degree of mental impairment is usually mild to moderate, with an average intelligence quotient (IQ) score of 35 to 70. Social skills in afected children are often higher than predicted by their IQ scores.

1	Data suggest that approximately 95 percent of liveborn infants with Down syndrome survive the irst year. The 10-year survival rate is at least 90 percent overall and is 99 percent if major malformations are absent (Rankin, 2012; Vendola, 2010). Several organizations ofer education and support for prospective parents faced with prenatal diagnosis of Down syndrome. These include the March of Dimes, National Down Syndrome Congress (ww.ndsccenter.org), and National Down Syndrome Society (ww.ndss.org).

1	Trisomy l8-Edwards Syndrome. he association between this constellation of abnormalities and an autosomal trisomy was irst described by Edwards (1960). In population-based series, prevalence of trisomy 18 approximates 1 in 2000 recognized pregnancies-including abortuses, stillbirths, and live births, and approximately 1 in 6600 liveborn neonates (Loane, 2013; Parker, 2010). he diference in prevalence is explained by the high in-utero lethality of the condition and the termination of many afected pregnancies. Perhaps not surprisingly, survival of liveborn neonates is likewise bleak. .10re than half die within the irst week, and the I-year survival rate approximates only 2 percent (Tennant, 2010; Vendola, 2010). The syndrome is three-to fourfold more common in females (Lin, 2006; Rosa, 2011) . Unlike Down and Patau syndromes, which involve acrocentric chromosomes and thus may stem from a robertsonian translocation, Edwards syndrome uncommonly results from a chromosomal rearrangement.

1	Virtually every organ system can be afected by trisomy 18. Common major anomalies include heart defects in more than 90 percent-particularly ventricular septal defects, as well as cerebellar vermian agenesis, myelomeningocele, diaphragmatic hernia, omphalocele, imperforate anus, and renal anomalies such as horseshoe kidney (Lin, 2006; Rosa, 2011; Yeo, 2003). Sonographic images of several of these are shown in Chapter 10.

1	Sonographic images of several of these are shown in Chapter 10. Cranial and extremity abnormalities are also frequent and include a prominent occiput, posteriorly rotated and malformed ears, micrognathia, clenched hands with overlapping digits, radial aplasia with hyperflexion of the wrists, and rockerbottom or clubbed feet (Fig. 13-5). A "strawberry-shaped" cranium is noted in approximately 40 percent of cases, abnormally wide cavum septum pellucidum in more than 90 percent, and choroid plexus cysts in up to 50 percent (Abele, 2013; Yeo, 2003). Importantly, isolated choroid plexus cysts are not associated with trisomy 18. These cysts only raise the risk for trisomy 18 if fetal structural abnormalities or an abnormal aneuploidy screening test result is also present (Reddy, 2014).

1	Pregnancies with trisomy 18 that reach the third trimester often develop fetal-growth restriction, and the mean birthweight is <2500 g (Lin, 2006; Rosa, 201l). Because abnormal fetal heart rate tracings are common during labor, mode of delivery and management of heart rate abnormalities should be discussed in advance. In older reports, more than half of undiagnosed fetuses underwent cesarean delivery for "fetal distress" (Schneider, 1981). Trisomy l3-Patau Syndrome. This constellation of fetal abnormalities and their association with an autosomal trisomy was irst described by Patau and colleagues (1960). The prevalence of trisomy 13 approximates 1 in 12,000 live births and 1 in 5000 recognized pregnancies, which includes abortuses and stillbirths (Loane, 2013; Parker, 2010). As with trisomy 18, trisomy 13 is highly lethal, and most afected fetuses are lost or terminated.

1	Approximately 80 percent of pregnancies with Patau syndrome result from trisomy 13. The remainder is caused by a robertsonian translocation involving chromosome 13. The most frequent structural chromosomal rearrangement is a translocation between chromosomes 13 and 14, der(l3; 14) (q 1 O;q 10). This translocation is carried by approximately 1 in 1300 individuals, although the risk of an afected liveborn neonate is less than 2 percent (Nussbaum, 2007). FIGURE 13-5 Trisomy 18-Edwards Syndrome. A. This transventricular sonographic view shows fetal choroid plexus cysts and an angulated "strawberry-shaped" skull. B. Radial clubhand is manifested as a single forearm bone (radius), with the hands in a fixed, hyperflexed position at right angles to the forearms. C. This three-dimensional (3-�) sonographic image shows the characteristic hand position of clenched fists with overlapping digits. D. 3-D sonographic image displays a rockerbottom foot.

1	Trisomy 13 is associated with abnormalities of virtually every organ system. One characteristic finding is holoprosencephaly (Fig. 10-15, p. 195). his is present in approximately two thirds of cases and may be accompanied by microcephaly, hypotelorism, and nasal abnormalities that range from a single nostril to a proboscis. Cardiac defects are found in up to 90 percent of fetuses with trisomy 13 (Shipp, 2002). Other abnormalities that suggest trisomy 13 include neural-tube defectsparticularly cephalocele, microphthalmia, cleft lip-palate, omphalocele, cystic renal dysplasia, polydactyly, rockerbottom feet, and areas of skin aplasia (Lin, 2007). For the fetus or newborn with a cephalocele, cystic kidneys, and polydactyly, the diferential diagnosis includes trisomy 13 and the autosomalrecessive Meckel-Gruber syndrome. Sonographic images of several of these are shown in Chapter 10 (p. 192).

1	Few fetuses with trisomy 13 survive until birth. Of those that do, the I-week survival rate approximates 40 percent, and I-year survival rate is only about 3 percent (Tennant, 2010; Vendola, 2010). Counseling regarding prenatal diagnosis and management options is similar to that described for trisomy 18. Unlike other aneuploidies, fetal trisomy 13 confers risk to the pregnant woman. Hyperplacentosis and preeclampsia develop in up to half of pregnancies with trisomy 13 carried beyond the second trimester (Tuohy, 1992). Chromosome 13 contains the gene for soluble fms-like tyrosine kinase-1 (sFlt-l), which is an anti angiogenic protein associated with preeclampsia (Chap. 40, p. 716). Investigators have documented overexpression of the slt-1 protein by trisomic 13 placentas and in serum of women with preeclampsia (Bdolah, 2006; Silasi, 2011).

1	Other Trisomies. In the absence of mosaicism, discussed later (p. 263), other autosomal trisomies rarely yield a liveborn neonate. Case of live births with trisomy 9 and with trisomy 22 have been noted (Kannan, 2009; Tinkle, 2003). Trisomy 16 is the most common trisomy found with first-trimester losses and accounts for 16 percent of these losses. However, it is not identiied later in gestation. Trisomy 1 has never been reported. Nondisjunction creates an equal number of nullisomic and disomic gametes. As a rule, missing chromosomal material is more devastating than extra chromosomal material, and almost all monosomic conceptuses are lost before implantation. he one exception is monosomy for the X chromosome (45,X), Turner syndrome, which is discussed subsequently. Despite the strong association between maternal age and trisomy, maternal age and monosomy are not linked.

1	This is an abnormal number of complete haploid chromosomal sets. Polyploidy accounts for approximately 20 percent of spon taneous abortions but is rare in later gestations.

1	Triploid pregnancies have three haploid sets-69 chromosomes. One parent must contribute two sets, and the phenotypic presentation difers according to the parent of origin. With diandric triploidy, also known as type I triploidy, the extra chromosomal set is paternal, resulting from fertilization of one egg by two sperm or by a single diploid-and thus abnormal-sperm. Diandric triploidy produces a partial molar pregnancy, discussed in Chapter 20 (p. 389). Diandric triploidy accounts for most triploid conceptions, but their first-trimester loss rate is extremely high. As a result, two thirds of triploid pregnancies identified beyond the first trimester are caused instead by diynic triploidy Q auniaux, 1999). With a digynic triploid pregnancy, also known as type II triploidy, the extra chromosomal set is maternal, and the egg fails to undergo the irst or second meiotic division before fertilization. Digynic triploid placentas do not develop molar changes. However, the fetus usually

1	chromosomal set is maternal, and the egg fails to undergo the irst or second meiotic division before fertilization. Digynic triploid placentas do not develop molar changes. However, the fetus usually displays asymmetrical growth restriction.

1	he prevalence of recognized triploid pregnancies approximates 1 in 5000 pregnancies (Zalel, 2016). Triploidy is a lethal aneuploidy, and more than 90 percent of fetuses with either the diandric or digynic form have multiple structural anomalies. hese include central nervous system defects-particularly involving the posterior fossa, as well as cardiac, renal, and extremity anomalies Qauniaux, 1999; Zalel, 2016). Counseling, prenatal diagnosis, and delivery options are similar to those for trisomies 18 and 13. he recurrence risk for a woman whose triploid fetus survived past the first trimester is 1 to 1.5 percent, and thus prenatal diagnosis is ofered in future pregnancies (Gardner, 1996). Tetraploid pregnancies have four haploid sets of chromosomes, resulting in either 92,XXor 92,YY. This suggests a postzygotic failure to complete an early cleavage division. The conceptus invariably succumbs, and the recurrence risk is minimal.

1	45, X-Turner Syndrome. First described by Turner (1938), this syndrome later was found to be caused by monosomy X (Ford, 1959). he prevalence of Turner syndrome is approximately 1 in 2500 liveborn girls (Cragan, 2009; Dolk, 2010). The missing X chromosome is paternally derived in 80 percent of cases (Cockwell, 1991; Hassold, 1990). Screening for Turner syndrome with cell-free DNA is discussed in Chapter 14 (p. 284). Turner syndrome is the only monosomy compatible with life, but it is also the most common aneuploidy in first-trimester losses, accounting for 20 percent. his is explained by the wide range in phenotype. Approximately 98 percent of afected conceptuses are so abnormal that they abort early in the first trimester. Of the remainder, many manifest large, septated cystic hygromas in the late irst or early second trimester (Fig. 10-22, p. 198). When cystic hygromas are accompanied by hydrops fetalis, fetuses nearly always die in utero (Chap 15, p. 309).

1	Less than 1 percent of pregnancies with Turner syndrome yield a liveborn neonate. And, only half of these actually have monosomy X. Approximately a fourth have mosaicism, such as 45,X/46,XX or 45,Xl46,XY, and another 15 percent have isochromosome X, that is, 46,X,i(Xq) (Milunsky, 2004; Nuss baum, 2007).

1	Abnormalities associated with Turner syndrome include left sided cardiac defects-such as coarctation of the aorta, hypo plastic left heart syndrome, or bicuspid aortic valve-in 30 to 50 percent; renal anomalies, particularly horseshoe kidney; and hypothyroidism. Other features are short stature, broad chest with widely spaced nipples, congenital lymphedema-pui ness over the dorsum of hands and feet, and a "webbed" pos terior neck resulting from cystic hygromas. Intelligence scores are generally in the normal range, but afected individuals are at risk for diiculties with visual-spatial organization, nonver bal problem solving, and interpretation of social cues Q ones, 2006). Growth hormone is typically administered in childhood to ameliorate short stature (Kappelgaard, 201i1). More than 90 starting just before adolescence. An exception is mosaicism involving the Y chromosome, as this confers risk for germ cell neoplasm-regardless of whether the child is phenotypically male or female.

1	starting just before adolescence. An exception is mosaicism involving the Y chromosome, as this confers risk for germ cell neoplasm-regardless of whether the child is phenotypically male or female. Accordingly, eventual prophylactic bilateral gonadectomy is indicated (Cools, 2011; Schorge, 2016).

1	47,XXX. Approximately 1 in 1000 female newborns has an additional X chromosome-47,XXX. The extra X is maternally derived in more than 90 percent of cases (Milunsky, 2004). Afected infants do not have a characteristic appearance, and in the past most children did not come to attention until school age. However, the incidence of 47,XXiis weakly associated with maternal age, and cell-free DNA screening has resulted in increased diagnoses (Table 14-5, p. 281). Frequent features include tall stature, hypertelorism, epicanthal folds, kyphoscoliosis, clinodactyly, and hypotonia (Tartaglia, 2010; Wigby, 2016). More than a third are diagnosed with a learning disability, half have attention deicit disorder, and overall cognitive scores are in the low-average range. No speciic pattern of malformations has been described, but genitourinary problems and seizure disorders are more common (Wigby, 2016). Pubertal development is unafected. Primary ovarian insuiciency has been reported (Holland, 2001).

1	has been described, but genitourinary problems and seizure disorders are more common (Wigby, 2016). Pubertal development is unafected. Primary ovarian insuiciency has been reported (Holland, 2001). Because of variable presentation and subtle abnormal indings, it is estimated that this diagnosis is ascertained clinically in only 10 percent of afected children.

1	Females with two or more extra X chromosomes-48,XXior 49,XXXX-are likely to have physical abnormalities apparent at birth. These abnormal X complements are associated with intellectual disability. For both males and females, the IQ score is lower with each additional X chromosome. 47,XXY-Klinefelter Syndrome. his is the most common sex chromosome abnormality and found in approximately 1 in 600 male infants. he additional X chromosome is maternally or paternally derived with equal propensity Qacobs, 1995; Lowe, 2001). The association with either advanced maternal or paternal age is weak (Milunsky, 2004).

1	Like 47,XX, newborns with 47,Y usually appear phenotypically normal and do not have a higher incidence of anomalies. As children, boys are typically taller than average and have normal prepubertal development. However, they have gonadal dysgenesis, do not undergo normal virilization, and require testosterone supplementation beginning in adolescence. They may develop gynecomastia. IQ scores usually lie in the average to low-average range, and many have delays in language development and reading (Boada, 2009; Girardin, 2011).

1	47,XY. This aneuploidy occurs in approximately 1 in 1000 male newborns. As with 47,XXand Y individuals, afected boys tend to be tall. A third have macrocephaly, nearly two thirds demonstrate hypotonia, and tremors are also common (Bardsley, 2013). Rates of major anomalies are not elevated, although hypertelorism and clinodactyly may be identified in more than hal. Pubertal development is normal, and fertility is unimpaired. ffected children carry risks for oral and written language impairments, attention deicit disorder is diagnosed in more than half, and the rate of autism spectrum disorder is also increased (Bardsley, 2013; Ross, 2009). Intelligence scores generally lie in the normal range. Males with more than two Y chromosomes-48Yor with both additional X and Y chromosomes-48,YYor 49,XYY-are more likely to have congenital abnormalities, medical problems, and intellectual disability (Tartaglia, 2011). • Abnormalities of Chromosome Structure

1	• Abnormalities of Chromosome Structure Structural chromosomal abnormalities include deletions, duplications, translocations, isochromosomes, inversions, ring chromosomes, and mosaicism (see Table 13-1). heir overall birth prevalence approximates 0.3 percent (Nussbaum, 2007). Identiication of a structural chromosomal abnormality raises two primary questions. First, what phenotypic abnormalities or later developmental abnormalities are associated with this finding? Second, is evaluation of parental karyotype indicated-speciically, are the parents at increased risk of carrying this abnormality? If so, what is their risk of having future afected ofspring?

1	A chromosomal deletion indicates that a portion of a chromosome is missing, whereas a duplication means that a portion has been included twice. Most deletions and duplications occur during meiosis and result from malalignment or mismatching during the pairing of homologous chromosomes. he misaligned segment may then be deleted, or if the mismatch remains when the two chromosomes recombine, it may result in a deletion in one chromosome and duplication in the other (Fig. 13-6). When a deletion or duplication is identified in a fetus or infant, parental karyotyping should be ofered, because if either parent carries a balanced translocation, the recurrence risk in subsequent pregnancies is signiicantly increased. Deletions involving DNA segments large enough to be seen with standard cytogenetic karyotyping are identiied in approximately 1 in 7000 births (Nussbaum, 2007). Common

1	FIGURE 13-6 A mismatch during pairing of homologous chromosomes may lead to a deletion in one chromosome and a duplication in the other. Del = deletion; Dupl duplication. deletions may be referred to by eponyms-for example, del 5p is called cri du chat syndrome.

1	Microdeletions and Microduplications. These chromosomal deletions or duplications-smaller than 3 to 5 million base pairs-are too small to be detected with standard karyotyping. However, prenatal chromosomal micro array analysis (CMA) , described later (p. 271), permits identification of syndromes associated with these microdeletion or duplications. When CMA is used, the region of DNA that is missing or duplicated is termed a genomic copy number variant. Despite the relatively small size, a microdeletion or duplication may involve a stretch of DNA that contains multiple genes-causing a contiguous gene syndrome, which can encompass serious but unrelated phenotypic abnormalities (Schmickel, 1986). In some cases, a micro duplication may involve the exact DNA region that causes a recognized microdeletion syndrome (Table 13-2). When a speciic microdeletion syndrome is suspected clinically, it is confirmed using either CMA or luorescence in situ hybridization.

1	22qlel.2 Microdeletion Syndrome. his syndrome is also known as DiGeorge syndrome, Shprintzen syndrome, and velocardiofacial syndrome. It is the most common microdeletion, with a prevalence of 1 in 3000 to 6000 births. Although inherited in an autosomal dominant fashion, more than 90 percent of cases arise from de novo mutations. The full deletion includes 3 million base pairs, encompasses 40 genes, may include 180 diferent features, and thus poses some counseling challenges (Shprintzen, 2008). Features can vary widely, even among afected family members. Previously, diferent constellations of features were thought to characterize the DiGeorge and Shprintzen phenotypes, but it is now accepted that they represent the same microdeletion (McDonald-McGinn, 2015). In approximately 75 percent of afected individuals, associated abnormalities include conotruncal cardiac anomalies, Prevalence reflects live births. Data from National Library of Medicine, 2017; Johns Hopkins University, 2017.

1	Prevalence reflects live births. Data from National Library of Medicine, 2017; Johns Hopkins University, 2017. such as tetralogy of Fallot, truncus arteriosus, interrupted aortic arch, and ventricular septal defects (McDonald-McGinn, 2015). Immune deiciency, such as T-cell lymphopenia, also develops in approximately 75 percent. More than 70 percent have velopharyngeal insuiciency or cleft palate. Learning disabilities, autism spectrum disorder, and intellectual disability are also common. Other manifestations include hypocalcemia, renal anomalies, esophageal dysmotility, hearing loss, behavioral disorders, and psychiatric illness-particularly schizophrenia. Short palpebral issures, bulbous nasal tip, micrognathia, short philtrum, and small or posteriorly rotated ears are characteristic facial features.

1	hese are DNA rearrangements in which a segment of DNA breas away from one chromosome and attaches to another. he rearranged chromosomes are called derivative (der) chromosomes. here are two types-reciprocal and robertsonian translocations.

1	Reciprocal Translocations. A double-segment or reciprocal translocation begins when breaks occur in two diferent chromosomes. The broken fragments are then exchanged, so that each afected chromosome contains a fragment of the other. If no chromosomal material is gained or lost in this process, the translocation is considered balanced. he prevalence of reciprocal translocations approximates 1 in 600 births (Nussbaum, 2007). Although the balanced translocation carrier is usually normal phenotypically, repositioning of specific genes within chromosomal segments can cause abnormalities. he risk of a major sttuctural or developmental abnormality in an apparent balanced translocation carrier is approximately 6 percent. Interestingly, using CMA technology, up to 20 percent of individuals who appear to have a balanced translocation are found instead to have missing or redundant DNA segments (Manning,i2010).

1	Balanced translocation carriers are at risk to produce unbalanced gametes, resulting in abnormal ofspring. As shown in FIGURE 13-7 A carrier of a balanced translocation may produce offspring who are also carriers ofthe balanced rearrangement (8), ofspring with unbalanced translocations (C, D), or ofspring with normal chromosomal complements (A). n.N

1	n.N Figure 13-7, if an oocyte or sperm contains a translocated chromosome, fertilization results in an unbalanced translocation-monosomy for part of one afected chromosome and trisomy for part of the other. he observed risk of a specific translocation can often be estimated by a genetic counselor. In general, translocation carriers identified after the birth of an abnormal child have a 5-to 30-percent risk of producing liveborn ofspring with an unbalanced translocation. Carriers identiied for other reasons, for example, during an infertility evaluation, have only a 5-percent risk. his is likely because the gametes are so abnormal that conceptions are nonviable.

1	Robertsonian Translocations. These involve only acrocentric chromosomes, which are chromosomes 13, 14, 15,i21, and 22. he acrocentric chromosomes have extremely short p arms. In a robertsonian translocation, the q arms of two acrocentric chromosomes fuse at one centromere to form a derivative chromosome. he other centromere and both sets of p arms are lost. Because the number of centromeres determines the chromosome count, a robertsonian translocation carrier has only 45 chromosomes. Fortunately, the p arms of the acrocentric chromosomes-the satellite regions-contain redundant copies of genes that code for ribosomal RNA. As these are present in multiple copies on other acrocentric chromosomes, their loss does not afect the translocation carrier, who is usually phenotypically normal. However, when the derivative chromosome is paired during fertilization with a haploid chromosome from the partner, resulting ofspring will be trisomic for that chromosome.

1	Robertsonian translocations are found in 1 in 1000 individuals. The incidence of abnormal ofspring approximates 15 percent if a robertsonian translocation is carried by the mother and 2 percent if carried by the father. Robertsonian translocations are not a major cause of miscarriage and are found in fewer than 5 percent of couples with recurrent pregnancy loss. When a fetus or child is found to have a translocation trisomy, both parents should be ofered karyotype analysis. If neither parent is a carrier, the recurrence risk is extremely low.

1	Balanced robertsonian carriers have reproductive diiculties for a number of reasons. Ifthe fused chromosomes are homologous, that is, from the same chromosome pair, the carrier can produce only unbalanced gametes. Each egg or sperm contains either both copies of the translocated chromosome, which would result in trisomy if fertilized, or no copy, which would result in monosomy. If the fused chromosomes are nonhomologous, four of the six possible gametes would be abnormal. he most common robertsonian translocation is der(13; 14) (ql0;ql0), which accounts for up to 20 percent of cases of Patau s) ndrome (p. 257). These abnormal chromosomes are composed of either two q arms or two p arms of one chromosome fused together.

1	Isochromosomes are thought to arise when the centromere breaks transversely instead of longitudinally during meiosis II or mitosis. They can also result from a meiotic error in a chromosome with a robertsonian translocation. An isochromosome containing the q arms of an acrocentric chromosome behaves like a homologous robertsonian translocation, and such a carrier can produce only abnormal unbalanced gametes. When an isochromosome involves non acrocentric chromosomes, with p arms containing important genetic material, the fusion and abnormal centromere break results in two isochromosomes. One is composed of both p arms, and one is composed of both q arms. It is likely that one of these isochromosomes would be lost during cell division, resulting in the deletion of all the genes located on the lost arm. Thus, a carrier is usually phenotypically abnormal and produces abnormal gametes. The most common isochromosome involves the long arm of the X chromosome, i(Xq), which is the etiology of

1	lost arm. Thus, a carrier is usually phenotypically abnormal and produces abnormal gametes. The most common isochromosome involves the long arm of the X chromosome, i(Xq), which is the etiology of 15 percent of cases of T umer syndrome.

1	When there are two breaks in the same chromosome, and the intervening genetic material is inverted before the breaks are repaired, the result is a chromosomal inversion. Although no genetic material is lost or duplicated, the rearrangement may alter gene function. There are two types-pericentric and paracentric inversions.

1	PericentricInversion. This results from breaks in both the p and q arms of a chromosome, such that the inverted material includes the centromere (Fig. 13-8). A pericentric inversion causes problems in chromosomal alignment during meiosis and confers signiicant risk for the carrier to produce abnormal gametes and abnormal ofspring. In general, the observed risk of abnormal ofspring in a peri centric inversion carrier is 5 to 10 percent if ascertainment was made after the birth of an abnormal child. But the risk is only 1 to 3 percent if prompted by another indication. An important exception is a pericentric inversion on chromosome 9. his is inv(9)(pllq12), which is a normal variant present in approximately 1 percent of the population.

1	Paracentric Inversion.If there are two breaks within one arm of a chromosome-either p or q-the inverted material does not include the centromere, and the inversion is paracentric (see Fig. 13-8). he carrier makes either normal balanced gametes or gametes that are so abnormal as to preclude fertilization. Thus, although infertility may be a problem, the risk of having an abnormal ofspring is extremely low. If there are deletions at each end of the same chromosome, the ends may come together to form a ring chromosome. he telomere regions, which are the ends of a chromosome, contain specialized nucleoprotein complexes that stabilize the chromosome. If just the telomeres are lost, all necessary genetic material is retained, and the carrier is essentially balanced. If a deletion extends more proximally than the telomere, the carrier is likely to be phenotypically abnormal. An example of this is a ring X chromosome, which may result in T umer syndrome.

1	centric inversion (one involving the centromere) or paracentric inversion (not involving the centromere). Individuals with pericen tric inversions are at increased risk for producing offspring with a duplication/deletion. Those with paracentric inversions are at increased risk for early pregnancy loss.

1	A mosaic individual has two or more cytogenetically distinct cell lines that are derived from a single zygote. Phenotypic expression of mosaicism depends on several factors, including whether the cytogenetically abnormal cells involve the fetus, part of the fetus, just the placenta, or some combination. Of amnionic luid cultures, mosaicism is found in approximately 0.3 percent but may not reflect the fetal chromosomal complement (Carey, 2014). When the abnormal cells are present in only a single lask of amnionic luid, the inding is likely pseudomosaicism, caused by cell-culture artifact (Bui, 1984; Hsu, 1984). When abnormal cells involve multiple cultures, however, true mosaicism is more likely, and further testing may be warranted. A second cell line is veriied in 60 to 70 percent of these fetuses (Hsu, 1984; Worton, 1984).

1	With chorionic villus sampling, studies demonstrate that up to 2 percent of placentas are mosaic, with the mosaicism conined to the in most of these cases (Bafero, 2012; Henderson, 1996). Amniocentesis should be ofered. In a series of more than 1000 pregnancies with mosaicism found from chorionic villus sampling, subsequent amniocentesis identiied true fetal mosaicism in 13 percent. Uniparental disomy, discussed later (p. 268), was found in 2 percent, and the remainder resulted from confined placental mosaicism (Malvestiti, 2015). If mosaicism is detected for a chromosome known to contain imprinted genes-such as chromosomes 6, 7, 11, 14, or 15testing for uniparental disomy should be considered, as there may be fetal consequences (Grati, 2014a).

1	lthough outcomes with conined placental mosaicism are generally good, fetal-growth restriction is more common, and the stillbirth risk is also higher (Reddy, 2009). Fetal-growth restriction may stem from impaired functioning of the aneuploid placental cells (Bafero, 2012). Placental mosaicism for trisomy 16 confers a particularly poor prognosis.

1	Mosaicism confined to the gonads likely arises from a mitotic error in cells destined to become the gonad, resulting in a population of abnormal germ cells. Because spermatogonia and oogonia divide throughout fetal life, and spermatogonia continue to divide throughout adulthood, gonadal mosaicism may also follow a meiotic error in previously normal germ cells. Gonadal mosaicism can account for de novo diseases in the ofspring of normal parents. Autosomal dominant examples are achondroplasia and osteogenesis imperfecta, and X-linked ones include Duchenne muscular dystrophy. Gonadal mosaicism also explains the 6-percent recurrence risk after the birth of a child with a disease caused by a "new" mutation.

1	A monogenic or mendelian disorder is caused by a mutation or alteration in a single locus or gene in one or both members of a gene pair. Types of mendelian inheritance include autosomal dominant, autosomal recessive, X-linked, and Y-linked. Other monogenic inheritance patterns, described subsequently, include mitochondrial inheritance, uniparental disomy, imprinting, and trinucleotide repeat expansion-also termed anticipation. By age 25, approximately 0.4 percent of the population exhibits an abnormality attributed to a monogenic disorder, and 2 percent will have at least one such disorder during their lifetime (Table 13-3).

1	When considering inheritance, it is the phenotype that is dominant or recessive, not the genotype. With a dominant disease, the normal gene may direct the production of normal protein, but the phenotype is abnormal because it is determined by protein produced by the abnormal gene. With a recessive disease, a heterozygous carrier may produce detectable levels of an abnormal gene product but have no features of the condition because the phenotype is directed by the product of the normal co-gene. For example, erythrocytes from carriers of sickle-cell anemia contain approximately 30 percent hemoglobin S, but because the other 70 percent is hemoglobin A, these cells do not usually sickle in vitro. Genetic heterogeneiy explains how diferent genetic mechanisms can result in the same phenotype. Locus heterogeneiy indicates

1	Genetic heterogeneiy explains how diferent genetic mechanisms can result in the same phenotype. Locus heterogeneiy indicates TABLE 13-3. Selected Monogenic (Mendelian) Disorders that a speciic disease phenotype can be caused by mutations in diferent genetic loci. It also explains why some diseases appear to follow more than one type of inheritance. An example is retinitis pigmentosa, which may develop following mutations in at least 35 diferent genes or loci and may result in autosomal dominant, autosomal recessive, or X-linked forms. Alelic heterogeneiy describes how diferent mutations of the same gene may afect presentation of a particular disease. For example, although only one gene has been associated with cystic ibrosis-the cystic ibrosis conductance transmembrane regulator gene-more than 2000 mutations in this gene have been described and result in variable disease severity (Chaps. 14 and 51, pp. 289 and 997).

1	can arise from diferent mutations in the same gene. As an example, mutations in the ibroblast growth actor receptor 3 (FGF3) gene may result in several diferent skeletal disorders, including achondroplasia and thanatophoric dysplasia, both of which are discussed in Chapter 10 (p. 210). If only one copy of a gene pair determines the phenotype, that gene is considered to be dominant. Carriers have a 50-percent chance of passing on the afected gene with each conception. A gene with a dominant mutation generally speciies the phenotype in preference to the normal gene. That said, not all individuals will necessarily manifest an autosomal dominant condition the same way. Factors that afect the phenotype of an autosomal dominant condition include penetrance, expressivity, and occasionally, presence of codominant genes.

1	his characteristic describes whether or not a dominant gene is expressed at all. A gene with recognizable phenotypic expression in all individuals is 100-percent penetrant, whereas penetrance is incomplete if some carriers express the gene but some do not. his may be quantitatively expressed-for example, a gene that is expressed in some way in 80 percent of individuals who have that gene is 80-percent penetrant. Importantly, incomplete penetrance explains why some autosomal dominant diseases may appear to "skip" generations. Individuals with the same autosomal dominant trait may manifest the condition diferently, even within the same family. Genes with variable expressivity can produce disease manifestations that range from mild to severe. Examples include neuroibromatosis, tuberous sclerosis, and adult polycystic kidney disease.

1	If two diferent alleles in a gene pair are both expressed in the phenotype, they are considered to be codominant. Blood type, for example, is determined by expression of dominant A and B red-cell antigens that can be expressed simultaneously. Another example of codominance is the group of genes responsible for hemoglobin production. An individual with one gene directing production of hemoglobin S and the other directing production of hemoglobin C will produce both Siand C hemoglobin (Chap. 56, p. 1082).

1	Paternal age older than 40 is associated with increased risk for spontaneous genetic mutations, particularly single base substitutions. This may result in ofspring with new autosomal dominant disorders or X-linked carrier states. In particular, advanced paternal age has been associated with mutations in the ibroblast growth actor receptor 2 (FGF2) gene, which may cause craniosynostosis syndromes such as Apert, Crouzon, and Pfei fer syndromes; mutations in the FGF3 gene, which may result in the ET proto-oncogene, which may cause multiple endo crine neoplasia syndromes Qung, 2003; Toriello, 2008). Using whole genome sequencing, described later (p. 272), Kong and utes to a rise in the rate of single-nucleotide polymorphisms among ofspring. his rate is approximately two mutations for each year of paternal age. Because individual autosomal domi nant disorders are uncommon, the actual risk for any speciic condition is low, and no screening or testing is speciically rec ommended.

1	Advanced paternal age has also been associated with a structural abnormalities (Grewal, 2012; Toriello, 2008; Yang, 2007). It is not generally considered to pose an elevated risk for other aneuploidies, probably because the aneuploid sperm cannot fertilize an egg.

1	Recessive diseases develop only when both gene copies are abnormal. Many enzyme deficiency diseases display autosomal recessive inheritance, and enzyme activity in the carrier is usually about half of normal. Unless carriers are screened for a specific disease, such as cystic ibrosis, they usually are recognized only after the birth of an afected child or the diagnosis of an afected family member (Chap. 14, p. 289). If a couple has a child with an autosomal recessive disease, the recurrence risk is 25 percent for each subsequent pregnancy. hus, 1/4 of ofspring will be homozygous normal, 2/4 will be heterozygous carriers, and 114 will be homozygous abnormal. In other words, three of four children will be phenotypically normal, and 2/3 of phenotypically normal siblings are actually carriers.

1	A heterozygous carrier of a recessive condition is only at risk to have afected children if his or her partner is heterozygous or homozygous for the disease. Genes for rare autosomal recessive conditions have low prevalence in the general population. Thus, the likelihood that a partner will be a gene carrier is small, unless there is consanguinity or the partner is a member of an at-risk group. Heterozygous carriers are usually undetectable clinically but may have biochemical test abnormalities that can be used for carrier screening. Other recessive conditions can be identified only by molecular genetic testing (Chap. 14, p. 288). Inborn Errors of Metabolism Most of these autosomal recessive diseases result from absence of a crucial enzyme, leading to incomplete metabolism of proteins, lipids, or carbohydrates. The metabolic intermediates that build up are toxic to various tissues and may result in intellectual disability or other abnormalities.

1	Phenylketonuria. Also known as phenylalanine hydroxylase (PAH) deficiency, this autosomal recessive disease is caused by mutations in the PAH gene. PAH metabolizes phenylalanine to tyrosine, and homozygotes have diminished or absent enzyme activity. This leads to abnormally high levels of phenylalanine, resulting in progressive intellectual impairment, autism, seizures, motor deficits, and neuropsychological abnormalities (Blau, 2010). Because phenylalanine competitively inhibits tyrosine hydroxylase-which is essential for melanin production, afected individuals also have hair, eye, and skin hypopigmentation. More than 500 PAH gene mutations have been characterized, and the carrier frequency is 1 in 60, such that the disease afects approximately 1 in 15,000 newborns (American College of Obstetricians and Gynecologists, 2017 c). Prompt diagnosis and restriction of dietary phenylalanine beginning early in infancy are essential to prevent neurological damage, and all states mandate

1	of Obstetricians and Gynecologists, 2017 c). Prompt diagnosis and restriction of dietary phenylalanine beginning early in infancy are essential to prevent neurological damage, and all states mandate newborn screening for phenylketonuria (PKU).

1	Phenylalanine restriction alone would result in inadequate protein consumption, and phenylalanine-free amino acid-based supplementation is required. lso, in 2007, a synthetic form of the P AH cofactor tetrahydrobiopterin (sapropterin) was approved for PKU treatment. Approximately 25 to 50 percent of afected individuals are sapropterin-responsive and may experience a significant decline in phenylalanine levels and improvement in neuropsychiatric symptoms (Vockley, 2014). Lifelong maintenance of phenylalanine concentrations in the range of 2 to 6 mg/dL (120 to 360 �mol/L) is necessary to prevent worsening neurocognitive and psychiatric problems (American College of Obstetricians and Gynecologists, 2017 c). Fortunately, even those who have previously discontinued therapy may experience improved neuropsychological function with treatment.

1	During pregnancy, women with PKU whose phenylalanine levels remain above the recommended range are at risk to have otherwise normal (heterozygous) ofspring who sustain in utero damage as a result of being exposed to toxic phenylalanine concentrations. Phenylalanine is actively transported to the fetus. Hyperphenylalaninemia raises the risk for miscarriage and for PKU embryopathy, characterized by intellectual disability, microcephaly, seizures, growth impairment, and cardiac anomalies. Among women on unrestricted diets, the risk to have a child with intellectual disability exceeds 90 percent, microcephaly occurs in more than 70 percent, and as many as 1 in 6 children have cardiac defects (Lenke, 1980). he Maternal Phenylketonuria Collaborative Study, which included 572 pregnancies followed more than 18 years, reported that maintenance of serum phenylalanine levels in the recommended range between 2 and 6 mg/dL signiicantly reduced the fetal abnormality risk and resulted in childhood

1	more than 18 years, reported that maintenance of serum phenylalanine levels in the recommended range between 2 and 6 mg/dL signiicantly reduced the fetal abnormality risk and resulted in childhood IQ scores in the normal range (Koch, 2003; Platt, 2000). Preconceptional counseling and consultation with providers from experienced PKU centers is recommended.

1	Two individuals are considered consanguineous if they have at least one recent ancestor in common. Although uncommon in Western countries, more than 1 billion people are estimated to live in countries in which 20 to 50 percent of marriages are consanguineous (Romeo, 2014). In medical genetics, a union is consanguineous if between second cousins or closer relatives. First-degree relatives share half of their genes, second-degree relatives share a fourth, and third-degree relatives-first cousins-share one eighth. Because of the potential for shared deleterious genes, consanguinity confers an increased risk to have ofspring with otherwise rare autosomal recessive diseases or multifactorial disorders. In population-based series, first cousins are reported to have a twofold risk for congenital anomalies (Sheridan, 2013; Stoltenberg, 1997). Consang�inity also is associated with a greater rate of stillbirth (Kapurubandara, 2016). Because CMA performed using a single-nucleotide polymorphism

1	anomalies (Sheridan, 2013; Stoltenberg, 1997). Consang�inity also is associated with a greater rate of stillbirth (Kapurubandara, 2016). Because CMA performed using a single-nucleotide polymorphism platform may identiy consanguinity, it is important that preprocedural counseling include this possibility.

1	Incest is defined as a sexual relationship between first-degree relatives such as parent-child or brother-sister and is universally illegal. Progeny of such unions carry the highest risk of abnormal outcomes, and older studies reported that up to 40 percent of ofspring were abnormal as a result of recessive and multifactorial disorders (Baird, 1982; Freire-Maia, 1984). Most X-linked diseases are recessive. Common examples include color blindness, hemophilia A and B, and Duchenne and Becker muscular dystrophy. Males with an X-linked recessive gene are usually afected by the disease it causes, because they lack a second X chromosome to express the normal dominant gene. A male with an X-linked disease cannot have afected sons because they cannot receive his X chromosome. When a woman carries a gene causing an X-linked recessive condition, each of her sons has a 50-percent risk of being afected, and each daughter has a 50-percent chance of being a carrier.

1	Women with an X-linked recessive gene are generally unaffected by the disease it causes. In some cases, however, the random inactivation of one X chromosome in each cell-termed lyonization-is skewed, and female carriers may have features of the condition. For example, approximately 10 percent of female carriers of hemophilia A will have factor VIII levels less than 30 percent of normal, and a similar proportion of female hemophilia B carriers have factor IX levels less than 30 percent. Levels below these thresholds confer a greater risk for abnormal bleeding when afected women give birth (Plug, 2006). Indeed, even with higher levels, carriers are reported to be at increased risk for bleeding complications (Olsson, 2014). Similarly, female carriers of Duchenne or Becker muscular dystrophy carry an elevated risk for cardiomyopathy, and periodic evaluation for cardiac dysfunction and neuromuscular disorders is recommended (American Academy of Pediatrics, 2008).

1	X-linked dominant disorders mainly afect females, because they tend to be lethal in males. Two examples are vitamin D-resistant rickets and incontinentia pigmenti. One exception is fragile X syndrome, which is discussed subsequently. he prevalence of Y-linked chromosomal disorders is low. This chromosome carries genes important for sex determination and various cellular functions related to spermatogenesis and bone development. Deletion of genes on the long arm of Y results in severe spermatogenic defects, whereas genes at the tip of the short arm are critical for chromosomal pairing during meiosis and for fertility.

1	Human cells contain hundreds of mitochondria, each with its own genome and associated replication system. Oocytes contain approximately 100,000 mitochondria. Sperm hold only about 100, and these are destroyed after fertilization. Each mitochon drion has multiple copies of a 16.5-kb circular DNA molecule that contains 37 genes. Mitochondrial DNA encodes peptides and transfer RNAs. Mitochondria are inherited exclusively from the mother.

1	Mitochondria are inherited exclusively from the mother. hus, although males and females both can be afected by a mitochondrial disorder, transmission is only through the mother. When a cell replicates, mitochondrial DNA sorts randomly into each of the daughter cells, a process termed replicative segregation. A consequence of replicative segregation is that any mitochondrial mutation will be propagated randomly into the daughter cells. Because each cell holds multiple copies of mitochondrial DNA, the mitochondrion may contain only normal or only abnormal DNA, termed homoplasmy. Alternatively, it may contain both normal and mutated DNA, namely heteroplasmy. If a heteroplasmic oocyte is fertilized, the relative proportion ofmutated DNA may afect whether the individual manifests a given mitochondrial disease. It is not possible to predict the potential degree of heteroplasmy among ofspring, and this poses challenges for genetic counseling.

1	As of 2016, 33 mitochondrial diseases or conditions with known molecular basis were described in Online Mendelian Inheritance in Man Qohns Hopkins University, 2017). Examples include myoclonic epilepsy with ragged red fibers (MERRF), Leber optic atrophy, Kearns-Sayre syndrome, Leigh syndrome, several forms of mitochondrial myopathy and cardiomyopathy, and susceptibility to chloramphenicol toxicity. Mendel's irst law is that genes are passed unchanged from parent to progeny, and barring new mutations, this is true for many genes or traits. However, certain genes are unstable, and their size, and thus function, may be altered during parentto-child transmission. This is manifested clinically by anticipation-a phenomenon in which disease symptoms seem to be more severe and to appear at an earlier age in each successive generation. Examples of some DNA triplet (trinucleotide) repeat diseases are shown in Table 13-4.

1	his is the most common inherited form of intellectual disability and afects approximately 1 in 3600 males and 1 in 4000 to 6000 females (American College of Obstetricians and Gynecologists, 2017a). Fragile X syndrome is caused by expansion TABLE 13-4. Some Disorders Caused by DNA Triplet Repeat Expansion of a repeated trinucleotide DNA segment-cytosine-guanineguanine (CGG)-at chromosome Xq27.3. When the CGG repeat number reaches a critical size-the full mutation-the ragile X mental retardation 1 (FMR1) gene becomes methyl ated. Methylation inactivates the gene, which halts expression of FMR1 protein. his protein is most abundant in nerve cells and is essential for normal cognitive development.

1	Although transmission of the syndrome is X-linked, both the sex of the afected individual and the number of CGG repeats determine the degree of clinical normalcy or impair ment. Intellectual disability is generally more severe in males, in whom average IQ scores are 35 to 45 (Nelson, 1995). Afected tion-deicit/hyperactivity disorder. Fragile X syndrome is also the most common known cause of autism or "autistic like" behavior. Associated phenotypic abnormalities become more prominent with age and include a narrow face with large jaw, prominent ears, connective tissue abnormalities, and macroor chidism in postpubertal males. Clinically, four groups have been described (American College of Obstetricians and Gyne •cologists, 2017a): Premutation-55 to 200 repeats Intermediate-45 to 54 repeats

1	Premutation-55 to 200 repeats Intermediate-45 to 54 repeats Full mutations are expressed (penetrant) in all males and many females. When a full mutation is present, males typically have significant cognitive and behavioral abnormalities and phenotypic features. In females, random X-inactivation, however, results in variable expression, and the disability may be much less severe. With rare exception, the parent of origin of repeat expansion that leads to a full mutation is female (Monaghan, 2013).

1	For individuals with a premutation, evaluation and counseling are more complex. A female with the fragile X premutation is at risk to have ofspring with the full mutation, depending on the repeat number. The risk ofa full mutation in an ofspring is 5 percent or less if the CGG repeat number is <70 but exceeds 95 percent with 100 to 200 CGG repeats (Nolin, 2003). Expansion is extremely unlikely in a male premutation carrier, but all of his daughters will carry the premutation. Among women with no risk factors, approximately 1 in 250 carries a fragile X premutation, and the risk approximates 1 in 90 in those with a family history of intellectual disability (Cronister, 2008). Premutation carriers may themselves experience significant health consequences. Males with the premutation are at increased risk for the fragile X tremor ataxia syndrome (FXTAS). his syndrome is characterized by memory loss, executive function deficits, anxiety, and dementia (Monaghan, 2013). Females are at risk for

1	risk for the fragile X tremor ataxia syndrome (FXTAS). his syndrome is characterized by memory loss, executive function deficits, anxiety, and dementia (Monaghan, 2013). Females are at risk for FXTAS as well, although less so. hey also have a 20-percent risk for fragile X-associated primary ovarian insuficiency.

1	he American College of Obstetricians and Gynecologists (2016c, 2017a) recommends carrier screening for women with a family histof) of fragile X syndrome; individuals with unexplained intellectual disability, developmental delay, or autism; and women with primary ovarian insuiciency. Prenatal diagnosis can be accomplished by amniocentesis or chorionic villus sampling. Specimens obtained by either can define the CGG repeat number, although chorionic villus sampling may not accurately determine FMRl gene methylation status.

1	his term describes some genes that are inherited but not expressed, depending on whether they are inherited from the mother or father. hus, the resulting phenotype varies according to the parent of origin. Imprinting afects gene expression by epigenetic control, which modiies genetic structure using methods other than altering the underlying nucleotide sequence. For example, methyl group addition may alter gene expression and thereby afect the phenotype without changing the genotype. Importantly, the efect may be reversed in a subsequent generation, because a female who inherits an imprinted gene from her father will pass it in her oocytes with a maternal-rather than paternal-imprint, and vice versa. Selected diseases that can involve imprinting are shown in

1	Selected diseases that can involve imprinting are shown in Table 13-5. A useful example includes two very diferent dis eases that afect the same region of DNA. First, Prader-Willi syndrome is characterized by obesity and hyperphagia; short stature; small hands, feet, and external genitalia; and mild mental retardation. In more than 70 percent of cases, PraderWilli syndrome is caused by microdeletion or disruption for the paternal 15q 11.2-q 13. The remaining cases are due to maternal uniparental disomy or due to maternal gene imprinting with the paternal gene inactivated.

1	In contrast, Angelman syndrome includes severe intellectual disability; normal stature and weight; absent speech; seizure disorder; ataxia and jerky arm movements; and paroxysms of inappropriate laughter. In approximately 70 percent of cases, Angelman syndrome is caused by microdeletion for the maternal 15q 11.2-q 13. In 2 percent, the syndrome is caused by paternal uniparental disomy, and another 2 to 3 percent is due to paternal gene imprinting with the maternal genes inactivated. here are other examples of imprinting important to obstetrics. Complete hydatidiform mole, with a paternally derived diploid chromosomal complement, is characterized by abundant TABLE 13-5. Some Disorders That Can Involve Imprinting Angelman lSq11.2-q13 Maternal Beckwith-Wiedemann 11 p1S.5 Paternal Prader-Willi lSq11l.2-q13 Paternal Pseudohypoparathyroidism 20q13.2 Variable Russell-Silver syndrome 7p1l1.2 Maternal

1	Angelman lSq11.2-q13 Maternal Beckwith-Wiedemann 11 p1S.5 Paternal Prader-Willi lSq11l.2-q13 Paternal Pseudohypoparathyroidism 20q13.2 Variable Russell-Silver syndrome 7p1l1.2 Maternal Data from Online Mendelian Inheritance in Man (Johns Hopkins University, 201l7.) placental growth with no fetal structures (Chap. 20, p. 389). Conversely, an ovarian teratoma, with a maternally derived diploid chromosomal complement, is characterized by the growth of various fetal but no placental tissues (Porter, 1993).

1	his occurs when both members of a chromosome pair are inherited from the same parent. Often, uniparental disomy does not have clinical consequences. Although both copies are inherited from one parent, they are not identical. However, if chromosomes 6, 7, 11, 14, or 15 are involved, ofspring are at increased risk for an abnormality because of parent-of-origin diferences in gene expression (Shafer, 2001). Several genetic mechanisms may cause uniparental disomy, the most common of which is trisomic rescue, shown in Figure 13-9. After a nondisjunction event produces a trisomic conceptus, one of the three homologues may be lost. his will result in uniparental disomy for that chromosome in approximately one third of cases.

1	Isodisomy is the unique situation in which an individual receives two identical copies of one chromosome in a pair from one parent. his mechanism explains some cases of cystic ibrosis, in which only one parent is a carrier but the fetus inherits two copies of the same abnormal chromosome from that parent (Spence, 1988; Spotila, 1992). It also has been implicated in abnormal growth related to placental mosaicism. Traits or diseases are considered to have multifactorial inheritance if they are determined by the combination of multiple genes and environmental factors (Table 13-6). Polygenic traits are determined by the combined efects of more than one gene. Most congenital and acquired conditions, as well as common traits, display multifactorial inheritance. Examples include malformations such as clefts and neural-tube defects, diseases such as diabetes and heart disease, and features or traits such as head TABLE 13-6. Characteristics of Multifactorial Diseases

1	TABLE 13-6. Characteristics of Multifactorial Diseases There is a genetic contribution: No mendelian pattern of inheritance No evidence of single-gene disorder Nongenetic factors are also involved in disease causation: Lack of penetrance despite predisposing genotype Monozygotic twins may be discordant Familial aggregation may be present: Relatives are more likely to have disease-predisposing alleles Expression more common among close relatives: Adapted from Nussbaum, 2007. Genetics 269 FIGURE 13-9 Mechanism of uniparental disomy arising from trisomic "rescue." A. In normal meiosis, one member of each pair of homologous chromosomes is inherited from each parent. B. If nondisjunction results in a trisomic conceptus, one homologue is sometimes lost. In a third of cases, loss of one homologue leads to uniparental disomy.

1	size or height. Abnormalities that display multifactorial inheritance tend to recur in families, but not according to a mendelian pattern. If a couple has had a child with a multifactorial birth defect, their empirical risk to have another afected child is 3 to 5 percent. This risk declines exponentially with successively more distant relationships. Multifactorial traits that have a normal distribution in the population are termed continuously variable. A measurement that is more than two standard deviations above or below the population mean is considered abnormal. Continuously variable traits tend to be less extreme in the ofspring of afected individuals, because of the statistical principle of regression to the mean.

1	Some multifactorial traits do not appear until a threshold is exceeded. Genetic and environmental factors that create propensity or liability for the trait are themselves normally distributed, and only individuals at the extreme of the distribution exceed the threshold and exhibit the trait or defect. Phenotypic abnormality is thus an all-or-none phenomenon. Examples include cleft lip-palate and pyloric stenosis.

1	Certain threshold traits have a clear male or female predominance. If an individual of the less common gender has the characteristic or defect, the recurrence risk is greater in his or her ofspring (Fig. 13-10). An example is pyloric stenosis, which is approximately four times more common in males (Krogh, 2012). A female with pyloric stenosis has likely inherited more predisposing genetic factors than are necessary to produce the defect in a male, and the recurrence risk for her children or siblings is thus higher than the expected 3 to 5 percent. Her male siblings or male ofspring would have the highest liability because they not only will inherit more than the usual number of predisposing genes but also are the more susceptible gender.

1	he recurrence risk for threshold traits is also greater if the defect is severe. For example, the recurrence risk after the birth of a child with bilateral cleft lip and palate is approximately 8 percent, but it is only about 4 percent following a child with unilateral cleft lip alone. Structural cardiac anomalies are the most common birth defects, with a birth prevalence of 8 cases per 1000. More than o.�c 5. o..= FIGURE 13-10 Schematic example of a threshold trait, such as pyloric stenosis, which has a predilection for males. Each gender is normally distributed, but at the same threshold, more males than females will develop the condition. 100 genes believed to be involved in cardiovascular morphogenesis have been identiied, including those directing production of various proteins, protein receptors, and transcription factors (Olson, 2006; Weismann, 2007).

1	he risk of having a child with a cardiac anomaly is approximately 5 to 6 percent if the mother has the defect and 2 to 3 percent if the father has the defect (Burn, 1998). Selected left-sided lesions, including hypoplastic left heart syndrome, coarctation of the aorta, and bicuspid aortic valve, may have recurrence risks four-to sixfold higher (Lin, 1988; Lupton, 2002; Nora, 1988). Observed recurrence risks for specific cardiac malformations are listed in Table 49-4 (p. 953).

1	These disorders are also classic examples of multifactorial inheritance. Development of neural-tube defects (NTDs) may be influenced by hyperthermia, hyperglycemia, teratogen exposure, ethnicity, family history, fetal gender, and various genes. Selected risks are more strongly associated with the specific defect location. Hyperthermia has been linked with anencephaly risk; pregestational diabetes with cranial and cervicalthoracic defects; and valproic acid exposure with lumbosacral defects (Becerra, 1990; Hunter, 1984; Lindhout, 1992). Sonographic features ofNTDs are described in Chapter 10 (p. 192), their prevention with folic acid is discussed in Chapter 9 (p. 169), and fetal therapy for myelomeningocele is reviewed in Chapter 16 (p. 319).

1	More than 50 years ago, Hibbard and Smithells (1965) postulated that abnormal folate metabolism was responsible for many NTDs. For a woman with a prior afected child, the recurrence risk of 3 to 5 percent is decreased by at least 70 percent-and potentially by as much as 85 to 90 percent-with periconceptional oral folic acid supplementation at a dosage of 4 mg/d (Grosse, 2007; MRC Vitamin Study Research Group, 1991). However, most NTD cases do not occur in the setting of maternal folic acid deiciency, and it has become clear that the gene-nutrient interactions underlying folate-responsive NTDs are complex. he NTD risk may be afected by genetic variation in folate transport or accumulation, impaired folate utilization via secondary nutrient deficiencies such as vitamin BI2 or choline deficiency, and genetic variation in activity of folate-dependent metabolic enzymes (Beaudin, 2009).

1	All pregnant women should have the option of prenatal aneuploidy screening and prenatal genetic diagnosis (American College of Obstetricians and Gynecologists, 2016b). Aneuploidy screening may be performed with serum analyte-based screening or with a DNA-based screen, namely, cell-free DNA found in the maternal circulation. Prenatal genetic screening of the parents also aids carrier status determination in at-risk individuals (Chap. 14, p. 288).

1	For prenatal genetic diagnosis, the most commonly used tests are cytogenetic analysis (karyotyping), luorescence in situ hybridization (FISH), and chromosomal microarray analysis. Testing may be performed on amnionic fluid or chorionic villi. In selected circumstances, whole genome or whole exome sequencing may be considered, but these are not recommended for routine use. To diagnose a specific disease whose genetic basis is known, DNA-based tests are often employed, typically using polymerase chain reaction (PCR) for rapid ampliication of DNA sequences. Karyotype analysis is commonly performed to test for chromosomal abnormalities. Any tissue containing dividing cells or cells that can be stimulated to divide is suitable for cytogenetic analysis. Karyotyping detects numerical abnormalities, that is, aneuploidy. It also identiies balanced or unbalanced structural rearrangements of at least 5 to 10 megabases in size. Karyotyping has diagnostic accuracy exceeding 99 percent.

1	The dividing cells are arrested in metaphase, and their chromosomes are stained to reveal light and dark bands. The most commonly used technique is Giemsa staining, which yields the G-bands shown in Figure 13-3. Each chromosome has a unique banding pattern that permits its identiication and detection of deleted, duplicated, or rearranged segments. he accuracy of cytogenetic analysis rises with the number of bands produced. High-resolution metaphase banding routinely yields 450 to 550 visible bands per haploid chromosome set. Banding of prophase chromosomes generally yields 850 bands.

1	Because only dividing cells can be evaluated, the rapidity with which results are obtained correlates with the rapidity of cell growth in culture. Amnionic fluid, which contains epithelial cells, gastrointestinal mucosal cells, and amniocytes, usually yields results in 7 to 10 days. Fetal blood cells may provide results in 36 to 48 hours but are rarely needed (Chap. 14, p. 294). If fetal skin fibroblasts are evaluated postmortem, stimulation of cell growth can be more diicult, and cytogenetic analysis may take 2 to 3 weeks (Chap. 35, p. 647).

1	This technique may be used for rapid identiication of a speciic chromosome abnormality and for veriication of suspected microdeletion or duplication syndromes, such as the 22q 11.2 microdeletion described earlier (p. 260). Because of its 1-to 2-day turnaround time, FISH is often selected for cases in which indings may alter pregnancy management. To perform FISH, cells are ixed onto a glass slide, and fluorescent-labeled probes are hybridized to the ixed chromosomes (Figs. 13-11 and 13-12). Each probe is a DNA sequence that is complementary to a region of the chromosome or gene being investigated. If the DNA sequence is present, hybridization is detected as a bright signal visible by microscopy. The number of signals indicates the number of chromosomes or genes of that type in the cell being analyzed. Findings are probe-speciic. Namely, FISH does not provide information on the entire chromosomal complement but merely the chromosomal or gene region of interest.

1	he most common prenatal application of FISH involves testing interphase chromosomes with DNA sequences speciic to chromosomes 21, 18, 13, X, and Y. Figure 13-12 shows an example of interphase FISH using a-satellite probes for chromosomes 18, X, and Y to conirm trisomy 18. In a review of FIGURE 13-1 1 Steps in fluorescence in situ hybridization (FISH). more than 45,000 samples, the concordance between FISH analysis and standard cytogenetic karyotyping was 99.S percent (Tepperberg, 2001). he American College of Obstetricians and Gynecologists (20 16b) recommends that clinical decision-making based on FISH incorporate clinical information consistent with the suspected diagnosis, such as an abnormal aneuploidy screening test result or sonographic inding, or incorporate a conirmatory diagnostic test such as karyotyping or CMA. his test is 100 times more sensitive than standard karyotyping and detects microduplications and microdeletions as small as

1	his test is 100 times more sensitive than standard karyotyping and detects microduplications and microdeletions as small as FIGURE 13-12 Interphase fluorescence in situ hybridization (FISH) using a-satellite probes for chromosomes 18, X, and Y. In this case, the three light blue signals, two green signals, and absence of red signals indicate that this is a female fetus with trisomy 18. (Used with permission from Dr. Frederick Elder.)

1	Fluorescent probe illuminates chromosome region of interest 50 to 100 kilobases. Direct CMA can yield results in 3 to 5 days, whereas if cultured cells are required, results may take 10 to 14 days (American College of Obstetricians and Gynecologists, 2016b). Microarrays use either a comparative genomic hybridization (CGH) platform, a single-nucleotide polymorphism (SNP) platform, or a combination of the two. he CGH microarray platform compares test specimen DNA with a normal control sample. Shown in Figure 13-13, the CGH chip contains reference DNA fragments of known sequenceoligonucleotides. Fetal DNA from the amniocentesis or chorionic villus sampling specimen is labeled with a fluorescent dye and then hybridized to the DNA on the chip. Normal control DNA is labeled with a diferent probe and also hybridized to the chip. hen, the intensity of the luorescent signals from the two samples is compared. With an SNP array, the chip contains known DNA sequence variants-single-nucleotide

1	and also hybridized to the chip. hen, the intensity of the luorescent signals from the two samples is compared. With an SNP array, the chip contains known DNA sequence variants-single-nucleotide polymorphisms. When fetal DNA is labeled and hybridized to the chip, the luorescent signal intensity indicates copy number variation.

1	Both types of platforms detect aneuploidy, unbalanced translocations, and microdeletions and microduplications. Neither type of array platform currently detects balanced chromosomal rearrangements. For this reason, couples with recurrent pregnancy loss should be ofered karyotyping as the first-line test (Society for Maternal-Fetal Medicine, 2016). In addition, SNP arrays are able to identiY triploidy and can detect absence of heterozygosiy. he latter can occur with uniparental disomy when both copies of a chromosome are inherited from one parent. Further, absence of heterozygosity may occur when there is consanguinity, and counseling prior to performance of an SNP array should include this possibility.

1	Arrays may be genome-wide or may be targeted to known genetic syndromes. Genome-wide arrays are typically used in research settings, for example, to identiY novel microdeletion syndromes in individuals with intellectual disability (Slavotinek, 200S). Targeted arrays are generally preferred prenatally because the likelihood of detecting a copy number variant of uncertain clinical signiicance is lower. In a systematic 1.28 cm A Actual size of chip Nonhybridized Thousands of identical nucleotide DNA strands on one cell B 500,000 cells on each chip C One cell on chip Labeled fetal DNA is presented to the cells

1	FIGURE 13-13 Chromosomal microarray analysis. A. Actual microarray chip size. B. Each chip contains thousands of cells (squares). C & D. Each cell contains thousands of identical oligonucleotides on its surface, and each cell is unique in its nucleotide content. E. During genetic analysis, a mixture containing tagged fetal DNA is presented to the chip. Complementary DNA sequences bind. F. If a laser is shined on the chip, DNA sequences that have bound will glow. This identifies a matching sequence. (Modified with permission from Doody J: Treatment of the infertile couple. In Hofman BL, Schorge JO, Schaffer JI, et al (eds): Williams Gynecology, 2nd ed. New York, McGraw-Hili, 2012.) review, Hillman and colleagues (2013) identified copy number variants of uncertain significance in 1 to 2 percent of prenatal specimens. Not unexpectedly, this may be a source of signiicant distress to families, even with comprehensive pretest counseling.

1	In pregnancies at increased risk for autosomal trisomy based on aneuploidy screening, karyotyping or FISH plus karyotyping should be ofered, and CMA should be made available (American College of Obstetricians and Gynecologists, 2016b). When the karyotype is normal, CMA has identiied clinically relevant copy number variants in approximately 6.5 percent of pregnancies with fetal abnormalities and in 1 to 2 percent with no obvious fetal abnormality (Callaway, 2013). he American College of Obstetricians and Gynecologists (2016b) and the Society for Maternal-Fetal Medicine (2016) recommend that CMA be ofered as a irst-tier test when fetal structural abnormalities are identiied, replacing fetal karyotyping in these cases. If a particular anomaly that strongly suggests a speciic aneuploidy is identiied, such as an endocardial cushion defect (trisomy 21) or alobar holoprosencephaly (trisomy 13), karyotyping or FISH may be ofered as the initial test. It is recommended that genetic counseling

1	such as an endocardial cushion defect (trisomy 21) or alobar holoprosencephaly (trisomy 13), karyotyping or FISH may be ofered as the initial test. It is recommended that genetic counseling include information about the beneits and limitations of both CMA and karyotyping, and that each be made available to women who elect prenatal diagnosis (Society for Maternal-Fetal Medicine, 2016). CMA may identiy instances of autosomal dominant genetic disorders that have not yet manifested in an afected parent, and it may also identiy instances of nonpaternity.

1	For stillbirth evaluation, CMA is more likely than standard karyotyping to provide a genetic diagnosis, in part because it does not require dividing cells. he Stillbirth Collaborative Research Network found that when karyotyping was uninformative, approximately 6 percent of cases had either aneuploidy or a pathogenic copy number variant identiied with CMA (Reddy, 2012). Overall, CMA yields results nearly 25 percent more often than standard karyotyping alone.

1	Most fetuses with structural abnormalities have a normal karyotype and a normal CMA result. Whole genome sequencing (WGS) is a technique for analyzing the entire genome. Whole exome sequencing (WES) analyzes just the DNA coding regions, which account for approximately 1 percent of the genome. These next-generation sequencing tools are increasingly used in the postnatal setting to evaluate suspected genetic syndromes and intellectual disability. The American College of Medical Genetics Board of Directors (2012) states that WGS and WES may be considered for evaluation of the fetus with a likely genetic disorder in which CMA has failed to arrive at a diagnosis. The American College of 0bstetricians and Gynecologists (201i6a) suggests that this be in only selected circumstances, for example, with recurrent or lethal anomalies in which other approaches have been noninformative. Importantly, WGS and WES have signiicant limitations in their current form, including turnaround times that may

1	with recurrent or lethal anomalies in which other approaches have been noninformative. Importantly, WGS and WES have signiicant limitations in their current form, including turnaround times that may be prohibitively long and a high rate of variants of uncertain signiicance (American College of �1edical Genetics, 2012; Atwal, 2014). As a result, the clinical utility of this promising technology for prenatal cases is currently limited. • Fetal DNA in the Maternal Circulation Fetal cells are present in maternal blood at a very low concentration, only 2 to 6 cells per milliliter (Bianchi, 2006). Sometimes, intact fetal cells may persist in the maternal circulation for decades following delivery. Persistent fetal cells may engraft in the mother and result in microchimerism, which has been implicated in maternal autoimmune diseases such as scleroderma, systemic lupus erythematosus, and Hashimoto thyroiditis. For prenatal diagnosis, the use of intact fetal cells from maternal blood is

1	implicated in maternal autoimmune diseases such as scleroderma, systemic lupus erythematosus, and Hashimoto thyroiditis. For prenatal diagnosis, the use of intact fetal cells from maternal blood is limited by low cell concentration, cell persistence into successive pregnancies, and diiculties in distinguishing fetal from maternal cells. In these cases, however, cell-free DNA overcomes these limitations. Cell-Free DNA hese DNA fragments are derived from maternal cells and from apoptotic placental trophoblast cells-although DNA from the latter is often termed "fetal." Cell-free DNA can be reliably genotyping Maternal Real-time quantitative PCR Detection of single-gene disorders Genetics 273 detected in maternal blood after 9 to 10 weeks' gestation (American College of 0bstetricians and Gynecologists, 20 17b). he proportion of cell-free DNA that is placental is called the fetal fraction, and it composes approximately 10 percent of the total circulating cell-free DNA in maternal plasma.

1	20 17b). he proportion of cell-free DNA that is placental is called the fetal fraction, and it composes approximately 10 percent of the total circulating cell-free DNA in maternal plasma. Unlike intact fetal cells, cell-free DNA is cleared within minutes from maternal blood. In research settings, cell-free DNA has been used to detect numerous single-gene disorders transmitted through paternally inherited alleles. These include myotonic dystrophy, achondroplasia, Huntington disease, congenital adrenal hyperplasia, cystic fibrosis, and a-thalassemia (Wright, 2009). Clinical applications of cell-free DNA are aneuploidy screening, fetal sex determination, and h D genotyping (Fig. 13-14). Aneuploidy Screening. Several diferent types of assays are used to screen for fetal autosomal trisomies and sex chromosomal aneuploidies. hese include whole-genome sequencing, which is also called massively parallel or shotgun sequencing; chromosome selective or targeted sequencing; and analysis of SNPs

1	sex chromosomal aneuploidies. hese include whole-genome sequencing, which is also called massively parallel or shotgun sequencing; chromosome selective or targeted sequencing; and analysis of SNPs (American College of Obstetricians and Gynecologists, 2016a,b). By simultaneously sequencing millions of DNA fragments, investigators can identiy whether the proportion or ratio of fragments from one chromosome is higher than expected. Sequences of fetal DNA are specific to individual chromosomes. hus, samples from women with a Down syndrome fetus have a larger proportion of DNA sequences from chromosome 21. The screening performance of cell-free DNA is excellent. In a metaanalysis of 37 studies of largely high-risk pregnancies, the pooled sensitivity to detect Down syndrome was 99 percent, and to identiy trisomies 18 and 13, 96 and 91 percent, respectively. For each, the speciicity was 99.9 percent (Gil, 2015). The false-positive rate is cumulative for each aneuploidy for which screening is

1	identiy trisomies 18 and 13, 96 and 91 percent, respectively. For each, the speciicity was 99.9 percent (Gil, 2015). The false-positive rate is cumulative for each aneuploidy for which screening is performed, but it is usually below 1 percent. As a result, cell-free DNA screening is recommended as !DNA isolation Cell-free fetal DNA determination Massively parallel genomic sequencing Trisomy 21, 18, and 13screening FIGURE 13-14 Cell-free DNA is actually derived from apoptotic trophoblast. The DNA is isolated from maternal plasma, and real-time quantitative polymerase chain reaction (PCR) may be used to target specific regions or sequences. This may be used for Rh D genotyping, detection of paternally inherited single-gene disorders, or fetal sex determination. Screening for autosomal trisomies and sex chromosomal aneuploidies is performed using whole-genome sequencing, chromosome selective or targeted sequencing, and analysis of single nucleotide polymorphisms.

1	a screening option in those at greater risk for fetal autosomal trisomy (American College of Obstetricians and Gynecologists, 2017b; Society for Maternal-Fetal Medicine, 2015). Unfortunately, cell-free DNA screens do not yield a result in 4 to 8 percent of cases. This may be due to assay failure, high assay variance, or low fetal fraction (Norton, 2012; Pergame nt, 2014; Quezada, 2015). Such pregnancies carry a greater risk for fetal aneuploidy. In addition, results may not relect the fetal DNA complement but rather may indicate conined placental mosaicism, early demise of an aneuploid cotwin, maternal mosaicism, or rarely occult maternal malignancy (Bianchi, 2015; Curnow, 2015; Grati, 2014b; Wang, 2014). Recommendations for counseling are discussed in Chapter 14 (p. 285).

1	Fetal Sex Determination. From the standpoint of genetic disease, fetal sex determination may be clinically useful if the fetus is at risk for an X-linked disorder. It may also be beneficial if the fetus is at risk for congenital adrenal hyperplasia because maternal corticosteroid therapy may be avoided if the fetus is male (Chap. 16, p. 317). In a metaanalysis of more than 6000 pregnancies by Devaney and associates (201i1), the sensitivity of cell-free DNA testing for fetal sex determination approximated 95 percent between 7 and 12 weeks' gestation and improved to 99 percent after 20 weeks. The test speciicity was 99 percent at both time periods, suggesting that cellfree fetal DNA is a reasonable alternative to invasive testing in selected cases.

1	Rh D Genotype In a predominantly white population, nearly 40 percent of fetuses of h D-negative women are themselves h D negative. Fetal h D genotype assessment from maternal blood can eliminate administration of anti-D immune globulin in these pregnancies, thereby reducing cost and potential risk. With h D alloimmunization, early identification of an h D-negative fetus might avoid unnecessary middle cerebral artery Doppler assessment or amniocentesis. Evaluation using cell-free DNA is done using real-time PCR to target several exons of the HD gene. These are typically exons 4,i5, and 7.

1	h D-genotyping is performed routinely with cell-free DNA in Denmark and the Netherlands (Clausen, 2012; de Haas, 2016). In a population-based study of more than 25,000 h D-negative women screened at 27 weeks, the false-negative rate-in which h D-negative status was missed-was only 0.03 percent. The false-positive rate-in which h immune globulin would be given unnecessarily-was less than 1 percent (de Haas, 2016). Similar results were reported from the United Kingdom, although the false-negative rate was higher in the irst trimester (Chitty, 2014). Investigators concluded that false-negative screening results might increase the alloimmunization risk, but by less than 1 case per million births (Chitty, 2014). h D alloimmunization is discussed in Chapter 15 (p. 301). Abele H, Babiy-Pachomow 0, Sonek J, et al: The cavum septum pellucidi in euploid and aneuploidy fetuses. Ultrasound Obstet Gynecol 2013; 42(2):156,o2013

1	Abele H, Babiy-Pachomow 0, Sonek J, et al: The cavum septum pellucidi in euploid and aneuploidy fetuses. Ultrasound Obstet Gynecol 2013; 42(2):156,o2013 American Academy of Pediatrics: Clinical report: cardiovascular health supervision for individuals afected by Duchenne or Becker muscular dystrophy. Pediatrics 116(6):1569,o2005, Reairmed December 2008 American College of Medical Genetics (ACMG) Board of Directors: Points to consider in the clinicl application of genomic sequencing. Genet Med 14(8):759,o2012 American College of Obstetricians and Gynecologists: Microarrays and nextgeneration sequencing technology: the use of advanced genetic diagnostic tools in obstetrics and gynecology. Committee Opinion No. 682, December 20o16a American College of Obstetricians and Gynecologists: Prenatal diagnostic testing for genetic disorders. Practice Bulletin No. 162, May 2016b

1	American College of Obstetricians and Gynecologists: Prenatal diagnostic testing for genetic disorders. Practice Bulletin No. 162, May 2016b American College of Obstetricians and Gynecologists: Primary ovarian insuficiency. Committee Opinion No. 605, July 2014, Reairmed 2016c American College of Obstetricians and Gynecologists: Screening for fetal aneuploidy. Practice Bulletin No. 163, May 2016d American College of Obstetricians and Gynecologists: Carrier screening for genetic conditions. Committee Opinion No. 69o1, March 20 17a American College of Obstetricians and Gynecologists: Cell free DNA screening for fetal aneuploidy. Committee Opinion No. 640, September 2015, Reafirmed 2017b American College of Obstetricians and Gynecologists: Management of women with phenylketonuria. Committee Opinion No. 636, June 20o15, Reairmed 20ol7c

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1	Reddy UM, Goldenberg R, Silver R, et al: Stillbirth classification-developing an international consensus for research: executive summary of a National Institute of Child Health and Human Development workshop. Obstet Gynecol 114(4):901,o2009 Reddy UM, Grier PP, Saade GR, et al: Karyotype versus microarray testing for genetic abnormalities ater stillbirth. N Engl J Med 367(23):2185,2012 Rehm HL, BergJS, Brooks LD, et al: ClinGen-he Clinical Genome Resource. N Engl J Med 372(23):2235, 2015 Romeo G, Bittles AH: Consanguinity in the contemporary world. Hum Hered 77(1):6,o2014 Rosa F, Rosa Re, Lorenzen MB, et al: Trisomy 18: experience of a reference hospital from the south of Brazil. Am J Med Genet A 155A(7):1529, 2011 Ross JL, Zeger MP, Kushner H, et al: An extra X or Y chromosome: contrasting the cognitive and motor phenotypes in childhood in boys with 47,yysyndrome or 47,XY Klinefelter syndrome. Dev Disabil Res Rev 15(4):309, 2009 Scharrer S, Stengel-Rutkowski S, Rodewald-Rudescu A, et

1	the cognitive and motor phenotypes in childhood in boys with 47,yysyndrome or 47,XY Klinefelter syndrome. Dev Disabil Res Rev 15(4):309, 2009 Scharrer S, Stengel-Rutkowski S, Rodewald-Rudescu A, et al: Reproduction in a female patient with Down's syndrome. Case report of a 46,y child showing slight phenotypical anomalies born to a 47,X, +21 mother. Humangenetik 26(3):207, 1975 Schmickel RD: Contiguous gene syndromes: a component of recognizable syndromes. J Pediatr 109(2):231,o1986 Schneider AS, Mennuti MT, Zackai EH: High cesarean section rate in trisomy 18 births: a potential indication for late prenatal diagnosis. m J Obstet GynecoIo140(4):367, 1981 Schorge JO: Ovarian germ cell and sex cord-stromal tumors. In Hofman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hill Education, 20o16 Shafer LG, Agan N, Goldberg JD, et al: American College of Medical Genetics Statement on diagnostic testing for uniparental disomy. Genet Med 3(3):206,o2001

1	Sheridan E, Wright J, Small N, et al: Risk factors for congenital anomaly in a multiethnic birth cohort: an analysis of the Born in Branford study. Lancet 382(9901):1350,o2013 Shin M, Besser LM, Kucik JE, et al: Prevalence of Down syndrome in children and adolescents in 10 regions of the United States. Pediatrics 124(6):1o565,o2009 Shipp TD, Benacerraf BR: Second trimester ultrasound screening for chromo somal abnormalities. Prenat Diagn 22(4):296, 2002 Shprintzen RJ: Velo-cardio-facial syndrome: 30 years of study. Dev Disabil Res Rev 14(1):3,o2008 Silasi M, Rana S, Powe e, et al: Placental expression of angiogenic factors in trisomy 13. Am J Obstet Gynecol 204(6):546.e1, 2011 Siavotinek AM: Novel micro deletion syndromes detected by chromosomal microarrays. Hum Genet 124(1):1, 2008 Society for Maternal-Fetal Medicine: Prenatal aneuploidy screening using cellfree DNA. SMFM Consult Series No. 36. June 2015

1	Society for Maternal-Fetal Medicine: Prenatal aneuploidy screening using cellfree DNA. SMFM Consult Series No. 36. June 2015 Society for Maternal-Fetal Medicine: The use of chromosomal micro array for prenatal diagnosis. SMHvl Consult Series No. 41. October 2016 Spence JE, Perciaccante RG, Greig FM, et al: Uniparental disomy as a mechanism for human genetic disease. Am J Hum Genet 42(2):217,o1988 Spotila LD, Sereda L, Prockop DJ: Partial isodisomy for maternal chromosome 7 and short stature in an individual with a mutation at the COLIA2 locus. m J Hum Genet 51(6):1396, 1992 Stevenson DA, Carey JC: Contribution of malformations and genetic disorders to mortality in a children's hospital. Am J Med Genet 126A(4):393, 2004 Stoltenberg C, Magnus P, Lie RT, et al: Birth defects and parental consanguin ity in Norway. m J Epidemiol 145(5):439, 1997

1	Tartaglia N, Ayari N, Howell S, et al: 48,YY, 48,XY, and 49,UXY syndromes: not just variants of Klinefelter syndrome. Acta Paediatrica 100(6):851,o2011 Tartaglia NR, Howell S, Sutherland A, et al: A review of trisomy X (47,XX). Orphanet J Rare Dis 5:8, 2010 Tennant PW, Pearce MS, Bythell M, et al: 20-year survival of children born with congenital anomalies: a population-based study. Lancet 375(9715):649, 2010 Tepperberg J, Pettenati MJ, Rao PN, et al: Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH): 2-year multi-center retrospective study and review of the literature. Prenat Diagn 21(4):293,o2001 Tinkle BT, Walker ME, Blough-Pfau I, et al: Unexpected survival in a case of prenatally diagnosed non-mosaic trisomy 22: clinical report and review of the natural history. Am J Med Genet A 118A(1):90, 2003 Toriello HV, Meck JM, Professional Practice and Guidelines Committee:

1	Toriello HV, Meck JM, Professional Practice and Guidelines Committee: Statement on guidance for genetic counseling in advanced paternal age. Genet Med 10(6):457, 2008 Tuohy JF, James DK: Pre-eclampsia and trisomy 13. BJOG 99(11):891, 1992 Turner HH: A syndrome of infantilism, congenital webbed neck and cubitus valgus. Endocrinol 23:566, 1938 Vendola e, Canfield M, Daiger SP, et al: Survival of Texas infants born with trisomies 21, 18, and 13. Am J Med Genet A 152A(2):360, 2010 Vintzileos Ni, Egan JF: Adjusting the risk for trisomy 21 on the basis of second-trimester ultrasonography. Am J Obstet GynecoIo172(3):837, 1995 Vockley J, ndersson He, Antshel KM, et al: ACMG Practice Guidelines: phenylalanine hydroxylase deficiency: diagnosis and management guideline. Genet Med 16(2):188,o2014 Wang Y, Chen Y, Tian F, et al: Maternal mosaicism is a significant contributor to discordant sex chromosomal aneuploidies associated with non-invasive prenatal testing. Clin Chen 60(1):251, 2014

1	Wang Y, Chen Y, Tian F, et al: Maternal mosaicism is a significant contributor to discordant sex chromosomal aneuploidies associated with non-invasive prenatal testing. Clin Chen 60(1):251, 2014 Weismann CG, Gelb BD: The genetics of congenital heart disease: a review of recent developments. Curr Opin Cardiol 22(3):200, 2007 Wellesley D, Dolk H, Boyd PA, et al: Rare chromosome abnormalities, prevalence, and prenatal diagnosis rates from population-based congenital anomaly registers in Europe. Eur J Human Genet 20(5):521,o2012 Wigby K, D'Epagnier e, Howell S, et al: Expanding the phenotype of triple X syndrome: a comparison of prenatal versus postnatal diagnosis. Am J Med Genet Part A 170(11):2870, 2016 Worton RG, Stern R: A Canadian collaborative study of mosaicism in amniotic fluid cell cultures. Prenat Diagn 4(7): 131, 1984

1	Worton RG, Stern R: A Canadian collaborative study of mosaicism in amniotic fluid cell cultures. Prenat Diagn 4(7): 131, 1984 Wou K, Hyun Y, Chitayat D, et al: Analysis of tissue from products of conception and perinatal losses using QF-PCR and microarray: a three-year retrospective study resulting in an eicient protocol. Eur J Med Genet 59(8):417, 2016 Wright CF, Burton H: he use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis. Hum Reprod Update 15(1):139,o2009 Yang Q, Wen SW, Leader A, et al: Paternal age and birth defects: how strong is the association? Human Reprod 22(3):696, 2007 Yeo L, Guzman ER, Day-Salvatore D, et al: Prenatal detection of fetal trisomy 18 through abnormal sonographic features. J Ultrasound Med 22(6):581, 2003 Zalel Y, Shapiro I, Weissmann-Brenner A, et al: Prenatal sonographic features of triploidy at 12-16 weeks. Prenat Diagn 36(7)650, 2016 HISTORICAL PERSPECTIVE ..e..e.e.e....e....e277

1	Zalel Y, Shapiro I, Weissmann-Brenner A, et al: Prenatal sonographic features of triploidy at 12-16 weeks. Prenat Diagn 36(7)650, 2016 HISTORICAL PERSPECTIVE ..e..e.e.e....e....e277 FIRST-TRIMESTER ANEUPLOIDY SCREENING ...e. 281 SECOND-TRIMESTER ANEUPLOIDY SCREENING .e282 INTEGRATED AND SEQUENTIAL SCREENING ...e.. 284 CELL-FREE DNA SCREENING ..e..e.e.e.e284 SONOGRAPHIC SCREENING ...e..e..e.e..e.e...e286 CARRIER SCREENING FOR GENETIC DISORDERS .e... 288 AMNIOCENTESIS AND CHORIONIC VILLUS SAMPLINGe. .e291 PREIMPLANTATION GENETIC TESTING .e.e.....e.. 295 Carul examination should ordinariy lead to a correct diagnosis of hydrocephalus in the last weeks of pregnancy. In many cases the dormiy can be detected by external papation. -]. Whitridge Williams (1903)

1	-]. Whitridge Williams (1903) In the initial edition of Williams Obstetrics, very few fetal disorders could be identiied before delivery. Now, more than 100 years later, prenatal diagnosis has become a separate field of its own. Strictly speaking, prenatal diagnosis is the science of identiying congenital abnormalities, aneuploidies, and other genetic syndromes in the fetus. It encompasses the diagnosis of structural malformations with specialized sonography; routine screening tests for aneuploidy and neural-tube defects; diagnostic tests such as karyotyping and chromosomal microarray analysis performed on chorionic villi and amniocentesis specimens; and additional screening and diagnostic tests ofered to those with pregnancies at risk for speciic genetic disorders. he goal of prenatal diagnosis is to provide accurate information regarding short-and long-term prognosis, recurrence risk, and potential therapy, thereby improving patient counseling and optimizing outcomes.

1	Management of an afected pregnancy, including whether a woman would elect pregnancy termination, may be incorporated into the discussion of screening and testing options. However, nondirective counseling is central to prenatal diagnosis. his practice provides the patient with unbiased knowledge regarding a diagnosis and preserves her autonomy (Flessel, 2011). Fetal imaging of congenital anomalies is discussed in Chapter 10, and pregnancy termination is discussed in Chapter 18.

1	More than 40 years ago, Brock (1972, 1973) observed that pregnancies complicated by neural-tube defects had higher levels of alpha-fetoprotein (AFP) in maternal serum and amnionic luid. his formed the basis for the irst maternal serum screening test for a fetal condition. The beginning of widespread serum screening came in 1977, after a collaborative trial from the United Kingdom established the association between elevated maternal serum AFP levels (MSAFP) and fetal open neural-tube defects (Wald, 1977). When screening was performed at 16 to 18 weeks' gestation, detection approached 90 percent for pregnancies with fetal anencephaly and 80 percent for those with myelomeningocele (spina biida). hese sensitivities are comparable to current testing (American College of Obstetricians and Gynecologists, 20 16a).

1	The terms level I and level II sonography were coined in this context. In the California MSAFP Screening Program of the 1980s and early 1990s, women received serum screening prior to sonography, and those with an elevated MSAFP level would undergo level I sonography to identiy an incorrect gestational age, mulrifetal gestation, or fetal demise (Filly, 1993). A third of pregnancies with an elevated MSAFP level had one of these three etiologies. Although birth defects were occasionally detected during level I sonography, this was not the expectation. If level I sonography did not identiy an etiology for the MSAFP level elevation, amniocentesis would be ofered. Then, only if the amnionic luid AFP concentration were elevated would the woman undergo level II sonography. his more detailed and comprehensive survey of fetal anatomy was performed to detect and characterize the fetal abnormality.

1	If the amnionic fluid AFP level was elevated, an assay for amnionic luid acetylcholinesterase was concurrently performed. his capitalized on the tendency of acetylcholinesterase to leak directly from exposed neural tissue into the amnionic luid. The presence of both analytes in the amnionic luid was considered diagnostic for neural-tube defects (American College of Obstetricians and Gynecologists, 2016a).

1	The overall sensitivity of amniocentesis to diagnose open neural-tube defects approximates 98 percent, with a false-positive rate of 0.4 percent (Milunsky, 2004). Importantly, other fetal abnormalities are associated with elevated amnionic fluid AFP levels and positive assay results for acetylcholinesterase. These include ventral wall defects, esophageal atresia, fetal teratoma, cloacal exstrophy, and skin abnormalities such as epidermolysis bullosa. hus, by current standards, these amnionic luid analytes would be considered ancillary screening tests, understanding that a positive result would prompt additional fetal imaging.

1	With current imaging technology, most neural-tube defects are detected with sonography, and targeted sonography is the diagnostic test of choice (Dashe, 2006). Pregnant women now have the option of neural-tube defect screening with either MSAFP or sonography (American College of Obstetricians and Gynecologists, 20 16c). Although level II is used as a synonym for targeted sonography, the former might best be removed from our lexicon. Namely, today's targeted sonography includes a much more comprehensive evaluation of fetal anatomy (Chap. 10, p. 187).

1	As MSAFP screening was being adopted, the designation «advanced maternal age" (AMA) became popular. A 1979 National Institutes of Health Consensus Development Conference recommended advising pregnant women who were 35 years and older about the possibility of amniocentesis for fetal karyotyping. he threshold was based on the greater risk for selected fetal chromosomal abnormalities with increasing maternal age and on the assumption that at that time, the loss rate attributable to amniocentesis was equivalent to the fetal Down syndrome risk at maternal age 35. Notaby, this is no longer the case, as discussed later (p. 293).

1	Serum aneuploidy screening soon became available for women who would be younger than 35 at delivery. In 1984, Merkatz and colleagues reported that MSAFP levels were lower in pregnancies with trisomies 21 and 18 at 15 to 21 weeks' gestation. Maternal age was incorporated into the calculation, such that a speciic risk could be assigned (DiMaio, 1987; New England Regional Geneties Group, 1989). The MSAFP screen detected approximately 25 percent of cases of fetal trisomy 21 when the threshold ratio for a positive result was set at 1 :270. This ratio relects the approximate second-trimester risk for Down syndrome at maternal age 35. This trisomy 21 FIGURE 14-1 Trends in the percentage of births to women aged 35 to 44 years. (Data from the Centers for Disease Control and Prevention, 201o5.) risk threshold and the associated 5-percent false-positive rate became standards that remain in use in some laboratories today.

1	For more than a decade after its introduction, serum aneuploidy screening was intended for women younger than 35, because it simply did not have suicient sensitivity to be ofered to women who had higher a priori risk. This is also no longer the case. And, because the prevalence of fetal aneuploidy rises sharply with maternal age, the positive-predictive value of all aneuploidy screening tests-whether analyte-based or cellfree DNA tests-is higher in women aged 35 years or older. Women 35 and older now make up more than 15 percent of deliveries in the United States (Fig. 14-1). At Parkland Hospital, this age group accounts for half of births with Down syndrome (Hussamy, 2017).

1	Aneuploidy is the presence of one or more extra chromosomes, usually resulting in trisomy, or loss of a chromosomemonosomy. Data from population-based registries that include births, fetal deaths, and pregnancy terminations indicate an overall prevalence of 4 such abnormalities per 1000 births (Wellesley, 2012). Aneuploidy accounts for more than 50 percent of first-trimester abortions, about 20 percent of second-trimester losses, and 6 to 8 percent of stillbirths and early-childhood deaths (Reddy, 2012; Stevenson, 2004; Wou, 2016). Of recognized pregnancies with chromosomal abnormalities, trisomy 21 composes approximately half of cases; trisomy 18 accounts for 15 percent; trisomy 13, for 5 percent; and the sex chromosomal abnormalities-45,X, 47,XX, 47,Y, and 47,XY-for approximately 12 percent (Wellesley, 2012).

1	The risk for fetal trisomy increases with maternal age, particularly after age 35 (Fig. 13-2, p. 255). When counseling, a provider includes specific maternal-age-related aneuploidy risks (Tables 14-1 and 14-2). Other important fetal aneuploidy risk factors include a numerical chromosomal abnormality or structural chromosomal rearrangement in the woman or her partner-such as balanced translocation-or a prior pregnancy with autosomal trisomy or triploidy. Broadly speaking, there are two types of aneuploidy screening tests, those that are traditional or analyte-based and those that are cell-free DNA-based. All pregnant women should be

1	Broadly speaking, there are two types of aneuploidy screening tests, those that are traditional or analyte-based and those that are cell-free DNA-based. All pregnant women should be TABLE 14-1. Maternal Age-Related Risk for Down Syndrome and Any Aneuploidy at Midtrimester and at Term in Singleton Pregnancies analysis provides information about genetic conditions that screening tests and karyotyping alone currently do not (American College of Obstetricians and Gynecologists, 2016b). This is discussed further on page 291 and in Chapter 13 (p. 271). 3. Is this a multetal gestation? All traditional (analyte-based) aneuploidy screening tests are significantly less efective in multifetal gestations, and cell-free DNA screening is not cur rently recommended with multifetal gestations (p. 285). 37 1/149 1/227 1/83 1/1l30 4. What method will be used or neural-tube deect screening?

1	37 1/149 1/227 1/83 1/1l30 4. What method will be used or neural-tube deect screening? 1/89 1/137 1/53 1/81 that does not include second-trimester serum analytes, 41 1/53 1/81 1131 1/50 arately, either with MSAFP assessment or with sonography 42 1/41 1/64 1/25 1/39 (American College of Obstetricians and Gynecologists, 43 1/31 1/50 1/19 1/30 2016c). 44 1/25 1/38 1/15 1/24 5. Does the etus have a major anomay? If so, diagnostic testing 1/19 1/30 1/12 1/19 is recommended rather than screening. Data from Hook EB, Cross PK, Schreinemachers DM: live-born infants, JAMA. 1983 Apr 15;249(15):2034-2038. ofered aneuploidy screening or diagnostic testing early in pregnancy (American College of Obstetricians and Gynecologists, 2016c). Considerations prior to screening are as follows: 1.

1	ofered aneuploidy screening or diagnostic testing early in pregnancy (American College of Obstetricians and Gynecologists, 2016c). Considerations prior to screening are as follows: 1. Has the patient elected screening? At least 20 percent of women elect not to receive aneuploidy screening, even when financial barriers are removed. Fewer than 40 percent of women with a positive screening result elect prenatal diagnosis (Dar, 2014; Kuppermann, 2014). 2. ould the patient preer prenatal diagnosis? Diagnostic testing is safe and efective, and chromosomal microarray TABLE 14-2. Maternal Age-Related Risk for Down Syndrome and Any Aneuploidy at Midtrimester and at Term in Dizygotic Twin Pregnanciesa

1	TABLE 14-2. Maternal Age-Related Risk for Down Syndrome and Any Aneuploidy at Midtrimester and at Term in Dizygotic Twin Pregnanciesa The American College of Obstetricians and Gynecologists (2016c) has airmed that screening for aneuploidy should be an informed patient choice, with an underlying foundation of shared decision making that its her clinical circumstances, values, interests, and goals. Elements of counseling prior to aneuploidy screening are listed in Table 14-3.

1	Aneuploidy screening can be challenging because the test characteristics of each option may vary with maternal age and with whether the test is analyte-based or cell-free DNA-based. The test sensitivity is the detection rate-that is, the proportion of aneuploid fetuses identiied by the screening test. Its converse, the false-negative rate, is the percentage of cases that the test is expected to miss. A first-trimester screening test with a sensitivity of 80 percent is expected to miss 1 in 5 cases. The sensitivity of screening tests for Down syndrome has increased steadily during the past 30 years, from just 25 percent with serum AFP alone to more than 90 percent with integrated or sequential screening.

1	Another key characteristic is the false-positive rate, the percentage of unafected pregnancies that will "falsely" screen positive. This approximates 5 percent for first-trimester screening, quadruple-marker screening, or integrated screening options (Baer, 2015; Kazerouni, 2011; Malone, 2005b; Nor 1/160 1/257 1/91 1/126 ton, 2015). The converse of false-posi tive rate is speciici ty percent of unafected pregnancies. lthough test sensitivity 1177 1/118 1/44 1/67 has improved, the false-positive rate has been held constant for many diferent aneuploidy screening tests (Table 14-4). 1/47 1172 1/27 1/44 Both statistics are relevant for counseling. An additional con aRisk applies to one or both fetuses. Data from Meyers C, Adam R, Dungan J, et al: Aneuploidy in twin gestations: when is maternal age advanced? Obstet Gynecol. 1997 Feb;89(2):248-251l.

1	Data from Meyers C, Adam R, Dungan J, et al: Aneuploidy in twin gestations: when is maternal age advanced? Obstet Gynecol. 1997 Feb;89(2):248-251l. sideration is that with all analyte-based screening tests, women 35 and older have higher rates of positive results (Kazerouni, 2011; Malone, 2005b). Importantly, neither sensitivity nor false-positive rate conveys individual risk. he statistic that patients and providers usually consider to be the test result is the positive-predictive value, which is the proportion of those with a positive screening result who actually have an aneuploid fetus. It may be expressed TABLE 14-3. Aneuploidy Screening Counseling Elements 1. All pregnant women have 3 options: screening, diagnostic testing, and no screening or testing. The purpose of a screening test is to provide information, not to dictate a course of action. Diagnostic testing is safe and effective and provides information that screening does not.

1	The purpose of a screening test is to provide information, not to dictate a course of action. Diagnostic testing is safe and effective and provides information that screening does not. 2. The diference between a screening test and a diagnostic test. Screening evaluates whether the pregnancy is at increased risk and estimates degree of risk. Detection rate, false-negative rate, and false-positive rate are provided. Cell-free DNA screening does not always provide a result. Irreversible management decisions should not be based on screening test results. With a positive screening result, a diagnostic test is recommended if the patient wants to know whether the fetus is affected. 3. Basic information is provided about each condition covered by the screening test (prevalence, associated abnormalities, prognosis), and screening test limitations. A benefit of diagnosis includes earlier identification of associated abnormalities.

1	A benefit of diagnosis includes earlier identification of associated abnormalities. In case of trisomy 18 or 13, diagnosis may affect pregnancy management if complications arise such as growth restriction or nonreassuring fetal heart rate. With sex chromosome aneuploidies, phenotypic expression varies widely. Several are so mild they would otherwise not be diagnosed. 4. The patient's a priori risk for fetal aneuploidy may affect her screening test options or election. Age-related risk information may be found in reference tables. If a patient has had a prior fetus with autosomal trisomy, robe sonian translocation, or other chromosomal abnorrnality, additional evaluation and counseling are suggested. Modified with permission from Dashe JS: Aneuploidy screening in pregnancy, Obstet Gynecol. 2016 Jul;128(1 ):181l-194.

1	Modified with permission from Dashe JS: Aneuploidy screening in pregnancy, Obstet Gynecol. 2016 Jul;128(1 ):181l-194. as a l:X ratio or as a percentage. he positive-predictive value value reported in a research trial is the proportion of women is directly afected by disease prevalence, so it is much higher with positive screening results who have afected fetuses for women aged 35 years and older than for younger women (see Table 14-4). Negative-predictive value is the proportion of (Table 14-5). Positive-predictive values can also be reported those with a negative screening test result who have unafected for cohorts of pregnancies. For example, the positive-predictive (euploid) fetuses. Because the prevalence of aneuploidy is so TABLE 14-4. Characteristics of Screening Tests for Trisomy 21 in Singletons Screening Test Rate Rate Predictive Valuea Quadruple screen: AFP, hCG, estriol, inhibin 80-82% 5% 3% First-trimester screen: NT, hCG, PAPP-A 80-84% 5% 3-4%

1	Screening Test Rate Rate Predictive Valuea Quadruple screen: AFP, hCG, estriol, inhibin 80-82% 5% 3% First-trimester screen: NT, hCG, PAPP-A 80-84% 5% 3-4% Sequential screening: Stepwise 92% 5.1l% 5% Contingent 91% 4.5% 5% Cell-free DNA screening: Positive result 99% 0.1% Table 14-5 aThe positive-predictive value represents the overall population studied and cannot be applied to any individual patient. NT = nuchal translucencyf PA.PP-A = pregnancy-associated plasma protein A. Data from Baer, 2015f Gil, 2015; Malone, 200Sb; j\lorton, 2015; 2014; Quezada, 2015; Dashe, 201l6. TABLE 14-5. Positive-Predictive Value of Cell-Free DNA Screening for Autosomal Trisomies and Selected Sex Chromosome Abnormalities, According to Maternal Age Maternal Age Trisomy 21 Trisomy 18 Trisomy 13 45,X 47,XXY NA = not available; ��IPT = non-invasive prenatal test.

1	Maternal Age Trisomy 21 Trisomy 18 Trisomy 13 45,X 47,XXY NA = not available; ��IPT = non-invasive prenatal test. Positive-predictive values were obtained using the NIPT/Cel1 Free DNA Screening Predictive Value Calculator from the Perinatal Quality Foundation, 2017. Calculations are based on prevalence at 16 weeks' gestation using sensitivities and specificities from Gil, 2015. low, the negative-predictive value of all aneuploidy screening tests generally exceeds 99 percent (Gil, 2015; Norton, 2015). are also called conventional or traditional to diferentiate them from cell-free DNA-based screening. There are three categories: irst-trimester screens, second-trimester screens, and combina tions of irst-and second-trimester screens. If the test has a irst trimester component, it almost always includes a measurement of the sonographic nuchal translucency, which is discussed in the next section.

1	Each maternal serum analyte is measured as a concentration-for example, nanograms per milliliter of AFP. he concentration is converted to a multiple of the median (MoM) by adjusting for maternal age, maternal weight, and gestational age. he nuchal translucency measurement increases with crown-rump length (CRL) , and thus its value is adjusted for CRL and also reported as an MoM. The AFP analyte is further adjusted for maternal race and ethnicity and for presence of diabetes, which all afect the calculation of neural-tube defect risk rather than of aneuploidy risk (Greene, 1988; Huttly, 2004). Reporting these results as an MoM of the unafected population normalizes the distribution of analyte levels and permits comparison of results from diferent laboratories and populations.

1	he analyte-based aneuploidy screening result is based on a composite likelihood ratio, and the maternal age-related risk is multiplied by this ratio. his principle similarly applies to modiication of fetal Down syndrome risk by selected sonographic markers, which are discussed later on page 286. Each woman is provided with a speciic risk for trisomy 21 and for trisomy 18-or in the irst trimester, for trisomy 18 or 13 in some cases. he result is expressed as a ratio that represents the positive-prediCtive value.

1	Importantly, each screening test also has a predetermined value at which or above which it is deemed "positive" or abnormal. For second-trimester tests, this threshold has traditionally been set at the risk for fetal Down syndrome in a woman aged 35 years-approximately 1 in 270 in the second trimester (see Table 14-1). he threshold selected for a positive screen relects the laboratory requirement but is somewhat problematic, as it may bear no relationship to patient preference. However, a positive screening result may afect whether the patient is deemed "high risk," receives formal genetic counseling, and is ofered diagnostic testing with chorionic villus sampling or amniocentesis. Thus, it behooves the provider to discuss the patient's preferences prior to screening.

1	Also called combined irst-trimester screening, this test combines two maternal serum analytes, human chorionic gonadotropin (hCG) and pregnancy-associated plasma protein A (PAPP-A), with the sonographic measurement of the nuchal translucency (NT). It is performed between 11 and 14 weeks' gestation. In cases of fetal Down syndrome, the irst-trimester serum free 3-hCG level is higher and the PAPP-A level is lower. With trisomy 18 and trisomy 13, levels of both analytes are lower (Cuckle, 2000; Malone, 2005b).

1	Nuchal Translucency. his is the maximum thickness of the subcutaneous translucent area between the skin and soft tissue overlying the fetal spine at the back of the neck (Fig. 14-2). An increased NT thickness is not a fetal abnormality but rather is a marker that confers increased risk. It is measured in the sagittal plane and is valid when the CRL value lies between 38 to 45 mm and 84 mm, with the lower limit varying according to the laboratory. Speciic criteria for NT measurement are listed in Table 10-4 (p. 186). Whenever possible, it is helpful to diferentiate increased NT from cystic hygroma, which is a venolymphatic malformation that appears as a septated hypoechoic space behind the neck, extending along the length of the back (Fig. 10-22, p. 198). Cystic hygroma confers a ivefold increased aneuploidy risk when identiied in the irst trimester (Malone, 2005a).

1	In addition to aneuploidy, an increased NT thickness is associated with other genetic syndromes and various birth defects, especially fetal cardiac anomalies (Simpson, 2007). And, if the NT measurement reaches 3 mm or more, the aneuploidy risk

1	FIGURE 14-2 Sagittal image of a normal, 12-week fetus demonstrating correct caliper placement (+) for nuchal translucency measurement. The fetal nasal bone and overlying skin are indicated. The nasal tip and the 3rd and 4th ventricles (asterisk), which are other landmarks that should be visible in the nasal bone image, are also shown. (Used with permission from Dr. Michael Zaretsky.) is unlikely to normalize using serum analyte assessment (Comstock, 2006). Because of this, if the NT measurement is at least 3 mm or exceeds the 99th percentile, the patient should receive counseling and be ofered targeted sonography with fetal echocardiography. In addition, she should be ofered cell-free DNA screening and prenatal diagnosis (American College of Obstetricians and Gynecologists, 2016c).

1	The NT must be imaged and measured with a high degree of precision for aneuploidy detection to be accurate. This has led to standardized training, certiication, and ongoing quality review programs. In the United States, training, credentialing, and monitoring are available through the Nuchal Translucency Quality Review program of the Perinatal Quality Foundation and through the Fetal Medicine Foundation.

1	Efficacy of First-Trimester Screening. Before first-trimester screening became widely adopted, four large prospective trials were conducted, together including more than 100,000 pregnancies (Reddy, 2006). When the false-positive rate was set at 5 percent, the overall rate for trisomy 21 detection was 84 percent, comparable to quadruple-marker screening (see Table 14-4). he detection rate is approximately 5 percent higher if performed at 11 weeks' compared with 13 weeks' gestation, and slightly lower-80 to 82 percent-when cases with cystic hygroma are analyzed separately (Malone, 2005a). In a recent multicenter trial, first-trimester screening detected approximately 80 percent of fetuses with trisomy 21, 80 percent with trisomy 18, and 50 percent with trisomy 13 (Norton, 2015).

1	As an isolated marker, NT detects approximately two thirds of fetuses with Down syndrome, with a false-positive rate of 5 percent (Malone, 2005b). However, NT is generally used as an isolated marker only in screening for multifetal gestations, in which serum screening is less accurate or may not be available. he NT distribution is similar in twins and singletons (Cleary-Goldman, 2005). In twin pregnancies, serum free 3-hCG and PAPP-A levels are approximately double the singleton values (Vink, 2012). Even with speciic curves, a normal dichorionic cotwin will tend to normalize screening results, and thus, the aneuploidy detection rate is at least 15-percent lower (Bush, 2005).

1	Maternal age afects the performance of first-trimester aneuploidy screening. Prospective trials have demonstrated Down syndrome detection rates of 67 to 75 percent in women younger than 35 years at delivery, which are 10 percent lower than the overall detection rates in these studies (Malone, 2005b; Wapner, 2003). Among women older than 35 at delivery, Down syndrome detection reached 90 to 95 percent, albeit at a higher false-positive rate of 15 to 22 percent. Unexplained Abnormalities of First-Trimester AnaIytes. There is a signiicant association between serum PAPPA levels below the 5th percentile and preterm birth, growth restriction, preeclampsia, and fetal demise (Cignini, 2016; Dugof, 2004; Jellife-Pawlowski, 2015). Similarly, low levels of free 3-hCG have been associated with fetal demise (Goetzl, 2004). he sensitivity and positive-predictive values of isolated markers are generally too low to make them clinically useful as screening tests.

1	There has been renewed interest in low-dose aspirin for prevention of early preeclampsia in women identified as at risk based on mean arterial pressure, uterine artery Doppler values, and PAPP-A levels. However, these observations are preliminary (Park, 2015).

1	Currently, the only second-trimester multiple marker test widely used in the United States is the quadruple marker or "quad" screening test. It is performed from 15 to 21 weeks' gestation, and inclusive gestational age ranges vary according to individual laboratories. Pregnancies with fetal Down syndrome are characterized by lower maternal serum AFP, higher hCG, lower unconjugated estriol, and higher dimeric inhibin levels. When the quad screen was initially described, the Down syndrome detection rate approximated 70 percent. But, by the early 2000s, the reported detection rate in two large prospective trials had improved to 81 to 83 percent, with a 5-percent screen-positive rate (Malone, 2005b; Wald, 1996, 2003). The improved detection rate is attributable, at least in part, to accurate gestational age assessment with sonography. In a review of more than 500,000 pregnancies receiving quadruple-marker screening through the statewide California Prenatal Screening Program, trisomy 21

1	gestational age assessment with sonography. In a review of more than 500,000 pregnancies receiving quadruple-marker screening through the statewide California Prenatal Screening Program, trisomy 21 detection was 78 percent with sonographic gestational age assessment but only 67 percent when the screen was calculated based on last menstrual period alone (Kazerouni, 2011). As with first-trimester screening, aneuploidy detection rates are lower in younger women and higher in women older than 35 years at delivery. If second-trimester serum screening is used in twin pregnancies, aneuploidy detection rates are significantly lower (Vink, 2012). With trisomy 18, the levels of the first three analytes are all decreased, and inhibin is not part of the calculation. Trisomy 18 detection is similar to that for Down syndrome, with a false-positive rate of only 0.5 percent (Benn, 1999).

1	Although the quadruple-marker screening test is used to screen for Down syndrome and trisomy 18, pregnancies with other chromosomal abnormalities may be identiied as well. The California Prenatal Screening Program found that the of those with triploidy, in 75 percent with Turner syndrome (45,X), in 44 percent with trisomy 13, and in more than 40 percent of those with other major chromosomal abnormalities (Kazerouni, 2011). Although a speciic risk for these aneuploi dies cannot be provided based on the test result, the informa tion may be relevant for women considering amniocentesis.

1	Quadruple-marker screening ofers no beneit over irsttrimester screening from the standpoint of trisomy 21 or trisomy 18 detection. As a stand-alone test, it is generally used if women do not begin care until the second trimester or if irst-trimester screening is not available. In 2011, women who initiated prenatal care beyond the irst trimester made up nearly 25 percent of pregnancies in the United States. As subsequently discussed, combining irst-and second-trimester screening yields an even greater aneuploidy detection rate.

1	Maternal Serum AFP Elevation: Neural-Tube Defect Screening. All pregnant women are ofered screening for fetal open neural-rube defects in the second trimester, either with MSAFP screening or with sonography (American College of Obstetricians and Gynecologists, 20 16c). Measurement of the MSAFP concentration between 15 and 20 weeks' gestation has been ofered as part of routine prenatal care for more than 30 years. Because AFP is the major protein in fetal serum, analogous to albumin in a child or adult, the normal concentration gradient between fetal plasma and maternal serum is on the order of 50,000: 1. Defects in fetal integument, such as neural-tube and ventral-wall defects, permit AFP to leak into the amnionic fluid, resulting in dramatically increased maternal serum levels. he AFP value rises by about 15 percent per week during the screening window (Knight, 1992). he MoM is generally recalculated if the irst-trimester CRL or second-trimester biparietal diameter difers from the

1	value rises by about 15 percent per week during the screening window (Knight, 1992). he MoM is generally recalculated if the irst-trimester CRL or second-trimester biparietal diameter difers from the stated gestational age by more than 1 week.

1	Using an MSAFP level of 2.5 MoM as the upper limit of normal, the neural-tube defect detection rate is at least 90 percent for anencephaly and 80 percent for spina biida, with a screen-positive rate of 3 to 5 percent (American College of Obstetricians and Gynecologists, 2016a; Milunsky, 2004). Higher screening threshold values are used in twin pregnancies (Cuckle, 1990).

1	Virtually all cases of anencephaly and many cases of spina biida may be detected or suspected during a standard second-trimester sonographic examination (Dashe, 2006). Most centers now use targeted sonography as the primary method to evaluate elevated MSAFP levels and as the prenatal diagnostic test of choice for neural-tube defects (Chap. 10, p. 192). If targeted sonography is not available and myelomeningocele cannot be excluded, amniocentesis may be considered for measurement of amnionic luid AFP and acetylcholinesterase levels. That said, we recommend additional imaging prior to establishing the diagnosis, with the understanding that other TABLE 14-6. Conditions Associated with an Elevated MSAFP Concentration Renal anomalies-polycystic kidneys, renal agenesis, congenital nephrosis, urinary tract obstruction Chorioangioma of placenta MSAFP = maternal serum alpha-fetoprotein.

1	Renal anomalies-polycystic kidneys, renal agenesis, congenital nephrosis, urinary tract obstruction Chorioangioma of placenta MSAFP = maternal serum alpha-fetoprotein. abnormalities or conditions can result in elevation of these amnionic luid analytes (Table 14-6). Sonographic indings characteristic of fetal neural-tube defects are reviewed in Chapter 10 (p. 192). Fetal surgery for myelomeningocele is discussed in Chapter 16 (p. 319). Unexplained Abnormalities of Second-Trimester Analytes.

1	Unexplained Abnormalities of Second-Trimester Analytes. he positive-predictive value of an elevated MSAFP value is only 2 percent. Approximately 98 percent of pregnancies with an MSAFP level exceeding 2.5 MoM have an etiology other than a neural-tube defect. hus, counseling is indicated not only to inform the patient about the beneits and limitations of targeted sonography for the diagnosis of neural-tube defects but also to review the numerous other conditions. Some of these include fetal anomalies, placental abnormalities, and adverse outcomes associated with MSAFP level elevation (see Table 14-6). he likelihood of one of these abnormalities or of an adverse pregnancy outcome in the absence of a recognized abnormality rises in proportion to the AFP level. Adverse outcomes include fetal-growth restriction, preeclampsia, preterm birth, fetal demise, and stillbirth. More than 40 percent of pregnancies may be abnormal if the MSAFP level is greater than 7 MoM (Reichler, 1994).

1	Second-trimester elevation of either hCG or dimeric inhibin alpha levels also shows signiicant association with adverse pregnancy outcomes. he outcomes reported are similar to those associated with MSAFP level elevation. Moreover, the likelihood of adverse outcome is augmented when levels of several markers are elevated (Dugof, 2005). Many of these complications are assumed to result from placental damage or dysfunction. However, the sensitivity and positive-predictive values of these markers are considered too low to be useful for screening or management. No speciic program of maternal or fetal surveillance has been found to favorably afect pregnancy outcomes (Dugof, 2010). At Parkland Hospital, prenatal care for these women is not altered unless a specific complication arises. Despite the extensive list of possible adverse outcomes, it is reassuring that most women with unexplained elevation of these analytes have normal outcomes.

1	Low Maternal Serum Estriol Level. A maternal serum estriol level less than 0.25 MoM has been associated with two uncommon but important conditions. he irst, Smith-Lemli-Opitz syndrome, is an autosomal recessive condition resulting from mutations in the 7 -dehydrocholesterol reductase gene. It is characterized by abnormalities of the central nervous system, heart, idney, and extremities; with ambiguous genitalia; and with fetal-growth restriction. For this reason, the Society for Maternal-Fetal Medicine has recommended that sonographic evaluation be performed if an unconjugated estriol level is <0.25 MoM (Dugof, 2010). If abnormalities are identified, an elevated amnionic luid 7 -dehydrocholesterol level can conirm the diagnosis.

1	he second condition is steroid suatase diciency, also known as X-linked ichthyosis. It is typically an isolated condition, but it may also occur in the setting of a contiguous gene deletion syndrome (Chap. 13, p. 260). In such cases, it may be associated with Kallmann syndrome, chondrodysplasia punctata, and/or mental retardation (Langlois, 2009). If the estriol level is <0.25 MoM and the fetus appears to be male, chromosomal microarray analysis or fluorescence in situ hybridization to assess the steroid sulfatase locus on the X-chromosome may be considered.

1	As shown in Table 14-4, if irst-trimester screening is combined with second-trimester screening, aneuploidy detection is signiicantly improved. Combined screening test options require coordination between the provider and laboratory. Specifically, if a second sample is required, it is obtained during the appropriate gestational age window, sent to the same laboratory, and linked to the first-trimester results. The irst-and secondtrimester components cannot be performed independently because if either component yields positive results, then providing accurate risk assessment would be problematic.

1	Three types of screening strategies are available: 1. Integrated screening combines results of irst-and secondtrimester tests. his includes a combined measurement of fetal NT and serum analyte levels at 11 to 14 weeks' gestation plus quadruple markers at approximately 15 to 21 weeks. n aneuploidy risk is then calculated from these seven parameters. As expected, integrated screening has the highest Down syndrome detection rate-94 to 96 percent, with a false-positive rate of 5 percent (see Table 14-4). If NT measurement is not available, then serum integrated screening includes all six serum markers to calculate risk. his screening, however, is less efective, and Down syndrome detection rates are 85 to 88 percent (Malone, 2005b).

1	2. Sequential screening involves performing first-trimester screening and informing the patient of the results. his is coupled with the understanding that if the calculated risk value lies above a speciied threshold, she will receive counseling and will be ofered diagnostic testing. here are two testing strategies in this category: With stepwise sequential screening, women with firsttrimester screen results that confer risk for Down syndrome above a particular threshold are ofered invasive testing, and the remaining women receive secondtrimester screening. Using data from the First-and Second-Trimester Evaluation of Risk (FaSTER) trial, when the first-trimester threshold is set at approximately 1 :30, and the overall threshold is set at 1 :270, stepwise sequential screening resulted in a 92-percent detection rate of Down syndrome pregnancies, with a false-positive rate of 5 percent (see Table 14-4) (Cuckle, 2008).

1	With contingent sequential screening, women are divided into high-, moderate-, and low-risk groups. Those at highest risk for Down syndrome-for example, riski> 1 :30, are counseled and ofered invasive testing. Women at moderate risk, between 1 :30 and 1: 1500, undergo second-trimester screening, whereas those at lowest risk of < 1: 1500 receive negative screening test results and have no further testing (Cuckle, 2008). Using this strategy, more than 75 percent of those screened are provided with reassuring results almost immediately, while still maintaining a high detection rate of about 91 percent, with a 5-percent false-positive rate (see Table 14-4). his option is lso more cost efective because a second-trimester test is obviated in most patients.

1	In a population-based review of 450,000 pregnancies from the California Prenatal Screening Program, integrated screening detected 94 percent of trisomy 21 fetuses and 93 percent with trisomy 18 (Baer, 2015). Additionally, the screening result was abnormal in 93 percent of cases of trisomy 13, in 91 percent with triploidy, and in 80 percent with Turner syndrome. Women considering options of integrated screening and cellfree DNA screening may ind this information helpful.

1	his was introduced in 2011 and has completely changed the prenatal screening paradigm. he test works by identiying DNA fragments that are derived primarily from apoptotic trophoblasts, which are placental cells undergoing programmed cell death. hus, the term cell-free etal DNA is somewhat of a misnomer. Screening is not gestational-age dependent and can be performed at any time ater 9 to 10 weeks' gestation. Results are available in 7 to 10 days (American College of Obstetricians and Gynecologists, 2017 c). hree types of assays are currently available: wholegenome sequencing, which is also called massively parallel or shotgun sequencing; chromosome selective or targeted sequencing; and analysis of single nucleotide polymorphisms.

1	he screening performance of cell-free DNA is excellent. In a metaanalysis of 37 studies of largely high-risk pregnancies, the pooled sensitivity to detect Down syndrome was 99 percent, and for trisomies 18 and 13, 96 percent and 91 percent, respectively. For each of these autosomal trisomies, the speciicity was 99.9 percent. Thus, most unafected pregnancies received a norml screening result. Cell-free DNA also detects 90 percent with Turner syndrome (45,X) and 93 percent with sex chromosome aneuploidies other than 45,X (Gil, 2015). The false-positive rate is cumulative for each aneuploidy for which screening is performed, but it is usually only 0.5 to 1 percent. s a result, cell-free DNA screening is recommended as a screening option in those at increased risk for fetal autosomal trisomy (American College of Obstetricians and Gynecologists, 2017c; Society for MaternalFetal Medicine, 2015). This includes the following categories: 1. 2. 3. Sonogram with a minor aneuploidy marker 4.

1	2. 3. Sonogram with a minor aneuploidy marker 4. Prior pregnancy with autosomal trisomy 5. Known carriage (patient or partner) of a balanced robertsonian translocation involving chromosome 21 or 13.

1	If cell-free DNA screening is performed as a seconday screen following a positive irst-or second-trimester analyte-based test result, a normal result is not quite as reassuring. The residual risk for chromosomal abnormality is estimated to be 2 percent (Norton, 2014). Compared with amniocentesis, use of cellfree DNA screening after an initial abnormal analyte-based test result is estimated to yield a 20-percent reduction in aneuploidy diagnoses. his takes into consideration false-negative diagnoses and aneuploidies not detectable with cell-free DNA screening (Davis, 2014; Norton, 2014). In addition, definitive diagnosis may be delayed, potentially afecting management. Concurrent or parallel screening is not recommended, and if an aneuploidy screening test of any type yields a negative result, additional screening is not indicated (American College of Obstetricians and Gynecologists, 2016b, 2017c).

1	he association between increased NT values and fetal structural and genetic abnormalities has raised questions about the role of a NT measurements following cell-free DNA screening. he College (20 16b) has stated that NT measurement is not necessary at the time of cell-free DNA screening but that sonography may help to conirm fetal number and viability and to assign gestational age. he Society for Maternal-Fetal Medicine (20i15) states that after a negative cell-free DNA screening test result, the additional clinical utility of NT measurement to detect other chromosomal or structural abnormalities is unknown but appears to be limited.

1	Most studies of cell-free DNA have been conducted in highrisk pregnancies. Pragmatically, chromosomal abnormalities are individually so rare that even large studies of low-risk pregnancies contain few afected cases. Available data suggest that the high sensitivity and speciicity for Down syndrome detection are preserved in low-risk pregnancies (Norton, 2015; Pergament, 2014; Zhang, 2015). Importantly, the positive-predictive value of cell-free DNA screening still depends greatly on maternal age and the specific aneuploidy in question (see Table 14-5). For a woman in her early 20s, the positive-predictive value is approximately 50 percent for fetal trisomy 21, 15 percent for trisomy 18, and < 10 percent for trisomy 13. Hence, decisions for irreversible medical intervention should not be based on the results of this or other screening test alone. Important caveats are considered with selection of cell-free

1	Important caveats are considered with selection of cell-free DNA aneuploidy screening. Because the cell-free DNA that is analyzed is maternal and placental, results may not relect the fetal DNA complement but rather may indicate con ined placental mosaicism, early demise of an aneuploid co twin, maternal mosaicism, or even occult maternal malignancy (Bianchi, 2015; Curnow, 2015; Grati, 2014; Wang, 2014). In addition, if a twin pregnancy is identiied sonographically, cell free DNA screening is not currently recommended because of limited evidence regarding eicacy.

1	Another limitation is that cell-free DNA testing does not yield a result in approximately 4 to 8 percent of screened pregnancies, due to assay failure, high assay variance, or low fetal fraction (Norton, 2012; Pergament, 2014; Quezada, 2015). Most cell-free DNA is maternal. he fetal fraction is the proportion derived from the placenta and generally is about 10 percent of the total. A low fetal fraction is usually defined as <4 percent of the total and confers signiicantly higher risk for fetal aneuploidy (Ashoor, 2013; Norton, 2015; Pergament, 2014). Women with a low fetal fraction or "no-call" results have fetal aneuploidy rates as high as 4 percent, a percentage comparable to the average predictive value conferred by a positive irst-trimester screening test result (see Table 14-4). The fetal fraction is not related to maternal age or analyte-based screening test results. However, it is lower earlier in pregnancy and appears to be reduced in women of greater weight (Ashoor, 2013).

1	Because of the increased risk for fetal aneuploidy in cases not generating a cell-free DNA screening result (no-call result), genetic counseling is indicated, and amniocentesis should be ofered. If the patient elects repeat screening, the risk for screen failure may exceed 40 percent (Dar, 2014; Quezada, 2015). Targeted sonography is recommended but is not a substitute for amniocentesis, because it is unclear what the residual risk would be with normal sonogram indings. (American College of Obstetricians and Gynecologists, 2016b, 2017 c). Pretest counseling should include the possibility of a low fetal fraction or no-call result and its clinical signiicance. Comparison with Analyte-Based Screening

1	Comparison with Analyte-Based Screening Cell-free DNA screening has obvious advantages, but it is not simply a "better" test-because no screening test is superior for all test characteristics (American College of Obstetricians and Gynecologists, 20 16c). Compared with analyte-based tests, beneits of cell-free DNA screening in women 35 years and older include the lower likelihood of a false-positive result, its higher positive-predictive value, and the fact that isolated minor aneuploidy markers are generally not a concern (p. 286). But, analyte-based tests are frequently positive with a large range of chromosomal abnormalities, whereas cell-free DNA TABLE 14-7. Aneuploidy Risk Associated with Selected Major Fetal Anomalies Cystic hygroma 1/5000 50-70 45,X; 21; 18; 13; triploidy Nonimmune hydrops 1/1500-4000 10-20 21, 18, 13, 45X, triploidy Ventricu Iomega Iy 1/1000-2000 5-25 13, 18,21/ triploidy Holoprosencephaly 1/10,000-15,000 30-40 13, 18, 22, triploidy

1	Nonimmune hydrops 1/1500-4000 10-20 21, 18, 13, 45X, triploidy Ventricu Iomega Iy 1/1000-2000 5-25 13, 18,21/ triploidy Holoprosencephaly 1/10,000-15,000 30-40 13, 18, 22, triploidy Dandy-Walker malformation 1/12,000 40 18/ 13/21, triploidy Clet lip/palate 1/1000 5-15 18,13 Cardiac defects 5-8/1000 10-30 21; 18; 13; 45/X; 22q 11.2 microdeletion Diaphragmatic hernia 1/3000-4000 5-15 18,l13,l21 Esophageal atresia 1/4000 10 18,l21 Duodenal atresia 1/10,000 30 21 Gastroschisis 112000-4000 No increase Omphalocele 1/4000 30-50 18, 13, 21, triploidy autosomal trisomies except where indicated. For example, 45,X indicates Turner syndrome. Data from Best, 201l2; Canfield, 2006; Colvin, 2005; Cragan, 2009; Dolk/ 201l0; Ecker, 2000; Gallot/ 2007; Long, 2006; Orioli, 2010; Pedersen/ 2012; Sharma/ 201l1l; Solomon, 2010; Walker/ 2001l.

1	screens are specific for individual aneuploidies (Baer, 2015; Kazerouni, 2011). Women younger than 35 are at lower risk for the speciic autosomal trisomies for which cell-free DNA screening is typically performed. hus, if the goal is to select a screening test that will identiY the highest proportion of fetuses with any chromosomal abnormality, the yield may be comparable or even slightly higher with integrated or sequentil screening than with current cell-free DNA screening (Baer, 2015; Norton, 2014).

1	Sonography can augment aneuploidy screening by providing accurate gestational age assessment, by detecting multifetal gestations, and by identiYing major structural abnormalities and minor sonographic markers. s shown in Table 14-7, with rare exceptions, the aneuploidy risk associated with any major abnormality is high enough to warrant ofering prenatal diagnosis. Generally, chromosomal microarray analysis is recommended as the irst-line test. Importantly, a fetus with one abnormality may have others that are less likely to be detected sonographically but that greatly afect the prognosis. Aneuploidy screening-including cell-free DNA-is not recommended if a major abnormality has been identified. The fetal risk cannot be normalized with a normal screening result, not merely because screening results can be falsely negative, but also because major anomalies confer risk for genetic syndromes not identiied through screening tests.

1	If a major abnormlity is identified, targeted sonography is indicated. Sonography is not an alternative to prenatal diagnosis, but the aneuploidy risk is urther increased if additional findings are identified. An earlier study reported that only 25 to 30 percent of second-trimester fetuses with Down syndrome had a major malformation that could be identiied sonographically (Vintzileos, 1995). When both major anomalies and minor aneuploidy markers are considered, it is estimated that 50 to 60 percent of Down syndrome pregnancies can be detected sonographiclly (American College of Obstetricians and Gynecologists, 2016c). Fortunately, most fetuses with aneuploidy that is likely to be lethal in uterosuch as trisomy 18 and 13 and triploidy-usully have sonographic abnormalities that can be seen by the second trimester.

1	For three decades, investigators have recognized that the sonographic detection of aneuploidy, particularly Down syndrome, may be improved by minor markers that are collectively referred to as "sot signs." Minor markers are normal variants rather than fetal abnormalities, and in the absence of aneuploidy or an associated abnormality, they do not significantly afect prognosis. They are present in at least 10 percent of unafected pregnancies (Bromley, 2002; Nyberg, 2003). Examples of these sonographic findings are listed in Table 14-8 and depicted in Figure 14-3. Findings are generally useful from 15 to 20 or 22 weeks' gestation. Six of these markers have been the focus of sonographic studies, in which likelihood ratios have been derived that allow a numerical aneuploidy risk to be calculated (Table 14-9). The risk rises steeply with the number of markers identiied. Alternatively, absence of a minor marker has been used to reduce the calculated risk (Agathokleous, 2013). This should be

1	(Table 14-9). The risk rises steeply with the number of markers identiied. Alternatively, absence of a minor marker has been used to reduce the calculated risk (Agathokleous, 2013). This should be done systematically, following a protocol that specifies the markers included in a model, the definition for what constitutes a inding, and positive-and negative-likelihood ratios (Reddy, 2014).

1	he nuchal skinod is measured in the trans cerebellar view of the fetal head, from the outer edge of the skull to the outer border of the skin (see Fig. 14-3A). A measurement �6 mm is typically considered abnormal (Benacerraf, 1985). This inding is present in approximately 1 per 200 pregnancies and confers a more than tenfold risk for Down syndrome (Bromley, 2002; Nyberg, 2001; Smith-Bindman, 2001). An echogenic intracardiac ocus is a focal papillary muscle calcification that is neither a structural nor functional cardiac abnormality. It is usually left-sided (see Fig. 14-3B). Such a focus is present in approximately 4 percent of fetuses, but it TABLE 14-8. Second-Trimester Sonographic Markers or "Sot Signs" Associated with Fetal Trisomy 21 a Clinodactyly (hypoplasia of the 5th digit middle phalanx) Echogenic bowel OUsted alphabetically.

1	TABLE 14-8. Second-Trimester Sonographic Markers or "Sot Signs" Associated with Fetal Trisomy 21 a Clinodactyly (hypoplasia of the 5th digit middle phalanx) Echogenic bowel OUsted alphabetically. may be found in up to 30 percent of Asian individuals (Shipp, 2000). As an isolated finding, this approximately doubles the risk for fetal Down syndrome (see Table 14-9). Bilateral echogenic foci are associated with trisomy 13 (Nyberg, 2001). Y1ild renal pelvis dilatation is usually transient or physiological and does not represent an underlying abnormality (Chap. 10, p. 208). The renal pelves are measured in a transverse image of the kidneys, anterior-to-posterior, with calipers placed aHigher in Asian individuals. Data from Bromley, 2002; Nyberg, 2001; Smith-Bindman, 2001l.

1	Data from Bromley, 2002; Nyberg, 2001; Smith-Bindman, 2001l. at the inner borders of the fluid collection (see Fig. 14-3C). A measurement �4 mm is found in about 2 percent of fetuses and approximately doubles the risk for Down syndrome. he degree of pelvic dilatation beyond 4 mm correlates with the likelihood of an underlying renal abnormality, and additional evaluation is generally performed at approximately 32 weeks. Echogenic etal bowel is deined as bowel that appears as bright as fetal bone (see Fig. 14-3D). It is identiied in approximately FIGURE 14-3 Minor sonographic markers that are associated with increased risk for fetal Down syndrome. A. Nuchal skinfold thickening (bracket). B. Echogenic intracardiac focus (arrow). c. Mild renal pelvis dilatation (pyelectasis) (arrows). D. Echogenic bowel (arrow). E. Clinodactyly-hypoplasia of the 5th finger middle phalanx creates an inward curvature (arrow). F. "Sandal-gap" (arrow).

1	E. Clinodactyly-hypoplasia of the 5th finger middle phalanx creates an inward curvature (arrow). F. "Sandal-gap" (arrow). 0.5 percent of pregnancies and most commonly represents small amounts of swallowed blood, frequently in the setting of maternal serum AFP level elevation. Although typically associated with normal outcomes, it raises the risk for Down syndrome approximately sixfold. Echogenic bowel has also been associated with fetal cytomegalovirus infection and cystic ibrosisrepresenting inspissated meconium in the latter.

1	The femur and humerus are slightly shorter in Down syndrome fetuses. The femur is considered "short" for Down syndrome screening if it measures below the 2.5th percentile or is shortened to :;90 percent of that expected based on the measured biparietal diameter (American College of Obstetricians and Gynecologists, 2016c; Benacerraf, 1987). As an isolated finding in an otherwise low-risk pregnancy, it is generally not considered to pose great enough risk to warrant counseling modification. Similarly, a humerus shortened to :;89 percent of that expected, based on a given biparietal diameter, has also been associated with an elevated risk for Down syndrome.

1	If an isolated minor marker is identified in a woman who has not yet received aneuploidy screening, screening should be ofered, and a minor marker is considered an indication to ofer cell-free DNA screening (American College of Obstetricians and Gynecologists, 2016c). If cell-free DNA screening has already been performed, the association between isolated minor markers and aneuploidy risk is no longer considered relevant (Reddy, 2014). And, if the cell-free DNA screening result is negative, the fetal aneuploidy risk is not modified by the marker. Conversely, if a cell-free DNA screening result is positive, the absence of minor markers is not considered reassuring.

1	Unlike second-trimester soft signs, which may be readily visible during standard sonography, irst-trimester findings associated with aneuploidy require specialized training. The fetal NT measurement has gained widespread use for aneuploidy screening. Other irst-trimester sonographic findings are not routinely used in the United States but may be available in specialized centers. he Perinatal Quality Foundation's Nuchal Translucency Quality Review Program ofers an education program in irsttrimester nasal bone assessment (see Fig. 14-2). he Fetal Medicine Foundation also provides online instruction and certification in first-trimester assessment of nasal bone, ductus venosus low, and tricuspid flow.

1	Other benefits of irst-trimester sonography in women who elect aneuploidy screening include accurate assessment of gestational age and early detection of multifetal gestation or fetal demise. As discussed in Chapter 10 (p. 186), first-trimester sonography may identiy selected major anomalies associated with aneuploidy, such as cystic hygroma.

1	Three types of carrier screening may be ofered: ethnicity-based screening, panethnic screening (performed regardless of ethnicity), and expanded carrier screening-which is a type of panethnic screening performed for a larger number of conditions, potentially 100 or more. The goal of screening is to provide individuals with meaningful information to guide pregnancy planning according to their values (American College of Obstetricians and Gynecologists, 2017 a). Each type of screening has beneits, risks, and limitations. For example, so many disorders are included in expanded carrier screening panels that more than 50 percent of those screened may be identified to be carriers for at least 1 condition. his can cause anxiety for families and may pose challenges if genetic counseling resources are limited. Recognizing that each type of screening is an acceptable strategy, it is recommended that obstetrical providers develop a standard approach to ofer one of these three types of carrier

1	are limited. Recognizing that each type of screening is an acceptable strategy, it is recommended that obstetrical providers develop a standard approach to ofer one of these three types of carrier screening to pregnant women and couples considering pregnancy (American College of 0bstetricians and Gynecologists, 2017 a). All carrier screening is optional and should be an informed choice.

1	Couples with a personal or family history of a heritable genetic disorder should be ofered genetic counseling. They are provided an estimated risk of having an afected newborn and given information concerning beneits and limitations of available prenatal testing options. Prenatal diagnosis may be available if the diseasecausing mutation or mutations are known. The publicly unded Genetic Testing Registry website contains detailed information regarding more than 10,000 genetic conditions and 48,000 genetic tests (www.ncbi.nlm.nih.gov/gtrl). hat said, many genetic disorders are characterized by a high degree of penetrance but variable expressivity. Thus, prediction of phenotype may not be possible, even when family members are afected. Common examples include neurofibromatosis, tuberous sclerosis, and Marfan syndrome. There are also conditions for which risk may be refined by detection of associated sonographic abnormalities or by gender determination if X-linked.

1	Ethniciy-based carrier screening is ofered for certain autosomal recessive disorders that are found in greater frequency in specific racial or ethnic groups (Table 14-10). The ounder eect occurs when an otherwise rare gene is found with greater frequency in a certain population and can be traced back to a single family member or small group of ancestors. his phenomenon may develop when generations of individuals procreate only within their own groups because of religious or ethnic prohibitions or geographical isolation. Because it is becoming increasingly diicult to assign a single ethnicity, a panethnic screening panel is another option. The American College of Obstetricians and Gynecologists (2017 a) has developed the following criteria for expanded carrier screening panels: 1. Conditions included in the panel should have a carrier frequency of at least 1: 1 00, which correlates with a population frequency, at minimum, of 1 :40,000. 2.

1	Conditions included in the panel should have a carrier frequency of at least 1: 1 00, which correlates with a population frequency, at minimum, of 1 :40,000. 2. Conditions should have a well-defined phenotype, detrimental efect on quality of life, cognitive or physical impairment, early onset, or require surgical or medical intervention. 3. Conditions primarily associated with disease of adult onset are not recommended for inclusion. 4. If an individual is at increased risk for a speciic condition, such as Tay-Sachs disease or 3-thalassemia, the provider should consider that the test included in the panel may not be the most sensitive one for that condition.

1	his disorder is caused by a mutation in the cystic ibrosis conductance transmembrane regulator (CFTR) gene, which is located on the long arm of chromosome 7 and encodes a chloride-channel protein. Although the most common CFTR gene mutation associated with classic cystic fibrosis (CF) is the .F508 mutation, more than 2000 mutations have been identiied (Cystic Fibrosis Mutation Database, 2016). CF may develop from either homozygosiy or compound heterozygosiy for mutations in the CFTR gene. In other words, one mutation must be present in each copy of the gene, but they need not be the same mutation. As expected, this results in a tremendous range of clinical disease severity. Median survival is approximately 37 years, but approximately 15 percent have milder disease and can survive for decades longer. Care for the pregnant woman with CF is discussed in Chapter 51 (p. 998).

1	he American College of Obstetricians and Gynecologists (2017a,b) recommends that all patients who are considering pregnancy or who are already pregnant should be ofered carrier screening for CF, regardless of ethnicity. The current recommended screening panel contains 23 panethnic CF gene mutations, selected because they are present in at least 0.1 percent of patients with classic CF (American College of Obstetricians and Gynecologists, 20 17b). The CF carrier frequency approximates 1 in 25 in non-Hispanic white Americans and those of Ashkenazi Jewish descent, who are from Eastern Europe. Thus, the incidence of CF in a child born to a non-Hispanic white couple approximates 4 X 1/25 X 125, or 1 :2500. As shown in 1i4-11i, both CF incidence and the sensitivity of the screening test are lower for other ethnicities.

1	lthough a negative screening test result does not preclude the possibility of carrying a less-common mutation, it reduces the risk substantively from the background rate. If both parents are carriers, chorionic villus sampling or amniocentesis can help determine whether the fetus has inherited one or both of the parental mutations. Counseling following identification of two disease-causing mutations is challenging, because phenotype prediction is reasonably accurate only for pancreatic disease, and then only for well-characterized mutations. Prognosis is most heavily afected by the degree of pulmonary disease, which varies considerably even among individuals with the most common genotype associated with classic disease, that is, those homozygous for the .F508 mutation. his likely relects the efect of genetic modiiers on protein function, which may further vary depending on the CFTR mutation and on exposure and susceptibility to environmental factors (Cutting, 2005; Drumm, 2005).

1	his autosomal recessive disorder results in spinal cord motor neuron degeneration that leads to skeletal muscle atrophy and generalized weakness. here is currently no efective treatment. The prevalence of spinal muscular atrophy (SA) is 1 in 6,000 to 10,000 live births. Types I, II, III, and IV are caused by mutations in the survival motor neuron (SMN1) gene, which is TABLE 14-1e1. Cystic Fibrosis Detection and Carrier Rates before and ater Testing Ashkenazi Jewish 94.0 1/24 1 in 384 Caucasian 88.3 '1125 1 in 206 Hispanic American 71.7 1/58 1 in 203 African American 64.5 1/61 1 in 171 Asian American 48.9 1/94 1 in 183 Data from American College of Medical Genetics, 2006.

1	Data from American College of Medical Genetics, 2006. located on the long arm of chromosome 5 (5q 13.2) and encodes the SMN protein. Types I and II account for 80 percent of cases and are both lethal (American College of Obstetricians and Gynecologists, 2017b). SMA type I, known as WerdnigHofmann, is the most severe. Disease onset is within the first 6 months, and afected children die of respiratory failure by age 2 years. Type II generally has onset before age 2 years, and the age at death can range from 2 years to the third decade of life. Type III also presents before age 2 years, with disease severity that is milder and more variable. Type IV does not present until adulthood.

1	The American College of Obstetricians and Gynecologists (20 17b) recommends that carrier screening for SMA be ofered to all women who are considering pregnancy or are currently pregnant. The SMA carrier frequency approximates 1:35 in those of non-Hispanic white (caucasian) ethnicity, 1:41 in Ashkenazi Jews, 1:53 in Asians, 1:66 in African Americans, and 1: 117 in those of Hispanic white ethnicity (Hendrickson, 2009). Carrier detection rates range from 90 to 95 percent for each race/ethnicity except African Americans, in whom it just exceeds 70 percent. Approximately 2 percent of individuals with SMNI mutations are not identiied with carrier screening. In addition, although there is usually one copy of the SMNI gene on each chromosome, approximately 3 to 4 percent of individuals have two copies of this gene on one chromosome and no copies on the other. These individuals are carriers for the disease. African mericans are more likely to have this genetic variation, which explains the

1	copies of this gene on one chromosome and no copies on the other. These individuals are carriers for the disease. African mericans are more likely to have this genetic variation, which explains the lower sensitivity of screening in this group. he American College of Obstetricians and Gynecologists (20 17b) recommends that prior to screening for SMA, providers counsel about its potential spectrum of severity, carrier frequency, and its detection rate. Posttest counseling should include the residual risk after a negative screening result, which difers according to the patient's ethnicity and also according to the number of SMNI copies detected. Most unafected individuals have two copies, but a small percentage has three copies and is at even lower risk. If the patient or her partner has a family history of SMA, or if carrier screening is positive, genetic counseling is recommended.

1	These include sickle-cell anemia, sickle-cell hemoglobin C disease, and sickle-cell 3-thalassemia. Their pathophysiology and inheritance are discussed in detail in Chapter 56 (p. 1081). African and African-American patients are at increased risk to carry hemoglobin S and other hemoglobinopathies and should be ofered preconceptional or prenatal screening. Of African Americans, 1 in 12 has sickle-cell trait, 1 in 40 carries hemoglobin C, and 1 in 40 carries the trait for 3-thalassemia. Hemoglobin S is also more common among individuals of Middle Eastern, and Asian Indian descent (Davies, 2000). The American College of Obstetricians and Gynecologists (2015) recommends that patients of African descent be ofered hemoglobin electrophoresis. If a couple is at risk to have a child with a sickle hemoglobinopathy, genetic counseling should be ofered. Prenatal diagnosis can be performed with either chorionic villus sampling or amniocentesis.

1	These syndromes are the most common single-gene disorders worldwide, and up to 200 million people carry a gene for one of these hemoglobinopathies (Chap. 56, p. 1084). Some individuals with thalassemia have microcytic anemia secondary to decreased synthesis of either a-or 3-hemoglobin chains. In general, deletions of a-globin chains cause a-thalassemia, whereas mutations in 3-globin chains cause 3-thalassemia. Less commonly, an a-globin chain mutation also causes a-thalassemia.

1	The number of a-globin genes that are deleted may range from one to all four. If two a-globin genes are deleted, both may be deleted from the same chromosome-cis coniguration (aal--), or one may be deleted from each chromosometrans configuration (a-/a-). Alpha-thalassemia trait is common among individuals of African, Mediterranean, Middle Eastern, West Indian, and Southeast Asian descent and results in mild anemia. he cis coniguration is more prevalent among Southeast Asians, whereas those of African descent are more likely to inherit the trans configuration. Clinically, when both parents carry cis deletions, ofspring are at risk for an absence of a-hemoglobin, called Hb Barts disease. This typically leads to hydrops and fetal loss as discussed in Chapter 15 (p. 309).

1	Detection of a-thalassemia or a-thalassemia trait is based on molecular genetic testing and is not detectable using hemoglobin electrophoresis. Because of this, routine carrier screening is not ofered. If there is microcytic anemia in the absence of iron deiciency and the hemoglobin electrophoresis is normal, then testing for a-thalassemia can be considered, particularly among individuals of Southeast Asian descent (American College of Obstetricians and Gynecologists, 2015). Mutations in 3-globin genes may cause reduced or absent production of 3-globin chains. If the mutation afects one gene, it results in 3-thalassemia minor. If both copies are afected, the result is either 3-thalassemia major-termed Cooley anemiaor 3-thalassemia intermedia. Because of reduced production of hemoglobin A among carriers, electrophoresis demonstrates elevated levels of hemoglobins that do not contain 3-chains. hese include hemoglobins F and A2.

1	3-Thalassemia minor is more common among individuals of African, Mediterranean, and Southeast Asian descent. The American College of Obstetricians and Gynecologists (2015) recommends that they be ofered carrier screening with hemoglobin electrophoresis, parti�ularly if found to have microcytic anemia in the absence of iron deiciency. Hemoglobin A2 levels exceeding 3.5 percent conirm the diagnosis. Other ethnicities at increased risk include those of Middle Eastern, West Indian, and Hispanic descent .

1	This autosomal recessive lysosomal-storage disease is characterized by absence of the hexosaminidase A enzyme. his leads to accumulation of GM2 gangliosides in the central nervous system, progressive neurodegeneration, and death in early childhood. Afected individuals have almost complete absence of the enzyme, whereas carriers are asymptomatic but have less than 55-percent hexosaminidase A activity. The carrier frequency of T ay-Sachs disease in Jewish individuals of Eastern European (Ashkenazi) descent approximates 1 in 30, but it is much lower, only about 1 in 300, in the general population. Other groups at greater risk for Tay-Sachs disease include those of French-Canadian and Cajun descent. An international Tay-Sachs carrier-screening campaign was initiated in the 1970s and met with unprecedented success in the Ashkenazi Jewish population. he incidence of T ay-Sachs disease subsequently declined more than 90 percent (Kaback, 1993). Most cases of Tay-Sachs disease now occur in

1	unprecedented success in the Ashkenazi Jewish population. he incidence of T ay-Sachs disease subsequently declined more than 90 percent (Kaback, 1993). Most cases of Tay-Sachs disease now occur in nonJewish individuals.

1	he American College of Obstetricians and Gynecologists (20 17b) has the following screening recommendations for T aySachs disease: 1. Screening should be ofered before pregnancy if both members of a couple are of Ashkenazi Jewish, French-Canadian, or Cajun descent, or if there is a family history ofTay-Sachs disease. 2. When only one member of the couple is of one of the above ethnicities, the high-risk partner may be screened first, and if found to be a carrier, the other partner also should be ofered screening. If there is a family history ofTay-Sachs disease, an expanded carrier screening panel may not be the best approach unless the familial mutation is included in the panel. 3. Molecular testing (DNA-based mutation analysis) is highly efective in Ashkenazi Jewish individuals and other high-risk groups, but the detection rate in low-risk groups is more limited. 4.

1	3. Molecular testing (DNA-based mutation analysis) is highly efective in Ashkenazi Jewish individuals and other high-risk groups, but the detection rate in low-risk groups is more limited. 4. Biochemical analysis of the hexosaminidase A serum level has a sensitivity of 98 percent and is the test that should be performed in individuals from low-risk ethnicities. Leukocyte testingmust be used if the woman is already pregnant or taking oral contraceptives. 5. Ifboth partners are found to be carriers ofTay-Sachs disease, genetic counseling and prenatal diagnosis should be ofered. Hexosaminidase activity may be measured from chorionic villus sampling or amniocentesis specimens.

1	The carrier rate among individuals of Eastern European (Ashkenazi) Jewish descent approximates 1 in 30 for T ay-Sachs disease, 1 in 40 for Canavan disease, and 1 in 32 for familial dysautonomia. Fortunately, the detection rate of screening tests for each is at least 98 percent in this population. Because of their relatively high prevalence and consistently severe and predictable phenotype, the American College of Obstetricians and Gynecologists (2017b) recommends that carrier screening for these three conditions be ofered to Ashkenazi Jewish individuals, either before conception or during early pregnancy. his is in addition to carrier screening for cystic fibrosis and spinal muscular atrophy, which are ofered to all women who are considering pregnancy or who are currently pregnant. Further, there are several other autosomal recessive conditions for which the College recommends that screening be considered (American College of Obstetricians and Gynecologists, 2017b).

1	As of 20 17, these include Bloom syndrome, familial hyperinsulinism, Fanconi anemia, Gaucher disease, glycogen storage disease type I (von Gierke disease), Joubert syndrome, maple syrup urine disease, mucolipidosis type IV, Niemann-Pick disease, and Usher syndrome. Gaucher disease difers from the other conditions listed in that it is has a wide range in phenotype-from childhood illness to absence of symptoms throughout life. Also, efective treatment is available in the form of enzyme therapy.

1	Diagnostic procedures used in prenatal diagnosis include amniocentesis, chorionic villus sampling (CVS), and rarely fetal blood sampling. hese enable characterization of an increasingly large number of genetic abnormalities before birth. Karyotype analysis has a diagnostic accuracy of more than 99 percent for aneuploidy and chromosomal abnormalities larger than 5 to 10 megabases. In the setting of a fetal structural abnormality, chromosomal microarray analysis (CMA) is recommended as the first-line genetic test performed, as it may detect clinically significant chromosomal abnormalities in approximately 6 percent of fetuses with normal standard karyotype (Callaway, 2013; de Wit, 2014). An exception would be if the structural abnormality strongly suggests a particular karyotype-such as endocardial cushion defect with trisomy 21 or holoprosencephaly with trisomy 13. In such cases, karyotyping with or without luorescence in-situ hybridization (FISH) may be ofered as the initial test

1	endocardial cushion defect with trisomy 21 or holoprosencephaly with trisomy 13. In such cases, karyotyping with or without luorescence in-situ hybridization (FISH) may be ofered as the initial test (American College of Obstetricians and Gynecologists, 2016b). Among those without evidence of a fetal structural abnormality and with a normal karyotype, CMA has detected additional chromosomal abnormalities (pathogenic copy number variants) in approximately 1 percent. It is therefore made available whenever a prenatal diagnostic procedure is performed (American College of Obstetricians and Gynecologists, 2016b; Callaway, 2013). Types of CMA platforms and their benefits and limitations are reviewed in Chapter 13 (p. 271).

1	Ironically, improvements in aneuploidy screening tests, in particular the widespread use of cell-free DNA screening, have resulted in a dramatic drop in the number of prenatal diagnostic procedures. Larion and colleagues (2014) reported a 70-percent decline in CVS procedures and a nearly 50-percent drop in amniocentesis procedures following introduction of cell-free DNA screening in 2012. This urther amplified the decrease in amniocentesis procedures that began following adoption of first-trimester screening (Warso, 2015). In addition, because so many disorders can be diagnosed from amnionic fluid specimens, fetal blood sampling is now rarely, if ever, indicated for genetic diagnoses.

1	This is the most common prenatal diagnostic procedure. T ransabdominal withdrawal of amnionic luid is generally done between 15 and 20 weeks but may be performed at any point later in gestation. Indications include diagnosis of fetal genetic disorders, congenital infections, and alloimmunization, as well as assessment of fetal lung maturity. The most common types of prenatal diagnostic tests are CMA to assess copy-number gains or losses, karyotype analysis to test for aneuploidy, and FISH to identiY gain or loss of specific chromosomes or chromosome regions (Chap. 13, p. 270). Because amniocytes must be cultured before fetal karyotype can be assessed, the time needed for karyotyping is 7 to 10 days. In contrast, FISH studies are usually completed within 24 to 48 hours. CMA can oten be performed directly on uncultured amniocytes with a turnaround time of only 3 to 5 days, and if amniocyte culture is required, turnaround time is 10 to 14 days (merican College of Obstetricians and

1	be performed directly on uncultured amniocytes with a turnaround time of only 3 to 5 days, and if amniocyte culture is required, turnaround time is 10 to 14 days (merican College of Obstetricians and Gynecologists, 20 16b).

1	Amniocentesis is performed using aseptic technique, with a 20or 22-gauge spinal needle and ultrasound guidance (Fig. 14-4). A standard spinal needle is 9 em long, and depending on patient habitus, a longer needle may be required. Measurement of the TABLE 14-12. Selected Tests Performed on Amnionic Fluid and Typical Volume of Fluid Required FIGURE 14-4 A. Amniocentesis. B. The amniocentesis needle is seen in the upper right portion of this sonogram. (Reproduced with permission from Mastrobattista JM, Espinoza J: Invasive prenatal diagnostic procedures. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. Ne

1	York, McGraw-Hili Education, 201o7.) sonographic distance from skin to amnionic luid pocket may aid needle selection. Sonography is used to identiY a pocket of amnionic luid that is close to the midline, being cognizant of uterine size and shape. he needle is inserted perpendicular to the skin and guided into the deepest portion of the luid pocket, avoiding fetal parts and umbilical cord. Eforts are made to puncture the chorioamnion rather than to push or "tent" it away from the uterine wall. he amnion usually uses with the adjacent chorion by 16 weeks' gestation, and the procedure is generally deferred until ater chorioamnion usion has occurred. Discomfort from the procedure is considered minor, and local anesthetic has not been found to be beneicial (Mujezinovic, 2011).

1	Following the procedure, the color and clarity of the luid are documented. Amnionic fluid should be clear and colorless or pale yellow. Blood-tinged luid is more frequent if there is transplacental passage of the needle. However, it generally clears with continued aspiration. The placenta implants along the anterior uterine wall in approximately half of pregnancies. In these cases, the placenta will be traversed by the needle approximately 60 percent of the time (Bombard, 1995). Needle passage through the placenta is avoided when possible, although fortunately this has not been associated with greater pregnancy loss rates (Marthin, 1997). Dark brown or greenish luid may represent a past episode of intraamnionic bleeding.

1	he volume of fluid generally needed for commonly performed analyses is shown in Table 14-12. Because the initial 1 to 2 mL of fluid aspirate may be contaminated with maternal cells, it is generally discarded. Approximately 20 to 30 mL of fluid is then collected for either fetal CMA or karyotyping before removing the needle. Sonography is used to observe the uterine puncture site for bleeding, and fetal cardiac motion is documented at the procedure's end. If the patient is h D-negative and unsensitized, anti-D immune globulin is administered following the procedure (Chap. 15, p. 305). Multifetal Pregnancy. When performing the procedure in a diamnionic twin gestation, careful attention is paid to the aThe volume of fluid needed for each test may vary according to individual laboratory specifications. bFluorescence in-situ (FISH) is typically performed for chromosomes 21, 18, 13, X, and Y. PCR = polymerase chain reaction.

1	bFluorescence in-situ (FISH) is typically performed for chromosomes 21, 18, 13, X, and Y. PCR = polymerase chain reaction. location of each sac and the dividing membrane. Until recently, a small quantity of dilute indigo carmine dye was often injected before removing the needle from the irst sac, with return of clear amnionic fluid anticipated following needle placement into the second sac. Because of widespread shortages of indigo carmine dye, most experienced providers ofer amniocentesis in multifetal gestations when indicated, without dye injection. Methylene blue dye is contraindicated because it has been associated with jejunal atresia and neonatal methemoglobinemia (Cowett, 1976; van der Pol, 1992).

1	he procedure-related loss rate following midtrimester amniocentesis has decreased with improvements in imaging technology. Based on single-center studies and metaanalysis data, the amniocentesis procedure-related loss rate approximates 0.1 to 0.3 percent when performed by an experienced providerabout 1 per 500 procedures (Akolekar, 2015; American College of Obstetricians and Gynecologists, 2016b; Odibo, 2008). he loss rate may be doubled in women with class 3 obesity, which is a body mass index (BMI) >40 kg/m2 (Harper, 2012). In twin pregnancies, Cahill and coworkers (2009) reported a loss rate of 1.8 percent attributable to amniocentesis.

1	he amniocentesis indication can influence loss rates, which can be greater with some fetal abnormalities, aneuploidies, and conditions such as hydrops. nd, some losses are due to abnor mal placental implantation or abruption, uterine abnormalities, or infection. Wenstrom and colleagues (1990) analyzed 66 fetal deaths following nearly 12,000 procedures and found that 12 per cent were associated with preexisting intrauterine infection. Other complications of amniocentesis include amnionic fluid leakage or transient vaginal spotting in 1 to 2 percent. Following leakage of amnionic luid, which generally occurs within 48 hours of the procedure, fetal survival exceeds 90 percent (Borgida, 2000). Needle injuries to the fetus are rare. Amnionic fluid culture is successul in more than 99 percent of cases, although cells are less likely to grow if the fetus is abnormal (Persutte, 1995).

1	This describes amniocentesis performed between 11 and 14 weeks' gestation. he technique is the same as for traditional amniocentesis, but sac puncture may be more challenging due to lack of membrane fusion to the uterine wall. Less luid is typically withdrawn-approximately 1 mL for each gestational week (Shulman, 1994; Sundberg, 1997). Early amniocentesis is associated with signiicantly higher rates of procedure-related complications than other fetal procedures. hese include development of talipes equinovarus (clubfoot), amnionic luid leakage, and fetal loss (Canadian Early and MidTrimester Amniocentesis Trial, 1998; Philip, 2004). Given these risks, the American College of Obstetricians and Gynecologists (20 16b) recommends that early amniocentesis not be performed.

1	Biopsy of chorionic villi is typically performed between 10 and 13 weeks' gestation. As with amniocentesis, the specimen is generally sent for karyotyping or CMA. The primary advantage of villus biopsy is that results are available earlier in pregnancy, permitting more time for decision-making and safer pregnancy termination, if desired. Very few analyses speciically require either amnionic luid or placental tissue.

1	Chorionic villi may be obtained transcervically or transabdominally, using aseptic technique. Both approaches are considered equally safe and efective (American College of Obstetricians and Gynecologists, 2016b). Transcervical CVS is performed using a specifically designed catheter made from lexible polyethylene that contains a blunt-tipped, malleable stylet. Transabdominal sampling is performed using an 18-or 20-gauge spinal needle. With either technique, transabdominal sonography is used to guide the catheter or needle into the early placentachorion rondosum, followed by aspiration of villi into a syringe containing tissue culture media (Fig. 14-5).

1	FIGURE 14-5 A. Transcervical chorionic villus sampling. B. Catheter entering the placenta is marked and labeled. (Reproduced with permission from Mastrobattista JM, Espinoza J: Invasive prenatal diagnostic procedures. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201o7.) Relative contraindications include vaginal bleeding or spotting, active genital tract infection, extreme uterine ante-or retrolexion, or body habitus precluding adequate visualization. If the patient ish D-negative and unsensitized, anti-D immune globulin is administered following the procedure.

1	he overal loss rate following CVS is higher than that following midtrimester amniocentesis. This is because of background spontaneous losses, that is, losses that would have occurred between the first and second trimester in the absence of a fetal procedure. he procedurerelated fetal loss rate is comparable with that of amniocentesis. Caughey and colleagues (2006) found that the overall loss rate following CVS was approximately 2 percent compared with less than 1 percent following amniocentesis. However, the adjusted procedure-related loss rate approximated 1 per 400 for either procedure. The indication for CVS will also afect the loss rate. For example, fetuses with increased NT thickness have a greater likelihood of demise. Finally, there is a learning curve associated with safe performance of CVS (Silver, 1990; Wijnberger, 2003).

1	An early problem with CVS was its association with limbreduction dects and oro mandibular limb hypogenesis, shown in Figre 14-6 (Firth, 1991, 1994; Hsieh, 1995). hese were subsequently found to be associated with procedures performed at 7 weeks' gestation (Holmes, 1993). When performed at : 10 weeks' gestation, the incidence of limb defects does not exceed the background population rate of about 1 per 1000 (Evans, 2005; Kuliev, 1996). Vaginal spotting is not uncommon following transcervical sampling, but it is self-limited and not associated with pregnancy loss. The incidence of infection is less than 0.5 percent (American College of Obstetricians and Gynecologists, 20 16c).

1	A limitation of CVS is that chromosomal mosaicism is identified in up to 2 percent of specimens (Malvestiti, 2015). In most cases, the mosaicism reflects confined placental mosaicism rather than a true second cell line within the fetus. his is discussed in Chapter 13 (p. 263). Amniocentesis should be ofered, and if the result is normal, the mosaicism is usually presumed to be confined to the placenta. Conined placental mosaicism has been associated with growth-restricted newborns (Bafero, 2012).

1	his procedure is also called cordocentesis or percutaneous umbilical blood sampling (PUBS). It was initially described for fetal transfusion of red blood cells in the setting of anemia from alloimmunization, and fetal anemia assessment remains the most common indication (Chap. 16, p. 304). Fetal blood sampling is also performed for assessment and treatment of platelet alloimmunization and for fetal karyotype determination, particularly in cases of mosaicism identified following amniocentesis or CVS. Fetal blood karyotyping can be accomplished within 24 to 48 hours. Thus, it is significantly quicker than the 7-to 10-day turnaround time with amniocentesis or CVS. Although fetal blood can be analyzed for virtually any test performed on neonatal blood, improvements in tests available with amniocentesis and CVS have eliminated the need for fetal venipuncture in most cases (Society for Maternal-Fetal Medicine, 2013).

1	FIGURE 14-6 Oromandibular limb hypogenesis is characterized by transverse limb deficiency and absence or hypoplasia of the tongue or mandible. This is hypothesized to result from vascular disruption with subsequent loss of tissue. A. Sonogram obtained at 25 weeks' gestation demonstrates a fetal limb reduction defect involving the right hand. B. Photograph of the right extremity of the same newborn. Chorionic villus sampling was not performed in this pregnancy. (Used with permission from Dr. Jamie Morgan.)

1	Under direct sonographic guidance, using aseptic technique, the operator introduces a 22-or 23-gauge spinal needle into the umbilical vein, and blood is slowly withdrawn into a heparinized syringe (Fig. 14-7). Adequate visualization of the needle is essential. 5 with amniocentesis, a longer needle may be required depending on patient habitus. Fetal blood sampling is often performed near the placental cord insertion site, where it may be easier to enter the cord if the placenta lies anteriorly. Alternatively, a free loop of cord may be punctured. Because fetal blood sampling requires more time than other fetal procedures, a local anesthetic may be administered. Prophylactic antibiotics are used at some centers, although no trils support this policy. Arterial puncture is avoided, because it may result in vasospasm and fetal bradycardia. ter the needle is removed, fetal cardiac motion is documented, and the site is observed for bleeding.

1	he procedure-related fetal loss rate following fetal blood sampling approximates 1.4 percent (Ghidini, 1993; Tongsong, 2001). The actual loss rate varies according to the procedure indication and the fetal status. Other complications may include cord vessel bleeding in 20 to 30 percent of cases, fetal-maternal bleeding in approximately 40 percent of cases in which the placenta is traversed, and fetal bradycardia in 5 to 10 percent (Boupaijit, 2012; Society for Maternal-Fetal Medicine,

1	FIGURE 14-7 Fetal blood sampling. A. Access to the umbilical vein varies depending on placental location and cord position. With an anterior placenta, the needle may traverse the placenta. Inset: With posterior placentation, the needle passes through amnionic fiuid before penetrating the umbilical vein. Alternatively, a free loop of cord may be accessed. B. Sonogram shows an anterior placenta with transplacental needle passage into the umbilical vein (U). (Reproduced with permission from Mastrobattista JM, Espinoza J: Invasive prenatal diagnostic procedures. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201/.) 2013). Most complications are transitory, with complete recovery, but some result in fetal loss.

1	In one series of more than 2000 procedures comparing fetal blood sampling near the placental cord insertion site with puncture of a free loop, rates of procedure success, pregnancy loss, visible bleeding from the cord, and fetal bradycardia did not difer. Time to complete the procedure was signiicantly shorter if the cord was sampled at the placental insertion site than in a free 100p-5 versus 7 minutes. However, sampling at the insertion site had a higher rate of maternal blood contamination (T angshewinsirikul, 2011).

1	For couples undergoing in vitro fertilization (IVF), genetic testing performed on oocytes or embryos before implantation may provide valuable information regarding the chromosomal complement and single-gene disorders. There are two separate categories of testing-preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS)-each with diferent indications. Comprehensive genetic counseling is required before consideration of these procedures. There are three techniques that are used for both categories: 1.

1	Polar body anaysis is a technique used to infer whether a developing oocyte is afected by a maternally inherited genetic disorder. The irst and second polar bodies are normally extruded from the developing oocyte following meiosis I and II, and their sampling should not afect fetal development. However, two separate micromanipulation procedures are required, and genetic abnormalities of paternal origin are not detected. his technique has been used to diagnose 146 mendelian disorders, and the reported accuracy exceeds 99 percent (Kuliev, 2011). 2.

1	2. Blastomere biopsy is done at the 6-to 8-cell (cleavage) stage when an embryo is 3 days old. his allows both maternal and paternal genomes to be evaluated. One cell is typically removed through a hole made in the zona pellucida (Fig. 14-8). A limitation of using this technique for aneuploidy assessment is that because of mitotic nondisjunction, mosaicism of the blastomeres may not reflect the chromosomal complement of the developing embryo (American Society for Reproductive Medicine, 2008). In addition, the implantation rate of normal embryos is slightly lower following this technique.

1	FIGURE 14-8 Blastomere biopsy. After a blastomere is selected, it is then drawn into the pipette. (Reproduced with permission from Doody J: Treatment of the infertile couple. Hoffman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.) 3. Trophectoderm biopsy involves removal of 5 to 7 cells from a 5-to 6-day blastocyst. An advantage is that because the trophectoderm cells give rise to the trophoblast-the placenta-no cells are removed from the developing embryo. Disadvantageously, because the procedure is performed later in development, if genetic analysis cannot be performed rapidly, then cryopreservation and embryo transfer during a later IVF cycle may be required.

1	A genetic abnormality-rather than infertility-may be a reason for a couple to elect IVF. With a known carrier(s) of a speciic genetic disease or a balanced chromosomal rearrangement, PGD may be performed to determine if an oocyte or embryo has the defect. Only embryos without the abnormality would be implanted. This procedure has numerous applications. It is used to diagnose single-gene disorders such as cystic fibrosis, 3-thalassemia, and hemophilia; to determine gender in X-linked diseases; to identiy mutations such as BRA-J that do not cause disease but confer signiicantly greater later cancer risk; and to match human leukocyte antigens for umbilical cord stem cell transplantation for a sibling (de Wert, 2007; Fragouli, 2007; Grewal, 2004; Rund, 2005; Xu, 2004).

1	Because typically only one or two cells are available for analysis and because rapid completion time is essential, this procedure is technically challenging. Risks include failure to ampliy the genetic region of interest, selection of a cell that does not contain a nucleus, and maternal cell contamination. Infrequently, afected embryos thought to be normal are implanted, and unaffected embryos are misdiagnosed as abnormal and discarded. Because of this, the American Society for Reproductive Medicine (2008) encourages urther prenatal diagnostic testing-either CVS or amniocentesis-to conirm PGD results. This term is used for aneuploidy screening that is performed on oocytes or embryos before IVF transfer. Such screening is used with couples who are not known to have or carry a genetic abnormality. Although PGS has obvious theoretical advantages, it has faced challenges in practice.

1	Mosaicism is common in cleavage-stage embryo blastomeres. However, it may not be clinically signiicant because it often does not relect the actual embryonic chromosomal complement. In addition, among women 35 years or older, pregnancy rates following PGS with FISH are significantly lower than rates observed following IVF without PGS (Mastenbroek, 2007,2011). Because the number of chromosome pairs per cell nucleus that can be evaluated with FISH is limited, more recent eforts have focused on the use of chromosomal comprehensive screening with CMA (Dahdouh, 2015). Agathokleous M, Chaveeva P, Poon LC, et al: Meta-analysis of second-trimester markers for trisomy 2l. Ultrasound Obstet GynecoIo41(3):247, 2013 Akolekar R, Beta J, Picciarelli G, et al: Procedure-related risk of miscarriage following amniocentesis and chorionic villus sampling: a systematic review and meta-analysis. Ultrasound Obstet GynecoIo45(1):16, 2015

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1	Tangshewinsirikul C, Wanapirak C, Piyamongkol W, et al: Efect of cord puncture site on cordocentesis at mid-pregnancy on pregnancy outcome. Prenat Diagn 31(9):861, 2011 Tongsong T, Wanapirak C, Kunavikatikul C, et al: Fetal loss rate associated with cordocentesis at midgestation. Am J Obstet Gynecol 184(4):719, 2001 van der Pol JG, Wolf H, Boer K, et al: Jejunal atresia related to the use of methylene blue in genetic amniocentesis in twins. BJOG 99(2): 141, 1992 Vink J, Wapner R, D'Alton ME: Prenatal diagnosis in twin gestations. Semin PerinatoIo36(3):169,o2012 Vintzileos AJ, Egan JF: Adjusting the risk for trisomy 21 on the basis of secondtrimester ultrasonography. Am J Obstet Gynecol 172(3):837, 1995 Wald NJ, Cuckle H, Brock JH, et al: Maternal serum-alpha-fetopro.tein measurement in antenatal screening for anencephaly and spina biida in early pregnancy. Report of UK Collaborative Study on alpha-fetoprotein in relation to neural-tube defects. Lancet 1 (8026): 1323, 1977

1	Wald NJ, Densem JW, George L, et al: Prenatal screening for Down's syndrome using inhibin-A as a serum marker. Prenat Diagn 16(2): 143, 1996 Wald NJ, Rodeck C, Hackshaw AK, et al: First and second trimester antenatal screening for Down's syndrome: the results of the Serum, Urine and Ultrasound Screening Study (SURUSS). Health Technol Assess 7(11):1, 2003 Walker SJ, Ball RH, Babcook q, et al: Prevalence of aneuploidy and additional anatomic abnormalities in fetuses and neonates with cleft lip with or without cleft palate. A population-based study in Utah. J Ultrasound Med 20(1o1):1o175,o2001 Wang Y, Chen Y, Tian F, et al: Maternal mosaicism is a significant contributor to discordant sex chromosomal aneuploidies associated with non-invasive prenatal testing. Clin Chen 60(1):251, 2014 Wapner R, Thon E, Simpson JL, et al: First-trimester screening for trisomies 21 and 18. N Engl J Med 349(15):1471,o2003

1	Wapner R, Thon E, Simpson JL, et al: First-trimester screening for trisomies 21 and 18. N Engl J Med 349(15):1471,o2003 Warsof SL, Larion S, Abuhamad Z: Overview of the impact of noninvasive prenatal testing on diagnostic procedures. Prenat Diagn 35(10):972, 2015

1	Warsof SL, Larion S, Abuhamad Z: Overview of the impact of noninvasive prenatal testing on diagnostic procedures. Prenat Diagn 35(10):972, 2015 Wellesley D, Dolk H, Boyd PA, et al: Rare chromosome abnormalities, prevaWou K, Hyun Y, Chitayat D, et al: Analysis of tissue from products of conception lence and prenatal diagnosis rates from population-based congenital anomand perinatal losses using F-PCR and microarray: a three-year retrospective aly registers in Europe. Eur J Hum Genet 20(5):521,o2012 study resulting in an eicient protocol. Eur J Med Genet 59(8):417,o2016 Wenstrom KD, Weiner CP, Williamson A, et al: Prenatal diagnosis of fetal Xu K, Rosenwaks Z, Beaverson K, et al: Preimplantation genetic diagnosis for hyperthyroidism using funipuncture. Obstet Gynecol 76(3 Pt 2):513, retinoblastoma: the irst reported liveborn. Am J OphthalmoIo137(1): 18,o2004 1990 Zhang H, Gao Y, Jiang F, et al: Non-invasive prenatal testing for trisomies

1	Wijnberger LD, van der Schouw T, Christiaens GC: Learning in medicine: 21, 18, and 13: clinical experience from 146,958 pregnancies. Ultrasound chorionic villus sampling. Prenat Diagn 20(3):241, 2003 Obstet Gynecol 45(5):530, 2015 CHAPTER 15 . FETAL ANEMIA .........e...e..e...e.. .....e....e.... 300 RED CEll AllOIMMUNIZATION ..e.............e.... 301 FETOMATERNAl HEMORRHAGE . . . . . . . . . . . . . . . . . . 306 FETAL THROMBOCYTOPENIA ...e..e..e...e...e.....e. 307 HYDROPS FETAlIS. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . 309 MIRROR SYNDROMEe. . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

1	HYDROPS FETAlIS. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . 309 MIRROR SYNDROMEe. . . . . . . . . . . . . . . . . . . . . . . . . . . . 312 General dropsy of the oetus is a rare condition in which the oetus and placenta are markedy oedematous. As the result of iniltration with serum the ormer may attain immense proportions and the latter may be increased to three or our times its normal size. Although a good deal has been written on the subject, no satiactory explanation of the anomay has as yet been arrived at. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) Little was written of fetal disorders in the first edition of this textbook. General dropsy described above is today known as hydrops etalis (p. 309). Hydrops is perhaps the quintessential fetal disorder, as it can be a manifestation of severe illness from a wide variety of etiologies. Fetal disorders may be acquired-such as alloimmunization, they may be genetic-congenital adrenal hyperplasia or aA-thalassemia, or they may be sporadic developmental abnormalities-like many structural malformations. In this chapter, fetal anemia and thrombocytopenia as well as immune and nonimmune fetal hydrops are reviewed. Fetal structural malformations are reviewed in Chapter 10, genetic abnormalities in Chapters 13 and 14, and conditions amenable to medical and surgical fetal therapies in Chapter 16. Because congenital infections arise as a result of maternal infection or colonization, they are considered in Chapters 64 and 65.

1	Of the many causes of fetal anemia, one of the most frequent is red cell alloimmunization, which results from transplacental passage of maternal antibodies that destroy fetal red cells. Alloimmunization leads to overproduction of immature fetal and neonatal red cells-erythroblastosis etalis-a condition now referred to as hemoytic disease of the etus and newborn (HDFN).

1	In addition, several congenital infections are also associated with fetal anemia, particularly parvovirus B19, discussed in Chapter 64 (p. 1216). In Southeast Asian populations, x4-thalassemia is a common cause of severe anemia and nonimmune hydrops. Fetomaternal hemorrhage occasionally creates severe fetal anemia and is discussed on page 306. Rare causes of anemia include red cell production disorders-such as Blackfan-Diamond anemia and Fanconi anemia; red cell enzymopathies-glucose-6-phosphate dehydrogenase deficiency and pyruvate kinase deficiency; red cell structural abnormalities-hereditary spherocytosis and elliptocytosis; and myeloproliferative disorders-leukemias. Anemia may be identified through fetal blood sampling, described in Chapter 14 (p. 294), or by Doppler evaluation of the fetal middle cerebral artery (MCA) peak systolic velocity, described on page 303.

1	Progressive fetal anemia from any cause leads to heart failure, hydrops fetalis, and ultimately death. Fortunately, the prevalence and the course of this otherwise devastating disorder have been dramatically changed by prevention and treatment. Prevention ofD alloimmunization is with anti-D immune globulin. Identiication and treatment of fetal anemia is with MCA Doppler studies and intrauterine transfusions, respectively. Severely anemic fetuses transfused in utero have survival rates exceeding 90 percent, and even in cases of hydrops fetalis, survival rates approach 80 percent (Lindenberg, 2013; Zwiers, 2017).

1	Currently, 33 diferent blood group systems and 339 red cell antigens are recognized by the International Society of Blood Transfusion (Storry, 2014). Although some of these are immunologically and genetically important, many are so rare as to be of little clinical signiicance. Any individual who lacks a specific red cell antigen may produce an antibody when exposed to that antigen. Such antibodies can prove harmful to that individual if she receives an incompatible blood transfusion. Accordingly, blood banks routinely screen for erythrocyte antigens. hese antibodies may also be harmful to a mother's fetus during pregnancy. As noted, maternal antibodies formed against fetal erythrocyte antigens may cross the placenta to cause fetal red cell lysis and anemia.

1	Typically, a fetus inherits at least one red cell antigen from the father that is lacking in the mother. hus, the mother may become sensitized if enough fetal erythrocytes reach her circulation to elicit an immune response. Even so, alloimmunization is uncommon for the following reasons: (1) low prevalence of incompatible red cell antigens; (2) insuicient transplacental passage of fetal antigens or maternal antibodies; (3) maternalfetal ABO incompatibility, which leads to rapid clearance of fetal erythrocytes before they elicit an immune response; (4) variable antigenicity; and (5) variable maternal immune response to the antigen. In population-based screening studies, the prevalence of red cell alloimmunization in pregnancy approximates 1 percent (Bollason, 2017; Koelewijn, 2008). Most cases of severe fetal anemia requiring antenatal transfusion are attributable to anti-D, anti-Kell, anti-c, or anti-E alloimmunization (de Haas, 2015).

1	At the first prenatal visit, a blood type and antibody screen are routinely assessed, and unbound antibodies in maternal serum are detected by the indirect Coombs test (Chap. 9, p. 160). When the result is positive, the specific antibodies are identified, their immunoglobulin subtype is determined as either immunoglobulin G (IgG) or M (IgM) , and the titer is quantiied. Only IgG antibodies are a concern because IgM antibodies do not cross the placenta. Selected antibodies and their potential to cause fetal hemolytic anemia are listed in Table he critical titer is the level at which significant fetal anemia could potentially develop. This may be diferent for each antibody, is determined individually by each laboratory, and usually ranges between 1:8 and 1 :32. If the critical titer for anti-D antibodies is 1: 16, a titer : 1: 16 indicates the possibility of severe hemolytic disease. An important exception is Kell sensitization, which is discussed on page 302.

1	he CDE system includes ive red cell proteins or antigens: C, c, D, E, and e. here is no "d" antigen, and D-negativity is deined as the absence of the D antigen. Although most people are D positive or negative, more than 200 D antigen variants exist (Daniels, 2013). h was formerly termed rhesus because of TABLE 15-1. Selected Red Cell Antigens and Their Relationship to Fetal Hemolytic Disease CDE (Rh) 0,E, Bea, Ce, Cw, Cx, ce, c Ow, Evans, e, G, Goal, Hr, Hro, JAL, HOFM, LOCR, Riv. Rh29, Rh32, Rh42, Rh46, STEM, Tar k, Kp3, Kpb, K1s1, K22 Ku, Jsa, Jsb, Ula Jkb, Jk3 MNS M, N, S, s, U, Mtd, Ena, Far, Hil, Hut, Mia, Mit, Mut, Mur, Mv, sO, Vw Colton Cd, C03 Severe disease risk Severe disease infrequent, mild disease risk Severe disease infrequent, mild disease risk Severe disease infrequent, mild disease risk Not associated with fetal hemolytic disease Severe disease infrequent, mild disease risk Mild disease possible Severe disease infrequent, mild disease risk

1	Severe disease infrequent, mild disease risk Severe disease infrequent, mild disease risk Severe disease infrequent, mild disease risk Dd, Gy3, Hy, Joa Mild disease possible , Ge3, Ge4 , Lsa Mild disease possible I I, i Not associated with fetal hemolytic disease Lewis Lea, Leb Not associated with fetal hemolytic disease From de Haas, 2015; Moise, 2008; Weinstein, 1982. a misconception that red cells from rhesus monkeys expressed human blood group antigen. In transfusion medicine, "rhesus" is no longer used (Sandler, 2017).

1	a misconception that red cells from rhesus monkeys expressed human blood group antigen. In transfusion medicine, "rhesus" is no longer used (Sandler, 2017). CDE antigens are clinically important. D-negative individuals may become sensitized after a single exposure to as little as 0.1 mL of fetal erythrocytes (Bowman, 1988). The two responsible genes-RHD and RHCE-are located on the short arm of chromosome 1 and are inherited together, independent of other blood group genes. The incidence of antigen positivity varies according to racial and ethnic origin. Nearly 85 percent of non-Hispanic white Americans are D-positive. The incidence approximates 90 percent for Native Americans, 93 percent for frican Americans and Hispanic Americans, and at least 99 percent for Asian individuals (Garratty, 2004).

1	The prevalence of D alloimmunization complicating pregnancy ranges from 0.5 to 0.9 percent (Koelewijn, 2008; \1artin, 2005). Without anti-D immune globulin prophylaxis, a D-negative woman delivered of a D-positive, ABO-compatible newborn has a 16-percent likelihood of developing alloimmunization. Two percent will become sensitized by the time of delivery, 7 percent by 6 months postpartum, and the remaining 7 percent will be "sensibilized"-producing detectable antibodies only in a subsequent pregnancy (Bowman, 1985). If there is ABO incompatibility, the D alloimmunization risk approximates 2 percent without prophylaxis (Bowman, 2006). The reason for the difering rates relative to ABO blood type results from erythrocyte destruction of ABO-incompatible cells, which thereby limits sensitizing opportunities. D sensitization also may occur following irst-trimester pregnancy complications, prenatal diagnostic procedures, and maternal trauma (Table 15-2).

1	The C, c, E, and e antigens have lower immunogenicity than the D antigen but can cause hemolytic disease. Sensitization to E, c, and C antigens complicates approximately 0.3 percent of pregnancies in screening studies and accounts for about 30 percent of red cell alloimmunization cases (Howard, 1998; Koelewijn, 2008). Anti-E alloimmunization is the most common, but the need for fetal or neonatal transfusions is greater with anti-c alloimmunization than with anti-E or anti-C (de Haas, 2015; Hackney, 2004; Koelewijn, 2008).

1	The Grandmother Efect. In virtually all pregnancies, small amounts of maternal blood enter the fetal circulation. Realtime polymerase chain reaction (PCR) has been used to identiy maternal D-positive DNA in peripheral blood from pre term and full-term D-negative newborns (Lazar, 2006). hus, it is possible for a D-negative female fetus exposed to maternal D-positive red cells to develop sensitization. When such an individual reaches adulthood, she may produce anti-D antibodies even before or early in her irst pregnancy. This mechanism is called the grandmother eect or theory because the fetus in the current pregnancy is jeopardized by maternal antibodies that were initially provoked by his or her grandmothers erythrocytes. Alloimmunization to Minor Antigens Because routine administration of anti-D immunoglobulin prevents anti-D alloimmunization, proportionately more cases of hemolytic disease are caused by red cell antigens

1	Because routine administration of anti-D immunoglobulin prevents anti-D alloimmunization, proportionately more cases of hemolytic disease are caused by red cell antigens TABLE 15-2. Causes of Fetomaternal Hemorrhage Associated with Red Cell Antigen Alloimmunizationa Procedures Chorionic villus sampling Amniocentesis Fetal blood sampling Evacuation of molar pregnancy aFor each of the above, anti-D immune globulin is recom mended. Expanded from American Academy of Pediatrics and American College of Obstetricians and Gynecologists, 2017; American College of Obstetricians and Gynecologists, 2017.

1	Expanded from American Academy of Pediatrics and American College of Obstetricians and Gynecologists, 2017; American College of Obstetricians and Gynecologists, 2017. other than D (American College of Obstetricians and Gynecologists, 2016; Koelewijn, 2008). hese are also known as minor antigens. Kell antigens are among the most frequent. Other antigens with potential to cause severe alloimmunization include Duy group A_Fya, MNS, and Kidd-Jka (de Hass, 2015; Moise, 2008). Most cases of sensitization to minor antigens result from incompatible blood transfusions. However, if an IgG red cell antibody is detected and there is any doubt as to its significance, the clinician should err on the side of caution, and the pregnancy should be evaluated for hemolytic disease.

1	Only a few blood group antigens pose no fetal risk. Lewis antibodies-Lea and Leb, as well as I antibodies, are cold agglutinins. They are predominantly IgVl and are not expressed on fetal red cells (American College of Obstetricians and Gynecologists, 2016). Another antibody that does not cause fetal hemolysis is Duy group B-Fl. Kell Alloimmunization. Approximately 90 percent of nonHispanic white Americans and up to 98 percent of African Americans are Kell negative. Kell type is not routinely determined. Transfusion history is important, as nearly 90 percent of Kell sensitization cases result from transfusion with Kellpositive blood. .

1	Kell sensitization may develop more rapidly and may be more severe than with sensitization to D and other blood group antigens. his is because Kell antibodies attach to erythrocyte precursors in the fetal bone marrow, thereby impairing the normal hemopoietic response to anemia. With fewer erythrocytes produced, there is less hemolysis, and severe anemia may not be predicted by the maternal Kell antibody titer. One option is to use a lower critical titer-l:8-for Kell sensitization (Moise, 2012). he American College of Obstetricians and Gynecologists (2016) has recommended that antibody titers not be used to monitor Kell-sensitized pregnancies.

1	Incompatibility for the major blood group antigens A and B is the most common cause of hemolytic disease in newborns, but it does not cause appreciable hemolysis in the fetus. Approxi mately 20 percent of newborns have ABO blood group incom patibility, yet only 5 percent are afected clinically. And in such cases, the resulting anemia is typically mild.

1	The condition difers from CDE incompatibility in several respects. First, ABO incompatibility is often seen in firstborn neonates, whereas sensitization to other blood group antigens is not. This is because most group 0 women have developed antiA and anti-B isoagglutinins before pregnancy from exposure to bacteria displaying similar antigens. Second, ABO alloimmunization rarely becomes more severe in successive pregnancies. Last, ABO incompatibility is considered a pediatric diseaserarely of obstetrical concern. his is because most anti-A and anti-B antibodies are IgM and do not cross the placenta. Fetal red cells also have fewer A and B antigenic sites than adult cells and are thus less immunogenic. Consequently, fetal surveillance and early delivery are not indicated in pregnancies with prior ABO incompatibility. Careful neonatal observation is essential, however, because hyperbilirubinemia may require treatment with phototherapy or occasionally transfusion (Chap. 33, p. 626).

1	• Management of the Alloimmunized An estimated 25 to 30 percent of fetuses from D-alloimmunized pregnancies will have mild-to-moderate hemolytic anemia. And without treatment, up to 25 percent will develop hydrops (Tannirandorn, 1990). If alloimmunization is detected and the titer is below the critical value, the titer is generally repeated every 4 weeks for the duration of the pregnancy (American College of Obstetricians and Gynecologists, 2016). Importantly, if a prior pregnancy was complicated by alloimmunization, serial titer assessment is not indicated, and the pregnancy is assumed to be at risk regardless of titer. Management of such pregnancies is discussed subsequently. In any pregnancy in which an antibody titer has reached a critical value, there is no beneit to repeating it. The pregnancy is at risk even if the titer drops, and further evaluation is still required.

1	Up to 40 percent of D-negative pregnant women carry a D-negative fetus. he presence of anti-D antibodies relects maternal sensitization but does not indicate whether the fetus is D-positive. If a woman became sensitized in a prior pregnancy, her antibody titer might rise to high levels during the current pregnancy even if the current fetus is D-negative, due to an amnestic response. In a non-Hispanic white couple in which the woman is D-negative, there is an 85-percent chance that the man is D-positive. But, in 60 percent of these cases, he will be heterozygous at the D-Iocus. And, if he is heterozygous, then half of his children will be at risk for hemolytic disease. Transfusion history is relevant. Alloimmunization to a red cell transfusion in the past, and if that antigen is not present on paternal erythrocytes, the pregnancy is not at risk.

1	Initial evaluation of alloimmunization begins with determining the paternal erythrocyte antigen status. Provided that paternity is certain, if the father is negative for the red cell antigen to which the mother is sensitized, the pregnancy is not at risk. In a D-alloimmunized pregnancy in which the father is D-positive, it is helpful to determine paternal zygosity for the D antigen using DNA-based analysis. If the father is heterozygous-or if paternity is not known-the woman should be ofered assessment of fetal genotype. Traditionally, this was done with amniocentesis and PCR testing of uncultured amniocytes, which has a positive-predictive value of 100 percent and negative-predictive value of approximately 97 percent (American College of Obstetricians and Gynecologists, 2016; Van den Veyver, 1996). Fetal testing for other antigens-such as E/e, C/c, Duy, Kell, Kidd, and �1/N-is also available with this method. Chorionic villus sampling is not recommended because of greater risk for

1	1996). Fetal testing for other antigens-such as E/e, C/c, Duy, Kell, Kidd, and �1/N-is also available with this method. Chorionic villus sampling is not recommended because of greater risk for fetomaternal hemorrhage and subsequent worsening of alloimmunization.

1	Noninvasive fetal D genotyping has been performed using cell-free DNA (cfDNA) from maternal plasma (Chap. 13, p. 273). The reported sensitivity exceeds 99 percent, the speciicity exceeds 95 percent, and positive-or negative-predictive values are similarly very high (de Haas, 2016; Johnson, 2017; Moise, 2016; Vivanti, 2016). Fetal D genotyping with cDNA is routinely used in parts of Europe. There are two potential indications in D-negative pregnant women: (1) in women with D alloimmunization, testing can identiY fetuses that are also D-negative and do not require anemia surveillance, and (2) in women without D alloimmunization, anti-D immune globulin might be withheld if the fetus is D negative. In the case of the latter, the American College of Obstetricians and Gynecologists (2017) does not recommend routine cDNA screening in D-negative pregnancies until it becomes cost-efective.

1	Management of the alloimmunized pregnancy is individualized and may consist of maternal antibody titer surveillance, sonographic monitoring of the fetal MCA peak systolic velocity, amnionic luid bilirubin studies, or fetal blood sampling. Accurate pregnancy dating is critical. The gestational age at which fetal anemia developed in prior pregnancies is important because anemia tends to occur earlier and be sequentially more severe. Middle Cerebral Artery Doppler Velocimetry. Serial measurement of the peak systolic velocity of the fetal MCA is the recommended test for detection of fetal anemia (Society for Maternal-Fetal Medicine, 20 15a). he anemic fetus shunts blood preferentially to the brain to maintain adequate oxygenation. he velocity rises because of increased cardiac output and decreased blood viscosity. The technique is discussed Fetus without anemia or with mild anemiac� nE 120 • Fetus with severe anemia .-u .�::100 u ..o� )� . ) 40n� �u

1	Fetus without anemia or with mild anemiac� nE 120 • Fetus with severe anemia .-u .�::100 u ..o� )� . ) 40n� �u FIGURE 15-1 Doppler measurements ofthe peak systolic velocity in the middle cerebral artery (MeA) in 165 fetuses at risk for severe anemia. The blue line indicates the median peak systolic velocity in normal pregnancies, and the red line shows 1.5 multiples ofthe median. (Reproduced with permission from Oepkes 0, Seaward PG, Vandenbussche et al: Doppler ultrasonography versus amniocentesis to predict fetal anemia, N Engl J Med. 2006 Jul 13;355(2):156-164.) in Chapter 10 (p. 214) and requires training and experience (American College of Obstetricians and Gynecologists, 2016).

1	In a landmark study, Mari and coworkers (2000) measured the MCA peak systolic velocity serially in 111 fetuses at risk for anemia and in 265 normal control fetuses. he threshold value of 1.5 multiples ofthe median (MoM) for gestational age correctly identiied all fetuses with moderate or severe anemia. his provided a sensitivity of 100 percent, with a false-positive rate of 12 percent.

1	he MCA peak systolic velocity is followed serially, and values are plotted on a curve like the one shown in Figure 15-1. If the velocity is between 1.0 and 1.5 MoM and the slope is rising-such that the value is approaching 1.5 Movf-surveillance is generally increased to weekly Doppler interrogation. If the MCA peak systolic velocity exceeds 1.5 \10M and the gestational age is younger than 34 or 35 weeks, fetal blood sampling should be considered and followed by fetal transfusion if needed (Society for Maternal-Fetal Medicine, 2015a). he false-positive rate of MCA peak systolic velocity increases signiicantly beyond 34 weeks, due to the normal augmentation in cardiac output that develops at this gestational age (Moise, 2008; Zimmerman, 2002).

1	Amnionic Fluid Spectral Analysis. This test is included for historical interest. More than 50 years ago, Liley (1961) demonstrated the utility ofamnionic fluid spectral analysis to measure bilirubin concentration and to thereby estimate hemolysis severity. Amnionic luid bilirubin concentration was measured by a spectrophotometer and was represented as the change in optical density absorbance at 450 nm-10D450. The likelihood of fetal anemia was determined by plotting the 10D45o value on a graph that was divided into zones. hese zones roughly correlated with fetal hemoglobin concentration, and thus with anemia severity. he original Liley graph was valid from 27 to 42 weeks' gestation and was subsequently modiied by Queenan (1993) to include gestational ages as early as 14 weeks. However, the amnionic luid bilirubin level is normally high in midpregnancy, limiting the reliability ofthis technique.

1	Middle cerebral artery velocimetry is more accurate than 10D45o assessment and does not confer risks for increased alloimmunization associated with amniocentesis. It has replaced 10D45o assessment for this purpose.

1	If there is evidence of severe fetal anemia, because of either elevated MCA peak systolic velocity or development of fetal hydrops, management is strongly inluenced by gestational age. Fetal blood sampling and intrauterine transfusion are generally performed prior to 34 to 35 weeks (Society for Maternal-Fetal Medicine, 2015a). Intravascular transfusion into the umbilical vein under sonographic guidance is the preferred method of fetal transfusion. Transfusion into the fetal peritoneal cavity may be necessary with severe, early-onset hemolytic disease in the early second trimester, a time when the umbilical vein is too narrow to readily permit needle entry. With hydrops, although peritoneal absorption is impaired, some prefer to transfuse into both the fetal peritoneal cavity and the umbilical vein.

1	Transfusion is generally recommended only if the fetal hematocrit is <30 percent (Society for Maternal-Fetal Medicine, 2015a). Once hydrops has developed, the hematocrit is generally 15 percent or lower. The red cells transfused are type 0, D-negative, cytomegalovirus-negative, packed to a hematocrit of approximately 80 percent to prevent volume overload, irradiated to prevent fetal graft-versus-host reaction, and leukocyte-poor. he fetal-placental volume allows rapid infusion ofa relatively large quantity ofblood. Before transfusion, a paralytic agent such as vecuronium may be given to the fetus to minimize movement. In a nonhydropic fetus, the target hematocrit is generally 40 to 50 percent. he volume transfused may be estimated by multiplying the estimated fetal weight in grams by 0.02 for each 10-percent rise in hematocrit needed (Giannina, 1998). In the severely anemic fetus at 18 to 24 weeks' gestation, less blood is transfused initially, and another transfusion may be planned

1	for each 10-percent rise in hematocrit needed (Giannina, 1998). In the severely anemic fetus at 18 to 24 weeks' gestation, less blood is transfused initially, and another transfusion may be planned for approximately 2 days later. Subsequent transfusions usually take place every 2 to 4 weeks, depending on the hematocrit.

1	The MCA peak systolic velocity threshold for severe anemia is higher following an initial transfusion-1.70 MoM rather than 1.50 MoYI (Society for Maternal-Fetal Medicine, 2015a). It is hypothesized that the change in threshold compensates for the contribution ofdonor cells in the initial transfusion, because donor cells (from adults) have a smaller mean corpuscular volume. Alternately, the timing ofsubsequent transfusions is based on anemia severity and posttransfusion hematocrit. Following transfusion, the fetal hematocrit generally drops by approximately 1 percent per day. A more rapid initial decline may be encountered in the setting of fetal hydrops.

1	Outcomes. Procedure-related complications have declined signiicantly at experienced centers in recent years, with overall survival rates exceeding 95 percent (Zwiers, 2017). Complications include fetal death in approximately 2 percent, need for emergent cesarean delivery in 1 percent, and infection and preterm rupture of membranes in 0.3 percent each, respectively. The stillbirth rate exceeds 15 percent if transfusion is required before 20 weeks (Lindenberg, 2013; Zwiers, 2017). Considering that fetal transfusion is potentially lifesaving in severely compromised fetuses, these risks should not dissuade therapy. Van Kamp (2001) reported that if hydrops had developed, the survival rate approached 75 to 80 percent. However, of the nearly two thirds with resolution of hydrops following transfu sion, more than 95 percent survived. he survival rate was < 40 percent if hydrops persisted.

1	Lindenberg (2012) reviewed long-term outcomes following intrauterine transfusion in a cohort of more than 450 alloimmunized pregnancies. Alloimmunization was secondary to anti-D in 80 percent, anti-Kell in 12 percent, and anti-c in 5 percent. Approximately a fourth of afected fetuses had hydrops, and more than half also required exchange transfusion in the neonatal period. Among nearly 300 children aged 2 to 17 years who participated in neurodevelopmental testing, fewer than 5 percent had severe impairments. hese included severe developmental delay in 3 percent, cerebral palsy in 2 percent, and deafness in 1 percent. • Prevention of Anti-D Alloimmunization

1	• Prevention of Anti-D Alloimmunization Anti-D immune globulin is one of the success stories of modern obstetrics. It has been used for nearly five decades to prevent D alloimmunization. In countries without access to anti-D immune globulin, up to 10 percent of D-negative pregnancies are complicated by hemolytic disease of the fetus and newborn (Zipursky, 2015). With immunoprophylaxis, however, the alloimmunization risk is reduced to <0.2 percent. Despite long-standing and widespread use, its mechanism of action is not completely understood.

1	As many as 90 percent of alloimmunization cases occur from fetomaternal hemorrhage at delivery. Routine postpartum administration of anti-D immune globulin to at-risk pregnancies within 72 hours of delivery lowers the alloimmunization rate by 90 percent (Bowman, 1985). Additionally, provision of anti-D immune globulin at 28 weeks' gestation reduces the third-trimester alloimmunization rate from approximately 2 percent to 0.1 percent (Bowman, 1988). Whenever there is doubt whether to give anti-D immunoglobulin, it should be given. If not needed, it will not cause harm, but failure to provide it when needed can have severe consequences.

1	Current preparations of anti-D immune globulin are derived from human plasma donated by individuals with hightiter anti-D immunoglobulin D antibodies. Formulations prepared by cold ethanol fractionation and ultrailtration must be administered intramuscularly because they contain plasma proteins that could result in anaphylaxis if given intravenously. However, formulations prepared using ion exchange chromatography may be administered either intramuscularly or intravenously. his is important for treatment of significant fetomaternal hemorrhage, which is discussed subsequently. Both preparation methods efectively remove viral particles, including hepatitis and human immunodeiciency viruses. Depending on the preparation, the half-life of anti-D immune globulin ranges from 16 to 24 days, which is why it is given both in the third trimester and following delivery. he standard intramuscular dose of anti-D immune globulin-300 I-Lg or 1500 IU-will protect the average-sized mother from a fetal

1	is why it is given both in the third trimester and following delivery. he standard intramuscular dose of anti-D immune globulin-300 I-Lg or 1500 IU-will protect the average-sized mother from a fetal hemorrhage of up to 30 mL of fetal whole blood or 15 mL of fetal red cells.

1	In the United States, anti-D immune globulin is given prophylactically to all D-negative, unsensitized women at approximately 28 weeks' gestation, and a second dose is given after delivery if the newborn is D-positive (American College of Obstetricians and Gynecologists, 2017). Before the 28-week dose of anti-D immune globulin, repeat antibody screening is recommended to identiy individuals who have become alloimmunized (American Academy of Pediatrics, 2017). Following delivery, anti-D immune globulin should be given within 72 hours. Recognizing that 40 percent of neonates born to D-negative women are also D negative, administration of immune globulin is recommended only after the newborn is conirmed to be D positive (American College of Obstetricians and Gynecologists, 2017). If immune globulin is inadvertently not administered following delivery, it should be given as soon as the omission is recognized, because there may be some protection up to 28 days postpartum (Bowman, 2006).

1	globulin is inadvertently not administered following delivery, it should be given as soon as the omission is recognized, because there may be some protection up to 28 days postpartum (Bowman, 2006). Anti-D immune globulin is also administered after pregnancyrelated events that could result in fetomaternal hemorrhage (see Table 15-2).

1	Anti-D immune globulin may produce a weakly positive-1: 1 to 1 :4-indirect Coombs titer in the mother. This is harmless and should not be confused with development of alloimmunization. Additionally, as the body mass index increases above 27 to 40 kg/m2, serum antibody levels decrease by 30 to 60 percent and may be less protective (MacKenzie, 2006; Woelfer, 2004). D-negative women who receive other types of blood products-including platelet transfusions and plasmapheresis-are also at risk of becoming sensitized, and this can be prevented with anti-D immune globulin. Rarely, a small amount of antibody crosses the placenta and results in a weakly positive direct Coombs test in cord and infant blood. Despite this, passive immunization does not cause signiicant fetal or neonatal hemolysis.

1	It is estimated that in 2 to 3 per 1000 pregnancies, the volume of fetomaternal hemorrhage exceeds 30 mL of whole blood (American College of Obstetricians and Gynecologists, 2017). A single dose of anti-D immune globulin would be insuicient in such situations. If additional anti-D immune globulin is considered only for women with risk factors such as those shown in Table 15-2, then hafof those who require additional immune globulin may be missed. For this reason, all D-negative women should be screened at delivery, typically with a rosette test, followed by quantitative testing if indicated (American College of Obstetricians and Gynecologists, 20i17).

1	he rosette test is a qualitative test that identiies whether fetal D-positive cells are present in the circulation of a D-negative woman. A sample of maternal blood is mixed with anti-D antibodies that coat any D-positive fetal cells present in the sample. Indicator red cells bearing the D-antigen are then added, and rosettes form around the fetal cells as the indicator cells attach to them by the antibodies. hus, if rosettes are visualized, there are fetal D-positive cells in that sample. In the setting of D incompatibility, or any time a large fetomaternal hemorrhage is suspected-regardless of antigen status, a Kleihauer-Betke test or flow cytometry test are used. These are discussed on page 307.

1	The dosage of anti-D immune globulin is calculated from the estimated volume of the fetal-to-maternal hemorrhage, as described on page 307. One 300-�g dose is given for each 15 mL of fetal red cells or 30 mL of fetal whole blood to be neutralized. If using an intramuscular preparation of anti-D immune globulin, no more than five doses may be given in a 24-hour period. If using an intravenous preparation, two ampules-totaling 600 �g-may be given every 8 hours. To determine if the administered dose was adequate, the indirect Coombs test may be performed. A positive result indicates that there is excess anti-D immunoglobulin in maternal serum, thus demonstrating that the dose was suicient. Alternatively, a rosette test may be performed to assess whether circulating fetal cells remain.

1	Formerly called D") these are the most common antigenic D variants in the United States and Europe. Serological weak D phenotypes have been further refined into two general categories using molecular analysis-RHD genotyping. Molecular weak D phenotypes carry reduced numbers of intact D antigens on the red cell surface. Those designated partial D types have protein deletions associated with abnormal D antigens that lack epitopes (Sandler, 2017). When this distinction is known, it can have clinical consequences in terms of sensitization risk and need for anti-D immune globulin.

1	Traditionally, serological weak D individuals have been considered to be D-positive or -negative depending on the clinical situation. For the purposes of blood donation, they are categorized as D-positive, whereas transfusion recipients with weak D are considered D-negative. In pregnancy, weak D has also been considered D-negative, so that individuals receive immune globulin and avoid potential sensitization (American College of Obstetricians and Gynecologists, 2017; Sandler, 2015).

1	Many non-Hispanic white Americans who test positive for weak D have weak D phenotypes 1, 2, or 3. Individuals with these phenotypes may be managed as though they are D-positive. Because they are not at risk for alloimmunization, anti-D immune globulin is not needed (Sandler 2015, 2017). In contrast, individuals with partial D antigens may be at risk for D-sensitization and do require immune globulin. Molecular RHD genotyping has been suggested for pregnant women with weak D phenotype, bur cost-benefit analysis of this strategy is presently lacking (American College of Obstetricians and Gynecologists, 2017). f molecular genetic testing has not been peormed in those with serologic weak D phenoype) D immunoprophylaxis should be administered to those with weak D phenotype.

1	A small amount of fetomaternal bleeding likely occurs in all pregnancies, and in two thirds, this may be suicient to provoke an antigen-antibody reaction. As shown in Figure 15-2, the incidence increases with advancing gestation and the 80 0.19 mL 70 )> 60;;)>C 50)C·uE 40 FIGURE 15-2 Incidence of fetal-to-maternal hemorrhage during pregnancy. The numbers at each data point represent total volume of fetal blood estimated to have been transferred into the maternal circulation. (Data from Choavaratana, 1997.) volume of fetal blood in the maternal circulation. Fortunately, a large blood loss-true fetomaternal hemorrhage-is rare. In one series of more than 30,000 pregnancies, fetomaternal hemorrhage ::150 mL occurred in 1 per 2800 births (de Almeida, 1994). he prevalence of fetomaternal hemorrhage of at least 30 mL-the volume of fetal blood covered by a standard 300 �g dose of anti-D immune globulin-is estimated to be 3 per 1000 pregnancies (Wylie, 2010).

1	Selected causes of fetomaternal hemorrhage are shown in Table 15-2. It also may occur with placenta previa, placental chorioangioma, or vasa previa (Giacoia 1997; Rubod, 2007). In each of these circumstances, however, fetomaternal hemorrhage is extremely uncommon if not rare. And, in more than 80 percent of cases, no cause is identified. With significant hemorrhage, the most common presenting complaint is decreased fetal movement (Bellussi, 2017; Wylie, 2010). A sinusoidal fetal heart rate pattern is infrequently seen but warrants immediate evaluation (Chap. 24, p. 464). Sonography may demonstrate elevated MCA peak systolic velocity, and indeed this is reported to be the most accurate predictor (Bellusi, 2017; Wylie, 2010). Hydrops is an ominous inding. If fetomaternal hemorrhage is suspected, an elevated MCA peak systolic velocity or sonographic evidence of hydrops prompts consideration of urgent fetal transfusion or delivery.

1	One limitation of quantitative tests for fetal cells in the maternal circulation is that they do not provide information regarding hemorrhage timing or chronicity (Wylie, 2010). In general, anemia developing gradually or chronically, as in alloimmunization, is better tolerated by the fetus than acute anemia. Chronic anemia may not produce fetal heart rate abnormalities until the fetus is moribund. In contrast, significant acute hemorrhage is poorly tolerated by the fetus and may cause profound fetal neurological impairment from cerebral hypoperfusion, ischemia, and infarction. In some cases, fetomaternal hemorrhage is identified during stillbirth evaluation (Chap. 35, p. 646). FIGURE 15-3 Kleihauer-Betke test demonstrating massive fetalto-maternal hemorrhage. After acid-elution treatment, fetal red cells rich in hemoglobin F stain darkly, whereas maternal red cells with only very small amounts of hemoglobin F stain lightly.

1	Once fetomaternal hemorrhage is recognized, the volume of fetal blood loss should be estimated. he volume is essential to calculate the appropriate dose of anti D-immune globulin if the woman is D-negative, and it may inluence obstetrical management. The most commonly used quantitative test for fetal red cells in the maternal circulation is the acid elution or Kleihauer-Betke (KB) test (Kleihauer, 1957). Fetal erythrocytes contain hemoglo bin F, which is more resistant to acid elution than hemoglobin

1	A. After exposure to acid, only fetal hemoglobin remains, such that after staining, the fetal erythrocytes appear red and adult erythrocytes appear as "ghosts" (Fig. 15-3). he fetal cells are then counted and expressed as a percentage of adult cells. he KB test is labor intensive. Importantly, there are two scenarios in which it may not be accurate: (1) maternal hemoglobinopathies such as �-thalassemia in which the fetal hemoglobin level is elevated and (2) pregnancies at or near term, when the fetus has already started to produce hemoglobin A. he volume of fetomaternal hemorrhage is calculated from the KB test result using the following formula:

1	One method is to estimate the maternal blood volume (MBV) as 5000 mL for a normal-size, normotensive women at term. Thus, for 1.7 -percent positive KB-stained cells in a woman of average size with a hematocrit of 35 percent and whose fetus has a hematocrit of 50 percent: 5000 X 0.35 X 0.017 0.5 he fetal-placental blood volume at term approximates 125 mLlkg. For a 3000-g fetus, that would equate to 375 mL. Thus, this fetus lost approximately 15 percent (60 -375 mL) of the fetal-placental volume. Because the hematocrit is 50 percent in a term fetus, this 60 mL of whole blood represents 30 mL of red cells lost into the maternal circulation. This loss should be well tolerated hemodynamically but would require two 300-lLg doses of anti-D immunoglobulin to prevent alloimmunization. A more precise method to estimate the maternal blood volume includes a calculation based on the maternal height, weight, and anticipated physiological maternal blood volume accrual (Table 41-1, p. 756).

1	Fetomaternal hemorrhage can also be quantified using low cytometry, which uses monoclonal antibodies to hemoglobin F or to the D antigen, followed by quantiication of luorescence (Chambers, 2012; Welsh, 2016). Flow cytometry is an automated test that can analyze a greater number of cells than the KB test. Further, it is unafected by maternal levels of fetal hemoglobin or by fetal levels of hemoglobin A. Flow cytometry has been reported to be more sensitive and accurate than the KB test, however, it uses specialized technology not routinely available in many hospitals (Chambers, 2012; Corcoran, 2014; Fernandes, 2007).

1	This condition is also referred to as neonatal alloimmune throm cytopenia (FNAIT). Alloimmune thrombocytopenia (AIT) is the most common cause of severe thrombocytopenia among term newborns, with a frequency of 1 to 2 per 1000 births (Kamphuis, 2010; Pacheco, 2013; Risson, 2012). FNAIT is caused by maternal alloimmunization to paternally inherited fetal platelet antigens. he resulting maternal antiplatelet anti bodies cross the placenta in a manner similar to red cell allo immunization (p. 301). Unlike immune thrombocytopenia, the maternal platelet count is normal with FNAIT. And, unlike anti-D alloimmunization, severe sequelae may afect the initial at-risk pregnancy.

1	Maternal platelet alloimmunization is most often against human platelet antigen-1a (HPA-1a). It accounts for 80 to 90 percent of cases and is associated with the greatest severity (Bussel, 1997; Knight, 2011; Tiller, 2013). his is followed in order of frequency by HPA-5b, HPA-1b, and HPA-3a. Alloimmunization to other antigens accounts for only 1 percent of reported cases.

1	Approximately 85 percent of non-Hispanic white individuals are HPA-1a positive. Two percent are homozygous for HPA-1b and thus at risk for alloimmunization. Importantly, however, only 10 percent of homozygous HPA-1 b mothers who carry an HPA-1a fetus will produce anti-platelet antibodies. Approximately a third of afected fetuses or neonates will develop severe thrombocytopenia, and 10 to 20 percent of those with severe thrombocytopenia sustain an intracranial hemorrhage (ICH) (Kamphuis, 2010). As a result, population-based screening studies have identified FNAIT-associated ICH in 1 per 25,000 to 60,000 pregnancies (Kamphuis, 2010; Knight, 2011).

1	FNAIT may present in various ways. In some cases, neonatal thrombocytopenia may be an incidental finding or the newborn may manifest petechiae. In the other extreme, a fetus or neonate may develop devastating ICH-often before birth. Of 600 pregnancies with FNAIT identified through a large international registry, fetal or neonatal ICH complicated 7 percent of cases (Tiller, 2013). Hemorrhage afected the irst-born child in 60 percent and occurred before 28 weeks' gestation in half. A third of afected children died soon after birth, and 50 percent of survivors had severe neurological disabilities. Bussel and coworkers (1997) evaluated fetal platelet counts before therapy in 107 fetuses with FNAIT. Thrombocytopenia severity was predicted by a prior sibling with perinatal ICH, and 98 percent of cases were identiied this way. he initial platelet count was <20,000/�L in 50 percent. In cases in which the platelet count was initially >80,000/�L, they noted that it dropped by more than 1

1	of cases were identiied this way. he initial platelet count was <20,000/�L in 50 percent. In cases in which the platelet count was initially >80,000/�L, they noted that it dropped by more than 1 O,OOO/�L each week in the absence of therapy.

1	Alloimmune thrombocytopenia is typically diagnosed following delivery of a neonate with severe and unexplained thrombocytopenia to a woman whose platelet count is normal. Rarely, the diagnosis is ascertained after identiYing fetal ICH. he condition recurs in 70 to 90 percent of subsequent pregnancies, is often severe, and usually develops earlier with each successive pregnancy. Traditionally, fetal blood sampling was performed to detect fetal thrombocytopenia and to tailor therapy, with transfusion of platelets if the fetal platelet count was < 50,000/�L. Because of procedure-related complications, however, experts recommend abandoning routine fetal platelet sampling in favor of empirical treatment with intravenous immune globulin (IVIG) and prednisone (Berkowitz, 2006; Pacheco, 2011).

1	herapy is stratified according to whether a prior afected pregnancy was complicated by perinatal ICH, and if so, at what gestational age (Table 15-3). Pioneering work by Bussel (1996) and Berkowitz (2006) and their colleagues demonstrated the eicacy of such treatment. In one series of 50 pregnancies with fetal thrombocytopenia secondary to FNAIT, IVIG raised the platelet count by approximately 50,000/�L, and no fetus developed ICH (Bussel, 1996). Among pregnancies at particularly high risk-based on a platelet count <20,000/�L or sibling with FNAIT-associated ICH-the addition of corticosteroids to IVIG increased the platelet count in 80 percent of cases (Berkowitz, 2006). Cesarean delivery has been recommended at or near term. A noninstrumental vaginal delivery is generally considered only if fetal blood sampling has demonstrated a platelet count > 100,000/�L (Pacheco, 2011).

1	Additional considerations include risks and costs associated with therapy. Side efects ofIVIG may include fever, headache, nausea/vomiting, myalgia, and rash. Maternal hemolysis also has been described (Rink, 2013). Costs for IVIG may exceed $70 per gram or nearly $10,000 for each weekly 2-g/kg infusion for an average-size pregnant woman (Pacheco, 2011).

1	lso known as immune or idiopathic thrombocytopenic 2urpura (ITP), this autoimmune disorder is characterized by antiplatelet IgG antibodies that attack platelet glycoproteins. In pregnancy, these antibodies may cross the placenta and cause fetal thrombocytopenia. Maternal ITP is discussed in Chapter 56 (p. 1086). Fetal thrombocytopenia is usually mild. However, neonatal platelet levels may fall rapidly after birth, with a nadir at 48 to 72 hours of life. Neither the maternal platelet count, identification of anti platelet antibodies, nor treatment with corticosteroids efectively predicts fetal or neonatal platelet counts (Hachisuga, 2014). Importantly, fetal platelet counts are usually adequate to allow vaginal delivery without an increased risk of ICH. In a recent review of more than 400 pregnancies TABLE 15-3. Fetal-Neonatal Alloimmune Thrombocytopenia (FNAIT) Treatment Recommendations

1	TABLE 15-3. Fetal-Neonatal Alloimmune Thrombocytopenia (FNAIT) Treatment Recommendations Maternal anti-HPA antibody screening and cross-matching with paternal platelets at 12,s24, and 32 weeks' gestation; no treatment for negative test results Beginning at 20 wks: IVIG 1 g/kg/wk and prednisone 0.5 mg/kg/d or IVIG 2 g/kg/wk Beginning at 32 weeks: IVIG 2 g/kg/wk and prednisone 0.5 mg/kg/d. Beginning at 12 wks: IVIG 1 g/kg/wk Beginning at 20 wks: either increase IVIG to 2 g/kg/wk or add prednisone 0.5 mg/kg/d Beginning at 28 wks: IVIG 2 g/kg/wk and prednisone 0.5 mg/kg/d. Beginning at 12 wks: IVIG 2 g/kg/wk Beginning at 20 wks: add prednisone 1 mg/kg/d Continue both until delivery HPA human platelet antigen; ICH intracerebral hemorrhage; IVIG intravenous immunoglobulin G. Data from Pacheco, 2011s.

1	Beginning at 20 wks: add prednisone 1 mg/kg/d Continue both until delivery HPA human platelet antigen; ICH intracerebral hemorrhage; IVIG intravenous immunoglobulin G. Data from Pacheco, 2011s. with ITP, there was no case of fetal or neonatal ICH and no infant with any central nervous system abnormality (Wyszynski, 2016). Fetal bleeding complications are considered rare, and fetal blood sampling is not recommended (Neunert, 201i1). Delivery mode is based on standard obstetrical indications.

1	This term refers to excessive accumulation of serous fluid. Strictly defined, hydrops etalis is edema of the fetus. Traditionally, the diagnosis was made after delivery of a massively edematous neonate, often stillborn (Fig. 1i5-4). With sonography, hydrops has become a prenatal diagnosis. It is deined as two or more fetal efusions-pleural, pericardial, or ascites-or one efusion plus anasarca. As hydrops progresses in severity, edema is invariably a component, and is usually accompanied by placentomegaly and hydramnios. Clinically significant edema is defined sonographically as skin thickness > 5 mm, and placentomegaly if the placenta thickness is at least 4 em in the second trimester or 6 em in the third trimester (Bellini, 2009; Society for Maternal-Fetal Medicine, 2015b). Hydrops may result from a wide range of conditions with varying pathophysiologies, each with the potential to make the fetus severely ill. It is divided into two categories. If found in association with red cell

1	result from a wide range of conditions with varying pathophysiologies, each with the potential to make the fetus severely ill. It is divided into two categories. If found in association with red cell alloimmunization, it is termed immune, otherwise, it is nonimmune.

1	he incidence of immune hydrops has dramatically declined with the advent of anti-D immune globulin, MCA Doppler studies for detection of severe anemia, and prompt fetal transfusion when needed (p. 304). However, fewer than 10 percent of hydrops cases are caused by red cell alloimmunization (Bellini, 2012; Santolaya, 1992). The pathophysiology underlying hydrops remains unknown. Immune hydrops is postulated to share several physiological abnormalities with nonimmune hydrops. As shown in

1	FIGURE 15-4 Hydropic, macerated stillborn infant and characteristically large placenta. The etiology was B1o9 parvovirus infection. (Used with permission from Dr. April Bleich.) 15-5, these include decreased colloid oncotic pres sure, increased hydrostatic (or central venous) pressure, and enhanced vascular permeability. Immune hydrops results from transplacental passage of maternal antibodies that destroy fetal red cells. The resultant anemia stimulates marrow erythroid hyperplasia and extramedullary hematopoiesis in the spleen and liver. The latter likely causes portal hypertension and impaired hepatic protein synthesis, which lowers plasma oncotic pressure (Nicolaides, 1985). Fetal anemia also may raise central venous pressure (Weiner, 1989). Finally, tissue hypoxia from anemia may increase capillary permeability, such that luid collects in the fetal thorax, abdominal cavity, andlor subcutaneous tissue. he degree of anemia in immune hydrops is typically severe.

1	he degree of anemia in immune hydrops is typically severe. In a series of 70 pregnancies with fetal anemia from red cell alloimmunization, Mari and coworkers (2000) found that all those with immune hydrops had hemoglobin values <5 g/dL. As discussed on page 304, immune hydrops is treated with fetal blood transfusions.

1	At least 90 percent of cases of hydrops are nonimmune (Bellini, 2012; Santolaya, 1992). The prevalence estimate is 1 per 1500 second-trimester pregnancies (Heinonen, 2000). The number of specific disorders that can lead to nonimmune hydrops is extensive. Etiologies and the proportion of births within each hydrops category from a review of more than 6700 afected pregnancies are summarized in Table 1i5-4. A cause is identified in at least 60 percent prenatally and in more than 80 percent postnatally (Bellini, 2009; Santo, 2011). Currently, approximately 20 percent of cases remain idiopathic (Bellini, 2015). As shown in Figure 15-5, several diferent pathophysiological processes are proposed to account for the inal common pathway of hydrops fetalis.

1	Importantly, the etiology of non immune hydrops varies according to when in gestation it is identified. Of those diagnosed prenatally, aneuploidy accounts for approximately 20 percent, cardiovascular abnormalities for 15 percent, and infections for 14 percent-the most common of these being parvovirus B19 (Santo, 2011). Overall, only 40 percent of pregnancies with nonimmune hydrops result in a liveborn neonate, and of these, the neonatal survival rate is only about 50 percent. Sohan and colleagues (2001) reviewed 87 pregnancies with hydrops and found that 45 percent of those diagnosed before 24 weeks' gestation had a chromosomal abnormality. The most frequent aneuploidy was 45,X-Turner syndrome, and in such cases, the survival rate was <5 percent (Chap. 13, p. 259). If hydrops is detected in the irst trimester, the aneuploidy risk is nearly 50 percent, and most have cystic hygromas (Fig. 10-22, p. 198).

1	Although the prognosis of nonimmune hydrops is guarded, it is heavily dependent on etiology. In large series from Thailand and Southern China, aA-thalassemia is the predominant cause of nonimmune hydrops, accounting for 30 to 50 percent of cases and conferring an extremely poor prognosis (Liao, 2007; Ratanasiri, 2009; Suwanrath-Kengpol, 2005). In contrast, treatable etiologies such as parvovirus, chylothorax, and tachyarrhythmias, which each comprise about 10 percent of Anemia Red cell alloimmunization Fetomaternal hemorrhage Hematological disorder Infection Selected fetal anomalies Placental abnormality Volume overload or Extramedullary hematopoiesis Impaired venous return Hepatic dysfunction, Metabolic disorders impaired protein synthesis Infection Tissue hypoxia ? Heart failure Increased hydrostatic pressure Lymphatic abnormality Decreased plasma oncotic pressure Increased capillary permeability Decreased lymphatic flow Increased interstitial fluid Hydrops fetalis

1	FIGURE 15�5 Proposed pathogenesis of immune and nonimmune hydrops fetalis. (Adapted from Bellini, 2009; Lockwood, 2009.) cases, can result in survival in two thirds of cases with fetal therapy (Sohan, 2001). Hydrops is readily detected sonographically. As noted, two efusions or one efusion plus anasarca are required for diagnosis. Edema may be particularly prominent around the scalp, or equally obvious around the trunk and extremities. Efusions are visible as luid outlining the lungs, heart, or abdominal viscera (Fig. 15-6). In many cases, targeted sonographic and laboratory evaluation will identiy the underlying cause of fetal hydrops. hese include cases due to fetal anemia, arrhythmia, structural abnormality, aneuploidy, placental abnormality, or complications of monochorionic twinning. Depending on the circumstances, initial evaluation includes the following: 1. 2. Targeted sonographic fetal and placental examination, including:

1	2. Targeted sonographic fetal and placental examination, including: A detailed anatomical survey to assess for the structural abnormalities listed in Table 15-4 MCA Doppler peak systolic velocity to assess for fetal anemia Fetal echocardiography with M-mode evaluation 3. Amniocentesis for fetal karyotype and for parvovirus B19, cytomegalovirus, and toxoplasmosis testing as discussed in Chapter 64. Consideration of chromosomal microarray analysis if fetal anomalies are present 4. Kleihauer-Betke test for fetomaternal hemorrhage if anemia is suspected, depending on findings and test results 5. Consideration of testing for alpha-thalassemia and/or inborn errors of metabolism.

1	Kleihauer-Betke test for fetomaternal hemorrhage if anemia is suspected, depending on findings and test results 5. Consideration of testing for alpha-thalassemia and/or inborn errors of metabolism. Isolated Efusion or Edema. Although one efusion or anasarca alone is not diagnostic for hydrops, the above evaluation should be considered if these are encountered, as hydrops may develop. For example, an isolated pericardial eusion may be the initial finding in fetal parvovirus B19 infection (Chap. 64, p. 1217). n isolated pleural efusion may represent a chylothorax, which is amenable to prenatal diagnosis, and for which fetal therapy may be lifesaving if hydrops develops (Chap. 16, p. 324). Isolated ascites also may be the initial inding in fetal parvovirus B 19 infection, or it may result from a gastrointestinal TABLE 15-4. Categories and Etiologies of Nonimmune Hydrops Fetalis

1	TABLE 15-4. Categories and Etiologies of Nonimmune Hydrops Fetalis Structural defects: Ebstein anomaly, Fallot tetralogy with absent pulmonary valve, hypoplastic left or right heart, premature closure of ductus arteriosus, arteriovenous malformation (vein of Galen aneurysm) Bradycardia, as may occur in heterotaxy syndrome with endocardial cushion defect or with anti-RolLa antibodies Cystic hygroma, systemic lymphangiectasis, pulmonary lymphangiectasis Parvovirus B19, syphilis, cytomegalovirus, toxoplasmosis, rubella, enterovirus, varicella, herpes simplex, coxsackievirus, listeriosis, leptospirosis, Chagas disease, Lyme disease Skeletal dysplasia with very small thorax Meconium peritonitis, gastrointestinal tract obstruction Congenital (Finnish) nephrosis, Bartter syndrome, mesoblastic nephroma Placental, Twin, and Cord Abnormalities 5

1	Meconium peritonitis, gastrointestinal tract obstruction Congenital (Finnish) nephrosis, Bartter syndrome, mesoblastic nephroma Placental, Twin, and Cord Abnormalities 5 Placental chorioangioma, twin-twin transfusion syndrome, twin reversed arterial perfusion sequence, twin anemia polycythemia sequence, cord vessel thrombosis Inborn errors of metabolism: Gaucher disease, galactosialidosis, GM1 gangliosidosis, sialidosis, mucopolysaccharidoses, mucolipidoses Tumors: sacrococcygeal teratoma, hemangioendothelioma with Kassabach-Merritt syndrome apercentages reflect the proportion within each category from a systematic review of 6775 pregnancies with nonimmune hydrops. Modified from Bellini, 2015.

1	Modified from Bellini, 2015. FIGURE 15-6 Hydropic features. A. This profile of a 23-week fetus with nonimmune hydrops secondary to B 19 parvovirus infection depicts scalp edema (arrowheads) and ascites (*). B. This 34-week fetus had hydrops secondary to an arteriovenous malformation in the brain, known as a vein of Galen aneurysm. In this coronal image, prominent pleural effusions (*) outline the lungs (L). Fetal ascites is also pres- ent (arrows), as is anasarca. C. This axial (transverse) image depicts a pericardial effusion (arrows) in a 23-week fetus with hydrops from B1o9 parvovirus infection. The degree of cardiomegaly is impressive, and the ventricular hypertrophy raises concern for myocarditis, which can accompany parvovirus infection. D. This axial (transverse) image depicts fetal ascites (*) in a 15-week fetus with hydrops secondary to large cystic hygromas. Anasarca is also seen (bracket).

1	abnormality such as meconium peritonms. Finally, isolated edema, particularly involving the upper torso or the dorsum of the hands and feet, may be found in Turner or Noonan syndrome or may represent congenital lymphedema syndrome (Chap. 13, p. 259). An association between fetal hydrops and development of maternal edema in which the fetus mirrors the mother is attributed to Ballantyne. He called the condition triple edema because the fetus, mother, and placenta all became edematous. he etiology of the hydrops is not related to development of mirror syndrome. It has been associated with hydrops from D alloimmunization, twin-twin transfusion syndrome, placental chorioangioma, and with fetal cystic hygroma, Ebstein anomaly, sacrococcygeal teratoma, chylothorax, bladder outlet obstruction, supraventricular tachycardia, vein of Galen aneurysm, and various congenital infections (Braun, 2010).

1	In a review of more than 50 cases of mirror syndrome, Braun (2010) found that approximately 90 percent of women had edema, 60 percent had hypertension, 40 percent had proteinuria, 20 percent had liver enzyme elevation, and nearly 15 percent had headache and visual disturbances. Based on these indings, it is reasonable to consider mirror syndrome a form of severe preeclampsia (Espinoza, 2006; Midgley, 2000). Others, however, have suggested that it is a separate disease process with hemodilution rather than hemoconcentration (Carbillon, 1997; Livingston, 2007).

1	Some reports describe the same imbalance of angiogenic and antiangiogenic factors that is observed with preeclampsia, and this suggests a common pathophysiology (Espinoza, 2006; Goa, 2013; Llurba, 2012). hese indings, which include elevated concentrations of soluble fms-like tyrosine kinase-1 (sFlt-l), decreased placental growth factor (PIG F) levels, and elevation of soluble vascular endothelial growth factor receptor-I (s VEG FR-1) concentrations, are discussed further Chapter 40 (p. 716).

1	In most cases with mirror syndrome, prompt delivery is indicated and followed by resolution of maternal edema and other findings (Braun, 2010). However, in isolated cases of fetal anemia, supraventricular tachycardia, hydrothorx, and bladder outlet obstruction, successful fetal treatment resulted in resolution of both fetal hydrops and maternal mirror syndrome (Goa, 2013; Livingston, 2007; Llurba, 2012; Midgley, 2000). Normalization of the angiogenic imbalance has also been described following fetal transfusion for parvovirus B 19 infection. Fetal therapy for these conditions is reviewed in Chapter 16. Given the parallels to severe preeclampsia, delaying delivery to efect fetal therapy should be considered only with caution. If the maternal condition deteriorates, delivery is recommended. American Academy of Pediatrics, American College of Obstetricians and Gynecologists: Guidelines for Perinatal Care. 8th ed. Elk Grove Village, MP, 201

1	American Academy of Pediatrics, American College of Obstetricians and Gynecologists: Guidelines for Perinatal Care. 8th ed. Elk Grove Village, MP, 201 American College of Obstetricians and Gynecologists: Management of alloimmunization during pregnancy. Practice Bulletin No. 75, August 2006, Reairmed 2016

1	American College of Obstetricians and Gynecologists: Prevention ofRh 0 alloimmunization. Practice Bulletin No. 181, August 20o17 Bellini C, Hennekam RC: Non-immune hydrops fetalis: a short review of etiology and pathophysiology. Am] Med Genet 158A(3):597, 2012 Bellini C, Hennekam RC, Fulcheri E, et al: Etiology of nonimmune hydrops fetalis: a systematic review. Amo] Med Genet A 149A(5):844, 2009 Bellini C, Donarini G, Paladini 0, et al: Etiology of non-immune hydrops fetalis: an update. Am ] Med Genet 167 A: 1082, 2015 Bellussi F, Perolo A, Ghi T, et al: Diagnosis of severe fetomaternal hemorrhage with fetal cerebral Doppler: case series and systematic review. Fetal Diagn Ther 41(1):1,o2017 Berkowitz L, Kolb A, McFarland ]G, et al: Parallel randomized trials of risk-based therapy for fetal alloimmune thrombocytopenia. Obstet Gynecol 107(1):91,o2006 Bollason G, Hjartardottir H, Jonsson T, et al: Red blood cell alloimmunization in pregnancy during the years 1996-2015 in Iceland: a

1	fetal alloimmune thrombocytopenia. Obstet Gynecol 107(1):91,o2006 Bollason G, Hjartardottir H, Jonsson T, et al: Red blood cell alloimmunization in pregnancy during the years 1996-2015 in Iceland: a nation-wide population study. Transfusion 57(1o1):2578, 2017

1	Bowmano]: Rh-immunoglobulin: Rh prophylaxis. Best Pract Res Clin HaematoIo19(1):27,o2006 Bowman ]M: Controversies in Rh prophylaxis: who needs Rh immune globulin and when should it be given? Am ] Obstet Gynecol 151 :289, 1985 Bowman ]M: he prevention of h immunization. Transfus Med Rev 2: 129, 1988 Braun T, Brauer M, Fuchs I, et al: Mirror syndrome: a systematic review of fetal associated conditions, maternal presentation, and perinatal outcome. Fetal Diagn Ther 27(4):191,o2010 Bussel ]B, Berkowitz L, Lynch L, et al: Antenatal management of alloimmune thrombocytopenia with intravenous gamma-globulin: a randomized trial of the addition oflow-dose steroid to intravenous gamma-globulin. Am ] Obstet GynecoIo174(5):1414, 1996 Bussel ]B, Zabusky MR, Berkowitz L, et al: Fetal alloimmune thrombocytopenia. N Englo] Med 337:22, 1997

1	Bussel ]B, Zabusky MR, Berkowitz L, et al: Fetal alloimmune thrombocytopenia. N Englo] Med 337:22, 1997 Carbillon L, Oury ]F, Guerin ]M, et al: Clinical biological features of Ballantyne syndrome and the role of placental hydrops. Obstet Gynecol Surv 52(5):310, 1997 Chambers E, Davies L, Evans S, et al: Comparison of haemoglobin F detection by the acid elution test, low cytometry and high-performance liquid chromatography in maternal blood samples analysed for fetomaternal haemorrhage. Transfus Med 22(3):199, 2012 Choavaratana R, Uer-Areewong S, Makanantakocol S: Fetomaternal transfusion in normal pregnancy and during delivery. ] Med Assoc Thai 80:96, 1997 Corcoran 0, Murphy 0, Donnelly], et al: The prevalence of maternal F cells in a pregnant population and potential overestimation of foeto-maternal haemorrhage as a consequence. Blood Transfus 12:570,o2014

1	Daniels G: Variants of RhO-current testing and clinical consequences. Br ] Haematol 161(4):461,o2013 de Almeida V, Bowman ]M: Massive fetomaternal hemorrhage: Manitoba experience. Obstet Gynecol 83:323, 1994 de Haas M, Thurik FF, Koelewijno]M et al: Haemolytic disease of the fetus and newborn. Vox Sang 109(2):99, 2015 de Haas M, Thurik FF, van der Ploeg CP, et al: Sensitivity of fetal RHO screening for safe guidance of targeted anti-D immunoglobulin prophylaxis: prospective cohort study of a nationwide programme in the Netherlands. BM] 89,o2016 Espinozao], Romero R, Nien ]K, et al: A role of the anti-angiogenic factor sVEGFR-1 in the "mirror syndrome" (Ballantyne's syndrome). ] Matern Fetal Neonatal Med 19(10):607,o2006 Fernandes B], von Dadelszen P, Fazal I, et al: Flow cytometric assessment of feto-maternal hemorrhage; a comparison with Betke-Kleihauer. Prenat Diagn 27(7):641, 2007

1	Fernandes B], von Dadelszen P, Fazal I, et al: Flow cytometric assessment of feto-maternal hemorrhage; a comparison with Betke-Kleihauer. Prenat Diagn 27(7):641, 2007 Garratty G, Glynn SA, McEntire R, et al: ABO and Rh(D) phenotype frequencies of diferent racial/ethnic groups in the United States. Transfusion 44(5):703, 2004 Giacoia GP. Severe fetomaternal hemorrhage: a review. Obstet Gynecol Surv 52:372, 1997 Giannina G, Moise K] ]r, Dorman K: A simple method to estimate the volume for fetal intravascular transfusion. Fetal Diagn Ther 13:94, 1998 Goa S, Mimura K, Kakigano A, et al: Normalisation of angiogenic imbalance after intra-uterine transfusion for mirror syndrome caused by parvovirus B19. Fetal Diagn her 6,o2013 Hachisuga K, Hidaka N, Fujita Y, et al: Can we predict neonatal thrombocytopenia in ofspring of women with idiopathic thrombocytopenic purpura? Blood 49(4):259,o2014

1	Hachisuga K, Hidaka N, Fujita Y, et al: Can we predict neonatal thrombocytopenia in ofspring of women with idiopathic thrombocytopenic purpura? Blood 49(4):259,o2014 Hackney ON, Knudtson E], Rossi KQ, et al: Management of pregnancies complicated by anti-c isoimmunization. Obstet Gynecol 103:24, 2004 Heinonen S, Ruynamen M, Kirkinen P: Etiology and outcome of second trimes ter nonimmunological fetal hydrops. Scando] Obstet Gynecol 79: 15, 2000 Howard H, Mardew V, McFadyen I, et al: Consequences for fetus and neonate of maternal red cell allo-immunization. Arch Dis Child Fetal Neonat Ed 78:F62, 1998 Johnson ]A, MacDonald K, Clarke G, et al: No. 343-Routine non-invasive prenatal prediction of fetal RHO genotype in Canada: the time is here. ] Obstet Gynaecol Can 39(5):366, 2017 Kamphuis MM, Paridaans N, Porcelijn L, et al: Screening in pregnancy for fetal or neonatal alloimmune thrombocytopenia: systematic review. B]OG 117(1o1):1335,o2010

1	Kamphuis MM, Paridaans N, Porcelijn L, et al: Screening in pregnancy for fetal or neonatal alloimmune thrombocytopenia: systematic review. B]OG 117(1o1):1335,o2010 Kleihauer B, Braun H, Betke K: Demonstration of fetal hemoglobin in erythrocytes of a blood smear. Klin Wochenschr 35(12):637, 1957 Knight M, Pierce M, Allen 0, et al: The incidence and outcomes of fetomaternal alloimmune thrombocytopenia: a UK national study using three data sources. Br] HaematoIo152(4):460, 2011 Koelewijn ]M, Vrijkotte TG, van der Schoot CE, et al: Efect of screening for red cell antibodies, other than anti-D, to detect hemolytic disease of the fetus and newborn: a population study in the Netherlands. Transfusion 48:941,o2008 Lazar L, Harmath AG, Ban Z, et al: Detection of maternal deoxyribonucleic acid in peripheral blood of premature and mature newborn infants. Prenat Diagn 26(2):168, 2006

1	Lazar L, Harmath AG, Ban Z, et al: Detection of maternal deoxyribonucleic acid in peripheral blood of premature and mature newborn infants. Prenat Diagn 26(2):168, 2006 Liao C, Weio], Li Q, et al: Nonimmune hydrops fetal is diagnosed during the second half of pregnancy in Southern China. Fetal Diagn Ther 22(4):302,o2007 Liley A W: Liquor amnii analysis in management of pregnancy complicated by rhesus sensitization. Amo] Obstet GynecoIo82:1359, 1961 Lindenburg I, van Kamp I, van Zwet E, et al: Increased perinatal loss after intrauterine transfusion for alloimmune anaemia before 20 weeks of gestation. B]OG 120:847,o2013 Lindenburg IT, Smits-Wintjens VE, van Klink ]M, et al: Long-term neurodevelopmental outcome after intrauterine transfusion for hemolytic disease of the fetus/newborn: the LOTUS study. Amo] Obstet Gynecol 206:o141. e1,o2012

1	Livingston ]C, Malik M, Crombleholme TM, et al: Mirror syndrome: a novel approach to therapy with fetal peritoneal-amniotic shunt. Obstet Gynecol 110(2 Pt 2):540,o2007 Llurba E, Marsal G, Sanchez 0, et al: Angiogenic and antiangiogenic factors before and after resolution of maternal mirror syndrome. Ultrasound Obstet Gynecol 40(3):367, 2012 Lockwood q, Nadel AS, King ME, et al: A 32-year old pregnant woman with an abnormal fetal ultrasound study. Case 16-2009. N Englo] Med 360(21):2225,o2009 MacKenzie IZ, Roseman F, Findlayo], et al: he kinetics of routine antenatal prophylactic intramuscular injections of polyclonal anti-D immunoglobulin. B]OG 113:97, 2006 Mari G, Deter L, Carpenter RL, et al: Noninvasive diagnosis by Doppler ultrasonography of fetal anemia due to maternal red-cell alloimmunization. N Englo] Med 342:9, 2000 Martin ]A, Hamilton BE, Sutton PD, et al: Births: final data for 2003. Natl Vital Stat Rep 54(2): 1, 2005

1	Martin ]A, Hamilton BE, Sutton PD, et al: Births: final data for 2003. Natl Vital Stat Rep 54(2): 1, 2005 Midgley DY, Hardrug K: The mirror syndrome. Euro] Obstet Gynecol Reprod Bioi 8:201, 2000 Moise K]: Fetal anemia due to non-Rhesus-D red-cell alloimmunization. Semin Fetal Neonatal Med ,o2008 Moise K], Argoti PS: Management and prevention of red cell alloimmunization in pregnancy. A systematic review. Obstet Gynecol 120(5): 1132, 20o12 Moise K], Gandhi M, Boring NH, et al: Circulating cell-free DNA to determine the fetal RHD status in all three trimesters of pregnancy. Obstet Gynecol 128(6):1340, 2016 Neunert C, Lim W, Crowther M, et al: The American Society of Hematology 2011 evidence-based practice guideline for immune thrombocytopenia. Blood 11 (16):4190,o2011 Nicolaides KH, Warenski ]c, Rodeck CH: The relationship of fetal plasma protein concentration and hemoglobin level to the development of hydrops in rhesus isoimmunization. Am] Obstet Gynecol 152:341, 1985

1	Oepkes 0, Seaward PG, Vandenbussche FP, et al: Doppler ultrasonography versus amniocentesis to predict fetal anemia. N Engl] Med 355: 156, 2006 Pacheco LD, Berkowitz RL, Moise K], et a1: Fetal and neonatal alloimmune thrombocytopenia. A management algorithm based on risk stratification. Obstet Gynecol 118(5):1157,2011 Queenan ]T, Thomas PT, Tomai TP, et al: Deviation in amniotic fluid optical density at a wavelength of 450 nm in Rh isoimmunized pregnancies from 14 to 40 weeks' gestation: a proposal for clinical management. Am ] Obstet Gynecol 168:1370, 1993 Ratanasiri T, Komwilaisak R, Sittivech A, et al: Incidence, causes, and pregnancy outcomes of hydrops fetalis at Srinagarind Hospital, 1996-2005: a 10-year review.o] Med Assoc hai 92(5):594, 2009 Rink BD, Gonik B, Chmait RH, et al: Maternal hemolysis after intravenous immunoglobulin treatment in fetal and neonatal alloimmune thrombocytopenia. Obstet GynecoIo121o(2):47o1, 20o13

1	Rink BD, Gonik B, Chmait RH, et al: Maternal hemolysis after intravenous immunoglobulin treatment in fetal and neonatal alloimmune thrombocytopenia. Obstet GynecoIo121o(2):47o1, 20o13 Risson DC, Davies MW, Williams BA: Review of neonatal alloimmune thrombocytopenia.o] Pediatr Child Health 48(9):816,o2012 Rubod C, Deruelle P, Le Gouef F, et al: Long-term prognosis for infants after massive fetomaternal hemorrhage. Obstet Gynecol 110(2 pt 1),o200 Sandler SG, Flegel WA, Westhof CM, et al: It's time to phase-in RHD genotyping for patients with a serological weak D phenotype. Transfusion 55(3):680,o2015 Sandler SG, Queenan ]T: A guide to terminology for Rh immunoprophylaxis. Obstet Gynecol 130(3):633,o2017 Santo S, Mansour S, Thilaganathan B, et al: Prenatal diagnosis of non-immune hydrops fetalis: what do we tell the parents? Prenat Diagn 31: 186, 2011 Santolayao], Alley 0, Jafe R, et al: Antenatal classiication of hydrops fetalis. Obstet 9:256, 1992

1	Obstet 9:256, 1992 Society for Maternal-Fetal Medicine, Mari G, Norton ME, et a1: Society for Maternal-Fetal Medicine (SMFM) Clinical Guideline #8: he fetus at risk for anemia-diagnosis and management. Am] Obstet Gynecol 212(6):697, 20o15a Society for Maternal-Fetal Medicine, Norton ME, Chauhan SP, et al: Society for Maternal-Fetal Medicine Clinical Guideline # : Nonimmune hydrops fetalis. Amo] Obstet Gynecol 212(2):127, 2015b Sohan K, Carroll SG, De La Fuente S, et al: Analysis of outcome in hydrops fetalis in relation to gestational age at diagnosis, cause, and treatment. Acta Obstet Gynecol Scand 80(8):726, 2001 Storry]R, Castilho L, Daniels G, et a1: International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology: Cancun report (2012). Vox Sang 107(1): 90, 2014

1	Suwanrath-Kengpol C, Kor-anantakul 0, Suntharasaj T, et a1: Etiology and outcome of non-immune hydrops fetalis in southern Thailand. Gynecol Obstet Invest 59(3): 134, 2005 Tannirandorn Y, Rodeck CH: New approaches in the treatment of haemolytic disease of the fetus. Ballieres Clin Haematol 3(2):289, 1990 Tiller H, Kamphuis MM, Flodmark 0, et al: Fetal intracranial hemorrhages caused by fetal and neonatal alloimmune thrombocytopenia: an observational cohort study of 43 cases from an international multicentre registry. BM] 3:e002490, 20o13 Van den Veyver lB, Moise K]: Fetal RhO typing by polymerase chain reaction in pregnancies complicated by rhesus alloimmunization. Obstet Gynecol 88:1061, 1996

1	Van den Veyver lB, Moise K]: Fetal RhO typing by polymerase chain reaction in pregnancies complicated by rhesus alloimmunization. Obstet Gynecol 88:1061, 1996 Van Kamp IL, lumper F], Bakkum RS, et al: he severity of immune fetal hydrops is predictive of fetal outcome after intrauterine treatment. Am ] Obstet Gynecol 185:668,o2001 Vivanti A, Benachi A, Huchet FX, et al: Diagnostic accuracy of fetal rhesus 0 genotyping using cell-free fetal DNA during the first trimester of pregnancy. Am] Obstet GynecoIo215:606.e1, 2016 Weiner CP, Pelzer GO, Heilskov], et al: he efect of intravascular transfusion on umbilical venous pressure in anemic fetuses with and without hydrops. Amo] Obstet Gynecol 161:1498, 1989 Weinstein L: Irregular antibodies causing hemolytic disease of the newborn: a continuing problem. Clin Obstet Gynecol 25(2):321, 1982

1	Weinstein L: Irregular antibodies causing hemolytic disease of the newborn: a continuing problem. Clin Obstet Gynecol 25(2):321, 1982 Welsh K], Bai Y, Education Committee of the Academy of Clinical Laboratory Physicians and Scientists: Pathology consultation on patients with a large h immune globulin dose requirement. Amo] Clin Pathol 145: 44, 2016 Woelfer B, Schuchter K, ]anisiw M, et al: Postdelivery levels of anti-D IgG prophylaxis in mothers depend on maternal body weight. Transfusion 44:512, 2004 Wylie B], D'Alton ME: Fetomaternal hemorrhage. Obstet Gynecol 115(5): 1039,o2010 Wyszynski OF, Carmen J, Cantor AB, et al: Pregnancy and birth outcomes among women with idiopathic thrombocytopenic purpura. ] Pregnancy 2016:8297407,o2016

1	Wyszynski OF, Carmen J, Cantor AB, et al: Pregnancy and birth outcomes among women with idiopathic thrombocytopenic purpura. ] Pregnancy 2016:8297407,o2016 Zimmerman R, Carpenter R] ]r, Durig P, et al: Longitudinal measurement of peak systolic velocity in the fetal middle cerebral artery for monitoring pregnancies complicated by red cell alloimmunization: a prospective multicenter trial with intention-to-treat. B]OG ):746,o2002 Zipursky A, Bhutani VK: Impact of Rhesus disease on the global problem of bilirubin-induced neurologic dysfunction. Semin Fetal Neonatal Med 20(1):2,o2015 Zwiers C, Lindenburg IT, Klumper F], et al: Complications of intrauterine intravascular blood transfusion: lessons learned ater 1678 procedures. Ultrasound Obstet Gynecol 50(2):180, 2017 MEDICAL THERAPY ....e.....e..e.... ...e......e...e..315 SURGICAL THERAPY ..e........e.... ...e....e..e..e... 318 OPEN FETAL SURGERY .....e....e..e.=...........e...319 FETOSCOPIC SURGERY ...........e.........e...... 321

1	SURGICAL THERAPY ..e........e.... ...e....e..e..e... 318 OPEN FETAL SURGERY .....e....e..e.=...........e...319 FETOSCOPIC SURGERY ...........e.........e...... 321 PERCUTANEOUS PROCEDURESe. . .. .............. 324 EX-UTERO INTRAPARTUM TREATMENT ............e. 327 Minor grades of hydramnios rarey require active treatment. On the other han, when the abdomen is immensey distended and respiration is seriousy hampere, the termination of pregnancy is urgenty indicate. In such cases, the symptoms can be prompty relieved by peorating the membranes through the cervix, after which the amniotic luid drains of and labour pains set in.

1	-J. Whitridge Williams (1903) he concept of fetal therapy-even amniocentesis-was not considered by Williams in his irst edition. Aside from a few destructive procedures to aid vaginal delivery, any type of fetal treatment is not mentioned as even a remote possibility. Again, fast forward to this 25th edition, when interventions developed during the past three decades have dramatically altered the course of selected fetal anomalies and conditions. Reviewed in this chapter are fetal disorders amenable to treatment with either maternal medication or surgical procedures. he management of fetal anemia and thrombocytopenia is reviewed in Chapter 15, and treatment of some fetal infections is discussed in Chapters 64 and 65. Fetal pharmacotherapy uses medications administered to the mother and then transported transplacentally to the fetus. s described here, it can be used to treat narray of serious conditions.

1	Fetal pharmacotherapy uses medications administered to the mother and then transported transplacentally to the fetus. s described here, it can be used to treat narray of serious conditions. Fetal cardiac rhythm disturbances may be broadly categorized as tachyarrhythmias, heart ratesi> 180 beats per minute (bpm); bradyarrhythmia, heart rate < 110 bpm; and ectopy, typically premature atrial contractions. If these are identiied, fetal M-mode sonography is performed to measure the atrial and ventricular rates and to clariY the relationship between atrial and ventricular beats, thereby diagnosing the type of rhythm disturbance.

1	This is by far the most common arrhythmia and is identiied in 1 to 2 percent of pregnancies (Hahurij, 2011; Strasburger, 2010). Generally a benign inding, premature atrial contractions represent immaturity of the cardiac conduction system, and they typically resolve later in gestation or in the neonatal period. If the premature atrial contraction is conducted, it sounds like an extra beat when auscultated with handheld Doppler or fetoscope. However, premature atrial contractions are more commonly blocked and sound like dropped beats.

1	In general, premature atrial contractions are not associated with major structural cardiac abnormalities, although they sometimes occur with an atrial septal aneurysm. As shown in Figure 10-34 (p. 205), M-mode evaluation demonstrates that the dropped beat is a compensatory pause following the premature atrial contraction. They may occur as frequently as every other beat, known as blocked atrial bigeminy. This results in an auscultated fetal ventricular rate as low as 60 to 80 bpm. Unlike other causes of bradycardia, atrial bigeminy is benign and does not require treatment (Strasburger, 2010).

1	Approximately 2 percent of fetuses with premature atrial contractions are later found to have a supraventricular tachycardia (Copel, 2000; Srinivasan, 2008). Given the importance of identiying and treating supraventricular tachyarrhythmias, a fetus with premature atrial contractions is often monitored with heart rate assessment every 1 to 2 weeks until the ectopy resolves. This requires neither sonography nor fetal echocardiography, as the rate and rhythm may be easily ascertained with handheld Doppler.

1	The two most common tachyarrhythmias are supraventricular tachycardia (ST) and atrial lutter. SVT is characterized by an abrupt increase in the fetal heart rate to 180 to 300 bpm with 1: 1 atrioventricular concordance. he typical range is 200 to 240 bpm. ST may develop secondary to an ectopic focus or to an accessory atrioventricular pathway leading to a reentrant tachycardia. Atrial lutter is characterized by a much higher atrial rate, generally 300 to 500 bpm, with varying degrees of atrioventricular block. As a result, the ventricular rate in a fetus with atrial flutter may range from below normal to approximately 250 bpm (Fig. 16-1). In contrast, fetal sinus tachycardia typically presents with a gradual heart rate rise to a rate that is only slightly above normal. With this, readily discernible causes may be maternal fever or hyperthyroidism, or rarely, fetal anemia or infection.

1	If a fetal tachyarrhythmia is identified, it is important to determine whether it is sustained-deined as present for at least 50 percent of the time. It may be necessary to monitor the fetal heart rate for 12 to 24 hours upon initial detection, and then periodically to reassess (Srinivasan, 2008). Unsustained or intermittent tachyarrhythmias generally do not require treatment, provided that fetal surveillance is reassuring.

1	Sustained fetal tachyarrhythmia with ventricular rates exceeding 200 bpm impairs ventricular illing to a degree that the risk for hydrops is significant. With atrial flutter, lack of coordinated atrioventricular contractions may further compound this risk. Maternal administration of antiarrhythmic agents that cross the placenta may convert the rhythm to normal or lower the baseline heart rate to forestall heart failure. Therapy may require dosages at the upper end of the therapeutic adult range. Thus, a maternal electrocardiogram is obtained before and during therapy.

1	Antiarrhythmic medications most commonly used include digoxin, sotalol (Betapace), flecainide (Tambocor), and procainamide (Pronestyl). heir selection depends on the type of tachyarrhythmia as well as provider familiarity and experience with the drug. Traditionally, digoxin has been the initial preferred treatment, although it may poorly transfer to the fetus after hydrops has developed. Many centers now use lecainide or sotalol as first-line therapy Gaeggi, 2011; Shah, 2012). In many cases, additional agents are needed, particularly if hydrops has developed. SVT is generally more likely than atrial flutter to convert to a normal rhythm. With either arrhythmia, however, the overall neonatal survival rate now exceeds 90 percent (Ekman-Joelsson, 2015; Jaeggi, 2011; van der Heijden, 2013).

1	FIGURE 16-1 Atrial flutter. In this M-mode image at 28 weeks' gestation, calipers mark the ventricular rate, which is approximately 225 bpm. There are two atrial beats (A) for each ventricular beat (), such that the atrial rate is approximately 450 bpm with 2:1 atrioventricular block. The most common etiology of pronounced fetal bradycardia is congenital heart block. Approximately 50 percent of cases occur in the setting of a structural cardiac abnormality involving the conduction system. hese include heterotaxy, in particular ltatrial isomerism; endocardial cushion deect; and less commonly corrected transposition of the great vessels (Srinivasan, 2008). he prognosis of heart block secondary to a structural cardiac anomaly is extremely poor, and fetal loss rates exceed 80 percent (Glatz, 2008; Strasburger, 2010).

1	In a structurally normal heart, 85 percent of atrioventricular block cases develop secondary to transplacental passage of maternal anti-SSA/Ro or anti-SSB/La antibodies (Buyon, 2009). Many of these women have, or subsequently develop, systemic lupus erythematosus or other connective tissue disease (Chap. 59, p. 1142). he risk of third-degree heart block with these antibodies is small-only about 2 percent. But, the risk may reach 20 percent if a prior infant has been afected. Immune-mediated congenital heart block confers a mortality rate of 20 to 30 percent, requires permanent pacing in two thirds of surviving children, and also poses a risk for cardiomyopathy (Buyon, 2009). If associated with efusions, bradyarrhythmias, or endocardial ibroelastosis, neonatal status may progressively worsen after birth (Cuneo, 2007). Initial research eforts focused on maternal corticosteroid therapy to potentially reverse fetal heart block or forestall it.

1	Initial research eforts focused on maternal corticosteroid therapy to potentially reverse fetal heart block or forestall it. Friedman and colleagues (2008, 2009) conducted a prospective multicenter trial of pregnancies with anti-SSA/Ro antibodies the PR Interval and Dexamethasone (PRIDE) study. Weekly sonographic surveillance was performed, and heart block was treated with maternal oral dexamethasone 4 mg daily. Unfortu nately, progression from second-to third-degree block was not prevented with maternal dexamethasone therapy, and third degree atrioventricular block was irreversible. In rare cases, there block. However, irst-degree block did not generally progress even without treatment. In a subsequent review of 156 preg nancies with isolated second-or third-degree fetal heart block, sion, need for pacemaker in the neonatal period, or overall sur vival rates (Izmirly, 2016). hus, dexamethasone use cannot be recommended for this indication.

1	More recent eforts have turned to potential therapy with hydroxychloroquine (Plaquenil), a mainstay of treatment for systemic lupus erythematosus (Chap. 59, p. 1142). In a multi center review of more than 250 pregnancies in women whose prior pregnancies had been complicated by neonatal lupus, recurrence of congenital heart block was signiicantly lower if the woman had been treated with hydroxychloroquine during pregnancy (Izmirly, 2012). Research in this area is ongoing. Maternal terbutaline has also been given to increase the fetal heart rate in cases with sustained bradycardia of any cause in which the fetal heart rate is below 55 bpm. Reversal of hydrops with this therapy has been reported (Cuneo, 2007, 2010).

1	Several autosomal recessive enzyme deiciencies cause impaired fetal synthesis of cortisol from cholesterol by the adrenal cortex. his results in congenital adrenal hyperplasia (CAH). CAH is the most common etiology of androgen excess in females with 46,X disorders of sex development, formerly female pseudohermaphroditism (Chap. 3, p. 41). Lack of cortisol stimulates adrenocorticotrophic hormone (ACTH) secretion by the anterior pituitary, and the resulting androstenedione and testosterone overproduction leads to virilization of female fetuses. Sequelae may include formation of labioscrotal folds, persistence of a urogenital sinus, or even creation of a penile urethra and scrotal sac.

1	More than 90 percent ofCAH cases are caused by 21-hydroxylase deficiency, which is found in classic and nonclassic forms. he incidence of classic CAH approximates 1: 15000 births overall and is higher in selected populations. For example, it has been reported in approximately 1 :300 Yupik Eskimos (Nimkarn, 2010). Among those with classic CAH, 75 percent are at risk for salt-wasting adrenal crises and require postnatal treatment with mineralocorticoids and glucocorticoids to prevent hyponatremia, dehydration, hypotension, and cardiovascular collapse. The remaining 25 percent with classic CAH have the simple virilizing ype and also require glucocorticoid supplementation. As discussed in Chapter 32 (p. 614), all states mandate newborn screening for CAH.

1	he eicacy of maternal dexamethasone treatment to suppress fetal androgen overproduction and either obviate or ameliorate virilization of female fetuses has been recognized for more than 30 years (David, 1984; New, 2012). Prenatal corti costeroid therapy is considered successful in 80 to 85 percent of cases (Miller, 2013; Speiser, 2010). The alternative is con sideration of postnatal genitoplasty, a complex and somewhat controversial surgical procedure (Braga, 2009). he tpical preventive regimen is oral dexamethasone given to the mother at a dosage of 20 Lg/kg/d-up to 1.5 mg per day, divided in three doses. The critical period for external genitlia development is 7 to 12 weeks' gestation, and treatment to pre vent virilization should be initiated by 9 wees-bore it is known whether the etus is at risk. Because this is n autosomal recessive condition, ffected females mke up only 1 in 8 at-risk conceptions.

1	Typically, carrier parents are identified after the birth of an afected child. Molecular genetic testing is clinically available, initially using sequence analysis of the CP21A2 gene, which encodes the 21-hydroxylase enzyme (Nimkarn, 2016). If this is uninformative, gene-targeted deletion/duplication analy sis is performed, and additional testing such as whole exome sequencing may be considered (Chap. 13, p. 272). A goal of prenatal diagnosis is to limit dexamethasone expo sure in males and in unafected females. Prenatal diagnosis villi-at 10 to 12 weeks' gestation-or on amniocytes after 15 weeks. Cell-free DNA testing of maternal serum has potential to replace invasive tests such as chorionic villus sampling and amniocentesis for CAH (Chap. 13, p. 273). Determination of (Devaney, 2011). In the research setting, cell-free DNA testing using hybridization probes lanking the CP21A2 gene can be efective as early as 5617 weeks' gestation (New, 2014).

1	Maternal treatment with dexamethasone has become a topic of signiicant controversy. The Endocrine Society recommends that treatment be given only in the context of research protocols (Miller, 2013; Speiser, 2010). It should be noted that if therapy is initiated shortly before 9 weeks, the dose of dexamethasone used is not considered to have significant teratogenic potential because organogenesis of major organs has already taken place (McCullough, 2010). Ongoing concerns, however, focus on the potential efects of either excess endogenous androgens or excess exogenous dexamethasone on the developing brain. Although maternal dexamethasone has been used for many years to prevent virilization of female fetuses with CAH, longterm safety data are relatively limited.

1	Sonographically, this malformation is a well-circumscribed lung mass that may appear solid and echogenic or may have one or multiple variably sized cysts (Fig. 10-24, p. 199). Lesions with cysts � 5 mm are termed macrocystic, whereas microcystic lesions have smaller cysts or appear solid (Adzick, 1985). Also called congenital pulmonary airway malformation (CPAM), it represents a hamartomatous overgrowth of terminal bronchioles. Therapy for macrocystic congenital cystic adenomatoid malformation (CCAM) is discussed later (p. 324).

1	Occasionally, a microcystic CCAM may demonstrate rapid growth, generally between 18 and 26 weeks' gestation. he mass may become so large that it causes mediastinal shift, which may compromise cardiac output and venous return, resulting in hydrops (Cavoretto, 2008). A CCAM-volume ratio (CVR) has been used to quantiy size and risk for hydrops in these severe cases (Crombleholme, 2002). his ratio is an estimate of the CCAM volume (length X width X height X 0.52) divided by the head circumference. In a series of 40 pregnancies with microcystic CCAM, the mean CVR was 0.5 at 20 weeks' gestation, peaking in size at 1.0 at 26 weeks, followed by a pronounced decline prior to delivery (Macardle, 2016). A third of fetuses had no increase in mass size. In the absence of a dominant cyst, a CVR exceeding 1.6 is associated with a hydrops risk as high as 60 percent. However, CCAM growth resulting in hydrops develops in fewer than 2 percent of cases if the initial CVR is below 1.6 (Ehrenberg-Buchner,

1	1.6 is associated with a hydrops risk as high as 60 percent. However, CCAM growth resulting in hydrops develops in fewer than 2 percent of cases if the initial CVR is below 1.6 (Ehrenberg-Buchner, 2013; Peranteau, 2016). Importantly, a CVR in the range of 1.6 indicates that the mass essentially fills the thorax, and thus it is not unexpected that ascites or hydrops may develop.

1	If the CVR exceeds 1.6 or if signs of hydrops develop, corticosteroid treatment has been used in an efort to improve outcome. Regimens include dexamethasone-6.25 mg every 12 hours for four doses, or betamethasone-12.5 mg intramuscularly every 24 hours for two doses. Following a single course of corticosteroids, hydrops resolved in approximately 80 percent of cases, and 90 percent of treated fetuses survived (Loh, 2012; Peranteau, 2016). Recently, multiple courses of steroids-generally twohave been advocated for fetuses with large CCAM lesions and with persistent or worsening hydrops or ascites despite a single course of medication (Derderian, 2015; Peranteau, 2016).

1	Identification of fetal thyroid disease is rare and usually prompted by sonographic detection of a fetal goiter. If a goiter is found, determination of fetal hyper-or hypothyroidism is essential, and thyroid hormone levels may be measured in amnionic luid or fetal blood. Traditionally, fetal blood sampling, described in Chapter 14 (p. 294), is preferred to amniocentesis for guiding treatment, although data are limited (Abuhamad, 1995; Ribault, 2009). Goals of therapy are correction of the physiological abnormality and diminished goiter size. The goiter may compress the trachea and esophagus to such a degree that severe hydramnios or neonatal airway compromise may develop. Hyperextension of the fetal neck by a goiter can create labor dystocia.

1	Untreated fetal thyrotoxicosis may present with goiter, tachycardia, growth restriction, hydramnios, accelerated bone maturation, and even heart failure and hydrops (Huel, 2009; Peleg, 2002). The cause is usually maternal Graves disease with transplacental passage ofIgG thyroid-stimulating immunoglobulins. Fetal blood sampling may confirm the diagnosis (Duncombe, 2001; Heckel, 1997; Srisupundit, 2008). Conirmed fetal thyrotoxicosis is followed by maternal antithyroid treatment. During this, if the mother develops hypothyroidism, she is given supplementalilevothyroxine (Hui, 2011). In a woman receiving medication for Graves disease, transplacental passage of methimazole or propylthiouracil may cause etal hypothyroidism (Bliddal, 2011 a). Other potential causes of fetal hypothyroidism resulting in goiter include transplacental passage of thyroid peroxidase antibodies, fetal thyroid dyshormonogenesis, and maternal overconsumption of iodine supple ments (Agrawal, 2002; Overcash, 2016).

1	Goitrous hypothyroidism may lead to hydramnios, neck hyperextension, and delayed bone maturation. If the mother is receiving antithyroid medication, discontinuation is generally recommended, along with intraamnionic levothyroxine injection. Numerous case reports describe intraamnionic levothyroxine treatment. However, optimal dosage and frequency have not been established, and reported dosages range from 50 to 800 �g every 1 to 4 weeks (Abuhamad, 1995; Bliddal, 2011b; Ribault, 2009) .

1	Also called maternaletal surgery, these procedures are ofered for selected congenital abnormalities in which the likelihood of fetal deterioration is so great that delaying treatment until after delivery would risk fetal death or substantially greater postnatal morbidity. Open fetal surgef) is a highly specialized intervention performed at relatively few centers in the United States and for only a few fetal conditions. Criteria for consideration of fetal surgery are listed in Table 16-1. In many cases, data regarding the safety and eicacy of these procedures are limited. TABLE 16-1. Guiding Principles for Fetal Surgical Procedures Accurate prenatal diagnosis for the defect is available, with staging if applicable The defect appears isolated, with no evidence of other abnormality or underlying genetic syndrome that would significantly worsen survival or quality of life

1	The defect appears isolated, with no evidence of other abnormality or underlying genetic syndrome that would significantly worsen survival or quality of life The defect results in a high likelihood of death or irreversible organ destruction, and postnatal therapy is inadequate The procedure is technically feaSible, and a multidisciplinary team is in agreement regarding the treatment plan Maternal risks from the procedure are well documented and considered acceptable There is comprehensive parental counseling It is recommended that there be an animal model for the defect and procedure Data from Deprest, 201s0; Harrison, 1982; Vrecenak, 2013; Walsh, 201s1. TABLE 16-2. Selected Fetal Abnormalities Amenable to Fetal Surgery Twin-twin transfusion: laser of placental anastomoses Diaphragmatic hernia: fetal endoscopic tracheal occlusion Posterior urethral valves: cystoscopic laser Congenital high airway obstruction: vocal cord laser Amnionic band release

1	Posterior urethral valves: cystoscopic laser Congenital high airway obstruction: vocal cord laser Amnionic band release Posterior-urethral valves/bladder outlet obstruction Pleural effusion: chylothorax or sequestration Dominant cyst in CCAM Twin-reversed arterial perfusion (TRAP) sequence Monochorionic twins with severe anomaly in 1 twin Chorioangioma Aortic or pulmonic valvuloplasty for stenosis Atrial septostomy for hypoplastic left heart with EXIT-to-resection: resection of fetal thoracic or mediastinal mass EXIT-to-extracorporeal membrane oxygenation (E(MO): congenital diaphragmatic hernia he Agency for Healthcare Research and Quality stresses that when considering fetal surgery, the overriding concern must be maternal and fetal safety. Accomplishing the fetal goals of the procedure is secondary (Walsh, 2011).

1	Some abnormalities amenable to fetal surgical treatment, antepartum or intrapartum, are shown in Table 16-2. An overview of these procedures, their indications, and complications is provided here to assist with initial patient evaluation and counseling. Additional content is also found in Cunningham and Gilstrap s Operative Obstetrics, 3rd edition.

1	These procedures require extensive preoperative counseling and multidisciplinary care. he mother must undergo general endotracheal anesthesia to suppress both uterine contractions and fetal responses. Using intraoperative sonographic guidance to avoid the placental edge, a low-transverse hysterotomy incision is made with a stapling device that seals the edges for hemostasis. To replace amnionic fluid losses, warmed luid is continuously infused into the uterus thorough a rapid infusion device. he fetus is gently manipulated to permit pulse oximetry monitoring and to establish venous access, in case luids or blood are emergently needed. The surgical procedure is then performed. After completion, the hysterotomy is closed and tocolysis begun. T ocolysis typically includes intravenous magnesium sulfate for 24 hours, oral indomethacin for 48 hours, and, at some centers, oral nifedipine until delivery (Wu, 2009). Prophylactic antibiotics are also administered and generally continued for 24

1	sulfate for 24 hours, oral indomethacin for 48 hours, and, at some centers, oral nifedipine until delivery (Wu, 2009). Prophylactic antibiotics are also administered and generally continued for 24 hours following the procedure. Cesarean delivery is needed later in gestation and for all future deliveries.

1	Morbidities associated with fetal surgery are well characterized. In a review of 87 open procedures, Golombeck and coworkers (2006) reported the following morbidities: pulmonary edema-28 percent, placental abruption-9 percent, blood transfusion-13 percent, premature rupture of membranes-52 percent, and preterm delivery-33 percent. Wilson and associates (2010) reviewed subsequent pregnancy outcomes following open fetal surgery and reported that 14 percent of women experienced uterine rupture and 14 percent had uterine dehiscence. Morbidities identiied in the recent Management of Myelomeningocele Study (MOMS) are shown in Table 16-3 (Adzick, 2011). Other potential risks include maternal sepsis and fetal death during or following the procedure, particularly if hydrops is present.

1	Even with postnatal repair, children with myelomeningocele generally have varying degrees of paralysis, bladder and bowel dys function, developmental delays, and brainstem dysfunction from the Arnold-Chiari II malformation (Chap. 10, p. 193). Damage is postulated to result from abnormal embryonic neurulation and from ongoing exposure of neural elements to amnionic luid (Adzick, 2010; Meuli, 1995, 1997). Fetal myelomeningocele meets the criteria listed in Table 16-1 and is the first nonlethal birth defect for which fetal surgery has been ofered (Fig. 16-2).

1	In preliminary reports, infants following antepartum defect repair were more likely to have reversal of the Arnold-Chiari II malformation and were less likely to require ventriculoperitoneal shunt placement (Bruner, 1999; Sutton, 1999). Spurred by this, the randomized, multicenter MOMS trial was conducted (Adzick, 2011). Criteria for trial participation included: (1) a singleton fetus at 19.0 to 25.9 weeks' gestation; (2) an upper myelomeningocele boundary between T1 and S 1 conirmed by fetal magnetic resonance (MR) imaging; (3) evidence of hindbrain herniation; and (4) a normal karyotype and no evidence of a fetal anomaly unrelated to the myelomeningocele. Women at risk for preterm birth or placental abruption, those with a contraindication to fetal surgery, and women with body mass indexi> 35 kg/m2 were excluded.

1	he MOMS indings demonstrated improved early childhood outcomes in the prenatal surgery cohort (see Table 16-3). Children who had undergone prenatal surgery were twice as likely to walk independently by 30 months. hey had signiicantly less hindbrain herniation and were only half Composite developmental scorea,b TABLE 16-3. Benefits and Risks of Fetal 68% 98% <0.001 40% 82% <0.001 149 ± 58 123 ± 57 0.007 64% 96% <0.001 20% 48% <0.001 42% 21% 0.01 6% 0 0.03 6% 0 0.03 9% 1% 0.03 21% 4% 0.001 34 ± 3 37 ± 1 <0.001 79% 15% <0.001 aEach primary outcome had two components. The perinatal death components of the primary outcomes as well as the Bayley Mental Development Index at 30 months did not differ between the two study cohorts. bScore derived from Bayley Mental Development Index and difference between functional and anatomical level of lesion (30 months). Data from Adzick, 201s1s.

1	FIGURE 16-2 Fetal myelomeningocele surgery. A. With the edges of both the laparotomy and hysterotomy incisions retracted, the skin around the defect is incised. Subsequently, the neural placode is sharply dissected from the arachnoid membrane. B. The dural membrane is reflected to the midline to cover the neural placode and is reapproximated using suture. In some cases a patch is needed (not shown). The fetal skin incision is subsequently sutured. Last, hysterotomy and laparotomy are then closed. (Reproduced with permission from Shamshirsaz AA, Ramin, SM, Belfort MA: Fetal therapy. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al: Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201o7.) as likely to undergo ventriculoperitoneal shunting by the age of 1 year. A primary outcome was a composite score that was derived from the Bayley Mental Development Index and from the diference between the functional and anatomical level of the lesion at 30

1	age of 1 year. A primary outcome was a composite score that was derived from the Bayley Mental Development Index and from the diference between the functional and anatomical level of the lesion at 30 months. This primary outcome was also signii cantly better in the prenatal surgery group.

1	When counseling prospective families, however, results are placed in perspective. For example, despite improvements in the proportion with independent ambulation, most children who received fetal surgery were not able to ambulate inde pendently, and nearly 30 percent were not able to ambulate at all. Prenatal surgery did not confer improvements in fetal or neonatal death rates or in the Bayley Mental Development Index score at age 30 months. And, as shown in Table 16-3, surgery was associated with a small but significant risk for pla cental abruption and maternal pulmonary edema. Moreover, nearly half were delivered before 34 weeks, which signiicantly increased the risk for respiratory distress syndrome (Adzick, 201l). Long-term surveillance data have only recently become cele repair prior to the MOMS trial. At a median follow-up of 10 years, these children have higher rates of behavioral problems and adverse executive functioning compared with population norms (Danzer, 2016).

1	Since publication of the MOMS findings, fetal myelomeningocele surgery rates have grown. Expansion of centers ofering this procedure has raised concerns about the importance of training and ongoing experience, adherence to the MOMS research criteria, and need for a registry to ensure that future eforts have similar success rates (Cohen, 2014; Vrecenak, 20l3).

1	In the past, if hydrops developed in a fetus with a large pulmonary sequestration or cystic adenomatoid malformation without a dominant cyst, open fetal surgery with lobectomy was the only treatment available other than preterm delivery. Most thoracic masses are small and have a benign prognosis, and larger masses are generally treated with corticosteroids (p. 318). Fetal surgery is generally reserved for cases prior to 32 weeks in which hydrops is developing, and in selected cases, the survival rate following open lobectomy approximates 60 percent (Vrecenak, 20l3). Use of the ex-utero intrapartum treatment procedure in the treatment of fetal lung masses at delivery is discussed later on page 327.

1	This germ cell tumor has a prevalence of approximately 1 per 28,000 births (Derikx, 2006; Swamy, 2008). Sonographically, a sacrococcygeal teratoma (SCT) is a solid and/or cystic mass that arises from the anterior sacrum (Fig. 16-3). Fetal MR imaging can aid evaluation of the extent of the internal tumor component. he mass may grow rapidly, usually extending inferiorly and externally (Fig. 10-18, p. 196). Hydramnios is common, and hydrops may develop from high-output cardiac failure, either as a consequence of tumor vascularity or secondary to bleeding within the tumor and resultant anemia. Mirror syndrome-maternal preeclampsia developing along with fetal hydrops-may occur in this setting (Chap. 15, p. 312). FIGURE 16-3 Fetal surgery for sacrococcygeal teratoma resection. Following laparotomy and hysterotomy, the caudal portion of the fetus has been delivered onto the surgical field. The tumor is held by the surgeon's hand. (Used with permission from Dr. Timothy M. Crombleholme.)

1	The perinatal mortality rate for cases of SCT diagnosed prenatally approximates 40 percent (Hedrick, 2004; Shue, 20l3). Poor prognostic factors include a solid component comprising more than 50 percent of the tumor mass and a tumor volume-to-fetal weight ratio (tumor volume divided by estimated fetal weight) exceeding 12 percent prior to 24 weeks' gestation (Akinkuotu, 2015). Fetal loss rates approach 100 percent if hydrops or placentomegaly develop (Vrecenak, 20l3). The group at the Children's Hospital of Philadelphia recommends consideration of open fetal surgery for SCT only in cases in which the tumor is completely external (Type I) and in which high cardiac output with early hydrops has developed in the second trimester (Vrecenak, 2013). For excision, hysterotomy is performed, and the external component is resected. he coccyx and any deep tumor are left in place for postnatal removal. Because tumor debulking interrupts the pathological vascular steal, normal fetal physiology may

1	external component is resected. he coccyx and any deep tumor are left in place for postnatal removal. Because tumor debulking interrupts the pathological vascular steal, normal fetal physiology may be restored.

1	As with open fetal surgeries, these procedures are performed at highly specialized centers, and some are considered investigational. To accomplish them, iberoptic endoscopes only 1 to 2 mm in diameter are used to penetrate the maternal abdominal wall, the uterine wall, and membranes. Instruments such as lasers fit through 3-to 5-mm cannulas that surround the endoscope. vIorbidities are generally lower than with open fetal surgery, but they still may be formidable, particularly if maternal laparotomy is required for access (Golombeck, 2006). Examples of some conditions treated by fetoscopy are listed in Table 16-2. Indications and Technique. As discussed in Chapter 45 (p. 879), fetoscopic laser ablation of placental anastomoses is the preferred management for severe twin-twin transfusion syndrome (TTTS). It is generally performed between 16 and

1	FIGURE 16-4 Selective laser photocoagulation for twin-twin transfusion syndrome. The fetoscope is inserted into the recipienttwin sac and positioned over the vascular equator, which lies in between the two placental cord insertion sites. Arteriovenous anastomoses along the placental surface are individually photocoagulated using the laser. (Reproduced with permission from Shamshirsaz AA, Ramin, SM, Belfort MA: Fetal therapy. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al: Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201o7.) 26 weeks' gestation for monochorionic-diamnionic twin pregnancies with stage II to stage IV TTTS. These categories of the Quintero Staging System are described in Chapter 45 (p. 879) (Quintero, 1999; Society for Maternal-Fetal Medicine, 2013).

1	For the procedure, a fetoscope is used to view the vascular equator that separates the placental cotyledons supplying each twin (Fig. 16-4). Arteriovenous anastomoses along the placental surface of the vascular equator are photocoagulated using a 600-�m diameter diode laser or a 400-�m neodymium:yttriumaluminum-garnet (Nd:YAG) laser (Fig. 16-5). he procedure is typically performed under epidural or local analgesia. At the end, amnioreduction is performed to decrease the single deepest pocket of amnionic luid to below 5 cm, and antibiotics are injected into the amnionic cavity.

1	With selective laser photocoagulation, anastomoses crossing between the twins along the vascular equator are individually coagulated (Ville, 1995). Unfortunately, residual anastomoses remain in up to a third of cases and may lead to TTTS recurrence or to the development of twin-anemia polycythemia sequence (TAPS). The latter is a feto-fetal transfusion characterized by large diferences in hemoglobin concentrations between a pair of monochorionic twins. To address these complications, the Solomon technique was developed. With this, after selective photocoagulation, the laser is used to coagulate the entire vascular equator, from one edge of the placenta to the other (Slaghekke, 2014a). he Solomon technique lowers the proportion of pregnancies with recurrent TTTS and TAPS in multiple trials. Also, placental dye-injection studies confirm a significant reduction in the number of residual anastomoses (Ruano, 2013; Slaghekke, 2014b).

1	Complications. Families should have reasonable expectations of procedural success and potential complications. Without treatment, the perinatal mortality rate for severe TTTS is 70 to 100 percent. Following laser therapy, the anticipated perinatal mortality rate approximates 30 to 50 percent, with a 5-to 20-percent risk for long-term neurological handicap (Society for Maternal-Fetal Medicine, 2013). Cystic periventricular leukomalacia and grade III to IV interventricular hemorrhage are identiied neonatally in up to 10 percent of laser-treated cases (Lopriore, 2006).

1	Procedure-related complications include preterm prematurely ruptured membranes in up to 25 percent, placental abruption in 8 percent, vascular laceration in 3 percent, amnionic band syndrome resulting from laser laceration of the membranes in 3 percent, and TAPS in 16 percent with photocoagulation and 3 percent with the Solomon modification (Habli, 2009; Robyr, 2006; Slaghekke, 2014b). Finally, most laser-treated TTTS pregnancies deliver before 34 weeks.

1	FIGURE 16-5 Fetoscopic photograph of laser photocoagulation for twin-twin transfusion syndrome. A. Vascular anastomoses (arrows) are shown before photocoagulation is performed. B. The ablation sites appear as blanched yellow-white areas (arrows). (Used with permission from Dr. Timothy M. Crombleholme.) he prevalence of congenital diaphragmatic hernia (CDH) is approximately 1 in 3000 to 4000 births, and the overall sur vival rate is 50 to 60 percent. Associated anomalies occur in 40 percent of cases and confer a considerably lower survival rate. he main causes of mortality among those with isolated CDH are pulmonary hypoplasia and pulmonary hypertension. And, the major risk factor is liver herniation, which complicates at least half of cases and is associated with a 30-percent reduction in the survival rate (vfullassery, 2010, Oluyomi-Obi, 2017).

1	Because of maternal and fetal risks associated with fetal surgical intervention, eforts have focused on identiying those least likely to survive with postnatal therapy alone. Fetuses with associated anomalies are typically excluded, as are those with out liver herniation. Prediction is further hampered because of improvements in neonatal care for newborns with CDH. These include permissive hypercapnia, "gentle ventilation" to avoid barotrauma, and delayed surgery.

1	Lung-to-Head Ratio. This sonographic ratio was developed to improve prediction of survival in fetuses with isolated leftsided CDH diagnosed before 25 weeks' gestation (Metkus, 1996). he lung-to-head ratio (LHR) is a measurement of the right lung area, taken at the level of the four-chamber view of the heart, divided by the head circumference (Fig. 10-23, p. 198). Investigators found that the survival rate was 100 percent if the LHR wasi> 1.35, and there were no survivors if it was <0.6. Nearly three fourths of pregnancies had values between 0.6 and 1.35, and prediction was diicult in this large group because the overall survival rate approximated 60 percent (Metkus, 1996).

1	As of 2017, trials underway have selected a threshold LHR of < 1.0 or an observed-to-expected LHR <25 percent for study inclusion. An observed LHR is obtained sonographically from the afected fetus, whereas rhe expected LHR is an established reference value from normal fetuses (Peralta, 2005). In a recent metaanalysis, the odds ratio for survival with an LHR < 1.0 was only 0.14 (Oluyomi-Obi, 2017). Similarly, with an observed-to-expected LHR <25 percent, survival rates ranged from 13 to 30 percent. In contrast, an observed-to-expected LHR > 35 percent was associated with survival rates ranging from 65 to 88 percent.

1	Magnetic Resonance Imaging. This has been used to estimate the total volume of lung tissue, both ipsilateral and contralateral to the diaphragmatic hernia, which may then be compared with a gestational age-matched reference. Mayer and coworkers (2011) performed a metaanalysis of 19 studies involving more than 600 pregnancies in which isolated CDH was evaluated with feral MR imaging. Factors signiicantly associated with neonatal survival included the side of the defect, total fetal lung volume, observed-to-expected lung volume, and fetal liver position.

1	Fetal MR imaging has also been used to quantiy the volume of herniated liver (Fig. 10-57, p. 219). Two reasons underlie the rationale for assessing liver volume. First, liver herniation is perhaps the strongest predictor of outcome in fetuses with isolated CDH. Second, liver volume might be a more reliable predictor because lungs are inherently more compressible than liver. Indeed, these MR parameters-lung volumes and degree of liver herniation-correlate well with postnatal survival rates and may be more useful predictors than sonographic parameters (Bebbington, 2014; Ruano, 2014; Worley, 2009).

1	Tracheal Occlusion. Early attempts to treat severe diaphragmatic herniation used open fetal surgery to reposition the liver into the abdomen, which unfortunately kinked the umbilical vein and led to fetal demise (Harrison, 1993). Knowledge that lungs normally produce luid and that fetuses with upper airway obstruction develop hyperplastic lungs formed the rationale for tracheal occlusion. The idea was to "plug the lung until it grows" (Hedrick, 1994). Initial eforts focused on occluding the trachea with an external clip (Harrison, 1993). Subsequently, a detachable silicone balloon was placed within the trachea endoscopically (Fig. 16-6).

1	The balloon technique-termed etal endoscopic tracheal occlusion (FETO)-uses a 3-mm operating sheath and fetoscopes as small as 1 mm (Depresr, 2011; Ruano, 2012). he procedure is generally performed between 27 and 30 weeks' gestation, with the goal of removing the balloon at approximately 34 weeks, either through a second fetoscopic procedure or by ultrasound-guided puncture Gimenez, 2017). If these are unsuccessful, the balloon is removed during an ex-utero intrapartum treatment procedure at delivery (p. 327). FIGURE 16-6 Fetoscopic tracheal occlusion (FETO). The endoscope enters the fetal oropharynx and advances down the trachea. Inset: The balloon is inflated to occlude the trachea, and then the endoscope is removed. (Reproduced with permission from Shamshirsaz AA, Ramin, SM, Belfort MA: Fetal therapy. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al: Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 2017.)

1	In 2003, a randomized trial of the FETO procedure in pregnancies with isolated CDH, liver herniation, and LHR < 1.4 did not identiY a benefit from fetal therapy (Harrison, 2003). Survival rates 90 days after birth were unexpectedly high in both groups and approximated 75 percent. Following this study, however, enthusiasm for the technique continued, particularly outside the United States. Using a lower LHR threshold of 1.0 in addition to liver herniation as prerequisites for inclusion, signiicantly higher postnatal survival rates have been reported. Rates improved from <25 percent with postnatal therapy to approximately 50 percent with FETO Gani, 2009; Ruano, 2012). In a recent metaanalysis of five trials that included 21i1 pregnancies, those treated with FETO were 13 times more likely to survive (Al-Maary, 2016). At present, FETO is available in the United States only in research trials.

1	After publication of the MOMS indings, research eforts focused on whether maternal morbidities associated with open fetal myelomeningocele repair might be mitigated if the procedure was accomplished endoscopically. Araujo Junior and associates (2016) conducted a systematic review that included 456 open cases and 84 endoscopic surgeries. he endoscopic procedures were generally performed by inserting instruments through the maternal abdominal wall and then through the uterine wall, with partial carbon dioxide insuiation of the uterus. he rate of maternal myometrial dehiscence or attenuation was only 1 percent following endoscopy compared with 26 percent following open procedures. However, endoscopy was associated with significantly increased rates of preterm delivery before 34 weeks-80 versus 45 percent, and of perinatal mortality-14 versus 5 percent.

1	Belfort and colleagues (2017) recently described their outcomes in 22 pregnancies with fetal myelomeningocele using a technique in which the maternal abdomen was opened, the uterus exteriorized, and the procedure then performed endoscopically using warmed carbon dioxide insuiation. In contrast with earlier endoscopic reports, most treated pregnancies were delivered at term, with no perinatal losses. Further, the proportion of infants requiring hydrocephalus treatment prior to 1 year of age-approximately 40 percent-was similar to that with open fetal surgery in the MOMS trial (Adzick, 2011; Belfort, 2017). Research eforts in this area will undoubtedly continue.

1	Sonographic guidance can be used to permit therapy with a shunt, radiofrequency ablation needle, or angioplasty catheter. With these procedures, desired instruments cross the maternal abdominal wall, uterine wall, and membranes to reach the amnionic cavity and fetus. Risks include maternal infection, preterm labor or prematurely ruptured membranes, and fetal injury or loss. A shunt placed from the fetal pleural cavity into the amnionic cavity may be used to drain pleural luid (Fig. 16-7). A large efusion may cause a significant mediastinal shift, resulting in pulmonary hypoplasia or in heart failure and hydrops. he most common etiology of a primary efusion is chylothoraxcaused by lymphatic obstruction. Pleural efusions may also form secondary to congenital viral infection or aneuploidy, or they may be associated with a malformation such as pulmonary

1	FIGURE 16-7 Thoracoamnionic shunt placement. A. A large, right-sided fetal pleural effusion (asterisks) and ascites were identified at 18 weeks' gestation. The effusion was drained but rapidly reaccumulated. The xanthochromic fluid contained 95-percent lymphocytes, consistent with chylothorax. B. A double-pigtail shunt (arrow) was inserted under ultrasound gUidance. Following shunt placement, the efusion and ascites resolved.

1	FIGURE 16-8 Vesicoamnionic shunt placement. A. After amnioinfusion is performed, a trocar is inserted into the distended fetal bladder under sonographic guidance. The pigtail catheter is threaded into the trocar. B. The double-pigtail shunt has been deployed down the trocar, and the trocar has been removed. The distal end of the shunt is coiled within the fetal bladder, and the proximal end is draining into the amnionic cavity. (Reproduced with permission from Shamshirsaz AA, Ramin, SM, Belfort MA: Fetal therapy. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al: Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201o7.) sequestration. Yinon and associates (2010) reported aneuploidy cent of cases.

1	Typically, the efusion is first drained using a 22-gauge needle with sonographic guidance. Tests for aneuploidy and infection are performed, as well as a cell count. A pleural-fluid cell count with greater than 80-percent lymphocytes, in the absence of infection, is diagnostic of chylothorax. If the luid reaccumulates, a trocar and cannula may be inserted through the fetal chest wall, and a double-pigtail shunt may be placed to drain the efusion. If the efusion is right-sided, the shunt is placed in the lower third of the chest to permit maximum expansion of the lung. If left-sided, the shunt is placed along the upper axillary line to allow the heart to return to normal position (Mann, 2010). he overall survival rate is 70 percent, and that for hydropic fetuses approximates 50 percent (Mann, 2010; Yinon, 2010). Shunt displacement into the amnionic cavity is not uncommon. If the shunt remains in place, it must be clamped immediately upon delivery of the newborn to avoid pneumothorax.

1	Shunts can also drain a dominant cyst in fetuses with macrocystic congenital cystic adenomatoid maormation. However, cysts rarely are large enough to pose a risk for hydrops or pulmonary hypoplasia. Shunt placement may improve survival to 90 percent in the absence of hydrops and to more than 75 percent with hydrops (Liwinska, 2017).

1	Vesicoamnionic shunts are used in selected fetuses with severe bladder-outlet obstruction in which diminished amnionic fluid portends a grim prognosis (Fig. 16-8). Distal obstruction of the urinary tract occurs more often in male fetuses. The most common etiology is posterior urethral valves, followed by urethral atresia and by prune bely syndrome, which is also called Eagle-Barrett syndrome. Sonographic indings include dilation of the bladder and proximal urethra, termed the "keyhole" sign, along with bladder wall thickening (Fig. 10-45, p. 210). Associated oligohydramnios before midpregnancy leads to pulmonary hypoplasia. Unfortunately, postnatal renal function may be poor even when amnionic fluid volume is normal.

1	Evaluation includes a careful search for concurrent anomalies, which may coexist in 40 percent of cases, and for aneuploidy, which has been reported in 5 to 8 percent of cases (Hayden, 1988; Hobbins, 1984; Mann, 2010). Fetal urine sampled at vesicocentesis may be used to perform genetic studies. As with other structural fetal abnormalities, chromosomal micro array analysis is recommended. Because visualization may be limited due to lack of amnionic fluid, counseling should include the increased likelihood that associated anomalies may be missed sonographically.

1	Potential candidates are fetuses without other severe anomalies or genetic syndromes and without sonographic features that confer poor prognosis, for example, renal cortical cysts. herapy is generally ofered only if the fetus is male, because in females, the underlying anomaly tends to be even more severe. Serial bladder drainage-vesicocentesis-performed under sonographic guidance at approximately 48-hour intervals is used to evaluate fetal urine electrolyte and protein content. Fetal urine is normally hypotonic due to tubular resorption of sodium and chloride, whereas isotonic urine in the setting of obstruction suggests renal tubular damage. Serial assessment permits classiication of the renal prognosis as good or poor and helps guide candidate selection (Table 16-4). TABLE 16-4. Fetal Urinary Analyte Values with Bladder Outlet Obstruction Good or poor prognosis is based on values from serial gestation, using the last specimen obtained. Data from Mann, 2010.

1	TABLE 16-4. Fetal Urinary Analyte Values with Bladder Outlet Obstruction Good or poor prognosis is based on values from serial gestation, using the last specimen obtained. Data from Mann, 2010. Shunt placement allows urine to drain from the bladder into the amnionic cavity. When successful, this often prevents pulmonary hypoplasia, however, renal function is not reliably preserved. Before shunting, amnioinfusion of warmed lactated Ringer solution is generally performed to aid catheter placement. Amnioinfusion also aids sonographic evaluation of fetal anatomy. A small trocar and cannula are then inserted into the fetal bladder under sonographic guidance. he shunt is placed as low as possible within the bladder to avoid dislodgement after bladder decompression. A double-pigtail catheter is used. The distal end lies within the fetal bladder, and the proximal end drains into the amnionic cavity.

1	Complications include displacement of the shunt out of the fetal bladder in up to 40 percent of cases, urinary ascites in about 20 percent, and development of gastroschisis in 10 percent (Freedman, 2000; Mann, 2010). Preterm delivery is common, and neonatal survival rates range from 50 to 90 percent (Biard, 2005; Walsh, 201l). A third of surviving children have required dialysis or renal transplantation, and almost half have respiratory problems (Biard, 2005). In one randomized trial, vesicoamnionic shunt placement was compared with conservative management in 31 cases (Morris, 2013). hose receiving shunts had higher survival rates. However, only two children had normal renal function at age 2 years.

1	With this procedure, high-frequency alternating current is used to coagulate and desiccate tissue. Radiofrequency ablation (RFA) has become a favored modality for treatment of twin-reversed arterial peusion (TAP) sequence, also known as acardiac twin (Chap. 45, p. 880). Without treatment, the mortality rate for the normal or pump twin in severe TRAP sequence exceeds 50 percent. he procedure is also used for selective termination with other monochorionic twin complications (Bebbington, 2012).

1	Under sonographic guidance, a 17-to 19-9auge RF A needle is directed into the base of the umbilical cord of the acardiac twin and inserted into its abdomen. After a 2-cm area of coagulation is achieved, color Doppler sonography is applied to veriy absent low into the acardius. In several centers, survival rates for the normal twin following RF A have signiicantly improved (Lee, 2007; Livingston, 2007). RF A was performed at approximately 20 weeks' gestation in 98 pregnancies with TRAP sequence reported by the North American Fetal Therapy Network (NAFTNet). The median gestational age at delivery was 37 weeks, and the neonatal survival rate was 80 percent. The major complication was prematurely ruptured membranes and pre term birth. Twelve percent were delivered at approximately 26 weeks (Lee, 2013).

1	RFA has generally been ofered for TRAP sequence when the volume of the acardiac twin is large. In the NFTNet series cited above, the median size of the acardius relative to the pump twin was 90 percent (Lee, 2013). Considering procedure-related risks, expectant management with close fetal surveillance is instead considered if the estimated weight of the acardius is below 50 percent of the pump twin Jelin, 2010). Finally, acardiac twins are more likely to complicate monoamnionic gestations. In one recent series, pump twin survival following RFA was 88 percent in monochorionic diamnionic pregnancies but only 67 percent in monoamnionic pregnancies (Sugibayashi, 2016).

1	Selected fetal cardiac lesions may worsen during gestation, further complicating or even obviating options for postnatal repair. Severe narrowing of a cardiac outflow tract may result in progressive myocardial damage in utero, and a goal of fetal intervention is to permit muscle growth and preserve ventricular function (Walsh, 2011). These innovative procedures include aortic valvuloplasy for critical aortic stenosis; atrial septostomy for hypoplastic left heart syndrome with intact interatrial septum; and pulmonary valvuloplasy for pulmonary atresia with intact interventricular septum.

1	Fetal aortic valvuloplasty is the most commonly performed cardiac procedure, accounting for 75 percent of cases reported by the International Fetal Cardiac Intervention Registry (MoonGrady, 2015). It is ofered for selected cases of critical aortic stenosis in which the left ventricle is either normal sized or dilated. he goal is to prevent progression to hypoplastic left heart and to permit postnatal biventricular repair (McElhinney, 2009). Under sonographic guidance, an I8-gauge cannula is inserted through uterus and fetal chest wall and into the left ventricle. Although the procedure is ideally performed percutaneouslythrough the maternal abdominal wall-laparotomy may be needed if the fetal position is unfavorable. The cannula tip is positioned in front of the stenotic aortic valve, and a 2.5-to 4.5-mm balloon catheter is then guided into the aortic annulus and inlated. Fetal bradycardia requiring treatment complicates a third of cases, and hemopericardium requiring drainage afects

1	a 2.5-to 4.5-mm balloon catheter is then guided into the aortic annulus and inlated. Fetal bradycardia requiring treatment complicates a third of cases, and hemopericardium requiring drainage afects approximately 20 percent (Moon-Grady, 2015).

1	From the first 100 cases at Boston Children's Hospital, 85 children survived, 38 of whom achieved biventricular circulation (Freud, 2014). Despite these successes, the mortality rate and risk for neurodevelopmental impairment in childhood appear to be similar to cases treated with postnatal repair (Laraja, 2017; Moon-Grady, 2015). Fetal atrial septostomy, also using a percutaneous balloon catheter, is ofered in select cases of hypoplastic left heart with an intact or highly restrictive interatrial septum. This condition has a postnatal mortality rate of nearly 80 percent (Glantz, 2007). In an efort to ensure patency, atrial septal stent placement has TABLE 16-5. Components of the Ex-Utero Intrapartum Treatment (EXIT) Procedure Comprehensive preoperative evaluation: specialized sonography, fetal echocardiography, magnetic resonance imaging, Uterine relaxation with deep general anesthesia and tocolysis

1	Comprehensive preoperative evaluation: specialized sonography, fetal echocardiography, magnetic resonance imaging, Uterine relaxation with deep general anesthesia and tocolysis Intraoperative sonography to confirm placental margin and fetal position and to visualize vessels at uterine entry Placement of stay-sutures followed by use of uterine stapling device to decrease uterine entry bleeding Maintenance of uterine volume during the procedure via continuous amnioinfusion of warmed physiological solution to help prevent placental separation Delivery of the fetal head, neck, and upper torso to permit access as needed Fetal injection of intramuscular vecuronium, fentanyl, and atropine Fetal peripheral intravenous access, pulse oximeter, and cardiac ultrasound Following procedure, umbilical lines placed prior to cord clamping Uterotonic agents administered as needed Data from Moldenhauer, 2013.

1	Data from Moldenhauer, 2013. also been performed. Of37 cases of atrial septostomy, survival to hospital discharge was almost 50 percent (Moon-Grady, 2015). Fetal pulmonary valvuloplasty has been ofered in cases of pulmonary atresia with intact interventricular septum to prevent development of hypoplastic right heart syndrome. Although success is achieved in approximately two thirds of cases, it is not yet clear whether outcomes are improved compared with standard postnatal repair (Arzt, 2011; McElhinney, 2010).

1	This procedure allows the fetus to remain perfused by the placenta after being partially delivered, so that lifesaving treatment can be performed before completing the delivery. The technique was irst developed to obtain an airway with fetal tumors involving the oropharynx and neck (Catalano, 1992; Kelly, 1990; Langer, 1992). An ex-utero intrapartum treatment (EXIT) procedure is performed by a multidisciplinary team, which may include an obstetrician, maternal-fetal medicine specialist, pediatric surgeon(s), pediatric otolaryngologist, pediatric cardiologist, anesthesiologists for the mother and fetus, and neonatologists, as well as specially trained nursing personnel. Components of the procedure are shown in Table 16-5.

1	Selected indications are listed in Table 16-2. EXIT is the preferred procedure for intrapartum management oflarge venolymphatic malformations of the neck such as the one shown in Figure 16-9. At the Children's Hospital of Philadelphia, criteria for EXIT with a cervical venolymphatic malformation include compression, deviation, or obstruction of the airway by the mass, and also involvement of the floor of the mouth (Laje, 2015). In a review of 112 pregnancies with fetal cervical venolymphatic malformations, only about 10 percent met these criteria. Other indications for EXIT include severe micrognathia and congenital high airway obstruction sequence (CHAOS), which are discussed in Chapter 10 (Figs. 10-20 and 10-26, p. 197). Criteria for an EXIT procedure for micrognathia include a fetal jaw measurement below the 5th percentile along with indirect evidence of obstruction, such as hydramnios, an absent stomach bubble, or glossoptosis (Morris, 2009b). Case selection for EIT procedures is

1	measurement below the 5th percentile along with indirect evidence of obstruction, such as hydramnios, an absent stomach bubble, or glossoptosis (Morris, 2009b). Case selection for EIT procedures is generally based on fetal 1R imaging findings (Chap. 10, p. 219).

1	FIGURE 16-9 Ex-utero intrapartum treatment (EXID procedure for a venolymphatic malformation. A. Upon delivery of the head, placental circulation was maintained and an airway was established over the course of 20 minutes by a team of pediatric subspecialists that included a surgeon, anesthesiologist, and otolaryngologist. B. Following a controlled intubation, the fetus was ready for delivery and transfer to the neonatal intensive care unit team. (Used with permission from Drs. Stacey Thomas and Patricia Santiago-Munoz.)

1	In some cases, an EIT procedure has been used as a bridge to other procedures. For example, resection of large thoracic masses may be accomplished by fetal thoracotomy performed with intact placental circulation. In a series of 16 fetuses with CCM volume ratiosi> 1.6 or hydrops, all of whom had mediastinal compression, Cass and colleagues (2013) reported that nine infants undergoing EIT-to-resection survived. In contrast, there were no survivors with urgent postnatal surgery alone. Similarly, Moldenhauer (2013) reported that 20 of 22 newborns treated with EXIT -to-resection for lung masses survived. he EIT procedure has also been used as a bridge to extracorporeal membrane oxygenation-EIT-to-ECMO-in pregnancies with severe congenital diaphragmatic hernia. However, it has not been found to clearly confer survival benefit in such cases (Morris, 2009a; Shieh, 2017; Stofan, 2012).

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1	New MI, Tong K, Yuen T, et al: Noninvasive prenatal diagnosis of congenital adrenal hyperplasia using cell-free DNA in maternal plasma. J Clin Endocrinol Metab 99(6):E1022, 2014 Nimkan S, Gangishetti PK, Yau M, et al: 21-Hydroxylase deicient congenital adrenal hyperplasia. In Pagon RA, Adam MP, Ardinger HH, et al (eds): GeneReviews. Seattle, University of Washington, 2016 Noah �IM, Norton ME, Sandberg P, et al: Short-term maternal outcomes that are associated with the EXIT procedure, as compared with cesarean delivery. Am J Obstet Gynecol 186(4):773,o2002 Overcash RT, Marc-Aurele KL, Hull AD, et l: Maternal iodine exposure: a case offetal goiter and neonatal hearing loss. Pediatrics 137(4):el, 2016 Oluyomi-Obi T, Kuret V, Puligandla P, et l: Antenatal predictors of outcome in prenatally diagnosed congenital diaphragmatic hernia (CDH). J Pediatr Surg 52(5):881, 2017

1	Oluyomi-Obi T, Kuret V, Puligandla P, et l: Antenatal predictors of outcome in prenatally diagnosed congenital diaphragmatic hernia (CDH). J Pediatr Surg 52(5):881, 2017 Peleg 0, Cada S, Peleg A, et al: he relationship between maternal serum thyroid-stimulating immunoglobulin and neonatal thyrotoxicosis. Obstet GynecoIo99(6):1040,o2002 Peralta CF, Cavoretto P, Csapo B, et al: Assessment of lung area in normal fetuses at 12-32 weeks. Ultrasound Obstet Gynecol 26(7):718, 2005 Peranteau H, Boelig MM, Khalek N, et al: Efect of single and multiple courses of maternal betamethasone on prenatal congenital lung lesion growth and fetal survival. J Pediatr Surg 51(1):28,o2016 Quintero RA, Morales J, Allen MH, et al: Staging of twin-twin transfusion syndrome. J Perinatol 19:550, 1999 Ribault V, Castanet M, Bertrand vI,et al: Experience with intraamniotic thyroxine treatment in nonimmune fetal goitrous hypothyroidism in 12 cases. J Clin Endocrinol Metab 94:3731, 2009

1	Ribault V, Castanet M, Bertrand vI,et al: Experience with intraamniotic thyroxine treatment in nonimmune fetal goitrous hypothyroidism in 12 cases. J Clin Endocrinol Metab 94:3731, 2009 Robyr R, Lewi L, Salomon LJ, et al: Prevalence and management of late fetal complications following successful selective laser coagulation of chorionic plate anastomoses in twin-to-twin transfusion syndrome. Am J Obstet Gynecol 194(3):796,o2006 Ruano R, Lazar DA, Cass DL, et al: Fetal lung volume and quantification of liver herniation by magnetic resonance imaging in isolated congenital diaphragmatic hernia. Ultrasound Obstet GynecoIo43(6):662, 2014 Ruano R, Rodo C, Peiro JL, et al: Fetoscopic laser ablation of placental anastomoses in twin-twin transfusion syndrome using "Solomon technique." Ultrasound Obstet GynecoIo42(4):434, 2013

1	Ruano R, Rodo C, Peiro JL, et al: Fetoscopic laser ablation of placental anastomoses in twin-twin transfusion syndrome using "Solomon technique." Ultrasound Obstet GynecoIo42(4):434, 2013 Ruano R, Yoshisaki CT, da Silva MM, et 1: A randomized controlled trial of fetal endoscopic tracheal occlusion versus postnatal management of severe isolated congenital diaphragmatic hernia. Ultrasound Obstet Gynecol 39(1):20, Shah A, Moon-Grady A, Bhogal N, et al: Efectiveness of sotalol as irstline therapy for fetal supraventricular tachyarrhythmias. Am J Cardiol 190(1o1):1614,o2012 Shamshirsaz A, Ramin, SM, Belfort A: Fetal therapy. In Yeomans ER, Hofman BL, Gilstrap LC III, et al: Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hill Education, 2017

1	Shamshirsaz A, Ramin, SM, Belfort A: Fetal therapy. In Yeomans ER, Hofman BL, Gilstrap LC III, et al: Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hill Education, 2017 Shieh HF, Wilson JM, Sheils CA, et a1: Does the ex utero intrapartum treatment to extracorporeal membrane oxygenation procedure change morbidity outcomes for high-risk congenital diaphragmatic hernia survivors? J Pediatr Surg 52(1):22,o2017 Shue E, Bolouri M, Jelin EB, et a1: Tumor metrics and morphology predict poor prognosis in prenatally diagnosed sacrococcygeal teratoma: a 25-year experience at a single institution. J Pediatr Surg 48(6):1225,o2013 Slaghekke F, Lewi L, Middeldorp JM, et al: Residual anastomoses in twintwin transfusion syndrome after laser: the Solomon randomized trial. Am J Obstet GynecoI211(3):285.el, 2014a

1	Slaghekke F, Lewi L, Middeldorp JM, et al: Residual anastomoses in twintwin transfusion syndrome after laser: the Solomon randomized trial. Am J Obstet GynecoI211(3):285.el, 2014a Slaghekke F, Lopriore E, Lewi L, et a1: Fetoscopic laser coagulation of the vascular equator versus selective coagulation for twin-to-twin transfusion syndrome: an open-label randomized controlled trial. Lancet 383(9935):2144, 2014b Society for Maternal-Fetal Medicine, Simpson LL: Twin-twin transfusion syndrome. Am J Obstet Gynecol 208(1):3, 2013 Speiser PW, Azziz, Baskin LS, et a1: Congenital adrenal hyperplasia due to steroid 21o-hydroxylase deficiency: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab 95(9):4133, 2010 Srinivasan S, Strasburger J: Overview of fetal arrhythmias. Curr Opin Pediatr 20:522, 2008 Srisupundit K, Sirichotiyakul S, Tongprasent F, et al: Fetal therapy in fetal thyrotoxicosis: a case report. Fetal Diagn her 23(2):1o14,o2008

1	Srisupundit K, Sirichotiyakul S, Tongprasent F, et al: Fetal therapy in fetal thyrotoxicosis: a case report. Fetal Diagn her 23(2):1o14,o2008 Stofan AP, Wilson J, Jennings RW, et al: Does the ex utero intrapartum treatment to extracorporeal membrane oxygenation procedure change outcomes for high-risk patients with congenital diaphragmatic hernia? J Pediatr Surg 47(6):1053,o2012 Strasburger JF, Wakai RT: Fetal cardiac arrhythmia detection and in utero therapy. Nat Rev (5):277,o2010 Sugibayashi R, Ozawa K, Sumie M, et a1: Forty cases of twin reversed arterial perfusion sequence treated with radio frequency ablation using the multistep coagulation method: a single-center experience. Prenat Diagn 36(5):437, 2016 Sutton LN, Adzick NS, Bilaniuk LT, et al: Improvement in hindbrain herniation demonstrated by serial fetal magnetic resonance imaging following fetal srgery for myelomeningocele. JAMA 282(19):1826, 1999

1	Swamy R, Embleton N, Hale J, et al: Sacrococcygeal teratoma over two decades: birth prevalence, prenatal diagnosis and clinical outcomes. Prenat Diagn 28: 1048, 2008 Van der Heijden LB, Oudijk MA, Manten GTR, et a1: Sotalol as irst-line treatment for fetal tachycardia and neonatal follow-up. Ultrasound Obstet GynecoIo42(3):285,o2013 Ville Y, Hyett J, Hecher K, et a1: Preliminary experience with endoscopic laser surgery for severe twin-twin transfusion syndrome. N Engl J Med 332(4):224, 1995 Vrecenak JD, Flake A W: Fetal surgical intervention: progress and perspectives. Pediatr Surg Int 29(5):407,o2013

1	Vrecenak JD, Flake A W: Fetal surgical intervention: progress and perspectives. Pediatr Surg Int 29(5):407,o2013 Walsh WF, Chescheir NC, Gillam-Krakauer M, et al: Maternal-fetal surgical procedures. Technical Brief No. 5. AHRQ Publication No.o1 0(1)-EHC059EF, Rockville, Agency for Healthcare Research and Quality, 201o1 Wilson RD, Lemerand K, Johnson MP, et a1: Reproductive outcomes in subsequent pregnancies after a pregnancy complicated by open maternal-fetal srgery (1996-2007). Am J Obstet Gynecol 203(3):209.el, 2010 Worley KC, Dashe JS, Barber RG, et al: Fetal magnetic resonance imaging in isolated diaphragmatic hernia: volume of herniated liver and neonatal outcome. Am J Obstet Gynecol 200:318.el, 2009 Wu D, Ball RH: The maternal side of maternal-fetal surgery. Clin Perinatol 36(2):247, 2009 Yinon Y, Grisaru-Granovsky S, Chaddha V, et al: Perinatal outcome following fetal chest shunt insertion for pleural efusion. Ultrasound Obstet Gynecol 36:58,o2010

1	Yinon Y, Grisaru-Granovsky S, Chaddha V, et al: Perinatal outcome following fetal chest shunt insertion for pleural efusion. Ultrasound Obstet Gynecol 36:58,o2010 FETAL MOVEMENTS............................ 331 FETAL BREATHING .............................. 333 CONTRACTION STRESS TESTING.................. 334 NONSTRESS TESTS .............................. 334 ACOUSTIC STIMULATION TESTS ................... 337 BIOPHYSICAL PROFILE.......................... 337 AMNIONIC FLUID VOLUME....................... 339 DOPPLERVELOCIMETRY......................... 339 ANTENATAL TESTING SUMMARY.................. 340 The rate of the oetal heart is subject to considerable variations, which aords us a airy reliable means of judging as to the well-being of the child. As a general rule, its lie should be considered in danger when the heart-beats all below 100 or exceed 160. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) More than 100 years ago, the approach to fetal assessment was rather primitive. Since that time, and especially since the 1970s, technology to evaluate the health of the fetus has advanced remarkably. Techniques employed today to forecast fetal wellbeing focus on fetal biophysical indings that include heart rate, movement, breathing, and amnionic luid production. These indings aid antepartum fetal surveillance to prevent fetal death and avoid unnecessary interventions, which are stated goals of the American College of Obstetricians and Gynecologists and the American Academy of Pediatrics (2017).

1	Most fetuses will be healthy, and usually a negativethat is, normal-antepartum test result is highly reassuring, because fetal deaths within 1 week of a normal test are rare. Indeed, negative-predictive values-a true negative test-for most of the tests described are 99.8 percent or higher. In contrast, estimates of the positive-predictive values-a true positive test-for abnormal test results are low and range between 10 and 40 percent. Importantly, fetal surveillance is primarily based on circumstantial evidence. No deinitive randomized clinical trials have been conducted for obvious ethical reasons (American College of Obstetricians and Gynecologists, 2016).

1	commences as early as 7 weeks' gestation and becomes more sophisticated and coordinated by the end of pregnancy (Sajapala, 2017; Vindla, 1995). Indeed, beyond 8 menstrual weeks, fetal body movements are never absent for periods exceeding 13 minutes (DeVries, 1985). Between 20 and 30 weeks' gestation, general body movements become organized, and the fetus starts to show rest-activity cycles (Sorokin, 1982). Fetal movement maturation continues until approximately 36 weeks, when behavioral states are established in most normal fetuses. Nijhuis and colleagues (1982) described four fetal behavioral states: State 1 F is a quiescent state-quiet sleep-with a narrow oscillatory bandwidth of the fetal heart rate. State 2F includes frequent gross body movements, continuous eye movements, and wider oscillation of the fetal heart rate. This state is analogous to rapid eye movement (EM) or active sleep in the neonate.

1	State 3F includes continuous eye movements in the absence ofbody movements and no heart rate accelerations. he existence of this state is disputed (Pillai, 1990a). State 4F is one ofvigorous body movement with continuous eye movements and heart rate accelerations. his state corresponds to the awake state in newborns.

1	State 4F is one ofvigorous body movement with continuous eye movements and heart rate accelerations. his state corresponds to the awake state in newborns. Fetuses spend most of their time in states IF and 2F. For example, at 38 weeks, 75 percent of time is spent in these two states. hese behavioral states-particularly IF and 2F, which correspond to quiet sleep and active sleep-have been used to develop an increasingly sophisticated understanding offetal behavior. In a study offetal urine production, bladder volumes increased during state IF quiet sleep (Fig. 17-1). Duringstate 2F, the fetal heart rate baseline bandwidth increased appreciably, and bladder volume was signiicantly diminished due to decreased urine production and infrequent fetal voiding. These phenomena were interpreted to represent reduced renal blood flow during active sleep.

1	n important determinant of fetal activity appears to be sleep-awake cycles, which are independent of maternal ones. Fetal sleep cyclicity varies from approximately 20 minutes to as much as 75 minutes. In one study, the mean length of the quiet or inactive state for term fetuses was 23 minutes (TimorTritsch, 1978). Patrick and associates (1982) measured gross fetal body movements with real-time sonography for 24-hour periods in 31 normal pregnancies and found the longest period ofinactivity to be 75 minutes. Amnionicluid volume is another important determinant of fetal activity. Sherer and colleagues (1996) assessed the number of fetal movements in 465 pregnancies during biophysical profile testing in relation to amnionic luid volume. hey observed decreased fetal activity with diminished amnionic volumes and suggested that a restricted uterine space might physically limit fetal movements.

1	with fetal heart rate (FHR) variation recorded in relation to 1 F or 2F behavior states. State 1 F fetal heart rate has a narrow bandwidth consistent with quiet sleep. State 2F heart rate shows wide oscillation ofthe baseline consistent with active sleep. (Modified with permission from Oosterhof H, vd Stege JG, Lander M, et al: Urine production rate is related to behavioural states in the near term human fetus, Br J Obstet Gynaecol. 1993 Oct;l 00(1 0):920-922.) �E > 400 .. ._ 100 FIGURE 17-2 Graph depicts averages offetal movements counted during 12-hour periods (mean ± SEM). (Data from Sadovsky, 1979a.)

1	._ 100 FIGURE 17-2 Graph depicts averages offetal movements counted during 12-hour periods (mean ± SEM). (Data from Sadovsky, 1979a.) Sadovsky and coworkers (1979b) classiied fetal movements into three categories according to both maternal perceptions and independent recordings using piezoelectric sensors. Weak, strong, and rolling movements were described, and their relative contributions to total weekly movements throughout the last half of pregnancy were quantiied. As pregnancy advances, the rate of weak movements decreases, more vigorous movements increase for severalweeks, and then rates of these subside at term. Presumably, declining amnionic luid and space account for diminished activity at term.

1	Figure 1i7-2 shows fetal movements during the last half ofgestation in 127 pregnancies with normal outcomes. he mean number of weekly movements calculated from 12-hour daily recording periods rose from approximately 200 at 20 weeks' gestation to a maximum of 575 movements at 32 weeks. Fetal movements then declined to an average of 282 at 40 weeks. Normal weekly maternal counts of fetal movements ranged between 50 and 950. Count showed large daily variations, with included counts as low as 4 to 10 per 12-hour period in normal pregnancies. Diminished fetal activity may be a harbinger ofimpending fetal death (Sadovsky, 1973). To quantiy fetal movement, clinical methods include use of a uterine contraction tocodynamometer, visualization with sonography, and maternal subjective perceptions.

1	Most, but not all, investigators report excellent correlation between maternally perceived fetal motion and movements documented by instrumentation. For example, Rayburn (1980) found that 80 percent of all movements observed during sonographic monitoring were perceived by the mother. In contrast, Johnson and colleagues (1992) reported that beyond 36 weeks, mothers perceived only 16 percent of fetal body movements. Fetal motions lasting more than 20 seconds were more likely to be identified than shorter episodes. Although several fetal-movement counting protocols have been used, neither the optimal number ofmovements nor the ideal duration for counting them has been defined. For example, in one method, perception of 10 fetal movements in up to 2 hours is considered normal (Moore, 1989). Commonly, women may present in the third trimester complaining of subjectively reduced fetal movement. Harrington and associates (1998) reported that 7 percent of nearly 6800 women presented with a

1	Commonly, women may present in the third trimester complaining of subjectively reduced fetal movement. Harrington and associates (1998) reported that 7 percent of nearly 6800 women presented with a complaint of decreased fetal movement. Fetal heart rate monitoring tests were employed if sonographic scans for fetal growth or Doppler velocimetry were abnormal. Pregnancy outcomes for women who complained of decreased fetal movement were not significantly diferent from those for women without this complaint. Scala and colleagues (2015) reported that 6 percent of women at term reported decreased fetal movements at 36 weeks or more. Women with two or more episodes of reduced fetal movements had greater risks of growth-restricted newborns and abnormal Doppler uterine artery low studies. However, stillbirth rates were not increased. Measurement ofthe myocardial performance index did not improve accuracy (Ho, 2017).

1	Grant and coworkers (1989) performed an unparalleled investigation of maternally perceived fetal movements and pregnancy outcome. More than 68,000 pregnancies were randomly assigned between 28 and 32 weeks' gestation. Women in the fetal movement arm of the study were instructed by specially employed midwives to record the time needed to feel 10 movements each day. his required an average of2.7 hours each day. Women in the control group were informally asked about movements during prenatal visits. Reports of decreased fetal motion were evaluated with tests of fetal well-being. Antepartum death rates for otherwise normal singleton fetuses were similar in the two study groups. Despite the counting policy, most stillborn fetuses were dead by the time the mothers reported for medical attention. Importantly, rather than concluding that maternal perceptions of fetal activity were meaningless, these investigators concluded that informal maternal perceptions were as valuable as formally

1	Importantly, rather than concluding that maternal perceptions of fetal activity were meaningless, these investigators concluded that informal maternal perceptions were as valuable as formally recorded fetal movement.

1	Saastad and associates (2011) reported a total of 1076 women who were randomly assigned to standardized fetal movement counting from gestational week 28 versus no counting. Growth-restricted fetuses were identiied before birth signiicantly more often when fetal movement counting was used. he rate of I-minute Apgar scores :;3 was signiicant reduced (0.4 versus 2.3 percent) when counting was used. Also, Warrander and coworkers (2012) described placental pathology in pregnancies complicated by diminished fetal movements. Decreased movement was associated with various placental abnormalities including infarction.

1	After decades of uncertainty as to whether the fetus normally breathes, Dawes and coworkers (1972) showed small inward and outward flows of tracheal fluid in fetal sheep, indicating thoracic movement. These chest wall movements difered from those following birth in that they were discontinuous. Another interesting feature of fetal respiration was paradoxical chest wal movement (Fig. 17-3). In the newborn or adult, the opposite occurs. One interpretation of the paradoxical respiratory motion might be coughing to clear amnionic fluid debris. Although the physiological basis for the

1	FIGURE 17-3 Paradoxical chest movement with fetal respiration. During inspiration (A), the chest wall paradoxically col/apses and the abdomen protrudes, whereas during expiration (8), the chest wall expands. (Adapted from Johnson, 1988.) breathing relex is not completely understood, such exchange of amnionic fluid appears to be essential for normal lung development (Chap. 7, p. 133). Dawes (1974) identiied two types of respiratory movements. The irst are gasps or sighs, which occurred at a frequency of 1 to 4 per minute. The second, irregular bursts ofbreathing, occurred at rates up to 240 cycles per minute. hese latter rapid respiratory movements were associated with rapid eye movement. Badalian and associates (1993) studied the maturation of normal fetal breathing using color low and spectral Doppler analysis of nasal fluid low as an index of lung function. hey suggested that fetal respiratory rate declined in conjunction with increased respiratory volume at 33 to 36 weeks and

1	spectral Doppler analysis of nasal fluid low as an index of lung function. hey suggested that fetal respiratory rate declined in conjunction with increased respiratory volume at 33 to 36 weeks and coincidental with lung maturation.

1	Many investigators have examined fetal breathing movements using sonography to determine whether chest wall movements might reflect fetal health. Several variables in addition to hypoxia were found to afect fetal respiratory movements. These included hypoglycemia, sound stimuli, cigarette smoking, amniocentesis, impending preterm labor, gestational age, the fetal heart rate itself, and labor-during which it is normal for respiration to cease. Because fetal breathing movements are episodic, interpretation of fetal health when respirations are absent may be tenuous. Patrick and associates (1980) performed continuous 24-hour observation using sonography to characterize fetal breathing patterns during the last 10 weeks of pregnancy. A total of 1224 hours of fetal observation in 51 pregnancies were collected, and ).>D C)..=20 Time of day

1	C)..=20 Time of day FIGURE 17-4 The percentage of time spent breathing by 11 fetuses at 38 to 39 weeks. There is a significant increase in fetal breathing activity after breakfast. Breathing activity diminished during the day and reached its minimum between 20:00 and 24:00 hours. There was a significant increase in the percentage of time spent breathing between 04:00 and 07:00 hours, when mothers were asleep. (Adapted with permission from Patrick J, Campbell K, Carmichael L, et al: Patterns of human fetal breathing during the last 10 weeks of pregnancy, Obstet Gynecol. 1980 Jul;56(1 ):24-30.)

1	Figure 17-4 displays the percentages of time spent breathing near term. Clearly, there is diurnal variation, because breathing substantively diminishes during the night. In addition, breathing activity increases somewhat following maternal meals. Total absence of breathing was observed in some of these normal fetuses for up to 122 minutes, indicating that fetal evaluation to diagnose absent respiratory motion may require long periods of observation. The potential for breathing activity to be an important marker of fetal health is unfulilled because of the multiplicity of factors that normally afect breathing. Most clinical applications have included assessment of other fetal biophysical indices, such as heart rate. As discussed subsequently, fetal breathing has become a component of the biophysical proile.

1	As amnionic luid pressure rises with uterine contractions, myometrial pressure exceeds collapsing pressure for vessels coursing through uterine muscle. his ultimately lowers blood flow to the intervillous space. Brief periods of impaired oxygen exchange result, and if utero placental pathology is present, these elicit late fetal heart rate decelerations (Chap. 24, p. 466). Contractions also may produce a pattern of variable decelerations as a result of cord compression, suggesting oligohydramnios, which is often a concomitant of placental insuiciency.

1	Ray and colleagues (1972) used this concept in 66 complicated pregnancies and developed the oytocin challenge test, which was later called the contraction stress test. Intravenous oxytocin is used to stimulate contractions, and the criterion for a positive test result, that is, an abnormal result, is uniform repetitive late fetal heart rate decelerations. These reflected the uterine contraction waveform and had an onset at or beyond the contraction acme. Such late decelerations could be the result of uteroplacental insuiciency. In their study, the tests were generally repeated on a weekly basis, and the investigators concluded that negative contraction stress test results, that is, normal results, forecasted fetal health. A major disadvantage is that the average contraction stress test requires 90 minutes to complete.

1	To perform the test, the fetal heart rate and uterine contractions are recorded simultaneously with an external monitor. If at least three spontaneous contractions of 40 seconds or longer are present in 10 minutes, no uterine stimulation is necessary (American College of Obstetricians and Gynecologists, 2016). Contractions are induced with either oxytocin or nipple stimulation if there are fewer than three in 10 minutes. For oxytocin use, a dilute intravenous inusion is initiated at a rate of 0.5 mU/min and doubled every 20 minutes until a satisfactory contraction pattern is established (Freeman, 1975). The results of the contraction stress test are interpreted according to the criteria shown in Table

1	Nipple stimulation to induce uterine contractions is usually successful for contraction stress testing (Huddleston, 1984). One method involves a woman rubbing one nipple through her clothing for 2 minutes or until a contraction begins. This 2-minute nipple stimulation ideally will induce a pattern of three contractions per 10 minutes. If not, after a 5-minute interval, she is instructed to retry nipple stimulation to achieve the desired pattern. If this is unsuccessful, then dilute oxytocin may be used. Advantages include reduced cost and shortened testing times. Some have reported unpredictable uterine hyperstimulation and fetal distress, whereas others did not find excessive activity to be harmful (Frager, 1987; Schellpfefer, 1985).

1	Freeman (1975) and Lee and colleagues (1975) introduced the nonstress test to describe fetal heart rate acceleration in response to fetal movement as a sign of fetal health. This test involved the use of Doppler-detected fetal heart rate acceleration coincident with fetal movements perceived by the mother. By the end of the 1970s, the nons tress test had become the primary method of testing fetal TABLE 17-1. Criteria for Interpretation of the Contraction Stress Test

1	Negative: no late or significant variable decelerations Positive: late decelerations following 50% or more of contractions (even if the contraction frequency is fewer than three in 10 minutes) Equivocal-suspicious: intermittent late decelerations or significant variable decelerations Equivocal-hyperstimulatory: fetal heart rate decelerations that occur in the presence of contractions more frequent than every 2 minutes or lasting longer than 90 seconds Unsatisfactory: fewer than three contractions in 10 minutes or an uninterpretable tracing health. he nonstress test was easier to perform, and normal results were used to urther discriminate false-positive contraction stress tests. Simplistically, the nonstress test is primarily a test ofetal condition, and it difers from the contraction stress test, which is considered a test of uteropacentalonction. Currently, nonstress testing is the most widely used primary testing method for assessment of fetal well-being. It has lso been

1	stress test, which is considered a test of uteropacentalonction. Currently, nonstress testing is the most widely used primary testing method for assessment of fetal well-being. It has lso been incorporated into the biophysical profile testing system, subsequently discussed.

1	Autonomic inluences are mediated by sympathetic or para sympathetic impulses from brainstem centers to normally raise or slow the fetal heart rate. Beat-to-beat variability is also under the control ofthe autonomic nervous system (Matsuura, 1996). Consequently, pathological loss of fetal heart rate acceleration may be seen in conjunction with signiicantly decreased beat to-beat variability (Chap. 24, p. 462). Loss of such reactivity, however, is most commonly associated with sleep cycles. It also rette smoking Oansson, 2005). The nonstress test is based on the hypothesis that the heart rate of a fetus that is not acidemic as a result of hypoxia or to fetal movement. Fetal movements during testing are identi ied by maternal perception and recorded. As hypoxia develops, these fetal heart rate accelerations diminish (Smith, 1988).

1	Gestational age inluences acceleration or reactivity ofthe fetal heart rate. Pillai and James (1990b) studied the development of fetal heart rate acceleration patterns during normal pregnancy. The percentage of body movements that is accompanied by accelerations and the amplitude of these accelerations both increase with gestational age (Fig. 17-5). Guinn and colleagues (1998) studied nonstress test results between 25 and 28 weeks' gestation in 188 normal fetuses. Only 70 percent of these normal fetuses demonstrated the required 15 beats per minute (bpm) or more of heart rate acceleration. Lesser degrees of acceleration, that is, 10 bpm, occurred in 90 percent ofthe fetuses.

1	The National Institute of Child Health and Human Development Fetal Monitoring Workshop defined normal acceleration based on gestational age (Macones, 2008). In fetuses at or beyond 32 weeks' gestation, the acceleration acme is 15 bpm or more above the baseline rate, and the acceleration lasts 15 seconds or longer but less than 2 minutes. Before 32 weeks, normal accelerations are defined as having an acme that is 10 bpm or more above baseline for 10 seconds or longer. Cousins and associates (2012) compared the Workshop criteria recommended before 32 weeks, that is, 10 bpm/10 seconds, with standard 15 bpm/15 seconds criteria in a randomized trial of 143 women. They found no diferences in perinatal outcomes. ):J=20

1	):J=20 FIGURE 17-5 Percentage offetuses tlith at least one acceleration of 15 bpm sustained for 15 seconds concurrent with fetal movement. (Redrawn from Pillai M, James 0: The development offetal heart rate patterns during normal pregnancy, Obstet Gynecol. 1990 Nov;76(5 Pt 1):812-816.)

1	Criteria to define normal nonstress test results difer. They vary regarding the number, amplitude, and duration of accelerations and the test duration. he definition recommended by the American College of Obstetricians and Gynecologists (2016) requires two or more accelerations peaking at 15 bpm or more above baseline, each lasting 15 seconds or more, and all occurring within 20 minutes of beginning the test (Fig. 17-6). It is also recommended that accelerations with or without fetal movements be accepted, and that a 40-minute or longer tracing-to account for fetal sleep cycles-should be performed before concluding that fetal reactivity is insuicient. Miller and coworkers (1996b) reviewed outcomes in fetuses with nons tress tests considered as nonreactive because there was only one acceleration. hey concluded

1	FIGURE 17-6 Reactive nonstress test. In the upper panel, notice the increase offetal heart rate by more than 15 beats/min for longer than 15 seconds following fetal movements, which are indicated by the vertical marks (lower panen. that one acceleration was just as reliable as two in predicting healthy fetal status. Although a normal number and amplitude of accelerations seems to reflect fetal well-being, their absence does 60 not invariably predict fetal compromise. Indeed, some investigators have reported 90-percent or higher false-positive rates (Devoe, 1986). Because healthy fetuses may not move for periods of up to 75 minutes, some have considered that a longer duration of nonstress testing might increase the positive-predictive value of an abnormal, that is, nonreactive, test (Brown, 1981). In this scheme, either the test became reactive during a period up to 80 minutes or the test remained nonreactive for 120 minutes, which indicated that the fetus was very ill.

1	Not only do deinitions of normal nonstress test results difer, but the reproducibility ofinterpretations is problematic (Hage, 1985). Thus, although nons tress testing is popular, the reliability of test interpretation needs improvement. Based on the foregoing, an abnormal nons tress test is not always ominous and can be seen with a sleeping fetus. Also, an abnormal test can revert to normal as the fetal condition changes, such as the example shown in Figure 17-7. Importantly, a normal nonstress test can become abnormal if the fetal condition deteriorates.

1	Figure 17-7. Importantly, a normal nonstress test can become abnormal if the fetal condition deteriorates. There are abnormal patterns that reliably forecast severe fetal jeopardy (Fig. 17-8). Devoe and coworkers (1985) concluded that nonstress tests that were nonreactive for 90 minutes were almost invariably-93 percent-associated with significant perinatal pathology. Hammacher and coworkers (1968) described tracings with what they termed a silent oscillatoy pattern that he considered dangerous. his pattern consisted of a fetal heart rate baseline that oscillated less than 5 bpm and presumably indicated absent acceleration and beat-to-beat variability.

1	Visser and associates (1980) described a terminal cardiotocoram, which included: (1) baseline oscillation ofless than 5 bpm, (2) absent accelerations, and (3) late decelerations with spontaneous uterine contractions. hese results were similar to experiences from Parkland Hospital in which absence of accelerations during an 80-minute recording period in 27 fetuses was associated consistently with evidence of uteroplacental pathology (Leveno, 1983). he latter included fetal-growth restriction in 75ipercent, oligohydramnios in 80 percent, fetal acidemia in 40 percent, meconium in 30 percent, and placental infarction in 93 percent.

1	FIGURE 17-7 Two antepartum fetal heart rate (FHR) tracings in a 28-week pregnant woman with diabetic ketoacidosis. A. FHR tracing (upper panel) and accompanying contraction tracing (second panen. Tracing, obtained during maternal and fetal acidemia, shows absence of accelerations, diminished variability, and late decelerations with weak spontaneous contractions. B. Fetal heart rate tracing shows return of normal accelerations and variability of the fetal heart rate following correction of maternal acidemia.

1	Set originally rather arbitrarily at 7 days, the interval between tests appears to have been shortened as experience evolved with nonstress testing. According to the American College of Obstetricians and Gynecologists (2016), more frequent testing is advocated by some investigators for women with postterm pregnancy, multifetal gestation, pregestational diabetes, fetalgrowth restriction, or pregnancy hypertension. In these circumstances, some investigators perform twice-weekly tests, with additional testing completed for maternal or fetal deterioration regardless of the time elapsed since the last test. Others perform nonstress tests daily or even more frequently, such as with severe preeclampsia remote from term.

1	Fetal movements commonly produce heart rate decelerations. Timor-Tritsch and associates (1978) reported this during nonstress testing in half to two thirds of tracings, depending on the vigor of the fetal motion. This high incidence of decelerations inevitably makes interpretation of their signiicance problematic. Indeed, Meis and coworkers (1986) reported that variable fetal TOCO=IT. FIGURE 17-8 Nonreactive nonstress test (left side of tracing) followed by contraction stress test showing mild, late decelerations (right side of tracing). Cesarean delivery was performed, and the severely acidemic fetus could not be resuscitated.

1	heart rate decelerations during nonstress tests were not a sign of fetal compromise. he American College of Obstetricians and Gynecologists (2016) has concluded that variable decelerations, if nonrepetitive and brief-less than 30 seconds-do not indicate fetal compromise or the need for obstetrical intervention. In contrast, repetitive variable decelerations-at least three in 20 minutes-even if mild, have been associated with a greater risk of cesarean delivery for fetal distress. Decelerations lasting 1 minute or longer have been reported to have an even worse prognosis (Bourgeois, 1984; Druzin, 1981; Pazos, 1982).

1	Hoskins and associates (1991) attempted to reine interpretation of testing that shows variable decelerations by adding sonographic estimation of amnionic luid volume. The incidence of cesarean delivery for intrapartum fetal distress progressively rose concurrently with the severity of variable decelerations and decline of amnionic luid volume. Severe variable decelerations during a nonstress test plus an amnionic luid index (AFI) ;5 em resulted in a 75-percent cesarean delivery rate. Fetal distress in labor, however, also frequently developed in those pregnancies with variable decelerations but with normal amounts of amnionic luid. Similar results were reported by Grubb and Paul (1992).

1	Smith and associates (1987) performed a detailed analysis of the causes of fetal death within 7 days of normal nons tress tests. The most common indication for testing was postterm pregnancy. he mean interval between testing and death was 4 days, with a range of 1 to 7 days. he single most common autopsy inding was meconium aspiration, oten associated with some type of umbilical cord abnormality. They concluded that an acute asphyxial insult had provoked fetal gasping. hey also concluded that nonstress testing was inadequate to preclude such an acute asphyxial event and that other biophysical characteristics might be beneicial. Importantly, assessment of amnionic luid volume was considered valuable. Other ascribed frequent causes of fetal death included intrauterine infection, abnormal cord position, malformations, and placental abruption.

1	Loud external sounds have been used to startle the fetus and thereby provoke heart rate acceleration-an acoustic stimulation nonstress test. A commercially available acoustic stimulator is positioned on the maternal abdomen, and a stimulus of 1 to 2 seconds is applied (Eller, 1995). his may be repeated up to three times for up to 3 seconds (American College of Obstetricians and Gynecologists, 2016). A positive response is deined as the rapid appearance of a qualiYing acceleration following stimulation (Devoe, 2008). In a randomized trial of 113 women undergoing nonstress testing, vibroacoustic stimulation shortened the average time of testing from 24 to 15 minutes (Perez-Delboy, 2002). Similar results were reported by Turitz and coworkers (2012). Laventhal and colleagues (2003) reported that fetal tachyarrhythmia could be provoked with vibroacoustic stimulation.

1	Manning and colleagues (1980) proposed the combined use of ive fetal biophysical variables as a more accurate means of assessing fetal health than a single element. Typically, these tests require 30 to 60 minutes of examiner time. Shown in Table 1i7-2 are the ive fetal biophysical components assessed: (1) heart rate acceleration, (2) breathing, (3) movements, (4) tone, and (5) amnionic luid volume. Normal variables were assigned a score of 2 each, and abnormal variables were given a score of O. Thus, the highest score possible for a normal fetus is 10. Maternal medications such as narcotics and sedatives can signiicantly lower the score (Kopecky, 2000). Ozkaya and associates (2012) found that biophysical test scores were higher if a test was performed in late evening-20:00 to 22:00 hours-compared with 08:00 to 10:00 hours. TABLE 17-2. Components and Scores for the Biophysical Profile

1	TABLE 17-2. Components and Scores for the Biophysical Profile Nonstress testa :2 accelerations of : 15 beats/min for : 15 sec within 20-40 min Fetal breathing : 1 episode of rhythmic breathing lasting ::30 sec within 30 min Fetal movement ::3 discrete body or limb movements within 30 min Fetal tone ::1 episode of extremity extension and subsequent retun to fiexion Amnionic fluid A pocket of amnionic fluid that measures at least 2 cm in two volumeb planes perpendicular to each other (2 x 2 cm pocket) o or 1 acceleration within 20-40 min <30 sec of breathing within 30 min <3 discrete movements o extension/flexion events Largest single vertical pocket :;2 cm aMay be omitted if all four sonographic components are normal. bFurther evaluation warranted, regardless of biophysical composite score, if largest vertical amnionic fluid pocket :;2 cm.

1	bFurther evaluation warranted, regardless of biophysical composite score, if largest vertical amnionic fluid pocket :;2 cm. Manning and colleagues (1987) tested more than 19,000 pregnancies using the biophysical profile interpretation and management shown in Table 17-3. More than 97 percent of the pregnancies tested had normal test results. They reported a false-normal test rate-defined by an antepartum death of a structurally normal fetus-of approximately 1 per 1000. he most common identiiable causes of fetal death after a normal biophysical profile include fetomaternal hemorrhage, umbilical cord accidents, and placental abruption (Dayal, 1999).

1	Manning and coworkers (1993) published a remarkable description of 493 fetuses in which biophysical scores were performed immediately before measurement of umbilical venous blood pH values obtained via antepartum cordocentesis. Approximately 20 percent of tested fetuses had growth restriction, and the remainder had alloimmune hemolytic anemia. As shown in Figure 17-9, a biophysical score of 0 was almost invariably associated with significant fetal acidemia, whereas a normal score of 8 or 10 was associated with normal pH. An equivocal test result-a score of 6-was a poor predictor of abnormal outcome. As the abnormal score dropped from 2 or 4 down to 0, this decline was a more accurate predictor of abnormal fetal outcome. hus overall, these scores provide poor sensitivity to predict cord blood pH.

1	Similar studies have substantiated these findings. Salvesen and associates (1993) concluded that the biophysical proile was of limited value in the prediction of fetal pH. Weiner and coworkers (1996) assessed 135 overtly growth-restricted fetuses and came to a similar conclusion. Kaur and colleagues (2008) performed daily biophysical proiles to ascertain the optimal delivery time in 48 growth-restricted preterm fetuses that weighed less than 1000 g. Despite scores of 8 in 27 fetuses and 6 in 13, there were six deaths and 21 acidemic fetuses. Lalor and associates (2008) performed a Cochrane review and concluded that there is insuicient evidence to support the use of the biophysical proile as a fetal well-being test in high-risk pregnancies.

1	Because the biophysical proile is labor intensive and requires a person trained in sonography, Clark and coworkers (1989) used an abbreviated biophysical proile as a irst-line screening test in 2628 singleton pregnancies. Speciically, a vibroacoustic nons tress test was performed twice weekly and combined with TABLE 17-3. Interpretation of Biophysical Profile Score 10 Normal, nonasphyxiated fetus 8/10 (Normal AFV) Normal, nonasphyxiated fetus 8/8 (NST not done) 8/10 (Decreased AFV) Chronic fetal asphyxia suspected 6 Possible fetal asphyxia o to 2 Almost certain fetal asphyxia If amnionic fluid volume abnormal, deliver If normal fluid at >36 weeks with favorable cervix, deliver If repeat test :;6, deliver If repeat test >6, observe and repeat per protocol Repeat testing same day; if biophysical profile score :;6, deliver Deliver AFV = amnionic fluid volume; NST = nonstress test.

1	If repeat test >6, observe and repeat per protocol Repeat testing same day; if biophysical profile score :;6, deliver Deliver AFV = amnionic fluid volume; NST = nonstress test. Reproduced with permission from Manning FA, Morrison I, Harman CR, et al: Fetal assessment based on fetal biophysical profile scoring: experience in 19,221 referred high-risk pregnancies. II. An analysis of false-negative fetal deaths, Am J Obstet Gynecol. 1987 Oct;157(4 Pt 1 ):880-884. I. ):J0 downstream impedance (Chap. 10, p. 213). For growth 7.30 7.25 0E:J E:J= 7.20 7.10 7.05 FIGURE 17-9 Mean umbilical vein pH (±2 SD) obtained by cordocentesis in relation to fetal biophysical profile score category. (Data from Manning, 1993.) API determination for which �5 cm was considered abnormal (Chap. 11, p. 230). his abbreviated biophysical proile required approximately 10 minutes to perform, and they concluded that it was a superb antepartum surveillance method because there were no unexpected fetal deaths.

1	Nageotte and colleagues (1994) also combined biweely nons tress tests with the AFI and considered measures �5 cm to be abnormal. They performed 17,429 modiied biophysical proiles in 2774 women and concluded that such testing was an excellent fetal surveillance tool. Miller and associates (1996a) reported results with more than 54,000 modiied biophysical profiles performed in 15,400 high-risk pregnancies. They described a false-negative rate of 0.8 per 1000 and a falsepositive rate of 1.5 percent. The American College of Obstetricians and Gynecologists (2016) has concluded that the modiied biophysical proile test is as predictive of fetal well-being as other approaches to biophysical fetal surveillance.

1	he importance of amnionic fluid volume estimation is indicated by its inclusion into virtually all schemes in which fetal health is assessed (Ff0en, 2008). This is based on the rationale that diminished utero placental perfusion may lead to lower fetal renal blood low, decreased urine production, and ultimately, oligohydramnios (Chap. 11, p. 231). The American College of Obstetricians and Gynecologists (2016) concludes that data available from randomized trials indicate that the use of the deepest vertical pocket measurement, as opposed to the AFI, to diagnose oligohydramnios is associated with a reduction in unnecessary interventions without an increase in adverse perinatal outcomes (Nabhan, 2008; Reddy, 2014).

1	restricted fetuses, several fetal vascular circuits including the umbilical artery, middle cerebral artery, and ductus venosus have been evaluated as diagnostic tools for fetal well-being (Chap. 44, p. 854). Maternal uterine artery Doppler velocimetry has also been assessed as a modality to predict placental dysfunction, with the goal to balance stillbirth against the risks of preterm delivery (Ghidini, 2007). Even the efects of sildenail in pregnant sheep have been evaluated using Doppler velocimetry (Alanne, 2017). he rationale is that sildenafil would improve placental blood low in the presence of placental insuiciency. This proved untrue, as sildenail was associated with detrimental efects on fetal cardiovascular dynamics.

1	Waveforms were irst studied in the umbilical arteries late in pregnancy, and abnormal waveforms correlated with placental villous hypovascularity. Of the small placental arterial channels, 60 to 70 percent need to be obliterated before the umbilical artery Doppler waveform becomes abnormal. Such extensive placental vascular pathology has a major efect on fetal circulation. According to Trudinger (2007), because more than 40 percent of the combined fetal ventricular output is directed to the placenta, obliteration of placental vascular channel increases aterload and leads to fetal hypoxemia. This in turn leads to ventricular dilation and redistribution of middle cerebral artery blood low. Ultimately, pressure rises in the ductus venosus due to afterload in the right side of the fetal heart (Baschat, 2004). Clinically, abnormal Doppler waveforms in the ductus venosus are a late inding in the progression of fetal deterioration due to chronic hypoxemia.

1	he umbilical artery systolic-diastolic (SID) ratio is considered abnormal if it is >95th percentile for gestational age or if diastolic low is either absent or reversed (Chap. 10, p. 213). Absent or reversed end-diastolic low signiies greater impedance to umbilical artery blood low (Fig. 44-8, p. 854). It is reported to result from poorly vascularized placental villi and is seen in extreme cases of fetal-growth restriction (T odros, 1999). According to Zelop and colleagues (1996), the perinatal mortali ty rate for absent end-diastolic flow was about 10 percent, and for reversed end-diastolic low, it approximated 33 percent. Spinillo and associates (2005) studied neurodevelopmental outcome at 2 years of age in 266 growth-restricted fetuses delivered between 24 and 35 weeks' gestation. Of infants who had shown absent or reversed umbilical artery low, 8 percent had evidence of cerebral palsy compared with 1 percent of those in whom Doppler low had been normal.

1	Doppler ultrasound of the umbilical artery has been subjected to more extensive assessment with randomized controlled trials than has any previous test of fetal health. Williams and colleagues (2003) randomized 1360 high-risk women to either nons tress testing or Doppler velocimetry. hey found a significantly higher incidence of cesarean delivery for fetal distress in the nonstress test group compared with that for those tested with Doppler velocimetry-8.7 versus 4.6 percent, respectively. One interpretation of this finding is that the nonstress test more frequently identiied fetuses in jeopardy. Conversely, Gonzalez and associates (2007) found that abnormal umbilical artery Doppler indings in a cohort of growth-restricted fetuses were the best predictors of perinatal outcomes.

1	he utility of umbilical artery Doppler velocimetry was reviewed by the American College of Obstetricians and Gynecologists (2016). They concluded that no beneit has been demonstrated other than in pregnancies with suspected fetal-growth restriction. Similarly, velocimetry has not proved valuable as a screening test for fetal compromise in the general obstetrical population. Various other fetal-maternal Doppler indices have been studied, including the fetal middle cerebral artery and ductus venosus and the uterine arteries. The American College of Obstetricians and Gynecologists (2016) concluded that Doppler investigations of other blood vessels besides the umbilical artery have not been shown to improve perinatal outcome.

1	As discussed, at this time, Doppler velocimetry interrogation of the middle cerebral artery (MCA) to detect fetal compromise is not recommended. Still, the technology has received particular attention because of observations that the hypoxic fetus attempts brain sparing by reducing cerebrovascular impedance and thus increasing blood flow. Such brain sparing in growthrestricted fetuses has been documented to undergo reversal (Konje, 2001). Investigators reported that 8 of 17 fetuses with this reversal died. Ott and coworkers (1998) randomized 665 women undergoing modified biophysical proile evaluation to either the profile alone or combined with middle cerebral and umbilical artery velocity low assessment. Pregnancy outcomes between these two study groups did not difer significantly.

1	Middle cerebral artery Doppler velocimetry has proven valuable to detect severe fetal anemia in 165 fetuses with D-antigen alloimmunization. Oepkes and colleagues (2006) prospectively compared serial amniocentesis for measurement of bilirubin levels with Doppler measurement of peak systolic velocity in the middle cerebral artery. These investigators concluded that Doppler could safely replace amniocentesis in the management of alloimmunized pregnancies. And as discussed in Chapter 15 (p. 303), this technique has been reported to be useul for detection and management of fetal anemia of any cause (Moise, 2008).

1	Doppler ultrasound has also been used to assess the fetal venous circulation. Bilardo and colleagues (2004) prospectively studied umbilical artery and ductus venosus Doppler velocimetry in 70 growth-restricted fetuses at 26 to 33 weeks' gestation. hey concluded that ductus venosus velocimetry was the best predictor of perinatal outcome. Importantly, negative or reversed low in the ductus venosus was a late finding because these fetuses had already sustained irreversible multiorgan damage due to hypoxemia. Also, gestational age at delivery was a major determinant of perinatal outcome independent of ductus venosus flow. Specifically, 36 percent of growth-restricted fetuses delivered between 26 and 29 weeks' gestation succumbed compared with only 5 percent delivered from 30 to 33 weeks.

1	Baschat and coworkers (2007) studied 604 growth-restricted fetuses using umbilical artery, middle cerebral artery, and ductus venosus Doppler velocimetry and reached similar conclusions. Speciically, absent or reversed flow in the ductus venosus was associated with profound generalized fetal metabolic collapse. hey too reported that gestational age was a powerful cofactor in ultimate perinatal outcome for growth-restricted fetuses delivered before 30 weeks. Put another way, by the time severely abnormal low is seen in the ductus venosus, it is too late because the fetus is already near death. Conversely, earlier delivery puts the fetus at risk for death due to preterm delivery. Ghidini (2007) concluded that these reports do not support routine use of ductus venosus Doppler in the monitoring of growth-restricted fetuses and recommended further study.

1	Vascular resistance in the uterine circulation normally decreases in the irst half of pregnancy due to invasion of maternal uterine vessels by trophoblastic tissue (Chap. 5, p. 92). This process can be detected using Doppler flow velocimetry, and uterine artery Doppler may be most helpful in assessing pregnancies at high risk of utero placental insuiciency (Abramowicz, 2008). Persistence or development of high-resistance patterns has been linked to various pregnancy complications (Lees, 2001; Yu, 2005). In a study of 30,519 unselected British women, Smith and colleagues (2007) assessed uterine artery velocimetry at 22 to 24 weeks' gestation. The risk of fetal death before 32 weeks, when associated with abruption, preeclampsia, or fetal-growth restriction, was significantly linked to high-resistance flow. This has led to suggestions for continued research of uterine artery Doppler velocimetry as a screening tool to detect pregnancies at risk for stillbirth (Reddy, 2008). Sciscione and

1	flow. This has led to suggestions for continued research of uterine artery Doppler velocimetry as a screening tool to detect pregnancies at risk for stillbirth (Reddy, 2008). Sciscione and Hayes (2009) reviewed the use of uterine artery Doppler flow studies in obstetrical practice. Because standards for the study technique and criteria for an abnormal test are lacking, they noted that uterine artery Doppler studies should not be considered standard practice in either low-or high-risk populations.

1	Antenatal forecasts of fetal health have clearly been the focus of intense interest, and several themes emerge. First, despite a continuous evolution of testing options, the precision or eicacy of any given method is limited. Second, the wide range of normal biological fetal variation makes interpretation of test results challenging. Last, despite the invention of increasingly complex testing methods, abnormal results are seldom reliable, prompting many clinicians to use antenatal testing to forecast fetal welness rather than ilness.

1	Platt and coworkers (1987) reviewed the eicacy of antenatal testing between 1971 and 1985 at Los Angeles County Hospital. During this 15-year period, more than 200,000 pregnancies were managed, and nearly 17,000 of these women underwent antepartum testing of various types. Fetal surveillance rose from < 1 percent of pregnancies in the early 1970s to 15 percent in the mid-1980s. hese authors concluded that such testing was clearly beneicial because the fetal death rate was signiicantly less in the tested high-risk pregnancies compared with the rate in those not tested. he study, however, did not consider other innovations incorporated into practice during those 15 years. Preliminary results from Ghana suggest that nonstress testing may be beneicial in low-resource countries (Lawrence, 2016). In an observational study of 316 pregnancies complicated by gestational hypertension, women undergoing nons tress testing had a nonsignificant decreased risk for stillbirth compared with those not

1	In an observational study of 316 pregnancies complicated by gestational hypertension, women undergoing nons tress testing had a nonsignificant decreased risk for stillbirth compared with those not tested-3.6 versus 9.2 percent, respectively.

1	The beneits of antenatal fetal testing have not been suficiently evaluated in randomized controlled trials according to hacker and Berkelman (1986). This was concluded after reviewing 600 reports, which included only four randomized trials that were not powered to permit detection of important beneits. From their review, Enkin and colleagues (2000) concluded that "despite their widespread use, most tests of fetal well-being should be considered of experimental value only rather than validated clinical tools." nother important and unanswered question is whether antepartum fetal surveillance identiies fetal asphyxia early enough to prevent brain damage. Manning and coworkers (1998) studied the incidence of cerebral palsy in 26,290 high-risk pregnancies managed with serial biophysical proile testing. These outcomes were compared with those of 58,657 low-risk pregnancies in which antepartum testing was not performed. The rate of cerebral palsy was 1.3 per 1000 in tested pregnancies

1	proile testing. These outcomes were compared with those of 58,657 low-risk pregnancies in which antepartum testing was not performed. The rate of cerebral palsy was 1.3 per 1000 in tested pregnancies compared with 4.7 per 1000 in untested women. Todd and coworkers (1992) attempted to correlate cognitive development in infants up to age 2 years following either abnormal umbilical artery Doppler velocimetry or nons tress test results. Only abnormal nonstress tests were associated with marginally poorer cognitive outcomes. These investigators concluded that by the time fetal compromise is diagnosed with antenatal testing, fetal damage has already been sustained. Low and associates (2003) reached a similar conclusion.

1	According to the American College of Obstetricians and Gynecologists (2016), a normal antepartum fetal test result is , highly reassuring that a stillbirth will not occur within 1 week. This conclusion was reached after an analysis of reports of stillbirth rates associated with the various antepartum fetal heart rate tests (Table 17-4). Note that these results are corrected to remove lethal anomalies and unpredictable catastrophes such TABLE 17-4. Stillbirth Rates within 1 Week of a Normal Antepartum Fetal Surveillance Test Nonstress test 1s.9 5861 Contraction stress test OJ 12,656 Biophysical profile 0.8 44,828 Modified biophysical profile 0.8 54,61s7 aCorrected fot lethal anomalies and unpredictable causes of fetal death such as abruption or cord accident. as placental abruption or cord accidents. The most important consideration in deciding when to begin antepartum testing is the prognosis for neonatal survival.

1	as placental abruption or cord accidents. The most important consideration in deciding when to begin antepartum testing is the prognosis for neonatal survival. The severity of maternal disease is another. In general, with most high-risk pregnancies, testing begins by 32 to 34 weeks' gestation. P�egnancies with severe complications might require testing as early as 26 to 28 weeks. he frequency for repeating tests has been arbitrarily set at 7 days, but more frequent testing is often done. Abramowicz JS, Sheiner E: Ultrasound of the placenta: a systemic approach. Part II: function assessment (Doppler). Placenta 29(11):921,o2008 Alanne L, Hofren J, Haapsamo M, et al: Efect of sildenail citrate on fetal central hemodynamics and placental volume blood low during hypoxemia in a chronic sheep model. Abstract No. 25. Presented at the 37th Annual Meeting of the Society for Maternal-Fetal Medicine. January 23-28, 2017

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1	Grubb OK, Paul RH: Amnionic luid index and prolonged antepartum fetal heart rate decelerations. Obstet Gynecol 79:558, 1992 Guinn DA, Kimberlin KF, Wigton TR, et al: Fetal heart rate characteristics at 25 to 28 weeks gestation. Am J Perinatol 15:507, 1998 Hage ML: Interpretation of nonstress tests. Am J Obstet Gynecol 153:490, 1985 Hammacher K, Huter A, Bokelmann J, et al: Foetal heart frequency and perinatal condition of the foetus and newborn. Gynaecologia 166:349, 1968 Harrington K, Thompson 0, Jorden L, et al: Obstetric outcomes in women who present with a reduction in fetal movements in the third trimester of pregnancy. J Perinat Med 26:77, 1998 Ho 0, Wang J, Homann Y, et a1: Use of the myocardial performance index in decreased fetal movement assessment: a case-control study. Fetal Diagn Ther June 15, 2017 [Epub ahead of print]

1	Ho 0, Wang J, Homann Y, et a1: Use of the myocardial performance index in decreased fetal movement assessment: a case-control study. Fetal Diagn Ther June 15, 2017 [Epub ahead of print] Hoskins IA, Frieden FJ, Young BK: Variable decelerations in reactive nonstress tests with decreased amnionic luid index predict fetal compromise. Am J Obstet Gynecol 165: 1094, 1991 Huddleston JF, Surlif JG, Robinson 0: Contraction stress test by intermittent nipple stimulation. Obstet Gynecol 63:669, 1984 Jansson LM, DiPietro J, Elko A: Fetal response to matenal methadone administration. Am J Obstet Gynecol 193:611,2005 Johnson MJ, Paine LL, Mulder HH, et al: Population diferences of fetal biophysical and behavioral characteristics. Am J Obstet Gynecol 166: 138, 1992 Kaur S, Picconi JL, Chadha R, et al: Biophysical proile in the treatment of intrauterine growth-restricted fetuses who weigh < 1000 g. Am J Obstet Gynecol 199:264.el, 2008

1	Kaur S, Picconi JL, Chadha R, et al: Biophysical proile in the treatment of intrauterine growth-restricted fetuses who weigh < 1000 g. Am J Obstet Gynecol 199:264.el, 2008 Konje JC, Bell SC, Taylor DT: Abnormal Doppler velocimetry and blood low volume in the middle cerebral artery in very severe intrauterine growth restriction: is the occurrence of reversal of compensatory low too late? BJOG 108:9 3,r2001 Kopecky EA, Ryan ML, Barrett JFR, et a1: Fetal response to maternally administered morphine. Am J Obstet Gynecol 183:424,r2000 Lalor JG, Fawole B, Alirevic Z, et al: Biophysical proile for fetal assessment in high risk pregnancies. Cochrane Database Syst Rev I :CD000038, 2008 Laventhal NT, Dildy GA III, Belfort MA: Fetal tachyarrhythmia associated with vibroacoustic stimulation. Obstet Gynecol 101: 116, 2003

1	Lawrence ER, Quarshie EL, Lewis KF, et al: Introduction of cardiotocograph monitoring improves birth outcomes in women with preeclampsia in Ghana. IntJ Gynaecol Obstet 132(1):103,r2016 Lee CY, DiLoreto Pc, O'Lane JM: A study of fetal heart rate acceleration patterns. Obstet GynecoIr45:142, Lees C, Parra M, Missfelder-Lobos H, et al: Individualized risk assessment for adverse pregnancy outcome by uterine artery Doppler at 23 weeks. Obstet Gynecol 98:369, 2001 Leveno KJ, Williams ML, DePalma RT, et al: Perinatal outcome in the absence of antepartum fetal heart rate acceleration. Obstet Gynecol 61 :347, 1983 Low JA, Killen H, Derrick EJ: Antepartum fetal asphyxia in the preterm preg nancy. Am J Obstet Gynecol 188:461, 2003 Macones GA, Hankins GO, Spong CY, et al: The 2008 National Institute of Child Health and Human Development workshop report on electronic fetal monitoring: update on deinitions, interpretation, and research guidelines. Obstet Gynecol 112:661, 2008

1	Manning FA, Bondagji N, Harman CR, et 1: Fetal assessment based on fetal biophysical proile scoring VIII: the incidence of cerebral palsy in tested and untested perinates. Am J Obstet Gynecol 178:696, 1998 Manning FA, Morrison 1, Harman CR, et a1: Fetal assessment based on fetal biophysical proile scoring: experience in 19,221 referred high-risk pregnancies, 2. n analysis of false-negative fetal deaths. Am J Obstet Gynecol 15 :880, 1987 Manning FA, Platt LD, Sipos L: Antepartum fetal evaluation: development of a fetal biophysical proile. Am J Obstet Gynecol , 1980 Manning FA, Snijders R, Harman CR, et al: Fetal biophysical proile score, VI. Correlation with antepartum umbilical venous fetal pH. Am J Obstet GynecoIr169:755, 1993 Matsuura M, Murata Y, Hirano T, et al: he efects of developing autonomous nervous system on FHR variabilities determined by the power spectral analysis. Am J Obstet Gynecol 174:380, 1996

1	Matsuura M, Murata Y, Hirano T, et al: he efects of developing autonomous nervous system on FHR variabilities determined by the power spectral analysis. Am J Obstet Gynecol 174:380, 1996 Meis PJ, Ureda JR, Swain M, et a1: Variable decelerations during nonstress tests are not a sign of fetal compromise. Am J Obstet Gynecol 154:586, 1986 Miller DA, Rabello YA, Paul RH: The modiied biophysical proile: antepar tum testing in the 1990s. Am J Obstet GynecoIr174:812, 1996a Miller F, Miller 0, Paul R, et al: Is one fetal heart rate acceleration during a nons tress test as reliable as two in predicting fetal status? Am J Obstet 4:337, 1996b Moise KJ Jr: he usefulness of middle cerebral artery Doppler assessment in the treatment of the fetus at risk for anemia. Am J Obstet Gynecol 198: 161. e1,r2008 Moore TR, Piaquadio K: A prospective evaluation of fetal movement screening to reduce the incidence of antepartum fetal death. Am J Obstet Gynecol 160: 1075, 1989

1	Moore TR, Piaquadio K: A prospective evaluation of fetal movement screening to reduce the incidence of antepartum fetal death. Am J Obstet Gynecol 160: 1075, 1989 Nabhan AF, Abdelmoula YA: Amniotic luid index versus single deepest vertical pocket as a screening test for preventing adverse pregnancy outcome. Cochran Database Syst Rev 3:CD006593, 2008

1	Nageotte MP, Towers CV, Asrat T, et al: Perinatal outcome with the modiied biophysical proile. Am J Obstet Gynecol 170: 2, 1994 Nijhuis JG, Prechtl HF, Martin CB J r, et al: Are there behavioural states in the human fetus? Early Hum Dev 6: , 1982 Oepkes 0, Seaward PG, andenbussche FP, et al: Doppler ultrasonography versus amniocentesis to predict fetal anemia. N Engl J Med 355: 156, 2006 Oosterhof H, vd Stege JG, Lander M, et al: Urine production rate is related to behavioural states in the near term human fetus. BJOG 100:920, 1993 Ott ('], Mora G, Arias F, et al: Comparison of the modiied biophysical proile to a "new" biophysical proile incorporating the middle cerebral artery to umbilical artery velocity low systolic/diastolic ratio. Am J Obstet Gynecol 178:1346,r1998 Ozkaya E, Baser E, Cinar M, et al: Does diurnal rhythm have an impact on fetal biophysical profile? J Maten Fetal Neonatal Med 25(4):335, 2012 Patrick J, Campbell K, Carmichael L, et al: Patterns of gross fetal body

1	E, Cinar M, et al: Does diurnal rhythm have an impact on fetal biophysical profile? J Maten Fetal Neonatal Med 25(4):335, 2012 Patrick J, Campbell K, Carmichael L, et al: Patterns of gross fetal body movements over 24-hour observation intervals during the last 10 weeks of pregnancy. Am J Obstet Gynecol 142:363, 1982 Patrick J, Campbell K, Carmichael L, et al: Patterns of human fetal breathing during the last 10 weeks of pregnancy. Obstet Gynecol 56:24, 1980 Pazos R, Vuolo K, Aladjem S, et al: Association of spontaneous fetal heart rate decelerations during antepartum nonstress testing and intrauterine growth retardation. Am J Obstet Gynecol 144:574, 1982 Perez-Oelboy A, Weiss J, Michels A, et al: A randomized trial of vibroacoustic stimulation for antenatal fetal testing. Am J Obstet Gynecol 187:S 146, 2002 Pillai M, James 0: Behavioural states in normal mature human fetuses. Arch Dis Child 65:39, 1990a Pillai M, James 0: The development of fetal heart rate patterns during normal

1	Gynecol 187:S 146, 2002 Pillai M, James 0: Behavioural states in normal mature human fetuses. Arch Dis Child 65:39, 1990a Pillai M, James 0: The development of fetal heart rate patterns during normal pregnancy. Obstet Gynecol 6:812, 1990b Platt LD, Paul RH, Phelan J, et al: Fifteen years of experience with antepartum fetal testing. Am J Obstet Gynecol 156:1509, 198 Ray M, Freeman R, Pine S, et al: Clinical experience with the oxytocin challenge test. Am J Obstet Gynecol 1r14: 1, 2 Raybun WF: Clinical signiicance of perceptible fetal motion. Am J Obstet Gynecol 138:210, 1980 Reddy UM, Abuhamad Z, Levine 0, et al: Fetal imaging: executive summary of a joint Eunice Kennedy Shriver National Institute of Child Health and Human Development, Sociery for Maternal-Fetal Medicine, American Institute of Ultrasound in Medicine, American College of Obstetricians and Gynecologists, American College of Radiology, Society for Pediatric Radiology, and Society of Radiologists in Ultrasound Fetal

1	of Ultrasound in Medicine, American College of Obstetricians and Gynecologists, American College of Radiology, Society for Pediatric Radiology, and Society of Radiologists in Ultrasound Fetal Imaging Workshop. Obstet Gynecol 123(5):10 0,r2014 Reddy UM, Filly A, Copel JA, et al: Prenatal imaging: ultrasonography and magnetic resonance imaging. Obstet Gynecol 112(1):145,r2008 Saastad E, Winje BA, Stray Penderson B, et al: Fetal movement counting improved identiication of fetal growth restriction and perinatal outcomesa multi-centre, randomized, controlled trial. PLoS One 6(12):e28482, 2011 Sadovsky E, Evron S, Weinstein 0: Daily fetal movement recording in normal pregnancy. Riv Obstet Ginecol Practica Med Perinatal 59:395, J 9a Sadovsky E, Laufer N, Allen JW: he incidence of diferent types of fetal movement during pregnancy. BJOG 86:10, 1979b Sadovsky E, Yafe H: Daily fetal movement recording and fetal prognosis. Obstet Gynecol 41:845,r1973

1	Sajapala S, AboElIail MA, Kanenshi K, et a1: 40 ultrasound study of fetal movement early in the second trimester of pregnancy. J Perinat Med 45(6):737,r2017 Salvesen DR, Freeman J, Brudenell JM, et al: Prediction of fetal acidemia in pregnancies complicated by maternal diabetes by biophysical scoring and fetal heart rate monitoring. BJOG , 1993 Scala C, Bhide A, Familiari A, et al: Number of episodes of reduced fetal movement at term: association with adverse perinatal outcome. Am J Obstet Gynecol 213(5):678.el, 2015 Schell pfefer MA, Hoyle 0, Johnson JWC: Antepartum uterine hypercontractiliry secondary to nipple stimulation. Obstet Gynecol 65:588, 1985 Sciscione AC, Hayes EJ: Uterine artery Doppler low studies in obstetric practice. Am J Obstet Gynecol 20r1(2):121, 2009 Sherer DM, Spong CY, Ghidini A, et al: In preterm fetuses decreased amniotic luid volume is associated with decreased fetal movements. Am J Obstet Gynecol 174:344, 1996

1	Sherer DM, Spong CY, Ghidini A, et al: In preterm fetuses decreased amniotic luid volume is associated with decreased fetal movements. Am J Obstet Gynecol 174:344, 1996 Smith CV, Nguyen HN, Kovacs B, et al: Fetal death following antepartum fetal heart rate testing: a review of 65 cases. Obstet Gynecol 70: 18, 1987 Smith Gc, Yu CK, Papageorghiou AT, et al: Maternal uterine artery Doppler low velocimetry and the risk of stillbirth. Obstet Gynecol 109: 144, 2007 Smith JH, Anand KJ, Cotes PM, et al: Antenatal fetal heart rate variation in relation to the respiratory and metabolic status of the compromised human fetus. BJOG 95:980, 1988 Sorokin Y, Bottoms SF, Dierker CJ, et al: he clustering of fetal heart rate changes and fetal movements in pregnancies berween 20 and 30 weeks gestation. Am J Obstet Gynecol 143:952, 1982

1	Sorokin Y, Bottoms SF, Dierker CJ, et al: he clustering of fetal heart rate changes and fetal movements in pregnancies berween 20 and 30 weeks gestation. Am J Obstet Gynecol 143:952, 1982 Spinillo A, Montanari L, Bergante C, et al: Prognostic value of umbilical artery Doppler studies in unselected preterm deliveries. Obstet Gynecol 105:613, 2005 hacker SB, Berkelman RL: Assessing the diagnostic accuracy and eicacy of selected antepartum fetal surveillance techniques. Obstet Gynecol Surv 41:121,r1986 Timor-Tritsch IE, Dierker LJ, Hertz RH, et al: Studies of antepartum behavioral state in the human fetus at term. Am J Obstet Gynecol 132:524, 1978 Todd AL, Tridinger BJ, Cole MJ, et al: Antenatal tests of fetal welfare and development at age 2 years. Am J Obstet Gynecol 167:66, 1992 Todros T, Sciarrone A, Piccoli E, et al: Umbilical Doppler waveforms and placental villous angiogenesis in pregnancies complicated by fetal growth restriction. Obstet Gynecol 93:499, 1999

1	Todros T, Sciarrone A, Piccoli E, et al: Umbilical Doppler waveforms and placental villous angiogenesis in pregnancies complicated by fetal growth restriction. Obstet Gynecol 93:499, 1999 Trudinger B: Doppler: more or less? Ultrasound Obstet Gynecol29 (3):243, 2007 Turitz AL, Bastek JA, Sam mel MD, et al: Can vibroacoustic stimulation improve the eiciency of a tertiary care antenatal testing unit? J Matern Fetal Neonatal Med 25(12):2645,r2012 VindJa S, James 0: Fetal behavior s a test oHetl well-being. BJOG 102:597, 1995 Visser GHA, Redman CWG, Huisjes HJ, et al: Nonstressed antepartum heart rate monitoring: implications of decelerations after spontaneous contractions. Am J Obstet Gynecol 138:429, 1980 Warrander LK, Batra G, Bernatavicius G, et al: Maternal perception of reduced fetal movements is associated with altered placental structure and function. PLoS One 7(4):e34851, 2012

1	Warrander LK, Batra G, Bernatavicius G, et al: Maternal perception of reduced fetal movements is associated with altered placental structure and function. PLoS One 7(4):e34851, 2012 Weiner Z, Divon MY, Katz N, et al: Multi-variant analysis of antepartum fetal test in predicting neonatal outcome of growth retarded fetuses. Am J Obstet Gynecol 174:338, 1996 Williams KP, Farquharson OF, Bebbington M, et al: Screening for fetal wellbeing in a high-risk pregnant population comparing the nonstress test with umbilical artery Doppler velocimetry: a randomized controlled clinical trial. Am J Obstet Gynecol 188: 1366, 2003 Yu CK, Smith GC, Papageorghiou AT, et al: An integrated model for the prediction of preeclampsia Llsing maternal factors and uterine artery Doppler velocimetry in unselected low-risk women. Am J Obstet Gynecol 193:429,r2005

1	Zelop CM, Richardson OK, Hefner LJ: Outcomes of severely abnormal umbilical artery Doppler velocimetry in structurally normal singleton fetuses. Obstet Gynecol 8 :434, 1996 NOMENCLATUREe. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 FIRST-TRIMESTER SPONTANEOUS ABORTION .e.e.e347 SPONTANEOUS ABORTION CLINICAL CLASSIFICATION .e348 RECURRENT MISCARRIAGE ..e......... ....e.. ..e352 MIDTRIMESTER ABORTIONe. . . . . . . . . . . . . . . . . . . . . . . 353 CERVICAL INSUFFICIENCY ..e..e..... . .... ....... 354 INDUCED ABORTION .e.. ....e.e...e..e...e...e...e. 357 FIRST-TRIMESTER ABORTION METHODS ..........e. 358 SECOND-TRIMESTER ABORTION METHODS .. .... . 362

1	INDUCED ABORTION .e.. ....e.e...e..e...e...e...e. 357 FIRST-TRIMESTER ABORTION METHODS ..........e. 358 SECOND-TRIMESTER ABORTION METHODS .. .... . 362 In the eary months of pregnancy spontaneous expulsion of the ovum is neary always preceded by the death of the oetus. For this reason the consideration of the aetioloy of abortion practicaly resolves itsef into determining the cause of oetal death. In the later months, on the other han, the oetus is frequenty born alive, and other actors must be looked or to explain its expulsion. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) In early pregnancy, miscarriage is a common event. Most early losses stem from genetic abnormalities or yet unidentified reasons. Thus, the opportunity for prevention is currently small. Women with later miscarriage or with recurrent miscarriage more likely have a repetitive cause that may be modiied. In contrast to these spontaneous losses, pregnancy termination may be elected. For both induced abortion and miscarriage, management has evolved to include surgical or medical options, and providers should have an understanding of these techniques and their potential complications.

1	Abortion is defined as the spontaneous or induced termination of pregnancy before fetal viability. It thus is appropriate that miscarriage and abortion are terms used interchangeably. However, popular use of abortion by laypersons implies intended pregnancy termination, and many prefer miscarriage for spontaneous loss. In contrast, induced abortion describes surgical or medical termination of a live fetus that has not reached viability.

1	Terms used to define fetal viability and thus an abortus vary among authoritative organizations. The National Center for Health Statistics, the Centers for Disease Control and Prevention, and the World Health Organization all deine abortion as pregnancy termination or loss before 20 weeks' gestation or with a fetus delivered weighing <500 g. hese criteria, however, are somewhat contradictory because the mean birthweight of a 20-week fetus is 320 g, whereas 500 g is the mean for 22 to 23 weeks (Moore, 1977). Further confusion may derive from criteria set by state laws that deine abortion even more widely.

1	Technological developments have added to current abortion terminology. For example, precise measurement of serum human chorionic gonadotropin (hCG) concentrations can identiY extremely early pregnancies. Also, transvaginal sonography allows greater inspection of failed pregnancies, but recommendations vary as to terms for: (1) early conceptions in which no products are seen sonographically, (2) pregnancies that display a gestational sac but no embryo, and (3) those in which a dead embryo is seen (Kolte, 2015; Silver, 2011). Further, incongruity exists for the term eary pregnancy loss itsel. Currently, the American College of Obstetricians and Gynecologists (2017 c) deines this as a nonviable, intrauterine pregnancy (IUP) with either an empty gestational sac or a ges activity within the irst 126' weeks of gestation. Of other clini cal terms, spontaneous abortion includes threatened, inevitable, incomplete, complete, and missed abortion. Septic abortion is used to further classiy any of

1	126' weeks of gestation. Of other clini cal terms, spontaneous abortion includes threatened, inevitable, incomplete, complete, and missed abortion. Septic abortion is used to further classiy any of these that are complicated further by infection. Recurrent pregnancy loss is variably deined but is meant to identiy women with repetitive miscarriage.

1	pic gestations. he term pregnancy of unknown location (PUL) confirmed sonographic location. In this context, ive categories are proposed for early pregnancies: definite ectopic pregnancy, probable ectopic, PUL, probable IUP, and definite IUP (Barn hart, 2011). Diagnostic and management options for ectopic gestation are described in Chapter 19 (p. 373).

1	More than 80 percent of spontaneous abortions occur within the irst 12 weeks of gestation. With irst-trimester losses, demise of the embryo or fetus nearly always precedes spontaneous expulsion. Death is usually accompanied by hemorrhage into the decidua basalis. This is followed by adjacent tissue necrosis that stimulates uterine contractions and expulsion. An intact gestational sac is usually filled with luid. An anembryonic miscarriage contains no identifiable embryonic elements. Less accurately, the term blighted ovum may be used (Silver, 201l). The others are embyonic miscarriages, which often display a developmental abnormality of the embryo, fetus, yolk sac, and, at times, the placenta. In contrast, in later pregnancy losses, the fetus usually does not die before expulsion, and other sources for abortion are sought.

1	Rates for miscarriage vary according to the study population. In pregnancies aged 5 to 20 weeks' gestation, the incidence ranges from 11 to 22 percent and is higher in earlier weeks (Ammon Avalos, 2012). To evaluate rates starting at conception, Wilcox and colleagues (1988) studied 221 healthy women trying to conceive through 707 menstrual cycles and found a miscarriage rate of 31 percent. This study found that two thirds of these losses are early and clinicaly silent. Currently, certain factors are known to inluence clinicaly apparent miscarriage. However, it is unknown if these same factors also afect clinically silent pregnancy loss.

1	Of all miscarriages, approximately half are euploid abortions, that is, carrying a normal chromosomal complement. The other half has a chromosomal abnormality. Initially determined by tissue karyotyping, this percentage appears to persist even when implementing newer cytogenetic techniques Qenderny, 2014). Notably, the American College of Obstetricians and Gynecologists (20 16d) does not recommend routine use of chromosomal microarray testing of irst-trimester fetal tissues. However, these organizations, and the American Society for Reproduc tive Medicine (2012), recognize its value if cytogenetic analysis alters future care. Both abortion and chromosomal anomaly rates decline with advancing gestational age (Ammon Avalos, 2012; Eiben, 1990).

1	Both abortion and chromosomal anomaly rates decline with advancing gestational age (Ammon Avalos, 2012; Eiben, 1990). Kajii and associates (1980) noted that 75 percent of chromo somally abnormal abortions occurred by 8 weeks' gestation. Of chromosomal abnormalities, 95 percent are caused by maternal gametogenesis errors, and 5 percent by paternal errors Qacobs, 1980). Most common abnormalities are trisomy, found in 50 to 60 percent; monosomy X, in 9 to 13 percent; and triploidy, in 11 to 12 percent (Eiben, 1980; Jenderny, 2014). Trisomies typically result from isolated nondisjunction, rates of which rise with maternal age (Bow§, 1975). Trisomies of chromosomes 13, 16, 18, 21, and 22 are most common. In contrast, balanced structural chromosomal rearrangements may originate from either parent and are found in 2 to 4 percent of couples with recurrent pregnancy loss.

1	Monosomy X (45,X) is the single most frequent specific chro mosomal abnormality. his is Turner syndrome, which usually results in abortion, but liveborn females are described in Chap ter 13 (p. 259). Conversely, autosomal monosomy is rare and incompatible with life. Triploidy is often associated with hydropic or molar placental degeneration (Chap. 20, p. 389). The fetus within a partial hydatidiform mole frequently aborts early, and the few carried longer are all grossly deformed. Advanced maternal and paternal ages do not increase the incidence of triploidy. Tetraploid fetuses most often abort early in gestation, and they are rarely liveborn. In chromosomally normal pregnancy losses, maternal influences play a role. The causes of euploid abortions are poorly understood, but various medical disorders, environmental conditions, and developmental abnormalities have been implicated.

1	Euploid pregnancies abort later than aneuploid ones. Specifically, the rate of euploid abortion peaks at approximately 13 weeks (Kajii, 1980). In addition, the incidence of euploid abortion rises dramatically after maternal age exceeds 35 years (Stein, 1980). Some common viruses, bacteria, and parasites that invade the normal human can infect the fetoplacental unit by blood-borne transmission. Others may infect locally through genitourinary infection or colonization. However, despite the numerous infections acquired in pregnancy and discussed in Chapters 64 and 65, these uncommonly cause early abortion.

1	Some disorders are possibly linked with higher rates of early pregnancy loss and are discussed in their respective chapters. Prominent risks are associated with poorly controlled diabetes mellitus, obesity, thyroid disease, and systemic lupus erythematosus. In these and others, inflammatory mediators may be an underlying theme (Kalagiri, 2016; Sjaarda, 2017). Although thrombophilias were initially linked to various pregnancy outcomes, most putative associations have been refuted (American College of Obstetricians and Gynecologists (2017e).

1	herapeutic doses of radiation are undeniably abortifacient. Doses that cause abortion are not precisely known, but suggested parameters are found in Chapter 46 (p. 906). Similarly, the efects of chemotherapy exposure in causing abortion are not well deined (Chap. 12, p. 242). Particularly worrisome are women with an ongoing pregnancy after early exposure to methotrexate, described later (p. 361). Of cancer survivors, those who were treated with abdominopelvic radiotherapy or chemotherapy may later be at greater risk for miscarriage, as discussed in Chapter 63 (p. 1192).

1	The risk of miscarriage caused by surgery is not well studied. But, as discussed in Chapter 46 (p. 901), uncomplicated surgical procedures performed during early pregnancy are unlikely to increase the abortion risk (Mazze, 1989). Of indications, ovarian tumors can generally be resected without inciting miscarriage. An important exception involves early removal of the corpus luteum or the ovary in which it resides. If performed before 10 weeks' gestation, supplemental progesterone should be given, and supplementation is enumerated in Chapter 63 (p.i1198). Trauma seldom causes first-trimester miscarriage, and although Parkland Hospital is a busy trauma center, this is an infrequent association. Major trauma-especially abdominalcan cause fetal loss, but is more likely as pregnancy advances (Chap. 47, p. 925).

1	Sole deficiency of one nutrient or moderate deiciency of all does not appear to increase risks for abortion. Even in extreme cases-for example, hyperemesis gravidarum-abortion is rare. Dietary quality may play a role, as miscarriage risk may be reduced in women who consume a diet rich in fruits, vegetables, whole grains, vegetable oils, and ish (Gaskins, 2015). With regard to maternal weight, underweight is not associated with a greater miscarriage risk (Balsells, 2016). However, as noted in Chapter 48 (p. 938), obesity does raise pregnancy loss rates. Lifestyle choices reputed to be associated with a higher miscarriage risk are most often related to chronic and especially heavy use oflegal substances. he most commonly used is alcohol, with its potent teratogenic efects discussed in Chapter 12 (p. 239). That said, an increased miscarriage risk is only seen with regular or heavy use (Avalos, 2014; Feodor Nilsson, 2014).

1	Approximately 10 percent of pregnant women admit to cigarette smoking (Centers for Disease Control and Prevention, 2016). It seems intuitive that cigarettes could cause early pregnancy loss (Pineles, 2014). Adverse efects of illicit drugs areidiscussed in Chapter 12 (p. 249).

1	Excessive cafeine consumption-not well defined-has been associated with a higher abortion risk. Reports link heavy intake of approximately ive cups of cofee per dayabout 500 mg of cafeine-with a slightly greater abortion risk (Cnattingius, 2000; Klebanof, 1999). Studies of "moderate" intake-less than 200 mg daily-did not indicate increased risk (Savitz, 2008; Weng, 2008). In contrast, in one prospective cohort of more than 5100 gravidas, cafeine was linked to miscarriage but not in a dose-response relationship (Hahn, 2015). Currently, the American College of Obstetricians and Gynecologists (2016e) has concluded that moderate consumption likely is not a major abortion risk and that any associated risk with higher intake is unsettled.

1	Environmental toxins suggested to have a possible link to miscarriage include bisphenol A, phthalates, polychlorinated biphenyls, and dichlorodiphenyltrichloroethane (DDT) (Krieg, 2016). Even fewer studies implicate occupational exposures. In a follow-up of the Nurses Health Study II, Lawson and associates (2012) reported slightly increased miscarriage risks in nurses exposed to sterilizing agents, x-rays, and antineoplastic drugs. Also, a higher miscarriage risk was found for dental assistants exposed to more than 3 hours of nitrous oxide daily if there was no gas-scavenging equipment (Boivin, 1997).

1	Increasing paternal age is signiicantly associated with an greater risk for abortion (de La Rochebrochard, 2003). In the Jerusalem Perinatal Study, this risk was lowest before age 25 years, after which it progressively increased at 5-year intervals (Kleinhaus, 2006). The etiology of this association is not well studied, but chromosomal abnormalities in spermatozoa likely play a role (Sartorius, 2010). his diagnosis is presumed when bloody vaginal discharge or bleeding appears through a closed cervical os during the first 20 weeks. This bleeding in early pregnancy must be diferentiated from that with implantation, which some women have at the time of their expected menses. Aside from this, almost one fourth of women develop bleeding during early gestation that may persist for days or weeks. It may be accompanied by suprapubic discomfort, mild cramps, pelvic pressure, or persistent low backache. Of symptoms, bleeding is by far the most predictive risk factor for pregnancy loss.

1	Even if miscarriage does not follow threatened abortion, rates of later adverse pregnancy outcomes are increased as shown in Table Of these, highest risks are for preterm delivery. Weiss and coworkers (2004) noted greater risks for adverse outcomes in later pregnancy if early bleeding was heavy rather than light. Compared with those without bleeding, women with first-trimester bleeding in an initial pregnancy have higher recurrence rates in their second (Lykke, 2010). TABLE 18-1. Adverse Outcomes That Are Increased in Women with Threatened Abortion Placenta previa Preterm ruptured membranes Placental abruption Preterm birth Manual removal of placenta Low-birthweight infant Cesarean delivery Fetal-growth restriction From Lykke, 2010; Saraswat, 2010; Weiss, 2004; Wijesiriwardana,s2006.

1	From Lykke, 2010; Saraswat, 2010; Weiss, 2004; Wijesiriwardana,s2006. Every woman with an early pregnancy, vaginal bleeding, and pain should be evaluated. he primary goal is prompt diagnosis of ectopic pregnancy, and serial quantitative serum S-hCG levels and transvaginal sonography are integral tools. Because these are not 100-percent accurate to confirm early embryo death or location, repeat evaluations are often necessary. With a robust uterine pregnancy, serum S-hCG levels should rise at least 53 to 66 percent every 48 hours (Barnhart, 2004c; Kadar, 1982). Although a less-used marker, serum progesterone concentrations <5 ng/mL suggest a dying pregnancy. Values >20 ng/mL support the diagnosis of a healthy one (Daily, 1994).

1	Transvaginal sonography is used to locate the pregnancy and determine viability. If this cannot be done, then a PUL is diagnosed, and serial surveillance is implemented for clinically stable women. The gestational sac-an anechoic fluid collection that represents the exocoelomic cavity-may be seen by 4.5 weeks (Fig. 9-3, p. 159). At this same time, S-hCG levels generally measure 1500 to 2000 mIU/mL (Barnhart, 1994; Timor-Tritsch, 1988). Connolly and colleagues (2013) observed that this value could be as low as 390 mIU/mL. However, they also noted that a threshold as high as 3500 mIU/mL may be needed to identiY the gestational sac in some cases that ultimately yield a viable singleton IUP.

1	Another caveat is that a gestational sac may appear similar to other intrauterine fluid accumulations-the so-called pseudogestational sac (Fig. 19-4, p. 375). his pseudosac may be blood derived from a bleeding ectopic pregnancy and is easier to exclude once a yolk sac is seen. Typically, the yolk sac is visible by 5.5 weeks and with a mean gestational-sac diameter of 10 mm. Thus, the diagnosis of an IUP should be made cautiously if the yolk sac is not yet seen (American College of Obstetricians and Gynecologists, 20 16h). For management of threatened abortion, observation is the norm. Acetaminophen-based analgesia will help relieve discomfort from cramping. Bed rest is often recommended but does not improve outcomes. The hematocrit and blood type is determined. If anemia or hypovolemia is signiicant, then pregnancy evacuation is generally indicated. In cases in which there is a live fetus, some instead may choose transfusion and further observation.

1	During abortion, bleeding follows partial or complete placental separation and dilation of the cervical os. Before 10 weeks' gestation, the fetus and the placenta are frequently expelled together, but later, they deliver separately. hus, tissue may remain entirely within the uterus or partially extrude through the cervix. Products lying loosely within the cervical canal can be easily extracted with ring forceps. In contrast, with incom plete expulsion, three management options include curettage, expectant management, or misoprostol (Cytotec), which is prostaglandin EI (PGEI) (Kim, 2017). he last two are deferred in clinically unstable women or those with uterine infection.

1	Each option has its own risks and benefits. With all three, infection and need for transfusion are uncommon. However, misoprostol and expectant care are associated with unpredictable bleeding, and some women will undergo unscheduled curettage. Expectant management of spontaneous incomplete abortion has failure rates that approximate 25 percent in randomized trials (Nadarajah, 2014; Nielsen, 1999; Trinder, 2006). Some observational studies have shown failure rates of 10 to 15 percent (Blohm, 2003; Casikar, 2012; Luise, 2002). Medical therapy carries failure rates of 5 to 30 percent (Dao, 2007; Shochet, 2012; Trinder, 2006). In many studies for this, an oral misoprostol dose of 600 1g has been used (American College of Obstetricians and Gynecologists, 2009). Alternatively, an 800-l1g vaginal or a 400-1g oral or sublingual misoprostol dose is suitable. Last, curettage usually results in a quick resolution that is 95-to 100-percent successful. However, it is invasive and not necessary for

1	or a 400-1g oral or sublingual misoprostol dose is suitable. Last, curettage usually results in a quick resolution that is 95-to 100-percent successful. However, it is invasive and not necessary for all women.

1	At times, complete expulsion of the entire pregnancy may ensue, and the cervical os subsequently closes. A history of heavy bleeding, cramping, and passage of tissue is typical. Patients are encouraged to bring in passed tissue, in which a complete gestation should be discerned from blood clots or a decidual cast. he latter is a layer of endometrium in the shape of the uterine cavity that when sloughed can appear as a collapsed sac (Fig. 19-2, p. 373).

1	If an expelled complete gestational sac is not identified, transvaginal sonography is performed to diferentiate a complete abortion from threatened abortion or ectopic pregnancy. Characteristic findings of a complete abortion include a minimally thickened endometrium without a gestational sac. However, this does not guarantee a recent uterine pregnancy. Condous and associates (2005) described 152 women with heavy bleeding, an empty uterus with endometrial thickness < 15 mm, and a diagnosis of completed miscarriage. Six percent were subsequently found to have an ectopic pregnancy. Thus, a complete abortion cannot be surely diagnosed unless: (1) true products of conception are seen grossly or (2) unless sonography confidently documents first an intrauterine pregnancy and then later an empty cavity. In unclear settings, serial serum hCG level measurements aid clariication. With complete abortion, these levels drop quickly (Table 18-2).

1	This describes dead products of conception that have been retained for days or weeks in the uterus with a closed cervical os. Diagnosis is imperative prior to intervention and avoids interruption of a potentially live IUP. Transvaginal sonography is the primary tool. TABLE 18-2. Percentage Decline of Initial Serum FIGURE 18-1 Transvaginal sonogram displays a large anechoic sac consistent with an anembryonic gestation. Calipers measure uterine length and anteroposterior thickness in a sagittal plane. aThe percentage decline is given as the expected decline. The minimum expected decline in parentheses is the 95th percentile value. Declines less than this minimum may reflect retained either intrauterine or extrauterine trophoblast. Data from Barnhart, 2004a; Chung, 2006.

1	Data from Barnhart, 2004a; Chung, 2006. At 5 to 6 weeks' gestation, a 1-to 2-mm embryo adjacent to the yolk sac can be seen (Daya, 1993). As listed in Table 18-3, absence of an embryo in a sac with a mean sac diameter (MSD) ::25 mm signiies a dead fetus (Fig. 18-1). Fetal cardiac activity can typically be detected at 6 to 6.5 weeks with a crown-rump length (CL) of 1 to 5 mm and an MSD of 13 to 18 mm (Goldstein, 1992; Levi, 1990). A threshold CL ::7 mm with absent cardiac activity is also used to diagnose nonviability (Doubilet, 2013). Preisler and associates (2015) implemented the guidelines in Table 18-3 and conirmed' these CL and MSD thresholds. However, for cases in which a gestational sac had no embryo or yolk sac and measured < 12 mm, they TABLE 18-3. Guidelines for Early Pregnancy Loss Diagnosisa

1	TABLE 18-3. Guidelines for Early Pregnancy Loss Diagnosisa CRL ::7 mm and no heartbeat MSD ::25 mm and no embryo An initial US scan shows a gestational sac with yolk sac, and after ::11 days no embryo with a heartbeat is seen An initial US scan shows a gestational sac without a yolk sac, and after ::2 weeks no embryo with a heatbeat is seen Transvaginal preferable to transabdominal US M-mode imaging used to document and measure heartbeat Doppler US not used to evaluate a normal early embryo aFrom the Society of Radiologists in Ultrasounds; American College of Radiology. CRL = crown-rump length; MSD = mean sac diameter; US = ultrasound. From Doubilet, Lane, 2013. recommended that to diagnose nonviability after 2 weeks, in addition to the lack of an embryo with a heartbeat, the MSD should have failed to double.

1	From Doubilet, Lane, 2013. recommended that to diagnose nonviability after 2 weeks, in addition to the lack of an embryo with a heartbeat, the MSD should have failed to double. During scanning, because of theoretical temperature elevation in tissues exposed to pulsed Doppler beam, this modality is applied only when needed for additional diagnostic purposes. M-mode should be used to document cardiac activity and measure the rate (Lane, 2013). Finding of an IUP and cardiac activity lowers subsequent miscarriage rates (Siddiqi, 1988).

1	In addition to the diagnostic parameters of Table 18-3, other softer sonographic markers may portend early pregnancy failure. Values for yolk sac diameters (measured inner-to-inner ring) for each gestational week in normal pregnancy have been established. Yolk sac diameters ::6 mm in pregnancies < 10 weeks' gestation are suspicious for pregnancy failure (Berdahl, 2010; Lindsay, 1992). The fetal heart rate in the irst trimester rises from 110 to 130 beats per minute (bpm) at 6 weeks' gestation to 160 to 170 bpm at 8 weeks (Achiron, 1991; Rauch, 2009). A slower heart rate is unfavorable, especially those <85 bpm (Laboda, 1989; Stefos, 1998). Even with cardiac activity, fetuses with a small MSD may presage embryonic loss. Speciically, a diference <5 mm between the MSD and CRL values raises concern (Bromley, 1991; Dickey, 1992). Last, subchorionic hematoma, that is, blood collected between the chorion and uterine wall, often accompanies threatened miscarriage. Studies are contradictory as

1	(Bromley, 1991; Dickey, 1992). Last, subchorionic hematoma, that is, blood collected between the chorion and uterine wall, often accompanies threatened miscarriage. Studies are contradictory as to its association with ultimate pregnancy loss (Pedersen, 1990; Stabile, 1989; Tuuli, 2011). Bennett and associates (1996) noted that miscarriage risk correlated with larger hematoma size, older maternal age, and bleeding at a gestational age :;8 weeks.

1	With rapid confirmation of embryonic or fetal death, surgicalior medical evacuation or expectant observation is an option. As with induced abortion, nonsurgical options balance their noninvasiveness against heavier procedural bleeding, longer completion times, and lower success rates. Of options, expectant care underperforms medical or surgical options, and failure rates range from 15 to 50 percent (Luise, 2002; Trinder, 2006; Zhang, 2005). Also, weeks may pass between pregnancy failure diagnosis and actual spontaneous miscarriage.

1	Alternatively, misoprostol can be given to hasten uterine evacuation. A single 800-l-1g dose vaginally is a common stan dard (American College of Obstetricians and Gynecologists, 20 16c). It may be repeated in 1 to 2 days, and one large trial reported that 22 percent of women required a second dose (Zhang, 2005). Overall, failure rates range from 15 to 40 per cent (Petersen, 2014; Trinder, 2006). Unlike induced abortion, adding mifepristone does not add value (Stockheim, 2006). Contraindications mirror those listed in the section describing induced abortion (p. 361). Confirmation of completion may include a history of heavy bleeding, cramping, and tissue passage followed by ebbing flow; ping serum hCG levels. That said, there is no consensus on intervention.

1	Confirmation of completion may include a history of heavy bleeding, cramping, and tissue passage followed by ebbing flow; ping serum hCG levels. That said, there is no consensus on intervention. Pre term premature rupture of membranes (PPRO\1) at a pre viable gestational age complicates 0.5 percent of pregnancies (Hunter, 2012). Rupture may be spontaneous or may follow an invasive procedure such as amniocentesis or fetal surgery. Risks for spontaneous rupture at a previable gestation are prior PPROM, prior second-trimester delivery, and tobacco use (Kilpatrick, 2006).

1	Risks for spontaneous rupture at a previable gestation are prior PPROM, prior second-trimester delivery, and tobacco use (Kilpatrick, 2006). A gush of vaginal fluid that is seen pooling during sterile speculum examination confirms the diagnosis. In suspect cases, amnionic luid will fern on a microscope slide or will have a pH >7, or oligohydramnios will be seen on sonography (Sugibayashi, 2013). lso, amnionic fluid proteins placental alpha microglobulin-1 and insulin growth factor binding protein-I, described in Chapter 22 (p. 235), can be assayed (Doret, 2013). In iatrogenic cases, defects are typically higher in the uterus and tend to self seal. Also, an occlusive plug-termed an amniopatch-can be created by intraamnionic instillation of autologous platelets and cryoprecipitate. Considered investigational, it is used to seal some surgical leaks (Richter, 2013).

1	Spontaneous rupture in the first trimester is nearly always followed by either uterine contractions or infection, and termination is typical. In some second-trimester cases not associated with pain, fever, or bleeding, fluid may have collected previously between the amnion and chorion. If this is documented, then diminished activity with observation is reasonable. After 48 hours, if no additional amnionic luid has escaped and if there is no bleeding, cramping, or fever, then a woman may resume ambulation and pelvic rest at home.

1	However, more typically with second-trimester spontaneous PPROM at a previable age, 40 to 50 percent of women will deliver within the first week, and 70 to 80 percent will do so after 2 to 5 weeks (American College of Obstetricians and Gynecologists, 2016f). Average latency is 2 weeks (Hunter, 2012; Kibel, 2016). Significant maternal complications attend previable PPROM and include chorioamnionitis, endometritis, sepsis, placental abruption, and retained placenta (Waters, 2009). With bleeding, cramping, or fever, abortion is considered inevitable, and the uterus is evacuated. Without these complications, expectant management is an option in the well-counseled patient (American College of

1	Without these complications, expectant management is an option in the well-counseled patient (American College of Obstetricians and Gynecologists, 20 17). Many will choose ter mination due to the just-described maternal risks and tenuous neonatal outcomes. In contemporary cohorts with PPROM at <24 weeks' gestation, only approximately 20 percent of fetuses survive until hospital discharge (Esteves, 2016; Everest, 2008; Muris, 2007). Of surviving infants, 50 to 80 percent sufer long-term sequelae (Miyazaki, 2012; Pristauz, 2008). Further stratification of outcomes by gestational age is described in

1	Muris, 2007). Of surviving infants, 50 to 80 percent sufer long-term sequelae (Miyazaki, 2012; Pristauz, 2008). Further stratification of outcomes by gestational age is described in Chapter 42 (p. 806). Overall, prognosis is improved if previa ble PPROM occurred at a later gestation, latency is longer, and oligohydramnios is absent. Neonatal mortality predominantly stems from pulmonary dysfunction, which has higher rates when oligohydramnios persists (Winn, 2000). Fetal deforma tions may also result from scant amnionic luid. Amnioinfusion has been investigated but is currently investigational (Roberts, 20i14). If expectant care is elected, management is described in Chapter 42 (p. 807). Antibiotics are considered and given for 7 days to extend latency. Other topics include lung-maturing corticosteroids, magnesium sulfate neuroprophylaxis, group

1	Chapter 42 (p. 807). Antibiotics are considered and given for 7 days to extend latency. Other topics include lung-maturing corticosteroids, magnesium sulfate neuroprophylaxis, group B streptococcus antibiotic prophylaxis, tocolytics, and neo natal resuscitation eforts. After initial hospitalization, the patient may be discharged home, with instruction for careful surveillance for complications until viability, at which time readmission is usual (American College of Obstetricians and Gynecologists, 20 16). In subsequent pregnancies, the risk for recurrent preterm birth is great, and in one cohort study, the rate neared 50 percent (Monson, 2016).

1	Gynecologists, 20 16). In subsequent pregnancies, the risk for recurrent preterm birth is great, and in one cohort study, the rate neared 50 percent (Monson, 2016). With abortion legalization, horriic infections and maternal deaths associated previously with criminal septic abortions are now rare. Still, with spontaneous or induced abortion, organisms may invade myometrial tissues and extend to cause parametritis, peritonitis, and septicemia. Most bacteria causing septic abortion are part of the normal vaginal lora. Particularly worrisome are severe necrotizing infections and toxic shock syndrome caused by group A streptococcus-S pyogenes (Daif, 2009).

1	Rare but severe infections with otherwise low-virulence organisms can complicate medical or spontaneous abortions. Deaths have been reported from toxic shock syndrome due to Clostridium peringens (Centers for Disease Control and Prevention, 2005). Similar infections are caused by Clostridium sordellii and have clinical manifestations that begin within a few days after an abortion. Women may be afebrile when first seen with severe endothelial injury, capillary leakage, hemoconcentration, hypotension, and a profound leukocytosis. Maternal deaths from these clostridial species approximate 0.58 per 100,000 medical abortions (Meites, 2010).

1	Management of clinical infection includes prompt administration of broad-spectrum antibiotics as discussed in Chapter 37 (p. 668). If there are retained products, then suction curettage is also performed. Most women respond to this treatment within 1 to 2 days and are discharged when afebrile. Follow-up oral antibiotic treatment is likely unnecessary (Savaris, 2011). In a very few women, severe sepsis syndrome develops, and intensive supportive care is essential. Although rare, clinical decline in the patient and widespread peritonitis despite curettage should raise concerns. Imaging that shows free air or air within the uterine wall typically prompts laparotomy (Eschenbach, 2015). If the uterus is necrotic, hysterectomy is indicated.

1	With spontaneous miscarriage, 2 percent of h D-negative women will become alloimmunized if not provided passive isoimmunization. With an induced abortion, this rate may reach 5 percent. The American College of Obstetricians and Gynecologists (2017g) recommends anti-Rho (D) immunoglobulin given as 300 �g intramuscularly (IY!) for all gestational ages. Doses may also be graduated, with 50 �g given Hvl for pregnancies :;12 weeks and 300 �g for ::13 weeks. This is administered immediately following surgical evacuation. For planned medical or expectant management, the injection is given within 72 hours of pregnancy failure diagnosis. With threatened abortion, immunoglobulin prophylaxis is controversial because of sparse evidence-based data (Hannafin, 2006). hat said, it is reasonable to administer anti-D immunoglobulin for a threatened abortion and a live fetus, and this is our practice.

1	Afecting approximately 1 percent of fertile couples, recurrent pregnancy loss (RPL) is classically deined as three or more consecutive pregnancy losses <20 weeks' gestation or with a fetal weight < 500 g. Mindful of this threshold, data from two large studies showed the risk for a subsequent miscarriage to be similar whether following two or three prior pregnancy losses (Bhattacharya, 2010; Brigham, 1999). And, the American Society for Reproductive Medicine (2013) now deines RPL as two or more failed pregnancies conirmed by sonographic or histopathological examination. Primary PL refers to multiple losses in a woman who has never delivered a liveborn, and secondary PL refers to multiple pregnancy losses in a patient with a prior live birth. Remarkably, the chances for a successful pregnancy arei> 50 percent even after five losses (Table 18-4). TABLE 18-4. Predicted Success Rate of Subsequent Pregnancy According to Age and Number of Previous Miscarriages

1	TABLE 18-4. Predicted Success Rate of Subsequent Pregnancy According to Age and Number of Previous Miscarriages No. of Previous 2 3 4 5 Miscarriages at Predicted Success of Subsequent Evaluation for RPL addresses the major etiologies, described next (American Society for Reproductive Medicine, 2012). Treatment considerations reach beyond the scope of this book, and interested readers are referred to Chapter 6 in Wiliams Gynecoloy, 3rd edition (Halvorson, 2016). hree widely accepted causes of RPL are parental chromosomal abnormalities, antiphospholipid antibody syndrome, and structural uterine abnormalities. First-trimester losses in PL have a signiicantly lower incidence of genetic abnormalities than sporadic miscarriage (Stephenson, 2002; Sullivan, 2004).

1	The timing of recurrent loss can ofer clues, and in some women, each miscarriage may occur near the same gestational age (Heuser, 2010). Genetic factors usually result in early embryonic losses, whereas autoimmune or uterine anatomical abnormalities more likely cause second-trimester losses (Schust, 2002). Approximately 40 to 50 percent of women have idiopathic RPL (Li, 2002; Stephenson, 1996). Although these account for only 2 to 4 percent of RPL cases, karyotyping of both parents is considered by many to be essential. Of abnormalities, reciprocal translocations are most common and followed by robertsonian translocations (Fan, 2016). heir genesis and reproductive sequelae are discussed in Chapter 13 (p. 261).

1	After thorough genetic counseling, couples with an abnormal karyotype can be ofered in vitro fertilization (IVF) followed by preimplantation genetic diagnosis (American Society for Reproductive Medicine, 2012; Society for Assisted Reproductive Technology, 2008). This technique is described in Chapter 14 (p. 295). However, in couples with RPL who are chromosomaly normal, PGD is not currently recommended. Several genital tract abnormalities have been implicated in RPL and other adverse pregnancy outcomes (Reichman, 2010). According to Devi Wold and colleagues (2006), 15 percent of women with three or more consecutive miscarriages will be found to have a congenital or acquired uterine anomaly.

1	Of acquired abnormalities, uterine synechiae-Asherman syndrome-usually result from destruction of large areas of endometrium. This can follow uterine curettage, hysteroscopic surgeries, or uterine compression sutures (Conforti, 2013; Rathat, 2011). Characteristic multiple illing defects are seen with hysterosalpingography or saline-infusion sonography. Treatment is hysteroscopic adhesiolysis. In many, this lowers miscarriage rates and improves live birth rates (Yu, 2008). Uterine leiomyomas are common and may cause miscarriage, especially iflocated near the placental implantation site. That said, data indicating them to be a significant cause ofRPL are not con 77 73 68 62 vincing (Saravelos, 2011). Uterine cavity distortion is apparently 69 64 58 52 not requisite for bad outcomes (Sunkara, 2010). But in women undergoing IVF, pregnancy outcomes are adversely afected by Data fror Brigham, 1999 submucous but not subserosal or intramural leiomyomas a un,

1	Data fror Brigham, 1999 submucous but not subserosal or intramural leiomyomas a un, TABLE 18-5. Clinical and Laboratory Criteria for Diagnosis of Antiphospholipid Antibody Syndromea Obstetric: One or more unexplained deaths of a morphologically normal fetus at or beyond 10 weeks, Severe preeclampsia or placental insufficiency necessitating delivery before 34 weeks, Vascular: One or more episodes of arterial, venous, r small vessel thrombosis in any tissue or organ Presence of lupus anticoagulant according to guidelines of the International Society on Thrombosis and Hemostasis, Medium or high serum levels of IgG or IgM anticardiolipin antibodies, IgG = immunoglobulin G; igM = immunoglobulin M. Modified from Branch, 2010; ETkan, 201s1; Miyakis, 2006. 2001; Ramzy, 1998). s discussed in Chapter 63 (p. 1197), most agree that excision of submucosal leiomyomas in women with RPL can be considered.

1	2001; Ramzy, 1998). s discussed in Chapter 63 (p. 1197), most agree that excision of submucosal leiomyomas in women with RPL can be considered. Congenital genital tract anomalies often originate from abnormal mlillerian duct formation. These have an overall incidence of approximately 1 in 200 women (Nahum, 1998). Depending on their anatomy, some may raise risks for early miscarriage, whereas others may cause midtrimester abortion or preterm delivery. Unicornuate, bicornuate, and septate uteri are associated with all three types of loss (Reichman, 2010). A fuller discussion of these anatomical abnormalities and their reproductive efects is found in Chapter 3 (p. 41).

1	Miscarriages are more common in women with systemic lupus erythematosus (Clowse, 2008). Many of these women, as well as some without lupus, carry antiphospholipid antibodies, a family of autoantibodies that bind to phospholipid-binding plasma proteins (Erkan, 201 1). Women with RPL have a higher frequency of these antibodies compared with normal controls (Branch, 2010). As shown in 1 able 18-5, the antiphospholipid antibody syndrome APS) is deined by these antibodies in combination with various forms of reproductive loss and substantively increased risks for venous thromboembolism (American College of Obstetricians and Gynecologists, 20 17b,i). Mechanisms that cause pregnancy loss are discussed along with treatment in Chapter 59 (p. 1144).

1	Regarding alloimmunity, one provocative theory suggests that normal pregnancy requires formation of blocking factors that avert maternal rejection of foreign fetal antigens that are paternally derived (Chap. 5, p. 95). Factors that prevent this tolerance may underlie RPL (Berger, 2010). However, proposed therapies using paternal or third-party leukocyte immunization or intravenous immunoglobulin (IlG) have not proved beneicial in aAt least one clinical and one laboratory criteria must be present for diagnosis. bThese tests must be positive on two or more occasions at least 12 weeks apart. women with idiopathic RPL (Christiansen, 2015; Stephenson, 2010).

1	women with idiopathic RPL (Christiansen, 2015; Stephenson, 2010). According to Arredondo and Noble (2006), 8 to 12 percent of recurrent miscarriages are caused by endocrine factors. Studies to evaluate these have been inconsistent and generally underpowered. Two examples, both controversial, are progesterone deficiency caused by a luteal-phase dect and poycystic ovarian syndrome (Bukulmez, 2004; Cocksedge, 2008). In contrast, the well-known abortifacient action of uncontrolled diabetes mellitus is detailed in Chapter 57 (p. 1099). Optimal periconceptional glycemic control will mitigate many of these losses.

1	In contrast, the well-known abortifacient action of uncontrolled diabetes mellitus is detailed in Chapter 57 (p. 1099). Optimal periconceptional glycemic control will mitigate many of these losses. Likewise, the efects of overt hypothyroidism and severe iodine deficiency on early pregnancy failure are well known and discussed in Chapter 58 (p. 1124). Correction with supplementation reverses these actions. Also, the influence of subclinical hypothyroidism and antithyroid antibodies are sporadic, and thus any efects on recurrent miscarriage rates have been debated (Garber, 2012). That said, two metaanalyses reported convincingly positive associations between these antibodies and a greater risk for sporadic and recurrent miscarriages (Chen, 2011; Thangaratinam, 2011). One ongoing randomized trial regarding potential beneits of treatment will help guide future management (Vissenberg, 2015).

1	he timespan that deines a mid trimester fetal loss extends from the end of the first trimester until the fetus weighs < 500 g or gestational age reaches 20 weeks. Less than in the irst trimester, TABLE 18-6. Some Causes of Midtrimester Spontaneous Abruption, previa the spontaneous loss rate in the second ranges from 1.5 to 3 percent and, after 16 weeks, is only 1 percent (Simpson, 2007; Wyatt, 2005). Unlike earlier miscarriages that frequently are caused by chromosomal aneuploidies, these later fetal losses are due to a multitude of causes (Table 18-6). One frequently overlooked factor is that many second-trimester abortions are medically induced because of fetal abnormalities detected by prenatal screening programs for chromosome aneuploidy and structural defects.

1	.Midtrimester abortions are classified similarly to first-trimester miscarriages (p. 348). Management is similar in many regards to that used for second-trimester induced abortion, described later (p. 362). One exception is cervical cerclage, which may be employed for cervical insuiciency. Also known as incompetent cervix, this is a discrete obstetrical entity characterized classically by painless cervical dilatation in the second trimester. It can be followed by prolapse and ballooning of membranes into the vagina, and ultimately, expulsion of an immature fetus. his sequence oten repeats in uture pregnancies.

1	Although the cause of insuiciency is obscure, previous cervical trauma has been implicated. One Norwegian cohort study of more than 15,000 women with prior cervical conization found a fourfold risk of pregnancy loss before 24 weeks' gestation (lbrechtsen, 2008). However, cerclage is not beneicial for women solely with this risk and without a preterm birth history (Zeisler, 199 ). Of other surgeries, dilation and evacuation carries a 5-percent risk of cervical injury, but neither it nor dilation and extraction raises the likelihood of cervical insuiciency (Chasen, 2005). In other instances, abnormal cervical development, including that following in utero exposure to diethylstilbestrol (DES), may playia role (Hoover, 2011). Last, cervical ripening changes, such as altered hyaluronan or collagen content, discussed in Chapter 21 (p. 409), may be contributory (Eglinton, 2011; Sundtoft, 2017).

1	Last, cervical ripening changes, such as altered hyaluronan or collagen content, discussed in Chapter 21 (p. 409), may be contributory (Eglinton, 2011; Sundtoft, 2017). For women with an unequivocal history of second-trimester painless delivery, prophylactic cerclage placement is an option and reinforces a weak cervix by an encircling suture. However, some women have a history and clinical findings that make it diicult to veriy-classic cervical insuiciency. In one randomized trial of almost 1300 women with an atypical history, cerclage was found to be only marginally beneicial-13 versus 17 percent-to prolong pregnancy past 33 weeks (MacNaughton, 1993). It seems likely that many of these women instead had preterm labor.

1	In addition to history, the physical inding of early dilation of the internal cervical os may be an indicator of insuiciency. In a systematic review, cerclages that were placed based on this finding provided superior perinatal outcomes compared with expectant management (Ehsanipoor, 2015).

1	Transvaginal sonography is yet another tool, and cervical length and the presence offunneling are sought. he latter is ballooning of the membranes into a dilated internal os, but with a closed external os. In women with these findings, early randomized trials were inconclusive in proving the clinical value of cerclage to prevent preterm birth (Rust, 2001; To, 2004). A multicenter randomized trial of 302 high-risk women with cervical length <25 mm reported that cerclage prevented birth before viability but not birth before 34 weeks (Owen, 2009). Subsequently, however, Berghella and coworkers (201i1) included ive trials in a metaanalysis and showed that cerclage for these high-risk women signiicantly reduced preterm birth before 24, 28, 32, 35, and 37 weeks.

1	Cervical length screening is now recommended by both the American College of Obstetricians and Gynecologists (2016b) and the Society for Maternal-Fetal Medicine (2015) for women with prior preterm birth. Between 16 and 24 weeks' gestation, sonographic cervical measurement is completed every 2 weeks. If an initial or subsequent cervical length is 25 to 29 mm, then a weekly interval is considered. If the cervical length measures <25 mm, cerclage is ofered to this group of women. Notably, for women without a history of preterm birth but with a short cervix incidentally identiied sonographically, progesterone therapy is ofered instead of cerclage. With twin gestations, one retrospective analysis found no improved outcomes in women with a cervical length <25 mm (Stoval, 2013). he College (2016b) does not recommend the use of cerclage in twin pregnancies.

1	Contraindications to cerclage usually include bleeding, contractions, or ruptured membranes, which substantially raises the likelihood of failure. Thus, prophylactic cerclage before dilatation is preferable. Surgery between 12 and 14 weeks' gestation allows this early intervention yet avoids surgery in a woman with a irst-trimester pregnancy destined for spontaneous loss. Preoperatively, screening for aneuploidy and obvious malformation is completed. Cervical secretions are tested for gonorrhea and chlamydial infection. hese and obvious cervical infections are treated.

1	At times, the cervix instead is found to be dilated, efaced, or both, and an emergent rescue cerclage is performed. However, there is debate as to how late this should be performed. The conundrum is that the more advanced the pregnancy, the greater the risk that surgical intervention will stimulate preterm labor or membrane rupture. At Parkland Hospital, cerclage procedures are generally not done once supposed fetal viability is reached after 23 to 24 weeks. Others, however, recommend placement later than this (Caruso, 2000; Terkildsen, 2003).

1	When outcomes of cerclage are evaluated, women with similar clinical presentations are ideally compared. For example, in the study of elective cerclage by Owen and associates (2009), approximately a third of women delivered before 35 weeks, and complications were few. By contrast, in a 10-year review of 75 women undergoing rescue cerclage procedures, Chasen and Silverman (1998) reported that only half were delivered after 36 weeks. Importantly, only 44 percent of those with bulging membranes at the time of cerclage reached 28 weeks. Terkildsen and associates (2003) had similar experiences. Caruso and coworkers (2000) described rescue cerclage done in 23 women with a dilated cervix and protruding membranes from 17 to 27 weeks' gestation. There were 11 liveborn neonates, and these researchers concluded that success was unpredictable. Our experiences at Parkland Hospital are that rescue cerclages have a high failure rate, and women are counseled accordingly.

1	If the clinical indication for cerclage is questionable, a woman may instead be observed. Most undergo cervical examinations weekly or every 2 weeks to assess efacement and dilatation. Unfortunately, rapid efacement and dilation can develop despite such precautions (Witter, 1984). Of the two vaginal cerclage operations, most use the simpler procedure developed by McDonald (1963) and shown in Figure 1i8-2. The more

1	Of the two vaginal cerclage operations, most use the simpler procedure developed by McDonald (1963) and shown in Figure 1i8-2. The more FIGURE 18-2 McDonald cerclage procedure for incompetent cervix. A. Start of the cerclage procedure with a no. 2 monofilament suture being placed in the body of the cervix very near the level of the internal os. B. Continuation of suture placement in the body of the cervix so as to encircle the os. C. Encirclement completed. D. The suture is tightened around the cervical canal sufficiently to reduce the diameter of the canal to 5 to 10 mm, and then the suture is tied. The efect of the suture placement on the cervical canal is apparent. A second suture placed somewhat higher may be of value if the first is not in close proximity to the internal os.

1	complicated operation is a modification of the procedure described by Shirodkar (1955) and shown in Figure 18-3. When either technique is performed prophylactically, women with a classic history of cervical incompetence have excellent outcomes (Caspi, 1990; Kuhn, 1977). For either vaginal or abdominal cerclage, there is insuicient evidence to recommend perioperative antibiotic prophylaxis (American College of Obstetricians and Gynecologist, 2016b,i). Thomason and coworkers (1982) found that perioperative tocolytics failed to arrest most labor. Regional analgesia is suitable and preferred. Ater this, the woman is placed in standard dorsal lithotomy position. he vagina and perineum are surgically prepared, and the bladder is drained. Some operators do not use potentially irritating antiseptic solution on the exposed amnionic membranes and instead use warm saline (Pelosi, 1990). Although steps are described subsequently, a thorough and illustrated review of cerclage technique is provided

1	solution on the exposed amnionic membranes and instead use warm saline (Pelosi, 1990). Although steps are described subsequently, a thorough and illustrated review of cerclage technique is provided by Hawkins (2017). For suturing, options include a no. 1 or 2 nylon or polypropylene monoilament suture or 5-mm Mersilene tape. During placement, the suture is placed as high as possible and into the dense cervical stroma. Two cerclage sutures do not appear more efective than one (Giraldo-Isaza, 2013).

1	FIGURE 18-3 Modified Shirodkar cerclage for incompetent cervix. A. A transverse incision is made in the mucosa overlying the anterior cervix, and the bladder is pushed cephalad. B. A 5-mm Mersilene tape on a swaged-on or Mayo needle is passed anterior to posterior. C. The tape is a cerclage in place with labor contractions. T ransvaginally placed cerclages are typically removed even with cesarean delivery to avoid rare long-term foreign-body complications (Hawkins, 2014). With scheduled cesarean delivery, the cerclage may be removed at 37 weeks or deferred until the time of regional analgesia and delivery. Again, the risk of labor ensuing before delivery must be considered. During extraction, particularly with a Shirodkar cerclage or a cerclage using Mersilene tape, analgesia helps ensure patient comfort B and adequate visualization.

1	B and adequate visualization. At times, suture placed at the uterine isthmus can be used and left until completion of childbearing. Because of significantly greater risks of bleeding and complications during placement, this approach is reserved for selected instances of severe cervical anatomical defects or prior transvagi c nal cerclage failure. Placement of a cervicoisthmic cerclage was originally described using laparotomy, but several reports additionally detail lapa then directed posterior to anterior on the other side of the cervix. Allis clamps are placed so as to bunch the cervical tissue. This diminishes the distance that the needle must travel submucosally cervicoisthmic cerclages. Steps are and aids tape placement. D. The tape is snugly tied anteriorly, after ensuring that all slack has been taken up. The cervical mucosa is then closed with continuous stitches of chromic suture. summarizedin Figure 18-4. Tulandi

1	Rescue cerclage with a thinned dilated cervix is more diicult and risks tissue tearing and membrane puncture. Replacement of the prolapsed amnionic sac back into the uterus will usually aid suturing (Locatelli, 1999). Options include steep Trendelenburg or filling the bladder with 600 mL of saline through an indwelling Foley catheter. However, these steps may carry the cervix cephalad and away from the operating field. Membrane reduction can also be achieved by pressure from a wide moist swab or by placing a Foley catheter through the cervix and inlating the 30-mL balloon to deflect the amnionie sac cephalad. The balloon is then deflated gradually as the cerclage suture is tightened around the catheter tubing, which is then removed. Simultaneous outward traction created by ring forceps placed on the cervical edges may be helpful. In some women with bulging membranes, transabdominal amnionic fluid aspiration to decompress the sac may be considered. If this is done, bacterial cultures

1	on the cervical edges may be helpful. In some women with bulging membranes, transabdominal amnionic fluid aspiration to decompress the sac may be considered. If this is done, bacterial cultures of the luid should be obtained.

1	For uncomplicated pregnancies without labor, the cerclage is usually cut and removed at 37 weeks' gestation. This balances the risk of preterm birth against that of cervical laceration from and coworkers (2014) evaluated 16 studies involving 678 pregnancies. Placement before pregnancy and during pregnancy was similar, whether performed laparoscopically or by laparotomy.

1	Zaveri and associates (2002) reviewed 14 observational studies in which a prior transvaginal cerclage had failed to prevent preterm delivery. The risk of perinatal death or delivery before 24 weeks' gestation was only slightly lower following transabdominal cerclage compared with the risk following repeat transvaginal cerclage-6 versus 13 percent, respectively. Importantly, 3 percent of women who underwent transabdominal cerclage had serious operative complications, whereas there were none in the transvaginal group. Whittle and coworkers (2009) described 31 women in whom transabdominal cervico isthmic cerclage was done laparoscopieally between 10 and 16 weeks. he procedure was converted to laparotomy in 25 percent, and there were four failures due to chorioamnionitis. Overall, fetal survival rate approximated 80 percent. Principal among these are membrane rupture, preterm labor, hemorrhage, infection, or combinations thereof. All are

1	FIGURE 18-4 Transabdominal cervicoisthmic cerclage. Following incision and sharp dissection in the vesicouterine space, the blad der is mobilized caudally. At the level of the internal os, a window is made in free space medial to the uterine vessels. This avoids vessel compression by the tightened cerclage. Care is also taken to avoid the ureter, which is lateral and posterior. The suture is passed anterior to posterior or vice versa. In this case, the knot is tied anteriorly, and the vesicouterine peritoneum is closed with absorbable suture in a running fashion. (Reproduced with permission from Hawkins JS: Lower genital tract procedures. In Yeomans ER, Hofman BL, Gilstrap LC III, et al: Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 2017.) uncommon with prophylactic cerclage. In the multicenter study by Owen and colleagues (2009), of 138 procedures, there was one instance each of ruptured membranes and bleeding. In the trial by MacNaughton

1	with prophylactic cerclage. In the multicenter study by Owen and colleagues (2009), of 138 procedures, there was one instance each of ruptured membranes and bleeding. In the trial by MacNaughton and associates (1993), membrane rupture complicated only 1 of more than 600 procedures done before 19 weeks. In our view, clinical infection mandates immediate removal of the suture with labor induced or augmented. Similarly, ¥lith imminent abortion or delivery, the suture should be removed at once because uterine contractions can tear through the uterus or cervix.

1	Following cerclage, if subsequent cervical thinning is detected by sonographic assessment, then some consider a reinforcement cerclage. In one retrospective study, however, reinforcing cerclage sutures placed later did not significantly prolong pregnancy (Contag, 2016). Membrane rupture during suture placement or within the irst 48 hours following surgery is considered by some to be an indication for cerclage removal because of the likelihood of serious fetal or maternal infection (Kuhn, 1977). That said, the range of management options includes observation, removal of the cerclage and observation, or removal of the cerclage and labor induction (O'Connor, 1999). he term induced abortion is defined as the medical or surgical termination of pregnancy before the time of fetal viability.

1	he term induced abortion is defined as the medical or surgical termination of pregnancy before the time of fetal viability. Deinitions used to evaluate these statistically include: (1) abortion ratio-the number of abortions per 1000 live births, and (2) abortion rate-the number of abortions per 1000 women aged 15 to 44 years. Overall, abortions most likely are underreported in the United States because clinics inconsistently list medically induced abortions. For example, the Guttmacher Institute found that 926,000 procedures were performed in 2014 Oones, 2017). But for 2013, only about 664,400 elective abortions were reported to the Centers for Disease Control and Prevention Oatlaoui, 2016). Of these, 66 percent were pregnancies aged ;8 weeks' gestation, and 92 percent of abortions were completed beforei; 13 weeks. he abortion ratio was 200 per 1000 live births, and the abortion rate was 12.5 per 1000 women.

1	herapeutic abortion refers to termination of pregnancy for medical indications. Inclusive medical and surgical disorders are diverse and discussed throughout this text. In cases of rape or incest, many consider termination. The most frequent indi cation currently is to prevent birth of a fetus with a signiicant anatomical, metabolic, or mental deformity. the interruption of pregnancy before viability at the request of the woman, but not for medical reasons. Most abortions done today are elective, and thus, it is one of the most frequently performed medical procedures. • Abortion in the United States

1	• Abortion in the United States The legality of elective abortion was established by the United States Supreme Court in the case ofRoe v. Wae. The Court defined the extent to which states might regulate abortion and ruled that firsttrimester procedures must be let to the medical judgment of the physician. ter this, the state could regulate abortion procedures in ways reasonably related to maternal health. Finally, subsequent to viability, the state could promote its interest in the potential of human life and regulate and even proscribe abortion, except for preservation of the life or health of the mother.

1	Other legislation soon followed. he 1976 Hyde Amendment forbids use of federal funds to provide abortion services except in case of rape, incest, or life-threatening circumstances. The Supreme Court in 1992 reviewed Planned Parenthood v. Casy and upheld the fundamental right to abortion, but established that regulations before viability are constitutional as long as they do not impose an "undue burden" on the woman. Subsequently, many states introduced counseling requirements, waiting periods, parental consent for minors, facility requirements, and funding restrictions. Such limits are often called targeted regulation of abortion providers (TRAP) laws. One major choice-limiting decision was the 2007 Supreme Court decision that reviewed Gonales v. Carhart and upheld the 2003 Partial-Birth Abortion Ban Act. This was problematic because there is no medically approved definition of partial-birth abortion according to the American College of Obstetricians and Gynecologists (20 14a). In

1	Abortion Ban Act. This was problematic because there is no medically approved definition of partial-birth abortion according to the American College of Obstetricians and Gynecologists (20 14a). In 2016, some TAP laws were dialed back by the Supreme

1	Court ruling in the case of Who/� Woman 5 Health v. Helerstedt. With this, the justices noted that abortion laws must confer health safety benefits that outweigh burdens on access. The American College of Obstetricians and Gynecologists (2014a, 2017 d) supports the legal right of women to obtain an abortion prior to fetal viability and advocates for improved access. he College also (2017 a) supports abortion training, and the Accreditation Council for Graduate Medical Education mandates that obstetrics and gynecology residency education must include access to experience with induced abortion. The Kenneth J. Ryan Residency Training Program was established in 1999 to work with residency programs to improve abortion and contraceptive training. Moreover, postresidency training in these techniques is available in formal 2-year Family Planning fellowships.

1	Other residency programs are less codified, but teach residents technical aspects through their management of early spontaneous abortions and pregnancy interruption for fetal death, severe fetal anomalies, and life-threatening medical or surgical disorders. he College (2016g) respects the need and responsibility of health-care providers to determine their individual positions on induced abortion. It also advocates for counseling and timely referral if providers have individual beliefs that preclude pregnancy termination. hree basic choices available to a woman considering an abortion are: (1) continued pregnancy with its risks and parental responsibilities; (2) continued pregnancy with arranged adoption; or (3) termination of pregnancy with its risks. Knowledgeable and compassionate counseling should objectively describe and provide information regarding these choices to permit informed decision-making (Templeton, 2011).

1	Pregnancy termination can be performed either medically or surgically by several methods. In the absence of serious maternal medical disorders, abortion procedures do not require hospitalization (Guiahi, 2012). However, outpatient surgical facilities should have the ability to provide emergency resuscitation and immediate transfer to a hospital (American College of Obstetricians and Gynecologists, 20 14b).

1	Surgical evacuation is performed transvaginally through an appropriately dilated cervix. For this, preoperative cervical ripening is favored by many and is typically associated with less manual intraoperative cervical dilation, a technically easier procedure, less pain, and shorter operative times (Kapp, 2010; Webber, 2015). On balance, cervical preparation adds a surgical delay and potential side efects. In a selective approach, some recommend cervical priming for irst-trimester suction curettage only for those at greater risk of complications from intraoperative cervical dilation, such as those with cervical stenosis and adolescents (Allen, 2016). Of note, surgical steps presented here apply to both induced abortion and miscarriage, discussed earlier (p. 347). FIGURE 18-5 Hygroscopic dilators. With each type, the dry unit (left) expands exponentially when exposed to water (right) as in the endocervical canal. A. Laminaria. B. Dilapan-S.

1	FIGURE 18-5 Hygroscopic dilators. With each type, the dry unit (left) expands exponentially when exposed to water (right) as in the endocervical canal. A. Laminaria. B. Dilapan-S. For ripening, hygroscopic dilators, also called osmotic dilators, are devices that draw water from surrounding tissues and expand to gradually dilate the endocervical canal. One type is derived from various species of Laminaria algae that are harvested from the ocean floor (Fig. 18-5). hese come in diferent diameters, which allow the number of inserted devices, also called tents, to be customized to a given cervix. Another device is Dilapan-S, which is composed of an acrylic-based gel. Each type expands to an ultimate diameter three to four times that of its dry state. However, Dilapan-S achieves this in 4 to 6 hours, which is faster than the 12 to 24 hours needed for laminaria (Fox, 2014).

1	With hygroscopic dilators, shallow insertion yields insuicient internal os dilation or tent expulsion, but deep placement risks dislodgement into the uterine cavity (Fig. 18-6). Accordingly, the numbers of sponges and dilators inserted are carefully counted and recorded in the patient's chart. Once tents are inserted, several gauze sponges at the external os help prevent spontaneous tent expulsion. Patients can ambulate, void, or stool without limitation. FIGURE 18-6 Insertion of laminaria before dilatation and curettage. A. Laminaria immediately after being appropriately placed with its upper end just through the internal os. B. Several hours later the laminaria is now swollen, and the cervix is dilated and softened. C. Laminaria inserted too far through the internal os; the laminaria may rupture the membranes.

1	C. Laminaria inserted too far through the internal os; the laminaria may rupture the membranes. Schneider and associates (1991) described 21 cases in which women who had a hygroscopic dilator placed changed their minds. Of 17 women who chose to continue their pregnancy, there were 14 term deliveries, two preterm deliveries, and one miscarriage 2 weeks later. None sufered infection-related morbidity, including three untreated women with cervical cultures positive for Chlamydia trachomatis. In similar circumstances with four second-trimester terminations, Siedhof and Cremer (2009) described two preterm and two term deliveries.

1	Instead of hygroscopic dilators, misoprostol is often used for cervical ripening. he typical dose is 400 �g administered sublingually, buccally, or placed into the posterior vaginal fornix 3 to 4 hours prior to surgery. Instead, oral administration proves less efective and may take longer (Allen, 2016). Another efective cervical-ripening agent is the antiprogestin mifepristone, 200 mg given orally 24 to 48 hours before surgery (Ashok, 2000). Its cost and greater delay to the procedure, however, typically favor misoprostol use instead. In comparing hygroscopic dilators and misoprostol for ripening, randomized studies show equal or slightly greater dilation with hygroscopic dilators. Other surgical parameters do not vary significantly (Bartz, 2013; Burnett, 2005; Macisaac, 1999). Hygroscopic dilators extend procedure time and can be uncomfortable, whereas misoprostol introduces fever, bleeding, and gastrointestinal side efects.

1	If not done as part of early prenatal care, hemoglobin level and h status are assessed. Screening for gonorrhea, for syphilis, and for human immunodeficiency virus, hepatitis B, and chlamydial infections is also completed. Obvious cervical infections are treated and resolved before elective procedures. To prevent postabortal infection after a first-or second-trimester surgical evacuation, prophylactic doxycycline, 100 mg orally 1 hour before and then 200 mg orally after, is provided (Achilles, 2011; American College of Obstetricians and Gynecologists, 20 16a). Prophylaxis specifically for infective endocarditis prevention in those with valvular heart disease is not required in the absence of active infection (Nishimura, 2017). No recommendations speciically address venous thromboembolism prophylaxis for suction curettage in low-risk gravidas. At our hospital, we encourage early ambulation.

1	lso called suction dilation and curettage or suction curettage, vacuum aspiration is a transcervical approach to surgical abortion. he cervix is first dilated and then products of conception are evacuated. For this, a rigid cannula is attached either to an electric-powered vacuum source or to a handheld GO-mL syringe for its vacuum source. hese are electric vacuum aspiration (EVA) or manual vacuum aspiration (MVA), respectively. Sharp dilation and curettage (D -C) in which contents are mechanically scraped out soley by a sharp curette is currently not recommended for pregnancy evacuation due to greater blood loss, pain, and procedural time (National Abortion Federation, 2016; World Health Organization, 2012). Importantly, this practice is distinguished from brief sharp curettage following initial aspiration. In one survey, this combination is employed by nearly 50 percent of abortion providers (O'Connell, 2009).

1	After bimanual examination is performed to determine uterine size and orientation, a speculum is inserted, and the cervix is swabbed with povidone-iodine or equivalent solution. he anterior cervical lip is grasped with a toothed tenaculum. The cervix, vagina, and uterus are richly supplied by nerves of Frankenhauser plexus, which lies within connective tissue lateral to the uterosacral and cardinal ligaments. Thus, vacuum aspiration at minimum requires intravenously or orally administered sedatives or analgesics, and some add a paracervical or intracervical blockade with lidocaine (llen, 2009; Renner, 2012). For local blocks, 5 mL of 1-or 2-percent lidocaine is most efective if placed immediately lateral to the insertion of the uterosacral ligaments into the uterus at 4 and 8 o'clock. An intracervical block with 5-mL aliquots of I-percent lidocaine injected at 12, 3, 6, and 9 o'clock was reported to be equally efective (Mankowski, 2009). Alternatively, general or regional anesthesia

1	intracervical block with 5-mL aliquots of I-percent lidocaine injected at 12, 3, 6, and 9 o'clock was reported to be equally efective (Mankowski, 2009). Alternatively, general or regional anesthesia may be elected.

1	Uterine sounding measures the depth and inclination of the cavity before other instrument insertion. If required, the cervix FIGURE 18-7 Dilatation of cervix with a Hegar dilator. Note that the fourth and fifth fingers rest against the perineum and buttocks, lateral to the vagina. This maneuver is an important safety measure because if the cervix relaxes abruptly, these fingers prevent a sudden and uncontrolled thrust of the dilator, a common cause of uterine perforation. is further dilated with Hegar, Hank, or Pratt dilators until a suction cannula of the appropriate diameter can be inserted. he degree of required cervical dilation roughly approximates gestational age. Hegar sizes reflect their diameter in millimeters. Pratt and Hank dilators are sized in French units, which can be converted to millimeters by dividing the French number by three.

1	With dilation, the fourth and fifth ingers of the hand introducing the dilator should rest on the perineum and buttocks as the instrument is pushed through the internal os (Fig. 18-7). his technique minimizes forceful expansion and provides a safeguard against uterine perforation.

1	Following dilation, for most first-trimester aspiration procedures, an 8-to 12-mm Karman cannula is appropriate. Small cannulas carry the risk of leaving retained intrauterine tissue postoperatively, whereas large cannulas risk cervical injury and more discomfort. To begin, the cannula is slowly moved toward the fundus until resistance is met. Suction is then activated. he cannula is gradually pulled back toward the os and is slowly turned circumferentially to cover the entire surface of the uterine cavity (Fig. 18-8). his is repeated until no more tissue is aspirated. A gentle sharp curettage can follow to remove any remaining tissue fragments (Fig. 18-9). Strong and consistent evidence supports high eicacy, safety, and patient acceptability for both MVA and EVA (Lichtenberg, 2013).

1	For abortion done at $6 weeks' gestation, a distinct drawback is that the pregnancy may be small and missed by the curette. To identiy placenta, the aspirated contents are rinsed in a strainer to remove blood, and then placed in a clear plastic container with saline and examined with back lighting (MacIsaac, 2000). Placental tissue macroscopically appears soft, luy, and feathery. A magniying lens, colposcope, or microscope can augment visualization. With gestations $7 weeks, the failed abortion rate approximates 2 percent (Kaunitz, 1985; FIGURE 18-8 A suction curette has been placed through the cervix into the uterus. The figure shows the rotary motion used to aspirate the contents. (Reproduced with permission from Hoffman BL, Corton MM: Surgeries for benign gynecologic disorders. In Hofman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.)

1	Paul, 2002). hus, if products are not clearly identiied, serial serum heG levels can be informative (Dean, 2015). In women undergoing abortion, complication rates rise with gestational age. Of these, uterine perforation and lower-genitaltract laceration are uncommon but potentially serious. In one

1	FIGURE 18-9 A sharp curette is advanced into the uterine cavity while the instrument is held with the thumb and forefinger as shown in Figure 18-7. In the movement of the curette, only the strength of these two fingers should be used. (Reproduced with permission from Hofman BL, Corton MM: Surgeries for benign gynecologic disorders. In Hofman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.) systematic review of irst-trimester abortion, the uterine per foration rate was � 1 percent, as was the cervical or vaginal laceration rate (White, 2015). Perforation is usually recognized when the instrument passes without resistance deep into the pelvis. isk factors include operator inexperience, prior cervi cal surgery or anomaly, adolescence, multiparity, and advanced gestational age (llen, 2016; Grimes, 1984). If the uterine per foration is small and fundal, as when produced by a uterine sound or narrow dilator, observation of vital

1	multiparity, and advanced gestational age (llen, 2016; Grimes, 1984). If the uterine per foration is small and fundal, as when produced by a uterine sound or narrow dilator, observation of vital signs and for uter ine bleeding is usually su.cient.

1	If a suction cannula or sharp curette passes into the peritoneal cavity, considerable intraabdominal damage can ensue. In these cases, laparotomy or laparoscopy to thoroughly examine the abdominal contents is oten the safest course. Uterine perforation is not a contraindication to completing the curettage under direct guidance during laparoscopy or laparotomy (Owen, 2017). Following curettage, uterine synechiae may form, and the risk of synechiae increases with the number of procedures. Most cases are mild and of unclear reproductive significance (Hooker, 2014). However, of Asherman syndrome cases, one series found that two thirds were linked to first-trimester curettage (Schenker, 1982).

1	Other irst-trimester abortion complications are hemorrhage, incomplete removal of products, and postoperative infections, and these are germane to both surgical and medical abortion techniques. Hemorrhage with abortion is variably defined. One supported by the Society for Family Planning is bleeding that prompts a clinical response or bleeding in excess of 500 mL (Kerns, 2013). For irst-trimester surgical abortions, hemorrhage complicates �1 percent (White, 2015). Atony, abnormal placentation, and coagulopathy are frequent sources, whereas surgical trauma is a rare cause. With medical abortion, bleeding is more common. In one study of more than 42,000 Finnish women undergoing pregnancy termination with pregnancies less 63 days, hemorrhage complicated 15 percent of medical abortion but only 2 percent of surgical cases (Niinimaki, 2009).

1	Infection is another risk. One review of surgical abortion found a cumulative rate of 0.5 percent in those given prophylaxis compared with 2.6 percent in those given placebo (Achilles, 2011). In another review of nearly 46,000 irst-trimester abortions, the postoperative infection rate was <0.3 percent for either surgical or medical methods (Upadhyay, 2015). Incomplete abortion may require reevacuation. For medical abortion, this neared 5 percent in one systematic review (Raymond, 2013). Reaspiration rates following surgical abortion are typically <2 percent (Ireland, 2015; Niinimaki, 2009). In sum, first-trimester surgical abortion ofers higher e.cacy rates (96 to 100 percent) than medical abortion (83 to 98 percent). Medical abortion also carries a greater cumulative risk of complications, although diferences are small (Lichtenberg, 2013). hese are balanced against the greater privacy of medical abortion and the more invasive steps of curettage.

1	In appropriately selected women, outpatient medical abortion is an acceptable option for pregnancies with a menstrual age <63 days (American College of Obstetricians and Gynecolo gists (20 16c). Although suitable at later gestational ages, success rates are lower. gestation, one third are completed medically in the United States Qatlaoui, 2016). hree medications are used alone or in combination: mifepristone, methotrexate, and misoprostol. Of these, mifepristone augments uterine contractility by reversing progesterone-induced myometrial quiescence, whereas misopro scol directly stimulates the myometrium. Both also ripen the cervix (Mahajan, 1997; Tang, 2007). Methotrexate acts on tro phoblast and halts implantation. It is used less frequently now due to current availability of the more efective mifepristone.

1	Contraindications to medical abortion have evolved from exclusion criteria that were used in initial clinical trials. Cau tions include current intrauterine device; severe anemia, coagu lopathy, or anticoagulant use; long-term systemic corticosteroid liver, renal, pulmonary, or cardiovascular disease; or uncon trolled hypertension (Guiahi, 2012). Of note, misoprostol is suitable for early pregnancy failure in those with prior uterine surgery (Chen, 2008). Methotrexate and misoprostol are both teratogens. Thus there must be a commitment to completing the abortion once these drugs are given (Aufret, 2016; Hyoun, 2012; Kozma, 2011). With mifepristone, for women who choose to continue their preg nancies after exposure, the ongoing pregnancy rate ranges from 10 to 46 percent (Grossman, 2015). The associated major mal formation rate was 5 percent in one series of 46 exposed preg nancies (Bernard, 2013).

1	Several dosing schemes are efective, and some are shown in Table 18-7. Because of its greater e.cacy, mifepristonel misoprostol combinations are favored. Presently, for gestations up to 63 days, the most widely accepted regimen is mifepristone, 200 mg given orally on day 0 and followed in 24 to 48 hours by misoprostol 800 �g, administered by a vaginal, buccal, or sublingual route (American College of Obstetricians and Gynecologists, 2016c). Another earlier regimen used a 600-mg oral mifepristone dose followed in 48 hours by a 400-�g oral misoprostol dose (Spitz, 1998). If desired, mifepristone and misoprostol may be self-administered at home (Chong, 2015). At Planned Parenthood clinics, for irst-trimester medical abortion, doxycycline 100 mg is taken orally daily for 7 days and begins with abortifacient administration (Fjerstad, 2009). he woman is then discharged home and appointed to return in 1 to 2 weeks.

1	Symptoms following misoprostol are common within 3 hours and include vomiting, diarrhea, fever, and chills. Bleeding and cramping with medical termination typically is signiicantly worse than with menses. Thus, adequate analgesia, usually including a narcotic, is provided. If bleeding soaks two or more pads per hour for at least 2 hours, the woman is instructed to contact her provider to determine whether she needs to be seen . At the follow-up appointment, routine postabortal sonographic examination is typically unnecessary (Clark, 2010). Instead, assessment of the clinical course along with bimanual pelvic examination is recommended. If sonography is indicated TABLE 18-7. Various Regimens for Medical Termination of Pregnancy aMifepristone, 200-600 mg orally; followed in 24-48 hr by: bMisoprostol, 200-600 �g orally or 400-800 �g vaginally, buccally, or sublingually

1	Methotrexate/Misoprostol dMethotrexate, 50 mg/m2 BSA intramuscularly or orally; followed in 3-7 days by: eMisoprostol, 800 �g vaginally. Repeat if needed 1 week after methotrexate initially given Mifepristone, 200 mg orally; followed in 24-48 hr byj Misoprostol, 400 �g vaginally or buccally every 3 hr up to 5 doses Misoprostol, 600-800 �g vaginally; followed by 400 �g vaginally or buccally every 3 hr up to 5 doses 50 units oxytocin in 500 mL of normal saline infused during 3 hr; then 1-hr diuresis (no oxytocin); then escalate sequentially in a similar fashion through 150,200,250, and finally 300 units oxytocin each in 500 mL normal saline aOoses of 200 versus 600 mg similarly effective. bOral route may be less effective and have more nausea and diarrhea. Sublingual route has more side effects than vaginal route.

1	3-12 hours given vaginally; 3-4 hours given sublingually. dEficacy similar for routes of administration. eSimilar efficacy when given on day 3 versus day 5. BSA = body surface area. Pymar, 2001; Raghavan, 2009; Schaff, 2000; Shannon, 2006; von Hertzen, 2003, 2007, 2009, 2010; Winikof, 2008.

1	due to concern for failed abortion or for bleeding, unnecessary surgery can be avoided if scans are interpreted appropriately. Specifically, if no gestational sac is seen and there is no heavy bleeding, then intervention is unnecessary. This is true even when, as is common, the uterus contains sonographically evident debris (Paul, 2000). vleasurements < 15 mm and < 30 mm have been used as thresholds to signal evacuation success (Nielsen, 1999; Zhang, 2005). Another study reported that a multilayered sonographic pattern indicated a successful abortion (Tzeng, 2013). Last, hCG values may be informative. Compared with preprocedural levels, Barnhart and coworkers (2004b) found declines of 88 percent at day 3 and 82 percent at day 8 following misoprostol administration correlated with a 95-percent rate of successful abortion completion.

1	In the second trimester, fetal anomaly or death, maternal health complications, inevitable abortion, or desired termination may be indications for uterine evacuation. As in the irst trimester, available options are medical or surgical. But, in the second trimester, dilation and evacuation (D & E) rather than suction D & C is dictated because of fetal size and bony structure. Of options, D & E is a common means of second-trimester induced abortion in the United States. Of legally obtained abortions in 2013, 9 percent were performed by D & E at gestational agesi> 13 weeks Oarlaoui, 2016). Many of the surgical and medical steps for second-trimester abortion mirror those in the irst trimester, and diferences are emphasized here.

1	With D & E, wide mechanical cervical dilation precedes evacuation of fetal parts. he degree needed rises with fetal gestational age, and inadequate dilatation risks cervical trauma, uterine perforation, or tissue retention (Peterson, 1983). Thus, presurgical cervical preparation is advised, and main options include hygroscopic dilators or misoprostol. With laminaria, overnight preparation ofers optimal cervical dilation (Fox, 2014). Uncommonly, laminaria may fail to adequately dilate the cervix, and serialilaminaria insertion with an increasing number of tents over several days is one option (Stubblefield, 1982). Supplementing laminaria with misoprosrol or mifeprisrone is another choice (Ben-Ami, 201i5). Dilapan-S is also suitable for cervical preparation. It may be preferable for same-day procedures as this device achieves its maximal efect in 4 to 6 hours (Newmann, 2014).

1	Dilapan-S is also suitable for cervical preparation. It may be preferable for same-day procedures as this device achieves its maximal efect in 4 to 6 hours (Newmann, 2014). Misoprostol can be used instead of hydroscopic dilators for cervical preparation. The typical dose is 400 1g given vaginally or buccally 3 to 4 hours prior to D & E. Randomized trials vary regarding the ability of misoprostol to achieve results equal to that with hydroscopic dilators (Bartz, 2013; Goldberg, 2005; Sagiv, 2015). vIisoprostol added to laminaria ofers small increases in dilation but also greater side efects (Edelman, 2006).

1	Fewer studies have evaluated mifepristone for cervical ripening. In one, mifepristone alone provided less dilation than hydroscopic dilators (BO'gatta, 2012). In another trial, mifepristone added 48 hours before misoprostol created greater cervical dilation compared with misoprostol alone (Carbonell, 2007). Last, Goldberg and associates (2015) compared hygroscopic dilation with or without added mifepristone. They found no diferences for gestations < 19 weeks, but the combination aided procedures for later ages. In sum, hygroscopic dilators are consistently efective for cervical preparation before D & E. For those desiring same-day procedures, Dilapan-S alone or misoprostol alone may ofer advantages. Layering agents may be most helpful for later gestations or for an inadequate response from initial hygroscopic dilators alone. Yet, layering adds cost and potential side efects (Shaw, 2016).

1	With elective abortion, some choose to induce fetal demise prior to D & E to avert a live birth or to avoid violating the Partial Birth Abortion Ban Act, cited later (Diedrich, 2010). For this, an intracardiac potassium chloride injection or a 1-mg intraamnionic or intrafetal digoxin injection is frequently used prior to cervical ripening (Sfakianaki, 2014; White, 2016).

1	During D & E, sonography can be used as an adjunct in all cases or selectively in more challenging ones. Perioperative antibiotic prophylaxis mirrors that for first-trimester procedures (p. 359). To reduce postprocedure bleeding, vasopressin, 2 to 4 units in 20 mL of saline or anesthetic, can be injected intracervically or as part of a paracervical block (Kerns, 2013; Schulz, 1985). Once adequate cervical dilation is achieved, the initial surgical step drains amnionic fluid with an 11-to 16-mm suction cannula or with amniotomy and gravity. his reduces the risk of amnionic fluid embolism and brings the fetus into the lower uterine segment for removal (Owen, 2017; Prager, 2009). For pregnancies beyond 16 weeks, the fetus is extracted, usually in parts, using Sopher forceps or other destructive instruments. With complete removal of the fetus, a large-bore vacuum curette is used to remove the placenta and remaining tissue.

1	Major complications are infrequent with D & E, and rates range from 0.2 to 2 percent in large series (Cates, 1982; Lederie, 2015; Peterson, 1983). These include uterine perforation, cervical laceration, uterine bleeding, and postabortal infection. Rare complications include disseminated intravascular coagulopathy or amnionic luid embolism (Ray, 2004; York,i2012). Placenta previa or the accrete syndromes can raise D & E risks. Once diagnosed, placenta accreta typically prompts hysterec tomy (Matsuzaki, 2015). For placenta previa, D & E is preferred to quickly evacuate the placenta, but the ability to transfuse present (American College of Obstetricians and Gynecologists, 2017h; Perriera, 2017). Medical induction may be elected, but the risk for transfusion is greater than with D & E (Nakayama, 2007; Ruano, 2004). Data are few, but predelivery uterine artery embolization may lower bleeding risks (Pei, 2017).

1	Prior cesarean delivey is not a contraindication for 0 & E and may be preferred over prostaglandins for those with multiple prior hysterotomies (Ben-Ami, 2009; Schneider, 1994). During medical abortion, the uterine rupture rate is 0.4 percent with one prior cesarean delivery (Berghella, 2009). From fewer data, the rate may reach 2.5 percent with two or more prior cesarean deliveries (Andrikopoulou, 2016). If a medical agent is elected in those with prior cesarean hysterotomy, misoprostol is an option. Prostaglandin E2 (PGE2) appears to pose similar risk (Ie Roux, 2001; Reichman, 2007).

1	Of these, dilation and extraction (D & ) is similar to 0 & E except that a suction cannula is used to evacuate the intracranial contents after delivery of the fetal body through the dilated cervix. This aids extraction and minimizes uterine or cervical injury from large instruments or fetal bones. 0 & X is also called an intact D & . In political parlance, this procedure has been termed partial birth abortion. In some women with second-trimester pregnancies who desire sterilization, hysterotomy with tubal ligation is reason able. If there is signiicant uterine disease, then hysterectomy may provide ideal treatment. In some cases of a failed second trimester medical induction, either of these may be considered.

1	Principal among noninvasive methods is a mifepristone plus misoprostol regimen or misoprostol alone (see Table 18-7). Of these two options, the combined regimen yields a shorter termination duration (Kapp, 2007; Ngoc, 201l). Hygroscopic dilators may speed the time to delivery with this combined regimen (Mazouni, 2009; Vincienne, 2017). In selecting misoprostol routes, oral administration leads to a longer time to delivery compared with vaginal or sublingual routes (Dickinson, 2014). Prophylactic antibiotics are not typically given, and infection surveillance during labor is instead applied (Achilles, 2011).

1	Another induction agent, PGE2, shows similar eicacy and side efects compared with misoprostol Gain, 1994; Jansen, 2008). Simultaneous administration of an antiemetic such as metoclopramide (Reglan), an antipyretic such as acetaminophen, and an antidiarrheal such as diphenoxylate/atropine (Lomotil) will help prevent or treat symptoms. Dinoprostone (Prostin) is an available PGE2 in the United States. However, its greater cost and poor pharmacologic stability at room temperature may make it less attractive than misoprostol. Of other agents, high-dose intravenous oxytocin in saline will result in second-trimester abortion in 80 to 90 percent of cases (see Table 18-7). However, by comparison, misoprostol leads to higher successful induction rates and faster delivery times (Alavi, 2013).

1	Rarely used, ethacridine lactate is an organic antiseptic that activates myometrial mast cells to release prostaglandins (Olund, 1980). Placed extraovularly, that is, extraamnionically, it is associated with longer times to delivery and greater complication rates compared with misoprostol (Boza, 2008). For second-trimester gestations, D & E or medical induction is suitable clinically and psychologically. Thus, patient input and clinical indication guide selection (Burgoine, 2005; Kerns, 2012). Once delivered, viewing and holding the fetus mayior may not be desired by the patient (Sloan, 2008).

1	Evaluation of a stillborn fetus is described in Chapter 35 (p. 646). One component is autopsy, which can also be valuable for second-trimester losses or terminations due to anomaly. For example, in a stud) of 486 women of all ages with secondtrimester miscarriage, fetal malformations were identiied in 13 percent 000, 2009). In another, a third of otherwise normal fetuses had associated chorioamnionitis that was judged to have preceded labor (Allanson, 2010). Indeed, according to Srinivas and associates (2008), 95 percent of placentas in midtrimester miscarriages are abnormal. Other abnormalities are vascular thromboses and infarctions. With either surgical or medical abortion, subsequent autopsy can yield information, but fragmented D & E specimens may provide less information than intact fetuses (Gawron, 2013; Lal, 2014). Karyotyping can be performed on samples from either method (Bernick, 1998).

1	Legally induced abortion in the United States has a low associated mortality rate, and from 2008 to 2012, the rate was < 1 death per 100,000 procedures Oatlaoui, 2016). Earlyiabortions are safer. For example, Zane and coworkers (2015) found a mortality rate of 0.3 deaths per 100,000 procedures at;8 weeks' gestation; a rate of2.5 at 14 to 17 weeks; and 6.7 at ::18 weeks. As emphasized by Raymond and Grimes (2012), mortality rates are 14-fold greater for pregnancies that are continued.

1	Data relating abortion to overall maternal health and to subsequent pregnancy outcome are limited. From studies, there is no evidence for excessive mental disorders (Biggs, 2017; Munk-Olsen, 2011). here are few data regarding subsequent reproductive health, although the rates of infertility or ectopic pregnancy are not increased. Exceptions may stem from postabortal infections, especially those caused by C trachomatis. Of subsequent adverse pregnancy outcomes, several studies note an approximate 1.5-fold greater incidence of preterm delivery following surgical evacuation (Lemmers, 2016; Makhlouf, 2014; Saccone, 2016). his risk accrues with the number of terminations (Hardy, 2013; Klemetti, 2012). Subsequent pregnancy outcomes are similar regardless of whether a prior induced abortion was completed medically or surgically (Mannisto, 2013; Virk, 2007).

1	After medical or surgical management of an early pregnancy termination or loss, ovulation na) resume as early as 8 days, but the average time is 3 weeks (Lahteenmaki, 1978; Stoddard, 2011). Thus, unless another pregnancy is imminently desired, efective contraception is initiated to help lower the unintended pregnancy rate, which was 45 percent in 2011 in the United States (Finer, 2016). In suitable candidates described in Chapter 38 (p. 685), an intrauterine device can be inserted after the procedure or medical abortion is completed (Bednarek, 2011; Korjamo, 2017). Alternatively, any of the various forms of hormonal contraception can be initiated at this time (Curtis, 2016).

1	For women who desire another pregnancy, conception need not be delayed. Specifically, Wong and colleagues (2015) found similar live-birth rates in groups conceiving within 3 months of first-trimester pregnancy loss compared with groups with later conception. Others have found similarly reassuring results using an interval-to-conception threshold of 6 months (Kangatharan, 2017; Love, 2010). Achilles SL, Reeves MF, Society of Family Planning: Prevention of infection after induced abortion: release date October 2010: SFP guideline 20102. Contraception 83(4):295, 201r1 Achiron R, Tadmor 0, Mashiach S: Heart rate as a predictor of first-trimester spontaneous abortion after ultrasound-proven viability. Obstet Gynecol 78(3 Pt 1):330 Alavi A, Rajaei M, mirian M, et al: Misoprostol versus high dose oxytocin and laminaria in termination of pregnancy in second-trimester pregnancies. Electron Physician 5(4):713, 2013

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1	Witter FR: Negative sonographic indings followed by rapid cervical dilatation due to cervical incompetence. Obstet Gynecol 64: 136, 1984 Wong LF, Schliep KC, Silver RM, et al: The efect of a very short interpregnancy interval and pregnancy outcomes following a previous pregnancy loss. Am 1 Obstet Gynecol 212(3):375.e1, 2015 World Health Organization: Safe Abortion: Technical and Policy Guidance for Health Systems, 2nd ed. Geneva, WHO, 2012 Wyatt PR, Owolabi T, Meier C, et al: Age-speciic risk of fetal loss observed in a second trimester serum screening population.r·Am 1 Obstet Gynecol 192:240,r2005 York S, Lichtenberg ES: Characteristics of presumptive idiopathic disseminated intravascular coagulation during second-trimester induced abortion. Contraception 85(5):489, 2012 Yu D, Wong YM, Cheong Y, et al: Asherman syndrome-one century later. Fertil Steril 89(4):759, 2008

1	Yu D, Wong YM, Cheong Y, et al: Asherman syndrome-one century later. Fertil Steril 89(4):759, 2008 Zane S, Creanga A, Berg Cl, et al: Abortion-related mortality in the United States: 1998-2010. Obstet GynecoIr126(2):258, 2015 Zaveri V, Aghajafari F, Amankwah K, et al: Abdominal versus vaginal cerclage after a failed transvaginal cerclage: a systematic review. Am 1 Obstet Gynecol 187:868,r2002 Zeisler H, laura A, Bancher-Todesca D, et al: Prophylactic cerclage in pregnancy. Efect in women with a history of conization. 1 Reprod Med 42(7):390, 1997 Zhang 1, Gilles 1M, Barnhart K, et al: A comparison of medical management with misoprostol and surgical management for early pregnancy failure. N Engll Med 353:761,r2005 TUBAL PREGNANCY. .e..e.....e..e....e...e....e....e..e.371 CLINICAL MANIFESTATIONS ........e.......e.e..e..e.372 MUL TIMODALITY DIAGNOSIS .e...........e... ...373 MEDICAL MANAGEMENT ........e..e..e....e...e..... 377

1	CLINICAL MANIFESTATIONS ........e.......e.e..e..e.372 MUL TIMODALITY DIAGNOSIS .e...........e... ...373 MEDICAL MANAGEMENT ........e..e..e....e...e..... 377 SURGICAL MANAGEMENT ..e...e....e.e....e....e..e... 378 INTERSTITIAL PREGNANCY .e.e..e.....e..e....e. ...e.380 CESAREAN SCAR PREGNANCYe. . . . . . . ............. 381 CERVICAL PREGNANCY. . . . . . . . . . . . . ............. 382 ABDOMINAL PREGNANCY . . . . . . . . . . ............. 383 OVARIAN PREGNANCYe. . . . . . . . . . . . . ............. 384 As soon as an unruptured extra-uterine pregnancy is positivey diagnosed, its immediate removal by laparotomy is urgenty indicate, since rupture may occur at any time and the patient die rom haemorrhage bore operative aid can be obtaine. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) Following fertilization and fallopian tube transit, the blastocyst normally implants in the endometrial lining of the uterine cavity. Implantation elsewhere is considered ectopic and accounts for 0.5 to 1.5 percent of all irst-trimester pregnancies in the United States (Hoover, 2011; Stulberg, 2014). his small proportion disparately accounts for 3 percent of all pregnancyrelated deaths (Creanga, 2017). Fortunately, urine and serum beta-human chorionic gonadotropin (�-hCG) assays and transvaginal sonography allow earlier diagnosis. As a result, both maternal survival rates and conservation of reproductive capacity are improved.

1	Nearly 95 percent of ectopic pregnancies are implanted in the various segments of the fallopian tube. hese segments are shown in Chapter 2 (Fig. 2-14, p. 29). he ampulla (70 percent) is the most frequent site, followed by isthmic (12 percent), imbrial (1i1 percent), and interstitial tubal pregnancies (2 percent) (Bouyer, 2002). The remaining 5 percent of nontubal ectopic pregnancies implant in the ovary, peritoneal cavity, cervix, or prior cesarean scar. Occasionally, a multifetal pregnancy contains one conceptus with normal uterine implantation that coexists with one implanted ectopically. he natural incidence of these heterotopic pregnancies approximates 1 per 30,000 pregnancies (Reece, 1983). However, with assisted reproductive technologies (ART), their incidence is 9 in 10,000 pregnancies (Perkins, 2015). Rarely, twin tubal pregnancy with both embryos in the same tube or with one in each tube has been reported (Eze, 2012; Goswami, 2015).

1	Regardless of location, D-negative women with an ectopic pregnancy who are not sensitized to D-antigen are given IgG anti-D immunoglobulin (American College of Obstetricians and Gynecologists, 2017). In irst-trimester pregnancies, a 50-�g or 300-�g dose is appropriate, whereas a standard 300-�g dose is used for later gestations (Chap. 15, p. 305).

1	Abnormal fallopian tube anatomy underlies many cases of tubal ectopic pregnancy. Surgeries for a prior tubal pregnancy, for fertility restoration, or for sterilization confer the highest risk. After one previous ectopic pregnancy, the chance of another is increased ivefold (Bhattacharya, 2012). Prior sexually transmitted disease or other tubal infection, which can distort normal tubal anatomy, is another factor. Speciically, one episode of salpingitis can be followed by a subsequent ectopic pregnancy in up to 9 percent of women (Westrom, 1992). Peritubal adhesions subsequent to salpingitis, appendicitis, or endometriosis can also increase chances. Sapingitis isthmica nodosa, which is a condition in which epithelium-lined diverticula extend into a hypertrophied muscularis layer, is another (Bolaji, 2015). Finally, congenital fallopian tube anomalies, especially those secondary to in utero diethylstilbestrol exposure, can predispose (Hoover, 2011).

1	Infertility, as well as the use of ART to overcome it, is linked to substantively increased risks for ectopic pregnancy (Clayton, 2006). With ART, the ectopic pregnancy rate in the United States between 2001 and 2011 was 1.6 percent (Perkins, 2015). And "atypical" implantations-cornual, abdominal, cervical, ovarian, and heterotopic pregnancy-are more frequent. Smoking is another known association, although the underlying mechanism is unclear (Hyland, 2015). Last, with any form of contraception, the absolute number of ectopic pregnancies is decreased because pregnancy occurs less often. However, with some contraceptive method failures, the relative number of ectopic pregnancies is increased. Examples include tubal sterilization, copper and progestin-releasing intrauterine devices (IUDs), and progestin-only contraceptives (Chap. 38, p. 680).

1	With tubal pregnany, because the fallopian tube lacks a submucosalilayer, the fertilized ovum promptly burrows through the epithelium. he zygote comes to lie near or within the muscularis, which is invaded by rapidly proliferating trophoblast. The embryo or fetus in an ectopic pregnancy is oten absent or stunted. Outcomes of ectopic pregnancy include tubal rupture, tubal abortion, or pregnancy failure with resolution. With rupture, the invading expanding conceptus and associated hemorrhage can tear rents in the fallopian tube (Fig. 19-1). Tubal ectopic pregnancies usually burst spontaneously but may occasionally rupture following coitus or bimanual examination.

1	FIGURE 19-1 Ruptured ampullary early tubal pregnancy. (Used with permission from Dr. Togas Tulandi.) lternatively, the pregnancy may pass out the distal fallopian tube. Tubal abortion frequency depends in part on the initial implantation site, and distal implantations are favored. Subsequently, hemorrhage may cease and symptoms eventually disappear. But bleeding can persist as long as products remain in the tube. Blood slowly trickles from the tubal fimbria into the peritoneal cavity and typically pools in the rectouterine cul-de-sac. If the fimbriated extremity is occluded, the fallopian tube may gradually become distended by blood, forming a hematosalpinx. Uncommonly, an aborted fetus will implant on a peritoneal surface and become an abdominal pregnancy, which is discussed on page 383. Last, an unknown number of ectopic pregnancies spontaneously fail and are reabsorbed. This may be documented now more regularly with the advent of sensitive 3-hCG assays.

1	Last, an unknown number of ectopic pregnancies spontaneously fail and are reabsorbed. This may be documented now more regularly with the advent of sensitive 3-hCG assays. Distinctions beween "acute" ectopic pregnancy just described and "chronic" ectopic pregnancy can be drawn. The more common acute ectopic pregnancies are those with a high serum 3hCG level and rapid growth, leading to a timely diagnosis. hese carry a higher risk of tubal rupture (Barnhart, 2003c). With chronic ectopic pregnancy, abnormal trophoblast dies early, and thus negative or low, static serum 3-hCG levels are found (Brennan, 2000). Chronic ectopic pregnancies typically rupture late, if at all, but commonly form a complex pelvic mass, which oten is the reason prompting diagnostic surgery (Cole, 1982; Ugur, 1996).

1	Earlier patient presentation and more precise diagnostic technology typically allow identiication before rupture. In these cases, symptoms and signs of ectopic pregnancy are often subtle or even absent. The woman does not suspect tubal pregnancy and assumes that she has a normal early pregnancy or is having a miscarriage.

1	With later diagnosis, the classic triad is delayed menstruation, pain, and vaginal bleeding or spotting. With tubal rupture, lower abdominal and pelvic pain is usually severe and frequently described as sharp, stabbing, or tearing. Abdominal palpation elicits tenderness. Bimanual pelvic examination, especially cervical motion, causes exquisite pain. The posterior vaginal fornix may bulge from blood in the rectouterine cul-de-sac, or a tender, boggy mass may be felt beside the uterus. The uterus can also be slightly enlarged due to hormonal stimulation. Symptoms of diaphragmatic irritation, characterized by neck or shoulder pain, especially on inspiration, develop in perhaps half of women with sizable hemoperitoneum.

1	Some degree of vaginal spotting or bleeding is reported by 60 to 80 percent of women with tubal pregnancy. Although profuse vaginal bleeding suggests an incomplete abortion, such bleeding occasionally is seen with tubal gestations. Moreover, tubal pregnancy can lead to signiicant intraabdominal hemorrhage. Responses to moderate bleeding include no change in vital signs, a slight rise in blood pressure, or a vasovagal response with bradycardia and hypotension. Blood pressure will fall and pulse will rise only if bleeding continues and hypovolemia FIGURE 19-2 This decidual cast was passed by a patient with a tubal ectopic pregnancy. The cast mirrors the shape of the endometrial cavity, and each arrow marks the portion of decidua that lined the cornua. becomes significant. Vasomotor disturbances develop, ranging from vertigo to syncope.

1	becomes significant. Vasomotor disturbances develop, ranging from vertigo to syncope. Even after substantive hemorrhage, hemoglobin or hematocrit readings may at irst show only a slight reduction. Hence, after an acute hemorrhage, a trending decline in hemoglobin or hematocrit levels over several hours is a more valuable index of blood loss than is the initial level. In approximately half of women with a ruptured ectopic pregnancy, varying degrees of leukocytosis up to 30,000/�L may be documented.

1	Decidua is endometrium that is hormonally prepared for pregnancy, and the degree to which the endometrium is converted with ectopic pregnancy is variable. Thus, in addition to bleeding, women with ectopic tubal pregnancy may pass a decidual cast. his is the entire sloughed endometrium that takes the form of the endometrial cavity (Fig. 19-2). Importantly, decidual sloughing may also occur with uterine abortion. hus, tissue is carefully evaluated visually by the provider and then histologically for evidence of a conceptus. If no clear gestational sac is seen or if no villi are identified histologically within the cast, then the possibility of ectopic pregnancy must still be considered.

1	he diferential diagnosis for abdominal pain coexistent with pregnancy is extensive. Pain may derive from uterine conditions such as miscarriage, infection, degenerating or enlarging leiomyomas, or round-ligament pain. Adnexal disease may include ectopic pregnancy; hemorrhagic, ruptured, or torsed ovarian masses; salpingitis; or tuboovarian abscess. Last, appendicitis, cystitis, renal stone, and gastroenteritis are more common nongynecological sources of lower abdominal pain in early pregnancy.

1	Several algorithms have been proposed to identiY ectopic pregnancy. Most include these key components: physical indings, transvaginal sonography (TVS), serum 3-hCG level measurement-both the initial and the subsequent pattern of rise or decline, and diagnostic surgery, which includes dilation and curettage (D&C), laparoscopy, and occasionally, laparotomy (Fig. 19-3). Algorithm use applies only to hemodynamically stable women, and those with presumed rupture undergo prompt surgical therapy. For a suspected unruptured ectopic pregnancy, all diagnostic strategies involve trade-ofs. Strategies that maximize detection of ectopic pregnancy may result in termination of a normal intrauterine pregnancy (IUP). Conversely, those that reduce the potential for normal pregnancy interruption will delay ectopic pregnancy diagnosis. Patient desires for the index pregnancy are also discussed and may inluence these trade-ofs.

1	apid and accurate determination of pregnancy is essential to iden tiY an ectopic pregnancy. Current pregnancy tests use enzyme linked immunosorbent ssays (ELISAs) for the beta subunit of hCG. With these assays, lower limits of detection are 20 to 25 mIU/mL for urine and :;5 mIU/mL for serum (Greene, 2015). With bleeding or pain and a positive pregnancy test result, an initial TVS is typically performed to identiY gestation loca tion. If a yolk sac, embryo, or fetus is identiied within the uterus or the adnexa, then a diagnosis can be made. In many cases, however, TVS is nondiagnostic, and tubal pregnancy is still a possibility. In these cases in which neither intrauterine nor extrauterine pregnancy is identiied, the term pregnancy of unknown location (PUL) is used until additional clinical infor mation allows determination of pregnancy location.

1	Levels above the Discriminatory Zone. Several investigators ure to visualize a uterine pregnancy indicates that the pregnancy either is not alive or is ectopic (Barnhart, 1994). Some institu tions set their discriminatory threshold at ::1500 mIU/mL, whereas others use ::2000 mIU/mL. Connolly and associates (2013) suggested an even higher threshold. hey noted that with live uterine pregnancies, a gestational sac was seen 99 percent of the time with a discriminatory level of >3510 mIU/mL.

1	If the initial 3-hCG level exceeds the set discriminatory level and no evidence for an IUP is seen with TVS, then ectopic pregnancy is a concern. The diagnosis is narrowed in most cases to a failing IUP, a recent complete abortion, or an ectopic pregnancy. Early multifetal gestation also remains a possibility. Without clear evidence for ectopic pregnancy, serial 3-hCG level assessment is reasonable, and a level is checked 48 hours later. This averts unnecessary methotrexate administration and avoids harming an early normal multifetal pregnancy. With greater concern for an ectopic gestation, D&C is another option to distinguish an ectopic from a failing IUP. Importantly, patient factors greatly influence these decisions.

1	Levels below the Discriminatoy Zone. If the initial 3-hCG level is below the set discriminatory value, pregnancy location is often not technically discernible with TVS. With these PULs, serial 3-hCG level assays are done to identiY patterns that indicate either a growing or failing IUP. Levels that rise or fall outside these expected parameters increase the concern for ectopic pregnancy. hus, appropriately selected women with a possible ectopic pregnancy, but whose initial 3-hCG level is below the discriminatory threshold, are seen 2 days later for further evaluation. Trends in levels aid diagnosis. IUP t Prenatal care FIGURE 19-3 One suggested algorithm for evaluation of a woman with a suspected ectopic pregnancy. aExpectant management, D&C, or medical regimens are suitable options. bMay consider repeat 3-hCG level if normal IUP suspected. 3-hCG = beta human chorionic gonadotropin; D&C = dilatation and curettage; IUP = intrauterine pregnancy; VS

1	Nondiagnostic t D&Cb Chorionic villi Absent t Treat ectopic pregnancy Present = transvaginal sonography. With early normal progressing IUPs, Barnhart and coworkers (2004b) reported a 53-percent 48-hour minimum rise with a 24-hour minimum rise of 24 percent. Seeber and associates (2006) found an even more conservative minimal 35-percent 48-hour rise in normal IUPs. With multifetal gestation, this same anticipated rate of rise is expected (Chung, 2006). Despite these guidelines, Silva and colleagues (2006) caution that a third of women with an ectopic pregnancy will have a 53-percent rise at 48 hours. They further reported that no single pattern characterizes ectopic pregnancy and that approximately half of ectopic pregnancies will show decreasing �-hCG levels, whereas the other half will have increasing levels. Also, despite a declining �-hCG level, a resolving ectopic pregnancy may rupture.

1	With a failing IUP, patterned rates of �-hCG level decline can also be anticipated. Following spontaneous abortion, rates decline by 21 to 35 percent at 48 hours and 68 to 84 percent at 7 days. Of note, these ranges relect that �-hCG percentages drop faster if the initial �-hCG level is higher (Barnhart, 2004a). With resolving PULs, Butts and coworkers (2013) found greater rates of decline that ranged from 35 to 50 percent at 48 hours and 66 to 87 percent at 7 days for starting hCG values between 250 and 5000 mIU/mL. In pregnancies without these expected rises or falls in �-hCG levels, distinction between a nonliving IUP and an ectopic pregnancy may be aided by additional �-hCG levels (Zee, 2014). Again delay is balanced against the risk from rupture. D&C is an option and provides a quicker diagnosis balanced against normal pregnancy interruption. Before curettage, a second TVS examination may be indicated and may display new informative indings.

1	A single serum progesterone measurement may clariy the diagnosis in a few cases (Stovall, 1989, 1992). A value exceeding 25 ng/mL excludes ectopic pregnany with 92-percent sensitivity (Lipscomb, 1999a; Pisarska, 1998). Conversely, values <5 ng/mL are found in only 0.3 percent of normal progressing IUPs (Mol, 1998; Verhaegen, 2012). hus, values <5 ng/mL suggest either a nonliving IUP or an ectopic pregnancy. Because in most ectopic pregnancies, progesterone levels range between 10 and 25 ng/mL, the clinical utility of this practice is limited. One caveat is that pregnancy achieved with assisted reproductive technology may be associated with higher than usual progesterone levels (Perkins, 2000).

1	Endometrial Findings. In a woman in whom ectopic pregnancy is suspected, TVS is performed to look for findings indicative of uterine or ectopic pregnancy. During endometrial cavity evaluation, an intrauterine gestational sac is usually visible between 4Y2 and 5 weeks. The yolk sac appears between 5 and 6 weeks, and a fetal pole with cardiac activity is irst detected at 5Y2 to 6 weeks (Fig. 9-3, p. 159). With transabdominal sonography, these structures are visualized slightly later. In contrast, with ectopic pregnancy, a trilaminar endometrial pattern can be diagnostic (Fig. 19-4). Its speciicity is 94 percent, but with a sensitivity of only 38 percent (Hammoud, 2005). In addition, Moschos and Twickler (2008b) determined sac within the endometrial cavity. Its cavity-conforming shape and central location are characteristic of these anechoic fluid col lections. Distal to this fluid, the endometrial stripe has a trilaminar pattern, which is a common finding with ectopic pregnancy.

1	(Reproduced with permission from Gala RB: Ectopic pregnancy. In Hoffman BL, Schorge JO, Bradshaw KD, et al: Williams Gynecology, 3rd ed. New York, McGraw-Hili Education; 2016. Photo contributor: Dr. Elysia Moschos.) in women with a pregnancy of unknown location at presenta tion that no normal IUPs had a stripe thickness <8 mm.

1	Dr. Elysia Moschos.) in women with a pregnancy of unknown location at presenta tion that no normal IUPs had a stripe thickness <8 mm. Anechoic luid collections, which might normally suggest an early intrauterine gestational sac, may also be seen with ectopic pregnancy. These include pseudogestational sac and decidual cyst. First, a pseudosac is a fluid collection between the endometrial layers and conforms to the cavity shape (see Fig. 19-4). If a pseudosac is noted, the risk of ectopic pregnancy is increased (Hill, 1990; Nyberg, 1987). Second, a decidual cyst is identiied as an anechoic area lying within the endometrium but remote from the canal and often at the endometrial-myometrial border. Ackerman and colleagues (1993 b) suggested that this finding represents early decidual breakdown and precedes decidual cast formation.

1	hese two indings contrast with the intradecidual sign seen with uterine pregnancies. With this, an early gestational sac is seen as an anechoic sac eccentrically located within one of the endometrial stripe layers (Dashefsky, 1988). The American College of Obstetricians and Gynecologists (2016) advises caution in diagnosing an IUP in the absence of a deinite yolk sac or embryo.

1	Adnexal Findings. he sonographic diagnosis of ectopic pregnancy rests on visualization of an adnexal mass separate from the ovary (Fig. 19-5). If fallopian tubes and ovaries are visualized and an extrauterine yolk sac, embryo, or fetus is identified, then an ectopic pregnancy is conirmed. In other cases, a hyperechoic halo or tubal ring surrounding an anechoic sac is seen (Nadim, 2017). Alternatively, an inhomogeneous adnexal mass is usually caused by hemorrhage within the ectopic sac. Overall, approximately 60 percent of ectopic pregnancies are seen as an inhomogeneous mass adjacent to the ovary; 20 percent appear as a hyperechoic ring; and 13 percent have an obvious gestational sac with a fetal pole (Condous, 2005). Importantly, not all adnexal masses represent an ectopic pregnancy, and integration of sonographic findings with other clinical information is necessary. adnexal mass. In this last image, color Doppler shows a classic "ring

1	adnexal mass. In this last image, color Doppler shows a classic "ring FIGURE 19-5 Various transvaginal sonographic findings with ectopic tubal pregnancies. For sonographic diagnosis, an ectopic mass should be seen in the adnexa separate from the ovary and may be seen as: (A) a yolk sac (shown here) and/or fetal pole with or without cardiac activity within an extrauterine sac, (B) an empty extrauterine sac with a hyperechoic ring, or (C) an inhomogeneous of fire," which reflects increased vascularity typical of ectopic pregnancies. LT OV = left ovary; SAG LT AD = sagittal left adnexa; UT = uterus. Placental blood low within the periphery of the complex adnexal mass-the ring ofire-can be seen with transvaginal color Doppler imaging. Although this can aid diagnosis, this finding can also be seen with a corpus luteum cyst, and differentiation can be challenging.

1	Hemoperitoneum. In afected women, blood in the peritoneal cavity is most often identified using sonography, but assessment can also be made by culdocentesis (Fig. 19-6). Sonographically, anechoic or hypoechoic luid initially collects in the dependent retrouterine cul-de-sac, and then additionally surrounds the uterus as it fills the pelvis. As much as 50 mL of blood can be seen in the cul-de-sac using TVS, and transabdominal imaging then is used to assess the hemoperitoneum extent. Importantly, however, a small amount of peritoneal luid is physiologically normal. With signiicant intraabdominal hemorrhage, blood will track up the pericolic gutters to fill Morison pouch near the liver. Free luid in this pouch typically is not seen until accumulated volumes reach 400 to 700 mL (Branney, 1995; Rodgerson, 2001; Rose, 2004). Diagnostically, peritoneal fluid in conjunction with an adnexal mass is highly predictive of ectopic pregnancy (Nyberg, 1991). Ascites from ovarian or other cancer is a

1	Rodgerson, 2001; Rose, 2004). Diagnostically, peritoneal fluid in conjunction with an adnexal mass is highly predictive of ectopic pregnancy (Nyberg, 1991). Ascites from ovarian or other cancer is a notable mimic.

1	Culdocentesis is a simple technique used commonly in the past. The cervix is pulled ourward and upward toward the symphysis with a tenaculum, and a long 18-gauge needle is inserted through the posterior vaginal fornix into the retrouterine culde-sac. If present, fluid can be aspirated. However, a failure to do so is interpreted only as unsatisfactory entry into the culde-sac. Fluid containing fragments of old clots or bloody fluid that does not clot suggests hemoperitoneum. In contrast, if the blood sample clots, it may have been obtained from an adjacent blood vessel or from a briskly bleeding ectopic pregnancy. Several studies have challenged its usefulness, and culdocentesis has been largely replaced by TVS (Glezerman, 1992; Vermesh, 1990).

1	Several endometrial changes accompany ectopic pregnancy, and all lack coexistent trophoblast. Decidual reaction is found in 42 percent of samples, secretory endometrium in 22 percent, and proliferative endometrium in 12 percent (Lopez, 1994). Some recommend that the absence of trophoblastic tissue be confirmed by D&C before methotrexate treatment is given (Chung, 201i1; Shaunik, 201l). Investigators found that the presumptive diagnosis of ectopic pregnancy is inaccurate in nearly 40 percent of cases without histological exclusion of a spontaneous pregnancy loss. Nevertheless, the risks of D&C are weighed against the limited maternal risks of methotrexate. Endometrial biopsy with a Pipelle catheter was studied as an alternative to D&C and found inferior (Barnhart, 2003b; Ries, 2000). By comparison, frozen section of curettage fragments to identiY products of conception is accurate in more than 90 percent of cases (Barak, 2005; Li, 2014b).

1	Direct visualization of the fallopian tubes and pelvis by laparoscopy ofers a reliable diagnosis in most cases of suspected FIGURE 19-6 Techniques to identiy hemoperitoneum. A.Transvaginal sonography of an anechoic fluid collection (arrow) in the retrouterine cul-de-sac. B. Culdocentesis: with a 16-to 18-gauge spinal needle attached to a syringe, the cul-de-sac is entered through the posterior vaginal fornix as upward traction is applied to the cervix with a tenaculum. (B, Reproduced with permission from Gala RB: Ectopic pregnancy. In Hofman BL, Schorge JO, Bradshaw KD, et al: Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.) ectopic pregnancy. This also permits a ready transition to definitive operative therapy, which is discussed on page 378.

1	Medical therapy traditionally involves the antimetabolite methotrexate (MTX). This drug is a folic acid antagonist. It tightly binds to dihydrofolate reductase, blocking the reduction of dihydrofolate to tetrahydrofolate, which is the active form of folic acid. As a result, de novo purine and pyrimidine synthesis is halted, which leads to arrested DNA, RNA, and protein synthesis. Thus, MTX is highly efective against rapidly proliferating tissue such as trophoblast. Overall, ectopic tubal pregnancy resolution rates approximate 90 percent with its use. The drawbacks, however, are that bone marrow, gastrointestinal mucosa, and respiratory epithelium can also be harmed. It is directly toxic to hepatocytes and is renally excreted. MTX is also a potent teratogen, and MTX embryopathy is notable for craniofacial and skeletal abnormalities and fetal-growth restriction (N urmohamed, 201i1). In addition, MTX is excreted into breast milk and may accumulate in neonatal tissues and interfere with

1	for craniofacial and skeletal abnormalities and fetal-growth restriction (N urmohamed, 201i1). In addition, MTX is excreted into breast milk and may accumulate in neonatal tissues and interfere with neonatal cellular metabolism (American Academy of Pediatrics, 2001; Briggs, 2015). Based on all these findings, a list of contraindications and pre therapy laboratory testing is found in Table

1	TABLE 19-1. Medical Treatment Protocols for Ectopic Pregnancy Sensitivity to MTX Tubal rupture Breastfeeding One dose; repeat if necessary Days 1 (baseline), 4, and 7 If seru m �-hCG level does not decline by 15% from day 4 to day 7 Less than 15% decline during Up to four doses of both drugs until serum �-hCG declines by 15% 1 mg/kg, days 1,s3,5, and 7 0.1 mg/kg days 2, 4, 6, and 8 Days 1 (baseline), 3, 5, and 7 If serum �-hCG level declines < 15%, give additional dose; repeat serum �-hCG in 48 hours and compare with previous value; maximum four doses Once 15% decline achieved, then weekly serum �-hCG levels until undetectable Intrauterine pregnancy Immunodeficiency Peptic ulcer disease Hepatic, renal, or hematologic dysfunction Active pulmonary disease BSA = body surface area; �-hCG =�-human chorionic gonadotropin; MTX = methotrexate; NA = not applicable. Data from American Society for Reproductive Medicine, 2013.

1	BSA = body surface area; �-hCG =�-human chorionic gonadotropin; MTX = methotrexate; NA = not applicable. Data from American Society for Reproductive Medicine, 2013. Of precautions, MTX is bound primarily to albumin, and its displacement by other medications such as phenytoin, tetracyclines, salicylates, and sulfonamides can increase MTX serum drug levels. Moreover, renal clearance of MTX may be impaired by nonsteroidal antiinlammatory drugs including aspirin, probenecid, or penicillins (Stika, 2012). Last, vitamins containing folic acid may lower MTX eicacy.

1	For ease and eicacy, intramuscular MTX administration is used most often for ectopic pregnancy medical resolution, and single-dose and multidose MTX protocols are available (see Table 19-1). As noted, MTX can lead to bone marrow depression. his toxicity can be blunted by early administration of leucovorin, which is olinic acid and has activity equivalent to folic acid. Thus, leucovorin, which is given within the multidose protocol, allows for some purine and pyrimidine synthesis to bufer side efects. In comparing these two protocols, trade-ofs are recognized. For example, single-dose therapy ofers simplicity, less expense, and less intensive posttherapy monitoring and does not require leucovorin rescue. However, some but not all studies report a higher success rate for the multidose regimen (Alleyassin, 2006; Barnhart, 2003a; Lipscomb, 2005). At our institution, we use single-dose MTX.

1	The best candidate for medical therapy is the woman who is asymptomatic, motivated, and compliant. With medical therapy, some classic predictors of success include a low initial serum 3-hCG level, small ectopic pregnancy size, and absent fetal cardiac activity. Of these, initial serum 3-hCG level is the single best prognostic indicator of successful treatment with single-dose MTX. Speciically, reported failure rates are 1.5 percent if the initial serum 3-hCG concentration is < 1000 mID/mL; 5.6 percent at 1000 to 2000 mID/mL; 3.8 percent at 2000 to 5000 mID/mL; and 14.3 percent when levels range between 5000 and 10,000 mID/mL (Menon, 2007). Interestingly, the initial serum 3-hCG value is not a valid indicator of the number of doses needed for successful resolution (NowakMarkwitz, 2009).

1	Many early trials also used "large size" as an exclusion criterion, although these data are less precise. Lipscomb and colleagues (1998) reported a 93-percent success rate with single-dose MTX when the ectopic mass was <3.5 cm. This compared with success rates between 87 and 90 percent when the mass wasi> 3.5 cm. Last, failure rates rise if cardiac activity is seen, with an 87 -percent success rate in such cases.

1	hese regimens are associated with minimal laboratory changes and symptoms, although occasional toxicity may be severe. Kooi and Kock (1992) reviewed 16 studies and reported that adverse efects resolved by 3 to 4 days ater MTX was discontinued. he most common were liver involvement-12 percent; stomatitis6 percent; and gastroenteritis-1 percent. One woman had bone marrow depression. Fortunately, MTX treatment does not diminish ovarian reserve (Boots, 2016; Dyar, 2013). Moreover, conceptions within the irst 6 months after MTX treatment for this indication are not associated with elevated rates of miscarriage or fetal malformations and growth restriction (Svirsy, 2009).

1	Importantly, 65 to 75 percent of women initially given MTX will have increasing pain beginning several days ater therapy. Thought to relect separation of the ectopic pregnancy from the tubal wall, this "separation pain" generally is mild and relieved by analgesics. In a series of 258 MTX-treated women by Lipscomb and colleagues (1999b), 20 percent had pain that merited evaluation in a clinic or emergency room. Dltimately, 10 of these 53 underwent surgical exploration. Said another way, 20 percent of women given single-dose MTX will have significant pain, and about 20 percent of these will require laparoscopy.

1	As shown in Table 19-1, monitoring single-dose therapy calls for serum 3-hCG determinations at days 4 and 7 following initial injection on day 1. After single-dose MTX, mean serum 3-hCG levels may rise or fall during the first 4 days and then gradually decline. If the level fails to drop more than 15 percent between days 4 and 7, then a second dose of MTX is required. This is necessary in 15 to 20 percent of women treated with single-dose therapy (Cohen, 2014a; Kirk, 2007). With multi dose MTX, levels are measured at 48-hour intervals until they fall more than 15 percent. D p to four doses may be given to one patient if required (Stovall, 1991).

1	With multi dose MTX, levels are measured at 48-hour intervals until they fall more than 15 percent. D p to four doses may be given to one patient if required (Stovall, 1991). Once appropriately dropping levels are achieved in either regimen, serum 3-hCG determinations are then measured weekly until undetectable. Outpatient monitoring is preferred, but if patient safety or compliance is questioned, the woman is hospitalized during initial surveillance. Lipscomb and colleagues (1998) used single-dose MTX to successfully treat 287 women and reported that the average time to resolutiondeined as a serum 3-hCG level < 15 mID/mL, was 34 days. Importantly, the longest time was 109 days. Failure is judged when the 3-hCG level plateaus or rises or the tube ruptures. Importantly, tubal rupture can occur even with declining 3-hCG levels. Lipscomb and associates (1998) described a 14-day mean time to rupture, but one woman had tubal rupture 32 days after single-dose MTX.

1	From one metaanalysis, the overall success rate for treatment with MX is 89 percent. The success for the multidose regiment is 92.7 percent, whereas that for single-dose is 88.1 percent (Barnhart, 2003a). Despite this diference, the single dose is more frequently used because of its simplicity and convenience.

1	Studies have compared laparotomy with laparoscopic surgery for ectopic pregnancy (Lundorf, 1991; Murphy, 1992; Vermesh, 1989). Overall, tubal patency and number of subsequent uterine pregnancies do not difer between these routes. hus, laparoscopy is the preferred surgical treatment for ectopic pregnancy unless a woman is hemodynamically unstable. As experience has accrued, cases previously managed by laparotomy-for example, ruptured tubal pregnancies with hemoperitoneum-can safely be managed laparoscopically by those with suitable expertise (Cohen, 2013; Sagiv, 2001). hat said, the lowered venous return and cardiac output associated with the pneumoperitoneum of laparoscopy must be factored into the decision to select mini mally invasive surgery for hypovolemic women. Before surgery, future fertility desires are discussed. In women desiring permanent sterilization, the unafected tube can be ligated or removed concurrently with salpingectomy for the afected fallopian tube.

1	Two procedures-salpingostomy or salpingectomy-are options. Two multicenter, randomized controlled trials have compared laparoscopic outcomes between the two procedures in women with a normal contralateral fallopian tube. he European Surgery in Ectopic Pregnancy (ESEP) study randomized 23i1 women to salpingectomy and 215 to salpingostomy. After surgery, subsequent rates of ongoing pregnancy by natural conception did not difer significantly between groups-56 versus 61 percent, respectively (Mol, 2014). Again, in the DEMETER trial, the subsequent 2-year rate for achieving a uterine pregnancy did not difer between groups-64 versus 70 percent, respectively (Fernandez, 2013). In women with an abnormalappearing contralateral tube, salpingostomy is a conservative option for fertility preservation.

1	his procedure is typically used to remove a small unruptured pregnancy. A 10-to 15-mm linear incision is made on the anti mesenteric border of the fallopian tube over the pregnancy. The products usually will extrude from the incision. These can be carefully removed or flushed out using high-pressure irrigation that more thoroughly removes the trophoblastic tissue (Al-Sunaidi, 2007). Small bleeding sites are controlled with needlepoint electrocoagulation, and the incision is left unsutured to heal by secondary intention. Serum 3-hCG levels are used to monitor response to both medical and surgical therapy. After linear salpingostomy, serum 3-hCG levels decline rapidly over days and then more gradually, with a mean resolution time of approximately 20 days.

1	Seldom performed today, salpingotomy is essentially the same procedure except that the incision is closed with delayedabsorbable suture. According to T ulandi and Guralnick (1991), prognosis does not difer with or without suturing, and laparoscopic suturing adds surgical time.

1	Tubal resection may be used for both ruptured and unruptured ectopic pregnancies. To minimize the rare recurrence of pregnancy in the tubal stump, complete excision of the fallopian tube is advised. With one laparoscopic technique, the afected fallopian tube is lifted and held with atraumatic grasping forceps (Thompson, 2016). One of several suitable bipolar grasping devices is placed across the fallopian tube at the uterotubal junction. Once desiccated, the tube is cut. The bipolar device is then advanced across the most proximal portion of mesosalpinx. Similarly, current is applied, and the desiccated tissue cut. This process moves serially from the proximal mesosalpinx to its distal extent under the tubal ampulla. Alternatively, an endoscopic suture loop can be used to encircle and ligate the knuckle of involved fallopian tube and its underlying vascular supply within the mesosalpinx. Two consecutive suture loops are placed, and the tube distal to these ligatures is then cut free

1	the knuckle of involved fallopian tube and its underlying vascular supply within the mesosalpinx. Two consecutive suture loops are placed, and the tube distal to these ligatures is then cut free with scissors. Salpingectomy during laparotomy is shown in Chapter 39 (p. 704).

1	Most tubal ectopic pregnancies are small and pliant. Accord ingly, they can be held firmly by grasping forceps and drawn up into one of the accessory site cannulas. Larger tubal ectopic pregnancies may be placed in an endoscopic sac to prevent frag mentation as they are removed through the laparoscopic port site. Importantly, to remove all trophoblastic tissue, the pelvis and abdomen should be irrigated and suctioned free of blood and tissue debris. Slow and systematic movement of the patient from Trendelenburg to reverse Trendelenburg positioning dur ing irrigation can also assist in dislodging stray tissue and luid. hese should be suctioned and removed from the peritoneal cavity.

1	hese should be suctioned and removed from the peritoneal cavity. After surgery, 3-hCG levels usually fall quickly and approxi mate 10 percent of preoperative values by day 12 (Hajenius, 1995; Vermesh, 1988). Persistent trophoblast is rare following salpingectomy, but complicates 5 to 15 percent of salpingos tomies (Kayatas, 2014; Pouly, 1986; Seifer, 1993). Rates are lower for laparotomy versus laparoscopic procedures (Hajenius, 1995). Other risk factors are debatable but may include greater serum 3-hCG levels and smaller ectopic size (Rabischong, 2010; Seifer, 1997). Bleeding caused by retained trophoblast is the most serious complication.

1	Incomplete removal of trophoblast can be identiied by stable or rising 3-hCG levels. v1onitoring approaches are not codified. One scheme measures serum 3-hCG levels on postoperative day 1, and values dropping < 50 percent of the preoperative value relect risk for persistent trophoblast (Spandorfer, 1997). Another measures weekly levels (Mol, 2008). With stable or increasing 3-hCG levels, additional surgical or medical therapy is necessary. Without evidence for tubal rupture, standard therapy for this is single-dose MTX, 50 mg/m2 X body surface area (BSA). Rupture and bleeding require surgical intervention.

1	Several randomized trials have compared methotrexate treatment with laparoscopic surgery. One multicenter trial compared a multidose MTX protocol with laparoscopic salpingostomy and found no diferences for tubal preservation and primary treatment success (Hajenius, 1997). In this same study group, however, health-related quality-of-life factors such as pain, posttherapy depression, and decreased perception of health were significantly impaired after systemic MX compared with laparoscopic salpingostomy (Nieuwkerk, 1998). In their randomized controlled trial, Fernandez and coworkers (2013) compared multidose medical therapy against salpingostomy and found that medical and conservative surgery provided similar 2-year rates of attaining a uterine pregnancy.

1	Evidence is conlicting when single-dose MTX is compared with surgical intervention. In two separate studies, single-dose MTX was overall less successful in resolving pregnancy than laparoscopic salpingostomy, although tubal patency and subsequent uterine pregnancy rates were similar between both groups (Fernandez, 1998; Sowter, 2001). Women treated with MTX had significantly better physical functioning immediately following therapy, but there were no diferences in psychological functioning. Krag NIoeller and associates (2009) reported the results from their randomized trial that had a median surveillance period of 8.6 years during which future pregnancy rates were evaluated. Ectopic-resolution success rates were not significantly diferent between those managed surgically and those treated with MTX. Moreover, cumulative spontaneous uterine pregnancy rates were not diferent between the MTX group (73 percent) and the surgical group (62 percent).

1	Based on these studies, we conclude that women who are hemodynamically stable and in whom there is a small tubal diameter, no fetal cardiac activity, and serum �-hCG concentrations <5000 mIU/mL have similar outcomes with medicalior surgical management. Despite lower success rates with medical therapy for women with larger tubal size, higher serum �-hCG levels, and fetal cardiac activity, medical management can be ofered to the motivated woman who understands the risks. In select cases, it is reasonable to observe very early tubal pregnancies that are associated with stable or falling serum �-hCG levels. Mavrelos and coworkers (2013) noted that almost one third of 333 tubal ectopic pregnancies measuring <3 cm and with �-hCG levels < 1500 mIU/mL resolved without intervention. Cohen and associates (20 14b) similarly followed 674 women with declining �-hCG levels to successul resolution. hese findings have been supported by smaller randomized trials Qurkovic, 2017; van Mello, 2013).

1	With expectant management, subsequent rates of tubal patency and intrauterine pregnancy are comparable with surgical or medical management. That said, compared with the established safety of medical and surgical therapy, the prolonged surveillance and risks of tubal rupture support the practice of expectant therapy only in appropriately selected and counseled women.

1	An interstitial pregnancy is one that implants within the proximal tubal segment that lies within the muscular uterine wall (Fig. 19-7). Incorrectly, they may be called cornual pregnancies, but this term describes a conception that develops in the rudimentary horn of a uterus with a miillerian anomaly (Moawad, 2010). Risk factors are similar to others discussed for tubal ectopic pregnancy, although previous ipsilateral salpingectomy is a specific risk factor for interstitial pregnancy (Lau, 1999). Undiagnosed interstitial pregnancies usually rupture following 8 to 16 weeks of amenorrhea, which is later than for more distal pregnancies. his is due to greater distensibility of the myometrium covering the interstitial fallopian tube segment. Because of the proximity of these pregnancies to the uterine and ovarian arteries, hemorrhage can be severe and associated with mortality rates as high as 2.5 percent (Tulandi, 2004).

1	With transvaginal sonography and serum �-hCG assays, interstitial pregnancy can now be diagnosed early in many cases, but diagnosis can be challenging. hese pregnancies sonographically can appear similar to an eccentrically implanted uterine pregnancy, especially in a uterus with a miillerian anomaly. Criteria that may aid diferentiation include: an empty uterus, a gestational sac seen separate from the endometrium and > 1 cm away from the most lateral edge of the uterine cavity, and a thin, < 5-mm myometrial mantle surrounding the sac (Timor-Tritsch, 1992). Moreover, an echo genic line, known as the "interstitial line sign," extending from the gestational sac to the endometrial cavity most likely represents the interstitial portion of the fallopian tube and is highly sensitive and specific (Ackerman, 1993a). In unclear cases, three-dimensional (3-D) sonography, magnetic resonance (MR) imaging, or diagnostic laparoscopy can help clariy anatomy (Parker, 2012; Tanaka, 2014).

1	and specific (Ackerman, 1993a). In unclear cases, three-dimensional (3-D) sonography, magnetic resonance (MR) imaging, or diagnostic laparoscopy can help clariy anatomy (Parker, 2012; Tanaka, 2014). Laparoscopically, an enlarged protuberance is found lying outside the round ligament and coexistent with a normal distal fallopian tube and ovary.

1	FIGURE 19-7 Interstitial ectopic pregnancy. A.This parasagittal view using transvaginal sonography shows an empty uterine cavity and a mass that is cephalad and lateral to the uterine fundus (calipers). B. Intraoperative photograph during laparotomy and before cornual resection of the same ectopic pregnancy. In this frontal view, the bulging right-sided interstitial ectopic pregnancy is lateral to the round ligament insertion and medial to the isthmic portion of the fallopian tube. (Used with permission from Drs. David Rogers and Elaine Duryea.)

1	Surgical management with either cornual resection or cornuostomy may be performed via laparotomy or laparoscopy, depending on patient hemodynamic stability and surgeon expertise (Hofman, 2016; Zuo, 2012). With either approach, intraoperative intramyometrial vasopressin injection may limit surgical blood loss, and 3-hCG levels should be monitored postoperatively to exclude remnant trophoblast. Cornual resection removes the gestational sac and surrounding cornual myometrium by means of a wedge excision (Fig. 19-8). Alternatively, cornuostomy involves incision of the cornua and suction or instrument extraction of the pregnancy. Both instances require myometrial closure.

1	With early diagnosis, medical management may be considered. But because of the low incidence, consensus regarding methotrexate regimens is lacking. In their small series, J ermy and associates (2004) reported a 94-percent success with systemic MTX using a dose of 50 mg/m2 X BSA. Others have described direct MTX injection into the gestational sac (Framarino-dei-Malatesta, 2014). Importantly, because these women typically have higher initial serum 3-hCG levels, longer surveillance is usually needed. The risk of uterine rupture with subsequent pregnancies following either medical or surgical management is unclear. hus, careful observation of these women during pregnancy, along with strong consideration of elective cesarean delivery, is warranted.

1	FIGURE 19-8 During cornual resection, the pregnancy, surrounding myometrium, and ipsilateral fallopian tube are excised en bloc. The incision is angled inward as it is deepened. This creates a wedge shape into the myometrium, which is then closed in layers with delayed-absorbable suture. The serosa is closed with subcuticular style suturing. (Reproduced with permission from Hofman BL, Corton MM: Surgeries for benign gynecologic conditions. In Hofman BL, Schorge JO, Bradshaw KD, et al: Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.)

1	Distinct from interstitial pregnancy, the term angular pregnany describes implantation within the endometrial cavity but at one cornu and medial to the uterotubal junction and round ligament. An angular pregnancy displaces the round ligament upward and outward, whereas an interstitial tubal pregnancy does not shift it (Arleo, 2014). This distinction is important because angular pregnancies can sometimes be carried to term but with increased risk of abnormal placentation and its consequences Qansen, 1981).

1	This term describes implantation within the myometrium of a prior cesarean delivery scar. Its incidence approximates 1 in 2000 normal pregnancies and has increased along with the cesarean delivery rate (Ash, 2007; Rotas, 2006). he pathogen esis of cesarean scar pregnancy (CSP) has been likened to that rhage (Timor-Tritsch, 2014a,b). It is unknown if the incidence increases with multiple cesarean deliveries or if it is afected by either one-or two-layer uterine incision closure during cesarean.

1	Women with CSP usually present early, and pain and bleed ing are common. Still, up to 40 percent of women are asymp tomatic, and the diagnosis is made during routine sonographic examination (Rotas, 2006). Sonographically, diferentiating be diicult (Moschos, 2008a; Timor-Tritsch, 2016). According to Godin (1997), four sonographic criteria should be satisied for the diagnosis and are described in Figure 19-9. Although TVS is the typical first-line imaging tool, MR imaging is useful when sonography is inconclusive (Huang, 2014; Osborn, 2012).

1	Treatment standards are lacking, and several options are available. Expected management is an option, and live birth rates were 57 percent in one review (Maheux-Lacroix, 2017). However, hemorrhage, placenta accreta, and uterine rupture are risks. Thus, hysterectomy is an acceptable initial choice in those desiring sterilization. It is sometimes necessary with heavy uncontrolled bleeding. Fertility-preserving options include systemic or locally injected methotrexate, either alone or combined with conservative surgery (Birch Petersen, 2016; Cheung, 2015). Surgical procedures include visually guided suction curettage, hysteroscopic removal, or isthmic excision done abdominally or vaginally. hese are completed solely or with adjunctive MTX Qurkovic, 2016; Li, 2014a; Wang, 2014; Yang, 2009). Often uterine artery embolization (UE) is used preoperatively to minimize hemorrhage risk (Zhang, 2012; Zhuang, 2009). Foley balloon catheter placement can be another option for procedure-associated

1	Often uterine artery embolization (UE) is used preoperatively to minimize hemorrhage risk (Zhang, 2012; Zhuang, 2009). Foley balloon catheter placement can be another option for procedure-associated bleeding (TimorTritsch,i2015a).

1	Following conservative treatment, subsequent pregnancies have good outcomes, but placenta accreta and recurrent CSP are risks (Gao, 2016; Wang, 2015). Uterine arteriovenous malformations are a potential long-term complication (TimorTritsch,i2015b).

1	FIGURE 19-9 Cesarean scar pregnancy. A. Transvaginal sonogram of a uterus with a cesarean scar pregnancy (CSP) in a sagittal plane. An empty uterine cavity is identified by a bright hyperechoic endometrial stripe (long, white arrow). An empty cervical canal is similarly identified (short, white arrow). Last, an intrauterine mass is seen in the anterior part of the uterine isthmus (red arrows). Myometrium between the bladder and gestational sac is absent or thinned (1 to 3 mm). Photo contributor: Dr. Elysia Moschos.) B. Hysterectomy specimen containing a cesarean scar pregnancy. C. This same hysterectomy specimen is transversely sectioned at the level of the uterine isthmus and through the gestational sac. The uterine body lies to the left, and the cervix is on the right. A metal probe is placed through the endocervical canal to show the eccentric development of this gestation. Only a thin layer of myometrium overlies this pregnancy, which pushes anteriorly through the uterine wall.

1	placed through the endocervical canal to show the eccentric development of this gestation. Only a thin layer of myometrium overlies this pregnancy, which pushes anteriorly through the uterine wall. (Reproduced with permission from Gala RB: Ectopic pregnancy. In Hoffman BL, Schorge JO, Bradshaw KD, et al: Williams Gynecology, 3rd ed. New York, McGraw-Hili Education; 2016. Photo contributors: Drs. Sunil Balgobin, Manisha Sharma, and Rebecca Stone.) his rare ectopic pregnancy is defined by cervical glands noted histologically opposite the placental attachment site and by all or part of the placenta found below the entrance of the uterine vessels or below the peritoneal relection on the anterior uterus. In a typical case, the endocervix is eroded by trophoblast, and the pregnancy develops in the fibrous cervical wall. Predisposing risks include ART and prior uterine curettage (Ginsburg, 1994; Jeng, 2007).

1	Painless vaginal bleeding is reported by 90 percent of women with a cervical pregnancy-a third of these have massive hemorrhage (Ushakov, 1997). As pregnancy progresses, a distended, thin-walled cervix with a partially dilated external os may be evident. Above the cervical mass, a slightly enlarged uterine fundus can be felt. Identification of cervical pregnancy is based on speculum examination, palpation, and TVS. Sonographic indings typical of cervical pregnancy are shown and described in Figure 19-10. MR imaging and 3-D sonography have also been used to confirm the diagnosis Q ung, 2001; Sherer, 2008).

1	Cervical pregnancy may be treated medically or surgically. Conservative management strives to minimize hemorrhage, resolve the pregnancy, and preserve fertility. In many centers, including ours, methotrexate has become the first-line therapy in stable women, and administration follows protocols listed in Table 19-1 (Verma, 2011; Zakaria, 2011). he drug has also been injected directly into the gestational sac, alone or with systemic doses Qeng, 2007; 1urji, 2015). Others describe MTX infusion combined with uterine artery embolization"chemoembolization" (Xiaolin, 2010). FIGURE 19-10 Cervical pregnancy. Transvaginal sonographic findings may include: (1) an hourglass uterine shape and ballooned cervical canal; (2) gestational tissue at the level of the cervix (black arrow); (3) absent intrauterine gestational tissue (white arrows); and (4) a portion of the endocervical canal seen interposed between the gestation and the endometrial canal. (Used with permission from Dr. Elysia Moschos.)

1	With MTX regimens, resolution and uterine preservation are achieved for gestations < 12 weeks in 91 percent of cases (Kung, 1997). In selecting appropriate candidates, Hung and colleagues (1i996) noted higher risks of systemic MTX treat ment failute in those with a gestational age >9 weeks, �-hCG levels > 10,000 mIU/mL, crown-rump length > 10 mm, and fetal cardiac activity. For this reason, many induce fetal death with intracardiac or intrathoracic injection of potas sium chloride. With a single-dose intramuscular protocol, an MTX dose between 50 and 75 mg/m2 X BSA is typical. To resolve fetal cardiac activity, a sonographically guided fetal intracardiac injection of 2 mL (2 mEq/mL) potassium chlo ride solution can be given (Verma, 2009). Song and associ ates (2009) described management of 50 cases and observed regression.

1	As an adjunct to medical or surgical therapy, uterine artery embolization has been described either as a response to bleeding or as a preprocedural preventive tool (Hirakawa, 2009; Zakaria, 2011). Also, in the event of hemorrhage, a 26F Foley catheter with a 30-mL balloon can be placed intracervically and inlated to efect hemostasis by vessel tamponade and to monitor uterine drainage. The balloon remains inlated for 24 to 48 hours and is gradually decompressed over a few days (Ushakov, 1997). Although conservative management is feasible for many women with cervical pregnancies, suction curettage or hysterectomy may be selected. Moreover, hysterectomy may be required with bleeding uncontrolled by conservative methods. Because of the close proximity of the ureters to the ballooned cervix, urinary tract injury rates are a concern during hysterectomy.

1	If cervical curettage is planned, intraoperative bleeding may be lessened by preoperative UAE, by intracervical vasopressin injection, or by a cerclage placed at the internal cervical os to compress feeding vessels (Chen, 2015; Fylstra, 2014; Wang, 2011). Also, cervical branches of the uterine artery can efectively be ligated with vaginal placement of hemostatic cervical sutures on the lateral aspects of the cervix at 3 and 9 o'clock (Bianchi, 2011). Following curettage, a Foley balloon can be placed to tamponade bleeding and is managed as described earlier. Suction curettage may be especially favored in rare cases of a heterotopic pregnancy composed of a cervical and a desired uterine pregnancy (Tsakos, 2015).

1	hese rare ectopic pregnancies are defined as an implantation in the peritoneal cavity exclusive of tubal, ovarian, or intraligamentous implantations. Although a zygote can traverse the tube and implant primarily in the peritoneal cavity, most abdominal pregnancies are thought to follow early tubal rupture or abortion with reimplantation. In cases of advanced extrauterine pregnancy, it is not unusual for the placenta to be still at least partially attached to the uterus or adnexa.

1	Diagnosis may be diicult. First, symptoms may be absent or vague. Laboratory tests are typically uninformative, although maternal serum alpha-fetoprotein levels can be elevated. Clinically, abnormal fetal positions may be palpated, or the cervix is displaced (Zeck, 2007). Sonographically, the diagnosis is often missed (Costa, 1991). Oligohydramnios is common but nonspecific. Other clues include a fetus seen separate from the uterus or eccentrically positioned within the pelvis; lack of myometrium beween the fetus and the maternal anterior abdominal wall or bladder; extrauterine placental tissue; or bowel loops surrounding the gestational sac (Allibone, 1981; Chukus, 2015). If additional anatomical information is needed, MR imaging can help confirm the diagnosis and provide maximal information concerning placental implantation (Bertrand, 2009; Mittal, 2012).

1	Treatment of an abdominal pregnancy depends on the ges tational age at diagnosis. Conservative management carries a maternal risk for sudden and dangerous hemorrhage. Moreover, in 20 percent. Thus, we believe that termination generally is indicated when the diagnosis is made. Certainly, before 24 weeks, conservative treatment rarely is justified. Despite this, some have described waiting until fetal viability with close sur veillance (Kim, 2013; Marcellin, 2014).

1	Once placental implantation has been assessed, several options to control intraoperative hemorrhage mimic those used for placenta accrete syndrome (Chap. 41, p. 792). he principal surgical objectives involve delivery of the fetus and careful assessment of placental implantation without provoking hemorrhage. Unnecessary exploration is avoided because the anatomy is commonly distorted and surrounding areas are extremely vascular. Importantly, placental removal may precipitate torrential hemorrhage because the normal hemostatic mechanism of myometrial contraction to constrict hypertrophied blood vessels is lacking. If it is obvious that the placenta can be safely removed or if there is already hemorrhage from its implantation site, then removal begins immediately. When possible, blood vessels supplying the placenta should be ligated irst.

1	Some advocate leaving the placenta in place as the lesser of two evils. It decreases the chance of immediate life-threatening hemorrhage, but at the expense of long-term sequelae. If let in the abdominal cavity, the placenta commonly becomes infected, with subsequent formation of abscesses, adhesions, intestinal or ureteral obstruction, and wound dehiscence (Bergstrom, 1998; Martin, 1988). In many of these cases, surgical removal becomes inevitable. If the placenta is let, its involution may be monitored using sonography and serum �-hCG levels (France, 1980; Martin, 1990). Color Doppler sonography can be used to assess changes in blood low. In some cases, and usually depending on its size, placental function rapidly declines, and the placenta is resorbed. But placental resorption may take years (Roberts, 2005; Valenzano, 2003).

1	If the placenta is left in place, postoperative methotrexate use is controversial. It has been recommended to hasten involution but has been reported to cause accelerated placental destruction with accumulation of necrotic tissue and infection with abscess formation (Rahman, 1982). It is diicult to envision a supporting role for the use of an antimetabolite for a senescent organ (Worley, 2008).

1	Ectopic implantation of the fertilized egg in the ovary is rare and is diagnosed if four clinical criteria are met. hese were outlined by Spiegelberg (1878): (1) the ipsilateral tube is intact and distinct from the ovary; (2) the ectopic pregnancy occupies the ovary; (3) the ectopic pregnancy is connected by the uteroovarian ligament to the uterus; and (4) ovarian tissue can be demonstrated histologically amid the placental tissue. Risk factors are similar to those for tubal pregnancies, but ART or IUD failure seems to be disproportionately associated (Zhu, 2014). Presenting complaints and findings mirror those for tubal ectopic pregnancy. Although the ovary can accommodate the expanding pregnancy more easily than the fallopian tube, rupture at an early stage is the usual consequence (Melcer, 2016).

1	Transvaginal sonography use has resulted in a more frequent diagnosis of unruptured ovarian pregnancies. Sonographically, an internal anechoic area is surrounded by a wide echogenic ring, which in turn is surrounded by ovarian cortex (Comstock, 2005). In their review of 49 cases, Choi and associates (2011) noted that the diagnosis may not be made until surgery, as many cases are presumed tubal ectopic pregnancy. Moreover, at surgery, an early ovarian pregnancy may be considered to be a hemorrhagic corpus luteum. Evidence-based management accrues mainly from case reports (Hassan, 2012; Scutiero, 2012). Classically, management for ovarian pregnancies has been surgical. Small lesions can be managed by ovarian wedge resection or cystectomy, whereas larger lesions require oophorectomy (Elwell, 2015; Melcer, 2015). With conservative surgery, 3-hCG levels should be monitored to exclude remnant trophoblast.

1	Pregnancy implanted toward the mesosalpinx may rupture into a space formed between the broad ligament leaves and become an intraligamentous or broad ligament pregnancy. Rents in prior cesarean sca's serve as another conduit (Rudra, 2013). hese are rare, and information accrues from case reports. Clinical indings and management mirror those for abdominal pregnancy. Although laparotomy is required in most instances, a few case reports describe laparoscopic excision of early small pregnancies (Apantaku, 2006; Cormio, 2006).

1	Ectopic placental implantations in less expected sites have been described in case reports and include the omentum, liver, and retroperitoneum, among others (Brouard, 2015; Liang, 2014; Watrowski, 2015). Also, intramural uterine implantations at sites other than a cesarean scar have been noted in women with prior uterine surgeries, ART, or adenomyosis (Memtsa, 2013; Wu, 2013). Although laparotomy is preferred by many for these ectopic sites, laparoscopic excision by those with suitable skills is gaining acceptance. Ackerman TE, Levi CS, Dashefsky SM, et al: Interstitial line: sonographic finding in interstitial (cornual) ectopic pregnancy. Radiology 189(1):83, 1993a Ackerman TE, Levi CS, Lyons A, et al: Decidual cyst: endovaginal sono graphic sign of ectopic pregnancy. Radiology 189(3):727, 1993b

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1	Worley KC, Hnat MD, Cunningham FG: Advanced extrauterine pregnancy: diagnostic and therapeutic challenges. Am J Obstet Gynecol 198:297e1, 2008 WU PJ, Han CM, Wang C], et al: Early detection and minimally invasive management of intramural pregnancy. J Minim Invasive GynecoI20(1): 123, 2013 Xiaolin Z, Ling L, Chengxin Y, et al: Transcatheter intraarterial methotrexate infusion combined with selective uterine artery embolization as a treatment option for cervical pregnancy. J Vase Interv RadioI21(6):836, 2010 Yang Q, Piao S, Wang G, et al: Hysteroscopic surgery of ectopic pregnancy in the cesarean section scar. J Minim Invasive Gynecol 16(4):432,o2009 Zakaria MA, Abdallah ME, Shavell VI, et al: Conservative management of cervical ectopic pregnancy: utility of uterine artery embolization. Ferti! Steril 95(3):872,o201o1 Zeck W, Kelters I, Winter R, et al: Lessons learned from four advanced abdominal pregnancies at an East African Health Center. J Perin at Med 35(4):278, 2007

1	Zeck W, Kelters I, Winter R, et al: Lessons learned from four advanced abdominal pregnancies at an East African Health Center. J Perin at Med 35(4):278, 2007 Zee J, Sammel MD, Chung K, et al: Ectopic pregnancy prediction in women with a pregnancy of unknown location: data beyond 48 h are necessary. Hum Reprod 29(3):441, 2014 Zhang B, Jiang ZB, Huang MS, et l: Uterine artery embolization combined with methotrexate in the treatment of cesarean scar pregnancy: results of a case series and review of the literature. J Vase Interv RadioI23(12): 1582,2012 Zhu Q, Li C, Zhao WH, et al: Risk factors and clinical features of ovarian pregnancy: a case-control study. BMJ Open 4(12):e006447, 2014 Zhuang Y, Huang L: Uterine artery embolization compared with methotrexate for the management of pregnancy implanted within a cesarean scar. Am J Obstet GynecoIo201(2):152.e1, 2009

1	Zhuang Y, Huang L: Uterine artery embolization compared with methotrexate for the management of pregnancy implanted within a cesarean scar. Am J Obstet GynecoIo201(2):152.e1, 2009 Zuo X, Shen A, Chen M: Successful management of unruptured interstitial pregnancy in 17 consecutive cases by using laparoscopic surgery. Aust N Z J Obstet Gynaecol 52(4):387,o2012 HYDATIDIFORM MOLE-MOLAR PREGNANCY . . . 388 PATHOGENESIS ..e....e....e. .. .. ...e...e...e.... 389 DIAGNOSIS......e.... ..e....e....e............ 391 ......e....e...e....e...e.. .. ...e.. 392 GESTATIONAL TROPHOBLASTIC NEOPLASIA ..e... .. 393 DIAGNOSIS, STAGING, AND PROGNOSTIC SCORING ... 393 HISTOLOGICAL CLASSIFICATION ..........e....e...e.394 TREATMENTe. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395

1	DIAGNOSIS, STAGING, AND PROGNOSTIC SCORING ... 393 HISTOLOGICAL CLASSIFICATION ..........e....e...e.394 TREATMENTe. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395 SUBSEQUENT PREGNANCY ....................... 396 . .. the terminal extremities of the chorionic vili are converted into transparent vesicles with clear, viscid contents. These vay in size from minute bodies a ew milimetres in diameter to cystic structures the size of hazel-nuts, and hang in clusters from the vilous stems, to which they are connected by thin pedicles, giving to the external suace of the chorion a grape-like appearance. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) Gestational trophoblastic disease (GTD) is the term used to encompass a group of tumors typiied by abnormal trophoblast proliferation. Trophoblast produces human chorionic gonadotropin (hCG), thus the measurement of this peptide hormone in serum is essential for GTD diagnosis, management, and surveillance. GTD histologically is divided into hydatidiorm moles, which are characterized by the presence of villi, and into nonmolar trophoblastic malignant neoplasms, which lack villi. Hydatidiform moles are excessively edematous immature placentas (Benirschke, 2012). These include the benign complete hydatidorm mole and partial hydatidiorm mole and the malignant invasive mole. Invasive mole is deemed malignant due to its marked penetration into and destruction of the myometrium and its ability to metastasize.

1	Nonmolar trophoblastic neoplasms include choriocarcinoma, placental site trophoblastic tumor, and epithelioid trophoblastic tumor. hese three are diferentiated by the type of trophoblast they contain. The malignant forms of gestational trophoblastic disease are termed gestational trophoblastic neoplasia (GTN). hese include invasive mole, choriocarcinoma, placental site trophoblastic tumor, and epithelioid trophoblastic tumor. Other terms applied to GTN are malignant gestational trophoblastic disease and persistent gestational trophoblastic disease. hese malignancies develop weeks or years following any type of pregnancy, but frequently follow a hydatidiform mole.

1	Each of the GTN malignancy types is histologically distinct and varies in its propensity to invade and metastasize. However, histological confirmation is typically not available. Instead, measurement of serum hCG levels combined with clinical indingsrather than a histological specimen-is used to diagnose and treat this malignancy. Accordingly, GTN is often identified and efectively treated as a group. In the past, these metastatic tumors had a prohibitively high mortality rate. However, with chemotherapy, most tumors currently are highly curable. Early-stage GTN is typically cured with single-agent chemotherapy, whereas laterstage disease usually responds to combination chemotherapy (Ngan,i2015). The classic histological findings of molar pregnancy include trophoblast proliferation and villi with stromal edema (Fig. 20-1).

1	The classic histological findings of molar pregnancy include trophoblast proliferation and villi with stromal edema (Fig. 20-1). FIGURE 20-1 Complete hydatidiform mole. A. Gross specimen with characteristic vesicles of variable size. (Used with permission from Dr. Brian Levenson.) B. Low-magnification photomicrograph shows generalized edema and cistern formation (black asterisks) within avascular villi. Haphazard trophoblastic hyperplasia is marked by a yellow asterisk on the right. (Used with permission from Dr. Erika Fong.) The degree of histological changes, karyotypic diferences, and the absence or presence of embryonic elements are used to clas siy them as either complete orpartial moles. hese two also vary and postevacuation GTN. Of the two, GTN more frequently follows complete hydatidiform mole.

1	A complete mole has abnormal chorionic villi that grossly appear as a mass of clear vesicles. These vary in size and often hang in clusters from thin pedicles. In contrast, a partial molar pregnancy has focal and less advanced hydatidiform changes and contains some fetal tissue. Both forms of moles usually fill the uterine cavity, but they rarely may be tubal or other forms of ectopic pregnancy (Hassadia, 2012; Sebire, 2005). An ethnic predisposition is seen with hydatidiform mole, which has increased prevalence in Asians, Hispanics, and American Indians (Drake, 2006; Lee, 2011; Smith, 2006). he incidence in the United States and Europe has been relatively constant at 1 to 2 per 1000 deliveries (Eysbollts, 2016; Lee, 2011).

1	The strongest risk factors are age and a prior hydatidiform mole. Women at both extremes of reproductive age are most vulnerable. Speciically, adolescents and women aged 36 to 40 years have a twofold risk, but those older than 40 have an almost tenfold risk (ltman, 2008; Sebire, 2002a). With a prior complete mole, the risk of another mole is 0.9 percent, and with a previous partial mole, the rate is 0.3 percent. Mter two prior complete moles, approximately 20 percent of women have a third mole (Eagles, 2015). Molar pregnancies typically arise from chromosomally abnormal fertilizations Figure 20-2. Complete moles most often have a diploid chromosomal composition (Table

1	Molar pregnancies typically arise from chromosomally abnormal fertilizations Figure 20-2. Complete moles most often have a diploid chromosomal composition (Table These usually are 46,X and result from androgenesis, meaning both sets of chromosomes are paternal in origin. The chromosomes of the ovum are either absent or inactivated. The ovum is fertilized by a haploid sperm, which then duplicates its own chromosomes after meiosis. Less commonly, the chromosomal pattern may be 46,Y or 46,X and due to fertilization by two sperm, that is, dispermic ertilization or dispermy (Lawler, 1991; Lipata, 2010). 23,Y FIGURE 20-2 Typical pathogenesis of complete and partial moles. A. A 46,XX complete mole may be formed if a 23,x-bearing haploid sperm penetrates a 23,X-containing haploid egg whose genes have been "inactivated." Paternal chromosomes then duplicate to create a 46,XX diploid complement solely of paternal origin.

1	B. A partial mole may be formed if two sperm-either 23,X-or 23,Y-bearing-both fertilize (dispermy) a 23,X-containing haploid egg whose genes have not been inactivated. The resulting fertilized egg is triploid with two chromosome sets being donated by the father. This paternal contribution is termed diandry .. aTypical karyotypes. bThese include anemia, hyperthyroidism, hyperemesis gravidarum, preeclampsia, and infection. GTN = gestational trophoblastic neoplasia; hCG = human chorionic gonadotropin.

1	aTypical karyotypes. bThese include anemia, hyperthyroidism, hyperemesis gravidarum, preeclampsia, and infection. GTN = gestational trophoblastic neoplasia; hCG = human chorionic gonadotropin. Partial moles usually have a triploid karyotype-69 ,XX, 69,Y-or much less commonly, 69,YY. These are each composed of two paternal haploid sets of chromosomes contributed by dispermy and one maternal haploid set (see Fig. 20-2B). Less frequently, a similar haploid egg may be fertilized by an unreduced diploid 46,Y sperm. These triploid zygotes result in some embryonic development, however, it ultimately is a lethal fetal condition a oergensen, 2014; Lakovschek, 2011). Fetuses that reach advanced ages have severe growth restriction, multiple congenital anomalies, or both.

1	Rarely, in some twin pregnancies, one chromosomally normal fetus is paired with a complete diploid molar pregnancy. Importantly, these cases must be distinguished from a single partial molar pregnancy with its associated abnormal fetus. Amniocentesis and fetal karyotyping aid confirmation. Several unique pregnancy problems complicate such twin pregnancies. And, many women may choose to terminate the gestation, if diagnosed early. In those with continuing pregnancy, survival of the normal fetus varies and depends on associated comorbidity from the molar component. The most worrisome are preeclampsia or hemorrhage, which frequently necessitate preterm delivery. Wee and Jauniaux (2005) reviewed outcomes in 174 women, of whom 82 chose termination. Of the remaining 92 pregnancies, 42 percent either miscarried or had a perinatal death; approximately 60 percent delivered preterm; and only 40 percent delivered at term.

1	Another concern for those continuing their pregnancy is the risk for developing subsequent GTN. However, most data indicate no significant diference between women who continue or terminate their pregnancy (Massardier, 2009; Sebire, 2002b). Postdelivery surveillance is conducted as for any molar pregnancy (p. 393). The presentation of women with a molar pregnancy has changed remarkably over the past several decades because prenatal care is sought much earlier and because sonography is virtually universal. Typically, 1 to 2 months of amenorrhea precede the diagnosis. For example, in 194 women with a complete mole, evacuation was completed at a median gestational age of 9 weeks and at 12 weeks for 172 patients with a partial mole (Sun, 2015b). As a result, most molar pregnancies are detected before complications ensue (Kerkmeijer, 2009; Mangili, 2008).

1	As gestation advances, symptoms tend to be more pronounced with complete compared with partial moles (Niemann, 2007). Untreated molar pregnancies will almost always cause uterine bleeding that varies from spotting to profuse hemorrhage. Bleeding may presage spontaneous molar abortion, but more often, it follows an intermittent course for weeks to months. In more advanced moles with considerable concealed uterine hemorrhage, moderate iron-deiciency anemia develops. Nausea and vomiting may be signiicant. Of physical findings, many women have uterine growth that is more rapid than expected, and the enlarged uterus is comparatively softer. Fetal heart motion is absent with complete moles. he ovaries can be fuller and cystic from multiple theca-lutein cysts (Fig. 20-3). These are more common with a complete mole and likely result from ovarian overstimulation by excessive hCG levels. Because theca-lutein cysts regress following pregnancy evacuation, expectant management is preferred.

1	with a complete mole and likely result from ovarian overstimulation by excessive hCG levels. Because theca-lutein cysts regress following pregnancy evacuation, expectant management is preferred. Occasionally a larger cyst may undergo torsion, infarction, and hemorrhage. However,

1	FIGURE 20-3 Sonographic image of an ovary with theca-lutein cysts in a woman with a hydatidiform mole. oophorectomy is not performed unless extensive infarction persists after untwisting. The thyrotropin-like efects of hCG frequently cause serum free thyroxine (T4) levels to be elevated and thyroid-stimulating hormone (TSH) levels to be decreased. Despite this, clinically apparent thyrotoxicosis is unusual and in our experience can be mimicked by bleeding and sepsis from infected products. Moreover, the serum free T4ilevels rapidly normalize after uterine evacuation. Despite this, cases of presumed "thyroid storm" have been reported (Koinas, 2015).

1	Severe preeclampsia and eclampsia are relatively common with advanced molar pregnancies. However, these are seldom seen today because of early diagnosis and evacuation. An exception is the case of a normal fetus coexisting with a complete mole, described earlier. In continuing twin gestations, severe preeclampsia frequently mandates preterm delivery. prompts pregnancy testing and sonography. Some women will present with spontaneous passage of molar tissue.

1	prompts pregnancy testing and sonography. Some women will present with spontaneous passage of molar tissue. With a complete molar pregnancy, serum �-hCG levels are commonly elevated above those expected for gestational age. With more advanced moles, values in the millions are not unusual. Importantly, these high values can lead to erroneous false-negative urine pregnancy test results. Termed a "hook efect," excessive �-hCG hormone levels oversaturate the assay's targeting antibody and create a falsely low reading (Cormano, 2016). In these cases, serum �-hCG determinations with or without sample dilution will clariy the conundrum. With a partial mole, �-hCG levels may also be significantly elevated, but more commonly concentrations fall into ranges expected for gestational age.

1	Although this is the mainstay of trophoblastic disease diagnosis, not all cases are confirmed initially. Sonographically, a com plete mole appears as an echogenic uterine mass with numerous anechoic cystic spaces but without a fetus or amnionic sac. The appearance is often described as a "snowstorm" (Fig. 20-4). A partial mole has features that include a thickened, multicystic placenta along with a fetus or at least fetal tissue. However, in early pregnancy, these sonographic characteristics are seen in fewer than half of hydatidiform moles. In the largest series of more than 1000 patients with molar pregnancy, the reported sensitivity and speciicity of sonography were 44 and 74 percent, respectively (Fowler, 2006). he most common mimics are incomplete or missed abortion. Occasionally, molar pregnancy may be confused for a multifetal pregnancy or a uterine leiomyoma with cystic degeneration.

1	FIGURE 20-4 Sonograms of hydatidiform moles. A. Sagittal view of a uterus with a complete hydatidiform mole. The characteristic "snowstorm" appearance is due to an echogenic uterine mass, marked by calipers, that has numerous anechoic cystic spaces. Notably, a fetus and amnionic sac are absent. B. In this image of a partial hydatidiform mole, the fetus is seen above a multicystic placenta. (Used with permission from Dr. Elysia Moschos.) Surveillance for subsequent neoplasia following molar pregnancy is crucial. Thus, moles must be distinguished from other types of pregnancy failure that have hydropic placental degeneration, which can mimic molar villous changes. Some distinguishing histological characteristics are shown in Table 20-1.

1	In pregnancies before 10 weeks, classic molar changes may not be apparent because villi may not be enlarged and molar stroma may not yet be edematous and avascular. Histopathologic evaluation can be enhanced by immunohistochemical staining for pS7 expression and by molecular genotyping (Banet, 2014). pS7IP2 is a nuclear protein whose gene is paternally imprinted and maternally expressed. his means that the gene product is produced only in tissues containing a maternal allele. Because complete moles contain only paternal genes, the pS7IP2 protein is absent in complete moles, and tissues do not pick up this stain (Merchant, 200S). In contrast, this nuclear protein is strongly expressed in normal placentas, in spontaneous pregnancy losses with hydropic degeneration, and in partial hydatidiform moles (Castrillon, 2001). Accordingly, immunostaining for pS7IP2 is an efective means to isolate complete mole from the diagnostic list. For distinction of a partial mole from a non molar hydropic

1	moles (Castrillon, 2001). Accordingly, immunostaining for pS7IP2 is an efective means to isolate complete mole from the diagnostic list. For distinction of a partial mole from a non molar hydropic abortus, both of which express pS7, molecular genotyping can be used. Molecular genotyping determines the parental source of alleles. Thereby, it can distinguish among a diploid diandric genome (complete mole), a triploid diandric-monogynic genome (partial mole), or biparental diploidy (nonmolar abortus).

1	Maternal deaths from molar pregnancies are rare because of early diagnosis, timely evacuation, and vigilant postevacuation surveillance for GTN. Preoperative evaluation attempts to identiy known potential complications such as preeclampsia, hyperthyroidism, anemia, electrolyte depletions from hyperemesis, and metastatic disease (Table 20-2) (Lurain, 2010). Most recommend chest radiography, whereas computed tomography (CT) and magnetic resonance (MR) imaging are not routinely done unless a chest radiograph shows lung lesions or unless other extrauterine disease is suspected.

1	Regardless of uterine size, molar evacuation by suction curettage is usually the preferred treatment. Preoperative cervical dilatation with an osmotic dilator is recommended if the cervix is minimally dilated. Intraoperative bleeding can be greater with molar pregnancy than with a comparably sized uterus containing nonmolar products. hus with large moles, adequate anesthesia, suicient intravenous access, and blood-banking support is imperative. he cervix is mechanically dilated to preferably allow insertion of a larger suction curette. Depending on uterine size, a 10-to 14-mm diameter is typical. As evacuation is begun, oxytocin is infused to limit bleeding. Intraoperative sonography is often recommended to help ensure complete uterine cavity emptying. When the myometrium has contracted, a thorough but gentle curettage with a sharp large-loop TABLE 20-2. Some Considerations for Management of Hydatidiform Mole

1	TABLE 20-2. Some Considerations for Management of Hydatidiform Mole Hemogram; serum 3-hCG, creatinine, electrolyte, and hepatic aminotransferase levels TSH, free T4 levels Oxytocin (Pitocin): 20 units in 1000 mL Ringer lactate for continuous infusion One or more other uterotonic agents may be added as needed: Methylergonovine (Methergine): 0.2 mg = 1 mL = 1 ampule 1M every 2 hr prn Carboprost tromethamine (PGF20) (Hemabate)k 250 .Lg = 1 mL = 1 ampule 1M every 15-90 min prn Misoprostol (PGE1) (Cytotec): 200 mg tablets for rectal administration, 800-1000 mg once Karman cannula-size 10 or 14 mm Consider sonography machine Serum hCG levels: within 48 hours of evacuation, weekly until undetectable, then monthly for 6 months alntrauterine devices are not suitable during surveillance. hCG = human chorionic gonadotropin; 1M = intramuscular; PG = prostaglandin; T4 = thyroxine; TSH = thyroid-stimulating hormone.

1	hCG = human chorionic gonadotropin; 1M = intramuscular; PG = prostaglandin; T4 = thyroxine; TSH = thyroid-stimulating hormone. Sims curette is performed. If bleeding continues despite uterine evacuation and oxytocin infusion, other uterotonic agents are given (see Table 20-2). In rare cases, pelvic arterial emboliza tion or hysterectomy may be necessary (Tse, 2007). Profuse management are discussed in Chapter 41 (p. 755).

1	Some volume of trophoblast is deported into the pelvic venous system during molar evacuation (Hankins, 1987). With large moles, the amount of tissue may be suicient to produce clinically apparent respiratory insuiciency, pulmonary edema, or even embolism. In our earlier experiences with substantial moles, these and their chest radiographic manifestations clear rapidly without specific treatment. However, fatalities have been described (Delmis, 2000). Because of deportation, there is concern that trophoblastic tissue will thrive within the lung parenchyma to cause persistent disease or even overt malignancy. Fortunately, no evidence suggests that this is a major problem.

1	Following curettage, anti-D immunoglobulin (Rhogam) is given to Rh D-negative women because fetal tissues with a partial mole may include red cells with D-antigen (Chap. 15, p. 305). Those with suspected complete mole are similarly treated because a deinitive diagnosis of complete versus partial mole may not be conirmed until histologicl evaluation of the evacuated products. Following evacuation, the long-term prognosis for women with a hydatidiform mole is not improved with prophylactic chemotherapy. Moreover, chemotherapy toxicity-including death-may be significant, and thus it is not recommended routinely (Gueye, 2014; Wang, 2017).

1	Methods other than suction curettage can be considered for select cases. Hysterectomy with ovarian preservation may be preferable for women with complete moles who have inished childbearing. Of women aged 40 to 49 years, 30 to 50 percent will subsequently develop GTN, and hysterectomy markedly reduces this likelihood (Bandy, 1984; Elias, 2010, 2012). Theca-lutein cysts seen at the time of hysterectomy do not require removal, and they spontaneously regress following molar termination. Labor induction or hysterotomy is seldom used for molar evacuation in the United States. Both will likely increase blood loss and theoretically may increase the incidence of persistent trophoblastic disease (American College of Obstetricians and Gynecologists, 2016; Tidy, 2000).

1	Close biochemical surveillance for persistent gestational neoplasia follows each hydatidiform mole evacuation. This monitoring is by serial measurement of serum 3-hCG to detect persistent or renewed trophoblastic proliferation. s a glycoprotein, hCG shows structural heterogeneity and exists in diferent isoforms. Thus for surveillance, an hCG assay that can detect all forms of hCG should be used (Harvey, 2010; Ngan, 2015). hese are different from those used for routine pregnancy testing (de Medeiros, 2009). he initial 3-hCG level is obtained within 48 hours ater evacuation. This serves as the baseline, which is compared with 3-hCG quantiication done thereater every 1 to 2 weeks until levels progressively decline to become undetectable.

1	he median time for such resolution is 7 weeks for partial moles and 9 weeks for complete moles. Once 3-hCG is undetectable, this is confirmed with monthly determinations for another 6 months (Lurain, 2010; Sebire, 2007). Concur rently, reliable contraception is imperative to avoid confusion caused by rising 3-hCG levels from a new pregnancy. Most recommend combination hormonal contraception, injectable depot medroxyprogesterone acetate, or progestin implant (Dantas, 2017). he latter two are particularly useful if poor patient compliance is anticipated. Intrauterine devices are not used until 3-hCG levels are undetectable because of the risk of uterine perforation if there is an invasive mole. lthough not recommended, if a woman conceives during surveillance, live birth rates and risk for congenital anomalies appear to mirror the general population (Tuncer, 1999a,b). Mter these 6 months, monitoring is discontinued and pregnancy allowed.

1	Importantly, during 3-hCG level surveillance, either increasing or persistently plateaued levels mandate evaluation for trophoblastic neoplasia. If the woman has not become pregnant, then these levels signiY increasing trophoblastic proliferation that is most likely malignant. Several factors predispose a patient to trophoblastic neoplasia following molar evacuation. Most important, complete moles have a 15 to 20 percent incidence of malignant sequelae, compared with 1 to 5 percent following partial moles. Surprisingly, with much earlier recognition and evacuation of molar pregnancies, the risk for neoplasia has not been lowered (Schorge, 2000; Sun, 2015a). Other risk factors are older maternal age, 3-hCG levelsi> 100,000 mIU/mL, uterine size that is large for gestational age, theca-lutein cysts >6 cm, and slow decline in 3-hCG levels (Berkowitz, 2009; Kang, 2012; Wolfberg, 2005).

1	This group includes invasive mole, choriocarcinoma, placental site trophoblastic tumor, and epithelioid trophoblastic tumor. These tumors almost always develop with or after some form of recognized pregnancy. Half follow hydatidiform mole, a fourth follow miscarriage or tubal pregnancy, and another fourth develop after a preterm or term pregnancy (Goldstein, 2012). Although these four tumor types are histologically distinct, they are usually diagnosed solely by persistently elevated serum 3-hCG levels because tissue is infrequently available for study. Criteria to diagnose postmolar GTN are shown in Table 20-3.

1	hese placental tumors are characterized clinically by their aggressive invasion into the myometrium and propensity to metastasize. he most common inding with GTN is irregular bleeding associated with uterine subinvolution. The bleeding may be continuous or intermittent, with sudden and sometimes massive hemorrhage. Myometrial perforation from trophoblastic growth may cause intraperitoneal hemorrhage. In some women, lower genital tract metastases are evident, whereas in others only distant metastases are found with no trace of uterine tumor. • Diagnosis, Staging, and Prognostic Scoring Consideration for the possibility of GTN is the most important factor in its recognition. Unusually persistent bleeding ater any type of pregnancy should prompt measurement of serum 3-hCG TABLE 20-3. Criteria for Diagnosis of Gestational Trophoblastic Neoplasia 1. Plateau of serum �-hCG level (± 10 percent) for four measurements during a period of 3 weeks or longer-days 1, 7, 14,s21 2.

1	Plateau of serum �-hCG level (± 10 percent) for four measurements during a period of 3 weeks or longer-days 1, 7, 14,s21 2. Rise of serum �-hCG levels> 10 percent during three weekly consecutive measurements or longer, during a period of 2 weeks or more-days '1, 7, 14 3. 4. levels and consideration for diagnostic curettage if levels are elevated. Uterine size is assessed along with careful examination for lower genital tract metastases, which usually appear as bluish vascular masses (Cagayan, 2010). Tissue diagnosis is unnecessary, thus biopsy is not required and may cause significant bleeding.

1	Once the diagnosis is veriied, in addition to a baseline serum J-hCG level and hemogram, a search for local disease and metastases includes tests of liver and renal function, transvaginal sonography, chest CT scan or radiograph, and brain and abdominopelvic CT scan or MR imaging. Less commonly, positron-emission tomographic (PET) scanning and cerebrospinal luid J-hCG level determination are used to identiy metastases (Lurain, 2011). GTN is staged clinically using the system of the International Federation of Gynecology and Obstetrics (FIGO) (2009). his includes a modiication of the World Health Organization (WHO) (1983) prognostic index score, with which scores of 0 to 4 are given for each of the categories shown in Table 20-4. Women with WHO scores of 0 to 6 are considered to have low-risk disease, whereas those with a score :::7 are considered in the high-risk group.

1	Again, it is stressed that the diagnosis of trophoblastic neoplasias is usually made by persistently elevated serum J-hCG levels without conirmation by tissue study. Clinical staging is assigned without regard to histological indings, even if available. Still, there are distinct histological types, described next. These are the most common trophoblastic neoplasms that follow hydatidiform moles, and almost all invasive moles arise from partial or complete moles. Previously known as chorioadenoma destruens, invasive mole is characterized by extensive tissue invasion by trophoblast and whole villi. There is penetration deep into the myometrium, sometimes with involvement of the peritoneum, adjacent parametrium, or vaginal vault. Although locally aggressive, invasive moles are less prone to metastasize. his is the most common type of trophoblastic neoplasm to follow a term pregnancy or a miscarriage, and only a third of

1	his is the most common type of trophoblastic neoplasm to follow a term pregnancy or a miscarriage, and only a third of TABLE 20-4. International Federation of Gynecology and Obstetrics (FIGO) Staging and Diagnostic Scoring System for Gestational Trophoblastic Neoplasia Age (years) <40 :::40 Antecedent pregnancy Mole Abortion Term <103 largest tumor size (including uterus) <3 cm 3-4 cm :::5 cm Site of metastases Spleen, kidney GI Liver, brain Number of metastases Previous failed chemotherapy drugs :::2 aAdapted by FIGO. blow risk = WHO score of 0 to 6; high risk = WHO score ofs:::7. J-hCG = beta human chorionic gonadotropin; GI = gastrointestinal; GTN = gestational trophoblastic neoplasia. Adapted with permission from FIGO Committee on Gynecologic Oncology: Current FIGO staging for cancer of the vagina, fallopian tube, ovary, and gestational trophoblastic neoplasia, Int J Gynaecol Obstet 2009 Apr;l 05(1 )j3-4. 4-6 7-12 >12 103 to 104 104 to 105 :::105

1	FIGURE 20-5 Metastatic choriocarcinoma. A. Chest radiograph demonstrates widespread metastatic lesions. B. Autopsy specimen with multiple hemorrhagic hepatic metastases. (Used with permission from Dr. Michael Conner.) cases follow a molar gestation (Soper, 2006). Choriocarcinoma is composed of cells reminiscent of early cytotrophoblast and syncytiotrophoblast, however, it contains no villi. his rapidly growing tumor invades both myometrium and blood vessels to create hemorrhage and necrosis. Myometrial tumor may spread outward and become visible on the uterine surface as dark, irregular nodules. Metastases often develop early and are generally blood-borne (Fig. 20-5). The most common sites are the lungs and vagina, but tumor may travel to the vulva, kidneys, liver, brain, ovaries, and bowel. Bleeding can complicated these metastases (Fatema, 2016; Wei, 2016; Zhang, 2017). Choriocarcinomas are commonly accompanied by ovarian theca-lutein cysts.

1	This rare tumor arises from intermediate trophoblasts at the placental site. hese tumors have associated serum �-hCG levels that may be only modestly elevated. However, they produce variant forms of hCG, and identiication of a high proportion of free �-hCG is considered diagnostic. Treatment of placental site trophoblastic tumor by hysterectomy is preferred because these locally invasive tumors are usually resistant to chemotherapy (Baergen, 2006). For higher-risk stage I and for later stages, adjuvant multidrug chemotherapy is also given (Schmid, 2009). This rare tumor develops from chorionic-type intermediate trophoblast. he uterus is the main site of involvement, and bleeding and low hCG levels are typical findings (Scott, 2012). Primary treatment is hysterectomy because this tumor is relatively resistant to chemotherapy. Metastatic disease is common, and combination chemotherapy is employed (Davis, 2015) .

1	Women with GTN are best managed by oncologists, and some (Kohorn, 2014). The prognosis is excellent with rare excep tions, and patients are routinely cured even in the presence of widespread disease. Chemotherapy alone is usually the primary treatment. Although controversial, some also consider a sec ond uterine evacuation to be an adjuvant therapeutic option in some GTN cases to avoid or minimize chemotherapy (Pezeshki, 2004; van Trommel, 2005). In other cases, suction curettage may infrequently be needed to resolve bleeding or remove a sub stantial amount of retained molar tissue. In speciic cases, hys terectomy may be primary or adjuvant treatment (Clark, 2010).

1	Single-agent chemotherapy protocols are usually suicient for nonmetastatic or low-risk metastatic neoplasia (Lawrie, 2016). In their review of 108 women with low-risk disease, Abrao and colleagues (2008) reported that monotherapy protocols with either methotrexate or actinomycin D were equally efective compared with a regimen containing both. In general, methotrexate is less toxic than actinomycin D (Chan, 2006; Secki, 2010). Regimens are repeated until serum �-hCG levels are undetectable. Combination chemotherapy is given for high-risk disease, and reported cure rates approximate 90 percent (Lurain, 2010). Several regimens have been used with success. One is EMA-CO, which includes �toposide, methotrexate, gctinomycin D, �yclophosphamide, and

1	Oncovin (vincristine). In selected cases, adjuvant surgical and radiotherapy may also be employed (Hanna, 2010). Frequent causes of death include hemorrhage from metastatic sites, respiratory failure, sepsis, and multiorgan failure due to widespread chemoresistant disease (Lybol, 2012; Neubauer, 2015). With either low-or high-risk disease, once serum �-hCG levels are undetectable, serosurveillance is continued for 1 year. During this time, efective contraception is crucial to avoid any teratogenic efects of chemotherapy to the fetus and to mitigate conusion from rising �-hCG levels caused by superimposed pregnany (Secki, 2010; Williams, 2014). For those who conceive despite this within the surveillance year following treatment, pregnancy may continue since most will have a favorable outcome (Tse, 2012; Woolas, 1998). Importantly, this group is advised of the low but important risk of delayed diagnosis if tumor recurs during the pregnancy (Blagden, 2002; Tuncer, 1999b).

1	A small number of women during surveillance, despite no evidence of metastases, will be found to have very low �-hCG levels that plateau. This phenomenon is called quiescent hCG and presumably is caused by dormant trophoblast. Close observation without therapy is recommended, but 20 percent will eventually have recurrent active and progressive trophoblastic neoplasia (Ngu, 2014). Women with prior hydatidiform mole generally do not have impaired fertility, and their pregnancy outcomes are usually normal Qoneborg, 2014; Matsui, 2011; Sebire, 2003). One concern is the 2-percent risk for developing trophoblastic disease in a subsequent pregnancy, which was described earlier. Sonographic evaluation is recommended in early pregnancy, and subsequently if indicated.

1	Women who have successfully completed GTN chemotherapy are advised to delay pregnancy for 12 months. Fertility and pregnancy outcomes are typically normal, and congenital anomaly rates are not increased (Berkowitz, 2000; Tse, 2012). One exception is an unexplained higher stillbirth rate of 1.5 percent compared with a background rate of 0.8 percent (Vargas, 2014). After hydatidiform mole or GTN treatment, in subsequent pregnancy, the placenta or products of conception are sent for pathological evaluation at delivery. A serum 3-hCG level is measured 6 weeks postpartum (Lurain, 2010). Abrao A, de Andrade ]M, Tiezzi DG, et al: Treatment for low-risk gestational trophoblastic disease: comparison of single-agent methotrexate, dactinomycin and combination regimens. Gynecol Oncol 108:149,o2008 Altman AD, Bently B, Murray S, et al: Maternal age-related rate of gestational trophoblastic disease. Obstet Gynecol 11o2:244, 2008

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1	Seck! MJ, Sebire NJ, Berkowitz RS: Gestational trophoblastic disease. Lancet 376(9742):717,o2010 Smith HO, Wiggins C, Verschraegen CF, et al: Changing trends in gestational trophoblastic disease. J Reprod Med 51 :777, 2006 Soper JT: Gestational trophoblastic disease. Obstet Gynecol 108: 176, 2006 Sun SY, Melamed A, Goldstein DP, et al: Changing presentation of complete hydatidiform mole at the New England Trophoblastic Disease Center over the past three decades: does early diagnosis alter risk for gestational trophoblastic neoplasia? Gynecol Oncol 138(1):46,o2015 Tidy JA, Gillespie M, Bright N, et al: Gestational trophoblastic disease: a study of mode of evacuation and subsequent need for treatment with chemotherapy. Gynecol Oncol 78 pp. 309,o2000 Tse Y, Chan KK, Tam F: 20-year experience of managing profuse bleeding in gestational trophoblastic disease. J Reprod Med (5):397, 200

1	Tse Y, Chan KK, Tam F: 20-year experience of managing profuse bleeding in gestational trophoblastic disease. J Reprod Med (5):397, 200 Tse Y, Ngan HY: Gestational trophoblastic disease. Best Pract Res Clin Obstet GynaecoIo26(3):357, 2012 Tuncer ZS, Bernstein MR, Goldstein DP, et al: Outcome of pregnancies occurring before completion of human chorionic gonadotropin follow-up in patients with persistent gestational trophoblastic tumor. Gynecol Oncol 73(3):345, 1999a Tuncer ZS, Bernstein MR, Goldstein DP, et al: Outcome of pregnancies occurring within 1 year of hydatidiform mole. Obstet Gynecol 94(4):588, 1999b van Trommel NE, Massuger LF, Verheijen H, et al: he curative efect of a second curettage in persistent trophoblastic disease: a retrospective cohort survey. Gynecol Oncol 99:6, 2005

1	Vargas R, Barroilhet LM, Esselen K, et al: Subsequent pregnancy outcomes after complete and partial molar pregnancy, recurrent molar pregnancy, and gestational trophoblastic neoplasia: an update from the New England Trophoblastic Disease Center. J Reprod Med 59(5-6):188, 2014 Wang Q, Fu J, Hu L, et al: Prophylactic chemotherapy for hydatidiform moleto prevent gestational trophoblastic neoplasia. Cochrane Database Syst Rev 9:CD007289, 2017 Wee L, Jauniaux E: Prenatal diagnosis and management of twin pregnancies complicated by a co-existing molar pregnancy. Prenat Diagn 25(9):772, 2005 Wei H, Zhang T, Uu B, et al: Choriocarcinoma of unknown origin with multiple organ metastasis and cerebral hemorrhage: a case report and literature review. Oncol Lett 11(6):3749,2016 Williams ], Short D, Dayal L, et al: Efect of early pregnancy following chemotherapy on disease relapse and fetal outcome in women treated for gestational trophoblastic neoplasia. J Reprod Med 59(5-6):248-54, 2014

1	Wolfberg AJ, Berkowitz RS, Goldstein DP: Postevacuation hCG levels and risk of gestational trophoblastic neoplasia in women with complete molar pregnancy. Obstet Gynecol 106(3):548,o2005 Woolas RP, Bower M, Newlands ES, et al: Influence of chemotherapy for gestational trophoblastic disease on subsequent pregnancy outcome. BJOG 105: 1032, 1998 World Health Organization Scientific Group: Gestational trophoblastic disease. WHO Tech Rep Ser 692:1,o1983 Zhang W, Uu B, Wu J, et al: Hemoptysis as primary manifestation in three women with choriocarcinoma with pulmonary metastasis: a case series. J Med Case Rep 11(1):110,o201 Physiology of Labor MATERNAL AND FETAL COMPARTMENTS .......... 400 SEX STEROID HORMONE ROLE . . ................ 401 PROSTAGLANDINS ROLE .......e..e......e..e. . ... 402 PHASE 1: UTERINE QUIESCENCE AND CERVICAL SOFTENING ................... .......... ... 403 PHASE 2: PREPARATION FOR LABOR ............... 408

1	PROSTAGLANDINS ROLE .......e..e......e..e. . ... 402 PHASE 1: UTERINE QUIESCENCE AND CERVICAL SOFTENING ................... .......... ... 403 PHASE 2: PREPARATION FOR LABOR ............... 408 PHASE 3: .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411 UTEROTONINS IN PARTURITION PHASE 3 ........... 416 PHASE 4: THE PUERPERIUM ... .................. 417 From time immemorial inquiring minds have sought an explanation or the act that labour usualy ensues about 280 days after the appearance of the last menstrual perio, but thus ar no satiactory universal cause has been discovered. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) The importance of labor physiology was highlighted in the irst edition of Wiliams Obstetrics, in which an entire section was devoted to the topic. Given the science at that time, those nine chapters were concerned with the mechanics of labor and delivery. However, the current understanding of labor includes a wide spectrum of preparedness even before the first regular contractions.

1	Labor is the last few hours of human pregnancy. It is characterized by forceful and painful uterine contractions that efect cervical dilation and cause the fetus to descend through the birth canal. Extensive preparations take place in both the uterus and cervix long before this. During the irst 36 to 38 weeks of normal gestation, the myometrium is in a preparatory yet unresponsive state. Concurrently, the cervix begins an early stage of remodeling yet maintains structural integrity. Following this prolonged uterine quiescence, a transitional phase follows during which myometrial unresponsiveness is suspended and the cervix undergoes ripening, efacement, and loss of structural cohesion.

1	The physiological processes that regulate parturition-the bringing forth of young-and the onset of labor continue to be deined. Three general contemporaneous theories describe labor initiation. Viewed simplistically, the irst is the functional loss of prenancy maintenance actors. he second focuses on synthesis of actors that induce parturition. The third suggests that the mature fetus is the source of the initial signal or parturition commencement. Current research supports a model that draws from all three themes. However, labor onset clearly represents the culmination of a series of biochemical changes in the uterus and cervix. hese result from endocrine and paracrine signals emanating from both mother and fetus. Their relative contributions vary between species, and it is these diferences that complicate elucidation of the exact factors that regulate human parturition. When parturition is abnormal, then preterm-labor, dystocia, or postterm pregnancy may result. Of these, preterm

1	that complicate elucidation of the exact factors that regulate human parturition. When parturition is abnormal, then preterm-labor, dystocia, or postterm pregnancy may result. Of these, preterm labor remains the major contributor to neonatal mortality and morbidity.

1	The myometrial layer of the uterus is composed of bundles of smooth muscle cells surrounded by connective tissue. In contrast to skeletal or cardiac muscle, the smooth muscle cell is not terminally diferentiated and therefore is readily adaptable to environmental changes. Varied stimuli such as mechanical stretch, inlammation, and endocrine and paracrine signals can modulate Physiology of Labor 401

1	Physiology of Labor 401 FIGURE 21-1 The amnion synthesizes prostaglandins, and late in pregnancy, synthesis is augmented by increased phospholipase A2 and prostaglandin H synthase, type 2 (PGHS-2) activity. During pregnancy, the transport of prostaglandins from the amnion to maternal tissues is limited by expression of the inactivating enzymes, prostaglandin dehydrogenase (PGDH), in the chorion. During labor, PGDH levels decline, and amnion-derived prostaglandins can influence membrane rupture and uterine contractility. The role of decidual activation in parturition is unclear but may involve local progesterone metabolism and higher prostaglandin receptor concentrations, thus enhancing uterine prostaglandin actions and cytokine production. (Redrawn from Smith R: Parturition. N Engl J Med. 2007 Jan 18;356(3):271-283.) the transition of the smooth muscle cell among phenotypes that provide cell growth, proliferation, secretion, and contractility.

1	In addition to this phenotypic plasticity, several smooth muscle qualities confer advantages for uterine contraction eiciency and fetal delivery. First, the degree of smooth muscle cell shortening with contractions may be one order of magnitude greater than that attained in striated muscle cells. Second, forces can be exerted in smooth muscle cells in multiple directions. his difers from the contraction force generated by skeletal muscle, which is always aligned with the axis of the muscle fibers. hird, smooth muscle is not organized in the same manner as skeletal muscle. In myometrium, the thick and thin ilaments are found in long, random bundles throughout the cells. his plexiform arrangement aids greater shortening and force-generating capacity. Last, greater multidirectional force generation in the uterine fundus compared with that of the lower uterine segment permits versatility in expulsive force directionality.

1	Lining the thick muscular uterine walls, the endometrium is transformed by pregnancy hormones and is then termed decidua. Composed of stromal cells and maternal immune cells, the decidua serves to maintain the pregnancy via unique immuno regulatory functions that suppress inlammatory signals during gestation. However, at the end of pregnancy, decidual activation ensues. With this, the decidua transitions to induce inflammatory signals and withdraw active immunosuppression, which contribute to parturition initiation. During pregnancy, the cervix has multiple functions that include: (1) maintenance of barrier function to protect the reproductive tract from infection, (2) maintenance of cervical competence despite greater gravitational forces as the fetus grows, and (3) orchestration of extracellular matrix changes that allow progressively greater tissue compliance.

1	In nonpregnant women, the cervix is closed and firm, and its consistency is similar to nasal cartilage. By the end of pregnancy, the cervix is easily distensible, and its consistency is similar to the lips of the oral cavity. Observations in threedimensional sonography and magnetic resonance imaging show increases in the cross-sectional area of the cervical canal and in the cervical stroma from early to late pregnancy (House, 2009; Lang, 2010). Concurrent with expansion of the stroma, the cervical epithelia proliferate and exert a pregnancy-specific immunoprotection.

1	In addition to providing the exchange of nutrients and waste between mother and fetus, the placenta is a key source of steroid hormones, growth factors, and other mediators that maintain pregnancy and potentially aid the transition to parturition. The fetal membranes-amnion and chorion and adjacent decidua-make up an important tissue shell around the fetus that serves as a physiological, immunological, and metabolic shield to protect against untimely parturition initiation.

1	he amnion provides virtually all of the fetal membranes' tensile strength to resist membrane tearing and rupture (Chap. 5, p. 95). This avascular tissue is highly resistant to penetration by leukocytes, microorganisms, and neoplastic cells (Fig. 21-1). It also constitutes a selective filter to prevent fetal particulatebound lung and skin secretions from reaching the maternal compartment. In this manner, maternal tissues are protected from amnionic fluid constituents that could prematurely accelerate decidual or myometrial activation or could promote adverse events such as amnionic luid embolism. The chorion is a primarily protective tissue layer and provides immunological acceptance. It is also enriched with enzymes that inactivate uterotonins, which are agents that stimulate contractions. Inactivating enzymes include prostaglandin dehydrogenase, oxytocinase, and enkephalinase (Cheung, 1990; Germain, 1994).

1	In many species, the role of sex steroid hormones is clear-estrogen promotes and progesterone inhibits the events leading to parturition. nd, the removal of progesterone, that is, progesterone withdrawa, directly precedes progression of parturition. In addition, providing progesterone to some species will delay parturition via a decline in myometrial activity and continued cervical competency (Challis, 1994). In humans, however, it seems most likely that both estrogen and progesterone are components of a broader molecular system that maintains uterine quiescence.

1	Plasma levels of estrogen and progesterone in normal pregnancy are enormous and in great excess of the ainity constants for their receptors. For this reason, it is diicult to comprehend how relatively subtle changes in the ratio of their concentrations could modulate physiological processes during pregnancy. The teleological evidence, however, for an increased progesteroneto-estrogen ratio in the maintenance of pregnancy and a decline in this ratio for parturition is overwhelming. In all species studied, including humans, administration of the progesteronereceptor antagonists miepristone (RU-48) or onapristone will promote some or all key features of parturition. These include cervical ripening, greater cervical distensibility, and augmented uterine sensitivity to uterotonins (Bygdeman, 1994; Chwalisz, 1994b; Wolf, 1993).

1	The exact role of estrogen in regulation of human uterine quiescence and cervical competency is less well understood. hat said, estrogen can advance progesterone responsiveness and, in doing so, promote uterine quiescence. At the end of pregnancy, estrogen aids processes that mediate uterine activation and cervical ripening. Both progesterone and estrogen bind to nuclear receptors that regulate gene transcription in a cell-and context-speciic pattern. Two nuclear receptors for estrogen are estrogen receptor a (ERa) and estrogen receptor 3 (ER3). Nuclear receptor isoforms of the progesterone receptor (PR-A and PR-B) are encoded by difering transcripts from a single gene (Patel, 2015).

1	Prostaglandins are lipid molecules with varied hormone-like actions. In parturition, they playia prominent role in myometrial contractility, relaxation, and inlammation. Prostaglandins interact with a family of eight diferent G-protein-coupled receptors (p. 406), several of which are expressed in myometrium and cervix (Konopka, 2015; Myatt, 2004).

1	The major synthetic pathways involved in prostaglandin biosynthesis are shown in Figure 21-2. Prostaglandins are produced using plasma membrane-derived arachidonic acid, which usually is released by the action of phospholipase A2 or C. Arachidonic acid can then act as substrate for both type 1 and 2 prostaglandin H synthase (PGHS-l and -2), which are also called cyclooxygenase-l and -2 (COX-l and -2). Both PGHS isoforms convert arachidonic acid to the unstable prostaglandin G2 and then to prostaglandin H2. hese enzymes are the target of many nonsteroidal antiinlammatory drugs (NSAIDs). Indeed, the tocolytic actions of speciic NSAIDs, as discussed in Chapter 42 (p. 826), were considered promising until they were shown to have adverse fetal efects (Loudon, 2003; Olson, 2003, 2007).

1	Through prostaglandin isomerases, prostaglandin H2 is converted to active prostaglandins. These include prostaglandins E2 (PGEJ, F2x (PGF2x), and 12 (PGI2). Isomerase expression is tissue-speciic and thereby controls the relative production of various prostaglandins. Another important control point for prostaglandin activity is its metabolism, which most often is

1	Phospholipase C with through the action of 15-hydroxyprostaglandin dehydrogenase (PGDH). Expression of this enzyme is up regulated during pregnancy in the uterus and cervix, which provides the important ability to rapidly inactivate prostaglandins (Giannoulias, 2002; Kishore, 2014). Thus, myometrial responses to prostaglandins stem from a balance between prostaglandin synthesis versus metabolism, from the relative expression of various prostaglandin receptors, or from a switch in receptor-signaling pathways (Kandola, 2014; Lyall, 2002; Olson, 2007; Smith, 2001). It is entirely possible that prostanoids contribute to myometrial relaxation at one stage of pregnancy and to myometrial contractions after parturition initiation (Myatt, 2004).

1	In addition to the myometrium, the amnion synthesizes several bioactive pep tides and prostaglandins that cause myometrial relaxation or contraction (see Fig. 21-1). Late in pregnancy, amnionic prostaglandin biosynthesis is increased, and phospholipase A2 and PGHS-2 show greater activity Oohnson, 2002). Accordingly, many hypothesize that prostaglandins regulate events leading to parturition. The amnion is likely the major source for amnionic fluid prostaglandins, and their role in the activation of cascades that promote membrane rupture is clear. he influence of amnion-derived prostaglandins on uterine quiescence and activation, however, is less delineated. his is because prostaglandin transport from the amnion through the chorion to access maternal tissues is limited by expression of PGDH. Physiology of Labor 403

1	Physiology of Labor 403 Importantly, the phases of parturition should not be confused with the clinical stages of labor, that is, the first, second, and third stages-which make up phase 3 of parturition (Fig. 2i1-4). arturitionContractile 1unresponsiveness, Uterine of labor cervical softening Phase 3 Stimulation Processes Uterine Phase4 Involution Parturient Uterine preparedness contraction, involution, for labo, cervical dilation, cervical repai, tion, a remarkably efective period of expUlsion (three myometrial quiescence is imposed. This stages of labor) Initiation of maintenance of cervical structural integ parturition Onset of labor FIGURE 21-3 The phases of parturition. PHASE 1: UTERINE QUIESCENCE AND CERVICAL SOFTENING

1	Initiation of maintenance of cervical structural integ parturition Onset of labor FIGURE 21-3 The phases of parturition. PHASE 1: UTERINE QUIESCENCE AND CERVICAL SOFTENING As shown in Figure 21-3, parturition can be arbitrarily divided into four overlapping phases that correspond to the major physiological transitions of the myometrium and cervix during pregnancy (Casey, 1993, 1997; Challis, 2000; Word, 2007). These phases of parturition include: (1) a prelude to it, (2) the preparation for it, (3) the process itself, and (4) recovery. Stages of Labor : Phase of of pregnancy and is characterized by

1	Stages of Labor : Phase of of pregnancy and is characterized by Delivery of uterine smooth muscle tranquility with rity (Fig. 21-5). All manner of molecular systems-neural, endocrine, paracrine, and autocrine-are likely called to implement and coordinate a state of relative uterine unresponsiveness. Moreover, a complementary "fail-safe" system that protects the uterus against agents that could perturb the tranquility of phase 1 also must be in place. During phase 1, the myometrial cells undergo a phenotypic modification to a noncontractile state, and uterine muscle is rendered unresponsive to natural stimuli. Concurrently, the uterus must initiate extensive changes in its size and vascularity to accommodate fetal growth and prepare for uterine :-6

1	FIGURE 21-4 Composite of the average dilation curve for labor in nulliparous women. The curve is based on analysis of data derived from a large, nearly consecutive series of women. The first stage is divided into a relatively flat latent phase and a rapidly progressive active phase. In the active phase, there are three identifiable parts: an acceleration phase, a linear phase of maximum slope, and a deceleration phase. (Redrawm from Friedman EA: Labor: Clinical Evaluation and Management, 2nd ed. New York, Appleton-Century-Crofts, 1978.) contractions. he myometrial unre sponsiveness of phase 1 continues until near the end of pregnancy. That said, tractions are felt during the quiescent phase, but they do not normally cause cervical dilation. hese contractions are common toward the end of preg nancy, especially in multiparas, and are referred to as Braxton Hicks contractions oralse labor (Chap. 4, p. 50).

1	The quiescence of phase 1 likely stems from: (1) actions of estrogen and progesterone via intracellular receptors, myometrial-cell plasma membrane receptor-mediated increases in cyclic adenosine monophosphate (cMIP), generation of cyclic guanosine monophosphate (cGMP), and (4) other systems, including modiication of myometrial-cell ion channels. The balance between myometrial relaxation and contraction is controlled by steroid-and peptide-hormone transcriptional regulation of key genes and their protein products. Quiescence is achieved in part by: (1) diminished intracellular crosstalk and reduced intracellular Ca2+ ([Ca2+J levels; (2) ionchannel regulation of cell membrane • Myometrium is major site of action • Cervix is major site • Both are sites of action Phase 1 Phase 2 Phase 3 Uterine stretch Gap junction receptors Fetal signals (SPA, PAF, CRH) Fetal membrane Prostaglandins Cortisol? Phase 4

1	Phase 1 Phase 2 Phase 3 Uterine stretch Gap junction receptors Fetal signals (SPA, PAF, CRH) Fetal membrane Prostaglandins Cortisol? Phase 4 FIGURE 21-5 The key factors thought to regulate the phases of human parturition. CRH = corticotropin-releasing hormone; hCG i.):D o:sD) CRH? potential; (3) activation of the uterine endoplasmic reticulum stress-unfolded protein response; and (4) uterotonin degradation. In contrast, contractility results from: (1) enhanced interactions between the actin and myosin proteins; (2) heightened excitability of individual myometrial cells; and (3) promotion of intracellular crosstalk that allows synchronous contractions to develop.

1	Actin and myosin proteins are essential to muscle contraction. For this, actin must be converted from a globular to a ilamentous form. Indeed, a potential mechanism for maintenance of relaxation is the promotion ofactin into a globular form rather than into ibrils, which are required for contraction (Fig. 21-6). Moreover, actin must be attached to the cytoskeleton at focal points in the cell membrane to allow tension to develop. Actin must partner with myosin, which is composed of multiple light and heavy chains. The coupling of myosin and actin activates adenosine triphosphatase (ATPase), hydrolyzes adenosine triphosphate, and generates force. This interaction is brought about by enzymatic phosphorylation of the 20-kDa light chain of myosin (Stull, 1998). his is catalyzed by the enzyme myosin light-chain kinase, which is activated by calcium. Calcium binds to calmodulin, a calcium-binding regulatory protein, which in turn binds to and activates myosin lightchain kinase.

1	hus, logically, uterine relaxation ordinarily is promoted by conditions that lower concentrations of (Ca2+)i. In contrast, prostaglandin dehydrogenase; SPA surfactant protein A.

1	agents that prompt contraction act on myometrial cells to augment (Ca2+)i levels. Or, they allow an inlux of extracellular calcium through ligand-or voltage-regulated calcium channels (see Fig. 21-6). Voltage-gated ion channels open, additional calcium ions move into the cell, and cellular depolarization follows. For example, prostaglandin F2ex and oxytocin bind their respective receptors during labor to open ligand-activated calcium channels. Activation of these receptors also releases calcium from the sarcoplasmic reticulum to lower electronegativity within the cell. Additionally, greater localization of nonselective cation channels on the cell membrane promotes Ca2+ entry (Ying, 2015). The rise in (Ca2+)i levels is often transient. But, contractions can be prolonged by inhibition of myosin phosphatase, an enzyme which dephosphorylates myosin (Woodcock, 2004). Regulation of Membrane Potentials

1	Regulation of Membrane Potentials As just noted, myocyte excitability is regulated in part by changes in the electrochemical potential gradient across the plasma membrane. Before labor, myocytes maintain a relatively high interior electronegativity. Maintenance of a hyperpolarized membrane potential attenuates smooth muscle cell excitation and is regulated by ion channels. Consistent with the importance of myometrial quiescence, numerous potassium channels control membrane potential. One key regulator is the large-conductance voltage-and Ca2+activated K channel (BKeJ (Perez, 1993). In normal physiology, the myometrial BKca channel plays dual and opposing CRH, �2-sympathomimetics, 8 Oxytocin, thrombin, Physiology of Labor 405

1	FIGURE 21 -6 Uterine myocyte relaxation and contraction. A. Uterine relaxation is maintained by factors that increase myocyte cyclic adenosine monophosphate (cAMP) levels. This activates protein kinase A (PKA) to promote phosphodiesterase activity with dephosphorylation of myosin light-chain kinase (MLCK). Other processes serve to maintain actin in a globular form and thus to prevent the fibril formation necessary for contractions. B. Uterine contractions result from reversal of these sequences. Actin now assumes a fibrillar form, and calcium enters the cell to combine with calmodulin to form complexes. These complexes activate MLCK to bring about phosphorylation of the myosin light chains. This generates ATPase activityto cause sliding of myosin over the actin fibrils, which is a uterine contraction. AC = + calcium; DAG = diacylglycerol; Gs and GQ = G-receptor proteins; IP3 = inositol triphosphate; LC20 = light chain 20; PIP3 = phosphatidylinositol 3,4,5-triphosphate;PLC =

1	a uterine contraction. AC = + calcium; DAG = diacylglycerol; Gs and GQ = G-receptor proteins; IP3 = inositol triphosphate; LC20 = light chain 20; PIP3 = phosphatidylinositol 3,4,5-triphosphate;PLC = phospholipase C; R-PKA inactive protein kinase. (Redrawn from Smith R: Parturition. N Engl J Med. 2007 Jan 18;356(3):271-283.) roles to maintain a balance between uterine quiescence and contractility. The Bca channel is abundantly expressed in the myometrium. For most of pregnancy, opening the BKca channel allows potassium to leave the cell to maintain interior electronegativity, thus preventing voltage-gated Ca2+ inlux and contraction. Enhancing Bca channel opening results in myometrial relaxation, whereas inhibition ofthe Bca channel augments myometrial contractility. he ability of Bca channel to regulate calcium dynamics and ultimately uterine contractility from early to late gestation may result from temporal changes in expression ofthe Bca channel and/or Bca interacting partners

1	channel to regulate calcium dynamics and ultimately uterine contractility from early to late gestation may result from temporal changes in expression ofthe Bca channel and/or Bca interacting partners (Wakle-Prabagaran, 2016).

1	Cellular signals that control myometrial contraction and relaxation can be efectively transferred between cells through intercellular junctional channels. Communication is established between myocytes by gap junctions, which aid the passage of electrical or ionic coupling currents as well as metabolite coupling. The transmembrane channels that make up the gap junctions consist of two protein "hemi-channels" (Saez, 2005). These connexons are each composed of six connexin subunit proteins (Fig. 21-7). Of these, connexin-43 is expressed in myometrium, and concentrations rise near labor onset. Pairs of connexons establish a conduit between coupled cells for the exchange of small molecules that can be nutrients, waste, metabolites, second messengers, or ions. Optimal numbers and types ofgap junctions are believed to be important for electrical myometrial synchrony.

1	Progesterone maintains uterine quiescence in part by mechanisms that lower expression of various key proteins needed for contractility. hese contraction-associated proteins (CAPs) include the oxytocin receptor, prostaglandin F receptor, and connexin-43. At the end of pregnancy, increased stretch along with greater estrogen dominance raises CAP levels. Integration ofdiverse regulatory pathways culminates in released inhibition ofconnexin-43 and oxytocin receptor levels to promote greater uterine contractility (Nadeem, 2016; Renthal, 2010; Williams, 2012b). As another potential mechanism, progesterone maintains uterine quiescence through support of myometrial caspase 3, which is an anticontractile agent Qeyasuria, 2009). his protein degrades both actin and the speciic gap junction protein, connexin-43 (Kyathanahalli, 2015).

1	called connexins. Six connexins form a hemichannel (connexon), nel. Connexons and gap junction channels can be formed from one or more connexin proteins. The composition of the gap junc tion channel is important for these channels' selectivity with regard to passage of molecules and communication between cells. In mice, myometrial caspase 3 activation is regulated by a pregnancy-induced endoplasmic reticulum stress response (ERSR). As background, the endoplasmic reticulum aids protein folding and transport. Functional irregularities cause misfolded proteins to accumulate and trigger the ERSR. he ERSR and its unolded-protein response (UPR) are cellular mechanisms that work to maintain homeostasis in the face of stimuli, such as stretch and inlammation. Prolonged ERSR promotes caspase 3 activation to preserve quiescence despite these stimuli.

1	Various cell surface receptors directly regulate myocyte contractility. Discussions thus far have described ion channel-linked receptors that regulate intracellular Ca2+ and membrane potential. In addition, numerous G-protein-coupled receptors appear to be modified during the phases of parturition. Several of these are present in myometrium and associated with Gos-mediated activation of adenylyl cyclase to yield higher cAMP levels. These receptors together with appropriate ligands may act with sex steroid hormones to maintain uterine quiescence (Price, 2000; Sanborn, 1998). Examples are the LH receptor and corticotropin-releasing hormone receptor 1 (CRHR1), both described in this section (Fig. 21-8). Other G-protein-coupled myometrial receptors, instead, are associated with G-protein-mediated activation of phospholipase C, which remember releases arachidonic acid. Ligands for the G-protein-coupled receptors include numerous neuropeptides, hormones, and autacoids. Many of these are

1	activation of phospholipase C, which remember releases arachidonic acid. Ligands for the G-protein-coupled receptors include numerous neuropeptides, hormones, and autacoids. Many of these are available to the myometrium during pregnancy in high concentration via endocrine or autocrine mechanisms.

1	3-Adrenoreceptors are prototypical examples of cAMP signaling causing myometrium relaxation. 3-Adrenergic receptors mediate Gs-stimulated increases in adenylyl cyclase, elevated

1	FIGURE 21-8 G-protein-coupled receptor signal transduction pathways. A. Receptors coupled to heterotrimeric guanosine-triphosphate (GTP)-binding proteins (G proteins) are integral transmembrane proteins tl13t transduce extracellular signals to the cell interior. G-protein-coupled receptors exhibit a common structural motif consisting of seven membrane-spanning regions. B. Receptor occupation promotes interaction between the receptor and the G protein on the interior surface of the membrane. This induces an exchange of guanosine diphosphate (GOP) for GTP on the G protein Q subunit and dissociation of the Q subunit from the 31 heterodimer. Depending on its isoform, the GTP-Q subunit complex mediates intracellular signaling either indirectly by acting on effector molecules such as adenylyl cyclase (AC) or phospholipase C (PLC), or directly by regulating ion channel or kinase function. cAMP cyclic adenosine monophosphate; DAG diacylglycerol; IP3 inositol triphosphate.

1	levels of cAMP, and myometrial cell relaxation. The rate-lim iting factor is likely the number of receptors expressed and the level of adenylyl cyclase expression. Agents binding to these receptors have been used for tocolysis of preterm labor and include ritodrine and terbutaline (Chap. 42, p. 826). LH and hCG hormones share the same receptor, and this smooth muscle and blood vessels (Ziecik, 1992). Levels of myometrial LH-hCG receptors during pregnancy are greater before than during labor. Chorionic gonadotropin acts to acti vate adenylyl cyclase by way of a plasma membrane receptor G's-linked system. This lessens contraction frequency and force and lowers the number of tissue-speciic myometrial cell gap junctions (Ambrus, 1994; Eta, 1994). hus, high circulating levels of hCG may be one mechanism of uterine quiescence. In the mouse, variations in FSH-receptor density also regulate myometrial contractile activity (Stilley, 2016).

1	In the mouse, variations in FSH-receptor density also regulate myometrial contractile activity (Stilley, 2016). four G-protein-coupled receptors. Speciically, prostaglandin E receptors 1 through 4 (EP ,-EP 4) are expressed in the myo metrium during pregnancy and with labor onset (Astle, 2005; Leonhardt, 2003). EP2 and EP4 act through G's to raise cAMP levels and maintain myometrial cell quiescence but switch to a G'q/1i1 calcium-activating pathway during labor (Kandola, 2014). EP] and EP3 receptors act through G'q and G'i to aug ment intracellular Ca2+ and contractility.

1	he peptide hormone relaxin binds to the G-protein-coupled receptor named reaxin amiy peptide receptor 1 (FP 1). Binding activates adenylyl cyclase in uterine smooth muscle cells. Adenylyl cyclase in turn prevents increased intracellular Ca2+ and thus promotes uterine quiescence (Downing, 1993; Meera, 1995). There are two separate human relaxin genes, designated HI and H2. Of these, HI is primarily expressed in the decidua, trophoblast, and prostate, whereas H2 is primarily expressed in the corpus luteum. Relaxin in plasma of pregnant women is believed to originate exclusively from corpus luteum secretion. Plasma levels peak at approximately 1 ng/mL between 8 and 12 weeks' gestation. Thereater, they decline to lower levels that persist until term.

1	Corticotropin-releasing hormone (CRH) is synthesized in the placenta and hypothalamus. Discussed on page 410, CRH plasma levels rise dramatically during the inal 6 to 8 weeks of normal pregnancy and are implicated in mechanisms that control the timing of human parturition (Smith, 2007; Wadhwa, 1998). CRH appears to promote myometrial quiescence during most of pregnancy but then aids myometrial contractions with parturition onset. Studies suggest that these opposing actions are achieved by diferential actions of CRH via its receptor CRHR1. In nonlaboring myometrium at term, the interaction of CRH with its CRHR1 receptor activates the Gs-adenylate cyclasecAMP signaling pathway. his results in inhibition of inositol triphosphate (IP3) and stabilization of (Ca2+)j levels (You, 2012). However, in term laboring myometrium, (Ca2+)j concentrations are augmented by CRH activation of G proteins Gq and Gi and prompts stimulation ofIP3 production and greater contractility.

1	As just described, cAvIP is an important mediator of myometrial relaxation. However, activation of guanylyl cyclase raises Physiology of Labor 407 intracellular cyclic guanosine monophosphate (cGMP) levels. This also promotes smooth muscle relaxation (Word, 1993). Intracellular cGMP levels are increased in the pregnant myometrium and can be stimulated by atrial natriuretic peptide (ANP) , brain natriuretic peptide (BNP) receptors, and nitric oxide (Telfer, 2001). All of these factors and their receptors are expressed in the pregnant uterus.

1	In addition to pregnancy-induced compounds that promote myometrial cell refractoriness, the activity of enzymes that degrade or inactivate endogenously produced uterotonins are strikingly increased in phase 1. Some of these degrading enzymes and their respective targets include PGDH and prostaglandins; enkephalinase and endothelins; oxytocinase and oxytocin; diamine oxidase and histamine; catechol O-methyltransferase and catecholamines; angiotensinases and angiotensin-II; and platelet-activating factor (P AF) and P AF acetylhydrolase. Levels of several of these enzymes decrease late in gestation (Germain, 1994). To ensure uterine quiescence, the synthesis in the decidua of prostaglandins, in particular PGF2', is markedly suppressed. Suppression of prostaglandin production here persists through out most of pregnancy, and suppression withdrawal is a prereq uisite for parturition (Norwitz, 2015).

1	Suppression of prostaglandin production here persists through out most of pregnancy, and suppression withdrawal is a prereq uisite for parturition (Norwitz, 2015). Phase 1 of parturition also promotes an environment of immune tolerance to protect the fetus. Namely, decidual stromal cells proactively ensure that fetal antigens do not elicit a maternal immune response. his stems from a reduced capacity to attract T cells. This limited ability derives in part from epigenetic silencing of T cell-attracting inlammatory chemokine genes (Erlebacher, 2013; Nancy, 2012; PrabhuDas, 2015).

1	The initial stage of cervical remodeling-termed soteningbegins in phase 1 of parturition. It is characterized by greater tissue compliance, yet the cervix remains irm and unyielding. Hegar (1895) irst described palpable softening of the lower uterine segment at 4 to 6 weeks' gestation, and this sign was once used to diagnose pregnancy. Clinically, the maintenance of cervical anatomical and structural integrity is essential for pregnancy to continue to term. Preterm cervical dilation, structural insuiciency, or both may forecast delivery.

1	Cervical softening results from increased vascularity, cellular hypertrophy and hyperplasia, and slow, progressive compositional and structural changes in the extracellular matrix (Mahendroo, 2012; Myers, 2015; Word, 2007). Key to matrix changes, collagen, which is the main structural protein in the cervix, undergoes conformational changes that alter tissue stifness and flexibility (Zhang, 2012). Speciically, collagen processing and the number or type of stable covalent cross-links between collagen triple helices is altered. Mature cross-links between newly synthesized collagen monomers are reduced due to diminished expression and activity of the cross-link-forming enzymes beginning in early pregnancy (Akins, 2011; Drewes, 2007; Yoshida, 2014). hese enzymes are lysyl hydroxylase and lysyl oxidase. Together, these early pregnancy changes contribute to greater tissue compliance.

1	Clinical evidence for the importance of matrix changes to cervical sotening is supported by in vivo mechanical evaluation of the cervix (Badir, 2013; Parra-Saavedra, 2011). he prevalence of cervical insuiciency is also higher in those with inherited defects in the synthesis or assembly of collagen or elastic ibers (Anum, 2009; Hermanns-Le, 2005; Rahman, 2003; Wang, 2006). Examples are Ehlers-Danlos and Marfan syndromes, discussed in Chapter 59 (p. 1151). Concurrent with matrix remodeling in the sotening period, genes involved in cervical dilation and parturition are actively repressed (Hari Kishore, 2012). PHASE 2: PREPARATION FOR LABOR To prepare for labor, the myometrial tranquility of phase 1 of parturition must be suspended-so-called uterine awakening or activation. This phase 2 of parturition is a progression of uterine changes during the last few weeks of pregnancy. Importantly, shifting events associated with phase 2 can cause either preterm or delayed labor.

1	Key factors in uterine activation are depicted in Figure 21-5. In species that exhibit progesterone withdrawal, parturition progression to labor can be blocked by administering progesterone to the mother. Whether progesterone administration in the absence of classic progesterone withdrawal in pregnant women can delay the timely onset of parturition or prevent preterm labor continues to be investigated. he possibility that progesterone-containing injections or vaginal suppositories may prevent preterm labor has been studied in several randomized trials conducted during the past 15 years. hese are discussed in Chapter 42 (p. 816), and their use in preventing recurrent preterm birth continues to be debated (Norman, 2016).

1	Classic progesterone withdrawal resulting from decreased secretion does not occur in human parturition. However, a mechanism for progesterone inactivation, whereby the myometrium and cervix become refractory to progesterone's inhibitory actions, is supported by studies using progesterone-receptor antagonists. Mifepristone is a classic steroid antagonist, acting at the level of the progesterone receptor. Although less efective in inducing abortion or labor in women later in pregnancy, mifepristone appears to have some efect on cervical ripening and on increasing myometrial sensitivity to uterotonins (Berkane, 2005; Chwalisz, 1994a).

1	The diverse mechanisms by which functional progesterone withdrawal or antagonism is achieved is an active area of research. These include: (1) changes in the relative expression of the nuclear progesterone-receptor isoforms, PR-A, PR-B, and PR-C; (2) diferential interaction of PR-A and PB-B with enhancers and inhibitors of gene expression; (3) alterations in PR activity through changes in the expression of coactivators or corepressors that directly influence receptor function; (4) local inactivation of progesterone by steroid-metabolizing enzymes or synthesis of a natural antagonist; and (5) microRNA regulation of progesterone-metabolizing enzymes and transcription factors that modulate uterine quiescence (Condon, 2003; Mahendroo, 1999; Mesiano, 2002; Nadeem, 2016; Renthal, 2010; Williams, 2012a). Taken together, these observations support the concept that multiple pathways exist for a functional progesterone withdrawal.

1	Phase 2 myometrial changes prepare it for labor contractions. his results from a shit in the expression of key proteins that control uterine quiescence to an expression of contraction-associated proteins, described earlier (p. 405) (Renthal, 2015). Of these CAPs, myometrial oxytocin receptors and gap junction proteins, such as connexin-43, markedly rise in number. These CAPs increase uterine irritability and responsiveness to uterotonins.

1	Another critical change in phase 2 is formation of the lower uterine segment from the isthmus. With this development, the fetal head often descends to or even through the pelvic inletso-called lightening. he abdomen commonly undergoes a shape change, sometimes described by women as "the baby dropped." It is also likely that the lower segment myometrium is unique from that in the upper uterine segment, resulting in distinct roles for each near term and during labor. This is supported by human studies that demonstrate diferential expression of prostaglandin receptors and CAPs within the upper-and lower-segment myometrial regions (Astle, 2005; Blanks, 2003; Sparey, 1999). Near term, elevated expression of the HoA13 gene in the lower myometrial segment compared with the upper segment also induces CAP expression and regionalized contractility of the lower segment (Li, 2016).

1	Because of its long-standing application for labor induction, it seemed logical that oxytocin must playia central role in spontaneous human labor. Myometrial oxytocin receptor levels do rise during phase 2 of parturition, and the level of oxytocin receptor mRNA in human myometrium at term is greater than that found in preterm myometrium (Wathes, 1999). However, it is unclear whether oxytocin plays a role in the early phases of uterine activation or whether its sole function is in the expulsive phase of labor. Most studies of regulation of myometrial oxytocin receptor synthesis have been performed in rodents. Disruption of the oxytocin receptor gene in the mouse does not afect parturition. This suggests that, at least in this species, multiple systems likely ensure that parturition occurs.

1	Progesterone and estradiol appear to be the primary regulators of oxytocin receptor expression. Estradiol treatment in vivo or in myometrial explants raises myometrial oxytocin receptor concentrations. This action, however, is prevented by simultaneous treatment with progesterone (Fuchs, 1983). Progesterone also may act within the myometrial cell to enhance oxytocin receptor degradation and inhibit oxytocin activation of its receptor at the cell surface (Bogacki, 2002). hese data indicate that one of the mechanisms whereby progesterone maintains uterine quiescence is through inhibition of a myometrial oxytocin response. Physiology of Labor 409

1	Physiology of Labor 409 Before contractions begin, the cervix must undergo extensive remodeling. his eventually leads to the cervix yielding and dilating from forceful uterine contractions. Cervical modiications during phase 2 principally involve connective tissue changes-termed cervical ripening. The transition from the softening to the ripening phase begins weeks or days before labor. During this transformation, the cervical matrix changes its total amounts of gycosaminogycans, which are large linear polysaccharides, and proteogycans, which are proteins bound to these glycosaminoglycans.

1	Many of the processes that aid cervical remodeling are controlled by the same hormones regulating uterine function. That said, the molecular events of each are varied because of diferences in cellular composition and physiological requirements. For example, the hormone relaxin regulates myometrial quiescence. It also regulates cervical ripening, but through cell proliferation and modulation of extracellular matrix components (Park, 2005; Soh, 2012). The uterine corpus is predominantly smooth muscle. In contrast, the cervix has a high ratio of ibroblasts to smooth muscle cells, and extracellular matrix contributes significantly to overall tissue mass. Recent studies in the nonpregnant human cervix report a spatial gradient of smooth muscle cells. Specifically, smooth muscle cells make up approximately 50 percent of stromal cells at the internal os but only 10 percent at the external os (Vink, 2016).

1	Collagen. The cervix is an extracellular-matrix-rich tissue. Constituents of the matrix include type I, III, and IV collagen, matricellular proteins, glycosaminoglycans, proteoglycans, and elastic ibers. Of these, collagen is largely responsible for the structural disposition of the cervix. During collagen assembly, multiple collagen triple-helical molecules are cross-linked to one another by the actions of lysyl oxidase to form ibrils. In addition, ibril size, packing, and organization determine the strength and mechanical properties of the cervix. These properties are regulated in part by collagen-binding proteoglycans such as decorin or biglycan, as well as matricellular proteins such as thrombospondin 2 (Fig. 21-9).

1	Higher turnover of collagen during pregnancy likely allows the gradual replacement of mature cross-linked collagen ibrils with poorly cross-linked ibrils, which yield greater collagen disorganization. his increased turnover, rather than loss of collagen to achieve cervical remodeling, is supported by mouse and human studies that document no changes in collagen content between nonpregnant states and term pregnancy (Akins, 201i1; Myers, 2008; Read, 2007; Yoshida, 2014). In further support, polymorphisms or mutations in genes required for collagen assembly are associated with an elevated incidence of cervical insuiciency (Anum, 2009; Rahman, 2003; Warren, 2007).

1	Glycosaminoglycans and Proteoglycans. Hyaluronan is a high-molecular-weight polysaccharide that functions alone, whereas most other glycosaminoglycans (GAGs) complex with proteins to form proteoglycans. Hyaluronan is a hydrophilic, space-illing molecule, and thus greater hyaluronan production during cervical ripening is thought to increase viscoelasticity, hydration, and matrix disorganization. Hyaluronan synthesis is carried out by hyaluronan synthase isoenzymes, and expression of these enzymes is elevated in the cervix during ripening (kgul, 2012; Straach, 2005).

1	Although not well deined, changes in proteoglycan composition are also suggested to accompany cervical ripening. At least three small leucinerich proteoglycans are expressed in the cervix-decorin, biglycan, and ibromodulin (Westergren-Thorsson, 1998). In other connective tissues, decorin interacts with collagen to regulate the packing, order, and strength of collagen fibrils (see Fig. 21-9) (Ameye, 2002). In addition to the cervix, these proteoglycans are expressed in the fetal membranes and uterus.

1	Inflammatory Changes. In phase 2, resident immune cells are localized to the cervical stroma, although a functional role for these cells in this phase of remodeling has been challenged. Microarray studies comparing gene to allow a gradual increase in mechanical compliance of the cervix, A collagen fiber is made up of many fibrils, Fibril size and packing are regulated in part by small proteoglycans such as ,,obefore and after cervical ripening decorln and by the denSity of collagen cross-links, In phase 1, fibril size is uniform and fibrils are veil organizedoalthough a decline in cross-link density aids softening, During cervical ripening show little rise in proinflammatory ,: ,o,

1	In phase 2, fibril size IS less uniform, and spacing between collagen fibrils and fibers is greater gene expression. In contrast, proinand disorganized, flammatory and immunosuppressive gene expression in the cervix after delivery increases markedly compared with that during cervical ripening (Bollapragada, 2009; Hassan, 2006, 2009). Further, detailed studies in mice provide evidence that leukocyte migration but not activation takes place before labor. Once labor is underway, activation of neutrophils, pro inflammatory M 1 macrophages, and tissue repair M2 macrophages in the cervix is augmented. This suggests a role for inlammatory cells in postpartum cervical remodeling and repair (Mahendroo, 2012). Induction of Cervical Ripening

1	Induction of Cervical Ripening No therapies prevent premature cervical ripening. In contrast, treatment to promote cervical ripening for labor induction includes direct application of prostaglandins PGE2 and PGF2'. Prostaglandins likely modiy extracellular matrix structure to aid ripening. Although the role of prostaglandins in the normal physiology of cervical ripening remains unclear, this property is useful clinically to assist labor induction (Chap. 26, p. 505). In some nonhuman species, the cascades of events that allow cervical ripening are induced by dropping serum progesterone concentrations. And in humans, administration of progesterone antagonists causes cervical ripening.

1	In addition to matrix changes, during pregnancy, endocervical epithelial cells proliferate such that endocervical glands account for a signiicant percentage of cervical mass. he endocervical canal is lined with mucus-secreting columnar and stratified squamous epithelia. These cells form both a mucosal barrier and a tight junctional barrier that protect against microbial invasion (Akgul, 2014; Blaskewicz, 2011; Timmons, 2007). The mucosal epithelium recognizes and deters pathogen invasion via expression of toll-like receptors that identiy pathogens and via antimicrobial pep tides and protease inhibitors. In addition, these epithelia express signals to underlying immune cells when a pathogenic challenge exceeds their protective capacity (Wira, 2005). • Fetal Contributions to Parturition

1	• Fetal Contributions to Parturition It is intriguing to envision that the mature human fetus provides the signal to initiate parturition, and evidence for fetal signaling is mounting (Mendelson, 2017). The fetus may give signals through blood-borne agents that act on the placenta or through secretion into the amnionic luid. Fetal growth is an important component in uterine activation in phase 2 of parturition. With uterine activation, stretch is required for induction of specific CAPs. Namely, stretch increases expression of connexin-43 and oxytocin receptors. Levels of gastrin-releasing peptide, a stimulatory agonist for smooth muscle, are also augmented by stretch in the myometrium (Tattersall, 2012).

1	Clinical clues for a role of stretch come from the observa preterm labor than singleton ones. And, preterm labor is also signiicantly more common in pregnancies complicated by hydramnios. Although the mechanisms causing preterm birth in these two examples are debated, a role for uterine stretch must be considered. Cell signaling systems that are inluenced by stretch to regulate the myometrial cell continue to be defined. his processmechanotransduction-may include activation of cell-surface receptors or ion channels, transmission of signals through extracellular matrix, or release of autocrine molecules that act directly on myometrium (Shynlova, 2007; Young, 2011).

1	The ability of the fetus to provide endocrine signals that initiate parturition has been demonstrated in several species. However, evidence suggests that it is not regulated in the same manner in humans. hat said, the human fetal hypothalamic-pituitaryadrenal-placental axis is considered a critical component of normal parturition. Moreover, premature activation of this axis is considered to prompt many cases of pre term labor (Challis, 2000, 2001). As in the sheep, steroid products of the human fetal adrenal gland are believed to have efects on the placenta and membranes that eventually transform the myometrium from a quiescent to a contractile state. A key component in the human may be the unique ability of the placenta to produce large amounts ofCRH 21-10).

1	A CRH hormone that is identical to maternal and fetal hypothalamic CRH is synthesized by the placenta in relatively large amounts (Grino, 1987; Saijonmaa, 1988). However, unlike hypothalamic CRH, which is under glucocorticoid negative feedback, cortisol instead stimulates placental CRH production. This ability makes it possible to create a feed-forward endocrine cascade that does not end until delivery. Maternal plasma CRH levels are low in the first trimester and rise from midgestation to term. In the last 12 weeks, CRH plasma levels rise exponentially, peak during labor, and then fall precipitously after delivery (Frim, 1988; Sasaki, 1987). Amnionic luid CRH concentrations similarly increase in late gestation. CRH is the only trophic hormone-releasing FIGURE 21-10 The placental-fetal adrenal endocrine cascade.

1	FIGURE 21-10 The placental-fetal adrenal endocrine cascade. In late gestation, placental corticotropin-releasing hormone (CRH) stimulates fetal adrenal production of dehydroepiandrosterone sul fate (DHEA-S) and cortisol. The latter stimulates production of placental CRH, which leads to a feed-forward cascade that enhances adrenal steroid hormone production. ACTH = adrenocorticotropic hormone. factor to have a speciic serum binding protein. During most of pregnancy, CRH-binding protein (CRH-BP) binds most maternal circulating CRH, and this inactivates it (Lowry, 1993). During later pregnancy, however, CRH-BP levels in both maternal plasma and amnionic luid decline, leading to markedly greater levels of bioavailable CRH (Perkins, 1995; Petraglia, 1997).

1	Petraglia, 1997). In pregnancies in which the fetus can be considered "stressed" from various complications, concentrations of CRH in fetal plasma, amnionic luid, and maternal plasma are greater than those seen in normal gestation (Berkowitz, 1996; McGrath, 2002). he placenta is the likely source of this elevated CRH concentration. For example, placental CRH content is four fold higher in placentas from women with preeclampsia than in those from normal pregnancies (Perkins, 1995).

1	Placental CRH is thought to play several roles in parturition regulation. It may enhance fetal cortisol production to provide positive feedback so that the placenta produces more CRH. Late in pregnancy-phase 2 or 3 of parturition-modiication in the CRH receptor favors a switch from cAMP formation to increased myometrial cell calcium levels via protein kinase C activation (You, 2012). Oxytocin acts to attenuate CRHstimulated accumulation of cAMP in myometrial tissue. CRH acts to augment myometrial contractile force in response to PGF2: (Benedetto, 1994). Finally, CRH stimulates fetal adrenal Cwsteroid synthesis, thereby increasing substrate for placental aromatization.

1	Some have proposed that the rising CRH level at the end of gestation relects a etal-placental clock (McLean, 1995). CRH concentrations vary greatly among women, and the rate of rise in maternal CRH levels is a more accurate predictor of pregnancy outcome than is a single measurement (Leung, 2001; McGrath, 2002). In this regard, the placenta and fetus, through endocrinological events, inluence the timing of parturition at the end of normal gestation.

1	Surfactant protein A (SP-A) produced by the fetal lung is required for lung maturation. SP-A is expressed by the human amnion and decidua, is present in the amnionic luid, and prompts signaling pathways in human myometrial cells (Garcia-Verdugo, 2008; Lee, 2010; Snegovskikh, 2011). he exact mechanisms by which SP-A activates myometrial contractility in women, however, remain to be clarified. One mode may be its efects on prostaglandins. Namely, SP-A selectively inhibits prostaglandin F2: in the term decidua, but SP-A levels drop in the amnionic luid at term (Chaiworapongsa, 2008). In addition to SP-A, the fetal lung makes the uterotonic agent platelet-activating factor (Frenkel, 1996; T oyoshima, 1995). This factor and SP-A playia role in fetal-maternal signaling for parturition (Gao, 2015).

1	Toward the end of pregnancy, fetal membranes undergo physiological aging termed cellular senescence (Menon, 2016). In human fetal membranes and animal models, stretch and oxidative stress induce senescent fetal membrane to manifest a form of sterile inflammation termed senescent-associated secretory Physiology of Labor 41s1 phenotype (SASP). This in turn propagates inlammatory signals that further weaken the fetal membrane and activate signals in the decidua and myometrium to initiate parturition. hus, as the functional necessity of fetal membranes declines at term, they are able to promote signals that contribute to parturition initiation.

1	Some evidence shows that pregnancies with markedly diminished estrogen production may be associated with prolonged gestation. These "natural experiments" include women with inherited placental sulfatase deiciency and fetal anencephaly with adrenal hypoplasia. The broad range of gestational length seen with these disorders, however, calls into question the exact role of estrogen in human parturition initiation. Other fetal abnormalities that prevent or severely reduce the entry of fetal urine or lung secretions into amnionic fluid do not prolong human pregnancy. Examples are renal agenesis and pulmonary hypoplasia, respectively. Thus, a fetal signal through the paracrine arm of the fetal-maternal communication system does not appear to be mandated for parturition initiation.

1	Some brain anomalies of the fetal calf, fetal lamb, and sometimes the human fetus delay the normal timing of parturition. More than a century ago, Rea (1898) observed an association between fetal anencephaly and prolonged human gestation. Malpas (1933) extended these observations and described a pregnancy with an anencephalic fetus that was prolonged to 374 days-53 weeks. He concluded that the association between anencephaly and prolonged gestation was attributable to anomalous fetal brain-pituitary-adrenal function. Indeed, the adrenal glands of the anencephalic fetus are very small and, at term, may be only 5 to 10 percent as large as those of a normal fetus. This is caused by developmental failure of the fetal zone that normally accounts for most of fetal adrenal mass and production of Cwsteroid hormones (Chap. 5, p. 104). Such pregnancies are associated with delayed labor and suggest that the fetal adrenal glands are important for the timely onset of parturition. PHASE 3: LABOR

1	This phase is synonymous with active labor, which is customarily divided into three stages. These compose the commonly used labor graph shown in Figure 21-4. he irst stage begins when spaced uterine contractions of suicient frequency, intensity, and duration are attained to bring about cervical thinning, termed facement. Several uterotonins may be important to the success of this stage of active labor (see Fig. 21-5). These have been shown to stimulate smooth muscle contraction through G-protein coupling. This labor stage ends when the cervix is fully dilated-about 10 cm-to allow passage of the term-sized fetus. The irst stage of labor, therefore, is the stage of cervical eacement and dilation. The second stage begins when cervical dilation is complete and ends with delivery. Thus, the second stage of labor is the stage of etal expulsion. Last, the third stage begins immediately after delivery of the fetus and ends with the delivery of the placenta. Thus, the third stage of labor is

1	stage of labor is the stage of etal expulsion. Last, the third stage begins immediately after delivery of the fetus and ends with the delivery of the placenta. Thus, the third stage of labor is the stage of placental separation and expulsion.

1	Body Anat.eI.O. Hist.eI.O. Active segmentI II E.O. � Pathological retraction ring (Sandi) Obliterated 1.0. Active segmentIII Passive segment PREGNANT UTERUS AT TERM UTERUS IN LABOR NORMAL EARLY FIRST STAGE UTERUS IN LABOR NORMAL SECOND STAGE UTERUS IN LABOR ABNORMAL SECOND STAGE -DYSTOCIA FIGURE 21-11 Sequence ofdevelopment ofthe segments and rings in the uterus at term and in labor. Note comparison between the uterus of a nonpregnant woman, the uterus at term, and the uterus during labor. The passive lower uterine segment is derived from the isthmus, and the physiological retraction ring develops at the junction ofthe upper and lower uterine segments. The pathological retraction ring develops from the physiological ring. Anat. 1.0. = anatomical internal os; E.O. = external os; Hist. 1.0. = histological internal os; Ph.R.R. physiological retraction ring. • First Stage: Clinical Onset of Labor

1	Ph.R.R. physiological retraction ring. • First Stage: Clinical Onset of Labor In some women, forceful uterine contractions that efect delivery begin suddenly. In others, labor initiation is heralded by spontaneous release of a small amount of blood-tinged mucus from the vagina. his extrusion of the mucus plug that had previously illed the cervical canal during pregnancy is referred to as "show" or "bloody show." Its passage indicates that labor is already in progress or likely will ensue in hours to days. Unique among physiological muscular contractions, those of uterine smooth muscle during labor are painful. Several possible causes have been suggested: (1) hypoxia of the contracted myometrium-such as that with angina pectoris; (2) compression of nerve ganglia in the cervix and lower uterus by contracted interlocking muscle bundles; (3) cervical stretching during dilation; and (4) stretching ofthe peritoneum overlying the fundus.

1	Of these, compression of nerve ganglia in the cervix and lower uterine segment by the contracting myometrium is an especially attractive hypothesis. Paracervical infiltration with local anesthetic usually produces appreciable pain relief with contractions (Chap. 25, p. 490). Uterine contractions are involuntary and, for the most part, independent of extrauterine control. Neural blockade from epidural analgesia does not diminish their frequency or intensity. In other examples, myometrial contractions in paraplegic women and in women after bilateral lumbar sympathectomy are normal but painless. Mechanical stretching of the cervix enhances uterine activity in several species, including humans. his phenomenon is the Ferguson relex (Ferguson, 1941). Its exact mechanism is unclear, and release of oxytocin has been suggested but not proven. Manipulation of the cervix and "stripping" the fetal membranes is associated with a rise in blood levels of prostaglandin F2. metabolites.

1	The interval between contractions narrows gradually from approximately 10 minutes at the onset of irst-stage labor to as little as 1 minute or less in the second stage. Periods ofrelaxation between contractions, however, are essential for fetal welfare. Unremitting contractions compromise uteroplacental blood flow suiciently to cause fetal hypoxemia. In active-phase labor, the duration ofeach contraction ranges from 30 to 90 seconds and averages 1 minute. Contraction intensity varies appreciably during normal labor. Speciically, amnionic fluid pressures generated by contractions during spontaneous labor average 40 mm Hg, but vary from 20 to 60 mm Hg (Chap. 24, p. 479).

1	Distinct Lower and Upper Uterine Segments. During active labor, the anatomical uterine divisions that were initiated in phase 2 ofparturition become increasingly evident (Figs. 21-11 and 21-12). By abdominal palpation, even before membrane rupture, the two segments can sometimes be diferentiated. he upper segment is firm during contractions, whereas the lower segment is softer, distended, and more passive. This mechanism is imperative because if the entire myometrium, including the lower uterine segment and cervix, were to contract simultaneously and with equal intensity, the net expulsive force would markedly decline. hus, the upper segment contracts, retracts, and expels the fetus. In response to these contractions, the segment -Passive segment--0evel of internal cervical os Cervix Level of external cervical os Vagina

1	FIGURE 21-12 The uterus at the time ofvaginal delivery. The active upper segment retracts around the presenting part as the fetus descends through the birth canal. In the passive lower segment, there is considerably less myometrial tone. form a greatly expanded, thinned-out tube through which the fetus can pass. he myometrium of the upper segment does not relax to its original length after contractions. Instead, it becomes relatively fixed at a shorter length. The upper active uterine segment con tracts down on its diminishing contents, but myometrial ten sion remains constant. he net efect is to take up slack, thus maintaining the advantage gained in expulsion of the fetus.

1	Concurrently, the uterine musculature is kept in irm contact with the uterine contents. As the consequence of retraction, left of. hus, the upper part of the uterine cavity becomes slightly smaller with each successive contraction. Because of the successive shortening of the muscular fibers, the upper active second-stage labor (see Fig. 21-11). This process continues and immediately after delivery. Clinically, it is important to understand that the phenomenon of upper segment retraction is contingent on a decrease in the volume of its contents. For this to happen, particularly early in labor when the entire uterus is virtually a closed sac with only minimal cervical dilation, the musculature of the lower segment must stretch. This permits a greater portion of the uterine contents to occupy the lower segment. The upper segment retracts only to the extent that the lower segment distends and the cervix dilates.

1	Relaxation of the lower uterine segment mirrors the same gradual progression of retraction. Recall that after each contraction of the upper segment, the muscles do not return to their previous length, but tension remains essentially the same. By comparison, in the lower segment, successive lengthening of the fibers with labor is accompanied by thinning, normally to only a few millimeters in the thinnest part. s a result of the lower segment thinning and concomitant upper segment thickening, a boundary between the two is marked by a ridge on the inner uterine surface-the physiological retraction ring. When the thinning of the lower uterine segment is extreme, as in obstructed labor, the ring is prominent and forms a pathological retraction ring. This abnormal condition is also known as the Bandl ring, which is discussed further in Chapter 23 (p. 455).

1	Changes in Uterine Shape. Each contraction gradually elongates the ovoid uterine shape and thereby narrows the horizontal diameter. This change in shape has important efects on the labor process. First, there is greater etal xis pressure, that is, the smaller horizontal diameter serves to straighten the fetal vertebral column. This presses the upper pole of the fetus firmly against the fundus, whereas the lower pole is thrust farther downward. The lengthening of the ovoid shape has been estimated at 5 to 10 cm. Second, with lengthening of the uterus, the longitudinal muscle ibers are drawn taut. As a result, the lower segment and cervix are the only parts of the uterus that are flexible, and these are pulled upward and around the lower pole of the fetus. After the cervix is dilated fully, the most important force in fetal expulsion is produced by maternal intraabdominal pressure. Physiology of Labor 413

1	After the cervix is dilated fully, the most important force in fetal expulsion is produced by maternal intraabdominal pressure. Physiology of Labor 413 Contraction of the abdominal muscles simultaneously with forced respiratory eforts with the glottis closed is referred to as pushing. The force is similar to that with defecation, but the intensity usually is much greater. The importance of intraabdominal pressure is shown by the prolonged descent during labor in paraplegic women and in those with a dense epidural block. And, although increased intraabdominal pressure is necessary to complete second-stage labor, pushing accomplishes little in the first stage. It exhausts the mother, and its associated elevated intrauterine pressures may be harmful to the fetus.

1	s the result of contraction forces, two fundamental changesefacement and dilation-occur in the ripened cervix. For an average-sized fetal head to pass through the cervix, its canal must dilate to a diameter of approximately 10 cm. At this time, the cervix is said to be completely or ully dilated. Although there may be no fetal descent during cervical efacement, most commonly the presenting fetal part descends somewhat as the cervix dilates. Cervical facement is "obliteration" or "taking up" of the cervix. It is manifest clinically by shortening of the cervical canal from a length of approximately 3 cm to a mere circular oriice with almost paper-thin edges. The muscular fibers at the level of the internal cervical os are pulled upward, or "taken up," into the lower uterine segment. The condition of the external os remains temporarily unchanged (Fig. 2i1-13). FIGURE 21-13 Schematic showing effacement and dilation.

1	FIGURE 21-13 Schematic showing effacement and dilation. A. Before labor, the primigravid cervix is long and undilated in contrast to that of the mUltipara, which has dilation of the internal and external os. B. As efacement begins, the multiparous cervix shows dilation and funneling of the internal os. This is less apparent in the primigravid cervix. C. As complete efacement is achieved in the primigravid cervix, dilation is minimal. The reverse is true in the multipara. Efacement may be compared to a funneling process in which the whole length of a narrow cylinder is converted into a very obtuse, flaring funnel with a small distal circular opening. Because of growing myometrial activity during uterine preparedness for labor, appreciable efacement of a softened cervix sometimes is accomplished before active labor begins. Efacement causes expulsion of the mucous plug as the cervical canal is shortened.

1	Because the lower segment and cervix have less resistance during a contraction, a centrifugal pull is exerted on the cervix and creates cervical dilation (Fig. 21-14). As uterine contractions cause pressure on the membranes, the hydrostatic action of the amnionic sac in turn dilates the cervical canal like a wedge. The process of cervical efacement and dilation causes formation of the orebag of amnionic luid. This is the leading portion of fluid and amnionic sac located in front of the presenting part. In the absence of intact membranes, the pressure of the presenting fetal part against the cervix and lower uterine segment is similarly efective. Early rupture of the membranes does not retard cervical dilation so long as the presenting fetal part is positioned to exert pressure against the cervix and lower segment.

1	Referring back to Figure 21-4, recall that cervical dilation is divided into latent and active phases. The active phase is subdivided further into the acceleration phase, the phase of maximum slope, and the deceleration phase (Friedman, 1978). The duration of the latent phase is more variable and sensitive to extraneous factors. For example, sedation may prolong the latent phase, and myometrial stimulation shortens it. The latent phase duration has little bearing on the subsequent course of labor, whereas the characteristics of the accelerated phase are usually predictive of labor outcome. The first stage ends when cervical dilation is complete. • Second Stage: Fetal Descent

1	• Second Stage: Fetal Descent In many nulliparas, engagement of the head is accomplished before labor begins. hat said, the head may not descend further until late in labor. In the descent pattern of normal labor, a typical hyperbolic curve is formed when the station of the fetal head is plotted as a function of labor duration. Station describes descent of the fetal biparietal diameter in relation to a line drawn between maternal ischial spines (Chap. 22, p. 436). Active descent usually takes place after dilation has progressed for some time (Fig. 21-15). During second-stage labor, the speed of descent is maximal and is maintained until the presenting part reaches the perineal floor (Friedman, 1978). In nulliparas, the presenting part typically descends slowly and steadily. In multiparas, however, particularly those of high parity, descent may be rapid.

1	he birth canal is supported and functionally closed by the pelvic loor (Chap. 2, p. 21). The most important component of the loor is the levator ani muscle and the ibromuscular connective tissue that covers its upper and lower surfaces. he biomechanical properties of these structures and of the vaginal wall A B c Internal cervical as External cervical os \ Internal cervical os cervical os cervical os External cervical os FIGURE 21-14 Hydrostatic action of membranes in effecting cervical effacement and dilation, With labor progression, note the changing relations of the internal and external os in (A), (B), and(C), Although not shown in this diagram, with membrane rupture, the presenting part, applied to the cervix and the forming lower uterine segment, acts Similarly,

1	Physiology of Labor 415 its diminishing contents. Normally, by the time the newborn is completely deliv ered, the uterine cavity is nearly obliter ated. he organ consists of an almost solid mass of muscle, several centimeters thick, above the thinner lower segment. he uterine fundus now lies just below the level of the umbilicus .

1	in the area of the placental implantation 5.�2 c0 site (Fig. 21-16). For the placenta to accommodate itself to this reduced area, it thickens, but because of limited placen tal elasticity, it is forced to buckle. The resulting tension pulls the weakest layer decidua spongiosa-from that site. Thus, FIGURE 21-15 Labor course divided on the basis of expected evolution of the dilatation placental separation follows the disproand descent curves into three functional divisions. The preparatory division includes the portion created between the relatively latent and acceleration phases. The dilatational division is the phase of maximum slope of unchanged placental size and the reduced dilatation. The pelvic division encompasses both the deceleration phase and the second implantation site size. stage, which is concurrent with the phase of maximum slope of fetal descent. (Redrawm

1	stage, which is concurrent with the phase of maximum slope of fetal descent. (Redrawm Cleavage of the placenta is aided from Friedman EA: Labor: Clinical Evaluation and Management, 2nd ed. New York, greatly by the loose structure of the Appleton-Century-Crofts, 1978.) spongy decidua. As detachment proceeds, a hematoma forms between the separating change markedly during parturition. These result from altered placenta and the adjacent decidua, which remains attached to extracellular matrix structure or composition (Alperin, 2015; the myometrium. The hematoma is usually the result rather Rahn, 2008; Lowder, 2007). The levator ani muscle closes the lower end of the pelvic cavity as a diaphragm. Thereby, a concave upper and a convex lower surface are presented. The posterior and lateral portions of the pelvic Boor, which are not spanned by the levator ani muscle, are occupied bilaterally by the piriformis and coccygeus muscles.

1	The levator ani muscle varies in thickness from 3 to 5 mm, although its margins encircling the rectum and vagina are somewhat thicker. During pregnancy, the levator ani usually undergoes hypertrophy, forming a thick band that extends backward from the pubis and encircles the vagina about 2 cm above the plane of the hymen. On contraction, the levator ani draws both the rectum and the vagina forward and upward in the direction of the symphysis pubis and thereby acts to close the vagina.

1	In the first stage of labor, the membranes, when intact, and the fetal presenting part serve to dilate the upper vagina. he most marked change consists of stretching levator ani muscle fibers. This is accompanied by thinning of the central portion of the perineum, which becomes transformed from a wedgeshaped, 5-cm-thick tissue mass to a thin, almost transparent membranous structure less than 1 cm thick. hen the perineum is distended maximally, the anus becomes markedly dilated and presents an opening that varies from 2 to 3 cm in diameter and through which the anterior wall of the rectum bulges . • Third Stage: Delivery of Placenta and Membranes This stage begins immediately after fetal delivery and involves separation and expulsion of the placenta and membranes. As FIGURE 21-16 Diminution in size of the placental site after birth of the newborn. A. Spatial relations before birth. B. Placental spatial relations after birth.

1	FIGURE 21-16 Diminution in size of the placental site after birth of the newborn. A. Spatial relations before birth. B. Placental spatial relations after birth. the neonate is born, the uterus spontaneously contracts around than the cause of the separation, because in some cases bleeding is negligible. FIGURE 21-17 Postpartum, membranes are thrown up into folds as the uterine cavity decreases in size. (Used with permission from Dr. Kelley S. Carrick.) The great decline in uterine cavity surface area simultaneously throws the fetal membranes-the amniochorion and the parietal decidua-into innumerable folds (Fig. 21-17). Membranes usually remain in situ until placental separation is nearly completed. These are then peeled of the uterine wall, partly by further contraction of the myometrium and partly by traction that is exerted by the separated placenta as it descends during expulsion.

1	After the placenta has detached, it can be expelled by increased abdominal pressure. Completion of the third stage is also accomplished by alternately compressing and elevating the fundus, while exerting minimal traction on the umbilical cord. The retroplacental hematoma either follows the placenta or is found within the inverted sac formed by the membranes. In this process, known as the Schultze mechanism of placental expulsion, blood from the placental site pours into the membrane sac and does not escape externally until after extrusion of the placenta. In the other form of placental extrusion, known as the Duncan mechanism, the placenta separates irst at the periphery and blood collects between the membranes and the uterine wall and escapes from the vagina. In this circumstance, the placenta descends sideways, and its maternal surface appears irst.

1	Late in pregnancy, during phase 2 of parturition, the number of myometrial oxytocin receptors grows appreciably (Fuchs, 1982; Kimura, 1996). his increase coincides with a greater uterine contractile responsiveness to oxytocin. Prolonged gestation is associated with a delay in the rise of these receptor levels (Fuchs, 1984). Oxytocin-literally, quick birt-was the irst uterotonin to be implicated in parturition initiation. his nanopeptide is synthesized in the magnocellular neurons of the supraoptic and paraventricular neurons. The prohormone is transported with its carrier protein, neurophysin, along the axons to the neural lobe of the posterior pituitary gland in membrane-bound vesicles for storage and later release. The prohormone is converted enzymatically to oxytocin during transport (Gainer, 1988; Leake, 1990).

1	In addition to its efectiveness in pharmacologically inducing labor at term, oxytocin is a potent uterotonin and occurs naturally in humans. Subsequent observations provide additional support for this theory: (1) the number of oxytocin receptors strikingly rises in myometrial and decidual tissues near the end of gestation; (2) oxytocin acts on decidual tissue to promote prostaglandin release; and (3) oxytocin is synthesized directly in decidual and extraembryonic fetal tissues and in the placenta (Chibbar, 1993; Zingg, 1995).

1	Although little evidence suggests a role for oxytocin in phase 2 of parturition, abundant data support its important role during second-stage labor and in the puerperium-phase 4 of parturition. Speciically, maternal serum oxytocin levels are elevated: (1) during second-stage labor, which is the end of phase 3 of parturition; (2) in the early puerperium; and (3) during breastfeeding (Nissen, 1995). Immediately after delivery of the fetus, placenta, and membranes, which completes parturition phase 3, irm and persistent uterine contractions are essential to prevent postpartum hemorrhage. Oxytocin likely causes persistent contractions.

1	Although their role in phase 2 of parturition in uncomplicated pregnancies is less well defined, a critical role for prostaglandins in phase 3 of parturition is clear (MacDonald, 1993). First, levels of prostaglandins-or their metabolites-in amnionic luid, maternal plasma, and maternal urine are increased during labor. Second, receptors for PGE2 and PGF2x are expressed in the uterus and cervix. Thus, if these tissues are exposed to prostaglandins, they will respond. Third, treatment of pregnant women with prostaglandins, by any of several administration routes, causes abortion or labor at all gestational ages. Moreover, administration of prostaglandin H synthase type 2 (PGHS-2) inhibitors to pregnant women will delay spontaneous labor onset and sometimes arrest preterm labor (Loudon, 2003). Last, prostaglandin treatment of myometrial tissue in vitro sometimes causes contraction, dependent on the prostanoid tested and the physiological status of the tissue treated.

1	During labor, prostaglandin production within the myometrium and decidua is an eicient mechanism of activating contractions. For example, prostaglandin synthesis is high and unchanging in the decidua during phase 2 and 3 of parturition. Moreover, the receptor level for PGF2x is augmented in the decidua at term, and this increase most likely is the regulatory step in prostaglandin action in the uterus. he fetal membranes and placenta also produce prostaglandins. Primarily PGE2, but also PGF2x, are detected in amnionic fluid at all gestational ages. As the fetus grows, prostaglandin levels in the amnionic fluid rise gradually. heir greatest

1	FIGURE 21-18 Sagittal view of the exposed forebag and attached decidual fragments after cervical dilation during labor. (Redrawn from MacDonald PC, Casey ML: Preterm birth. Sci Am 3:42, 1996.) elevation in concentration within amnionic luid, however, is demonstrable after labor begins. hese higher levels likely result as the cervix dilates and exposes decidual tissue (Fig. 21-18). hese higher levels in the forebag, compared with those in the upper compartment, are believed to follow an inflammatory response that signals the events leading to active labor. Together, the rise in cytokine and prostaglandin concentrations further degrade the extracellular matrix, thus weakening fetal membranes.

1	The endothelins are a family of 21-amino-acid pep tides that powerfully induce myometrial contraction (Word, 1990). The endothelin A receptor is preferentially expressed in smooth muscle, and when activated, it efects a rise in intracellular calcium. Endothelin-1 is produced in myometrium of term gestations and is able to induce synthesis of other contractile mediators such as prostaglandins and inflammatory mediators (Momohara, 2004; Sutclife, 2009). The requirement of endothelin-1 in normal parturition physiology remains to be established. Two G-protein-linked angiotensin II receptors are expressed in the uterus-AT1 and AT2. In nonpregnant women, the AT2 receptor predominates, but the ATI receptor is preferentially expressed in gravidas (Cox, 1993). Angiotensin II binding to the plasma-membrane receptor evokes contraction. During pregnancy, the vascular smooth muscle that expresses the AT2 receptor is refractory to the pressor efects of infused angiotensin II (Chap. 4, p. 63).

1	Physiology of Labor 41 7 PHASE 4: THE PUERPERIUM Immediately and for about an hour after delivery, the myometrium remains persistently contracted. This directly compresses large uterine vessels and allows thrombosis of their lumens to prevent hemorrhage. his is typically augmented by endogenous and pharmacological uterotonic agents (Chap. 27, p. 526). Uterine involution and cervical repair are prompt remodeling processes that restore these organs to the nonpregnant state. hese protect the reproductive tract from invasion by commensal microorganisms and restore endometrial responsiveness to normal hormonal cyclicity. During the early puerperium, lactogenesis and milk let-down begin in mammary glands (Chap. 36, p. 656). Reinstitution of ovulation signals preparation for the next pregnancy. Ovulation generally occurs within 4 to 6 weeks after birth. However, it is dependent on the duration of breastfeeding and lactationinduced, prolactin-mediated anovulation and amenorrhea.

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1	Smith GC, Wu X, Nathanielsz PW: Efects of gestational age and labor on expression of prostanoid receptor genes in baboon uterus. Bioi Reprod 64(4): 1131,o2001 Smith R: Parturition. N Engl J Med 356(3):271, 2007 Snegovskikh V, Bhandari V, Wright JR, et al: Surfactant protein-A (SP A) selectively inhibits prostaglandin F2alpha (PGF2alpha) production in term decidua: implications for the onset of labor. J Clin Endocrinol Metab 96(4):E624,o2011 Soh YM, Tiwari A, Mahendroo M, et al: Relaxin regulates hyaluronan synthesis and aquaporins in the cervix of late pregnant mice. Endocrinology 153(12):6054,o2012 Sparey C, Robson SC, Bailey J, et al: The diferential expression of myometrial connexin-43, cyclooxygenase-1 and -2, and Gs alpha proteins in the upper and lower segments of the human uterus during pregnancy and labor. J Clin Endocrinol Metab 84(5): 1705, 1999

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1	Telfer ]F, Itoh H, Thomson AJ, et al: Activity and expression of soluble and particulate guanylate cyclases in myometrium from nonpregnant and pregnant women: down-regulation of soluble guanylate cyclase at term. J Clin Endocrinol Metab 86(12):5934, 2001 Timmons Be, Mahendroo M: Processes regulating cervical ripening difer from cervical dilation and postpartum repair: insights from gene expression studies. Reprod Sci 14(8 Suppl):53, 2007 Toyoshima K, Narahara H, Furukawa M, et al: Platelet-activating factor. Role in fetal lung development and relationship to normal and premature labor. Clin Perinatolo22(2):263, 1995 ink JY, Qin S, Brock CO, et al: A new paradigm for the role of smooth muscle cells in the human cervix. Am J Obstet Gynecol 215(4):478.el, 2016 Wadhwa PD, Porto M, Garite T], et al: Maternal corticotropin-releasing hormone levels in the early third trimester predict length of gestation in human pregnancy. Am] Obstet GynecoIo179(4):1079, 1998

1	Wakle-Prabagaran M, Lorca A, vla X, et al: BKCa channel regulates calcium oscillations induced by alpha-2-macroglobulin in human myometrial smooth muscle cells. Proc Nat! Acad Sci USA 113(16):E2335, 2016 Wang H, Parry S, Macones G, et al: A functional SNP in the promoter of the SERPINH 1 gene increases risk of preterm premature rupture of membranes in African Americans. Proc Nat! Acad Sci USoA 103(36):1o3463,o2006 Warren ]E, Silver M, Dalton ], et al: Collagen 1Alphal and transforming growth factor-beta polymorphisms in women with cervical insuiciency. Obstet Gynecol 110(3):619,o2007 Wathes DC, Borwick Se, Timmons PM, er al: Oxyrocin receptor expression in human term and preterm gestational tissues prior to and following the onset of labour. ] EndocrinoIo161(l):143, 1999

1	Wathes DC, Borwick Se, Timmons PM, er al: Oxyrocin receptor expression in human term and preterm gestational tissues prior to and following the onset of labour. ] EndocrinoIo161(l):143, 1999 Westergren-horsson G, Norman M, Bjornsson 5, et al: Diferential expressions of mRNA for proteoglycans, collagens and transforming growth factor-beta in the human cervix during pregnancy and involution. Biochim Biophys Acta 1406(2):203, 1998 Williams KC, Renthal NE, Condon JC, et al: MicroNA-200a serves a key role in the decline of progesterone receptor function leading to term and preterm labor. Proc Natl Acad Sci USA 109(19):7529, 2012a Williams KC, Renthal NE, Gerard D, et al: he microRNA (miR)-199a/214 cluster mediates opposing efects of progesterone and estrogen on uterine contractility during pregnancy and labor. Mol Endocrinolo26(11):1857, 2012b

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1	Word A, Li XH, Hnat M, et al: Dynamics of cervical remodeling during pregnancy and parturition: mechanisms and current concepts. Semin Reprod Med 25(1):69, 2007 Word A, Stull JT, Casey ML, et al: Contractile elements and myosin light chain phosphorylation in myometrial tissue from nonpregnant and pregnant women. J Clin Invest 92(1):29, 1993 Ying L, Becard M, Lyell D, et al: The transient receptor potential vanilloid 4 channel modulates uterine tone during pregnancy. Sci Transl Med 7(319):319ra204,o2015 Yoshida K, Jiang H, Kim M, et al: Quantitative evaluation of collagen crosslinks and corresponding tensile mechanical properties in mouse cervical tissue during normal pregnancy. PLoS One 9(11):el12391, 2014 You X, Gao L, Liu J, et al: CRH activation of diferent signaling pathways results in diferential calcium signaling in human pregnant myometrium before and during labor. J Clin Endocrinol Metab 97(10):E1851, 20o12

1	Young RC, Goloman G: Mechanotransduction in rat myometrium: coordination of contractions of electrically and chemically isolated tissues. Reprod Sci 18(1):64,2011 Zhang Y, Akins tvlL, Murari K, et al: A compact fiber-optic SHG scanning endomicroscope and its application to visualize cervical remodeling during pregnancy. Proc Nat! Acad Sci USA 109(32):12878, 2012 Ziecik AJ, Derecka-Reszka K, Rzucidlo SJ: Extragonadal gonadotropin receptors, their distribution and function. J Physiol Pharmacol 43(4 Suppl 1):33, 1992 Zingg HH, Rozen F, Chu K, et al: Oxytocin and oxytocin receptor gene expression in the uterus. Recent Prog Horm Res 50:255, 1995 MECHANISMS OF LABOR .e.........e..e.........e... 421 FETAL POSITION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423 OCCIPUT ANTERIOR PRESENTATION ..e.......e..... 427 NORMAL LABOR CHARACTERISTICS . . . . . . . . . . . . . . . 431 ......................... 432 SECOND STAGE OF LABOR ..e...e.......e.....e..e..e. 434

1	NORMAL LABOR CHARACTERISTICS . . . . . . . . . . . . . . . 431 ......................... 432 SECOND STAGE OF LABOR ..e...e.......e.....e..e..e. 434 MANAGEMENT OF NORMAL LABOR ...... ........ 434 MANAGEMENT OF FIRST-STAGE LABOR ...e.....e...e. 436 MANAGEMENT OF SECOND-STAGE LABOR ... ..e..e. 438 LABOR MANAGEMENT PROTOCOLS . . . . . .. . . . . . . . . 438 It olows that some process of adaptation or accommodation of suitable portions or the head to the various pelvic planes is necessary to insure the completion of childbirth. his is brought about by certain movement of the presenting part, which belong to what is termed the mechanism of labour. -J. Whitridge Williams (1903) Labor is the process that leads to childbirth. It begins with the onset of regular uterine contractions and ends with delivery of the newborn and expulsion of the placenta. Pregnancy and birth are physiological processes, and thus, labor and delivery should be considered normal for most women.

1	Many adaptive changes are required for pregnancy and for labor and delivery. According to Nygaard (2015), vaginal delivery is a traumatic event. To assess this in part, Staer-] ensen and colleagues (2015) obtained transperineal sonographic measurements of the pelvic loor muscles at 21 weeks' and 37 weeks' gestation, and again at 6 weeks, 6 months, and 12 months postpartum. In 300 nulliparas, they measured bladder neck mobility and the area within the urogenital hiatus during Valsalva. This hiatus is the U-shaped opening in the pelvic loor muscles through which the urethra, vagina, and rectum pass (Chap. 2, p. 19). In this study, the levator hiatus area was signiicantly larger at 37 weeks' gestation and at 6 weeks postpartum compared with earlier pregnancy. hen, by 6 months postpartum, the hiatus had improved and narrowed to return to an area comparable to that at 21 weeks' gestation. However, no further improvement was noted by 12 months postpartum. Of note, hiatal area enlargement

1	hiatus had improved and narrowed to return to an area comparable to that at 21 weeks' gestation. However, no further improvement was noted by 12 months postpartum. Of note, hiatal area enlargement was only seen in those who delivered vaginally.

1	These indings demonstrate antepartum changes in pelvic loor structure that may relect adaptations needed to permit vaginal delivery (Nygaard, 2015). Additional pelvic loor changes are discussed in Chapter 4 (p. 52), and the contributions of pregnancy and delivery to later pelvic organ prolapse and incontinence are described in Chapter 30 (p. 568). At the onset of labor, the position of the fetus with respect to the birth canal is critical to the route of delivery and thus should be determined in early labor. Important relationships include fetal lie, presentation, attitude, and position. TABLE 22-1 . Fetal Presentation in 68,097 Singleton Pregnancies at Parkland Hospital Cephalic 96.8 Breech 2.7 1s:36 Transverse lie 0.3 1 :335 Compound 0.1 1:1000 Face 0.05 1 :2000 Brow 0.01 1 :10,000

1	Cephalic 96.8 Breech 2.7 1s:36 Transverse lie 0.3 1 :335 Compound 0.1 1:1000 Face 0.05 1 :2000 Brow 0.01 1 :10,000 Fetal lie describes the relationship of the fetal long axis to that of the mother. In more than 99 percent of labors at term, the fetal lie is longitudinal. A transverse lie is less frequent, and predisposing factors include multiparity, placenta previa, hydramnios, and uterine anomalies (Chap. 23, p. 452). Occasionally, the fetal and maternal axes may cross at a 45-degree angle, forming an oblique lie. his is unstable and becomes longitudinal or transverse during labor.

1	The presenting part is the portion of the fetal body that is either foremost within the birth canal or in closest proximity to it. It typically can be felt through the cervix during vaginal examination. Accordingly, in longitudinal lies, the presenting part is either the fetal head or the breech, creating cephalic and breech presentations, respectively. When the fetus lies with the long axis transversely, the shoulder is the presenting part. Table 22-1 describes the incidences of these various presentations.

1	These presentations are classified according to the relationship between the head and body of the fetus (Fig. 22-1). Ordinarily, the head is lexed sharply so that the chin contacts the thorx. The occipital fontanel is the presenting part, and this presentation is referred to as a vertex or occiput presentation. Much less often, the fetal neck may be sharply extended so that the occiput and back come into contact, and the face is foremost in the birth canal-oce presentation. The fetal head may assume a position between these extremes. When the neck is only partly lexed, the anterior (large) fontanel may present-sincput presentation. When the neck is only partially extended, the brow may emerge-brow presentation. These latter two are usually transient. As labor progresses, sinciput and brow presentations almost always convert into vertex or face presentations by neck lexion or extension, respectively. Failure to do so can lead to dystocia, discussed in Chapter 23 (p. 452).

1	FIGURE 22-1 Longitudinal lie. Cephalic presentation. Differences in attitude of the fetal body in (A) vertex, (B) sinciput, (C) brow, and (0) face presentations. Note changes in fetal attitude in relation to fetal vertex as the fetal head becomes less flexed.

1	The term fetus usually presents with the vertex, most logically because the uterus is piriform or pear shaped. Although the fetal head at term is slightly larger than the breech, the entire podalic pole of the fetus-that is, the breech and extremities-is bulkier and more mobile than the cephalic pole. The cephalic pole is composed of the fetal head only. Until approximately 32 weeks, the amnionic cavity is large compared with the fetal mass, and the fetus is not crowded by the uterine walls. Subsequently, however, the ratio of amnionic luid volume declines relative to the growing fetal mass. As a result, the uterine walls are apposed more closely to the fetal parts. The fetus orients its polarity to make use of the roomier fundus for its bulkier and more mobile podalic pole. The high incidence of breech presentation in hydrocephalic fetuses is in accord with this theory, as the larger fetal cephalic pole requires more room than its podalic pole.

1	The incidence of breech presentation drops with gestational age and approximates 3 percent at term. When the fetus presents breech, the three general configurations arerank, complete, and ootling presentations, described in Chapter 28 (p. 539). Breech presentation may result from circumstances that prevent normal version from taking place. One example is a septum that protrudes into the uterine cavity (Chap. 3, p. 45). Variances of fetal attitude, particularly extension of the vertebral column as seen in frank breeches, also may prevent the fetus from turning. If the placenta is implanted in the lower uterine segment, it may distort normal intrauterine anatomy and result in a breech presentation.

1	In the later months of pregnancy, the fetus assumes a characteristic posture described as attitude or habitus (see Fig. 22-1). As a rule, the fetus forms an ovoid mass that corresponds roughly to the shape of the uterine cavity. The fetus becomes folded upon itself to create a convex back. The head is sharply lexed; the chin is almost in contact with the chest; the thighs are lexed over the abdomen; and the legs are bent at the knees. In all cephalic presentations, the arms usually lie across the thorax or parallel to the sides. The umbilical cord ills the space between the FIGURE 22-2 Longitudinal lie. Vertex presentation. A. Left occiput anterior (LOA). B. Left occiput posterior (LOP). extremities. his characteristic posture results from the mode of fetal growth and its accommodation to the uterine cavity.

1	extremities. his characteristic posture results from the mode of fetal growth and its accommodation to the uterine cavity. Abnormal exceptions to this attitude occur as the fetal head becomes progressively more extended from the vertex to the face presentation. This results in a progressive change in fetal attitude from a convex (flexed) to a concave (extended) contour of the vertebral column. Position refers to the relationship of an arbitrarily chosen portion of the fetal presenting part to the right or left side of the birth canal. Accordingly, with each presentation, there may be two positions-right or left. he fetal occiput, chin (mentum), and sacrum are the determining points in vertex, face, and breech presentations, respectively (hgs. 22 2 to Because the presenting part may be in either the left or right position, there are left and right occipital (LO and RO), left and right mental (LM and RM), and left and right sacral (LS and RS) designations.

1	Further, the relationship of a given portion of the presenting part to the anterior (A), transverse (T), or posterior (P) portion of the maternal pelvis is considered. As shown in Figures 22-2 to 22-6, there are six varieties of each of the three presentations. Thus, in an occiput presentation, the presentation, position, and variety may be abbreviated in clockwise fashion as: Approximately two thirds of all vertex presentations are in the let occiput position, and one third in the right.

1	Approximately two thirds of all vertex presentations are in the let occiput position, and one third in the right. In shoulder presentations, the acromion (scapula) is the portion of the fetus arbitrarily chosen for orientation with the maternal pelvis. One example of the terminology sometimes employed for this purpose is illustrated in Figure 22-7. The acromion or back of the fetus may be directed either posteriorly or anteriorly and superiorly or inferiorly. Because it is impossible to diferentiate exactly the several varieties of shoulder presentation by clinical examination and because such speciic diferentiation serves no practical purpose, it is customary FIGURE 22-3 Longitudinal lie. Vertex presentation. A. Right occiput posterior (RaP). B. Right occiput transverse (Ran.

1	FIGURE 22-3 Longitudinal lie. Vertex presentation. A. Right occiput posterior (RaP). B. Right occiput transverse (Ran. to refer to all transverse lies simply as shoulder presentations. Another term used is transverse lie, with back up or back down, which is clinically important when deciding incision type for cesarean delivery (Chap. 23, p. 453) . Several methods can be used to diagnose fetal presentation and position. Abdominal examination can be conducted systematically employing the four maneuvers described by Leopold in 1894 and shown in Figure 22-8. he mother lies supine and comfortably positioned with her abdomen bared. hese maneuvers may be diicult if not impossible to perform and interpret if the patient is obese, if amnionic luid volume is excessive, or if the placenta is anteriorly implanted.

1	he irst maneuver assesses the uterine fundus. It permits identification of fetal lie and determination of which fetal pole-that is, cephalic or podalic-occupies the fundus. he breech gives the sensation of a large, nodular mass, whereas the head feels hard and round and is more mobile. he second maneuver is accomplished as the palms are placed on either side of the maternal abdomen, and gentle but deep pressure is exerted. On one side, a hard, resistant structure is felt-the back. On the other, numerous small, irregular, mobile parts are felt-the fetal extremities. By noting whether anterior (ROA). orientation can be determined. FIGURE 22-4 Longitudinal lie. Vertex presentation. Right occiput the back is directed anteriorly, transversely, or posteriorly, fetal FIGURE 22-5 Longitudinal lie. Face presentation. Left and right mentum anterior and right mentum posterior positions.

1	FIGURE 22-5 Longitudinal lie. Face presentation. Left and right mentum anterior and right mentum posterior positions. FIGURE 22-6 Longitudinal lie. Breech presentation. Left sacrum FIGURE 22-7 Transverse lie. Right acromiodorsoposterior (RADP). The posterior (LSP). shoulder of the fetus is to the mother's right, and the back is posterior. and colleagues (1993), experienced clinicians have accurately identified fetal malpresentation using Leopold maneuvers with a high sensitivity-88 percent, speciicity-94 percent, positive-predictive value-74 percent, and negative-predictivevalue-97 percent. With experience, it is possible to estimate the size of the fetus with these maneuvers (Field, 1995). However, and especially with an obese woman, estimates by palpation and actual birth weights often correlate poorly (Fox, 2009; Goetzinger, 2014; Noumi, 2005).

1	Before labor, the diagnosis of fetal presentation and position by vaginal examination is often inconclusive because the presenting part must be palpated through a closed cervix and lower uterine segment. With the onset of labor and after cervical dila tion, vertex presentations and their positions are recognized by palpation of the various fetal sutures and fontanels. Face and breech presentations are identiied by palpation of facial features or the fetal sacrum and perineum, respectively. During this vaginal examination, it is advis able to pursue a deinite routine, comprising four movements. First, the examiner inserts two fingers into the vagina and the presenting part is found. Diferentiation of vertex, FIGURE 22-8 Leopold maneuvers (A-D) performed in fetus with a longitudinal lie in the left face, and breech is then accomocciput anterior position (LOA).

1	The third maneuver aids confirmation of fetal presentation. The thumb and ingers of one hand grasp the lower portion of the maternal abdomen just above the symphysis pubis. If the presenting part is not engaged, a movable mass will be felt, usually the head. The diferentiation between head and breech is made as in the first maneuver. The fourth maneuver helps determine the degree of descent. he examiner faces the mother's feet, and the ingertips of both hands are positioned on either side of the presenting part. They exert inward pressure and then slide caudad along the axis of the pelvic inlet. In many instances, when the head has descended into the pelvis, the anterior shoulder or the space created by the neck may be diferentiated readily from the hard head.

1	Abdominal palpation can be performed throughout the latter months of pregnancy and during and between the contractions of labor. At least in the past, according to Lydon-Rochelle plished readily. Second, if the vertex is presenting, the fingers are directed posteriorly and then swept forward over the fetal head toward the maternal symphysis (Fig. 22-9). During this movement, the fingers necessarily cross the sagittal suture, and its linear course is delineated. Next, the positions of the two fontanels, found at either end of the sagittal suture, are ascertained. For this, fingers are passed to the most anterior extension of the sagittal suture, and the fontanel encountered there is examined and identiied. hen, the fingers pass along the suture to the other end of the head until the other fontanel is felt and diferentiated (Fig.

1	10). Last, the station, or extent to which the presenting part has descended into the pelvis, can also be established at this time (p. 436). Using these maneuvers, the various sutures and fontanels are determined (Fig. 29-1, p. 554). identification, especially in obese women or in women with muscular FIGURE 22-9 Locating the sagittal suture by vaginal examination. abdominal walls. Compared with digital examinations, sonography for fetal head position determination during secondstage labor is more accurate (Ramphul, 2014; Wiafe, 2016).

1	abdominal walls. Compared with digital examinations, sonography for fetal head position determination during secondstage labor is more accurate (Ramphul, 2014; Wiafe, 2016). In most cases, the vertex enters the pelvis with the sagittal suture lying in the transverse pelvic diameter. The fetus enters the pelvis in the let occiput transverse (LO) position more commonly than right occiput transverse (RO) position (Caldwell, 1934). In occput anterior positions-LOA or ROA-either the head enters the pelvis with the occiput rotated 45 degrees anteriorly from the transverse position, or this rotation occurs subsequently. The mechanism oflabor in all these presentations is usually similar. The positional changes of the presenting part required to navigate the pelvic canal constitute the mechanisms of labor. The cardinal movements of labor are engagement, descent, lexion, internal rotation, extension, external rotation, and expulsion

1	During labor, these movements not only are sequential but also show great temporal overlap. For example, as part of engagement, there is both flexion and descent of the head. It is impossible for the movements to be completed unless the presenting part descends simultaneously. Concomitantly, uterine contractions efect important modifications in fetal attitude, or habitus, especially ater the head has descended into the pelvis. These changes consist principally of fetal straightening, with loss of dorsal convexity and closer application of the extremities to the body. As a result, the fetal ovoid is transformed into a cylinder, with the smallest possible cross section typically passing through the birth canal. FIGURE 22-10 Differentiating the fontanels by vaginal examination.

1	FIGURE 22-10 Differentiating the fontanels by vaginal examination. he mechanism by which the biparietal diameter-the greatest transverse diameter in an occiput presentation-passes through the pelvic inlet is designated engagement. he fetal head may engage during the last few weeks of pregnancy or not until after labor commencement. In many multiparas and some nulliparas, the fetal head is freely movable above the pelvic inlet at labor onset. In this circumstance, the head is sometimes referred to as "floating." A normal-sized head usually does not engage with its sagittal suture directed anteroposteriorly. Instead, as discussed, the fetal head usually enters the pelvic inlet either transversely or obliquely. Segel and coworkers (2012) analyzed labor in 5341 nulliparous women and found that fetal head engagement before labor onset did not afect vaginal delivery rates in either spontaneous or induced labor.

1	The fetal head tends to accommodate to the transverse axis of the pelvic inlet, whereas the sagittal suture, while remaining parallel to that axis, may not lie exactly midway between the symphysis and the sacral promontory. The sagittal suture frequently is deflected either posteriorly toward the promontory or anteriorly toward the symphysis (Fig. 22-12). Such lateral delection to a more anterior or posterior position in the pelvis is called asynclitism. If the sagittal suture approaches the sacral promontory, more of the anterior parietal bone presents itself to the examining fingers, and the condition is called anterior asynclitism. If, however, the sagittal suture lies close to the symphysis, more of the posterior parietal bone will present, and the condition is called posterior asynclitism. With extreme posterior asynclitism, the posterior ear may be easily palpated. Moderate degrees of asynclitism are the rule in normal labor.

1	Moderate degrees of asynclitism are the rule in normal labor. However, if severe, the condition is a common reason for cepha lopelvic disproportion even with an otherwise normal-sized pelvis. Successive fetal head shiting from posterior to anterior asynclitism aids descent. This movement is the first reqUISIte for birth of the newborn. In nulliparas, engagement may take place before the onset oflabor, and further descent may not follow until the onset of the second stage. In multiparas, descent usually begins with engagement. Descent is brought about by one or more of four forces: (1) pressure of the amnionic fluid, (2) direct pressure of the fundus upon the breech with contractions, (3) bearing-down eforts of maternal abdominal muscles, and (4) extension and straightening of the fetal body. 1.Head floating, before engagement 5.Complete extension 2.Engagement, descent, flexion 6.Restitution (external rotation) 3.Further descent, internal rotation 7.Delivery of anterior shoulder

1	1.Head floating, before engagement 5.Complete extension 2.Engagement, descent, flexion 6.Restitution (external rotation) 3.Further descent, internal rotation 7.Delivery of anterior shoulder FIGURE 22-11 Cardinal movements of labor and delivery from a left occiput anterior position. 4.Complete rotation, beginning extension 8.Delivery of posterior shoulder Anterior asynclitism Normal synclitism Posterior asynclitism '\ '\, ,/�>,I '.-Occipito-Sagittal � frontal plane -_.. Anterior parietal suture Pelvic inlet plane Posterior parietal FIGURE 22-12 Synclitism and asynclitism. As soon as the descending head meets resistance, whether from the cervix, pelvic walls, or pelvic loor, it normally lexes. With this movement, the chin is brought into more intimate contact with the fetal thorax, and the appreciably shorter suboccipitobregmatic diameter is substituted for the longer occipitofrontal diameter (Fig. 22-13).

1	his movement turns the occiput gradually away from the transverse axis. Usually the occiput rotates anteriorly toward the symphysis pubis, but less commonly, it may rotate posteriorly FIGURE 22-13 Lever action produces flexion of the head. Conversion from occipitofrontal (left) to suboccipitobregmatic (right) diameter typically reduces the anteroposterior diameter from nearly 12 to 9.5 cm. toward the hollow of the sacrum (Figs. 22-14 and 22-15). Internal rotation is essential for completion of labor, except when the fetus is unusually small.

1	toward the hollow of the sacrum (Figs. 22-14 and 22-15). Internal rotation is essential for completion of labor, except when the fetus is unusually small. Calkins (1939) studied more than 5000 women in labor to ascertain the time of internal rotation. He concluded that in approximately two thirds, internal rotation is completed by the time the head reaches the pelvic loor; in about another fourth, internal rotation is completed shortly after the head reaches the pelvic loor; and in the remaining 5 percent, rotation does not take place. When the head fails to turn until reaching the pelvic floor, it typically rotates during the next one or two contrac tions in multiparas. In nulliparas, rotation usually occurs dur ing the next three to ive contractions.

1	Ater internal rotation, the sharply flexed head reaches the vulva and undergoes extension. If the sharply flexed head, on reaching the pelvic floor, did not extend but was driven farther downward, it would impinge on the posterior portion of the perineum and would eventually be forced through the perineal tissues. When the head presses on the pelvic floor, however, two forces come into play. he irst force, exerted by the uterus, acts more posteriorly, and the second, supplied by the resistant pelvic floor and the symphysis, acts more anteriorly. he resultant vector is in the direction of the vulvar opening, thereby causing head extension. his brings the base of the occiput into direct contact with the inferior margin of the symphysis pubis (see Fig. 22-14).

1	With progressive distention of the perineum and vaginal opening, an increasingly large portion of the occiput gradually appears. The head is born as the occiput, bregma, forehead, nose, mouth, and inally the chin pass successively over the anterior margin of the perineum. Immediately after its delivery, the head drops downward so that the chin lies over the maternal anus. he delivered head next undergoes restitution (see Fig. 22-11). If the occiput was originally directed toward the left, it rotates FIGURE 22-14 Mechanism of labor for the left occiput transverse position, lateral view. A. Engagement with posterior asynclitism at the pelvic brim. During descent, the sagittal suture is then deflected toward the sacrum. B. This leads to anterior asynclitism. C. Internal rotation and descent. D. Further internal rotation and descent with extension of the neck. FIGURE 22-15 Mechanism of labor for right occiput posterior position showing anterior rotation.

1	FIGURE 22-15 Mechanism of labor for right occiput posterior position showing anterior rotation. toward the left ischial tuberosity. If it was originally directed toward the right, the occiput rotates to the right. Restitution of the head to the oblique position is followed by external rotation completion to again reach a transverse position. This movement corresponds to rotation of the fetal body and serves to bring its bisacromial diameter into relation with the anteroposterior diameter of the pelvic outlet. hus, one shoulder is anterior behind the symphysis and the other is posterior. his movement apparently is brought about by the same pelvic factors that produced internal rotation of the head.

1	lmost immediately after external rotation, the anterior shoulder appears under the symphysis pubis, and the perineum soon becomes distended by the posterior shoulder. After delivery of the shoulders, the rest of the body quickly passes. When the anterior shoulder is tightly wedged beneath the symphysis, then shoulder dystocia is diagnosed, which is described in Chapter 27 (p. 520). In approximately 20 percent of labors, the fetus enters the pelvis in an occiput posterior (OP) position (Caldwell, 1934). The right occiput posterior (RaP) is slightly more common than the left (LOP). It appears likely from radiographic evidence that posterior positions are more often associated with a narrow forepelvis. They also are more commonly seen in association wi th anterior placentation (Gardberg, 1994a).

1	In most occiput posterior presentations, the mechanism of labor is identical to that observed in the transverse and anterior varieties, except that the occiput has to internally rotate to the symphysis pubis through 135 degrees, instead of 90 and 45 degrees, respectively (see Fig. 22-15).

1	Efective contractions, adequate head lexion, and average fetal size together permit most posteriorly positioned occiputs to rotate promptly as soon as they reach the pelvic loor, and labor is not lengthened appreciably. In perhaps 5 to 10 percent of cases, however, rotation may be incomplete or may not take place at all, especially if the fetus is large (Gardberg, 1994b). Poor contractions, faulty head lexion, or epidural analgesia, which diminishes abdominal muscular pushing and relaxes pelvic floor muscles, may predispose to incomplete rotation. If rotation is incomplete, transverse arrest may result. If no rotation toward the symphysis takes place, the occiput may remain in the direct occiput posterior position, a condition known as persistent occiput posterior. Both can lead to dystocia and cesar ean delivery. Techniques to manually rotate from OP to OA positions are illustrated in Chapter 29 (p. 560).

1	In vertex presentations, labor forces alter fetal head shape. In prolonged labors before complete cervical dilation, the portion of the fetal scalp immediately over the cervical os becomes edematous. his swelling is known as the caput succedaneum (rig. 22-16). It usually attains a.thickness of only a few millimeters, but in prolonged labors it may be suiciently extensive FIGURE 22-16 Considerable molding of the head and caput succedaneum formation in a recently delivered newborn. to prevent diferentiation of the various sutures and fontanels. More commonly, the caput is formed when the head is in the lower portion of the birth canal and frequently only after the resistance of a rigid vaginal outlet is encountered. Because it develops over the most dependent area of the head, one may deduce the original fetal head position by noting the location of the caput succedaneum.

1	Molding refers to changes in the bony fetal head shape as a result of external compressive forces (see Fig. 22-16). Possibly related to Braxton Hicks contractions, some molding develops before labor. Most studies indicate that there is seldom overlapping of the parietal bones. A "locking" mechanism at the coronal and lambdoidal sutures actually prevents such overlapping (Carlan, 1991). Molding results in a shortened suboccipitobregmatic diameter and a lengthened mentovertical diameter. These changes are of greatest importance in women with contracted pelves or asynclitic presentations. In these circumstances, the degree to which the head is capable of molding may make the diference between spontaneous vaginal delivery and an operative delivery. Some older literature cited severe head molding as a cause for possible cerebral trauma. Because of the multitude of associated factors, for example, prolonged labor with fetal sepsis and acidosis, it is impossible to link molding to any

1	molding as a cause for possible cerebral trauma. Because of the multitude of associated factors, for example, prolonged labor with fetal sepsis and acidosis, it is impossible to link molding to any alleged fetal or neonatal neurological sequelae. .10st cases of molding resolve within the week following delivery, although persistent cases have been described (Graham, 2006). Diferentiation of molding, caput succedaneum, and cephalohematoma is discussed in Chapter 33 (p. 628).

1	The greatest impediment to understanding normal labor is recognizing its start. he strict deinition oflabor is: uterine contractions that bring about demonstrable facement and dilation of the cervix. This does not easily aid the clinician in determining when labor has actually begun, because this diagnosis is conirmed only retrospectively. Several methods may be used to mark its start. One defines onset as the clock time when painful contractions become regular. Unfortunately, uterine activity that causes discomfort, but that does not represent true labor, may develop FIGURE22-17 Average labor curves for women with singleton term pregnancies presenting in spontaneous labor with vaginal delivery for nulliparas from 1959-1966 compared with those from 2002-2008.

1	FIGURE22-17 Average labor curves for women with singleton term pregnancies presenting in spontaneous labor with vaginal delivery for nulliparas from 1959-1966 compared with those from 2002-2008. (Redrawn from Laughon SK, Branch W, Beaver J, et al: Changes in labor patterns over 50 years. Am J Obstet Gynecol 206:419.e1.9, 2012.) at any time during pregnancy. False labor often stops spontaneously, or it may proceed rapidly into efective contractions.

1	A second method deines the onset of labor as beginning at the time of admission to the labor unit. In the United States, admission for labor is frequently based on the extent of cervical dilation accompanied by painful contractions. If a woman has intact membranes, then a cervical dilation of3 to 4 cm or greater is presumed to be a reasonably reliable threshold for the diagnosis of labor. In this case, labor onset commences with the time of admission. his presumptive method obviates many of the uncertainties in diagnosing labor during earlier stages of cervical dilation. Laughon and associates (2012) compared the duration of spontaneous labor at term in nulliparas delivered in the United States between 1959 and 1966 to that of those delivered from 2002 to 2008. As shown in Fiure 22-17, during those 50 years, the length oflabor increased by approximately 2 hours. • First Stage of Labor

1	For labor, Friedman (1954) described a characteristic sigmoid pattern by graphing cervical dilationagainst time. his graphical approach, which was based on statistical observations, changed labor management. Friedman developed the concept of three functional labor divisions to describe the physiological objectives ofeach division (Fig. 22-18). First, during the preparatoy division, although the cervix dilates little, its connective tissue components change considerably (Chap. 21, p. 413). Sedation and conduction analgesia are capable of arresting this labor division. he dilationaldivision, duringwhich dilation proceeds at its most rapid rate, is unafected by sedation. Last, the pelvic division commences with the deceleration phase of cervical dilation. The classic labor mechanisms that involve the cardinal fetal movements of the cephalic presentation take place principally during this pelvic division. In actual practice, however, the onset of the pelvic division is seldom clearly

1	involve the cardinal fetal movements of the cephalic presentation take place principally during this pelvic division. In actual practice, however, the onset of the pelvic division is seldom clearly identiiable.

1	As shown in Figure 22-18, the pattern of cervical dilation during the preparatory and dilational divisions of normal labor is a sigmoid curve. Two phases of cervical dilation are deined. he latent phase corresponds to the preparatory division, and 0i::'a� ofdilatation and descent curves into: (1) a preparatory division, including latent and acceleration phases; (2) a dilatational division, occupying the phase of maximum slope; and (3) a pelvic division, encompassing both deceleration phase and second stage concurrent with the phase of maximum slope of descent. (Redrawn from Friedman EA: Labor: Clinical Evaluation and Management, 2nd ed. New York, Appleton-Century-Crofts, 1978.) the activephase to the dilational division. Friedman further subdivided the active phase into the acceleration phase, thephase of mximum slope, and the deceleration phase (Fig. 22-19).

1	he onset of latent labor, as deined by Friedman (1972), is the pointatwhich the motherperceives regularcontractions. helatent phase for most women ends once dilation of3 to 5 cm is achieved. his threshold may be clinically useul, for it deines dilation limits beyond which active labor can be expected. More recently, a Consensus Committee of the American College of Obstetricians and ::0'a� 0 1)':D FIGURE 22-19 Composite ofthe average dilatation curve for nulliparous labor. The first stage is divided into a relatively flat latent phase and a rapidly progressive active phase. In the active phase, there are three identifiable component parts that include an acceleration phase, a phase ofmaximum slope, and a deceleration phase. (Redrawn from Friedman EA: Labor: Clinical Evaluation and Management, 2nd ed. New York, Appleton-Century-Crofts, 1978.) -Hendricks, Brenner & Kraus Ledger Univ. of Michigan -Rodesch et al. Temple Univ.

1	Univ. of Michigan -Rodesch et al. Temple Univ. FIGURE 22-20 Progress of labor in primigravid women from the time of admission. When the starting point on the abscissa begins with admission to the hospital, a latent phase is not observed. Gynecologists and Society for Maternal-Fetal Medicine (2016c) has redefined active labor to begin at 6 cm. A uller discussion of these labor changes is found in Chapter 23 (p. 445). his concept of a latent phase has great signiicance in understanding normal human labor, because labor is considerably longer when a latent phase is included. To better illustrate this, labor was diagnosed beginning with their admission, rather than with the onset of regular contractions. When labor is deined similarly, individual labor curves are remarkably comparable.

1	Aprolonged latent phase was defined by Friedman and Sachtleben (1963) as one exceeding 20 hours in the nullipara and 14 hours in the multipara. hese times corresponded to the 95th percentiles. Factors that afected latent phase duration include excessive sedation or epidural analgesia; unfavorable cervical condition, that is, thick, unefaced, or undilated; and false labor. Of women who had been administered heavy sedation, 85 percent eventually entered active labor. In another 10 percent, uterine contractions ceased, suggesting that they had false labor. The remaining 5 percent experienced persistence of an abnormal latent phase and required oxytocin stimulation. Amniotomy was discouraged because of the 10-percent incidence of false labor. Sokol and associates (1977) reported a 3-to 4-percent incidence of prolonged latent phase, regardless of parity. Friedman (1972) reported that latent phase prolongation did not adversely inluence fetal or maternal morbidity or mortality rates.

1	4-percent incidence of prolonged latent phase, regardless of parity. Friedman (1972) reported that latent phase prolongation did not adversely inluence fetal or maternal morbidity or mortality rates. However, Chelmow and coworkers (1993) disputed the long-held belief that prolongation of the latent phase is benign.

1	The progress of labor in nulliparas has particular significance because these curves all reveal a rapid change in the slope of cervical dilation rates between 3 and 5 cm (see Fig. 22-20). Thus, cervical dilation of3 to 6 cm or more, in the presence of uterine contractions, can be taken to reliaby represent the threshold or active labor. Similarly, these curves provide useful guideposts for labor management.

1	Turning again to Friedman (1955), the mean duration of active-phase labor in nulliparas was 4.9 hours. But, the standard deviation of 3.4 hours is large, hence, the active phase was reported to have a statistical maximum of 11.7 hours. Indeed, rates of cervical dilation ranged from a minimum of 1.2 up to 6.8 cm/hr. Friedman (1972) also found that multiparas progress somewhat faster in active-phase labor, with a minimum normal rate of 1.5 cm/hr. His analysis of active-phase labor concomitantly describes rates of fetal descent and cervical dilation (see Fig. 22-18). Descent begins in the later stage of active dilation, commencing at 7 to 8 cm in nulliparas and becoming most rapid after 8 cm.

1	Hendricks and coworkers (1970) challenged Friedman's conclusions about the course of normal human labor. Their principal diferences included: (1) absence of a latent phase, (2) no deceleration phase, (3) brevity ofilabor, and (4) dilation at similar rates for nulliparas and multiparas after 4 cm. They disputed the concept of a latent phase because they observed that the cervix dilated and efaced slowly during the 4 weeks preceding labor. They contended that the latent phase actually progressed over several weeks. hey also reported that labor was relatively rapid. Specifically, the average time from admission to complete dilation was 4.8 hours for nulliparas and 3.2 hours for multiparas.

1	Others have reassessed the Friedman labor curves. Zhang and associates (2010) studied electronic labor records from 62,415 parturients with spontaneous labor at term and vaginal birth. For nulliparas, the median time to progress from 4 to 5 cm was 1.3 hours, from 5 to 6 cm 0.8 hours, and thereafter, additional centimeters were gained approximately each 0.5 hours. They found that normal labor may take more than 6 hours to progress from 4 to 5 cm and more than 3 hours to progress from 5 to 6 cm dilation. Rates for multiparas were similar from 4 to 6 cm. Then, labor accelerated much faster in multiparas. Data from this study form the foundation for new guidelines regarding cesarean delivery indications for labor arrest put forth in the Obstetric Care Consensus document by the American College of Obstetrics and Gynecology and Society for Maternal-Fetal Medicine (2016c) and described in Chapter 23 (p. 444).

1	In a study performed at Parkland Hospital, epidural analgesia was found to lengthen the active phase of the Friedman labor curve by 1 hour (lexander, 2002). his increase was the result of a slight but significant decline in the rate of cervical dilation-1.4 cm/hr in women given epidural analgesia compared with 1.6 cm/hr in those without such analgesia. Several other reports also note that maternal obesity lengthens the irst stages oflabor by 30 to 60 minutes (Chin, 2012; Kominiarek, 2011). Finally, Adams and coworkers (2012) found that maternal fear prolonged labor by approximately 45 minutes. Active-phase abnormalities have been reported to occur in 25 percent of nulliparous and 15 percent of multiparous labors (Sokol, 1977). Friedman (1972) subdivided active-phase problems into protraction and arrest disorders. Abnormal labor patterns, diagnostic criteria, and treatment methods are summarized in Chapter 23 (p. 442).

1	• Second Stage of Labor his stage begins with complete cervical dilation and ends with fetal delivery. The median duration is approximately 50 minutes for nulliparas and about 20 minutes for multiparas, but it is highly variable (Kilpatrick, 1989). In a woman of higher parity with a previously dilated vagina and perineum, two or three expulsive eforts after full cervical dilation may suice to complete delivery. Conversely, in a woman with a contracted pelvis, with a large fetus, or with impaired expulsive eforts from conduction analgesia or sedation, the second stage may be longer. Higher maternal body mass index does not interfere with second-stage labor length (Carlhall, 2013; Robinson, 2011). Abnormalities of this labor stage are described in Chapter 23 (p. 446).

1	he normal duration of labor may be clouded by the many clinical variables that afect the conduct of labor in modern obstetrical units. Kilpatrick and Laros (1989) reported that the mean length of irst-and second-stage labor was approximately 9 hours in nulliparas without regional analgesia, and that the 95th percentile upper limit was 18.5 hours. Corresponding times for multiparas were a mean of 6 hours and a 95th percentile maximum of 13.5 hours. These authors defined labor onset as the time when a woman recalled regular, painful contractions every 3 to 5 minutes that led to cervical change.

1	Spontaneous labor was analyzed in nearly 25,000 women delivered at term at Parkland Hospital in the early 1990s. Almost 80 percent of women were admitted with a cervical dilation of 5 cm or less. Parity-nulliparous versus multiparous-and cervical dilation at admission were significant determinants of the length of spontaneous labor. The median time from admission to spontaneous delivery for all parturients was 3.5 hours, and 95 percent of all women delivered within 10.1 hours. These results suggest that normal human labor is relatively short. • Summary of Normal Labor

1	• Summary of Normal Labor Labor is characterized by brevity and considerable biological variation. Active labor can be reliably diagnosed when cervical dilation is :3 cm in the presence of uterine contractions. Once this cervical dilation threshold is reached, normal progression to delivery can be expected, depending on parity, in the ensuing 4 to 6 hours. Anticipated progress during a 1-to 3-hour second stage is monitored to ensure fetal safety. Finally, most women in spontaneous labor, regardless of parity, if left unaided, will deliver within approximately 10 hours after admission for spontaneous labor. Insuicient uterine activity is a common and correctable cause of abnormal labor progress. Thereore, when the length of otherwise normal labor exceeds the expected norm, interventions other than cesarean delivery-or example, oxytocin administration-must be irst considere.

1	The ideal management of labor and delivery requires two potentially opposing viewpoints on the part of clinicians. First, birthing should be recognized as a normal physiological process that most women experience without complications. Second, intrapartum complications, often arising quickly and unexpectedly, should be anticipated. Thus, clinicians must simultaneously make every woman and her supporters feel comfortable, yet ensure safety for the mother and newborn if complications suddenly develop. he American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) have collaborated in the development of Guidelines or Perinatal Care. These provide detailed information on the appropriate content of intrapartum care, including both personnel and facility requirements (Table 22-2). Labor and delivery outside the hospital is elected by some parturients. This option and its risks and benefits are discussed in Chapter 27 (p. 524).

1	Labor and delivery outside the hospital is elected by some parturients. This option and its risks and benefits are discussed in Chapter 27 (p. 524). Congress enacted EMT ALA in 1986 to ensure public access to emergency services regardless of the ability to pay. All Medicare-participating hospitals with emergency services must provide an appropriate screening examination for any pregnant woman experiencing contractions and presenting to the emergency department for evaluation.

1	The deinition of an emergency condition makes specific reference to a pregnant woman who is having contractions. Labor is defined as "the process of childbirth beginning with the latent phase of labor continuing through delivery of the placenta. A woman experiencing contractions is in true labor unless a physician certifies that after a reasonable time of observation the woman is in false labor." A woman in true labor is considered "unstable" for interhospital transfer purposes until the newborn and placenta are delivered. A stable woman may, however, be transferred at the direction of the patient or by a physician who certifies that the benefits of treatment at another facility outweigh the transfer risks. Physicians and hospitals violating these federal requirements are subject to civil penalties and termination from participation in the Medicare program. • Identification of Labor

1	• Identification of Labor Pregnant women are urged to report early in labor rather than to procrastinate until delivery is imminent for fear that they might be experiencing false labor. Early admittance is especially important if during antepartum care the woman, her fetus, or both are found to have risk factors for intrapartum complica tions.

1	Although the diferentiation between false and true labor is diicult at times, the diagnosis usually can be clariied by contraction frequency and intensity and by cervical dilation. Pates and associates (2007) studied one commonly used recommendation given to pregnant women. Namely, in the absence of ruptured membranes or bleeding, uterine contractions 5 minutes apart for 1 hour-that is, ::12 contractions in 1 hourmay signiY labor onset. Among 768 women in this study at Parkland Hospital, active labor deined as cervical dilation ::4 cm was diagnosed within 24 hours in three fourths of women with ::12 contractions per hour. Bailit and coworkers (2005) compared labor outcomes of 6121 women who presented in active labor deined as uterine contractions plus cervical dilation ::4 cm with those of 2697 women who presented in the latent phase. Women admitted during latent-phase labor had more active-phase arrest, more frequent need for oxytocin labor stimulation, and higher rates of

1	those of 2697 women who presented in the latent phase. Women admitted during latent-phase labor had more active-phase arrest, more frequent need for oxytocin labor stimulation, and higher rates of chorioamnionitis. It was concluded that physician interventions in women presenting in the latent phase may have been the cause of subsequent labor abnormalities.

1	In those instances when a diagnosis of labor cannot be established with certainty, observation for a longer period is often wise. Women who present to Parkland Hospital for labor 24°/7 ,. .

1	symptoms at weeks gestatIon or greater are routmely evaluated in a labor triage unit contiguous to our labor and delivery unit. All women in the triage area are evaluated by nurse practitioners and certiied nurse midwives using written protocols. Women with uncomplicated pregnancies with intact membranes and cervical dilation < 4 cm receive continuous external fetal monitoring for up to 2 hours. Women diagnosed with labor by either cervical change or persistent uterine contractions are admitted. After review by a physician, women without cervical change or with abatement of contractions return home with a diagnosis of false labor. In a recent study, a total of 3949 women with uncomplicated pregnancies between and 416r weeks' gestation were diagnosed with false labor. The mean inteval from hospital discharge to when they again presented was 4.9 days (Nelson, 2017). Within this protocol, hospital discharge with false labor at term was not associated with higher rates of adverse neonatal

1	hospital discharge to when they again presented was 4.9 days (Nelson, 2017). Within this protocol, hospital discharge with false labor at term was not associated with higher rates of adverse neonatal outcomes or cesarean delivery. he American College of Obstetricians and Gynecologists (2016a) has endorsed hospital-based obstetrical triage units.

1	Maternal blood pressure, temperature, pulse, and respiratory rate are recorded. Fetal heart rate is evaluated using a portable Doppler device, sonography, or fetoscope. The pregnancy record is promptly reviewed to identiY complications. Problems identiied or anticipated during prenatal care should be displayed prominently in the pregnancy record. Most often, unless there has been bleeding in excess of bloody show, a cervical examination is performed. he gloved index and second ingers are introduced into the vagina while avoiding the anal region. During prenatal care, the woman is instructed to be aware of luid leakage from the vagina and to report such an event promptly. Rupture of the membranes is signiicant for three reasons. First, if the presenting part is not ixed in the pelvis, the umbilical cord can prolapse and be compressed. Second, labor is likely to begin soon if the pregnancy is at or near term.

1	hird, if delivery is delayed after membrane rupture, intrauter ine and neonatal infection is more likely as the time interval increases (Herbst, 2007).

1	During sterile speculum examination, ruptured membranes are diagnosed if amnionic luid pools in the posterior fornix or clear fluid lows from the cevical canal. lthough several diagnostic tests for [he detection of ruptured membranes have been recommended, none is completely reliable. If the diagnosis remains uncertain, another method involves pH determination of vaginal luid. The pH of vaginal secretions normally ranges from 4.5 to 5.5, whereas that of amnionic luid is usually > .0. The use of the indicator nitrazine to identiY ruptured membranes is a simple and fairly reliable method. Test papers are impregnated with the dye, and the color of the reaction between these paper strips and vaginal luids is interpreted by comparison with a standard color chart. A pH above 6.5 is consistent with ruptured membranes. False-positive test results may occur with coexistent blood, semen, or bacterial vaginosis, whereas false-negative tests may result with scant luid.

1	Other tests to identiY amnionic luid include arborization or ferning of vaginal luid, which suggests amnionic rather than cervical luid. Amnionic luid crystallizes to form a fernlike pattern due to its relative concentrations of sodium chloride, proteins, and carbohydrates. Detection of alpha-fetoprotein in the vaginal vault has been used to identiY amnionic luid (Yamada, 1998). Although rarely required, identiication may also follow injection of indigo carmine into the amnionic sac via abdominal amniocentesis. Last, speciic amnionic luid proteins can be sought using point-of-care assays. These include AmniSure, which binds placental alpha microglobulin-1, and ROM Plus, which detects insulin growth factor binding protein-1 plus alpha-fetoprotein (Doret, 2013; Igbinosa, 2017).

1	The degree of cervical facement relects the length of the cervical canal compared with that of an unefaced cervix. When the length of the cervix is reduced by one half, it is 50-percent efaced. When the cervix becomes as thin as the adjacent lower uterine segment, it is completely, or 100-percent, efaced. Cervical dilation is determined by estimating the average diameter of the cervical opening by sweeping the examining inger from the margin of the cevical opening on one side to that on the opposite side. The diameter traversed is estimated in centimeters. The cervix is said to be fully dilated when the diameter measures 10 cm, because the presenting part of a term-size newborn usually can pass through a cervix this widely dilated.

1	he position of the cervix is determined by the relationship of the cervical os to the fetal head and is categorized as posterior, midposition, or anterior. Along with position, the consistency of the cervix is determined to be soft, irm, or intermediate between these two. heetal station, that is, the level of the presenting fetal part in the birth canal, is described in relationship to the ischial spines. These spines lie halway between the pelvic inlet and the pelvic outlet. When the lowermost portion of the presenting fetal part is at the level of the spines, it is designated s being at zero (0) station.

1	In the past, the long axis of the birth canal above and below the ischial spines was arbitrarily divided into thirds by some and into fifths (approximately 1 em) by other groups. In 1989, the American College of Obstetricians and Gynecologists adopted the classiication of station that divides the pelvis above and below the spines into ifths. Each ifth represents 1 em above or below the spines. hus, as the presenting fetal part descends from the inlet toward the ischial spines, the designation is -5, -4, -3, -2, -1i, then 0 station. Below the spines, as the presenting fetal part descends, it passes +1, +2, +3, +4, and +5 stations to delivery. Station + 5 em corresponds to the fetal head being visible at the introitus.

1	If the leading part of the fetal head is at 0 station or below, most often the fetal head has engaged-thus, the biparietal plane has passed through the pelvic inlet. f the head is unusualy moded or f caput succedaneum ormation is extensive, or both, engagement might not have taken place although the head appears to be at 0 station.

1	In a study done at five teaching centers in Denver, residents, nurses, and faculty were surveyed to determine what definitions were being used to describe fetal station (Carollo, 2004). Four diferent deinitions were in use. Disturbingly, these investigators found that few caregivers were aware that others were using diferent deinitions of station! Dupuis and associates (2005) tested the reliability of clinical estimations of station using the position of the leading part in centimeters above or below the spines. A birth simulator was used in which station could be precisely measured and compared with the vaginal examination done by clinicians. hey reported that the clinical examiners were incorrect a third of the time.

1	hese ive characteristics-cervical dilation, efacement, consistency, position, and fetal station-are assessed when tabulating the Bishop score. his score is commonly used to predict labor induction outcome and is discussed in Chapter 26 (p. 505). Taken together, these factors suggest the subjective "favorability" of the cervix for induction success.

1	When a woman is admitted in labor, most often the hematocrit or hemoglobin concentration is checked. he hematocrit can be measured easily and quickly. At Parkland Hospital, blood is collected in a standard collection tube with anticoagulant. From this, a heparinized capillary tube is filled to spin in a microhematocrit centrifuge in the labor and delivery unit. his provides a hematocrit value within 3 minutes. he initial collection tube is also sent to the hematology laboratory for evaluation if the point-of-care hematocrit is <30 volume percent. Another labeled tube of blood is allowed to clot and sent to the blood bank for blood type and antibody screen, if needed. A final sample is collected for syphilis and human immunodeficiency virus (HIV) serology. In some labor units, a cleancatch voided specimen is examined in all women for protein and glucose. At Parkland Hospital, however, we obtain a urine specimen for protein determination in hypertensive women only (Table 40-1, p. 712).

1	Women with no prenatal care are considered to be at risk for syphilis, hepatitis B, and HIV, and laboratory screening studies for these, as well as a blood type and antibody screen, are performed (American Academy of Pediatrics and American College of Obstetricians and Gynecologists, 2017). Some states, for example, Texas, require routine testing for syphilis, hepatitis B, and HIV in all women admitted to labor and delivery units, even if these were done during prenatal care. • Management of First-Stage Labor As soon as possible after admittance, the remainder of a general examination is completed. Whether a pregnancy is normal can best be determined when all examinations, including record and laboratory review, are completed. A rational plan for monitoring labor can then be established based on the needs of the fetus and the mother. Because labor lengths vary markedly among individuals, precise statements regarding anticipated labor duration are unwise.

1	In general, pain relief should depend on the needs and desires of the woman. The American College of Obstetricians and Gynecologists (2017) has speciied optimal goals for anesthesia care in obstetrics. his is discussed in detail in Chapter 25. In some units, women can choose to spend part of irststage labor in a large water tub. isks and benefits are described in Chapter 27 (p. 524).

1	This is discussed in detail in Chapter 24. Briely, the American Academy of Pediatrics and American College of Obstetricians and Gynecologists (2017) recommend that during irst-stage labor, in the absence of any abnormalities, the fetal heart rate should be checked immediately after a contraction at least every 30 minutes and then every 15 minutes during the second stage. If continuous electronic monitoring is used, the tracing is evaluated at least every 30 minutes during the irst stage and at least every 15 minutes during second-stage labor. For women with pregnancies at risk, fetal heart auscultation is performed at least every 15 minutes during irst-stage labor and every 5 minutes during the second stage. Continuous electronic monitoring may be used with evaluation of the tracing every 15 minutes during the first stage of labor, and every 5 minutes during the second stage.

1	Temperature, pulse, and blood pressure are evaluated at least every 4 hours. If membranes have been ruptured for many hours before labor onset or if there is a borderline temperature elevation, the temperature is checked hourly. Although uterine contractions are usually assessed with electronic monitoring, they can be quantitatively and qualitatively evaluated manually (Chap. 24, p. 478). With the palm of the hand resting lightly on the uterus, the time of contraction onset is determined. Its intensity is gauged from the degree of irm ness the uterus achieves. At the acme of efective contractions, the finger or thumb cannot readily indent the uterus during a "irm" contraction. he time at which the contraction disap pears is noted next. his sequence is repeated to evaluate the frequency, duration, and intensity of contractions.

1	During the first stage of labor, the need for subsequent vaginal examinations to monitor cervical change and present ing part position will vary considerably. When the membranes rupture, an examination to exclude cord prolapse is performed expeditiously if the fetal head was not deinitely engaged at the previous examination. he fetal heart rate is also checked immediately and during the next uterine contraction to help detect occult umbilical cord compression. At Parkland Hos pital, periodic pelvic examinations are typically performed at 2-to 3-hour intervals to evaluate labor progress. Evidence implicating the number of vaginal examinations in infection related morbidity is conlicting (Cahill, 2012; Soper, 1989).

1	Food and liquids with particulate matter should be withheld during active labor and delivery. Gastric emptying time is remarkably prolonged once labor is established and analgesics are administered. As a consequence, ingested food and most medications remain in the stomach and are not absorbed. Instead, they may be vomited and aspirated (Chap. 25, p. 499). According to the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017), oral intake of moderate amounts of clear liquids is reasonable for women with uncomplicated labor. Modest amounts of clear liquids such as water, clear tea, black cofee, carbonated beverages, Popsicles, and pulp-free fruit juices are allowed in uncomplicated laboring women. In those with appreciable risks for aspiration or those with significant risks for cesarean delivery, further restriction may be instituted. For example, for those with planned cesarean delivery, liquids are halted 2 hours before and solids are

1	or those with significant risks for cesarean delivery, further restriction may be instituted. For example, for those with planned cesarean delivery, liquids are halted 2 hours before and solids are stopped 6 to 8 hours prior to surgery (American College of Obstetricians and Gynecologists, 2016b).

1	Although an intravenous (IV) infusion system is often routinely established early in labor, real need for this in the normal pregnant woman is limited, at least until analgesia is administered. However, venous access is advantageous during the immediate puerperium to administer oxytocin prophylactically and at times therapeutically when uterine atony persists. Moreover, with longer labors, the administration of glucose, sodium, and water to the otherwise fasting woman at the rate of 60 to 120 mLlhr prevents dehydration and acidosis. Shrivastava and associates (2009) noted shorter labors in nulliparas delivering vaginally who were provided an intravenous normal saline with dextrose solution compared with those given saline solution only. In another study, 195 women in labor received lactated Ringer or isotonic sodium chloride solution at a rate of either 125 or 250 mLlhr. The mean volume of total IV luid was 2008 mL in the 125 mL/hr group and 2487 mL in the 250 mL/hr group (Garite,

1	Ringer or isotonic sodium chloride solution at a rate of either 125 or 250 mLlhr. The mean volume of total IV luid was 2008 mL in the 125 mL/hr group and 2487 mL in the 250 mL/hr group (Garite, 2000). Labor lasted more than 12 hours in signiicantly more of the women given a 125 mLlhr infusion compared with those given 250 mLlhr-26 versus 13 percent, respectively. In yet another study, 31i1 nulliparas with uncomplicated pregnancies in spontaneous labor at term received one of three IV infusions (Edwards, 2014). Group 1 was given 125 mLlhr of lactated Ringer solution with 5 percent dextrose (D5LR), Group 2 received 250 mLihour of the same solution (D5LR), and Group 3 was administered 25 mL/hr of D5LR. Groups 1 and 2 were allowed ice chips, Popsicles, and hard candy, and Group 3 also received Gatorade. Oral intake was limited in Groups 1 and 2 but was ad libitum in Group 3. The authors concluded that any of the regimens studied was safe but none was superior for labor performance.

1	In bed, the laboring woman may assume the position she finds most comfortable, and often this will be lateral recumbency. Lying supine is typically avoided to avert aortocaval compression and its potential to lower uterine perfusion (Chap. 4, p. 62). However, the normal laboring woman need not be conined to bed early in labor. A comfortable chair may be beneicial psychologically and perhaps physiologically. Others encourage ambulation.

1	Proponents of walking report that it shortens labor, lowers rates of oxytocin augmentation, diminishes the need for analgesia, and decreases the frequency of operative vaginal delivery (Flynn, 1978; Read, 1981). In their Cochrane review, Lawrence and associates (2013) found that labor in ambulant or upright positions shortened irst-stage labor by about 1 hour and lowered cesarean delivery and epidural analgesia rates. Lupe and Gross (1986) concluded, however, that no conclusive evidence supports assertions that upright maternal posture or ambulation improves labor. hey reported that women preferred to lie on their side or sit in bed. Few chose to walk, fewer to squat, and none wanted the knee-chest position. Parturients tended to assume fetal positions in later labor. Most women enthusiastic about ambulation returned to bed when active labor began (Carlson, 1986; Williams, 1980).

1	Bloom and colleagues (1998) conducted a randomized trial to study the efects of walking during irst-stage labor. In 1067 women with uncomplicated term pregnancies delivered at Parkland Hospital, these investigators reported that ambulation did not afect labor duration. mbulation did not reduce the need for analgesia, nor was it harmul to the newborn. Because of these observations, we give women without complications the option to select either recumbency or supervised mbulation during labor. Rupture of Membranes

1	Rupture of Membranes If the membranes are intact, temptation is great, even during normal labor, to perform amniotomy. The presumed beneits are more rapid labor, earlier detection of meconium-stained amnionic luid, and the opportunity to apply an electrode to the fetus or insert a pressure catheter into the uterine cavity for monitoring. he advantages and disadvantages of amniotomy are discussed in Chapter 26 (p. 511). Importantly, the fetal head must be well applied to the cervix and not be dislodged from the pelvis during the procedure to avoid umbilical cord prolapse. In cases with prolonged membrane rupture, deined as greater than 18 hours, antimicrobial administration for prevention of group B streptococcal infections is recommended. his is discussed in Chapter 64 (p. 1221). his practice similarly lowers rates of chorioamnionitis and endometritis (Saccone, 2015).

1	Distention of the bladder can hinder descent of the fetal presenting part and lead to subsequent bladder hypotonia and infection. Periodically during labor, the suprapubic region is inspected and palpated to detect distention. If the bladder is readily seen or palpated above the symphysis, the woman should be encouraged to void. At times, those who may be unable to void on a bedpan may be able to ambulate with assistance to a toilet and successfully void. If the bladder is distended and voiding is not possible, catheterization is indicated. Carley and coworkers (2002) found that 51 of 11,332 vaginal deliveries (1 in 200) were complicated by postpartum urinary retention. Most women resumed normal voiding before discharge from the hospital. Musselwhite and associates (2007) reported retention in 4.7 percent of women who had labor epidural analgesia. Risk factors for retention were primiparity, oxytocin-induced or augmented labor, perineal lacerations, operative vaginal delivery,

1	retention in 4.7 percent of women who had labor epidural analgesia. Risk factors for retention were primiparity, oxytocin-induced or augmented labor, perineal lacerations, operative vaginal delivery, catheterization during labor, and labor durationi> 10 hours.

1	• Management of Second-Stage Labor With full cervical dilation, which signiies the onset of the second stage, a woman typically begins to bear down. With descent of the presenting part, she develops the urge to defecate. Uterine contractions and the accompanying expulsive forces may now last 1 minute and recur at an interval no longer than 90 seconds. As discussed earlier, the median duration of the second stage is 50 minutes in nulliparas and 20 minutes in multiparas, although the interval can vary. Monitoring intervals of the fetal heart rate were discussed on page 436, and interpretation of second-stage electronic fetal heart rate patterns is discussed in Chapter 24 (p. 469).

1	In most cases, bearing down is reflexive and spontaneous during second-stage labor. Occasionally, a woman may not employ her expulsive forces to good advantage and coaching is desirable. Her legs should be half-flexed so that she can push with them against the mattress. When the next uterine contraction begins, she is instructed to exert downward pressure as though she were straining at stool. A woman is not encouraged to push beyond the completion of each contraction. Instead, she and her fetus are allowed to rest and recover. During this period of actively bearing down, the fetal heart rate auscultated during the contraction is likely to be slow but should recover to normal range before the next expulsive efort. Fetal and obstetrical outcomes appear to be unafected whether pushing is coached or uncoached during second-stage labor (Bloom, 2006; Tuuli, 2012). Bloom and colleagues (2006) studied efects of actively coaching expulsive eforts in women without epidural analgesia. hey

1	is coached or uncoached during second-stage labor (Bloom, 2006; Tuuli, 2012). Bloom and colleagues (2006) studied efects of actively coaching expulsive eforts in women without epidural analgesia. hey reported that although the second stage was slightly shorter in coached women, no other maternal advantages were gained.

1	Several positions during the second stage have been recommended to augment pushing eforts. Eason and colleagues (2000) reviewed various posltlons and their efect on the incidence of perineal trauma. They found that the supported upright position had no advantages over the recumbent one. Upright positions include sitting, kneeling, squatting, or resting with the back at a 30-degree elevation. In women with regional analgesia, one recent randomized trial found higher vaginal delivery rates in those in a recumbent position compared with an upright one-41 versus 35 percent (The Epidural and Position Trial Collaborative Group, 2017). In women without epidural analgesia, Gupta (2017) in their review compared upright positions with supine or lithotom) positions and their efect on labor. Upright positions ofered a slightly shorter interval to delivery as well as fewer episiotomies and operative vaginal deliveries. However, rates of blood loss > 500 mL and perhaps of second-degree lacerations

1	positions ofered a slightly shorter interval to delivery as well as fewer episiotomies and operative vaginal deliveries. However, rates of blood loss > 500 mL and perhaps of second-degree lacerations were increased. Berghella and coworkers (2008) hypothesized that parity, less intense aortocaval compression, improved fetal alignment, and larger pelvic outlet diameters might explain these indings. In an earlier study, a 20-to 30-percent increase in the area of the pelvic outlet was noted with squatting compared with the supine position (Russell, 1969). Finally, Babayer and associates (1998) cautioned that prolonged sitting or squatting during the second stage may cause neuropathy of the common fibular (formerly common peroneal) nerve.

1	As the head descends through the pelvis, the perineum begins to bulge and the overlying skin becomes stretched. Now the scalp of the fetus may be visible through the vulvar opening. At this time, the woman and her fetus are prepared for delivery, which is described in Chapter 27 (p. 516). An orderly and systematic approach to labor management results in reproducible beneficial maternal and perinatal outcomes (lthabe, 2008). Several labor management protocols are subsequently presented. These include those from the National Maternity Hospital in Dublin, from the World Health Organization, and from Parkland Hospital.

1	In Dublin more than 30 years ago, O'Driscoll and associates (1984) pioneered the concept that a disciplined, standardized labor management protocol reduced the number of cesarean deliveries for dystocia. Their overall cesarean delivery rate was 5 percent in the 1970s and 1980s with such management. The approach is now referred to as active management of labor. Two of its components-amniotomy and oxytocinhave been widely used, especially in English-speaking countries outside the United States. With this protocol, labor is diagnosed when painful contractions are accompanied by complete cervical efacement, bloody "show," or ruptured membranes. Women with such indings are committed to delivery within 12 hours. Pelvic examination is performed each hour for the next 3 hours, and thereafter at 2-hour intervals. When dilation has not increased by at least 1 cm/hr, amniotomy is performed. Progress is again assessed at 2 hours, and high-dose oxytocin infusion, described in Chapter 26 (p. 509),

1	intervals. When dilation has not increased by at least 1 cm/hr, amniotomy is performed. Progress is again assessed at 2 hours, and high-dose oxytocin infusion, described in Chapter 26 (p. 509), is started unless dilation of at least 1 cm/hr is attained. Women are constantly attended by midwives. If membranes rupture before admission, oxytocin is begun for no progress at the I-hour mark.

1	such active management with their "traditional" approach to cago. hey randomly assigned 705 nulliparas with uncompli cated pregnancies in spontaneous labor at term. he cesarean delivery rate was significantly lower with active versus tradi tional management-10.5 versus 14.1 percent, respectively. Subsequent studies did not show this. Wei and associates tion in cesarean delivery rates when active management of labor was compared with standard care. Frigoletto and coworkers (1995) reported another randomized trial with 1934 nullipa rous women at Brigham and Women's Hospital in Boston. ened labor, it did not afect the cesarean delivery rate. hese observations have since been reported by others (Brown, 2013).

1	A partograph was designed by the World Health Organization (WHO) for use in developing countries (Dujardin, 1992). According to Orji (2008), the partograph is similar for nulliparas and multiparas. Labor is divided into a latent phase, which should last no longer than 8 hours, and an active phase. The active phase starts at 3 cm dilation, and progress should be no slower than 1 cm/hr. A 4-hour wait is recommended before intervention when the active phase is slow. Labor is graphed, and analysis includes use of alert and action lines. Lavender and colleagues (2006) randomized 3000 nulliparous women to labor interventions at 2 hours versus 4 hours as recommended by WH O. Their cesarean delivery rate was unafected, and they concluded that interventions such as amniotomy and oxytocin were needlessly increased using the 2-hour time interval. From their Cochrane Database review, Lavender and associates (2013) do not recommend use of the partograph for standard labor management.

1	At Parkland Hospital, women are admitted if active labor is diagnosed or if ruptured membranes are conirmed. Labor is defined as cervical dilation of 3 to 4 cm or more in the presence of uterine contractions. Management guidelines direct that a pelvic examination be performed approximately every 2 hours. Inefective labor is suspected when the cervix does not dilate within approximately 2 hours of admission. Amniotomy is then performed, and labor progress determined at the next 2-hour evaluation. In women whose labors do not progress, an intrauterine pressure catheter is placed to assess uterine function. Hypotonic contractions and no cervical dilation after an additional 2 to 3 hours result in stimulation of labor using the high-dose oxytocin regimen described in Chapter 26 (p. 509). The goal is uterine activity of 200 to 250 Montevideo units for 2 to 4 hours before dystocia can be diagnosed. If hypotonic contractions are strongly suspected, internal monitors may be placed with

1	The goal is uterine activity of 200 to 250 Montevideo units for 2 to 4 hours before dystocia can be diagnosed. If hypotonic contractions are strongly suspected, internal monitors may be placed with amniotomy and again cervical change and contraction pattern are assessed in 2 hours. Conirmation of deicient Montevideo units at that time may prompt oxytocin augmentation for maternal or fetal indications.

1	Dilation rates of 1 to 2 cm/hr are accepted as evidence of progress after satisfactory uterine activity has been established with oxytocin. This can require up to 8 hours or more before cesarean delivery is performed for dystocia. The cumulative time required to efect this stepwise management approach permits many women to establish efective labor. This management protocol has been evaluated in more than 20,000 women with uncomplicated pregnancies. Importantly, these labor interventions and the relatively infrequent use of cesarean delivery did not jeopardize the fetus-newborn. Adams SS, Eberhard-Gran M, Eskild A: Fear of childbirth and duration of labour: a study of 2206 women with intended vaginal delivery. BJOG 119(10):1238,r2012 Alexander JM, Sharma SK, Mcintire DD, et al: Epidural analgesia lengthens the Friedman active phase of labor. Obstet Gynecol 100:46,r2002

1	Alexander JM, Sharma SK, Mcintire DD, et al: Epidural analgesia lengthens the Friedman active phase of labor. Obstet Gynecol 100:46,r2002 Althabe F, Buekens P, Bergel E, et al: A behavioral intervention to improve obstetrical care. N Engl J Med 358:1929, 2008 American Academy of Pediatrics and the American College of Obstetricians and Gynecologists: Guidelines for Perinatal Care, 8th ed. Elk Grove Village, AAP, 2017 American College of Obstetricians and Gynecologists: Obstetric forceps. Committee Opinion 71, August 1989 American College of Obstetricians and Gynecologists: Hospital-based triage of obstetric patients. Committee Opinion No. 667, July 2016a American College of Obstetricians and Gynecologists: Oral intake during labor. Committee Opinion No. 441, September 2009, Reairmed 2016b American College of Obstetricians and Gynecologists: Obstetric analgesia and anesthesia. Committee Opinion No. 177, April 2017

1	American College of Obstetricians and Gynecologists, Society for MaternalFetal Medicine: Safe prevention of the primaty cesarean delivery. Obstetric Care Consensus No. I, March 2014, Reairmed 2016c Babayer M, Bodack MP, Creatura C: Common peroneal neuropathy secondary to squatting during childbirth. Obstet Gynecol 91 :830, 1998 Bailit JL, Dierker L, Blanchard MH, et al: Outcomes of women presenting in active versus latent phase of spontaneous labor. Obstet Gynecol 105:77, 2005 Berghella V, Baxter JK, Chauhan SP: Evidence-based labor and delivery management. Am J Obstet Gynecol 199:445,2008 Bloom SL, Casey BM, Schafer JI, et al: A randomized trial of coached versus uncoached maternal pushing during the second stage of labor. Am J Obstet Gyneco1r194:10,r2006 Bloom SL, Mcintire DO, Kelly MA, et al: Lack of efect of walking on labor and delivery. N Engl J Med 339:76, 1998

1	Bloom SL, Mcintire DO, Kelly MA, et al: Lack of efect of walking on labor and delivery. N Engl J Med 339:76, 1998 Brown He, Paranjothy S, Dowswell T, et al: Package of care for active management in labour for reducing caesarean section rates in low-risk women. Cochrane Database Syst Rev 9:CD004907, 2013 Cahill AG, DuY CR, Odibo AO, et al: Number of cervical examinations and risk of intrapartum maternal fever. Obstet Gynecol 119(6): 1096, 2012 Caldwell WE, Moloy HC, D'Esopo DA: A roentgenologic study of the mecha nism of engagement of the fetal head. Am J Obstet Gynecol 28:824, 1934 Calkins LA: he etiology of occiput presentations. Am J Obstet Gynecol 37:618, 1939 Carlan 5J, Wyble L, Lense J, et al: Fetal head molding: diagnosis by ultrasound and a review of the literature. J Perinatal 11r: 1 05, 1991 Carley ME, Carley JM, Vasdev G, et al: Factors that are associated with clinically overt postpartum urinary retention after vaginal delivery. Am J Obstet Gynecol 187:430,r2002

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1	Chin JR, Henry E, Holmgren CM, et al: Maternal obesity and contraction strength in the first stage ofrlabor. m J Obstet :129.e1,r2012 Doret M, Cartier R, Miribel J, et al: Premature preterm rupture of the membrane diagnosis in early pregnancy: PAMG-l and IGFBP-l detection in amniotic fluid with biochemical tests. Clin Biochem 46(18): 1816, 2013 Dujardin B, De Schampheleire I, Sene H, et al: Value of the alert and action lines on the partogram. Lancet 339: 1336, 1992 Dupuis 0, Silveira R, Zentner A, et al: Birth simulator: Reliability of transvaginal assessment of fetal head station as defined by the American College of Obstetricians and Gynecologists classification. Am J Obstet Gynecol 192:868, 2005 Eason E, Labrecque M, Wells G, et al: Preventing perineal trauma during childbirth: a systematic review. Obstet Gynecol 95:464, 2000

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1	Lavender T, Hart A, Smyth M: Efect of partogram use on outcomes for women in spontaneous labour at term. Cochrane Database Syst Rev 7:CD005461,o20o13 Lawrence A, Lewis L, Hofmeyr G], et al: Maternal positions and mobility during irst stage labour. Cochrane Database Syst Rev 10:CD003934, 2013 Leopold]: Conduct of normal births through external examination alone. Arch GynaekoIo45:337, 1894 L6pez-Zeno ]A, Peaceman M, Adashek ]A, et al: A controlled trial of a program for the active management of labor. N Engl] Med 326:450, 1992 Lupe P], Gross TL: Maternal upright posture and mobility in labor: a review. Obstet GynecoIo67:727, 1986 Lydon-Rochelle M, Albers L, Gowoda], et al: Accuracy of Leopold maneuvers in screening for malpresentation: a prospective study. Birth 20: 132, 1993 Musselwhite L, Faris P, Moore K, et al: Use of epidural anesthesia and the risk of acute postpartum urinary retention. Amo] Obstet Gynecol 196:472, 2007

1	Musselwhite L, Faris P, Moore K, et al: Use of epidural anesthesia and the risk of acute postpartum urinary retention. Amo] Obstet Gynecol 196:472, 2007 Nelson DB, McIntire DO, Leveno K]: False labor at term in singleton pregnancies: Discharge after a standardized assessment and perinatal outcomes. Obstet GynecoIo130(1):139, 2017 Noumi G, Collado-Khoury F, Bombard A, et al: Clinical and sonographic estimation of fetal weight performed in labor by residents. Am ] Obstet GynecoIo192:1407,o2005 Nygaard I: Pelvic loor recovery ater childbirth. Obstet Gynecol 125(3):529, 20o15 O'Driscoll K, Foley M, MacDonald 0: Active management of labor as an alternative to cesarean section for dystocia. Obstet Gynecol 63:485, 1984 Orji E: Evaluating progress of labor in nulliparas and multiparas using the modiied WHO partograph. Into] Gynaecol Obstet 102:249,o2008 Pates ]A, McIntire DO, Leveno K]: Uterine contractions preceding labor. Obstet Gynecol 110:566, 2007

1	Pates ]A, McIntire DO, Leveno K]: Uterine contractions preceding labor. Obstet Gynecol 110:566, 2007 Ramphul M, Ooi PV, Burke G, et al: Instrumental delivery and ultrasound: a multicentre randomised controlled trial of ultrasound assessment of the fetal head position versus standard care as an approach to prevent morbidity at instrumental delivery. B]OG 121(8):1029,o2014 Read ]A, Miller FC, Paul RH: Randomized trial of ambulation versus oxytocin for labor enhancement: a preliminary report. Am ] Obstet Gynecol 139(6):669, 1981 Robinson BK, Mapp DC, Bloom SL, et al: Increasing maternal body mass index and characteristics of the second stage of labor. Obstet Gynecol 118:1309,o2011 Russell ]G: Moulding of the pelvic outlet. ] Obstet Gynaecol Br Commonw 76:817,o1969 Saccone G, Berghella V: Antibiotic prophylaxis for term or near-term premature rupture of membranes: metaanalysis of randomized trials. Amo] Obstet Gynecol. 2015 May;212(5):627.e1

1	Saccone G, Berghella V: Antibiotic prophylaxis for term or near-term premature rupture of membranes: metaanalysis of randomized trials. Amo] Obstet Gynecol. 2015 May;212(5):627.e1 Segel SY, Carreno CA, Weiner MS, et al: Relationship between fetal station and successful vaginal delivery in nulliparous women. Am ] Perinatol 29:723,o2012 Shrivastava K, Garite T], Jenkins SM, et al: A randomized, double-blinded, controlled trial comparing parenteral normal saline with and without dextrose on the course oflabor in nulliparas. Amo] Obstet Gynecol 200(4):379. el,o2009 Sokol R], Stojkov ], Chik L, et al: Normal and abnormal labor progress: 1. A quantitative assessment and survey of the literature. ] Reprod Med 18:47, 1977 Soper DE, Mayhall CG, Dalton HP: Risk factors for imraamniotic infection: a prospective epidemiologic study. Am] Obstet GynecoIo161(3):562, 1989

1	Soper DE, Mayhall CG, Dalton HP: Risk factors for imraamniotic infection: a prospective epidemiologic study. Am] Obstet GynecoIo161(3):562, 1989 Staer-]ensen ], Siafarikas F, Hilde G, et al: Postpartum recovery of levator hiatus and bladder neck mobility in relation to pregnancy. Obstet Gynecol 125(3):531,o2015 Tuuli MG, Frey A, Odibo AO, et al: Immediate compared with delayed pushing in the second stage oflabor. Obstet Gynecol 120:660, 2012 Wei S, Wo BL, Qi HP, et al: Early amniotomy and early oxytocin for prevention of, or therapy for, delay in irst stage spontaneous labour compared with routine care. Cochrane Database Syst Rev 8:CD006794, 2013 Wiafe YA, Whitehead B, Venables H, et al: he efectiveness of intrapartum ultrasonography in assessing cervical dilatation, head station and position: a systematic review and meta-analysis. Ultrasound 24(4) :222, 2016

1	Williams M, hon MH, Studd ]W: A study of the beneits and acceptability of ambulation in spontaneous labor. B]OG 87: 122, 1980 Yamada H, Kishida T, Negishi H, et al: Silent premature rupture of membranes, detected and monitored serially by an AFP kit. ] Obstet Gynaecol Res 24:103, 1998 Zhango], Landy H], Branch OW, et al: Contemporary patterns of spontaneous labor with normal neonatal outcomes. Obstet Gynecol 116: 1281, 2010 DYSTOCIA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441 ABNORMALITIES OF THE EXPULSIVE FORCES ........ 442 PREMATURELY RUPTURED MEMBRANES AT TERM ... 447 PRECIPITOUS LABOR AND DELIVERY ............... 448 FETOPELVIC DISPROPORTION . . . . . . . . . . . . . . . . . . . . 448 PELVIC CAPACITY .............................. 448 FACE PRESENTATION . . . . . . . . . . . . . . . . . . . . . . . . . . . 450

1	FETOPELVIC DISPROPORTION . . . . . . . . . . . . . . . . . . . . 448 PELVIC CAPACITY .............................. 448 FACE PRESENTATION . . . . . . . . . . . . . . . . . . . . . . . . . . . 450 BROW PRESENTATION . . . . . . . . . . . . . . . . . . . . . . . . . . 452 TRANSVERSE LIE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452 COMPOUND PRESENTATION ..................... 454 COMPLICATIONS WITH DYSTOCIA. . . . . . . . . . . . . . . . . 454 ... pains become less requent and less intense, although giving rise to quite as much or even more suering than previousy. At the same time, the cervix, which was becoming obliterated and dilated in a satiactory manner, ceases to make urther proress and labour apparenty comes to a stanstill. -J.

1	he term dystocia as described by Williams in the irst edition of this text still applies today. It literally means dficult labor and is characterized by abnormally slow labor progress. Similar to the factors described by Williams, dystocia arises from three distinct abnormality categories. First, uterine contractions may be insuiciently strong or inappropriately coordinated to eface and dilate the cevix-uterine dyunction. lso, voluntary maternal muscle efort during second-stage labor may be inadequate. Second, fetal abnormalities of presentation, position, or anatomy may slow progress. Last, structural changes can contract the maternal bony pelvis. Or, soft tissue abnormalities of the reproductive tract may form an obstacle to fetal descent. More simply, these alterations can be mechanistically simplified into three categories that include abnormalities of the powers-uterine contractility and maternal expulsive efort; of the passenger-the fetus; and of the passage-the pelvis and lower

1	simplified into three categories that include abnormalities of the powers-uterine contractility and maternal expulsive efort; of the passenger-the fetus; and of the passage-the pelvis and lower reproductive tract.

1	23-1 oten interact singly or in combination to produce dysfunctional labor. Commonly used expressions today such as cephalopelvic disproportion and ailure to progress are used to describe inefective labors. Of these, TABLE 23-1. Common Clinical Findings in Women with Ineffective Labor Inadequate cervical dilation or fetal descent: Fetopelvic disproportion:

1	Inadequate pelvic capacity Malpresentation or pOSition of the fetus Abnormal fetal anatomy cephalopelvic disproportion is a term that came into use before the 20th century to describe obstructed labor resulting from disparity between the fetal head size and maternal pelvis. But, the term originated at a time when the main indication for cesarean delivery was overt pelvic contracture due to rickets (0lah, 1994). Such absolute disproportion is now rare, and most cases result from malposition of the fetal head within the pelvis (asynclitism) or from inefective uterine contractions. True disproportion is a tenuous diagnosis because many women who undergo cesarean delivery for this reason subsequently deliver even larger newborns vaginally in subsequent pregnancies. A second phrase, ailure to progress in either spontaneous or stimulated labor, has become an increasingly popular description of inefectual labor. his term reflects lack of progressive cervical dilation or lack of fetal

1	to progress in either spontaneous or stimulated labor, has become an increasingly popular description of inefectual labor. his term reflects lack of progressive cervical dilation or lack of fetal descent. Neither of these two terms is specific.

1	• Mechanisms of Dystocia At the end of pregnancy, the fetal head encounters a relatively thick lower uterine segment and undilated cervix. With the . onset of labor, the factors influencing progress are uterine contractions, cervical resistance, and the forward pressure exerted by the leading fetal part. Ater complete cervical dilation, the mechanical relationship between the fetal head size and position and the pelvic capacity, namelyetopelvic proportion, becomes clearer as the fetus attempts to descend. Because of this, abnormalities in fetopelvic proportions become more apparent once the second stage is reached. Uterine muscle malfunction can result from uterine overdistention, obstructed labor, or both. Thus, infective labor is generaly accepted as a possible warning sign of etopelvic disproportion.

1	Uterine muscle malfunction can result from uterine overdistention, obstructed labor, or both. Thus, infective labor is generaly accepted as a possible warning sign of etopelvic disproportion. Although artificial separation of labor abnormalities into pure uterine dysfunction andetopelvic disproportion simplifies classification, it is an incomplete characterization because these two abnormalities are closely interlinked. Indeed, the bony pelvis rarely limits vaginal delivery. In the absence of objective means of precisely distinguishing these two causes of labor failure, clinicians must rely on a trial of labor to determine if labor can be successful in efecting vaginal delivery.

1	Cervical dilation as well as propulsion and expulsion of the fetus are brought about by uterine contractions. During second-stage labor, these contractions are then reinforced by voluntary or involuntary muscular action of the abdominal wall-"pushing." The diagnosis of uterine dysfunction in the latent phase is diicult and sometimes can be made only in retrospect. Women who are not yet in active labor commonly are erroneously treated for this dysfunction. Beginning in the 1960s, at least three significant advances have aided treatment of uterine dysfunction. First is the realization that undue labor prolongation may contribute to maternal and perinatal morbidity and mortality rates. Second, dilute intravenous infusion of oxytocin is administered to treat certain types of uterine dysfunction. Last, cesarean delivery is selected rather than diicult midforceps delivery when oxytocin fails or its use is inappropriate. • Types of Uterine Dysfunction

1	Reynolds and coworkers (1948) emphasized that uterine contractions of normal labor are characterized by a gradient of myometrial activity. These forces are greatest and last longest at the fundus-considered fundal dominance-and they diminish toward the cervix. Caldeyro-Barcia and colleagues (1950) from Montevideo, Uruguay, inserted small balloons into the myometrium at various levels (Chap. 24, p. 478). hey reported that in addition to a gradient of activity, the onset of the contractions difered in the fundus, midzone, and lower uterine segments. Larks (1960) described the stimulus as starting in one cornu and then several milliseconds later in the other. he excitation waves then join and sweep over the fundus and down the uterus. Normal spontaneous contractions often exert pressures approximating 60 mm Hg (Hendricks, 1959). Even so, the Montevideo group ascertained that the lower limit of contraction pressure required to dilate the cervix is 15 mm Hg.

1	From these observations, two physiological types of uterine dysfunction are defined. In the more common hypotonic uterine dyunction, there is no basal hypertonus, and uterine contractions have a normal gradient pattern (synchronous). However, pressure during a contraction is insuicient to dilate the cervix. In the second type, hypertonic uterine dysunction or incoordinate uterine dyunction, either basal tone is elevated appreciably or the pressure gradient is distorted. Gradient distortion may result from more forceful contraction of the uterine midsegment than the fundus, from complete asynchrony of the impulses originating in each cornu, or from a combination of these two.

1	hese just-described types of uterine dysfunction can in turn lead to labor abnormalities (Table 23-2). First, the latent phase may be prolonged, which is defined as exceeding 20 hours in the nullipara and 14 hours in the multipara. In some, uterine contractions cease, suggesting false labor. In the remainder, an abnormally long latent phase persists and is often treated with oxytocin stimulation. In active labor, disorders are divided into slower-than-normal progress-a protraction disorder-or complete cessation of progress-an arrest disorder. Terms presented in Table 23-2 and their diagnostic criteria more precisely describe abnormal labor. To be diagnosed with either of these, a woman must be in the active phase of labor, which is defined by cervical change.

1	hese criteria and management of abnormal labor have recently undergone a sea change. In 2014, the American College of Obstetricians and Gynecologists and the Society for vIaternal-Fetal Medicine issued their irst Obstetric Care Consensus titled Sae Prevention of the Primary Cesarean Delivery. It was reairmed in 2016. his consensus statement was a response to concerns that cesarean delivery was overused in the United States. Namely, approximately one in three women who give birth each year undergoes this surgery (Fig. 31-1, p. 592). New recommendations from the Consensus Committee are based on TABLE 23-2. Abnormal Labor Patterns, Diagnostic Criteria, and Methods of Treatment Arrest Disorders Prolonged deceleration phase Secondary arrest of dilation Arrest of descent Failure of descent CPo = cephalopelvic disproportion. Modified from Cohen, 1983. <l.2scm/hr 1.5 cm/hr Expectant and Cesarean delivery for CPD >2 hr >2 hr Evaluate for cpo: > 1 hr > 1 hr CPO: cesarean

1	CPo = cephalopelvic disproportion. Modified from Cohen, 1983. <l.2scm/hr 1.5 cm/hr Expectant and Cesarean delivery for CPD >2 hr >2 hr Evaluate for cpo: > 1 hr > 1 hr CPO: cesarean No descent in deceleration No CPD: oxytocin "more recent data used to revise the definition of contemporary normal labor progress" and reflect a signiicant revision of the preexisting understanding of abnormal labor.

1	Active-Phase Protraction. Of active-phase disorders, protraction disorders are less well described, and the time necessary before diagnosing slow progress is undefined. he Wodd Health Organization (1994) has proposed a labor management partograph in which protraction is defined as < 1 cm/ hr cervical dilation for a minimum of 4 hours. hese criteria were adapted from those of Cohen and Friedman (1983) and shown in Table 23-2. For this disorder, observation for further progress is appropriate treatment. If insuicient Montevideo units are noted, oxytocin augmentation is initiated. According to the Consensus Committee (2016), slow but progressive irststage labor should not be an indication for cesarean delivery.

1	Active-Phase Arrest. Handa and Laros (1993) diagnosed active-phase arrest, deined as no dilation for 2 hours or more, in 5 percent of term nulliparas. This incidence has not changed since the 1950s (Friedman, 1978). Inadequate uterine contractions, deined as less than 180 Montevideo units, calculated as shown in Figure 23-1, were diagnosed in 80 percent of women with active-phase arrest. Hauth and coworkers (1986, 1991) FIGURE 23-' Montevideo units are calculated by subtracting the baseline uterine pressure from the peak contraction pressure for each contraction in a 1 O-minute window and adding the pressures generated by each contraction. In the example shown, there were five contractions, producing pressure changes of 52,50,47,44, and 49 mm Hg, respectively. The sum of these five contractions is 242 Montevideo Final study sample n 62,41e5

1	Final study sample n 62,41e5 FIGURE 23-2 Study cohort for the analysis of spontaneous labor in the Consortium on Safe Labor study. NICU = neonatal intensive care unit. (Data from Zhang, 2010.) reported that when labor is efectively induced or augmented with oxytocin, 90 percent of women achieve 200 to 225 Montevideo units, and 40 percent achieve at least 300 Montevideo units. hese results suggest that certain minimums of uterine activity should be achieved before performing cesarean delivery for dystocia.

1	Other criteria should also be met. First, the latent phase should be completed, and the cervix is dilated ::4 cm. Also, a uterine contraction pattern of 200 Montevideo units or more in a 10-minute period has been present for 2 or more hours without cervical change. Rouse and associates (1999) challenged the "2-hour rule" on the grounds that a longer time, that is, at least 4 hours, is necessary before concluding that the active phase of labor has failed. We agree. The Consensus Committee (2016) has expanded the criteria, as described next. Obstetric Care Consensus Committee. There are four recom mendations by the Consensus Committee (2016) applicable to management of the irst-stage labor. The irst admonishes against cesarean delivery in the latent phase of labor. Speciically, a prolonged latent phase is not an indication for cesarean delivery. This guideline is not new and is traceable to the work of Friedman (1954), on which traditional tenets are based.

1	The second directive, too, is conventional practice. It recommends against cesarean delivery if labor is progressive but slow-aprotraction disorder. This instance is typically managed with observation, assessment of uterine activity, and stimulation of contractions as needed. A third instruction addresses the cervical dilation threshold that serves to herald active labor. Namely, a cervical dilation of 6 cm-not 4 cm-is now the recommended threshold. Thus, before this threshold, standards for active-phase progress should not be applied. A fourth stipulation notes that cesarean delivery for activephase arrest "should be reserved for women at or beyond 6 cm of dilation with ruptured membranes who fail to progress despite 4 hours of adequate uterine activity, or at least 6 hours of oxytocin administration with inadequate contractions and no cervical change."

1	According to the Consensus Committee (2016), the "6-cm rule" stems from labor data from the Consortium on Safe Labor study (Zhang, 2010). This study derived its numbers from a retrospective observational dataset built from abstracted labor and delivery information from 19 hospitals across the United States. Various statistical methods and heavy manipulations of these numbers were used (Cohen, 2015b). As shown in Figure 23-2, a total of 62,415 women were analyzed after excluding all women with cesarean deliveries or asphyxiated newborns. The Consensus Committee (2016) was explicit that "the Consortium on Safe Labor data, rather than the standards proposed by Friedman should inform evidence-based labor management." Described in Chapter 21 (p. 432), the latter are labor curves that have been used since irst proposed by Friedman (1955).

1	Critics of the Consensus Committee (2016) recommendations note that the Consortium on Safe Labor data were derived from clinical settings with a net cesarean rate of 30 percent. hus, adherence to the new recommendations may fail to achieve desired cesarean rate reductions. Also, the study lacked a focus on neonatal safety, given that all the asphyxiated neonates were excluded. Supporters note that the study of prolonged first-stage labors by Cheng and coworkers (2010) found higher rates of cesarean delivery and chorioamnionitis but not higher rates of neonatal morbidity. However, Harper and associates (2014) analyzed maternal and neonatal adverse outcomes related to first-stage labor lengths. In 5030 women, first-stage labor durations were divided into those <90th percentile or those ::90th percentile, with incremental increases thereafter. These authors concluded that a longer irst-stage labor was associated with maternal and neonatal complications and that these should be balanced

1	percentile, with incremental increases thereafter. These authors concluded that a longer irst-stage labor was associated with maternal and neonatal complications and that these should be balanced against the risks of cesarean delivery. his concern for adverse fetal and maternal efects resulting from the new Consensus Committee guidelines was echoed by Cohen and Friedman (2015a,b).

1	Spontaneous onset of labor, Singletons, cephalic n = 98,169 Excluded: 23,280 cesarean births Vaginal delivery n = 74,889 women TABLE 23-3. Comparison of Study Populations Analyzed to Define Normal Labor Curves Year of data collection Early 1950s 1992-1996 Caudal/epidural analgesia (%) 8 48 Oxytocin augmentation (%) 9 50 aTripler Army Hospital. Data from Friedman, 1955; Zhang, 2002. TABLE 23-4. Rate of Change at Each Stage of Cervical Dilation Cervical Dilation Rate of Cervical Dilation 2 0.3 (0.1, 1.8) 3 0.4 (0.1,s1.8) 4 0.6 (0.2, 1 .8) 5 1.2 (0.3, 5.0) 6 1.7 (0.5, 6.3) 7 2.2 (0.7, 7.1) 8 2.4 (0.8, 7.7) 9 2.4 (0.7, 8.3) aMedian (5th and 95th percentiles). Data from Zhang, 2002.

1	Another caveat notes that the eicacy of these recommendations to achieve their primary goal is limited. In support, one retrospective cohort study of 200 women undergoing induction or augmentation before and 200 ater guideline changes found a cesarean delivery rate drop from 35 to 25 percent (WilsonLeedy, 2016). However, although the study was not powered to assess adverse neonatal outcomes, nonsigniicant but higher rates of umbilical artery gases with pH <7 and base deficit > 12 mmollL were noted in the postguideline group (Marte, 2016). In another evaluation of outcomes before and ater guideline implementation, cesarean delivery rates were unchanged. Overall, there were no associations between length of arrest and maternal or neonatal morbidity, but neonatal respiratory morbidity rates rose in newborns of women with longer periods of arrested dilation (Rosenbloom, 2017). Thus, additional studies are needed to define risks and beneits of the new guidelines.

1	Background for the 6-cm Rule. Review of selected publications helps explain the Consensus Committee (2016) evolution to a 6-cm rule. First, Zhang and coworkers (2002) compared study populations from one report by Friedman (1955) with one from 1992 to 1996 at TripIer Army Hospital, Hawaii (Table 23-3). The Friedman labor curves relected women in spontaneous labor with infrequent use of neuraxial labor analgesia or �: 6.2 0).;; Duration of labor (hours)

1	Duration of labor (hours) FIGURE 23-3 Cervical dilation curves. (From Friedman (1955) and Zhang (2002).) oxytocin augmentation. In contrast, in the TripIer cohort, approximately 50 percent of women had neuraxial analgesia or augmentation. The rate of cervical change for the ensuing hour at each cervical dilation between 2 and 9 cm in the T ripIer group is shown in Table 23-4. Progress is slow between 4 and 6 cm but accelerates thereafter. his could reasonably be interpreted as the active phase beginning at 6 cm. Shown in Figure 23-3 are the labor curves for Friedman (1955) compared to the TripIer cohort. These difer in a flattening of the active phase beginning at 3 to 4 cm in the TripIer group. his is consistent with the labor results obtained in the Safe Labor Consortium study (Zhang, 2010). Namely, the 6-cm rule for active labor derives from a slowing of the rate or lattening of the slope of cervical change in irst-stage labor.

1	N euraxial analgesia delays the active phase of spontaneous labor and flattens the slope. For example, Gambling and associates (1998) compared combined spinal epidural (CSE) labor analgesia against intermittent intravenous (IV) boluses of 50 mg meperidine in 1223 nulliparas in spontaneous labor at term. The active phase of labor was diagnosed when cervical dilation was 4 cm in the presence of regular uterine contractions. s shown in Table 23-5, mean cervical dilation at first pain aAdministered as intermittent intravenous boluses. bMedian value. CPresented as number (%). dpresented as mean ± standard deviation. Data from Gambling, 1998. TABLE 23-6. 9000 Women Undergoing Primary Cesarean Delivery for Dystocia Percentage of Women with Noted Median (1st, 3rd 6 cm (5,s8) Augmentation to delivery time 9.3 hr (6, 13) Data from Alexander, 2003.

1	Percentage of Women with Noted Median (1st, 3rd 6 cm (5,s8) Augmentation to delivery time 9.3 hr (6, 13) Data from Alexander, 2003. relief was 5 cm in both study groups. Oxytocin augmentation rates were signiicantly greater in the CSE group. Also, the first analgesia-to-delivery interval was lengthened (5 versus 4 hours). However, cesarean delivery rates did not difer significantly. Importantly, the efect of neuraxial analgesia to slow activephase labor should not negatively afect use of neuraxial pain relie. As described in Chapter 25 (p. 495), investigators at Parkland Hospital completed five randomized trials involving a total of 2703 nulliparas in spontaneous labor at term (Sharma, 2004). Various neuraxial techniques were compared with meperidine for pain relief. First-stage labor was significantly lengthened with neuraxial analgesia (8.1 versus 6.6 hours) but cesarean delivery rates for dystocia were unafected (9.i1 versus 8.1 percent).

1	In sum, neuraxial analgesia slows the active phase of firststage labor. his currently is empirically corrected by augmentation of uterine contractions. Thus, recommendations of the Consensus Committee regarding protraction disorders are correct. Second, the Consensus Committee (2016) suggests that cesarean deliveries for dystocia are being done before 6 cm cervical dilation. However, there is good reason to believe that all of the Committee's first-stage recommendations are actually already empirically in use but are concurrent with an overall cesarean delivery rate in excess of 30 percent. For example, Table 23-6 lists labor characteristics in 9000 women with primary cesarean deliveries for dystocia at 13 university hospitals between 1999 and 2000 (Alexander, 2003). Notably, the median cervical dilation at the time ofcesarean for dystocia was 6 cm. Moreover, Figure 23-4 depicts primary cesarean delivery rates for dystocia at Parkland Hospital between 1988 and 2017. The rate has not

1	cervical dilation at the time ofcesarean for dystocia was 6 cm. Moreover, Figure 23-4 depicts primary cesarean delivery rates for dystocia at Parkland Hospital between 1988 and 2017. The rate has not changed significantly in 28 years. Thus, Consensus Committee (2016) guidelines may fail to prevent additional cesareans for dystocia. Again, additional studies are needed.

1	Fetal descent largely follows complete dilation. Moreover, the second stage incorporates many of the cardinal movements necessary for the fetus to negotiate the birth canal (Chap. 22, p. 428). Accordingly, disproportion ofthe fetus and pelvis frequently becomes apparent during second-stage labor. Similar to irst-stage labor, time boundaries have been supported to limit second-stage duration to minimize adverse maternal and fetal outcomes. he second stage in nulliparas is limited to 2 hours and extended to 3 hours when regional analgesia is used. For multiparas, 1 hour is the limit, extended to 2 hours with regional analgesia.

1	Cohen (1977) investigated the fetal efects of second-stage labor length at Beth Israel Hospital. He included 4403 term nulliparas in whom electronic fetal heart rate monitoring was performed. he neonatal mortality rate was not increased in women whose second-stage labor exceeded 2 hours. Epidural analgesiawas used commonly, and this likely accounted for the large number of pregnancies with a prolonged second stage. These data inluenced decisions to permit an additional hour for the second stage when regional analgesia is used. Menticoglou and coworkers (1995a,b) also challenged the prevailing dicta on second-stage duration. Their concern stemmed from grave neonatal injuries associated with forceps rotations to shorten second-stage labor. As a result, they allowed a longer second stage to decrease the operative vaginal delivery rate. Between 1988 and 1992, second-stage labor exceeded 2 hours in a fourth of 6041 nulliparas at term. Labor epidural 4 0)) 3.�i0C)E 20:0E)0). 0

1	Year of delivery FIGURE 23-4 Primary caesarean delivery rates for dystocia in lowrisk women at term at Parkland Hospital from 1988 to 2017. analgesia was used in 55 percent. he length of the second stage, even in those lasting up to 6 hours or more, was not related to neonatal outcome. hese results were attributed to careful use of electronic monitoring and scalp pH measurements. hese investigators concluded that there is no compelling reason to intervene with a possibly diicult forceps or vacuum extraction because a certain number of hours have elapsed. hey observed, however, that after 3 hours in the second stage, delivery by cesarean or other operative method increased progressively. By 5 hours, the prospects for spontaneous delivery in the subse quent hour were only 10 to 15 percent.

1	Newer guidelines have been promoted by the Consensus Com mittee (2016) for second-stage labor. These recommend allowing a nullipara to push for at least 3 hours and a multipara to push for at least 2 hours before second-stage labor arrest is diagnosed. One caveat is that maternal and fetal status should be reassuring. hese authors provide options to these times before cesarean deliv ery is performed. Namely, longer durations may be appropriate as long as progress is documented. Also, a speciic maximal length of undergo operative delivery has not been identiied.

1	Intuitively, the goal to lower cesarean delivery rates is best balanced with one to ensure neonatal safety. And, it is problematic that no robust data on neonatal outcomes support the safety of allowing prolonged second-stage labor. Data from many evaluations reveal that serious newborn consequences attend second-stage labors longer than 3 hours (Allen, 2009; Bleich, 2012; Laughon, 2014; Leveno, 2016; Rosenbloom, 2017). Other data, when adjusted for labor variables, show no diference in neonatal complications for these longer second stages (Cheng, 2004; Le Ray, 2009; Rouse, 2009). Grobman and colleagues (2016) have argued that the absolute number of such adverse outcomes is small and "overall outcomes remain good." hat said, some of the complications are severe. Thus, to fully ascertain specific efect of these guidelines on morbidity rates, randomized controlled trials are needed.

1	It is possible that prolonged first-stage labor presages that with the second stage. Nelson and associates (20l3) studied the relationships between the lengths of the irst and second stages of labor in 12,523 nulliparas at term delivered at Parkland Hospital. The second stage signiicantly lengthened concomitantly with increasing irst-stage duration. The 95th percentile was 15.6 and 2.9 hours for the first and second stages, respectively. Women with first stages lasting longer than 15.6 hours (>95th percentile) had a 16-percent rate of second-stage labor lasting 3 hours (95th percentile). his compared with a 4.5-percent rate of prolonged second stages in women with irst-stage labors lasting <95th percentile.

1	With ull cervical dilation, most women cannot resist the urge to "bear down" or "push" each time the uterus contracts (Chap. 22, p. 438). he combined force created by contractions of the uterus and abdominal musculature propels the fetus downward. At times, force created by abdominal musculature is compromised suiciently to slow or even prevent spontaneous vaginal delivery. Heavy sedation or regional analgesia may reduce the reflex urge to push and may impair the ability to contract abdominal muscles efectively. In other instances, the inherent urge to push is overridden by the intense pain created by bearing down. Two approaches to second-stage pushing in women with epidural analgesia have yielded contradictory results. The irst advocates pushing forcefully with contractions ater complete dilation, regardless of the urge to push. With the second, analgesia inusion is stopped and pushing begins only ater the woman regains the sensory urge to bear down. Fraser and coworkers (2000)

1	dilation, regardless of the urge to push. With the second, analgesia inusion is stopped and pushing begins only ater the woman regains the sensory urge to bear down. Fraser and coworkers (2000) found that delayed pushing reduced diicult operative deliveries, whereas Manyonda and associates (1990) reported the opposite. Hansen and colleagues (2002) randomly assigned 252 women with epidural analgesia to one of the two approaches. No adverse maternal or neonatal outcomes were linked to delayed pushing despite signiicantly prolonging second-stage labor. Plunkett and coworkers (2003), in a similar study, confirmed these indings.

1	Descent of the leading edge of the presenting part to the level of the ischial spines (0 station) is deined as engagement. A higher station at the onset of labor is signiicantly linked with subsequent dystocia (Friedman, 1965, 1976; Handa, 1993). Roshanfekr and associates (1999) analyzed fetal station in 803 nulliparas at term in active labor. At admission, the third with the fetal head at or below 0 station had a 5-percent cesarean delivery rate. This compared with a 14-percent rate for those with higher stations. The prognosis for dystocia, however, was not related to incrementally higher fetal head stations above the pelvic midplane (0 station). Importantly, 86 percent of nulliparous women without fetal head engagement at diagnosis of active labor delivered vaginally. hese observations apply especially for parous women because the head typically descends later in labor.

1	Various labor factors have been implicated as causes of uterine dysfunction. As described, neuraxial analgesia can slow labor and has been associated with lengthening both first and second stages of labor and slowing the rate of fetal descent.

1	Chorioamnionitis is associated with prolonged labor, and some clinicians have suggested that this maternal intrapartum infection itself contributes to abnormal uterine activity. Satin and coworkers (1992) studied the efects of chorioamnionitis on oxytocin stimulation in 266 pregnancies. Infection diagnosed late in labor was found to be a marker of cesarean delivery performed for dystocia. Specifically, 40 percent of women developing chorioamnionitis after requiring oxytocin for dysfunctional labor later required cesarean delivery for dystocia. However, this was not a marker in women diagnosed as having chorioamnionitis early in labor. It is likely that uterine infection in this clinical setting is a consequence of dysfunctional, prolonged labor rather than a cause of dystocia.

1	Membrane rupture at term without spontaneous uterine contractions complicates approximately 8 percent of pregnancies. In the past, labor stimulation was initiated if contractions did not begin ater 6 to 12 hours. Practice-changing research included that of Hannah (1996) and Peleg (1999) and their associates, who enrolled a total of 5042 pregnancies with ruptured membranes in a randomized investigation. hey measured the efects of induction versus expectant management and also compared induction using intravenous oxytocin with that using prostaglandin E2 gel. There were approximately 1200 pregnancies in each of the four study arms. They concluded that labor induction with intravenous oxtocin was preferred management. This was based on signiicantly fewer intrapartum and postpartum infections in women whose labor was induced. There were no significant differences in cesarean delivery rates. Subsequent analysis by Hannah and coworkers (2000) indicated higher rates of adverse outcomes when

1	in women whose labor was induced. There were no significant differences in cesarean delivery rates. Subsequent analysis by Hannah and coworkers (2000) indicated higher rates of adverse outcomes when expectant management at home was compared with in-hospital observation. Mozurkewich and associates (2009) reported lower rates of chorioamnionitis, metritis, and neonatal intensive care unit admissions for women with term ruptured membranes whose labors were induced compared with those managed expectantly. At Parkland Hospital, labor is induced soon ater admission when ruptured membranes are conirmed at term. In those with hypotonic contractions or with advanced cervical dilation, oxytocin is selected to lower potential hyperstimulation risk. In those with an unfavorable cervix and no or few contraction, prostaglandin E1 (misoprostol) is chosen to promote cervical ripening and contractions. he benefit of prophylactic antibiotics in women with ruptured membranes before labor at term is

1	contraction, prostaglandin E1 (misoprostol) is chosen to promote cervical ripening and contractions. he benefit of prophylactic antibiotics in women with ruptured membranes before labor at term is unclear (Passos, 2012). However, in those with membranes ruptured longer than 18 hours, antibiotics are instituted for group B streptococcal infection prophylaxis (Chap. 64, p. 1221).

1	Labor can be too slow, but it also can be abnormally rapid. Precipitous labor and delivery is extremely rapid labor and delivery. It may result from an abnormally low resistance of the soft parts of the birth canal, from abnormally strong uterine and abdominal contractions, or rarely from the absence of painful sensations and thus a lack of awareness of vigorous labor. Precipitous labor terminates in expulsion of the fetus in less than 3 hours. Using this deinition, 25,260 live births-3 percent-were complicated by precipitous labor in the United States in 2013 (Martin, 2015). Despite this incidence, little published information describes maternal and perinatal outcomes.

1	For the mother, precipitous labor and delivery seldom are accompanied by serious maternal complications if the cervix is efaced appreciably and compliant, if the vagina has been stretched previously, and if the perineum is relaxed. Conversely, vigorous uterine contractions combined with a long, irm cervix and a noncompliant birth canal may lead to uterine rupture or extensive lacerations of the cervix, vagina, vulva, or perineum (Sheiner, 2004). It is in these latter circumstances that amnionic-luid embolism most likely develops (Chap. 41, p. 785). Precipitous labor is frequently followed by uterine atony. he uterus that contracts with unusual vigor bore delivery is likey to be hypotonic ater delivey. In one report of 99 term pregnancies, short labors were more common in multiparas who typically had contractions at intervals less than 2 minutes. Precipitous labors have been linked to cocaine abuse and associated with placental abruption, meconium, postpartum hemorrhage, and low Apgar

1	had contractions at intervals less than 2 minutes. Precipitous labors have been linked to cocaine abuse and associated with placental abruption, meconium, postpartum hemorrhage, and low Apgar scores (Mahon, 1994).

1	For the neonate, adverse perinatal outcomes from rapid labor may be increased considerably for several reasons. The tumultuous uterine contractions, often with negligible intervals of relaxation, prevent appropriate uterine blood flow and fetal oxygenation. Resistance of the birth canal may rarely cause intracranial trauma. Acker and coworkers (1988) reported that Erb or Duchenne brachial palsy was associated with such labors in a third of cases. Finally, during an unattended birth, the newborn may fall to the loor and be injured, or it may need resuscitation that is not immediately available.

1	As treatment, analgesia is unlikely to modiy these unusually forceful contractions to a signiicant degree. The use of tocolytic agents such as magnesium sulfate or terbutaline is unproven in these circumstances. Use of general anesthesia with agents that impair uterine contractibility such as isoflurane is often excessively heroic. Certainly, any oxytocin being administered should be stopped immediately. Fetopelvic disproportion arises from diminished pelvic capacity, from abnormal fetal size or presentation, or more usually from both. The pelvic inlet, midpelvis, or pelvic outlet may be contracted solely or in combination. Any contraction of the pelvic diameters that diminishes pelvic capacity can create dystocia during labor. Normal pelvic dimensions are additionally discussed and illustrated in Chapter 2 (p. 30).

1	Using clinical measures, it is important to identiy the shortest anteroposterior diameter through which the fetal head must pass. Before labor, the fetal biparietal diameter averages from 9.5 to as much as 9.8 cm. Therefore, it might prove diicult or even impossible for some fetuses to pass through a pelvic inlet that has an anteroposterior diameter < 10 cm. Mengert (1948) and Kaltreider (1952), employing x-ray pelvimetry, demonstrated that the incidence of diicult deliveries rises when either the anteroposterior diameter of the inlet is < 10 cm or the transverse diameter is < 12 cm. As expected, when both diameters are contracted, dystocia rates are much greater than when only one is contracted. Either of these measures is used to consider a pelvis contracted.

1	The anteroposterior diameter of the inlet, which is the obstetrical conjugate, is commonly approximated by manually measuring the diagonal conjugate, which is approximately 1.5 cm greater. Ascertainment of these measures is described in Chapter 2 (p. 30). Therefore, inlet contraction usually is defined as a diagonal conjugate < 11.5 cm. A small woman is likely to have a small pelvis, but she is also likely to have a small neonate. Thoms (1937) studied 362 nulliparas and found that the mean birth weight of their ofspring was significantly lower-280 g-in women with a small pelvis than in those with a medium or large pelvis.

1	Normally, cervical dilation is aided by hydrostatic action of the unruptured membranes or after their rupture, by direct application of the presenting part against the cervix. In con tracted pelves, however, because the head is arrested in the pelvic inlet, the entire force exerted by the uterus acts directly on the portion of membranes that contact the dilating cervix. Consequently, early spontaneous rupture of the membranes is more likely.

1	Consequently, early spontaneous rupture of the membranes is more likely. After membrane rupture, absent pressure by the head against the cervix and lower uterine segment predisposes to less efec tive contractions. Hence, further dilation may proceed very slowly or not at all. Cibils and Hendricks (1965) reported that the mechanical adaptation of the fetal passenger to the bony passage plays an important part in determining the eiciency of contractions. The better the adaptation, the more eicient the contractions. hus, cervical response to labor provides a prog nostic view of labor outcome in women with inlet contraction.

1	A contracted inlet also plays an important part in the pro duction of abnormal presentations. In nulliparas with normal pelvic capacity, the presenting part at term commonly descends into the pelvic cavity before labor onset. When the inlet is con tracted considerably or there is marked asynclitism, descent usually does not take place until after labor onset, if at all. Cephalic presentations still predominate, but the head loats freely over the pelvic inlet or rests more laterally in one of the iliac fossae. Accordingly, very slight influences may cause the fetus to assume other presentations. In women with contracted pelves, face and shoulder presentations are encountered three times more frequently, and the cord prolapses four to six times more often. his inding is more common than inlet contraction. It frequently causes transverse arrest of the fetal head, which potentially can lead to a diicult midforceps operation or to cesarean delivery.

1	his inding is more common than inlet contraction. It frequently causes transverse arrest of the fetal head, which potentially can lead to a diicult midforceps operation or to cesarean delivery. The obstetrical plane of the midpelvis extends from the inferior margin of the symphysis pubis through the ischial spines and touches the sacrum near the junction of the fourth and ifth vertebrae. A transverse line theoretically connecting the ischial spines divides the midpelvis into anterior and posterior portions (Fig. 2-16, p. 30). he former is bounded anteriorly by the lower border of the symphysis pubis and laterally by the ischiopubic rami. The posterior portion is bounded dorsally by the sacrum and laterally by the sacrospinous ligaments, forming the lower limits of the sacrosciatic notch.

1	Average midpelvis measurements are as follows: transverse, or interischial spinous, 10.5 cm; anteroposterior, from the lower border of the symphysis pubis to the junction of 54-5, 11.5 cm; and posterior sagittal, from the midpoint of the interspinous line to the same point on the sacrum, 5 cm. he deinition of midpelvic contractions has not been established with the same precision possible for inlet contractions. Even so, the midpelvis is likely contracted when the sum of the interspinous and posterior sagittal diameters of the midpelvis-normally, 10.5 plus 5 cm, or 15.5 cm-falls to 13.5 cm or less. his concept was emphasized by Chen and Huang (1982) in evaluating possible midpelvic contraction. Nlidpelvic contraction is suspected whenever the interspinous diameter is < 10 cm. When it mea sures < 8 cm, the midpelvis is contracted.

1	Although no precise manual method permits measure of midpelvic dimensions, a suggestion of contraction sometimes can be inferred if the spines are prominent, the pelvic sidewalls converge, or the sacrosciatic notch is narrow. Moreover, Eller and Mengert (1947) noted that the relationship between the intertuberous and interspinous diameters of the ischium is suf ficiently constant that narrowing of the interspinous diameter can be anticipated when the intertuberous diameter is narrow. A normal intertuberous diameter, however, does not always exclude a narrow interspinous diameter.

1	A normal intertuberous diameter, however, does not always exclude a narrow interspinous diameter. This inding usually is defined as an interischial tuberous diam eter of 8 cm or less. The pelvic outlet may be roughly likened to two triangles, with the interischial tuberous diameter con stituting the base of both. The sides of the anterior triangle are the pubic rami, and its apex is the inferoposterior surface of the symphysis pubis. The posterior triangle has no bony sides but is limited at its apex by the tip of the last sacral vertebra-not the tip of the coccyx. Diminution of the intertuberous diam eter with consequent narrowing of the anterior triangle must inevitably force the fetal head posteriorly. Floberg and asso almost 1 percent of more than 1400 unselected nulliparas with term pregnancies. A contracted outlet may cause dystocia not tion. Oulet contraction without concomitant midplane contrac tion is rare.

1	Although the disproportion between the fetal head and the pelvic outlet is not suiciently great to give rise to severe dystocia, it may play an important part in perineal tears. With increased narrowing of the pubic arch, the occiput cannot emerge directly beneath the symphysis pubis but is forced farther down upon the ischiopubic rami. The perineum, consequently, becomes increasingly distended and thus exposed to risk of laceration. Vallier (2012) reviewed experiences with pelvic fractures and pregnancy. Trauma from automobile collisions was the most common cause. Moreover, they note that fracture pattern, minor malalignment, and retained hardware are not absolute indications for cesarean delivery. In determining suitability for vaginal delivery, fracture healing requires 8 to 12 weeks and thus recent fracture merits cesarean delivery (Amorosa, 2013). A history of pelvic fracture warrants careful review of previous radiographs and possible imaging pelvimetry later in pregnancy.

1	The techniques for clinical evaluation using digital examination of the bony pelvis during labor are described in detail in Chapter 2 (p. 30). The value of radiological imaging to assess pelvic capacity has also been examined. First, with x-ray pelvimetry alone, the prognosis for successful vaginal delivery in any given pregnancy with cephalic presentation cannot be established (Mengert, 1948). Similarly, one systematic review found insufficient evidence to support the use of x-ray pelvimetry with cephalic presentations (Pattinson, 2017). Advantages of pelvimetry with computed tomography (CT) compared with those of conventional x-ray pelvimetry include greater accuracy and easier performance. With either method, costs are comparable, and x-ray exposure is small (Chap. 46, p. 906). Depending on the machine and technique employed, fetal doses with CT pelvimetry may range from 250 to 1500 mrad (Moore, 1989).

1	Advantages of magnetic resonance (MR) pelvimetry include lack of ionizing radiation, accurate measurements, complete fetal imaging, and the potential for evaluating soft tissue dystocia (McCarthy, 1986; Stark, 1985). Zaretsky and colleagues (2005) used l\1R imaging to measure pelvic and fetal head volume to identiy those women at greatest risk of undergoing cesarean delivery for dystocia. Signiicant associations were found between some of the measures and cesarean delivery for dystocia. However, these researchers could not with accuracy predict which individual woman would require cesarean delivery. Others have reported similar indings (Sporri, 1997).

1	Fetal size alone is seldom a suitable explanation for failed labor. Even with current technology, a fetal size threshold to predict fetopelvic disproportion is still elusive. Most cases of disproportion arise in fetuses whose weight is well within the range of the general obstetrical population. As shown in Figure 23-5, two thirds of neonates who required cesarean delivery after failed forceps delivery weighed <3700 g. hus, other factorsfor example, malposition of the head-obstruct fetal passage through the birth canal. hese include asynclitism, occiput posterior position, and face or brow presentation. For fetal head size estimation, clinical and radiographical methods to predict fetopelvic disproportion have proved �0 �O >� � x:5•x3 xO FIGURE 23-5 Birthweight distribution of 362 newborns born by cesarean delivery after a failed forceps attempt at Parkland Hospital from 1989-1•999. Only 12 percent (n 44) of the newborns weighed >4000 g (dark bars).

1	disappointing. Mueller (1885) and Hillis (1930) described a clinical maneuver to predict disproportion. The fetal brow and the suboccipital region are grasped through the abdominal wall with the fingers, and firm pressure is directed downward in the axis of the inlet. If no disproportion exists, the head readily enters the pelvis, and vaginal delivery can be predicted. Thorp and coworkers (1993) performed a prospective evaluation of this Mueler-Hillis maneuver. They found no relationship between failed descent during the maneuver and subsequent labor dystocia.

1	Measurements of fetal head diameters using plain radiographical techniques are not used because of parallax distortions. he biparietal diameter and head circumference can be measured sonographically, and investigators have attempted to use this information in the management of dystocia. hurnau and colleagues (1991) used the etal-pelvic index to identiy labor complications. Unfortunately, the sensitivity of such measurements to predict cephalopelvic disproportion is poor (Ferguson, 1998; Korhonen, 2015). We believe that no current method of measurement satisfactorily predicts fetopelvic disproportion based on head size.

1	With this presentation, the neck is hyperextended so that the occiput is in contact with the fetal back, and the chin (mentum) is presenting (Fig. 23-6). The fetal face may present with the chin (mentum) anteriorly or posteriorly, relative to the maternal symphysis pubis (Chap. 22, p. 425). Although some mentum posterior presentations persist, most convert spontaneously to anterior even in late labor (Duf, 1981). If not, the fetal brow (bregma) is pressed against the maternal symphysis pubis. his position precludes lexion of the fetal head necessary to negotiate the birth canal. hus, a mentum posterior presentation is undeliverable except with a very preterm fetus. FIGURE 23-6 Face presentation. The occiput is the longer end of the head lever. The chin is directly posterior. Vaginal delivery is impossible unless the chin rotates anteriorly. 450FIGURE 23-7 Mechanism of labor for right mentoposterior position with subsequent rotation of the mentum anteriorly and delivery.

1	450FIGURE 23-7 Mechanism of labor for right mentoposterior position with subsequent rotation of the mentum anteriorly and delivery. Face presentation is diagnosed by vaginal examination and palpation of facial features. A breech may be mistaken for a face presentation. Namely, the anus may be mistaken for the mouth, and the ischial tuberosities for the malar prominences. Digital diferentiation is described in Chapter 28 (p. 540). Radiographically, demonstration of the hyperextended head with the facial bones at or below the pelvic inlet is characteristic. Cruikshank and White (1973) reported an incidence of 1 in 600, or 0.17 percent. As shown in Table 22-1 (p. 422), among more than 70,000 singleton newborns delivered at Parkland Hospital, approximately 1 in 2000 had a face presentation at delivery.

1	Causes of face presentations are numerous and include conditions that favor extension or prevent head lexion. Preterm fetuses, with their smaller head dimensions, can engage before conversion to vertex position (Shafer, 2006). In exceptional instances, marked enlargement of the neck or coils of cord around the neck may cause extension. Bashiri and associates (2008) reported that fetal malformations and hydramnios were risk factors for face or brow presentations. Anencephalic fetuses naturally present by the face. Extended neck positions develop more frequently when the pelvis is contracted or the fetus is very large. In a series of 141 face presentations studied by Hellman and coworkers (1950), the incidence of inlet contraction was 40 percent. his high incidence of pelvic contraction should be kept in mind when considering management.

1	High parity is a predisposing factor for face presentation (Fuchs, 1985). In these cases, a pendulous abdomen permits the back of the fetus to sag forward or laterally, often in the same direction in which the occiput points. his promotes extension of the cervical and thoracic spine. Mechanism of Labor

1	Mechanism of Labor Face presentations rarely are observed above the pelvic inlet. Instead, the brow generally presents early and is usually converted to present the face after further extension of the neck during descent. he mechanism of labor in these cases consists of the cardinal movements of descent, internal rotation, and flexion, and the accessory movements of extension and external rotation (Fig. 23-7). Descent is brought about by the same factors as in cephalic presentations. Extension results from the relation of the fetal body to the delected head, which is converted into a two-armed lever, the longer arm of which extends from the occipital condyles to the occiput. When resistance is encountered, the occiput must be pushed toward the back of the fetus while the chin descends.

1	The objective of internal rotation of the face is to bring the chin under the symphysis pubis. Only in this way can the neck traverse the posterior surface of the symphysis pubis. If the chin rotates directly posteriorly, the relatively short neck cannot span the anterior surface of the sacrum, which measures about 12 cm in length. Moreover, the fetal brow (bregma) is pressed against the maternal symphysis pubis. This position precludes lexion necessary to negotiate the birth canal. Hence, as discussed earlier, birth of the head from a mentum posterior position is impossible unless the shoulders enter the pelvis at the same time, an event that is impossible except when the fetus is extremely small or macerated. Internal rotation results from the same factors as in vertex presentations.

1	After anterior rotation and descent, the chin and mouth appear at the vulva, the undersurface of the chin presses against the symphysis, and the head is delivered by lexion. The nose, eyes, brow (bregma), and occiput then appear in succession over the anterior margin of the perineum. ter birth of the head, the occiput sags backward toward the anus. Next, the chin rotates externally to the side toward which it was originally directed, and the shoulders are born as in cephalic presentations. Edema may sometimes significantly distort the face. At the same time, the skull undergoes considerable molding, manifested by an increase in length of the occipitomental diameter of the head.

1	Edema may sometimes significantly distort the face. At the same time, the skull undergoes considerable molding, manifested by an increase in length of the occipitomental diameter of the head. In the absence of a contracted pelvis and with efective labor, successful vaginal delivery usually will follow. Fetal heart rate monitoring is probably better done with external devices to avoid damage to the face and eyes. Because face presentations among term-size fetuses are more common when there is some degree of pelvic inlet contraction, cesarean delivery frequently is indicated. Attempts to convert a face presentation manually into a vertex presentation, manual or forceps rotation of a persistently posterior chin to a mentum anterior position, and internal podalic version and extraction are dangerous and should not be attempted. Low or outlet forceps delivery of a mentum anterior face presentation can be completed and is described in Chapter 29 (p. 562).

1	This rare presentation is diagnosed when that portion of the fetal head between the orbital ridge and the anterior fontanel presents at the pelvic inlet. As shown in Figure 23-8, the fetal head thus occupies a position midway between full lexion (occiput) and extension (face). Except when the fetal head is small or the pelvis is unusually large, engagement of the fetal head and subsequent delivery cannot take place as long as the brow presentation persists. The causes of persistent brow presentation are the same as those for face presentation. A brow presentation is commonly unstable and often converts to a face or an occiput presentation (Cruikshank, 1973). The presentation may be recognized by abdominal palpation when both the occiput and chin can be palpated easily, but vaginal examination is usually necessary. The frontal sutures, large anterior fontanel, orbital ridges, eyes, and root of the nose are felt on vaginal examination, but neither the mouth nor the chin is palpable.

1	FIGURE 23-8 Brow posterior presentation. With a very small fetus and a large pelvis, labor is generally easy. With a larger fetus, it is usually diicult. his is because engagement is impossible until there is marked molding that shortens the occipitomental diameter or more commonly, until the neck either lexes to an occiput presentation or extends to a face presentation. The considerable molding essential for vaginal delivery of a persistent brow characteristically deforms the head. he caput succedaneum is over the forehead, and it may be so extensive that identiication of the brow by palpation is impossible. In these instances, the forehead is prominent and squared, and the occipitomental diameter is diminished. In transient brow presentations, the prognosis depends on the ultimate presentation. If the brow persists, prognosis is poor for vaginal delivery unless the fetus is small or the birth canal is large. Principles of management are the same as those for a face presentation.

1	In this position, the long axis of the fetus is approximately perpendicular to that of the mother. When the long axis forms an acute angle, an oblique lie results. The latter is usually only transitory, because either a longitudinal or transverse lie commonly results when labor supervenes. For this reason, the oblique lie is called an unstable lie in Great Britain. FIGURE 23-9 Leopold maneuver performed on a woman with a fetal transverse lie, right acromiodorsoanterior position. In a transverse lie, the shoulder is usually positioned over the pelvic inlet. The head occupies one iliac fossa, and the breech the other. This creates a shoulder presentation in which the side of the mother on which the acromion rests determines the designation of the lie as right or let acromial. And because in either position the back may be directed anteriorly or posteriorly, superiorly or inferiorly, it is customary to distinguish varieties as dorsoanterior and dorsoposterior (Fig. 23-9).

1	A transverse lie is usually recognized easily, often by inspection alone. The abdomen is unusually wide, whereas the uterine fundus extends to only slightly above the umbilicus. No fetal pole is detected in the fundus, and the ballottable head is found in one iliac fossa and the breech in the other. The position of the back is readily identifiable. When the back is anterior, a hard resistance plane extends across the front of the abdomen. When it is posterior, irregular nodulations representing fetal small parts are felt through the abdominal wall. On vaginal examination, in the early stages of labor, if the side of the thorax can be reached, it may be recognized by the "gridiron" feel of the ribs. With further dilation, the scapula and the clavicle are distinguished on opposite sides of the thorax. The position of the axilla indicates the side of the mother toward which the shoulder is directed.

1	(0.3 percent) at both the Mayo Clinic and the University of Iowa Hospital (Cruikshank, 1973; Johnson, 1964). This is remarkably similar to the incidence at Parkland Hospital of approximately 1 in 335 singleton fetuses. Some of the more common causes of transverse lie include: (1) abdominal wall relaxation from high parity, (2) preterm fetus, (3) placenta previa, (4) abnormal uterine anatomy, (5) hydramnios, and (6) contracted pelvis. Women with four or more deliveries have a tenfold incidence of transverse lie compared with nulliparas. A relaxed and pendulous abdomen allows the uterus to fall forward, delecting the long axis of the fetus away from the axis of the birth canal and into an oblique or transverse position. Placenta previa and pelvic contraction act similarly. A transverse or oblique lie occasionally develops in labor from an initial longitudinal position. Mechanism of Labor

1	Mechanism of Labor Spontaneous delivery of a fully developed newborn is impossible with a persistent transverse lie. After rupture of the membranes, if labor continues, the fetal shoulder is forced into the pelvis, and the corresponding arm frequently prolapses (Fig. 23-10). After some descent, the shoulder is arrested by the margins of the pelvic inlet. As labor continues, the shoulder is impacted irmly in the upper part of the pelvis. he uterus then contracts vigorously in an unsuccessful attempt to overcome the obstacle. With time, a retraction ring rises increasingly higher and becomes more marked. With this neglected transverse lie, the uterus will eventually rupture. Even without this complication, morbidity is increased because of the frequent association

1	FIGURE 23-10 Neglected shoulder presentation. A thick cular band forming a pathological retraction ring has developed just above the thin lower uterine segment. The force generated during a uterine contraction is directed centripetally at and above the level of the pathological retraction ring. This serves to stretch further and possibly to rupture the thin lower segment below the retraction ring. with placenta previa, the increased likelihood of cord prolapse, and the necessity for major operative eforts.

1	with placenta previa, the increased likelihood of cord prolapse, and the necessity for major operative eforts. If the fetus is small-usually < 800 g-and the pelvis is large, spontaneous delivery is possible despite persistence of the abnormal lie. The fetus is compressed with the head forced against its abdomen. A portion of the thoracic wall below the shoulder thus becomes the most dependent part, appearing at the vulva. he head and thorax then pass through the pelvic cavity at the same time. he fetus, which is doubled upon itself and thus sometimes referred to as condupLicato corpore, is expelled.

1	Active labor in a woman with a transverse lie is usually an indication for cesarean delivery. Before labor or early in labor, with the membranes intact, attempts at external version are worthwhile in the absence of other complications. If the fetal head can be maneuvered by abdominal manipulation into the pelvis, it should be held there during the next several contractions in an attempt to ix the head in the pelvis. With cesarean delivery, because neither the feet nor the head of the fetus occupies the lower uterine segment, a low transverse incision into the uterus may lead to diicult fetal extraction. his is especially true of dorsoanterior presentations. herefore, a vertical hysterotomy incision is often indicated .

1	With this, an extremity prolapses alongside the presenting part, and both present simultaneously in the pelvis (Fig. 23-11). Goplerud and Eastman (1953) identiied a hand or arm prolapsed alongside the head once in every 700 deliveries. Much less common was prolapse of one or both lower extremities alongside a cephalic presentation or a hand alongside a breech. At Parkland Hospital, compound presentations were identiied in only 68 of more than 70,000 singleton fetuses-an incidence of approximately 1 in 1000. Causes of compound presentations are conditions that prevent complete occlusion of the pelvic inlet by the fetal head, including preterm labor.

1	In most cases, the prolapsed part should be left alone, because most often it will not interfere with labor. If the arm is prolapsed alongside the head, the condition should be observed closely to ascertain whether the arm retracts out of the way with descent of the presenting part. If it fails to retract and if it appears to prevent descent of the head, the prolapsed arm should be pushed gently upward and the head simultaneously downward by fundal pressure. In general, rates of perinatal mortality and morbidity are increased as a result of concomitant preterm delivery, prolapsed cord, and traumatic obstetrical procedures. Serious injury to the forearm is rare (Kwok, 2015; Tebes, 1999). Dystocia, especially if labor is prolonged, is associated with a higher incidence of several common obstetrical and neonatal complications. Inection, either intrapartum chorioamnionitis or postpartum pelvic infection, is more common with desultory

1	FIGURE 23-11 Compound presentation. A. The left hand is lying in front of the vertex. With further labor, the hand and arm may retract from the birth canal, and the head may then descend normally. B. Photograph of a small 34-week fetus with a compound presentation that delivered uneventfully with the hand presenting first. (Used with permission from Dr. Elizabeth Mosier.) and prolonged labors. Pospartum hemorrhage rates from atony are increased with prolonged and augmented labors. Uterine tears with hysterotomy also occur at greater incidence if the fetal head is impacted in the pelvis.

1	Uterine rupture is another risk. Abnormal thinning of the lower uterine segment creates a serious danger during prolonged labor, particularly in women of high parity and in those with a prior cesarean delivery. When disproportion is so pronounced that there is no engagement or descent, the lower uterine segment becomes increasingly stretched, and rupture may follow. In such cases, the normal contraction ring is usually exaggerated, like that shown in Figure 23-10.

1	Such pathological retraction rings are localized constrictions of the uterus that develop in association with prolonged obstructed labors. Seldom encountered today, the pathological retraction ring o/Bandl is associated with marked stretching and thinning of the lower uterine segment. In contemporary practice, after birth of a irst twin, a pathological ring may still develop occasionally as an hourglass constriction of the uterus. he band may be seen clearly as a uterine indentation and signifies impending rupture of the lower uterine segment. he ring can sometimes be relaxed and delivery efected with appropriate general anesthesia, but for the second twin (Chap. 45, p. 890).

1	Fistula ormation may result from dystocia, as the presenting part is irmly wedged into the pelvic inlet. Tissues of the birth canal lying between the leading part and the pelvic wall may be subjected to excessive pressure. Because of impaired circula tion, necrosis can result and become evident several days after delivery as vesicovaginal, vesicocervical, or rectovaginal istulas. Most often, pressure necrosis follows a very prolonged second stage. Such fistulas are rarely seen today except in undeveloped countries.

1	Most often, pressure necrosis follows a very prolonged second stage. Such fistulas are rarely seen today except in undeveloped countries. recent attention. The pelvic floor is exposed to direct compres sion from the fetal head and to downward pressure from mater nal expulsive eforts. These forces stretch and distend the pelvic floor, resulting in functional and anatomical alterations in the muscles, nerves, and connective tissues. Accumulating evidence suggests that such efects on the pelvic floor during childbirth can afect urinary or anal continence and pelvic support. hese relationships are discussed in Chapter 30 (p. 568).

1	Lower extremiy nerve injuy in the mother can follow prolonged second-stage labor. Wong and colleagues (2003) reviewed neurological injury involving the lower extremities in association with labor and delivery. The most common mechanism is external compression of the common fibular (formerly common peroneal) nerve. This is usually caused by inappropriate leg positioning in stirrups, especially during prolonged second-stage labor. hese and other injuries are discussed in Chapter 36 (p. 661). Fortunately, symptoms resolve within 6 months of delivery in most women. Similar to the mother, the incidence of peripartum fetal sepsis rises with longer labors. Caput succedaneum and molding develop commonly and may be impressive (Fig. 22-16, p. 431) (Buchmann, 2008). Mechanical trauma such as nerve injury, fractures, and cephalohematoma are also more frequent and are discussed further in Chapter 33 (p. 627). Acker DB, Gregory KD, Sachs BP, et al: Risk factors for Erb-Duchenne palsy.

1	Acker DB, Gregory KD, Sachs BP, et al: Risk factors for Erb-Duchenne palsy. Obstet Gynecol 71:389, 1988 Alexander J: MFMU Cesarean Registry: labor characteristics of women undergo ing cesarean delivery for dystocia. Am J Obstet Gynecol 189(6):5138,2003 Allen VM, Baskett TF, O'Connell CM, et al: Maternal and perinatal outcomes with increasing duration of the second stage oflabor. Obstet Gynecol 113(6):1248,r2009 American College of Obstetricians and Gynecologists, Society for Matenal Fetal Medicine: Safe prevention of the primary cesarean delivery. Obstetric Care Consensus No.r1, March 2014, Reairmed 2016 Amorosa LF, Amorosa JH, Wellman OS, et al: Management of pelvic injuries in pregnancy. Orthop Clin North Am 44(3):301,r2013 Bashiri A, Burstein E, Bar-David J, et al: Face and brow presentation: independent risk factors. ] Matern Fetal Neonatal Med 21 (6):357, 2008

1	Bashiri A, Burstein E, Bar-David J, et al: Face and brow presentation: independent risk factors. ] Matern Fetal Neonatal Med 21 (6):357, 2008 Bleich AT, Alexander ]M, McIntire DO, et al: An analysis of second-stage labor beyond 3 hours in nulliparous women. Am] PerinatoI29:717, 2012 Buchmann E], Libhaber E: Sagittal suture overlap in cephalopelvic disproportion: blinded and non-participant assessment. Acta Obstet Gynecol Scand 87(7):731,r2008 Caldeyro-Barcia R, Alvarez H, Reynolds SR: A better understanding of uterine contractility through simultaneous recording with an internal and a seven channel external method. Surg Obstet Gynecol 91:641, 1950 Chen HY, Huang SC: Evaluation of midpelvic contraction. Int Surg 67:516, 1982 Cheng YW, Hopkins LM, Caughey AB: How long is too long: does a prolonged second stage oflabor in nulliparous women afect matenal and neonatal outcomes? Am] Obstet GynecoIr191(3):933, 2004

1	Cheng YW, Hopkins LM, Caughey AB: How long is too long: does a prolonged second stage oflabor in nulliparous women afect matenal and neonatal outcomes? Am] Obstet GynecoIr191(3):933, 2004 Cheng YW, Shafer BL, Bryant AS, et al: Length of the irst stage of labor and associated perinatal outcomes in nulliparous women. Obstet Gynecol 116(5):1127,r2010 Cibils LA, Hendricks CH: Normal labor in vertex presentation. Am J Obstet GynecoIr91:385, 1965 Cohen W: InAuence of the duration of second stage labor on perinatal outcome and puerperal morbidity. Obstet Gynecol 49:266, 1977 Cohen W, Friedman EA: Management of Labor. Baltimore, University Park Press, 1983 Cohen WR, Friedman EA: Misguided guidelines for managing labor. Am J Obstet Gynecol 212(6):753.el, 2015a Cohen WR, Friedman EA: Perils of the new labor management guidelines. Am J Obstet Gynecol 212(4):420, 2015b Cruikshank DP, White CA: Obstetric malpresentations: twenty years' experience. Am J Obstet Gynecol 116:1097, 1973

1	Cruikshank DP, White CA: Obstetric malpresentations: twenty years' experience. Am J Obstet Gynecol 116:1097, 1973 Duf P: Diagnosis and management of face presentation. Obstet Gynecol 57:105,r1981 Eller We, Mengert WF: Recognition of mid-pelvic contraction. Am ] Obstet Gynecol 53:252, 1947 Ferguson ]E, Newberry YG, DeAngelis GA, et al: he fetal-pelvic index has minimal utility in predicting fetal-pelvic disproportion. Am J Obstet Gynecol 179: 1186, 1998 Floberg], Belfrage P, Ohlsen H: InAuence of pelvic outlet capacity on labor. A prospective pelvimetry study of 1,429 unselected primiparas. Acta Obstet Gynecol Scand 66: 121, 1987 Fraser WD, Marcoux 5, Krauss I, et al: Multicenter, randomized, controlled trial of delayed pushing for nulliparous women in the second stage of labor with continuous epidural analgesia. Am J Obstet Gynecol 182: 1165,r2000

1	Friedman E: The graphic analysis of labor. Am] Obstet Gynecol68: 1568, 1954 Friedman EA: Labor. Clinical Evaluation and Management, 2nd ed. New York, Appleton-Century-Crofts, Friedman EA: Primigravid labor; a graphicostatistical analysis. Obstet Gynecol 6(6):567, 1955 Friedman EA, Sachtleben MR: Station of the fetal presenting part 11: effect on the course oflabor. Am] Obstet Gynecol 93:530, 1965 Friedman EA, Sachtleben MR: Station of the fetal presenting part IV: arrest of descent in nulliparas. Obstet :129,r1976 Fuchs K, Peretz BA, Marcovici R, et al: The grand multipara-is it a problem? Int J Gynaecol Obstet 73:321, 1985 Gambling DR, Sharma SK, Ramin SM, et al: A randomized study of combined spinal-epidural analgesia versus intravenous meperidine during labor: impact on cesarean delivery rate. Anesthesiology 89(6): 1336, 1998 Goplerud ], Eastman N]: Compound presentation: survey of 65 cases. Obstet Gynecol 1:59, 1953

1	Goplerud ], Eastman N]: Compound presentation: survey of 65 cases. Obstet Gynecol 1:59, 1953 Grobman WA, Bailit], Lai Y, et al: Association of the duration of active pushing with obstetric outcomes. Obstet Gynecol 12 (4):667, 2016 Handa VL, Laros RK: Active-phase arrest in labor: predictors of cesarean delivery in a nulliparous population. Obstet Gynecol 81 :758, 1993 Hannah M, Ohlsson A, Farine 0, et al: International Term PROM Trial: a RCT of induction of labor for prelabor rupture of membranes at term. Am J Obstet Gynecol 174:303, 1996 Hannah ME, Hodnett ED, Willan A, et al: Prelabor rupture of the membranes at term: expectant management at home or in hospital? Obstet Gynecol 96:533, 2000 Hansen SL, Clark SL, Foster JC: Active pushing versus passive fetal descent in the second stage of labor: a randomized controlled trial. Obstet Gynecol 99:29,r2002

1	Hansen SL, Clark SL, Foster JC: Active pushing versus passive fetal descent in the second stage of labor: a randomized controlled trial. Obstet Gynecol 99:29,r2002 Harper LM, Caughey AB, Roehl A, et al: Deining an abnormal irst stage of labor based on maternal and neonatal outcomes. m J Obstet Gynecol 210(6):536.el,r2014 Hauth )C, Hankins GO, Gilstrap LC III: Uterine contraction pressures achieved in parturients with active phase arrest. Obstet Gynecol 78:344, 1991 Hauth )C, Hankins GO, Gilstrap LC III, et al: Uterine contraction pressures with oxytocin induction/augmentation. Obstet GynecoI68:305, 1986 Hellman LM, Epperson )W, Connally F: Face and brow presentation: the experience of the Johns Hopkins Hospital, 1896 to 1948. Am ) Obstet Gynecol 59:831, 1950 Hendricks CH, Quilligan E), Tyler AB, et al: Pressure relationships between intervillous space and amniotic fluid in human term pregnancy. Am ) Obstet Gynecol77:1028, 1959

1	Hendricks CH, Quilligan E), Tyler AB, et al: Pressure relationships between intervillous space and amniotic fluid in human term pregnancy. Am ) Obstet Gynecol77:1028, 1959 Hillis OS: Diagnosis of contracted pelvis by the impression method. Surg Gynecol Obstet 51:857,o1930 Johnson CE: Transverse presentation of the fetus. )MA 187:642,o1964 Kaltreider OF: Criteria of midplane contraction. Am) Obstet GynecoI63:392, 1952 Korhonen U, Taipale P, Heinonen S: Fetal pelvic index to predict cephalopelvic disproportion-a retrospective clinical cohort study. Acta Obstet Gynecol Scand 94(6):615, 2015 Kwok CS, Judkins CL, Sherratt M: Forearm injury associated with compound presentation and prolonged labour.o) Neonatal Surg 4(3):40,o2015 Larks SO: Electrohysterography. Springfield, Thomas, 1960 Laughon SK, Berghella V, Reddy UM, et a1: Neonatal and maternal outcomes with prolonged second stage oflabor. Obstet Gynecol 124(1):57,2014

1	Laughon SK, Berghella V, Reddy UM, et a1: Neonatal and maternal outcomes with prolonged second stage oflabor. Obstet Gynecol 124(1):57,2014 Le Ray C, Audibert F, Goinet F, et al: When to stop pushing: efects of duration of second-stage expulsion eforts on maternal and neonatal outcomes in nulliparous women with epidural analgesia. Amo) Obstet Gynecol 201(4):361.e1,o2009 Leveno K), Nelson DB, McIntire DO: Second-stage labor: how long is too long? Am) Obstet Gynecol 214(4):484, 2016 Mahon TR, Chazotte C, Cohen WR: Short labor: characteristics and outcome. Obstet Gynecol 84:47, 1994 Manyonda IT, Shaw DE, Drife )0: he efect of delayed pushing in the second stage of labor with continuous lumbar epidural analgesia. Acta Obstet Gynecol Scand 69:291, 1990 Marte K, Voutsos L: Reduction in the cesarean delivery rate after obstetric care consensus guideline implementation. Obstet Gynecol 128(6):1445,o2016 Martin )A, Hamilton BE, Osterman M), et al: Births: final data for 2013. Nat!

1	Vital Stat Rep 64(1):1, 2015 McCarthy S: Magnetic resonance imaging in obstetrics and gynecology. Magn Reson Imaging 4:59, 1986 Mengert F: Estimation of pelvic capaciry.o)1A 138:169, 1948 Menticoglou SM, Manning F, Harman C, et 1: Perinatal outcomes in relation to second-stage duration. Am ) Obstet Gynecol 173:906, 1995a Menticoglou SM, Perlman M, Manning FA: High cervical spinal cord injury in neonates delivered with forceps: report of 15 cases. Obstet Gynecol 86:589, 1995b Moore MM, Shearer DR: Fetal dose estimates for CT pelvimetry. Radiology 171:265,o1989 Mozurkewich E, Chilimigras ), Koepke E, et al: Indications for induction of labour: a best-evidence review. B)OG 116(5):626,o2009 Mueller P: About the prognosis for delivery with a narrow pelvis. Arch GynaekoIo27:311, 1885

1	Mueller P: About the prognosis for delivery with a narrow pelvis. Arch GynaekoIo27:311, 1885 Nelson DB, McIntire DO, Leveno K): Relationship of the length of the first stage oflabor to the length of the second stage. Obstet GynecoIo122:27, 2013 Olah KS, Neilson ): Failure to progress in the management of labour. B)OG 101:1, 1994 Passos F, Cardose K, Coelho M, et a1: Antibiotic prophylaxis in premature rupture of membranes at term. Obstet GynecoIo120:1045, 2012 Pattinson RC, Cuthbert A, Vannevel V: Pelvimetry for fetal cephalic presentations at or near term for deciding on mode of delivery. Cochrane Database Syst Rev 3:CD000161, 2017 Peleg 0, Hannah ME, Hodnett ED, et 1: Predictors of cesarean delivery after prelabor rupture of membranes at term. Obstet Gynecol 93: 1 03o1, 1999 Plunkett BA, Lin A, Wong CA, et a1: Management of the second stage oflabor in nulliparas with continuous epidural analgesia. Obstet Gynecol 102:109, 2003

1	Plunkett BA, Lin A, Wong CA, et a1: Management of the second stage oflabor in nulliparas with continuous epidural analgesia. Obstet Gynecol 102:109, 2003 Reynolds SR, Heard 00, Bruns P, et 1: A multichannel strain-gauge tocodynamometer: an instrument for studying patterns of uterine contractions in pregnant women. Bull Johns Hopkins Hosp 82:446, 1948 Rosenbloom )1, Stout M), Tuuli MG, et al: New labor management guidelines and changes in cesarean delivery patterns. m)Obstet Gynecol October 14, 2017 [Epub ahead of print] Roshanfekr 0, Blakemore K), Leeo), et 1: Station at onset of active labor in nulliparous patients and risk of cesarean delivery. Obstet Gynecol 93:329, 1999 Rouse D), Oweno), Hauth ]C: Active-phase labor arrest: oxytocin augmenta tion for at least 4 hours. Obstet Gynecol 93:323, 1999 Rouse D), Weiner S), Bloom SL, et a1: Second-stage labor duration in nulliparous women: relationship to maternal and perinatal outcomes. Am ) Obstet GynecoIo201(4):357.e1,o2009

1	Rouse D), Weiner S), Bloom SL, et a1: Second-stage labor duration in nulliparous women: relationship to maternal and perinatal outcomes. Am ) Obstet GynecoIo201(4):357.e1,o2009 Satin A), Maberry lC, Leveno ), et al: Chorioamnionitis: a harbinger of dystocia. Obstet Gynecol 79:913, 1992 Shafer BL, Cheng W, Vargas ]E, et a1: Face presentation: predictors and delivery route. Am] Obstet Gynecol 194(5):elO, 2006 Sharma SK, McIntire DO, Wiley), et al: Labor analgesia and cesarean delivery: an individual patient meta-analysis of nulliparous women. Anesthesiology 100(1):142,o2004 Sheiner E, Levy A, Mazor M: Precipitate labor: higher rates of maternal complications. Euro] Obstet Gynecol Reprod BioI 116(1):43, 2004 Sporri S, Hanggi W, Brahetti A, et a1: Pelvimetry by magnetic resonance imaging as a diagnostic tool to evaluate dystocia. Obstet Gynecol 89:902, 1997 Stark DO, McCarthy SM, Filly A, et a1: Pelvimetry by magnetic resonance imaging. Am) RadioIo144:947, 1985

1	Tebes CC, Mehta P, Calhoun DA, et 1: Congenital ischemic forearm necrosis associated with a compound presentation. ] Matern Fetal Med 8:281, 1999 Thoms H: he obstetrical signiicance of pelvic variations: a study of 450 primiparous women. Bl] 2:210, 1937 Thorpo)M )r, Pahel-Short L, Bowes WAo)r: The Mueller-Hillis maneuver: can it be used to predict dystocia? Obstet Gynecol 82:5o19, 1993 Thurnau GR, Scates DH, Morgan MA: he fetal-pelvic index: a method of identiying fetal-pelvic disproportion in women attempting vaginal birth after previous cesarean delivery. Am) Obstet Gynecol 165:353, 1991 Vallier A, Cureton BA, Schubeck 0: Pregnancy outcomes ater pelvic ring injury.o) Orthop Trauma 26(5):302,o2012 Wilson-Leedy ]G, DiSilvestro A), Repke ]T, et al: Reduction in the cesarean delivery rate after Obstetric Care Consensus guideline implementation. Obstet GynecoIo128(1):145, 2016

1	Wilson-Leedy ]G, DiSilvestro A), Repke ]T, et al: Reduction in the cesarean delivery rate after Obstetric Care Consensus guideline implementation. Obstet GynecoIo128(1):145, 2016 Wong CA, Scavone BM, Dugan S, et 1: Incidence of postpartum lumbosacral spine and lower extremiry nerve injuries. Obstet Gynecol 101o:279,o2003 World Health Organization: Partographic management of labour. Lancet 343:1399, 1994 Zaretsky MV, Alexander ]M, McIntire DO, et al: Magnetic resonance imaging pelvimetry and the prediction of labor dystocia. Obstet Gynecol 106:919, 2005 Zhango), Landy H), Branch OW, et al: Contemporary patterns of spontaneous labor with normal neonatal outcomes. Obstet Gynecol 116: 1281, 2010 Zhang], Troendle ]F, Yancey MK: Reassessing the labor curve in nulliparous women. m) Obstet GynecoIo187(4):824, 2002 ELECTRONIC FETAL MONITORING ................. 457 OTHER INTRAPARTUM ASSESSMENT TECHNIQUES ... 470 NONREASSURING FETAL STATUS ................. 472

1	ELECTRONIC FETAL MONITORING ................. 457 OTHER INTRAPARTUM ASSESSMENT TECHNIQUES ... 470 NONREASSURING FETAL STATUS ................. 472 MECONIUM IN THE AMNIONIC FLUID .............. 474 FETAL HEART RATE PAIERNS AND BRAIN INJURY ... 476 CURRENT RECOMMENDATIONS ................... 478 INTRAPARTUM SURVEILLANCE OF UTERINE ACTIVIY ... 478 To study the orces exerted by labour, a rubber bag was inserted into the uterus which was connected with a manometer. In this way it was ound that the intra-uterine pressure, in the intervals between the contractions, was represented by a column of mercury 20 millimeters high, 5 of which were due to the toniciy of the wals and 15 to its contents. During the pains, however, the mercury rose consideraby, reaching a height of from 80 to 250 milimeters. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) Little is written in the first edition of this textbook concerning monitoring of the fetus during labor. Much later, periodic auscultation of the fetal heartbeat with a fetoscope was adopted. These practices were eclipsed in the late 1960s and early 1970s by the development of electronic fetal monitoring (Hon, 1958). It was hoped that the continuous graph-paper portrayal of the fetal heart rate was potentially diagnostic in assessing pathophysiological events afecting the fetus. When first introduced, electronic fetal heart rate monitoring was used primarily in complicated pregnancies but gradually became used in most pregnancies. Now, more than 85 percent of all live births in the United States undergo electronic fetal monitoring (Ananth, 2013). Direct fetal heart measurement is accomplished by attaching a bipolar spiral electrode directly to the fetus (Fig.

1	Direct fetal heart measurement is accomplished by attaching a bipolar spiral electrode directly to the fetus (Fig. The wire electrode penetrates the fetal scalp, and the second pole is a metal wing on the electrode. The electrical fetal cardiac signal-P wave, QRS complex, and T wave-is ampliied and must be applied because air conducts ultrasound waves poorly. he device Mother 50 mm/sec FIGURE 24-1 Internal electronic fetal monitoring. Schematic representation of a bipolar electrode attached to the fetal scalp for detection of fetal QRS complexes (F). Also shown is the maternal heart and corresponding electrical complex (M) that is detected. is held in position by an elastic belt.

1	is held in position by an elastic belt. FIGURE 24-3 The top tracing shows standard fetal monitor tracing of heart rate using a fetal scalp electrode, Spiking ofthe fetal rate in the monitor tracing is due to premature atrial con entiation of fetal cardiac motion from tractions, The second panel displays accompanying contractions, The bottom two tracings repmaternal arterial pulsations (Neilson, resent cardiac electrical complexes detected from fetal scalp and maternal chest wall electrodes, 2008). ECG = electrocardiogram; F = fetus; M = mother; PAC = fetal premature atrial contraction, fed into a cardiotachometer for heart rate calculation. he peak R-wave voltage is the portion of the fetal electrocardiogram (ECG) most reliably detected.

1	n example of the method of fetal heart rate processing employed when a scalp electrode is used is shown in Figure 24-2. Time (t) in milliseconds between fetal R waves is fed into a cardiotachometer, where a new fetal heart rate is set with the arrival ofeach new R wave. As also shown in Figure 24-2, a premature atrial contraction is computed as a heart rate acceleration because the interval (t2) is shorter than the preceding one (t1). he phenomenon of continuous R-to-R wave fetal heart rate computation is known as beat-to-beat variabiliy.

1	Electrical cardiac complexes detected by the electrode include those generated by the mother. However, the amplitude of the maternal ECG signal is diminished when recorded through the fetal scalp electrode and is masked by the fetal ECG. Shown in Figure 24-3 are simultaneous recordings of maternal chest wall ECG signals and fetal scalp electrode ECG signals. This fetus is experiencing premature atrial contractions, which cause the cardiotachometer to rapidly and erratically seek new heart rates, resulting in the "spiking" shown in the standard fetal monitor tracing. Importantly, when the fetus is dead, the maternal R waves are still detected by the scalp electrode as the next best signal and are counted by the cardiotachometer (Fig. 24-4).

1	Although membrane rupture may be avoided, external monitoring does not provide the precision of fetal heart rate measurement aforded by internal monitoring (Nunes, 2014). In some women-for example, those who are obese-external monitoring may be dificult (Brocato, 2017). With external monitoring, the fetal heart rate is detected through the maternal abdominal wall using the ultrasound Doppler principle. Ultrasound waves undergo a shift in frequency as they are reflected from moving fetal heart valves and from pulsatile blood ejected during systole (Chap. 10, p. 213). he unit consists of a transducer that emits ultrasound and a sensor to detect a shift in frequency of the reflected sound. The transducer is placed on the maternal abdomen at a site where fetal heart action is best detected. A coupling gel

1	FIGURE 24-2 Schematic representation offetal electrocardiographic signals used to compute continuing beat-to-beat heart rate with scalp electrodes, Time intervals (tIl t21 t3) in milliseconds between successive fetal R waves are used by a cardiotachometer to compute instantaneous fetal heart rate, ECG = electrocardiogram; PAC = premature atrial contraction, contractions. FIGURE 24-4 Placental abruption. In the upper panel, the fetal scalp electrode first detected the heart rate of the dying fetus. After fetal death, the maternal electrocardiogram complex is detected and recorded. The second panel displays an absence of uterine

1	Ultrasound Doppler signals are edited electronically before fetal heart rate data are printed onto monitor paper. Relected ultrasound signals from moving fetal heart valves are analyzed through a microprocessor that compares incoming signals with the most recent previous signal. This process, called autocorrelation, is based on the premise that the fetal heart rate has regularity, whereas "noise" is random and without regularity. Several fetal heart motions must be deemed electronically acceptable by the microprocessor before the fetal heart rate is printed. Such electronic editing has greatly improved the tracing quality of the externally recorded fetal heart rate. Other features of current fetal monitors include the capability to monitor twin fetuses, monitor concurrent maternal heart rate, display the fetal ECG, and record maternal pulse oximetry values. Many fetal monitors are capable of interfacing with archival storage systems, which obviates maintaining actual paper tracings.

1	Technological advances now allow fetal heart rate monitoring from a remote, centralized location. Theoretically, the ability to monitor several patients simultaneously was hoped to improve neonatal outcomes. That said, only one study on centralized fetal monitoring has been reported. Anderson and colleagues (2011) measured the ability of 12 individuals to detect critical signals in fetal heart rate tracings on one, two, or four monitors. The results showed that detection accuracy declined as the number of displays increased. The interpretation of fetal heart rate patterns can be problematic without deinitions and nomenclature. In one example, Blackwell and colleagues (2011) asked three .1aternal-Fetal Medicine specialists to independently interpret 154 fetal heart rate tracings. Interobserver agreement was poor for the most ominous tracings and moderate for less severe patterns.

1	The National Institute of Child Health and Human Development (NICHD) Research Planning Workshop (1997) brought together investigators with exper tise in the ield to propose standardized, tion of fetal heart rate patterns during labor. This workshop reconvened in 2008. he deinitions proposed as a result of this second workshop are used in this chapter and have been adopted by the American College of Obstetricians and Gynecolo Importantly, interpretation of electronic fetal heart rate data is based on the visual pattern of the heart rate as portrayed on chart recorder graph paper. Thus, the choice of vertical and horizontal scaling greatly afects the appearance of the fetal heart rate. Scal ing factors recommended by the NICHD

1	Workshop are 30 beats per minute (beats/ min or bpm) per vertical cm (range, 30 to paper speed. Fetal heart rate variation is falsely displayed at the slower 1 cm/ min paper speed compared with that of the smoother baseline recorded at 3 cm/min. hus, pattern recognition can be con siderably distorted depending on the scaling factors used. This refers to the modal characteristics that prevail apart from periodic accelerations or decelerations associated with uterine contractions. Descriptive characteristics of baseline fetal heart activity include rate, beat-to-beat variabili, etal arrhythmia, and distinct patterns such as sinusoidal or saltatory fetal heart rates.

1	With increasing fetal maturation, the heart rate decreases. This continues postnatally such that the average rate is 85 bpm by age 8 years (Tintinalli, 2016). Pillai and James (1990) reported that the baseline fetal heart rate declined an average of 24 bpm between 16 weeks' gestation and term, or approximately 1 bpm per week. his normal gradual slowing of the fetal heart rate is thought to correspond to maturation of parasympathetic (vagal) heart control (Renou, 1969).

1	The baseline fetal heart rate is the approximate mean rate rounded to increments of 5 bpm during a 10-minute tracing segment. In any 10-minute window, the minimum interpretable baseline duration must be at least 2 minutes. If the baseline fetal heart rate is less than 110 bpm, it is termed bradycardia . If the baseline rate is greater than 160 bpm, it is called tachycardia. The average fetal heart rate is considered the result of tonic balance between accelerator and decelerator influences on pacemaker cells. In this concept, the sympathetic system is the accelerator influence, and the parasympathetic system is the decelerator factor mediated by vagal slowing of heart rate (Dawes, 1985). Heart rate also is under the control of arterial chemoreceptors such that both hypoxia and hypercapnia can modulate rate. More severe and prolonged hypoxia, with a TABLE 24-' . Electronic Fetal Monitoring Definitions

1	TABLE 24-' . Electronic Fetal Monitoring Definitions Baseline • The mean FHR rounded to increments of 5 bpm during a 1 O-min segment, excluding: -Periods of marked FHR variability -Segments of baseline that difer by more than 25 bpm The baseline must be for a minimum of 2 min in any 1 O-min segment or the baseline for that time period is indeterminate. In this case, one may refer to the prior 1 O-min window for determination of baseline. Normal FHR baseline: 11s0-1s60 bpm Tachycardia: FHR baselines is greater than 160 beats per minute Bradycardia: FHR baseline is less than 11s0 beats per minute

1	Normal FHR baseline: 11s0-1s60 bpm Tachycardia: FHR baselines is greater than 160 beats per minute Bradycardia: FHR baseline is less than 11s0 beats per minute Baseline • Fluctuations in the baseline FHR that are irregular in amplitude and frequency variability • Variability is visually quantified as the amplitude of peak-to-trough in beats per minute -Absent: amplitude range undetectable -Minimal: amplitude range detectable but 5 beats per minutes or fewer -Moderate (normal): amplitude range 6-25 beats per minute -Marked: amplitude range greater than 25 beats per minute Acceleration • A visually apparent abrupt increase (onset to peak in less than 30 seconds) in the FHR At 32 weeks of gestation and beyond, an acceleration has a peak of 15 bpm or more above baseline, with a duration of 15 sec or more but less than 2 minutes from onset to return

1	At 32 weeks of gestation and beyond, an acceleration has a peak of 15 bpm or more above baseline, with a duration of 15 sec or more but less than 2 minutes from onset to return Before 32 weeks, an acceleration has a peak of 10 bpm or more above baseline, with a duration of 10 seconds or more but less than 2 minutes from onset to return If an acceleration lasts 10 minutes or longer, it is a baseline change Early deceleration • Visually apparent usually symmetrical gradual decrease and return of the FHR associated with a A gradual FHR decrease is defined as from the onset to the FHR nadir of 30 seconds or more The decrease in FHR is calculated from the onset to the nadir of the deceleration The nadir of the deceleration occurs at the same time as the peak of the contraction

1	The decrease in FHR is calculated from the onset to the nadir of the deceleration The nadir of the deceleration occurs at the same time as the peak of the contraction In most cases the onset, nadir, and recovery of the deceleration are coincident with the beginning, peak, and ending of the contraction, respectively Late deceleration • Visually apparent usually symmetrical gradual decrease and return of the FHR associated with a A gradual FHR decrease is defined as from the onset to the FHR nadir of 30 seconds or more The decrease in FHR is calculated from the onset to the nadir of the deceleration The deceleration is delayed in timing, with the nadir of the deceleration occurring after the peak of the contraction In most cases the onset, nadir, and recovery of the deceleration occur after the beginning, peak, and ending of the contraction, respectively deceleration • An abrupt FHR decrease is defined as from the onset of the deceleration to the beginning of the

1	FHR nadir of less than 30 seconds The decrease in FHR is calculated from the onset to the nadir of the deceleration The decrease in FHR is 15 beats per minute or greater, lasting 15 seconds or greater, and less than 2 minutes in duration When variable decelerations are associated with uterine contraction, their onset, depth, and duration commonly vary with successive uterine contractions Prolonged • Visually apparent decrease in the FHR below the baseline deceleration • Decrease in FHR from the baseline that is 15 per minute or more, and less than 2 minutes in duration • If a deceleration last 10 minutes or longer, it is a baseline change Sinusoidal pattern • Visually apparent, smooth, sine wave-line undulating pattern in FHR baseline with a cycle frequency of 3-5 per minute which persists for 20 dinutes or more FHR = fetal heart rate. Data from Macones, 2008.

1	FHR = fetal heart rate. Data from Macones, 2008. FIGURE 24-6 Schematic representation of short-term beat-tobeat variability measured by a fetal scalp electrode. t time interval between successive fetal R waves. (Adapted with permission from Klavan M, Laver AT, Boscola MA: Clinical concepts of fetal heart rate monitoring. Waltham, Hewlett-Packard, 1977.) FIGURE 24-5 Fetal bradycardia measured with a scalp electrode (upper paneD in a pregnancy complicated by placental abruption and subsequent fetal death. Concurrent uterine contractions are shown in the lower panel. rising blood lactate level and severe metabolic acidemia, induces a prolonged fall in heart rate (hakor, 2009).

1	rising blood lactate level and severe metabolic acidemia, induces a prolonged fall in heart rate (hakor, 2009). Bradycardia. In the third trimester, the normal mean baseline fetal heart rate has generally been accepted to range between 120 and 160 bpm. But, pragmatically, a rate between 100 and 119 bpm, in the absence of other changes, usually is not considered to represent fetal compromise. Such low but potentially normal baseline heart rates also have been attributed to head compression from occiput posterior or transverse positions, particularly during second-stage labor (Young, 1976). Such mild bradycardias were observed in 2 percent of monitored pregnancies and averaged approximately 50 minutes in duration. Freeman and associates (2003) have concluded that bradycardia within the range of 80 to 120 bpm and with good variability is reassuring. Interpretation of rates less than 80 bpm is problematic, and such rates generally are considered nonreassuring.

1	Some causes of fetal bradycardia include congenital heart block and serious fetal compromise 0aeggi, 2008; Larma, 2007). Figure 24-5 shows bradycardia in a fetus dying from placental abruption. Maternal hypothermia under general anesthesia for repair of a cerebral aneurysm or during maternal cardiopulmonary bypass for open-heart surgery can also cause fetal bradycardia. Sustained fetal bradycardia in the setting of severe pyelonephritis and maternal hypothermia also has been reported (Hankins, 1997). Involved fetuses apparently are not harmed by several hours of such bradycardia.

1	Tachycardia. Fetal tachycardia is deined as a baseline heart rate greater than 160 bpm. The most common explanation for fetal tachycardia is maternal fever from chorioamnionitis, although fever from any source can produce this. In some cases, fetal tachycardia may precede overt maternal fever (Gilstrap, 1987). Fetal tachycardia caused by maternal infection typically is not associated with fetal compromise unless there are associated periodic heart rate changes or fetal sepsis. Other causes of fetal tachycardia include fetal compromise, cardiac arrhythmias, and maternal administration of parasympathetic inhibiting (atropine) or sympathomimetic (terbutaline) drugs. Prompt relief of the compromising event, such as correction of maternal hypotension caused by epidural analgesia, can result in fetal recovery. he key feature to distinguish fetal compromise in association with tachycardia seems to be concomitant heart rate decelerations.

1	Wandering Baseline. This baseline rate is unsteady and "wanders" between 120 and 160 bpm (Freeman, 2003). This rare finding is suggestive of a neurologically abnormal fetus and may occur as a preterminal event. In contrast, changes of the normal baseline are common in labor and do not predict morbidity (Yang, 2017).

1	Baseline variability is an important index of cardiovascular function and appears to be regulated largely by the autonomic nervous system (Kozuma, 1997). That is, a sympathetic and parasympathetic "push and pull" mediated via the sinoatrial node produces moment-to-moment or beat-to-beat oscillation of the baseline heart rate. Such heart rate change is deined as baseline variability. Variability can be further analyzed over the short term and long term, although these terms have fallen out of use. Short-term variabiliy relects the instantaneous change in fetal heart rate from one beat-or R wave-to the next. This variability is a measure of the time interval between cardiac systoles (Fig. 24-6). Short-term variability can most reliably be determined to be normally present only when electrocardiac cycles are measured directly with a scalp electrode. Long-term variability is used to describe the oscillatory changes during 1 minute and result in the waviness of the baseline (Fig. 24-7).

1	FIGURE 24-7 Schematic representation of long-term beat-to-beat variability of the fetal heart rate ranging between 125 and 135 bpm. (Adapted with permission from Klavan M, Laver AT, Boscola MA: Clinical concepts of fetal heart rate monitoring. Waltham, HewlettPackard, 1977.) he normal frequency of such waves is three to ive cycles per minute (Freeman, 2003).

1	It should be recognized that precise quantitative analysis of both short-and long-term variability presents several frustrating problems due to technical and scaling factors (Parer, 1985). hus, most clinical interpretation is based on visual analysis with subjective judgment of the smoothness or latness of the baseline. According to Freeman and associates (2003), no evidence suggests that the distinction between short-and long-term variability has clinical relevance. Similarly, the NICHD Workshop (1997) did not recommend diferentiating short-and long-term variability because in actual practice they are visually determined as a unit. The workshop panel defined baseline variability as those baseline luctuations of two cycles per minute or greater. They recommended the criteria shown in Figure 24-8 for quantification of variability. Normal beatto-beat variability was accepted to be 6 to 25 bpm.

1	Increased Variability. Several physiological and pathological processes can afect beat-to-beat variability. Greater variability accompanies fetal breathing and body movements (Dawes, 1981; Van Geijn, 1980). Pillai and James (1990) reported increased baseline variability with advancing gestation. Up to 30 weeks, baseline characteristics were similar during both fetal rest and activity. After 30 weeks, fetal inactivity was associated with diminished baseline variability, but fetal activity enhanced it. Last, the baseline fetal heart rate becomes more physiologically ixed (less variable) as the rate rises. This phenomenon presumably relects less cardiovascular physiological wandering as beat-to-beat intervals shorten with a higher heart rate.

1	Decreased Variability. A common cause of diminished beatto-beat variability is administration of analgesic drugs during labor (Chap. 25, p. 487). Various central nervous system depressant drugs can cause transient diminished beat-to-beat variability. Included are narcotics, barbiturates, phenothiazines, tranquilizers, and general anesthetics. Corticosteroids also dampen variability (Knaven, 2017). As one specific example, variability regularly diminishes within 5 to 10 minutes following intravenous meperidine administration, and the efects may last up to 60 minutes or longer (Hill, 2003; Petrie, 1993). Butorphanol given intravenously has similar efects (Schucker, 1996). And, chronically administered buprenorphine suppresses fetal heart rate and movement (Jansson, 2017).

1	Magnesium suate, widely used in the United States for tocolysis or management of hypertensive gravidas, is associated with diminished beat-to-beat variability. In a study of nearly 250 term gestations, magnesium sulfate administration led to decreased variability but without evidence of adverse neonatal efects (Duy, 2012). Others have echoed these findings (Hallak, 1999; Lin, 1988). With magnesium sulfate tocolysis of preterm labor, variability was also diminished in most reviewed studies (Nensi, 2014; Verdurmen, 2017). Of greatest concern, diminished beat-to-beat variability can be an ominous sign indicating a seriously compromised fetus. Paul and coworkers (1975) reported that loss of variability in combination with decelerations was associated with etal acidemia. Decreased variability was deined as an excursion of the baseline of;5 bpm (see Fig. 24-8). Severe maternal acidemia can also lower fetal beat-to-beat variability, for example, in a mother with diabetic ketoacidosis.

1	According to Dawes (1985), metabolic acidemia that causes depression of the fetal brainstem or the heart itself creates the loss of variability. hus, diminished beat-to-beat variability, when it relects fetal compromise, likely relects acidemia rather than hypoxia. Indeed, mild degrees of fetal hypoxemia have been reported actually to enhance variability, at least initially (Murotsuki, 1997).

1	Reduced baseline heart rate variabiliy is the single most reliable sign of etal compromise. Smith and coworkers (1988) performed a computerized analysis of beat-to-beat variability in growth-restricted fetuses before labor. Diminished variability (;4.2 bpm) maintained for 1 hour was diagnostic of developing acidemia and imminent fetal death. In contrast, Samuelof and associates (1994) evaluated variability in 2200 consecutive deliveries and concluded that variability by itself could not be used as the only indicator of fetal well-being. They also warned that good variability should not be interpreted as necessarily reassuring. Blackwell and associates (2011) found that even experts often disagreed as to whether variability was absent or minimal (;5 bpm).

1	In sum, beat-to-beat variability is afected by fetal physiology, and its meaning difers depending on the clinical setting. Decreased variability in the absence of decelerations is unlikely to reflect fetal hypoxia (Davidson, 1992). A persistently flat fetal heart rate baseline-absent variability-within the normal baseline rate range and without decelerations may relect a previous fetal insult that has resulted in neurological damage (Freeman, 2003). When fetal cardiac arrhythmias are irst suspected using electronic monitoring, indings can include baseline bradycardia, tachycardia, or most commonly in our experience, abrupt baseline spiking (Fig. 24-9). An arrhythmia can only be documented, practically speaking, when scalp electrodes are used. Some fetal monitors can be adapted to output the scalp electrode signals into an ECG recorder. Because only a single lead is obtained, analysis and interpretation of rhythm and rate disturbances are severely limited.

1	Southall and associates (1980) studied fetal cardiac rate and rhythm disturbances in 934 normal pregnancies between 30 and 40 weeks. Arrhythmias, episodes of bradycardia < 100 bpm, or tachycardia > 180 bpm were encountered in 3 percent. Most supraventricular arrhythmias are of little signiicance during labor unless there is coexistent fetal heart failure as evidenced by hydrops. Many supraventricular arrhythmias disappear in the immediate neonatal period, although some are associated with structural cardiac defects (Api, 2008). Intermittent baseline bradycardia is frequently due to congenital heart block. Conduction defects, most often complete atrioventricular (A V) block, usually are found in association with maternal connective tissue diseases (Chap. 59, p. 1142). Antepartum evaluation of the fetus with an identiied arrhythmia and potential treatment options are discussed in Chapter 16 (p. 315).

1	FIGURE 24-8 Grades of baseline fetal heart rate variability shown in the following five panels. 1. Undetectable, absent variability. 2. Minimal variability, ;5 bpm. 3. Moderate (normal) variability, 6 to 25 bpm. 4. Marked variability, >25 bpm. 5. Sinusoidal pattern. This differs from variability in that it has a smooth, sinelike pattern of regular fluctuation and is excluded in the definition of fetal heart rate variability. (Adapted with permission from National Institute of Child Health and Human Development Research Planning Workshop, 1997.) FIGURE 24-9 Internal fetal monitoring at term demonstrated occasional abrupt beat-to-beat fetal heart rate spiking due to erratic extrasystoles shown in the corresponding fetal electrocardiogram. The normal newborn was delivered spontaneously and had normal cardiac rhythm in the nursery.

1	Most fetal arrhythmias without comorbid fetal hydrops are inconsequential during labor, but they may hinder interpretation of fetal heart rate tracings. Sonographic evaluation of fetal anatomy and echo cardiography can be useful. Generally, in the absence of fetal hydrops, neonatal outcome is not measurably improved by pregnancy intervention. At Parkland Hospital, intrapartum fetal cardiac arrhythmias, especially those associated with clear amnionic fluid, are typically managed conservatively.

1	A true sinusoidal pattern such as that shown in panel 5 of Figure 24-8 can be observed with fetal intracranial hemorrhage, with severe fetal asphyxia, and with severe feral anemia. he last may stem from anti-D alloimmunization, fetomaternal hemorrhage, twin-twin transusion syndrome, fetal parvoviral infection, or vasa previa with bleeding. Insigniicant sinusoidal patterns have been reported following administration of meperidine, morphine, alphaprodine, and butorphanol (Angel, 1984; Egley, 1991; Epstein, 1982). Shown in Figure 24-10 is a sinusoidal pattern seen with maternal meperidine administration. An important characteristic of this pattern when due to narcotics is the sine frequency of 6 cycles per minute. A sinusoidal pattern also has been described with chorioamnionitis, fetal distress, and umbilical cord occlusion (lvlurphy, 1991). Young (1980a) and Johnson (1981) with their coworkers concluded that intrapartum sinusoidal fetal heart patterns were not generally associated with

1	and umbilical cord occlusion (lvlurphy, 1991). Young (1980a) and Johnson (1981) with their coworkers concluded that intrapartum sinusoidal fetal heart patterns were not generally associated with fetal compromise. hus, management is usually dictated by the clinical setting. Modanlou and Freeman (1982), based on their extensive review, proposed adoption of a strict definition: 1.

1	Stable baseline heart rate of 120 to 160 bpm with regular oscillations 2. Amplitude of 5 to 15 bpm (rarely greater) FIGURE 24-10 Sinusoidal fetal heart rate pattern associated with maternal intravenous meperidine administration. Sine waves are occurring at a rate of 6 cycles per minute. 3. Long-term variability frequency of 2 to 5 cycles per minute 4. 5. Oscillation of the sinusoidal waveform above or below a baseline 6. Absent accelerations. Although these criteria were selected to deine a sinusoidal pattern that is most likely ominous, they observed thar the pattern associated with alphaprodine is indistinguishable. Other investigators have proposed a classiication of sinusoidal heart rate patterns into mild-amplitude 5 to 15 bpm, intermediate-16 to 24 bpm, and major-::25 bpm to quantiy fetal risk (Murphy, 1991; Neesham, 1993).

1	Some have defined intrapartum sine wavelike baseline variation with periods of acceleration as pseudosinusoidal. Murphy and colleagues (1991) reported that pseudosinusoidal patterns were seen in 15 percent of monitored labors. Mild pseudosinusoidal patterns were associated with use of meperidine and epidural analgesia. Intermediate pseudosinusoidal patterns were linked to fetal sucking or transient episodes of fetal hypoxia caused by umbilical cord compression. Egley and associates (1991) reported that 4 percent of fetuses demonstrated sinusoidal patterns transiently during normal labor. hese authors observed patterns persisting for up to 90 minutes in some cases.

1	The pathophysiology of sinusoidal patterns is unclear, in part due to various deinitions. There seems to be general agreement that antepatum sine wave baseline undulations portend severe fetal anemia. Still, few anti-D alloimmunized fetuses develop this pattern (Nicolaides, 1989). The sinusoidal pattern has been reported to develop or disappear ter fetal transusion (Del Valle, 1992; Lowe, 1984). Ikeda and associates (1999) proposed that the pattern is related to waves of arterial blood pressure, reflecting oscillations in the baroreceptor-chemoreceptor feedback mechanism.

1	These refer to deviations from baseline that are temporally related to uterine contractions. Acceleration refers to a rise in fetal heart rate above baseline, and deceleration is a drop below the baseline rate. The nomenclature most commonly used in the United States is based on the timing of the deceleration in relation to contractions-thus, eary, late, or variable. he waveform of these decelerations is also significant for pattern recognition. In early and late decelerations, the slope of fetal heart rate change is gradual, resulting in a curvilinear and uniform or symmetrical waveform. With variable decelerations, the slope of fetal heart rate change is abrupt and erratic, giving the waveform a jag ged appearance. The NICHD Workshop (1997) proposed that decelerations be defined as recurrent if they accompanied �50 percent of contractions in any 20-minute period.

1	Another system now used less often to describe decelera tions is based on the pathophysiological events considered most likely to underlie the pattern. In this system, early decelera tions are termed head compression, late decelerations are termed uteroplacental insuiciency, and variable decelerations are cord compression patterns. hese are abrupt heart rate increases above the fetal heart rate baseline and defined by n onset-to-peak rise within 30 seconds (American College of Obstetricians and Gynecologists, 2017a). At 32 weeks' gestation and beyond, an acceleration has a peak � 15 bpm above baseline. Its duration is � 15 sec but <2 minutes from onset to baseline return (see Table 24-1). Before 32 weeks, a peak �10 bpm for 10 seconds to 2 minutes is considered normal. Prolonged acceleration is defined as �2 minutes but < 10 minutes.

1	According to Freeman and coworkers (2003), accelerations most often occur antepartum, in early labor, and in association with variable decelerations. Proposed mechanisms for intrapartum accelerations include fetal movement, stimulation by uterine contractions, umbilical cord occlusion, fetal stimulation during pelvic examination, scalp blood sampling, and acoustic stimulation. Accelerations are common during labor. These are virtually always reassuring and almost always conirm that the fetus is not acidemic at that time.

1	As with beat-to-beat variability, accelerations represent intact neurohormonal cardiovascular control mechanisms linked to fetal behavioral states. Krebs and colleagues (1982) analyzed electronic heart rate tracings in nearly 2000 fetuses and found sporadic accelerations during labor in 99.8 percent. Fetal heart rate accelerations during the irst or last 30 minutes during labor, or both, were a favorable sign for fetal well-being. The absence of such accelerations during labor, however, is not necessarily an unfavorable sign unless coincidental with other nonreassuring changes. he chance of acidemia in the fetus that fails to respond to stimulation in the presence of an otherwise nonreassuring pattern approximates 50 percent (Clark, 1984; Smith, 1986). his physiological response shows a gradual fetal heart rate decline and then return to baseline associated with a contraction (Fig.

1	his physiological response shows a gradual fetal heart rate decline and then return to baseline associated with a contraction (Fig. Freeman and associates (2003) defined early decelerations as those generally seen in active labor between 4 and 7 cm cervical dilation. In their deinition, the degree of deceleration is generally proportional to the contraction strength and rarely falls below 100 to 110 bpm or 20 to 30 bpm below baseline. Such decelerations are common during active labor and not associated with tachycardia, loss of variability, or other fetal heart rate changes. Importantly, early decelerations are not associated with fetal hypoxia, acidemia, or low Apgar scores. FIGURE 24-11 Features of early fetal heart rate deceleration. Characteristics include a gradual decline in the heart rate with both onset and recovery coincident with the onset and recovery of the contraction. The nadir of the deceleration is 30 seconds or more after the deceleration onset.

1	Head compression probably causes vagal nerve activation as a result of dural stimulation, and this mediates the heart rate deceleration (Paul, 1964). Ball and Parer (1992) concluded that fetal head compression is a likely cause not only of the deceleration shown in Figure 24-11 but also of those shown in Figure 24-12, which typically occur during second-stage labor. FIGURE 24-12 Two diferent fetal heart rate patterns during second-stage labor that are likely both due to head compression (upper pane0. Maternal pushing eforts (lower pane0 correspond to the spikes with uterine contractions. Fetal heart rate deceleration () is consistent with the pattern of head compression shown in Figure 24-1o1. Deceleration (B), however, is "variable" in appearance because of its jagged configuration and may alternatively represent cord occlusion.

1	FIGURE 24-13 Features of late fetal heart rate deceleration. Characteristics include gradual decline in the heart rate with the contraction nadir, and recovery occurring after the end ofthe contraction. The nadir ofthe deceleration occurs 30 seconds or more after the onset of the deceleration. Indeed, they observed that head compression is the likely cause of many variable decelerations classically attributed to cord compression.

1	The fetal heart rate response to uterine contractions can reflect uterine perfusion or placental function. A late deceleration is a smooth, gradual, symmetrical decline in fetal heart rate beginning at or after the contraction peak and returning to baseline only after the contraction has ended. his deceleration reaches its nadir within 30 seconds of its onset. In most cases, the onset, nadir, and recovery of the deceleration occur after the beginning, peak, and ending of the contraction, respectively (Fig. 24-13). The magnitude of late decelerations is seldom more than 30 to 40 bpm below baseline and typically not more than 10 to 20 bpm. Late decelerations usually are not accompanied by accelerations. Myers and associates (1973) studied monkeys in which they compromised uteroplacenral perfusion by lowering maternal aortic blood pressure. The interval or lag from the contraction onset until the late deceleration onset was directly related to basal fetal oxygenation. hey demonstrated

1	by lowering maternal aortic blood pressure. The interval or lag from the contraction onset until the late deceleration onset was directly related to basal fetal oxygenation. hey demonstrated that the length of the lag was predictive of the fetal P02 but not fetal pH. he lower the fetal P02 before contractions, the shorter the lag to the onset of late decelerations. his lag relected the time necessary for the fetal P02 to fall below a critical level necessary to stimulate arterial chemoreceptors, which mediated the decelerations.

1	Murata and coworkers (1982) also showed that a late deceleration was the irst fetal heart rate consequence ofuteroplacental-induced hypoxia. During the course of progressive hypoxia that led to death over 2 to 13 days, monkey fetuses invariably exhibited late decelerations before development of acidemia. Variability of the baseline heart rate disappeared as acidemia developed. Generally, any process that produces maternal hypotension, excessive uterine activity, or placental dysfunction can induce late decelerations. The two most common sources are hypotension from epidural analgesia and uterine hyperactivity from onset : sece: 1 ... FIGURE 24-14 Features ofvariable fetal heart rate decelerations. Characteristics include an abrupt decline in the heart rate, and onset that commonly varies with successive contractions. The deceleration measures : 15 bpm for : 15 seconds and has an onset-to-nadir phase of <30 seconds. Total duration is <2 minutes.

1	oxytocin stimulation. Maternal diseases such as hypertension, diabetes, and collagen vascular disorders can cause chronic placental dysfunction. Placental abruption can produce acute late decelerations. he most frequent deceleration patterns encountered during labor are variable decelerations attributed to umbilical cord occlusion. In a study of more than 7000 monitor tracings, variable decelerations were identified in 40 percent when labor had progressed to 5 cm dilation and in 83 percent by the end of first-stage labor (Melchior, 1985). A variable deceleration is defined as an abrupt drop in the fetal heart rate beginning with the onset of the contraction and reaching a nadir in less than 30 seconds. he decrease must last between 15 seconds and 2 minutes and must be :::15 bpm in amplitude. The onset of deceleration typically varies with successive contractions (Fig. 24-14).

1	Hon (1959) tested the efects of umbilical cord compression on fetal heart rate (Fig. 24-15). In experimental animals, complete occlusion of the umbilical cord produces abrupt, jagged-appearing deceleration of the fetal heart rate 24-16). Concomitantly, fetal aortic pressure rises. Itskovitz and colleagues (1983) observed that variable decelerations in fetal lambs occurred only after umbilical blood flow was reduced by at least 50 percent.

1	Two types ofvariable decelerations are shown in Figure 24-17. The deceleration denoted by "A" is very much like that seen with complete umbilical cord occlusion in experimental animals (see Fig. 24-16). Deceleration "B," however, has a diferent configuration because of the "shoulders" of acceleration before and ater the deceleration component. Lee and coworkers (1975) proposed that this form of variable deceleration was caused by difering degrees of partial cord occlusion. In this physiological scheme, occlusion of only the vein reduces fetal blood return, thereby triggering a baroreceptor-mediated c'Eii. 40

1	FIGURE 24-15 A. The effects oF 25-second cord compression compared with those of 40 seconds in panel (8). (Redrawn with permission from Hon EH: The fetal heart rate patterns preceding death in utero, Am J Obstet Gynecol. 1959 Jul;78(l ):47-56.) acceleration. With increasing intrauterine pressure and subsequent complete cord occlusion, fetal systemic hypertension develops due to obstruction of umbilical artery flow. This stimulates a baroreceptor-mediated deceleration. Presumably, the aftercoming shoulder of the acceleration represents the same events occurring in reverse (Fig. 24-18).

1	Ball and Parer (1992) concluded that variable decelerations are mediated vagally and that the vagal response may be due to chemoreceptor or baroreceptor activity or both. Partial or complete cord occlusion produces an increase in afterload (baroreceptor) and a drop in fetal arterial oxygen content (chemoreceptor). These both result in vagal activity leading to deceleration. In fetal monkeys, the baroreceptor relexes appear to operate during the first 15 to 20 seconds of umbilical cord occlusion followed by decline in P02 at approximately 30 seconds, which then serves as a chemoreceptor stimulus (MuellerHeubach, 1982).

1	Thus, variable decelerations represent fetal heart rate reflexes that relect either blood pressure changes due to interruption of umbilical flow or changes in oxygenation. It is likely that most fetuses have experienced brief but recurrent periods of hypoxia due to umbilical cord compression during gestation. The frequency and inevitability of cord occlusions undoubtedly have provided the fetus with these physiological mechanisms as a means of coping. The great dilemma for the obstetrician in managing variable fetal heart rate decelerations is determining when variable decelerations are pathological. According to the American College of 0bstetricians and Gynecologists (2017 a), recurrent variable decelerations with minimal-to-moderate beat-to-beat variability are indeterminate, whereas those with absent variability are abnorma.

1	FIGURE 24-16 Total umbilical cord occlusion (arrow) in the sheep fetus is accompanied by an increase in fetal aortic blood pressure. Blood pressure changes in the umbilical vessels are also shown. (Redrawn with permission from Kunzel W: Fetal heat rate alterations in partial and total cord occlusion. In Kunzel W (ed): Fetal Heart Rate Monitoring: Clinical Practice and Pathophysiology. Berlin, Springer, 1985.) FIGURE 24-17 Varying (variable) fetal heart rate decelerations. Deceleration (8) exhibits "shoulders" of acceleration compared with deceleration (A).

1	Fetal heart rate Uterine contraction occlusion Complete occlusion IIIIIIIIII I: Partial occlusion Umbilical vein Umbilical artery Fetal systolic BP Umbilical cord FIGURE 24-18 Schematic representation ofthe fetal heart rate efects with partial and complete umbilical cord occlusion. Uterine pressures generated early in a contraction cause cord compres-sion predominantly ofthe thin-walled umbilical vein. The resulting decrease in fetal cardiac output leads to an initial compensatory rise in fetal heart rate. As cord compression intensifies, umbilical arteries are then also compressed. The resulting rise in fetal systolic blood pressure leads to a vagal-mediated fetal heart rate deceleration. As the contraction abates and compression is relieved first on the umbilical arteries, elevated fetal systolic blood pressures drop and the deceleration resolves. A final increase in fetal heart rate is seen as a result of persistent umbilical vein occlusion. With completion of the uterine

1	fetal systolic blood pressures drop and the deceleration resolves. A final increase in fetal heart rate is seen as a result of persistent umbilical vein occlusion. With completion of the uterine contraction and cord compression, the fetal heart rate returns to baseline. BP = blood pressure. (Adapted with permission from Lee (V, DiLaretto pc, Lane JM: A study offetal heart rate acceleration patterns, Obstet Gynecol. 1975 Feb;45(2):142-146.) Other fetal heart rate patterns have been associatedwith umbilical cord compression. Saltatoy baseline heart rate (Fig. 24-19) was first linked o umbilical cord complications during labor (Hammacher, 1968). he pattern consists ofrapidlyrecurringcouplets of acceleration and deceleration causing relatively large oscillations of the baseline fetal heart rate. We also observed a relationship between cord occlusion and the saltatory pattern in postterm pregnancies (Leveno, 1984). In the absence of other fetal heart rate finings, these do not signal fetal

1	We also observed a relationship between cord occlusion and the saltatory pattern in postterm pregnancies (Leveno, 1984). In the absence of other fetal heart rate finings, these do not signal fetal compromise. Lamba is a pattern involving an acceleration followed by a variable deceleration with no acceleration at the end ofthe deceleration. This pattern typically is seen in early labor and is not ominous (Freeman, 2003). This lambda pattern may result from mild cord compression or stretch. Overshootis a variable deceleration followed by acceleration. The clinical significance ofthis pattern is controversial (Westgate, 2001). Prolonged Deceleration This pattern, which is shown in Figure 24-20, is deined as an isolated deceleration ::15 bpm that lasts ::2 minutes but

1	FIGURE 24-19 Saltatory baseline fetal heart rate showing rapidly recurring couplets ofacceleration combined with deceleration. < 10 minutes from onset to return to baseline. Prolonged decelerations are diicult to interpret because they are seen in many diferent clinical situations. Some of the more frequent causes are cervical examination, uterine hyperactivity, cord entanglement, and maternal supine hypotension. Epidural, spinal, or paracervical analgesia may induce a prolonged deceleration (Eberle, 1998). Hill and associates (2003) observed prolonged deceleration in 1 percent of women given epidural analgesia during labor at Parkland Hospital. Other causes of prolonged deceleration include maternal hypoperfusion or hypoxia from any cause, placental abruption, umbilical

1	FIGURE 24-20 Prolonged fetal heart rate deceleration due to uterine hyperactivity. Approximately 3 minutes of the tracing are shown, but the fetal heart rate returned to normal after uterine hypertonus resolved. Vaginal delivery later ensued. Loss of variability pH 6.9 FIGURE 24-21 Cord-compression fetal heart rate decelerations in second-stage labor associated with tachycardia and loss of variability. The umbilical cord arterial pH was 6.9. cord knots or prolapse, maternal seizures including eclampsia and epilepsy, application of a fetal scalp electrode, impending birth, or maternal Valsalva maneuver. In one example, Ambia and colleagues (2017) described prolonged decelerations lasting 2 to 10 minutes following an eclamptic seizure.

1	he placenta is efective in resuscitating the fetus if the original insult does not recur immediately. Occasionally, such self-limited prolonged decelerations are followed by loss of beat-to-beat variability, baseline tachycardia, and even a period of late decelerations, all of which resolve as the fetus recovers. Freeman and colleagues (2003) emphasize that the fetus may die during prolonged decelerations. hus, management of prolonged decelerations can be extremely tenuous. Management of isolated prolonged decelerations is based on bedside clinical judgment, which inevitably will sometimes be imperfect given the unpredictability of these decelerations.

1	Decelerations are virtually ubiquitous during the second stage of labor. In one study, only 1.4 percent of more than 7000 deliveries lacked decelerations during second-stage labor (Melchior, 1985). Both cord and fetal head compressions have been implicated as causes of decelerations and baseline bradycardia in this stage. Profound, prolonged fetal heart rate deceleration in the 10 minutes preceding vaginal delivery has been described (Boehm, 1975). And, similar prolonged second-stage decelerations were associated with a stillbirth and neonatal death (Herbert, 1981). hese experiences attest to the unpredictability of the fetal heart rate during second-stage labor.

1	Spong and associates (1998) analyzed the characteristics of second-stage variable fetal heart rate decelerations in 250 deliveries. hey found that as the total number of decelerations <70 bpm increased, the 5-minute Apgar score decreased. Of other patterns in second-stage labor, Picquard and coworkers (1988) reported that loss of beat-to-beat variability and baseline fetal heart rate < 90 bpm predicted fetal acidemia. Krebs and associates (1981) also found that persistent or progressive baseline bradycardia or baseline tachycardia was associated with lower Apgar scores. Gull and colleagues (1996) observed that abrupt fetal heart rate deceleration to < 100 bpm associated with loss of beat-to-beat variability for 4 minutes or longer was predictive of fetal acidemia. Thus, abnormal baseline heart rate-either bradycardia or tachycardia, absent beat-tobeat variability, or both-in the presence of deep second-stage decelerations is associated with a greater risk for fetal compromise (Fig.

1	With this approach, women with low-risk pregnancies are monitored for a short time on admission for labor. In one study, 3752 low-risk women in spontaneous labor at admission were randomly assigned either to auscultation of the fetal heart or to 20 minutes of electronic fetal monitoring (Mires, 2001). Use of admission electronic fetal monitoring did not improve neonatal outcome. Moreover, its use resulted in a greater number of interventions, including operative delivery. A similar study echoed these neonatal outcomes (Impey, 2003). More than half of the women enrolled in these studies eventually required continuous monitoring. A review by Devane and associates (2017) found that admission fetal monitoring programs for low-risk pregnancy are associated with a higher risk for cesarean delivery. Somewhat related, with the increasing rate of scheduled cesarean deliveries in the United States, clinicians and hospitals must decide whether fetal monitoring is required before the procedure in

1	Somewhat related, with the increasing rate of scheduled cesarean deliveries in the United States, clinicians and hospitals must decide whether fetal monitoring is required before the procedure in low-risk women.

1	Fetal heart rate pattern interpretations are subjective. hus, the potential for computer assistance to enhance the precision of identiying abnormal patterns appeared promising. he INFANT Collaborative Group (2017) studied whether the addition of computer-based decision-support software for interpretation of fetal heart rate patterns lowered the number of poor neonatal outcomes. In this trial, 23,515 women were randomized to computer-assisted interpretation compared with 23,055 women in a conventional clinical interpretation arm. Perinatal outcomes such as intrapartum stillbirth, early neonatal death, and neonatal encephalopathy were not improved by computer assistance. Cesarean delivery rates were similar in both groups. Moreover, a 2-year follow-up of a subset of the surviving children showed no diferences in their neurological development.

1	According to the American College of Obstetricians and Gynecologists (2017a), measurements of the pH in capillary scalp blood may help identiy the fetus in serious distress. However, this group also emphasizes that neither normal nor abnormal scalp pH results are predictive of neonatal outcome. Notably, the procedure is now used uncommonly and is not available at most hospitals in the United States. With sampling, an illuminated endoscope is inserted through the dilated cervix after membrane rupture and is pressed irmly against the fetal scalp (Fig. 24-22). The skin is wiped clean with a cotton swab and coated with a silicone gel, which allows fetal blood to accumulate s discrete globules. An incision is made through the fetal scalp to a depth of 2 mm with a special blade on a long handle. As a drop of blood forms on the surface, it is immediately collected into a heparinized glass capillary tube . The pH of the blood is measured promptly.

1	FIGURE 24-22 The technique of fetal scalp sampling using an amnioscope. The end of the endoscope is displaced from the fetal vertex approximately 2 cm to show the disposable blade against the fetal scalp before incision. The pH of fetal capillary scalp blood is usually lower than that of umbilical venous blood and approaches that of umbilical arterial blood. In one algorithm, if the pH is :7.25, labor is observed, and if between 7.20 and 7.25, the pH measurement is repeated within 30 minutes (Zalar, 1979). If the pH is <7.20, another scalp blood sample is collected immediately, and the mother is taken to an operating room and prepared for surgery. Delivery is performed promptly if the low pH is conirmed. Otherwise, labor is allowed to continue, and scalp blood samples are repeated periodically.

1	he only beneits reported for scalp blood pH testing are fewer cesarean deliveries for fetal distress (Young, 1980b). However, Goodwin and coworkers (1994) showed a decrease in the scalp pH sampling rate from approximately 1.8 percent in the mid-1980s to 0.03 percent by 1992. This drop in sampling rate was not associated with a higher cesarean delivery rate for fetal distress. They concluded that scalp blood pH sampling was unnecessary. Kruger and colleagues (1999) have advocated the use of fetal scalp blood lactate concentration as an adjunct to pH. WibergItzel and associates (2008) randomly assigned 1496 fetuses to scalp blood pH analysis and 1496 to scalp blood lactate analysis. They found either to be equivalent in predicting fetal acidemia. The advantage of lactate measurement was that a smaller amount of blood was needed, which led to a lower procedural failure rate compared with scalp blood sampling for pH.

1	Clark and coworkers (1984) have suggested that fetal scalp stimulation is an alternative to scalp blood sampling. his proposal was based on the observation that heart rate acceleration in response to pinching the fetal scalp with an Allis clamp just before obtaining blood was invariably associated with a normal pH. Conversely, failure to provoke acceleration was not uniformly predictive of fetal acidemia. Later, Elimian and associates (1997) reported that of 58 cases in which the fetal heart rate accelerated > 10 bpm after 15 seconds of gentle digital stroking of the scalp, 100 percent had a scalp blood pH of > 7.20. Without an acceleration, however, only 30 percent had a scalp blood pH > 7.20. Following a prospective cohort study, Tahir Mahmood and coworkers (2017) concluded that fetal scalp stimulation was a reliable alternative to scalp blood pH determination.

1	Fetal heart rate acceleration in response to vibroacoustic stimulation has been recommended as a substitute for fetal scalp blood sampling (Edersheim, 1987). The technique uses an electronic artificial larynx placed approximately 1 cm from or directly onto the maternal abdomen (Chap. 17, p. 337). Response to vibroacoustic stimulation is considered normal if a fetal heart rate acceleration of at least 15 bpm for at least 15 seconds occurs within 15 seconds after the stimulation and with prolonged fetal movements (Sherer, 1994). 0 0 80 40 o ,, 15 1111111111111111111111-1-"."." ".".".".'." 45 60 75 FIGURE 24-23 Frequency distribution of umbilical artery oxygen saturation values in 1281 vigorous newborn infants. Dotted line indicates normal distribution. (Redrawn with permission from Arikan GM, Scholz HS, Petru E, et al: Cord blood oxygen saturation in vigorous infants at birth: what is normal? BJOG. 2000 Aug;l07(8):987-994.)

1	Lin and colleagues (2001) prospectively studied vibroacoustic stimulation in 113 women in labor with either moderateto-severe variable or late fetal heart rate decelerations. hey concluded that this technique is an efective predictor of fetal acidosis in the setting ofvariable decelerations. The predictability for fetal acidosis, however, is limited in the setting of late decelerations. Other investigators have reported that vibroacoustic stimulation in second-stage labor did not predict neonatal outcome or enhance labor management (Anyaegbunam, 1994).

1	Skupski and coworkers (2002) performed a metaanalysis of reports on intrapartum fetal stimulation tests published between 1966 and 2000. Four types of fetal stimulation were analyzed and included fetal scalp puncture for blood pH testing, llis clamp pinching of the fetal scalp, vibroacoustic stimulation, and digital strokingofthe fetal scalp. Results were similar for all four methods. These investigators concluded that intrapartum stimulation tests were useful to exclude fetal acidemia. They cautioned, however, that these tests are "less than perfect."

1	Using technology similar to that of adult pulse oximetry, this instrumentation allows assessment offetal oxyhemoglobin saturation once membranes are ruptured. A unique padlike sensor is inserted through the cervix and positioned against the fetal face. he transcervical device reliably registers fetal oxygen saturation in 70 to 95 percent ofwomen throughout 50 to 88 percent of their labors (Yam, 2000). Using fetal pulse oximetry, the lower limit for normal fetal oxygen saturation is generally considered to be 30 percent (Gorenberg, 2003; Stiller, 2002). However, when measured in umbilical arterial blood, fetal oxygen saturation normally varies greatly, as shown in Figure 24-23. Bloom u:

1	J:Jr� and associates (1999) reported that brief, transient fetal oxygen saturations <30 percent were common during labor because such values were observed in 53 percent offetuses with normal outcomes. When persistent for 2 minutes or longer, however, saturation values <30 percent were associated with a greater risk of potential fetal compromise. women with term pregnancies and in whom predeined abnor mal fetal heart rate patterns developed. Patients received either continuous fetal pulse oximetry. he use of fetal pulse oxim etrysigniicantlyreduced the cesarean delivery rate for nonreas suring fetal status from 10.2 to 4.5 percent. Alternatively, the cesarean delivery rate for dystocia rose significantly from 9 to 19 percent when pulse oximetry was used. No neonatal ben eits or adverse efects were associated with fetal pulse oximetry. Based on these observations, the Food and Drug Administra tion approved marketing of the Nellcor N-400 Fetal Oxygen Monitoring System.

1	Based on these observations, the Food and Drug Administra tion approved marketing of the Nellcor N-400 Fetal Oxygen Monitoring System. Since then, three other randomized trials have compared fetal pulse oximetry with standard care. In all three trials, neo natal outcomes were similar between the two study arms. East and coworkers (2006) reported that the addition of oximetry fetal heart rate pattern. However, Bloom (2006) and lauser (2005), each with their colleagues, found no diference in cesar ean delivery rates between the two study groups. Because of these indings, in 2005, the manufacturer discontinued sale of the fetal oximeter system in the United States.

1	As fetal hypoxia worsens, the fetal ECG changes. Namely, the mature fetus exposed to hypoxemia develops an elevated ST segment and a progressive rise in the T-wave height that can be expressed as a T:QRS ratio (Fig. 24-24). Increasing T:QRS ratios are thought to relect the fetal cardiac ability to adapt to hypoxia and appear before neurological damage. Further worsening of hypoxia then leads to progressively negative ST-segment delection that takes on a biphasic form (Fig. 24-25). It is reasonable to consider that ST-segment abnormalities might occur late in the course of fetal compromise. Indeed, it has been hypothesized that ST-segment changes relect myocardial tissue hypoxia.

1	Because of these indings, several investigators have assessed the value of analyzing these parameters as an adjunct to conventional fetal monitoring. The technique requires internal fetal heart monitoring and special equipment to process the fetal ECG. In 2005, the manufacturer-Neoventa Medicalreceived Food and Drug Administration approval for their ST analysis program named the STAN system. Several studies have evaluated ST-segment changes with fetal monitoring. In one randomized trial of 2400 pregnancies, neonatal outcomes were not improved compared with those in which conventional fetal monitoring alone was used (Westgate, 1993). However, the cesarean delivery rate for fetal distress declined in those with ST-segment analysis. Amer-Wahlin and colleagues (2001, 2007) found that the addition ofST-segment analysis to conventional fetal monitoring significantly lowered cesarean delivery rates for fetal distress and reduced metabolic acidemia in umbilical artery blood.

1	A Normal ST• Aerobic metabolism B Generation ofT:QRS ratios• Positive energy balance raw-ECG Average of 30 ECG complexes IncreasedT-wave amplitude• Hypoxia/anaerobic metabolism• Adrenaline surge R T ratio FIGURE 24-24 A. ST segment changes in normal and hypoxic conditions. B. Generation ofT:QRS ratios. (Redrawn with permission from Devoe L: ECG analysis: the next generation in electronic fetal monitoring? Contemporary Ob/Gyn, September 15,o2006.) Subsequently, Doria and associates (2007) introduced STAN as a clinical practice and reported no changes in the incidence of operative delivery or neonatal encephalopathy. And, one metaanalysis of ive randomized trials comprising 15,352 patients found that ST-segment analysis did not lower rates of cesarean delivery or fetal metabolic acidemia at birth (Becker, 2012).

1	Last, in a trial by the NICHD, 5532 women were randomly assigned to an ST-segment analysis arm (the open group) and 5576 to standard intrapartum management (the masked group). he primary outcome was a composite of one or more of seven events associated with fetal compromise (Belfort, 2015). In the open group, clinical practice was directed to some degree by predetermined ST-segment analysis guidelines. hese stipulated that intervention should be withheld, that is, expectant management adopted, for at least 60 minutes despite the presence of minimal variability; variable decelerations lasting �60 seconds or dropping to �60 bpm; recurrent late decelerations; or prolonged decelerations lasting >2 minutes, so long as no ST-eventwas present. hese guidelines did not pertain to the standard usual management group. Notably, in the open group, 55 women were delivered when STAN guidelines indicated that labor should continue. his composed 20 percent ofthe total 287 cesarean deliveries performed

1	management group. Notably, in the open group, 55 women were delivered when STAN guidelines indicated that labor should continue. his composed 20 percent ofthe total 287 cesarean deliveries performed for fetal distress in this group. Clearly, the attending physicians abandoned the open group protocol that stipulated nonintervention. They likely perceived the fetal heart rate patterns to reflect those formerly accepted in their usual practice as nonreassuring.

1	FIGURE 24-25 Biphasic ST-segment waveform with progressive fetal hypoxia. (Adapted with permission from Devoe L: ECG analysis: the next generation in electronic fetal monitoring? Contemporary Ob/Gyn, September 15,o2006.) The results of this trial showed that STAN had no efect on neonatal outcome or cesarean delivery rates (Belfort, 2015). In their review, Neilson and colleagues (2015) reached similar conclusions. hese results have essentially eliminated use ofSTsegment analysis in the United States, but this technology is still used in Europe. Doppler interrogation of the umbilical artery has been studied as another potential adjunct to conventional fetal monitoring. Further described in Chapter 10 (p. 213), abnormal Doppler waveforms may signiy pathological umbilical-placental vessel resistance. From their review, Farrell and associates (1999) concluded that this technique, used intrapartum, was a poor predictor of adverse perinatal outcomes.

1	The termetal distress is too broad and vague to be applied with any precision to clinical situations (American College ofObstetricians and Gynecologists, 2014). Uncertainty regarding the diagnosis based on interpretation of fetal heart rate patterns has given rise to descriptions such as reassuring or nonreassuring. he term "reassuring" suggests a restoration of confidence in the health of the fetus by a particular pattern. In contrast, a "nonreassuring" designation suggests inability to remove doubt. hese patterns during labor are dynamic, and they can rapidly change from reassuring to nonreassuring and vice versa. These assessments are subjective clinicaljudgments that are inevitaby subject to impeection and must be reconized as such.

1	These assessments are subjective clinicaljudgments that are inevitaby subject to impeection and must be reconized as such. The diiculty in assigning a nonreassuring label to fetal heart rate patterns stems in part from the fact that these patterns are more a reflection of fetal physiology than of pathology. Physiological control of heart rate includes various interconnected mechanisms that depend on blood flow and oxygenation. Moreover, the activity of these control mechanisms is inluenced by the preexisting state of fetal oxygenation, for example, as seen with chronic placental insuiciency. Importantly, the fetus is tethered by an umbilical cord, whereby blood flow is constantly in jeopardy. Moreover, normal labor is a process of increasing acidemia (Rogers, 1998). hus, normal labor is a process of repeated fetal hypoxic events that can infrequently lead to signiicant acidemia.

1	Identiication of "fetal distress" based on fetal heart rate patterns is imprecise and controversial. Experts in interpretation of these patterns often disagree with each other. Ayres-de-Campos and colleagues (1999) investigated interobserver agreement of fetal heart rate pattern interpretation and found that agreement-or conversely, disagreement-was related to whether the pattern was normal, suspicious, or pathological. Speciically, experts agreed on 62 percent of normal patterns, 42 percent of suspicious patterns, and only 25 percent of pathological patterns. Keith and coworkers (1995) asked each of 17 experts to review 50 tracings on two occasions, at least 1 month apart. Approximately 20 percent changed their own interpretations, and approximately 25 percent did not agree with the interpretations of their colleagues.

1	To develop standardized and unambiguous deinitions of fetal heart rate (FHR) tracings, the NICHD (1997) held a succession of workshops in 1995 and 1996 and published recommendations for interpreting these patterns. As previously shown in Table 24-1, a second workshop was convened to reevaluate these recommendations and clariy terminology (Macones, 2008). A major result was the recommendation of a three-tier system for classification of FHR patterns (1 able 24-2). The American College of Obstetricians and Gynecologists (2017b) has recommended use of this tiered system.

1	A few studies have assessed this three-tiered system. J ackson and coworkers (201i1) studied 48,444 women in labor and found that category I (normal FHR) patterns were observed during labor in 99.5 percent of tracings. Category II (indeterminate FHR) patterns were found in 84.i1 percent of tracings, and category III (abnormal FHR) patterns were seen in 0.1 percent (54 women). Most-84 percent of women-had a mix of categories during labor. Cahill and colleagues (2012) retrospectively correlated the incidence of umbilical cord acidemia (pH :;7.10) with fetal heart rate characteristics during the 30 minutes preceding delivery. None of the three categories demonstrated a significant association with cord blood acidemia. The American College of Obstetricians and Gynecologists and the American Academy of Pediatrics (2014) concluded that a category I or II tracing with a 5-minute Apgar scorei> 7 or with normal arterial blood acid-base values was not consistent with an acute hypoxic-ischemic

1	Academy of Pediatrics (2014) concluded that a category I or II tracing with a 5-minute Apgar scorei> 7 or with normal arterial blood acid-base values was not consistent with an acute hypoxic-ischemic event.

1	TABLE 24-2. Three-Tier Fetal Heart Rate Interpretation System Include all of the following: • Baseline rate: 110-160 bpm Baseline FHR variability: moderate Early decelerations: present or absent Accelerations: present or absent Include all FHR tracings not categorized as Category I or III. of those encountered in clinical care. Examples include any of the following: Absence of induced accelerations after fetal stimulation Periodic or episodic decelerations Prolonged deceleration :2 min but < 1 0 min Variable decelerations with other characteristics, such as slow return to baseline, "overshoots,"

1	Prolonged deceleration :2 min but < 1 0 min Variable decelerations with other characteristics, such as slow return to baseline, "overshoots," Include either: • Absent baseline FHR variability and any of the following: bpm = beats per minute; FHR = fetal heart rate. Reproduced with permission from Macones GAl Hankins GO, Spong CY, et al: The 2008 National Institute of Child Health and Human Development workshop report on electronic fetal monitoring: update on definitions, interpretation, and research guidelines, Obstet Gynecol. 2008 Sep;1s12(3))661-666.

1	Sholapurkar (2012) challenged the validity of the threetier system because most abnormal fetal heart rate patterns fall into the indeterminate category II. It was further suggested that this resulted from most fetal heart rate decelerations being inappropriately classiied as variable decelerations due to cord compression. A group of 19 experts led by Clark (2013) observed that more than 80 percent of fetuses have FHR patterns in tier II. hey proposed a management algorithm for these fetuses, however, their hypothetical algorithm was not clinically tested.

1	Parer and King (2010) compared this situation in the United States with that of other countries in which a consensus on classiication and management has been reached by several professional societies. Some of these include the Royal College of Obstetricians and Gynaecologists, the Society of Obstetricians and Gynaecologists of Canada, the Royal Australian and New Zealand College of Obstetricians and Gynaecologists, and the Japan Society of Obstetrics and Gynecology. These authors further comment that the NICHD three-tier system is inadequate because category II-an indeterminate FHR pattern-contains a vast heterogenous mixture of patterns that prevents development of a management strategy.

1	Parer and Ikeda (2007) had previously proposed a colorcoded ive-tier system for both FHR interpretation and management. Two subsequent reports have compared the five-and three-tier systems. Bannerman and associates (2011) found that the two systems were similar in fetal heart rate interpretations for tracings that were either very normal or very abnormal. Coletta and coworkers (2012) found that the ive-tier system had better sensitivity than the three-tier system. Elliott and colleagues (2010) used computerization to measure the performance of a ive-tier classiication system but failed to successfully analyze and categorize 2472 fetal heart recordings. It is apparent that, after 50 years of continuous electronic fetal heart rate monitoring use, there is not a consensus on interpretation and management recommendations for FHR patterns (Parer, 2011). • Meconium in the Amnionic Fluid

1	• Meconium in the Amnionic Fluid Obstetricians have long realized that meconium during labor is problematic in the prediction of fetal distress or asphxia. Indeed, although 12 to 22 percent oflabors are complicated by meconium, only a few are linked to neonatal mortality. In an investigation from Parkland Hospital, meconium was found to be a "low-risk" obstetrical hazard because the perinatal mortality rate attributable to meconium was only 1 death per 1000 live births (Nathan, 1994).

1	Three theories regarding fetal passage of meconium may explain, in part, the tenuous connection between its detection and neonatal mortality. First, fetuses may pass meconium in response to hypoxia, and meconium therefore signals fetal compromise (Walker, 1953). Second, in utero passage of meconium may represent normal gastrointestinal tract maturation under neural control (Mathews, 1979). A inal theory posits that meconium passage follows vagal stimulation from common but transient umbilical cord entrapment with resultant increased bowel peristalsis (Hon, 1961).

1	Ramin and associates (1996) studied almost 8000 pregnancies with meconium-stained amnionic fluid delivered at Parkland Hospital. Meconium aspiration syndrome was signiicantly associated with fetal acidemia at birth. Other signiicant correlates of aspiration included cesarean delivery, forceps to expedite delivery, intrapartum heart rate abnormalities, depressed Apgar scores, and need for assisted ventilation at delivery. Analysis of the type of fetal acidemia based on umbilical blood gases suggested that the fetal compromise associated with meconium aspiration syndrome was an acute event. his is because most acidemic fetuses had abnormally increased PC02 values rather than a pure metabolic acidemia. Dawes and coworkers (1972) observed that such hypercarbia in fetal lambs induces gasping and resultant increased amnionic fluid inhalation. Jovanovic and Nguyen (1989) observed that meconium gasped into the fetal lungs caused aspiration syndrome only in asphyxiated animals.

1	Ramin and colleagues (1996) hypothesized that the pathophysiology of meconium aspiration syndrome includes, but is not limited to, fetal hypercarbia, which stimulates fetal respiration leading to aspiration of meconium into the alveoli. Lung parenchymal injury is secondary to acidemia-induced alveolar cell damage. In this pathophysiological scenario, meconium in amnionic fluid is a fetal environmental hazard rather than a marker of preexistent compromise. This proposed pathophysiological sequence is not all-inclusive, because it does not account for approximately half of the cases of meconium aspiration syndrome in which the fetus is not acidemic at birth.

1	Thus, it was concluded that the high incidence of meconium observed in the amnionic fluid during labor often represents fetal passage of gastrointestinal contents in conjunction with normal physiological processes. lthough normal, such meconium becomes an environmental hazard when fetal acidemia supervenes. Importantly, such acidemia occurs acutely, and therefore meconium aspiration is unpredictable and likely unpreventable. Moreover, Greenwood and colleagues (2003) showed that clear amnionic fluid was also a poor predictor. In a prospective study of 8394 women with clear amnionic fluid, they found that clear fluid was an unreliable sign of fetal wellbeing.

1	Growing evidence indicates that many newborns with meconium aspiration syndrome have suffered chronic hypoxia before birth (Ghidini, 2001). Blackwell and associates (2001) found that 60 percent of neonates diagnosed with meconium aspiration syndrome had umbilical artery blood pH ::7.20, implying that the syndrome was unrelated to the neonatal condition at delivery. Similarly, markers of chronic hypoxia, such as elevated fetal erythropoietin levels and increased nucleated red blood cell counts in newborns, suggest that chronic hypoxia is involved in many meconium aspiration syndrome cases (Dollberg, 2001; Jazayeri, 2000).

1	In the recent past, routine obstetrical management of a newborn with meconium-stained amnionic fluid included intrapartum suctioning of the oropharynx and nasopharynx. In 2005, management guidelines were signiicantly modiied. Now, the American College of Obstetricians and Gynecologists (2017c) recommends that newborns with meconium-stained amnionic fluid, regardless of their vigor, should no longer routinely receive intrapartum suctioning. Suctioning is reserved for those with airway obstruction. hey also recommend that an appropriately credentialed team with full resuscitation skills be available (Chap. 32, p. 607). TABLE 24-3. Some Resuscitative Measures for Category II or Category III Tracings Tachysystole with category II or III tracing Discontinue oxytocin or prostaglandins; tocolytics: terbutaline, magnesium sulfate

1	Tachysystole with category II or III tracing Discontinue oxytocin or prostaglandins; tocolytics: terbutaline, magnesium sulfate Recurrent variable decelerations Reposition mother; amnioinfusion; with cord prolapse, manually elevate Prolonged decelerations or bradycardia the presenting part while preparing for immediate delivery aSimultaneous evaluation of the suspected cause(s) is also an important step in management of abnormal FHR tracings. The combination of multiple interventions Simultaneously may be appropriate and potentially more effective than doing them individually or serially. FHR = fetal heart rate.

1	FHR = fetal heart rate. Principal management options for variant fetal heart rate patterns consist of correcting any fetal insult, if possible. Suggestions are listed in Table 24-3. he woman is moved to a lateral position, and supplemental oxygen is provided by mask. Correcting maternal hypotension caused by regional analgesia and discontinuing oxytocin both serve to improve uteroplacental perfusion. Vaginal examination excludes a prolapsed cord or impending delivery. Simpson and James (2005) assessed the beneits of three maneuvers in 52 women with fetal oxygen saturation sensors already in place. They used intravenous hydration-500 to 1000 mL of lactated Ringer solution given over 20 minutes; lateral versus supine positioning; and administration of supplemental oxygen at lOiLl min using a nonrebreathing mask. Each of these maneuvers signiicantly raised fetal oxygen saturation levels.

1	Terbutaline sulfate given to relax the uterus can be a temporizing maneuver in the management of nonreassuring fetal heart rate patterns during labor. A single 250-�g intravenous or subcutaneous injection is used to inhibit uterine contractions and thereby improve fetal oxygenation. Cook and Spinnato (1994) described their 10-year experiences with terbutaline tocolysis in 368 pregnancies. Such resuscitation improved fetal scalp blood pH values, although all fetuses underwent cesarean delivery. hese investigators concluded that although the studies were small and rarely randomized, most reported favorable results with terbutaline tocolysis for nonreassuring patterns. Small intravenous doses of nitroglycerin-60 to 180 �g-also have been reported to be beneicial (Mercier, 1997). Bullens and associates (2015) concluded in their review that tocolysis was beneicial. Still, the American College of Obstetricians and Gynecologists (20i1i7b) cites that evidence is insuicient to recommend

1	and associates (2015) concluded in their review that tocolysis was beneicial. Still, the American College of Obstetricians and Gynecologists (20i1i7b) cites that evidence is insuicient to recommend tocolysis for nonreassuring fetal heart rate patterns.

1	Such therapy improved the heart rate pattern in half of the women studied. Later, .Miyazaki and Nevarez (1985) randomly assigned 96 nulliparas in labor with cord compression patterns and found that those who were treated with amnioinfusion required cesarean delivery for fetal distress less often. Based on many of these early reports, transvaginal amnioinfusion has been extended into three clinical areas (Dad, 2016). hese include: (1) treatment of variable or prolonged decelerations; (2) prophylaxis for women with oligohydramnios, as with prolonged ruptured membranes; and (3) attempts to dilute or wash out thick meconium (Chap. 33, p. 620).

1	Many diferent amnioinusion protocols have been reported, but most provide a 500-to 800-mL bolus of warmed normal saline followed by a continuous inusion of approximately 3 mLimin (Owen, 1990; Pressman, 1996). In another study, Rinehart and colleagues (2000) gave either a 500-mL bolus of normal saline at room temperature alone or a similar bolus plus a continuous inusion at 3 mLimin. heir study included 65 women with variable decelerations, and the investigators found neither method to be superior. Wenstrom and associates (1995) surveyed use of mnioinusion in teaching hospitals in the United States. The procedure was used in 96 percent of the 186 centers surveyed, and it was estimated that 3 to 4 percent of llwomen delivered at these centers received such inusion. Potential complications of mnioinusion are summarized in Table 24-4. TABLE 24-4. Complications Associated with Amnioinfusion from a Survey of 186 Obstetrical Units Data from Wenstrom, 1995.

1	TABLE 24-4. Complications Associated with Amnioinfusion from a Survey of 186 Obstetrical Units Data from Wenstrom, 1995. able or prolonged decelerations attributed to cord entrapment. For variable decelerations, Hofmeyr and Lawrie (2012) reviewed the efects of amnioinfusion in the management of fetal heart rate patterns associated with umbilical cord compression. They concluded that amnioinfusion appeared to be useful in reducing the occurrence of variable decelerations, improving neonatal outcome, and lowering cesarean delivery rates. The American College of Obstetricians and Gynecologists (2016) has concluded that amnioinfusion is a reasonable approach in the treatment of repetitive variable decelerations regardless of meconium status.

1	For oligohydramnios, amnioinfusion has been used prophylactically to avoid intrapartum fetal heart rate patterns from cord occlusion. Nageotte and coworkers (1991) found that this resulted in signiicantly fewer and less severe variable decelerations in labor. However, the cesarean delivery rate or condition of term newborn was not improved. In a randomized investigation, Macri and colleagues (1992) studied prophylactic amnioinfusion in 170 term and postterm pregnancies complicated by both thick meconium and oligohydramnios. Amnioinfusion significantly reduced meconium aspiration syndrome rates and cesarean delivery rates for fetal distress. In contrast, Ogundipe and associates (1994) randomly assigned 116 term pregnancies with an amnionic luid index < 5 cm to receive prophylactic amnioinfusion or standard obstetrical care. Overall cesarean delivery rates, delivery rates for fetal distress, or umbilical cord acid-base studies did not difer significantly between groups.

1	For meconium-stained amnionic luid, Pierce and associates (2000) reviewed 13 prospective trials of intrapartum amnioinfusion for 1924 women with meconium-stained fluid. In the amnioinfusion group, newborns were signiicantly less likely to have meconium below the vocal cords, and meconium aspiration syndrome rates were lower. he cesarean delivery rate was also reduced in the amnioinfusion group. Similar results were reported by Rathore and coworkers (2002).

1	In contrast, several investigators were not supportive of amnioinfusion for meconium staining. For example, Usta and associates (1995) reported that amnioinfusion was not feasible in half of women with moderate or thick meconium who were randomized to this treatment. These investigators were unable to demonstrate improved neonatal outcomes with this treatment. Spong and coworkers (1994) also concluded that although prophylactic amnioinfusion did dilute meconium, it did not improve perinatal outcome. Last, Fraser and colleagues (2005) randomized amnioinfusion in 1998 women with thick meconium-stained amnionic fluid in labor and found no beneits. Hofmeyr and associates (2014) reported mixed results from their review. Because of these indings, the American College of Obstetricians and Gynecologists (2016) does not recommend amnioinfusion to dilute meconium-stained amnionic fluid.

1	Studies that have attempted to correlate fetal heart rate patterns with brain injury primarily have examined infants identiied in medicolegal actions. Phelan and Ahn (1994) reported that among 48 fetuses later found to be neurologically impaired, a persistent nonreactive fetal heart rate tracing was already present at the time of admission in 70 percent. They concluded that fetal neurological injury occurred predominately before arrival to the hospital. When they looked retrospectively at heart rate patterns in 209 brain-injured newborns, they concluded that there was not a single unique pattern associated with fetal neurological injury (Ahn, 1996). Graham and associates (2006) reviewed the world literature published between 1966 and 2006 on the efect of fetal heart rate monitoring to prevent perinatal brain injury and found no beneit.

1	Fetal heart rate patterns necessary for perinatal brain damage have been studied in experimental animals. .1yers (1972) described the efects of complete and partial asphyxia in rhesus monkeys. Complete asphyxia was produced by total occlusion of umbilical blood low that led to prolonged deceleration (Fig. 24-26). Fetal arterial pH did not drop to 7.0 until approximately 8 minutes after complete cessation of oxygenation and umbilical low. At least 10 minutes of such prolonged deceleration was required before there was evidence of brain damage in surviving fetuses. Myers (1972) also produced partial asphyxia in rhesus monkeys by impeding maternal aortic blood flow. This resulted in ...A �EEI0). /0 5 1005 10 Asphyxia (min) 0. .Cordclamp010 120 20 40 0)0 .. :JQ) .0

1	.Cordclamp010 120 20 40 0)0 .. :JQ) .0 FIGURE 24-26 Prolonged deceleration in a rhesus monkey shown with blood pressure and biochemical changes during total occlusion of umbilical cord blood flow. (Data from Myers, 1972.) analgesia was used in 55 percent. The length of the second stage, even in those lasting up to 6 hours or more, was not related to neonatal outcome. These results were attributed to careful use of electronic monitoring and scalp pH measurements. These investigators concluded that there is no compelling reason to intervene with a possibly diicult forceps or vacuum extraction because a certain number of hours have elapsed. They observed, however, that after 3 hours in the second stage, delivery by cesarean or other operative method increased progressively. By 5 hours, the prospects for spontaneous delivery in the subse quent hour were only 10 to 15 percent.

1	Newer guidelines have been promoted by the Consensus Com mittee (2016) for second-stage labor. These recommend allowing a nullipara to push for at least 3 hours and a multipara to push for at least 2 hours before second-stage labor arrest is diagnosed. One caveat is that maternal and fetal status should be reassuring. These authors provide options to these times before cesarean deliv ery is performed. Namely, longer durations may be appropriate as long as progress is documented. Also, a specific maximal length of undergo operative delivery has not been identiied.

1	Intuitively, the goal to lower cesarean delivery rates is best balanced with one to ensure neonatal safety. And, it is problematic that no robust data on neonatal outcomes support the safety of allowing prolonged second-stage labor. Data from many evaluations reveal that serious newborn consequences attend second-stage labors longer than 3 hours (Allen, 2009; Bleich, 2012; Laughon, 2014; Leveno, 2016; Rosenbloom, 2017). Other data, when adjusted for labor variables, show no diference in neonatal complications for these longer second stages (Cheng, 2004; Le Ray, 2009; Rouse, 2009). Grobman and colleagues (2016) have argued that the absolute number of such adverse outcomes is small and "overall outcomes remain good." That said, some of the complications are severe. Thus, to fully ascertain specific efect of these guidelines on morbidity rates, randomized controlled trials are needed.

1	It is possible that prolonged first-stage labor presages that with the second stage. Nelson and associates (2013) studied the relationships between the lengths of the irst and second stages of labor in 12,523 nulliparas at term delivered at Parkland Hospital. The second stage signiicantly lengthened concomitantly with increasing irst-stage duration. The 95th percentile was 15.6 and 2.9 hours for the first and second stages, respectively. Women with irst stages lasting longer than 15.6 hours (>95th percetile) had a 16-percent rate of second-stage labor lasting 3 hours (95th percentile). his compared with a 4.5-percent rate of prolonged second stages in women with irst-stage labors lasting <95th percentile.

1	With full cervical dilation, most women cannot resist the urge to "bear down" or "push" each time the uterus contracts (Chap. 22, p. 438). The combined force created by contractions of the uterus and abdominal musculature propels the fetus downward. At times, force created by abdominal musculature is compromised suiciently to slow or even prevent spontaneous vaginal delivery. Heavy sedation or regional analgesia may reduce the reflex urge to push and may impair the ability to contract abdominal muscles efectively. In other instances, the inherent urge to push is overridden by the intense pain created by bearing down. Two approaches to second-stage pushing in women with epidural analgesia have yielded contradictory results. The irst advocates pushing forcefully with contractions after complete dilation, regardless of the urge to push. With the second, analgesia inusion is stopped and pushing begins only ater the woman regains the sensory urge to bear down. Fraser and coworkers (2000)

1	dilation, regardless of the urge to push. With the second, analgesia inusion is stopped and pushing begins only ater the woman regains the sensory urge to bear down. Fraser and coworkers (2000) found that delayed pushing reduced diicult operative deliveries, whereas Manyonda and associates (1990) reported the opposite. Hansen and colleagues (2002) randomly assigned 252 women with epidural analgesia to one of the two approaches. No adverse maternal or neonatal outcomes were linked to delayed pushing despite signiicantly prolonging second-stage labor. Plunkett and coworkers (2003), in a similar study, confirmed these indings.

1	Descent of the leading edge of the presenting part to the level of the ischial spines (0 station) is deined as engagement. A higher station at the onset of labor is signiicantly linked with subsequent dystocia (Friedman, 1965, 1976; Handa, 1993). Roshanfekr and associates (1999) analyzed fetal station in 803 nulliparas at term in active labor. At admission, the third with the fetal head at or below 0 station had a 5-percent cesarean delivery rate. This compared with a 14-percent rate for those with higher stations. The prognosis for dystocia, however, was not related to incrementally higher fetal head stations above the pelvic midplane (0 station). Importantly, 86 percent of nulliparous women without fetal head engagement at diagnosis of active labor delivered vaginally. These observations apply especially for parous women because the head typically descends later in labor.

1	Various labor factors have been implicated as causes of uterine dysfunction. As described, neuraxial analgesia can slow labor and has been associated with lengthening both first and second stages of labor and slowing the rate of fetal descent.

1	Chorioamnionitis is associated with prolonged labor, and some clinicians have suggested that this maternal intrapartum infection itself contributes to abnormal uterine activity. Satin and coworkers (I992) studied the efects of chorioamnionitis on oxytocin stimulation in 266 pregnancies. Infection diagnosed late in labor was found to be a marker of cesarean delivery performed for dystocia. Speciically, 40 percent of women developing chorioamnionitis after requiring oxytocin for dysfunctional labor later required cesarean delivery for dystocia. However, this was not a marker in women diagnosed as having chorioamnionitis early in labor. It is likely that uterine infection in this clinical setting is a consequence of dysfunctional, prolonged labor rather than a cause of dystocia.

1	Membrane rupture at term without spontaneous uterine contractions complicates approximately 8 percent of pregnancies. In the past, labor stimulation was initiated if contractions did not begin ater 6 to 12 hours. Practice-changing research included that of Hannah (1996) and Peleg (1999) and their associates, who enrolled a total of 5042 pregnancies with ruptured membranes in a randomized investigation. They measured the efects of induction versus expectant management and also compared induction using intravenous oxytocin with that using prostaglandin E2 gel. There were approximately 1200 pregnancies in each of the four study arms. They concluded that labor induction with intravenous oxytocin was preferred management. This was based on signiicantly fewer intrapartum and postpartum infections in women whose labor was induced. There were no signiicant differences in cesarean delivery rates. Subsequent analysis by Hannah and coworkers (2000) indicated higher rates of adverse outcomes when

1	in women whose labor was induced. There were no signiicant differences in cesarean delivery rates. Subsequent analysis by Hannah and coworkers (2000) indicated higher rates of adverse outcomes when expectant management at home was compared with in-hospital observation. Mozurkewich and associates (2009) reported lower rates of chorioamnionitis, metritis, and neonatal intensive care unit admissions for women with term ruptured membranes whose labors were induced compared with those managed expectantly. At Parkland Hospital, labor is induced soon ater admission when ruptured membranes are confirmed at term. In those with hypotonic contractions or with advanced cervical dilation, oxytocin is selected to lower potential hyperstimulation risk. In those with an unfavorable cervix and no or few contraction, prostaglandin E\ (misoprostol) is chosen to promote cervical ripening and contractions. he beneit of prophylactic antibiotics in women with ruptured membranes before labor at term is

1	few contraction, prostaglandin E\ (misoprostol) is chosen to promote cervical ripening and contractions. he beneit of prophylactic antibiotics in women with ruptured membranes before labor at term is unclear (Passos, 2012). However, in those with membranes ruptured longer than 18 hours, antibiotics are instituted for group B streptococcal infection prophylaxis (Chap. 64, p. 1221).

1	Labor can be too slow, but it also can be abnormally rapid. Precipitous labor and delivey is extremely rapid labor and delivery. It may result from an abnormally low resistance of the soft parts of the birth canal, from abnormally strong uterine and abdominal contractions, or rarely from the absence of painful sensations and thus a lack of awareness of vigorous labor. Precipitous labor terminates in expulsion of the fetus in less than 3 hours. Using this definition, 25,260 live births-3 percent-were complicated by precipitous labor in the United States in 2013 (Martin, 2015). Despite this incidence, little published information describes maternal and perinatal outcomes.

1	For the mother, precipitous labor and delivery seldom are accompanied by serious maternal complications if the cervix is efaced appreciably and compliant, if the vagina has been stretched previously, and if the perineum is relxed. Conversely, vigorous uterine contractions combined with a long, firm cervix and a noncompliant birth canal may lead to uterine rupture or extensive lacerations of the cervix, vagina, vulva, or perineum (Sheiner, 2004). It is in these latter circumstances that amnionic-luid embolism most likely develops (Chap. 41, p. 785). Precipitous labor is frequently followed by uterine atony. The uterus that contracts with unusual vigor bore delivery is likey to be hypotonic ater delivey. In one report of 99 term pregnancies, short labors were more common in multiparas who typically had contractions at intervals less than 2 minutes. Precipitous labors have been linked to cocaine abuse and associated with placental abruption, meconium, postpartum hemorrhage, and low Apgar

1	had contractions at intervals less than 2 minutes. Precipitous labors have been linked to cocaine abuse and associated with placental abruption, meconium, postpartum hemorrhage, and low Apgar scores (Mahon, 1994).

1	For the neonate, adverse perinatal outcomes from rapid labor may be increased considerably for several reasons. he tumultuous uterine contractions, often with negligible intervals of relaxation, prevent appropriate uterine blood low and fetal oxygenation. Resistance of the birth canal may rarely cause intracranial trauma. Acker and coworkers (1988) reported that Erb or Duchenne brachial palsy was associated with such labors in a third of cases. Finally, during an unattended birth, the newborn may fall to the floor and be injured, or it may need resuscitation that is not immediately available.

1	As treatment, analgesia is unlikely to modiy these unusually forceful contractions to a significant degree. he use of tocolytic agents such as magnesium sulfate or terbutaline is unproven in these circumstances. Use of general anesthesia with agents that impair uterine contractibility such as isoflurane is often excessively heroic. Certainly, any oxytocin being administered should be stopped immediately. Fetopelvic disproportion arises from diminished pelvic capacity, from abnormal fetal size or presentation, or more usually from both. he pelvic inlet, midpelvis, or pelvic outlet may be contracted solely or in combination. ny contraction of the pelvic diameters that diminishes pelvic capacity can create dystocia during labor. Normal pelvic dimensions are additionally discussed and illustrated in Chapter 2 (p. 30).

1	Using clinical measures, it is important to identiy the shortest anteroposterior diameter through which the fetal head must pass. Before labor, the fetal biparietal diameter averages from 9.5 to as much as 9.8 cm. Therefore, it might prove diicult or even impossible for some fetuses to pass through a pelvic inlet that has an anteroposterior diameter <10 cm. Mengert (1948) and Kaltreider (1952), employing x-ray pelvimetry, demonstrated that the incidence of diicult deliveries rises when either the anteroposterior diameter of the inlet is < 10 cm or the transverse diameter is < 12 cm. As expected, when both diameters are contracted, dystocia rates are much greater than when only one is contracted. Either of these measures is used to consider a pelvis contracted.

1	he anteroposterior diameter of the inlet, which is the obstetrical conjugate, is commonly approximated by manually measuring the diagonal conjugate, which is approximately 1.5 cm greater. Ascertainment of these measures is described in Chapter 2 (p. 30). herefore, inlet contraction usually is defined as a diagonal conjugate < 11.5 cm. A small woman is likely to have a small pelvis, but she is also likely to have a small neonate. homs (1937) studied 362 nulliparas and found that the mean birthweight of their ofspring was significantly lower-280 g-in women with a small pelvis than in those with a medium or large pelvis.

1	as the rise in this pressure above a resting pressure baseline. hese investigators also introduced the concept of Montevideo units to deine uterine activity (Chap. 23, p. 443). With this deinition, uterine performance is the product of contraction intensity in mm Hg multiplied by the number of contractions in a 10-minute span. For example, three contractions in 10 minutes, each of 50 mm Hg intensity, would equal 150 Montevideo units. During the irst 30 weeks of pregnancy, uterine activity is comparatively quiescent. Contractions are seldom greater than 20 mm Hg, and these have been equated with those irst described by John Braxton Hicks. Uterine activity increases gradually after 30 weeks, and it is noteworthy that these Braxton Hicks contractions also increase in intensity and frequency. Uterine activity is further enhanced during the last weeks of pregnancy. During this phase, the cervix ripens (Chap. 21, p. 409).

1	According to Caldeyro-Barcia and Poseiro (1960), clinical labor usually commences when uterine activity reaches values between 80 and 120 Montevideo units. his translates into approximately three contractions of 40 mm Hg every 10 minutes. Importantly, no clear-cut division marks labor onset, which is a gradual and progressive transition.

1	In irst-stage labor, uterine contractions progressively grow in intensity from approximately 25 mm Hg at labor commencement to 50 mm Hg at its end. At the same time, the frequency advances from three to ive contractions per 10 minutes, and uterine baseline tone rises from 8 to 12 mm Hg. Uterine activity is further enhanced during second-stage labor, aided by maternal pushing. Indeed, contraction intensity of 80 to 100 mm Hg is typical, and the uterus contracts as frequently as ive to six times each 10 minutes. Hauth and coworkers (1986) quantiied uterine contraction pressures in 109 women at term who received oxytocin for labor induction or augmentation. Most of these women achieved 200 to 225 Montevideo units, and 40 percent had up to 300 units to efect delivery. The authors suggested that these levels of uterine activity should be sought before consideration of cesarean delivery for presumed dystocia (Chap. 23, p. 443).

1	Interestingly, the duration of uterine contractions-60 to 80 seconds-does not lengthen appreciably from early active labor through the second stage (Bakker, 2007; Pontonnier, 1975). Presumably, this duration constancy serves fetal respiratory gas exchange. During a uterine contraction, as the intrauterine pressure exceeds that of the intervillous space, respiratory gas exchange is halted. This leads to functional fetal "breath holding," which has a 60-to 80-second limit that remains relatively constant.

1	readily be depressed by the inger. At greater intensities, the uterine wall then becomes so hard that it resists easy depression. Uterine contractions usually are not associated with pain until their strength exceeds 15 mm Hg. Presumably, this is the minimum pressure required to distend the lower uterine segment and cervix. It follows that Braxton Hicks contractions exceeding 15 mm Hg may be perceived as uncomfortable because distention of the uterus, cervix, and birth canal is generally thought to produce discomfort.

1	Hendricks (1968) observed that "the clinician makes great demands upon the uterus." The uterus is expected to remain well relaxed during pregnancy, to contract efectively but intermittently during labor, and then to remain in a state of almost constant contraction for several hours postpartum. Figure 24-28 demonstrates an example of normal uterine activity during labor. Uterine activity progressively and gradually rises from early through late labor. Interestingly, uterine contractions after birth are identical to those resulting in delivery of the newborn. Logically, the uterus that performs poorly before delivery is also prone to atony and puerperal hemorrhage. J.A. 5-26-60 Intrauterine pressure (mm Hg) Delivery 2:54 PM. 8:45 A.M. 10:20 A.M. 1:10ePM. 2:30 PM. 3:30 PM. 5:20 P.M.

1	J.A. 5-26-60 Intrauterine pressure (mm Hg) Delivery 2:54 PM. 8:45 A.M. 10:20 A.M. 1:10ePM. 2:30 PM. 3:30 PM. 5:20 P.M. Caldeyro-Barcia and Poseiro (1960) also observed empirically that uterine contractions are clinically palpable only after their intensity exceeds 10 mm Hg. Moreover, until the intensity of contractions reaches 40 mm Hg, the uterine wall can FIGURE 24-28 Intrauterine pressure recorded through a single catheter. A. Prelabor. B. Early labor. C. Active labor. D. Late labor. E. Spontaneous activity Y2 hour postpartum. F. Spontaneous activity 2Y2 hours postpartum. (Redrawn from Hendricks (H: Uterine contractility changes in the early puerperium, (lin Obstet Gynecol. 1968 Mar;11 (1 ):125-144.) , I of the --contraction Amnionic pressure[1 0 Tonus -.A

1	Amnionic pressure[1 0 Tonus -.A FIGURE 24-29 Schematic representation of the normal contractile wave of labor. Large uterus on the let shows the four points at which intramyometrial pressure was recorded with microballoons. Four corresponding pressure tracings are shown in relation to each other by shading on the small uteri at top. (Adapted with permission from (aldeyro-Barcia R, Poseiro JJ: Physiology ofthe uterine contraction. (lin Obstet Gynecol 1960 3:386.) • Origin and Propagation of Contractions

1	The normal contractile wave of labor originates near the uterine end of one of the fallopian tubes. hus, these areas act as "pacemakers" (Fig. 24-29). he right pacemaker usually predominates over the left and starts most contractile waves. Contractions spread from the pacemaker area throughout the uterus at 2 em/sec, and the whole organ is depolarized within 15 seconds. his depolarization wave propagates downward toward the cervix. Intensity is greatest in the fundus, and it diminishes in the lower uterus. his phenomenon is thought to relect the reduced myometrial thickness from the fundus to the cervix. Presumably, this descending pressure gradient serves to direct fetal descent toward the cervix and to eface the cer vix. Importantly, all parts of the uterus are synchronized and reach their peak pressure almost simultaneously, giving rise to the curvilinear waveform shown in Figure 24-29. Young and Zhang (2004) have shown that the initiation of each contraction is triggered by a

1	their peak pressure almost simultaneously, giving rise to the curvilinear waveform shown in Figure 24-29. Young and Zhang (2004) have shown that the initiation of each contraction is triggered by a tissue-level bioelectrical event.

1	The pacemaker theory also serves to explain the varying intensity of adjacent coupled contractions shown in panels A and B of Figure 24-28. Such coupling was termed incoordination by Caldeyro-Barcia and Poseiro (1960). A contractile wave begins in one cornual-region pacemaker but does not synchronously depolarize the entire uterus. As a result, another contraction begins in the contralateral pacemaker and produces the second contractile wave ofthe couplet. hese small contractions alternating with larger ones appear to be typical of early labor. Indeed, labor may progress with such uterine activity, albeit at a slower pace. hese authors also observed that labor would progress slowly ifregular contractions were hypotonicthat is, contractions with intensity <25 mm Hg or frequency <2 per 10 minutes.

1	Terms for the description and quantiication of uterine contractions have been recommended by the American College of Obstetricians and Gynecologists (2017b). Normal uterine activiy is defined as five or fewer contractions in 10 minutes, averaged during a 30-minute span. Tachysystole is more than ive contractions in 10 minutes, averaged over 30 minutes. Tachysystole can be applied to spontaneous or induced labor. he term hyperstimulation was abandoned. Stewart and associates (2012) prospectively studied uterine tachysystole in 584 women undergoing labor induction with misoprostol at Parkland Hospital. A higher rate of adverse neonatal outcomes was not associated with an increasing number of contractions per 10 minutes or per 30 minutes. Counts of six or more contractions in 10 minutes, however, were significantly associated with fetal heart rate decelerations.

1	Electrodes for fetal heart rate evaluation and catheters for uterine contraction measurement are both associated with infrequent but potentially serious complications. Rarely, an intrauterine pressure catheter during placement may lacerate a fetal vessel in the placenta. Also with insertion, placental and possibly uterine perforation can cause hemorrhage, abruption, serious morbidity, and spurious recordings that have resulted in inappropriate management. Severe cord compression has been described from entanglement with the pressure catheter. Injury to the fetal scalp or breech by a heart rate electrode is rarely severe. However, application at some other site-such as the eye in face presentations-can be serious.

1	Both the fetus and the mother may be at greater risk of infection from internal monitoring (Faro, 1990). Scalp wounds from the electrode may become infected, and subsequent cranial osteomyelitis has been reported (Brook, 2005; Eggink, 2004; McGregor, 1989). The American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) have recommended that certain maternal infections, including human immunodeficiency virus (HIV), herpes simplex virus, and hepatitis Biand C virus, are relative contraindications to internal fetal monitoring. Adamsons K, Myers E: Late decelerations and brain tolerance of the fetal monkey to intrapartum asphyxia. Am J Obstet Gyneco1o128:893, 197 Ahn MO, Korst L, Phelan JP: Intrapartum fetal heart rate patterns in 209 brain damaged infants. Am J Obstet GynecoIo174:492, 1996

1	Ahn MO, Korst L, Phelan JP: Intrapartum fetal heart rate patterns in 209 brain damaged infants. Am J Obstet GynecoIo174:492, 1996 Alfirevic Z, Devane 0, Gyte GM, et al: Continuous cardiotocography (CTG) as a form of electronic fetal monitoring (EFM) for fetal assessment during labour. Cochrane Database Syst Rev 2:CD006066, 2017 Ambia M1, Yule SS, Wells E: Does fetal bradycardia during eclamptic seizure necessitate cesarean delivery? Unpublished data, 20o17 Amer-Wihlin I, Arulkumaran S, Hagberg H, et al: Fetal electrocardiogram: ST waveform analysis in intrapartum surveillance. BJOG 114: 1191,2007 Amer-Wahlin 1, Hellsten C, Noren H, et al: Cardiotocography only versus cardiotocography plus ST analysis of fetal electrocardiogram for intrapartum fetal monitoring: a Swedish randomized controlled trial. Lancet 358:534, 200o1 American Academy of Pediatrics and the American College of Obstetricians and Gynecologists: Guidelines for Perinatal Care, 8th ed. Elk Grove Village, AAP, 2017

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1	Paul WM, Quilligan E], MacLachlan T: Cardiovascular phenomena associated with fetal head compression. Am J Obstet Gynecol 90:824, 1964 Petrie RH: Dose/response efects of intravenous meperidine in fetal heart rate variability. ] Matern Fetal Med 2:215, 1993 Phelan ]P, Ahn MO: Perinatal observations in forty-eight neurologically impaired term infants. Amr] Obstet GynecoIr171:424, 1994 Picquard F, Hsiung R, Mattauer M, et al: The validity of fetal heart rate monitoring during the second stage oflabor. Obstet Gynecol 2:746, 1988 Pierce ], Gaudier FL, Sanchez-Ramos L: Intrapartum amnioinfusion for meconium-stained fluid: meta-analysis of prospective clinical trials. Obstet Gynecol 95:1051, 2000 Pillai M, James 0: he development of fetal heart rate pattens during normal pregnancy. Obstet Gynecol 76:812, 1990 Pontonnier G, Puech F, Grandjean H, et al: Some physical and biochemical parameters during normal labour. Fetal and maternal study. BioI Neonate 26:159,r1975

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1	Sherer OM: Blunted fetal response to vibroacoustic stimulation associated with matenal intravenous magnesium sulfate therapy. Amr] Perinatol 11r:401, 1994 Sholapurkar SL: he conundrum of vanishing early decelerations in British obstetrics, a step backwards? Detailed appraisal of British and American classiications of fetal heart rate decelerationsBfallacies of emphasis on waveform and putative aetiology.r] Obstet Gynaecol 32(6):505, 2012 Simpson KR, James DC: Eicacy of intrauterine resuscitation techniques in improving fetal oxygen status during labor. Obstet Gynecolr105: 1362,2005 Skupski DW, Rosenberg CR, Eglinton GS: Intrapartum fetal stimulation tests: a meta-analysis. Obstet Gynecol 99: 129, 2002 Smith CV, Nguyen HN, Phelan JP, et al: Intrapartum assessment of fetal wellbeing: a comparison of fetal acoustic stimulation with acid-base determinations. Am J Obstet GynecoIr155:726, 1986

1	Smith CV, Nguyen HN, Phelan JP, et al: Intrapartum assessment of fetal wellbeing: a comparison of fetal acoustic stimulation with acid-base determinations. Am J Obstet GynecoIr155:726, 1986 Smith JH, Anand KJ, Cotes PM, et al: Antenatal fetal heart rate variation in relation to the respiratory and metabolic status of the compromised human fetus. BJOG 95:980, 1988 Southall DP, Richards J, Hardwick A, et al: Prospective study of fetal heart rate and rhythm patterns. Arch Dis Child 55:506, 1980 Spong CY, Ogundipe OA, Ross MG: Prophylactic amnioinfusion for meconium-stained amniotic fluid. Am J Obstet Gynecolr1 1:931, 1994 Spong CY, Rasul C, CoHea JV, et al: Characterization and prognostic signiicance of variable decelerations in the second stage of labor. Am J Perinatol 15:369, 1998 Stewart RD, Bleich AT, Lo JY, et al: Deining uterine tachysystole: how much is too muc,? Am J Obstet Gynecol 207:290.el, 2012

1	Stewart RD, Bleich AT, Lo JY, et al: Deining uterine tachysystole: how much is too muc,? Am J Obstet Gynecol 207:290.el, 2012 Stiller R, von Mering R, Konig V, et al: How well does reflectance pulse oximetry reflect intrapartum fetal acidosis? Am J Obstet Gynecol 186: 1351, 2002 Tahir Mahmood U, O'Gorman C, vfarchocki Z, et al: Fetal scalp stimulation (FSS) versus fetal blood sampling (FBS) for women with abnormal fetal heart rate monitoring in labor: a prospective cohort study. J Matern Fetal Neonatal Med May 19, 2017 [Epub ahead of print] Thakor AS, Giussani DA: Efects of acute acidemia on the fetal cardiovascular defense to acute hypoxemia. Am J Physiol Regul Integr Comp Physiol 296(1):R90, 2009 Tintinalli JE, Stapczynski JS, Ma OJ, et al: Tintinalli's Emergency Medicine:

1	Tintinalli JE, Stapczynski JS, Ma OJ, et al: Tintinalli's Emergency Medicine: A Comprehensive Study Guide, 8th ed. New York, McGraw-Hill, 2016 Tooley JR, Satas S, Porter H, et al: Head cooling with mild systemic hypothermia in anesthetized piglets is neuroprotective. Ann Neurol 53(1):65, 2003 Usta 1M, Mercer BM, Aswad NK, et al: The impact of a policy of amnioinfusion for meconium-stained amniotic fluid. Obstet Gynecol 85:237, 1995 Van Geijn HP, Jongsma HN, deHaan J, et al: Heart rate as an indicator of the behavioral state. Am J Obstet Gynecol 136: 1 061, 1980 Verdurmen l,Hulsenboom AD, van Laar JO, et al: Efect of tocolytic drugs on fetal heart rate variability: a systematic review. J Matern Fetal Neonatal Med 30(20):2387, 2017 Wagner BP, N edelcu J, Vlartin E: Delayed postischemic hypothermia improves long-term behavioral outcome after cerebral hypoxia-ischemia in neonatal rats. Pediatr Res 51 (3):354, 2002

1	Wagner BP, N edelcu J, Vlartin E: Delayed postischemic hypothermia improves long-term behavioral outcome after cerebral hypoxia-ischemia in neonatal rats. Pediatr Res 51 (3):354, 2002 Walker J: Foetal anoxia. J Obstet Gynaecol Br Commonw 61:162, 1953 � enstrom K, Andrews W, Maher JE: Amnioinfusion survey: prevalence protocols and complications. Obstet Gynecol 86:572, 1995 Westgate J, Harris M, Curnow JSH, et al: Plymouth randomized trial of cardiotocogram only versus ST waveform plus cardiotocogram for intrapartum monitoring in 2400 cases. Am J Obstet GynecoIr169:1151, 1993 Westgate JA, Bennet L, De Haan HH, et al: Fetal heart rate overshoot during repeated umbilical cord occlusion in sheep. Obstet Gynecol 97:454, 2001 Wiberg-Itzel E, Lipponer C, Norman M, et al: Determination of pH or lactate in fetal scalp blood in management of intrapartum fetal distress: randomised controlled multicenter trial. BMJ 336: 1284, 2008

1	Yam J, Chua S, Arulkumaran S: Intrapartum fetal pulse oximetty. Part I: principles and technical issues. Obstet Gynecol Surv 55: 163, 2000 Yang M, Stout MJ, Lopez ]D, et al: Association of fetal heart rate baseline change and neonatal outcomes. Am] PerinatoI34(9):879, 2017 Young BK, Katz M, Wilson SJ: Sinusoidal fetal heart rate, 1. Clinical significance. Am] Obstet GynecoIr136:587, 1980a Young BK, Weinstein HM: Moderate fetal bradycardia. Am J Obstet Gynecol 126:271, 1976 Young DC, Gray ]H, Luther ER, et al: Fetal scalp blood pH sampling: its value in an active obstetric unit. Am J Obstet GynecoIr136:276, 1980b Young RC, Zhang P: Functional separation of deep cytoplasmic calcium from subplasmalemmal space calcium in cultured human uterine smooth muscle cells. Cell Calcium 36(1):11, 2004 Zalar RW, Quilligan EJ: The influence of scalp sampling on the cesarean section rate for fetal distress. Am J Obstet Gynecol 135:239, 1979

1	Zalar RW, Quilligan EJ: The influence of scalp sampling on the cesarean section rate for fetal distress. Am J Obstet Gynecol 135:239, 1979 GENERAL PRINCIPLES .s..s...........s....s....s..s... 486 ANALGESIA AND SEDATION DURING LABOR ........ 487 REGIONAL ANALGESIA ..........s...s...s..s..s....s.. 488 SPINAL (SUBARACHNOID) BLOCK .....s............ 490 EPIDURAL ANALGESIA .s...s...s.....s.....s......... 492 LOCAL INFILTRATION FOR CESAREAN DELIVERY ....s. 497 GENERAL ANESTHESIA ......s..............s....s.. 498 POSTPARTUM ANALGESIA .s.......s...s....s....s..s.. 500

1	LOCAL INFILTRATION FOR CESAREAN DELIVERY ....s. 497 GENERAL ANESTHESIA ......s..............s....s.. 498 POSTPARTUM ANALGESIA .s.......s...s....s....s..s.. 500 We are indebted to Sir James .Simpson, the discoverer of chloroorm, or the introduction of anaesthesia into obstetrical practice. He employed ether or this purpose in 184, and replaced it by chloroorm. Every one agrees as to the marked beneits derived from anaesthesia when operative procedures are to be undertaken, but there is stil considerable dference of opinion as to the advisabiliy of its routine employment in normal labour. -]. Whitridge Williams (1903)

1	-]. Whitridge Williams (1903) As cited by Williams, anesthetic techniques were a most welcome addition to obstetrics. That said, obstetrical anesthesia presents unique challenges. Labor begins without warning, and anesthesia may be required within minutes of a full meal. Vomiting with potential aspiration of the gastric contents is a constant threat due to delayed gastric emptying during pregnancy. Disorders of pregnancy such as preeclampsia, placental abruption, or sepsis further compound provision of obstetrical anesthesia.

1	Of all anesthesia-related deaths in the United States from 1995 to 2005,i3.6 percent were in pregnant women (Li, 2009). Creanga and colleagues (2017) analyzed deaths of women during or within 1 year of pregnancy in the United States from 2011 through 2013. Of these deaths, they found that 3 of 2009 (0.2 percent) were attributable to anesthesia complications. As shown in Table 25-1, between 1979 and 2002, anesthesia-related maternal mortality rates decreased nearly 60 percent, and currently approximately ive deaths per million live births are attributed to anesthesia complications.

1	About two thirds of deaths associated with general anesthesia are caused by intubation failure or induction problems during cesarean delivery. Deaths associated with regional analgesia are caused by high spinal or epidural blocks-26 percent; respiratory failure-19 percent; and drug reaction-19 percent. he improved case-fatality rate for general anesthesia is especially notable considering that such anesthesia is now used for the highest-risk patients and the most hurried emergencies, that is, decision-to-incision intevals < 15 minutes (Bloom, 2005). 25-1 . Case-Fatality Rates and Rate Ratios of Anesthesia-Related Deaths During Cesarean Delivery by Type of Anesthesia in the United States, ,979-2002 aDeaths per million general or regional anesthetics. (I = confidence interval. Data from Hawkins, 201s1.

1	Data from Hawkins, 201s1. The most signiicant factor linked to lower maternal mortality rates is the greater use of regional analgesia (Hawkins, 2011). In-house anesthesia coverage that is available around the clock is certainly another contributing factor. Logically, with increased use of regional analgesia, there are now reports of complications with these techniques. Indeed, compared to pre-1990 data, post-1990 obstetrical anesthesia was associated with more legal claims involving regional analgesia (Davies, 2009). In a recent analysis of 466,442 obstetrical hospital discharges, complications associated with regional analgesia accounted for 81 percent of anesthesia-related adverse events (Guglielminotti, 2015).

1	For the fetus, recent human studies suggest that single, relatively short exposure to general anesthetic and sedation is unlikely to have negative efects on subsequent behavior or learning. his evidence is presented in Chapter 46 (p. 901). That said, in 2016, the Food and Drug Administration (FDA) warned that repeated or lengthy use of general anesthetic and sedation drugs in pregnant women during their third trimester may afect fetal brain development. Listed drugs include inhalation agents used in general anesthesia as well as lorazepam, ketamine, propofol, and midazolam. Notably, the American College of Obstetricians and Gynecologists (2016a) and the Society for Obstetric Anesthesia and Perinatology (2017) have voiced concerns with this statement and cited the lack of signiicant human data, especially in pregnant women, to underpin this warning.

1	he American College of Obstetricians and Gynecologists (2017a) recognizes that a woman's request for labor pain relief is suicient medical indication for its provision. Identification of any of the risk factors shown in Table 25-2 should prompt consultation with anesthesia personnel to permit a joint management plan. This plan should include strategies to minimize the need for emergency anesthesia. Goals for optimizing obstetrical anesthesia services have been established by the American College of Obstetricians and Gynecologists (2017 a) and the American Society of Anesthesiologists (2016) and include: TABLE 25-2. Maternal Factors That May Prompt Anesthetic Consultation Obstructive lesions: edema, anatomical abnormalities, trauma Decreased range of motion in opening the mouth or small Obstetrical complications with a high risk of operative delivery Matenal medical complications such as cardiopulmon 1.

1	Obstetrical complications with a high risk of operative delivery Matenal medical complications such as cardiopulmon 1. Availability of a licensed practitioner who is credentiled to administer n appropriate anesthetic whenever necessary and to maintain support of vital unctions in an obstetrical emergency. 2. Availability of anesthesia personnel to permit the start of a cesarean delivery within 30 minutes of the decision to perform the procedure. 3. Anesthesia personnel immediately available to perform an emergency cesarean delivery during the active labor of a woman attempting vaginal birth ater cesarean (Chap. 31, p. 597). 4. Appointment of a qualified anesthesiologist to be responsible for all anesthetics administered. 5. Availability of a qualified physician with obstetrical privileges to perform operative vaginal or cesarean delivery during administration of anesthesia. 6.

1	5. Availability of a qualified physician with obstetrical privileges to perform operative vaginal or cesarean delivery during administration of anesthesia. 6. Availability of equipment, facilities, and support personnel equal to that provided in any surgical suite. 7. Immediate availability of personnel, other than the surgical team, to assume responsibility for resuscitation of a depressed newborn (Chap. 32, p. 606).

1	7. Immediate availability of personnel, other than the surgical team, to assume responsibility for resuscitation of a depressed newborn (Chap. 32, p. 606). To meet these goals, 24-hour, in-house anesthesia coverage is usually necessary. Providing such service in smaller facilities is more challenging-a problem underscored by the fact that approximately a third of all hospitals providing obstetrical care perform fewer than 500 deliveries per year. The financial burden incurred to provide 24/7 obstetrical anesthesia coverage may result in cost deicits (Bell, 2000). Compounding this burden, some third-party payers have denied reimbursement for epidural analgesia in the absence of a specific medical indication-an approach repudiated by the American College of Obstetricians and Gynecologists (2017a). Regarding obstetricians, they should be proficient in local and pudendal analgesia. These may be administered in appropriately selected circumstances described on page 489.

1	Regarding obstetricians, they should be proficient in local and pudendal analgesia. These may be administered in appropriately selected circumstances described on page 489. Hawkins (2010) emphasized that labor pain is a highly individual response to variable stimuli that are uniquely received and interpreted (Fig.

1	Hawkins (2010) emphasized that labor pain is a highly individual response to variable stimuli that are uniquely received and interpreted (Fig. These stimuli are modified by emotional, motivational, cognitive, social, and cultural circumstances. Labor pain caused by uterine contractions and cervical dilation is transmitted through visceral aferent sympathetic nerves entering the spinal cord from Tto through Lt. Later in labor, perineal stretching transmits painful stimuli through the pudendal nerve and sacral nerves S2 through 54' Cortical responses to pain and anxiety during labor are complex and may be inluenced by maternal expectations for childbirth, her age, preparation through education, emotional support, and other factors. Pain perception is heightened by fear and the need to move into various positions. A woman may be motivated to have a certain type of birthing experience, and these opinions will influence her judgment regarding pain management.

1	Maternal physiological responses to labor pain can influence maternal and fetal well-being and labor progress. For example, hyperventilation may induce hypocarbia. A greater metabolic rate augments oxygen consumption. Increases in cardiac output and vascular resistance may raise maternal blood pressure. Pain, stress, and anxiety trigger release of stress hormones such as Cortical responses to pain pain, anxle y, age, FIGURE 25-' Sources of pain during labor and maternal physiological responses. (Reproduced with permission from Hawkins JL: Epidural analgesia for labor and delivery, N Engl J Med. 2010 Apr 22;362(16):1503-1510.) cortisol and �-endorphins. The sympathetic nervous system response to pain leads to a marked elevation in circulating catecholamines that can adversely afect uterine activity and uteroplacental blood low. Efective analgesia attenuates or eliminates these responses.

1	If uterine contractions and cervical dilatation cause discomfort, pain relief is ofered. If neuraxial analgesia is contraindicated or unavailable or is declined, a narcotic from Table 25-3 plus one of the tranquilizer-antiemetic drugs such as promethazine (Phenergan) is usually appropriate. With a successful program of analgesia and sedation, the mother ideally rests quietly between contractions. In this circumstance, discomfort usually is felt at the acme of an efective uterine contraction.

1	Meperidine, 50 to 100 mg, with promethazine, 25 mg, may be administered intramuscularly at intervals of 2 to 4 hours. A more rapid efect is achieved by giving meperidine intravenously in doses of 25 to 50 mg every 1 to 2 hours. Whereas analgesia is mximal 30 to 45 minutes after an intramuscular injection, it develops almost immediately following intravenous administration. Meperidine readily crosses the placenta and can have a prolonged half-life in the newborn (American College of Obstetricians and Gynecologists, 20l7a). Its depressant efect in the fetus follows closely behind the peak maternal analgesic efect. TABLE 25-3. Some Parenteral Analgesic Agents for Labor Pain Meperidine 25-50 mg (IV) Q 1-2 hr 5 min (IV) �.1A8-20 hr 50-100 mg (1M) Q 2-4 hr 30-45 min (1M) �60 hr Fentanyl 50-100 tg (IV) Q 1 hr 1 min �5 hr Morphine 2--5 mg (IV) Q 4 hr 5 min (IV) �.7 hr IV = intravenously, 1M = intramuscularly; Q = every.

1	According to Bricker and Lavender (2002), meperidine is the most common opioid used worldwide for pain relief during labor. In one randomized study at Parkland Hospital, patientcontrolled intravenous analgesia with meperidine was found to be an inexpensive and reasonably efective method for labor analgesia (Sharma, 1997). Women randomized to self-administered analgesia were given a 50-mg meperidine plus 25-mg promethazine dose intravenously as an initial bolus. hereafter, an infusion pump was set to deliver 15 mg of meperidine every 10 minutes as needed until delivery. Neonatal sedation, as measured by the need for naloxone treatment in the delivery room, was identiied in 3 percent of newborns. Both meperidine and its metabolite, normeperidine, are lipophilic and readily cross the placenta. Analgesia with meperidine was associated with lower Apgar scores in comparison to epidural analgesia (Sharma, 2004). Normeperidine is a strong respiratory depressant that has a significantly longer

1	Analgesia with meperidine was associated with lower Apgar scores in comparison to epidural analgesia (Sharma, 2004). Normeperidine is a strong respiratory depressant that has a significantly longer half-life than meperidine and is likely responsible for the fetal side efects of meperidine.

1	his synthetic opioid receptor agonist-antagonist analgesic, given in 1-to 2-mg intravenous doses, compares favorably with 40 to 60 mg of meperidine. Its major side efects are somnolence, dizziness, and dysphoria. Neonatal respiratory depression is reported to be less than with meperidine. Importantly, the two drugs are not given contiguously because butorphanol antagonizes the narcotic efects of meperidine. Butorphanol has been associated with transient sinusoidal fetal heart rate patterns (Hatjis, 1986). This is another mixed opioid receptor agonist-antagonist analgesic. It can be given intramuscularly, intravenously, or subcutaneously. he usual dose is 10 to 20 mg, administered every 4 to 6 hours irrespective of the route of administration. Small doses of nalbuphine may also be used to treat pruritus associated with neuraxial opioids.

1	This short-acting and potent synthetic opioid may be given in doses of 50 to 1 00 �g intravenously every hour. Its main disadvantage is its short duration of action, which requires frequent dosing or use of a patient-controlled intravenous infusion pump. This is a synthetic opioid with an extremely rapid onset of action. It is hydrolyzed rapidly, resulting in a half-life of 3.5 minutes (Ohashi, 2016). Although it readily crosses the placenta, it is quickly metabolized or redistributed within the fetus (Kan, 1998). Various dosing regimens have been studied, and single boluses appear to mirror the periodic uterine contraction pattern. Infusions, on the other hand, have been reported to cause maternal apnea (Waring, 2007). Due to the aforementioned risks, only trained personnel should administer it, and only under strictly controlled circumstances. Efficacy and Safety of Parenteral Agents

1	Efficacy and Safety of Parenteral Agents Hawkins and colleagues (1997) reported that four of 129 maternal anesthetic-related deaths were from parenteral sedation-one from aspiration, two from inadequate ventilation, and one from overdosage. Opioids used during labor may cause newborn respiratory depression. Naloxone is a narcotic antagonist capable of reversing this respiratory depression. It acts by displacing the narcotic from specific receptors in the central nervous system. Withdrawal symptoms may be precipitated in recipients who are physically dependent on narcotics. For this reason, naloxone is contraindicated in a newborn of a narcotic-addicted mother.

1	Inhaled nitrous oxide has a rapid onset and ofset that provides analgesia during episodic contractions. It can be self-administered as a mixture of 50-percent nitrous oxide and 50-percent oxygen premixed in a single cylinder (Entonox) or using a blender that mixes the two gases from separate tanks (Nitronox). he gases are connected to a breathing circuit through a one-way valve that opens only during inspiration. he use of intermittent nitrous oxide for labor pain is generally regarded s safe for the mother and newborn, but pain control is less efective than epidural analgesia (Barbieri, 2014; Likis, 2014). In many cases, nitrous oxide simply serves to delay more deinitive neuraxial analgesia. For maximal eicacy, nitrous oxide is inhaled 30 seconds prior to the start of a contraction, although this prevents adequate rest for the mother. Nitrous oxide is also associated with nausea and vomiting. The environmental and health risk of its use without proper scavenging remains to be

1	although this prevents adequate rest for the mother. Nitrous oxide is also associated with nausea and vomiting. The environmental and health risk of its use without proper scavenging remains to be carefully evaluated (King, 2014).

1	Various nerve blocks have been developed over the years to provide pain relief during labor and/or delivery. These include pudendal, paracervical, and neuraxial blocks such as spinal, epidural, and combined spinal-epidural techniques. Some of the more commonly used nerve block anesthetics, along with their usual concentrations, doses, and durations of action, are summarized in Table 25-4. he dose of each agent varies widely and is dependent on the particular nerve block and physical status of the woman. he onset, duration, and quality of analgesia can be enhanced by raising the volume and/or concentration. his can be done safely only by incrementally administering small-volume boluses of the agent and by careully monitoring early warning signs of toxicity. Administration of these agents must be followed by appropriate monitoring for adverse reactions. Equipment and personnel to manage these reactions must be immediately available.

1	Most often, serious toxicity follows inadvertent intravenous injection. Systemic toxicity from local anesthetics typically manifests in the central nervous and cardiovascular systems. For this reason, when epidural analgesia is initiated, dilute epinephrine is sometimes added and given as a test dose. A sudden significant rise in the maternal heart rate or blood pressure immediately after administration suggests intravenous catheter placement. his should halt further injection and should prompt catheter repositioning. Local anesthetic agents are manufactured in more than one concentration and ampule size, which raises the potential for dosing errors. aWithout epinephrine. bEsters are hydrolyzed by plasma cholinesterases and amides by hepatic clearance. D&C = dilatation and curettage. Data from Liu SS, Lin Y: Local anesthetics. In Barash P, Cullen B, Stoeling R, et al (eds): Clinical Anesthesia, 6th ed. Philadelphia, Lippincott Williams & Wilkins, 2009.

1	Data from Liu SS, Lin Y: Local anesthetics. In Barash P, Cullen B, Stoeling R, et al (eds): Clinical Anesthesia, 6th ed. Philadelphia, Lippincott Williams & Wilkins, 2009. Early symptoms are those of stimulation, but as serum levels rise, depression follows. Symptoms may include light-headedness, dizziness, tinnitus, metallic taste, and numbness of the tongue and mouth. Patients may show bizarre behavior, slurred speech, muscle fasciculation and excitation, and ultimately, generalized convulsions, followed by loss of consciousness.

1	hese manifestations generally develop later than those of cerebral toxicity. Moreover, no symptoms may develop because signs are usually induced by higher serum drug levels. he notable exception is bupivacaine, which is associated with neurotoxicity and cardiotoxicity at virtually idenricalilevels (Mulroy, 2002). Because of its toxicity risk, use of a 0.75-percent solution of bupivacaine for epidural injection has been proscribed by the FDA. Similar to neurotoxicity, cardiovascular toxicity is characterized first by stimulation and then by depression. Accordingly, hypertension and tachycardia are soon followed by hypotension, cardiac arrhythmias, and impaired uteroplacental perfusion. • Management of Local Anesthetic

1	• Management of Local Anesthetic Seizures and severe ventricular arrhythmias can follow large doses of local anesthetics that are given inadvertently. Labor and delivery units should be stocked with a 20-percent lipid emulsion solution (Intralipid). It is administered as a rapid intravenous bolus followed by an infusion upon the first sign of local anesthetic systemic toxicity (Neal, 2012). Controlling seizures and securing the airway are essential to prevent aspiration and hypoxemia. Benzodiazepines, such as midazolam or lorazepam, may be used to help control seizures, particularly if lipid emulsions are not available. Magnesium sulfate also controls convulsions (Chap 40, p. 736). Abnormal fetal heart follow and stem from maternal hypoxia. With proper manage ment, including supportive measures, the fetus usually recovers. herefore, it is best for the fetus and mother to delay delivery until the mother is stabilized.

1	herefore, it is best for the fetus and mother to delay delivery until the mother is stabilized. With proper treatment of local anesthetic systemic toxicity (LAST) with lipid emulsions, vital signs usually return to normal. The woman, however, should be monitored, placed in the lateral decubitus position to avoid aortocaval compression, and provided continued supportive care. Vasopressors can be used to support blood pressure. With cardiac arrest, emergency cesarean delivery is considered if maternal vital signs have not been restored within 5 minutes (Chap, 47, p. 931). As with convulsions, however, the fetus is likely to recover more quickly in utero once maternal cardiac output is reestablished.

1	Pain with vaginal delivery arises from stimuli from the lower genital tract. These are transmitted primarily through the pudendal nerve, the peripheral branches of which provide sensory innervation to the perineum, anus, vulva, and clitoris. The pudendal nerve passes beneath the sacrospinous ligament just as the ligament attaches to the ischial spine. Sensory nerve fibers of the pudendal nerve are derived from ventral branches of the S2 through S4 nerves.

1	The pudendal nerve block is a relatively safe and simple method of providing analgesia for spontaneous delivery. As shown in Figure 25-2, a tubular introducer is used to sheathe and guide a 15-cm-long 22-gauge needle into position near the pudendal nerve. The end of the introducer is placed against the vaginal mucosa just beneath the tip of the ischial spine. he in troducer allows 1.0 to 1.5 em of needle to at the injection site. he infection may spread posteriorly to the hip joint, into the gluteal musculature, or into the retropsoas space (Svancarek, 1977). his block usually provides satisfactory pain relief during irst-stage labor. How ever, because the pudendal nerves are not blocked during paracervical blockade, addi Ischial spine tional analgesia is required for delivery. For

1	Ischial spine tional analgesia is required for delivery. For Pudendal nerve paracervical blockade, usually 5 to 10 mL of lidocaine (1 to 2 percent) or chloroprocaine (3 percent) is injected into the cervix later ally at 3 and 9 o'clock. Because these anes thetics are relatively short acting, this block may have to be repeated during labor. Fetal bradycardia is a worrisome com plication that occurs with approximately 15 percent of paracervical blocks (Rosen, FIGURE 25-2 Local infiltration of the pudendal nerve. Transvaginal technique showing 2002). Bradycardia usually develops within the needle extended beyond the needle guard and passing through the sacrospinous 10 minutes and may last up to 30 minutes. ligament to reach the pudendal nerve.

1	ligament to reach the pudendal nerve. protrude beyond its tip, and the needle is pushed beyond the introducer tip into the mucosa. A mucosal wheal is made with 1 mL of I-percent lidocaine solution or an equivalent dose of another local anesthetic (see Table 25-4). To guard against intravascular infusion, aspiration is attempted before this and all subsequent injections. The needle is then advanced until it touches the sacrospinous ligament, which is iniltrated with 3 mL of lidocaine. he needle is advanced farther through the ligament. As the needle pierces the loose areolar tissue behind the ligament, resistance against the plunger drops. Another 3 mL of solution is injected in this region. Next, the needle is withdrawn into the introducer, which is moved to a point just above the ischial spine. The needle is inserted through the mucosa and a inal 3 mL is deposited. The procedure is then repeated on the other side.

1	Within 3 to 4 minutes of injection, a successful pudendal block will allow pinching of the lower vagina and posterior vulva bilaterally without pain. If delivery occurs before the pudendal block becomes efective and an episiotomy is indicated, then the fourchette, perineum, and adjacent vagina can be iniltrated with 5 to 10 mL of I-percent lidocaine solution directly at the planned episiotomy site. By the time of repair, the pudendal block usually has become efective. Pudendal block usually does not provide adequate analgesia when delivery requires extensive obstetrical manipulation. Moreover, such analgesia is usually inadequate for women in whom complete visualization of the cervix and upper vagina or manual exploration of the uterine cavity is indicated.

1	Infrequently, complications may follow this block. As previously described, intravascular injection of a local anesthetic agent may cause serious systemic toxicity. Hematoma formation from perforation of a blood vessel is most likely when there is a coagulopathy (Lee, 2004). Rarely, severe infection may originate Doppler studies have shown a rise in the pulsatility index of the uterine arteries following paracervical blockade. These observations support the hypothesis of drug-induced arterial vasospasm as a cause of fetal bradycardia (Manninen, 2000). For these reasons, paracervical block is not used in situations of potential fetal compromise.

1	Epidural, spinal, or combined spinal-epidural techniques are the most common methods used for pain relief during labor and delivery. In the United States in 2008, epidural analgesia was used in nearly 70 percent of mothers during labor and had a success rate of 98.8 percent. Neuraxial analgesia was used even more often in operative vaginal deliveries and supported 84 percent of forceps deliveries and 77 percent of vacuum extractions (Osterman, 2011).

1	Anesthetic in this block can be given as a single dose, can be partnered with an epidural catheter as combined spinalepidural analgesia, or can be administered as a continuous infusion. Injection of a local anesthetic into the subarachnoid space to efect analgesia has long been used for delivery. Advantages include rapid analgesia onset, short duration of action, and high success rate. he subarachnoid space during pregnancy is smaller, which likely results from internal vertebral venous plexus engorgement. Thus, in parturients, the same amount of anesthetic agent in the same volume of solution produces a much higher blockade than in nonpregnant women.

1	The irst stage of labor requires a sensory block to the level of the umbilicus (Tlo)' During the second stage of labor and for operative vaginal delivery, a sensory block of 52 through S4 is usually adequate to cover pain from perineal stretching and/ or instrumentation. Analgesic options include continuous lumbar epidural analgesia, combined spinal-epidural, continuous spinal analgesia, and other blocks such as pudendal and paracervical blocks.

1	Local anesthetic agents are usually given to establish a sensory block to the desired dermatome level. They are almost exclusively used in conjunction with neuraxial opioids. he mechanism of action is a function of the administration route and lipid solubility. Analgesia is induced by absorption into the vascular system (supraspinal), actions on the dorsal horns, and direct spread in the cerebrospinal fluid to the brainstem. Highly-soluble lipid opioids such as fentanyl and sufentanil have a rapid onset of action. But, because they are absorbed into lipid membranes and the epidural vasculature, their duration of action is short. Hydrophilic solutions such as morphine, on the other hand, provide extended analgesia (Lavoie, 2013). he major advantages of using such a combination are the rapid onset of pain relie, a decrease in shivering, and less dense motor blockade. Side efects are common and include pruritus and urinary retention. Nalbuphine, 2.5 to 5 mg intravenously, can be used to

1	onset of pain relie, a decrease in shivering, and less dense motor blockade. Side efects are common and include pruritus and urinary retention. Nalbuphine, 2.5 to 5 mg intravenously, can be used to treat pruritis without diminishing the analgesic efect.

1	A level of sensory blockade extending to the T 4 dermatome is desired for cesarean delivery. Depending on maternal size, 10 to 12 mg of bupivacaine in a hyperbaric solution or 50 to 75 mg of lidocaine hyperbaric solution is administered. The addition of opioid increases the rapidity of blockade onset, reduces shivering, and minimizes referred pain and other symptoms such as nausea and vomiting. The addition of a preservative-free morphine (Duramorph or Astramorph), 0.1 to 0.3 mg intrathecal or 2 to 4 mg epidural, provides pain control up to 24 hours postoperatively. Hypotension. Shown in Table 25-5 are some of the more common adverse events associated with neuraxial analgesia. Importantly, obese women have signiicantly impaired ventilation, and thus close clinical monitoring is imperative (Vricella, 2011). TABLE 25-5. Complications of Regional Analgesia Uncommon Inadvertant intrathecal, subdural, or intravascular injection of local anesthetic drugs Neurologic injury

1	TABLE 25-5. Complications of Regional Analgesia Uncommon Inadvertant intrathecal, subdural, or intravascular injection of local anesthetic drugs Neurologic injury Hypotension is a common complication that may develop soon after injection of the local anesthetic agent. It is the consequence of vasodilatation from sympathetic blockade and is compounded by obstructed venous return due to uter ine compression of the great vessels. In the supine position, even in the absence of maternal hypotension measured in the brachial artery, placental blood flow may still be significantly reduced. Treatment includes uterine displacement by left lat eral patient positioning, intravenous crystalloid hydration, and intravenous bolus injections of ephedrine or phenyleph rine.

1	Ephedrine is a sympathomimetic drug that binds to X-and 0-receptors but also indirectly enhances norepinephrine release. It raises blood pressure by raising heart rate and cardiac output and by variably elevating peripheral vascular resistance. In early animal studies, ephedrine preserved uteroplacental blood flow during pregnancy compared with ai-receptor agonists. Accordingly, it had been the preferred vasopressor for obstetrical use. Phenylephrine is a pure a-agonist and elevates blood pressure solely through vasoconstriction. A metaanalysis of seven randomized trials by Lee (2002a) suggests that the safety profiles of ephedrine and phenylephrine are comparable. Following their systematic review of 14 reports, Lee (2002b) questioned whether routine prophylactic ephedrine is needed for elective cesarean delivery. Although fetal acidemia has been reported with prophylactic ephedrine use, this was not observed with prophylactic phenylephrine use (Ngan Kee, 2004).

1	High or Total Spinal Blockade. Most often, high or total spinal blockade follows administration of an excessive dose of local anesthetic or inadvertent injection into the subdural or subarachnoid space. Subdral injection manifests as a high but patchy block even with a small dose of local anesthetic agent, whereas subarachnoid injection typically leads to complete spinal blockade with hypotension and apnea. hese conditions must be immediately treated to prevent cardiac arrest. In the undelivered woman: (1) the uterus is immediately displaced laterally to minimize aortocaval compression; (2) efective ventilation is established, preferably with tracheal intubation; and (3) intravenous fluids and vasopressors are given to correct hypotension. If chest compressions are to be performed, the woman is placed in the left-lateral position to allow left uterine displacement.

1	Postdural Puncture Headache. Leakage of cerebrospinal fluid (CSF) from the dura mater puncture site can lead to postdural puncture or "spinal headache." Presumably, when the woman sits or stands, the diminished CSF volume creates traction on pain-sensitive central nervous system structures. Another mechanism may be the compensatory cerebral vasodilation in response to the loss of CSF-the Monro-Kellie doctrine (Mokri, 2001).

1	Rates of this complication can be reduced by using a small-gauge spinal needle and avoiding multiple punctures. In a prospective, randomized study of ive diferent spinal needles, Vallejo and associates (2000) concluded that Sprotte and Whitacre needles had the lowest risks of postdural puncture headaches. Sprigge and Harper (2008) reported that the incidence of postdural puncture headache was 1 percent in more than 5000 women undergoing spinal analgesia. Postdural puncture headaches are much less frequent with epidural blockade because the dura mater is not intentionally punctured. he incidence of inadvertent dural puncture with epidural analgesia approximates 0.2 percent (Introna, 2012; Katircioglu, 2008). here is no good evidence that placing a woman absolutely lat on her back for several hours is efective in preventing this headache.

1	Once headache develops, it is managed aggressively, as expectant management increases hospital-stay lengths and subsequent emergency-room visits (Angle, 2005). Conservative management, such as luid administration and bed rest, is largely inefective. If not efectively treated, postdural puncture headache can persist as a chronic headache (Webb, 2012). Epidural blood patch is considered the gold standard for treatment. Typically, 10 to 20 mL of autologous blood obtained aseptically by venipuncture is injected into the epidural space. Further CSF leakage is halted by either mass efect or coagulation. Relief is almost always immediate, and complications are uncommon. The initial success rate of an epidural blood patch ranges from 61 to 73 percent (Paech, 2011). Performing a "prophylactic" blood patch is debatable and is thought not to be as efective as if performed after the headache develops (Scavone, 2004, 2015).

1	If a headache does not have the pathognomonic postural characteristics or persists despite treatment with a blood patch, other diagnoses are considered. Chisholm and Campbell (2001) described a case of superior sagittal sinus thrombosis that manifested as a postdural headache. Smarkusky and colleagues (2006) described pneumocephalus, which caused immediate cephalgia. Finally, intracranial and intraspinal subarachnoid hematomas have developed after spinal analgesia (Dawley, 2009; Liu, 2008). Convulsions. In rare instances, postdural puncture cephalgia is associated with temporary blindness and convulsions. Shearer and associates (1995) described eight such cases associated with 19,000 regional analgesic procedures done at Parkland Hospital. It is presumed that these too are caused by CSF hypotension. Immediate treatment of seizures and a blood patch were usually efective in these cases.

1	Bladder Dysfunction. With neuraxial analgesia, bladder sensa tion is likely to be obtunded and bladder emptying impaired for several hours after delivery. As a consequence, bladder distention is a frequent postpartum complication, especially if appreciable volumes of intravenous luid are given. Millet and colleagues (2012) randomized 146 women with neuraxial analgesia to either intermittent or continuous bladder catheterizations and found that the intermittent method was associated with signiicantly higher rates of bacteriuria. That said, we do not recommend routine postpartum use of indwelling catheters following uncomplicated vaginal delivery. Arachnoiditis and Meningitis. Local anesthetics are no longer preserved in alcohol, formalin, or other toxic solutes, TABLE 25-6. Absolute Contraindications to Neuraxial Analgesia

1	Arachnoiditis and Meningitis. Local anesthetics are no longer preserved in alcohol, formalin, or other toxic solutes, TABLE 25-6. Absolute Contraindications to Neuraxial Analgesia Skin infection over site of needle placement and disposable equipment is usually used. hese practices, coupled with aseptic technique, have made meningitis and arachnoiditis rare (Centers for Disease Control and Prevention, 2010). Contraindications to Neuraxial AnalgeSia Shown in Table 25-6 are absolute contraindications. Obstetrical complications that are associated with maternal hypovolemia and hypotension-for example, severe hemorrhage-are contraindications (Kennedy, 1968).

1	Disorders of coagulation and defective hemostasis also preclude neuraxial analgesia use. Although no randomized studies guide the management of anticoagulation at the time of delivery, consensus opinion suggests that women given subcutaneous unfractionated heparin or low-molecular-weight heparin should be instructed to stop therapy when labor begins (Krivak, 2007). Subarachnoid puncture is also contraindicated if cellulitis involves the planned needle entry site. Many consider neurological disorders to be a contraindication, if for no other reason than that exacerbation of the neurological disease might be erroneously attributed to the anesthetic agent. Other maternal conditions, such as aortic stenosis or pulmonary hypertension, are also relative contraindications (Chap. 49, p.i948).

1	Severe preeclampsia is another comorbid condition in which markedly decreased blood pressure can be predicted when neuraxial analgesia is used. Wallace and associates (1995) randomly assigned 80 women with severe preeclampsia undergoing cesarean delivery at Parkland Hospital to receive general anesthesia or either epidural or combined spinal-epidural analgesia. Maternal and neonatal outcomes did not difer. Still, 30 percent of women given epidural analgesia and 22 percent of those given spinal-epidural blockade developed hypotension. he average reduction in mean arterial pressure ranges between 15 and 25 percent.

1	Relief of labor and childbirth pain, including cesarean delivery, can be accomplished by injection of a local anesthetic agent into the epidural or peridural space 25-3). his potential space contains areolar tissue, fat, lymphatics, and the internal vertebral venous plexus. This plexus becomes engorged during pregnancy such that the volume of the epidural space is appreciably reduced. Entry for obstetrical analgesia is usually through a lumbar intervertebral space. Although only Spinal needle punctures the dura mater for injection 1 I: FIGURE 25-3 Neuraxial analgesia: A. Combined spinal-epidural analgesia. B. Epidural analgesia.

1	Spinal needle punctures the dura mater for injection 1 I: FIGURE 25-3 Neuraxial analgesia: A. Combined spinal-epidural analgesia. B. Epidural analgesia. one injection may be elected, usually an indwelling catheter is placed for subsequent agent boluses or infusion via a volumetric pump. he American College of Obstetricians and Gynecologists (2017 a) concludes that under appropriate physician supervision, labor and delivery nursing personnel who have been specifically trained in the management of epidural infusions should be able to adjust dosage and also discontinue infusions.

1	Complete analgesia for the pain of labor and vaginal delivery necessitates a block from the TIO to the S5 dermatomes (see Fig. 25-1). For cesarean delivery, a block extending from the T4 to the S1 derma tomes is desired. The efective spread of anesthetic depends on the catheter tip location; the dose, concentration, and volume of anesthetic agent used; and whether the mother is head-down, horizontal, or head-up (Setayesh, 2001). Individual variations in anatomy or presence of synechiae may preclude a completely satisfactory block. Finally, the catheter tip may migrate from its original location during labor.

1	One example of the sequential steps and techniques for performance of epidural analgesia is detailed in Table 25-7. Before injection of the local anesthetic therapeutic dose, a test dose is given. The woman is observed for features of toxicity from intravascular injection and for signs of high or total blockade from subdural or subarachnoid injection. If these are absent, only then is a full dose given. Analgesia is maintained by intermittent boluses of similar volume or by small volumes delivered continuously by infusion pump (Halpern, 2009). Current pumps used for epidural analgesia ofer a programmed intermittent epidural bolus (PIEB) mode, which reduces the required concentration of local anesthetics, the degree of lower extremity motor blockade, and rates of operative vaginal delivery (Capogna, 2011). he addition of small doses of a short-acting narcotic-fentanyl or sufentanil-has been shown to improve analgesic eicacy while avoiding motor blockade (Chestnut,

1	TABLE 25-7. Technique for Labor Epidural Analgesia Informed consent is obtained, and the obstetrician consulted Monitoring includes the following: Blood pressure every 1 to 2 minutes for 15 minutes ater giving a bolus of local anesthetic Continuous matenal heart rate monitoring during analgesia induction Hydration with 500 to 1000 mL of lactated solution The woman assumes a lateral decubitus or sitting position The epidural space is identified with a loss-of-resistance technique The epidural catheter is threaded 3 to 5 cm into the epidural space A test dose of 3 mL of 1.5% lidocaine with 1 :200,000 epinephrine or 3 mL of 0.25% bupivacaine with 1 :200,000 epinephrine is injected after careful aspiration to avert injection and after a uterine contraction. This minimizes the chance of confusing tachycardia that results from labor pain with tachycardia from intravenous injection of the test dose.

1	If the test dose is negative, 10-15 mL of 0.0625-0.125% bupivacaine are injected to achieve a sensory TlO level. Ater 15 to 20 minutes, the block is assessed using loss of sensation to cold or pinprick. If no block is evident, the catheter is replaced. If the block is asymmetrical, the epidural catheter is withdrawn 0.5 to 1s.0 cm and an additional 5 to 10 mL of 0.0625-0.125% bupivacaine is injected. If the block remains inadequate, the catheter is replaced. The woman is positioned in the lateral or semilateral position to avoid aortocaval compression. Subsequently, maternal blood pressure is recorded every 5 to 15 minutes. The fetal heart rate is monitored continuously. The level of analgesia and intensity of motor blockade are assessed at least hourly. Reproduced with permission from Glosten B: Local anesthetic techniques. In Chestnut DH (ed): Obstetric Anesthesia: 2nd ed. St Louis, Mosby, 1999.

1	Reproduced with permission from Glosten B: Local anesthetic techniques. In Chestnut DH (ed): Obstetric Anesthesia: 2nd ed. St Louis, Mosby, 1999. 1988). As with spinal blockade, close monitoring, including the level of analgesia, is imperative and must be performed by trained personnel. Appropriate resuscitation equipment and drugs must be available during administration of epidural analgesia. Higher or Total Spinal Blockade. In general, complications with epidural analgesia are similar to those with spinal analgesia (see Table 25-5). Dural puncture with inadvertent subarachnoid injection may cause total spinal blockade. Sprigge and Harper (2008) cited an incidence of 0.91 percent recognized accidental dural punctures at the time of epidural analgesia in more than 18,000 women. Personnel and facilities must be immediately available to manage this complication as described earlier (p. 491). In other aspects, however, complications are unique and inherent to epidural analgesia use.

1	Inefective Analgesia. Using currently popular continuous epidural infusion regimens such as 0.125-percent bupivacaine with 2-�g/mL fentanyl, 90 percent of women rate their pain relief as good to excellent (Sharma, 1997). lternatively, a few women ind epidural analgesia to be inadequate for labor. In a study of almost 2000 parturients, Hess and associates (2001) found that approximately 12 percent complained of three or more episodes of pain or pressure. Risk factors for such breakthrough pain included nullipariry and heavier fetal weights. Dresner and colleagues (2006) also reported that epidural analgesia was more likely to fail as body mass index increased. If epidural analgesia is allowed to dissipate before another Injection of anesthetic drug, subsequent pain relief may be delayed, incomplete, or both.

1	In some women, epidural analgesia is insuicient for cesarean delivery. For example, in a Maternal Fetal Medicine Units (MFMU) Network study, 4 percent of women initially given epidural analgesia required a general anesthetic for cesarean delivery (Bloom, 2005). Also at times, perineal analgesia for delivery is diicult to obtain, especially with the lumbar epidural technique. When this situation is encountered, pudendal block or systemic analgesia or rarely general anesthesia may be added.

1	Hypotension. Sympathetic blockade from epidurally injected analgesic agents can cause hypotension and decreased cardiac output. Despite precautions, hypotension is the most frequent side efect and is severe enough to require treatment in a third of women (Sharma, 1997). According to Miller and coworkers (2013), hypotension is more common-20 percent-in women with an admission pulse pressure <45 mm Hg, compared with 6 percent in those whose pulse pressure is >45 mm Hg. In normal gravidas, hypotension induced by epidural analgesia usually can be prevented by rapid infusion of 500 to 1000 mL of crystalloid solution as described for spinal analgesia. Maintaining a lateral position also minimizes hypotension.

1	Maternal Fever. Fusi and colleagues (1989) observed that the mean temperature rose in laboring women given epidural analgesia. Subsequently, several randomized and retrospective cohort studies have confirmed that some women develop intrapartum fever following this procedure. Many studies are limited by inabiliry to control for other risk factors such as labor length, duration of ruptured membranes, and number of vaginal examinations. With this in mind, the frequency of intrapartum fever associated with epidural analgesia was found by Lieberman and O'Donoghue (2002) to be 10 to 15 percent above the baseline rate.

1	The two general theories concerning the etiology of maternal hyperthermia are maternaletal inection or dysregulation of body temperature. Dashe and coworkers (1999) studied placental histopathology in laboring women given epidural analgesia and identified intrapartum fever only when there was placental inflammation. This suggests that fever is due to infection. he other proposed mechanisms include alteration of the hypothalamic thermoregulatory set point; impairment of peripheral thermoreceptor input to the central nervous system, with selective blockage of warm stimuli; or imbalance between heat production and heat loss. Sharma (2014) randomized 400 nulliparas with labor epidural analgesia to receive cefoxitin 2 g prophylactically versus placebo. It was hypothesized that epidural-related fever was due to infection and that prophylactic antimicrobial use should significantly reduce the rate of fever. Approximately equal proportions-about 40 percentof women developed fever > 38°C

1	fever was due to infection and that prophylactic antimicrobial use should significantly reduce the rate of fever. Approximately equal proportions-about 40 percentof women developed fever > 38°C during labor. This suggests that infection is unlikely to be the cause of fever.

1	Back Pain. An association between epidural analgesia and subsequent back pain has been reported by some but not all. In a prospective cohort study, Butler and Fuller (1998) reported that back pain after delivery was common with epidural analgesia, however, persistent pain was uncommon. Based on their systematic review, Lieberman and O'Donoghue (2002) concluded that available data do not support an association between epidural analgesia and development of de novo, long-term backache. Miscellaneous Complications. A spinal or epidural hematoma is a rare complication of an epidural catheter (Grant, 2007). Epidural abscesses are equally infrequent (Darouiche, 2006). And uncommonly, the plastic epidural catheter can be sheared of (Noblett, 2007).

1	Most studies, including the ive from Parkland Hospital, report that epidural analgesia prolongs labor and increases the use of oxytocin stimulation (Table 25-8). Alexander and associates (2002) examined the efects of epidural analgesia on the Friedman (1955) labor curve described in Chapter 22 (p. 432). Compared with original Friedman criteria, epidural analgesia prolonged the active phase of labor by 1 hour. As further shown in Table 25-8, epidural analgesia also increased the need for operative vaginal delivery because of prolonged second-stage labor. But importantly, this led to no greater rates of adverse neonatal efects.

1	This association among epidural analgesia and prolonged second-stage labor and operative vaginal delivery has been attributed to anesthesia-induced motor blockade and resultant impaired matenal expulsive eforts. Craig and colleagues (2015) randomized 310 nulliparous women with labor epidural analgesia to bupivacaine plus fentanyl or fentanyl alone during second-stage labor. Epidural bupivacaine analgesia did cause motor blockade during the second stage, however, the duration of the second stage was not increased. TABLE 25-8. Selected Labor Events in 2703 Nulliparous Women Randomized to Epidural Analgesia First-stage duration 8.1 ± 5 7.5 ± 5 0.01s1 (hr)b Second-stage 60 ± 56 47 ± 57 <0.001 duration (min) Oxytocin ater 641 (48) 546 (40) <0.001 Type of delivery SVD 1027 (77) 11s22 (82) <0.001 Forceps 172 (l3) 101 (7) <0.001 Cesarean 140 (10.5) 141 (10.3) 0.92 aData are presented as n (%) or mean ± SD. bFirst stage = initiation of analgesia to complete cervical dilatation.

1	bFirst stage = initiation of analgesia to complete cervical dilatation. SVD = spontaneous vaginal delivery. Adapted with permission from Sharma SK, Mcintire DD, Wiley J, et al: Labor analgesia and cesarean delivery. An individual patient meta-analysis of nulliparous women, Anesthesiology. 2004 Jan;l 00(1 ):142-148. Fetal Heart Rate. Hill and associates (2003) examined the efects of epidural analgesia with 0.25-percent bupivacaine on fetal heart rate patterns. Compared with intravenous meperidine, no deleterious efects were identified. Reduced beat-co-beat variability and fewer accelerations were more frequent sequelae in fetuses whose mothers received meperidine (Chap. 24, p. 462). Based on their systematic review, Reynolds and coworkers (2002) reported that epidural analgesia was associated with improved neonatal acid-base status compared with meperidine.

1	Cesarean Delivery Rates. A contentious issue in the past was whether epidural analgesia increased the risk for cesarean delivery. Supporting evidence for this view came from the era when dense blocks of local anesthetic agents were used that impaired motor function and therefore likely did contribute to higher cesarean delivery rates. As techniques were refined, however, many investigators came to believe that epidural administration of dilute anesthetic solutions did not increase cesarean delivery rates. Several studies conducted at Parkland Hospital were designed to answer this and related questions. From 1995 to 2002, a total of 2703 nulliparas at term and in spontaneous labor were enrolled in ive trials to evaluate epidural analgesia techniques compared with methods of intravenous meperidine administration. he results from these are summarized in Figure 25-4 and show that epidural analgesia does not signiicantly raise cesarean delivery rates. Timing of Epidural Placement

1	Timing of Epidural Placement In several retrospective studies, epidural placement in early labor was linked to an increased risk of cesarean delivery Ramin et ai, 1995 Sharma et ai, 1997 Gambling et ai, 1998 Lucas et ai, 2001 Sharma et ai, 2002 I OR (95% el) Force on Obstetrical Anesthesia (2016).I Epidural hematomas are rare, and inci 1.01 (0.79,e1.30) dence of nerve damage from a hematoma is estimated to be 1 in 150,000 (Grant, 2007). he American College of Obstetri 1.20 (0.73, 1.97) cians and Gynecologists (2016b) has conI cluded that selected women with platelet 0.77 (0.31e, 1.91e) counts of 80,000 to 100,000/LL may be

1	I candidates for regional analgesia. Caveats --: -1.13 (0.65, 1.97) include a stable platelet count, no acquired or congenital coagulopathy, normal plateI let function, no antiplatelet-specific drugs, 1.05 (0.68, 1.63) and anticoagulation parameters, describedI next, that are met. Counts between 50,000 0.81 (0.41e, 1.61e) and 80,000 require an individualizedI decision on risks and benefits (van Veen, 1.04 (0.81e,e1.34) 2010). Single-shot spinal anesthesia with a 25-gauge needle is less traumatic than epiI dural or combined spinal-epidural anesthe sia with a 17-or 18-gauge epidural needle 0.0 0.4 0.8 1.2 1.6 2.0 platelets in this range. FIGURE 25-4 Results offive studies comparing the incidence of cesarean delivery in women given either epidural analgesia or intravenous meperidine. The individual

1	FIGURE 25-4 Results offive studies comparing the incidence of cesarean delivery in women given either epidural analgesia or intravenous meperidine. The individual Anticoagulation. Women recelvmg odds ratios (ORs) with 95-percent confidence intervals (Cis) for each randomized study, coagulation therapy who are given regional as well as overall crude and adjusted ORs with 95-percent Cis, are shown. An OR <1o.0 analgesia are at increased risk for spinal cord favored epidural over meperidine analgesia. (Reproduced with permission from Sharma

1	SK, Mcintire DO, Wiley J, et al: Labor analgesia and cesarean delivery. An individual patient meta-analysis of nulliparous women, Anesthesiology. 2004 Jan;l00(1):142-148.) sion (Chap. 52, p. 1014). Our practice pat (Lieberman, 1996; Rogers, 1999; Seyb, 1999). These observations prompted at least ive randomized trials, which showed that timing of epidural placement has no efect on the risk of cesarean birth, forceps delivery, or fetal malposition (Chestnut, 1994a,b; Ohel, 2006; Wong, 2005, 2009). Thus, withholding epidural placement until some arbitrary cervical dilation has been attained is unsupportable and serves only to deny women maximal labor pain relief.

1	he relative safety of epidural analgesia is relected by the extraordinary earlier experiences reported by Crawford (1985) from the Birmingham Maternity Hospital in England. Similarly, there were no anesthesia-related maternal deaths among nearly 20,000 women who received epidural analgesia in the MFMU Network study cited earlier (Bloom, 2005). And, Ruppen and associates (2006) reviewed data from 27 studies involving 1.4 million pregnant women who received epidural analgesia. hey calculated risks of 1: 145,000 for deep epidural infection, 1: 168,000 for epidural hematoma, and 1 :240,000 for persistent neurological injury. Thrombocytopenia. For epidural analgesia, contraindications are similar to those with spinal analgesia (see Table 25-6). Although low platelet counts are intuitively worrisome, the level at which epidural bleeding might develop is unknown according to the American Society of Anesthesiologists Task tern includes the following: 1.

1	Women receiving unfractionated heparin therapy should be able to receive regional analgesia if they have a normal activated partial thromboplastin time (aPTT). 2. Women receiving prophylactic doses of unfractionated heparin or low-dose aspirin are not at increased risk and can be ofered regional analgesia. 3. For women receiving once-daily, low-dose low-molecularweight heparin, regional analgesia should not be placed until 12 hours after the last injection. 4. Low-molecular-weight heparin should be withheld for at least 2 hours after epidural catheter removal. 5. he safety of regional analgesia in women receiving twicedaily low-molecular-weight heparin has not been studied suiciently. It is not known whether delaying regional analgesia for 24 hours after the last injection is adequate.

1	Severe Preeclampsia-Eclampsia. Potential concerns with epidural analgesia in women with severe preeclampsia include hypotension as well as hypertension from pressor agents given to correct hypotension. Additionally, pulmonary edema following infusion of large volumes of crystalloid is a potential risk. These are outweighed by disadvantages of general anesthesia. Tracheal intubation may be diicult because of upper airway edema. Moreover, general anesthesia can lead to severe, sudden hypertension that can cause pulmonary or cerebral edema or intracranial hemorrhage. With improved techniques for infusion of dilute local anesthetics into the epidural space, most obstetricians and obstetrical anesthesiologists have come to favor epidural block ade for labor and delivery in women with severe preeclamp sia. here seems to be no argument that epidural analgesia for women with severe preeclampsia-eclampsia can be safely used cians (Lucas, 2001).

1	Women with severe preeclampsia have remarkably diminished intravascular volumes compared with unafected gravidas (Zeeman, 2009). Conversely, extravascular volume is increased because of the capillary leak caused by endothelial cell activation (Chap. 40, p. 717). his imbalance is manifested as pathological peripheral edema, proteinuria, ascites, and total lung water. For all of these reasons, aggressive volume replacement increases the risk for pulmonary edema, especially in the irst 72 hours postpartum. In one study, Hogg and associates (1999) reported that 3.5 percent of women with severe preeclampsia developed pulmonary edema when preloaded without a protocol limitation to volume. Importantly, this risk can be reduced or obviated with judicious prehydration-usually with 500 to 1000 mL of crystalloid solution. Speciically, in the study by Lucas and colleagues (2001), there were no instances of pulmonary edema among the women in whom the crystalloid preload was limited to 500 mL.

1	mL of crystalloid solution. Speciically, in the study by Lucas and colleagues (2001), there were no instances of pulmonary edema among the women in whom the crystalloid preload was limited to 500 mL. Moreover, vasodilation produced by epidural blockade is less abrupt if the analgesia level is achieved slowly with dilute solutions of local anesthetic agents. This allows maintenance of blood pressure while simultaneously avoiding infusion of large crystalloid volumes.

1	he combination of spinal and epidural techniques has increased in popularity and may provide rapid and efective analgesia for labor and for cesarean delivery. An introducer needle is irst placed in the epidural space. A small-gauge spinal needle is then introduced through the epidural needle into the subarachnoid space-this is called the needle-throughneedle technique (see Fig. 25-3). A single bolus of an opioid, sometimes in combination with a local anesthetic, is injected into the subarachnoid space. The spinal needle is withdrawn, and an epidural catheter is then placed through the introducer needle. A subarachnoid opioid bolus results in the rapid onset of profound pain relief with virtually no motor blockade. he epidural catheter permits repeated analgesia dosing. Miro and associates (2008) compared epidural analgesia with combined spinal-epidural analgesia for labor in 6497 women and found the overall outcomes and complications to be similar for the two techniques. In a

1	(2008) compared epidural analgesia with combined spinal-epidural analgesia for labor in 6497 women and found the overall outcomes and complications to be similar for the two techniques. In a randomized comparison, however, Abrao and colleagues (2009) reported that combined spinal-epidural analgesia was associated with a greater incidence of fetal heart rate abnormalities related to uterine hypertonus. Beamon and coworkers (2014) reported similar results .

1	here is emerging interest in continuous spinal analgesia for relief of labor pain. Arkoosh (2008) randomized 429 laboring women to either continuous spinal or conventional epidural analgesia. Complication rates between these two neuraxial techniq ues did not difer. Tao and colleagues (2015) reported their experiences with 113 women. With a dilute bupivacaine solution for analgesia, they found no cases of peripheral nerve injury and a headache rate of2.6 percent. he utility of continuous spinal analgesia in labor and delivery remains to be further studied. A local block is occasionally useful to augment an inadequate or "patchy" regional block that was given emergently. Rarely, local iniltration may be needed to perform an emergent cesarean delivery to save the life of a fetus in the absence of anesthesia support (Young, 2012).

1	In one technique, the skin is iniltrated along the proposed incision, and the subcutaneous, muscle, and rectus sheath layers are injected as the abdomen is opened. Up to a total of 70 mL of 0.5-percent lidocaine with 1 :200,000 epinephrine is prepared for iniltration. Injection of large volumes into the fatty layers, which are relatively devoid of nerve supply, is avoided to limit the total dose of local anesthetic needed. A second technique involves a ield block of the major branches supplying the abdominal wall, to include the 10th, 11 th, and 12th intercostal nerves and the ilioinguinal and genitofemoral nerves (Nandagopal, 2001). As shown in 25-5, the former group of nerves is located at a point midway

1	FIGURE 25-5 Local anesthetic block for cesarean delivery. The first injection site is halfway between the costal margin and iliac crest in the midaxillary line to block the 10th, 11 th, and 12th intercostal nerves. A second injection at the external inguinal ring blocks branches of the genitofemoral and ilioinguinal nerves. These two sites are infiltrated bilaterally. The fifth and final site is along the line of proposed skin incision.

1	between the costal margin and iliac crest in the midaxillary line. he latter group is found at the level of the external inguinal ring. Only one skin puncture is made at each of the four sites (right and left sides). At the intercostal block site, the needle is directed medially, and injection is carried down to the fascia, avoiding injection of the subcutaneous fat. Approximately 5 to 8 mL of 0.5-percent lidocaine is injected. The procedure is repeated at a 45-degree angle cephalad and caudad to this line. he other side is then injected. At the ilioinguinal and genitofemoral sites, the injection is started at a site 2 to 3 cm lateral from the pubic tubercle at a 45-degree angle. Finally, the skin overlying the planned incision is injected.

1	Trained personnel and specialized equipment including alternative airways, video laryngoscopes, and fiberoptic intubation scopes are mandatory for the safe use of general anesthesia. A common cause of death cited for general anesthesia is failed intubation. This occurs in approximately 1 of every 400 general anesthetics administered to pregnant women (Kinsella, 2015). There is a growing trend to continue surgery with a supraglottic airway device, such as a laryngeal mask airway, in the event of a failed intubation (Mushambi, 2015). Because of these relatively greater morbidity and mortality rates, neuraxial analgesia is the preferred method of pain control and should be used unless contraindicated (see Table 25-6). Indeed, in two reports from the MFMU Network, 93 percent of more than 54,000 cesarean deliveries were performed using neuraxial analgesia (Bloom, 2005; Brookfield, 2013). A higher incidence of general anesthesia use for nonwhite women has been reported (Butwick,i2014).

1	Before anesthesia induction, several steps are taken to help minimize complication risks: 1. Antacid administration shortly before anesthesia induction has probably lowered mortality rates from general anesthesia more than any other single practice. The American Society of Anesthesiologists Task Force on Obstetrical Anesthesia (2016) recommends timely administration of a nonparticulate antacid, an Hrreceptor antagonist, or metoclopramide. For many years, we have administered 30 mL of Bicitra-sodium citrate with citric acid-a few minutes before anesthesia induction by either general or major neuraxial block. If more than 1 hour has passed ater the first dose was given and anesthesia has not yet been induced, then a second dose is given. 2.

1	2. Lateral uterine displacement is also provided, as the uterus may compress the inferior vena cava and aorta when the mother is supine. With uterine displacement, the duration of general anesthesia has less efect on neonatal condition than if the woman remains supine. 3. Preoxygenation is done because functional reserve lung capacity is reduced and the pregnant woman becomes hypoxemic more rapidly during periods of apnea. Obesity exacerbates this tendency (McClelland, 2009). To minimize hypoxia between the time of muscle relaxant injection and intubation, oxygen is introduced into the lungs in place of nitrogen. 1bis preoxygenation is accomplished by administering 100-percent oxygen via face mask for 2 to 3 minutes before anesthesia induction. In an emergency, four vital capacity breaths of 100-percent oxygen via a tight breathing circuit will provide similar beneit (Norris, 1985).

1	Almost all parturients are considered to have a full stomach, which necessitates a rapid-sequence induction. Namely, an intravenous anesthetic and rapid-onset muscle relaxant are simultaneously administered while cricoid pressure is applied by an assistant.

1	Of anesthetics, intravenous propofol or etomidate is widely used and ofers a smooth, rapid induction. Propofol is associated with a quick onset and recovery, and it may lower the incidence of nausea and vomiting. Since thiopental is no longer available, propofol is used as the primary agent for induction of general anesthesia with a reasonable safety record. Etomidate is the induction agent of choice for hemodynamically unstable parturients. Alternatively, ketamine can be used but is avoided in hypertensive women. For muscle relaxation, succinylcholine is an ultrafast-onset, short-acting agent commonly used in obstetrics. It ofers intense muscle relaxation to aid endotracheal intubation but also allows for the rapid return of spontaneous respiration in the case of failed intubation. Rocuronium is an alternative muscle relaxant if succinylcholine is contraindicated or unavailable. Its duration is much longer than succinylcholine unless its efect is reversed by sugammadex (Bridion), a

1	is an alternative muscle relaxant if succinylcholine is contraindicated or unavailable. Its duration is much longer than succinylcholine unless its efect is reversed by sugammadex (Bridion), a specific binding agent recently approved by the FDA. To decrease the incidence of fetal respiratory depression, an intermediate or long-acting opioid is usually avoided upon induction of general anesthesia. The intense stimulation from direct laryngoscopy may worsen hypertension and tachycardia in certain women. Remifentanil, an ultrashort-acting narcotic, has been used during induction for cesarean deliveries with favorable maternal hemodynamics and fetal outcome (Heesen, 2013).

1	During induction and intubation, cricoid pressure is applied by a trained assistant to occlude the esophagus and thereby minimize regurgitation of the gastric contents-the Sellick maneuver. Positive mask ventilation during rapid sequence induction is typically avoided to lower the risk of increased intragastric pressure, which raises the risk of vomiting. Surgery should begin only after an airway is secured or, depending on the status of the mother and fetus, efective ventilation has been established.

1	Although uncommon, failed intubation is a major cause of anesthesia-related maternal mortality. A history of prior difficult intubation and a careful anatomical assessment of the neck and maxillofacial, pharyngeal, and laryngeal structures may help predict intubation complications. Even in cases in which the initial airway assessment was unremarkable, edema may develop intrapartum and present considerable challenges. Morbid obesity is another major factor for failed or diicult intubation. The American Society of Anesthesiologists Task Force on Obstetrical Anesthesia (2016) stresses the importance of appropriate preoperative preparation. This includes the immediate availability of specialized equipment such as diferent-shaped laryngoscopes, laryngeal mask airways, a fiberoptic bronchoscope, and a transtracheal ventilation set, as well as liberal use of awake oral intubation techniques.

1	Ideally, an operative procedure is initiated only after it has and that adequate ventilation can be accomplished. Even with an abnormal fetal heart rate pattern, cesarean delivery initiation will only serve to complicate matters if there is diicult or failed intubation. Frequently, the woman must be allowed to awaken and a diferent technique used, such as an awake intubation or regional analgesia.

1	Following failed intubation, the woman is ventilated by mask and cricoid pressure is applied to reduce the aspiration risk. Surgery may proceed with mask ventilation, or the woman may be allowed to awaken. In those cases in which the woman has been paralyzed and ventilation cannot be reestablished by insertion of an oral airway, by laryngeal mask airway, or by use of a iberoptic laryngoscope to intubate the trachea, then a life-threatening emergency exists. To restore ventilation, percuraneous or even open cricothyrotomy is performed and jet ventilation begun. Failed intubation drills have been recommended to optimize the response to such an emergency.

1	With the endotracheal tube secured, anesthesia is maintained with a halogenated agent, typically mixed with air or nitrous oxide. he most commonly used inhalational anesthetics in the United States include deslurane and sevolurane. Both have low solubility in blood and fat. As a result, they ofer faster onset and clearance than more traditional gases such as isolurane. In addition to providing amnesia, they produce profound uterine relaxation when given in high concentrations. his is advantageous when relaxation is a requisite, such as for internal podalic version of the second twin, for breech decomposition, or for replacement of the acutely inverted uterus. That said, unless the woman is alread) under general anesthesia, intravenous nitroglycerine is preferred by many in such situations.

1	he endotracheal tube may be safely removed only if the woman is conscious to a degree that enables her to follow commands and is capable of maintaining oxygen saturation with spontaneous respiration. Consideration is given to emptying the stomach via a nasogastric tube before extubation. As induction has become safer, extubation may now be relatively more perilous. Of 15 anesthesia-related deaths of pregnant women from 1985 to 2003 in Michigan, none occurred during induction. Five resulted from hypoventilation or airway obstruction during emergence, extubation, or recovery (Mhyre, 2007). Massive gastric acidic inhalation may cause pulmonary insufficiency from aspiration pneumonitis. In the past, this was the most common cause of anesthetic deaths in obstetrics and therefore deserves special attention. To minimize this risk, antacids are given rourinely, intubation is accompanied by cricoid pressure, and regional analgesia is employed when possible.

1	According to the American Society of Anesthesiologists Task Force on Obstetrical Anesthesia (2016) and the merican College of Obstetricians and Gynecologists (2017b), data are insuicient regarding fasting times for clear liquids and the risk of pulmonary aspiration during labor. Recommendations are that modest amounts of clear liquids such as water, clear tea, black cofee, carbonated beverages, and pulp-free fruit juices be allowed in uncomplicated laboring women (Chap 22, p. 437). Obvious solid foods are avoided. A fasting period of 6 to 8 hours for solid food is recommended for uncomplicated parturients prior to undergoing elective cesarean delivery or puerperal tubal ligation.

1	O'Sullivan (2009) randomized 2426 low-risk nulliparas to consume either water and ice chips alone or small amounts of bread, biscuits, vegetables, fruits, yogurt, soup, and fruit juice. Approximately 30 percent of women in each arm of the study underwent cesarean delivery. No cases of aspiration occurred during the study, although approximately a third of women in each study arm vomited during labor or delivery. Epidural analgesia during labor was used in this study, although the authors did not report the type of anesthesia used for cesarean deliveries. Presumably, neuraxial analgesia was used, and this greatly minimized the pulmonary aspiration risk. Given the low prevalence of aspiration, this trial was not powered to measure whether feeding during labor was safe (Sperling, 2016).

1	In 1952, Teabeaut demonstrated experimentally that if the pH of aspirated fluid was <2.5, severe chemical pneumonitis developed. It was later demonstrated that the pH of gastric juice in nearly half of women tested intrapartum was <2.5 (Taylor, 1966). The right mainstem bronchus usually ofers the simplest pathway for aspirated material to reach the lung parenchyma, and therefore, the right lower lobe is most often involved. In severe cases, there is bilateral widespread involvement. The woman who aspirates may develop evidence of respiratory distress immediately or several hours after aspiration, depending in part on the material aspirated and the severity of the response. Aspiration of a large amount of solid material causes obvious airway obstruction. Smaller particles without acidic liquid may lead to patchy atelectasis and later to bronchopneumonia.

1	When highly acidic liquid is inspired, decreased oxygen saturation along with tachypnea, bronchospasm, rhonchi, rales, atelectasis, cyanosis, tachycardia, and hypotension are likely to develop. At the injury sites, there is pulmonary capillary leakage and exudation of protein-rich fluid containing numerous erythrocytes into the lung interstitium and alveoli. his causes decreased pulmonary compliance, shunting of blood, and severe hypoxemia. Radiographic changes may not appear immediately, and these may be variable, although the right lung most often is afected. hus, chest radiographs alone should not be used to exclude aspiration.

1	he methods recommended for treatment of aspiration have changed appreciably in recent years, indicating that previous therapy was not very successful. Suspicion of aspiration of gastric contents demands close monitoring for evidence of pulmonary damage. Respiratory rate and oxygen saturation as measured by pulse oximetry are the most sensitive and earliest indicators of injury.

1	Inhaled luid should be immediately and thoroughly wiped from the mouth and removed from the pharynx and trachea by suction. Saline lavage may further disseminate the acid throughout the lung and is not recommended. If large particulate matter is inspired, bronchoscopy may be indicated to relieve airway obstruction. No convincing evidence supports that corticosteroid therapy or prophylactic antimicrobial administration is beneicial (Marik, 2001). If infection develops, however, then vigorous treatment is given. If acute respiratory failure develops, mechanical ventilation with positive end-expiratory pressure may be lifesaving (Chap. 47, p. 919).

1	patient satisfaction, minimizing side efects, aiding unctional capacity, and preventing prolonged hospital stays (Lavoie, 2013). In a prospective study, 96 percent of women reported pain immediately ater delivery (Eisenach, 2008). he incidence of persistent pain 1 and 2 years following cesarean delivery was reported to approximate 20 percent (Hannah, 2004; Kainu, 2010).

1	he American Society of Anesthesiologists (2016) recommends neuraxial opioids for postoperative analgesia. Although most cesarean deliveries in the United States are performed under neuraxial anesthesia, in certain situations a peripheral nerve block such as a transversus abdominis plane (TAP) block may be considered (McDonnell, 2007). hese include cases in which the parturient did not receive neuraxial opioids, underwent general anesthesia, or has persistent pain following neuraxial anesthesia. It is usually performed under ultrasound guidance and involves injection of a local anesthetic into the transversus abdominis plane between the internal oblique and transversus abdominis muscles. he nerves lying in this plane supply the anterior abdominal wall at the T 6 to L1 dermatomes. A metaanalysis of 31 controlled trials showed that ultrasoundguided TAP block marginally reduced opioid consumption at 6 hours following abdominal surgery (Baeriswyl, 2015).

1	Abrao KC, Francisco RP, Miyadahira S, et al: Elevation of uterine basal tone and fetal heart rate abnormalities after labor analgesia: a randomized controlled trial. Obstet Gynecol 113(10):41,2009 Alexander JM, Sharma SK, McIntire DD, et al: Epidural analgesia lengthens the Friedman active phase ofolabor. Obstet Gynecol 100:46,o2002 American College of Obstetricians and Gynecologists: Practice advisory: FDA warnings regarding use of general anesthetics and sedation drugs in young children and pregnant women. 2016a. Available at: http://www.acog.org/ About-ACOG/News-Room/Practice-Advisories/FDA-Warnings-Rega rding-Use-of-General-Anesthetics-and-Sedation-Drugs. Accessed January 30, 20o17 American College of Obstetricians and Gynecologists: hrombocytopenia in pregnancy. Practice Bulletin 166, September 20o16b American College of Obstetricians and Gynecologists: Obstetric analgesia and anesthesia. Practice Bulletin 177, Aprilo20o17a

1	American College of Obstetricians and Gynecologists: Oral intake during labor. Committee Opinion No. 44o1, September 2009, Reairmed 20o17b American Society of Anesthesiologists: Task Force on Obstetrical Anesthesia: practice guidelines for obstetrical anesthesia. Anesthesiology 124:270, 20o16 Angle P, Tang SL, Thompson D, et al: Expectant management of postdural puncture headache increases hospital length of stay and emergency room visits. Can J Anaesth 52(4):397, 2005

1	Arkoosh V, Palmer C, Yun E, et al: A randomized, double-masked, multicenter comparison of the safety of continuous intrathecal labor analgesia using a 28-gauge catheter versus continuous epidural labor analgesia. Anesthesiology 108(2):286, 2008 Baeriswyl M, Kirkham KR, Kern C, et al: he analgesic eicacy of ultrasoundguided transversus abdominis plane block in adult patients: a meta-analysis. AnesthAnalg 121(6):1640,2015 Barbieri L, Camann W, McGovern C: Nitrous oxide for labor pain. OBG Manag 26(12):10,o2014 Beamon C, Stuebe A, Edwards L, et al: Efect of mode of regional anesthesia on neonatal outcomes in preeclamptic patients. Am J Obstet Gynecol 210:S173,o2014 Bell ED, Penning DH, Cousineau EF, et al: How much labor is in a labor epidural? Manpower cost and reimbursement for an obstetric analgesia service in a teaching institution. Anesthesiology 92:851, 2000 Bloom SL, Spong CY, Weiner SJ, et al: Complications of anesthesia for cesarean delivery. Obstet Gynecol 106:28o1, 2005

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1	Capogna G, Camorcia M, Stirparo S, et al: Programmed intermittent epidural bolus versus continuous epidural infusion for labor analgesia: the efects on maternal motor function and labor outcome. A randomized double-blind study in nulliparous women. Anesth Analg 113(4):826, 2011 Centers for Disease Control and Prevention: Bacterial meningitis after intrapartum anesthesia-New York and Ohio, 2008-2009. MMWR 59(3):65, 2010 Chestnut DH, McGrath JM, Vincent RD Jr, et al: Does early administration of epidural analgesia afect obstetric outcome in nulliparous women who are in spontaneous labor? Anesthesiology 80: 1201, 1994a Chestnut DH, Owen CL, Bates IN, et al: Continuous infusion epidural analgesia during labor: a randomized, double-blind comparison of 0.625% bupivacaine/0.0002% fentanyl versus 0.125% bupivacaine. Anesthesiology 68:754, 1988

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1	Lee A, Posner KL, Domino KB, et al: Injuries associated with regional anesthesia in the 1980s and 1990s: a closed claims analysis. Anesthesiology 101:143,r2004 Li G, Warner M, Lang BH et al: Epidemiology of anesthesia-related mortality in the United States, 1999-2005. Anesthesiology 110(4):759,r2009 Lieberman E, Lang JM, Cohen A, et al: Association of epidural analgesia with cesarean delivery in nulliparas. Obstet Gynecol 88:993, 1996 Lieberman E, O'Donoghue C: Unintended efects of epidural analgesia during labor: a systematic review. Am J Obstet Gynecol 186:531, 2002 Likis FE, Andrews Je, Collins MR, et al: Nitrous oxide for the management of labor pain: a systematic review. Anesth Analg 118(1):153, 2014 Liu SS, Lin Y: Local anesthetics. In Barash , Cullen B, Stoeling R, et al (eds): Clinical Anesthesia, 6th ed. Philadelphia, Lippincott Williams & Wilkins,

1	Liu SS, Lin Y: Local anesthetics. In Barash , Cullen B, Stoeling R, et al (eds): Clinical Anesthesia, 6th ed. Philadelphia, Lippincott Williams & Wilkins, Liu x'H, Lin JH, Lin Je, et al: Severe intracranial and intraspinal subarachnoid hemorrhage after lumbar puncture: a rare case report. Am J Emerg Med 26:633, 2008 Lucas MJ, Sharma SK, McIntire DD, et al: A randomized trial oflabor analgesia in women with pregnancy-induced hypertension. Am J Obstet Gynecol 185:970,r2001 Manninen T, Aantaa R, Salonen M, et al: A comparison of the hemodynamic efects of paracervical block and epidural anesthesia for labor analgesia. Acta Anaesthesiol Scand 44:441, 2000 Marik PE: Aspiration pneumonitis and aspiration pneumonia. N Engl J Med 344:665, 2001 McClelland SH, Bogod DG, Hardman JG: Pre-oxygenation and apnoea in pregnancy: changes during labour and with obstetric morbidity in a computational simulation. Anaesthesia 64(4):371, 2009

1	McClelland SH, Bogod DG, Hardman JG: Pre-oxygenation and apnoea in pregnancy: changes during labour and with obstetric morbidity in a computational simulation. Anaesthesia 64(4):371, 2009 McDonnell JG, O'Donnell B, Curley G, et al: he analgesic eicacy of transversus abdominis plane block after abdominal s urge ty: a prospective randomized controlled trial. Anesth Analg 104(1):193,r200 Mhyre JM, Riesner MN, Polley LS, et al: A series of anesthesia-related maternal deaths in Michigan, 1985-2003. Anesthesiology 106:1096,r2007 Miller N, Cypher R, Thomas S, et al: Admission pulse pressure is a novel predictor of fetal heart rate abnormalities initial dosing of a labour epidural: a retrospective cohort study. Abstract No. 333. Am J Obstet Gynecol 208(1 Suppl):S149, 2013 Millet L, Shaha S, Bartholomew ML: Rates of bacteriuria in laboring with epidural analgesia: continuous vs intermittent bladder catheterization. Am J Obstet Gynecol 206:316, 2012

1	Millet L, Shaha S, Bartholomew ML: Rates of bacteriuria in laboring with epidural analgesia: continuous vs intermittent bladder catheterization. Am J Obstet Gynecol 206:316, 2012 Miro M, Guasch E, Gilsanz F: Comparison of epidural analgesia with combined spinal-epidural for labor: a retrospective study of 6497 cases. Int J Obstet Anesth 17: 15, 2008 Mokri B: The Monro-Kellie hypothesis: application in CSF volume depletion. Neurology 56(12):1746,r2001 Mulroy MF: Systemic toxicity and cardiotoxicity from local anesthetics: incidence and preventive measures. Reg Anesth Pain Med 27:556, 2002 Mushambi MC, Kinsella SM: Obstetric Anaesthetists' Association/Diicult Airway Society diicult and failed tracheal intubation guidelines-the way forward for the obstetric airway. Br J Anaesth 115(6):815,2015 Nandagopal M: Local anesthesia for cesarean section. Tech Reg Anesth Pain Manag 5(1):30,r2001

1	Nandagopal M: Local anesthesia for cesarean section. Tech Reg Anesth Pain Manag 5(1):30,r2001 Neal JM, Mulroy MF, Weinberg GL, et al: American Society of Regional Anesthesia and Pain Medicine checklist for managing local anesthetic systemic toxicity: 2012 version. Reg Anesth Pain Med 37(1):16,r2012 Ngan Kee WD, Khaw KS, Ng FF, et al: Prophylactic phenylephrine infusion for preventing hypotension during spinal anesthesia for cesarean delivery. Anesth Analg 98:815,r2004 Noblett K, McKinney A, Kim R: Sheared epidural catheter during an elective procedure. Obstet Gynecol 109:566,r2007 Norris Me, Dewan DM: Preoxygenation for cesarean section: a comparison of rwo techniques. Anesthesiology 62:827, 1985 Ohashi Y, Baghirzada L, Sumikura H, et al: Remifentanil for labor analgesia: a comprehensive review. J Anesth 30(6):1020, 2016

1	Ohashi Y, Baghirzada L, Sumikura H, et al: Remifentanil for labor analgesia: a comprehensive review. J Anesth 30(6):1020, 2016 Ohel G, Gonen R, Vaida S, et al: Early versus late initiation of epidural analgesia in labor: does it increase the risk of cesarean section? A randomized trial. m J Obstet Gynecol 194:600, 2006 Osterman MJ, Martin JA: Epidural and spinal anesthesia use during labor, 2008. Nat! Vital Stat Rep 59(5):1, 2011 O'Sullivan G, Liu B, Hart D, et al: Efect of food intake during labour on obstetric outcome: randomised controlled trial. BMJ 338:b784, 2009 Paech MJ, Doherty DA, Christmas T, et al: The volume of blood for epidural patch in obstetrics: a randomized blinded clinical trial. Anesth Analg 13(1):126,r2011 Ramin SM, Gambling DR, Lucas MJ, et al: Randomized trial of epidural versus intravenous analgesia during labor. Obstet Gynecol 83, 1995

1	Ramin SM, Gambling DR, Lucas MJ, et al: Randomized trial of epidural versus intravenous analgesia during labor. Obstet Gynecol 83, 1995 Reynolds F, Sharma SK, Seed PT: Analgesia in labour and fetal acid-base balance: a meta-analysis comparing epidural with systemic opioid analgesia. BJOG 109:1344,r2002 Rogers R, Gilson G, Kammerer-Doak D: Epidural analgesia and active management of labor: efects on length of labor and mode of delivery. Obstet Gynecol 93:995, 1999 Rosen /1A: Paracervical block for labor analgesia: a brief historic review. Am J Obstet Gynecol 186:S , 2002 Ruppen W, Derry S, McQuay H, et al: Incidence of epidural hematoma, infection, and neurologic injury in obstetric patients with epidural analgesia/ anesthesia. Anesthesiology 105:394,r2006 Scavone BM: Timing of epidural blood patch: clearing up the confusion. Anaesthesia 70(2):119,r2015

1	Scavone BM: Timing of epidural blood patch: clearing up the confusion. Anaesthesia 70(2):119,r2015 Scavone BM, Wong CA, Sullivan JT, et al: Eicacy of a prophylactic epidural blood patch in preventing post dural puncture headache in parturients after inadvertent dural puncture. Anesthesiology 101: 1422, 2004

1	Setayesh AR, Kholdebarin AR, Moghadam MS, et al: he Trendelenburg position increases the spread and accelerates the onset of epidural anesthesia for cesarean section. Can J Anaesth 48:890,r2001 Seyb ST, Berka RJ, Socol ML, et al: Risk of cesarean delivery with elective induction of labor at term in nulliparous women. Obstet Gynecol 94:600, 1999 Sharma SK, Alexander JM, Messick G, et al: Cesarean delivery: a randomized trial of epidural analgesia versus intravenous meperidine analgesia during labor in nulliparous women. Anesthesiology 96:546, 2002 Sharma SK, McIntire DD, Wiley J, et al: Labor analgesia and cesarean delivery. An individual patient meta-analysis of nulliparous women. Anesthesiology 100:142,r2004 Sharma SK, Rogers BB, Alexander JM, et al: A randomized trial of the efects of antibiotic prophylaxis on epidural related fever in labor. Anesth Analg 118(3):604,r2014 Sharma SK, Sidawi JE, Ramin SM, et al: Cesarean delivery: a randomized trial of epidural versus

1	the efects of antibiotic prophylaxis on epidural related fever in labor. Anesth Analg 118(3):604,r2014 Sharma SK, Sidawi JE, Ramin SM, et al: Cesarean delivery: a randomized trial of epidural versus patient-controlled meperidine analgesia during labor. Anesthesiology 87 :487, 1997 Shearer VE, Jhaveri HS, Cunningham FG: Puerperal seizures after post-dural puncture headache. Obstet Gynecol 85:255, 1995 Smarkusky L, DeCarvalho H, Bermudez A, et al: Acute onset headache complicating labor epidural caused by intrapartum pneumocephalus. Obstet Gynecol 108:795, 2006

1	Society for Obstetric Anesthesia and Perinarology: Response to the FDA Med Watch December 16, 2016. 2017. Available at: https://soap.org/asaresponse-fda-soapl-20-17.pd. Accessed February 2,r2017 Sperling JD, Dahlke JD, Sibai BM: Restriction of oral intake during labor: whither are we bound? Am J Obstet GynecoI214(5):592, 2016 Sprigge JS, Harper SJ: Accidental dural puncture and post dural puncture headache in obstetric anaesthesia: presentation and management: a 23-year survey in a district general hospital. Anaesthesia 63:36, 2008 Svancarek W, Chirino 0, Schaefer G Jr, et al: Retropsoas and subgluteal abscesses following paracervical and pudendal anesthesia. JAMA 237:892, 1977 Tao W, Grant EN, Craig MG, et al: Continuous spinal analgesia for labor and delivery: an observational study with a 23-gauge spinal catheter. Anesth Analg 121(5):1290, 2015

1	Tao W, Grant EN, Craig MG, et al: Continuous spinal analgesia for labor and delivery: an observational study with a 23-gauge spinal catheter. Anesth Analg 121(5):1290, 2015 Taylor G, Pryse-Davies J: The prophylactic use of antacids in the prevention of the acid pulmonary aspiration syndrome (Mendelson's syndrome). Lancet 1:288, 1966 Teabeaut JR II: Aspiration of gastric contents: an experimental study. Am J Pathol 28:51, 1952 Vallejo Me, Mandell GL, Saba DP, et al: Postdural puncture headache: a randomized comparison of five spinal needles in obstetric patients. Anesth Analg 91:916,r2000 van Veen J], Nokes TJ, Makris M: The risk of spinal haematoma following neuraxial anaesthesia or lumbar puncture in thrombocytopenic individuals. Br J HaematoIr148(I):15, 2010 Vricella LK, Louis JM, Mercer BM, et al: Impact of morbid obesity on epidural anesthesia complications in labor. Am J Obstet Gynecol 205:307, 2011

1	Vricella LK, Louis JM, Mercer BM, et al: Impact of morbid obesity on epidural anesthesia complications in labor. Am J Obstet Gynecol 205:307, 2011 Wallace DH, Leveno KJ, Cunningham FG, et al: Randomized comparison of general and regional anesthesia for cesarean delivery in pregnancies complicated by severe preeclampsia. Obstet Gynecol 86: 193, 1995 Waring J, Mahboobi SK, Tyagaraj K, et al: Use of remifentanil for labor analgesia: the good and the bad. Anesth Analg 1 04( 46): 1616, 2007 Webb CA, Weyker PD, Zhang L, et I: Unintentional dural puncture with a Tuohy needle increases risk of chronic headache. Anesth Analg 115(1): 124, 2012 Wong CA: Epidural and spinal analgesia/anesthesia for labor and vaginal delivery. In Chestnut's Obstetrical Anesthesia: Principles and Practice, 5th ed. Philadephia, Saunders, 2014 Wong CA, McCarthy RJ, Sullivan JT, et al: Early compared with late neuraxial analgesia in nulliparous labor induction. Obstet Gynecoirl13(5):1066, 2009

1	Wong CA, McCarthy RJ, Sullivan JT, et al: Early compared with late neuraxial analgesia in nulliparous labor induction. Obstet Gynecoirl13(5):1066, 2009 Wong CA, Scavone BM, Peaceman AM, et al: The risk of cesarean delivery with neuraxial analgesia given early versus late in labor. N Engl J Med 352:655, 2005 Young MJ, Gorlin AW, Modes VE, et al: Clinical implications of the transversus abdominis plane block in adults. Anesthesiol Res Pract 2012:731645,r2012 Zeeman GG, Cunningham FG, Pritchard JA: The magnitude of hemoconcentration with eclampsia. Hypertens Preg 28(2): 127, 2009 Induction and Augmentation of labor LABOR INDUCTION ......e...........e......e....e. 503 PREINDUCTION CERVICAL RIPENING ..e....e.....e.... 505 PHARMACOLOGICAL TECHNIQUESe. . . . . . . . . . . . . . . . 506 MECHANICAL TECHNIQUES .e........e....e...e..e... 507 METHODS OF INDUCTION AND AUGMENTATION . .. 508 PROSTAGLANDIN E, .. .......... ......... .... 508

1	MECHANICAL TECHNIQUES .e........e....e...e..e... 507 METHODS OF INDUCTION AND AUGMENTATION . .. 508 PROSTAGLANDIN E, .. .......... ......... .... 508 OXYTOCIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509 AMNIOTOMY FOR INDUCTION AND AUGMENTATION . 511 In other cases, if inteerence becomes imperative, the introduction of a bougie into the uterus, or the employment of a smal Champetier de Ribes rubber bag acts as an eective uterine irritant and brings about complete dilatation. -J. No efective means of labor induction were available when Williams wrote the first edition of this book. Labor augmentation methods were largely inefective, and manual cervical dilation was performed as a last resort. Contrast with today, when several pharmacological agents permit labor induction or augmentation, and ironically the use of a "bougie" has come back into vogue.

1	Induction implies stimulation of contractions before the spontaneous onset of labor, with or without ruptured membranes. When the cervix is closed and unefaced, labor induction will often commence with cervical ripening, a process that generally employs prostaglandins to soften and open the cervix. Augmentation refers to enhancement of spontaneous contractions that are considered inadequate because of failed cervical dilation and fetal descent-inertia uteri-as described by Williams (1903).

1	Augmentation refers to enhancement of spontaneous contractions that are considered inadequate because of failed cervical dilation and fetal descent-inertia uteri-as described by Williams (1903). In the United States, the incidence ofilabor induction rose 2.5-fold from 9.5 percent in 1991 to 23.8 percent in 2015 (Martin, 2017). he incidence varies between practices. At Parkland Hospital, approximately 35 percent of labors are induced or augmented. By comparison, at the University of Alabama at Birmingham Hospital, labor is induced in approximately 20 percent of women, and another 35 percent are given oxytocin for augmentation-a total of 55 percent. This chapter discusses indications for labor induction and augmentation and various techniques to efect preinduction cervical ripening.

1	Induction is indicated when the beneits to either mother or fetus outweigh those of pregnancy continuation. The more common indications include membrane rupture without labor, gestational hypertension, oligohydramnios, nonreassuring fetal status, postterm pregnancy, and various maternal medical conditions such as chronic hypertension and diabetes (American College of Obstetricians and Gynecologists, 20i16). Methods to induce or augment labor are contraindicated by most conditions that preclude spontaneous labor or delivelY. The few maternal contraindications are related to prior uterine incision type, contracted or distorted pelvic anatomy, abnormally implanted placentas, and uncommon conditions such as active genital herpes infection or cervical cancer. Fetal factors include appreciable macrosomia, severe hydrocephalus, malpresentation, or nonreassuring fetal status.

1	Oxytocin has been used for decades to induce or augment labor. Other efective methods include prostaglandins, such as misoprostol and dinoprostone, and mechanical methods that encompass membrane stripping, artiicial rupture of membranes, extraamnionic saline infusion, transcervical balloons, and hygroscopic cervical dilators. Importantly, and as recommended in Guidelines or Perinatal Care, each obstetrical department should have its own written protocols that describe administration of these methods for labor induction and augmentation (American Academy of Pediatrics, 2017).

1	Maternal complications associated with labor induction are cesarean delivery, chorioamnionitis, uterine rupture, and postpartum hemorrhage from uterine atony. Of these, labor induction carries a two-to threefold greater risk for cesarean delivery (Hofman, 2003; Maslow, 2000; Smith, 2003). his risk is particularly higher among nulliparas (Luthy, 2004; Wolfe, 2014; Yeast, 1999). More recently, this association has been questioned (Macones, 2009; Melamed, 2016; Miller, 2015; Saccone, 2015). Indeed, Darney and colleagues (2012) reported the cesarean delivery risk was actually lower for women with labor induction at 39 weeks' gestation compared with that in women expectantly managed. In their review, Little and Caughey (2015) found a reduced cesarean delivery rate when women undergoing labor induction were compared with women expectantly managed, as opposed to women spontaneously laboring. Currently, this is the topic of a randomized trial by the 1aternal-Fetal Medicine Units (MFMU)

1	induction were compared with women expectantly managed, as opposed to women spontaneously laboring. Currently, this is the topic of a randomized trial by the 1aternal-Fetal Medicine Units (MFMU) NetworkA Randomized Trial of Induction Versus Expectant v1anagement-ARRIVE (National Institutes of Health, 2015).

1	Amniotomy is often selected to augment labor (p. 511). Women whose labor is managed with amniotomy have a higher incidence of chorioamnionitis compared with those in spontaneous labor (American College of Obstetricians and Gynecologists, 2016). Rupture of a prior uterine incision during labor in women with a history of prior uterine surgery can be catastrophic (Chap. 31, p. 598). The MFMU Network reported a threefold greater risk of uterine scar rupture with oxytocin, and this was even higher with prostaglandin use (Landon, 2004). he American College of Obstetricians and Gynecologists (20 17b) recommends against the use of prostaglandins for preinduction cervical ripening or labor induction in women with a prior uterine incision.

1	Uterine atony and associated postpartum hemorrhage are more common in women undergoing induction or augmentation (Chap. 41, p. 759). And, atony with intractable hemorrhage, especially during cesarean delivery, is a frequent indication for peripartum hysterectomy. In a study from Parkland Hospital, labor induction was associated with 17 percent of 553 emergency peripartum hysterectomies (Hernandez, 2013). In the United States, the postpartum hysterectomy rate rose 15 percent between 1994 and 2007 (Bateman, 2012). his was largely attributed to increased rates of atony associated with more medical labor inductions and more primary and repeat cesarean deliveries. In another analysis, elective induction was also linked with a threefold higher rate of hysterectomy (Bailit, 2010).

1	Until recently, elective induction for convenience had become increasingly prevalent. Clark and coworkers (2009) described 14,955 deliveries at �37 weeks' gestation. hey noted that 32 percent were elective deliveries, and 19 percent were elective labor inductions.

1	he American College of Obstetricians and Gynecologists (2016) does not endorse this once widespread practice. Occasional exceptions might include logistical and other reasons such as a risk of rapid labor, a woman who lives a long distance from the hospital, or psychosocial indications. Because of the greater risks for adverse maternal outcomes, we are also of the opinion that routine elective induction at term is not justified. Elective delivery before 39 completed weeks is also associated with signiicant adverse neonatal morbidity (Chiossi, 2013; Clark, 2009; Salemi, 2016; Tita, 2009). If elective induction is considered at term, inherent risks must be discussed, informed consent obtained, and guidelines followed as promulgated by the American College of Obstetricians and Gynecologists (2016), which are detailed in Chapter 31 (p. 597).

1	Guidelines to discourage elective inductions have been described by Fisch (2009) and Oshiro (20l3) and their associates. Both groups reported signiicant declines in elective delivery rates following guideline initiation. In 201i1, the Texas Medicaid program began to deny payment for elective induction prior to 39 weeks' gestation. his resulted in a 14-percent drop in such early-term deliveries and a rise in birthweights (Dahlen, 2017). A program in Oregon also reduced early-term deliveries, but maternal and fetal outcomes were not improved (Snowden, 2016).

1	Several factors afect the ability of labor induction to achieve vaginal delivery. Favorable factors include younger age, multiparity, body mass index (BMI) <30, favorable cervix, and birthweight <3500 g (Gibson, 2015; Roland, 2017; Sievert, 2017). In many cases, the uterus is simply poorly prepared for labor. One example is an "unripe cervix." Indeed, investigators with the Consortium on Sae Labor reported that elective induction resulted in vaginal delivery in 97 percent of multiparas and 76 percent of nulliparas, but that induction was more often successful with a ripe cervix (Laughon, 2012).

1	he greater cesarean delivery risk associated with induction is likely also strongly influenced by the induction attempt duration, especially with an unfavorable cervix (Spong, 2012). In one study, labor duration to reach the active phase and to complete dilation was adversely afected by a higher BMI (Kominiarek, 2011). Similar indings were reported for women with diabetes (Hawkins, 2017). Simon and Grobman (2005) concluded that a latent phase as long as 18 hours allowed most women undergoing labor induction to achieve a vaginal delivery without a signiicantly increased risk of maternal or neonatal morbidity. Rouse and associates (2000) recommend a minimum of Induction and Augmentation of Labor 505 TABLE 26-1 . Some Commonly Used Regimens for Preinduction Cervical Ripening and/or Labor Induction Prostaglandin E2 Oinoprostone gel, Cervical 0.5 mg; repeat in 1. Shorter 1-0 times with oxytocin infusion 0.5 mg (Prepidil) 6 hr; permit 3 doses total than oxytocin alone

1	Prostaglandin E2 Oinoprostone gel, Cervical 0.5 mg; repeat in 1. Shorter 1-0 times with oxytocin infusion 0.5 mg (Prepidil) 6 hr; permit 3 doses total than oxytocin alone Oinoprostone insert, Posterior 10 mg 1. Insert has shorter 1-0 times than gel 10 mg (Cervidil) 2. 6-12 hr interval from last insert to oxytocin aOf-label use. bTablets must be divided for 25-and 50-�g dose, but drug is evenly dispersed. EASI = extraamnionic saline infusion at 30-40 mUhr; 1-0 = induction-to-delivery. 12 hours of uterine stimulation with oxytocin after membrane rupture, whereas Kawakita and coworkers (2016) recommend up to 15 hours for multiparas. As discussed, the condition of the cervix-described as cervical "ripeness" or "favorability"-is important to successful labor induction. However, at least some estimates of favorability are highly subjective (Peltovich, 2017). hat said, pharmacological and mechanical methods can enhance cervical favorabilityalso termed preinduction cervical ripening.

1	Some of the techniques described may have benefits when compared with oxytocin induction alone (Table Some are also quite successful for initiating labor. However, few data support the premise that any of these techniques lower cesarean delivery rates or lessen maternal or neonatal morbidity compared with women in whom these methods are not used. One quantifiable method used to predict labor induction outcomes is the score described by Bishop (1964) and presented in Table 26-2. As favorability or Bishop score declines, the rate of induction to efect vaginal delivery also decreases. A Bishop score of 9 conveys a high likelihood for a successful induction. Por research purposes, a Bishop score of 4 or less identifies an unfavorable cervix and may be an indication for cervical ripening. TABLE 26-2. Bishop Scoring System Used for Assessment of Inducibility Score Dilatation (cm) Efacement (%) Station (-3 to +2) Consistency Position �80 +1, +2 From Bishop, 1964.

1	TABLE 26-2. Bishop Scoring System Used for Assessment of Inducibility Score Dilatation (cm) Efacement (%) Station (-3 to +2) Consistency Position �80 +1, +2 From Bishop, 1964. Laughon and coworkers (201i1) attempted to simpliy the Bishop score by performing a regression analysis on 5610 singleton, uncomplicated deliveries between 37°/7 and 416/7 weeks' gestation in nulliparas. Only cervical dilation, station, and efacement were signiicantly associated with successful vaginal delivery. hus, a simplified Bishop score, which incorporated only these three parameters, had a similar or improved positiveor negative-predictive value compared with that of the original Bishop score. Other investigators have reported similar findings when consistency and position are omitted (Ivars, 2016; Raghuraman,i2016).

1	Transvaginal sonographic measurement of cervical length is the only biophysical marker that has been evaluated as a Bishop score alternative (Feltovich, 2017). In one metaanalysis of trials in which cervical length was used to predict successful induction, study criteria heterogeneity precluded the authors from reaching a summary answer (Hatfield, 2007). A subsequent metaanalysis of 31 trials found overall low sensitivity and speciicity and limited predictive utility for sonographic cervical length and "wedging" to predict successful labor induction (Verhoeven, 2013). Unfortunately, women frequently have an indication for induction but also have an unfavorable cervix. Several techniques are available, and these can also stimulate contractions and thereby aid subsequent labor induction or augmentation. Methods most commonly used for preinduction cervical ripening and induction include several prostaglandin analogues.

1	Dinoprostone is a synthetic analogue of prostaglandin E2 (PGE2). It is commercially available in three forms: a gel, a timerelease vaginal insert, and a 20-mg suppository (Table 26-1). he gel and time-release vaginal insert formulations are indicated only for cervical ripening before labor induction. However, the 20-mg suppository is not indicated for cervical ripening. It instead is used for pregnancy termination between 12 and 20 weeks' gestation and for evacuation of the uterus after fetal demise up to 28 weeks. Local application of its gel form-Prepidil-is available in a 2.5-mL syringe for an intracervical application of 0.5 mg of dinoprostone. With the woman supine, the tip of a prefilled syringe is placed intracervically, and the gel is deposited just below the internal cervical os. After application, the woman remains reclined for at least 30 minutes. Doses may be repeated every 6 hours, with a maximum of three doses recommended in 24 hours.

1	A 10-mg dinoprostone vaginal insert-Cervidil-is also approved for cervical ripening. This is a thin, flat, rectangular polymeric wafer held within a small, white, mesh polyester sac (Fig. 26- The sac has a long attached tail to allow easy removal from the vagina. The insert provides slower release of medication-0.3 mg/hr-than the gel form. Cervidil is used as a single dose placed transversely in the posterior vaginal fornix. Lubricant is used sparingly, if at all, because it can coat the device and hinder dinoprostone release. Following insertion, the woman remains recumbent for at least 2 hours. he insert is removed after 12 hours or with labor onset and at least 30 minutes before the administration of oxytocin. FIGURE 26-1 Cervidil vaginal insert contains 10 mg of dinoprostone designed to release approximately OJ mg/hr during a1 O-hour period.

1	Most metaanalyses of dinoprostone eicacy report a reduced time-to-delivery within 24 hours, however, they do not consistently show a reduction in the cesarean delivery rate. Thomas and colleagues (2014) provided a Cochrane review of 70 trials and 11i,487 women given vaginal prostaglandins or either placebo or no treatment. They noted a higher vaginal delivery rate within 24 hours when prostaglandins were used. hey also reported a threefold greater risk of tachysystole accompanied by fetal heart rate changes, but cesarean delivery rates were not signiicantly decreased. Similar results were noted in another Cochrane review of intracervical dinoprostone gel (Boulvain, 2008). Compared with placebo or no treatment, a reduced risk of cesarean delivery was found only in a subgroup of women with an unfavorable cervix and intact membranes. Finally, the Foley catheter versus vaginal PGE2 gel for induction of labor at term-PROBAAT-P and -M trials-were un blinded, randomized trials comparing

1	an unfavorable cervix and intact membranes. Finally, the Foley catheter versus vaginal PGE2 gel for induction of labor at term-PROBAAT-P and -M trials-were un blinded, randomized trials comparing these two options Qozwiak, 2011, 2013, 2014). he cesarean delivery rate did not difer, a inding consistent with accompanying metaanalyses.

1	Side Efects. Uterine tachysystole follows vaginally administered PGE2 in 1 to 5 percent of women (Hawkins, 2012). Although deinitions of abnormal uterine activity vary among studies, most use the definition recommended by the American College of Obstetricians and Gynecologists (2017a): 1. Uterine tachysystole is deined as > 5 contractions in a 10-minute period. It should always be qualiied by the presence or absence of fetal heart rate abnormalities. 2. Uterine hypertonus, hyperstimulation, and hypercontractiliy are terms no longer deined, and their use is not recommended. Because uterine tachysystole associated with fetal compromise may develop when prostaglandins are used with preexisting spontaneous labor, such use is not recommended. If tachysystole follows the 10-mg insert, its removal by pulling on the tail of the surrounding net sac will usually reverse this efect. Irrigation to remove the gel preparation has not been shown to be helpful.

1	The manufacturers recommend caution when these preparations are used in women with ruptured membranes. This concern is also extended to women with glaucoma or asthma. However, in a review of 189 women with asthma, dinoprostone was not associated with asthma worsening or exacerbation (Towers, 2004). Other contraindications listed by the manufacturers include a history of dinoprostone hypersensitivity, suspicion of fetal compromise or cephalopelvic disproportion, unexplained vaginal bleeding, women already receiving oxytocin, those with six or more previous term pregnancies, those with a contraindication to vaginal delivery, or women with a contraindication to oxytocin or who may be endangered by prolonged uterine contractions, for example, those with a history of cesarean delivery or uterine surgery.

1	Administration. PGE2 preparations should only be administered in or near the delivery suite. Moreover, uterine activity and fetal heart rate should be monitored (American College of Obstetricians and Gynecologists, 2016). These guidelines stem from the risk of uterine tachysystole. When contractions begin, they are usually apparent in the irst hour and show peak activity in the irst 4 hours. According to manufacturer guidelines, oxytocin induction that follows prostaglandin use for cervical ripening should be delayed for 6 to 12 hours following PGE2 gel administration or for at least 30 minutes after removal of the vaginal insert. Prostaglandin E,

1	Prostaglandin E, Misoprostol-Cytotec-is a synthetic prostaglandin EI (PGE1) that is approved as a 100-or 200-�g tablet for peptic ulcer prevention. It has been used "of label" for preinduction cervical ripening and may be administered orally or vaginally. he tablets are stable at room temperature. Although widespread, the of-label use of misoprostol has been controversial (Wagner, 2005; Weeks, 2005). Specifically, G. D. Searle & Company notiied physicians that misoprostol is not approved for labor induction or abortion (Cullen, 2000). Still, the American College of Obstetricians and Gynecologists (2016) reairmed its recommendation for use of the drug because of proven safety and eicacy. It currently is the preferred prostaglandin for cervical ripening at Parkland Hospital. In one review of 234 women administered misoprostol, no instances of asthma exacerbation were associated with its use, and the risk of this was calculated to be <2 percent (Rooney Thompson, 2015).

1	Vaginal Administration. Compared with intracervical or intravaginal PGE2, vaginally administered misoprostol tablets ofer equivalent or superior eicacy for cervical ripening or labor induction. A metaanalysis of 121 trials also conirmed these indings (Hofmeyr, 2010). Compared with oxytocin or with intravaginal or intracervical dinoprostone, vaginal misoprostol increased the vaginal delivery rate within 24 hours. In this review, although the uterine tachysystole rate rose, this did not afect cesarean delivery rates. Moreover, compared with dinoprostone, misoprostollowered the need for oxytocin induction,

1	Induction and Augmentation of Labor 507 but it increased the frequency of meconium-stained amni onic fluid. Higher doses of misoprostol are associated with a decreased need for oxytocin but with more uterine tachysystole, with and without fetal heart rate changes. The American Col lege of Obstetricians and Gynecologists (2016) recommends a 25-�g vaginal dose-a fourth of a 1 OO-�g tablet. he drug is evenly distributed among these quartered tablets. Wing and colleagues (2013) described use of a vaginal polymer insert containing 200 �g of PGE1• hey compared its eicacy with 10-mg dinoprostone inserts, and preliminary observations are favorable.

1	Oral Administration. PGE1 tablets are also efective when given orally. One Cochrane metaanalysis of76 trials reported that oral misoprostol compared with placebo signiicantly raised the rate of vaginal birth within 24 hours, while decreasing the need for oxytocin and lowering the cesarean delivery rate. Comparisons of oral misoprostol and oxytocin and of oral misoprostol and dinoprostone also found signiicantly reduced rates of cesarean delivery with misoprostol. Similar eicacy was noted between oral misoprostol and vaginal administration, although oral administration was associated with significantly higher Apgar scores and less postpartum hemorrhage (Alirevic, 2014). Thorbionson and associates (2017) also reported lower rates of cesarean delivery for oral misoprostol compared with vaginal dinoprostone.

1	stimulate nitric oxide (NO) production locally (Chanrachakul, 2000). First, NO is likely a mediator of cervical ripening. Also, cervical NO metabolite concentrations are increased at the beginning of uterine contractions. And, cervical NO produc tion is very low in postterm pregnancy (Vaisanen-T ommiska, 2003, 2004).

1	Bullarbo and colleagues (2007) reviewed rationale and use of two NO donors, isosorbide mononitrate and gyceyl trinitrate. Isosorbide mononitrate induces cervical cyclooxygenase 2 (COX-2), and it also initiates cervical ultrastructure rearrangement similar to that seen with spontaneous cervical ripening (Ekerhovd, 2002, 2003). Despite this, NO donors are less efective clinically than prostaglandins, either PGE2 or misoprostol, for cervical ripening. In one large metaanalysis, the rate of cesarean delivery was not reduced in those given NO donors compared with those given placebo, intravaginal or intracervical prostaglandins, intravaginal misoprostol, or intracervical catheter (Ghosh, 2016). However, NO donors were associated with signiicantly more headaches, nausea, and vomiting.

1	These include transcervical placement of a Foley catheter, with or without extraamnionic saline infusion; hygroscopic cervical dilators; and membrane stripping. In their metaanalysis, Jozwiak and associates (2012) reported that mechanical techniques reduced the risk of uterine tachysystole compared with prostaglandins, although cesarean delivery rates were unchanged. Trials comparing mechanical techniques with oxytocin found a lower rate of cesarean delivery with mechanical methods. Trials comparing mechanical techniques with dinoprostone found a higher rate of multiparas undelivered at 24 hours with mechanical techniques. In another metaanalysis comparing Foley catheter placement with intravaginal dinoprostone inserts, rates of cesarean delivery were similar, but uterine tachysystole was less frequent with catheter use Qozwiak, 2013).

1	Generally, these techniques are only used when the cervix is unfavorable because the catheter tends to come out as the cervix opens. It is suitable for women with intact or ruptured membranes. In most cases, a Foley catheter is placed through the internal cervical os, and downward tension is created by taping the catheter to the thigh (Mei-Dan, 2014). A modification of this-extraamnionic saline inusion (EASI}-adds a constant saline infusion through the catheter into the space between the internal os and placental membranes (Fig. 26-2). Karjane and coworkers (2006) reported that chorioamnionitis was signiicantly less frequent when infusion was done compared with no infusion-6 versus 16 percent. Similarly, in a large metaanalysis, trans cervical catheters were not associated with higher rates of maternal or fetal infection (McMaster, 2015).

1	As discussed above, transcervical catheters do not reduce the cesarean delivery rate compared with prostaglandins. The PROBAAT trials (-I, -P, -M, and II), in which cervical ripening with a Foley catheter was compared with vaginal dinoprostone gel, dinoprostone vaginal inserts, and vaginal or oral misoprostol, reported similar outcomes between the mechanical technique and the prostaglandin agents. Also, fewer overall cases of cardiotocographic changes were seen in the mechanical technique group Qozwiak, 2011, 2013, 2014; Ten Eikelder, 2016). FIGURE 26-2 Extraamnionic saline infusion (EASI) through a 26F Foley catheter that is placed through the cervix. The 30-mL balloon is inflated with saline and pulled snugly against the internal os, and the catheter is taped to the thigh. Room-temperature normal saline is infused through the catheter port of the Foley at 30 or 40 mUhour by intravenous infusion pump.

1	Similar cesarean delivery rate results are found in other comparison studies. Schoen and coworkers (2017) observed that concurrent oxytocin with a transcervical Foley catheter shortened the median time-to-delivery compared with a Foley catheter followed by oxytocin. However, rates of cesarean delivery were unchanged. Connolly and associates (2016) reported similar findings for women within intact membranes undergoing labor induction. Amorosa and colleagues (2017) found no beneit for transcervical catheter coupled with oxytocin compared against oxytocin alone for women with ruptured membranes. Other studies of concurrent misoprostol reported reduced timeto-delivery without afecting cesarean delivey rates (Carbone, 2013; Levine, 2016). Finally, the concurrent addition of tension does not appear to enhance catheter eicacy. Fruhman and coworkers (2017) randomized 140 women to trans cervical Foley catheter with and without tension, and reported similar vaginal delivery rates within 24

1	appear to enhance catheter eicacy. Fruhman and coworkers (2017) randomized 140 women to trans cervical Foley catheter with and without tension, and reported similar vaginal delivery rates within 24 hours or overall.

1	Cervical dilation can be accomplished using hygroscopic osmotic cervical dilators, as described for early pregnancy termination (Chap. 18, p. 358). Intuitive concerns of ascending infection have not been veriied, and their use appears to be safe. Placement generally requires a speculum and positioning of the woman on an examination table. Several studies performed in the 1990s compared hygroscopic cervical dilators and prostaglandins and found few benefits of this mechanical technique. And, more recent studies veriied these conclusions (Maier, 2017).

1	Labor induction has primarily been efected with the use of amniotomy, prostaglandins, and oxytocin, alone or in combination. Because preinduction cervical ripening frequently eventuates in labor, studies to determine induction eicacy for some of these agents have produced sometimes confusing results. he use of prostaglandins for labor augmentation has generally been considered experimental due to their high rates of uterine tachysystole. • Prostaglandin E,

1	Both vaginal and oral misoprostol are used for either cervical ripening or labor induction. For labor induction in women at or near term with either prematurely ruptured membranes or a favorable cervix, 100 I1g of oral or 25 1g of vaginal misoprostol has similar eicacy compared with intravenous oxytocin. From these studies, evidence supports that oral misoprostol may be superior (Alirevic, 2014; Hofmeyr, 2010; Lo, 2003). Misoprostol may be associated with a greater rate of uterine tachysystole, particularly at higher doses. Also, induction with PGEI may prove inefective and require subsequent induction or augmentation with oxtocin. hus, although there are trade-ofs regarding the risks, costs, and ease of administration of each drug, either is suitable for labor induction. At Parkland Hospital, we administer an initial oral 100-l1g dose, which may be repeated ater 6 hours for inadequate labor. Six hours ater the second dose or in those with tachysystole, an oxytocin infusion is begun,

1	we administer an initial oral 100-l1g dose, which may be repeated ater 6 hours for inadequate labor. Six hours ater the second dose or in those with tachysystole, an oxytocin infusion is begun, if needed, for hypotonic labor. Dobert and colleagues (2017) have described preliminary use of a misoprostol vaginal insert.

1	For labor aumentation, results of a randomized controlled trial showed oral misoprostol, 75 ILg given at 4-hour intervals for a maximum of two doses, to be safe and efective (Bleich, 2011). he 75-Lg dose was based on a previous dose-inding study (Villano, 2011). Although there was more uterine tachy systole among women with labor augmented with misoprostol, the frequency of nonreassuring fetal status or cesarean delivery did not difer between oxytocin and misoprostol.

1	In many instances, preinduction cervical ripening and labor induction are simply a continuum. hus, "ripening" can also stimulate labor. If not, induction or augmentation may be continued with solutions of oxytocin given by infusion pump. Its use in augmentation is a key component in the active management oflabor, described in Chapter 22 (p. 438). With oxytocin use, the American College of Obstetricians and Gynecologists (2016) recommends fetal heart rate and uterine contraction monitoring. Contractions can be monitored either by palpation or by electronic means.

1	he goal of induction or augmentation is to efect uterine activity suicient to produce cervical change and fetal descent, while avoiding development of a nonreassuring fetal status. In general, oxytocin is discontinued if the number of contractions persists with a frequency of more than ive in a 10-minute period or more than seven in a 15-minute period or with a persistent nonreassuring fetal heart rate pattern. Oxytocin discontinuation nearly always rapidly lowers contraction frequency. When oxytocin is stopped, its concentration in plasma rapidly falls because the half-life is approximately 3 to 5 minutes. Seitchik and associates (1984) found that the uterus contracts within 3 to 5 minutes of beginning an oxytocin infusion and that a plasma steady state is reached in 40 minutes. Response is highly variable and depends on preexisting uterine activity, cervical status, pregnancy duration, and individual biological diferences. Caldeyro-Barcia and Poseiro (1960) reported that the uterine

1	is highly variable and depends on preexisting uterine activity, cervical status, pregnancy duration, and individual biological diferences. Caldeyro-Barcia and Poseiro (1960) reported that the uterine response to oxytocin increases from 20 to 30 weeks' gestation and rises rapidly at term (Chap. 24, p. 479).

1	Oxytocin Dosage. A 1-mL ampule containing 10 units of oxytocin usually is diluted into 1000 mL of a crystalloid solution and administered by infusion pump. A typical infusate consists of 10 or 20 units, which is 10,000 or 20,000 mU or one or two 1-mL vials, respectively, mixed into 1000 mL oflactated Ringer solution. his mixture results in an oxytocin concentration of 10 or 20 mU/mL, respectively. To avoid bolus administration, the infusion should be inserted into the main intravenous line close to the venipuncture site. Oxytocin is generally very successful when used to stimulate labor. In one large Cochrane metaanalysis, oxytocin was compared with expectant management, and fewer women-8 versus 54 percent-failed to deliver vaginally within 24 hours Induction and Augmentation of Labor 509 TABLE 26-3. Various Low-and High-Dose Oxytocin Regimens Used for Labor Induction

1	Induction and Augmentation of Labor 509 TABLE 26-3. Various Low-and High-Dose Oxytocin Regimens Used for Labor Induction Low-dose 0.5-1.5 15-40 2 15 4,8,12,s16,20,25,30 4.5 15-30 4.5 aUterine tachysystole is more common with shorter intervals. bWith uterine tachysystole and after oxytocin infusion is discontinued, It is restarted at one half the previous dose and then increased at 3 mU/min incremental doses. Data from Merrill, 1999; Satin, 1992, 1994; Xenakis, 1995. with oxytocin (Alfirevic, 2009). This analysis studied diferent oxytocin dosing regimens.

1	Data from Merrill, 1999; Satin, 1992, 1994; Xenakis, 1995. with oxytocin (Alfirevic, 2009). This analysis studied diferent oxytocin dosing regimens. Oxytocin Regimens. Several evidence-based regimens for labor stimulation are now recommended by the American College of Obstetricians and Gynecologists (2016). hese and others are shown in Table 26-3. Initially, only variations of low-dose protocols were used in the United States. Subsequently, O'Driscoll and colleagues (1984) described their Dublin protocol for the active management of labor that called for oxytocin at a starting dosage of 6 mU/min and advanced in 6-mU/min increments. Subsequent comparative trials during the 1990s studied high-dose (4 to 6 mU/min) versus conventional low-dose (0.5 to 1.5 mU/min) regimens, both for labor induction and for augmentation.

1	From Parkland Hospital, Satin and associates (1992) evaluated an oxytocin regimen using an initial and incremental dosage of 6 mU/min compared with one using 1 mU/min. Increases at 20-minute intervals were provided as needed. Among 1112 women undergoing induction, the 6-mU/min regimen resulted in a shorter mean admission-to-delivery time, fewer failed inductions, and no cases of neonatal sepsis. Among 1676 women who had labor augmentation, those who received the 6-mU/min regimen had a shorter duration-to-delivey time, fewer forceps deliveries, fewer cesarean deliveries for dystocia, and lower rates of intrapartum chorioamnionitis or neonatal sepsis. With this protocol, uterine tachysystole was managed by oxytocin discontinuation followed by resumption when indicated and at half the stopping dosage. hereafter, the dosage was increased at 3 mU/min when appropriate, instead of the usual 6-mU/min increase used for women without tachysystole. No adverse neonatal efects were observed.

1	Xenakis and coworkers (1995) reported beneits using an incremen tal oxytocin regimen starting at 4 m U / min. In another study, 816 women were randomly assigned for labor induction and 816 for augmentation with incremental oxytocin gihen at either 1.5 or 4.5 mU/min (Merrill, 1999). Women randomized to the 4.5 mU/min dosage had signiicantly shorter mean durations of induction-to-second-stage labor and inductionto-delivery times. Nulliparas randomized to the 4.5 mU/min dosage had a signiicantly lower cesarean delivery rate for dystocia compared with those given 1.5 mU/min dosage-6 versus 12 percent. Thus, benefits favor higher-dose regimens of 4.5 to 6 mU/min compared with lower dosages of 0.5 to 1.5 mU/min.

1	In 1990 at Parkland Hospital, routine use of the 6-mU/min oxytocin beginning and incremental dosage was incorporated and continues through today. In other labor units, a 2-mU/min beginning and incremental oxytocin regimen is preferred and administered. With either regimen, dosages are employed for either labor induction or augmentation. Although a Cochrane metaanalysis of randomized and quasi-randomized trials comparing high-dose versus low-dose regimens for labor induction at term reported no benefit of higher dosing, the metaanalysis included studies judged to have high potential bias. The authors concluded that the results might be confounded by these poorquality studies (Budden, 2014).

1	Interval between Incremental Dosing. Intervals to increase oxytocin doses vary from 15 to 40 minutes (see Table 26-3). Satin and associates (1994) addressed this aspect with a 6-mU/ min regimen providing increases at either 20-or 40-minute intervals. Women assigned to the 20-minute interval regimen for labor augmentation had a significantly reduced cesarean delivery rate for dystocia compared with that for the 40-minute interval regimen-8 versus 12 percent. As perhaps expected, uterine tachysystole was significantly more frequent with the 20-minute escalation regimen.

1	Other investigators reported even more frequent incremental increases. F rigoletto (1995) and Xenakis (1995) and their coworkers gave oxytocin at 4 mU/min with increases as needed every 15 minutes. Merrill and Zlatnik (1999) started with 4.5 mU/min doses and increased this every 30 minutes. L6pez-Zeno and associates (1992) used 6 mU/min doses and 15-minute intervals. Thus, there are several acceptable oxytocin protocols that at least appear dissimilar. But, a comparison of protocols from two institutions indicates that this is not so: 1. The Parkland Hospital protocol uses a starting dose of oxytocin at 6 mU/min, which is increased by 6-mU/min every 40 minutes, and employs flexible dosing based on uterine tachysystole. 2. The University of Alabama at Birmingham Hospital protocol begins oxytocin at 2 mU/min and increases it as needed every 15 minutes to 4,8, 12, 16,i20,25, and 30 mU/min.

1	2. The University of Alabama at Birmingham Hospital protocol begins oxytocin at 2 mU/min and increases it as needed every 15 minutes to 4,8, 12, 16,i20,25, and 30 mU/min. hus, although the regimens at first appear disparate, if there is no uterine activity, either regimen is delivering 12 mU/ min by 45 minutes into the infusion. Maximal OxytOcin Dosage. The maximal efective dose of oxytocin to achieve adequate contractions in all women is diferent. Wen and colleagues (2001) studied 1151 consecutive nulliparas and found that the likelihood of progression to vaginal delivery decreased at and beyond an oxytocin dosage of 36 mU/min. Still, at a dosage of 72 mU/min, half of the nulliparas were delivered vaginally. Thus, if contractions are not adequate-less than 200 Montevideo units-and if the fetal status is reassuring and labor has arrested, an oxytocin infusion dose greater than 48 mU/min has no apparent risks.

1	Unless the uterus is scarred, uterine rupture associated with oxytocin infusion is rare, even in parous women. Flannelly and associates (1993) reported no cases of uterine ruptures, with or without oxytocin, in 27,829 nulliparas. here were eight instances of overt uterine rupture during labor in 48,718 parous women. Only one of these was associated with oxytocin use. A population-based retrospective review from Denmark reported a rupture rate of 3.3 per 100,000 women without prior cesarean, with the highest risk among multiparas (This ted, 2015). Our experiences from Parkland Hospital are that oxytocin induction and augmentation are associated with uterine rupture (Happe, 2017). During an 8-year period in which there were about 95,000 births, 15 women sufered a primary uterine rupture, and 14 of these cases were associated with oxytocin use. In half of these women, prostaglandins were also given before augmentation with oxytocin.

1	Oxytocin has amino-acid homology similar to arginine vasopressin and has signiicant antidiuretic action. When infused at doses of 20 mU/min or more, renal free water clearance drops markedly. If aqueous luids are infused in appreciable amounts along with oxytocin, water intoxication can lead to convulsions, coma, and even death. In general, if oxytocin is to be administered in high doses for a considerable period of time, its concentration should be increased rather than raising the flow rate of a more dilute solution. Consideration also should be given to use of crystalloids-either normal saline or lactated Ringer solution. Contraction forces in spontaneously laboring women range from 90 to 390 Montevideo units (Chap. 24, p. 479). Caldeyro-Barcia (1950) and Seitchik (1984) with their coworkers found that the mean or median spontaneous uterine contraction pattern between 140 and 150 Montevideo units resulted in progression to vaginal delivery.

1	In the management of active-phase arrest, and with no contraindication to intravenous oxytocin, decisions must be made with knowledge of the safe upper range of uterine activity. Hauth and colleagues (1986) described an efective and safe protocol for oxytocin augmentation for active-phase arrest. With it, more than 90 percent of women achieved an average of at least 200 to 225 Montevideo units. They later reported that nearly all women in whom active-phase arrest persisted despite oxytocin generated more than 200 Montevideo units (Hauth, 1991). Importantly, despite no labor progression, no adverse maternal or perinatal efects were noted in those ultimately requiring cesarean delivery. here are no data regarding safety and eicacy of contraction patterns in women with a prior cesarean delivery, with twins, or with an overdistended uterus.

1	First-stage arrest of labor is deined as a completed latent phase and contractions exceeding 200 NIontevideo units for more than 2 hours without cervical change. Some have attempted to deine a more accurate duration for active-phase arrest (Spong, 2012). Arulkumaran and coworkers (1987) extended the 2-hour limit to 4 hours and reported a 1.3-percent cesarean delivery rate in women who continued to have adequate con tractions and progressive cervical dilation of at least 1 cm/hr. In another 4 hours of labor, half required cesarean delivery.

1	Rouse and colleagues (1999) prospectively managed 542 women at term with active-phase arrest and no other complications. Their protocol was to achieve a sustained pattern of at least 200 Montevideo units for a minimum of 4 hours. This time frame was extended to 6 hours if activity of 200 Montevideo units or greater could not be sustained. Almost 92 percent of these women were delivered vaginally. As discussed in Chapter 23 (p. 443), these and other studies support the practice of allowing an active-phase arrest of 4 hours (Rouse, 2001).

1	Zhang and coworkers (2002) analyzed labor duration from 4 cm to complete dilatation in 1329 nulliparas at term. They found that before dilation of 7 cm was reached, lack of progress for more than 2 hours was not uncommon in those who delivered vaginally. Alexander and associates (2002) reported that epidural analgesia prolonged active labor by 1 hour compared with duration of the active phase as deined by Friedman (1955). Consideration of these changes in the management oflabor, especially in nulliparas, may safely reduce the cesarean delivery rate.

1	As data have accrued, investigators have increasingly questioned the thresholds for labor arrest disorders established by Friedman and others in the 1960s. In particular, investigators with the Consortium on Sai Labor reported that half of cases of dystocia after labor induction occurred before 6 cm of cervical dilation (Boyle, 2013; Zhang, 2010c). Even for women with spontaneous labor, these researchers found that active-phase labor was more likely to occur at 6 cm, and after slow progress between 4 and 6 cm (Zhang, 2010a). Additionally, they reported that a 2-hour threshold for diagnosing arrest disorders may be too brief when cervical dilation is <6 em (Zhang, 201Ob). This is discussed in detail in Chapter 23 (p. 444). Importantly, however, these studies of data from the Collaborative Perinatal Project included only singleton term gestation with spontaneous onset of labor, vaginal delivery, and a normal perinatal outcome. By excluding abnormal outcomes, cesarean deliveries, and

1	Perinatal Project included only singleton term gestation with spontaneous onset of labor, vaginal delivery, and a normal perinatal outcome. By excluding abnormal outcomes, cesarean deliveries, and those who were more than 6 cm dilated upon arrival, the above studies that sought to redeine the labor curve

1	Induction and Augmentation of Labor 511 have been faulted for introducing biases that limit general use of these findings (Cohen, 20 15a, b). Elective amniotomy with the intention of accelerating labor is often performed. Shown in Table 26-4, amniotomy at approximately 5-cm dilation accelerated spontaneous labor by 1 to 1 Y2 hours. Importantly, neither the need for oxytocin stimulation nor the overall cesarean delivery rate was increased. Although the incidences of mild and moderate cord compression patterns were raised following amniotomy, cesarean delivery rates for fetal distress were not higher. Most importantly, there were no adverse perinatal efects.

1	For labor induction, artificial rupture of the membranessometimes called surgical induction-can be used and always implies a commitment to delivery. The main disadvantage of amniotomy used alone for labor induction is the unpredictable and occasionally long interval until labor onset. hat said, in a randomized trial, Bakos and Backstrom (1987) found that amniotomy alone or combined with oxytocin was superior to oxytocin alone. Mercer and colleagues (1995) randomly assigned 209 women undergoing oxytocin induction to either early amniotomy at 1 to 2 cm or late amniotomy at 5 cm. Early amniotomy was associated with a 4-hour reduction in labor duration. With early amniotomy, however, the incidence of chorioamnionitis was elevated.

1	For labor augmentation, amniotomy is commonly performed when labor is abnormally slow. Rouse and associates (1994) found that amniotomy with oxytocin augmentation for arrested active-phase labor shortened the time to delivery by 44 minutes compared with that of oxytocin alone. Although amniotomy did not alter the delivery route, one drawback was that it significantly increased the incidence of chorioamnionitis. Regardless of the indication, amniotomy is associated with a risk of cord prolapse. To minimize this risk, disengagement of the fetal head during amniotomy is avoided. Toward this goal, fundal or suprapubic pressure or both may be helpful. Some clinicians prefer to rupture membranes during a contraction. If the vertex is not well applied to the lower uterine segment, aNo effect on overall rate; cesarean delivery for fetal distress significantly increased. blncreased mild and moderate umbilical cord compression patterns. NA = not assessed.

1	NA = not assessed. a gradual egress of amnionic fluid can sometimes be accomplished by several membrane punctures with a 26-gauge needle held with a ring forceps and with direct visualization using a vaginal speculum. In many of these, however, membranes tear and luid is lost rapidly. Because of the risk of cord prolapse or rarely abruption, the fetal heart rate is assessed before and immediately after amniotomy.

1	Labor induction by membrane "stripping" is a frequent practice. Several studies have suggested that membrane stripping is safe and lowers the incidence of postterm pregnancy without consistently raising the incidence of ruptured membranes, infection, or bleeding. Authors of one large metaanalysis found that membrane stripping reduced the number of women remaining undelivered after 41 weeks without elevating the infection risk. They concluded that eight women would need to undergo membrane stripping to avoid one labor induction. Downsides are discomfort and associated bleeding (Boulvain, 2005). Alexander ]M, Sharma SK, McIntire 0, et al: Epidural analgesia lengthens the Friedman active phase oflabor. Obstet Gynecol 100(1):46,o2002 Alirevic Z, Maifel N, Weeks A: Oral misoprostol for induction of labour. Cochrane Database Syst Rev 6:CD001338 2014

1	Alirevic Z, Maifel N, Weeks A: Oral misoprostol for induction of labour. Cochrane Database Syst Rev 6:CD001338 2014 Alirevic Z, Kelly A], Dowswell T: Intravenous oxytocin alone for cervical ripening and induction of labour. Cochrane Database Syst Rev 4:CD003246, 2009 American Academy of Pediatrics, American College of Obstetricians and Gynecologists: Guidelines for Perinatal Care, 8th ed. Elk Grove Village, P, 2017 American College of Obstetricians and Gynecologists: Induction of labor. Practice Bulletin No. 107, August 2009, Reairmed 2016 American College of Obstetricians and Gynecologists: Intrapartum fetal heart rate monitoring: nomenclature, interpretation, and general management principles. Practice Bulletin No. 106,o] uly 2009, Reairmed 20o17 a American College of Obstetricians and Gynecologists: Vaginal birth after previous cesarean delivery. Practice Bulletin 115, August 2010, Reairmed 20 17b

1	American College of Obstetricians and Gynecologists: Vaginal birth after previous cesarean delivery. Practice Bulletin 115, August 2010, Reairmed 20 17b Amorosa ]M, Stoneo], Factor SH, et al: A randomized trial of Foley bulb for labor induction in premature rupture of membranes in nulliparas (FLIP). Am] Obstet GynecoI217(3):360.e1

1	Arulkumaran S, Koh CH, Ingemarsson I, et al: Augmentation of labourmode of delivery related to cervi metric progress. Aust N Z ] Obstet GynaecoIo27:304, 1987 Bailit ]L, Gregory KD, Reddy UM, et al: Maternal and neonatal outcomes by labor onset type and gestational age. Am ] Obstet Gynecol 202(3):245. el,o2010 Bakos 0, Backstrom T: Induction of labor: a prospective, randomized study into amniotomy and oxytocin as induction methods in a total unselected population. Acta Obstet Gynecol Scand 66:537, 1987 Bateman BT, Mhyre ]M, Callaghan WM, et al: Peripartum hysterectomy in the United States: nationwide 14 year experience. Amo] Obstet Gynecol 206(1):63.el,o2012 Bishop EH: Pelvic scoring for elective induction. Obstet GynecoIo24:266, 1964 Bleich AT, Villano KS, Lo ]Y, et al: Oral misoprostol for labor augmentation: a randomized controlled trial. Obstet GynecoIo118(6):1255, 2011 Boulvain M, Kelly A, Irion 0: Intracervical prostaglandins for induction of labour. Cochrane Database Syst Rev

1	for labor augmentation: a randomized controlled trial. Obstet GynecoIo118(6):1255, 2011 Boulvain M, Kelly A, Irion 0: Intracervical prostaglandins for induction of labour. Cochrane Database Syst Rev 1:CD006971, 2008 Boulvain M, Stan C, Irion 0: Membrane sweeping for induction of labour. Cochrane Database Syst Rev 1 :CD00045o1, 2005 Boyle A, Reddy UM, Landy H], et al: Primary cesarean delivery in the United States. Obstet GynecoIo122(1):33, 2013 Budden A, Chen L], Henry A: High-dose versus low-dose oxytocin infusion regimens for induction of labour at term. Cochrane Database Syst Rev 10:CD009701,o2014 Bullarbo M, Orrskog ME, Andersch B, et al: Outpatient vaginal administration of the nitric oxide donor isosorbide mononitrate for cervical ripening and labor induction postterm: a randomized controlled study. Amo] Obstet GynecoIo196:50.e1,o2007

1	Caldeyro-Barcia R, Alvarez H, Reynolds SR: A better understanding of uterine contractility through simultaneous recording with an internal and a seven channel external method. Surg Obstet Gynecol 91 :641, 1950 Caldeyro-Barcia R, Poseiro JJ: Physiology of the uterine contraction. Clin Obstet Gynecol 3:386, 1960 Carbone ]F, Tuuli MG, Fogertey P], et al: Combination of Foley bulb and vaginal misoprostol compared with vaginal misoprostol alone for cervical ripening and labor induction: a randomized controlled trial. Obstet Gynecol. 121:247,2013 Chanrachakul B, Herabutya Y, Punyavachira P: Potential eicacy of nitric oxide for cervical ripening in pregnancy at term. Int ] Gynaecol Obstet ,2000 Chiossi G, Lai Y, Landon MB, et al: Timing of delivery and adverse outcomes in term singleton repeat cesarean deliveries. Obstet Gynecol 121 (3) :561, 2013

1	Clark SL, Miller DO, Belfort MA, et al: Neonatal and maternal outcomes associated with elective term delivery. Amo] Obstet Gynecol 200(2): 156. el,o2009 Cohen WR, Friedman EA: Misguided guidelines for managing labor. Am ] Obstet GynecoIo212(6):753.e1, 2015a Cohen R, Friedman A: Perils of the new labor management guidelines. Am ] Obstet Gynecolo212(4):420, 2015b Connolly A, Kohari KS, Rekawek P, et al: A randomized trial of Foley balloon induction oflabor trial in nulliparas (FIAT-N). Am] Obstet Gynecol. 215:392.el,2016 Cullen M: Important drug warning concerning unapproved use of intravaginal or oral misoprostol in pregnant women for induction of labor or abortion. 2000. Available at: http://www.fda.gov/ohrms/dockets/dailys/OO/ NovOO/111500/cpOOO1.pdf. Accessed July 28,o2017 Dahlen HM, McCullough ]M, Fertig AR, et al: Texas Medicaid payment reform: fewer early elective deliveries and increased gestational age and birthweight. Health ff(Millwood) 36(3):460, 20o17

1	Dahlen HM, McCullough ]M, Fertig AR, et al: Texas Medicaid payment reform: fewer early elective deliveries and increased gestational age and birthweight. Health ff(Millwood) 36(3):460, 20o17 Darney BG, Snowden ]M, Cheng W, et al: Elective induction of labor at term compared with expectant management: maternal and neonatal outcomes. Obstet GynecoIo122:761, 2013 Dobert M, Brandsetter A, Heinrich W, et al: he misoprostol vaginal insert compared with oral misoprostol for labor induction in term pregnancies: a pair-matched case-control study. ] Perinatal Med June 26, 20o17 [Epub ahead of print] Ekerhovd E, Bullarbo M, Andersch B, et al: Vaginal administration of the nitric oxide donor isosorbide mononitrate for cervical ripening at term: a randomized controlled study. Amo] Obstet GynecoIo189:o1692, 2003

1	Ekerhovd E, Weijdegard B, Brannstrom I, et al: Nitric oxide induced cervical ripening in the human: involvement of cyclic guanosine monophosphate, prostaglandin F2a, and prostaglandin E2• Amo] Obstet GynecoIo186:745, 2002 Feltovich H: Cevical evaluation. From ancient medicine to precision medi cine. Obstet GynecoIo130:51, 2017 Fisch ]M, English 0, Pedaline S, et al: Labor induction process improvement: a patient quality-of-care initiative. Obstet Gynecol 113(4):797,o2009 Flannelly GM, Turner M], Rassmussen M], et al: Rupture of the uterus in Dublin: an update.o] Obstet GynaecoIo13:440, 1993 Fraser W, Marcoux S, Moutquin ]M, et al: Efect of early amniotomy on the risk of dystocia in nulliparous women. N Engl] Med 328:1145, 1993 Friedman A: Primigravid labor: a graphicostatistical analysis. Obstet Gynecol 6:567, 1955 Frigoletto FD, Lieberman E, Lang ]M, et al: A clinical trial of active management oflabor. N Engl] Med 333:745, 1995

1	Frigoletto FD, Lieberman E, Lang ]M, et al: A clinical trial of active management oflabor. N Engl] Med 333:745, 1995 Fruhman G, Gavard ]A, Amon E, et al: Tension compared to no tension on a Foley trans cevical catheter for cervical ripening: a randomized controlled trial. Am] Obstet GynecoI216:67.e1, 2017 Garite T], Porto M, Carlson N], et al: he inluence of elective amniotomy on fetal heart rate patterns and the course of labor in term patients: a randomized study. Am] Obstet GynecoIo168:1827, 1993

1	Ghosh A, Lattey R, Kelly A]: Nitric oxide donors for cervical ripening and induction of labour. Cochrane Database Syst Rev 12:CD00690 1, 2016 Gibson KS, Waters TP: Measures of success: prediction of successful labor induction. Semin PerinatoIo39:475, 2015 Happe SK, Yule CS, Wells CE: Outcomes in pregnancies complicated by intrapartum uterine rupture. Unpublished data, 2017 Hatield AS, Sanchez-Ramos L, Kaunitz M: Sonographic cervical assessment to predict the success of labor induction: a systematic review with metaanalysis. Amo] Obstet Gynecol 197: 186, 2007 Hauth]C, Hankins GO, Gilstrap LC III: Uterine contraction pressures achieved in parturients with active phase arrest. Obstet Gynecol 78:344, 1991 Hauth ]c, Hankins GO, Gilstrap LC III: Uterine contraction pressures with oxytocin induction/augmentation. Obstet GynecoIo68:305, 1986 Hawkins ]S, Stephenson M, Powers B, et al: Diabetes mellitus: an independent predictor of duration of prostaglandin labor induction. ] Perinatol 37:488,

1	Obstet GynecoIo68:305, 1986 Hawkins ]S, Stephenson M, Powers B, et al: Diabetes mellitus: an independent predictor of duration of prostaglandin labor induction. ] Perinatol 37:488, 20o17

1	Hawkins ]S, Wing DA: Current pharmacotherapy options for labor induction. Expert Opin Pharmacother 13(14):2005,2012 Hernandez ]S, Wendel GO ]r, Sheield]S: Trends in emergency peripartum hysterectomy at a single institution: 1988-2009. Amr] PerinatoIr30(5):365, 2013 Hofman MK, Sciscione AC: Elective induction with cervical ripening increases the risk of cesarean delivery in multiparous women. Obstet GynecoIr101:7S, 2003 Hofmeyr G], G.ilmezoglu M, Pileggi C: Vaginal misoprostol for cervical ripening and induction of labour. Cochrane Database Syst Rev 1 0:CD000941 , 2010 Ivars J, Garabedian C, Devos P, et al: Simpliied Bishop score including parity predicts successful induction of labor. Eur ] Obstet Gynecol Reprod Bioi 203:309,r2016 Jozwiak M, Bloemenkamp KW, Kelly AJ, et al: Mechanical methods for induction of labour. Cochrane Database Syst Rev 3:CD001233, 2012

1	Jozwiak M, Bloemenkamp KW, Kelly AJ, et al: Mechanical methods for induction of labour. Cochrane Database Syst Rev 3:CD001233, 2012 Jozwiak M, Oude Rengerink K, Benthem M, et al: Foley catheter versus vaginal prostaglandin E2 gel for induction of labour at term (PROBAAT trial): an open-label, randomised controlled trial. Lancet 378(9809):2095,r201r1 Jozwiak M, Oude Rengerink K, Ten Eikelder ML, et al: Foley catheter or prostaglandin E2 inserts for induction of labour at term: an open-label randomized controlled trial (PROBAAT-P trial) and systematic review of literature. Eur J Obstet Gynecol Reprod Bioi 170(1):137, 2013 Jozwiak M, Ten Eikelder M, Rengerink KO, et al: Foley catheter versus vaginal misoprostol: randomized controlled trial (PROBAAT-M Study) and systematic review and meta-analysis of literature. Am J Perinatol 31: 145, 2014

1	Karjane NW, Brock EL, Walsh SW: Induction of labor using a Foley balloon, with and without extra-amniotic saline infusion. Obstet Gynecol 107:234, 2006 Kawakita T, Reddy UM, Iqbal SN, et al: Duration of oxytocin and rupture of membranes before diagnosing a failed induction of labor. Obstet Gynecol 128:373,r2016 Kominiarek A, Zhang J, Vanveldhuisen P, et al: Contemporaty labor patterns: the impact of maternal body mass index. Am J Obstet Gynecol 205(3):244.e1,r2011 Landon MB, Hauth JC, Leveno KJ, et al: Maternal and perinatal outcomes associated with a trial of labor ater prior cesarean delivery. N Engl J Med 351(25):2581,r2004 Laughon SK, Branch OW, Beaverr], et al: Changes in labor patterns over 50 years. Am J Obstet GynecoI206(5):419.e1, 2012 Laughon SK, Zhang J, Troendle ], et al: Using a simplified Bishop score to predict vaginal delivery. Obstet GynecoIr117(4):805, 2011

1	Laughon SK, Zhang J, Troendle ], et al: Using a simplified Bishop score to predict vaginal delivery. Obstet GynecoIr117(4):805, 2011 Levine LD, Downes L, Elovitz A, et al: Mechanical and pharmacologic methods of labor induction: a randomized controlled trial. Obstet Gynecol 128:1357,r2016 Little SE, Caughey AB: Induction of labor and cesarean: what is the true relationship? Clin Obstet Gynecol 58:269, 2015 Lo JY, Alexander JM, McIntire DO, et al: Ruptured membranes at term: randomized, double-blind trial of oral misoprostol for labor induction. Obstet Gynecolr101:685,r2003 Lopez-Zeno JA, Peaceman M, Adashek ]A, et al: A controlled trial of a program for the active management oflabor. N Engl J Med 326:450, 1992 Luthy DA, Malmgren ]A, Zingheim RW: Cesarean delivery after elective induction in nulliparous women: the phYSician efect. Amr] Obstet Gynecol 191:1511,r2004 Macones GA: Elective induction of labor: waking the sleeping dogma? Ann Intern Med 151 (4):281, 2009

1	Macones GA: Elective induction of labor: waking the sleeping dogma? Ann Intern Med 151 (4):281, 2009 Maier JT, Metz M, Watermann N, et al: Induction of labor in patients with an unfavorable cervix after a cesarean using an osmotic dilator versus vaginal prostaglandin. J Perinatal Med June 26, 2017 [Epub ahead of print] Martin JA, Hamilton BE, Osterman MJ, et al: Births: final data for 2015. Natl Vital Stat Rep 66(1):1, 2017 Maslow AS, Sweeny AL: Elective induction of labor as a risk factor for cesarean delivery among low-risk women at term. Obstet GynecoIr95:917, 2000 McMaster K, Sanchez-Ramos L, Kaunitz M: Evaluation of a trans cevical Foley catheter as a source of infection: a systematic review and meta-analysis. Obstet GynecoIr126:539, 2015 Mei-Dan E, Walisch A, Valencia C, et al: Making cervical ripening EASI: a prospective controlled comparison of single versus double balloon catheters. J Matern Fetal Neonatal Med 27:1765,r2014

1	Mei-Dan E, Walisch A, Valencia C, et al: Making cervical ripening EASI: a prospective controlled comparison of single versus double balloon catheters. J Matern Fetal Neonatal Med 27:1765,r2014 Melamed N, Ray JG, Geary M, et al: Induction of labor before 40 weeks is associated with lower rate of cesarean delivery in women with gestational diabetes mellitus. Am J Obstet GynecoI214:364, 2016 Mercer BM, McNanley T, O'Brien JM, et al: Early versus late amniotomy for labor induction: a randomized trial. Am J Obstet Gynecol 173: 1371, 1995 Merrill DC, Zlatnik FJ: Randomized, double-masked comparison of oxytocin dosage in induction and augmentation of labor. Obstet Gynecol 94:455, 1999 Induction and Augmentation of Labor 513 Miller NR, Cypher L, Foglia LM, et al: Elective induction oflabor compared with expectant management of nulliparous women at 39 weeks of gestation: a randomized controlled trial. Obstet GynecoIr126:1258, 2015

1	National Institutes of Health: ClinicaITrials.gov: a randomized trial of induction versus expectant management (ARRIVE). 2015. Available at: https:!1 c1inicaltrials.gov/ct2/show/NCT01990612. Accessed July 28,r2017 O'Driscoll K, Foley M, MacDonald 0: Active management of labor as an alternative to cesarean section for dystocia. Obstet Gynecol 63:485, 1984 Oshiro BT, Kowalewski L, Sappenfield W, et al: A multistate quality improvement program to decrease elective deliveries before 39 weeks of gestation. Obstet Gynecol 121(5):1025,r2013 Raghuraman N, Stout MJ, Young OM: Utility of the simpliied Bishop score in spontaneous labor. Am ] Perinatol 33: 1176, 2016

1	Raghuraman N, Stout MJ, Young OM: Utility of the simpliied Bishop score in spontaneous labor. Am ] Perinatol 33: 1176, 2016 Roland e, Warshak CR, DeFranco EA: Success of labor induction for preeclampsia at preterm and term gestational ages. J Perinatol 37(6):636, 2017 Rooney Thompson M, Towers CV, Howard Be, et l: The use of prostaglandin El in peripartum patients with asthma. m J Obstet Gynecol 212:392.e 1, 2015 Rouse 0], McCullough e, Wren AL, et al: Active-phase labor arrest: a randomized trial of chorioamnion management. Obstet GynecoIr83:937, 1994 Rouse OJ, Owen J, Hauth J C: Active-phase labor arrest: oxytocin augmenta tion for at least 4 hours. Obstet Gynecol 93:323, 1999 Rouse OJ, Owen J, Hauth JC: Criteria for failed labor induction: prospective evaluation of a standardized protocol. Obstet Gynecol 96:671, 2000 Rouse OJ, Owen J, Savage KG, et al: Active phase labor arrest: revisiting the 2-hour minimum. Obstet Gynecol 98:550, 2001

1	Rouse OJ, Owen J, Savage KG, et al: Active phase labor arrest: revisiting the 2-hour minimum. Obstet Gynecol 98:550, 2001 Saccone G, Berghella V: Induction of labor at full term in uncomplicated singleton gestations: a systematic review and metaanalysis of randomized controlled trials. Am J Obstet GynecoIr213:629, 2015 Salemi ]L, Pathak EB, Salihu HM: Infant outcomes ater elective early-term delivery compared with expectant management. Obstet Gynecol 127:657, 2016 Satin AJ, Leveno KJ, Sherman ML, et al: High-dose oxytocin: 20-versus 40-minute dosage interval. Obstet Gynecol 83:234, 1994 Satin A], Leveno KJ, Sherman ML, et al: High-versus low-dose oxytocin for labor stimulation. Obstet Gynecol 80: 111, 1992 Schoen CN, Grant G, Berghella V, et al: Intracevical Foley catheter with and without oxytocin for labor induction: a randomized controlled trial. Obstet GynecoIr129:1046,r2017

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1	Wagner M: Of-label use of misoprostol in obstetrics: a cautionary tale. BjOG 112: 266,r2005 Weeks AD, Fiala C, Safar P: Misoprostol and the debate over of-label drug use. BjOG 112: 269, 2005 Wen T, Beceir A, Xenakis E, et al: Is there a maximum efective dose of Pitocin? Am j Obstet Gynecol 185:5212,r2001 Williams JW: Obstetrics: a Text-book for the Use of Students and Practitioners. New York, o. Appleton and Co., 1903 Wing OA, Brown R, Plante A, et al: Misoprostol vaginal insert and time to vaginal delivery. A randomized controlled trial. Obstet Gynecolrl22(2 pt 1): 201, 2013 Wolfe H, Timofeev j, Tefera E, et al: Risk of cesarean in obese nulliparous women with unfavorable cervix: elective induction vs expectant management at term. Am j Obstet Gynecol. 211:53.e1, 2014 Xenakis EM, Langer 0, Piper jM, et al: Low-dose versus high-dose oxytocin augmentation of labor-a randomized trial. m j Obstet Gynecol 3: 1874, 1995

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1	As soon as the head appears at the vulva the physician should be ready to restrain its progress. He should hold his hand in such a manner as to be able to bring it immediatey into action, or in many instances the resistance of the vulva is unexpectedy overcome, and a single pain may be suicient to push the head suddeny through it with a resulting perineal tea. -J. hitridge Williams (1903)

1	-J. hitridge Williams (1903) As described by Williams, the natural culmination of secondstage labor is controlled vaginal delivery of a healthy neonate with minimal trauma to the mother. Vaginal delivery is the preferred route of delivery for most fetuses, although various clinical settings may favor cesarean delivery. Of delivery routes, spontaneous vaginal vertex delivery poses the lowest risk of most maternal comorbidity, and comparisons with cesarean delivery are found in Chapter 30 (p. 568). Delivery is usually spontaneous, although some maternal or fetal complications may warrant operative vaginal delivery, described in Chapter 29 (p. 553). Last, a malpresenting fetus or multifetal gestation in many cases may be delivered vaginally but requires special techniques. These are described in Chapters 28 (p. 543) and 45 (p. 888).

1	he end of second-stage labor is heralded as the perineum begins to distend, the overlying skin becomes stretched, and the fetal scalp is seen through the separating labia. Increased perineal pressure from the fetal head creates reflexive bearing-down eforts, which are encouraged when appropriate. At this time, preparations are made for delivery. If the bladder is distended, catheterization may be necessary. Continued attention is also given to fetal heart rate monitoring. s one example, a nuchal cord oten tightens with descent and may lead to deepening variable decelerations.

1	During second-stage labor, pushing positions may vary. But for delivery, the dorsal lithotomy position is most common and often the most satisfactory. For better exposure, leg holders or stirrups are used. Corton and associates (2012) found no increased rates of perineal lacerations with or without their use. With positioning, legs are not separated too widely or placed one higher than the other. Within the leg holder, the popliteal region should rest comfortably in the proximal portion and the heel in the distal portion. he legs are not strapped into the stirrups, thereby allowing quick flexion of the thighs backward onto the abdomen should shoulder dystocia develop. Legs may cramp during second-stage pushing, and cramping is relieved by repositioning the afected leg or by brief massage.

1	Preparation for delivery includes vulvar and perineal cleansing. If desired, sterile drapes may be placed in such a way that only the immediate area around the vulva is exposed. Scrubbing, gowning, gloving, and donning protective mask and eyewear protect both the laboring woman and accoucheur from infectious agents. • Delivery of the Head By the time of perineal distention, the position of the occiput is usually known. In some cases, however, molding and caput formation may have precluded early accurate identification. At this time, careful assessment is again performed as described in Chapter 22 (p. 426). In most cases, position is directly occiput anterior (OA) or is rotated slightly oblique. But, in perhaps 5 percent, occiput posterior (OP) positioning persists.

1	With each contraction, the vulvovaginal opening is dilated by the fetal head to gradually form an ovoid and inally, an almost circular opening (Fig. 27-1). his encirclement of the largest head diameter by the vulvar ring is termed crowning. he perineum thins and may spontaneously lacerate. he anus becomes greatly stretched, and the anterior wall of the rectum can easily be seen through it.

1	Routine episiotomy is no longer recommended, and selective use aims to enlarge the vaginal opening for specific indications (p. 529). To limit spontaneous vaginal laceration, some perform antenatal massage of the perineal body to increase perineal distensibility or intrapartum perineal massage to widen the introitus for head passage. During massage with a lubricant, the perineum is grasped in the midline by both hands using the thumb and opposing ingers. Outward and lateral stretching to thin the perineum is repeatedly performed. But in randomized studies, this technique did not significantly prevent perineal laceration (Beckmann, 2013; Mei-dan, 2008; Stamp, 2001). Antepartum use of the Epi-No intravaginal pump balloon has a similar aim, but it also fails to prevent perineal trauma or levator injury (Brito, 2015; Kamisan Atan, 2016).

1	When the head distends the vulva and perineum enough to open the vaginal introitus to a diameter of 5 cm or more, a gloved hand may be used to support the perineum (Fig. 27-2). he other hand is used to guide and control the fetal head to deliver the smallest head diameter through the introitus and to FIGURE 27-2 Delivery of the head. The mouth appears over the perineum. avoid expulsive delivery. Slow delivery of the head may decrease lacerations (Laine, 2008). Overall, bracing the perineum lowers rates of anal sphincter injury compared with a "hands of' approach to delivery (Bulchandani, 2015; McCandlish, 1998).

1	Alternatively, if expulsive eforts are inadequate or expeditious delivery is needed, the modied Ritgen maneuvermay be employed or an episiotomy cut. With the modified maneuver, gloved ingers beneath a draped towel exert forward pressure on the fetal chin through the perineum just in front of the coccyx. Concurrently, the other hand presses against the occiput (Fig. 27-3). Originally described in 1855, the maneuver allows controlled fetal head delivery (Cunningham, 2008). It also favors neck extension so that the head passes through the introitus and over the perineum with its smallest diameters. Comparing the Ritgen maneuver with simple perineal support in 1623 women, Jonsson and colleagues (2008) found a similar incidence of third-and fourth-degree tears, defined later (p. 528). FIGURE 27-1 Perineum is supported as the head crowns.

1	FIGURE 27-1 Perineum is supported as the head crowns. FIGURE 27-3 Modified Ritgen maneuver. Moderate upward pressure is applied to the fetal chin by the posterior hand covered by a sterile towel. The other hand applies occipital pressure. • Delivery of the Shoulders Following delivery of the fetal head, a inger is passed across the fetal neck to determine whether it is encircled by one or more umbilical cord loops. he nuchal cord incidence increases with gestational age and is found in nearly 25 percent of deliveries at term (Larson, 1997; Ogueh, 2006). If an umbilical cord coil is felt, it is slipped over the head if loose enough. If applied too tightly, the loop is cut between two clamps. Tight nuchal cords complicate approximately 6 percent of all deliveries but are not associated with worse neonatal outcome than those without a cord loop (Henry, 2013).

1	Following its delivery, the fetal head falls posteriorly, bringing the face almost into contact with the maternal anus. he occiput promptly turns toward one of the maternal thighs, and the head assumes a transverse position. This external rotation indicates that the bisacromial diameter, which is the distance between the shoulders, has rotated into the anteroposterior diameter of the pelvis. Most often, the shoulders appear at the vulva just after external rotation and are born spontaneously. If delayed, extraction aids controlled delivery. The sides of the head are grasped with two hands, and genle downward traction is applied until the anterior shoulder appears under the pubic arch (Fig. 27-4). Next, by an upward movement, the posterior shoulder is delivered. During delivery, abrupt or powerful force is avoided to avert fetal brachial plexus injury.

1	he rest of the body almost always follows the shoulders without diiculty. With prolonged delay, however, its birth may be hastened by moderate outward traction on the head and moderate pressre on the uterine fundus. Hooking the fingers in the axillae is avoided. This can injure upper extremity nerves and produce a transient or possibly permanent paralysis. Immediately after delivery of the newborn, a gush of amnionic luid that is often blood-tinged but not grossly bloody usually follows.

1	Previously, immediate nasopharyngeal bulb suctioning of the newborn was routine to remove secretions. It was found, however, that suctioning of the nasopharynx may lead to neonatal bradycardia (Gungor, 2006). he current American Heart Association neonatal resuscitation recommendations eschew most suctioning immediately following birth-even with meconium present (Chap. 33, p. 620). And with meconium-stained luid, routine intubation for tracheal suction is not recommended for vigorous or for nonvigorous neonates. Suctioning is reserved for neonates who have obvious obstruction to spontaneous breathing or who require positive-pressure ventilation (Wyckof, 2015). For suctioning, options are bulb syringe or suction catheter aspiration and may include intubation and suctioning if the airway is obstructed.

1	FIGURE 27-4 Delivery of the shoulders. A. Gentle downward traction to effect descent of the anterior shoulder. B. Delivery of the anterior shoulder completed. Gentle upward traction to deliver the posterior shoulder. The umbilical cord is cut between two clamps placed 6 to 8 cm from the fetal abdomen, and later an umbilical cord clamp is applied 2 to 3 cm from its insertion into the fetal abdomen. For term neonates, the timing of umbilical cord clamping remains debatable. Delayed umbilical cord clamping transfers a greater volume of blood to the newborn. A delay for up to 60 seconds may increase total body iron stores, expand blood volume, and decrease anemia incidence in the neonate (Andersson, 2011; Yao, 1974). his practice may be particularly valuable in populations in which iron deficiency is prevalent (Kc, 2017; World Health Organization, 2014).

1	Conversely, a higher hemoglobin concentration increases risks for hyperbilirubinemia and extended hospitalization for neonatal phototherapy (McDonald, 2013). Delayed cord clamping may also hinder timely and needed neonatal resuscitation. That said, early pilot studies are assessing the value of resuscitating newborns at the bedside to permit delayed clamping (Katheria, 2017; Winter 2017). Fortunately, in general, delayed umbilical cord clamping compared with early clamping does not worsen Apgar scores, umbilical cord pH, or respiratory distress caused by polycythemia. Regarding maternal outcomes, rates of postpartum hemorrhage are similar between early and delayed clamping groups (Andersson, 2013). Fewer data are available regarding cord "milking," in which the opera tor pushes blood through the cord toward the newborn. his clamping is clinically indicated (Upadhyay, 2013).

1	For the preterm neonate, delayed cord clamping has sev eral benefits. hese include higher red cell volume, decreased need for blood transfusion, and lower rates of intraventricu lar hemorrhage and of necrotizing enterocolitis (Backes, 2014; Rabe, 2012). For neonates who require expedited resuscitation, cord milking may have beneits to quickly transfer volume (Al-Wassia, 2015; Katheria, 2015; Patel, 2014). Still, because of rapid blood volume changes, the American Heart Associa tion currently suggests against the routine use of cord milking for neonates born <29 weeks' gestation (Wyckof, 2015). The American College of Obstetricians and Gynecologists (2017a) notes suicient evidence to support delayed umbili 30 to 60 seconds after birth. This opinion is also endorsed by the American Academy of Pediatrics (2017 a). The American Heart Association guidelines advise that the practice may have resuscitation at birth (Wyckof, 2015).

1	In the absence of a pelvic architecture abnormalit) or asynclitism, the occiput transverse (OT) position is usually transitoly. Thus, unless contractions are hypotonic, the head usually spontaneously rotates to an OA posItion. If rotation ceases because of poor expulsive forces, vaginal deliveY usually can be accomplished readily in several ways. he easiest is manual rotation of the occiput either anteriorly to OA or less commonly, posteriorly to occiput posterior. If either is successful, Le Ray and coworkers (2007) reported a 4-percent cesarean delivery rate compared with a 60-percent rate in women in whom manual rotation was not successful. Some recommend rotation with Kielland forceps for the persistent OT position as outlined in Chapter 29 (p. 561). These forceps are used to rotate the occiput to the anterior position, and delivery is accomplished with the same forceps or by substitution with Simpson, Tucker-McLane, or similar forceps.

1	In some cases, pelvic shape leads to a persistent OT position that is not easily overcome. For example, a platypelloid pelvis is lattened anteroposteriorly and an android pelvis is heart shaped. With these, space may be inadequate for occipital rotation to either an anterior or posterior position (Fig. 2-17, p. 31). Because of these concerns, undue force is avoided if forceps delivery is attempted. Approximately 2 to 10 percent of singleton term cephalic fetuses deliver in an occiput posterior (OP) position (Cheng, 2010). Many fetuses delivering OP are OA in early labor and relect malrotation during labor. Predisposing risks include epidural analgesia, nulliparity, greater fetal weight, and prior delivery with OP positioning (Cheng, 2006a; Gardberg, 2004; Lieberman, 2005). Regarding pelvic shape, an anthropoid pelvis and narrow subpubic angle can predispose (Barth, 2015; Ghi, 2016).

1	Women with a persistent OP position have higher associated rates of prolonged second-stage labor, cesarean delivery, and operative vaginal delivery. For women who deliver vaginally, rates of blood loss and of third-and fourth-degree lacerations are increased (Senecal, 2005).

1	plication rates then those born positioned OA. Cheng and coworkers (2006b) compared outcomes of 2591 women under going delivery with a persistent OP position with those of28,801 women whose newborns were delivered OA. Virtually every possible delivery complication was found more frequently with persistent OP position. Only 46 percent of these women deliv ered spontaneously, and the remainder accounted for 9 percent of cesarean deliveries performed. hese investigators also found that an OP position at delivery was associated with more adverse cord gases, birth trauma, Apgar scores <7, and intensive care nursery admission, among others. Similar results were reported by Ponkey (2003) and Fitzpatrick (2001) and their associates.

1	Methods to prevent persistent OP position and its associated morbidity have been investigated. First, digital examination for identiication of fetal head position can be inaccurate, and transabdominal sonography can be used to increase accuracy (Dupuis, 2005; Zahalka, 2005). The transducer is placed transversely just cephalad to the maternal mons pubis. In the sonogram, fetal orbits and nasal bridge lie ventrally, whereas the occiput apposes the lower sacrum. Such information may provide an explanation for prolonged second-stage labor or may identiy suitable candidates for rotation. Of other possible interventions, varying maternal position either antepartum or during labor does not appear to lower rates of persistent OP position (Desbriere, 2013; Kariminia, 2004; Le Ray, 2016).

1	The fetus in an OP position may be delivered either spontaneously or by operative vaginal delivery. First, if the bony pelvic outlet is roomy and the perineum is somewhat relaxed from prior deliveries, rapid spontaneous OP delivery will often take place. Conversely, if the perineum is resistant to stretch, secondstage labor may be appreciably prolonged. During each expulsive efort, the head is driven against the perineum to a much greater degree than when the head position is OA. This leads to greater rates of third-and fourth-degree lacerations (Groutz, 2011; Melamed, 2013).

1	In some cases, spontaneous vaginal delivery from an OP position does not appear feasible or expedited delivery is needed. Here, manual rotation with spontaneous delivery from an OA position may be preferred. This technique is described fully in Chapter 29 (p. 560). Successful rotation rates range from 47 to 90 percent. And, as would be expected, lower rates of cesarean delivery, vaginal laceration, and maternal blood loss follow rotation to OA position and vaginal delivery (Le Ray, 2005; Sen, 2013; Shafer, 2006, 2011). Disadvantageously, manual rotation is linked with higher cervical laceration rates. Thus, careful inspection of the cervix following rotation is mandatory.

1	For exigent delivery, forceps or vacuum device can be applied to a persistent OP position. This is often performed in conjunction with an episiotomy. Also, if the head is engaged, the cervix fully dilated, and the pelvis adequate, forceps rotation may be attempted for those with suitable skills. These operative vaginal techniques are detailed in Chapter 29 (p. 561). Infrequently, protrusion of fetal scalp through the introitus is the consequence of marked elongation of the fetal head from molding combined with formation of a large caput succedaneum. In some cases, the head may not even be engaged-that is, the biparietal diameter may not have passed through the pelvic inlet. In these, labor is characteristically long and descent of the head is slow. Careful palpation above the symphysis may disclose the fetal head to be above the pelvic inlet. Prompt cesarean delivery is appropriate.

1	At Parkland Hospital, spontaneous delivery or manual rotation is preferred for management of persistent OP position. When needed, either manual rotation to OA position followed by forceps delivery or forceps delivery from the OP position is used. If neither can be completed with ease and safety, cesarean delivery is performed. Following complete emergence of the fetal head during vaginal delivery, the remainder of the body may not rapidly follow. The anterior fetal shoulder can become wedged behind the symphysis pubis and fail to deliver using normally exerted downward traction and maternal pushing. Because the umbilical cord is compressed within the birth canal, this dystocia is an emergency. Several maneuvers, in addition to downward traction on the fetal head and neck, may be performed to free the shoulder. This requires a team approach, in which efective communication and leadership are critical.

1	Consensus regarding a speciic definition of shoulder dystocia is lacking. Some focus on whether maneuvers to free the shoulder are needed, whereas others use the head-to-body delivery time interval as deining (Beall, 1998). Spong and coworkers (1995) reported that the mean head-to-body delivery time in normal births was 24 seconds compared with 79 seconds in those with shoulder dystocia. These investigators proposed that a head-to-body delivery time >60 seconds be used to define shoulder dystocia. Currently, however, the diagnosis continues to rely on the clinical perception that the normal downward traction needed for fetal shoulder delivery is inefective.

1	Because of these difering deinitions, the incidence of shoulder dystocia varies. One recent review cites a clinically useful average of 1 percent of all deliveries (Ouzounian, 2016). The incidence has increased in recent decades, likely due to increasing fetal birthweight (MacKenzie, 2007; 0verland, 2014). Increased identiication and documentation may also raise the incidence (Kim, 2016).

1	In general, shoulder dystocia poses greater risk to the fetus than to the mother. The main maternal risks are serious perineal tears and postpartum hemorrhage, usually from uterine atony but also from lacerations (Gauthaman, 2016; Rahman, 2009). In contrast, significant neonatal neuromusculoskeletal injury and asphyxia are concerns. These specific injuries are described in Chapter 33 (p. 630). In one review of 1177 shoulder dystocia cases, brachial plexus injury was diagnosed in 11 percent and clavicular or humeral fracture in 2 percent (Chauhan, 2014). MacKenzie and associates (2007) reviewed 514 cases. Of the neonates, 7 percent showed evidence of acidosis at delivery, and 1.5 percent required cardiac resuscitation or developed hypoxic ischemic encephalopathy (HIE). In another review of 200 cases, rates of severe fetal acidosis and HIE were each 0.5 percent if delivery was completed within 5 minutes. These rates rose to 6 and 24 percent, respectively, with delivery delays �5 minutes

1	200 cases, rates of severe fetal acidosis and HIE were each 0.5 percent if delivery was completed within 5 minutes. These rates rose to 6 and 24 percent, respectively, with delivery delays �5 minutes (Leung, 2011a).

1	Fetal macrosomia, maternal obesity, prolonged second-stage labor, and a prior event raise risks for shoulder dystocia (Mehta, 2004; Overland, 2009; Schummers, 2015). Although these factors are clearly associated with this complication, identification of individual instances before the fact has proved to be impossible. The American College of Obstetricians and Gynecologists (2017 c) reviewed studies and concluded that: 1. Most cases of shoulder dystocia cannot be accurately predicted or prevented. 2. Elective induction of labor or elective cesarean delivery for all women suspected of having a macrosomic fetus is not appropriate. 3. Planned cesarean delivery may be considered for the nondiabetic woman with a fetus whose estimated fetal weight is > 5000 g or for the diabetic woman whose fetus is estimated to weigh >4500 g.

1	There is a corresponding rise in the incidence of shoulder dystocia with increasing birthweight (Acker, 1985; 0verland, 2012; Stotland, 2004). Commonly cited maternal characteristics associated with increased fetal birthweight are obesity, postterm pregnancy, multiparity, and diabetes (Jolly, 2003; Koyanagi, 2013). The combination of fetal macrosomia and maternal diabetes mellitus escalates the frequency of shoulder dystocia (Langer, 1991; Nesbitt, 1998). This predisposition may stem from the fact that fetuses of diabetic women have larger shoulder and extremity circumferences and greater shoulder-to-head and chest-to-head size diferences relative to comparable-weight fetuses of nondiabetic mothers (McFarland, 1998; Modanlou, 1982). That said, translating these specific measurements into stand-alone sonographic clinical thresholds has shown poor predictive sensitivity (Burkhardt, 2014).

1	Preventively, early labor induction has yielded conflicting results. In one study, approximately 800 women with suspected macrosomic fetuses were randomized either to early induction between 37 and 39 weeks or to expectant care (Boulvain, 2015). Dystocia rates were lowered by two thirds in the intervention group, and neither group sufered brachial plexus injury. Although not measured, this practice is balanced against morbidity of early delivery. Moreover, the poor accuracy of antepartum fetal weight prediction should be considered as well (Hoopmann, 2010; Mlin, 2016; Noumi, 2005). In contrast, an earlier randomized study of 284 women showed that rates of shoulder dystocia were not lowered by early induction at 38 weeks (Gonen, 1997).

1	As previously discussed, cesarean delivery may be considered to prevent shoulder dystocia. hat said, Rouse and Owen (1999) concluded that a prophylactic cesarean delivery policy for macrosomic fetuses would require more than 1000 cesarean deliveries with attendant morbidity to avert a single permanent brachial plexus injury. he risk of recurrent shoulder dystocia ranges from 1 to 13 percent (Bingham, 2010; Moore, 2008; Ouzounian, 2013). For many women with prior shoulder dystocia, a trial of labor may be reasonable. he American College of Obstetricians and Gynecologists (2017 c) recommends that estimated fetal weight, gestational age, maternal glucose intolerance, and severity of prior neonatal injury be evaluated and risks and benefits of cesarean delivery discussed with any woman with a history of

1	FIGURE 27-5 The McRoberts maneuver. The maneuver consists of removing the legs from the stirrups and sharply flexing the thighs up toward the abdomen. The assistant is also providing suprapubic pressure simultaneously (arrow). shoulder dystocia. After discussion, either mode of delivery may be appropriate.

1	shoulder dystocia. After discussion, either mode of delivery may be appropriate. Because shoulder dystocia cannot be accurately predicted, clinicians should be well versed in its management principles. Because of ongoing cord compression with this dystocia, one goal is to reduce the head-to-body delivery time. This is balanced against the second goal, which is avoiding fetal and maternal injury from aggressive manipulations. Accordingly, an initial gentle attempt at traction, assisted by maternal expulsive eforts, is recommended. Adequate analgesia is certainly ideal. Some clinicians advocate performing a large episiotomy to provide room for manipulations. Episiotomy itself does not lower brachial plexus injury rates but raises third-and fourthdegree laceration rates (Gurewitsch, 2004; Paris, 2011; SagiDain, 2015). Episiotomy may be elected to complete needed maneuvers.

1	After gentle traction, various techniques can be used to free the anterior shoulder from its impacted position behind the symphysis pubis. A more detailed discussion of these and the topic is found in Cunningham and Gilstrap s Operative Obstetrics, 3rd edition (Cunningham, 2017). Of these, moderate suprapubic pressure can be applied by an assistant, while downward trac tion is applied to the fetal head. Pressure is applied with the heel of the hand to the anterior shoulder wedged above and behind the symphysis. The anterior shoulder is thus either depressed or rotated, or both, so the shoulders occupy the oblique plane of the pelvis. Here, the anterior shoulder can be freed.

1	The McRoberts maneuver is often selected next if additional steps are needed. The maneuver consists of removing the legs from the stirrups and sharply lexing them up toward the abdomen. Suprapubic pressure is often concurrently applied (Fig. 27-5). Gherman and associates (2000) analyzed the McRoberts maneuver using x-ray pelvimetry. hey found that the procedure caused straightening of the sacrum relative to the lumbar vertebrae, rotation of the symphysis pubis toward the maternal head, and a decrease in the angle of pelvic inclination. Although this does not increase pelvic dimensions, pelvic rotation cephalad tends to free the impacted anterior shoulder. Gonik and coworkers (1989) tested the McRoberts position objectively with laboratory models and found that the maneuver reduced the forces needed to free the fetal shoul der. If unsuccessful, most move next either to free the posterior shoulder or to rotate the bisacromial diameter into one of the oblique diameters of the maternal

1	needed to free the fetal shoul der. If unsuccessful, most move next either to free the posterior shoulder or to rotate the bisacromial diameter into one of the oblique diameters of the maternal pelvis.

1	With delivey of the posterior shoulder, the accoucheur carefully sweeps the posterior arm of the fetus across its chest, followed by deliveY of the arm (Fig. 27-6). If possible, the operator's ingers are aligned parallel to the long axis of the fetal humerus to lower bone fracture risks. The shoulder girdle is then rotated into one of the oblique diameters of the pelvis with subsequent delivery of the anterior shoulder.

1	Of rotational maneuvers, Woods (1943) reported that by progressively rotating the posterior shoulder 180 degrees in a corkscrew fashion, the impacted anterior shoulder could be released. his is frequently referred to as the Woods corkscrew maneuver (Fig. 27-7). Rubin (1964) recommended two maneuvers. First, the fetal shoulders are rocked from side to side by applying force to the maternal abdomen. If this is not successful, the pelvic hand reaches the most easily accessible fetal shoulder, which is then pushed toward the anterior surface of the chest. This maneuver most oten abducts both shoulders, which in turn produces a smaller bisacromial diameter. This permits displacement of the anterior shoulder from behind the symphysis (Fig. 27-8).

1	If the above are initially unsuccessful, they may be repeated, and finally other methods may be elected. With an allours maneuver, also called the Gaskin maneuver, the parturient rolls onto her knees and hands. Here, downward traction against the head and neck attempts to free the posterior shoulder (Bruner, 1998). Challenges with this include immobility from regional analgesia and time lost in patient repositioning. In some, the posterior arm is inaccessible for delivery. Cluver and Hofmeyr (2009) describedposterior axila sling traction to deliver the posterior arm. With this alternative method, a suction catheter is threaded under the axilla and both ends are brought together above the shoulder. Upward and outward traction on the catheter loop delivers the shoulder. In a small series of 19 cases, this maneuver was successful in 18 cases. However, neonatal injury included three cases of humeral fracture and one permanent and four transient cases ofErb palsy (Cluver, 2015).

1	FIGURE 27-6 Delivery of the posterior shoulder for relief of shoulder dystocia. A. The operator's hand is introduced into the vagina along the fetal posterior humerus. B.The arm is splinted and swept across the chest, keeping the arm flexed at the elbow. C. The fetal hand is grasped and the arm extended along the side of the face. The posterior arm is delivered from the vagina. ....�. i" Jr-.. � FIGURE 27-7 Woods maneuver. The hand is placed behind the posterior shoulder of the fetus. The shoulder is then rotated in a corkscrew manner so that the impacted anterior shoulder is released. FIGURE 27-8 The second Rubin maneuver. A. The bisacromial diameter is aligned vertically. B. The more easily accessible fetal shoulder (the anterior is shown here) is pushed toward the anterior chest wall of the fetus (arrow). Most often, this results in abduction of both shoulders, which reduces the bisacromial diameter and frees the impacted anterior shoulder.

1	Deliberate fracture of the anterior clavicle using the thumb to press it toward and against the pubic ramus can be attempted to free the shoulder impaction. In practice, however, deliberate fracture of a large neonate's clavicle is diicult. If successful, the fracture will heal rapidly and is usually trivial compared with brachial nerve injury, asphyxia, or death.

1	he Zavanelli maneuver involves replacement of the fetal head into the pelvis followed by cesarean delivery (Sandberg, 1985). Terbutaline, 0.25 mg, is given subcutaneously to produce uterine relaxation. he irst part of the maneuver consists of returning the head to an OA or OP position. The operator flexes the head and slowly pushes it back into the vagina. Cesarean delivery is then performed. Sandberg (1999) reviewed 103 reported cases. It was successful in 91 percent of cephalic cases and in all cases of breech head entrapments. Despite successful replacement, fetal injuries were common bur may have resulted from the multiple manipulations used before the Zavanelli maneuver (Sandberg, 2007).

1	Symphysiotomy, in which the intervening symphyseal cartilage and much of its ligamentous support is cut to widen the symphysis pubis, is described in Chapter 28 (p. 548). It has been used successfully for shoulder dystocia (Goodwin, 1997; Hartield, 1986). Maternal morbidity can be signiicant due to urinary tract injury. Cleidotomy consists of cutting the clavicle with scissors or other sharp instruments and is usually done for a dead fetus (Schramm, 1983). Hernandez and Wendel (1990) suggest use of a shoulder dystocia drill to better organize emergency management: 1. Call for help-mobilize assistants and anesthesia and pediatric personnel. Initially, a gentle attempt at traction is made. Drain the bladder if it is distended. 2. A generous episiotomy may be desired at this time to aford room posteriorly. 3.

1	2. A generous episiotomy may be desired at this time to aford room posteriorly. 3. Suprapubic pressure is used initially by most practitioners because it has the advantage of simplicity. Only one assistant is needed to provide suprapubic pressure, while normal downward traction is applied to the fetal head. 4. The McRoberts maneuver requires two assistants. Each assistant grasps a leg and sharply lexes the maternal thigh toward the abdomen. These maneuvers will resolve most cases of shoulder dystocia. If the above listed steps fail, the following steps may be attempted, and any of the maneuvers may be repeated: 5. Delivery of the posterior arm is attempted. With a fully extended arm, however, this is usually diicult to accomplish. 6. Woods screw maneuver is applied. 7. Rubin maneuver is attempted.

1	Delivery of the posterior arm is attempted. With a fully extended arm, however, this is usually diicult to accomplish. 6. Woods screw maneuver is applied. 7. Rubin maneuver is attempted. The American College of Obstetricians and Gynecologists (2017c) has concluded that no one maneuver is superior to another in releasing an impacted shoulder or reducing the chance of injury. Performance of the McRoberts maneuver, however, is deemed a reasonable initial approach. In one review of more than 2000 cases, Hofman and colleagues (2011) noted an 84-percent success rate with posterior shoulder delivery and comparable rates of neonatal injury compared with other standard methods. This contrasts with a review of 205 cases, in which posterior shoulder delivery yielded greater neonatal injury rates than rotational methods (Leung, 201i1b). Spain and associates (2015) found that duration rather than a speciic maneuver increased neonatal injury.

1	Importantly, progression from one maneuver to the next should be organized and methodical. As noted, the urgency to relieve the dystocia should be balanced against potentially lnJurious traction forces and manipulations. Lerner and coworkers (201l) in their evaluation of 127 shoulder dystocia cases reported that all neonates without sequelae from shoulder dystocia were born by 4 minutes. Conversely, most depressed neonates-57 percent-had head-to-body delivery intervals >4 minutes. The percentage of depressed neonates rose sharply after 3 minutes.

1	Shoulder dystocia training and protocols using simulationbased education and drills has evidence-based support. These tools improve performance and retention of drill steps (Buerkle, 2012; Crofts, 2008; Grobman, 201l). Their use has translated into improved neonatal outcome in some, but not all, investigations (Crofts, 2016; Fransen, 2017; Kim, 2016; Walsh, 201l). The American College of Obstetricians and Gynecologists (2012) also has created a Patient Safety Checklist to guide the documentation process with shoulder dystocia.

1	In 2014, 0.7 percent of deliveries in the United States were planned home births and 0.2 percent were unplanned (MacDorman, 2016). Of unplanned births in a 15-year epoch in Norway, 69 of 6027 or 1.1 percent resulted in fetal or neonatal death. This high rate was attributable to infection, prematurity, and placental abruption (Gunnarsson, 2017). Multiparity and distance from the hospital were ascribed risks (Gunnarsson, 2014). In the United States, youth, lack of prenatal care, minority race, and lower educational attainment were associated risks for unplanned home birth (Declercq, 2010).

1	In contrast, the demographics of women choosing planned home birth in the United States favor those who are white, nonsmoking, self-pay, college-educated, and multiparous (MacDorman, 2016). As perceived beneits, planned delivery at home for those with low-risk pregnancies results in fewer medical interventions that include labor augmentation, episiotomy, operative vaginal delivery, and cesarean delivery (Bolten, 2016; Cheyney, 2014). Regarding the safety of planned home birth, data from randomized trial are lacking, and large observational studies derive from heterogeneous care systems, whose results may not be generalizable. For example, several developed countries deliver at home a large volume of carefully screened women, delivered by midwives with substantial training and in a setting closely integrated with the local health-care system (Birthplace in England Collaborative Group, 2011; de Jonge, 2015; Hutton, 2016). The level of such coordination in the United States is less

1	closely integrated with the local health-care system (Birthplace in England Collaborative Group, 2011; de Jonge, 2015; Hutton, 2016). The level of such coordination in the United States is less uniform.

1	Overall, risks of home births in the United States are small but greater than those of hospital delivery. Midwife-attended home births carry a neonatal mortality risk of 1.3 per 1000 births. This is a nearly fourfold greater rate compared with midwife-attended hospital births. The most common underlying causes of death are those attributed to labor and delivery events, to congenital anomalies, and to infection. Of neonatal injuries, rates of neonatal seizure and serious neurological dysfunction are similarly elevated in home-birth groups (Grunebaum, 2013, 2014, 2017; Snowden, 2015; Wasden, 2016). Importantly, substantial risks attend home birth for those with prior cesarean delivery, with breech presentation, and with multifetal gestation (Cheyney, 2014; Cox, 2015). The American College of Obstetricians and Gynecologists (2017b) considers these to be absolute contraindications. Further, the College considers accredited hospitals and birthing centers to be the safest site for birth but

1	Obstetricians and Gynecologists (2017b) considers these to be absolute contraindications. Further, the College considers accredited hospitals and birthing centers to be the safest site for birth but recognizes the autonomy of the well-counseled patient.

1	As one option for pain relief, some women choose to spend part of first-stage labor in a large water tub. With this practice, one Cochrane review found lower rates of anesthesia block use and no greater adverse neonatal or maternal efects compared with traditional labor (Cluett, 2009).

1	For delivery, however, water birth carries greater concern for neonatal harm and without proven benefits. Case reports describe aspiration leading to fresh-water drowning (Pinette, 2004). The risk of cord avulsion during water birth approximates 3 per 1000 births, and stems primary from abruptly bringing the newborn out of the water (Schafer, 2014). Last, case reports also enumerate serious infections, which emphasize the need for rigorous sanitizing protocols. hat said, in most large studies comparing land and water births, overall maternal or neonatal infection rates are not increased (Bovbjerg, 2016; Burns, 2012; Thoeni, 2005). In sum, several reviews comment on study shortcomings and isolated complications but do not identiy definitive evidence for overall greater rates of neonatal harm from water birth in low-risk populations (Davies, 2015; Taylor, 2016). However, given the paucity of robust data and potential for serious complications, the American College of Obstetricians and

1	from water birth in low-risk populations (Davies, 2015; Taylor, 2016). However, given the paucity of robust data and potential for serious complications, the American College of Obstetricians and Gynecologists (2016a) currently recommend that "birth occur on land, not in water."

1	This practice refers to medically unnecessary vulvar and perineal modification. In the United States, it is a federal crime to perform unnecessary genital surgery on a girl younger than 18 years. That said, forms of female genital mutilation are practiced in countries throughout Africa, the Middle East, and Asia. As many as 200 million women worldwide have undergone one of these procedures, and approximately 513,000 girls in the United States were at risk for this practice in 2012 (Goldberg, 2016; UNICEF, 2016). Cultural sensitivity is imperative, because many women may be ofended by the suggestion that they have been assaulted or mutilated (merican College of Obstetricians and Gynecologists, 2014).

1	The World Health Organization (2008) classifies genital mutilations into four types (Table 27-1). Long-term complications from surgery and its associated scarring include infertility, genital pain, diminished sexual quality of life, and propensity for urogenital infection (Almroth, 2005; Andersson, 2012; Nour, 2015). In general, women with significant symptoms following type III procedures are candidates for corrective surgery. Specifically, division of midline scar tissue to reopen the vulva is termed deinibulation. Female genital mutilation has been associated with some adverse maternal and neonatal complications. The World TABLE 27-1. World Health Organization Classification of Female Genital Mutilation Type I Partial or total removal of the clitoris and/or prepuce Type II Partial or total removal of the clitoris and the labia minora, with or without labia majora excision

1	Type I Partial or total removal of the clitoris and/or prepuce Type II Partial or total removal of the clitoris and the labia minora, with or without labia majora excision Type III Partial or total labial minora and/or majora excision, followed by fusion of the wound, termed infibulation, to cover and narrow the vagina. With or without clitoridectomy Type IV Pricking, piercing, incising, scraping, cautery, or other injury to female genitalia Adapted from the World Health Organization, 2008. Health Organization (2006, 2008) estimated that these proce dures increased perinatal morbidity rates by 10 to 20 per 1000. Small increased risks for prolonged labor, cesarean delivery, and postpartum hemorrhage are also found (Berg, 2014; Chibber, 2011; Wuest, 2009). Importantly, the psychiatric consequences can be profound.

1	To prevent obstetrical complications, deinibulation can be performed either antepartum or intrapartum (Fig. 27-9) (Esu, 2017). In women not undergoing deinibulation, anal sphincter tear rates with vaginal delivery may be increased (Berggren, 2013; Rodriguez, 2016). In our experiences, intrapartum deinibulation in many cases allows successful vaginal delivery without major complications.

1	hese surgeries are performed with increasing frequency in reproductive-aged women, and thus pregnancy following these procedures is not uncommon. Logically, there are concerns for symptom recurrence following vaginal delivery, and high-quality data to aid evidenced-based decisions are limited. For women with prior stress urinary incontinence surgery, slightly greater protection against postpartum incontinence is gained by elective cesarean delivery (Pollard, 2012; Pradhan, 2013). Stated another way, most women with prior corrective surgery for incontinence can be delivered vaginally without symptom recurrence. Also, cesarean delivery is not always protective. Obviously, symptom recurrence and the need for additional vaginal surgery should be weighed against the surgical risk of cesarean delivery (Groenen, 2008). In those with prior surgeries for anal incontinence or pelvic organ prolapse, only scant information regarding outcomes is available. Such cases require individualization.

1	Rarely, delivery can be obstructed by extreme hydrocephaly, by body stalk anomaly, or by massive fetal abdominal enlargement from a greatly distended bladder, ascites, or organomegaly (Costa, 2012; Sikka, 2011). With milder forms of hydrocephaly, if the biparietal diameter is < 10 cm or if the head circumference is <36 cm, then vaginal delivery may be permitted (Anteby, 2003).

1	FIGURE 27-9 Deinfibulation. Although not shown here, lidocaine is first infiltrated along the planned incision if regional analgesia is not in place already. As protection, two fingers of one hand are insinuated behind the shelf created by fused labia but in front of the urethra and crowning head. The shelf is then incised in the midline. After delivery, the raw edges are sutured with rapidly absorbable material to secure hemostasis. (Reproduced with permission from Hawkins JS: Lower genital tract procedures. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201o7.)

1	In rare cases in which neonatal death has occurred or is certain due to associated anomalies, vaginal delivery may be reasonable, but the head or abdomen must be reduced in size for delivery. Removal of luid by cephalocentesis or paracentesis with sonographic guidance can be performed intrapartum. As described on page 523, cleidotomy can shorten the bisacromial diameter. For hydrocephalic fetuses that are breech, cephalocentesis can be accomplished suprapubically when the aftercoming head enters the pelvis. Currently, these practices are more germane in developing countries. • Delivery of the Placenta hird-stage labor begins immediately after fetal birth and ends with placental delivery. Goals include delivery of an intact placenta and avoidance of uterine inversion or postpartum hemorrhage. The latter two are grave intrapartum complications and constitute emergencies, as described in Chapter 41 (p. 755).

1	Immediately after newborn birth, uterine fundal size and consistency are examined. If the uterus remains irm and there is no unusual bleeding, watchful waiting until the placenta separates is the usual practice. Neither massage nor downward fundal pressure is employed, but the fundus is frequently palpated to ensure that it does not become atonic and illed with blood from placental separation. To prevent uterine inversion, umbilical cord traction must not be used to pul the placenta rom the uterus. Signs of separation include a sudden gush of blood into the vagina, a globular and firmer fundus, a lengthening of the umbilical cord as the placenta descends into the vagina, and elevation of the uterus into the abdomen. With the last, the placenta, having separated, passes down into the lower uterine segment and vagina. Here, its bulk pushes the uterine body upward.

1	These signs appear within minutes after newborn delivery, and the median time ranges from 4 to 12 minutes (Combs, 1991; Frolova, 2016; Shinar, 2016b). Once the placenta has detached from the uterine wall, the mother may be asked to bear down, and the intraabdominal pressure often expels the placenta into the vagina. These eforts may fail or may not be possible because of analgesia. After ensuring that the uterus is contracted irmly, the umbilical cord is kept slightly taut but is not pulled. Pressure is exerted by a hand wrapped around the fundus to propel the detached placenta into the vagina (Fig. 27-10). Concurrently, the heel of the hand exerts downward pressure between the symphysis pubis and the uterine fundus. This also aids inversion prevention. Once the placenta passes through the introitus, pressure on the uterus is relieved. The placenta is then gently lifted away. Care is taken to prevent placental membranes from being torn of and left behind. If the membranes begin to

1	the introitus, pressure on the uterus is relieved. The placenta is then gently lifted away. Care is taken to prevent placental membranes from being torn of and left behind. If the membranes begin to tear, they are grasped with a clamp and removed by gentle teasing (Fig. 27-11).

1	FIGURE 27-10 Expression of placenta. Note that the hand is not trying to push the fundus of the uterus through the birth canal! As the placenta leaves the uterus and enters the vagina, the uterus is elevated by the hand on the abdomen while the cord is held in position. The mother can aid in the delivery of the placenta by bearing down. As the placenta reaches the perineum, the cord is lifted, which in turn lifts the placenta out of the vagina. FIGURE 27-11 Membranes that were somewhat adhered to the uterine lining are separated by gentle traction with ring forceps. • Management of the Third Stage

1	FIGURE 27-11 Membranes that were somewhat adhered to the uterine lining are separated by gentle traction with ring forceps. • Management of the Third Stage Practices within the third stage of labor may be broadly considered as either expectant or active management. Expectant management involves waiting for placental separation signs and allowing the placenta to deliver either spontaneously or aided by nipple stimulation or gravity (World Health Organization, 2012). In contrast, active management of third-stage labor consists of early cord clamping, controlled cord traction during placental delivery, and immediate administration of prophylactic oxytocin. The goal of this triad is to limit postpartum hemorrhage (Begley, 2015; Jangsten, 2011; Westhof, 2013).

1	As noted earlier, delayed cord clamping does not increase postpartum hemorrhage rates, and thus early clamping is a less important component of this trio. Similarly, cord traction may also be less critical (Deneux-haraux, 2013; Du, 2014; Giilmezoglu, 2012). Uterine massage following placental delivery is recommended by many, but not all, to prevent postpartum hemorrhage. We support this with the caveat that evidence for this practice is not strong (Abdel-Aleem, 2010).

1	Therefore, utero tonics play an essential role to decrease postpartum blood loss. Choices include oxytocin (Pitocin), misoprostol (Cytotec), carboprost (Hemabate), and the ergots, namely ergonovine (Ergotrate) and methylergonovine (Methergine). In addition, a combination agent of oxytocin and ergonovine (Syntometrine) is used outside the United States. Also in other countries, carbetocin (Duratocin), a long-acting oxytocin analogue, is available and efective for hemorrhage prevention during cesarean delivery (Attilakos, 2010; Su, 2012). Of these, the World Health Organization (2012) recommends oxytocin as a first-line agent. Ergot-based drugs and misoprostol are alternatives in settings that lack oxytocin.

1	Utero tonics may be given before or after placental expulsion without afecting rates of postpartum hemorrhage, placental retention, or third-stage labor length (Soltani, 2010). If they are given before delivery of the placenta, however, they may entrap an undiagnosed, undelivered second (Win. hus, abdominal palpation should conirm no additional fetuses. Notably, this concern is less relevant with current widespread sonography use.

1	Synthetic oxytocin is identical to that produced by the posterior pituitary. Its action is noted at approximately 1 minute, and it has a mean half-life of 3 to 5 minutes. When given as a bolus, oxytocin can cause profound hypotension. Secher and coworkers (1978) reported that an intravenous bolus of 10 units of oxytocin caused a marked transient fall in blood pressure with an abrupt increase in cardiac output. Svansrrom and associates (2008) conirmed those findings. hese hemodynamic changes could be dangerous for women hypovolemic from hemorrhage or those with certain types of cardiac disease. Thus, oxytocin should be given as a dilute solution by continuous intravenous infusion or as an intramuscular injection.

1	Water intoxication can result from the antidiuretic action of high-dose oxytocin if administered in a large volume of electro lyte-free dextrose solution (Whalley, 1963). Thus, if oxytocin is to be administered in high doses for a considerable period of time, its concentration should be increased rather than increas ing the infusion flow rate.

1	Despite the routine use of oxytocin, no standard prophylactic dose has been established for its use following either vaginal or cesarean delivery. Our practice is to add 20 units (2 mL) of oxytocin per liter of infusate. his solution is administered after delivery of the placenta at a rate of 10 to 20 mUmin-200 to 400 mU/min-for a few minutes until the uterus remains irmly contracted and bleeding is controlled. he infusion rate then is reduced to 1 to 2 mUmin until the mother is ready for transfer from the recovery suite to the postpartum unit. The infusion is usually then discontinued. For women without intravenous access, 10 units of intramuscular oxytocin are injected.

1	Ergonovine and methylergonovine have similar activity levels in myometrium, and only methylergonovine is currently manufactured in the United States. These ergot alkaloid agents do not provide superior protection against postpartum hemorrhage compared with oxytocin. \10reover, safety and tolerability are greater with oxytocin (Liabsuetrakul, 2011). For these reasons, ergot alkaloid agents are considered second-line for prevention of postpartum hemorrhage. If selected, a 0.2-mg dose of methylergonovine is slowly given intravenously in a period not less than 60 seconds to avoid sudden hypertension (Novartis, 2012). Methylergonovine is relatively contraindicated in the hypertensive woman.

1	Misoprostol is a prostaglandin E, analogue, which has proved inferior to oxytocin for postpartum hemorrhage prevention (Tun:alp, 2012). However, in resource-poor settings that lack oxytocin, misoprostol is suitable for hemorrhage prophylaxis and is given as a single oral 600-�g dose (Mobeen, 2011; World Health Organization, 2012). Notably, although oxytocin is preferred for prevention of hemorrhage, ergot alkaloids and prostaglandins playia greater role in postpartum hemorrhage treatment, discussed in Chapter 41 (p. 759). • Manual Removal of Placenta

1	• Manual Removal of Placenta In approximately 2 percent of singleton births, the placenta may not deliver promptly (Cheung, 2011). Three possibilities include pacenta adherens, in which uterine contractions are insu.cient to detach the placenta; lower uterine segment constriction and a detached but trapped placenta; or a morbidly adherent placenta. Consistent risks for retained placenta include stillbirth, prior cesarean delivery, prior retention, and preterm delivery (Belachew, 2014; Coviello, 2015; Endler, 2014; Nikolajsen, 2013). For the last, in one study with nearly 46,000 deliveries, analysis predicted that 90 percent of placentas would spontaneously deliver by 180 minutes for gestations at 20 weeks; 21 minutes at 30 weeks; and 14 minutes at 40 weeks (Dombrowski, 1995).

1	Postpartum hemorrhage can complicate a retained placenta, and bleeding risk accrues with third-stage length. Thus, in the absence of bleeding, some recommend expectant management for 30 minutes, whereas others use a 15-minute threshold (Cummings, 2016; Deneux-Tharaux, 2009; Shinar, 2016a). he World Health Organization (2012) cites a 60-minute threshold. Notably, if brisk bleeding ensues and the placenta cannot be delivered by standard technique, manual removal of the placenta is indicated (Fig. 27-12). When performed, some administer a single dose of intravenous antibiotics, however, one systematic review of observational studies found no beneits (Chibueze, 2015). Although the American College of Obstetricians and Gynecologists (2016c) concludes that data neither support nor refute this practice, the World Health Organization (2012) recommends prophylaxis. At our institution, we administer a single dose to women not already receiving antibiotics.

1	The hour immediately following delivery of the placenta is critical. During this time, lacerations are repaired. Although utero tonics are administered, postpartum hemorrhage as the result of uterine atony is most likely at this time. Hematomas may expand. Consequently, uterine tone and the perineum are frequently evaluated. The American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (20 17b) recommend that maternal blood pressure and pulse be recorded immediately after delivery and every 15 minutes for the first 2 hours. The placenta, membranes, and umbilical cord are examined for completeness and for anomalies, as described in Chapter 6 (p. 111).

1	Lower genital tract lacerations may involve the cervix, vagina, or perineum. Those of the cervix and vagina are described in Chapter 41 (p. 763). Those of the perineum oten follow vaginal delivery, and most are irst-and second-degree lacerations. Lacerations are classified by their depth, and complete deinitions and visual examples are given in Figure 27-13. Of these, third-degree lacerations relect anal sphincter injury and are now subcategorized as: (3c) EAS plus internal anal sphincter (lAS) tears. FIGURE 27-12 Manual removal of placenta. A. One hand grasps the fundus and the other hand is inserted into the uterine cavity and the fingers are swept from side to side as they are advanced. B. When the placenta detaches, it is grasped and removed.

1	FIGURE 27-13 1. First-degree perineal laceration: injury to only the vaginal epithelium or perineal skin. 2. Second-degree laceration: injury to perineum that spares the anal sphincter complex but involves the perineal muscles, which are the bulbospongiosus and superficial transverse perineal muscles. 3a.Third-degree laceration: <50 percent of the external anal sphincter (EAS) is torn. 3b.Third-degree laceration: >50 percent of the EAS is torn, but the internal anal sphincter (lAS) remains intact. 3c.Third-degree laceration: EAS and lAS are torn. 4. Fourth-degree laceration: the perineal body, entire anal sphincter complex, and anorectal mucosa are lacerated. (Reproduced with permission from Kenton K, Mueller M: Episiotomy and obstetric anal sphincter lacerations. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds):

1	Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201o7.) hird-and fourth-degree lacerations are considered obstetrical anal sphincter injuries (OASIS), and their combined incidence varies from 0.5 to 5 percent (Blondel, 2016; Friedman, 2015). ity, midline episiotomy, persistent OP position, operative vaginal delivery, Asian race, short perineal length, and increasing fetal birthweight (Ampt, 2013; Dua, 2009; Gurol-Urganci, 2013; Landy, 2011). Mediolateral episiotomy is protective in most, but not all, studies Ganga, 2014; Raisanen, 2011; Shmueli, 2016).

1	Landy, 2011). Mediolateral episiotomy is protective in most, but not all, studies Ganga, 2014; Raisanen, 2011; Shmueli, 2016). Morbidity rates rise as laceration severity increases. Compared with simpler lacerations, anal sphincter injuries are associated with greater blood loss and puerperal pain. Wound disruption and infection rates are other risks (Goldaber, 1993; Lewicy-Gaupp, 2015). Stock and coworkers (2013) reported that approximately 7 percent of 909 OASIS lacerations had complications. Long term, anal sphincter injuries are linked with approximately doubled rates of fecal incontinence compared with vaginl delivery without OASIS (Evers, 2012; Gyhagen, 2014). Data on long-term dyspareunia are limited, and rates are increased in some but not all studies (MoS, 2008; Otero, 2006; Salim, 2014; Sundquist, 2012).

1	To ensure appropriate repair, identiication and correct categorization is essential. Diagnosis rates of OASIS improve with clinical experience (ndrews, 2006). Intrapartum endoanal ultrasound, performed in research studies, also boosts detection, and rates of clinically occult tears in primiparas range from 6 to 12 percent (Corton, 2013; Faltin, 2005; Ozyurt, 2015). hat said, few data currently support routine intrapartum endoanal sonography, and the American College of Obstetricians and Gynecologists (20 16b) does not recommended i t (Walsh, 2015).

1	Women with a prior OASIS have a higher recurrence rate compared with multiparas without prior OASIS (Baghestan, 2012; Edozien, 2014; Elfaghi, 2004). That said, the risk mirrors that of primiparas in the general population and is low (Basham, 2013; Boggs, 2014; Priddis, 2013). Fetal macrosomia and operative vaginal delivery are notable risks in this cohort of parturients and can influence counseling in future pregnancies. Speciically, patients may choose to deliver by cesarean to avoid repeat OASIS. his consideration may be most pertinent for those with prior postpartum anal incontinence, with OASIS complications requiring corrective surgery, or with psychological trauma (American College of Obstetricians and Gynecologists, 2016b). However, planned cesarean delivery is balanced against its associated operative risks discussed in Chapter 30 (p. 568).

1	In contrast to spontaneous lacerations, perineotomy is intended incision of the perineum. Episiotomy is incision of the pudendum-the external genital organs. In common parlance, however, the term episiotomy often is used synonymously with perineotomy, a practice that we follow here. Obstetrical textbooks and organizational guidelines difer considerably in their description of episiotomy techniques. Kalis and associates (2012) have presented a classification, and we agree with the need for terminology standardization.

1	Midline and mediolateral episiotomies are the two main types and vary by the angle of perineal incision. Involved structures mirror those found with second-degree laceration, and their repairs are analogous. The midline episiotomy begins at the fourchette, incises the perineal body in the midline, and ends well before the external anal sphincter is reached. he incision length varies from 2 to 3 cm depending on perineal length and degree of tissue thinning. The mediolateral episiotomy begins at the midline of the fourchette and is directed to the right or left at an angle 60 degrees of the midline (Fig. 27-14). This angle accounts for perineal anatomy distortion during crowning and ultimately yields an incision 45 degrees of the midline for suturing (El-Din, 2014; Kalis, 2011). The lateral episiotomy begins at point 1 to 2 cm lateral from the midline. It too is angled toward either the right or the left ischial tuberosity.

1	Before episiotomy, analgesia may be provided by existing labor regional analgesia, by bilateral pudendal nerve blockade, or by local infiltration of I-percent lidocaine. Some instead advocate 2.5-percent lidocaine-prilocaine cream (EMLA cream), but this requires application an hour before expected delivery, which may be logistically diicult (Franchi, 2009; Kargar, 2016). If episiotomy is performed unnecessarily early, incisional bleeding may be considerable before delivery. If it is performed too late, lacerations will not be prevented. Typically, episiotomy is completed when the head is visible during a contraction to a diameter of approximately 4 cm, that is, crowning. When used in conjunction with forceps delivery, most perform an episiotomy after application of the blades.

1	Few data directly compare midline and mediolateral types. As noted, midline episiotomy has a greater likelihood of associated anal sphincter lacerations (Coats, 1980; de Leeuw, 2001). Shortterm rates of self-perceived pain and dyspareunia are similar or increased with mediolateral episiotomy (Fodstad, 2013, 2014; Sartore, 2004). head crowns. Fingers are insinuated between the perineum and head. The incision begins in the midline and is directed toward the ipsilateral ischial tuberosity at an angle 60 degrees of the midline. (Reproduced with permission from Kenton K, Mueller M: Episiotomy and obstetric anal sphincter lacerations. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 2017.)

1	Even fewer studies compare lateral episiotomy to either mediolateral or midline. One randomized trial compared lateral and mediolateral tpes in nulliparas. Groups did not difer in pain scores, in sexual qulity of life, or in vaginal or perineal trauma, including OASIS (Karbanova, 2014a,b; Necesalova, 2016). The authors also reported that mediolateral episiotomies required less time and suture for the repair. Thus, among the three, mediolateral episiotomy may be the preferred incision to reduce OASIS rates. In the past, routine episiotomy was practiced to avoid a ragged laceration and to limit postoperative pain and anal sphincter injury rates. But, a Cochrane review of randomized trials showed lower rates of severe perineal/vaginal trauma in women managed with a restrictive, that is, selective use of episiotomy for spontaneous delivery rather than with routine episiotomy Qiang, 2017). Importantly, this review did not discern between midline and mediolateral episiotomies.

1	The American College of Obstetricians and Gynecologists (20 16b) has concluded that restricted use of episiotomy is preferred to routine use. We are of the view that the procedure should be applied selectively for appropriate indications. Thus, episiotomy can be considered for indications such as shoulder dystocia, breech delivery, fetal macrosomia, operative vaginal deliveries, persistent OP positions, markedly short perineal length, and other instances in which failure to perform an episiotomy will result in signiicant perineal rupture. The final rule is that there is no substitute for surgical judgment and common sense.

1	FIGURE 27-15 Mediolateral episiotomy repair. A. The vaginal epithelium and deeper tissues are closed with a single, continuous, locking suture. The angle seems less acute now (approximately 45°) since the perineum is no longer distended. B. Ater the vaginal component of the laceration is repaired, deeper perineal tissues are reapproximated by a single, continuous, nonlocking suture. Small episiotomies may not require this deeper layer. C. With a similar continuous, non locking technique, the superficial transverse perineal and bulbospongiosus muscles are reapproximated. D. Last, the perineal skin is closed using a subcuticular stitch. (Reproduced with permission from Kenton K, Mueller M: Episiotomy and obstetric anal sphincter lacerations. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201•7.) With this new approach, epIsIotomy rates have dropped.

1	With this new approach, epIsIotomy rates have dropped. Oliphant and coworkers (2010) used the National Hospital Discharge Survey to analyze episiotomy rates between 1979 and 2006 in the United States. hey noted a 75-percent decline in the age-adjusted episiotomy rate. In the United States in 2012 episiotomy was performed in approximately 12 percent of vaginal births (Friedman, 2015). Typically, perineal repairs are deferred until the placenta has been delivered. his policy permits undivided attention to the signs of placental separation and delivery. A further advantage is that the repair is not interrupted or disrupted by placenta delivery. his is especially true if manual removal must be performed. The major disadvantage is continuing blood loss until the repair is completed. Direct pressure from an applied gauze sponge will help to limit this volume.

1	For suitable repair, an understanding of perineal support and anatomy is necessary and is discussed in Chapter 2 (p. 19). Adequate analgesia is imperative, and women without regional analgesia can experience high levels of pain during perineal suturing. Again, locally injected lidocaine can be used solely or asia supplement to bilateral pudendal nerve blockade. In those with epidural analgesia, additional dosing may be necessary. First-degree lacerations do not always require repair, and sutures are placed to control bleeding or restore anatomy. Here, few data guide suture selection, and ine-gauge absorbable or delayed-absorbable suture or adhesive glue is suitable.

1	Second-degree laceration correction as well as midline and mediolateral episiotomy repairs include similar steps. Namely, these close the vaginal epithelium and reapproximate the bulbospongiosus and supericial transverse perineal muscles during restoration of the perineal body (Figs. 27-15 and 27-16). For this, most studies support a continuous

1	FIGURE 27-16 Midline episiotomy repair. A. An anchor stitch is placed above the wound apex to begin a running, locking closure with 2-0 suture to close the vaginal epithelium and deeper tissues and reapproximate the hymeneal ring. B. A transition stitch redirects suturing from the vagina to the perineum. C. The superficial transverse perineal and bulbospongiosus muscles are reapproximated using a continuous, non locking technique with the same length of suture. This aids restoration of the perineal body for long-term support. D. The continuous suture is then carried upward as a subcuticular stitch. The final knot is tied proximal to the hymeneal ring. (Reproduced with permission from Kenton K, Mueller M: Episiotomy and obstetric anal sphincter lacerations. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201•7.) suturing method, which is faster than placing interrupted sutures and, with

1	LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201•7.) suturing method, which is faster than placing interrupted sutures and, with few exceptions, yields less pain (Grant, 2001; Kettle, 2012; Kindberg, 2008; Valenzuela, 2009). Blunt needles are suitable and likely decrease the incidence of needle-stick injuries (El-Refaie, 2012; Mornar, 2008). Commonly used suture materials are 2-0 polyglactin 910 (Vicryl) or chromic catgut. With the former, a decrease in postsurgical pain and lower risk of wound dehiscence are cited as major advantages Qallad, 2016; Kettle, 2010). Closures with traditional polyglactin 910, however, occasionally require removal of residual suture from the repair site because of pain or dyspareunia. This disadvantage may be reduced by using a rapidly absorbed polyglactin 910 (Vicryl Rapide) (Bharathi, 2013; Kettle, 2002; Leroux, 2006).

1	For third-degree laceration repair, two methods are available to repair the external anal sphincter. The irst is an endto-end technique, which we prefer, and is shown in Figure 27-17. Initially, the cut ends of the external anal sphincter, which often retract, are isolated and brought to the midline. Importantly, the strength of this closure is derived from the connective tissue surrounding the sphincter-often called the capsule-and not the striated muscle. Thus, serial interrupted

1	FIGURE 27-17 In overview, with end-to-end approximation of the external anal sphincter (EAS), a suture is placed through the EAS muscle, and four to six simple interrupted 2-0 or 3-0 sutures of polyglactin 910 are placed at the 3, 6, 9, and 12 o'clock positions through the perisphincter connective tissue. To begin, disrupted ends of the striated EAS muscle and capsule are identified and grasped. The first suture is placed posteriorly to maintain clear exposure. Another suture is then placed inferiorly at the 6 o'clock position. The sphincter muscle fibers are next reapposed by a figure-of-eight stitch. Last, the remainder of the fascia is closed with a stitch placed anterior to the sphincter cylinder and again with once placed superior to it. (Reproduced with permission from

1	Kenton K, Mueller M: Episiotomy and obstetric anal sphincter lacerations. In Yeomans ER, Hofman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative ObstetriCS, 3rd ed. New York, McGraw-Hili Education, 201o7.) sutures incorporate sphincter fibers and perisphincter connective tissue, to bring sphincter ends together. here are few evidence-based data to guide suture selection for sphincter repair, but delayed-absorbable material can provide sustained tensile strength during healing. This theory is supported by the above study by J all ad and coworkers (2016), which showed higher perineal breakdown rate following OASIS repair with chromic gut.

1	With the overlapping technique, the ends of the external anal sphincter are brought to the midline and lie atop one another. This method is only suitable for type 3c lacerations-those involving the external and internal anal sphincter. Two rows of mattress sutures travel through both sphincter ends to recreate the anal ring. In comparing the two methods, neither yields superior long-term anatomical or functional results (Farrell, 2012; Fernando, 2013; Fitzpatrick, 2000). Also with type 3c lacerations, the lAS is repaired before the EAS and is described next.

1	With fourth-degree laceration repairs, the torn edges of the rectal mucosa are reapproximated (Fig. 27-18). At a point 1 cm proximal to the wound apex, sutures are placed approximately 0.5 cm apart in the rectal muscularis and do not enter the ano rectal lumen. Clinicians oten use 4-0 polyglactin 910 or chromic gut for this running suture line. Some recommend a second reinforcing layer above this (Hale, 2007). If this is not done, then the next layer to cover the anorectal mucosa is formed by reapproximation of the internal anal sphincter. his running, nonlocking closure is completed with 3-0 or 4-0 suture (see Fig. 27-18B). Following any repair, needle and sponge counts are reconciled and recorded in the delivery note.

1	For reduction of infectious morbidity associated with anal sphincter lacerations, a single dose of antibiotic at the time of repair is recommended by the American College of Obstetricians and Gynecologists (20 16c). his practice is supported by evidence (Buppasiri, 2014; Duggal, 2008; Lewicky-Gaupp, 2015; Stock, 2013). A single dose of a secondgeneration cephalosporin is suitable, or clindamycin for penicillin-allergic women. With OASIS, postoperatively, stool softeners are prescribed for a week, and enemas and suppositories are avoided. Unfortunately, normal function is not always ensured even with correct and complete surgical repair. Some women may experience continuing fecal incontinence caused by injury to the innervation of the pelvic Boor musculature (Roberts, 1990).

1	FIGURE 27-18 A. Suturing of the anorectal mucosa begins above the laceration apex using a continuous, non locking method with finegauge absorbable suture such as 3-0 or 4-0 chromic gut or polyglactin 910. Sutures are placed through the anorectal submucosa approximately 0.5 cm apart down to the anal verge. B. A second reinforcing layer uses 3-0 delayed-absorbable suture in a continuous, nonlocking fashion. This incorporate the torn ends of the internal anal sphincter (lAS), which can be identified as the glistening white fibrous structure lying between the anal canal submucosa and the fibers of the external anal sphincter. In many cases, the lAS retracts laterally and must be sought and retrieved for repair. (Reproduced with permission from Kenton K, Mueller M: Episiotomy and obstetric anal sphincter lacerations. In Yeomans ER, Hofman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201o7.)

1	Initially, locally applied ice packs help reduce swelling and allay discomfort (de Souza Bosco Paiva, 2016). In subsequent days, warm sitz baths aid comfort and hygiene. Additionally, a small squirt botde of warm water can cleanse the site after voiding or stooling. For pain, topical application of 5-percent lidocaine ointment was not efective in relieving episiotomy or perineal laceration discomfort in one randomized trial (Minassian, 2002). Oral analgesics containing codeine provide considerable relie. For lesser degree of discomfort, NSAID tablets can be given. Because pain may signal a large vulvar, paravaginal, or ischiorectal fossa hematoma or perineal cellulitis, these sites should be examined carefully if pain is severe or persistent. Management of these complications is discussed in Chapters 37 and 41 (pp. 674 and 764). In addition to pain, urinary retention may complicate episiotomy recovery (Mulder, 2012, 2016). Its management is described in Chapter 36 (p. 660).

1	For those with second-degree lacerations or anal sphincter tears, intercourse is usually proscribed until after the irst puerperal visit at 6 weeks. Compared with women with intact perineum, those with perineal trauma show higher rates of delayed intercourse at 3 and 6 months, but not at 1 year (McDonald, 2015; Rldestad, 2008; Signorello, 2001). Abdel-Aleem H, Singata M, Abdel-Aleem M, et al: Uterine massage to reduce postpartum hemorrhage after vaginal delivery. Int ] Gynaecol Obstet 111(1):32,r2010 Acker DB, Sachs BP, Friedman EA: Risk factors for shoulder dystocia. Obstet Gynecol 66(6):762, 1985 Almroth L, Elmusharaf S, El Hadi N, et al: Primary infertility ater genital mutilation in girlhood in Sudan: a case-control study. Lancet 366:385, 2005 Al-Wassia H, Shah PS: Eicacy and safety of umbilical cord milking at birth: a systematic review and meta-analysis.]AMA Pediatr 169(1):18, 2015 American Academy of Pediatrics: Delayed umbilical cord clamping after birth.

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1	Wuest 5, Raio L, Wyssmueller D, et al: Efects of female genital mutilation on birth outcomes in Switzerland. B]OG 116(9):1204,r2009 Wyckof MH, Aziz K, Escobedo MB, et al: Part 13: Neonatal resuscitation: 2015 American Heart Association guidelines update for cardiopulmonaIY resuscitation and emergency cardiovascular care. Circulation 132(18 Suppl 2):5543,r2015 Yao AC, Lind J: Placental transfusion. Am] Dis Child :128, 1974 Zahalka N, Sadan 0, Malinger G, et al: Comparison of transvaginal sonography with digital examination and transabdominal sonography for the determination of fetal head position in the second stage of labor. Am ] Obstet Gynecol 193:381,r2005 CLASSIFICATION OF BREECH PRESENTATIONS ....... 539 DIAGNOSIS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 540 ROUTE OF DELIVERYe. . . . . . . . . . . . . . . . . . . . . . . . . . . . 540 LABOR AND DELIVERY MANAGEMENTe. . . . . . . . . . . . . 543 PARTIAL BREECH EXTRACTION ................... 544

1	LABOR AND DELIVERY MANAGEMENTe. . . . . . . . . . . . . 543 PARTIAL BREECH EXTRACTION ................... 544 TOTAL BREECH EXTRACTIONe. . . . . . . . . . . . . . . . . . . . . 548 EXTERNAL CEPHALIC VERSION. . . . . . . . . . . . . . . . . . . . 549 extremities and buttocks. With a frank breech, lower extremities are flexed at the hips and extended at the knees, and thus the feet lie close to the head With a complete breech, both hips are lexed, and one or both knees are also lexed (Fig. 28-2). With an incomplete breech, one or both hips are extended. As a result, one or both feet or knees lie below the breech, such that a foot or knee is lowermost in the birth canal (Fig. 28-3). A footling breech is an incomplete breech with one or both feet below the breech. FIGURE 28-1 Frank breech presentation.

1	FIGURE 28-1 Frank breech presentation. The essential prerequisite or the successful peormance of breech extraction lies in the complete dilatation of the cervix and the absence of any serious mechanical obstacle. It is true that in a certain number of cases extraction through an impeecty dilated cervix is possible, but this is usualy fected ony at the cost of deep cervical tears. -J. Whitridge Williams (1903) Near term, the fetus typically has spontaneously assumed a cephalic presentation. Conversely, if the fetal buttocks or legs enter the pelvis before the head, the presentation is breech. This fetal lie is more common remote from term, as earlier in pregnancy each fetal pole has similar bulk. At term, breech presentation persists in approximately 3 to 5 percent of singleton deliveries (Cammu, 2014; Lyons, 2015; Macharey, 2017). he categories of frank, complete, and incomplete breech presentations difer in their varying relations between the lower

1	he categories of frank, complete, and incomplete breech presentations difer in their varying relations between the lower FIGURE 28-2 Complete breech presentation. FIGURE 28-3 Incomplete breech presentation. Of term breech fetuses, the neck may be extremely hyperextended in perhaps 5 percent, and the term stargazing etus is used (Cimmino, 1975). With these, fetal or uterine anomalies may be more prevalent and are sought if not previously identified (Phelan, 1983). With this hyperextension, vaginal delivery can result in injury to the cervical spinal cord. hus, if identified at term, this is an indication for cesarean delivery (Westgren, 1981). hat said, flexion itself may be implicated, s cses of spinal cord injury have been reported following uneventul cesarean delivery of such fetuses (Hernandez-Marti, 1984). With transverse lie and similar hyperextension of the fetal neck, the term iyingetus is applied.

1	Understanding the clinical settings that predispose to breech presentation can aid early recognition. Other than early gestational age, risk factors include extremes of amnionic fluid volume, multifetal gestation, hydrocephaly, anencephaly, structural uterine abnormalities, placenta previa, pelvic tumors, and prior breech delivery. One study found that following one breech delivery, the recurrence rate for a second breech presentation was 10 percent, and for a subsequent third breech it was 28 percent (Ford, 2010). Leopold maneuvers to ascertain fetal presentation are discussed in Chapter 22 (p. 424). With the irst maneuver, the hard, round fetal head occupies the fundus. he second maneuver identifies the back to be on one side of the abdomen and the small parts on the other. With the third maneuver, if not engaged, the softer breech is movable above the pelvic inlet.

1	After engagement, the fourth maneuver shows the breech to be beneath the symphysis. he accuracy of this palpation varies (Lydon-Rochelle, 1993; Nassar, 2006). hus, with suspected breech presentation-or any presentation other than cephalic-sonographic evaluation is indicated.

1	During cervical examination with a frank breech, no feet are appreciated, but the fetal ischil tuberosities, sacrum, and anus are usually palpable. Ater urther fetal descent, the external genitalia may also be distinguished. When labor is prolonged, the fetal buttocks may become markedly swollen, rendering digital diferentiation of a face and breech diicult. In some cases, the anus may be mistaken for the mouth and the ischial tuberosities for the malar eminences. With careul examination, however, the inger encounters muscular resistance with the anus, whereas the hard, less yielding jaws are felt through the mouth. The finger, upon removal from the anus, may be stained with meconium. The mouth and malar eminences form a triangular shape, whereas the ischial tuberosities and anus lie in a straight line. With a complete breech, the feet may be felt alongside the buttocks. In footling presentations, one or both feet are inferior to the buttocks.

1	The fetal sacrum and its spinous processes are palpated to establish position. As with cephalic presentations, fetal position is designated to reflect the relations of the fetal sacrum to the maternal pelvis. Positions include left sacrum anterior (LSA) , right sacrum anterior (RSA), left sacrum posterior (LSP), right sacrum posterior (RSP), and sacrum transverse (ST). Multiple factors aid determination of the best delivery route for a given mother-fetus pair. These include fetal characteristics, maternal pelvic dimensions, coexistent pregnancy complications, provider experience, patient preference, hospital capabilities, and gestational age. Compared with their term counterparts, preterm breech fetuses have distinct complications related to their small size and immaturity. For example, rates of head entrapment, birth trauma, and perinatal mortality can be greater. Accordingly, separate dis cussions of term and preterm breech fetuses are more appropriate.

1	Current obstetrical thinking regarding vaginal delivery of the term breech fetus has been tremendously inluenced by results of the Term Breech Trial (Hannah, 2000). his trial included 1041 women randomly assigned to planned cesarean and 1042 to planned vaginal delivery. In the planned vaginal delivery group, 57 percent were actually delivered vaginally. Planned cesarean delivery was associated with a lower risk of perinatal mortality compared with planned vaginal delivery-3 per 1000 versus 13 per 1000. Cesarean delivery was also associated with a lower risk of "serious" neonatal morbidity-1.4 versus 3.8 percent. Short term maternal morbidity was similar between groups. Critics of the Term Breech Trial emphasize that fewer than 10 percent of candidates underwent radiological pelvimetry. Also, most of the outcomes included in the "serious" neonatal morbidity composite did not actually portend long-term infant disability (Whyte, 2004).

1	Since that trial, however, additional data favoring cesarean delivery has come from the World Health Organization (Lumbiganon, 2010). From their evaluation of more than 100,000 deliveries from nine participating Asian countries, they reported improved perinatal outcomes for the term breech fetus with planned cesarean compared with planned vaginal delivery. Other studies have evluated neonatal outcome with cesarean delivery and also found lowered neonatal morbidity and mortality rates (Hartnack Tharin, 2011; Lyons, 2015; Rietberg, 2005; Vistad, 2015). From their metaanalysis, Berhan and Haileamlak (2016) calculate absolute risk of perinatal mortality to be 0.3 percent and of fetal birth trauma or neurological morbidity to be 0.7 percent.

1	In contrast, other studies support vaginal delivery as a suitable option at term (Hofmeyr, 2015a). he Presentation et Mode dAccouchement-PEMODA study-which translates as presentation and mode of delivery-showed no diferences in corrected neonatal mortality rates and neonatal outcomes according to delivery mode (Goinet, 2006). his French prospective observational study involved more than 8000 women with term breech singletons. Strict criteria were used to select 2526 of these for planned vaginal delivery, and 71 percent of that group were delivered vaginally. Similarly, data from the Lille Breech Study Group in France showed no excessive morbidity in term breech singletons delivered vaginally provided strict fetal biometric and maternal pelvimetry parameters were applied (Michel, 2011). Other smaller studies support these findings as long as guidelines are part of the selection process (Alarab, 2004; Giuliani, 2002; Toivonen, 2012).

1	Long-term evidence in support of vaginal breech delivery comes from Eide and associates (2005). These investigators analyzed intelligence testing scores of more than 8000 men delivered breech and ing vaginal or cesarean delivery. Also, a 2-year follow up from the velopmental delay between delivery groups (Whyte, 2004). Despite evidence on both sides of the debate, at least in the United States, rates of planned vaginal delivery attempts con tinue to decline. And as predicted, the number of skilled pro viders able to safely select and vaginally deliver breech fetuses continues to dwindle (Chinnock, 2007). Moreover, obvious ies diicult. In response, some institutions have developed birth simulators to improve resident competence in vaginal breech delivery (Deering, 2006; Maslovitz, 2007).

1	In contrast to the term breech fetus, there are no randomized trials regarding delivery of the preterm breech fetus. Moreover, study comparisons are oten made diicult by lumping, splitting, or overlapping of preterm gestational age groups. All that said, it would appear that for the preterm breech fetus, planned cesar ean delivery confers a survival advantage compared with planned vaginal delivery. Reddy and associates (2012) reported data from deliveries between 24 and 32 weeks' gestation. For breech fetuses within these gestational ages, attempting vaginal delivery yielded a low success rate, and those completed were associated with higher neonatal mortality rates compared with planned cesar ean delivery. Other investigations have reported similar findings (Bergenhenegouwen, 2014; Demirci, 2012; Muhuri, 2006).

1	For preterm fetuses in younger subgroups-23 to 28 weeksthe data are more conflicting, and some studies describe no improved survival rate with planned cesarean delivery (Bergenhenegouwen, 2015; Kayem, 2015; Thomas, 2016). Forperiviable etuses, deined by them as 20 to 256/7 weeks, a consensus workshop of perinatal organizations concluded that "available data do not consistently support routine cesarean delivery to improve perinatal mortality or neurological outcomes for early preterm infants" (Raju, 2014). A subsequent joint statement by the American College of Obstetricians and Gynecologists and the Society for Maternal-Fetal Medicine (2017) suggested consideration for cesarean delivery for periviable fetuses beginning at 23°/7 weeks, with a recommendation for cesarean delivery at 25°1 weeks.

1	For more mature preterm breech fetuses, that is, between 32 and 37 weeks, again there are sparse data to guide delivery route selection. Bergenhenegouwen and coworkers (2015) studied more than 6800 breech deliveries in a subgroup between 32 and 37 weeks. With planned cesarean delivery, they found similar perinatal mortality rates but less composite mortality and severe morbidity. It appears in this subgroup that fetal weight rather than gestational age is likely more important. he Maternl-Fetal Nfedicine Committee of the Society of Obstetricians and Gynaecologists of Canada (SOGC) states that vaginal breech delivery is reasonable when the estimated fetal weight is >2500 g (Kotaska, 2009). There are especial concerns for delivery of the second noncephalic-presenting twin fetus that are discussed in Chapter 45 (p. 888).

1	In the United States, all these findings shape practice, and cesarean delivery is almost uniformly favored for the preterm breech fetus for which resuscitation is planned.

1	Increased rates of maternal and perinatal morbidity can be anticipated with breech presentations. For the mother, with either cesarean or vaginal delivery, genital tract laceration can be problematic. With cesarean delivery, added stretching of the lower uterine segment by forceps or by a poorly molded fetal head can extend hysterotomy incisions. With vaginal delivery, especially with a thinned lower uterine segment, delivery of the atercoming head through an incompletely dilated cervix or application of forceps may cause vaginal wall or cervical lacerations, and even uterine rupture. Manipulations may also extend an episiotomy, create deep perineal tears, and increase infection risks. Anesthesia suicient to induce appreciable uterine relaxation during vaginal delivery may cause uterine atony and in turn postpartum hemorrhage. Maternal death is rare, but rates appear higher in those with planned cesarean delivery for breech presentation-a case fatality rate of 0.47 maternal deaths per

1	and in turn postpartum hemorrhage. Maternal death is rare, but rates appear higher in those with planned cesarean delivery for breech presentation-a case fatality rate of 0.47 maternal deaths per 1000 births (Schutte, 2007). Last, the risks associated with vaginal breech delivery are balanced against general cesarean delivery risks described in Chapter 30 (p. 568). Long-term, cesarean risks include those associated with repeated hysterotomy or with vaginal birth ater cesarean-VBAC-further described in Chapter 31 (p. 593).

1	For the fetus, prematurity and its complications are frequently comorbid with breech presentation. Rates of congenital anomalies are also greater (Cammu, 2014; Mostello, 2014). Compared with cephalic presentation, umbilical cord prolapse is more frequent with breech fetuses (Behbehani, 2016; Obeidat, 2010). Birth trauma can include fractures of the humerus, clavicle, and femur (Canpolat, 2010; Matsubara, 2008). In some cases, traction may separate scapular, humeral, or femoral epiphyses (Lamrani, 2011). Trauma is more common with vaginal births, but fetal trauma is also seen with cesarean deliveries.

1	Rare traumatic injuries may involve soft tissues. Brachial plexus injury and paralysis is one example (Foad, 2008). The spinal cord may be injured or even severed, or vertebrae fractured, especially if great force is employed (Vialle, 2007). Hematomas of the sternocleidomastoid muscles occasionally develop after delivery but usually disappear spontaneously. Last, genital injury may follow breech delivery (Saroha, 2015). Some perinatal outcomes may be inherent to the breech position rather than delivery. For example, development of hip dysplasia is more common in breech compared with cephalic presentation and is unafected by delivery mode (de Hundt, 2012; Fox, 2010; Ortiz-Neira, 2012).

1	In many fetuses-especially those that are preterm-the breech is smaller than the atercoming head. Moreover, unlike cephalic presentations, the head of a breech-presenting fetus does not undergo appreciable molding during labor. Thus, if vaginal delivery is considered, fetal size, type of breech, and degree of neck lexion or extension are evaluated. In addition, pelvic dimensions are assessed to avoid head entrapment from cephalopelvic disproportion. Sonography and fetal pelvimetry are options. Sonographic fetal evaluation will have been performed in most cases as part of prenatal care. If not, gross fetal abnormalities, such as hydrocephaly or anencephaly, can be rapidly ascertained with sonography. This will identiy many fetuses not suitable for vaginal delivery. It will also help to ensure that a cesarean delivery is not performed under emergency conditions for an anomalous fetus with no chance of survival.

1	Head flexion can usually also be determined sonographically, and for vaginal delivery, the fetal head should not be extended (Fontenot, 1997; Rojansky, 1994). If imaging is uncertain, then simple two-view radiography of the maternal abdomen is useful to define fetal head inclination. Sonographic identification of a nuchal arm may warrant cesarean delivery to avoid neonatal harm (Sherer, 1989). he accuracy of fetal weight estimation by sonography is not altered by breech presentation (McNamara, 2012). Although variable, many protocols use fetal weights >2500 g and <3800 to 4000 g or evidence of growth restriction as exclusion criteria for planned vaginal delivery (Azria, 2012; Kotaska, 2009). Similarly, a biparietal diameter (BPD) >90 to 100 mm is often considered exclusionary (Giuliani, 2002; Roman, 2008).

1	Pelvimetry assesses the maternal bony pelvis before vaginal delivery, and one-view computed tomography (CT) , magnetic resonance (MR) imaging, or plain film radiography is suitable. Comparative data among these modalities for pelvimetry are lacking, but CT is favored due to its accuracy, low radiation dose, and widespread availability (homas, 1998). At Parkland Hospital, we use CT pelvimetry when possible to assess the critical dimensions of the pelvis (Chap. 2, p. 30). Although variable, some suggest specific measurements to permit a planned vaginal delivery: inlet anteroposterior diameter : 10.5 cm; inlet transverse diameter : 12.0 cm; and midpelvic interspinous distance : 10.0 cm (Azria, 2012; Vendittelli, 2006). Some have recommended maternalfetal biometry correlation. Appropriate values include: the sum of the inlet obstetrical conjugate minus the fetal BPD is : 15 mm; the inlet transverse diameter minus the BPD is :25 mm; and the midpelvis interspinous distance minus the BPD is

1	include: the sum of the inlet obstetrical conjugate minus the fetal BPD is : 15 mm; the inlet transverse diameter minus the BPD is :25 mm; and the midpelvis interspinous distance minus the BPD is :0 mm (Michel, 2011). With MR imaging, Hofmann and colleagues (2016) found vaginal delivery success rates of 79 percent in selected candidates if the interspinous distance exceeded 11 cm.

1	Currently, the American College of Obstetricians and Gynecologists (2016b) recommends that "the decision regarding the mode of delivery should depend on the experience of the health-care provider" and that "planned vaginal delivery of a term singleton breech fetus may be reasonable under hospitalspecific protocol guidelines." These guidelines have been echoed by other obstetrical organizations (Kotaska, 2009; Royal College of Obstetricians and Gynaecologists, 2006). Risks versus beneits are weighed and discussed with the patient. If possible, this is preferably done before admission. A diligent search is made for other complications, actual or anticipated, that might warrant cesarean delivery. Common circumstances are listed in Table

1	For a favorable outcome with any breech delivery, at the very minimum, the birth canal must be suiciently large to allow passage of the fetus without trauma. he cervix must be fully dilated, and if not, then a cesarean delivery nearly always is the more appropriate method of delivery if suspected fetal compromise develops. TABLE 28-1 . Factors Favoring Cesarean Delivery of the Breech Fetus Lack of operator experience Patient request for cesarean delivery Large fetus: >3800 to 4000 g Apparently healthy and viable fetus either with active labor or with indicated delivery Severe fetal-growth restriction Fetal anomaly incompatible with vaginal delivery Prior perinatal death or neonatal birth trauma Incomplete or footling breech presentation Hyperextended head Pelvic contraction or unfavorable pelvic shape determined clinically or with pelvimetry Prior cesarean delivery

1	The conduct of both labor and delivery difer between cephalic and breech presentations. First, breech labor in general proceeds more slowly, but steady cervical progress is a positive indicator of adequate pelvic proportions (Lennox, 1998). Vaginal breech delivery is accomplished by one of three methods. With spontaneous breech delivery, the fetus is expelled entirely without any traction or manipulation other than support of the newborn. With partial breech extraction, the fetus is delivered spontaneously as far as the umbilicus, but the remainder of the body is delivered by provider traction and assisted maneuvers, with or without maternal expulsive eforts. With total breech extraction, the entire fetal body is extracted by the provider.

1	As with many other aspects of breech position, induction or augmentation of labor is controversial. Here again, data are limited and mostly retrospective. With labor induction, Burgos and coworkers (2017) reported equivalent vaginal delivery rates compared with spontaneous labor. With induction, however, they reported higher rates of neonatal intensive care unit admission. But, others have found similar perinatal outcome and cesarean delivery rates Qarniat, 2017; Marzouk, 2011). Finally, others described greater cesarean delivery rates with induction but similar neonatal outcomes (Macharey, 2016).

1	In many studies, successful vaginal delivery is associated with orderly labor progression. hus, some protocols avoid augmentation for the breech-presenting fetus, whereas others recommend it only for hypotonic contractions (Alarab, 2004; Kotaska, 2009). In women with a viable fetus, at Parkland Hospital, we attempt amniotomy induction but prefer cesarean delivery instead of pharmacological labor induction or augmentation.

1	On arrival to the labor unit, surveillance offetal heart rate and uterine contractions begins, and immediate recruitment of necessary staf includes: (1) a provider skilled in the art of breech extraction, an associate to assist with the delivery, (3) anesthesia personnel who can ensure adequate analgesia or anesthesia when needed, and an individual trained in newborn resuscitation. For the mother, intravenous access is obtained. his allows, if needed, emergency induction of anesthesia or maternal resuscitation following hemorrhage from lacerations or from uterine atony.

1	At admission, the status of the membranes and progression of labor are assessed. Knowledge regarding cervical dilatation, cervical efacement, and presenting part station is essential for preparation. If labor is too far advanced, pelvimetry may be unsafe if fetal expulsion in the radiology department is a pos sibility. This alone, however, should not force the decision for cesarean delivery. As mentioned, stepwise labor progression itself is a good indicator of pelvic adequacy (Biswas, 1993). Sonographic assessment, described earlier, is completed. Ulti mately, the choice of abdominal or vaginal delivery is based on factors discussed earlier and listed in Table 28-1.

1	Sonographic assessment, described earlier, is completed. Ulti mately, the choice of abdominal or vaginal delivery is based on factors discussed earlier and listed in Table 28-1. During labor, one-on-one nursing is ideal because of cord prolapse risks, and physicians must be readily available for such emergencies. Guidelines for monitoring the high-risk fetus are applied (Chap. 24, p. 478). For first-stage labor, while most clini cians prefer continuous electronic monitoring, the fetal heart rate is recorded at a minimum of every 15 minutes. A scalp electrode can be safely aixed to the buttock, but genitalia are avoided. If a nonreassuring fetal heart rate pattern develops, then a decision must be made regarding the necessity of cesarean delivery.

1	When membranes rupture, either spontaneously or artificially, the cord prolapse risk is appreciable and is increased when the fetus is small or when the breech is not frank. Therefore, vaginal examination is performed immediately following rupture, and special attention is directed to the fetal heart rate for the irst 5 to 10 minutes thereafter. For women in labor with a breech presentation, continuous epidural analgesia is advocated by some. This may increase the need for labor augmentation and prolong second-stage labor (Chadha, 1992; Confino, 1985). hese potential disadvantages are weighed against the advantages of better pain relief and increased pelvic relaxation should extensive manipulation be required. Analgesia must be suicient for episiotomy, for breech extraction, and for Piper forceps application. Nitrous oxide plus oxygen inhalation can provide further relief from pain. If general anesthesia is required, it must be induced quickly.

1	Similar to vertex delivery, spontaneous expulsion of a breech fetus entails sequential cardinal movements. First, engagement and descent of the breech usually take place with the bitrochanteric diameter in one of the oblique pelvic diameters. The anterior hip usually descends more rapidly than the posterior hip, and when the resistance of the pelvic Boor is met, internal rotation of 45 degrees usually follows, bringing the anterior hip toward the pubic arch and allowing the bitrochanteric diameter to occupy the anteroposterior diameter of the pelvic outlet. If the posterior extremity is prolapsed, however, it, rather than the anterior hip, rotates to the symphysis pubis.

1	After rotation, descent continues until the perineum is distended by the advancing breech, and the anterior hip appears at the vulva. By lateral lexion of the fetal body, the posterior hip then is forced over the perineum, which retracts over the fetal buttocks, thus allowing the fetus to straighten out when the anterior hip is born (Fig. 28-4). he legs and feet follow the breech and may be born spontaneously or require aid. FIGURE 28-4 The hips of the frank breech are delivering over the perineum. The anterior hip usually delivers first.

1	FIGURE 28-4 The hips of the frank breech are delivering over the perineum. The anterior hip usually delivers first. After the birth of the breech, there is slight external rotation, with the back turning anteriorly as the shoulders are brought into relation with one of the oblique diameters of the pelvis. The shoulders then descend rapidly and undergo internal rotation, with the bisacromial diameter occupying the anteroposterior plane. Immediately following the shoulders, the head, which is normally sharply flexed on the thorax, enters the pelvis in one of the oblique diameters and then rotates to bring the posterior portion of the neck under the symphysis pubis. The head is then born in flexion.

1	The breech may engage in the transverse diameter of the pelvis, with the sacrum directed anteriorly or posteriorly. he mechanism oflabor in the transverse position difers only in that internal rotation is through an arc of 90 rather than 45 degrees. Infrequently, rotation renders the back of the fetus to lie posteriorly instead of anteriorly. Such rotation is prevented if possible. Although the head can be delivered by allowing the chin and face to pass beneath the symphysis, the slightest traction on the body may cause extension of the head, which increases the diameter of the head that must pass through the pelvis. With breech delivery, successively larger and less compressible parts are born. Thus, spontaneous expulsion is the exception, and vaginal delivery typically requires skilled provider participation for the fetus to navigate the birth canal. Noteworthy clinical pearls are provided by Yeomans (2017) in the third edition of Cunningham and Gilstrap s Operative Obstetrics.

1	First, with all breech deliveries, unless the perineum is considerably lax, an episiotomy is made and is an important adjunct to delivery. As discussed in Chapter 27 (p. 529), mediolateral episiotomy may be preferred for its lower associated risk of anal FIGURE 28-5 To deliver the left leg, two fingers of the provider's left hand are placed beneath and parallel to the femur. The thigh is then slightly abducted and pressure from the fingertips in the popliteal fossa should induce knee flexion and bring the foot within reach. The foot is then grasped to gently deliver the entire leg outside the vagina. A similar procedure is followed on the right. (Figures 28-5 though 28-8: Reproduced with permission from Yeomans ER: Vaginal breech delivery. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New (ork, McGraw-Hili Education, 201o7.)

1	FIGURE 28-6 To deliver the body, thumbs are placed over the sacrum, and each index finger wraps over the top of the corresponding fetal iliac crest. Gentle downward traction is applied until the scapulas are clearly visible. (Reproduced with permission from Yeomans ER: Vaginal breech delivery. In Yeomans ER, Hofman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 2017.)

1	FIGURE 28-7 A. After delivery of the first arm, 180-degree rotation of the fetal body brings the sacrum to a right sacrum transverse (RST) position. B. Fingers of the provider's hand extended over the right shoulder and parallel to the humerus. These sweep the arm downward across the chest and out. (Reproduced with permission from Yeomans ER: Vaginal breech delivery. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201o7.) sphincter lacerations. Ideally, the breech is allowed to deliver spontaneously to the umbilicus. Delivery of the breech draws the umbilicus and attached cord into the pelvis. Therefore, once the breech has passed beyond the vaginal introitus, the abdomen, thorax, arms, and head must be delivered promptly either spontaneously or assisted.

1	The posterior hip will deliver, usually from the 6 o'clock position, and oten with suicient pressure to evoke passage of thick meconium (see Fig. 28-4). he anterior hip then delivers, followed by external rotation to a sacrum anterior position. The mother is encouraged to continue to push as the fetus descends until the legs are accessible. The legs are sequentially delivered by splinting the femur with the operator's fingers positioned parallel to the long axis of the femur, and by exerting pressure upward and laterally to sweep each leg away from the midline (Fig. 28-5). Following delivery of the legs, the fetal bony pelvis is grasped with both hands. The fingers should rest on the anterior superior iliac crests and the thumbs on the sacrum. his minimizes the chance of fetal abdominal soft-tissue injury (Fig. 28-6). Maternal expulsive eforts are again used in conjunction with downward traction to afect delivery.

1	A cardinal rule in successul breech extraction is to employ steady, gentle, downward traction until the lower halves of the scapulas are delivered, making no attempt at delivery of the shoulders and arms until one axilla becomes visible. It makes little difference which shoulder is delivered irst, and two methods are suitable for their delivery. In the first method, with the scapulas visible, the trunk is rotated either clockwise or counterclocwise to bring the anterior shoulder and arm into view (Fig. 28-7). During delivery of the arm, fingers and hand are aligned parallel to the humerus and act to splint and prevent humeral fracture. he body of the fetus is then rotated 180 degrees in the reverse direction to bring the other shoulder and arm into position for delivery.

1	he second method is employed if trunk rotation is unsuccessful. With this maneuver, the posterior shoulder is delivered first. For this, the feet are grasped in one hand and drawn upward over the inner thigh of the mother (Fig. 28-8). The

1	FIGURE 28-8 Infrequently, the posterior arm must be delivered first. For this, the lower half of the fetal body is raised up and over the maternal groin. The provider's fingers are inserted under the posterior shoulder and aligned with the humerus. (Reproduced with permission from Yeomans ER: Vaginal breech delivery. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 2017.) hand enters over the shoulder, fingers are aligned parallel to the long axis of the humerus, and the fetal arm is swept upward. The posterior shoulder slides out over the perineal margin and is usually followed by the arm and hand. Then, by depressing the body of the fetus, the anterior shoulder emerges beneath the pubic arch, and the arm and hand usually follow spontaneously. After both shoulders are delivered, the back of the fetus tends to rotate spontaneously to the symphysis. Delivery of the head may then be

1	arch, and the arm and hand usually follow spontaneously. After both shoulders are delivered, the back of the fetus tends to rotate spontaneously to the symphysis. Delivery of the head may then be accomplished.

1	During delivery, one or both fetal arms occasionally may lie across the back of the neck and become impacted at the pelvic inlet. With such a nuchal arm, delivery is more diicult and can be aided by rotating the fetus through a half circle in such a direction that the friction exerted by the birth canal will draw the elbow toward the face (Fig. 28-9). With a right nuchal arm, the body should be rotated counterclockwise, which rotates the fetal back toward the maternal right. With a left nuchal arm, the rotation is clockwise. If rotation fails to free the nuchal arm, it may be necessary to push the fetus upward to a roomier part of the pelvis. If the rotation is still unsuccessul, the nuchal arm oten is extracted by hooking a inger(s) over it and forcing the arm over the shoulder, and down the ventral surface for delivery of the arm. In this event, fracture of the humerus or clavicle is common. Delivery of the Aftercoming Head

1	Delivery of the Aftercoming Head Mauriceau Maneuver. The fetal head is normally extracted with forceps or by one of several maneuvers. With any of these techniques, hyperextension of the fetal neck is avoided. With the Mauriceau maneuver, the index and middle inger of one hand are applied over the maxilla, to flex the head, while FIGURE 28-10 A. Delivery of the aftercoming head using the Mauriceau maneuver. Note that as the fetal head is being delivered, flexion of the head is maintained by suprapubic pressure provided by an assistant. B. Pressure on the maxilla is applied Simultaneously by the operator as upward and outward traction is exerted.

1	FIGURE 28-9 Reduction of a right nuchal arm is accomplished by rotating the fetal body 180 degrees counterclockwise, which directs the fetal back to the maternal right. Friction exerted by the birth canal will draw the elbow toward the face. (Reproduced with permission from Yeomans ER: Vaginal breech delivery. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201•7.) the fetal body rests on the palm of the same hand and forearm (Fig. 28-10). Fetal legs straddle the forearm. Two ingers of the other hand then are hooked over the fetal neck and grasp the shoulders. Downward traction is concurrently applied until the suboccipital region appears under the symphysis. Gentle suprapubic pressure simultaneously applied by an assistant helps keep the head flexed. The body then is slightly elevated toward the maternal abdomen, and the mouth, nose, brow, and eventually the occiput emerge successively

1	applied by an assistant helps keep the head flexed. The body then is slightly elevated toward the maternal abdomen, and the mouth, nose, brow, and eventually the occiput emerge successively over the perineum. With this maneuver, the provider uses both hands simultaneously to exert continuous downward gentle traction while balancing forces between the fetal neck and maxilla to avoid neck hyperextension.

1	Forceps. Specialized forceps can be used to deliver the aftercoming head. Piper forceps, shown in Figure 28-11, or Laufe-Piper forceps may be applied electively or when the Mauriceau maneuver cannot be accomplished easily. The blades FIGURE 28-11 Piper forceps for delivery of the aftercoming head. A. The fetal body is held elevated using a warm towel and the left blade of forceps is applied to the aftercoming head. B.The right blade is applied with the body still elevated. C. Forceps delivery of the aftercom ing head. Note the direction of movement shown by the arrow. of the forceps are not applied to the aftercoming head until it has been brought into the pelvis by gentle traction, combined with suprapubic pressure, and is engaged. Suspension of the body of the fetus in a towel efectively holds the fetus up and helps keep the arms and cord out of the way as the forceps blades are applied.

1	Because the forceps blades are directed upward from the level of the perineum, some choose to apply them from a one-knee kneeling position. Piper forceps have a downward arch in the shank to accommodate the fetal body and lack a pelvic curve. This shape permits direct application of the cephalic curve of the blade along the length of the maternal vagina and fetal parietal bone. The blade to be placed on the maternal left is held in the provider's left hand. The right hand slides between the fetal head and left maternal vaginal sidewall to guide the blade inward and around the parietal bone. he opposite blade mirrors this application.

1	Once in place, the blades are articulated, and the fetal body rests across the shanks. The head is delivered by pulling gently outward and slightly raising the handle simultaneously. This rolls the face over the perineum, while the occiput remains beneath the symphysis until after the brow delivers. Ideally, the head and body move in unison to minimize neck hyperextension. Modified Prague Maneuver. Rarely, the back of the fetus fails to rotate to the symphysis. The fetus still may be delivered using the modiied Prague maneuver. With this, two fingers of one hand grasp the shoulders of the back-down fetus from below while the other hand draws the feet up and over the maternal abdomen (Fig. 28-12). FIGURE 28-12 Delivery of the aftercoming head using the modified Prague maneuver necessitated by failure of the fetal trunk to rotate anteriorly.

1	FIGURE 28-12 Delivery of the aftercoming head using the modified Prague maneuver necessitated by failure of the fetal trunk to rotate anteriorly. FIGURE 28-13 Duhrssen incision being cut at 2 o'clock, which is followed by a second incision at 10 o'clock. Infrequently, an additional incision is required at 6 o'clock. The incisions are so placed as to minimize bleeding from the laterally located cervical branches of the uterine artery. After delivery, the incisions are repaired as described in Chapter 41 (p.o763).

1	Head Entrapment. his emergency reflects either an incompletely dilated cervix or cephalopelvic disproportion. First, especially with a small preterm fetus, an incompletely dilated cervix can constrict around the neck and impede delivery of the aftercoming head. At this point, signiicant cord compression must be assumed, and time management is essential. With gentle traction on the fetal body, the cervix, at times, may be manually slipped over the occiput. If unsuccessful, then Diihrssen incisions may be necessary (Fig. 28-13). General anesthesia with halogenated agents or intravenous nitroglycerin is another option to aid lower uterine segment relaxation. As an extreme measure, replacement of the fetus higher into the vagina and uterus, followed by cesarean delivery, can rescue an entrapped breech fetus. This Zavaneli maneuver is classically performed to relieve intractable shoulder dystocia (Sandberg, 1988). However, case reports also have described its use for an entrapped

1	entrapped breech fetus. This Zavaneli maneuver is classically performed to relieve intractable shoulder dystocia (Sandberg, 1988). However, case reports also have described its use for an entrapped aftercoming head (Sandberg, 1999; Steyn, 1994).

1	In cases with cephalopelvic disproportion and arrest of aftercoming head, the Zavanelli maneuver or symphysiotomy are options (Sunday-Adeoye, 2004; Wery, 2013). Using local analgesia, symphysiotomy surgically divides the intervening symphyseal cartilage and much of its ligamentous support to widen the symphysis pubis up to 2.5 cm (Basak, 2011). Lack of provider training and potentially serious maternal pelvic or urinary tract injury explain its rare use in the United States. That said, if cesarean delivery is not possible, symphysiotomy may be lifesaving for both mother and baby (Hofmeyr, 2012).

1	At times, total extraction of a complete or incomplete breech may be required. A hand is introduced through the vagina, and both fetal feet are grasped. he ankles are held with the middle finger lying between them. With gentle traction, the feet are brought through the introitus (Fig. 28-14). As the legs begin to emerge through the vulva, downward gentle traction is continued. As the legs emerge, successively higher portions are grasped, first the calves and then the thighs. When the breech appears at the vaginal outlet, gentle traction is applied until the hips are delivered. The thumbs are then placed over the sacrum and the fingers over the iliac crests. Breech extraction is then completed, as described for partial breech extraction (p. 544). FIGURE 28-14 Complete breech extraction begins with traction on the feet and ankles.

1	FIGURE 28-14 Complete breech extraction begins with traction on the feet and ankles. If only one foot can be grasped, it can be brought down into the vagina and held with the appropriate hand, right hand for right foot and left hand for left foot (Yeomans, 2017). With the irst foot secure, the opposite hand is introduced, passed upward along the leg, and guided to locate the other foot. If the remaining hip is extended, the second foot is usually easily grasped and brought down. If the hip is lexed and knee extended, a finger is hooked into that groin, and traction will bring the lower half of the fetus down until the leg can be reached. For cesarean delivery, these total breech extraction maneuvers can be used to deliver a complete, incomplete, or footling breech through the hysterotomy incision.

1	During complete extraction of a frank breech, moderate traction is exerted by a finger in each groin and aided by a generous episiotomy. Once the breech is pulled through the introitus, the steps described for partial breech extraction are then completed (p. 544). These maneuvers are also used during cesarean delivery of a frank breech through a hysterotomy incision.

1	Rarely during vaginal delivery, a frank breech will require decomposition inside the uterine cavity. Attributed to Pinard (1889), this procedure converts a frank breech into a footling breech. It is accomplished more readily if the membranes have ruptured only recently. It becomes extremely diicult if amnionic luid is scant and the uterus is tightly contracted around the fetus. Pharmacological relaxation by general anesthesia or intravenous magnesium sulfate, nitroglycerin, or a betamimetic agent may be required. To begin, two ingers are carried up along one leg to externally rotate the hip by pressing on the medial side of the thigh parallel to the femur. Simultaneously, pressure in the popliteal fossa should prompt spontaneous knee flexion, which brings the corresponding foot into contact with the back of the provider's hand. he fetal foot then may be grasped and brought down.

1	With version, fetal presentation is altered by physically substituting one pole of a longitudinal presentation for the other, or converting an oblique or transverse lie into a longitudinal presentation. Manipulations performed through the abdominal wall that yield a cephalic presentation are termed external cephalic version. Manipulations accomplished inside the uterine cavity that yield a breech presentation are designated intenal podalic version. This latter procedure is reserved for delivery of a second twin and described in Chapter 45 (p. 890). External cephalic version (ECy) reduces the rate of noncephalic presentation at birth (Hofmeyr, 2015b). For breech fetuses near term, the American College of Obstetricians and Gynecologists (2016a,b) recommends that version be ofered and attempted whenever possible. Its success rate averages about 60 percent (de Hundt, 2014). For women with a transverse lie, the overall success rate is significantly higher.

1	In general, ECV is attempted before labor in a woman who has reached 37 weeks' gestation. Before this time, breech pre sentation still has a high likelihood of correcting spontaneously. And, if ECV is performed too early, time may allow a rever sion back to breech (Bogner, 2012). Last, if attempts at version cause a need for immediate delivery, complications of iatro genic late-preterm delivery generally are not severe. Absolute contraindications to external version are few. It is contraindicated if vaginal delivery is not an option, such as with placenta previa. Another is multifetal gestation. Relative contraindications are early labor, oligohydramnios or rupture of membranes, known nuchal cord, structural uterine abnor malities, fetal-growth restriction, and prior abruption or its risks (Rosman, 2013). While many consider a prior cesarean delivery a contraindication, a few small studies found ECV was not asso ciated with uterine rupture (Burgos, 2014; Keepanasseril, 2017;

1	Weill, 2017). At Parkland Hospital, we do not attempt version in these women. More data from clinical studies are needed. Several factors can improve the chances of a successful attempt. These include multiparity, unengaged presenting part, nonanterior placenta, nonobese patient, and abundant amnionic fluid (Kok, 2009, 2011; Velzel, 2015). To augment the last parameter, Burgos and coworkers (2014) administered a preprocedurali2-L intravenous luid bolus. While this improved amnionic luid volume, it did not increase version success rates.

1	Patient counseling includes a discussion regarding small but real risks for placental abruption, preterm labor, and fetal compromise. Rarely, uterine rupture, fetomaternal hemorrhage, alloimmunization, amnionic luid embolism, and even death may also complicate attempts at external version. hat said, fetal deaths are rare, serious complication rates are typically very low, and emergent cesarean rates are 0.5 percent or less (Grootscholten, 2008; Rodgers, 2017). And even after successful EC, several reports suggest that the cesarean delivery rate does not completely revert to the baseline for vertex presentations. Specifically, dystocia, malpresentation, and nonreassuring fetal heart patterns may be more common in these fetuses completing successful version (Chan, 2004; de Hundt, 2014; Vezina, 2004).

1	ECV should be carried out in an area that has ready access to a facility equipped to perform emergency cesarean delivery (American College of Obstetricians and Gynecologists, 20 16a). Because of the risk for surgical intervention, intravenous access is obtained, and patients abstain from eating for 6 or more hours. Sonographic examination is performed to conirm nonvertex presentation, document amnionic fluid volume adequacy, exclude obvious fetal anomalies if not done previously, and identiy placental location and fetal spine orientation. Preprocedural external monitoring is performed to assess fetal heart rate reactivity. Anti-D immune globulin is given to Rh-D negative women. Tocolysis and regional analgesia may be elected, and rationale for these is provided in subsequent sections.

1	he woman is placed in left lateral tilt to aid utero placental perfusion, and Trendelenburg positioning helps during elevation of the breech. During the procedure, we prefer to monitor fetal heart motion sonographically. An abundant abdominal coating of ultrasound gel permits this and also minimizes painful skin friction (Vallikkannu, 2014).

1	A forward roll of the fetus usually is attempted first. One or two providers may participate, and one hand grasps the head. he fetal buttocks are then elevated from the maternal pelvis and displaced laterally (Fig. 28-15). The buttocks are then gently guided toward the fundus, while the head is simultaneously directed toward the pelvis. If the forward roll is unsuccessful, a backward flip is attempted. ECV attempts are discontinued for excessive discomfort, persistently abnormal fetal heart rate, or after multiple failed attempts. Failure is not always absolute. Ben-Meir and colleagues (2007) reported a spontaneous version rate of 7 percent among 226 failed versions-2 percent among nulliparas and 13 percent among multiparas.

1	If ECV is successful, a nons tress test is repeated until a normal test result is obtained. If version is completed before 39 weeks' gestation, then awaiting spontaneous labor and fetal maturity is preferred. In some studies, immediate labor induction is linked to higher cesarean delivery rates (Burgos, 2015; Kuppens, 2013). FIGURE 28-15 External cephalic version. With an attempted forward roll, clockwise pressure is exerted against the fetal poles.

1	To relax the uterus prior to an ECV attempt, existing evidence supports the use of tocolysis (American College of Obstetricians and Gynecologists, 2016a). Most data support the use of the beta-mimetics terbutaline and ritodrine (Cluver, 2015). In one such trial, Fernandez and coworkers (1996) reported that the success rate with subcutaneous terbutaline-52 percentwas significantly higher than without-27 percent. Our policy at Parkland Hospital is to administer 250 I1g of terbutaline subcutaneously to most women before attempted ECV. When maternal tachycardia-a known side efect of terbutaline-is noted, the attempt is begun. Data are limited and, in some cases nonsupportive, for alternate agents that include calciumchannel blockers, such as nifedipine; nitric oxide donors, such as nitroglycerin; the oxytocin-receptor antagonist atosiban; and another betamimetic salbutamol (Burgos, 2010; Hilton, 2009; Kok, 2008; Vani, 2009; Velzel, 2017; Wilcox, 2011).

1	Epidural analgesia coupled with tocolysis has been reported to increase version success rates compared with tocolysis alone (Goetzinger, 2011; Magro-Malosso, 2016). Moreover, rates of complications that include fetal heart rate aberrations, emergency cesarean delivery, or placental abruption were not greater with regional analgesia. Of randomized trials, spinal and epidural have both shown success (Khaw, 2015; Weiniger, 2010). Currently, the superior technique and best drugs to administer are unclear. In contrast, from limited data, intravenous sedation does not appear to improve success rates (Burgos, 2016; Khaw, 2015).

1	This is a traditional Chinese medicine technique that burns a cigarette-shaped stick of ground Artemisia vugaris-which is also known as mugwort or in Japanese as moxa. At the BL 67 acupuncture point, the stick is directly placed against the skin or indirectly heats an acupuncture needle at the site to increase fetal movement and promote spontaneous breech version (Ewies, 2002). It is performed usually between 33 and 36 weeks' gestation to permit a trial of ECV if not successful. Results from randomized controlled studies are conflicting (Bue, 2016; Coulon, 2014; Coyle, 2012; Sananes, 2016; Vas, 2013). larab M, Regan C, O'Connell MP, et al: Singleton vaginal breech delivery at term: still a safe option. Obstet Gynecol 103:407, 2004 American College of Obstetricians and Gynecologists: External cephalic version. Practice Bulletin No. 161, February 2016a

1	American College of Obstetricians and Gynecologists: Mode of term in singleton breech delivery. Committee Opinion No. 340, July 2006, Reairmed 2016b American College of Obstetricians and Gynecologists, Society for MaternalFetal Medicine: Periviable birth. Obstetric Care Consensus No.r6, October 2017 Azria E, Le Meaux JP, Khoshnood B, et al: Factors associated with adverse perinatal outcomes for term breech fetuses with planned vaginal delivery. Am J Obstet Gynecol 207(4):285.e1, 2012 Basak S, Kanungo S, Majhi C: Symphysiotomy: is it obsolete? J Obstet Gynaecol Res 37(7):770,r201r1 Behbehani S, Patenaude V, Abenhaim HA: Maternal risk factors and outcomes of umbilical cord prolapse: a population-based study. J Obstet Gynaecol Can 38(1):23, 2016 Ben-Meir A, Elram T, Tsafrir A, et al: The incidence of spontaneous version after failed external cephalic version. Am J Obstet Gynecol 196(2): 157,200

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1	Maslovitz S, Barkai G, Lessing JB, et al: Recurrent obstetric management mistakes identiied by simulation. Obstet Gynecol 109(6): 1295, 2007 Matsubara S, Izumi A, Nagai T, et al: Femur fracture during abdominal breech delivery. Arch Gynecol Obstet 278(2):195,r2008 McNamaraJM, Odibo AO, Macones GA, et al: The efect of breech presentation on the accuracy of estimated fetal weight. Am J PerinatoIr29(5):353, 2012 Michel S, Drain A, Closset E, et al: Evaluation of a decision protocol for type of delivery of infants in breech presentation at term. Eur J Obstet Gynecol Reprod Bioi 158(2):194, 2011 Mostello 0, Chang JJ, Bai F, et al: Breech presentation at delivery: a marker for congenital anomaly? J Perinatol 34(1): 11, 2014 Muhuri PK, Macdorman MF, Menacker F: Method of delivery and neonatal mortaliry among very low birth weight infants in the United States. Matern Child Health J 10:47,r2006

1	Muhuri PK, Macdorman MF, Menacker F: Method of delivery and neonatal mortaliry among very low birth weight infants in the United States. Matern Child Health J 10:47,r2006 Nassar N, Roberts CL, Cameron CA, et al: Diagnostic accuracy of clinical examination for detection of non-cephalic presentation in late pregnancy: cross sectional analytic study. BMJ 333:578, 2006 Obeidat N, Zayed F, Alchalabi H, et al: Umbilical cord prolapse: a lO-year retrospective study in two civil hospitals, North Jordan. J Obstet Gynaecol 30(3):257,r2010 Ortiz-Neira CL, Paolucci EO, Donnon T: A meta-analysis of common risk factors associated with the diagnosis of developmental dysplasia of the hip in newborns. Eur J RadioIr81(3):e344, 2012 Phelan JP, Bethel M, DeVore G, et al: Use of ultrasonography in the breech presentation with hyperextension of the fetal head. J Ultrasound Med 2(8):373, 1983 Pinard A: On version by external maneuvers. In Traite du Palper Abdominal. Paris, Lauwereyns, 1889

1	Raju TN, Mercer BM, Burchfield OJ, et al: Periviable birth: executive summary of a joint workshop by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, Society for Maternal-Fetal Medicine, American Academy of Pediatrics, and merican College of Obstetricians and Gynecologists. Obstet GynecoIr123(5):1083, 2014 Reddy UM, Zhang J, Sun L, et al: Neonatal mortality by attempted route of delivery in early preterm birth. Am J Obstet Gynecol 207(2): 117.e1, 2012 Rietberg CC, Elferink-Stinkens PM, Visser GH: The efect of the Term Breech Trial on medical intervention behaviour and neonatal outcome in he Netherlands: an analysis of35,453 term breech infants. BJOG 112(2):205,r2005 Rodgers R, Beik N, Nassar N, et al: Complications of external cephalic version: a retrospective analysis of 1121 patients at a tertiary hospital in Sydney. BJOG 124(5):767,r201 Rojansky N, Tanos V, Lewin A, et al: Sonographic evaluation of fetal head extension and maternal pelvis in

1	analysis of 1121 patients at a tertiary hospital in Sydney. BJOG 124(5):767,r201 Rojansky N, Tanos V, Lewin A, et al: Sonographic evaluation of fetal head extension and maternal pelvis in cases of breech presentation. Acta Obstet Gynecol Scand 73:607, 1994 Roman H, Carayol M, Watier L, et al: Planned vaginal delivery of fetuses in breech presentation at term: prenatal determinants predictive of elevated risk of cesarean delivery during labor. Eur J Obstet Gynecol Reprod Bioi 138(1):14,r2008

1	Rosman AN, Guijt A, Vlemmix F, et al: Contraindications for external cephalic version in breech position at term: a systematic review. Acta Obstet Gynecol Scand 92(2):137,r2013 Royal College of Obstetricians and Gynaecologists: he management of breech presentation. RCOG Green Top Guidelines, No. 20b. London, 2006 Sananes N, Roth GE, issi GA, et al: Acupuncture version of breech presentation: a randomized sham-controlled single-blinded trial. Eur J Obstet Gynecol Reprod Bioi 204:24, 2016 Sandberg EC: The Zavanelli maneuver: 12 years of recorded experience. Obstet GynecoIr93:312, 1999

1	Sandberg EC: he Zavanelli maneuver extended: progression of a revolutionary concept. AmJ Obstet Gynecolr158(6 Pt 1):1347,r1988 Saroha M, Batra P, Dewan P, et al: Genital injuries in neonates following breech presentation. J Neonatal Perinatal Med 8(4):421, 2015 Schutte JM, Steegers A, Santema JG, et al: Maternal deaths after elective cesarean section for breech presentation in the Netherlands. Acta Obstet Gynecol Scand 86(2):240, 2007 Sherer OM, Menashe M, Palti Z, et al: Radiologic evidence of a nuchal arm in the breech-presenting fetus at the onset of labor: an indication for abdominal delivery. Am J PerinatoI6(3):353, 1989

1	Steyn W, Pieper C: Favorable neonatal outcome after fetal entrapment and partially successful Zavanelli maneuver in a case of breech presentation. Am J Perinatolrll:348, 1994 Sunday-Adeoye 1M, Okonta P, Twomey 0: Symphysiotomy at the Mater Misericordiae Hospital Afikpo, Ebonyi State of Nigeria (1982-1999): a review of 1013 cases. J Obstet GynaecoI24(5):525, 2004 Thomas PE, Petersen SG, Gibbons K: he influence of mode of birth on neonatal survival and maternal outcomes at extreme prematurity: a retrospective cohort study. Aust N Z J Obstet Gynaecol 56(1):60, 2016 homas SM, Bees NR, Adam EJ: Trends in the use of pelvimetry techniques. Clin RadioIr53(4):293, 1998 Toivonen E, Palomaki 0, Huhtala H, et al: Selective vaginal breech delivery at term-still an option. Acta Obstet Gynecol Scand 91 (10): 1177, 2012 Vallikannu N, Nadzratulaiman N, Omar SZ, et al: Talcum powder or aqueous gel to aid external cephalic version: a randomised controlled trial. BMC Pregnancy Childbirth 14:49,2014

1	Vallikannu N, Nadzratulaiman N, Omar SZ, et al: Talcum powder or aqueous gel to aid external cephalic version: a randomised controlled trial. BMC Pregnancy Childbirth 14:49,2014 Vani S, Lau SY, Lim BK, et al: Intravenous salbutamol for external cephalic version. Int J Gynecol Obstet 104(1):28, 2009 Vas J, Aranda-Regules JM, Modesto M, et al: Using moxibustion in primary healthcare to correct non-vertex presentation: a multicentre randomised controlled trial. Acupunct Med 31(1):31,r2013 Velzel J, Vlemmix F, Opmeer Be, et al: Atosiban versus fenoterol as a uterine relaxant for external cephalic version: a randomised controlled trial. BMJ 26:356,r2017 Velzel J, de Hundt M, Mulder FM, et al: Prediction models for successful external cephalic version: a systematic review. Eur J Obstet Gynecol Reprod BioI 195:160,r2015

1	Vendittelli F, Pons Je, Lemery 0, et al: he term breech presentation: neonatal results and obstetric practices in France. Eur J Obstet Gynecol Reprod BioI 125(2):176,r2006 Vezina Y, Bujold E, Varin J, et al: Cesarean delivery after successful external cephalic version of breech presentation at term: a comparative study. Am J Obstet Gynecol 190:763,r2004 Vialle R, Pietin-Vialle C, Ilharreborde B, et al: Spinal cord injuries at birth: a multicenter review of nine cases. J Matern Fetal Neonatal Med 20(6):435, 2007 Vistad I, Klungs0yr K, Albrechtsen S, et al: Neonatal outcome of singleton term breech deliveries in N otway from 1991 to 2011. Acta Obstet Gynecol Scand 94(9):997, 2015

1	Weill Y, Pollack N: he eicacy and safery of external cephalic version after a previous caesarean delivery. Aust N Z J Obstet Gynaecol 57(3):323, 2017 Weiniger CF, Ginosar Y, Elchalal U, et al: Randomized controlled trial of external cephalic version in term multiparae with or without spinal analgesia. Br J Anaesth 104(5):613,r2010 Wery E, Le Roch A, Subtil 0: Zavanelli maneuver performed in a breech presentation. Int J Gynaecol Obstet 120(2):193,2013 Westgren M, Grundsell H, Ingemarsson I, et al: Hyperextension of the fetal head in breech presentation. A study with long-term follow-up. BJOG 88(2):101, 1981 Whyte H, Hannah ME, Saigal S, et al: Outcomes of children at 2 years after planned cesarean birth versus planned vaginal birth for breech presentation at term: the International Randomized Term Breech Trial. Am J Obstet GynecoIr191(3):864,r2004 Wilcox CB, Nassar N, RobertS CL: Efectiveness of nifedipine tocolysis to facilitate external cephalic version: a systematic review. BJOG

1	Term Breech Trial. Am J Obstet GynecoIr191(3):864,r2004 Wilcox CB, Nassar N, RobertS CL: Efectiveness of nifedipine tocolysis to facilitate external cephalic version: a systematic review. BJOG 118(4):423,2011 Yeomans ER: Vaginal breech delivery. In Yeomans ER, Hofman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hill Education, 2017

1	The most important function of orceps is traction exercised or the purpose of drawing the head through the genital tract. In not a ew cases, however, particulary in occipito-posterior presentations, its employment as a rotator is attended by most happy results. -J. Whitridge Williams (1903) Operative deliveries are vaginal deliveries accomplished with the use of forceps or a vacuum device. Once either is applied to the fetal head, outward traction generates forces that augment maternal pushing to deliver the fetus vaginally. he most important function of both devices is traction. In addition, forceps may also be used for rotation, particularly from occiput transverse and posterior positions.

1	According to the birth certiicate data from the National Vital Statistics Report, forceps-or vacuum-assisted vaginal delivery was used for 3.2 percent of births in the United States in 2014. his is a decline from 9.0 percent in 1990 (Hamilton, 2015). For these deliveries, a vacuum is disproportionately selected, and the vacuum-to-forceps delivery ratio is nearly 5: 1 (Merriam, 2017). In general, most of these attempts are successful. In 2006, only 0.4 percent of forceps trials in the United States and 0.8 percent of vacuum extraction attempts failed to result in vaginal delivery (Osterman, 2009).

1	If it is technically feasible and can be safely accomplished, termination of second-stage labor by traction instruments is indicated in any condition threatening the mother or fetus that is likely to be relieved by delivery. Some fetal indications include nonreassuring fetal heart rate pattern and premature placental separation (Schuit, 2012). In the past, forceps delivery was believed to be somewhat protective of the fragile preterm infant head. However, outcomes for neonates who 500 to 1500 g do not signiicantly difer if delivered spontaneously or by outlet forceps (Fairweather, 1981; Schwartz, 1983).

1	Some maternal indications include heart disease, pulmonary compromise, intrapartum infection, and certain neurological conditions. The most common are exhaustion and prolonged second-stage labor. However, a speciic maximum length beyond which all women should be considered for operative vaginal delivery has not been identiied (American College of Obstetricians and Gynecologists, 2016). Operative delivery is generally performed from either a low or outlet station. Additionally, forceps or vacuum delivery should not be used electivey until the criteria for an outlet delivery have been met. In these circumstances, operative delivery is a simple and safe operation, although with some risk of maternal lower reproductive tract injury (Yancey, 1999). Classiication for operative vaginal delivery is summarized in Table It emphasizes that the two most important discriminators of risk for both mother and neonate are station and rotation. Station is measured in centimeters, -5 to 0 to +5.

1	It emphasizes that the two most important discriminators of risk for both mother and neonate are station and rotation. Station is measured in centimeters, -5 to 0 to +5. TABLE 29-' . Operative Vaginal Delivery Prerequisites and Classification According to Station and Rotationa Outlet forceps Scalp is visible at the introitus without separating the labia Fetal head is at or on perineum Head is OA or OP or Head is right or left OA or OP position but rotation ;45 degrees Low forceps (2 types) Leading point offetal skull is at station �+2 cm, and not on the pelvic floor, and: aClassification for the vacuum delivery system is the same as forforceps except that vacuum is used for traction but not rotation. bForceps, but not vacuum extractor, may be used for delivery of a face presentation with mentum anterior. (Piper forceps may be used to deliver the head during breech delivery.

1	(Piper forceps may be used to deliver the head during breech delivery. Zero station relects a line drawn between the ischial spines. Deliveries are categorized as outlet, low, and midpelvic procedures. High orceps, in which instruments are applied above 0 station, have noplace in contemporay obstetrics. Once station and rotation are assessed, several prerequisites are met and are listed in Table 29-1. For vacuum extraction, fetuses should also be at least 34 weeks' gestation, and although infrequently used in the United States, fetal scalp blood sampling should not have been recently performed. Of requisites, ascertaining correct head position is essential, and the anatomy of the fetal skull is described in Figure In unclear cases, sonography is useful to identiy fetal orbits and nasal bridge to aid orientation (Malvasi, 2014). occiput posterior; OVD operative vaginal delivery.

1	In unclear cases, sonography is useful to identiy fetal orbits and nasal bridge to aid orientation (Malvasi, 2014). occiput posterior; OVD operative vaginal delivery. Regional analgesia or general anesthesia is preferable for low forceps or midpelvic procedures, although pudendal blockade may prove adequate for outlet forceps. As discussed in Chapter 25 (p. 495), regional analgesia during labor does not appear to increase the risk for operative delivery (Halpern, 2004; Marucci, 2007; Wassen, 2014). The bladder is emptied to provide additional pelvic space and minimize bladder trauma. Urinary retention and bladder dysunction are oten short-term efects offorceps and vacuum deliveries (Mulder, 2012; Pifarotti, 2014). Notably, episiotomy and epidural analgesia, both common associates, are also identified risks for urinary retention. Symptoms are brief and typically resolve with 24 to 48 hours ofpassive Foley catheter bladder drainage. Biparietal diam.eter = 9.5 cm

1	Biparietal diam.eter = 9.5 cm Coronal suture --=./ FIGURE 29-1 Fetal head (A, B) at term showing fontanels, sutures, and various dimensions. There is an increased risk of certain morbidities for both mother and fetus when operative delivery is employed. In general, these are related to the ease with which the delivery is accomplished.

1	There is an increased risk of certain morbidities for both mother and fetus when operative delivery is employed. In general, these are related to the ease with which the delivery is accomplished. In general, a higher station and/or greater degrees of rotation increase the chance of maternal or fetal injury. Morbidity is most properly compared with morbidity from cesarean delivery, and not with that from spontaneous vaginal delivery. his is the most appropriate comparator because the alternative to indicated operative delivery is cesarean delivery. For example, postpartum wound or uterine infection is more frequent in women following cesarean compared with operative vaginal delivery (Bailit, 2016; Halscott, 2015). Moreover, in a study of more than 1 million births, Spiliopoulos and associates (201i1) reported cesarean delivery, but not operative vaginal delivery, as a risk for peripartum hysterectomy.

1	he very conditions that lead to indications for operative delivery also increase the need for episiotomy and the likelihood of lacerations (de Leeuw, 2008). That said, forceps and vacuum deliveries are associated with higher rates of third-and fourthdegree lacerations as well s vaginal wall and cervical lacerations (Gurol-Urganci, 2013; Hirayama, 2012; Landy, 2011; Pergialiotis, 2014). These appear to occur more frequently with forceps compared with vacuum extraction, and especially if there is a midline episiotomy (Kudish, 2006; O'Mahony, 2010). Hagadorn-Freathy and coworkers (1991) reported a 13-percent rate of third-and fourth-degree episiotomy extensions and vaginal lacerations for outlet forceps, 22 percent for low forceps with less than 45-degree rotation, 44 percent for low forceps with more than 45-degree rotation, and 37 percent for midforceps deliveries.

1	In an efort to lower rates of third-and fourth-degree lacerations, and coincident with overall eforts to reduce routine episiotomy use, many advocate only indicated episiotomy with operative delivery. If episiotomy is required, a protective efect against these more extensive perineal lacerations may be aforded by mediolateral episiotomy (de Leeuw, 2008; de Vogel, 2012; Hirsch, 2008). Early disarticulation of forceps and cessation of maternal pushing during disarticulation can also be protective. Last, these injuries are more common with an occiput posterior position (Damron, 2004). Thus, manual or forceps rotation to an occiput anterior position and then subsequent traction delivery may decrease rates oflower reproductive tract injury (Bradley, 2013).

1	his term encompasses urinary incontinence, anal incontinence, and pelvic organ prolapse. Operative vaginal delivery has been suggested as a possible risk for each of these. Proposed mechanisms include structural compromise and/or pelvic loor denervation secondary to forces exerted during delivery. Parity and speciically vaginal delivery are risk factors for urinary incontinence (Gyhagen, 2013; Rortveit, 2003). But, many studies do not support an increased risk compared with vaginal delivery alone (Gartland, 2016; Leijonhuvud, 2011; MacArthur, 2016; Tahtinen, 2016).

1	MacArthur, 2016; Tahtinen, 2016). Evidence linking anal incontinence with operative vaginal delivery is conlicting. Some studies show that anal sphincter disruption caused by higher-order episiotomy, but not delivery mode, is the main etiological factor strongly associated with anal incontinence (BoIs, 2010; Evers, 2012; Nygaard, 1997). In con trast, others directly link operative delivery with this complica tion (Dolan, 2010; Marthur, 2013). But, these studies may not be incongruous-recall that operative delivery is associated with increased rates of higher-order episiotomy. Importantly, several studies and reviews have not found cesarean delivery to be protec tive for anal incontinence (Nelson, 2010). Last, evidence linking pelvic organ prolapse with operative delivery also indicates mixed results (Gyhagen, 2013; Handa, 2012; Vo1l0yhaug, 2015).

1	hese injuries are more frequent with operative vaginal delivery than with cesarean delivery or spontaneous vaginal delivery. Injuries may be seen with either method. hey are more common with vacuum extraction, and types associated with this device include cephalohematoma, subgaleal hemorrhage, retinal hemorrhage, neonatal jaundice secondary to these hemorrhages, shoulder dystocia, clavicular fracture, and scalp lacerations. Cephalohematoma and subgaleal hemorrhage are both extracranial lesions described in Chapter 33 (p. 628). Forceps-assisted vaginal delivery has higher rates of facial nerve injury, brachial plexus injury, depressed skull fracture, and corneal abrasion (merican College of Obstetricians and Gynecologists, 2015; Demissie, 2004; Dupuis, 2005). For intracranial hemorrhage, some studies have associated vacuum extraction with higher rates, whereas others show similar rates with either of the two methods (Towner, 1999; Wen, 2001; Werner, 201l).

1	Comparing operative vaginal with cesarean delivery, rates of extracranial hematoma, skull fracture, facial nerve or brachial plexus injury, retinal hemorrhage, and facial or scalp laceration are lower with cesarean deliveY, and shoulder dystocia is eliminated. Importantly, however, fetal acidemia rates are not increased with operative delivery (Contag, 2010; Walsh, 2013). Intracranial hemorrhage rates are similar among newborns delivered by vacuum extraction, forceps, or cesarean delivery during labor (Towner, 1999). But, these rates are higher than among those delivered spontaneously or by cesarean delivery before labor. These authors suggest that the common risk factor for intracranial hemorrhage is abnormal labor. Werner and associates (2011), in their evaluation of more than 150,000 singleton deliveries, reported that forceps-assisted delivery was associated with fewer total neurological complications compared with vacuum-assisted or cesarean delivery. However, as a subset,

1	singleton deliveries, reported that forceps-assisted delivery was associated with fewer total neurological complications compared with vacuum-assisted or cesarean delivery. However, as a subset, subdural hemorrhage was signiicantly more frequent in both instrumental cohorts compared with neonates in the cesarean delivery group.

1	Comparing rotational operative delivery and second-stage cesarean delivery, maternal and neonatal morbidity rates are similar (Aiken, 2015; Bahl, 2013; Stock, 2013). For example, in their large series, Tempest and associates (2013) found similar morbidity rates among malpositioned fetuses during second-stage labor that underwent Kielland rotation, rotational vacuum extraction, or emergency cesarean delivery.

1	Comparing midforceps and cesarean delivery, reports of neonatal morbidity rates are from older studies and are conflicting. In the study by Towner and colleagues (1999), similar risks were reported for intracranial hemorrhage. Bashore and associates (1990) observed comparable Apgar scores, cord blood acid-base values, neonatal intensive care unit admission, and birth trauma between these two. In another study, however, Robertson and coworkers (1990) reported signiicantly higher rates of these adverse outcomes in the midforceps group. Hagadorn-Freathy and colleagues (1991) reported an increased risk for facial nerve palsy-9 percent-with midforceps delivery. In a recent report comparing low and midforceps procedures, Ducarme and coworkers (2015) found comparable morbidity rates.

1	Mechanisms of Acute Injury he types of fetal injury with operative delivery can usually be explained by the forces exerted. In cases of cephalohematoma or subgaleal hemorrhage, suction and perhaps rotation during vacuum extraction may lead to a primary vessel laceration. Intracranial hemorrhage may result from skull fracture and vessel laceration or from vessel laceration alone due to exerted forces. With facial nerve palsy, one of the forceps blades may compress the nerve against the facial bones. he higher rates of shoulder dystocia seen with vacuum extraction may result from the angle of traction. With the vacuum, this angle creates vector forces that actually pull the anterior shoulder into the symphysis pubis (Caughey, 2005). To explain brachial plexus injury, Towner and Ciotti (2007) proposed that as the fetal head descends down the birth canal, the shoulders may stay above the pelvic inlet. Thus, similar to shoulder dystocia at the symphysis, this "shoulder dystocia at the

1	proposed that as the fetal head descends down the birth canal, the shoulders may stay above the pelvic inlet. Thus, similar to shoulder dystocia at the symphysis, this "shoulder dystocia at the pelvic inlet" is overcome by traction forces but with concomitant stretch on the brachial plexus.

1	Evidence regarding long-term neurodevelopmental outcomes in children born by operative delivery is reassuring. In an older study, Seidman and colleagues (1991) evaluated more than 52,000 Israeli Defense Forces dratees at age 17 years and found that regardless of delivery mode, rates of physical or cognitive impairments were similar. Wesley and associates (1992) noted similar intelligence scores among 5-year-olds following spontaneous, forceps, or vacuum deliveries. Murphy and coworkers (2004) found no association between forceps delivery and epilepsy in a cohort of more than 21,000 adults. In their epidemiological review, O'Callaghan and colleagues (2011) found no association between cerebral palsy and operative delivery. Last, Bahl and associates (2007) noted that the incidence of neurodevelopmental morbidity was similar in those undergoing successul forceps delivery, failed forceps with cesarean delivery, or cesarean delivery without forceps.

1	Data regarding midforceps deliveries are for the most part reassuring. Broman and coworkers (1975) reported that infants delivered by midforceps had slightly higher intelligence scores at age 4 years compared with those of children delivered spontaneously. Using the same database, however, Friedman and associates (1977, 1984) analyzed intelligence scores at or after age 7 years. They concluded that children delivered by midforceps had lower mean intelligence quotients compared with those delivered by outlet forceps. In yet another report from this database, Dierker and colleagues (1986) compared long-term outcomes of children delivered by midforceps with those delivered by cesarean after dystocia. he strength of this study is the appropriateness of the control group. hese investigators reported that delivery by midforceps was not associated with neurodevelopmental disability. Last, Nilsen (1984) evaluated 18-year-old men and found that those delivered by Kielland forceps had higher

1	reported that delivery by midforceps was not associated with neurodevelopmental disability. Last, Nilsen (1984) evaluated 18-year-old men and found that those delivered by Kielland forceps had higher intelligence scores than those delivered spontaneously, by vacuum extraction, or by cesarean.

1	If an attempt to perform an operative delivery is expected to be diicult, then it should be considered a trial. Moving the woman to an operating room for this attempt, which could be followed by immediate cesarean delivery if operative delivery fails, has merit. If forceps cannot be satisfactorily applied, then the procedure is stopped and either vacuum extraction or cesarean delivery is performed. With the former, if the fetus does not descend with traction, the trial should be abandoned and cesarean delivery performed.

1	With such caveats, cesarean delivery after an attempt at operative vaginal delivery was not associated with adverse neonatal outcomes if there was a reassuring fetal heart rate tracing (Alexander, 2009). A similar study evaluated 122 women who had a trial of midcavity forceps or vacuum extraction in a setting with full preparations for cesarean delivery (Lowe, 1987). Investigators found no significant diference in immediate neonatal or maternal morbidity compared with that of 42 women delivered for similar indications by cesarean but without such a trial. Conversely, in 61 women who had "unexpected" vacuum or forceps failure in which there was no prior preparation for immediate cesarean delivery, neonatal morbidity was higher.

1	Some factors associated with operative delivery failure are persistent occiput posterior position and birthweight >4000 g (Ben-Haroush, 2007; Verhoeven, 2016). However, Palatnik and associates (2016) found that risk factors poorly predicted success. In general, to avert morbidity with failed forceps or vacuum delivery, the American College of Obstetricians and Gynecologists (2015) cautions that these trials should be attempted only if the clinical assessment suggests a successful outcome. We also emphasize proper training.

1	Sequential instrumentation most often involves an attempt at vacuum extraction followed by one with forceps. his most likely stems from the higher completion rate with forceps compared with vacuum extraction noted earlier. This practice significantly increases risks for fetal trauma (Dupuis, 2005; Gardella, 2001; Murphy, 2011). Because of these adverse outcomes, the American College of Obstetricians and Gynecologists (2015) recommends against the sequential use of instruments unless there is a "compelling and justifiable reason." As the rate of operative vaginal delivery has declined, so have opportunities for training (Fitzwater, 2015; Kyser, 2014). In FIGURE 29-2 Simpson forceps have fenestrated blades, parallel shanks, and English lock. The cephalic curve accommodates the fetal head.

1	FIGURE 29-2 Simpson forceps have fenestrated blades, parallel shanks, and English lock. The cephalic curve accommodates the fetal head. many programs, training in even low and outlet forceps proce dures has reached critically low levels. For residents completing training in 2015, the Accreditation Council for Graduate Med ical Education reported a median of only five forceps deliveries, and that for vacuum deliveries was 16.

1	Because traditional hands-on training has evolved, residency programs should have readily available skilled operators to teach these procedures by simulation as well as through actual cases (Skinner, 2017; Spong, 2012). And, the efectiveness of simulation training has been reported (Dupuis, 2006, 2009; Leslie, 2005). In one program, maternal and neonatal morbidity rates with operative delivery decreased after the implementation of a formal education program that included a manikin and pelvic model (Cheong, 2004). In another, a 59-percent increase in forceps deliveries over 2 years was related to a single experienced and proactive instructor assigned to teach forceps to residents in labor and delivery (Solt, 2011).

1	Forceps refers to the paired instrument, and each member of this pair is called a branch. Branches are designated left or right according to the side of the maternal pelvis to which they are applied (Fig. 29-2). Each branch has four components: blade, shank, lock, and handle (Fig. 29-3). Each blade has a toe, a heel, and two curves. Of these, the outward cephalic curve conforms to the round fetal head, whereas the upward pelvic curve corresponds more or less to the curve of the birth canal. Some blades have an opening within or a depression along the blade surface and are termed enestrated or pseudoenestrated, respectively. True fenestration reduces the degree of head slippage during forceps rotation. Disadvantageously, it can increase friction between the blade and vaginal wall. With pseudo fenestration, the forceps blade is smooth on the outer maternal side but indented on the inner fetal surface. he goal is to reduce head slipping yet improve the ease and safety of application and

1	fenestration, the forceps blade is smooth on the outer maternal side but indented on the inner fetal surface. he goal is to reduce head slipping yet improve the ease and safety of application and removal of forceps compared with pure fenestrated blades. In general, fenestrated blades are used for a fetus with a molded head or for rotation. In most situations, however, despite these subtle diferences any are appropriate.

1	he blades are connected to shanks, which may be parallel or overlapping. Locks are found on all forceps and help to connect FIGURE 29-3 Luikart forceps have pseudofenestrated blades, overlapping shanks, sliding lock, tongue groove handles. The pelvic curve is marked in this example by the black line. the right and left branches and stabilize the instrument. They can be located at the end of the shank nearest to the handles (English lock), at the ends of the handles (pivot lock), or along the shank (sliding lock). Although varied in design, handles, when squeezed, raise compression forces against the fetal head. hus, forces to consider include traction and compression.

1	Forceps blades grasp the head and are applied according to fetal head position. If the head is in an occiput anterior (OA) position, two or more fingers of the right hand are introduced inside the left posterior portion of the vulva and then into the vagina beside the fetal head. he handle of the left branch is grasped between the thumb and two fingers of the left hand (Fig. 29-4). FIGURE 29-4 For OA or LOA positions, the left handle of the forceps is held in the left hand. The blade is introduced into the left side of the pelvis between the fetal head and the fingers of the operator's right hand. FIGURE 29-5 Insertion arc of the blade. Importantly, the thumb of the right hand, guides the blade during placement, as shown in Figure 29-6.

1	FIGURE 29-5 Insertion arc of the blade. Importantly, the thumb of the right hand, guides the blade during placement, as shown in Figure 29-6. he blade tip is then gently passed into the vagina between the fetal head and the palmar surface of the ingers of the right hand (Fig. 29-5). For application of the right blade, two or more ingers of the left hand are introduced into the right posterior portion of the vagina to serve as a guide for the right blade. his blade is held in the right hand and introduced into the vagina. With each blade, the thumb is positioned behind the heel, and most of the insertion force comes from this thumb (Fig. 29-6). If the head is positioned in a let OA (LOA) or right OA (ROA) position, then the lower of the two blades is typically placed irst. Mter positioning, the branches are articulated.

1	he blades are constructed so that their cephalic curve is closely adapted to the sides of the fetal head (Fig. 29-7). The fetal head is perfectly grasped only when the long axis of the blades corresponds to the occipitomental diameter (see Fig. 29-1). As a result, most of the blade lies over the lateral face. If the fetus is in an OA positi�n, then the concave arch of the blades is directed toward the sagittal suture. If the fetus is in an occiput posterior (OP) position, then the concave arch is directed toward the midline face.

1	Suboptimal blade placement can increase morbidity (Ramphul, 2015). For OA position, appropriately applied blades are equidistant from the sagittal suture, and each blade is equidistant from its adjacent lambdoidal suture. In the OP position, the blades are equidistant from the midline of the face and brow. Also for OP position, blades are symmetrically placed relative to the sagittal suture and each coronal suture. Applied in this way, the forceps should not slip, and traction may be applied most advantageously. With most forceps, if one blade is applied over the brow and the other over the occiput, the instrument cannot be locked, or if locked, the blades will slip of when traction is applied (Fig. 29-8). With both branches in place, it should be an easy matter to articulate the handles, engage the lock, and correct asynclitism if present. Asynclitism is resolved by pulling and/or pushing each branch along the long axis of the instrument until the

1	FIGURE 29-6 In applying the second blade, insertional force is generated mainly by the thumb. (Reproduced with permission from Yeomans ER: Operative vaginal delivery. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201•7.) inger guards align. If necessary, rotation to OA position is performed before traction is applied (Fig. 29-9). When it is certain that the blades are placed satisfactorily, then gentle, intermittent, downward and outward traction is exerted concurrent with maternal eforts until the perineum FIGURE 29-7 A. The forceps are symmetrically placed and articulated. B.The vertex is OA. (Reproduced with permission from Yeomans ER: Operative vaginal delivery. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201•7.)

1	FIGURE 29-8 Incorrect application of forceps. A. One blade over the occiput and the other over the brow. Forceps cannot be locked. B.With incorrect placement, blades tend to slip of with traction. begins to bulge. When the head is at 0 to + 2 of + 5 sta tion, the initial direction of traction is quite posterior, almost toward the Roor. With head descent, the vector of forces changes continuously (Fig. 29-10). As a teaching tool for this, a Bill axis traction device can be attached over the finger guards of most forceps. he instrument has an arrow and indicator line. When the arrow points directly to the line, traction is FIGURE 29-9 A. If LOA, the vertex is rotated (arrow) from this position to OA (B). (Reproduced with permission from Yeomans ER: Operative vaginal delivery. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201o7.)

1	FIGURE 29-10 With low forceps, the direction of gentle traction for delivery of the head is indicated (arrow). The vector changes with fetal descent. along the path of least resistance. With traction, as the vulva is distended by the occiput, an episiotomy may be performed if indicated. Additional horizontal traction is applied, and the handles are then gradually elevated. As the handles are raised, the head is extended. During the birth of the head, mechanisms of spontaneous delivery should be simulated as closely as possible.

1	The force produced by the forceps on the fetal skull is a function of both traction and compression by the forceps, as well as friction produced by maternal tissues. It is impossible to ascertain the amount of force exerted by forceps for an individual patient. Traction should therefore be intermittent, and the head should be allowed to recede between contractions, as in spontaneous labor. Except when urgently indicated, as in severe fetal bradycardia, delivery should be suiciently slow, deliberate, and gentle to prevent undue head compression. It is preferable to apply traction only with each uterine contraction. Maternal pushing will augment these eforts.

1	After the vulva has been well distended by the head, the delivery may be completed in several ways. Some clinicians keep the forceps in place to control the head. If this is done, however, the blade volume adds to vulvar distention, thus from that in which they were originally placed. The fingers of the right hand, covered by a sterile towel, bolster the perineum. The thumb is placed directly on the head to prevent sudden egress. (Reproduced with permission from Yeomans ER: Operative vaginal delivery. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201o7.) increasing the likelihood of laceration or necessitating a large episiotomy. To prevent this, the forceps may be removed, and delivery is then completed by maternal pushing (Fig. 29-11). Importantly, if blades are disarticulated and removed too early, the head may recede and lead to a prolonged delivery. Delivery in some cases may be

1	then completed by maternal pushing (Fig. 29-11). Importantly, if blades are disarticulated and removed too early, the head may recede and lead to a prolonged delivery. Delivery in some cases may be aided by addition of the modiied Ritgen maneuver.

1	Prompt delivery may at times become necessary when the small occipital fontanel is directed toward one of the sacroiliac synchondroses. In these right OP (ROP) or left OP (LOP) positions, the fetal head is often imperfectly lexed. With OP positions, second-stage labor can be lengthened. In these cases, the head may spontaneously deliver OP, may be manually or instrumentally rotated to an OA position, or may be delivered OP by forceps or vacuum. With manual rotation, an open hand is inserted into the vagina. The palm straddles the sagittal suture of the fetal head. The operator's ingers wrap around one side of the fetal face and thumb extends along the other side. If the occiput is ROP, rotation is clockwise to bring it to an ROA or OA position (Fig. 29-12).

1	/�(, _A.V\..IA...._ ./�...._/' B FIGURE 29-12 A. Manual rotation using the left hand, palm-up, to rotate from ROP. B. The head is flexed and destationed during clockwise rotation to reach an OA pOSition. (Reproduced with permission from Yeomans ER: Operative vaginal delivery. In Yeomans ER, Hofman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201o7.) FIGURE 29-14 Kielland forceps. The characteristic features are minimal pelvic curvature (A), sliding lock (8), and light weight.

1	FIGURE 29-14 Kielland forceps. The characteristic features are minimal pelvic curvature (A), sliding lock (8), and light weight. With LOr position, rotation is counterclockwise. hree actions are performed simultaneously between contractions. The irst is fetal head flexion to provide a smaller diameter for rotation and subsequent descent. Second, slight destationing of the fetal head moves the head to a level in the maternal pelvis with sufficient room to complete the rotation. Importantly, destationing should not be confused with disengaging the fetal head, which is proscribed. Concurrently, some prefer to also place the other hand externally on the corresponding side of the maternal abdomen to pull the fetal back up toward the midline in synchrony with the internal rotation. Le Ray and colleagues (2007, 2013) reported a success rate of greater than 90 percent with manual rotation. Barth (2015) provides an excellent summary of this technique.

1	Manual rotations are most easily completed in multiparas. If manual rotation cannot be easily accomplished, application of forceps blades to the head in the posterior position and delivery from an or position may be the safest procedure. In many cases, the cause of a persistent or position and of the diiculty in accomplishing rotation is an anthropoid pelvis. his architecture opposes rotation and predisposes to posterior delivery (Fig. 2-17, p. 31). With forceps delivery from an or position, downward and outward traction is applied until the base of the nose passes under the symphysis (Fig. 29-13). he handles are then slowly elevated until the occiput gradually emerges over the upper margin of the perineum. he forceps are directed downward again, and the nose, mouth, and chin successively emerge from the vulva.

1	or delivery causes greater distention of the vulva, and a large episiotomy may be needed. or deliveries have a higher incidence of severe perineal lacerations and extensive episiotomy compared with OA positions (de Leeuw, 2008; Pearl, 1993). Also, newborns delivered from or positions have a higher incidence ofErb and facial nerve palsies, 1 and 2 percent, respectively, than those delivered from OA positions. As expected, rotations to OA ultimately decrease perineal delivery trauma (Bradley, 2013). Last, for forceps rotations from an or to OA position, the Kielland instruments are preferred because they have a less pronounced pelvic curve (Fig. 29-14). Cunningham and Gilstrapis FIGURE 29-13 Outlet forceps delivery from an OP position. The head should be flexed ater the bregma passes under the symphysis. Operative Obstetrics, 3rd edition, ofers a more detailed description of this Kielland forceps procedure (Yeomans, 2017).

1	Operative Obstetrics, 3rd edition, ofers a more detailed description of this Kielland forceps procedure (Yeomans, 2017). With occiput transverse (OT) positions, rotation is required for delivery. With experienced operators, high success rates with minimal maternal morbidity can be achieved (Burke, 2012; Stock, 2013). Either standard forceps, such as Simpson, or specialized forceps, such as Kielland, are employed. With Kielland forceps, each handle has a small knob, and branches are placed so that this knob faces the occiput. he station of the fetal head must be accurately determined to be at, or preferably below, the level of the ischial spines, especially in the presence of extreme molding.

1	Kielland described two methods of applying the anterior blade. In our example, placement with a left OT (LOT) position is described. With the wandering method, the anterior blade is first introduced into the posterior pelvis (Fig. 29-15). The blade is then arched around the face to an anterior position. To permit this sweep of the blade, the handle is held close to the maternal left buttock throughout the maneuver. he second blade is introduced directly posteriorly, and the branches are locked.

1	After checking the application, the handles of the Kielland forceps are pulled slightly to the patient's right to increase fetal head lexion and create a smaller diameter for rotation. he irst and second fingers of the left hand are placed over the inger guards with the palm against the handles. his palm faces the maternal left. Concurrently, the first two ingers of the operator's right hand are placed against the anterior lambdoid suture. he fetal head is then destationed approximately 1 cm. For rotation in a counterclockwise direction, the wrist of the left hand supinates, to direct this palm upward. Simultaneously, two ingers of the right hand press on the edge of the right parietal bone that borders the lambdoid suture. This ensures that the fetal head turns with the blades and does not slip. he second type of blade application introduces the anterior blade with its cephalic curve directed upward to curve under

1	he second type of blade application introduces the anterior blade with its cephalic curve directed upward to curve under A B c FIGURE 29-15 A. Application of the right branch of the Kielland forceps to a head in LOT position. The knob on this branch (colored blue) will ultimately face the occiput. B. The right branch is wandered to its final position behind the symphysis. C. Insertion ofthe left branch of the Kielland forceps directly posterior along the hollow of the sacrum. This branch is inserted to the maternal right of the anterior branch to aid in engaging the sliding lock. (Reproduced with permission from Yeomans ER: Operative vaginal delivery. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201•7.) the symphysis. After it has been advanced far enough toward the upper vagina, it is turned on its long axis through 180 degrees to adapt the cephalic curvature to the head.

1	With either application, after rotation completion, the operator may choose from two acceptable methods for delivery. In one, the operator applies traction on the Kielland forceps using a bimanual grip described previously for conventional forceps (p. 559). When the posterior fontanel has passed under the subpubic arch, the handles can be elevated to the horizontal. Raising the handles above the horizontal may cause vaginal sulcus tears because of the reverse pelvic curve (Dennen, 1955). Alternatively, the Kielland forceps can be removed after rotation and replaced with conventional forceps. With this approach, moderate traction is first employed to seat the head before switching instruments.

1	With a mentum anterior face presentation, forceps can be used to efect vaginal delivery. The blades are applied to the sides of the head along the occipitomental diameter, with the pelvic curve directed toward the neck. Downward traction is exerted until the chin appears under the symphysis. hen, by an upward movement, the face is slowly extracted, with the nose, eyes, brow, and occipur appearing in succession over the anterior margin of the perineum. Forceps should not be applied to the mentum posterior presentation because vaginal delivey is impossible except in vey smal ttuses.

1	With vacuum delivery, suction is created within a cup placed on the fetal scalp such that traction on the cup aids fetal expulsion. In the United States, vacuum extractor is the preferred term, whereas in Europe it is commonly called a vento use (Fig. 29-16). heoretical beneits of this tool compared with forceps include simpler requirements for precise positioning on the fetal head and avoidance of space-occupying blades within the vagina, thereby mitigating maternal trauma.

1	Vacuum devices contain a cup, shaft, handle, and vacuum generator. Vacuum cups may be metal or hard or soft plastic, and they may also difer in their shape, size, and reusability. In the United States, nonmetal cups are generally preferred, and there are two main types. he soft cup is a pliable bell-shaped dome, whereas the rigid type has a firm flattened mushroomshaped cup and circular ridge around the cup rim (Table 29-2). When compared, rigid mushroom cups generate significantly more traction force (Hofmeyr, 1990; Muise, 1993). With or positions or with asynclitism, the flatter cup also permits improved placement at the flexion point, which is typically less accessible with these head positions. The trade-of is that the flatter cups have higher scalp laceration rates. Thus, many manufacturers recommend soft bell cups for more straightforward OA deliveries.

1	Several investigators have compared outcomes with various rigid and soft cups. Metal cups provide higher success rates but greater rates of scalp injuries, including cephalohematomas -�..--A FIGURE 29-16 Vacuum delivery systems. A. The Kiwi OmniCup contains a handheld vacuum-generating pump, which is attached via flexible tubing to a rigid plastic mushroom cup. B. The Mityvac Mystic II MitySoft Bell Cup has a soft bell cup attached by a semirigid shaft to a handheld pump. TABLE 29-2. Vacuum Cups for Operative Vaginal Delivery "Reusable cups. bSuitable for occiput posterior positions or asynclitism. (For extractions through a hysterotomy incision during cesarean delivery.

1	(O'Mahony, 2010). In another study, Kuit and coworkers (1993) found that the only advantage of the soft cups was a lower incidence of scalp injury. hey reported a 14-percent episiotomy extension rate with both rigid and pliable cups. In a review, Vacca (2002) concluded that there were fewer scalp lacerations with the soft cup, but that the rate of cephalohematomas and subgaleal hemorrhage was similar between soft and rigid cups. Importantly, high-pressure vacuum generates large amounts of force regardless of the cup used (Duchon, 1998) . Aside from the cup, the shaft that connects the cup and handle may be lexible or semiflexible. Tubing-like flexible shafts may be preferred for OP positions or asynclitic presentation to permit better seating of the cup. Last, the vacuum generator may be handheld and actuated by the operator or may be held and operated by an assistant.

1	An important step in vacuum extraction is proper cup placement over the lexion point. his pivot point maximizes traction, minimizes cup detachment, lexes but averts twisting the fetal head, and delivers the smallest head diameter through the pelvic oudet. This improves success rates, lowers fetal scalp injury rates, and lessens perineal trauma because the smallest fetal head diameter distends the vulva (Baskett, 2008). The lexion point is found along the sagittal suture, approximately 3 cm in front of the posterior fontanel and approximately 3em 6em /II -_ ...._-\ o\ I\ I\\ ........"------,///� FIGURE 29-17 Drawing demonstrates correct cup placement at the flexion point. Along the sagittal suture, this spot lies 3 cm from the posterior fontanel and 6 cm from the anterior fontanel.

1	FIGURE 29-17 Drawing demonstrates correct cup placement at the flexion point. Along the sagittal suture, this spot lies 3 cm from the posterior fontanel and 6 cm from the anterior fontanel. 6 cm from the anterior fontanel. Because cup diameters range from 5 to 6 cm, when properly placed, the cup rim lies 3 cm from the anterior fontanel (Fig. 29-17). Placement of the cup more anteriorly on the fetal cranium-near the anterior fontanel-should be avoided as it leads to cervical spine extension during traction unless the fetus is small. Such placement also delivers a wider fetal head diameter through the vaginal opening. Last, asymmetrical placement relative to the sagittal suture may worsen asynclitism. For elective use, cup placement in OA positions is seldom diicult. In contrast, when the indication for delivery is failure to descend caused by occipital malpositionwith or without asynclitism or deflexion-cup positioning can be diicult.

1	During cup placement, maternal soft tissue entrapment predisposes the mother to lacerations and virtually ensures cup dislodgement, colloquially called a "pop of." Thus, the entire cup circumference should be palpated both before and after the vacuum has been created and again prior to traction to exclude such entrapment. Gradual vacuum creation is advocated by some and is generated by increasing the suction in increments of 0.2 kg/cm2 every 2 minutes until a total negative pressure of 0.8 kg/cm2 is reached 29-3). hat said, other studies have shown that negative pressure can be increased to 0.8 kg/cm2 in <2 minutes without a signiicant diference in eicacy or in maternal and fetal outcomes (Suwannachat, 2011, 2012) . TABLE 29-3. Vacuum Pressure Conversions 100 10 3.9 1.9 0.l3 200 20 7.9 3.9 0.27 300 30 11.8 5.8 OA1 400 40 15.7 7.7 0.54 500 50 19.7 9.7 0.68 600 60 23.6 11s.6 0.82

1	Once suction is created, the instrument handle is grasped, and traction is initiated. Similar to forceps delivery, traction angles mirror that in Figure 29-10. Eforts are intermittent and coordinated with maternal expulsive eforts. Manual torque to the cup is avoided as it can cause cup displacement or cephalohematomas and, with metal cups, "cookie-cutter"-type scalp lacerations. hus, OA oblique positions are corrected not by rotation, but solely by downward outward traction. During pulls, the operator should place the nondominant hand within the vagina, with the thumb on the extractor cup and one or more ingers on the fetal scalp. So positioned, descent of the presenting part can be judged and the traction angle can be adjusted with head descent. In addition, the relationship of the cup edge to the scalp can be assessed to help detect cup separation.

1	Between contractions, some physicians will lower the suction levels to decrease rates of scalp injury, whereas others will maintain suction in cases with a nonreassuring fetal heart rate to aid rapid delivery. No diferences in maternal or fetal outcome were noted if the level of vacuum was decreased between contractions or if an efort was made to prevent fetal loss of station (Boill, 1997). Once the head is extracted, the vacuum pressure is relieved and the cup removed.

1	Vacuum extraction should be considered a trial. Without early and clear evidence of descent toward delivery, an alternative delivery approach should be considered. As a general guideline, progressive descent should accompany each traction attempt. Neither data nor consensus are available regarding the number of pulls required to efect delivery, the maximum number of cup pop-ofs that can be tolerated, or optimal total duration of the procedure. Some manufacturers have recommendations regarding these in their instructional literature (Clinical Innovations, 2016; CooperSurgical, 2011).

1	During a vacuum extraction trial, cup dislodgement due to technical failure or less than optimal placement should not be equated with dislodgement under ideal conditions of exact cup placement and optimal vacuum maintenance. hese cases may merit either additional attempts at placement or, alternatively, a trial of forceps (Ezenagu, 1999; Williams, 1991). he least desirable cases are those in which traction without progress or multiple disengagements occur following correct cup application and appropriate traction. As with forceps, clinicians should embrace a willingness to abandon attempts at vacuum extraction if satisfactory progress is not made (American College of Obstetricians and Gynecologists, 2015). Accreditation Council for Graduate Medical Education: Obstetrics and

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1	Gurol-Urganci I, Cromwell DA, Edozien LC, et al: hird-and fourth-degree perineal tears among primiparous women in England between 2000 and 2012: time trends and risk factors. BJOG 120(12):1516,r2013 Gyhagen M, Bullarbo M, Nielsen T, et al: The prevalence of urinary incontinence 20 years after childbirth: a national cohort study in singleton primiparae after vaginal or caesarean delivery. BJOG 120:144,r2013 Hagadorn-Freathy AS, Yeomans ER, Hankins GO: Validation of the 1988 ACOG forceps classiication system. Obstet Gynecol 77:356, 1991 Halpern SH, Muir H, Breen W, et al: A multicenter randomized controlled trial comparing patient-controlled epidural with intravenous analgesia for pain relief in labor. Anesth Analg 99(5):1532,2004 Halscott TL, Reddy UM, Landy HJ, et al: Maternal and neonatal outcomes by attempted mode of operative delivery from a low station in the second stage of labor. Obstet GynecoIr126(6):1265, 2015

1	Halscott TL, Reddy UM, Landy HJ, et al: Maternal and neonatal outcomes by attempted mode of operative delivery from a low station in the second stage of labor. Obstet GynecoIr126(6):1265, 2015 Hamilton BE, Martin JA, Osterman MJ, et al: Births: final data for 2014. Nat! Vital Stat Rep 64(12):1, 2015 Handa L, Blomquist JL, McDermott KC, et al: Pelvic floor disorders after vaginal birth: efect of episiotomy, perineal laceration, and operative birth. Obstet Gynecol 119(2 Pt 1):233,2012 Hirayama F, Koyanagi A, Mori R, et al: Prevalence and risk factors for thirdand fourth-degree perineal lacerations during vaginal delivery: a multicountry study. BJOG 119(3):340,2012 Hirsch E, Haney EI, Gordon TE, et al: Reducing high-order perineal laceration during operative vaginal delivery. AmJ Obstet GynecoIr198(6):668.e1, 2008 Hofmeyr GJ, Gobetz L, Sonnendecker EW, et al: New design rigid and soft vacuum extractor cups: a preliminary comparison of traction forces. BJOG 97(8):681, 1990

1	Hofmeyr GJ, Gobetz L, Sonnendecker EW, et al: New design rigid and soft vacuum extractor cups: a preliminary comparison of traction forces. BJOG 97(8):681, 1990 Kudish B, Blackwell S, Mcneeley SG, et al: Operative vaginal delivery and midline episiotomy: a bad combination for the perineum. Am J Obstet Gynecol 195(3):749,r2006 Kuit JA, Eppinga HG, Wallenburg HCS, et al: A randomized comparison of vacuum extraction delivery with a rigid and a pliable cup. Obstet Gynecol 82:280, 1993 Kyser L, Lu X, Santillan 0, et al: Forceps delivery volumes in teaching and nonteaching hospitals: are volumes suicient for physicians to acquire and maintain competence? Acad Med 89(1):71, 2014 Landy HJ, Laughon SK, Bailit JL, et al: Characteristics associated with severe perineal and cervical lacerations during vaginal delivery. Obstet Gynecol 117(3):627,r201r1

1	Landy HJ, Laughon SK, Bailit JL, et al: Characteristics associated with severe perineal and cervical lacerations during vaginal delivery. Obstet Gynecol 117(3):627,r201r1 Leijonhuvud A, Lundholm C, Cnattingius S, et al: Risks of stress urinary incontinence and pelvic organ prolapse surgery in relation to mode of childbirth. Am J Obstet GynecoI204(1):70.e1, 2011 Le Ray C, Deneux-haraux C, Khireddine I, et al: Manual rotation to decrease operative delivery in posterior or transverse positions. Obstet Gynecol 122(3):634,r2013 Le Ray C, Serres P, Schmitz T, et al: Manual rotation in occiput posterior or transverse positions. Obstet Gynecol 110:873, 200 Leslie KK, Dipasqule-Lehnerz P, Smith M: Obstetric forceps training using visual feedback and the isometric strength testing unit. Obstet Gynecolr1 05:377,2005 Lowe B: Fear of failure: a place for the trial of instrumental delivery. BJOG 94:60, 1987

1	MacArthur C, Wilson 0, Herbison P, et al: Faecal incontinence persisting after childbirth: a 12-year longitudinal study. BJOG 120(2):169,r2013 MacArthur C, X ilson 0, Herbison P, et al: Urinary incontinence persisting after childbirth: extent, delivery history, and efects in a 12-year longitudinal cohort study. Prolong Study Group. BJOG 123(6):1022,r2016 Malvasi A, Tinelli A, Barbera A, et al: Occiput posterior position diagnosis: vaginal examination or intrapartum-sonography? A clinical review. J Matern Fetal Neonatal Med 27(5):520,r2014 Marucci M, Cinnella G, Perchiazzi G, et al: Patient-requested neuraxial analgesia for labor: impact on rates of cesarean and instrumental vaginal delivery. Anesthesiology 106(5):1035, 2007 Merriam A, Ananth CV, Wright JD, et al: Trends in operative vaginal delivery, 2005-2013: a population-based study. BJOG 124(9):1365,2017

1	Merriam A, Ananth CV, Wright JD, et al: Trends in operative vaginal delivery, 2005-2013: a population-based study. BJOG 124(9):1365,2017 Muise L, Duchon A, Brown RH: he efect of artificial caput on performance of vacuum extractors. Obstet GynecoI81(2):170, 1993 Mulder F, Schofelmeer M, Hakvoort R, et al: Risk factors for postpartum urinary retention: a systematic review and meta-analysis. BJOG 119(12): 1440, 2012 Murphy OJ, Libby G, Chien P, et al: Cohort study offorceps delivery and the risk of epilepsy in adulthood. Am J Obstet Gynecol 191 :392, 2004 Murphy OJ, Macleod M, Bahl R, et al: A cohort study of maternal and neonatal morbidiry in relation to use of sequential instruments at operative vaginal delivery. Eur J Obstet Gynecol Reprod Bioi 156(1):41,r2011

1	Nelson L, Furner SE, Westercamp M, et al: Cesarean delivery for the prevention of anal incontinence. Cochrane Database Syst Rev 2:CD006756, 2010 Nilsen ST: Boys born by forceps and vacuum extraction examined at 18 years of age. Acta Obstet Gynecol Scand 63:549, 1984 Nygaard IE, Rao SS, Dawson JD: Anal incontinence after anal sphincter disruption: a 30-year retrospective cohort study. Obstet Gynecol 89:896, 199 O'Callaghan ME, MacLennan AH, Gibson CS, et al: Epidemiologic associations with cerebral palsy. Obstet GynecoIr118(3):576, 2011 O'Mahony F, Hofmeyr GJ, Menon V: Choice of instruments for assisted vaginal delivery. Cochrane Database Syst Rev 11:CD005455, 2010 Osterman MJ, Martin JA, Menacker F: Expanded health data from the new birth certificate, 2006. Nat! Vital Stat Rep 58(5):1, 2009 Palatnik A, Grobman WA, Hellendag MG, et al: Predictors offailed operative vaginal delivery in a contemporary obstetric cohort. Obstet Gynecol 127(3): 501,r2016

1	Palatnik A, Grobman WA, Hellendag MG, et al: Predictors offailed operative vaginal delivery in a contemporary obstetric cohort. Obstet Gynecol 127(3): 501,r2016 Pearl ML, Roberts JM, Laros RK, et al: Vaginal delivery from the persistent occiput posterior position: influence on maternal and neonatal morbidiry. J Reprod Med 38:955, 1993 Pergialiotis V, Vlachos 0, Protopapas A, et al: Risk factors for severe perineal lacerations during childbirth. Int J Gynaecol Obstet 125(1):6,r2014 Pifarotti P, Gargasole C, Folcini C, et al: Acute post-partum urinary retention: analysis of risk factors, a case-control study. Arch Gynecol Obstet 289(6):1249,r2014 Ramphul M, Kennelly MM, Burke G, et al: Risk factors and morbidiry associated with suboptimal instrument placement at instrumental delivery: observational study nested within the Instrumental Delivery & Ultrasound randomised controlled trial ISRCTN 72230496. BJOG 122(4):558,r2015

1	RobertSon PA, Laros RK, Zhao L: Neonatal and maternal outcome in lowpelvic and mid-pelvic operative deliveries. Am J Obstet Gynecol 162: 1436, 1990 Rorrveit G, Dalrveit AK, Hannestad YS, et al: Urinary incontinence after vaginal delivery or cesarean section. N Engl J Med 348:9000, 2003 Schuit E, Kwee A, Westerhuis ME, et al: A clinical prediction model to assess the risk of operative delivery. BJOG 119(8):915,r2012 Schwartz DB, Miodovnik M, Lavin JP Jr: Neonatal outcome among low birth weight infants delivered spontaneously or by low forceps. Obstet Gynecol 62:283, 1983 Skinner S, Davies-Tuck M, Wallace E, et al: Perinatal and maternal outcomes after training residents in forceps before vacuum instrumental birth. Obstet Gynecol 130(1):151,r2017 Seidman OS, Laor A, Gale R, et al: Long-term efects of vacuum and forceps deliveries. Lancet 337:1583, 1991 Solt I, Jackson S, Moore T, et al: Teaching forceps: the impact of proactive faculry. Am J Obstet Gynecol 204(5):448.e1, 2011

1	Solt I, Jackson S, Moore T, et al: Teaching forceps: the impact of proactive faculry. Am J Obstet Gynecol 204(5):448.e1, 2011 Spiliopoulos M, Kareti A, Jain NJ, et al: Risk of peripartum hysterectomy by mode of delivery and prior obstetric history: data from a population-based study. Arch Gynecol Obstet 283(6):1261, 2011 Spong CY, Berghella V, Wenstrom KD, et al: Preventing the first cesarean delivery: summary of a Joint Eunice Kennedy Shriver National Institute of Child Health and Human Development, Society for Maternal-Fetal Medicine, and American College of Obstetricians and Gynecologists X orkshop. Obstet GynecoIr120(5):1181, 2012 Stock SJ, Josephs K, Farquharson S, et al: Matenal and neonatal outcomes of successful Kielland's rotational forceps delivery. Obstet GynecoIr121(5): 1032,r2013

1	Stock SJ, Josephs K, Farquharson S, et al: Matenal and neonatal outcomes of successful Kielland's rotational forceps delivery. Obstet GynecoIr121(5): 1032,r2013 Suwannachat B, Laopaiboon M, Tonmat S, et al: Rapid versus stepwise application of negative pressure in vacuum extraction-assisted vaginal delivery: a multicentre randomised controlled non-inferiority trial. BJOG 118(10): 1247,r2011 Suwannachat B, Lumbiganon P, Laopaiboon M: Rapid versus stepwise negative pressure application for vacuum extraction assisted vaginal delivery. Cochrane Database Syst Rev 8:CD006636, 2012 Tahtinen RNf, Cartwright R, Tsui JF, et al: Long-term impact of mode of delivery on stress urinary incontinence and urgency urinary incontinence: a systematic review and meta-analysis. Eur Urol 70(1):148, 2016

1	Tempest N, Hart A, Walkinshaw S, et al: A re-evaluation of the role of rotational forceps: retrospective comparison of maternal and perinatal outcomes following diferent methods of birth for malposition in the second stage of labor. BJOG 7,r2013 Towner 0, Castro NlA, Eby-Wilkens E, et al: Efect of mode of delivery in nulliparous women on neonatal intracranial injuY. N Engl J /Ied 341: 1 09, 1999 Towner DR, Ciotti MC: Operative vaginal delivery: a cause of birth injury or is it? Clin Obstet Gynecol 50(3):563, 2007 Vacca A: Vacuum-assisted delivery. Best Pract Res Clin Obstet Gynaecol 16:17,r2002 Verhoeven CJ, Nuij C, Janssen-RolfCR, et al: Predictors for failure of vacuumassisted vaginal delivery: a case-control study. Eur J Obstet Gynecol Reprod Bioi 200:29, 2016

1	Verhoeven CJ, Nuij C, Janssen-RolfCR, et al: Predictors for failure of vacuumassisted vaginal delivery: a case-control study. Eur J Obstet Gynecol Reprod Bioi 200:29, 2016 Voll0yhaug I, M0rkved S, Salvesen 0, et al: Forceps delivery is associated with increased risk of pelvic organ prolapse and muscle trauma: a cross-sectional study 16-24 years after irst delivery. Ultrasound Obstet Gynecol 46(4): 487,r2015 Walsh CA, Robson M, McAulife FM: Mode of delivery at term and adverse neonatal outcomes. Obstet Gynecol 121 (1): 122, 2013 Wassen MM, Hukkelhoven CW, Scheepers He, et al: Epidural analgesia and operative delivery: a ten-year population-based cohort study in The Netherlands. Eur J Obstet Gynecol Reprod Bioi 183: 125, 2014 Wen SW, Liu S, Kramer MS, et al: Comparison of maternal and infant outcomes between vacuum extraction and forceps deliveries. Am J Epidemiol 153(2):103,r2001

1	Wen SW, Liu S, Kramer MS, et al: Comparison of maternal and infant outcomes between vacuum extraction and forceps deliveries. Am J Epidemiol 153(2):103,r2001 Werner EF, Janevic TM, IIIuzzi J, et al: Mode of delivery in nulliparous women and neonatal intracranial injury. Obstet Gynecol 118(6): 1239,r2011 Wesley B, Van den Berg B, Reece EA: The effect of operative vaginal delivery on cognitive development. Am J Obstet Gynecol 166:288, 1992 Williams MC, Knuppel A, O'Brien WF, et al: A randomized comparison of assisted vaginal delivery by obstetric forceps and polyethylene vacuum cup. Obstet Gynecol 78:789,r1991 Yancey MK, Pierce B, Schweitzer 0,et al: Observations on labor epidural analgesia and operative delivery rates. Am J Obstet Gynecol 180(2 Pt 1):353, 1999 Yeomans ER: Operative vaginal delivery. In Yeomans ER, Hofman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 2017

1	Yeomans ER: Operative vaginal delivery. In Yeomans ER, Hofman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 2017 CESAREAN DELIVERY IN THE UNITED STATES . .. ... 567 CESAREAN DELIVERY RISKS ......... . . .... .. 568 PATIENT PREPARATION .... .. . . .. ... ... 569 CESAREAN DELIVERY TECHNIQUE . . . ........... 571 LAPAROTOMY ........ .... . .... ... .. .. 571 HYSTEROTOMY .. ..... .. ... ..... .. . .. . 573 PERIPARTUM HYSTERECTOMY ... ......... ...... 580 POSTOPERATIVE CARE . . . . . . . . . . . . . . . . . . . . . . . . . 585 ?he anterior suace of the uterus is opened longitudinaly along its midline. ?his is best accomplished by making an incision a ew centimetres long with a scapel, and then rapidy enlarging it with the scissors to 16 or 18 centimetres. ?he membranes are then rupture, the child is seized by one oot and rapidy extracted -]. Whitridge Williams (1903)

1	From the above description, cesarean technique has evolved during the past century. For example, preference for classical hysterotomy has given way to low transverse incision. Evidence-based data now guide many surgical steps and are presented throughout this chapter. Of deinitions, cesarean delivey defines the birth of a fetus via laparotomy and then hysterotomy. This definition is not applied to removal of the fetus from the abdominal cavity in the case of uterine rupture or with abdominal pregnancy. Rarely, hysterotomy is performed in a woman who has just died or in whom death is expected soon-postmortem orperimortem cesarean delivery (Chap. 47, p. 931).

1	In some instances, abdominal hysterectomy is indicated following delivery. When performed at the time of cesarean delivery, the operation is termed cesarean hysterectomy. If done within a short time after vaginal delivery, it is termedpospartum hysterectomy. Peripartum hysterectomy is a broader term that combines these two. In most cases, hysterectomy is total, but supracervical hysterectomy is an option. The adnexa are not usually removed. In most instances, a simple or type I hysterectomy is performed. However, for women with invasive cervical cancer, radical hysterectomy removes the uterus, parametrium, and proximal vagina to achieve tumor excision with negative margins. Also, for cases of placenta percreta that extend toward the pelvic sidewall, similar radical excision of the parametrium may be needed.

1	In the United States, the cesarean delivery rate rose from 4.5 percent in 1970 to 32.9 percent in 2009. Following this peak, the rate has trended slightly downward, and it was 32.0 percent in 2015 (Martin, 2017). Some indications for performing cesarean delivery are shown in Table More than 85 percent of these operations are performed for four reasollSprior cesarean delivery, dystocia, fetal jeopardy, or abnormal fetal presentation. The latter three compose the main indications for primary cesarean delivery (Barber, 2011; Boyle, 2013). The reasons for persistently significant cesarean rates are not completely understood, but some explanations include the following: 1. Women are having fewer children, thus, a greater percentage of births are among nulparas, who are at increased risk for cesarean delivery. 2. The average maternal age is rising, and older women, especially nulliparas, have a higher risk of cesarean delivery. TABLE 30-1 . Some Indications for Cesarean Delivery

1	2. The average maternal age is rising, and older women, especially nulliparas, have a higher risk of cesarean delivery. TABLE 30-1 . Some Indications for Cesarean Delivery HIV human immunodeficiency virus; HSV herpes simplex virus. 3. he use of electronic etal monitoring is widespread. This practice is associated with an increased cesarean delivery rate compared with intermittent fetal heart rate auscultation. Fetal distress accounts for only a minority of all cesareans. In many more cases, concern for an abnormal or "nonreassuring" fetal heart rate tracing prompts cesarean delivery. 4. Most fetuses presenting breech are now delivered by cesarean. 5. he frequency of operative vaginal delivery has declined. 6. Rates of labor induction continue to rise, and induced labor, especially among nulliparas, raises the cesarean delivery rate. 7. Obesi, which is a cesarean delivery risk, has reached epidemic proportions. 8.

1	7. Obesi, which is a cesarean delivery risk, has reached epidemic proportions. 8. Rates of cesarean delivery in women with preeclampsia have increased, whereas labor induction rates for these patients have declined. 9. he rate of vaginal birth ater cesarean-BAC-has decreased from a high of 28 percent in 1996 and was 11 percent in 2014 (Hamilton, 2015). 10. Elective cesarean deliveries are increasingly being performed for various indications that include maternal request, concern for pelvic loor injury associated with vaginal birth, and reduction of etal injury rates. 11. Assisted reproductive technoloy is more widely used than in the past and is associated with greater cesarean delivery rates (Reddy, 2007). 12. Mapractice litigation related to fetal injury during spontaneous or operative vaginal delivery continues to contribute to the present cesarean delivery rate.

1	12. Mapractice litigation related to fetal injury during spontaneous or operative vaginal delivery continues to contribute to the present cesarean delivery rate. To provide accurate informed consent, understanding both maternal and neonatal risks and beneits with surgery is essential. In broad terms, cesarean delivery has higher maternal surgical risks for the current and subsequent pregnancies compared with spontaneous vaginal birth. his is balanced against lower rates of perineal injury and short-term pelvic floor disorders. For the neonate, cesarean delivery ofers lower rates of birth trauma and stillbirth but greater rates of initial respiratory diiculties.

1	For the mother, death attributable solely to cesarean delivery is rare in the United States. Even so, numerous studies attest to increased mortality risks. Clark and colleagues (2008), in a review of nearly 1.5 million pregnancies, found maternal mortality rates of 2.2 per 100,000 cesarean deliveries compared with 0.2 per 100,000 vaginal births. In a metaanalysis of 203 studies, Guise and coworkers (2010) reported a maternal mortality rate of 13 per 100,000 with elective repeat cesarean delivery compared with 4 per 100,000 women undergoing a trial of labor after prior cesarean.

1	Similar to mortality rates, the frequencies of some maternal complications are increased with all cesarean compared with vaginal deliveries. Villar and associates (2007) reported that maternal morbidity rates increased twofold with cesarean compared with vaginal delivery. Principal among these are infection, hemorrhage, and thromboembolism. In addition, anesthetic complications, which also rarely include death, have a greater incidence with cesarean compared with vaginal delivery (Cheesman, 2009; Hawkins, 201l). Adjacent organs infrequently may be injured, which is described in detail on page 583. Women who undergo a cesarean delivery are much more likely to be delivered by a repeat operation in subsequent pregnancies. For women undergoing subsequent cesarean, the maternal risks just described are even greater (Cahill, 2006; Marshall, 2011; Silver, 2006).

1	As an advantage, cesarean delivery is associated with lower rates of urinary incontinence and pelvic organ prolapse than is vaginal birth (Glazener, 2013; Gyhagen, 2013a,b; Handa, 2011; Leijonhuvud, 2011). Rates of anal incontinence appear uninluenced by delivery route (Fritel, 2007; Nelson, 2010). Protective advantages persist to some degree over time, but cesarean delivery is not totally protective. Moreover, longitudinal studies suggest that initial pelvic floor advantages gained from cesarean delivery are lost as women age (Dolan, 2010; MacArthur, 2011, 2013; Nelson, 2010). To address this, the National Institutes of Health (2006) held a conference on cesarean delivery on maternal request. It summarized that stress urinary incontinence rates after elective cesarean delivery are lower than those following vaginal delivery. However, the duration of this protection is unclear, particularly in older and multiparous populations. This same panel considered the evidence implicating

1	than those following vaginal delivery. However, the duration of this protection is unclear, particularly in older and multiparous populations. This same panel considered the evidence implicating vaginal delivery in other pelvic loor disorders to be weak and not favoring either delivery route.

1	Cesarean delivery is associated with a lower rate of fetal trauma (Linder, 2013; Moczygemba, 2010). lexander and colleagues (2006) found that fetal injury complicated 1 percent of cesarean deliveries. Skin laceration was most common, but others included cephalohematoma, clavicular fracture, brachial plexopathy, skull fracture, and facial nerve palsy. Cesarean deliveries following a failed operative vaginal delivery attempt had the highest injury rate, whereas the lowest rate-0.5 percentoccurred in the elective cesarean delivery group. hat said, Worley and colleagues (2009) noted that approximately a third of women who were delivered at Parkland Hospital entered spontaneous labor at term, and 96 percent of these delivered vaginally without adverse neonatal outcomes.

1	Some evidence shows higher asthma and allergy rates in those delivered by cesarean. With the hope to improve neonatal microbiota, swabbing the newborn mouth with a gauze that was incubated in the maternal vagina 1 hour before surgery is described in preliminary studies. However, the American College of Obstetricians and Gynecologists (2017 e) does not encourage this practice due to few data and the potential for transmission of harmful organisms. Some women request elective cesarean delivery. Data regarding the true incidence of cesarean delivey on matenal request (CDMR) are poor. Rate estimates range from 1 to 8 percent in the United States (Barber, 2011; Declercq, 2005; Gossman, 2006; Menacker, 2006).

1	Reasons for the request include pelvic floor protection, convenience, fear of childbirth, and reduced risk of fetal injury. Data to address these concerns are slowly accruing. One study of more than 66,000 Chinese parturients compared outcomes of those who elected planned vaginal or primary cesarean delivery (Liu, 2015). Short-term serious maternal morbidity and neonatal mortality rates were similar. For the newborns, rates of birth trauma, infection, and hypoxic ischemic encephalopathy were low in both groups but statistically lower with cesarean delivery. Respiratory distress syndrome rates were greater in the CMDR cohort. A smaller study comparing these two routes of delivery support these findings (Larsson, 2011).

1	he debate surrounding CDMR includes these medical points, the concept of informed free choice by the woman, and the autonomy of the physician in ofering CMDR. During the National Institutes of Health panel (2006) cited above, participants noted that most of the maternal and neonatal outcomes examined had insuicient data to permit recommendations. Despite this, the panel was able to draw a few conclusions, which are echoed by the American College of Obstetricians and Gynecologists (2017a). Namely, CMDR should not be performed before 39 weeks' gestation unless fetal lung maturity is conirmed. Cesarean delivery is ideally avoided in women desiring several children because of placental implantation abnormalities and cesarean hysterectomy risks. Finally, CMDR should not be motivated by the unavailability of efective pain management.

1	No nationally recognized standard of care currently dictates the acceptable time interval to begin cesarean delivery. Previously, a 30-minute decision-to-incision interval was recommended. In studying this, Bloom and coworkers (2001) found that 69 percent of 7450 cesareans performed in labor commenced more than 30 minutes after the decision to operate. In a second study, Bloom and colleagues (2006) evaluated cesarean deliveries performed for emergency indications. They reported that failure to achieve a cesarean delivery decision-to-incision time of less than 30 minutes was not associated with a negative neonatal outcome. A subsequent systematic review echoed this inding (ToIcher, 2014). Despite this, when faced with an acute, catastrophic deterioration in fetal condition, cesarean delivery usually is indicated as rapidly as possible, and thus purposeful delays are inappropriate. he American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017)

1	usually is indicated as rapidly as possible, and thus purposeful delays are inappropriate. he American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) recommend that facilities giving obstetrical care should have the ability to initiate cesarean delivery in a time frame that best incorporates maternal and fetal risks and beneits.

1	Obtaining informed consent is a process and not merely a medical document (American College of Obstetricians and Gynecologists, 2015). The conversation should enhance a woman's awareness of her diagnosis and contain a discussion of medical and surgical care alternatives, procedure goals and limitations, and surgical risks. For women with a prior cesarean delivery, the option of a trial of labor should be included for suitable candidates. lso, in those desiring permanent sterilization or intrauterine device insertion, consenting for these can be completed concurrently. n informed patient may decline a particular recommended intervention, and a woman's decision-making autonomy must be respected. In the medical record, clinicians should document her reasons for refusal and should note that the intervention's value and the health consequences of not proceeding with it have been explained.

1	For Jehovah's Witnesses, informed consent discussions regarding blood products ideally begin early in pregnancy. Acceptable blood products vary widely among individual women, and a preoperative checklist of approved products allows superior preparation (Hubbard, 2015; Husarova, 2016). In general, red cells, white cells, platelets, and plasma are viewed as primary blood components and are eschewed. However, certain clotting factors or cell fractions may be acceptable (Lawson, 2015). Before and after surgery, iron, folate, and, if necessary, erythropoietin are accepted agents to help maximize hemoglobin levels. Perioperatively, phlebotomy should be limited, and pediatric collection tubes are preferable. Intraoperative options include treatment of atony to limit blood loss; topical hemostatic agents, tranexamic acid, and desmopressin to promote clot formation; red blood cell salvage or acute normovolemic hemodilution to provide autologous donation; and controlled hypotensive anesthesia,

1	agents, tranexamic acid, and desmopressin to promote clot formation; red blood cell salvage or acute normovolemic hemodilution to provide autologous donation; and controlled hypotensive anesthesia, uterine artery embolization, occlusive vascular balloons, and temporary aortic compression for uncontrolled bleeding (Belfort, 2011; vlason, 2015).

1	• Timing of Scheduled Cesarean Delivery Adverse neonatal sequelae from neonatal immaturity with elective delivery before 39 completed weeks are appreciable (Clark, 2009; Tita, 2009). To avoid these, assurance of fetal maturity before scheduled elective surgery is essential as outlined by the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) and discussed in Chapter 31 (p. 597). To assist with this and other components of cesarean delivery planning, the American College of Obstetricians and Gynecologists (2011, 20 14b) has created Patient Safety Checklists to be completed before the planned surgery.

1	If cesarean delivery is scheduled, a sedative may be given at bedtime the night before surgery. In general, no other sedatives, narcotics, or tranquilizers are administered until after the fetus is born. In one small randomized trial, no benefits were gained from a presurgical enema (Lurie, 2012). Solid food intake is stopped at least 6 to 8 hours before the procedure. Uncomplicated patients may have moderate amounts of clear liquids up to 2 hours before surgery (American Society of Anesthesiologists, 2016). his comports with Enhanced Recovery After Surgery (ERAS) protocols that strive to maintain anabolic homeostasis and advocate clear carbohydrate drinks up to 2 hours before scheduled surgery and early postoperative feeding (Ljungqvist, 2017). Although evidence supports an ERAS approach for many procedures, data specifically addressing this for cesarean delivery are scarce (Wrench, 2015).

1	The woman scheduled for repeat cesarean delivery typically is admitted the day of surgery and evaluated by the obstetrical and anesthesia teams. Recently performed hematocrit and indirect Coombs test are reviewed. If the latter is positive, then availability of compatible blood must be ensured.

1	As discussed in Chapter 25 (p. 490), regional analgesia is preferred for cesarean delivery. An antacid is given shortly before regional analgesia or induction of general anesthesia. One example is Bicitra, 30 mL orally in a single dose. This minimizes the lung injury risk from gastric acid aspiration. Once the woman is supine, a wedge beneath the right hip and lower back creates a left lateral tilt to aid venous return and avoid hypotension. Data are insuicient to determine the value of fetal monitoring before scheduled cesarean delivery in women without risk factors. Our practice is to obtain a 5-minute tracing prior to elective cases. At minimum, fetal heart sounds should be documented in the operating room prior to surgery.

1	Of urther preparations, hair removal at the surgical site does not lower surgical site infection (SSI) rates (Kowalski, 2016). However, if hair is obscuring, it is removed the day of surgery by clipping, which is associated with fewer SSIs than shaving (Tanner, 2011). Chemical depilation the night before surgery compared with clipping has similar SSI rates (Lefebvre, 2015). An electrosurgical grounding pad is placed near the surgical incision and typically on the lateral thigh. n indwelling bladder catheter is typically placed at Parkland Hospital to collapse the bladder away from the hysterotomy incision, to avert urinary retention secondary to regional analgesia, and to allow accurate postoperative urine measurement. Small studies show that catheterization may be withheld in hemodynamically stable women to minimize urinary infections (Abdel-Aleem, 2014; Li, 2011; Nasr, 2009).

1	he risk of venous thromboembolism is increased with pregnancy and almost doubled in those undergoing cesarean delivery Oames, 2006). Accordingly, for all women not already receiving thromboprophylaxis, the American College of Obstetricians and Gynecologists (2017d) recommends initiation of pneumatic compression hose before cesarean delivery. These are usually discontinued once the woman ambulates. Recommendations between organizations vary, and the American College of Chest Physicians suggests only early ambulation for women without risk factors who are undergoing cesarean delivery (Bates, 2012). For women already receiving prophylaxis or those with increased risk factors, they support escalation of prophylaxis. Last, the Royal College of Obstetricians and Gynaecologists (2015) are the most conservative and suggest pharmacological prophylaxis for the largest proportion of patients. hese various methods and recommendations are discussed in Chapter 52 and are shown in Table 52-6 (p.

1	most conservative and suggest pharmacological prophylaxis for the largest proportion of patients. hese various methods and recommendations are discussed in Chapter 52 and are shown in Table 52-6 (p. 1020).

1	Some women scheduled for cesarean delivery have concurrent comorbidity that requires specific management in anticipation of surgery. Among others, these include insulin-requiring or gestational diabetes, coagulopathy or thrombophilia, chronic corticosteroid use, and signiicant reactive airway disease. Surgical preparations are discussed in the respective chapters covering these topics.

1	Cesarean delivery is considered a clean contaminated case, and postoperative febrile morbidity is common. Numerous goodquality trials show that a single dose of an antibiotic given at the time of cesarean delivery signiicantly decreases infectious morbidity (Smaill, 2014). Although more obvious for women undergoing unscheduled cesarean delivery, this practice also pertains to women undergoing elective surgery (American College of Obstetricians and Gynecologists, 2016). Depending on drug allergies, most recommend a single intravenous dose of a 3-lactam antibiotic-either a cephalosporin or extended-spectrum penicillin. A 1-g dose of cefazolin (Ancd) is an eicacious and cost-efective choice. Additional doses are considered in cases with blood loss > 1500 mL or with duration longer than 3 hours. Recommen dations for the best dose in obese parturients are conflicting (Ahmadzia, 2015; Maggio, 2015; Swank, 2015; Young, 2015).

1	levels with a 2-g dose for cesarean deliveries lasting 1.5 hours. women if surgeries were longer (Grupper, 2017). A growing body of evidence supports extending the antibiotic spectrum (Andrews, 2003; Tita, 2008). One large randomized trial added azithromycin, 500 mg intravenously, to standard prophylaxis prior to cesarean delivery for women in labor or with ruptured membranes (Tita, 2016). Rates of wound infection and endometritis were signiicantly lower in the extended-spectrum group compared with those in the standard prophylaxis cohort. In pregnant women with a history of infection with methicillin-resistant Staphylococcus aureus (MRSA), a single dose of vancomycin added to the standard prophylaxis for cesarean deliveries can be elected. Decolonization plays a limited role but may be considered prior to a planned cesarean delivery in women with known MRSA colonization (American College of Obstetricians and Gynecologists, 2016).

1	Significant penicillin or cephalosporin allergy, which manifests by anaphylaxis, angioedema, respiratory distress, or urticaria, merits prophylaxis with a single 600-mg intravenous dose of clindamycin combined with a weight-based dose of an aminoglycoside as an alternative. A 900-mg clindamycin dose is used for obese patients. Antibiotic administration before surgical incision lowers postoperative infection rates without adverse neonatal efects compared with drug administration after umbilical cord clamping (Mackeen, 2014b; Sullivan, 2007; Witt, 2011). Prophylaxis is ideally administered within the 60 minutes prior to the start of planned cesarean delivery. For emergent delivery, antibiotics are given as soon as feasible.

1	Preoperative preparation of the abdominal wall skin is efective to prevent wound infection. Either chlorhexidine or povidone-iodine solutions are suitable (Hadiati, 2014; Ngai, 2015; Springel, 2017). In studies that found a diference, chlorhexidine was favored, and this is our practice (Menderes, 2012; Tuuli, 2016a). In addition, preoperative vaginal cleansing with a povidone-iodine scrub has been evaluated in small randomized trials (Haas, 2014; Caissutti, 2017). Some showed lower rates of metritis, especially for those with ruptured membranes or active labor, but not lower rates of wound infection (Haas, 2010; Memon, 2011; Yildirim, 2012). Some recommend preoperative vaginal cleansing, bur we do not do this at Parkland Hospital.

1	Antibiotic prophylaxis against infective endocarditis is not recommended for most cardiac conditions-exceptions are women with cyanotic heart disease, prosthetic valves, or both (American College of Obstetricians and Gynecologists, 2016). Regimens selected for routine cesarean infection prophylaxis will also serve as appropriate endocarditis coverage (Chap. 49, p. 965).

1	Glycemic control in diabetics lowers wound infection rates and is emphasized in Chapter 57 (p. 1105). Smoking is another modiiable risk, and its mitigation is especially helpful for morbidly obese women (Alanis, 2010; Avila, 2012; Shree, 2016). Intraoperative normothermia lowers wound infection rates in general surgery and is a Surgical Care Improvement Project measure (Kurz, 1996; he Joint Commission, 2016). his tenet might logically be extrapolated to cesarean delivery, although deinitive studies are lacking (Carpenter, 2012). Perioperative supplementation with high-concentration inspired oxygen does not lower wound infection rates (Duggal, 2013; Klingel, 2013).

1	he Joint Commission (2013) established a protocol to prevent surgical errors. For cesarean delivery, all relevant documents are veriied immediately before surgery, and a "time out" is completed. The "time out" requires attention of the entire team to confirm that the patient, site, and procedure are correct. Important discussions also include introduction of the patient-care team members, veriication of prophylactic antibiotics, estimation of procedure length, and communication of anticipated complications. Additionally, requests for special instrumentation should be addressed preoperatively to prevent potential patient compromise and intraoperative delays. An instrument, sponge, and needle count before and after surgery is crucial to surgical safety. If counts are not reconciled, radiographic imaging for retained foreign objects is obtained (American College of Obstetricians and Gynecologists, 20 14a).

1	With minor variations, surgical performance of cesarean delivery is comparable worldwide. Most steps are founded on evidence-based data, and these have been reviewed by Dahlke and associates (2013). As with all surgery, a clear understanding of relevant anatomy is essential, and this is described and illustrated in Chapter 2 (p. 14). In obstetrics, a suprapubic transverse incision or a midline vertical one is chosen for laparotomy. Transverse abdominal entry is by either Pfannenstiel or Maylard incisions. Of all these, the Pfannenstiel incision is selected most frequently for cesarean delivery. Transverse incisions follow Langer lines of skin tension.

1	Thus, compared with vertical ones, Pfannenstiel incisions ofer superior cosmesis and lower incisional hernia rates. Use of the Pfannenstiel incision, however, is often discouraged for cases in which a large operating space is essential or in which access to the upper abdomen may be needed. With transverse incisions, because of the layers created during incision of the internal and external oblique aponeuroses, purulent fluid can collect between these. Therefore, some favor a midline vertical incision for cases with high infection risks. Emergent entry is typically faster with vertical incision during primary and repeat cesarean delivery (Wylie, 2010). Last, neurovascular structures, which include the ilioinguinal and iliohypogastric nerves and supericial and inferior epigastric vessels, are often encountered with transverse inCISIons. Logically, bleeding, wound hematoma, and neurological disruption may more frequently complicate these incisions compared with vertical ones. he best

1	are often encountered with transverse inCISIons. Logically, bleeding, wound hematoma, and neurological disruption may more frequently complicate these incisions compared with vertical ones. he best incision for the morbidly obese parturient is unclear (Smid, 2016). As discussed in Chapter 48 (p. 943), our preference with very obese women is a periumbilical midline vertical incision.

1	he Maylard incision difers mainly from the Pfannenstiel in that the bellies of the rectus abdominis muscle are transected horizontally to widen the operating space. It is technically more diicult due to its required muscle cutting and isolation and ligation of the inferior epigastric arteries, which lie laterally to these muscle bellies. Once access is gained, metal handheld retractors provide exposure for hysterotomy. A few small randomized studies have evaluated postcesarean wound infection rates with a disposable plastic barrier retractor (Alexis-O). Results showing benefit are contradictory (Hinkson, 2016; Scolari Childress, 2016; heodoridis, 2011).

1	With the Pfannenstiel incision, the skin and subcutaneous tissue are incised using a low, transverse, slightly curvilinear incision. his is made at the level of the pubic hairline, which is typically 3 cm above the superior border of the symphysis pubis. The incision is extended laterally suiciently to accommodate delivery-12 to 15 cm is typical. Sharp dissection is continued through the subcutaneous layer to the fascia. The supericial epigastric vessels can usually be identiied halway between the skin and fascia, several centimeters from the midline, and are coag ulated. If lacerated, these may be suture ligated with 3-0 plain gut suture or coag ulated with an electro surgical blade.

1	he fascia is then incised sharply at the midline. he anterior abdominal fascia is typically composed of two visible layers, the aponeurosis from the external oblique muscle and a fused layer containing aponeuroses of the internal oblique and transverse abdominis muscles. Ideally, the two layers are individually incised during lateral extension of the fascial incision. The inferior epigastric vessels usually lie outside the lateral border of the rectus abdominis muscle and beneath the fused aponeuroses of the internal oblique and transverse abdominis muscles. hus, although infrequently required, extension of the fascial incision further laterally may cut these vessels. With extension, these vessels are best identiied and coagulated or ligated to prevent bleeding and vessel retraction.

1	Once the fascia is incised, the inferior fascial edge is grasped with Kocher clamps and elevated by an assistant as muscle either bluntly or sharply until the superior border of the symphysis pubis is reached. Next, the superior fascial edge is grasped and again, separation of fascia from the rectus muscle is completed. Blood vessels coursing between the sheath and muscles are clamped, cut, and ligated, or they are coagulated with an electrosurgery blade. Meticulous hemostasis is imperative to lower rates of incisional hematoma and infection. he fascial separation progresses cephalad and laterally to create a semicircular area above the transverse incision with a radius of approximately 8 cm. This will vary depending on fetal size. he rectus abdominis and pyramidalis muscles are then separated in the midline, irst superiorly and then inferiorly, by sharp and blunt dissection to expose the transversalis fascia and peritoneum.

1	he transversalis fascia and preperitoneal fat are bluntly dissected away to reach the underlying peritoneum. The peritoneum near the upper end of the incision is opened carefully, either bluntly or by elevating it with two hemostats placed approximately 2 cm apart. his upper site lowers cystotomy risks. The tented fold of peritoneum between the clamps is examined and palpated to ensure that omentum, bowel, or bladder is not adjacent. he peritoneum is then incised. The peritoneal incision is extended superiorly to the upper pole of the fascial dissection and downward to just above the peritoneal relection over the bladder. Importantly, in women with prior intraabdominal surgery, including cesarean delivery, omentum or bowel may be adhered to the undersurface of the peritoneum. In women with obstructed labor, the bladder may be pushed cephalad almost to the level of the umbilicus.

1	the operator separates the fascial sheath FIGURE 30-1 The loose peritoneum above the bladder reflection is grasped with forceps from the underlying rectus abdominis and incised with Metzenbaum scissors, This incision begins 2 to 3 cm above the superior margin of the symphysis. It should suiciently long to allow fetal delivery, and 12 to 15 cm is typical. Sharp or electrosurgical blade dissection through the subcutaneous layers ultimately exposes the anterior rectus sheath. A small opening is made sharply with scalpel in the upper half of the linea alba. Placement here helps avoid potential cystotomy. Index and middle ingers are placed beneath the fascia to elevate it, and the fascial incision is extended irst superiorly and then inferiorly with scissors. Midline separation of the rectus muscles and pyramidalis muscles and peritoneal entry are similar to those with the Pfannenstiel incision.

1	Most often, the lower uterine segment is incised transversely as described by Kerr in 1921. Occasionally, vertical incision conined solely to the lower uterine segment may be elected (Kronig, 1912). In contrast, a classical incision begins as a low-vertical incision, which is then extended cephalad into the active portion of the uterine corpus. Last, a fundal or even posterior incision may be selected for cases with placental accrete syndromes. For most cesarean deliveries, this incision is preferred. Compared with a classical incision, it is easier to repair, causes less incision-site bleeding, and promotes less bowel or omentum adherence to the myometrial incision. Located in the inactive segment, it also is less likely to rupture during a subsequent pregnancy.

1	Before any hysterotomy, the surgeon palpates the fundus to identiy degrees of uterine rotation. The uterus may be rotated so that one round ligament is more anterior and closer to the midline. In such cases, the uterus can be FIGURE 30-2 This peritoneal edge is elevated and incised laterally. bladder is gently separated from the underlying lower uterine segment with blunt or sharp dissection within this vesicouterine space (Fig. 30-3). This bladder lap creation efectively moves the bladder away from the planned hysterotomy site. It also helps prevent bladder laceration if an unintended inferior hysterotomy extension occurs during fetal delivery. In general, this caudad separation of bladder does not exceed 5 cm and usually is less. However, in instances in which cesarean hysterectomy is planned or anticipated, extended caudad dissection is recommended to aid total hysterectomy and decrease the risk of cystotomy.

1	manually reoriented and held to permit centering of the incision. This avoids incision extension into and laceration of the adjacent uterine artery. A moist sponge may be used to pack protruding bowel away from the operative ield. he relection of peritoneum at the upper margin of the bladder and overlying the lower uterine segment is grasped in the midline with forceps and incised transversely with scissors (Fig. 30-1). Following this initial incision, scissors are inserted between peritoneum and lower uterine segment. Open scissors are pushed laterally from the midline on each side. This transverse peritoneal incision extends almost the full length of the lower uterine segment. As the lateral margin on each side is approached, the scissors are directed slightly cephalad (Fig. 30-2). The lower FIGURE 30-3 Cross section shows blunt dissection of the bladder of the uterus to edge of peritoneum is elevated, and the expose the lower uterine segment.

1	FIGURE 30-4 The myometrium is incised with shallow strokes to avoid cutting the fetal head. FIGURE 30-5 After entering the uterine cavity, the incision is extended laterally with fingers or with bandage scissors (inset). Some surgeons do not create a bladder lap. The main advantage is a shorter skin incision-to-delivery time. However, data supporting this practice are limited (O'Neill, 2014; Tuuli, 2012). Uterine Incision. The uterus is entered through the lower uterine segment. Digital palpation to find the physiological border between irmer upper segment myometrium and the more flexible lower segment can guide placement. The bladder flap incision can also serve as a guide, and a hysterotomy site near this line is often selected.

1	For women with advanced or complete cervical dilatation, the hysterotomy is placed relatively higher. Failure to adjust increases the risk of lateral extension of the incision into the uterine arteries. It may also lead to incision of the cervix or vagina rather than the lower uterine segment. Such incisions into the cervix can distort postoperative cervical anatomy.

1	The uterus can be incised by various techniques. Each is initiated by using a scalpel to transversely incise the exposed lower uterine segment for 1 to 2 cm in the midline (Fig. 30-4). Repetitive shallow strokes avoid fetal laceration. As the myometrium thins, a fingertip can then bluntly enter the uterine cavity. Once the uterus is opened, the hysterotomy is lengthened by simply spreading the incision, using lateral and slightly upward pressure applied with each index inger 30-5). Some evidence also supports widening the lower-uterine-segment incision instead with ingers pulling in opposition in a cephalocaudad direction (Cromi, 2008; Xodo, 2016).

1	Alternatively, if the lower uterine segment is thick and unyielding, cutting laterally and then slightly upward with bandage scissors will lengthen the incision. Importantly, when scissors are used, the index and midline fingers of the nondominant hand should be insinuated beneath the myometrium and above fetal parts to prevent fetal laceration. Comparing blunt and sharp expansion of the initial uterine incision, blunt stretch is associated with fewer unintended incision extensions, shorter operative time, and less blood loss. However, the rates of infection and need for transfusion do not difer (ASIClOglu, 2014; Saad, 2014). The uterine incision is made large enough to allow delivery of the fetus without tearing into the uterine vessels that course along the lateral uterine margins. If the placenta is encountered in the incision line, it must be either detached or incised. Placental function is thereby compromised, and thus delivery is performed expeditiously.

1	At times, a low transverse hysterotomy is selected but provides inadequate room for delivery. In such instances, one corner of the hysterotomy incision is extended cephalad into the contractile portion of the myometrium-a J incision. If this is completed bilaterally, a U incision is formed. Last, some prefer instead to extend in the midline-a T incision. As expected, each has higher intraoperative blood loss (Boyle, 1996; Patterson, 2002). Moreover, as these extend into the contractile portion, a trial of labor is more likely to be complicated by uterine rupture in future pregnancies. Delivey of the Fetus. In a cephalic presentation, a hand is slipped into the uterine cavity between the symphysis and fetal head. The head is elevated gently with the ingers and palm through the incision. Once the head enters the incision, delivery may be aided by modest transabdominal fundal pressure (Fig. 30-6).

1	After a long labor with cephalopelvic disproportion, the fetal head may be tightly wedged in the birth canal. Release of an impacted fetal head raises the risk of hysterotomy extension, of associated blood loss, and of fetal skull fracture. In FIGURE 30-6 Delivery of the fetal head. this situation, there are three considerations for delivery. First, a "push" method may be used. With this, upward pressure exerted by a hand in the vagina by an assistant will help to dislodge the head and allow its delivery above the symphysis. If this is anticipated, a patient in frog-leg position may allow easier vaginal access. Second, as an alternative, a "pull" method grasps the fetal legs to bring them through the hysterotomy. he fetus is then delivered by traction as one would complete a breech extraction.

1	Second, as an alternative, a "pull" method grasps the fetal legs to bring them through the hysterotomy. he fetus is then delivered by traction as one would complete a breech extraction. ized trials and retrospective cohort studies (Berhan, 2014; J eve, 2016; Nooh, 2017). A low vertical hysterotomy incision, which will give more room for the "pull" technique, may be selected. If a low transverse incision has already been made, then this can be extended to a J-, U-, or T-incision as previously discussed. The third method is use of the "fetal pillow," which is a distensible intravaginal balloon that when inflated, elevates the fetal head. The device is available outside the United States, but evidence for its eicacy is limited (Safa, 2016; Seal, 2016).

1	Conversely, in women without labor, the fetal head may be unmolded and without a leading cephalic point. The round head may be diicult to lift through the uterine incision in a relatively thick lower segment that is unattenuated by labor. In such instances, either forceps or a vacuum device may be used to deliver the fetal head (Fig. 30-7).

1	fter head delivery, a inger should be passed across the fetal neck to determine whether it is encircled by one or more umbilical cord loops. If present, these are slipped over the head. The head is rotated to an occiput transverse position, which aligns the fetal bisacromial diameter vertically. The sides of the head are grasped with two hands, and gentle downward traction is applied until the anterior shoulder enters the hysterotomy incision (Fig. 30-8). Next, by upward movement, the posterior shoulder is delivered. During delivery, abrupt or powerful force is avoided to avert brachial plexus injury. With steady outward traction, the rest of the body then readily follows. Gentle fundal pressure may aid this.

1	With some exceptions, current American Heart Association neonatal resuscitation recommendations eschew suctioning immediately following birth, even with meconium present (Wyckof, 2015). A fuller discussion of this and delayed umbilical cord clamping is found in Chapter 27 (p. 518). he umbilical cord is clamped, and the newborn is given to the team member who will conduct resuscitative eforts as needed.

1	Comparing elective cesarean under neuraxial anesthesia and spontaneous vaginal deliveries, studies show that the need for neonatal resuscitation is not practically signiicant between the two (Atherton, 2006; Gordon, 2005; Jacob, 1997). he American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) recommend that "a qualiied person who is skilled in neonatal resuscitation should be in the delivery room." At Parkland Hospital, pediatric nurse practitioners attend uncomplicated, scheduled cesarean deliveries. Notably, as anticipated neonatal risks rise, so too should the resuscitative skills of the attendants (Wyckof, 2015). To promote breastfeeding, the American College of Obstetricians and Gynecologists (2017b) recommends skin-to-skin contact between newborn and mother in the delivery room. FIGURE 30-7 A.The first cesarean forceps blade is placed. B. Slight upward and outward traction is used to lit the head through the incision.

1	FIGURE 30-7 A.The first cesarean forceps blade is placed. B. Slight upward and outward traction is used to lit the head through the incision. Although most randomized trials focus on vaginal birth, several small studies support such contact following cesarean delivery, and this our practice (Moore, 2016; Stevens, 2014).

1	After birth, an intravenous infusion containing two ampules or 20 units of oxytocin per liter of crystalloid is infused at 10 mLlmin. Some prefer higher infusion dosages, however, nondilute boluses are avoided because of associated hypotension (Roach, 2013). Once the uterus contracts satisfactorily, the rate can be reduced. An alternative is carbetocin-a longer-acting oxytocin derivative that is not available in the United Statesthat provides suitable, albeit more expensive, hemorrhage prophylaxis Qin, 2016). Ergot-alkaloids are second-tier agents and carry hypertensive side efects. Carboprost, a IS-methyl derivative of prostaglandin F2> is another second-tier agent used to treat uterine atony. Some but certainly not all studies indicate that misoprostol appears to perform similarly to oxytocin (Chaudhuri, 2014; Conde-Agudelo, 2013). Finally, some recommend the use of tranexamic acid added to a standard oxytocin infusion to decrease blood loss (Simonazzi, 2016; Wang, 2015). Its

1	to oxytocin (Chaudhuri, 2014; Conde-Agudelo, 2013). Finally, some recommend the use of tranexamic acid added to a standard oxytocin infusion to decrease blood loss (Simonazzi, 2016; Wang, 2015). Its antifibrinolytic action and efects on thromboembolism rates in pregnant surgical patients are unclear. Larger trials are needed before widespread use. Additional discussions of all these agents are found in Chapter 41 (p. 759).

1	Delivey of the Placenta. he uterine incision is observed for any vigorously bleeding sites. These should be quickly clamped with Pennington or ring forceps. Although some surgeons may prefer , manual removal of the placenta, spontaneous delivery prompted by some cord traction may reduce the risk of operative blood loss and infection (Anorlu, 2008; Baksu, 2005). Fundal massage may FIGURE 30-8 The anterior (A) and then the posterior (B) shoulder are delivered. begin as soon as the fetus is delivered to hasten placental separation and delivery (Fig. 30-9).

1	FIGURE 30-8 The anterior (A) and then the posterior (B) shoulder are delivered. begin as soon as the fetus is delivered to hasten placental separation and delivery (Fig. 30-9). Immediately after delivery and quick gross inspection of the placenta, the uterine cavity is suctioned and wiped out with a gauze sponge to remove avulsed membranes, vernix, and clots. In the past, double-gloved fingers or ring forceps placed through the hysterotomy incision were used to dilate an ostensibly closed cervix. his practice does not reduce infection rates from potential hematometra and is not recommended (Kirscht, 2017; Liabsuetrakul, 2011).

1	Uterine Repair. Ater placental delivery, the uterus is lited through the incision and onto the draped abdominal wall, and the fundus is covered with a moistened laparotomy sponge. We favor this and believe a relaxed, atonic uterus can be recognized quickly and massage applied. Incision and bleeding points are more easily visualized and repaired, especially if there have been extensions. Adnexal exposure is superior, and thus, tubal sterilization is easier. Instead, some clinicians prefer to close the hysterotomy with the uterus in situ. Comparing these two approaches, febrile morbidity, pain, and blood loss are not signiicantly diferent (Walsh, 2009; Zaphiratos, 2015).

1	Before hysterotomy closure, previously clamped large vessels may be ligated separately or incorporated within the running incision closure. IUD insertion, if planned, is completed prior to hysterotomy closure (Chap. 38, p. 685). One angle of the uterine incision is grasped to stabilize and maneuver the incision. he uterine incision is then closed with one or two layers of continuous 0-or no. 1 absorbable suture (Fig. 30-10). Chromic catgut suture is used by many, but some prefer synthetic delayed-absorbable polyglactin 910 (Vicryl). In subsequent pregnancy, neither suture type has been shown superior by mitigating against greater rates of adverse pregnancy outcomes such as uterine incision rupture (CORONIS Collaborative Group, 2016). Single-layer closure is typically faster and is not associated with higher rates of infection or transfusion (CAESAR Study Collaborative Group, 2010; Dodd, 2014; Roberge, 2014). Moreover, most studies observed that the number of layers does not

1	is not associated with higher rates of infection or transfusion (CAESAR Study Collaborative Group, 2010; Dodd, 2014; Roberge, 2014). Moreover, most studies observed that the number of layers does not significantly afect complication rates in the next pregnancy (Chapman, 1997; CORONIS Collaborative Group, 2016; Durnwald, 2003; Roberge, 2011).

1	At Parkland Hospital, we use a one-layer uterine closure with chromic catgut. he initial suture is placed just beyond one angle of the uterine incision. A continuous, locking suture line for hemostasis is then performed, with each suture penetrating the full thickness of the myometrium. he suture line then extends to a point just beyond the opposite incision angle. If approximation is not satisfactory after a single layer or if bleeding sites persist, then more sutures are required. Either another layer of running suture is placed to achieve approximation and hemostasis, or individual bleeding sites can be secured with targeted figure-of-eight or mattress stitches. FIGURE 30-9 Placenta bulging through the uterine incision as the uterus contracts. A hand gently massages the fundus to help aid spontaneous placental separation.

1	FIGURE 30-9 Placenta bulging through the uterine incision as the uterus contracts. A hand gently massages the fundus to help aid spontaneous placental separation. Traditionally, the peritoneum in the anterior cul-de-sac is approximated with a continuous 2-0 chromic catgut suture line. Multiple randomized trials suggest that omission of this step causes no postoperative complications (Grundsell, 1998; Irion, 1996; Nagele, 1996). If tubal sterilization is to be performed, it is completed as described in Chapter 39 (p. 702). FIGURE 30-10 The cut edges of the uterine incision are approximated with a running, locking suture.

1	FIGURE 30-10 The cut edges of the uterine incision are approximated with a running, locking suture. Following cesarean delivery, adhesions commonly form within the vesicouterine space or between the anterior abdominal wall and uterus. And, with each successive pregnancy, the percentage of afected women and adhesion severity rise (Morales, 2007; Tulandi, 2009). Adhesions can significantly lengthen incision-to-delivery time and total operative time (Rossouw, 2013; Sikirica, 2012). Although occurring infrequently, rates of cystotomy and bowel injury are also increased because of adhesive disease (Rahman, 2009; Silver, 2006).

1	Intuitively, scarring can be reduced by handling tissues delicately, achieving hemostasis, and minimizing tissue ischemia, infection, and foreign-body reaction. Most recent data on shortand long-term outcomes show no benefit to peritoneal closure (CAESAR Study Collaborative Group, 2010; CORONIS Collaborative Group, 2013, 2016; Kapustian, 2012). Similarly, most studies show no benefit from placement of an adhesion barrier at the hysterotomy site (Edwards, 2014; Kiefer, 2016). Any laparotomy sponges are removed, and the paracolic gutters and cul-de-sac are gently suctioned of blood and amnionic fluid. Some surgeons irrigate the gutters and cul-de-sac, especially in the presence of infection or meconium. Routine irrigation in low-risk women, however, leads to greater intraoperative nausea but not to lower postoperative infection rates (Eke, 2016; Viney, 2012).

1	Prior to abdominal closure, correct sponge and instrument counts are verified. he rectus abdominis muscle bellies are allowed to fall into place. With significant diastasis, the rectus muscles may be approximated with one or two figure-of-eight sutures of 0 or no. 1 chromic gut suture. The overlying rectus fascia is closed by a continuous, nonlocking technique with a delayed-absorbable suture. In patients with a higher risk for infection, there may be theoretical value in selecting a monoilament suture here rather than braided material.

1	The subcutaneous tissue usually need not be closed if it is less than 2 em thick. With thicker layers, however, closure is recommended to minimize seroma and hematoma formation, which can lead to wound infection and/or disruption (Bohman, 1992; Chelmow, 2004). One recent metaanalysis found lower rates of seroma formation and of developing any wound complication with closure, but hematoma and wound infection rates were unafected (Pergialiotis, 2017). Addition of a subcutaneous drain does not prevent significant wound complications (Hellums, 2007; Ramsey, 2005).

1	Skin is closed with a running subcuticular stitch of 4-0 delayed-absorbable suture, with adhesive glue, or with staples. In comparison, final cosmetic results and infection rates appear similar, skin suturing takes longer, but wound separation rates are higher with metal staples (Basha, 2010; Figueroa, 2013; Mackeen, 2014a, 2015). Poliglecaprone 25 (NIonocryl) or polyglactin 910 (Vieryl) are both suitable (Tuuli, 2016b). Outcomes with 2-octyl cyanoacrylate adhesive (Dermabond) were equivalent to sutures for Pfannenstiel incisions (Daykan, 2017; Siddiqui, 2013). A sterile thin abdominal wound dress ing is suicient. In morbidly obese women, application of a prophylactic negative-pressure device atop the closed skin incision to prevent seroma and subsequent infection does not appear to lower wound complication rates (Hussamy, 2018; Smid, 2017).

1	The Pfannenstiel-Kerr technique just described has been used for decades. More recently, Joel-Cohen and Misgav Ladach techniques have been added (Holmgren, 1999). These difer from traditional Pfannenstiel-Kerr entry mainly by their initial incision placement and greater use of blunt dissection.

1	he Joel-Cohen technique creates a straight 10-cm transverse skin incision 3 em below the level of the anterior superior iliac spines (Olofson, 2015). he subcutaneous tissue layer is opened sharply 2 to 3 em in the midline. This is carried down, without lateral extension, to the fascia. A small transverse incision is made in the fascia, and curved Mayo scissors are pushed laterally on each side and beneath intact subcutaneous fat to incise the fascia. With this incision completed, an index finger from each hand is inserted between the rectus abdominis muscle bellies and beneath the fascia. One finger is moved cranially and the other caudally, in opposition, to separate the bellies and further open the fascial incision. hen, a finger from each hand hooks under each belly to stretch the muscles laterally. The peritoneum is entered sharply, and this incision is sharply extended cephalocaudad. Entry with the Misgav Ladach technique difers in that the peritoneum is entered bluntly

1	the muscles laterally. The peritoneum is entered sharply, and this incision is sharply extended cephalocaudad. Entry with the Misgav Ladach technique difers in that the peritoneum is entered bluntly (Holmgren, 1999).

1	Modiications to the Joel-Cohen method abound. For emergency delivery, we begin along a line somewhat lower on the abdomen. For speed, we extend the fascial incision bluntly by hooking index fingers in the fascial incision's lateral angles and pulling laterally (Hofmeyr, 2009; Olofson, 2015). Index fingers insinuated between the rectus bellies then move cephalocaudad in opposition to stretch the incision. Blunt index-finger dissection enters the peritoneum, and again, cranial and caudad opposing stretch opens this layer. Last, all the layers of the abdominal wall are grasped manually and pulled laterally in opposition to further open the operating space. These techniques have been associated with shorter operative times and with lower rates of intraoperative blood loss and postoperative pain (Mathai, 2013). They may, however, prove diicult for women with anterior rectus fibrosis and peritoneal adhesions (BoIze, 2013).

1	Indications. This incision is usually avoided because it encompasses the active upper uterine segment and thus is prone to rupture with subsequent pregnancies. Some indications stem from diiculty in exposing or safely entering the lower uterine segment. For example, a densely adhered bladder from previous surgery is encountered; a leiomyoma occupies the lower uterine segment; the cervix has been invaded by cancer; or massive maternal obesity precludes safe access to the lower uterine segment. A classical incision is also preferred for placenta previa with anterior implantation, especially those complicated by placenta accrete syndromes. In extreme cases of this, the typical classical hysterotomy may be placed even higher in the uterine body or posteriorly to avoid the placenta. As such, fetuses with cephalic presentation are then delivered in a manner similar to total breech extraction (Chap. 28, p. 548).

1	In other instances, fetal indications dictate the need. Transverse lie of a large etus, especially if the membranes are ruptured and the shoulder is impacted in the birth canal, usually necessitates a classical incision. A fetus presenting as a back-down transverse lie is particularly di.cult to deliver through a transverse uterine incision. In instances when the fetus is very small and breech, a classical incision may be preferable (Osmundson, 2013). In such cases, the poorly developed lower uterine segment provides inadequate space for the manipulations required for breech delivery. Or, less commonly, the small fetal head may become entrapped by a contracting uterine fundus following membrane rupture. Last, with multiple fetuses, a classical incision again may provide suitable room for extraction of fetuses that may be malpositioned or preterm (Osmundson, 2015).

1	Uterine Incision and Repair. A vertical uterine incision is initiated with a scalpel beginning as low as possible and preferably within the lower uterine segment (Fig. 30-11). If adhesions, insu.cient exposure, a tumor, or placenta percreta preclude development of a bladder flap, then the incision is made above the level of the bladder. Once the uterus is entered with a scalpel, the incision is extended cephalad with bandage scissors until it is long enough to permit delivery of the fetus. With scissor use, the fingers of the nondominant hand are insinuated between the myometrium and fetus to prevent fetal laceration. As the incision is opened, numerous large vessels that bleed profusely are commonly encountered within the myometrium. The remainder of fetal and placental delivery mirrors that with a low transverse hysterotomy. For incision closure, one method employs a layer of 0-or no. 1 chromic catgut with a running stitch to approximate the

1	For incision closure, one method employs a layer of 0-or no. 1 chromic catgut with a running stitch to approximate the FIGURE 30-11 An initial small vertical hysterotomy incision is made in the lower uterine segment. Fingers are insinuated between the myometrium and fetus to avoid fetal laceration. Scissors extend the incision cephalad as needed for delivery. (Reproduced with permission from Johnson DD: Cesarean delivery. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201o7.) deeper length of the incision (Fig. 30-12). he outer layer of myometrium is then closed along its length with similar suture and with a running suture line. To achieve good approximation and to prevent the suture from tearing through the myometrium,

1	FIGURE 30-12 Classical incision closure. The deeper half (left) and superficial half (middle) of the incision are closed in a running fashion. The serosa is then closed (right). (Reproduced with permission from Johnson DD: Cesarean delivery. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili Education, 201o7.) it is helpul to have an assistant compress the uterus on each side of the wound toward the midline as each stitch is placed.

1	Hysterectomy is most commonly performed to arrest or prevent hemorrhage from intractable uterine atony or abnormal placentation (Bateman, 2012; Hernandez, 2012; Owolabi, 2013). It is more often completed during or after cesarean delivery but may be needed following vaginal birth. If all deliveries are considered, the peripartum hysterectomy rate in the United States approximates 1 per 1000 births and has risen signiicantly during the past few decades (Bateman, 2012; Govindappagari, 2016). During a 25-year period, the rate of peripartum hysterectomy at Parkland Hospital was 1.7 per 1000 births (Hernandez, 2012). Most of this rise is attributed to the increasing rates of cesarean delivery and its associated complications in subsequent pregnancy (Bateman, 2012; Bodelon, 2009; Flood, 2009; Orbach, 2011). Of hysterectomies, approximately one half to two thirds are total, whereas the remaining cases are supracervical (Rossi, 2010; Shellhaas, 2009).

1	Major complications of peripartum hysterectomy include greater blood loss and risk of urinary tract damage. Blood loss is usually appreciable because hysterectomy is being performed for hemorrhage that frequently is torrential, and the procedure itself is associated with substantial bleeding. lthough many cases with hemorrhage cannot be anticipated, those with abnormal implantation are oten identiied antepartum. Preoperative preparations for placenta accreta are discussed in Chapter 41 (p. 781) and have also been outlined by the Society for Maternal-Fetal FIGURE 30-14 The posterior leaf of the broad ligament adjacent to the uterus is perforated just beneath the fallopian tube, uteroovarian ligaments, and ovarian vessels. FIGURE 30-13 The round ligaments are clamped, doubly ligated, FIGURE 30-15 The uteroovarian ligament and fallopian tube are and transected bilaterally. clamped and cut. The lateral pedicle is doubly ligated.

1	Medicine (2010) and American College of Obste tricians and Gynecologists (2017 c). An important factor afecting the cesarean hysterectomy complication rate is whether the operation is performed electively or emergently. tomy, rates of blood loss, blood transfusion, and urinary tract complications are lower that with emergent procedures (Briery, 2007; Glaze, 2008).

1	tomy, rates of blood loss, blood transfusion, and urinary tract complications are lower that with emergent procedures (Briery, 2007; Glaze, 2008). Total or supracervical hysterectomy is performed using standard operative techniques. Adequate exposure is essential, but initially, placement of a self-retaining retractor such as a Balfour is not necessary. Rather, satisfactory exposure is obtained with cephalad traction on the uterus by an assistant, along with handheld Richardson or Deaver retractors. he bladder lap is deflected downward to the level of the cervix if possible to permit total hysterectomy. In cases in which cesarean hysterectomy is planned or strongly suspected, extended bladder flap dissection is ideally completed before initial hysterotomy. Later attempts at bladder dissection may be obscured by bleeding, or excess blood may be lost while this dissection is performed.

1	After cesarean delivery, the placenta is typically removed. In cases of placenta accrete syndrome for which hysterectomy is already planned, the placenta is usually left undisturbed in situ. In either situation, if the hysterotomy incision is bleeding appreciably, it can be sutured or Pennington or sponge forceps can be applied for hemostasis. If bleeding is minimal, neither maneuver is necessary.

1	he round ligament is divided close to the uterus between clamps, and each pedicle is ligated (Fig. 30-13). Either 0 or no. 1 suture can be used in either chromic gut or delayedabsorbable material. he anterior leaf of the broad ligament is incised downward to meet the former bladder flap incision. he posterior leaf of the broad ligament adjacent to the uterus is bluntly or sharply perforated just beneath the fallopian tube, uteroovarian ligament, and ovarian vessels (Fig. 30-14). hese structures together are then divided between sturdy clamps placed close to the uterus 30-15). he lateral pedicle is doubly ligated. The medial clamp remains and is removed later with the entire uterine specimen. he posterior leaf of the broad ligament is incised toward the uterosacral ligaments (Fig. 30-16). Next, the bladder and attached peritoneal flap are further deflected and dissected as needed. If the bladder flap is unusually adhered, as it may be after previous hysterotomy incisions, careful sharp

1	the bladder and attached peritoneal flap are further deflected and dissected as needed. If the bladder flap is unusually adhered, as it may be after previous hysterotomy incisions, careful sharp dissection may be necessary (Fig. 30-17).

1	Posterior leaf of broad ligament Uterosacral :n�s � FIGURE 30-16 The posterior leaf of the broad ligament is divided inferiorly toward the uterosacral ligament. -. .k7{ FIGURE 30-17 The bladder is dissected sharply from the lower uterine segment. ::=:\!i ..., FIGURE 30-18 The uterine vessels are clamped, and a third medial clamp helps prevent "back bleeding." Once divided, the lateral vascular pedicle is doubly ligated to ensure hemostasis.

1	Special care is required from this point on to avoid injury to the ureters, which pass beneath the uterine arteries. To help accomplish this, an assistant places constant traction to pull the uterus in the direction away from the side on which the uterine vessels are being ligated. he ascending uterine artery and veins on either side are identiied. These vessels are then clamped adjacent to the uterus. For security, some may prefer two lateral clamps as shown in Figure 30-18. he most medial clamp helps prevents back bleeding from the uterus and remains for later removal with the specimen. The uterine vessels are divided, and the lateral tissue pedicle is doubly suture ligated. After securing the uterine vessels on one side, the round ligament, adnexal pedicle, and uterine vessels are then addressed on the contralateral side.

1	With cesarean hysterectomy, it may be more advantageous in cases of profuse hemorrhage to rapidly double clamp and divide all of the vascular pedicles between clamps to gain hemostasis. The surgical team can then return to ligate all of the pedicles. Even if total hysterectomy is planned, we find it technically easier in many cases to finish the operation after amputating the uterine fundus and placing Ochsner or Kocher clamps on the cervical stump for traction and hemostasis. Self-retaining retractors also may be placed at this time. To remove the cervix, the bladder is mobilized further if needed. This carries the ureters caudad as the bladder is retracted beneath the symphysis and will prevent laceration or suturing of the bladder during cervical excision and vaginal cuf closure. FIGURE 30-19 The cardinal ligaments are clamped, incised, and ligated.

1	FIGURE 30-19 The cardinal ligaments are clamped, incised, and ligated. The cardinal ligament, the uterosacral ligaments, and the many large vessels these ligaments contain are clamped systematically with sturdy Heaney-type curved or straight clamps (Fig. 30-19). he clamps are placed as close to the cervix as possible, taking care not to include excessive tissue in each clamp. The tissue between the pair of clamps is incised, and the lateral pedicle is suture ligated. hese steps are repeated caudally and bilaterally until the level of the lateral vaginal fornix is reached on each side. In this way, the descending branches of the uterine vessels are clamped, cut, and ligated as the cervix is separated from the cardinal ligaments.

1	If the cervix is efaced and dilated considerably, its softness may obscure palpable identiication of the cervicovaginal junction. he junction location can be ascertained through a vertical uterine incision made anteriorly in the midline, either through the open hysterotomy incision or through an incision created at the level of the ligated uterine vessels. A finger is directed inferiorly through the incision to identiy the free margin of the dilated, efaced cervix. The contaminated glove is replaced. Another useful method to identiy the cervical margins in cases of planned hysterectomy is to transvaginally place four metal skin clips or brightly colored sutures at 12, 3, 6, and 9 o'clock positions on the cervical edges.

1	Immediately below the level of the cervix, a curved clamp is placed across the lateral vaginal fornix on each side, and the vagina is incised above the clamp (Fig. 30-20).The cervix is inspected to ensure that it has been completely removed. A transfixing suture is used for vaginal cuf closure as each clamp is removed. Interrupted stitches may be added to approximate the middle portion FIGURE 30-20 A curved clamp is placed across the lateral vaginal fornix below the level of the cervix, and the tissue incised medially to the point of the clamp.

1	FIGURE 30-20 A curved clamp is placed across the lateral vaginal fornix below the level of the cervix, and the tissue incised medially to the point of the clamp. of the cuf Each lateral vaginal fornix is secured to the uterosac raliligaments to mitigate later vaginal prolapse. For cuf closure, some surgeons instead prefer to close the vagina by apposing the anterior and posterior vaginal walls with interrupted figure-of ll sites are examined carefully for bleeding. One technique is to perform a systematic bilateral survey from the fallopian tube and ovarian ligament pedicles to the vaginal vault and bladder lap. Bleeding sites are ligated with care to avoid the ureters. The abdominal wall normally is closed in layers, as previously described for cesarean delivery (p. 578).

1	To perform a subtotal nysterectomy, the uterine body is amputated immediately above the level of uterine artery ligation. The cervical stump may be closed with continuous or interrupted chromic catgut suture. Subtotal hysterectomy is often all that is necessary to stop hemorrhage. It may be preferred for women who would benefit from a shorter surgery or for those with extensive adhesions that threaten significant urinary tract injury. Because of the large adnexal vessels and their close proximity to the uterus, it may be necessalY to remove one or both adnexa to obtain hemostasis. Briery and colleagues (2007) reported unilateral or bilateral oophorectomy in a fourth of cases. Preoperative counseling for anticipated hysterectomy should include this possibility. hese injuries are rare during cesarean delivery. The bladder laceration rate approximates 2 per 1000 cesarean deliveries, FIGURE 30-21 A running-lock suture approximates the vaginal wall edges.

1	hese injuries are rare during cesarean delivery. The bladder laceration rate approximates 2 per 1000 cesarean deliveries, FIGURE 30-21 A running-lock suture approximates the vaginal wall edges. whereas that for ureteral trauma nears 0.3 per 1000 cases (Giingordiik, 2010; Oliphant, 2014; Rajasekar, 1997). Bowel is damaged in about 1 in 1000 cesarean deliveries (Silver, 2006). Bladder laceration most commonly occurs during blunt or sharp dissection in the vesicouterine space to create the bladder lap, during peritoneal cavity entry, and during hysterotomy (Phipps, 2005; Rahman, 2009). Risks are prior cesarean delivery; emergency cesarean delivery; comorbid adhesive disease; cesarean hysterectomy, especially cases with morbidly adherent placenta; and surgery in second-stage labor compared with irst-stage (Alexander, 2007; Silver, 2006; Yossepowitch, 2004).

1	Bladder injury is typically identified intraoperatively, and initially, a clear-luid gush or the Foley bulb may be seen. If cystotomy is suspected, it can be conirmed with retrograde instillation of infant formula or methylene-blue-stained saline through a Foley catheter into the bladder. Leakage of opaque milk or methylene blue aids in identiication of the laceration as well as delineation of its borders. The dome is lacerated in 95 percent of cases, and injuries at the trigone form the remainder (Phipps, 2005). Prior to cystotomy repair, ureters are examined, and surveillance for urine jets from each oriice follows. This can be done directly through the cystotomy, if at the dome, or through a separate diagnostic extraperitoneal or retropubic cystotomy, if injury nears the trigone. Jet visualization can be assisted by 50 mg of methylene blue administered intravenously.

1	Once ureteral patency is conirmed, the bladder may be closed with a two-or three-layer running closure using a 3-0 absorbable or delayed-absorbable suture (Fig. 30-22). he first layer inverts the mucosa into the bladder. The bladder is then illed with a marker luid to demonstrate integrity of the repair. Leaking defects can be closed with interrupted reinforcing stitches. Subsequent layers reapproximate the bladder muscularis. Postoperative care requires continuous bladder drainage for 7 to 14 days to permit healing and minimize the risk of fistula formation. U ropathogen prophylaxis during this drainage is not required. Also, cystourethrography need not be routinely performed prior to catheter removal for a simple, �ingle laceration (Davis, 1999).

1	FIGURE 30-22 Cystotomy repair, A. The primary layer inverts the bladder mucosa with running or interrupted sutures of 3-0 delayedabsorbable or absorbable suture, B. Second and possibly a third layer approximate the bladder muscularis to reinforce the incision closure. Larger lacerations in or near the trigone require careful attention. Specialists may be consulted, and in preparation, ureteral stents can be assembled. In these cases, ureteral oriices are directly inspected to document jets from both. If not seen, then stents may be passed through the cystotomy and into each oriice to conirm patency. Once this is confirmed, repair should not disrupt the ureteral orifices, and stents may remain to ensure ureteral patency.

1	U nrepaired cystotomy can manifest as hematuria, oliguria, abdominal pain, ileus, ascites, peritonitis, fever, urinoma, or istula. For diagnosis, retrograde cystography or abdominal computed tomography (CT) with cystography can be used (Tarney, 2013). Cystoscopy is also an option but may require an operating room. Once identified, prompt repair is indicated (Balgobin, 2017).

1	hese injuries occur most oten during repair of hysterotomy extensions into the broad ligament or vagina (Eisenkop, 1982). If ureteral injury is suspected, methylene blue is administered. The pelvis is directly inspected for dye extravasation, which suggests ureteral transection. Next, brisk dye-stained urine jets are sought from each oriice to exclude ureteral kinking or ligation. Oriice viewing may be via cystoscopy, if available; through a comorbid traumatic cystotomy; or through a diagnostic cystotomy. With sluggish or absent jets, consultation with a specialist is typically requested. A ureteral catheter is irst threaded to identiY a potential obstruction site and guide ureterolysis. Kinked or ligated ureters can be relieved by release of ensnaring sutures. Crush injuries are inspected to ensure vital tissue. In these cases, stents are let to avert ureteral stricture. A Foley cath�ter remains for 7 to 10 days, and the ureteral catheters are removed via cystoscopy ater 14 days.

1	to ensure vital tissue. In these cases, stents are let to avert ureteral stricture. A Foley cath�ter remains for 7 to 10 days, and the ureteral catheters are removed via cystoscopy ater 14 days. Intravenous pyelography (IVP) is usually not necessary before removal of the stent if it was placed as a precautionary measure ater relatively minor injury (Davis, 1999).

1	Crush injuries with devascularization, thermal injury, or transection require more extensive repair. If a healthy-appearing ureter can be reimplanted into the bladder without undue tension, then ureteroneocystostomy is preferable. For more proximal injuries, ureteroureterostomy, psoas hitch, or Boari lap creation may be needed. An explanation of these more extensive procedures is found in Cunningham and Gistrap s Operative Obstetrics, 3rd edition (Balgobin, 2017). Unrecognized ureteral injury can mimic those of cystotomy with the addition of possible costovertebral angle tenderness. CT urography is a preferred initial diagnostic tool (Sharp, 2016). he duration of time from injury to identification directs repair. hose identified early are often suitable for immediate repair.

1	Serosal tears represent weak points in the small bowel. If obstruction develops postoperatively, these weak spots may perforate, leading to peritonitis. If serosal tears are few in number, they can be oversewn with either a ine absorbable or nonabsorbable suture (Davis, 1999). More signiicant lacerations are often repaired in consultation with a general surgeon or gynecologic oncologist.

1	During and after cesarean delivery, requirements for intravenous luids can vary considerably. Administered fluids consist of either lactated Ringer solution or a similar crystalloid solution with 5-percent dextrose. Typically, at least 2 L is infused during surgery. Blood loss with uncomplicated cesarean delivery approximates 1000 mL. he average-sized woman with a hematocrit of 30 percent or more and with a normally expanded blood and extracellular luid volume most often will tolerate blood loss up to 2000 mL without diiculty. Unappreciated bleeding through the vagina during the procedure, bleeding concealed in the uterus after its closure, or both commonly lead to underestimation.

1	Blood loss averages 1500 mL with elective cesarean hysterectomy, although this is variable (Pritchard, 1965). Most peripartum hysterectomies are unscheduled, and blood loss in these cases is correspondingly greater. Thus, in addition to close monitoring of vital signs and urine output, the hematocrit should be determined intra-or postoperatively as indicated.

1	he amount of vaginal bleeding is closely monitored for at least an hour in the immediate postoperative period. he uterine fundus is also identiied frequently by palpation to ensure that the uterus remains irmly contracted. Unfortunately, as conduction analgesia fades or the woman awakens from general anesthesia, abdominal palpation is likely to produce pain. A patient-controlled analgesia (PCA) pump can be efective. Once regional analgesia begins to fade or the woman becomes fully awake following general anesthesia, criteria for transfer to the postpartum ward include minimal bleeding, stable vital signs, and adequate urine output. Analgesia, Vital Signs, Intravenous Fluids

1	Analgesia, Vital Signs, Intravenous Fluids Several schemes are suitable for postoperative pain control. One PCA regimen uses intravenous morphine given as needed as a I-mg dose with a 6-minute lockout interval and maximum dose of 30 mg in 4 hours. An additional 2-mg booster dose is permitted for a maximum of 2 doses. Alternatively, intramuscular (1M) meperidine, 50 to 75 mg every 3 to 4 hours, or 1M morphine, 10 to 15 mg every 3 to 4 hours, is suitable. In a trial using these options, Yost and associates (2004) found that morphine provided superior pain relief to meperidine and was associated with significantly higher rates of breastfeeding and continuation of newborn rooming in. Breastfeeding can be ini tiated the day of surgery. If the mother elects not to breastfeed, a binder that supports the breasts without marked compression usually will minimize discomfort.

1	After transfer to her room, the woman is assessed at least hourly for 4 hours, and thereafter at intervals of 4 hours. Deep breathing and coughing are encouraged to prevent atelectasis. Vital signs, uterine tone, urine output, and bleeding are evalu ated. The hematocrit is routinely measured the morning after surgery. It is checked sooner if there was unusual blood loss or if there is hypotension, tachycardia, oliguria, or other evidence to suggest hypovolemia. If the hematocrit is decreased signii cantly from the preoperative level, the measurement is repeated and a search is instituted to identiy the cause. If the hematocrit stabilizes, the mother can be allowed to ambulate, and if there is little likelihood of further blood loss, iron therapy is preferred to transfusion.

1	Postpartum, the patient begins to mobilize and excrete her physiologically expanded extravascular volume. Thus, mainte sistent oral intake is reestablished. If urine output falls below 30 mLlhr, however, the woman should be reevaluated promptly. The cause of the oliguria can range from unrecognized blood loss to an antidiuretic efect from infused oxytocin. Women undergoing unscheduled cesarean delivery may have pathological retention or constriction of the extracellular fluid compartment caused by severe preeclampsia, sepsis syndrome, vomiting, prolonged labor without adequate fluid intake, or increased blood loss. Women with these complications are generally observed in the recovery room until stabilization is assured.

1	The Foley catheter most often can be removed by 12 hours postoperatively, or more conveniently, the morning after surgery. he prevalence of urinary retention following cesarean delivery approximates 3 to 7 percent (Chap. 36, p. 660). Failure to progress in labor and postoperative narcotic analgesia are identified risks (Chai, 2008; Kandadai, 2014; Liang, 2007).

1	In uncomplicated cases, liquids or solid food may be ofered within hours of surgery and advanced as tolerated (Guo, 2015). Some degree of adynamic ileus follows virtually every abdominal operation, but in most cases of cesarean delivery, it is negligible. Postoperative ileus symptoms include abdominal distention, gas pains, and an inability to pass flatus or stool. With persistent nausea and vomiting or with prolonged bowel function delay, radiological imaging may aid exclusion of bowel obstruction. A plain abdominal radiograph is a frequent first choice. However, in the general population, this study is diagnostic in only 50 to 60 percent of small bowel obstruction cases (Maglinte, 1997). Thus, a radiograph may best serve as a triage tool in cases in which ileus is the suspected diagnosis. Notably, an enlarged postpartum uterus can compress the rectosigmoid and prevent it from illing with gas. Thus, indings suggesting a distal colonic obstruction may confuse true cases of transient

1	Notably, an enlarged postpartum uterus can compress the rectosigmoid and prevent it from illing with gas. Thus, indings suggesting a distal colonic obstruction may confuse true cases of transient ileus (Kammen, 2000). In comparison, CT with intravenous contrast provides greater accuracy for small bowel obstruction. Oral contrast is concurrently given when SBO is a consideration (Katz, 2013). Last, although uncommon, an unrecognized bowel injury may be responsible for otherwise unexplained fever and poor bowel function. Here, CT may be most diagnostic of potential etiologies.

1	As treatment of ileus, intravenous luids compensate for poor oral intake and losses from emesis. Electrolyte imbalances are corrected to improve smooth muscle activity and avoid bowel edema. Nasogastric decompression is necessary only with persistent vomiting or severe distention. For prevention, intraoperative goals strive to minimize bowel manipulation, avoid excess intravenous luids or profound hypovolemia, and limit surgery length (Bragg, 2015). Postoperatively, gum chewing enhances early bowel function recovery by nearly 7 hours after cesarean delivery (Zhu, 2014). Among studies, chewing was initiated immediately or up to 12 hours later, lasted 15 to 60 minutes, and was repeated in at least three sessions daily (Pereira Gomes Morais, 2016).

1	As discussed earlier, women undergoing cesarean delivery have an increased risk of venous thromboembolism compared with those delivering vaginally. Early ambulation lowers the thromboembolism risk. Walking to the bathroom begins, initially with assistance. Brief walks are encouraged, and ambulation can be timed so that a recently administered analgesic will minimize discomfort. lthough not evidence based, we remove the surgical dressing after 24 hours and inspect the incision daily. One small randomized trial showed no wound healing diferences if removed at 6 hours (Peleg, 2016). By the third postpartum day, showering is not harmful to the incision. Prior to this, a plastic cover can maintain dryness during showers. If used, staples often are removed on the fourth day. Once removed, dressing strips (Steri-Strips) can be placed as needed for 1 week to reinforce skin edge integrity. If there is concern for supericial wound separation, staples remain in place for 7 to 10 days.

1	For uncomplicated cesarean delivery, the average hospitalization length is three to four days (Buie, 2010). Data from studies suggest that earlier discharge is feasible for properly selected women and newborns (Bayoumi, 2016; Tan, 2012). Protocols ideally include earlier reevaluation for neonatal jaundice.

1	Activities during the irst week should be restricted to selfcare and newborn care with assistance. Driving can be resumed when pain does not limit the ability to brake quickly and when narcotic medications are not in use. In women with cesarean delivery, intercourse was resumed in 44 percent by 6 weeks postpartum, in 81 percent by 3 months, and 97 percent at 1 year (McDonald, 2013). After the puerperium, the quality of sexual functioning does not difer between those undergoing spontaneous vaginal delivery or cesarean (Chang, 2015; Fehniger, 2013; Rogers, 2014). Rerurn to work is variable. Six weeks is commonly cited, although many women use the Family and Medical Leave Act to allow up to 12 weeks for recovery and newborn bonding. Abdel-Aleem H, Aboelnasr MF, Jayousi TM, et al: Indwelling bladder catheterisation as part of intraoperative and postoperative care for caesarean section. Cochrane Database Syst Rev 4:CDO 1 0322, 2014

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1	American College of Obstetricians and Gynecologists: Optimizing support for breastfeeding as part of obstetric practice. Committee Opinion No. 658, February 2016, Reairmed 2017b American College of Obstetricians and Gynecologists: Placenta accreta. Committee Opinion No. 529, July 2012, Reairmed 2017c American College of Obstetricians and Gynecologists: Thromboembolism in pregnancy. Practice Bulletin No. 123, September 201r1, Reairmed 201 d American College of Obstetricians and Gynecologists: Vaginal seeding. Committee Opinion No. 725, November 201/e American Society of Anesthesiologists: Task Force on Obstetrical Anesthesia: practice guidelines for obstetrical anesthesia. Anesthesiology 124:270, 2016 Andrews WW, Hauth JC, Cliver SP, et al: Randomized clinical trial of extended spectrum antibiotic prophylaxis with coverage for Ureaplasma urealyticum to reduce post-cesarean delivery endometritis. Obstet Gynecol 101(6):1183,r2003

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1	Walsh CA, Walsh SR: Extraabdominal vs intraabdominal uterine repair at cesarean delivery: a metaanalysis. Amr] Obstet Gynecol 200(6):625. e1,r2009 Wang HY, Hong SK, Duan Y, et al: Tranexamic acid and blood loss during and after cesarean section: a meta-analysis.r] PerinatoIr35(10):818, 2015 Witt A, Doner M, Petricevic L, et al: Antibiotic prophylaxis before surgery vs after cord clamping in elective cesarean delivery: a double-blind, prospective, randomized, placebo-controlled trial. Arch Surg 146(12): 1404, 2011 Worley KC, Mcintire DD, Leveno K]: he prognosis for spontaneous labor in women with uncomplicated term pregnancies: implications for cesarean delivery on maternal request. Obstet Gynecol 113(4):812,r2009 Wrench I], Allison A, Galimberti A, et al: Introduction of enhanced recovery for elective caesarean section enabling next day discharge: a tertiary centre experience. Int] Obstet Anesth 24(2):124,2015

1	Wyckof MH, Aziz K, Escobedo MB, et 1: Part 13: Neonatal resuscitation: 2015 American Heart Association guidelines update for cardiopulmonary resuscitation and emergency cardiovascular care. Circulation 132(18 SuppI2):S543, 2015 Wylie B], Gilbert S, Landon MB, et al: Comparison of transverse and vertical skin incision for emergency cesarean delivery. Obstet GynecoIr115(6):1134, 2010 Xodo S, Saccone G, Cromi A, et a1: Cephalad-caudad versus transverse blunt expansion of the low transverse uterine incision during cesarean delivery. Eur] Obstet Gynecol Reprod BioI 202:75, 2016 Yildirim G, Gungorduk K, AsIClOgiu 0, et 1: Does vaginal preparation with povidone-iodine prior to caesarean delivery reduce the risk of endometritis? A randomized controlled trial.] Matern Fetal Neonatal Med 25(11):2316,r2012 Yossepowitch 0, Baniel ], Livne PM: Urological injuries during cesarean section: intraoperative diagnosis and management.r] UroIr172(1):196, 2004

1	Yost NP, Bloom SL, Sibley MK, et al: A hospital-sponsored quality improvement study of pain management after cesarean delivery. Amr] Obstet Gynecol 190:1341, 2004 Young OM, Shaik IH, Twedt R, et al: Pharmacokinetics of cefazolin prophylaxis in obese gravidae at time of cesarean delivery. Am ] Obstet Gynecol 213(4):541.e1,r2015 Zaphiratos V, George RB, Boyd ]c, et al: Uterine exteriorization compared with in situ repair for cesarean delivery: a systematic review and meta-analysis. Can] Anaesth 62(11):1209,r2015 Zhu P, Wang WJ, Zhang SL, et al: Efects of gum chewing on postoperative bowel motility after caesarean section: a meta-analysis of randomised controlled trials. B]OG 121(7):787,r2014 100 YEARS OF CONTROVERSY .e....e...e....e....e...e. 591 INFLUENCING FACTORS ....e. .e.. .e....e......e. 592 DELIVERY ROUTE RISKS . . ........e..... ..e... 593 CANDIDATES FOR TRIAL OF LABOR. . . . . . . . . . . . . . . 594 LABOR AND DELIVERY CONSIDERATIONS ..e..e...e.. 597

1	DELIVERY ROUTE RISKS . . ........e..... ..e... 593 CANDIDATES FOR TRIAL OF LABOR. . . . . . . . . . . . . . . 594 LABOR AND DELIVERY CONSIDERATIONS ..e..e...e.. 597 UTERINE SCAR ......e..e...e........ ... 598 MULTIPLE REPEAT CESAREAN DELIVERIESe. . . . . .. . . . 599 VAGINAL BIRTH AFTER CESAREAN-2017 . ...... 600 The occurrence of pregnancy ater a Caesarean section, however, is not always devoid of danger, cases have been reported in which the uterine cicatrix ruptured in the latter part of a subsequent gestation. It is also stated that the adhesions that sometimes orm between the uterus and the abdominal wall occasionaly exert a deleterious inluence in subsequent pregnancies. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) From the above, there was an early appreciation for some of the major problems encountered in women with a prior cesarean delivery. Few issues in modern obstetrics have been as controversial as the management of these women. Indeed, the dangers associated with uterine rupture led to the oft-quoted remark by Cragin in 1916: "Once a cesarean, always a cesarean." As we reach the 100-year mark of Cragin's pronouncement, the issue remains largely unsettled.

1	By the beginning of the 20th century, cesarean delivery had become relatively safe. But, as women survived the first operation and conceived again, they were now at risk for rupture of the uterine scar. Still, the specter of rupture did not did not result in strict adherence to repeat cesarean delivery. Indeed, Eastman (1950) described a 30-percent postcesarean vaginal delivery rate at Johns Hopkins Hospital. he uterine rupture incidence was 2 percent and associated with a 1 O-percent maternal mortality rate. During the 1960s, observational studies suggested that vaginal delivery was a reasonable option (Pauerstein, 1966, 1969). Germane to this is that through the 1960s, the overall cesarean delivery rate approximated only 5 percent. Since then, as the primary cesarean rate escalated, the rate for repeat cesarean delivery followed (Rosenstein, 2013).

1	During the 1980s, a National Institutes of Health (NIH) Consensus Development Conference (1981) was convened, and it questioned the necessity of routine repeat cesarean delivery. With support and encouragement from the American College of Obstetricians and Gynecologists (1988, 1994), enthusiastic attempts were begun to increase the use of vaginal birth after cesa rea n-VBA. These attempts were highly successful, and VBAC rates increased from 3.4 percent in 1980 to a peak of 28.3 percent in 1996. These rates, along with a concomitant decline in total cesarean delivery rates for the United States, are shown in Figure 31-1.

1	As the vaginal delivery rate increased, so did reports of uterine rupture-related maternal and perinatal morbidity and mortality 1996; Sachs, 1999). These complications dampened prevailing enthusiasm for a trial of labor after cesarean section (TOLAC) and stimulated the American College of Obstetricians and Gynecologists (1998) to caution that such trials should be attempted only j:.:c)�00 .j!a:

1	FIGURE 31-1 Total, primary, and low-risk cesarean delivery (CS) rates and vaginal birth after previous cesarean (VBAC) rates in the United States, 1989-2015. Epochs denoted within rectangles represent contemporaneous ongoing events related to these rates. ACOG = American College of Obstetricians and Gynecologists; NIH = National Institutes of Health; PB = practice bulletin. (Data from Hamilton, 2015,2016; National Institutes of Health: NIH Consensus Development Conference, 2010.) in appropriately equipped institutions with physicians readiy available to provide emergency care. Less than a year later, the College (1999) recommended that physicians should be immediatey available. Many believe that this change ofone wordfrom readiy to immediatey available-was in large part responsible for the decade-long decline in national VBAC rates illustrated in Figure 31-1 (Cheng, 2014; Leeman, 2013).

1	Uddin and colleagues (2013) reported the proportion of women with a prior cesarean delivery who underwent TOLAC. This number peaked in 1995, when slightly more than half of all of these women chose this option. Since that time,

1	FIGURE 31-2 Percentage of births with trial of labor among all deliveries with a prior cesarean delivery and percentage ofsuccessful trials oflabor among all trials oflabor ater caesarean delivery in the United States, 1990-2009. TOLAC = trial of labor ater cesarean delivery; VBAC = vaginal birth after cesarean delivery. (Data from Uddin SFG, Simon AE: Rates and success rates oftrial of labor ater cesarean delivery in the United States, 1990-2009. Matern Child Health J 17:1309,2013.) the Oice of Medical Applications of Research (0MAR) convened an NIH Consensus Development Conference Panel (2010) to study the issues of VBAC. The panel report included a contemporaneous summary concerning the risks and benefits of repeat cesarean versus vaginal delivery. These indings are subsequently described along with summaries of current recommendations by various professional organizations. Importantly, data from California indicate that VBAC rates have not perceptibly increased since the 2010 NIH

1	along with summaries of current recommendations by various professional organizations. Importantly, data from California indicate that VBAC rates have not perceptibly increased since the 2010 NIH Consensus Conference (Barger, 2013).

1	For the woman who has had a previous cesarean delivery, planning for future pregnancies and the delivery route should begin with preconceptional counseling and be addressed once again early in prenatal care. Importantly, any decision is subject to continuing revisions as dictated by exigencies that arise during pregnancy. Assuming no mitigating circumstances, there are two basic choices. First, a TOAC ofers the goal of achieving VBA. If cesarean delivery becomes necessary during the trial, then it is termed a "failed trial oflabor." A second choice is elective repeat cesarean delivey (ERCD). his includes scheduled cesareandelivery as well as unscheduled but planned cesarean delivery for spontaneous labor or another indication. he ultimate decision should weigh clinical factors known to influence TOLAC success as well as benefits and risks. As expected, these rates vary between institutions and providers. Factors that inluence a successful TOLAC are listed in Table

1	Finally, economic, staing, and medicolegal factors may shape the decision to ofer TOLAC. the proportion of women attempting TOLAC declined to a nadir in 2006 of about 16 percent and has subsequently increased to 20 to 25 percent through 2009. These investigators further reported that the percentage of VBACs reached its peak in 2000 with approximately 70 percent ofwomen being successful, but this has subsequently declined to a nadir of 38 percent in 2008 (Fig. 31-2). In reality, several other interrelated factors-both medical and nonmedicalhave undoubtedly contributed to declining VBAC rates. Because of their complexity and importance, the Eunice Kennedy Shriver National Institute of Child Health and Human Devel TABLE 31-1. Some Factors That Influence a Successful Trial of Labor in a Woman with Prior Cesarean Delivery aMost consider these absolute contraindications. EGA = estimated gestational age.

1	TABLE 31-1. Some Factors That Influence a Successful Trial of Labor in a Woman with Prior Cesarean Delivery aMost consider these absolute contraindications. EGA = estimated gestational age. As evidence mounted that the risk of uterine rupture might be greater than expected, the American College of Obstetricians and Gynecologists (1988, 1998, 1999, 2017a) issued updated Practice Bulletins supporting labor trials but also urging a more cautious approach. It is problematic that both options have risks and beneits to mother and fetus but that these are not always congruent.

1	Rates of uterine rupture and associated complications clearly are increased with TO LAC. Uterine rupture typically is classified as either (1) complete, when all layers of the uterine wall are separated, or (2) incomplete, when the uterine muscle is separated but the visceral peritoneum is intact. Incomplete rupture is also commonly referred to as uterine dehiscence. It is these risks that underpin most of the angst in attempting TOLAC. Despite this, some have argued that these factors should weigh only minimally in the decision because their absolute risk is low. One systematic review by Guise and colleagues (2010) concluded that the risk of uterine rupture was significantly elevated in women undergoing TOLAC-absolute risk of 0.47 percent and relative risk of 20.7-compared with those choosing ERCD.

1	he Maternal-Fetal Medicine Units Network conducted a prospective study at 19 academic centers (Landon, 2004). The outcomes of nearly 18,000 women attempting TOLAC were compared with more than 15,000 gravidas undergoing ERCD. The absolute risk of uterine rupture was 0.7 percent compared with no reported uterine ruptures in the ERCD cohort (Table 31-2). .10st studies suggest that the maternal mortaliy rate does not difer significantly between these two groups (Landon, 2004; Mozurkewich, 2000). But, the aforementioned systematic review by Guise (2010) found the Single mother Classical or T incision Increased maternal age Prior rupture Macrosomic fetus Patient refusal Obesity Transfundal surgery Breech Obstetrical contraindication,

1	Single mother Classical or T incision Increased maternal age Prior rupture Macrosomic fetus Patient refusal Obesity Transfundal surgery Breech Obstetrical contraindication, Multifetal pregnancy e.g., previa Preeclampsia Inadequate facilities risk of maternal death to be signiicantly reduced for women undergoing TOLAC compared with ERCD. In a retrospective Canadian cohort study, the maternal death rate for women undergoing ERCD was 5.6 per 100,000 cases compared with 1.6 per 100,000 for those attempting TOLAC (Wen, 2005).

1	Estimates of maternal morbidiy are also conflicting. he review by Guise (2010) observed no significant diferences in the risk of hysterectomy or transfusion. But, another metaanalysis reported that women undergoing TOLAC were approximately half as likely to require a blood transfusion or hysterectomy compared with those undergoing ERCD (Mozurkewich, 2000). Conversely, in the Network study, investigators observed that the risks of transfusion and infection were significantly greater for women attempting TOLAC (Landon, 2004). This disparity is also found among other studies. Notably, compared with a successful TOLAC, the risk of these major complications was ivefold greater with an attempted vaginal delivery that failed (Babbar, 2013; Rossi, 2008).

1	TOLAC is associated with significantly higher perinatal mortaliy rates compared with ERCD. he perinatal rate with TOLAC is 0.13 compared with 0.05 percent for ERCD, and the neonatal mortality rates are 0.i11 versus 0.06 percent, respectively (Guise, 2010). In another study of nearly 25,000 women with a prior cesarean delivery, the vaginal-delivery-related perinatal death risk was 1.3 per 1000 among 15,515 women electing TOLAC. Although this absolute risk is small, it is 11 times greater than the risk found in 9014 women with ERCD (Smith, 2002). TOLAC also appears to be associated with a higher risk of hypoxic ischemic encephalopathy (HIE) than ERCD. The Network study reported the incidence of HIE at term to be 46 per 100,000 TOLACs compared with zero cases in women undergoing ERCD (Landon, 2004). Uterine dehiscence 11s9 (0.7)

1	Uterine dehiscence 11s9 (0.7) Hysterectomy 41 (0.2) 76 (0.5) 1.38 (1s.04-1.85) 0.77 (0.51-1s.17) .03 .22 aDenominator is 15,338 for the trial of labor group and 15,014 for the elective repeat cesarean delivery group. confidence interval; HIE hypoxic ischemic encephalopathy; NA not applicablesNICHD = National Institute of Child Health and Human Development; NS = not significant. Adapted from Landon, 2004.

1	confidence interval; HIE hypoxic ischemic encephalopathy; NA not applicablesNICHD = National Institute of Child Health and Human Development; NS = not significant. Adapted from Landon, 2004. In the systematic review, the absolute risk of transient tachypnea of the newborn was slightly higher with ERCD compared with TOAC-4.2 versus 3.6 percent (Guise, 2010). But, neonatal bag and mask ventilation were used more often in newborns delivered following TOAC than in those delivered by ERCD-5.4 versus 2.5 percent. Finally, there are no signiicant diferences in 5-minute Apgar scores or neonatal intensive care unit admission rates for newborns delivered by TO AC compared with those delivered by ERCD. Birth trauma from lacerations is more commonly seen in neonates born by ERCD.

1	Few high-quality data are available to guide selection of TO AC candidates. In a population-based cohort study of 41,450 women he type and number of prior cesarean deliveries are overriding factors in recommending TOAC. Women with one prior low-transverse hysterotomy have the lowest risk of symptomatic scar separation (Table 31-3). The highest risks are with prior vertical incisions extending into the fundus, such as that

1	TABLE 31 -3. Types of Prior Uterine Incisions and Estimated Risks for Uterine Rupture delivering in California hospitals, Gregory and colleagues (2008) Classical reported a TOAC success rate of74 percent when no maternal, T-shaped fetal, or placental complications were present. Several algorithms and nomograms have been developed to aid prediction, but none has demonstrated reasonable prognostic value (Grobman, 2007b, 2008,i2009; Macones, 2006; Metz, 2013; Srinivas, 2007). A predictive model for failed trial of labor, however, was found to be somewhat predictive of uterine rupture or dehiscence (Stanhope, 2013). Despite these limitations for precision, several points are pertinent to candidate evaluation and are described in the next sections. Current recommendations of the American College of Obstetricians and Gynecologists (2017 a) are that most women with one previous low-transverse hysterotomy are candidates, and if appropriate, they should be counseled regarding TOAC and ERCD

1	of Obstetricians and Gynecologists (2017 a) are that most women with one previous low-transverse hysterotomy are candidates, and if appropriate, they should be counseled regarding TOAC and ERCD options. Although not our practice, those with two prior low-transverse incisions may be considered.

1	aSee text for definition. Data from the American College of Obstetricians and Gynecologists, 201s7a; Cahill, 20s1 Ob; Chauhan, 2002; Landon, 2006; Macones, 2005a,b; Martin, 1997; Miller, 1994; Sciscione, 2008; Society for Maternal-Fetal Medicine, 2012; Tahseen, 201s0. FIGURE 31-3 Ruptured vertical cesarean delivery scar (arrow) identified at time of repeat cesarean delivery early in labor. The two black asterisks to the left indicate some sites of densely adhered omentum. shown in Figure 3i1-3. Importantly, in some women, a classical scar will rupture before labor onset, and this can happen several weeks before term. In a review of 157 women with prior classical cesarean delivery, one woman had a complete uterine rupture before labor onset, whereas 9 percent had a uterine dehiscence (Chauhan, 2002).

1	he risk of uterine rupture in women with a prior vertical incision that did not extend into the fundus is unclear. Martin (1997) and Shipp (1999) and their coworkers reported that these low-vertical uterine incisions did not have an increased risk for rupture compared with low-transverse incisions. The American College of Obstetricians and Gynecologists (2017a) concluded that although evidence is limited, women with a prior vertical incision in the lower uterine segment without fundal extension may be candidates for TOLAC. This is in contrast to prior classical or T -shaped uterine incisions, which are considered by most as contraindications to labor.

1	Although there are few indications for a primary classical incision, 53 percent of women undergoing cesarean delivery between 24°/7 weeks and 256r weeks have such an incision (Osmundson, 20l3). By 28 weeks' gestation, the risk drops to 35 percent and declines to < 10 percent by 32 weeks. he likelihood of classical uterine incision is also increased by noncephalic presentations. In those instances-for example, preterm breech fetus with an undeveloped lower segment-the "low vertical" incision almost invariably extends into the active segment. Prior preterm cesarean delivery may result in a twofold increased risk for rupture (Sciscione, 2008). This may be in part explained by the greater likelihood with a preterm fetus of upward uterine incision extension. Lannon and coworkers (2015) compared 456 women with a prior periviable cesarean delivery with more than 10,000 women whose prior cesarean delivery occurred at term. hey observed uterine rupture in 1.8 percent in the prior periviable

1	456 women with a prior periviable cesarean delivery with more than 10,000 women whose prior cesarean delivery occurred at term. hey observed uterine rupture in 1.8 percent in the prior periviable group versus 0.4 percent in the prior term group. Of the uterine ruptures in the periviable group, half were in women whose prior uterine incision was described as low transverse. Harper and associates (2009) did not confirm these findings.

1	There are also special considerations for women with uterine malformations who have undergone cesarean delivery. Earlier reports suggested that the uterine rupture risk in a subsequent pregnancy was greater than the risk in those with a prior low transverse hysterotomy and normally formed uterus (Ravasia, 1999). But, in a study of 103 women with miillerian duct anomalies, there were no cases of uterine rupture (Erez, 2007). Given the wide range of risk for uterine rupture associated with the various uterine incision types, it is not surprising that most fellows of the American College of Obstetricians and Gynecologists consider the type of prior incision to be the most important factor when considering a TOLAC (Coleman, 2005).

1	Gynecologists consider the type of prior incision to be the most important factor when considering a TOLAC (Coleman, 2005). As discussed in Chapter 30 (p. 577), the low-transverse hysterotomy incision can be sutured in either one or two layers. A metaanalysis by Roberge and colleagues (2014) compared single-versus double-layer closure and locking versus unlocking suture for uterine closure. They reported that rates for uterine dehiscence or uterine rupture for these closures did not difer signiicantly. Single-layer closure and locked irst layer, however, was associated with a reduced myometrial thickness during subsequent sonographic measurement. In contrast, Bennich and coworkers (2016) reported that a double-layer closure did not increase the residual myometrial thickness when saline contrast sonography was done several months postpartum. At Parkland Hospital, we routinely close the lower-segment incision with one running, locking suture line. Number of Prior Cesarean Incisions

1	Number of Prior Cesarean Incisions At least three studies report a doubling or tripling of the rupture rate in women with two compared with one prior transverse hysterotomy (.1acones, 2005a; Miller, 1994; Tahseen, 2010). In contrast, analysis of the Network database by Landon and associates (2006) did not conirm this. Instead, they reported an insigniicant diference in the uterine rupture rate in 975 women with multiple prior cesarean deliveries compared with 16,915 women with a single prior operation-0.9 versus 0.7 percent, respectively. As discussed on page 599, other serious maternal morbidity increases along with the number of prior cesarean deliveries (Marshall, 2011). Imaging of Prior Incision

1	Sonographic measurement of a prior hysterotomy incision has been used to predict the likelihood of rupture. Large defects in a nonpregnant uterus forecast a greater risk for subsequent rupture (Osser, 201i1). Naji and coworkers (20l3a,b) found that the residual myometrial thickness decreased as pregnancy progressed and that rupture correlated with a thinner scar. In a systematic review, women with a prior low-transverse cesarean incision underwent third-trimester sonographic evaluation (Jastrow, 2010a). Investigators concluded that the thickness of the lower uterine segment was a strong predictor for a uterine scar defect in women with prior cesarean delivery. They deined this segment as the smallest measurement between urine in the maternal bladder and amnionic luid. hat said, they could not ind an ideal threshold value to recommend TOLAC. his same group subsequently recruited 1856 women contemplating vaginal birth after a single low-transverse incision, and they sonographically

1	not ind an ideal threshold value to recommend TOLAC. his same group subsequently recruited 1856 women contemplating vaginal birth after a single low-transverse incision, and they sonographically measured lower uterine segment thickness by between 34 weeks and 39 weeks G as trow, 2016). hey grouped women into three risk categories for uterine rupture during TOLAC based on the measured segment value: high risk <2.0 mm; intermediate risk 2.0-2.4 mm; and low risk �2.5 mm. he TOAC rates were 9,42, and 61 percent in the three categories, respectively. Of the 984 TOLACs, there were no symptomatic uterine ruptures. Overall, data are limited, and this evaluation is currently not part of our routine practice.

1	Women who have previously sustained a uterine rupture are at greater risk for recurrence. As shown in Table 31-3, those with a previous low-segment rupture have up to a 6-percent recurrence risk, whereas prior upper segment uterine rupture confers a 9-to 32-percent risk (Reyes-Ceja, 1969; Ritchie, 1971). Fox and associates (2014) reported 14 women with prior uterine rupture and 30 women with prior uterine dehiscence. In 60 subsequent pregnancies, they reported no uterine ruptures or severe complications if women were managed in a standardized manner with cesarean delivery prior to labor onset.

1	Magnetic resonance imaging studies of myometrial healing suggest that complete uterine involution and restoration of anatomy may require at least 6 months (Dicie, 1997). To explore this further, Shipp and coworkers (2001) examined the relationship between interdelivery interval and uterine rupture in 2409 women with one prior cesarean delivery. There were 29 women with a uterine rupture-l.4 percent. Interdelivery intervalsi:;18 months were associated with a threefold greater risk of symptomatic rupture during a subsequent TOLAC compared with intervalsi> 18 months. Similarly, Stamilio and associates (2007) noted a threefold augmented risk of uterine rupture in women with an interpregnancy interval <6 months compared with one �6 months.

1	Prior vaginal delivery, either before or after a cesarean birth, improves the prognosis for a subsequent vaginal delivery with either spontaneous or induced labor (Aviram, 2017; Grinstead, 2004; Hendler, 2004; Mercer, 2008). Prior vaginal delivery also lowers the risk of subsequent uterine rupture and other morbidities (Cahill, 2006; Hochler, 2014; Zelop, 1999). Women with a nonrecurring indication-for example, breech presentation-have the highest VBAC rate of nearly 90 percent (Wing, 1999). Those with a prior cesarean delivery for fetal compromise have an approximately 80-percent VBAC rate, and for those done for labor arrest, VBAC rates approximate 60 percent (Bujold, 2001; Peaceman, 2006). Prior second-stage cesarean delivery can be associated with second-stage uterine rupture in a subsequent pregnancy Gastrow, 2013).

1	Most studies show that increasing fetal size is inversely related to VBAC rates. he risk for uterine rupture is less robustly linked. Zelop and associates (2001) studied outcomes of almost 2750 women undergoing TOAC, and the rate of uterine rupture increased-albeit not signiicantly-with rising fetal weight. he rate was 1.0 percent for fetal weight <4000 g, 1.6 percent for >4000 g, and 2.4 percent for >4250 g. Similarly, Jastrow and colleagues (201 Ob) in a retrospective report of 2586 women with a prior low-transverse uterine incision, observed an elevated risk for a failed trial of labor, uterine rupture, shoulder dystocia, and perineal laceration associated with rising birthweights. Conversely, Baron and coworkers (2013) did not ind higher uterine rupture rates with birthweights >4000 g. With a preterm fetus, women who attempt a TOLAC have higher VBAC rates and lower rupture rates (Durnwald, 2006; Quinones, 2005).

1	Data supporting external cephalic version (ECV) for breech presentation are limited and are derived from small studies (Burgos, 2014; Weill, 2017). From thes;, ECV success and adverse event rates appear comparable to women without prior cesarean. The American College of Obstetricians and Gynecologists (2016) acknowledges this lack of robust data. At Parkland Hospital, we do not attempt ECV in those with a prior cesarean delivery. Twin pregnancy does not appear to increase the risk of uterine rupture. Ford and associates (2006) analyzed 1850 women with twins and reported a 45-percent successful VBAC rate and a rupture rate of 0.9 percent. Similar studies by Cahill (2005) and Varner (2007) and their colleagues reported rupture rates of 0.7 to 1.1 percent and VBAC rates of 75 to 85 percent. According to the American College of Obstetricians and Gynecologists (2017a), women with twins and a prior low-transverse hysterotomy can safely undergo TO LAC.

1	Multiple studies have reported an inverse relationship between prep regnancy body mass index (BMI) and VBAC rates. Hibbard and coworkers (2006) reported the following rates: 85 percent with a normal BMI, 78 percent with a BMI between 25 and 30, 70 percent with a BMI between 30 and 40, and 61 percent with a BMI �40. Similar findings were reported by Juhasz and associates (2005).

1	Most women with a prior cesarean delivery and fetal death in the current pregnancy would prefer a vaginal delivery. Although fetal concerns are obviated, available data suggest that maternal risks are increased. Nearly 46,000 women with a prior cesarean delivery in the Network database had a total of209 fetal deaths at an average gestational age of32.8 weeks (Ramirez, 2010). There were 158 women who elected TOLAC, with a VBAC rate of 87 percent. In the entire TOLAC group, the uterine rupture rate was 2.4 percent. Of the 116 women who underwent an induction oflabor, there were five uterine ruptures (3.4 percent).

1	The American College of Obstetricians and Gynecologists and the Society for Maternal-Fetal Medicine (2017b) recommend delaying nonmedically indicated deliveries until 39 completed weeks of gestation or beyond. As shown in Figure 31-4, signiicant and appreciable adverse neonatal morbidity has been reported with elective delivery before 39 completed weeks (Chiossi, 2013; Clark, 2009). Thus, if ERCD is planned, it is essential that the fetus be mature. The American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) have established the following guidelines for timing an elective cesarean delivery, and accurate gestational dating is suitable using any of these criteria. 1. Sonographic measurements taken before 20 weeks' gestation support a gestational age :39 weeks. 2. Fetal heart sounds have been documented for 30 weeks by Doppler ultrasound. 3.

1	1. Sonographic measurements taken before 20 weeks' gestation support a gestational age :39 weeks. 2. Fetal heart sounds have been documented for 30 weeks by Doppler ultrasound. 3. A positive serum or urine �-human chorionic gonadotropin (hCG) test result has been documented for :36 weeks.

1	Because of uterine rupture risks for women undergoing TOLAC, the American Academy of Pediatricians and the American College of Obstetricians and Gynecologists (2017) recommend that such trials be undertaken only in facilities with staf immediately available to provide emergency care. Moreover, these centers should have a plan and resources for managing uterine rupture. Some argue that these provisions deny women full access to choices. For example, in an earlier survey of Ohio hospitals, 15 percent of Level I, 63 percent of Level II, and 100 percent of Level III institutions met these requirements (Lavin, 2002). Moreover, an obstetrical anesthesia workforce survey reported that due to stafing limitations, TOLAC was allowed in only 88 percent of hospitals with : 1500 annual deliveries, in 59 percent of those with 500 to 1499 deliveries, and in 43 percent of those with < 500 deliveries (Traynor, 2016). In some cases, women choose to attempt TOLAC at a birthing center or at home

1	in 59 percent of those with 500 to 1499 deliveries, and in 43 percent of those with < 500 deliveries (Traynor, 2016). In some cases, women choose to attempt TOLAC at a birthing center or at home (Shields, 2017).

1	Labor induction is associated with a higher failure rate during TOLAC. The risks for uterine rupture, however, are less clear with induction or augmentation, with the exception of prostaglandin E1-misoprostol-which is contraindicated (American College of Obstetricians and Gynecologists, 2017a). Although most institutions are not so conservative, we do not induce or augment labor pharmacologically in women electing TOLAC at Parkland Hospital. Instead, we attempt induction only by amniotomy. Other considerations are to avoid induction or augmentation in women with an unknown prior incision type, an unfavorable cervix, or pregnancy >40 weeks.

1	Induction or augmentation of labor with oxytocin has been implicated in increased rates of uterine rupture in women undergoing TOLAC (Zelop, 1999). In the Network study reported by Landon and colleagues (2004), uterine rupture was more frequent in women induced with oxytocin alone-I.l percentthan in those in spontaneous labor-O.4 percent. Augmentation of labor was associated with uterine rupture in 0.9 percent. Among women in this trial without a prior vaginal delivery, the uterine rupture risk associated with oxytocin induction was 1.8 percent-a fourfold greater risk compared with spontaneous labor (Grobman, 2007a). In contrast, in one case-control study, induction was not associated with a higher risk for rupture (Harper, 2012a). Cahill (2008) and Goetzl (2001) and their coworkers reported a dose-related risk of rupture with oxytocin.

1	Various prostaglandin preparations commonly employed for cervical ripening or labor induction are discussed in Chapter 26 (p. 506). s a group, their safe use in women with a prior cesarean delivery is unclear because of conlicting data. With misoprostol (PGE1), Wing and colleagues (1998)

1	With misoprostol (PGE1), Wing and colleagues (1998) FIGURE 31-4 Neonatal morbidity rates seen with 13,258 elective repeat cesarean deliveries. Any adverse outcome includes death. Sepsis includes suspected and proven. RDS = respiratory distress syndrome; IN = transient tachypnea of the newborn. (Data from Tita AT, Landon MB, Spong CY, et al: Timing of elective repeat cesarean delivery at term and neonatal outcomes. N Engl J Med 360(2):1o11,o2009.) compared it versus oxytocin for labor induction in women with a prior cesarean delivery. They terminated their trial after two of the irst 17 women assigned to misoprostol developed a uterine rupture. Other studies confirmed this, and most consider misoprostol to be contraindicated (American College of Obstetricians and Gynecologists, 2017 a).

1	Of other prostaglandins, studies to evaluate their use for induction are contradictory. Ravasia and coworkers (2000) compared uterine rupture in 172 women given PGE2 gel with 1544 women in spontaneous labor. The rupture rate was significantly greater in women treated with PGE2 gel-2.9 percent compared with 0.9 percent in those with spontaneous labor. Lydon-Rochelle and associates (2001) found similar results. However, in the Network study cited previously, the uterine rupture rate was 1.4 percent when any prostaglandin was used in combination with oxytocin (Landon, 2004). But, in the subgroup of 227 women in whom labor was induced with a prostaglandin alone, there were no ruptures. Similar findings were reported with intravaginal prostaglandins, which were not associated with a greater uterine rupture risk (Macones, 2005b). These latter investigators, along with Kayani and colleagues (2005), found that sequential use of a prostaglandin followed by oxytocin was associated with a

1	uterine rupture risk (Macones, 2005b). These latter investigators, along with Kayani and colleagues (2005), found that sequential use of a prostaglandin followed by oxytocin was associated with a threefold greater risk of rupture compared with spontaneous labor.

1	Studies concerning the use of a transcervical Foley catheter for cervical ripening and induction of labor in women with a prior cesarean delivery are limited (Ben-Aroya, 2002; Jozwiak, 2014). In a retrospective study of 2479 women with prior cesarean delivery, the uterine rupture risk using a transcervical Foley catheter for labor induction (1.6 percent) was not significantly greater than that with spontaneous labor (1.1 percent) or with using amniotomy with or without oxytocin (1.2 percent) (Bujold, 2004). In contrast, Hofman (2004) described 138 women who underwent preinduction cervical ripening with a Foley catheter compared with 536 women who entered labor spontaneously. They observed a significant and inordinately high uterine rupture risk during labor following Foley catheter cervical ripening compared with spontaneous onset of labor-6.5 versus 1.9 percent.

1	Concerns that epidural analgesia for labor might mask the pain of uterine rupture have not been veriied. Fewer than 10 percent of women with scar separation experience pain and bleeding, and fetal heart rate decel erations are the most likely sign (ieser, 2002). That said, Cahill and coworkers (2010a) documented that more frequent episodes of epidural dosing were associated with increasing uterine rupture rates. VBAC rates are similar, and in some cases higher, among women with labor epidural analgesia compared with those using other forms of analgesia (Aviram,

1	VBAC rates are similar, and in some cases higher, among women with labor epidural analgesia compared with those using other forms of analgesia (Aviram, Following VBAC, some clinicians routinely document the integrity of a prior scar by placing a hand through the dilated cervix and along the inner surface of the lower uterine segment. But routine uterine exploration is considered by others to be unnecessary. In a longitudinal study of 3469 women who had a VBAC, seven uterine dehiscences and one uterine rupture yielded an overall event rate of 0.23 percent (Silberstein, 1998). They concluded that trans cervical evaluation need only be performed in symptomatic patients.

1	Currently, the benefits of routine scar evaluation in the asymptomatic woman are unclear, however, surgical correction of a dehiscence is necessary if significant bleeding is encountered. Our practice is to routinely examine these prior hysterotomy sites. Any decision for laparotomy and repair takes into consideration the extent of the tear, whether the peritoneal cavity has been entered, and the presence of active bleeding.

1	Progress of labor in women attempting TOLAC is similar to normal labor, and no speciic pattern presages uterine rupture (Graseck, 2012; Harper, 2012b; Sondgeroth, 2017). Before hypovolemic shock develops, symptoms and physical findings in women with uterine rupture may appear bizarre unless the possibility is kept in mind. For example, hemoperitoneum from a ruptured uterus may result in diaphragmatic irritation with pain referred to the chest. This may direct one to a diagnosis of pulmonary or amnionic fluid embolism instead of uterine rupture. As shown in Figure 31-5, the most common sign of uterine rupture is a nonreassuring fetl heart rate pattern with variable decelerations that may evolve into late decelerations and bradycardia. In 36 cases of such rupture during TOLAC, there were fetal signs of uterine rupture in 24, maternal signs in eight, and a combination of maternal and fetal in three (Holmgren, 2012). Few women experience cessation of contractions following uterine

1	were fetal signs of uterine rupture in 24, maternal signs in eight, and a combination of maternal and fetal in three (Holmgren, 2012). Few women experience cessation of contractions following uterine rupture, and the use of intrauterine pressure catheters does not assist reliably in the diagnosis (Rodriguez, 1989).

1	2017; Shmudi, 2017). Perhaps related, almost a fourth of VBAC deliveries were completed with either forceps or vacuum (Inbar, 2017). he merican Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) have concluded that epidural analgesia while pushing. The rupture apparently stimulated a reflex push, after which uterine tone may safely be used during TOLAC. diminished and fetal bradycardia worsened. FIGURE 31-6 Maternal-Fetal Medicine Units Network: rates of some complications with increasing number of repeat cesarean deliveries. (Data from Silver RM, Landon MB, Rouse OJ, et al: Maternal morbidity associated with mUltiple repeat cesarean deliveries. Obstet Gynecol 207:1o226, 2006.)

1	In some women, the clinical appearance of uterine rupture mirrors that of placental abruption. In most, however, there is remarkably little appreciable pain or tenderness. Also, because most women in labor are treated for discomfort with either narcotics or epidural analgesia, pain and tenderness may not be readily apparent. The condition usually becomes evident because of fetal distress and occasionally because of maternal hypovolemia from concealed hemorrhage. If the fetal presenting part has already entered the pelvis with labor, loss of station may be detected by pelvic examination. If the fetus is partly or totally extruded from the uterine rupture site, abdominal palpation or vaginal examination may be help ful to identiY the presenting part, which will have moved away from the pelvic inlet. A irm contracted uterus may at times be felt alongside the fetus. Sonography may be helpful.

1	With rupture and expulsion of the fetus into the peritoneal cavity, the chances for intact fetal survival are dismal, and reported mortality rates range from 50 to 75 percent. Fetal condition depends on the degree to which placental implantation remains intact, although this can change within minutes. With rupture, the only chance of fetal survival is aforded by immediate delivery-most often by laparotomy-otherwise, hypoxia is inevitable. If rupture is followed by total placental separation, then very few neurologically intact fetuses will be salvaged.

1	Thus, even in the best of circumstances, some etal outcomes wil be impaire. he Utah experiences are instructive here (Holmgren, 2012). Of the 35 laboring patients with uterine rupture, the decision-to-delivery time was < 18 minutes in 17, and none of these infants had an adverse neurological outcome. Of the 18 born > 18 minutes from decision time, the three infants with long-term neurological impairments were delivered at 31, 40, and 42 minutes. There were no deaths, thus severe neonatal neurological morbidity developed in 8 percent of this group of 35 women with uterine rupture. In a study using the Swedish Birth Registry, Kaczmarczyk and coworkers (2007) found that the risk of neonatal death following uterine rupture was 5 percent. In the Network study cited earlier, seven of the 114 uterine ruptures associated with TOAC-6 percent-were complicated by development of neonatal HIE (Spong, 2007).

1	Maternal deaths from uterine rupture are uncommon. Of 2.5 million women who gave birth in Canada between 1991 and 2001, there were 1898 cases of uterine rupture, and four of these-0.2 percent-resulted in maternal death (Wen, 2005). In other regions of the world, however, maternal mortality rates are much higher. From rural India, the maternal mortality rate associated with uterine rupture was 30 percent (Chatterjee, 2007).

1	With complete rupture during TOAC, hysterectomy may be required. In selected cases, however, suture repair with uterine preservation may be performed. Sheth (1968) described outcomes from a series of 66 women in whom repair of a uterine rupture was elected rather than hysterectomy. hirteen of the 41 mothers who did not have tubal sterilization had a total of 21 subsequent pregnancies. Uterine rupture recurred in four of these-approximately 20 percent. Usta and associates (2007) reported similar results. In another study, however, women with a uterine dehiscence were not more likely to have a subsequent uterine rupture (Baron, 2014).

1	Because of the aforementioned concerns with TOAC, most women in the United States undergo ERCD. his choice has several signiicant maternal complications, and rates of these rise in women who have multiple repeat operations. The incidences of some common complications for women with one prior transverse cesarean delivery who undergo an ERCD were shown in Table 31-2. Of note, half of cesarean hysterectomies done at Parkland Hospital are in women with one or more prior cesarean deliveries (Hernandez, 2013).

1	The Network addressed issues of increased morbidity in a cohort of30,132 women who had from one to six repeat cesarean deliveries (Silver, 2006). he rates of some of the more frequent or serious complications are depicted in Figure 31-6. In addition, rates of bowel or bladder injury, admission to an intensive care unit or need for ventilator therapy, and maternal mortality, as well as operative and hospitalization length, showed significantly rising trends. Similar results have been reported by others (Nisenblat, 2006; Usta, 2005). More diicult to quantiY are risks for bowel obstruction and pelvic pain from peritoneal �.� c0 4 ..A 2�): E0u First Second Third � Fifth (6201) (15,808) (6324) (347) Number of repeat cesarean deliveries (Number of women) TABLE 31-4. Some Recommendations of Professional Societies Concerning a Trial of Labor to Attempt VBAC CD cesarean delivery; VBAC vaginal birth after cesarean.

1	TABLE 31-4. Some Recommendations of Professional Societies Concerning a Trial of Labor to Attempt VBAC CD cesarean delivery; VBAC vaginal birth after cesarean. adhesive disease, both of which increase with each successive cesarean delivery (Andolf, 2010; Mankuta, 2013). Cook and colleagues (2013) from the United Kingdom Obstetric Surveillance System (UKOSS) described adverse sequelae of women with ive or more cesarean deliveries. These women had signiicantly higher rates of morbidity. Namely, the major hemorrhage rate increased 18-fold; visceral damage, 17 -fold; critical care admissions, IS-fold; and delivery <37 weeks, sixfold. Much of this morbidity was in the 18 percent who had a placenta previa or an accrete syndrome (Chap. 41, p. 773).

1	For providers and their patients, unfortunately, no large rndomized trials have compared outcomes of women with an intent to pursue either TOAC or ERCD. Most studies to date have compared actual routes of delivey rather than the intended route of delivery. hus, we agree with Scott (2011) regarding a "commonsense" approach. The woman-and her partner if she wishes-are encouraged to actively participate with her provider in informed consent. Counseling should include documentation of the prior uterine incision and discussion of risks, benefits, and success rates of TO AC or ERCD. This includes consideration of risks involving uture pregnancies. Ideally, counseling begins preconceptionally and continues throughout pregnancy, with lexible options extending up to delivery. For women who desire TOAC despite a factor that increases their speciic risk, additions to the consent form are recommended by the merican College of Obstetricians and Gynecologists (2017 a). Bonanno and colleagues (2011)

1	despite a factor that increases their speciic risk, additions to the consent form are recommended by the merican College of Obstetricians and Gynecologists (2017 a). Bonanno and colleagues (2011) have provided such an example. Brief synopses of professional society guidelines are shown in Table 31-4. Guidelines that tend to be more conservative are shown in Table 31-5.

1	TABLE 31 -5. Conservative Guidelines to Approach a Trial of Labor Following Cesarean Delivery Early during prenatal care Develop preliminary plan Revisit at least each trimester Be willing to alter decision Have facilities availability Review relative and absolute contraindications Reconsider risks as pregnancy progresses Tread carefully: > 1 prior transverse CD, unknown incision, twins, macrosomia Cautions for induction-unfavorable cervix, high station Consider AROM Respect oxytocin-know when to quit Beware of abnormal labor progress Respect EFM pattern abnormalities Know when to abandon a trial of labor ACOG American College of Obstetricians and Gynecologists; AROM = artificial rupture of membranes; American College of Obstetricians and Gynecologists (20l7a) Society of Obstetricians and Gynaecologists of Canada (2005) Royal College of Obstetricians and Gynaecologists (2007)

1	American College of Obstetricians and Gynecologists (20l7a) Society of Obstetricians and Gynaecologists of Canada (2005) Royal College of Obstetricians and Gynaecologists (2007) Offer to most women with one prior low-transverse incision; consider for t.IO prior low-transverse incisions Offer to wornen with one prior transverse lowsegment cesarean delivery; withs> 1 prior CD then VBAC likely successful but increased risks Discuss VB,\C option with women with prior low-segment cesarean delivery; decision between obstetrician and patient Safest with ability for immediate cesarean delivery; patients should be allowed to accept increased risk when not available Should deliver in hospital in which timely cesarean delivery is available; approximate timeframe of 30 minutes Suitable delivery suite with continuous care and monitoring; immediate cesarean delivery capability Not precluded: twins, macrosomia, prior lowvertical or unknown type of incision

1	Suitable delivery suite with continuous care and monitoring; immediate cesarean delivery capability Not precluded: twins, macrosomia, prior lowvertical or unknown type of incision Oxytocin or Foley catheter induction safe, but prostaglandins should not be used; macrosomia, diabetes, postterm pregnancy, twins are not contrai nd ications Caution with twins and macrosomia American Academy of Pediatrics, American College of Obstetricians and Gynecologists: Guidelines for Perinatal Care, 8th ed. Elk Grove Village, 2017 American College of Obstetricians and Gynecologists: Guidelines for vaginal delivery ater a previous cesarean birth. Committee Opinion No. 64, October 1988

1	American College of Obstetricians and Gynecologists: Guidelines for vaginal delivery ater a previous cesarean birth. Committee Opinion No. 64, October 1988 American College of Obstetricians and Gynecologists: Vaginal delivery after previous cesarean birth. Committee Opinion No. 143, October 1994 American College of Obstetricians and Gynecologists: Vaginal birth ater previous cesarean delivery. Practice Bulletin No.r2, October 1998 American College of Obstetricians and Gynecologists: Vaginal birth after previous cesarean delivery. Practice Bulletin No.r5, ] uly 1999 American College of Obstetricians and Gynecologists: External cephalic version. Practice Bulletin No. 161, February 2016

1	American College of Obstetricians and Gynecologists: Vaginal birth after cesarean delivery. Practice Bulletin No. 184, November 2017a merican College of Obstetricians and Gynecologists, Society for MaternalFetal Medicine: Nonmedically indicated early-term deliveries. Committee Opinion No. 561, April 2013, Reairmed 2017b Andolf E, Thorsell M, Kallen K: Cesarean delivery and risk for postoperative adhesions and intestinal obstruction: a nested case-control study of the Swedish Medical Birth Registry. Am] Obstet Gynecol 203:406.el, 2010 Aviram A, Hadar E, Gabbay-Benziv R, et al: Successful tolac in a population with a high success rate-what are the diferences? Abstract No. 923. Am ] Obstet GynecoIr216:S526, 2017 Babbar S, Chauhan S, Hammas I, et al: Failed trial of labor after cesarean delivery: indications for failure and peripartum complications. Abstract No. 818, Am] Obstet Gynecol208 (1 Suppl):S342, 2013

1	Barger MK, Dunn ]T, Bearman S, et al: A survey of access to trial of labor in California hospitals in 2012. BMC Pregnancy Childbirth 13:83,r2013 Baron], Weintraub AY, Eshkoli T, et al: he consequences of previous uterine scar dehiscence and cesarean delivery on subsequent births. Intr] Gynaecol Obstet 126(2):120,r2014 Baron], Weintraub A, Sergienko R, et al: Is vaginal delivery of a macrosomic infant after cesarean section really so dangerous? Abstract No. 799, Am ] Obstet Gynecolr208(1 Suppl):S335, 2013 Ben-Aroya Z, Hallak M, Segal 0, et al: Ripening of the uterine cervix in a post-cesarean parturient: prostaglandin E2 versus Foley catheter. ] Matern Fetal Neonatal Med 12(1):42r2002 Bennich G, Rudnicki M, Wilken-Jensen C, et al: Impact of adding a second layer to a single unlocked closure of a cesarean uterine incision: randomized controlled trial. Ultrasound Obstet GynecoIr47(4):417, 2016

1	Bonanno C, Clausing M, Berkowitz R: VBAC: a medicolegal perspective. Clin Perinatol 38:217, 2011 Bujold E, Blackwell SC, Gauthier R]: Cervical ripening with trans cervical Foley catheter and the risk of uterine rupture. Obstet Gynecol 103(1): 18 2004 Bujold E, Gauthier R]: Should we allow a trial oflabor after a previous cesarean for dystocia in the second stage of labor? Obstet Gynecol 98:652, 2001 Burgosr], Cobos P, Rodriguez L, et al: Is external cephalic version at term contraindicated in previous caesarean section? A prospective comparative cohort study. B]OG 121:230,r2014 Cahill A, Stamilio OM, Pare E, et al: Vaginal birth ater cesarean (VBAC) attempt in twin pregnancies: is it safe? Am] Obstet Gynecol 193: 1 050, 2005 Cahill AG, Odibo AO, Allswroth ]E, et al: Frequent epidural dosing as a marker for impending uterine rupture in patients who attempt vaginal birth after cesarean delivery. Amr] Obstet Gynecol 202:355.e1, 2010a

1	Cahill AG, Stamilio DM, Odibo A, et al: Is vaginal birth after cesarean (VBAC) or elective repeat cesarean safer in women with a prior vaginal delivery? Am ] Obstet Gynecol 195:1143,r2006 Cahill AG, Tuuli M, Odibo AO, et al: Vaginal birth ater caesarean for women with three or more prior caesareans: assessing safety and success. B]OG 117:422,r201Ob Cahill AG, Waterman BM, Stamilio DM, et al: Higher maximum doses of oxytocin are associated with an unacceptably high risk for uterine rupture in patients attempting vaginal birth after cesarean delivery. Am ] Obstet Gynecol 199:32.e1, 2008 Chatterjee SR, Bhaduri S: Clinical analysis of 40 cases of uterine rupture at Durgapur Subdivisional Hospital: an observational study. ] Indian Med Assoc 105:510,r2007 Chauhan SP, Magann EF, Wiggs CD, et al: Pregnancy after classic cesarean delivery. Obstet Gynecol 100:946, 2002

1	Chauhan SP, Magann EF, Wiggs CD, et al: Pregnancy after classic cesarean delivery. Obstet Gynecol 100:946, 2002 Cheng Y, Snowden], Cottrell E, et al: Trends in proportions of hospitals with VBAC: impact of ACOG guidelines. Am] Obstet Gynecol 21 0:S241, 2014 Chiossi G, Lai Y, Landon MB, et al: Timing of delivery and adverse outcomes in term singleton repeat cesarean deliveries. Obstet Gynecol 121 :561, 2013 Clark SL, Miller DO, Belfort A, et al: Neonatal and maternal outcomes associated with elective term delivery. Am ] Obstet Gynecol 200(2): 156. el,r2009 Coleman VH, Erickson K, Schulkinr], et al: Vaginal birth after cesarean delivery.r] Reprod Med 50:261, 2005 Cookr], ]avis S, Knight M, et al: Multiple repeat caesarean section in the UK: incidence and consequences to mother and child. A national, prospective, cohort study. B]OG 120(1):85, 2013 Cragin E: Conservatism in obstetrics. N Y Med] 104: 1, 1916

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1	CY, et al: he IvfFMU Cesarean Registry: factors afecting the success of trial of labor after previous cesarean delivery. Am J Obstet Gynecol 193:1016,r2005 Landon MB, Spong CY, Thon E, et al: Risk of uterine rupture with a trial of labor in women with multiple and single prior cesarean delivery. Obstet Gynecol 108: 12, 2006 Lannon SM, Guthrie A, Vanderhoeven JP, et al: Uterine rupture risk after periviable cesarean delivery. Obstet Gynecol 125: 1 095, 2015 Lavin JP, DiPasquale L, Crane S, et al: A state-wide assessment of the obstetric, anesthesia, and operative team personnel who are available to manage the labors and deliveries and to treat the complications of women who attempt vaginal birth after cesarean delivery. Am J Obstet Gynecol 187:611, 2002 Leeman Uvl, Beagle M, Espey E, et al: Diminishing availability of trial of labor ater cesarean delivery in New Mexico hospitals. Obstet GynecoIr122:242, 2013 Lydon-Rochelle M, Holt VL, Easterling TR, et al: Risk of uterine rupture

1	et al: Diminishing availability of trial of labor ater cesarean delivery in New Mexico hospitals. Obstet GynecoIr122:242, 2013 Lydon-Rochelle M, Holt VL, Easterling TR, et al: Risk of uterine rupture during labor among women with a prior cesarean delivery. N Engl J Med 345:3,r2001 Macones GA, Cahill A, Pare E, et al: Obstetric outcomes in women with two prior cesarean deliveries: is vaginal birth after cesarean delivery a viable option? Am J Obstet GynecoIr192:1223, 2005a Macones GA, Cahill AG, Stamilio DM, et al: Can uterine rupture in patients attempting vaginal birth ater cesarean delivery be predicted? Am J Obstet Gynecol 195:1148,2006 Macones GA, Peipert J, Nelson DB, et al: Maternal complications with vaginal birth after cesarean delivery: a multicenter study. Am J Obstet Gynecol 193: 1656, 2005b Mankuta D, Mansour M, Alon SA: Ivlaternal and fetal morbidity due to abdominal adhesions after repeated cesarean section. Abstract No. 792, Am J Obstet Gynecol 208(1 Suppl):S332, 2013

1	1656, 2005b Mankuta D, Mansour M, Alon SA: Ivlaternal and fetal morbidity due to abdominal adhesions after repeated cesarean section. Abstract No. 792, Am J Obstet Gynecol 208(1 Suppl):S332, 2013 Marshall NE, Fu R, Guise JM: Impact of multiple cesarean deliveries on maternal morbidity: a systematic review. Am J Obstet GynecoI205:262.e1, 2011 Martin IN, Perry KG, Roberts WE, et al: The care for trial of labor in the patients with a prior low-segment vertical cesarean incision. Am J Obstet Gynecol 177: 144, 1997 McMahon MJ, Luther ER, Bowes WA J r, et al: Comparison of a trial of labor with an elective second cesarean section. N Engl J Med 335:689, 1996 Mercer BM, Gilbert S, Landon MB, et al: Labor outcomes with increasing number of prior vaginal births after cesarean delivery. Obstet Gynecol 111:285,r2008 Metz TD, Stoddard GJ, Henry E, et al: Simple, validated vaginal birth after cesarean delivery prediction model for use at the time of admission. Obstet Gynecol 122:571,r2013 Miller

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1	Osser OV, Valentin L: Clinical importance of appearance of cesarean hysterotomy scar at transvaginal ultrasonography in nonpregnant women. Obstet Gynecol 117:525,r2011 Pauerstein CJ: Once a section, always a trial of labor? Obstet Gynecol 28:273, 1966 Pauerstein CJ, Karp L, Muher S: Trial oflabor after low segment cesarean section. S Med J 62:925, 1969 Peace man v!, Gersnoviez R, Landon MB, et al: he MFMU cesarean registry: impact of fetal size on trial of labor success for patients with previous cesarean for dystocia. Am J Obstet Gynecol 195:1127,2006 Quinones IN, Stamilio OM, Pare E, et al: he efect of prematurity on vaginal birth after cesarean delivery: success and maternal morbidity. Obstet Gynecol 105:519,r2005 Ramirez MM, Gilbert S, Landon MB, et al: Mode of delivery in women with antepartum fetal death and prior cesarean delivery. Am J Perinatol 27:825, 2010

1	Ramirez MM, Gilbert S, Landon MB, et al: Mode of delivery in women with antepartum fetal death and prior cesarean delivery. Am J Perinatol 27:825, 2010 Ravasia OJ, Brain PH, Pollard JK: Incidence of uterine rupture among women with mullerian duct anomalies who attempt vaginal birth after cesarean delivery. Am J Obstet Gynecol 7, 1999 Ravasia OJ, Wood SL, Pollard JK: Uterine rupture during induced trial of labor among women with previous cesarean delivery. Am J Obstet Gynecol 183:1176,r2000

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1	rates. Obstet Gynecol 122:lO10, 2013 Rossi AC, D'Addario V: Maternal morbidity following a trial of labor after cesarean section vs elective repeat cesarean delivery: a systematic review with metaanalysis. Am J Obstet Gynecol 199(3):224,2008 Royal College of Obstetricians and Gynaecologists: Birth after previous caesarean birth. Green-top Guideline No. 45, February 2007 Sachs BP, Koblin C, Castro A, et al: The risk of lowering the cesarean-delivery rate. N Engl J Med 340:5, 1999 Sciscione AC, Landon MB, Leveno KJ, et al: Previous preterm cesarean delivery and risk of subsequent uterine rupture. Obstet Gynecol 111 :648, 2008 Scott JR: Vaginal birth after cesarean delivery: a common-sense approach. Obstet Gynecol 118:342, 2011 Sheth SS: Results of treatment of rupture of the uterus by suturing. J Obstet Gynaecol Br Commonw 75:55, 1968 Shields M, Zwerling B, Cheng W: Outcomes of hospital versus out-ofhospital birth in vaginal birth after cesarean. Abstract No. 827. Am J Obstet Gynecol

1	J Obstet Gynaecol Br Commonw 75:55, 1968 Shields M, Zwerling B, Cheng W: Outcomes of hospital versus out-ofhospital birth in vaginal birth after cesarean. Abstract No. 827. Am J Obstet Gynecol 216:S474, 2017 Shipp TO, Zelop CM, Repke JT, et al: Interdelivery interval and risk of symptomatic uterine rupture. Obstet Gynecol 97: 175, 2001 Shipp TO, Zelop CM, Repke JT, et al: Intrapartum uterine rupture and dehiscence in patients with prior lower uterine segment vertical and transverse incisions. Obstet Gynecol 94:735, 1999 Shmueli A, Salman L, Nassie 01, et al: he intriguing association between epidural anesthesia and mode of delivery among women in trial of labor after cesarean delivery. Abstract No. 949. Am J Obstet Gynecol 216:S536, 2017 Silberstein T, Wiznitzer A, Katz M, et al: Routine revision of uterine scar after cesarean section: has it ever been necessary? Eur J Obstet Gynecol Reprod BioI 78:29, 1998

1	Silver M, Landon MB, Rouse 0], et al: Maternal morbidity associated with multiple repeat cesarean deliveries. Obstet Gynecol 207: 1226, 2006 Smith GC, Pell ]P, Cameron AD, et al: Risk of perinatal death associated with labor after previous cesarean delivery in uncomplicated term pregnancies. ]AMA 287:2684, 2002 Society for Matenal-Fetal Medicine: Counseling and management of women with prior classical cesarean delivery. Conremp OB/GYN 57(6):26, 2012 Society of Obstetricians and Gynaecologists of Canada: SOGC clinical practice guidelines. Guidelines for vaginal birth after previous caesarean birth. Number 155 (replaces guideline Number 147), February 2005. Intr] Gynaecol Obstet 89(3):319, 2005 Sondgeroth KE, Stout M], Tuuli MG, et al: Does uterine resting tone have any clinical value in trial oflabor (TOLAC)? Abstract No. 829. Am] Obstet Gynecol 216:5475, 201

1	Sondgeroth KE, Stout M], Tuuli MG, et al: Does uterine resting tone have any clinical value in trial oflabor (TOLAC)? Abstract No. 829. Am] Obstet Gynecol 216:5475, 201 Spong CY, Landon MB, Gilbert 5, et al: Risk of uterine rupture and adverse perinatal outcome at term after cesarean delivery. Obstet Gynecol 110:801, 2007 Srinivas SK, Stamilio OM, Stevens E], et al: Predicting failure of a vaginal birth attempt after cesarean delivery. Obstet Gynecol 109:800, 2007 Stamilio OM, DeFranco E, Pare E, et al: Short inrerpregnancy inrerval. Risk of uterine rupture and complications of vaginal birth after cesarean delivery. Obstet Gynecol 110, 1075,2007 Stanhope T, El-Nasher 5, Garrett A, et al: Prediction of uterine rupture or dehiscence during trial of labor after cesarean delivery: a cohort study. Abstract No. 821, Amr] Obstet Gynecol 208(1 Suppi):S343, 2013

1	Tahseen 5, Griiths M: Vaginal birth after two caesarean sections (VBAC-2)a systematic review with meta-analysis of success rate and adverse outcomes of VBAC-2 versus VBAC-1 and repeat (third) caesarean sections. B]OG 117:5,r2010 Tita AT, Landon MB, Spong CY, et al: Timing of elective repeat cesarean delivery at term and neonatal outcomes. N Engl ] Med 360(2): Ill, 2009 Traynor A], Aragon M, Ghosh 0, et al: Obstetric Anesthesia Workforce Survey: a 30-year update. Anesth Analg 122(6):1939, 2016 Uddin SFG, Simon E: Rates and success rates of trial oflabor ater cesarean delivery in the United States, 1990-2009. Maten Child Health] 17:1309,2013 Usta 1M, Hamdi MA, Abu Musa A, et al: Pregnancy outcome in patients with previous uterine rupture. Acta Obstet Gynecol 86: 172, 2007 Usta 1M, Hobeika EM, Abu-Musa A, et al: Placenra previa-accreta: risk factors and complications. Am] Obstet Gynecol 193:1045,2005

1	Usta 1M, Hobeika EM, Abu-Musa A, et al: Placenra previa-accreta: risk factors and complications. Am] Obstet Gynecol 193:1045,2005 Varner MW, Thon E, Spong CY, et al: Trial oflabor after one previous cesarean delivery for multifetal gestation. Obstet Gynecol 110:814, 2007 Weill Y, Pollack RN: The eicacy and safety of external cephalic version after a previous caesarean delivery. Aust N Z ] Obstet Gynaecol 57(3):323, 20r17 Wen SW, Huang L, Liston R, et al: Severe maternal morbidity in Canada, 1991-2001. CMA] 173:759,r2005 Wing DA, Lovett K, Paul RH: Disruption of prior uterine incision following misoprostol for labor induction in women with previous cesarean delivery. Obstet Gynecol 91 :828, 1998 Wing DA, Paul RH: Vaginal birth after cesarean section: selection and management. Clin Obstet GynecoIr42:836, 1999

1	Wing DA, Paul RH: Vaginal birth after cesarean section: selection and management. Clin Obstet GynecoIr42:836, 1999 Zelop CM, Shipp TO, Repke ]T, et al: Outcomes of trial of labor following previous cesarean delivery among women with fetuses weighing >4000 g. Am] Obstet Gynecol 185:903,2001 Zelop CM, Shipp TO, Repke ]T, et al: Uterine rupture during induced or augmented labor in gravid women with one prior cesarean delivery. Am ] Obstet Gynecol 181r:882, 1999 The Newborn TRANSITION TO AIR BREATHING .................. 606 CARE IN THE DELIVERY ROOM .................... 607 EVALUATION OF NEWBORN CONDITION ............ 610 PREVENTIVE CARE.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613 ROUTINE NEWBORN CARE ....................... 614 Normaly the newy born child begins to cry almost immediatey ater its exit from the vulva. This act indicates the establishment of respiration, which is accompanied by important modications in the circulatory system.

1	-J. Whitridge Williams (1903) In most instances at delivery, the newborn is healthy and vigorous, but at times, special care may be needed. For this reason, the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (20 17b) recommend that every birth should be attended by at least one qualified individual. This person should be skilled in the initial steps of newborn care and positive-pressure ventilation, and their only responsibility is management of the newborn. his usually is a pediatrician, nurse practitioner, anesthesiologist, nurse anesthetist, or specially trained nurse. However, in their absence, the responsibility for neonatal resuscitation falls to the obstetrical attendant. hus, obstetricians should be well versed in measures for immediate care of the newborn.

1	The number and qualiications of personnel who attend the delivery will vary depending on the anticipated risk, the number of babies, and the hospital setting. A qualiied team with full resuscitation skills should be present for high-risk deliveries and immediately available for every resuscitation (Wyckof, 2015). This team should not be on call at home or in a remote area of the hospital. Moreover, team training through frequent simulation practice is recommended for all who may be called to attend deliveries (Perlman, 2015).

1	Immediately following birth, the newborn must promptly convert from placental to pulmonary gas exchange. Pulmonary vascular resistance must fall, pulmonary perfusion must rapidly rise, and unique fetal vascular shunts must begin to close to separate the systemic and pulmonary circulations (Rudolph, 1979). These shunts include the patent ductus arteriosus and patent foramen ovale, described in Chapter 7 (p. 129). Lung aeration is not only critical for pulmonary gas exchange. Recent studies suggest that it is signiicantly responsible for initiating cardiovascular changes at birth (Hooper, 2016).

1	In utero, the fetal lungs are illed with amnionic luid, which must be cleared quickly for air breathing. This clearance occurs through various means, and the contributions of these mechanisms may depend on gestational age and mode of delivery. First, a large release of fetal adrenaline late in labor stimulates pulmonary epithelial cells to stop secreting and instead to start reabsorbing lung liquid as a result of sodium-channel activation (te Pas, 2008). The contribution of this mechanism is unlikely to be major, as blockade of the receptors for sodium channel activation reduces or delays but does not prevent lung liquid clearance at birth (O'Brodovich, 1990).

1	As a second method, mechanical forces aid lung fluid clearance during labor. Early reports described compression of the fetal thorax and abdomen as they passed through the birth canal leading to lung liquid expulsion (Karl berg, 1962; Saunders, 1978). By this mechanism, up to a third of lung liquid is expelled in a jet of luid from the nose and mouth once the respiratory tract is exposed to the lower outside pressure. The Newborn 607 However, it may be that uterine contractions force a change in fetal posture leading to compression of the thorax and increased intrathoracic pressures. his prompts expulsion of lung liquid early in labor more so than the "vaginal squeeze" theory (Lines, 1997; te Pas, 2008; Vyas, 1981).

1	In a third mechanism, a significant amount of lung liquid is cleared after birth (Hooper, 2016). In animal studies, most lung aeration occurs during inspiration-within three to ive breaths after birth. But, no liquid clears between breaths (Hooper, 2007). Speciically, the transpulmonary pressure gradient during inspiration promotes movement of fluid into the interstitial tissue. From here, it is gradually cleared, probably by the pulmonary circulation and lymphatic vessls. It is possible for lung interstitial tissue pressure to rise to a point that luid can actually move back into the airspaces during expiration unless positive end-expiratory pressure opposes liquid reentry (Siew, 2009a,b). This may be a contributing factor in the development of transient tachypnea of the newborn.

1	As fluid is replaced by air, compression of the pulmonary vasculature is reduced considerably, and in turn, resistance to blood flow is lowered. With the fall in pulmonary arterial blood pressure, the ductus arteriosus normally closes. High, negative intrathoracic pressures are required to permit the initial entry of air into the fluid-illed alveoli. Normally, from the irst breath after birth, progressively more residual air accumulates in the lung. And, with each successive breath, lower pulmonary opening pressure is required. In the normal mature newborn, by approximately the ifth breath, pressurevolume changes achieved with each respiration are very similar to those of the adult. hus, the breathing pattern shifts from shallow episodic inspirations characteristic of the fetus to regular, deeper inhalations (Chap. 17, p. 333).

1	As a last mechanism, surfactant, which is synthesized by the type II pneumocytes, lowers alveolar surface tension and helps maintain lung inlation by preventing alveolar collapse. Insuficient surfactant, which is common in preterm neonates, leads promptly to respiratory distress syndrome (Chap. 34, p. 636). In utero, umbilical venous retun is the main source of preload for the left ventricle, particularly as fetal pulmonary blood flow is very low due to high pulmonary vascular resistance and is unable to provide suicient venous return to maintain left ventricular output (Hooper, 2015).

1	Clamping the umbilical cord reduces preload for the left ventricle and thus reduces cardiac output. Until the lungs aerate and pulmonary blood low increases, the reduced cardiac output will manifest as bradycardia. If cord clamping is delayed until after the lungs have aerated, the transition is smoother and cardiac ouput does not fall (Bhatt, 2013). This understanding has led to interest in delayed (physiological) cord clamping, especially if it can be done after successful inflation of the lung. Randomized trials are currently underway. The International Liaison Committee on Resuscitation (ILCOR) updated its scientiic review for neonatal delivery room care and resuscitation (Perlman, 2015). The ILCOR scientiic review is used by the American Academy of Pediatrics and the American Heart Association to develop the neonatal resuscitation guidelines for North America (Wyckof, 2015).

1	Before and during delivery, careful consideration must be given to several determinants of neonatal well-being. hese include: (1) maternal health status; (2) prenatal complications, including any suspected fetal malformations; (3) gestational age; (4) labor complications; (5) duration of labor and ruptured membranes; (6) type and duration of anesthesia; (7) diiculty of delivery; and (8) medications given during labor and their dosages, administration routes, and timing.

1	When risk factors are present, neonatal resuscitation providers should be present for the delivery. This team readies equipment, ensures that adequate personnel are present, delegates roles and responsibilities, and considers contingency plans to stabilize the newborn. Four questions a neonatal provider will ask pertain to expected gestional age, amnionic fluid color, fetal number, and additional fetal risks. Several conditions are associated with a nonvigorous presentation. These may include immaturity, hypoxemia or acidosis from any cause, sepsis syndrome, recent drugs administered to the mother, and central nervous system developmental abnormalities. Those related to the respiratory tract are lung abnormalities, upper airway obstruction, pneumothorax, and meconium aspiration.

1	Ideally, obstetrical and pediatric teams discuss plans regarding umbilical cord management. Delayed cord clamping provides transfusion of placental blood to the newborn. In term infants, delay of cord clamping by 30 to 60 seconds raises hemoglobin levels at birth, improves iron stores during infancy, and enhances neurodevelopment at 4 years of age (Katheria, 2017). As discussed in Chapter 33 (p. 625), the only reported negative outcome of delayed cord clamping is hyperbilirubinemia, leading to a higher rate of phototherapy (American College of Obstetricians and Gynecologists, 2017a). In preterm neonates, delayed cord clamping reduces rates of blood transfusion, intraventricular hemorrhage, and necrotizing enterocolitis.

1	Delayed cord clamping should be performed in preterm and term newborns who do not require resuscitation at birth (American Academy of Pediatrics, 2017 a; American College of Obstetricians and Gynecologists, 2017a; Perlman, 2015). here should be no delay if a newborn requires resuscitation or if the placental circulation is disrupted by abruption, cord avulsion, or bleeding placenta previa or vasa previa. Approximately 10 percent of newborns require some degree of active resuscitation to stimulate breathing, and 1 percent need extensive care. Perhaps not coincidentally, the risk of death for newborns delivered at home compared with those delivered in hospitals is increased two-to threefold (American College of Obstetricians and Gynecologists, 2017 d). When deprived of adequate gas exchange, either before or after birth, neonates demonstrate a well-deined sequence of

1	When deprived of adequate gas exchange, either before or after birth, neonates demonstrate a well-deined sequence of FIGURE 32-1 Physiological changes associated with primary and secondary apnea in the newborn. bpm = beats per minute; HR = heart rate; MAP mean arterial pressure. (Adapted with permission from Kattwinkel J: Textbook of Neonatal Resuscitation, 6th ed. Elk Grove Village, American Academy of Pediatrics and American Heart Association, 2010.) events leading to apnea (Fig.

1	With oxygen deprivation and carbon dioxide (C02) elevation, there is a transient period of rapid breathing, and if it persists, breathing stops, which is termed primay apnea. This stage is accompanied by a fall in heart rate and loss of neuromuscular tone. Simple stimulation will usually reverse primary apnea. If oxygen deprivation and asphyxia persist, however, the newborn will develop deep gasping respirations, followed by seconday apnea. This latter stage is associated with a further decline in heart rate, fall in blood pressure, and loss of neuromuscular tone. Neonates in secondary apnea will not respond to stimulation and will not spontaneously resume respiratory eforts. Unless ventilation is assisted, death follows. Clinically, primary and secondary apneas are indistinguishable, and thus, secondary apnea must be assumed. And, when a response to stimulation is not immediate, resuscitation with efective ventilation of the apneic newborn must be started quickly.

1	Immediately after birth and usually during the delay for umbilical cord clamping, newborn tone, respiratory efort, and heart rate are evaluated (Fig. 32-2). :Most term neonates are vigorous by 10 to 30 seconds after birth (Ersdal, 2012). For these, initial steps of warming the newborn can be done on the mother's chest or abdomen. Direct skin-to-skin contact with the mother and drying and covering the newborn with a warm blanket will help maintain euthermia (36.5 to 37.5°C). A vigorously crying newborn does not require routine oral suctioning (Carrasco, 1997; Gungor, 2006). Instead, bulb suctioning to remove secretions is best reserved for those who cannot clear secretions on their own due to apnea or copious secretions. Additional routine care steps include drying, gentle stimulation by rubbing the newborn's back, and continued observation during the transition period.

1	If not vigorous or if preterm, the neonate is carried to a prewarmed radiant warmer for the initial newborn care steps. The initial wet birth blanket is removed to allow newborn drying. Cold stress is associated with multiple neonatal morbidities and mortality. Preterm infants are particularly vulnerable, and special steps to maintain euthermia include providing a warmer delivery room (>25°C), covering the neonatal head with either a plastic or wool hat, application of polyethylene plastic "ponchos" or wraps to slow evaporative heat losses, use of chemically activated thermal mattresses to reduce conductive heat loss, and administration of warm, humidified respiratory gases during respiratory stabilization (Perlman, 2015).

1	At the radiant warmer, newborns must be positioned to maximally open the airway, with mild extension of the neck. If the newborn is apneic or has copious secretions that it cannot clear, a bulb syringe or suction catheter may be used to clear the mouth and then the nose. Routine intubation and suctioning of meconium-stained amnionic luid is no longer recommended for the nonvigorous newborn (American College of Obstetricians and Gynecologists, 2017b; Perlman, 2015). Intubation and suction are reserved for suspected airway obstruction. After completion of the initial stabilization steps, apnea, gasping respirations, or heart rate � 100 beats per minute (bpm) should prompt immediate administration of positivepressure ventilation with room air (Fig. 32-3). his should be started by 60 seconds of life, if not sooner, once the initial steps are completed.

1	Assisted ventilation by facemask at a rate of 40 to 60 breaths per minute is recommended. Oxygen saturation is monitored by pulse oximetry. Supplemental oxygen can be given in graduated, rising percentages to maintain oxygen saturation values within a normal range per minute of life. Adequate ventilation is best indicated by an improved heart rate. Colorimetric end-tidal carbon dioxide (ETC02) monitoring placed between the positive-pressure device and facemask serves as a helpful adjunct for detection of successful gas exchange during mask ventilation (Weiner, 2016).

1	If the heart rate remains � 100 bpm after 5 to 10 positive pressure breaths, the attempted ventilation is inadequate and corrective steps must be taken. These can be remembered by the pneumonic MR. SOPA (Table 32-1). The two most common problems are mask leak due to an inefective seal and malposition of the airway (Schmolzer, 2011). If corrective steps do not improve the heart rate, either intubation with an endotracheal tube or placement of a laryngeal mask airway is required.

1	If mask ventilation is inefective or prolonged, an alternative airway is placed. For tracheal intubation, a laryngoscope with a straight blade-size 0 for a preterm newborn and size 1 for a term neonate-is used. Gentle cricoid pressure may be useful. An increasing heart rate and ETC02 detection after several breaths are the primary methods of conirming intubation of the trachea and not the esophagus. One can also look for symmetrical chest wall motion; auscultate for equal breath sounds, especially in the axillae; and auscultate for the absence of breath sounds or gurgling over the stomach. The Newborn 609

1	The Newborn 609 Once in place, the tube is used for tracheal suctioning only for a suspected obstructed airway. Otherwise, an appropriate positive-pressure device is attached to the endotracheal tube. Air pufs are delivered at a rate of 40 to 60 per minute with a force adequate to stabilize the heart rate. In term infants, opening pressures of 30 to 40 cm H20 typically will expand the alveoli without causing barotrauma. Once the lung is inflated, less pressure is typically needed (20 to 25 cm H20). For preterm infants, pressures of 20 to 25 cm Term? Good tone? Breathing/crying? H20 are typically used. An increase in heart rate and peripheral oxygen saturation (Sp02) levels within acceptable ranges reflect a positive response.

1	Term? Good tone? Breathing/crying? H20 are typically used. An increase in heart rate and peripheral oxygen saturation (Sp02) levels within acceptable ranges reflect a positive response. FIGURE 32-2 Algorithm for resuscitation of the newborn based on the International Liaison Committee on Resuscitation scientific review and recommended by the American Academy of Pediatrics and American Heart Association (Perlman, 2015; Wyckoff, 2015). bpm = beats per minute; CPAP = continuous positive airway pressure; ECG = electrocardiogram; ETT = endotracheal tube; HR = heart FIGURE 32-3 Correct use of bag-and-mask ventilation. The head rate; IV = intravenous; PPV = positive-pressure ventilation; Sp02= should be in a sniffing position with the tip of the nose pointing to peripheral oxygen saturation; UVC = umbilical venous catheter. the ceiling. The neck should not be hyperextended.

1	Epinephrine measures (American Academy of Pediatrics, 2017). s shown in Table 32-2, each of five easily identiiable characteristics Intravenously administered epinephrine is indicated if the heart heart rate, respiratory efort, muscle tone, relex irritability, andrate remains ;60 bpm after adequate ventilation and chest color-is assessed and assigned a value of 0, 1, or 2. In the curcompressions. The recommended intravenous dose is 0.01 to rently recommended expanded form, concurrent resuscitation 0.03 mg/kg. Epinephrine may be given through the endotracheal interventions are also recorded over time. he total score, based on tube if venous access has not been established, but its action is the sum of the five components, is determined in all neonates at 1less reliable (Kapadia, 2017). If given through the endotracheal and 5 minutes ater delivery. In those with a score <7, the scoretube, higher doses are employed-0.05 to 0.1 mg/kg.

1	may be calculated at urther 5-minute intervals until a 20-minute Discontinuation of Resuscitation Apgar score is assigned or resuscitation eforts are halted. In an analysis of more than 150,000 newborns delivered at ILCOR concludes that it is reasonable to discontinue resusciParkland Hospital, Casey and associates (2001 b) assessed thetative eforts for a neonate who remains without a heartbeat signiicance of the 5-minute score for predicting survival durdespite at least 10 minutes of continuous and adequate resusciing the first 28 days of life. They found that in term neonates,tative eforts. Notably, the decision to continue or discontinue the risk of neonatal death was approximately 1 in 5000 for resuscitative eforts must be individualized (Perlman, 2015).

1	those with Apgar scores of 7 to 10. his risk compares with a mortality rate of 25 percent for term newborns with 5-minute scores ;3. Low 5-minute scores were comparably predictive of neonatal death in preterm neonates. These investigators con • Apgar Score cluded that the Apgar scoring system remains relevant for the he scoring system described by Dr. Virginia Apgar in 1953 prediction of neonatal survival. remains a useul clinical tool to classiY newborn health immeThere have been attempts to use Apgar scores to define diately after birth and to assess the efectiveness of resuscitative asphxial injury and to predict subsequent neurological TABLE 32-2. 20-Minute Expanded Apgar Score Respiration Absent Weak cry; hypo-Good, crying Comments: Resuscitation CPAP = continuous positive airway pressure; En = endotracheal tube; PPV = positive-pressure ventilation. Data from Weiner, 2016.

1	Comments: Resuscitation CPAP = continuous positive airway pressure; En = endotracheal tube; PPV = positive-pressure ventilation. Data from Weiner, 2016. outcome-uses for which the Apgar score was never intended (Chap. 33, p. 624). Such associations are diicult to mea sure with reliability given that both asphyxial injury and low Apgar scores are infrequent outcomes. For example, according to United States birth certiicate records for 2010, only 1.8 percent of newborns had a 5-minute score below 7 (Martin, 2012). Similarly, in a population-based study of more than 1 million term newborns in Sweden between 1988 and 1997, the incidence of 5-minute Apgar scores of �3 approximated 2 per 1000 (Thorngren-Jerneck, 2001).

1	Previously, many groups established erroneous definitions of asphyxia based solely on low Apgar scores. hese prompted the American College of Obstetricians and Gynecologists and American Academy of Pediatrics (2017f) to issue a series of joint opinions with important caveats regarding Apgar score limitations. Certain elements of the Apgar score are partially dependent on the physiological maturity of the newborn, and a healthy, preterm neonate may receive a low score only because of immaturity. Other influencing factors include fetal malformations, maternal medications, and infection. Therefore, it is inappropriate to use an Apgar score alone to diagnose asphyxia. Moreover, the Apgar score alone cannot establish hypoxia as the cause of cerebral palsy, as discussed in Chapter 33 (p. 624).

1	Blood taken from umbilical vessels may be used for acid-base studies to assess the metabolic status of the neonate. Blood collection is performed following delivery by immediately isolating a 10-to 20-cm segment of cord with two clamps placed near the neonate and another two clamps positioned nearer the placenta. The cord is then cut between the two proximal clamps and then the two distal clamps (Blickstein, 2007).

1	Arterial blood is drawn from the isolated cord segment into a 1-to 2-mL commercially prepared plastic syringe containing lyophilized heparin or a similar syringe that has been flushed with a heparin solution containing 1000 U/mL. Once sampling is completed, the needle is capped and the syringe transported, on ice, to the laboratory. Although eforts should be made for prompt transport, neither the pH nor partial pressure of CO2 (pC02) values change significantly in blood kept at room temperature for up to 60 minutes (Lynn, 2007). Mathematical models have been developed that allow reasonable prediction of birth acid-base status in properly collected cord blood samples analyzed as late as 60 hours after delivery (Chauhan, 1994). Acid-base measurements can show signiicant variances between diferent analyzing devices (vlokarami,r2012).

1	he fetus produces both carbonic and organic acids. Carbonic acid (H2C03) is formed by oxidative metabolism of CO2. he fetus usually rapidly clears CO2 through the placental circulation. If CO2 clearance is lowered, then carbonic acid levels rise. This often follows impaired placental exchange. When H2C03 The Newborn 61 1 rently rise, the result is respiratoy acidemia.

1	The Newborn 61 1 rently rise, the result is respiratoy acidemia. In contrast, organic acids primarily include lactic and 3-hydroxybutyric acids. Levels of these increase with persistent placental exchange impairment, and they result from anaero bic glycolysis. These organic acids are cleared slowly from fetal blood. When they accumulate, without a concurrent increase in H2C03, the result is metabolic acidemia. With the develop ment of metabolic acidemia, bicarbonate (HC03 -) levels drop because it is used to bufer the organic acid. A rise in H2C03 concentrations accompanied by greater organic acid levels, relected by decreased HC03 -levels, causes mixed respiratoy metabolic acidemia.

1	In the fetus, respiratory and metabolic acidemia and ultimately tissue acidosis are most likely part of a progressively worsening continuum. his is diferent from adult pathophysiology, in which distinct conditions result either in respiratory acidosis-for example, pulmonary disease, or in metabolic acidosis-for example, diabetes. In the fetus, the placenta serves as both the lungs and, to a certain degree, the kidneys. One principal cause of fetal acidemia is a drop in uteroplacental perfusion. This creates retention of CO2, that is, respiratory acidemia, and if protracted and severe enough, yields a mixed or metabolic acidemia. Assuming that maternal pH and blood gases are normal, the actual pH of fetal blood is dependent on the proportion of carbonic and organic acids and the amount of bicarbonate, which is the major bufer in blood. This can best be illustrated by the Henderson-Hasselbalch equation: [base]pH = pK + Iog -or, pH = pK + Iog

1	For clinical purposes, HC03-represents the metabolic component and is reported in mEq/L. The H2C03 concentration reflects the respiratory component and is reported as the pC02 in mm Hg. Thus: The result of this equation is a pH value. Because pH is a logarithmic term, it does not give a linear measure of acid accumulation. For example, a change in hydrogen ion concentration associated with a fall in pH from 7.0 to 6.9 is almost twice that which is associated with a fall in pH from 7.3 to 7.2. For this reason, the change in base-termed delta base-ofers a more linear measure of the degree of accumulation of metabolic acid (Armstrong, 2007). The delta base is a calculated number used as a measure of the change in bufering capacity of bicarbonate (HC03 -). he formula for calculating the base excess (BE) is as follows:

1	BE = 0.02786 X pC02 X lO(pH -6.,1) X 13.77 X pH -124.58 32-4 is a nomogram developed from which these can be calculated if only two parameters are known. For example, the HC03 -concentration declines with a metabolic acidemia as it is consumed to maintain a normal pH. A base deicit develops when the HC03-concentration drops below 6.8 reported for preterm neonates (Dick8 pH 015 ? ,:00 ,0ED 70 inson, 1992; Ramin, 1989; Riley, 7.2 7.1 Fetal oxygenation and pH generally decline during the course of normal 7.0 labor. Normal umbilical cord blood pH and blood gas values at delivery 6.9 60 in term newborns are summarized in

1	Table 32-3. Similar values have been 6.7 1993). he lower limits of normal pH in the newborn have been found to range from 7.04 to 7.10 (horp, 6.6 1996). Thus, these values should be 6 considered to deine neonatal acidemia. Even so, most fetuses will tol erate intrapartum acidemia with a 110 pH as low as 7.00 without incurring neurological impairment (Freeman, 1988; Gilstrap, 1989). That said, in FIGURE 32-4 Nomogram for determining the delta base. (Adapted with permission from Siggaarda study of newborns with a pH <7.0 Anderson 0: Blood acid-base alignment nomogram, Scand J (lin Lab Invest. 1963;15:21o1-7.) from Parkland Hospital, there were TABLE 32-3. Umbilical Cord Blood pH and Blood Gas Values in Normal Term Newborns

1	TABLE 32-3. Umbilical Cord Blood pH and Blood Gas Values in Normal Term Newborns Ramin, 1989a Riley, 1993b Kotaska, 201 Ob Kotaska, 2010e pH 7.28 (0.07) 7.27 (0.069) 7.26 (7.01n-7.39) 7.3 (7.05-7.39) Pco2 (mm Hg) 49.9 (14.2) 50.3(11n.1) 51 (30.9-85.8) 54 (37.5-79.5) HC03 -(mEq/L) 23.1 (2.8) 22.0 (3.6) Base excess (mEq/L) -3.6 (2.8) -2.7 (2.8) pH 7.34 (0.063) 7.31 (7.06-7.44) 7.34 (7.10-7.42) PC02 (mm Hg) 40.7 (7.9) 41 (24.9-70.9) 44 (29.1n-70.2) HC03 -(mEq/L) 21.4 (2.5) Base excess (mEq/L) -2.4 (2) aNewborns of selected women with uncomplicated vaginal deliveries. bNewborns of unselected women with vaginal deliveries. (Data shown as mean (SO). dOata shown as range with 2.5 or 97.5 percentile. eCesarean delivery-labor not stated. From Centers for Disease Control and Prevention, 201n2; Watson, 2006.

1	(Data shown as mean (SO). dOata shown as range with 2.5 or 97.5 percentile. eCesarean delivery-labor not stated. From Centers for Disease Control and Prevention, 201n2; Watson, 2006. normal levels, and a base excess occurs when HC03 -values are above normal. Importantly, a mixed respiratory-metabolic acidemia with a large base deicit and a low HC03 -, for example 12 mmollL, is more often associated with a depressed neonate than is a mixed acidemia with a minimal base deficit and a more nearly normal HC03 -level.

1	The Newborn 61 3 inordinate proportions of neonatal deaths-8 percent, intensive care admissions-39 percent, intubations-14 percent, and seizures-13 percent (Goldaber, 1991). And, in a study from Oxford of more than 51,000 term newborns, the incidence of neonatal encephalopathy in those with a birth pH <7.0 was 3 percent (Yeh, 2012). Even those with who had normalrS-minute Apgar scores but an arterial cord pH values <7.0 had a significantly higher risk of morbidity that included respiratory distress, neonatal intensive care unit admission, and sepsis (Sabol, 2016). The speed of acidemia resolution after birth is associated with outcome (Casey, 2001a). Acute interruption in placental gas exchange is accompanied by subsequent CO2 retention and respiratory acidemia. The most common antecedent factor is transient umbilical cord compres sion. Generally, respiratory acidemia is not harmful to the fetus (Low, 1994).

1	he degree to which pH is afected by pC02-the respira tory component of the acidosis-can be calculated. First, the upper normal neonatal pC02 of about 50 mm Hg is subtracted from the cord blood gas pC02 value. Each additional 10 mm Hg pC02 increment will lower the pH by 0.08 units (Eisen berg, 1987). hus, in a mixed respiratory-metabolic acidemia, the benign respiratory component can be calculated. As an example, acute cord prolapse during labor prompts cesarean delivery of a neonate 20 minutes later. The umbilical artery blood gas pH was 6.95 and the pC02 was 90 mm Hg. The degree to which the cord compression and subsequent impair ment of CO2 exchange afected the pH is calculated using the relationship given earlier and shown below. To correct pH: (40 -10) X 0.08 = 0.32; 6.95 + 0'.32 = 7.27 Therefore, the pH before cord prolapse was approximately 7.27, well within normal limits. Thus, the low pH resulted from respiratory acidosis.

1	The fetus begins to develop metabolic acidemia when oxygen deprivation is suiciently long and severe to require anaerobic metabolism for cellular energy needs. Low and associates (1997) defined fetal acidosis as a base deicit ::12 mmoUL, and severe fetal acidosis as a base deicit ::16 mmoUL. In the Parkland study of more than 150,000 newborns cited earlier, metabolic acidemia was defined using umbilical cord blood gas thresholds that were two standard deviations below the mean (Casey, 2001 b). Thus, metabolic acidemia was an umbilical artery blood pH <7.00 accompanied by a pC02 ;76.3 mm Hg, with higher values indicating a respiratory component; HC03-concentration ;17.7 mmoUL; and base deficit ::10.3 mEq/L. From the standpoint of possible neurological injury, the American College of Obstetricians and Gynecologists (2014) defines metabolic acidosis as umbilical arterial pH <7.0 and a base deficit ::12 mmoUL.

1	Metabolic acidemia is associated with a high rate of multiorgan dysfunction. In rare cases, such hypoxia-induced metabolic logical impairment-hypoxic-ischemic encephalopathy (Chap. 33, p. 621). In fact, a fetus without such acidemia cannot by definition have sufered recent hypoxic-induced injury. hat said, severe metabolic acidosis is poorly predictive of subse quent neurological impairment in the term neonate (King, 1998; Socol, 1994). In very-Iow-birthweight neonates, that is, those < 1000 g, newborn acid-base status may be more closely linked to intraventricular hemorrhage and possibly long-term neurological outcome (Lavrijsen, 2005; Salhab, 2005; Victory, 2003).

1	Casey and coworkers (2001 b) described the association between metabolic acidemia, low Apgar scores, and neonatal death in term and preterm newborns. Regarding term neonates, the risk of neonatal death was more than 3200-fold greater in term neonates with metabolic acidemia and 5-minute scores ;3 compared with those with a 5-minute Apgar score ::7. In some centers, cord gas analysis is performed in all neonates at birth (Casey, 2001 b; Sabol, 2016). Cost-efectiveness analysis potential cost savings (White, 2010, 2016). It seems reasonable of cesarean delivery for fetal compromise, abnormal fetal heart rate tracing, fever, and low 5-minute Apgar score. Multifetal gestation and severely growth-restricted fetuses are others. are poorly predictive of either immediate or long-term adverse neurological outcome, they provide the most objective evidence of the fetal metabolic status at birth.

1	are poorly predictive of either immediate or long-term adverse neurological outcome, they provide the most objective evidence of the fetal metabolic status at birth. Ophthalmia neonatorum is mucopurulent conjunctivitis of newborns. Some form of conjunctivitis afects 1 to 12 percent of all neonates, and gonococcal and chlamydial infections are among the most common (Zuppa, 201r1).

1	Neisseria gonorrhoeae infection acquired at birth was a common cause of childhood blindness in the past. However, the practice of instilling a I-percent ophthalmic solution of silver nitrate largely eliminated this. Various other antimicrobial agents have also proven efective, and gonococcal prophylaxis is now mandatory for all neonates in most states (American Academy ofPediatrics, 20 17b). Forprophyxis soon ater delivery, recommendations include a single application of either I-percent silver nitrate solution or 0.5-percent erythromycin ointment. In North America, a previously used I-percent tetracycline ophthalmic ointment is no longer available (Mabry-Hernandez, 2010; Moore, 2015). For a neonate born to a mother with untreated gonorrhea, treatment of presumptive neonatal gonococcal conjunctivitis is a single ceftriaxone dose, 100 mg/kg, given either intramuscularly or intravenously. Before treatment, testing for both gonococcal and chlamydia infections should be obtained.

1	With chamydial conjunctivitis, adequate neonatal prophylaxis is complex. Ideally, prenatal screening and treatment for Chamydia trachomatis obviates conjunctival infection (Hammerschlag, 2011). In neonates delivered vaginally of mothers with an active chlamydial infection, 12 to 25 percent will develop conjunctivitis up to 20 weeks ater birth (Teoh, 2003). Prophyactic topical eye treatments do not reliaby reduce the incidence of chlamydial conjunctivitis. In a study from Kenya, 2.5-percent povidone-iodine solution was reported to be superior to either I-percent silver nitrate solution or 0.5-percent erythromycin ointment in preventing chlamydial conjunctivitis (Isenberg, 1995). In another study from Iran, povidone-iodine eye drops were twice as efective in preventing clinical conjunctivitis as erythromycin drops-9 versus 18 percent failure rate, respectively (Ali, 2007).

1	Conjunctivitis in a newborn up to age 3 months should prompt consideration for chlamydial infection (Moore, 2015). Treatment for pediatric chlamydial infection is with oral azithromycin for 5 days or oral erythromycin for 14 days. Routine immunization with thimerosal-free vaccine against hepatitis B before hospital discharge is standard practice for all medically stable newborns with birthweights greater than 2000 g (merican Academy of Pediatrics, 20 17b). If the mother is seropositive for hepatitis B surface antigen, then the neonate is also passively immunized with hepatitis B immune globulin. As discussed in Chapter 55 (p. 1064), some advocate treatment of high-risk or even all seropositive women with antiviral nucleoside or nucleotide analogues during pregnancy to minimize fetal transmission (Dusheiko, 2012; Tran, 2012).

1	his virus is primarily spread by mosquito bites. Infection is asymptomatic in most people but can cause severe birth defects (Chap. 64, p. 1219). Screening begins with an interrogation for recent travel to endemic areas. For women at risk, serological screening is then completed. All newborns of mothers who have laboratory evidence of Zika virus infection during pregnancy should receive a comprehensive examination, a neurological assessment, postnatal head ultrasound, standard newborn hearing screen before hospital discharge, and Zika virus laboratory testing (Reynolds, 2017). Supplemental vitamin K injection will prevent vitamin K-dependent hemorrhagic disease of the newborn (Chap. 33, p. 626). A single intramuscular dose of vitamin K, 0.5 to 1 mg, is given within 1 hour of birth (American Academy of Pediatrics, 2017b).

1	Numerous mass-screening tests are now available for 29 newborn conditions. Shown in Table 32-4, many are mandated by various state laws (American College of Obstetricians and Gynecologists, 2017 c). lost states require that all tests in the core panel be performed. Supplemental conditions-secondary targets-are also listed on the Maternal and Child Health Bureau website. Some states require some of these in addition to their mandated core panel. Each practitioner should be familiar with their individual state requirements, which are available at http:// genes-r-us.uthscsa.edu/resources/consumer/statemap.htm. Newborn gestational age can be estimated very soon after delivery. The relationship between gestational age and birthweight can identiy neonates at risk for complications. For example, (cobalamin A, B) Propionic 3-Ketothiolase aDetermined by tandem mass spectrometry. bAdded after 2006. From Centers for Disease Control and Prevention, 20r12; Watson, 2006.

1	From Centers for Disease Control and Prevention, 20r12; Watson, 2006. The Newborn 61 5 neonates who are either small or large for gestational age are at greater risk for hypoglycemia and polycythemia, and measurements of blood glucose and hematocrit are indicated. • Care of Skin and Umbilical Cord All excess vernix, blood, and meconium is gently wiped of after delivery while keeping the newborn warm. Any remaining ver nix is readily absorbed and disappears within 24 hours. he first bath is postponed until the neonate's temperature is stable.

1	Aseptic precautions are observed in the immediate care of the cord. The American Academy of Pediatrics has concluded that keeping the cord dry is suicient care (Stewart, 2016). he umbilical cord begins to lose water from Wharton jelly shortly after birth. Within 24 hours, the cord stump loses its characteristic bluish-white, moist appearance and soon becomes dry and black. Within several days to weeks, the stump sloughs and leaves a small, granulating wound, which after healing forms the umbilicus. Separation usually takes place within the irst 2 weeks. The range is 3 to 45 days (Novack, 1988). he umbilical cord dries more quickly and separates more readily when exposed to air. Thus, a dressing is not recommended.

1	In resource-poor countries, local antimicrobial prophylaxis is reasonable (Salam, 2014). Triple-dye applied to the cord was reported to be superior to soap and water care in preventing colonization and exudate formation Qanssen, 2003). In a Nepalese study, cleaning the cord stump with 4-percent chlorhexidine reduced severe infection by 75 percent compared with soap and water (Mullany, 2006). Likewise, O.l-percent chlorhexidine powder was superior to dry cord care (Kapellen, 2009). he World Health Organization (2014) recommends cleansing with chlorhexidine.

1	Despite precautions, a serious umbilical infection-omphaLitis-sometimes develops. In a German study of more than 750 newborns with aseptic cord care, 1.3 percent sufered such infections (Kapellen, 2009). he most likely ofending organisms are StaphyLococcus aureus, Escherichia coLi, and group B streptococcus. Typical signs of cellulitis and stump discharge usually aid diagnosis. Mild erythema and some bleeding at the stump site with cord detachment is also common, but some cases present with no outward signs.

1	In 2016, 81 percent of U.S. newborns were initially breastfed, 52 percent were still breastfed at 6 months, and 31 percent at 1 year (Centers for Disease Control and Prevention, 2016). According to the American College of Obstetricians and Gynecologists (2017 e), exclusive breastfeeding is preferred until 6 months. In many hospitals, breastfeeding begins in the delivery room. Most term newborns thrive best when fed 8 to 12 times daily for approximately 15 minutes each episode. Preterm or growth-restricted newborns require feedings at shorter intervals. Breastfeeding is discussed further in Chapter 36 (p. 656).

1	Because most neonates actually receive little nutriment for the first 3 or 4 days of life, they progressively lose weight until the flow of maternal milk is established or other feeding is instituted. Preterm neonates lose relatively more weight and regain their birthweight more slowly. Conversely, growth-restricted but otherwise healthy newborns regain their initial weight more quickly than those born preterm. With proper nourishment, birthweight of term newborns usually is regained by 10 days. For the irst 2 or 3 days after birth, the colon contains sot, brown-green meconium. his consists of desquamated epithelial cells from the intestinal tract, mucus, epidermal cells, and lanugo (fetal hair) that have been swallowed along with amnionic luid. The characteristic color results from bile pigments. During fetal life and for a few hours after birth, the intestinal contents are sterile, but bacteria quickly colonize the bowel contents.

1	The characteristic color results from bile pigments. During fetal life and for a few hours after birth, the intestinal contents are sterile, but bacteria quickly colonize the bowel contents. Meconium stooling is seen in 90 percent of newborns within the irst 24 hours, and most of the rest within 36 hours. Usually, newborns irst void shortly ater birth but may not until the second day. Meconium and urine passage indicates patency of the gastro intestinal and urinary tracts, respectively. Failure of the newborn to stool or urinate ater these times suggests a congenital defect, such as Hirschsprung disease, imperforate anus, or posterior urethral valve. Ater the third or fourth day, as a result of milk ingestion, meconium is replaced by light-yellow, soter, homogenous feces.

1	Between the second and fifth day of life approximately one third of all neonates develop physiological jaundice of the newborn. It has special significance considering most hospitals have policies for early discharge. Guidelines regarding standard phototherapy equipment and monitoring, as well as treatment recommendations per gestational age, hour of life, and risk factors are used (Bhutani, 2011; Maisels, 2009). Hyperbilirubinemia is discussed further in Chapter 33 (p. 626).

1	Neonatal circumcision of male infants has been a controversial topic in the United States for at least 30 years. Even so, scientific evidence supports several medical beneits that include prevention of phimosis, paraphimosis, and balanoposthitis. Circumcision also lowers the incidence of penile cancer and of cervical cancer among their sexual partners. Previously, the American Academy of Pediatrics Task Force on Circumcision (1999) concluded that existing evidence was insuicient to recommend routine neonatal circumcision. It seems that this policy has had only a negligible efect on practices in this country. Speciically, the Centers for Disease Control and Prevention (2011) estimated that the newborn male circumcision rate declined during a 12-year period from approximately 60 percent in 1999 to only 55 percent in 2010. Other studies have endorsed health beneits of circumcision.

1	Other studies have endorsed health beneits of circumcision. In large randomized trials from regions of Africa with a high prevalence of human immunodeiciency virus (HIV), male circumcision was found to lower the risk of HIV acquisition in the adult by half (Bailey, 2007; Gray, 2007). And, male circumcision was also reported to decrease adult incidences of HIV, HPV, and herpes infections (Tobian, 2009). In its subsequent policy statement, the American Academy of Pediatrics Task Force on Circumcision (2012) concluded that health beneits of newborn male circumcision outweigh the risks. hus, access to the procedure is justified for families who choose it. The Task Force stopped short of recommending circumcision for all newborns. Circumcision is performed only in a healthy neonate. Other contraindications include any genital abnormalities such as hypospadias and a family history of a bleeding disorder, unless excluded in the newborn.

1	The Task Force (2012) recommends procedural analgesia. Various pain relief techniques include lidocaine-prilocaine topical cream, local analgesia iniltration, dorsal penile nerve block, or ring block (Arnett, 1990; Stang, 1988). The dorsal penile nerve block or the ring block is superior to topical analgesia (Hardwick-Smith, 1998; Lander, 1997; Taddio, 1997). he use of a paciier dipped in sucrose is a useful adjunct to these methods (Kaufman, 2002). After appropriate penile cleansing, the ring block places a wheal of I-percent lidocaine at the base of the penis and then advances the needle in a 180-degree arc around the base of the penis. The needle is advanced first to one side and then to the other to achieve a circumferential ring of analgesia. he maximum dose of lidocaine is 1.0 mL. No vasoactive compounds such as epinephrine should ever be added to a local analgesic agent or circumcision.

1	The most commonly used instruments are shown in Figure 32-5 and are Gomco and Mogen clamps and the Plastibell device. Compared with the Gomco procedure, Kaufman and colleagues (2002) reported that the Mogen technique required FIGURE 32-5 Three different tools used for circumcision. A.Mogen clamp. The arms of the clamp open to a 3-mm maximum width. B. Gomco clamp, assembled. C. Plastibell device.

1	FIGURE 32-5 Three different tools used for circumcision. A.Mogen clamp. The arms of the clamp open to a 3-mm maximum width. B. Gomco clamp, assembled. C. Plastibell device. less time to perform and was associated with less apparent discomfort for the newborn. Regardless of the method used, the goal is to remove enough shaft skin and inner preputial epithelium so that the glans is exposed suiciently to prevent phimosis. In all techniques: (1) the amount of external skin to be removed must be accurately estimated, (2) the preputial oriice must be dilated to visualize the glans and ensure that it is normal, (3) the inner preputial epithelium must be freed from the glans epithelium, and (4) the circumcision device must be left in place long enough to produce hemostasis before amputating the prepuce (Lerman, 2001).

1	The risks for bleeding, infection, and hematoma formation are low (Christakis, 2000). Unusual complications include distal glans amputation, acquisition of HIV infection or other sexually transmitted disease, meatal stenosis, penile denudation, penile destruction with electrosurgical coagulation, subsequent epidermal inclusion cyst and urethrocutaneous fistula, and ischemia following the inappropriate use of lidocaine with epinephrine (Amukele, 2003; Neulander, 1996; Nicoll, 1997; Pippi-Salle, 2013; Upadhyay, 1998). Hospital rooming in places newborns in their mothers' rooms instead of central nurseries. This practice attempts to make all phases of childbearing as natural as possible and to foster early mother-child relationships. By 24 hours, the mother is generally fully ambulatory. hereafter, with rooming-in, she can usually provide routine care for herself and her newborn. n obvious advantage of this is her ability to assume full care when she arrives home.

1	Traditionally, the newborn is discharged with its mother, and in most cases, maternal stay has determined that of the neonate. From 1970 to the mid-1990s, average maternal postpartum length of stay declined steadily, and many mothers were discharged before 48 hours. The World Health Organization (2014) cites a minimal stay of only 24 hours. Although it is clear that most newborns can be safely discharged within 48 hours, this is not uniformly true. For example, in more than 2.1 million neonates in Canada, Liu and associates (2000) examined readmission rates following initial neonatal discharge. As the length of hospital stay dropped from 4.2 days in 1990 to 2.7 days in 1997, the readmission rate rose from 27 to 38 per 1000. Dehydration and jaundice accounted for most of these readmissions. Using Washington state data, Malkin and coworkers (2000) found that the 28-day mortality rate was increased fourfold in newborns discharged within 30 hours of birth, and the I-year mortality rate

1	Using Washington state data, Malkin and coworkers (2000) found that the 28-day mortality rate was increased fourfold in newborns discharged within 30 hours of birth, and the I-year mortality rate grew twofold. Safe discharge for latepreterm newborns has special concerns (Whyte, 2012).

1	Because of the increased scrutiny regarding short hospital stays, federal legislation-he Newbons' and Mothers' Health Protection Act of 199-was enacted to prohibit insurers from restricting hospital stays for mothers and newborns to less than 2 days for vaginal delivery or 4 days for cesarean delivery. In an analysis of more than 662,000 births in California, Datar and Sood (2006) found that readmission rates declined by 9, 12, and 20 percent, respectively, at 1, 2, and 3 years after the legislation was implemented. li A, Khadije D, Elahe A, et al: Prophylaxis of ophthalmia neonatorum comparison of Betadine, erythromycin and no prophylaxis. J Trop Pediatr 53(6):388, 2007 American Academy of Pediatrics: Delayed umbilical cord clamping after birth. Pediatrics 139(6), 2017a American Academy of Pediatrics, American College of Obstetricians and Gynecologists: Care of the newborn. In Guidelines for Perinatal Care, 8th ed. Elk Grove Village, AAP, 2017b

1	American Academy of Pediatrics, American College of Obstetricians and Gynecologists: Care of the newborn. In Guidelines for Perinatal Care, 8th ed. Elk Grove Village, AAP, 2017b American Academy of Pediatrics Task Force on Circumcision: Circumcision policy statement. Pediatrics 103:686, 1999 American Academy of Pediatrics Task Force on CircumCIsIOn: CircumCIsion policy statement. Pediatrics 130(3):585,t2012 American College of Obstetricians and Gynecologists: Executive summary: neonatal encephalopathy and neurologic outcome, second edition. Report of the American College of Obstetricians and Gynecologists' Task Force on Neonatal Encephalopathy. Obstet Gynecol 123(4):896,t2014 American College of Obstetricans and Gynecologists: Delayed umbilical cord clamping after birth. Committee Opinion No. 684, January 2017a American College of Obstetricians and Gynecologists: Delivery of a newbor� with meconium-stained amniotic fluid. Committee Opinion No. 689, Apnl 2017b

1	American College of Obstetricians and Gynecologists: Delivery of a newbor� with meconium-stained amniotic fluid. Committee Opinion No. 689, Apnl 2017b American College of Obstetricians and Gynecologists: Newborn screening and the role of the obstetrician-gynecologist. Committee Opinion No. 616, January 2015, Reairmed 2017c American College of Obstetricians and Gynecologists: Planned home birth. Committee Opinion No. 697, Aprilt2017d American College of Obstetricians and Gynecologists: Optimizing support for breastfeeding as part of obstetric practice. Committee Opinion No. 658, February 2016, Reairmed 2017 e American College of Obstetricians and Gynecologists. American Academy of Pediatrics: The Apgar score. Committee Opinion No. 644, October 2015, Reairmed 2017f Amukele SA, Lee GW, Stock JA, et al: 20-year experience with iatrogenic penile injury. J Urolt170:1691, 2003 . Apgar V: A proposal for a new method of evaluation of the newborn mfant.

1	Amukele SA, Lee GW, Stock JA, et al: 20-year experience with iatrogenic penile injury. J Urolt170:1691, 2003 . Apgar V: A proposal for a new method of evaluation of the newborn mfant. Curr Res Anesth Analg 32:260, 1953 Armstrong L, Stenson BJ: Use of umbilical cord blood gas analysis in the assessment of the newborn. Arch Dis Child Fetal Neonatal Ed 92:430, 2007 Arnett M, Jones JS, Horger EO III: Efectiveness of 1% lidocaine dorsal penile nerve block in infant circumcision. Am J Obstet Gynecol 163:1074, 1990 Bailey RC, Moses S, Parker CB, et al: Male circumcision for HIV prevention in young men in Kismu, Kenya: a randomized controlled trial. Lancet 369:643, 2007 Bhatt S, Alison BJ, Wallace EM, et al: Delaying cord clamping until ventilation onset improves cardiovascular function at birth in preterm lambs. J Physiol 591(8):2113,t2013

1	Bhatt S, Alison BJ, Wallace EM, et al: Delaying cord clamping until ventilation onset improves cardiovascular function at birth in preterm lambs. J Physiol 591(8):2113,t2013 Bhutani K,Committee on Fetus and Newborn, American Academy ofPediatrics: Phototherapy to prevent severe neonatal hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics 128(4):el046, 2011 Blickstein 1, Green T: Umbilical cord blood gases. Clin Perinatol 34(3):451, 2007 Carrasco M, Martell M, Estol PC: Oronasopharyngeal suction at birth: efects on arterial oxygen saturation. J Pediatr 130(5):832, 1997 . Casey BM, Goldaber KG, Mcintire DD, et al: Outcomes among term mfants when two-hour postnatal pH is compared with pH at delivery. Am J Obstet Gynecol 184:447, 2001a Casey BM, Mcintire DD, Leveno KJ: he continuing value of the Apgar score for the assessment of newborn infants. N Engl J Med 344:467, 2001b

1	Casey BM, Mcintire DD, Leveno KJ: he continuing value of the Apgar score for the assessment of newborn infants. N Engl J Med 344:467, 2001b Centers for Disease Control and Prevention: Breastfeeding report card: progressing toward national breastfeeding goals-United States 2016. Available at: http://www.cdc.gov/breastfeeding/data/reportcard2.htm. Accessed June 2017 Centers for Disease Control and Prevention: CDC grand rounds: newborn screening and improved outcomes. MMWR 61(21)390,t2012 Centers for Disease Control and Prevention: Trends in in-hospital newborn male circumcision-United States, 1999-2010. MMWR 60(34):1167, 2011 Chauhan SP, Cowan BD, Meydrech EF, et al: Determination of fetal acidemia at birth from a remote umbilical arterial blood gas analysis. Am J Obstet Gynecol 170: 1705, 1994 Christakis DA, Harvey E, Zerr DM, et al: A trade-of analysis of routine newborn circumcision. Pediatrics 105:246,t2000

1	Christakis DA, Harvey E, Zerr DM, et al: A trade-of analysis of routine newborn circumcision. Pediatrics 105:246,t2000 Datar A, Sood N: Impact of postpartum hospital-stay legislation on newborn length of stay, readmission, and mortality in California. Pediatrics 118:63, 2006 The Newborn 617 Dickinson JE, Eriksen NL, Meyer BA, et al: The efect of preterm birth on umbilical cord blood gases. Obstet Gynecol 79:575, 1992 Dusheiko G: Interruption of mother-to-infant transmission of hepatitis B: time to include selective antiviral prophylaxis? Lancet 379(9830):2019, 2012 Eisenberg MS, Cummins RO, Ho MT: Code Blue: Cardiac Arrest and Resus citation. Philadelphia, Saunders, 1987 Ersdal HL, Mduma E, Svensen E, et al: Early initiation of basic resuscitation interventions including face mask ventilation may reduce birth asphyxia related mortality in low-income countries: a prospective descriptive observa tional study. Resuscitation 83(7):869, 2012 . .

1	Freeman JM, Nelson KB: Intrapartum asphyxia and cerebral palsy. Pediatrics 82:240, 1988 Gilstrap LC III, Leveno KJ, Burris J, et al: Diagnosis of birth asph�ia on the basis of fetal pH, Apgar score, and newborn cerebral dysfunction. Am J Obstet Gynecol 161:825, 1989 Goldaber KG, Gilstrap LC III, Leveno KJ, et al: Pathologic fetal acidemia. Obstet Gynecol 78:1103, 1991 Gray RH, Kigozi G, Serwadda D, et al: Male circumcision for HIV prevention in Rakai, Uganda: a randomized trial. Lancet 369:657,t2007 Gungor S, Kurt E, Teksoz E, et al: Oronasopharyn�eal suction vers�s no suction in normal and term infants delivered by elective cesarean section: a prospective randomized controlled trial. Gynecol Obstet Invest 61 (1):9, 200? Hammerschlag MR: Chlamydial and gonococcal infections in infants and children. Clin Infect Dis 53(3):S99, 2011 Hardwick-Smith S, Mastrobattista JM, Wallace PA, et al: Ring block for neonatal circumcision. Obstet Gynecol 91 :930, 1998

1	Hardwick-Smith S, Mastrobattista JM, Wallace PA, et al: Ring block for neonatal circumcision. Obstet Gynecol 91 :930, 1998 Hooper SB, Kitchen MJ, Wallace MJ, et al: Imaging lung aeration and lung liquid clearance at birth. FASEB J 21(12):3329, 2007 Hooper SB, te Pas AB, Kitchen MJ: Respiratory transition in the newborn: a three-phase process. Arch Dis Child Fetal Neonatal Ed 101(3):F266, 201? Hooper SB, te Pas AB, Lang J, et al: Cardiovascular transition at birth: a phySIological sequence. Pediatr Res 77(5):608, 2015 Isenberg SJ, Apt L, Wood M: A controlled trial of povidone-iodine as prophy laxis against ophthalmia neonatorum. N Engl J Med 332:562, 1995 . Janssen PA, Selwood BL, Dobson SR, et al: To dye or not to dye: a rando�lIzed clinical trial of a triple dye/alcohol regime versus dry cord care. PedJatflcs 111:15, 2003 Kapadia V, Wyckof MH: Chest compressions for bradycardia or asystole in neonates. Clin Perinatol 39(4):833, 2012

1	Kapadia V, Wyckof MH: Chest compressions for bradycardia or asystole in neonates. Clin Perinatol 39(4):833, 2012 Kapadia VS, Wyckof MH: Epinephrine use during newborn resuscitation. Front Pediatr 5:97, 2017 Kapellen TM, Gebauer CM, Brosteanu 0, et al: Higher rate of cord-related adverse events in neonates with dry umbilical cord care compared to chlorhexidine powder. Results of a randomized controlled study to compare eicacy and safely of chlorhexidine powder versus dry care in umbilical cord care of the newborn. Neonatology 96(1): 13, 2009 Karlberg P, Adams FH, Geubelle F, et al: Alteration of the infant's thorax dur ing vaginal delivery. Acta Obstet Gynecol Scand 41 :223, 1962 . Katheria AC, Lakshminrusimha S, Rabe H, et al: Placental transfUSIOn: a review. J Perinatol 37(2): 105, 2017 Kattwinkel J: Textbook of Neonatal Resuscitation, 6th ed. Elk Grove Village, American Academy of Pediatrics and American Heart Association, 2010

1	Kattwinkel J: Textbook of Neonatal Resuscitation, 6th ed. Elk Grove Village, American Academy of Pediatrics and American Heart Association, 2010 Kaufman GE, Cimo S, Miller L W, et al: An evaluation of the efects of sucrose on neonatal pain with 2 commonly used circumcision methods. Am J Obstet Gynecol 186:564, 2002 King TA, Jackson GL, Josey AS, et al: The efect of profound umbil�cal artery acidemia in term neonates admitted to a newborn nursery. J Pedlatr 132(4):624, 1998 Kotaska K, Urinovska R, Klapkova E, et al: Re-evaluation of cord blood arterial and venous reference ranges for pH, pOz, pCOz, according to spontaneous or cesarean delivery. J Clin Lab Anal 24(5):300, 2010 Lander J, Brady-Fryer B, Metcalfe JB, et al: Comparison of ring block, dorsal penile nerve block, and topical anesthesia for neonatal circumcision: a randomized controlled trial. JAMA 278:2157, 1997

1	Lander J, Brady-Fryer B, Metcalfe JB, et al: Comparison of ring block, dorsal penile nerve block, and topical anesthesia for neonatal circumcision: a randomized controlled trial. JAMA 278:2157, 1997 Lavrijsen SW, Uiterwaal CS, Stigter RH, et al: Severe umbilical co:d acidemia and neurological outcome in preterm and full-term neonates. BIOI Neonate 88(1):27, 2005 Lerman SE, Liao JC: Neonatal circumcision. Pediatr Clin North Am 48: 1539, 200t1 Lines A, Hooper SB, Harding R: Lung liquid production rates and v?lunes do not decrease before labor in healthy fetal sheep. J Appl PhyslOl (1985)82(3):927, 1997 Liu S, Wen SW, McMillan D, et al: Increased neonatal readmission rate associated with decreased length of hospital stay at birth in Canada. Can J Public Health 91 :46, 2000 Low JA, Lindsay BG, Derrick EJ: Threshold of metabolic acidosis associated with newborn complications. Am J Obstet GynecoIt177:1391, 1997

1	Low JA, Lindsay BG, Derrick EJ: Threshold of metabolic acidosis associated with newborn complications. Am J Obstet GynecoIt177:1391, 1997 Lowt]A, Panagiotopoulos C, Derrick E]: Newborn complications after intrapartum asphyxia with metabolic acidosis in the term fetus. Am ] Obstet Gynecol 170: 1081, 1994 Lynn A, Beeby P: Cord and placenta arterial gas analysis: the accuracy of delayed sampling. Arch Dis Child Fetal Neonatal Ed 92(4):F281, 2007 Mabry-Henandez I, Oliverio-Hofman R: Ocular prophylaxis for gonococcal ophthalmia neonatorum: evidence update for the u.s. Preventive Services Task Force reairmation recommendation statement. AHRQ Publication No. 10-05146. Rockville, Agency for Healthcare Research and Quality, 2010 Maisels M], Bhutani VK, Bogen D, et al: hyperbilirubinemia in the newborn infant ::35 weeks' gestation: an update with clariications. Pediatrics 124(4):1193,t2009

1	Maisels M], Bhutani VK, Bogen D, et al: hyperbilirubinemia in the newborn infant ::35 weeks' gestation: an update with clariications. Pediatrics 124(4):1193,t2009 Malkin ]D, Garber 5, Broder MS, et a1: Infant mortality and early postpartum discharge. Obstet Gynecol 96: 183, 2000 Martin ]A, Hamilton BE, Ventura 5], et al: Births: inal data for 2010. Natl Vital Stat Rep 61(1), 2012 Mokarami P, Wiberg N, Olofsson P: An overlooked aspect on metabolic acidosis at birth: blood gas analyzers calculate base deicit diferently. Acta Obstet Gynecol Scand 91(5):5 4,2012 Moore DL, MacDonald NE, Canadian Paediatric Society Infectious Disease and Immunization Committee: Preventing ophthalmia neonatorum. Paediatr Child Health 20(2):93, 2015 Mullany LC, Darmstadt GL, Khatry SK, et al: Topical applications of chlorhexidine to the umbilical cord for prevention of omphalitis and neonatal mortality in southern Nepal: a community-based, cluster randomized trial. Lancet 367:910,t2006

1	Neulander E, Walisch 5, Kaneti ]: Amputation of distal penile glans during neonatal ritual circumcision-a rare complication. Br] Urol 77:924, 1996 Nicoll A: Routine male neonatal circumcision and risk of infection with HIV-l and other sexually transmitted diseases. Arch Dis Child 77:t194,t1997 Novack AH, Mueller B, Ochs H: Umbilical cord separation in the normal newborn. Amt] Dis Child 142:220, 1988 O'Brodovich H, Hannam V, Seear M, et al: Amiloride impairs lung water clearance in newborn guinea pigs.t] Appl Physiol (1985)68(4): 1758, 1990 Perlman ]M, Wyllie], Kattwinkel], et al: Part 7: neonatal resuscitation: 2015 international consensus on cardiopulmonary resuscitation and emergency cardiovascular care science with treatment recommendations. Pediatrics 136 Suppl 2:5120, 2015

1	Pippi-Salle ]L, Jesus LE, Lorenzo A], et a1: Glans amputation during routine neonatal circumcision: mechanism of injuty and strategy for prevention. ] Pediatr Urol 9:763, 2013 Ramin SM, Gilstrap LC, Leveno K], et a1: Umbilical artery acid-base status in the preterm infant. Obstet Gynecol 74:256, 1989 Reynolds MR, Jones AM, Petersen EE, et al: Vital signs: update on Zika virus-associated birth defects and evaluation of all U.S. infants with congenital Zika virus exposure-U.S. Zika Pregnancy Registry, 2017 MMWR 66(13):366,t2017 Riley R], Johnson ]W: Collecting and analyzing cord blood gases. Clin Obstet Gynecol 36: 13, 1993 Rudolph M: Fetal and neonatal pulmonary circulation. Annu Rev Physiol 41 :383, 1979 Sabol BA, Caughey AB: Acidemia in neonates with a 5-minute Apgar score of7 or greater-what are the outcomes? Am] Obstet GynecoI215(4):486 e481, 2016

1	Sabol BA, Caughey AB: Acidemia in neonates with a 5-minute Apgar score of7 or greater-what are the outcomes? Am] Obstet GynecoI215(4):486 e481, 2016 Salam A, Mansoor T, Mallick D, et a1: Essential childbirth and postnatal interventions for improved maternal and neonatal health. Reprod Health 11 Suppl 1:53,t2014 Salhab WA, Perlman ]M: Severe fetal acidemia and subsequent neonatal encephalopathy in the larger premature infant. Pediatr Neurol 32(1):25, 2005 Saunders A, Milner AD: Pulmonary pressure/volume relationships during the last phase of delivery and the irst postnatal breaths in human subjects. J Pediatr 93:667, 1978 Schmolzer GM, Dawson ]A, Kamlin CO, et a1: Airway obstruction and gas leak during mask ventilation of preterm infants in the delivery room. Arch Dis Child Fetal Neonatal Ed 96(4):F254, 2011

1	Schmolzer GM, Dawson ]A, Kamlin CO, et a1: Airway obstruction and gas leak during mask ventilation of preterm infants in the delivery room. Arch Dis Child Fetal Neonatal Ed 96(4):F254, 2011 Siew ML, te Pas AB, Wallace M], et al: Positive end-expiratory pressure enhances development of a functional residual capacity in preterm rabbits ventilated from birth.] Appl Physiol (1985)106(5):1487, 2009a Siew ML, Wallace M], Kitchen M], et al: Inspiration regulates the rate and temporal pattern of lung liquid clearance and lung aeration at birth. ] Appl Physiol (1985)106(6):1888, 2009b Siggaard-Anderson 0: Blood acid-base alignment nomogram. Scand ] Clin Lab Invest 15:211, 1963 Socol ML, Garcia PM, Riter 5: Depressed Apgar scores, acid-base status, and neurologic outcome. Am] Obstet Gynecol 170:991, 1994 Stang H], Gunnar MR, Snellman L, et al: Local anesthesia for neonatal circumcision: efects on distress and cortisol response. ]AMA 259: 1507, 1)88

1	Stang H], Gunnar MR, Snellman L, et al: Local anesthesia for neonatal circumcision: efects on distress and cortisol response. ]AMA 259: 1507, 1)88 Stewart D, Benitz W, AAP Committee on Fetus and Newborn: Umbilical cord care in the newborn infant. Pediatrics 138(3):e20162149, 2016 Taddio A, Stevens B, Craig K, et al: Eicacy and safety of lidocaineprilocaine cream for pain during circumcision. N Engl ] Med 336: 1197, 1997 Teoh D, Reynolds 5: Diagnosis and management of pediatric conjunctivitis. Pediatr Emerg Care 19:48,t2003 te Pas AB, Davis PG, Hooper SB, et a1: From liquid to air: breathing after birth.] Pediatr 152(5):607,t2008 Thongren-]eneck K, Herbst A: Low 5-minute Apgar score: a populationbased register study of 1 million term births. Obstet Gynecol 98:65, 200t1 horp ]A, Dildy GA, Yeomans ER, et a1: Umbilical cord blood gas analysis at delivery. Am] Obstet Gynecolt175(3 Pt 1):517,t1996

1	Tobian A, Serwadda D, Quinn TC, et al: Male circumcision for the prevention of HSV-2 and HPV infections and syphilis. N Engl ] Med 360(13): 1298, 2009 Tran TT: Hepatitis B: treatment to prevent perinatal transmission. Clin Obstet Gynecol 55(2):541, 2012 Upadhyay V, Hammodat HM, Pease PW: Post circumcision meatal stenosis: 12 years' experience. N Z Medt] Ill:57, 1998 Victory R, Penava D, da Silva 0, et a1: Umbilical cord pH and base excess values in relation to neonatal morbidity for infants delivered preterm. Am ] Obstet Gynecol 189(3):803,t2003 Vyas H, Milner AD, Hopkins IE: Intrathoracic pressure and volume changes during the spontaneous onset of respiration in babies born by cesarean section and by vaginal delivery.t] Pediatr 99(5):787, 1981 Watson MS, Mann MY, Lloyd-Puryear MA, et a1: Newborn screening: towards a uniform screening panel and system. Executive summary. Genet Med 8(Suppl 5):15, 2006

1	Watson MS, Mann MY, Lloyd-Puryear MA, et a1: Newborn screening: towards a uniform screening panel and system. Executive summary. Genet Med 8(Suppl 5):15, 2006 Weiner GM: Textbook of Neonatal Resuscitation. 7th ed. Elk Grove Village, American Academy of Pediatrics, 2016 White CR, Doherty DA, Cannon ]W, et a1: Cost efectiveness of universal umbilical cord blood gas and lactate analysis in a tertiary level matenity unit.] Perinat Med 44(5):573,t2016 White CR, Doherty DA, Henderson J], et a1: Beneits of introducing universal umbilical cord blood gas and lactate analysis into an obstetric unit. Aust N Z J Obstet Gynaecol 50(4):318, 2010 Whyte RK: Neonatal management and safe discharge of late and moderate preterm infants. Semin Fetal Neonatal Med 17(3):153,t2012 World Health Organization: Postnatal care of the mother and newborn, 2013. Geneva, WHO, 2014

1	World Health Organization: Postnatal care of the mother and newborn, 2013. Geneva, WHO, 2014 Wyckof MH, Aziz K, Escobedo MB, et al: Part 13: neonatal resuscitation: 20t15 American Heart Association guidelines update for cardiopulmonary resuscitation and emergency cardiovascular care. Pediatrics 136 Suppl 2:5196,t2015 Yeh P, Emary K, Impey L: The relationship between umbilical cord arterial pH and serious adverse neonatal outcome: analysis of 51,519 consecutive validated samples. B]OG 119(7):824,t2012 Zuppa A, D'Andrea V, Catenazzi P, et al: Ophthalmia neonatorum: what time of prophylaxis? ] Matern Fetal Neonatal Med 24(6):769, 2011 Diseases and Injuries of the Term Newborn In a small number of cases tactures of the skul are met with. his accident usualy olows violent attempts at delivey, though occasionaly it may occur spontaneousy. -]. Whitridge Williams (1903)

1	In a small number of cases tactures of the skul are met with. his accident usualy olows violent attempts at delivey, though occasionaly it may occur spontaneousy. -]. Whitridge Williams (1903) In the irst edition of this book, Williams wrote very little of the disorders of the term newborn. That said, it is well known that these neonates are susceptible to a wide variey of illnesses and injuries. In many instances, clinical manifestations of these disorders are extensions of pathological efects already incurred by the fetus. A common example is the newborn who is depressed and acidotic because of intrapartum septicemia. Because many of these disorders manifest diferently, those more common in term newborns are considered here. hose more frequent in preterm neonates are discussed in Chapter 34. Speciic disorders that are the direct consequence of maternal diseases are discussed in pertinent chapters.

1	At the time of delivery, the newborn must convert rapidly to air breathing as described in Chapter 32 (p. 606). With inspiration, there is alveolar expansion, fluid clearance, and surfactant secretion by type II pneumocytes to prevent alveolar collapse. Interference with these functions can create respiratory insuiciency with hypoxemia and compensatory tachypnea, nasal flaring, retractions, and grunting (Reuter, 2014). In preterm infants, this is caused by lung immaturity and insuicient surfactantrespiratory distress syndrome (DS}-and variants may be seen in severely ill older children and adults (Chap. 47, p. 918). All of these have some element of surfactant deiciency because the inciting agent damages alveolar epithelium. As fetuses approach term, surfactant deiciency as a cause of respiratory distress diminishes. he leading causes in term newborns are transient tachypnea of the newborn, RDS, meconium aspiration syndrome, pneumonia, persistent pulmonaY hypertension, and

1	of respiratory distress diminishes. he leading causes in term newborns are transient tachypnea of the newborn, RDS, meconium aspiration syndrome, pneumonia, persistent pulmonaY hypertension, and hypoxic-ischemic encephalopathy (Lin, 2015).

1	In a report from Beijing that described 125 term infants with RDS, the most frequent causes were perinatal infection with sepsis syndrome in 50 percent, elective cesarean delivery in 27 percent, severe asphyxia in 10 percent, and meconium aspiration in 7 percent (Liu, 2010). Notably, even with a low incidence in term infants, RDS from surfactant deiciency is not rare (Berthelot-Ricou, 2012). Chorioamnionitis, male gender, and white race are independent risks (Anadkat, 2012; Higgins, 2016). lso, mutations of genes that encode for surfactant protein synthesis may augment the deiciency (Wambach, 2012). Regardless of etiology, when surfactant secretion is diminished, the pulmonary pathophysiology, clinical course, and management are similar to that for preterm infants. Treatment includes mechanical ventilation and replacement of surfactant (Chap. 34, p. 637). Evidence now supports that antenatal maternal corticosteroid treatment will enhance surfactant synthesis in late-preterm fetuses,

1	ventilation and replacement of surfactant (Chap. 34, p. 637). Evidence now supports that antenatal maternal corticosteroid treatment will enhance surfactant synthesis in late-preterm fetuses, that is, those 34 to 37 weeks' gestation (Gyami-Bannerman, 2016). At Parland Hospital, corticosteroids are not given for this indication in the late-preterm period. Neonatal hypoglycemia is a concern with such treatment, and long-term efects are unknown. However, data indicate that hypoglycemia, if promptly treated, creates no adverse sequelae (McKinlay, 2015). he prognosis in term newborns with RDS largely depends on the cause, severity, and response to treatment.

1	The physiology of meconium passage and amnionic luid contamination is considered in Chapter 24 (p. 474). In some instances, inhalation of meconium-stained fluid at or near delivery causes acute airway obstruction, chemical pneumonitis, surfactant dysfunction or inactivation, and pulmonary hypertension (Lee, 2016; Lindenskov, 2015). If severe, hypoxemia may lead to neonatal death or long-term neurological sequelae in survivors.

1	Given the high incidence-10 to 20 percent-of meconium-stained amnionic fluid in laboring women at term, one may reasonably assume that meconium aspiration must be relatively common. Fortunately, severe aspiration leading to overt respiratory failure is much less frequent. And although the exact incidence of meconium aspiration syndrome is unknown, Singh and associates (2009) reported it to complicate 1.8 percent of all deliveries. In a French study of nearly 133,000 term newborns, the prevalence of severe aspiration syndrome was 0.07 percent, and this rose progressively from 37 to 43 weeks' gestation (Fischer, 2012). Mortality rates depend on severity.

1	Fetal morbidity is more often associated with thicker meconium content. Presumably, in most cases, amnionic luid is ample to dilute the meconium to permit prompt clearance by normal fetal physiological mechanisms. Meconium aspiration syndrome still occasionally develops with light staining. Many newborns are afected after a normal labor and uncomplicated delivery. However, some associated obstetrical factors include postterm pregnancy and fetal-growth restriction. hese fetuses are at highest risk because diminished amnionic fluid and labor with cord compression or utero placental insuiciency are often comorbid. These can enhance the likelihood of meconium passage that is thick and undiluted (Leveno, 1984).

1	Previously, aspiration was thought to be stimulated by fetal hypoxic episodes, and fetal heart rate tracing abnormalities were used to identiy fetuses at greatest risk during labor. Unfortunately, this was found to be an unreliable predictor (Dooley, 1985). As another potential prevention, oropharyngeal suctioning was standard care for a time. However, this was abandoned when evidence failed to support a reduction in syndrome incidence or severity (Davis, 1985; Wiswell, 1990). At the same time, reports described that pulmonary hypertension caused by aspirated meconium was characterized by abnormal arterial muscularization beginning well before birth. These findings led some to conclude that only chronically asphyxiated fetuses developed meconium aspiration syndrome (Katz, 1992). But, correlation was not found between meconium aspiration and markers of acute asphyxia-for example, umbilical artery acidosis (Bloom, 1996; Richey, 1995). Others, however, have reported that thick meconium

1	was not found between meconium aspiration and markers of acute asphyxia-for example, umbilical artery acidosis (Bloom, 1996; Richey, 1995). Others, however, have reported that thick meconium is an independent risk factor for neonatal acidosis (Maisonneuve, 201r1).

1	In response to conlicting results regarding suctioning, an II-center randomized trial was designed to compare suctioning with no suctioning (Vain, 2004). There was an identical 4-percent incidence of meconium aspiration syndrome in both groups. Subsequently, a committee that represented the American Heart Association updated its guidelines (Wyckof, 2015) . Adopted by the American College of Obstetricians and Gynecologists (2017c) and the World Health Organization (2012), these recommend against routine intrapartum oro-and nasopharyngeal suctioning at delivery. For vigorous newborns, no treatment is required. For depressed newborns, management includes intervention to support ventilation and oxygenation, and intubation is used as indicated (Chap. 32, p. 609).

1	Intrapartum amnioinusion has been used successully in laboring women with diminished amnionic fluid volume and frequent variable fetal heart rate decelerations (Chap. 24, p. 475). Earlier, it was studied as a preventive measure in labors complicated by meconium staining. This practice failed to lower meconium aspiration syndrome rates because fetuses usually inhaled meconium before labor (Bryne, 1987; Wenstrom, 1995). To urther settle this issue, a trial was conducted with almost 2000 women at 36 weeks' gestation or later and in whom labor was complicated by thick meconium (Fraser, 2005). he perinatal death rate with and without amnioinusion was 0.05 percent in both groups. Rates of moderate or severe meconium aspiration were also not significantly diferent-4.4 percent with and 3.1 percent without amnioinusion. Finally, cesarean delivery rates were similar-32 versus 29 percent, respectively. Currently, the American College of Obstetrics and Gynecologists (2016a) does not recommend

1	without amnioinusion. Finally, cesarean delivery rates were similar-32 versus 29 percent, respectively. Currently, the American College of Obstetrics and Gynecologists (2016a) does not recommend amnioinusion to reduce meconium aspiration syndrome.

1	Ventilatory support and intubation are carried out as needed (Wyckof, 2015). Because some aspects of meconium aspiration syndrome are caused by surfactant deficiency, replacement therapy is beneficial (N atarajan, 20 16a). Also, inhaled corticosteroids may ameliorate the severity (Garg, 2016). Extracorporeal membrane oxygenation-ECMO-therapy is reserved for neonates who remain poorly oxygenated despite maximal ventilatory assistance (Hirakawa, 2017). In their review of randomized trials, EI Shahed and colleagues (2014) found that surfactant replacement may reduce the need for ECMO but did not lower the mortality rate. he proportion that requires ECMO treatment varies. In a report by Singh and coworkers (2009),r1.4 percent of7518 term newborns with the syndrome required such treatment, and these had a 5-percent mortality rate. Ramachandrappa and associates (2011) reported a higher mortality rate in late-preterm neonates with meconium aspiration compared with afected term newborns.

1	and these had a 5-percent mortality rate. Ramachandrappa and associates (2011) reported a higher mortality rate in late-preterm neonates with meconium aspiration compared with afected term newborns. Finally, pulmonary lavage with surfactant is being evaluated (Choi, 2012).

1	Few events evoke more apprehension in parents and obstetricians than the specter of "brain injury," which immediately

1	Diseases and Injuries of the Term Newborn 621 prompts concerns for disabling cerebral palsy and intellectual disability. Although most brain disorders or injuries are less profound, history has helped to perpetuate the more dismal outlook. In his irst edition of this textbook, Williams (1903) limited discussions of brain injury to those sustained from birth trauma. When later editions introduced the concept that asphyxia neonatorum was another cause of cerebral palsy, this too was linked to traumatic birth. Even as brain damage caused by traumatic delivery became uncommon during the ensuing decades, the belief-albeit erroneous-was that intrapartum events caused most neurological disability. his was a major reason for the escalating cesarean delivery rate beginning in the 1970s. Unfortunately, because in most cases the genesis of cerebral palsy occurs long before labor, this did little to mitigate risks for cerebral palsy (O'Callaghan, 2013).

1	These realizations stimulated scientiic investigations to determine the etiopathogenesis of fetal brain disorders, including those leading to cerebral palsy. Seminal observations include those of Nelson and Ellenberg (1984, 1985, 1986a), discussed subsequently. hese investigators are appropriately credited with proving that these neurological disorders are due to complex multifactorial processes caused by a combination of genetic, physiological, environmental, and obstetrical factors. Importantly, these studies showed that few neurological disorders were associated with peripartum events. Continuing international interest was garnered to codiy the potential role of intrapartum events. In 2000, a task force of the American College ofObstetricians and Gynecologists was appointed to study the �icissitudes of neonatal encephalopathy and cerebral palsy. he multispecialty coalition reviewed contemporaneous data and provided criteria to deine various neonatal brain disorders. heir indings

1	the �icissitudes of neonatal encephalopathy and cerebral palsy. he multispecialty coalition reviewed contemporaneous data and provided criteria to deine various neonatal brain disorders. heir indings were promulgated by the American Academy of Pediatrics and American College of Obstetricians and Gynecologists (2003).

1	Ten years later, a second task force of these organizations updated the indings (American College of Obstetricians and Gynecologists, 2014c). he 2014 Task Force findings are more circumspect in contrast to the earlier ones. Speciically, more limitations are cited in identiying cause(s) of peripartum hypoxic-ischemic encephalopathy (HIE) compared with other etiologies of neonatal encephalopathy. he 2014 Task Force recommends multidimensional assessment of each afected infant. hey add the caveat that no one strategy is infallible, and thus, no single strategy will achieve 100-percent certainty in attributing a cause to neonatal encephalopathy.

1	he 20 14 Task Force defined neonatal encephalopathy as a syndrome of neurological dysfunction identiied in the earliest days of life in neonates born at :35 weeks' gestation. It is manifested by subnormal levels of consciousness or seizures and oten accompanied by diiculty with initiating and maintaining respiration and by depressed tone and relexes. he incidence of encephalopathy has been cited to be 0.27 to 1.1 per 1000 term liveborn neonates, and it is much more frequent in preterm newborns (Ensing, 2013; Plevani, 2013; Takenouchi, 2012; Wu, 2011). Although the 2014 Task Force concluded that there are many causes of encephalopathy and cerebral palsy, it focused on HIE and those that were thought to be incurred intrapartum. To identiy afected infants, a thorough evaluation is necessary and includes maternal history, obstetrical antecedents, intrapartum factors, placental pathology, and newborn course. hese are complemented by laboratory and neuroimaging indings.

1	There are three clinically deined levels. Mild encephalopathy is characterized by hyperalertness, irritability, jitteriness, and hypertonia and hypotonia. Moderate encephalopathy is manifest by lethargy, severe hypertonia, and occasional seizures. Severe encephalopathy is manifest by coma, multiple seizures, and recurrent apnea. The 20 14 Task Force also concluded that of the several forms of cerebral palsy, only the spastic quadriplegic type can result from acute peripartum ischemia. Other forms-hemiparetic or hemiplegic cerebral palsy, spastic diplegia, and ataxia-are unlikely to result from an intrapartum event. Purely dyskinetic or ataxic cerebral palsy, especially when accompanied by a learning disorder, usually has a genetic origin (Nelson, 1998).

1	he 2014 Task Force radically revised its 2003 criteria used to deine an acute peripartum event that is consistent with an HIE and neonatal encephalopathy. hese are outlined in Table 33-1 and are considered with the following caveats. TABLE 33-1 . Findings Consistent with an Acute Peripartum or Intrapartum Event Leading to Hypoxic-Ischemic Encephalopathy Apgar score: <5 at 5 and 10 minutes Umbilical arterial acidemia: pH <7.0 and/or base deficit :12 mmol/L Neuroimaging evidence of acute brain injury: MR imaging or MRS consistent with HIE Multisystem involvement consistent with HIE Type and Timing of Contributing Factors Fetal heart rate monitor patterns consistent with an acute peripartum or intrapartum event HIE = hypoxic ischemic encephalopathy; MR = magnetic resonance; MRS = magnetic resonance spectroscopy. Summarized from the American College of Obstetricians and Gynecologists, 2014b.

1	HIE = hypoxic ischemic encephalopathy; MR = magnetic resonance; MRS = magnetic resonance spectroscopy. Summarized from the American College of Obstetricians and Gynecologists, 2014b. First, Apgar Scores that are low at 5 and 10 minutes are associated with greater risk for neurological impairment. Low scores stem from many causes, and most of these infants will not develop cerebral palsy. With a 5-minute Apgarr? 7, it is unlikely that peripartum HIE caused cerebral palsy. Acid-base study results deine a second HIE criterion. Low pH and base deficit levels raise the likelihood that neonatal encephalopathy was caused by HIE. Decreasing levels form a continuum of increasing risk, but most acidemic neonates will be neurologically normal (Wayock, 2013). A cord artery pH ?7.2 is very unlikely to be associated with HIE.

1	Magnetic resonance (MR) imaging or MR spectroscopy (MRS) is the best modality with which to visualize findings consistent with HIE. The 2014 Task Force concludes that cranial sonography and computed tomography (CT) lack sensitivity in the term newborn. Normal imaging indings after the first 24 hours of life, however, efectively exclude a hypoxic-ischemic cause of encephalopathy. MR imaging between 24 and 96 hours may be more sensitive for the timing of peripartum cerebral injury, and MR imaging at 7 to 21 days following birth is the best technique to delineate the full extent of cerebral injury. Last, multisystem involvement of injury is consistent with HIE. hese include renal, gastrointestinal, hepatic, or cardiac injury; hematological abnormalities; or combinations of these. The severity of neurological injury does not necessarily correlate with injuries to these other systems.

1	The 20 14 Task Force also found that certain contributing factors may be consistent with an acute peripartum event. Of these, sentinel events are considered adverse obstetrical events that may lead to catastrophic clinical outcomes. Examples include ruptured uterus, severe placental abruption, cord prolapse, and amnionic fluid embolism. Ivfartinez-Biarge and associates (2012) studied almost 58,000 deliveries and identified 192 cases with one of these sentinel events. Of these 192 fetus/ newborns, 6 percent died intrapartum or in the early neonatal period, and 10 percent developed neonatal encephalopathy. Other risk factors for neonatal acidosis include prior or emergent cesarean delivery, maternal age ?35 years, thick meconium, chorioamnionitis, and general anesthesia (Ahlin, 2016; Johnson, 2014; Nelson, 2014).

1	Diferentiating an abnormal etal heart rate (FHR) tracing on presentation versus one that develops subsequently was also emphasized by the 20 14 Task Force. A category 1 or 2 FHR tracing associated with Apgar scores ?7 at 5 minutes, normal cord gases (± 1 SD), or both are not consistent with an acute HIE event (Graham, 2014). An FHR pattern at the time of presentation with persistently minimal or absent variability and lacking accelerations, with duration ?60 minutes, and even without decelerations is suggestive of an already compromised fetus (Chap. 24, p. 462). The 2014 Task Force further recommended that if fetal well-being cannot be established with these findings present, the woman should be evaluated for the method and timing of delivery.

1	Most prophylactic measures for neonatal encephalopathy have been evaluated in preterm infants (Chap. 42, p. 824). One of these-postnatally induced hypothermia-may prevent death and mitigate moderate to severe neurological disability in term newborns (Garfinkle, 2015; Nelson, 2014; Shankaran, 2012). MR imaging studies have demonstrated a slowing of difusional abnormalities and fewer infarctions with hypothermia (Bednarek, 2012; Natarajan, 2016b). Most randomized trials have shown improved outcomes with induced hypothermia in those born at 36 weeks' gestation or older (Azzopardi, 2014; Guillet, 2012; Jacobs, 2011). In a metaanalysis of more than 1200 newborns, Tagin and colleagues (2012) concluded that hypothermia improves survival rates and neurodevelopment. Clinical trials to evaluate concomitant neonatal erythropoietin therapy for neuroprophylaxis have reported conlicting results (Fauchere, 2015; Ivf all a, 2017). Preliminary data from one multicenter trial of maternal alopurinol

1	concomitant neonatal erythropoietin therapy for neuroprophylaxis have reported conlicting results (Fauchere, 2015; Ivf all a, 2017). Preliminary data from one multicenter trial of maternal alopurinol therapy indicate some mitigation of cerebral damage caused by hypoxia and ischemia (Kaandorp,r2013).

1	This term refers to a group of nonprogressive disorders of movement or posture caused by abnormal development or damage to brain centers for motor control. Cerebral palsy is further classiied by the type of neurological dysfunction-spastic, dyskinetic, or ataxic-and by the number and distribution of limbs involved-quadriplegia, diplegia, hemiplegia, or monoplegia. Together, the major types are spastic quadriplegia-the most common-which has a strong association with mental retardation and seizure disorders; diplegia, which is common in preterm or low-birthweight infants; hemiplegia; choreoathetoid ypes; and mixed varieties. Although epilepsy and mental retardation frequently accompany cerebral palsy, these two disorders seldom are associated with perinatal asphyxia in the absence of cerebral palsy.

1	According to Nelson and coworkers (2015), the prevalence of cerebral palsy in the United States averages 2 of every 1000 children. t is crucial to emphasize that this rate is derived rom al children-including those born preterm. Because of the remarkably greater survival rates of the latter currently, and despite the elevated cesarean delivery rate, the overall rate of cerebral palsy has remained essentially unchanged (Fig. 33-1). For example, follow-up studies of more than 900,000 Norwegian nonanomalo us term infants cite an incidence of 1 per 1000, but the incidence was 91 per 1000 for those born at 23 to 27 weeks (Moster, 2008). Similar indings have been reported for Australian births (Smithers-Sheedy, 2016). In absolute numbers, term newborns comprise half of cerebral palsy cases because there are proportionately far fewer preterm births. It is again emphasized that most studies of cerebral palsy rates have not made distinctions between term and preterm infants.

1	As noted earlier, Nelson and Ellenberg (1984, 1985, 1986a) made many fundamental observations concerning cerebral palsy. Their initial studies emanated from data from the Collaborative Perinatal Project. This included children from almost 54,000 pregnancies who were followed until age 7. hey found that the most frequently associated risk factors for cerebral palsy were: (1) evidence of genetic abnormalities Diseases and Injuries of the Term Newborn 623 20 2003 Task Force applied these criteria to more -18 contemporaneous outcomes and determined ) that only 1.6 cases of cerebral palsy per 10,000 ) deliveries are attributable solely to intrapartum hypoxia. This finding is supported by a study ) ) U. 1975 to 1980 (Stanley, 1991). Other stud :)0 (Phelan, 1996; Strijbis, 2006). Year of birth Despite persistent attempts to validate con

1	Elective and emergency cesarean deliveries and live births with cerebral palsy. (Reproduced with permission from Nelson KB, Blair E: Prenatal factors in singletons ing as efective to prevent adverse perinatal with cerebral palsy born at or near term, N Engl J Med. 201o5 Sep 3;373(10):946-953.) outcomes, evidence does not support its ability to predict or reduce cerebral palsy risk such as maternal mental retardation or fetal congenital malformations; (2) birthweight <2000 g; (3) birth before 32 weeks; and (4) perinatal infection. hey also found that obstetrical complications were not strongly predictive, and only a fifth of afected children had markers of perinatal asphyxia. For the irst time, there was solid evidence that the cause of most cases of cerebral palsy was unknown, and importanty, ony a small proportion was caused by neonatal HI. Equally importantly, there was no foreseeable single intervention that would likely prevent a large proportion of cases.

1	Numerous studies have since confirmed many of these findings and identiied an imposing list of other risk factors that are shown in Table 33-2. As expected, preterm birth continues to be the single most important risk factor (Nelson, 2015; (Clark, 2003; hacker, 1995). Importantly, no speciic fetal heart rate patterns predict cerebral palsy. Further, no relationship has been found between the clinician's response to abnormal patterns and neurological outcome. And, eforts using assisted computer analysis of fetal TABLE 33-2. Perinatal Risk Factors Reported to Be Increased in Children with Cerebral Palsy Hydramnios 6.9 1n.0-49.3 Placental abruption 7.6 2.7-21.1 Interval between pregnancies 3.7 1.0-4.4 1.7-6.7

1	Thorngren-Jerneck, 2006). Small-for-gestational-age neonates Spontaneous preterm labor are also at higher risk. Stoknes and associates (2012) showed that in more than 90 percent of growth-restricted newborns, cerebral palsy was due to antepartum factors. Many other placental and neonatal risk factors have been correlated with neurodevelopmental abnormalities (Ahlin, 20 l3; Avagliano, 2010; Blair, 2011; Redline, 2008). Some placental factors are discussed further in Chapter 6 (p. 114). One example is the substantively greater risk from chorioamnionitis (Gilbert, 2010; Shatrov, 2010). An example of a neonatal cause is arterial ischemic stroke, which may be associated with inherited fetal thrombophilias (Harteman, 2013; Kirton, 2011). Also, newborns with isolated congenital heart lesions have an elevated risk for microcephaly, possibly due to chronic fetal hypoxemia (Barbu, 2009). Other miscellaneous etiologies of cerebral palsy include fetal anemia, twin-twin transfusion syndrome,

1	have an elevated risk for microcephaly, possibly due to chronic fetal hypoxemia (Barbu, 2009). Other miscellaneous etiologies of cerebral palsy include fetal anemia, twin-twin transfusion syndrome, intrauterine transfusions, and fetal alcohol syndrome (Dejong, 2012; Lindenburg, 2013; O'Leary, 2012; Rossi, 2011; Spruijt, 20r12).

1	Apart from these causes, intrapartum hypoxemia was linked to only a minority of cerebral palsy cases by the National Collaborative Perinatal Project. However, because the study was carried out in the 1960s, there were inconsistent criteria to accurately assign cause. The contribution of HIE to subsequent neurological disorders is discussed in detail on page 621. The Preterm delivery at 23-27 weeks 78.9 56.5-110 Breech or face presentation, 3.8 1.6-9.1 Severe birth defect 5.6 8.1n-30.0 Nonsevere birth defect 6.1 3.1-11.8 Time to cry >5 minutes 9.0 4.3-18.8 Obesity 1.2-2 1.1-2.8 Low placental weight 3.6 1.5-8.4 Placental infarction 2.5 1n.2-5.3 Clinical 2.4 1.5-3.8

1	Nonsevere birth defect 6.1 3.1-11.8 Time to cry >5 minutes 9.0 4.3-18.8 Obesity 1.2-2 1.1-2.8 Low placental weight 3.6 1.5-8.4 Placental infarction 2.5 1n.2-5.3 Clinical 2.4 1.5-3.8 Histological 1.8 1.2-2.9 alncludes respiratory distress syndrome, meconium aspiration, emergent cesarean or operative vaginal delivery, hypoglycemia, gestational hypertension, hypotension, advanced maternal age, genetic factors, twins, thrombotic states, nighttime delivery, seizures, fetal-growth restriction, male gender, and nulliparity. CI = confidence interval. From Ahlin, 2013; Blair, 2011; Mcintyre, 2013; Moster, 2008; Nelson, 2015; O'Callaghan, 2011; Shatrov, 2010; Takenouchi, 2012; Torfs, 1990; Villamor, 2017; Wu, 2012.

1	CI = confidence interval. From Ahlin, 2013; Blair, 2011; Mcintyre, 2013; Moster, 2008; Nelson, 2015; O'Callaghan, 2011; Shatrov, 2010; Takenouchi, 2012; Torfs, 1990; Villamor, 2017; Wu, 2012. heart tracings have not enhanced predictability (Alirevic, 2017; INFANT Collaborative Group, 2017). Indeed, an abnormal heart rate pattern in fetuses that ultimately develop cerebral palsy may reflect a preexisting neurological abnormality (Phelan, 1994). Because of these studies, the American College of Obstetricians and Gynecologists (20r17 a,d) has concluded that electronic fetal monitoring does not reduce the incidence of long-term neurological impairment. his is discussed further in Chapter 24 (p. 477).

1	In general, 1-and 5-minute Apgar scores are poor predictors of long-term neurological impairment (American College of Obstetricians and Gynecologists, 2017e). When the 5-minute Apgar score is $3, however, neonatal death or the risk of neurological sequelae rises substantially (Dijxhoorn, 1986; Nelson, 1984). In a Swedish study, 5 percent of such children subsequently required special schooling (Stuart, 2011). In a Norwegian study, the incidence of these low Apgar scores was 0.1 percent in more than 235,000 newborns. lmost a fourth of those with such scores died, and 10 percent of survivors devel oped cerebral palsy (Moster, 2001).

1	Persistence past 5 minutes of these extremely low scores correlates strongly with a higher risk for neurological morbidity and death (Grtinebaum, 2013). his of course is not absolute, and the 2003 Task Force cited a 10-percent risk for cerebral palsy for infants with 10-minute scores of 0 to 3. For 15-minute scores $2, there is a 53-percent mortality rate and a 36-percent cerebral palsy rate. For 20-minute scores $2, mortality rate is 60 percent, and a cerebral palsy rate is 57 percent. Some outcomes in the Norwegian Study of infants with these low 5-minute Apgar scores are shown in Table33-3. Survivors who had Apgar scores of 0 at 10 minutes have even worse outcomes. In a review of 94 such newborns, 78 died, and al survivors assessed had long-term disabilities (Harrington, 2007). TABLE 33-3. Comparison of Mortality and Morbidity in Norwegian Infants Weighing >2500 g According to 5-Minute Apgar Scores Number 292 233,n500 Neonatal 16.5 0.05 386 (270-552)

1	TABLE 33-3. Comparison of Mortality and Morbidity in Norwegian Infants Weighing >2500 g According to 5-Minute Apgar Scores Number 292 233,n500 Neonatal 16.5 0.05 386 (270-552) Infant 19.2 0.3 76 (56-103) 1-8 yr 3 0.2 18 (8-39) Cerebral palsy 6.8 0.09 81 (48-128) Menta I reta rdation 1.3 0.1 9 (3-29) Other neurological 4.2 0.5 9 (5-17) As outlined on page 621, objective evidence for metabolic acidosis-cord arterial blood pH <7.0 and base deicit � 12 mmollL-is a risk factor for encephalopathy and for cerebral palsy. he risk accrues as acidosis worsens. From their review of 51 studies, Malin and coworkers (2010) found that low cord arterial pH correlates with greater risk for neonatal encephalopathy and cerebral palsy. When used alone, however, these determinations are not accurate in predicting long-term neurological sequelae (Dijxhoorn, 1986; Yeh, 2012).

1	Data from several studies corroborate that a pH <7.0 is the threshold for clinically signiicant acidemia (Gilstrap, 1989; Goldaber, 1991). he likelihood of neonatal death grows as the cord artery pH falls to 7.0 or less. Casey and colleagues (2001) reported that when the pH was $6.8, the neonatal mortality rate rose 1400-fold. When the cord pH was $7.0 and the 5-minute Apgar score was 0 to 3, the risk of neonatal death was increased 3200-fold. In the study from Oxford, adverse neurological outcomes were 0.36 percent with pH <7.1 and 3 percent with pH <7.0 (Yeh, 2012). As mentioned, newborn complication rates rise coincident with increasing severity of acidemia at birth. In a Swedish study, researchers observed that cord blood lactate levels may prove to be superior to base deicit for prognostication of neurological disorders (Wiberg, 2010).

1	Both immature red cells and lymphocytes enter the circulation of term newborns in response to hypoxia or hemorrhage. During the past two decades, quantification of these cells has been proposed as a measure of hypoxia, but most studies do not support this premise (Boskabadi, 2017; Silva, 2006; Walsh, 2011, 2013). Various neuroimaging techniques have provided important insight into the etiology and evolution of perinatal HIE and later cerebral palsy (p. 621). Importantly, indings are highly dependent on fetal age. he preterm neonatal brain responds quite diferently to an ischemic episode compared with that of a term newborn. Other factors include insult severity and duration as well as restoration of cerebrovascular hypoperfusion. hus, precise timing of an injuy with neuroimaging studies is not a realistic goal. Moreover, the grade of neonatal encephalopathy, that is, mild, moderate, or severe, does not correlate with MR imaging indings (Walsh, 2017).

1	Regarding early use, the 2014 Task Force concluded that these imaging techniques provide the following information: 1. Sonographic studies are generally normal on the day of birth. With injury, increasing echogenicity in the thalami and basal 2.0 2 (0.8-5.5) ganglia is seen beginning at approximately 24 hours. This progresses over 2 to 3 days and persists for 5 to 7 days. CI = confidence interval. 2. Computed tomography scans are usually normal the first Data from Moster, 2001 . day in term infants. With injury, decreased density in the Diseases and Injuries of the Term Newborn 625 thalami or basal ganglia is seen beginning at about 24 hours and persists for 5 to 7 days.

1	day in term infants. With injury, decreased density in the Diseases and Injuries of the Term Newborn 625 thalami or basal ganglia is seen beginning at about 24 hours and persists for 5 to 7 days. 3. Magnetic resonance imaging will detect some abnormalities on the irst da. Within 24 hours, MR imaging may show restricted water difusion that peaks at approximately 5 days and disappears within 2 weeks. Acquisitions with Tl-and T2-weighted images show variable abnormalities, which have an onset from less than 24 hours to several days. In a study of 175 term neonates with acute encephalopathy, it was reported that MR imaging showing basal ganglia lesions accurately predicted motor impairment at 2 years of age (Martinez-Biarge, 2012). he 2014 Task Force concluded that for term newborns, imaging studies are helpful in timing an injury, but they provide only a window in time that is imprecise. In one study, the optimal range was 3 to 10 days (Lee, 2017).

1	Neuroimaging in Older Children with Cerebral Palsy Imaging studies performed in children diagnosed with cerebral palsy frequently show abnormal indings. Wu and associates (2006) used CT or MR imaging to study 273 children who were born after 36 weeks' gestation and who were diagnosed later in childhood with cerebral palsy. lthough a third of these studies were normal, focal arterial infarction was seen in 22 percent; brain malformations in 14 percent; and periventricular white-matter injuries in 12 percent. In another study of 351 children with cerebral palsy-approximately half were born near term-MR imaging indings were abnormal in 88 percent (Bax, 2006). Similar findings were reported in an Australian study (Robinson, 2008).

1	CT and v1R imaging techniques have also been used in older children to help deine the timing of fetal or perinatal cerebral injury. Wiklund and coworkers (l991a,b) studied 83 children between ages 5 and 16 years who were born at term and who developed hemiplegic cerebral palsy. Nearly 75 percent had abnormal CT indings, and these investigators concluded that more than half had CT changes that suggested a prenatal injury. Approximately 20 percent were attributed to a perinatal injury. In a similar study, Robinson and associates (2008) used MR imaging. hey reported pathological indings in 84 percent of children with spastic quadriplegia. Remember, this is the neurological lesion that the 20 14 Task Force concluded correlated with neonatal encephalopathy.

1	The term intellectual disabiliy describes a spectrum of disabilities and seizure disorders that frequently accompany cerebral palsy. But, when either of these manifests alone, they are seldom caused by perinatal hypoxia (Nelson, 1984, 1986a,b). Severe mental disability has a prevalence of 3 per 1000 children, and its most frequent causes are chromosomal, gene mutation, and other congenital malformations. Finally, preterm birth is a common association for these (Moster, 2008). he major predictors of seizure disorders are fetal malformations-cerebral and noncerebral; family history of seizures; and neonatal seizures (Nelson, 1986b). Neonatal encephalopathy causes a small proportion of seizure disorders. Reports from the Neonatal Research Network and other studies concluded that increasing severity of encephalopathy correlates best with seizures (Glass, 2011; Kwon, 201l).

1	According to the Centers for Disease Control and Prevention, the frequency of autism spectrum disorders is 14.6 per 1000 in 8-year-old children (Christensen, 2016). Although these may be associated with maternal metabolic conditions, none has been linked convincingly to peripartum events (Krakowiak, 2012). his is a drug-withdrawal syndrome that most commonly fol lows in utero exposure to maternal opioids. It also may com plicate exposure to ethanol or benzodiazepines. The syndrome is characterized by hypertonia, autonomic instability, irritabil ity, poor sucking relex, and seizures (Finnegan, 1975). he incidence of abstinence syndrome has risen six-to sevenfold during the past decade, coincidental with the growing opioid use described in Chapter 1 (p. 9). For example, Tolia and col leagues (2015) reported that 4 percent of all neonatal intensive care unit (NICU) days in 2013 were attributed to care of these afected newborns.

1	Afected neonates undergo close observation, and pharmacotherapy is usually given. In addition to morphine and methadone, other treatment may include phenobarbital, benzodiazepines, and clonidine (Tolia, 2015). More recently, buprenorphine compared with morphine was reported to result in shorter lengths of stay (Kraft, 2017). Consensus is lacking regarding the most efective regimen. he American College of Obstetricians and Gynecologists and the American Society of Addiction Medicine (2017f) have taken the lead in screening, intervention, and treatment of opioid use disorders in pregnant women (Chap. 12, p. 248). here are a few neonatal disorders of erythrocytes, platelets, and coagulation with which the obstetrician should be familiar. As is the case for most other conditions manifest by the newborn shortly after birth, many of these hematological problems were manifest by the fetus and persist in the newborn.

1	fter 35 weeks' gestation, the mean cord hemoglobin concentration approximates 17 g/dL, and values below 14 g/dL are considered abnormal. he American College of Obstetricians and Gynecologists (20 17b) now recommends a 30-to 60-second delay in cord clamping in all healthy newborns. A review of nearly 4000 deliveries found that this delayed cord clamping was associated with a mean neonatal hemoglobin rise of 1.5 g/dL (McDonald, 2013). At the same time, this practice almost doubled the incidence of hyperbilirubinemia requiring phototherapy.

1	Fetal anemia results from many causes (Colombatti, 2016; Yaish, 2017). .Many of these are discussed in more detail in Chapter 15 (p. 300). Acute anemia with hypovolemia is seen with deliveries in which the placenta is cut or torn, if a fetal vessel is perforated or lacerated, if there is recent fetal-maternal hemorrhage, or if the newborn is held well above the level of the placenta for some time before cord clamping. Intracranial or extracranial injury or trauma to fetal intraabdominal organs can also cause hemorrhage with acute anemia (kin, 2011; McAdams, 2017).

1	Neonatal polycythemia with hyperviscosity can be associated with chronic hypoxia in utero, twin-twin transusion syndrome, placental-and fetal-growth restriction, fetal macrosomia from maternal diabetes, and transusion at delivery. When the hematocrit rises above 65, blood viscosity markedly increases and may cause neonatal plethora, cyanosis, or neurologicl aberrations. Because of the shorter life span of macrocytic fetal erythrocytes, hyperbilirubinemia commonly accompanies polycythemia. Other findings include thrombocytopenia, fragmented erythrocytes, and hypoglycemia. Cui and associates (2017) reported a case of unilateral macular hemorrhage in a newborn with polycythemia and platelets of 1 million/IlL Partial exchange transusion may be necessary in some neonates.

1	Even in term fetuses, hepatic maturation is not complete, and thus some unconjugated bilirubin-either albumin bound or free-is cleared by placental transfer to be conjugated in the maternal liver (Chap. 7, p. 135). Fetal protection from unconjugated bilirubin is lost after delivery if not cleared rapidly. Because clearance is totally dependent on neonatal hepatic function, varying degrees of neonatal hyperbilirubinemia result. Even in the mature newborn, serum bilirubin levels usually rise for 3 to 4 days to reach up to 10 mg/ dL. After this, concentrations usually fall rapidly. In one large study, 1 to 2 percent of neonates delivered at 35 weeks' gestation or later had a maximum serum bilirubin level >20 mg/ dL (Eggert, 2006). Concomitant glucose-6-phosphate deiciency worsens hyperbilirubinemia (Chang, 2017). In approximately 15 percent of term newborns, bilirubin levels cause clinically visible skin yellowing termed physiological jaundice (Burke, 2009). As expected, in preterm

1	(Chang, 2017). In approximately 15 percent of term newborns, bilirubin levels cause clinically visible skin yellowing termed physiological jaundice (Burke, 2009). As expected, in preterm neonates, the bilirubin elevation is greater and more prolonged.

1	Excessive serum bilirubin levels can be neurotoxic for newborns (Dijk, 2012; Watchko, 2013). he pathogenesis is complex, and toxicity has two forms. Acute bilirubin encephalopathy is encountered in the irst days of life and is characterized by hypotonia, poor feeding, lethargy, and abnormal auditory-evoked responses (Kaplan, 2011). Immediate recognition and treatment'will usually mitigate progressive neurotoxicity. he chronic form is termed kernicterus. With this, neurotoxicity follows bilirubin deposition and staining of the basal ganglia and hippocampus and is further characterized by profound neuronal degeneration. Survivors have spasticity, muscular incoordination, and varying degrees of mental deiciencies (Frank, 2017). Although there is a positive correlation between kernicterus and unconjugated bilirubin levels above 18 to 20 mg/dL, it can develop at much lower concentrations, especially in very preterm neonates (Sgro, 2011). Continuing hemolysis is a risk factor for

1	and unconjugated bilirubin levels above 18 to 20 mg/dL, it can develop at much lower concentrations, especially in very preterm neonates (Sgro, 2011). Continuing hemolysis is a risk factor for kernicterus (EI Houchi, 2017; Vandborg, 2012).

1	Various forms of phototherapy are used to prevent and treat neonatal hyperbilirubinemia (Ree, 2017). These «bili-lights" emit a spectrum of 460 to 490 nm, which augments bilirubin oxidation to enhance its renal clearance and lower serum levels. Sunlight iltered to remove ultraviolet light has been used in resource-poor countries (Slusher, 2015). Light that penetrates the skin also increases peripheral blood low, which further enhances photo-oxidation. It is problematic that available devices are not standardized (Bhutani, 2011). Another advantage is that exchange transfusions are seldom required with phototherapy. Studies in both preterm and term newborns attest to phototherapy eicacy (Watchko, 2013). A Neonatal Research Network study reported that aggressive phototherapy in low-birthweight neonates reduced rates of neurodevelopmental impairment (Newman, 2006). Similar reductions were reported from Canada after implementation of 2007 guidelines (Sgro, 2016).

1	For term newborns, the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) stress early detection and prompt phototherapy to prevent bilirubin encephalopathy. Despite these measures, bilirubin encephalopathy persists, and this is somewhat related to early hospital discharges (Gazzin, 2011; Kaplan, 2011; Sgro, 2011). According to Burke and coworkers (2009), hospitalizations for kernicterus in term newborns were 5.1 per 100,000 in 1988. Since then, however, this rate has dropped to 0.4 to 2.7 cases per 100,000 births (Watchko, 2013). This may be due in part to legislation, discussed in Chapter 36 (p. 662), to minimize brief postpartum hospital stays.

1	• Hemorrhagic Disease of the Newborn his disorder is characterized by spontaneous internal or external bleeding beginning any time after birth. Most hemorrhagic disease results from abnormally low levels of the vitamin K-dependent clotting factors-V, VII, IX, X, prothrombin, and proteins C and S (Zipursky, 1999). Newborns whose mothers took anticonvulsant drugs are at higher risk because these suppress maternal hepatic synthesis of some of these factors. Classic hemorrhagic disease is usually apparent 2 to 5 days after birth in neonates not given vitamin K prophylaxis at delivery (Busield, 2013). Delayed hemorrhage may occur at 2 to 12 weeks in exclusively breastfed infants because breast milk contains little vitamin K. Other causes of neonatal hemorrhage not related to vitamin K include hemophilia, congenital syphilis, sepsis, thrombocytopenia purpura, erythroblastosis, and intracranial hemorrhage.

1	The American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) recommend routine prophylaxis for hemorrhagic disease with a 0.5-to 1-mg dose of vitamin K\ (phytonadione) given intramuscularly. Oral administration is not efective, and maternal vitamin K administration results in very little transport to the fetus (Sankar, 2016).

1	Abnormally low platelet concentrations in term newborns may be due to various etiologies such as immune disorders, infections, drugs, or inherited platelet defects, or they may be part of a congenital syndrome (American College of Obstetricians and Gynecologists, 20 16b). In many, thrombocytopenia is an extension of a fetal disorder such as infection with B 19 parvovirus, cytomegalovirus, toxoplasmosis, and others discussed in Chapters 64 and 65. Neonatal thrombocytopenia has been reported with maternal antiretroviral therapy for human immunodeiciency virus (HIV) infection (Smith, 2016) . Term newborns admitted to NICUs, especially those with sepsis, have accelerated platelet consumption (Eissa, 2013).

1	In women with an autoimmune disorder such as systemic lupus erythematosus or immunological thrombocytopenia, maternal antiplatelet IgG is transferred to the fetus and can cause accelerated platelet destruction. Most cases are mild, and platelet levels usually reach a nadir at 48 to 72 hours. Maternal corticosteroid therapy generally has no efect on fetal platelets. Fetal blood sampling for platelet determination is seldom necessary, and platelets are usually adequate to prevent fetal hemorrhage during delivery (Chap. 56, p. 1086). Alloimmune thrombocytopenia (AIT) or neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal-fetal platelet antigen disparity. If maternal alloimmunization is stimulated, then transplacental antiplatelet IgG antibodies cause severe fetal thrombocytopenia and severe bleeding (Winkelhorst, 2017). his is considered in detail in Chapter 15 (p. 307).

1	Maternal platelet function and destruction can be severely afected in women with severe preeclampsia. That said, fetal or neonatal thrombocytopenia is rarely caused by the preeclampsia syndrome even when the mother has severe thrombocytopenia. Findings from the large study of mother-infant pairs delivered at Parland Hospital dispelled earlier reports of an association of neonatal thrombocytopenia with preeclampsia (Pritchard, 1987). Instead, neonatal thrombocytopenia was found to be associated with preterm delivery and its numerous complications (Chap. 34, p. 636). Birth injuries can potentially complicate any delivery. hus, although some are more likely associated with operative delivery by forceps or vacuum, others are seen with otherwise uncomplicated vaginal or cesarean delivery. In this section, some injuries are discussed in general, but specific injuries are Diseases and Injuries of the Term Newborn 627

1	Diseases and Injuries of the Term Newborn 627 TABLE 33-4. Incidence of Major and Minor Birth Trauma-Nova Scotia, 1988-2001 Type of Delivery Birth Trauma Spontaneous (14) 88,324 1.2 Vacuum (71) 3175 3.7 67 Forceps (58) 10,478 5.2 53 Vacuum (1n05) 609 8.3 100 Forceps (56) 714 7.0 50 Cesarean (8.6) 16,132 OJ 8.3 Labor (12) 10)31 0.4 11.9 No labor (1.2) 5401 0.2 1.1 All (19.5) 119,432 1.6 18 aMajor trauma = depressed skull fracture, intracranial hemorrhage, brachial plexopathy, or combination. bMinor trauma = linear skull fracture, other fractures, facial palsy, cephalohematoma, or combination. Data from Baskett, 2007. described elsewhere in connection with their associated obstetrical complications.

1	Data from Baskett, 2007. described elsewhere in connection with their associated obstetrical complications. In three population studies that included more than 8 million term newborns, the overall incidence of birth trauma was 20 to 26 per 1000 deliveries (Baskett, 2007; Linder, 2012; Moczygemba, 2010). Data from Nova Scotia show an overall trauma risk of 19.5 per 1000 deliveries (Table 33-4). Only 1.6 cases of major trauma per 1000 were found, and these rates were highest with failed forceps or vacuum delivery and lowest with cesarean delivery without labor. hus, most traumatic injuries were minor, and these had an incidence of 18 per 1000 deliveries.

1	Trauma associated with cesarean delivery from a vIaternalFetal Medicine Units Network study was described by Alexander and coworkers (2006). There were 400 injuries identiied from a total of 37,100 operations-a rate of 11 per 1000 cesarean deliveries. Although skin lacerations predominated-7 per 1000-more serious injuries in these 400 infants included 88 cephalohematomas, 11 clavicular fractures, 11 facial nerve palsies, nine brachial plexopathies, and six skull fractures.

1	Traumatic head injuries that are associated with labor or delivery can be external and obvious, such as a skull or mandibular fracture; they can be intracranial; and in some, they are covert. The fetal head has considerable plasticity and can undergo appreciable molding. Rarely, severe molding can result in tearing of veins. hese may be the bridging cortical veins that empty into the sagittal sinus, the internal cerebral veins, the vein of Galen, or those of the tentorium itself. As a result, intracranial, subdural, and even epidural hemorrhage can be seen after an apparently uneventful vaginal delivery (Scheibl, 2012). Bleeding may also be asymptomatic. Conversely, subgaleal hemorrhages associated with forceps of vacuum delivery can be life threatening (Doumouchtsis, 2008; Swanson, 2012). In rare severe head trauma cases, fetal brain tissue can embolize to the heart or lungs (Cox, 2009).

1	Most aspects of neonatal intracranial hemorrhage are related to gestational age. Speciically, most hemorrhage in the preterm neonate results from hypoxia and ischemia. However, in term newborns, trauma is the most frequent cause. Some varieties are shown in Table 33-5. Importanty, in some newborns, a putative cause is not ound. Intracranial hemorrhage is asymptomatic in many cases. The reported incidence varies, but it is highest with operative deliveries-both vaginal and cesarean deliveries. In the study by Moczygemba and colleagues (2010), for more than 8 million singleton deliveries, the overall intracranial hemorrhage rate approximated 0.2 per 1000 births. In another study, Werner and associates (201rl) cited a combined incidence in more than 120,000 nulliparous singleton operative deliveries of 0.12 percent, or about 1 in 750 procedures. he rates of intracranial hemorrhage were 1 :385 with vacuum delive)a; 1: 515 with forceps, and 1: 1210 with cesarean delivery. In another study,

1	of 0.12 percent, or about 1 in 750 procedures. he rates of intracranial hemorrhage were 1 :385 with vacuum delive)a; 1: 515 with forceps, and 1: 1210 with cesarean delivery. In another study, its incidence was nearly 1 percent following vacuum-assisted deliveries (Simonson, 2007).

1	According to the American College of Obstetricians and Gynecologists (2015), the incidence of intracranial hemorrhage from birth trauma has been substantively lowered by elimination of diicult instrumented vaginal deliveries. This was veriied in a report of carefully conducted Kielland forceps deliveries (Burke, 2012). The prognosis after hemorrhage depends on its location and extent (see Table 33-5). For example, subdural and subarachnoid hemorrhage seldom results in neurological abnormalities, whereas large hematomas are serious. Any bleeding into the parenchyma from intraventricular or intracerebellar hemorrhage often causes serious permanent damage or death. Periventricular hemorrhage rarely causes the type of sequelae that are common in those born preterm (Chap. 34, p. 639).

1	Newborns who have traumatic subdural or infratentorial hemorrhage tears will have neurological abnormalities from the time of birth (Volpe, 1995). Those most severely afected have stupor or coma, nuchal rigidity, and opisthotonos that worsen over minutes to hours. Some newborns who are born depressed appear to improve until about 12 hours of age, when drowsiness, apathy, feeble cry, pallor, failure to nurse, dyspnea, cyanosis, vomiting, and convulsions become evident. Spontaneous intracranial hemorrhage has also been documented in healthy term neonates (Rutherford, 2012; Shah, 2016). In a prospective MR imaging study, Whitby and coworkers (2004) found that 6 percent of those delivered spontaneously and 28 percent of those delivered by forceps had a subdural hemorrhage. None of these had clinical indings, and hematomas resolved by 4 weeks in all infants.

1	These blood collections accumulate outside the calvarium and are categorized as a cephalohematoma or subgaleal hemorrhage (Fig. 33-2). From its most superficial surface inward, the scalp is composed of skin, subcutaneous tissue, galea aponeurotica, subgaleal space, and calvarium periosteum. The galea aponeurotica is dense fibrous tissue, whereas the subgaleal space contains TABLE 33-5. Major Types of Neonatal Intracranial Hemorrhage Trauma-tentorial, falx, or venous (sinus) laceration Uncommon but potentially serious; symptom causing hematoma onset is variable depending on hematoma expansion, but usually <24 hours: irritability, lethargy, and brainstem compression Possibly due to trauma or hypoxia-excludes SAH Common but almost always benign associated with subdural, intraventricular, intracerebral (AVM, aneurysm), or intracerebellar hemorrhage

1	Possibly due to trauma or hypoxia-excludes SAH Common but almost always benign associated with subdural, intraventricular, intracerebral (AVM, aneurysm), or intracerebellar hemorrhage Trauma and perhaps hypoxia-most cases in preterm Uncommon but serious infants Trauma and hypoxia (no discernible cause in Uncommon but serious; symptoms as for 25 percent)-hemorrhage usually from choroid plexus subdural hemorrhage Trauma with epidural or intracerebral hemorrhage Depends on cause Hemorrhagic infarction-embolism or thrombosis in AVM = arteriovenous malformation; SAH = subarachnoid hemorrhage. Data from Volpe, 1995. FIGURE 33-2 Schematic of extracranialolesions in the neonate that include caput succedaneum, subgaleal hemorrhage, and cephalohematoma.

1	FIGURE 33-2 Schematic of extracranialolesions in the neonate that include caput succedaneum, subgaleal hemorrhage, and cephalohematoma. loose, ibroareolar tissue. Traversing across the subgaleal space are large, valveless emissay veins, which connect the dural sinuses inside the skull with supericial scalp veins. Both the galea aponeurotica and subgaleal space span across the occipital, parietal, and frontal bones. In contrast, periosteum invests each individual skull bone and does not cross suture lines. Cephalohematomas are cranial subperiosteal hematomas.

1	These develop from shearing forces during labor and delivery that lacerate the emissary or diploic veins. Fortunately, the densely adhered periosteum impedes rapid enlargement and limits inal hematoma size. Hemorrhage can be over one or both parietal bones, but palpable edges can be appreciated as the blood reaches the limits of the periosteum. These hematomas must be diferentiated from caput succedaneum, also shown in Figure 33-2. A cephalohematoma may not be apparent until hours after delivery, when bleeding suicient to raise the periosteum has occurred. After it is identiied, it often grows larger and persists for weeks or even months, and bleeding may be suicient to cause anemia as discussed on page 625. By contrast, with caput succedaneum, swelling of the scalp is from soft-tissue edema that overlies the periosteum. The caput is maximal at birth, rapidly grows smaller, and usually disappears within hours or a few days. Occasionally it becomes infected, and an abscess may form

1	edema that overlies the periosteum. The caput is maximal at birth, rapidly grows smaller, and usually disappears within hours or a few days. Occasionally it becomes infected, and an abscess may form (Kersten, 2008).

1	Cephalohematomas are common, and in the study from Nova Scotia shown in Table 33-3, these accounted for 80 percent of traumatic injuries with an incidence of 16 per 1000 (Baskett, 2007). They rarely develop in the absence of birth trauma, and an II-percent incidence was reported in 913 term Diseases and Injuries of the Term Newborn 629 newborns delivered by vacuum extraction (Simonson, 2007). In the Network study of cesarean delivery outcomes cited above, the incidence of cephalohematoma was 2.4 per 1000 operations (Alexander, 2006). Others have reported lower incidences, although cephalohematoma is more common with vacuum compared with forceps deliveries-0.8 versus 2.7 per 1000 operative deliveries (Werner, 2011).

1	Subgaleal hemorrhage results from laceration of one of the emissary veins, with bleeding between the galea aponeurotica and the skull periosteum (Shah, 2016). Although most common with operative deliveries, cases with spontaneous vaginal delivery have been described (Uu, 2017). Because of its loose areolar tissue and large surface area, significant blood volumes can collect in this potential space and can extend from the neck to the orbits and laterally to the temporal fascia above the ears (Modanlou, 2016). Resulting hypotension can lead to signiicant morbidity, and cited mortality rates range from 12 to 18 percent (Chang, 2007; Kilani, 2006).

1	These are rare but are especially worrisome because of their association with the serious intracranial hemorrhages. Volpe (1995) considers three types of skull injuries to be fractures-linear and depressed fractures and occipital osteodiastasis. In a French study of nearly 2 million deliveries from 1990 to 2000, the incidence of skull fractures was reported to be 3.7 per 100,000 births, and 75 percent were associated with instrumented vaginal deliveries (Dupuis, 2005). These are occasionally seen with spontaneous or cesarean delivery (Fig. 33-3). These latter fractures are more common when the head is tightly wedged in the pelvis. In such cases, there are at least three possible causes. A fracture may result from skull compression against the sacral promontory, by hand pressure used to lift the head at cesarean delivery, or from transvaginally applied upward hand pressure by an assistant. Fractures are managed with surgical decompression, although spontaneous resolution can follow

1	lift the head at cesarean delivery, or from transvaginally applied upward hand pressure by an assistant. Fractures are managed with surgical decompression, although spontaneous resolution can follow (Basaldella, 2011).

1	FIGURE 33-3 Depressed skull fracture evident immediately after cesarean delivery. Labor had progressed, and the head was deep in the pelvis. Dislodgment of the head from the birth canal was performed by an assistant using manual pressure upward through the vagina. (Used with permission from Dr. Kimberly M Spoonts.) Overstretching of the spinal cord and associated hemorrhage and edema are rare. They are usually caused by excessive longitudinal or lateral traction of the spine or by torsion during delivery. In some cases, vertebrae are fractured or dislocated. Menticoglou and associates (1995) described 15 neonates with this type of high cervical spinal cord injury and found that all of the injuries were associated with forceps rotations. Spinal cord injury also can occur during breech delivery. Ross and coworkers (2006) described C5-6 vertebral dislocation associated with a Zavanelli maneuver done because of shoulder dystocia (Chap. 27, p. 523).

1	Traumatic injuries to nerves can be serious and distressing, especially if permanent. Injury can involve a single nerve, or it can afect a nerve root, plexus, or trunk (Volpe, 1995). Injuries to the brachial plexus are relatively common. hey are identiied in 1 to 3 per 1000 term births (Baskett, 2007; Lindqvist, 2012; Wall, 2014). In the study reported by Moczygemba and colleagues (2010), the incidence of brachial nerve injury was 1.5 per 1000 vaginal deliveries and 0.17 per 1000 cesarean deliveries. he incidence among 366,408 neonates born at Parkland Hospital was 3.5 per 1000 births (Wall, 2014). Breech delivery and shoulder dystocia are risks for this trauma. However, severe plexopathy may also occur without risk factors (Torki, 2012).

1	With plexopathy, the injury damages the nerve roots that supply the brachial plexus-C5_8 and T ,. With hemorrhage and edema, axonal function may be temporarily impaired, but the recovery chances are good. However, with avulsion, the prognosis is poor. In 90 percent of cases, damage to the C5-6 nerve roots causes Erb or Duchenne paraysis (Volpe, 1995). Injuries with breech delivery are normally of this type, whereas the more extensive lesions follow diicult cephalic deliveries (Ubachs, 1995). The C5-6 roots join to form the upper trunk of the plexus, and injury leads to paralysis of the deltoid, infraspinatus, and lexor muscles of the forearm. he afected arm is held straight and internally rotated, the elbow is extended, and the wrist and ingers lexed. Finger function usually is retained. Because lateral traction on the fetal head is frequently employed to efect delivery of the shoulders in normal vertex presentations, most cases of Erb paralysis follow deliveries that do not appear

1	Because lateral traction on the fetal head is frequently employed to efect delivery of the shoulders in normal vertex presentations, most cases of Erb paralysis follow deliveries that do not appear diicult.

1	Damage to the C8-T, roots supplying the lower plexus results in Klumpke paraysis, in which the hand is laccid. Total involvement of all brachial plexus nerve roots results in laccidity of the arm and hand, and with severe damage, there may also be Horner syndrome. Because of its importance, the American College of Obstetricians and Gynecologists (2014a) convened a task force to review extant studies. his Task Force concluded that shoulder dystocia cannot be accurately predicted, but in most cases, axonal death does not occur and the prognosis is good. Lindqvist and associates (2012) reported complete recovery in 86 percent of children with C5-6 trauma, which was the most common injury, and in 38 percent of those with C5-7 damage. However, those with global C5-8-T, inj uries always had permanent disability. Associated clavicular fracture is somewhat protective (Wall, 2014). Surgical exploration and possible repair may improve function if there is persistent paralysis (Malessy, 2009).

1	FIGURE 33-4 Left facial nerve injury. This was almost completely resolved two days after delivery.

1	Trauma to the facial nerve commonly occurs as it emerges from the stylomastoid foramen, and this can cause facial paralysis (Fig. 33-4). The incidence, which ranges from 0.2 to 7.5 per 1000 term births, is likely inluenced by the vigor with which the diagnosis is sought (Al Tawil, 2010; Moczygemba, 2010). Facial paralysis may be apparent at delivery or may develop shortly after birth. It most frequently is associated with uncomplicated vaginal delivery. However, in one series, a fourth of cases followed cesarean delivery (Alexander, 2006; Al Tawil, 2010). Facial nerve damage is likely more common with low forceps (Levine, 1984). It is possible that damage is caused by pressure exerted by the posterior blade when forceps have been placed obliquely on the fetal head. In these cases, forceps marks indicate the cause of injury. Spontaneous recovery within a few days is the rule, however, permanent paralysis has been described (Al Tawil, 2010).

1	Most long-bone fractures follow diicult deliveries, however, this is not always the case. At minimum, palpation of the clavicles and long bones is indicated for all newborns after a diicult delivery. Crepitation or unusual irregularity should prompt radiographic examination. Clavicular ractures are common, unpredictable, and unavoidable complications of normal birth. heir incidence averages 5 to 10 per 1000 live births (Linder, 2012; Moczygemba, 2010). Other than female gender, no speciic risk factorsincluding birthweight and mode of delivery-have been identiied. Clavicular fractures protect against brachial plexopathy when there is shoulder dystocia (Wall, 2014). ion No. 11, August 2017f r� haudharitBt, eased risk for respira-

1	ion No. 11, August 2017f r� haudharitBt, eased risk for respira- HumeraL ractures are infrequent, and 70 percent follow an uneventful birth (T urpenny, 1993). Others are associated with diicult delivery of the shoulders in cephalic deliveries and of an extended arm in breech deliveries. Radiographically, they are often of the greenstick type, although complete fractures and distal humeral epiphyseal fractures can occur (harakan, 2016). FemoraL ractures are rare and usually are associated with vag inal breech delivery. hey occasionally follow cesarean delivery, and in one report, they were bilateral (Cebesoy, 2009). Because most breech-presenting fetuses now undergo cesarean delivery, most of these fractures are associated with this mode (Alexan der, 2006; Cebesoy, 2009).

1	MandibuLar .ractures have been reported, are rare, and have been reviewed by Vasconcelos and coworkers (2009). he rare cases of cervicaL vertebraL disLocation in fetuses delivered as breech or after the Zavanelli maneuver were discussed earlier (Ross, 2006). Finally, rib ractures are occasionally encountered (Khan, 2016). Sternocleidomastoid muscle injury in the past was usually seen with vaginal breech delivery. Hematomas of the muscle or the fascial sheath may resolve slowly with cicatricial contraction. With normal neck growth, the less-elastic damaged muscle does not elongate appropriately. As a result, the head is gradually turned toward the side of the injuy-torticoLis.

1	Conceivably, any fetal organ or part could be injured with either vaginal or cesarean delivery. Some of these include subcapsular hepatic hematomas that presented as inguinal and scrotal hematoma. In such cases, ecchymoses of the inguinal region are termed StabLer sign, and those of the scrotum are termed Bryant sign (Heyman, 2011; Saroha, 2015). hymic gland traumatic hemorrhage in those with underlying hyperplasia or cyst has been described before, during, and after delivery (EHinger, 2007; Saksenberg, 2001). Inj uries to the sixth cranial nerve with resultant lateral rectus ocular muscle paralysis have also been reported (Galbraith, 1994).

1	before delivery. One is the amnionic band syndrome caused when a free strip of amnion forms a focal ring around an extremity or digit. Eventually, deformation or amputation may the uterus. he genesis of such bands is debated and discussed in Chapter 6 (p. 116). A similar anomaly is a Limb-reduction deect associated with chorionic villus sampling performed before 9 weeks' gestation (Chap. 14, p. 294). mechanical factors. Examples of the latter include chronic oli gohydramnios, as well as restricted fetal movement imposed by an abnormally shaped or small uterine cavity or by the presence of additional fetuses. Some mechanical deformations include Diseases and Injuries of the Term Newborn 631 talipes equinovarus (clubfoot), scoliosis, and hip dislocation (Miller, 1981). Talipes and other positional foot abnormalities are associated with membrane rupture from early amniocentesis between 11 and 13 weeks' gestation (Chap. 14, p. 293).

1	hlin K, Himmelmann K, Hagberg G, et al: Cerebral palsy and perinatal infection in children born at term. Obstet Gynecol 122:41, 2013 Ahlin K, Himmelmann K, Nisson S, et al: Antecedents of cerebral palsy according to severity of motor impairment. Acta Obstet Gynecol Scand 95(7):793,t2016 kin MA, Coban D, Doganay S, et al: Intrahepatic and adrenal hemorrhage as a rare cause of neonatal anemia. J Perinat Med 39(3):353,2011 Al Tawil K, Saleem N, Kadri H, et al: Traumatic facial nerve palsy in newborns: is it always iatrogenic? Am J Perinatol 27:71t1, 2010 Alexander JM, Leveno KJ, Hauth J, et al: Fetal injury associated with cesarean delivery. Obstet Gynecol 108:885,t2006 Alfirevic Z, Devane D, Gyte GM, et al: Continuous cardiotocography (CTG) as a form of electronic fetal monitoring (EFM) for fetal assessment during labour. Cochrane Database System Rev 2:CD006066, 2017

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1	Australian Cerebral Palsy Register, birth years 1993 to 2006. Dev Med Child Neurol 58(Suppl 2):5, 2016 Spruijt M, Steggerda S, Rath M, et al: Cerebral injury in twin-twin transfusion syndrome treated with fetoscopic laser surgery. Obstet Gynecol 120(1):15, 2012 Stanley F], Blair E: Why have we failed to reduce the frequency of cerebral palsy? Med ] Aust 154:623, 1991 Stoknes M, Andersen GL, Dahlseng MO, et al: Cerebral palsy and neonatal death in term singletons born small for gestational age. Pediatrics 130(6):el629,t2012 Strijbis EM, Oudman I, van Essen P, et al: Cerebral palsy and the application of the inrernational criteria for acute intrapartum hypoxia. Obstet Gynecol 107:t1t357,t2006 Stuart A, Olausson PO, Kallen K: Apgar scores at 5 minutes after birth in relation to school performance at 16 years of age. Obstet Gynecol 11t8 (2 Pt 1):201, 2011 Swanson AE, Veldman A, Wallace EM, et al: Subgaleal hemorrhage: risk factors and outcomes. Acta Obstet Gynecol Scand 91 (2):260, 2012

1	Tagin A, Woolcott CG, Vincer M], et al: Hypothermia for neonatal hypoxic ischemic encephalopathy: an updated systematic review and meta-analysis. Arch Pediatr Adolesc vIed 166(6):558,t2012 Takenouchi T, KasdorfE, Engel M, et al: Changing pattern of perinatal brain injury in term infants in recent years. Pediatr Neurol 46(2): 106, 2012 hacker SB, Stroup OF, Peterson HB: Eicacy and safety of intrapartum electronic fetal monitoring: an update. Obstet Gynecol 86:613, 1995 harakan S], Lee R], White M, et al: Distal humeral epiphyseal separation in a newborn. Orthopedics 39(4):e764, 2016 horngren-]erneck K, Herbst A: Perinatal factors associated with cerebral palsy in children born in Sweden. Obstet Gynecolt108:1499, 2006 Tolia VN, Patrick SW, Bennett MM, et al: Increasing incidence of the neonatal abstinence syndrome in U.S. neonatal ICUs. N Engl] Med 372(22):2118, 2015

1	Tolia VN, Patrick SW, Bennett MM, et al: Increasing incidence of the neonatal abstinence syndrome in U.S. neonatal ICUs. N Engl] Med 372(22):2118, 2015 Torfs CP, van den Berg B, Oechsli FW, et al: Prenatal and perinatal factors in the etiology of cerebral palsy. ] Pediatr 116:615, 1990 Torki M, Barton L, Miller 0, et al: Severe brachial plexus palsy in women without shoulder dystocia. Obstet Gynecol 120(3):539,t2012 Turpenny PO, Nimmo A: Fractured clavicle of the newborn in a population with a high prevalence of grand-multiparity: analysis of78 consecutive cases. B]OG 100:338, 1993 Ubachs ]M, Sloof AC, Peeters LL: Obstetric antecedents of surgically treated obstetric brachial plexus injuries. B]OG 102:813, 1995 Vain NE, Szyld EG, Prudent LM, et al: Oropharyngeal and nasopharyngeal suctioning of meconium-stained neonates before delivery of their shoulders: multicentre, randomized controlled trial. Lancet 364:597, 2004

1	Vandborg PK, Hansen BM, Greisen G, et al: Follow-up of neonates with total serum bilirubin levels �25 mg/dL: a Danish population-based study. Pediatrics 130(1):61,t2012 Vasconcelos BC, Lago CA, Nogueira RV, et al: Mandibular fracture in a premature infant: a case report and review of the literature. ] Oral Maxillofac Surg 67(1):218,t2009 Villamor E, Tedrof K, Peterson M, et al: Association between matenal body mass index in early pregnancy and incidence of cerebral palsy. ]AMA 317(9):925,t2017 Volpe JJ: Neurology of the Newborn, 3rd ed. Philadelphia, Saunders, 1995 Wall LB, Mills ]K, Leveno K], et al: Incidence and prognosis of neonatal brachial plexus palsy with and without clavicle fractures. Obstet Gynecol 123(6):1288,t2014 Walsh B, Boylan G, Dempsey E, et al: Association of nucleated red blood cells and severity of encephalopathy in normothermic and hypothermic infants. Acta Paediatr 102(2):e64, 2013

1	Walsh B, Boylan G, Dempsey E, et al: Association of nucleated red blood cells and severity of encephalopathy in normothermic and hypothermic infants. Acta Paediatr 102(2):e64, 2013 Walsh BH, Bovian GB, Murray OM: Nucleated red blood cells and early EEG: predicting Sarnat stage and two year outcome. Early Hum Dev 87(5):335, 201t1 Walsh BH, Neil], Morey], et al: he frequency and severity of magnetic resonance imaging abnormalities in infants with mild neonatal encephalopathy. ] Pediatr 187:26, 2017 Wambach ]A, Wegner 0], Depass K, et al: Single ABCA3 mutations increase risk for neonatal respiratory distress syndrome. Pediatrics 130(6):e 15 75, 2012 Watchko ]F, Tiribelli C: Bilirubin-induced neurologic damage-mechanisms and management approaches. N Englt] Med 369:21,t2013 Wayock CP, Meserole RL, Saria S, et al: Perinatal risk factors for severe injury in neonates treated with whole-body hypothermia for encephalopathy. Am ] Obstet GynecoIt21t1(1):41.el, 2014

1	Wayock CP, Meserole RL, Saria S, et al: Perinatal risk factors for severe injury in neonates treated with whole-body hypothermia for encephalopathy. Am ] Obstet GynecoIt21t1(1):41.el, 2014 Wenstrom KD, Andrews W, Maher ]E: Amnioinfusion survey: prevalence, protocols, and complications. Obstet Gynecol 86:572, 1995 Wener EF, ]anevic TM, Illuzzi], et al: Mode of delivery in nulliparous women and neonatal intracranial injury. Obstet Gynecol 118(6): 1239,t201t1 Whitby EH, Griiths PO, Rutter S, et al: Frequency and natural history of subdural haemorrhages in babies and relation to obstetrical factors. Lancet 363:846, 2004 Wiberg N, Kallen K, Herbst A, et al: Relation between umbilical cord blood pH, base deicit, lactate, 5-minute Apgar score and development of hypoxic ischemic encephalopathy. Acta Obstet Gynecol Scand 89: 1263, 2010

1	Wiklund LM, Uvebrant P, Flodmark 0: Computed tomography as an adjunct in etiological analysis of hemiplegic cerebral palsy, 1. Children born preterm. Neuropediatrics 22:50, 1991 a Wiklund LM, Uvebrant P, Flodmark 0: Computed tomography as an adjunct in etiological analysis of hemiplegic cerebral palsy, 2. Children born at term. Neuropediatrics 22: 121) 1991 b Williams ]W: Obstetrics: a Text-book for the Use of Students and Practitioners. New York, Appleton, 1903 Winkelhorst 0, Murphy MF, Greinacher A, et al: Antenatal managemet in fetal and neonatal alloimmune thrombocytopenia: a systematic review. Blood 129(11):1538,t2017 Wiswell TE, Tuggle JM, Turner BS: Meconium aspiration syndrome: have we made a diference? Pediatrics 85:715, 1990 World Health Organization: Guidelines on Basic Newborn Resuscitation. Geneva, World Health Organization, 2012

1	World Health Organization: Guidelines on Basic Newborn Resuscitation. Geneva, World Health Organization, 2012 Wu YW, Bauer A, Ballard A, et al: Erythropoietin for neuroprotection in neonatal encephalopathy: safety and pharmacokinetics. Pediatrics 130(4):683,t2012 Wu W, Croen A, Shah SJ, et al: Cerebral palsy in a term population: risk factors and neuroimaging findings. Pediatrics 1t18:691,t2006 Wu W, Plum TN, Danielsen B, et al: Nighttime delivery and risk of neonatal encephalopathy. Am J Obstet GynecoI204(l):37.e1, 2011 Diseases and Injuries of the Term Newborn 635 Wyckof MH, Aziz K, Escobedo MB, et al: Part 13: Neonatal resuscitation: 2015 American Heart Association guidelines update for cardiopulmonary resuscitation and emergency cardiovascular care. Pediatrics 136 (Suppl 2): SI96,t2015 Yaish HM, Christensen RD, Lemmons RS: Neonatal nonimmune hemolytic anemia. Curr Opin Pediatr 29(1): 12,t2017

1	SI96,t2015 Yaish HM, Christensen RD, Lemmons RS: Neonatal nonimmune hemolytic anemia. Curr Opin Pediatr 29(1): 12,t2017 Yeh P, Emary K, Impey L: The relationship between umbilical cord arterial pH and serious adverse neonatal ourcome: analysis of 51,519 consecurive validated samples. BlOG 119(7):824,t2012 Zipursky A: Prevention of vitamin K deficiency bleeding in newborns. Br J Haematol 104:430, 1999 CHAPTER 34 . The Preterm Newborn RESPIRATORY DISTRESS SYNDROME. .. .. ... 636 NECROTIZING ENTEROCOLITIS . .... ... 638 RETINOPATHY OF PREMATURITY ..639 BRAIN DISORDERS .. ........ . 639 INTRACRANIAL .......... 639 CEREBRAL PALSY . .... ............. 640 !he prognosis or the child depends, of course, upon the degree of development, as wel as the pathological condition or which premature delivery is undertaken. Generaly speaking, in the case of children born bore the thiry-second week, the chances of surviving are vey small. -]. Whitridge Williams (1903)

1	-]. Whitridge Williams (1903) At the time of this textbook's irst edition, preterm delivery of a living newborn was frequently followed by neonatal death. Contrast this with today's technological advances that have advanced the threshold of viability to 22 to 24 weeks' gestation. Even so, the preterm newborn is susceptible to various serious medical complications both early and later in life (Table 34-1). A less commonly cited cause of morbidity and mortality is congenital malformations, which are much more prevalent in preterm births. These complications of prematurity can be placed in perspective in terms of overall neonatal outcomes. In 2009, two thirds of all infant deaths in the United States were in the 12 percent born before 37 weeks (Mathews, 2013). Fortunately, during the past decade, rates of preterm birth have declined from approximately 12 percent in 2007 to 10 percent in 2014. This is in part due to a decline in births to teen mothers (Ferre, 2016).

1	The seminal complication of the preterm newborn is respiratory distress syndrome (DS). This results from immature lungs that are unable to sustain necessary oxygenation. Resulting hypoxia is an underlying associated cause of neurological damage such as cerebral palsy. In addition, hyperoxia, a side efect of DS treatmentacontributes to morbidities such as bronchopulmonary dysplasia, pulmonary hypertension, necrotizing enterocolitis, periventricular leukomalacia, and retinopathy of prematurity. To provide blood gas exchange immediately following delivery, the lungs must rapidly fill with air while being cleared of fluid. Concurrently, pulmonary arterial blood low must rise remarkably. Although some of the luid is expressed as the chest is compressed during vaginal delivery, most is absorbed through the TABLE 34-1. Complications of Prematurity Retinopathy of prematurity (ROP) The Preterm Newborn 637

1	TABLE 34-1. Complications of Prematurity Retinopathy of prematurity (ROP) The Preterm Newborn 637 Chapter 32 (p. 606). Suicient surfactant, synthesized by type II pneumocytes, is essential to stabilize the air-expanded alveoli. It expiration (Chap. 7, p. 133). If surfactant is inadequate, hyaline membranes form in the distal bronchioles and alveoli, and RDS develops. Although respiratory distress syndrome is generally a disease of preterm neonates, it does develop in term newborns, especially with sepsis or meconium aspiration. In these cases, of meconium (Chap. 33, p. 619).

1	With inadequate surfactant, alveoli are unstable, and low pressures cause collapse at end expiration. Pneumocyte nutrition is compromised by hypoxia and systemic hypotension. Partial persistence of the fetal circulation may lead to pulmonary hypertension and a relative right-to-Ieft shunt. Eventually, alveolar cells undergo ischemic necrosis. When oxygen therapy is initiated, the pulmonary vascular bed dilates, and the shunt reverses. Protein-filled fluid leaks into the alveolar ducts, and the cells lining the ducts slough. Hyaline membranes composed of ibrin-rich protein and cellular debris line the dilated alveoli and terminal bronchioles. he epithelium underlying the membrane becomes necrotic. At autopsy, with hematoxylin-eosin staining of lung tissue, these membranes appear amorphous and eosinophilic, like hyaline cartilage. Because of this, respiratory distress syndrome is also termed hyaline membrane disease.

1	In typical RDS, tachypnea develops, the chest wall retracts, and expiration is accompanied by nostril flaring and by gruntingin an attempt to provide a positive end-expiratory pressure to prevent lung collapse. Shunting of blood through nonventilated lung contributes to hypoxemia and to metabolic and respiratory acidosis. Poor peripheral circulation and systemic hypotension may be evident. The chest radiograph shows a difuse reticulogranular infiltrate and an air-illed tracheobronchial tree-air bronchogram. As discussed further in Chapter 33 (p. 619), respiratory insuiciency can also be caused by sepsis, pneumonia, meconium aspiration, pneumothorax, persistent fetal circulation, heart failure, and malformations involving thoracic structures, such as diaphragmatic hernia. Common mutations in surfactant protein production and the phospholipid transporter (ABCA3) contribute to RDS (Beers, 2017; Tredano, 2003; Wert, 2009).

1	An important factor inluencing survival is neonatal intensive care. Although hypoxemia prompts supplemental oxygen, excess oxygen can damage the pulmonary epithelium, retina, and other immature tissues. Despite this, advances in mechanical ventilation technology have improved neonatal survival rates. For example, continuous positive airway pressure (CPAP) prevents the collapse of unstable alveoli. This allows high inspired-oxygen concentrations to be reduced, thereby minimizing its toxicity. In an attempt to minimize the need for tracheal intubation and intermittent positive-pressure ventilation, CPAP has been studied in well-designed multicenter trials (Morley, 2008; SUPPORT Study Group, 2010b). An initial CPAP strategy with subsequent selective surfactant use is a beneficial alternative to immediate intubation and surfactant for many neonates of extremely early gestational age (American Academy of Pediatrics, 2014).

1	Academy of Pediatrics, 2014). rates but is an important factor in the genesis of chronic lung dis ease of prematurity-bronchopulmonary dypasia (BPD). Namely, and volutrauma. Moreover, hyperoxia can create reactive oxy gen species that trigger inflammation. Infection can also be con tributory. In afected newborns, alveolar and pulmonary vascular development is disrupted and leads to hypoxia, hypercarbia, and chronic oxygen dependence (Davidson, 2017; Kair, 2012). As prevention, hightequency oscilatory ventilation has been evaluated. However, beneits and risks varied considerably between studies (Cools, 2015). Treatment of the ventilator-dependent neonate with gluco corticoids was also used previously to prevent BPD. he Ameri can Academy of Pediatrics now recommends against routine steroid use because of limited beneits and greater rates of in exposed neonates (Doyle, 20a14a,b; Watterberg, 2010).

1	In other eforts for BPD prevention, early animal studies demonstrated significant improvements in lung function with weeks of inhaled nitric oxide (McCurnin, 2005). Despite initial enthusiasm, clinical trials failed to demonstrate a consistent benefit. A National Institutes of Health (NIH) consensus statement and the American Academy of Pediatrics (2014) concluded that the available data do not support its use to prevent or treat BPD (Cole, 2011). Caeine has been used widely to treat apnea of prematurity, but it also has bronchodilatory efects. One large randomized trial of cafeine versus placebo showed lower BPD rates, improved neurodevelopmental outcomes during early childhood, and good evidence of safety up to 11 years (Schmidt, 2006, 2012, 2017). This therapy is now widely used for newborns weighing ; 1250 g.

1	The antioxidant vitamin A is necessary for normal lung growth and the integrity of respiratory tract epithelial cells. Preterm newborns have low vitamin A levels at birth, and this has been associated with a greater risk of developing BPD. Randomized trials support the use of vitamin A to achieve a modest reduction in BPD rates for very-Iow-birthweight neonates weighing <1500 g (Darlow, 2016) . Exogenous surfactant products are delivered via endotracheal tube to help prevent RDS. They contain biological or animal surfactants such as bovine-Survanta, calf-Inasu,or porcine-Curosuf Synthetic surfactants such as irst-generation Exosuf and second-generation Suaxin R are equivalent but not superior to animal-derived surfactant (Moya, 2007). In a Cochrane review, Ardell and coworkers (2015) found that animal-derived surfactants led to better outcomes than synthetic surfactants, which do not contain important surfactant proteins. here are currently no synthetic surfactants available.

1	Surfactant replacement was established decades ago as an efective and safe therapy for RDS. Treatment reduces rates of mortality and pneumothorax and improves survival without BPD (Polin, 2014). It has been used for prophyxis of preterm, at-risk newborns and for rescue of those with established disease. Given together, antenatal corticosteroids and surfactant result in an even greater reduction in the overall death rate. However, randomized trials indicate that in populations with high use of antenatal steroids and routine use of CP P in the delivery room, prophylactant surfactant is no longer beneficial and is associated with more risk of death or BPD (Rojas-Reyes, 2012; Sardesai, 2017). Exploration of diferent, less invasive ways to deliver rescue surfactant to spontaneously breathing preterm neonates is currently underway. Potential routes include surfactant application into the pharynx, surfactant nebulization, or application via laryngeal mask or via a thin catheter placed in the

1	neonates is currently underway. Potential routes include surfactant application into the pharynx, surfactant nebulization, or application via laryngeal mask or via a thin catheter placed in the trachea (Kribs, 2016).

1	The NIH (1994, 2000) has concluded that a single course of antenatal corticosteroid therapy reduces RDS and intraventricular hemorrhage rates in preterm neonates born between 24 and 34 weeks' gestation (p. 640). he American College of Obstetricians and Gynecologists (2016a) considers all women at risk for preterm birth in this gestational-age range to be potential candidates for therapy. It also may be considered for pregnant women starting at 23 weeks' gestation who are at risk of preterm delivery within 7 days. his is discussed further is Chapter 42 (p. 823). More recently, administration of antenatal corticosteroids to women at risk for late-preterm delivery (34 to 36 weeks' gestation) was found to significantly reduce the rate of neonatal respiratory complications (Gyami-Bannerman, 2016). Amniocentesis to Assess Fetal Lung Maturity

1	Amniocentesis to Assess Fetal Lung Maturity In some instances, when gestational age is uncertain, knowledge of fetal lung maturity may inluence plans for delivery. One example is the woman with a prior classical cesarean delivery in whom repeat operation is planned and gestational age cannot be confirmed. Several tests are used to ensure fetal pulmonary maturity by analysis of amnionic fluid obtained by sonographically guided amniocentesis. At Parkland Hospital, we still find an occasional indication for such testing, however, the American College of Obstetricians and Gynecologists (2017 a, b) counsels against its use in most of these cases. Instead the College recommends late-term delivery at "41 weeks' gestation" using the best clinical estimate of gestational age (Chap. 10, p. 183).

1	If amniocentesis is elected, luid acquisition is similar to that described for second-trimester amniocentesis (Chap. 14, p. 292). Complications requiring urgent delivery are rare (Zalud, 2008). Following analysis, the probability of RDS developing in a given newborn depends on the test used and fetal gestational age. Importantly, administration of corticosteroids to induce pulmonary maturation has variable efects on some of these tests. Varner and colleagues (2013) have provided a review of testing options. Of biochemical tests, the labor-intensive lecithin-sphingomyelin (LIS) ratio for many years was the gold-standard test. 2•..J• .-E c.o .-8 8c .0ll,� 2 FIGURE 34-1 Changes in mean concentrations of lecithin and sphingomyelin in amnionic fluid during gestation in normal pregnancy. (Modified with permission from Gluck L, Kulovich MV: Lecithin-sphingomyelin ratios in amniotic fluid in normal and abnormal pregnancy, Am J Obstet Gynecol. 1973 Feb 15;115(4):539-546.)

1	Lecithin-sphingomyelin ratios in amniotic fluid in normal and abnormal pregnancy, Am J Obstet Gynecol. 1973 Feb 15;115(4):539-546.) Dipalmitoylphosphatidylcholine (DPPC), that is, lecithin, and phingomyelin are surfactant components. Before 34 weeks, both are present in amnionic fluid in similar concentrations. At 32 to 34 weeks, the concentration of lecithin relative to sphingomyelin begins to rise (Fig. 34-1). The risk of neonatal RDS is slight whenever the concentration of lecithin is at least twice that of sphingomyelin-LIS ratio >2 (Gluck, 1971). Previously, RDS was thought to develop despite an LIS ratio > 2 in newborns of women with diabetes. Some recommend that phosphatidygycerol, another surfactant phospholipid, be documented in amnionic fluid of these women. Based on current evidence, it is unclear if either diabetes, per se, or its level of control causes false-positive phospholipid test results for fetal lung maturity (De Luca, 2009).

1	Of biophysical tests, the luorescence poariation test is an automated assay that measures the surfactant-to-albumin ratio in uncentriuged amnionic luid and gives results in less than an hour. Investigators found the TDx-FLM to be equal or superior to the LIS ratio, foam stability index, or phosphatidylglycerol assessment. his included testing in diabetic pregnancies (Karcher, 2005; Varner, 2013). he modified TDx-FLM Ilis used by many hospitals as their primary test of pulmonary maturity. Thresholds vary by gestational age (Bennasar, 2009). he oam stabiliy or shake test relies on the ability of surfactant in amnionic luid, when mixed appropriately with ethanol, to generate stable foam at the air-liquid interface (Clements, 1972). Problems include errors caused by slight contamination and frequent false-negative test results. Of other tests, the Lumadex-FSI test, luorescent poariation (microviscomery), and amnionic luid absorbance at 650-nm waveength have all been used with variable

1	frequent false-negative test results. Of other tests, the Lumadex-FSI test, luorescent poariation (microviscomery), and amnionic luid absorbance at 650-nm waveength have all been used with variable success.

1	The lamelar body count is a rapid, simple, and accurate method of assessing fetal lung maturity and is comparable to TDx-FLM and LIS ratio accuracy (Karcher, 2005; Varner, 2013). This newborn bowel disorder has clinical findings that include abdominal distention, emesis, ileus, bilious gastric aspirates,

1	This newborn bowel disorder has clinical findings that include abdominal distention, emesis, ileus, bilious gastric aspirates, The Preterm Newborn 639 and bloody stools. here is often radiological evidence of bacteria. Other classic imaging findings include hepatobiliary gas and pneumoperitoneum. Bowel perforation may prompt resection. Necrotizing enterocolitis (NEC) is seen primarily in low-birthweight newborns but occasionally is encountered in mature neonates. Various hypothesized causes include peri natal hypotension, hypoxia, sepsis, umbilical catheterization, exchange transfusions, blood transfusions, and the feeding of cow milk and hypertonic solutions (Neu, 2010). he patho physiology is thought to be multifactorial, and genetic disposi tion, intestinal immaturity, imbalance in microvascular tone, abnormal microbial colonization in the intestine, exposure to enteral feeds, and highly immunoreactive intestinal mucosa play potential roles (Caplan, 2017; Neu, 2010).

1	Medical treatment includes abdominal decompression, bowel rest, broad-spectrum antibiotics, and parenteral nutrition. Surgery is reserved for neonates with intestinal perforation or deteriorating clinical or biochemical status. Possible surgical procedures include drain placement, exploratory laparotomy with resection of diseased bowel, or enterostomy with creation of a stoma (Neu, 2010). By 1950, this condition, formerly known as retrolental ibroplasia, became the largest single cause of blindness in this country. Mter the discovery that the disease resulted from hyperoxemia, its frequency declined but began to rise again with the increasing survival rates of extremely pre term newborns.

1	Normally, the fetal retina vascularizes centrifugally from the optic nerve starting at approximately the fourth month and continues until shortly after birth. During vascularization, excessive oxygen induces severe retinal vasoconstriction with endothelial damage and vessel obliteration. his is followed by subsequent aberrant neovascularization, in which the new vessels penetrate the retina and extend into the vitreous. Here, they are prone to leak proteins or burst with subsequent hemorrhage. Adhesions can form to detach the retina. Vascular endothelial growth factor (VEGF) plays an important role in normal angiogenesis and is up regulated during retinopathy of prematurity (ROP) development (Sharma, 2017). his understanding has opened new avenues of treatment with anti-VEGF therapies.

1	Precise levels of hyperoxemia that can be sustained without causing ROP are unknown. Mter birth, there is a "relative" hyperoxia compared with in utero oxygen content, even in newborns not exposed to higher inspired oxygen concentrations. To better understand the oxygen saturation threshold necessary to minimize ROP without raising rates of other adverse outcomes, the Neonatal Research Network performed a randomized trial of oxygenation in 1316 neonates born between 24 and 27 weeks' gestation (SUPPORT Study Group, 2010a). he two target ranges of oxygen saturation were 85 to 89 percent in one arm and 91 to 95 percent in the other arm. hese targets were both commonly employed in neonatal intensive care units. Death before discharge occurred signiicantly more frequently in the lower-oxygen saturation group-20 versus 16 percent. However, severe ROP among survivors developed signiicantly less often in the lower-oxygen saturation group 8.6 versus 17.9 percent.

1	roanatomical sequelae in preterm newborns compared with those at term (Chap. 33, p. 621). In preterm neonates, cerebral orrhage, cerebellar hemorrhage, periventricular hemorrhagic infarction, cystic periventricular leukomalacia, and difuse white matter injury. All of these are strongly associated with adverse neurodevelopmental outcomes (Kwon, 2014).

1	Cranial sonography remains the preferred approach for events. It is readily available and reliable for detecting common abnormalities and monitoring brain growth. Because cystic inju ries may take 2 to 5 weeks to evolve, serial scans are obtained dur ing this time. In those whose findings are transient and resolve in the neonatal period, prognosis is improved compared with infants whose lesions remain and evolve. At the same time, how ever, between 4 and 10 percent of prematurely born children may develop cerebral palsy (CP) in the absence of lesions. Put another way, 90 to 96 percent of preterm newborns with CP have cerebral lesions that are detectable using cranial sonography.

1	here are ive major categories of intracranial hemorrhage in the neonate (Volpe, 2008). Primay subarachnoid hemorrhage is more common in those born preterm and is frequently benign. Cerebelar hemorrhage is also more frequent in preterm neonates and is increasingly recognized as a cause of serious sequelae. Intraventricular hemorrhage IVH) is almost exclusively seen in preterm newborns, is relatively common, and can have serious efects. Subdural hemorrhages are more frequent in term newborns and can be serious. Miscelaneous intraparenchymal hemorrhage is also more frequent in those born at term and is of variable concern.

1	In preterm infants, the germinal matrix capillary network is fragile for several reasons. First, the subependymal germinal matrix provides poor support for the vessels coursing through it. Second, venous anatomy in this region causes stasis and congestion, which makes vessels susceptible to bursting if intravascular pressure rises. Third, vascular autoregulation is impaired in the preterm neonate (Matsuda, 2006; Verhagen, 2014).

1	If fragile capillaries in the germinal matrix rupture, blood escapes into surrounding tissues and may extend into the ventricular system and brain parenchyma. This type of hemorrhage is common in preterm neonates, especially those born before 32 weeks. However, it can also develop at later gestational ages and even in term neonates. Most hemorrhages develop within 72 hours of birth, but they have been observed as late as 24 days (Whitelaw, 20ll). Because IVH usually is recognized within 3 days of delivery, its genesis is often erroneously attributed to birth events. It is important to realize that prelabor IVH can also occur (Achiron, 1993; Nores, 1996).

1	The pathogenesis of IVH is multifactorial and includes hypoxic-ischemic events, carbon dioxide elevations, anatomical factors, blood pressure instability, coagulopathy, genetic factors, and many others (McCrea, 2008; Ment, 2016). Moreover, preterm birth is frequently associated with infection, which further predisposes to endothelial activation, platelet adherence, and thrombi (Redline, 2008). Respiratory distress and mechanical ventilation are commonly associated factors (Sarkar, 2009).

1	lmost half of hemorrhages are clinically silent. Most small germinal matrix hemorrhages and those confined to the cerebral ventricles resolve without impairment. But, nearly half do show some sign of neurological impairment (Patra, 2006). Survivors of extensive periventricularlintraventricular hemorrhage can have major neurodevelopmental handicaps (Mukerji, 2015). Large lesions can result in hydrocephalus or in degenerated cystic areas termed periventricular leukomalacia (PVL), discussed below. Importantly, the extent ofPVL correlates with CP risk (Bassan, 2006).

1	Ventricular hemorrhage incidences depends on gestational age at birth. From the Neonatal Research Network, approximately 65 percent of all neonates born before 28 weeks' gestation demonstrated some evidence of hemorrhage or PVL (Stoll, 2010). he incidence ranged from 60 percent in those born at 23 weeks to only 23 percent in those at 28 weeks. Importantly, grade IV intraventricular hemorrhage was documented in 21 percent of 23-week-old neonates but in only 3 percent of those at 28 weeks. The severity of IVH can be assessed by neuroimaging studies. Papile and coworkers (1978) devised the mostwidelyused grading scheme to quantiY the extent ofa lesion and estimate prognosis: Grade I-hemorrhage limited to the germinal matrix Grade Ill-hemorrhage with ventricular dilation Grade IV-parenchymal extension of hemorrhage.

1	Grade I-hemorrhage limited to the germinal matrix Grade Ill-hemorrhage with ventricular dilation Grade IV-parenchymal extension of hemorrhage. If given at least 24 hours before delivery, corticosteroids prevent or reduce the incidence and severity ofIVH (Wei, 2016). A Consensus Development Conference of the NIH (1994) concluded that such therapy reduced rates of mortality, RDS, and IVH in preterm neonates born between 24 and 32 weeks' gestation. A second consensus statement by the NIH (2000) recommended that repeated courses of corticosteroids not be given (Chap. 42, p. 823).

1	Subsequently, the Maternal-Fetal Medicine Units Network reported that repeated corticosteroid courses were associated with some improved preterm neonatal outcomes, but also with reduced birthweight and increased risk for fetal-growth restriction (Wapner, 2006). Surveillance of this cohort through age 2 to 3 years found that children exposed to repeated versus singledose steroid courses did not difer signiicantly in physical or neurocognitive measures (Wapner, 2007). It was worrisome, however, that there was a nonsignificant 5.7-fold relative risk of CP in infants exposed to multiple steroid courses.

1	At the same time, the 2-year follow-up ofthe Australian CollaborativeTrialwas reported byCrowtherand coworkers (2007). In more than 1100 newborns, the incidence of CP was almost identical4.2 versus 4.8 percent-in those given repeated versus single-course steroids, respectively. More recently, it was reported that for those born before 28 weeks' gestation, if 10 days or more had passed since betamethasone administration, the incidence of severe IVH was higher (Liebowitz, 2016).

1	he most recent recommendations from the American College of0bstetricians and Gynecologists (2016a) are for a single course of corticosteroids for pregnant women between 24°/ weeks and 336/ weeks' gestation who are at risk for preterm delivery. They further note that those given their initial course more than 14 days prior and who have imminent risk of preterm delivery may receive a second "rescue" course. Antenatal corticosteroids are "considered" for 23°/ to 236/7 weeks and not recommended for pregnancies <23 weeks (American College of Obstetricians and Gynecologists, 2017c).

1	Although antenatal magnesium suate for those at risk for preterm delivery does not reduce the incidence ofIVH, it does ofer protection from neurodevelopmental impairment (Crowther, 2007; Doyle, 2009). The American College of Obstetricians and Gynecologists (2016b) recommends its use for this indication, as discussed further in Chapter 42 (p. 824). he eicacy of antenatal vitamin K and phenobarbital, as well as postnatal phenobarbital, have not been shown to consistently reduce the incidence ofIVH (Crowther, 2010a,b; Smit, 2013). Although vitamin E reduced IVH rates, the associated risk for sepsis was increased (Brion, 2003). One metaanalysis of the many randomized trials ofpostnatal indomethacin showed a reduction in IVH rates, but no improvement in rates of death or neurodevelopmental impairment (Fowlie, 2010).

1	he beneits of cesarean delivery compared with vaginal birth to lower IVH rates remains controversial. One metaanalysis reported that cesarean delivery for very-Iow-birthweight neonates had no efect on rates of severe IVH but did reduce overall IVH rates (Barzilay, 2016). Delayed cord clamping compared with immediate cord clamping has been reported to reduce the risk for IVH in preterm newborns (Rabe, 2012). This pathological description refers to cystic areas deep in brain white matter that develop after hemorrhagic or ischemic infarction. Tissue ischemia leads to regional necrosis. Because brain tissue does not regenerate and the preterm neonate has minimal gliosis, these irreversibly damaged areas appear as echolucent cysts in neuroimaging studies. Generally, they require at least 2 weeks to form but may develop as long as 4 months after the initial insult. hus, their presence at birth may help to determine the timing of an inciting event.

1	This term refers to a group of conditions that are characterized by chronic movement or posture abnormalities that are cerebral in onglll, arise early in life, and are nonprogressive (Nelson, 2003). Epilepsy and mental retardation frequently accom pany CP. Its cause(s) are diferent in preterm and term infants (Chap. 33, p. 622). CP is commonly classified by the type of neurological dys function-spastic, dyskinetic, or ataxic-as well as the num ber and distribution of limbs involved-quadriplegia, diplegia, hemiplegia, or monoplegia. The major types and their frequen their associates: Spastic quadrplegia, which has a strong association with developmental retardation and seizure disorders-20 percent Diplegia, which is common in preterm or low-birthweight Mixed varieties.

1	Spastic quadrplegia, which has a strong association with developmental retardation and seizure disorders-20 percent Diplegia, which is common in preterm or low-birthweight Mixed varieties. According to the Centers for Disease Control and Prevention (2016), the prevalence of CP in the United States approximates 3 in 1000 children. In some countries, the incidence has risen because advances in care of very preterm newborns have improved their survival but not their neurological prognosis (O'Callaghan, 2011). For example, Moster and coworkers (2008) presented long-term follow-up of more than 900,000 births in Norway. he CP rate was 0.1 percent in nonanomalous term newborns but was 9.1 percent in those born at 23 to 27 weeks.

1	Various clinical and pathological data link CP with associated severe IVH (grade III or IV) and resulting PVL. In one study of nearly 1500 neonates born before ;28 weeks, the rate of CP was fivefold greater in those who had grade III or IV hemorrhage compared with those who sufered no IVH (Bolisetty, 2014).

1	Preterm newborns are most susceptible to brain ischemia and PVL. Before 32 weeks' gestation, the vascular anatomy of the brain is composed of two systems. One penetrates into the cortex-the ventrieulopedal system. The other reaches down to the ventricles, but then curves to flow outward-the ventrieulofugal system (Weindling, 1995). There are no vascular anastomoses connecting these two systems. As a result, the area between these systems, through which the pyramidal tracts pass near the lateral cerebral ventricles, is a watershed area vulnerable to ischemia. Vascular insuiciency before 32 weeks leading to ischemia would afect this watershed area first. Resulting damage of the pyramidal tracts may cause spastic diplegia. After 32 weeks, vascular flow shifts toward the cortex, and hypoxic injury after this time primarily damages the cortical region. The Preterm Newborn 641

1	The Preterm Newborn 641 PL is associated with infection and inflammation. Zupan and colleagues (1996) studied 753 infants born between 24 and 32 weeks, 9 percent of whom developed PVL. Those born before 28 weeks, those who had inflammatory events during the last days to weeks before delivery, and those who had both were at highest risk. In another study, PVL was strongly associated with prolonged membrane rupture, chorioamnionitis, and neonatal hypotension (Perlman, 1996). Bailis and coworkers (2008) reported that chronic-and not acute-placental inlammation was associated with PVL.

1	Fetal infection may be a key element in the pathway between preterm birth and CP (Burd, 2012; Leviton, 2010). As discussed in Chapter 42 (p. 810), chorioamnionitis is a major cause of spontaneous preterm delivery. In the pathway proposed in Figure 34-2, antenatal reproductive tract infection evokes the production of cytokines such as tumor necrosis factor and interleukins-1, -6, and -8. hese in turn stimulate prostaglandin production and preterm labor. Preterm fetal intracranial blood vessels are susceptible to rupture and damage, and the cytokines that stimulate preterm labor also have direct toxic efects on oligodendrocytes and myelin. Vessel rupture, tissue hypoxia, and cytokine-mediated damage result in massive neuronal cell death. Glutamate is released, stimulating membrane receptors to allow excess calcium to enter the neurons. High intracellular calcium levels are toxic to white matter, and glutamate may be directly toxic to oligodendrocytes (hwaja, 2008).

1	Many studies have shown that infection and cytokines can directly damage the immature brain (Chau, 2014; Yoon, 1997a). Tumor necrosis factor and interleukin-6 were more frequently found in the brains of infants who died with PVL (Yoon, 1997b). Cytokines are strongly linked to white matter lesions even when organisms cannot be demonstrated (Y oon, 2000). FIGURE 34-2 Schematic representation of the hypothesized pathway between maternal or intrauterine infection and preterm birth or periventricular leukomalacia. Both potentially lead to cerebral palsy. LPS = lipopolysaccharide; PG = prostaglandin.

1	Andrews and colleagues (2008) provided data that raise questions regarding a higher incidence of adverse neurodevelopmental outcomes related to exposure to chorioamnionitis. In a cohort born between 23 and 32 weeks, they studied several surrogate indicators and direct markers of in utero inflammation. These included clinical findings, cytokine levels, histological findings, and microbial culture results. Infants undergoing comprehensive psychoneurological testing had similar incidences of CP, intelligence quotient (IQ) scores <70, or both, regardless of these markers. The researchers interpreted their findings to support current practices that employ eforts to delay delivery with preterm pregnancies in the absence of overt intrauterine infection. Importantly, this does not apply to preterm pregnancy in which clinical chorioamnionitis is diagnosed. Of 3094 singletons born before 33 weeks' gestation, 15 percent had evidence of clinical chorioamnionitis (Soraisham, 2009). Compared with

1	pregnancy in which clinical chorioamnionitis is diagnosed. Of 3094 singletons born before 33 weeks' gestation, 15 percent had evidence of clinical chorioamnionitis (Soraisham, 2009). Compared with noninfected infants, cases complicated by infection had significantly higher rates of early-onset sepsis-4.8 versus 0.9 percent-and ofaIH-22 versus 12 percent.

1	The beneits of antenatal magnesium sulfate and corticosteroids have already been described. Few specific treatments have been identified to reduce or prevent brain injury in the vulnerable preterm newborn. One potential neuroprotective therapy is with erythropoiesis stimulating agents (ESAs) such as erythropoietin and darbepoetin. In addition to stimulating erythropoiesis, ESAs are protective in the developing brain in animal models (Wassink, 2017). Preliminary clinical studies are encouraging, and large trials are now underway (Beirer, 2014). Achiron R, Pinchas OH, Reichman B, et al: Fetal intracranial haemorrhage: clinical significance of in-utero ultrasonic diagnosis. BJOG 100:995, 1993 American Academy of Pediatrics: Respiratory support in preterm infants at birth. Pediatrics 133:171,t2014 American College of Obstetricians and Gynecologists, Society for MaternalFetal Medicine: Antenatal corticosteroid therapy for fetal maturation. Committee Opinion No. 677, October 2016a

1	American College of Obstetricians and Gynecologists, Society for MaternalFetal Medicine: Antenatal corticosteroid therapy for fetal maturation. Committee Opinion No. 677, October 2016a American College of Obstetricians and Gynecologists, Society for MaternalFetal Medicine: Magnesium sulfate before anticipated preterm birth for neuroprotection. Committee Opinion No. 455, March 2010, Reairmed 2016b American College of Obstetricians and Gynecologists: Medically indicated late-preterm and early-term deliveries. Committee Opinion No. 560, April 2013, Reairmed 2017a American College of Obstetricians and Gynecologists, Society for MaternalFetal Medicine: Management of suboptimally dated pregnancies. Committee Opinion No. 688, March 2017b American College of Obstetricians and Gynecologists: With the Society for Maternal-Fetal Medicine: Peri viable birth. Obstetric Care Consensus No. 6, October 2017 c

1	American College of Obstetricians and Gynecologists: With the Society for Maternal-Fetal Medicine: Peri viable birth. Obstetric Care Consensus No. 6, October 2017 c Andrews W, Cliver SP, Biasini F, et al: Early preterm birth: association between in utero exposure to acute inlammation and severe neurodevelopmental disability at 6 years of age. Am J Obstet Gynecol 198:466, 2008 Ardell S, Pister RH, Soli R: Animal derived surfactant extract versus protein free synthetic surfactant for the prevention and treatment of respiratory distress syndrome. Cochrane Database Syst Rev (5):CD000144, 2015 Bailis A, Maleki Z, Askin F, et al: Histopathological placental features associated with development of periventricular leukomalacia in preterm infants. Am J Obstet Gynecol 199(6):S43, 2008 Barzilay E, Gadot Y, Koren G. Safety of vaginal delivery in very low birthweight vertex singletons: a meta-analysis. J Matern Fetal Neonatal Med 29(22):3724,t2016

1	Barzilay E, Gadot Y, Koren G. Safety of vaginal delivery in very low birthweight vertex singletons: a meta-analysis. J Matern Fetal Neonatal Med 29(22):3724,t2016 Bassan H, Venson CB, Limperopoulos C, et al: Ultrasonographic features and severity scoring of periventricular hemorrhagic infarction in relation to risk factors and outcome. Pediatrics 117:2111, 2006 Beers MF, Mulugeta S: he biology of the ABCA3 lipid transporter in lung health and disease. Cell Tissue Res 367(3):481, 2017 Beirer R, Peceny MC, Hartenberger CH, et al: Erythropoietin concentrations and neurodevelopmental outcome in preterm infants. Pediatrics 118:635, 2006 Bennasar M, Figueras F, Palacio M et al: Gestational age-specific cutof levels ofTDx-FLM II for the prediction of neonatal respiratory distress syndrome. Fetal Diagn Ther 25:392, 2009 Bolisetty S, Dhawan A, Abdel-Latif M, et al: Intraventricular hemorrhage and neurodevelopmental outcomes in extreme preterm infants. Pediatrics 133(1):55,t2014

1	Bolisetty S, Dhawan A, Abdel-Latif M, et al: Intraventricular hemorrhage and neurodevelopmental outcomes in extreme preterm infants. Pediatrics 133(1):55,t2014 Brion LP, Bell EF, Raghuveer TS: Vitamin E supplementation for prevention of morbidity and mortality in preterm infants. Cochrane Database Syst Rev 4:CD003665, 2003 Burd I, Balakrishnan B, Kannan S: Models of fetal brain injury, intrauterine inlammation, and preterm birth. Am J Reprod Immunol 67(4):287, 2012 Caplan MS, Fanarof A. Necrotizing enterocolitis: a historical perspective. Semin PerinatoIt41(lt):2, 201 Centers for Disease Control and Prevention: Data and statistics for cerebral palsy. 2016. Available at: https:llwww.cdc.gov/ncbddd/cp/data.html. October 23, 2017 Chau V, McFadden DE, Poskitt KJ, et al: Chorioamnionitis in the pathogenesis of brain injury in preterm infants. Clin PerinatoIt41(1):83, 2014

1	Chau V, McFadden DE, Poskitt KJ, et al: Chorioamnionitis in the pathogenesis of brain injury in preterm infants. Clin PerinatoIt41(1):83, 2014 Clements JA, Platzker ACG, Tierney OF, et al: Assessment of the risk of respiratory distress syndrome by a rapid test for surfactant in amniotic fluid. N Engl J Med 286: 1 07 , 1972 Cole FS, Alleyne C, Barks JD et al: NIH Consensus Development Conference statement: inhaled nitric-oxide therapy for premature infants. Pediatrics 127:363,t201t1 Cools F, Ofringa M, Askie LM: Elective high frequency oscillatory ventilation versus conventional ventilation for acute pulmonary dysfunction in preterm infants. Cochrane Database Syst Rev 3:CDOOO 1 04, 2015 Crowther CA, Crosby DO, Henderson-Smart OJ: Phenobarbital prior to preterm birth for preventing neonatal periventricular haemorrhage. Cochrane Database Syst Rev 1 :CDOOO 164, 201 Oa

1	Crowther CA, Crosby DO, Henderson-Smart OJ: Phenobarbital prior to preterm birth for preventing neonatal periventricular haemorrhage. Cochrane Database Syst Rev 1 :CDOOO 164, 201 Oa Crowther CA, Crosby DO, Henderson-Smart OJ: Vitamin K prior to preteI'm birth for preventing neonatal peri ventricular haemorrhage. Cochrane Database Syst Rev 1:CD000229, 2010b Crowther CA, Doyle L W, Haslam RR, et al: Outcomes at 2 years of age after repeat doses of antenatal corticosteroids. N Engl J Med 357: 9, 2007 Darlow BA, Graham PJ, Rojas-Reyes MX: Vitamin A supplementation to prevent mortality and short-and long-term morbidity in very low birth weight infants. Cochrane Database Syst Rev 8:CD00050 1, 2016 Davidson LM, Berkelhamer SK: Bronchopulmonary dysplasia: chronic lung disease of infancy and long-term pulmonary outcomes. J Clin Med 6(1), 2017

1	Davidson LM, Berkelhamer SK: Bronchopulmonary dysplasia: chronic lung disease of infancy and long-term pulmonary outcomes. J Clin Med 6(1), 2017 De Luca AK, Nakazawa CY, Azevedo BC, et al: Influence of glycemic control on fetal lung maturity in gestations afected by diabetes or mild hyperglycemia. Acta Obstet Gynecol Scand 88(9):1036, 2009 Doyle LW, Crowther CA, Middleton P, et al: Antenatal magnesium sulfate and neurologic outcome in preterm infants: a systematic review. Obstet Gynecol 113: 1327, 2009

1	Doyle LW, Crowther CA, Middleton P, et al: Antenatal magnesium sulfate and neurologic outcome in preterm infants: a systematic review. Obstet Gynecol 113: 1327, 2009 Doyle L W, Ehrenkranz A, Halliday HL: Early « 8 days) postnatal corticosteroids for preventing chronic lung disease in preterm infants. Cochrane Database Syst Rev 5:CDOOI146, 2014a Doyle L W, Ehrenkranz A, Halliday HL: Late (> days) postnatal corticosteroids for chronic lung disease in preterm infants. Cochrane Database Syst Rev 5:CDOOI145, 2014b Ferre C, Callaghan W, Olson C et al: Efects of maternal age and age-speciic preterm birth rates on overall preterm birth rates-United States, 2007 and 2014. MMWR 65:1t181,t2016 Fowlie PW, Davis PG, McGuire W: Prophylactic intravenous indomethacin for preventing mortality and morbidity in preterm infants. Cochrane Database Syst Rev 7:CD000174, 2010 Freeman JM, Nelson KB: Intrapartum asphyxia and cerebral palsy. Pediatrics 82:240, 1988

1	Freeman JM, Nelson KB: Intrapartum asphyxia and cerebral palsy. Pediatrics 82:240, 1988 Gluck L, Kulovich MV: Lecithin-sphingomyelin ratios in amniotic fluid in normal and abnormal pregnancy. Am J Obstet Gynecol 115:539, 1973 Gluck L, Kulovich MV, Borer RC J r, et al: Diagnosis of the respiratory distress syndrome by amniocentesis. Am J Obstet Gynecol 109:440, 1971 The Preterm Newborn 643 Gyami-Bannerman C, Thon EA, Blackwell Sc, et al: Antenatal betamethasone for women at risk for late preterm delivery. N Engl J Med 3 4(14):1311, 2016 Kair LR, Leonard DT, Anderson JM: Bronchopulmonary dysplasia. Pediatr Rev 33(6):255,t2012 Karcher R, Sykes E, Batton D, et al: Gestational age-speciic predicted risk of neonatal respiratory distress syndrome using lamellar body count and surfactant-to-albumin ratio in amniotic fluid. Am J Obstet Gynecol 193:1680, 2005

1	Khwaja 0, Volpe JJ: Pathogenesis of cerebral white matter injury of prematurity. Arch Dis Child Fetal Neonatal Ed 93(2):F153, 2008 Kribs A: Minimally invasive surfactant therapy and noninvasive respiratory support. Clin Perinatol 55,t2016 Kwon SH, Vasung L, Ment LR et al: The role of neuroimaging in predicting neurodevelopmental outcomes of preterm neonates. Clin Perinatol 41(1):257,t2014 Leviton A, Allred EN, Kuban KC, et al: Microbiologic and histologic characteristics of the extremely preterm infant's placenta predict white matter damage and later cerebral palsy. The ELGAN study. Pediatr Res 67:95, 2010 Liebowitz M, Clyman R: Antenatal betamethasone: a prolonged time interval from administration to delivery is associated with an increased incidence of intraventricular hemorrhage before 28 weeks gestation. J Pediatr 177: 114,2016 Mathews TJ, MacDorman MF: Infant mortality statistics from the 2009 period linked birth/infant death data set. Natl Vital Stat Rep 61(8):1, 2013

1	Mathews TJ, MacDorman MF: Infant mortality statistics from the 2009 period linked birth/infant death data set. Natl Vital Stat Rep 61(8):1, 2013 Matsuda T, Okuyama K, Cho K, et al: Cerebral hemodynamics during the induction of antenatal periventricular leukomalacia by hemorrhagic hypotension in chronically instrumented fetal sheep. Am J Obstet Gynecol 194:1057,t2006 McCrea HJ, Ment LR: he diagnosis, management, and postnatal preven tion of intravetricular hemorrhage in the preterm neonate. Clin Perinatol 7,t2008 McCurnin DC, Pierce A, Chang LY, et al: Inhaled NO improves early pulmonary function and modifies lung growth and elastin deposition in a baboon model of neonatal chronic lung disease. Am J Physiol Lung Cell Mol Physiol 288:L540, 2005 Ment LR, Aden U, Bauer CR, et al: Genes and environment in neonatal intraventricular hemorrhage. Semin PerinatoI39(8):592, 2016

1	Ment LR, Aden U, Bauer CR, et al: Genes and environment in neonatal intraventricular hemorrhage. Semin PerinatoI39(8):592, 2016 Morley J, Davis PG, Doyle LW et al: Nasal CPAP or intubation at birth for very preterm infants. N Engl J Med 358:700, 2008 Moster D, Lie RT, Markestad T: Long-term medical and social consequences of preterm birth. N Engl J Med 359:262, 2008 Moya F, Sinha S, Gadzinowski j, et al: One year follow-up of very preterm infants who received lucinactat for prevention of respiratory distress syndrome: results from 2 multicenter randomized controlled trials. Pediatrics 119(6):e1361,t2007

1	Mukerji A, Shah V, Shah PS: Periventricularlintraventricular hemorrhage and neurodevelopmental ourcomes: a meta-analysis. Pediatrics 136(6):1132,t2015 National Institutes of Health: Antenatal corticosteroids revisited: repeat courses. NIH Consensus Statement 17(2):1, 2000 National Institutes of Health: The efects of corticosteroids for fetal maturation on perinatal outcomes. NIH Consensus Statement 12(2): 1, 1994 Nelson KB: Can we prevent cerebral palsy? N Engl J Med 349: 1765, 2003 Neu j, Walker WA: Necrotizing enterocolitis. N Engl j Med 364:255,t2010 Nores j, Roberts A, Carr S: Prenatal diagnosis and management of fetuses with intracranial hemorrhage. Am j Obstet Gynecol 174:424, 1996 O'Caliaghan ME, MacLennan AH, Gibson CS, et al: Epidemiologic associations with cerebral palsy. Obstet Gynecol 118:576,2011

1	Papile LA, Burstein J, Burstein R, et al: Incidence and evolution of subependymal and intraventricular hemorrhage: a study of infants with birth weights less than 1500 gm. J Pediatr 92:529, Patra K, Wilson-Costello D, Taylor HG, et al: Grades I-II intraventricular hemorrhage in extremely low birth weight infants: efects on neurodevelopment. J Pediatr 149: 169,t2006 Perlman ]M, Risser R, Broyles RS: Bilateral cystic leukomalacia in the premature infant: associated risk factors. Pediatrics 97:822, 1996 Polin A, Carlo WA, Committee on Fetus and Newborn of the American Academy of Pediatrics: Surfactant replacement therapy for preterm and term neonates with respiratory distress. Pediatrics 133(1):156,t2014 Rabe H, Diaz-Rossello JL, Duley L, et al: Efect of timing of umbilical cord clamping and other strategies to influence placental transfusion at preterm birth on maternal and infant outcomes. Cochrane Database Syst Rev 8:CD003248,t2012

1	Redline RW: Placental pathology: a systematic approach with clinical correlations. Placenta 22:S86, 2008 Rojas-Reyes MX, Morley CJ, Soli R: Prophylactic versus selective use of surfactant in preventing morbidity and mortality in preteI'm infants. Cochrane Database Syst Rev 3:CD00051 0, 2012 Rosen MG, Dickinson JC: he incidence of cerebral palsy. Am J Obstet Gynecol 167:417, 1992 Sardesai S, Biniwale M, Wertheimer F, et al: Evolution of surfactant therapy for respiratory distress syndrome: past, present, and future. Pediatr Res 81(1-2):240,t2017 Sarkar S, Bhagat I, Dechert R, et al: Severe intraventricular hemorrhage in preterm infants: comparison of risk factors and short-term neonatal morbidities between grade 3 and grade 4 intraventricular hemorrhage. Am J Perinatol 26:419,t2009 Schmidt B, Anderson Pj, Doyle LW, et al: Survival without disability to age 5 years after neonatal cafeine therapy for apnea of prematurity. JAMA 307(3):275,t2012

1	Schmidt B, Anderson Pj, Doyle LW, et al: Survival without disability to age 5 years after neonatal cafeine therapy for apnea of prematurity. JAMA 307(3):275,t2012 Schmidt B, Roberts RS, Anderson PJ, et al: Academic performance, motor function, and behavior 11 years after neonatal cafeine citrate therapy for apnea of prematuri ty: an I I-year follow-up of the CAP Randomized Clinical Trial. JAMA Pediatr 1(6):564,t2017 Schmidt B, Roberts RS, Davis P, et al: Cafeine therapy for apnea of prematurity. N Engl J Med 354(20):21t12,t2006 Sharma M, VanderVeen D: Identiication and treatment of retinopathy of prematurity: update 2017. New Reviews 18(2):e85, 2017 Smit E, Odd D, Whitelaw A: Postnatal phenobarbital for the prevention of intraventricular haemorrhage in preteI'm infants. Cochrane Database SYSt Rev 8:CDOOI691, 2013 Soraisham AS, Singhal Nalini, McMillan DD, et al: A multicenter study on the clinical outcome of chorioamnionitis in preterm infants. Am j Obstet 2.e1,t2009

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1	Varner S, Sherman C, Lewis D et al: Amniocentesis for fetal lung maturity: will it become obsolete? Rev Obstet Gynecol 6(3/4): 126, 2013 Verhagen EA, Hummel LA, Bos AF, et al: Near-infrared spectroscopy to detect absence of cerebrovascular autoregulation in preteI'm infants. Clin Neurophysiol 125(1):47,t2014 Volpe JJ: Neurology of the Newborn, 5th ed. Philadelphia, Saunders, 2008 Wapner Rj, Sorokin Y, Mele L, et al: Long-term outcomes after repeated doses of antenatal corticosteroids. N Engl J Med 357: 1190, 2007 Wapner RJ, Sorokin Y, Thom EA, et al: Single versus weekly courses of antenatal corticosteroids: evaluation of safety and eicacy. Am J Obstet Gynecol 195:633,t2006 Wassink G, Davidson jO, Dhillon SK, et al: Partial white and grey matter protection with prolonged infusion of recombinant human erythropoietin after asphyxia in preterm fetal sheep. J Cereb Blood Flow Metab 37(3):1080, 201

1	Watterberg KL, American Academy of Pediatrics Committee on Fetus and Newborn: Policy statement-postnatal corticosteroids to prevent or treat bronchopulmonary dysplasia. Pediatrics 126(4):800,t2010 Wei JC, Catalano R, Proit J, et al: Impact of antenatal steroids on intraventricular hemorrhage in very-low-birth weight infants. j Perinatol 36(5):352, 2016 Weindling M: Periventricular haemorrhage and periventricular leukomalacia. BJOG 102(4):278, 1995 Wert SE, Whitsett JA, Nogee LM: Genetic disorders of surfactant dysfunction. Pediatr Dev Pathol 12(4):253, 2009 Whitelaw A: Core concepts: intraventricular hemorrhage. NeoReviews 12(2):e94,t2011 Yoon BH, Kim Cj, Romero R, et al: Experimentally induced intrauterine infection causes fetal brain white matter lesions in rabbits. Am j Obstet Gynecol 177:797, 1997a Yoon BH, Romero R, Kim CJ, et al: High expression of tumor necrosis factoralpha and interleukin-6 in periventricular leukomalacia. Am J Obstet Gynecol 177:406, 1997b

1	Yoon BH, Romero R, Kim CJ, et al: High expression of tumor necrosis factoralpha and interleukin-6 in periventricular leukomalacia. Am J Obstet Gynecol 177:406, 1997b Yoon BH, Romero R, Park JS, et al: Fetal exposure to an intra-amniotic inflammation and the development of cerebral palsy at the age of three years. Am J Obstet Gynecol 182:6 5,t2000 Zalud I, Janas S: Risks of third trimester amniocentesis. J Reptod Med 53(1):45, 2008 Zupan V, Gonzalez P, Lacaze-Masmonteil T, et al: Periventricular leukomalacia: risk factors revisited. Dev Med Child NeuroIt38:1061, 1996 DEFINITION OF FETAL MORTALITY ................. 645 CAUSES OF FETAL DEATH ......................... 645 RISK FACTORS ....................................... 646 EVALUATION OF THE STILLBORN FETUS .............. 646 PSYCHOLOGICAL ASPECTS. . . . . . . . . . . . . . . . . . . . . . . 648 PRIOR STILLBIRTH .............................. 648 CHANGES IN STILLBIRTH RATES ................... 649

1	PSYCHOLOGICAL ASPECTS. . . . . . . . . . . . . . . . . . . . . . . 648 PRIOR STILLBIRTH .............................. 648 CHANGES IN STILLBIRTH RATES ................... 649 In the latter months of pregnancy, the disappearance of oetal movements usualy directs the attention of the patient to the possibiliy of oetal death. The diagnosis of this condition, however, can be considered absolute ony after repeated examinations, when one has ailed to hear the oetal heart or perceive the movements of the child. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) In Williams' time, absolute documentation of fetal death was frustrating for both patient and obstetrician. Now, sonography provides prompt conirmation, which allows expedient induction of labor and delivery. However, epidemiologically, defining and reporting fetal deaths was-and continues to be-a challenge. In response, eforts to standardize the deinition of stillbirth and analyze varying reports for application into clinical practice and public health policy are now being emphasized. Moreover, stillbirth research and prevention within the United States and abroad has expanded. Global public health eforts were stimulated in part by a six-part series in The Lancet.

1	his compilation was considered a call to action after the recognition that an estimated 2.65 million stillbirths occur each year and that 98 percent of stillborn fetuses are from low-and middle-income countries (The Lancei s Stillbirth Series Steering Committee, 201 la-). Unfortunately, progress in improving these rates has been slow, as outlined in The Lancet's subsequent five-part progress report, which emphasized the need for dedicated leadership, measured efects of interventions, and investigation into knowledge gaps (The Lancets Ending Preventable Stillbirths Series Study Group, 20 l6a-e).

1	In the United States, an estimated 1 million fetal losses are reported each year, and most occur before 20 weeks' gestation. Fetal mortality data from the National Vital Statistics system are usually presented for fetal deaths after the 20-week threshold (MacDorman, 2015). Using this definition, numbers of fetal deaths in the United States in 2013 slightly surpassed numbers of infant deaths (Fig. 35-1). As shown in Figure 35-2, 24.9% Undereage 28days 33.8% 16.1% FIGURE 35-1 Percent distribution of fetal deaths at 20 weeks' gestation or more and of infant deaths: United States, 2013. (Data from MacDorman MF, Reddy UM, Silver RM: Trends in stillbirth by gestational age in the United States, 2006-201o2, Obstet Gynecol. 2015 Dec;126(6):1146-1150.) 0.7 0.6 0.5 ) 0.4�: 0.3 0.2 0.1 0.0 weeks of gestation: United States, 2013. (Redrawn from MacDorman

1	MF, Reddy UM, Silver RM: Trends in stillbirth by gestational age in the United States, 2006-2012, Obstet Gynecol. 2015 Dec;126(6): 1146-1o150.) fetal death rates are highest at the earliest and latest gestational ages, which suggests etiological diferences.

1	he current definition of fetal death adopted by the Centers for Disease Control and Prevention National Center for Health Statistics is based on a deinition recommended by the World Health Organization (MacDorman, 2015). It states that "Fetal death means death prior to complete expulsion or extraction .rom the mother of a product of human conception irrespective of the duration of pregnancy and which is not an induced termination of pregnancy. The death is indicated by the act that after such expulsion or extraction, the etus does not breathe or show any other evidence of lie such as beating of the heart, pulsation of the umbilical cor, or deinite movement of voluntary muscles. Heartbeats are to be distinguished rom transient cardiac contractions; respirations are to be distinguished .rom fleeting respiratory eorts or gasps."

1	Reporting requirements for fetal deaths in the United States are determined by each state, and thus, criteria difer signiicantly (Chap. 1, p. 3). Most states mandate reporting of deaths of fetuses that are 20 weeks' gestation or older or have a minimum birthweight of 350 g (roughly equivalent to 20 weeks) or some combination of these two. However, several states require reporting of fetal deaths at all periods of gestation, and one sets the threshold at 16 weeks. lternatively, two states require reporting of deaths for fetuses with birthweights of 500 g, which approximates that at 22 weeks. here is substantial evidence that not all fetal deaths for which reporting is required are actually recorded (MacDorman, 2015). This is most likely for those at earlier gestational ages.

1	Comparisons of rates among countries are limited by incomplete fetal death data. Namely, internationally, less than 5 percent of neonatal deaths have formalized documentation (The Lancet's Ending Preventable Stillbirths Series Study Group, 2016d). Further, comparative analyses using birthweight versus gestational age among countries do not provide equivalent results. For example, in the United States, if stillbirth would be deined by a birthweight �500 g, the stillbirth rate would be reduced by 40 percent compared with FIGURE 35-3 Fetal mortality rates by period of gestation: United States, 2000-2013. (Data from MacDorman MF, Reddy UM, Silver RM: Trends in stillbirth by gestational age in the United States, 2006-2012, Obstet Gynecol. 2015 Dec;126(6):1146-1150.) a 22-week-age deined cohort (Blencowe, 2016). To address nomenclature diferences, some have called for changes to the current deini tion a oseph, 2015).

1	Overall, fetal mortality rates in the United States have remained relatively unchanged since 2006. However, the infant mortality rate has declined 11 percent, and both rates are now essentially equal (MacDorman, 2015). Three fetal mortality epochs are generally described: early (less than 20 completed weeks' gestation); intermediate (20 to 27 weeks); and late (28 weeks or more). The fetal mortality rate at 20 to 27 weeks in 2013 declined 3 percent from the prior year. Between 2006 and 2012, rates were essentially unchanged in this age group. The late fetal mortality rate has been relatively unchanged since 2006 35-3).

1	The Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) created the Stillbirth Collaborative Research Network to ascertain stillbirth causes in racially and geographically diverse populations in the United States. From this, the Stillbirth Collaborative Research Network Writing Group (201a1b) examined reasons for death at 20 weeks' gestation or later between 2006 and 2008 in 59 tertiary care and community hospitals in ive states. Standardized evaluations included autopsy, placental histology, and testing of maternal or fetal bloodltissues, including fetal karyotyping. Evaluations were performed in 500 women with 512 stillbirths. Of these losses, 83 percent were before labor. Causes of stillbirth were divided into eight categories shown in Table hese categories were then classified as probable, possible, or unknown. As an example, diabetes was considered a probable cause if the fetus had diabetic embryopathy with lethal anomalies or the mother had

1	were then classified as probable, possible, or unknown. As an example, diabetes was considered a probable cause if the fetus had diabetic embryopathy with lethal anomalies or the mother had diabetic ketoacidosis. It was a possible cause if the mother had poor glycemic control and the fetus had abnormal growth. Overal, a probable or possible source was identiied in 76 percent of cases.

1	This Network study is unprecedented in the United States for several reasons. It was a population-based cohort of TABLE 35-1. Causes of 512 Stillbirths in the Stillbirth Collaborative Research Network Study Percentages are rounded and total more than 100 percent because some stillbirths had more than one cause. Overall, a cause was identified in 76 percent of stillbirths. Data from Stillbirth Research Network Writing Group, 201n1 b.

1	Data from Stillbirth Research Network Writing Group, 201n1 b. stillbirths, in which all underwent systematic and thorough evaluation. Each assigned cause of fetal death is reasonably straightforward and comprehensible except for "placental abnormalities." This category contains "uteroplacental insufficiency" and a few other less clearly defined placental entities. his aside, the leading reasons for fetal death were obstetrical and primarily included abruption, multifetal gestation complications, and spontaneous labor or ruptured membranes before viability. Importantly, this study illustrated that systematic evaluation may identiy a likely cause in approximately three fourths of stillbirths. his rate is considerably higher than those in most prior analyses and serves to emphasize the importance of careful examination.

1	Many factors are associated with an increased risk of stillbirth. Among others, these include advanced maternal age; AfricanAmerican race; smoking; illicit drug use; maternal medical diseases-such as overt. diabetes or chronic hypertension; assisted reproductive technology; nulliparity; obesity; and prior adverse pregnancy outcomes-such as prior preterm birth or growth-restricted newborn (Reddy, 2010; Varner, 2014).

1	Two major studies have assessed whether stillbirth risk factors could be identiied either before or shortly after pregnancy confirmation. In the first, Reddy and colleagues (2010) analyzed data from the NICHD Consortium on Safe Labor. Briely, the pregnancy outcomes of 206,969 women delivered between 2002 and 2008 at 19 hospitals in the United States were analyzed. When the distribution of stillbirths according to gestational age was studied, the tragedy of stillbirth occurred primarily in term pregnancies. hese investigators concluded that their results did not support routine antenatal surveillance for any demographic risk factors.

1	The second analysis of stillbirth risk factors was included in the Stillbirth Collaborative Research Network study described earlier. he validity of stillbirth prediction was assessed based on risks identified in early pregnancy. They found that pregnancy factors known at the start of pregnancy accounted for only a small proportion of stillbirth risk. Except for prior stillbirth or pregnancy loss from causes such as preterm birth or fetal-growth restriction, other risks had limited predictive value (Stillbirth Collaborative Research Network Writing Group, 2011a). he importance of prior stillbirth as a risk for recurrence has been emphasized by Sharma and associates (2006). Specifically, the stillbirth risk was ivefold higher in women with a prior stillbirth. From another report, prior preterm birth, fetal-growth restriction, preeclampsia, and placental abruption were strongly associated with subsequent stillbirth (Rasmussen, 2009). Table 35-2 lists estimates of stillbirth risk

1	preterm birth, fetal-growth restriction, preeclampsia, and placental abruption were strongly associated with subsequent stillbirth (Rasmussen, 2009). Table 35-2 lists estimates of stillbirth risk according to maternal factors.

1	Determining the cause of fetal death aids maternal coping, helps assuage any perceived guilt, permits more accurate counseling regarding recurrence risk, and may prompt therapy or intervention to prevent a similar outcome in subsequent pregnancies (American College of Obstetricians and Gynecologists, 20 16a). Identification of inherited syndromes also provides useful information for other family members. Important tests in stillbirth evaluation are neonatal autopsy, chromosomal analysis, and examination of the placenta, cord, and chorioamnionic membranes (Pinar, 2014). Page and coworkers (2017) found placental pathology and fetal autopsy to be the most useful. One algorithm from the American College of Obstetricians and Gynecologists (2016a) is shown in

1	Figure 35-4. Findings are documented in the medical record, and relevant prenatal events are delineated. Photographs are taken whenever possible, and a full radiograph of the fetusa etogram-may be performed. Postnatal magnetic resonance (MR) imaging or sonography may be especially important in providing anatomical information if parents decline a full autopsy (McPherson, 2017; Shruthi, 2017). If autopsy and chromosomal studies are performed, up to 35 percent of stillborn fetuses are discovered to have major TABLE 35-2. Estimated Maternal Risk Factors and Risk Inspect fetus and placenta: Weight, head circumference, and length of fetus Weight of placenta Photographs of fetus and placenta Frontal and profile photographs of whole body, face, extremities, palms, and any abnormalities Document finding and abnormalities Obtain consent from parents for cytologic specimens:

1	Frontal and profile photographs of whole body, face, extremities, palms, and any abnormalities Document finding and abnormalities Obtain consent from parents for cytologic specimens: Obtain cytologic specimens with sterile techniques and instruments -Amniotic fluid obtained by amniocentesis at time of prenatal diagnosis of demise: paticularly valuable if delivery is not expected imminently -Placental block (1 x 1) cm taken from below the cord insertion site on the unfixed placenta -Umbilical cord segment (1e.5 cm) -Internal fetal tissue specimen, such as costochondral junction or patella; skin is not recommended Place specimens in a sterile tissue culture medium of lactated Ringer's solution and keep at room temperature when transported to cytology laboratory

1	Place specimens in a sterile tissue culture medium of lactated Ringer's solution and keep at room temperature when transported to cytology laboratory Fetal autopsy and placental If no consent is given for pathology (may include autopsy, send placenta fetal whole-body X-ray) alone for pathology (Chap. 13, p. 271). CMA does not require dividing cells and is reported to be more useful in the evaluation of fetal death. his is especially true because the culturing of macerated fetal tissue is frequently unsuccessful (Reddy, 2012). Both the American College of Obstetricians and Gynecologists (2016c) and the Society for Maternal-Fetal Medicine (2016) now endorse the use of CMA for stillborn fetuses.

1	Appropriate consent must be obtained to take fetal samples, including tissue or luid obtained postmortem by needle aspiration. As recently outlined by the American College of Obstetricians and Gynecologists (2016c), any type of fetal or placental tissue or amnionic fluid can be submitted for genetic testing by CMA. Contamination with maternal tissue or blood is ideally avoided. If fetal blood cannot be obtained from the umbilical cord or by cardiac puncture, the American College of Obstetricians and Gynecologists (20a16a) recommends at least one of the following samples: (1) a placental block measuring about 1 X 1 cm taken below the cord insertion site in the unfixed specimen; (2) umbilical cord segment approximately 1.5 cm long; or (3) internal fetal tissue specimen such as costochondral junction or patella. Tissue is washed with sterile saline before Cholestasis of pregnancy

1	Cholestasis of pregnancy BMI 25-29.9 kg/m2 ::12 yr) ::40 yr 6.4 4.0-5.5 1n.0 0.86 1.5-2.7 1n.2-4.0 1n.8-4.4 1n.2-2.2 1.7-7.0 2.2-30 2.2-3.0 2.8-5.0 1n.8-4.4 1n.7-3.0 1n.9-2.7 2.1n-2.8 1.6-2.0 2-4.6 1.4-3.2 1.8-2.2 1n.8-3.3 2.0-2.2 Twins Triplets Maternal age 12 1n.0-2.8 34 2.8-3.7 FIGURE 35-4 Flow chart for fetal and placental evaluation. (Modified with permission from ACOG Practice Bulletin No.o1 02: management of stillbirth, Obstet Gynecol. 2009 Mar;1o13(3):748-761o.) aOdds ratio of the factor being present compared with the risk factor being absent. BMI = body mass index; IUGR = intrauterine growth restriction; PIH = pregnancy-induced hypertension; SLE = systemic lupus erythematosus. Adapted from Fretts, 2005.

1	structural anomalies (Faye-Petersen, 1999). Approximately 20 percent have dysmorphic features or skeletal anomalies, and 8 percent have chromosomal abnormalities (Pauli, 1994; Saller, 1995). In the absence of anatomic dysmorphology, up to 5 percent of stillborn fetuses will have a chromosomal abnormality (Korteweg, 2008). Although the American College of Obstetricians and Gynecologists (20a1a6a) previously recommended karyotyping all stillborn fetuses, technological advancements of high-resolution, whole-genome sequencing-such as with chromosomal microarray anaysis ()-are now replacing standard karyotyping for chromosomal analysis of stillborn fetuses being placed in lactated Ringer solution or sterile cytogenetic medium. Notably, placement in formalin or alcohol kills viable cells. If conventional karyotyping is the only test available and the timing of death is recent, amnionic luid can be obtained by amniocentesis, as those cells obtained in a sterile fashion provide a greater

1	conventional karyotyping is the only test available and the timing of death is recent, amnionic luid can be obtained by amniocentesis, as those cells obtained in a sterile fashion provide a greater likelihood of cell growth and eventual result compared with tissue obtained after delivery. Maternal blood is obtained for Kleihauer-Betke staining; for anti phospholipid antibody and lupus anticoagulant testing if indicated; and for serum glucose measutement to exclude overt diabetes (Silver, 20al3).

1	In cases with significant growth restriction, with a family or personal history of thrombosis, or with severe placental pathology, testing for factor V Leiden mutation, prothrombin mutation, antithrombin level, and protein C and S activity may provide some information that could afect future pregnancy management (American College of Obstetricians and Gynecologists, 2016a). Our interpretation of relevant placental pathology includes derangements that stem from maternal vessel obstruction, which are described in Chapter 6. Although some have recommended routine evaluation of heritable thrombophilias, no evidence supports the clinical or financial eiciency of screening in an unselected population. Silver and colleagues (2016), with data from the Stillbirth Collaborative Research Network, found that most maternal and fetal thrombophilias were not associated with stillbirth and recommended against routine testing.

1	Parents are ofered and encouraged to allow a full autopsy. That said, valuable information can still be obtained from limited studies. Pinar and coworkers (2012) described the autopsy protocol used by the Stillbirth Collaborative Research Network. As an alternative, gross external examination combined with photography, radiography, MR imaging, bacterial cultures, and selective use of chromosomal and histopathological studies often aids determination.

1	A complete autopsy is more likely to yield valuable data. An analysis of 400 consecutive fetal deaths in Wales showed that autopsy altered the presumed cause of death in 13 percent and provided new information in another 26 percent (Cartlidge, 1995). Other investigators have found that autopsy results changed the recurrence risk estimates and parental counseling in 25 to 50 percent of cases (Faye-Petersen, 1999; Silver, 2007). For example, Miller and associates (2016) recently demonstrated that placental examination with autopsy altered future medical management in 45 percent of cases.

1	According to the survey by Goldenberg and coworkers (2013), most hospitals do not audit stillbirths. In other centers, however, maternal records and autopsy findings are reviewed on a monthly basis by a stillbirth committee composed of obstetricians, maternal-fetal medicine specialists, neonatologists, clinical geneticists, and perinatal pathologists. If possible, the cause of death is assigned based on available evidence. Most importantly, parents are then contacted and ofered counseling regarding the reason for the death, the potential recurrence risk, and possible strategies to avoid recurrence.

1	Fetal death is psychologically traumatic for a woman and her family. Further stressors are an interval of more than 24 hours between the diagnosis of fetal death and labor induction, not seeing her infant for as long as she desires, having no tokens of remembrance, and poor communication (Radestad, 1996; Siassakos, 2017). The importance of seeing and holding a stillborn fetus for parental psychological well-being was recently summarized by Kingdon and colleagues (2015). As discussed in Chapter 61 (p. 1176), a woman experiencing a stillbirth or early miscarriage is at increased risk for depression and should be closely monitored (Nelson, 2013).

1	Nuzum and coworkers (2014) reported that few obstetrical providers receive formal training in perinatal bereavement care. At Parkland Hospital, this care includes time with the infant, keepsake items, photographs, chaplaincy consultation, and bereavement support information. Care is coordinated through a dedicated nursing team ailiated with labor and delivery.

1	Table 35-3 lists an outlined approach for women with prior stillbirth. Importantly, these recommendations are based primarily on limited or inconsistent scientiic evidence or on expert opinions. Unfortunately, few studies address management of afected women. Those with modiiable risk factors for stillbirth, such as hypertension or diabetes, warrant speciic prevention strategies. Given that obesity has been identiied as a risk factor for stillbirth and other obstetrical complications, preconceptional weight loss would seem prudent. Logically, women with a prior fetal death due to placental vascular events, that is, placental insuiciency, are also at increased risk for subsequent adverse perinatal outcomes (Monari, 2016). According to Reddy (2007), because almost half of fetal deaths are associated with growth restriction, fetal sonographic anatomical assessment beginning at midpregnancy is recommended. his is followed by serial growth studies beginning at 28 weeks. Supplementation with

1	with growth restriction, fetal sonographic anatomical assessment beginning at midpregnancy is recommended. his is followed by serial growth studies beginning at 28 weeks. Supplementation with vitamin C or E in pregnancy has not been demonstrated to reduce the risk of fetal death (Rumbold, 2015a,b).

1	Weeks and associates (1995) evaluated fetal biophysical testing in 300 women whose only indication was prior stillbirth. There was one subsequent stillbirth, and only three fetuses had abnormal testing results before 32 weeks. Notably, no relationship was found between the gestational age of the previous stillborn fetus and the incidence or timing of abnormal test results or fetal jeopardy in the subsequent pregnancy. These investigators concluded that antepartum surveillance should begin at 32 weeks or later in the otherwise healthy woman with a history of stillbirth. This recommendation is supported by the American College of Obstetricians and Gynecologists (2016a) with the caveat that it increases the iatrogenic preterm delivery rate. Although fetal movement counting strategies are routinely employed as described in Chapter 17 (p. 332), few data guide its use in clinical practice for those with a prior stillbirth (Mangesi, 2015).

1	Delivery at 39 weeks' gestation is recommended. Labor induction is suitable, and cesarean delivery is elected for those with a contraindication to induction. his timing minimizes TABLE 35-3. Management of Subsequent Pregnancy after Stillbirth Review evaluation of prior stillbirth Determination of recurrence risk Discuss recurrence of comorbid obstetric complications Thrombophilia screen: antiphospholipid antibodies First-trimester screen: pregnancy-associated plasma protein A, human chorionic gonadotropin, and nuchal Sonographic screening for fetal-growth restriction, Delivery before 39 weeks only with documented fetal aprovides risk modification but does not alter management. Modified from Reddy, 2007. fetal mortality rates, although the degree of risk reduction may be greater for older women (Page, 2013).

1	Following declines between 2000 and 2006, the United States fetal mortality rate has been relatively unchanged since 2006 (MacDorman, 2015). Interpretation of these fetal mortality rates in the context of changing national healthcare strategies has spawned considerable debate. One example is the efort to prevent non-medically indicated deliveries before 39 weeks and its subsequent efect on term stillbirth rates. he value of this practice for neonatal outcome is described in Chapter 26 (p. 504). To analyze whether implementation of this "39week rule" has altered the term stillbirth rate, Nicholson and coworkers (2016) examined data from 45 states and the District of Columbia during a 7 -year period. The proportion of births before 39 weeks progressively declined from 2007 and 2013, but the term stillbirth rate rose. his suggested that the 39-week rule may cause unintended harm. MacDorman and associates (2015) also evaluated trends in stillbirth rates by gestational ages in the United

1	term stillbirth rate rose. his suggested that the 39-week rule may cause unintended harm. MacDorman and associates (2015) also evaluated trends in stillbirth rates by gestational ages in the United States between 2006 and 2012. They used a "traditional stillbirth rate," which was calculated using a denominator composed of the live-birth number plus the stillbirth number at a given gestational age. They found increased rates at 24 to 27, 34 to 36, and 38 weeks' gestation. Alternatively, no diferences were found in "prospective stillbirth rates." These rates were calculated using a denominator composed of the number of women who are pregnant at a given gestational age for weeks 21 through 42. he discrepancies in stillbirth rates appear to be primarily due to the decline in the number of births in the preterm and early-term gestational ages.

1	To summarize, implementation of the 39-week rule has reduced the number of elective births before 39 weeks' gestation, although an unintended consequence may be an increase in term stillbirths-especially among women with medical complications. he importance of induction at less than 39 weeks in pregnant women with complications to prevent stillbirth is underscored by Little and colleagues (2015). These authors performed a retrospective multistate analysis of earlyterm deliveries (37°/7 to 386/7 weeks) from 2005 to 2011a. They noted a decline in the number of early-term deliveries during this time but not a signiicant change in the term stillbirth rates. There was, however, a 25-percent rise in the rate of term, singleton stillbirths among women with diabetes, and this was attributed to clinicians misapplying early-term delivery policies to high-risk women. Undoubtedly, continued surveillance of stillbirth rates is warranted for both high-and low-risk pregnancies at a state and

1	to clinicians misapplying early-term delivery policies to high-risk women. Undoubtedly, continued surveillance of stillbirth rates is warranted for both high-and low-risk pregnancies at a state and national level.

1	American College of Obstetricians and Gynecologists: Management of still birth. Practice Bulletin No. 102, March 2009, Reairmed 2016a American College of Obstetricians and Gynecologists: Microarrays and next-generation sequencing technology: the use of advanced genetic diagnostic tools in obstetrics and gynecology. Committee Opinion No. 682, December 2016b American College of Obstetricians and Gynecologists: Prenatal diagnostic test ing for genetic disorders. Practice Bulletin No. 162, May 2016c Blencowe H, Cousens 5, Bianchi JF, et al: National, regional, and worldwide estimates of stillbirth rates in 2015, wi th trends from 2000: a systematic analysis. Lancet Glob Health 4(2):e98, 2016 Cartlidge PH, Stewart JH: Efect of changing the stillbirth deinition on evalu ation of perinatal mortality rates. Lancet 346:486, 1995 Faye-Petersen OM, Guinn DA, Wenstrom KD: Value of perinatal autopsy.

1	Obstet Gynecol 94(6):915, 1999 FrettS RC: Etiology and prevention of stillbirth. Am J Obstet Gynecol 193(6):1923,t2005 Goldenberg L, Farrow V, McClure EM, et l: Stillbirth: knowledge and practice among U.S. obstetrician-gynecologists. Am J Perinatol30(l 0):813, 2013 Joseph KS, Kinniburgh B, Hutcheon JA, et al: Rationalizing deinitions and stillbirth. Obstet GynecoIt125(4):784, 2015 Kingdon C, Givens JL, O'Donnell E, et al: Seeing and holding Baby: systematic review of clinical management and parental outcomes ater stillbirth. Birth 42(3):206,t2015 Korteweg FJ, Bouman K, Erwich J,et al: Cytogenetic analysis after evaluation of 750 fetal deaths. Obstet Gynecol 111 :865, 2008 Little SE, Zera CA, Clapp A, et al: A multi-state analysis of early-term delivery trends and the association with term stillbirth. Obstet Gynecol 126(6):1138,t2015 MacDorman MF, Gregory EC: Fetal and perinatal mortaliry, United States, 2013. Natl Vital Stat Rep 64(8):1, 2015

1	MacDorman MF, Gregory EC: Fetal and perinatal mortaliry, United States, 2013. Natl Vital Stat Rep 64(8):1, 2015 MacDorman MF, Reddy UM, Silver M: Trends in stillbirth by gestational age in the United States, 2006-2012. Obstet GynecoIt126(6):1146, 2015 Mangesi L, Hofmeyr GJ, Smith V, et al: Fetal movement counting for assess ment offetal wellbeing. Cochrane Database Syst Rev 1O:CD004909, 2015 McPherson E, Nestoridi E, Heinke D, et al: Alternatives to autopsy for fetl and early neonatal (perinatal) deaths: insights from the Wisconsin stillbirth service program. Birth Defects Res September 12, 2017 [Epub ahead of print] Miller ES, Minturn L, Linn R, et al: Stillbirth evaluation: a stepwise assessment of placental pathology and autopsy. Am J Obstet Gynecol 214: 115, 2016 Monari F, Pedrielli G, Vergani P, et al: Adverse perinatal outcome in subsequent pregnancy after stillbirth by placental vascular disorders. PLoS One 11(5):e0155761,t2016

1	Monari F, Pedrielli G, Vergani P, et al: Adverse perinatal outcome in subsequent pregnancy after stillbirth by placental vascular disorders. PLoS One 11(5):e0155761,t2016 Nelson DB, Freeman MP, Johnson NL, et al: A prospective study of postpartum depression in 17,648 parturients. J Matern Fetal Neonatal Med 26(12):1155,t2013 Nicholson JM, Kellar LC, Ahmad S, et al: US term stillbirth rates and the 39-week rule: a cause for concern? Am J Obstet Gynecol 214:621, 2016 Nuzum D, Meaney S, O'Donoghue K: he impact of stillbirth on consultant obstetrician gynaecologists: a qualitative study. BJOG 121:1020,t2014 Page JM, Christiansen-Lindquist L, horsten V, et al: Diagnostic Tests for Evaluation of Stillbirth: Results From the Stillbirth Collaborative Research Network. Obstet Gynecolt129(4):699, 2017 Page JM, Snowden JM, Cheng W, et al: he risk of stillbirth and infant death by each additional week of expectant management stratiied by maternal age. Am J Obstet GynecoI209(4):375.e1, 2013

1	Page JM, Snowden JM, Cheng W, et al: he risk of stillbirth and infant death by each additional week of expectant management stratiied by maternal age. Am J Obstet GynecoI209(4):375.e1, 2013 Pauli M, Reiser CA: Wisconsin Stillbirth Service Program: II. Analysis of diagnoses and diagnostic categories in the irst 1,000 referrals. Am J Med Genet 50: 135, 1994 Pinar H, Goldenberg L, Koch MA, et al: Placental indings in singleton stillbirths. Obstet Gynecol 123:325,t2014 Pinar H, Koch A, Hawkins H, et al: The stillbirth collaborative research network postmortem examination protocol. Am J Perinatol 29:187, 2012 Radestad I, Steineck G, Nordin C, et al: Psychological complications after stillbirth-influence of memories and immediate management: population based study. BMJ 312:1505, 1996 Rasmussen S, Irgens LM, Skjaerven R, et al: Prior adverse pregnancy outcome and the risk of stillbirth. Obstet Gynecol 114(6): 1259, 2009

1	Rasmussen S, Irgens LM, Skjaerven R, et al: Prior adverse pregnancy outcome and the risk of stillbirth. Obstet Gynecol 114(6): 1259, 2009 Reddy UM: Prediction and prevention of recurrent stillbirth. Obstet Gynecol 110:1151,t2007 Reddy UM, Laughon SK, Sun L, et al: Prepregnancy risk factors for antepartum stillbirth in the United States. Obstet Gynecol 116:11t19,t2010 Reddy UM, Page GP, Saade GR, et al: Karyotype versus microarray testing for genetic abnormalities after stillbirth. N Engl J Med 367(23):2185,t2012 Rumbold A, Ota E, Hori H, et al: Vitamin E supplementation in pregnancy. Cochrane Database Syst Rev 9:CD004069, 2015a Rumbold A, Ota E, Nagata C, et al: Vitamin C supplementation in pregnancy. Cochrane Database Syst Rev 9:CD004072, 2015b Saller DN Jr, Lesser KB, Harrel U, et al: he clinical utility of the perinatal autopsy. JAMA 273:663, 1995 Sharma PP, Salihu HM, Oyelese Y, et al: Is race a determinant of stillbirth recurrence? Obstet Gynecol 107(2 Pt 1):391,2006

1	Sharma PP, Salihu HM, Oyelese Y, et al: Is race a determinant of stillbirth recurrence? Obstet Gynecol 107(2 Pt 1):391,2006 Shruthi M, Gupta N, Jana M, et al: Comparative study of conventional and virtual autopsy using postmortem MRI in the phenotypic characterization of stillbirths and malformed fetuses. Ultrasound Obstet Gynecol March 13, 2017 [Epub ahead of print] Siassakos D, Jackson S, Gleeson K, et al: ll bereaved parents are entitled to good care after stillbirth: a mixed-methods multicentre study (INSIGHT). B JOG July 31, 2017 [Epub ahead of print] Silver M:Fetal death. Obstet Gynecol 109: 153, 2007 Silver RM, Parker CB, Reddy UM, et al: Antiphospholipid antibodies in stillbirth. Obstet GynecoIt122(3):641, 2013 Silver RM, Saade GR, Thorsten V, et al: Factor V Leiden prothrombin G20210A, and methylene tetrahydrofolate reductase mutations and stillbirth: the Stillbirth Collaborative Research Network. Am J Obstet Gynecol 215:468,t2016

1	Society for Maternal-Fetal Medicine: he use of chromosomal micro array for prenatal diagnosis. Sociery for Maternal-Fetal Medicine (SMFM) Consult Series No. 41, October 2016 Stillbirth Collaborative Research Network Writing Group: Association between stillbirth and risk factors known at pregnancy conirmation. JAMA 306(22):2469,t2011a Stillbirth Collaborative Research Network Writing Group: Causes of death among stillbirths. JAMA 306(22):2459, 2011b The Lancet's Ending Preventable Stillbirths Series Study Group: Stillbirths: economic and psychosocial consequences. Lancet 387:604, 2016a The Lancet's Ending Preventable Stillbirths Series Study Group: Stillbirths: ending preventable deaths by 2030. Lancet 387:703, 2016b The Lancet's Ending Preventable Stillbirths Series Study Group: Stillbirths: progress and uninished business. Lancet 387:574, 2016c

1	The Lancet's Ending Preventable Stillbirths Series Study Group: Stillbirths: progress and uninished business. Lancet 387:574, 2016c The Lancet's Ending Preventable Stillbirths Series Study Group: Stillbirths: rates, risk factors, and acceleration towards 2030. Lancet 387:587, 2016d The Lance's Ending Preventable Stillbirths Series Study Group: Stillbirths: recall to action in high-income countries. Lancet 387: 691, 2016e The Lancet's Stillbirths Series Steering Committee: Stillbirths: how can health systems deliver for mothers and babies? Lancet 377:1610, 20lla The Lancet's Stillbirths Series Steering Committee: Stillbirths: the vision for 2020. Lancet 3 7:1798,2011b The Lancet's Stillbirths Series Steering Committee: Stillbirths: the way forward in high-income countries. Lancet 7:1703,t2011c The Lancet's Stillbirths Series Steering Committee: Stillbirths: what diference can we make and at what cost? Lancet :1523, 2011d

1	The Lancet's Stillbirths Series Steering Committee: Stillbirths: what diference can we make and at what cost? Lancet :1523, 2011d The Lancet's Stillbirths Series Steering Committee: Stillbirths: where? When? Why? How to make the data count? Lancet 377: 1448, 2011e The Lancet's Stillbirths Series Steering Committee: Stillbirths: why they matter. Lancet 377:1353, 2011f Varner JW, Silver M, Rowland Hogue CJ, et al: Association between stillbirth and illicit drug use and smoking during pregnancy. Obstet Gynecol 123:113,t2014 Weeks JW, Asrat T, Morgan MA, et al: Antepartum surveillance for a history of stillbirth: when to begin? Am J Obstet Gynecol 172:486, 1995 The Puerperi um REPRODUCTIVE TRACT INVOLUTION ................ 652 PLACENTAL SITE INVOLUTION.. . . . . . . . . . . . . . . . .. 654 URINARY TRACT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 654 PERITONEUM AND ABDOMINAL WALL ............. 655 BLOOD AND BLOOD VOLUME .........0............ 655

1	PERITONEUM AND ABDOMINAL WALL ............. 655 BLOOD AND BLOOD VOLUME .........0............ 655 LACTATION AND BREASTFEEDING ................ 656 HOSPITAL CARE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659 HOME CARE ................................... 663 Although the changes occurring during the puerperium are considered as physiological, they border vey closey upon the pathological, in as much as under no other circumstances does such marked and rapid tissue metabolism occur without a departure rom a condition of health. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) The word puerperium is derived from Latin-puer, child + parus, bringing forth. Currently, it defines the time following delivery during which pregnancy-induced maternal anatomical and physiological changes return to the nonpregnant state. Its duration is understandably inexact, but is considered to be between 4 and 6 weeks. Although much less complex compared with pregnancy, the puerperium has appreciable changes as stated above by Williams (1903), and some of these may be either bothersome or worrisome for the new mother. Kanotra and colleagues (2007) analyzed challenges that women faced from 2 to 9 months following delivery. The Pregnancy Risk Assessment Surveillance System-PRAMS-of the Centers for Disease Control and Prevention (2016) listed concerns of new mothers that are shown in Table 36-1. At least a third of these women felt the need for social support, and 25 percent had concerns with breastfeeding.

1	Return of the tissues in the birth canal to the nonpregnant state begins soon ater delivery. he vagina and its outlet gradually diminish in size but rarely regain their nulliparous dimensions. Rugae begin to reappear by the third week but are less prominent than before. he hymen is represented by several small tags of tissue, which scar to form the mytom caunces. The vaginal epithelium reflects the hypoestrogenic state, and it does not begin to proliferate until 4 to 6 weeks. This timing is usually coincidental with resumed ovarian estrogen production. Lacerations or stretching of the perineum during delivery can lead to vaginal outlet relaxation. Some damage to the pelvic loor may be inevitable, and parturition predisposes to urinary incontinence and pelvic organ prolapse.

1	The massively increased uterine blood flow necessary to maintain pregnancy is made possible by signiicant hypertrophy and remodeling of pelvic vessels. After delivery, their caliber gradually diminishes to approximately that of the prep regnant state. Within the puerperal uterus, larger blood vessels become obliterated by hyaline changes. They are gradually resorbed and replaced by smaller ones. Minor vestiges of the larger vessels, however, may persist for years. During labor, the margin of the dilated cervix, which corresponds to the external os, may be lacerated. The cervical opening contracts slowly, and for a few days immediately after The Puerperium 653 TABLE 36-1 . Pregnancy Risk Assessment Surveillance System-PRAMSa Concerns Raised by Women in the First 2-9 Months Postpartum

1	The Puerperium 653 TABLE 36-1 . Pregnancy Risk Assessment Surveillance System-PRAMSa Concerns Raised by Women in the First 2-9 Months Postpartum Help with postpartum depression 10 aCenters for Disease Control and Prevention, 201n6. Data from Kanotra 5, D'Angelo 0, Phares TM, et al: Challenges faced by new mothers in the early postpartum period: an analysis of comment data from the 2000 Pregnancy Risk Assessment Monitoring System (PRAMS) survey. Matern Child Health J 11 (6):549,n2007.

1	labor, it readily admits two ingers. By the end of the first week, this opening narrows, the cervix thickens, and the endocervical canal re-forms. The external os does not completely resume its pregravid appearance. It remains somewhat wider, and typically, ectocervical depressions at the site of lacerations become permanent. These changes are characteristic of a parous cervix (Fig. 36-1). Cervical epithelium also undergoes considerable remodeling. This actually may be salutary because almost half of women have regression of high-grade dysplasia following delivery (Ahdoot, 1998; Kaneshiro, 2005).

1	Ater delivery, the fundus of the contracted uterus lies slightly below the umbilicus. It consists mostly of myometrium covered by serosa and internally lined by decidua. The markedly attenuated lower uterine segment contracts and retracts, but not as forceully as the uterine corpus. During the next few weeks, the lower segment is converted from a clearly distinct substructure large enough to accommodate the fetal head to a barely discernible uterine isthmus located between the corpus and internal cervical os. Immediately postpartum, the anterior and posterior walls, which lie in close apposition, are each 4 to 5 cm thick (Buhimschi, 2003). At this time, the uterus weighs approximately 1000 g.

1	Myometrial involution is a truly remarkable feat of destruction or deconstruction that begins as soon as 2 days ater delivery (Williams, 1931). The total number of myocytes does not decrease appreciably-rather, their size decreases markedly. As emphasized by Hytten (1995), the quality of studies that describe the degree of decreasing uterine weight postpartum are (8) cervices. 12.8 12.4 10 9 Cavity length 7.6 6.7 AP diameter 6.4 E) 10 12 ;:) 8 6 FIGURE 36-2 Sonographic measurements of uterine involution during the first 9 days postpartum. AP = anteroposterior. (Data from Hytten F: The Clinical Physiology of the Puerperium. London, Farrand Press, 1995.) poor. Best estimates show that the uterus weighs approximately 500 g by 1 week postpartum, about 300 g by 2 weeks, and at 4 weeks, involution is complete and the uterus weighs approximately 100 g. Ater each successive delivery, the uterus is usually slightly larger than before the most recent pregnancy.

1	Uterine involution and rapid dissipation of size progresses in the first week (Fig. 36-2). Sonographically, the uterus and endometrium return to pregravid size by 8 weeks postpartum (Bae, 2012; Steinkeler, 2012). In a study of 42 normal puerperas, Tekay and Jouppila (1993) identiied fluid in the endometrial cavity in 78 percent of women at 2 weeks, 52 percent at 3 weeks, 30 percent at 4 weeks, and 10 percent at 5 weeks. Belachew and coworkers (2012) used three-dimensional sonography and visualized intracavitary tissue matter in a third on day 1, in 95 percent on day 7, in 87 percent on day 14, and in 28 percent on day 28. By day 56, the small cavity was empty. Sohn and associates (l9��) described Doppler ultrasound results showing continuously increasing uterine artery vascular resistance during the first 5 days postpartum. Weintraub and colleagues (2013) posited that uterine involution may be diferent in preeclamptic women because they more likely had early diastolic notches seen on

1	during the first 5 days postpartum. Weintraub and colleagues (2013) posited that uterine involution may be diferent in preeclamptic women because they more likely had early diastolic notches seen on uterine artery velocimetry.

1	Because separation of the placenta and membranes involves the spongy layer, the decidua basalis is not sloughed. The in situ decidua varies markedly in thickness, it has an irregular jagged border, and it is infiltrated with blood, especially at the placental site. Within 2 or 3 days ater delivery, the remaining decidua becomes diferentiated into two layers. he superficial layer becomes necrotic and is sloughed in the lochia. The basal layer adjacent to the myometrium remains intact and is the source of new endometrium. Endometrial regeneration is rapid, except at the placental site. Within a week or so, the free surface becomes covered by epithelium, and Sharman (1953) identified ully restored endometrium in all biopsy specimens obtained from the 16th day onward. Histological endometritis is part of the normal reparative process.

1	vforeover, microscopic inflammatory changes characteristic of acute salpingitis are seen in almost half of women between 5 and 15 days, however, these findings are not thought to reflect infection (Andrews, 1951). Afterpains. Several clinical findings arise with uterine involution. In primiparas, the uterus tends to remain tonically contracted following delivery. In multiparas, it often contracts vigorously at intervals and gives rise to aterpains, which are similar to but milder than labor contractions. hese are more pronounced as parity increases and worsen when the newborn suckles, likely because of oxytocin release (Holdcroft, 2003). Usually, afterpains decrease in intensity and become mild by the third day. We have encountered unusually severe and persistent afterpains in women with postpartum uterine infections.

1	Lochia. Early in the puerperium, sloughing of decidual tissue results in a vaginal discharge of variable quantity. he discharge is termed lochia and contains erythrocytes, shredded decidua, epithelial cells, and bacteria. For the irst few days ater delivery, there is blood suicient to color it red-lochia rubra. Ater 3 or 4 days, lochia becomes progressively pale in color-lochia serosa. ter approximately the 10th day, because of an admixture of leukocytes and reduced fluid content, lochia assumes a white or yellow-white color-lochia alba. he average duration of lochial discharge ranges from 24 to 36 days (Fletcher, 2012). Because of this expected leukocyte component, saline preparations of lochia for microscopic evaluation in cases of suspected puerperal metritis are typically uninformative and not recommended.

1	Complete extrusion of the placental site takes up to 6 weeks. Immediately after delivery, the placental site is approximately palm-sized. Within hours of delivery, it normally contains many thrombosed vessels that ultimately undergo organization. By the end of the second week, it is 3 to 4 cm in diameter. Placental site involution is an exfoliation process, which is prompted in great part by undermining of the implantation site by new endometrial proliferation (Williams, 1931). Thus, involution is not simply absorption in situ. Exfoliation consists of both extension and "downgrowth" of endometrium from the margins of the placental site, as well as development of endometrial tissue from the glands and stroma left deep in the decidua basalis after placental separation. Anderson and Davis (1968) concluded that placental site exfoliation results from sloughing of infarcted and necrotic supericial tissues followed by a remodeling process.

1	In some cases, uterine involution is hindered because of infection, retained placental fragments, or other causes. Such subinvolution is accompanied by varied intervals of prolonged lochia as well as irregular or excessive uterine bleeding. During bimanual examination, the uterus is larger and soter than would be expected. With bleeding, pelvic sonography may help exclude retained placenta or, less-commonly, vascular malformations as the source (lraha, 2017). Methylergonovine (Methergine), 0.2 mg orally every 3 to 4 hours for 24 to 48 hours, is recommended by many, but its eicacy is questionable. If there is infection, antimicrobial therapy usually leads to a good response. In an earlier study, Wager and coworkers (1980) reported that a third of these late cases of postpartum metritis are caused by Chamydia trachomatis. For mild infection, empirical therapy with azithromycin or doxycycline usually prompts resolution regardless of bacterial etiology. At our institution, common oral

1	are caused by Chamydia trachomatis. For mild infection, empirical therapy with azithromycin or doxycycline usually prompts resolution regardless of bacterial etiology. At our institution, common oral options taken for 7 to 10 days include doxycycline, 100 mg twice daily; azithromycin, 500 mg twice daily; or ampicillin-clavulanate (Augmentin), 875 mg twice daily. Serious metritis is treated with intravenous broad-spectrum antibiotics listed in Table 37-2.

1	Another cause of subinvolution is incompletely remodeled uteroplacental arteries (Andrew, 1989; Kavalar, 2012). hese noninvoluted vessels are illed with thromboses and lack an endothelial lining. Perivascular trophoblasts are also identiied in the vessel walls, which suggests an aberrant interaction between uterine cells and trophoblasts.

1	Seconday pospartum hemorrhage is deined as bleeding 24 hours to 12 weeks ater delivery. Clinically worrisome uterine hemorrhage develops within 1 to 2 weeks in perhaps 1 percent of women. Such bleeding most oten is the result of abnormal involution of the placental site. It occasionally is caused by retention of a placental fragment or by a uterine artery pseudoaneurysm. Usually, retained products undergo necrosis with ibrin deposi tion and may eventually form a so-called placental poyp. As the eschar of the polyp detaches from the myometrium, hemorrhage may be brisk. As discussed in Chapter 56 (p. 1090), delayed postpartum hemorrhage may also be caused by von Willebrand disease or other inherited coagulopathies (Lipe, 2011).

1	In our experiences, few women with delayed hemorrhage are found to have retained placental fragments. hus, we and others do not routinely perform curettage (Lee, 1981). Another concern is that curettage may worsen bleeding by avulsing part of the implantation site. Thus, in a stable patient, if sonographic examination shows an empty cavity, then oxytocin, methylergonovine, or a prostaglandin analogue is given. Suitable dosing is found in Table 20-2 (p. 392). Antimicrobials are added if uterine infection is suspected. If large clots are seen in the uterine cavity with sonography, then gentle suction curettage is considered. Otherwise curettage is carried out only if appreciable bleeding persists or recurs after medical management.

1	Normal pregnancy-induced glomerular hyperiltration persists during the puerperium but returns to prepregnancy baseline by 2 weeks (Hladunewich, 2004). Dilated ureters and renal pelves return to their prep regnant state by 2 to 8 weeks postpartum. Because of this dilated collecting system, coupled with residual urine and bacteriuria in a traumatized bladder, symptomatic urinary tract infection remains a concern in the puerperium. Funnell and colleagues (1954) used cystoscopy immediately postpartum and described varying degrees of submucosal hemorrhage and edema. Bladder trauma is associated most

1	Funnell and colleagues (1954) used cystoscopy immediately postpartum and described varying degrees of submucosal hemorrhage and edema. Bladder trauma is associated most The Puerperium 655 closely with labor length and thus to some degree is a normal accompaniment of vaginal delivery. Postpartum, the bladder has an increased capacity and a relative insensitivity to intravesical pressure. hus, overdistention, incomplete emptying, and excessive residual urine are frequent (Buchanan, 2014; Mulder, 2014). Acute urinary retention is also more common with narcotic analgesia (Kandadai, 2014). heir management is discussed on page 660. It is unusual for urinary incontinence to manifest during the puerperium. hat said, much attention has been given to the potential for subsequent development of urinary incontinence and other pelvic loor disorders in the years following delivery. A more detailed discussion is found in Chapter 30 (p. 568).

1	The broad and round ligaments require considerable time to recover from stretching and loosening during pregnancy. As a result of ruptured elastic ibers in the skin and prolonged distention by the pregnant uterus, the abdominal wall remains soft and laccid. If the abdomen is unusually labby or pendulous, an ordinary girdle is often satisfactory. n abdominal binder is another temporary measure. Several weeks are required for these structures to return to normal, and recovery is aided by exercise. hese may be started anytime following vaginal delivery. Ater cesarean delivery, a 6-week interval to allow fascia to heal and abdominal soreness to diminish is reasonable. Silvery abdominal striae commonly develop as striae graviarum (Chap. 4, p. 53). Except for these, the abdominal wall usually resumes its prep regnancy appearance. When muscles remain atonic, however, the abdominal wall also remains lax. Marked separation of the rectus abdominis muscles-diastasis recti-may result.

1	Marked leukocytosis and thrombocytosis may occur during and after labor. The white blood cell count sometimes reaches 30,000/�L, with the increase predominantly due to granulocytes. There is a relative lymphopenia and an absolute eosinopenia. Normally, during the irst few postpartum days, hemoglobin concentration and hematocrit luctuate moderately. We routinely check these on the irst postpartum day or earlier if indicated. If they fall much below the levels present just before labor, a considerable amount of blood has been lost.

1	By the end of pregnancy, laboratory values that assess coagulation are altered (Kenny, 2014). These changes are discussed in Chapter 4 (p. 59) and listed in the Appendix (p. 1256). Many persist variably in the puerperium. For example, a markedly increased plasma ibrinogen level is maintained at least through the irst week and hence so is an elevated sedimentation rate. Hypercoagulability appears to be greater and is relected by the likelihood of deep-vein thrombosis and pulmonary embolism in the 12 weeks following childbirth (Kamel, 2014). his is depicted in Figure 36-3 and is discussed further in Chapter 52 (p. 1004). FIGURE 36-3 Risk of deep-vein thrombosis or pulmonary embolism following childbirth. (Data from Kamel H, Navi S, Sriram N, et al: Risk of a Thrombotic Event after the 6-week postpartum period. N Engl J Med 370:1307,o2014.)

1	When the amount of blood attained by normal pregnancy hypervolemia is lost as postpartum hemorrhage, the woman almost immediately regains her nonpregnant blood volume (Chap. 41, p. 756). If less has been lost at delivery, blood volume generally nearly returns to its nonpregnant level by 1 week after delivery. Cardiac output usually remains elevated for 24 to 48 hours postpartum and declines to nonpregnant values by 10 days (Robson, 1987). Heart rate changes follow this pattern, and blood pressure similarly returns to nonpregnant values (Fig. 36-4). Correspondingly, systemic vascular resistance remains in the lower range characteristic of pregnancy for 2 days postpartum and then begins to steadily increase to normal nonpregnant values (Hibbard, 2014). Despite this, Morris and coworkers (2015) found that reduced arterial stifness persists following pregnancy. They suggest a signiicant favorable

1	FIGURE 36-4 During the early puerperium, blood pressure normally rises toward nonpregnant values. MAP = mean arterial pressure. efect of pregnancy on maternal cardiovascular remodeling, which may represent a mechanism by which preeclampsia risk is reduced in subsequent pregnancies. Normal pregnancy is associated with an appreciable increase in extracellular sodium and water retention, and postpartum diuresis is a physiological reversal of this process. Chesley and coworkers (1959) demonstrated a decrease in sodium space of approximately 2 L during the first week postpartum. his also corresponds with loss of residual pregnancy hypervolemia. In preeclampsia, pathological retention of luid antepartum and its diuresis postpartum may be prodigious (Chap. 40, p. 744).

1	Postpartum diuresis results in relatively rapid weight loss of 2 to 3 kg, which is additive to the 5 to 6 kg incurred by delivery and normal blood loss. Weight loss from pregnancy itself is likely to be maximal by the end of the second week postpartum. It follows that any residual increased weight compared with prepregnancy values probably represents fat stores that will persist. According to Schauberger and associates (1992), women approach their self-reported prepregnancy weight 6 months after delivery but still retain an average surplus of 1.4 kg (3 Ib).

1	Each mature mammary gland or breast is composed of 15 to 25 lobes. hey are arranged radially and are separated from one another by varying amounts of fat. Each lobe consists of several lobules, which in turn are composed of numerous alveoli. Each alveolus is provided with a small duct that joins others to form a single larger duct for each lobe as shown in Figure 36-5. These lacterous ducts open separately on the nipple, where they may be distinguished as minute but distinct orifices. he alveolar secretory epithelium synthesizes the various milk constituents.

1	After delivery, the breasts begin to secrete colostrum, which is a deep lemon-yellow liquid. It usually can be expressed from the nipples by the second postpartum day. Compared with mature milk, colostrum is rich in immunological components and contains more minerals and amino acids (Ballard, 2013). It also has more protein, much of which is globulin, but less sugar and fat. Secretion persists for 5 days to 2 weeks, with gradual conversion from "transitional" to mature milk by 4 to 6 weeks. The colostrum content of immunoglobulin A (IgA) ofers the newborn protection against enteric pathogens. Other host resistance factors found in colostrum and milk include complement, macrophages, lymphocytes, lactoferrin, lactoperoxidase, and lysozymes.

1	Mature milk is a complex and dynamic biological luid that includes fat, proteins, carbohydrates, bioactive factors, minerals, vitamins, hormones, and many cellular products (Table 36-2). The concentrations and contents of human milk change even during a single feed and are inluenced by maternal diet and by newborn age, health, and needs. A nursing mother easily produces 600 mL of milk daily, and maternal gestational FIGURE 36-5 Schematic of the alveolar and ductal system during lactation. Note the myoepithelial fibers (M) that surround the outside of the uppermost alveolus. The secretions from the glandular elements are extruded into the lumen of the alveoli (A) and ejected by the myoepithelial cells into the ductal system (0), which empties through the nipple. Arterial blood supply to the alveolus is identified by the upper right arrow and venous drainage by the arrow beneath.

1	PUFA = Polyunsaturated fatty acids. The Puerperium 657 weight gain has little impact on its quantity or quality. Milk is isotonic with plasma, and lactose accounts for half of the osmotic pressure. Essential amino acids are derived from blood, and nonessential amino acids are derived in part from blood or synthesized in the mammary gland. Most milk proteins are unique and include alpha-lactalbumin, beta-lactoglobulin, and casein. Fatty acids are synthesized in the alveoli from glucose and are secreted by an apocrine-like process. Most vitamins are found in human milk, but in variable amounts. Vitamin K is virtually absent, and thus, an intramuscular dose is given to the newborn (Chap. 33, p. 626). Vitamin D content is low 22 IU/mL, and newborn supplementation is also recommended by the American Academy of Pediatrics (Wagner, 2008).

1	Whey is milk serum and has been shown to contain large amounts of interleukin-6 (Saito, 1991). Human milk has a whey-to-casein ratio of 60:40, considered ideal for absorption. Prolactin appears to be actively secreted into breast milk. Epidermal growth octor (EGF) has been identiied, and because it is not destroyed by gastric proteolytic enzymes, it may be absorbed to promote growth and maturation of newborn intes tinal mucosa (McCleary, 1991). Other critical components in human milk include lactoferrin, melatonin, oligosaccharides, and essential fatty acids. • Endocrinology of Lactation

1	• Endocrinology of Lactation The precise humoral and neural mechanisms involved in lactation are complex. Progesterone, estrogen, and placental lactogen, as well as prolactin, cortisol, and insulin, appear to act in concert to stimulate the growth and development of the milksecreting apparatus (Stuebe, 2014). With delivery, the maternal serum levels of progesterone and estrogen decline abruptly and profoundly. This drop removes the inhibitory inluence of progesterone on alpha-lactalbumin production and stimulates lactose synthase to increase milk lactose. Progesterone withdrawal also allows prolactin to act unopposed in its stimulation of alpha-lactalbumin production. Activation of calcium-sensing receptors (CaSR) in mammary epithelial cells downregulates parathyroid hormone-related protein (PTHrP) and increases calcium transport into milk (Vanhouten, 2013). Serotonin is also produced in mammary epithelial cells and has a role in maintaining milk production (Collier, 2012).

1	The intensity and duration of subsequent lactation are controlled, in large part, by the repetitive stimulus of nursing and emptying of milk from the breast. Prolactin is essential for lactation, and women with extensive pituitary necrosisSheehan syndrome�o not lactate (Chap. 58, p. 1133). Although plasma prolactin levels fall after delivery to levels lower than during pregnancy, each act of suckling triggers a rise in levels (Pang, 2007). Presumably a stimulus from the breast curtails the release of dopamine, also known as prolactin-inhibiting octor, from the hypothalamus. In turn, this transiently induces increased prolactin secretion.

1	The posterior pituitary secretes oxytocin in pulsatile fashion. This stimulates milk expression from a lactating breast by causing contraction of myoepithelial cells in the alveoli and small milk ducts (see Fig. 36-5). Milk ejection, or letting down, is a relex initiated especially by suckling, which stimulates the posterior pituitary to liberate oxytocin. he reflex may even be provoked by an infant cry and can be inhibited by maternal fright or stress (Stuebe, 2014). Human milk contains several protective immunological substances, including secretory IgA and growth factors. The antibodies in human milk are speciically directed against maternal environmental antigens such as Escherichia coli (Iyengar, 2012). According to the Centers for Disease Control and Prevention (Perrine, 2015), breastfeeding decreases the incidence of ear, respiratory, and gastrointestinal infections; necrotizing enterocolitis; and sudden infant death syndrome.

1	Much attention has been directed to the role of maternal breast milk lymphocytes in neonatal immunological processes. Milk contains both T and B lymphocytes, but the T lymphocytes appear to difer from those found in blood. Speciically, milk T lymphocytes are almost exclusively composed of cells that exhibit speciic membrane antigens. These memory T cells appear to be an avenue for the neonate to beneit from the maternal immunological experience.

1	Human milk is ideal food for newborns in that it provides agespeciic nutrients, immunological factors, and antibacterial substances. Milk also contains factors that act as biological signals for promoting cellular growth and diferentiation. A list of the advantages of breastfeeding is shown in Table 36-3. For both mother and infant, the beneits of breastfeeding are long-term. For example, women who breastfeed have a lower risk of breast and reproductive cancer, and their children have increased adult intelligence independent of a wide range of possible confounding factors (J ong, 2012; Kramer, 2008). Breastfeeding is associated with decreased postpartum weight retention (Baker, 2008). In addition, rates of sudden-infant-death syndrome are signiicantly lower among breastfed infants. Bartek and colleagues (2013) estimate that a 90-percent breastfeeding rate for 12 months would save more than $3 billion annually in excess infant and maternal morbidity costs. For all these reasons, the

1	TABLE 36-3. Advantages of Breastfeeding Data from American Academy of Pediatrics and the American College of Obstetricians and Gynecologists: Guidelines for Perinatal Care, 8th ed, Elk Grove Village, AAP, 2017. TABLE 36-4. Ten Steps to Successful Breastfeeding 1n. Have a written breastfeeding policy that is regularly communicated to all health-care staff 2. Train all staf in skills necessary to implement this policy 3. Inform all pregnant about the beneits and management of breastfeeding 4. Help mothers initiate breastfTeding within an hour of birth 5. Show mothers how to breastfeed and how to sustain lactation, even if they should be separated from their infants 6. Feed newborns nothing but breast milk, unless medi cally indicated, and under no circumstances provide breast milk substitutes, feeding bottles, or paCiiers free of charge or at low cost 7. Practice rooming-in, which allows mothers and newborns to remain together 24 hours a day 8. 9.

1	Practice rooming-in, which allows mothers and newborns to remain together 24 hours a day 8. 9. Give no artiicial paCiiers to breastfeeding newborns 10. Help start breastfeeding support groups and refer mothers to them Adapted with permission from World Health Organization: Protecting, promoting and supporting breast-feeding: the special role of maternity services. Geneva, World Health Organization, 1989. American Academy of Pediatrics (2017) and American College of Obstetricians and Gynecologists (2016a, 20 17b) supports the World Health Organization (201r1) recommendations of exclusive breastfeeding for up to 6 months.

1	The Surgeon General of the u.S. Department of Health and Human Services (2011) lists some barriers to breastfeeding and suggests practical means of overcoming them. Educational initiatives that include father and peer counseling may improve these rates (Pisacane, 2005; Wolfberg, 2004). The Baby Friendy Hospital Initiative is an international program to increase rates of exclusive breastfeeding and to extend its duration. It is based on the World Health Organization (1989) Ten Steps to Successul Breaseeding (Table 36-4). Worldwide, almost 20,000 hospitals are designated as "baby-friendly," however, only 10 to 15 percent of hospitals in the United States are so designated (Centers for Disease Control and Prevention, 2014; Perrine, 2015). Forrester-Knauss and coworkers (2013) described successul trends toward exclusive breastfeeding in Switzerland during 9 years in which a Baby-Friendly Hospital Initiative was implemented. In a large population-based study done in the United States,

1	trends toward exclusive breastfeeding in Switzerland during 9 years in which a Baby-Friendly Hospital Initiative was implemented. In a large population-based study done in the United States, fewer than two thirds of term neonates were exclusively breastfed at the time of discharge (McDonald, 2012).

1	Various individual resources available are available for breastfeeding mothers that include online information from the American Academy of Pediatrics (http://www.aap.org) and La Leche League International (http://ww.llli.org). • Care of Breasts

1	The nipples require little attention other than cleanliness and attention to skin issures. Fissured nipples render nursing painful, and they may have a deleterious influence on milk production. These cracks also provide a portal of entry for pyogenic bacteria. Because dried milk is likely to accumulate and irritate the nipples, washing the areola with water and mild soap is helpful before and ater nursing. When the nipples are irritated or fissured, some recommend topical lanolin and a nipple shield for 24 hours or longer. Although specific evidence supporting this practice is lacking, nipple pain usually subsides by 10 days (Dennis, 2014). If issuring is severe, the newborn should not be permitted to nurse on the afected side. Instead, the breast is emptied regularly with a pump until the lesions are healed. Poor latching of the neonate to the breast can create such issures. For example, the newborn may take into its mouth only the nipple, which is then is forced against the hard

1	the lesions are healed. Poor latching of the neonate to the breast can create such issures. For example, the newborn may take into its mouth only the nipple, which is then is forced against the hard palate during suckling. Ideally, the nipple and areola are both taken in to evenly distribute suckling forces. Moreover, the force of the hard palate against the lactiferous sinuses aids their eicient emptying, while the nipple is thereby positioned closer to the soft palate.

1	• Contraindications to Breastfeeding

1	Nursing is contraindicated in women who take street drugs or do not control their alcohol use; have an infant with galactosemia; have human immunodeiciency virus (HIV) infection; have active, untreated tuberculosis; take certain medications; or are undergoing breast cancer treatment (American Academy of Pediatrics, 2017; Faupel-Badger, 2013). Breastfeeding has been recognized for some time as a mode of HIV transmission and is proscribed in developed countries in which adequate nutrition is otherwise available. Other viral infections do not contraindicate breastfeeding. For example, with maternal cytomegalovirus infection, both virus and antibodies are present in breast milk. And, although hepatitis B virus is excreted in milk, breastfeeding is not contraindicated if hepatitis B immune globulin is given to the newborns of afected mothers. Maternal hepatitis C infection is not a contraindication because breastfeeding has not been shown to transmit infection (Society for MaternalFetal

1	is given to the newborns of afected mothers. Maternal hepatitis C infection is not a contraindication because breastfeeding has not been shown to transmit infection (Society for MaternalFetal Medicine, 2017).Women with active herpes simplex virus may sucle their infants if there are no breast lesions and if particular care is directed to hand washing before nursing.

1	Most drugs given to the mother are secreted in breast milk, although the amount ingested by the infant typically is small. Many factors influence drug excretion and include plasma concentration, degree of protein binding, plasma and milk pH, degree of ionization, lipid solubility, and molecular weight (Rowe, 2013). The ratio of drug concentration in breast milk to that in maternal plasma is the milk-to-plasma drug-concentration ratio. Ideally, to minimize infant exposure, medication selection should favor drugs with a shorter halflife, poorer oral absorption, and lower lipid solubility. If multiple daily drug doses are required, then each is taken by the mother ater the closest feed. Single daily-dosed drugs may be taken just before the longest infant sleep interval-usually at bedtime (Spencer, 2002).

1	Only a few drugs are absolutely contraindicated while breastfeeding (Berlin, 2013; Bertino, 2012). Cytotoxic drugs may interfere with cellular metabolism and potentially cause immune suppression or neutropenia, afect growth, and at least theoretically, increase the risk of childhood cancer. Examples include cyclophosphamide, cyclosporine, doxorubicin, metho trexate, and mycophenolate. If a medication presents a con cern, then the importance of therapy should be ascertained. It should be determined whether there is a safer alternative or whether neonatal exposure can be minimized if the medication dose is taken immediately after each breastfeeding (American Academy of Pediatrics, 2017). Finally, recreational drugs such of Obstetricians and Gynecologists, 2017 a). Data on individual drugs are available through the National Institutes of Health website, LactMed, which can be found at toxnet.nlm.nih.gov.

1	Radioactive isotopes of copper, gallium, indium, iodine, sodium, and technetium rapidly appear in breast milk. Con sultation with a nuclear medicine specialist is recommended before performing a diagnostic study with these isotopes (Chap. 46, p. 908). he goal is to use a radionuclide with the shortest excretion time in breast milk. The mother should pump her breasts before the study and store enough milk in a freezer to feed the infant. After the study, she should pump her breasts to maintain milk low but discard all milk produced during the time that radioactivity is present. his ranges from 15 hours to 2 weeks, depending on the isotope used. Importantly, radioactive iodine concentrates and persists in the thyroid. Its special considerations are discussed in Chapter 63 (p. 1201).

1	his is common in women who do not breastfeed. It is typified by milk leakage and breast pain, which peak 3 to 5 days after delivery (Spitz, 1998). Up to half of afected women require analgesia for breast pain relief, and as many as 10 percent report severe pain for up to 14 days. Evidence is insuicient to irmly support any speciic treatment (Mangesi, 2016). hat said, breasts can be supported with a well-itting brassiere, breast binder, or sports bra. Cool packs and oral analgesics for 12 to 24 hours aid discomfort. Pharmacological or hormonal agents are in general not recommended to suppress lactation.

1	Fever caused by breast engorgement was common before the renaissance of breastfeeding. In one study, Almeida and Kitay (1986) reported that 13 percent of puerperas had fever from engorgement that ranged from 37.8 to 39°C. Fever seldom persists for longer than 4 to 16 hours. The incidence and severity of engorgement and of the fever associated with it are much lower if women breastfeed. Other causes of fever, especially those due to infection, must be excluded. Of these, mastitis is infection of the mammary parenchyma. I t is relatively common in lactating women and is discussed in Chapter 37 (p. 675). • Other Issues with Lactation With inverted nipples, lactiferous ducts open directly into a depression at the center of the areola. With these depressed nipples, nursing is diicult. If the depression is not deep, milk sometimes can be drawn out by a breast pump. If instead the

1	The Puerperium 659 nipple is greatly inverted, daily attempts are made during the last few months of pregnancy to draw or "tease" the nipple out with the fingers. Extra breasts-poymastia, or extra nipples-poythelia, may develop along the former embryonic mammary ridge. Also termed the milk line, this line extends from the axilla to the groin bilaterally. In some women, rests of accessory breast tissue can be found in the mons pubis or vulva (Wagner, 2013). In the general population, the incidence of accessory breast tissue ranges from 0.22 to 6 percent (Loukas, 2007). hese breasts may be so small as to be mistaken for pigmented moles, or if without a nipple, for lymphadenopathy or lipoma. Polymastia has no obstetrical significance, although occasion ally enlargement of these accessory breasts during pregnancy and anxiety.

1	Polymastia has no obstetrical significance, although occasion ally enlargement of these accessory breasts during pregnancy and anxiety. Galactocele is a milk duct that becomes obstructed by inspis sated secretions. The amount is ordinarily limited, but an excess pressure symptoms and have the appearance of an abscess. It may resolve spontaneously or require aspiration. Among individuals, the volume of milk secreted varies markedly. This depends not on general maternal health but on breast glandular development. Rarely, there is complete lack of mammary secretion-agalactia. Occasionally, mammary secretion is excessive-poygalactia.

1	For 2 hours after delivery, blood pressure and pulse are taken every 15 minutes, or more frequently if indicated. Temperature is assessed every 4 hours for the first 8 hours and then at least every 8 hours subsequently (American Academy of Pediatrics, 2017). The amount of vaginal bleeding is monitored, and the fundus palpated to ensure that it is well contracted. If relaxation is detected, the uterus should be massaged through the abdominal wall until it remains contracted. U terotonics are also sometimes required. Blood can accumulate within the uterus without external bleeding. his may be detected early by uterine enlargement during fundal palpation in the irst postdelivery hours. Because the likelihood of signiicant hemorrhage is greatest immediately postpartum, even in normal births, the uterus is closely monitored for at least 1 hour after delivery. Postpartum hemorrhage is discussed in Chapter 41 (p. 758). If regional analgesia or general anesthesia was used for labor or

1	the uterus is closely monitored for at least 1 hour after delivery. Postpartum hemorrhage is discussed in Chapter 41 (p. 758). If regional analgesia or general anesthesia was used for labor or delivery, the mother should be observed in an appropriately equipped and stafed recovery area.

1	Women are out of bed within a few hours after delivery. n attendant should be present for at least the irst time, in case the woman becomes syncopal. he many conirmed advantages of early ambulation include fewer bladder complications, less frequent constipation, and reduced rates of puerperal venous thromboembolism. As discussed on page 655, deep-vein thrombosis and pulmonary embolism are common in the puerperium (see Fig. 36-3). In an audit of puerperal women at Parkland Hospital, the frequency of venous thromboembolism was found to be 0.008 percent after a vaginal birth and 0.04 percent following cesarean delivery. We attribute this low incidence to early ambulation. Risk factors and other measures to diminish the frequency of thromboembolism are discussed in Chapter 52 (p. 1004).

1	here are no dietary restrictions for women who have been delivered vaginally. Two hours after uncomplicated vaginal delivery, a woman is allowed to eat. With breastfeeding, the level of calories and protein consumed during pregnancy are increased slightly as recommended by the Food and Nutrition Board of the National Research Council (Chap. 9, p. 167). If the mother does not breastfeed, dietary requirements are the same as for a nonpregnant woman. We recommend oral iron supplementation for at least 3 months after delivery and hematocrit evaluation at the first postpartum visit. As noted earlier, profound drops in estrogen levels follow removal of the placenta. Reminiscent of the menopause, postpartum women may experience hot lushes, especially at night. Importantly, the patientr's temperature is assessed to diferentiate these physiological vasomotor events from infection.

1	In women with migraines, dramatic hypoestrogenism may trigger headaches. Importantly, severe headaches should be differentiated from spinal headache or hypertensive complications. Care varies depending on migraine severity. Mild headaches may respond to analgesics such as ibuprofen or acetaminophen. lternatively, Midrin combines isometheptene mucate, which is a sympathomimetic agent; dichloralphenazone, which is a mild sedative; and acetaminophen and is compatible with breastfeeding. For more severe headaches, oral or systemic narcotics can be used. Instead of Midrin, a triptan, such as sumatriptan (Imitrex), can efectively relieve headaches by causing intracranial vasoconstriction.

1	he woman is instructed to clean the vulva from anterior to posterior-the vulva toward the anus. A cool pack applied to the perineum may help reduce edema and discomfort during the irst 24 hours if there is a perineal laceration or an episiotomy. Most women also appear to obtain a measure of relief from the periodic application of a local anesthetic spray. Severe perineal vaginal, or rectal pain always warrants carul inspection and papation. Severe discomfort usually indicates a problem, such as a hematoma within the irst day or so and infection ater the third or fourth day (Chap. 37, p. 674 and Chap. 41, p. 764). Beginning approximately 24 hours after delivery, moist heat as provided by warm sitz baths can be used to reduce local discomfort. Tub bathing after uncomplicated delivery is allowed. The episiotomy incision normally is firmly healed and nearly asymptomatic by the third week.

1	Rarely, the cervix, and occasionally a portion of the uterine body, may protrude from the vulva following delivery. his is accompanied by variable degrees of anterior and posterior vaginal wall prolapse. Symptoms include a palpable mass at or past the introitus, voiding diiculties, or pressure. Puerperal procidentia typically improves with time as the weight of the uterus lessens with involution. As a temporizing measure in those with pronounced prolapse, the uterus can be replaced and held in position with a suitable pessary. Hemorrhoidal veins are often congested at term. Thrombosis is common and may be promoted by second-stage pushing. Treatment includes topically applied anesthetics, warm soaks, and stool-softening agents. Nonprescription topical preparations containing corticosteroids, astringents, or phenylephrine are often used, but no randomized studies support their eicacy compared with conservative management.

1	In most delivery units, intravenous luids are inused during labor and for an hour or so ater delivery. Oxytocin, in doses that have an antidiuretic efect, is typically inused postpartum, and rapid bladder filling is common. Moreover, both bladder sensation and capability to empty spontaneously may be diminished by local or conduction analgesia, by trauma to the bladder, by episiotomy or lacerations, or by operative vaginal delivery. Thus, urinary retention and bladder overdistention is common in the early puerperium. he incidence in more than 5500 women studied with a bladder scanner was 5.1 percent (Buchanan, 2014). In another study, Musselwhite and coworkers (2007) reported retention in 4.7 percent of women who had labor epidural analgesia. Risk factors that increased the likelihood of retention were primiparity, cesarean delivery, perineal laceration, oxytocin-induced or augmented labor, operative vaginal delivery, catheterization during labor, and labor durationr> 10 hours.

1	Prevention of bladder overdistention demands observation ater delivery to ensure that the bladder does not overill and that it empties adequately with each voiding. he enlarged bladder can be palpated suprapubically, or it is evident abdominally indirectly as it elevates the fundus above the umbilicus. he use of an automated bladder scanner sonography system has been studied to detect high bladder volumes and thus postpartum urinary retention (Buchanan, 2014; Van Os, 2006).

1	If a woman has not voided within 4 hours ater delivery, it is likely that she cannot. If she has trouble voiding initially, she also is likely to have further trouble. n examination for perineal and genital-tract hematomas is completed. With an overdistended bladder, an indwelling catheter should be left in place until the factors causing retention have abated. Even without a demonstrable cause, it usually is best to leave the catheter in place for at least 24 hours. his prevents recurrence and allows recovery of normal bladder tone and sensation.

1	When the catheter is removed, a voiding trial is completed to demonstrate an ability to void appropriately. If a woman cannot void after 4 hours, she should be catheterized and the urine volume measured. If more than 200 mL, the bladder is not functioning appropriately, and the catheter is left for another 24 hours. Although rare, if retention persists after a second voiding trial, an indwelling catheter and leg bag can be elected, and the patient returns in 1 week for an outpatient voiding trial. Intermittent sel-catheterization is another option (Mulder, 2017). During a voiding trial, if less than 200 mL of urine is obtained, the catheter can be removed and the bladder subsequently monitored clinically as described earlier. Harris and coworkers (1977) reported that 40 percent of such women develop bacteriuria, and thus a single dose or short course of antimicrobial therapy against uropathogens is reasonable after the catheter is removed. • Pain, Mood, and Cognition

1	• Pain, Mood, and Cognition Discomfort and its causes following cesarean delivery are con sidered in Chapter 30 (p. 585). During the irst few days after vaginal delivery, the mother may be uncomfortable because of afterpains, episiotomy and lacerations, breast engorgement, and at times, postdural puncture headache. Mild analgesics contain ing codeine, aspirin, or acetaminophen, preferably in combina tions, are given as frequently as every 4 hours during the irst few days.

1	It is important to screen the postpartum woman for depression (American College of Obstetricians and Gynecologists, 2016b). It is fairly common for a mother to exhibit some degree of depressed mood a few days after delivery. Termed pospartum blues, this likely is the consequence of several factors. hese include emotional letdown that follows the excitement and fears experienced during pregnancy and delivery, discomforts of the early puerperium, fatigue from sleep deprivation, anxiety over the ability to provide appropriate newborn care, and body image concerns. In most women, efective treatment includes anticipation, recognition, and reassurance. This disorder is usually mild and self-limited to 2 to 3 days, although it sometimes lasts for up to 10 days. Should these moods persist or worsen, an evaluation for symptoms of major depression is done (Chap. 61, p. 1176). Suicidal or infanticidal ideation is dealt with emergently. Because major postpartum depression recurs in at least a

1	an evaluation for symptoms of major depression is done (Chap. 61, p. 1176). Suicidal or infanticidal ideation is dealt with emergently. Because major postpartum depression recurs in at least a fourth of women in subsequent pregnancies, some recommend pharmacological prophylaxis beginning in late pregnancy or immediately postpartum.

1	Last, postpartum hormonal changes in some women may afect brain function. Bannbers and colleagues (2013) compared a measure of executive function in postpartum women and controls and observed a functional decline in postpartum subjects.

1	Pressure on branches of the lumbosacral nerve plexus during labor may manifest as complaints of intense neuralgia or cramplike pains extending down one or both legs as soon as the head descends into the pelvis. If the nerve is injured, pain may continue after delivery, and variable degrees of sensory loss or muscle paralysis can result. In some cases, there is footdrop, which can be secondary to injury at the level of the lumbosacral plexus, sciatic nerve, or common fibular (peroneal) nerve (Bunch, 2014). Components of the lumbosacral plexus cross the pelvic brim and can be compressed by the fetal head or by forceps. The common ibular nerves may be externally compressed when the legs are positioned in stirrups, especially during prolonged second-stage labor.

1	Obstetrical neuropathy is relatively infrequent. Wong and associates (2003) evaluated more than 6000 puerperas and found that approximately 1 percent had a conirmed nerve injury. Lateral femoral cutaneous neuropathies were the most The Puerperium 661 common (24 percent), followed by femoral neuropathies (14 percent). A motor deicit accompanied a third of injuries. Nul liparity, prolonged second-stage labor, and pushing for a long duration in the semi-Fowler position were risk factors. The median duration of symptoms was 2 months, and the range was 2 weeks to 18 months. Nerve injuries with cesarean delivery include the iliohypo gastric and ilioinguinal nerves (Rahn, 2010). These are dis cussed further in Chapter 2 (p. 15).

1	Nerve injuries with cesarean delivery include the iliohypo gastric and ilioinguinal nerves (Rahn, 2010). These are dis cussed further in Chapter 2 (p. 15). Pain in the pelvic girdle, hips, or lower extremities may follow stretching or tearing injuries sustained at normal or diicult delivery. Magnetic resonance (MR) imaging is often informative (Miller, 2015). One example is the piriformis muscle hematoma shown in Figure 36-6. Most injuries resolve with antiinlammatory agents and physical therapy. Rarely, there may be septic pyomyositis such as with iliopsoas muscle abscess (Nelson, 2010; Young, 2010).

1	Separation of the symphysis pubis or one of the sacroiliac synchondroses during labor leads to pain and marked interference with locomotion (Fig. 36-7). Estimates of the frequency of this event vary widely from 1 in 600 to 1 in 30,000 deliveries (Reis, 1932; Taylor, 1986). In our experiences, symptomatic separations are uncommon. heir onset of pain is often acute during delivery, but symptoms may manifest either antepartum or up to 48 hours postpartum (Snow, 1997). In suspected cases, radiography is typically selected. The normal distance of the symphyseal joint is 0.4 to 0.5 cm, and symphyseal separation > 1 cm is diagnostic for diastasis. Treatment is generally conservative, with rest in a lateral decubitus position and an appropriately itted pelvic binder (Lasbleiz, 2017). Surgery is occasionally necessary in some symphyseal separations of more than 4 cm (Kharrazi, 1997). The recurrence risk is high in subsequent pregnancy, and Culligan and coworkers (2002) recommend consideration

1	necessary in some symphyseal separations of more than 4 cm (Kharrazi, 1997). The recurrence risk is high in subsequent pregnancy, and Culligan and coworkers (2002) recommend consideration for cesarean delivery.

1	FIGURE 36-6 Inhomogeneous mass of the right piriformis muscle consistent with a hematoma (yellow cursor measurements) is compared with the normal-appearing left piriformis muscle (yellow arrow), postpartum day following vaginal delivery of a 2840-g newborn. The patient had pain over the pubic bone and pain with ambulation. A shuffling gait was noted, and she had difficulty with leg elevation when supine. The patient was treated with physical therapy and analgesics. A pelvic binder was applied, and a rolling walker was provided. She improved quickly and was discharged home on postoperative day S.

1	In rare cases, fractures of the sacrum or pubic ramus are caused by even uncomplicated deliveries (Alonso-Burgos, 2007; Speziali, 2015). As discussed in Chapter 58 (p. 1129), the latter are more likely with osteoporosis associated with heparin or corticosteroid therapy (Cunningham, 2005). In rare bur serious cases, bacterial osteomyelitis-osteitis pubis-can be devastating. Lawford and coworkers (2010) reported such a case that caused massive vulvar edema.

1	he D-negative woman who is not isoimmunized and whose newborn is D-positive is given 300 -1g of anti-D immune globulin shortly after delivery (Chap. IS, p. 305). Women who are not already immune to rubella or varicella are excellent candidates for vaccination before discharge (Swamy, 2015). hose who have not received a tetanus/diphtheria or inluenza vaccine should be given these (American College of Obstetricians and Gynecologists, 2017 c). Morgan and colleagues (2015) reported that implementation of a best-practices alert in the electronic medical record was associated with a tetanus/diphtheria immunization rate of 97 percent at Parkland Hospital. Vaccination is also discussed in Chapter 9 (p. 171).

1	Following uncomplicated vaginal delivery, hospitalization is seldom warranted for more than 48 hours. A woman should receive instructions concerning anticipated normal physiological puerperal changes, including lochia patterns, weight loss from diuresis, and milk let-down. She also should receive instructions concerning fever, excessive vaginal bleeding, or leg pain, swelling, or tenderness. Persistent headaches, shortness of breath, or chest pain warrant immediate concern. Hospital-stay length following labor and delivery is now regulated by federal law (Chap. 32, p. 616). Currently, the norms are hospital stays up to 48 hours following uncomplicated vaginal delivery and up to 96 hours following uncomplicated cesarean delivery (American Academy of Pediatrics, 2017; Blumenfield, 2015). Earlier hospital discharge is acceptable for appropriately selected women if they desire it .

1	During the hospital stay, a concerted efort is made to provide family planning education. Various forms of contraception are discussed throughout Chapter 38 and sterilization procedures in Chapter 39.

1	Women not breastfeeding have return of menses usually within 6 to 8 weeks. At times, however, it is diicult clinically to assign a speciic date to the first menstrual period after delivery. A minority of women bleed small to moderate amounts intermittently, starting soon after delivery. Ovulation occurs at a mean of 7 weeks, but ranges from 5 to 11 weeks (Perez, 1972). hat said, ovulation before 28 days has been described (Hytten, 1995). hus, conception is possible during the artiicially deined 6-week puerperium. Women who become sexualy active during the puerperium, and who do not desire to conceive, should initiate contraception. Kelly and associates (2005) reported that by the third month postpartum, 58 percent of adolescents had resumed sexual intercourse, but only 80 percent of these were using contraception. Because of this, many recommend long-acting reversible contraceptives-LARC (Baldwin, 2013).

1	Women who breastfeed ovulate much less frequently compared with those who do not, but variation is great. Timing of ovulation depends on individual biological variation and the intensity of breastfeeding. Lactating women may first menstruate as early as the second or as late as the 18th month after delivery. Campbell and Gray (1993) analyzed daily urine specimens to determine the time of ovulation in 92 lactating women. As shown in Figure 36-8, breastfeeding in general delays resumption of ovulation, although as already empha 8..) 50 FIGURE 36-8 Cumulative proportion of breastfeeding women who ovulated during the first 70 weeks following delivery. (Data from Campbell OM, Gray RH: Characteristics and determinants of postpartum ovarian function in women in the United States. Am J Obstet Gynecol 169:5S, 1993.) sized, it does not invariably forestall it. Other findings in their study included the following: 1.

1	Resumption of ovulation was frequently marked by return of normal menstrual bleeding. 2. Breastfeeding episodes lasting 15 minutes seven times daily delayed ovulation resumption. 3. Ovulation can occur without bleeding. 4. Bleeding can be anovulatory. 5. The risk of pregnancy in breastfeeding women was approximately 4 percent per year. For the breastfeeding woman, progestin-only contraceptives, such as progestin pills, depot medroxyprogesterone, or progestin implants, do not afect the quality or quantity of milk. Success with the progesterone-releasing vaginal ring has also been described (Carr, 2016). hese may be initiated any time during the puerperium. Estrogen-progestin contraceptives likely reduce the quantity of breast milk, but under the proper circumstances, they too can be used by breastfeeding women. hese hormonal methods are discussed in Chapter 38.

1	No evidence-based data guide resumption of coitus after delivery, and practices are individualized (Minig, 2009). After 2 weeks, coitus may be resumed based on desire and comort. Barrett and colleagues (2000) reported that almost 90 percent of 484 primiparous women resumed sexual activity by 6 months. And although 65 percent of these reported problems, only 15 percent discussed them with a health-care provider. Intercourse too soon may be unpleasant, if not frankly painful, and this may be related to episiotomy incisions or severe lacerations. In a study of women without an episiotomy, only 0.4 percent of those with a first-or second-degree tear had dyspareunia (Ventolini, 2014). Conversely, in primiparas with an episiotomy, 67 percent had sexual dysfunction at 3 months, 31 percent at 6 months, and 15 percent at 12 months (Chayachinda, 2015). Dyspareunia was also common following cesarean delivery (McDonald, 2015).

1	Postpartum, the vulvovaginal epithelium is thin, and very little lubrication follows sexual stimulation. his stems from the hypoestrogenic state following delivery, which lasts until ovulation resumes. It may be particularly problematic in breastfeeding women who are hypoestrogenic for many months postpartum (Palmer, 2003). For treatment, small amounts of topical estrogen cream can be applied daily for several weeks to vulvar tissues. Additionally, vaginal lubricants may be used with coitus. This same thinning of the vulvovaginal epithelium can lead to dysuria. Topical estrogen can again be ofered once cystitis is excl uded. Taken together, major and minor maternal morbidity are surprisingly common in the months following childbirth. In a survey of 1249 British mothers followed for up to 18 months, The Puerperium 663 Gin 87 percent of all women, at least one symptom was reported. Data from Glazener CM, Abdalla M, Stroud P, et al:

1	The Puerperium 663 Gin 87 percent of all women, at least one symptom was reported. Data from Glazener CM, Abdalla M, Stroud P, et al: Postnatal maternal morbidity: extent, causes, prevention and treatment. BJOG 102:282, 1995. 3 percent required hospital readmission within 8 weeks (Glazener, 1995; hompson, 2002). Milder health problems during the first 8 weeks were reported by 87 percent (Table 36-5). Moreover, almost three fourths continued to have various problems for up to 18 months. Practitioners should be aware of these potential issues in their convalescing patients.

1	By discharge, women who had an uncomplicated vaginal delivery can resume most activities, including bathing, driving, and household functions. Jimenez and Newton (1979) tabulated cross-cultural information on 202 societies from various international geographical regions. Following childbirth, most societies did not restrict work activity, and approximately half expected a return to full duties within 2 weeks. Wallace and coworkers (20 l3) reported that 80 percent of women who worked during pregnancy resume work by 1 year ater delivery. Despite this, Tulman and Fawcett (1988) reported that only half of mothers regained their usual level of energy by 6 weeks. Women who delivered vaginally were twice as likely to have normal energy levels at this time compared with those with a cesarean delivery. Ideally, the care and nurturing of the infant should be provided by the mother with ample help from the father.

1	The American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) recommend a postpartum visit between 4 and 6 weeks. his has proven quite satisfactory to identiy abnormalities beyond the immediate puerperium and to initiate contraceptive practices. Ahdoot 0, Van Nostrand M, Nguyen NJ, et al: he efect of route of deliv ery on regression of abnormal cervical cytologic indings in the postpartum period. Am J Obstet Gynecol 178: 1116, 1998 Almeida 00 Jr, Kitay DZ: Lactation suppression and puerperal fever. m J Obstet Gynecol 154:940, 1986 Alonso-Burgos A, Royo P, Diaz L, et al: Labor-related sacral and pubic frac tures. J Bone Joint Surg 89:396, 2007 TABLE 36-5. Puerperal Morbidity Reported by 8 Weeks Morbidity PercentQ American Academy of Pediatrics: Breastfeeding and the use of human milk. Pediatrics 129(3):e827, 2012

1	TABLE 36-5. Puerperal Morbidity Reported by 8 Weeks Morbidity PercentQ American Academy of Pediatrics: Breastfeeding and the use of human milk. Pediatrics 129(3):e827, 2012 American Academy of Pediatrics, American College of Obstetricians and Gynecologists: Guidelines for Perinatal Care, 8th ed. Elk Grove Village, AAP, 2017 American College of Obstetricians and Gynecologists: Breastfeeding in underserved women: increasing initiation and continuation of breastfeeding. Committee Opinion No. 570, August 2013, Reairmed 2016a American College of Obstetricians and Gynecologists: Screening for perinatal depression. Committee Opinion No. 630, May 2015, Reairmed 2016b American College of Obstetricians and Gynecologists: Marijuana use during pregnancy and lactation. Committee Opinion No. 722, October 2017a American College of Obstetricians and Gynecologists: Optimizing support for breastfeeding as part of obstetric practice. Committee Opinion No. 658, February 2016, Reairmed 2017b

1	American College of Obstetricians and Gynecologists: Optimizing support for breastfeeding as part of obstetric practice. Committee Opinion No. 658, February 2016, Reairmed 2017b American College of Obstetricians and Gynecologists: Update on immunizationand pregnancy: tetanus, diphtheria, and pertussis vaccination. Committee Opinion No. 718, September 2017c Anderson R, Davis J: Placental site involution. Am J Obstet Gynecol 102:23, 1968 Andrew AC, Bulmer IN, Wells M, et al: Subinvolution of the uteroplacental arteries in the human placental bed. Histopathology 15:395, 1989 Andrews MC: Epithelial changes in the puerperal fallopian tube. Am J Obstet Gyneco1 62:28, 1951 Bae HS, m KH, Oh MJ, et al: Postpartum uterine involution: sonographic changes in the endometrium between 2 and 6 weeks postpartum related to delivery mode and gestational age at delivery. Ultrasound Obstet Gynecol 39(6):727, 2012

1	Baker JL, Gamborg M, Heitmann BL, et al: Breastfeeding reduces postpartum weight retention. Am J Clin Nutr 88(6):1543, 2008 Baldwin MK, Edelman AB: he efect of long-acting reversible contraception on rapid repeat pregnancy in adolescents: a review. J Adolesc Health 52(4 Suppl):S47, 2013 Ballard 0, Morrow L: Human milk composition: nutrients and bioactive factors. Pediatr Clin North Am 60(1):49, 2013 Bannbers E, Gingnell M, Engman J, et al: Prefrontal activity during response inhibition decreases over time in the postpartum period. Behav Brain Res 241:132, 2013 Barrett G, Pendry E, Peacock J, et al: Women's sexual health after childbirth. BJOG 107:186, 2000 Bartek MC, Stuebe M, Schwarz EB, et al: Cost analysis of maternal disease associated with suboptimal breastfeeding. Obstet Gynecol 122:111, 2013

1	BJOG 107:186, 2000 Bartek MC, Stuebe M, Schwarz EB, et al: Cost analysis of maternal disease associated with suboptimal breastfeeding. Obstet Gynecol 122:111, 2013 Belachew J, Axelsson 0, Mulic-Lutvica A, et al: Longitudinal study of the uterine body and cavity with three-dimensional ultrasonography in the puerperium. Acta Obstet Gynecol Scand 91(10):1184, 2012 Berlin CM Jr, van den Anker IN: Safety during breastfeeding: drugs, foods, environmental chemicals, and maternal infections. Semin Fetal Neonatal Med 18(1):13, 2013 Bertino E, Varalda A, Di Nicola P, et al: Drugs and breastfeeding: instructions for use. J Matern Fetal Neonatal Med 25(Supp1 4):78, 2012 Blumenfield J, El-sayed Y, Lyell DJ, et al: Risk factors for prolonged postpartum length of stay following cesarean delivery. Am J Perinatol 32:825, 2015 Buchanan J, Beckmann M: Postpartum voiding dysfunction: identiying the risk factors. Aust N Z J Obstet Gynaecol 54(1):41, 2014

1	Buhimschi CS, Buhimschi A, Malinow M, et al: Myometrial thickness during human labor and immediately postpartum. Am J Obstet Gynecol 188:553, 2003 Bunch K, Hope E: An uncommon case of bilateral peroneal nerve palsy following delivery: a case report and review of the literature. Case Rep Obstet Gyneco1 2014:746480, 2014 Campbell OM, Gray RH: Characteristics and determinants of postpartum ovarian function in women in the United States. Am J Obstet Gynecol 169:55, 1993 Carr SL, Gaield ME, Dragoman MV, et al: Safety of the progesterone-releasing vaginal ring (PVR) among lactating women. Contraception 94(3):253, 2016 Centers for Disease Control and Prevention: Breastfeeding Report CardUnited States, 2014. Available at: http://ww.cdc.gov/breastfeeding/data/ reportcard.htm. Accessed March 27, 2016 Centers for Disease Control and Prevention: PRAMS, the Pregnancy Risk Assessment Monitoring System. 2016. Available at: http://ww.cdc.gov/ PRAMS/index.htm. Accessed March 27, 2016

1	Centers for Disease Control and Prevention: PRAMS, the Pregnancy Risk Assessment Monitoring System. 2016. Available at: http://ww.cdc.gov/ PRAMS/index.htm. Accessed March 27, 2016 Chayachinda C, Titapant V, UngkanungdechaA: Dyspareunia and sexual dysfunction ater vaginal delivery in hai primiparous women with episiotomy. J Sex Med 12(5):1275, 2015 Chesley LC, Valenti C, Uichano L: Alterations in body fluid compartments and exchangeable sodium in early puerperium. Am J Obstet Gynecol 77: 1054, 1959 Collier RJ, Hernandez LL, Horseman ND: Serotonin as a homeostatic regulator of lactation. Domest Anim Endocrino1 43(2):161, 2012 Culligan P, Hill S, Heit M: Rupture of the symphysis pubis during vaginal delivery followed by two subsequent uneventful pregnancies. Obstet Gynecol 100:1114, 2002 Cunningham FG: Screening for osteoporosis. N Engl J Med 353(18):1975, 2005

1	Dennis CL, Jackson K, Watson J: Interventions for treating painful nipples among breastfeeding women. Cochrane Database Syst Rev 12:CD007366, 2014 Faupel-Badger JM, Arcaro F, Balkam J], et al: Postpartum remodeling, lactation, and breast cancer risk: summary of a National Cancer Institute-sponsored workshop. J Nat! Cancer Inst 105(3):166, 2013 Fletcher S, Grotegut CA, James AH: Lochia patterns among normal women: a systematic review. J Womens Health (Larchmt) 21(12):1290, 2012 Forrester-Knauss C, Merten S, Weiss C, et al: The Baby-Friendly Hospital Initiative in Switzerland: trends over a 9-year period. J Hum Lact 29(4):510, 2013 Funnell JW, Klawans AH, Cottrell TL: he postpartum bladder. Am J Obstet Gyneco1 67:1249, 1954 Glazener CM, Abdalla M, Stroud P, et al: Postnatal maternal morbidity: extent, causes, prevention and treatment. BJOG 102:282, 1995 Harris E, homas L, Hui GW: Postpartum suveillance for urinary tract infection: patients at risk of developing pyelonephritis after

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1	Taylor RN, Sonson RD: Separation of the pubic symphysis. An underrecognized peripartum complication. J Reprod Med 31 :203, 1986 Tekay A, Jouppila P: A longitudinal Doppler ultrasonographic assessment of the alterations in peripheral vascular resistance of uterine arteries and ultrasonographic indings of the involuting uterus during the puerperium. Am J Obstet Gynecol 168(1 Pt 1): 190, 1993 hompson JF, Roberts CL, Currie M, et al: Prevalence and persistence of health problems after childbirth: associations with parity and method of birth. Birth 29:83, 2002 Tulman L, Fawcett J: Return of functional ability after childbirth. Nurs Res 37:77, 1988 U.S. Department of Health and Human Services. Executive summary: the Surgeon General's call to action to support breastfeeding. 20n11. Available at: http://ww.surgeongeneral.gov/library/calls/breastfeeding/executivesummary. pdf. Accessed March 27, 2016

1	Van Os AF, Van der Linden PJ: Reliability of an automatic ultrasound system in the post partum period in measuring urinary retention. Acta Obstet Gynecol Scand 85:604, 2006 Vanhouten IN, Wysolmerski JJ: he calcium-sensing receptor in the breast. Best Prac Res Clin Endocrinol Metab 27(3)403, 2013 Ventolini G, Yaklic JL, Galloway ML, et al: Obstetric vulvar lacerations and postpartum dyspareunia. ] Reprod Med 59(11-12):560, 2014 Wager GP, Martin DH, Koutsky L, et al: Puerperal infectious morbidity: relationship to route of delivery and to antepartum Chlamydia trachomatis infection. Am J Obstet GynecoIn138:1028, 1980 Wagner CL, Greer FR, American Academy of Pediatrics Section on Breastfeeding: Prevention of rickets and vitamin D deficiency in infants, children, and adolescents. Pediatrics 122(5): 1142, 2008 Wagner IJ, Damitz A, Carey E, et al: Bilateral accessory breast tissue of the vulva: a case report introducing a novel labiaplasty technique. Ann Plast Surg 70(5):549,n2013

1	Wagner IJ, Damitz A, Carey E, et al: Bilateral accessory breast tissue of the vulva: a case report introducing a novel labiaplasty technique. Ann Plast Surg 70(5):549,n2013 Wallace M, Saurel-Cubizolles M], EDEN mother-child cohort study group: Returning to work one year after childbirth: data from the mother-child cohort EDEN. Matern Child Health] 17(8): 1432,n2013 Weintraub AY, Aricha-Tamir B, Steiner N, et al: Postpartum uterine artery Doppler velocimetry among patients following a delivery complicated with preeclampsia. Hypenens Pregnancy 32(4):450,n2013 Williams JW: Obstetrics. New York, D. Appleton, 1903 Williams JW: Regeneration of the uterine mucosa after delivery with especial reference to the placental site. Am J Obstet Gynecol 22:664, 1931 Wolfberg A], Michels KB, Shields W, et al: Dads as breastfeeding advocates: results from a randomized controlled trial of an educational intervention. Am J Obstet GynecoIn191n:708, 2004

1	Wolfberg A], Michels KB, Shields W, et al: Dads as breastfeeding advocates: results from a randomized controlled trial of an educational intervention. Am J Obstet GynecoIn191n:708, 2004 Wong CA, Scavone BM, Dugan S, et al: Incidence of postpartum lumbosacral spine and lower extremity nerve injuries. Obstet Gynecol 101:279,n2003 World Health Organization: Exclusive breastfeeding for six months best for babies everywhere. 2011. Available at: http://www.who.int/mediacentre/news/statemenrs/20111breastfeeding..,20110115/en.l Accessed March ,2016 World Health Organization: Protecting, promoting and supporting breastfeeding: the special role of maternity services. Geneva, World Health Organization, 1989 Young OM, Werner E, Sfakianaki AK: Primary psoas muscle abscess after an uncomplicated spontaneous vaginal delivery. Obstet Gynecol 116(2 Pt 2):

1	Young OM, Werner E, Sfakianaki AK: Primary psoas muscle abscess after an uncomplicated spontaneous vaginal delivery. Obstet Gynecol 116(2 Pt 2): PUERPERAL FEVER ............................. 666 UTERINE INFECTION............................ 667 ABDOMINAL INCISIONAL INFECTIONS............. 670 ADNEXAL ABSCESSES AND PERITONITIS ........... 671 PARAMETRIAL PHLEGMON ...................... 672 SEPTIC PELVIC THROMBOPHLEBITIS............... 673 PERINEAL INFECTIONS.......................... 674 TOXIC SHOCK SYNDROME ....................... 675 BREAST INFECTIONS............................ 675 One cannot ail to be impressed with the very large proportion of patients whose troubles have originated rom ibrile aictions during the puerperium, which in many cases were cleary due to the neglect of aseptic precautions on the part of the obstetrician or midwe. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) Although the woman who recently gave birth is susceptible to several potentially serious complications, pelvic infection continues to be the most important source of maternal morbidity and mortality. Other infections include mastitis and breast abscesses. hat said, puerperal complications include many of those encountered during pregnancy. For example, as discussed in Chapter 52 (p. 1004), venous thromboembolism during the short 6-week puerperium is as frequent as during all 40 antepartum weeks. Other puerperal issues and care are discussed in Chapter 36.

1	Traditionally, the term puerperal iniction describes any bacterial infection of the genital tract after delivery. hese infections as well as preeclampsia and obstetrical hemorrhage formed the lethal triad of maternal death causes before and during the 20th century. Fortunately, because of efective antimicrobials, maternal mortality from infection has become uncommon. Creanga and associates (2017) reported results from the Pregnancy Mortality Surveillance System, which contained 2009 pregnancy-related maternal deaths in the United States from 2011 through 2013. Infection caused 12.7 percent of pregnancy-related deaths and was the second leading cause. In a similar analysis of the North Carolina population from 1991 through 1999, Berg and colleagues (2005) reported that 40 percent of infection-related maternal deaths were preventable.

1	Several infective and noninfective factors can cause puerperal fever-a temperature of 38.0°C (100.4°F) or higher. Most persistent ivers ater childbirth are caused by genital tract iniction. Using this conservative deinition of fever, Filker and Monif (1979) reported that only about 20 percent of women febrile within the first 24 hours after vaginal delivery were subsequently diagnosed with pelvic infection. his was in contrast to 70 percent of those after cesarean delivery. It must be emphasized that spiking fevers of 39°C or higher that develop within the first 24 hours postpartum may be associated with virulent pelvic infection caused by group A streptococcus, discussed on page 667.

1	Other causes of puerperal fever include breast engorgement; infections of the urinary tract, of perineal lacerations, and of episiotomy or abdominal incisions; and respiratory complications ater cesarean delivery (Nlaharaj, 2007). Approximately 15 percent of women who do not breastfeed develop postpartum fever from breast engorgement. As discussed in Chapter 36 (p. 659), the incidence of fever is lower in breastfeeding women. "Breast fever" rarely exceeds 39°C in the irst few postpartum days and usually lasts <24 hours. Urinary inections are uncommon post partum because of the normal diuresis encountered then. Acute pyelonephritis has a variable clinical picture. The irst sign of renal infection may be fever, followed later by costovertebral angle ten derness, nausea, and vomiting. Atelectasis following abdominal delivery is caused by hypoventilation and is best prevented by gery. Fever associated with atelectasis is thought to stem from normal flora that proliferate distal to

1	Atelectasis following abdominal delivery is caused by hypoventilation and is best prevented by gery. Fever associated with atelectasis is thought to stem from normal flora that proliferate distal to obstructing mucus plugs.

1	Postpartum uterine infection or puerperal sepsis has been called variously endometritis, endomyometritis, and endoparametritis. Because infection involves not only the decidua but also the myometrium and parametrial tissues, we prefer the inclusive term metritis with pelvic celulitis. The route of delivery is the single most signiicant risk factor for the development of uterine infection (Burrows, 2004; Koroukian, 2004). In the French Confidential Enquiry on Maternal Deaths, Deneux-haraux and coworkers (2006) cited a nearly 25-fold increased infection-related mortality rate with cesarean versus vaginal delivery. Rehospitalization rates for wound complications and metritis were increased significantly in women undergoing a planned primary cesarean delivery compared with those having a planned vaginal birth (Declercq, 2007).

1	Women delivered vaginally at Parkland Hospital have a Ito 2-percent incidence of metritis. For women at high risk for infection because of membrane rupture, prolonged labor, and multiple cervical examinations, the frequency of metritis after vaginal delivery is 5 to 6 percent. If intrapartum chorioamnionitis is present, the risk of persistent uterine infection increases to 13 percent (Maberry, 1991). These igures are similar to those reported from a cohort of more than 11r5,000 women by the Maternal Fetal Medicine Units Network in whom the overall pelvic infection rate approximated 5 percent (Grobman, 2015).

1	Because of the significant morbidity following hysterotomy, single-dose perioperative antimicrobial prophylxis is recommended for all women undergoing cesarean delivery (American College of Obstetricians and Gynecologists, 2016b). Antimicrobial prophylaxis has done more to decrease the incidence and severity of postcesarean delivery infections than any other intervention in the past 30 years. Such practices decrease the puerperal pelvic infection risk by 65 to 75 percent (Smaill, 2010).

1	he magnitude of the risk is exemplified from earlier reports that predate antimicrobial prophylaxis. Cunningham and associates (1978) described an overall incidence of 50 percent in all women undergoing cesarean delivery at Parland Hospital. Important risk factors for infection following surgery included prolonged labor, membrane rupture, multiple cervical examinations, and internal fetal monitoring. Women with all these factors who were not given perioperative prophylaxis had a 90-percent serious postcesarean delivery pelvic infection rate (DePalma, 1982). It is generally accepted that pelvic infection is more frequent in women of lower socioeconomic status (Maharaj, 2007). Except in extreme cases usually not seen in this country, it is likely uncommon that anemia or poor nutrition predispose to infection. Bacterial colonization of the lower genital tract with certain microorganisms-for example, group B streptococcus,

1	Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma ureayticum, and Gardnerela vaginalis-has been associated with an increased postpartum infection risk (Andrews, 1995; Jacobsson, 2002; Watts, 1990). Other factors associated with an increased infection risk include general anesthesia, cesarean delivery for multifetal gestation, young maternal age and nulliparity, prolonged labor induction, obesity, and meconium-stained amnionic fluid (Acosta, 2012; Leth, 2011; Siriwachirachai, 2014; Tsai,r2011).

1	Most female pelvic infections are caused by bacteria indigenous to the genital tract. Over the past 25 years, there have been reports of group A 3-hemolytic streptococcus causing toxic shock-like syndrome and life-threatening infection (Castagnola, 2008; Nathan, 1994). Prematurely ruptured membranes are a prominent risk factor in these infections (Anteby, 1999). In reviews by Crum (2002) and Udagawa (1999) and their colleagues, women in whom group A streptococcal infection was manifested before, during, or within 12 hours of delivery had a maternal mortality rate of almost 90 percent and fetal mortality rater> 50 percent. In the past 10 years, skin and sot-tissue infections due to community-acquired methicillin-resistant Staphylococcus aureus (A-MRSAJ have become common (Chap. 64, p. 1223). Although this variant is not a frequent cause of puerperal metritis, it is oten implicated in abdominal incisional infections (Anderson, 2007; Patel, 2007). Rotas and coworkers (2007) reported a

1	Although this variant is not a frequent cause of puerperal metritis, it is oten implicated in abdominal incisional infections (Anderson, 2007; Patel, 2007). Rotas and coworkers (2007) reported a woman with episiotomy cellulitis from CA-MRSA and hematogenously spread necrotizing pneumonia.

1	Common Pathogens. Bacteria responsible for most female genital tract infections are listed in Table Most of these infections are polymicrobial, which enhances bacterial synergy. TABLE 37-1 . Bacteria Commonly Responsible for Female Genital Infections Gram-positive cocci-group A, B, and D streptococci, enterococcus, Staphylococcus aureus, Staphylococcus Gram-negative bacteria-Escherichia coll� Klebsiella, Mycoplasma and Chlamydia, Neisseria gonorrhoeae Cocci-Peptostreptococcus and Peptococcus species Others-Clostridium, Bacteroides, Fusobacterium,

1	Other factors that promote virulence are hematomas and devitalized tissue. Although the cervix and vagina routinely harbor such bacteria, the uterine cavity is usually sterile before rupture of the amnionic sac. As the consequence of labor and delivery and associated manipulations, the amnionic fluid and uterus become contaminated with anaerobic and aerobic bacteria. Intraamnionic cytokines and C-reactive protein are also markers of infection (Combs, 2013; Marchocki, 2013). In studies done before the use of antimicrobial prophylaxis, Gilstrap and Cunningham (1979) cultured amnionic fluid obtained at cesarean delivery in women in labor with membranes ruptured more than 6 hours. All had bacterial growth, and on average, each specimen contained 2.5 organisms. Anaerobic and aerobic organisms were identified in 63 percent, anaerobes alone in 30 percent, and aerobes alone in only 7 percent. Anaerobes included Peptostreptococcus and Peptococcus species in 45 percent, Bacteroides species in 9

1	identified in 63 percent, anaerobes alone in 30 percent, and aerobes alone in only 7 percent. Anaerobes included Peptostreptococcus and Peptococcus species in 45 percent, Bacteroides species in 9 percent, and Clostridium species in 3 percent. Aerobes included Enterococcus in 14 percent, group B streptococcus in 8 percent, and Escherichia coli in 9 percent of isolates. Sherman and coworkers (1999) later showed that bacterial isolates at cesarean delivery correlated with those taken from women with metritis at 3 days postpartum. Group B streptococci, E coli, and enterococci are some of the more common blood culture isolates with metritis (Cape, 2013; O'Higgins, 2014). Although important because of the severity of infections they cause, clostridial species rarely cause puerperal infections (Chong, 2016).

1	he role of other organisms in the etiology of these infections is unclear. Observations of Chaim and colleagues (2003) suggest that when cervical colonization of U ureayticum is heavy, it may contribute to the development of metritis. To add evidence to these observations, Tita and associates (2016) recently reported that azithromycin-based extended-spectrum antimicrobial prophylaxis reduced postoperative cesarean delivery infections from 12 to 6 percent compared with j-Iactam agents given alone. Chlamydial infections have been implicated in late-onset, indolent metritis (Ismail, 1985). Finally, Jacobsson and associates (2002) reported a threefold risk of puerperal infection in a group of Swedish women in whom bacterial vaginosis was identified in early pregnancy (Chap. 65, p. 1245).

1	Bacterial Cultures. Routine genital tract cultures obtained before treatment serve little clinical use and add significant costs. Similarly, routine blood cultures seldom modiy care. In two earlier studies done before perioperative prophylaxis was used, blood cultures were positive in 13 percent of women with postcesarean metritis at Parkland Hospital and 24 percent in those at Los Angeles County Hospital (Cunningham, 1978; DiZerega, 1979). In a later Finnish study, Kankuri and associates (2003) identified bacteremia in only 5 percent of almost 800 women with puerperal sepsis. Blood cultures might be reasonable in women with exceedingly high temperature spikes that may signiy virulent infection with group A streptococci.

1	Puerperal infection following vaginal delivery primarily involves the placental implantation site, decidua and adjacent myometrium, or cervicovaginal lacerations. The pathogenesis of uterine infection following cesarean delivery is that of an infected surgical incision. Bacteria that colonize the cervix and vagina gain access to amnionic fluid during labor. Postpartum, they invade devitalized uterine tissue. Parametrial cellulitis next follows with infection of the pelvic retroperitoneal fibroareolar connective tissue. With early treatment, infection is contained within the parametrial and paravaginal tissue, but it may extend deeply into the pelvis.

1	Fever is the most important criterion or the diagnosis of postpartum metritis. Intuitively, the degree of fever is believed proportional to the extent of infection and sepsis syndrome. Temperatures commonly are 38 to 39°C. Chills that accompany fever suggest bacteremia or endotoxemia. Women usually complain of abdominal pain, and parametrial tenderness is elicited on abdominal and bimanual examination. Leukocytosis may range from 15,000 to 30,000 cells/�L, but recall that delivery itself increases the leukocyte count (Hartmann, 2000). Although an ofensive odor may develop, many women have foul-smelling lochia without evidence for infection, and vice versa. Some other infections, notably those caused by group A j-hemolytic streptococci, may be associated with scant, odorless lochia (Anderson, 2014).

1	If nonsevere metritis develops following vaginal delivery, then treatment with an oral or intramuscular antimicrobial agent may be suicient (Meaney-Delman, 2015). For moderate to severe infections, however, intravenous therapy with a broad-spectrum antimicrobial regimen is indicated. Improvement follows in 48 to 72 hours in nearly 90 percent of women treated with one of several regimens discussed below. Persistent fever after this interval mandates a careful search for causes of refractory pelvic infection. hese include a parametrial phlegmon-an area of intense cellulitis; an abdominal incisional or pelvic abscess or infected hematoma; and septic pelvic thrombophlebitis. In our experience, persistent fever is seldom due to antimicrobial-resistant bacteria or due to drug side efects. he woman may be discharged home after she has been afebrile for at least 24 hours, and further oral antimicrobial therapy is not needed (French, 2004; Nlackeen, 2015).

1	Choice of Antimicrobials. Although therapy is empirical, ini tial treatment following cesarean delivery is directed against elements of the mixed lora shown in Table 37-1. For infections following vaginal delivery, as many as 90 percent of women respond to regimens such as ampicillin plus gentamicin. In contrast, anaerobic coverage is included for infections following cesarean delivery (Table 37-2). In 1979, DiZerega and colleagues compared the efectiveness of clindamycin plus gentamicin with that of penicillin G plus gentamicin for treatment of pelvic infections following cesarean delivery. Women given the clindamycin-gentamicin regimen had a 95-percent response rate, and this regimen is still considered by most to be the standard by which others are measured (F rench, 2004; Mackeen, 2015). Because enterococcal cultures may be persistently positive despite this standard TABLE 37-2. Antimicrobial Regimens for Pelvic Infections Following Cesarean Delivery

1	TABLE 37-2. Antimicrobial Regimens for Pelvic Infections Following Cesarean Delivery Clindamycin + gentamicin "Gold standard," 90-97% eficacy, once-daily gentamicin dosing acceptable Ampicillin added to regimen with sepsis syndrome or suspected enterococcal infection Clindamycin + aztreonam Gentamicin substitute for renal insuficiency Extended-spectrum penicillins Piperacillin, piperacillin tazobactam, ampicillin/sulbactam, ticarcillin/clavulanate Cephalosporins Cefotetan, cefoxitin, cefotaxime Vancomycin Added to other regimens for suspected Staphylococcus aureus infections Metronidazole + Metronidazole has excellent anaerobic coverage ampicillin + gentamicin Carbapenems Imipenemlcilastatin, meropenem, ertapenem .eserved for special indications therapy, some add ampicillin to the clindamycin-gentamicin regimen, either initially or if there is no response by 48 to 72 hours (Brumfield, 2000).

1	Many authorities recommend that serum gentamicin levels be periodically monitored. At Parkland Hospital, we do not routinely do so if the woman has normal renal function. Oncedaily dosing and multiple-dosing with gentamicin both provide adequate serum levels, and either method has similar cure rates (Livingston, 2003). Because of potential nephrotoxicity and ototoxicity with gentamicin in the event of diminished glomerular iltration, some have recommended a combination of clindamycin and a second-generation cephalosporin to treat such women. Others recommend a combination of clindamycin and aztreonam, which is a monobactam compound with activity similar to the aminoglycosides.

1	The spectra of 3-lactam antimicrobials include activity against many anaerobic pathogens. Some examples include cephalosporins such as cefoxitin, cefotetan, cefotaxime, and ceftriaxone, as well as extended-spectrum penicillins such as piperacillin, ticarcillin, and mezlocillin. 3-Lactam antimicrobials are inherently safe and, except for allergic reactions, are free of major toxicity. The 3-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam have been combined with ampicillin, amoxicillin, ticarcillin, and piperacillin to extend their spectra. Metronidazole has superior in vitro activity against most anaerobes. This agent given with ampicillin and an aminoglycoside provides coverage against most organisms encountered in serious pelvic infections. Metronidazole is also used to treat Clostridium dficile colitis.

1	lmpenem and similar antimicrobials are in the carbapenem family. hese ofer broad-spectrum coverage against most organisms associated with metritis. Imipenem is used in combination with cilastatin, which inhibits its renal metabolism. Preliminary indings with ertapenem indicated suboptimal outcomes (Brown, 2012). It seems reasonable from both a medical and an economic standpoint to reserve these drugs for serious nonobstetrical infections. Vancomycin is a glycopeptide antimicrobial active against gram-positive bacteria. It is used in lieu of 3-lactam therapy for a patient with a type 1 allergic reaction and given for suspected infections due to Staphylococcus aureus and to treat C diicile colitis (Chap. 54, p. 1048).

1	The use of periprocedural antimicrobial prophylaxis is com mon in obstetrics. Even so, no rigorous studies have evalu delivery or manual removal of the placenta (Chongsomchai, 2014; Liabsuetrakul, 2017). But, as discussed, antimicrobial prophylaxis at the time of cesarean delivery has remarkably reduced the postoperative pelvic and wound infection rates. bials reduce the pelvic infection rate by 70 to 80 percent (Chelmow, 2001; Dinsmoor, 2009; Smaill, 2014). he observed benefit applies to both elective and nonelective incision infection rates.

1	bials reduce the pelvic infection rate by 70 to 80 percent (Chelmow, 2001; Dinsmoor, 2009; Smaill, 2014). he observed benefit applies to both elective and nonelective incision infection rates. Single-dose prophylaxis with a 2-g dose of ampicillin or a first-generation cephalosporin is ideal. Both equal the eicacy of broad-spectrum agents or multiple-dose regimens (American College of Obstetricians and Gynecologists, 2016b). For obese women, evidence supports a 3-g dose of cefazolin to reach optimal tissue concentrations (Swank, 2015). Extendedspectrum prophylaxis with azithromycin added to standard single-dose prophylaxis further reduced postcesarean metritis rates (Sutton, 2015; Ward, 2016). As noted earlier, Tita and colleagues (2016) reported that postoperative uterine infection was decreased from 12 to 6 percent with the addition of azithromycin to cefazolin. Women known to be colonized with MRSA are given vancomycin in addition to a cephalosporin (Chap. 64, p. 1223).

1	It is controversial whether the infection rate is lowered further if the antimicrobial is given before the skin incision compared with after umbilical cord clamping (Baaqeel, 2013; Macones, 2012; Sun, 2013). The American College of Obstetricians and Gynecologists (2016b) has concluded that the evidence favors predelivery administration. Abdominal preoperative skin preparation with chlorhexidine-alcohol is superior to iodine-alcohol for preventing surgical-site infections (Tuuli, 2016). Additive salutary efects may be gained by preoperative vaginal cleansing with povidone-iodine rinse or application of metronidazole gel (Haas, 2014; Reid, 2011; Yildirim, 2012).

1	Other Methods of Prophylaxis. Several studies have addressed the value of prenatal cervicovaginal cultures. These are obtained in the hope of identiying pathogens that might be eradicated to decrease incidences of preterm labor, chorioamnionitis, and puerperal infections. Unfortunately, treatment of asymptomatic vaginal infections has not been shown to prevent these complications. Carey and coworkers (2000) reported no beneficial efects for women treated for asymptomatic bacterial vaginosis. Klebanof and colleagues (2001) reported a similar postpartum infection rate in women treated for second-trimester asymptomatic Trichomonas vaginalis infection compared with that of placebo-treated women.

1	Technical maneuvers done to alter the postpartum infection rate have been studied with cesarean delivery. For example, allowing the placenta to separate spontaneously compared with removing it manually lowers the infection risk. However, changing gloves by the surgical team after placental delivery does not (Atkinson, 1996). Exteriorizing the uterus to close the hysterotomy may decrease febrile morbidity (Jacobs-Jokhan, 2004). Postdelivery mechanical lower segment and cervical dilatation has not been shown to be efective (Liabsuetrakul, 2011). No diferences were found in postoperative infection rates when single-and two-layer uterine closures were compared (Hauth, 1992). Similarly, infection rates are not appreciatively afected by closure versus nonclosure of the peritoneum (Bamigboye, 2014; Tulandi, 2003). Importantly, although closure of subcutaneous tissue in obese women does not lower the wound infection rate, it does decrease the wound separation incidence (Chelmow, 2004).

1	2014; Tulandi, 2003). Importantly, although closure of subcutaneous tissue in obese women does not lower the wound infection rate, it does decrease the wound separation incidence (Chelmow, 2004). Similarly, skin closure with staples versus suture has a higher incidence of noninfectious skin separation (Mackeen, 2012; Tuuli, 2011).

1	• Complications of Uterine and Pelvic Infections In more than 90 percent of women, metritis responds to antimicrobial treatment within 48 to 72 hours. In some of the remainder, any of several complications may arise. These include wound infections, complex pelvic infections such as phlegmons or abscesses, and septic pelvic thrombophlebitis (Jaiyeoba, 2012). As with other aspects of puerperal infections, the incidence and severity of these complications are remarkably decreased by perioperative antimicrobial prophylaxis.

1	Wound infection is a common cause of persistent fever in women treated for metritis. Incisional infection risk factors include obesity, diabetes, corticosteroid therapy, immunosuppression, anemia, hypertension, and inadequate hemostasis with hematoma formation. If prophylactic antimicrobials are given, the incidence of abdominal wound infection following cesarean delivery ranges from 2 to 10 percent depending on risk factors (Andrews, 2003; Chaim, 2000). From our experiences at Parkland Hospital, the incidence is closer to 2 percent. Incisional abscesses that develop following cesarean delivery usually cause persistent fever or fever that begins on about the fourth day. In many cases, antimicrobials had been given to treat pelvic infection, yet fever persisted. The wound is FIGURE 37-1 Secondary abdominal wound closure technique. (Reproduced with permission from Worley KC: Postoperative complications. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds):

1	FIGURE 37-1 Secondary abdominal wound closure technique. (Reproduced with permission from Worley KC: Postoperative complications. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw Hill Education, 201o7.) erythematous and drains pus. Although organisms that cause wound infections are generally the same as those isolated from amnionic luid at cesarean delivery, hospital-acquired pathogens may also be causative (Owen, 1994).

1	Treatment includes antimicrobials and surgical drainage and debridement of devitalized tissue. his typically requires spinal analgesia or general anesthesia. The fascia is carefully inspected to document integrity. Local wound care thereafter is completed twice daily. Before each dressing change, procedural analgesia is tailored to wound size and location, and oral, intramuscular, or intravenous dosage routes are suitable. Topical lidocaine may also be added. Necrotic tissue is removed, and the wound is repacked with moist gauze. At 4 to 6 days, healthy granulation tissue is typically present, and secondary en bloc closure of the open layers can usually be accomplished (Wechter, 2005). As shown in Figure 37-1, a polypropylene or nylon suture of appropriate gauge enters 2 to 3 cm from one wound edge. It crosses the wound to incorporate the full wound thickness and emerges 3 cm from the other wound edge. hese are placed in series to close the opening. In most cases, sutures may be

1	one wound edge. It crosses the wound to incorporate the full wound thickness and emerges 3 cm from the other wound edge. hese are placed in series to close the opening. In most cases, sutures may be removed on postprocedural day 10.

1	his system was designed to apply negative pressure to a foamwound interface that would promote wound healing. The technique is variably referred to as vacuum-assisted closure-VAC; topical negative pressure-TNP; and negative-pressure wound therapy-NPWT. Several systems are available and widely (Echebiri, 2015; Rouse, 2015; Swift, 2015). In obstetrics, disrupted and infected abdominal wounds are a major indica tion for vacuum-assisted closure. Closure of perineal wounds resulting from infected episiotomies, hematomas, or abscesses is another (Aviki, 2015). hese devices are also used for the "open surgical abdomen," which is occasionally encountered in obstetrics. Negative-pressure wound therapy has also been used to prevent wound infections in those closed to heal by primary intention.

1	wound closure with conventional wound care (Semsarzadeh, 2015). Likewise, its cost efectiveness has not been thoroughly studied, although provider time is decreased substantially (Lewis, 2014). From their review, Moues and colleagues (201l) wounds because of scarce data. Other reviewers conclude that vacuum therapy is the most eicient method of temporary abdominal closure for patients with open abdominal wounds (Bruhin, 2014; Quyn, 2012).

1	Wound disruption or dehiscence refers to separation of the fascial layer. This is a serious complication and requires secondary closure of the incision in the operating room. McN eeley and associates (1998) reported a fascial dehiscence rate of approximately 1 per 300 operations in almost 9000 women undergoing cesarean delivery. Other than wound infection, obesity may be a risk factor (Subramaniam, 2014). Most disruptions manifested on about the fifth postoperative day and were accompanied by a serosanguinous discharge. Two thirds of 27 fascial dehiscences identified in this study were associated with concurrent fascial infection and tissue necrosis.

1	This uncommon, severe wound infection is associated with high mortality rates. In obstetrics, necrotizing fasciitis may involve abdominal incisions, or it may complicate episiotomy or other perineal lacerations. As the name implies, tissue necrosis is significant. Of the risk factors for fasciitis summarized by Owen and Andrews (1994), three of these-diabetes, obesity, and hypertension-are relatively common in gravidas. Like pelvic these wound complications usually are polymicrobial and are caused by organisms that make up the normal vaginal lora. In some cases, however, infection is caused by a single virulent bacterial species such as group A O-hemolytic streptococcus (Anderson, 2014; Rimawi, 2012). Occasionally, necrotizing infections are caused by rarely encountered pathogens (Chong, 2016; Swartz, 2004).

1	Goepfert and coworkers (1997) reviewed their experiences with necrotizing fasciitis. Nine cases complicated more than 5000 cesarean deliveries, a frequency of 1.8 per 1000. In two women, the infection was fatal. In another report, Schorge and colleagues (1998) described ive women with fasciitis following cesarean delivery. None of these women had predisposing risk factors, and none died. FIGURE 37-2 Necrotizing fasciitis involving the abdominal wall and Pfannenstiel incision. The skin rapidly became dusky and gangrenous, and pus is seen exuding from the left angle of the incision. Extensive debridement and supportive therapy were lifesaving. Infection may involve skin, supericial and deep subcu taneous tissues, and any of the abdominopelvic fascial layers (Fig. 37-2). In some cases, muscle is also involved-myoosciitis.

1	Infection may involve skin, supericial and deep subcu taneous tissues, and any of the abdominopelvic fascial layers (Fig. 37-2). In some cases, muscle is also involved-myoosciitis. Although some virulent infections-for example, those caused tum, most of these necrotizing infections do not cause symp toms until 3 to 5 days after delivery. Clinical findings vary, and it is frequently diicult to diferentiate more innocuous superi cial wound infections from an ominous deep fascial one. A high index of suspicion, with surgical exploration if the diagnosis is uncertain, may be lifesaving (Goh, 2014). We aggressively pursue early exploration. Certainly, if myofasciitis progresses, the woman may become ill from septicemia (Chap. 47, p. 921).

1	Early diagnosis, surgical debridement, antimicrobials, and intensive care are paramount to successfully treat necrotizing soft-tissue infections (Gallup, 2002; Goh, 2014). Surgery includes extensive debridement of all infected tissue, leaving wide margins of healthy bleeding tissue. This may include extensive abdominal or vulvar debridement with unrooing and excision of abdominal, thigh, or buttock fascia. Death is virtually universal without surgical treatment, and rates approach 25 percent even if extensive debridement is performed. With extensive resection, synthetic mesh may ultimately be required later to close the fascial incision (Gallup, 2002; McNeeley, 1998). An ovarian abscess rarely develops in the puerperium. These are presumably caused by bacterial invasion through a rent in the ovarian capsule (Wetchler, 1985). The abscess is usually unilateral, and women typically present 1 to 2 weeks after delivery. Rupture is common, and peritonitis may be severe.

1	Peritonitis is infrequent following cesarean delivery. It almost invariably is preceded by metritis, especially cases with uterine incisional necrosis and dehiscence. However, it may stem from a ruptured adnexal abscess or an inadvertent intraoperative bowel injury.

1	Peritonitis is rarely encountered ater vaginal delivery, and many such cases are due to virulent strains of group A 3-hemolytic streptococci or similar organisms. Importantly in postpartum women, abdominal rigidity may not be prominent with puerperal peritonitis because of physiological abdominal wall laxity from pregnancy. Pain may be severe, but frequently, the irst symptoms of peritonitis are those of adynamic ileus. Marked bowel distention may develop, which is unusual ater uncomplicated cesarean delivery. If the infection begins in an intact uterus and extends into the peritoneum, antimicrobial treatment alone usually sufices. Conversely, peritonitis caused by uterine incisional necrosis as discussed subsequently, or from bowel perforation, must be treated promptly with surgical intervention .

1	For some women in whom metritis develops following cesarean delivery, parametrial cellulitis is intensive and forms an area of induration-a phlemon-within the leaves of the broad ligament (Fig. 37-3). hese infections are considered when fever persists longer than 72 hours despite intravenous antimicrobial therapy (Brown, 1999; DePalma, 1982). Phlegmons are usually unilateral, and they frequently are limited to the parametrium at the base of the broad ligament. FIGURE 37-3 Left-sided parametrial phlegmon: cellulitis causes induration in the parametrium adjacent to the hysterotomy incision. (Reproduced with permission from Worley KC: Postoperative complications. In Yeomans ER, Hofman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw Hill Education, 201o7.)

1	FIGURE 37-4 Pelvic computed tomography scan showing necrosis of the uterine incision with gas in the myometrium (arrows). There is also a large right-sided parametrial abscess (a). If the inflammatory reaction is more intense, cellulitis extends along natural lines of cleavage. he most common form of extension is laterally along the broad ligament, with a tendency to extend to the pelvic sidewall. Occasionally, posterior extension may involve the rectovaginal septum, producing a firm mass posterior to the cervix. In most women with a phlegmon, clinical improvement follows continued treatment with a broad-spectrum antimicrobial regimen. Typically, fever resolves in 5 to 7 days, but in some cases, it persists longer. Absorption of the induration may require several days to weeks.

1	In some women, severe cellulitis of the uterine incision may ultimately lead to necrosis and separation (T reszezamsky, 201r1). Extrusion of purulent material as shown in Figure 37-4 causes intraabdominal abscess formation and peritonitis as described above. Surgery is reserved for women in whom uterine incisional necrosis is suspected because of ileus and peritonitis. For most, hysterectomy and surgical debridement are needed and are predictably diicult because the cervix and lower uterine segment are involved with an intense inflammatory process that extends to the pelvic sidewall. he adnexa are seldom involved, and one or both ovaries can usually be conserved. Blood loss is often appreciable, and transfusion is usually necessary.

1	Persistent puerperal infections can be evaluated using computed tomography (CT) or magnetic resonance (MR) imaging. Brown and associates (1991) used CT imaging in74 women in whom pelvic infection was refractory to antimicrobial therapy given for 5 days. hey found at least one abnormal radiological inding in 75 percent of these women, and in most, these were nonsurgical lesions. In most cases, imaging can be used to dissuade surgical exploration. Uterine incisional dehiscence such as shown in Figure 37-4 can sometimes be confirmed based on CT scanning images. hese indings must be interpreted within the clinical context because apparent uterine incisional defects thought to represent edema can be seen even after uncomplicated cesarean delivery (Twickier, 1991). Shown in 37-5 is a necrotic hysterotomy incision that leaked into the peritoneal cavity.

1	FIGURE 37-5 Necrotic hysterotomy infection. Severe cellulitis of the uterine incision resulted in dehiscence with subsequent leakage into the peritoneal cavity. Hysterectomy was required for sufficient debridement of necrotic tissue. Occasionally, a parametrial phlegmon may suppurate, forming a fluctuant broad ligament mass that may point above the inguinal ligament. hese abscesses can dissect anteriorly as shown in Figure 37-4 and be amenable to CT-directed needle drainage. Occasionally they dissect posteriorly to the rectovaginal septum, where surgical drainage is easily efected by colpotomy. Apsoas abscess is rare, and despite antimicrobial therapy, percutaneous drainage may be required to efectively treat it (Shahabi, 2002; Swanson, 2008).

1	Suppurative thrombophlebitis was a frequent complication in the preantibiotic era, and septic embolization was common. However, with the advent of antimicrobial therapy, the mortality rate and need for surgical therapy for these infections diminished. Septic phlebitis arises as an extension along venous routes and may cause thrombosis as shown in Figure 37-6. Lymphangitis often coexists. he ovarian veins may then become involved because they drain the upper uterus and therefore the placental implantation site. The experiences of Witlin and Sibai (1995) and Brown and coworkers (1999) suggest that puerperal septic thrombophlebitis is likely to involve one or both ovarian venous plexuses. In a fourth of women, the clot extends into the inferior vena cava and occasionally to the renal vein. The incidence of septic phlebitis has varied in several reports.

1	The incidence of septic phlebitis has varied in several reports. In a 5-year survey of 45,000 women who were delivered at Parkland Hospital, Brown and associates (1999) found an incidence of septic pelvic thrombophlebitis in 1 per 9000 gravidas following vaginal delivery and 1 per 800 after cesarean delivery. he overall incidence of 1 per 3000 deliveries was similar to the 1 per 2000 reported by Dunnihoo and colleagues (1991). In large studies of women with cesarean delivery, the incidence was 1 in 400 to 1 in 1000 surgeries (Dotters-Katz, 2017; Rouse 2004). Chorioamnionitis, endometritis, and wound complications were other risks. Women with septic thrombophlebitis usually have symptomatic improvement with antimicrobial Inferior vena cava treatment, however, they continue to have fever. Although pain occasionally is noted in one or both lower quadrants, patients are usually asymptomatic except for chills. As shown in

1	Common iliac vein Figure 37-7, the diagnosis can be con firmed by pelvic CT or MR imaging (Klima, 2008). Using either, Brown and colleagues (1r999) found that 20 percent of 69 women with metritis who had fever despite >5 days of appropriate antimicrobial therapy Internal iliac vein had septic pelvic thrombophlebitis.

1	It has been disproven that intravenous heparin causes fever to dissipate with septic phlebitis (Brown, 1986; Witlin, 1995). And although Garcia and coworkers (2006) and Klima and Snyder (2008) advocate heparin therapy, we do not recommend anticoagulation. In a randomized study of (1999), the addition of heparin to to any pelvic vessel as well as the inferior vena cava. The clot in the right common iliac vein extends from the uterine and internal iliac veins and into the inferior vena cava. The ovarian antimicrobial therapy for septic pelvein septic thrombosis extends halfway to the vena cava. vic thrombophlebitis did not hasten FIGURE 37-7 Septic ovarian vein thrombosis-contrast-enhanced computed tomography scan. A.Enlarged right ovarian vein filled with low-density thrombus (black arrow). Contrast is seen in ureter (white arrow). R = lower pole, right kidney. B. Coronal image demonstrates enlarged right ovarian vein filled with low-density thrombus (arrows). (Reproduced with

1	arrow). Contrast is seen in ureter (white arrow). R = lower pole, right kidney. B. Coronal image demonstrates enlarged right ovarian vein filled with low-density thrombus (arrows). (Reproduced with permission from Worley KC: Postoperative complications. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds):

1	Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw Hill Education, 201o7.) recovery or improve outcome. Certainly, no evidence supports long-term anticoagulation. Episiotomy infections are not common because the operation is performed much less frequently now than in the past (American College of Obstetricians and Gynecologists, 20 16a). Reasons for this are discussed further in Chapter 27 (p. 530). In an older study, Owen and Hauth (1990) described only 10 episiotomy infections in 20,000 women delivered vaginally. With infection, however, dehiscence is a concern. Ramin and colleagues (1992) reported an episiotomy dehiscence rate of 0.5 percent at Parkland Hospital-80 percent of these were infected. Uygur and associates (2004) reported a I-percent dehiscence rate and attributed two thirds to infection. No data suggest that dehiscence is related to faulty repair.

1	When the anal sphincter is disrupted at delivery, the subsequent infection rate is higher and is likely influenced by intrapartum antimicrobial treatment (Buppasiri, 2014; Stock, 2013). Lewicky-Gaupp and colleagues (2015) reported a 20-percent infection rate. Infection of a fourth-degree laceration can be even more serious. Goldaber and coworkers (1993) described fourth-degree lacerations in 390 parturients, of whom 5.4 percent had morbidity. In these women, 2.8 percent had infection and dehiscence, 1.8 percent had only dehiscence, and 0.8 percent only infection. Although life-threatening septic shock is rare, it may still occur as a result of an infected episiotomy. Occasionally also, necrotizing fasciitis develops as discussed on page 671.

1	Episiotomy dehiscence is most commonly associated with infection. Other factors include coagulation disorders, smoking, and human papillomavirus infection (Ramin, 1994). Local pain and dysuria, with or without urinary retention, are frequent symptoms. Ramin and colleagues (1992), evaluating a series of 34 women with episiotomy dehiscence, reported that the most common indings were pain in 65 percent, purulent discharge in 65 percent, and fever in 44 percent. In extreme cases, the entire vulva may become edematous, ulcerated, and covered with exudate.

1	Vaginal lacerations may also become infected directly or by extension from the perineum. he epithelium becomes red and swollen and may then become necrotic and slough. Parametrial extension can lead to lymphangitis. Cervical lacerations are common but seldom are noticeably infected, which may manifest as metritis. Deep lacerations that extend directly into the base of the broad ligament may become infected and cause lymphangitis, parametritis, and bacteremia. Treatment. Infected episiotomies are managed similar to other infected surgical wounds. Drainage is established, and in most cases, sutures are removed and the infected wound debrided. In women with obvious cellulitis but no purulence, close observation and broad-spectrum antimicrobial therapy alone may be appropriate. With dehiscence, local wound care is continued along with intravenous antimicrobials.

1	Early Repair of Infected Episiotomy. Hauth and colleagues (1986) were the irst to advocate early episiotomy repair after infection subsided, and other studies have confirmed the eicacy of this approach. Hankins and coworkers (1990) described early repair in 31 women with an average duration of 6 days from dehiscence to repair. All but two had a successful repair. Each of the two women with failures developed a pinpoint rectovaginal istula that was treated successfully with a small rectal lap. With episiotomy dehiscence due to infection, Ramin and coworkers (1992) reported successful early repair in 32 of TABLE 37-3. Preoperative Protocol for Early Repair of Episiotomy Dehiscence Open wound, remove sutures, begin intravenous or general anesthesia may be necessary for the first Scrub wound twice daily with a povidone-iodine solution

1	Open wound, remove sutures, begin intravenous or general anesthesia may be necessary for the first Scrub wound twice daily with a povidone-iodine solution Closure when afebrile and pink, healthy granulation tissue 34 women (94 percent), and Uygur and colleagues (2004) noted a similarly high percentage. Rarely, intestinal diversion may be required to allow healing (Rose, 2005).

1	Before performing early repair, diligent preparation is essential as outlined in Table 37-3. The surgical wound must be properly cleaned and cleared of infection. Once the surface of the episiotomy wound is free of infection and exudate and covered by pink granulation tissue, secondary repair can be accomplished. The tissue must be adequately mobilized, with special attention to identiy and mobilize the anal sphincter muscle. Secondary closure of the episiotomy is accomplished in layers, as described for primary episiotomy closure (Chap. 27, p. 531). Postoperative care includes local wound care, stool softeners, and nothing per vagina or recrum until healed.

1	This acute febrile illness with severe multisystem derangement has a case-fatality rate of 10 to 15 percent. Fever, headache, mental confusion, difuse macular erythematous rash, subcutaneous edema, nausea, vomiting, watery diarrhea, and marked hemoconcentration are usual findings. Renal failure followed by hepatic failure, disseminated intravascular coagulopathy, and circulatory collapse may follow in rapid sequence. During recovery, the rash-covered areas undergo desquamation. For some time, Staphylococcus aureus was recovered from almost all aHicted persons. Specifically, a staphylococcal exotoxin, termed toxic shock syndrome toxin-l (TSST-l), was found to cause the clinical manifestations by provoking profound endothelial injury. A very small amount ofTSST-1 has been shown to activate T cells to create a "cytokine storm" as described by Que (2005) and Heying (2007) and their coworkers.

1	During the 1990s, sporadic reports of virulent group A 3-hemolytic streptococcal infection began to appear (Anderson, 2014). Heavy colonization or infection is complicated in some cases by streptococcal toxic shock syndrome, which is produced when pyrogenic exotoxin is elaborated. Serotypes MI and M3 are particularly virulent (Beres, 2004; Okumura, 2004). Finally, almost identical indings of toxic shock were reported by Robbie and associates (2000) in women with Clos tridium sordellii colonization. Thus, in some cases of toxic shock syndrome, infection is not apparent and colonization of a mucosal surface is the pre sumed source. At least 10 to 20 percent of pregnant women have vaginal colonization with S aureus. And Clostridium petingens and sordellii are cultured from 3 to 10 percent of asymptomatic women (Chong, 2016). Thus, it is not surprising that the disease develops in postpartum women when growth of vaginal bacteria is luxuriant (Chen, 2006; Guerinot, 1982).

1	Delayed diagnosis and treatment may be associated with maternal mortality (Schummer, 2002). Crum and colleagues shock syndrome. Principal therapy is supportive, while allow ing reversal of capillary endothelial injury. Ant�microbial ther apy that includes staphylococcal and streptococcal coverage is given. With evidence of pelvic infection, antimicrobial therapy must also include agents used for polymicrobial infections. Women with these infections may require extensive wound debridement and possibly hysterectomy. Because the toxin is so potent, the mortality rate is correspondingly high (Hotch kiss, 2003).

1	Parenchymal infection of the mammary glands is a rare antepartum complication but is estimated to develop in up to a third of breast feeding women (Barbosa-Cesnik, 2003). Excluding breast engorgement, in our experiences, as well as that of Lee and associates (2010), the incidence of mastitis is much lower and probably approximates 3 percent. No evidence supports any of several prophylactic measures to prevent breast infection (Crepinsek, 2012). Risk factors include diiculties in nursing, cracked nipples, and oral antibiotic therapy (BranchElliman, 2012; Mediano, 2014). Symptoms of suppurative mastitis seldom appear before the end of the first week postpartum and, as a rule, not until the third or fourth week. Infection almost invariably is unilateral, and marked engorgement usually precedes inlammation. Symptoms include chills or actual rigors, which are soon followed by fever and tachycardia. Pain is severe, and the breast(s) becomes hard and red (Fig. 37-8). Approximately 10 percent

1	inlammation. Symptoms include chills or actual rigors, which are soon followed by fever and tachycardia. Pain is severe, and the breast(s) becomes hard and red (Fig. 37-8). Approximately 10 percent of women with mastitis develop an abscess. Detection of fluctuation may be diicult, and sonography is usually diagnostic.

1	Staphylococcus au reus, especially MRSA, is the most commonly isolated organism in breast infections. Matheson and coworkers (1988) found it in 40 percent of women with mastitis. Other commonly isolated organisms are coagulase-negative staphylococci and viridans streptococci. The immediate source of organisms that cause mastitis is almost always the infant's nose and throat. Bacteria enter the breast through the nipple at fissures or small abrasions. he infecting organism can usually be cultured from milk. Toxic shock syndrome from mastitis caused by S aureus has been reported (Demey, 1989; Fujiwara, 2001). FIGURE 37-8 Puerperal mastitis with breast abscess. A.Photograph shows indurated, erythematous skin overlying area of right breast infection. B. Sonographic picture of this 5-cm abscess. (Used with permission from Dr. Emily Adhikari.)

1	At times, suppurative mastitis reaches epidemic levels among nursing mothers. Such outbreaks most often coincide with the appearance of a new strain of antibiotic-resistant staphylococcus. A contemporaneous example is CA-MRSA, which has rapidly become the most commonly isolated staphylococcal species in some areas (Berens, 2010; Klevens, 2007). At Parkland Hospital from 2000 to 2004, Laibl and associates (2005) reported that a fourth of CA-MRSA isolates were from pregnant or postpartum women with puerperal mastitis. Hospital-acquired MRSA may cause mastitis when the newborn becomes colonized ater contact with nursery personnel who are colonized (Centers for Disease Control and Prevention, 2006). Staford and colleagues (2008) found a higher incidence of recurrent abscess in those with CA-MRSA-associated mastitis.

1	Provided that appropriate therapy for mastItiS is started before suppuration begins, the infection usually resolves within 48 hours. As discussed, abscess formation is more common with Saureus infection (Matheson, 1988). Most recommend that milk be expressed from the afected breast onto a swab and cultured before therapy is begun. Bacterial identification and antimicrobial sensitivities provide information mandatory for a successful program of nosocomial infection surveillance (Lee, 2010).

1	The most efective treatment has not been reported Oahanfar, 2013). hus, the initial antimicrobial choice is influenced by the current experience with staphylococcal infections at a given institution. Dicloxacillin, 500 mg orally four times daily, may be started empirically. Erythromycin is given to women who are penicillin sensitive. If the infection is caused by resistant, penicillinase-producing staphylococci or if resistant organisms are suspected while awaiting the culture results, then vancomycin, clindamycin, or trimethoprim-sulfamethoxazole is given (Sheield, 2013). Although clinical response may be prompt, treatment is recommended for 10 to 14 days.

1	Marshall and coworkers (1975) demonstrated the importance of continued breastfeeding. hey reported that of 65 women with mastitis, the only three who developed abscesses were among the 15 women who quit breastfeeding. Vigorous milk expression may be suicient treatment alone (homsen, 1984). Sometimes the infant will not nurse on the inlamed breast. his probably is not related to any changes in the milk taste but is secondary to engorgement and edema, which can make the areola harder to grip. Pumping can alleviate this. When nursing bilaterally, it is best to begin suckling on the uninvolved breast. This allows let-down to commence before moving to the tender breast.

1	In resource-poor countries, breastfeeding in women infected with the human immunodeficiency virus (HIV) is not contraindicated. In the setting of mastitis or breast abscess, it is recommended to stop feeding from the infected breast. his is because HIV RNA levels increase in afected breast milk. These levels return to baseline after symptoms resolve (Semrau, 2013).

1	In a population-based study of nearly 1.5 million Swedish women, the incidence of breast abscess was 0.1 percent (Kvist, 2005). An abscess should be suspected when defervescence does not follow within 48 to 72 hours of mastitis treatment or when a mass is palpable. Again, sonographic imaging is valuable. Breast abscesses can be large, and in one case report, 2 L of pus were released (Martic, 2012). Traditional therapy is surgical drainage, which usually requires general anesthesia. he incision ideally is placed along Langer skin lines for a cosmetic result (Stehman, 1990). In early cases, a single incision over the most dependent portion of luctuation is usually suicient. Multiple abscesses, however, require several incisions and disruption of loculations. The resulting cavity is loosely packed with gauze, which should be replaced at the end of 24 hours by a smaller pack.

1	A more recently used technique that is less invasive is sonographically guided needle aspiration using local analgesia. his has an 80-to 90-percent success rate (Geiss, 2014; Schwarz, 2001). In a randomized trial, Naeem and colleagues (2012) compared surgical drainage and aspiration. They found aspiration resulted in quicker healing at 8 weeks, 77 versus 93 percent, respectively. Acosta CD, Bhattacharya S, Tufnell D, et al: Maternal sepsis: a Scottish population-based case-control study. BJOG 119(4):474,2012 American College of Obstetricians and Gynecologists: Prevention and management of obstetric lacerations at vaginal delivery. Practice Bulletin No. 165, July 2016a . . American College of Obstetricians and Gynecologists: Use of prophylaCtIc antibiotics in labor and delivery. Practice Bulletin No. 120, June 2011, Reafirmed 20n16b Anderson BL: Puerperal group A streptococcal infection: beyond Semmelweis. Obstet GynecoIn123(4):874, 2014

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1	Sutton AL, Acosta EP, Larson KB, et al: Perinatal pharmacokinetics of azithromycin for cesarean prophylaxis. Amn] Obstet GynecoIn212(6):812.el, 2015 Swank ML, Wing DA, Nicolau DP, et al: Increased 3-gram cefazolin dosing for cesarean delivety prophylaxis in obese women. Am ] Obstet Gynecol 213(3):415.e1,2015 Swanson A, Lau KK, Kornman T, et al: Primary psoas muscle abscess in pregnancy. Aust N Z] Obstet Gynaecol 48(6):607, 2008 Swartz MN: Cellulitis. N Engln] Med 350:904, 2004 Swift SH, Zimmerman MB, Hardy-Fairbanks A]: Efect of Single-use negative pressure wound therapy on postcesarean infections and wound complications for high-risk patients.] Reprod Med 60(5-6):211,n2015 Pue.peral Complications 679 Tita AT, Szychowski ]M, Boggess K, et al: Adjunctive azithromycin prophylaxis for cesarean delivery. N Engl] Med 375(13):1231,n2016

1	Pue.peral Complications 679 Tita AT, Szychowski ]M, Boggess K, et al: Adjunctive azithromycin prophylaxis for cesarean delivery. N Engl] Med 375(13):1231,n2016 Thomsen AC, Espersen T, Maigaard S: Course and treatment of milk stasis, noninfectious inflammation of the breast, and infectious mastitis in nursing women. Am] Obstet GynecoIn149:492, 1984 Treszezamsky AD, Feldman 0, Sarabanchong VO: Concurrent postpartum uterine and abdominal wall dehiscence and Streptococcus anginosus infection. Obstet GynecoIn118(2):449, 2011 Tsai PS, Hsu CS, Fan YC: General anaesthesia is associated with increased risk of surgical site infection after cesarean delivery compared with neuraxial anaesthesia: a population-based study. Br] Anaesth 107(5):757,2011 Tulandi T, Al-]aroudi 0: Nonclosure of peritoneum: a reappraisal. Am ] Obstet Gynecol 189:609,n2003 Tuuli MG, Liu], Stout M], et al: A randomized trial comparing skin antiseptic agents at cesarean delivery. N Engl] Med 4(7):647-55,n2016

1	Tuuli MG, Liu], Stout M], et al: A randomized trial comparing skin antiseptic agents at cesarean delivery. N Engl] Med 4(7):647-55,n2016 Tuuli MG, Rampersad \1, Carbone ]F, et al: Staples compared with subcuticular suture for skin closure after cesarean delivety: a systematic review and meta-analysis. Obstet Gynecol 117(3):682,n2011 Twickler OM, Setiawan AT, Harrell RS, et al: CT appearance of the pelvis after cesarean section. A]R Am ] Roentgenol 156:523, 1991 Udagawa H, Oshio Y, Shimizu Y: Serious group A streptococcal infection around delivery. Obstet Gynecol 94: 153, 1999 Uygur 0, Yesildaglar N, Kis S, et al: Early repair of episiotomy dehiscence. Aust N Z] Obstet Gynaecol 44:244, 2004 Ward E, Duf P: A comparison of 3 antibiotic regimens for prevention of postcesarean endometritis: an historical cohort study. Amn] Obstet Gynecol 214(6):751.e1,n2016

1	Ward E, Duf P: A comparison of 3 antibiotic regimens for prevention of postcesarean endometritis: an historical cohort study. Amn] Obstet Gynecol 214(6):751.e1,n2016 Watts DH, Krohn A, Hillier SL, et al: Bacterial vaginosis as a risk factor for post-cesarean endometritis. Obstet Gynecol 75:52, 1990 Wechter ME, Pearlman MD, Hartmann KE: Reclosure of the disrupted laparotomy wound. A systematic review. Obstet Gynecol 106:376,n2005 Wetchler S], Dunn L]: Ovarian abscess. Report of a case and a review of the literature. Obstet Gynecol Surv 40:476, 1985 Widin AG, Sibai BM: Postpartum ovarian vein thrombosis ater vaginal delivery: a report of 11 cases. Obstet Gynecol 85:775, 1995 Worley KC: Postoperative complications. In Yeomans ER, Hofman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hill Education, 2017

1	Worley KC: Postoperative complications. In Yeomans ER, Hofman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hill Education, 2017 Yildirim G, Gungorduk K, Asicioglu 0, et al: Does vaginal preparation with povidone-iodine prior to cesarean delivery reduce the risk of endometritis? A randomized controlled trial. ] Maten Fetal Neonatal Med 25(11):2316, 2012 INTRAUTERINE DEVICES ..n........................ 681 PROGESTIN IMPLANTS .n..............n............. 685 PROGESTIN-ONLY CONTRACEPTIVES .............n. 689 HORMONAL CONTRACEPTIVES.. . . . . . . . . . . . . . . . . . . 689 BARRIER METHODS ............................. 693 FERTILITY AWARENESS-BASED METHODS .......... 695 SPERMICIDES ....n........n...........n.........n....... 695 EMERGENCY CONTRACEPTION . . . . . . . . . . . . . . . . . . . 696 PUERPERAL CONTRACEPTION.. . . . . . . . . . . . . . . . . . . . 697

1	SPERMICIDES ....n........n...........n.........n....... 695 EMERGENCY CONTRACEPTION . . . . . . . . . . . . . . . . . . . 696 PUERPERAL CONTRACEPTION.. . . . . . . . . . . . . . . . . . . . 697 From the evidence available, it appears to be toleraby satiactoriy demonstrated that in women who copulate at requent intervals the tube must be regarded as a species of receptaculum seminis, in which spermatozoa are always present and waiting or the ovum, and that ertilization usualy occurs in the tubes and ony rarey in the uterus. -J. Whitridge Williams (1903) Nearly half of all pregnancies each year in the United States are unintended (Finer, 2016). hese may follow contraceptive method failure or stem from lack of contraceptive use. In 2011 to 2013, 7 percent of sexually active fertile women in the United States not pursuing pregnancy did not use any birth control method (Daniels, 2015).

1	TABLE 38-1. Contraceptive Failure Rates During the First Year of Method Use in Women in the United States Top Tier: Most Effective Intrauterine devices: Levonorgestrel system 0.2 0.2 T 380A copper 0.6 0.8 Levonorgestrel implants 0.05 0.05 Female sterilization 0.5 0.5 Male sterilization 0.1 0.1n5 Second Tier: Very Effective DMPA 0.2 6 Third Tier: Effective Diaphragm with spermicides 6 12 Fertility-awareness 24 Sym ptotherma I 0.4 Fourth Tier: Least Effective For those seeking contraception, efective contraceptive No contraception methods are available and variably selected (Table 38-1). With aMethods organized according to tiers of efficacy. these methods, estimated failure rates of perfect and typical use during the irst year difer widely. To reflect these failure WHO = World Health Organization. rates, the World Health Organization (WHO) has grouped Data from Trussell, 201n1 a.

1	WHO = World Health Organization. rates, the World Health Organization (WHO) has grouped Data from Trussell, 201n1 a. methods into eicacy tiers (see Table 38-1). Implants and intrauterine devices are found in the top tier. They efectively lower unintended pregnancy rates and are considered long-act ing reversible contraception (ARC). he American College tiers, recommends counseling on al options, and encourages highly efective LARC for appropriate candidates. No contraceptive method is completely without side efects, but contraception usually poses less risk than pregnancy.

1	No contraceptive method is completely without side efects, but contraception usually poses less risk than pregnancy. However, some disorders or medications can raise the risks from certain contraceptives. The World Health Organization (2015) has provided and updated evidence-based guidelines, termed Medical Eigibiliy Criteria, for the use of all highly efective reversible contraceptive methods by women with vari ous health conditions. Individual countries have subsequently modiied these guidelines. The United States Medical Eligibiliy Criteria (US MEC) was updated in 2016 by the Centers for Disease Control and Prevention (Curtis, 2016b). These docu ments are available at: http://ww.cdc.govlreproductivehealth/ U nin tendedPregnancy/ Contraception_ Guidance.h tm.

1	Disease Control and Prevention (Curtis, 2016b). These docu ments are available at: http://ww.cdc.govlreproductivehealth/ U nin tendedPregnancy/ Contraception_ Guidance.h tm. In the United States MEC, reversible contraceptive methods are organized into six groups by their similarity: combination hormonal contraceptives (CHCs), progestin-only pills (POPs), depot medroxyprogesterone acetate (DMPA), implants, levonorgestrel-releasing intrauterine system (LNGIUS), and copper intrauterine devices (Cu-IUDs). For a given health condition, each method is categorized 1 through 4 (Table 38-2). he score describes the safety proile for a typical woman with that condition: (1) no restriction of method use, (2) method advantages outweigh risks, (3) method risks outweigh advantages, and (4) method poses an unacceptably high health risk.

1	lternatively, depending on the underlying disorder or patient desire, male or female sterilization may be a preferred or recommended permanent contraceptive method (American College of Obstetricians and Gynecologists, 20 17b). hese options are fully discussed in Chapter 39.

1	Globally, 14 percent of reproductive-aged women use intrauterine contraception, and in the United States, 10 percent of contracepting women use this method (Buhling, 2014; Daniels, 2015). he five intrauterine devices (IUDs) currently approved for use in the United States are chemicaly active and continually elute either copper or levonorgestrel. These all have arT-shaped frame of polyethylene that is compounded with barium to render them radiopaque. Of devices, Mirena and Liletta both measure 32 X 32 mm and contain a 52-mg levonorgestrel-releasing cylinder reservoir in the stem of the T (Fig. 38-1). Two trailing strings that are tan (Mirena) or blue (Liletta) are attached to the distal stem to aid eventual device removal. Skyla-known asJaydess in some countries-contains 13.5 mg of levonorgestrel. It has smaller dimensions-28 X 30 mm-and was sized to more appropriately it a nulliparous uterus (Gemzell-Danielsson, 2012). yleena has the same dimensions but contains 19.5 mg of the same

1	It has smaller dimensions-28 X 30 mm-and was sized to more appropriately it a nulliparous uterus (Gemzell-Danielsson, 2012). yleena has the same dimensions but contains 19.5 mg of the same progestin. Two trailing strings of Skyla are tan, and those of yleena are blue. Skyla and yleena can be diferentiated from Mirena and

1	Liletta visually and sonographically by a silver ring near the junction of their stem and arms. Mirena and yleena are cur rently approved for 5 years of use following insertion, whereas Skyla and Liletta are approved for 3 years. he third device, the T380A IUD named ParaGar, is wound with copper, and two strings extend from the stem base. Originally blue, the strings are now white. It is currently approved for 10 years of use following insertion (T eva Wom en's Health, 2014). In addition to these ive currently marketed IUDs, women may retain discontinued brands. A Lippes Loop has two "S" shapes stacked one on the other. he Dalkon Shield has a crab form, whereas a Copper 1 mirrors that number. Progestasert is an early T -shaped progestin-releasing IUD. Last, various metaleluting ring devices are common in Asia.

1	All these IUDs are efective. Failure rates are well below 1 percent and similar overall to those of tubal sterilization (Thonneau, 2008; Trussell, 2011 b). heir mechanisms have not been precisely deined, but prevention of fertilization is now favored.

1	With the LNG-IUS, long-term progestin release leads to endometrial atrophy, which hinders normal implantation (Silverberg, 1986). Moreover, progestins create scant viscous cervical mucus that obstructs sperm motility (Apter, 2014; Moraes, 2016). Within the uterus, an intense local endometrial inlammatory response is induced, especially by coppercontaining devices. Cellular and humoral components of this inlammation are expressed in endometrial tissue and in luid illing the uterine cavity and fallopian tubes. These lead to decreased sperm and egg viability (Ortiz, 2007). Also, in the unlikely event that fertilization does occur, the same inlammatory actions are directed against the blastocyst. Also, with the Cu-IUD speciically, copper levels rise in the cervical mucus of users and act to decrease sperm motility and viability (Jecht, 1973). he above efects are considered primary for contraception because ovulation inhibition is inconsistent with the LNG-IUS and lacking with the Cu-IUD

1	decrease sperm motility and viability (Jecht, 1973). he above efects are considered primary for contraception because ovulation inhibition is inconsistent with the LNG-IUS and lacking with the Cu-IUD (Nilsson, 1984).

1	FIGURE 38-1 Intrauterine devices (IUDs). A.ParaGard T 380A copper IUD. B. Mirena levonorgestrel-releasing intrauterine system. (Reproduced with permission from Stuart GS: Contraception and sterilization. In Hoffman BL, Schorge JO, Bradshaw KD, et al: Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 201o6.) TABLE 38-2. Contraindications and Cautions with Specific Contraceptive Methods In this table, a blank space indicates the method is category 1 or 2 Smoking & age ::35 yr 3/4c Active breast cancer 4 4 4 4 4 Breast disease free ::5 yr 3 3 3 3 Systolic BP ::160 or diastolic BP ::100 4 3 Surgery with long immobilization 4 MS with immobilization 3 DM with end-organ disease 3/4d 3 Cirrhosis (severe, decompensated) 4 3 3 3 3 Liver tumors9 4 3 3 3 3 iStartlContinue (S/C). Current r history of ISHD 4 2/3 3 2/3 2/3 Stroke 4 2/3 3 2/3

1	DM with end-organ disease 3/4d 3 Cirrhosis (severe, decompensated) 4 3 3 3 3 Liver tumors9 4 3 3 3 3 iStartlContinue (S/C). Current r history of ISHD 4 2/3 3 2/3 2/3 Stroke 4 2/3 3 2/3 Migraine with aura 4 2/3 2/3 2/3 2/3 Unexplained vaginal bleedingl 3 3 4/2 4/2 aCombination hormone contraception (CHC) group includes pills, vaginal ring, and patch. bAssociated risks that increase category score include: age :35, transfusion at delivery, BMI :30, postpartum hemorrhage, cesarean delivery, smoking, preeclampsia. (Smoking : 15 cigarettes/day increases risk category to 4 in this age group. dRisk category score is modified by associated risk factors and disease severity. elncluding superficial thrombosis. fOral agents only. Ring and patch are category 1. gBenign hepatic adenoma or hepatocellular cancer. Focal nodular hyperplasia compatible with hormone use. hThese include phenytoin, barbiturates, carbamazepine, oxcarbazepine, primidone, topiramate.

1	gBenign hepatic adenoma or hepatocellular cancer. Focal nodular hyperplasia compatible with hormone use. hThese include phenytoin, barbiturates, carbamazepine, oxcarbazepine, primidone, topiramate. lin those methods under Start/Continue columns, the first US MEC number refers to whether a method may be initiated in an afected patient. For patients who initially develop the condition while using a specific method, the second number refers to risks for continuing that method. JPrior to evaluation. kThose on chronic corticosteroids and at risk for bone fracture.

1	JPrior to evaluation. kThose on chronic corticosteroids and at risk for bone fracture. = antiretroviral; BP = blood pressure; BMI = body mass index; CHTN = chronic hypertension; Cu-IUD = copper intrauterine device; DM = diabetes mellitus; DMPA = depot medroxyprogesterone acetate; GC/CT = gonorrhea/chlamydial infection; GTD = gestational trophoblastic disease; ISHD = ischemic heart disease; LNG-IUS = levonorgestrel-releasing intrauterine system; MS = multiple sclerosis; PI = protease inhibitor; PID = pelvic inflammatory disease; POP = progestin-only pills; SLE = system lupus erythematosus; VTE = venous thromboembolism. Compiled from Curtis, 2016b; Merck, 2015; Teva Women's Health, 2014.

1	Contraindications to IUD use are few and shown in Table 38-2. In the past, IUDs were perceived to increase the risk of ectopic pregnancy, but this has since been clariied. Speciically, IUDs provide efective contraception and lower the absolute number of ectopic pregnancies by half compared with the rate in noncontracepting women (World Health Organization, 1985, 1987). But, the IUD mechanisms of action are more efective in preventing intrauterine implantation. hus, if an IUD fails, a higher proportion of pregnancies are likely to be ectopic (Backman, 2004; Furlong, 2002).

1	Expulsion of an IUD from the uterus is most common during the first month. hus, women are examined approximately 1 month following IUD insertion, usually after menses, to identiy the tails trailing from the cervix. Following this, a woman is instructed to palpate the strings each month after menses. Regardless of IUD type, the cumulative 3-year expulsion rate approximates 10 percent (Madden, 2014; Simonatto, 2016). This rate is higher in those :;25 years Qatlaoui, 2017).

1	If the tail of an IUD cannot be visualized, the device may have been expelled, may have perforated the uterus, or may be malpositioned. Alternatively, the device may be normally positioned with its tails folded within the endocervical canal or uterine cavi ty. To investigate, after excluding pregnancy, a cytological brush can be twirled within the endocervical canal to entangle the strings and bring them gently into the vagina. If unsuccessful, the uterine cavity is probed gently with a Randall stone clamp or with a specialized rod with a terminal hook to snare the strings or device.

1	One should not assume that a device has been expelled unless it was seen. Thus, if tails are not visible and the device is not felt by gentle probing of the uterine cavity, transvaginal sonography can be used to ascertain if the device lies within the uterus. lthough traditional sonography will document IUD position adequately in most cases, three-dimensional sonography ofers improved visualization, especially with the LNG-IUS (Moschos, 201l). If sonography is inconclusive or if no device is seen, then a plain radiograph of the abdominopelvis is taken. Computed tomography (CT) scanning or, less commonly, magnetic resonance (MR) imaging is an alternative (Boortz, 2012). It is safe to perform MR imaging at 1.5 and 3 Tesla (T) with an IUD in place (Ciet, 2015).

1	During uterine sounding or IUD insertion, the uterus may be perforated, which is identiied by the tool traveling farther than the expected uterine length based on initial bimanual examination. Rates approximate 1 per 1000 insertions, and risks include puerperal insertion, lactation, provider inexperience, and extremes of uterine lexion (Harrison-Woolrych, 2003; Heinemann, 2015). Although devices may migrate spontaneously into and through the uterine wall, most perforations occur, or at least begin, at the time of insertion (Ferguson, 2016).

1	With acute perforation, the fundus is the more common site, and bleeding is typically minimal due to myometrial contraction around the puncture hole. If no brisk or persistent bleeding is noted from the os following instrument or device removal, then patient observation alone is reasonable. Rarely, acute lateral perforations may lacerate the uterine artery, and subsequent brisk bleeding may prompt laparoscopy or laparotomy for control. Following any perforation, although this is not irmly evidence-based, a single dose of broad-spectrum antibiotic may mitigate infection.

1	With chronic perforation, a device can penetrate the muscular uterine wall to varying degrees. A patient may be asymptomatic but abdominal pain, uterine bleeding, or missing strings can be clues (Kaislasuo, 2013). hose with a predominantly intrauterine location are usually managed by hysteroscopic IUD removal. In contrast, devices that have nearly or completely perforated through the uterine wall are more easily removed laparoscopically. Notably, an extrauterine Cu-IUD frequently induces an intense local inlammatory reaction and adhesions (Kho, 2014). Laparotomy may be necessary, and bowel preparation is considered. Sigmoid and bladder perforations and small-bowel obstruction have been reported remote from insertion (Sano, 2017; Xu, 2015; Zeino, 2011).

1	Dysmenorrhea and bleeding irregularities can complicate IUD use (Aoun, 2014; Grunloh, 2013). These can be treated with some degree of success by nonsteroidal antiinlammatory drugs (NSAIDs) or tranexamic acid, which is an antiibrinolytic (Godfrey, 2013; Madden, 2012; S0rdal, 2013). Of the two IUDs, heavy bleeding more often complicates Cu-IUD use and may cause iron-deiciency anemia, for which oral iron salts are given. With the LNG-IUS, irregular spotting for up to 6 months after placement often gives way to progressive amenorrhea, which is reported by 30 percent of women after 2 years and by 60 percent after 12 years (Ronnerdag, 1999). his is frequently associated with improved dysmenorrhea.

1	he risk of upper genital tract device-related infection is greatest during the irst months following IUD insertion (Farley, 1992; Turok, 2016). Pathogens include Neisseria gonorrhoeae, Chlamydia trachomatis, and vaginal lora. Women at risk for sexually transmitted diseases (STDs) should be screened either before or at the time of IUD insertion (Centers for Disease Control and Prevention, 2015; Sufrin, 2012). That said, device insertion need not be delayed while awaiting sexually transmitted disease or Pap test results in asymptomatic women (Birgisson, 2015). If these bacteria are subsequently found and the patient is without symptoms, then the IUD may remain and treatment prescribed as detailed in Chapter 65 (p. 1240). Importantly, routine antimicrobial prophylaxis before insertion is not recommended (Grimes, 2012; Walsh, 1998). And the American Heart Association does not recommend bacterial endocarditis prophylaxis with insertion (Nishimura, 2014).

1	Mter the irst month, infection risk is not increased in IUD users who would otherwise be at low risk of sexually transmitted infections. Correspondingly, IUDs appear to cause little, if any, increase in infertility rates in these low-risk patients (H ubacher, 2001). The American College of 0bstetricians and Gynecologists (20 15c, 2016a) recommends that women at low risk for STDs, including adolescents, be considered good candidates for IUDs. he IUD is also safe and efective in women infected with human immunodeiciency virus (HIV) and may be used in others who are immunosuppressed (Centers for Disease Control and Prevention, 2015; Tepper, 2016a).

1	If infection does develop, it may take several forms and typically requires broad-spectrum antimicrobials. Pelvic inlammatoy disease (PID) without abscess is treated with antibiotics on an outpatient or inpatient basis, depending on infection severity. here are theoretical concerns that a coexistent IUD may worsen the infection or delay resolution. A provider may choose to remove an IUD in this setting, although some evidence supports allowing a device to remain during treatment in those hospitalized with mild or moderate PID (Centers for Disease Control and Prevention, 2015; Tepper, 2013). Ifinfection fails to improve during 48 to 72 hours of treatment, the device is removed. Tuboovarian abscess can complicate PID and is treated aggressively with intravenous broad-spectrum antibiotics and IUD removal. Last, septic abortion mandates immediate uterine evacuation and antibiotics.

1	Actinomyces israelii is a gram-positive, slow-growing, anaerobic indigenous vaginal bacterium that rarely causes suppurative infection. Some have found it more frequently in the vaginal lora or on the Pap smears of IUD users (Curtis, 1981; Kim, 2014). Current recommendations advise that an asymptomatic woman may retain her IUD and does not require antibiotic treatment (American College of Obstetricians and Gynecologists, 2017c; Lippes, 1999; Westhof, 2007a). However, if signs or symptoms of infection develop in a woman who harbors Actinomyces species, then the device is removed and antibiotics are given. Early indings with infection include fever, weight loss, abdominal pain, and abnormal uterine bleeding or discharge. Actinomyces species are sensitive to antibiotics with gram-positive coverage, notably the penicillins. Pregnancy with an IUD

1	Pregnancy with an IUD For women who conceive while using an IUD, ectopic pregnancy should be excluded. With intrauterine pregnancy, if the tail is seen, it should be grasped and the IUD removed by gentle outward traction. his action reduces complications such as abortion, chorioamnionitis, and preterm birth (Fulkerson Schaefer, 2017; Kim, 2010). Speciically, in one cohort, a 54-percent abortion rate and 17 -percent preterm delivery rate was noted if the device remained in situ. More favorably, rates of 25 percent and 4 percent, respectively, resulted from prompt Cu-IUD removal (Tatum, 1976). Few data guide management with the LNG-IUS, and most practice extrapolates from copper devices.

1	If the tail is not visible, attempts to locate and remove the device may result in abortion. Although not our practice, some case reports and small series describe sonography or hysteroscopy to assist in diicult device removals (Perez-Medina, 2014; Schiesser, 2004). Ater fetal viability is reached, it is unclear whether it is better to remove an IUD whose strings are visible and accessible or to leave it in place. Fetal malformation rates are not greater with a device let in situ (Tatum, 1976; Vessey, 1979).

1	Second-trimester miscarriage with an IUD in place is more likely to be infected (Vessey, 1974). Sepsis may be fulminant and fatal. Pregnant women with a device in utero who demonstrate any evidence of pelvic infection are treated with broadspectrum antibiotics and prompt uterine evacuation. Because of these risks, a woman should be given the option of early pregnancy termination if the device cannot be removed early in pregnancy. Last, in women who give birth with a device in place, appropriate steps should be taken at delivery to identiy and remove the IUD. To reduce expulsion rates and perforation risks, IUD insertion 6 weeks after delivery. Women delivered at Parkland Hospital are seen 3 weeks postpartum, and IUDs are inserted 6 weeks postpartum or sooner if involution is complete.

1	Alternatively, immediately ater miscarriage, surgical abortion, or delivery, an IUD may be inserted in the absence of overt infection (Lopez, 2015a; Okusanya, 2014). Also, "immediate" insertion 1 week ater mifepristone and completed medical abortion has been described (Saav, 2012; Shimoni, 2011). The risk ofIUD expulsion is slightly higher if it is placed immediately following any of these recent pregnancies (Whitaker, 2017). However, in studies, the number of women in immediate-placement groups who ultimately receive and retain an IUD is greater than in groups scheduled for traditionally timed placement, some of whose members do not return for insertion (Bednarek, 2011; Chen, 2010). With immediate insertion, techniques depend on uterine size. After irst-trimester evacuation, the IUD can be placed using the manufacturer's standard instructions. If the uterine cavity is larger, the IUD can be placed using ring forceps with sonographic guidance (Drey, 2009; Fox, 2011).

1	Immediately following vaginal or cesarean delivery, an IUD can be placed by a hand, by its inserter tube, or by ring forceps (Levi, 2015; Xu, 1996). With any of these methods, the arms of the IUD need not be folded into the inserter tube prior to insertion. During cesarean delivery placement, the hand or inserter travels through the unsutured open hysterotomy to deposit the device at the fundus. A second hand cupping the outer fundus can provide back pressure and stabilize the uterus during insertion. Strings are then gently directed toward the cervix. For instrumented insertion following vaginal delivery, the clinician resterilizes the vulva and changes gloves after placental delivery but before perineal repairs. he anterior lip of the loppy cervix is held with ring forceps. A second ring forceps grasp the IUD stem and guides it through the uterine cavity to the fundus. For manual insertion following vaginal delivery, the provider secures the IUD between the index and middle fingers

1	forceps grasp the IUD stem and guides it through the uterine cavity to the fundus. For manual insertion following vaginal delivery, the provider secures the IUD between the index and middle fingers to deposit the device. In either case, back pressure against the fundus by an abdominal hand can guide positioning (Stuart, 2017; The ACQUIE Project, 2008).

1	For placement not related to pregnancy, insertion near the end of normal menstruation, when the cervix is usually softer and somewhat more dilated, may be easier and also helps exclude early pregnancy. But, insertion is not limited to this time. For the woman who is sure she is not pregnant and does not want to be pregnant, insertion is done at any time. Before insertion, contraindications are sought. Candidates are counseled, and written consent obtained. n oral NSAID, with or without codeine, can be used to allay cramps ater insertion (Ngo, 2015). But NSAIDs, misoprostol, or even paracervical during device placement (Bednarek, 2015; Hubacher, 2006;

1	Mody, 2012; Pergialiotis, 2014). Of topical lidocaine products, 2-percent gel is inefective, but a newer gel and a spray both show promise (Aksoy, 2016; Lopez, 2015b; Tornblom-Paulander, 2015). Bimanual pelvic examination delineates uterine position and size. Abnormalities are evaluated, as they may contraindi cate insertion. Mucopurulent cervicitis or signiicant vaginitis is appropriately treated and resolved before IUD insertion. The cervical surface is cleansed with an antiseptic solution, and sterile instruments and a sterile IUD are used. A tenaculum is placed on the cervical lip, and the canal and uterine cavity are straightened by applying gentle outward traction. The cav ity is then probed by a uterine sound to identiy its direction and depth. Speciic steps of ParaGard and Mirena insertion are tive package inserts.

1	Following insertion, only the threads should be visible trailing from the cervix. These are trimmed to allow 3 to 4 cm to protrude into the vagina, and their length is recorded. If improper device positioning is suspected, placement should be confirmed, using sonography if necessary. If the IUD is not positioned completely within the uterus, it is removed and replaced with a new device. An expelled or partially expelled device should not be reinserted.

1	Contraception can be provided by thin, pliable progestincontaining cylinders that are implanted subdermally and release hormone over many years. One of these, Nepanon is a single-rod implant with 68 mg of etonogestrel covered by an ethylene vinyl acetate copolymer cover. The implant is placed subdermally on the medial surface of the upper arm 8 to 10 cm from the elbow in the biceps groove and is aligned with the long axis of the arm. It may be used as contraception for 3 years, removed, and then replaced at the same site or in the opposite arm (Merck, 2016a).

1	Nexplanon is radiopaque, and its inserter device is designed to assist with subdermal positioning and avert deeper placement. This device replaced Implanon, which is not radiopaque. A misplaced Implanon may be identiied with sonography using a 10-to IS-MHz linear array transducer (Shulman, 2006). In some cases, MR imaging may be required if supplemental information is needed despite sonography (Correia, 2012). Both implants are similarly shaped and pharmacologically identical. They are highly efective, and the mechanism of action for progestin-only products is described later (p. 689) (Croxatto, 1998; Mommers, 2012). Implanon is safe and still approved by Flange 1.5cm After slider moved back, inserter tube removed

1	FIGURE 38-2 Insertion of the Mirena intrauterine system. Initially, threads from behind the slider are first released to hang freely. The slider found on the handle should be positioned at the top ofthe handle nearest the device. The IUD arms are oriented horizontally. A flange on the outside ofthe inserter tube is positioned from the IUD tip to reflect the depth found with uterine sounding. A.As both free threads are pulled, the Mirena IUD is drawn into the inserter tube. The threads are then tightly fixed from below into the handle's cleft. In these depictions, the inserter tube has been foreshortened. The inserter tube is gently inserted into the uterus until the flange lies 1.5 to 2 cm from the external cervical os to allow the arms to open. B. While holding the inserter steady, the IUD arms are released by pulling the slider back to reach the raised horizontal mark on the handle, but no further. C. The inserter is then gently guided into the uterine cavity until its flange

1	the IUD arms are released by pulling the slider back to reach the raised horizontal mark on the handle, but no further. C. The inserter is then gently guided into the uterine cavity until its flange touches the cervix. D. The device is released by holding the inserter firmly in position and pulling the slider down all the way. The threads will be released automatically from the clet. The inserter may then be removed, and IUD strings trimmed. (Reproduced with permission from Stuart GS: Contraception and sterilization. In Hoffman BL, Schorge JO, Bradshaw KD, et al: Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.)

1	Tip of IUD inserter rod Inserter tube Flange A B FIGURE 38-3 Insertion of ParaGard T 380A. The uterus is sounded, and the IUD is loaded into its inserter tube not more than 5 minutes before insertion. A blue plastic flange on the outside of the inserter tube is positioned from the IUD tip to reflect uterine depth. The IUD arms should lie in the same plane as the flat portion of the oblong blue flange. A.The inserter tube, with the IUD loaded, is passed into the endometrial cavity. A long, solid, white inserter rod abuts the base of the IUD. When the blue flange contacts the cervix, insertion stops.

1	B. To release the IUD arms, the solid white rod within the inserter tube is held steady, while the inserter tube is withdrawn no more than 1 cm. C. The inserter tube, not the inserter rod, is then carefully moved upward toward the top of the uterus until slight resistance is felt. At no time during insertion is the inserter rod advanced forward. D. First, the solid white rod and then the inserter tube are withdrawn individually. At completion, only the threads should be visible protruding from the cervix. These are trimmed to allow 3 to 4 cm to extend into the vagina. (Reproduced with permission from Stuart GS: Contraception and sterilization. In Hoffman BL, Schorge JO, Bradshaw KD, et al: Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 2016.) the Food and Drug Administration (FDA), but it is no longer distributed by the manufacturer.

1	he irst progestin implants contained levonorgestrel (LNG), and systems are still available outside the United States. Jadelle, originally named Norplant-2, provides LNG and contraception for 5 years through two subdermally implanted Silastic rods. Ater this time, rods may be removed and if desired, new rods inserted at the same site (Bayer Group, 2015). Jadele is approved by the FDA, however, it is not marketed or distributed in the United States. Sino-implant II is a two-rod system with the same amount (150 mg) of LNG and same mechanism of action asJadele but provides 4 years of contraception. Sinoimplant II is manufactured in China and approved for use by 20 countries in Asia and Mrica (FHI 360, 2012).

1	Like the etonogestrel implant, these systems are placed subdermally on the inner arm approximately 8 cm from the elbow and have similar removal steps. Implants vary regarding their insertion technique, and manufacturer instructions should be consulted. Both implant systems are highly efective (Sivin, 1998; Steiner, 2010). he forerunner of these implants was the Norpant System, which provided LNG in six Silastic rods implanted subdermally. The manufacturer stopped distributing the system in 2002. Few data compare the LNG and etonogestrel implants. In one, eicacy and discontinuation rates by 2.5 years of use were similar (Bahamondes, 2015).

1	Few data compare the LNG and etonogestrel implants. In one, eicacy and discontinuation rates by 2.5 years of use were similar (Bahamondes, 2015). Unscheduled bleeding is common with progestin-only methods and described on page 689. Device-specific adverse efects derive mainly from malpositioning. First, branches of the medial antebrachial cutaneous nerve can be injured during implant or needle insertion that is too deep or during exploration for a lost implant. Clinically, numbness and paresthesia over the anteromedial aspect of the forearm are noted (Wechselberger, 2006).

1	Second, nonpalpable devices are not uncommon and require radiological imaging for localization. As an adjunct, one group adopted the hook-wire tagging method used in breast tumor surgery to allow deep-lying implants to be marked prior to extraction (Nouri, 2013). If imaging fails to locate an implant, etonogestrel blood level determination can help veriy that the implant is indeed in situ. his special assay must be coordinated with the manufacturer (1-877-467-5266).

1	For those not currently using hormonal contraception, the etonogestrel implant is ideally inserted within 5 days of menses onset. With LNG-releasing implants, contraception is established within 24 hours if inserted within the first 7 days of the menstrual cycle (Sivin, 1997; Steiner, 2010). For transitioning methods, an implant is placed on the day of the first placebo combination oral contraceptive (COC) pill; on the day that the next depot-medroxyprogesterone injection would be due; or within 24 hours of taking the last POP (Merck, 2016a). In women certain that they are not pregnant, insertion at other times of the cycle is followed by alternative contraception for 7 days. Related to pregnancy, an implant may be inserted before discharge following delivery or abortion (Sothornwit, 2017).

1	With the patient lying down, her nondominant arm, forearm, and hand are outstretched on the bed with the inner aspects of each exposed upward, and the elbow is flexed. The insertion site is marked with a sterile pen 8 to 10 cm proximal to the medial condyle of the humerus. A second mark is placed 4 cm proximally and delineates the inal path of the implant. he Nexplanon is inserted using sterile technique. The area is cleansed aseptically, and a I-percent lidocaine anesthetic track is injected beneath the sin along the planned insertion path. The implant is then placed as shown in Figure 38-4. Ater placement, both

1	FIGURE 38-4 Nexplanon insertion. A sterile pen marks the insertion site, which is 8 to 10 cm proximal to the medial humeral condyle. A second mark is placed 4 cm proximally along the arm's long axis. The area is cleaned aseptically, and a l-percent lidocaine anesthetic track is injected along the planned insertion path. A.The insertion device is grasped at its gripper bubbles found on either side, and the needle cap is removed outward. The device can be seen within the needle bore. The needle bevel then pierces the skin at a 30-degree angle.

1	B. Once the complete bevel is subcutaneous, the needle is quickly angled downward to lie horizontally. C. Importantly, the skin is tented upward by the needle as the needle is slowly advanced horizontally and subdermally. D. Once the needle is completely inserted, the lever on the top of the device is pulled backward toward the operator. This retracts the needle and thereby deposits the implant. The device is then lifted away from the skin. After placement, both patient and operator should palpate the 4-cm implant. patient and provider should palpate and identiY both ends of the 4-cm implant. To minimize bruising at the site, a pressure ban dage is created around the arm and is removed the following day.

1	patient and provider should palpate and identiY both ends of the 4-cm implant. To minimize bruising at the site, a pressure ban dage is created around the arm and is removed the following day. With implant extraction, the removal site is irst cleansed with antiseptic. he proximal end of the implant is depressed with a finger to allow the distal end to bulge up toward the skin. Ater anesthetizing the skin over this bulge, the skin is incised 2 mm toward the elbow along the long axis of the arm. he proximal butt of the implant is then pushed toward this incision. Once vis ible, the distal end of the implant is grasped with a hemostat and removed. If present, supericial adhesions surrounding an implant may be dissected away with hemostat tips placed into the incision.

1	Progestin-only contraceptives include the implants just described, injectables, and pills. s their primary contraceptive action, these progestins suppress luteinizing hormone (LH) and in turn block ovulation. As other efects, cervical mucus is thickened to retard sperm passage, and atrophy renders the endometrium unfavorable for implantation. Fertility is restored rapidly following cessation of progestin-only contraception. n exception is DMPA, as described on page 693 (Mansour, 201l).

1	For all progestin-only methods, irregular or heavy uterine bleeding is a distinct disadvantage. It is the most frequently reported adverse event leading to method discontinuation. Often, counseling and reassurance is suicient. Troublesome bleeding may be improved by one to two cycles of combination oral contraceptives, by a 1-to 3-week course of estrogen alone, or by a short course of NSAIDs combined with the established method (Abdel-Aleem, 2013). Fortunately, with prolonged use, progestins induce endometrial atrophy, which leads to sustained amenorrhea. For the well-counseled patient, this is often an advantage.

1	Most progestin-only contraceptive methods do not signiicantly afect lipid metabolism, glucose levels, hemostatic factors, liver function, thyroid function, or blood pressure (DorRinger, 2002). However, the increased low-density lipoprotein (LDL) cholesterol and decreased high-density lipoprotein (HDL) cholesterolrlevels seen with DMPA may be less desirable for women with cardiac or vascular disease risks (Kongsayreepong, 1993).

1	Progestin-only methods do not impair milk production and are an excellent choice for lactating women. here are no increased risks of genital tract, liver, or breast neoplasia (Samson, 2016; Wilailak, 2012; World Health Organization, 1991a,b, 1992). Weight gain and bone fracture are not prominent side efects of this contraceptive group, except for depot progesterone, discussed on page 693 (Lopez, 2012a, 2013a). Functional ovarian cysts develop with a greater frequency in women using progestin-only agents, although they do not usually necessitate intervention (European Society of Human Reproduction and Emblyology, 2001; Hidalgo, 2006; Nahum, 2015). Last, an association between depression and DMPA or POPs is unclear (Civic, 2000; Pagano, 2016; Svendal, 2012; Westhof, 1995). Women with depression may be prescribed these methods, but surveillance following initiation is reasonable.

1	These methods are ideal for most women, but contraindications and cautions are associated with a few conditions listed in Table 38-2. Current breast cancer and pregnancy are the only two absolute contraindications. In a few instances, manufacturer restrictions difer from the US MEC. First, manufacturer prescribing information lists thrombosis or thromboembolic disorders as contraindications (Merck, 2016a; Pizer, 2015a,b). However, for individuals with these disorders, US MEC considers progestin-containing methods category 2. Moreover, evidence does not link progestin-only methods with thromboembolism, stroke, or cardiovascular disease (Mantha, 2012; Tepper, 2016b; World Health Organization, 1998). Second, for many progestin products, manufacturers note prior ectopic pregnancy as a contraindication. his is secondary to progesterone's efect of slowing fallopian tube motility and thereby delaying fertilized egg transport to the endometrial cavity. hat said, efective contraception lowers

1	his is secondary to progesterone's efect of slowing fallopian tube motility and thereby delaying fertilized egg transport to the endometrial cavity. hat said, efective contraception lowers pregnancy rates overall. Thus, for those with prior ectopic pregnancy, US MEC considers progestin injectables and implants category 1, and progestin-only pills are category 2.

1	These currently are available in forms that contain both estrogen and progestin or contain only progestin. Progestin-only injectables and pills are considered very efective, yet secondtier agents, due to the need for increased patient compliance. Similarly, products containing both estrogen and progestin, often termed combination hormonal contraception (CHC), are considered in this tier. These may be supplied as pills, transvaginal rings, or transdermal patches. • Combination Hormonal Contraceptives Mechanism of Action

1	• Combination Hormonal Contraceptives Mechanism of Action Actions of combination hormonal contraceptives are multiple, but the most important efect is suppression of hypothalamic gonadotropin-releasing factors. his in turn blocks pituitary secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and thereby inhibits ovulation. The progestin component of CHCs provides ovulation prevention by suppressing LH; it thickens cervical mucus and thereby retards sperm passage; and it renders the endometrium unfavorable for implantation. Estrogen blocks ovulation by suppressing FSH release. To promote cycle control, estrogen stabilizes the endometrium, which prevents intermenstrual bleeding-also known as breakthrough bleeding. The net efect is an extremely efective yet highly reversible method (Mansour, 2011).

1	hese pills are the most frequently used reversible birth control method in the United States. In a 2006 to 2010 survey, 16 percent of contracepting women in the United States were using these (Daniels, 2015). COCs are marketed in a wide variety of estrogen and progestin combinations. lvlost are available as generics, and the FDA (2016) confirms the bioequivalence of COC generics. The merican College of Obstetricians and Gynecologists (2015a) supports the use of either branded or generic preparations. Pharmacologically, ethinyl estradiol is the most common estrogen present in COC formulations in the United States. Less frequently, mestranol or estradiol valerate is used. Unwanted efects most often attributed to the estrogen component include breast tenderness, weight gain, nausea, and headache.

1	COCs also contain one of several progestins that are structurally related to progesterone, testosterone, or spironolactone. hus, these progestins bind variably to progesterone, androgen, glucocorticoid, and mineralocorticoid receptors. hese ainities explain many pill-related side efects and are often used to compare one progestin with another. Most progestins used in COCs are related to testosterone and may impart androgenic side afects such as acne and adverse HDL and LDL levels. To avoid these efects, antiandrogenic progestins have been introduced and include dienogest and nomegestroL acetate. he latter is used in a COC approved outside the United States. Despite these pharmacological differences, the true advantage of one progestin over another is less apparent clinically (Lawrie, 2011; Moreau, 2007).

1	Another progestin, drospirenone, is structurally similar to spironolactone. he doses in currently marketed COCs have efects similar to 25 mg of this diuretic (Seeger, 2007). Drospirenone displays antiandrogenic activity, provides an antialdosterone action to minimize water retention, and has antimineralocorticoid properties that may, in theory, cause potassium retention and hyperkalemia (Krattenmacher, 2000). hus, it is avoided in women with renal or adrenal insuiciency or with hepatic dysfunction. Moreover, serum potassium level monitoring is recommended in the first month for patients chronically treated concomitantly with any drug associated with potassium retention. hese include NSAIDs, angiotensinconverting enzyme (ACE) inhibitors, angiotensin II antagonists, heparin, aldosterone antagonists, and potassium-sparing diuretics (Bayer HealthCare Pharmaceuticals, 2015).

1	Since the development of COCs, their estrogen and progestin content has dropped remarkably to minimize adverse efects. Currently, the lowest acceptable dose is limited by the ability to prevent pregnancy and to avoid unacceptable breakthrough bleeding. hus, the daily estrogen content varies from 10 to 50 1g of ethinyl estradiol, and most contain 35 j.1g or less. In a few COCs, inert placebo pills have been replaced by tablets containing iron. These have the suix Fe added to their name. In addition, Beyaz has a form of folate-levomefolate calcium-within both its active and placebo pills.

1	With COCs termed monophasic piLs, the progestin dose remains constant throughout the cycle. In others, the dose frequently is varied, and term biphasic, triphasic, or quadriphasic piL is used depending on the number of dose changes within the cycle. In some formulations, the estrogen dose also varies. In general, phasic pills were developed to reduce the total progestin content per cycle without sacrificing contraceptive eicacy or cycle control. he theoretical advantage of a lower total progesterone dose per cycle, however, has not been borne out clinically (Moreau, 2007). Cycle control also appears to be comparable among mono-through triphasic pills (van Vliet, 2011a,b,c). Hormones are taken daily for a specified time (21 to 81 days) and then replaced by placebo for a speciied time (4 to 7 days), which is called the "pill-free interval." During these pill-free days, withdrawal bleeding is expected.

1	With the trend toward lower estrogen doses to minimize side efects, follicular development and ovulation may occur. To counter this, the active-pill duration in some formulations is extended to 24 days. In comparison, these 24/4 regimens perform similarly to higher-estrogen-dose 21/7 regimens (Anttila, 2011; Marr, 2012). Alternatively, longer durations of active hormone, designed to minimize the number of withdrawal episodes, have similar eicacy and safety proiles as more traditional administration (Edelman, 2014). These extended-cycle products produce a 13-week cycle, that is, 12 weeks of hormone use, followed by a week for withdrawal menses. The product Amethyst provides continuous active hormone pills for 365 days each year. Such extended or continuous regimens may be especially suited for women with significant menstrual symptoms (Mendoza, 2014).

1	For general initiation, women ideally begin COCs on the first day of a menstrual cycle. In such cases, a supplementary contraceptive method is unnecessary. With the more traditional "Sunday start," women begin pills on the irst Sunday that follows menses onset, and an additional method is needed for 1 week to prevent conception. If menses begin on a Sunday, then pills are begun that day and no supplemental method is required. Alternatively, with the "quick start" method, COCs are started on any day, commonly the day prescribed, regardless of cycle timing. An additional method is used during the first week (Westhof, 2002, 2007b). If the woman is unknowingly already pregnant during quick start initiation, COCs are not teratogenic (Lammer, 1986; Rothman, 1978; Savolainen, 1981). However, a missed menses following COC initiation should prompt pregnancy testing. Similar same-day initiation can be implemented with the contraceptive vaginal ring or patch (Murthy, 2005; Schafer, 2006).

1	For maximum eiciency, pills are best taken at the same time each day. If one dose is missed, the missed pill is taken immediately; the scheduled dose for that day is taken on time; and then daily pills are continued. If two or more doses are missed, the most recent missed pill is taken immediately; the scheduled dose for that day is taken on time; and an efective barrier technique used for 7 days while daily pills are then continued (Curtis, 2016a). If withdrawal bleeding fails to occur during the pill-free interval, a woman should continue her pills but seek attention to exclude pregnancy. With initiation of COCs, spotting or bleeding is common. It does not relect contraceptive failure and typically resolves within one to three cycles. If unscheduled bleeding persists, those with bleeding during the first part of a pill pack may beneit from an increase in the estrogen dose, whereas those with bleeding during the second part may improve with a higher progestin dose (Nelson, 2011).

1	Some drugs decrease COC efectiveness, and choosing another contraceptive method is preferable. However, if a COC is selected for concurrent use in these instances, a preparation containing a minimum of 30 �g ethinyl estradiol is ideally chosen. Conversely, some COCs interfere with the actions of certain drugs (see Table 38-2). In obese women, COCs are efective (Lopez, 2016). Some studies point to lowered hormone bioavailability, but over all eicacy remains high (Nakajima', 2016; Westhof, 2010; Yamazaki, 2015). With the transdermal patch method, how ever, evidence is more robust that obesity may alter pharmaco kinetics and lower eicacy, as discussed on page 692.

1	Yamazaki, 2015). With the transdermal patch method, how ever, evidence is more robust that obesity may alter pharmaco kinetics and lower eicacy, as discussed on page 692. Combination oral contraceptives alter lipid synthesis and in general raise serum levels of triglycerides and of total cholesterol, HDL, and very-low density lipoprotein (VLDL) cholesterol. Estrogen lowers LDL cholesterol concentrations. Oral contraceptives are not atherogenic, and their efect on lipids is clinically inconsequential for most women (Wallach, 2000). In women with dyslipidemias, limited data suggest that COCs increase the risk for myocardial infarction and minimally so for venous thromboembolism or stroke (Dragoman, 2016). For those with multiple additional risk factors for vascular disease, alternative contraceptive methods are recommended.

1	With COCs, protein metabolism is afected, and estrogens boost hepatic production of various globulins. First, fibrinogen and many of the clotting factor levels rise in direct proportion to the estrogen dose and may lead to thrombosis. Angiotensinogen production is also augmented by COCs, and its conversion by renin to angiotensin I may be associated with "pill-induced hypertension," discussed subsequently. Last, COCs elevate sex hormone-binding globulin (SHBG) levels, which in turn lower concentrations of bioavailable testosterone and lessen androgenic side efects. Regarding carbohydrate metabolism, current low-dose formulations have minimal efects in women who do not have diabetes (Lopez, 2014). And, the risk of developing diabetes is not increased (Kim, 2002). For diabetic women, COCs may be used in nonsmokers with disease duration <20 years and without associated vascular disease, nephropathy, retinopathy, or neuropathy (Curtis, 2016b).

1	Of other metabolic changes, thyroid-binding globulin and thyroid-stimulating hormone (TSH) levels are elevated, but free plasma thyroxine (FT4) levels are unchanged (Raps, 2014). Studies have not supported a connection between COCs and weight gain (Gallo, 2014). Despite increased plasma angiotensinogen (renin substrate) levels, women using low-dose COC formulations rarely develop clinically significant hypertension (Chasan-Taber, 1996). However, it is common practice for patients to return 8 to 12 weeks ater COC initiation for evaluation of blood pressure and other symptoms. During initial contraception selection, a history of gesta tional hypertension does not preclude subsequent COC use.

1	During initial contraception selection, a history of gesta tional hypertension does not preclude subsequent COC use. Among women with well-controlled hypertension, COC use is linked to greater risks than nonusers for stroke, acute myo cardial infarction, and peripheral arterial disease, and in these women, COCs are considered US MEC category 3 (Curtis, 2016b). Severe forms of hypertension, especially those with end-organ involvement, preclude COC use. For nonsmoking women younger than 35, the risk of stroke is extremely low (World Health Organization, 1996). COCs are associated with a small increased risk for ischemic stroke (Chan, 2004; Lidegaard, 2012). Rates increase signiicantly for women who have hypertension, who smoke, or who have migraine headaches with visual aura or other focal neurological changes and use COCs (MacClellan, 2007; Tepper, 2016c).

1	The evidence for stroke risk in migraineurs without aura is less clear (Etminan, 2005; Schurks, 2009). COC initiation may be considered for women with preexisting migraines without aura if they are otherwise healthy, younger, normotensive nonsmok ers. For women with prior stroke, COCs should not be consid ered due to risks for repeat events. For women with prior myocardial infarction, COCs should not be considered. Also, in women with multiple cardiovas cular risk factors, which include smoking, hypertension, older age, and diabetes, the risk for myocardial infarction outweighs the beneits of this method. However, for those without these risks, low-dose oral contraceptives are not associated with an increased risk of myocardial infarction (Margolis, 2007; World Health Organization, 1997).

1	The risks for deep-vein thrombosis and pulmonary embolism are increased in women who use COCs (Stadel, 1981). These clearly are estrogen-dose related, and rates have substantively declined with lower-dose formulations containing 10 to 35 �g of ethinyl estradiol. he general-population risk of venous thromboembolism (VTE) is 4 to 5 events per 100,000 woman-years. he incidence ofVTE with COC use increases three-to fivefold compared with nonusers (Shaw, 2013; van Hylckama Vlieg, 2009). Obesity raises the VTE risk, which is compounded by COCs (Horton, 2016; Suchon, 2016). Accordingly, in an obese woman, COCs are considered a US MEC category 2. VTEs are signiicantly increased in women older than 35 years who smoke, and COCs are not recommended. Those most at risk for VTE include women with thrombophilias (ESHRE Capri Workshop Group, 2013). Nloreover, COC use during the month before a major operative procedure appears to double the risk for postoperative VTE (Robinson, 1991). Thus, the

1	thrombophilias (ESHRE Capri Workshop Group, 2013). Nloreover, COC use during the month before a major operative procedure appears to double the risk for postoperative VTE (Robinson, 1991). Thus, the merican College of Obstetricians and Gynecologists (2016d) recommends balancing the risks ofVTE and the degree of postoperative immobility with the risk of unintended pregnancy during the 4 to 6 weeks required to reverse the thrombogenic efects of COCs before surgery. In the early puerperium, VTE risks are also increased, and COCs are not recommended for women within the irst 4 weeks after delivery.

1	Certain progestins within COC are linked with greater rates of thromboembolism. A slightly higher VTE risk with drospirenone-containing COCs has been shown in two studies. In response, an assessment of benefits and VTE risks in users of these pills has been emphasized (Food and Drug Administration, 2012; Jick, 2011; Parkin, 2011). Desogestrel and gestodene are also implicated and carry similarly elevated risks (Stegeman, 2013; Vinogradova, 2015).

1	Most studies indicate that COCs overall are not associated with an increased risk of cancer (Cibula, 2010). In fact, a protective efect against ovarian and endometrial cancer has been shown (Collaborative Group on Epidemiological Studies of Ovarian Cancer, 2008; Tsilidis, 201r1). As an exception, the relative risk of cervical dysplasia and cervical cancer is higher in current COC users, but this declines after use is discontinued. Following 10 or more years, risk returns to that of never users (International Collaboration of Epidemiological Studies of Cervical Cancer, 2007). It is unclear whether COCs contribute to breast cancer development. Major studies show no risk or a small risk among current users, which drops with time following cessation (Collaborative Group on Hormonal Factors in Breast Cancer, 1996; Hannaford, 2007; Marchbanks, 2002).

1	Although COC use in the past was linked to development of hepatic ocal nodular hyperplasia and benign hepatic adenoma, large studies do not support this (Heinemann, 1998). Moreover, no evidence supports concern for greater risk of hepatocellular cancer (Maheshwari, 2007). For women with known tumors, COCs may be used in those with focal nodular hyperplasia, but avoided in those with benign hepatic adenoma and hepatocellular carcinoma (Kapp, 2009b). Rates of colorectal cancer appear to be reduced in ever users (Bosetti, 2009; Luan, 2015).

1	Cholestasis and cholestatic jaundice are uncommon, but they resolve when COCs are discontinued. In women who have active hepatitis, COCs should not be initiated, but these may be continued in women who experience a flare of their liver disease while already taking COCs. Use of progestin-only contraception in these women is not restricted. Moreover, there is no reason to withhold COCs from women who have recovered. Mild compensated cirrhosis does not limit the use of COCs or progestin-only methods. But in those with severe decompensated disease, all hormonal methods are avoided (Kapp, 2009a).

1	Chloasma, which is hyperpigmentation of the face and forehead, is more likely in women who demonstrated such a change during pregnancy (Chap. 4, p. 53). This is less common with lowdose estrogen formulations. Although previously used for treating unctional ovarian cysts, low-dose COC formulations have been shown to have no efects related to cyst resolution or prevention (European Society of Human Reproduction and Embryology, 2001; Grimes, 2014).

1	Many noncontraceptive beneits are associated with COC use (American College of Obstetricians and Gynecologists, 20 16c). And indeed, COCs may be used for these efects, even in those without contraceptive needs. Dysmenorrhea and heavy menstrual bleeding lessen with COC use. Another action is to improve androgenic conditions such as acne and hirsutism. For women with premenstrual dysphoric disorder (PMDD), several studies have shown symptom improvement in those who use the drospirenone-containing COC Yaz (Lopez, 2012b; Pearlstein, 2005; Yonkers, 2005).

1	he Ortho Evra patch contains ethinyl estradiol and the progestin norelgestromin. It has an inner layer containing an adhesive and hormone matrix, and a water-resistant outer layer. Thus, women can wear the patch in bathtubs, showers, swimming pools, saunas, and whirlpools without decreased eicacy. he patch may be applied to buttocks, upper outer arm, lower abdomen, or upper torso, but the breasts are avoided. Because the hormones are combined with the adhesive, improper skin adherence will lower hormone absorption and eicacy. herefore, if a patch is so poorly adhered that it requires reinforcement with tape, it should be replaced. Initiation of the patch is the same as for COCs, and a new patch is applied weekly for 3 weeks, followed by a patch-free week to allow withdrawal bleeding. Although a patch is ideally worn no longer than 7 days, hormone levels remain in an efective range for up to 9 days. This afords a 2-day window for patch-change delays (Abrams, 2001).

1	In general, the transdermal patch and vaginal ring produce metabolic changes, side efects, and eicacy rates comparable to those with COC pills. However, the patch has been associated with a higher thromboembolism risk in some but not all studies (Cole, 2007; Jick, 2010; Lidegaard, 2011). In response, the Food and Drug Administration (2015 b) approved labeling for the patch to state that the risk for VTE may be increased compared with other COCs, and relative risk estimates range from 1.2 to 2.2. Obesity-90 kg or greater-may be associated with a higher risk for patch contraceptive failure (Janssen Pharmaceuticals, 2015; Zieman, 2002). Finally, applicationsite reaction and breast tenderness are more frequent during initial cycles in patch wearers (Urdl, 2005).

1	The NuvaRing is yet another form of combination hormonal contraception and is a lexible intravaginal ring. he ring is constructed of ethinyl vinyl acetate, and it measures 54 mm in diameter and 4 mm in cross section (Fig. 38-5). During FIGURE 38-5 NuvaRing: estrogen-progestin-releasing vaginal contraceptive ring. insertion, the ring is compressed and threaded into the vagina, but no specific final orientation within the vagina is required. Its core releases ethinyl estradiol and the progestin etonogestrel, which are absorbed across the vaginal epithelium. Before being dispensed, the rings are refrigerated, and once dispensed, their shelflife is 4 months. he ring is placed within 5 days of menses onset and, after 3 weeks of use, is removed for 1 week to allow withdrawal bleeding. Contraception will still be aforded if a ring is left in place for a fourth week (Merck, 2016b).

1	Patient satisfaction is high with this method, although vagi nitis, ring-related events, and leukorrhea are more common (Lopez, 2013b; Oddsson, 2005). Despite this, no deleterious been found (Lete, 2013; Veres, 2004). A ring may be used con currently with vaginal medications or with a tampon (Haring, 2003; Verhoeven, 2004a,b). Approximately 70 percent of part ners feel the ring during intercourse (Dieben, 2002). If this is bothersome, the ring may be removed for intercourse but should be replaced within 3 hours to maintain eicacy.

1	Both intramuscular depot medroxyprogesterone acetate-DepoProvera (DMPA)-150 mg every 3 months, and norethisterone enanthate, 200 mg every 2 months, are injectable progestin contraceptives used worldwide. Of the two, DMPA is available in the United States. DMPA is injected into the deltoid or gluteus muscle, but massage is avoided to ensure that the drug is released slowly. lternatively, a subcutaneous version, depo-subQprovera 104, is also available and is injected into the subcutaneous tissue of the anterior thigh or abdomen every 3 months.

1	DMPA is efective, and as with other progestin-only methods, contraception is provided by ovulation inhibition, greater cervical mucus viscosity, and creation of an endometrium unfavorable for ovum implantation. Initial injection is given within the irst 5 days following menses onset. Serum levels suicient for contraception are observed by 24 hours. Thus, no additional contraceptive method is required for initiation within this window. Alternatively, limited data support a "quick weart," or initiation of DMPA regardless of cycle day. If so implemented, investigators recommend an initial negative pregnancy test result before injection, a supplemental contraceptive method during the 7 days following injection, and a second pregnancy test after 3 to 6 weeks to identiy an early pregnancy (Rickert, 2007; Sneed, 2005). Pregnancies conceived during DMPA use are not associated with a higher risk of fetal malformation (Katz, 1985). For women who present for intramuscular DMPA reinjection more

1	2007; Sneed, 2005). Pregnancies conceived during DMPA use are not associated with a higher risk of fetal malformation (Katz, 1985). For women who present for intramuscular DMPA reinjection more than 13 weeks or for subcutaneous DMPA reinjection more than 14 weeks ater the prior dose, the manufacturer recommends exclusion of pregnancy before reinjection (Pfizer, 2015a,b).

1	Injected progestins ofer the convenience of a 3-month dosing schedule, contraceptive eicacy comparable with or better than COCs, and minimal to no lactation impairment. Irondeiciency anemia is less likely in long-term users because of amenorrhea, which develops in up to 50 percent after 1 year and in 80 percent after 5 years. Similar to other progestin-only contraceptive, irregular menstrual bleeding is common, and a fourth of women dis continued DVIPA in the irst year because of this (Cromer, 1994). Unique to DMPA, prolonged anovulation can follow discontinuation, which results in delayed fertility resumption. After injections are stopped, a fourth of patients do not resume regular menses for up to 1 year (Gardner, 1970). Accordingly, DMPA may not be ideal for women who plan to use birth control only brieRy before attempting conception.

1	DMPA may not be ideal for women who plan to use birth control only brieRy before attempting conception. As with other progestins, DMPA has not been associ ated with cardiovascular events or stroke in otherwise healthy women. However, in those with severe hypertension, a higher risk of stroke has been found in DMPA users (World Health Organization, 1998). Moreover, the US MEC authors express levels from DMPA in women with vascular disease or multiple risks for cardiovascular disease.

1	Weight gain is generally attributed to DMPA, and these increases are comparable between the two depot forms (Bahamondes, 2001; Vickery, 2013; Westhof, 2007c). In long-term users, loss of bone mineral density is also a potential problem (Petitti, 2000; Scholes, 1999). In 2004, the FDA added a black box warning to DMPA labeling, which notes that this concern is probably most relevant for adolescents, who are building bone mass, and perimenopausal women, who will soon have increased bone loss during menopause. That said, World Health Organization (1998) and American College of Obstetricians and Gynecologists (20 16b) believe that DMPA should not be restricted in those high-risk groups. And, it seems prudent to reevaluate overall risks and beneits during extended use. It is somewhat reassuring that bone loss appears to be reversible after discontinuation of therapy, although reversal is still not complete after 18 to 24 months (Clark, 2006; Scholes, 2002).

1	So-called mini-pils are progestin-only contraceptives that are taken daily. hese contraceptives have not achieved widespread popularity and are used by only 0.4 percent of reproductive-aged American women (Hall, 2012). Unlike COCs, they do not reliably inhibit ovulation. Rather, their efectiveness depends more on cervical mucus thickening and endometrial atrophy. Because mucus changes are not sustained longer than 24 hours, mini-pills should be taken at the same time every day to be maximally efective. If a progestin-only pill is taken even 4 hours late, a supplemental form of contraception must be used for the next 48 hours. Progestin-only pills are contraindicated in women with known breast cancer or pregnancy. Other cautions are listed in Table 38-2. For many years, male and female condoms, vaginal diaphragms, and periodic abstinence have been used for contraception with variable success (see Table 38-2). When used properly, condoms

1	For many years, male and female condoms, vaginal diaphragms, and periodic abstinence have been used for contraception with variable success (see Table 38-2). When used properly, condoms FIGURE 38-6 FC2 Female Condom insertion and positioning. A. diaphragm. B. The inner ring is pushed inward with an index finger. provide considerable but not absolute protection against a broad range of sexually transmitted diseases, including HIV (Eaton, 2014). Contraceptive eicacy of the male condom is enhanced appreciably by a reservoir tip and probably by the addition of a spermicide. Such agents, as well as those used for lubrication, should be water-based because oil-based products degrade latex condoms and diaphragms.

1	For individuals sensitive to latex, condoms made from lamb intestines are efective, but they do not provide infection protection. Fortunately, nonallergenic condoms have been developed that are made of polyurethane or of synthetic elastomers. Polyurethane condoms are efective against STDs but have a higher breakage and slippage rate compared with latex condoms (Gallo, 2012a).

1	The only female condom available in the United States is marketed as the FC2 Female Condom. It is a synthetic nitrile sheath with one lexible polyurethane ring at each end. Its open ring remains outside the vagina, whereas its closed internal ring is fitted under the symphysis like a diaphragm (Fig. 38-6). The female condom can be used with both water-based and oilbased lubricants. Male condoms should not be used concurrently because simultaneous use may cause friction that leads to condom slipping, tearing, and displacement. Following use, the female condom outer ring should be twisted to seal the condom so that no semen spills. As an added value, the female condom may ofer some protection against STDs (Minnis, 2005). The diaphragm consists of a circular latex dome of variable diameter supported by a circumferential latex-covered metal spring. It is efective when used in combination with spermicidal

1	The inner ring is squeezed for insertion. The sheath is inserted similarly to a jelly or cream. The spermicide is applied into the dome cup and along the device rim. The diaphragm is then positioned so that the cup faces the cervix and that the cervix, vaginal fornices, and anterior vaginal wall are partitioned efectively from the remainder of the vagina and the penis. In this fashion, the centrally placed spermicide is held against the cervix. When appropriately positioned, one rim is lodged deep in the posterior vaginal fornix, and the opposite rim its behind the inner surface of the symphysis and immediately below the urethra (Fig. 38-7). If a diaphragm is too small, it will not remain in place. If it is too large, it is uncomfortable when forced into position. A coexistent cystocele or uterine prolapse typically leads to instability and expulsion. Because size and spring flexibility must be individualized, the diaphragm is itted by

1	FIGURE 38-7 A diaphragm in place creates a physical barrier between the vagina and cervix. providers and available only by prescription. For use, the diaphragm and spermicide can be inserted hours before intercourse. If more than 6 hours elapse, the diaphragm can remain but additional spermicide is placed in the upper vagina for maximum protection. Spermicide is reapplied before each subsequent coital episode. The diaphragm is not removed for at least 6 hours after intercourse. Because toxic shock syn drome has been described following its use, it may be worth while to remove the diaphragm at 6 hours, or at least the next morning, to minimize this rare event. Diaphragm use is associ ated with a slightly greater rate of urinary infections, presumably from urethral irritation by the ring under the symphysis.

1	FemCap is currently the only available cervical cap in the United States. Made of silicone rubber, it has a sailor-cap shape with a dome that covers the cervix and a lared brim, which allows the cap to be held in place by the muscular walls of the upper vagina. Available in 22-, 26-, and 30-mm sizes, it is used with a spermicide applied once at insertion to both sides of the dome cup. For contraception, it should remain in place for 6 hours following coitus and may remain for up to 48 hours. Even with proper itting and correct use, pregnancy rates with this method are higher that with the diaphragm (Gallo, 2012b; Mauck, 1999).

1	These family planning methods attempt to identiY the fertile days each cycle and advise sexual abstinence during these days. Their major drawback is their limited eicacy, which is shown in Table 38-1r. Common forms of these fertility awarenessbased (FAB) methods include Standard Days, Temperature Rhythm, Cervical Mucus, and Symptothermal Methods. Some smartphone applications aim to assist these practices (Fehring, 2013). he Standard Days Method counsels women to avoid unprotected intercourse during cycle days 8 through 19. For successful use, women must have regular monthly cycles of 26 to 32 days. hose who use this method can mark a calendar or can use Cycle-Beads, which is a ring of counting beads, to keep track of their days.

1	he Temperature Rhythm Method relies on a sustained O.4°F rise in the basal body temperature, which usually precedes ovulation. For maximum eicacy, the woman must abstain from intercourse from'the irst day of menses through the third day after the temperature increase. he Cervical Mucus Method, also called the Two-Day Method or Billings Method, relies on awareness of vaginal "dryness" and "wetness." hese reflect changes in the amount and quality of cervical mucus at diferent times in the menstrual cycle. With the Billings Method, abstinence is required from the beginning of menses until 4 days after slippery mucus is identified. With the T wo-Day Method, intercourse is considered safe if a woman did not note mucus on the day of planned intercourse or the day prior.

1	he Symptothermal Method combines changes in cervical mucus-onset of fertile period; changes in basal body temperature-end of fertile period; and calculations to estimate the time of ovulation. This method is more complex to learn and apply, but it does not appreciably improve eicacy. These contraceptives are marketed variously as creams, jellies, suppositories, films, and aerosol foam. \10st can be purchased without a prescription. They are considered a less efective method (see Table 38-1). If pregnancy does occur, they are not teratogenic (Briggs, 2015).

1	Typically, spermicides function by providing a physical barrier to sperm penetration and a chemical spermicidal action. The active ingredient is nonoxynol-9 or octoxynol-9. Although these are spermicidal, they do not provide STD protection. Ideally, spermicides must be deposited high in the vagina in contact with the cervix shortly before intercourse. Their duration of maximal efectiveness is usually no more than 1 hour, and thereafter, they must be reinserted before repeat intercourse. Douching is avoided for at least 6 hours after coitus.

1	The Today contraceptive sponge is an over-the-counter, onesize-fits-all device. The nonoxynol-9-impregnated polyurethane disc is 2.5 cm thick and 5.5 cm wide and has a dimple on one side and satin loop on the other (Fig. 38-8). The sponge can be inserted up to 24 hours prior to intercourse, and while in place, it provides contraception regardless of coital frequency. It should remain in place for 6 hours after intercourse. Pregnancy is prevented primarily by the spermicide nonoxynol-9 and to a lesser extent by covering the cervix and absorbing semen. FIGURE 38-8 Today sponges. The sponge is moistened with tap water and gently squeezed to create light suds. It is then tioned with the dimple directly against the cervix. The fabric loop trails within the vagina and can be hooked with a finger to later extract the sponge. TABLE 38-3. Methods Available for Use as Emergency Contraception Pills per Number of COC Pillsb,c aDoses taken 12 hours apart if multiple.

1	TABLE 38-3. Methods Available for Use as Emergency Contraception Pills per Number of COC Pillsb,c aDoses taken 12 hours apart if multiple. bOther COC brands with formulations identical to those above may also be used. (Use of an antiemetic agent before taking the medication will lessen the risk of nausea, which is a common side effect. COC = combination oral contraceptive; EE = ethinyl estradiol; IUD = intrauterine device; LNG = levonorgestrel; PRM = progesterone-receptor modulator.

1	COC = combination oral contraceptive; EE = ethinyl estradiol; IUD = intrauterine device; LNG = levonorgestrel; PRM = progesterone-receptor modulator. Although the sponge is possibly more convenient than the diaphragm or condom, it is less efective than either (Kuyoh, 2013). Most common causes for method discontinuance are pregnancy, irritation, discomfort, or vaginitis (Beckman, 1989). Although toxic shock syndrome has been reported with the contraceptive sponge, it is rare, and evidence suggests that the sponge may actually limit production of the responsible staphylococcal exotoxin (Remington, 1987). Still, it is recommended that the sponge not be used during menses or the puerperium.

1	Following unprotected sexual intercourse, many women present for contraceptive care. Several emergency contraception (EC) regimens substantially lower the likelihood of an unwanted pregnancy when used correctly. Current methods include COCs, progestins, progesterone antagonists, and copper-containing IUDs (Table 38-3). Overall, the IUD is most efective, and ulipristal acetate is the most eicient oral regimen (American College of Obstetricians and Gynecologists, 2017a). Patients can obtain information regarding emergency contraception by calling 1-888-NOT-2-LATE or accessing The Emergency Contraception Website: http://not-2-late.com.

1	Except for allergy to a particular component, no conditions in the US MEC contraindicate hormonal EC methods. With progestin-only regimens, levonorgestrel is taken as a single, onetime 1.5-mg dose (Arowojolu, 2002). his is now recommended instead of two 0.75-mg doses separated by 12 or 24 hours (Ngai, 2005). Dosing begins ideally within 72 hours of unprotected coitus but may be given up to 120 hours. Notably, the singledose regimen is available over-the-counter without a prescription to all reproductive-aged women (Food and Drug Administration, 2013,r2015a). One progesterone-receptor modulator currently available for EC is ulipristal acetate and is marketed as Ela. It is taken as a single 30-mg tablet up to 120 hours after unprotected intercourse (Brache, 2010; Watson, 2010).

1	Also known as the Yuzpe method, this older EC method provides a minimum of 100 jLg of ethinyl estradiol and 0.5 mg of levonorgestrel in each of two doses. As shown in Table 38-3, a suicient dose may be achieved by two or more pills. he first dose is taken ideally within 72 hours of intercourse but may be given up to 120 hours. The initial dose is followed 12 hours later by a second equivalent dose. The major mechanism with all hormonal regimens is inhibition or delay of ovulation. Of oral methods, failure rates are lowest with ulipristal (1 to 2 percent) and greatest with the Yuzpe method (2 to 3.5 percent) (Cleland, 2014). IfEC fails to prevent pregnancy or is mistimed, no associations with major congenital malformation or pregnancy complications have been noted with these hormonal methods (Jatlaoui, 2016; Levy, 2014).

1	With EC administration, nausea and vomiting can be an important side efect (American College of Obstetricians and Gynecologists, 2015b; Gemzell-Danielsson, 2013). Accordingly, an oral antiemetic may be prescribed at least 1 hour before each dose (Rodriguez, 2013). If a woman vomits within 2 hours of a dose, the dose is repeated. For women who are candidates, Cu-IUD insertion is the most efective emergency contraceptive method and provides an efective 10-year method of contraception (Cheng, 2012). If an IUD is placed up to 5 days after unprotected coitus, the failure rate approximates only 0.1 percent (Cleland, 2012; Wu, 2010).

1	For mothers who are nursing exclusively, ovulation during the irst 10 weeks after delivery is unlikely. Nursing, however, is not a reliable method of family planning for women whose infants are on a daytime-only feeding schedule. Moreover, waiting for first menses involves a risk of pregnancy, because ovulation usually antedates menstruation. Certainly, after the first menses, contraception is essential unless the woman desires pregnancy. As shown in Table 38-2, all methods may be suitable for nursing mothers after the initial weeks, during which thromboembolism risks are still great. With all hormonal methods, very small quantities are excreted in breast milk, but no adverse efects on infants have been reported (Phillips, 2015; World Health Organization, 1988). Although not robust, some older studies link decreased infant weight gain or milk volume with early initiation of combination oral contraceptives before 6 weeks postpartum (Lopez, 2015c; Tepper, 2016a).

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1	Sufrin CB, Postlethwaite 0, Armstrong A, et al: Neisseria gonorrhea and Chlamydia trachomatis screening at intrauterine device insertion and pelvic inflammatory disease. Obstet GynecoIn120(6):1314, 2012 Svendal G, Berk M, Pasco JA, et al: The use of hormonal contraceptive agents and mood disorders in women. J Afect Disord 140(1):92,n2012 Tatum HJ, Schmidt FH, Jain AK: Management and outcome of pregnancies associated with Copper-T intrauterine contraceptive device. Am J Obstet Gynecol 126:869, 1976 Tepper NK, Curtis KM, Nanda K, et al: Safety of intrauterine devices among women with HIV: a systematic review. Contraception 94(6):713, 2016a Tepper NK, Phillips SJ, Kapp N, et al: Combined hormonal contraceptive use among breastfeeding women: an updated systematic review. Contraception 94(3):262n2016b Tepper NK, Steenland MW, Gaield ME, et al: Retention of intrauterine devices in women who acquire pelvic inflammatory disease: a systematic review. Contraception 87(5):655, 2013

1	Tepper NK, Steenland MW, Gaield ME, et al: Retention of intrauterine devices in women who acquire pelvic inflammatory disease: a systematic review. Contraception 87(5):655, 2013 Tepper NK, Whiteman MK, Marchbanks PA, et al: Progestin-only contraception and thromboembolism: a systematic review. Contraception 94(6):678, 2016c Tepper NK, Whiteman MK, Zapata LB, et al: Safety of hormonal contraceptives among women with migraine: a systematic review. Contraception 94(6):630,n2016d Teva Women's Health: ParaGard T 380A intrauterine copper contraceptive: prescribing information, 2014. Available at: http://paragard.com/Pdf/ParaGard-PLpdf. Accessed October 10, 2016 he ACQUIRE Project: The Postpartum Intrauterine Device: a Training Course for Service Providers. New York, EngenderHealth, 2008 honneau PF, Almont TE: Contraceptive eicacy of intrauterine devices. Am J Obstet GynecoIn198:248, 2008

1	Tornblom-Paulander S, Tingaker BK, Werner A, et al: Novel topical formulation of lidocaine provides signiicant pain relieffor intrauterine device insertion: pharmacokinetic evaluation and randomized placebo-controlled trial. Ferril Steriln103(2):422, 2015 Trussell J: Contraceptive eicacy. In Hatcher A, Trussell J, Nelson AL, et al (eds): Contraceptive Technology, 20th ed. New York, Ardent Media, 2011a Trussell J: Contraceptive failure in the United States. Contraception 70:89, 2011b Tsilidis KK, Allen NE, Key TJ, et al: Oral contraceptive use and reproductive factors and risk of ovarian cancer in the European Prospective Investigation into Cancer and Nutrition. Br J Cancer 105(9): 1436,n2011 Turok OK, Eisenberg DL, Teal SB, et al: A prospective assessment of pelvic infection risk following same-day sexually transmitted infection testing and levonorgestrel intrauterine system placement. Am J Obstet Gynecol 215(5):599.el, 2016

1	Urdl W, Apter 0, Alperstein A, et al: Contraceptive eicacy, compliance and beyond: factors related to satisfaction with once-weekly transdermal compared with oral contraception. Eur J Obstet Gynecol Reprod Bioi 121n:202, 2005 van H ylckama Vlieg A, Helmerhorst FM, Vandenbroucke JP, et al: The venous thrombotic risk of oral contraceptives, efects of oestrogen dose and progestogen type: results of the MEGA case-control study. BMJ 339:b2921, 2009 Van Vliet A, Grimes DA, Helmerhorsr FM, et al: Biphasic versus mono phasic oral contraceptives for contraception. Cochrane Database Syst Rev 6:CD002032, 2006, Reairmed 2011a Van Vliet HA, Grimes DA, Lopez LM, et al: Triphasic versus monophasic oral contraceptives for contraception. Cochrane Database Syst Rev 11:CD003553,n2011b Van Vliet HA, Raps M, Lopez LM, et al: Quadriphasic versus monophasic oral contraceptives for contraception. Cochrane Database Syst Rev 1ln:CD009038,n2011c

1	Van Vliet HA, Raps M, Lopez LM, et al: Quadriphasic versus monophasic oral contraceptives for contraception. Cochrane Database Syst Rev 1ln:CD009038,n2011c Veres S, Miller L, Burington B: A comparison between the vaginal ring and oral contraceptives. Obstet Gynecol 104:555, 2004 Verhoeven CH, Dieben TO: he combined contraceptive vaginal ring, NuvaRing, and tampon co-usage. Contraception 69(3): 197, 2004a Verhoeven CH, van den Heuvel MW, Mulders TM, et al: The contraceptive vaginal ring, NuvaRing, and antimycotic co-medication. Contraception 69(2): 129, 2004b Vessey MP, Johnson B, Doll R, et al: Outcome of pregnancy in women using intrauterine devices. Lancet 1 :495, 1974 Vessey MP, Meisler L, Flavel R, et al: Outcome of pregnancy in women using diferent methods of contraception. Br J Obstet Gynaecol 86:548, 9 Vickery Z, Madden T, Zhao Q, et al: Weight change at 12 months in users of three progestin-only contraceptive methods. Contraception 88(4):503, 2013

1	Vinogradova Y, Coupland C, Hippisley-Cox J: Use of combined oral contraceptives and risk of venous thromboembolism: nested case-control studies using the QResearch and CPRD data-bases. BMJ 350:h2135, 2015 Wallach M, Grimes DA (eds): Modern Oral Contraception. Updates from he Contraception Report. Totowa, Emron, 2000 Walsh T, Grimes 0, Frezieres R, et al: Randomised controlled trial of prophylactic antibiotics before insertion of intrauterine devices. IUD Study Group. Lancet 351:1005, 1998 Watson: Ella prescribing information. 2010. Available at: http://www.access data. fda.govl drugsatfda_docs/labell20 1 01022474s0001bl.pdf. Accessed December 27,n2016 Wechselberger G, Wolfram 0, Piilzl P, et al: Nerve injury caused by removal of an implantable hormonal contraceptive. Am J Obstet Gynecol 195(1):323, 2006 Westhof C: IUDs and colonization or infection with Actinomyces. Contraception 75:S48, 2007a

1	Westhof C: IUDs and colonization or infection with Actinomyces. Contraception 75:S48, 2007a WesthofC, Heartwell S, Edwards S, et al: Initiation of oral contraceptive using a quick start compared with a conventional start: a randomized controlled trial. Obstet Gynecoln109:1270, 2007b Westhof C, Jain JK, Milson, et al: Changes in weight with depot medroxyprogesterone acetate subcutaneous injection 104 mg/0.65 mL. Contraception 75:261,n2007c Westhof C, Kerns J, Morroni C, et al: Quick start: novel oral contraceptive initiation method. Contraception 66: 141, 2002 WesthofC, Wieland 0, Tiezzi L: Depression in users of depo-medroxyprogesterone acetate. Contraception 51(6):351,n1995 Westhof CL, T orgal AH, Mayeda ER, et al: Pharmacokinetics of a combined oral contraceptive in obese and normal-weight women. Contraception 81(6):474,n2010

1	Westhof CL, T orgal AH, Mayeda ER, et al: Pharmacokinetics of a combined oral contraceptive in obese and normal-weight women. Contraception 81(6):474,n2010 Whitaker AK, Chen BA, Borgatta L: Society of Family Planning Guidelines: postplacental insertion of intrauterine devices. Contraception October 5, 20n17 [Epub ahead of print) Wilailak S, Vipupinyo C, Suraseranivong V, et al: Depot medroxyprogesterone acetate and epithelial ovarian cancer: a multicentre case-control study. BJOG 119(6):672,2012 World Health Organization: A multinational case-control study of ectopic pregnancy. Clin Reprod Ferriln3:131, 1985 World Health Organization: Acute myocardial infarction and combined oral contraceptives: results of an international multi-center case-control study. Lancet 349:1202, 1997

1	World Health Organization: Acute myocardial infarction and combined oral contraceptives: results of an international multi-center case-control study. Lancet 349:1202, 1997 World Health Organization: Cardiovascular disease and use of oral and injectable progestogen-only contraceptives and combined injectable contraceptives. Resulrs of an international, multicenter, case-control study. Contraception 57:315, 1998 World Healrh Organization: Depot-medroxyprogesterone acetate (DMPA) and risk of endometrial cancer. Int J Cancer 49: 186, 1991a World Health Organization: Depot-medroxyprogesterone acetate (DMPA) and risk of invasive squamous cell cervical cancer. Contraception 45(4): 299, 1992 World Health Organization: Depot-medroxyprogesterone acetate (DMPA) and risk of liver cancer. Int J Cancer 49(2):182, 1991b World Health Organization: Efects of hormonal contraceptives on breast milk composition and infant growth. Stud Fam Plann 19/361, 1988

1	World Health Organization: Efects of hormonal contraceptives on breast milk composition and infant growth. Stud Fam Plann 19/361, 1988 World Health Organization: Ischaemic stroke and combined oral contraceptives: results of an international, multi-center case-control study. Lancet 348:498, 1996 World Health Organization: Mechanism of action, safety and eicacy of intrauterine devices. Technical Reporr No. 753, Geneva, Switzerland, WHO, 1987 World Health Organization: vledical Eligibility for Contraceptive Use, 5th ed. Geneva, World Health Organization, 2015 Wu S, Godfrey EM, Wojdyla 0, et al: Copper T380A intrauterine device for emergency contraception: a prospective, multicentre, cohort clinical trial. BJOG 117(10):1205,2010 XU JX, Remedios E, Duthie A, et al: Intrauterine contraceptive device: cause of small bowel obstruction and ischaemia. ANZ J Surg May 26, 2015 [Epub ahead of print)

1	XU JX, Remedios E, Duthie A, et al: Intrauterine contraceptive device: cause of small bowel obstruction and ischaemia. ANZ J Surg May 26, 2015 [Epub ahead of print) XU JX, Rivera R, Dunson TR, et al: A comparative study of two techniques used in immediate postplacental insertion (IPPI) of the Copper T-380A IUD in Shanghai, People's Republic of China. Contraception 54(1):33, 1996 Yamazaki M, Dwyer K, Sobhan M, et al: Efect of obesity on the efectiveness of hormonal contraceptives: an individual participant data meta-analysis. Contraception 92(5):445, 2015 Yonkers A, Brown C, Pearlstein TB, et al: Eicacy of a new low-dose oral contraceptive with drospirenone in premenstrual dysphoric disorder. Obstet Gynecol 106:492, 2005 Zeino MY, Wietfeldt ED, Advani V, et al: Laparoscopic removal of a copper intrauterine device from the sigmoid colon. JSLS 15(4):568,n2011

1	Zeino MY, Wietfeldt ED, Advani V, et al: Laparoscopic removal of a copper intrauterine device from the sigmoid colon. JSLS 15(4):568,n2011 Zieman M, Guillebaud J, Weisberg E, et al: Contraceptive eicacy and cycle control with the Orrho Evra/Evra transdermal system: the analysis of pooled data. Fertil Steril 77:S13, 2002 PUERPERAL TUBAL STERILIZATION.................702 NON PUERPERAL TUBAL STERILIZATION............ 704 COMPLICATIONS. . . . . . . ............. 704 TRANSCERVICAL STERILIZATION................... 705 VASECTOMY....................................706 In order, therore, to render a woman permanenty sterile by an operation upon the tubes, they must be excised by wedge-shaped incisions at the cornua of the uterus and the wounds closed by sutures. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) Sterilization is a popular choice of contraception for millions of men and women. Among women using contraception, one third rely on either male or female sterilization (Daniels, 2015). This procedure is indicated in those requesting sterilization and who clearly understand its permanence and its diicult and often unsuccessful reversal. All persons considering sterilization should also be counseled regarding alternative contraceptive choices (American College of Obstetricians and Gynecologists, 2017a,c). Female sterilization is usually accomplished by occlusion, excision, or division of the fallopian tubes. Puerperal sterilization procedures follow cesarean or vaginal delivery and approximately 7 percent of all live births in the United States (Moniz, 2017). Nonpuerperal tubal sterilization is done at a time unrelated to recent pregnancy and is also termed interval sterilization.

1	For several days postpartum, the uterine fundus lies at the level of the umbilicus, and fallopian tubes are accessible directly beneath the abdominal wall. Moreover, abdominal laxity allows easy repositioning of the incision over each uterine cornu.

1	On our service, puerperal tubal ligation is performed by a surgical team dedicated to this role the morning after delivery. This timing minimizes hospital stay but lowers the likelihood that postpartum hemorrhage would complicate recovery following surgery. In addition, the status of the newborn can be better ascertained before surgery. In contrast, some prefer to perform sterilization immediately following delivery and use neuraxial analgesia already placed for labor. In this model, barriers to sterilization can be lessened by designating these postpartum surgeries as urgent, especially in high-volume labor and delivery units, which usually prioritize limited operating-room availability for intrapartum procedures (American College of Obstetricians and Gynecologists, 2016; Potter, 2013).

1	Various techniques are now used to disrupt tubal patency. In general, a midtubal segment of fallopian tube is excised, and the severed ends seal by fibrosis and peritoneal regrowth. Commonly used methods of puerperal sterilization include the Parkland, Pomeroy, and modiied Pomeroy techniques (American College of Obstetricians and Gynecologists, 2017a). Less often, Filshie clips are used (Madari, 2011). Irving and Uchida techniques or Kroener fimbriectomy are rarely used because of their increased required dissection or unfavorably high failure rates. Also, in the absence of uterine or other pelvic disease, hysterectomy solely for sterilization at the time of cesarean delivery, early in the puerperium, or even remote from pregnancy is diicult to justiy. It carries significantly increased surgical morbidity compared with tubal sterilization.

1	Evidence suggests that the fallopian tube may be the origin of pelvic serous carcinomas, especially those of the ovary. With this knowledge, the Society of Gynecologic Oncologists (2013) and American College of Obstetricians and Gynecologists (2017b) recommend consideration of salpingectomy to lower cancer risks. Speciically, for women at average risk of ovarian cancer, risk-reducing salpingectomy should be discussed and considered with patients at the time of abdominal or pelvic surgery, at hysterectomy, or in lieu of tubal ligation. Spinal analgesia is typically selected for cases scheduled for the first postpartum day. If done more proximate to delivery, the same epidural catheter used for labor analgesia can be used for sterilization analgesia. Notably, for those with preeclampsia, HELLP (hemolysis, �levated liver enzyme levels, low 2latelet count) syndrome, or gestational thrombocytopenia, platelet levels should be > 100,000 for spinal blockade (Chap. 25, p.

1	HELLP (hemolysis, �levated liver enzyme levels, low 2latelet count) syndrome, or gestational thrombocytopenia, platelet levels should be > 100,000 for spinal blockade (Chap. 25, p. 496). General anesthesia may be less desirable due to residual pregnancy-related airway vulnerabilities (Bucklin, 2003). The bladder is emptied before surgery to avoid its laceration. A full bladder can also push the fundus above the umbilicus.

1	A small infraumbilical incision is ideal for several reasons. As noted, the fundus in most cases lies near the umbilicus. Second, the umbilicus usually remains the thinnest portion of the anterior abdominal wall and requires less subcutaneous dissection to reach the linea alba fascia. hird, an infraumbilical incision ofers fascia with suicient integrity to provide a closure that has minimal risk for later incisional hernia. Last, incisions that follow the natural curve of the lower umbilical skin fold yield suitable cosmesis. A 2-to 4-cm transverse or vertical skin incision is usually suicient for normal-weight women. For obese women, a 4-to 6-cm incision may be needed for adequate abdominal access.

1	Beneath this incision, the subcutaneous tissue is bluntly separated to reach the linea alba fascia. For this, an Allis clamp can be opened and closed as downward pressure is exerted. Similarly, the blades of two army-navy retractors both pulling in downward yet opposite directions can part the subcutaneous layer. Clearing this fatty tissue away from the fascia isolates the fascia for incision and for later closure without intervening fat, which may impede wound healing.

1	he fascial incision may be transverse or vertical and follows the same orientation of the skin incision. For this, once the linea alba is reached, it is grasped with two Allis clamps-one placed on either side of the planned fascial incision. The purchase of tissue with each clamp should be substantial and creates a small roll of fascia to be incised. Often, the peritoneum is incorporated simultaneously and entered. If not, the peritoneum is grasped with two hemostats and sharply cut. Others may prefer to bluntly enter with a single index finger. Notably, if the initial fascial incision is too small, it can be extended with curved Mayo scissors.

1	Adequate exposure is critical, and army-navy or appendiceal retractors are suitable. For obese women, a slightly larger incision and narrow deeper retractors may be required. If bowel or omentum is obstructing, Trendelenburg position can help displace these cephalad. Digitally packing with a single, moist, fanned-out piece of surgical gauze can also be used, but a hemostat should always be attached to the distal end to avert

1	FIGURE 39-1 Parkland method. A.An avascular site in the meso salpinx adjacent to the fallopian tube is perforated with a small hemostat. The jaws are opened to separate the fallopian tube from the adjacent mesosalpinx for approximately 2.5 cm. B. The freed fallopian tube is ligated proximally and distally with O-chromic suture. The intervening segment of approximately 2 cm is excised, and the excision site is inspected for hemostasis. This method was designed to avoid the initial intimate proximity of the cut ends of the fallopian tube inherent with the Pomeroy procedure. (Reproduced with permission from Hoffman BL, Corton MM: Surgeries for benign gynecologic conditions. In Hoffman BL, Schorge JO, Bradshaw KD, et al: Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 201o6.) its retention. At times, tilting the entire table to the opposite side of the tube being exposed assists tube isolation.

1	he fallopian tube is identiied and grasped at its midportion with a Babcock clamp, and the distal fimbria conirmed. his prevents confusing the round ligament with the midportion of the tube. A common reason for sterilization failure is ligation of the wrong structure, typically the round ligament. Therefore, identiication and isolation of the distal tube prior to ligation is necessary. Whenever the tube is inadvertently dropped, it is mandatory to repeat this identification process. Surgical steps for ligation are outlined in Figures 39-1 and 39-2. FIGURE 39-2 Pomeroy method. During ligation of a tubal loop, plain catgut is used to ensure prompt absorption of the ligature and subsequent separation of the severed tubal ends. (Reproduced with permission from Hoffman BL, Corton MM: Surgeries for benign gynecologic conditions. In Hoffman BL, Schorge JO, Bradshaw KD, et al: Williams Gynecology, 3rd ed. New York, McGraw-Hili Education, 201o6.)

1	Steps of salpingectomy are shown in Figure 39-3. he umbilical incision generally will need to be larger to allow an adequate view of the tube and mesosalpinx and to place clamps. With total salpingectomy, the entire mesosalpinx must be divided to free the fallopian tube. In two small cohorts undergoing salpingectomy ater vaginal birth, surgical times were longer than for tubal occlusion, and in one report, blood loss was increased (Danis, 2016; Powell, 2017). With salpingectomy and cesarean delivery, total blood loss rates were not statistically higher (Powell, 2017; Shinar, 2017). After surgery, diet is given as tolerated. Ileus is infrequent and should prompt concern for bowel injury, albeit rare. Most women have an uncomplicated course and are discharged on the first postoperative day.

1	These techniques and other modiications basically consist of (1) ligation and resection at laparotomy as described earlier for puerperal sterilization; (2) application of permanent rings, clips, or inserts to the fallopian tubes by laparoscopy or hysteroscopy; or (3) electrocoagulation of a tubal segment, usually through a laparoscope. A detailed description and illustration of these can be found in Wiliams Gynecoloy, 3rd edition (hompson, 2016).

1	In the United States, a laparoscopic approach to interval tubal sterilization is the most common. The procedure is frequently performed in an ambulatory surgical setting under general anesthesia. In almost all cases, the woman can be discharged within several hours. Minilaparotomy using a 3-cm suprapubic incision is also popular, especially in resource-poor countries. Major morbidity is rare with either minilaparotomy or laparoscopy. lthough not often used, the peritoneal cavity can be entered through the posterior vaginal fornix via colpotomy to perform tubal interruption. Pregnancy following sterilization is infrequent. The Collaborative Review of Sterilization (CREST) study followed 10,863 women who had undergone tubal sterilization from 1978 through 1986 (Peterson, 1996). he cumulative failure rate for the various tubal procedures was 18.5 per 1000 or approximately

1	FIGURE 39-3 A. With salpingectomy, the mesosalpinx is sequentially clamped, cut, and ligated. B. At the cornu, clamps are placed across the fallopian tube and its adjacent mesosalpinx prior to tubal transection. (Reproduced with permission from Stuart GS: Puerperal sterilization. In Yeomans ER, Hoffman BL, Gilstrap, III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili, 201o7.) 0.5 percent. The study found puerperal sterilization to be highly efective. The 5-year failure rate was 5 per 1000, and for 12 years, it was 7 per 1000. Puerperal sterilization fails for two major reasons. First, sur gical errors occur and include transection of the round ligament or only partial transection of the tube. For this reason, both tubal segments are submitted for pathological confirmation. Second, a fistulous tract or spontaneous reanastomosis may form between the severed tubal stumps.

1	Second, a fistulous tract or spontaneous reanastomosis may form between the severed tubal stumps. Approximately 30 percent of pregnancies that follow a failed tubal sterilization procedure are ectopic. his rate is 20 percent for those following a postpartum procedure (Peterson, 1996, 1997). hus, any symptoms of pregnancy in a woman after tubal sterilization must be investigated, and an ectopic preg nancy excluded. Overall, risks for ovarian cancer decline and for breast cancer are unafected following sterilization (Gaudet, 2013; Pearce, 2015). Women who have undergone tubal sterilization are highly unlikely to subsequently have salpingitis (Levgur, 2000). For menorrhagia and intermenstrual bleeding following tubal sterilization, most studies of the risk have found no association (DeStefano, 1985; Peterson, 2000; Shy, 1992).

1	Less objective but important psychological sequelae of sterilization have also been evaluated. In the CREST study, Costello (2002) found that tubal ligation did not change sexual interest or pleasure in 80 percent of women. In most of the 20 percent of women who did report a change, positive efects were 10 to 15 times more likely. Invariably, a number of women express regrets regarding sterilization, and this is especially true if it is performed at a younger age (Curtis, 2006; Kelek:i, 2005). In the CREST study, Jamieson (2002) reported that 7 percent of women who had undergone tubal ligation had regrets by 5 years. his is not limited to their own sterilization, because 6.1 percent of women whose husbands had undergone vasectomy had similar regrets.

1	No woman should undergo tubal sterilization believing that subsequent fertility is guaranteed either by surgery or by assisted reproductive techniques. Both approaches are technically dificult, expensive, and not always successful. In general, pregnancy rates after tubal reversal favor women with age younger than 35 years, with 7 cm of remaining tube, with a short time from antecedent sterilization, and with isthmic-isthmic repairs. With reanastomosis via laparotomy, rates of live births range from 44 to 82 percent (Deieux, 2011; Malacova, 2015). The rate of ectopic pregnancy is 2 to 10 percent after reanastomosis (American Society for Reproductive Medicine, 2015). With reanastomosis to reverse Essure sterilization, only 27 percent of women had subsequent live births (Monteith, 2014).

1	Devices can be inserted via hysteroscopy to occlude the proximal fallopian tubes. he Essure micro insert has a ine stainless-steel inner coil enclosed in polyester fibers and an expandable outer coil of Nitinol-a nickel and titanium alloy (Fig. 39-4). he outer coil expands after placement, allowing the inner fibers to expand. These synthetic fibers incite a chronic inflammatory response to prompt local tissue ingrowth that leads to complete tubal lumen occlusion. For hysteroscopic placement, sedation, paracervical block, or both may be used, and in-oice insertion is often elected. Devices cannot be placed in all women, and some do not tolerate the procedure while awake (DuY, 2005). Bilateral placement is achieved in 81 to 98 percent of cases on a first attempt (la Chapelle, 2015).

1	FIGURE 39-4 Essure microinsert placement hysteroscopically and ingrowth of tissue. (Reproduced with permission from Thompson M, Kho K: Minimally invasive surgery. In Hoffman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hill,o201o6.) Since the introduction of Essure, cited adverse events include abnormal bleeding, perforation of the uterus or fallopian tubes from device migration, and allergy or hypersensitivity reactions, especially to the nickel component (Al-Sai, 2013; IIao, 2015). Some events resulted in device removal that requires abdominal surgery (Casey, 2016; Lazorwitz, 2017). To provide more information of the risks and beneits, the Food and Drug Administration (2016) has drafted a black box warning and patient-decision checklist to aid counseling.

1	Because complete tubal blockage is not 100 percent, it must be conirmed by hysterosalpingography (HSG) 3 months following surgery (Bayer Healthcare, 2002). With such confirmation, eicacy rates for these devices reach 98 to 99 percent (Chudnof, 2015; Munro, 2014). In real-world settings, pregnancies following transcervical sterilization are most frequently attributed to conception before insertion or HSG and to noncompliance with HSG or its misinterpretation. Although data are limited to small case series, pregnancies conceived with Essure in place do not appear to be at increased risk from the device (Arora, 2014; Veersema, 2014). Another insert, Adiana, also stimulates tissue ingrowth for tubal occlusion using a cylindrical, nonabsorbent silicone elastomer matrix. However, for inancial reasons, production of this device has now been discontinued by the manufacturer

1	Vasectomy is safer than tubal sterilization because it is less invasive and is performed with local analgesia (American College of Obstetricians and Gynecologists, 2017a). In a review to compare the two, Hendrix and associates (1999) found that, compared with vasectomy, female tubal sterilization rate, a 10-to 37 -fold failure rate, and costs three times as much. Incision on left side of One disadvantage is that sterilizascrotum, and in tunica tion following vasectomy is not immediate. Complete release of sperm stored V2" of duct removed Incision on in the reproductive tract beyond the right side Ends are sealed interrupted vas deferens takes approximately 3 months or 20 ejaculations.Epididymis The American Urological Association recommends a postprocedural semen analysis at 8 to 16 weeks to document sterility (Sharlip, 2012). During the period before azoospermia is documented, another form of contraception should be used.

1	FIGURE 39-5 Anatomy of male reproductive system showing procedure for vasectomy. The failure rate for vasectomy dur (Hologic, 2012). hat said, patients with these inserts may be encountered and can consider their device efective. lthough not currently available in the United States, quinacrine pellets cause sclerosis at the tubal ostia. Placement at the uterine fundus with an IUD-type inserter method allows pellet migration into the tubal ostia. Of drawbacks, prior cancer associations have been disproved (Sokal, 201 Oa,b). Eicacy appears enhanced by technique modification. In one early cohort of 1335 treated women, pregnancy rates at 10 years were 12 percent (Sokal, 2008). Following insertion technique improvement, a 2-year failure rate of 1.2 percent was calculated by Lippes (2015).

1	Currently, up to a half million men in the United States undergo vasectomy each year (Barone, 2006; Eisenberg, 2010). And 5 percent of women rely on this method for contraception (Daniels, 2015). For sterilization, the vas deferens lumen is disrupted to block the passage of sperm from the testes. Most commonly, a no-scapel vasectomy (NS) accomplishes this with one specialized instrument that grasps the vas deferens and surrounding skin together. A second dissector tool punctures the skin and then isolates the vas (Rogers, 2013). As clariied by the American Urological Association, minimaly invasive vasectomy includes any vas isolation procedure, including the no-scalpel technique, which uses a skin incision measuringr:;1 cm and requires minimal vas dissection (Fig. 39-5) (Sharlip, 2012). Compared with conventional vasectomy that employs incisions > 1 cm and greater dissection, the no-scalpel technique is associated with fewer minor surgical complications, but each is equally efective

1	Compared with conventional vasectomy that employs incisions > 1 cm and greater dissection, the no-scalpel technique is associated with fewer minor surgical complications, but each is equally efective (Cook, 2014).

1	ing the first year is 9.4 per 1000 procedures and 11.4 per 1000 at 2,r3, and 5 years Qamieson, 2004). Failures result from unprotected intercourse too soon after ligation, incomplete occlusion of the vas deferens, or recanalization (Awsare, 2005; Deneux-haraux, 2004). Other than regrets, long-term consequences are rare. One is troublesome chronic scrotal pain, which develops in up to 15 percent of men (Leslie, 2007; vlanikandan, 2004). Previous concerns for atherogenesis, immune-complex mediated disease, testicular cancer, and prostate cancer have been allayed by a number of investigators (Bernal-Delgado, 1998; Giovannucci, 1992; Goldacre, 1983; M0ller, 1994).

1	Reanastomosis of the vas deferens can be completed most efectively using microsurgical techniques. In general, conception rates following reversal are adversely afected by longer duration from vasectomy, poor sperm quality found at reversal, and type of reversal procedure required (American Society for Reproductive Medicine, 2008). Al-Safi ZA, Shavell VI, Hobson DT, et al: Analysis of adverse events with Essure hysteroscopic sterilization reported to the Manufacturer and User Facility Device Experience database. J Minim Invasive Gynecol 20(6):825, 2013 American College of Obstetricians and Gynecologists: Access to postpartum sterilization. Committee Opinion No. 530, July 2012, Reairmed 2016 American College of Obstetricians and Gynecologists: Benefits and risks of sterilization. Practice Bulletin No. 133, February 2013, Reairmed 2017a

1	American College of Obstetricians and Gynecologists: Salpingectomy for ovarian cancer prevention. Committee Opinion No. 620, January 2015b, Reairmed 2017b American College of Obstetricians and Gynecologists: Sterilization of women: ethical issues and considerations. Committee Opinion No. 695, Apri 20l7c American Society for Reproductive Medicine: Vasectomy reversal. Fertil Steril 90:S78, 2008 American Society for Reproductive Medicine: Role of tubal surgety in the era of assisted reproductive technology: a committee opinion. Fertil Steril 103(6):e37,n2015 Arora P, Arora RS, Cahill 0: Essure for management of hydrosalpinx prior to in vitro fertilisation-a systematic review and pooled analysis. B]OG 121(5):527,n2014 Awsare N, Krishnan ], Boustead GB, et al: Complications of vasectomy. Ann R Coli Surg Engl :406, 2005 Barone A, Hutchison PL, Johnson CH, et al: Vasectomy in the Unites States, 2002. ] U rol 176:232, 2006

1	Bayer Healthcare: Essure: instruction for use. 2002. Available at: http://www. hcp.essure-us.com/ assets/ pdf/Link%20 Essure%20 IFU. pdf. Accessed April 28, 2016 Benal-Delgado E, Latour-Perez ], Pradas-Arnal F, et al: The association between vasectomy and prostate cancer: a systematic review of the literature. Fertil Steril 0:201, 1998 Bucklin BA: Postpartum tubal ligation: timing and other anesthetic consideradons. Clin Obstet Gynecol 46(3):657, 2003 Casey], Aguirre F, Yunker A: Outcomes of laparoscopic removal of the Essure sterilization device for pelvic pain: a case series. Contraception 94(2): 190, 2016 Chudnof SG, Nichols JE ]r, Levie M: Hysteroscopic Essure inserts for permanent contraception: extended follow-up results of a phase III multicenter international study. J Minim Invasive Gynecol 22(6):95n1, 2015 Cook A, Pun A, Gallo MF, et al: Scalpel versus no-scalpel incision for vasectomy. Cochrane Database SYSt Rev 3:CD004112, 2014

1	Cook A, Pun A, Gallo MF, et al: Scalpel versus no-scalpel incision for vasectomy. Cochrane Database SYSt Rev 3:CD004112, 2014 Costello C, Hillis S, Marchbanks P, et al: he efect of interval tubal sterilizadon on sexual interest and pleasure. Obstet Gynecol 100:3, 2002 Curtis M, Mohllajee AP, Peterson HB: Regret following female sterilization at a young age: a systematic review. Contraception 3:205, 2006 Daniels K, Daugherty ], Jones ], et al: Current contraceptive use and variation by selected characteristics among women aged 15-44: United States, 2011-2013. Nat! Health Stat Report 86:1,n2015 Danis RB, Della Badia CR, Richard SD: Postpartum permanent sterilization: could bilateral salpingectomy replace bilateral tubal ligation? J Minim Invasive GynecoIn23(6):928, 2016 Deieux X, Morin Surroca M, Faivre E, et al: Tubal anastomosis after tubal sterilization: a review. Arch Gynecol Obstet 83(5):1n149,n201n1

1	Deieux X, Morin Surroca M, Faivre E, et al: Tubal anastomosis after tubal sterilization: a review. Arch Gynecol Obstet 83(5):1n149,n201n1 Deneux-Tharaux C, Kahn E, Nazerali H, et al: Pregnancy rates after vasectomy: a survey of U.S. urologists. Contraception 69:401, 2004 DeStefano F, Perlman ]A, Peterson HB, et al: Long term risk of menstrual disturbances after tubal sterilization. Am J Obstet GynecoIn152:835, 1985 DuY S, Marsh F, Rogerson L, et al: Female sterilization: a cohort controlled comparative study of Essure versus laparoscopic sterilization. BJOG 112: 1522, 2005 Eisenberg ML, Lipshultz LI: Estimating the number of vasectomies performed annually in the United States: data from the National Survey of Family Growth. J Urol 184(5):2068,n2010

1	Eisenberg ML, Lipshultz LI: Estimating the number of vasectomies performed annually in the United States: data from the National Survey of Family Growth. J Urol 184(5):2068,n2010 Food and Drug Administration: Labeling for permanent hysteroscopicallyplaced tubal implants itended for sterilization. 2016. Available at: http:// ww.fda.gov/downloads/MedicaIDevices/DeviceRegulationandGuidance/ GuidanceDocuments/UCM488020.pdf. Accessed April 28, 2016 Gaudet MM, Patel AV, Sun J, et al: Tubal sterilization and breast cancer incidence: results from the cancer prevention study II nuuition cohort and meta-analysis. Am J Epidemiol 7(6):492,n2013 Giovannucci E, Tosteson TD, Speizer FE, et al: A long-term study of mortality in men who have undergone vasectomy. N Engl ] Med 326: 1392, 1992 Goldacre ]M, Holford TR, Vessey MP: Cardiovascular disease and vasectomy. N Engl ] Med 308:805, 1983 Hendrix NW, Chauhan SP, Morrison JC Sterilization and its consequences. Obstet Gynecol Surv 66, 1999

1	N Engl ] Med 308:805, 1983 Hendrix NW, Chauhan SP, Morrison JC Sterilization and its consequences. Obstet Gynecol Surv 66, 1999 Hofman BL, Corton MM: Surgeries for benign gynecologic conditions. In Hofman BL, Schorge JO, Bradshaw KD, et al: Williams Gynecology, 3rd ed. New York, McGraw-Hill Education, 2016 Hologic: Hologic announces second quarter iscal 2012 operating results. 2012. Available online at: ile:/ / /C/Users/bhofm/Downloads/Hologic-AnnouncesSecond-Quarter-Fiscal-20 12-0perating-Results. pdf. Accessed May 19, 2016 Jamieson DJ, Costello C, Trussell ], et al: The risk of pregnancy after vasectomy. Obstet Gynecol 103:848, 2004 Jamieson DJ, Kaufman SC, Costello C, et al: A comparison of women's regret after vasectomy versus tubal sterilization. Obstet Gynecol 99: I073, 2002

1	Jamieson DJ, Kaufman SC, Costello C, et al: A comparison of women's regret after vasectomy versus tubal sterilization. Obstet Gynecol 99: I073, 2002 Kelek:i S, Erdemoglu E, Kuduk S, et al: Risk factors for tubal ligation: regret and psychological efects. Impact of Beck Depression Inventoty. Contraception ,2005 la Chapel Ie CF, Veersema S, Brolmann HA, et al: Efectiveness and feasibility of hysteroscopic sterilization techniques: a systematic review and metaanalysis. Ferril Steriln103(6):1516, 2015 Lazorwitz A, Tocce K: A case series of removal of nickel-titanium sterilization microinserts from the uterine cornua using laparoscopic electrocautety for salpingectomy. Contraception 96(2):96, 2017 Leslie TA, Illing RO, Cranston DW, et al: he incidence of chronic scrotal pain after vasectomy: a prospective audit. BJU Int 100:n1330,n2007 Levgur M, Duvivier R: Pelvic inAammatory disease after tubal sterilization: a review. Obstet Gynecol Surv 55:41, 2000

1	Levgur M, Duvivier R: Pelvic inAammatory disease after tubal sterilization: a review. Obstet Gynecol Surv 55:41, 2000 Lippes J: Quinacrine sterilization (QS): time for reconsideration. Contraception 92(2):91, 2015 Madari S, Varma R, Gupta J: A comparison of the modified Pomeroy tubal ligation and Filshie clips for immediate postpartum sterilisation: a systematic review. Eur] Contracept Reprod Health Care 16(5):341,n2011 Malacova E, Kemp-Casey A, Bremner A, et al: Live delivety outcome after tubal sterilization reversal: a population-based study. Ferril Steril 104(4):92, 2015 Manikandan R, Srirangam SJ, Pearson E, et al: Early and late morbidity after vasectomy: a comparison of chronic scrotal pain at 1 and 10 years. BJU Int 93:571,n2004 Mao], pfeifer S, Schlegel P, et al: Safety and eicacy of hysteroscopic sterilization compared with laparoscopic sterilization: an observational cohort study. BM] 351:h5162, 2015

1	Mao], pfeifer S, Schlegel P, et al: Safety and eicacy of hysteroscopic sterilization compared with laparoscopic sterilization: an observational cohort study. BM] 351:h5162, 2015 M01ler H, Knudsen LB, Lynge E: Risk of testicular cancer after vasectomy: cohort study of over 73,000 men. BM] 309:295, 1994 Moniz MH, Chang T, Heisler M, et al: Inpatient postpartum long-acting reversible conuaception and sterilization in the United States, 2008-2013. Obstet Gynecol 129(6): 1078,n20] Monteith CW, Berger GS, Zerden ML: Pregnancy success after hysteroscopic sterilization reversal. Obstet Gynecol 124(6):1183,n2014 Munro MG, Nichols ]E, Levy B, et al: Hysteroscopic sterilization: lO-year retrospective analysis of worldwide pregnancy reportS. ] Minim Invasive Gynecol 21(2):245, 2014 Pearce CL, Stram DO, Ness RB, et al: Population distribution of lifetime risk of ovarian cancer in the United States. Cancer Epidemiol Biomarkers Prev 24(4):671,n2015

1	Pearce CL, Stram DO, Ness RB, et al: Population distribution of lifetime risk of ovarian cancer in the United States. Cancer Epidemiol Biomarkers Prev 24(4):671,n2015 Peterson HB, ]eng G, Folger SG, et al: he risk of menstrual abnormalities after tubal sterilization. N Engl ] Med 343: 1681, 2000 Peterson HB, Xia Z, Hughes ]M, et al: The risk of ectopic pregnancy after tubal sterilization. U.S. Collaborative Review of Sterilization Working Group. N Engl J Med 336(11):762, 1997 Peterson HB, Xia Z, Hughes ]M, et al: The risk of pregnancy after tubal sterilization: findings from the U.S. Collaborative Review of Sterilization. m ] Obstet Gynecol 1 4: 1161, 1996 Potter JE, Stevenson A], White K, et al: Hospital variation in postpartum tubal sterilization rates in California and Texas. Obstet Gynecol 121 (1): 152,n2013

1	Potter JE, Stevenson A], White K, et al: Hospital variation in postpartum tubal sterilization rates in California and Texas. Obstet Gynecol 121 (1): 152,n2013 Powell CB, Alabaster A, Simmons S, et al: Salpingectomy for sterilization: change in practice in a large integrated health care system, 2011-2016. Obstet Gynecol 130(5):961,n2017 Rogers MD, Kolettis PN: Vasectomy. Urol Clin North Am 40(4):559, 2013 Sharlip 10, Belker AM, Honig S, et al: Vasectomy: AUA guideline. Outcomes of microsurgical vasovasostomy for vasectomy reversal: a meta-analysis and systematic review.n] Urol 188(6 Suppi):2482, 2012 Shinar S, Blecher Y, Alpern S, et al: Total bilateral salpingectomy versus partial bilateral salpingectomy for permanent sterilization during cesarean delivety. Arch Gynecol Obstet295(5):1185, 2017 Shy KK, Stergachis A, Grothaus LG, et al: Tubal sterilization and risk of subsequent hospital admission for menstrual disorders. Am ] Obstet Gynecol 166:n1698,n1992

1	Shy KK, Stergachis A, Grothaus LG, et al: Tubal sterilization and risk of subsequent hospital admission for menstrual disorders. Am ] Obstet Gynecol 166:n1698,n1992 Society of Gynecologic Oncologists: SGO Clinical Practice Statement: Salpingectomy for ovarian cancer prevention. Available at: https://www.sgo.org/ cl inical-practice/ gu idel i nes/ sgo-cl inical-p ractice-statemen t -sal pi ngecto m yfor-ovarian-cancer-prevention.l Accessed December 13,n2013 Sokal DC, Hieu do T, Loan NO, et al: Contraceptive efectiveness of two insertions of quinacrine: results from 10-year follow-up in Vietnam. Con 8:61, 2008 Sokal DC, Trujillo V, Guzman SC, et al: Cancer risk after sterilization with transcervical quinacrine: updated indings from a Chilean cohort. Contraception 81(1): 5,n2010a Sokal DC, Vach TH, Nanda K, Quinacrine sterilization and gynecologic cancers: a case-control study in northen Vietnam. Epidemiology 21 (2): 164, 201 Ob

1	Sokal DC, Vach TH, Nanda K, Quinacrine sterilization and gynecologic cancers: a case-control study in northen Vietnam. Epidemiology 21 (2): 164, 201 Ob Stuart GS: Puerperal sterilization. In Yeomans ER, Hofman BL, Gilstrap, III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hill, 201 Thompson M, Kho K: Minimally invasive surgery. In Hofman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw-Hill, 2016 Veersema S, Mijatovic V, Dreyer K, et al: Outcomes of pregnancies in women with hysteroscopically placed micro-inserts in situ. ] Minim Invasive Gynecol 21(3):492, 2014 TERMINOLOGY AND DIAGNOSIS .................. 710 INCIDENCE AND RISK FACTORS ................... 713 ETIOPATHOGENESIS ............................ 713 PATHOPHYSIOLOGY ............................ 717 PREDICTION.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725

1	ETIOPATHOGENESIS ............................ 713 PATHOPHYSIOLOGY ............................ 717 PREDICTION.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725 PREVENTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 726 PREECLAMPSIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 728 ECLAMPSIA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734 MANAGEMENT CONSIDERATIONS ................. 738 LONG-TERM CONSEQUENCES .................... 744 An eclamptic convulsion sometimes occurs without warning, "like a bolt from a clear sky': in women who are apparenty in peect health. In the majoriy of cases, however, the outbreak is preceded or a longer or shorter period by premonitoy symptoms indicative of toxemia of pregnancy, among the more common being oedema, headache, epigastric pain, and possiby disturbances of vision. -J. Whitridge Williams (1903)

1	At the time of this textbook's irst edition, it was accepted that "toxemia" preceded most cases of eclampsia. he central role of hypertension had not yet been discovered, and after many years, it became apparent that preeclampsia was a syndrome of which hypertension was only one important facet. Still, the mechanisms by which pregnancy incites or aggravates hypertension remain unsolved. Indeed, hypertensive disorders remain among the most signiicant and intriguing unsolved problems in obstetrics. hese disorders complicate 5 to 10 percent of all pregnancies, and together they are one of the deadly triad-along with hemorrhage and infection-that contributes greatly to maternal morbidity and mortality rates. Of hypertensive disorders, the preeclampsia syndrome, either alone or superimposed on chronic hypertension, is the most dangerous. As subsequently discussed, new-onset hypertension during pregnancy-termed gestational hypertension-is followed by signs and symptoms of preeclampsia

1	on chronic hypertension, is the most dangerous. As subsequently discussed, new-onset hypertension during pregnancy-termed gestational hypertension-is followed by signs and symptoms of preeclampsia almost half the time, and preeclampsia is identiied in 4 to 5 percent of all pregnancies (Martin, 2012).

1	he World Health Organization (WHO) systematically reviews maternal mortality worldwide, and in developed countries, 16 percent of maternal deaths were attributed to hypertensive disorders (han, 2006). In the United States from 201r1 to 2013, 7.4 percent of2009 pregnancy-related maternal deaths were caused by preeclampsia or eclampsia (Creanga, 2017). A similar rate was 10 percent in France from 2003 through 2007 (Saucedo, 2013). Importantly, more than half of these hypertension-related deaths were deemed preventable (Berg, 2005). To update and codiy the terminology and classiication of hypertensive disorders of pregnancy, a Task Force of the American College of Obstetricians and Gynecologists (2013) has provided evidence-based recommendations for clinical practice. The previous basic classiication was retained and describes four types of hypertensive disease: 1. 2. Chronic hypertension of any etiology 3.

1	2. Chronic hypertension of any etiology 3. 4. Gestational hypertension-definitive evidence for the preeclampsia syndrome does not develop and hypertension resolves by 12 weeks postpartum. Importantly, this classification diferentiates the preeclampsia syndrome from other hypertensive disorders because it is potentially more ominous. • Diagnosis of Hypertensive Disorders

1	Importantly, this classification diferentiates the preeclampsia syndrome from other hypertensive disorders because it is potentially more ominous. • Diagnosis of Hypertensive Disorders Hypertension is diagnosed empirically when appropriately taken blood pressure exceeds 140 mm Hg systolic or 90 mm Hg diastolic. Korotkofphase V is used to define diastolic pressure. Previously, incremental increases of 30 mm Hg systolic or 15 mm Hg diastolic above blood pressure values taken at midpregnancy had also been used as diagnostic criteria, even when absolute values were < 140/90 mm Hg. These incremental changes are no longer used to define hypertension, but it is recommended that such women be observed more closely because eclamptic seizures develop in some whose blood pressures have stayed below 140/90 mm Hg (Alexander, 2006). Also, a sudden rise in mean arterial pressure but still in a normal range-"delta hypertension"-may signiy preeclampsia (Macdonald-Wallis, 2012; Zeeman, 2007).

1	he systolic and diastolic blood pressure levels of 140/90 mm Hg have been arbitrarily used since the 1950s to define "hypertension" in nonpregnant individuals. However, these levels were selected by insurance companies to characterize a group of middle-aged men. It seems more realistic to deine normalrange blood pressures that fall between an upper and lower limit for a particular population-such as young, healthy, pregnant women. A schematic example using arbitrary mean arterial blood pressure readings is shown in Figure 40-1. Data curves for both women show blood pressure measurements near the ...Patient A ...Patient B )..:J, C,

1	C, FIGURE 40-1 Schematic shows normal reference ranges for mean arterial blood pressure changes across pregnancy. Patient A (blue) has mean blood pressures near the 20th percentile throughout pregnancy. Patient B (red) has a similar pattern with mean pressures at the 25th percentile until approximately 36 weeks when her blood pressure begins to rise. By term, it is substantively higher and in the 75th percentile, but she is still considered "normotensive." 25th percentile until 32 weeks. These begin to rise in patient B, who by term has substantively higher blood pressures. However, her pressures are still < 140/90 mm Hg, and thus she is considered to be "normotensive." We use the term delta hypertension to describe this rather acute rise in blood pressure. Some of these women will go on to have obvious preeclampsia, and some even develop eclamptic seizures or HELLP (hemolysis, devatedliver enzyme levels, low 2latelet count) syndrome while still normotensive.

1	his diagnosis is made in women whose blood pressures reach 140/90 mm Hg or greater for the irst time after midpregnancy, but in whom proteinuria is not identiie. Almost half of these women subsequently develop preeclampsia syndrome. Even so, when blood pressure increases appreciably, it is dangerous to both mother and fetus to ignore this rise only because proteinuria has not yet developed. As Chesley (1985) emphasized, 10 percent of eclamptic seizures develop before overt proteinuria can be detected. Finally, gestational hypertension is reclassiied by some as transient hypertension if evidence for preeclampsia does not develop and the blood pressure returns to normal by 12 weeks postpartum.

1	Preeclampsia is best described as a pregnancy-speciic syndrome that can aect virtualy evey organ system. In addition, it heralds a higher incidence of cardiovascular disease later in life (p. 744). Although preeclampsia is much more than simply gestational hypertension with proteinuria, appearance ofproteinuria remains an important diagnostic criterion. Thus, proteinuria is an objective marker and relects the system-wide endothelial leak that characterizes the preeclampsia syndrome. In some women with the preeclampsia syndrome, neither overt proteinuria nor fetal-growth restriction are features (Sibai, 2009). Because of this, the Task Force (2013) suggests other diagnostic criteria, which are shown in Table Evidence of multiorgan involvement may include thrombocytopenia, renal dysfunction, hepatocellular necrosis, central nervous system perturbations, or pulmonary edema. • Indicators of Preeclampsia Severity

1	• Indicators of Preeclampsia Severity The markers listed in Table 40-1 are also used to classiy preeclampsia syndrome severity. lthough many use a dichotomous "mild" and "severe" classiication, the Task Force (2013) discourages the use of "mild preeclampsia." It is problematic that there are criteria for the diagnosis of "severe" preeclampsia, but the default classiication is either implied or speciically termed as "mild," "less severe," or "nonsevere" (Alexander, 2003; Lindheimer, 2008b). here are no generally agreed-on criteria for "moderate" preeclampsia-an elusive third category. We use the criteria listed in Table 40-2, which are categorized as "severe" versus "nonsevere." Some symptoms are considered ominous. Headaches or visual disturbances such as scotomata can precede eclampsia, which is a convulsion not attributable to another cause. The TABLE 40-1. Classification and Diagnosis of Pregnancy-Associated Hypertension

1	TABLE 40-1. Classification and Diagnosis of Pregnancy-Associated Hypertension Gestational hypertension Preeclampsia: Hypertension plus • 2300 mg/24 h, or Urine protein: creatinine ratio 20.3, or Platelet count < 1 OO,OOO/.LL 1.1 mg/dL or doubling of baselineb Headache, visual disturbances, convulsions aRecommended only if sole available test. bNo prior renal disease. CAST (aspartate transaminase) or ALT (alanine transaminase). BP = blood pressure. Modified with permission from American College ofObstetricians and Gynecologists; Task Force on Hypertension in Pregnancy: Hypertension in pregnancy. Report ofthe American College ofObstetricians and Gynecologists'Task Force on

1	Hypertension in Pregnancy, Obstet Gynecol. 201n3 Nov;122(S):n seizures are generalized and may appear before, during, or after labor. The proportion that develops seizures later, after 48 hours postpartum, approximates 10 percent (Sibai, 2005; Zwart, 2008). Another symptom, epigastric or right upper quadrantpain, frequently accompanies hepatocellular necrosis, ischemia, and edema that ostensibly stretches Glisson capsule. This characteristic pain is frequently accompanied by elevated serum TABLE40-2. Indicators of Severity of Gestational (< 1 OO,OOO/LL) None to positive None to positive aCompare with criteria in Table 40-1. blncludes "mild" and "moderate" hypertension not specifically defined. cMost disregard degrees of proteinuria to classify nonsevere or severe. BP = blood pressure.

1	blncludes "mild" and "moderate" hypertension not specifically defined. cMost disregard degrees of proteinuria to classify nonsevere or severe. BP = blood pressure. hepatic transaminase levels. Finally, thrombocytopenia also signifies worsening preeclampsia. It represents platelet activation and aggregation as well as microangiopathic hemolysis. Other factors indicative ofsevere preeclampsia include renal or cardiac involvement, obvious fetal-growth restriction, and early-onset disease. he more profound these signs and symptoms, the less likely it is that they can be temporized, and the more likely that delivery will be required. A caveat is that dierentiation between nonsevere and severe gestational hypertension or preeclampsia can be misleading because what might be apparenty mild disease may progress rapidy to severe disease.

1	Regardless of its cause, any chronic hypertensive disorder predisposes a woman to develop superimposed preeclampsia syndrome. Chronic underlying hypertension is diagnosed in women with documented blood pressures > 140/90 mm Hg before pregnancy or before 20 weeks' gestation, or both. Hypertensive disorders can create diicult problems with diagnosis and management in women who are not irst seen until after midpregnancy. This is because blood pressure normally drops during the second and early third trimesters in both normotensive and chronically hypertensive women (see Fig. 40-1). Thus, a woman with previously undiagnosed chronic vascular disease who is seen before 20 weeks frequently has blood pressures within normal range. During the third trimester, however, as blood pressures return to their originally hypertensive levels, it may be diicult to determine whether hypertension is chronic or induced by pregnancy. Even a careful search for evidence of preexisting end-organ damage may be

1	their originally hypertensive levels, it may be diicult to determine whether hypertension is chronic or induced by pregnancy. Even a careful search for evidence of preexisting end-organ damage may be futile, as many ofthese women have mild disease and no evidence ofventricular hypertrophy, retinal vascular changes, or renal dysfunction.

1	In some with chronic hypertension, blood pressure rises to obviously abnormal levels, typically after 24 weeks' gestation. If new-onset or worsening baseline hypertension is accompanied by new-onset proteinuria or other indings listed in Table 40-1, then superimposed preeclampsia is diagnosed. Compared with "pure" preeclampsia, superimposed preeclampsia commonly develops earlier in pregnancy. It also tends to be more severe and more often is accompanied by fetal-growth restriction. he same criteria shown in Table 40-2 are also used to further characterize severity of superimposed preeclampsia.

1	Young and nulliparous women are particularly vulnerable to developing preeclampsia, whereas older women are at greater risk for chronic hypertension with superimposed preeclampsia. he incidence is markedly inluenced by race and ethnicity-and thus by genetic predisposition. In one study by the Maternal-Fetal Medicine Units (MFMU) Network, the incidence of preeclampsia was 5 percent in white, 9 percent in Hispanic, and 11 percent in African-American women (Myatt, 2012a,b). In addition, black women have greater morbidity (Shahul, 2015). In several worldwide studies reviewed by Staf and coworkers (2015), the incidence of preeclampsia in nulliparous populations ranged from 3 to 10 percent. he incidence of preeclampsia in multiparas also varies and ranges from 1.4 to 4 percent (Fisher, 2015).

1	Bartsch and associates (2016) extracted data from more than 25 million pregnancies and calculated relative risks for several clinical factors shown in Table 40-3. Others include the metabolic syndrome and hyperhomocysteinemia (Karumanchi, 2016a; Masoudian, 2016; Scholten, 2013). Pregnancies with a male fetus are also at slightly higher risk a askolka, TABLE 40-3. Selected Clinical Risk Factors for Preeclampsia APA = antiphospholipid antibody; ART = assisted reproductive technology; BMI = body mass index; CHTN = chronic hypertension; CKD = chronic kidney disease; SLE = systemic lupus erythematosus. Data from Ba rtsch, 201 6. 2017). Although smoking during pregnancy causes various adverse pregnancy outcomes, ironically, it carries a reduced risk for hypertension during pregnancy (Bainbridge, 2005; Kraus, 2014). Other factors are human immunodeficiency virus (HIV) seropositivity and sleep-disordered breathing (Facco, 2017; Sansone, 2016).

1	For eclampsia, the incidence has declined in areas where health care is more readily available. In the United States in 1998, it afected 1 in 3250 births (Ventura, 2000). Except for Iceland, which has an extremely low rate, in countries with adequate resources the incidence averages 1 in 2000 to 3000 deliveries (Andersgaard, 2006; Jaatinen, 2016; O'Connor, 2013; Royal College of Obstetricians and Gynaecologists, 2006; Zwart, 2008). Any satisfactory theory concerning the origins of preeclampsia must account for the observation that gestational hypertensive disorders are more likely to develop in women with the following characteristics: Are exposed to chorionic villi for the irst time Are exposed to a superabundance of chorionic villi, as with twins or hydatidiform mole Have preexisting conditions associated with endothelial cell activation or inflammation, such as diabetes, obesity, cardiovascular or renal disease, immunological disorders, or hereditary inluences

1	Have preexisting conditions associated with endothelial cell activation or inflammation, such as diabetes, obesity, cardiovascular or renal disease, immunological disorders, or hereditary inluences Are genetically predisposed to hypertension developing during pregnancy. A fetus is not a requisite for preeclampsia to develop. And, although chorionic villi are essential, they need not be intrauterine. For example, preeclampsia can develop with an abdominal pregnancy (Worley, 2008). Regardless of precipitating etioloy, the cascade of events leading to the preeclampsia syndrome is characterized by abnormalities that result in systemic vascular endothelial damage with resultant vasospasm, transudation ofplasma, and ischemic and thrombotic sequelae.

1	• Phenotypic Expression of he preeclampsia syndrome varies widely in its clinical phenotypic expression. But, at least two major subtypes are diferentiated by whether or not remodeling of uterine spiral arterioles by endovascular trophoblasts is defective. his concept has given rise to the "two-stage disorder" theory of preeclampsia pathogenesis. According to Redman and coworkers (20 15a), stage 1 is caused by faulty endovascular trophoblastic remodeling that downstream causes the stage 2 clinical syndrome. Importantly, stage 2 can be modified by preexisting maternal conditions that are also manifest by endothelial cell activation or inlammation and are listed in the third prior bullet. Such staging is artiicial, and it seems logical that preeclampsia syndrome presents clinically as a spectrum of worsening disease. Moreover, evidence is accruing that many "isoforms" exist as discussed subsequently. Examples include diferences in

1	FIGURE 40-2 Schematic representation of normal placental implantation shows proliferation of extravillous trophoblasts from an anchoring villus. These trophoblasts invade the decidua and extend into the walls of the spiral arteriole to replace the endothelium and muscular wall to create a dilated low-resistance vessel. With preeclampsia, defective implantation is characterized by incomplete invasion of the spiral arteriolar wall by extravillous trophoblasts. This results in a small-caliber vessel with high resistance to flow. maternal and fetal characteristics, placental indings, and earlyversus late-onset disease (Phillips, 2010; Valensise, 2008; van der Merwe, 2010). n imposing number of mechanisms have been proposed to explain the cause of preeclampsia. Those currently considered important include: 1. Placental implantation with abnormal trophoblastic invasion of uterine vessels 2. Immunological maladaptive tolerance between maternal, paternal (placental), and fetal tissues 3.

1	Placental implantation with abnormal trophoblastic invasion of uterine vessels 2. Immunological maladaptive tolerance between maternal, paternal (placental), and fetal tissues 3. Maternal maladaptation to cardiovascular or inlammatory changes of normal pregnancy 4. Genetic factors including inherited predisposing genes and epigenetic inluences. Discussed in Chapter 5 (p. 88), normal implantation is characterized by extensive remodeling of the spiral arterioles within the decidua basalis (Fig. 40-2). Endovascular trophoblasts replace the vascular endothelial and muscular linings to enlarge the vessel diameter (Zhou, 1997). The veins are invaded only superficially.

1	In some cases of preeclampsia, however, trophoblastic invasion may be incomplete. With this, decidual vessels, but not myometrial vessels, become lined with endovascular trophoblasts. The deeper myometrial arterioles thus do not lose their endothelial lining and musculoelastic tissue, and their mean external diameter is only half that of corresponding vessels in normal placentas (Fisher, 2015). In general, the magnitude of defective trophoblastic invasion correlates with the severity of the hypertensive disorder (Madazli, 2000). And importantly, it is more prevalent in women with early-onset preeclampsia (Khodzhaeva, 2016). cMahon and associates (2014) found that lower levels of soluble antiangiogenic growth factors may be involved in this faulty endovascular remodeling.

1	From placental electron microscopy studies, early preeclamptic changes include endothelial damage, insudation of plasma constituents into vessel walls, proliferation of myointimal cells, and medial necrosis (De Wolf, 1980). Hertig (1945) referred to lipid accumulation in myointimal cells and macro phages as atherosis. These indings are more common in placentas from women diagnosed with preeclampsia before 34 weeks (Nelson, 2014b). Acute placental vascular atherosis may also identiy a group of women at greater risk for later atherosclerosis and cardiovascular disease (Staf, 2015). In pregnancy, the abnormally narrow lumen of spiral arterioles likely impairs placental blood flow. Diminished perfusion and a hypoxic environment eventually lead to release of placental debris or microparticles.

1	At this point, these changes incite a systemic inlammatory response, which is stage 2 of the preeclampsia syndrome (Lee, 2012; Redman, 2012). Defective placentation is posited to further cause the susceptible woman to develop gestational hypertension, the preeclampsia syndrome, preterm delivery, a growth-restricted fetus, and/or placental abruption (Brosens, 2011; Labarrere, 2017; Nelson, 2014b). IVlaternal immune tolerance to paternally derived placental and fetal antigens is discussed in Chapter 5 (p. 95). Loss of this tolerance is another cited theory for preeclampsia (Erlebacher, 2013). Certainly, the histological changes at the maternalplacental interface are suggestive of acute graft rejection.

1	Inferential data also suggest that preeclampsia is an immunemediated disorder. For example, the risk of preeclampsia is appreciably enhanced in circumstances in which formation of blocking antibodies to placental antigenic sites might be impaired. In this scenario, the irst pregnancy would carry a higher risk. Tolerance dysregulation might also explain an ele vated risk when the paternal antigenic load is increased, that is, with two sets of paternal chromosomes-a "double dose." Namely, women with molar pregnancies have a high incidence of early-onset preeclampsia. Women with a trisomy 13 fetus also have a 30-to 40-percent incidence of preeclampsia. hese women have elevated serum levels of antiangiogenic factors. he gene for one of these factors, soluble ins-like tyrosine kinase 1, is on chromosome 13 (Bdolah, 2006). Conversely, women previ ously exposed to paternal antigens, such as a prior pregnancy with the same partner, are "immunized" against preeclampsia.

1	his phenomenon is not as apparent in women with a prior abortion (Strickland, 1986). Multiparas impregnated by a new consort have a greater risk of preeclampsia (Mostello, 2002).

1	Redman and colleagues (2015a) reviewed the possible role of immune maladaptation in preeclampsia pathophysiology. In women destined to be preeclamptic, extravillous trophoblasts early in pregnancy express reduced amounts of immunosuppressive nonclassic human leukocyte antigen G (HLA G). Black women more commonly have the 1597 �C gene allele that further predisposes to preeclampsia (Loisel, 2013). hese changes may contribute to the defective placental vascularization in stage 1 of the preeclampsia syndrome. As discussed in Chapter 4 (p. 59), T-helper (h) lymphocytes during normal pregnancy are produced so that type 2 activity is increased in relation to type 1-so-called ype 2 bias (Redman, 2012, 2015a). h2 cells promote humoral immunity, whereas Th1 cells stimulate inlammatory cytoine secretion. Beginning in the early second trimester in women who develop preeclampsia, Th1 action is increased.

1	Inflammatory changes are believed to be a continuation of stage 1 alterations. In response to ischemia or other inciting causes, placental factors are released and begin a cascade of events (Davidge, 2015). Thus, antiangiogenic and metabolic factors and other inflammatory leukocyte mediators are thought to provoke systemic endothelial cell injury, which is used synonymously here with endothelial cel activation or dyfunction.

1	Endothelial cell dysfunction may result from an extreme activated state of leukocytes in the maternal circulation (Faas, 2000; Gervasi, 2001). Briefly, cytokines such as tumor necrosis factor-a (TNF-a) and the interleukins may contribute to the systemic oxidative stress associated with preeclampsia. This is characterized by reactive oxygen species and free radicals that lead to formation of self-propagating lipid peroxides (Manten, 2005). hese peroxides in turn generate highly toxic radicals that injure systemic vascular endothelial cells, modiy nitric oxide production by these cells, and interfere with prostaglandin balance. Other consequences of oxidative stress include production of the lipid-laden macrophage foam cells seen in placental atherosis, activation of systemic microvascular coagulation manifested by thrombocytopenia, and greater systemic capillary permeability relected by edema and proteinuria.

1	Preeclampsia appears to be a multifactorial, polygenic disorder. In one study of almost 1.2 million Swedish births, a genetic Data from Buurma, 2013; Staines-Urias, 2012; Triche, 2014; Ward, 2014; Ye, 2016. association for gestational hypertension and for preeclampsia was found (Nilsson, 2004). Ward and Taylor (2015) cite an incident risk for preeclampsia of 20 to 40 percent for daughters of preeclamptic mothers; 11 to 37 percent for sisters of preeclamptic women; and 22 to 47 percent for twins. Ethnoracial factors are important, as evidenced by the high incidence of preeclampsia in African-American women. It may be that Latina women have a lower incidence because of interactions of American Indian and white race genes (Shahabi, 2013).

1	he hereditary predisposition for preeclampsia likely stems from interactions of literally hundreds of inherited genesboth maternal and paternal-that control myriad enzymatic and metabolic functions throughout every organ system (Triche, 2014). Plasma-derived factors may induce some of these genes in preeclampsia (Mackenzie, 2012). Thus, the clinical manifestation in any given woman with the preeclampsia syndrome will occupy a spectrum. In this regard, phenotypic expression will difer among similar genotypes depending on interactions with environmental components (Yang, 2013).

1	Hundreds of genes have been studied for their possible association with preeclampsia (Buurma, 2013; Sakowicz, 2016; Ward, 2015). Several that may have a significant association with the syndrome are listed in Table 40-4. However, because of the complex phenotypic expression of preeclampsia, it is doubtful that anyrone candidate gene will be found responsible. Indeed, Majander and associates (2013) have linked preeclampsia predisposition to evenetal genes on chromosome 18.

1	The concept of vasospasm with preeclampsia has been advanced for a century (Volhard, 1918). Systemic endothelial activation causes vasospasm that elevates resistance to produce subsequent hypertension. Concurrently, systemic endothelial cell injury promotes interstitial leakage, and blood constituents, including platelets and ibrinogen, are deposited subendothelially. Endothelial junctional proteins are also disrupted, and the subendothelial region of resistance arteries undergoes ultrastructural change (Suzuki, 2003; Wang, 2002). The much larger venous circuit is similarly involved. With diminished blood low because of maldistribution from vasospasm and interstitial leakage, ischemia of the surrounding tissues can lead to necrosis, hemorrhage, and other end-organ disturbances characteristic of the syndrome. One important clinical correlate to this is the markedly attenuated blood volume seen in women with severe preeclampsia (Zeeman, 2009).

1	Injury to systemic endothelial cells is now a centerpiece of preeclampsia pathogenesis (Davidge, 2015). In this scheme, protein factor(s)-likely placental-are secreted into the maternal circulation and provoke activation and dysfunction of the systemic vascular endothelium. Many facets of the clinical syndrome of preeclampsia are thought to result from these widespread endothelial cell changes.

1	Intact endothelium has anticoagulant properties. lso, systemic endothelial cells, by releasing nitric oxide, blunt the response of vascular smooth muscle to agonists. Injured or activated endothelial cells may produce less nitric oxide and may secrete substances that promote coagulation and greater sensitivity to vasopressors. Further evidence of endothelial activation includes the characteristic changes in glomerular capillary endothelial morphology, greater capillary permeability, and elevated blood concentrations of substances associated with endothelial activation. Likely, multiple factors in the plasma of preeclamptic women combine to exert these vasoactive efects (Myers, 2007; Walsh, 2009).

1	As discussed in Chapter 4 (p. 63), pregnant women normally develop refractoriness to infused vasopressors (AbdulKarim, 1961). Women with early preeclampsia, however, have enhanced vascular reactivity to infused norepinephrine and angiotensin II (Raab, 1956; Talledo, 1968). Moreover, increased sensitivity to angiotensin II clearly precedes the onset of gestational hypertension (Gant, 1974). Paradoxically, women who develop preterm preeclampsia have lower circulating levels of angiotensin II (Chase, 2017).

1	Several prostaglandins are thought to be central to preeclampsia syndrome pathophysiology. Specifically, the blunted pressor response seen in normal pregnancy is at least partially due to diminished vascular responsiveness mediated by endothelial prostaglandin synthesis. For example, compared with normal pregnancy, endothelial prostacyclin (PGI2) production is lower in preeclampsia. This action appears to be mediated by phospholipase A2 (Davidge, 2015). At the same time, thromboxane A2 secretion by platelets is increased, and the prostacyclin:thromboxane A2 ratio declines. The net result favors greater sensitivity to infused angiotensin II and, ultimately, vasoconstriction (Spitz, 1988). hese changes are apparent as early as 22 weeks' gestation in gravidas who later develop preeclampsia (Chavarria, 2003).

1	Nitric oxide is a potent vasodilator synthesized from I-arginine by endothelial cells. Inhibition of nitric oxide synthesis raises mean arterial pressure, lowers heart rate, and reverses the pregnancy-induced refractoriness to vasopressors. In humans, nitric oxide likely is the compound that maintains the normal low-pressure vasodilated state characteristic of fetoplacental perfusion (Myatt, 1992; Weiner, 1992). he efects of nitric oxide production in preeclampsia are unclear. It appears that the syndrome is associated with decreased endothelial nitric oxide synthase expression, thus resulting in lower nitric oxide activity (Davidge, 2015).

1	Endothelins are 21-amino-acid pep tides and potent vasoconstrictors. Endothelin-1 (ET -1) is the primary isoform produced by human endothelium (Karumanchi, 2016b). Plasma ET-1 levels are elevated in normotensive pregnant women, but women with preeclampsia have even higher levels (Ajne, 2003). According to Taylor and Roberts (1999), the placenta is not the source of increased ET -1 concentrations, and they likely arise from systemic endothelial activation. Interestingly, treatment of preeclamptic women with magnesium sulfate lowers ET -1 concentrations (Sagsoz, 2003). And, in animal studies, sildenafil reduces ET-1 concentrations (Gillis, 2016).

1	Placental vasculogenesis is evident by 21 days after conception. The list of pro-and antiangiogenic substances involved in placental vascular development is extensive, and the families of vascular endothelial growth factor (VEGF) and angiopoietin are the most studied. Angiogenic imbalance describes excessive amounts of antiangiogenic factors, which are thought to be stimulated by worsening hypoxia at the uteroplacental interface. Trophoblast of women destined to develop preeclampsia overproduces at least two antiangiogenic pep tides that enter the maternal circulation (Karumanchi, 20 16a).

1	First, soluble ins-like yrosine kinase i {sFlt-i} is a receptor for VEGF. As depicted in Figure 40-3, elevated maternal sFlt-1 levels inactivate and reduce circulating free placental growth factor (PIGF) and VEGF concentrations, leading to endothelial dysfunction (Maynard, 2003). Importantly, sFlt-1 levels begin to rise in maternal serum months before preeclampsia is evident (Fig. 40-4). These high levels in the second trimester are associated with a doubling of the risk for preeclampsia (Haggerty, 2012). This divergence from normal levels appears to develop even sooner with early-onset preeclampsia (Vatten, 2012). These factors are also operative in pregnancies complicated by fetal-growth restriction (Herraiz, 2012).

1	A second antiangiogenic peptide, soluble endoglin (sEng), inhibits various transforming growth factor beta (TGF-3) isoforms from binding to endothelial receptors (see Fig. 40-3). Endoglin is one of these receptors. Decreased binding to endoglin diminishes endothelial nitric oxide-dependent vasodilatation. Serum levels of sEng also begin to rise months before clinical preeclampsia develops (Haggerty, 2012). Interestingly, metformin reduces antiangiogenic secretion from human tissues (Brownfoot, 2016). In one systematic review, third-trimester elevation of sFlt-1 levels and lower PIGF concentrations correlate with and soluble endoglin (sEng). preeclampsia development after 25 weeks' gestation (Widmer, 2007). Subsequently, Haggerty and coworkers (2012) reported that doubling of expressions of sFlt-1 and sEng increased the preeclampsia risk by 39 and 74 percent, respectively. The cause :J• :JE

1	FIGURE 40-4 Angiogenic and antiangiogenic factors in normotensive (N) and preeclamptic (PE) women across pregnancy. Both pairs of factors are significantly divergent by 23 to 26 weeks' gestation. sFlt = soluble fms-like tyrosine kinase 1; PIGF = placental growth factor. (Data from Myatt, 201o3.) of placental overproduction of anti angiogenic proteins remains an enigma. here is a racial-ethnic diference in their secretion (Yang, 2016). Concentrations of the soluble forms are not higher in fetal circulation or amnionic fluid of preeclamptic women, and their levels in maternal blood dissipate after delivery (Staf, 2007). Clinical research aims to employ antiangiogenic proteins in the prediction and diagnosis of preeclampsia. One preliminary report described therapeutic apheresis to reduce sFlt-1 levels (Thadhani, 2016).

1	Evidence for preeclampsia manifestation begins early in pregnancy with covert pathophysiological changes that gain momentum across gestation and eventually become clinically apparent. Unless delivery supervenes, these changes ultimately lead to multiorgan involvement with a clinicl spectrum ranging from meager findings to one of cataclysmic deterioration. As discussed, these are thought to be a consequence of endothelial dysfunction, vasospasm, and ischemia. Although the many maternal consequences of the preeclampsia syndrome are usually described in terms of individual organ systems, they frequently are multiple and overlap .

1	Cardiovascular disturbances are common with preeclampsia syndrome. hese are related to: (1) greater cardiac afterload caused by hypertension; (2) cardiac preload, which is reduced by a pathologically diminished volume expansion during pregnancy and which is increased by intravenous crystalloid or oncotic solutions; and (3) endothelial activation leading to interendothelial extravasation of intravascular fluid into the extracellular space and, importantly, into the lungs.

1	The cardiovascular aberrations of pregnancy-related hypertensive disorders vary depending on several modifiers. These factors include preeclampsia severity, hypertension severity, presence of underlying chronic disease, and the part of the clinical spectrum in which these are studied. In some women, these cardiovascular changes may precede hypertension (De Paco, 2008; Easterling, 1990; Khalil, 2012; Melchiorre, 2013). Nevertheless, with the clinical onset of preeclampsia, cardiac outpur declines, due at least in part to greater peripheral resistance. When assessing cardiac function in preeclampsia, consideration is given to echo cardiographic measures ofmyocardial function and to clinically relevant ventricular function. blood loss incurred at delivery. Anemia may also partially result from greater erythrocyte destruction as subsequently described.

1	blood loss incurred at delivery. Anemia may also partially result from greater erythrocyte destruction as subsequently described. The platelet count is routinely measured in women with any form of gestational hypertension. Decreased platelet concentra tions with eclampsia were described more than 100 years ago.

1	The frequency and intensity of thrombocytopenia vary and are dependent on the severity and duration of the preeclampsia syn drome (Heilmann, 2007; Hupuczi, 2007). Overt thrombocy topenia-deined by a platelet count < 100,000/�L-indicates severe disease (see Table 40-2). In general, the lower the platelet count, the higher the rates of maternal and fetal morbidity and mortality (Leduc, 1992). In most cases, delivery is advisable because worsening thrombocytopenia usually ensues. Mter delivery, the platelet count may continue to decline for the irst day or so. It then usually rises progressively to reach a normal level within 3 to 5 days. As discussed later (p. 722), in some instances with HELLP syndrome, the platelet count continues to fall after delivery. If these do not reach a nadir until 48 to 72 hours, then preeclampsia syndrome may be incorrectly attrib uted to one of the thrombotic microangiopathies discussed in Chapter 56 (p. 1088).

1	Chapter 56 (p. 1088). Myriad other platelet alterations are attributed to the preeclampsia syndrome. hese were reviewed by Kenny and coworkers (2015) and include platelet activation with increased .-degranulation producing �-thromboglobulin, factor 4, and enhanced clearance. Paradoxically, in most studies, in vitro platelet aggregation is reduced compared with the normal increase that is characteristic of pregnancy. This likely is due to platelet "exhaustion" following in vivo activation. Although the cause is unknown, immunological processes or simply platelet deposition at sites of endothelial damage may be implicated. Levels of platelet-bound and circulating platelet-bindable immunoglobulins are elevated, which suggests platelet surface alterations.

1	Abnormally low platelets do not develop in the fetuses or neonates born to preeclamptic women despite severe maternal thrombocytopenia (Kenny, 2015; Pritchard, 1987). Thus, maternal thrombocytopenia in a hypertensive woman is not a etal indication or cesarean delivery. Severe preeclampsia is frequently accompanied by hemolysis, which manifests as elevated serum lactate dehydrogenase levels and reduced haptoglobin levels. Other evidence comes from schizocytosis, spherocytosis, and reticulocytosis in peripheral blood (Cunningham, 1985; Pritchard, 1954, 1976). hese derangements result in part from microangiopathic hemoysis caused by endothelial disruption with platelet adherence and ibrin deposition. Cunningham and coworkers (1995) postulated that erythrocyte morphology was partially caused by serum lipid alterations. Related, substantively decreased longchain fatty acid content is found in erythrocytes of preeclamptic women (Mackay, 2012).

1	Mter early reports of hemolysis and thrombocytopenia with severe preeclampsia, descriptions were added of abnormally elevated serum liver transaminase levels that indicated hepatocellular necrosis (Chesley, 1978). Weinstein (1982) referred to this combination of events as the HELLP syndrome-and this term now is used worldwide. Also, facets of the HELLP syndrome are included in criteria that diferentiate severe from nonsevere preeclampsia (see Table 40-2). he HELLP syndrome is discussed further in that section (p. 722).

1	Subtle changes consistent with intravascular coagulation, and less often erythrocyte destruction, commonly are found with preeclampsia and especially eclampsia (Cunningham, 2015; Kenny, 2015). Some of these changes include elevated factor VIII consumption, increased levels of ibrinopeptides A and B and of D-dimers, and reduced levels of regulatory proteinsantithrombin III and proteins C and S. Coagulation aberrations generally are mild and are seldom clinically signiicant (Kenny, 2015; Pritchard, 1984). Unless placental abruption is comorbid, plasma ibrinogen levels do not difer remarkably from levels found in normal pregnancy. Fibrin degradation products such as D-dimers are minimally elevated. As preeclampsia worsens, so do abnormal indings with thromboelastography (Pisani-Conway, 2013). Despite these changes, routine laboratory assessments of coagulation, such as prothrombin time (PT), activated partial thromboplastin time (aPTT), and plasma ibrinogen level, are not required in the

1	Despite these changes, routine laboratory assessments of coagulation, such as prothrombin time (PT), activated partial thromboplastin time (aPTT), and plasma ibrinogen level, are not required in the management of pregnancy-associated hypertensive disorders.

1	Plasma levels of renin, angiotensin II, angiotensin 1-, aldosterone, deoxycorticosterone, and atrial natriuretic peptide (ANP) are substantively augmented during normal pregnancy. ANP is released during atrial wall stretching from blood volume expansion, and it responds to cardiac contractility (Chap. 4, p. 63). Levels of serum ANP rise in pregnancy, and its secretion is further enhanced in women with preeclampsia (Luft, 2009). Levels of its precursor-proatrial natriuretic peptide-are also increased in preeclampsia (Sugulle, 2012). Vasopressin levels are similar in nonpregnant, normally pregnant, and preeclamptic women even though the metabolic clearance is elevated in the latter two (Durr, 1999) .

1	In women with severe preeclampsia, the volume of extracellular luid, manifest as edema, is usually much greater than that in normal pregnant women. As discussed, the mechanism responsible for pathological luid retention is endothelial injury (Davidge, 2015). In addition to generalized edema and proteinuria, these women have reduced plasma oncotic pressure. his reduction creates a iltration imbalance and further displaces intravascular luid into the surrounding interstitium. Electrolyte concentrations do not difer appreciably in women with preeclampsia compared with those of normal pregnant women. Following an eclamptic convulsion, the serum pH and bicarbonate concentration are lowered due to lactic acidosis and compensatory respiratory loss of carbon dioxide. he intensity of acidosis relates to the amount of lactic acid producedmetabolic acidosis-and the rate at which carbon dioxide is exhaled-respiratory acidosis.

1	During normal pregnancy, renal blood flow and glomerular filtration rate rise appreciably (Chap. 4, p. 65). With preeclampsia, several reversible anatomical and pathophysiological changes ensue. Of clinical importance, renal perfusion and glomerular iltration are reduced. Levels that are much less than normal nonpregnant values are infrequent and are the consequence of severe disease. Most of the decrement in glomerular filtration is from higher renal aferent arteriolar resistance that may be elevated up to fivefold (Conrad, 2015; Comelis, 201r1). Morphological changes are characterized by glomerular endotheliosis, which blocks the barrier that allows filtration. Diminished iltration causes serum creatinine levels to rise to values seen in nonpregnant individuals, that is, 1 mg/ mL, and sometimes higher (Lindheimer, 2008a). Abnormal values usually begin to normalize 10 days or later after delivery (Comelis, 2011; Spaan, 2012a).

1	In most preeclamptic women, the urine sodium concentration is elevated. Urine osmolality rises, urine:plasma creatinine ratio is elevated, and fractional excretion of sodium is low, which all indicated that a prerenal mechanism is involved. Sodium-containing crystalloid infusion raises left ventricular illing pressure, and although oliguria temporarily improves, rapid infusions may cause clinically apparent pulmonary edema. Intensive intravenous fluid therapy is not indicated as "treatment" for preeclamptic women with oliguria unless urine output is diminished from hemorrhage or luid loss from vomiting or fever.

1	Plasma uric acid concentration is typically elevated in preeclampsia. The elevation exceeds that attributable to the reduction in glomerular filtration rate and likely is also due to enhanced tubular reabsorption (Chesley, 1945). At the same time, preeclampsia is associated with diminished urinary excretion of calcium, perhaps because of greater tubular reabsorption (Taufield, 1987). As shown in Table 40-1, detection of proteinuria helps to establish the diagnosis of preeclampsia. Abnormal protein excretion is empirically deined by 24-hour urinary excretion exceeding 300 mg; a urine protein:creatinine ratio �0.3; or persistent protein values of 30 mg/ dL (1 + dipstick) in random urine samples. Although worsening or nephrotic-range proteinuria has been considered by most to be a sign of severe disease, this does not appear to be the case (Airoldi, 2007). Certainly, this concept was not accepted by the 20r13 Task Force.

1	Problematically, the optimal method of establishing abnormal levels of either urine protein or albumin remains to be defined. For a 24-hour quantitative specimen, the "consensus" threshold value used is �300 mg/24 h (American College of Obstetricians and Gynecologists, 2013). Using a urinary protein excretion threshold of 165 mg in a 12-hour sample shows equivalent eicacy (Stout, 2015; Tun, 2012).

1	Determination of urinary protein:creatmme ratio may supplant the cumbersome 24-hour quantiication (Kyle, 2008; Morris, 2012). Chen and associates (2008) found that clean-catch and catheterized urine specimens correlate well. In one systematic review, random urine protein:creatinine ratios below 130 to 150 mg/ g, that is, 0.13 to 0.15, indicate a low likelihood of proteinuria exceeding 300 mg/ d (Papanna, 2008). Ratios <0.08 or > 1.19 have negative-or positive-predictive values of86 and 96 percent, respectively (Stout, 2013). However, midrange ratios, that is, 300 mg/g or 0.3, have poor sensitivity and specificity. Thus, many recommend that with midrange ratio values, 24-hour protein excretion should be quantified. With urine dipstick assessment, determinations depend on urine concentration and are notorious for false-positive and -negative results. hus, assessment may show a dipstick value of 1 + to 2+ from concentrated urine specimens from women who excrete < 300 mg/ d.

1	Importantly, proteinuria may develop late, and some women may already be delivered or have had an eclamptic convulsion before it appears. For example, 10 to 15 percent of women with HELLP syndrome do not have proteinuria at presentation (Sibai, 2004). In one report, 17 percent of eclamptic women did not have proteinuria by the time of seizures (Zwart, 2008). Sheehan and Lynch (1973) frequently found changes identifiable at autopsy by light and electron microscopy in the kidneys of eclamptic women. Glomeruli are enlarged by approximately 20 percent, they are "bloodless," and capillary loops variably are dilated and contracted. Endothelial cells are swollen-termed glomerular capillay endotheliosis (Spargo, 1959). Endothelial cells are often so swollen that they block or partially block the capillary lumens (Fig. 40-7) (Hecht, 2017). Homogeneous subendothelial deposits of proteins and fibrin-like material are seen.

1	Endothelial swelling may result from angiogenic protein "withdrawal" caused by the complexing of free angiogenic proteins with a compatible circulating antiangiogenic protein receptor (see Fig. 40-3). The angiogenic proteins are crucial for podocyte health, and their inactivation leads to podocyte dysfunction and endothelial swelling (Conrad, 2015; Karumanchi, 2009). Also, eclampsia is characterized by greater excretion of these epithelial podocytes (Wagner, 2012; White, 2014).

1	Although mild degrees of acute kidney injury are encountered, clinically apparent acute tubular necrosis is almost invariably induced by comorbid hemorrhage with hypovolemia and hypotension (Chap. 41, p. 755). his is usually caused by severe obstetrical bleeding-especially placental abruption-coupled with inadequate blood replacement. Drakeley and coworkers (2002) described 72 women with preeclampsia and renal failure. Half had HELLP syndrome, and a third had placental abruption. In one review of 183 women with HELLP syndrome, 5 percent had kidney injury (Haddad, 2000). Of those with renal injury, half had placental abruption, and most had postpartum hemorrhage. Last, irreversible renal cortical necrosis develops rarely (Chap. 53, p. 1037).

1	Ofwomen with preeclampsia, serial echocardiographic studies document diastolic dysfunction in 40 to 45 percent (Guirguis, 2015; Melchiorre, 2012). With this dysfunction, ventricles do not properly relax and cannot ill properly. In some of these women, functional diferences persist up to 4 years after delivery (Evans, 2011; Orabona, 2017). Diastolic dysfunction stens from ventricular remodeling, which is judged to be an adaptive response to maintain normal contractility despite the increased afterload ofpreeclampsia. High levels ofantiangiogenic proteins may be contributory (Shahul, 2016). In the otherwise healthy pregnant woman, these changes are usually clinically inconsequential. But when combined with underlying ventricular dysfunction-for example, concentric ventricular hypertrophy from chronic hypertension-further diastolic dysfunction may cause cardiogenic pulmonary edema (Wardhana, 2017). his is discussed further in Chapters 47 (p. 918) and 49 (p. 964).

1	Despite the relatively high frequency of diastolic dysfunction with preeclampsia, clinical cardiac function in most afected women is appropriate (Hibbard, 2015). In some preeclamptic women, cardiac troponin levels are slightly elevated, and amino-terminal pro-brain natriuretic peptide (Nt pro-BNP) levels are elevated with severe preeclampsia (Pergialiotis, 2016; Zachary, 2017). Importantly, both normally pregnant women and those with preeclampsia syndrome can have normal or slightly hyperdynamic ventricular function (Fig. 40-5). hus, both have a cardiac output that is appropriate for leftsided illing pressures. Filling pressures are dependent on the volume of intravenous fluids. Thus, aggressive hydration results in overtly hyperdynamic ventricular function. his is .J,

1	FIGURE 40-5 Ventricular function in normally pregnant women (striped area) and in women with eclampsia (boxed area) is plotted on a Braunwald ventricular function cur e. Normal values are from Clark (1989), and those for eclampsia are from Hankins (1984). PCWP = pulmonary capillary wedge pressure; LVSWI = left ventricular stroke work index. accompanied by elevated pulmonary capillary wedge pressures, and pulmonary edema may develop despite normal ventricular function. This is because of an alveolar endothelial-epithelialrleak, and it is compounded by decreased oncotic pressure from a low serum albumin concentration. In sum, aggressive fluid administration to otherwise normal women with severe preeclampsia substantially elevates normal leftsided filling pressures and raises a physiologically normal cardiac output to hyperdynamic levels.

1	Hemoconcentration is a hallmark ofeclampsia. This concept was precisely quantiied by Zeeman and colleagues (2009), who expanded the prior observations of Pritchard and associates (1984). hey showed in eclamptic women that the normally expected pregnancy blood volume expansion is severely curtailed (Fig. 40-6). Women ofaverage size have a blood volume of 3000 mL, and during the last several weeks of a normal pregnancy, this averages 4500 mL. With eclampsia, however, much or all of the anticipated 1500 mL excess is lost. Such hemoconcentration results from generalized vasospasm that follows endothelial activation and leakage of plasma into the interstitial space. In women with preeclampsia, and depending on its severity, hemoconcentration is usually not as marked.

1	hese changes have substantial clinical consequences. Importantly, women with severe hemoconcentration are unduly sensitive to blood loss at delivery that otherwise may be considered normal. Vasospasm and endothelial leakage of plasma persist for a variable time ater delivery as the endothelium is restored to normalcy. u this takes place, vasoconstriction reverses, and s the blood volume reexpands, the hematocrit usully falls. Importanty} a substantive cause ofthisall in hematocrit} however} is usualy the :J0 FIGURE 40-6 Total blood volumes in normotensive women compared with those with eclampsia. The vertical extensions are one standard deviation from the mean. In eclamptic women, blood volume is minimally increased compared with a subsequent normotensive pregnancy. (Data from Zeeman, 2009.)

1	Liver involvement with preeclampsia may clinically display at least three manifestations. Narrow First, pain is considered a sign of interspace severe disease. It typically manifests by moderate-to-severe right upper quadrant or midepigasEndothelial tric pain and tenderness. Such swelling women usually have elevated serum aspartate transaminase (AST) or alanine transaminase (AL T) levels. In some cases, however, the amount of hepatic tissue involved with infarction may be surprisingly extensive yet still clinically insignificant (N elson, 2017). In our experiences, infarction may be worsened by FIGURE 40-7 Schematic showing glomerular capillary endotheliosis. The capillary of the normal hypotension from obstetrical glomerulus shown on the left has wide endothelial fenestrations, and the pedicels emanating hemorrhage, and it occasion from the podocytes are widely spaced (arrow). The illustration on the right is of a glomerulus with changes induced by the preeclampsia syndrome. The

1	pedicels emanating hemorrhage, and it occasion from the podocytes are widely spaced (arrow). The illustration on the right is of a glomerulus with changes induced by the preeclampsia syndrome. The endothelial cells are swollen and their fenes called shock liver (Alexander, trae narrowed, as are the pedicels that now abut each other.

1	2009; Yoshihara, 2016). Second, elevations of serum • Liver AST and ALT levels are markers for severe preeclampsia. Values seldom exceed 500 U/L, but levels reaching more than 2000 The characteristic hepatic lesions with eclampsia are regions of U/L have been reported (Chap. 55, p. 1058). In general, serumperiportal hemorrhage in the liver periphery (Hecht, 2017). concentrations inversely follow platelet levels, and they bothHowever, lesions as extensive as those shown in Figure 40-8 are usually normalize within 3 days following delivery.

1	unusual. Sheehan and Lynch (1973) described that some degree As a third presentation, hemorrhagic infarction may extend of hepatic infarction accompanied hemorrhage in almost half of to form a hepatic hematoma. This in turn can extend to form women who died with eclampsia. hese findings corresponded a subcapsular hematoma that may rupture. Computed tomogwith reports during the 1960s that described elevated serum raphy (CT) scanning or magnetic resonance (MR) imaginghepatic transaminase levels. Along with the earlier observations greatly aids diagnosis (Fig. 40-9). Unruptured hematomasby Pritchard and associates (1954), who described hemolysis are probably more common than clinically suspected and areand thrombocytopenia with eclampsia, this constellation of more likely to be found with HELLP syndrome. Althoughhemolysis, hepatocellular necrosis, and thrombocytopenia was later termed HELLP syndrome.

1	FIGURE 40-8 Gross liver specimen from a woman with preeclampsia who died from aspiration Periportal hemorrhagic necrosis was seen microscopically. (Reproduced with permission from Cunningham FG: Liver disease complicating pregnancy. Williams Obstetrics, 19th ed. (Suppl 1), Norwalk, Appleton & Lange, 1993.) a woman with severe HELLP syndrome and right-upper quadrant pain. A large subcapsular hematoma (asterisk) is seen confluent with intrahepatic infarction and hematoma (arrowhead). Numerous flameshaped hemorrhages are seen at the hematoma interface (arrows).

1	FIGURE 40-11 Composite illustration showing location of cerebral hemorrhages and petechiae in women with eclampsia. Insert shows the level of the brain from which the main image was constructed. (Data from Sheehan, 1973.) once considered a surgical condition, current management of a hepatic hematoma usually consists of observation unless bleeding is ongoing. In some cases, however, prompt surgical intervention or angiographic embolization may be lifesaving. In one review of 180 cases of hepatic hematoma or rupture, 94 percent of afected gravidas had HELLP syndrome, and in 90 percent of the total, the capsule had ruptured (Vigil-De Gracia, 2012). The maternal mortality rate was 22 percent, and the perinatal mortality rate was 31 percent. In rare cases, liver transplantation is necessary (Hunter, 1995; Wicke, 2004).

1	Acute fatty liver of pregnancy is sometimes confused with preeclampsia (Nelson, 2013; Sibai, 2007a). It too has an onset in late pregnancy, and often there is accompanying hypertension, elevated serum transaminase and creatinine levels, and thrombocytopenia. However, the hallmark of acute fatty liver is significant liver dysfunction, and Table 55-1 (p. 1059) highlights these clinical diferences. Last, no convincing data link pancreatic involvement with preeclampsia syndrome (Sheehan, 1973). Thus, the occasional case of concurrent hemorrhagic pancreatitis is likely unrelated (Lynch, 2015; Swank, 2012). In our experiences from Parkland Hospital, amylase levels were seldom elevated in preeclamptic women (Nelson, 2014a).

1	There is no universally accepted strict deinition of HELLP syndrome, and thus its incidence varies by investigator. In the previously noted study of 183 women with HELLP syndrome, 40 percent had adverse outcomes, and two mothers died (Haddad, 2000). Complications included eclampsia in 6 percent, placental abruption-10 percent, acute kidney injury-5 percent, and pulmonary edema-10 percent. Stroke, hepatic hematoma, coagulopathy, acute respiratory distress syndrome, and sepsis were other serious complications.

1	Women with preeclampsia and HELLP syndrome typically have worse outcomes than preeclamptic women without the HELLP constellation (Kozic, 2011; Martin, 2012, 2013). In one review of693 women with HELLP syndrome, 10 percent had concurrent eclampsia (Keiser, 2011). Sep and associates (2009) described a significantly higher risk for complications in women with HELLP syndrome compared with those with "isolated preeclampsia." These included eclampsia-15 versus 4 percent; preterm birth-93 versus 78 percent; and perinatal mortality rate-9 versus 4 percent, respectively. Because of these marked clinical diferences, it has been postulated that HELLP syndrome has a distinct pathogenesis (Reimer, 2013; Vaught, 2016). Headaches and visual symptoms are common with severe preeclampsia, and associated convulsions deine eclampsia. The earliest anatomical descriptions of brain involvement came from autopsy specimens, but CT and MR imaging and Doppler studies have added many important insights.

1	FIGURE 40-10 This autopsy brain slice shows a fatal hypertensive hemorrhage in a primigravida with eclampsia.

1	From early anatomical descriptions, brain pathology accounted for only about a third of fatal cases such as the one shown in Figure 40-10. In fact, most deaths were from pulmonary edema, nd brain lesions were coincidental. Thus, lthough gross intracerebral hemorrhage was seen in up to 60 percent of eclamptic women, it was fatal in only half of these (Melrose, 1984; Richards, 1988; Sheehan, 1973). s shown in Figure 40-11, other principal lesions found at autopsy of eclamptic women were cortical and subcortical petechial hemorrhages. The classic microscopic vascular lesions consist of fibrinoid necrosis of the arterial wall and perivascular micro infarcts and hemorrhages. Other frequently described major lesions include subcortical edema, multiple nonhemorrhagic areas of "sotening" throughout the brain, and hemorrhagic areas in the white matter (Hecht, 2017). here also may be hemorrhage in the basal ganglia or pons, sometimes with rupture into the ventricles.

1	Clinical, pathological, and neuroimaging findings have led to two general theories to explain cerebral abnormalities with eclampsia. Importantly, endothelial cell dysfunction that characterizes the preeclampsia syndrome likely is a key in both. The first theory suggests that in response to acute and severe hypertension, cerebrovascular overregulation leads to vasospasm (Trommer, 1988). In this scheme, diminished cerebral blood low is hypothesized to result in ischemia, cytotoxic edema, and eventually tissue infarction. Little objective evidence supports this mechanism.

1	he second theory is that sudden elevations in systemic blood pressure exceed the normal cerebrovascular autoregulatory capacity (Hauser, 1988; Schwartz, 2000). Regions of forced vasodilation and vasoconstriction develop, especially in arterial boundary zones. At the capillary level, disruption of end-capillary pressure causes increased hydrostatic pressure, hyperperfusion, and extravasation of plasma and red cells through endothelial tight-junction openings. his leads to vasogenic edema. he recent description of a central nervous system lymphatic vasculature lends credibility to this theory (Louveau, 2015). he most likely mechanism is a combination of the two.

1	he most likely mechanism is a combination of the two. hus, a preeclampsia-associated interendothelial cell leak develops at blood pressure (hydraulic) levels much lower than those that usually cause vasogenic edema and is coupled with a loss of upper-limit autoregulation (Fugate, 2015; Zeeman, 2009). With imaging studies, these manifest as theposterior reversible encephalopathy syndrome (Fig. 40-12) (Fugate, 2015; Hinchey, 1996). he lesions of this syndrome principally involve the posterior brain-the occipital and parietal cortices. But, in at least a third of cases, other areas are involved (Edlow, 2013; Zeeman, 2004a). FIGURE 40-12 Cranial magnetic-resonance imaging in a nullipara with eclampsia. Multilobe T2-FLAIR high-signal lesions are apparent. FLAIR = fluid-attenuated inversion recovery. (Used with permission from Dr. Gerda Zeeman.)

1	Autoregulation is the mechanism by which cerebral blood flow sion pressure. Remember that cerebral perfusion pressure is the sure. In nonpregnant individuals, this autoregulation protects the brain from hyperperfusion when mean arterial pressures rise to as high as 160 mm Hg. hese are pressures far greater than those seen in all but a very few women with eclampsia. Thus, to explain eclamptic seizures, it was theorized that autoregu lation must be altered by pregnancy. Studies by Cipolla and colleagues (2007, 2009, 2015) have convincingly shown that autoregulation is unchanged across pregnancy in rodents. But, some investigators have provided evidence of impaired auto regulation in women with preeclampsia a anzarik, 2014; van Veen, 2013).

1	Veen, 2013). Zeeman and associates (2003) showed that cerebral blood flow during the irst two trimesters of normal pregnancy is similar to nonpregnant values. But during the last trimester, low significantly drops by 20 percent. his group also found greater cerebral blood low in this trimester in women with severe preeclampsia compared with that in normotensive pregnant women (Zeeman, 2004b). Taken together, these findings suggest that eclampsia occurs when cerebral hyperperfusion forces capillary luid interstitially because of endothelial damage. This leak leads to perivascular edema characteristic of the preeclampsia syndrome. Several neurological manifestations typiy the preeclampsia syndrome. Each signiies severe involvement and requires immediate attention.

1	Several neurological manifestations typiy the preeclampsia syndrome. Each signiies severe involvement and requires immediate attention. First, headache and scotomata are thought to arise from cerebrovascular hyperperusion that has a predilection for the occipital lobes. Up to 75 percent of women have headaches, and 20 to 30 percent have visual changes preceding eclamptic convulsions (Sibai, 2005; Zwart, 2008). The headaches may be mild to severe and intermittent to constant. In our experiences, they are unique in that they do not usually respond to traditional analgesia, but they frequently improve ater magnesium sulfate infusion. Convulsions are diagnostic for eclampsia. These are caused by excessive release of excitatory neurotransmitters-especially glutamate; massive depolarization of network neurons; and bursts of action potentials (Meldrum, 2002). Clinical and experimental evidence suggests that extended seizures can cause signiicant brain injury and later brain dysfunction.

1	Blindness is rare with preeclampsia alone, but it complicates eclamptic convulsions in up to 15 percent of women (Cunningham, 1995). Blindness may develop up to a week or more following delivery (Chambers, 2004). There are at least two ypes of blindness, as discussed subsequently. Generalized cerebral edema may develop and is usually manifest by mental status changes that vary from confusion to coma. This situation is particularly dangerous because fatal transtentorial herniation can result. Last, women with eclampsia have been shown to have some cognitive decline when studied 5 to 10 years following an eclamptic pregnancy. This is discussed further in the inal section (p. 745).

1	Last, women with eclampsia have been shown to have some cognitive decline when studied 5 to 10 years following an eclamptic pregnancy. This is discussed further in the inal section (p. 745). With CT imaging, localized hypodense lesions at the gray-and white-matter junction, primarily in the parietooccipital lobes, are typically found in eclampsia. Such lesions may also be seen in the frontal and inferior temporal lobes, the basal ganglia, and thalamus (Brown, 1988). These hypodense areas correspond to petechial hemorrhages and local edema. Edema of the occipital lobes or difuse cerebral edema may cause symptoms such as blindness, lethargy, and confusion (Cunningham, 2000). Widespread edema can appear as marked compression or even obliteration of the cerebral ventricles. Such women may develop signs of impending life-threatening trans tentorial herniation.

1	Several MR imaging acquisitions are used to study eclamptic women. Common findings are hyperintense T2lesions-namely, posterior reversible encephalopathy syndrome (PRES)-in the subcortical and cortical regions of the parietal and occipital lobes (see Fig. 40-12). Also, the basal ganglia, brainstem, and cerebellum are relatively commonly involved (Brewer, 2013; Zeeman, 2004a). Again, these lesions represent focal cerebral edema. Although these PRES lesions are almost universal in women with eclampsia, their incidence in women with preeclampsia approximates 20 percent (Mayama, 2016). Lesions are more likely in women who have severe disease and who have neurological symptoms. And although usully reversible, a fourth of these hyperintense lesions represent cerebral infarctions that have persistent indings (Loureiro, 2003; Zeeman, 2004a).

1	Scotomata, blurred vision, or diplopia are common with severe preeclampsia and eclampsia. hese usually improve with magnesium sulfate therapy and/or lowered blood pressure. Blindness is less common, is usually reversible, and may arise from three potential areas. These are the visual cortex of the occipital lobe, the lateral geniculate nuclei, and the retina. In the retina, pathological lesions may be ischemia, infarction, or detachment (Handor, 2014; Roos, 2012). Occipital blindness is also called amaurosis-from the Greek dimming. With imaging, afected women usually have evidence of extensive occipital lobe vasogenic edema. Of 15 women cared for at Parkland Hospital, occipital blindness lasted from 4 hours to 8 days, but it resolved completely in all cases (Cunningham, 1995). Rarely, extensive cerebral infarctions may result in total or partial visual defects.

1	Blindness from retinal lesions is caused either by serous retinal detachment or rarely by retinal infarction, which is termed Purtscher retinopathy 40-13). Serous retinal detachment is usually unilateral and seldom causes total visual loss. In fact, asymptomatic serous retinal detachment is relatively common with preeclampsia (Saito, 1998). In most cases of eclampsiaassociated blindness, visual acuity subsequently improves. However, if blindness is caused by retinal artery occlusion, vision may be permanently impaired (Lara-Torre, 2002; Moseman, 2002; Roos, 2012). Clinical manifestations suggesting widespread cerebral edema are worrisome. During 13 years at Parkland Hospital, 10 of 175 women (6 percent) with eclampsia were diagnosed with

1	FIGURE 40-1 3 Purtscher retinopathy caused by choroidal ischemia and infarction in preeclampsia syndrome. Ophthalmoscopy shows scattered yellowish, opaque lesions of the retina (arrows). (Reproduced with permission from Lam OS, Chan W: Images in clinical medicine. Choroidal ischemia in preeclampsia. N Engl J Med 344(1o0):739,o2001o.) symptomatic cerebral edema (Cunningham, 2000). Symptoms ranged from lethargy, confusion, and blurred vision to obtundation and coma. In most cases, symptoms waxed and waned. Mental status changes generally correlated with the degree of involvement seen with CT and MR imaging studies. These women are very susceptible to sudden and severe blood pressure elevations, which can acutey worsen the already widespread vasogenic edema. Thus, careful blood pressure control is essential. In the 10 women with generalized edema, three became comatose and had imaging indings of trans tentorial herniation, from which one died. Consideration is given for treatment with

1	is essential. In the 10 women with generalized edema, three became comatose and had imaging indings of trans tentorial herniation, from which one died. Consideration is given for treatment with mannitol or dexamethasone.

1	Compromised utero placental perfusion is almost certainly a major culprit in the greater perinatal morbidity and mortality rates seen with preeclampsia (Harmon, 2015). Defects in endovascular trophoblastic invasion with the preeclampsia syndrome were discussed earlier (p. 714). hus, measurement of uterine, intervillous, and placental blood low would likely be informative. Attempts to assess these in humans have been hampered by several obstacles that include inaccessibility of the placenta, the complexity of its venous eiuent, and the need for radioisotopes or invasive techniques.

1	Measurement of uterine artery blood low velocity has been used to estimate resistance to uteroplacental blood low (Chap. 17, p. 339). Vascular resistance is estimated by comparing arterial systolic and diastolic velocity waveforms. By the completion of placentation, impedance of uterine artery blood low is markedly decreased, but with abnormal placentation, abnormally high resistance persists (Everett, 2012; Ghidini, 2008; Napolitano, 2012). Earlier studies were done to assess this by measuring peak systolic:diastolic velocity ratios from uterine and umbilical arteries in preeclamptic pregnancies. In some cases, but certainly not all, there was higher resistance (Fleischer, 1986; Trudinger, 1990).

1	Another Doppler waveform-uterine artery "notching"has been associated with elevated risks for preeclampsia or fetal-growth restriction (Groom, 2009). In the MFMU Network study reported by Myatt and colleagues (2012a), however, notching had a low predictive value except for early-onset severe disease.

1	Resistance in uterine spiral arteries has also been measured. Impedance was higher in peripheral than in central vessels-a "ring-like" distribution (Matijevic, 1999). Mean resistance values were greater in all women with preeclampsia compared with those in normotensive controls. One study used MR imaging and other techniques to assess placental perfusion ex vivo in myometrial arteries removed from women with preeclampsia or fetal-growth restriction (Ong, 2003). In both conditions, myometrial arteries exhibited endothelium-dependent vasodilatory response. Moreover, other pregnancy conditions are also associated with increased resistance (Urban, 2007). One major adverse efect, fetal-growth restriction, is discussed in Chapter 44 (p. 847).

1	de Almeida Pimenta and colleagues (2014) assessed placental vascularity using a three-dimensional power Doppler histogram and described a placental vasculariy index. This index value was reduced in women with any pregnancy-associated hypertensive disorders-1l.1 percent compared with 15.2 percent in normal controls. Despite these indings, evidence for compromised uteroplacental circulation is found in only a few women who go on to develop preeclampsia. Indeed, when preeclampsia develops during the third trimester, only a third of women with severe disease have abnormal uterine artery velocimetry (Li, 2005). In a study of 50 women with HELLP syndrome, only a third had abnormal uterine artery waveforms (Bush, 2001). In general, the extent of abnormal waveforms correlates with severity of fetal involvement (Ghidini, 2008; Groom, 2009).

1	Various biological markers implicated in the preeclampsia syndrome have been measured to help predict its development. Although most have been evaluated in the irst half of pregnancy, some have been tested as predictors of severity in the third trimester (Chaiworapongsa, 2013; Lai, 2013; Mosimann, 2013). Others have been used to forecast recurrent preeclampsia (Demers, 2014; Eichelberger, 2015). Some of these tests are listed in Table 40-5, which is by no means all inclusive. Overall, these eforts have resulted in testing strategies with poor sensitivity and with poor positive-predictive values for preeclampsia (Conde-Agudelo, 2015; Odibo, 2013). Currenty, no screening tests or preecampsia are predictaby reliable, vali, and economical However, combinations of tests, some yet to be adequatelyevaluated, may be promising (Gallo, 2016; Olsen, 2012).

1	Most tests in this category are cumbersome, time consuming, and overall inaccurate. To evaluate blood pressure changes, three tests assess the blood pressure rise in response to a stimulus. In one, women at 28 to 32 weeks' gestation rest in the left lateral decubitus position and then roll to the supine position. With this rol-over test, increased blood pressure with this maneuver signifies a positive test. he isometric exercise TABLE 40-5. Predictive Tests for Development of the Preeclampsia Syndrome Roll-over test, isometric handgrip or cold pressor test, pressor response to aerobic exercise, angiotensin-II infusion, midtrimester mean arterial pressure, platelet angiotensin-II binding, renin, 24-hour ambulatory blood pressure monitoring, uterine artery r fetal transcranial Doppler velocimetry

1	Human chorionic gonadotropin (hCG), alpha-fetoprotein (AFP), estriol, pregnancy-associated protein A (PAPP A), inhibin A, activin A, placental protein 13, procalcitonin, corticotropinreleasing hormone, A disintegrin, ADAM-12, kisspeptin Serum uric acid, microalbuminuria, urinary calcium or kallikrein, microtransferrinuria, N-acetyl-3-glucosaminidase, cystatin C, podocyturia, podocalyxin Platelet count and activation, fibronectin, endothelial adhesion molecules, prostaglandins, prostacyclin, MMP-9, thromboxane, C-reactive protein, cytokines, endothelin, neurokinin B, homocysteine, lipids, insulin resistance, resistin, antiphospholipid antibodies, plasminogen activator-inhibitor (PAl), leptin, p-selectin, angiogenic and antiangiogenic factors such as placental growth factor (PIGF), vascular endothelial growth factor (VEGF), fms-like tyrosine kinase receptor-1 (sFlt-1), endoglin

1	Antithrombin-III(AT-3), atrial natriuretic peptide (ANP), 32-microglobulin, haptoglobin, transferrin, ferritin, 25-hydroxyvitamin 0, genetic markers, cell-free fetal DNA, serum and urine proteomics and metabolomic markers, hepatic aminotransferases ADAM 12 = ADAM metallopeptidase domain 12; MMP = matrix metalloproteinase. Adapted from Conde-Agudelo, 2015, Duckworth, 2016. test employs the same principle by squeezing a handball. The angiotensin 11 infusion test is performed by giving incrementally increasing doses intravenously, and the hypertensive response is quantiied. In an updated metaanalysis, sensitivities of all three tests were reported to range from 55 to 70 percent, and specificities approximated 85 percent (CondeAgudelo, 2015).

1	Uterine artey Doppler velocimety is posited to relect faulty trophoblastic invasion of the spiral arteries. his failure results in diminished placental perfusion and upstream greater uterine artery resistance. Increased uterine artery velocimetry determined by Doppler ultrasound in the first two trimesters might provide indirect evidence of this process and thus serve as a predictive test for preeclampsia (Dar, 2010; Groom, 2009). Elevated low resistance results in an abnormal vessel waveform represented by an exaggerated diastolic notch. hese indings have value for prediction of fetal-growth restriction but not preeclampsia (American College of Obstetricians and Gynecologists, 2015). Several low velocity waveforms have been investigated for preeclampsia prediction, however, none is suitable for clinical use (CondeAgudelo, 2015; Kleinrouweler, 2012; Myatt, 2012a).

1	Several serum analytes have been proposed to help predict preeclampsia (see Table 40-5). Newer ones are continually added. In general, none of these tests are clinically beneicial for hypertension prediction. Hyperuricemia likely results from reduced uric acid clearance from diminished glomerular filtration, increased tubular reabsorption, and decreased secretion. Cnossen and coworkers (2006) reported that its sensitivity to detect preeclampsia ranged from 0 to 55 percent, and speciicity was 77 to 95 percent. Isolated gestational proteinuria is a risk factor for preeclampsia Qayaballa, 2015; Morgan, 2016; Yamada, 2016). As a predictive test for preeclampsia, microalbuminuria has sensitivities that range from 7 to 90 percent and specificities that span 29 to 97 percent (Conde-Agudelo, 2015).

1	Endothelial activation and inflammation are major participants in the pathophysiology of the preeclampsia syndrome. As a result, compounds such as those listed in Table 40-5 are found to be elevated in circulating blood of afected women, and some have been assessed for their predictive value. First, ibronectins are high-molecular-weight glycoproteins released from endothelial cells and extracellular matrix following endothelial injury. However, in one systematic review, neither cellular nor total ibronectin levels were clinically useful to predict preeclampsia (Leeflang, 2007).

1	Thrombocytopenia and platelet dysfunction are integral features of preeclampsia. Platelet activation causes augmented destruction and lower concentrations. Mean platelet volume rises because of platelet immaturity (Kenny, 2015). Although markers of coagulation activation, described earlier (p. 719), are elevated, the substantive overlap with levels in normotensive pregnant women stultiies their predictive value. Markers of oxidative stress were also hoped to predict preeclampsia. Namely, associated higher levels of lipid peroxides coupled with decreased antioxidant activity raised this possibility. Other markers include iron, transferrin, and ferritin; resistin; hyperhomocysteinemia; blood lipids, including triglycerides, free fatty acids, and lipoproteins; and antioxidants such as ascorbic acid and vitamin E (Christiansen, 2015; CondeAgudelo, 2015; D'Anna, 2004; Mackay, 2012; Mignini, 2005). However, these have not been found to be predictive.

1	Last, an imbalance in antiangiogenic factors is linked to preeclampsia etiopathogenesis. For example, serum levels ofVEGF and PIGF begin to drop before clinical preeclampsia develops. And, recall from Figure 40-4 that at the same time, levels of some antiangiogenic factors, such as sFlt-1 and sEng, begin to rise (Karumanchi, 2016a; Maynard, 2008). With some of these factors, sensitivities for all cases of preeclampsia ranged from 30 to 50 percent, and specificity approximated 90 percent (Conde-Agudelo, 2015). heir predictive accuracy is higher for early-onset preeclampsia (Redman, 2015b; Tsiakkas, 2016). Determination of the sFlt-lIPIGF ratio in women admitted near 37 weeks' gestation to exclude preeclampsia was useful as a predictive factor (Baltajian, 2016; Zeisler, 2016a,b). These results suggest a clinical role for preeclampsia prediction, especially later in pregnancy (Ducworth, 2016; Gallo, 2016). They may also predict adverse pregnancy outcomes in women with lupus and comorbid

1	suggest a clinical role for preeclampsia prediction, especially later in pregnancy (Ducworth, 2016; Gallo, 2016). They may also predict adverse pregnancy outcomes in women with lupus and comorbid anti phospholipid antibodies (Kim, 2016).

1	As discussed in Chapter 13 (p. 273), cell-free DNA (cfDNA) can be detected in maternal plasma. It is hypothesized that cDNA is released in preeclampsia by accelerated apoptosis of cytotrophoblasts (DiFederico, 1999). One MFMU Network study found no correlation between total cfDNA levels and preeclampsia (Silver, 2017). Proteomic, metabolomic, and transcriptomic technologies can be employed to study serum and urinary proteins and cellular metabolites. These have opened new vistas for preeclampsia prediction, and preliminary studies indicate that these may become useful (Bahado-Singh, 2013; Carty, 2011; Ma, 2014; Myers, 2013). Various strategies used to prevent or modiy preeclampsia severity have been evaluated. Some are listed in Table 40-6. In general, none of these has been found to be convincingly and reproducibly efective.

1	Dietary "treatment" for preeclampsia has produced some interesting abuses (Chesley, 1978). A low-salt diet was one of the earliest research eforts to prevent preeclampsia (De Snoo, 1937). This was followed by years of inappropriate diuretic therapy. Although these practices were discarded, it ironically TABLE 40-6. Some Methods to Prevent Preeclampsia That Have Been Evaluated in Randomized Trials Dietary manipulation-low-salt diet, calcium or fish oil Exercise-physical activity, stretching Cardiovascular drugs-diuretics, antihypertensive drugs Antioxidants-ascorbic acid (vitamin C), a-tocopherol (vitamin E), vitamin D Antithrombotic drugs-low-dose aspirin, aspirin/ dipyridamole, aspirin + heparin, aspirin + ketanserin Modified from Staff, 2015. was not until relatively recently that the irst randomized trial was done and showed that a sodium-restricted diet was inefective in preventing preeclampsia (Knuist, 1998).

1	was not until relatively recently that the irst randomized trial was done and showed that a sodium-restricted diet was inefective in preventing preeclampsia (Knuist, 1998). Regular exercise during pregnancy is linked to a lower risk of developing preeclampsia (Barakat, 2016; Morris, 2017). Also, in one systematic review, a trend toward risk reduction with exercise was noted (Kasawara, 2012). Only a few studies have been randomized, and thus, more research is needed (Staf, 2015).

1	Somewhat related, Abenhaim and coworkers (2008) reported a retrospective cohort study of 677 nonhypertensive women hospitalized for bed rest because of threatened preterm delivery. When outcomes of these women were compared with those of the general obstetrical population, bed rest was associated with a signiicantly reduced relative risk-0.27-of developing preeclampsia. From two small randomized trials, prophylactic bed rest for 4 to 6 hours daily at home was successful in signiicantly lowering the incidence of preeclampsia in women with normal blood pressures (\1eher, 2006).

1	Calcium supplementation has been studied in several trials, including one by the National Institute of Child Health and Human Development (NICHD) that included more than 4500 low-risk nulliparas (Levine, 1997). Calcium supplementation did not prevent preeclampsia or pregnancy-associated hypertension. In one metaanalysis, increased calcium intake in high-risk women lowered the risk for preeclampsia (Patrelli, 2012). However, in aggregate, most of these trials have shown that unless women are calcium deficient, supplementation has no salutary efects (Sanchez-Ramos, 2017; Staf, 2015).

1	Cardioprotective aty acids found in some fatty fishes are plentiful in diets of Scandinavians and American Eskimos. Because supplementation with these fatty acids likely prevents inflammatory-mediated atherogenesis, it was posited that they might also prevent preeclampsia. Unfortunately, randomized trials conducted thus far have shown no such beneits from ish oil supplementation (Makrides, 2006; Olafsdottir, 2006; Zhou, 2012).

1	Because of the putative efects of sodium restriction for preeclampsia prevention, diuretic therapy became popular with the introduction of chlorothiazide in 1957 (Finnerty, 1958; Flowers, 1962). In one metaanalysis of nine randomized trials with more than 7000 pregnancies, women given diuretics had a lower incidence of edema and hypertension but not of preeclampsia (Churchill, 2007). Because women with chronic hypertension are at high risk for preeclampsia, several randomized trials have evaluated various antihypertensive drugs to reduce the incidence of superimposed preeclampsia (Chap. 50, p. 980). A critical analysis of these trials by Staf and coworkers (2015) failed to demonstrate benefits for this goal.

1	Data imply that an imbalance between oxidant and antioxidant activity plays a role in preeclampsia pathogenesis. Thus, naturally occurring antioxidants-vitamins C, D, and Emight reduce such oxidation. Several randomized studies have assessed antioxidant vitamin supplementation for women at high risk for preeclampsia (Poston, 2006; Rumbold, 2006; Villar, 2009). he Combined Antioxidant and Preeclampsia Prediction Studies (CAPPS) by the MFMU Network included almost 10,000 low-risk nulliparas (Roberts, 2010). None of these studies showed reduced preeclampsia rates in women provided vitamins C and E compared with those given placebo. Statins were proposed to prevent preeclampsia because they stimulate hemoxygenase-1 expression, which inhibits sFlt-1 release. Preliminary animal data suggest that statins may prevent hypertensive disorders of pregnancy (Lewis, 2017). The MFMU Network plans a randomized trial to test pravastatin for this purpose (Costantine, 2013, 2016).

1	Meormin inhibits hypoxic inducible actor 1. by lowering mitochondrial electron transport chain activity. It reduces sFlt-1 and sEng activity and thus has potential to prevent preeclampsia (Brownfoot, 2016). However, clinical studies are lacking. As noted earlier (p. 715), preeclampsia is characterized by vasospasm, endothelial cell dysfunction, and inlammation, as well as activation of platelets and the coagulation-hemostasis system. Other sequelae include placental infarction and spiral artery thrombosis (Nelson, 2014b). Thus, antithrombotic agents have been evaluated to reduce the incidence of preeclampsia. Low-molecular-weight heparin for prophylaxis has been studied in several randomized trials. Rodger and colleagues (2016) performed a metaanalysis using individual patient data from 963 women. The risk for recurrent preeclampsia, abruption, or fetal-growth restriction was similar in women receiving heparin or placebo.

1	Aspirin, in low oral doses of 50 to 150 mg daily, efectively inhibits platelet thromboxane A2 biosynthesis but has minimal efects on vascular prostacyclin production (Wallenburg, 1986). Still, several clinical trials have shown limited benefits in preeclampsia prevention. For example, a randomized trial from the MFMU Network found that risks for adverse outcomes were not significantly reduced with aspirin therapy (Caritis, 1998). Some combined reports, however, are more favorable. The Paris Collaborative Group performed a metaanalysis that included 31 randomized trials involving 32,217 women (Askie, 2007). For women assigned to receive antiplatelet agents, the relative risk for preeclampsia, superimposed preeclampsia, preterm delivery, or any adverse pregnancy outcome was significantly decreased by 10 percent. Other metaanalyses report marginal benefits of low-dose aspirin for prevention of severe preeclampsia (Roberge, 2012; Villa, 2013). Recently, one randomized trial of more than

1	by 10 percent. Other metaanalyses report marginal benefits of low-dose aspirin for prevention of severe preeclampsia (Roberge, 2012; Villa, 2013). Recently, one randomized trial of more than 1600 women at high risk for preterm preeclampsia provided low-dose aspirin from 11 to 14 weeks' gestation until 36 weeks to prevent recurrence. The rate of preterm recurrence was 1.6 percent in the aspirin group compared with 4.3 percent in the placebo arm (Rolnik, 2017).

1	In recent dueling metaanalyses, Roberge and colleagues (2017) found that aspirin prophylaxis initiated before 16 weeks' gestation was associated with a signiicant risk reductionabout 60 percent-for preeclampsia and fetal-growth restriction. Moreover, they found a dose-response efect. At the same time, Meher and associates (2017) performed an individual participant data metaanalysis and reported a much lower-about 10 percent-risk reduction that was significant whether therapy was initiated before or after 16 weeks.

1	Meanwhile, the U.S. Preventive Services Task Force recommends low-dose aspirin prophylaxis for women at high risk for preeclampsia (Henderson, 2014). Because of this, the American College of Obstetricians and Gynecologists (2016b) issued a Practice Advisory that recommends low-dose aspirin be given between 12 and 28 weeks' gestation to help prevent preeclampsia in high-risk women. This includes those with a history of preeclampsia and those with twins, chronic hypertension, overt diabetes, renal disease, and autoimmune disorders. These results have also raised the question as to whether al pregnant women should be given aspirin (Mone, 2017). At this time, our answer is "no."

1	Low-dose aspirin coupled with heparin mmgates thrombotic sequelae in women with lupus anticoagulant (Chap. 59, p. 1145). Because of a similarly high prevalence of placental thrombotic lesions found with severe preeclampsia, trials have assessed the possible merits of such treatments for women with prior preeclampsia. In two randomized trials, women with a history of early-onset preeclampsia were given an aspirin therapy or an enoxaparin plus aspirin regimen (Groom, 2017; Haddad, 2016). Outcomes were similar. From their reviews, Sergis and associates (2006) reported better pregnancy outcomes in women with prior severe preeclampsia given low-molecularweight heparin plus low-dose aspirin compared with those given low-dose aspirin alone. Similar indings were reported by de Vries and coworkers (2012).

1	Pregnancy complicated by gestational hypertension is managed based on its severity, presence of preeclampsia, and gestational age. Preeclampsia cannot always be diagnosed definitively. hus, the Task Force (2013) recommends more frequent prenatal visits if preeclampsia is "suspected." Increases in systolic and diastolic blood pressure can be either normal physiological changes or signs of developing patholoy. Heightened surveillance permits more prompt recognition of ominous changes in blood pressure, critical laboratory findings, and clinical signs and symptoms (Macdonald-Wallis, 2015).

1	The basic management objectives for any pregnancy complicated by preeclampsia are: (1) termination of pregnancy with the least possible trauma to mother and fetus, (2) birth of a healthy newborn that subsequently thrives, and (3) complete restoration of health to the mother. In many women with preeclampsia, especially those at or near term, all three objectives are served equally well by induction of labor. One of the most important clinical questions or succesul management is precise knowledge of etal age. • Early Diagnosis of Preeclampsia

1	• Early Diagnosis of Preeclampsia Traditionally, the frequency of prenatal visits is increased during the third trimester, and this aids early detection of preeclampsia. Women without overt hypertension, but in whom eary developing preeclampsia is suspected during routine prenatal visits, are seen more frequenty. For many years at Parkland Hospital, women with new-onset diastolic blood pressures >80 mm Hg but <90 mm Hg or with sudden abnormal weight gain of more than 2 pounds per week have, at minimum, returned for visits at 7 -day intervals. Outpatient surveillance is continued unless overt hypertension, proteinuria, headache, visual disturbances, or epigastric pain supervenes. Women with overt new-onset hypertension-either diastolic pressures �90 mm Hg or systolic pressures � 140 mm Hg-are admitted to determine if the increase is due to preeclampsia, and if so, to evaluate its severity. With hospitalization, a systematic evaluation is instituted to include:

1	With hospitalization, a systematic evaluation is instituted to include: Detailed examination, which is coupled with daily scrutiny for clinical indings such as headache, visual disturbances, epigastric pain, and rapid weight gain Quantification of proteinuria or urine protein:creatmme Blood pressure readings with an appropriate-size cuf every 4 hours, except between 2400 and 0600 unless previous readings are elevated Measurements of plasma or serum creatinine and hepatic transaminase levels and a hemogram that includes a platelet count. The frequency of testing is determined by hypertension severity. Although some recommend measurement of serum uric acid and lactate dehydrogenase levels and coagulation studies, their value has been questioned (CondeAgudelo, 2015; Thangaratinam, 2006). Evaluation of fetal size and well-being and amnionic luid volume, by either physical examination or sonography.

1	Evaluation of fetal size and well-being and amnionic luid volume, by either physical examination or sonography. Reduced physical activity throughout much of the day is likely beneficial, but as the 2013 Task Force concluded, absolute bed rest is not desirable. Ample protein and calories are included in the diet, and sodium and fluid intake are not limited or forced. In sum, goals of evaluation include early identification of preeclampsia or worsening of the syndrome and development of a management plan for timely delivery. Fortunately, many cases are suiciently mild and near enough to term that they can be managed conservatively until labor commences spontaneously or until the cervix becomes favorable for labor induction. Complete abatement 0/ al signs and symptoms, however, is uncommon until ater delivery. If severe preeclampsia is diagnosed using the criteria in Table 40-2, further management is subsequently described.

1	Termination o/pregnancy is the ony cure or preeclampsia. Head ache, visual disturbances, or epigastric pain are indicative that convulsions may be imminent, and oliguria is another ominous sign. Severe preeclampsia demands anticonvulsant and often antihypertensive therapy, followed by delivery. Treatment for eclampsia is identical. The prime objectives are to forestall con vulsions, to prevent intracranial hemorrhage and serious dam age to other vital organs, and to deliver a healthy newborn. his is true even when the cervix is unfavorable (T ajik, 2012). Labor induction is carried out, usually with preinduc tion cervical ripening with a prostaglandin or osmotic dilator (Chap. 26, p. 505).

1	Concerns stemming from an unfavorable cervix, a perceived sense of urgency because of preeclampsia severity, and a need to coordinate neonatal intensive care have led some to advocate cesarean delivery. Alexander and colleagues (1999) reviewed 278 singleton liveborn neonates weighing 750 to 1500 g delivered of women with severe preeclampsia at Parkland Hospital. In half of the women, labor was induced, and the remainder underwent cesarean delivery without labor. Induction was successful in accomplishing vaginal delivery in a third, and it was not harmful to very-low-birthweight neonates. Others have reported similar observations (Alanis, 2008; Roland, 2017). However, whenever it appears that induction almost certainly will not succeed or attempts have failed, then cesarean delivery is indicated.

1	For a woman near term, with a soft, partially efaced cervix, even a milder degree of preeclampsia probably carries more risk to the mother and her fetus-newborn than does induction of labor (Tajik, 2012). A randomized trial of 756 women with mild preeclampsia supported delivery after 37 weeks' gestation (Koopmans, 2009).

1	When the fetus is preterm, the tendency is to temporize in the hope that additional weeks in utero will reduce the risk of neonatal death or serious morbidity from prematurity. Such a policy certainly is justified in milder cases. Assessments of fetal well-being and placental function are performed, especially when the fetus is immature. Most recommend frequent performance of nonstress testing or biophysical proiles to assess fetal well-being (American College of Obstetricians and Gynecologists, 20 16a). Several tests can be used to provide evidence of lung maturity (Chap. 34, p. 638). An sFlt-lIPlGF ratio <38 is predictive of the short-term absence of preeclampsia, but this ratio testing is still investigational (Zeisler, 2016a,b). Also, women with higher ratios tend to have more adverse outcomes (Baltajian, 2016).

1	The decision to deliver late-preterm fetuses is less clear. Barton and coworkers (2011) reported excessive neonatal morbidity in women delivered before 38 weeks despite having stable, mild, nonprotein uric hypertension. The Netherlands study of 4316 newborns delivered, between 34°/7 and 36617 weeks also described substantive neonatal morbidity in these cases (Langenveld, 201l). Another Dutch study-HYPITAT-II-randomly assigned women with nonsevere hypertension between 34 and 37 weeks to immediate delivery or to expectant management (Broekhuijsen, 2015). Immediate delivery reduced the risks for adverse maternal outcomes-I. 1 versus 3.1 percent. However, it increased the risk for neonatal respiratory distress syndrome-5.7 versus 1. 7 percent. For women with mild-to-moderate stable hypertension is continued. During surveillance, reduced physical activity throughout much of the day, at least intuitively, seems beneicial. That said, complete bed rest is not recommended by the 2013

1	That said, complete bed rest is not recommended by the 2013 Task Force. First, this is pragmatically unachievable because of the severe restrictions it places on otherwise well women. Also, it likely predisposes to thromboembolism (Knight, 2007). To reduce activity, several studies have addressed the beneits of inpatient care and outpatient management.

1	he concept of prolonged hospitalization for women with hypertension arose during the 1970s. At Parkland Hospital, an inpatient antepartum unit was established in 1973 by Dr. Peggy Whalley in large part to provide care for such women. Initial results from this unit were reported by Hauth (1976) and Gilstrap (1978) and their coworkers. Most hospitalized womenJJ have a beneficial response characterized by amelioration or improvement of hypertension. These women are not "cure, and neary 90 percent have recurrent hypertension bore or during labor. By 2016, more than 10,000 nulliparas with mild-to-moderate, early-onset hypertension during pregnancy had been managed successfully in this unit. Provider costsnot charges-for this relatively simple physical facility, modest nursing care, no drugs other than iron and folate supplements, and few essential laboratory tests are minimal compared with the cost of neonatal intensive care for a preterm neonate. Importantly, none of these women have

1	other than iron and folate supplements, and few essential laboratory tests are minimal compared with the cost of neonatal intensive care for a preterm neonate. Importantly, none of these women have sufered thromboembolic disease.

1	Many clinicians believe that further hospitalization is not warranted if hypertension abates within a few days, and this has legitimized third-party payers to deny hospitalization reimbursement. Consequently, many women with mild-tomoderate hypertension are managed at home. Outpatient management may continue as long as preeclampsia syndrome does not worsen and fetal jeopardy is not suspected. Sedentary activity throughout the greater part of the day is recommended. These women are instructed in detail to report symptoms. Home blood pressure and urine protein monitoring or frequent evaluations by a visiting nurse may prove beneficial.

1	To assess this approach, 11r82 nulliparas with mild gestational hypertension-20 percent had proteinuria-were managed with home health care (Barton, 2002). Their mean gestational ages were 32 to 33 weeks at enrollment and 36 to 37 weeks at delivery. Severe preeclampsia developed in approximately 20 percent, about 3 percent developed HELLP syndrome, and two Day Unit 24 57 23 36 0 39.8 1n.1 3320 0 Usual Care 30 54 21 36.5 21 39 5.1 3340 0 Turnbull (2004) 374b Hospitalization 125 63 0 35.9 22 39 S.5 3330 3.S 0 Day Unit 249 62 0 36.2 22 39.7 7.2 3300 2.3 0 aExciuded women with proteinuria at study entry. blncluded women with sl+ proteinuria. BW = birthweight; EGA = estimated gestational age; HTN = hypertension; Parao= nulliparas; PMR = perinatal mortality rate; Prot = proteinuria; SGA = small for gestational age.

1	BW = birthweight; EGA = estimated gestational age; HTN = hypertension; Parao= nulliparas; PMR = perinatal mortality rate; Prot = proteinuria; SGA = small for gestational age. women had eclampsia. Perinatal outcomes were generally good. In approximately 20 percent, there was fetal-growth restriction, and the perinatal mortality rate was 4.2 per 1000 births.

1	Several studies have compared continued hospitalization and outpatient care. In a pilot study from Parkland Hospital, 72 nulliparas with new-onset hypertension from 27 to 37 weeks were assigned either to continued hospitalization or to outpatient care (Horsager, 1995). he only significant diference was that women in the home care group developed severe preeclampsia significantly more frequently than hospitalized women42 versus 25 percent. In another trial, ater hospital evaluation, 218 women with mild gestational nonprotein uric hypertension were similarly divided (Crowther, 1992). As shown in Table 40-7, the mean hospital duration was 22.2 days for women with inpatient management compared with only 6.5 days in the home care group. Preterm delivery before 34 and before 37 weeks' gestation was increased twofold in the outpatient group. However, maternal and newborn outcomes were otherwise similar.

1	Another approach, popular in Europe, is day care (Milne, 2009). In one study, 54 women with hypertension after 26 weeks' gestation were assigned to either day care or routine outpatient management (see Table 40-7) (Tufnell, 1992). Progression to overt preeclampsia and labor inductions were signiicantly greater in the routine outpatient management group. In another, 395 women participated in either day care or inpatient management (Turnbull, 2004). Almost 95 percent had mild-to-moderate hypertension. Of enrolled women, 288 lacked proteinuria, and 86 had � 1 + proteinuria. There were no perinatal deaths, and none of the women developed eclampsia or HELLP syndrome. Costs for either scheme were not signiicantly diferent, and general satisfaction favored day care.

1	In sum, either inpatient or close outpatient management is appropriate for a woman with mild de novo hypertension, including those with nonsevere preeclampsia. Most of these studies were carried out in academic centers with dedicated management teams. hat said, the key to success is close surveillance and a conscientious patient with good home support. he use of antihypertensive drugs to prolong pregnancy or modiY perinatal outcomes in pregnancies complicated by various hypertensive disorders has been of considerable interest. Treatment for women with chronic hypertension complicating pregnancy is discussed in detail in Chapter 50 (p. 980).

1	Drug treatment for early mild preeclampsia has been disappointing (Table 40-8). Sibai and colleagues (1987a) reported that women given labetalol had significantly lower mean blood pressures. However, mean pregnancy prolongation, gestational age at delivery, and birthweight did not difer between groups. he cesarean delivery rate and the number of newborns admitted to special-care nurseries were also similar. The requency of growth-restricted neonates was doubled in women given labetalol-19 versus 9 percent. The three other studies listed in Table 40-8 compared labetalol or the calcium-channel blockers nifedipine and isradipine against placebo. Except for fewer episodes of severe hypertension, none of these studies showed any benefits from antihypertensive treatment (Magee, 2015). Similar conclusions were reached by Abalos and associates (2014), who reviewed 49 randomized trials of active antihypertensive therapy compared with either no treatment or placebo given to women with

1	Similar conclusions were reached by Abalos and associates (2014), who reviewed 49 randomized trials of active antihypertensive therapy compared with either no treatment or placebo given to women with mild-to-moderate gestational hypertension.

1	Up through the early 1990s, the prevailing\practice was that women with severe preeclampsia were usually delivered without delay. However, another approach for women with preterm severe preeclampsia has also been advocated. This approach calls for "conservative" or "expectant" management with the aA11 women had preeclampsia. blncludes postpartum hypertension. cp < .05 when study drug compared with placebo. HTN = hypertension; NS = not stated. aim of improving neonatal outcome without compromising maternal safety. Aspects of such management always include careful daily-and usually more frequent-inpatient monitoring of the mother and her fetus. Expectant Management of Preterm Severe Preeclampsia

1	Expectant Management of Preterm Severe Preeclampsia Theoretically, antihypertensive therapy has potential application when severe preeclampsia develops before intact neonatal survival is likely. Such management is controversial, and it may be dangerous. In one of the irst studies, Sibai and the Memphis group (1985) attempted to prolong pregnancy because of fetal immaturity in 60 women with severe preeclampsia between 18 and 27 weeks. The results were disastrous. The perinatal mortaliy rate was 87 percent. Although no mothers die, 13 su.ired placental abruption, 10 had eclampsia, three developed renal ailure, two had hypertensive encephalopathy, one had an intracerebral hemorrhage, and another had a ruptured hepatic hematoma.

1	Because of their early study, the Memphis group redeined criteria and performed a randomized trial of aggressive versus expectant management for 95 women who had severe preeclampsia but with more advanced gestations of 28 to 32 weeks (Sibai, 1994). Women with HELLP syndrome were excluded rom this tria. Aggressive management included glucocorticoid administration for fetal lung maturation followed by delivery in 48 hours. Expectantly managed women were observed at bed rest and given either labetalol or nifedipine orally for severe hypertension. In this study, pregnancy was prolonged for a mean of 15.4 days in the expectant management group. An overall improvement in neonatal outcomes was also reported.

1	Following these experiences, expectant management became more commonly practiced, but with the caveat that women with HELLP syndrome or growth-restricted fetuses were usually excluded. But in a subsequent follow-up observational study, the Memphis group compared outcomes in 133 preeclamptic women with and 136 without HELLP syndrome who presented between 24 and 36 weeks (Abramovici, 1999). Women were subdivided into three study groups. The first group included those with complete HELLP syndrome. The second group included women with partial HELLP syndromedeined as either one or two but not all three of the deining laboratory values. The third group included women who had severe preeclampsia without HELLP syndrome. Perinatal outyrcomes were similar in each group, and importantly, outcomes were not improved with procrastination. Despite this, the investigators concluded that women with partial HELLP syndrome and those with severe preeclampsia alone could be managed expectantly. Those with

1	not improved with procrastination. Despite this, the investigators concluded that women with partial HELLP syndrome and those with severe preeclampsia alone could be managed expectantly. Those with fetal-growth restriction generally have shorter interval-to-delivery durations (McKinney, 2016).

1	Sibai and Barton (2007b) reviewed expectant management of severe preeclampsia from 24 to 34 weeks. More than 1200 women were included, and although the average time gained ranged from 5 to 10 days, the maternal morbidity rates were formidable. Serious complications in some of these and in later studies included placental abruption, HELLP syndrome, pulmonary edema, renal failure, and eclampsia (Table 40-9). Moreover, perinatal mortality rates averaged 90 per 1000 births. Fetal-growth restriction was common, and in the studies from The Netherlands, it was an astounding 94 percent (Ganzevoort, 2005a,b). Perinatal mortality rates are disproportionately high in these growth-restricted neonates, but maternal outcomes are not appreciably diferent (Haddad, 2007; Shear, 2005). The MEXPRE Latin Study was a multicenter trial that randomly assigned 267 women with severe preeclampsia at 28 to 32 weeks to prompt delivery or to expectant management (VigilDe Gracia, 2013). The perinatal mortality

1	Study was a multicenter trial that randomly assigned 267 women with severe preeclampsia at 28 to 32 weeks to prompt delivery or to expectant management (VigilDe Gracia, 2013). The perinatal mortality rate approximated

1	Ganzevoort (2005a, b) 216 11 1.8 18 3.6 NS 1.8 Bombrys (2009) 66 5 11 8 9 3 0 27 1.5 Abdel-Hady (2010) 211 12 3.3 7.6 0.9 6.6 0.9 NS 48 Vigil-De Gracia (2013) 131 10.3 7.6 14 1.5 4.5 0.8 22 8.7 Range 91n0 5-1n2 1.8-23 4.6-27 0.9-3.9 2.3-6.6 0.9-1n8 27-94 1.5-48 alncludes one maternal death. AKI = acute kidney injury; EGA = estimated gestational age; FGR = fetal-growth restriction; HELLP = hemolysis, elevated liver enzyme levels, low platelet count syndrome; NS = not stated; PMR = perinatal mortality rate; Pulm. = pulmonary. 9 percent in each group, the composite neonatal morbidity outcome was not improved with expectant management. On the other hand, fetal-growth restriction-22 versus 9 percent-and placental abruption-7.6 versus 1.5 percent-were significantly higher in the group managed expectantly. Expectant Management of Midtrimester Severe Preeclampsia

1	Expectant Management of Midtrimester Severe Preeclampsia Several small studies have focused on expectant management of severe preeclampsia syndrome bore 28 weeks. In their review, Bombrys and coworkers (2008) found eight such studies that included nearly 200 women with severe preeclampsia with an onset <26 completed weeks. Maternal complications were common. Because no neonates survived when delivered before 23 weeks, the Task Force (2013) recommends pregnancy termination in these cases. For women with slightly more advanced pregnancies, however, the decision is less clear. For example, at 23 weeks' gestation, the perinatal survival rate was 18 percent, but long-term perinatal morbidity is yet unknown. For women with pregnancies at 24 to 26 weeks, perinatal survival approached 60 percent, and it averaged almost 90 percent for those at 26 weeks.

1	At least five observational studies of women with severe midtrimester preeclampsia who were managed expectantly have been published since 2005 (AbdelHady, 2010; Belghiti, 2011; Bombrys, Admit to L&D Contraindications to conservative management Eclampsia, pulmonary edema, HELLP syndrome Significant renal dysfunction, coagulopathy Frequent evaluation: vital signs, UOP Serial lab evaluation of re n.aI function and for HELLP syndrome Daily fetal assessment and evaluation of serial growth and amnionic fluid

1	Frequent evaluation: vital signs, UOP Serial lab evaluation of re n.aI function and for HELLP syndrome Daily fetal assessment and evaluation of serial growth and amnionic fluid FIGURE 40-14 Clinical management algorithm for severe preeclampsia at <34 weeks. HELLP = hemolysis, elevated liver enzyme levels, low platelet count; L&D = labor and delivery; MgS04= magnesium sulfate; UOP = urine output. (Adapted from the Society for 2008; Budden, 2006; Gaugler-Senden, Maternal-Fetal Medicine, 2011.) 2006). Maternal complications developed in 60 percent, and there was one maternal death. he perinatal mortality rate was 650 per 1000 births. At this time, no comparative studies attest to perinatal benefits of such expectant treatment versus early delivery in the face of serious maternal complications, which approach rates of 50 percent. We do not recommend such management.

1	To enhance fetal lung maturation, glucocorticoids have been administered to women with severe hypertension who are remote from term. Treatment does not seem to worsen maternal hypertension, and a lower incidence of respiratory distress and improved fetal survival rates have been cited. hat said, only one randomized trial has evaluated corticosteroids given to hypertensive women for fetal lung maturation. his trial included 218 women with severe preeclampsia between 26 and 34 weeks' gestation who were randomly assigned to betamethasone or placebo administration (Amorim, 1999). Rates of neonatal complications that included respiratory distress, intraventricular hemorrhage, and death were reduced significantly when betamethasone was given compared with placebo. On the heaviy weighted negative side, there were two maternal deaths and 18 stillbirths. We add these indings to buttress our unenthusiastic acceptance of attempts to prolong gestation in many of these women (Alexander, 2015;

1	side, there were two maternal deaths and 18 stillbirths. We add these indings to buttress our unenthusiastic acceptance of attempts to prolong gestation in many of these women (Alexander, 2015; Bloom, 2003).

1	Taken in toto, these studies do not show overwhelming beneits compared with maternal risks for expectant management of severe preeclampsia in women with gestations from 24 to 32 weeks. Despite these caveats, the Society for MaternalFetal Medicine (201r1) has determined that such m�nagement is a reasonable alternative in selected women with severe preeclampsia before 34 weeks (Fig. 40-14). The Task Force (2013) supports this recommendation. As shown in Table 40-10, such management calls for in-hospital maternal and fetal surveillance with delivery prompted by evidence for worsening severe preeclampsia or maternal or fetal compromise. Although attempts are made for vaginal delivery in most cases, the likelihood of cesarean delivery rises with decreasing gestational age.

1	Our view is more conservative. Undoubtedly, the overriding reason to terminate pregnancies with severe preeclampsia is maternal safety. Indeed, it seems obvious that a delay to prolong gestation in women with severe preeclampsia may have serious maternal consequences (see Table 40-9). These observations are even more pertinent when considered with the absence of convincing evidence that perinatal outcomes are markedly improved by the average prolongation of pregnancy by approximately 1 week. If undertaken, the caveats that mandate delivery shown in Table 40-10 should be strictly heeded. • Corticosteroids to Ameliorate HELLP Syndrome At least three randomized trials have evaluated the beneits of glucocorticoids given to improve the laboratory abnormalities TABLE 40-1 0. Indications for Delivery in Women Delivery ater Maternal Stabilization: Delay Delivery 48 hr If Possible: Thrombocytopenia < 1 OO,OOO/�L

1	TABLE 40-1 0. Indications for Delivery in Women Delivery ater Maternal Stabilization: Delay Delivery 48 hr If Possible: Thrombocytopenia < 1 OO,OOO/�L Hepatic transaminase levels twice upper limit of normal alnitial dose only, do not delay delivery. From the Society for Maternal-Fetal Medicine, 2011, and the Task Force of the American College of Obstetricians and Gynecologists, 201n3.

1	associated with HELLP syndrome. First, Fonseca and associates (2005) randomly assigned 132 women with HELLP syndrome to either dexamethasone or placebo administration. Outcomes assessed included hospitalization length, recovery time of abnormal laboratory test results, resolution of clinical parameters, and complications that included acute renal fail ute, pulmonary edema, eclampsia, and death. None of these was signiicantly diferent between the two groups. In another study, 105 postpartum women with HELLP syndrome were assigned to dexamethasone or placebo treatment (Katz, 2008). Outcomes were analyzed similarly to the Fonseca study, and no advantage to dexamethasone was found (Fig. 40-15). In the third study, preeclamptic women were given either placebo or methylprednisolone if their platelet count was between 50,000 and 150,000/�L (Pourrat, 2016). No benefits were gained from corticosteroid therapy. Because of these indings, the 2013 Task Force does not recommend corticosteroid

1	platelet count was between 50,000 and 150,000/�L (Pourrat, 2016). No benefits were gained from corticosteroid therapy. Because of these indings, the 2013 Task Force does not recommend corticosteroid treatment for thrombocytopenia with HELLP syndrome.

1	In several preliminary studies, therapies have attempted to lower serum levels or mitigate the action of antiangiogenic factors. Some of these include therapeutic apheresis, done to lower sFlt-1 levels (Thadhani, 2016). Pravastatin has been given for preeclampsia prevention (Cleary, 2014). Sildenail citrate, a phosphodiesterase inhibitor, has been provided to promote vasodilation (Trapani, 2016; Vigil-De Gracia, 2016). In a recent randomized trial of 120 women with early-onset preeclampsia, an recombinant antithrombin infusion compared and occasionally anuria develops. There may be hemoglobinuria, but hemoglobinemia is rare. Often, facial and peripheral edema is pronounced, but it may be absent (Fig. 40-17). FIGURE 40-1 7 Severe edema in a young nullipara with antepartum preeclampsia. (Used with permission from Dr. Nidhi Shah.)

1	FIGURE 40-1 7 Severe edema in a young nullipara with antepartum preeclampsia. (Used with permission from Dr. Nidhi Shah.) As with severe preeclampsia, urinary output rises after delivery and is usually an early sign of improvement. With renal dysfunction, serum creatinine levels are serially monitored. Proteinuria and edema ordinarily disappear within a week postpartum. In most cases, blood pressure returns to normal within a few days to 2 weeks after delivery (Berks, 2009). As subsequently discussed, persisting and severe hypertension likely predicts underlying chronic vascular disease (Podymow, 2010).

1	In antepartum eclmpsia, labor may begin spontaneously shortly ater convulsions ensue and may progress rapidly. If the convulsions occur during labor, contractions may increase in frequency and intensity, and the duration oflabor may be shortened. Because of maternal hypoxemia and lactic acidemia caused by convulsions, fetal bradycardia oten follows a seixure (Fig. 40-18). he fetal heart rate usually recovers within 2 to 10 minutes (Ambia, 2018). If it persists more than about 10 minutes, another cause of bradycardia, such as placental abruption or imminent delivery, should be considered.

1	Pulmonary edema may follow shortly after eclamptic convulsions or to several hours later. This up usually is caused by aspiration pneumonitis from gastric-content inhalation during vomiting that frequently accompanies convulsions. In some women, pulmonary edema may be caused by ventricular failure from increased aterload that results from severe hypertension. Both pulmonary edema and hypertension can be further aggravated by vigorous intravenous fluid administration (Dennis, 2012b). Such pulmonary edema from ventricular failure is more common in morbidly obese women and in those with previously unappreciated chronic hypertension. Occasionally, sudden death occurs synchronously with an eclamptic convulsion, or it follows shortly thereafter. Most often in these cases, a massive cerebral hemorrhage is the cause (see Fig. 40-10). Hemiplegia may result from sublethal hemorrhage. Cerebral hemorrhages are more likely in older women with underlying chronic hypertension.

1	In approximately 10 percent of eclamptic women, some degree of blindness follows a seizure. The causes of blindness or impaired vision were discussed earlier (p. 724). Blindness with severe preeclampsia without convulsions usually stems from retinal detachment (Vigil-De Gracia, 2011). Conversely, blindness with eclampsia is typically due to occipital lobe edema (Cunningham, 1995). In both instances, however, the prognosis for return to normal function is good and is usually complete within 1 to 2 weeks postpartum. Up to 5 percent of women with eclampsia have substantively altered consciousness, including persistent coma, following a seizure. This is due to extensive cerebral edema, and associated trans tentorial herniation may cause death (p. 723).

1	Rarely, eclampsia is followed by psychosis, and the woman becomes violent. This may last for several days to 2 weeks. The prognosis for return to normal function is good, provided there was no preexisting mental illness. It is presumed to be similar to postpartum psychosis discussed in Chapter 61 (p. 1179). Antipsychotic medications have proven efective in the few cases of posteclampsia psychosis treated at Parkland Hospital. Generally, eclampsia is more likely to be diagnosed too frequently rather than overlooked. Epilepsy, encephalitis, meningitis, brain rumor, neurocysticercosis, amnionic fluid embolism, postdural puncture cephalalgia, and ruptured cerebral aneurysm during late pregnancy or in the puerperium may simulate eclampsia. Until other such causes are excluded, however, all pregnant women with convulsions should be considered to have eclampsia.

1	FIGURE 40-18 Fetal heart rate tracing shows fetal bradycardia following an intrapartum eclamptic convulsion. Bradycardia resolved and beat-to-beat variability returned approximately 5 minutes following the seizure. • Management of Eclampsia Magnesium sulfate is highly efective to prevent convulsions in women with preeclampsia and to stop them in those with eclampsia. In his review, Chesley (1978) cited observational data by Pritchard and colleagues (1955, 1975) from Parkland Hospital and from his own institution. At that time, most eclampsia regimens in the United States adhered to a similar philosophy, and it is still in use today: 1. Control of convulsions using an intravenously administered loading dose of magnesium sulfate that is followed by a maintenance dose, usually intravenous, of magnesium sulfate 2. Intermittent administration of an antihypertensive medication to lower blood pressure whenever it is considered dangerously high 3.

1	Intermittent administration of an antihypertensive medication to lower blood pressure whenever it is considered dangerously high 3. Avoidance of diuretics unless pulmonary edema is obvious, limitation of intravenous luid administration unless fluid loss is excessive, and avoidance of hyperosmotic agents 4. Delivery of the fetus to resolve preeclampsia. • Magnesium Sulfate to Control Convulsions Magnesium sulfate administered parenterally is an efective anticonvulsant that avoids producing central nervous system depression. It may be given intravenously by continuous inusion or intramuscularly by intermittent injection (Table 40-11). The dosages for severe preeclampsia are the same as for eclampsia. Because labor and delivery is a more likely time for convulsions to develop, women with preeclampsia-eclampsia usually are given magnesium sulfate during labor and for 24 hours postpartum.

1	In the United States, magnesium sulfate is almost universally administered intravenously. Of concern, magnesium sulfate solutions, although inexpensive to prepare, are not readily available in all parts of the developing world. And even when the solutions are available, the technology to infuse them may not be. herefore, it should not be overlooked that the drug can be administered intramuscularly and that this route is as efective as intravenous administration (Salinger, 20l3). In two reports from India, intramuscular regimens were nearly equivalent in preventing recurrent convulsions and maternal deaths in women with eclampsia (Chowdhury, 2009; lana, 20l3). hese observations comport with earlier ones from Parkland Hospital (Pritchard, 1975, 1984).

1	Magnesium suate is not given to treat hypertension. Magnesium most likely exerts a specific anticonvulsant action on the cerebral cortex. Typically, the mother stops convulsing after the initial 4-g loading dose. By an hour or two, she regains consciousness suiciently to be oriented to place and time.

1	he magnesium sulfate dosage regimens presented in Table 40-11 usually result in plasma magnesium levels illustrated in Figure 40-19. When magnesium sulfate is given to arrest eclamptic seizures, 10 to 15 percent of women will have a subsequent convulsion. If so, an additional 2-g dose of magnesium sulfate in a 20-percent solution is slowly administered intravenously. In a small woman, this additional 2-g dose may be used once, but it can be given twice if needed in a larger woman. In only 5 of 245 women with eclampsia at Parkland Hospital was it necessary to use alternative supplementary anticonvulsant medication to control convulsions (Pritchard, 1984). For these, an intravenous barbiturate is given slowly. Midazolam or lorazepam may also be given in a small single dose, but prolonged use is avoided because it is associated with a higher mortality rate from aspiration pneumonia (Royal College of Obstetricians and Gynaecologists, 2006).

1	TABLE 40-1 1. Magnesium Sulfate Dosage Schedule for Severe Preeclampsia and EclampSia Give 4-to 6-g loading dose of magnesium sulfate diluted in 100 mL of IV fluid administered over 15-20 min Begin 2 g/hr in 100 mL of IV maintenance infusion. Some recommend 1 g/hr Monitor for magnesium toxicity: Assess deep tendon reflexes periodically Some measure serum magnesium level at 4-6 hr and adjust infusion to maintain levels between 4 and 7 mEq/L (4.8 to 8.4 mg/dL) Measure serum magnesium levels if serum creatininen::1.0 mg/dL Magnesium sulfate is discontinued 24 hr ater delivery Give 4 g of magnesium sulfate (MgS04·7H20 USP) as a 20% solution intravenously at a rate not to exceed 1 g/min

1	Give 4 g of magnesium sulfate (MgS04·7H20 USP) as a 20% solution intravenously at a rate not to exceed 1 g/min Follow promptly with 109 of 50% magnesium sulfate solution, one half (5 g) injected deeply in the upper outer quadrant of each buttock through a 3-inch-long 20-gauge needle. (Addition of 1.0 mL of 2% lidocaine minimizes discomfort.) If convulsions persist ater 15 min, give up to 2 g more intravenously as a 20% solution at a rate not to exceed 1 g/min. If the woman is large, up to 4 g may be given slowly. Every 4 hr thereater, give 5 g of a 50% solution of magnesium sulfate injected deeply in the upper outer quadrant of alternate buttocks, but only after ensuring that: The patellar reflex is present, Respirations are not depressed, and Urine output the previous 4 hr exceeded 100 mL Magnesium sulfate is discontinued 24 hr after delivery :J01500 250 deficits, prolonged coma, or atypical eclampsia (Sibai,r2012) . 200 • Clinical Findings with Eclampsia

1	200 • Clinical Findings with Eclampsia Eclamptic seizures may be violent, and the woman must be protected, especially her air150 C way. So forceful are the muscular movements J,.. U :L,that the woman may throw herself out of her -0 bed, and if not protected, her tongue is bitten aby the violent action of the jaws (Fig. 40-16). his phase, in which the muscles alternately contract and relax, may last approximately a minute. Gradually, the muscular movements become smaller and less frequent, and finally the woman lies motionless. After a seizure, the woman is postictal, but in some, a coma of variable duration ensues. When the convulsions are infrequent, the

1	After a seizure, the woman is postictal, but in some, a coma of variable duration ensues. When the convulsions are infrequent, the Days after treatment woman usually recovers some degree of con sciousness ater each attack. As the woman arouses, a semiconscious combative state may nase (ASn levels in women with HELLP syndrome assigned to receive treatment with dexamethasone or placebo. (Data from Katz, 2008.) with saline aforded the same interval-to-delivery timing (Sibai,r2017).

1	Preeclampsia complicated by generalized tonic-clonic convulsions appreciably raises the risk to both mother and fetus. In an earlier report, Mattar and Sibai (2000) described outcomes in 399 consecutive women with eclampsia from 1977 through 1998. Major maternal complications included placental abruption-10 percent, neurological deicits-7 percent, aspiration pneumonia-7 percent, pulmonary edema-5 percent, cardiopulmonary arrest-4 percent, and acute renal failure4 percent. Moreover, 1 percent of these women died. Several subsequent reports similarly described excessive maternal morbidity and mortality rates with eclampsia that also included HELLP syndrome, pulmonary embolism, and stroke (Andersgaard, 2006; Knight, 2007). In he Netherlands, there were three maternal deaths among 222 eclamptic women (Zwart, 2008). Data from Ireland and Australia are similar (O'Connor, 2013; Thornton, 2013). In perspective, this is a thousandfold increase above the overall maternal death rates for these

1	women (Zwart, 2008). Data from Ireland and Australia are similar (O'Connor, 2013; Thornton, 2013). In perspective, this is a thousandfold increase above the overall maternal death rates for these countries.

1	Almost without exception-but at times unnoticed-preeclampsia precedes the convulsion onset. Eclampsia is most common in the last trimester and becomes increasingly frequent as term approaches. In more recent years, the incidence of postpartum eclampsia has declined. his is presumably related to improved access to prenatal care, earlier detection of antepartum preeclampsia, and prophylactic use of magnesium sulfate (Chames, 2002). Importantly, other diagnoses should be considered in women with convulsions more than 48 hours postpartum or in women with focal neurological ensue. In severe cases, coma persists from one convulsion to another, and death may result. In rare instances, a single convulsion may be followed by coma from which the woman may never emerge. As a rule, however, death does not occur until after frequent convulsions. Finally and also rarely, convulsions continue unabatedstatus epilepticus-and require deep sedation and even general anesthesia to obviate anoxic

1	death does not occur until after frequent convulsions. Finally and also rarely, convulsions continue unabatedstatus epilepticus-and require deep sedation and even general anesthesia to obviate anoxic encephalopathy.

1	he respiratory rate ater an eclamptic convulsion is usually increased and may reach 50 or more per minute in response to hypercarbia, lactic acidemia, and transient hypoxia. Cyanosis may be observed in severe cases. High fever is a grave sign as it likely emanates from cerebrovascular hemorrhage. Proteinuria is usually, but not always, present as discussed earlier (p. 720). Urine output may be diminished appreciably, FIGURE 40-16 Hematoma of tongue from laceration during an eclamptic convulsion. Thrombocytopenia may have contributed to the bleeding. :s, FIGURE 40-19 Serum magnesium concentration in normotensive and preeclamptic women following a 4-g loading dose of magnesium sulfate and 2 g/h infusion. (Data from Brookfield, 2016.)

1	:s, FIGURE 40-19 Serum magnesium concentration in normotensive and preeclamptic women following a 4-g loading dose of magnesium sulfate and 2 g/h infusion. (Data from Brookfield, 2016.) Maintenance magnesium sulfate therapy has traditionally been continued for 24 hours after delivery. For eclampsia that develops postpartum, magnesium sulfate is administered for 24 hours after the onset of convulsions. A few investigators have truncated this therapy duration to 12 hours and found no seizures (Anjum, 2016; Ehrenberg, 2006; Kashanian, 2016). And more recently, Ludmir and colleagues (2017) described salutary outcomes when magnesium sulfate therapy was stopped after delivery. That said, these studies are small, and the abbreviated magnesium regimen needs further study before being routinely implemented.

1	Using United States Pharmacopeia (USP) standards, magnesium sulfate USP is MgS04·7H20, and it contains 8.12 mEq magnesium per 1 g. Parenterally administered magnesium is cleared almost totally by renal excretion, and magnesium intoxication is unusual when the glomerular iltration rate is normal or only slightly reduced. Adequate urine output usualy correlates with preserved glomerular iltration rates. That said, magnesium excretion is not urine Row dependent, and urinary volume per unit time does not, per se, predict renal unction. Thus, serum creatinine leves must be mesured to etect a decreased glomeruar iltration rate.

1	Eclamptic convulsions are almost always prevented or arrested by plasma magnesium levels maintained at 4 to 7 mEq/L, 4.8 to 8.4 mg/dL, or 2.0 to 3.5 mmollL. But, one review of magnesium pharmacokinetics showed that most regimens result in much lower serum magnesium levels (Okusanya, 2016). This was especially true if only 1 g/hr was infused (Yefet, 2017). Importantly, the obesity epidemic has afected these observations (Cunningham, 2016). Tudela and colleagues (2013) described our observations from Parkland Hospital with magnesium administration to obese women. More than 60 percent of women whose body mass index (BMI) exceeded 30 kg/mrand who were receiving the 2 g/hr dose had sub therapeutic levels at 4 hours. hus, obese women would require 3 gl hr to maintain efective plasma levels. That said, most currently do not recommend routine magnesium level measurements (American College of Obstetricians and Gynecologists, 2013; Royal College of Obstetricians and Gynaecologists, 2006).

1	Patellar reRexes disappear when the plasma magnesium level reaches 10 mEq/L-about 12 mgl dL-presumably because of a curariform action. This sign serves to warn of impending magnesium toxicity. When plasma levels rise above 10 mEq/L, breathing becomes weakened. At 12 mEq/L or higher levels, respiratory paralysis and respiratory arrest follow (Somjen, 1966). Treatment with cacium gluconate or cacium chloride, 1 g intravenousy, along with discontinuation of forther manesium suate, usualy reverses mid-to-moderate respiratory depression. One of these agents should be readily available whenever magnesium is being infused. Unfortunately, the efects of intravenously administered calcium may be short-lived if there is a steady-state toxic level. For severe respiratory depression and arrest, prompt tracheal intubation and mechanical ventilation are lifesaving. Direct toxic efects on the myocardium from high levels of magnesium are uncommon (McCubbin, 1981; Morisaki, 2000).

1	Because magnesium is cleared almost exclusivey by renal excre . tion, the dosages described wil become excessive if glomerular il tration is substantialy decreased. The initial 4-g loading dose of magnesium sulfate can be safely administered regardless of renal function. It is important to administer the standard load ing dose and not to reduce it under the mistaken conception that diminished renal function requires it. This is because after distribution, a loading dose achieves the desired therapeutic level, and the infusion maintains the steady-state level. Thus, ony the maintenance infusion rate should be altered with dimin ished glomerular iltration rate. Renal function is estimated by measuring plasma creatinine. Whenever plasma creatinine lev els arer> 1.0 mg/mL, serum magnesium levels are determined to guide the infusion rate.

1	After a 4-g intravenous dose administered over 15 minutes, mean arterial pressure falls slightly, accompanied by a 13-percent rise in cardiac index (Cotton, 1986b). Thus, magnesium lowers systemic vascular resistance and mean arterial pressure. At the same time, cardiac output is increased. These indings are coincidental with transient nausea and Rushing, and the cardiovascular efects persist for only 15 minutes despite continued magnesium infusion. Thurnau and associates (1987) showed that magnesium therapy led to a small but signiicant rise in the total magnesium concentration in the cerebrospinal Ruid. The magnitude of the elevation was directly proportional to the corresponding serum concentration.

1	Magnesium has anticonvulsant and neuroprotective efects in several animal models. Some proposed mechanisms of action include: (1) reduced presynaptic release of the neurotransmitter glutamate, (2) blockade of glutamatergic N-methyl-D-aspartate (NMDA) receptors, (3) potentiation of adenosine action, (4) improved calcium bufering by mitochondria, and (5) blockage of calcium entry via voltage-gated channels (Arango, 2008; Wang, 2012). In the uterus, relatively high serum magnesium concentrations depress myometrial contractility both in vivo and in vitro. With the suggested regimen, myometrial depression has not been observed, except for a transient decline in activity during and immediately ater the initial intravenous loading dose (Leveno, TABLE 40-12. Randomized Comparative Trials of Magnesium Sulfate Versus Phenytoin and Diazepam to Prevent Recurrent Eclamptic Convulsions

1	TABLE 40-12. Randomized Comparative Trials of Magnesium Sulfate Versus Phenytoin and Diazepam to Prevent Recurrent Eclamptic Convulsions Recurrent seizuresa 60/453 (1n3%) 126/452 (28%) 22/388 (5.6%) 66/389 (1n7%) Matenal deathsb 10/388 (2.6%) 20/387 (5.2%) 17/453 (3.8%) 24/452 (5.3%) aA11 comparisons p < 0.01n. blndividual comparisons nonsignificant, combined comparison p < .05. Data from Eclampsia Trial Collaborative Group, 1995. 1998; Szal, 1999; Witlin, 1997). Blood loss at delivery is not increased by standard magnesium treatment (Graham, 2016). However, inhibition of uterine contractility is magnesium dose dependent, and serum levels of at least 8 to 10 mEq/L are necessary to inhibit uterine contractions (Watt-Morse, 1995).

1	Magnesium administered parenterally promptly crosses the placenta to achieve equilibrium in fetal serum and less so in amnionic fluid (Hallak, 1993). Levels in amnionic luid rise with the duration of maternal infusion (Gortzak-Uzan, 2005). Magnesium sulfate has small but signiicant efects on the fetal heart rate pattern-specifically beat-to-beat variability (Hallak, 1999). Duy and associates (2012) reported a lower heart rate baseline that was within the normal range; decreased variability; and fewer prolonged decelerations. They noted no adverse outcomes.

1	Overall, maternal magnesium therapy appears safe for perinates (Drassinower, 2015). One MFMU Network study of more than 1500 exposed preterm neonates found no association between the need for neonatal resuscitation and cord blood magnesium levels Qohnson, 2012). Still, a few neonatal adverse events are associated with its use. In a Parkland Hospital study of 6654 mostly term, exposed newborns, 6 percent had hypotonia (Abbassi-Ghanavati, 2012). In addition, exposed neonates had lower 1-and 5-minute Apgar scores, a higher intubation rate, and more admissions to the special care nursery. The study showed that neonatal depression occurs only if hypermagnesemia at delivery is severe.

1	Observational studies suggest a protective efect of magnesium against the development of cerebral palsy in very-Iowbirthweight newborns (Nelson, 1995; Schendel, 1996). At least ive randomized trials have also assessed neuroprotective efects in preterm neonates. These findings are discussed in detail in Chapter 42 (p. 824). Nguyen and colleagues (2013) expanded this possibility to include term newborn neuroprotection, but data were insuicient to draw conclusions. Last, in cases of preterm labor, magnesium has been given for several days for tocolysis (Chap. 42, p. 826). Administration in these instances has been associated with neonatal osteopenia (American College of Obstetricians and Gynecologists, 20 16c).

1	The multinational Eclampsia Trial Collaborative Group study (1995) involved 1687 women with eclampsia randomly allocated to one of three diferent anticonvulsant regimens: magnesium sulfate, diazepam, or phenytoin (Table 40-12). In aggregate, magnesium sulfate therapy was associated with a significantly lower incidence of recurrent seizures (9.7 percent) compared with women given phenytoin (28 percent) or diazepam (17 percent). Importantly, the aggregate maternal death rate of 3.2 percent with magnesium sulfate was signiicantly lower than that of 5.2 percent for the other two regimens.

1	In their review of more than 9500 treated women, Smith and coworkers (2013) reported the overall rate of absent patellar tendon reflexes to be 1.6 percent; respiratory depression, 1.3 percent; and calcium gluconate administration, 0.2 percent. Only one mother died due to magnesium toxicity. Our experiences are similar. In the more than 60 years of magnesium use at Parkland Hospital, only one woman has died from an overdose (P ri tchard, 1984). Dangerous hypertension can cause cerebrovascular hemorrhage and hypertensive encephalopathy, and it can trigger eclamptic convulsions in women with preeclampsia. Other complications include placental abruption and congestive heart failure induced by elevated hypertensive afterload.

1	Because of these serious sequelae, the working group for the National High Blood Pressure Education Program (NHBPEP) (2000) and the 2013 Task Force recommend treatment to lower systolic pressures to or below 160 mm Hg and diastolic pressures to or below llO mm Hg. Martin and associates (2005, 2016) reported provocative observations that highlight the importance of treating systolic hypertension. They described 28 selected women with severe preeclampsia who sufered an associated stroke. Most (93 percent) were hemorrhagic strokes, and all women had systolic pressures > 160 mm Hg before sufering their stroke. By contrast, only 20 percent of these same women had diastolic pressures > 110 mm Hg.

1	From other observations, it seems likely that at least half of serious hemorrhagic strokes associated with preeclampsia are in women with chronic hypertension (Cunningham, 2005). Long-standing hypertension results in development of CharcotBouchard aneuysms in the deep penetrating arteries of the lenticulostriate branch of the middle cerebral arteries. hese vessels supply the basal ganglia, putamen, thalamus, and adjacent deep white matter, as well as the pons and deep cerebellum. These unique aneurysmal weakenings predispose these small arteries to rupture during sudden hypertensive episodes.

1	Several drugs are available to rapidly lower dangerously elevated blood pressure in women with pregnancy-associated hypertension. he three most commonly employed are hydralazine, labetalol, and nifedipine. For years, parenteral hydralazine was the only one of these three available. But when parenteral labetalol was later introduced, it was proven to be equally efective for obstetrical use. Orally administered nifedipine has since also gained popularity. ll three of these are recommended as first-line agents by the American College of Obstetricians and Gynecologists (2017 a).

1	This is probably still the most commonly used antihypertensive agent in the United States for treatment of women with severe gestational hypertension. Hydralazine is administered intravenously with a 5-to 10-mg initial dose, and this is followed by 10-mg doses at 15-to 20-minute intervals until a satisfactory response is achieved. Although we will administer a third dose, the American College of Obstetricians and Gynecologists (2017 a) recommends labetalol therapy if severe hypertension persists after the second dose. Antepartum or intrapartum, the target response is a decline in systolic pressure to < 160 mm Hg and diastolic blood pressure to 90 to 110 mm Hg. Lower diastolic pressures risk compromised placental perfusion. Hydralazine has proven remarkably efective to prevent cerebral hemorrhage. Its onset of action can be as rapid as 10 minutes. Although repeated administration every 15 to 20 minutes may theoretically lead to undesirable hypotension, this has not been our experience

1	Its onset of action can be as rapid as 10 minutes. Although repeated administration every 15 to 20 minutes may theoretically lead to undesirable hypotension, this has not been our experience when given in these 5-to 10-mg increments.

1	At Parkland Hospital, between 5 and 10 percent of all women with intrapartum hypertensive disorders are given a parenteral antihypertensive agent. Antepartum, we usually give hydralaxine as described. We do not limit the total dose, and seldom is a second antihypertensive agent needed. We estimate that by severe superimposed preeclampsia, and hydralazine was injected more frequently than recommended. Her blood pressure in less than 1 hour dropped from 240-270/130-150 mm Hg to 1r10/80 mm Hg. Fetal heart rate decelerations characteristic of utero placental insuiciency became evident. Decelerations persisted until her blood pressure was increased with rapid crystalloid infusion. In some cases, this fetal response to diminished uterine perfusion may be confused with placental abruption and may result in unnecessary and potentially dangerous emergency cesarean delivery.

1	This efective intravenous antihypertensive agent is an Q-and nonselective 3-blocker. Some prefer its use over hydralazine because of fewer side efects. At Parkland Hospital, we give 10 mg intravenously initially. If the blood pressure has not decreased to the desirable level in 10 minutes, then 20 mg is given. The next 10-minute incremental dose is 40 mg and is followed by another 40 mg if needed. If a salutary response is not achieved, then an 80-mg dose is given. Sibai (2003) recommends 20 to 40 mg every 10 to 15 minutes as needed and a maximum dose of 220 mg per treatment cycle. The American College of Obstetricians and Gynecologists (2017a) recommends starting with a 20-mg intravenous bolus. If not efective within 10 minutes, this is followed by 40 mg, then 80 mg every 10 minutes. If hypertension persists, hydralazine is then given.

1	Comparative studies of hydralazine versus labetalol show equivalent results (Umans, 2015). In one trial, labetalol lowered blood pressure more rapidly, and associated tachycardia was minimal. But, hydralazine lowered mean arterial pressures to safe levels more efectively (Mabie, 1987). In another trial, maternal and neonatal outcomes were similar (Vigil-De Gracia, 2007). Hydralazine causes significantly more maternal tachycardia and palpitations, whereas labetalol more frequently leads to maternal hypotension and bradycardia. Both drugs have been associated with a reduced frequency of fetal heart rate accelerations (Cahill, 2013). Labetalol is not given to asthmatic women.

1	FIGURE 40-20 Hydralazine was given at 5-minute intervals instead of 15-minute intervals. The there is a tendency to give a larger initial dose of hydralazine. But, this must be avoided. The response 100 not be predicted by hypertension severity. Thus, our protocol is to o 15 30 45 15 always administer 5 mg as the ini-Time (hours) tial dose. n adverse response to exceeding this initial dose is shown mean arterial pressure dropped from 180 to 90 mm Hg within 1 hour and was associated with in Figure 40-20. This woman had fetal bradycardia. Rapid crystalloid infusion raised the mean pressure to 115 mm Hg, and the fetus chronic hypertension complicated recovered.

1	his orally administered calcium-channel blocking agent has become popular because of its eicacy to control acute pregnancy-related hypertension. he American College of Obstetricians and Gynecologists (2017a), the NHBPEP Working Group (2000), and the Royal College of Obstetricians and Gynaecologists (2006) recommend a 10-mg initial immediate-release oral dose to be followed in 20 to 30 minutes with 10 to 20 mg if necessary. If not satisfactory, this is followed by labetalol. Nedipine given sublingualy is no longer recommended. his route is associated with dangerously rapid and extensive efects. Randomized trials that compared nifedipine with labetalol found neither drug definitively superior, but nifedipine lowered blood pressure more quickly (Scardo, 1999; Shekhar, 2016; Vermillion, 1999). Finally, nifedipine does not potentiate magnesium-related efects (Magee, 2015).

1	A few other generally available antihypertensive agents have been tested in clinical trials but are not widely used (Umans, 2015). These include verapamil, nitroglycerin, nitroprusside, ketanserin, nicardipine, and nimodipine (Belfort, 1990, 2003; Bolte, 2001; Cornette, 2016). There are also experimental antihypertensive drugs that may become useful for preeclampsia treatment (Lam, 2013). Potent loop diuretics can further compromise placental perfusion. Immediate efects include redistribution of the intravascular volume, which most oten is already reduced in severe preeclampsia (p. 718). herefore, before delivery, diuretics are not used to lower blood pressure (Zeeman, 2009; Zondervan, 1988). We use antepartum furosemide or similar drugs solely to treat pulmonary edema.

1	Lactated Ringer solution is administered routinely at a rate between 60 and 125 mL per hour, unless luid loss is unusual from vomiting, diarrhea, or diaphoresis, or, more likely, excessive blood loss with delivery. Oliguria is common with severe preeclampsia. hus, coupled with the knowledge that maternal blood volume is likely constricted compared with that of normal pregnancy, it is tempting to administer intravenous fluids more vigorously. But controlled, conservative luid administration is preferred for the typical woman with severe preeclampsia who already has excessive extracellular luid that is inappropriately distributed between intravascular and extravascular spaces. Infusion of large luid volumes enhances the maldistribution and thereby appreciably elevates the risk of pulmonary and cerebral edema (Dennis, 2012a; Sciscione, 2003; Zinaman, 1985). hus, for preeclamptic women with anuria, small incremental boluses can be given to maintain urine output above 30 mL per hour.

1	and cerebral edema (Dennis, 2012a; Sciscione, 2003; Zinaman, 1985). hus, for preeclamptic women with anuria, small incremental boluses can be given to maintain urine output above 30 mL per hour. Diminished intravascular volume from hemorrhage or luid loss from vomiting or fever can similarly be replaced by gradual incremental boluses. For labor analgesia with neuraxial analgesia, crystalloid solutions are infused slowly in graded amounts (Chap. 25, p. 496).

1	Women with severe preeclampsia who develop pulmonary edema most often do so postpartum (Cunningham, 1986, 2012; Zinaman, 1985). With suspected pulmonary edema in the eclamptic woman, aspiration of gastric contents, which may be the result of convulsions, anesthesia, or oversedation, should be excluded. here are three common causes of pulmonary edema in women with severe preeclampsia syndromepulmonary capillary permeability edema, cardiogenic edema, or a combination of the two.

1	Some women with severe preeclampsia-especially if given vigorous fluid replacement-will have mild pulmonary congestion secondary to permeability edema. his is caused by normal pregnancy changes magnified by the preeclamp�ia syndrome. Importantly, plasma oncotic pressure drops appreciably in normal term pregnancy because of decreased serum albumin concentration, and oncotic pressure falls even more with preeclampsia (Zinaman, 1985). Moreover, both increased extravascular luid oncotic pressure and increased capillary permeability are found in women with preeclampsia (Brown, 1989; 0ian, 1986).

1	Knowledge concerning cardiovascular and hemodynamic pathophysiological alterations associated with severe preeclampsia-eclampsia has accrued from studies done using invasive monitoring and a low-directed pulmonary artery catheter (see Fig. 40-5). Two conditions frequently cited as indications are preeclampsia associated with oliguria and that associated with pulmonary edema (Clark, 2010). Somewhat ironically, it is usually vigorous treatment of the former that results in most cases of the latter. The Task Force (2013) recommends against routine invasive monitoring. Such monitoring is best reserved for severely preeclamptic women with accompanying cardiac disease, renal disease, or both or with refractory hypertension, oliguria, and pulmonary edema.

1	Because the preeclampsia syndrome is associated with hemoconcentration, some have inused various fluids, starch polymers, albumin concentrates, or combinations thereof to expand blood volume (Ganzevoort, 2004). Older observational studies describe serious complications-especially pulmonary edema-with volume expansion (Benedetti, 1985; LOpez-Llera, 1982; Sibai, 1987b).

1	he Amsterdam randomized study reported by Ganzevoort and coworkers (2005a,b) was a well-designed investigation done to evaluate volume expansion. A total of216 women with severe preeclampsia were enrolled between 24 and 34 weeks' gestation. he study included women whose preeclampsia was complicated by HELLP syndrome, eclampsia, or fetal-growth restriction. In the group randomly assigned to volume expansion, each woman was given 250 mL of 6-percent hydroxyethyl starch infused over 4 hours twice daily. heir outcomes were compared with a control group, and none of these outcomes were significantly diferent (Table 40-13). Importantly, serious maternal morbidity and a substantive perinatal mortality rate accompanied their "expectant" management (see Table 40-9). Pulmonary edema 2.9 Placental abruption 3.8 l.0 Prolongation of pregnancy (mean) 11n.6 d 6.7 d EGA at death (mean) 26.7 wk 26.3 wk Prolongation of pregnancy (mean) 10.5 d 7.4 d EGA at delivery (mean) 31.6 wk 31.4 wk

1	Prolongation of pregnancy (mean) 11n.6 d 6.7 d EGA at death (mean) 26.7 wk 26.3 wk Prolongation of pregnancy (mean) 10.5 d 7.4 d EGA at delivery (mean) 31.6 wk 31.4 wk Neonatal death (%) 7.6 8.1 0.05. EGA = estimated gestational age; HELLP = hemolysis, elevated liver enzyme levels, low platelet count; RDS = respiratory distress syndrome. Data from Ganzevoort, 2005a, b. • Neuroprophylaxis-Prevention of Seizures aL studies, magnesium suate was reported to be superior to the Several randomized trials have tested the eicacy of seizure comparator agent to prevent eclampsia. Four of the larger studies prophylxis for women with gestational hypertension, with or are summarized in Table 40-14. In the study from Parland without proteinuria. In most of these, magnesium sulfate was Hospital, magnesium sulfate therapy was superior to phenycompared with another anticonvulsant or with a placebo. In toin to prevent eclamptic seizures in women with gestational

1	TABLE 40-14. Randomized Comparative Trials of Prophylaxis with Magnesium Sulfate and Placebo or Another Anticonvulsant in Women with Gestational Hypertension No. with Seizures/Total No. Treated (%) Gestational hypertensionb 0/1n049 (0) 10/1n089 (0.9) P < 0.001 Severe preeclampsia 1/345 (0.3) 1n1/340 (3.2) RR = 0.09 (0.1-0.69) Severe preeclampsia 40/5055 (0.8) 96/5055 (1n.9) RR = 0.42 (0.26-0.60) Severe preeclampsia 7/831 (0.8) 21/819 (2.6) RR = 0.33 (0.14-0.77) aAIl comparisons significant p < 0.05. blncluded women with and without proteinuria and those with all severities of preeclampsia. (Magpie Trial Collaboration Group, 2002. RR = relative risk.

1	blncluded women with and without proteinuria and those with all severities of preeclampsia. (Magpie Trial Collaboration Group, 2002. RR = relative risk. hypertension or preeclampsia (Lucas, 1995). In another, magnesium sulfate and nimodipine-a calcium-channel blocker with specific cerebral vasodilator activity-were compared in 1650 women with severe preeclampsia (Belfort, 2003). The rate of eclampsia was more than threefold higher for women allocated to the nimodipine group-2.6 versus 0.8 percent.

1	he largest comparative study was Manesium Suate or Prevention of Eclampsia reported by the Magpie Trial Collaboration Group (2002). More than 10,000 women with severe preeclampsia from 33 countries were randomly allocated to treatment with magnesium sulfate or placebo. Women given magnesium had a 58-percent significantly lower risk of eclampsia than those given placebo. In follow-up data of infants born to these mothers given magnesium sulfate, child behavior at approximately 18 months did not difer in those exposed compared with those not exposed to magnesium sulfate (Smyth, 2009). Who Should Be Given Magnesium Sulfate?

1	Who Should Be Given Magnesium Sulfate? Magnesium will prevent proportionately more seizures in women with correspondingly worse disease. However, severity is diicult to quantiy, and thus deciding which individual woman might benefit most from neuroprophylaxis is diicult. The 2013 Task Force recommends that women with either eclampsia or severe preeclampsia should be given magnesium sul ate prophylaxis. Again, criteria that establish "severity" are not totally uniform (see Table 40-2). At the same time, however, the 2013 Task Force suggests that women with "mild" preeclampsia do not need magnesium sulfate neuroprophylaxis. The conundrum is whether or not to give neuroprophylaxis to any of these women with "nonsevere" gestational hypertension or preeclampsia (Alexander, 2006).

1	In most other countries, and principally following dissemination of the Magpie Trial Collaboration Group (2002) study results, magnesium sulfate is now recommended for women with severe preeclampsia. In some, however, debate continues concerning whether therapy should be reserved for women who have an eclamptic seizure. We believe that eclamptic seizures are dangerous (pp. 722 and 745). Maternal mortality rates of up to 5 percent have been reported even in recent studies. Moreover, perinatal mortality rates are substantially increased (Abd EI Aal, 2012; Knight, 2007; Ndaboine, 2012; Schutte, 2008; von Dadelszen, 2012). Finally, the possibility of adverse longterm neuropsychological and vision-related sequelae of eclampsia have raised additional concerns that eclamptic seizures are not "benign."

1	Because of the foregoing, there is uncertainty about which women with nonsevere gestational hypertension should be given magnesium sulfate neuroprophylaxis. An opportunity to address these questions was aforded by a change in our prophylaxis protocol at Parkland Hospital. Before this time, the risk of eclampsia without magnesium prophylaxis was approximately 1 in 100 for women with mild preeclampsia (Lucas, 1995). Up until 2000, all women with gestational hypertension were given magnesium prophylaxis intramuscularly. Mter 2000, we instituted a standardized protocol for intravenously administered magnesium sulfate (Alexander, 2006). At the same time, we TABLE 40-15. Selective versus Universal Magnesium Sulfate Prophylaxis: Parkland Hospital Criteria to Define Severe Gestational In a woman with new-onset proteinuric hypertension, at least one of the following criteria is required: Systolic BP : 160 r diastolic BP : 110 mm Hg Proteinuria :2+ by dipstick in a catheterized urine

1	Systolic BP : 160 r diastolic BP : 110 mm Hg Proteinuria :2+ by dipstick in a catheterized urine Serum creatininen> 1n.1 mg/dL Platelet count < 1OO,OOO/1L above upper limit of normal range BP = blood pressure. Criteria based on those from [Jational High Blood Pressure Education Program Working Group, 2000; American (ollege of Obstetricians and Gynecologists, 2012; cited by Alexander, 2006. also changed our practice of universal seizure prophylaxis for all women with gestational hypertension to one of selective prophylaxis given only to women who met our criteria for severe gestational hypertension. These criteria, shown in Table 40-15, included women with :2+ proteinuria measured by dipstick in a catheterized urine specimen.

1	Following this protocol change, 60 percent of 6518 women with gestational hypertension during a 4Y2-year period were given magnesium sulfate neuroprophylaxis. The remaining 40 percent with nonsevere hypertension were not treated, and of these, 27 women developed eclamptic seizures-1 in 92. The seizure rate was only 1 in 358 for 3935 women with criteria for severe disease who were given magnesium sulfate, and thus these cases were treatment failures.

1	To assess morbidity, outcomes in 87 eclamptic women were compared with outcomes in all 6431 noneclamptic severely hypertensive women (Alexander, 2006). lthough most maternal outcomes were similar, almost a fourth of women with eclampsia who underwent emergent cesarean delivery required general anesthesia. his is a great concern because eclamptic women have laryngotracheal edema and are at a higher risk for failed intubation, gastric acid aspiration, and death. Neonatal outcomes were also a concern because the composite morbidity was increased tenfold in eclamptic compared with noneclamptic women-12 versus 1 percent, respectively. These outcomes included cord artery pH <7.0; 5-minute Apgar score <4; or unanticipated admission of a term newborn to an intensive care nursery.

1	Thus, if one uses the Parkland criteria for nonsevere gestational hypertension, approximately 1 of 100 such women who are not given magnesium sulfate prophylaxis can be expected to have an eclamptic seizure. A fourth of these women likely will require emergent cesarean delivery with attendant maternal and perinatal morbidity and mortality from general anesthesia. From this, the major question regarding management of nonsevere gestational hypertension remains whether it is acceptable to avoid unnecessary treatment of 99 women to risk eclampsia in one? he answer appears to be yes as suggested by the 20 13 Task Force. At Parkland Hospital, we only give magnesium neuroprophylxis to women with severe criteria.

1	During the past 20 years, the use of conduction analgesia for women with preeclampsia syndrome has proven ideal. Initial problems with this method included hypotension and diminished uterine perfusion caused by sympathetic blockade in preeclamptic women, with already attenuated hypervolemia. But pulmonary edema was mitigated by techniques that used slow induction of epidural analgesia with dilute solutions of anesthetic agents. his countered the need for rapid infusion of large volumes of crystalloid or colloid to correct maternal hypotension from neural blockade (Hogg, 1999; Wallace, 1995). These techniques are described in detail in Chapter 25 (p. 496). Importantly, epidural blockade avoids general anesthesia, in which the stimulation of tracheal intubation may cause sudden severe hypertension. Such blood pressure spikes, in turn, can cause pulmonary edema, cerebral edema, or intracranial hemorrhage. Finally, tracheal intubation may be particularly diicult and thus hazardous in

1	Such blood pressure spikes, in turn, can cause pulmonary edema, cerebral edema, or intracranial hemorrhage. Finally, tracheal intubation may be particularly diicult and thus hazardous in women with airway edema due to preeclampsia (American College of Obstetricians and Gynecologists, 20 17b).

1	At least three randomized studies have been performed to compare these methods of analgesia and anesthesia. Wallace and colleagues (1995) studied 80 women at Parkland Hospital with severe preeclampsia who were to undergo cesarean delivery. They had not been given labor epidural analgesia and were randomly assigned to receive general anesthesia, epidural analgesia, or combined spinal-epidural analgesia. heir average preoperative blood pressures approximated 170/110 mm Hg, and all had proteinuria. Maternal and perinatal outcomes in each group were similar. Maternal hypotension resulting from regional analgesia was managed with judicious intravenous fluid administration. In women undergoing general anesthesia, maternal blood pressure was managed to avoid severe hypertension. here were no serious maternal or fetal complications attributable to any of the three anesthetic methods. It was concluded that all three are acceptable for use in women with pregnancies complicated by severe

1	no serious maternal or fetal complications attributable to any of the three anesthetic methods. It was concluded that all three are acceptable for use in women with pregnancies complicated by severe preeclampsia if steps are taken to ensure a careful approach to the selected method.

1	Another randomized study included 70 women with severe preeclampsia receiving spinal analgesia versus general anesthesia for cesarean delivery (Dyer, 2003). Their maternal and fetal outcomes were equivalent. In a third study, 116 women with severe preeclampsia received either epidural or patient-controlled intravenous meperidine analgesia during labor (Head, 2002). More women-9 percent-from the group assigned to epidural analgesia required ephedrine for hypotension. As expected, pain relief was superior in the epidural group. Maternal and neonatal complications were similar between groups, and one woman in each group developed pulmonary edema. Importantly, epidural analgesia is not considered treatment of preeclampsia (Lucas, 2001; Ray, 2017).

1	Judicious fluid administration is essential in severely preeclamptic women who receive regional analgesia. Vigorous crystalloid infusion with epidural blockade in women with severe preeclampsia elevates pulmonary capillary wedge pressures (Newsome, 1986). Aggressive volume replacement in preeclamptic women raises their risk for pulmonary edema, especially in the first 72 hours postpartum (Clark, 1985; Cotton, 1986a). Finally, most cases of pharyngolaryngeal edema are related to aggressive volume therapy (Heller, 1983). Hemoconcentration or lack of norml pregnancy-induced hyper volemia is an almost predictable feature of severe preeclampsia eclampsia (see Fig. 40-7) (Zeeman, 2009). These women, who consequenty ack nomal pregnany hypervoemia, are much less toer ant of even normal blood oss than are nomotensive pregnant women.

1	Importantly, an appreciable fall in blood pressure soon ater delivery most oten means excessive blood loss and not sudden resolution of vasospasm and endothelial damage. When oliguria follows delivery, the hematocrit should be evaluated frequently to help detect excessive blood loss. If identiied, hemorrhage should be treated appropriately by crystalloid and blood transusion.

1	Although severe postpartum hypertension usually follows labor and delivery complicated by hypertension, 8 percent of women develop de novo hypertension postpartum (Goel, 2015). In either case, if diiculty arises in controlling severe hypertension or if intravenous hydralazine or labetalol are being used repeatedly, then oral regimens can be given. Examples include labetalol or another 3-blocker, or nifedipine or another calcium-channel blocker (Sharma, 2017). Women so treated are less likely to require readmission (Hirshberg, 2016). Persistent or refractory hypertension is likely aggravated by mobilization of pathological interstitial fluid and redistribution into the intravenous compartment, underlying chronic hypertension, or usually both (Sibai, 2012; Tan, 2002). Chronic, but not sporadic, administration of some nonsteroidal antiinflammatory drugs, namely ibuprofen, may aggravate postpartum hypertension in those with preeclampsia (Vigil-De Gracia, 2017; Viteri, 2017). In women with

1	administration of some nonsteroidal antiinflammatory drugs, namely ibuprofen, may aggravate postpartum hypertension in those with preeclampsia (Vigil-De Gracia, 2017; Viteri, 2017). In women with chronic hypertension and let-ventricular hypertrophy, severe postpartum hypertension can cause pulmonary edema from cardiac failure (Cunningham, 1986,2012; Sibai, 1987a).

1	Because persistence of severe hypertension corresponds to the onset and length of diuresis and extracellular fluid mobilization, it seems logical that furosemide-augmented diuresis might serve to hasten blood pressure control. One randomized trial included 264 postpartum preeclamptic women who, ater onset of spontaneous diuresis, were assigned to 20-mg oral furosemide given daily or to no therapy (Ascarelli, 2005). Women with mild disease had similar blood pressure control regardless of whether they received treatment or placebo. However, ater 2 days, women with severe preeclampsia who were treated, compared with those receiving placebo, had a lower mean systolic blood pressure-142 versus 153 mm Hg. They also less frequently required supplemental antihypertensive therapy during the remainder of hospitalization-14 versus 26 percent, respectively. In a recent randomized study, Veena and colleagues (2017) treated severe postpartum eclampsia with nifedipine plus furosemide or nifedipine

1	of hospitalization-14 versus 26 percent, respectively. In a recent randomized study, Veena and colleagues (2017) treated severe postpartum eclampsia with nifedipine plus furosemide or nifedipine alone. They reported that this prophylactic therapy signiicantly lowered the need for an additional antihypertensive-26 versus 8 percent, respectively.

1	We use a simple method to estimate excessive extracellular! interstitial luid. he posparum weight is compared with the most recentprenatal weight, either from the last clinic visit or on admission for delivery. Typically, soon ater delivery, maternal weight should be reduced by at least 10 to 15 pounds depending on newborn and placental weight, amnionic luid volume, and blood loss. Because of various interventions, especially intravenous crystalloid inusions given with labor epidural analgesia or during operative vaginal or cesarean delivery, women with severe preeclampsia oten have an immediate postpartum weight in excess of their st prenatal weight. If this weight increase is associated with severe persistent postpartum hypertension, then diuresis with intravenous urosemide is usually helpul in controlling blood pressure.

1	Occasionally, women have an atypical syndrome in which severe preeclampsia-eclampsia persists despite delivery. Martin and colleagues (1995) described 18 such women whom they encountered during a 10-year period. hey advocate single or multiple plasma exchange for these women. In some cases, 3 L of plasma was exchanged three times-a 36-to 45-donor unit exposure for each patient-before a response was forthcoming. Others have described plasma exchange performed in postpartum women with HELLP syndrome (Forster, 2002; Obeidat, 2002). In all of these cases, however, the distinction between HELLP syndrome and thrombotic thrombocytopenic purpura or hemolytic uremic syndrome was not clear (Tsai, 2016).

1	In our experiences with more than 50,000 women with gestational hypertension among nearly 450,000 pregnancies cared for at Parkland Hospital through 2017, we have encountered very few women with persistent postpartum hypertension, thrombocytopenia, and renal dysfunction who were diagnosed as having a thrombotic micro angiopathy (Dashe, 1998). hese latter syndromes complicating pregnancy were reviewed by Martin (2008) and George (2013) and their colleagues, who conclude that a rapid diagnostic test for ADAMTS-13 enzyme activity might be helpful to diferentiate most of these syndromes.

1	his is another cause of persistent hypertension, "thunderclap" headaches, seizures, and central nervous system indings. It is a form of postpartum angiopathy. Reversible cerebral vasoconstriction syndrome is characterized by difuse segmental constriction of cerebral arteries and may be associated with ischemic and hemorrhagic strokes. his syndrome has several inciting causes that include pregnancy, and particularly preeclampsia (Ducros, 2012). It is more common in women, and in some cases, vasoconstriction may be so severe as to cause cerebral ischemia and infarction. he appropriate management is not known at this time (Edlow, 2013).

1	Defective remodeling of the spiral arteries in some placentas is posited as a cause of at least one preeclampsia phenotype. Specifically, lack of deep placentation is linked with preeclampsia, placental abruption, fetal-growth restriction, and preterm birth (Wikstrom, 2011). With this type of "overlap syndrome," hypertensive disorders may serve as markers for subsequent preterm labor and fetal-growth restriction. For example, even in subsequent nonhypertensive pregnancies, women who had preterm preeclampsia are at higher risk for preterm birth and growth-restricted neonates (Bramham, 2011; Connealy, 2014; Palatnik, 2016).

1	In addition, women who have had either gestational hypertension or preeclampsia risk developing hypertension in future pregnancies (Lykke, 2009b). Generally, the earlier preeclampsia is diagnosed during the index pregnancy, the greater the likelihood of recurrence. And, the recurrence risk for preeclampsia is elevated further in women with the metabolic syndrome (Stekkinger, 2015). Sibai and colleagues (1986, 1991) found that nulliparas diagnosed with preeclampsia before 30 weeks had a recurrence risk as high as 40 percent during a subsequent pregnancy. In a prospective study of 500 women previously delivered for preeclampsia at 37 weeks, the recurrence rate in a subsequent gestation was 23 percent (Bramham, 2011).

1	As perhaps expected, women with HELLP syndrome have a substantive risk for recurrence in subsequent pregnancies. In two studies, the risk ranged from 5 to 26 percent, but the true recurrence risk likely lies beween these two extremes (Habli, 2009; Sibai, 1995). Even if HELLP syndrome does not recur with subsequent pregnancies, again incidences of preterm delivery, fetal-growth restriction, placental abruption, and cesarean delivery are increased (Habli, 2009; Hnat, 2002).

1	Evidence has accrued that the preeclampsia syndrome is a marker for subsequent long-term cardiovascular and related morbidity and mortality (Table 40-16). hus, women with hypertension identified during pregnancy should be evaluated during the first several months postpartum. he working group of the NHBPEP (2000) concluded that hypertension attributable to pregnancy should resolve within 12 weeks of delivery. Hypertension persisting beyond this time is considered chronic (Chap. 50, p. 976). he Magpie Trial Follow-Up Collaborative Group (2007) reported that 20 percent of 3375 preeclamptic women seen at a median of 26 months postpartum had hypertension. Importantly, even if hypertension does not persist in the short term, convincing evidence suggests a higher risk for long-term cardiovascular morbidity.

1	Any hypertension during pregnancy is a risk marker for morbidity and mortality in later life (American College of Obstetricians and Gynecologists, 2013; Bellamy, 2007). In a case-control study from Iceland, Arnadottir and associates (2005) reported TABLE 40-16. Some Long-Term Consequences in Women with Preeclampsia Syndrome the prevalences of ischemic heart disease-24 versus 15 percent, and stroke-9.5 versus 6.5 percent, were significantly increased in women who had gestational hypertension compared with normotensive controls. In a Swedish population study of more than 400,000 women, those with recurrent preeclampsia have systolic dysfunction and a greater incidence of ischemic heart disease (Valensise, 2016). Diastolic dysfunction is also more common (Bokslag, 2017). Preeclampsia is also a risk for coronary artery calciication and idiopathic cardiomyopathy (Behrens, 2016; White, 2016).

1	Lyke and associates (2009a) cited indings from a Danish registry of more than 780,000 nulliparas. After a mean followup of almost 15 years, the incidence of chronic hypertension was fivefold higher in those who had gestational hypertension, 3.5-fold greater after mild preeclampsia, and sixfold higher after severe preeclampsia. After two hypertensive pregnancies, this incidence rose sixfold. Moreover, these women with pregnancy-associated hypertension are at increased risk for ype 2 diabetes (Rice, 2016). And, preeclampsia is a risk factor for later diabetic retinopathy and retinal detachment (Auger, 2017; Beharier,r2016). As emphasized by several investigators, other cofactors or comorbidities are related to acquisition of these long-term adverse outcomes (Gastrich, 2012; Harskamp, 2007; Hermes, 2012; Spaan, 2012b). These include the metabolic syndrome, diabetes, obesity, dyslipidemia, and atherosclerosis (Kajantie, 2017; Orabona, 2016; Stekkinger, 2015).

1	Individuals who are born pre term have greater ventricular mass later in life (Lewandowski, 20l3). And, women who have preeclampsia and who develop chronic hypertension later in life have an increased ventricular mass index before they become hypertensive (Ghossein-Doha, 2013). Finally, in at least some of these women, hypertensive cardiovascular pathologies appear to have begun near the time of their own births. A similar phenomenon is associated with preterm birth and with fetal-growth disorders.

1	Preeclampsia is also a marker for subsequent renal disease. lmost 15 percent of previously preeclamptic women have renal dysfunction (Lopes van Balen, 2017). In a 40-year study of Norwegian birth and end-stage renal disease linked registries, although the absolute risk of renal failure was small, preeclampsia was associated with a fourfold greater risk (Vikse, 2008). Women with recurrent preeclampsia had an even higher risk. These data need to be considered in light of the indings that 15 to 20 percent of women with preeclampsia who undergo renal biopsy have evidence of chronic renal disease (Chesley, 1978). In another long-term study, Spaan and coworkers (2009) compared formerly preeclamptic women with a cohort of women who were normotensive at delivery. At 20 years following delivery, preeclamptic women were signiicantly more likely to be chronically hypertensive-55 versus 7 percent-compared with control women. They also had higher peripheral vascular and renovascular resistance and

1	women were signiicantly more likely to be chronically hypertensive-55 versus 7 percent-compared with control women. They also had higher peripheral vascular and renovascular resistance and decreased renal blood low. These data do not permit conclusions as to cause versus efect.

1	Until recently, eclamptic seizures were believed to have no signiicant long-term sequelae. However, this may not be the case (heilen, 2016). Recall that almost all eclamptic women have multifocal areas of perivascular edema, and approximately a fourth also have areas of cerebral infarction (Zeeman, 2004a).

1	In several long-term follow-up studies in women with severe preeclampsia and eclampsia, brain white-matter lesions that followed eclamptic convulsions persist (Aukes, 2007, 2009, 2012). Speciically, when studied with MR imaging at a mean of 7 years, 40 percent of formerly eclamptic women had more numerous and larger aggregate white matter lesions compared with 17 percent of normotensive control women. These investigators later also observed these white-matter lesions in preeclamptic women without convulsions (Aukes, 2012). And, Siepmann and associates (2017) documented temporal lobe white matter changes and reduced cortical volume in previously preeclamptic women. In studies designed to assess clinical relevance, formerly eclamptic women had subjectively impaired cognitive functioning (Postma, 2014). Wiegman and associates (2012) reported that formerly eclamptic women at approximately 10 years had lower vision-related quality of life compared with control subjects. This likely

1	(Postma, 2014). Wiegman and associates (2012) reported that formerly eclamptic women at approximately 10 years had lower vision-related quality of life compared with control subjects. This likely coincides with an elevated risk for retinopathy described by Auger and colleagues (2017). Because no baseline studies were done before these women sufered from preeclampsia or eclampsia, the investigators appropriately concluded that a cause versus an efect of these white-matter lesions remains unknown.

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1	Zeeman GG, Alexander ]M, McIntire DO, et al: Homocysteine plasma concentration levels for the prediction of preeclampsia in women with chronic hypertension. Am] Obstet Gynecol 189:574,n2003 Zeeman GG, Cunningham FG, Pritchard ]A: he magnitude of hemoconcentration with eclampsia. Hypertens Pregnancy 28(2): 127, 2009 Zeeman GG, Fleckenstein ]L, Twickler OM, et al: Cerebral infarction in eclampsia. Am] Obstet Gynecol 190:714, 2004a Zeeman GG, Hatab M, Twickler OM: Increased cerebral blood flow in preeclampsia with magnetic resonance imaging. Am ] Obstet Gynecol 191(4):1425,n2004b Zeisler H, L1urba E, Chantraine F, et al: Predictive value of the sFlt-1:PIGF ration in women with suspected preeclampsia. N Engl] Med 374:l3, 2016a Zeisler H, L1urba E, Chantraine F, et al: Soluble fms-like tyrosine kinase-1-toplacental growth factor ratio and time to delivery in women with suspected preeclampsia. Obstet Gynecol 128(2):261, 2016b

1	Zhou 5], Yelland L, McPhee A], et al: Fish-oil supplementation in pregnancy does not reduce the risk of gestational diabetes or preeclampsia. Am ] Clin Nutr 95(6):l378, 2012 Zhou Y, Damsky CH, Fisher 5]: Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype. ] C1in Invest 99(9):2152, 1997 Zinaman M, Rubin ], Lindheimer MD: Serial plasma oncotic pressure levels and echoencephalography during and after delivery in severe preeclampsia. Lancet 1:1245,n1985 Zondervan HA, Oosting ], Smorenberg-Schoorl ME, et al: Maternal whole blood viscosity in pregnancy hypertension. Gynecol Obstet Invest 25:83, 1988 Zwart J], Richters A, Ory F, et al: Eclampsia in he Netherlands. Obstet Gynecol 112:820, 2008 GENERAL CONSIDERATIONS ......................755 UTERINE ATONY.................................758 UTERINE INVERSION.............................761

1	GENERAL CONSIDERATIONS ......................755 UTERINE ATONY.................................758 UTERINE INVERSION.............................761 INJURIES TO THE BIRTH CANAL....................763 PUERPERAL HEMATOMAS........................764 UTERINE RUPTURE.............................. 765 PLACENTAL ABRUPTION..........................767 PLACENTA PREVIA. . . .. . . . . . . . . . . . . . . . . . . . . . .... 773 MORBIDLY ADHERENT PLACENTA..................777 OBSTETRICAL COAGULOPATHIES..................782 MANAGEMENT OF HEMORRHAGE..................787 ADJUNCTIVE SURGICAL PROCEDURES.............. 792 A profuse hemorrhage occurring prior to or shorty ater the birth of the child is always dangerous and not inrequenty a atal complication. -J. Whitridge Williams (1903)

1	As in Williams' time, obstetrical hemorrhage continues along with hypertension and infection to be one part of the infamous "triad" of maternal death causes. It also is a leading reason for admission of pregnant women to intensive care units (Chantry, 2015; Crozier, 2011; De Greve, 2016; Guntupalli, 2015). Hemorrhage was a direct cause of 11r.4 percent of 5367 pregnancy-related maternal deaths from 2006 to 2013 in the United States (Creanga, 2015, 2017). Similarly, 16 percent of1102 maternal deaths recorded in the Nationwide Inpatient Sample were caused by hemorrhage (Kuriya, 2016). In developing countries, hemorrhage's contribution is even more striking, and it is the single most important cause of maternal death worldwide (Gofman, 2016; Oladapo, 2016; Thomas, 2016). Despite these numbers, a declining maternal mortality rate from hemorrhage in the United States has been a seminal achievement. But, as discussed in Chapter 1 (p. 5), it seems unlikely that deaths from hemorrhage have

1	a declining maternal mortality rate from hemorrhage in the United States has been a seminal achievement. But, as discussed in Chapter 1 (p. 5), it seems unlikely that deaths from hemorrhage have reached an irreducible minimum.

1	• Mechanisms of Normal Hemostasis A major concept in understanding the pathophysiology and management of obstetrical hemorrhage is the mechanism by which hemostasis is achieved after normal delivery. Recall that near term an incredible amount of blood-at least 600 mLi min-flows through the intervillous space (Pates, 2010). his prodigious flow circulates through the spiral arteries, which average 120 in number. Also, recall that these vessels have no muscular layer because of their remodeling by trophoblasts, which creates a low-pressure system. With placental separation, these vessels at the implantation site are avulsed, and hemostasis is achieved first by myometrial contraction, which compresses this formidable number of large vessels. Compression is followed by clotting and obliteration of vessel lumens. If, after delivery, the myometrium contracts vigorously, fatal hemorrhage from the placental implantation site is unlikely.

1	If, after delivery, the myometrium contracts vigorously, fatal hemorrhage from the placental implantation site is unlikely. Importanty, an intact coagulation system is not necessary or postpartum hemostasis unless there are lacerations in the uterus, birth cana, or perineum. At the same time, however, fatal postpartum hemorrhage can result from uterine atony despite normal coagulation. 70 he blood volume of a pregnant D < 500 mL woman with normal pregnancy-induced hypervolemia usually rises by half, but individual increases range from 30 to 60 D 1000-1e500 mL percent, that is, 1500 ) • > 2500 mL average-sized woman (Pritchard, 1965).

1	D 1000-1e500 mL percent, that is, 1500 ) • > 2500 mL average-sized woman (Pritchard, 1965). ) 40 The equation to calculate blood volume is shown in Table It is axiomatic ) ates, without any decrease in postpartum ..e hematocrit, blood loss at delivery that approaches the volume of blood that she added during pregnancy. Thus, if blood loss is less than the pregnancy-added vol ume, the hematocrit remains the same acutely and during the irst several days delivery delivery with hysterectomy postpartum. It then rises as nonpregnant FIGURE 41-1 Blood loss associated with vaginal delivery, repeat cesarean delivery, and plasma volume normalizes during the repeat cesarean delivery plus hysterectomy. (Data from Pritchard, 1962.)

1	Traditionally, postpartum hemorrhage is defined as the loss of ::500 mL of blood after completion of the third stage of labor. This is problematic because almost half of all women delivered vaginally shed that amount of blood or more when losses are carefully measured (Pritchard, 1962). These results are depicted in Figure 41-1 and show further that approximately 5 percent of women delivering vaginally lose more than 1000 mL of blood. According to the American College of Obstetricians and Gynecologists (2017d), postpartum hemorrhage is deined as cumulative blood lossr> 1000 mL accompanied by signs and symptoms ofhypovolemia. And, almost a third of women undergoing cesarean delivery have blood loss that exceeds 1000 mL. These studies show that estimated blood loss is commony ony approximatey hafthe actual loss. Because of this, estimated blood loss in excess of "average" should alert the obstetrician to possible excessive bleeding. Whether quantiication of blood loss improves accuracy

1	hafthe actual loss. Because of this, estimated blood loss in excess of "average" should alert the obstetrician to possible excessive bleeding. Whether quantiication of blood loss improves accuracy is controversial (Hamm, 2017; Toledo, 2007).

1	TABLE 41 -1. Calculation of Maternal Total Blood Volume next week or so. Whenever thepospartum hematocrit is lower than one obtained on admission or deivery, blood loss can be estimated as the sum of the calculated pregnancy-added volume plus 500 mL or each 3 volume percent decline ofthe hematocrit. Excessive blood loss has been estimated by several methods. Sosa and colleagues (2009) used specially constructed drapes and reported that 10.8 percent of women had hemorrhage in excess of 500 mL with vaginal delivery, whereas 1.9 percent lost > 1000 mL. Compared with the indings of Figure 41-1, these estimates likely are too low. Tita and associates (2012) used a 6-volume percent drop in the postpartum hematocrit to define clinically signiicant blood loss with vaginal delivery. This decline easily signifies a > 1000-mL blood loss in the averaged-sized woman. hey documented this amount in a fourth ofwomen, which agrees with Figure 41-1.

1	Another marker used to estimate hemorrhage incidence is the transfusion rate. In the study by Tita just cited, more than 6 percent of women who delivered vaginally underwent blood transfusions. In a study of more than 66,000 women delivered at Parkland Hospital, 2.3 percent overall were given Nonpregnant blood volumea: [Height (inches) x 50J + [Weight (pounds) x 25J = Blood volume (mL) Pregnancy blood volume: Average increase is 30 to 60 percent of calculated nonpregnant Increases across gestational age and plateaus at approximately 34 weeks Usually larger with low normal-range hematocrit (-30) and smaller with high normal-range hematocrit (-40) Average increase is 40 to 80 percent with multifetal gestation Average increase is less with preeclampsia-volumes vary inversely with severity Postpartum blood volume with serious hemorrhage:

1	Average increase is less with preeclampsia-volumes vary inversely with severity Postpartum blood volume with serious hemorrhage: Assume acute return to nonpregnant total volume after fluid resuscitation Pregnancy hypervolemia cannot be restored postpartum aFormula arrived at by measuring blood volume and blood loss in more than 100 women using 51Cr-labeled erythrocytes. Data from Hernandez, 201n2. blood transfusions for hypovolemia (Hernandez, 2012). Half 25 of these women had undergone cesarean delivery. Importantly, for those transfused, these investigators calculated blood loss to E average approximately 3500 mLl Finally, Green and coworkers (2016) reported that the incidence of massive tranfusion for From the foregoing, it is apparent that significant blood loss � t postpartum hemorrhage was 23 per 100,000 births.

1	From the foregoing, it is apparent that significant blood loss � t postpartum hemorrhage was 23 per 100,000 births. accompanies up to a fourth of vaginal deliveries. The amounts and proportions for cesarean delivery are much greater. And, � £ hemorrhage is underreported. For example, data from the O. postpartum hemorrhage incidences of only 2.0 and 2.6 per cent for two epochs in the United States (Berg, 2009). Similar incidences have been reported by others (Kramer, 2013; Mehrabadi, 2013; Patterson, 2014). Numerous clinical circumstances raise the risks for obstetrical hemorrhage. he imposing list shown in Table 41-2 illustrates that hemorrhage can manifest at any time throughout pregnancy, delivery, and the puerperium. Thus, any description TABLE 41 -2. Obstetrical Hemorrhage: Causes, Predisposing Factors, and Vulnerable Patients Anesthesia or analgesia Halogenated agents Conduction analgesia with Previous uterine atony Parity: primiparity, high parity

1	Anesthesia or analgesia Halogenated agents Conduction analgesia with Previous uterine atony Parity: primiparity, high parity Acute fatty liver Anticoagulant treatment Congenital coagulopathies Amnionic fluid embolism Prolonged retention of Cause of hemorrhage FIGURE 41-2 Contributions to maternal death from various causes of obstetrical hemorrhage. Percentages are approximations because of different classification schemata used. DIC = nated intravascular coagulopathy. (Data from AI-Zirqi, 2008; Berg, 201o0; Creanga, 201o5; Zwart, 2008.) of obstetrical hemorrhage should include gestational age. Contributions to maternal death from some of these causes of are shown in Figure 41-2.

1	Obstetrical hemorrhage is traditionally classified as antepartumsuch as with placenta previa or placental abruption, or as pospartum-commonly caused by uterine atony or genital tract lacerations. In individual women, however, these terms are nonspeciic, and it is reasonable to speciy the cause and gestational age as descriptors. to its cause. Many aspects of bleeding during the first half of pregnancy from abortion or ectopic pregnancy are covered in Chapters 18 and 19. Discussions that follow concern pregnan cies with a viable-size fetus. In these cases, rapid assessment should always consider the deleterious etal fects ofmaternal hemorrhage.

1	Chapters 18 and 19. Discussions that follow concern pregnan cies with a viable-size fetus. In these cases, rapid assessment should always consider the deleterious etal fects ofmaternal hemorrhage. During active labor, slight vaginal bleeding is common. This "bloody show" is the consequence of efacement and dilation of the cervix, with tearing of small vessels. Uterine bleeding above the cervix, however, is concerning. It may follow some separation of a placenta previa implanted in the immediate vicinity of the cervical canal, or it may be from a placental abruption or uterine tear. In some women, especially with a placenta previa, cervical varicosities may bleed (O'Brien, 2013). Rarely, there may be velamentous insertion of the umbilical cord, and the involved placental vessels may overlie the cevix-vasa previa. In this case, serious fetal hemorrhage follows laceration of these vessels at the time of membrane rupture (Swank, 2016).

1	N ear term in many women, the source of uterine bleeding is not identified, bleeding ceases, and no apparent anatomical cause is found at delivery. In most of these cases, bleeding likely originated from a slight marginal placental separation. Despite this, any pregnancy with antepartum bleeding remains at higher risk or an adverse outcome even though bleeding has stopped and placenta previa has been excluded sonographicaly. Injuries to the Birth Canal

1	Injuries to the Birth Canal Bleeding after midpregnancy is associated with several adverse outcomes. he Canadian Perinatal Network described 806 women with hemorrhage between 22 and 28 weeks' gestation (Sabourin, 2012). Placental abruption (32 percent), previa (21 percent), and cervical bleeding (6.6 percent) were the most frequent causes identified. In a third, no cause was found. Of all women, 44 percent were delivered before 29 weeks' gestation. In more than 68,000 women in Scotland, the incidence of antepartum hemorrhage after the irst trimester was 11 percent (Bhandari, 2014). hese women were at signiicantly higher risk for preterm birth, labor induction, and postpartum hemorrhage.

1	In most cases, the source of postpartum hemorrhage can and should be determined. Frequent causes are uterine atony with placental site bleeding, genital tract trauma, or both. Postpartum hemorrhage is usually obvious. Important exceptions are unrecognized intrauterine and intravaginal blood accumulation and uterine rupture with intraperitoneal or retroperitoneal bleeding. Another consideration is an expanding vulvar or vaginal hematoma (p. 764). Initial evaluation attempts to diferentiate uterine atony from genital tract lacerations. For this, risk factors are sought, the lower genital tract is examined, and uterine tone is assessed. Atony is identiied by a boggy, soft uterus during bimanual examination and by expression of clots and hemorrhage during uterine massage.

1	Persistent bleeding despite a firm, well-contracted uterus suggests that hemorrhage most likely is from lacerations. Bright red blood further suggests arterial bleeding. To conirm that lacerations are a source of bleeding, carful inspection of the vagina, cervix, and uterus is essential. Sometimes bleeding may be caused by both atony and trauma, especially after forceps or vacuum-assisted vaginal delivery. Examination is easier if conduction analgesia was given. If there are no lower genital tract lacerations and the uterus is contracted, yet supracervical bleeding persists, then manual exploration of the uterus is done to exclude a uterine tear (Kaplanoglu, 2016). his also is completed routinely after internal podalic version, breech extraction, or successful vaginal birth after cesarean.

1	Late pospartum hemorrhage describes bleeding after the irst 24 hours. Found in up to 1 percent of women, it may be serious and is discussed in Chapter 37 (American College of Obstetricians and Gynecologists, 2017 d). As noted, visual estimates are notoriously inaccurate, especially with excessive bleeding. Instead of sudden massive hemorrhage, postpartum bleeding is frequently steady. If atony persists, bleeding may appear to be only moderate at any given instant but may continue until serious hypovolemia develops. Bleeding from an episiotomy or a vaginal laceration can also appear to be only minimal to moderate. But, constant seepage can lead to enormous blood loss relatively quickl)r . In some cases, ater placental separation, blood may not escape vaginally but instead may collect within the uterine cavity, which can become distended by 1000 mL or more of blood. In others, postpartum uterine massage is applied to a roll of abdominal fat mistaken for the uterus.

1	All of these factors can lead to an underappreciation of the magnitude of hemorrhage over time. he efects of hemorrhage depend to a considerable degree on the maternal nonpregnant blood volume and the corresponding degree of pregnancyinduced hypervolemia. For this and other reasons, hypovolemia may not be recognized until very late. A treacherous eature of pospartum hemorrhage is the ailure of the pulse and blood pressure to undergo more than moderate alterations until large amounts of blood have been lost. The normotensive woman initially may actually become somewhat hypertensive from catecholamine release in response to hemorrhage. And importantly, women with preeclampsia may become "normotensive" despite remarkable hypovolemia.

1	Some gravidas may be particularly susceptible to hemorrhage because their blood volume expansion is less than expected. This situation is most commonly encountered in small womeneven those with normal pregnancy-induced hypervolemia. Women with severe preeclampsia or eclampsia are also more vulnerable to hemorrhage because they frequently do not have a normal blood volume accrual. Specifically, Zeeman and associates (2009) documented a mean increase above nonpregnant volume of only 10 percent in eclamptic women (Chap. 40, p. 718). A third example is the moderate-to-severe curtailing of pregnancy-induced volume expansion in women with chronic renal insuiciency (Chap. 53, p. 1034). When excessive hemorrhage is suspected in these high-risk women, cystalloid and blood are prompty administered or suspected hypovolemia.

1	The most frequent cause of obstetrical hemorrhage is failure of the uterus to contract suiciently after delivery and to arrest bleeding from vessels at the placental implantation site (p. 755). That said, some bleeding is inevitable during thirdstage labor as the placenta begins to separate. Blood from the implantation site may escape into the vagina immediately-the Duncan mechanism of placental separation, or it remains concealed behind the placenta and membranes until the placenta is delivered-the Schultze mechanism. After signs of placental separation, the uterus should be massaged if it is not contracted irmly, and placental descent is indicated by a slack umbilical cord. Importanty, separation and delivey of the placenta by cord traction, especialy when the uterus is atonic, may cause uterine inversion.

1	If heavy bleeding persists after delivery of the newborn and while the placenta remains partially or totally attached, then manual placental removal is indicated (Cummings, 2016; Frolova, 2016). For this, adequate analgesia is mandatory, and aseptic surgical technique should be used. As illustrated in Figure 41-3, the fingertips of one hand, with fingers approxi mated, are insinuated between the uterine wall and placenta. A sweeping forward motion in this plane will peel the placenta of its uterine attachmenc. After its removal, trailing membranes are carefully teased free from the decidua using ring forceps as needed. Another method to clear membranes is to wipe out the uterine cavity with a gauze-wrapped hand. Most recommend FIGURE 41 -3 Manual removal of placenta. A. One hand grasps the fundus. The other hand is inserted into the uterine cavity, and the fingers are swept from side to side as they are advanced. B. When the placenta has become detached, it is grasped and removed.

1	removal (World Health Organization, 2015). he fundus is always palpated following placental delivery to conirm that the uterus is well contracted. If it is not irm, hemorrhage from atony (Hofmeyr, 2013). Simultaneously, 20 units of oxytocin in 1000 mL of crystalloid solution will of 200 m U / min. Higher concentrations are minimally more efective (Tita, 2012). Oxytocin is never given as an undiluted bolus dose because serious hypotension or cardiac arrhythmias can develop.

1	In many women with known risks, uterine atony can at least be anticipated well in advance of delivery. In one study, however, up to half of women with atony after cesarean deliveY had no risk factors (Rouse, 2006). The magnitude of risk for atony imposed by each of the factors shown in Table 41-2 varies considerably between reports. Primiparity and high parity are risk factors (Driessen, 2011). In one study, the incidence of postpartum hemorrhage rose from 0.3 percent in women of low parity to 1.9 percent with parity of four or greater. It was 2.7 percent with parity of seven or greater (Babinszki, 1999). The overdistended uterus is prone to hypotonia after delivery, and thus women with a large fetus, multiple fetuses, or hydramnios are at greater risk. Labor abnormalities predispose to atony and include hyper-or hypotonic labor. Similarly, labor induction or augmentation with either prostaglandins or oxytocin is more likely to be followed by atony (Driessen, 2011). he frequency of

1	atony and include hyper-or hypotonic labor. Similarly, labor induction or augmentation with either prostaglandins or oxytocin is more likely to be followed by atony (Driessen, 2011). he frequency of hemorrhage increases with prolongation of the third stage (Frolova, 2016). Finally, the woman who has had aprior postpartum hemorrhage is at risk for recurrence.

1	With immediate postpartum hemorrhage, careful inspection is done to exclude birth canal laceration. Because bleeding can be caused by retained placental fragments, inspection of the placenta after delivery should be routine. If a defect is seen, the uterus should be manually explored and the fragment removed. Occasionally, retention of a succenturiate lobe may cause postpartum hemorrhage (Chap. 6, p. 112). During examination for lacerations and causes of atony, the uterus is massaged and uterotonic agents are administered.

1	Several compounds can prompt the postpartum uterus to contract (Chap. 27, p. 527). One of these is routinely selected and given to prevent postpartum bleeding by ensuring uterine contractions. Nfost of these same agents are also used to treat uterine atony with bleeding. Moreover, because many trials combine results from atony prophylaxis and treatment, their evaluation is problematic. For example, oxytocin has been used for more than 70 years, and in most cases, it is infused intravenously or given intramuscularly after placental delivery. Neither route has been shown to be superior (Dagdeviren, 2016). his or other utero tonics given prophylactically will prevent most cases of uterine atony.

1	To treat uterine atony, ergot alkaloids have been used for centuries. If atony persists despite oxytocin and other preventive measures, ergot derivatives can be used for secondline treatment. Ergot preparations include methylergonovine (Methergine) and ergonovine, however, only methylergonovine is currently manufactured in the United States. Given parenterally, these drugs rapidly stimulate tetanic uterine contractions and act for approximately 45 minutes (Schimmer, 2011). A common regimen is 0.2 mg of either drug given intramuscularly. Methergine can be repeated at 2-to 4-hour intervals as needed. A caveat is that ergot agents, especialy given intravenousy, may cause dangerous hypertension, especialy in women with preeclampsia. Severe hypertension is also seen with concomitant use of protease inhibitors given for human immunodeiciency viral (HIV) infection. These adverse efects notwithstanding, it is speculative whether ergot derivatives ofer superior therapeutic efects compared with

1	inhibitors given for human immunodeiciency viral (HIV) infection. These adverse efects notwithstanding, it is speculative whether ergot derivatives ofer superior therapeutic efects compared with oxytocin.

1	In cases of atony refractory to one agent, an agent from a diferent group can be added. At least two randomized studies have addressed combined ergot-oxytocin regimens. In one, ergometrine plus oxytocin was compared with ergometrine alone to prevent postpartum hemorrhage (Koen, 2016). The overall need for transfusion was signiicantly lower with the combination regimen. Another comparable study reairmed these indings (�enti.irk, 2016).

1	During the past 40 years, other second-line agents for atony have included the E-and F-series prostaglandins. Carboprost tromethamine (Hemabate) is the 15-methyl derivative of prostaglandin F2a• It is approved for uterine atony treatment in a dose of 250 Lg (0.25 mg) given intramuscularly. This dose can be repeated if necessary at 15-to 90-minute intervals up to a maximum of eight doses. Observational data indicate an 88-percent success rate (Oleen, 1990). Carboprost causes side efects in approximately 20 percent of women. These include, in descending order of frequency, diarrhea, hypertension, vomiting, fever, flushing, and tachycardia. Another pharmacological efect is pulmonary airway and vascular constriction. hus, carboprost should not be used for asthmatic women and those with suspected amnionic fluid embolism (p. 785). We have occasionally encountered severe hypertension with carboprost given to women with preeclampsia. It has also been reported to cause arterial oxygen

1	suspected amnionic fluid embolism (p. 785). We have occasionally encountered severe hypertension with carboprost given to women with preeclampsia. It has also been reported to cause arterial oxygen desaturation that averaged 10 percent (Hanins, 1988). Relative contraindications to carboprost include renal, liver, and cardiac disease (American College of Obstetricians and Gynecologists, 2017 d).

1	E-series prostaglandins can also prevent or treat atony. Dinoprostone-prostaglandin E2-may be used of label and is given as a 20-mg suppository per rectum or per vaginum every 2 hours. It typically causes diarrhea, which is problematic for the rectal route, whereas vigorous vaginal bleeding may preclude its use per vaginum. Hypotension, which is commonly encountered with hemorrhage, is considered a contraindication by some. Intravenous prostaglandin E2-suprostone-is used in Europe, but it is not available in the United States (Schmitz, 2011).

1	Misoprostol-Cytotec-is a synthetic prostaglandin Ej analogue that is used for prevention and treatment of atony (AbdelAleem, 2001; Ugwu, 2016). Most studies have addressed prevention and have conflicting conclusions. In a Cochrane review, Mousa and associates (2014) reported no added benefits for misoprostol use compared with oxytocin or ergonovine for treatment. Derman and coworkers (2006) compared a 600-l-Lg oral dose given preventively at delivery against placebo. They found that the drug lowered the incidence of hemorrhage from 12 to 6 percent and that of severe hemorrhage from 1.2 to 0.2 percent. In another study, Gerstenfeld and Wing (2001) concluded that 400 ILg misoprostol administered rectally was not superior to intravenous oxytocin given to prevent postpartum hemorrhage. From a systematic review, Villar (2002) found that oxytocin and ergot preparations administered after delivery were more efective than misoprostol for prevention of postpartum hemorrhage (Chap. 27, p. 527).

1	systematic review, Villar (2002) found that oxytocin and ergot preparations administered after delivery were more efective than misoprostol for prevention of postpartum hemorrhage (Chap. 27, p. 527). If misoprostol is used to treat atony, the American College of Obstetricians and Gynecologists (2017 d) recommends a dose of 600 to 1000 ILg rectally, orally, or sublingually.

1	Bleeding Unresponsive to Uterotonic Agents If bleeding persists after initial measures for atony have been implemented, then the following management steps are performed immediately and simultaneously: 1. Begin bimanual uterine compression, which is easily done and controls most cases of continuing hemorrhage (Fig. 41-4). This technique is not simply fundal massage. The posterior uterine wall is massaged by one hand on the abdomen, while the other hand is made into a fist and placed into the vagina. his fist kneads the anterior uterine wall through the anterior vaginal wall and the uterus is also compressed between the two hands. 2. Immediately mobilize the emergent-care obstetrical team to the delivery room and call for whole blood or packed red cells. 3. Request urgent help from the anesthesia team. 4.

1	2. Immediately mobilize the emergent-care obstetrical team to the delivery room and call for whole blood or packed red cells. 3. Request urgent help from the anesthesia team. 4. Secure at least two large-bore intravenous catheters so that crystalloid with oxytocin can be continued simultaneously with blood products. Insert an indwelling Foley catheter for continuous urine output monitoring. 5. Begin volume resuscitation with rapid intravenous infusion of crystalloid (p. 788). 6. With sedation, analgesia, or anesthesia established and now with optimal exposure, once again manually explore the uterine cavity for retained placental fragments and for uterine abnormalities, including lacerations or rupture. FIGURE 41-4 Bimanual compression for uterine atony. The uterus is positioned with the fist of one hand in the anterior fornix pushing against the anterior wall, which is held in place by the other hand on the abdomen. The abdominal hand is also used for uterine massage. 7.

1	7. Thoroughly inspect the cervix and vagina again for lacerations that may have escaped attention. 8. If the woman is still unstable or if there is persistent hemorrhage, then blood transfusions are given (p. 788). At this juncture, after causes other than atony have been excluded and after hypovolemia is reversed, several other measures are considered if bleeding continues. Their use depends on several factors such as parity, desire for sterilization, and experience with each method.

1	Balloon Tamponade. Uterine packing to treat refractory uterine atony fell from favor because of concerns regarding concealed bleeding and infection (Gilstrap, 2017). Newer techniques of balloon tamponade help alleviate some of these concerns (Sentilhes, 2016; Zelop, 201l). In one technique, the tip of a 24F to 30F Foley catheter with a 30-mL balloon is guided into the uterine cavity and illed with 60 to 80 mL of saline. The open tip permits continuous drainage of blood from the uterus. We have experienced balloon rupture when more than 50 mL was instilled into the balloon, thus a 34F Foley with a 60-ml balloon can be used. If bleeding subsides, the catheter is typically removed ater 12 to 24 hours. Similar devices for tamponade include SegstakenBlakemore, Rusch, and ebb balloons and condom catheters (Antony, 2017; Georgiou, 2009).

1	Enthusiasm has developed for specially constructed intrauterine balloons to treat hemorrhage from uterine atony and other causes. A Bakri Pospartum Baloon or BT-Cath may be inserted and inlated to tamponade the endometrial cavity and stop bleeding (Fig. 41-5). Insertion requires two or three team members. he irst performs abdominal sonography during the procedure. The second places the delated balloon into the uterus and stabilizes it. The third member instills luid to inlate the balloon, rapidly infusing at least 150 mL followed by further instillation over a few minutes for a total of 300 to 500 mL to arrest hemorrhage. It is reasonable to remove the balloon after approximately 12 hours (Einerson, 2017). In prospective studies, nearly 150 women have been managed for postpartum hemorrhage with these uterine balloons FIGURE 41 -5 Intrauterine Bakri balloon for postpartum hemorrhage.

1	In prospective studies, nearly 150 women have been managed for postpartum hemorrhage with these uterine balloons FIGURE 41 -5 Intrauterine Bakri balloon for postpartum hemorrhage. (Gronvall, 2013; Kaya, 2016; Vintejoux, 2015). Perhaps a fourth of cases were caused by uterine atony. For all causes, the success rate was noted to be approximately 85 percent. Combinations of balloon tamponade and uterine compression sutures have also been described (Diemert, 2012; Yoong, 2012). Failures for all of these require various surgical methods including hysterectomy. These include uterine compression sutures, pelvic vessel ligation, angiographic embolization, and hysterectomy. hese are discussed on page 792.

1	These include uterine compression sutures, pelvic vessel ligation, angiographic embolization, and hysterectomy. hese are discussed on page 792. Puerperal inversion of the uterus is one of the classic hemorrhagic disasters encountered in obstetrics. Unless promptly recognized and managed appropriately, associated bleeding often is massive. Risk factors include alone or in combination: (1) fundal placental implantation, (2) uterine atony, (3) cord traction applied bore placental separation, and (4) abnormally adhered placentation such as with the accrete syndromes (p. 777). Depending on which of these factors are contributory, the incidence and severity of uterine inversion varies. There is progressive severity of inversion as shown in Figure 41-6. The worst scenario is complete inversion with the uterus protruding from the birth canal (Fig. 41-7).

1	The incidence of uterine inversion ranges from 1 in 2000 to 1 in 20,000 vaginal deliveries (Coad, 2017; Ogah, 2011; Rana, 2009; Witteveen, 2013). Our experiences at Parkland Hospital comport with the higher 1 :2000 incidence. his is despite our policy of discouraging placental delivery by cord traction alone, and before certainty of its separation. It is unknown if active management o/third-stage labor with cord traction applied ostensiblyater signs of placental separation raises the likelihood of uterine inversion (Deneux-Tharaux, 2013; Gulmezoglu, 2012; Prick, 2013). Immediate recognition of uterine inversion improves the chances of a quick resolution and good outcome (Furukawa, 20 15b). If initially unrecognized, continued hemorrhage likely FIGURE 41-6 Progressive degrees of uterine inversion. FIGURE 41 -9 Surgical anatomy of a completely inverted uterus viewed from above at laparotomy.

1	FIGURE 41-6 Progressive degrees of uterine inversion. FIGURE 41 -9 Surgical anatomy of a completely inverted uterus viewed from above at laparotomy. In some cases, the uterus will again invert almost immediately after repositioning. With this problem, uterine compression sutures can be used to prevent another inversion (Matsubara, 2009; Mondal, 2012). Occasionally, chronic puerperal uterine inversion may become apparent weeks after delivery. Childbirth is invariably associated with trauma to the birth canal, which includes the uterus and cervix, vagina, and perineum. Injuries sustained during labor and delivery range from minor mucosal tears to lacerations that create life-threatening hemorrhage or hematomas. According to the American College of Obstetricians and Gynecologists (2016b), up to 80 percent of women sustain some type of laceration at vaginal delivery. hese may lie proximally or distally along the lower genital tract.

1	First, small tears of the anterior vaginal wall near the urethra are relatively common. hey are often supericial with little to no bleeding, but they occasionally require sutures for hemostasis. Those large enough to require extensive repair are typically associated with short-term voiding diiculty, and an indwelling bladder catheter will obviate this. Deeper perineal lacerations are usually accompanied by varying degrees of injury to the outer third of the vaginal vault. Some extend to involve the anal sphincter or varying depths of the vaginal walls. Repair of these perineal lacerations is detailed in Chapter 27 (p. 531).

1	Lacerations involving the middle or upper third of the vaginal vault usually are comorbid with injuries of the perineum or cervix. hese sometimes are missed unless inspection is thorough. Those that extend upward usually are longitudinal. They may follow spontaneous delivery but frequently result from injries sustained during operative vaginal delivery. Most niicant hemorrhage, which is controlled by suture repair. For this, efective analgesia or anesthesia, clear visualization, capable assistance, and suicient resuscitation of hypovolemia are man datory. search for evidence of retroperitoneal hemorrhage or of perito neal perforation with hemorrhage. Also, intrauterine explora tion is considered to exclude uterine tears or rupture (Conrad, 2015). If peritoneal perforation or uterine rupture is strongly suspected, laparotomy is considered (Rai, 2010). As discussed later (p. 793), imaging and potential embolization may be suit able for large retroperitoneal hematomas.

1	Supericial lacerations of the cervix can be seen on close inspec tion in more than half of all vaginal deliveries. Most of these measure <0.5 cm and seldom require repair. Deeper lacera tions are less frequent, but even these may be unnoticed. Due to ascertainment bias, variable incidences are described. For example, with close inspection, the incidence of cervical lacera tions in the Consortium on Safe Labor database was 1 percent in nulliparas and 0.5 percent in multiparas (Landy, 2011). But, the overall incidence in a study of more than 81 ,000 Israeli women was only 0.16 percent (Melamed, 2009). Such lacera tions are more likely to be associated with vacuum-or forceps assisted vaginal delivery (Fong, 2014).

1	Cervical lacerations are not usually problematic unless they cause hemorrhage or extend to the vagina. Rarely, the cervix may be entirely or partially avulsed from the vagina in the anterior, posterior, or lateral fornices, an injury termed coporrhxis. Another rare injury is when the entire vaginal portion of the cervix is avulsed-annular or circular detachment. These injuries sometimes follow forceps deliveries performed through an incompletely dilated cervix with the blades applied over the cer vix. In some women, cervical tears reach into the lower uterine segment and involve the uterine artery and its major branches. They occasionally extend into the peritoneal cavity. More severe lacerations usually manifest as external hemorrhage or as a hematoma, however, they may occasionally be unsuspected. In the Israeli study just cited, almost 11 percent of women with a cervical laceration required blood transfusions (Melamed, 2009).

1	At times, the edematous anterior cervical lip is compressed berween the fetal head and maternal symphysis pubis. This usually is of little consequence and resolves spontaneously. Rarely, this causes severe ischemia, and the anterior lip may undergo necrosis and subsequently separate from the rest of the cervix. As with vulvovaginal lacerations, cervical tears can be more fully appreciated with adequate exposure, which may be best attained with transfer to an operating room. An assistant applies irm downward pressure on the uterus, while the operator exerts gentle traction on the lips of the cervix with ring forceps. A second assistant can provide even better exposure with right-angle vaginal wall retractors or Breisky vaginal retractors. Use of suction devices can also aid viewing.

1	In general, cervical lacerations of 1 and even 2 cm are not repaired unless they are bleeding. Such tears heal rapidly and ultimately create an irregular, sometimes stellate appearing, external cervical os that indicates previous delivery. FIGURE 41 -10 Repair of cervical laceration with appropriate surgical exposure. Continuous absorbable sutures are placed beginning at the upper angle of the laceration.

1	FIGURE 41 -10 Repair of cervical laceration with appropriate surgical exposure. Continuous absorbable sutures are placed beginning at the upper angle of the laceration. Deep cervical tears usually require surgical repair. When the laceration is limited to the cervix or even when it extends somewhat into the vaginal fornix, satisfactory results are obtained by suturing the cervix after bringing it into view as depicted in Figure 41-10. While cervical lacerations are repaired, any associated vaginal lacerations or an episiotomy may be tamponaded with gauze packs to arrest their bleeding. Because hemorrhage usually comes from the upper angle of the wound, the first suture using 2-0 chromic or polyglactin is placed in tissue above the angle. Subsequently, either interrupted or continuous locking sutures are serially placed outward toward the operator. If the uterus is involved and hemorrhage persists, some of the methods described later (p. 792) may be necessary to obtain hemostasis.

1	Pelvic hematomas can have several anatomical manifestations following childbirth. One classification is anatomical and describes vulvar, vulvovaginal, paravaginal, and retroperitoneal hematomas. Vulvar hematomas may involve the vestibular bulb or branches of the pudendal artery, which are the inferior rectal, perineal, and clitoral arteries (Fig. Paravaginal hematomas may involve the descending branch of the uterine artery. In some cases, a torn vessel lies above the pelvic fascia, and a supralevator hematoma develops. These can extend into the upper portion of the vaginal canal and may almost occlude its lumen. Continued bleeding may dissect retroperitoneally to form a mass palpable above the inguinal ligament. In some cases, it may even dissect up behind the ascending colon to the hepatic flexure (Rai, 2010).

1	Risks for puerperal hematomas include vaginal or perineal laceration, episiotomy, or an operative delivery (Iskender, 2016). Any hematoma can also develop following stretch and rupture of a blood vessel without an associated laceration (Nelson, 2012). This may be especially true with forceps delivery. Occasionally, they are associated with an underlying coagulopathy (p. 782).

1	Perineal, vulvar, and paravaginal hematomas can develop rapidly and frequently cause excruciating pain (Fig. 41-12). A tense, tender swelling of varying size rapidly develops, encroaches on the vaginal lumen, and causes overlying skin or epithelium to become ecchymotic. A paravaginal hematoma may escape detection initially. However, symptoms of pelvic pressure, pain, or inability to void should prompt evaluation. Others may go undetected until other measures of hypovolemia become evident. When there is a supralevator extension, the hematoma extends upward in the paravaginal space and between the leaves of the broad ligament. The hematoma may escape detection until it can be felt on abdominal palpation or until hypovolemia develops. Imaging with sonography or computed tomographic scanning may be useful (Cichowski, 2017; Kawamura, 2014; Takeda, 2014).

1	Small hematomas often remained contained and show minimal expansion. In others, the tissues overlying an expanding hematoma may rupture from pressure necrosis. In some, profuse hemorrhage may follow, but in other cases, the hematoma drains in the form of large clots and old blood. In those that involve the paravaginal space and extend above the levator sling, retroperitoneal bleeding may be massive and occasionally fatal. Finally, we have encountered a few that rebled up to 2 weeks postpartum (Cunningham, 2017 a).

1	Vulvovaginal hematomas are managed according to their size, location, duration since delivery, and expansion. If bleeding ceases, then small-to moderate-sized hematomas may be treated expectantly until absorbed. But, if pain is severe or if the hematoma continues to enlarge, sutgical exploration is preferable. Blood loss with arge puerperal hematomas is neary always consideraby more than the clinical estimate. Hypovolemia is common, and transfusions are frequently required when surgical repair is necessary.

1	For repair, an incision is made at the point of maximal distention, blood and clots are evacuated, and bleeding points ligated. The cavity may then be obliterated with absorbable sutures. Often, no sites of bleeding are identified. Nonetheless, the evacuated hematoma cavity is surgically closed, and the vagina is packed for 12 to 24 hours. Supralevator hematomas are more diicult to treat. Although some can be evacuated by vulvar or vaginal incisions, laparotomy or interventional embolization, described next, is a consideration if bleeding continues. Angiographic embolization has become popular for management of some puerperal hematomas. This is especially true FIGURE 41 -7 Maternal death during home delivery caused by exsanguination from uterine inversion and a fundally implanted placenta accreta.

1	FIGURE 41 -7 Maternal death during home delivery caused by exsanguination from uterine inversion and a fundally implanted placenta accreta. will prompt closer examination of the birth canal. Although complete inversion is usually evident, the partially inverted uterus can be mistaken for a uterine myoma, and sonography can aid diferentiation (Pan, 2015; Smulian, 2013). Many cases are associated with immediate life-threatening hemorrhage, and a fourth require blood replacement (eoad, 2017). Once any degree of uterine inversion is recognized, several steps must be implemented urgently and simultaneously: 1. Immediate assistance is summoned, including obstetrical and anesthesia personnel. 2. Blood is brought to the delivery suite for potential use. 3. he woman is evaluated for emergency general anesthesia. Large-bore intravenous infusion systems are secured to begin rapid crystalloid infusion to treat hypovolemia while awaiting arrival of blood products. 4.

1	Large-bore intravenous infusion systems are secured to begin rapid crystalloid infusion to treat hypovolemia while awaiting arrival of blood products. 4. If the recently inverted uterus has not contracted and retracted completely and if the placenta has already separated, then the uterus may often be replaced simply by pushing up on the inverted fundus with the palm of the hand and ingers in the direction of the long axis of the vagina (Fig. 41-8). Some use two fingers rigidly extended to push the center of the fundus upward. Care is taken not to appy so much pressure as to peorate the uterus with the ingertips. 5.

1	5. If the placenta is still attached, then attempts are made to reposition the uterus with the placenta in situ. Many recommend a trial of an intravenously administered tocolytic drug such as terbutaline, magnesium sulfate, or nitroglycerin for uterine relaxation and repositioning (You, 2006). If these fail to provide suicient relaxation, then a rapidly acting halogenated inhalational agent is administered. After the uterus is replaced, the placenta is carefully manually removed. 6. If uterine repositioning fails with the placenta attached, then it is peeled of and steady pressure with the fist, palm, or ingers is applied to the inverted fundus in an attempt to push it up into and through the dilated cervix as described in Step 4. FIGURE 41-8 Incomplete uterine inversion repositioned by using the abdominal hand for palpation of the crater-like depression while simultaneously gently pushing the inverted fundus upward.

1	FIGURE 41-8 Incomplete uterine inversion repositioned by using the abdominal hand for palpation of the crater-like depression while simultaneously gently pushing the inverted fundus upward. 7. Once the uterus is restored to its normal coniguration, tocolysis is stopped. Oxytocin is then infused, and other utero tonics may be given as described for atony (p. 759). Meanwhile, the operator maintains the fundus in its normal anatomical position while applying bimanual compression to control further hemorrhage until the uterus is well contracted (see Fig. 41-4). The operator continues to monitor the uterus transvaginally for evidence of subsequent inversion. A Bakri balloon has been used to maintain the repositioned uterus (Haeri, 2015; Ida, 2015).

1	In most cases, the inverted uterus can be restored to its normal position by the techniques just described. Occasionally, manual replacement fails. One cause is a dense m) ometrial constriction ring. At this point, laparotomy is imperative. The anatomical coniguration found at surgery can be confusing as shown in

1	Figure 41-9. With agents given for tocolysis, a combined efort is made to reposition the uterus by simultaneously pushing upward from below and pulling upward from above. Application of atraumatic clamps to each round ligament and upward traction may be helpul-the Huntinton procedure. In some cases, placing a deep traction suture in the inverted fundus or grasping it with tissue forceps may be of aid. Either or both of these may be technically diicult. If a constriction ring still prohibits repositioning, a sagittal surgical cut-Haultain incisionis made posteriorly through the muscular ring to release it. he exposed fundus can then be reinverted (Sangwan, 2009). After uterine replacement, tocolytics are stopped, oxytocin and other utero to nics are given, and the uterine incision is repaired. Risks of separation of this posterior hysterotomy incision during subsequent pregnancy, labor, and delivery are unknown. Further illustration and discussion is found in Cunningham and

1	Gilstrapis Operative Obstetrics, 3rd edition (Zahn, 2017). Clitoral crus Batholin gland , Bulbospongiosus m. A Colles fascia Ischioanal fossa hematoma c B Pudendal artery Vulvar hematoma branches Colles fascia cut edge Ischiocavenosus m. Dorsal atery of the clitoris Bulbospongiosus m. Perineal a. Superficial transvese perineal n. External anal sphinter n. Inferior rectal a. Levator ani m. --Gluteus maxinus m. FIGURE 41-11 Schematic drawing showing types of puerperal hematomas. A. Coronal view showing a supralevator hematoma. B. Coronal view showing an anterior perineal triangle hematoma. C. Perineal view showing posterior perineal triangle anatomy and an ischioanal fossa hematoma. (Reproduced with permission from Cunningham FG: Genital tract lacerations and hematomas. In Yeomans ER, Hofman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd edition. New York, McGraw-Hili Education, 201o7a.)

1	FIGURE 41-12 Let-sided anterior perineal triangle hematoma associated with a vaginal laceration following spontaneous delivery in a woman with consumptive coagulopathy from acute fatty liver of pregnancy. for supralevator or retroperitoneal hematomas. Embolization can be used primarily, or more likely secondarily, if surgical attempts at hemostasis have failed or if the hematoma is difficult to access surgically (Distefano, 2013; Lee, 2012; Poujade, 2012). he use of a Bakri balloon for a paracervical hematoma has also been described (Gizzo, 2013; Gronvall, 2013). Finally, ultrasound-guided drainage of a recurrent supralevator hematoma has been reported (Mukhopadhyay, 2015) .

1	Uterine rupture frequently is catastrophic. It may be primay, deined as occurring in a previously intact or unscarred uterus, or may be seconday and associated with a preexisting incision, injury, or anomaly of the myometrium. Some of the etiologies associated with uterine rupture are presented in Table 41-3. Importantly, the contribution of each of these underlying causes has changed remarkably during the past 50 years. Speciically, before 1960, when the cesarean delivery rate was much TABLE 41 -3. Some Causes of Uterine Rupture Surgery involving the myometrium: Myomectomy incision through or to the endometrium Deep cornual resection of interstitial fallopian tube Metroplasty Coincidental uterine trauma: Abortion with instrumentation-sharp or suction curette, sounds Sharp or blunt trauma-assaults, accidents, bullets, knives Congenital: Before delivery:

1	Coincidental uterine trauma: Abortion with instrumentation-sharp or suction curette, sounds Sharp or blunt trauma-assaults, accidents, bullets, knives Congenital: Before delivery: Persistent, intense, spontaneous contractions Labor stimulation-oxytocin or prostaglandins Intraamnionic instillation-saline or prostaglandins Perforation by internal uterine pressure catheter External trauma-sharp or blunt Uterine overdistention-hydramnios, multifetal pregnancy During delivery: Fetal anomaly distending lower segment Vigorous uterine pressure during delivery Difficult manual removal of placenta Acquired:

1	Fetal anomaly distending lower segment Vigorous uterine pressure during delivery Difficult manual removal of placenta Acquired: Sacculation of entrapped retroverted uterus lower and women of great parity were numerous, primary uterine rupture predominated. As the incidence of cesarean delivery rose and especially as a subsequent trial oflabor in these women became prevalent through the 1990s, uterine rupture through the cesarean hysterotomy scar became the preeminent cause (Gibbins, 2015; Mone, 2016). However, concurrent with the diminished enthusiasm for a trial of labor in women with a prior cesarean delivery, incidence trends for the two types of rupture have again changed. In a study of 3942 cases of uterine rupture in more than 15 million women, approximately half were in women with a prior cesarean delivery (Yao, 2017). In 40 cases of rupture at Parkland Hospital from 2009 to 2016, 15 events (37 percent) were primary, and 25 (63 percent) were secondary (Happe, 2017).

1	Additional risks for rupture include other previous operations or manipulations that traumatize the myometrium. Examples are uterine curettage or perforation, endometrial ablation, myomectomy, or operative hysteroscopy (Kieser, 2002; Pelosi, 1997). In a study by Porreco and colleagues (2009), seven of21 women without a prior cesarean delivery had undergone prior uterine surgery.

1	In developed countries, the incidence of rupture is 1 in 4800 deliveries (Getahun, 2012). During a 40-year period in Norway, the uterine rupture rate rose significantly to about 1 in 1560 deliveries (Al-Zirqi, 2016). he frequency of primary rupture, however, approximates 1 in 10,000 to 15,000 births (Porreco, 2009). As discussed, one reason is a decreased incidence of women of great parity. Another is that excessive or inappropriate uterine stimulation with oxytocin-previously a frequent cause-has mostly disappeared. Maggio and associates (2014) found no association between the number of Montevideo units and secondary uterine rupture. In addition, in a recent analysis of three trials comparing high-versus low-dose oxytocin regimens, the rate of uterine rupture did not difer between groups (Budden, 2014). The rate of rupture is elevated with sequential induction of labor with prostaglandins and oxytocin (Al-Zirqi, 2017). At Parkland Hospital, we too have encountered primary uterine

1	(Budden, 2014). The rate of rupture is elevated with sequential induction of labor with prostaglandins and oxytocin (Al-Zirqi, 2017). At Parkland Hospital, we too have encountered primary uterine rupture in a disparate number of women in whom labor was induced with prostaglandin E1•

1	Blunt abdominal trauma can precipitate uterine rupture. Although the distended pregnant uterus is surprisingly resistant, pregnant women sustaining such trauma should be watched carefully for signs of a ruptured uterus (Chap. 47, p. 927). In one study of 13 cases of primary uterine rupture, trauma accounted for three cases (Miller, 1996). Other causes of traumatic rupture that are uncommon today are those due to internal podalic version and extraction, diicult forceps delivery, breech extraction, and unusual fetal enlargement such as with hydrocephaly. Uncommon associations of rupture are uterine anomalies or multifetal pregnancy (Bankada, 2015; Tarney, 2013; Tola, 2014). Occasionally, focal inherent weakness in the myometrium predisposes to rupture. Examples include anatomical anomalies, leiomyomas, adenomyosis, choriocarcinoma, and connective-tissue defects such as Ehlers-Danlos syndrome (Arici, 2013; Nikolaou, 2013; Noh, 2013; Ramskill, 2014; Sun, 2016).

1	Rupture of the previously intact uterus during labor most often involves the thinned-out lower uterine segment. When the rent is in the immediate vicinity of the cervix, it frequently extends transversely or obliquely. When the rent forms in the portion of the uterus adjacent to the broad ligament, the tear FIGURE 41-13 Supracervical hysterectomy specimen showing uterine rupture during spontaneous labor with a vertical tear at the left lateral edge of lower uterine segment.

1	FIGURE 41-13 Supracervical hysterectomy specimen showing uterine rupture during spontaneous labor with a vertical tear at the left lateral edge of lower uterine segment. is usually longitudinal. Although these tears develop primarily in the lower uterine segment, they can extend upward into the active segment or downward through the cervix and into the vagina (Fig. 41-13). In some cases, the bladder may also be lacerated. If the rupture is of suicient size, the uterine contents will usually escape into the peritoneal cavity. If the presenting fetal part is firmly engaged, however, then only a portion of the fetus may be extruded from the uterus. Fetal prognosis is largely dependent on the degree of placental separation and magnitude ;f !ii�tei!�l he!ii;iih�be �!d hypovolemia. In some cases, the overlying peritoneum remains intact, and this usually is accompanied by hemorrhage that extends into the broad ligament to cause a large retroperitoneal hematoma.

1	Following vaginal delivery in an unscarred uterus, we and others have occasionally encountered cases of an incomplete tear on the inside of the uterus that extends vertically into the active segment and is a source of profuse hemorrhage (Conrad, 2015). These tears are usually not visible from below but are found at the time of hysterectomy for intractable bleeding despite a contracted uterus. Hemorrhage with this type of tear can be torrential, and bleeding is usually not slowed until the uterine artery pedicles are clamped bilaterally.

1	The varied clinical presentations of uterine rupture and its management are discussed in detail in Chapter 31 (p. 598). In the most recent maternal mortality statistics from the Centers for Disease Control and Prevention, uterine rupture accounted for almost 10 percent of deaths caused by hemorrhage (Creanga, 2015, 2017). Maternal morbidity includes hysterectomy that may be necessary to control hemorrhage. Rates of perinatal mortality and morbidity, which may include severe neurological impairment, are also high (Gibbins, 2015; Porreco, 2009). Maternal obesity comorbid with uterine rupture is associated with increased rates of adverse neonatal outcomes (Yao, 2017).

1	Maternal obesity comorbid with uterine rupture is associated with increased rates of adverse neonatal outcomes (Yao, 2017). Separation of the placenta-either partially or totally-from its implantation site before delivery is described by the Latin term abruptio placentae. Literally translated, this refers to "rending asunder of the placenta," which denotes a sudden accident that is a clinical characteristic of most cases. In the purest sense, the cumbersome-and thus seldom used-term premature separation of the normaly implanted placenta is most descriptive because it excludes separation of a placenta previa.

1	Placental abruption is initiated by hemorrhage into the decidua basalis. he decidua then splits, leaving a thin layer adhered to the myometrium. Consequently, the process begins as a decidual hematoma and expands to cause separation and compression of the adjacent placenta. Inciting causes of many cases have been posited. he phenomenon of impaired tropho blastic invasion with subsequent atherosis is related in some cases of preeclampsia complicated by abruption (Brosens, 2011). Inflammation or infection may be contributory (Mhatre, 2016; Nath, 2007). Histological findings cannot be used to determine timing of the abruption (Chen, 2017). Abruption likely begins with rupture of a decidual spi ral artery and then an expanding retroplacental hematoma.

1	Abruption likely begins with rupture of a decidual spi ral artery and then an expanding retroplacental hematoma. In the early stages of placental abruption, clinical symptoms may be absent. Even with continued bleeding and placen tal separation, placental abruption can still be either total or partial (Fig. 41-14). With either, bleeding typically insinuates itself between the membranes and uterus, ultimately escaping through the cervix to cause external hemorrhage. Less often, the blood is retained between the detached placenta and the uterus, leading to concealed hemorrhage and delayed diagnosis. The delay translates into greater maternal and fetal hazards. Also with concealed hemorrhage, the likelihood of consumptive coagulopathy is elevated. This is because increased pressure within the intervillous space, caused by the expanding retroplacental clot, forces more placental thromboplastin into the maternal circulation (p. 784).

1	Nfost blood in the retroplacental hematoma in a nontraumatic placental abruption is maternal. his is because hemorrhage derives from separation within the maternal decidua, and placental villi are usually initially intact. In 78 women at Parkland Hospital with a nontraumatic placental abruption, fetal-tomaternal hemorrhage was documented in only 20 percent-and all of these had < 10 mL fetal blood loss (Stettler, 1992). Atkinson and colleagues (2015) identiied fetal cells in peripheral blood in only 4 percent of 68 women with a placental abruption.

1	When clinically suspected, an abruption is seen on a freshly delivered placenta as a circumscribed depression on the maternal surface. hese usually measure a few centimeters in diameter and are covered by dark, clotted blood. Because several minutes are required for these anatomical changes to materialize, a very recently separated placenta may appear totally normal at delivery. Our experiences are like those of fetal complications such as nonreassuring fetal status, growth restriction, or death; or neonatal outcomes that include death, preterm delivery, or growth restriction.

1	External trauma-usually from motor vehicle accidents or aggravated assault-can cause placental separation. he frequency of abruption originating from trauma varies. Kettel (1988) and Staford (1988) and their associates have appropriately stressed that abruption can stem from relatively minor trauma. he clinical presentation and consequences of these abruptions difer somewhat from spontaneous cases. For example, associated fetomaternal hemorrhage, while seldom clinically signiicant with most spontaneous abruptions, is more common with trauma because of concomitant placental tears or "fractures" (Chap. 47, p. 928). Fetal abruption with concealed hemorrhage. To the right is a partial abruption with blood a third of women with a traumatic abrup and clots dissecting between membranes and decidua to the internal cervical os and tion reported by Pearlman (1990). In eight

1	FIGURE 41-14 Schematic of placental abruption. Shown to left is a total placental bleeding that averaged 12 mL was noted in then externally into the vagina. Benirschke and associates (2012) in that the "age" of the retroplacental clot cannot be determined exactly. In the example shown in Figure 41-15, a large dark clot is well formed, it has depressed the placental bulk, and it likely is at least several hours old. Deining severity of placental abruption is problematic. We have considered abruption severe when the fetus dies, however, maternal and fetal complications can be serious even with a liveborn fetus. Ananth and coworkers (2016) have deined severe abruption as displaying one or more of the following: (1) maternal sequelae that include disseminated intravascular coagulation, shock, transfusion, hysterectomy, renal failure, or death; women cared for at Parkland Hospital, we FIGURE 41-15 Partial placental abruption with a dark adherent clot.

1	FIGURE 41-15 Partial placental abruption with a dark adherent clot. found fetal-to-maternal hemorrhage of 80 to 100 mL in three of eight cases of traumatic placental abruption (Stettler, 1992). Importantly, in some cases of trauma, a nonreassuring fetal heart rate tracing may not be accompanied by other evidence of placental separation. A sinusoidal tracing is one example. Traumatic abruption is considered in more detail in Chapter 47 (p. 928).

1	Some cases of chronic placental separation begin early in pregnancy. Dugof and coworkers (2004) observed an association between some abnormally elevated maternal serum aneuploidy markers and subsequent abruption. Other have correlated irstand second-trimester bleeding with third-trimester placental abruption (Ananth, 2006; Weiss, 2004). In some cases of a chronic abruption, subsequent oligohydramnios developschronic abruption-oligohydramnios sequence-CAGS (Elliott, 1998). Even later in pregnancy, hemorrhage with retroplacental hematoma formation is occasionally arrested completely without delivery. hese women may have abnormally elevated serum levels of alpha-fetoprotein or placenta-specific RNAs as markers of the event (Miura, 2016; Ngai, 2012) .

1	he reported incidence of placental abruption varies because of diferent criteria used for diagnosis. hat said, its frequency averages 0.5 percent or 1 in 200 deliveries. From one database of almost 28 million births from 2006 through 2012, the incidence of placental abruption was nearly 1 percent (Ananth, 2016). From a cohort of more than 1.57 million births in the Netherlands, Ruiter and coworkers (2015) found the frequency was 0.22 percent-1 in 450. In more than 250,000 0.....) 5. FIGURE 41-16 Frequency of placental abruption and placenta previa by maternal age at Parkland Hospital from 2000 through 2015. deliveries at Parkland Hospital from 2000 through 2015, the incidence of placental abruption averaged 0.35 percent or 1 in 290 (Fig. 41-16).

1	deliveries at Parkland Hospital from 2000 through 2015, the incidence of placental abruption averaged 0.35 percent or 1 in 290 (Fig. 41-16). he frequency ofplacental abruption has risen in this country, and most ofthis increase is in blackwomen (Ananth, 2005, 2016). At Parkland Hospital, however, the frequency ofsevere abruption has declined. This discrepancy may be explained in part by the variations in management ofearly-onset preeclampsia (Chap. 40, p. 728). Specifically, with placental abruption so extensive as to kill theetus, the incidence was 0.24 percent or 1 in 420 births from 1956 through 1967 (Pritchard, 1967). As the number of high-parity women giving birth declined along with improved availability of prenatal care and emergency transportation, the frequency of abruption causing fetal death dropped to 0.12 percent through 1989 in our obstetrical population. And, most recently through 2015, it declined to 0.05 percent or 1 in 2060.

1	Overall, perinatal outcomes are influenced by gestational age, and the frequency of placental abruption rises across the third trimester. As seen in Figure 41-17, more than half of the placental abruptions at Parkland Hospital developed at gestational ages ::37 weeks. Perinatal mortality and morbidity, however, are more common with earlier abruptions (Furukawa, 2015a). Ofother related factors, major fetal congenital anomalies have greater association with placental abruption (Riihimaki, 20l3).

1	Although the rates of fetal death have declined, the contribution of abruption as a cause of stillbirth remains prominent because other causes have also decreased. For example, since the early 1990s, 10 to 12 percent of all third-trimester stillbirths at Parkland Hospital have been the consequence of placental abruption. Others have documented high perinatal mortality rates caused by placental abruption. Salihu and colleagues (2005) analyzed more than 15 million singleton births between 1995 and 1998. The perinatal mortality rate associated with placental abruption was 11r9 per 1000 births compared with 8 per 1000 for the general obstetrical population. 22.7 FIGURE 41-17 Frequency of placental abruption by gestational age at Parkland Hospital. Neonatal deaths are common following placental abruption.

1	22.7 FIGURE 41-17 Frequency of placental abruption by gestational age at Parkland Hospital. Neonatal deaths are common following placental abruption. At Parkland Hospital, 15 percent ofliveborn neonates died. Perinatal morbidity-oten severe-is also common in surviving newborns (Abdella, 1984). Studies by Matsuda and coworkers (2003, 2013) reported that 20 percent of survivors developed cerebrl palsy. These observations are similar to ours from Parkland Hospital. Notably, 20 percent ofliveborn neonates ofwomen with an abruption had severe acidemia, deined by a cord arterial blood pH <7.0 or base deficit of::12 mmollL. One review conirmed the associated risk for cerebral palsy (Downes, 2017). Even so, nanth and coworkers (2017) attribute adverse neurodevelopmental outcomes to be largely attributable to preterm delivery.

1	Several predisposing factors raise the risk for placental abruption, and some are listed in Table 41-4.Advancing matenalage is one, although data are conlicting regarding women of reat pariy TABLE 41 -4. Risk Factors for Placental Abruption Increased age and parity 1.3-2.3 Preeclampsia 2.1-4.0 Chronic hypertension 1n.8-3.0 Chorioamnionitis 3.0 Preterm ruptured membranes 2.4-4.9 Low birthweight 14.0 Cigarette smoking 1.4-1.9 Single umbilical artery 3.4 NA = not available. Data from Ananth/ 1999a/b/ 2004/ 2007; Aviram/ 2015; Gutvirtz/ 2016; Morgan/ 2016; Nath/ 2007/ 2008; Ruiter/ 2015.

1	Cigarette smoking 1.4-1.9 Single umbilical artery 3.4 NA = not available. Data from Ananth/ 1999a/b/ 2004/ 2007; Aviram/ 2015; Gutvirtz/ 2016; Morgan/ 2016; Nath/ 2007/ 2008; Ruiter/ 2015. (Okby, 2017; Pritchard, 1991). ace or ethnicity also appears to be important. In almost 366,000 deliveries at Parkland Hospital, abruption severe enough to kill the fetus was most common in black and white women-1 in 200, less so in Asian women-1 in 300, and least common in Latin-American women-1 in 350 (Pritchard, 1991). Aomilial association was found in an analysis of a Norwegian population-based registry (Rasmussen, 2009). If a woman had a severe abruption, the risk for her sister was doubled.

1	Some form of hypertension is the most frequent condition associated with placental abruption. his includes gestational hypertension, preeclampsia, chronic hypertension, or a combination thereof. In a report by Pritchard and colleagues (1991) that described 408 women with placental abruption and fetal demise, hypertension was apparent in half once hypovolemia was corrected. Half of these latter women-a fourth of all 408-had chronic hypertension. Looked at another way, one Maternal-Fetal Medicine Units (MFMU) Network study found that 1.5 percent of pregnant women with chronic hypertension sufered placental abruption (Sibai, 1998). As discussed in Chapter 50 (p. 978), at Parkland Hospital, the frequency of placental abruption in treated chronically hypertensive women was almost 1 percent, which was threefold higher than the 0.3-percent baseline C..10rgan, 2016).

1	Chronic hypertension with superimposed preeclampsia or with fetal-growth restriction confers an even greater risk (Ananth, 2007). Even so, the severity of hypertension does not necessarily correlate with abruption incidence (Morgan, 2016; Zetterstrom, 2005). The long-term efects of these associations are apparent from the signiicantly elevated cardiovascular mortality risk in women with prior abruption, with or without chronic hypertension (DeRoo, 2016; Pariente, 2013). Observations from the Magpie Trial Collaborative Group suggest that women with preeclampsia, with or without chronic hypertension, given magnesium sulfate may have a reduced risk for abruption (Altman, 2002).

1	he abruption risk substantially rises when placental membranes rupture before term (American College of Obstetricians and Gynecologists, 2016a; Hackney, 2016). Major and colleagues (1995) reported that 5 percent of 756 women with ruptured membranes between 20 and 36 weeks' gestation developed an abruption. It was 17 percent with previable prematurely ruptured membranes (Kibel, 2016). he risk for abruption with preterm rupture is further increased with comorbid infection (Ananth, 2004). In these cases, inlammation and infection as well as preterm delivery may be primary causes leading to abruption (Nath, 2007, 2008). Somewhat related, Aviram and coworkers (2015) found an eightfold higher abruption risk in pregnancies �34 weeks if hydramnios was comorbid. Abrupt uterine decompression during membrane rupture may be an inciting factor.

1	Many of the predisposing factors are chronic, and in these cases, placental abruption has a high recurrence rate. Pritchard and associates (1970) identiied a recurrence rate of 12 percentand half of these caused another fetal death. Furuhashi and colleagues (2002) reported a 22-percent recurrence rate-half recurred at a gestational age 1 to 3 weeks earlier than the first abruption. In the Dutch study mentioned previously, Ruiter and coworkers (2015) cited a recurrence risk of 5.8 percent. Looked at a second way, Tikkanen and associates (2006) found that of 114 parous women who experienced an abruption, 9 percent had a prior abruption. A third perspective is provided by a population-based study of 767,000 pregnancies reported by Rasmussen and Irgens (2009). They found a 6.5-fold higher risk for recurrence of a "mild" abruption and 11.5-fold risk for a "severe" abruption. For women who had two severe abruptions, the risk for a third was increased 50-fold.

1	Management of a pregnancy subsequent to an abruption is diicult because another separation may suddenly occur, even remote from term. In many of these recurrences, fetal wellbeing is almost always reassuring beforehand. hus, antepartum fetal testing is usually not predictive. Because term abruptions tend to be recurrent, Ruiter and coworkers (2015) recommend labor induction at 37 weeks. Our practice at Parkland Hospital is to induce labor at 38 weeks if other complications do not develop beforehand. Cigarette smoking is linked to an elevated risk for abruption (vIisra, 1999; Naeye, 1980). Results of a metaanalysis of 1.6 million pregnancies included a twofold risk for abruption in smokers (Ananth, 1999b). This risk was five-to eightfold if smokers had chronic hypertension, severe preeclampsia, or both. Similar findings are reported by others (Hogberg, 2007; Kaminsky, 2007). Antepartum Vitamin C and E were reported to be protective for abruption in smokers (Abramovici, 2015).

1	Cocaine abuse is linked with an alarming frequency of placental abruption (Addis, 2001; Cressman, 2014). Bingol and colleagues (1987) described 50 women who abused cocaine during pregnancy-eight had a stillbirth caused by placental abruption. Uterine leiomyomas, especially if located near the mucosal surface behind the placental implantation site, can predispose to placental abruption. This was reviewed recently by Ezzedine and Norwitz (2016). Isolated single umbiical artey is associated with a 3.4-fold increased risk for placental abruption (Gutvirtz, 2016). Twins resulting from infertility treatments also carry greater risk (Okby, 2017). Subclinical hypothyroidism or high levels of antithyroid antibodies have been associated with a two-to threefold higher risk for abruption (Abbassi-Ghanavati, 2010; Casey, 2014; Maraka, 2016).

1	Women afected by some of the thrombophilias have higher associated rates of thromboembolic disorders during pregnancy. However, the link with placental abruption is less clear (American College of Obstetricians and Gynecologists, 2017 a,b). Lupus anticoagulant is associated with maternal floor infarction of the placenta but is less so with typical abruptions. No convincing evidence supports a role for thrombophilias and placental abruption. Most women with a placental abruption have sudden-onset abdominal pain, vaginal bleeding, and uterine tenderness. In a prospective study, Hurd and colleagues (1983) reported that 78 percent with placental abruption had vaginal bleeding, 66 percent had uterine tenderness or back pain, and 60 percent had a nonreassuring fetal status. Other indings included fre quent contractions and persistent hypertonus. In a ifth of these women, preterm labor was diagnosed, and abruption was not suspected until fetal distress or death followed.

1	Importantly, the signs and symptoms of placental abruption can vary considerably. In some women, external bleeding can be profuse, yet placental separation may not be so extensive as to compromise the fetus. In others, there may be no external bleeding, but the placenta is suiciently sheared of that the fetus is dead-a concealed abruption. In one unusual case, a multiparous woman cared for at Parkland Hospital presented with a nosebleed. She had no abdominal or uterine pain, tenderness, or vaginal bleeding. Her fetus was dead, however, and her blood did not clot. he plasma ibrinogen level was 25 mg/dL. Labor was induced, and a total abruption was conirmed at delivery.

1	With severe placental abruption, the diagnosis generally is obvious. From the previous discussion, it follows that less severe, more common forms of abruption cannot always be recognized with certainty. hus, the diagnosis is one of exclusion. Unfortunately, no laboratory tests or other diagnostic methods accurately conirm lesser degrees of placental separation. Sonography has limited use because the placenta and fresh clots may have similar imaging characteristics. Glantz and Purnell (2002) reported only 24-percent sensitivity for sonography in 149 consecutive women with a suspected placental abruption. Important, negative indings with sonographic examination do not exclude placental abruption. Conversely, magnetic resonance (MR) imaging is highly sensitive for placental abruption and should be considered if the diagnostic information would change management (Masselli, 2011).

1	With abruption, some degree of intravascular coagulation is almost universal. hus, elevated serum levels of D-dimers may be suggestive, but this has not been adequately tested. Preliminary data show that serum alpha-fetoprotein levels > 280 �g/L have a positive-predictive value of 97 percent (Ngai, 2012).

1	hus, in the woman with vaginal bleeding and a live fetus, it is often necessary to exclude placenta previa and other causes of bleeding by clinical and sonographic evaluation. It has long been taught-perhaps with some justiication-that painful uterine bleeding signiies placental abruption, whereas painless uterine bleeding is indicative of placenta previa. he diferential diagnosis is usually not this straightforward, and labor accompanying previa may cause pain suggestive of placental abruption. On the other hand, pain from abruption may mimic normal labor, or it may be painless, especially with a posterior placenta. At times, the cause of the vaginal bleeding remains obscure even after delivery.

1	Placental abruption is one of several notable obstetrical entities that may be complicated by massive and sometimes torrential hemorrhage. Hypovolemic shock is caused by maternal blood loss. In an earlier report from Parkland Hospital, Pritchard and Brekken (1967) described 141 women with abruption so severe as to kill the fetus. Blood loss in these women often amounted to at least half of their pregnant blood volume. Importantly, massive blood loss and shock can develop with a concealed abruption. Prompt treatment of hypotension with crystalloid and blood infusion is essential, and resuscitation steps are described later (p. 788). onic fluid embolism-led to the initial recognition of di brination syndrome. his syndrome is currently referred to as lation, which later is described more broadly on page 782.

1	onic fluid embolism-led to the initial recognition of di brination syndrome. his syndrome is currently referred to as lation, which later is described more broadly on page 782. is intravascular activation of clotting. Abruption is the most common cause of clinically profound consumptive coagulop athy in obstetrics-and indeed, probably in all of medicine (Cunningham, 2015).

1	An important consequence of intravascular coagulation is the activation of plasminogen to plasmin, which lyses ibrin microemboli to maintain microcirculatory patency. With placental abruption severe enough to kill the fetus, there are always pathological levels of ibrinogen-ibrin degradation products and D-dimers in maternal serum (Erez, 2015). heir quantiication is not clinically useful. In a third of women with an abruption severe enough to kill the fetus, the plasma ibrinogen level will be < 150 mg/ dL. hese levels are dependent on the maternal preabruption ibrinogen level, and thus higher levels are "protective" (Cunningham, 2015; Wang, 2016). Clinically signiicant low levels may cause troublesome surgical bleeding. Levels of several other coagulation factors are also variably decreased. In addition, thrombocytopenia, sometimes profound, may accompany severe hypoibrinogenemia initially and becomes common after repeated blood transfusions.

1	Consumptive coagulopathy is more likely with a concealed abruption because intrauterine pressure is higher. his forces more thromboplastin into the large veins draining the implantation site. With a partial abruption and a live fetus, severe coagulation defects are less common. Our experience has been that if serious coagulopathy develops, it is usually evident by the time abruption symptoms appear. At the time of cesarean delivery, it is not uncommon to ind widespread extravasation of blood into the uterine musculature and beneath the serosa (Fig. 41-18). It is named after Couvelaire, who in the early 1900s termed it uteroplacental apoplexy. hese myometrial hemorrhages seldom cause uterine atony, and alone they are not an indication for hysterectomy. Efusions of blood are also seen beneath the tubal serosa, between the leaves of the broad ligaments, in the substance of the ovaries, and free in the peritoneal cavity.

1	FIGURE 41-18 Couvelaire uterus from total placental abruption ater cesarean delivery. Blood markedly infiltrates the myometrium to reach the serosa, especially at the cornua. The small serosaloleio myoma seen on the lower anterior uterine surface is an incidental finding. (Used with permission from Dr. Angela Fields Walker.)

1	Acute kidney injury AI) is a general term describing renal dysfunction from many causes (Chap. 53, p. 1036). Delayed or incomplete treatment of hypovolemia with severe placental abruption can be one. However, even with abruption complicated by severe disseminated intravascular coagulation, prompt and vigorous treatment of hemorrhage with blood and crystalloid solution usually prevents clinically signiicant renal dysfunction. The risk for renal injury with abruption is magnified when preeclampsia coexists (Alexander, 2015; Drakeley, 2002). Most cases of AKI are reversible and not so severe as to require dialysis. Generally, long-term outcomes are good (Arazi, 2015). That said, irreversible acute cortical necrosis encountered in pregnancy can be associated with abruption (Gopalakrishnan, 2015).

1	Rarely, pituitary failure-Sheehan syndrome-follows severe intrapartum or early postpartum hemorrhage. Described in Chapter 58 (1133), the exact pathogenesis is not well understood, especially because endocrine abnormalities are infrequent even in women who sufer catastrophic hemorrhage (Matsuwaki, 2014; Robalo, 2012).

1	Treatment of the woman with a placental abruption varies depending on her clinical condition, gestational age, and the amount of associated hemorrhage. With a living viable-aged fetus, and with vaginal delivery not imminent, emergency cesarean delivery is chosen by most. In some women, fetal compromise will be evident as shown in Figure 41-19. When evaluating fetal status, sonographic conirmation of fetal heart activity may be necessary because sometimes an electrode applied directly to a dead fetus will provide misleading information by recording the maternal heart rate. If the fetus has died or if it is not considered suiciently mature to live outside the uterus, then FIGURE 41-19 Placental abruption with fetal compromise. Lower panel: Uterine hypertonus with a baseline pressure of 20 to 25 mm Hg and frequent contractions peaking at approximately 75 mm Hg. Upper panel: The fetal heart rate demonstrates baseline bradycardia with repetitive late decelerations.

1	vaginal delivery is preferable. In either case, prompt and intensive resuscitation with blood plus crystalloid is begun to replace blood lost from retroplacental and external hemorrhage. hese measures are lifesaving for the mother and hopefully for her fetus. If the diagnosis of abruption is uncertain and the fetus is alive and without evidence of compromise, then close observation may be warranted provided that immediate intervention is available. Colon and coworkers (2016) performed a randomized trial and found no beneits to magnesium sulfate tocolysis given to women with a preterm "nonsevere" abruption at 24 to 34 weeks' gestation.

1	The compromised fetus is usually best served by cesarean delivery, and the speed of response is an important factor in perinatal outcomes. Kayani and coworkers (2003) studied this relationship in 33 singleton pregnancies with a clinically overt placental abruption and fetal bradycardia. Of the 22 neurologically intact survivors, 15 were delivered within a 20-minute decision-to-delivery interval. However, eight of 11 infants who died or developed cerebral palsy were delivered with intervals > 20 minutes. A major hazard to cesarean delivery is imposed by clinically signiicant consumptive coagulopathy. Preparations include plans for blood and component replacement and assessment of coagulation-especially fibrinogen levels.

1	If the fetus has died, then vaginal delivery is usually preferred. As reviewed earlier, hemostasis at the placental implantation site depends primarily on myometrial contraction and not blood coagulability. Thus, after vaginal delivery, uterotonic agents and uterine massage are used to stimulate myometrial contractions. Uterine muscle ibers compress placental site vessels and prompt hemostasis even if coagulation is defective. In some instances, vaginal delivery may not be preferable, even with a dead fetus. One example is brisk hemorrhage that cannot be successfully managed by vigorous blood replacement. Others are the myriad obstetrical complications that prohibit vaginal delivery in general. hese are listed in Table 30-1 (p. 568).

1	In some women with extensive placental abruption, labor tends to be rapid because the uterus is usually persistently hypertonic. This can magniy fetal compromise. In some cases, baseline intraamnionic pressures reach 50 mm Hg or higher, and with contractions, pressures may attain levels exceeding 100 mm Hg. Overall, however, irst-and second-stage labor do not appear to be shortened (Downes, 2016).

1	Early amniotomy has long been championed in the manage ment of placental abruption. his ostensibly achieves better spi ral artery compression to diminish implantation site bleeding and reduce thromboplastin infusion into the maternal vascular system. Although evidence supporting this theY is lacking, membrane ruprure may hasten delivery. However, if the fetus is small, the intact sac may be more eicient in promoting cervical dilation. If rhythmic uterine contractions are not superimposed on baseline hypertonus, then oxytocin is given in standard doses. No data indicate that oxytocin augments thromboplas tin escape into the maternal circulation to worsen coagulopathy (Clark, 1995; Pritchard, 1967). In light of hypertonus associ ated with abruption, misoprostol may be a less favored induc tion agent due to its association with uterine tachysystole.

1	In the past, some had set arbitrary time limits to permit vaginal delivery. Instead, experiences illustrate that maternal outcome depends on the diligence with which adequate luid and blood replacement therapy are pursued rather than on the interval to delivery. Observations from Parkland Hospital described by Pritchard and Brekken (1967) are similar to those from the University of Virginia reported by Brame and associates (1968). Specifically, women with severe abruption who were transfused during 18 hours or more before delivery had similar outcomes to those in whom delivery was accomplished sooner. Expectant Management with a Preterm Fetus

1	Expectant Management with a Preterm Fetus If possible, delaying delivery may benefit an immature fetus. Bond and colleagues (1989) expectantly managed 43 women with placental abruption before 35 weeks' gestation, and 31 of them were given tocolytic therapy. he mean interval-todelivery for all 43 was approximately 12 days. Cesarean delivery was performed in 75 percent, and there were no stillbirths. As discussed earlier, women with a very early abruption may develop chronic abruption-oligohydramnios sequence. In one report, Elliott and coworkers (1998) described four women with an abruption at a mean gestational age of 20 weeks who developed oligohydramnios and delivered at an average gestational age of 28 weeks. In a description of 256 women with an abruption at <28 weeks' gestation, Sabourin and colleagues (2012) reported that a mean of 1.6 weeks was gained. Of the group, 65 percent were delivered <29 weeks, and half of all women underwent emergent cesarean delivery.

1	Unfortunately, even continuous fetal heart rate monitoring does not guarantee universally good outcomes. For example, a normal tracing may precede sudden further separation with instant fetal compromise. In some of these, if the separation is suicient, the fetus will die before it can be delivered. Tocolysis is advocated by some for suspected abruption if the fetus does not display compromise. Some investigators have observed that tocolysis improved outcomes in a highly selected cohort of women with preterm pregnancies (Bond, 1989; Combs, 1992; Sholl, 1987). In another study, Towers and coworkers (1999) administered magnesium sulfate, terbutaline, or both to 95 of 131 women with abruption diagnosed before 36 weeks. The perinatal mortality rate was 5 percent in both groups with or without tocolysis. Similar results were reported from a randomized trial (Colon, 2016). We are of the opinion that suspected placental abruption contraindicates use of tocolytic agents.

1	The Latin previa means going bore-and in this sense, the placenta goes before the fetus into the birth canal. In obstetrics, placenta previa describes a placenta that is implanted somewhere in the lower uterine segment, either over or very near the internal cervical os. Because these anatomical relationships cannot always be precisely deined, and because they frequently change across pregnancy, terminology can sometimes be confusing. Beginning with the use of sonography in obstetrics, the term placental migration was coined to describe the apparent movement of the placenta away from the internal os (King, 1973). Obviously, the placenta does not move per se, and the mechanism of apparent movement is not completely understood. To begin with, migration is clearly a misnomer, because decidual invasion anchors chorionic villi at the cervical os. Explanations of placental migration are likely additive.

1	Explanations of placental migration are likely additive. First, apparent movement of the low-lying placenta relative to the internal os is related to the imprecision of two-dimensional sonography. Second, as pregnancy progresses, growth of the lower and upper uterine segments difers. With greater blood low in the upper uterus, placental growth is more likely directed toward the fundus-trophotropism. Many of those placentas that "migrate" most likely never were circumferentially implanted with true villous invasion that reached the internal cervical os. Importanty, a low-ying placenta or placenta previa is less likey to "migrate JJ if there is a prior cesarean delivey scar.

1	he frequency of placental migration has been quantiied. Sanderson and Milton (1991) studied 4300 women at midpregnancy and found that 12 percent had a low-lying placenta. Of placentas not covering the internal os, previa did not persist, and none subsequently had placental hemorrhage. Conversely, approximately 40 percent of placentas that covered the os at midpregnancy continued to do so until delivery. Thus, placentas that lie close to but not over the internal os up to the early third trimester are unlikely to persist as a previa by term (Heller, 2014; Parrott, 2015). However, other evidence from Bohrer and associates (2012) showed that a second-trimester low-lying placenta was associated with antepartum admission for hemorrhage and increased blood loss at delivery.

1	The likelihood that placenta previa persists after being identiied sonographically at given epochs before 28 weeks' gestation is shown in Figure 41-20. For twin pregnancies, similar findings are reported until 23 weeks, after which the previa persistence rate is much higher (Kohari, 2012). Staford and coworkers FIGURE 41 -21 Placenta previa showing that copious hemorrhage could be anticipated with any cervical dilatation. • Previa, prior CD ) • Previa, no prior CD � • 1-5 mm,prior CD )S 75 • 1-5 mm,no prior CD

1	• Previa, prior CD ) • Previa, no prior CD � • 1-5 mm,prior CD )S 75 • 1-5 mm,no prior CD FIGURE 41-20 Likelihood of persistence of placenta previa or low-lying placenta 1 to 5 mm from the internal os at delivery. These are shown as a function of sonographic diagnosis at three pregnancy epochs. CD = cesarean delivery. (Data from Oyelese, 2006.) (2010), but not Trudell and colleagues (2013), found that a previa and a third-trimester cervical length <30 mm elevated the risks for hemorrhage, uterine activity, and preterm birth. Friszer and associates (2013) showed that women admitted for bleeding had a greater chance of delivery in the subsequent 7 days when the cervical length was <25 mm, although Trudell (2013) again did not confirm this.

1	Terminology for placenta previa has evolved, and from a Fetal Imaging Workshop sponsored by the National Institutes of Health (NIH), the following classiication was recommended: • Placenta previa-the internal os is covered partially or completely by placenta (Figs. 41-21 and 41-22). In the past, these were further classiied as either total or partial previa. FIGURE 41-22 On speculum examination, placenta is visible protruding through the cervical os. (Used with permission from Dr. Maureen E. Flowers.) • Low-ying placenta-implantation in the lower uterine segment is such that the placental edge does not cover the internal os but lies within a 2-cm wide perimeter around the os. A previously used term, marginal previa, described a placenta that was at the edge of the internal os but did not overlie it (Reddy, 2014).

1	Clearly, the classification of some cases of previa will depend on cervical dilation at the time of assessment (Dashe, 2013; Reddy, 2014). For example, a low-lying placenta at 2-cm dilation may become a partial placenta previa at 4-cm dilation because the cervix has opened to expose the placental edge. Conversely, a placenta previa that appears to be total before cervical dilation may become partial at 4-cm dilation because the cervical opening now extends beyond the edge of the placenta. Digital papation in an attempt to ascertain these changing relations between the placental edge and intenal os as the cervix dilates usualy causes severe hemorrhage! With any degree of placenta previa, a certain amount of spontaneous placental separation is inevitable during lower uterine segment remodeling and cervical dilation. Although this frequently causes bleeding, and thus technically constitutes a placental abruption, this term is usually not applied in these instances.

1	Somewhat but not always related is vasa previa, in which fetal vessels course through membranes and present at the cervical os (Catanzarite, 2016). Vasa previa was recently reviewed by the Society for Maternal-Fetal Medicine (2015) and is discussed in Chapter 6 (p. 118). he incidence of placenta previa has risen during past 30 years. Reported incidences average 0.3 percent or 1 case per 300 to 400 deliveries. he frequency at Parkland Hospital from 1988 through 2003 for nearly 250,000 births was 2.6 per 1000. For the 2004 to 2015 epoch, it rose to 3.8 per 1000. Similar frequencies have been reported from Austria, Finland, and Israel (Kollmann, 2016; Raisanen, 2014; Rosenberg, 2011). Several demographic factors may contribute to this higher risk for placenta previa. First, maternal age raises the frequency of placenta previa (Biro, 2012; Roberts, 2012). In the First-and

1	Several demographic factors may contribute to this higher risk for placenta previa. First, maternal age raises the frequency of placenta previa (Biro, 2012; Roberts, 2012). In the First-and Second-Trimester E val uation of Risk (FASTER) trial, which included more than 36,000 women, the frequency of previa was 0.5 percent for women <35 years compared with 1.1 percent in those :35 years (Cleary-Goldman, 2005). At Parkland Hospital, this incidence difered from a low rate of approximately 0.65 per 1000 births for women ; 19 years to almost 10 per 1000 births for women older than 35 (see Fig. 41-16). Multpariy also elevates the risk for previa (Raisanen, 2014). Obviously, the efects of advancing maternal age and parity are confounding. Still, Babinszki and colleagues (1999) reported that the 2.2-percent incidence in women with parity of five or greater was significantly higher than that of women with lower parity. he interpregnancy interval does not afect this rate (Fox, 2015).

1	he interpregnancy interval does not afect this rate (Fox, 2015). Cigarette smoking increases the relative risk of placenta previa at least twofold (Usta, 2005). It has been postulated that carbon monoxide hypoxemia causes compensatory placental hypertrophy and more surface area. Smoking may also be related to decidual vasculopathy. Last, uterine leiomyomas are a risk factor for previa Oenabi, 2017).

1	Several clinical characteristics also raise previa risks. Foremost, women with one or more prior cesarean deliveries are at greater risk for subsequent placental disorders that include placenta previa, abruption, or morbidly adherent placenta (Gibbins, 2018; Klar, 2014). The cumulative risks for placenta previa that accrue with the increasing number of cesarean deliveries are extraordinary. The risk rises even further if there was a prior prelabor cesarean delivery (Downes, 2015). In one MFMU Network study of 30,132 women undergoing cesarean delivery, the incidence was 1.3 percent for those with only one prior cesarean delivery, but it was 3.4 percent if there were six or more prior cesareans (Silver, 2006). In a retrospective cohort of nearly 400,000 women who were delivered of two consecutive singletons, those with a cesarean delivery for the first pregnancy had a 1.6-fold greater risk for previa in the second pregnancy (Gurol-Urganci, 2011). hese same investigators reported a

1	consecutive singletons, those with a cesarean delivery for the first pregnancy had a 1.6-fold greater risk for previa in the second pregnancy (Gurol-Urganci, 2011). hese same investigators reported a 1.5-fold higher risk from six similar population-based cohort studies. The likelihood of previa is increased more than eightfold in women with parity greater than four and who have more than four prior cesarean deliveries (Gesteland, 2004; Gilliam, 2002).

1	Importantly, women with a prior uterine incision and placenta previa have an elevated likelihood that cesarean hysterectomy will be necessary because of an associated morbidly adherent placenta (Wei, 2014). In one study, 6 percent of women with a primary cesarean delivery for previa required a hysterectomy. his rate was 25 percent for women with a previa undergoing repeat cesarean delivery (Frederiksen, 1999). Maternal serum aphaetoprotein (MSAFP) levels, if abnormally elevated for otherwise unexplained reasons during prenatal screening, raise the risk for previa and a host of other abnormalities. Moreover, women with a previa and comorbid MSAFP level :2.0 multiples of the median (MoM) at 16 weeks' preterm birth (Chap. 14, p. 283).

1	Last, assisted reproductive technoloy AR) used for concep tion elevates previa risks. Some of this association may derive from overlapping efects. For example, older women comprise a signiicant portion of ART patients (Luke, 2017). In addition, multifetal gestation is a well-known risk of both in vitro fertil ization and previa. However, even adjusting for these overlap ping elements, ART is still associated with higher previa rates (Romundstad, 2006).

1	Painless bleeding is the most characteristic event with placenta previa. Bleeding usually does not develop until near the end of the second trimester or later, but it can begin even before mid pregnancy. And undoubtedly, some late abortions are caused by an abnormally located placenta. Bleeding from a previa usu in a woman who has had an uneventful prenatal course. his so-called sentinel bleed is rarely so profuse as to prove fatal. Usu ally it ceases, only to recur. However, in perhaps 10 percent of women, particularly those with a placenta implanted near but not over the cervical os, there is no bleeding until labor onset. Bleeding at this time varies from slight to profuse, and it may clinically mimic placental abruption.

1	Bleeding at this time varies from slight to profuse, and it may clinically mimic placental abruption. A speciic sequence of events leads to bleeding in cases in which the placenta is located over the internal os. First, the uterine body remodels to form the lower uterine segment. With this, the internal os dilates, and some of the implanted placenta inevitably separates. Bleeding that ensues is augmented by the inherent inability of myometrial fibers in the lower uterine segment to contract and thereby constrict torn vessels. Similarly, bleeding from this lower segment implantation site also frequently continues ater placentl delivery. Last, there may be lacerations in the friable cervix and lower segment. These may be especially problematic following manual removal of a somewhat adhered placenta.

1	Morbidy adherent placentas are a frequent and serious complication associated with placenta previa. Described later (p. 777), this abnormally firm placental attachment derives in part from poorly developed decidua that lines the lower uterine segment. Biswas and coworkers (1999) performed placental bed biopsies in 50 women with a previa and in 50 control women. Trophoblastic giant-cell iniltration of spiral arterioles-rather than endovascular trophoblast cells-was found in half of previa specimens. In contrast, only 20 percent of biopsies from normally implanted placentas had these changes. In another study of 514 cases of previa, abnormal placental attachment was identiied in 7 percent (Frederiksen, 1999). As discussed, previa overlying a prior cesarean incision conveys a particularly high risk for morbidly adherent placenta.

1	Coagulation dects are rare complications of placenta previa, even when implantation site separation is extensive (Cunningham, 2015). Placental thromboplastin, which incites the intravascular coagulation seen with placental abruption, is presumed to readily escape through the cervical canal rather than be forced into the maternal circulation. he paucity of large myometrial veins in this area may also be protective.

1	Whenever there is uterine bleeding after midpregnancy, placenta previa or abruption are always considered. In the Canadian Perinatal Network study discussed earlier (p. 758), placenta previa accounted for 21 percent of women admitted from 22 to 28 weeks' gestation with vaginal bleeding (Sabourin, 2012). Previa should not be excluded until sonographic evaluation has clearly proved its absence. If sonography is not readily available, diagnosis by clinical examination is done using the double set-up technique because it requires that a inger be passed through the cervix and the placenta palpated. A digital examination should not be performed unless delivery is planned. A cervical digital examination is done with the woman in an operating room and with preparations or immediate cesarean delivey. Even the gentlest examination can cause torrential hemorrhage. Fortunately, double set-up examination is rarely necessary because placental location can almost always be ascertained

1	cesarean delivey. Even the gentlest examination can cause torrential hemorrhage. Fortunately, double set-up examination is rarely necessary because placental location can almost always be ascertained sonographically.

1	Quick and accurate localization can be accomplished using standard sonographic techniques (American Institute of Ultrasound in Medicine, 2013). his is usually done with transabdominal sonography. If the placenta clearly overlies the cervix or if it lies away from the lower uterine segment, the examination has excellent sensitivity and negative-predictive value (Olive, 2006; Quant, 2014). Obese women may have limitations of visualization of the lower uterine segment. Also, a full bladder may artiicially elongate the cervix and compress the lower uterine segment to give the impression that the placenta overlies the cervix. If placental location remains in question, then transvaginal sonography is the most accurate method of assessment (Fig. 41-23). It is safe, even when there is bleeding.

1	Accuracy depends on the sonographic technique used. In a comprehensive study, the internal os was visualized in all cases with transvaginal sonography but in only 30 percent with transabdominal sonography (Farine, 1988). As discussed, according to the Fetal Imaging Workshop, if the placental edge is <2 cm from the internal os, but not covering it, the placenta is considered low lying (Reddy, 2014). In the absence of any other indication, sonography need not be frequently repeated simply to document placental position. At Parkland, women with a placenta previa identiied at 18 to 22 weeks' gestation with a prior cesarean delivery are evaluated again at 28 weeks and those without at 32 weeks. Restriction of activity is not necessary unless a previa persists beyond 28 weeks or if clinical findings such as bleeding or contractions develop before this time. At 32 weeks' gestation, if the placental edge is still <2 cm from the os, then transvaginal sonography is repeated at 36 weeks.

1	Using MR imaging, several investigators have reported excellent results in visualizing placental abnormalities. hat said, it is unlikely that this technique will replace sonography for routine evaluation anytime soon. However, MR imaging has proved useful for evaluation of morbidly adherent placenta (p. 780).

1	Women with a placenta previa are managed based on their individual clinical circumstances. Three prominent factors include fetal age and maturity, labor, and bleeding severity. In one study of 214 women with a previa, 43 percent had an emergency delivery, and half of these were preterm (Ruiter, 2015). But, if the fetus is immature and active bleeding subsides, close observation in an obstetrical unit is indicated. Data are sparse regarding tocolytic administration for uterine contractions. Although robust randomized trials are lacking, Bose and colleagues (201r1) recommend that if tocolytics are given, they be limited to 48 hours of administration. We categorically recommend against their use in this setting.

1	Ater bleeding has ceased for approximately 2 days and the fetus is judged to be healthy, a woman can usually be discharged home with instructions for "pelvic rest." Importantly, the woman and her family must ully appreciate the possibility of recurrent bleeding and be prepared for immediate transport back to the hospital. In other cases, prolonged hospitalization may be ideal.

1	FIGURE 41 -23 Placenta previa. A. In this transvaginal image at 34 weeks' gestation, the anterior placenta completely covers the internal cervical os outlined by arrows. B.This transvaginal image at 34 weeks' gestation depicts a posterior placenta (arrow) that just reaches the level of the internal cervical os. (Reproduced with permission from Cunningham FG: Placenta previa and morbidly adherent placenta. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd edition. New York, McGraw-Hili Education,o201o7b.) he frequency of emergency delivery in women with pla centa previa ranges from 25 to 40 percent (Gibbins, 2018;

1	Kassir, 2017). But, in properly selected patients, long-term inpatient care does not appear to add beneits compared with outpatient management (Neilson, 2003). In one random ized study of 53 women who had a bleeding previa at 24 to 36 weeks' gestation, maternal or fetal morbidity rates did not difer between management method (Wing, 1996). Of all study women, 60 percent had recurrent bleeding, and half eventually required expeditious cesarean delivery.

1	For women who are near term and who are not bleeding, plns re made for scheduled cesarean delivery. Timing balances fetal immaturity risks against antepartum hemorrhage. One NIH workshop suggested elective delivery at 36 to 37 completed weeks' gestation (Spong, 2011). he Society for Maternal-Fetal Medicine (2017) recommends delivery between 34 and 37 weeks. At Park land Hospital, we usually perform elective cesarean delivery at 38 weeks. With a suspected morbidly adherent placenta, delivery is recommended at 34 to 35 completed weeks by the NIH workshop (p. 781). Our practice is to schedule delivery at 36 completed weeks.

1	Practically all women with placenta previa undergo cesarean delivery. Many surgeons recommend a vertical laparotomy incision to provide rapid entry in cases with torrential bleeding or operating space if hysterectomy is required. As discussed, cesarean delivery is emergently performed in more than half because of hemorrhage, for which about a fourth require blood transfusion (Boyle, 2009; Sabourin, 2012). Although a low transverse hysterotomy is usually possible, this may cause fetal bleeding if the placenta is implanted anteriorly and the placenta is incised. In such cases, fetal delivery should be expeditious (Silver, 2015a). A vertical uterine incision may be preferable in some instances. In either case, even when the incision extends through the placenta, maternal or fetal outcomes are rarely compromised.

1	Following placental removal, the placenta site may bleed uncontrollably due to poorly contracted smooth muscle, which is characteristic of the lower uterine segment. If hemostasis at the placental implantation site cannot be obtained by adequate uterotonic administration and pressure, it can be oversewn with O-chromic sutures. Cho and associates (1991) described interrupted O-chromic sutures at I-em intervals to form a circle around the bleeding portion of the lower segment to control hemorrhage. Others have reported success with compression sutures that traversed and compressed the anterior and posterior uterine wall (Kayem, 2011; Penotti, 2012).

1	Of other methods, Bakri or Foley balloon tamponade used alone or coupled with compression sutures has been described (Albayrak, 2011; Diemert, 2012; Kumru, 2013). Law and coworkers (2010) successfully used a hemostatic gel. Other surgical options are bilateral uterine or internal iliac artery ligation, illustrated later (p. 792). Finally, pelvic artery embolization has also gained acceptance. If these more conservative methods fail and bleeding is brisk, hysterectomy is necessary. Placenta previa-especially with an abnormally adherent placenta-currently is the most frequent and other institutions Qakobsson, 2015; Wong, 2011). When there is no associated accrete syndrome, the reported incidence of hysterectomy is 2 percent (Gibbins, 2018).

1	hus, it is not possible to accurately estimate the efect on the hysterectomy rate from previa alone without considering the associated accrete syndromes. Again, or women whose pla centa previa is implanted anteriory at the site of a prior uterine incision, the likelihood of an associated morbidy adherent placenta and need or hysterectomy is increased. In a study of 318 peri partum hysterectomies performed in the United Kingdom, 40 percent were done for abnormal placentation (Knight, 2007). Similar results were reported for 211 hysterectomies from the Nordic Obstetric Surveillance Study Qakobsson, 2015). At Parkland Hospital, 44 percent of cesarean hysterectomies were placenta (Wortman, 2015). The technique for peripartum hys terectomy is described in Chapter 30 (p. 580). ute substantively to maternal morbidity and mortality rates. he maternal mortality ratio is increased approximately threefold for women with a placenta previa (Gibbins, 2018; Oyelese, 2006).

1	ute substantively to maternal morbidity and mortality rates. he maternal mortality ratio is increased approximately threefold for women with a placenta previa (Gibbins, 2018; Oyelese, 2006). In another report of 5367 maternal deaths in the United States from 2006 to 2013, placenta previa alone accounted for nearly 3 percent of deaths from hemorrhage (Creanga, 2015, 2017).

1	In another report of 5367 maternal deaths in the United States from 2006 to 2013, placenta previa alone accounted for nearly 3 percent of deaths from hemorrhage (Creanga, 2015, 2017). The report from the Consortium on Safe Labor emphasizes the ongoing perinatal morbidity with placenta previa (Lai, 2012). Preterm delivery continues to be a major cause of perinatal death (N0rgaard, 2012). In deliveries with placenta previa in the United States in 1997, the neonatal mortality rate was threefold higher than that in unafected pregnancies and stemmed primarily from preterm delivery (Salihu, 2003). Ananth and colleagues (2003) reported a comparably elevated risk of neonatal death even for fetuses who delivered at term. This is at least partially related to the fetal anomaly rate, which is two-to threefold higher in pregnancies with placenta previa (Crane, 1999).

1	The association of fetal-growth restriction with placenta previa is likely minimal ter controlling for gestational age. In a populationbased cohort of more than 500,000 singleton births, Ananth and associates (2001) found that most low-birthweight newborns associated with placenta previa resulted from preterm birth. Harper and coworkers (2010) reported similar indings from a cohort of nearly 58,000 women. In contrast, at least two studies reported a greater risk for fetal-growth restriction (iisanen, 2014; Weiner, 2016). The term morbidy adherent placenta describes aberrant placentation characterized by abnormally implanted, invasive, or adhered placenta. We also refer to these disorders collectively as accrete syndromes and use these terms interchangeably. Derivation of accrete comes from the Latin ac-+ crescere-to adhere or become attached to (Benirschke, 2012).

1	In the accrete syndromes, abnormal placental adherence to the myometrium stems in part from partial or total absence of the decidua basalis and imperfect development of the ibrinoid or Nitabuch layer, described in Chapter 5 (p. 86). If the decidual spongy layer is lacking either partially or totally, then the physiological line of cleavage is absent, and some or all cotyledons are densely anchored. Microscopically, placental villi attach to smooth muscle ibers rather than to decidual cells. This decidual deiciency then prevents normal placental separation after delivery. he surface area of the implantation site involved and the depth of trophoblastic tissue ingrowth are variable between women, but all afected placentas can potentially cause signiicant hemorrhage.

1	Substantiated data now suggest that accrete syndromes are not solely caused by this anatomical layer deficiency (Duzyj, 2017; T antbirojn, 2008). Indeed, the cytotrophoblasts may control decidual invasion through factors such as angiogenesis (Duzyj, 2015; Goh, 2016; Wehrum, 2011). Also, accrete syndrome tissue specimens show "hyperinvasiveness" (Pri-Paz, 2012) . Myometrial ibers attached to the basal plate in an antecedent pregnancy are predictive markers for a subsequent FIGURE 41-24 Morbidly adherent placentas: A. Placenta accreta. B. Placenta increta. C. Placenta percreta. placenta accreta (Linn, 2015; Nliller, 2016). his implies an antecedent "constitutional endometrial defect" in most cases. The greater risk conveyed by previous surgical uterine trauma may be partially explained by an enhanced vulnerability to trophoblast invasion (Garmi, 2012; Gill, 2015; Jauniaux, 2017).

1	his association with prior trauma is reinforced by the close relationship between cesarean-scar pregnancy (CSP) and later development of placenta accreta in the same pregnancy. Indeed, accruing evidence suggests that CSP and accrete syndromes lie on a spectrum and that CSP is a precursor, as both share the same histopathology (Happe, 2018; Timor-Tritsch, 2014). CSP frequency has been reported to approximate 1 in 2000 pregnancies (Berhie, 2015; Rotas, 2006). Described in Chapter 19 (p. 381), early rupture and hemorrhage are not uncommon with CSP, and women often elect pregnancy-terminating interventions to avoid these (Michaels, 2015; Timor-Tritsch, 2015). Variants of the morbidly adherent placenta are classified by the depth of trophoblastic growth (Figs. 41-24 and 41-25).

1	Variants of the morbidly adherent placenta are classified by the depth of trophoblastic growth (Figs. 41-24 and 41-25). FIGURE 41-25 Varying degrees of myometrial invasion with the accrete syndromes. Incisions begin on the serosal surface and extend through to the placenta. A. In this case, the myometrium (M) shows minimal invasion by the placenta (P). S = uterine serosa. B. A greater degree of myometrial invasion is seen here. C. In this example, the placenta (backets) extends to the serosal edge, held by the surgeon's hand. No myometrium remains at this site. (Reproduced with permission from Dr. C. Edward Wells in Cunningham FG: Placenta previa and morbidly adherent placenta. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd edition. New York, McGraw-Hili Education, 2017b.)

1	Placenta accreta indicates that villi are attached to the myome trium. With placenta in creta , villi actually invade the myome trium, and placenta percreta defines villi that penetrate through the myometrium and to or through the serosa (Bailit, 2015; Silver, 20 15a). In clinical practice, these three variants are encountered in an approximate ratio of 80:15:5, respectively (Wong, 2008). In all three varieties, abnormal adherence may involve all lobules-total placenta accreta. If all or part of a single lobule is abnormally attached, it is described as a ocal placenta accreta. Histological diagnosis cannot be made from the placenta alone, and myometrial samples are necessary for confirmation (Benirschke, 2012) .

1	he frequency of accrete syndromes was 1 in 20,000 births almost 100 years ago (McKeogh, 1951). As late as 1971, Hell man and Pritchard in the 14th edition of Williams Obstetrics described accreta to be the subject of case reports. Since then, the incidence has grown remarkably in direct relationship to the rising cesarean delivery rate. For example, incidence was 1 in 2500 births in the 1980s, but it was 1 per 731 births in the report from the MFMU Network comprising 115,502 women (Bailit, 2015). And a Canadian study of more than 570,000 births found an incidence of 1 in 700 deliveries (Mehrabadi, 2015). In the Nationwide Inpatient Sample, the prevalence of accreta was 3.7 per 1000 births-1 per 270 (Mogos, 2016).

1	This rising frequency has made accrete syndromes one of the most formidable problems in obstetrics. In one review of 5367 pregnancy-related maternal deaths in the United States from 2006 to 2013, 13 percent were due to hemorrhage caused by accrete syndromes (Creanga, 2015, 2017). In addition, they are a leading cause of hemorrhage and emergency peripartum hysterectomy (Awan, 2011; Eller, 2011; Rossi, 2010). The American College of Obstetricians and Gynecologists (2017 c) and the Society for Maternal-Fetal Medicine (2010) have taken the lead to address and optimize management. In subsequent pregnancies following placenta accreta, recurrence risks are high. Women in whom hysterectomy is avoided have an estimated 20-percent incidence of recurrence (Cunningham, 2016; Roeca, 2017). In addition, some evidence shows that these women have greater risks for previa, uterine rupture, and hysterectomy (Eshkoli, 2013).

1	hese are similar in many aspects to those for placenta previa (p. 774). That said, the two most important risk factors are an associated previa, a prior cesarean delivery, and more likely a combination of the two (Kia', 2014). A classical hysterotomy incision has a higher risk for a subsequent accrete placenta (Gyami-Bannerman, 2012). In fact, almost half of women with a prior cesarean delivery had myometrial fibers seen microscopically adhered to the placenta (Hardardottir, 1996; Miller, 2016). An associated previa confers an even higher risk. his is shown in Figure 41-26, and the astonishing increase in frequency of associated accrete syndromes is apparent with a concomitant previa. FIGURE 41-26 Frequency of morbidly adherent placenta in women with 1 to 5 prior cesarean deliveries (CDs) now with a previa. (Data from Silver, 2006.)

1	FIGURE 41-26 Frequency of morbidly adherent placenta in women with 1 to 5 prior cesarean deliveries (CDs) now with a previa. (Data from Silver, 2006.) Dysfunctional decidual formation also may follow any other type of myometrial trauma such as curettage or endometrial ablation (Benirschke, 2012; Gill, 2015). Even without a prior hysterotomy, coexisting placenta previa is additive to frequency, and in one study, 10 percent ofsuch women with a previa had an associated accrete syndrome. A shorter cervical length with placenta accrete syndromes did not confer a greater risk for preterm delivery (Rae, 2017).

1	Another risk marker became apparent with widespread use of MSAFP and human chorionic gonadotropin (hCG) screening for neural-tube defects and aneuploidies. In one study of more than 9300 women screened at 14 to 22 weeks' gestation, the risk for accrete syndromes was eightfold higher with MSAFP levels >2.5 110M, and it was increased fourfold with maternal serum free �-hCG levels >2.5 MoM (Hung, 1999). In cases offirst-and second-trimester accrete syndromes, there is usually hemorrhage that is the consequence ofcoexisting placenta previa. Such bleeding will typically prompt evaluation and management. In some women who do not have an associated previa, accreta may not be identiied until third-stage labor when an adhered placenta is encountered. Unfortunately, imaging modalities are less than perfect to identiy all of these placentas early.

1	Ideally, sonography is used for antepartum identiication of abnormal placental ingrowth (Chantraine, 2013; Jauniaux, 2016; Reddy, 2014; Tam Tam, 2012). Happe and colleagues (2018) found that first-trimester measurement of the smallest myometrial thickness can be used to predict the necessity for peripartum hysterectomy with an accrete syndrome. Other indings include loss of the normal hypoechoic retroplacental zone between the placenta and uterus, placental vascular lacunae, and placental bulging into the posterior bladder wall (Fig. 41-27). Using these criteria, Warshak and associates (2006) calculated the following values: sensitivity of 77 percent; specificity of 96 percent; positive-predictive creta shows mUltiple and massive placental "lakes" or "lacunae". (Reproduced with permission from Dr. Martha Rac in Cunningham FG: Placenta previa and morbidly adherent placenta. In Yeomans ER, Hofman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd

1	from Dr. Martha Rac in Cunningham FG: Placenta previa and morbidly adherent placenta. In Yeomans ER, Hofman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd edition. New York, McGraw-Hili Education, 201o7b.) value of 98 percent. Similar values are cited by the American College ofObstetricians and Gynecologists (2017c) and others (Chalubinski, 2013; Elhawary, 2013; Maher, 2013).

1	Despite these findings, some investigators report less spectacular results with sonography aauniaux, 2016; Primo, 2014). Bowman and colleagues (2014)r.described the sensitivity of sonography to be 54 percent; specificity, 88 percent; positivepredictive value, 82 percent; negative-predictive value, 65 percent; and accuracy, 65 percent. Location afects sonographic accuracy. In one study, the detection rate was 90 percent for anterior placenta accreta compared with 50 percent for posterior wall ones (Pilloni, 2016). Nageotte (2014) concluded that identiication of the morbidly adherent placenta with sonography should be interpreted along with clinical and operative indings.

1	Better results have been reported by some using threedimensional (3-D) sonography and power Doppler (Collins, 2015; Doyle, 2015). We too have found that the addition of Doppler color low mapping is highly predictive ofmyometrial invasion (Fig. 41-28). This is suspected if the distance between the uterine serosa-bladder wall interface and the retroplacental vessels measures < 1 mm and if there are large intraplacental lacunae (Rae, 2015a; Twicler, 2000). Similarly, Cali and associates (2013) reported that hypervascularity of the uterine serosa-bladder wall interface had the highest positive-and negative-predictive values for placenta percreta.

1	MR imaging can be added to outline anatomy and to identiy invasion of adjacent structures, including possible ureteral involvement (Chalubinski, 2013; Reddy, 2014). Although gadolinium is usually not added during pregnancy, this contrast may enhance images (Millischer, 2017). Lax and coworkers (2007) described three MR imaging findings that suggest accreta: uterine bulging, heterogeneous signal intensity within the placenta indicative of lacunae, and dark intraplacental bands on T2-weighted imaging. Some recommend use of MR imaging if sonography results are inconclusive or there is a posterior previa (American College of Obstetricians and Gynecologists, 2017c; Silver, 2015a). FIGURE 41-28 Transvaginal sonogram of placental invasion with a morbidly adherent placenta. Retroplacental vessels (white arrows) invade the myometrium and obscure the bladder-serosal interface. Abnormal intraplacental venous lakes (black arrowheads) are commonly seen in this setting.

1	Preoperative assessment ideally begins once a possible accrete syndrome is recognized antenatally (Fitzpatrick, 2014; Sentilhes, 2013). A major decision concerns the timing of and the ideal aciliy or delivey. Considerations include appropriate surgical, anesthesia, intensive care, and blood banking capabilities. n obstetrical surgeon or gynecological oncologist and surgical, urological, and interventional radiological consultants should be available (Brennan, 2015; Shamshirsaz, 2015). he American College of Obstetricians and Gynecologists (2017c) and the Society for Maternal-Fetal Medicine (2010) recommend planned delivery in a tertiary-care facility. In some of these, specially designed teams have been assembled and are on call (AI-Khan, 2014; Erfani, 2017a; Smulian, 2017; Walker, 2013). Silver and colleagues (2015b) have provided criteria for accreta centers of excellence. Shown in Table 41-5 are some

1	Silver and colleagues (2015b) have provided criteria for accreta centers of excellence. Shown in Table 41-5 are some TABLE 41-5. Criteria for Consideration of Delivery in an Accrete Center of Excellence Placenta previa with abnormal ultrasound appearance Placenta previa with ::3 prior cesarean deliveries Inability to adequately evaluate or exclude placenta accreta Any other reason to suspect morbidly adherent placenta Reproduced with permission from Silver, 201Sb. criteria to consider transfer to a higher level-of-care facility. Women who refuse blood or its derivatives pose especially diicult management dilemmas (Barth, 201r1). If possible, delivery is best scheduled for peak availability of all resources and team members. Even so, a third of cases require unscheduled delivery, and contingency plans should be ready (Pettit, 2017) .

1	Timing of Delivery maternal consequences of emergency cesarean delivety (Stephen son, 2016). The American College of Obstetricians and Gynecolo gists (2017 c) recommends individualization of delivery timing. It fetal lung maturity testing ater 34 completed weeks (Robinson, 2010). he Society for Maternal-Fetal Medicine (2017) recom mends delivery between 34 and 37 weeks. Two recent surveys weeks or later (Esako, 2012; Wright, 2013). At Parkland Hos pital, we generally schedule these procedures ater 36 completed weeks but are prepared also to manage them in nonelective situ ations (Rac, 2015b). Perlman and colleagues (2017) recommend individualization based on speciic risk criteria.

1	In some cases, placenta accrete syndrome is not recognized until laparotomy. If there are inadequate resources to surgically manage the percreta, and if the woman is stable and not bleed ing, then the fetus is not delivered, the abdominal incision is closed, and she is transferred to a tertiary-care facility . In cases that may involve one or both ureters, catheterization may aid in dissection or identification and repair of injury. Some, but not all, advocate preoperative ureteral catheterization (Eller, 2011; Society for Maternal-Fetal Medicine, 2010; Tam Tam, 2012).

1	Balloon-tipped intraarterial catheters to mitigate blood loss and thereby enhance surgical visibility have also gained supporters. Catheters are advanced preoperatively into the internal iliac arteries, and then after delivery, they are inflated to occlude pelvic blood low (Ballas, 2012; Desai, 2012). Alternatively, the catheters can be used to deliver occluding emboli to bleeding arterial sites. Others have concluded that these procedures ofer borderline eicacy and have serious risks (Salim, 2015; Sentilhes, 2009). Complications have included thromboses of the common and left iliac arteries (Bishop, 2011). At this time, the American College of Obstetricians and Gynecologists (2017 c) concludes that a irm recommendation cannot be made for or against intraarterial catheter use. Similarly, there are no obvious benefits to internal artery ligation (Eller, 2011; Po, 2012).

1	Before commencing with delivery, the risk of hysterectomy to prevent exsanguination should be estimated. Some of these abnormal placentations, especially if partial, may be amenable to placental delivery with hemostatic suture placement. Conirmation of a percreta or increta almost always mandates hysterectomy. Because the scope of invasion may not be apparent before delivery of the fetus, we complete many dissection steps early. This also minimizes blood loss during potentially tedious dissection after hysterotomy. Thus, we usually attempt to create a wide bladder flap bore making the hysterotomy incision (Cunningham, 2017b). The round ligaments are divided, and the lateral edges of the peritoneal relection are dissected downward. If possible, these incisions are extended to encircle the entire placental implantation site that visibly occupies the prevesical space and posterior bladder wall. Following this, a classical hysterotomy or transverse fundal incision is made to avoid the

1	entire placental implantation site that visibly occupies the prevesical space and posterior bladder wall. Following this, a classical hysterotomy or transverse fundal incision is made to avoid the placenta (Kotsuji, 2013).

1	Ater etal delivery, the extent of placental invasion is assessed without attempts at manual placental removal. In a report from the United Kingdom, attempts for partial or total placental removal prior to hysterectomy were associated with twice as much blood loss (Fitzpatrick, 2014). Generally speaking, with obvious percreta or increta, hysterectomy is usually the best course, and the placenta is left in situ (Eller, 2011). With more extensive placental ingrowth, there may be little or no bleeding until manual placental removal is attempted. Unless there is spontaneous separation with bleeding that mandates emergency hysterectomy, the operation begins after full assessment is made. With bleeding, successful treatment depends on immediate blood replacement therapy and other measures that can include uterine or internal iliac artery ligation, balloon occlusion, or embolization.

1	he group at Baylor College of .Medicine has described a modified radical hysterectomy for surgical management of the morbidly adherent placenta (Shamshirsaz, 2015). For a description of this technique, refer to Cunningham and Gilstrap s Operative Obstetrics (Yeomans, 2017). At Parkland Hospital, we have had cases in which a traditional radical hysterectomy was necessary to excise all abnormally implanted placenta. Occasionally, it may be possible to trim the umbilical cord, repair the hysterotomy incision, leave the placenta in situ, and not pursue hysterectomy. This option may be used for women in whom abnormal placentation was not suspected before cesarean delivery and in whom uterine closure stops bleeding. After this, she can be transferred to a higher-level facility for definitive management. Another consideration is the woman with a strong desire for fertility and who has received extensive counseling.

1	Conservative management was reviewed by Perez-Delboy (2014) and Fox (2015) and their colleagues. In some of these cases, the placenta spontaneously resorbed between 1 and 12 months with a mean of6 months. Numerous complications can occur and include sepsis, disseminated intravascular coagulation, pulmonary embolism, and arteriovenous malformation (Fox, 2015; Judy, 2015; Roach, 2015).

1	In some of these women, a subsequent hysterectomyeither planned or prompted by bleeding or infection-is performed days to weeks postpartum when blood loss might be lessened (l-Khan, 2014; Sentilhes, 2009). In one study, only 21 percent of such women ultimately required hysterectomy (Bretelle, 2007). In other reports, however, up to 60 percent eventually required emergency hysterectomy (Clausen, 2013; Pather, 2014). Evidence that treatment with methotrexate aids resorption is lacking. Last, for women in whom the placenta is left in situ, serial serum �-hCG measurements are not informative, and serial sonographic or MR imaging is recommended (Timmermans, 2007; Worley, 2008). At this time, we agree with the American College of Obstetricians and Gynecologists (2017 c) that leaving the placenta in situ is seldom indicated. Exceptions are for temporization to permit transfer to a higher level of care.

1	In sum, these syndromes can have disastrous outcomes for both mother and fetus. Although the depth of placental invasion does not correspond with perinatal outcome, it is of paramount maternal significance (Seet, 2012). Shown in Table 41-6 are outcomes from reports of women from tertiary-care hospitals and in whom the diagnosis of morbidly adherent placenta was made preoperatively. Despite these advantages, a litany of complications included hemorrhage, urinary tract injury, intensive care unit admission, and secondary surgical procedures. Some of these reports chronicle outcomes in a second cohort of women in whom care was not given at a tertiary-care facility or in whom the diagnosis of percreta was not made until delivery, or both. In these cohorts, morbidity was higher, and there was one maternal death.

1	The terms consumptive coagulopathy, deibrination syndrome, or disseminated intravascular coagulation (DIC) are often used interchangeably, but there is an important distinction in these terms. An event related to actual consumption of pro coagulants within the intravascular tree results in a consumptive coagulopathy. In contrast, massive loss of procoagulants from hemorrhage results in a dilutional coagulopathy. Semantics aside, the clinicopathological coagulation disturbances with consumptive coagulopathy culminate in a systemic intravascular activation that completely disrupts natural hemostasis. As a result, an inefective balance of natural anticoagulant mechanisms leads to widespread fibrin deposition that can cause multiorgan failure (Levi, 2013).

1	Because of the many deinitions and variable severity, citing an accurate incidence for consumptive coagulopathy in pregnant women is problematic, but it ranges from 0.03 to 0.35 percent (Erez, 2014; Rattray, 2012). For example, some degree of significant coagulopathy is found in virtually all cases of placental abruption and amnionic fluid embolism. Other instances in which frequently occurring but less recognized degrees of coagulation activation can be found include sepsis, thrombotic microangiopathies, acute kidney injury, acute fatty liver, severe preeclampsia, and hemolysis, elevated liver enzyme levels, low platelet count (HELLP) syndrome (Cunningham, 2015). he overall contribution of each of these obstetrical disorders also varies depending on the population studied (Erez, 2015). JOutcomes shown as mean ± 1 SO; median (range). hOata from Warshak, 2010. (Data from Eller, 201n1n. dOata from Walker, 2013. eOata from AI-Khan, 2014. fData from Erfani, 2017b; Shamshirsaz, 2015.

1	JOutcomes shown as mean ± 1 SO; median (range). hOata from Warshak, 2010. (Data from Eller, 201n1n. dOata from Walker, 2013. eOata from AI-Khan, 2014. fData from Erfani, 2017b; Shamshirsaz, 2015. FFP = fresh-frozen plasma; ICU = intensive care unit; LOS = length of stay; NS = not stated; RBC = red blood cells.

1	When consumptive coagulopathy is severe, the likelihood of and ibrinogen-ibrin degradation products, which includes maternal and perinatal morbidity and mortality is increased. In D-dimers. long with lower concentrations of anticoagulant one study of 49 cases, antecedent causes included those listed protein S, hypercoagulability, and decreased ibrinolysis, there above, and 59 percent received blood transfusions, 18 percent is augmented-yet compensated-intravascular coagulation underwent hysterectomy, 6 percent were dialyzed, and three that may function to maintain the utero placental interface. mothers died (Rattray, 2012). he perinatal mortality rate was 30 percent. Callaghan and associates (2012) reviewed data from • Activation of Normal Coagulation the Nationwide Inpatient Sample and found a rising prevalence Instead of the "waterfall" sequential activation of clotting, a curofDIC from 1998 to 2009. And, from 2010 to 2011, DIC was rent theory proposes that tissue factor-an integral

1	a rising prevalence Instead of the "waterfall" sequential activation of clotting, a curofDIC from 1998 to 2009. And, from 2010 to 2011, DIC was rent theory proposes that tissue factor-an integral membrane the second most common severe maternal morbidity indicator glycoprotein-serves as the principal initiator of coagulation(Creanga, 2014). Notably, DIC was associated with nearly a (Levi, 2010b). Coagulation then moves forward but incorpofourth of maternal deaths during this study period. Despite these rates a feedback loop. To begin, tissue factor forms complexesstatistics, consumptive coagulopathy as the sole cause of maternal with factor VII/VIla to activate factors IX and X. Tissue factor death is relatively uncommon and accounts for only 0.2 percent is found in highly vascularized organs such as the brain, lungs, of pregnancy-related deaths in the United States (Creanga, 2015).

1	(Kuczynski, 2002; 0sterud, 2006; Uszynski, 2001).

1	Tissue factor-factor VIla complexes ultimately generate During normal pregnancy, extensive changes in coagulation activated factor X (Xa) to initiate clotting. Subsequently, the and fibrinolysis develop to create a procoagulant state. Some previously labeled "intrinsic" pathway amplifies this process. of these include appreciable increases in the plasma concentraSpecifically, the initial thrombin produced directly activates tions of factors I (fibrinogen), VII, VIII, IX, and X. A partial factor XI by providing a feedback amplification loop. This list of these normal values is found in the Appendix (p. 1256). primary role of tissue factor-factor VIla complex in coaguAt the same time, plasminogen levels rise considerably, but lation and consequent ampliication loop of thrombin is levels of plasminogen activator inhibitor-1 and 2 (PAI-1 and depicted in Figure 41-29 (Rapaport, 1995). he end result PAI-2) also grow. hus, plasmin activiy usually declines until of this ampliied coagulation

1	plasminogen activator inhibitor-1 and 2 (PAI-1 and depicted in Figure 41-29 (Rapaport, 1995). he end result PAI-2) also grow. hus, plasmin activiy usually declines until of this ampliied coagulation process is fibrin formation. This after delivery (Hale, 2012; Hui, 2012). he mean platelet count is then counterbalanced by the ibrinolytic system, in which drops by 10 percent during pregnancy, and platelet activation is plasminogen is activated. As shown in Figure 41-29, even enhanced (Kenny, 2015). this process is tied initially to tissue factor. he inal result is

1	The net results of these changes include greater levels production of fibrinogen/fibrin degradation products, which of fibrinopeptide A, �-thromboglobulin, platelet factor 4, include D-dimers. Plasminogen ---�, FIGURE 41 -29 Schematic of coagulation pathway. FSP = fibrin split products; PAl = plasminogen activator inhibitor;TFPI = tissue factor pathway inhibitor. Activation of Pathological Coagulation he initiation of DIC begins with the release of tissue factor by pathological entities. Tissue factor is released by subendothelial tissue and stimulated monocytes, which in turn provoke release of cytokines from the endothelium. With generalized endothelial activation, difuse activation of coagulation follows. his pathological cycle ofcoagulation and ibrinolysis becomes clinically important when coagulation factors and platelets are suiciently depleted to create consumptive coagulopathy.

1	Several obstetrical syndromes can trigger consumptive coagulopathy. The best known and most common is placental abruption with its significant release of thromboplastin. Another is embolization of amnionic luid and debris into the maternl circulation. This causes activation of factor X by abundant mucin found in fetal squames. Other causes include endotoxins from gram-negative bacteriaand exotoxins from gram-positive bacteria.

1	Bioassay is an excellent method to detect or suspect clinically signiicant coagulopathy. Excessive bleeding at sites of modest trauma characterizes defective hemostasis. Examples include persistent bleeding from venipuncture sites, nicks from shaving the perineum or abdomen, trauma from bladder catheterization, and spontaneous bleeding from the gums, nose, or gastrointestinal tract. Purpura or petechiae at pressure sites such as sphygmomanometer cufs or tourniquets suggest signiicant thrombocytopenia. Any surgical procedure provides the ultimate bioassay and elicits generalized oozing from abdominal wall layers, the retroperitoneal space, the episiotomy, or incisions and dissections for cesarean delivery or hysterectomy.

1	Ofaboratory tests, ibrinogen, fibrin, and degradation product leves can be informative. In late pregnancy, plasma fibrinogen levels typicallyhave risen to 300 to 600 mg/dL. Even with severe consumptive coagulopathy, levels may sometimes be suiciently high to protect against clinically signiicant hypofibrinogenemia. For example, deibrination caused by a placental abruption might lower an initial fibrinogen level of 600 mg/dL to 250 mg/dL. Although this would indicate massive ibrinogen consumption, levels are still adequate to promote clinical coagulationusually about 150 mg/dL. If serious hypoibrinogenemia-less than 50 mg/dL-is present, the clot formed from whole blood in a glass tube may initially be sot but not necessarily remarkably reduced in volume. Then, over the next half hour or so, as platelet-induced clot retraction develops, the clot becomes quite small. When many ofthe erythrocytes are extruded, the volume ofliquid in the tube clearlyexceeds that ofclot.

1	As depicted in Figure 41-29, fibrinolysis cleaves ibrin and ibrinogen into various fibrin degradation products that are detected by several sensitive assays. There are many fragment types, and monoclonal antibodies in assay kits usually measure D-dimers speciic for that assay. These values are always abnormally high with clinically significant consumptive coagulopathy. At least in obstetrical disorders, quantiication has not been correlated with outcomes. Examples of the magnitude of ibrin split product elevations in various obstetrical coagulopathies is shown in Figure 41-30. Thrombocytopenia is likely if petechiae are abundant or if clotted blood fails to retract within an hour or so. Conirmation is provided by a low platelet count. Ifsevere preeclampsia syndrome is comorbid, there may also be qualitative platelet dysfunction (Chap. 40, p. 719). are standard coagulation tests. Prolongation may stem from very 256 ... . j .� ):l0 " 128 ..,.., j .. .S ::•64 ... • . 32 . .

1	are standard coagulation tests. Prolongation may stem from very 256 ... . j .� ):l0 " 128 ..,.., j .. .S ::•64 ... • . 32 . . 16 • .. ... 8 .. FIGURE 41 -30 Quantification offibrin-split products in various obstetrical syndromes that cause disseminated intravascular coagulation. AFLP = Acute fatty liver of pregnancy; HELLP = hemolysis, elevated liver enzyme levels, low platelet count. (Reproduced with permission from Cunningham FG, Nelson DB: Disseminated intravascular coagulation syndromes in obstetrics. Obstet Gynecol. 2015 Nov;126(5):999-101o1.) low fibrinogen concentrations, from appreciably reduced levels of the procoagulants needed to generate thrombin, or from large amounts of circulating fibrinogen-ibrin degradation products. Thromboelastomety and thromboelastography are point-of care tests used as adjuncts to conventional laboratory studies (Abdul-Kadir, 2014). Their current role may serve to guide blood product replacement, discussed later (p. 791).

1	Using many of these tests, several organizations have attempted to establish a more uniform deinition of DIC. One is the International Society on Thrombosis and Haemostasis (ISTH) scoring system. The score is used ony ater a condition known to cause intravascular coagulation is identiied and is calculated using a combination of laboratory tests. Composite ISTH-DIC scores <5 suggest nonovert DIC, whereas scores �5 are compatible with overt DIC. Other than one report of acute fatty liver of pregnancy, this scoring system has not been applied widely in obstetrics (Nelson, 2014).

1	To halt ongoing deibrination, prompt identiication and removal of the inciting source of the coagulopathy is a priority. With surgical incisions or extensive lacerations accompanied by severe hemorrhage, rapid replacement of procoagulants is usually indicated. Vigorous restoration and maintenance of the circulation to treat hypovolemia cannot be overemphasized. Adequate perfusion restores hepatic and endothelial synthesis of procoagulants and permits prompt removal of activated coagulation factors, ibrin, and ibrin degradation products by the reticuloendothelial system.

1	Aside from these fundamental steps, few other agents have proven soundly efective. Although seemingly counterintuitive, unfractionated heparin had been recommended but has now been abandoned. Other examples include use of antiibrinolytic agents-either tranexamic acid or epsilon-aminocaproic acid (micar) (American College of Obstetricians and Gynecologists, 2017 d; Pacheco, 2017). Currently, use of these two agents is not recommended because the ibrinolytic system is necessary for dissolution of widespread ibrin thromboses caused by generalized intravascular coagulation (Hunt, 2014). Discussed later (p. 790), recombinant factor VIla (rFVIIa) has been used to help control severe obstetrical hemorrhage from other causes. However, current clinical evidence is insuicient to make irm recommendations on its administration for obstetrical coagulopathies.

1	Placental abruption is the most common cause of severe consumptive coagulopathy in obstetrics and is discussed more fully on page 767. Typical quantiied levels of ibrin-split products with abruption are shown in Figure 41-30. With preeclampsia, eclampsia, and HELLP syndrome, endothelial activation is a hallmark and is discussed in Chapter 40 (p. 715). In general, the clinical severity of preeclampsia is directly correlated with thrombocytopenia and ibrinogen-ibrin degradation products (Kenny, 2015; Levi, 2010b). As shown in Figure 41-30, intravascular coagulation is seldom severe enough to be clinically worrisome (Pritchard, 1976). Consumptive coagulopathy associated with prolonged reten tion of a dead fetus is unusual today because fetal death can be easily conirmed and there are highly efective methods for labor induction. With singleton pregnancies, if the dead fetus is unde livered, most women enter spontaneous labor within 2 weeks.

1	Gross disruption of maternal coagulation rarely develops before 4 weeks (Pritchard, 1959, 1973). After 1 month, however, almost a fourth will develop consumptive coagulopathy. in a multifetal pregnancy in which one fetus dies while the other survives (Chescheir, 1988; Landy, 1989). his situation is uncommon, and in one study of 22 such pregnancies, none developed a coagulopathy (Petersen, 1999). Most cases are seen in monochorionic twins with shared circulations, which are described in Chapter 45 (p. 877).

1	he classic triad of abrupt hemodynamic and respiratory compromise along with DIC underpins its diagnosis (Clark, 2016). Most reports describe a frequency of 1 in 40,000 to 1 in 50,000 (Clark, 2014; Knight, 2010; Kramer, 2012). The case-fatality rate in all of these studies ranges from 11 to 43 percent. From another perspective, amnionic fluid embolism was the cause of 5 to 15 percent of all pregnancy-related deaths in the United States and Canada (Berg, 2003, 2010; Creanga, 2015; Kramer, 2012).

1	Predisposing conditions are rapid labor, meconium-stained luid, and tears into uterine and other large pelvic veins that permit an exchange of luids between the maternal and fetal compartment (Society for Maternal-Fetal Medicine, 2016). Other commonly cited risks include older maternal age; postterm pregnancy; labor induction or augmentation; eclampsia; cesarean, forceps, or vacuum delivery; placental abruption or previa; and hydramnios (Knight, 2010, 2012; Kramer, 2012). The association of uterine hypertonus appears to be the fectrather than the cause because uterine blood low ceases when intrauterine pressures exceed 35 to 40 mm Hg. Thus, a hypertonic contraction would be the least likely circumstance for amnionic luid and other debris to enter uterine veins (Clark, 1985). For this reason, hypertonus from oxytocin is not implicated.

1	Diagnosis. Proposed criteria for diagnosis of amnionic luid embolism are shown in Table 41-7. he classic example is dramatic, and a woman in the late stages of labor or immediately postpartum begins gasping for air. Seizures or cardiorespiratory arrest rapidly follows accompanied by massive hemorrhage from consumptive coagulopathy. Clinical manifestations are variable. For example, we and others have managed several women in whom otherwise uncomplicated vaginal or cesarean delivery was followed by severe acute consumptive coagulopathy without overt cardiorespiratory diiculties. In those women, consumptive coagulopathy appears to be the orme ruste of amnionic luid embolism (Kramer, 2012; Porter, 1996).

1	Because of this clinical variability, other sources of acute cardiac or respiratory failure should be considered. These include myocardial infarction, pulmonary or air embolism, high spinal blockade, eclampsia, and anaphylactic shock. In some cases, the temporal relationship of events aids diagnosis. Unortunatey, no FIGURE 41 -31 Fatal amnionic fluid embolism. A.Autopsy TABLE 41 -7. Diagnostic Criteria for Amnionic Fluid Embolism Abrupt onset of cardiorespiratory arrest, or both hypotension and respiratory compromise. Documentation of overt disseminated intravascular coagulation. Coagulopathy must be detected prior to loss of sufficient blood to cause dilutional or shock-related consumptive coagulopathy. Clinical onset during labor or within 30 minutes of placental delivery. No fever �38°C. Adapted from Clark, 201n6.

1	specic diagnostic laboratoy test conirms or refutes the diagnosis Management. The initial period of systemic and pulmonary of amnionic luid embolism, and it remains a clinical diagnosis. hypertension with amnionic fluid embolism is transient. Thus, Importantly, women sufering from excessive blood loss and immediate high-quality cardiopulmonary resuscitation and resulting coagulopathy may be misdiagnosed with amnionic advanced cardiac life support must be initiated without delay fluid embolism, when the true culprit is unrecognized or under(Society for Maternal-Fetal 1edicine, 2016). These are disappreciated hemorrhage (Clark, 2016). In either event, a woman cussed in detail in Chapter 47 (p. 931). with cardiopulmonary compromise should receive immediate If resuscitation is successful, hemodynamic instability is resuscitation (Society for Maternal-Fetal Medicine, 2016). common in survivors. Both fever and hyperoxia will worsen ischemia-reperfusion injury to the brain, and thus both are

1	instability is resuscitation (Society for Maternal-Fetal Medicine, 2016). common in survivors. Both fever and hyperoxia will worsen ischemia-reperfusion injury to the brain, and thus both are Pathophysiology.

1	The mechanism of injury from amnionic avoided. A suitable goal for temperature is 36°C and for mean luid embolism has evolved. Early theories proposed that amniarterial pressure is 65 mm Hg (Society for Maternal-Fetal onic fluid and debris entered maternal circulation and obstructed pulmonary artery flow, which led to hypoxia, right heart failure, and death. However, during normal delivery, amnionic fluid commonly enters the maternal circulation through venous channels at the placental implantation site or from small lacerations. Accordingly, squames, fetal cells, and trophoblasts can oten be identiied in maternal peripheral blood at delivery (Clark, 1986; Lee, 1986). And, infused amnionic luid is generally innocuous, even in large amounts (Adamsons, 1971; Stolte, 1967).

1	Current explanations describe disruption of the maternal-fetal interface, which allows material from the fetal compartment to enter maternal circulation. This leads to abnormal activation of proinflammatory mediator systems, similar to the systemic inflammatory response syndrome (SIRS), and causes initial, transient pulmonary vasoconstriction and hypertension. Acute right ventricular failure is then followed by hemodynamic collapse from right ventricular infarction coupled with interventricular 300A Platelets x 103/lL septum displacement to the let and ultimately decreased let sided cardiac output. This right and now let ventricular dysfunc250 tion is followed by cardiogenic pulmonary edema and systemic hypotension. Concurrently in this process, acute respiratory fail ure with severe hypoxemia from shunting develops. Notably, the resulting multiorgan dysfunction is an interrelated process, with both the cardiac and pulmonary systems afecting each other. Women who survive beyond these

1	from shunting develops. Notably, the resulting multiorgan dysfunction is an interrelated process, with both the cardiac and pulmonary systems afecting each other. Women who survive beyond these first phases invariably have the third component of the classic triad-a consumptive coagulopathy. Similar to the coagulation process described earlier, the material from the fetal compartment containing tissue 50 factor activates factor VII. This leads to the development of DIC (see Fig. 41-29).

1	In those who succumb, postmortem histopathological ind1100 1200 ings may be obvious (Fig. 41-31). However, detection of such B Time(hours) material may require special stains, and even then, debris may not be seen. In one study, fetal elements were detected in 75 findings of fetal squames (arrows) packed into a small pulmonary percent of autopsies and in 50 percent of specimens prepared artery. B. Results of coagulation studies from the same woman from concentrated buy coat aspirates taken antemortem from with abruptly decreased fibrinogen levels and platelets and a pulmonary artery catheter (Clark, 1995). simultaneously increased fibrin split products. Medicine, 2016). Additional supportive care measures such as intubation are usually necessary. During the phase of right ventricular failure, inotropic agents such as dobutamine may improve right heart output, and later systemic hypotension should be treated with vasopressors such as norepinephrine.

1	Excess fluid administration is discouraged due to risks of wors ening dilation of an already engorged right ventricle, which of the inteventricular septum. lapse or during the ensuing phases of injly, a coagulopathy develops in most cases from activation of factor VII and X. This may be exacerbated by ongoing hemorrhage. A common source of obstetrical bleeding is uterine atony. herefore, immediate evaluation of coagulation parameters is prudent with concur rent clinical management of bleeding.

1	Clinical Outcomes. Most reports describe dismal outcomes with amnionic fluid embolism. his is likely influenced by underdiagnosis and reporting biases that favor the most severe cases with the highest mortality rates. Several reports are illustrative. From a California database of 1.1 million deliveries, the mortality rate with amnionic fluid embolism was 60 percent (Gilbert, 1999). In a report of34 mothers from China, 90 percent died (Weiwen, 2000). Death can be amazingly rapid, and 12 of the 34 died within 30 minutes. he mortality rate was somewhat better in the largest study from Canada. Of 120 women with an amnionic luid embolism, only a fourth died. Survivors commonly have profound neurological impairment. Clark (1995) observed that only 8 percent of women who lived despite cardiac arrest survived neurologically intact. Overall, prognosis appears to be more associated with disease severity and the attendant cardiac arrest than with any speciic treatment modality (Clark, 2014).

1	As perhaps expected, perinatal outcomes are also poor and are inversely related to the maternal cardiac arrest-to-delivery interval. Even so, neonatal survival rate is 70 percent, but unfortunately, up to half of survivors sufer residual neurological impairment. In the Canadian study, 28 percent of infants were considered to be asphyxiated at birth (Kramer, 2012).

1	Various infections that are accompanied by endo-or exotoxin release can lead to sepsis syndrome. Although a feature of this syndrome includes activation of coagulation, seldom does sepsis alone cause massive procoagulant consumption. Escherichia coli bacteremia is frequently seen with antepartum pyelonephritis and puerperal infections, however, accompanying consumptive coagulopathy is usually not severe. Some notable exceptions are septicemia associated with puerperal infection or septic abortion caused by exoroxins released from infecting organisms such as group A Streptococcus pyogenes, Staphylococcus aureus, or Clostridium peringens, C sordelii, or C noyi (Herrera, 2016). Treatment of sepsis syndrome and septic shock is discussed in Chapter 47 (p. 921).

1	his severe-often lethal-form of consumptive coagulopathy is caused by micro thrombi in small blood vessels leading to skin necrosis and sometimes vasculitis. Debridement of large areas of skin over the extremities and buttocks frequently requires treatment in a burn unit. Purpura fulminans usually complicates sepsis in women with heterozygous protein C deiciencies and low protein C serum levels (Levi, 2010b). Note that homozygous protein C or S deiciency results in fatal neonatal purpurafulminans (Chap. 52, p. 1007). Septic abortion-especially associated with the organisms just discussed-can incite coagulation and worsen hemorrhage, especially with midtrimester abortions. Indeed, sepsis syn 25 percent of abortion-related deaths (Saraiya, 1999). In the past, especially with illegal abortions, infections with C perin gens were a frequent cause of intense intravascular hemolysis at

1	Parkland Hospital (Pritchard, 1971). More recently, however, septic abortions from infection with C sordelii have emerged as important causes (Chap. 18, p. 351). cular coagulation even in the absence of sepsis. Ben-Ami and associates (2012) described a 1.6-percent incidence in 1249 evacuation. Two thirds were done for fetal demise, which may have been contributory to coagulopathy. Another source of intense coagulation is from instillation of hypertonic solutions to efect mid trimester abortions. These are not commonly used currently for pregnancy terminations. he mechanism is thought to initiate coagulation by thromboplastin release into maternal circulation from the placenta, fetus, and decidua by the necrobiotic efect of hypertonic solutions (Burkman, 1977).

1	Recognition of obstetrical hemorrhage severity is crucial to its management. Visual estimation of blood loss, especially when excessive, is notoriously inaccurate, and true blood loss is often two to three times the clinical estimate. Consider also that in obstetrics, part and sometimes even all of the lost blood may be concealed. Estimation is further complicated in that peripartum hemorrhage also includes the pregnancy-induced augmented blood volume. After pregnancy hypevolemia is lost at delivery, blood loss can be estimated by calculating 500 mL loss for each 3 volume percent drop in hematocrit. he hematocrit nadir depends on the speed of resuscitation with intravenous crystalloids. ith acute blood loss, the real-time hematocrit is at its maximum whenever measured in the delivey, operating, or recovey room.

1	A prudent rule is that any time blood loss is considered more than average, then the hematocrit is determined and plans are made for close observation for potential physiological deterioration. Urine output measured hourly is one of the most important "vital signs." Unless diuretic agents are given-and these are seldom indicated with active bleeding-accuratey measured urine low relects renal pefusion, which in turn rlects pefusion of other vital organs. Urine low of at least 30 mL, and preferably :50 mL per hour, should be maintained. Shock from hemorrhage evolves through several stages. Early in the course of massive bleeding, mean arterial pressure, stroke volume, cardiac output, central venous pressure, and pulmonary capillary wedge pressure decline. Increases in arteriovenous oxygen content diference relect a relative rise in tissue oxygen extraction, although overall oxygen consumption falls.

1	Blood low to capillary beds in various organs is controlled by arterioles. These are resistance vessels that are partially controlled by the central nervous system. However, approximately 70 percent of total blood volume is contained in venules which are passive resistance vessels controlled by humoral factors. Catecholamine release during hemorrhage prompts greater venular tone, which provides an auto transfusion from this capacitance reservoir (Barber, 1999). his is accompanied by compensatory rises in heart rate, systemic and pulmonary vascular resistance, and myocardial contractility. In addition, cardiac output and blood volume are redistributed from the efect of selective, centrally mediated arteriolar constriction or relaxation-autoregulation. hus, although perfusion to the kidneys, splanchnic beds, muscles, skin, and uterus is diminished, relatively more blood flow is diverted to the heart, brain, and adrenal glands.

1	When the blood volume deicit exceeds approximately 25 percent, compensatory mechanisms usually are inadequate to maintain cardiac output and blood pressure. Importantly, additional small losses of blood will now cause rapid clinical deterioration. Following an initial augmented total oxygen extraction by maternal tissue, maldistribution of blood flow results in local tissue hypoxia and metabolic acidosis. This creates a vicious cycle of vasoconstriction, organ ischemia, and cellular death.

1	Another important clinical efect of hemorrhage is activation of lymphocytes and monocytes, which in turn causes endothelial cell activation and platelet aggregation. These promote release of vasoactive mediators that occlude small vessels and further impair microcirculatory perfusion. Other common obstetrical syndromes-preeclampsia and sepsis-also lead to loss of capillary endothelial integrity, additional loss of intravascular volume into the extracellular space, and platelet aggregation. These then can incite DIC.

1	he pathophysiological events just described create important but often overlooked extracellular luid and electrolyte shifts involved in both the genesis and successful treatment of hypovolemic shock. hese include changes in the cellular transport of various ions such as sodium and water into skeletal muscle as well as potassium loss. Replacement of extracellular luid and intravascular volume are both necessary. Survival is enhanced in acute hemorrhagic shock if blood plus cystalloid solution is given compared with blood tranusions alone.

1	Whenever excessive blood loss is suspected in a pregnant woman, steps are simultaneously taken to identiy the bleeding source and to begin resuscitation. If she is undelivered, restoration of blood volume is beneficial to mother and fetus, and it also prepares for emergent delivery. If she is postpartum, it is essential to immediately identiy uterine atony, retained placental fragments, or genital tract lacerations. At least one and preferably more large-bore intravenous infusion systems are established promptly with rapid administration of crystalloid solutions, while blood is made available. An operating room is readied, and a surgical and anesthesia team are assembled immediately. Specific management of hemorrhage is further dependent on its etiology.

1	It cannot be overemphasized that treatment of serious hemorrhage demands prompt and adequate reilling of the intravascular compartment with crystalloid solutions. These rapidly equilibrate into the extravascular space, and only 20 percent of crystalloid remains intravascularly in critically ill patients after 1 hour (Zuckerbraun, 2010). Because of this, initial luid is infused in a volume two to three times the estimated blood loss. Resuscitation of hypovolemic shock with colloid versus crystalloid solutions has been debated. In a Cochrane review of resuscitation of nonpregnant critically ill patients, Perel and coworkers (2013) found equivalent benefits but concluded that colloid solutions were more expensive. Similar results were found in the Saline versus Albumin Fluid Evaluation (SAFE) randomized trial of almost 7000 nonpregnant patients (Finfer, 2004). We concur with Zuckerbraun and colleagues (2010) that acute volume resuscitation is preferably done with crystalloid and blood.

1	The hematocrit level or hemoglobin concentration that mandates blood transfusion is controversial. Cardiac output does not substantively drop until the hemoglobin concentration falls to approximately 7 g/dL or hematocrit of 20 volume percent. At this level, several organizations recommend consideration for red cell transfusions (Carson, 2017). Also, Military Combat Trauma Units in Iraq used a target hematocrit of 21 volume percent (Barbieri, 2007). In general, with ongoing obstetrical hemorrhage, we recommend rapid blood infusion when the hematocrit is <25 volume percent. This decision is dependent on whether the fetus has been delivered; surgery is imminent or ongoing operative blood loss is expected; or acute hypoxia, vascular collapse, or other factors are present.

1	Scant clinical data elucidate these issues. In a study from the Canadian Critical Care Trials Group, nonpregnant patients were randomly assigned to restrictive red cell transfusions to maintain hemoglobin concentrationr>7 gl dL or to liberal transfusions to maintain the hemoglobin level at 10 to 12 gl dL. he 30-day mortality rate was similar-19 versus 23 percent in the restrictive versus liberal groups, respectively (Hebert, 1999). Transfusion therapy in nonpregnant patients with septic shock had similar mortality rates when 7 g/dL was compared with 9 gl dL as targets for transfusions (Holst, 2014). he number of units transused in a given woman to reach a target hematocrit depends on her body mass and on expectations of additional blood loss. Contents and efects of transfusion of various blood components are shown in Table 41-8. Compatible whole blood is ideal TABLE 41 -8. Blood Products Commonly Transfused in Obstetrical Hemorrhage

1	Contents and efects of transfusion of various blood components are shown in Table 41-8. Compatible whole blood is ideal TABLE 41 -8. Blood Products Commonly Transfused in Obstetrical Hemorrhage Whole blood About 500 mL; Hct RBCs, plasma, 600-700 mg Restores blood volume and �40 percent fibrinogen, no platelets fibrinogen, increases Hct Packed RBCs 250-300 mL; RBCs, minimal fibrinogen, no Increases Hct 3-4 volume percent Fresh-frozen About 250 mL; 30-minute Colloid, 600-700 mg fibrinogen, no Restores circulating volume and plasma (FFP) thaw platelets fibrinogen 3-4 g will increase �Cryoprecipitate About 15 mL, frozen One unit �200 mg fibrinogen, 15-20 units or other clotting factors, no platelets baseline fibrinogen 150 mg/dL Platelets About 50 mL, stored at One unit raises platelet count about 6-10 units transfused: single-donor room temperature 5000/�L; single-donor apheresis bag preferable to raise platelets Hct = hematocrit; RBCs = red blood cells.

1	Hct = hematocrit; RBCs = red blood cells. or treatment of hypovolemia rom catastrophic hemorrhage. It has a shelf life of 40 days, and 70 percent of the transfused red cells function for at least 24 hours following transfusion. One unit raises the hematocrit by 3 to 4 volume percent. Important for obstetrical hemorrhage, whole blood replaces many coagulation factors in obstetrics-especially fibrinogen-and its plasma treats hypovolemia. A collateral derivative is that women with severe hemorrhage are resuscitated with fewer blood donor exposures than with packed red cells and components (Shaz, 2009).

1	Evidence supports the preferable use of whole blood for massive hemorrhage, including our experiences at Parkland Hospital (Alexander, 2009; Hernandez, 2012). Of more than 66,000 deliveries, women with obstetrical hemorrhage treated with whole blood had signiicantly lower incidences of renal failure, acute respiratory distress syndrome, pulmonary edema, hypoibrinogenemia, intensive care unit admissions, and maternal death compared with those given packed red cells and component therapy. Freshly donated whole blood has also been used successfully for life-threatening massive hemorrhage at combat support hospitals (Murdock, 2014; Stubbs, 2016).

1	In most institutions today, however, whole blood is rarely available. hus, most women with obstetrical hemorrhage and ongoing massive blood loss are given packed red cells and crystalloid. In these instances, no data support a 1: 1 plasma: red cell transfusion ratio. As subsequently discussed, many institutions use massive tranfusion protocols designed to anticipate all facets of massive obstetrical hemorrhage. These "recipes" commonly contain a combination of red cells, plasma, cryoprecipitate, and platelets (Cunningham, 2015; Pacheco, 2011; Shields, 201l).

1	Several studies have assessed plasma:red cell ratio with massive transfusion protocols used in civilian trauma units and military combat hospitals (Borgman, 2007; Gonzalez, 2007; Hardin, 2014; Johansson, 2007). Patients undergoing massive transfusion-deined as 10 or more units of blood-had much higher survival rates as the ratio of plasma to red cell units neared 1: 1.4, that is, one unit of plasma given for each 1.4 units of packed red cells. By way of contrast, the highest mortality group had a ratio of 1 :8. Most of these studies ound that component replacement is rarey necessay with acute replacement of 5 to 10 units of packed red cels.

1	F rom the foregoing, when red cell replacement exceeds five units or so, evaluation of platelet count, clotting studies, and plasma fibrinogen concentration is reasonable. In the woman with obstetrical hemorrhage, the platelet count should be maintained > 50,000/�L by the infusion of platelet concentrates. A fibrinogen level < 150 mg/dL or a suiciently prolonged PT or PTT in a woman with surgical bleeding is an indication for replacement. Fresh-frozen plasma is administered in doses of 10 to 15 mLlkg, or alternatively, cryoprecipitate is infused (see Table 41-8).

1	A major drawback of treatment for massive hemorrhage with crystalloid solutions and packed red blood cells is depletion of platelets and clotting factors. his can lead to a dilutional coagulopathy that is clinically indistinguishable from DIe (Hossain, 2013). Thrombocytopenia is the most frequent coagulation defect found with blood loss and multiple transfusions (Counts, 19 9). In addition, packed red cells have only very small amounts of soluble clotting factors, and stored whole blood is deicient in platelets and in factors V, VIII, and XI. As discussed, massive replacement with red cells only and without factor replacement can also cause hypoibrinogenemia and prolongation of the PT and PTT. Because many causes of obstetrical hemorrhage also cause consumptive coagulopathy, the distinction between dilutional and consumptive coagulopathy can be confusing. Fortunately, treatment for both is similar.

1	A blood type and antibody screen should be performed for any woman at significant risk for hemorrhage. Screening involves mixing maternal serum with standard reagent red cells that carry antigens to which most of the common clinically signiicant antibodies react. Crossmatching involves the use of actual donor erythrocytes rather than the standardized red cells. This process is eicient, and only 0.03 to 0.07 percent of patients identiied as having no antibodies are subsequently found to have antibodies (Boral, 1979). Importanty, administration of screened blood rarey results in adverse clinical sequelae. One unit of packed erythrocytes is derived from one unit of whole blood to have a hematocrit of 55 to 80 volume percent. One unit will increase the hematocrit by 3 to 4 volume percent.

1	One unit of packed erythrocytes is derived from one unit of whole blood to have a hematocrit of 55 to 80 volume percent. One unit will increase the hematocrit by 3 to 4 volume percent. With surgical delivery or with lacerations, platelet transfusions are considered with ongoing obstetrical hemorrhage when the platelet count falls below 50,000/�L (Kenny, 2015). In the nonsurgical patient, bleeding is rarely encountered if the platelet count is 10,000/�L or higher (Murphy, 2010). The preferable source of platelets is one "bag" obtained by single-donor apheresis. This contains the equivalent of six units from six individual donors. Depending on maternal size, each single-donor apheresis six-unit bag raises the platelet count by approximately 20,000/�L (Schlicter, 2010). If these bags are not available, then individual-donor platelet units are used, and six to eight such units are generally transfused one at a time.

1	Importantly, the donor plasma in platelet units must be compatible with recipient erythrocytes. Further, because some red blood cells are invariably transfused along with the platelets, only units from D-negative donors should be given to D-negative recipients. If it is necessary to give these, however, adverse sequelae are unlikely (Lin, 2002). This component is prepared by separating plasma from whole blood and then freezing it. Approximately 30 minutes are required for frozen plasma to thaw. It is a source of all stable and labile clotting factors, including fibrinogen. Thus, it is often used for treatment of women with consumptive or dilutional coagulopathy. Plasma is not appropriate or use as a volume expander in the absence of speciic clotting actor deiciencies. It should be considered in a bleeding woman with a fibrinogen level < 150 mg/dL or with an abnormal PT or PTT.

1	An alternative to frozen plasma is liquid plasma (LQP). This never-frozen plasma is stored at 1 to 6°C for up to 26 days, and in vitro, it appears to be superior to thawed plasma (Matijevic, 2013),. Each unit of cryoprecipitate is prepared from one unit of fresh-frozen plasma. Each 10-to 15-mL unit contains at least 200 mg of fibrinogen along with factor VIII:C, factor VIII:von Willebrand factor, factor XIII, and ibronectin (American Association of Blood Banks, 2014). It is usually given as a "pool" or "bag" using an aliquot of ibrinogen concentrate taken from 8 to 120 donors. Cryoprecipitate is an ideal source of ibrinogen when levels are dangerously low and there is oozing from surgical incisions. Another alternative is virus-inactivated ibrinogen concentrate. Each gram of this raises the plasma fibrinogen level approximately 40 mg/ dL (Ahmed, 2012; Kikuchi, 2013).

1	This synthetic vitamin K-dependent protein is available as NovoSeven. It binds to exposed tissue factor at the site of injury to generate thrombin that activates platelets and the coagulation cascade. Since its introduction, rFVIla has been used to help control hemorrhage from surgery, trauma, and obstetrical causes (Goodnough, 2016; Murakami, 2015). Most Level I trauma centers include it in their massive transfusion protocols, and it is included in the one used at Parkland Hospital. Importantly, rFVIIa will not be efective if the plasma fibrinogen level is <50 mg/dL or the platelet count is <30,000/�L. One major concern with rFVIla use is arterial-and to a lesser degree venous-thrombosis. In a review of 35 randomized trials with nearly 4500 subjects, arterial thromboembolism developed in 55 percent (Levi, 2010a). A second concern is that it was found to be only marginally efective (Pacheco, 2011).

1	This antiibrinolytic drug has been used for traumatic and obstetrical hemorrhage. Tranexamic acid inhibits clot lysis to help forestall bleeding by preventing plasmin from degrading ibrin. Its use has been associated with a higher incidence of renal cortical necrosis (F rimat, 2016). The evidence supporting its use as an adjunct in obstetrical hemorrhage is limited, and its routine use for prophylaxis is not recommended American College of Obstetricians and Gynecologists, 2017 d; Pacheco, 2017).

1	These function to speed blood product delivery to the bedside or operating room, which permits product infusion early in the resuscitation process. he rationale is to prevent adverse efects of aggressive resuscitation solely with crystalloid and packed red blood cells. That said, it is not necessary to activate massive transfusions until at least four to ive units of red cells have been given within 2 hours or so. Once activated, red cells, plasma, platelets, and fibrinogen are given by protocol in amounts shown in Table 41-9. Some protocols include rFVIIa and others include tranexamic acid. As expected, studies attesting to the superiority for survival with massive transfusion protocols are limited. Most TABLE 41-9. Parkland Hospital Obstetrical Massive Transfusion Protocol No. 5 Units 3 Units 6-pack 1 Unit 2 mg rVlla = recombinant activated factor VII (NovoSeven).

1	TABLE 41-9. Parkland Hospital Obstetrical Massive Transfusion Protocol No. 5 Units 3 Units 6-pack 1 Unit 2 mg rVlla = recombinant activated factor VII (NovoSeven). reports describe nonpregnant Normal coagulation trauma victims, but some obser vational studies address obstetrical hemorrhage (Green, 2016; 60 60 Pacheco, 2016). More data with 40 40 use of these protocols is needed. 20 20 rotational thromboelastometry (ROTEM) are point-of-care tests 10 20 30 40 50 min 10 20 30 40 50 min that assess coagulation in whole A EXTEM B FIBTEM blood during massive transfu FIGURE 41-32 TEG/ROTEM based viscoelastic assays of coagulation profiles in a pregnant sions. These tests work by ana woman. A. EXTEM clot profile: CT = clotting time; A5-20 = clot amplified at 5, 10, 15,o20 min;

1	MCF = maximum clot firmness. B. FIBTEM clot profile showing excellent fibrin-based clot qualbreakdown in a whole blood samity. (Reproduced with permission from Solomon C, Collis RE, Collins PW: Haemostatic monitorple from a given patient. Testing ing during postpartum haemorrhage and implications for management, Br J Anaesth. 201o2 produces a proile of coagulation Dec;1o09(6):851-863.) dynamics, and displayed values indicate the speed and quality of clot formation (Fig. 41-32). hese assays provide information regarding time to clot formation, clot strength, and fibrinolysis. Currently, they guide blood product replacement in trauma, liver transplant, and cardiac surgery patients. Studies of TEG and ROTEM techniques in pregnant women have conirmed the hypercoagulable state of pregnancy and provide reference ranges for use in this population (Butwick, 2015; de Lange, 2014; Solomon, 2012).

1	Although these point-of-care tests appear promising, they also have several limitations. For example, they cannot be used to detect disorders of primary hemostasis (Solomon, 2012). Additionally, these tests cannot diagnose coagulopathies stemming from platelet dysfunction or anti platelet drugs. A major drawback is the risk of misinterpretation when tests are used by inadequately trained personnel. Further study is necessary before these tests are widely applied for treatment of obstetrical hemorrhage. Several agents can be used to control persistent surgical oozing. These were recently reviewed by Miller and colleagues (2015). Other than for cesarean hysterectomy, these are seldom used in obstetrical hemorrhage.

1	Preoperative patient phlebotomy and autologous blood storage for transfusion has been disappointing. Exceptions are women with a rare blood type or with unusual antibodies. Most have concluded that autologous transfusions are not cost efective (Etchason, 1995; Pacheco, 2011, 2013).

1	Intraoperative blood salvage with reinfusion is considered to be a safe intervention in obstetrical patients. As discussed in Chapter 30 (p. 569), this practice may be helpful for women declining transfusion. Prior concern centered on amnionic fluid contamination and embolism (Dhariwal, 2014; Goucher, 2015; Pacheco, 2011). A recent randomized trial involving 3028 women compared routine cell salvage use against routine care, in which salvage was employed only for bleeding indications. he rate of nonautologous donor blood transfusion was reduced in the cell salvage group-2.5 versus 3.5 percent, but this was not a signiicant diference (han, 2017). Similar to prior reports, no cases of amnionic luid embolism were reported.

1	Of serious known risks, transfusion of an incompatible blood component may result in acute hemolysis. If severe, this can cause DIC, acute kidney injury, and death. Preventable errors responsible for most of such reactions frequently include mislabeling of a specimen or incorrectly transfusing a patient not slated for those products. The rate of such errors in the United States is estimated to be 1 in 14,000 units, but these events are likely underreported (Lerner, 2010). A transfusion reaction is characterized by fever, hypotension, tachycardia, dyspnea, chest or back pain, lushing, severe anxiety, and hemoglobinuria. Immediate supportive measures include stopping the transfusion, treating hypotension and hyperkalemia, provoking diuresis, and alkalinizing the urine.

1	Transfusion-related acute lung injury (TAIl) is the most common cause of transfusion-related mortality. The syndrome is characterized by severe dyspnea, hypoxia, and noncardiogenic pulmonary edema that develop within 6 hours of transfusion (Peters, 2015). TRALI is estimated to complicate at least 1 in 12,000 transfusions (Carson, 2017). Although the pathogenesis is incompletely understood, injury to the pulmonary capillaries may arise from anti-human leukocyte antigen (HA) and neutrophil (HNA) antibodies in donor plasma (Lerner, 2010). A delayed form ofTRALI has been reported to begin 6 to 72 hours following transfusion (Marik, 2008). Management is supportive and may include mechanical ventilation (Chap. 47, p. 919).

1	Bacterial inection from transfusion of a contaminated blood component is unusual because organism growth is discouraged by refrigeration. The most often implicated contaminants of red cells include Yersinia, Pseudomonas, Serratia, Acinetobacter, and Escherichia species. The more important risk is from bacterial contamination of platelets, which are stored at room temperature. Current estimates are that 1 in 1000 to 2000 platelet units are contaminated. Death from transfusion-related sepsis is 1 per 17,000 for single-donor platelets and 1 per 61,000 for apheresis-donor packs (Lerner, 2010).

1	iral inection risks from transfusion have been curtailed. he risk of HIV or hepatitis C virus infection in screened blood is estimated to be 1 case per 1 to 2 million units transfused (Carson, 2017; Stramer, 2004). The risk for HIV-2 infection is less. Other viral infections include hepatitis B transmission, which is estimated to be < 1 per 100,000 transused units Qackson, 2003). Because of its high prevalence, cytomegalovirus-infected leukocytes are often transfused. hus, precautions are taken for immunosuppressed recipients, keeping in mind that this includes the fetus. Also, risks for transmitting West Nile virus, human T -lymphotropic virus type I, parvovirus B 19, and toxoplasmosis are slight (American Association of Blood Banks, 2013;

1	Also, risks for transmitting West Nile virus, human T -lymphotropic virus type I, parvovirus B 19, and toxoplasmosis are slight (American Association of Blood Banks, 2013; Foroutan-Rad, 2016). Finally, Zika virus has emerged as another relevant transfusion-transmitted infection (Motta, 2016). The Food and Drug Administration (2016) revised recommendations for collection of all whole blood components to include testing for Zika virus. This practice has been airmed by the Centers for Disease Control and Prevention (2016). Several invasive procedures can help arrest postpartum hemorrhage. A report from the Agency for Healthcare Research and Quality concluded that most studies addressing these methods are of poor quality (Likis, 2015). In one study of 6660 women with postpartum hemorrhage, 4.4 percent underwent an invasive procedure, and 1.1 percent had a hysterectomy (Kayem, 2016). he failure rate of conservative measures was 15 percent in surgical and embolization procedures.

1	The technique for unilateral or bilateral uterine artery ligation is used primarily for lacerations at the lateral part of a h) ster otomy incision (Fig. 41-33). In our experiences, this procedure is less helpful for hemorrhage from uterine atony.

1	his surgical technique uses a no. 2 chromic suture to compress the anterior and posterior uterine walls together (B-Lynch, 1997). Because they give the appearance of suspenders, they are also called braces (Fig. 41-34). Several modiications of the B-Lynch technique have been described (Cho, 2000; Hayman, 2002; Matsubara, 2013; Nelson, 2007). Indications vary for its application, and this will afect the success rate. For example, B-Lynch (2005) cited 948 cases with only seven failures. Conversely, Kayem and associates (2011) described 211 women who had an overall failure rate of 25 percent, which did not difer between B-Lynch sutures and their modiications. In another series, the failure rate was 20 percent (Kaya, 2016). From their review, Sathe and coworkers (2016) reached similar conclusions.

1	FIGURE 41-33 Uterine artery ligation. The suture goes through the lateral uterine wall anteriorly, curves around posteriorly, then re-enters anteriorly. When tied, it encompasses the uterine artery. Some unique complications can rarely follow compression sutures (Matsubara, 2013). Most involve variations of uterine ischemic necrosis with peritonitis (Gottlieb, 2008; Joshi, 2004; Ochoa, 2002; Treloar, 2006). In one case, total uterine necrosis followed B-Lynch sutures that were placed in combination with bilateral ligation of uterine, uteroovarian, and round ligament arteries (Friederich, 2007). In most cases, subsequent pregnancies are uneventful if compression sutures are used (n, 2013). A few women, however, with B-Lynch or Cho sutures developed uterine wall defects (Akoury, 2008). Another longterm complication is uterine cavity synechiae (Alouini, 2011; Ibrahim, 2013; Poujade, 2011).

1	For years, ligation of one or both internal iliac arteries has been used to reduce pelvic hemorrhage. Drawbacks are that the procedure may be technically diicult and is only successful half of the time (American College of Obstetricians and Gynecologists, 2017d). It is not particularly helpful for abating hemorrhage with postpartum atony (Clark, 1985).

1	For ligation, adequate exposure is obtained by opening the peritoneum over the common iliac artery and dissecting down to the bifurcation of the external and internal iliac arteries (Fig. 41-35). Branches distal to the external iliac arteries are palpated to veriY pulsations at or below the inguinal area. ligation of the internal iliac artery 5 cm distal to the common iliac bifurcation will usually avoid the posterior division branches (Bleich, 2007). The areolar sheath of the artery is incised longitudinally, and a right-angle clamp is carefully passed just beneath the artery from lateral to medial. Care must be taken not to perforate contiguous large veins, especially the internal

1	FIGURE 41-34 Uterine compression suture or "brace." The B-Lynch suture technique is illustrated from an anterior view of the uterus in Figures A, B, and 0 and a posterior view in Figure C. The numbers denote the sequential path of the suture and are shown in more than one figure. Step 1. Beginning below the incision, the needle pierces the lower uterine segment to enter the uterine cavity. Step 2. The needle exits the cavity above the incision. The suture then loops up and around the fundus to the posterior uterine surface. Step 3. The needle pierces the posterior uterine wall to reenter the uterine cavity. The suture then traverses to the opposite side within the cavity. Step 4. The needle exits the uterine cavity through the posterior uterine wall. From the back of the uterus, the suture loops up and around the fundus to the front of the uterus. Step 5. The needle pierces the myometrium above the incision to reenter the uterine cavity. Step 6. The needle exits below the incision and

1	loops up and around the fundus to the front of the uterus. Step 5. The needle pierces the myometrium above the incision to reenter the uterine cavity. Step 6. The needle exits below the incision and the sutures at points 1 and 6 are tied below the incision. The hysterotomy incision is then closed in the usual fashion.

1	iliac vein. Suture-usually nonabsorbable-is passed under the artery with a clamp, and the vessel is then securely ligated. he most important mechanism of action with internal iliac artery ligation is an 85-percent reduction in pulse pressure in those arteries distal to the ligation (Burchell, 1968). his converts an arterial pressure system into one with pressures approaching those in the venous circulation. his creates vessels more amenable to hemostasis via pressure and clot formation. Even bilateral internal iliac artery ligation does not appear to interfere with subsequent reproduction. Nizard and colleagues (2003) reported follow-up in 17 women who had bilateral artery ligation. From a total of 21 pregnancies, 13 were normal, three ended with miscarriage, three were terminated, and two were ectopic.

1	This modality is now used for many causes of intractable hemorrhage when surgical access is diicult. In more than 500 women reported, embolization was 90-percent efective (Gronvall, 2014; Lee, 2012; Poujade, 2012; Zhang, 2015). After his review, Rouse (2013) concluded that embolization can be used to arrest refractory postpartum hemorrhage. Other reports have been less enthusiastic. Fertility is not impaired, and many subsequent successful pregnancies have been reported (Chauleur, 2008; Fiori, 2009; Kolomeyevskaya, 2009). An important caveat or these procedures is that women with hemodynamic instabiliy related to active bleeding should not be removed rom the operating room. FIGURE 41 -35 Ligation of the right internal iliac artery. Unembalmed cadaveric dissection shows the right-angle clamp passing underneath the anterior division of the internal iliac artery just distal to its posterior division. (Used with permission from Dr. Marlene Corton.)

1	Complications of embolization are relatively uncommon but can be severe. Case reports detail instances of iatrogenic iliac artery rupture, uterine ischemic necrosis, and uterine infection (Gronvall, 2014; Katakam, 2009; Nakash, 2012). Finally, l-hunyan and coworkers (2012) described a woman with massive buttock necrosis and paraplegia following bilateral internal iliac artery embolization. In a few instances, massive blood loss and diicult surgical dissection is anticipated. The use of balloon-tipped catheters preoperatively inserted into the iliac or uterine arteries was described earlier in management of placenta accrete syndromes (p. 781).

1	For significant bleeding refractory to suture or topical hemostats, pelvic packing with gauze and termination of the operation may be considered. Rolls of gauze are packed to provide constant local pressure. This may serve as a temporizing step prior to interventional embolization. In other cases, packing alone may be left for 24 to 48 hours. If the patient is stable and bleeding appears to have stopped, packing is removed.

1	The umbrela orparachute pack uses a similar concept (Logothetopulos, 1926). Although seldom used today, it can be lifesaving if all other measures have failed, especially in low-resource areas (Dildy, 2006; Howard, 2002). The pack is constructed of a sturdy sterile plastic bag that is illed with gauze rolls that are unwound and knotted together. Suicient rolls are used to provide enough volume to fill the pelvis. he pack is introduced transabdominally with the stalk exiting the vagina. Mild traction is applied by tying the stalk to a I-liter fluid bag, which is hung over the foot of the bed. The umbrella pack is removed vaginally after 24 hours.

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1	Zelop CM: Postpartum hemorrhage. Becoming more evidence-based. Obstet Gynecoln11 (1):3,n201n1 Zetterstrom K, Lindeberg SN, Haglund B, et al: Maternal complications in women with chronic hypertension: a population-based cohort study. Acta Obstet Gynecol Scand 84:419, 2005 Zhang E, Liu L, Owen R: Pelvic artery embolization in the management of obstetrical hemorrhage: predictive factors for clinical outcomes. Cardiovasc Intervent Radiol 38(6):1477, 2015 Zuckerbraun BS, Peitzman AB, Billiar TR: Shock. In Brunicardi FC, Andersen OK, Billiar TR, et al (eds): Schwartz's Principles of Surgery, 9th ed. New York, McGraw-Hill, 20LO Zwart J], Richters ]M, OY F, et al: Severe maternal morbidity during pregnancy, delivery and puerperium in the Netherlands: a nationwide population-based study of 371 ,000 pregnancies. B]OG 115:842, 2008

1	It is generaly admitted that there exists in the medulla a centre or uterine contractions, which can be stimulated by an excess of carbon dioxide in the bloo, by anaemia and the presence of various toxic substances; and it seems highy probable that the requency of premature labour in cases of renal insuiciency and eclampsia may be due to the action of metabolic poisons upon the centre. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) In this textbook's first edition, very little was mentioned regarding preterm birth. Indeed, preterm birth was not incorporated as a stand-alone topic until the 13th edition in 1966. And, this content totaled only three sentences that cited use ofisoxsuprine as a tocolytic agent. In contrast, present-day research now produces more than 3000 articles published annually. Data derive from study of animal models, translational research, clinical trials, and genetic investigations. Despite eforts, elucidating the biology of human parturition and the subsequent eforts to prevent preterm birth remain elusive (Martin, 2017).

1	Low birthweight deines neonates who are born too small. Preterm or premature birth describes neonates who are born too early. With respect to gestational age, a newborn may be preterm, term, or postterm. With respect to size, a newborn may be normally grown and appropriate or gestational age; undersized, thus, smaL or gestational age; or overgrown and consequently, large or gestational age. Small for gestational age categorizes newborns whose birthweight is <10th percentile for gestational age. Other frequently used terms have included etal-growth restriction or intrauterine growth restriction. he term large for gestational age describes newborns whose birthweight is > 90th percentile for gestational age. The term appropriate or gestational age designates newborns whose weight is between the 10th and 90th percentiles.

1	Thus, neonates born before term can be small or large for gestational age, but still preterm by deinition. Low birthweight refers to neonates weighing 1500 to 2500 g; vey low birthweight are those between 1000 and 1500 g; and extremey low birthweight refers to those between 500 and 1000 g. Before the 15th edition of this textbook, apreterm orpremature newborn was deined by a birthweight <2500 g. With that edition, preterm neonates were considered to be those delivered before 37 completed weeks, that is, <366r weeks (Pritchard, 1976). This definition, which has now been in use for more than 40 years, was first promulgated in 1976 by the World Health Organization (WHO) and the International Federation

1	FIGURE 42-1 Percentage of preterm births in the United States according to method of assessment of gestational age. LMP = date of last normal menses. (Adapted with permission from Martin JA, Osterman MJ, Kirmeyer SE, et al: Measuring gestational age in vital statistics data: transitioning to the obstetric estimate. Natl Vital TABLE 42-1 . Infant Mortality Rates in the United States in 201e3 No. (%) (per 1000 births) Total infants 3,932,1n81 (1n00) 23,446 (6) Gestational age: <34 weeks 133,503 (3) 13,284 (100) 34-36 weeks 313,858 (8) 2268 (7) <37 weeks 447,361 (1n1) 15,552 (35) 37-38 weeks 974,162 (25) before 39 weeks (Spong, 2011). An unintended consequence of this health-care strategy has been a rise in stillbirth rates in the United States. One concern is that the rule may be misapplied to gestations with true medical indications for early delivery (Hill, 2017; Nicholson, 2016). Spong (2016) has emphasized the need to perform necessary obstetrical interventions when indicated.

1	39-41 weeks 2,291,468 (58) 4218 (2) ::42 weeks 215,510 (5) 515 (2) Data from Matthews, 2015. of Gynecology and Obstetrics (FIGO). The definition derived from a statistical analysis of gestational age distribution at birth (Steer, 2005). Importantly, the denotation lacks a specific functional basis and should be clearly distinguished from the concept ofprematuriy. Prematurity represents incomplete development of various organ systems at birth. For example, the lungs are particularly afected, leading to the respiratory distress syndrome (RDS) (Chap. 34, p. 636).

1	In 2013 in the United States, 23,446 infants died in their first year oflife, and a third ofinfants died from preterm-related causes (Matthews, 2015). Gestational age at deliveryand the risk of neonatal morbidity and mortality are inversely related (Frey, 2016). Namely, neonates born in the early-preterm period make up the smallest proportion of births, but these infants experience disproportionately higher rates of prematurity-related complications (Table 42-1).

1	Beginning in 2005, in recognition that neonates born between 34°/7 weeks and 366/7 weeks experience morbidities and mortality characteristic of premature newborns, preterm births were subdivided. Those before 336/ weeks are labeled earypreterm, and those occurring between 34 and 36 completed weeks are late preterm. Indeed, compared with births at weeks through 40617 weeks, these late-preterm infants experience morbidities that are also associated with prematurity (Spong, 2013). Recently, this concepthas expanded to births 37°/7 weeks through 386/ weeks, which are now defined as eary term, and those 39°/7 weeks through weeks, which are deined as term.

1	his revised terminology has led some to redefine a short gestation as those <39°/ weeks. By doing so, more than a third of live births in the United States in 2015 would be defined as having a shortened period of gestation (Martin, 2017). One implication is that only 65 percent of births in the United States occurred during the optimal 39 to 41 weeks' gestation. This emphasizes the realization that fetal maturation in humans is a continuum that is completed later in human pregnancy than previously appreciated. As a result, adverse neonatal sequelae from neonatal immaturity with elective delivery before 39 completed weeks are appreciable (Reddy, 2009; Tita, 2009).

1	This knowledge resulted in the development and application ofthe "39-week rule" to deter nonmedically indicated deliveries In the United States, the preterm birth rate rose slightly from 9.57 percent in 2014 to 9.63 percent for 2015 (Martin, 2017). This marks the irst rise in this percentage since 2007. Although concerning, some argue that the drop in preterm birth rates from 2007 to 2014 reflected systematic bias associated with changes in obstetrical dating (Frey, 2016).

1	Speciically, beginningwith the 2014 data year, the National Vital Statistics Reports from the National Center for Health Statistics transitioned to a new standard for estimating newborn gestational age for birth certificate completion (Martin, 2015). The new measure-obstetrical estimate of gestational age at delivery-replaced calculations based on the date of the last normal menses (Chap. 44, p. 846). As shown in Figure 42-1, these measures difer and do not provide equivalent absolute numerical comparisons ofpreterm birth rates. For example, the 2015 obstetrical estimate-based preterm birth rate was 9.6 percent compared with the last menstrual period-based rate of 11.3 percent (Martin, 2017). hus, current national data are now not directy comparable to previousy reported rates ofpreterm birth due to diering gestational age caculation methodologies. he national data are now reported starting with year 2007, which coincides with the year that this information became available. 12.68

1	12.68 J.., Stat Rep. 2015 Jun 1 ;64(5):1-20.) tem data showed that the overall reduction of preterm birth before 2014 was in part due to the changes in maternal age dis tribution. Speciically, teen birth rates declined. This translated into a drop in preterm birth rates over the same epoch and could explain the lower infant mortality rates (Callaghan, 2017).

1	One disturbing aspect of preterm birth rate trends in the United States is persistent racial and ethnic disparities. Rates of pre term birth among black women are markedly elevated above those for white and Hispanic women in every year recorded (Martin, 2017). Moreover, rates of births before 32 completed weeks in black women are higher than those in white and Hispanic women combined. Some investigators attribute this disparity to socioeconomic circumstances (Collins, 2007; Leveno, 2009). Internationally, the rates of preterm birth in the United States are also higher compared with those in other industrialized countries (Ananth, 2009; Delnord, 2017; Martin, 2017).

1	Newborns born before 37 weeks sufer various morbidities, largely due to organ system immaturity (Table 42-2). hat said, remarkable strides have been made in neonatal survival for those born preterm. his is especially true for neonates born after 28 weeks. In a study of more than 18,000 newborns weighing between 400 and 1500 g or aged between 22 and 32 weeks' gestation, survival rates were analyzed as a function of both birthweight and gestational age (Fanarof, 2007). Mter achieving a birthweight of : 1000 g or a gestational age of 28 weeks for females, or 30 weeks for males, survival rates reach 95 percent. • Threshold of Viability Births once considered to be "abortuses" because the fetus weighed <500 g are now classified as live births. In the United States in 2014, 5863 live births <500 g were recorded (Martin, 2017). For those newborns delivered before 33 weeks' gesta tion, perinatal and neonatal care has advanced tremendously.

1	States in 2014, 5863 live births <500 g were recorded (Martin, 2017). For those newborns delivered before 33 weeks' gesta tion, perinatal and neonatal care has advanced tremendously. As a result, the threshold of viability, which is the lower limit of fetal maturation compatible with extrauterine survival, has been reassessed. Currently, the threshold of viability lies between 20 and 26 weeks' gestation.

1	Neonates born in this periviable period have been described as fragile and vulnerable because of their immature organ systems. Many of these are described in Chapter 34 (p. 639) and include brain injury from hypoxic-ischemic injury and sepsis. In this setting, hypoxia and sepsis start a cascade of events that lead to brain hemorrhage, to white-matter injury that causes periventricular leukomalacia, and to poor subsequent brain growth eventuating in neurodevelopmental impairment. Associated morbidities include intellectual disability, cerebral palsy, blindness, seizures, and spastic quadriparesis that can result in the need for a lifetime of medical care (Annas, 2004). Because active brain development normally occurs throughout the second and third trimesters, those born <25 weeks are believed to be especially vulnerable to brain injury.

1	To clariY obstetrical care of these fetuses, the Society for Maternal-Fetal Medicine, the Eunice Kennedy Shriver National Institute for Child Health and Human Development (NICHD), the American Academy of Pediatrics, and the American College of Obstetricians and Gynecologists convened a joint workshop in 2013 (Raju, 2014). he executive summary statement from this meeting served as the underpinnings for an Obstetric Care Consensus document from the American College of Obstetricians and Gynecologists (2017 e). TABLE 42-2. Major Short-and Long-Term Problems in Very-Low-Birthweight Infants Respiratory distress syndrome, air leak, bronchopulmonary dysplasia, apnea of prematurity Hyperbilirubinemia, feeding intolerance, necrotizing enterocolitis, growth failure Hospital-acquired infection, immune deficiency, perinatal infection Intraventricular hemorrhage, periventricular leukomalacia, hydrocephalus Retinopathy of prematurity

1	Hospital-acquired infection, immune deficiency, perinatal infection Intraventricular hemorrhage, periventricular leukomalacia, hydrocephalus Retinopathy of prematurity Hypotension, patent ductus arteriosus, pulmonary hypertension Water and electrolyte imbalance, acid-base disturbances Iatrogenic anemia, need for frequent transfusions, anemia of prematurity Hypoglycemia, transiently low thyroxine levels, cortisol deficiency Bronchopulmonary dysplasia, reactive airway disease, asthma Failure to thrive, short-bowel syndrome, cholestasis Respiratory syncytial virus infection, bronchiolitis Cerebral palsy, hydrocephalus, cerebral atrophy, neurodevelopmental delay, hearing loss Blindness, retinal detachment, myopia, strabismus Pulmonary hypertension, hypertension in adulthood Impaired glucose regulation, increased insulin Data from Eichenwald, 2008.

1	Pulmonary hypertension, hypertension in adulthood Impaired glucose regulation, increased insulin Data from Eichenwald, 2008. TABLE 42-3. Outcomes at 2V2 Years Corrected Age 80 by Gestational Age at Birth in Sweden, 2004-2007 )o 40 • Ishii (2013) .D • Stoll (2010) Outcome 22 23 24 25 26 Total o (no.) Survived to 10 53 67 82 85 70

1	FIGURE 42-2 Neonatal survival rates according to condition at birth and gestational age. Ishii (201o3) data curve reflects liveborn survival rates; Stoll (201o0) curve reflects liveborn survival rates; Mild 40 19 33 29 34 31 Rysavy (2015) curve reflects overall survival rates. Moderate 20 30 21 17 10 16 • %of infants who died • %of infants who survived with he Obstetric Care Consensus summary provides a review of outcomes for those born in the periviable period. Delivery before 23 weeks typically results in death, and survival rates approximate only 5 percent (Fig. 42-2). Among those that live, morbidity is nearly universal. Notably, the authors highlight the wide variation in practices regarding active resuscitation and suggest that these variations may explain the difering perinatal outcomes among diferent institutions. An important caveat, however, is ascertainment bias. For example, the mean survival rate is 45 percent if the denominator is all live births compared with 72 percent

1	among diferent institutions. An important caveat, however, is ascertainment bias. For example, the mean survival rate is 45 percent if the denominator is all live births compared with 72 percent if the denominator is only newborns admitted to neonatal intensive care (Guillen, 2011). Another source of bias is use of multicenter datasets with considerable diferences in obstetrical and early neonatal interventions, particularly at 22 and 23 weeks' gestation (Stoll, 2010).

1	To evaluate contemporaneous outcomes of neonates born at 22 to 24 weeks, the NICHD Neonatal Research Network reported both survival and neurodevelopmental outcomes assessed across consecutive birth-year epochs of 2000 to 2003, 2004 to 2007, and 2008 to 2011 in infants aged 18 to 22 months (Younge, 2017). The percentage of infants who survived rose signiicantly from 30 percent in 2000 to 2003 to 36 percent in 2008 to 2011r. he percentage of infants who survived without neurodevelopmental impairment also significantly grew from 16 percent to 20 percent during the same time period (Fig. 42-3). Although rates of survival without neurodevelopmental impairment increased over time among infants born at 23 and apercentage with disability at 2V2 years corrected age. The overall rate of disabilities includes performance on the Bayley III assessments, mental development delay, cerebral palsy. and visual and hearing disabilities.

1	Bayley III assessments, mental development delay, cerebral palsy. and visual and hearing disabilities. Data from Serenius F, I allen K, Blennow M, et al: infants at 2.5 years after active perinatal care in Sweden, JAMA 201 3 May 1;309( 1 7): '181n0-1n820. 24 weeks, only 1 percent of infants born at 22 weeks survived without neurodevelopmental impairment (Younge, 2017). Somewhat similar results were published from Sweden (Serenius, 2013). his report details a national populationbased prospective study of all neonates born before 27 weeks. Shown in Table 42-3 are the survival and disability rates for 707 Swedish infants born alive from 22 to 26 weeks' gestation between 2004 and 2007 in Sweden. Compared with rates in the United States, rates of survival without neurodevelopmental impairment were higher in the Swedish cohort for infants born at 24 weeks during 2004 to 2007.

1	he Obstetric Care Consensus document also addresses management options based on the clinical characteristics of a given pregnancy. Nonmodiiable factors are fetal gender, weight, and plurality. Potentially modifiable antepartum and intrapartum factors include the location of delivery, intent to intervene FIGURE 42-3 Mortality and neurodevelopmental outcomes at 18 to 22 months of corrected age by birth epoch in neonates born at 22 to 24 weeks. (Data from Younge N, Goldstein RF, Bann eM, et al: Survival and neurodevelopmental outcomes among periviable infants, N Engl J Med. 2017 Feb 16;376(7):617-628.) EFM = electronic fetal monitoring; GBS = group B streptococcus; PPROM = preterm premature rupture of membranes. Data from the American College of Obstetricians and Gynecologists, 2017e; Raju, 2014.

1	EFM = electronic fetal monitoring; GBS = group B streptococcus; PPROM = preterm premature rupture of membranes. Data from the American College of Obstetricians and Gynecologists, 2017e; Raju, 2014. by cesarean delivery or labor induction, and administration of antenatal corticosteroids and magnesium sulfate. Postnatal management addresses the initiation or withdrawal of intensive care after birth. Areas of general guidance were then reviewed for each week of gestation (Table 42-4). Mode of delivery represents another dilemma because cesarean delivery at the threshold of viability is controversial. For example, if the fetus-neonate is perceived to be too immature for aggressive support, then cesarean delivery for common indications such as breech presentation or nonreassuring fetal heart rate patterns might be preempted. Moreover, observational studies have failed to document a benefit of cesarean delivery for the sole indication of periviability (Alirevic, 2013).

1	In a study of 2906 singletons between 24°17 and weeks eligible for attempted vaginal birth, 84 percent of cephalic presenting fetuses were delivered vaginally (Reddy, 2012). Neonatal mortality rates did not difer compared with those associated with planned cesarean delivery. For breech presentations, however, relative risk for mortality was threefold higher with attempted vaginal delivery. In another study, Werner and colleagues (2013) analyzed 20,231 newborns delivered at 24 to 34 weeks. Cesarean delivery did not protect against poor outcomes such as neonatal death, intraventricular hemorrhage, seizures, respiratory distress, and subdural hemorrhage. From these indings, the Obstetric Care Consensus proposes that cesarean delivery be considered for fetal indications at 23°(; to weeks. However, before 22 weeks, this route is reserved only for maternal indications.

1	It is diicult to summarize the current practices of obstetrical care in the management of the periviable pregnancy given its rapid evolution. For example, since January 2016, the American College of Obstetricians and Gynecologists has penned three iterations of its Practice Bulletin "Management of Preterm Labor," and three versions of the "Obstetric Care Consensus" document have been published since November 2015. In this uncertain environment, individualized, patientcentered care with a multidisciplinary team remain as bedrock for the clinician.

1	While we do not have the answers, we describe our strategies from Parkland Hospital as one approach to management. Our policies were developed in conjunction with the Division of Neonatal Medicine. Importantly, the decision not to perform cesarean delivery does not necessarily imply that care for the fetus is discounted. Neonatologists are consulted before delivery, and a trilateral discussion of survival and morbidity rates ensues with the woman and her family. A neonatologist attends each delivery and determines subsequent management. In our institution, traditional fetal indications for cesarean delivery are practiced in women at 25°/7 weeks or beyond. Cesarean delivery is not ofered for fetal indications before 24°/7 weeks. At 240r weeks, cesarean delivery is not ofered unless fetal weight is estimated at 750 g or greater. Aggressive obstetrical management is practiced in cases of growth restriction, wherein gestational age is used to guide management rather than fetal size.

1	Neonates born between 34 and 36 weeks account for more than 70 percent of all preterm births (Fig. 42-4). This group has been the fastest rising proportion of singleton preterm births in the United States (Raju, 2006). Nationally, the late-preterm 32-33 weeks (12.1%) 34-36 weeks (71e.3%) (late preterm) FIGURE 42-4 Distribution of preterm births by gestational age in the United States in 201o5. (Data from Martin JA, Hamilton BE, Osterman MJ: Births: final data for 2015. Natl Vital Stat Rep 66(1):1, 2017.) Spontaneous rupture ofPlacental abruption, 1 % membranes Placenta previa, 1 % 35% Fetalcomplications, 2% Other, 3% FIGURE 42-5 Obstetrical complications associated with 21,771 late-preterm births at Parkland Hospital. (Data from Mcintire DO, Leveno J: Neonatal mortality and morbidity rates in later preterm births compared with births at term, Obstet Gynecol. 2008 Jan;loll (1 ):35-41o.) birth rate rose from 6.82 percent to 6.87 percent from 2014 to 2015 (Martin, 2017).

1	To estimate the risks associated with late-preterm births, investigators analyzed neonatal mortality and morbidity rates at 34, 35, and 36 weeks compared with those of births at term between 1988 and 2005 at Parkland Hospital (McIntire, 2008). Approximately 3 percent of all births during the study period were between 24 and 32 weeks, and 9 percent were during the late-preterm weeks. hus, and similar to the national rates, late-preterm births accounted for three fourths of all preterm births. Approximately 80 percent of these resulted from 1.6 1.5a 1.4 1.2 1.0 0.8 0.6 0.4 0.2

1	FIGURE 42-6 Neonatal death rates at Parkland Hospital from 34 to 40 weeks' gestation in singleton infants without malformations. ap <0.001 compared with 39 weeks as the referent. bp = 0.02 compared with 39 weeks as the referent. (Reproduced with permission from Mcintire DO, Leveno J: Neonatal mortality and morbidity rates in later preterm births compared with births at term, Obstet Gynecol. 2008 Jan;lolol (1 ):35-41.) idiopathic spontaneous preterm labor or prematurely ruptured membranes (Fig. 42-5). Other obstetrical complications were implicated in the remaining 20 percent of cases. Rates of morbidity and mortality were greater in these late-preterm newborns compared with rates in term ones (Table 42-5 and Fig. 42-6). Similarly, T omashek (2017) also reported higher neonatal mortality rates for late-preterm newborns. Rates of TABLE 42-5. Neonatal Morbidity Rates at Parkland Hospital in Live Births Delivered Late Preterm Compared with 39 Weeks Grades 1,2 Grades 3,r4 Sepsis

1	TABLE 42-5. Neonatal Morbidity Rates at Parkland Hospital in Live Births Delivered Late Preterm Compared with 39 Weeks Grades 1,2 Grades 3,r4 Sepsis One or more of the above aData presented as n (%). bp <.001 compared with 39 weeks referent. cp <.05 compared with 39 weeks referent. Reproduced with permission from Mcintire DO, Leveno IJ: Neonatal mortality and morbidity rates in later preterm births compared with births at term, Obstet Gynecol. 2008 Jan;lnll (1 ):35-41r. adverse neurodevelopment outcomes are also increased in these late-preterm infants (Petrini, 2009). Taken together, these findings suggest that a health-care focus on prematurity should include these late-pre term births.

1	Taken together, these findings suggest that a health-care focus on prematurity should include these late-pre term births. Moreover, approximately 80 percent of afected women begin labor spontaneously-similar to births before 34 weeks-and attempts to interrupt preterm labor have been insuicient (Institute of Medicine, 2007). Because of this, a national strat egy aimed at prevention of late-preterm births is unlikely to provide discernible benefit without new developments in the prevention and management of preterm labor. In the mean time, the American College of Obstetricians and Gynecolo for delivery exists.

1	Four direct causes for preterm births in the United States include: (1) spontaneous unexplained preterm labor with intact membranes, (2) idiopathic preterm premature rupture of membranes (PPROM), (3) delivery for maternal or fetal indications, and (4) twins and higher-order multi fetal births. Of all preterm births, 30 to 35 percent are indicated, 40 to 45 percent are due to spontaneous preterm labor, and 30 to 35 percent follow preterm membrane rupture (Goldenberg, 2008). Indeed, much of the increase in the singleton preterm birth rate in the United States is explained by rising numbers of indicated preterm births (Ananth, 2005). Last, more than one of every two twins and more than nine of every ten triplets are born preterm or with low birthweight in the United States (Chap. 45, p. 873) (Martin, 2017). Reasons for preterm birth have multiple, often interacting, antecedents and contributing factors (Esplin, 2016). This is particularly true for PPROM and spontaneous preterm labor.

1	Reasons for preterm birth have multiple, often interacting, antecedents and contributing factors (Esplin, 2016). This is particularly true for PPROM and spontaneous preterm labor. Analogous to other complex disease processes, multiple coexistent genetic alterations and environmental factors may lead to preterm birth (Esplin, 2005; Velez, 2008; Ward, 2008). For example, inherited mutations in genes regulating collagen assembly may predispose to cervical insuiciency or prematurely ruptured membranes (Anum, 2009; Wang, 2006; Warren, 2007). And, whole blood gene expression and proteonomic biomarkers are now being used to help identiy predictors of preterm birth (Cantonwine, 2016; Heng, 2016).

1	For both clinical and research purposes, pregnancies with spontaneous preterm labor yet intact fetal membranes must be distinguished from those complicated by preterm prematurely ruptured membranes. Even so, those with spontaneous preterm labor do not constitute a homogeneous group. Among the more common associated findings are multi fetal pregnancy, intrauterine infection, bleeding, placental infarction, premature cervical dilation, cervical insuiciency, hydramnios, uterine fundal abnormalities, and fetal anomalies. Severe maternal illness from infections, autoimmune diseases, and gestational hypertension also raises preterm labor risks.

1	Despite their diversity, these processes culminate in a common end point-premature cervical dilation and efacement and premature activation of uterine contractions. Importantly, the actual process of preterm labor should be considered a final step stemming from progressive or acute changes that could be initiated days or even weeks before labor onset. Indeed, many forms of spontaneous preterm labor that result from premature initiation of phase 2 of parturition may be viewed in this light (Chap. 21, p. 408). Although the end result in preterm birth is the same as at term with cervical ripening and myometrial activation, recent studies in animal models support the idea that preterm birth is not always an acceleration of the normal process. Diverse pathways to instigate parturition exist and are dependent on the etiology of preterm birth. Four major causes include uterine distention, maternal-fetal stress, premature cervical changes, and infection.

1	Multifetal pregnancy and hydramnios are well-recognized risks for preterm birth. Early uterine distention likely acts to initiate expression of contraction-associated proteins (CAPs) in the myometrium. he CAP genes that are influenced by stretch include those coding for gap-junction proteins such as connexin 43, for oxytocin receptors, and for prostaglandin synthase (Korita, 2002; Lyall, 2002; Sooranna, 2004). More recent reports suggest that levels of gastrin-releasing pep tides (GRPs) are increased with stretch to promote myometrial contractility. GRP antagonists can inhibit uterine contractility (Tattershell, 2012). Also, a stretch-induced potassium channel-TREK-1is up regulated during gestation and downregulated in labor. his suggests a potential role in uterine relaxation during pregnancy (Buxton, 2010).

1	Excessive uterine stretch also leads to early activation of the placental-fetal endocrine cascade shown in Figure 21-10 (p. 410). The resultant early rise in maternal corticotropinreleasing hormone and estrogen levels can further enhance the expression of myometrial CAP genes (Warren, 1990; Wolfe, 1988). Finally, the influence of uterine stretch should be considered with regard to the cervix. Prematurely increased stretch and endocrine activity may initiate events that shift the timing of uterine activation, including premature cervical ripening.

1	Stress is deined as a condition or adverse circumstance that disturbs the normal physiological or psychological functioning of an individual. Examples of stressors are nutrient restriction, obesity, infection, and diabetes. Psychological duress can include racial discrimination, childhood stress, depression, or posttraumatic stress syndrome (Gillespie, 2017; Goldstein, 2017; Shaw, 2017). A quantitative measure of "stress" is difficult. Yet, considerable evidence shows a correlation between some degree of maternal stress and adverse birth outcomes that include stillbirth, preterm birth, and abnormal fetal development (Hobel, 2003; Ruiz, 2003). Factors that activate this cascade likely are broad and inluence the stress response.

1	One potential mechanism for stress-induced preterm birth is premature activation of the placental-adrenal endocrine axis. One trigger may be elevations in cortisol from maternal psychological stress (Lockwood, 1999; Petraglia, 2010; Wadhwa, 2001). Activation of this axis yields rising maternal serum levels of placentalderived corticotropin-releasing hormone (CRH). This raises adult and fetal adrenal steroid hormone production and promotes early loss of uterine quiescence (Fig. 21-10, p. 410). If preterm delivery is associated with early activation of the fetal adrenal-placental endocrine axis, maternal estrogen levels would likely be prematurely elevated. Indeed, an early rise in serum estriol concentrations is noted in women with subsequent preterm labor (Heine, 2000; McGregor, 1995). Physiologically, this premature rise in estrogen levels may alter myometrial quiescence and accelerate cervical ripening.

1	Another mechanism by which stress may translate to preterm birth is premature cellular senescence. As part of normal physiology, aging of fetal and decidual cells precipitates the release of uterotonic signals for uterine activation at term (Menon, 20 14a). Animal studies supported by correlative studies in women demonstrate that accelerated senescence of the decidua results in preterm birth (Cha, 2013; Hirota, 2010). In addition, premature cellular senescence may contribute to PPROM (Menon, 2016).

1	In most cases, premature cervical remodeling precedes premature labor onset. In some instances, cervical dysfunction of either the epithelia or stromal extracellular matrix is the underlying cause. For example, an intact cervical epithelial barrier is critical to prevent ascending infection. Disruption of this barrier, such as that in mice lacking the glycosaminoglycan hyaluronan, predisposes mice to ascending infection and preterm birth (Akgul, 2014). Interestingly, the enhanced risk of preterm birth from group B streptococcal colonization may be in part due to the bacteria's ability to secrete hyaluronidase. his enzyme degrades hyaluronic acid in the cervicovaginal epithelia to aid bacterial ascension (V ornhagen, 2017).

1	Second, the mechanical competence of the cervix can be reduced. For example, genetic mutations in components of collagen and elastic fibers or proteins required for their assembly are risk factors for cervical insuiciency, PPROM, and preterm birth (Anum, 2009; Nallasamy, 2017; Pyeritz, 2000).

1	A patent female reproductive tract, although essential for conception and delivery, is theoretically problematic during phase 1 of parturition. Bacteria can gain access to intrauterine tissues through: (1) transplacental transfer of maternal systemic infection, (2) retrograde flow of infection into the peritoneal cavity via the fallopian tubes, or (3) ascending infection with bacteria from the vagina and cervix. Because the lower pole of the fetal membrane-decidual junction is contiguous with the cervical canal oriice, this anatomical arrangement provides a passageway for microorganisms. Ascending infection is considered to be the most common entry route. Ascending microorganisms colonize the cervix, decidua, and possibly the membranes, where they then may enter the amnionic sac.

1	Intraamnionic infection as a primary cause of preterm labor in pregnancies with intact membranes accounts for 25 to 40 percent of preterm births (Goncalves, 2002; lams, 1987). In some instances, histological evidence of inflammation is found in the fetal membranes, decidua, or umbilical cord. Other cases are deemed "subclinical." Current data suggest that microbial invasion of the reproductive tract is suicient to induce infection-mediated preterm birth. ffected women are more likely to develop clinical chorioamnionitis and PPROM compared with women with sterile cultures. Moreover, their neonates are also more likely to have perinatal complications that include RDS, intraventricular hemorrhage, and necrotizing enterocolitis (Hitti, 2001). Although the clinical course is more severe when intraamnionic infection is obvious, inlammation in the absence of detectable intraamnionic microorganismstermed sterile intraamnionic inlammation-is also a risk factor for an inlammatory response,

1	intraamnionic infection is obvious, inlammation in the absence of detectable intraamnionic microorganismstermed sterile intraamnionic inlammation-is also a risk factor for an inlammatory response, described in the next section (Lee, 2007, 2008; Romero, 2014). In sum, the earlier the onset of preterm labor, the greater the likelihood of underlying infection (Goldenberg, 2000; Goncalves, 2002; Watts, 1992).

1	he incidence of culture-positive amnionic luid collected by amniocentesis during spontaneous term labor is similar to that with preterm labor (Gomez, 1994; Romero, 1993). It has been suggested that at term, amnionic luid is iniltrated by bacteria as a consequence oflabor, whereas in preterm pregnancies, bacteria represent an inciting cause of labor. hus, fetal infection, as deined by bacteria detected within the amnionic luid, has both difering etiologies and consequences.

1	Despite these observations, considerable data associate chorioamnionitis with preterm labor (Goldenberg, 2002; Ustiin, 2001). With chorioamnionitis, microbes may invade maternal tissue only and not amnionic fluid. Despite this, endotoxins can stimulate amnionic cells to secrete cytokines that enter amnionic luid. his scenario may serve to explain the apparently contradictory observations concerning an association between amnionic luid cytokines and preterm labor in cases in which microbes are not detected in the amnionic luid.

1	Inflammatory Responses. Inlammatory responses drive the pathogenesis of infection-induced preterm labor. lipopolysaccharide (LPS) or other toxins elaborated by bacteria are recognized by pattern-recognition receptors such as toll-like receptors (Janssens, 2003). hese receptors are present on mononuclear phagocytes, decidual cells, cervical epithelia, and trophoblasts (Chuang, 2000; Gonzalez, 2007; Holmlund, 2002). Loss of specific toll-like receptors results in delayed parturition in mouse models (Montalbano, 2013). Instead, activation of tolllike receptors induces a signaling cascade that activates production of chemokines such as interleukin 8 (IL-8) and cytokines such as IL-13. Activation also recruits immune cells into the reproductive tract. Cytokines are produced by immune cells and by cells within the cervix, decidua, membranes, or fetus itsel.

1	LPS-induced production ofIL-13 in turn promotes a series of responses that include: (1) increased synthesis of others, that is, IL-6, IL-8, and tumor-necrosis factor alpha (TNF-.); (2) proliferation, activation, and migration of leukocytes; (3) modiications in extracellular matrix proteins; and (4) mitogenic and cytotoxic efects such as fever and acute-phase response (EI-Bastawissi, 2000). Also, in many tissues, including myometrium, decidua, and amnion, IL-1 3 promotes prostaglandin formation that induces cervical ripening and loss of myometrial quiescence (Casey, 1990; Challis, 2002; Keelan, 2003). he importance of prostaglandins to infection-mediated pre term birth is supported by the observation that prostaglandin inhibi tors can reduce the rate of LPS-induced preterm birth in both the mouse and nonhuman primate (Gravett, 2007; Timmons, 2014). Inhibition of cyclooxygenase-2 prevents inlammation mediated preterm labor in the mouse. And, immunomodula intraamnionic infection in a

1	the mouse and nonhuman primate (Gravett, 2007; Timmons, 2014). Inhibition of cyclooxygenase-2 prevents inlammation mediated preterm labor in the mouse. And, immunomodula intraamnionic infection in a nonhuman primate model.

1	Proteases such as matrix metalloproteinases (MMPs) are also induced by IL-1 3 and function to break down extracel lular matrix components such as collagen or elastic ibers. This disrupts the structural integrity of fetal membranes and the cer vix. Current evidence from animal and human studies suggests that many aspects of infection-mediated preterm birth difer from pathways that regulate term parturition (Hamilton, 2012; Holt, 2011; Shynlova, 2013a,b; Timmons, 2014). Origin of Cytokines. Uterine cytokines are likely important for preterm labor. For example, it appears that cytokines produced side, whereas cytokines produced in the membranes or in cells within the amnionic fluid will not be transferred to maternal tissues. he transfer of cytokines such as IL-1 3 from decidua across the membranes into amnionic luid, however, appears to be severely limited. Additionally, the human myometrium expresses chemokine receptors that decline during labor (Hua, 2013).

1	he requirement of leukocytes for initiation of term labor in women remains inconclusive. In general, resident and invading leukocytes produce the bulk of cytokines in cases of inlammation resulting from infection. Indeed, with infection, leukocytesmainly neutrophils, macrophages, and T lymphocytes-iniltrate the cervix, lower uterine segment, fundus, and membranes at the time of labor. Invading leukocytes and certain parenchymal cells produce cytokines and appear to be the primary source of myometrial cytokines (Young, 2002). By contrast, in the decidua, both stromal cells and leukocytes likely contribute. In the cervix, glandular and surface epithelial cells appear to produce cytokines.

1	he presence of cytokines in amnionic fluid and their association with preterm labor is well documented. But, their exact cellular origin-with or without recoverable microorganismsis not well deined. Amnionic fluid cytokines are most likely secreted by mononuclear phagocytes or neutrophils activated and recruited into the amnionic luid. Thus, the amount of amnionic luid IL-1 3 would be determined by the number of leukocytes recruited, their activational status, or the efect of amnionic fluid constituents on their IL-1 3 secretion rate.

1	Vaginal Microbiota. Factors that predispose to ascending infection and then preterm birth are a key focus of ongoing basic research. \1ucosal immunity and barrier function of the cervicovaginal epithelia, the microbiota composition in the vaginal tract, and their interplay are a major topic (Smith, 2017). From animal studies, mucosal immunity of the lower reproductive tract can be disrupted by viral infection with a subsequent enhanced susceptibility to ascending bacterial infection (Racicot, 2013, 2017). Along with the ability of the cervico vaginal epithelia to respond to environmental insults, the com position of the microbe ecosystem in the vaginal tract may also determine susceptibility to ascending infection. that the nonpregnant vaginal tract hosts a complex microbial community (Gajer, 2012; White, 201l). Also described in

1	that the nonpregnant vaginal tract hosts a complex microbial community (Gajer, 2012; White, 201l). Also described in Chapter 65 (p. 1245), these community state types can difer widely among women who are all healthy. And, the vaginal microbiome changes during normal pregnancy (Aagaard, 2012; Stout, 2017). Namely, the diversity and richness of microbe populations are reduced during pregnancy and become more stable. Compared with nonpregnant controls, Lactobacillus species show an enhanced dominance. Some but not all stud ies report an increased population of certain microbes-for example, Gardnerella vaginalis and Ureaplasma ureayticumqi in women with preterm birth (Donders, 2009; Nelson, 2014). However, diferences in populations of pregnant women stud ied, in deinitions of preterm birth, and in data analysis may complicate interpretation of these data.

1	Some speciic microorganisms are detected more frequently than others in amnionic fluid of women with preterm labor (Gerber, 2003; Hillier, 1988; Yoon, 1998). hese include G vaginalis, Fusobacterium species, Mycoplasma hominis, and U ureayticum. Their identification was interpreted by some as presumptive evidence that speciic microorganisms are more commonly involved as pathogens in the induction of preterm labor. Another interpretation, however, is that given direct access to the membranes after cervical dilation, selected micro organisms, such as fusobacteria, are more capable of burrowing through these exposed tissues and will do so. Fusobacteria are found in the vaginal luid of only 9 percent of women but in 28 percent of positive amnionic luid cultures from pregnancies with preterm labor and intact membranes (Chaim, 1992).

1	• Preterm Premature Rupture of Membranes his term deines spontaneous rupture of the fetal membranes before 37 completed weeks and before labor onset (American College of Obstetricians and Gynecologists, 20 16d). Such rupture likely has various causes, but intrauterine infection, oxidative stress-induced DNA damage, and premature cellular senescence are major predisposing events (Dutta, 2016; Gomez, 1997; Mercer, 2003). Associated risk factors include lower socioeconomic status, body mass index < 19.8, nutritional deiciencies, and cigarette smoking. Women with PPROM carry an enhanced risk for recurrence during a subsequent pregnancy (Bloom, 2001). Despite these known risk factors, none is identified in most cases of pre term rupture.

1	Increased apoptosis or necroptosis of membrane cellular components and greater levels of specific proteases in membranes and amnionic luid are related to PPROM. Most tensile strength of the membranes is provided by the amnionic extracellular matrix and interstitial amnionic collagens that are produced in mesenchymal cells (Casey, 1996). Thus, collagen degradation has been a focus of research. he MMP family is involved with normal tissue remodeling and particularly with collagen degradation. Some members are found in higher concentrations in amnionic luid from pregnancies with PPROM (Maymon, 2000; Park, 2003; Romero, 2002). MMP activity is in part regulated by tissue inhibitors of matrix metalloproteinases-TI\1Ps. Several of these inhibitors are found in lower concentrations in amnionic fluid from women with ruptured membranes. Elevated MMP levels found at a time when protease inhibitor expression declines urther supports that their expression alters amnionic tensile strength.

1	Studies of amniochorion explants show greater MMP expression following treatment with certain cytokines (Fortunato, 1999a,b, 2002). With membrane rupture, thrombin activity rises, which activates MMPs and prostaglandin synthesis. Studies by Mogami (2013) provide a mechanism b) which bacterial endotoxin or TNF-. elicits release of fetal fibronectin (FN) by amnion epithelial cells. The FN then binds toll-like receptor 4 in the amnion mesenchymal cells to activate signaling cascades. hese result in augmented prostaglandin E (PGEJ synthesis and elevated activity of MJlIPs. Higher prostaglandin levels promote cervical ripening and uterine contractions. Greater MMP concentrations allow collagen breakdown in the fetal membranes resulting in premature rupture.

1	In pregnancies with PPROM, the amnion exhibits a higher degree of cell death and more apoptosis markers than that in term amnion (Arechavaleta-Velasco, 2002; Fortunato, 2003). In vitro studies indicate that apoptosis is likely regulated by bacterial endotoxin, IL-13, and TNF-.. In addition, oxidative stress initiated by events other than infection can induce DNA damage, premature senescence, and subsequent inflammation and proteolysis that leads to PPROM (Menon, 2014a,b). Last, there are proteins involved in the synthesis of mature crosslinked collagen or matrix proteins that bind collagen and thereby promote tensile strength. These proteins are altered in membranes with premature rupture (Wang, 2006).

1	Several studies have investigated the incidence of infectioninduced PPROM. Bacterial cultures of amnionic luid support a role for infection in a significant proportion. One review of 18 studies and almost 1500 women with PPROM found that bacteria were isolated from amnionic fluid in a third of cases (Goncalves, 2002). Accordingly, some have given antimicrobial treatment to women in spontaneous preterm labor with intact membranes, however, results have been disappointing as discussed later on page 825 (Kenyon, 2008b). The inflammatory response that leads to membrane weakening and mediators of this process are currently areas of research. One goal is to identiy early risk markers for PPROM.

1	The inflammatory response that leads to membrane weakening and mediators of this process are currently areas of research. One goal is to identiy early risk markers for PPROM. Twins and higher-order multifetal births account for approximately 3 percent of neonates born in the United States (Martin, 2017). Preterm delivery continues to be the major cause of the excessive perinatal morbidity and mortality with multifetal pregnancies. The efects of uterine stretch discussed on page 809 are obvious. Many of these interrelationships are discussed in Chapter 45 (p. 885).

1	Several genetic and environmental factors afect the frequency of preterm labor. Of these, threatened abortion in early pregnancy is associated with higher rates of later adverse outcomes. Weiss (2004) reported outcomes with vaginal bleeding at 6 to 13 weeks' gestation in nearly 14,000 women. Both light and heavy bleeding were associated with subsequent preterm labor, placental abruption, and pregnancy loss before 24 weeks. Birth defects in the fetus may also predispose to preterm birth. In a secondary analysis of data from the First-and Second-Trimester Evaluation of Risk (FASTER) Trial, birth defects were associated with preterm birth and low birthweight neonates (Dolan, 2007).

1	Cigarette smoking, inadequate maternal weight gain, and illicit drug use afect the incidence and outcome of low-birthweight neonates (Chap. 44, p. 849). Extremes of maternal weight-both underweight and obese mothers-have an enhanced risk of preterm birth (Cnattingius, 2013; Girsen, 2016). Other maternal factors implicated include young or advanced maternal age, poverty, short stature, and vitamin C deficiency (Casanueva, 2005; Gielchinsy, 2002; Kramer, 1995; Leveno, 2009; Meis, 1995). As discussed on page 809, psychological factors such as depression, anxiety, and chronic stress are associated with preterm birth (Hofman, 2016; Venkatesh, 2016). In one review of more than 50 studies, Donovan and coworkers (2016) found a signiicant link between low birthweight and preterm birth in women injured by physical abuse (Chap. 47, p. 925).

1	Studies of work and physical activity related to preterm birth have yielded conlicting results (Goldenberg, 2008). Some evidence suggests that working long hours and hard physical labor are probably linked to a higher risk of preterm birth (Luke, 1995). However, aerobic exercise in normal-weight women with uncomplicated singleton pregnancies appears to be safe and not associated with preterm birth (American College of Obstetricians and Gynecologists, 2017d; Di Mascio, 2016). One metaanalysis of physical activity found that leisure-time physical activity was associated with a reduced risk of preterm birth (Aune, 2017).

1	he recurrent, familial, and racial nature of preterm birth suggests that genetics may playra causal role. Accumulating literature on genetic variants buttresses this concept (Gibson, 2007; Hampton, 2006; Macones, 2004; Velez, 2009). Several such studies have also implicated immunoregulatory genes in potentiating chorioamnionitis in cases of preterm delivery due to infection (Varner, 2005). Gum inflammation is a chronic anaerobic inflammation that afects as many as 50 percent of pregnant women in the United

1	Gum inflammation is a chronic anaerobic inflammation that afects as many as 50 percent of pregnant women in the United States (Goepfert, 2004). Vergnes and Sixou (2007) performed a metaanalysis of 17 studies and concluded that periodontal disease was signiicantly associated with preterm birth. To better study this relationship, Michalowicz (2006) randomly assigned 813 pregnant women between 13 and 17 weeks' gestation who had periodontal disease to treatment during pregnancy or postpartum. Treatment during pregnancy improved periodontal disease and was safe. However, treatment failed to significantly alter preterm birth rates. his position was reafirmed by a joint workshop of the European Federation of Periodontology and the American Academy of Periodontology (Sanz, 2013).

1	he intervals between pregnancies are linked with adverse perinatal outcomes. In a metaanalysis, intervals < 18 months and > 59 months were associated with greater risks for both preterm birth and small-for-gestational age newborns (Conde-Agudelo, 2006). he causal efect of short interpregnancy intervals, however, has been questioned (Ball, 2014).

1	he most important risk factor for preterm labor is a prior preterm delivery. Data from nearly 16,000 women delivered at Parkland Hospital are instructive. Namely, the recurrent preterm delivery risk for women with a preterm first delivery was threefold greater than that of women whose first neonate was born at term. More than a third of women whose irst two newborns were preterm subsequently delivered a third preterm newborn. Most-70 percent-of the recurrent births in this study occurred within 2 weeks of the gestational age of the prior preterm delivery. he causes of prior preterm delivery also recurred. Although women with prior preterm births are clearly at risk for recurrence, they represented only 10 percent of the total preterm births in this study. Expressed another way, 90 percent of the preterm births at Parkland Hospital could not be predicted based on a history of preterm birth. Laughon and associates (2014) confirmed the importance of prior spontaneous preterm birth.

1	of the preterm births at Parkland Hospital could not be predicted based on a history of preterm birth. Laughon and associates (2014) confirmed the importance of prior spontaneous preterm birth. Notably, these investigators also found that prior indicated preterm birth was strongly associated with subsequent spontaneous preterm birth. Variations in the deinition of spontaneous and indicated used may explain this association.

1	Ultimately, risk of recurrent preterm birth is influenced by three factors: the frequency of prior preterm deliveries, severity as measured by gestational age, and the order in which the prior preterm delivery occurred (McManemy, 2007). That is, an individual woman's risk for recurrent preterm birth is inluenced by her past number and sequence of preterm and term births. For example, a risk of recurrent preterm birth for a gravida 3 para 2 woman with a prior preterm birth followed by a term birth difers from a woman with a prior term birth followed by preterm birth. Thus, the inluence of reproductive history has a profound prognostic significance for risk of recurrence. Moreover, this may also inluence the supposed benefit attributed to various interventions described later.

1	As discussed on page 810, a link between some cases of preterm birth and infection seems irrefutable (Goldenberg, 2008). In several studies, antimicrobial treatment has been given to prevent preterm labor thought to be due to microbial invasion. These strategies especially targeted Mycoplasma species. Morency and colleagues (2007) performed a metaanalysis of 61 articles and suggested that antimicrobials given in the second trimester may prevent subsequent preterm birth. Andrews and associates (2006) reported results of a randomized trial in which they gave a course of azithromycin plus metronidazole every 4 months to 241 nonpregnant women whose last pregnancy resulted in spontaneous delivery before 34 weeks. Approximately 80 percent of the women with subsequent pregnancies had received study drug within 6 months of their subsequent conception. Such interconceptional antimicrobial treatment did not reduce the rate of recurrent preterm birth. Using a subgroup analysis of these same

1	study drug within 6 months of their subsequent conception. Such interconceptional antimicrobial treatment did not reduce the rate of recurrent preterm birth. Using a subgroup analysis of these same women, Tita and coworkers (2007) concluded that such use of antimicrobials may be harmful. In another randomized study, 2661 women received placebo or metronidazole plus erythromycin between 20 and 24 weeks' gestation followed by ampicillin plus metronidazole during labor (Goldenberg, 2006). his antimicrobial regimen did not reduce the rate of preterm birth or that of histological chorioamnionitis. At this time, antibiotic prophylaxis to prevent preterm birth is not recommended in women with preterm labor and intact membranes (Flenady,r2013).

1	In this condition, normal, hydrogen peroxide-producing, lactobacillus-predominant vaginal flora is replaced with anaerobes (Hillier, 1995; Nugent, 1991). Its diagnosis and management are discussed in Chapter 65 (p. 1245). Using Gram staining, relative concentrations of the bacterial morphotypes characteristic of bacterial vaginosis are determined and graded by the Nugent score or assessed clinically with Amsel criteria.

1	Bacterial vaginosis has been associated with spontaneous abortion, preterm labor, PPROM, chorioamnionitis, and amnionic fluid infection (Hillier, 1995; Kurki, 1992; Leitich, 2003a,b). Environmental factors appear to be important in bacterial vaginosis development. Exposure to chronic stress, ethnic diferences, and frequent or recent douching are associated with higher rates of the condition (Culhane, 2002; Ness, 2002). A gene-environment interaction has also been described (Macones, 2004). Women with bacterial vaginosis and a susceptible TNF-a genotype had a ninefold increased incidence of preterm birth.

1	From all of these studies, adverse vaginal flora seems associated with spontaneous preterm birth. Unfortunately, to date, screening and treatment have not prevented preterm birth. These are discussed further in Chapter 65 (p. 1246). Indeed, microbial resistance or antimicrobial-induced change in the vaginal flora results from regimens intended to eliminate bacterial vaginosis (Beigi, 2004; Carey, 2005).

1	Early diferentiation between true and false labor is diicultespecially before demonstrable cervical efacement and dilation. Uterine activity alone can be misleading because of Braxton Hicks contractions. These irregular, nonrhythmical contractions can cause considerable confusion in the diagnosis of true labor. Not infrequently, women who deliver before term have uterine activity that is attributed to Braxton Hicks contractions that prompt an incorrect diagnosis of false labor. Accordingly, the American College of Obstetricians and Gynecologists (2016b) defines preterm labor to be regular contractions before 37 weeks that are associated with cervical change. In addition to contractions, pelvic pressure, menstrual-like cramps, watery vaginal discharge, and lower back pain have been empirically associated with impending preterm birth. Such complaints are thought by some to be common in normal pregnancy and are therefore often minimized by patients and obstetrical care providers.

1	he importance of these symptoms as a harbinger of labor has been emphasized by some but not all investigators (lams, 1990; Kragt, 1990). lams and coworkers (1994) found that the signs and symptoms signaling preterm labor, including uterine contractions, appeared only within 24 hours of preterm labor.

1	Chao (2011) prospectively studied 843 women with a singleton fetus who presented to Parkland Hospital with preterm labor symptoms between 24°/7 and 336/7 weeks, intact membranes, and cervical dilation <2 em. hose whose cervix remained <2 em were sent home with a diagnosis of false preterm labor. When analyzed against the general obstetrical population, women sent home had a similar rate of birth before 34 weeks-2 versus 1 percent. However, these women did have significantly higher rates of birth between 34 and 36 weeks-5 percent compared with 2 percent. Women with cervical dilation of 1 em at discharge were signiicantly more likely to deliver before 34 weeks compared with women without cervical dilation-5 percent versus 1 percent. Importantly, almost 90 percent of the I-em group delivered within 21 days of the initial presentation.

1	Researchers have evaluated asymptomatic cervical changes that may predict preterm labor. Asymptomatic cervical dilation after midpregnancy is suspected to be a risk factor for preterm delivery, although some clinicians consider it to be a normal anatomical variant. Moreover, study results have suggested that parity alone is not suicient to explain cervical dilation discovered early in the third trimester.

1	Cook (1996) longitudinally evaluated cervical status with transvaginal sonography between 18 and 30 weeks in nulliparas and multiparas who all subsequently gave birth at term. Cervical length and diameter were identical in both groups throughout these critical weeks. In a study from Parkland Hospital, routine digital cervical examinations were performed between 26 and 30 weeks in 185 asymptomatic women. Approximately 25 percent of women whose cervix was dilated 2 or 3 em delivered before 34 weeks (Leveno, 1986a). Other investigators have verified cervical dilation as a predictor of increased preterm delivery risk (Copper, 1995) .

1	Although women with dilation and efacement in the third trimester are at greater risk for preterm birth, detection does not necessarily improve pregnancy outcome. Buekens and associates (1994) randomly assigned 2719 women to undergo routine cervical examinations at each prenatal visit and compared them with 2721 women in whom serial examinations were not performed. Knowledge of antenatal cervical dilation did not afect any pregnancy outcome related to preterm birth or the frequency of interventions for preterm labor. The investigators also reported that cervical examinations were not related to PPROM. hus, it seems that prenatal cervical examinations in asymptomatic women are neither beneficial nor harmful.

1	An external tocodynamometer belted around the abdomen and connected to an electronic waist recorder allows a woman to ambulate while uterine activity is recorded. Results are transmitted via telephone daily. Women are educated concerning signs and symptoms of preterm labor, and clinicians are kept apprised of their progress. The 1985 approval of this monitor by the Food and Drug Administration (FDA) prompted its widespread clinical use. Subsequently, it was proven that the use of this expensive and time-consuming system does not reduce preterm birth rates (Collaborative Home Uterine Monitoring Study Group, 1995; lams, 2002; Urquhart, 2017). Despite improvements in technology with the internet and cellular telephones, use of such monitoring is discouraged (merican College of Obstetricians and Gynecologists, 20 16c).

1	his glycoprotein is produced in 20 diferent molecular forms by various cell types, including hepatocytes, fibroblasts, endothelial cells, and fetal amnion cells. Present in high concentrations in maternal blood and amnionic luid, fFN is thought to function in intercellular adhesion during implantation and in maintenance of placental adherence to uterine decidua (Leeson, 1996). Detected in cervicovaginal secretions in women who have normal pregnancies with intact membranes at term, fFN appears to reflect stromal remodeling of the cervix before labor.

1	Lockwood (1991) reported that FN detection in cervicovaginal secretions before membrane rupture was a possible marker for impending preterm labor. Qualitative and quantitative FN levels are measured using enzyme-linked immunosorbent assays, and values exceeding 50 ng/mL are considered positive. Sample contamination by amnionic luid and maternal blood should be avoided. Interventional studies based on the use of FN screening in asymptomatic women have not demonstrated improved perinatal outcomes (Andrews, 2003; Esplin, 2017; Grobman, 2004). The American College of Obstetricians and Gynecologists (2016c) does not recommend screening with FN tests. Its use in conjunction with cervical length measurement is discussed next.

1	are associated with increased rates of preterm birth (lams, 1996). he technique to measure cervical length with sonog raphy is described in Chapter 10 (p. 189). When performed by trained operators, cervical length analysis using transvagi nal sonography is safe, highly reproducible, and more sensitive lege of Obstetricians and Gynecologists, 20 16c). The Society for the performance of proper cervical length measurement. obtain speciic training in the acquisition and interpretation of cervical length imaging through accreditation programs. Transvaginal cervical sonography is not afected by maternal obesity, cervix position, or shadowing from the fetal presenting part. Because of the inability to easily distinguish the lower uterine segment from the cervix in early gestation, transvaginal cervical length assessment is typically performed ater 16 weeks' gestation.

1	Such interrogation is currently limited to singleton gestations and not recommended for multifetal gestations outside of research tri als (American College of Obstetricians and Gynecologists, 2016c). Indications for cervical length measurement are somewhat controversial. For those women with a history of prior spontaneous preterm birth, the Society for Maternal-Fetal Medicine (20 16b) recommends transvaginal cervical length screening. But, the American College of Obstetricians and Gynecologists (2016c) only recommends consideration of screening for this indication. In women with singleton pregnancies but without a history of prior preterm birth, the Society for Maternal-Fetal Medicine (20 16b) views cervical length screening as reasonable yet acknowledges that this remains an area of debate.

1	A irst concern with screening surrounds the eicacy of interventions to improve perinatal outcomes once cervical length screening has isolated at-risk gravidas. Of interventions, cervical cerclage and administration of vaginal progesterone have both been evaluated. Vaginal progesterone for this indication is discussed on page 817. For prophylactic cerclage in women with prior preterm births and shortened cervices, many studies have failed to show superior primary outcomes. But, piecemeal subgroup analyses from the trials have been subsequently used as the basis for recommendations in current practice guidelines regarding cervical length assessment and consideration for cerclage placement. A second concern is the accuracy and utility of the screening test, especially in low-risk women, who represent most of the population with preterm birth (p. 813).

1	To address this issue in low-risk women, Esplin and colleagues (2017) prospectively studied 9410 nulliparas with singleton pregnancies. Universal screening of sonographically measured cervical length and quantitative measurement of vaginal fFN levels were evaluated as predictors of women who would spontaneously deliver before 37 weeks. hese measures had poor predictive performance as a screening test. In fact, all screening modalities had relative low sensitivity and low positive-predictive values. Based on these indings, routine use of these screening tests in this low-risk population is not recommended. Bloom and Leveno (2017) subsequently critiqued the use of transvaginal cervicalrlength screening in low-risk women and the promulgation of consensus guidelines. As described in Chapter 1 (p. 7), they highlighted the staggering costs encumbering the health-care system in the United States as a result of such strategies.

1	Prevention of preterm birth remains an elusive goal. Still, may be achievable. Of options, cerclage placement may be used to prevent pre term birth in at least three circumstances. First, the procedure may beneit women who have a history of recurrent midtrimester losses and who are diagnosed with cervical insuiciency. A second instance is the woman identiied during sonographic examination to have a short cervix. The third indication is a "rescue" cerclage, done emergently when cervical incompetence is recognized in women with threatened preterm labor.

1	As is the case for virtually all obstetrical conditions, an accurate history is critical for management decisions. For recurrent abortion from cervical incompetence, historical clues are outlined in Chapter 18 (p. 354). For women with a short cervix incidentally detected by sonography, the beneit of cerclage placement appears directly related to whether the woman has a history of prior preterm birth. In those without a prior preterm birth, cerclage for a sonographically detected short cervix alone ofers no advantage. To and associates (2004) screened 47,123 women and randomly assigned the 253 women with cervices < 15 mm, with or without a history of preterm birth, to cerclage or no cerclage. he frequency of preterm delivery before 33 weeks did not difer signiicantly. In contrast, women with a sonographically diagnosed short cervix and a history of preterm birth may beneit. Owen and colleagues (2009) randomly assigned 302 women with prior preterm birth and with a short cervix-deined as

1	a sonographically diagnosed short cervix and a history of preterm birth may beneit. Owen and colleagues (2009) randomly assigned 302 women with prior preterm birth and with a short cervix-deined as length <25 mm-to cerclage or no procedure. The primary study outcome was not supported by the intervention. However, women with a cervical length < 15 mm delivered before 35 weeks signiicantly less oten following cerclage compared with women with no cerclage-30 versus 65 percent. his study suggested that recurrent preterm birth could be prevented in a subset of women with asymptomatic singleton gestations with both previous pre term birth and short cervical length.

1	These findings prompted a reassessment by Berghella and coworkers (201r1), who performed a metaanalysis using individual patient data (Fig. 42-7). he primary outcomes from the included trials did not support cerclage placement. However, these investigators concluded that cerclage signiicantly prevented preterm birth and improved composite perinatal mortality and morbidity in women with prior spontaneous preterm birth, singleton gestation, and cervical length <25 mm. One caveat in the interpretation of this cerclage data is the inluence of obstetrical history. For example, all of the trials comprising the metaanalysis included preterm birth as early as 16 to 17 weeks' gestation. Deining these early second trimester losses as preterm births, rather than cervical incompetence, is problematic. Thus, it is diicult to distinguish whether these women were treated in the context of cervical incompetence or of preterm labor at 16 weeks. Nonetheless, based on these

1	Risk Ratio StudyorSubgroup M-H, Fixed, 95% CI 0.01 0.1 10 100 FIGURE 42-7 Cerclage versus no cerclage for prevention of recur rent preterm birth in women with a cervical length <25 mm. Forest plot analysis of composite perinatal mortality and morbidity. CI = confidence index. (Adapted with permission from Berghella V, Rafael TJ, Szychowski JM, et al: Cerclage for short cervix on ultrasonography in women with singleton gestations and previous preterm birth: a meta-analysis, Obstet Gynecol 117(3):663,2011.) indings, the American College of Obstetricians and Gynecologists (2016c) concluded that in women with a singleton pregnancy, prior spontaneous preterm birth before 34 weeks, cervical length <25 mm, and gestational age <24 weeks, cerclage placement may be considered. • Prophylaxis with Progestogen Compounds

1	• Prophylaxis with Progestogen Compounds In most mammals, progesterone withdrawal is considered to be a parturition-triggering event. During human parturition, however, maternal, fetal, and amnionic fluid progesterone levels remain elevated. It has been proposed that human parturition involves functional progesterone withdrawal mediated by decreased activity of progesterone receptors (Chap. 21, p. 401). It follows conceptually that the administration of progesterone may block preterm labor. This hypothesis has stimulated several studies of both 17 -alpha hydroxyprogesterone caproate (17OHP-C) and vaginally administered progesterone in women with varying risks for preterm birth.

1	At present, the reported benefits of either of these progestogen therapies are limited to women with singleton pregnancies. Progesterone prophylaxis speciically in multifetal gestations has not lowered preterm birth rates (Caritis, 2009; Rouse, 2007). Accordingly, both the American College of Obstetricians and Gynecologists (2016c) and the Society for Maternal-Fetal Medicine (2017 a) approve the use of progestogen therapy for prevention of preterm birth in select women with singleton pregnancies. Criteria are a history of prior preterm birth or no prior preterm birth but a sonographically identified short cervix.

1	17 -OHP-C is a synthetic progestogen, and the first and only drug approved by the FDA for prevention of recurrent preterm birth. The approval in 2011 was supported by a study by the Maternal-Fetal Medicine Units (MFNIU) Network (Meis, 2003). In this trial, 463 women with a prior preterm birth were randomly assigned to receive weekly intramuscular injections of inert oil or 17-0HP-C from 16 through 36 weeks' gestation. They reported a significantly reduced recurrence of preterm birth in 36 percent of women receiving 17-0HP-C compared with 55 percent of those given placebo.

1	This MFMU study has been challenged because of the unexpectedly high preterm delivery rate in the placebo arm (Romero, 2013). One explanation for this high rate in the placebo group was asymmetry in the risks of recurrence. Indeed, 41 percent of the control group had ::2 prior preterm births compared with only 28 percent in the 17-0HP-C group. Another concern was that the injection dosage of 17-0HP-C, which was 250 mg weekly, was empirically chosen (Caritis, 2014). Only later reports described the pharmacokinetics of 17-0HP-C (Caritis, 2012). Nonetheless, the Society for Maternal-Fetal Medicine (2017a) recently reairmed the use of 17-0HP-C, rather than vaginal progesterone, for prevention of recurrent preterm birth.

1	Sharma and associates (2008) reported that the metabolism of 17-0HP-C was predominantly mediated by the CYP3A enzymatic system. Thus, other agents that induce or inhibit this enzymatic system as well as hepatic impairment may alter drug levels. They also showed that 17-0 HP -C is not converted after administration to the primary progesterone metabolite, 17a-hydroxyprogesterone. The relative binding ainity of 17-0 HP -C to progesterone receptors approximates only 30 percent of that by progesterone (Attardi, 2007). Because synthetic 17-0HP-C is not converted to a naturally occurring progestogen and is not superior to progesterone in eliciting a hormonal response via the classic steroid-receptor mediated pathway, alternative pathways are now being considered to explain its eicacy (Manuck, 2011).

1	Caritis and colleagues (2012) examined 61 women receiving 17-0HP-C therapy and found that the hlf-life was relatively long (median 16.2 days). Pharmacokinetic parameters were ffected by maternal body habitus and varied widely between subjects. In addition, 17 -OHP-C crossed the placental barrier and was detectible in cord plasma 44 days ater the last maternal injection (Caritis, 2012). Despite this, evidence to date suggests that 17-0HP-C is safe for the fetus. No abnormalities, including abnormal genitlia, were found in a 48-month follow-up study of infants exposed in the 2003 MFMU Network trial (Northen, 2007).

1	here are special concerns involving 17-0HP-C and subsequent price-gouging claims (Cohen, 2011; Romero, 2013). In 2011, the FDA gave temporary approval to V Pharmaceutical to market 17 -OHP-C under the brand name Makena. Because regulations prohibited compounding, there was no competitor for this relatively inexpensive drug, and Makena was priced at $1500 per injection. This caused widespread concern because the cumulative cost of Makena would be more than $30,000 per pregnancy. Use of 17-0HP-C at Parkland Hospital A program for implementation of 17-0HP-C was incorporated at Parkland Hospital in 2012. Given the concerns above, Preterm Birth 81.7 TABLE 42-6. Prior Obstetrical History of 430 Women with Births ;35 weeks and Recurrence Rates After 17-0HP-C at Parkland Hospital Prior Birth <35 Weeks Recurrence Ratea No. No. Rate p valueb Overall 16.8% 430 106 25% 1.0 Para 1 18% 141 44 31 % 1.0 Para 2: Only 2nd birth :;35 weeks 17% 48 20 42% 0.49 11 21 % 0.84

1	Prior Birth <35 Weeks Recurrence Ratea No. No. Rate p valueb Overall 16.8% 430 106 25% 1.0 Para 1 18% 141 44 31 % 1.0 Para 2: Only 2nd birth :;35 weeks 17% 48 20 42% 0.49 11 21 % 0.84 Only 1 st birth :;35 weeks Para 3+: 11 % 5% 0.18 All :;35 weeks 45% 12 44% 0.56 Other sequences of :;35 weeks 12% 123 17 14% 0.78 aRecurrence rate is derived from the Parkland obstetrical population for 1988-201n1 prior to introduction of 17-0HP-C. bp values are one-sided. Reproduced with permission from Nelson, 2017.

1	a local compounding pharmacy provided 250-mg, single-dose From the foregoing studies, evidence to support the use of vials of 17-0HP-C in sesame oil at a cost of $25 per dose. 17-0HP-C to prevent recurrent preterm birth is problematic Nelson and colleagues (2017) recently reported their indings (Young, 2017). he mechanism of action remains unknown, from this program in a prospective study of 430 women given and the pharmacological properties have yet to be established. compounded 17-0HP-C. Use of 17-0HP-C was inefective Evidence of clinical efectiveness has yet to be replicated. A confor prevention of recurrent preterm birth at 35 weeks or less dition of the FDA approval for 17-0HP-C was that a conircompared with a historical cohort from Parkland. As shown matory multicenter, double-blind randomized controlled trial in Table 42-6, 17-0HP-C did not signiicantly reduce the be conducted with a preferred primary end point of delivery rates of recurrent preterm birth regardless of prior

1	randomized controlled trial in Table 42-6, 17-0HP-C did not signiicantly reduce the be conducted with a preferred primary end point of delivery rates of recurrent preterm birth regardless of prior preterm <35 weeks. This international trial-PRO LONG-is currently birth number or sequence. Moreover, plasma concentrations of underway with scheduled completion in 2018 and with an esti17-0HP-C were not diferent at 24 weeks or 32 weeks between mated enrollment of 1707 participants (PROLONG, 2014). women delivered :;35 weeks and those delivered later. Interestingly, levels were consistent with those previously reported using castor oil as a vehicle (Caritis, 2014). Last, the gestational age interval at which preterm birth recurred did not difer after use of 17-0HP-C. A side efect of 17-0HP-C was a signifiThree randomized trials are at the center of whether progestocant increase in the rate of gestational diabetes. Taken together, gen therapy should be used in women without prior preterm 17

1	a signifiThree randomized trials are at the center of whether progestocant increase in the rate of gestational diabetes. Taken together, gen therapy should be used in women without prior preterm 17 -0 HP -C use was inefective to prevent recurrent preterm births. hese trials, shown in Table 42-7, hinge on sonographbirth and was associated with a significant side efect. ically determined cervical length. In the irst trial, Fonseca and

1	TABLE 42-7. Randomized Trials of Progestogen Compounds Given Prophylactically to Prevent Preterm Labor Fonseca (2007) n = 250; 5% nulliparous, 10% <15 mm Progesterone, 200-mg Delivery <34 weeks: twins, 15% prior PTB; 8 hospitals: vaginal capsules daily 19% vs 34%, P = .02 UK, Greece, Brazil, Chile Hassan (201l) n = 465; singletons only; 55% 10-20 mm Progesterone, 90-mg Delivery <33 weeks: 9% nulliparous; 13% prior PTB; vaginal gel daily vs 16%, p = .02 Grobman (2012) n = 657; singletons only; nulliparous <30nmm 17-0HP-C, 250 mg 1M Delivery <37 weeks: only; 14 centers across US weekly 25% vs 24%, p = NS Determined sonographically. 17-0HP-C = 17-hydroxyprogesterone caproate; 1M = intramuscularly; NS = nonsignificant; PTB = preterm birth; UK = United Kingdom; US = United States.

1	Determined sonographically. 17-0HP-C = 17-hydroxyprogesterone caproate; 1M = intramuscularly; NS = nonsignificant; PTB = preterm birth; UK = United Kingdom; US = United States. colleagues (2007) randomly assigned 250 women with short cervices measuringr:;15 mm identified during routine prenatal care. Women were given nightly 200-mg micronized progesterone vaginal capsules or placebo from 24 to 34 weeks' gestation. Spontaneous delivery <34 weeks was significantly reduced by progesterone therapy. Importantly, this trial included not only nulliparas but also those with twins or prior pre term birth. In the second trial, Hassan and coworkers (2011) randomly assigned 465 women with a short cervix-IO to 20 mm-to vaginal progesterone gel, 90 mg daily, or placebo. his trial also included nulliparas and women with prior preterm births.

1	From these studies, the FDA rejected progesterone gel for use because the results did not meet the level of statistical significance required to show eicacy in the subjects recruited in the United States. According to Likis and colleagues (2012), the heterogeneity of these first two studies that included women with varied indications for progestogen treatment, combined with the fact that outcomes were not reported by risk factors such as nulliparity, made it impossible to interpret the eicacy of progesterone for speciic indications. The third study randomly assigned administration of 17 -0 HP -C intramuscular injection or placebo between 16 and 223/7 weeks to nulliparas with a singleton gestation and a cervical length < 30 mm detected sonographically (Table 42-8) (Grobman, 2012). Treatment with 17-0HP-C given weekly did not reduce the frequency of preterm birth before 37 weeks. Regardless of cervical length, 17-0 HP -C was inefective.

1	hus, vaginal progesterone, but not 17 -OHP-C, appears to benefit women with a sonographically measured short cervix. Romero and Stanczyk (2013) provided a review to explain the conlicting evidence and argued that naturally occurring progesterone, which is used in the vaginal preparations, is not the same as synthetic 17-0HP-C. Likewise, Furcron and coworkers (2015) found that 17-0 HP -C did not have local antiinflammatory efects at the maternal-fetal interface or cervix. Further, 17 -OHP-C did not protect against endotoxin-induced preterm birth. From all these studies, the merican College of Obstetricians and Gynecologists (2016c) concluded that universal cervical length screening in women without a prior preterm birth is not mandatory. However, this screening strategy could be considered in the context of treatment with vaginal progesterone.

1	The OPPTIMUM Study his study of 1228 high-risk women with singleton pregnancies is the largest to date for vaginal progesterone prophylaxis (N orman, 2016). his randomized trial of vaginal progesterone, 200 mg daily from 22-24 weeks to 34 weeks of gestation, was termed the OPPTIMUM study-dQes Erogesterone Erophyxis Io prevent preterm labor prove High-risk women were defined as those with a prior spontaneous birth :;34 weeks or with a cervical length :;25 mm or a positive FN test result combined with other clinical risk factors for preterm birth.

1	he primary outcomes of OPPTHvIUM were unique in that both immediate obstetrical and childhood outcomes were examined. These were fetal death or birth <34 weeks; a composite of death, brain injury, or bronchopulmonary dysplasia; and a standardized cognitive score at 2 years of age. Contrary to earlier reports, vaginal progesterone was not associated with a lower risk of preterm birth or composite neonatal adverse outcomes. In children at 2 years of age, vaginal progesterone also had no long-term benefit or harm.

1	hus, evidence is conflicting as to the eicacy of progestogens across the spectrum of the various specific indications. Some have attempted to resolve these issues through systematic review and metaanalysis (Prior, 2017; Romero, 2016, 2017). s outlined in this entire section, virtually all evidence supporting use of progestogens for a specific indication can be challenged in some way. We agree with the conclusions from the OPPTIMUM trial (Norman, 2016) that the results of recent studies should prompt a major review of progesterone use for preterm birth prophylaxis, a search to identiy specific women who might specifically benefit, and a redoubling of eforts to find alternative strategies to prevent preterm birth in women at risk. TABLE 42-8. Comparison of 17-0HP-C versus Placebo to Prevent Preterm Birth at Preterm birth <37 wk: Cervical length, mm Preterm birth <34 wk: Cervical length, mm ap value for Breslow-Day interaction term. Data are presented as n/N (%).

1	Preterm birth <37 wk: Cervical length, mm Preterm birth <34 wk: Cervical length, mm ap value for Breslow-Day interaction term. Data are presented as n/N (%). CI = confidence interval; RR = relative risk; 17-0HP-C = 17-hydroxyprogesterone caproate. Data from Grobman, 201n2. FIGURE 42-8 Percentage of births before 37 weeks' gestation at Parkland Hospital from 1988 to 2006 compared with that in the United States from 1996 to 2002. Analysis in both cohorts was limited to singleton liveborn infants �500 g who received prenatal care. (Reproduced with permission from with permission from Leveno J, Mcintire DO, Bloom SL, et al: Decreased preterm births in an innercity public hospital, Obstet Gynecol. 2009 Mar;1o13(3):578-584.)

1	Awell-organized prenatal system lowers the preterm birth rate in high-risk indigent populations (Creasy, 1980). One example is the Parkland Hospital prenatal clinic system (Leveno, 2009). As shown in Figure 42-8, the declining preterm birth rate between 1988 and 2006 coincided with a substantial rise in prenatal care attendance. In the early 1990s, a concerted efort was made to improve access to prenatal care by creating seamless care that began with antenatal enrollment and extended through delivery and the puerperium. Prenatal clinics were placed strategically throughout Dallas County to provide convenient access for our patients. Prenatal protocols are used by nurse practitioners at all clinic sites to guarantee homogeneous care. Women with highrisk pregnancy complications are referred to our hospital-based central clinic system. Here, Maternal-Fetal Medicine clinics operate each weekday and are stafed by residents and midwives and supervised by fellows and faculty.

1	hus, prenatl care is considered one component of a comprehensive and orchestrated public health-care system that is community-based. We believethat the drop in preterm births experienced at our inner-city hospitl is at least partially attributable to a geographically based public health-care program specifically targeting minority populations ofpregnant women. A similar obstetrical care system for indigent women at the University of Alabama at Birmingham has also produced salutary results (Tita, 2011).

1	Methods used to diagnose ruptured membranes are detailed in Chapter 22 (p. 435). A history of vaginal leakage of luid, either as a continuous stream or a gush, should prompt a speculum examination to visualize gross vaginal pooling ofamnionic fluid, clear fluid from the cervical canal, or both. Conirmation of PPROM is usually accompanied by sonographic examina tion to assess amnionic fluid volume, to identiY the presenting part, and if not previously determined, to estimate gestational age. Once PPROM is identified, the general scheme shown in Table 42-9 can guide management.

1	Table 42-9 can guide management. Cox and associates (1988) describedpregnancy outcomes of298 consecutive womenwho gave birth following spontaneously ruptured membranes between 24 and 34 weeks' gestation at Parkland Hospital. Although this complication was identified in only 1.7 percent of pregnancies, it contributed to 20 percent of all perinatal deaths. By the time they presented, 76 percent of the women were already in labor, and 5 percent were delivered for other complications. Thus, only 19 percent initially were permitted expectant management. Ultimately, deliverywas delayed 48 hours or more ater membrane rupture in only 7 percent of the total study cohort. There was beneit noted from delayed delivery, however, as none of the neonates died in this group. This contrasted with a neonatal death rate of 80 per 1000 in preterm newborns delivered within 48 hours ofmembrane rupture. Nelson and colleagues later (1994) reported similar results.

1	The time from PPROM to delivery is inversely proportional to the gestational age when rupture occurs (Carroll, 1995). s shown in Figure 42-9, very few days are gained when membranes ruptured during the third trimester compared with midpregnancy. Most clinicians hospitalize women with ruptured membranes. Concerns regarding the costs oflengthy hospitalizations are usually moot, because most women enter labor within a week or less after membrane rupture. Carlan and coworkers (1993) randomly assigned 67 women with ruptured membranes ro home or hospital management. No benefitswere found for hospitalization, and maternal hospital stays were reduced by 50 percent in those sent home-14 versus 7 days. Importantly, the investigators emphasized that this study was too small to conclude that home management was safe in regard to umbilical cord prolapse.

1	Before the mid-1970s, labor was usually induced in women with preterm ruptured membranes because of fear of sepsis. Maternal infection risk and fetal prematurity risk vary according to the gestational age at membrane rupture, and management decisions hinge on this balance. With regard ro periviable pregnancy, 110rales (1993b) expectantly managed 94 singleton pregnancies with ruptured membranes before 25 weeks. he average time gained was 11 days. Although 41 percent ofinfants survived to age 1 year, only 2 percent of the original cohort were neurologically normal. Similar results were reported by Farooqi (1998) and Winn (2000) and their colleagues. Management of these early pregnancies is discussed on page 807. For PPROM in general, two randomized trials in the 1990s compared labor induction with expectant management (Cox, 1995; Mercer, 1993). In both of these studies, the balance of risk and beneit was diicult to ascertain, as neither immediate • •I I . .•.,. . .• I · .......

1	FIGURE 42-9 Relationship of time between preterm membrane rupture and delivery in 172 singleton pregnancies. (Reproduced with permission from Carroll SG, Blott M, Nicolaides KH: of membranes before 37 weeks' gesPreterm prelabor amniorrhexis: Outcome of live births, Obstet Gynecol 1995 Jul;86(1 ):18-25.) tation without contraindications to TABLE 42-9. Management of Preterm Premature Rupture of Membranes 34 weeks or more Plan delivery: labor induction unless contraindicated Group B streptococcal Single corticosteroid course may be considered up to 366/7 weeksb 32 weeks to 33 completed weeks Expectant management Group B streptococcal prophylaxisa Single corticosteroid coursec Antimicrobials to prolong latency 24 weeks to 31 completed weeks Expectant management T ocolytics: no consensus Antimicrobials to prolong latency <24 weeks Expectant management or induction of labore Group B streptococcal prophylaxis is not recommendedf Single corticosteroid course may be considerede,f f

1	Group B streptococcal prophylaxis is not recommendedf Single corticosteroid course may be considerede,f f Tocolytics: no consensuse,n Antimicrobials: may be considerede,9 aFigure 64-7 (p. 1222) outlines group B streptococcal prophylaxis for preterm gestations. bMay be considered between 34°17 and 366/7 weeks in those who have not received a previous course of antenatal corticosteroids. (Repeat, or rescue, course of corticosteroids with preterm rupture of membranes is controversial. dMagnesium sulfate for neuroprotection in accordance with one of the larger studies. eSee Periviable Neonatal Survival (p. 806) to aid patient counseling and decision making. flntervention not recommended before viability but may be considered as early as 23°(7 weeks of gestation. 9May be considered as early as 20°/7 weeks of gestation. Data from American College of Obstetricians and Gynecologists 2016a, d, 2017a, e.

1	Data from American College of Obstetricians and Gynecologists 2016a, d, 2017a, e. delivery nor expectant management were proven to be superior prolonged latency after membrane rupture was not associated for neonatal outcomes. Lieman and associates (2005) found that with a greater incidence of fetal neurological damage. n imporneonatal outcomes did not improve with expectant management tant correlate is that infection-specifically chorioamnionitisbeyond 33 weeks. McElrath and coworkers (2003) found that is recognized as a risk factor for development of neonatal neurological injury (Gaudet, 2001; Wu,r2000). •Deaths from prematurity complications recently compared planned early 150 birth with expectant management ..Deaths from pulmonary hypoplasia for women with PPROM before 37 weeks' gestation. They evaluated women and 3628 newborns. No �90 • ..0• clinically important diferences in ..,..0. . D • the incidence of neonatal sepsis

1	D • the incidence of neonatal sepsis C .•. • between women who immediately ..0. . . • . . • delivered and those managed expec ..,..0. . tantly were identified. Although the .,incidence of chorioamnionitis was ..,:I · : lower, neonates of women random ized to early birth were more likely to be born at an earlier gestational Gestation at amniorrhexis (weeks) perinatal sequelae. The authors con cluded that in women with rupture continuing the pregnancy, a policy of expectant management with careful monitoring was associated with better outcomes for both the mother and newborn.

1	he American College of Obstetricians and Gynecologists (2016d) has recognized the controversies of immediate deliv ery compared with expectant management. Clearly, gestational age is an important consideration. At 24°/7 to weeks, expectant management in the absence of nonreassuring fetal status, clinical chorioamnionitis, or placental abruption is rec ommended. At 34°/7 weeks of gestation or greater, delivery is still recommended by the College for all women with ruptured membranes. Our current practices at Parkland Hospital are consistent with these recommendations. • Considerations with Expectant Management

1	• Considerations with Expectant Management Several scenarios during expectant management merit consideration. One is performance of digital cervical examination. Alexander and colleagues (2000) analyzed findings in women with PPROM expectantly managed between 24 and 32 weeks' gestation. hey compared those who had one or two digital cervical examinations with women who were not examined. Those who were examined had a rupture-to-delivery interval of 3 days compared with 5 days in those not examined. This diference did not worsen maternal or neonatal outcomes.

1	Rupture of membranes following second-trimester amniocentesis is uncommon (Chap. 14, p. 293). Compared with women with spontaneous rupture during the second trimester, Borgida and associates (2000) found that pregnancies complicated by PPROM after genetic amniocentesis resulted in signiicantly better perinatal outcomes. The perinatal survival rate was 91 percent. After counseling, afected women are typically managed expectantly as outpatients with serial surveillance of amnionic fluid volume (American College of Obstetricians and Gynecologists, 2016d). In the series cited above, the mean time to documentation of a normal amnionic fluid volume after amniocentesis approximated 2 weeks.

1	T ocolysis has been used in few studies. In women with ruptured membranes and lack of labor, prophylactic tocolysis does not improve neonatal outcomes but is associated with greater rates of chorioamnionitis (Mackeen, 2014). Similarly, therapeutic tocolysis-for those with ruptured membranes and labor-has also not provided signiicant perinatal beneit (Garite, 1987). There is uncertainty regarding PPROM in the woman who has undergone cervical cerclage. McElrath and associates (2002) studied 114 women with a cerclage in place who later had ruptured membranes before 34 weeks. They were compared with 288 controls. Pregnancy outcomes were equivalent in both groups. Cerclage retention for more than 24 hours after preterm rupture of membranes may be associated with pregnancy prolongation, however, there is risk of intrauterine infection and its consequences (Giraldo-Isaza, 2011r; Laskin, 2012). As discussed in Chapter 18 (p. 357), such management is controversial.

1	With PPROM in general, the volume of amnionic luid remaining after rupture appears to have prognostic importance in pregnancies before 26 weeks. Hadi and colleagues (1994) described 178 pregnancies with ruptured membranes between 20 and 25 weeks. Almost 40 percent developed oligohydram nios, defined by the absence of fluid pockets measuring 2 em or more. Virtually all women with oligohydramnios delivered before 25 weeks, whereas 85 percent with adequate amnionic luid volume were delivered in the third trimester. Carroll and coworkers (1995) observed no cases of pulmonary hypoplasia in fetuses born after membrane rupture at 24 weeks or beyond. This suggests that 23 weeks or less is the threshold for devel opment of lung hypoplasia (Chap. 7, p. 133). Further, when contemplating early expectant management, consideration is also given to oligohydramnios and resultant limb compression deformities (Chap. 11, p. 232).

1	Other risk factors have also been evaluated. First, in neo nates born to women with active herpetic lesions who were expectantly managed, the infectious morbidity risk appeared to be outweighed by risks associated with preterm delivery (Major, 2003). Second, Lewis and associates (2007) found that expectant management of women with PPROM and nonce phalic presentation was associated with a higher rate of umbili cal cord prolapse, especially before 26 weeks.

1	As discussed, infection is a major concern with membrane rupture. While some cases remain subclinical, if chorioamnionitis is diagnosed, prompt eforts to efect delivery, preferably vaginally, are initiated. Because maternal leukocytosis alone is not a consistent inding, ever is the ony reliable indicator or the diagnosis of chorioamnionitis. Institutional practices and protocols vary in deining the temperature threshold. T raditionally, a temperature �38°C (100.4°F) accompanying ruptured membranes has implied infection. At Parkland Hospital, we still adhere to this criterion.

1	In 2015, a workshop sponsored by the NICHD was convened and suggested renaming this condition "intraamnionic infection and inflammation" (Higgins, 2016). The merits and clinical utility of this novel "triple I" terminology have been questioned (Barth, 2016). Nonetheless, the American College of Obstetricians and Gynecologists (20 17b) recently revised both the definitions and temperature thresholds for intraamnionic infection. Using these new deinitions, the diagnosis of suspected intraamniotic infection is made when the maternal temperature is �39.0°C or when the maternal temperature is 38.0 to 38.9°C and one additional clinical risk factor is present. Suggested factors include low parity, multiple digital examinations, use of internal uterine and fetal monitors, meconiumstained amnionic fluid, and the presence of certain genital tract pathogens. Examples are group B streptococcus and sexually transmitted agents. Isolated maternal fever is deined as any maternal temperature between

1	fluid, and the presence of certain genital tract pathogens. Examples are group B streptococcus and sexually transmitted agents. Isolated maternal fever is deined as any maternal temperature between 38.0°C and 38.9°C with no additional risk factors present, and with or without persistent temperature elevation.

1	With chorioamnionitis, fetal and neonatal morbidity are substantively increased. lexander and colleagues (1998) studied 1367 very-Iow-birthweight neonates delivered at Parkland Hospital. Approximately 7 percent were born to women with overt chorioamnionitis, and their outcomes were compared with similar newborns without clinical infection. Those in the infected group had higher incidences of sepsis, RDS, earlyonset seizures, intraventricular hemorrhage, and periventricular leukomalacia. The investigators concluded that these very-Iowbirthweight newborns were vulnerable to neurological injury attributable to chorioamnionitis. Yoon and colleagues (2000) found that intraamnionic infection in preterm neonates was related to increased rates of cerebral palsy. Petrova and associates (2001) studied more than 11 million singleton live births in the United States from 1995 to 1997. During labor, 1.6 percent of all women had fever, and this was a strong predictor of infection-related death in

1	more than 11 million singleton live births in the United States from 1995 to 1997. During labor, 1.6 percent of all women had fever, and this was a strong predictor of infection-related death in both term and preterm neonates.

1	The proposed microbial pathogenesis for spontaneous preterm labor or ruptured membranes has prompted investigators to give various antimicrobials to forestall delivery. Mercer and associates (1995) reviewed 13 randomized trials performed before 35 weeks. heir metaanalysis indicated that only three of 10 outcomes werepossiby beneited: (1) fewer women developed chorioamnionitis, (2) fewer newborns developed sepsis, and (3) pregnancy was more often prolonged 7 days in women given antimicrobials. Rates of neonatal survival, necrotizing enterocolitis, RDS, or intracranial hemorrhage, however, were unafected.

1	To further address this issue, the MFMU Network designed a trial to study expectant management combined with placebo or with a 7 -day antibiotic regimen. Treatment included intravenous ampicillin plus erythromycin every 6 hours for 48 hours, which was followed by oral amoxicillin plus erythromycin, every 8 hours for 5 days. The women had membrane rupture between 24 and 32 weeks' gestation. Neither tocolytics nor corticosteroids were given. Antimicrobial-treated women had signiicantly fewer newborns with RDS, necrotizing enterocolitis, and composite adverse outcomes (Mercer, 1997). The latency period was also significantly longer. Specifically, 50 percent of women given an antimicrobial regimen remained undelivered after 7 days of treatment compared with only 25 percent of those given placebo. lso, a significantly greater number of treated pregnancies were undelivered at 14 and 21 days. Cervico vaginal group B streptococcal colonization did not alter these results.

1	Other studies have examined the eicacy of shorter treatment lengths and diferent antimicrobial combinations. hreeday treatments compared with 7 -day regimens using either ampicillin or ampicillin-sulbactam appear equally efective in regard to perinatal outcomes (Lewis, 2003; Segel, 2003). Similarly, erythromycin compared with placebo ofered a range of significant neonatal benefits. The amoxicillin-clavulanate regimen was not recommended, however, because of its association with an increased incidence of neonatal necrotizing enterocolitis (Kenyon, 2004).

1	Some predicted that prolonged antimicrobial therapy in such pregnancies might have unwanted consequences (Carroll, 1996; Mercer, 1999). Stoll and associates (2002) studied 4337 neonates weighing from 400 to 1500 g and born from 1998 to 2000. Their outcomes were compared with those of 7606 neonates of similar birthweight born from 1991 to 1993 and prior to the practice of antibiotic prophylaxis. The overall rate of early-onset sepsis did not change between these two epochs. But, the rate of group B streptococcal sepsis dropped from 5.9 per 1000 births in the 1991 to 1993 group to 1.7 per 1000 births in the 1998 to 2000 group. Comparing these same epochs, the rate of Escherichia coli sepsis, however, rose from 3.2 to 6.8 per 1000 births. Almost 85 percent of coliform isolates from the more recent cohort were resistant to ampicillin. Neonates with early-onset sepsis were more likely to die, especially if they were infected with coliforms. Long term, Kenyon and coworkers (2008a) found

1	recent cohort were resistant to ampicillin. Neonates with early-onset sepsis were more likely to die, especially if they were infected with coliforms. Long term, Kenyon and coworkers (2008a) found that antimicrobials given for women with PPROM had no efect on the health of children at age 7 years .

1	• Corticosteroids to Accelerate The use of antenatal corticosteroids in the setting of PPROM was once considered controversial as the magnitude of the beneits was not as great as when the membranes were intact. A single course of corticosteroids, however, is now recommended for pregnant women with ruptured membranes between 24°/7 and 34°17 weeks' gestation (American College of Obstetricians and Gynecologists, 2017 a). As with periviability (p. 807), a single course of corticosteroids as early as 23°/7 weeks in those who are at risk for preterm delivery within 7 days may be considered (American College of Obstetricians and Gynecologists, 2017e). A similar controversy is found at the other end of the gestational age spectrum, wherein corticosteroid administration in the late-pre term period is also under consideration (p. 823).

1	Tissue sealants are used for various purposes in medicine, including achieving surgical hemostasis. As discussed in Chap. 18 (p. 351), there are limited reports of sealants in the repair of fetal membranes. Crowley and coworkers (2016) recently reviewed the available evidence and concluded that data are currently insuicient to evaluate sealing procedures for ruptured membranes. At Parkland Hospital, we do not currently use these agents for this indication. Women with signs and symptoms of preterm labor with intact membranes are managed similarly to those with PPROM. If possible, delivery before 34 weeks' gestation is delayed. Drugs used to abate or suppress preterm uterine contractions are subsequently discussed. • Amniocentesis to Detect Infection

1	• Amniocentesis to Detect Infection Several tests have been used to diagnose intraamnionic infection (Andrews, 1995; Romero, 1993; Yoon, 1996). Although such infection can be conirmed with a positive test result, there is little utility for routine amniocentesis (American College of Obstetricians and Gynecologists (2017b). Because glucocorticosteroids were found to accelerate lung maturation in preterm sheep fetuses, Liggins and Howie (1e972) evaluated them to treat women. Corticosteroid therapy was efective in lowering the incidence of RDS and neonatal initiation of betamethasone. Infants exposed to corticosteroids in these early studies have now been followed to age 31 years with no ill efects detected. In 1995, a National Institutes of ened preterm birth. In a subsequent meeting, another NIH

1	Conference (2000) concluded that data were insuicient to by hypertension, diabetes, multifetal gestation, fetal-growth restriction, or fetal hydrops. It was concluded, however, that it was reasonable to administer corticosteroids to these women. A recent metaanalysis by Roberts and associates (20e17) of 30 studies totaling 7774 women and 8158 infants quantiied the beneit of a single course of corticosteroids. Treatment was associated with lower rates of perinatal death, neonatal death, RDS, intraventricular hemorrhage, necrotizing enterocolitis, need for mechanical ventilation, and systemic infection in the first 48 hours of life. No obvious beneits were gained for chronic lung disease, death in childhood, or neurodevelopmental delay in childhood. Therapy was not associated with chorioamnionitis. Parenthetically, corticosteroids given prophylactically to women at risk of preterm birth in low-and middle-income countries actually increase perinatal mortality rates (Althabe, 2015).

1	A single course of corticosteroids is currently recommended by the American College of Obstetricians and Gynecologists (2017a) for women between 24 and 34 weeks who are at risk for delivery within 7 days. This recommendation for premature twins has been challenged (Viteri, 20e16). Boundaries of gestational age for corticosteroid administration are also now being explored. For pregnancies at 23 weeks and at risk of delivery within 7 days, a single course of corticosteroids may be considered (p. 807). Administration of corticosteroids during the periviable period is linked to a family's decision regarding resuscitation and should be considered in that context (American College of Obstetricians and Gynecologists, 20e1e7e).

1	Betamethasone and dexamethasone appear to be equivalent for fetal lung maturation (Murphy, 2007). These two drugs are comparable in reducing rates of major neonatal morbidities in preterm newborns (Elimian, 2007). A treatment course may be two 12-mg doses of betamethasone, and each dose is given intramuscularly 24 hours apart. With dexamethasone, 6-mg doses are given intramuscularly every 12 hours for four doses. Because treatment for less than 24 hours may be beneicial and reduce neonatal morbidity and mortality rates, a first dose of antenatal corticosteroids is administered regardless of the ability to complete additional doses before delivery (American College of Obstetricians and Gynecologists, 20e1e7a). The MFMU Network conducted a randomized trial to assess whether administration of antenatal betamethasone to women who were likely to deliver in the late-pre term period would

1	The MFMU Network conducted a randomized trial to assess whether administration of antenatal betamethasone to women who were likely to deliver in the late-pre term period would Bannerman, 2016). Even though only 60 percent of the study cohort of 283e1 women received both injections, the rate of respiratory complications measured as a composite outcome was lower with corticosteroid use compared with placebo-1 1.6 versus 14.4 percent. Because of these indings, consideration for administration of a single course of betamethasone for women the American College of Obstetricians and Gynecologists (2017a) and the Society for Maternal-Fetal Medicine (2016a).

1	Adoption of this practice has not been universal. Both shortand long-term neonatal safety are concerns (Crowther, 2016; Kamath-Rayne, 2016). Specifically, in the newborns receiving betamethasone, rates of hypoglycemia were signiicantly greater (Gyami-Bannerman, 20e16) . Neonatal hypoglycemia is particularly worrisome for possible adverse long-term consequences that include developmental delay (Kerstjens, 2012). Another caveat is that the largest efects of betamethasone included a reduction in transient tachypnea of the newborn-a self-limited condition with little clinical signiicance (KamathRayne, 2016). Speciically, the rates of transient tachypnea of the newborn were 6.7 and 9.9 percent in those given betamethasone and placebo, respectively. These rates are three-to fourfold higher than those reported by the Consortium on Safe Labor (20e10), which was a retrospective, observational study that abstracted detailed labor and delivery information from 19 hospitals across the United

1	than those reported by the Consortium on Safe Labor (20e10), which was a retrospective, observational study that abstracted detailed labor and delivery information from 19 hospitals across the United States on 233,844 deliveries. Because of these issues, we do not provide corticosteroids beyond 34 weeks at Parkland Hospital at this time.

1	Single versus additional courses of intramuscular corticosteroids for lung maturation has been the topic of two major trials. Although both found repeated courses to be beneicial in reducing neonatal respiratory morbidity rates, the long-term consequences were much diferent. In one randomized study by Crowther and associates (2007), all women at risk for preterm birth were given a primary course of betamethasone. Women were then given serial weekly doses of 11e.4 mg of betamethasone for persistent risk or were given placebo. hese investigators found no adverse efects in the infants followed to age 2 years. Wapner and coworkers (2007) studied infants born to 495 women who were randomly assigned to receive a single corticosteroid course that contained two doses or assigned to repeated courses that were given weekly. A nonsignificant rise in the cerebral palsy rate was identified in infants exposed to repeated courses. The doubled betamethasone dose in this study was worrisome because

1	that were given weekly. A nonsignificant rise in the cerebral palsy rate was identified in infants exposed to repeated courses. The doubled betamethasone dose in this study was worrisome because some experimental evidence supports the view that adverse efects are dose dependent (Bruschettini, 2006). Stiles (2007) summarized these two studies as "early gain, long-term questions." We agree, and at Parkland Hospital, we follow the recommendation by the American College of Obstetricians and Gynecologists (2017a) for singlecourse therapy.

1	his refers to administration of a second corticosteroid dose when delivery becomes imminent and more than 7 days have elapsed since the initial dose. In one randomized trial, 326 women received placebo or a single 12-mg dose of betamethasone (Peltoniemi, 2007). Paradoxically, the rescue dose of betamethasone increased the risk of RDS. In another randomized study of 437 women with gestations <33 weeks, Garite and associates (2009) reported significantly lower rates of respiratory complications and neonatal composite morbidity with rescue corticosteroids versus placebo. Rates of perinatal mortality and other morbidities, however, did not difer. Last, McEvoy and colleagues (2010) found that treated infants had improved respiratory compliance.

1	Garite and coworkers (2009) randomly assigned 437 women with singletons or twins <33 weeks' gestation and with intact membranes to one rescue course of either betamethasone or dexamethasone or placebo. These women had all previously completed a single course of corticosteroids before 30 weeks' gestation and at least 14 days before the rescue course. RDS developed in 41 percent of the newborns given rescue corticosteroids compared with 62 percent of those randomized to placebo. Rates of other morbidities attributable to prematurity did not difer. In a metaanalysis, Crowther and colleagues (201e1) concluded that a single course of corticosteroids should be considered in women whose prior course was administered at least 7 days previously and who were <34 weeks' gestation. The American College of Obstetricians and Gynecologists (2017 a) has taken the position that a single rescue course of antenatal corticosteroids be considered in women before 34 weeks whose prior course was

1	College of Obstetricians and Gynecologists (2017 a) has taken the position that a single rescue course of antenatal corticosteroids be considered in women before 34 weeks whose prior course was administered at least 7 days previously. Efects of rescue therapy beyond 34 weeks are currently unknown. At Parkland Hospital, we currently do not provide additional courses of corticosteroids beyond the initial single-course therapy.

1	Very-Iow-birthweight neonates whose mothers were treated with magnesium sulfate for preterm labor or preeclampsia were found to have a reduced incidence of cerebral palsy at 3 years (Grether, 2000; Nelson, 1995). Because of this, randomized trials were designed to investigate this hypothesis. In one trial, 1063 women at risk of delivery before 30 weeks were given magnesium sulfate or placebo (Crowther, 2003). Magnesium exposure improved some perinatal outcomes. Namely, rates of both neonatal death and cerebral palsy were lower in the magnesium-treated group-but this study was not suiciently powered. The multicenter French trial reported by Marret and associates (2008) had similar problems.

1	More convincing evidence for magnesium neuroprotection came from the MFMU Network study-Beneicial Eects of Antenatal Magnesium Suote-BM-Study (Rouse, 2008). This was a placebo-controlled trial in 224e1 women at imminent risk for preterm birth between 24 and 31 weeks. Women randomized to magnesium sulfate were given a 6-g bolus over 20 to 30 minutes followed by a maintenance infusion of 2 g per hour. Magnesium sulfate was actually infusing at the time of delivery in approximately half of the treated women. A 2-year follow-up was available for 96 percent of the children. Results are shown in Table 42-10. his trial can be interpreted diferently depending on statistical methodologies employed. Some interpret these indings to mean that magnesium infusion prevents cerebral palsy regardless of the gestational age at which therapy is given. Those with a difering view conclude that this trial only supports use of magnesium sulfate for prevention of cerebral palsy before 28 weeks.

1	Subsequent to these studies, Doyle and associates (2009) reviewed five randomized trials to assess neuroprotective efects. A total of 6145 infants were studied, and these reviewers concluded that magnesium exposure compared with no exposure significantly lowered risks for cerebral palsy. Rates of other neonatal morbidity did not difer significantly. It was calculated that treatment given to 63 women would prevent one case of cerebral plsy.

1	Controversy surrounding magnesium eicacy for neuroprotection prompted a debate at the 20e11 annual meeting of the Society for Maternal-Fetal Medicine. Rouse (2011) spoke for the beneits of magnesium sulfate, whereas Sibai (2011) challenged that the reported benefits were false positive due to random statistical error in the metaanalysis by Doyle (2009). Another peculiarity is the apparent lack of dose-response for eicacy (McPherson, 2014). Because none of the individual studies found a benefit from magnesium sulfate for fetal neuroprotection, the American College of Obstetricians and Gynecologists (20e1e6a) concluded that those electing prophylaxis should develop specific guidelines. To guide such therapy, the American College of Obstetricians and Gynecologists (20e12) TABLE 42-10. Magnesium Sulfate for the Prevention of Cerebral Palsya Infants with 2-year follow-up

1	TABLE 42-10. Magnesium Sulfate for the Prevention of Cerebral Palsya Infants with 2-year follow-up Moderate or severe cerebral palsy: aSelected results from the Beneficial Efects of Antenatal Magnesium Sulfate (BEAM) Study. bWeeks' gestation at randomization. CI = confidence index. Data from Rouse, 2008. issued a Patient Saey Checklist for use of magnesium sulfate for neuroprotection. For those with PPROM, prophylaxis may similarly be considered. At Parkland Hospital, we provide magnesium sulfate for neuroprotection with threatened preterm delivery from 24°/7 to 276/7 weeks.

1	Results have been disappointing in studies of antimicrobials given to arrest preterm labor. From one Cochrane metaanalysis, antimicrobial prophylaxis given to women with intact membranes did not reduce preterm birth rates or afect other clinically important short-term outcomes (Flenady, 2013). However, rates of short-and longer-term harm were higher for children of mothers exposed to antibiotics. Kenyon (2001) reported the ORACLE Collaborative Group study of 6295 women with spontaneous preterm labor and intact membranes, but without evidence of infection. Women were randomly assigned to receive antimicrobial or placebo therapy. The primary outcomes of neonatal death, chronic lung disease, and major cerebral abnormality were similar in both groups. In a follow-up of the ORACLE II trial, fetal exposure to antimicrobials in this clinical setting was associated with an increased cerebral palsy rate at age 7 years compared with that in children without fetal exposure (Kenyon, 2008b).

1	fetal exposure to antimicrobials in this clinical setting was associated with an increased cerebral palsy rate at age 7 years compared with that in children without fetal exposure (Kenyon, 2008b). Importantly, antimicrobial use described here is distinct from that given for group B streptococcal prophylaxis (Chap. 64, p. 1222).

1	This is one of the most often prescribed interventions during pregnancy, yet one of the least studied. One systematic review concluded that evidence neither supported nor refuted bed rest for prevention of preterm birth (Sosa, 2004). Goulet and coworkers (2001) randomly assigned 250 Canadian women to either home care or hospitalization after treatment of an acute episode of preterm labor and found no beneits. There have, however, been reports of possible harm. Kovacevich and associates (2000) reported that bed rest for 3 days or more increased thromboembolic complications to 16 per 1000 women compared with only 1 per 1000 with normal ambulation. Promislow and colleagues (2004) observed significant bone loss in pregnant women prescribed outpatient bed rest. More recently, Grobman and associates (20e13) noted that women with activity restriction were nearly 2.5 times more likely to have a preterm birth before 34 weeks. his finding, however, may reflect ascertainment bias. hat is, women

1	(20e13) noted that women with activity restriction were nearly 2.5 times more likely to have a preterm birth before 34 weeks. his finding, however, may reflect ascertainment bias. hat is, women with restricted activity may have been assigned to bed rest because they were viewed to be at more imminent risk of preterm delivery. McCall and coworkers (2013) summarized the literature on bed rest, and they found insuicient evidence to support its use. The American College of Obstetricians and Gynecologists (20e17 d) suggests that, although frequently prescribed, bed rest is only rarely indicated, and ambulation should be considered in most cases.

1	Silicone rings, such as the Arabin pessay, are being used to support the cervix in women with a sonographically short cervix. For 385 Spanish women with a cervical length ;25 mm, Goya and associates (2012) provided a silicone pessary or expectant management. Newborns spontaneous delivered before 34 weeks' gestation in 6 percent of women in the pessary group compared with 27 percent in the expectant management group. Another trial randomly assigned almost 100 women with a cervix <25 mm at 20 to 24 weeks to silicone pessaries or expectant management (Hui, 2013). The pessary did not lower the rate of delivery < 34 weeks. Similar indings were reported by Nicolaides and colleagues (2016). he Society for Maternal-Fetal Medicine (2017b) recently recognized the conflicting published reports and lack of an FDA-approved pessary for the indication of preterm birth prevention. They currently recommend pessary prophylaxis only within research protocols.

1	Some evidence supports the concept that cervical incompetence and preterm labor lie along a spectrum leading to preterm delivery. Consequently, investigators have evaluated cerclage placement after preterm labor begins to manifest clinically. Althuisius and colleagues (2003) randomly assigned 23 women with cervical incompetence before 27 weeks to bed rest, with or without emergency McDonald cerclage. Delivery delay was significantly greater in the cerclage group compared with those assigned to bed rest-54 versus 24 days, respectively. Terkildsen and coworkers (2003) studied 116 women who underwent second-trimester emergency cerclage. Nulliparity, membranes extending beyond the external cervical os, and cerclage before 22 weeks were associated with a signiicantly decreased chance of signiicant pregnancy continuation. For women facing a poor pregnancy prognosis due to cervical dilation at midgestation, it seems reasonable to ofer emergency or rescue cerclage with appropriate counseling.

1	pregnancy continuation. For women facing a poor pregnancy prognosis due to cervical dilation at midgestation, it seems reasonable to ofer emergency or rescue cerclage with appropriate counseling. However, it is unclear if such interventions truly confer a beneit or merely increase the risk of membrane rupture and infection (Hawkins, 2017).

1	• Tocolysis to Treat Preterm Labor Although several drugs and other interventions have been used to prevent or inhibit preterm labor, none is completely efective. The American College of Obstetricians and Gynecologists (2016b) has concluded that tocolytic agents do not markedly prolong gestation but may delay delivery in some women for up to 48 hours. This may allow transport to an obstetrical center with higher-level neonatal care and permit time for a course of corticosteroid therapy. Although delivery may be delayed to administer corticosteroids, treatment has not improved perinatal outcome rates (Gyetvai, 1999).

1	Beta-adrenergic agonists, magnesium sulfate, calciumchannel blockers, or indomethacin are the recommended tocolytic agents for this short-term use. The gestational age range for tocolytic use is debatable. But, because corticosteroids are not generally used after 34 weeks, and because the perinatal outcomes in preterm neonates are generally good after this time, most do not recommend use of tocolytics after 33 weeks' gestation (Goldenberg, 2002). In many women, tocolytics stop contractions temporarily but rarely prevent preterm birth. he College (2016b) notes that maintenance therapy with tocolytics is inefective for preventing preterm birth. Importantly, no trial has ever convincingly shown reductions in rates of any important adverse outcome by a tocolytic drug compared with placebo (Walker, 2016). Maintenance tocolysis after acute therapy is not recommended.

1	Several compounds react with 3-adrenergic receptors to reduce intracellular ionized calcium levels and prevent activation of myometrial contractile proteins (Chap. 21, p. 406). Of 3-mimetic drugs in the United States, ritodrine and terbutaline have been used in obstetrics, but only ritodrine is approved for preterm labor by the FDA. Ritodrine was voluntarily withdrawn from the United States market in 2003, but a discussion of ritodrine is included here to present issues with 3-mimetic drug use. In one early trial, neonates whose mothers were treated with ritodrine for threatened preterm labor had lower rates of preterm birth and its complications (NIerkatz, 1980). In a randomized trial at Parkland Hospital, intravenous ritodrine delayed delivery for 24 hours but without other beneits (Leveno, 1986b). Additional studies confirmed a delivery delay up to 48 hours (Canadian Preterm Labor Investigators Group, 1992).

1	3-Agonist drug infusion has resulted in serious and even fatal maternal side efects. Pulmonary edema is a special concern, and its contribution to morbidity is discussed in Chapter 47 (p. 917). In one early study, tocolysis was the third most common cause of acute respiratory distress and death in pregnant women during a 14-year period in Mississippi (Perry, 1998). The cause of pulmonary edema is multifactorial. isk factors include tocolytic therapy with 3-agonist drugs, multifetal gestation, concurrent corticosteroid therapy, tocolysis for more than 24 hours, and intravenous infusion of large volumes of crystalloid. 3-Agonist agents cause retention of sodium and water, and with time-usually 24 to 48 hours-these can cause volume overload (Hankins, 1988). The drugs have been implicated in increased capillary permeability, cardiac rhythm disturbances, and myocardial ischemia.

1	Terbutaline is commonly used in the United States to forestall preterm labor. Like ritodrine, it may cause pulmonary edema (Angel, 1988). Low-dose terbutaline can be administered longterm by subcutaneous pump (Lam, 1988; Perry, 1995). But, randomized trials have shown no beneit for terbutaline pump therapy (Guinn, 1998; Wenstrom, 1997). Oral terbutaline given to prevent preterm delivery is also inefective (How, 1995; Parilla, 1993). In one trial, 203 women with arrested preterm labor at 24 to 34 weeks' gestation were randomly assigned to receive 5-mg terbutaline tablets or placebo every 4 hours (Lewis, 1996). Of outcomes, delivery rates at 1 week, median latency duration, mean gestational age at delivery, and incidence of preterm labor relapse were similar in both groups. Because of reports of serious maternal side efects, the FDA (2011) issued a warning regarding the use of terbutaline to treat preterm labor. The American College of Obstetricians and Gynecologists (2016b) recommends

1	of serious maternal side efects, the FDA (2011) issued a warning regarding the use of terbutaline to treat preterm labor. The American College of Obstetricians and Gynecologists (2016b) recommends only short-term inpatient use of terbutaline as a tocolytic or as acute therapy of uterine tachysystole. Subcutaneous dosages of 0.25 mg are commonly used for the latter indication. T erbutaline, used as a tocolytic prior to external cephalic version, is discussed in Chapter 28 (p. 550).

1	Ionic magnesium in a suiciently high concentration n alter myometrial contractility. Its role is presumably that of a calcium antagonist, and when given in pharmacological doses, it may inhibit labor. Intravenous magnesium sulfate, given s a 4-g loading dose and followed by a continuous inusion of 2 g/hr, usually arrests labor (Steer, 1977). Like 3-mimetic agents, magnesium treatment can cause pulmonary edema (Samol, 2005). However, this has not been our experience at Parkland Hospitl in the treatment of tens of thousands of preeclamptic women with intravenous magnesium sulfate. Pharmacology and toxicology of magnesium are considered in more detail in Chapter 40 (p. 737).

1	Only two randomized studies have evaluated tocolysis with magnesium sulfate. Cotton and colleagues (1984) compared magnesium sulfate, ritodrine, and placebo in 54 women with preterm labor. They identified few diferences in outcomes. Cox and coworkers (1990) randomly assigned 156 women to receive magnesium sulfate or infusions of normal saline. Magnesium-treated women and their neonates had identical outcomes compared with those given placebo. Because of these findings, this method of tocolysis was abandoned at Parkland Hospital. Similarly, Crowther and associates (2014) reviewed magnesium sulfate as a tocolytic agent and concluded it was inefective and potentially harmful. Last, the FDA (2013) has warned against prolonged use of magnesium sulfate given to arrest preterm labor because of bone thinning and fractures in fetuses exposed for more than 5 to 7 days. This was attributed to low calcium levels in the fetus.

1	hese compounds are intimately involved in contractions of normal labor (Chap. 21, p. 416). Antagonists act by inhibiting prostaglandin synthesis or by blocking their action on target organs. A group of enzymes collectively termed prostaglandin synthase is responsible for the conversion of free arachidonic acid to prostaglandins. Several drugs block this system, including acetylsalicylate and indomethacin.

1	Indomethacin, a nonselective cyclooxygenase inhibitor, was first used as a tocolytic in one study of 50 women (Zuckerman, 1974). Studies that followed reported the eicacy of indomethacin in halting contractions and delaying preterm birth (Muench, 2003; Niebyl, 1980). Morales and coworkers (1989, 1993a), however, compared indomethacin with either ritodrine or magnesium sulfate and found no diference in their eicacy to forestall preterm delivery. Berghella and associates (2006) reviewed four trials of indomethacin given to women with a sonographically determined short cervix and found such therapy to be inefective. Indomethacin is administered orally or rectally. Most studies have limited indomethacin use to 24 to 48 hours because of concerns for oligohydramnios, which can develop with therapeutic doses. If amnionic luid is monitored, oligohydramnios can be detected early, and it is reversible with drug discontinuation.

1	In a study of neonates born before 30 weeks, Norton and coworkers (1993) identiied necrotizing enterocolitis in 30 percent of 37 indomethacin-exposed newborns compared with 8 percent of 37 control newborns. Higher incidences of intraventricular hemorrhage and patent ductus arteriosus were also documented in the indomethacin group. Several investigators have challenged the association between indomethacin exposure and necrotizing enterocolitis (Muench, 2001; Parilla, 2000). Similarly, Gardner (1996) and Abbasi (2003) and their colleagues found no link between indomethacin use and intraventricular hemorrhage, patent ductus arteriosus, sepsis, necrotizing enterocolitis, or neonatal death. Two metaanalyses of the efects of antenatal indomethacin on neonatal outcomes had conflicting indings (Amin, 2007; Loe, 2005). Reinebrant and colleagues (2015) in a review of 20 studies reported no clear benefit from cyclooxygenase inhibitors, including indomethacin, compared with placebo or any other

1	2007; Loe, 2005). Reinebrant and colleagues (2015) in a review of 20 studies reported no clear benefit from cyclooxygenase inhibitors, including indomethacin, compared with placebo or any other tocolytic agent.

1	These potent smooth-muscle relaxants afect the vasculature, gut, and uterus. In randomized clinical trials, nitroglycerin administered orally, transdermally, or intravenously was ineffective or showed no superiority over other tocolytics. In addition, maternal hypotension was a common side efect (Bisits, 2004; EI-Sayed, 1999; Lees, 1999). Discussed in Chapter 21 (p. 404), myometrial aCtlVIty is directly related to cytoplasmic free calcium, and reduced calcium concentrations inhibit contractions. Calcium-channel blockers act to inhibit, by various mechanisms, calcium entry through cell membrane channels. Although they were developed to treat hypertension, their ability to arrest pre term labor has been evaluated.

1	From study results, calcium-channel blockers, especially nifedipine, are safer and more efective tocolytic agents than 3-agonist drugs (King, 2003; Papatsonis, 1997). Lyell and colleagues (2007) randomized 192 women at 24 to 33 weeks' gestation to either magnesium sulfate or nifedipine and found no substantial diferences in eicacy or adverse efects. In another randomized study, 145 women with preterm labor between 24 and 33 weeks received nifedipine or atosiban. Neither proved superior to delay delivery, and neonatal morbidity was equivalent (Salim, 2012).

1	Flenady and coworkers (20 14b) reviewed 38 trials of calciumchannel blockers (mainly nifedipine) for preterm labor. These investigators suggested that calcium-channel blockers have beneits compared with placebo or no treatment. But, this conclusion stemmed from a trial with unclear risk of selection bias and a three-arm study of 84 women that was not blinded (Ara, 2008; Zhang, 2002). We are currently performing a randomized, double-blind, placebo-controlled trial of nifedipine for acute tocolysis of preterm labor at Parkland Hospital.

1	Importantly, the combination of nifedipine with magnesium for tocolysis is potentially dangerous. Ben-Ami (1994) and Kurtzman (1993) and their coworkers reported that nifedipine enhances the neuromuscular blocking efects of magnesium, which can interfere with pulmonary and cardiac function. In one small study of 54 women with preterm labor who received either magnesium sulfate plus nifedipine or no tocolytic, neither beneit nor harm was found (How, 2006).

1	This nonapeptide oxytocin analogue is an oxytocin-receptor antagonist (ORA). Goodwin and colleagues (1995) described its pharmacokinetics in pregnant women. In randomized clinical trials, atosiban failed to improve relevant neonatal outcomes and was linked with signiicant neonatal morbidity (Moutquin, 2000; Romero, 2000). he FDA has denied approval of atosiban because of concerns regarding eicacy and fetal-newborn safety. Further, in 2014, a metaanalysis did not demonstrate superiority of ORAs (largely atosiban) as a tocolytic compared with placebo, 3-mimetic drugs, or calcium-channel blockers in terms of pregnancy prolongation or neonatal outcomes. But, ORAs were associated with fewer maternal adverse efects (Flenady, 20 14a). Recently, van Vliet and coworkers (2016) conducted a randomized trial comparing nifedipine with atosiban in 510 women with threatened preterm birth. Using a composite of adverse perinatal outcomes, no diferences were reported between the two study groups.

1	Whether preterm labor is induced or spontaneous, abnormalities of fetal heart rate and uterine contractions are sought. We prefer continuous electronic monitoring. Fetal tachycardia, especially with ruptured membranes, is suggestive of sepsis. Some evidence supports that intrapartum acidemia may intensiy some of the neonatal complications usually attributed to preterm delivery. For example, Morgan and associates (2017) found that metabolic acidemia signiicantly raised the risks related to prematurity in neonates delivered prior to 34 weeks' gestation. Low and colleagues (1995) observed that intrapartum acidosis-umbilical artery blood pH <7.0-had an important role in neonatal complications (Chap. 33, p. 621). Group B streptococcal infections are common and dangerous in the preterm neonate, and antimicrobial prophylaxis should be provided (Chap. 64, p. 1222).

1	In the absence of a relaxed vaginal outlet, an episiotomy for delivery may be necessary once the fetal head reaches the perineum. Perinatal outcome data do not support routine episiotomy or forceps delivery to protect the "fragile" preterm fetal head. Staf proicient in resuscitative techniques commensurate with the gestational age and fully oriented to any speciic problems should be present at delivery. Principles of resuscitation described in Chapter 32 (p. 608) are applicable. The importance of specialized personnel and facilities for preterm newborn care is underscored by the improved survival rates of these neonates when delivered in tertiary-care centers. • Prevention of Intracranial Hemorrhage

1	• Prevention of Intracranial Hemorrhage Preterm newborns frequently have intracranial germinal matrix bleeding that can extend to more serious intraventricular hemorrhage (Chap. 34, p. 639). It was hypothesized that cesarean delivery to obviate trauma from labor and vaginal delivery might prevent these complications. his has not been validated by subsequent studies. Malloy (1991) analyzed 1765 newborns with birthweights < 1500 g and found that cesarean delivery did not lower the risk of mortality or intracranial hemorrhage. Anderson and colleagues (1988), however, made an interesting observation regarding the role of cesarean delivery in intracranial hemorrhage prevention. These hemorrhages correlated with exposure to active-phase labor. However, they emphasized that avoidance of active-phase labor is impossible in most preterm births because decisions for delivery route are not required until active labor is irmly established.

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1	Alirevic Z, Milan S], Livio S: Caesarean section versus vaginal delivery for preterm birth in singletons. Cochrane Database Syst Rev 9:CD000078, 2013 Althabe F, Belizan ]M, McClure EM, et al: A population-based, multifaceted strategy to implement antenatal corticosteroid treatment versus standard care for the reduction of neonatal mortaliry due to preterm birth in lowincome and middle-income countries: the ACT cluster-randomised trial. Lancet 385(9968):629,n2015 Althuisius SM, Dekker G, Hummel P, et al: Cervical incompetence prevention randomized cerclage trial: emergency cerclage with bed rest versus bed rest alone. Am] Obstet Gynecol 189:907,n2003 Althuisius SM, Dekker GA, Hummel P, et al: Final results of the Cervical Incompetence Prevention Randomized Cerclage Trial (CIPRACT): therapeutic cerclage with bed rest versus bed rest alone. Am ] Obstet Gynecol 185:1106,n2001

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1	Ward K: Genetic factors in common obstetric disorders. Clin Obstet Gynecol 51t:74,t2008 Warren ]E, Silver i, Daltont], et al: Collagen 1A1 and transforming growth factor-3 polymorph isms in women with cervical insuiciency. Obstet Gynecol 110:619, 2007 Warren B, Goland RS, Wardlaw SL, et al: Elevated maternal plasma corticotropin releasing hormone levels in twin gestation. ] Perinat Med 18:39, 1990 Watts DH, Krohn A, Hillier SL, et al: he association of occult amniotic luid infection with gestational age and neonatal outcome among women in preterm labor. Obstet Gynecol 9:35t1,t1992 Weiss ]L, Malone FD, Vidaver ], et al: hreatened abortion: a risk factor for poor pregnancy outcome, a population-based screening study. Am ] Obstet Gynecol 190:745,t2004 Wenstrom K, Weiner CP, Merrill D, et al: A placebo controlled randomized trial of the terbutaline pump for prevention of preterm delivery. Amt] PerinatoIt14:87, 1997

1	Wenstrom K, Weiner CP, Merrill D, et al: A placebo controlled randomized trial of the terbutaline pump for prevention of preterm delivery. Amt] PerinatoIt14:87, 1997 Werner EF, Han CS, Savitz DA, et al: Health outcomes for vaginal compared with cesarean delivery of appropriately grown preterm neonates. Obstet Gynecol 121:1195,t2013 White BA, Creedon D], Nelson E, et al: The vaginal microbiome in health and disease. Trends Endocrinol Metab 22(10):389, 2011 Winn HN, Chen M, Amon E, et al: Neonatal pulmonary hypoplasia and perinatal mortality in patients with mid-trimester rupture of amniotic membranes-a critical analysis. Am ] Obstet Gynecol 182:1638, 2000 Wolfe CD, Patel SP, Linton EA, et al: Plasma corticotrophin-releasing factor (CRF) in abnormal pregnancy. B]OG 95:1003, 1988 Wu W, Colfordt]M ]r: Chorioamnionitis as a risk factor for cerebral palsy: a meta-analysis. ]AMA 284 (11): 1417, 2000

1	Wu W, Colfordt]M ]r: Chorioamnionitis as a risk factor for cerebral palsy: a meta-analysis. ]AMA 284 (11): 1417, 2000 Yoon BH, Romero R, Park]S, et al: Fetal exposure to an intra-amniotic inflammation and the development of cerebral palsy at the age of three years. Am ] Obstet Gynecol 182:675,t2000 Yoon BH, Romero R, Park ]S, et al: Microbial invasion of the amniotic cavity with Ureaplasma urealyticum is associated with robust host response in fetal, amniotic, and maternal compartments. Am ] Obstet Gynecol 179: 1254, 1998 Yoon BH, Yang SH, ]un ]K, et al: Maternal blood C-reactive protein, white blood cell count, and temperature in preterm labor: a comparison with amniotic luid white blood cell count. Obstet GynecoIt87:23t1, 1996 Young A, Thomson A], Ledingham M, et al: Immunolocalization of proinlammatory cytokines in myometrium, cervix, and fetal membranes during human parturition at term. BioI Reprod 66:445, 2002

1	Young A, Thomson A], Ledingham M, et al: Immunolocalization of proinlammatory cytokines in myometrium, cervix, and fetal membranes during human parturition at term. BioI Reprod 66:445, 2002 Young D: Clinical trials and tribulations: 170HPC and preventing recurrent preterm birth. Am] Obstet GynecoI216(6):543, 2017 Younge N, Goldstein RF, Bann CM, et al: Survival and neurodevelopmental outcomes among periviable infants. N Engl ] Med 376(7):617, 2017 Zhang X, Liu M: Clinical observations on the prevention and treatment of premature labor with nifedipine. [Chinese] Hua i Yi Ke Da Xue Xue Bao 33(2):288, 2002 Zuckerman H, Reiss U, Rubinstein I:

1	Zuckerman H, Reiss U, Rubinstein I: Inhibition of human premature labor by indomethacin. Obstet , 1974 t must be admitted that the duration ofpregnancy not infrequenty exceeds 280 days rom the last menstrual period and that when it lasts much longer large children are developed which are frequenty delivered ony after great dficuly. Thus, whenever the menstrual history of the patient indicates that she has passed much beyond the tenth and is approaching the eleventh lunar month, we should consider the pro priey of the induction of labour, provided that examination shows the child is larger than usual. -J. Whitridge Williams (1903) he above passage from Williams shows that pregnancies exceeding the expected normal length were problematic more than 100 years ago. These postterm pregnancies remain so today.

1	The adjectives postterm, prolonged, postdates, and postmature are often loosely used interchangeably to describe pregnancies that have exceeded a duration considered to be the upper limit of normal. We eschew use of the term postdates because the real issue in many postterm pregnancies is "post-what dates?" Post mature is reserved for the relatively uncommon speciic clinical fetal syndrome in which the newborn has recognizable features indicating a pathologically prolonged pregnancy. herefore, postterm or prolonged pregnancy is our preferred expression for an extended pregnancy.

1	The international definition of prolonged pregnancy, endorsed by the American College of Obstetricians and Gynecologists (2016b,d) is one that exceeds 42°/7 weeks, namely, 294 days or more from the first day of the last menstrual period. Importantly, this is 42 "completed weeks," as pregnancies between 41 weeks 1 day and 41 weeks 6 days, although in the 42nd week, do not complete 42 weeks until the seventh day has elapsed. The method that we use widely in this book is to divide the 42nd week into 7 days, that is, 42°/7 through 426' weeks.

1	The current deinition of postterm pregnancy assumes that the last menses was followed by ovulation 2 weeks later. hat said, some pregnancies may not actually be postterm. Instead, the because of faulty menstrual date recall or delayed ovulation. Thus, the two categories of pregnancies that reach 42 completed weeks are those truly 40 weeks past conception and those of less-advanced gestation but with inaccurately estimated gestational age. Even with exactly recalled menstrual dates, there still is imprecision, and the American College of Obstetricians and Gynecologists (2016d, 20 17b) considers first-trimester sonography to be the most accurate method to establish or confirm gestational age. Several clinical studies support this practice (Bennett, 2004; Blondel, 2002; Joseph, 2007).

1	Of the 3.93 million neonates born in the United States during 2015, 0.4 percent were delivered at 42 weeks or later (11artin, 2017). In the past, the proportion was much higher. This trend suggests earlier intervention, however, the added accuracy from earlier sonographic dating ofgestational age is another factor. To identiy potential predisposing factors for postterm pregnancy, Olesen and associates (2006) analyzed various characteristics in the Danish Birth Cohort. Only prepregnancy body mass index (BMI) :25 and nulliparity were signiicantly associated with prolonged pregnancy. Mission (2015) and Arrowsmith (2011) and their coworkers also reported similar associations. In nulliparas, those whose cervical length at midpregnancy is longer, that is, in the third or fourth quartile, are twice as likely to deliver after 42 weeks (van der Ven, 2016).

1	The tendency for some mothers to have repeated postterm births suggests that some prolonged pregnancies are biologically determined. Oberg and colleagues (2013) reported that when mother and daughter had a prolonged pregnancy, the risk for the daughter to have a subsequent postterm pregnancy was signiicantly increased. Laursen and associates (2004) found that maternal, but not paternal, genes inluenced prolonged pregnancy. As discussed in Chapter 5 (p. 105), rare fetal-placental factors that predispose to postterm pregnancy include anencephaly, adrenal hypoplasia, and X-linked placental sulfatase deiciency (Ayyavoo, 2014; MacDonald, 1965).

1	Rates of stillbirth, neonatal death, and infant morbidity all rise after the expected due date has passed. This is best seen when perinatal mortality rates are analyzed from times before widespread intervention for postterm pregnancies. In two large Swedish studies shown in Figure 43-1, after reaching a nadir at � 7.2 4.0 c 2.32.4 ing to gestational age of all births in Sweden during 1943-1952 compared with those during 1977-1o978. The partially compressed scale is used for convenience in depiction. (Adapted from Bakketeig, 1991; Lindell, 1956.) TABLE 43-1 . Adverse Maternal and Perinatal Outcomes Associated with Postterm Pregnancy NICU = neonatal intensive care unit. 39 to 40 weeks, the perinatal mortality rate rose as pregnancy duration exceeded 41 weeks. his trend is also reported for the United States (Cheng, 2008; MacDorman, 2009). As shown in

1	39 to 40 weeks, the perinatal mortality rate rose as pregnancy duration exceeded 41 weeks. his trend is also reported for the United States (Cheng, 2008; MacDorman, 2009). As shown in Table 43-1, the major cause of death in these studies includes gestational hypertension, prolonged labor with cephalopelvic disproportion, birth injuries, and hypoxic-ischemic encephalopathy. Similar outcomes were reported by Olesen and colleagues (2003) in 78,022 women with postterm pregnancies delivered before routine labor induction was adopted in Denmark. Moster and associates (2010) found higher rates of cerebral palsy in postterm births, and Yang and coworkers (2010) reported lower intelligence quotient (IQ) scores at age 6.5 years in children born :42 weeks' gestation. Conversely, autism was not associated with postterm birth (Gardener, 2011).

1	Alexander and colleagues (2000a) reviewed 56,317 consecutive singleton pregnancies delivered at :40 weeks between 1988 and 1998 at Parkland Hospital. Labor was induced in 35 percent of pregnancies completing 42 weeks. he rate of cesarean delivery for dystocia and fetal distress was signiicantly greater at 42 weeks compared with earlier deliveries. More newborns of postterm pregnancies were admitted to intensive care units. Importantly, the incidence of neonatal seizures and deaths was doubled at 42 weeks. Smith (2001) has challenged analyses such as these because the population at risk for perinatal mortality in a given week consists ofall ongoing pregnancies rather than just the births in a givenweek. He calculated perinatal mortality rates calculated using only births in a given week of gestation from 37 to 43 completedweeks comparedwith the cumulative probabilitythe perinatal index-of death when all ongoing pregnancies are included in the denominator. Using this computation,

1	of gestation from 37 to 43 completedweeks comparedwith the cumulative probabilitythe perinatal index-of death when all ongoing pregnancies are included in the denominator. Using this computation, delivery at 38 weeks had the lowest risk index for perinatal death.

1	The postmature newborn is unique, and features include wrinkled, patchy, peeling skin; a long, thin body suggesting wasting; and advanced maturity in that the infant is open-eyed, unusually alert, and appears old and worried 43-2). Skin wrinkling can be particularly prominent on the palms and soles. The nails are typically long. Most postmature neonates are not technically FIGURE 43-2 Postmaturity syndrome. Neonate delivered at 43 weeks' gestation with thick, viscous meconium coating the desquamating skin. Note the long, thin appearance and wrinkling of the hands. growth restricted because their birthweight seldom falls below the 10th percentile for gestational age (Chap. 44, p. 847). On the other hand, severe growth restriction-which logically must have preceded completion of 42 weeks-may be present.

1	he incidence of postmaturity syndrome in newborns at 41, 42, or 43 weeks, respectively, has not been conclusively determined. From data, the syndrome complicates 10 to 20 percent of pregnancies at 42 completed weeks (American College of Obstetricians and Gynecologists, 2016d). Associated oligohydramnios substantially raises the likelihood of postmaturity. Trimmer and associates (1990) reported that 88 percent of fetuses were postmature if there was oligohydramnios deined by a sonographic maximal vertical amnionic fluid pocket that measuredr; 1 cm at 42 weeks. Many believe that postterm pregnancy is an abnormal state. Redman and Staf (2015) posit that limited placental capacity, which is characterized by dysfunctional syncytiotrophoblast, explains the greater risks of the postmaturity syndrome.

1	Cliford (1954) proposed that the associated skin changes were due to loss of the protective efects of vernix caseosa. He also attributed the postmaturity syndrome to placental senescence, although he did not ind placental degeneration histologically. Still, the concept that postmaturity stems from placental insuiciency has persisted despite an absence of morphological or signiicant quantitative findings (Larsen, 1995; Redman, 2015; Rushton, 1991). here are indings that the

1	FIGURE 43-3 Mean daily fetal growth during previous week of gestation. (Redrawn from Hendricks CH: Patterns of fetal and placental growth: the second half of pregnancy. Obstet Gynecol 24:357, 1964.) rate of placental apoptosis-programmed cell death-is significantly greater at 41 to 42 completed weeks compared with that at 36 to 39 weeks (Smith, 1999). Several proapoptotic genes such as kisspeptin are upregulated in postterm placental explants compared with the same genes in term placental explants (Torricelli, 2012). The clinical significance of such apoptosis is currently unclear.

1	Jazayeri and coworkers (1998) investigated cord blood erythropoietin levels in 124 appropriately grown newborns delivered from 37 to 43 weeks. he only known stimulator of erythropoietin is decreased partial oxygen pressure. Thus, they sought to assess whether fetal oxygenation was compromised due to placental aging in postterm pregnancies. All women had an uncomplicated labor and delivery. These investigators found that cord blood erythropoietin levels were significantly higher in pregnancies reaching 41 weeks or more. Although Apgar scores and acid-base studies were normal, these researchers concluded that fetal oxygenation was decreased in some postterm gestations.

1	Another scenario is that the postterm fetus may continue to gain weight and thus be unusually large at birth. This at least suggests that placental function is not severely compromised. Indeed, continued fetal growth is the norm-albeit at a slower rate beginning at 37 completed weeks (Fig. 43-3). Nahum and colleagues (1995) conirmed that fetal growth continues until at least 42 weeks. However, Link and associates (2007) showed that umbilical blood low did not increase concomitantly. The principal reasons for increased risks to postterm fetuses were described by Leveno and associates (1984). Both antepartum fetal jeopardy and intrapartum fetal distress were found to be the consequence of cord compression associated with oligohydramnios. In their analysis of 727 postterm pregnancies, intrapartum fetal distress detected with electronic monitoring was not associated with late decelerations characteristic of uteroplacental insuiciency. Instead, one or more prolonged 1008060 -.0 100f80 -0

1	FIGURE 43-4 A. Prolonged fetal heart rate deceleration before emergency cesarean delivery in a postterm pregnancy with oligohydramnios. B. Severe-less than 70 bpm for 60 seconds or longer-variable decelerations in a postterm pregnancy with oligohydramnios. C. Saltatory baseline fetal heart rate showing oscillations exceeding 20 bpm and associated with oligohydramnios in a postterm pregnancy. (Reproduced with permission from Leveno J, Quirk JG, Cunningham FG, et al: Prolonged pregnancy, I. Observations concerning the causes of fetal distress, Am J Obstet Gynecol. 1984 Nov 1 ;1o50(5 Pt 1 ):465-473.) decelerations such as shown in of emergency cesarean deliveries for nonreassuring fetal heart rate tracings. In all but two cases, there were also variable decelerations. Another common fetal heart rate pattern, although not ominous by itself, was the saltatory baseline. As described in Chapter 24 (p. 468), these indings are consistent with cord occlusion as the proximate cause of the

1	heart rate pattern, although not ominous by itself, was the saltatory baseline. As described in Chapter 24 (p. 468), these indings are consistent with cord occlusion as the proximate cause of the nonreassuring tracings. Other correlates included oligohydramnios and viscous meconium. Schafer and colleagues (2005) implicated a nuchal cord in abnormal intrapartum fetal heart rate patterns, meconium, and compromised newborn condition in prolonged pregnancies.

1	he volume of amnionic luid normally continues to decline ater 38 weeks and may become problematic. Moreover, meconium release into an already reduced amnionic luid volume results in thick, viscous meconium that may cause meconium aspiration syndrome (Chap. 33, p. 620). Trimmer and coworkers (1990) sonographically measured hourly fetal urine production using sequential bladder volume measurements in 38 postterm pregnancies. Diminished urine production was found to be associated with oligohydramnios. They hypothesized that decreased fetal urine flow was likely the result of preexisting oligohydramnios that limited fetal swallowing. Oz and associates (2002), using Doppler waveforms, concluded that fetal renal blood low is reduced in those postterm pregnancies complicated by oligohydramnios. s a possible cause, again, the study by Link and associates (2007) showed that umbilical blood flow did not increase past term.

1	In the late 19905, the clinical significance of fetal-growth restriction in the otherwise uncomplicated pregnancy became more fully appreciated. Divon (1998) and Clausson (1999) and their coworkers analyzed births between 1991 and 1995 in the National Swedish Medical Birth Registry. Stillbirths were more common among growth-restricted newborns who were delivered after 42 weeks. Indeed, a third of postterm stillborn neonates were growth restricted. During this time in Sweden, labor induction and antenatal fetal testing usually commenced at 42 weeks. In a study from Parkland Hospital, Alexander and colleagues (2000d) analyzed outcomes for 355 neonates from pregnancies ::42 weeks and whose birthweights were <3rd percentile. hey compared these with outcomes of 14,520 similarly aged newborns above the 3rd percentile and found that morbidity and mortality rates were significantly increased in the growth-restricted neonates. Notably, a fourth of all stillbirths associated with prolonged

1	above the 3rd percentile and found that morbidity and mortality rates were significantly increased in the growth-restricted neonates. Notably, a fourth of all stillbirths associated with prolonged pregnancy were in this comparatively small number of growth-restricted fetuses.

1	In the event of a medical or other obstetrical complication, it is generally not recommended that a pregnancy be allowed to continue past 42 weeks. Indeed, in many such instances, earlier delivery is indicated. Common examples include gestational hypertensive disorders, prior cesarean delivery, and diabetes. Other clinically important factors include amnionic luid volume and potential fetal macrosomia.

1	Most clinical studies support theview that diminished amnionic fluid determined by various sonographic methods identifies a postterm fetus with increased risks. Indeed, decreased amnionic luid in any pregnancy signiies increased fetal risk (Chap. 11r, p. 231). Unfortunately, lack of an exact method to define "decreased amnionic luid" has limited investigators, and many diferent criteria for sonographic diagnosis have been proposed. Fischer and colleagues (1993) attempted to determine which criteria were most predictive of normal versus abnormal outcomes in postterm pregnancies. As shown in Figure 43-5, the smaller the amnionic luid pocket, the greater the likelihood that there was clinically signiicant oligohydramnios. Importantly, normal amnionic luid volume did not preclude abnormal outcomes. lfirevic and coworkers (1997) randomly assigned 500 women with postterm pregnancies to assessment using either the amnionic luid index (AFI) or the deepest vertical pocket described in Chapter

1	lfirevic and coworkers (1997) randomly assigned 500 women with postterm pregnancies to assessment using either the amnionic luid index (AFI) or the deepest vertical pocket described in Chapter 11 (p. 226). They concluded that the AFI overestimated the number of abnormal outcomes in postterm pregnancies.

1	Regardless of the criteria used to diagnose oligohydramnios in postterm pregnancies, most investigators have found a higher incidence of some measure of "fetal distress" during labor. Thus, oligohydramnios by most definitions is a clinically meaningful finding. Conversely, reassurance of continued fetal well-being in the presence of "normal" amnionic luid volume is tenuous. This may be related to how quickly pathological oligohydramnios develops. Although such cases are unusual, Clement and coworkers (1987) described six postterm pregnancies in which amnionic luid volume diminished abruptly over 24 hours, and in one of these, the fetus died. : 80 00 20 : 6.7em< 5em< 2em< 1 em

1	FIGURE 43-5 Comparison of the prognostic value of various pregnancies. Abnormal outcomes include cesarean or operative vaginal delivery for fetal jeopardy, 5-minute Apgar score �6, umbili cal arterial blood pH <7.1, or admission to the neonatal intensive care unit. (Redrawn from Fischer RL, McDonnell M, Bianculli KW, et al: Amniotic fluid volume estimation in the postdate pregnancy: a comparison oftechniques. Obstet Gynecol 81 :698, 1993.) he velocity of fetal weight gain peaks at approximately 37 weeks (see Fig. 43-3). Although growth velocity slows at that time, most fetuses continue to gain weight. For example, the percentage of fetuses born in 2009 whose birthweight exceeded 4000 g was 8.2 percent at 37 to 41 weeks and increased to 11.0 percent at 42 weeks or more (Martin, 2011). According to Duryea and associates (2014), the 95th percentile at 42 weeks is 4475 g. Even so, in some studies, brachial plexus injurywas not related to postterm gestation (Walsh, 2011). Intuitively, it

1	to Duryea and associates (2014), the 95th percentile at 42 weeks is 4475 g. Even so, in some studies, brachial plexus injurywas not related to postterm gestation (Walsh, 2011). Intuitively, it seems that both maternal and fetal morbidity associated with macrosomia would be mitigated with timely induction to preempt urther growth. This, however, does not appear to be the case. The American College ofObstetricians and Gynecologists (2016c) has concluded that current evidence does not support such a practice in women at term with suspected fetal macrosomia. Moreover, the College concluded that in the absence of diabetes, vaginal delivery is not contraindicated for women with an estimated fetal weight up to 5000 g (Chap. 27, p. 520). Obvious problems with all such recommendations are substantive variations in fetal weight estimation.

1	Although some intervention is indicated for prolonged pregnancies, the method and timing of this are not unanimous. he decision focuses on whether labor induction is warranted or if expectant management with fetal surveillance is best. In a survey done more than 10 years ago, Cleary-Goldman and associates (2006) reported that 73 percent of members of the American College ofObstetricians and Gynecologists routinely induced women at 41 weeks. Most of the remainder performed twice weekly fetal testing until 42 weeks.

1	Although all obstetricians know what an "unfavorable cervix" is, the term unfortunately defies precise objective deinition. Thus, investigators have used difering criteria for studies of prolonged pregnancies. Harris and coworkers (1983) defin�d an unfavorable cervix by a Bishop score <7 and reported thiS m 92 percent of women at 42 weeks (Chap. 26, p. 505). Hannah and colleagues (1992) found that 40 percent of 3407 women with a 41-week pregnancy had an "undilated cervix." In a study of 800 women undergoing induction for postterm pregnancy at Parkland Hospital, Alexander and associates (2000b) reported that women in whom there was no cervical dilation had a twofold higher cesarean delivery rate fore" dystocia. " Yang and coworkers (2004) found that cervical length 53 em measured with transvaginal sonography was predictive of successful induction. In a similar study, Vankayalapati and associates (2008) found that cervical length 525 mm was predictive of spontaneous labor or successful

1	sonography was predictive of successful induction. In a similar study, Vankayalapati and associates (2008) found that cervical length 525 mm was predictive of spontaneous labor or successful induction.

1	Several investigators have evaluated prostaglandin E2 (PGE2) and E1 (PGE1) for induction in women with an unfavorable cervix and prolonged pregnancies. A study by the MaternalFetal Medicine Units Network (1994) found that PGE2 gel was not more efective than placebo. Alexander and associates (2000c) treated 393 women with a postterm pregnancy with PGE2, regardless of cervical "favorability," and reported that almost half of the 84 women with cervical dilatation of 2 to 4 em entered labor with PGE2 use alone. In another study, mifepristone was reported to increase uterine activity without uterotonic agents in women beyond 41 weeks (Fasset, 2008). Prostaglandins and other agents used for cervical ripening are discussed in Chapter 26 (p. 506).

1	Sweeping or stripping of the membranes to induce labor and thereby prevent postterm pregnancy was studied in 15 randomized trials during the 1990s. Boulvain and coworkers (2005) performed a metaanalysis of these and found that membrane stripping at 38 to 40 weeks lowered the frequency of postterm pregnancy. Although maternal and neonatal infection rates were not increased, this practice did not modiy the cesarean delivery rate. Since then, randomized trials by Wong (2002), Kashanian (2006), Hill (2008), and their coworkers found that sweeping membranes did notereduce the need to induce labor. Drawbacks of membrane stripping included pain, vaginal bleeding, and irregular contractions without labor.

1	Drawbacks of membrane stripping included pain, vaginal bleeding, and irregular contractions without labor. The station of the fetal head within the pelvis is another predictor of successful postterm pregnancy induction. Shin and colleagues (2004) studied 484 nulliparas who underwent induction after 41 weeks. The cesarean delivery rate was directly related to station. The rate was 6 percent if the vertex before induction was at -1 station; 20 percent at -2 station; 43 percent at -3 station; and 77 percent at -4 station.

1	Because of the marginal benefits from induction with an unfavorable cervix, as just discussed, some clinicians prefer instead to implement a strategy of fetal testing beginning at 41 completed weeks. For example, in a Canadian study, 3407 women were randomly assigned at 41 or more weeks to induction or to fetal testing (Hannah, 1992). In the surveillance group, evaluation included: (1) counting fetal movements during a 2-hour period each day, (2) nons tress testing three times weekly, and (3) amnionic fluid volume assessment two to three times weekly, with pockets <3 em considered abnormal. Labor induction resulted in a small but signiicant reduction in the cesarean delivery rate compared with fetal testing-2e1 versus 24 percent, respectively. This diference was due to fewer procedures for fetal distress. here were only two stillbirths in the fetal testing group.

1	The !v1aternal-Fetal Medicine Network performed a randomized trial of induction versus fetal testing beginning at 41 weeks (Gardner, 1996). Fetal surveillance included nonstress testing and sonographic estimation of amnionic fluid volume performed twice weekly in 175 women. Perinatal outcomes were compared with those of 265 women also at 41 weeks randomly assigned to induction with or without cervical ripening. There were no perinatal deaths, and the cesarean delivery rate was not diferent between management groups. he results of this study could be used to support the validity of either management scheme. In an analysis of 22 trials, Gulmezoglu and colleagues (2012) found that induction after 41 weeks rather than surveillance was associated with significantly fewer perinatal deaths and meconium aspiration syndrome cases and a lower cesarean delivery rate. In a review of two metaanalyses and a randomized study, similar conclusions were reached (Mozurkewich, 2009).

1	In most studies, labor induction at 42°/7 weeks has a higher cesarean delivery rate compared with spontaneous labor. From Parkland Hospital, Alexander and coworkers (2001) evaluated pregnancy outcomes in 638 such women in whom labor was induced and compared them with outcomes of 687 women with postterm pregnancies who had spontaneous labor. Cesarean delivery rates were signiicantly increased-19 versus 14 percent-in the induced group because of failure to progress. When these investigators corrected for risk factors, however, they concluded that intrinsic maternal factors, rather than the induction itself, led to the higher rate. These factors included nulliparity, an unfavorable cervix, and epidural analgesia.

1	A large study from Denmark by Zizzo and associates (20e17) is also instructive. In 2011e, the Danish national guidelines were changed from labor induction at 42°/7 weeks with no fetal sur veillance to labor induction at 41e/7 to 416/7 weeks with fetal surveillance beginning at 41°/7 weeks. They compared two 3-year epochs-one before and one ater 20 II-and the results are shown in Table 43-2. The rate of pregnancies that progressed past 42°/7 weeks decreased from 2.85 to 0.62 percent. Concu�rently, as expected, the induction rate rose signiicantly, and thiS was accompanied by a drop in the perinatal mortality rate-22 to 13 per 1000 births. The cesarean delivery rate was not changed. A similar before-and-ater observational study reported that induction at ::42 weeks was associated with a signiicantly lower cesarean delivery rate-15 versus 19.4 percent (Bleicher, 2017).

1	From the foregoing, evidence to substantiate interventionwhether induction or fetal testing-commencing at 41 versus 42 weeks is limited. Most evidence used to justiy intervention TABLE 43-2. National Death Cohort Study of 102,1e67 Pregnancies that Reached 41e°/7 Weeks' Gestation EGA >42°/7 2.85% 0.62% Stillbirths 9/1000 5/1000 0.018 Neonatal deaths 13/1000 811000 .033 Vacuum delivery 11.3% 10.2% <0.001 Induction 28% 43% <0.001 aNational guidelines changed between epochs as described in text. NS = not significant. Data from Zizzo, 201n7. at 41 weeks is from the randomized Canadian and American investigations cited earlier. No randomized studies have specifically assessed intervention at 41 weeks versus an identical intervention used at 42 weeks. A large Swedish multicenter randomized trial of more than 10,000 women at 41°1 weeks has been designed to address the question (Elden, 2016).

1	he American College of Obstetricians and Gynecologists (2016a) deines postterm pregnancies as having completed 42 weeks, namely, beyond 42°/7 weeks. There is insuicient evidence to mandate a management strategy between 40 and 42 completed weeks. hus, although not considered mandatory, initiation of fetal surveillance at 41 weeks is a reasonable option. After completing 42 weeks, recommendations are for labor induction as summarized in Figure 43-6. When gestational age is uncertain, the American College of Obstetricians and Gynecologists (2017b) recommends delivery at 41 weeks' gestation using the best clinical estimate of gestational age. he College also recommends against amniocentesis for fetal lung maturity. At Parkland Hospital, based on results from the trials just discussed, we consider 41-week pregnancies without other

1	FIGURE 43-6 Algorithm for management of postterm pregnancy. (Summarized from American College of Obstetricians and Gynecologists,o201o6d.) complications to be normal. hus, no interventions are practiced solely based on fetal age until 42 completed weeks. With complications such as hypertension, decreased fetal movement, or oligohydramnios, labor induction is carried out. It is our view that large, randomized trials should be performed before otherwise uncomplicated 41-week gestations are routinely considered pathologically prolonged. In women in whom a certain gestational age is known, labor is induced at the completion of 42 weeks. Almost 90 percent of such women are induced successfully or enter labor within 2 days of induction. For those who do not deliver with the first induction, a second induction is performed within 3 days. lmost all women are delivered using this management plan, but in the unusual few who are not delivered, management decisions involve a third-or even

1	a second induction is performed within 3 days. lmost all women are delivered using this management plan, but in the unusual few who are not delivered, management decisions involve a third-or even more-induction versus cesarean delivery. Women classiied as having uncertain postterm pregnancies are managed with weekly nonstress fetal testing and assessment ofamnionic luid volume. Women with an AFI ;5 cm or with reports ofdiminished fetal movement undergo labor induction.

1	Labor is a particularly dangerous time for the postterm fetus. Therefore, women whose pregnancies are known or suspected to be postterm ideally come to the hospital as soon as they suspect labor. While being evaluated for active labor, we monitored electronically for variations consistent with fetal compromise. During labor, the decision to perform amniotomy is problematic. Further reduction in luid volume following amniotomy can enhance the possibility of cord compression. Conversely, after membrane rupture, a scalp electrode and an intrauterine pressure catheter can be placed. These usually provide more precise data concerning fetal heart rate and uterine contractions. Amniotomy also aids identification of thick meconium.

1	Thick meconium in the amnionic luid is particularly worrisome. The viscosity probably signiies the lack of liquid and thus oligohydramnios. Aspiration of thick meconium may cause severe pulmonary dysfunction and neonatal death (Chap. 33, p. 620). Because of this, amnioinfusion during labor has been proposed as a way of diluting meconium to lower the incidence of aspiration s) ndrome (Wenstrom, 1989). As discussed in Chapter 24 (p. 475), the benefits of amnioinfusion remain controversial. In a large randomized trial by Fraser and colleagues (2005), amnioinfusion did not reduce the risk of meconium aspiration syndrome or perinatal death. According to the American College of Obstetricians and Gynecologists (2016a), amnioinfusion does not prevent meconium aspiration, however, it remains a reasonable treatment approach for repetitive variable decelerations.

1	he likelihood of a successful vaginal delivery is reduced appreciably for the nullipara who is in early labor with thick, meconium-stained amnionic fluid. Therefore, if the woman is remote from delivery, strong consideration should be given to prompt cesarean delivery, especially when cephalopelvic disproportion is suspected or either hypotonic or hypertonic dysfunctional labor is evident. Some practitioners choose to avoid oxytocin use in these cases. Until recently, it was taught-including at Parkland Hospital -that aspiration of meconium could be minimized but not eliminated by suctioning the pharynx as soon as the head was delivered. According to the American Heart Association guidelines, this in no longer recommended (Wyckof, 2015). The merican College of Obstetricians and Gynecologists (2017a) does not recommend routine intrapartum suctioning. Alternatively, if the depressed newborn has meconium-stained fluid, then intubation is carried out.

1	Alexander JM, McIntire DO, Leveno KJ: Forty weeks and beyond: pregnancy outcomes by week of gestation. Obstet Gynecol 96:291, 2000a lexander JM, McIntire DO, Leveno KJ: Postterm pregnancy: does induction increase cesarean rates? J Soc Gynecol Invest 7:79A, 2000b lexander JM, McIntire DO, Leveno KJ: Postterm pregnancy: is cervical "ripening" being used in the right patients? J Soc Gynecol Invest 7:247 A, 2000c Alexander JM, McIntire DO, Leveno KJ: Prolonged pregnancy: induction of labor and cesarean births. Obstet Gynecol 97:9t11t, 2001 Alexander JM, McIntire DO, Leveno KJ: he efect of fetal growth restriction on neonatal outcome in postterm pregnancy. Abstract No. 463. Am J Obstet Gynecol 182:S148, 2000d Alfirevic Z, Luckas M, Walkinshaw SA, et al: A randomized comparison between amniotic fluid index and maximum pool depth in the monitoring of postterm pregnancy. Br J Obstet Gynaecol 104:207, 1997

1	Alfirevic Z, Luckas M, Walkinshaw SA, et al: A randomized comparison between amniotic fluid index and maximum pool depth in the monitoring of postterm pregnancy. Br J Obstet Gynaecol 104:207, 1997 American College of Obstetricians and Gynecologists: Amnioinfusion does not prevent meconium aspiration syndrome. Committee Opinion No. 346, October 2006, Reairmed 2016a American College of Obstetricians and Gynecologists Qoint with the Society for Maternal-Fetal Medicine): Deinition of term pregnancy. Committee Opinion No. 579, November 20t13, Reairmed 20t16b American College of Obstetricians and Gynecologists: Fetal macrosomia. Practice Bulletin No. 173, November 20t16c American College of Obstetricians and Gynecologists: Management of lateterm and postterm pregnancies. Practice Bulletin No. 146, August 20t14, Reairmed 2016d

1	American College of Obstetricians and Gynecologists: Management of lateterm and postterm pregnancies. Practice Bulletin No. 146, August 20t14, Reairmed 2016d American College of Obstetrics and Gynecologists: Management of delivery of a newborn with meconium-stained amniotic fluid. Committee Opinion No. 689, March 20t17a American College of Obstetricians and Gynecologists: Management of suboptimally dated pregnancies. Committee Opinion No. 688, March 2017b Arrowsmith S, Wray S, Quenby S: Maternal obesity and labour complications following induction of labour in prolonged pregnancy. BJOG 118(5):578, 2011 Ayyavoo A, Derraik JG, Hofman PL, et al: Postterm births: are prolonged pregnancies roo long? J Pediatr 164(3):647,t2014 Bakketeig LS, Bergsj0 P: Post-term pregnancy: magnitude of the problem. In Chalmers I, Enkin M, Keirse M (eds): Efective Care in Pregnancy and Childbirth. Oxford, Oxford University Press, 1991, p 765

1	In Chalmers I, Enkin M, Keirse M (eds): Efective Care in Pregnancy and Childbirth. Oxford, Oxford University Press, 1991, p 765 Bennett A, Crane JM, O'Shea P, et al: First trimester ultrasound screening is efective in reducing postterm labor induction rates: a randomized controlled trial. Am J Obstet Gynecol 190: 107 , 2004 Bleicher I, Vinter 0, Iofe A, et al: When should pregnancies that extended beyond term be induced? J Matern Fetal Neonatal Med 30(2):219,t2017 Blondel B, Morin I, Platt RW, et al: Algorithms for combining menstrual and ultrasound estimates of gestational age: consequences for rates of preterm and postterm birth. Br J Obstet Gynaecol 109: 18, 2002 Boulvain M, Stan CM, Irion 0: Membrane sweeping for induction of labour. Cochrane Database Syst Rev 1 :CD00045t1, 2005 Cheng W, Nicholson JM, Nakagawa S, et al: Perinatal outcomes in low-risk term pregnancies: do they difer by week of gestation? Am J Obstet Gynecol 0.e1,t2008

1	Cheng W, Nicholson JM, Nakagawa S, et al: Perinatal outcomes in low-risk term pregnancies: do they difer by week of gestation? Am J Obstet Gynecol 0.e1,t2008 Clausson B, Cnattingus S, Axelsson 0: Outcomes of postterm births: the role of fetal growth restriction and malformations. Obstet Gynecol 94:758, 1999 Cleary-Goldman J, Bettes B, Robinon IN, et al: Postterm pregnancy: practice patterns of contemporary obstetricians and gynecologists. Am J Perinatol 23:15,t2006 Clement 0, Schifrin BS, Kates RB: Acute oligohydramnios in postdate pregnancy. Am J Obstet Gynecol 157:884, 1987 Cliford SH: Postmaturity with placental dysfunction. Clinical syndromes and pathologic indings. J Pediatr 44: 1, 1954 Divon MY, Haglund B, Nisell H, et al: Fetal and neonatal mortality in the postterm pregnancy: the impact of gestational age and fetal growth restriction. Am J Obstet Gynecol 178:726, 1998

1	Divon MY, Haglund B, Nisell H, et al: Fetal and neonatal mortality in the postterm pregnancy: the impact of gestational age and fetal growth restriction. Am J Obstet Gynecol 178:726, 1998 Duryea EL, Hawkins JS, McIntire DO, et al: A revised birth weight reference for the United States. Obstet GynecoIt124:16, 2014 Elden H, Hagberg H, Wessberg A, et al: Study protocol of SWEPIS a Swedish multicentre register based randomised controlled trial to compare induction of labour at 41 completed gestational weeks versus expectant management and induction at 42 completed gestational weeks. BMC Pregnancy Childbirth 16:49,t2016 Fasset MJ, Wing DA: Uterine activity after oral mifepristone administration in human pregnancies beyond 41 weeks' gestation. Gynecol Obstet Invest 65(2): ll2, 2008 Fischer RL, McDonnell M, Bianculli W, et al: Amniotic luid volume estimation in the postdate pregnancy: a comparison of techniques. Obstet Gynecol 81t:698, 1993

1	Fischer RL, McDonnell M, Bianculli W, et al: Amniotic luid volume estimation in the postdate pregnancy: a comparison of techniques. Obstet Gynecol 81t:698, 1993 Fraser WD, Hofmeyr J, Lede R, et al: Amnioinfusion for the prevention of the meconium aspiration syndrome. New Engl J Med 353:909, 2005 Gardener H, Spiegelman D, Buka SL: Perinatal and neonatal risk factors for autism: a comprehensive meta-analysis. Pediatrics 128:344,t2011 Gardner M, Rouse 0, Goldenberg R, et al: Cost comparison of induction of labor at 41 weeks versus expectant management in the postterm pregnancy. Am J Obstet 4:351, 1996 Gulmezoglu M, Crowther CA, Middleton P, et l: Induction of labour for improving birth outcomes for women at or beyond term. Cochrane Database Syst Rev 6:CD004945, 2012 Hannah ME, Hannah J, Hellman J, et al: Induction of labor as compared with serial antenatal monitoring in post-term pregnancy. N Engl J vIed 326:1587, 1992

1	Hannah ME, Hannah J, Hellman J, et al: Induction of labor as compared with serial antenatal monitoring in post-term pregnancy. N Engl J vIed 326:1587, 1992 Harris BA Jr, Huddleston JF, Sutlif G, et al: he unfavorable cervix in prolonged pregnancy. Obstet Gynecol 62: 1, 1983 Hendricks CH: Patterns of fetal and placental growth: the second half of pregnancy. Obstet Gynecol 24:357, 1964 Hill MJ, McWilliams GC, Garcia-Sur, et al: he efect of membrane sweeping on prelabor rupture of membranes: a randomized controlled trial. Obstet Gynecoltll1(6):1313,t2008 Jazayeri A, Tsibris JC, Spellacy N: Elevated umbilical cord plasma erythropoietin levels in prolonged pregnancies. Obstet Gynecol 92:6t1, 1998 Joseph KS, Huang L, Liu S, et l: Reconciling the high rates of preterm and postterm birth in the United States. Obstet Gynecol 109(4):798,t200

1	Kashanian M, Aktarian A, Baradaron H, et al: Efect of membrane sweeping at term pregnancy on duration of pregnancy and labor induction: a randomized trial. Gynecol Obstet Invest 62:41,t2006 Larsen LG, Clausen HV, Andersen B, et al: A stereologic study of postmature placentas fixed by dual perfusion. Am J Obstet Gynecol 2:500, 1995 Laursen M, Bille C, Olesen A W, et al: Genetic inluence on prolonged gestation: a population-based Danish twin study. Am J Obstet Gynecol 190:489, 2004 Leveno KJ, Quirk JG, Cunningham FG, et al: Prolonged pregnancy, 1. Observations concerning the causes of fetal distress. Am J Obstet Gynecol 150:465, 1984 Lindell A: Prolonged pregnancy. Acta Obstet Gynecol Scand 35: 136, 1956 Link G, Clark KE, Lang U: Umbilical blood low during pregnancy: evidence for decreasing placental perfusion. Am J Obstet Gynecol 196(5)489.e1, 2007 MacDonald PC, Siiteri PK: Origin of estrogen in women pregnant with an anencephalic fetus. J Clin Invest 44:465, 1965

1	MacDonald PC, Siiteri PK: Origin of estrogen in women pregnant with an anencephalic fetus. J Clin Invest 44:465, 1965 MacDorman MF, Kirmeyer S: Fetal and perinatal mortality, United States, 2005. Natl Vital Stat Rep 57(8):1, 2009 Martin JA, Hamilton BE, Osterman MJK, et al: Births: inal data for 2013. Natl Vital Stat Rep 64:t1,t2015 Martin JA, Hamilton BE, Sutton PO, et al: Births: inal data for 2015. Natl Vital Stat Rep 66(1):1,t2017 Maternal-Fetal Medicine Units Network: A clinical trial of induction of labor versus expectant management in postterm pregnancy. Am J Obstet Gynecol 170:716,t1994 Mission JF, Marshall NE, Caughey AB: Pregnancy risks associated with obesity. Obstet Gynecol Clin North Am 42:335,t2015 Moster 0, Wilcox AJ, Vollset SE, et al: Cerebral palsy among term and postterm births. JAMA 304(9):976,t2010 Mozurkewich E, Chilimigras J, Koepke E, et al: Indications for induction of labour: a best-evidence review. BJOG 116(5):626, 2009

1	Mozurkewich E, Chilimigras J, Koepke E, et al: Indications for induction of labour: a best-evidence review. BJOG 116(5):626, 2009 Nahum GG, Stanislaw H, Hufaker B]: Fetal weight gain at term: linear with minimal dependence on maternal obesity. Amt] Obstet Gynecol 172: 1387, 1995 Oberg AS, Frisell T, Svensson AC, et al: Matenal and fetal genetic contributions to postterm birth: familial clustering in a population-based sample of 475,429 Swedish births. Amt] Epidemiol 177(6):531,t2013 Olesen A W, Westergaard ]G, Olsen ]: Perinatal and maternal complications related to postterm delivery: a national register-based study, 1978-1993. Amt] Obstet Gynecol 189:227,t2003 Olesen AW, Westergaard ]G, Olsent]: Prenatal risk indicators of a prolonged pregnancy. The Danish Birth Cohort 1998-2001. Acta Obstet Gynecol Scand 85: 1338, 2006 Oz AU, Holub B, Mendilcioglu 1, et aI: Renal artery Doppler investigation of the etiology of oligohydramnios in postterm pregnancy. Obstet Gynecol 100:715,t2002

1	Oz AU, Holub B, Mendilcioglu 1, et aI: Renal artery Doppler investigation of the etiology of oligohydramnios in postterm pregnancy. Obstet Gynecol 100:715,t2002 Redman CW, Staf AC: Preeclamptic biomarkers, syncytiotrophoblast stress, and placental capacity. Amt] Obstet Gynecol 213 (4 Suppl):S9.e 1, 2015 Rushton DI: Pathology of placenta. In Wigglesworth ]5, Singer DB (eds): Textbook of Fetal and Perinatal Pathology. Boston, Blackwell, 1991, P 171 Schafer L, Burkhardt T, Zimmerman R, et al: Nuchal cords in term and postterm deliveries-do we need to know? Obstet Gynecol 106:23,t2005 Shin KS, Brubaker L, Ackerson LM: Risk of cesarean delivery in nulliparous women at greater than 41 weeks' gestational age with an unengaged vertex. Am] Obstet Gynecol 190:129,t2004 Smith GC: Life-table analysis of the risk of perinatal death at term and post term in singleton pregnancies. Amt] Obstet Gynecol 184:489,t2001

1	Smith GC: Life-table analysis of the risk of perinatal death at term and post term in singleton pregnancies. Amt] Obstet Gynecol 184:489,t2001 Smith Sc, Baker PN: Placental apoptosis is increased in postterm pregnancies. B]OG 106:861, 1999 Torricelli M, Novembri R, Conti N, et al: Correlation with kisspeptin in postterm pregnancy and apoptosis. Reprod Sci 19(10): 1133, 2012 Trimmer K], Leveno K], Peters MT, et al: Observation on the cause of oligohydramnios in prolonged pregnancy. Am] Obstet Gynecol 163: 1900, 1990 Van del' Ven Aj, van Os MA, Kleinrouweler CE, et al: Midpregnancy cervical length in nulliparous women and its association with postterm delivery and intrapartum cesarean delivery. Am ] Perinatol 33 (1) :40, 2016 Vankayalapati P, Sethna F, Roberts N, et al: Ultrasound assessment of cervical length in prolonged pregnancy: prediction of spontaneous onset of labor and successful vaginal delivery. Ultrasound Obstet Gynecol 31 (3):328, 2008

1	Walsh ]M, Kandamany N, Shuibhne NN, et al: Neonatal brachial plexus injury: comparison of incidence and antecedents between 2 decades. Amt] Obstet Gynecol 204:324, 201t1. Wenstrom KD, Parsons MT: The prevention of meconium aspiration in labor using amnioinfusion. Obstet Gynecol 73:647, 1989 Wong SF, Hui SK, Choi H, et al: Does sweeping of membranes beyond 40 weeks reduce the need for formal induction of labour? Br] Obstet Gynaecol 109:632, 2002 Wyckof MH, Aziz K, Escobedo MB, et al: Part 13: neonatal resuscitation. 2015 American Heart Association guidelines for cardiopulmonaty resuscitation and emergency cardiovascular care. Circulation 132:5543, 20t15 Yang 5, Platt RW, Kramer MS: Variation in child cognitive ability by week of gestation among healthy term births. Am] Epidemiol 171 :399, 2010 Yang SH, Roh CR, Kim ]H: Transvaginal ultrasonography for cervical assessment before induction of labor. Obstet Gynecol Surv 59:577, 2004

1	Yang SH, Roh CR, Kim ]H: Transvaginal ultrasonography for cervical assessment before induction of labor. Obstet Gynecol Surv 59:577, 2004 Zizzo AR, Kirkegaard I, Pin borg A, et al: Decline in stillbirths and perinatal mortality after implementation of a more aggressive induction policy in post-date pregnancies: a nationwide register study. Acta Obstet Gynecol Scand 96(7):862, 2017 FETAL GROWTH. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 844 FETAL-GROWTH RESTRICTION.................... 847 RISK FACTORS AND ETIOLOGIES.................. 849 FETAL-GROWTH RESTRICTION RECOGNITION....... 852 MANAGEMENT................................. 854 FETAL OVERGROWTH......................... 856 RISK FACTORS ................................. 857 MANAGEMENT. ............................... 858

1	MANAGEMENT................................. 854 FETAL OVERGROWTH......................... 856 RISK FACTORS ................................. 857 MANAGEMENT. ............................... 858 When inants are of large size and abundant, they may mechanicaly throw out of function so great a portion of the placenta as seriousy to inteere with the nutrition of the oetus, and sometimes cause its death. Excessive development of the oetus can usualy be traced to prolongation of pregnancy, large size of one or both parents, advancing age, or multipariy of the mother. -J. Whitridge Williams (1903) he concept of excessive or impaired fetal growth was not considered in detail by Williams in his irst edition. Abnormally diminished fetal growth was attributed to placental lesions and fetal infections. Conversely, a large fetus was of obvious concern because of associated dystocia. Currently, fetal-growth disorders at both ends of the spectrum are major problems in obstetrics.

1	Nearly 20 percent of the almost 4 million neonates born in the United States are at the low and high extremes of fetal growth.

1	In 2015, 8.1 percent of newborns weighed <2500 g at birth, whereas 8.0 percent weighed >4000 g. And, although almost 70 percent of low-birthweight neonates are born preterm, the balance of low-birthweight newborns accounted for approximately 3 percent of term births in 2015 (Martin, 2017). Between 1990 and 2006, the proportion of newborns with birthweights <2500 g grew by more than 20 percent when the rate peaked at 8.3 percent (Martin, 2012). his trend toward smaller babies has slowed since the mid-to late-2000s and might partly be explained by the concurrent movement toward fewer deliveries prior to 39 weeks' gestation (Richards, 2016). In contrast, between 1990 and 2006, the incidence of birth weights >4000 g declined approximately 30 percent to a nadir of 7.6 percent in 2010 (Martin, 2012). This trend away from the upper extreme is diicult to explain because it coincides with the epidemic prevalence of obesity, a known cause of macrosomia (Morisaki, 2013).

1	Human fetal growth is characterized by sequential patterns of tissue and organ growth, diferentiation, and maturation. However, the "obstetrical dilemma" postulates a conlict between the need to walk upright-requiring a narrow pelvis-and the need to think-requiring a large brain, and thus a large head. Some speculate that evolutionary pressures restrict growth late in pregnancy (Mitteroecker, 2016). Thus, the ability to growth restrict may be adaptive rather than pathological. Fetal growth has been divided into three phases. The initial phase of hyperplasia occurs in the irst 16 weeks and is characterized by a rapid increase in cell number. The second phase, which extends up to 32 weeks' gestation, includes both cellular hyperplasia and hypertrophy. After 32 weeks, fetal mass accrues by cellular hypertrophy, and it is during this phase that most fetal fat and glycogen are accumulated. The corresponding c 5..,

1	FIGURE 44-1 Increments in fetal weight gain in grams per day from 24 to 42 weeks' gestation. The black line represents the mean and the outer blue lines depict ±2 standard deviations. (Data from pregnancies managed at Parkland Hospital.) levels below the threshold for diabetes. Overgrowth does occur in the fetuses of euglycemic women. Its etiology is thus likely more complicated than merely dysregulated glucose metabolism (Catalano, 2011). Genetic factors, including genomic imprinting and epigenetic modifications via gene methylation, are also important and emphasize the potential role of inheritance (Begemann, 2015; Nawathe, 2016).

1	Excessive transfer of lipids may also lead to fetal overgrowth (Higa, 2013). Free or nonesteriied fatty acids in maternal plasma may be transferred to the fetus via facilitated diusion or ater liberation of fatty acids from triglycerides by trophoblastic lipases (Gil-SGnchez, 2012). Generally speaking, lipolytic activity is augmented in pregnancy, and fatty acid levels are increased in nonobese women during the third trimester (Diderholm, 2005). fetal-growth rates during these three phases are 5 g/d at 15 weeks' gestation, 15 to 20 g/d at 24 weeks', and 30 to 35 g/d at 34 weeks' (Williams, 1982). As shown in Figre 44-1, the velocity of fetal growth varies considerably.

1	Fetal development is determined by maternal provision of substrate and placental transfer of these, whereas fetal growth potentil is governed by the genome. The precise cellular and molecular mechanisms by which normal fetal growth ensues are incompletely understood. That said, considerable evidence support an important role for insulin and insulin-like growth factors (IGFs) in regulation offetal growth and weight gain (Luo, 2012). hese growth factors are produced by virtually all fetal organs and are potent stimulators of cell division and diferentiation.

1	Other hormones implicated in fetal growth have been identified, particularly hormones derived from adipose tissue. These hormones are known broadly as adipokines and include leptin, the protein product of the obesiy gene. Fetal leptin concentrations rise during gestation, and they correlate both with birthweight and with neonatal fat mass (Brifa, 2015; Logan, 2017; Simpson, 2017). Other adipokines possibly involved include adiponectin, ghrelin, follistatin, resistin, visfatin, vaspin, omentin-I, apelin, and chemerin.

1	Fetal growth is also dependent on an adequate supply of nutrients. As discussed in Chapter 7 (p. 138), both excessive and diminished maternal glucose availability afect fetal growth. Reduced maternal glucose levels may result in a lower birthweight. Still, growth-restricted neonates do not typically show pathologically low glucose concentrations in cord blood (Pardi, 2006). Fetal-growth restriction in response to glucose deprivation generally results only after long-term severe maternal caloric deprivation (Lechtig, 1975). Conversely, excessive glycemia produces macrosomia. Varying levels of glucose afect fetal growth via insulin and its associated IGFs. he Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) Study Cooperative Research Group (2008) found that elevated cord C-peptide levels, which relect fetal hyperinsulinemia, are associated with greater birthweight. his relationship was noted even in women with maternal glucose

1	Independent of prepregnancy body mass index (BMI), higher free fatty acid levels during the latter half of pregnancy correlate with birthweight (Crume, 2015). Other studies have correlated mater nal triglyceride levels with birthweight (Di Cianni, 2005; Vrijkotte, 2011). Greater intake of certain fatty acids, particularly omega-3, is also associated with greater birthweight (Calabuig-Navarro, 2016).

1	Placental fatty acid metabolism and transfer may be dysregulated in fetal-growth restriction and in maternal conditions associated with fetal overgrowth. For example, levels of endothelial lipase are reduced with deicient fetal growth, and this enzyme is overexpressed in placentas of women with diabetes (Gauster, 2007, 2011). Others have reported that diabetes and obesity are associated with altered placental lipid-transport gene expression (Radaelli, 2009). Obesity is also linked with greater expression of fatty acid binding/transport proteins within the trophoblast (Myatt, 2016; Scifres, 2011). he end result of these alterations is an abnormal accumulation of lipids that can result in pathological placental inlammation and dysfunction (Calabuig-Navarro, 2016; Myatt, 2016; Yang, 2016).

1	mino acids undergo active transport, which explains the normally higher fetal concentrations compared with maternal levels. In growth restriction, this pattern is reversed. One possible mechanism is altered transport of these amino acids. Remember, amino acids that reach the fetus must first cross the microvillus membrane at the maternal interface. Amino acids then traverse the trophoblastic cell, and inally cross the basal membrane into fetal blood (Chap. 5, p. 90). In human placentas, fetal growth correlates with peroxisome proliferator activator receptor gamma (PPAR-,) activity, which governs placental regulation of L-type amino acid (A T) receptors 1 and 2 (Chen, 2015b). Additional modulation is from rap amycin complex (mTORC) 1 and 2 receptors (Rosario, 2013). Placental mTORC activity is reduced in fetal-growth restriction. Others have shown that increasing birthweight and maternal BMI are linked to expression and activity of particular aminoacid transporters at the microvillus

1	is reduced in fetal-growth restriction. Others have shown that increasing birthweight and maternal BMI are linked to expression and activity of particular aminoacid transporters at the microvillus membrane (Jansson, 2013).

1	Normative data for fetal growth based on birthweight vary with ethnicity and geographic region. Accordingly, researchers have developed fetal-growth curves using various populations and geographic locations throughout the United States (Brenner, 1976; Ott, 1993; Overpeck, 1999; Williams, 1975). Because

1	TABLE 4-1. 2011 Gestational Age Birthweight (g) Percentiles for 3,252,01e1 Singleton Live Births in the United States (Duryea, 2014). hese current curves plot birthweight against a gestational age based on n obsteical estimate, formed in part by sonography. hese curves are thought to be more accurate and reflect more precise pregnancy dating. Older curves used gestational age derived rom a last menstrual period. Comparing birthweights rom 1991 to data from 2011, the more recent growth curves indicate that the earlier assessments overestimated birthweihts in the ase of preterm birth. In particular, the 50th percentile for fetal growth that previ ously corresponded to 31 to 32 weeks' gestation now corresponds to 33 to 34 weeks' when improved obstetrical dating is used.

1	The curves by Alexander (1996) and Duryea (2014) and their associates are most accurately termed a population rerence, rather than a standard. A population reference incorporates pregnancies of varying risks, along with the resulting outcomes, both normal and abnormal. In contrast, a stanard incorporates normal 1414 1573 2053 2579 2861 pregnancies with normal outcomes. Because population refer 34 1632 1793 2296 2846 3093 ences include preterm births, which are more likely to be growth 1871 2030 2549 3119 3345 restricted, it has been argued that the associated birthweight data 36 2117 2270 2797 3380 3594 overestimate deficient fetal growth (Mayer, 2013; Zhang, 2010). 37 2353 2500 3025 361n2 381n8 One recent project sought to define regional standards in health and socioeconomic conditions. Growth trajectories from the International Fetal and Newborn Growth Consortium for these curves are based on speciic ethnic or regional groups, they do not represent the entire population.

1	To address this, birthweights such as those shown in Table 4-1 are derived nationwide in the United States. Shown in Figure 4-2 are growth curves rom more thn 3.2 million mothers with singleton livebon neonates in the United States during 1991 and 2011 5,000 -By last menstrual period 4,000 impaired growth. hus, they cannot be used to identiy the fetus that fails to achieve an expected size but whose birthweight is above the 21st Century-INTERGROWTH 21 were similar in these eight: China, India, Kenya, Brazil, Oman, Italy, the United Kingdom, and the United States (Villar, 2014). However, an international standard based on the healthiest women is of questionable value (Hanson, 2015). Most of what is known regarding normal and abnormal human fetal growth is actually based on birthweights that are assembled as references for fetal growth at particular gestational ages. his is problematic, however, because birth weight does not deine the rate of fetal growth.

1	his is problematic, however, because birth weight does not deine the rate of fetal growth. Indeed, such birthweight curves reveal com- the of � the 10th percentile. For example, a fetus with 3,000 ) a birthweight in the 40th percentile may not have achieved its genomic growth potential for a birthweight in the 80th percentile. : 2,000 m The rate or velociy of fetal growth can be estimated by serial sonographic anthropometry. For example, Milovanovic (2012) 1,000 demonstrated that the growth rate of intrin borns (those below the 10th percentile) 22 24 26 28 30 32 34 36 38 40 42 44 approximates that of appropriate-for-gestaGestational age (weeks) tional-age neonates. However, diminished growth velocity may be linked to perinatal FIGURE 44-2 Fetal-growth curves for births in the United States in 2011. Curves vary depending on whether gestational age was calculated from the last menstrual changes that are independent of birthweight.

1	period or from an improved obstetrical estimate, derived in part using sonography. (Modified with permission from Duryea EL, Hawkins JS, Mcintire DO, et al: A revised Recently, Sovio and colleageus (2015) demonbirth weight reference for the United States. Obstet Gynecol. 2014 Jul;124(1 ):16-22.) strated that growth velocity of the abdominal circumference in the lowest decile distinguishes SGA newborns who sufer increased morbidity. Conversely, an excessive fetalgrowth velocity, particularly ofthe abdominal circumferencewhich may be correlated with increased hepatic blood low-is associated with an overgrown neonate (American College of Obstetricians and Gynecologists, 2016a). Several conditions or disorders can adversely afect the normal growth of a fetus. It is important clinically to distinguish between fetal-growth restriction and constitutional low birth weight. u

1	Fetal-Growth Disorders 100 -Morbidity 175 90 -Mortality) 150U) 80D �S s· C 70 > 0 100g0 60 30 0-E �� 75 50 -30 � u 0S 20 0E 259J) 10 >1010987654321<1 Birthweightpercentile FIGURE 44-3 Relationship between birthweight percentile and perinatal mortality and morbidity rates in 1560 small-for-gestationalage fetuses. A progressive increase in both mortality and morbidity rates is observed as birthweight percentile decreases.. (Data from Manning, 1995.) • Symmetrical versus Asymmetrical Growth Restriction Campbell and Thoms (1977) described the use of the sonographically determined head-to-abdomen circumerence ratio (HCIA) to diferentiate growth-restricted fetuses. Those who were symmetrical were proportionately small, and those who were asymmetrical had disproportionately lagging abdominal growth compared with head growth. The onset or etiology of a particular fetal insult is hypothetically linked to either type of growth restriction. In the instance ofsymmetricalgrowth restriction, an

1	growth compared with head growth. The onset or etiology of a particular fetal insult is hypothetically linked to either type of growth restriction. In the instance ofsymmetricalgrowth restriction, an early insult could result in a relative decrease in cell number and size. For example, early global insults such as those from chemical exposure, viral infection, or cellular maldevelopment with aneuploidy may cause a proportionate reduction of both head and body size. Asymmetricalgrowth restriction might follow a later pregnancy insult such as placental insuiciency from hypertension. In this variation, resultant diminished glucose transfer and hepatic storage would primarily afect cell size and not number. Thereby, fetal abdominal circumferencewhich relects liver size-would be reduced. Brain Sparing Such somatic growth restriction is proposed to result from preferential shunting of oxygen and nutrients to the brain. This allows normal brain and head growth, that is-brain sparing.

1	Brain Sparing Such somatic growth restriction is proposed to result from preferential shunting of oxygen and nutrients to the brain. This allows normal brain and head growth, that is-brain sparing. Accordingly, the ratio of brain weight to liver weight during the last 12 weeks-usually about 3 to I-may be increased to 5 to 1 or more in severely growth-restricted infants. Because of brainsparing efects, asymmetrical fetuses were thought to be preferentially protected from the full efects ofgrowth restriction. Considerable evidence has since accrued that fetal growth patterns are much more complex. For example, fetuses with 847 .�..,40

1	Lubchenco and coworkers (1963) published detailed comparisons of gestational ages with birthweights to derive norms for expected fetal size at a given gestational week. Battaglia and Lubchenco (1967) then classiied smalor-gestational-age neonates as those whose weights were below the 10th percentile for their gestational age. Low-birthweight newborns who are small for gestational age are often designated as having etalgrowth restriction. Such infants were shown to be at increased risk for neonatal death. For example, the mortality rate of SGA neonates born at 38 weeks was 1 percent compared with 0.2 percent in those with appropriate birthweights.

1	Importantly, manyneonates with birthweights < 10th percentile are not pathologically growth restricted, but instead are small simply because of normal biological factors. s many as 70 percent ofsuch SGA infants have normal outcomes and are thought to be appropriately grown when maternal ethnic group, parity, weight, and height are considered (Unterscheider, 2015). These small but normal infants also do not show evidence ofthe postnatal metabolic derangements commonly associated with deficient fetal growth. Moreover, intrinsically SGA newborns remain significantly smaller during surveillance to 2 years compared with appropriate-for-gestational age neonates, but they do not show diferences in measures ofmetabolic risk (Milovanovic, 2012).

1	Because of these disparities, other classifications have been developed. Usher and McLean (1969) suggested that fetal growth standards should be based on mean weights-for-age, with normal limits deined by ±2 standard deviations. This definition would limit SGA infants to 3 percent of births instead of 10 percent. In a population-based analysis of 122,754 births at Parkland Hospital, McIntire and colleagues (1999) showed this definition to be clinically meaningful. Also, as shown in Figure 44-3, most adverse outcomes are in newborns smaller than the 3rd percentile. The importance ofthis cut-ofhas been independently confirmed in a prospective study by Unterscheider and colleagues (2013a).

1	More recently, individual or customized fetal-growth potential is proposed to replace a population-based threshold. In this model, a fetus that deviates from its individual optimal size at a given gestational age is considered either overgrown or growth restricted (Chiossi, 2017). Such optimal projections are based on maternal race or ethnicity. But, the superiority of customized growth curves has not been established (Chiossi, 2017; Costantine, 2013; Grobman, 2013; Zhang, 2011). aneuploidy typically have disproportionately large head sizes and thus are asymmetricaLy growth restricted, which is contrary to contemporaneous thinking (Nicolaides, 1991). Moreover, most preterm neonates with growth restriction due to preeclampsia and associated utero placental insuiciency are found to have more symmetrical growth impairment-again, a departure from accepted principles (Salaia, 1995).

1	More evidence of the complexity of growth patterns was presented by Dashe and associates (2000). These investigators analyzed 8722 consecutive liveborn singletons who had undergone sonographic examination within 4 weeks of delivery. Although only 20 percent of growth-restricted fetuses demonstrated sonographic head-to-abdomen asymmetry, these fetuses were at greater risk for intrapartum and neonatal complications. Symmetrically growth-restricted fetuses were not at increased risk for adverse outcomes compared with those appropriately grown. These investigators concluded that asymmetrical fetalgrowth restriction represented signiicantly disordered growth, whereas symmetrical growth restriction more likely represented normal, genetically determined small stature. Other data further challenge the concept of brain sparing.

1	Roza and associates (2008) found that fetuses with circulatory redistribution-brain sparing-had a higher incidence of later behavioral problems. In another study, evidence of brain sparing was found in half of 62 growth-restricted fetuses with birthweights < 10th percentile and who showed abnormal middle cerebral artery Doppler low studies (Figueras, 2011). Compared with controls, these neonates had significantly lower neurobehavioral scores in multiple areas, suggesting profound brain injury. Zhu and coworkers (2016) prospectively compared late-onset growth restriction in 14 fetuses with that in 26 non-growth-restricted fetuses using magnetic resonance imaging to analyze hemodynamic flow. Despite the concept of brain sparing, growth-restricted infants had significantly smaller brains than controls. he complex efects of such insults-with respect to timing and severity-on brain structure, connectivity, and neurobehavioral outcomes have been recently reviewed by Miller and colleagues

1	controls. he complex efects of such insults-with respect to timing and severity-on brain structure, connectivity, and neurobehavioral outcomes have been recently reviewed by Miller and colleagues (2016).

1	Fetal-growth restriction is one of the "major obstetrical syndromes" associated with defects in early placentation (Brosens, 2015). Rogers and coworkers (1999) concluded that implantation-site disorders may be both a cause and consequence of hypoperfusion at the placental site. This comports with the association of certain placental angiogenic factors with pregnancy hypertensive disorders (Chap. 40, p. 716). Thus, it may be that placentas from pregnancies complicated by hypertension elaborate these angiogenic factors in response to placental-site hypoperfusion, whereas pregnancies complicated by fetal-growth restriction without hypertension do not Qeyabalan, 2008).

1	Mechanisms leading to abnormal trophoblastic invasion are likely multifactorial, and both vascular and immunological etiologies have been proposed. For example, atriaL natriuretic peptide converting enzyme, also known as corin, plays a critical role in trophoblastic invasion and remodeling of the uterine spiral arteries (Cui, 2012). hese processes are impaired in corin-deficient mice, which also develop evidence of preeclampsia. Moreover, mutations in the gene for corin have been reported in women with preeclampsia (Chen, 2015a).

1	Several immunological abnormalities are associated with fetalgrowth restriction. This raises the prospect of maternal rejection of ther" paternal semiallograft." Rudzinski and colleagues (2013) studied C4d, a component of complement that is associated with humoral rejection of transplanted tissues. They found this to be highly associated with chronic villitis-88 percent of cases versus only 5 percent of controls-and with reduced placental weight. In a study of 10,204 placentas, chronic villitis was associated with placental hypoperfusion, fetal acidemia, and fetal-growth restriction and its sequelae (Greer, 2012). im and coworkers (2015) extensively reviewed chronic inflammatory placental lesions and their association with fetal-growth restriction, preeclampsia, and preterm birth.

1	Several short-term and long-term adverse sequelae are linked with fetal-growth restriction. First, perinatal morbidity and mortality rates are substantive (see Fig. 44-3). Rates of stillbirth and adverse neonatal outcomes that include birth asphyxia, meconium aspiration, hypoglycemia, and hypothermia are all increased, as is the prevalence of abnormal neurological development. This is true for both term and preterm growth-restricted newborns. In one analysis of nearly 3000 newborns born before 27 weeks' gestation, those weighing < 10th percentile had a nearly fourfold higher risk of neonatal death or neurodevelopmental impairment and a 2.6-fold increased risk of cerebral palsy compared with non-SGA neonates (De Jesus, 2013). In another analysis of more than 91,000 uncomplicated pregnancies, newborns with weights < 5th percentile had a higher risk of low 5-minute Apgar score, respiratory distress, necrotizing enterocolitis, and neonatal sepsis than appropriate-weight neonates. The

1	newborns with weights < 5th percentile had a higher risk of low 5-minute Apgar score, respiratory distress, necrotizing enterocolitis, and neonatal sepsis than appropriate-weight neonates. The risks of stillbirth and neonatal death were sixfold and fourfold higher, respectively .\1endez-Figueroa, 2016).

1	The most severely growth-impaired newborns also have the worst outcomes. In one study of more that 44,561 neonates, only 14 percent of those weighing < 1st percentile at birth survived to discharge (Griin, 2015). For those infants who survive, the risks of adverse neurodevelopmental outcomes are substantial, especially for growth-impaired fetuses with either brain sparing or a major birth defect (Meher, 2015; Nelson, 2015b). Poor motor, cognitive, language and attention, and behavioral outcomes in growth-restricted newborns unfortunately persist into early childhood and adolescence (Baschat, 2014; Levine, 2015; Rogne, 2015).

1	Barker (1992) hypothesized that aduLt mortality and morbidity are related to fetal and infant health. This includes both under-and overgrowth. In the context of fetal-growth restriction, numerous reports describe a relationship between suboptimal fetal nutrition and an increased risk of subsequent adult hypertension, atherosclerosis, type 2 diabetes, and metabolic derangement (Burton, 2016; J ornayvaz, 2016). he degree to which low birthweight mediates adult disease is controversial, as weight gain in early life also appears important (Breij, 2014; Kerhof, 2012; McCloskey, 2016).

1	Kerhof, 2012; McCloskey, 2016). Mounting evidence suggests that fetal-growth restriction may afect organ development, particularly that of the heart. Individuals with low birthweight demonstrate cardiac structural changes and dysfunction persisting through childhood, adolescence, and adulthood. In one study, 80 infants who were born SGA before 34 weeks' gestation were compared at 6 months with 80 normally grown children (Cruz-Lemini, 2016). The heart in the SGA children had a more globular ventricle that resulted in systolic and diastolic dysfunction. In another study, echocardiography in 418 adolescents showed that low birthweight was associated with a thicker let ventricular posterior wall (Hietalampi, 2012). In their review, Cohen and colleagues (2016) concluded, however, that these findings have unclear long-term signiicance.

1	Deficient fetal growth is also associated with postnatal structural and functional renal changes. In a review by Luyckx and Brenner (2015), birthweight abnormalities were evaluated for linkage with disordered nephrogenesis, renal dysfunction, chronic kidney disease, and hypertension. Both low and high birthweight, as well as maternal obesity and gestational diabetes, afect in-utero development of the kidney and its health into adulthood. However, other variables that include childhood nutrition, acute kidney injury, excessive childhood weight gain, and obesity also worsen long-term renal function.

1	Particularly in women with diabetes and elevated cord blood levels of IGF-1, fetal overgrowth is associated with greater neonatal fat mass and morphological heart changes. Pedersen (1954) irst proposed that hyperglycemia leads to fetal hyperinsulinemia and fetal overgrowth. This has been extended to organ dysmorphia, for example, increased interventricular septal thickness in neonates of mothers with gestational diabetes (nan, 2011; Garcia-Flores, 2011). he cardiopulmonary vasculature is also adversely afected by diabetes in pregnancy. In 3277 cases of persistent pulmonary hypertension of the newborn (PPHN), maternal obesity, diabetes, and both deficient and excessive fetal growth were independent risk factors (Steurer, 2017). Longterm consequences of fetal overgrowth from obesity and diabetes are discussed in Chapters 48 (p. 941) and 57 (p. 1097).

1	Numerous reports have described accelerated fetal pulmonary maturation in complicated pregnancies associated with growth restriction (Perelman, 1985). One possible explanation is that the fetus responds to a stressed environment by augmenting adrenal glucocorticoid secretion, which leads to accelerated fetal lung maturation (Laatikainen, 1988). Although this concept pervades modern perinatal thining, evidence to support it is negligible.

1	To examine this hypothesis, Owen and associates (1990) analyzed perinatal outcomes in 178 women delivered because of hypertension. hey compared these with outcomes in newborns of 159 women delivered because of spontaneous preterm labor or ruptured membranes. They concluded that a "stressed" pregnancy did not confer an appreciable survival advantage. Similar findings were described by Friedman and colleagues (1995) in women with severe preeclampsia. Two studies from Parkland Hospital also substantiate that the preterm infant accrues no apparent advantages from fetal-growth restriction (McIntire, 1999; Tyson, 1995).

1	Risk factors for impaired fetal growth include potential abnormalities in the mother, fetus, and placenta. hese three "compartments" are depicted in Figure 44-4. Some of these factors are known causes of fetal-growth restriction and may afect more than one compartment. For instance, cytomegalovirus infections can afect the fetus directly. In contrast, bacterial infections such as tuberculosis may have signiicant maternal efects that lead to poor fetal growth. Similarly, malaria, a protozoal infection, is a recognized cause of fetal-growth restriction (Briand, 2016). Importantly, many causes of diminished fetal growth are prospectively considered risk factors, because impaired fetal growth is not consistent in all afected women.

1	It is axiomatic that small women typically have smaller newborns. As discussed subsequently, both prepregnancy weight and gestational weight gain modulate this risk. Durie and colleagues (2011) showed that the risk of delivering an SGA neonate was highest among underweight women who gained less weight than recommended by the Institute of Medicine (Chap. 9, p. 166). Also, both maternal and paternal size inluences birthweight. In a Swedish study of 137,538 term births, it was estimated that the maternal and paternal birthweights explained 6 and 3 percent of variance in birthweight, respectively (Mattsson, 2013). In the study by Durie (2011) cited above, gestational weight gain during the second and third trimesters that was less than FIGURE 44-4 Risk factors and causes of impaired fetal growth centering on the mother, her fetus, and the placenta.

1	FIGURE 44-4 Risk factors and causes of impaired fetal growth centering on the mother, her fetus, and the placenta. that recommended by the Institute of Medicine was associated with SGA neonates in women of all weight categories except class II or III obesity. Conversely, excessive gestational weight gain was associated with an overgrown newborn in all weight categories (Blackwell, 2016).

1	As perhaps expected, eating disorders are linked with significantly higher risks for low birthweight and preterm birth (Micali, 2016). Marked weight gain restriction ater midpregnancy should not be encouraged even in obese women (Chap. 48, p. 941). Even so, it appears that food restriction to < 1500 kcal/d adversely afects fetal growth minimally (Lechtig, 1975). The best documented efect of famine on fetal growth was in the Hunger Winter of 1944 in Holland. For 6 months, the German occupation army restricted dietary intake to 500 kcal/ d for civilians, including pregnant women. This resulted in an average birthweight decline of only 250 g (Stein, 1975).

1	It is unclear whether undernourished women may beneit from micronutrient supplementation. In one study, almost 32,000 Indonesian women were randomly assigned to receive micronutrient supplementation or only iron and folate tablets (Prado, 2012). Ofspring of those receiving the supplement had lower risks of early infant mortality and low birthweight and had improved childhood motor and cognitive abilities. Conversely, Liu and coworkers (2013) randomly assigned 18,775 nulliparas to folic acid alone; folic acid and iron; or folic acid, iron, and 13 other micronutrients. Folic acid and iron, with or without the additional micro nutrients, resulted in a 30-percent reduction in risk of third-trimester anemia. But, supplementation did not afect other maternal or neonatal outcomes. A Cochrane database review of 19 trials involving 138,538 women concluded that supplementation of iron and folic acid improved birth outcomes, including lower risks of low birthweight and SGA (Haider, 2017). he

1	database review of 19 trials involving 138,538 women concluded that supplementation of iron and folic acid improved birth outcomes, including lower risks of low birthweight and SGA (Haider, 2017). he importance of antenatal vitamins and trace metals is further discussed in Chapter 9 (p. 168).

1	Exercise in pregnancy may be beneficial for optimal fetal growth. One metaanalysis of28 studies involving 5322 women concluded that exercise reduces the risk of fetal overgrowth without raising the risk of poor growth (Wiebe, 2015). Another metaanalysis concluded that aerobic exercise did not result in low-birthweight neonates (Di Mascio, 2016). The efect of social deprivation on birthweight is interconnected with lifestyle factors such as smoking, alcohol or other substance abuse, and poor nutrition. With appropriate modiying interventions, women with psychosocial factors were signiicantly less likely to deliver a low-birthweight infant and also had fewer preterm births and other pregnancy complications (Coker, 2012).

1	Women who are immigrants may be at particular risk for poor fetal growth. In one study of 56,443 singleton pregnancies in Rotterdam, social deprivation was associated with adverse perinatal outcomes that included SGA newborns (Poeran, 2013). That said, a similar linkage was not found in socially deprived women of non-Western origin. The efect of immigration, however, is complex and dependent on the population studied (Howell, 2017; Sanchez-Vaznaugh, 2016).

1	Especially when complicated by superimposed preeclampsia, chronic vascular disease commonly causes growth restriction (Chap. 50, p. 980). In a study of more than 2000 women, vascular disease as evidenced by abnormal uterine artery Doppler velocimetry early in pregnancy was associated with higher rates of preeclampsia, SGA neonates, and delivery before 34 weeks (Groom, 2009). Using Washington state birth certiicate data, Leary and colleagues (2012) found that maternal ischemic heart disease was linked to SGA infants in 25 percent of 186 births. Roos-Hesselink and coworkers (2013) described similar pregnancy outcomes in 25 women with ischemic heart disease. Chronic renal insuiciency is frequently associated with underlying hypertension and vascular disease. Nephropathies are commonly accompanied by restricted fetal growth (Cunningham, 1990; Feng, 2015; Saliem, 2016). hese relationships are considered further in Chapter 53 (p. 1034).

1	Fetal-growth restriction in the newborns of women with diabetes may be related to congenital malformations or may follow substrate deprivation from advanced maternal vascular disease (Chap. 57, p. 1100). Also, the likelihood of restricted growth increases with worsening White classification, particularly nephropathy (Klemetti, 2016). hat said, the prevalence of serious vascular disease associated with diabetes in pregnancy is low, and the primary efect of overt diabetes, especially type 1, is fetal overrowth. For example, in a prospective study of 682 consecutive pregnancies complicated by diabetes, women with type 1 diabetes were significantly more likely than women with type 2 diabetes to have a neonate weighing above the 90th and 97.7th percentiles (Murphy, 2011). Additionally, women with type 1 diabetes were significantly less likely to deliver an SGA newborn. In a recent study of 375 term singleton pregnancies complicated by type 1 diabetes, the risk of fetal overgrowth

1	women with type 1 diabetes were significantly less likely to deliver an SGA newborn. In a recent study of 375 term singleton pregnancies complicated by type 1 diabetes, the risk of fetal overgrowth correlated with rising third-trimester glycemic values (C yganek, 2017) . Nearly a fourth of neonates were macrosomic. And, third-trimester hemoglobin Al c and fasting glucose values were independent predictors for the risk of macrosomia.

1	Conditions associated with chronic uteroplacental hypoxia include preeclampsia, chronic hypertension, asthma, maternal cyanotic heart disease, smoking, and high altitude. When exposed to a chronically hypoxic environment, some fetuses have signiicantly reduced birthweight. In more than 1.8 million births in Austria, the birthweight declined 150 g for each 1000meter rise in altitude (Waldhoer, 2015). In 63,620 Peruvian live births, the mean birthweight was significantly decreased at higher compared with lower altitudes-3065 g ± 475 g versus 3280 g ± 525 g (Gonzales, 2009). In this study, the rate of birth weights <2500 g was 6.2 percent at low altitudes, and it was 9.2 percent at high altitudes. In contrast, the rate of birthweights >4000 g was 6.3 percent at low altitudes and 1.6 percent at high altitudes.

1	In most cases, maternal anemia does not restrict fetal growth. Exceptions include sickle-cell disease and some other inherited anemias (Desai, 2017; Thame, 2016). Importantly, curtailed maternal blood-volume expansion is linked to fetal-growth restriction (de Haas, 2017; Stott, 2017). This is further discussed in Chapter 40 (p. 718).

1	Adverse obstetrical outcomes including fetal-growth restriction have been associated with three species of antiphospholipid antibodies: anticardiolipin antibodies, lupus anticoagulant, and anti-32 gycoprotein-I antibodies. Mechanistically, a "wohit" hypothesis suggests that initial endothelial damage is then followed by intervillous placental thrombosis. More specifically, oxidative damage to certain membrane proteins such as 32 glycoprotein-I is followed by antiphospholipid antibody binding, which leads to immune complex formation and ultimately to thrombosis (Giannakopoulos, 2013). his syndrome is considered in detail in Chapters 52 (p. 1008) and 59 (p. 1143). Pregnancy outcomes in women with these antibodies may be poor and include fetal-growth restriction and fetal demise (Cervera, 2015). he primary autoantibody that predicts obstetrical antiphospholipid syndrome appears to be lupus anticoagulant (Yelnik,r2016).

1	It is controversial whether pregnancies in women with prior infertility with or without treatment have an increased risk of SGA newborns (Zhu, 2007). Dickey and colleagues (2016) compared birthweight curves for singletons conceived by in vitro fertilization to the birthweight curves of Duryea (2014), described on page 846. hey found no reduction in fetal growth. Kondapalli and Perales-Puchalt (2013) reviewed possible links between low birthweight and infertility with its various interventions and concluded that any association remains unexplained for singletons. Placental, Cord, and Uterine Abnormalities

1	Placental, Cord, and Uterine Abnormalities Several placental abnormalities may cause poor fetal growth. These are discussed further throughout Chapter 6 and include chronic placental abruption, extensive chorioangioma, velamentous cord insertion, placenta previa, and umbilical artery thrombosis. Growth failure in these cases is presumed secondary to uteroplacental insuiciency. Abnormal placental implantation leading to endothelial dysfunction may also limit fetal growth (Brosens, 2015). This pathology is implicated in pregnancies complicated by preeclampsia (Chap. 40, p. 714). If the placenta is implanted outside the uterus, the fetus is usually growth restricted (Chap. 19, p. 383). Finally, some uterine malformations have been linked to impaired fetal growth (Chap. 3, p. 44).

1	Pregnancy with two or more fetuses is more likely to be complicated by diminished growth of one or more fetuses compared with that ofnormal singletons. This is illustrated in Figure 44-5 and discussed in Chapter 45 (p. 872). Drugs with Teratogenic and Fetal Effects Several drugs and chemicals are capable oflimiting fetal growth. Some are teratogenic and afect the fetus before organogenesis is ::,1500 FIGURE 44-5 Birthweight and gestational age relationships in multifetal gestations without malformations delivered at Parkland Hospital.

1	FIGURE 44-5 Birthweight and gestational age relationships in multifetal gestations without malformations delivered at Parkland Hospital. complete. Some exert-or continue to exert-fetal efects after embryogenesis ends at 8 weeks. Many ofthese are considered in detail in Chapter 12, and examples include anticonvulsants and antineoplastic agents. Cigarette smoking, opiates and related drugs, alcohol, and cocaine may also cause growth restriction, either primarily or by decreasing maternal food intake. he link with cafeine use and fetal-growth restriction remains speculative (American College of Obstetricians and Gynecologists, 2016b). In contrast, Cyganek and colleagues (2014) studied growth restriction in pregnancies complicated by renal and liver transplants and concluded that common immunosuppressive drugs-prednisone, azathioprine, cyclosporine A, and tacrolimus-did not significantly afect fetal-growth rates.

1	Viral, bacterial, protozoan, and spirochetal infections have been implicated in up to 5 percent of fetal-growth restriction cases and are discussed throughout Chapters 64 and 65. The best known of these are rubela and cytomegalovirus inection. Both promote calcifications in the fetus that are associated with cell death, and infection earlier in pregnancy correlates with worse outcomes. Toda and colleagues (2015) described a Vietnamese epidemic in which 39 percent of 292 term newborns with congenital rubella syndrome were low birthweight. In one study of 238 primary cytomegalovirus infections, no severe cases were observed when infection occurred after 14 weeks' gestation (Picone, 2013). hese investigators later identified sonographic findings in 30 of 69 cases of congenital infection, and growth restriction was noted in 30 percent of these 30 cases (Picone, 2014).

1	Tuberculosis and syphilis have also both been associated with poor fetal growth. Both extrapulmonary and pulmonary tuberculosis are linked with low birthweight (Chap. 51, p. 995). Sobhy (2017) analyzed 13 studies that included a total of3384 women with active tuberculosis. The odds ratio was 1.7 for low birthweight. The etiology is uncertain, however, the adverse efects on maternal health, compounded by efects of poor nutrition and poverty, are important Gana, 2012). Congenital syphilis is more common, and paradoxically, the placenta is almost always larger and heavier due to edema and perivascular inflammation (Chap. 65, p. 1237). Congenital syphilis is strongly linked with preterm birth and thus low-birthweight newborns (Sheield, 2002).

1	Toxoplasma gondii can also cause congenital infection, and Paquet and Yudin (2013) describe its classic association with fetal-growth restriction. Capobiango (2014) described 31 Brazilian pregnancies complicated by congenital toxoplasmosis. Only 13 percent were treated antepartum for toxoplasmosis, and low birthweight complicated nearly 40 percent of all the pregnancies. Congenital maaria also causes low birthweight and poor fetal growth. Briand and colleagues (2016) emphasize the importance of prophylaxis early in pregnancy for women at risk.

1	In a study of more than 13,000 fetuses with major structural anomalies, 22 percent had accompanying growth restriction (Khoury, 1988). In one study of 111 pregnancies complicated by fetal gastroschisis, a third had birthweights < 10th percentile (Nelson, 20 15a). As a general rule, the more severe the malformation, the more likely it is that the fetus will be SGA. This is especially evident in fetuses with chromosomal abnormalities or those with serious cardiovascular malformations.

1	Depending on which chromosome is redundant, fetuses with autosomal trisomies may display poor fetal growth. For example, in trisomy 21, fetal-growth restriction is generally mild. By contrast, fetal growth in trisomy 18 is virtually always significantly limited. he crown-rump length in fetuses with trisomy 18 and 13, unlike that with trisomy 21, is typically shorter than expected (Bahado-Singh, 1997; Schemmer, 1997). By the second trimester, long-bone measurements usually are below the 3rd percentile. In one group of 174 children with trisomy 13, the mean birthweight with trisomy 13 was 2500 g, and in 254 children with trisomy 18, it was 1800 g (Nelson, 2016).

1	Poor fetal growth also complicates Turner syndrome, and the severity correlates with increasing haploinsuiciency of the short arm of the X chromosome (Fiot, 2016). In contrast, poor growth is not characteristic of an increased number of X chromosomes (Ottesen, 2010; Wigby, 2016). As discussed in Chapter 13 (p. 263), aneuploidic patches in the placentaconined placental mosaicism (CPM)-is a recognized cause of fetal-growth restriction. Evidence suggests that aneuploidy afecting both the cytotrophoblast and mesenchymal core of the placenta, which is type 3 CPM, is associated with fetalgrowth restriction (Toutain, 2010).

1	First-trimester prenatal programs that screen for fetal aneuploidy'may incidentally identiy pregnancies at risk for fetalgrowth restriction unrelated to karyotype. In their analysis of 8012 women, the risk for growth restriction was higher in eukaryotic fetuses with extremely low free 3-human chorionic gonadotropin (3-hCG) and pregnancy-associated plasma protein-A (PAPP-A) levels (Krantz, 2004). From her review, Dugof (2010) concluded that a low PAPP-A level is strongly associated with poor fetal growth, but studies of free 3-hCG are conflicting.

1	Second-trimester analytes, including elevated alpha-fetoprotein and inhibin A levels and low unconjugated serum estriol concentrations, are significantly associated with birthweight below the 5th percentile. An even greater risk of poor growth is linked with certain combinations of these analytes. Still, these markers are poor screening tools for complications such as fetalgrowth restriction due to low sensitivity and positive-predictive values (Dugof, 2010). Nuchal translucency is also not predictive of fetal-growth restriction. he role of all these markers in aneuploidy screening is discussed in Chapter 14 (p. 281).

1	Identification of the inappropriately growing fetus remains a challenge. Eary establishment of gestational age, ascertainment of maternal weight gain, and careful measurement of uterine fundal growth throughout pregnancy will identiy many cases of abnormal fetal growth in low-risk women. Risk factors, including aprior growth-restricted etus, raise the recurrence risk to nearly 20 percent (American College of Obstetricians and Gynecologists, 2015). In women with risk factors, serial sonographic evaluation is considered. Although examination frequency varies depending on indications, an initial early dating examination followed by an examination at 32 to 34 weeks, or when otherwise clinically indicated, will identiy many growthrestricted fetuses. Even so, deinitive diagnosis frequently cannot be made until delivery.

1	According to one systematic review, insuicient evidence supports the utility of fundal height measurement to detect fetalgrowth restriction (Robert Peter, 2015). Nonetheless, carefully performed serial fundal height measurements are recommended as a simple, safe, inexpensive, and reasonably accurate screening method to detect growth-restricted fetuses. As a screening tool, its principal drawback is imprecision. Haragan and coworkers (2015) reported sensitivities of 71 and 43 percent for detecting excessive or deficient fetal growth. Specificities were 85 and 66 percent, respectively. he method used by most for fundal height measurement is described in Chapter 9 (p. 164). Between 18 and 30 weeks' gestation, the uterine fundal height in centimeters coincides within 2 weeks of gestational age. Thus, if the measurement is more than 2 to 3 cm from the expected height, inappropriate fetal growth is suspected and sonography is considered.

1	One supporting point for routine sonographic evaluation of all pregnancies is the opportunity to diagnose growth restriction. Typically, such routine screening incorporates an early initial sonographic examination-usually at 16 to 20 weeks' gestation. Increasingly, a first-trimester examination is added to establish gestational age and identiy anomalies. Some then recommend repeat sonographic evaluation at 32 to 34 weeks to evaluate fetal growth. First-trimester sonography has limited accuracy to predict SGA newborns. For example, Croverro and associates (2017) reported detection rates of 35 and 42 percent with false-positive rates of 5 and 10 percent, respectively. From nearly 9000 screened pregnancies, Tuuli and colleagues (2011) concluded

1	FIGURE 44-6 Correlation of sonographic fetal weight estimation using abdominal circumference (A C) and actual birthweight. (Data from pregnancies managed at Parkland Hospital.) that second-trimester sonography is superior to first-trimester scans for predicting SGA neonates. At Parkland Hospital, we provide midpregnancy sonographic screening examination of all pregnancies. Additional sonographic evaluations of fetal growth are performed as clinically indicated.

1	With sonography, the most common method for identiying poor fetal growth is estimation of weight using multiple fetal biometrical measurements. Combining head, abdomen, and femur dimensions provides optimum accuracy, whereas little incremental improvement is gained by adding other biometrical measurements (Platz, 2008). Of the dimensions, femur length measurement is technically the easiest and the most reproducible. Biparietal diameter and head circumference measurements are dependent on the plane of section and may also be afected by deformative pressures on the skull. Last, abdominal circumference measurements are more variable. However, these are most frequently abnormal with fetal-growth restriction because soft tissue predominates in this dimension (Fig. 44-6). Shown in Figure 44-7 is an example of a severely growth-restricted newborn.

1	Some studies have reported a significant predictive value for small abdominal circumference with respect to lagging fetal growth. One study screened nearly 4000 pregnancies using either clinically indicated or universal sonography in the third trimester (Sovio, 2015) . Universal sonography raised the rate of detection of SGA from 20 percent to 57 percent. Importantly, however, the neonatal morbidity rate was increased only if the abdominal circumference growth velocity was in the lowest decile.

1	Sonographic estimates of fetal weight and actual weight may be discordant by 20 percent or more, leading to both false-positive and false-negative indings. Dashe and associates (2000) studied 8400 live births at Parkland Hospital in which fetal sonographic evaluation had been performed within 4 weeks of delivery. They reported that 30 percent of growth-restricted fetuses were not detected. In a study of 2586 women with low-risk pregnancies randomly assigned to sonography at 32 or 36 weeks' gestation, sensitivity to identiy grow restriction was improved at the later gestational age (Roma, 2015). Still, nearly 40 percent of cases of growth restriction deined as birthweight <3rd percentile were missed. A Cochrane database analysis of 13 trials with 34,980 women concluded that routine late pregnancy ultrasound for a low-risk or an unselected population is not associated with maternal or fetal benefit (Bricker, 2015).

1	FIGURE 44-7 A 36-week newborn with severe fetal-growth restriction. (Used with permission from Dr. Roxane Holt.) An association between pathological fetal-growth restriction and oligohydramnios has long been recognized. Petrozella and associates (201r1) reported that decreased amnionic fluid volume between 24 and 34 weeks' gestation was signiicantly associated with malformations. In the absence of malformations, a birthweight < 3rd percentile was seen in 37 percent of pregnancies with oligohydramnios, in 21 percent with borderline amnionic fluid volume, but in only 4 percent with normal volumes. Also, from a recent metaanalysis of 15 studies involving more than 35,000 pregnancies, high-risk pregnancies with oligohydramnios were more likely to be complicated by low birthweight compared with low-risk pregnancies with oligohydramnios (Rabie, 2017). Hypoxia and diminished renal blood low are proposed explanations for oligohydramnios.

1	With this technique, early changes in placenta-based growth restriction are detected in peripheral vessels such as the umbilical and middle cerebral arteries. Late changes are characterized by reversal of umbilical artery flow and by abnormal flow in the ductus venosus and fetal aortic and pulmonary outlow tracts.

1	Of these, abnormal umbilical artery Doppler velocimetry indings-characterized by absent or reversed end-diastolic low-are uniquely linked with fetal-growth restriction (Chap. 10, p. 213). hese abnormalities highlight early versus severe growth restriction and represent the transition from fetal adaptation to failure. Thus, persistently absent or reversed end-diastolic flow, such as that shown in Figure 44-8, has long been correlated with hypoxia, acidosis, and fetal death. In one prospective sonographic examination of 1116 fetuses with estimated fetal weights < 10th percentile, only 1.3 percent of fetuses with normal umbilical artery Doppler studies had adverse outcomes compared with 11.5 percent of those with Doppler abnormalities (O'Dwyer, 2014). Unterscheider and associates (2013a) reported that abnormal umbilical artery Doppler velocimetry combined with an estimated fetal weight <3rd percentile is most strongly associated with poor obstetrical outcome.

1	FIGURE 44-8 Doppler velocity waveforms. A. Normal waveform with normal SID ratio. B. Increased impedance to flow with abnormally elevated SID ratio. C. Absent end-diastolic flow. D. Reversed end-diastolic flow. Because of these findings, umbilical artery Doppler velocimetry is considered standard in the evaluation and management of the growth-restricted fetus. he American College of Obstetricians and Gynecologists (2015) has concluded that umbilical-artery Doppler velocimetry improves clinical outcomes. It is recommended in the management of fetal-growth restriction as an adjunct to standard surveillance techniques such as nonstress testing and biophysical proile.

1	Other Doppler assessments are still investigational. Interrogation of the ductus venosus was evaluated in a series of 604 fetuses <33 weeks' gestation who had an abdominal circumference <5th percentile (Baschat, 2007). Ductus venosus Doppler parameters were the primary cardiovascular factor in predicting neonatal outcome. These late changes are felt to relect myocardial deterioration and acidemia, which are major contributors to adverse perinatal and neurological outcome. In another study of 46 growth-restricted fetuses, Doppler flow abnormalities of the aortic valve isthmus preceded those in the ductus venosus by 1 week (Figueras, 2009). In their evaluation of several fetal vessels, Turan and associates (2008) described the seq uence of changes characteristic of mild placental dysfunction, progressive placental dysfunction, and severe, early-onset placental dysfunction. However, Unterscheider and colleagues (2013b) questioned whether a predictable progression of Doppler indices

1	progressive placental dysfunction, and severe, early-onset placental dysfunction. However, Unterscheider and colleagues (2013b) questioned whether a predictable progression of Doppler indices actually exists in fetal-growth restriction.

1	Fetal-growth restriction prevention ideally begins before conception. Maternal medical conditions, medications, and nutrition are optimized, and smoking cessation is critical. Other risk factors are tailored to the maternal condition, such as antimalarial prophylaxis for women living in endemic areas and correction of nutritional deficiencies. Of note, treatment of mild-to-moderate hypertension does not reduce the incidence of growth-restricted newborns (Chap. 50, p. 981).

1	Accurate dating is essential during early pregnancy. Serial sonographic evaluations are typically used, but the best interval between assessments has not been clearly established. Given that a prior SGA newborn is associated with other adverse outcomes in a subsequent pregnancy, particularly stillbirth and preterm birth, surveillance during a subsequent pregnancy may be beneicial (Mendez-Figueroa, 2016; Spong, 2012). he American College of Obstetricians and Gynecologists (2015) notes that if growth is normal during a pregnancy following a prior pregnancy complicated by fetal-growth restriction, then Doppler velocimetry and fetal surveillance are not indicated. A recent metaanalysis of 45 trials involving 20,909 women reported that low-dose aspirin initiated prior to 16 weeks' gestation was associated with a significantly lower risk of fetal-growth restriction (Roberge, 2017). Moreover, they described a dose-response efect. he American College of Obstetricians and Gynecologists (2015)

1	associated with a significantly lower risk of fetal-growth restriction (Roberge, 2017). Moreover, they described a dose-response efect. he American College of Obstetricians and Gynecologists (2015) has not endorsed prophylaxis with low-dose aspirin for women with a prior growth-restricted fetus.

1	If fetal-growth restriction is suspected, then eforts are made to conirm the diagnosis, assess fetal condition, and search for possible causes. Early-onset growth restriction is especially problematic. In pregnancies in which fetal anomalies are suspected, patient counseling and prenatal diagnostic testing are indicated (American College of Obstetricians and Gynecologists, 2015). Ifnoindications for immediate delivery, Ifnoindications for immediate delivery: begin antepartum fetal surveillance: -Antepartum fetal surveillance-BPP, NST, etc. -Regular fetal testing -Umbilical artery Doppler velocimetry weekly -Weekly umbilical artery Doppler velocimetry -Amnionic fluid evaluation weekly -Weekly evaluation of amnionic fluid surveillance until delivery 38 weeks, then deliver delivery 34 weeks, then begin FIGURE 44-9 Algorithm for management of fetal-growth restriction at Parkland Hospital. BPP = biophysical profile; NST = nonstress test.

1	One management algorithm is shown in Figure 44-9. In pregnancies with suspected fetal-growth restriction, antepartum fetal surveillance includes periodic Doppler velocimetry of the umbilical arteries in addition to more frequent fetal testing. At Parkland Hospital, for women whose fetus measures ;3rd percentile and has reached a viable age, we encourage hospitalization on our High-Risk Pregnancy Unit. Daily fetal heart rate tracings, weekly Doppler velocimetry, and sonographic assessment of fetal growth every 3 to 4 weeks are initiated. Other modalities of Doppler velocimetry, such as middle cerebral arteries or ductus venosus assessment, are considered experimental. he American College of Obstetricians and Gynecologists (2015) recommends that antenatal corticosteroids for pulmonary maturation be given to pregnancies complicated by fetal-growth restriction and at risk for birth before 34 weeks' gestation.

1	he timing of delivery is crucial, and the risks of fetal death versus the hazards of preterm birth must be considered. Several multicenter studies address these problems, but unfortunately, none have elucidated the optimal timing of delivery. For the preterm fetus, the only randomized trial of delivery timing is the Growth Restriction Intervention Trial (GRIT) (hornton, 2004). This trial involved 548 women between 24 and 36 weeks' gestation with clinical uncertainty regarding delivery timing. Women were randomly assigned to immediate delivery or to delayed delivery until the situation worsened. he primary outcome was perinatal death or disability after reaching age 2 years. Mortality rates did not difer through 2 years of age. Moreover, children aged 6 to 13 years did not show clinically signiicant diferences between the two groups (Walker, 2011).

1	In the Trial of Randomized Umbilical and Fetal Flow in Europe (TRUFFLE), ductus venosus Doppler evaluation was compared with fetal heart rate monitoring. There were 310 pregnancies between 26 and 32 weeks' gestation with fetuses displaying an abdominal circumference < 10th percentile and an umbilical artery pulsatility index >95th percentile (Lees, 2015). Delivery timing was determined by the results of three difering antenatal fetal assessment arms that were: short-term fetal heart rate variability, early ductus venosus Doppler velocimetry changes, or late ductus changes. he proportion of children with neuroimpairment at 2 years of age was not diferent among the groups. Of note, only 32 percent of the newborns overall were delivered according to this randomization. Safety net criteria and other maternal/fetal indications prompted these protocol deviations (Visser, 2016). In a post-hoc analysis, these authors concluded that before 32 weeks, delaying delivery until ductus venosus

1	and other maternal/fetal indications prompted these protocol deviations (Visser, 2016). In a post-hoc analysis, these authors concluded that before 32 weeks, delaying delivery until ductus venosus Doppler or fetal heat rate abnormalities occur is likely safe and possibly beneits long-term outcome (Ganzevoort, 2017).

1	he Disproportionate Intrauterine Growth Intervention Trial at Term (DIGITAT) study examined the delivery timing of growth-restricted fetuses who were 36 weeks' gestation or older. In these 321 women who were randomized to induction or to expectant management, composite neonatal morbidity did not difer, except that neonatal admissions were lower after 38 weeks in a secondary analysis (Boers, 2010, 2012). Another secondary analysis of DIGITAT did not identiy a clear subgroup that beneited from labor induction (T ajik, 2014). Other secondary analyses included assessment of neurodevelopmental and behavioral outcomes at age 2, and these also were similar between the randomized groups (Van Wyk, 2012). Management of the Near-Term Fetus

1	As shown in Figure 44-9, delivery of a suspected growthrestricted fetus with normal umbilical artery Doppler velocimetry, normal amnionic luid volume, and reassuring fetal heart rate testing can likely be deferred until 38 weeks' gestation. Said another way, uncertainty regarding the diagnosis should preclude intervention until fetal lung maturity is assured. Expectant management can be guided using antepartum fetal surveillance techniques described in Chapter 17. Most clinicians, however, recommend delivery at 34 weeks or beyond if there is clinically signiicant oligohydramnios. Consensus statements by the Society for Maternal-Fetal Medicine (Spong, 2011) and the American College of Obstetricians and Gynecologists (2017a) are similar. These recommend delivery between 34 and 37 weeks when there are comorbid conditions such as oligohydramnios. With a reassuring fetal heart rate pattern, vaginal delivery is planned. Notably, some of these fetuses do not tolerate labor.

1	Management of the Fetus Remote from Term If growth restriction is identiied in an anatomically normal fetus before 34 weeks, and amnionic luid volume and fetal surveillance indings are normal, observation is recommended. Screening for toxoplasmosis, cytomegalovirus infection, rubella, herpes, and other infections is recommended by some. However, we and others have not found this to be productive (Yamamoto, 2013).

1	As long as interval fetal growth and fetal surveillance test results are normal, pregnancy is allowed to continue until fetal lung maturity is reached (see Fig. 44-9). Reassessment of fetal growth is typically made no sooner than 3 to 4 weeks. Weekly assessment of umbilical artery Doppler velocimetry and amnionic fluid volume is combined with periodic nons tress testing, although the optimal frequency has not been determined. As mentioned, we hospitalize these women in our High-Risk Pregnancy Unit and monitor their fetuses daily. If interval growth, amnionic luid volume, and umbilical artery Doppler velocimetry are normal, then the mother is discharged home and seen intermittently for outpatient surveillance.

1	With growth restriction remote from term, no speciic treatment ameliorates the condition. For example, evidence does not support diminished activity or bed rest to accelerate growth or improve outcomes. Despite this, many clinicians intuitively advise a program of modiied rest. Nutrient supplementation, attempts at plasma volume expansion, oxygen therapy, antihypertensive drugs, heparin, and aspirin are all inefective (American College of Obstetricians and Gynecologists, 2015).

1	In most cases diagnosed before term, neither a precise etiology nor a specific therapy is apparent. Management decisions hinge on assessment of the relative risks of fetal death during expectant management versus the risks from preterm delivery. lthough reassuring fetal testing may allow observation with continued maturation, long-term neurological outcome is a concern (Baschat, 2014; Lees, 2015; Thornton, 2004). Baschat and associates (2009) showed that neurodevelopmental outcome at 2 years in growth-restricted fetuses was best predicted by birthweight and gestational age. Doppler abnormalities are generally not associated with poor childhood cognitive developmental scores among low-birthweight fetuses delivered in the third trimester (Llurba, 20l3). hese indings emphasize that adverse neurodevelopmental outcomes cannot always be predicted.

1	Fetal-growth restriction is commonly the result of placental insuiciency due to faulty maternal perfusion, reduction of functional placenta, or both. If present, these conditions are likely aggravated by labor. Equally important, diminished amnionic luid volume raises the likelihood of cord compression during labor. For these and other reasons, the frequency of cesarean delivery is increased. Accordingly, a woman with a suspected growth-restricted fetus should undergo "high-risk" intrapartum monitoring (Chap. 24, p. 478).

1	The risk of neonatal hypoxia or meconium aspiration is also greater. hus, care for the newborn should be provided immediately by an attendant who can skillfully clear the airway and ventilate a neonate as needed (Chap. 32, p. 608). he severely growth-restricted newborn is particularly susceptible to hypothermia and may also develop other metabolic derangements such as hypoglycemia, polycythemia, and hyperviscosity. In addition, low-birthweight newborns are at higher risk for motor and other neurological disabilities. Risk is greatest at the lowest extremes of birth weight (Baschat, 2009, 2014; Llurba, 2013).

1	The term macrosomia is used rather imprecisely to describe a very large fetus or newborn. Although there is general agreement among obstetricians that neonates weighing < 4000 g are not excessively large, a similar consensus has not been reached for the definition of macrosomia. Newborn weight rarely exceeds 11 pounds (5000 g), and excessively large infants are a curiosity. The largest newborn cited in the Guinness Book of World Records was a 23-lb 12-oz (10,800 g) infant boy born in 1879 to a Canadian woman (Barnes, 1957). In the United States in 2015, of more than 4 million births, 6.9 percent weighed 4000 to 4499 g; 1 percent weighed 4500 to 4999 g; and 0.1 percent were born weighing 5000 g or more 4500-4649 3221 (Martin, 2017) . To be sure, the incidence of excessively large 4750-4999 1146 0.3 134 12 infants grew during the 20th century. According to Williams 5000-5249 385 0.1 57 15 (1903), at the beginning of the 20th century, the incidence 5250-5499 127 0.04 31

1	TABLE 44-2. Birthweight Distribution of 354,509 Liveborn Infants at Parkland Hospital between 1988 and 2012 (g) No. % No. /0 500-3999 322,074 90.9 13,365 4 4000-4249 19,106 5.4 1043 5 4250-4499 8391 2.4 573 7 of birthweight > 5000 g was 1 to 2 per 10,000 births. his 5500 or more 0.02 14 24 compares with 16 per 10,000 births at Parland Hospital from 1988 through 2008 and with 11 per 10,000 in the United States in 2010. he influence of increasing maternal obesity rates is overwhelming, and its association with diabetes is well known. Of Parkland mothers with newborns weighing > 5000 g, more than 15 percent were diabetic. Several terms currently describe pathological fetal overgrowth. he most common ofthese-macrosomia-is defined by birthweights that exceed certain percentiles for a given population. Another commonly used scheme is to deine macrosomia by an empirical birthweight threshold.

1	Macrosomia is frequently deined based on mathematical distributions of birthweight. Those newborns exceeding the 90th percentile for a given gestational week are usually used as the threshold for macrosomia or large-for-gestational age (LGA) birthweight. For example, the 90th percentile at 39 weeks is 4000 g. If, however, birthweights that are 2 standard deviations above the mean are used, then thresholds lie at the 97th percentile. Thus, substantially larger newborns are considered macrosomic compared with those at the 90th percentile. Specifically, the birthweight threshold at 39 weeks to be macrosomic would be approximately 4500 g for the 97th percentile rather than 4000 g for the 90th percentile.

1	Newborn weight exceeding 4000 g (8 lb 13 oz) is also a frequently used threshold to deine macrosomia. Others use 4250 g or even 4500 g (10 lb). As shown in Table 44-2, birthweights ::4500 g are uncommon. During a 30-year period at Parkland Hospital, during which there were more than 350,000 singleton births, only 1.4 percent of newborns weighed 4500 g or more. We are of the view that the upper limit of fetal growth, above which growth can be deemed abnormal, is likely two standard deviations above the mean, representing perhaps 3 percent of births. At 40 weeks, such a threshold would correspond to approximately 4500 g. The American College of Obstetricians and Gynecologists (2016a) concludes that the term macrosomia was an appropriate appellation for newborns who weigh 4500 g or more at birth. Total 354,509 15,501 Some factors associated with fetal overgrowth are listed in Table 44-3. Many are interrelated and thus likely are additive.

1	Total 354,509 15,501 Some factors associated with fetal overgrowth are listed in Table 44-3. Many are interrelated and thus likely are additive. For example, advancing age is usually related to multiparity and diabetes, and obesity is related to diabetes. In one study, the incidence of macrosomia exceeded 24 percent in China among obese women, and macrosomia rates were also signiicantly higher (approximately 2.5-fold) for prolonged pregnancy and gestational diabetes (Wang, 2017). Of these, maternal diabetes is an important risk factor for fetal overgrowth (Chap. 57, p. 1100). s shown in Table 44-2, the incidence of maternal diabetes grows s birthweights >4000 g rise. It should be emphasized, however, that maternal diabetes is associated with only a small percentage of the total number of such large newborns.

1	The adverse consequences of excessive fetal growth are considerable. Neonates with a birthweight of at least 4000 g have cesarean delivery rates > 50 percent. This is particularly true with maternal obesity or diabetes or with birthweights >5000 g (Cordero, 2015; Crosby, 2017; Gaudet, 2014; Hehir, 2015). One study found a higher risk for traumatic neonatal morbidity in LGA neonates compared with normal-weight ones (Chauhan, 2017). Rates of shoulder dystocia vary greatly and can reach nearly 30 percent for macrosomic neonates when maternal diabetes is comorbid (Cordero, 2015). In general obstetrical populations that include diabetic mothers, dystocia rates are at least 5 percent for neonates with birthweights ::5000 g (Crosby, TABLE 44-3. Risk Factors for Fetal Overgrowth Large size of parents Advancing maternal age Previous macrosomic infant Racial and ethnic factors

1	TABLE 44-3. Risk Factors for Fetal Overgrowth Large size of parents Advancing maternal age Previous macrosomic infant Racial and ethnic factors TABLE 4-4. Maternal and Fetal Outcomes for 208,090 Pregnancies Delivered at Parkland Hospital from 1998 through 201e2 <4000 9 4000-4499 9 4500-4999 9 �5000 9 Outcomea n = 187,1 19 n = 17,750 n = 2849 n = 372 P value Cesa rea n totaI 46,577 (25) 5,362 (30) 1204 (42) 224 (60) <0.001 Scheduled 12,564 (7) 1,481 (8) 316 (11) 65 (17) <0.001 Dystocia 7589 (4) 1388 (8) 337 (1n2) 46 (1n2) <0.001 Shou Ider dystocia 437 (0) 366 (2) 192 (7) 56 (15) <0.001 3rd-or 4th-degree laceration 7296 (4) 932 (5) 190 (7) 37 (1n0) <0.001 Labor induction 26,118 (13) 2499 (14) 420 (15) 39 (10) 0.141 Prolonged second stage 6905 (4) 899 (5) 147 (5) 14 (4) <0.001 Chorioamnionitis 13,448 (7) 1778 (1n0) 295 (1n0) 35 (9) <0.001 pH <7.0 925 (0.5) 96 (0.6) 20 (0.7) 4 (1.1) 0.039 Apgar <7 @ 5 minutes 1898 (1n.0) 80 (0.5) 22 (0.8) 10 (2.7) <0.001

1	Apgar <7 @ 5 minutes 1898 (1n.0) 80 (0.5) 22 (0.8) 10 (2.7) <0.001 ICN admission 4266 (2.2) 123 (0.7) 36 (1n.3) 9 (2.4) <0.00'1 Fractured clavicle 1880 (1.0) 616 (3.5) 125 (4.4) 16 (4.3) <0.001 Mechanical ventilation 2305 (1n.2) 54 (0.3) 11 (0.4) 9 (2.4) <0.001 Hypoglycemia 480 (0.2) 89 (0.5) 31 (1.1) 12 (3.2) <0.001 Hyperbilirubinemia 5829 (3.0) 305 (1.7) 60 (2.1) 12 (3.2) <0.001 Erb palsy 470 (0.2) 224 (1n.3) 74 (2.6) 22 (5.9) <0.001 Neonatal death 402 (0.2) 3 (0) 2 (0.1) 1 (0.3) <0.001 aOutcome data presented as n (%). ICN = intensive care nursery. 2017; Hehir, 2015). Rates of postpartum hemorrhage, perineal laceration, and maternal infection, which are related complications, are also higher in mothers delivering overgrown newborns. Maternal and neonatal outcomes by birthweight for large babies >4000 g delivered at Parkland Hospital are shown in Table 44-4.

1	Because current methods fail to accurately estimate excessive fetal size, macrosomia cannot be deinitively diagnosed until delivery. Inaccuracy in clinical estimates of fetal weight by physical examination is often attributable, at least in part, to maternal obesity. Numerous attempts have been made to improve the accuracy of sonographic fetal-weight estimation. Several formulas have been proposed to calculate fetal weight using measurements of the head, femur, and abdomen. Estimates provided by these computations are reasonably accurate for predicting the weight of small, preterm fetuses but are less valid in predicting the weight of large fetuses. In one study of 248 LGA and 655 non-LGA newborns from pregnancies complicated by diabetes, only 23 percent of women diagnosed with an LGA fetus before delivery actually delivered an LGA infant (Scifres, 2015). his resulted in a more than threefold rise in the cesarean delivery rate for suspected LGA birthweights.

1	From the foregoing, it is apparent that sonographic estimation of fetal weight is unreliable, and its routine use to identiy macrosomia is not recommended. Indeed, the American College of Obstetricians and Gynecologists (2016a) concludes that clinical fetal weight estimates are just as accurate as sonographic ones.

1	Several interventions have been proposed to interdict fetal overgrowth. Some include prophylactic labor induction for poorly deined indications such as "impending macrosomia," or elective cesarean delivery to avoid diicult delivery and shoulder dystocia. For women with diabetes in pregnancy, insulin therapy and close attention to good glycemic control reduces birthweight. However, this has not consistently translated into reduced cesarean delivery rates. Furthermore, as noted above, erroneous diagnosis of fetal overgrowth among women with diabetes raises cesarean delivery rates (Scifres, 2015). Also previously mentioned, fetal overgrowth irrespective of the diagnosis of diabetes mellitus is strongly associated with maternal obesity and excessive gestational weight gain (Durie, 2011; Durst, 2016; Harper, 2015). Dietary intervention to limit fetal overgrowth by curbing gestational weight gain, for example, is an active area of research. Currently recommended weight gains for pregnancy

1	2016; Harper, 2015). Dietary intervention to limit fetal overgrowth by curbing gestational weight gain, for example, is an active area of research. Currently recommended weight gains for pregnancy according to maternal BMI are described in Chapter 9 (p. 165).

1	Some clinicians induce labor when fetal macrosomia is suspected in nondiabetic women. his approach is suggested to obviate further fetal growth and thereby reduce potential delivery complications. Such prophylactic induction should theoretically reduce the risk of shoulder dystocia and cesarean delivery. In one systematic review of 11 studies of expectant management versus labor induction for suspected macrosomia, labor induction increased cesarean delivery rates without improving perinatal outcomes (Sanchez-Ramos, 2002). In contrast, Magro-Malosso and colleagues (2017) performed a metaanalysis of four randomized trials involving 1r190 women and concluded that labor induction at 38 or more weeks for suspected macrosomia significantly reduces the frequency of fetal overgrowth and fractures. In one of these studies, 822 women with suspected LGA fetuses were randomly assigned either to early term delivery (37°/7 to weeks) or to expectant management (Boulvain, 2015). here was a higher

1	In one of these studies, 822 women with suspected LGA fetuses were randomly assigned either to early term delivery (37°/7 to weeks) or to expectant management (Boulvain, 2015). here was a higher rate of vaginal delivery that was marginally significant and a lower composite measure of morbidity. These authors cautioned that any beneits should be balanced with the risks of earlyterm labor induction and delivery. Namely, a review of earlyterm births indicates that elective delivery before 39 weeks' gestation does not improve maternal outcomes and is associated with worse neonatal outcomes (Tita, 2016). We agree with the American College of Obstetricians and Gynecologists (2016a, 2017a,b) that current evidence does not support a policy for early labor induction or delivery before 39 weeks' gestation. It remains unclear whether delivery or induction for suspected macrosomia at term is better than expectan t management.

1	With the delivery of macrosomic infants, shoulder dystocia and its attendant risks described in Chapter 27 (p. 520) are major concerns. That said, the American College of Obstetricians and Gynecologists (20 17b) concluded that fewer than 10 percent of all shoulder dystocia cases result in a persistent brachial plexus injury, and 4 percent of these injuries still follow cesarean delivery. For prevention, planned cesarean delivery on the basis of suspected macrosomia to prevent brachial plexopathy is an unreasonable strategy in the general population (Chauhan, 2005). Ecker and coworkers (1997) analyzed 80 cases of brachial plexus injury in 77,616 consecutive infants born at Brigham and Women's Hospital. They concluded that an excessive number of otherwise unnecessary cesarean deliveries would be needed to prevent a single brachial plexus injury in neonates born to women without diabetes. Rouse and colleagues (1996) echoed these sentiments in their analysis of nondiabetic mothers.

1	Conversely, planned cesarean delivery may be a reasonable strategy for diabetic women with an estimated fetal weight >4250 or >4500 g. Conway and Langer (1998) described a protocol of routine cesarean delivery for sonographic estimates of �4250 g in diabetic women. This management significantly lowered the shoulder dystocia rate from 2.4 to 1.1 percent. In summaly, we agree with the College that elective delivery for the fetus that is suspected to be overgrown is inadvisable, particularly before 39 weeks' gestation. Finally, we also conclude that elective cesarean delivery is not indicated when estimated fetal weight is <5000 g among women without diabetes and < 4500 g among women with diabetes (American College of Obstetricians and Gynecologists, 2016a, 2017b).

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1	Zhang J, Merialdi M, Platt LO, et al: Deining normal and abnormal fetal growth: promises and challenges. Am J Obstet Gynecol 202(6):522, 2010 Zhang J, Mikolajczyk R, Grewal J, et al: Prenatal application of the individual ized fetal growth reference. Am J Epidemiol 173(5):539,t2011 Zhu JL, Obel C, Hammer Bech B, et al: Infertility, infertility treatment, and fetal growth restriction. Obstet Gynecol 110(6):1326,200 Zhu MY, Milligan N, Keating S, et al: The hemodynamics of late-onset intrauterine growth restriction by MRI. Am J Obstet Gynecol 214:367.e1, 2016 MECHANISMS OF MULTIFETAL GESTATIONS ......... 864 DIAGNOSIS OF MULTIFETAL GESTATION ........... 869 MATERNAL PHYSIOLOGICAL ADAPTATIONS ........ 870 PREGNANCY COMPLICATIONS ....................i. 871 UNIQUE FETAL COMPLICATIONS ................... 873 DISCORDANT GROWTH OF TWIN FETUSES .......... 881

1	PREGNANCY COMPLICATIONS ....................i. 871 UNIQUE FETAL COMPLICATIONS ................... 873 DISCORDANT GROWTH OF TWIN FETUSES .......... 881 FETAL DEMISE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 882 PRENATAL CARE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 884 PRETERM BIRTH ................................ 885 LABOR AND DELIVERY ......................... 887 SELECTIVE REDUCTION OR TERMINATION .......... 891 In single-ovum twins, there is always a certain area of the placenta in which there is anastomosis between vascular systems which is never present in the fused placenta of doubleovum twins. Thus, if at an eary period the heart of one embryo is consideraby stronger than that of the other, a gradualy increasing area of the communicating portion of the placenta is monopolized by the ormer, so that its heart increases rapidy in size, whilst that of the latter receives less blood and eventualy atrophies.

1	-J. Whitridge Williams (1903) In Williams' time, a great deal concerning the embryological and morphological development of multifetal pregnancies was unknown. These pregnancies may result from two or more fer tilization events, from a single fertilization followed by a split ting of the zygote, or from a combination of both. Multifetal today for both the mother and her fetuses. For example, in this country, approximately a fourth of very-low-birthweight gestations (Martin, 2017).

1	Fueled largely by infertility therapy, both the rate and the number of twins and higher-order multi fetal births grew dramatically during the 1980s and 1990s in the United States. National data from Martin and coworkers (2017) presented here is informative. he twinning rate rose 76 percent from 18.9 per 1000 live births in 1980 to 33.2 in 2009. During the same time, the number of higher-order multifetal births peaked in 1998 at a rate of 1.9 per 1000 total births. Since then, however, evolving infertility management has lowered rates of higher-order multifetal births-especially among nonHispanic white women. For example, the rate of triplets or more declined by more than 50 percent from 1998 to 2015 in this demographic group. And, in 2015, the overall m ultifetal birth rate was 34.5 per 1000, with twins representing nearly 97 percent of these births.

1	These rates of multi fetal pregnancies have a direct efect on the rates of preterm birth and its comorbidities. In addition, the risks for congenital malformation and its consequences are greater with multifetal gestations. Importantly, this increased risk applies to each fetus and is not simply the result of more fetuses. In sum, in 2013 in the United States, multifetal births accounted for 3 percent of all live births but for 15 percent of all infant deaths. Moreover, the risk of infant death rose proportionally with the number of fetuses in the pregnancy (Matthews, 2015). Speciically, the infant mortality rate for twins was more than four times the rate for single births. In the same year, the infant mortality rate for triplets was nearly 12 times the rate for singletons, and for quadruplets, it was a staggering 26 times that TABLE 45-1. Selected Outcomes in Singleton and Twin Outcome Singletons (No.) Twins (No.) Pregnancies 202,306 241n2 Birthsa 202,306 4824

1	TABLE 45-1. Selected Outcomes in Singleton and Twin Outcome Singletons (No.) Twins (No.) Pregnancies 202,306 241n2 Birthsa 202,306 4824 Stillbirths 1 011 (5.0) 114 (23.6) Neonatal deaths 590 (2.9) 92 (1n9.5) Perinatal deaths 1601 (7.9) 206 (42.7) Very low birthweight 1927 (9.6) 507 (1n07.6) aBirth data are represented as number (per 1000). bDenominator for neonatal deaths and very low birthweight is liveborn infants. Data from Dr. Don Mcintire. for singletons! From Parkland Hospital, a comparison of singleton and twin outcomes is shown in Table These risks are magnified further with higher-order births.

1	for singletons! From Parkland Hospital, a comparison of singleton and twin outcomes is shown in Table These risks are magnified further with higher-order births. he mother may also experience higher obstetrical morbidity and mortality rates. hese rates lso rise with the number of fetuses (Mhyre, 2012; Young, 2012). In one study of more than 44,000 multifetal pregnancies, the risks for preeclampsia, postpartum hemorrhage, and maternal death were twofold higher than these rates in singleton gestations (Walker, 2004). The risk for peripartum hysterectomy is also greater. Francois and associates (2005) reported this to be threefold for twins and 24-fold for triplets or quadruplets. Last, compared with women with a singleton pregnancy, these mothers are at increased risk for depression as well as parental divorce (Choi, 2009; Jenna, 2011).

1	Twin fetuses usually result from fertilization of two separate ova, which yields dizygotic orratenal twins. Less often, twins arise from a single fertilized ovum that then divides to create monozygotic or identical twins. Either or both processes may be involved in the formation of higher numbers. Quadruplets, for example, may arise from as few as one to as many as four ova. These traditional models of twinning discussed in the next sections have been taught for more than 50 years and remain the widely accepted theory. More recently, Herranz (2015) ofered a provocative alternative hypothesis, which posits that monozygotic twinning occurs with splitting at the postzygotic twocell stage. Notably, data are not robust in support of either the traditional or the newly proposed model (Denker, 2015).

1	Dizygotic twins are not in a strict sense true twins because they result from the maturation and fertilization of two ova during a single ovulatory cycle. Moreover, from a genetic perspective, dizygotic twins are like any other pair of siblings.

1	On the other hand, monozygotic or identical twins, although they have virtually the same genetic heritage, are usually not identical. Namely, the division of one fertilized zygote into two does not necessarily result in equal sharing of protoplasmic material. Monozygotic twins may actually be discordant for genetic mutations because of a postzygotic mutation, or may have the same genetic disease but with marked variability in expression. In female fetuses, skewed lyonization can produce diferential expression of X-linked traits or diseases. Further, the process of monozygotic twinning is in a sense a teratogenic event, and monozygotic twins have a higher incidence of often discordant malformations (Glinianaia, 2008). For example, in one study of 926 monozygotic twins, the prevalence of congenital heart defects was 12-fold greater than the general population rate, but 68 percent of afected infants had a normal sibling (Pettit, 20l3). From any of these mechanisms, dizygotic twins of the

1	heart defects was 12-fold greater than the general population rate, but 68 percent of afected infants had a normal sibling (Pettit, 20l3). From any of these mechanisms, dizygotic twins of the same sex may appear more nearly identical at birth than monozygotic twins.

1	• Genesis of Monozygotic Twins The developmental mechanisms underlying monozygotic twinning are poorly understood. The incidence of monozygotic twins is increased two-to fivefold in pregnancies conceived using assisted reproductive technology (ART). he predisposition to splitting may stem from specimen handling, growth media, or sperm DNA microinjection or may arise from intrinsic abnormalities associated with infertility (McNamara, 2016).

1	The outcome of the monozygotic twinning process depends on when division oCCuts. If zygotes divide within the irst 72 hours after fertilization, two embryos, two amnions, and two chorions develop, and a diamnionic, dichorionic twin pregnancy evolves (Fig. 45-1). Two distinct placentas or a single, fused placenta may develop. If division occurs between the fourth and eighth day, a diamnionic, monochorionic twin pregnancy results. By approximately 8 days after fertilization, the chorion and the amnion have already diferentiated, and division results in two embryos within a common amnionic sac, that is, a monoamnionic, monochorionic twin pregnancy. Conjoined twins result if twinning is initiated later.

1	It has long been accepted that monochorionicity incontrovertibly indicated monozygosity. Rarely, however, monochorionic twins may in fact be dizygotic (Hackmon, 2009). Mechanisms for this are speculative, but in one review of 14 cases, nearly all had been conceived after ART procedures (Ekelund, 2008) . McNamara and colleagues (2016) ofer an excellent review of the mechanisms and evidence for both typical and atypical twinning.

1	In supeetation, an interval as long as or longer than a menstrual cycle intervenes between fertilizations. Superfetation requires ovulation and fertilization during the course of an established pregnancy, which is theoretically possible until the uterine cavity is obliterated by fusion of the decidua capsularis to the decidua parietalis. Although known to occur in mares, superfetation is not known to occur spontaneously in humans. Lantieri and associates (2010) reported a case ater ovarian hyperstimulation and intrauterine insemination in the presence of an undiagnosed tubal pregnancy. Most authorities believe that alleged cases of human superfetation result from markedly FIGURE 45-1 Mechanism of monozygotic twinning. Black boxing and blue arrows in columns A, B, and C indicates timing of division.

1	FIGURE 45-1 Mechanism of monozygotic twinning. Black boxing and blue arrows in columns A, B, and C indicates timing of division. A. At 0 to 4 days postfertilization, an early conceptus may divide into two. Division at this early stage creates two chorions and two amnions (dichorionic, diamnionic). Placentas may be separate or fused. B. Division between 4 to 8 days leads to formation of a blastocyst with two separate embryoblasts (inner cell masses). Each embryoblast will form its own amnion within a shared chorion (monochorionic, diamnionic). C. Between 8 and 12 days, the amnion and amnionic cavity form above the germinal disc. Embryonic division leads to two embryos with a shared amnion and shared chorion (monochorionic, monoamnionic). D. Difering theories explain conjoined twin development. One describes an incomplete splitting of one embryo into two. The other describes fusion of a portion of one embryo from a monozygotic pair onto the other.

1	unequal growth and development of twin fetuses with the same setting of paternity lawsuits (Girela, 1997). Given that superfegestational age. cundation may also occur with ART, women should be advised Supeecundation refers to fertilization of two ova within the to consider avoiding intercourse after embryo transfer (McNasame menstrual cycle but not at the same coitus, nor necesmara, 2016; Peigne, 201r1). sarily by sperm from the same male. An instance of superfecundation or heteropaternity, documented by Harris (1982), is demonstrated in Figure 45-2. he mother was delivered of • Factors Afecting Twinning a black neonate whose blood type was A and a white neonate Dizygotic twinning is much more common than monozygotic whose blood type was O. The blood type of the mother and her splitting of a single oocyte, and its incidence is influenced by husband was O. More recent cases have been reported in the race, heredity, maternal age, parity, and, especially, fertility )c::0)�000 5.)�:

1	FIGURE 45-2 An example ofdizygotic twin boys as the consequence of superfecundation. treatment. By contrast, the frequency of monozygotic twin births is relatively constant worldwide-approximately 1 set per 250 births, and this incidence is generally independent of demographic factors. One exception is that rates of zygotic splitting are increased following ART (Aston, 2008). Among diferent races and ethnic groups, the frequency of multifetal births varies significantly. In one analysis of more than 8 million births in the United States between 2004 and 2008, the rate oftwinning was 3.5 percent in black women and 3 percent in whites (Abel, 2012). Hispanic, Asian, and Native merican women had comparatively lowerrates than white women. In one rural community in Nigeria, twinning occurred once in every 20 births (nox, 1960)! hese marked diferences in twinning frequency may be the consequence ofracial variations in levels of follicle-stimulating hormone-FSH (Nylander, 1973).

1	Maternal age is another important risk factor for multifetal pregnancies (Fig. 45-3). Dizygotic twinning frequency rises almost fourfold between the ages of 15 and 37 years (Painter, 2010). As such, there is a paradox of declining fertility but increasing twinning rates with advancing maternal age (Beemsterboer, 2006). Another explanation for the dramatic rise in twinning with advancing maternal age may be a higher use of ART in older women (Ananth, 2012). Paternal age has also been linked to twinning frequency, but its efect is felt to be small (Abel, 2012). Although twin pregnancy is associated with greater risks for most adverse perinatal outcomes, McLennan and associates (2017) did not find advanced maternal age to be an additional risk factor for fetal and infant death. From this population-based study of the United States, they concluded that women in their 30s may be counseled that their age is not a major additional risk factor for adverse obstetric outcomes in the setting

1	population-based study of the United States, they concluded that women in their 30s may be counseled that their age is not a major additional risk factor for adverse obstetric outcomes in the setting oftwin pregnancy.

1	Increasing pariy independently raises the incidence of twinning in all populations studied. During a 30-year period, Antsaklis and coworkers (2013) noted a progressively increasing positive correlation between multiparity and twinning. However, they FIGURE 45-3 Multifetal birth rates in the United States according to maternal age and race, 2015. (Data from Martin, 201o7.) cautioned that greater use ofRT may be partially contributory. In a two-year study from Nigeria, where such technology is not commonly available, Olusanya (2012) calculated the efects of multiparity compared with primiparity. They found an eightfold rise in multifetal gestation rates when parity was ;4, and a 20-fold rise when parity was :5.

1	As a determinant of twinning, the family history ofthe mother supersedes that of the father. One study of 4000 genealogical records showed that women who themselves were a dizygotic twin gave birth to twins at a rate of 1 set per 58 births (White, 1964). Women who were not a twin, but whose husbands were a dizygotic twin, gave birth to twins at a rate of 1 set per 116 pregnancies. Painter and associates (2010) performed genomewide linkage analyses on more than 500 families of mothers of dizygotic twins and identified four potential linkage peaks. he highest peak was on the long arm ofchromosome 6, and other suggestive peaks were on chromosomes 7, 9, and 16. That said, the contribution of these variants to the overall incidence of twinning is likely small (Hoekstra, 2008).

1	In animals, the litter size number grows in proportion to nutritional suiciency. Evidence from various sources indicates that this occurs in humans as well. Nylander (1971) showed an increasing gradient in the twinning rate related to greater nutritional status as relected by maternal size. Taller, heavier women had a twinning rate 25 to 30 percent greater than short, nutritionally deprived women. Likewise, Reddy and associates (2005) found an association of maternal weight and dizygotic twinning in the United States, in the absence of fertility drugs. Indeed, the influ ence of maternal weight s a factor for twinning will continue to rise in importance s the percentage of obese women in the United States continues to grow.

1	States continues to grow. Evidence acquired during and after World War II suggested that twinning correlated more with nutrition than with body size. Widespread undernourishment in Europe during those years was associated with a marked fall in the dizygotic twinning rate (Bulmer, 1959). Several investigators have reported a greater prevalence of twinning among women who have taken supplementary folic acid (Ericson, 2001; Haggarty, 2006). Conversely, in a systematic review, Muggli and Halliday (2007) were unable to demonstrate a significant association. Analysis of twinning rate in Texas after folic acid fortiication of cerealgrain products also failed to demonstrate an independent increase in twinning rates (Waller, 2003).

1	he common factor linking race, age, weight, and fertility to multifetal gestation may be FSH levels (Benirschke, 1973). This theory is supported by the fact that greater fecundity and a higher rate of dizygotic twinning have been reported in women who conceive within 1 month after stopping oral contraceptives, but not during subsequent months (Rothman, 1977). This may be due to the sudden release of pituitary gonadotropin in amounts greater than usual during the irst spontaneous cycle after stopping hormonal contraception. Indeed, the paradox of declining fertility but increasing twinning with advancing maternal age can be explained by an exaggerated pituitary release of FSH in response to decreased negative feedback from impending ovarian failure (Beemsterboer, 2006).

1	Ovulation induction with FSH plus human chorionic gonadotropin (hCG) or clomiphene citrate remarkably enhances the likelihood of multiple concurrent ovulations. In their review of this practice, McClamrock and coworkers (2012) reported rates of twins and higher-order multifetal pregnancies as high as 28.6 percent and 9.3 percent, respectively. Rates this high remain a major concern. Two ongoing multicenter trialsAssessment of Multiple Gestations from Ovarian Stimulation (AMIGOS) and Pregnancy in Polycystic Ovary Syndrome II (PPCOSII)-are designed to provide guidance on achieving maximum pregnancy rates while minimizing multifetal gestation rates (Diamond, 2015; Legro, 2014).

1	In general with in vitro fertilization (IVF), the greater the number of embryos that are transferred, the greater the risk of twins and other multifetal gestations. In 2014, ART contributed to 1.6 percent of all newborns in the United States and to 18.3 percent of all neonates in multifetal gestations (Sunderam, 2017). The American Society for Reproductive Medicine (2017) recently revised their age-related guidelines regarding the number of cleavage-stage embryos or blastocysts to transfer during IVF. This efort aims to reduce the incidence of higher-order multifetal pregnancies. Based on these new recommendations, women younger than 35 years are encouraged to receive a single-embryo transfer, regardless of embryo stage. hese practices have efectively lowered multifetal rates, and the rate of triplet or higher-order multifetal pregnancy has declined every year since 2009 (Kulkarni, 2013; Martin, 2017).

1	In humans, as the number of fetuses per pregnancy rises, the percentage of male conceptuses declines. Strandskov and coworkers (1946) found the percentage of males in 31 million singleton births in the United States was 51.6 percent. For twins, it was 50.9 percent; for triplets, 49.5 percent; and for quadruplets, 46.5 percent. Swedish birth data spanning 135 years reveals the number of males per 100 female newborns was 106 among singletons, 103 among twins, and 99 among triplets (Fellman, 2010). Females predominate even more in twins from late twinning events. For example, 68 percent of thoracopagus conjoined twins are female (Mutchinick, 2011). Two explanations have been ofered. First, beginning in utero and extending throughout the life cycle, mortality rates are lower in females. Second, female zygotes have a greater tendency to divide.

1	Twins of opposite sex are almost always dizygotic. In rare instances, due to somatic mutations or chromosome aberrations, the karyotype or phenotype of a monozygotic twin gestation can be diferent (Turpin, 1961). Most reported cases describe postzygotic loss of the Y chromosome in one 46,XY twin resulting in a phenotypically female twin with Turner syndrome (45,X). Zech and coworkers (2008) found a rare case of a 47,Y zygote that underwent postzygotic loss of the X chromosome in some cells and loss of the Y chromosome in other cells. The phenotype of the resultant twins was one male and one female. Karyotype analyses revealed both to be 46,XXJ46,XY genetic mosaics.

1	he risk for twin-speciic complications varies in relation to both zygosity and chorionicity-the number of chorions. Shown in Table 45-2, the latter is the more important determinant. Speciically, perinatal mortality and neurological injury rates are greater in monochorionic diamnionic twins compared with dichorionic diamnionic pairs (Hack, 2008; Lee, 2008). In one retrospective analysis of more than 2000 twins, the risk of fetal demise in one or both monochorionic twin(s) was twice that in dichorionic multifetal gestations (McPherson, 2012). Moreover, the prospective risk of antepartum stillbirth is higher for monochorionic than for dichorionic twins at all preterm gestational ages. he highest risk is before 28 weeks' gestation (Glinianaia, 2011). In contrast, chorionicity diferences do not signiicantly afect maternal outcomes (Carter, 2015).

1	This has become an integral tool to assist in multifetal pregnancy management. Indeed, the diagnosis and evaluation of a multifetal gestation is now considered a recognized indication for irst-trimester sonography (Reddy, 2014). In addition, the North American Fetal Therapy Network (NAFTNet)-a TABLE 45-2. Overview of the Incidence of Twin Pregnancy Zygosity and Corresponding Twin-Specific Complications Rates of Twin-Specific Complications in Percent consortium of 30 medical institutions in the United States and Canada-have provided recommendations for determination of chorionicity using sonography (Emery, 2015).

1	Sonographic features used to evaluate chorionicity vary according to gestational age. Accuracy is greatest in the irst trimester and diminishes as gestational age advances. Namely, chorionicity can be determined sonographically with 98-percent accuracy in the irst trimester but may be incorrect in up to 10 percent of second-trimester examinations (Emery, 2015; Lee, 2006). Moreover, for sonographic evaluations between 15 and 20 weeks' gestation, the odds of chorionicity misclassiication rise by approximately 10 percent for each week of advancing gestational age in pregnancies compared with those completed before 14 weeks (Blumenfeld, 2014). Overall, chorionicity can be correctly determined with sonography before 24 weeks in approximately 95 percent of cases (Lee, 2006).

1	Early in the first trimester, the number of chorions equates to the number of gestational sacs. A thick band of chorion separating two gestational sacs signals a dichorionic pregnancy, whereas monochorionic twins have a single gestational sac. If the gestation is monochorionic diamnionic, it may be diicult to visualize the thin intervening amnion before 8 weeks' gestation (Emery, 2015). If the intervening membrane is diicult to visualize, the number of yolk sacs usualy correlates with the number of amnions. However, the number of yolk sacs as a predictor of amnionicity may not always be accurate (Shen, 2006). Although uncommonly seen early, cord entanglement identifies a monoamnionic gestation. When chorionicity is uncertain, additional later sonographic examinations are performed.

1	Ater 10 to 14 weeks' gestation, sonographic assessment of chorionicity may be determined using four features. These are the number of placental masses, thickness of the membrane dividing the sacs, presence of an intervening membrane, and fetal gender (Emery, 2015). First, two separate placentas suggest dichorionicity. The converse is not necessarily true, such as cases with a single fused placental mass. Second, identiication of a thick dividing membrane-generally �2 mm-supports a presumed diagnosis of dichorionicity. In a dichorionic pregnancy, this visualized membrane is composed of a total of four layers-two amnion and two chorion. Also, the twin peak sign-also called lambda or delta sign-is seen by examining the point of origin of the dividing membrane on the placental surface. he peak appears as a triangular projection of placental tissue extending a short distance between the layers of the dividing membrane (Fig. 45-4).

1	FIGURE 45-4 A. Sonographic image of the "twin-peak" sign, also termed the "lambda sign," in a 24-week gestation. At the top of this sonogram, tissue from the anterior placenta is seen extending downward between the amnion layers. This sign confirms dichorionic twinning. B. The "tWin-peak" sign is seen at the bottom of this schematic diagram. The triangular portion of placenta insinuates between the amniochorion layers. FIGURE 45-5 A. Sonographic image of the '" sign in a monochorionic diamnionic gestation at 30 weeks. B. Schematic diagram of the '" sign. Twins are separated only by a membrane created by the juxtaposed amnion of each twin. A '" is formed at the point at which amnions meet the placenta.

1	In contrast, monochorionic pregnancies have a dividing membrane that is so thin (generally <2 mm) that it may not be seen until the second trimester. The relationship between the membranes and placenta without apparent extension of placenta between the dividing membranes is called the T sign (Fig. 45-5). Evaluation of the dividing membrane can establish chorionicity in more than 99 percent of pregnancies in the first trimester (Miller, 2012). Lack of a dividing membrane signals a monochorionic monoamnionic gestation. Last, twins with difering gender indicates a dichorionic (and dizygotic) gestation (Emery, 2015). A rare exception to this scenario would be a heterokaryotypic monochorionic gestation, described earlier (p. 867). If both twins are the same gender, additional measures are necessary.

1	A carefully performed visual examination of the placenta and membranes ater delivery serves to establish zygosity and chorionicity promptly in approximately two thirds of cases. The following systematic examination is recommended. As the irst neonate is delivered, one damp is placed on a portion of its FIGURE 45-6 Dichorionic diamnionic twin placenta. The membrane partition that separated twin fetuses is elevated and consists of chorion (c) between two amnions (0).

1	FIGURE 45-6 Dichorionic diamnionic twin placenta. The membrane partition that separated twin fetuses is elevated and consists of chorion (c) between two amnions (0). cord. Cord blood is generally not collected until after deliv ery of the other twin. As the second neonate is delivered, two damps are placed on that cord, and so on as necessary. lter natively, in higher-order deliveries, color-tagged damps can be simpler. Until the delivery of the last fetus, each cord segment must remain damped to prevent fetal hypovolemia and anemia caused by blood leaving the placenta via anastomoses and then through an undamped cord. At this time, evidence is insuicient in multifetal gestations (American College of Obstetricians and Gynecologists, 2017a). At Parkland Hospital, we currently do not perform delayed cord damping in these pregnancies.

1	Gynecologists, 2017a). At Parkland Hospital, we currently do not perform delayed cord damping in these pregnancies. The placenta is carefully delivered to preserve the attachment of the amnion and chorion. With one common amnionic sac or with juxtaposed amnions not separated by chorion, the fetuses are monozygotic (see Fig. 45-1). If adjacent amnions are separated by chorion, the fetuses could be either dizygotic or monozygotic, but dizygosity is more common (Fig. 45-6). If the neonates are of the same sex, blood typing of cord blood samples may be helpful. Diferent blood types conirm dizygosity, although demonstrating the same blood type in each fetus does not conirm monozygosity. For deinitive diagnosis, more complicated techniques such as DNA ingerprinting can be used. However, these tests are generally not performed at birth unless medical indications dictate a need.

1	During physical examination, accurate fundal height measurement, described in Chapter 9 (p. 164), is essential. With multifetal pregnancies, uterine size is typically larger during the second trimester than expected for a singleton. Rouse and associates (1993) reported fundal heights in 336 well-dated twin pregnancies. Between 20 and 30 weeks' gestation, fundal heights averaged approximately 5 cm greater than expected for singletons of the same fetal age. FIGURE 45-7 Sonograms of first-trimester twins. A. Dichorionic diamnionic twin pregnancy at 6 weeks' gestation. Note the thick dividing chorion (yellow arrow). One of the yolk sacs is indicated (blue arrow). B. Monochorionic diamnionic twin pregnancy at 8 weeks' gestation. Note the thin amnion encircling each embryo, resulting in a thin dividing membrane (blue arrow).

1	Diagnosing twins by palpation of fetal parts before the third trimester is diicult. Even late in pregnancy, this may be challenging, especially if one twin overlies the other, if the woman is obese, or if there is hydramnios. Palpating two fetal heads, often in diferent uterine quadrants, strongly supports a twin diagnosis. Late in the first trimester, two fetal heartbeats may be diferentiated with Doppler ultrasonic equipment if their rates are clearly distinct from each other and from that of the mother.

1	Overall, however, using clinical criteria alone to diagnose multifetal gestations is unreliable. For example, in the Routine Antenatal Diagnostic Imaging with Ultrasound (RADIUS) trial, for 37 percent of women who did not have a screening ultrasound examination, their twin pregnancies were not diagnosed until 26 weeks' gestation. And, in 13 percent of unscanned women, their multifetal gestations were only diagnosed during their admission for delivery (American College of Obstetricians and Gynecologists, 2016; LeFevre, 1993).

1	Sonographic examination should detect practically all sets of twins. And, given the increased frequency of sonographic examinations during the first trimester, early detection of a twin pregnancy is common. Sonography can also be used to determine fetal number, estimated gestational age, chorionicity, and amnionicity. With careful examination, separate gestational sacs, if present, can be identiied early in twin pregnancy (Fig. 45-7). Subsequently, each fetal head should be seen in two perpendicular planes so as not to mistake a cross section of the fetal trunk for a second fetal head. Ideally, two fetal heads or two abdomens should be seen in the same image plane to avoid scanning the same fetus twice and interpreting it as twins.

1	Higher-order multifetal gestations are more challenging to evaluate. Even in the irst trimester, it can be diicult to identiY the actual number of fetuses and their position. This determination is especially important if pregnancy reduction or selective termination is considered (p. 891). Abdominal radiography can be used if fetal number in a higherorder multifetal gestation is uncertain. However, radiographs generally have limited utility and may lead to an incorrect diagnosis if fetuses move during the exposure or if exposure time is inadequate. Additionally, fetal skeletons before 18 weeks' gestation are insuiciently radiopaque and may be poorly seen.

1	Although not typically used to diagnose multifetal pregnancy, magnetic resonance (MR) imaging may help delineate complications in monochorionic twins (Hu, 2006). In one review of 17 complicated twin gestations evaluated by both sonographic and MR imaging, the latter provided a more detailed assessment of twin pathology (Bekiesinska-Figatowska, 2013). This was particularly helpful in cases of conjoined twins. No biochemical test reliably identifies multifetal gestations. Serum and urine levels of 3-hCG and maternal serum levels of alpha-fetoprotein (MSAFP) are generally higher with twins compared with those in singletons. However, levels may vary considerably and overlap with those of singletons.

1	he various physiological burdens of pregnancy and the likelihood of serious maternal complications are typically greater with multifetal gestations than with a singleton pregnancy. his is considered, especially when counseling a woman whose health is compromised and in whom a multifetal gestation is recognized early. Similar consideration is given to the woman who is not pregnant but is considering infertility treatment.

1	Beginning in the first trimester, and temporarily associated with higher serum 3-hCG levels, women with a multifetal gestation oten have nausea and vomiting in excess of that with a singleton pregnancy. In women carrying more than one fetus, blood volume expansion is greater and averages 50 to 60 percent compared with 40 to 50 percent in those with a singleton (Pritchard, 1965). This augmented hypervolemia teleologically ofsets blood loss with vaginal delivery of twins, which is twice that with a single fetus. Although red cell mass also accrues, it does so proportionately less in twin pregnancies. Combined with greater iron and folate requirements, this predisposes to anemia.

1	Women carrying twins also have a typical pattern of arte rial blood pressure change. MacDonald-Wallis and coworkers (2012) analyzed serial blood pressures in more than 13,000 singleton and twin pregnancies. As early as 8 weeks' gestation, the diastolic blood pressure in women with twins was lower than that with singleton pregnancies but generally rose by a greater degree at term. An earlier study demonstrated that this rise was at least 15 mm Hg in 95 percent of women with twins compared with only 54 percent of women with a singleton (Campbell, 1986).

1	Hypervolemia along with decreased vascular resistance has an impressive efect on cardiac function. In one study of 119 women with a twin pregnancy, cardiac output rose another 20 percent above that in women with a singleton pregnancy (Kametas, 2003). Similarly, Kuleva and coworkers (201l) using serial echocardiography found a greater increase in cardiac output in 20 women with uncomplicated twin pregnancies. Both studies found the augmented cardiac output was predominantly due to greater stroke volume rather than higher heart rate. Vascular resistance was signiicantly lower in twin gestations throughout pregnancy compared with singleton ones. In a study of 30 uncomplicated twin pregnancies, this same group of investigators using echocardiography later identified progressive diastolic dysfunction from the irst to third trimester. The dysfunction subsequently normalized after delivery (Ghi, 2015).

1	Uterine growth in a multifetal gestation is substantively greater than in a singleton pregnancy. The uterus and its nonfetal contents may achieve a volume of 10 L or more and weigh in excess of 20 pounds. Especially with monozygotic twins, excessive amounts of amnionic luid may rapidly accumulate. In these circumstances, maternal abdominal viscera and lungs can be appreciably compressed and displaced by the expanding uterus. As a result, the size and weight of the large uterus may preclude more than a sedentary existence for these women.

1	Ifhydramnios develops, maternal renal function can become seriously impaired, most likely as the consequence of obstructive uropathy (Quigley, 1977). With severe hydramnios, therapeutic amniocentesis may provide relief for the mother, may improve obstructive uropathy, and possibly may lower the preterm delivery risk that follows preterm labor or prematurely ruptured membranes. Unfortunately, hydramnios is often characterized by acute onset remote from term and by rapid reaccumulation despite amniocentesis. Miscarriage is more likely with multifetal gestation. In one 16-year study, the spontaneous abortion rate per live birth in singleton pregnancies was 0.9 percent compared with 7.3 percent in multifetal ones (Joo, 2012). Also, twins achieved through ART are at greater risk for abortion compared with those conceived spontaneously (Szymusik, 2012).

1	In some cases, one fetus may be spontaneously lost rather than the entire gestation. As a result, the incidence of twins in the irst trimester is much greater than the incidence of twins at birth. It has been estimated that 1 in 80 births are multifetal, whereas 1 in 8 pregnancies begin multifetal but are spontane ously reduced (Corsello, 2010). Sonography studies in the first trimester have shown that one twin is spontaneously reduced or "vanishes" before the second trimester in up to 10 to 40 percent of all twin pregnancies (Brady, 20l3). The incidence is higher following ART conception. Also, monochorionic twins have a significantly greater risk of spontaneous reduction than dicho rionic twins (Sperling, 2006). Undoubtedly, some threatened abortions are the result of death and resorption of one embryo from an unrecognized twin gestation.

1	tion in 709 multifetal pregnancies. Before 12 weeks, one or more embryos died in 36 percent of twin pregnancies, in 53 percent of triplet pregnancies, and in 65 percent of quadru plet pregnancies. Interestingly, ultimate pregnancy duration and birthweight were inversely related to the initial gestational sac number regardless of the final number of fetuses at deliv ery. This efect was most pronounced in twins who started as quadruplets. Chasen and coworkers (2006) reported that spon taneous reduction of an IVF twin pregnancy to a singleton pregnancy was associated with perinatal outcomes intermediate did not undergo spontaneous reduction. Evidence for adverse immediate and long-term efects of twin spontaneous reduction on the remaining pregnancy is conflicting (MeN amara, 2016).

1	Notably, spontaneous reduction of a twin gestation may afect prenatal screening results. In one study of ART-conceived gestations, Gjerris and colleagues (2009) compared 56 twin pregnancies with a single early demise and 897 singleton gestations. They found no diferences in irst-trimester serum marker concentrations as long as the embryonic loss was identiied before 9 weeks' gestation. If diagnosed ater 9 weeks, the serum markers were higher and less precise in gestations with an early demise of one twin than in the singleton gestations. With a vanishing twin, first-trimester maternal serum levels of the pregnancy associated plasma protein-A (PAPP-A) can be elevated. Second-trimester MSAFP and dimeric inhibin A levels can also be higher (Huang, 2015). his phenomenon may also afect noninvasive prenatal testing using cell-free DNA (cDNA). In one report, this efect was thought to be responsible for 15 percent of the false-positive results from quantitative counting methods (Futch, 20l3).

1	prenatal testing using cell-free DNA (cDNA). In one report, this efect was thought to be responsible for 15 percent of the false-positive results from quantitative counting methods (Futch, 20l3). The recent development of single nucleotide polymorphism technology for cDNA testing appears to hold promise in better identiYing these cases (Curnow, 2015). Regardless, the diagnosis of a spontaneously reduced abortus is ideally excluded to help avoid confusion with results from aneuploidy and neural-tube defect screening.

1	As noted earlier, the incidence of congenital malformations is appreciably higher in multifetal gestations compared with that in singleton pregnancies. In one survey-based study, the congenital malformation rate was 406 per 10,000 twins compared with 238 per 10,000 singletons (Glinianaia, 2008). The malformation rate in monochorionic twins was almost twice that of dichorionic twin gestations. This increase has been attributed to the higher incidence of structural defects in monozygotic twins. Indeed, one large population-based study between 1998 and 2010 found that twins had a 73-percent greater risk of congenital heart disease than singletons. The risk was substantially higher among monochorionic twins (Best, 2015). But, from a 30-year European registry of multifetal births, structural anomaly rates rose steadily from 2.16 percent in 1987 to 3.26 percent in 2007 (Boyle, 2013). Yet, during this time, the proportion of dizygotic twins grew by 30 percent, whereas the proportion of

1	anomaly rates rose steadily from 2.16 percent in 1987 to 3.26 percent in 2007 (Boyle, 2013). Yet, during this time, the proportion of dizygotic twins grew by 30 percent, whereas the proportion of monozygotic twins remained stable. his higher risk of congenital malformations in dizygotic twins over time correlated with increased availability of AR. An increase in rates of birth defects related to ART has been reported repeatedly (Boulet, 2016; Talauliker, 2012).

1	Multifetal gestations are more likely to be low birthweight than singleton pregnancies due to restricted fetal growth and preterm delivery. From 1988 to 2012 at Parkland Hospital, data were collected from 357,205 singleton neonates without malformations and from 3714 normal twins who were both liveborn. Birthweights in twins closely paralleled those of singletons until 28 to 30 weeks' gestation. Thereafter, twin birthweights progressively lagged (Fig. 45-8). Beginning at 35 to 36 weeks' gestation, twin birthweights clearly diverge from those of singletons.

1	In general, the degree of growth restriction increases with fetal number. The caveat is that this assessment is based on growth curves established for singletons. Several authorities argue that fetal growth in twins is diferent from that of singleton pregnancies. And thus, abnormal growth should be diagnosed only when fetal size is less than expected for multietal gestation. Accordingly, twin and triplet growth curves have been developed (Kim, 2010; Odibo, 2013; Vora, 2006). At Parkland, we use the standards of birthweight in twin gestations stratified by placental chorionicity for identification of suspected fetal-growth restriction (Ananth, 1998). FIGURE 45-8 Birthweight percentiles (25th to 75th) for 357,205 singleton neonates compared with the 50th birthweight percentile for 3714 twins, Parkland Hospital 1988-2012. Infants with major

1	FIGURE 45-8 Birthweight percentiles (25th to 75th) for 357,205 singleton neonates compared with the 50th birthweight percentile for 3714 twins, Parkland Hospital 1988-2012. Infants with major FIGURE 45-9 Marked growth discordance in monochorionic twins. (Used with permission from Dr. Laura Greer.) he degree of growth restriction in monozygotic twins is likely to be greater than that in dizygotic pairs (Fig. 45-9). With monochorionic embryos, allocation of blastomeres may not be equal, vascular anastomoses within the placenta may cause unequal distribution of nutrients and oxygen, and discordant structural anomalies resulting from the twinning event itself may afect growth. For example, the quintuplets shown in Figure 45-10 represent three dizygotic and two monozygotic fetuses. When delivered at 31 weeks, the three neonates from separate ova weighed 1420, 1530, and 1440 g, whereas the two derived from the same ovum weighed 990 and 860 g.

1	In the third trimester, the larger fetal mass leads to accelerated placental maturation and relative placental insuiciency. In dizygotic pregnancies, marked size discordancy usually results from unequal placentation, with one placental site receiving more perfusion than the other. Size diferences may also reflect diferent genetic fetal-growth potentials. Discordancy can also result from fetal malformations, genetic syndromes, infection, or umbilical cord abnormalities such as velamentous insertion ' marginal insertion, or vasa previa (Chap. 44, p. 849).

1	Pregnancy-related hypertensive disorders are more likely to develop with multifetal gestations. The exact incidence attributable to twin pregnancy is diicult to determine because these gestations are more likely to deliver preterm and before preeclampsia usually develops. Also, women with twin pregnancies are often older and multiparous, qualities associated with lower rates of preeclampsia (Francisco, 2017). The incidence of pregnancy-related hypertension in women with twins is 20 percent at Parkland Hospital. In their analysis of 513 twin malformations, pregnancies complicated by stillbirth, and twin gestations with >25 percent discordance were also excluded. (Data from Dr. Don Mcintire.) pregnancies, Fox and coworkers (2014) identified 15 percent of parturients with preeclampsia. Another study compared 257 women with twins and gestational diabetes against 277 nondiabetic women carrying twins. hese researchers found a twofold greater risk of preeclampsia in women diagnosed with

1	study compared 257 women with twins and gestational diabetes against 277 nondiabetic women carrying twins. hese researchers found a twofold greater risk of preeclampsia in women diagnosed with gestational diabetes (Gonzalez, 2012). Conversely, no specific zygosity confers a greater rate of hypertensive disorder in twin pregnancies (Lucovnik, 2016). Finally, from the National Center for Health Statistics, Luke and associates (2008) analyzed 316,696 twin, 12,193 triplet, and 778 quadruplet pregnancies. hese investigators noted that the risk for pregnancy-associated hypertension was significantly increased for triplets and quadruplets (11 and 12 percent, respectively) compared with that for twins (8 percent).

1	FIGURE 45-10 Davis quintuplets at 3 weeks following delivery. The first, second, and fourth newborns from the left each arose from separate ova, whereas the third and fifth neonates are from the same ovum.

1	hese data suggest that fetal number and placental mass are involved in preeclampsia pathogenesis. Women with twin pregnancies have levels of antiangiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) that are twice that of singletons. Levels are seemingly related to greater placental mass rather than primary placental pathology (Bdolah, 2008; Maynard, 2008). Rana and coworkers (2012) measured antiangiogenic sFlt-1 and proangiogenic placental growth factor (PIGF) in 79 women with twins referred for evaluation of preeclampsia. In the 58 women identiied with either gestational hypertension or preeclampsia, there was an incremental rise in sFlt-1 concentrations, decline in PIGF levels, and increase in sFlt-lIPIGF ratios compared with normotensive twin pregnancies. With multifetal gestation, hypertension not only develops more often but also tends to develop earlier and be more severe. In the analysis of angiogenic factors mentioned above, more than half of afected women presented before

1	hypertension not only develops more often but also tends to develop earlier and be more severe. In the analysis of angiogenic factors mentioned above, more than half of afected women presented before 34 weeks, and their sFlt-lIPIGF ratio rise was more striking (Rana, 2012). This relationship is discussed in Chapter 40 (p. 716).

1	The duration of gestation shortens with accruing fetal number. More than five of every 10 twins and nine of 10 triplets born in the United States in 2015 were delivered preterm (Martin, 2017). Prematurity is sixfold and tenfold greater in twins and triplets, respectively (Giufre, 2012). One review showed that approximately 60 percent of preterm births in twins are indicated, about a third result from spontaneous labor, and 10 percent follow prematurely ruptured membranes (Chauhan, 2010). In another analysis of almost 300,000 live births, the proportion of preterm birth associated with premature membrane rupture rose with gestational plurality from 13 percent with singletons to 20 percent with triplets or more (Pakrashi, 2013).

1	Although the causes of preterm delivery in twins and singletons may be diferent, neonatal outcome is generally the same at similar gestational ages (Kilpatrick, 1996; Ray, 2009; Salem, 2017). However, outcomes for pre term twins who are markedly discordant may not be comparable with those for singletons because whatever caused the discordance may have long-lasting efects (Yinon, 2005) . Historically, twins have been considered cognitively delayed compared with singletons (Record, 1970; Ronalds, 2005). However, in cohort studies evaluating normal-birthweight term newborns, cognitive outcomes between twins and singletons are similar (Lorenz, 2012). Christensen and associates (2006) found similar national standardized test scores in the ninth grade in 341r1 twins and 7796 singletons born between 1986 and 1988.

1	In contrast, among normal-birthweight neonates, the cerebral palsy risk is higher among twins and higher-order multiples. For example, the cerebral palsy rate has been reported to be 2.3 per 1000 in singletons, 12.6 per 1000 in twins, and 44.8 per 1000 in triplets (Giufre, 2012). Greater risks of fetalgrowth restriction, congenital anomalies, twin-twin transfusion syndrome, and fetal demise of a cotwin are suggested contributors to these diferences (Lorenz, 2012). Several unique complications arise in multifetal pregnancies. These are described in twins but can be found in higher-order multifetal gestations. Most fetal complications due to the twinning process itself are seen with monozygotic twins. heir pathogenesis is best understood after reviewing the possibilities shown in Figure 45-1.

1	Only about 1 percent of all monozygotic twin gestations will share an amnionic sac, and approximately 1 in 20 monochorionic twin gestations are monoamnionic (Hall, 2003; Lewi, 2013). Diamnionic twins can become monoamnionic if the dividing membrane spontaneous or iatrogenically ruptures. Their morbidity and mortality rates then mirror those of monoamnionic twins.

1	Historical mortali ty rates in monoamnionic twins were reported to be as high as 70 percent. Contemporary outcomes are improved, yet the demise rate after viability remains elevated (Post, 2015). Of those fetuses alive before 16 weeks' gestation, less than half survive until the neonatal period. Fetal abnormalities and spontaneous miscarriage contribute to most losses (Prefumo, 2015). After 20 weeks, the perinatal mortality rate for monoamnionic twin pregnancies approximates 15 percent (Shub, 2015). A high fetal death rate is attributable to preterm birth, congenital anomalies, twin-twin transfusion syndrome, or cord entanglement.

1	Congenital anomaly rates in monoamnionic twins reach 18 to 28 percent (Post, 2015). Since concordance of anomalies is found in only approximately one quarter of cases, the finding of normal anatomy in one twin does not negate the need for a thorough evaluation in the second. Also, because of the higher risk of cardiac anomalies, fetal echo cardiography is indicated in these pregnancies. Of note, monoamnionic twins are by definition monozygotic and thus presumed to be genetically identical. Consequently, either both or none of the fetuses have chromosomal abnormalities except in rare cases of discordance (Zwijnenburg, 2010). Indeed, the risk for Down syndrome in each fetus of the monozygotic pair is similar to or lower than the risk in maternal age-matched singletons (Sparks, 2016). he standard methods for Down syndrome screening in these pregnancies can be applied (Chap. 14, p. 281).

1	The rate of twin-twin transfusion syndrome in monoamnionic twins is lower than the rate reported in monochorionic diamnionic pregnancies. This may be due to the near universal presence in monoamnionic twins of arterioarterial anastomoses, which are presumed to be protective (Hack, 2009b; Post, 2015). Nonetheless, twin-twin transfusion syndrome surveillance is recommended and described on page 879. Umbilical cords frequently entangle (Fig.

1	Umbilical cords frequently entangle (Fig. lorbid cord entanglement appears to occur early, and monoamnionic pregnancies that have successfully reached 30 to 32 weeks' gestation are at reduced risk. In one Dutch series, the incidence of intrauterine demise dropped from 15 percent after 20 weeks to 4 percent at gestational ages >32 weeks (Hack, 2009a). Although color-low Doppler sonography is used to diagnose entanglement (Fig. 45-12), factors that lead to pathological umbilical vessel constnctton are unknown. A consequence is that fetal death from cord entanglement is unpredictable. Unfortunately, monitoring for this is relatively inefective. In one study, after analysis of more than 10,000 hours of fetal tracing from 17 sets of monoamnionic twins, Quinn and colleagues (2011) concluded that monitoring was physically FIGURE 45-1 1 Monozygotic twins in a single amnionic sac. The smaller fetus apparently died first, and the second subsequently succumbed when umbilical cords entwined.

1	FIGURE 45-1 1 Monozygotic twins in a single amnionic sac. The smaller fetus apparently died first, and the second subsequently succumbed when umbilical cords entwined. FIGURE 45-12 Monochorionic monoamnionic cord entanglement. A. Despite marked knotting of the cords, vigorous twins were delivered by cesarean. B. Preoperative sonogram of this pregnancy shows entwined cords. C. This finding is accentuated with application of color Doppler. (Used with permission from Dr. Julie Lo.) FIGURE 45-13 Possible outcomes of monozygotic twinning. The asymmetrical category contains twinning types in which one twin complement is substantially smaller and incompletely formed. possible in only 50 percent of cases. An abnormal fetal heart rate tracing prompted delivery in only six cases.

1	One proposed management scheme is based on a study by Heyborne and coworkers (2005), who reported no stillbirths in 43 twin pregnancies of women admitted at 26 to 27 weeks' gestation for daily fetal surveillance. However, in 44 women managed as outpatients and admitted only for obstetrical indications, there were 13 stillbirths. Because of this report, women with monoamnionic twins are recommended to undergo 1 hour of daily fetal heart rate monitoring, either as an outpatient or inpatient, beginning at 26 to 28 weeks' gestation. With initial testing, a course of betamethasone is given to promote pulmonary maturation (Chap. 42, p. 823). If fetal testing remains reassuring and no other intervening indications arise, cesarean delivery is performed at 32 to 34 weeks. A second course of betamethasone can be given before this (American College of Obstetricians and Gynecologists, 2016). his management scheme is used at Parkland Hospital and resulted in the successful 34-week delivery of the

1	can be given before this (American College of Obstetricians and Gynecologists, 2016). his management scheme is used at Parkland Hospital and resulted in the successful 34-week delivery of the twins depicted in Figure 45-12.

1	Of monoamnionic twins just described, one interesting subset derives from embryonic splitting on postfertilization day 9. These "mirror image twins" are genetically identical but have mirror image features such as handedness and hair whorls (Post, 2015). More seriously, several aberrations in monozygotic twinning result in a spectrum of fetal malformations. hese are traditionally ascribed to incomplete splitting of an embryo into two separate twins. However, it is possible that they may result from early secondary fusion of two separate embryos. These separated embryos are either symmetrical or asymmetrical, and the spectrum of anomalies is shown in Figure 45-13.

1	In the United States, united or conjoined twins have been referred to as Siamese twins-after Chang and Eng Bunker of Siam (Thailand), who were displayed worldwide by P. T. Barnum. Joining of the twins may begin at either pole and produce characteristic forms depending on which body parts are joined or shared (Fig. 45-14). Of these, thoracopagus is the most common (Mutchinick, 2011). he frequency of conjoined twins is not well established. In Singapore, Tan and coworkers (1971) identified seven cases of conjoined twins among more than 400,000 deliveries-an incidence of 1 in 60,000.

1	Conjoined twins can frequently be identiied using sonography at midpregnancy (McHugh, 2006). his provides an opportunity for parents to decide whether to continue the pregnancy. As shown in Figure 45-15, identification of cases during the first trimester is also possible. During sonographic interrogation, fetal poles are closely associated and do not change relative position from one another. A targeted examination, including a careful evaluation of the organs involved, is necessary before counseling can be provided. As shown in Figure 45-16, MR imaging is a valuable adjunct to clariY shared organs. Compared with sonography, MR imaging can provide superior views, especially in later pregnancy when amnionic luid is diminished and fetal crowding is greater (Hibbeln, 2012).

1	Surgical separation of an almost completely joined twin pair may be successful if essential organs are not shared (O'Brien, 2015; Tannuri, 20l3). Conjoined twins may have discordant structural anomalies that further complicate decisions about whether to continue the pregnancy. Consultation with a pediatric surgeon often assists parental decision making. A recent FIGURE 45-14 Types of conjoined twins. (Modified with permission from Spencer R: Theoretical and analytical embryology of conjoined . Parapagus Parapagus twins: part I: embryogenesis, (lin Anat. 2000;13(1 ):36-53.) series in Seminars in Pediatric Surgey with a preface by Spitz (2015) provide an excellent reference regarding postnatal management.

1	Viable conjoined twins should be delivered by cesarean. For the purpose of pregnancy termination, however, vaginal delivery is possible because the union is most often pliable (Fig. 45-17). Still, dystocia is common, and if the fetuses are mature, vaginal delivery may be traumatic to the uterus or cervix. his is a grossly defective fetus or merely fetal parts, attached externally to a relatively normal twin. A parasitic twin usually consists of externally attached supernumerary limbs, often with some viscera. Classically, however, a functional heart or brain is absent. Attachment mirrors those sites described earlier for conjoined twins (see Fig. 45-14). Parasites are believed to result from demise of the defective twin. Its surviving tissue attaches to and receives vascularity from the normal cotwin (Spencer, 2001). In one large epidemiological study, parasitic twins accounted for 4 percent of all conjoined twins and occurred more frequently in male fetuses (Mutchinick, 2011).

1	FIGURE 45-15 Sonogram of a conjoined twin pregnancy at 13 weeks' gestation. These thoracoomphalopagus twins have two heads but a shared chest and abdomen. Early in development, one embryo may be enfolded within its twin. Normal development of this rare parasitic twin usually arrests in the first trimester. As a result, norml spatial arrangement of and presence of many organs is lost. Classically, vertebral or xial bones are found in the fetiform mass, whereas a heart and brain are absent. hese masses are believed to represent a monozygotic, monochorionic diamnionic twin gestation and are typically supported by large parasitic vessels to the host (McNamara, 2016; Spencer, 2000). Malignant degeneration is rare (Kaufman, 2007). All monochorionic placentas likely share some anastomotic connections. And, with rare exceptions, anastomoses between

1	All monochorionic placentas likely share some anastomotic connections. And, with rare exceptions, anastomoses between FIGURE 45-16 Magnetic resonance imaging of conjoined twins. This T2-weighted HASTE sagittal image demonstrates fusion from the level of the xiphoid process to just below the level of the umbilicus, that is, omphalopagus twins. Below the fused liver (L)/ there is a midline cystic mass (arrow) within the tissue connecting the twins. An omphalomesenteric cyst was favored given the location within the shared tissue. (Used with permission from Dr. April Bailey.)

1	Dr. April Bailey.) FIGURE 45-17 Conjoined twins aborted at 17 weeks' gestation. (Used with permission from Dr. Jonathan Willms.) twins are unique to monochorionic twin placentas. However, the number, size, and direction of these seemingly haphazard connections vary markedly (Fig. 45-18). In one analysis of more than 200 monochorionic placentas, the median number of anastomoses was 8, with an interquartile range of 4 to 14 (Zhao, 2013).

1	Artery-to-artery anastomoses are most frequent and are identiied on the chorionic surface of the placenta in up to 75 percent of monochorionic twin placentas. Vein-to-vein and artery-to-vein communications are each found in approximately half. One vessel may have several connections, sometimes to both arteries and veins. In contrast to these supericial vascular connections on the surface of the chorion, deep arteryto-vein communications can extend through the capillary bed of a given villus (Fig. 45-19). hese deep arteriovenous anastomoses create a common villous compartment or "third circulation" that has been identiied in approximately half of monochorionic twin placentas.

1	FIGURE 45-19 Anastomoses between twins may be artery-tovein (AV), artery-to-artery (AA), or vein-to-vein (W). Schematic representation of an AV anastomosis in twin-twin transfusion syndrome that forms a "common villous district" or "third circulation" deep within the villous tissue. Blood from a donor twin may be transferred to a recipient twin through this shared circulation. This transfer leads to a growth-restricted discordant donor twin with markedly reduced amnionic fluid, causing it to be "stuck." Whether these anastomoses are dangerous to either twin depends on the degree to which they are hemodynamically balanced. In those with signiicant pressure or low gradients, a shunt will develop between fetuses. his chronic fetofetal transfusion may result in several clinical syndromes that include

1	FIGURE 45-18 Shared placenta from pregnancy complicated by twin-twin transfusion syndrome. The following color code was applied for injection. Left twin: yellow = artery, blue = vein; right twin: red = artery, green = vein. A. Part of the arterial network of the right twin is filled with yellow dye, due to the presence of a small artery-to-artery anastomosis (arrow). B. Close-up of the lower portion of the placenta displays the yellow dye-filled anastomosis. (Reproduced with permission from De Paepe ME, DeKoninck P, Friedman RM: Vascular distribution patterns inmonochorionic twin placentas, Placenta. 2005 Jul;26(6):471-475.) FIGURE 45-20 These serial sonograms depict an interventricular hemorrhage with parenchymal extension and eventual porencephaly that developed following cotwin demise in a monochorionic pregnancy. From left to right, these images were obtained 1 week,o5 weeks, and 8 weeks following demise of the cotwin.

1	twin-twin transfusion syndrome (TTTS), twin anemia poycythemia sequence (TAPS), and acardiac twinning. In this syndrome, blood is transfused from a donor twin to its recipient sibling such that the donor may eventually become anemic and its growth may be restricted. In contrast, the recipient becomes polycythemic and may develop circulatory overload manifest as hydrops. Classically, the donor twin is pale, and its recipient sibling is plethoric. Similarly, one portion of the placenta often appears pale compared with the remainder. The recipient neonate may also have circulatory overload from heart failure and severe hypervolemia and hyperviscosity. Occlusive thrombosis is another concern. Finally, polycythemia in the recipient twin may lead to severe hyperbilirubinemia and kernicterus (Chap. 33, p. 626). he prevalence of TTTS approximates 1 to 3 cases per 10,000 births (Society for Maternal-Fetal Medicine, 2013).

1	Chronic TTTS results from unidirectional low through deep arteriovenous anastomoses. Deoxygenated blood from a donor placental artery is pumped into a cotyledon shared by the recipient (see Fig. 45-19). Once oxygen exchange is completed in the chorionic villus, the oxygenated blood leaves the cotyledon via a placental vein of the recipient twin. Unless compensatedtypically through superficial arterioarterial anastomoses-this unidirectional low leads to an imbalance in blood volumes (Lewi, 2013). Clinically important TTTS frequently is chronic and results from significant vascular volume diferences between the twins. Even so, the pathogenesis is more complex than a net transfer of red blood cells from one twin to another. Indeed, in most monochorionic twin pregnancies with the syndrome, hemoglobin concentrations between the donor and recipient twin do not difer (Lewi, 2013).

1	TTTS typically presents in midpregnancy when the donor fetus becomes oliguric from decreased renal perfusion (Society for Maternal-Fetal Medicine, 2013). This fetus develops oligohydramnios, and the recipient fetus develops severe hydramnios, presumably due to increased urine production. Virtual absence of amnionic fluid in the donor sac prevents fetal motion, giving rise to the descriptive term stuck twin or poyhydramnios-oligohydramnios syndrome-'poy-oli. " This amnionic fluid imbalance is associated with growth restriction, contractures, and pulmonary hypoplasia in the donor twin, and premature rupture of the membranes and heart failure in the recipient.

1	Fetal Brain Damage. Cerebral palsy, microcephaly, porencephaly, and multicystic encephalomalacia are serious complications associated with placental vascular anastomoses in multifetal gestation. he exact pathogenesis of neurological damage is not fully understood but is likely caused by ischemic necrosis leading to cavitary brain lesions (Fig. 45-20). In the donor twin, ischemia results from hypotension, anemia, or both. In the recipient, ischemia develops from blood pressure instability and episodes of profound hypotension (Lopriore, 2011). Cerebral lesions may also be due to postnatal injury associated with preterm delivery (Chap. 34, p. 639). In one review of315 liveborn fetuses from pregnancies with TTTS, cerebral abnormalities were found in 8 percent (Quarello, 2007).

1	If one twin of an afected pregnancy dies, cerebrl pathology in the survivor probably results from acute hypotension. A less likely cause is emboli of thromboplastic material originating from the dead fetus. Fusi and coworkers (1990, 1991) observed that with the death of one twin, acute twin-twin anastomotic transusion from the high-pressure vessels of the living twin to the lowresistance vessels of the dead twin leads rapidly to hypovolemia and ischemic antenatal brain damage in the survivor. In one review of 343 twin pregnancies complicated by single fetal demise, the risk of neurodevelopmental morbidity in monochorionic twins was 26 percent compared with 2 percent in dichorionic twins (Hillman, 2011). This morbidity was related to the gestational age at the death of the cotwin. If the death occurred between 28 and 33 weeks' gestation, monochorionic twins had an almost eightfold risk of neurodevelopmental morbidity compared with dichorionic twins of the same gestational age. With

1	death occurred between 28 and 33 weeks' gestation, monochorionic twins had an almost eightfold risk of neurodevelopmental morbidity compared with dichorionic twins of the same gestational age. With fetal death ater 34 weeks, the likelihood dramaticlly decreased-odds ratio 1.48.

1	he acuity of hypotension following the death of one twin with TTTS makes successful intervention for the survivor nearly impossible. Even with delivery immediately ater a cotwin demise is recognized, the hypotension that occurs at the moment of death has likely already caused irreversible brain damage (Langer, 1997; Wada, 1998). s such, immediate delivery is not considered beneficial in the absence of another indication.

1	Diagnosis. he criteria used to diagnose and classiy varying severities of TTTS have dramatically changed. Previously, weight discordancy and hemoglobin diferences in monochorionic twins were calculated. However, in many cases, these are late findings. According to the Society for Maternal-Fetal Medicine (2013), TTTS is diagnosed based on two so nographic criteria. First, a monochorionic diamnionic pregnancy is identified. Second, hydramnios defined by a largest vertical pocket > 8 em in one sac and oligohydramnios defined by a largest vertical pocket <2 em in the other twin is found. Only 15 percent of pregnancies complicated by lesser degrees of luid imbalance progress to TTTS (Huber, 2006). Although growth discordance or growth restriction may be found with TTTS, these per se are not considered diagnostic criteria.

1	Organizations that include the American College of Obstetricians and Gynecologists (2016), Society for Maternal-Fetal Medicine (2013), and North American Fetal herapy Network (Emery, 2015) recommend sonography surveillance of pregnancies at risk for TTTS. To aid earlier identification of amnionic luid abnormalities and other complications of monochorionic twins, these examinations begin at approximately 16 weeks' gestation, and subsequent studies are considered every 2 weeks. Once identiied, TTTS is typically classiied by the Quintero (1999) staging system (Fig. 45-21): Stage I-discordant amnionic fluid volumes as described in the earlier paragraph, but urine is still visible sonographically within the bladder of the donor twin Stage II-criteria of stage I, but urine is not visible within the donor bladder Stage III-criteria of stage II and abnormal Doppler studies of the umbilical artery, ductus venosus, or umbilical vein Stage V-demise of either fetus.

1	Stage III-criteria of stage II and abnormal Doppler studies of the umbilical artery, ductus venosus, or umbilical vein Stage V-demise of either fetus. In addition to these criteria, evidence suggests that cardiac function of the recipient twin correlates with fetal outcome (Crombleholme, 2007). Although fetal echocardiographic indings are not part of the Quintero staging system, many centers routinely perform fetal echocardiography for TTTS. heoretically, earlier diagnosis of cardiomyopathy in the recipient twin may identiy pregnancies that would beneit from early intervention. One system for evaluating cardiac function-the myocardial peormance index (MPI) or Tei index-is a Doppler index of ventricular function calculated for each ventricle (Michelfelder, 2007). lthough scoring systems that include assessment of cardiac function have been developed, their usefulness to predict outcomes remains controversial (Society for Maternal-Fetal Medicine, 2013).

1	Management and Prognosis. he prognosis for mulrifetal gestations complicated by TTTS is related to Quintero stage and gestational age at presentation. More than three fourths of stage I cases have been reported to remain stable or regress without intervention. Conversely, outcomes in those identiied at stage III or higher are much worse, and the perinatal loss rate is 70 to 100 percent without intervention (Society for Maternal-Fetal Medicine, 2013). At Parkland Hospital, among expectantly managed pregnancies with TTTS, most had early disease at diagnosis, and 50 percent of stage I cases progressed (Duryea, 2016).

1	Several therapies are available for TTTS and include amnioreduction, laser ablation of vascular placental anastomoses, selective feticide, and septosromy. Described further in Chapter 11 (p. 230), amnioreduction describes needle drainage of excess amnionic fluid. Septostomy is intentionally creating a hole in the dividing amnionic membrane but has largely been abandoned as treatment (Society for Maternal-Fetal Medicine, 2013). Comparative data from randomized trials for some of these other techniques are discussed below.

1	The Eurofetus trial included 142 women with severe TTTS diagnosed before 26 weeks. Participants were randomly assigned to laser ablation of vascular anastomoses or to serial amnioreduction (Senat, 2004). A higher survival rate to age 6 months for at least one twin was found in pregnancies undergoing laser ablation-76 versus 51 percent, respectively. Moreover, analyses of randomized studies confirm better neonatal outcomes with laser therapy compared with selective amnioreduction (Roberts, 2008; Rossi, 2008, 2009). In contrast, Crombleholme and associates (2007), in a randomized trial of 42 women, found equivalent rates of 30-day survival of one or both twins treated with either amnioreduction or selective fetoscopic laser ablation-75 versus 65 percent, respectively. Furthermore, evaluation of twins from the Eurofetus trial through 6 years of age did not demonstrate an additional survival benefit beyond 6 months or improved neurological outcomes in those treated with laser (Salomon,

1	of twins from the Eurofetus trial through 6 years of age did not demonstrate an additional survival benefit beyond 6 months or improved neurological outcomes in those treated with laser (Salomon, 2010). At this time, laser ablation of anastomoses is preferred for severe TTTS (stages II-IV). Optimal therapy for stage I disease is controversial.

1	FIGURE 45-21 A. Sonogram of stage I ms at 19 weeks' gestation. Oligohydramnios in the donor twin sac causes the membrane to essentially wrap around the "stuck twin" and suspend it from the anterior uterine wall. B. In this same pregnancy, hydramnios is seen in the recipient twin sac. The measured pocket exceeds 10 cm. C. Stage II ms in a donor twin at 17 weeks' gestation. Color Doppler highlights the arteries that outline the fetal bladder, which contains no urine. After laser therapy, close ongoing surveillance is necessary.

1	After laser therapy, close ongoing surveillance is necessary. Robyr and colleagues (2006) reported that a fourth of 101 pregnancies treated with laser required additional invasive therapy because of either recurrent TTTS, or middle cerebral artery (MCA) Doppler evidence of anemia or polycythemia. Recently, in a comparison of selective laser ablation of individual anastomoses versus ablation of the entire surface of the chorionic plate along the vascular equator, Baschat and coworkers (2013) found that equatorial photocoagulation reduced the likelihood of recurrence.

1	Selective fetal reduction has generally been considered if severe amnionic luid and growth disturbances develop before 20 weeks. In such cases, both fetuses typically will die without intervention. Any substance injected into one twin may afect the other twin because of shared circulations. hus, for the fetus chosen for reduction, feticidal techniques include methods that occlude the umbilical vein or umbilical cord of using radiofrequency ablation, fetoscopic ligation, or coagulation with laser, monopolar, or bipolar energy (Challis, 1999; Chang, 2009; Parra-Cordero, 2016). Even after these procedures, however, the risks to the remaining fetus are still appreciable (Rossi, 2009). This topic is further discussed on page 891.

1	This form of chronic fetofetal transfusion, referred to as TAPS, is characterized by significant hemoglobin diferences between donor and recipient twins. However, TAPS lacks the discrepancies in amnionic luid volumes typical of TTTS (Slaghekke, 2010). It is diagnosed ante natally by M CA peak systolic velocity (PSV) > 1.5 multiples of the median (MoM) in the donor and < 1.0 MoM in the recipient twin (Society for MaternalFetal Medicine, 2013). The spontaneous form of TAPS reportedly complicates 3 to 5 percent of monochorionic pregnancies, and it occurs in up to 13 percent of pregnancies after laser photocoagulation of the placenta. Spontaneous TAPS usually occurs after 26 weeks' gestation, and iatrogenic TAPS develops within 5 weeks of a procedure (Lewi, 2013). Although a staging system has been proposed by Slaghekke and colleagues (2010), further studies are necessary to better elucidate the natural history of TAPS and its management. In brief, evidence of fetal compromise or greater

1	been proposed by Slaghekke and colleagues (2010), further studies are necessary to better elucidate the natural history of TAPS and its management. In brief, evidence of fetal compromise or greater diferences in MCA PSV between twins raise the stage.

1	lso known as an acardiac twin, this is a rare but serious complication of monochorionic multifetal gestation. n estimated incidence is 1 case in 35,000 births. In the classic twin reversedarterial-perfusion (TRAP) sequence, there is a normally formed donor twin that shows features of heart failure and a recipient twin that lacks a heart (acardius) and other structures. In one theory, the TRAP sequence is caused by a large artery-to-artery placental shunt, often also accompanied by a vein-to-vein shunt

1	FIGURE 45-22 Twin reversed-arterial-perfusion sequence. In the TRAP sequence, there is usually a normally formed donor twin that has features of heart failure, and a recipient twin that lacks a heart. It has been hypothesized that the TRAP sequence is caused by a large artery-to-artery placental shunt, often also accompanied by a vein-to-vein shunt. Within the single, shared placenta, perfusion pressure of the donor twin overpowers that in the recipient twin, who thus receives reverse blood flow from its twin sibling. The "used" arterial blood that reaches the recipient twin preferentially goes to its iliac vessels and thus perfuses only the lower body. This disrupts growth and development of the upper body.

1	(Fig. 45-22). Within the single, shared placenta, arterial perfusion pressure of the donor twin exceeds that in the recipient twin, who thus receives reverse blood low containing deoxygenated arterial blood from its cotwin (Lewi, 2013). his "used" arterial blood reaches the recipient twin through its umbilical arteries and preferentially goes to its iliac vessels. Thus, only the lower body is perfused, and therefore disrupted growth and development of the upper body results. In these cases, failed head growth is called acardius acephalus; a partially developed head with identifiable limbs is called acardius myelacephalus; and failure of any recognizable structure to form is acardius amorphous, which is shown in Figure 45-23 (Faye-Petersen, 2006). Because of this vascular connection, the normal donor twin must not only support its own circulation but also pump blood through the underdeveloped acardiac recipient. This may lead to cardiomegaly and high-output heart failure in the normal

1	donor twin must not only support its own circulation but also pump blood through the underdeveloped acardiac recipient. This may lead to cardiomegaly and high-output heart failure in the normal twin (Fox, 2007).

1	In the past, the mortality rate among the pump twins exceeded 50 percent. This stemmed largely from complications of prematurity or from a prolonged high-output state leading to cardiac failure (Dashe, 2001). Risk appears to be directly related to size of the acardiac twin. One sonographic method to estimate acardiac twin size uses the volume of an ellipse: length X width X height x \/6. When the acardiac twin volume is < 50 percent of that of the pump twin, expectant management may be reasonable given the inherent risks of fetal intervention (Chap. 15, p. 326)(Jelin, 2010). When the volume of the

1	FIGURE 45-23 Photograph of an acardiac twin weighing 475 grams. The underdeveloped head is indicated by the black arrow, and its details are shown in the inset. A yellow clamp is seen on its umbilical cord. Its viable donor cotwin was delivered vaginally at 36 weeks and weighed 2325 grams. (Used with permission from Dr. Michael D. Hnat.) acardiac twin is large, however, treatment has generally been ofered. Radiofrequencyrablation (RFA) is the preferred modality of therapy, and contemporary reports now suggest improved perinatal outcomes. he North American Fetal Therapy Network reviewed their experiences with 98 cases from 1998 to 2008 in which RF A of the umbilical cord was performed (Lee, 2013). Median gestational age at delivery was 37 weeks, and 80 percent of neonates survived (Lee, 20l3). he average gestational age at the time of the RF A was 20 weeks, and the estimated acardius-to-pump twin volume on average was 90 percent. Major complications were prematurely ruptured membranes

1	he average gestational age at the time of the RF A was 20 weeks, and the estimated acardius-to-pump twin volume on average was 90 percent. Major complications were prematurely ruptured membranes and preterm birth.

1	Interestingly, TRAP sequences can also occur within monoamnionic pregnancies. he perinatal outcomes of such pregnancies appear to be worse than that of monochorionic diamnionic cases. Sugibayashi and associates (2016) in a review of 40 cases recently reported that pump twin survival following RF A was 88 percent in monochorionic diamnionic pregnancies but only 67 percent in monoamnionic pregnancies. • Hydatidiform Mole with Coexisting Normal Fetus his unique gestation contains one normal fetus, and its cotwin is a complete molar pregnancy. Reported prevalence rates range from 1 in 22,000 to 1 in 100,000 pregnancies (Dolapcioglu, 2009). It must be diferentiated from a partial molar pregnancy, in which an anomalous singleton fetususually triploid-is accompanied by molar tissue (Fig. 20-4, p. 391). At times, a twin pregnancy may occur with a normal twin in one sac and a partial mole in the other sac (McN amara, 2016). Diagnosis is usually made in the first half of pregnancy.

1	Sonographically, a normal-appearing twin is accompanied by its cotwin, which is a large placenta containing multiple small anechoic cysts (Fig. 20-4, p. 391). Often, these pregnancies are terminated, but pregnancy continuation is increasingly adopted. First, the pregnancy prognosis is not as poor as previously thought, and live birth rates range between 20 and 40 percent (Dolapcioglu, 2009; McNamara, 2016). Second, the risk of persistent trophoblastic disease is similar whether the pregnancy is terminated or not (Massardier, 2009; Sebire, 2002). That said, given the limited number of cases, robust data for irm recommendations are lacking. Importantly, complications of expectant management include vaginal bleeding, hyperemesis gravidarum, thyrotoxicosis, and early-onset preeclampsia (McNamara, 2016). Many of these complications result in preterm birth with its attendant adverse perinatal sequelae as well as perinatal loss. Logically, close surveillance is needed for those continuing

1	2016). Many of these complications result in preterm birth with its attendant adverse perinatal sequelae as well as perinatal loss. Logically, close surveillance is needed for those continuing the pregnancy.

1	Fetal size inequality develops in approximately 15 percent of twin gestations and may relect pathological growth restriction in one fetus (Lewi, 2013; Miller, 2012). Generally, as the weight diference within a twin pair rises, the perinatal mortality rate increases proportionately. If it develops, restricted growth of one twin fetus, often termed selective etal-growth restriction, usually develops late in the second and early third trimester. Earlier discordancy indicates higher risk for fetal demise in the smaller twin. Speciically, when discordant growth is identified before 20 weeks, fetal death occurs in approximately 20 percent of the growth-restricted fetuses (Lewi, 2013).

1	he cause of birthweight inequality in twin fetuses is often unclear, but the etiology in monochorionic twins likely difers from that in dichorionic twins. Because the single placenta is not always equally shared in monochorionic twins, these twins have greater rates of discordant growth outside of TTTS than dichorionic twins. Discordancy in monochorionic twins is usually attributed to placental vascular anastomoses that cause hemodynamic imbalance between the twins. Reduced pressure and perfusion of the donor twin can cause diminished placental and fetal growth. Even so, unequal placental sharing is probably the most important determinant of discordant growth in monochorionic twins (Lewi, 2013). Occasionally, monochorionic twins are discordant in size because they are discordant for structural anomalies.

1	Discordancy in dichorionic twins may result from various factors. Dizygotic fetuses may have diferent genetic growth potential, especially if they are of opposite genders. Second, because the placentas are separate and require more implantation space, one placenta might have a suboptimal implantation site. Bagchi and associates (2006) observed that the incidence of severe discordancy is twice as great in triplets as it is in twins. This finding lends credence to the view that in utero crowding is a factor in multifetal growth restriction. Placental pathology may playra role as well. In one study of 668 twin placentas, a strong relationship between histological placental abnormalities and birthweight discordancy was observed in dichorionic, but not monochorionic, twin pregnancies (Kent, 2012).

1	Size discordancy between twins can be determined sonographically. hat said, diferences in crown-rump length are not reliable predictors for birthweight discordance (Miller, 2012). hus, most begin surveillance for discordancy ater the first trimester. One common method uses sonographic fetal biometry to compute an estimated weight for each twin (Chap. 10, p. 184). The weight of the smaller twin is then compared with that of the larger twin. hus, percent discordancy is calculated as the weight of the larger twin minus the weight of the smaller twin, then divided by the weight of the larger twin. Alternatively, given that abdominal circumference (AC) relects fetal nutrition, some use the sonographic AC value of each twin.

1	With these methods, some diagnose selective fetal-growth restriction if the AC measurements difer more than 20 mm or if the estimated fetal weight diference is 20 percent or more. That said, several diferent weight disparities between twins have been used to deine discordancy. Accumulated data suggest that weight discordancy greater than 25 to 30 percent most accurately predicts an adverse perinatal outcome. At Parkland, Hollier and coworkers (1999) retrospectively evaluated 1370 delivered twin pairs and stratiied twin weight discordancy in 5-percent increments within a range of 15 to 40 percent. They found that the incidence of respiratory distress syndrome, intraventricular hemorrhage, seizures, periventricular leukomalacia, sepsis, and necrotizing enterocolitis rose directly with the degree of weight discordancy. Rates of these conditions grew substantially if discordancy exceeded 25 percent. The relative risk of fetal death increased significantly to 5.6 if discordancy was more

1	degree of weight discordancy. Rates of these conditions grew substantially if discordancy exceeded 25 percent. The relative risk of fetal death increased significantly to 5.6 if discordancy was more than 30 percent and rose to 18.9 if it was greater than 40 percent.

1	Sonographic monitoring of twin growth has become a mainstay in management. Monochorionic twins are generally monitored more frequently. his is because their risk of death is higher-3.6 percent versus 1.1 percent-and the risk of neurological damage in the surviving twin is substantial compared with those risks in dichorionic twins (Hillman, 2011; Lee, 2008). Thorson and colleagues (2011) retrospectively analyzed 108 monochorionic twin pregnancies and found that a sonographic evaluation interval > 2 weeks was associated with a higher Quintero stage at the time of TTTS diagnosis. hese indings have led some to recommend serial sonographic examination every 2 weeks in monochorionic twins (Simpson, 2013; Society for MaternalFetal Medicine, 2013). However, there have been no randomized trials of the optimal frequency of sonographic surveillance in monochorionic twin pregnancies. At Parkland Hospital, monochorionic twins undergo sonographic evaluation to assess interval growth every 4 weeks.

1	the optimal frequency of sonographic surveillance in monochorionic twin pregnancies. At Parkland Hospital, monochorionic twins undergo sonographic evaluation to assess interval growth every 4 weeks. In addition, a specific ultrasound examination to search for TTTS is completed at each intervening 2-week mark between these sonograms.

1	For dichorionic pregnancies, a recent report suggests that sonographic evaluation every 2 weeks would identiY more abnormalities prompting delivery (Corcoran, 2015). It has yet to be determined if this practice would improve perinatal outcomes. At our institution, dichorionic twins are sonographically evaluated every 6 weeks. Depending on the degree of discordancy and the gestational age, fetal surveillance may be indicated, especially if one or both fetuses exhibit restricted growth. Nonstress testing, biophysical profile, and umbilical artery Doppler assessment have all been recommended in the management of twins. However, none has been assessed in appropriately sized prospective trials (Miller, 2012).

1	If discordancy is identiied in a monochorionic twin pregnancy, umbilical artery Doppler studies in the smaller fetus may help guide management (Gratac6s, 2007). Namely, investigators have correlated umbilical artery Doppler results with placental indings and with the degree of selective fetal-growth restriction to predict fetal outcome (Gratac6s, 2012). hese correlations have yielded categories of selective fetal-growth restriction. Type I is characterized by positive end-diastolic low, a smaller degree of weight discordance, and a relatively benign clinical course. Type II displays persistently absent end-diastolic flow in the smaller twin and carries a high risk of deterioration and demise. Type III is intermittently absent or reversed end-diastolic flow. Because of large artery-to-artery anastomoses associated with the placentas in this category, type III is associated with a lower risk of deterioration than type II. In all evaluated cases, unequally shared placenta was noted to

1	anastomoses associated with the placentas in this category, type III is associated with a lower risk of deterioration than type II. In all evaluated cases, unequally shared placenta was noted to some degree.

1	With uncomplicated dichorionic multifetal gestations, use of antepartum surveillance has not improved perinatal outcomes. In sum, the American College of Obstetricians and Gynecologists (2016) recommends that antepartum testing be performed in multi fe tal gestations for indications similar to those for singleton fetuses (Chap. 17, p. 331). At Parkland, all women with twin discordancy :25 percent undergo daily monitoring as an inpatient. Data are limited to establish the optimal timing of delivery of twins for size discordancy alone. For those at advanced gestational ages, delivery can be pursued. • Death of One Fetus At any time during multifetal pregnancy, one or more fetuses may die, either simultaneously or sequentially. Causes and incidence of fetal death are related to zygosity, chorionicity, and growth concordance.

1	In some pregnancies, one fetus dies remote from term, but pregnancy continues with one or more live fetuses. When this occurs early in pregnancy, it may manifest as a vanishing twin, discussed on page 871. In a slightly more advanced pressed against the fetal membranes. Anatomical parts can be identified as marked. Demise of this twin had been noted during sonographic examination performed at 17 weeks' gestation. Its viable cotwin delivered at 40 weeks. (Used with permission from Dr. Michael V. Zaretsky.) gestation, fetal death may go undetected until delivery. In this case, delivery ofa normal newborn is followed by expulsion of a dead fetus that is barely identiiable. It may be compressed appreciably-'tus compressus, or it may be lattened remarkably through desiccation-'tuspapyraceus (Fig. 45-24).

1	As shown in Figure 45-25, the risk ofstillbirth is related to gestational age in all twins but is much higher for monochorionic twin pregnancies before 32 weeks' gestation. In a review of 9822 twin pregnancies, Morikawa and associates (2012) reported that 2.5 percent of monochorionic diamnionic twins �o 0: .� 15 ..0 FIGURE 45-25 Prospective risk of stillbirth among women who reached a given gestational week (per 1000 women). (Reproduced with permission from Morikawa M, Yamada 1, Yamada T, et al: Prospective risk of stillbirth: monochorionic diamniotic twins vs dichorionic twins, J Perinat Med. 2012 Jan 10;40(3):245-249.) greater than 22 weeks had a death of one or both twins. This compared with 1.2 percent of dichorionic twins. In this same review, women with monochorionic diamnionic twins who lost one twin were 16 times more likely to experience death of the cotwin than women with dichorionic twins who lost one twin. Other investigations have found similar trends (Danon, 2013;

1	Other investigations have found similar trends (Danon, 2013; Hillman, 2011; Mahony, 2011).

1	Hillman, 2011; Mahony, 2011). Other factors that afect the prognosis for the surviving twin include gestational age at the time of the demise and duration between the demise and delivery of the surviving twin. With a vanishing twin, the risk of death after the irst trimester is not increased for the survivor. However, when a fetus dies in the second trimester or later, the efect ofgestational age at the time ofdeath and the mortality risk to the cotwin are less clear. In an analysis by Hillman and colleagues (2011), cotwin demise rates were unafected regardless of whether the irst death occurred at 13 to 27 weeks' gestation or at 28 to 34 weeks. In cases with the death ofone twin after the irst trimester, however, the odds of spontaneous and iatrogenic preterm delivery of the remain ing living twin were increased (Hillman, 2011). Preterm birth gestation. Ifthe fetus died after 34 weeks, preterm delivery rates were similar.

1	The neurological prognosis for a surviving cotwin depends almost exclusively on chorionicity. In their comprehensive review, Ong and coworkers (2006) found an 18-percent rate of neurological abnormality in twins with monochorionic placentation compared with only 1 percent in those with dichorionic placentation. In another review, in twin pregnancies complicated by a single fetal demise before 34 weeks, a ivefold higher risk ofneurodevelopmental morbidity was identiied in monochorionic twins compared with dichorionic twins. If the one fetus died after 34 weeks, the likelihood ofneurological deicits was essentially the same between monochorionic and dichorionic twin pregnancies (Hillman, 201r1).

1	Later in gestation, the death ofone ofmultiple fetuses could theoretically trigger coagulation defects in the mother. Only a few cases of maternal coagulopathy after a single fetal death in a twin pregnancy have been reported. This is probably because the surviving twin is usually delivered within a few weeks ofthe demise (Eddib, 2006). That said, we have observed transient, spontaneously corrected consumptive coagulopathy in multifetal gestations in which one fetus died and was retained in utero along with its surviving twin. he plasma ibrinogen concentration initially decreased but then increased spontaneously, and the level of serum ibrinogen-ibrin degradation products increased initially but then returned to normal levels. At delivery, the portions of the placenta that supplied the living fetus appeared normal. In contrast, the part that had once provided for the dead fetus was the site of massive ibrin deposition.

1	Decisions should be based on gestational age, the cause of death, and the risk to the surviving fetus. First-trimester losses require no additional surveillance for this speciic indication. If the loss occurs after the irst trimester, the risk ofdeath or damage to the survivor is largely limited to monochorionic twin gestations. Morbidity in the monochorionic twin survivor is almost always due to vascular anastomoses, which often cause the demise of one twin followed by sudden hypotension in the other (p. 878). For this reason, if one fetus of a monochorionic twin gestation dies ater the first trimester but before viability, pregnancy termination can be considered (Blickstein, 2013). Occasionally, death of one but not all fetuses results from a maternal complication such as diabetic ketoacidosis or severe preeclampsia with abruption. Pregnancy management is based on the diagnosis and the status of both the mother and surviving fetus. If the death of one dichorionic twin is due to a

1	or severe preeclampsia with abruption. Pregnancy management is based on the diagnosis and the status of both the mother and surviving fetus. If the death of one dichorionic twin is due to a discordant congenital anomaly in the irst trimester, it should not afect the surviving twin.

1	Single fetal death during the late second and early third trimesters presents the greatest risk to the surviving twin. Although the risks of subsequent death or neurological damage to the survivor are comparatively higher for monochorionic twins at this gestational age, the risk of preterm birth is equally increased in mono-and dichorionic twins (Ong, 2006). Delivery generally occurs within 3 weeks of diagnosis of fetal demise, thus antenatal corticosteroids for survivor lung maturity should be considered (Blickstein, 2013). Regardless, unless the intrauterine environment is hostile, the goal is to prolong the preterm pregnancy.

1	Timing of elective delivery after conservative management of a late second-or early third-trimester single fetal death is debatable. Dichorionic twins can probably be safely delivered at term. Monochorionic twin gestations are more diicult to manage and are often delivered between 34 and 37 weeks' gestation (Blickstein, 2013). In cases of single fetal death at term, especially when the etiology is unclear, most opt for delivery instead of expectant management. The American College of Obstetricians and Gynecologists (2016) also endorse an individualized approach to such cases. • Impending Death of One Fetus

1	• Impending Death of One Fetus During antepartum surveillance tests of well-being, abnormal results in one twin, but not the other, pose a particular dilemma. Delivery may be the best option for the compromised fetus yet may result in death from immaturity of the cotwin. If fetal lung maturity is conirmed, salvage of both the healthy fetus and its jeopardized sibling is possible. Unfortunately, ideal management if twins are immature is problematic but should be based on the chances of intact survival for both fetuses. Often the compromised fetus is severely growth restricted or anomalous. Thus, performing amniocentesis for fetal chromosomal analysis in women of advanced maternal age carrying twin pregnancies is advantageous, even for those who would continue their pregnancies regardless of the diagnosis. Chromosomal abnormality identification in one fetus allows rational decisions regarding interventions.

1	With prenatal management of multifetal pregnancy, primary goals aim to prevent or interdict complications as they develop. A major imperative is to prevent preterm delivery of markedly immature neonates. At Parkland Hospital, women with multifetal gestations are seen every 2 weeks beginning at 22 weeks' gestation. A digital cervical examination is performed at each visit to screen for cervical shortening or dilation. Identification of other unique complications discussed earlier may also lead to interventions including admission or early delivery.

1	long with more frequent prenatal VISItS, the maternal diet should provide additional requirements for calories, protein, minerals, vitamins, and essential fatty acids. he Institute of Medicine (2009) recommends a 37-to 54-lb weight gain for women with twins and a normal BMI. In their review, Goodnight and Newman (2009) endorse supplementation of micronutrients such as calcium, magnesium, zinc, and vitamins C, D, and E. his is based on upper intake levels from the Food and Nutrition Board of the Institute of Medicine. he daily recommended augmented caloric intake for women with twins is 40 to 45 kcal/kg/d. Diets contain 20 percent protein, 40 percent carbohydrate, and 40 percent fat divided into three meals and three snacks daily.

1	As noted earlier (p. 882), serial sonographic examinations are usually performed throughout the third trimester to search for abnormal fetal growth and assess amnionic fluid volume. Associated oligohydramnios may indicate utero placental pathology and should prompt further evaluation of fetal well-being. That said, quantiYing amnionic luid volume in multifetal gestation is sometimes diicult. Some measure the deepest vertical pocket in each sac or assess the luid subjectively. Magann and coworkers (2000) compared subjective assessment and several objective methods of assessing amnionic fluid volume in 23 sets of twins. They found all methods to be equally poor in predicting abnormal volumes in diamnionic wins. At Parkland Hospital, the single deepest vertical pocket is measured in each sac. A measurement < 2 em is considered oligohydramnios, and a measurement > 8 cm is considered hydramnios (Duryea, 2017; Hernandez, 2012).

1	Of surveillance methods, the nons tress test or biophysical profile is often selected for twin or higher-order multifetal gestations. Because of the complex complications associated with these gestations and the potential technical diiculties in differentiating fetuses during antepartum testing, the usefulness of these methods appears limited. According to DeVoe (2008), the few exclusive studies of nonstress testing in twins suggest that the method performs the same as in singleton pregnancies. Elliott and Finberg (1995) used the biophysical profile as the primary method for monitoring higher-order multifetal gestations. They reported that four of 24 monitored pregnancies had a poor outcome despite reassuring biophysical proile scores. Although biophysical testing is commonly performed in multifetal gestations, there are insuicient data to determine its eicacy (DeVoe, 2008).

1	Similar indings have been reported with the addition of umbilical artery Doppler velocimetry in twins with concordant growth. For example, when umbilical artery Doppler velocime try was added to management compared with fetal testing based on fetal-growth parameters alone in the absence of growth dis cordance, perinatal outcomes were not improved (Giles, 2003). Likewise, Hack and associates (2008) investigated the utility mortality rates using pulsatility indices of the umbilical artery. tons, and data suggest that testing in multifetal gestations per forms no better. In cases of abnormal testing in one twin and normal results in another, iatrogenic preterm delivery remains a major concern. Options are similar to those described in the management of impending fetal death (p. 884).

1	Preterm labor is common in multifetal pregnancies and may complicate up to 50 percent of twin, 75 percent of triplet, and 90 percent of quadruplet pregnancies (Elliott, 2007). Similar to singleton preterm labor, intraamnionic infection is docu mented in approximately one third of twin pregnancy cases (Oh,r2017). In twins, the proportion of preterm births varies widely from 40 to 70 percent (Giufre, 2012). For example, black women have disparately higher risks for preterm delivery (Grant, 2017). • Prediction of Preterm Birth

1	A major goal of multi fetal prenatal care is accurate prediction of women likely to experience preterm delivery. Within the past decade, cervical length has been shown to be a potent predictor of preterm labor and delivery. To and associates (2006) so no graphically measured cervical length in 1163 twin pregnancies at 22 to 24 weeks' gestation. Rates of preterm delivery before 32 weeks were 66 percent in those with cervical lengths of 10 mm; 24 percent for lengths of 20 mm; and only 1 percent for 40 mm. In one review, Conde-Agudelo and coworkers (2010) concluded that a cervical length <20 mm was most accurate for predicting birth before 34 weeks, with a speciicity of 97 percent and positive likelihood ratio of 9.0. Kindinger and colleagues (2016) noted that prediction depended on both cervical length and gestational age at ascertainment. One study compared serial cervical length measurements with a single midgestation measurement. hese authors found that multiple assessments were more

1	length and gestational age at ascertainment. One study compared serial cervical length measurements with a single midgestation measurement. hese authors found that multiple assessments were more accurate to determine the risk of pre term twin birth in asymptomatic women (Melamed, 2016a). In another study, a change in cervical length :0.2 cm identified pregnancies at risk for delivery before 35 weeks (Moroz, 2017). Interestingly, a closed internal os by digital examination was found to be as predictive of postponed delivery as was the combination of a normal sonographically measured cervical length and negative fetal fibronectin test result (McMahon, 2002). Unfortunately, cervical length assessment in twin pregnancies has not been associated with improved outcomes (Gordon, 2016).

1	• Prevention of Preterm Birth Several schemes have been evaluated to prevent preterm labor and delivery. In recent years, some have been shown to decrease the risk of pre term delivery, but only in subgroups of singleton pregnancies. In general, most have been disappointingly ineffective for both singleton and multifetal pregnancies (American College of Obstetricians and Gynecologists, 2016). The bulk of evidence suggests that routine hospitalization does not prolong multifetal pregnancy. In one metaanaly sis, the practice did not reduce the risk of preterm birth or perinatal mortality (Crowther, 2010). At Parkland Hospital, elective hospitalization was compared with outpatient manage ment, and no advantages were found (Andrews, 1991). Impor tantly, however, almost half of women managed as outpatients required admission for specific indications such as hypertension or threatened preterm delivery.

1	Limited physical activity, early work leave, more frequent health-care visits and sonographic examinations, and structured advocated to reduce pre term birth rates in women with mul tiple fetuses. However, little evidence suggests that these mea sures substantially change outcome. his has not been studied extensively in multifetl pregnancies. In one review of prophylactic oral beta-mimetic therapy that included 374 twin pregnancies, treatment did not reduce the rate of twins delivering before 37 or before 34 weeks' gestation (Ymasmit, 2015). In light of the Food and Drug Administration warning against the use of oral terbutaline because of maternal side efects, the prophylactic use of beta-mimetic drugs in multifetal gestations seems unwarranted.

1	lthough somewhat efective in reducing recurrent preterm birth in women with a singleton pregnancy, weekly injections of 17 alpha-hydroxyprogesterone caproate (17 -OHP-C) are not efective for multifetal gestations (Caritis, 2009; Rouse, 2007). hese results were corroborated in a randomized trial of 240 twin pregnancies (Combs, 2011). Moreover, women carrying twins and having a cervical length <36 mm (25th percentile) did not beneit despite their greater risk for preterm birth (Durnwald, 2010). Senat and colleagues (2013) assigned 165 asymptomatic women with twins and a cervical length <25 mm to 17-0HP-C and also found no reduction in delivery rate before 37 weeks. Last, in an evaluation of plasma drug concentrations, higher concentrations of 17 -OHP-C were associated with earlier gestational age at delivery (Caritis, 2012). he authors concluded that 17-0HP-C may adversely lower the gestational age at delivery in women with twin gestations. In sum, administration of intramuscular 17

1	age at delivery (Caritis, 2012). he authors concluded that 17-0HP-C may adversely lower the gestational age at delivery in women with twin gestations. In sum, administration of intramuscular 17 -OHP-C to women with twin pregnancies, even to those with a shortened cervix, does not lower the preterm birth risk.

1	Micronized progesterone administered vaginally to women with twins to prevent preterm birth has provided conlicting results.

1	Cetingoz and coworkers (2011) gave 100 mg of micronized progesterone intravaginally daily from 24 to 34 weeks' gestation. his practice reduced rates of delivery before 37 weeks from 79 to 51 percent in 67 women with twins. In contrast, several studies have failed to demonstrate any preterm birth rate reduction in women receiving various formulations of vaginal progesterone. In the Prevention of Preterm Delivery in Twin Gestations (PREDICT) trial, 677 women with twins were randomly assigned to receive prophylactic, 200-mg progesterone pessaries or placebo pessaries (Rode, 2011). Progesterone failed to reduce delivery rates before 34 weeks. In a subgroup analysis that included only women with a short cervix or a history of prior preterm birth, also no benefit was found (Klein, 2011). Norman and colleagues (2009) also noted no lower rates of delivery before 34 weeks with progesterone gel treatment.

1	Romero and colleagues (2017) performed a metaanalysis of individual patient data for 303 women with twin gestation and a short cervix randomized to receive either vaginal progesterone or no treatment. They reported a significantly reduced risk of preterm birth before 30 weeks' gestation and improved composite perinatal outcomes in the treated women. Currently at Parkland Hospital, management of women with multifetal gestations does not typically include progestetone in any formulation. The Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) is currently enrolling patients into a randomized, placebo-controlled trial to further evaluate the use of micronized vaginal progesterone or the Arabin pessary, describe subsequently (PROSPECT, 2015). The primary outcome is delivery prior to 35 weeks or fetal loss.

1	Prophylactic cerclage does not improve perinatal outcome in women with multifetal pregnancies. Studies have included women who were not specially selected but also those who were selected because of a shortened cervix that was identified sonographically (Houlihan, 2016; Newman, 2002; Rebarber, 2005). Indeed, in the latter group, cerclage may actually worsen outcomes (Berghella, 2005; Roman, 2013). Rescue cerclage in women with a second-trimester twin gestation and a dilated cervix may be beneficial. Roman and coworkers (2016) reported a retrospective cohort study in which women undergoing rescue cerclage had significantly better neonatal outcomes than those without cerclage.

1	A vaginal pessary that encircles and theoretically compresses the cervix, alters the inclination of the cervical canal, and relieves direct pressure on the internal cervical os has been proposed as an alternative to cerclage. One of the most popular is the silicone Arabin pessary. In a study of its use in women with a short cervix between 18 and 22 weeks' gestation, a subgroup analysis of 23 women with twins showed a signiicant reduction in the delivery rate before 32 weeks compared with the rate in 23 control pregnancies (Arabin, 2003). In another randomized trial, women treated with a cervical pessary had signiicantly fewer births before 34 weeks (Goya, 2016).

1	Other studies have been less favorable. In the randomized Pessaries in Multiple Pregnancy as a Prevention of Preterm Birth (ProTWIN) trial, 813 un selected women with twins received either the Arabin pessary between 12 and 20 weeks or no treatment (Liem, 2013). The pessary failed to reduce preterm birth overall but did decrease delivery rates before 32 weeks-29 versus 14 percent-in a subset of women with a cervical length <38 mm. Similar results were reported from a randomized multicenter trial with a total of 11r80 twin pregnancies (Nicolaides, 2016). A smaller randomized study using a Bioteque cup pessay showed no diference in outcomes (Berghella, 2017). At this time, pessary use is not recommended by the merican College of Obstetricians and Gynecologists (2016). As noted above, results from the ongoing PROSPECT trial are anticipated to provide more data. • Treatment of Preterm Labor

1	Although many advocate their use, therapy with tocolytic agents to forestall preterm labor in multifetal pregnancy does not result in measurably improved neonatal outcomes (Chauhan, 2010; Gyetvai, 1999). Another caveat is that tocolytic therapy in women with a multifetal pregnancy entails higher risks than in singleton pregnancy. This stems in part from augmented pregnancy-induced hypervolemia, which raises cardiac demands and increases the susceptibility to iatrogenic pulmonary edema (Chap. 47, p. 917). Gabriel and colleagues (1994) compared outcomes of 26 twin and six triplet pregnancies with those of 51 singletons-all treated with a beta-mimetic drug for preterm labor. Women with a multifetal gestation had significantly more cardiovascular complications-43 versus 4 percent-including three gravidas with pulmonary edema. In a retrospective analysis, Derbent and coworkers (2011) evaluated nifedipine tocolysis in 58 singleton and 32 twin pregnancies. hese authors reported higher

1	three gravidas with pulmonary edema. In a retrospective analysis, Derbent and coworkers (2011) evaluated nifedipine tocolysis in 58 singleton and 32 twin pregnancies. hese authors reported higher incidences of side efects such as maternal tachycardia in women with twins-19 versus 9 percent.

1	Administration of corticosteroids to stimulate fetal lung maturation has not been well studied in multifetal gestation. However, these drugs logically should be as beneicial for multiples as they are for singletons (Roberts, 2006). In a large retrospective study evaluating betamethasone therapy eicacy in preterm twin versus preterm singleton pregnancies, no diferences in neonatal morbidity between the two groups were identiied (Melamed, 2016b). Gyamfi and associates (2010) evaluated betamethasone concentrations in women receiving weekly antenatal corticosteroids and found no diferences in levels between twins and singletons. Conversely, another study found lower cord/maternal ratios of dexamethasone in twin versus singleton pregnancies (Kim, 2017). hese treatments are discussed in Chapter 42 (p. 823). At this time, guidelines for the use of these agents do not difer from those for singleton gestations (American College of Obstetricians and Gynecologists, 2016).

1	The frequency of preterm premature rupture of membranes (PPROM) rises with increasing plurality. In a populationbased study of more than 290,000 live births, the proportion of preterm birth complicated by premature rupture was 13.2 percent in singletons (Pakrashi, 2013). This rate compared with rates of 17, 20, 20, and 100 percent in twins, triplets, quadruplets, and even higher-order multiples, respectively. Multifetal gestations with PPROM are managed expectantly similar to singleton pregnancies (Chap. 42, p. 820). Ehsanipoor and colleagues (2012) compared outcomes of 41 twin and 82 singleton pregnancies, both with ruptuted membranes between 24 and 32 weeks. They found the median number of days to subsequent delivery was overall shorter for twins-3.6 days compared with 6.2 days for singletons. This latency diference was significant in pregnancies after 30 weeks-l.7 days and 6.9 days. Importantly, latency beyond 7 days approximated 40 percent in both groups.

1	• Delayed Delivery of Second Twin

1	Infrequently, after preterm birth of the presenting fetus, it may be advantageous for undelivered fetus(es) to remain in utero. Trivedi and Gillett (1998) reviewed 45 case reports of asynchronous birth in multifetal gestations. Although reported outcomes may relect bias, pregnancies with a surviving retained twin or triplet continued for an average of 49 days. No advantage was gained by management with tocolytics, prophylactic antimicrobials, or cerclage. In their 10-year experience, Roman and associates (2010) reported a median latency of 16 days in 13 twin and ive triplet pregnancies with delivery of the irst fetus between 20 and 25 weeks' gestation. Survival of the irstborn neonate was 16 percent. Although 54 percent of the retained fetuses survived, only 37 percent of survivors did so without major morbidity. Livingston and coworkers (2004) described 14 pregnancies in which an active attempt was made to delay delivery of 19 fetuses after delivery of the irst neonate. Only one

1	so without major morbidity. Livingston and coworkers (2004) described 14 pregnancies in which an active attempt was made to delay delivery of 19 fetuses after delivery of the irst neonate. Only one fetus survived without major sequelae, and one mother developed sepsis syndrome with shock. Arabin and van Eyck (2009) reported better outcomes in a few of the 93 twin and 34 triplet pregnancies that qualiied for delayed delivery in their center during a 17 -year period.

1	If asynchronous birth is attempted, there must be careful evaluation for infection, abruption, and congenital anomalies. The mother must be thoroughly counseled, particularly regarding the potential for serious, life-threatening infection. The range of gestational age in which the beneits outweigh the risks for delayed delivery is likely narrow. Avoidance of delivery from 23 to 26 weeks would seem most beneficial. In our experience, good candidates for delayed delivery are rare. A litany of complications may be encountered during labor and delivery of multiple fetuses. In addition to preterm birth, rates of uterine contractile dysfunction, abnormal fetal presentation, umbilical cord prolapse, placenta previa, placental abruption, emergent operative delivery, and postpartum hemorrhage from uterine atony are higher. All of these must be anticipated, and thus certain precautions and special arrangements are prudent. These should include the following. 1.

1	1. An appropriately trained obstetrical attendant should remain with the mother throughout labor. Continuous electronic monitoring is preferable. If membranes are ruptured and the cervix dilated, the presenting fetus is monitored internally. 2. An intravenous infusion system capable of delivering fluid rapidly is established. In the absence of hemorrhage, lactated Ringer or an aqueous dextrose solution is infused at a rate of 60 to 125 mLlhr. 3. Blood for transfusion is readily available if needed. 4. n obstetrician skilled in intrauterine identification of fetal parts and in intrauterine manipulation of a fetus should be present. 5. A sonography machine is readily available to evaluate the presentation and position of the fetuses during labor and to image the remaining fetus(es) after delivery of the irst. 6.

1	5. A sonography machine is readily available to evaluate the presentation and position of the fetuses during labor and to image the remaining fetus(es) after delivery of the irst. 6. An anesthesia team is immediately available in the event that emergent cesarean delivery is necessary or that intrauterine manipulation is required for vaginal delivery. 7. For each fetus, at least one attendant who is skilled in resuscitation and care of newborns and who has been appropriately informed of the case should be immediately available. 8. he delivery area should provide adequate space for the nursing, obstetrical, anesthesia, and pediatric team members to work efectively. Equipment must be on site to provide emergent anesthesia, operative intervention, and maternal and neonatal resuscitation. • Timing of Delivery

1	• Timing of Delivery Several factors afect this timing and include gestational age, fetal growth, lung maturity, and presence of maternal complications. As measured by determination of the lecithin-sphingomyelin ratio, pulmonary maturation is usually synchronous in twins (Leveno, 1984). Moreover, although this ratio usually does not exceed 2.0 until 36 weeks in singleton pregnancies, it oten exceeds this value by approximately 32 weeks in multifetal pregnancies. Similar increased values of surfactant have been noted in twins after 31 weeks' gestation (McElrath, 2000). In a comparison of respiratory morbidity in 100 twins and 241 singleton newborns delivered by cesarean before labor, Ghi and associates (2013) found less neonatal respiratory morbidity in twins, especially those delivered <37 weeks' gestation. In some cases, however, pulmonary function may be markedly diferent, and the smallest, most stressed twin fetus is typically more mature.

1	At the other end of the spectrum, Bennett and Dunn (1969) suggested that a twin pregnancy of 40 weeks or more should be considered postterm. Twin stillborn neonates delivered at 40 weeks or beyond commonly had features similar to those of postmature singletons (Chap. 43, p. 836). From an analysis of almost 300,000 twin births, at and beyond 39 weeks, the risk of subsequent stillbirth was greater than the risk of neonatal mortality (Kahn, 2003).

1	From their guidelines, the American College of Obstetricians and Gynecologists (2016) recommends delivery at 38 weeks for uncomplicated dichorionic twin pregnancies. Women with uncomplicated monochorionic diamnionic twin pregnancies can undergo delivery between 34 and weeks. And, for women with monoamnionic twin pregnancies, delivery is recommended at 32 to 34 weeks. At Parkland Hospital, we generally follow these recommendations but do not routinely deliver monochorionic diamnionic twin pregnancies before 37 weeks unless another obstetrical indication develops. • Evaluation of Fetal Presentation

1	In addition to the standard preparations for the conduct oflabor and delivery discussed in Chapter 22, there are special considerations for women with a multifetal pregnancy. First, the positions and presentations of fetuses are best confirmed sonographically. Although any possible combination of positions may be encountered, those most common at admission for delivery are cephalic-cephalic, cephalic-breech, and cephalic-transverse. At Parkland Hospital between 2008 and 2013, 71 percent of twin pregnancies had a cephalic presentation of the irst fetus at the time of admission to labor and delivery. Importantly, with perhaps the exception of cephalic-cephalic presentations, these are all unstable before and during labor and delivery. Accordingly, compound, face, brow, and footling breech presentations are relatively common, and even more so if fetuses are small, amnionic luid is excessive, or maternal parity is high. Cord prolapse is also frequent in these circumstances.

1	After this initial evaluation, if active labor is conirmed, then a decision is made to attempt vaginal delivery or to proceed with cesarean delivery. The latter is usually chosen because of fetal presentations. In general, cephalic presentation of the irst fetus in a laboring woman with twins may be considered for vaginal delivery (American College of Obstetricians and Gynecologists, 2016). The proportion of women undergoing an attempted vaginal delivery varies greatly depending on the skills of the delivering physician (de Castro, 2016; Easter, 2017; Schmitz, 2017). Still, the cesarean delivery rate is high. For example, of the 547 women with the irst twin presenting cephalic who were admitted to Parkland Hospital during 5 years, only 32 percent were delivered spontaneously. And, the overall cesarean delivery rate in twin pregnancies during those years was 77 percent. Notably, 5 percent of cesareans performed were for emergent delivery of the second twin following vaginal delivery of

1	cesarean delivery rate in twin pregnancies during those years was 77 percent. Notably, 5 percent of cesareans performed were for emergent delivery of the second twin following vaginal delivery of the first twin. The desire to avoid this obstetrical dilemma has contributed to the rising cesarean delivery rate in twin pregnancies across the United States (Antsalis, 2013).

1	After a comparison of 891 twins with more than 100,000 singleton pregnancies included in the Consortium of Safe Labor, Leftwich and colleagues (2013) concluded that active labor progressed more slowly in both nulliparas and multiparas with twins. Provided women with twins meet all criteria for oxytocin administration, it may be used as described in Chapter 26 (p. 509). Wolfe and associates (2013) evaluated the success of labor induction and concluded that oxytocin alone or in combination with cervical ripening can safely be used in twin gestations. Taylor and coworkers (2012) reported similar results. Conversely, Razavi and colleagues (2017) found that maternal morbidity was increased with labor induction. In an analysis of twin births in the United States, induction rates of twin pregnancies have decreased from a maximum of 13.8 percent in 1999 to 9.9 percent in 2008 (Lee, 2011). Generally, at Parkland Hospital we do not induce or augment labor in women with a multifetal gestation.

1	have decreased from a maximum of 13.8 percent in 1999 to 9.9 percent in 2008 (Lee, 2011). Generally, at Parkland Hospital we do not induce or augment labor in women with a multifetal gestation. In suitable candidates with a strong desire for vaginal birth, amniotomy induction has been one option.

1	During labor and delivery of multiple fetuses, decisions regarding analgesia and anesthesia may be complicated by problems imposed by preterm labor, preeclampsia, desultory labor, need for intrauterine manipulation, and postpartum uterine atony and hemorrhage. Labor epidural analgesia is ideal because it provides excellent pain relief and can be rapidly extended cephalad if internal podalic version or cesarean delivery is required. If general anesthesia becomes necessary for intrauterine manipulation, uterine relxation can be accomplished rapidly with one of the halogenated inhalation agents discussed in Chapter 25 (p. 499). Some clinicians use intravenous or sublingual nitroglycerin or intravenous terbutaline to achieve uterine relaxation yet avoid the risks associated with general anesthetics. hese agents are usually best administered by the anesthesia team.

1	Regardless of fetal presentation during labor, obstetricians must be ready to deal with any change of fetal position during delivery. This is especially true following delivery of the first twin. Importantly, related to delivery method, second twins at term have worse composite neonatal outcomes compared with outcomes of their cotwin regardless of delivery method (Muleba, 2005; Smith, 2007; Thorngren-Jerneck, 2001). If the first twin presents cephalic, delivery can usually be accomplished spontaneously or with forceps. According to D'Alton (2010), there is general consensus that a trial oflabor is reasonable in women with cephalic-cephalic twins. From their review, Hogle and associates (2003) found that planned cesarean delivery does not improve neonatal outcome when both twins are cephalic. The randomized trial by Barrett and coworkers (2013) airms this conclusion.

1	The optimal delivery route for cephalic-noncephalic twin pairs remains controversial. Patient selection is crucial, and options include cesarean delivery of both twins, or less commonly, vaginal delivery with intrapartum external cephalic version of the second twin. Longer intertwin delivery time has been shown in some studies to be associated with poorer second twin outcome (Edris, 2006; Stein, 2008). Thus, breech extraction may be preferable to version. Least desirable, vaginal delivery of the first but cesarean delivery of the second twin may be required due to intrapartum complications such as umbilical cord prolapse, placental abruption, contracting cervix, or fetal distress. ·Most but not all studies report the worst composite fetal out comes for this scenario (Alexander, 2008; Rossi, 2011; Wen, 2004).

1	Several reports attest to the safety of vaginal delivery of second noncephalic twins whose birthweight is > 1500g. A French multicenter study of 5915 twin pregnancies illustrates this (Schmitz, 2017). Of these, 25 percent had a planned cesarean delivery. The other 75 percent with a first twin cephalic and gestational age > 32 weeks had a planned trial of vaginal delivery, which was successful in 80 percent. Interestingly, perinatal mortality and morbidity rates were significantly higher in the planned cesarean delivery group delivered <37 weeks-5.2 versus 3.0 percent, respectively. Fox and colleagues (2014) reported outcomes in 287 diamnionic twin pregnancies, of which 130 underwent a planned vaginal delivery. Only 15 percent of the planned vaginal delivery group underwent a cesarean delivery. Perinatal outcomes were similar in both groups. hese two studies included only those fetuses with estimated weightsr> 1500 g. Notably, comparable or even better fetal outcomes with vaginal

1	delivery. Perinatal outcomes were similar in both groups. hese two studies included only those fetuses with estimated weightsr> 1500 g. Notably, comparable or even better fetal outcomes with vaginal delivery have been reported with neonates weighing < 1500 g compared with those weighing > 1500 g (Caukwell, 2002; Davidson, 1992).

1	Other investigators advocate cesarean delivery for both members of a cephalic-noncephalic twin pair (Armson, 2006; Hofmann, 2012). Yang and coworkers (2005a,b) studied 15,185 cephalicnoncephalic twin pairs. he risks of asphyxia-related neonatal deaths and morbidity were higher in the group in which both twins were delivered vaginally compared with the group in which both twins underwent cesarean delivery.

1	To add insight into the clinical complexities just discussed, a randomized trial was designed by the Twin Birth Study Collaborative Group from Canada. he study results described by Barrett and associates (2013) included 2804 women carrying a presumed diamnionic twin pregnancy with the irst fetus presenting cephalic. Women were randomly assigned between 32 and 38 weeks' gestation to planned cesarean or vaginal delivery. The time from randomization to delivery-12.4 versus 13.3 days, the mean gestational age at delivery-36.7 versus 36.8 weeks, and use of regional analgesia-92 versus 87 percent, were similar in both groups. Salient maternal and perinatal outcomes are shown in Table 45-3. No significant diferences in outcomes were noted between the two groups of women. Although risks to mother or fetuses with planned vaginal delivery in these circumstances were not increased, Greene (2013) posited that this trial would have only modest efects on the cesarean delivery rate of women with

1	or fetuses with planned vaginal delivery in these circumstances were not increased, Greene (2013) posited that this trial would have only modest efects on the cesarean delivery rate of women with twins.

1	Breech Presentation of First Twin Problems with the first twin presenting as a breech are similar to those encountered with a singleton breech fetus. hus, major diiculties may develop in the following settings. First, the fetus may be large, and the aftercoming head is larger than the birth canal. Second, the fetal body can be small, and delivery of the extremities and trunk through an inadequately efaced and dilated cervix causes the relatively larger head to become trapped above the cervix. his is more likely when there is significant disproportion between the head and body. Examples are preterm or growth-restricted fetuses or those with Serious morbidity 1.3% 1.3% Possible 0.5% 0.4% Intubation 1.0% 0.6% alncludes coma; stupor; hyperalert, drowsy or lethargic; or ::2 seizu res. Data from Barrett, 20ln3. macrocephaly from hydrocephaly. Last, umbilical cord prolapse is an ever-present risk.

1	Data from Barrett, 20ln3. macrocephaly from hydrocephaly. Last, umbilical cord prolapse is an ever-present risk. If these problems are anticipated or identiied, cesarean delivery is often preferred with a viable-sized fetus. But even without these problems, many obstetricians perform cesarean delivery if the irst twin presents as breech. This is despite data that support the safety of vaginal delivery. Specifically, Blickstein and associates (2000) reported experiences from 13 European centers with 613 twin pairs and the first twin presenting breech. Vaginal delivery was attempted in 373 of these cases and was successful in 64 percent. Cesarean delivery of the second twin was done in 2.4 percent. here was no diference in the rate of 5-minute Apgar scores <7 or of mortality in breech-presenting first twins who weighed at least 1500 g. Details of techniques for delivery of a breech presentation are described in Chapter 28 (p. 544).

1	Twin fetuses may become locked together during delivery if the irst presents breech and the second cephalic. As the breech of the irst twin descends through the birth canal, the chin locks between the neck and chin of the second cephalic-presenting cotwin. This phenomenon is rare, and Cohen and coworkers (1965) described it only once in 817 twin gestations. Cesarean delivery should be considered when the potential for locking is identiied. • Vaginal Delivery of the Second Twin

1	• Vaginal Delivery of the Second Twin Following delivery of the first twin, the presenting part of the second twin, its size, and its relationship to the birth canal should be quickly and careully ascertained by combined abdominal, vaginal, and at times, intrauterine examination. Sonography is a valuable aid. If the fetal head or the breech is fixed in the birth canal, moderate undal pressure is applied and membranes are ruptured. Immediately aterward, digital examination of the cervix is repeated to exclude cord prolapse. Labor is allowed to resume. If contractions do not begin within approximately 10 minutes, dilute oxytocin may be used to stimulate contractions.

1	In the past, the safest interval between delivery of the irst and second twins was frequently cited as <30 minutes. Rayburn and colleagues (1984) and others have shown that if continuous fetal monitoring is used, a good outcome is usually achieved even if this interval is longer. A direct correlation between worsening umbilical cord blood gas values and increasing time between delivery of irst and second twins has been shown (Leung, 2002; Stein, 2008). From review of 239 twin gestations, Gourheux and associates (2007) determined that mean umbilical arterial pH was signiicantly lower after the delivery interval exceeded 15 minutes. In a study of more than 175,000 twin pairs, Cheng and colleagues (2017) reached similar conclusions for maternal and perinatal morbidity.

1	If the occiput or breech presents immediately over the pelvic inlet, but is not ixed in the birth canal, the presenting part can often be guided into the pelvis by one hand in the vagina, while a second hand on the uterine fundus exerts moderate pressure caudally. A presenting shoulder may be gently converted into a cephalic presentation. Alternatively, with abdominal manipulation, an assistant can guide the presenting part into the pelvis. Sonography can aid guidance and allow heart rate monitoring. Intrapartum external version of a noncephalic second twin has also been described.

1	If the occiput or breech is not over the pelvic inlet and cannot be so positioned by gentle pressure or if appreciable uterine bleeding develops, delivery of the second twin can be problematic. To obtain a favorable outcome, an obstetrician skilled in intrauterine fetal manipulation and anesthesia personnel skilled in providing anesthesia to efectively relax the uterus for vaginal delivery of a noncephalic second twin are essential (American College of Obstetricians and Gynecologists, 2016). To take maximum advantage of the dilated cervix before the uterus contracts and the cervix retracts, delay should be avoided. Prompt cesarean delivery of the second fetus is preferred if no one present is skilled in the performance of internal podalic version or if anesthesia that will provide efective uterine relaxation is not immediately available.

1	With internal podalic version, a fetus is turned to a breech presentation using the hand placed into the uterus (Fig. 45-26). he obstetrician grasps the fetal feet to then efect delivery by breech extraction (Chap. 28, p. 548). As mentioned earlier, Fox and associates (2010) described a strict protocol for management of the delivery of the second twin, which included internal podalic version. hey reported that none of the 110 women who delivered the irst twin vaginally underwent a cesarean delivery for the second twin. Chauhan and coworkers (1r995) compared outcomes of 23 second twins delivered by internal podalic version and breech extraction with those of 21 who underwent external cephalic version. Breech extraction was considered superior to external version because less fetal distress developed. Additional information and illustrations of this procedure are found in Cunningham and Gilstrap 5 Operative Obstetrics, 3rd edition (Yeomans, 2017).

1	FIGURE 45-26 Internal podalic version. Upward pressure on the head by an abdominal hand is applied as downward traction is exerted on the feet. Any attempt to deliver twins vaginally in a woman who has previously undergone one or more cesarean deliveries should be carefully considered. Some studies support the safety of attempting a vaginal birth after cesarean delivery (VBAC) for selected women with twins (Cahill, 2005; Ford, 2006; Varner, 2005). According to the American College of Obstetricians and Gynecologists (2017 c), no evidence currently suggests an increased risk of uterine rupture, and women with twins and one previous cesarean delivery with a low transverse incision may be considered candidates for trial of labor. At Parkland Hospital, we recommend repeat cesarean delivery.

1	Several unusual intraoperative problems can arise during cesarean delivery of twins or higher-order multiples. Supine hypotension is common, and thus gravidas are positioned in a left lateral tilt to delect uterine weight of the aorta (Chap. 4, p. 63). A low transverse hysterotomy is preferable if the incision can be made large enough to allow atraumatic delivery of both fetuses. Piper forceps can be used if the second twin is presenting breech (Fig. 28-11, p. 547). In some cases, a vertical hysterotomy beginning as low as possible in the lower uterine segment may be advantageous. For example, if a fetus is transverse with its back down and the arms are inadvertently delivered irst, it is much easier and safer to extend a vertical uterine incision upward than to extend a transverse incision laterally or to make a "T" incision vertically.

1	Fetal heart rate monitoring during labor with triplet pregnancies is challenging. A scalp electrode can be attached to the presenting fetus, but it is diicult to ensure that the other two fetuses are each being monitored separately. With vaginal deliv ery, the first neonate is usually born with little or no manipula tion. Subsequent fetuses, however, are delivered according to the presenting part. his often requires complicated obstetri cal maneuvers such as total breech extraction with or without internal podalic version or even cesarean delivery. Associated with malposition of fetuses is an increased incidence of cord prolapse. Moreover, reduced placental perfusion and hemor rhage from separating placentas are more likely during delivery. For all these reasons, many clinicians believe that preg cesarean delivery (American College of Obstetricians and

1	For all these reasons, many clinicians believe that preg cesarean delivery (American College of Obstetricians and Gynecologists, 2016). Vaginal delivery is reserved for those cir cumstances in which survival is not expected because fetuses are delivery hazardous to the mother. Others believe that vaginal delivery is safe under certain circumstances. Grobman and asso delivery completion rates of 88 and 84 percent, respectively, in women carrying triplets who underwent a trial of labor. Neo natal outcomes did not difer from those of a matched group of triplet pregnancies undergoing elective cesarean delivery. Con versely, in one review of more than 7000 triplet pregnancies, vaginal delivery was associated with a higher perinatal mortality rate (Vintzeleos, 2005). Lappen and coworkers (2016) reported similar results from the database of the Consortium on Safe

1	Labor. They recommended prelabor cesarean delivery for trip lets. Importantly, the overall cesarean delivery rate among trip lets was 95 percent. In some cases of higher-order multi fetal gestation, reduction of the fetal number to two or three improves survival of the remaining fetuses. Selective reduction implies early pregnancy intervention, whereas selective termination is performed later. he procedure should be performed by an operator skilled and experienced in sonographically guided procedures. Reduction of a selected fetus or fetuses in a multichorionic multifetal gestation may be chosen as a therapeutic intervention to enhance survival of the remaining fetuses (American College of Obstetricians and Gynecologists, 20 17b). One metaanalysis of nonrandomized prospective studies indicates that pregnancy reduction to twins compared with expectant management is associated with lower rates of maternal complications, preterm birth, and neonatal death (Dodd, 2004, 2012).

1	Pregnancy reduction can be performed transcervically, transvaginally, or transabdominally, but the transabdominal route is usually easiest. Transabdominal fetal reductions are typically performed between 10 and 13 weeks' gestation. This gestational age is chosen because most spontaneous abortions have already occurred, the remaining fetuses are large enough to be evaluated sonographically, the amount of devitalized fetal tissue remaining after the procedure is small, and the risk of aborting the entire pregnancy as a result of the procedure is low. The smallest fetuses and any anomalous fetuses are cho sen for reduction. Potassium chloride is then injected under sonographic guidance into the heart or thorax of each selected fetus. Care is used to avoid entry or traverse the sacs of fetuses selected for retention.

1	pregnancies from 1995 to 1998. he pregnancy loss rate var ied from a low of 4.5 percent for triplets that were reduced to twins. The loss rate rose with each addition to the starting num ber of fetuses and peaked at 15 percent for six or more fetuses. Operator skill and experience are believed responsible for the low and declining rates of pregnancy loss. With the identification of multiple fetuses discordant for struc tural or genetic abnormalities, three options are available: abor tion of all fetuses, selective termination of the abnormal fetus, or pregnancy continuation. Because anomalies are typically not discovered until the second trimester, selective termination is greater risk. This procedure is therefore usually not performed unless the anomaly is severe but not lethal. In some cases, ter mination is considered because the abnormal fetus may jeopar dize the normal one.

1	Prerequisites to selective termination include a precise diagnosis for the anomalous fetus and absolute certainty of fetal location. Unless a special procedure such as umbilical cord interruption is used, selective termination should be performed only in multichorionic multifetal gestations to avoid damaging the surviving fetuses (Lewi, 2006). Roman and coworkers (2010) compared 40 cases of bipolar umbilical cord coagulation with 20 cases of radiofrequency ablation for treatment of complicated monochorionic multifetal gestations at midpregnancy. hey found similar survival rates of 87 and 88 percent, and a median gestational age > 36 weeks at delivery in both. Prefumo and colleagues (2013) reported their preliminary experience with microwave ablation of the umbilical cord for selective termination in two monochorionic twin pregnancies. One pregnancy aborted within 7 days, and the other resulted in a term singleton delivered at 39 weeks' gestation.

1	Evans and coworkers (1999) have provided the most comprehensive results to date on second-trimester selective termination for fetal abnormalities. A total of 402 cases were analyzed from eight centers worldwide. Included were 345 twin, 39 triplet, and 18 quadruplet pregnancies. Selective termination using potassium chloride resulted in delivery of a viable neonate or neonates in more than 90 percent of cases, with a mean age of 35.7 weeks at delivery. The entire pregnancy was lost in 7 percent of pregnancies reduced to singletons and in 13 percent of those reduced to twins. The gestational age at the time of the procedure did not appear to afect the pregnancy loss rate.

1	Before selective termination or reduction, a discussion should include the morbidity and mortality rates expected if the pregnancy is continued; the morbidity and mortality rates expected with surviving twins or triplets; and the risks of the procedure itself (American College of Obstetricians and Gynecologists, 2017b). Speciic risks of selective termination or reduction are: abortion of the remaining fetuses; (2) abortion or retention of the wrong fetus(es); (3) damage without death to a fetus; preterm labor; (5) discordant or growth-restricted fetuses; and (6) maternal complications. he last includes potential infection, hemorrhage, or disseminated intravascular coagulopathy because of retained products of conception. The final decision to continue the pregnancy without intervention, to terminate the entire pregnancy, or to elect selective termination is solely the patient' s (Chervenak, 2013).

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1	Roman A, Papanna R, Johnson A, et aI: Selective reduction in complicated monochorionic pregnancies: radiofrequency ablation vs bipolar cord coagulation. Ultrasound Obstet Gynecol 36:37, 2010 Roman A, Rochelson B, Martinelli P, et aI: Cerclage in twin pregnancy with dilated cervix between 16 to 24 weeks of gestation: retrospective cohort study. mJ Obstet GynecoIt215(1):98.e1, 2016 Roman AS, Saltzman DH, Fox N, et al: Prophylactic cerclage in the management of twin pregnancies. mJ PerinatoIt30(9):751, 2013 Romero R, Conde-Agudelo A, EI-Refaie W, et aI: Vaginal progesterone decreases preterm birth and neonatal morbidity and mortality in women with a twin gestation and a short cervix: an updated meta-analysis of individual patient data. Ultrasound Obstet GynecoIt49(3):303, 2017 Ronalds GA, De Stavola BL, Leon DA: he cognitive cost of being a twin: evidence from comparisons within families in the Aberdeen children of the 1950s cohort study. BMJ 331(7528):1306, 2005

1	Ronalds GA, De Stavola BL, Leon DA: he cognitive cost of being a twin: evidence from comparisons within families in the Aberdeen children of the 1950s cohort study. BMJ 331(7528):1306, 2005 Rossi AC, D'Addario V: Laser therapy and serial amnioreduction as treatment for twin-twin transfusion syndrome: a metaanalysis and review of literature. mJ Obstet Gynecol 198:147,t2008 Rossi AC, D'Addario V: Umbilical cord occlusion for selective feticide in complicated monochorionic twins: a systematic review of literature. Am J Obstet GynecoIt200(2):123,t2009 Rossi AC, Mullin PM, Chmait RH: Neonatal outcomes of twins according to birth order, presentation and mode of delivery: a systematic review and meta-analysis. BJOG 118(5):523,t2011 Rothman KJ: Fetal loss, twinning and birthweight ater orl contraceptive use. N Engl J Med 297:468, 1977 Rouse DJ, Caritis SN, Peaceman M, et al: A trial of 17alpha-hydroxyprogesterone caproate to prevent prematurity in twins. N Engl J Med 357:454,t2007

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1	Schmitz T, Prunet C, Azria E, et aI: Association between planned cesarean delivery and neonatal mortality and morbidity in twin pregnancies. Obstet GynecoIt129(6):986,t2017 Sebire NJ, Foskett M, Paradinas FJ, et al: Outcome of twin pregnancies with complete hydatidiform mole and healthy co-twin. Lancet 359:2165,t2002 Senat MV, Deprest J, Boulvain M, et al: Endoscopic laser surgery versus serial amnioreduction for severe twin-to-twin transfusion syndrome. N Engl J Med 351: 136, 2004 Senat MV, Porcher R, Winer N, et al: Prevention of preterm delivery by 17a1pha-hydroxyprogesterone caproate in asymptomatic twin pregnancies with a short cervix: a randomized controlled trial. Am J Obstet Gynecol 208(3):194.e1,t2013 Shen 0, Samuelof A, Beller U, et I: Number of yolk sacs does not predict amnionicity in early first-trimester monochorionic multiple gestations. Ultrasound Obstet Gynecol 27(1):53, 2006

1	Shen 0, Samuelof A, Beller U, et I: Number of yolk sacs does not predict amnionicity in early first-trimester monochorionic multiple gestations. Ultrasound Obstet Gynecol 27(1):53, 2006 Shub A, Walker SP: Planned early delivery versus expectant management of monoamniotic twins. Cochran Database Syst Rev 4:CD008820, 2015 Simpson LL: Ultrasound in twins: dichorionic and monochorionic. Semin PerinatoIt37(5):348,t2013 Slaghekke F, Kist ], Oepkes D, et al: Twin anemia-polycythemia sequence: diagnostic criteria, classiication, perinatal management and outcome. Fetal Diagn Ther 27(4):181,t2010 Smith Gc, Fleming M, White IR: Birth order of twins and risk of perinatal death related to delivety in England, Northern Ireland, and Wales, 19942003: retrospective cohort study. BMJ 334(7593):576, 2007

1	Society for Maternl-Fetal Medicine, Simpson LL: Twin-twin transusion syndrome. Am J Obstet Gynecol 208(1):3, 2013 Sparks TN, Norton ME, Flessel M, et aI: Observed rate of Down syndrome in twin pregnancies. Obstet GynecoIt128(5):1127, 2016 Spencer R: Parasitic conjoined twins: externl, internal (fetuses in fetu and teratomas), and detached (acardiacs). Clin Anat 14:428,2001 Spencer R: Theoretical and analytical embryology of conjoined twins: part I: embryogenesis. Clin Anat 13:36, 2000 Sperling L, Kiil C, Larsen LU, et I: Naturlly conceived twins with monochorionic placentation have the highest risk of fetal loss. Ultrasound Obstet Gynecol 28:644, 2006 Spitz L: Seminars in pediatric surgery: The management of conjoined twins: the great Ormond Street experience. Preface. Semin Pediatr Surg 24(5):201, 2015

1	Spitz L: Seminars in pediatric surgery: The management of conjoined twins: the great Ormond Street experience. Preface. Semin Pediatr Surg 24(5):201, 2015 Stein W, Misselwitz B, Schmidt 5: Twin-to-twin delivery time interval: influencing factors and efect on short term outcome of the second twin. Acta Obstet Gynecol Scand 87(3):346, 2008 Strandskov HH, Edelen EW, Siemens GJ: Analysis of the sex ratios among single and plural births in the total white and colored U.S. populations. m J Phys Anthropol 4:491, 1946 Sugibayashi R, Ozawa K, Sumie M: et aI: Forty cases of twin reversed arterial perfusion sequence treated with radio frequency ablation using the multistep coagulation method: a single-center experience. Prenat Diagn 36(5):437, 2016 Sunderam 5, Kissin DM, Crawford SB, et al: Assisted reproductive technology surveillance-United States, 2014. MMWR 66(6):1,t2017

1	Sunderam 5, Kissin DM, Crawford SB, et al: Assisted reproductive technology surveillance-United States, 2014. MMWR 66(6):1,t2017 Szymusik I, Kosinska-Kaczynska K, Bomba-Opon D, et al: IVG versus spontaneous twin pregnancies-which are at higher risk of complications? J Matern Fetal Neonatal Med 25(12):2725,t2012 Talauliker VS, Arulkumaran 5: Reproductive outcomes after assisted conception. Obstet Gynecol Surv 67(9):566, 2012 Tan L, Goon SM, Salmon Y, et I: Conjoined twins. Acta Obstet Gynecol Scand 50:373, 1971 Tannuri A, Batatinha J, Velhote M, et aI: Conjoined twins-twenty years' experience at a reference center in Brazil. Clinics 68(3):371, 2013

1	Tannuri A, Batatinha J, Velhote M, et aI: Conjoined twins-twenty years' experience at a reference center in Brazil. Clinics 68(3):371, 2013 Taylor M, Rebarber A, Saltzman DH, et aI: Induction of labor in twin compared with singleton pregnancies. Obstet Gynecol 120(2):297,t2012 horngren-Jerneck K, Herbst A: Low 5-minute Apgar score: a populationbased register study of 1 million term births. Obstet GynecoI98(1):65, 2001 horson HL, Ramaeker DM, Emery ST: Optimal interval for ultrasound surveillance in monochorionic twin gestations. Obstet Gynecol 117(1):131, 2011 To MS, Fonseca EB, Molina FS, et al: Maternal characteristics and cervical length in the prediction of spontaneous early preterm delivery in twins. m J Obstet GynecoIt194(5):1360, 2006 Trivedi N, GillettR: he retained twinltriplet following a preterm deliveryan analysis of the literature. Aust N Z J Obstet GynaecoI38:461, 1998

1	Trivedi N, GillettR: he retained twinltriplet following a preterm deliveryan analysis of the literature. Aust N Z J Obstet GynaecoI38:461, 1998 Turpin R, Lejeune J, Lafourcade J, et I: Presumption of monozygotism in spite of sexul dimorphism: Y male subject and haploid X neuter subject. C R Hebd Seances Acad Sci 252:2945, 1961 Varner MW, Leindecker S, Spong CY, et al: he Maternal-Fetal Medicine Unit Cesarean Registry: trial of labor with a twin gestation. mJ Obstet Gynecol 193:135,t2005 Vintzileos AM, Ananth CV, Kontopoulos E, et al: Mode of delivety and risk of stillbirth and infant mortality in triplet gestations: United States, 1995 through 1998. m J Obstet Gynecol 192:464,t2005 Vora NL, Ruthazer R, House M, et al: Triplet ultrasound growth parameters. Obstet GynecoIt107:694, 2006 Wada H, Nunogami K, Wada T, et aI: Difuse brain damage caused by acute twin-twin transfusion during late pregnancy. Acta Paediatr Jpn 40:370, 1998

1	Obstet GynecoIt107:694, 2006 Wada H, Nunogami K, Wada T, et aI: Difuse brain damage caused by acute twin-twin transfusion during late pregnancy. Acta Paediatr Jpn 40:370, 1998 Walker MC, Murphy E, Pan 5, et I: Adverse maternal outcomes in multifetal pregnancies. BJOG Ill:1294, 2004 Waller DK, Tita TN, Annegers JF: Rates of twinning before and ater fortiication of foods in the U.S. with folic acid, Texas, 1996 to 1998. Paediatr Perinat EpidemioIt17(4):378, 2003 Wen SW, Demissie K, Yang , et al: Maternal morbidity and obstetric complications in triplet pregnancies and quadruplet and higher-order multiple pregnancies. mJ Obstet Gynecol 191 :254, 2004 White C, Wyshak G: Inheritance in human dizygotic twinning. N Engl J Med 271:1003, 1964 Wolfe MD, de la Torre L, Moore LE, et al: Is the protocol for induction of labor in singletons applicable to twin gestations? J Reprod Med 58(304): 137, 2013

1	Wolfe MD, de la Torre L, Moore LE, et al: Is the protocol for induction of labor in singletons applicable to twin gestations? J Reprod Med 58(304): 137, 2013 Yamasmit W, Chaithongwongwatthana 5, Tolosa JE, et al: Prophylactic oral betamimetics for reducing preterm birth in women with a twin pregnancy. Cochrane Database Syst Rev 12:CD004733, 2015 Yang Q, Wen SW, Chen Y, et al: Occurrence and clinical predictors of operative delivery for the vertex second twin after normal vaginal delivery of the irst twin. Am J Obstet Gynecol 192(1): 178, 2005a Yang Q, Wen SW, Chen Y, et al: Neonatal death and morbidity in vertexnonvertex second twins according to mode of delivery and birth weight. Am J Obstet GynecoIt192(3):840, 2005b Yeomans ER: Delivery of twin gestations. In Yeomans ER, Hofman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hill Education, 2017

1	Yeomans ER: Delivery of twin gestations. In Yeomans ER, Hofman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hill Education, 2017 Yinon Y, Mazkereth R, Rosentzweig N, et l: Growth restriction as a determinant of outcome in preterm discordant twins. Obstet Gynecol 105(1):80,t2005 Young BC, Wylie BJ: Efects of twin gestation on maternal morbidity. Semin Perinatol 36(3):162, 2012 Zech NH, Wisser J, Natalucci G, et al: Monochorionic-diamniotic twins discordant in gender form a naturally conceived pregnancy through postzygotic sex chromosome loss in a Y zygote. Prenat Diagn 28:759, 2008 Zhao DP, de Villiers SF, Slaghekke F, et al: Prevalence, size, number and localization of vascular anastomoses in monochorionic placentas. Placenta 34:589,t2013

1	Y zygote. Prenat Diagn 28:759, 2008 Zhao DP, de Villiers SF, Slaghekke F, et al: Prevalence, size, number and localization of vascular anastomoses in monochorionic placentas. Placenta 34:589,t2013 Zwijnenburg PJ, Meijiers-Heijboer H, Boomsma DI: Identical but not the same: the value of discordant monozygotic twins in genetic research. Am J Med Genet B Neuropsychiatr Genet 153B(6):1134, 2010 MEDICATIONS AND SURGERIES ................... 901 LAPAROSCOPIC SURGERY ....................... 901 RADIOGRAPHY ................................. 904 DIAGNOSTIC RADIATION . . . . . . . . . . . . . . . . . . . . . . . . 906 SONOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 909 MAGNETIC RESONANCE IMAGING ................. 909 As a rule, al diseases which subject the organism to a considerable strain are much more serious when occurring in a pregnant woman. -J. Whitridge Williams (1903)

1	As a rule, al diseases which subject the organism to a considerable strain are much more serious when occurring in a pregnant woman. -J. Whitridge Williams (1903) As reviewed by Williams in 1903, pregnant women are susceptible to any medical and surgical disorder that can afect childbearing-aged women. Some of these, especially those that are chronic, more often precede pregnancy. But, they can acutely complicate an otherwise normal pregnancy. Of estimates, one managed-care population had an overall antenatal hospitalization rate of 10.1 per 100 deliveries (Gazmararian, 2002). Approximately one third was for nonobstetrical conditions that included renal, pulmonary, and infectious diseases. In another study from the 2002 Nationwide Inpatient Sample, the injury hospitalization rate was 4.1 women per 1000 deliveries (Kuo, 2007). Last, approximately 1 in every 635 pregnant women will undergo a nonobstetrical surgical procedure (Corneille, 2010; Kizer, 2011).

1	Obstetricians should have a working knowledge of the wide-ranging medical disorders common to childbearing-aged women. Many of these are within the purview of the general obstetrician. Other disorders, however, will warrant consultation, and still others require a multidisciplinary team. The latter may include maternal-fetal medicine specialists, hospitalists, internists and medical subspecialists, surgeons, anesthesiologists, and numerous other disciplines (Levine, 2016). The American College of Obstetricians and Gynecologists and the Society for Maternal-Fetal Medicine (2014, 2017b) has redefined aspects of maternal care and proposed required levels of specialized care. It should be axiomatic that a woman must never be penalized because she is pregnant. To ensure this, several questions should be addressed: What management would be recommended if the woman were not pregnant? If the proposed management is diferent because the woman is pregnant, can this be justiied?

1	What management would be recommended if the woman were not pregnant? If the proposed management is diferent because the woman is pregnant, can this be justiied? What are the risks versus benefits to the mother and her fetus, and are they counter to each other? Can an individualized management plan be devised that balances beneits versus risks of any alterations? Such an approach allows individualized care for women with most medical and surgical disorders complicating pregnancy.

1	Such an approach allows individualized care for women with most medical and surgical disorders complicating pregnancy. Pregnancy induces physiological changes in virtually all organ systems. In turn, results of numerous laboratory tests are altered, and some values would, in the nonpregnant woman, be considered abnorml. Conversely, some may appear to be within a normal range but are decidedly abnormal for the gravida. These changes may ampliy or obuscate evaluation of coexisting conditions. he wide range of pregnany efects on normal physiology and laboratory values are discussed in the chapters that follow in this section and are listed in the Appendix (p. 1255). Fortunately, most medications needed to treat frequently with relative safety. That said, notable exceptions are considered in Chapter 12 and throughout this text.

1	Fortunately, most medications needed to treat frequently with relative safety. That said, notable exceptions are considered in Chapter 12 and throughout this text. Regarding surgery, the risk of an adverse pregnancy out come is not appreciably increased in most women who undergo an uncomplicated operative procedure. With complications, however, risks likely are increased. For example, perforative appendicitis with feculent peritonitis has significant maternal and anesthetic techniques are flawless. Conversely, procedure related complications may adversely afect outcomes. For exam ple, a woman who has uncomplicated removal of an inflamed appendix may sufer aspiration of acidic gastric contents dur ing tracheal intubation or extubation. Still, compared with nonpregnant women undergoing similar procedures, pregnant women do not appear to have excessive complications (Silvestri, 2011). In a study of the American College of Surgeons'

1	National Surgical Quality Improvement Program, outcomes in pregnant women were compared with matched nonpregnant controls (Moore, 2015). The investigators reported similar out comes in the two cohorts, each with 2539 patients. In a smaller study, however, women undergoing nonobstetrical surgery after 23 weeks' gestation had a high rate of subsequent preterm delivery (Baldwin, 2015).

1	The most extensive data regarding anesthetic and surgical risks for the gravida and her fetus are from the Swedish Birth and described by Mazze and Kallen (1989). The efects on pregnancy outcomes of 5405 nonobstetrical surgical procedures performed in 720,000 pregnant women from 1973 to 1981 were analyzed. For approximately half of these procedures, general anesthesia was used and commonly involved nitrous oxide supplemented by another inhalation agent or intravenous medications. These procedures were performed in 41 percent of women in the first trimester, 35 percent in the second, and 24 percent in the third. Overall, 25 percent were abdominal operations, and 20 percent were gynecological or urological procedures. Laparoscopy was the most frequently performed operation, and appendectomy was the most common second-trimester procedure.

1	Excessive perinatal morbidity associated with nonobstetrical surgery is attributable in many cases to the disease itself rather than to adverse efects of surgery and anesthesia. he Swedish Birth Registry again provides valuable data (Table Importantly, the incidences of congenital malformations or of stillbirths were not significantly diferent from those of nonexposed control newborns. However, incidences of low birthweight, preterm birth, and neonatal death in infants born to women who had undergone surgery were significantly greater. TABLE 46-1. Birth Outcomes in 5405 Pregnant Women Undergoing Nonobstetrical Surgery Major malformation 1.9% NS Stillbirth 7 per 1000 NS Neonatal death by 7 days 10.5 per 1 000 <0.05 Preterm <37 wk 7.5% <0.05 Birthweight < 1500 g 1.2% <0.05 Birthweight <2500 g 6.6% <0.05 aCompared with 720,000 pregnancies in women without surgery. NS = not significant. Data from Mazze, 1989.

1	Preterm <37 wk 7.5% <0.05 Birthweight < 1500 g 1.2% <0.05 Birthweight <2500 g 6.6% <0.05 aCompared with 720,000 pregnancies in women without surgery. NS = not significant. Data from Mazze, 1989. Increased neonatal death rates were largely due to preterm birth. In two other studies, the preterm delivery rate in women undergoing nonobstetrical surgery was also elevated (Baldwin, 2015; Hong, 2006). Rates of fetal abnormalities with maternal surgery in early pregnancy do not appear increased. Kallen and Mazze (1990) scrutinized 572 operations performed at 4 to 5 weeks' gestation and reported a nonsignificant relationship with elevated neural-tube defect rates. In a similar study from a Hungarian database, Czeizel and colleagues (1998) found no evidence that anesthetic agents were teratogenic.

1	Laparoscopy has become the most common first-trimester procedure used for diagnosis and management of several surgical disorders. In 2017, the Society of American Gastrointestinal and Endoscopic Surgeons (SAGES) updated its recommendations concerning laparoscopy use in pregnant women (Table 46-2).

1	Information regarding surgical approach selection in pregnancy comes from the American College of Surgeons database (Silvestri, 2011). During the 5-year period ending in 2009, almost 1300 pregnant women were studied who had undergone either appendectomy or cholecystectomy. Open appendectomy was performed in 36 percent of 857 gravidas compared with only 17 percent of those not pregnant. Of those undergoing cholecystectomy, an open procedure was used in 10 percent of 436 pregnant women compared with 5 percent of nonpregnant women. No randomized trials compare laparoscopic with open surgery, however, most reviews report equally satisfactory outcomes (Bunyavejchevin, 2013; Cox, 2015; Fatum, 2001). The most frequently performed procedures were cholecystectomy, adnexal surgery, and appendectomy. For adnexal mass surgery in pregnancy, laparoscopy is preferred, and several studies conirm its relative safety (Daykan, 2016; Hoover, 2011; Webb, 2015). At first, 26 to 28 weeks became the upper

1	For adnexal mass surgery in pregnancy, laparoscopy is preferred, and several studies conirm its relative safety (Daykan, 2016; Hoover, 2011; Webb, 2015). At first, 26 to 28 weeks became the upper gestational-age limit recommended, but as experience has accrued, many now describe laparoscopic surgery performed in the third trimester (Kizer, 2011). In one report of 59 gravidas undergoing laparoscopic cholecystectomy or appendectomy, a third were > 26 weeks'

1	TABLE 46-2. Some Guidelines from the Society of American Gastrointestinal Endoscopic Surgeons (SAGES) for Laparoscopic Surgery in Pregnant Women Indications-same as for nonpregnant women Investigation of acute abdominal processes Appendectomy, cholecystectomy, nephrectomy, adrenalectomy, splenectomy Position: lateral recumbent Entry: open technique, careful Veress needle, or optical trocar; fundal height may alter insertion site selection Trocars: direct visualization for placement; fundal height may alter insertion site selection CO2 insuflation pressures: 10-15 mm Hg Monitoring: capnography intraoperatively, FHR assessment pre-and postoperatively Perioperative pneumatic compression devices and early postoperative ambulation CO2 = carbon dioxide; FHR = fetal heart rate. Data from Pearl, 2017.

1	CO2 = carbon dioxide; FHR = fetal heart rate. Data from Pearl, 2017. gestation (Rollins, 2004). No serious adverse sequelae are In women, cardiorespiratory changes are generally not linked to these procedures. In addition, laparoscopic splesevere if insulation pressures are kept below 15 mm Hg. With nectomy, adrenalectomy, and nephrectomy have also been noninvasive hemodynamic monitoring in women at midpregdescribed in pregnant women (Asizare, 2014; Dong, 2014; nancy, the cardiac index decreased 26 percent by 5 minutes of Gernsheimer, 2007; Miller, 2012; Stroup, 2007). insulation and 21 percent by 15 minutes (Steinbrook, 2001). Despite this, mean arterial pressures, systemic vascular resistance, and heart rate did not change significantly.

1	Despite this, mean arterial pressures, systemic vascular resistance, and heart rate did not change significantly. changes that are summarized in Table 46-3. Reedy and associ ates (1995) studied baboons at the human equivalent of 22 to Laparoscopic surgery frequently is ideal for the obese woman 26 weeks' gestation. No substantive physiological changes were (Sisodia, 2015). However, some outcomes may be adversely found with insulation pressures of 10 mm Hg, but 20 mm afected in obese gravidas compared with normal-weight

1	Hg caused significant maternal cardiovascular and respiratory patients. Of these, higher conversion rates to laparotomy, changes after 20 minutes. hese included increased respiratory longer operating times, and longer hospitalizations have rate, respiratory acidosis, diminished cardiac output, and been reported. Also, adequate ventilation is more diicult, increased pulmonary artery and capillary wedge pressures. and greater pneumoperitoneal pressures are needed to create aEffects intensified when insufflation pressure >20 mm Hg in baboons (Reedy, 1995). bData primarily from animal studies. CO2 =carbon dioxide; CSF = cerebrospinal fluid; PC02 = patial pressure of CO2, Data from O'Rourke, 2006; Reynolds, 2003. suitable operating space. here is anatomical distortion of the abdominal wall with displaced landmarks. Finally, the risk of developing hernias at port sites is greater.

1	suitable operating space. here is anatomical distortion of the abdominal wall with displaced landmarks. Finally, the risk of developing hernias at port sites is greater. Because precise efects of laparoscopy in the human fetus are unknown, animal studies are informative. In early studies of pregnant ewes, various investigators reported that uteroplacental blood low declines when intraperitoneal insulation pressure exceeded 15 mm Hg (Barnard, 1995; Hunter, 1995). This was the result of decreased perfusion pressure and increased placental vessel resistance (see Table 46-3). The previously cited baboon studies by Reedy and coworkers (1995) produced similar findings.

1	Perinatal outcomes in women are limited to observational studies. Reedy and colleagues (1997) used the updated Swedish Birth Registry database to analyze a 20-year period with more than 2 million deliveries. Of 2181 laparoscopic procedures, most were performed during the irst trimester. Perinatal outcomes for these women were compared with those of all women in the database and those undergoing open surgical procedures. These investigators confirmed the earlier indings of an increased risk of low birthweight, preterm delivery, and fetal-growth restriction. Diferences were not found, however, in outcomes of women undergoing laparoscopy versus laparotomy. n observational study of 262 women undergoing surgery for an adnexal mass noted similar findings (Koo, 2012). The following description is an overview of laparoscopic techniques in pregnancy. For a detailed description refer to Chapter 15 in Cunningham and Gistrap s Operative Obstetrics, 3rd edition (Kho, 2016).

1	Preparation for laparoscopy difers little from that used for laparotomy. Bowel cleansing is not needed but may aid visualization and manipulations by emptying the large intestine. N asogastric or orogastric decompression reduces the risk of stomach trocar puncture and aspiration. Aortocaval compression is avoided by a left-lateral tilt. Positioning of the lower extremities in boot-type stirrups maintains access to the vagina for fetal sonographic assessment or manual uterine displacement. Intrauterine manipulators are logically avoided. Most reports describe the use of general anesthesia after tracheal intubation with monitoring of end-tidal carbon dioxideEtC02 (Hong, 2006; Ribic-Pucelj, 2007). With controlled ventilation, EtC02 is maintained at 30 to 35 mm Hg.

1	Beyond the irst trimester, technical modifications of standard pelvic laparoscopic entry are required to avoid uterine puncture or laceration (Fig. 46-1). Many recommend open enuy techniques to avoid perforations of the uterus, pelvic vessels, and adnexa (Kizer, 2011; Koo, 2012). The abdomen is incised at or above the umbilicus, and the peritoneal cavity entered under direct visualization (Fig. 46-2). At this point, the cannula is then connected to the insulation system, and a 12-mm Hg pneumoperitoneum is created. The initial insulation should be conducted slowly to allow for prompt assessment and reversal of any untoward pressure-related efects. Gas leakage around the cannula is managed by tightening the surrounding skin with a towel clamp. Insertion of secondary trocars into the abdomen is most safely performed under direct laparoscopic viewing. Single-port surgery has also been described (Dursun, 20l3).

1	In more advanced pregnancies, direct entry through a left upper quadrant port in the midclavicular line, 2 cm beneath the costal margin, may better avoid the fundus (Donkervoort, 2011; Stepp, 2004). Known as Palmer point, this entry site is also used in gynecological laparoscopy because visceroparietal adhesions infrequently form here (Vilos, 2007). FIGURE 46-1 Pregnant uterus at 10,o20, and 36 weeks' gestation depicting distortion of other intraperitoneal organs. (Reproduced with permission from Kho KA: Diagnostic and operative laparoscopy. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics 3rd ed, New York McGraw-Hili Education, 201o7.)

1	FIGURE 46-2 Open entry technique for laparoscopic instrument placement. A. Fascia grasped with two Allis clamps and elevated prior to sharp incision. B.Two fascial stitches incorporate the peritoneum and fascia. C. These fascial sutures are wrapped around holders of the Hasson cannula to anchor it in place. (Reproduced with permission from Kho KA: Diagnostic and operative laparoscopy. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics 3rd ed, New York McGraw-Hili Education, 201o7.) Gasless laparoscopy is a less commonly selected alternative approach that uses a rod with intraabdominal fan-blade-shaped retractors. When opened, these allow the abdominal wall to be lifted upward. It avoids the typical laparoscopic cardiovascular changes because the pneumoperitoneum is created by retraction rather than insuiation (Phupong, 2007).

1	Risks inherent to any abdominal endoscopic procedure are probably increased slightly during pregnancy. The obvious unique complication is perforation of the pregnant uterus with either a trocar or Veress needle (Azevedo, 2009; Kizer, 2011; Mala, 2014). hat said, reported complications are infrequent (Fatum, 2001; Koo, 2012). One Cochrane database review noted a need for randomized trials to deduce comparative benefits and risks of laparoscopy versus laparotomy during pregnancy (Bunyavejchevin, 2013). Pragmatically, this seems unfeasible, and common sense should dictate the approach.

1	Imaging modalities are used as adjuncts for diagnosis and therapy during pregnancy. Options include sonography, radiography, and magnetic resonance (MR) imaging. Of these, radiography is the most problematic. Inevitably, some radiographic procedures are performed before recognition of early pregnancy, usually because of trauma or serious illness. Fortunately, most diagnostic radiographic procedures are associated with minimal fetal risks. As with drugs and medications, however, these procedures may lead to litigation if pregnancy outcome is adverse. And, x-ray exposure may lead to a needless therapeutic abortion because of patient or physician anxiety.

1	Since 2007, the American College of Radiology (ACR) has addressed the growing concern of radiation dose in all ields of medicine (Amis, 2007). Some of its goals were to limit exposure through radiation safety practices and promote lifelong accumulated records of exposures in any given patient. Task Force recommendations included additional considerations for special radiosensitive populations, such as children and pregnant and potentially pregnant women. At our institutions, special recommendations are made for gravidas. Radiation exposure values and duration are recorded and monitored in high-exposure areas such as computed tomography (CT) and luoroscopy.

1	he term radiation literally refers to energy transmission and thus is often applied not only to x-rays, but also to microwaves, ultrasound, diathermy, and radio waves. Of these, x-rays and gamma rays have short wavelengths with very high energy and are ionizing radiation forms. The other four energy forms have rather long wavelengths and low energy (Brent, 1999b, 2009). Ionizing radiation refers to waves or particles-photons-of signiicant energy that can change the structure of molecules such as those in DNA or that can create free radical or ions capable of secondarily damaging tissue (Hall, 1991; National Research Council, 1990). Methods of measuring the efects of x-rays are summarized in Table 46-4. The standard terms used are exposure TABLE 46-4. Some Measures of Ionizing Radiation Exposure Number of ions produced by x-rays per kg Dose Amount of energy deposited per kg of tissue

1	TABLE 46-4. Some Measures of Ionizing Radiation Exposure Number of ions produced by x-rays per kg Dose Amount of energy deposited per kg of tissue Relative Amount of energy deposited per kg effective of tissue normalized for biological (in air), dose (to tissue), and reative fictive dose (to tissue). In the range of energies for diagnostic x-rays, the dose is now expressed in grays (Gy) , and the relative efective dose is expressed in sieverts (Sv). hese can be used interchangeably. For consistency, all doses discussed subsequently are expressed in contemporane ously used units of gray (1 Gy = 100 rad) or sievert (1 Sv = 100 rem) . To convert, 1 Sv = 100 rem = 100 rad. As noted, the biological efects of x-rays are caused by an electrochemical reaction that can damage tissue. According to

1	As noted, the biological efects of x-rays are caused by an electrochemical reaction that can damage tissue. According to Brent (1999a, 2009), x-and gamma-radiation at high doses can create two types of biological efects and reproductive risks in the fetus. These are deterministic eicts and stochastic focts, which are both described in the next sections. One potential harmful efect of radiation exposure is determin istic, which may result in abortion, growth restriction, congeni tal malformations, microcephaly, or mental retardation. hese deterministic efects are threshold efects, and the threshold level is the NOAEL-No Observed Adverse Eict Level (Brent, 2009). lthough controversial, the NOEL concept supports that there is no risk below a threshold dose (0.05 Gy or 5 rad). It also suggests that the threshold for gross fetal malformations is more likely to be 0.2 Gy (20 rad).

1	It also suggests that the threshold for gross fetal malformations is more likely to be 0.2 Gy (20 rad). The harmful deterministic efects of ionizing radiation have been extensively studied for cell damage with resultant disordered embryogenesis. These have been assessed in animal models, as well as in Japanese atomic bomb survivors and the Oxford Survey of Childhood Cancers (Sorahan, 1995). Additional sources have confirmed prior observations and provided more information (Groen, 2012). One is a 2003 International Commission on Radiological Protection publication that describes biological fetal efects from prenatal irradiation. Another is the Biological Efects of Ionizing Radiation-BEIR II Phase 2 report of the National Research Council (2006), which discusses health risks from exposure to low levels of ionizing radiation.

1	In the mouse model, the lethality risk is highest during the preimplantation period-up to 10 days postconception (Kanter, 2014). This is likely due to blastomere destruction caused by chromosomal damage (Hall, 1991). During organogenesis, high-dose radiation-1 Gy or 100 rad-is more likely to cause malformations and growth restriction and less likely to have lethal efects in the mouse. Studies of brain development suggest efects on neuronal development and a window of cortical sensitivity in early and midfetal periods. That said, acute lowdose ionizing radiation appears to have no deleterious efects (Howell,r2013). Data on adverse human efects of high-dose ionizing radiation mostly derive from the atomic bomb survivors of Hiroshima and Nagasaki (Greskovich, 2000; Otake, 1987). The International Commission on Radiological Protection (2003) confirmed initial studies showing that the increased risk of severe mental retardation was greatest between 8 and 15 weeks' gestation.

1	There may be a lower-threshold dose of 0.3 Gy (30 rad), which is a range similar to the window of cortical sensitivity in the mouse model discussed earlier. he mean decrease in intelligence quo tient (IQ) scores was 25 points per Gy or 100 rad. here appears to be linear dose response, but it is not clear whether there is a threshold dose. Most estimates err on the conservative side by assuming a linear nonthreshold hypothesis. In a study of fetuses exposed to low radiation doses, Choi and colleagues (2012) did not find an increased risk for congenital anomalies. Finally, an increased risk of mental retardation in humans < 8 weeks' or > 25 weeks' gestation has not been documented, even with doses exceeding 0.5 Gy or 50 rad (International

1	Finally, an increased risk of mental retardation in humans < 8 weeks' or > 25 weeks' gestation has not been documented, even with doses exceeding 0.5 Gy or 50 rad (International Commission on Radiological Protection, 2003). Reports have described high-dose radiation used to treat women for malig nancy, menorrhagia, and uterine myomas. Dekaban (1r968) described 22 infants with microcephaly, mental retardation, or both following exposure in the first half of pregnancy to an estimated 2.5 Gy or 250 rad. Summary of Fetal Radiation Exposure From 8 to 15 weeks, the fetus is most susceptible to radiation induced mental retardation. It has not been resolved whether this is a threshold or nonthreshold linear function of dose. he

1	From 8 to 15 weeks, the fetus is most susceptible to radiation induced mental retardation. It has not been resolved whether this is a threshold or nonthreshold linear function of dose. he Committee on Biological Efects (1990) estimates the risk of severe mental retardation to be as low as 4 percent for 0.1 Gy (10 rad) and as high as 60 percent for 1.5 Gy (150 rad). But recall that these doses are 2 to 100 times higher than those considered maximal from diagnostic radiation. Importantly, cumulative doses from multiple procedures may reach the harmful range, especially at 8 to 15 weeks' gestation. At 16 to 25 weeks, the risk is less. And again, there is no proven risk before 8 weeks or after 25 weeks.

1	Importantly, embryofetal risks from low-dose diagnostic radiation appear to be minimal. Current evidence suggests that risks for malformations, growth restriction, or abortion are not increased from a radiation dose of less than 0.05 Gy (5 rad). Indeed, Brent (2009) concluded that gross congenital malformations would not be increased with exposure to less than 0.2 Gy (20 rad). Because diagnostic x-rays seldom exceed 0.1 Gy (10 rad) , Strzelczyk and associates (2007) concluded that these procedures are unlikely to cause deterministic efects. As emphasized by Groen and coworkers (2012), 0.1 Gy is the radiation equivalent to that from more than 1000 chest x-rays!

1	These efects refer to random, presumably unpredictable oncogenic or mutagenic efects of radiation exposure. Stochastic efects concern associations between fetal diagnostic radiation exposure and increased risk of childhood cancers or genetic diseases. According to Doll and Wakeford (1997), as well as the National Research Council (2006) BEIR VII Phase 2 report, excess cancers can result from in utero exposure to doses as low as 0.01 Svror 1 rad. Stated another way by Hurwitz and colleagues (2006), the estimated risk of childhood cancer following fetal exposure to 0.03 Gy or 3 rad doubles the background risk of 1 in 600 to that of 2 in 600.

1	In one report, in utero radiation exposure was determined for 10 solid cancers in adults from age 17 to 45 years. here was a dose-response relationship as previously noted at the 0.r1 Sv or 10 rem threshold. hese cancers likely are associated with a complex series of interactions between DNA and ionizing radiation. They also make it more problematic to predict cancer risk from low-dose radiation of less than 0.1 Sv or 10 rem. Importantly, below doses of 0.1 to 0.2 Sv, there is no convincing evidence of a carcinogenic efect (Brent, 2009, 2014; Preston, 2008; Strzelczyk, 2007).

1	Estimates of dose to the uterus and embryo for various frequently used radiographic examinations are summarized in Table 46-5. Imaging of maternal body parts farthest from the uterus results in a very small dose of radiation scatter to the embryo or fetus. The size of the woman, radiographic technique, and equipment performance are other variables (Wagner, 1997). hus, data in the table serve only as guidelines. When the radiation dose for a specific individual is required, a medical physicist should be consulted. Brent (2009) recommends consulting the Health Physics Society website (ww.hps.org) to view some examples of questions and answers posed by patients exposed to radiation.

1	he Radiation Therapy Committee Task Group of the American Association of Physics in Medicine (Stovall, 1995) emphasizes careful individualization of radiotherapy for the pregnant woman (Chap. 63, p. 1191). For example, in some cases, shielding of the fetus and other safeguards can be employed (Fenig, 2001; Nuyttens, 2002). In other instances, the fetus will be exposed to dangerous radiation doses, and a carefully designed plan must be improvised (Prado, 2000). Examples include models that estimate the fetal dose given during maternal brain radiotherapy or tangential breast irradiation (Mazonakis, 1999, 2003). The harmful efects of radiotherapy on future fertility and pregnancy outcomes were reviewed by Wo and Viswanathan (2009) and others and are detailed in Chapter 63 (p. 1192).

1	To estimate fetal risk, approximate x-ray dosimetry must be known. According to the merican College of Radiology, no single diagnostic procedure results in a radiation dose signiicant enough to threaten embryo-fetal well-being (Hall, 1991).

1	For standard radiographs, dosimetry is presented in Table 46-5. In pregnancy, the AP-view chest radiograph is the most commonly used study, and fetal exposure is exceptionally small-0.0007 Gy or 70 mrad. With one abdominal radiograph, because the embryo or fetus is directly in the x-ray beam, the dose is higher-O.OO 1 Gy or 100 mrad. he standard intravenous pyelogram may exceed 0.005 Gy or 500 mrad because of several exposures. The one-shot pyelogram described in Chapter 53 (p. 1030) is useful when urolithiasis or other causes of obstruction are unproven by sonography but still suspected. Most "trauma series," such as radiographs of an extremity, skull, or rib series, deliver low doses because of the fetal distance from the target area (Shakerian, 2015).

1	Dosimetry calculations are much more diicult with these procedures because of variations in the number of radiographs obtained, total fluoroscopy time, and luoroscopy time in which the fetus is in the radiation field. As shown in Table 46-6, the range is variable. he Food and Drug Administration (FDA) limits the exposure rate for conventional fluoroscopy such as barium studies, however, special-purpose systems such as angiography units have the potential for much higher exposure. Dosea per View Study View (mGy) No. Filmsb Dose (mGy) TABLE 6-5. Dose to the Uterus for Common Radiologic Procedures SkuW AP, PA, Lat <0.0001 Chest AP, PN, Latd <0.0001n-0.0008 <0.0005 1.5 0.0002-0.0007 Mammogramd Cc, Lat <0.0003-0.0005 4.0 0.0007 -0.002 Lumbosacral spinee AP, Lat 1.14-2.2 3.4 1.76-3.6 Abdomene AP 1.0 0.8-1n.63 Intravenous pyelograme 3 views 5.5 6.9-14 Hipb (single) AP 0.7-1.4 2.0 1-2

1	Hipb (single) AP 0.7-1.4 2.0 1-2 Lat 0.18-0.51 aCalculated for x-ray beams with half-value layers ranging from 2 to 4 mm aluminum equivalent using the methodology of Rosenstein, 1988. bBased on data and methods reported by Laws, 1978. (Entrance exposure data from Conway, 1989. dEstimates based on compilation of above data. eBased on NEXT data reported in National Council on Radiation Protection and Measurements, 1989. AP = anterior-posterior; CC = cranial-caudal; Lat = lateral; PA = posterior-anterior. TABLE 46-6. Estimated X-Ray Doses to the Uterus/ Embryo from Common Fluoroscopic Procedures Dose to Fluoroscopic Cerebral angiographl <0.1 Cardiac angiographyb,( 0.65 223 (± 11n8) Single-vessel PTCAb,c 0.60 1023 (± 952) Double-vessel PTCAb,c 0.90 1186 (± 593) Upper gastrointestinal seriesd 0.56 136 ,e 0.06 192 Barium enemab,f,9 20-40 289-311 aWagner, 1997. Calculations based on data of Gorson, 1984. cFinci, 1987. dSuleiman, 1991n.

1	Upper gastrointestinal seriesd 0.56 136 ,e 0.06 192 Barium enemab,f,9 20-40 289-311 aWagner, 1997. Calculations based on data of Gorson, 1984. cFinci, 1987. dSuleiman, 1991n. eBased on female data from Rowley, 1987. fAssumes embryo in radiation field for entire examination. 9Bednarek, 1983. SD = standard deviation. Angiography and vascular embolization may occasionally be necessary for trauma and for serious maternal disorders, especially renal disease (Wortman, 2013). As before, a greater distance from the embryo or fetus lowers the exposure and risk. These x-ray images are usually performed by obtaining a spiral of 360-degree images that are postprocessed in multiple planes. Of these, the axial image remains the most commonly obtained

1	These x-ray images are usually performed by obtaining a spiral of 360-degree images that are postprocessed in multiple planes. Of these, the axial image remains the most commonly obtained CT (MDCT) images are now standard for common clinical indications. The most recent detectors have 16 or 64 channels, and multidetector protocols may result in increased dosimetry compared with traditional CT imaging. Several imaging parameters have an efect on exposure (Brenner, 2007). These include pitch, kilovoltage, tube current, collimation, number of slices, tube rotation, and total acquisition time. If a study is performed with and without contrast, the dose is doubled because twice as many images are obtained. Fetal exposure is also dependent on factors such as maternal size as well as fetal size and position. And as with plain radiography, the closer the target area is to the fetus, the greater the delivered dose.

1	Cranial CT scanning is the most commonly performed study in gravidas. It is used for neurological disorders as discussed in Chapter 60 (p. 1156) and with eclampsia as noted in Chapter 40 (p. 724). Nonenhanced CT scanning is commonly used to detect acute hemorrhage within the epidural, subdural, or subarachnoid spaces (Fig. 46-3). Because of the distance from the fetus, radiation dosage is negligible (Goldberg-Stein, 2012). Abdominal procedures are more problematic. Hurwitz and associates (2006) employed a 16-channel multidetector FIGURE 46-3 A 37-year-old with intrapartum eclampsia at term. renchymal hematoma (H) with intraventricular extension (arrow heads). The midline (arrow) is shifted to the right due to mass effect from the hematoma. (Reproduced with permission from Kho KA: Diagnostic and operative laparoscopy. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap's Operative

1	Obstetrics 3rd ed, New York McGraw-Hili Education, 2017.) scanner to calculate fetal exposure at 0 and 3 months' gestation using a phantom model. Calculations were made for three commonly requested procedures in pregnant women (Table 46-7). he pulmonary embolism protocol has the same dosimetry exposure as the ventilation-perfusion (V/Q) lung scan discussed on page 908. Because of the pitch used, the appendicitis protocol has the highest radiation exposure, however, it is very useful clinically when MR imaging is not available. Using a similar protocol in 67 women with suspected appendicitis, Lazarus and coworkers (2007) reported sensitivity of 92 percent, specificity of 99 percent, and a negative-predictive value of 99 percent. Here, dosimetry was markedly decreased compared with standard appendiceal imaging because of a different pitch. his is discussed further in Chapter 54 (p. 1052). Last, for suspected urolithiasis, the multidetector-scan protocol is used if sonography is

1	standard appendiceal imaging because of a different pitch. his is discussed further in Chapter 54 (p. 1052). Last, for suspected urolithiasis, the multidetector-scan protocol is used if sonography is nondiagnostic. Using a similar protocol, White and colleagues (2007) identified urolithiasis in 13 of 20 women at an average of 26.5 weeks. Finally, as shown in

1	TABLE 46-7. Estimated Radiation Dosimetry with , 6-Channel Multidetector Computed Pulmonary embolism 0.20-0.47 0.61-0.66 Data from Hurwitz, 2006. FIGURE 46-4 This woman in her third trimester was involved in a high-speed motor vehicle accident. A. Maximum intensity projection acquired for maternal indications readily identifies fetal skull fractures (arrows). B.3-D reformatted CT image in a bone algorithm demonstrates the fetal skeleton from data acquired during the maternal examination. Again, the arrow marks one fracture site. (Reproduced with permission from Bailey AA, Twickler OM: Perioperative imaging. In Yeomans ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap'S Operative Obstetrics 3rd ed. New York: McGraw-Hili Education; 201o7. Photo contributor: Dr. Travis Browning.) Figure 46-4, abdominal tomography is performed if indicated in the pregnant woman with severe trauma (Matzon, 2015; Shakerian, 2015).

1	Most experience with chest CT scanning is with suspected pulmonary embolism. The most recent recommendations for its use in pregnancy from the Prospective Investigation of Pulmonary Embolism Diagnosis-PIOPED-II investigators were summarized by Stein and associates (2007). They found that pulmonary scintigraphy-the V/Q scan-was recommended at that time for pregnant women by 70 percent of radiologists and chest CT angiography by 30 percent. Indeed, scintigraphy is still recommended by the American Thoracic Society for gravidas with a normal chest x-ray (Leung, 2012). hat said, most agree that multidetector-CT angiography has improved accuracy because of increasingly faster acquisition times (Brown, 2014). Others have reported a higher use rate for CT angiography and emphasize that dosimetry is similar to that with V/Q scintigraphy (Brenner, 2007; Greer, 2015; Hurwitz, 2006). Controversies on this topic continue, recognizing that fetal radiation doses are lower with CT angiography

1	is similar to that with V/Q scintigraphy (Brenner, 2007; Greer, 2015; Hurwitz, 2006). Controversies on this topic continue, recognizing that fetal radiation doses are lower with CT angiography compared with the V/Q scan, but maternal chest radiation doses are substantially higher with CT scanning (van Mens, 2017). We prefer MDCT scanning initially for suspected pulmonary embolism (Chap. 52, p. 1016).

1	CT pelvimetry is used by some before attempting breech vaginal delivery (Chap. 28, p. 542). he fetal dose approaches 0.015 Gyor 1.5 rad, but use of a low-exposure technique may reduce this to 0.0025 Gy or 0.25 rad. hese can be given intravenously or taken orally. Intravenous contrast agents are considered category B by the FDA. The types of intravenous contrast employed for imaging today are iodinated and low osmolality, thus, they cross the placenta to the fetus. With water-soluble iodinated contrast, no cases of neonatal hypothyroidism or other adverse efects have been documented (American College of Radiology, 2015). Oral contrast preparations, typically containing iodine or barium, have minimal systemic absorption and are unlikely to afect the fetus.

1	hese studies are performed by "tagging" a radioactive element to a carrier that can be injected, inhaled, or swallowed. For example, the radioisotope technetium-99m may be tagged to red blood cells, sulfur colloid, or pertechnetate. he method used to tag the agent determines fetal radiation exposure. he amount of placental transfer is obviously important, but so is renal clearance because of fetal proximity to the maternal bladder. Measurement of radioactive technetium is based on its decay, and the units used are the curie (Ci) or the becquerel (Bq). Dosimetry is usually expressed in millicuries (mCi). he efective tissue dose is expressed in sievert units (Sv) with conversion as discussed in Table 46-4: 1 Sv = 100 rem = 100 rad.

1	Depending on the physical and biochemical properties of a radioisotope, an average fetal exposure can be calculated (Wagner, 1997; Zanzonico, 2000). Commonly used radiopharmaceuticals and estimated absorbed fetal doses are given in Table 46-8. The radionuclide dose should be kept as low as possible (Adelstein, 1999; Zanotti-Fregonara, 2017). Exposures vary with gestational age and are greatest earlier in pregnancy for most radiopharmaceuticals. One exception is the later efect ofiodine-131 on the fetal thyroid (Wagner, 1997). Discussed earlier, some still use the ventilation-perfusion lung scan for suspected pulmonary embolism. It is also used if CT angiography is nondiagnostic. Perfusion is measured with injected 99T c-macroaggregated albumin, and ventilation is measured with inhaled xenon-127 or xenon-133. Fetal exposure with either is negligible (Chan, 2002; 110untford, 1997).

1	Thyroid scanning with iodine-123 or iodine-131 seldom is indicated in pregnancy. With trace diagnostic doses used, however, fetal risk is minimal. Importantly, therapeutic radioiodine in doses to treat Graves disease or thyroid cancer may cause fetal thyroid ablation and cretinism. amCi = millicuries. To convertto mrad, mUltiply by 100. bExposures are generally greater prior to 12 weeks compared with increasing gestational ages. cSome measurements account for placental transfer. dThe uptake and exposure of 1 increases with gestational age. Tc04 = pertechnetate; TI = thallium. Data from Adelstein, 1999; Schwartz, 2003; Stather, 2002; Wagner, 1997; Zanzonico, 2000.

1	dThe uptake and exposure of 1 increases with gestational age. Tc04 = pertechnetate; TI = thallium. Data from Adelstein, 1999; Schwartz, 2003; Stather, 2002; Wagner, 1997; Zanzonico, 2000. he sentinel lymphoscintigram, which uses 99mT c-sulfur indispensable in everyday practice. Its wide-ranging clinical colloid to detect the axillary lymph node most likely to have uses are further discussed in Chapter 10 and in most other secmetastases from breast cancer, is a commonly used preoperative tions of this book. study in nonpregnant women (Newman, 2007; Spanheimer, 2009; Wang, 2007). As shown in Table 46-8, the calculated dose is approximately 0.014 mSv or 1.4 mrad, which should MAGNETIC RESONANCE IMAGING not preclude its use during pregnancy.

1	Magnetic resonance technology does not use ionizing radiation, and its application is cited throughout this book. Advantages include high soft-tissue contrast, ability to characterize tissue, Of all of the major advances in obstetrics, the development of and acquisition of images in any plane-particularly axial, sagsonography for study of the fetus and mother certainly is one of ittal, and coronal. An entire section in Chapter 10 (p. 215) is the greater achievements. The technique has become virtually devoted to mechanisms that generate MR images. The most recent update of the expert panel on MR safety of the American College of Radiology was summarized by Kanal and colleagues (20l3). The panel concluded that no harmful human efects are reported from MR imaging. Similar conclusions were reached by the Canadian Task Force on Preventive Health Care (Patenaude, 2014).

1	Early studies found diferences in blastocyst formation of early murine embryos exposed to MR imaging with 1.5 T (T = tesla) (Chew, 2001). When operated within standardized limits, maternal and fetal imaging can be safely performed at clinical magnet strengths-3 T and below. MR imaging can be used, regardless of trimester: (1) if the information cannot be obtained with another nonionizing modality, namely sonography, (2) if the results of the study will guide maternal or fetal management during pregnancy, and (3) if the imaging cannot be delayed until the woman is no longer pregnant. he decision to use a magnetic field strengthr> 1.5 T may be made for specific maternal indications. Early work also suggests imaging at 3 T can improve fetal assessment (Victoria, 2016). A magnetic ield strength up to 4 T appears safe in animals (Magin, 2000). Vadeyar and associates (2000) reported no demonstrable fetal heart rate pattern changes during MR imaging of gravidas. Studies evaluating children

1	up to 4 T appears safe in animals (Magin, 2000). Vadeyar and associates (2000) reported no demonstrable fetal heart rate pattern changes during MR imaging of gravidas. Studies evaluating children exposed in utero have shown no deleterious efects (Clements, 2000; Kok, 2004; Reeves, 2010).

1	Contraindications to MR imaging include internal cardiac pacemakers, neurostimulators, implanted defibrillators and infusion pumps, cochlear implants, shrapnel or other metal in biologically sensitive areas, some intracranial aneurysm clips, and any metallic foreign body in the eye. Of more than 51,000 nonpregnant patients scheduled for MR imaging, Dewey and colleagues (2007) found that only 0.4 percent had an absolute contraindication to the procedure. Elemental gadolinium cheates are used to create paramagnetic contrast. hese cross the placenta and are found in the fetus, placenta, and nnionic fluid (Oh, 2015). In doses approximately 10 times the human

1	FIGURE 46-5 Multipara at 29 weeks' gestation with suspected appendicitis. A. Coronal T2-weighted magnetic resonance image demonstrates severe hydronephrosis (arrow) and perinephric fluid (white arrowhead) suggesting caliceal rupture. The normal appendix is seen laterally (black arrowhead). B. Coronal computed tomography (CT) better defines the source of obstruction as an 8-mm radiopaque stone at the ureteropelvic junction (blue arrow) and distal to the hydronephrosis (white arrow). C and D. In the same woman, more anterior coronal and sagittal CT images show the expected displacement of a noninflamed appendix yellow arrow) into the upper abdomen with advancing gestation. FIGURE 46-6 Nullipara at 27 weeks' gestation. A. Axial T2-weighted MR image demonstrates mild fetal unilateral ventriculomegaly involving the left lateral ventricle (arrow). B. Sagittal T2-weighted MR image demonstrates normal development of the corpus callosum (arrowheads) and vermis (arrow).

1	B. Sagittal T2-weighted MR image demonstrates normal development of the corpus callosum (arrowheads) and vermis (arrow). TABLE 6-9. Guidelines for Diagnostic Imaging During Pregnancy and Lactation Sonography and magnetic resonance (MR) imaging are not associated with fetal risk and are preferred options for imaging in pregnancy In general, radiation exposure during radiography, computed tomography (C), or nuclear medicine imaging delivers a dose much lower than that associated with fetal harm. If needed to supplement sonography or MR imaging or if more readily available, these should not be withheld With MR imaging, gadolinium contrast use should be restricted unless it significantly improves diagnostic accuracy to benefit fetal or matenal outcome Data from American College of Obstetricians and Gynecologists, 20ln7a.

1	Data from American College of Obstetricians and Gynecologists, 20ln7a. dose, a gadolinium-based contrast agent caused slight developmental delay in rabbit fetuses. De Santis and associates described 26 women given a gadolinium derivative in the first trimester without adverse fetal efects (Kanal, 2013). According to Briggs and colleagues (2015), the American College of Obstetricians and Gynecologists (2017a), and the American College of Radiology (2015), routine use of gadolinium is not recommended unless there are potential benefits that outweigh fetal risks. his recommendation stems from a possible dissociation of the toxic gadolinium ion from its ligand in amnionic Ruid and potential prolonged exposure of the fetus.

1	In some cases, MR imaging may be complementary to CT, and in others, MR imaging is preferable. Maternal central nervous system abnormalities, such as brain tumors or spinal trauma, are more clearly seen with MR imaging. As discussed in Chapter 40 (p. 723), MR imaging has provided valuable insights into the pathophysiology of preeclampsia (Twickler, 2007; Zeeman, 2003, 2014). It is invaluable in the diagnosis of neurological emergencies (Edlow, 2013). To evaluate the maternal abdomen and retroperitoneal space, MR imaging is a superb technique. It is chosen by many to determine the degree and extent of placenta accreta and its variants (Chap. 41, p. 780). It has been employed for detection and localization of adrenal tumors, renal lesions, gastrointestinal lesions, and pelvic masses in pregnancy. For evaluating neoplasms of the chest, abdomen, and pelvis in pregnancy, it has particular value (Boyd, 2012; Tica, 2013). MR urography has been used successfully for renal urolithiasis

1	in pregnancy. For evaluating neoplasms of the chest, abdomen, and pelvis in pregnancy, it has particular value (Boyd, 2012; Tica, 2013). MR urography has been used successfully for renal urolithiasis (Mullins, 2012). As discussed in Chapter 37 (p. 672), CT and MR imaging are useful for evaluation of puerperal infections, but MR imaging provides better visualization of the bladder Rap area following cesarean delivery (Brown, 1999; Twickler, 1997). MR imaging now includes evaluation of right lower quadrant pain in pregnancy, specifically appendicitis (Fig. 46-5) (Baron, 2012; Dewhurst, 2013; Furey, 2014; Pedrosa, 2009; Tsai, 2017). Investigators have also found other disorders of the gastrointestinal tract to be easily diagnosed with MR imaging (Chap. 54, p. 1043). Finally, cardiac MR imaging has shown promise in investigating normal physiology, complex defects, and cardiomyopathies (Kramer, 2015; Nelson, 2015; Stewart, 2016).

1	Fetal MR imaging provides a complement to sonography (LaiferNarin, 2007; Sandrasegaran, 2006). According to Zaretsky and associates (2003a), MR imaging can be used to image almost all elements of the standard fetal anatomical survey. The most frequent fetal indications for MR imaging are evaluation of complex abnormalities of the brain, chest, and genitourinary system (Williams, 2017). Reichel (2003), Twickler (2002), and others have validated its use for fetal central nervous system anomalies and biometry (Fig. 46-6). Caire and coworkers (2003) reported its merits for fetal genitourinary anomalies. Hawkins and colleagues (2008) described MR imaging in 21 fetuses with renal anomalies and oligohydramnios. Zaretsky and associates (2003b) noted that fetal-weight estimation was more accurate using MR imaging than with sonography. Fetal movement is less problematic with faster acquisitions. Morphology is primarily assessed with fast T2-weighted sequences such as HASTE-Half-Fourier

1	using MR imaging than with sonography. Fetal movement is less problematic with faster acquisitions. Morphology is primarily assessed with fast T2-weighted sequences such as HASTE-Half-Fourier Acquisition Single Shot Turbo Spin Echo, or SSFSE-Single hot Fast Spin Echo. Fetal indications and indings of MR imaging are discussed more extensively in Chapter 10 (p. 217) and throughout this book.

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1	Wortman A, Miller DL, Donahue TF, et al: Embolization of renal hemorrhage in pregnancy. Obstet Gynecol 121(Pt 2 Supplt1):480, 2013 Zanotti-Fregonara P, Hindie E: Performing nuclear medicine examinations in pregnant women. Phys Med 43:159,t2017 Zanzonico PB: Internal radionuclide radiation dosimetry: a review of basic concepts and recent developments. J Nucl Med 41t:297,t2000 Zaretsky M, McIntire 0, Twickler OM: Feasibility of the fetal anatomic and maternal pelvic survey by magnetic resonance imaging at term. Am J Obstet Gynecol 189:997, 2003a Zaretsky M, Reichel TF, McIntire DO, et al: Comparison of magnetic resonance imaging to ultrasound in the estimation of birth weight at term. Am J Obstet GynecoIt189:1017, 2003b Zeeman GG, Cipolla MJ, Cunningham FG: Cerebrovascular (patho)physiology in preeclampsia. In Taylor RN, Roberts JM, Cunningham FG, et al (eds): Chesley's Hypertensive Disorders in Pregnancy, 4th ed. Amsterdam, Elsevier, 2014, p 269

1	Zeeman GG, Hatab M, Twickler D: Maternal cerebral blood Row changes in pregnancy. Am J Obstet Gynecol 189:968,t2003 OBSTETRICALINTENSIVE CARE ....................915 ACUTE PULMONARY EDEMA ......................917 ACUTE RESPIRATORY DISTRESS SYNDROME ........918 SEPSIS SYNDROME.............................. 921 TRAUMA....................................... 925 THERMAL INJURY............................... 930 CARDIOPULMONARY RESUSCITATION.............931 The pregnant women is exposed to the same possibiliy of injuy as at other times, the prognosis not being naturaly altered except that abortion tequenty occurs.

1	-J. Whitridge Williams (1903) hese observations made more than a century ago are less applicable toda) to critically ill pregnant women because of current intensive care capabilities. For example, severe medical, surgical, and obstetrical disorders complicating pregnancy are frequently managed by a multidisciplinary team for optimal care. It is axiomatic that obstetricians and other members of the health-care team must have a working knowledge of the unique considerations for pregnant women. Some of those discussed in Chapter 46 include pregnancy-induced physiological changes, alterations in normal laboratory values, and consideration for the second patient-the fetus. Because these critically ill women are usually young and in good health, their prognosis is generally better than that of many other patients admitted to intensive care units (ICUs) (Gafney, 2014).

1	In the United States each year, 1 to 3 percent of pregnant women require critical care services, and the risk of death during such admissions ranges from 2 to 11 percent (American College of Obstetricians and Gynecologists, 2017b). hose with pregnancy-associated complications-especiall) hemorrhage and hypertension-have the greatest need for intensive care (Chantry, 2015; Gafney, 2014; Guntupalli, 2015a,b). That said, many antepartum admissions are for nonobstetrical reasons, and these include diabetes, pneumonia or asthma, heart disease, chronic hypertension, pyelonephritis, and thyrotoxicosis (Guntupalli, 2015b; Zeeman, 2006). Additionally, intrapartum and postpartum critical care for hypertensive disorders, hemorrhage, sepsis, or cardiopulmonary complications is often required. In instances of life-threatening hemorrhage, surgical procedures may be necessary, and close proximity to a delivery-operating room is paramount. For women who are undelivered, fetal well-being is also better

1	of life-threatening hemorrhage, surgical procedures may be necessary, and close proximity to a delivery-operating room is paramount. For women who are undelivered, fetal well-being is also better served by this close proximity, especially because many are delivered preterm (Kilpatrick,r2016).

1	• Organization of Critical Care he concept and development of critical care for all aspects of medicine and surgery began in the 1960s. The National Institutes of Health held a Consensus Conference (1983) and the Society of Critical Care Medicine (1988, 1999) subsequently established guidelines for ICUs. Especially pertinent to obstetrics, these costly units prompted the evolution of a step-down intermediate care unit. hese latter units were designed for patients who did not require intensive care, but who needed a higher level of care than that provided on a general ward. The American College of Critical Care Medicine and the Society of Critical Care Medicine (1998) have published guidelines for these units (Table TABLE 47-1 . Guidelines for Conditions That Could Qualify for Intermediate Care

1	TABLE 47-1 . Guidelines for Conditions That Could Qualify for Intermediate Care Cardiac: for possible infarction, stable infarction, stable arrhythmias, mild-to-moderate congestive heart failure, hypertensive urgency without end-organ damage Pulmonary: stable patients for weaning and chronic ventilation, patients potential for respiratory failure who are otherwise stable Neurological: stable central nervous system, neuromuscular, or neurosurgical conditions that close monitoring Drug overdose: hemodynamically stable Gastrointestinal: stable bleeding, liver failure with stable vital signs Endocrine: diabetic ketoacidosis, thyrotoxicosis that requires frequent monitoring Surgical: postoperative from major procedures or complications that require close monitoring Miscellaneous: early sepsis, patients who require closely titrated intravenous fluids, pregnant women with severe Data from Nasraway, 1998 .

1	Data from Nasraway, 1998 . Although the evolution of critical care for obstetrical patients has generally followed developments just described, there are no speciic guidelines. Most hospitals employ a blend of these concepts, and in general, units can be divided into three types.

1	First, in most hospitals, severely ill women are transferred to medical or surgical ICUs that are operated by specialists oten certified in critical care medicine. Admissions or transfers to these units are situation-speciic and based on the acuity of care needed and on the ability of the facility to provide it. For example, pregnant women who require ventilatory support, invasive monitoring, or pharmacological support of circulation are typically transferred to an ICU (Chantry, 2015). Another example is the neurological ICU (Sheth, 2012). In an earlier review of more than 25 tertiarycare referral institutions, approximately 0.5 percent of obstetrical patients were transferred to these types ofICUs (Zeeman, 2006).

1	A second type is the obstetrical intermediate care unit, sometimes referred to as a high-dependency care unit (HDU). One example is found at Parkland Hospital. Located within the labor and delivery unit, it has designated rooms stafed by experienced personnel. The two-tiered system incorporates the guidelines for intermediate and intensive care. Care is provided by maternal-fetal medicine specialists and nurses with experience in critical care obstetrics. As needed, this team is expanded to include other obstetricians and anesthesiologists, hospitalists, gynecological oncologists, pulmonologists, cardiologists, surgeons, and other medical and surgical subspecialists (Stevens, 2015). Many tertiary-care centers have developed similar intermediate care units and use selected triage to ICUs. Guidelines

1	Last, obstetrical ICUs are full-care ICUs but are operated by obstetrical and anesthesia personnel in the labor and delivery unit. Only a few obstetrical sevices have these capabilities (Zeeman, 2003, 2006). For smaller hospitls, transfer to a medical or surgical ICU is usually preferable, and sometimes transfer to another hospital is necessary. s discussed, indications for admission to these types of critical care units vary, however, patient mix for these units is similar (Table 47-2). he American College of Obstetricians and Gynecologists (2017b) has summarized criticl obstetrical care implementation depending on hospital size and technical facilities. Data obtained during pregnancy with pulmonary artery catheterization (PAC) have contributed immensely to the understanding of normal pregnancy hemodynamics and pathophysiology of common obstetrical conditions. These include preeclampsia-eclampsia, acute respiratory distress syndrome,

1	TABLE 47-2. Comparison of Acuity of Patient Mix for Obstetrical Critical Care Shown in Percent Care Unit Surgical.lCU Factor {n = 483)a {n = 81.3)b for such transfers must follow the federal Emergency Medical Hypertension Treatment and Labor Act (EMTALA) guidelines. According to the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017), the minimal monitoring required for a critically ill patient during transport includes continuous pulse oximetry, electrocardiography, and regular assessment of vital signs. They must have secure venous access, and those who are mechanically ventilated must have endotracheal tube position confirmed and secured. Left uterine displacement and supplemental oxygen is applied routinely during transport of antepartum patients. Continuous fetal heart rate or tocodynamic monitoring is individualized. Preg na ncy-Related 0.2 2 aData from Zeeman, 2003.

1	Preg na ncy-Related 0.2 2 aData from Zeeman, 2003. bData from Baskett, 2009; Keizer, 2006; Paxton, 2014; Small, 201n2; Stevens, 2006; Vasquez, 2007. (Columns of indications do not total 100 percent because some diagnoses are not listed. and amnionic-fluid embolism (Clark, 1988, 1995, 1997; Cunningham, 1986, 1987; Hankins, 1984, 1985). Also, because of these studies, most have concluded that such monitoring is seldom necessary (American College of Obstetricians and Gynecologists, 2013; Gidwani, 2013; Magder, 2015). In nonobstetrical patients, randomized trials of nearly 5000 subjects have shown no benefits with PAC (Harvey, 2005; National Heart, Lung, and Blood Institute, 2006; Sandham, 2003). According to a Cochrane Database review, no randomized trials have used PAC for preeclampsia management (Li, 2012). he overall mechanisms, benefits, and risks were recently reviewed by Magder (2015).

1	Formulas for deriving some hemodynamic parameters are shown in Table 47-3. hese measurements can be indexed for body size by dividing by body surface area (BSA). Normal values for nonpregnant adults are used, but with the caveat that these may not necessarily reflect changes induced by the more "passive" uteroplacental perfusion. In a landmark investigation, Clark and colleagues (1989) used PAC to obtain cardiovascular measurements in healthy pregnant women and again in these same women when nonpregnant (Chap. 4, p. 62). Because increased blood volume and cardiac output are compensated by decreased vascular resistance and increased pulse rate, ventricular performance remains within the normal range at term.

1	Cardiac complications are a common indication for ICU admission of pregnant women (Guntupalli, 2015b). Evaluation of cardiac unction is frequently performed using echo cardiography. his technology is indispensable in interrogating cardiac anatomy and especially right-ventricular unction (Krishnan, 2015; hiele, 2015). It is considered in more detail in Chapter 49 (p. 951), and some norml values are listed in the Appendix (p. 1261). A working knowledge of cardiovascular physiology in pregnancy is paramount to understanding the pathophysiology of gestational complications discussed later in this chapter and throughout the book. TABLE 47-3. Formulas for Deriving Various Cardiopulmonary Parameters

1	TABLE 47-3. Formulas for Deriving Various Cardiopulmonary Parameters BSA = body surface area (m2); CVP = central venous pressure (mm Hg); DBP = diastolic blood pressure; HR = heart rate (beats/min); MAP = mean systemic arterial pressure (mm Hg); MPAP = mean pulmonary artery pressure (mm Hg); PCWP = pulmonary capillary wedge pressure (mm Hg); SBP = systolic blood pressure. Critical Care and Trauma 91.7

1	Critical Care and Trauma 91.7 The incidence of pulmonary edema complicating pregnancy averages 1 in 500 deliveries at tertiary referral centers. The two general causes are: (1) cardiogenic, namely, hydrostatic edema caused by high pulmonary capillary hydraulic pressures, and (2) noncardiogenic, that is, permeability edema caused by capillary endothelial and alveolar epithelial damage. In pregnancy, noncardiogenic pulmonary edema is more common. Taken in toto, studies in gravidas indicate that more than half who develop pulmonary edema have some degree of sepsis syndrome in conjunction with tocolysis, severe preeclampsia, or obstetrical hemorrhage combined with vigorous luid therapy (O'Dwyer, 2014; hornton, 2011).

1	lthough cardiogenic pulmonary edema is less frequent, common precipitating causes include resuscitation for hemorrhage and vigorous treatment of preterm labor. In one study, the causes in 51 women with pulmonary edema were cardiac failure, tocolytic therapy, iatrogenic luid overload, and preeclampsia (Sciscione, 2003). In another study, more than half of cases were associated with preeclampsia, and the other three causes had equal distribution (Hough, 2007). In still another study of 53 cases, 83 percent were caused by hypertensive disorders, 11 percent cardiac, and 6 percent sepsis (O'Dwyer, 2015). Although used less commonly today, tocolytic therapy with �-mimetic drugs at one time was the cause of up to 40 percent of pulmonary edema cases (DiFederico, 1998; Gandhi, 2014; Jenkins, 2003).

1	Endothelial activation is the common denominator that is associated with preeclampsia, sepsis syndrome, and acute hemorrhage-or frequently combinations thereof-and they are the most common predisposing factors to pulmonary edema (T able 47-4). As discussed, these clinical scenarios are often associated with corticosteroids given to induce fetal lung TABLE 47-4. Some Causes and Associated Factors for Pulmonary Edema in Pregnancy Noncardiogenic permeability edema: endothelial activation with capillary-alveolar leakage Sepsis syndrome: pyelonephritis, metritis Tocolytic therapy: �-mimetics, MgS04

1	Noncardiogenic permeability edema: endothelial activation with capillary-alveolar leakage Sepsis syndrome: pyelonephritis, metritis Tocolytic therapy: �-mimetics, MgS04 Cardiogenic pulmonary edema: myocardial failure with maturation along with vigorous luid replacement and tocolytic therapy (Thornton, 2011). Parenteral 3-agonists are indisputably linked to pulmonary edema. Studies have also associated magnesium sulfate given for preeclampsia (Gandhi, 2014; Wilson, 2014; Xiao, 2014). Combined therapy is also causative. In one study of nearly 800 women given magnesium sulfate for preterm labor, 8 percent developed pulmonary edema, and half of this afected group had also received terbutaline (Samol, 2005).

1	Ventricular failure causing pulmonary edema in pregnancy is usually associated with some form of gestational hypertension. Although it can be due to congenital or acquired anatomical defects, diastolic dysfunction is frequently from chronic hypertension, obesity, or both (Jessup, 2003; Kenchaiah, 2002). In these women, acute systolic hypertension exacerbates diastolic dysfunction and causes pulmonary edema (Dennis, 2012; Gandhi, 2001). Of note, concentric and eccentric hypertrophy is two-to threefold more common in black women compared with white women (Drazner, 2005). In a case-control study of 28 gravidas with preeclampsia and pulmonary edema, half of them were undelivered (Gandhi, 2014).

1	In women with an underlying cardiomyopathy, heart failure is commonly precipitated by preeclampsia, hypertension, hemorrhage and anemia, and puerperal sepsis (Cunningham, 1986; Sibai, 1987). In many of these, when echocardiography is done later, systolic function is normal as measured by ejection fraction, but evidence for diastolic dysunction can often be found (Aurigemma, 2004). The use of brain natriuretic peptide (BNP) has not been evaluated extensively in pregnancy (Seror, 2014). his neurohormone is secreted from ventricle myocytes and fibroblasts with distention seen in heart failure. In nonpregnant patients, values < 100 pgl mL have an excellent negative-predictive value, and levels >500 pg/mL have an excellent positivepredictive value. It is problematic that levels frequently are 100 to 500 pg/mL, and thus nondiagnostic (Ware, 2005). Values for N-terminal BNP and atrial natriuretic peptide (ANP) are both elevated with preeclampsia (Szabo, 2014; Tihtonen, 2007). his is

1	are 100 to 500 pg/mL, and thus nondiagnostic (Ware, 2005). Values for N-terminal BNP and atrial natriuretic peptide (ANP) are both elevated with preeclampsia (Szabo, 2014; Tihtonen, 2007). his is discussed in greater detail in Chapter 4 (p. 63), and normal values for pregnancy are given in the Appendix (p. 1259).

1	Acute pulmonary edema requires emergency care. Furosemide is given in 20-to 40-mg intravenous doses along with therapy to control severe hypertension. Further treatment depends on whether a woman is ante-or postpartum. A live fetus prohibits the use of cardioactive drugs that might rapidly lower peripheral resistance and in turn severely diminish uteroplacental circulation. he cause of cardiogenic failure is determined by echocardiography, which will help direct further therapy. Acute pulmonary edema is not, per se, an indication for emergency cesarean delivery.

1	Acute lung injury that causes a form of severe permeability pulmonary edema and respiratory failure is termed acute respiratory distress syndrome (ARDS). This is a pathophysiological continuum from mild pulmonary insuiciency to dependence on high inspired oxygen concentrations and mechanical ventilation. Uniform criteria for its diagnosis are lacking, and thus the incidence is variably reported for pregnancy. In one survey of the Nationwide Inpatient Sample, 2808 pregnant women with ARDS were identified (Rush, 2017). he incidence ranged from 36 to 60 cases per 100,000 births, and the maternal mortality rate was 9 percent. In its most extreme form, requiring ventilatory support, the associated mortality rate is 45 percent. This rate can be as high as 90 percent if caused or complicated by sepsis (Phua, 2009). Although gravidas are younger and usully healthier thn the overall population, they still have mortality rates of 25 to 40 percent (Catanzarite, 2001; Cole, 2005). Finally, if

1	by sepsis (Phua, 2009). Although gravidas are younger and usully healthier thn the overall population, they still have mortality rates of 25 to 40 percent (Catanzarite, 2001; Cole, 2005). Finally, if ARDS develops antepartum, the perinatal mortality rate is correspondingly high.

1	Most investigators define ARDS as radiographically documented pulmonary iniltrates, a ratio of arterial oxygen tension to the fraction of inspired oxygen (Pao2:Fio2) <200, and no evidence of heart failure (Mallampalli, 2010; hompson, 2017). Revised by international consensus, the Berlin Dinition was described by the ARDS Definition Task Force (2012) and includes categories of mild, moderate and severe. To date, for most interventional studies, a working diagnosis of acute lung injuy is made when the Pao2:Fio2 ratio is <300 and is coupled with dyspnea, tachypnea, oxygen desaturation, and radiographic pulmonary infiltrates (Wheeler, 2007). ARDS is a pathophysiological description that begins with an acute lung injury from various causes (Table 47-5). In pregnant TABLE 47-5. Some Causes of Acute Lung Injury and Respiratory Failure in Pregnant Women Pneumonia: bacterial, viral, aspiration Sepsis syndrome: chorioamnionitis, pyelonephritis, puerperal infection, septic abortion

1	Pneumonia: bacterial, viral, aspiration Sepsis syndrome: chorioamnionitis, pyelonephritis, puerperal infection, septic abortion Hemorrhage: shock, massive transfusion, transfusion- Embolism: amnionic fluid, trophoblastic disease, air, fat Data from Cole, 2005; Duarte, 2014; Golombeck, 2006; Lapinsky, 2015; Martin, 2006; Sheffield, 2005; Sibai, 20104; Snyder, 201n3; Zeeman, 2003, 2006. women, sepsis and difuse infectious pneumonia are the two most common single-agent ARDS causes. Pyelonephritis, puer peral pelvic infection, and chorioamnionitis are the most frequent causes of sepsis. As discussed on page 917, severe preeclampsia and obstetrical hemorrhage are also commonly associated with permeability edema. Importantly, more than half of pregnant women with ARDS have some combination of sepsis, hemor rhage, shock, and luid overload. The contribution of tranosion related acute lung injuy (TAIl) is unclear (Chap. 41, p. 791).

1	Endothelial injury in the lung capillaries releases cytokines that recruit neutrophils to the inflammation site. Here, they elaborate more cytokines to worsen tissue injury. There are three stages of ARDS development. First, the exudative phase follows widespread injury to microvascular endothelium, including the pulmonary vasculature, and there is also alveolar epithelial injury. These result in increased pulmonary capillary perme ability, surfactant loss or inactivation, diminished lung volume, and vascular shunting with resultant arterial hypoxemia. Next, the ibroprolierative phase usually begins 3 to 4 days later and lasts up to day 21. Last, theibrotic phase results from healing, and despite this, the long-term prognosis for pulmonary func tion is surprisingly good (Herridge, 2003; Levy, 2015).

1	With pulmonary injury, the clinical condition depends largely on the insult magnitude, the ability to compensate for it, and the disease stage. For example, soon after the initial injury, physical findings are absent except perhaps hyperventilation. And at irst, arterial oxygenation usually is adequate. Pregnancy-induced mild metabolic alkalosis may be accentuated by hyperventilation. With worsening, clinical and radiological evidence for pulmonary edema, decreased lung compliance, and increased intrapulmonary blood shunting become apparent. Progressive alveolar and interstitial edema develop with extravasation of inflammatory cells and erythrocytes.

1	Ideally, pulmonary injury is identified at this early stage, and speciic therapy is directed at the underlying insult. Further progression to acute respiratory failure is characterized by marked dyspnea, tachypnea, and hypoxemia. Additional lung volume loss results in worsening of pulmonary compliance and increased shunting. Difuse abnormalities are heard by auscultation, and a chest radiograph characteristically demonstrates bilateral lung involvement (Fig. At this phase, the injury ordinarily would be lethal in the absence of ventilatory support. When shunting exceeds 30 percent, severe refractory hypoxemia develops along with metabolic and respiratory acidosis that can result in myocardial irritability, dysfunction, and cardiac arrest.

1	Reduced ARDS mortality rates have resulted from advances in care of the critically ill (Levy, 2015). his requires close attention to: (1) recognizing and treating underlying medical and surgical disorders, (2) minimizing procedures and their complications, (3) administering prophylaxis against venous thromboembolism, gastrointestinal bleeding, aspiration, and central venous catheter infection, (4) promptly diagnosing nosocomial infections, and (5) providing adequate nutrition. FIGURE 47-1 Anteroposterior chest radiograph of a secondtrimester pregnant woman with marked bilateral parenchymal and pleural opacification secondary to acute respiratory distress syndrome (ARDS) due to pyelonephritis.

1	In cases of severe acute lung injury, providing adequate oxygenation of peripheral tissues is balanced against maneuvers that further aggravate lung injury. At least intuitively, increasing oxygen delivery should produce a corresponding increase in tissue uptake, but this is diicult to measure. Support of systemic perfusion with intravenous crystalloid and blood is imperative. As discussed earlier, the trial conducted by the National Heart, Lung, and Blood Institute (2006) showed that pulmonary artery catheter use did not improve outcomes. Because sepsis is commonplace in lung injury, vigorous antimicrobial therapy is given for infection, and any necrotic tissues are debrided. Oxygen delivery can be greatly improved by correction of anemia. Speciically, each gram of hemoglobin carries 1.25 mL of oxygen when 90-percent saturated. By comparison, increasing the arterial P02 from 100 to 200 mm Hg results in the transport of only 0.1 mL of additional oxygen for each 100 mL of blood.

1	Reasonable goals in caring for the woman with severe lung injury are to attain a Pao2 of 60 mm Hg or 90-percent oxygen saturation using an inspired oxygen content < 50 percent and positive end-expiratory pressures < 15 mm Hg. With regard to the pregnancy, it remains controversial whether delivery of the fetus improves maternal oxygenation (Mallampalli, 2010). In a study of 29 women undergoing mechanical ventilation, 10 were delivered while intubated (Lapinsky, 2015). This was associated with a modest improvement in respiratory function in perhaps half, but no factors were identiied that predicted a better outcome.

1	Noninvasive ventilation, that is, positive pressure ventilation by face mask, may be efective in some women in early stages of pulmonary insuiciency (Duarte, 2014). Early intubation is preferred in the gravida if respiratory failure is more likely than not, and especially if it appears imminent. Many successful formulas for mechanical ventilation are employed, and initially a tidal volume :;6 mLlkg is optimal (Levy, 2015; Schwaiberger, 2016). High-frequency oscillation ventilation (HFOV) is controversial in ARDS (Ferguson, 2013; Slutsy, 2013). Adjustments are made to obtain a Paoz>60 mm Hg or a hemoglobin oxygen saturation 290 percent and a Pacoz of 35 to 45 mm Hg. Lower levels for Paoz are avoided, because placental perfusion may be impaired (Levinson, 1974).

1	For women who require ventilation for any length of time, the maternal mortality rate is 10 to 20 percent. In a study of 51 such women, almost half had severe preeclampsia, and most required intubation postpartum. Eleven were delivered while being ventilated, and another six were discharged undelivered (Jenkins, 2003). here were two maternal deaths, including a woman who died as a complication of tocolytic treatment. In three other reports, maternal mortality rates ranged from 10 to 25 percent (Chen, 2003; Lapinsky, 2015; Schneider, 2003). In most cases, delivery did not improve maternal outcome.

1	With severe lung injury and high intrapulmonary shunt fractions, it may not be possible to provide adequate oxygenation with usual ventilatory pressures, even with 100-percent oxygen. Positive end-expiratory pressure is usually successful in decreasing the shunt by recruiting collapsed alveoli. At low levels of 5 to 15 mm Hg, positive pressure can typically be used safely. At higher levels, impaired right-sided venous return can result in decreased cardiac output, lowered uteroplacental perfusion, alveolar overdistention, falling compliance, and barotrauma (Schwaiberger, 2016; Slutsky, 2013).

1	As discussed in Chapter 33 (p. 620), extracorporeal membrane oxygenation (ECM 0) has been successfully used for neonatal meconium aspiration syndrome. Preliminary observation suggests that it may be useful in adults with ARDS (Brodie, 2011; Levy, 2015; Peek, 2009). ECMO use has been reported in pregnant women. In one study, 12 patients with inluenza-induced lung failure were treated with ECMO, and of four maternal deaths, three were due to anticoagulation-related hemorrhage (Nair, 2011). In another study, the duration of support in four survivors was 2 to 28 days (Cunningham, 2006). In a review of 29 treated gravidas, 80 percent of cases were due to ARDS, and the maternal and perinatal mortality rate was 28 percent (Anselmi, 2015). Technical aspects of ECMO were reviewed by Brodie and Bacchetta (2011).

1	he propensity of the hemoglobin molecule to release oxygen is described by the oyhemoglobin dissociation curve (Fig. 47-2). For clinical purposes, the curve can be divided into an upper oxygen association curve representing the alveolar-capillary environment and a lower oxygen dissociation portion representing the tissue-capillary environment. Shifts of the curve have their greatest efect at the steep portion because they afect oxygen delivery. A rightward shit is associated with decreased hemoglobin ainity for oxygen and hence increased tissue-capillary oxygen :0 60= :l FIGURE 47-2 Oxyhemoglobin dissociation curve. With higher oxygen tension (Pao2) in the pulmonary alveoli, adult hemoglobin is maximally saturated compared with that at the lower oxygen tension in the tissue capillaries. Note that at any given oxygen tension, fetal hemoglobin carries more oxygen than adult hemoglobin, as indicated by percent saturation.

1	interchange. Rightward shifts are produced by hypercapnia, metabolic acidosis, fever, and increased 2,3-diphosphoglycerate levels. During pregnancy, the erythrocyte concentration of 2,3-diphosphoglycerate is increased by approximately 30 percent. This favors oxygen delivery to both the fetus and maternal peripheral tissues.

1	Fetal hemoglobin has a higher oxygen ainity than adult hemoglobin. As seen in Figure 47-2, its curve is positioned to the left of the adult curve. To achieve 50-percent hemoglobin saturation, the Paoz must be 27 mm Hg in the mother compared with only 19 mm Hg in the fetus. Under normal physiological conditions, the fetus is constantly on the dissociation, or tissue, portion of the curve. Even with severe maternal lung disease and very low Paoz levels, oxygen displacement to fetal tissues is favored. Another example of this comes from pregnant women who live at high altitudes. Here, despite a maternal Paoz of only 60 mm Hg, the fetal Paoz is equivalent to that of fetuses at sea level (Subrevilla, 1971).

1	Although mortality outcomes are similar, conservative rather than liberal fluid management is associated with fewer days of mechanical ventilation (Wiedemann, 2006). Some pregnancyinduced physiological changes predispose to a greater risk of permeability edema from vigorous fluid therapy. Colloid oncotic pressure (COP) is determined by serum albumin concentration, and 1 gl dL exerts approximately 6 mm Hg pressure. As discussed in Chapter 4 (p. 68), serum albumin concentrations normally drop in pregnancy. This results in a decline in oncotic pressure from 28 mm Hg in the nonpregnant woman to 23 mm Hg at term and to 17 mm Hg in the puerperium (Benedetti, 1979; Dennis, 2012). With preeclampsia, endothelial activation with leakage causes extravascular albumin loss and lowered serum albumin levels. As a result in these cases, 14 mm Hg postpartum (Zinaman, 1985). hese changes have a significant clinical efect on the colloid oncotic pressure/wedge pressure gradient. Normally, this gradient

1	As a result in these cases, 14 mm Hg postpartum (Zinaman, 1985). hese changes have a significant clinical efect on the colloid oncotic pressure/wedge pressure gradient. Normally, this gradient exceeds 8 mm Hg. However, when it is 4 mm Hg or less, the risk for pulmonary edema rises. No benefits are gained by albumin rather than crystalloid infusions in these women (Uhlig, 2014). hese associations were reviewed by Dennis and Solnordal (2012).

1	No long-term follow-up studies address gravidas who recover from ARDS. In nonpregnant subjects, risks for impaired global cognitive function at 3 and 12 months are significant (Pandharipande, 2013). Data from nonpregnant patients indicate a 1-to 2-year hiatus before basic normal activity is restored in all. In a 5-year follow-up study, Herridge and associates (201r1) reported normal lung function but signiicant exercise limitation, physical and psychological sequelae, decreased physical quality of life, and increased use of health-care services.

1	This syndrome is induced by a systemic inlammatory response to bacteria or viruses or their by-products such as endotoxins or exotoxins. The severity of the syndrome is a continuum or spectrum 47-3). According to the Centers for Disease Control and Prevention (CDC), sepsis caused 6.2 percent of pregnancy-related deaths in the United States from 201r1 to 2013 (Creanga, 2017). It was also a signiicant cause of maternal mortality in Michigan and the United Kingdom (Bauer, 2015; 110hamed-Ahmed, 2015; Nair, 2015).

1	Infections that most commonly cause sepsis syndrome in obstetrics are pyelonephritis (Chap. 53, p. 1028), chorioamnionitis and puerperal sepsis (Chap. 37, p. 667), septic abortion (Chap. 18, p. 351), and necrotizing fasciitis (Chap. 37, p. 671). With severe sepsis, the mortality rate in nonpregnant patients is 20 to 35 percent and is 40 to 60 percent with septic shock (Angus, 2013; Munford, 2015). With shock, the mortality rate in pregnancy has been reported to be 30 percent (Mabie, 1997; Snyder, 2013). That said, the maternal mortality risk from sepsis is significantly underestimated (Bauer, 2015; Chebbo, 2016; Mohamed-Ahmed,r2015).

1	Most of what is known concerning sepsis pathogenesis comes from study of lipopolysaccharide-LPS or endotoxin (Munford, 2015). The lipid A moiety is bound by mononuclear blood cells, becomes internalized, and stimulates release of mediators and a series of complex downstream perturbations. Clinical aspects of the sepsis syndrome are manifest when cytokines are released that have endocrine, paracrine, and autocrine actions (Angus, 2013; Singer, 2016).

1	Although the sepsis syndrome in obstetrics may be caused by several pathogens, most cases represent a small group. For example, pyelonephritis complicating pregnancy caused by Escherichia coli and Klebsiela species commonly is associated with bacteremia and sepsis syndrome (Cunningham, 1987; Snyder, 2013). And although pelvic infections are usually polymicrobial, bacteria that cause severe sepsis syndrome are frequently endotoxin-producing Enterobacteriaceae, most commonly E coli (Eschenbach, 2015). Other pelvic pathogens are aerobic and anaerobic streptococci, Bacteroides species, and Clostridium species. Some strains of group A 3-hemolytic ) SIRS-fever, tachycardia, tachypnea, leukocytosis, leukopenia t::, Organ system dysfunctionacidosis, encephalopathy, oliguria, hypoxemia, coagulopathy Spectrum of Severity FIGURE 47-3 The sepsis syndrome begins with a systemic inflammatory response syndrome (SIRS) in response to infection that may progress to septic shock.

1	Spectrum of Severity FIGURE 47-3 The sepsis syndrome begins with a systemic inflammatory response syndrome (SIRS) in response to infection that may progress to septic shock. streptococci and Staphylococcus aureus-including communityacquired methicillin-resistant strains (CA-MRSA)-produce a superantigen that activates T cells to rapidly cause all features of the sepsis syndrome-toxic shock syndrome (Moellering, 2011; Soper, 2011). his is discussed further in Chapter 37 (p. 675).

1	drome. Examples include exotoxins from Clostridium pertingens or sordellii, toxic-shock-syndrome toxin-1 (TSST -1) from S au reus, and toxic shock-like exotoxin from group A 3-hemolytic streptococci (Daif, 2009; Soper, 2011). These last exotoxins cause rapid and extensive tissue necrosis and gangrene, especially of the postpartum uterus, and may cause profound cardiovascular collapse and maternal death (Nathan, 1993; Sugiyama, 2010). In a review discussed subsequently, the maternal mortality rate from these infections was 58 percent (Yamada, 2010).

1	Thus, the sepsis syndrome begins with an inflammatory response that is directed against microbial endotoxins and exotoxins (Angus, 2013). CD4 T cells and leukocytes are stimulated to produce proinflammatory compounds that include tumor necrosis factor-a (TNF-a), several interleukins, other cytokines, proteases, oxidants, and bradykinin that result in a "cytokine storm" (Russell, 2006). Many other cellular reactions then follow that include stimulation of pro-and antiinflammatory compounds, procoagulant activity, gene activation, receptor regulation, and immune suppression (Filbin, 2009; Moellering, 2011). It is also likely that interleukin-6 (IL-6) mediates myocardial suppression (Pathan, 2004).

1	he pathophysiological response to this cascade is selective vasodilation with maldistribution of blood low. Leukocyte and platelet aggregation cause capillary plugging. Worsening endothelial injury causes profound permeability, capillary leakage, and interstitial fluid accumulation (Fig. 47-4). Depending on the degree of injury and inflammatory response, a pathophysiological and clinical continuum evolves as depicted in Figure 47-3. The clinical syndrome begins with subtle signs of Cytokines and other inflammatory mediators lead to endothelial junction separation FIGURE 47-4 Endothelial permeability. The normal interendothelial interface is shown in the left inset. Cytokines and other inflammatory mediators disassemble the cellular junctions, resulting in microvascular leaks (right).

1	sepsis from infection and terminates with septic shock, which is defined by hypotension unresponsive to intravenous hydration. In its early stages, clinical shock results primarily from decreased systemic vascular resistance that is not compensated fully by increased cardiac output. Hypoperfusion results in lactic acidosis, decreased tissue oxygen extraction, and end-organ dysfunction that includes acute lung and kidney injury. he sepsis syndrome has myriad clinical manifestations that, at least in part, are dependent on the specific invading microorganism and its particular endo-or exotoxins. Some of the general efects of LPS are as follows: 1. Central nervous system: confusion, delirium, somnolence, coma, combativeness, fever 2. Cardiovascular: tachycardia, hypotension 3. Pulmonary: tachypnea, arteriovenous shunting with dysoxia and hypoxemia, exudative iniltrates from endothelialalveolar damage, pulmonary hypertension 4.

1	Cardiovascular: tachycardia, hypotension 3. Pulmonary: tachypnea, arteriovenous shunting with dysoxia and hypoxemia, exudative iniltrates from endothelialalveolar damage, pulmonary hypertension 4. Gastrointestinal: gastroenteritis-nausea, vomiting, and diarrhea; ileus; hepatocellular necrosis-jaundice, transaminitis 5. Renal: prerenal oliguria, azotemia, acute kidney injury, proteinuria 6. Hematological: leukocytosis or leukopenia, thrombocytopenia, activation of coagulation with disseminated intravascular coagulopathy 7. Endocrine: hyperglycemia, adrenal insuiciency 8. Cutaneous: acrocyanosis, erythroderma, bullae, digital gangrene.

1	Endocrine: hyperglycemia, adrenal insuiciency 8. Cutaneous: acrocyanosis, erythroderma, bullae, digital gangrene. Thus, although capillary leakage initially causes hypovolemia, if intravenous crystalloid is given at this point, then sepsis hemodynamically can be described as a high cardiac output, low systemic vascular resistance condition (Fig. 47-5). Concomitantly, pulmonary hypertension develops, and despite the high cardiac output, severe sepsis also causes myocardial depression (Munford, 2015; Ognibene, 1988). This is often referred to as the warm phase of septic shock. hese indings are the most common cardiovascular manifestations of early sepsis, but they can be accompanied by some of the other clinical or laboratory aberrations listed above.

1	The response to initial intravenous hydration may be prognostic. Most pregnant women who have early sepsis show a salutary response with crystalloid and antimicrobial therapy, and if indicated, debridement of infected tissue. Conversely, if hypotension is not corrected following vigorous luid infusion, then the prognosis is more guarded. At this juncture, if there also is no response to 3-adrenergic inotropic agents, this indicates severe and unresponsive extracellular luid extravasation with vascular insuiciency, overwhelming myocardial depression, or both. Oliguria and continued peripheral vasoconstriction characterize a secondary, cold phase of septic shock that is rarely survived. Another poor prognostic sign is continued renal, pulmonary, and cerebral dysfunction once hypotension has been corrected (Angus, 2013; Chebbo, 2016). The average risk of death increases by 15 to 20 percent with failure of each organ system. With three systems, mortality rates are 70 percent (Martin,

1	has been corrected (Angus, 2013; Chebbo, 2016). The average risk of death increases by 15 to 20 percent with failure of each organ system. With three systems, mortality rates are 70 percent (Martin, 2003; Wheeler, 1999).

1	FIGURE 47-5 Hemodynamic efects of sepsis syndrome. Values for normal women at term are shown by dots. With early sepsis, there is high cardiac output and low vascular resistance. With fluid resuscitation, cardiac output increases even more, but so does capillary hydraulic pressure. With continued sepsis, there may be myocardial depression to further increase capillary hydraulic pressure. Decreased plasma oncotic pressure (serum albumin [g] x 6 mm Hg) contributes to interstitial lung fluid and endo/epithelialoleak causes alveolar flooding. LVSWI = left ventricular stroke work index; PCWP = pulmonary capillary wedge pressure.

1	In 2004, an international consensus efort was launched as the Surviving Sepsis Campaign (Dellinger, 2013). he cornerstone of management is eary goal-directed management, and it stresses prompt recognition of serious bacterial infection and close monitoring of vital signs and urine flow. It remains controversial if institution of this protocol has improved survival rates (ARISE Investigators, 2014; Mouncey, 2015; ProCESS Investigators, 2014). Similar conclusions were reached with sets of early warning systems in obstetrics (Edwards, 2015; Mhyre, 2014). Albright and associates (2017) have validated the Sepsis in Obstetrics Score to identiy the risk ofICU admission for sepsis.

1	An algorithm for management of sepsis syndrome is shown in 47-6. The three basic steps are performed as simultaneously as possible and include evaluation of the sepsis source and its sequelae, cardiopulmonary function assessment, and immediate management. The most important step in sepsis management is rapid infusion of 2 L and sometimes as many as 4 to 6 L of crystalloid luids to restore renal perfusion in severely afected women (Vincent, 2013). Simultaneously, appropriately chosen broad-spectrum antimicrobials are begun. Because hemoconcentration is caused by the capillary leak, if anemia coexists, then blood is given. Maintaining the hemoglobin concentration at : 9 g/ dL did not have superior outcomes compared with that of :7 g/dL (Holst, 2014). hat said, fetal oxygenation is improved by the higher concentration.

1	The use of colloid solution such as hetastarch is controversial (Angus, 2013; Ware, 2000). One randomized trial comparing tality rate with the starch solution (Perner, 2012). Another study found equivalent results with 6-percent hydroxyethyl starch compared with normal saline (Myburgh, 2012). Albumin was not found to be superior to crystalloids (Caironi, 2014). Aggressive volume replacement ideally is promptly followed by urinary output of at least 30 and preferably 50 mL/hr, as well as other indicators of improved perfusion. If not, then con sideration is given for vasoactive dtug therapy (Pacheco, 2014). Mortality rates are high when sepsis is further complicated by respiratory or renal failure. With severe sepsis, damage to pul alveolar looding and pulmonary edema. This may occur even with low or normal pulmonary capillary wedge pressures, as with the ARDS discussed on page 918 and depicted in Figure 47-1.

1	Broad-spectrum antimicrobials are chosen empirically based on the probable source of infection. They are given promptly in maximal doses after appropriate cultures are taken of blood, urine, or exudates not contaminated by normal flora. In severe sepsis, appropriate empirical coverage results in better survival rates (Barochia, 2010; MacArthur, 2004). In obstetrics acute pyelonephritis is usually caused by Enterobacteriaceae, as discussed in Chapter 53 (p. 1028). For pelvic infections, empirical coverage with regimens such as ampicillin plus gentamicin plus clindamycin generally suices (Chap. 37, p. 668). Associated incisional and other soft-tissue infections are increasingly likely to be caused by methicillin-resistant S aureus, thus vancomycin therapy may be added (Klevens, 2007; Rotas, 2007). With a septic abortion, a Gram-stained smear may be helpful in identiying Clostridium species or group A streptococcal organisms. his is also true for deep fascial infections.

1	Continuing sepsis may prove fatal, and debridement of necrotic tissue or drainage of purulent material is crucial (Nelson, 2015; Pacheco, 2014). In obstetrics, the major causes of sepsis are infected abortion, pyelonephritis, and puerperal pelvic infections, which include metritis and infections of perineal lacerations or of hysterotomy or laparotomy incisions. With a septic abortion, uterine contents must be removed promptly by curettage as described in Chapter 18 (p. 351) . Hysterectomy is seldom indicated unless gangrene has resulted.

1	For women with pyelonephritis, continuing sepsis should prompt a search for obstruction caused by calculi or by a perinephric or intrarenal phlegmon or abscess. Renal sonography or "one-shot" pyelography can help diagnose obstruction and calculi. With obstruction, ureteral catheterization, percutaneous nephrostomy, or flank exploration may be lifesaving (Chap. 53, p. 1029). Computed tomography (CT) or magneticresonance imaging aids in identiying a phlegmon or abscess. Puerperal Infections. Most cases of puerperal pelvic sepsis are clinically manifested in the first several days postpartum, and FIGURE 47-6 Algorithm for evaluation and management of sepsis syndrome. Rapid and aggressive implementation is paramount for success. The three steps-Evaluate, Assess, and Manage-are carried out as simultaneously as possible.

1	intravenous antimicrobial therapy without tissue debridement cases, there is bacteremia and widespread tissue invasion, but with is generally curative. here are at least three exceptions. an intact uterus and abdominal incision. If uterine necrosis can

1	First, massive uterine myonecrosis can be caused by group A be excluded-usually by CT scanning-then in our experiences, 3-hemolytic streptococcal or clostridial infections (Soper, 2011; as well as in others, hysterectomy may not be necessary (Soper, Sugiyama, 2010; Yamada, 2010). hose with early-onset disease 2011). Still, these infections are highly lethal (Yamada, 2010). present with findings listed in Table 47-6. The mortality rate in As a second exception, necrotizing fasciitis of the episiotomy these women with gangrene s shown in Figure 47-7 is high, and site or of the abdominal surgical incision is a surgical emergency. prompt hysterectomy may be lifesaving (Mabie, 1997; Nathan, As described by Gallup and coworkers (2002), these infections 1993). Group A 3-hemolytic streptococci and clostridial coloare aggressively managed as discussed in Chapter 37 (p. 671). nization or infection also cause toxic-shock syndrome without Sinha and colleagues (2015) described a woman with

1	and clostridial coloare aggressively managed as discussed in Chapter 37 (p. 671). nization or infection also cause toxic-shock syndrome without Sinha and colleagues (2015) described a woman with Fournier obvious gangrene (Mason, 2012). These are due to either strepgangrene who required radical debridement and colostomy. tococcal toxic-shock-syndrome-like toxin or clostridial exotoxin As a inal exception, persistent or aggressive postpartum that evolved from 5 aureus (Chap. 37, p. 675). In many of these uterine infection with necrosis, uterine incision dehiscence, and severe peritonitis may lead to sepsis (Chap. 37, p. 672). In this regard, women following cesarean delivery who are suspected of having peritonitis should be carefully evaluated for uterine incisional necrosis or bowel perforation. These infections tend to be less aggressive than necrotizing group A streptococcal infections and develop later postpartum. CT imaging of the abdomen and pelvis can frequently disclose these.

1	These infections tend to be less aggressive than necrotizing group A streptococcal infections and develop later postpartum. CT imaging of the abdomen and pelvis can frequently disclose these. If either is suspected, then prompt surgical exploration is indicated. With incisional necrosis, hysterectomy is usually necessary (Fig. 37-5, p. 673). Finally, peritonitis and sepsis much less commonly may result from a ruptured parametrial, intraabdominal, or ovarian abscess (Chap. 37, p. 671).

1	As shown in Figure 47-6, a woman with severe sepsis syndrome is supported with continuing crystalloid infusion, blood FIGURE 47-7 A fatal case of group A 3-hemolytic Streptococcus pyogenes puerperal infection following an uncomplicated vaginal delivery at term. The infection caused uterine gangrene and overwhelming sepsis syndrome. Arrows point to overtly "balloonedout" black gangrenous areas of the postpartum uterus at the time of laparotomy for hysterectomy. transusions, and ventilation. In some cases, other measures may be necessary. Vasoactive drugs are not given unless aggressive luid treatment fails to correct hypotension and perfusion abnor malities. First-line vasopressors are norepinephrine, epinephrine, dopamine, dobutamine, or phenylephrine (Vincent, 2013).

1	he use of corticosteroids remains controversial. Some stud ies, but not all, show a salutary efect of corticosteroid admin istration. It is thought that critical ilness-related corticosteroid sion. hus, corticosteroids may be considered for use in vaso pressor-dependent patients (Angus, 2013; Munford, 2015). Endotoxin stimulates endothelial cells to up regulate tis sue factor and thus procoagulant production (Cunningham, 2015). Consumptive coagulopathy associated with sepsis is dis cussed in Chapter 41 (p. 782). At the same time, it decreases the anticoagulant action of activated protein C. Several agents developed to block coagulation, however, did not improve outcomes. Some include recombinant activated protein C, anti thrombin II, platelet-activating actor antagonist, and tissue ac tor pathway inhibitor (Munford, 2015; Wenzel, 2012).

1	Depending on deinitions used, 10 to 20 percent of gravidas suffer physical trauma (Jain, 2015; Lucia, 2016). Moreover, injuryrelated deaths are the most commonly identiied nonobstetrical cause of maternal mortality (Brown, 2013a; Horon, 2001). In a California study of4.8 million pregnancies, almost 1 in 350 women were hospitalized for injuries from assaults (El Kady, 2005). From Parkland Hospital, motor vehicle accidents and falls accounted for 85 percent of injuries sustained by 1682 pregnant women (Hawkins, 2007). From the National Violent Death Reporting System, Palladino and colleagues (2011) found 2.0 pregnancyassociated suicides per 100,000 live births. he rate was 2.9 per 100,000 for pregnancy-associated homicides. Notably, intimatepartner violence may be linked to these suicides (Martin, 2007). Finally, injury prevention and education of high-risk patients may help to decrease morbidity (Chisolm, 2017; Lucia, 2016).

1	According to the CDC, llltlmate-partner violence describes physical, sexual, or psychological harm by a current or former partner or spouse (Breiding, 2015). Such violence afects 1 in 5 women each year. One goal in violence prevention for Healthy People 2010 was the reduction of physical abuse directed at women by male partners. The Pregnancy Risk Assessment Monitoring Systems (PRAMS) report showed some improvement in these areas (Suellentrop, 2006). Even more appalling is that physical violence directed at women continues during pregnancy. Abuse is linked to poverty, poor education, and use of tobacco, alcohol, and illicit drugs (Centers for Disease Control and Prevention, 2008). Unfortunately, abused women tend to remain with their abusers, and the major risk factor for intimate-partner homicide is prior domestic violence (Campbell, 2007). Finally, women seeking pregnancy termination have a higher incidence of intimatepartner violence (Bourassa, 2007).

1	exposure is high aFor nonpregnant women, doxycycline, 100 mg orally twice daily for 7 days, can be given instead. bpregnancy category C. HIV = human immunodeficiency virus; 1M = intramuscularly. Data from Centers for Disease Control and Prevention, 2015. The woman who is physically abused tends to present late, if at all, for prenatal care. In one study, pregnant women hospitalized in California as a result of assault had signiicantly increased perinatal morbidity rates (El Kady, 2005). Immediate sequelae included uterine rupture, preterm delivery, and maternal and perinatal death. Subsequent outcomes included increased rates of placental abruption, preterm and low-birthweight newborns, and other adverse outcomes. Silverman and associates (2006) reported similar results from PRAMS, which included more than 118,000 pregnancies in 26 states.

1	Preventatively, the American College of Obstetricians and Gynecologists (2012) recommend universal screening for intimate-partner violence at the initial prenatal visit, during each trimester, and again at the postpartum visit (Chap. 9, p. 162). Others recommend a case-finding approach based on clinical suspicion (Robertson-Blackmore, 2013). According to the National Intimate Partner and Sexual Violence Survey (Black, 2014), an estimated 1.2 million women will be sexually assaulted each year. Satin and coworkers (1992) reviewed more than 5700 female sexual assault victims in Dallas County and reported that 2 percent were pregnant. Associated physical trauma is common (Sugar, 2004). From a forensic standpoint, the evidence collection protocol is not altered (Linden, 2011).

1	In addition to attention to physical injuries, exposure to sexually transmitted diseases must be considered. The CDC (2015) recommends antimicrobial prophylaxis against gonorrhea, chlamydial infection, bacterial vaginosis, and trichomoniasis (Table 47-7). If the woman is not pregnant, another very important aspect is emergency contraception, as recommended by the American College of Obstetricians and Gynecologists (2016; 2017a) and discussed in Chapter 38 (p. 696). Finally, the importance of psychological counseling for the rape victim and her family cannot be overemphasized. A 30-to 35-percent lifetime risk each for posttraumatic stress disorder, major depression, and suicide contemplation follows sexual assault (Linden, 201r1) .

1	At least 3 percent of pregnant women are involved in motor vehicle accidents each year in the United States. Using data from PRAMS, Sirin and colleagues (2007) estimated that 92,500 gravidas are injured annually. Motor-vehicle crashes are the most common causes of serious, life-threatening, or fatal blunt FIGURE 47-8 Illustration showing correct use of three-point automobile restraint. The upper belt is above the uterus, and the lower belt fits snugly across the upper thighs and well below the uterus.

1	trauma during pregnancy (Brown, 2013a; Mendez-Figueroa, 2013,r2016; Vladutiu, 2013). Mattox and Goetzl (2005) report these accidents to be the leading cause of traumatic fetal deaths as well. This was also true from our experiences from Parkland Hospital (Hawkins, 2007). Traic crashes are most frequent in the second trimester (Redel meier, 2014). As with all motor vehicle crashes, alcohol use is often associated. But sadly, as many as half of accidents occur without seat-belt use, and many of these deaths would likely be preventable by the three-point restraints shown in Figure 47 8 (Luley, 2013; Schuster, 2016). Seat belts prevent contact with the steering wheel, and they reduce abdominal impact pressure (Motozawa, 2010).

1	Original concerns regarding injuries caused by airbag deployment have been somewhat allayed (Luley, 2013; Matsushita, 2014). One study included 30 such women from 20 to 37 weeks' gestation whose airbag deployed in accidents with a median speed of35 mph (Metz, 2006). A third did not use seat belts, and there was one fetal death from the single case of placental abruption. In a retrospective cohort study that included 2207 pregnant women in crashes with airbag deployment, perinatal outcomes were not clinically diferent from 1141 controls without airbags (Schif, 2010). Importantly, 96 percent of both groups used seat belts. Thus, it appears that injuries with airbag deployment are related to the severity of the crash (Mendez-Figueroa, 2016).

1	Some other common causes of blunt trauma are falls and aggra vated assaults. In the California review reported by El Kady and associates (2005), intentionally inflicted injuries were present in approximately a third of pregnant women who were hospitalized for trauma. Less common are blast or crush injury (Sela, 2008). With blunt trauma, intraabdominal injuries can be serious. Even so, bowel injuries are less frequent because of the protective efect of a large uterus. Still, diaphragmatic, splenic, liver, and kidney damage may also be sustained. Par ticularly worrisome is the specter of amnionic-fluid embolism, which has been reported with even mild trauma (Ellingsen, 2007; Pluymakers, 2007). Retroperitoneal hemorrhage is pos sibly more common than in nonpregnant women (T akehana, 2011).

1	Orthopedic injuries are also encountered with some regular ity (Desai, 2007). From the Parkland Hospital trauma unit, 6 percent of 1682 pregnant women evaluated had orthope dic injuries. his subset was also at increased risk for placen tal abruption, preterm delivery, and perinatal mortality. In a review of 101 pelvic fractures during pregnancy, there was a 9-percent maternal and 35-percent fetal mortality rate (Leggon, 2002). In another study of pelvic and acetabular fractures dur ing 15 pregnancies, there was one maternal death, and four of 16 fetuses died (Almog, 2007). Finally, head trauma and neu rosurgical care raise unique issues (Qaiser, 2007).

1	Perinatal death rates increase with the severity of maternal injuries. Fetal death is more likely with direct fetoplacental injury, maternal shock, pelvic fracture, maternal head injury, or hypoxia (Ikossi, 2005; Pearlman, 2008). Motor vehicle accidents caused 82 percent of fetal deaths from trauma. Death was caused by placental injury in half and by uterine rupture in 4 percent (Weiss, 2001).

1	Although uncommon, fetal skull and brain injuries are more likely if the head is engaged and the maternal pelvis is fractured (Palmer, 1994). Conversely, fetal head injuries, presumably from a contrecoup efect, may be sustained in unengaged vertex or nonvertex presentations. Fetal skull fractures are rare and best seen using CT imaging (Sadro, 2012). One example is Figure 46-8 (p. 908). Other sequelae include intracranial hemorrhage (Gherman, 2014; Green-hompson, 2005). A newborn with paraplegia and contractures associated with a motor vehicle accident sustained several months before birth was described by Weyerts and colleagues (1992). Other injuries have included fetal decapitation or incomplete midabdominal fetal transection at midpregnancy (Rowe, 1996; Weir, 2008).

1	Catastrophic events that occur with blunt trauma include placental injuries-abruption or placental tears (Fig. 47-9). Placental separation from trauma is likely caused by deformation of the elastic myometrium around the relatively inelastic placenta (Crosby, 1968). his may result from a deceleration injury as the large uterus meets the immovable steering wheel or seat belt. Some degree of abruption complicates 1 to 6 percent of p. 768). Kettel and coworkers (1988) emphasized that traumatic abruption may be occult and unaccompanied by uterine pain, tenderness, or bleeding. In our experiences with 13 such women at Parkland Hospital, 11 had uterine tenderness, but only five had vaginal bleeding. Because traumatic abruption is more likely to be concealed and generate higher intrauterine pressures, associated coagulopathy is more likely than with nontraumatic abruption (Cunningham, 2015). Partial separation may also generate uterine activity, which is described more fully on page 930.

1	associated coagulopathy is more likely than with nontraumatic abruption (Cunningham, 2015). Partial separation may also generate uterine activity, which is described more fully on page 930. Other features are evidence of fetal compromise such as fetal tachycardia, sinusoidal pattern, late decelerations, acidosis, and fetal death.

1	FIGURE 47-9 Mechanism of placental tear or "fracture" caused by a deformationreformation injury. Placental abruption is seen as blood collecting in the retroplacental space. Inset. From here, blood can be forced into placental bed venules and enter maternal circulation. Such fetomaternal hemorrhage may be identified with Kleihauer-Betke testing.

1	If the abdominal force associated with trauma is considerable, then the placenta can be torn or "fractured" (see Fig. 47-9). If so, then life-threatening fetal hemorrhage may be encountered either into the amnionic sac or by fetomaternal hemorrhage (Pritchard, 1991). he tear is linear or stellate and is caused by rapid deformation and reformation (Fig. 47-10). Especially if there is ABO compatibility, fetomaternal hemorrhage is quantified using a Kleihauer-Betke stain of maternal blood. A small amount of fetal-maternal bleeding has been described in up to a third of trauma cases, and in 90 percent of these, the volume is < 15 mL (Goodwin, 1990; Pearlman, 1990). Parenthetically, nontraumatic placental abruption is much less often associated with signifi minor injuries and up to 50 percent of major injuries (Pearlcant fetomaternal hemorrhage because only minimal fetal blood man, 1990; Schif, 2002). Abruption was found to be more enters into the intervillous space. With traumatic

1	of major injuries (Pearlcant fetomaternal hemorrhage because only minimal fetal blood man, 1990; Schif, 2002). Abruption was found to be more enters into the intervillous space. With traumatic abruption, likely if vehicle speed exceeded 30 mph (Reis, 2000). however, massive fetomaternal hemorrhage may follow. In one Clinical indings with traumatic abruption may be simistudy, the risk of associated uterine contractions and preterm lar to those for spontaneous placental abruption (Chap. 41, labor was a 20-fold if there was evidence for a fetomaternal

1	FIGURE 47-10 A. Partial placental abruption in which the adherent blood clot has been removed. Note the laceration of the placenta (arrow), which caused fetal death from massive fetomaternal hemorrhage. B. Kleihauer-Betke stain of a peripheral smear of maternal blood. The dark cells that constituted 4.5 percent of red blood cells are fetal in origin, whereas the empty cells are maternal. bleed (Muench, 2004). With severe fetal bleeding, long-term adverse neurological outcomes are frequent (Kadooka, 2014).

1	Blunt trauma leads to uterine rupture in < 1 percent of severe cases (American College of Obstetricians and Gynecologists, 20 17b). Rupture is more likely in a previously scarred uterus and is usually associated with a direct impact of substantial force. Decelerative forces following a 25-mph collision can generate up to 500 mm Hg of intrauterine pressure in a properly restrained woman (Crosby, 1968). Clinical indings may be identical to those for placental abruption with an intact uterus, and maternal and fetal deterioration are soon inevitable. Pearlman and Cunningham (1996) described uterine fundal "blowout" with fetal decapitation in a 20-week pregnancy following a high-speed collision. Similarly, Weir and colleagues (2008) described supracervical uterine avulsion and fetal transection at 22 weeks. CT scanning may be useful to diagnose uterine rupture with a dead fetus or placental separation (Kopelman, 2013; Manriquez, 2010; Sadro, 2012).

1	In a study of 321 pregnant women with abdominal trauma, Petrone (2011) reported a 9-percent incidence of penetrating injuries. Of these, 77 percent were gunshot wounds and 23 percent were stab wounds. he incidence of maternal visceral injury with penetrating trauma is only 15 to 40 percent compared with 80 to 90 percent in nonpregnant individuals (Stone, 1999). When the uterus sustains penetrating wounds, the fetus is more likely than the mother to be seriously injured. Indeed, although the fetus sustains injury in two thirds of cases with penetrating uterine injuries, maternal visceral injuries are seen in only 20 percent. Still, their seriousness is underscored in that maternal-fetal mortality rates are signiicantly higher than those seen with blunt abdominal injuries in pregnancy. Specifically, maternal mortality rates were 7 versus 2 percent, and fetal mortality rates were 73 versus 10 percent, respectively . • Management of Trauma

1	• Management of Trauma Maternal and fetal outcomes are directly related to the severity of injury. That said, commonly used methods of severity scoring do not take into account significant morbidity and mortality rates related to placental abruption and thus to pregnancy outcomes. In a study of 582 pregnant women hospitalized for injuries, the injury severity score did not accurately predict adverse pregnancy outcomes (Schif, 2005). Importantly, relatively minor injuries were associated with preterm labor and placental abruption. Others have reached similar conclusions (Biester, 1997; Ikossi, 2005). In a study of 317 women at 24 weeks' gestation or more who had "minor trauma," 14 percent had clinically significant urerine contractions requiring extended fetal evaluation past 4 hours (Cahill, 2008).

1	With few exceptions, treatment priorities in injured pregnant women are multidisciplinary (Barraco, 2010; Mendez-Figueroa, 2016). Primary goals are evaluation and stabilization of maternal injuries. Attention to fetal assessment during the acute evaluation (merican College of Obstetricians and Gynecologists, 2017b; Brown, 2009). Basic rules of resuscitation include ventilation, arrest of hemorrhage, and treatment of hypovolemia with crys talloid and blood products. After midpregnancy, the large uterus is positioned of the great vessels to diminish its efect on vessel compression and cardiac output (Nelson, 2015).

1	Following emergency resuscitation, evaluation is continued for fractures, internal injuries, bleeding sites, and placental, uterine, and fetal trauma. Radiography is not proscribed, but special attention is given each indication. Not surprisingly, one report observed that pregnant trauma victims had less radiation exposure than nonpregnant controls (Ylagan, 2008). Some advocate screening abdominal sonography followed by CT scanning for positive sonographic findings (Brown, 2005; Saphier, 2014). Procedures used include the FAST scan ocused 4ssessment with �onography or t.rauma. his examination is ars-minute, four-to six-view imaging study that evaluates perihepatic, perisplenic, pelvic, and pericardial views (MendezFigueroa, 2016). In general, if fluid is seen in any of these views, then the volume is >500 mL (Fig. 47-11). Importantly, this amount has not been corroborated for pregnancy. In some cases, open peritoneal lavage may be informative (Tsuei, 2006).

1	Penetrating injuries in most cases must be evaluated using radiography. Because clinical response to peritoneal irritation is blunted during pregnancy, an aggressive approach to exploratory laparotomy is pursued. Whereas exploration is mandatory for abdominal gunshot wounds, some clinicians advocate close observation for selected stab wounds. Diagnostic laparoscopy has also been used (Chap. 46, p. 901). The necessity for cesarean delivery depends on several factors. Laparotomy itself is not an indication for hysterotomy. FIGURE 47-1 1 Fast scan. Upper quadrant scan shows anechoic free fluid (asterisk) between the liver edge (arrow) and kidney (Morison pouch). The patient had 2500 mL of blood in the peritoneal cavity. (Reproduced with permission from Mendez-Figueroa H, Rouse OJ: Trauma in pregnancy. In Yeomans ER, Hoffman BL, Gilstrap, III, et al (eds): Cunningham and Gilstrap's Operative Obstetrics, 3rd ed. New York, McGraw-Hili, 2017.)

1	Some considerations include gestational age, fetal condition, extent of uterine injury, and whether the large uterus hinders adequate management of other intraabdominal injuries (Tsuei, 2006).

1	Because fetal well-being may relect the status of the mother, fetal monitoring is another "vital sign" that helps evaluate the extent of maternal injuries. Even if the mother is stable, electronic monitoring may suggest placental abruption. In a study by Pearlman and coworkers (1990), no woman had an abruption if uterine contractions were less often than every 10 minutes within the 4 hours after trauma was sustained. lmost 20 percent ofwomen who had contractions more frequently than every 10 minutes in the irst 4 hours had an associated placental abruption. In these cases, abnormal tracings were common and included fetal tachycardia and late decelerations. Conversely, no adverse outcomes were reported in women who had normal monitor tracings (Connolly, 1997). Importantly, if tocolytics are used for these contractions, they may obfuscate findings, and we do not recommend them.

1	Because placental abruption usually develops early following trauma, fetal monitoring is begun as soon as the mother is stable. The ideal duration ofposttrauma monitoring is not precisely known. From data cited above, observation for 4 hours is reasonable with a normal tracing and no other sentinel indings such as contractions, uterine tenderness, or bleeding. Certainly, monitoring should be continued as long as there are uterine contractions, nonreassuring fetal heart patterns, vaginal bleeding, uterine tenderness or irritability, serious maternal injury, or ruptured membranes (American College ofObstetricians and Gynecologists, 2017b). In rare cases, placental abruption has developed days after trauma (Higgins, 1984).

1	It is unclear whether routine use of the Kleihauer-Betke or an equivalent test in pregnant trauma victims might modiy adverse outcomes associated with fetal anemia, cardiac arrhythmias, and death (Pak, 1998). In a retrospective review of 125 pregnant women with blunt injuries, the Kleihauer-Betke test was judged to be of little value during acute trauma management (Towery, 1993). Others have reached similar conclusions, although a positive test with fetal cells of0.1 percent was predictive of uterine contractions or preterm labor (Connolly, 1997; Muench, 2003, 2004). For the woman who is D-negative, administration ofanti-D immunoglobulin should be considered. his may be omitted if a test for fetal bleeding is negative. Even with anti-D immunoglobulin, alloimmunization may still develop if the fetalmaternal hemorrhage exceeds 15 mL of fetal cells (Chap. 15, p.r306).

1	For the pregnant trauma patient, confirmation of current tetanus immunization status is pertinent. When indicated, a dose oftetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine (Tdap) is preferred for its neonatal pertussis immunity beneits (Chap. 9, p. 171). Fetal* Maternal... FIGURE 47-1 2 Maternal and fetal mortality rates by burn severity in nearly 400 women. (Data from Akhtar, 1994; Amy, 1985; Mabrouk, 1977; Maghsoudi, 2006; Parikh, 2015; Rayburn, 1984; Rode, 1990.)

1	Treatment ofthe burned gravida is similar to that for nonpregnant patients (Mendez-Figueroa, 2016). With treatment, it is generally agreed that pregnancy does not alter maternal outcome from thermal injury compared with that of nonpregnant women ofsimilar age. As perhaps expected, maternal and fetal survival parallels the percentage of burned surface area (Parikh, 2015). Karimi and colleagues (2009) reported higher mortality rates for both with suicidal attempts and with inhalational injuries. The composite mortality rate for nearly 400 women from seven studies increased in a linear fashion as the percent of burned body surface area increased 47-12). For 20-, 40-, and 60-percent burns, the maternal mortality rates were approximately 4, 30, and 93 percent, respectively. he corresponding fetal mortality rates were 20, 48, and 96 percent, respectively. With severe burns, the woman usually enters labor spontaneously within a few days to a week and often delivers a stillborn. Contributory

1	mortality rates were 20, 48, and 96 percent, respectively. With severe burns, the woman usually enters labor spontaneously within a few days to a week and often delivers a stillborn. Contributory factors are hypovolemia, pulmonary injury, septicemia, and the intensely catabolic state (Radosevich, 2013).

1	Following serious abdominal burns, skin contractures that develop may be painful during a subsequent pregnancy and may even require surgical decompression and split-skin autografts (Mitsukawa, 2015; Radosevich, 2013). Loss or distortion of nipples may cause problems in breastfeeding. Mitsukawa and associates (2015) reported that contracture release was indicated with scars spanning more thanr>75 percent of the total abdominal area. Alternatively, normal abdominal tissue expansion due to pregnancy appears to be an excellent source for obtaining skin grafts postpartum to correct scar deformities at other body sites (Del Frari, 2004).

1	Earlier case reports suggested a high fetal mortality rate with electric shock (Fatovich, 1993). In a prospective cohort study, however, Einarson and coworkers (1997) showed similar perinatal outcomes in 31 injured women compared with those of noninjured controls. They concluded that traditional 110volt North American electrical current likely is less dangerous than the 220-volt currents available in Europe. A woman with been related to a mild electrical shock at 22 weeks was described (Sozen, 2004). Another woman with brain death from cardiac arrest was reported (Sparic, 2014). Thermal burns with electro cution may be extensive. The pathophysiological efects of lightning injuries can be devastating. Garda Gutierrez and coworkers (2005) reviewed a 50-percent stillbirth rate.

1	According to estimates from the Nationwide Inpatient Sample, cardiac arrest complicates approximately 1 in 12,000 delivery admissions (Mhyre, 2014) . The most common underlying causes were hemorrhage, heart failure, amnionic-Buid embolism, and sepsis. General topics regarding planning and equipment have been reviewed by the American College of Obstetricians and Gynecologists (20 17b) and the Society for Obstetric Anesthesia and Perinatology (Lipman, 2014). Special considerations for cardiopulmonary resuscitation (CPR) conducted in the second half of pregnancy are outlined in the American Heart Association 2010 guidelines (Jeejeebhoy, 2015). he committee acknowledges the following as standards for critically ill gravidas: (1) relieve possible vena caval compression by left lateral uterine displacement, (2) administer 100-percent oxygen, (3) establish intravenous access above the diaphragm, (4) assess for hypotension that warrants therapy, which is defined as systolic blood pressure <

1	displacement, (2) administer 100-percent oxygen, (3) establish intravenous access above the diaphragm, (4) assess for hypotension that warrants therapy, which is defined as systolic blood pressure < 100 mm Hg or < 80 percent of baseline, and (5) review possible causes of critical illness and treat conditions as early as possible.

1	he position of the heart for external compressions is not diferent from that in nonpregnant women (Holmes, 2015). In nonpregnant women, external chest compression results in a cardiac output approximately 30 percent of normal. In late pregnancy, this may be even less with compressions because of uterine aortocaval compression (Clark, 1997; Nelson, 2015). Thus, it is paramount to accompany other resuscitative eforts with uterine displacement. This can be accomplished by tilting the operating table laterally, by placing a wedge under the patient's right hip, or by pushing the uterus to the left manually (Rees, 1988; Rose, 2015). If no equipment is available, an individual may kneel on the Boor with the maternal back on his or her thighs to form a "human wedge" (Whitty, 2002).

1	During maternal resuscitation, because of pregnancy-induced hindrances on CPR eforts, emergent perimortem cesarean delivey for fetal salvage and improved maternal resuscitation may be considered. Some have stated that cesarean delivery is indicated within 4 to 5 minutes of beginning CPR if the fetus is viable (Drukker, 2014). In women delivered by perimortem cesarean, neurologically intact neonatal survival and the cardiac arrest-todelivery interval are inversely related (Katz, 2012). Speciically, of newborns delivered within 5 minutes of arrest, 98 percent are neurologically intact; within 6 to 15 minutes, 83 percent are intact; within 16 to 25 minutes, 33 percent are intact; and within 26 to 35 minutes, only 25 percent are intact (Clark, 1997). This, coupled with some evidence that delivery may also enhance maternal resuscitation, has led the American College of Obstetricians and Gynecologists (2017b) to recommend consideration for cesarean delivery to begin within 4 minutes of

1	delivery may also enhance maternal resuscitation, has led the American College of Obstetricians and Gynecologists (2017b) to recommend consideration for cesarean delivery to begin within 4 minutes of cardiac arrest in these cases.

1	his serious and sometimes contentious issue is far from evi dence based. To wit, Katz and associates (2005) reviewed 38 perimortem cesarean deliveries with a "large selection bias." of maternal cardiac arrest improves maternal and fetal outcomes.

1	Even so, as emphasized by Clark (1997) and Rose (2015) and their coworkers, and in our experiences, these goals rarely can be met in actual practice. For example, most cases of cardiac arrest occur in uncontrolled circumstances, and thus, the time to CPR initiation alone would require the irst 5 minutes. hus "crash" cesarean delivery would supersede resuscitative eforts, would necessarily be done without appropriate anesthesia or surgical equipment, and more likely than not, would lead to maternal death. Moreover, the distinction between a peri mortem versus postmortem cesarean operation is imperative (Katz, 2012; Rose, 2015). Last, in the balance, any choice may favor survival of the mother over the fetus, or vice versa, and thus there are immediate unresolvable ethical concerns. Katz (2012) has provided a scholarly review of peri mortem cesarean delivery.

1	Occasionally, a pregnant woman with a supposedly healthy intact fetus will be kept on somatic support to await fetal viability or maturity. This is discussed in Chapter 60 (p. 1168). According to their review, Brown and coworkers (2013b) reported that clinically significant envenomations in pregnant women are from snakes, spiders, scorpions, jellyish, and hymenoptera such as bees, wasps, hornets, and ants. Adverse outcomes are related to maternal efects. These investigators conclude that limited evidence supports the use of a venomspeciic approach that includes symptomatic care, antivenom administration when appropriate, anaphylaxis treatment, and fetal assessment. One management scheme for North American snakebites was provided by Lei and associates (2015).

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1	Ylagan MV, Trivedi N, Basu T, et al: Radiation exposure in the pregnant trauma patient: implications for fetal risk counseling. Abstract No. 320. Am J Obstet Gynecol 199(6):5100,t2008 Zeeman GG: Obstetric critical care: a blueprint for improved outcomes. Crit Care Med 34:5208, 2006 Zeeman GG, Wendel GO Jr, Cunningham FG: A blueprint for obstetric critical care. Am J Obstet Gynecol 188:532, 2003 Zinaman M, Rubin J, Lindheimer MD: Serial plasma oncotic pressure levels and echoencephalography during and after delivery in severe preeclampsia. Lancet 1:1245, 1985 I recenty saw a patient who imagined hersef in the last month of pregnancy, and who, while talking to me, exclaimed at the violence of the movements, but on examination I ound that her uterus was normal in size, and that her enlarged abdomen was due to a rapidy increasing deposit of at. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) At the beginning of the last century, obesity was not terribly problematic, and with few exceptions, Williams did not refer to its adverse obstetricl efects. Fast forward to today when excessive weight is a major health problem in many aiuent societies (GBD 2015 Obesity Collaborators, 2017). Indeed, by 2014, more than a third of all adults in the United States were obese (Ogden, 2015). The adverse health aspects of obesity are staggering and include risks for diabetes mellitus, heart disease, hypertension, stroke, and osteoarthritis. Obese gravidas and their fetuses are predisposed to various serious pregnancy-related complications and to higher long-term morbidity and mortality rates.

1	Of systems to classiy obesity, the body mass index (EM), also known as the Quetelet index, is most oten used. The BMI is calculated as weight in kilograms divided by the square of the height in meters (kg/mr). Calculated BMI values are available in various chart and graphic forms (Fig. 48-1). The National Institutes of Health (2000) classiies adults according to BMI as follows: normal is 18.5 to 24.9 kg/mr, overweight is 25 to 29.9 kg/mr, and obese is �30 kg/mr. Obesity is urther divided into: class 1 is 30 to 34.9 kg/mr, cass 2 is 35 to 39.9 kg/mr, and cass 3 is �40 kg/mr. Class 3 obesity is oten referred to as morbid obesity, with super-morbid obesiy describing a BMI �50 kg/mr.

1	Using these deinitions, from 2011 to 2014, slightly more women than men were designated obese-36 versus 34 percent (Ogden, 2015). Among girls and women, the prevalence of obesity rises with age and varies among ethnicities 48-2). lthough obesity is now common among all socioeconomic levels, the overall severity advances with increasing poverty (Bilger, 2017). Also, a genetic predisposition has been identiied from several gene loci (Locke, 2015; Shungin, 2015).

1	Fat tissue is much more complex than merely its energy storage function. Many fat tissue cells communicate with all other tissues via endocrine and paracrine factors, which are cytokines speciically termed adipocytokines. Also simply called adipokines, some of these with metabolic functions include adiponectin, leptin, tumor necrosis factor-a (TNF-a), interleukin 6 (IL-6), resistin, visfatin, apelin, vascular endothelium growth factor (VEGF), lipoprotein lipase, and insulin-like growth factor. A principal adipokine is adiponectin, which is a 30-kDa protein. It enhances insulin sensitivity, blocks hepatic glucose release, and has cardioprotective efects on circulating plasma lipids. An adiponectin deficit is linked with diabetes, hypertension, endothelial cell activation, and cardiovascular disease.

1	Cytokines that result in insulin resistance are leptin, resistin, TNF-a, and IL-6, and higher levels of these are found during pregnancy. Indeed, adipokines, especially the inflammatory cytokines, may be the primary stimulant of insulin resistance

1	Weight (pounds) 90 110 130 150 170 190 210 230 250 270 290 310 330 350 2 , 1 1 6'6, 1 I, , 1 I I II ' , 1 I I II 1 1 I, , 1 I I I, , I 1 I I Underwei�ht 11BMI < 185 I /•BMI > I 1.9 I _ 1 I 6'3., 1 1 1 I , i 1 I 1 I, , 1 1 1, , 1 1 I I , , . 1 1 1I, , I 1 1 I1 1 , , 1 1 1 I n, , 1 1 1 1 , , 1 1 1 1 )1.8 5'1e1 c n 1 1 0, , 1 1 1 Cm 1 I, , 1 1 1 I3 1 1 1 1 01 1 C s I 1 1 1 l1 1 : ' 1 I 3, 1 I �) , 1 I') 1.7 1,-, 1 5'71 :I ''i 1 1I )I '), , I , , I, , 1 , , I , , 11 1.6 ' , 1 5'3t --, 1 , 1 , I1, 11I11 1.5 4'1e1 I· 40 50 60 70 80 90 100 110 120 130 140 150 160 Weight (kg) FIGURE 48-1 Chart for estimating body mass index (8M I). To find the 8Ml category for a particular subject, locate the point at which the height and weight intersect. (Al-Badri, 2015; Yang, 20r16). Conversely, adiponectin has antiinflammatory and insulin-sensitizing roles and is negatively regulated by fat mass. As one example of the discordant efects of these adipokines, gestational diabetes is associated with lower

1	has antiinflammatory and insulin-sensitizing roles and is negatively regulated by fat mass. As one example of the discordant efects of these adipokines, gestational diabetes is associated with lower adiponectin but higher leptin levels. Placental production of these adipokines is also important and related to fetal growth • Metabolic Syndrome Given its multifaceted endocrine and paracrine functions, the detrimental efects of excessive adipose tissue are not surprising (Cornier, 201r1; Gilmore, 2015). Obesity interacts with inherited factors to cause insulin resistance. This resistance is characterized by impaired glucose metabolism and a predisposition to type 2 diabetes. Insulin resistance also causes several subclinical abnormalities that predispose to cardiovascular disease and accelerate its onset. The most important among these are type 2 diabetes, dyslipidemia, and hypertension, which are constituents of the metabolic syndrome. Criteria to define this syndrome are found in Table

1	its onset. The most important among these are type 2 diabetes, dyslipidemia, and hypertension, which are constituents of the metabolic syndrome. Criteria to define this syndrome are found in Table 48-1 (Alberti, 2009). Waist circumference is the preferred measurement for screening, but any three of five factors listed are suicient to diagnosis the metabolic syndrome. N otably, most patients with type 2 diabetes have metabolic syndrome according to these criteria. Also, obese women with hypertension typically demonstrate elevated plasma insulin levels. hese are even higher in women with central obesity (Fu, 2015). he National Health Nutrition Examination Survey (NHANES) of the Centers for Disease Control and Prevention documented an overall 34-percent prevalence of the metabolic syndrome in the United States by 2012 (Moore, 2017). As expected, the prevalence rose with age. It was 20 percent for those aged 18 to 29 years and was 36 percent for those aged 30 to 49 years.

1	and adiposity by mechanisms yet to be defined (Sartori, 2016). 60 46 36 Girls Women FIGURE 48-2 Prevalence of obesity in girls and women in the United States for 2009-201o4. (Data from Ogden, 201o5.) FIGURE 48-3 Estimated hazard ratios (95% (I) for death due to cardiovascular disease according to body mass index among 1.46 million white adult men and women. (Data from de Gonzalez, 2010.) TABLE 48-1 . Criteria for Diagnosis of the Metabolic Syndrome Patients with three or more of the following: Elevated triglyceridesb: ::150 mg/dL Reduced high-density lipoprotein cholesterolb: <50 mg/dL in Elevated blood pressureb: systolicn::130 mm Hg and/or diastolic ::85 mm Hg Elevated fasting glucoseb: ::100 mg/dL country-and population-specific thresholds. bThose with normal values while taking medications are considered to meet these criteria. Data from Alberti, 2009.

1	bThose with normal values while taking medications are considered to meet these criteria. Data from Alberti, 2009. Generally speaking, visceral adiposity correlates with hepatic fat content (Cornier, 201l). With obesity, excessive fat accumulates in the liver-hepatic steatosis, which is also called nonalcoholic fatty liver disease (NAFLD). In persons with the metabolic syndrome, steatosis can progress to nonalcoholic steatohepatitis (NASH) and cirrhosis, as well as hepatocellular carcinoma. Indeed, one fourth of chronic liver disease cases worldwide are caused by NAFLD (Younossi, 2016). \10reover, NAFLD is strongly associated with both fatal and nonfatal cardiovascular disease (T argher, 2016).

1	Obese individuals sufer well-known consequences such as glucose intolerance, hypertension, dyslipidemia, and metabolic syndrome. Furthermore, metabolic syndrome and obesity are linked with cardiovascular disease, including myocardial infarction, atrial ibrillation, heart failure, and stroke (Long, 2016). Insulin resistance and metabolic syndrome cause structural cerebral changes and lower executive functioning and memory in adults. Similar consequences are also found in adolescents, suggesting that metabolic syndrome's efects on neurocognitive function are independent of signiicant occlusive vascular disease (Rusinek, 2014).

1	Obesity is associated with higher rates of all-cause early mortality (Fontaine, 2003; Peeters, 2003). Cardiovascular mortality data from 19 prospective studies are shown in Figure 48-3. In these and other studies, mortality risk from cardiovascular disease and cancer grew proportionally with increasing BM!. Importantly, however, an obesiy paradox-whereby certain groups actually derive a survival advantage from being obese-has been hypothesized (Hainer, 2013). Despite this, the health beneits of weight normalization are well documented (Cheung, 2017).

1	Weight loss is tremendously diicult for obese individuals. If achieved, long-term maintenance poses equally daunting challenges. Obstetrician-gynecologists are encouraged to aid weight 4.42 loss in obese adult women. Successful approaches include behavioral, pharmacological, and surgical techniques or a combination of these methods (Dixon, 2016). Dietary changes and exercise reduce weight and rates of the associated metabolic syndrome (Garvey, 2016; Martin, 2016). When used in conjunction with bariatric surgery, glucose control in those with type 2 diabetes is improved (Schauer, 2014). However, both surgical and medical interventions are associated with appreciable long-term failure rates-up to 50 percent in patients with type 2 diabetes undergoing bariatric surgery (Mingrone, 2015).

1	Obese women unequivocally have reproductive disadvantages (American Society for Reproductive Medicine, 2015).r1his translates into diiculty in achieving pregnancy, early and recurrent pregnancy loss, preterm delivery, and several obstetrical, medical, and surgical complications with pregnancy, labor, delivery, and the puerperium (American College of Obstetricians and Gynecologists, 2015). Also, oral contraceptive failure may be more likely in overweight women (Chap. 38, p. 691). Last, infantsand later, adult children-of obese mothers have correspondingly higher morbidity rates (Godfrey, 2017; Reynolds, 2013). Obesity complicating pregnancy has grown substantially in this country. Our experience at Parkland Hospital over three epochs is shown in Figure 48-4.

1	Obesity complicating pregnancy has grown substantially in this country. Our experience at Parkland Hospital over three epochs is shown in Figure 48-4. For overweight women, higher rates of adverse outcomes complicate pregnancy (Schummers, 2015). Shown in Table 48-2 are results from ive studies including more than 1 million singleton pregnancies. Although not as magniied as in the obese cohort, rates of almost all complications are signiicantly greater in overweight women than in those whose BMI is normal. obesity and the metabolic syndrome are charac terized by insulin resistance, which causes low grade inlammation and endothelial activation (Ma, 2016). hese latter efects playra central role in preeclampsia (Chap. 40, p. 715). he overwhelming evidence between rising maternal BMI and the incidence of preeclampsia is depicted in Figure 48-6. Similar observations were reported from a large Canadian study and by the Safe Labor Consortium (im, 2016; Schummers, 2015).

1	Obesity and hypertension are common cofactors in peripartum heart failure (Cunningham, 1986,r2012). Stewart and colleagues (2016) prospectively studied the efect of obesity on cardiac remodeling in pregnancy among 14 normal and 9 overweight or obese women (Fig. 4-8, p. 61). FIGURE 48-4 Increasing prevalence of obesity during four epochs in pregnant women classified at the time of their first prenatal visit at Parkland Hospital. regressed to normal by 3 months postpartum. or women (Fig. 48-7). This, however,

1	or women (Fig. 48-7). This, however, For obese women, deinitions used in studies of adverse out comes vary widely, and BMIs from >30 kg/mrto >50 kg/mrhave served as thresholds (Crane, 2013; Denison, 2008; Stamilio, 2014). Of outcomes, Mariona (2017) reviewed maternal deaths in Michigan and found that the risk of a maternal death was nearly fourfold higher in obese women. Women with supermorbid obesity experience very high rates of maternal and neonatal complications including preeclampsia, fetal overgrowth, and cesarean delivery, with even higher rates of meconium aspiration, ventilator support, and neonatal death (Marshall, 2014; Smid, 2016). Data from one large study is shown in Figure 48-5. Especially striking are the markedly elevated rates of hypertension and gestational diabetes. As discussed previously,

1	Especially striking are the markedly elevated rates of hypertension and gestational diabetes. As discussed previously, Obesity and gestational diabetes are inextricably linked as shown in Table 48-2. heir coexistence with and adverse efects on pregnancy outcomes are discussed in Chapter 57 (pp. 1097 and 1111). Nonalcoholic fatty liver disease is associated with several adverse pregnancy outcomes. In a cohort of 110 women with NAFLD, risks for preeclampsia, preterm birth, low-birthweight neonates, cesarean delivery, and gestational diabetes were elevated (Hagstrom, 2016). In one prospective study of 476 pregnancies, first-trimester sonographic evidence of maternal NAFLD was strongly associated with gestational diabetes (De Souza, 2016a,b). Meyer and associates (2013) found that overweight and obese gravidas had a higher proportion of low-density lipoprotein III (LDL-III) compared with that of

1	TABLE 48-2. Adverse Pregnancy Effects in Overweight and Obese Women 18.5-24.9 n = 621,048 2.3 2.7 3.u 20.9 6.6 25.2 2.0 Prevalence (%) with Odds Ratio Overweight 8MI 25-29.9 Obese 8MI > 30 n = 228,945 4.3 (OR 1n.91n, 1.86-1n.96) 4.3 (OR 1n.60, 1.56-1n.64) 4.1 (OR 1.09, 1.05-1.13) 23.8 (OR 1.19, 1.17-1n.21) 8.3 (OR 1.28, 1.26-1n.31) 31.5 (OR 1.37, 1.34-1.39) 2.4 (OR 1.22, 1n.17-1.28) n = 78,043 8.6 (OR 4.04,n3.94-4.15) 8.1 (OR 3.17, 3.0u-3.25) 4.8 (OR 1.28, 1.23--1n.34) 29.7 (OR 1.60, 1.57--1.64) 11.5 (OR 1.85, 1.81-1.89) 39.3 (OR 1.92, 1.88-1n.96) 2.3 (OR 1.14, 1.08-1.21) 8.4 (OR 1.29, 1.26-1.31) 8.7 (OR 1.34, 1.31-1.37) Pelvic infection 0.6 0.7 (OR 1.16, 1.06-1.26) 0.8 (OR 1.28, 1.15-1n.43) Wound infection or complication 0.4 0.5 (OR 1.42, 1.28-1.58) 1.0 (OR 2.70, 2.42-3.01) Large for gestational age 8.7 13.1 (OR 1.57, 1.54-1.61) 16.3 (OR 2.04, 1.99-2.10)

1	Wound infection or complication 0.4 0.5 (OR 1.42, 1.28-1.58) 1.0 (OR 2.70, 2.42-3.01) Large for gestational age 8.7 13.1 (OR 1.57, 1.54-1.61) 16.3 (OR 2.04, 1.99-2.10) Macrosomia 2.0 3.6 (OR 1.81, 1.74-1.88) 5.1 (OR 2.60, 2.50-2.71) Stillbirth 0.3 1.8 (OR 5.89, 5.57-6.22) 0.5 (OR 1.71, 1.56-1.87) aOdds ratios with 95% CI are significant when compared to normal BMI group. BMI = body mass index. Data from Kim, 2016; Lisonkova, 2017; Ovesen, 20n1n1; Schummers, 2015; Sebire, 2001n. normal-weight women. LDL-III predominance is a hallmark of ectopic liver fat accumulation that is typical of NAFLD. At Parkland Hospital, we are now frequently encountering obese gravidas who have NAFLD and evidence of steatohepatitis manifest by elevated serum hepatic transaminase levels. In rare cases, liver biopsy is necessary to exclude other causes.

1	In addition to these metabolic complications, quality-of-life measures are also negatively afected by obesity during pregnancy (Amador, 2008; Ruhstaller, 2017). One systematic review found significantly higher risks ofdepression in overweight and obese women during and after pregnancy (Molyneaux, 2014). Obese women were also significantly more likely to experience anxiety during pregnancy. Stillbirths are more prevalent as the degree of obesity accrues (Ovesen, 2011; Schummers, 2015). In a review of almost 100 -15-:))..)S 10)):)0'3E 5 0 30 20 FIGURE 48-6 Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study: frequency of preeclampsia according to BMI. (Data from the HAPO Study Cooperative Research Group, 2008.) D Controls (BMI <30) (n 3752) D Obese (BMI 30-35) (n = 1473) • Morbidly obese (BMI >35) (n = 877) 4.8 8.3

1	D Controls (BMI <30) (n 3752) D Obese (BMI 30-35) (n = 1473) • Morbidly obese (BMI >35) (n = 877) 4.8 8.3 FIGURE 48-5 Incidence of selected pregnancy outcomes in 16,102 women enrolled in the FASTER (First-and Second-Trimester Evaluation of Risk) trial according to BMI. (Data from Weiss, 2004.) studies, obesity was the highest ranking modifiable risk factor for stillbirth (Flenady, 201l). In super-morbidly obese compared with normal-weight gravidas, Yao and associates (2014) found 5.7 and l3.6-fold higher stillbirth rates at 39 and 41 weeks' gestation, respectively. Remarkably, 25 percent of term stillbirths in this study involved obese women. Chronic hypertension with superimposed preeclampsia associated with obesity is one cause of excessive stillbirths.

1	Evaluating perinatal death rates, Lindam and coworkers (2016) reported that high maternal BMI in early pregnancy was a risk factor. The risk ofneonatal death is also greater for obese women 0ohansson, 2014; Meehan, 2014). Finally, Cnattingius and Villamor (2016) noted that accruingweight between pregnancies is a risk factor for perinatal mortality, whereas weight loss between pregnancies for overweightwomen lowers this risk. Both fetal and neonatal complications are increased in obese women. Two important and interrelated cofactors that 0.85 • Obese J 0.80 0.75 .J,0.70 .J,0.65 0.60

1	Both fetal and neonatal complications are increased in obese women. Two important and interrelated cofactors that 0.85 • Obese J 0.80 0.75 .J,0.70 .J,0.65 0.60 FIGURE 48-7 Geometric changes of ventricular remodeling across pregnancy in obese and normal-weight women. LVM = left ventricular mass, LVEDV = left ventricular end-diastolic volume. (Data from Stewart, 2016.) contribute to excessive rates of perinatal morbidity are chronic hypertension and diabetes, both of which are associated with maternal obesity. hese comorbidities each may playra role in the higher rates of fetal-growth restriction and indicated pre term birth that are seen in obese women (Schummers, 2015). Pregestational diabetes also raises the birth defect rate, and ges tational diabetes is complicated by excessive numbers of large for-gestational-age and macrosomic fetuses (Chap. 44, p. 857). Even when diabetes is not considered, the prevalence of macrosomic newborns is greater in obese women (Kim, 2016;

1	Even when diabetes is not considered, the prevalence of macrosomic newborns is greater in obese women (Kim, 2016; Ovesen, 2011; Schummers, 2015). he group from Metro prepregnancy obesity, gestational weight gain, and diabetes and their relationship to adverse pregnancy outcomes and to greater newborn weight and fat mass (Catalano, 2009, 2015; Lassance, 2015; Ma, 2016; Yang, 2016). Although each of these variables is associated with larger and more corpulent newborns, pre gene expression has the strongest influence on the prevalence of macrosomic neonates.

1	Rates of birth defects are also higher with comorbid obesity (Stothard, 2009). For neural-tube defects, elevated risks of 1.2-, 1.7-, and 3. I-fold are found in overweight, obese, and severely obese women, respectively (Rasmussen, 2008). he National Birth Defect Prevention Study reported a correlation between BMI and congenital heart defects (Gilboa, 2010). However, this may be related to diabetes as a cofactor (Biggio, 2010). Importantly, obesity is detrimental to the accuracy of obstetrical sonographic examination and to antepartum identiication of birth defects (Adekola, 2015; Dashe, 2009; Weichart, 2011).

1	Obese women beget obese children, who themselves become obese adults. Catalano and coworkers (2009) studied ofspring at a mean age of 9 years and found a direct association with maternal prepregnancy obesity and childhood obesity. hey also reported associations with central obesity, elevated systolic blood pressure, increased insulin resistance, and lipid abnormalities-all elements of the metabolic syndrome. Reynolds and associates (2013) reported higher rates of cardiovascular disease and all-cause mortality in 37,709 adult ofspring of overweight and obese mothers. Similar cardiometabolic health efects in ofspring were echoed by Gaillard and colleagues (2016). Other data support that excessive maternal weight gain in pregnancy may predict obesity in adult ofspring (Lawrence, 2014; Reynolds, 2010). Last, rates of glucose intolerance and metabolic syndrome are higher among ofspring of obese women (Gaillard, 2016; Tan, 2015).

1	he potential biological mechanisms of these associations are unclear. But such studies raise the possibility ofetal programming, that is, the fetal environment may lead to adverse adult health outcomes. Elucidation is limited by insuicient data on potential maternal and genetic predisposing factors and on the environment of the infant and child in relation to diet and activity. he science of epigenetics has provided some support for the possibility that perturbations of the maternal-fetal environment can adversely alter postdelivery events (Kitsiou-T zeli, 2017). Also possible are contributions of the maternal-child environment subsequent to birth (Gluck, 2009). These and other factors regarding fetal programming are discussed in Chapter 44 (p. 849).

1	The Institute of Medicine (2009) has updated its previous maternal weight gain determinants (Table 9-4, p. 166). For overweight women, weight gain of 15 to 25 pounds is sug gested. For obese women, the Institute advocates a gain of 11 to 20 pounds. Intuitively, maternal weight must increase suf iciently to provide for fetal and placental tissue accrual and for amnionic fluid and maternal blood volume expansion. Thus, maternal weight loss is during pregnancy is discouraged. The American College of Obstetricians and Gynecologists (2015) endorses these Institute guidelines.

1	American College of Obstetricians and Gynecologists (2015) endorses these Institute guidelines. However, these recommendations were issued without irm scientific evidence to support them, and their value remains unproven (Rasmussen, 2010). For example, recent studies differ with respect to the efect of insuicient weight gain for obese women. Bodnar and colleagues (2016) reported no greater risk for low-birthweight or small-for-gestational-age newborns among 47,494 obese women who had inadequate weight gain during pregnancy. Bogaerts and associates (2015) found that even weight loss among obese women did not yield poor fetal growth. In contrast, however, Hannaford and coworkers (2017) reported that obese women who gained less than the Institute recommendations were almost three times more likely to deliver a small-for-gestational-age neonate. Another study similarly found an almost twofold greater risk among obese women who lost weight during pregnancy (Cox Bauer, 2016).

1	Apart from inadequate weight gain, excessive gestational weight gain may portend greater risks for the obese mother. Berggren and coworkers (2016) noted that overweight and obese women accrued maternal fat rather than lean mass with excessive gestational weight gain. From another analysis, overall higher rates of hypertensive disorders, cesarean delivery, and fetal overgrowth as well as lower rates of spontaneous preterm birth and fetal undergrowth were found among women gaining more than recommended Oohnson, 2013). However, when analyzed according to BMI category, significantly higher rates of preeclampsia, cesarean delivery, and fetal overgrowth were identified among the 1937 overweight women, but not for the 1445 obese women, who gained excess weight.

1	During pregnancy, overweight and obese women gain more weight than recommended compared with normal-weight gravidas (Endres, 2015). Moreover, overweight and obese women have excessive postpartum weight retention at 1 year, and one third retain at least 20 pounds more than their prep regnancy weight.

1	Several dietary interventions can help limit and achieve the weight-gain targets listed in the previous section. Options include lifestyle interventions and physical activity. In one randomized trial of exercise in 300 overweight women, risks for gestational diabetes were lowered (Wang, 2017). That said, in another trial, 75 overweight women were randomly assigned to routine care or to a 16-week moderate-intensity stationary cycling program starting after midpregnancy. NIaternal and neonatal outcomes did not difer between groups (Seneviratne, 2016). Also, a Cochrane database analysis of 11,444 women suggests that lifestyle interventions confer only a modest reduction in maternal weight gain, and their benefits for fetal overgrowth, cesarean delivery rate, and adverse neonatal outcome are not significant (Muktabhant, 2015). Regarding neonatal outcomes, the poor success of lifestyle interventions during pregnancy has been attributed to their late introduction, that is, after early gene

1	not significant (Muktabhant, 2015). Regarding neonatal outcomes, the poor success of lifestyle interventions during pregnancy has been attributed to their late introduction, that is, after early gene expression within the placenta has already been programmed (Catalano, 2015).

1	Close prenatal monitoring detects most early signs of diabetes or hypertension. Standard screening tests for fetal anomalies are suicient, while remembering the sonographic limitations for fetal anomaly detection in this group. Accurate fetal growth surveillance in obese women usually requires serial sonographic assessment. Antepartum external fetal heart rate monitoring is likewise more diicult. Obese women are at increased risk for multiple labor or intrapartum complications. These include postterm pregnancy or labor abnormalities (Carpenter, 2016). In one study of 143,519 women, the odds of spontaneous labor at term in obese women was approximately half that of normal-weight women (Denison, 2008). In an analysis of more than 5000 parturients, women with a Bh11 >30 kg/mrhad a longer duration of and slower early progression in first-stage labor (Norman, 2012).

1	Compared with normal-weight women, obese women are twice as likely to undergo labor induction (Denison, 2008). Unfortunately, obese women are also twice as likely to experience a failed induction, and this risk rises with greater degrees of obesity (Wolfe, 2011). In a retrospective analysis of 470 nul liparous women with a BMI >30 kg/mrand an unfavorable cervix, those who underwent labor induction at 39 weeks' gestation were compared with those expectantly managed beyond 39 weeks (Wolfe, 2014). Two thirds of pregnancies expectantly managed either labored or had spontaneously ruptured membranes. Compared with this cohort, those who underwent planned labor induction had an elevated cesarean delivery rate-26 versus 40 percent. Moreover, their newborns were more frequently admitted to the neonatal intensive care unit-6 versus 18 percent. Conversely, Lee and associates (2016) reviewed statistics from 74,725 deliveries in obese women and reported that elective induction at 37 to 39 weeks in

1	intensive care unit-6 versus 18 percent. Conversely, Lee and associates (2016) reviewed statistics from 74,725 deliveries in obese women and reported that elective induction at 37 to 39 weeks in nulliparas and especially multiparas was actually associated with a lower cesarean delivery rate. hese conflicting results highlight the diiculties faced by obstetrical providers as they contemplate the seemingly competing interests of the fetus and the obese mother. To address this, the Maternal-Fetal Medicine Units Network is conducting a randomized trial of planned labor induction at 39 weeks' gestation in nulliparous women.

1	Obese women present anesthesia challenges that include diicult epidural and spinal analgesia placement and complications from failed or diicult intubations. Evaluation of super-morbidly obese gravidas by the anesthesiologist is recommended during prenatal care or upon arrival to the labor unit (American College of Obstetricians and Gynecologists, 2017). Although the rationale for antepartum anesthesia consultation and early epidural analgesia access seems logical, little published data truly demonstrates benefits from these practices (Eley, 2016) .

1	Regional analgesia for morbidly obese women is associated with longer neuraxial procedure times and more failed placement attempts (Tonidandel, 2014). Importantly, however, spinal analgesia in obese women for cesarean delivery does not appear to have benefits over combined spinal-epidural. For example, Ross and colleagues (2014) compared single-shot spinal analgesia with combined spinal-epidural analgesia and found that both methods could be placed with equal expediency and function similarly in morbidly obese patients.

1	Obese women who undergo regional analgesia that is complicated by relative hypotension more frequently have neonates with umbilical artery cord blood acidemia, probably due to delayed delivery. Edwards and colleagues (2013) studied 5742 obese women and found that pH significantly dropped, and base deficit rose, with increasing BMI. The rate of pH <7.1 doubled from 3.5 percent for a BMI <25 kg/mrto 7.1 percent for a BMI ::40 kg/mr. Anesthetic risks and complications are discussed in more detail in Chapter 25.

1	These rates are significantly greater in obese women. In one study, the primary rate was 33.8 percent for obese and 47.4 percent for morbidly obese women. These values compare strikingly with the rate of only 20.7 percent for normal-weight gravidas (Weiss, 2004). In an analysis of 226,958 women, cesarean delivery rates rose signiicantly for overweight (34 percent), class I (38 percent), class II (43 percent), and class III (50 percent) obesity (Schummers, 2015). In the same study, rates of gestational diabetes, itself a risk factor for cesarean delivery, increased from 6 percent for women with a BMI <25 kg/mrto 21 percent for those with a BMI of ::40 kg/mr. More worrisome is that obese women also have higher rates of emergency cesarean delivery, and obesity lengthens times for decision-to-incision and for delivery (O'Dwyer, 2013; Pulman, 2015). Girsen and associates (2014) found significantly increased incision-to-delivery times for both emergent and nonemergent cases.

1	Discussed in Chapter 31 (p. 596), the incidence of failed trial oflabor after cesarean is higher in obese women (Grasch, 2017; Hibbard, 2006). Women who gain weight between pregnancies also have signiicantly lower rates of vaginal birth after cesarean. IIIII \I.' I FIGURE 48-8 Abdominal incision for the obese woman. A. Frontal view. The dotted line indicates an appropriate skin incision for abdominal entry relative to the panniculus. As shown by the uterus in the background, selection of this periumbilical site permits access to the lower uterine segment. B. Sagittal view.

1	For cesarean delivery, forethought is given to optimal placement and type of abdominal incision to allow access to the fetus and to efect the best wound closure. We prefer a vertical incision in obese women to provide the most direct access (Fig. 48-8). Others prefer a low transverse abdominal incision, with or without rostral taping of the pendulous abdomen. Individual diferences in maternal body habitus preclude naming anyrone approach as superior (tfcLean, 2012; Turan, 2016). Some observational studies have compared wound outcomes associated with vertical and transverse skin incisions, but results are conflicting as to a superiority of either (Brocato, 2013; Marrs, 2014; McClean, 2012; Sutton, 2016; Thornburg, 2012).

1	he frequency of abdominal wound infections is directly related to BM!. Conner and associates (2014) found the risk of wound infection is threefold higher for super-morbidly obese women compared with nonobese women-23 versus 7 percent. Among women with a BMI >45 kg/mr, wound complication rates range from 14 to 19 percent (Smid, 2015; Stamilio, 2014). Comorbid diabetes apparently raises this risk (Leth, 201l). Other studies describe wound complication rates ranging from 2 to >40 percent in obese women (Conner, 2014; vIarrs, 2014; Smid, 2015; Thornburg, 2012).

1	Several interventions maybe preventive. Closure ofsubcuta tion rates (Tipton, 2011). Studies have also examined the use of higher doses of perioperative prophylactic antibiotics. Phar macokinetic studies indicate that tissue concentrations of pro phylactic antibiotics are lower with increasing BMI (Pevzner, 2011; Young, 2015). One prospective study showed that a 3-g dose of cefazolin resulted in higher tissue concentrations com pared with a 2-g dose (Swank, 2015). hat said, a retrospective analysis of 335 women with a median weight of 310 pounds found that the higher dose of cefazolin did not result in few surgical site infections (Ahmadzia, 2015). In one recent study, phylaxis had a surgical infection rate of 13.4 percent compared with a rate of 6.4 percent for those given a 2-day course of oral cephalexin and metronidazole in addition to perioperative prophylaxis (Valent, 2017).

1	used prophylactically (Mark, 2014). To address this, Hussamy and colleagues (2018) designed a randomized trial of NPWT going cesarean delivery. Such therapy did not signiicantly lower the postoperative wound complication rate compared with routine care-19 versus 17 percent, respectively. To lower thromboembolic complications, graduated compression stockings, hydration, and early mobilization after cesarean delivery in obese women are recommended by the American College of Obstetricians and Gynecologists (2015). Some also recommend "mini-dose" heparin prophylaxis, but we do not routinely use this (Chap. 52, p. 1019).

1	Several surgical procedures are designed to treat morbid obesity either by diminishing gastric volume-restrictive, or by bypassing gastrointestinal absorption-restrictive nalabsorptive. In nonpregnant patients, these procedures improve or resolve diabetes, hyperlipidemia, hypertension, and obstructive sleep apnea and reduce risks of myocardial infarction and death (Beamish, 2016). Of options, the two approved laparoscopic adjustable silicone gastric banding (LASGB) procedures-LAPBAND and REALIZE-place a band 2 cm below the gastroesophageal junction to create a small stomach pouch above the ring. he ring diameter is controlled by a saline reservoir in the band. hese procedures can have positive efects on pregnancy outcomes. For example, Dixon and colleagues (2005) compared TABLE 8-3. Pregnancy Outcomes Following Bariatric Surgery Outcomea (n = 651) (n = 361.) aData not reported identically-frequencies are approxi mations. bData from Adams, 201n5; Bar-Zohar, 2006; Carelli, 201n1;

1	Outcomea (n = 651) (n = 361.) aData not reported identically-frequencies are approxi mations. bData from Adams, 201n5; Bar-Zohar, 2006; Carelli, 201n1; Dixon, 2005; Ducarme, 2013; Facchiano, 2012; Lapolla, 2010; Pilone, 2014; Sheiner, 2009; Skull, 2004. (Data from Adams, 2015; Ducarme, 2013; Facchiano, 2012; Gonzalez, 2015; Sheiner, 2009. pregnancy outcomes in bariatric surgery patients against their preprocedural outcomes and those of a matched cohort of obese women. Following banding, the incidences of gestational hypertension-1 0 versus 45 percent-and gestational diabetes-6 versus 15 percent-were significantly lower in the bariatric surgery patients compared with their preprocedural pregnancies. The results from these and other studies are shown in Table 48-3.

1	Deflation of the band during pregnancy afects maternal and fetal weight gain. Pilone and coworkers (2014) studied 22 pregnancies after band placement and reported that all women underwent full delation of the band in the first trimester and gained an average of 14.7 kg during pregnancy. In another study, 42 women underwent deflation of the band, whereas 54 women maintained band inlation. A deflated band was associated with higher mean weight gain-15.4 kg versus 7.6 kg, increased birthweight-3712 versus 3380 g, and a twofold greater risk of macrosomia compared with an inflated one (Cornthwaite, 2015). Rarely, the band may slip from nausea and vomiting, especially with advancing gestation or postpartum (Pilone, 2014; Schmitt, 2016; Sufee, 2012). One fatal fetal cerebral hemorrhage developed from maternal vitamin K deiciency secondary to prolonged vomiting due to band slippage that created a gastric outlet obstruction (Van lieghem, 2008).

1	The laparoscopically performed Roux-en-Y gastric bypass is the most commonly used procedure for gastric restriction and selective malabsorption. Its surgical steps are described in Figure 48-9. As with other bariatric procedures, pregnancy outcomes are changed remarkably following Roux-en-Y bypass (Adams, 2015). As shown in Table 48-3, rates of hypertension, gestational diabetes, and fetal macrosomia are reduced. Serious complications are uncommon, however, upper abdominal pain is frequent in pregnancy and often associated with internal FIGURE 48-9 Roux-en-Y bypass. With this, the proximal stomach is completely transected to leave a 30-mL pouch. The proximal end of the distal jejunum is then connected to the small pouch. This bypasses a large part of the stomach and duodenum. At a site 60 cm distal to this gastrojejunostomy, a Roux-en-Y enteroenterostomy is also completed to allow drainage of secretions from the unused stomach and duodenum.

1	herniation, which is protrusion of the bowel through a mesentery defect. Petersen and associates (2017) described outcomes in a birth cohort including 139 pregnancies. Upper abdominal pain complicated 46 percent, and a third of these had internal herniation. The preterm birth rate was 14 of 64 among those with upper abdominal pain versus 1 of75 in those without pain. Intussusception and small bowel obstruction can develop from internal herniation, and maternal deaths from herniation and obstruction have been reported (Moore, 2004; Renault, 2012). Bowel obstruction is notoriously diicult to diagnose (Vannevel, 2016; Wx, 2013).

1	Because of its associated health successes, bariatric surgery is popular, and many women subsequently become pregnant (Narayanan, 2016). From observational studies, fertility rates improve and obstetrical complication rates decline in women after bariatric surgery compared with morbidly obese controls (Kominiarek, 2017; Yi, 2015). In one these studies, despite surgical treatment, almost half of 670 women were still obese at the time of their first pregnancy after bypass Qohansson, 2015). Nevertheless, the frequency of large-for-gestational-age newborns dropped from 22 to 8.6 percent and of small-forgestational-age neonates rose from 7.6 to 15.6 percent. In a systematic review, Yi and colleagues (2015) conirmed these fetal weight trends after bariatric surgery. Also, risks for diabetes and preeclampsia were reduced.

1	Currently, the American College of Obstetricians and Gynecologists (2015) recommends that women who have undergone bariatric surgery be assessed for vitamin and nutritional suiciency. When indicated, vitamins Bl2 and D, folic acid, and calcium supplementation are given. Vitamin A deficiency is also possible (Chagas, 2013). Women with a gastric band should be monitored by their bariatric team during pregnancy because adjustments of the band may be necessary. Finally, special vigilance is appropriate for signs of internal herniation with intestinal obstruction (Stuart, 2017; Wx, 2013). Adams TD, Hammoud AO, Davidson LE, et al: Maternal and neonatal outcomes for pregnancies before and after gastric bypass surgery. Int JtObes (Lond) 39:686, 2015 Adekola H, Soto E, Dai J, et al: Optimal visualization of the fetal four-chamber and outlow tract views with transabdominal ultrasound in the morbidly obese: are we there yet? J Clin Ultrasound 43:548, 2015

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1	PHYSIOLOGICAL CONSIDERATIONS IN PREGNANCY.. . 948 DISEASE ................... 949 PERIPARTUM MANAGEMENT CONSIDERATIONS ..... 951 SURGICALLY DISEASE.. . . . . . . . . . 954 VALVULAR HEART DISEASE ...................... 955 CONGENITAL HEART DISEASE . . . . . . . . . . . . . . . . . . . . 958 PULMONARY HYPERTENSION . . . . . . . . . . . . . . . . . . . . 960 CARDIOMYOPATHIES ........................... 962 HEART FAILURE ................................ 964 INFECTIVE ENDOCARDITIS ....................... 965 ARRHYTHMIAS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 965 DISEASES OF THE AORTA ........................ 967 ISCHEMIC HEART DISEASE . . . . . . . . . . . . . . . . . . . . . . . 968 Some authorities recommend that women suering rom heart lesions be dissuaded rom marriage. This, however, appears to be an extreme view, though, of course, when the lesion is serious and the compensation auly, the dangers of childbearing should be caruly explaine.

1	-J. Whitridge Williams (1903) As Williams recognized more than a century ago, pregnancy in those with signiicant heart disease can be extremely hazardous and may lead to decompensation and death. In an analysis of maternal mortality in the United States between 2011 and 2013, the causes previously responsible for most maternal deaths-hemorrhage, hypertensive disorders, and embolism-continued to show declining rates. In contrast, deaths attributable to cardiovascular diseases were responsible for approximately 26 percent of all pregnancy-related deaths (Creanga, 2017). Cardiovascular diseases also account for significant maternal morbidity and are a prominent reason for obstetrical intensive care unit admissions (Small, 2012).

1	The rising prevalence of cardiovascular diseases complicating pregnancy is likely multifactorial and includes the higher rates of obesity, hypertension, and diabetes (Klingberg, 2017). Indeed, according to the National Center for Health Statistics, almost half of adults aged 20 and older have at least one risk factor for cardiovascular disease (Fryar, 2012). Another related reason is delayed childbearing. Last, as discussed subsequently (p. 958), an increasing number of women with congenital heart disease are now becoming pregnant.

1	he marked pregnancy-induced anatomical and functional changes in cardiac physiology can have a profound efect on underlying heart disease (Chap. 4, p. 60). Some of these changes are listed in Table 49-1. Importantly, cardiac output increases approximately 40 percent during pregnancy. Almost half of this total takes place by 8 weeks' gestation and is maximal by midpregnancy (Capeless, 1989). his early rise stems from augmented stroke volume, which results from lowered vascular resistance. Later in pregnancy, resting pulse and stroke volume are even higher because of greater end-diastolic ventricular volume that results from pregnancy hypervolemia. hese changes translate to a cardiac output that enlarges across pregnancy to TABLE 49-'. Hemodynamic Changes in 10 Normal Pregnant Women at Term Compared with Repeat Values Obtained 12 Weeks pregnant women because they do not take into account the spherical remodeling characteristic of normal pregnancy (Savu, 2012; Stewart, 2016).

1	Adjusting for these geometrical changes, Melchiorre and coworkers (2016) studied normal cardiac echocardiographic indings in 559 nulliparas at four points during pregnancy and again at 1 year postpartum. At term, signiicant chamber diastolic dysfunction and impaired myocardial relaxation were evident in approximately 18 and 28 percent of the women Data from Clark, 1989. average 40 percent higher at term. hese adaptations are even more profound in multifetal pregnancies (Kametas, 2003; Kuleva, 2011). Importantly, intrinsic left ventricular contractility does not change. hus, normal left ventricular function is maintained during pregnancy. Namely, pregnancy is not characterized by hyperdynamic function or a high cardiac-output state.

1	Women with underlying cardiac disease may not always accommodate these changes, and ventricular dysfunction leads to cardiogenic heart failure. A few women with severe cardiac dysfunction can experience evidence of heart failure before midpregnancy. In others, heart failure may develop after 28 weeks' gestation, when pregnancy-induced hypervolemia and cardiac output reach their maximum. In most, however, heart failure develops peripartum, when labor, delivery, and several common obstetrical conditions add undue cardiac burdens. Some of these include preeclampsia, hemorrhage and anemia, and sepsis.

1	Ventricular volumes and mass accrue to accommodate pregnancy-induced hypervolemia. his is reflected by greater end-systolic and end-diastolic dimensions. At the same time, however, septal thickness and ejection fraction are unchanged. his is because these alterations are accompanied by substantive ventricular remodeling-plasticiy-which is characterized by eccentric expansion of left-ventricular mass that averages 30 to 35 percent near term. All of these adaptations return to prepregnancy values within a few months postpartum.

1	Certainly for clinical purposes, ventricular function during pregnancy is normal as estimated by the Braunwald ventricular function graph depicted in Figure 4-9 (p. 61). For given illing pressures, there is appropriate cardiac output so that cardiac function during pregnancy is eudynamic. In nonpregnant subjects with a normal heart who sustain a high-output state, the left ventricle undergoes longitudinal remodeling, and echocardiographic functional indices of its deformation provide normal values. In pregnancy, there instead appears to be spherical remodeling, and these calculated indices that measure longitudinal deformation are depressed. Thus, normal nonpregnant indices are likely inaccurate when used to assess function in studied, respectively. Moreover, a significant proportion of women studied demonstrated a drop in stroke volume index and a tendency toward eccentric remodeling. hese indings suggest cardiovascular maladaptation to the increased volume demands in a substantial

1	women studied demonstrated a drop in stroke volume index and a tendency toward eccentric remodeling. hese indings suggest cardiovascular maladaptation to the increased volume demands in a substantial proportion of apparently normal pregnancies. Of note, signiicant dyspnea at rest was reported by 7.4 percent of the women at term, most of whom had chamber diastolic dysfunction. Cardiac function and all signs of dyspnea fully recovered at 1 year postpartum.

1	Cardiac magnetic resonance (MR) imaging increasingly is used to evaluate cardiac structure and function. Stewart and associates (2016) performed cardiac MR imaging studies in 23 women longitudinally across pregnancy and at 12 weeks postpartum. Compared with studies performed at 12 to 16 weeks' gestation, left ventricular mass grew significantly for both normal-weight and overweight women. he calculated geometrical ratio of left ventricular mass to left ventricular end-diastolic volume demonstrated concentric remodeling throughout gestation, which resolved by 12 weeks' postpartum. he right ventricle also remodels (Martin, 2017) . Taken together, these observations likely mean that pregnancy causes a mixture of eccentric and concentric ventricular remodeling.

1	he physiological adaptations of normal pregnancy can induce symptoms and alter clinical indings that may confound the diagnosis of heart disease. For example, in normal pregnancy, functional systolic heart murmurs are common, respiratory efort is accentuated, edema frequently accrues in lower extremities after midpregnancy, and fatigue and exercise intolerance often develop. Some systolic flow murmurs can be loud, and normal changes in the various heart sounds depicted in Figure 49-1 may erroneously suggest cardiac disease. In con trast, clinical findings that are more likely to suggest heart disease are listed in Table 49-2. Noninvasive cardiovascular studies such as electrocardiography, chest radiography, and echocardiography will provide the data necessary for evaluation in most women.

1	Noninvasive cardiovascular studies such as electrocardiography, chest radiography, and echocardiography will provide the data necessary for evaluation in most women. In the electrocardiogram (ECG) , an average 15-degree leftaxis deviation is found as the diaphragm is elevated in advancing pregnancy. Other indings, depicted in Figure 49-2, include a reduced PR interval, inverted or flattened T waves, and a Q wave in lead DIll (Angeli, 2014). Pregnancy does not alter voltage indings. Atrial and ventricular premature contractions are relatively frequent (Carruth, 1981). FIGURE 49-1 Normal cardiac examination findings in the pregnant woman. Sl = first sound; M1 = mitral first sound; S2 = second sound; P2 = pulmonary second sound. (Data from Gei, 2001; Hytten, 1991o.) TABLE 49-2. Clinical Indicators of Heart Disease During Pregnancy �� + Syncope Leftward deviation of the mean QRS axis .' I,,l, Clubbing of fingers

1	TABLE 49-2. Clinical Indicators of Heart Disease During Pregnancy �� + Syncope Leftward deviation of the mean QRS axis .' I,,l, Clubbing of fingers Q wave in lead 0'1" (Reproduced with permission from Angeli F, Angeli E, Verdecchia P: Electrocardiographic changes in hypertensive disorders of pregnancy, Hypertens Res. 2014 Nov;37(11 ):973-975.) 0001 ...0001 ",' FIGURE 49-2 Normal electrocardiograph (ECG) adaptations durPersistent tachycardia and/or arrhythmia ing pregnancy, including a reduced mean PR interval, increased Persistent split second sound heart rate, left axis deviation, inverted or flattened T waves and a With radiography, anteroposterior (AP) and lateral chest radiographs are useful, and when a lead apron shield is used, fetal radiation exposure is minimal. Gross cardiomegaly can usually be excluded, but slight heart enlargement cannot be detected accurately because the heart silhouette normally is larger in pregnancy. his is accentuated further with a portable

1	AP chest radiograph. Echocardiography is now widely used and permits accurate diagnosis of most heart diseases during pregnancy. Some nor the dimensions of all cardiac chambers, a slight but signii cant growth in left ventricular mass, and greater tricuspid and mitral valve regurgitation (Grewal, 2014). Of note, systolic function normally does not change. Savu (2012) and Vitarelli diographic parameters for pregnancy, which are listed in the Appendix (p. 1261). In some situations, such as complex con genital heart disease, transesophageal echo cardiography may be useful. Cardiovascular MR imaging, compared with echocardiography, is associated with higher reproducibility and is less hindered by ventricular geometry and body habitus. The right ventricle can also be assessed (Nelson, 2017). Ducas and associates (2014) have published normal reference values for pregnancy.

1	Of other studies, albumin or red cells tagged with technetium-99m are rarely needed during pregnancy to evaluate ventricular function. That said, the estimated fetal radiation exposure from nuclear medicine studies of myocardial perfusion is negligible. It is safe to perform cardiac catheterization with limited fluoroscopy time. During coronary angiography, the mean radiation exposure to the unshielded abdomen is 1.5 mGy, and less than 20 percent of this reaches the fetus (European Society of Cardiology, 2011). Shortening the fluoroscopic time may help to minimize radiation exposure (Raman, 2015; Tuzcu, 2015). In women with clear indications, any minimal theoretical fetal risk is outweighed by maternal beneits (Chap. 46, p. 906). • Classification of Functional Heart Disease

1	• Classification of Functional Heart Disease No clinically applicable test accurately measures functional cardiac capacity. The clinical classification of the New York Heart Association (NYHA) is based on past and present disability and is uninfluenced by physical signs: • Class I. Uncompromised-no limitation of physical activiy: These women do not have symptoms of cardiac insuiciency or experience anginal pain. Class II. Slight limitation of physical activiy: These women are comfortable at rest, but if ordinary physical activity is undertaken, discomfort in the form of excessive fatigue, palpitation, dyspnea, or anginal pain results. Class III. Marked limitation of physical activiy: These women are comfortable at rest, but less than ordinary activity causes excessive fatigue, palpitation, dyspnea, or anginal pain.

1	Class III. Marked limitation of physical activiy: These women are comfortable at rest, but less than ordinary activity causes excessive fatigue, palpitation, dyspnea, or anginal pain. Class IV. Severey compromised-inabiliy to peorm any physical activity without discomort: Symptoms of cardiac insufficiency or angina may develop even at rest. If any physical activity is undertaken, discomfort is increased. Cardiovascular Disorders 95.1

1	Cardiovascular Disorders 95.1 Siu and associates (2001 b) expanded the NYHA classification and developed a scoring system for predicting cardiac complications during pregnancy. The system derives from a Canadian prospective analysis of 562 pregnant women with heart disease during 617 pregnancies. Predictors of cardiac complications included: (1) prior heart failure, transient ischemic attack, arrhythmia, or stroke; (2) baseline NYHA class III or IV or cyanosis; (3) left-sided obstruction defined as mitral valve area <2 cmr, aortic valve area < 1.5 cmr, or peak left tion fraction <40 percent. The risk of pulmonary edema, sus tained arrhythmia, stroke, cardiac arrest, or cardiac death was substantially elevated with one of these factors and even more so with two or more. Khairy and colleagues (2006) reported similar indings.

1	An even more comprehensive risk stratification system is the tion of Cardiovascular Disease and Pregnancy (Table 49-3). It ceptional counseling. Lu (2015) and Pijuan-Domenech (2015) and their associates in their analyses concluded that the modi ied WHO classification provides the greatest predictive accu racy for cardiac complications during pregnancy.

1	Women with severe heart disease will beneit immensely from counseling before pregnancy, and they usually are referred for maternal-fetal medicine or cardiology consultation (Clark, 2012; Seshadri, 2012). Maternal mortality rates generally correlate directly with functional classification, and this relationship may change as pregnancy progresses. From the previously described Canadian study, Siu and colleagues (2001 b) observed signiicant worsening of NHA class in 4.4 percent of 579 pregnancies in which the baseline class was I or II. As described later, some women have life-threatening cardiac abnormalities that can be reversed by corrective surgery, and subsequent pregnancy becomes less dangerous. In other cases, such as women with mechanical valves taking warfarin, fetal teratogenic concerns predominate. Last, many congenital heart lesions are inherited as polygenic characteristics (Chap. 13, p. 268). Because of this, some women with congenital heart lesions give birth to similarly

1	predominate. Last, many congenital heart lesions are inherited as polygenic characteristics (Chap. 13, p. 268). Because of this, some women with congenital heart lesions give birth to similarly afected neonates, and the risk varies widely (Table 49-4).

1	In most instances, management involves a team approach with an obstetrician, cardiologist, anesthesiologist, and other specialists as needed. With complex lesions or other high-risk cases, evaluation by a multidisciplinary team is recommended early in pregnancy. Within this framework, both prognosis and management are influenced by the type and severity of the speciic lesion and by the maternal functional classiication. In some, pregnancy termination may be advisable. With rare exceptions, women in NYHA class I and most in class II negotiate pregnancy without morbidity. Special TABLE 49-3. World Health Organization (WHO) Risk Classification of Cardiovascular Disease and Pregnancy with Management Recommendations WHO l-Risk no higher than general population Uncomplicated, small, or mild: Mitral valve prolapse with no more than trivial mitral regurgitation Successfully repaired simple lesions:

1	WHO l-Risk no higher than general population Uncomplicated, small, or mild: Mitral valve prolapse with no more than trivial mitral regurgitation Successfully repaired simple lesions: WHO 2-Small increase in risk of maternal mortality If otherwise uncomplicated: • Individualized care similar to WHO categories 2 or 3 depending on lesion and disease severity WHO 3-Significantly increased risk of maternal Mechanical valve mortality or expert cardiac and obstetrical care Systemic right ventricle-congenitally corrected transposition, required simple transposition post Mustard or Senning repair WHO 4-Very high risk of maternal mortality or Pulmonary arterial hypertension termination discussed Previous peripartum cardiomyopathy with any residual impairment Marfan syndrome with aorta dilated >40 mm Pregnancy contraindicated. If pregnancy occurs, monthly or bimonthly cardiac and obstetrical monitoring

1	Marfan syndrome with aorta dilated >40 mm Pregnancy contraindicated. If pregnancy occurs, monthly or bimonthly cardiac and obstetrical monitoring Summarized from European Society of Gynecology, 201a1; Nanna, 2014; Thorne, 2006; World Health Organization, 201a0.

1	attention is directed toward both prevention and early recogCigarette smoking is prohibited. Illicit drug use may be parnition of heart failure. Of speciic risks, infection with sepsis ticularly harmful, an example being the cardiovascular efects syndrome can precipitate this. Moreover, bacterial endocardiof cocaine or amphetamines. In addition, intravenous drug use tis is a deadly complication of valvular heart disease (p. 965). raises the risk of infective endocarditis. Each woman is instructed to avoid contact with persons who Fortunately, women in NA class III and IV are uncomhave respiratory infections, including the common cold, and to mon today. In the prior Canadian study, only 3 percent of the report at once any evidence for infection. Pneumococcal and approximately 600 pregnancies were complicated by NA inluenza vaccines are recommended (Chap. 9, p. 172). class III heart disease, and no women had class IV when irst

1	TABLE 49-4. Risks for Fetal Heart Lesions Related to Afected Family Members Atrial septal defect 2.5 1.5 5-11 Patent ductus arteriosus 3 2.5 4 Coarctation of the aorta NS NS 14 Fallot tetralogy 2.5 1.5 2-3 NS = not stated. Data from Lupton, 2002. seen (Siu, 2001b). If a woman chooses pregnancy, she must understand the risks and is encouraged to be compliant with planned care. In some women, prolonged hospitalization or bed rest is often necessary. In general, vaginal delivery is preferred, and labor induction is usually safe (Thurman, 2017). From the large Registry on Pregnancy and Cardiac Disease, Ruys and coworkers (2015) planned cesarean delivery. Planned cesarean delivery conferred no advantage for maternal or neonatal outcome.

1	Pregnancy and Cardiac Disease, Ruys and coworkers (2015) planned cesarean delivery. Planned cesarean delivery conferred no advantage for maternal or neonatal outcome. Cesarean delivery is usually limited to obstetrical indications, and considerations are given for the speciic cardiac lesion, overall maternal condition, and availability of experienced anesthesia personnel and hospital capabilities. Some of these women tolerate major surgical procedures poorly and are best delivered in a unit experienced with management of complicated cardiac disease. Occasionally, pulmonary artery catheterization may be needed for hemodynamic monitoring (Chap. 47, p. 916). In our experiences, however, invasive monitoring is rarely indicated.

1	Based on her review, Simpson (2012) recommends cesarean delivery for women with the following: (1) dilated aortic root >4 cm or aortic aneurysm; (2) acute severe congestive heart failure; (3) recent myocardial infarction; (4) severe symptomatic aortic stenosis; (5) warfarin administration within 2 weeks of delivery; and (6) need for emergency valve replacement immediately after delivery. Although we agree with most of these, we have some caveats. For example, we prefer aggressive medical stabilization of pulmonary edema followed by vaginal delivery if possible. Also, warfarin anticoagulation can be reversed with vitamin K, plasma, or prothrombin concentrates.

1	During labor, the mother with signiicant heart disease should be kept in a semirecumbent position with a lateral tilt. Vital signs are taken frequently between contractions. Increases in pulse rate much above 100 beats per minute (bpm) or respiratory rate above 24 per minute, particularly when associated with dyspnea, may suggest impending ventricular failure. For evidence of cardiac decompensation, intensive medical management must be instituted immediately. Delivery itself does not necessarily improve the maternal condition and, in fact, may worsen it. Moreover, emergency cesarean delivery may be particularly hazardous. Clearly, both maternal and fetal status must be considered in the decision to hasten delivery under these circumstances.

1	Relief from pain and from apprehension is important. Although women, continuous epidural analgesia is recommended for most. The major problem with conduction analgesia is mater nal hypotension (Chap. 25, p. 491). This is especially danger ous in women with intracardiac shunts in whom low may be reversed. Hypotension can also be life-threatening if there is pulmonaY arterial hypertension or aortic stenosis because ven tricular output is dependent on adequate preload. In women with these conditions, narcotic regional analgesia or general anesthesia may be preferable.

1	For vaginal delivery in women with only mild cardiovas cular compromise, epidural analgesia given with intravenous sedation oten suices. his has been shown to minimize intra uum-assisted delivery. Subarachnoid blockade is not generally recommended in women with significant heart disease due to associated hypotension. For cesarean delivery, epidural analge sia is preferred by most clinicians with caveats for its use with pulmonary arterial hypertension (p. 962).

1	Cardiovascular decompensation during labor may manifest as pulmonary edema with hypoxia or as hypotension, or both. The proper therapeutic approach depends on the speciic hemodynamic status and the underlying cardiac lesion. For example, decompensated mitral stenosis with pulmonary edema due to luid overload is often best treated with aggressive diuresis. If precipitated by tachycardia, heart rate control with �-blocking agents is preferred. Conversely, the same treatment in a woman sufering decompensation and hypotension due to aortic stenosis could prove fatal. Unless the underlying pathophysiology is understood and the cause of the decompensation is clear, empirical therapy may be hazardous.

1	Women who have shown little or no evidence of cardiac compromise during pregnancy, labor, or delivery may still decompensate postpartum. Fluid mobilized into the intravascular compartment and reduced peripheral vascular resistance place higher demands on myocardial performance. Therefore, meticulous care is continued into the puerperium (Keizer, 2006; Zeeman, 2006). Postpartum hemorrhage, anemia, infection, and thromboembolism are much more serious complications with heart disease. Indeed, these factors often act in concert to precipitate postpartum heart failure. In addition, sepsis and severe preeclampsia cause or worsen pulmonary edema because of endothelial activation and capillary-alveolar leakage (Chap. 47, p. 917).

1	For puerperal tubal sterilization after vaginal delivery, the procedure can be delayed up to several days to ensure that the mother has normalized hemodynamically and that she is afebrile, not anemic, and ambulating normally. Alternatively, for those desiring future fertility, detailed contraceptive advice is available in the u. Medical Eligibiliy Criteria or Contraceptive Use guidelines (Curtis, 2016). Most clinically signiicant congenital heart lesions are repaired during childhood. Examples of those frequently not diagnosed until adulthood include atrial septal defects, pulmonic stenosis, bicuspid aortic valve, and aortic coarctation (Brickner, 2014). In some cases, the defect is mild and surgery is not required. In others, a significant anomaly is amenable to corrective surgery, performed ideally before pregnancy. In rare instances, surgical corrections are necessary during pregnancy.

1	Numerous reports describe subsequent pregnancy outcomes in women who have a prosthetic mitral or aortic valve. The type of valve, either mechanical or biological, is paramount. From one review, the overall estimated maternal mortality rate was 1.2 percent. he rate was 1.8 percent in the mechanical valve subgroup and 0.7 percent in the bioprosthetic subgroup (Lawley, 2015). Using the Registry of Pregnancy and Cardiac Disease, the maternal mortality rate was 1.4 percent in women with a mechanical heart valve and 1.5 percent in women with a tissue heart valve (van Hagen, 2015). Mechanical heart valve thrombosis complicated 4.7 percent. In total, only 58 percent with a mechanical heart valve had a pregnancy free of serious adverse events compared with 79 percent of patients with a tissue heart valve (Table 49-5). Because of thrombosis risks, anticoagulation may be requisite, but its complications are described in the next section. Thus, pregnancy is undertaken only ater serious

1	heart valve (Table 49-5). Because of thrombosis risks, anticoagulation may be requisite, but its complications are described in the next section. Thus, pregnancy is undertaken only ater serious consideration for women with a prosthetic mechanical valve.

1	Bouhout and coworkers (2014) reported the outcomes of 27 pregnancies in 14 women who underwent an aortic valve replacement prior to pregnancy. Seven of the 27 pregnancies occurred in five women with a mechanical prosthesis. Complications in this group included two embolic myocardial infarctions and one each of miscarriage, postpartum hemorrhage, placental abruption, and preterm birth. In the bioprosthetic group, nine miscarriages, two hospitalizations for syncope, and one preterm birth were noted.

1	Porcine tissue valves are safer during pregnancy, primarily because thrombosis is rare and anticoagulation is not required (see Table 49-5). Despite this, valvular dysfunction with cardiac deterioration poses a serious risk. Another drawback is that bioprostheses are less durable than mechanical ones, and valve replacement longevity averages 10 to 15 years. Cleuziou and colleagues (2010) concluded that pregnancy does not accelerate the risk for replacement. But, Nappi and associates (2014) found an association between pregnancy and valve deterioration in women with cryopreserved mitral homograft valves. his is critical for women with mechanical prosthetic valves . Unfortunately, warfarin is the most efective anticoagulant for preventing maternal thromboembolism but causes harmful fetal efects (Chap. 12, p. 247). Anticoagulation with heparin is less hazardous for the fetus, however, the risk of maternal thromboembolic complications is much higher (McLintock, 2011).

1	Warfarin is teratogenic and causes miscarriage, stillbirths, and fetal malformations. In one study of 71 women given warfarin throughout pregnancy, the rates of miscarriage were 32 percent; stillbirth, 7 percent; and embryopathy, 6 percent (Cotrufo, 2002). The risk was highest when the mean daily dose of warfarin exceeded 5 mg. Similarly, the American College of Cardiology and the American Heart Association estimate that the risk of embryopathy is dose dependent, with a lower risk-less than 3 percent-if the dose of warfarin is :;5 mg/d (Nishimura, 2014). If the dosage is >5 mg/d, the risk of embryopathy exceeds 8 percent.

1	Anticoagulation for mechanical valves using low-dose unfractionated heparin is definitely inadequate and carries a high associated maternal mortality rate (Chan, 2000; Iturbe-Alessio, 1986). Even ful anticoagulation with either unfractionated heparin (UFH) or one of the low-molecular-weight heparins (LMWH) is associated with valvular thrombosis (Leyh, 2002, 2003; Rowan, 2001). But, compliance with twice-daily dosing and therapeutic monitoring may have contributed (McLintock, 2014). hus, if full anticoagulation with dose-adjusted UFH or LMWH is used, meticulous monitoring is recommended. The activated partial thromboplastin time (aPTT) should be at least 2 times control or anti-Xa levels should be 0.8 to 1.2 U/mL at aData from the Registry of Pregnancy and Cardiac Disease. Data from van Hagen, 2015. 4 to 6 hours postdose (Nishimura, 2014).

1	Several diferent treatment options-none of which are completely ideal-are principally based on consensus opinion. Two are from the American College of Chest Physicians and the other jointly from the merican College of Cardiology and the American Heart Association (Bates, 2012; Nishimura, 2014). Any of four regimens is recommended. First, adjusted-dose LMWH is given twice daily, with a peak anti-Xa level drawn 4 hours ater dosing. In another, adjusted UFH is dosed every 12 hours to keep the midinterval aPTT twice control or anti Xa level between 0.35 0.70 U/mL. As a third option, LMWH or UFH is given as just described until 13 weeks, and then warfarin is substituted until near delivery, at which time it is replaced by LMWH or UFH. Last, in women judged to carry a high risk of thrombosis and for whom the eicacy and safety of heparins are concerns, warfarin is suggested throughout pregnancy. Heparin is then substituted close to delivery. In addition, aspirin, 75 to 100 mg, is given

1	and for whom the eicacy and safety of heparins are concerns, warfarin is suggested throughout pregnancy. Heparin is then substituted close to delivery. In addition, aspirin, 75 to 100 mg, is given daily.

1	Heparin is discontinued just before delivery. If delivery supervenes while the anticoagulant is still efective, and extensive bleeding is encountered, then protamine sulfate is given intravenously. nticoagulant therapy with warfarin or heparin may be restarted 6 hours following vaginal delivery, usually with no problems. Following cesarean delivery, full anticoagulation is withheld, but the optimal duration is not exactly nown. The American College of Obstetricians and Gynecologists (2017) advises resuming unfractionated or low-molecular-weight heparin 6 to 12 hours ater cesarean delivery. It is our practice, however, to wait at least 24 hours following a major surgical procedure. Because warfarin, LMWH, and UFH do not accumulate in breast milk, they do not induce an anticoagulant efect in the newborn. hese anticoagulants are compatible with breastfeeding (American College of Obstetricians and Gynecologists, 2017).

1	Although usually postponed until after delivery, valve replacement or other cardiac surgery during pregnancy may be lifesaving. Several reviews conirm that such surgery is associated with major maternal and fetal morbidity and mortality. At the Mayo Clinic between 1976 and 2009,r21 pregnant women underwent cardiothoracic surgery requiring cardiopulmonary bypass Oohn, 2011). The procedures included valve replacements, myxoma excisions, aneurysm repairs, patent foramen ovale closure, prosthetic aortic valve thrombectomy, and septal myectomy. Median cardiopulmonary bypass time was 53 minutes, with a range of 16 to 185 minutes. One woman died 2 days ater surgery, three fetuses died, and 52 percent were delivered before 36 weeks' gestation. Elassy and associates (2014) described 23 women who underwent urgent open cardiac surgery for severe valve malfunction. Two women and 10 fetuses-all at a gestational age below 28 weeks-died before hospital discharge. Only six fetuses were delivered at

1	underwent urgent open cardiac surgery for severe valve malfunction. Two women and 10 fetuses-all at a gestational age below 28 weeks-died before hospital discharge. Only six fetuses were delivered at term. To optimize outcomes, Chandrasekhar and coworkers (2009) recommend that surgery be elective when possible, pump low rate should remain >2.5 Llmin/m2, normothermic perfusion pressure should exceed 70 mm Hg, and hematocrit should be keptr> 28 volumes percent.

1	Many successful pregnancies have followed cardiac transplantation (Abdalla, 2014; Vos, 2014). The current recommendations from The International Society of Heart and Lung Transplantation do not discourage pregnancy in stable heart transplant recipients who are more than 1 year posttransplant (Costanzo, 2010). Obviously, a highly specialized level of care with a mul tidisciplinary team is necessary.

1	he transplanted heart appears to responds normally to preg nancy-induced alterations (Key, 1989; Kim, 1996). Despite this, complications are common during pregnancy (Dashe, 1998). Of 53 pregnancies in 37 heart recipients, almost half developed hypertension, and 22 percent sufered at least one rejection episode during pregnancy (Armenti, 2002; Miniero, 2004). They were delivered-usually by cesarean-at a mean of 37 to 38 weeks' gestation. hree fourths of neonates were liveborn. At follow-up, at least ive women had died more than 2 years postpartum. Another analysis of 25 such women with 42 pregnancies found no maternal deaths. Major complica tions included two rejections during the early puerperium, two cases of renal failure, and 11 spontaneous abortions (Estensen, 2011). Five women died 2 to 12 years after delivery. And from the United Kingdom, Mohamed-Ahmed and colleagues (2014) identiied 14 women with transplants between 2007 and 2011r.

1	Graft rejections occurred in two women, one of whom died. Rheumatic fever is uncommon in the United States because of less crowded living conditions, penicillin availability, and evolution of nonrheumatogenic streptococcal strains. Still, it remains the chief cause of serious mitral valvular disease in women of childbearing age in the nonindustrialized world (Nanna, 2014; Roeder, 2011) . Rheumatic endocarditis causes most mitral stenosis lesions. The normal mitral valve surface area is 4.0 cmr, and when stenosis 2 narrows this to <2.5 cmr, symptoms usually develop (Desai, 2000). The contracted valve impedes blood low from the left atrium to the ventricle.

1	With more severe stenosis, the left atrium dilates, let atrial pressure is chronically elevated, and signiicant passive pulmonary hypertension develops (Table 49-6). These women have a relatively ixed cardiac output, and thus the increased preload of normal pregnancy and other factors that raise cardiac output may cause ventricular failure and pulmonary edema. Indeed, a fourth of women with mitral stenosis have cardiac failure for the irst time during pregnancy (Caulin-Glaser, 1999). he resulting pulmonary venous hypertension and pulmonary edema create symptoms of dyspnea, fatigue, palpitations, cough, and hemoptysis. The classic murmur may not be heard in some women, and this clinical picture at term may be confused with idiopathic peripartum cardiomyopathy (Cunningham, 1986,r2012).

1	Also with signiicant stenosis, tachycardia shortens ventricular diastolic illing time and elevates the mitral gradient. his too may lead to pulmonary edema. Thus, sinus tachycardia is often treated prophylactically with 3-blocking agents. Atrial tachyarrhythmias, including ibrillation, are common in mitral stenosis and are treated aggressively. Atrial ibrillation also predisposes to mural thrombus formation and cerebrovascular embolization that can cause stroke (Chap. 60, p. 1161). Atrial thrombosis can develop despite a sinus rhythm (Hameed, 2005). Pregnancy Outcomes 66 newborns who were delivered at term had normal growth and development.

1	Pregnancy Outcomes 66 newborns who were delivered at term had normal growth and development. In general, complications are directly associated with the degree2 Labor and delivery are particularly stressful for women withofvalvular stenosis. Women with a mitral-valve area <2 cmrare symptomatic mitral stenosis (Fig. 49-3). Uterine contractionsat greatest risk (Siu, 2001b). In one study of 46 gravidas with increase cardiac output by increasing circulating blood volume. mitral stenosis, 43 percent developed heart failure, and 20 percent Pain, exertion, and anxiety cause tachycardia with possible ratedeveloped arrhythmias (Hameed, 2001). Fetal-growth restriction 2rrelated heart failure. Epidural analgesia for labor is ideal, but was more common in women with a mitral valve area < 1.0 cmr• fluid overload is avoided. Abrupt expansion in preload mayPrognosis is also related to maternal functional capacity.

1	raise pulmonary capillary wedge pressure and cause pulmonaryAmong 486 pregnancies complicated by rheumatic heart disedema. Wedge pressures rise immediately postpartum. Oneease-predominantly mitral stenosis-8 of 10 maternal deaths hypothesis for this suggests that the loss of the low-resistance were in women in NYHA classes III or IV (Sawhney, 2003). placental circulation couples with venous "autotransfusion"

1	placental circulation couples with venous "autotransfusion" Limited physical activity is generally recommended in women with mitral stenosis. If symptoms of pulmonary congesJ tion develop, activity is further reduced, dietary sodium is restricted, and diuretics are given (Siva, 2005). Also, �-blocker drug therapy slows the ventricular response to activity. If new onset atrial ibrillation develops, intravenous verapamil, 5 to 10 mg, is given, or electrocardioversion is performed. For chronic :) 15 ibrillation, digoxin, a �-blocker, or a calcium-channel blocker ; E can slow ventricular response. Therapeutic anticoagulation is = E 10 indicated with persistent ibrillation, left atrial thrombus, and/ ) A B C or a history of embolism (Nanna, 2014). ..m 5 0 Surgical intervention is considered for women with sympc tomatic severe mitral stenosis and in those with lesser degrees E0 of mitral stenosis-mitral-valve area 1.5 to 2.0 cm2-compli.

1	hypertension. Balloon valvuloplasty is preferred if the valve is pliable (Bui, 2014). In one review of 71 pregnant women with surements (red graph line) in eight women with mitral valve stenosis. Shaded yellow and blue boxes are mean (± 1 SO) pres neous valvuloplasty, 98 percent were either NYHA class I or sures in nonlaboring normal women at term. A. First-stage labor. II at delivery (Esteves, 2006). At a mean of 44 months, the B. Second-stage labor 15 to 30 minutes before delivery. C. Postpartotal event-free maternalsurvival rate was 54 percent. However, tum 5 to 15 minutes. D. Postpartum 4 to 6 hours. E. Postpartum 18 eight women required another surgical intervention. All of the to 24 hours. (Data from Clark, 1985, 1989.) from a now-empty, contracted uterus and from the lower extremities and pelvis (Clark, 1985). Most prefer vaginal delivery in women with mitral stenosis.

1	Most prefer vaginal delivery in women with mitral stenosis. Elective induction is reasonable so that labor and delivery are attended by a scheduled, experienced team. With severe steno sis and chronic heart failure, insertion of a pulmonary artery catheter may help guide management.

1	A trivial degree of mitral insuiciency is found in most normal patients. But if mitral valve lealets align improperly during systole, abnormal degrees of mitral regurgitation can develop. his is eventually followed by left ventricular dilation and eccentric hypertrophy (see Table 49-6). Acute mitral insuiciency is caused by chordae tendineae rupture, papillary muscle infarction, or leaflet perforation from infective endocarditis. Chronic mitral regurgitation, in contrast, may derive from rheumatic fever, connective tissue diseases, mitral valve prolapse, or left ventricular dilation of any etiology-for example, dilated cardiomyopathy. Less common causes include a calcified mitral annulus, possibly some appetite suppressants, and in older women, ischemic heart disease. Mitral valve vegetations-Libman-Sacks endocarditis-are relatively common in women with antiphospholipid antibodies (Shrof, 2012). These sometimes coexist with systemic lupus erythematosus.

1	In nonpregnant patients, symptoms from mitral valve insufficiency are rare, and valve replacement is seldom indicated unless infective endocarditis develops. During pregnancy, mitral regurgitation is similarly well tolerated, probably because the lowered systemic vascular resistance yields less regurgitation. Heart failure rarely develops during pregnancy, and occasionally tachyarrhythmias or severely depressed systolic function require treatment. This diagnosis implies the presence of a pathological connective tissue disorder-often termed myxomatous degenerationwhich may involve the valve lealets themselves, the annulus, or the chordae tendineae. Mitral insuiciency may develop. Most women with mitral valve prolapse are asymptomatic and are diagnosed during routine examination or echocardiography. he few women with symptoms have anxiety, palpitations, atypical chest pain, dyspnea with exertion, and syncope (Guy, 2012).

1	Pregnant women with mitral valve prolapse rarely have cardiac complications. Hypervolemia may even imptove alignment of the mitral valve, and women without pathological myxomatous degeneration generally have excellent pregnancy outcomes (LdniakSobelga, 2004). For women who are symptomatic, �-blocking drugs diminish sympathetic tone, relieve chest pain and palpitations, and reduce the risk of life-threatening arrhythmias.

1	Usually a disease of aging, aortic stenosis in younger women is most likely a congenital lesion. Since the decline in rheumatic disease incidence, aortic stenosis is less common, and the most frequent cause in the United States is a bicuspid valve (Friedman, 2008). A normal aortic valve has an area of 3 to 4 cmr, with a pressure gradient <5 mm Hg. If the valve area is < 1 cmr, there is severe obstruction to flow and a progressive pressure overload on the left ventricle (Roeder, 2011). Concentric left ventricular hypertrophy follows, and if it is severe, enddiastolic pressures become elevated, ejection fraction declines, and cardiac output is reduced (see Table 49-6). Characteristic manifestations develop late' and include chest pain, syncope, heart failure, and sudden death from arrhythmias. Life expectancy averages only 5 years after exertional chest pain develops, and valve replacement is indicated for symptomatic patients.

1	Clinically significant aortic stenosis is infrequent during pregnancy. Mild-to-moderate degrees of stenosis are well tolerated, however, severe disease is life-threatening. he principal underlying hemodynamic problem is the ixed cardiac output associated with severe stenosis. During pregnancy, several common events acutely lower preload further and thus aggravate the ixed cardiac output. hese include vena caval occlusion from the gravid uterus, regional analgesia, and hemorrhage. Importantly, these also decrease cardiac, cerebral, and uterine perfusion. It follows that severe aortic stenosis may be extremely dangerous during pregnancy. From the earlier-cited Canadian study, complication rates were higher if the aortic valve area measured < 1.5 cmr(Siu, 2001 b). And in the report by Hameed and associates (2001), the maternal mortality rate with aortic stenosis was 8 percent. Women with valve pressure gradients > 100 mm Hg appear to be at greatest risk.

1	For asymptomatic women with aortic stenosis, no treatment except close observation is required. Management of a symptomatic woman includes strict limitation of activity and prompt treatment of infections. If symptoms persist despite bed rest, surgical intervention may be considered. Catheter-based valvuloplasty is associated with risks to both the mother and fetus and is not very efective in the long term (Pessel, 2014; Reich, 2004). The aortic valve can again narrow or new aortic regurgitation may develop. he alternative surgical approach-valve replacement-is associated with significant risk of fetal demise due to the efects of cardiac bypass (Dart, 2010). Accordingly, the American College of Cardiology, the American Heart Association, and the European Society of Cardiology recommend delaying conception until after surgical correction for severe aortic stenosis (Bonow, 2008).

1	For women with critical aortic stenosis, intensive monitoring during labor is essential. Pulmonary arteY catheterization may be helpful because of the narrow margin separating fluid overload from hypovolemia. Women with aortic stenosis are dependent on adequate end-diastolic ventricular filling pressures to maintain cardiac output and systemic perfusion. Abrupt drops in end-diastolic volume may result in hypotension, syncope, myocardial infarction, and sudden death. hus, avoiding diminished ventricular preload and maintaining cardiac output are key. During labor and delivery, afected women are best managed on the "wet" side. his provides a margin of safety in intravascular volume in anticipation of possible hemorrhage. In women with a competent mitral valve, pulmonary edema is rare.

1	During labor, narcotic epidural analgesia seems ideal and avoids potentially hazardous hypotension. Easterling and coworkers (1988) studied the efects of epidural analgesia in ive women with severe stenosis and demonstrated immediate and profound efects from decreased illing pressures. Xia and associates (2006) emphasize slow administration of dilute local anesthetic agents into the epidural space. In hemodynamically stable women, forceps or vacuum delivery is used for standard obstetrical indications.

1	Aortic valve regurgitation or insuiciency allows diastolic Bow of blood from the aorta back into the let ventricle. Frequent causes of abnormal insuiciency are rheumatic fever, connective tissue abnormalities, and congenital lesions. With Marfan syndrome, the aortic root may dilate and create regurgitation (p. 967). Acute insuiciency may also develop with bacterial endocarditis or aortic dissection (pp. 965 and 967). Last, aortic and mitral vlve insuiciency have both been linked to the appetite suppressants fenBuramine and dexfenBuramine and to the ergot-derived dopamine agonists cabergoline and pergolide (Gardin, 2000; Schade, 2007; Zanettini, 2007). With chronic insuiciency, let ventricular hypertrophy and dilation develop and are followed by slow-onset fatigue, dyspnea, and pulmonary edema, although rapid deterioration usually follows (see Table 49-6).

1	Aortic insuiciency is generally well tolerated during pregnancy. Like mitral valve insuiciency, diminished vascular resistance is thought to improve hemodynamic function. If symptoms of heart failure develop, diuretics are given and bed rest is encouraged. his lesion is usually congenital and also may be associated with Fallot tetralogy or Noonan syndrome. The greater hemodynamic burden of pregnancy can precipitate right-sided heart failure or atrial arrhythmias in women with severe stenosis. Surgical correction ideally is done before pregnancy, but if symptoms progress, a balloon valvuloplasty may be necessary antepartum (Galal, 2015; Siu, 2001a).

1	In studying pregnancy outcomes, Drenthen and colleagues (2006) found infrequent cardiac complications in a group of 81 pregnancies in 51 Dutch women with pulmonic stenosis. The NHA classification worsened in two women, and nine experienced palpitations or arrhythmias. No changes in pulmonary valvular function or other adverse cardiac events were reported. However, non cardiac complication rates were significant-17 percent had preterm delivery, 15 percent had hypertension, and 4 percent developed thromboembolism.

1	The incidence of congenital heart disease in the United States approximates 11 per 1000 liveborn neonates (Egbe, 2014). With modern surgeries, approximately 90 percent of those born with congenital heart disease survive to childbearing age, and it is now the most common type of heart disease encountered during pregnancy (Brickner, 2014; Lindley, 2015). Specifically, analysis from the United States Nationwide Inpatient Sample database shows a linear rise in the prevalence of congenital heart disease between 2000 and 2010-from 6.4 to 9.0 per 10,000 women admitted for delivery (Thompson, 2015).

1	Of pregnancy outcomes in women with congenital heart disease compared with those without, the odds of cardiovascular and obstetrical complications were 10.5 to 35.5 and 1.2 to 2.1 times higher, respectively (Thompson, 2015). Moreover, maternal mortality rates were greater for women with congenital heart disease than for unafected gravidas-17.8 and 0.7 per 10,000 deliveries, respectively. Opotowsky and coworkers (2012) reported similar risks.

1	Approximately one fourth of all adults has a patent foramen ovale (Miller, 2015). Most atrial septal defects (ASDs) are asymptomatic until the third or fourth decade. he secundumtype defect accounts for 70 percent, and associated mitral valve myxomatous abnormalities with prolapse are common. Most recommend repair if ASD is discovered in adulthood. Pregnancy is well tolerated unless pulmonary hypertension has developed, but this is uncommon (Geva, 2014). Treatment of ASD during pregnancy is indicated for congestive heart failure or an arrhythmia. Based on their review, liaga and colleagues (2003) concluded that the risk of endocarditis with an ASD is negligible.

1	With the potential to shunt blood from right to let, aparadoxical embolism, that is, entry of a venous thrombus through the septal defect and into the systemic arterial circulation, is possible and may cause an embolic stroke (Erkut, 2006; Miller, 2015). In asymptomatic women with ASD, thromboembolism prophylaxis is problematic, and the heterogeneous recommendations have been summarized by Kizer and Devereux (2005). Compression stockings and prophylactic heparin have also been recommended for a pregnant woman with an ASD who is immobile or has another risk factor for thromboembolism (Head, 2005).

1	These lesions close spontaneously during childhood in 90 percent of cases. Most defects are paramembranous, and the degree of left-to-right shunt and associated physiological derangements are related to lesion size. In general, if the defect mea sures < 1.25 cmr, pulmonary hypertension and heart failure do not develop. If the efective defect size exceeds that of the aortic valve orifice, symptoms rapidly develop. For these reasons, most children undergo surgical repair before pulmonary hypertension develops. Adults with unrepaired large defects develop left ventricular failure and pulmonary hypertension and have a high incidence of bacterial endocarditis (Brickner, 2000, 2014).

1	Pregnancy is well tolerated with small-to-moderate sized shunts. If pulmonary arterial pressures reach systemic levels, however, there is reversal or bidirectional Bow-Eisenmenger syndrome (p. 960). When this develops, the maternal and fetal mortality rates are signiicantly higher, and thus, pregnancy is not generally advisable. Bacterial endocarditis is more common with unrepaired defects, and antimicrobial prophylaxis is often required (p. 965). As shown in Table 49-4, 10 to 16 percent of ofspring born to these women also have a ventricular septal defect.

1	These account for approximately 3 percent of all congenital cardiac malformations and are distinct from isolated atrial or ventricular septal defects. An atrioventricular (A V) septal defect is characterized by a common, ovoid A V junction. his defect is associated with aneuploidy, Eisenmenger syndrome, and other malformations (Altin, 2015). Compared with simple septal defects, complications are more frequent during pregnancy. In a review of 48 pregnancies in 29 afected women, complications included persistent deterioration of NA class in 23 percent, signiicant arrhythmias in 19 percent, and heart failure in 2 percent (Drenthen, 2005b). Congenital heart disease was identiied in 15 percent of the ofspring.

1	The ductus connects the proximal left pulmonary artery to the descending aorta just distal to the left subclavian artery. Functional closure of the ductus from vasoconstriction occurs shortly after term birth. he physiological consequences with its persistence are related to its size. Most signiicant lesions are repaired in childhood, but for individuals who do not undergo repair, the mortality rate is high after the ifth decade (Brickner, 2014). In some younger women with an unrepaired ductus during pregnancy, however, pulmonary hypertension, heart failure, or cyanosis will develop if systemic blood pressure falls and leads to shunt reversal of blood from the pulmonary artery into the aorta (Vashisht, 2015). A sudden blood pressure decline at delivery-such as with regional analgesia or hemorrhage-may lead to fatal collapse. Accordingly, hypotension is ideally avoided but treated vigorously if it develops. Prophylaxis for bacterial endocarditis is indicated at delivery for unrepaired

1	lead to fatal collapse. Accordingly, hypotension is ideally avoided but treated vigorously if it develops. Prophylaxis for bacterial endocarditis is indicated at delivery for unrepaired defects (p. 965). As shown in Table 49-4, the incidence of inheritance approximates 4 percent.

1	When congenital heart lesions produce right-to-Ieft shunting of blood past the pulmonary capillary bed, cyanosis develops. The classic and most commonly encountered lesion in adults and during pregnancy is the Fallot tetraloy (Lindley, 2015). his is characterized by a large ventricular septal defect, pulmonary stenosis, right ventricular hypertrophy, and an overriding aorta that receives blood from both the right and left ventricles. The magnitude of the shunt varies inversely with systemic vascular resistance. Hence, during pregnancy, when peripheral resistance declines, the shunt increases and cyanosis worsens.

1	Generally, women with cyanotic heart disease do poorly during pregnancy. With uncorrected Fallot tetralogy, maternal mortality rates approach 10 percent. here is a relationship between chronic hypoxemia, polycythemia, and pregnancy outcomes such as miscarriage and perinatal morbidity. When hypoxemia is intense enough to stimulate a rise in hematocrit above 65 vol umes percent, pregnancy wastage is virtually 100 percent.

1	Although not all cyanotic lesions are repairable, with satis factory surgical correction before pregnancy, maternal and fetal outcomes are much improved. In a review of 197 pregnancies in 99 women with surgically corrected Fallot tetralogy, preg nancy was usually well tolerated, and no mothers died. Still, almost 9 percent of pregnancies were complicated by adverse and heart failure (Balci, 2011; Kamiya, 2012). For women with a pulmonary valve replacement, pregnancy does not adversely afect graft function (Oosterhof, 2006). Prior to or after con ception, women with Fallot tetralogy are ofered genetic coun seling and evaluation for 22q 11 deletion syndrome (Lindley, 2015).

1	Some women with Ebstein anomay, characterized by a mal positioned and malformed tricuspid valve, may reach reproduc tive age. Right ventricular failure from volume overload and cyanosis are common during pregnancy. In the absence of cya nosis, heart failure, or signiicant arrhythmias, afected women usually tolerate pregnancy well (Brickner, 2014). Transposition of the Great Vessels

1	Pregnancy following surgical correction of transposition has prominent risks. Canobbio (2006) and Drenthen (2005a), each with their colleagues, described outcomes of 119 pregnancies in 68 women-90 percent had a prior Mustard procedure and 10 percent a previous Senning procedure. During pregnancy, one fourth had arrhythmias, 12 percent developed heart failure, and one subsequently required cardiac transplantation. One woman died suddenly a month after delivery, and another died 4 years later. A third of the newborns were delivered preterm. In another report of 60 pregnancies in 34 women who had undergone transposition repair, approximately a fourth ended in miscarriage or abortion, and another fourth delivered preterm (Trigas, 2014). Deterioration in functional class occurred in seven women, and documented deterioration in systolic function occurred in four women. Finally, two women required resuscitation during delivery, and one experienced supraventricular tachycardia during labor.

1	and documented deterioration in systolic function occurred in four women. Finally, two women required resuscitation during delivery, and one experienced supraventricular tachycardia during labor. Of other defects, in women with previously repaired truncus arteriosus and doubleoutlet right ventricle, successful-although eventful-pregnancies have also been described (Drenthen, 2008; Hoendermis, 2008).

1	With hypoplastic lt heart syndrome, almost 70 percent of afected women are now expected to survive into adulthood and frequently become pregnant (Feinstein, 2012). Those who have undergone a Fontan repair are at particularly high risk for complications. In brief, this procedure involves diverting blood via a surgical anastomosis from the vena cava to the pulmonary artery without passing through the right ventricle. Blood Bows passively to the pulmonary vasculature. Thus, patients with a Fontan palliation are very preload dependent (Lindley, 2015). lead to pulmonary

1	lead to pulmonary FIGURE 49-4 Eisenmenger syndrome due to a ventricular septal defect (VSD). A. Substantialolet-to-right shunting through the VSD leads to morphological changes in the smaller pulmonary arteries and arterioles. Specifically, medial hypertrophy, intimal cellular proliferations, and fibrosis lead to narrowing or closure of the vessel lumen. These vascular changes create pulmonary hypertension and a resultant reversal of the intracardiac shunt (8). With sustained pulmonary hypertension, extensive atherosclerosis and calcification often develop in the large pulmonary arteries. Although a VSD is shown here, Eisenmenger syndrome may also develop in association with a large atrial septal defect or patent ductus arteriosus.

1	Of outcomes, one review of 14 women conceiving after a Fontan repair found that six spontaneously aborted all pregnancies, and eight others carried 14 pregnancies to viability (Cauldwell, 2016). Cardiac complications included arrhythmias and thromboembolism. Ten newborns delivered preterm, and eight neonates were small for gestational age. Similar complications attend a maternal systemic right ventricle, that is, one in which the right ventricle rather than the left pumps blood to the systemic circulation (Khan, 2015).

1	his describes secondary pulmonary hypertension that arises from any cardiac lesion. The syndrome develops when pulmonary vascular resistance exceeds systemic resistance and leads to concomitant right-to-Ieft shunting. The most common underlying defects are atrial or ventricular septal defects and persistent ductus arteriosus (Fig. 49-4). Patients are asymptomatic for years, but eventually pulmonary hypertension becomes severe enough to cause this shunting (Greutmann, 2015).

1	Pregnant women with Eisenmenger syndrome tolerate hypotension poorly, and death usually is caused by right ventricular failure with cardiogenic shock. In a review of 44 cases through 1978, maternal and perinatal mortality rates approximated 50 percent (Gleicher, 1979). In a later review of 73 pregnancies, Weiss and associates (1998) cited a 36-percent maternal death rate. Three of 26 deaths were antepartum, and the remainder of women died intrapartum or within a month of delivey. In a subsequent study of 13 gravidas, one mother died 17 days after delivery, and there were ive perinatal deaths (Wang, 2011). Given such poor outcomes or both mother and etus, Eisenmenger syndrome is considered to be an absolute contraindication to pregnancy (Brickner, 2014; Lindley, 2015; Meng, 2017; Warnes, 2015). Management of those who do become pregnant has recently been detailed by Broberg (2016) and is discussed in the next section.

1	Normal resting mean pulmonary artery pressure is 12 to 16 mm Hg. Pulmonary vascular resistance in late pregnancy approximates 80 dyne/sec/cm-5, which is 34-percent less than the nonpregnant value of 120 dyne/sec/cm-5 (Clark, 1989). Pulmonay hypertension is deined in nonpregnant individuals as a resting mean pulmonary pressure >25 mm Hg. he current clinical classification system, shown in Table 49-7, includes five groups of disorders that cause pulmonary hypertension (Galie, 2016). There are important prognostic and therapeutic distinctions between group 1 pulmonary arterial hypertension and the other groups. Group 1 indicates that a specific disease afects pulmonary arterioles. It includes idiopathic or primary pulmonary arterial hypertension as well as those cases secondary to a known cause such as connective tissue disease. For example, approximately one third of women with scleroderma and 10 percent with systemic lupus erythematosus

1	TABLE 49-7. Comprehensive Clinical Classification of Pulmonary Hypertension 1. Pulmonary arterial hypertension Drug and toxin induced Associated with connective tissue disease, HIV infections, portal hypertension, congenital heart diseases, schistosomiasis Drugs, toxins and radiation induced Associated with connective tissue disease, HIV infection I" Persistent pulmonary hypertension of the newborn 2. Pulmonary hypertension due to let heart disease 3. Pulmonary hypertension due to lung diseases and/or hypoxia Other pulmonary diseases with mixed restrictive and obstructive pattern Sleep-disoriented breathing Chronic exposure to high altitude 4. Chronic thromboembolic pulmonary hypertension/other pulmonary artery obstructions Other pulmonary artery obstructions, i.e., tumors, arteritis, pulmonary stenosis, parasites 5. Pulmonary hypertension with unclear and/or multifactorial mechanisms

1	Other pulmonary artery obstructions, i.e., tumors, arteritis, pulmonary stenosis, parasites 5. Pulmonary hypertension with unclear and/or multifactorial mechanisms Hematological disorders: chronic hemolysis, myeloproliferative disorders, splenectomy Systemic disorders: sarcoidosis, pulmonary histiocytosis, neurofibromatosis Metabolic disorders: glycogen storage disease, Gaucher disease, thyroid disorders Others: fibrosing mediastinitis, chronic renal failure HIV = human immunodeficiency virus. Adapted from Galie, 2016. have pulmonary hypertension (Rich, 2005). Other causes in young women are human immunodeiciency virus (HI) infection, sickle-cell disease, and thyrotoxicosis (Newman, 2015; Sheield, 2004).

1	have pulmonary hypertension (Rich, 2005). Other causes in young women are human immunodeiciency virus (HI) infection, sickle-cell disease, and thyrotoxicosis (Newman, 2015; Sheield, 2004). In pregnant women, group 2 disorders are the most common. hese are secondary to pulmonary venous hypertension caused by left-sided atrial, ventricular, or valvular disorders. A typical example is mitral stenosis discussed earlier (p. 955). In contrast, groups 3 through 5 are seen infrequently in young healthy women .

1	Symptoms may be vague, and dyspnea with exertion is the most frequent. With group 2 disorders, orthopnea and nocturnal dyspnea are also usually present. Angina and syncope occur when right ventricular output is ixed, and they suggest advanced disease. Chest radiography often shows enlarged pulmonary hilar arteries and attenuated peripheral markings. It also may disclose parenchymal causes of hypertension. N oninvasive echocardiography can provide an estimate of pulmonary artery pressures, although cardiac catheterization remains the standard for measurement. In two studies with a combined 51 pregnant women who underwent both echo cardiography and cardiac catheterization, pulmonary artery pressures were significantly overestimated by echocardiography in approximately one third of cases (Penning, 2001; Wylie, 2007).

1	Regardless of the etiology, the inal common pathway of pulmonary hypertension is right heart failure and death. he average survival length after diagnosis is <4 years (Krexi, 2015). That said, longevity depends on the severity and cause of pulmonary hypertension at discovery. As discussed later, some disorders respond to medical interventions, which may improve quality of life. Preconceptional and contraceptive counseling are imperative (Gei, 2014).

1	he maternal mortality rate is appreciable in afected women, and this is especially so with idiopathic pulmonary hypertension. In the past, there were frequently poor distinctions in identiying both causes and severity of hypertension. hus, although most severe cases of idiopathic pulmonary arterial hypertension had the worst prognosis, it was erroneously assumed that all types of pulmonary hypertension were equally dangerous. With widespread use of echocardiography, lesssevere lesions with a better prognosis are now discernible. Bedard and coworkers (2009) reported that maternal mortality rate statistics improved during the decade ending in 2007 (25 percent) compared with those for the decade ending in 1996 (38 percent). Importantly, almost 80 percent of the deaths were during the first month postpartum. More recently, Meng and associates (2017) reported mortality rates of 23 percent with group 1 and 5 percent with the other groups. Mortality was related to pulmonary hypertension

1	month postpartum. More recently, Meng and associates (2017) reported mortality rates of 23 percent with group 1 and 5 percent with the other groups. Mortality was related to pulmonary hypertension severity.

1	As discussed, pregnancy is contraindicated with severe disease, especially in women with pulmonary arterial changesmost cases in group 1. With milder disease from other causes-group 2 being the most common-the prognosis is better (Meng, 2017). With the more frequent use of echocardiography and pulmonary artery catheterization in young women with heart disease, we have identified women with mild-to-moderate pulmonary hypertension who tolerate pregnancy, labor, and delivery well. One example described by Sheield and Cunningham (2004) is that of pulmonary hypertension that develops with thyrotoxicosis but is reversible with treatment. Similarly, Boggess and colleagues (1995) described nine women with interstitial and restrictive lung disease with varying degrees of pulmonary hypertension, and all tolerated pregnancy reasonably well.

1	Treatment of symptomatic pregnant women includes activity limitation and avoidance of the supine position later in gestation. Diuretics, supplemental oxygen, and pulmonary vasodilator drugs are standard therapy for symptoms. Some recommend anticoagulation (Hsu, 2011). Several reports describe the successful use of intravenous pulmonary artery vasodilators (Badalian, 2000; Garabedian, 2010; Goya, 2014). Prostacyclin analogues that can be administered parenterally include epoprostenol and treprostinil, whereas iloprost is inhaled. Each has been used in gravidas. Inhaled nitric oxide is an option that has been employed in cases of acute cardiopulmonary decompensation (Lane, 2011). As reviewed by Obiean and Cleary (2014), phosphodiesterase-5 inhibitors, such as sildenail, cause vasodilation of both the pulmonary and systemic vascular beds and have an inotropic efect on the hypertrophic right ventricle. his also has been used to advantage during pregnancy (Goland, 2010; Hsu, 2011; Meng,

1	of both the pulmonary and systemic vascular beds and have an inotropic efect on the hypertrophic right ventricle. his also has been used to advantage during pregnancy (Goland, 2010; Hsu, 2011; Meng, 2017). Bosentan, an endothelin-receptor antagonist, is teratogenic in mice and contraindicated in pregnancy (Obican, 2014).

1	During labor and delivery, these women are at greatest risk when venous return and right ventricular filling are diminished. To avoid hypotension, assiduous attention is given to epidural analgesia induction and to blood loss prevention and treatment at delivery (Meng, 2017).

1	The American Heart Association defines these as a heterogeneous group of myocardial diseases associated with mechanical and/ or electrical dysfunction. Afected women usually-but not invariably-have inappropriate ventricular hypertrophy or dilation. Cardiomyopathies stem from varied causes, some of which are genetic (Maron, 2006). Of the two major divisions, primay cardiomyopathies are solely or predominantly conined to heart muscle. Examples are hypertrophic cardiomyopathy, dilated cardiomyopathies, and peripartum cardiomyopathy. Secondary cardiomyopathies result from generalized systemic disorders that produce pathological myocardial involvement. Diabetes, systemic lupus erythematosus, chronic hypertension, and thyroid disorders are representative conditions.

1	Epidemiological studies suggest that this disorder is common, afecting approximately 1 in 500 adults (Herrey, 2014; Maron, 2004). Characterized by cardiac hypertrophy, myocyte disarray, and interstitial ibrosis, the condition is caused by mutations in anyrone of more than a dozen genes that encode cardiac sarcomere proteins in up to 60 percent of afected patients. In such cases, inheritance is autosomal dominant, and genetic screening is complex (Elliott, 2014). Other genetic and nongenetic causes account for 5 to 10 percent of cases, and the cause is unknown in approximately 25 percent. The myocardial muscle abnormality is typified by let ventricular myocardial hypertrophy with a pressure gradient against let ventricular outflow. Diagnosis is established by echocardiographic identification of a hypertrophied and nondilated left ventricle in the absence of other cardiovascular conditions.

1	Most afected women are asymptomatic, but dyspnea, anginal or atypical chest pain, syncope, and arrhythmias may develop. Complex arrhythmias may progress to sudden death, which is the most frequent cause of death. Asymptomatic patients with runs of ventricular tachycardia are especially prone to sudden death. Symptoms usually worsen with exercise. lthough limited reports suggest that pregnancy is well tolerated, adverse cardiac events are frequent. In one analysis of 271 pregnancies in 127 afected women, there were no maternal deaths. However, more than a fourth had at least one adverse cardiac symptom-including dyspnea, chest pain, or palpitations (Thaman, 2003). Based on one systematic review that included 237 women with hypertrophic cardiomyopathy who had a combined 408 pregnancies, Schinkel (2014) calculated a maternal mortality rate of 0.5 percent. Worsening of symptoms or other complications occurred in 29 percent, and 26 percent delivered preterm.

1	Management is similar to that for aortic stenosis. Strenuous exercise is prohibited during pregnancy. Abrupt positional changes are avoided to prevent reflex vasodilation and decreased preload. Likewise, drugs that evoke diuresis or diminish vascular resistance are generally not used. If symptoms develop, especially angina, 3-adrenergic or calcium-channel blocking drugs are given. The delivery route of the fetus is determined by obstetrical indications. Choice of anesthesia is controversial, and some authors consider general anesthesia the safest (Pitton, 2007). Neonates rarely demonstrate inherited lesions at birth.

1	This is characterized by left and/or right ventricular enlargement and reduced systolic function in the absence of coronary, valvular, congenital, or systemic disease known to cause myocardial dysfunction. Although there are many known causes of dilated cardiomyopathy-both inherited and acquired, the etiology remains undefined in approximately half of cases (Stergiopoulos, 2011). Some result from viral infections, including myocarditis and HIV (Barbaro, 1998; Felker, 2000). Other causes, which are potentially reversible, include alcoholism, cocaine abuse, and thyroid disease. Watkins and coworkers (2011) reviewed the many complex genetic mutations associated with inherited forms of dilated cardiomyopathy.

1	This disorder is similar to other forms of nonischemic dilated cardiomyopathy except for its unique relationship with pregnancy (Pyatt, 2011). Indeed, peripartum cardiomyopathy shares a genetic predisposition with both familial and sporadic idiopathic dilated cardiomyopathy (Ware, 2016). Currently, it is a diagnosis of exclusion following a concurrent evaluation for peripartum heart failure. Although the term peripartum cardiomyopathy has been used widely, at least until recently, little evidence supported a unique pregnancy-induced cardiomyopathy. Pearson (2000) reported findings of a workshop of the National Heart, Lung, and Blood Institute that established the following diagnostic criteria: 1. Development of cardiac failure in the last month of pregnancy or within 5 months after delivery, 2. Absence of an identifiable cause for the cardiac failure, 3. Absence of recognizable heart disease prior to the last month of pregnancy, and 4.

1	Absence of an identifiable cause for the cardiac failure, 3. Absence of recognizable heart disease prior to the last month of pregnancy, and 4. Left ventricular systolic dysfunction demonstrated by classic echocardiographic criteria, such as depressed ejection fraction or fractional shortening along with a dilated left ventricle (Fig. 49-5). he etiology of peripartum cardiomyopathy remains unknown, and proposed causes include viral myocarditis, abnormal immune response to pregnancy, aberrant response to the greater hemodynamic burden of pregnancy, hormonal interactions, malnutrition, inflammation, and apoptosis (Elkayam, 2011). Another suggests that oxidative stress during late pregnancy leads to the proteolytic cleavage of prolactin (Hilfiker-Kleiner, 2014). The resulting 16-kDa prolactin

1	FIGURE 49-5 Peripartum cardiomyopathy with mild pulmonary edema. Anterior-posterior projection chest radiograph of a woman with an abnormally enlarged heart and mild perihilar opacification consistent with dilated cardiomyopathy. fragment is cardiotoxic and can impair cardiomyocyte metab olism and contractility. Bromocriptine therapy has been suggested because it inhibits prolactin secretion. In one pre liminary study, bromocriptine improved recovery of afected women, and a randomized trial is currently recruiting patients (Haghikia, 2015; Sliwa, 2010).

1	Hypertensive disorders frequently coexist with peripartum cardiomyopathy, and another proposed mechanism links peripartum cardiomyopathy to preeclampsia (Cunningham, 2012; Fong, 2014; Patten, 2012). Antiangiogenic factors-already known to be associated with preeclampsia-can induce peripartum cardiomyopathy in susceptible mice. hus, cardiomyopathy may be precipitated by antiangiogenic factors in a host made susceptible because of insuicient proangiogenic factors. Several investigators describe a common pathway linking these suggested etiologies (Arany, 2016; Hiliker-Kleiner, 2014). Specifically, unbalanced oxidative stress and a high level of prolactin leads to production of the 16-kDa prolactin fragment that seems to both initiate and propagate the disease. The fragment, which mainly afects the endothelium, together with additional antiangiogenic factors might disturb the angiogenic balance during the puerperium and thereby impair cardiac function.

1	With no proven etiology, the diagnosis of peripartum cardiomyopathy currently requires that other causes of cardiac dysfunction be excluded. Biiltmann and coworkers (2005) studied endomyocardial biopsy specimens from 26 women with peripartum cardiomyopathy and reported that more than half had histological evidence of "borderline myocarditis." hey noted viral genomic material for parvovirus B 19, human herpesvirus 6, Epstein-Barr virus, and cytomegalovirus. From Parland Hospital, otherwise "idiopathic" heart failure was found to be caused by hypertensive heart disease, clinically silent mitral stenosis, obesity, or viral myocarditis (Cunningham, 1986). Indeed, 20 percent of women with treated chronic hypertension in pregnancy have concentric hypertrophy (Ambia, 2017).

1	N tusi and associates (2015) analyzed the clinical features of 30 women with peripartum cardiomyopathy compared with 53 women with hypertensive heart failure. With cardiomyopathy, the symptoms began postpartum in all women, whereas symptoms began antepartum in 85 percent of women with hypertensive heart failure. Peripartum cardiomyopathy was significantly linked with twin gestation, smoking, and echo cardiographic abnormalities. In contrast, hypertensive heart failure patients more often had a family history of hypertension, hypertension and preeclampsia in a prior pregnancy, tachycardia, and left ventricular hypertrophy on echo cardiography.

1	he incidence of peripartum cardiomyopathy varies considerably and depends on the diligence for the search of a cause. In a review of the Nationwide Inpatient Sample database, the incidence rose from 1 in 1181 live births in 2004 to 1 in 849 in 2011 (Kolte, 2014). Two other large population-based studies cite a frequency of 1 in 2000 to 2800 live births (Gunderson, 2011; Harper, 2012). In an earlier study from Parkland Hospital, we identified idiopathic cardiomyopathy in only approximately 1 in 15,000 deliveries-an incidence similar to that of idiopathic cardiomyopathy in young nonpregnant women (Cunningham, 1986).

1	Approximately half of women sufering from peripartum cardiomyopathy recover baseline ventricular function within 6 months of delivery. But in those with persistent cardiac failure, the mortality rate approaches 85 percent over 5 years (Moioli, 2010). In a group of 100 women with newly diagnosed peripartum cardiomyopathy, 72 percent had a left ventricular ejection fraction ::50 percent, and 93 percent had event-free survival (McNamara, 2015). However, six women experienced nine major events that included four deaths, four left ventricular assist device implantations, and one heart transplantation. Recovery to a left ventricular ejection fraction ::50 percent occurred in almost 90 percent of women whose baseline ejection fraction was at least 30 percent. This compared with <40 percent in women whose baseline ventricular ejection fraction was < 30 percent. Recovery was also related to the baseline left ventricular end-diastolic diameter. Li and colleagues (2016) also found that a

1	in women whose baseline ventricular ejection fraction was < 30 percent. Recovery was also related to the baseline left ventricular end-diastolic diameter. Li and colleagues (2016) also found that a baseline left ventricular ejection fraction <34 percent and a brain natriuretic peptide (BNP) level > 1860 pg/ mL were associated with an approximately threefold greater risk of persistent left ventricular systolic dysfunction.

1	From the largest studies on the topic, approximately one third of women with a history of peripartum cardiomyopathy will sufer relapse with worsening of symptoms and deterioration of left ventricular function during another pregnancy (Elkayam, 20 14a). The risk of relapse in women with persistent left ventricular dysfunction is substantially higher than in those who have developed normal ventricular function before a subsequent pregnancy (Hiliker-Kleiner, 2017). But, normalization of let ventricular function does not guarantee an uncomplicated pregnancy, because approximately 20 percent of these women are at risk for deterioration in left ventricular function.

1	Arrhythmogenic right ventricular dysplasia is a unique cardiomyopathy defined histologically by progressive replacement of right ventricular myocardium with adipose and fibrous tissue. It has an estimated population prevalence of 1 in 5000, predisposes to ventricular tachyarrhythmias, and is a cause of sudden death, particularly in younger individuals (Agir, 2014; Elliott, 2008). The additional risk of pregnancy in women with arrhythmogenic right ventricular cardiomyopathy is unknown, however, based on their systematic review, Krul and coworkers (2011) counsel against pregnancy.

1	Restrictive cardiomyopathy is probably the least common type. This inherited cardiomyopathy is characterized by a ventricular illing pattern in which worsening myocardial stifness raises ventricular pressure precipitously and al�ows only a small filling volume (Elliott, 2008). Because of the severe clinical course and poor prognosis in general, pregnancy is not advised (Krul, 2011). Takotsubo cardiomyopathy is a rare form of acute reversible left ventricular apical wall ballooning (Kraft, 2017).

1	Regardless of the underlying condition that causes cardiac dysfunction, women who develop peripartum heart failure almost always have obstetrical complications that either contribute to or precipitate heart failure. For example, preeclampsia is common and may precipitate afterload failure. Indeed, indings from the Registry on Pregnancy and Cardiac Disease indicate that women with preexisting heart disease who develop preeclampsia have a 30-percent risk of developing heart failure during pregnancy (Ruys, 2014). Moreover, high-output states caused by hemorrhage and acute anemia elevate cardiac workload and magniy the physiological efects of compromised ventricular function. Similarly, infection and sepsis syndrome raise cardiac output and oxygen utilization and depress myocardial function.

1	In many populations, chronic hypertension with superimposed preeclampsia is the most frequent cause of heart failure in pregnancy. Many of these women have concentric let ventricular hypertrophy (Ambia, 2017). In some, mild antecedent undiagnosed hypertension causes covert cardiomyopathy, and when superimposed preeclampsia develops, together they may cause otherwise inexplicable peripartum heart failure. Obesity is a frequent cofactor with chronic hypertension, and it too is associated with ventricular hypertrophy (Kenchaiah, 2002).

1	Congestive heart failure can have a gradual onset or may present as acute "flash" pulmonary edema. Heart failure onset is most likely at the end of the second/beginning of the third trimester and peripartum (Ruys, 2014). Of symptoms, dyspnea is universal and others are orthopnea, palpitations, substernal chest pain, a sudden decline in the ability to complete usual duties, and nocturnal cough. Clinical findings include persistent basilar rales, hemoptysis, progressive edema, tachypnea, and tachycardia (Sheield, 1999). Hallmark radiographic findings usually are cardiomegaly and pulmonary edema (see Fig. 49-5). Acutely, there is usually systolic failure, and echocardiography shows an ejection fraction <0.45 or a fractional shortening <30 percent, or both, and an end-diastolic dimensionr> 2.7 cm/m2 (Hibbard, 1999). Coincidental diastolic failure may also be found, depending on the underlying cause (Redfield, 2016).

1	with diuretic administration to reduce preload. Hypertension is common, and afterload reduction is accomplished with hydralazine or another vasodilator. Because of marked fetal efects, angiotensin-converting enzyme inhibitors are withheld until after delivery. With chronic heart failure, the incidence of associated thromboembolism is high, and thus prophylactic heparin is often recommended. Lt ventricular assist devices are now employed more fre quently for acute and chronic heart failure treatment. A few reports describe their use during pregnancy (LaRue, 2011; Sims, 2011). Extracorporeal membrane oxygenation (ECMO) was reported to be lifesaving in a woman with fulminating peri partum cardiomyopathy, and it may be used in women with pulmonary hypertension (Meng, 2017; Smith, 2009).

1	In the United States, those at greatest risk for endocarditis are those with congenital heart lesions, intravenous drug use, degenerative valve disease, and intracardiac devices (Karchmer, 2015). Subacute bacterial endocarditis usually stems from a low-virulence bacterial infection superimposed on an underlying structural lesion. These are usually native valve infections. Organisms that cause indolent endocarditis are most often viridans-group streptococci or Staphylococcus or Enterococcus species. Among intravenous drug abusers and those with catheter-related infections, Staphylococcus aureus predominates. With prosthetic valve infections, Staphylococcus epidermidis is a frequent cause. Streptococcus pneumoniae and Neisseria gonorrhoeae may occasionally cause acute, fulminating disease. Others have reported Neisseria sicca and N mucosa, group B streptococcus, and Escherichia coli endocarditis during pregnancy or peripartum (Cox, 1988; Deger, 1992; Kangavari, 2000; Kulas, 2006).

1	Infective endocarditis symptoms vary and often develop insidiously. Fever, often with chills, is seen in 80 to 90 percent of cases, a murmur is heard in up to 85 percent, and anorexia, fatigue, and other constitutional symptoms are common (Karchmer, 2015). Clinical clues are anemia, proteinuria, and manifestations of embolic lesions that include petechiae, focal neurological changes, chest or abdominal pain, and ischemia in an extremity. In some cases, heart failure develops. Symptoms may persist for several weeks before the diagnosis is found, and a high index of suspicion is necessary.

1	Diagnosis is made using the Duke criteria, which include positive blood cultures for typical organisms and evidence of endocardial involvement (Hoen, 2013; Pierce, 2012). Echocardiography may be diagnostic, but lesions <2 mm or those on the tricuspid valve may be missed. If uncertain, transesophageal echocardiog raphy is accurate and informative. Importantly, a negative echo cardiographic study does not exclude endocarditis. Treatment is primarily medical, and ascertainment of the infecting organism and its sensitivities is imperative for antimi crobial selection. Guidelines for appropriate antibiotic treatment (Habib, 2015; Karchmer, 2015). Recalcitrant bacteremia and heart failure due to valvular dysfunction are but a few reasons that persistent valvular infection may require replacement.

1	Infective endocarditis is uncommon during pregnancy and the puerperium. During a 7 -year period, the incidence of endocarditis at Parkland Hospital approximated 1 in 16,000 births, and two of seven women died (Cox, 1988). Matenal and fetal mortality rates range from 25 to 35 percent (Habib, 2015; Seaworth, 1986). In a systematic review of infective endocarditis during pregnancy, risk factors were intravenous drug use (14 percent), congenital heart disease (12 percent), and rheumatic heart disease (12 percent) (Kebed, 2014). he most common pathogens were streptococcal (43 percent) and staphylococcal (26 percent) species. Among 51 pregnancies, the maternal mortality rate was 11 percent.

1	For years, patients with heart valve problems were given peri procedural antibiotics for endocarditis prophylaxis. Currently, however, recommendations are more stringent. The American Heart Association recommends prophylaxis for dental procedures in those with: (1) a prosthetic valve or prosthetic material used in a valve repair, (2) prior endocarditis, (3) unrepaired cyanotic heart defect or repaired lesion with residual defect at prosthetic sites, and (4) valvulopathy ater heart transplantation (Nishimura, 2017). The American College of Obstetricians and Gynecologists (2016) does not recommend endocarditis prophylaxis for either vaginal or cesarean delivery in the absence of pelvic infection except with the lesions cited above. Women at highest risk for endocarditis are those with cyanotic cardiac disease, prosthetic valves, or both. When indicated, and for women not already receiving intrapartum antimicrobial therapy for another indication that would also provide coverage against

1	cardiac disease, prosthetic valves, or both. When indicated, and for women not already receiving intrapartum antimicrobial therapy for another indication that would also provide coverage against endocarditis, prophylactic regimens are shown in Table 49-8. hese are administered as close to 30 to 60 minutes before the anticipated delivery time as is feasible.

1	Both preexisting and new-onset cardiac arrhythmias are often encountered during pregnancy, labor, delivery, and the puerperium Qoglar, 2014; Knotts, 2014). In a study of73 women with a history of supraventricular tachycardia (ST) , paroxysmal atrial flutter or fibrillation, or ventricular tachycardia, recurrence rates during pregnancy were 50, 52, and 27 percent, respectively (Silversides, 2006). The mechanism(s) responsible for the higher incidence of arrhythmias during pregnancy are TABLE 49-8. Antibiotic Prophylaxis for Infective Endocarditis in High-Risk Patients American College of Obstetricians and Gynecologist (2016) Standard (IV): ampicillin 2 9 or cefazolin or ceftriaxone 1 9 Penicillin-allergic (IV): cefazolin or ceftriaxone 1 9 or clindamycin 600 mg Oral: amoxicillin 2 9 American Heart Association/European Society of Cardiology (Karchmer, 201n5)a

1	American Heart Association/European Society of Cardiology (Karchmer, 201n5)a Standard: amoxicillin 2 9 PO or ampicillin 2 9 IV or 1M Penicillin-allergic: clarithromycin or azithromycin 500 mg PO; cephalexin 2 gnPO; clindamycin 600 mg PO, IV, or 1M; or cefazolin or ceftriaxone 1 9 IV or 1M 1M = intramuscularly; IV = intravenously. aCefazolin r ceftriaxone given 30 minutes and all others given 1 hour prior to procedure. not well elucidated. From some studies, estradiol and progesterone are proarrhythmic. Estrogen augments the number of adrenergic receptors in the myocardium, and adrenergic responsiveness seems to be greater in pregnancy (Enriquez, 2014). Perhaps the normal but mild hypokalemia of pregnancy and/or the physiological rise in heart rate serves to induce arrhythmias. Alternatively, detection of arrhythmias may be greater because of the frequent visits typical of routine prenatal care.

1	Slow heart rhythms, including complete heart block, are compatible with a successful pregnancy outcome (Keepanasseril, 2015). Some women with complete heart block have syncope during labor and delivery, and occasionally temporary cardiac pacing is necessary (Hidaka, 2006). In our experiences and from others, women with permanent artificial pacemakers usually tolerate pregnancy well (Hidaka, 2011; Jafe, 1987). With ixed-rate devices, cardiac output apparently is increased by augmented stroke volume.

1	Patients with pacemakers or other electrical implants require special precautions during surgery. Stray current may be interpreted as an intracardiac signal by the implanted device and lead to pacing changes. In addition, myocardial burns may result from conduction of current through the pacing electrode rather than through the grounding pad (Pinski, 2002). With these devices, preventive steps include cardiology consultation; bipolar electrosurgery or Harmonic scalpel use rather than mono polar current; if needed, minimal monopolar settings; continuous cardiac and pulse oximetry monitoring; contingency plans for arrhythmias; and close proximity of electrosurgery active and return electrodes (Crossley, 2011).

1	The most common arrhythmia seen in reproductive-aged women is paroxysmal SVT. The prevalence during pregnancy is 24 cases per 100,000 hospital admissions, and approximately 20 percent will experience symptomatic exacerbations during pregnancy (Enriquez, 2014). Interestingly, the mean heart rate of pregnant women with paroxysmal SVT is faster compared with nonpregnant women-184 versus 166 bpm, respectively (Yu, 2015). From Hungary, BGnhidy and associates (2015) found that approximately half of women with paroxysmal SVT had an initial onset during pregnancy. Notably, maternal paroxysmal SVT was associated with a twofold greater risk of septal cardiac defects, particularly secundum atrial septal defects, in their ofspring.

1	In contrast, rarely do atrial ibrilation and atrial lutter present for the irst time during pregnancy. Indeed, new-onset atrial ibrillation should prompt a search for underlying etiologies that include cardiac anomalies, hyperthyroidism, pulmonary embolism, drug toxicity, and electrolyte disturbances (DiCarloMeacham, 2011). Major complications include embolic stroke, and when associated with mitral stenosis, pulmonary edema may develop in later pregnancy if the ventricular rate is increased.

1	For acute treatment, vagal maneuvers, which include Valsalva maneuver, carotid sinus massage, bearing down, and immersion of the face in ice water, raise vagal tone and block the atrioventricular node (Link, 2012; Page, 2015). Intravenous adenosine is a short-acting endogenous nucleotide that also blocks atrioventricular nodal conduction. Our experiences are similar to those of others in that adenosine is safe and efective for cardioversion in hemodynamically stable gravidas (Page, 2015; Robins, 2004). Transient fetal bradycardia has been described with adenosine (Dunn, 2000).

1	If pharmacological therapy is inefective or contraindicated, the merican College of Cardiology and the merican Heart Association recommend synchronized cardioversion in pregnant women with hemodynamically unstable SVT (Page, 2015). And although electrical cardioversion with standard energy settings is not contraindicated in pregnancy, vigilance is important. Barnes and colleagues (2002) described a case in which direct current cardioversion led directly to a sustained uterine contraction and fetal bradycardia. As an aside, pregnancy has no efect on the operation of implantable cardioverter-defibrillator devices (Boule, 2014). If cardioversion fails or is unsafe because of concurrent thrombus, then long-term anticoagulation and heart rate control with medication are necessary (DiCarlo-1eacham, 2011). Other treatment options recommended by the American College of Cardiology and the American Heart Association (Page, 2015) include:

1	Intravenous metoprolol or propranolol when adenosine is inefective or contraindicated Intravenous verapamil when adenosine and 3-blocking agents are inefective or contraindicated Intravenous amiodarone with potentially life-threatening SVT and when other therapies are inefective or contraindicated. Pregnancy may predispose otherwise asymptomatic women with WoffParkinson-White (WP) syndrome to exhibit arrhythmias. In a study of 25 women who had SVT diagnosed before pregnancy, three of 12 women with WPW syndrome and six of 13 without the condition developed SVT during pregnancy (Pappone, 2003). In some patients, accessory pathway ablation may be indicated. Driver and coworkers (2015) have provided a review.

1	This form of arrhythmia is uncommon in healthy young women without underlying heart disease. Brodsky and associates (1992) described seven pregnant women with new-onset ventricular tachycardia and reviewed 23 reports. Most of these women were not found to have structural heart disease. In 14 cases, tachycardia was precipitated by physical exercise or psychological stress. Abnormalities found included two cases of myocardial infarction, two of prolonged QT interval, and one of anesthesia-provoked tachycardia. They concluded that pregnancy events precipitated the tachycardia and recommended 3-blocking agents for control. As previously discussed (p. 964), arrhythmogenic right ventricular dysplasia will result occasionally in ventricular tachyarrhythmias (Lee, 2006). If unstable, emergency cardioversion is indicated, and standard adult energy settings are adequate Qeejeebhoy, 2011; Lin, 2015).

1	This conduction anomaly may predispose individuals to a potentially fatal ventricular arrhythmia known as torsades de pointes (Roden, 2008) . Two studies comprised of 502 pregnant women with long QT syndrome both reported a signiicant increase in cardiac events postpartum but not during pregnancy (Rashba, 1998; Seth, 2007). The normal rise in heart rate during pregnancy may be partially protective. Paradoxically, 3-blocking agents-preferably propranolol-lower the risk of torsades de pointes in patients with long QT syndrome and should be continued throughout pregnancy and the puerperium (Enriquez, 2014; Seth, 2007). Importantly, many medications, including some used during pregnancy such as azithromycin, erythromycin, and clarithromycin, may predispose to QT prolongation (Ray, 2012; Roden, 2004).

1	vlarfan syndrome and coarctation are two aortic diseases that place the pregnant woman at increased risk for aortic dissection (Russo, 2017). Indeed, half of dissection cases in young women are related to pregnancy (O'Gara, 2004). Other risk factors are bicuspid aortic valve and Turner or Noonan syndrome. A high rate of aortic dissection or rupture is also reported in patients with Ehlers-Danlos syndrome (Murray, 2014; Pepin, 2000). Although the mechanism(s) involved are unclear, the initiating event is a tear in the intimal layer of the aorta, followed by hemorrhage into the media, and finally rupture.

1	In most cases, aortic dissection presents with severe chest pain described as ripping, tearing, or stabbing. Diminution or loss of peripheral pulses coupled with a recently acquired aor tic insuiciency murmur is an important physical inding. he diferential diagnosis of aortic dissection in pregnancy includes myocardial infarction, pulmonary embolism, pneumothorax, aortic valve rupture, and obstetrical catastrophes, especially placental abruption and uterine rupture. More than 90 percent of patients with aortic dissection have an abnormal chest radiograph. Aortic angiography is the most definitive method for diagnosis confirmation. However, sonog raphy, computed tomography, and MR imaging are used more frequently depending on the urgency of the clinical situation. Initial medical treatment is given to lower blood pressure.

1	Initial medical treatment is given to lower blood pressure. Proximal dissections most often need to be resected, and the aortic valve replaced if necessary. Distal dissections are more complex, and many may be treated medically. Among non pregnant patients, survival is not improved by immediate elec tive repair compared with surveillance and delayed repair of abdominal aortic aneurysms <5.5 cm. But, Karthikesalingam and colleagues (2016) suggest that the size threshold for aneu rysm repair should be revisited.

1	This autosomal dominant connective tissue disorder has an incidence of 2 to 3 cases per 10,000 individuals and is without racial or ethnic predilection (Ammash, 2008). As discussed in Chapter 59 (p. 1151), Marfan syndrome is characterized by generalized tissue weakness that can result in dangerous cardiovascular complications. Because all tissues are involved, other defects are frequent and include joint laxity and scoliosis. Progressive aortic dilation causes aortic valve insuiciency, and there may be infective endocarditis and mitral valve prolapse with insuiciency. Aortic dilation and dissecting aneurysm are the most serious abnormalities. Early death is due either to valvular insuiciency and heart failure or to a dissecting aneurysm.

1	Of outcomes, a study using the Nationwide Inpatient Sample from 2003 to 2010 found 339 deliveries in women with Marfan syndrome. There was one maternal death and six (1.8 percent) aortic dissections (Hassan, 2015). Russo and associates (2017) used Texas obstetrical discharge data and found that eight of 47 women with aortic dissection had Marfan syndrome. A study from the United ingdom reported similar results (Curry, 2014).

1	The aortic root usually measures approximately 2 cm, and during normal pregnancy, it expands slightly (Easterling, 1991). With Marfan syndrome, aortic root repair is recommended at diameters of 4.0 to 4.5 cm (Smok, 2014). The guidelines of the American College of Cardiology, the American Heart Association, and the merican Association of horacic Surgeons advise prophylactic aortic repair in women considering pregnancy if the diameter of the ascending aorta exceeds 4 cm (Hiratzka, 2010). The guidelines of the European Society of Cardiology (2011) advise repair of the aorta at diameters :::4.5 cm. Because shorter patients have dissection at a smaller diameter, surgical repair is also considered using a formula indexed to height (Bradley, 2014; Smok, 2014).

1	For pregnant women with known thoracic aortic root or ascending aortic dilation, monthly or bimonthly echocardiographic measurements of the ascending aortic dimensions are recommended to detect expansion (Hiratzka, 2010). Prophylactic 3-blocking agents have become standard for pregnant women with �1arfan syndrome because they reduce hemodynamic stress on the ascending aorta and slow the dilation rate (Simpson, 2012). Ideally, pregnant women with aortic aneurysms are delivered at facilities in which cardiothoracic surgery is available. Vaginal delivery with regional analgesia and an assisted second stage seem safe for women with an aortic root diameter < 4 cm.

1	When the aortic root measures 4 to 5 cm or greater, elective cesarean delivery is recommended with consideration of postpartum replacement of the proximal aorta with a prosthetic graft (Simpson, 2012). Successful aortic root replacement during pregnancy has been described, but the surgery has also been associated with fetal hypoxic-ischemic encephalopathy (Mul, 1998; Seeburger, 2007). Several case reports describe emergency cesarean deliveries in women with acute type A dissections that were repaired successfully at the time of delivery (Guo, 2011; Haas, 2011; Papatsonis, 2009). To evaluate obstetrical outcomes, investigators for one study of 63 women with Marfan syndrome analyzed their 142 pregnancies. Of 111 pregnancies progressing past 20 weeks' gestation, 15 percent delivered preterm, and 5 percent had preterm prematurely ruptured membranes (vleijboom, 2006). here were eight perinatal deaths, and half of the neonatal survivors were subsequently diagnosed with Marfan syndrome.

1	In this relatively rare lesion, the aorta is abnormally narrowed and is often accompanied by abnormalities of other large arteries. A fourth of afected patients have a bicuspid aortic valve, and another 10 percent have cerebral artery aneurysms. Other associated lesions are persistent ductus arteriosus, septal defects, and Turner syndrome. he collateral circulation arising above the coarctation remodels and expands, often strikingly, to cause localized erosion of rib margins by hypertrophied intercostal arteries. Typical indings include hypertension in the upper extremities but normal or reduced pressures in the lower extremities. Authors have described diagnosis during pregnancy using MR imaging (Sherer, 2002; Zwiers, 2006). Moreover, Jimenez-Juan and associates (2014) found that aortic diameter measured by �1R and the risk of adverse events during pregnancy were inversely correlated. Of note, no adverse outcomes occurred if the minimum diameter at the coarctation exceeded 15 mm.

1	Major complications with aortic coarctation include congestive heart failure after long-standing severe hypertension, bacterial endocarditis of the bicuspid aortic valve, and aortic rupture. Because hypertension may worsen in pregnancy, antihypertensive therapy using 3-blocking drugs is usually required. Aortic rupture is more likely late in pregnancy or early puerperium. Cerebral hemorrhage from circle of Wiis aneurysms may also occur.

1	Cerebral hemorrhage from circle of Wiis aneurysms may also occur. Of outcomes from 188 pregnancies, a third of women had hypertension that was related to significant coarctation gradients, and one woman died from dissection at 36 weeks' gestation (Beauchesne, 2001). Of nearly 700 deliveries in women with coarctation from the Nationwide Inpatient Sample, hypertensive complications of pregnancy were increased threeto fourfold (rieger, 2011). Importantly, almost 5 percent of women with coarctation had an adverse cardiovascular outcome-maternal death, heart failure, arrhythmia, cerebrovascular or other embolic event-compared with only 0.3 percent of controls. Of women with coarctation, 41 percent underwent cesarean delivery compared with 26 percent of controls.

1	Congestive heart failure demands vigorous eforts to improve cardiac function and may warrant pregnancy interruption. Some authors recommend that resection of the coarctation be undertaken during pregnancy to protect against the possibility of a dissecting aneurysm and aortic rupture. This poses significant perfusion risk, especially for the fetus, because all the arterial collaterals must be clamped for variable periods.

1	Pregnant women with coronary artery disease commonly have the classic risk factors of diabetes, smoking, hypertension, hyperlipidemia, and obesity Games, 2006). Although relatively rare, the risk of acute myocardial infarction is approximately threefold higher in pregnant women compared with nonpregnant women of similar age (Elkayam, 2014b). From more than 50 million hospitalizations in the United States between 1998 and 2009, rates of acute myocardial infarction approximated 2 per 100,000 delivery hospitalizations and 4 per 100,000 postpartum hospitalizations (Callaghan, 2012). Ladner and colleagues (2005) reported a similar rate of 2.7 per 100,000 deliveries.

1	The mortality rate with myocardial infarction in pregnancy is higher compared with age-matched nonpregnant women. In a Nationwide Inpatient Sample study totaling 859 pregnancies complicated by acute infarction, the death rate was 5.1 percent Games, 2006). Women who sustain an infarction <2 weeks before delivery are at especially high risk of death due to the greater myocardial demand of labor and delivery (Esplin, 1999).

1	In a systematic review of 150 cases, most women developed an acute myocardial infarction (MI) during the third trimester or postpartum (Elkayam, 2014b). Approximately three fourths presented with ST segment-elevation MI (STEMI). he leading mechanisms of acute infarction included spontaneous coronary dissection (43 percent) and atherosclerotic disease (27 percent). Signiicant complications included heart failure/cardiogenic shock (38 percent), recurrent angina or infarction (19 percent), and ventricular arrhythmias (12 percent). he maternal and fetal mortality rates were 7 and 5 percent, respectively. Of other potential antecedents, coronary artery occlusion in two pregnant smokers with hypercholesterolemia has been described following ergometrine administration (Mousa, 2000; Ramzy, 2015; Sutaria, 2000). Schulte-Sasse (2000) reported myocardial ischemia associated with prostaglandin Ervaginal suppositories given for 1 labor induction.

1	Diagnosis of acute myocardial infarction during pregnancy does not difer from that in nonpregnant patients and is based on clinical presentation, characteristic ECG changes, and evidence of myocardial necrosis relected by elevated serum troponin levels (Pacheco, 2014). Of note, troponin I levels are undetectable near term in normal pregnancy and do not rise following either vaginal or cesarean delivery (Koscica, 2002; Shivvers, 1999). Importantly, however, troponin I levels are higher in preeclamptic women compared with normotensive controls (Atalay, 2005; Yang, 2006). With spontaneous coronary artery dissection, establishing the diagnosis requires a high index of suspicion in the gravida presenting with chest pain (Codsi, 2016). For this condition, coronary angiography is considered the diagnostic gold standard and should be expediently performed if acute coronary syndrome-deined as myocardial infarction or unstable angina-is present.

1	Treatment of acute myocardial infarction is similar to that for nonpregnant patients (Pacheco, 2014). An algorithm summarizing one approach to its management during pregnancy is shown in Figure 49-6. Several reports describe successful percutaneous transluminal coronary angioplasty and stent placement during pregnancy (Balmain, 2007; Duarte, 2011; FIGURE 49-6 Initial management of acute myocardial infarction during pregnancy. Risk stratification refers to the risk ofdeveloping recurrent symptoms despite optimal medical management. (Adapted with permission from Pacheco LO, Saade GR, Hankins GO: Acute myocardial infarction during pregnancy, (lin Obstet Gynecol. 2014 Oec;S7(4):83S-843.) Dwyer, 2005). Cardiopulmonary resuscitation may be required, as described in Chapter 47 (p. 931). If the infarct has healed suiciently, cesarean delivery is reserved for obstetrical indications, and epidural analgesia is ideal for labor (Esplin, 1999).

1	• Pregnancy with Prior Ischemic Heart Disease he advisability of pregnancy after a myocardial infarction is unclear. Ischemic heart disease is characteristically progressive, and because it is usually associated with hypertension or diabetes, pregnancy in most of these women seems inadvisable. In a review of 30 pregnancies in women who had sustained an inarction remoterompregnancy, none of the women died, four had congestive heart failure, and four had worsening angina during pregnancy (Vinatier, 1994). Pombar and coworkers (1995) evaluated outcomes of women with diabetes-associated ischemic heart disease and infarction. Three had undergone coronary artery bypass grafting before pregnancy. Of 17 women, eight died during pregnancy. Certainly, pregnancy raises cardiac workload, and these investigators concluded that ventricular performance should be assessed using ventriculography, radionuclide studies, echocardiography, or coronary angiography before conception. Without signiicant

1	investigators concluded that ventricular performance should be assessed using ventriculography, radionuclide studies, echocardiography, or coronary angiography before conception. Without signiicant ventricular dysfunction, pregnancy will likely be tolerated. For the woman who becomes pregnant before these studies are performed, echocardiography is done. Exercise tolerance testing may be indicated, and radionuclide ventriculography exposes the fetus to minimal radiation.

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1	Zeeman GG: Obstetric critical care: a blueprint for improved outcomes. Crit Care Med 34:S208, 2006 Zwiers WJ, Blodgett TM, Vallejo MC, et al: Successful vaginal delivery for a parturient with complete aortic coarctation.) Clin Anesth 18:300,t2006 GENERAL CONSIDERATIONS ..................... 975 DIAGNOSIS AND EVALUATION IN PREGNANCY ...... 977 ADVERSE PREGNANCY EFFECTS ...................978 ANTIHYPERTENSIVE DRUGS ....i....i....i....i.......... 980 ANTIHYPERTENSIVE TREATMENT IN PREGNANCY ... 981 SUPERIMPOSED PREECLAMPSIA .................. 983 FETAL ASSESSMENT ....i.........i............... 983 DELIVERY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983 POSTPARTUM CARE ............................ 984 A smal proportion of women suiringrom chronic nephritis had eclampsia. For the most part, autopsy wil reveal the presence of renal changes usualy of acute nephritis, though occasionaly it may be engrafied upon a chronic process.

1	-J. Whitridge Williams (1903) At the time of Wiliams Obstetrics' first edition, little attention was paid to blood pressure changes, even with "toxemia." At that time, chronic hypertension was designated "senile" and thought to develop only in older individuals (Lindheimer, 2015). Indeed, chronic hypertension is not mentioned, per se, in Williams' 1903 textbook, except for some deference given to chronic anatomical renal changes occasionally associated with eclampsia. It is now apparent that chronic hypertension is one of the most common serious complications encountered during pregnancy. his is not surprising because, according to the National the Centers for Disease Control and Prevention (2011), the prevalence of hypertension in women aged 18 to 39 years approximates 7 percent.

1	he incidence of chronic hypertension complicating pregnancy varies depending on population vicissitudes. In a study of more than 56 million births from the Nationwide Patient Sample, the incidence was 1.8 percent (Bateman, 2012). And, in more than 878,000 pregnancies from the Medicaid Analytic Extract, 2.3 percent were complicated by chronic hypertension (Bateman, 2015). Despite this substantive prevalence, optimal management has not been well studied. It is known that chronic hypertension usually improves during early pregnancy. his is followed by variable behavior later in pregnancy and, importantly, by the unpredictable development of superimposed preeclampsia. The latter carries increased risks for maternal and perinatal morbidity and mortality.

1	To define chronic hypertension, the range of normal blood pressure must irst be established. This is not a simple task because, like all polygenically determined biological variants, blood pressure norms difer between populations. And, within these norms, wide variations are found between individuals. Moreover, numerous epigenetic factors inluence presentation. For example, not only do blood pressures vary between races and genders, but pressures-especially systolic-rise directly with increasing age and weight. Thus, pragmatically, normal adults have a broad range of blood pressures, but so do those with chronic hypertension. And finally, resting blood pressure measurements do not reflect daily activities.

1	After these variables are acknowledged, important considerations for any population are the attendant risks of chronic hypertension. It is a leading cause of death and accounts for nearly 15 percent of mortality worldwide. Approximately 65 million Americans have hypertension, and this number is growing concurrently with epidemic obesity (Kotchen, 2015). Hypertension increases substantively the risk of cardiovascular disease, coronary heart disease, congestive heart failure, stroke, renal failure, and peripheral arterial disease (Forouzanfar, 2017).

1	For the foregoing reasons, chronic hypertension would logically be deined as some level of sustained resting blood pressure that is associated with acute or long-term adverse efects. In this regard, most consider 140/90 mm Hg as the upper limit of normal for blood pressure values. But, in the United States, these values are based primarily on actuarial tables constructed using data derived from white adult males and compiled by life insurance companies. These "norms" disregard interrelated factors such as ethnicity, gender, and other important covariants. he importance of race, for example, was emphasized by Kotchen (2015), who cites the incidence of hypertensiondeined as blood pressurea> 140/90 mm Hg-to be 34 percent in blacks, 29 percent in whites, and 21 percent in Mexican Americans.

1	For many years, the Joint National Committee has promulgated guidelines for diagnosis, classification, and management of chronic hypertension. In 2008, the National Heart, Lung, and Blood Institute discontinued these guidelines, and the Joint National Committee 8 GNC 8) was instead asked to provide an evidence-based review Games, 2014). Findings pertinent to caring for young women with chronic hypertension are summarized in Table

1	Proven benefits accrue with treatment of otherwise normal adults who have sustained hypertension. Numerous studies evaluating many combinations of antihypertensive therapy have been conducted. Importantly, these trials evaluated monotherapy versus combination therapeutic regimens and their ethnospecific beneits. Most evaluated cardiovascular outcomes, but many also confirmed risk reductions in rates of cerebrovascular accident, renal insuficiency, and mortality. Because of these incontrovertible beneits, the JNC 8 recommends the management outlined in Table 50-1.

1	Thus, even for mildly elevated blood pressure, interventions to reduce these sequelae are beneficial (SPRINT Research Group, 2015). Moreover, antihypertensive therapy in nonpregnant reproductive-aged women with sustained diastolic pressures ::90 mm Hg is considered standard. Not clear from these observations, however, is what constitutes the best management for women being treated who contemplate pregnancy, for those undergoing treatment who become pregnant, or for those first identified to have chronic hypertension during pregnancy (August, 2015). In these women, the benefits and safety of instituting antihypertensive therapy are less clear, as subsequently discussed on page 981.

1	Women with chronic hypertension are ideally counseled before pregnancy. The duration of hypertension, degree of blood pressure control, and current therapy are ascertained. hose women who require multiple medications for control or those who are poorly controlled carry greater risk for adverse pregnancy outcomes. Home measurement devices are checked for accuracy. General health, daily activities, and dietary habits are also assessed (Table 50-2). For hypertensive women with disease lasting longer than 5 years or with comorbid diabetes, cardiovascular and renal function is assessed (August, 2015; Gainer, 2005). Women with evidence for organ dysfunction or those with prior adverse events such as a stroke, myocardial infarction (MI), arrhythmias, or ventricular failure are at markedly higher risk for a recurrence or worsening dysfunction during pregnancy. Renal function is evaluated by serum creatinine measurement. Also, if

1	TABLE 50-1. Eighth Joint National Committee (JNC 8}-2014 Chronic Hypertension Guidelines and Recommendations Lifestyle modifications endorsed from the Lifestyle Work Group (Eckel, 2013) Recommend selection among four specific medication classes: angiotensin-converting enzyme inhibitors (ACE-I), angiotensin-receptor blockers (ARB), calcium-channel blockers, or diuretics: General population: <60 years old-initiate pharmacological therapy to lower diastolic pressure :;90 mm Hg and systolic pressuren:;140 mm Hg Diabetics: lower pressure to < 140/90 mm Hg Chronic kidney disease: lower pressure to < 140/90 mm Hg. Also add ACE-lnor ARB to improve outcomes

1	Chronic kidney disease: lower pressure to < 140/90 mm Hg. Also add ACE-lnor ARB to improve outcomes General non black population: initial therapy should include thiazide-type diuretic, calcium-channel blocker, ACE-I, or ARB General black population: primary antihypertensive therapy should include thiazide-type diuretic or calcium-channel blocker Assess monthly, and after 1 month, if goals not met, then increase primary drug dose or add second drug. If no response, increase either or add third drug; then if no response, refer to hypertension specialist Summarized from James, 201a4. TABLE 50-2. Lifestyle Modifications for Hypertensive Patients Consume a dietary pattern that emphasizes intake of vegetables, fruits, and whole grains; includes low-fat dairy products, poultry, fish, legumes, nontropical vegetable oils, and nuts; limits sweets and red meats. Examples are DASH, USDA Food Pattern, or the AHA Diet

1	Engage in aerobic physical activity three to four sessions per week, lasting on average 40 minutes per session, and involving moderate-to-vigorous intensity physical activity Moderation of alcohol consumption AHA = American Heart Association; DASH = Dietary Approaches to Stop Hypertension; USDA = United States Department of Agriculture. Summarized from Eckel, 2013; Kotchen, 2015. a urine spot protein/creatinine ratio is abnormally high (>0.3), proteinuria is further quantified with a 24-hour urine collection (Hladunewich, 2011; Kuper, 2016; Morgan, 2016a). The Working Group Report on High Blood Pressure in Pregnancy (2000) of the National Heart, Lung, and Blood Institute concluded that the risks of fetal loss and accelerated renal disease deterioration are increased if the serum creatinine level is above 1.4 mg/dL (Chap. 53, p. 1034).

1	Although pregnancy is considered by many to be contraindicated in women with severe, poorly controlled hypertension, there is no consensus. Certainly, women who maintain persistent diastolic pressures ::110 mm Hg despite therapy; require multiple antihypertensives; have a serum creatinine level > 2 mg/ dL; or have a history of prior stroke, MI, or cardiac failure must be counseled as to the marked risks to themselves and to their pregnancy outcome.

1	he hypertensive disorders that uniquely complicate pregnancy are discussed in Chapter 40 (p. 710). Women are diagnosed with chronic hypertension if it is documented to precede pregnancy or ifhypertension is identified before 20 weeks' gestation. Evidence also supports that prehypertension may herald adverse outcomes similar to those in women with chronic hypertension (Rosner, 2017). In some women without overt chronic hypertension, a history of repeated pregnancies complicated by gestational hypertension, with or without the preeclampsia syndrome, may be elicited. Each is a risk marker for latent chronic hypertension, and this is especially so for preeclampsia, and in particular early-onset preeclampsia. In many ways, gestational hypertension is analogous to gestational diabetes in that such women have a chronic hypertensive diathesis, in which heredity and environment playaa major role.

1	Although uncommon, secondary causes of hypertension are always a possibility in afected women. Thus, consideration is given to underlying chronic renal disease, connective-tissue disease, primary aldosteronism, Cushing syndrome, pheochromocytoma, and myriad other causes. That said, most pregnant women with antecedent hypertension will have otherwise uncomplicated disease. Several factors increase the likelihood that pregnant women will have chronic hypertension. hree of those most frequently cited are ethnicity, obesity, and diabetes. As previously dis cussed, chronic hypertension has a population incidence that women (Kotchen, 2015). Related to this, hundreds of blood identified, including candidate genes for preeclampsia and chronic hypertension (Cowley, 2006; Ward, 2015).

1	The metabolic syndrome is a clinical cluster that includes hypertension, high blood sugar, excess fat at the waist, and abnormal cholesterol or triglyceride levels. This constellation is a risk marker for superimposed preeclampsia and for persistent postpartum hypertension Qeyabalan, 2015; Spaan, 2012). This is not surprising because obesity may increase the prevalence of chronic hypertension tenfold (Chap. 48, p. 938). In addition, obese women are more likely to develop superimposed preeclampsia. Diabetes is also prevalent in chronically hypertensive women, and its interplay with obesity and preeclampsia is overwhelming (Leon, 2016). In aforementioned study from the Nationwide Patient Sample, the most frequent comorbidities associated with chronic hypertension were pregestational diabetes-6.6 percent, thyroid disorders-4.1 percent, and collagen-vascular disease-0.6 percent (Bateman, 2012). Similar comorbidities were described by Cruz and associates (201a1). • Efects of Pregnancy on

1	• Efects of Pregnancy on Blood pressure drops in early pregnancy in most women with chronic hypertension, and it rises again during the third trimester (Fig. According to studies by Tihtonen and coworkers (2007), women with chronic hypertension have persistently elevated vascular resistance and possibly reduced intravascular volume expansion. Adverse outcomes in these women are dependent largely on whether superimposed preeclampsia develops. This may be related to observations reported by Hibbard and colleagues (2005, 2015) that arterial mechanical properties are most marked in women with superimposed preeclampsia. .120 Chronic hypertension )..:ln 100 n (yellow) compared with blood pressures across pregnancy in 4589 healthy nulliparas (blue). (Data from August, 2015; Levine, 1997; Sibai, 1990a.)

1	.120 Chronic hypertension )..:ln 100 n (yellow) compared with blood pressures across pregnancy in 4589 healthy nulliparas (blue). (Data from August, 2015; Levine, 1997; Sibai, 1990a.) Chronic hypertension is associated with several adverse maternal and perinatal outcomes listed in Table 50-3. In sum, these adversities are directly related to severity and duration of hypertension before pregnancy and whether superimposed preeclampsia develops, especially early in gestation. Importantly, in women with mild chronic hypertension, outcomes are also related to blood pressure levels during pregnancy. At this time, however, there are no proven benefits of "tight" versus "lesstight" control of chronic hypertension during pregnancy, as discussed later (p. 982) (Magee, 2015).

1	Most women whose chronic hypertension is well controlled with therapy before pregnancy will do well. Even these women, however, are at increased risk for adverse outcomes. Complications are more likely with severe baseline hypertension and TABLE 50-3. Some Adverse Efects of Chronic Hypertension on Maternal and Perinatal Outcomes HELLP = hemolYSiS, elevated liver enzyme levels, low platelet cou nt.

1	TABLE 50-3. Some Adverse Efects of Chronic Hypertension on Maternal and Perinatal Outcomes HELLP = hemolYSiS, elevated liver enzyme levels, low platelet cou nt. especially with documented end-organ damage (Czeizel, 2011; Odibo, 2013). In a study of pregnancy outcomes in nearly 30,000 chronically hypertensive women, Gilbert and associates (2007) reported markedly increased rates of maternal morbidity that included stroke, pulmonary edema, and renal failure. hese observations were verified in the report from the Nationwide Patient Sample by Bateman and colleagues (2012). In this latter study, hypertension complications included stroke-2.7 per 1000, acute renal failure-5.9 per 1000, pulmonary edema-1.5 per 1000, mechanical ventilation-3.8 per 1000, and in-house maternal mortality-O.4 per 1000. he contribution of hypertension to pregnancy-related strokes is discussed in Chapter 60 (p. 1160) and to hypertensive and idiopathic peripartum cardiomyopathy in Chapter 49 (p. 963).

1	Pregnancy-aggravated hypertension may be due to gestational hypertension or to superimposed preeclampsia. In either instance, blood pressures can be dangerously elevated. As emphasized by Clark and Hankins (2012), systolic pressure :::160 mm Hg or diastolic pressure :::110 mm Hg will rapidly cause renal or cardiopulmonary dysfunction or cerebral hemorrhage. With superimposed severe preeclampsia or eclampsia, the maternal prognosis is poor unless the pregnancy is ended. Placental abruption is a common and serious complication (Chap. 41, p. 767). In addition to hypertensive heart failure mentioned above, aortic dissection was described by Weissman-Brenner and coworkers (2004) and is discussed in Chapter 49 (p. 967).

1	In aggregate, chronic hypertension is associated with a fivefold risk for maternal death (Gilbert, 2007). his is emphasized by the report by Creanga and colleagues (2015) describing 3358 pregnancy-related deaths in the United States from 2006 through 2010. Hypertensive disorders, including chronic hypertension and preeclampsia syndrome, accounted for 9.4 percent of these deaths. Undoubtedly related were other causes of death such as cardiovascular conditions-14.6 percent, cerebrovascular conditions-6.2 percent, and cardiomyopathy-11.8 percent. Moodley (2007) reported similar indings with 3406 maternal deaths from South Africa.

1	Because superimposed preeclampsia is not precisely defined in women with chronic hypertension, the reported incidence varies from 13 to 40 percent (American College of Obstetricians and Gynecologists, 2013; Bramham, 2016; Kim, 2016b; Moussa, 2017). August and colleagues (2015) posit that this predilection may stem from similar genetic, biochemical, and metabolic abnormalities. For example, the risk for superimposed preeclampsia is directly related to the severity of baseline hypertension (Ankumah, 2014; Morgan, 2016b). In a Maternal-Fetal Medicine Units (MFMU) Network trial, Caritis and coworkers (1998) identiied superimposed preeclampsia in 25 percent of hypertensive gravidas. he rate was 29 percent in a California database study (Yanit, 2012). And, women whose hypertension becomes severe enough to warrant chronic antihypertensive therapy during pregnancy are at inordinately high risk for superimposed preeclampsia (Morgan, 2016a). And, this risk is even higher if there is baseline

1	enough to warrant chronic antihypertensive therapy during pregnancy are at inordinately high risk for superimposed preeclampsia (Morgan, 2016a). And, this risk is even higher if there is baseline proteinuria. Finally, and women at high risk for preeclampsia (Henderson, 2014). The recommendation to initiate 81 mg between 12 and 28 weeks' gestation and continue therapy until delivery was adopted by the American College of Obstetricians and Gynecologists (2016b). In addition to chronic hypertension, indications for aspirin prophylaxis for those at high-risk of preeclampsia include a history ofpreeclampsia, multifetal gestation, diabetes, renal disease, and autoimmune disease.

1	Antioxidants to prevent preeclampsia have been studied. with chronic hypertension to treatment with vitamins C and E or with a placebo. A similar number in both groups developed preeclampsia-17 versus 20 percent, respectively. FIGURE 50-2 Blood pressure trends in treated, chronically hypertensive women with and without superimposed preeclampsia. Mean maternal pressures (MAPs) at entry (p = 0.002) and throughout gestation (p <0.001) are significantly diferent for each group. MAP nadir at 23.3 weeks (95% CI, 22.5-24.1) for superimposed preeclampsia versus 26.4 weeks (95% CI, 22.5-27.6) for those without preeclampsia is significant (3.1 weeks, 95% (I, 2.3-4.3). (Data from Morgan, 2016a.) shown in Figure 50-2, chronically hypertensive women destined to develop severe superimposed preeclampsia have higher initial blood pressures that nadir earlier than those of women who do not develop severe disease.

1	Thus far, clinical prognostic and predictive tests for superimposed preeclampsia have been disappointing (CondeAgudelo, 2015). Di Lorenzo and colleagues (2012) studied serum markers for Down syndrome to predict preeclampsia and calculated a sensitivity of 60 percent, with a 20-percent false-positive rate. Similar results were found using antiangiogenic factors to discriminate among chronic hypertension, gestational hypertension, and preeclampsia (Costa, 2016; Sibai, 2008). According to Anton and coworkers (2013), microRNA assays may prove valuable as predictors of pregnancy-associated hypertension. Trials ofvarious medications to prevent preeclampsia in women with chronic hypertension have generally been disappointing and

1	Trials ofvarious medications to prevent preeclampsia in women with chronic hypertension have generally been disappointing and Chronic hypertension augments the risk two-to threefold for premature placental separation. he general obstetrical population risk is 1 in 200 to 300 pregnancies, and this rises to 1 in 60 to 120 pregnancies in women with chronic hypertension (Ankumah, 2014; Cruz, 2011; Magee, 2015). he abruption risk is elevated further if the woman smokes. Most abruptions are in women with worsening gestational hypertension or superimposed preeclampsia. The abruption risk is highest with severe hypertension, and Vigil-De Gracia and colleagues (2004) reported it to be 8.4 percent. From medical record data from the Norwegian Birth Registry, folic acid and/or multivitamin supplements slightly lowered the abruption incidence in women with chronic hypertension (Nilsen, 2008).

1	Rates of almost all adverse perinatal outcomes are greater in women with chronic hypertension than in nonafected controls. As expected, for the entire group of hypertensive women, those who developed preeclampsia have substantially higher adverse outcome rates compared with those without preeclampsia. As shown in Figure 50-3, adverse outcome rates rise incrementally with rising blood pressures. Evidence also supports that chronic hypertension-treated or untreated-is associated with show little or no benefit. Low-dose aspirin has been evaluated 55

1	FIGURE 50-3 Frequency of selected adverse maternal and perinaLa! uuLcomes by blood stratification in women with mild chronic hypertension. SGA = small for gestational age. (Data Ankumah,o2014.) most frequently (Mol, 2016; Sta, 2015). In the MFMU Network study by Caritis (1998) cited above, the incidence ofsuperimposed preeclampsia, fetal-growth restriction, or both is similar in women given low-dose aspirin or placebo. Using the same database, Moore and associates (2015) found that early administration of low-dose aspirin « 17 weeks' gestation) resulted in a significant 41-percent lower frequency of superimposed preeclampsia in chronically hypertensive women-18 versus 31 percent. Duley (2007) and Meads (2008) and their colleagues performed systematic reviews and noted that low-dose aspirin was beneicial in some high-risk women. Moderate beneits were also found from a metaanalysis by Askie and coworkers (2007). In a secondary analysis, Poon and associates (2017) noted that aspirin was

1	was beneicial in some high-risk women. Moderate beneits were also found from a metaanalysis by Askie and coworkers (2007). In a secondary analysis, Poon and associates (2017) noted that aspirin was inefective to reduce the incidence ofpreterm preeclampsia.

1	he U.S. Preventive Services Task Force recommends treatment with low-dose aspirin for chronically hypertensive congenital anomalies. Bateman and coworkers (2015) from the Medicaid Analytic Extract cited earlier found an elevated risk for severe congenital malformations-especially cardiac defects. Moreover, severe hypertension and fetal esophageal atresia or stenosis have been associated (Binhidy, 2011; Van Gelder, 2015).

1	The stillbirth frequency with chronic hypertension is substantively greater in most reports (Chap. 35, p. 646). In the Nationwide Patient Sample study, the stillbirth rate was 15.1 per 1000 births (Bateman, 2012). This is similar to that of 18 per 1000 from a Norwegian study by Ahmad and coworkers (2012) and of 24 per 1000 births from a Network study reported by Ankumah and colleagues (2014) and described on page 982. Low-birthweight neonates are also common. hey are due to fetal-growth restriction, preterm delivery that is largely clinically indic�ted, or both (see Fig. 50-3). In the California database study noted earlier, a fourth of fetuses were delivered preterm (Yanit, 2012).

1	These and other studies attest to the elevated risk for fetalgrowth restriction, and the incidence averages 20 percent. Zetterstrom and coworkers (2006) reported a 2.4-fold risk for fetal-growth restriction in 2754 chronically hypertensive Swedish women compared with the risk in normotensive women. Broekhuijsen and associates (2012) found a 1.3-fold increased risk for 1609 Dutch nulliparas with chronic hypertension compared with that in normotensive controls. As with other complications, fetal-growth dysfunction is more likely in chronically hypertensive women who develop superimposed preeclampsia. In one study, the incidence of growth-restricted fetuses born to women with superimposed preeclampsia was almost 50 percent compared with only 21 percent in chronically hypertensive women without preeclampsia (Chappell, 2008). Finally, women with chronic hypertension severe enough to warrant treatment had an II-percent incidence of fetal-growth restriction to a degree yielding birthweights

1	preeclampsia (Chappell, 2008). Finally, women with chronic hypertension severe enough to warrant treatment had an II-percent incidence of fetal-growth restriction to a degree yielding birthweights ;3rd percentile (Morgan, 2016a). For all of these reasons, neonates born to these women have a correspondingly high rate of intensive-care nursery admission.

1	All of these adverse perinatal efects of chronic hypertension contribute to the greater perinatal mortality rate, which is threeto fourfold higher than the rate in nonafected gravidas (American College of Obstetricians and Gynecologists, 2013). In the Network study by Ankumah (2014) referenced in Figure 50-3, the perinatl death rate was 31 per 1000 births with mild hypertension, 72 per 1000 births with moderate disease, and 100 per 1000 births in women with severe chronic hypertension. And, in the study from Parkland Hospital by Morgan (20 16a), the perinatal mortality rate was 32 per 1000 births in women who were treated for their chronic hypertension. Again, as expected, the highest rates are in women who develop superimposed preeclampsia, for whom the risk doubled from 4 to 8 percent. Finally, if diabetes coexists with chronic hypertension, then preterm delivery, fetalgrowth restriction, and perinatal mortality rates are increased even more (Gonzalez-Gonzalez, 2008; Yanit, 2012).

1	he diagnosis of chronic hypertension in pregnancy should be confirmed. The American College of Obstetricians and Gynecologists (2013) recommends use of ambulatory monitoring to exclude suspected white-coat hypertension before initiating antihypertensive therapy. Goals for chronic hypertension management include rate reductions of adverse maternal or perinatal outcomes just discussed. Treatment is targeted to prevent moderate or severe hypertension and to delay or dampen the severity of pregnancy-aggravated hypertension. To some extent, these goals can be achieved pharmacologically. Blood pressure self-monitoring is encouraged, but for accuracy, automated devices must be properly calibrated (Brown, 2004; Staessen, 2004). Personal health modification includes dietary counseling and reduction of behaviors such as tobacco, alcohol, cocaine, or other substance use (see Table 50-2). A low-sodium diet is not required (American College of Obstetricians and Gynecologists, 2013).

1	Some women-especially those with long-term or untreated hypertension-have complications that increase the risk of adverse pregnancy events. For example, in one study, a fourth of gravidas with chronic hypertension also had concentric ventricular hypertrophy (Ambia, 2017; Kim, 20 16a). Thus, if not already accomplished, assessment during pregnancy is done for the cardiovascular and renal systems (Morgan, 2016a,b).

1	As concluded by the American College of Obstetricians and Gynecologists (2013, 2016a), treatment of hypertension during pregnancy has included every drug class, but information is still limited regarding safety and eicacy (Czeizel, 2011; Podymow, 2011). lthough many studies indicate greater perinatal adverse efects in gravidas requiring treatment, it is still not known whether this is due to cause or efect (Orbach, 2013). The following summary of antihypertensive drugs is abstracted from several sources, including the 2016 Physicians' Desk Rf erence. Many of these drugs are also discussed throughout Chapter 12 (p. 241) and have been reviewed by Umans and associates (2015).

1	PeripheraLy acting 3-adrenergic-receptor blockers cause a generalized decline in sympathetic tone and decreased cardiac output. Examples are propranolol, metoprolol, and atenolol. Labetalol is a popular and commonly used QJ3-adrenergic blocker that is considered safe. Some adrenergic-blocking drugs act centraLy by reducing sympathetic outflow to efect a generalized decreased vascular tone. These include clonidine and a-methyldopa. Drugs in this class most frequently used in pregnancy to treat hypertension are methyldopa or an a-or 3-receptor blocking agent such as labetalol.

1	hese drugs are divided into three subclasses based on their modiication of calcium entry into cells and interference with binding sites on voltage-dependent calcium channels. Common agents include nifedipine-a dihydropyridine, and verapamil-a phenylalkyl amine derivative. These agents have negative inotropic efects and thus can worsen ventricular dysfunction and congestive heart failure. Theoretically, they may potentiate the vasoactive actions of magnesium sulfate that is given for eclampsia neuroprophylaxis. Although data are limited regarding their use during pregnancy, they appear to be safe therapy for chronic hypertension (Briggs, 2015; U mans, 2015).

1	hiazide diuretics are sulfonamides, and these were the first drug group used to successfully treat chronic hypertension (Beyer, 1982). hese agents and loop-acting diuretics such as furosemide are commonly used in nonpregnant hypertensive patients. In the short term, they provide sodium and water diuresis with volume depletion. But with time, there is sodium escape, and volume depletion is partially corrected. Some aspect of lowered peripheral vascular resistance likely contributes to their efectiveness in reducing long-term morbidity (Umans, 2015).

1	Thiazide drugs may be mildly diabetogenic, and expected volume expansion may be curtailed in pregnant women. Sibai and colleagues (1984) showed that plasma volume expanded only about 20 percent over time in hypertensive pregnant women who continued diuretic therapy compared with a 50-percent expansion in women who discontinued treatment. lthough perinatal outcomes were similar in these women, such concerns have led to practices of withholding diuretics as irst-line therapy for chronic hypertension, particularly after 20 weeks' gestation (Working Group Report, 2000). Even so, in a Cochrane review, Churchill and associates (2007) reported no diferences in perinatal outcomes in 1836 nonhypertensive women randomly assigned to a thiazide diuretic or placebo for primary preeclampsia prevention. Overall, thiazide diuretics are considered safe in pregnancy (Briggs, 2015). But for preeclampsia treatment, they are considered to be inefective (Umans, 2015).

1	Hydralazine relaxes arterial smooth muscle and has been used parenterally for decades to safely treat severe peripartum hypertension (Chap. 40, p. 739). Oral hydralazine mono therapy for chronic hypertension is not generally used because of its weak antihypertensive efects and resultant tachycardia. It may be an efective adjunct for long-term use with other antihypertensives, especially if there is chronic renal insuiciency. In one study, vasodilator treatment of chronically hypertensive women was associated with a twofold rise in rates of low-birthweight and growth-restricted neonates (S u, 2013).

1	These drugs inhibit the conversion of angiotensin-I to the potent vasoconstrictor angiotensin-II. They can cause severe fetal malformations when given in the second and third trimesters. These include oligohydramnios, hypocalvaria, and renal dysfunction (Chap. 12, p. 241). Some preliminary studies also suggest teratogenic efects, and because of this, they are not recommended at any time during pregnancy (Briggs, 2015; Podymow, 2011). Angiotensin-receptor blockers act in a similar manner. But, instead of blocking the production of angiotensin-II, they inhibit binding to its receptor. They are presumed to have the same fetal efects as angiotensin-converting enzyme inhibitors and thus are also contraindicated.

1	The prognosis for pregnancy outcome with chronic hyperten sion is somewhat dependent on the severity of disease ante dating pregnancy. This may be related to indings that many women with severe hypertension have renal disease-as either cause or efect (Cunningham, 1990; Morgan, 2016a). It fol lows that women whose hypertension is severe enough to require antihypertensive therapy are at inordinately high risk for superimposed preeclampsia.

1	pregnancies in women whose blood pressure at 6 to 11 weeks' gestation was 2170/110 mm Hg. All were given oral treat ment with a-methyldopa and hydralazine to maintain pres sures < 160/1a10 mm Hg. Of the 44 pregnancies, superimposed preeclampsia developed in half, and all adverse perinatal out comes were in this group. Moreover, all neonates of women in the superimposed group were delivered preterm, nearly 80 percent were also growth restricted, and 48 percent sufered perinatal death. Conversely, those women with severe chronic had reasonably good outcomes. There were no perinatal deaths, and only 5 percent of fetuses were growth restricted. Webster and colleagues (2017) found labetalol and nifedipine to be equally efective for chronic hypertension in pregnant women.

1	Morgan and coworkers (2016a) reported 447 women whose chronic hypertension required treatment beginning prior to 20 weeks. More than half of these women developed superimposed severe preeclampsia. he rate of preeclampsia was 53 percent for those whose 24-hour protein excretion was <300 mg. But for those with antecedent baseline proteinuriaa> 300 mg/day, 79 percent developed severe preeclampsia. Continuing prepregnancy antihypertensive treatment during pregnancy is debatable for those with mild or moderate hypertension. Although blood pressure reduction certainly beneits the mother long term, it at least theoretically can reduce uteroplacental perfusion. In older observational reports, most pregnancy outcomes in women with mild to moderate hypertension generally were good without treatment and unless superimposed preeclampsia developed (Chesley, 1978; Umans, 2015).

1	N ewer data are accruing that address potential salutary efects on pregnancy outcomes by simply lowering blood pressure. Earlier studies were relatively small and had widely varying inclusion and outcome criteria. In a Cochrane review of 49 of these studies that included a total of 4723 women with mild to moderate hypertension, Abalos and coworkers (2014) conirmed that the risk for subsequent severe hypertension was lowered with therapy. Compared with untreated women, the frequencies of superimposed preeclampsia, eclampsia, abruption, preterm birth, fetal-growth restriction, and perinatal or maternal mortality did not difer. This latter Cochrane review raised concerns for fetal-growth restriction with 3-blocking TABLE 50-4. Selected Pregnancy Outcomes in Women with Chronic Hypertension Treated During Pregnancy with and without Baseline Proteinuriaa

1	TABLE 50-4. Selected Pregnancy Outcomes in Women with Chronic Hypertension Treated During Pregnancy with and without Baseline Proteinuriaa Superimposed preeclampsia 79% 49% <0.001 Abruption 0 1% 0.45 EGA at delivery (mean)b 35.1 ± 4.3 wks 37.2 ± 3.3 wks <0.001 ;30 weeks 18% 6% 0.001 ;34 weeks 34% 17% 0.005 ;37 weeks 48% 26% 0.002 Birthweight (mean)b 2379 ± 1028 g 2814 ± 807 g <0.001 ;3rd percentile 20% 9% 0.01 ; 10th percentile 41 % 22% <0.001 Perinatal mortality 36/1000 31/1000 0.47 aDefined as ::300 mgld protein excretion before 20 weeks' gestation. bMean ± standard deviations. EGA = estimated gestational age. Data from Morgan, 2016b.

1	Perinatal mortality 36/1000 31/1000 0.47 aDefined as ::300 mgld protein excretion before 20 weeks' gestation. bMean ± standard deviations. EGA = estimated gestational age. Data from Morgan, 2016b. drugs, ,notably atenolol. It is not resolved, however, because diminished placental perfusion secondary to lowered maternal blood pressure is confounded by the fact that worsening blood pressure itself is associated with abnormal fetal growth. Some also posit that the drugs have a direct fetal action (Umans, 2015). In two of the larger randomized trials, however, the incidence of growth restriction was not altered in women randomly assigned to treatment (Gruppo di Studio Ipertensione in Gravidanza, 1998; Sibai, 1990a).

1	he observations of Morgan and colleagues (20 16a) support the findings of the Cochrane review by Abalos. Specifically, they reported that despite therapy for chronic h) pertension, there was frequent superimposed preeclampsia, fetal-growth restriction, preterm delivery, and perinatal mortality. Moreover, and as shown in Table 50-4, women with baseline proteinuria >300 mg/ d had even worse obstetrical outcomes.

1	During the past decade, the concept of tight control of blood pressure has been espoused as a means of optimizing maternal and perinatal outcomes. Such control is analogous to that of glycemic control for management of the pregnant diabetic patient. he observational study by Ankumah (2014) noted earlier lends credence to tighter control of blood pressure. hese investigators showed that the risk of adverse pregnancy outcomes in 759 women with chronic hypertension was lower when blood pressures before 20 weeks were < 140 mm Hg compared with higher pressure categories and increasing blood pressures. Unfortunately, this did not hold up when less-tight was compared with tight control. Speciically, Magee and coworkers (2015) randomized 987 women with chronic hypertension or gestational hypertension to either one of these two management schemes. Except for a lower rate of severe hypertension in the tightly controlled group, they found no signiicant diferences between these two groups' other

1	to either one of these two management schemes. Except for a lower rate of severe hypertension in the tightly controlled group, they found no signiicant diferences between these two groups' other adverse pregnancy outcomes (Table 50-5). Tight control was also not more costly (Ahmed, 2016). These and similar findings prompted an ongoing randomized controlled trial-Project CHAP (ClinicaITrials.gov, 2016)-to answer this question.

1	Until there are data to conirm any salutary efects of treatment of uncomplicated mild to moderate chronic hypertension in pregnancy, it seems reasonable to follow the guidelines of the American College of Obstetricians and Gynecologists (2013) and the Society for Maternal-Fetal Medicine (2015). Pregnant women with severe hypertension must be treated for maternal neuro-, cardio-, and renoprotection. Treatment is ap <0.001, all other comparisons p >0.05. HELLP = hemolysis, elevated liver enzyme levels, low platelet count. Data from Magee, 201n5. also mandatory for women with prior adverse outcomes such as strokes, MIs, and evidence for cardiac or renal dysfunction. With end-organ dysfunction, treatment to diastolic pressure level ;90 mm Hg is reasonable to mitigate further organ damage. For most women with mild to moderate hypertension, the

1	With end-organ dysfunction, treatment to diastolic pressure level ;90 mm Hg is reasonable to mitigate further organ damage. For most women with mild to moderate hypertension, the College recommends that treatment be withheld as long as systolic blood pressure is < 160 mm Hg and diastolic blood pressure is < 105 mm Hg. Some find it reasonable to begin women with persistent systolic pressuresa> 150 mm Hg or dia stolic pressures of95 to 100 mm Hg or greater (August, 2015; Working Group Report, 2000). At Parkland Hospital we initi ate treatment with antihypertensive agents for blood pressures of 150/100 mm Hg or higher. Our preferred regimens include mono therapy with a 3-blocking drug such as labetalol or a calcium-channel blocking agent such as amlodipine. For women in the first half of pregnancy, therapy with a thiazide diuretic seems reasonable. This is especially true in black women, in whom there is a high prevalence of salt-sensitive chronic hypertension.

1	It is controversial whether or not women who present early in pregnancy and who are already taking antihypertensive drugs should continue to take these (Rezk, 2016). According to the American College of Obstetricians and Gynecologists (2013) and the Society for Maternal-Fetal Medicine (2015), for women with mild to moderate hypertension, it is reasonable to discontinue medications during the irst trimester and to restart them if blood pressures approach the severe range. Our practice at Parkland Hospital is to continue treatment if the woman is already taking drugs when she presents for prenatal care. Exceptions are discontinuation of angiotensin-converting enzyme inhibitors and receptor blockers.

1	Some women will have persistently worrisome hypertension despite usual therapy (Samuel, 2011; Sibai, 1990a). In these women, primary attention is given to the likelihood of pregnancy-aggravated hypertension, with or without superimposed preeclampsia. Other possibilities include inaccurate blood-pressure measurements, suboptimal treatment, and antagonizing substances such as chronic ingestion of nonsteroidal antiinlammatory drugs (NSAIDs) (Moser, 2006; Sowers, 2005). As discussed, the frequency of superimposed preeclampsia for women with chronic hypertension varies depending on the study population and hypertension severity (Ankumah, 2014). Importantly, in 40 to 50 percent of chronically hypertensive women, superimposed preeclampsia develops before 37 weeks (Chappell, 2008; Harper, 2016). This proportion is even higher in women who required hypertension treatment during pregnancy (Morgan, 20 16a).

1	The diagnosis may be diicult to make, especially in women with hypertension who have underlying renal disease with chronic proteinuria (Cunningham, 1990; Morgan, 2016b). As discussed in Chapter 40 (p. 712), conditions that support the diagnosis of superimposed preeclampsia include worsening hypertension, new-onset proteinuria, neurological symptoms such as severe headaches and visual disturbances, generalized edema, oliguria, and certainly, convulsions or pulmonary edema. Making the diagnosis based on worsening proteinuria in women with baseline proteinuria is problematic. Supporting laboratory abnormalities are rising serum creatinine or hepatic transaminase levels, thrombocytopenia, or any of the facets of

1	HELLP (hemolysis, �levated liver enzyme levels, low 2latelet count) syndrome. For women with chronic hypertension and superimposed preeclampsia with severe features, magnesium sulfate for maternal neuroprophylaxis is recommended (Ameri can College of Obstetricians and Gynecologists, 2013). Severe hypertension is treated as described in Chapter 40 (p. 738). Some pregnant women with chronic hypertension have worsening hypertension with no other findings of superim posed preeclampsia. This is most commonly encountered near the end of the second trimester. In the absence of other supporting criteria for superimposed preeclampsia, this likely represents the higher end of the normal blood-pressure curve shown in Figure 50-1. In such women, if preeclampsia can be confidently excluded, it is reasonable to begin or to increase the dose of antihypertensive therapy.

1	Women with well-controlled chronic hypertension who have no complicating factors can generally be expected to have a good pregnancy outcome. Because even those with mild hypertension have a greater risk of superimposed preeclampsia and fetal-growth restriction, serial antepartum assessment of fetal well-being is recommended by many. That said, according to the American College of Obstetricians and Gynecologists (2013), with the exception of sonographic fetal-growth monitoring, described in Chapter 44 (p. 852), no conclusive data address either benefit or harm associated with various antepartum surveillance strategies. • Expectant Management of Early-Onset

1	• Expectant Management of Early-Onset Given that many women with chronic hypertension develop superimposed preeclampsia before term, considerations for expectant management may be reasonable in some cases. In a study from Magee-Women's Hospital, 41 careully selected women with a median gestational age of 31.6 weeks were expectantly managed (Samuel, 2011). Despite liberal criteria to mandate delivery, 17 percent developed either placental abruption or pulmonary edema. The latency period was extended by a mean of 9.7 days. There were no perinatal deaths, however, salutary outcomes were similar. These investigators recommend randomized trials to study expectant management before this becomes usual care.

1	For chronically hypertensive women who have complications such as fetal-growth restriction or superimposed preeclampsia, the decision to deliver is made by clinical judgment. The route of delivery is dictated by obstetrical factors. Certainly, most women with superimposed severe preeclampsia are better delivered even when the fetus is markedly preterm. Increased risk for placental abruption, cerebral hemorrhage, and peripartum heart failure attend delivery delays (Cunningham, 1986, 2005; Martin, 2005). For women with chronic hypertension without preeclampsia, expectant management at later gestational ages was reported recently by Harper and colleagues (2016). They concluded that expectant management beyond 39 weeks' gestation was associated with an increasing incidence of severe preeclampsia and that planned delivery before 37 weeks was associated with a rise in rates of adverse neonatal outcomes.

1	For women with mild to moderate chronic hypertension who continue to have an uncomplicated pregnancy, the merican College of Obstetricians and Gynecologists (2013) recommends delivery not be pursued until 38°17 weeks. The consensus committee indings by Spong and associates (2011) recommend consideration for delivery at 38 to 39 weeks, that is, :::37 completed weeks. A trial of labor induction is preferable, and many of these women respond favorably and will be delivered vaginally (Alexander, 1999; Atkinson, 1995).

1	For women with severe preeclampsia, peripartum management is the same as described in Chapter 40 (p. 729). Epidural analgesia for labor and delivery is optimal with the caveat that it is not given to treat hypertension (Lucas, 2001). hat said, women with severe superimposed preeclampsia are more sensitive to the acute hypotensive efects of epidural analgesia (Vricella, 2012). Also in this group, magnesium sulfate neuroprophylaxis is initiated for prevention of eclampsia. Severe hypertension-diastolic blood pressure :::110 mm Hg or systolic pressurea:::160 mm Hg-is treated with either intravenous hydralazine or labetalol. Some prefer to treat women when the diastolic pressure reaches 100 to 105 mm Hg. Vigil-De Gracia and colleagues (2006) randomly assigned 200 women to intravenous hydralazine or labetalol to acutely lower severe high blood pressure in pregnancy. Outcomes were similar except for significantly more maternal palpitations and tachycardia with hydralazine and signiicantly

1	or labetalol to acutely lower severe high blood pressure in pregnancy. Outcomes were similar except for significantly more maternal palpitations and tachycardia with hydralazine and signiicantly more neonatal hypotension and bradycardia with labetalol.

1	In most respects, postpartum observation, prevention, and management of adverse complications are similar in women with severe chronic hypertension and in those with severe preeclampsia-eclampsia. For persistent severe hypertension, consideration is given for a cause such as pheochromocytoma or Cushing disease (Sibai, 2012). And, in women with chronic end-organ damage, certain complications are more common. These include cerebral or pulmonary edema, heart failure, renal dysfunction, or cerebral hemorrhage, especially within the irst 48 hours after delivery (Martin, 2005; Sibai, 1990b, 2012). hese frequently are preceded by sudden elevations"spikes"-of mean arterial blood pressure and of the systolic component (Cunningham, 2000, 2005).

1	Following delivery, as maternal peripheral resistance rises, left ventricular workload also grows. This elevation is further aggravated by appreciable and pathological amounts of interstitial luid that are mobilized to be excreted as endothelial disruption from preeclampsia resolves. In these women, sudden hypertension-either moderate or severe-may exacerbate diastolic dysfunction, cause systolic dysfunction, and lead to pulmonary edema (Cunningham, 1986; Gandhi, 2001). Prompt hypertension control, along with furosemide-evoked diuresis, usually quickly resolves pulmonary edema.

1	he antihypertensive regimen given antepartum can be restarted in the puerperium. It is also possible in many women to forestall postpartum hypertension by administering intravenous or oral furosemide to augment the normal postpartum diuresis. In one study, 20-mg oral furosemide given daily for 5 days to postpartum women with severe preeclampsia aided blood pressure control (Ascarelli, 2005). Daily weights are helpful in this regard. On average, a woman should weigh 15 pounds less immediately after delivery. Excessive extracellular fluid can then be estimated. Other studies are in progress to determine aspects of postpartum blood pressure management (Cursino, 2015). Some evidence supports that chronic ingestion ofNSAIDs in the puerperium elevates blood pressure in women with severe preeclampsia (Vigil-De Gracia, 2017). his may not be problematic if these drugs are given only as needed (Wasden, 2014).

1	Women with chronic hypertension have special considerations for contraceptive and sterilization choices. These are discussed in detail throughout Chapters 38 and 39. Ultimately, women with chronic hypertension are at high risk for lifetime cardiovascular complications, especially when accompanied by diabetes, obesity, and the metabolic syndrome. Recent evidence also suggests that these women are at greater risk to develop cardiomyopathy remote from pregnancy (Behrens, 2016). Abalos E, Duley L, Steyn OW, et al: Antihypertensive drug therapy for mild to moderate hypertension during pregnancy. Cochrane Database Syst Rev 2:CD002252,t2014 Ahmad AS, Samuelsen SO: Hypertensive disorders in pregnancy and fetal death at diferent gestational lengths: a population study of2 121 371 pregnancies. BJOG 119(12):1521,t2012 Ahmed RJ, Gafni A, Hutton EK, et al: The cost implications ofless tight versus tight control of hypertension in pregnancy (CHIPS Trial). Hypertension 68(4):1049,t2016

1	Ahmed RJ, Gafni A, Hutton EK, et al: The cost implications ofless tight versus tight control of hypertension in pregnancy (CHIPS Trial). Hypertension 68(4):1049,t2016 Alexander JM, Bloom SL, Mcintire DO, et al: Severe preeclampsia and the very low birth weight infant: is induction of labor harmful? Obstet Gynecol 93:485, 1999 Ambia M, Morgan JL, Wilson L, et al: Frequency and consequences of ventricular hypertrophy in pregnant women with treated chronic hypertension. Am J Obstet GynecoIt217:467.e1, 2017 American College of Obstetricians and Gynecologists: Chronic hypertension in pregnancy and superimposed preeclampsia. In: Hypertension in Pregnancy. Report of the American College of Obstetricians and Gynecologists' Task Force on Hypertension in Pregnancy. 2013

1	American College of Obstetricians and Gynecologists: Hypertension. In Clinical Updates in Women's Health Care, Volume V, No. I, January 2016a American College of Obstetricians and Gynecologists: Practice advisory on lowdose aspirin and prevention of preeclampsia: updated recommendations. 2016b. Available at: http://ww.acog.org/About-ACOG/News-Room/ Practice-Advisories/Practice-Advisory-Low-Dose-Aspirin-and-Preventionof-Preeclampsia-U pdated-Recommendations. Accessed January 5, 2017 Ankumah NA, Cantu J, Jauk V, et al: Risk of adverse pregnancy outcomes in women with mild chronic hypertension before 20 weeks of gestation. Obstet Gynecol 123(5):966,t2014 Anton L, Olarerin-George AO, Schwartz N, et al: miR-210 inhibits trophoblast invasion and is a serum biomarker for preeclampsia. Am J Pathol 183(5):1437,t2013 Ascarelli MH, Johnson V, McCreary H, et al: Postpartum preeclampsia management with furosemide: a randomized clinical trial. Obstet Gynecol 105(1):29,t2005

1	Ascarelli MH, Johnson V, McCreary H, et al: Postpartum preeclampsia management with furosemide: a randomized clinical trial. Obstet Gynecol 105(1):29,t2005 Askie LM, Duley L, Henderson-Smart OJ, et al: Antiplatelet agents for prevention of pre-eclampsia: a meta-analysis of individual patient data. Lancet 369(9575):1791,t2007 Atkinson MW, Guinn 0, Owen J, et al: Does magnesium sulfate afect the length of labor induction in women with pregnancy-associated hypertension? Am J Obstet GynecoIt173(4):1219, 1995 August P, Jeyabalan A, RobertS JM: Chronic hypertension and pregnancy. In: Taylor N, RobertS JM, Cunningham FG, et al (eds): Chesley's Hypertensive Disorders in Pregnancy. Amsterdam, Academic Press, 2015 Banhidy F, Acs N, Puh6 EH, et al: Chronic hypertension with related drug treatment of pregnant women and congenital abnormalities in their ofspring: a population-based study. Hypertens Res 34(2):257, 2011

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1	Broekhuijsen K, Langeveld J, van den Berg P, et al: Maternal and neonatal outcomes in pregnancy in women with chronic hypertension. Am J Obstet GynecoIt206:S344,t20t12 Brown M, McHugh L, Mangos G, et al: Automated self-initiated blood pressure or 24-hour ambulatory blood pressure monitoring in pregnancy? BJOG 111 :38, 2004 Caritis S, Sibai B, Hauth J, et al: Low-dose aspirin to prevent preeclampsia in women at high risk. N EnglJ Med 338(11):701,t1998 Centers for Disease Control and Prevention: Vital signs: prevalence, treatment, and control of hypertension-United States, 1999-2002 and 2005-2008. MMWR 60(4):1,t2011 Chappell LC, Enye S, Seed P, et al: Adverse perinatal outcomes and risk factors for preeclampsia in women with chronic hypertension: a prospective study. Hypertension 51(4):1002, 2008

1	Chappell LC, Enye S, Seed P, et al: Adverse perinatal outcomes and risk factors for preeclampsia in women with chronic hypertension: a prospective study. Hypertension 51(4):1002, 2008 Chesley LC: Superimposed preeclampsia or eclampsia. In Chesley LC (ed): Hypertensive Disorders in Pregnancy. New York, Appleton-Century-Crots, 1978 Churchill 0, Beevers GO, Meher S, et al: Diuretics for preventing pre-eclamp sia. Cochrane Database Syst Rev 1 :CD004451, 200 Clark SL, Hankins GO: Preventing maternal death. 10 clinical diamonds. Obstet Gynecol 119(2):360,t2012 ClinicalTrials.gov: Chronic Hypertension and Pregnancy (CHAP) Project. 2016. Available at: https:llclinicaltrials.gov/ct2/show/NCT02299414. Accessed JanualY 5,t2017 Conde-Agudelo A, Romero R, RobertS ltvl: Tests to predict preeclampsia. In Taylor N, RobertS JM, Cunningham FG, et al (eds): Chesley's Hypertensive Disorders in Pregnancy, 4th ed. Amsterdam, Academic Press, 20t15

1	Costa A, Hoshida MS, Alves A, et al: Preeclampsia and superimposed preeclampsia: the same disease? he role of angiogenic biomarkers. Hypertens Pregnancy 35(2): 139,t2016 Cowley AW Jr: he genetic dissection of essential hypertension. Nat Rev Genet 7:829,t2006 Creanga A, Berg CJ, Syverson C, et al: Pregnancy-related mortality in the United States, 2006-2010. Obstet Gynecol 125(1):5,t2015 Cruz MO, Gao W, Hibbard JU: Obstetrical and perinatal outcomes among women with gestational hypertension, mild preeclampsia, and mild chronic hypertension. Am J Obstet GynecoIt205:260.el, 2011 Cunningham FG: Severe preeclampsia and eclampsia: systolic hypertension is also important. Obstet Gynecol 105:237,t2005 Cunningham FG, Cox SM, Harstad W, et al: Chronic renal disease and pregnancy outcome. Am J Obstet Gynecol 163:453, 1990

1	Cunningham FG, Cox SM, Harstad W, et al: Chronic renal disease and pregnancy outcome. Am J Obstet Gynecol 163:453, 1990 Cunningham FG, Pritchard JA, Hankins GO, et al: Idiopathic cardiomyopathy or compounding cardiovascular events? Obstet GynecoI67:157, 1986 Cunningham FG, Twickler 0: Cerebral edema complicating eclampsia. Am J Obstet Gynecol 182(1):94,t2000 Cursino T, Katz L, Coutinho I, et al: Diuretics vs. placebo for postpartum blood pressure control in preeclampsia (DIUPRE): a randomized clinical trial. Reprod Health 12:66,t2015 Czeizel AE, Banhidy F: Chronic hypertension in pregnancy. Curr Opin Obstet Gynecol 23(2):76, 2011 Di Lorenzo G, Ceccarello M, Cecotti V, et al: First trimester maternal serum PIGF, free b-hCG, PAPP-A, PP-13, uterine artery Doppler and maternal history for the prediction of preeclampsia. Placenta 33(6):495, 2012

1	Duley L, Henderson-Smarr OJ, Meher S, et al: Antiplatelet agents for preventing pre-eclampsia and its complications. Cochrane Database Syst Rev 2:CD004659, 2007 Eckel RH, Jakicic JM, Ard JD, et al: 2013 AHAIACC guidelines on lifestyle management to reduce cardiovascular risk: a report of the American College of Cardiologyl American Heart Association Task Force on Practice Guidelines. Circulation 129(25 Suppl 2):S76, 2013 Forouzanfar MH, Liu P, Roth GA, et al: Global burden of hypertension and sysrolic blood pressure of at least 110 to 115 mm Hg, 1990-2015. JM1A 317:165,t2017 Gainer J, Alexander J, Mclntire 0, et al: Maternal echocardiogram findings in pregnant patients with chronic hypertension. Presented at the 25th Annual Meeting of the Society for Maternal-Fetal Medicine, Reno, February 7-12, 2005

1	Gandhi SK, Powers JC, Nomeir A, et al: The pathogenesis of acute pulmonary edema associated with hypertension. N Engl J Med 344(1): 17, 2001 Gilbert M, YoungAL, Danielsen B: Pregnancy-outcomes in women with chronic hypertension: a population-based study. J Reprod Med 52(11): 1 046, 2007 Gonzalez-Gonzalez NL, Ramirez 0, Mozas J, et al: Factors inluencing pregnancy outcomes in women with type 2 versus type 1 diabetes mellitus. Acta Obstet Gynecol Scand 87(1):43, 2008 Gruppo di Studio Iperrensione in Gravidanza: Nifedipine versus expectant management in mild to moderate hypertension in pregnancy. BJOG 105(7): 718, 1998 Harper LM, Biggio JR, Anderson S, et al: Gestational age of delivery in pregnancies complicated by chronic hypertension. Obstet Gynecol 127(6):1101,t2016

1	Harper LM, Biggio JR, Anderson S, et al: Gestational age of delivery in pregnancies complicated by chronic hypertension. Obstet Gynecol 127(6):1101,t2016 Henderson JT, Whitlock EP, O'Connor E, et al: Low-dose aspirin for prevention of morbidity and mortality from preeclampsia: a systematic evidence review for the U.S. Preventive Services Task Force. Ann Intern Med 160(10): 695,t2014 Hibbard JU, Korcarz CE, Nendaz GG, et al: The arterial system in pre-eclampsia and chronic hypertension with superimposed pre-eclampsia. BJOG 112(7):897,t2005 Hibbard JU, Shrof SG, Cunningham FG: Cardiovascular alterations in pregnancy and preeclamptic pregnancy. In Taylor RN, Roberts JM, Cunningham FG, et al (eds): Chesley's Hypertensive Disorders in Pregnancy. Amsterdam, Academic Press, 2015 Hladunewich MA, Schaefer F: Proteinuria in special populations: pregnant women and children. Adv Chronic Kidney Dis 18(4):267,t2011

1	Hladunewich MA, Schaefer F: Proteinuria in special populations: pregnant women and children. Adv Chronic Kidney Dis 18(4):267,t2011 James PA, Oparil S, Carter BL, et al: 2014 evidence-based guidelines for the management of high blood pressure in adults. Report from the panel members appointed to the Eighth Joint National Committee ONC 8). JAMA 311(5):507,t2014 Jeyabalan A, Hubel CA, Roberts JM: Metabolic syndrome and preeclampsia. In Taylor RN, RobertS JM, Cunningham FG, et al (eds): Chesley's Hyper tensive Disorders in Pregnancy, 4th ed. Amsterdam, Academic Press, 2015 Kim MJ, Seo J, Cho Kl, et al: Echocardiographic assessment of structural and hemodynamic changes in hypertension-related pregnancy. J Cardiovasc Ultrasound 24:28, 2016a Kim SA, Park JB: OS 23-03 Midtrimester risk prediction of superimposed pre-eclampsia in pregnant women with chronic hypertension. J Hypertens 34 Suppl 1:e241, 2016b

1	Kim SA, Park JB: OS 23-03 Midtrimester risk prediction of superimposed pre-eclampsia in pregnant women with chronic hypertension. J Hypertens 34 Suppl 1:e241, 2016b Kotchen TA: Hypertensive vascular disease: In Kasper DL, Fauci AS, Hauser SL, et al (eds): Harrison's Principles of Internal Medicine, 19th ed. New York, McGraw-Hill Education, 20t15 Kuper SG, Tita AT, Youngstrom ML, et al: Baseline renal function tests and adverse outcomes in pregnant patients with chronic hypertension. Obstet Gynecol 128:93,t2016 Leon MG, Moussa HN, Longo M, et al: Rate of gestational diabetes mellitus and pregnancy outcomes in patients with chronic hypertension. Am J PerinatoIt33(8):745,t2016 Levine RJ, Hauth JC, Curet LB, et al: Trial of calcium to prevent preeclampsia. N Engl J Med (2):69, 199

1	Levine RJ, Hauth JC, Curet LB, et al: Trial of calcium to prevent preeclampsia. N Engl J Med (2):69, 199 Lindheimer MD, Taylor RN, Roberts JM et al: Introduction, history, controversies, and deinitions. In Taylor RN, Roberts JM, Cunningham FG, et al (eds): Chesley's Hypertensive Disorders in Pregnancy, 4th ed. Amsterdam, Academic Press, 2015 Lucas M), Sharma SK, McIntire DD, et al: A randomized trial of labor analgesia in women with pregnancy-induced hypertension. Amt) Obstet Gynecol 185(4):970,t2001 Magee A, von Dadelszen P, Rey E, et al: Less-tight versus tight control of hypertension in pregnancy. N Engl) Med 2(5):407,t2015 Martin )N )r, Thigpen BD, Moore RC, et al: Stroke and severe preeclampsia and eclampsia: a paradigm shift focusing on systolic blood pressure. Obstet Gynecol 105(2):246, 2005

1	Martin )N )r, Thigpen BD, Moore RC, et al: Stroke and severe preeclampsia and eclampsia: a paradigm shift focusing on systolic blood pressure. Obstet Gynecol 105(2):246, 2005 Meads CA, Cnossen )S, Meher S, et al: Methods of prediction and prevention of pre-eclampsia: systematic reviews of accuracy and efectiveness literature with economic modelling. Health Technol Assess 12(6):1,t2008 Mol BW, Roberts CT, Thangaratinam S, et al: Pre-eclampsia. Lancet 387(10022):999,t2016 Moodley): Maternal deaths due to hypertensive disorders in pregnancy: Saving Mothers report 2002-2004. Cardiovasc) Afr 18:358,t2007 Moore GS, Allshouse A, Post AL. et al: Early initiation of/ow-dose aspirin for reduction in preeclampsia risk in high-risk women: a secondary analysis of the MFMU high-risk aspirin study.) Perinatol 35(5):328,t2015 Morgan )L, Nelson DB, RobertS SW, et al: Blood pressure profiles across pregnancy in women with chronic hypertension. Am) PerinatoI33(12): 1128, 2016a

1	Morgan )L, Nelson DB, RobertS SW, et al: Blood pressure profiles across pregnancy in women with chronic hypertension. Am) PerinatoI33(12): 1128, 2016a Morgan )L, Nelson DB, Roberts SW, et al: The association of baseline proteinuria and adverse pregnancy outcomes in pregnant women with treated chronic hypertension. Obstet Gynecol 128:270, 2016b Moser M, Setaro )F: Resistant or diicult-to-control hypertension. N Engl ) Med 355:385, 2006 Moussa HN, Leon MG, Marti A, et al: Pregnancy outcomes in women with preeclampsia superimposed on chronic hypertension with and without severe features. Am) PerinatoIt34(4):403, 2017 Nilsen RM, Vollset SE, Rasmussen SA, et al: Folic acid and multivitamin supplement use and risk of placental abruption: a population-based registry study. Am) Epidemiol 167(7):867,t2008

1	Nilsen RM, Vollset SE, Rasmussen SA, et al: Folic acid and multivitamin supplement use and risk of placental abruption: a population-based registry study. Am) Epidemiol 167(7):867,t2008 Odibo I, Zilberman D, Apuzzio ), et al: Utiliry of posterior and septal wall thickness in predicting adverse pregnancy outcomes in patients with chronic hypertension. Abstract No. 624, Am) Obstet Gynecol 208:S265, 2013 Orbach H, Matok I, Gorodischer R, et al: Hypertension and antihypertensive drugs in pregnancy and perinatal outcomes. Am ) Obstet Gynecol 208(4):301.e1,t2013 Physicians' Desk Reference, 70th ed. Chestertown, PDR Network, 2016 Podymow T, August P: Antihypertensive drugs in pregnancy. Semin Nephrol 31(1):70,t2011

1	Physicians' Desk Reference, 70th ed. Chestertown, PDR Network, 2016 Podymow T, August P: Antihypertensive drugs in pregnancy. Semin Nephrol 31(1):70,t2011 Poon LC, Wright D, Rolnik DL, et al: Aspirin for evidence-based preeclampsia prevention trial: efect of aspirin in prevention of preterm preeclampsia in subgroups of women according to their characteristics and medical and obstetrical history. Am ) Obstet Gynecol August 4, 2017 [Epub ahead of print] Rezk M, Eliakwa H, Gamal A, Emara M: Maternal and fetal morbidiry following discontinuation of antihypertensive drugs in mild to moderate chronic hypertension: a 4-year observational study. Pregnancy Hypertens 6:291, 2016 Rosner )Y, Gutierrez M, Dziadosz M, et al: Prehypertension in early pregnancy: what is the signiicance? Am) PerinatoIt34(2):1l7, 2017

1	Rosner )Y, Gutierrez M, Dziadosz M, et al: Prehypertension in early pregnancy: what is the signiicance? Am) PerinatoIt34(2):1l7, 2017 Samuel A, Lin C, Parviainen K, et al: Expectant management of preeclmpsia superimposed on chronic hypertension. ) Matern Fetal Neonatal Med 24(7) :907, 2011 Sibai BM: Etiology and management of postpartum hypertension-preeclampsia. Amt) Obstet Gynecol 206(6):470, 2012 Sibai BM, Anderson GD: Pregnancy outcome of intensive therapy in severe hypertension in irst trimester. Obstet Gynecol 67(4):517, 1986 Sibai BM, Grossman A, Grossman HG: Efects of diuretics on plasma volume in pregnancies with long-term hypertension. Am ) Obstet Gynecol 150(7):831, 1984 Sibai BM, Koch vA, Freire S, et al: Serum inhibin A and angiogenic factor levels in pregnancies with previous preeclampsia andlor chronic hypertension: are they useful markers for prediction of subsequent preeclampsia? Am ) Obstet Gynecol 199(3):268.e 1, 2008

1	Sibai BM, Mabie We, Shamsa F, et al: A comparison of no medication versus methyldopa or labetalol in chronic hypertension during pregnancy. Am ) Obstet Gynecol 162(4):960, 1990a Sibai BM, Villar A, Mabie BC: Acute renal failure in hypertensive disorders of pregnancy. Pregnancy outcome and remote prognosis in thirry-one consecutive cases. Am) Obstet GynecoIt162(3): 7, 1990b Sociery for Maternal-Fetal Medicine: SMFM statement: beneit of antihypertensive therapy for mild-to-moderate chronic hypertension during pregnancy remains uncertain. Am) Obstet GynecoIt213(1):3, 2015 Sowers )R, White WB, Pitt B, et al: he efects of cyclooxygenase-2 inhibitors and nonsteroidal anti-inflammatory therapy on 24-hour blood pressure in patients with hypertension, osteoarthritis, and rype 2 diabetes mellitus. Arch Intern Med 165(2):161,t2005 Spaan JJ, Sep S), van Balen VL, et al: Metabolic syndrome as a risk factor for hypertension after preeclampsia. Obstet Gynecol 120(2 Pt 1):311, 2012

1	Spaan JJ, Sep S), van Balen VL, et al: Metabolic syndrome as a risk factor for hypertension after preeclampsia. Obstet Gynecol 120(2 Pt 1):311, 2012 Spinnato)A 2nd, Freire S, Pinto ESilva)L, et al: Antioxidant therapy to prevent preeclampsia: a randomized controlled trial. Obstet Gynecol 11 O(6): 131t1, 2007 Spong CY, Mercer BM, D'lton M, et al: Timing of indicated late-preterm and early-term birth. Obstet Gynecolt118(2 Pt 1):323,t2011 SPRINT Research Group, Wright )T )r, Williamson )D, et al: A randomized trial of intensive versus standard blood-pressure control. N Engl ) Med 373(22):2103,t2015 Staessen )A, Den Hond E, Celis H, et al: Antihypertensive treatment based on blood pressure measurement at home or in the physician's oice: a randomized controlled trial. JAMA 291(8):955,t2004

1	Staessen )A, Den Hond E, Celis H, et al: Antihypertensive treatment based on blood pressure measurement at home or in the physician's oice: a randomized controlled trial. JAMA 291(8):955,t2004 Staf CA, Sibai BM, Cunningham FG: Prevention of preeclampsia and eclampsia. In Taylor RN, Roberts )M, Cunningham FG, et al (eds): Chesley's Hypertensive Disorders in Pregnancy, 4th ed. Amsterdam, Academic Press, 2015 Su CY, Lin HC, Cheng HC, et al: Pregnancy outcomes of anti-hypertensives for women with chronic hypertension: a population-based study. PLoS One 8(2):e53844,t2013 Tihtonen K, Koobi T, Huhtala H, et al: Hemodynamic adaptation during pregnancy in chronic hypertension. Hypertens Pregnancy 26(3):315, 2007 Umans )G, Abalos E, Cunningham FG: Antihypertensive treatment. In Taylor RN, Roberts )M, Cunningham FG, et al (eds): Chesley's Hypertensive Disorders in Pregnancy, 4th ed. Amsterdam, Academic Press, 2015

1	Van Gelder MM, Van Bennekom CM, Louik C, et al: Maternal hypertensive disorders, antihypertensive medication use, and the risk of birth defects: a case control-study. B)OG 122(7):1002,t2015 Vigil-De Gracia P, Lasso M, Montufar-Rueda C: Perinatal outcome in women with severe chronic hypertension during the second half of pregnancy. Intt) Gynaecol Obstet 85(2): 139,t2004 Vigil-De Gracia P, Lasso M, Ruiz E, et al: Severe hypertension in pregnancy: hydralazine or labetalol a randomized clinical trial. Eur ) Obstet Gynecol Reprod BioI 128(1-2):157, 2006 Vigil-De Gracia P, Solis V, Ortega N: Ibuprofen versus acetaminophen as a post-partum analgesic for women with severe pre-eclampsia: randomized clinical study.t) Matern Fetal Neonatal Med 30(11):1279,t2017 V ricella LK, Louis )M, Mercer BM, et al: Epidural-associated hypotension is more common among severely preeclamptic patients in labor. Amt) Obstet GynecoIt207(4):335.e1,t2012

1	V ricella LK, Louis )M, Mercer BM, et al: Epidural-associated hypotension is more common among severely preeclamptic patients in labor. Amt) Obstet GynecoIt207(4):335.e1,t2012 Ward K, Taylor RN: Genetic factors in the etiology of preeclampsia/eclampsia. In Taylor RN, Roberts )M, Cunningham FG, et al (eds): Chesley's Hypertensive Disorders in Pregnancy, 4th ed. Amsterdam, Academic Press, 2015 Wasden SW, Ragsdale ES, Chasen ST, et al: Impact of non-steroidal antiinflammatory drugs on hypertensive disorders of pregnancy. Pregnancy Hypertens 4:259,t2014 Webster LM, Myers )E, Nelson-Piercy C, et al: Labetalol versus nifedipine as antihypertensive treatment for chronic hypertension in pregnancy: a randomized controlled trial. Hypertension 70:915, 2017 Weissman-Brenner A, Schoen R, Divon MY: Aortic dissection in pregnancy. Obstet Gynecol 103: 1110, 2004

1	Weissman-Brenner A, Schoen R, Divon MY: Aortic dissection in pregnancy. Obstet Gynecol 103: 1110, 2004 Working Group Report on High Blood Pressure in Pregnancy: Report of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy. Am) Obstet Gynecol 183:S1, 2000 Yanit E, Snowden )M, Cheng W, et al: he impact of chronic hypertension and pregestational diabetes on pregnancy outcomes. Am ) Obstet Gynecol 207(4):333.e1,t2012 Zetterstrom K, Lindeberg SN, Haglund B, et al: Chronic hypertension as a risk factor for ofspring to be born small for gestational age. Acta Obstet Gynecol Scand 85(9):1046,t2006 ASTHMA................i.............i.....i.....988 ACUTE BRONCHITIS ...i.....i......i...............991 PNEUMONIA.i...............i.......i.......i..... 992 TUBERCULOSIS.. . .................i............ 995 SARCOIDOSIS.....................i............997 CYSTIC FIBROSIS.. . ................i............ 997

1	TUBERCULOSIS.. . .................i............ 995 SARCOIDOSIS.....................i............997 CYSTIC FIBROSIS.. . ................i............ 997 CARBON MONOXIDE POISONING.. . ..i............ 999 A lung which is partialy destroyed or thrown out of function may suice or the respiration of a normal individual, but be unable to respond to the added demands of pregnancy, particulary in the latter months, when the enlarged restricts the mobiliy of the diaphragm. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) As indicated by the above, it has long been appreciated that women in advanced pregnancy tolerate lung disease poorly. Nevertheless, pulmonary disorders are frequently encountered during pregnancy. Chronic asthma or an acute exacerbation is the most common and afects up to 8 percent of pregnant women. Moreover, asthma along with community-acquired pneumonia accounted for almost 10 percent of nonobstetrical antepartum hospitalizations in one managed care plan (Gazmararian, 2002). Pneumonia is also a frequent postpartum complication requiring readmission (Belfort, 2010). These and other pulmonary disorders are superimposed on several important pregnancy-induced changes of ventilatory physiology. For example, pregnant women, especially those in the last trimester, are susceptible to complications of severe acute pneumonitis as evidenced by the disparate number of maternal deaths during influenza pandemics.

1	The important and sometimes marked changes in the respira tory system induced by pregnancy are reviewed in Chapter 4 (p. 64), and values for associated tests can be found in the Appendix (p. 1260). Lung volumes and capacities that are measured directly to assess pulmonary pathophysiology may be signiicantly altered. In turn, these change gas concentrations and acid-base values in blood. Some of the physiological alterations induced by pregnancy were summarized by Wise and associates (2006): 1. ital capaciy and inspiratory capaciy increase by approximately 20 percent by late pregnancy. 2. Expiratory reserve volume decreases from 1300 mL to approximately 1100 mL. 3. idal volume increases approximately 40 percent as a result of respiratory stimulation by progesterone. 4. Minute ventilation increases 30 to 40 percent due to increased tidal volume. As a result, arterial P02 increases from 100 to 105 mm Hg. 5.

1	4. Minute ventilation increases 30 to 40 percent due to increased tidal volume. As a result, arterial P02 increases from 100 to 105 mm Hg. 5. Increasing metabolic demands cause a 30-percent rise in carbon dioxide (C02) production. But, because of its concomitantly increased difusion capacity and hyperventilation, the arterial Peo2 decreases from 40 to 32 mm Hg. 6. Residual volume decreases approximately 20 percent from 1500 mL to approximately 1200 mL. 7. Chest wal compliance is reduced by a third by the expanding uterus and increased intraabdominal pressure. This causes a 10-to 25-percent decrease in functional residual capacithe sum of expiratory reserve and residual volumes.

1	In a longitudinal cohort study, Grindheim and colleagues (2012) also showed that forced vital capacity and peak expiratory flow rose progressively across pregnancy after 14 to 16 weeks' gestation. The end result of these pregnancy-induced changes is substantively increased ventilation due to deeper but not more frequent breathing. hese are thought to be stimulated by basal oxygen consumption as it rises incrementally from 20 to 40 mLimin in the second half of pregnancy.

1	Reactive airway disease is seen frequently in young women and therefore oten complicates pregnancy. Asthma prevalence grew steadily in many countries beginning in the mid-1970s but may have plateaued in the United States with a prevalence in adults of approximately 10 percent (Barnes, 2015; Centers for Disease Control and Prevention, 201Oc, 2013). The estimated asthma prevalence during pregnancy ranges between 4 and 8 percent, and this appears to be rising (Kelly, 2015; Racusin, 2013). Finally, evidence is accruing that fetal and neonatal environmental exposures may contribute to the origins or mitigation of asthma (Grant, 2016; Litonjua, 2016; Spiegel, 2016).

1	Asthma is a chronic inlammatory airway syndrome with a major hereditary component. Increased airway responsiveness and persistent subacute inflammation are associated with polymorphism genes on chromosomes 5q that include cytokine gene clusters, 3-adrenergic and glucocorticoid receptor genes, and the T-cell antigen receptor gene (Barnes, 2015). Asthma is etiologically and clinically heterogeneous, and an environmental allergic stimulant such as influenza or cigarette smoke serves as a promoter for susceptible individuals (Bel, 2013).

1	he hallmarks of asthma are reversible airway obstruction from bronchial smooth muscle contraction, vascular congestion, tenacious mucus, and mucosal edema. Mucosal inlammation is characterized by infiltration with eosinophils, mast cells, and T lymphocytes. hese causes airway inflammation and increased responsiveness to numerous stimuli that include irritants, viral infections, aspirin, cold air, and exercise. Several inflammatory mediators produced by these and other cells include histamine, leukotrienes, prostaglandins, cytokines, IgE, and many others. Importantly, because F-series prostaglandins and ergonovine exacerbate asthma, these commonly used obstetrical drugs should be avoided if possible.

1	Pulmonary unction changes are more pronounced in asthmatics compared with healthy women (Zairina, 2015). Asthma manifestations range from mild wheezing to severe bronchoconstriction, which obstructs airways and decreases airflow. his lowers the forced expiratory volume in 1 second/forced vital capacity (FEVJFVC) ratio and the peak expiratory flow (PEF). The work ofbreathing progressively increases, and patients note chest tightness, wheezing, or breathlessness. Subsequent alterations in oxygenation primarily reflect ventilation-perfusion mismatching because the distribution of airway narrowing is uneven. Varied manifestations of asthma have led to a simple classiication that considers severity, onset, and duration of symptoms (Table 51-1). With persistent or worsening bronchial obstruction, clinical stages progress as shown in Figure

1	Hypoxia initially is mitigated by hyperventilation, which maintains arterial P02 within a normal range but lowers Peo2' creating respiratory alkalosis. As airway narrowing worsens, ventilation-perusion defects increase, and arterial hypoxemia ensues. With severe obstruction, ventilation becomes impaired as fatigue causes early CO2 retention. Because ofhyperventilation, this may only be seen initially as an arterial Peo2 returning to the normal range. With continuing obstruction, respiratory failure follows from fatigue. Although these changes are generally reversible and well tolerated by the healthy nonpregnant individual, even early asthma stages may be dangerous for the pregnant woman and her fetus. This is because smaller functional residual capacity and increased pulmonary shunting render the woman more susceptible to hypoxia and hypoxemia. Effects of Pregnancy on Asthma

1	Effects of Pregnancy on Asthma Pregnancy has an unpredictable efect on underlying asthma. In their review of six prospective studies of more than 2000 gravidas, Gluck and Gluck (2006) reported that approximatelyaa third each improved, remained unchanged, or clearly TABLE 51-1. Classification of Asthma Severity FIGURE 51-1 Clinical stages of asthma. FEV1 = forced expiratory volume in 1 second. worsened. Exacerbations are more common with severe disease (Ali, 2013). In a study by Schatz and associates (2003), baseline severity correlated with asthma morbidity during pregnancy. With mild disease, 13 percent of women had an exacerbation and 2.3 percent required admission; with moderate disease, these numbers were 26 and 7 percent; and for severe asthma, 52 and 27 percent. Others have reported similar observations (Charlton, 2013; Hendler, 2006). Finally, morbidity rates are disproportionately increased in black compared with white women.

1	Up to 20 percent of women with mild or moderate asthma have been reported to have an intrapartum exacerbation (Schatz, 2003). Conversely, Wendel and associates (1996) reported exacerbations at the time of delivery in only 1 percent of women. Mabie and coworkers (1992) reported an 18-fold increased exacerbation risk following cesarean versus vaginal delivery. Women with asthma have had improved pregnancy outcomes during the past 20 years. he incidence of spontaneous abortion in women with asthma may be slightly increased (Blais, 2013).

1	Women with asthma have had improved pregnancy outcomes during the past 20 years. he incidence of spontaneous abortion in women with asthma may be slightly increased (Blais, 2013). Maternal and perinatal outcomes for nearly 30,000 pregnan cies in asthmatic women are shown in Table 51-2. Findings are not consistent among these studies. For example, in some, but not all, the incidences of preeclampsia, preterm labor, growth restricted infants, and perinatal mortality are slightly increased (Murphy, 2011). Other reports cited a small rise in the incidence of placental abruption and previa, preterm rupture of mem branes, and gestational diabetes (Getahun, 2006; Wang, 2014). But, in a European report of 37,585 pregnancies of women with asthma, the risks for most obstetrical complications were not increased (Tata, 2007). Finally, Cossette and coworkers (2013) and increasing inhaled-corticosteroid dosage.

1	Increased morbidity appears to be signiicantly linked to severe disease, poor control, or both. In the study by the Maternal-Fetal Medicine Units (MFMU) Network, delivery before 37 weeks' gestation was not increased among the 1687 pregnancies of asthmatic women compared with those of 881 controls (Dombrowski, 2004a). But for women with severe asthma, the rate was increased approximately twofold. In a prospective evaluation of 656 asthmatic pregnant women and 1052 pregnant controls, Triche and coworkers (2004) found that women with moderate to severe asthma, regardless of treatment, are at increased risk of preeclampsia. Finally, the MFMU Network study suggests a direct relationship of baseline pregnancy FEV] with birthweight and an inverse relationship with rates of gestationl hypertension and preterm delivery (Schatz, 2006).

1	Maternal morbidity includes life-threatening complications from status asthmaticus. his causes muscle fatigue with respiratory arrest, pneumothorax, pneumomediastinum, acute cor pulmonale, and cardiac arrhythmias. Not surprisingly, maternal and perinatal mortality rates rise substantively when mechanical ventilation is required. TABLE 51-2. Maternal and Perinatal Outcomes in Pregnancies Complicated by Asthma blncidence not significantly diferent compared with control group or generan l obstetrical population. clncidence significantly greater than control group or general obstetrical population. NS = not stated.

1	clncidence significantly greater than control group or general obstetrical population. NS = not stated. As discussed, with reasonable asthma control, perinatal outcomes are generally good. In the Network study cited above, rates of adverse neonatal sequelae caused by asthma were not signiicantly increased (Dombrowski, 2004a). he caveat is that severe asthma was uncommon in this closely monitored group. When respiratory alkalosis develops, earlier animal and human studies suggest that fetal hypoxemia develops well before the alkalosis compromises maternal oxygenation (Rolston, 1974). It is hypothesized that the fetus is jeopardized by decreased uterine blood flow, decreased maternal venous return, and an alkaline-induced letward shit of the oxyhemoglobin dissociation curve (Chap. 47, p. 920).

1	The fetal response to maternal hypoxemia is decreased umbilical blood flow, increased systemic and pulmonary vascular resistance, and decreased cardiac output. Observations by Bracken and colleagues (2003) confirm that the incidence of fetal-growth restriction increases with asthma severity. Because the fetus may be seriously compromised as asthma severity increases, the need for aggressive management is underscored. Monitoring the fetal response is, in efect, an indicator of maternal status. Possible teratogenic or adverse fetal efects of drugs given to control asthma have been a concern. Several reports show a slightly greater risk for varied abnormalities such as cleft lip and palate and autism spectrum disorders. However, not all studies have veriied this (Eltonsy, 2016; Gidaya, 2016; Murphy, 2013b; Wang, 2014). It is worrisome that up to half of these women discontinue essential treatment between 5 and 13 weeks' gestation (Enriquez, 2006).

1	he subjective severity of asthma frequently does not correlate with objective measures of airway function or ventilation. Although clinical examination can also be an inaccurate predictor, useful signs include labored breathing, tachycardia, pulsus paradoxus, prolonged expiration, and use of accessory muscles. Signs of a potentially fatal attack include central cyanosis and altered consciousness.

1	Arterial blood gas analysis provides objective assessment of maternal oxygenation, ventilation, and acid-base status. With this information, the severity of an acute attack can be assessed (see Fig. 51-1). That said, in a prospective evaluation, Wendel and associates (1996) found that routine arterial blood gas analysis did not help to manage most pregnant women who required admission for asthma control. If used, the results must be interpreted in relation to normal values for pregnancy. For example, a Pco2>35 mm Hg with a pH <7.35 is consistent with hyperventilation and CO2 retention in a pregnant woman.

1	Pulmonary function testing should be routine in the management of chronic and acute asthma. Sequential measurement of the FEV1 or of the peak expiratoy low rate-PEFR-is the best measure of severity. n FEV1 less than 1 L, or less than 20 percent of predicted value, correlates with severe disease defined by hypoxia, poor response to therapy, and a high relapse rate. The PEFR correlates well with the FEVl, and it can be measured reliably with inexpensive portable meters. It is advantageous for each woman to determine her own baseline when asymptomatic to compare with values when symptomatic. • Management of Chronic Asthma Asthma management by an experienced team produces the most salutary outcomes (Bonham, 2017; Lim, 2014; Wendel, 1996). Management guidelines include: 1. Patient education-general asthma management and its efect on pregnancy. 2. Environmental precipitating factors-avoidance or control.

1	Patient education-general asthma management and its efect on pregnancy. 2. Environmental precipitating factors-avoidance or control. Viral infections that include the common cold are frequent triggering events (Ali, 2013; Murphy, 2013a). 3. Objective assessment of pulmonary function and fetal status-monitor with PEFR or FEVl. 4. Pharmacological therapy-in appropriate combinations and doses to provide baseline control and treat exacerbations. Compliance may be a problem, and periodic medication reviews are helpful (Sawicki, 2012). In general, women with moderate to severe asthma ideally measure and record either their FEV1 or PEFR twice daily. The FEVl ideally is >80 percent of predicted. For PEFR, predicted values range from 380 to 550 L/min. Each woman has her own baseline value, and therapeutic adjustments can be made using this (American College of Obstetricians and Gynecologists, 2016a; Rey, 2007).

1	Treatment depends on disease severity. No therapeutic regimen for management of pregnant asthmatics is universally accepted (Bain, 2014). 3-Agonists help abate bronchospasm, and corticosteroids treat inflammation. Regimens recommended for outpatient management are listed in Figure 51-2. For mild asthma, inhaled 3-agonists as needed are usually suicient. For persistent asthma, inhaled corticosteroids are administered every 3 to 4 hours. The goal is to reduce the use of 3-agonists for symptomatic relie. A case-control study from Canada with a cohort of more than 15,600 nonpregnant women with asthma showed that inhaled corticosteroids reduced hospitalizations by 80 percent (Blais, 1998). At Parkland Hospital, Wendel and

1	FIGURE 51-2 Stepwise approach to asthma treatment.oICS = inhaled corticosteroids; LABA = long-acting 3-agonists; OCS = oral coticosteroids. (Modified from Barnes PJ: Asthma. In Kasper D, Fauci A, Hauser SL, et al (eds): Harrison's Principles of Internal Medicine, 19th ed. New York, McGraw-Hili Education, 2015, p 1669.) sions for severe exacerbations with inhaled steroids. corticosteroids became available. Minimal beneit is gained with use of these compounds and they have a high rate of side efects. However, some theophylline derivatives are considered useful for oral maintenance therapy if the initial response to inhaled corticosteroids and 3-agonists is not optimal (Dom browski, 2004b).

1	zileuto n, zairlukast, and montelukast. hese drugs are given orally or by inhalation for prevention, but they are not efec tive for acute disease (Barnes, 2015). For maintenance, they are used in conjunction with inhaled corticosteroids to allow minimal dosing. Approximately half of asthmatics will improve with these drugs. hese agents are not as efective as inhaled corticosteroids, and there is little experience with their use in pregnancy (Fanta, 2009). mast cell degranulation. They are inefective for acute asthma and are used primarily to treat childhood asthma. • Management of Acute Asthma

1	mast cell degranulation. They are inefective for acute asthma and are used primarily to treat childhood asthma. • Management of Acute Asthma Treatment of acute asthma during pregnancy is similar to that for the nonpregnant asthmatic. Importantly, the threshold for hospitalization is signiicantly lower. Intravenous (IV) hydration may help clear pulmonary secretions, and supplemental oxygen is given by mask. The therapeutic aim is to maintain the P02 >60 mm Hg, and preferably normal, along with 90to 95-percent oxygen saturation. Baseline pulmonary function testing includes FEV) or PEFR. Continuous pulse oximetry and electronic fetal monitoring, depending on gestational age, may provide useful information. Antibiotics are not given unless there is concomitant pneumonitis, which is caused by the same organisms discussed on page 992 (T erraneo, 2014).

1	First-line therapy for acute asthma includes a short-acting 3-adrenergic agonist, such as terbutaline, albuterol, isoetharine, epinephrine, isoproterenol, or metaproterenol, which is given subcutaneously, taken orally, or inhaled. In severely ill women, these drugs can be given IV (Barnes, 2015). They bind to speciic cell-surface receptors and activate adenylyl cyclase to increase intracellular cyclic AMP and modulate bronchial smooth muscle relaxation. Long-acting preparations are used for outpatient therapy.

1	If not previously given for maintenance, inhaled corticosteroids are commenced. A nebulized anticholinergic drug may be added if the response at this point is unsatisfactory (Barnes, 2015). Also, for severe exacerbations, IV magnesium sulfate or theophylline may prove eicacious. Corticosteroids are given early to all patients with severe acute asthma. Unless the response to bronchodilator and inhaled corticosteroid therapy is prompt, oral or parenteral corticosteroids are given (Lazarus, 2010). One regimen is oral prednisone or prednisolone or IV methylprednisolone in a dose of 30 to 45 mg daily for 5 to 10 days without tapering (Barnes, 2015). Because their onset of action is several hours, corticosteroids are given initially along with 3-agonists for severe acute asthma.

1	At this juncture, further management depends on the severity and response to therapy. If initial therapy with 3-agonists is associated with improvement of FEV, or PEFR to above 70 percent of baseline, then discharge can be considered. Some women may beneit from longer observation. Alternatively, for the woman with obvious respiratory distress, or if the FEV) or PEFR is <70 percent of predicted after three doses of 3-agonist, admission is usually advisable (Lazarus, 2010). Intensive therapy is continued with inhaled 3-agonists, IV corticosteroids, and close observation for worsening respiratory distress or fatigue in breathing (Racusin, 2013). The woman is cared for in the delivery unit or an intermediate or intensive care unit (ICU) (Dombrowski, 2006; Zeeman, 2003).

1	Severe asthma of any type not responding after 30 to 60 minutes of intensive therapy is termed status asthmaticus. This has been termed by some as critical asthma symptoms (Kenyon, 2015). Generally, management of nonpregnant patients with status asthmaticus in an intensive care setting results in a good outcome. Consideration should be given to early intubation when maternal respiratory status worsens despite aggressive treatment (see Fig. 51-1). Fatigue, CO2 retention, and hypoxemia are indications for mechanical ventilation (Chan, 2015). Although Lo and colleagues (2013) described a woman with status asthmaticus in whom cesarean delivery was necessary to efect ventilation, Andrews (2013) cautioned that such clinical situations are uncommon.

1	For the laboring asthmatic, maintenance medications are continued through delivery. Stress-dose corticosteroids are administered to any woman given systemic corticosteroid therapy within the preceding 4 weeks. The usual dose is 100 mg of hydrocortisone given IV every 8 hours during labor and for 24 hours ater delivery. The PEFR or FEV) is determined on admission, and serial measurements are taken if symptoms develop. Oxytocin or prostaglandins E) or E2 are used for cervical ripening and induction. A nonhistamine-releasing narcotic such as fentanyl may be preferable to meperidine for labor, and epidural analgesia is ideal. For surgical delivery, conduction analgesia is preferred because tracheal intubation can trigger severe bronchospasm. Postpartum hemorrhage is treated with oxytocin or prostaglandin E) or E2. Prostaglandin F2: or ergotamine derivatives are contraindicated because they may cause signiicant bronchospasm.

1	Infection of the large airways is manifest by cough without pneumonitis. It is common in adults, especially in winter months. Infections are usually caused by viruses, and of these, influenza A and B, parainluenza, respiratory syncytial, coronavirus, adenovirus, and rhinovirus are frequent isolates (Wenzel, 2006). Bacterial agents causing community-acquired pneumonia are rarely implicated. The cough of acute bronchitis persists for 10 to 20 days (mean 18 days) and occasionally lasts for a month or longer. According to the 2006 guidelines of the American College of Chest Physicians, routine antibiotic treatment is not indicated (Smith, 2014).

1	his is a leading cause of death in the United States (Heron, 2016). Current classiication includes communiy-acquired pneumonia (CAP), which is typically encountered in otherwise healthy young women, including during pregnancy. Healthcare-associated pneumonia (HAP) develops in patients in outpatient care facilities and more closely resembles hospitalacquired pneumonia (HAP). In most cases of community-acquired pneumonia, the ofending pathogen is not identified. In a recent study from the Centers for Disease Control and Prevention (CDC), pathogens were identified in only 38 percent of nearly 2S00 adults with pneumonia Gain, 201S). hese included viruses in 23 percent, bacteria in 11 percent, both in 3 percent, and ungi or protozoa in 1 percent. Half of bacterial isolates were Sreptococcus pneumoniae.

1	Pneumonia in pregnant women is relatively common (Brito, 2011; Sheield, 2009). Gazmararian and coworkers (2002) reported that pneumonia accounts for 4.2 percent of antepartum admissions for nonobstetrical complications. Pneumonia is also a frequent indication for postpartum readmission (Belfort, 2010). During inluenza season, admission rates for respiratory illnesses double compared with rates in the remaining months (Cox, 2006). Regardless of etiology, mortality from pneumonia is infrequent in young women, but during pregnancy severe pneumonitis with appreciable loss of ventilatory capacity is not as well tolerated (Callaghan, 201S; Rogers, 2010). Hypoxemia and acidosis are also poorly accommodated by the fetus and frequently stimulate preterm labor after midpregnancy. Because many cases of pneumonia follow viral upper respiratory illnesses, worsening or persistence of symptoms may represent developing pneumonia. Any gravida suspected of having pneumonia should undergo chest

1	cases of pneumonia follow viral upper respiratory illnesses, worsening or persistence of symptoms may represent developing pneumonia. Any gravida suspected of having pneumonia should undergo chest radiography.

1	Many bacteria that cause community-acquired pneumonia, such as Streptococcus pneumoniae, are part of the normal resident lora. Some factors that perturb the symbiotic relationship between colonizing bacteria and mucosal phagocytic defenses include acquisition of a virulent strain or bacterial infections following a viral infection. Cigarette smoking and chronic bronchitis favor colonization with S pneumoniae, HaemophiIus inluenzae, and Legionella species. Other risk factors include asthma, binge drinking, and human immunodeficiency virus (HIV) infection (Sheield, 2009).

1	Pregnancy itself does not appear to predispose to pneumonia. Jin and colleagues (2003) reported the antepartum hospitalization rate for pneumonia in Alberta, Canada, to be 1.S per 1000 deliveries-almost identical to the rate of 1.47 per 1000 for nonpregnant women. Likewise, Yost and associates (2000) reported an incidence of 1.S per 1000 for pneumonia complicating 7S,000 pregnancies cared for at Parkland Hospital. s discussed, at least hlf are caused by viruses. A fourth are bacterial, and S pneumoniae causes half of the latter. Over the past few years community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has emerged as a common pathogen that may cause necrotizing pneumonia (Mandell, 201S; Moran, 2013). Occasionally, Legionnaires disease is encountered (Close, 2016). FIGURE 51-3 Chest radiograph in a pregnant woman with right lower lobe and left upper lobe pneumonia. Rounded right basilar and let apical infiltrates are consistent with the diagnosis.

1	Typical symptoms of pneumonia include cough, dyspnea, sputum production, and pleuritic chest pain. Mild upper respiratory symptoms and malaise usually precede these symptoms, and mild leukocytosis is usually present. Chest radiography is essential for diagnosis (Fig. SI-3). Radiographical findings do not accurately predict the etiology, and as discussed, the responsible pathogen is identiied in fewer than half of cases. According to the Infectious Diseases Society of America (IDSA) and the American horacic Society (A TS), tests to identiy a specific agent are optional (Mandell, 2007). hus, sputum cultures, serological testing, cold agglutinin identiication, and tests for bacterial antigens are not routinely recommended. The one exception to this may be rapid serological testing for inluenza A and B (Sheield, 2009).

1	Although many otherwise healthy young adults can be safely treated as outpatients, at Parkland Hospital we hospitalize all pregnant women with radiographically proven pneumonia. Another option is outpatient therapy or 23-hour observation, which is reasonable with optimal follow-up. At least for nonpregnant patients, the pneumonia severity index (PSI) and the CURB-6S scoring system are used as a guide to admission (Mandell, 201S). Neither has been studied in pregnancy. Given that, risk factors shown in Table 5a1-3 should prompt consideration for hospitalization. With severe disease, admission to an intensive or intermediate care unit is advisable. Approximately 20 percent of pregnant women admitted to Parkland Hospital for pneumonia require this level of care (Zeeman, 2003). Severe pneumo TABLE 51-3. Criteria for Severe Community-Acquired Pneumoniaa Leukopenia: WBC <4000/-LL Thrombocytopenia-platelets < 1 OO,OOO/-LL aCriteria of the Infectious Diseases Society of America/

1	TABLE 51-3. Criteria for Severe Community-Acquired Pneumoniaa Leukopenia: WBC <4000/-LL Thrombocytopenia-platelets < 1 OO,OOO/-LL aCriteria of the Infectious Diseases Society of America/ American Thoracic Society. Pao/Fio2 = partial pressure oxygen in arterial blood/ fraction of inspired oxygen; WBC = white blood cell. Adapted from Mandell, 2007. nia is a relatively common cause of acute respiratory distress syndrome (ARDS) during pregnancy, and mechanical ventilation may become necessary (Chap. 47, p. 919). Indeed, of the 51 gravidas who required mechanical ventilation in the review by Jenkins and coworkers (2003), 12 percent had pneumonia.

1	Initial antimicrobial and antiviral treatment is empirical (Mandell, 2015). Because most adult bacterial pneumonias are caused by pneumococci, mycoplasma, or chlamydophila, monotherapy initially is with a macrolide-azithromycin, clarithromycin, or erythromycin (Table 51-4). Yost and colleagues (2000) reported that erythromycin mono therapy, given IV and then orally, was efective in all but one of 99 pregnant women with uncomplicated pneumonia. During inluenza season, we routinely administer oseltamivir treatment along with empirical therapy for bacterial pneumonia.

1	For women with severe disease according to criteria in Table 51-3, Mandell and associates (2007) summarized IDSAI ATS guidelines, which call for either: (1) a respiratory luoroquinolone-Ievofloxacin, moxifloxacin, or gemiloxacin; or (2) a macrolide plus a preferred �-lactam-either high-dose amoxicillin or amoxicillin-clavulanate. �-Lactam alternatives include ceftriaxone, cefpodoxime, or cefuroxime (see Table 51-4). In areas in which the resistance of pneumococcal isolates to macrolides is great, these latter regimens are preferred. he teratogenicity risk of luoroquinolones is low, and these should be given if indicated (Briggs, 2015). IfCA-MRSA is suspected, then vancomycin or linezolid is added (Mandell, 2015; Moran, 2013; Wunderink, 2013).

1	Clinical improvement is usually evident in 48 to 72 hours with resolution of fever in 2 to 4 days. Radiographic abnormalities may take up to 6 weeks to completely resolve (Torres, 2008). Worsening disease is a poor prognostic feature, and subsequent radiography is recommended if fever persists. Even with improvement, however, approximately 20 percent of women develop a pleural eusion. Treatment of uncomplicated pneumonia is recommended for 5 to 7 days (Musher, 2014). Treatment failure may occur in up to 15 percent of cases, and a wider antimicrobial regimen and more extensive diagnostic testing is warranted. During the preantimicrobial era, as many as a third of pregnant women with pneumonia died (Finland, 1939). Although much improved, maternl and perinatl morbidity and mortality remain formidable. In ive studies with a total of 632 women published ater 1990, almost 7 percent required intubation and mechanical ventilation, and the maternal mortality rate was 0.8 percent.

1	Prematurely ruptured membranes and preterm delivery are frequent complications and have been reported in up to a third of cases of acute lung infection (Getahun, 2007; Shariatzadeh, 2006). Likely related are older studies reporting a twofold increase in low-birthweight neonates (Sheield, 2009). In one populationbased study from Taiwan of nearly 219,000 births, incidences of preterm and growth-restricted newborns and of preeclampsia and cesarean delivery were significantly increased (Chen, 2012). Two pneumococcal vaccines, a 23-serotype older preparation and a newer 13-serotype vaccine, are used in children (Swamy, 2015). he 23-serotype vaccine is 60-to 70-percent protective, and its use lowers emergence of drug-resistant pneumococci (Kyaw, 2006). he 13-serotype vaccine is not recommended for TABLE 51-4. Empirical Antimicrobial Treatment for Community-Acquired Pneumonia Uncomplicated, otherwise health' Macrolidesb: clarithromycin or azithromycin

1	TABLE 51-4. Empirical Antimicrobial Treatment for Community-Acquired Pneumonia Uncomplicated, otherwise health' Macrolidesb: clarithromycin or azithromycin Respiratory fluoroquinolones: moxifloxacin, gemifloxacin, or levofloxacin 3-lactams: amoxicillin/clavulanate, ceftriaxone, cefotaxime, or cefuroxime plus a macrolide aUse as inpatient or outpatient regimen. bDoxycyciine may be given instead if postpartum. (See Table 51-3 for criteria. otherwise healthy pregnant women. It is, however, recommended for women who are immunocompromised, including those with HIV infection; significant smoking history; diabetes; cardiac, pulmonary, or renal disease; and asplenia, such as with sicklecell disease (Table 9-7, p. 173). Protection against pneumococcal infection in women with chronic diseases may be less eicacious than in healthy patients (Moberley, 2013).

1	Each year, 10 percent of pregnant women develop influenza (Cantu, 2013). Influenza A and B are RNA viruses that cause respiratory infection, including pneumonitis, that is epidemic in the winter months. The virus is spread by aerosolized droplets and quickly infects ciliated columnar epithelium, alveolar cells, mucus gland cells, and macrophages. Disease onset is 1 to 4 days following exposure. Common symptoms include fever, cough, myalgia, and chills (Sokolow, 2015). In most healthy adults, infection is self-limited. Pneumonia is the most frequent complication of inluenza and mimics bacterial pneumonia. According to the CDC (201 Oa), infected pregnant women are more likely to be hospitalized and admitted to an ICU. Others have corroborated these observations (Mertz, 2017). At Parkland Hospital during the 2003 to 2004 inluenza season, pneumonia developed in 12 percent of gravidas with influenza (Rogers, 2010).

1	he 2009 to 2010 pandemic with the influenza (NHINI) pdm09 strain was particularly severe (Rasmussen, 2014). In an MFMU Network study, 10 percent of pregnant or postpartum women admitted with HINI inluenza were cared for in an ICU, and 11 percent of these patients died (Varner, 201I). Risk factors included late pregnancy, smoking, and chronic hypertension. Overall, inluenza accounted for 12 percent of pregnancy-related deaths during the 2009 to 2010 pandemic (Callaghan, 2015). During the 2013 to 2014 inluenza season, a fourth of pregnant women admitted to California ICUs died (Louie, 2015). Of865 pregnant women with influenza, Oboho and coworkers (2016) noted that 7 percent of their cohort had severe disease, and four women died. If inluenza causes ARDS, extracorporeal membrane oxygenation (ECMO) may be lifesaving (Anselmi, 2015; Saad, 2016).

1	Primary influenza pneumonitis is characterized by sparse sputum production and radiographic interstitial infiltrates (Cohen, 2015). More commonly, either secondary or mixed pneumonia develop from bacterial superinfection by streptococci or staphylococci after 2 to 3 days of initial clinical improvement. he CDC (2007) reported several cases in which CA-MRSA caused influenza-associated pneumonitis with a case-fatality rate of 25 percent. Other possible adverse efects of inluenza A and B on pregnancy outcome are discussed in Chapter 64 (p. 1213).

1	Supportive treatment is recommended for uncomplicated influenza, and early antiviral treatment is efective Gamieson, 2011; Oboho, 2016). Hospitalization is considered for severely ill women, and for those with pneumonia. As discussed, inluenza hospitalization rates for those with advanced pregnancy are increased compared with nonpregnant women (Dodds, 2007; Schanzer, 2007). he CDC (2016b) recommends neuraminidase inhibitors given within 2 days of symptom onset for chemoprophylaxis and treatment of influenza A and B (Chap. 64, p. 1214). he drugs interfere with release of progeny virus from infected host cells and thus prevent infection of new host cells (Cohen, 2015). Oseltamivir is given orally, 75 mg twice daily, or zanamivir is given by inhalation, 10 mg twice daily. Recommended treatment duration with either is 5 days. The drugs shorten the course of illness by 1 to 2 days, and they probably reduce the risk for pneumonitis (Belgi, 2014; Muthuri, 2014). Our practice is to treat all

1	duration with either is 5 days. The drugs shorten the course of illness by 1 to 2 days, and they probably reduce the risk for pneumonitis (Belgi, 2014; Muthuri, 2014). Our practice is to treat all pregnant women with influenza whether or not pneumonitis is identiied (Rasmussen, 2014). Few data guide use of these agents in pregnant women, but the drugs are not teratogenic in animal studies and are considered low risk (Briggs, 2015).

1	A major potential concern for viral resistance comes from avian inluenza isolates. "Bird lu" with HPAI, H5N8, H5N2, and H5Nl isolates has been reported in the United States by the CDC Ohung, 2015). In Asia, human infection has been documented with some of these, and mortality rates are high. Preventively, vaccination for inluenza A is recommended by the American College of Obstetricians and Gynecologists (2016b) and the CDC (2016b). Vaccination is discussed in detail in Chapter 64 (p. 1214). Prenatal vaccination also afords some temporary protection for infants (Madhi, 2014; Tita, 2016). During the 2014 to 2015 lu season, the CDC reported that only half of pregnant women received influenza vaccine (Ding,a2015). Infection with varicella-zoster virus, the same agent responsible for chicken pox, results in pneumonitis in 5 percent of gravidas (Harger, 2002). Diagnosis and management are considered in Chapter 64 (p. 1212).

1	Fungal and parasitic pulmonary infections are usually of greatest consequence in immunocompromised hosts, especially in women with acquired immunodeficiency syndrome (AIDS). Of these, lung infection with Pneumoystis jiroveci, formerly called Pneumoystis carinii, is a common complication. The opportunistic fungus causes interstitial pneumonia characterized by dry cough, tachypnea, dyspnea, and difuse radiographic infiltrates. Although this organism can be identiied by sputum culture, bronchoscopy with lavage or biopsy may be necessary.

1	In an earlier report from the AIDS Clinical Trials Centers, Stratton and colleagues (1992) described pneumocystis pneumonia as the most frequent HIV-related disorder in pregnant women. In some cases, tracheal intubation and mechanical ventilation may be required. Ahmad and coworkers (200 I) reviewed 22 cases during pregnancy and cited a 50-percent mortality rate. Treatment is with trimethoprim-sulfamethoxazole for 14 to 21 days (Masur, 2015). Alternative agents include the more toxic pentamidine (Walzer, 2005). For prophylaxis, several international health agencies recommend one double-strength trimethoprim-sulfamethoxazole tablet orally daily for certain groups of HIV-infected pregnant women. These include women with CD4+ T-lymphocyte counts <2001LL, those whose CD4+ T lymphocytes constitute < 14 percent, or if there is an AIDS-defining illness, particularly oropharyngeal candidiasis (Centers for Disease Control and Prevention, 2016a).

1	Any of several fungi can cause pneumonia. In pregnancy, this is usually seen in women with HIV infection or who are oth erwise immunocompromised. Infection is usually mild and self-limited. It is characterized initially by cough and fever, and dissemination is infrequent (Mansour, 2015).

1	Histoplasmosis and blastomycosis do not appear to be more frequent or more severe during pregnancy (Youssef, 2013). Data concerning coccidioidomycosis are conlicting (Bercovitch, 2011; Patel, 2013). In a case-control study from an endemic area, Rosenstein and associates (2001) reported that pregnancy was a signiicant risk factor for disseminated disease. In another study, however, Caldwell and coworkers (2000) identified 32 serologically confirmed cases during pregnancy and documented dissemination in only three cases. Women with associated erythema nodosum have a better prognosis, whereas mediastinal lymphadenopathy may more likely relect disseminated disease (Caldwell, 2000; Mayer, 2013). Finally, Crum and Ballon-Landa (2006) reviewed 80 cases of antepartum coccidioidomycosis and found that almost all women diagnosed in the third trimester had disseminated disease. Although the overall maternal mortality rate was 40 percent, it was only 20 percent for 29 cases reported since 1973.

1	that almost all women diagnosed in the third trimester had disseminated disease. Although the overall maternal mortality rate was 40 percent, it was only 20 percent for 29 cases reported since 1973. Spinello (2007) and Bercovitch (201a1) with their colleagues have provided reviews of coccidioidomycosis in pregnancy.

1	Most cases of cryptococcosis reported during pregnancy manifest as meningitis. Otherwise healthy pregnant women occasionally have cryptococcal pneumonia (Asadi Gharabaghi, 2014; Ely, 1998). Diagnosis is diicult because clinical presentation is similar to that of other community-acquired pneumonias. Treatment. The 2007 IDSNATS guidelines recommend itraconazole as preferred therapy for disseminated fungal infections (Mandell, 2007). Pregnant women have also been given IV amphotericin B or ketoconazole (Paranyuk, 2006; Pilmis, 2015). Amphotericin B has been used extensively in pregnancy with no embryofetal efects. Because of evidence that fluconazole, itraconazole, and ketoconazole may be embryo toxic in large doses in early pregnancy, Briggs and coworkers (2015) recommend that irst-trimester use should be avoided if possible.

1	hree echinocandin derivatives-caspofongin, micaongin, and anidulafungin-are efective for invasive candidiasis (Pilmis, 2015; Reboli, 2007). They are embryotoxic and teratogenic in laboratory animals, and use in human pregnancies has not been reported (Briggs, 2015). his coronaviral respiratory infection was first identiied in China in 2002, but no new cases have been reported since 2005. It caused atypical pneumonitis with a case-fatality rate of approximately 10 percent (Dolin, 2012). SARS in pregnancy had a case-fatality rate of up to 25 percent (Lam, 2004; Wong, 2004). Ng and coworkers (2006) reported that the placentas from 7 of 19 cases showed abnormal intervillous or subchorionic ibrin deposition in three, and extensive fetal thrombotic vasculopathy in two. Tuberculosis is still a major worldwide concern. Indeed, it is estimated that a third of the world population is infected (Getahun, 2015). However, it is uncommon in the United

1	Tuberculosis is still a major worldwide concern. Indeed, it is estimated that a third of the world population is infected (Getahun, 2015). However, it is uncommon in the United States. The incidence of active tuberculosis in this country has plateaued since 2000 (Scott, 2015). More than half of active cases are in immigrants (Centers for Disease Control and Pre vention, 2009b). Persons born in the United States have newly acquired infection, whereas foreign-born persons usually have reactivation of latent infection. In this country, tuberculosis is a disease of the elderly, the urban poor, minority groups especially black Americans, and patients with HIV infection (Khan, 2013; Raviglione, 2015).

1	Infection is via inhalation of Mycobacterium tuberculosis, which incites a granulomatous pulmonary reaction. In more than 90 percent of patients, infection is contained and is dor mant for long periods (Getahun, 2015; Zumla, 2013). In some, especially those who are immunocompromised or who have other diseases, tuberculosis becomes reactivated to cause clini cal disease. Manifestations usually include cough with minimal sputum production, low-grade fever, hemoptysis, and weight loss. Various iniltrative patterns are seen on chest radiograph, ated. Acid-fast bacilli are seen on stained smears of sputum in approximately two thirds of culture-positive individuals. Forms of extrapulmonary tuberculosis include lymphadenitis, pleural, genitourinary, skeletal, meningeal, gastrointestinal, and miliary or disseminated (Raviglione, 2015).

1	Resistance to antituberculosis drugs in the United States in the early 1990s was associated with emergence of strains of multidrug-resistant tuberculosis (MDR-TB). Because of this, the CDC (2009a) now recommends a multidrug regimen for initial empirical treatment of patients with symptomatic tuberculosis. Isoniazid, rifampin, pyrazinamide, and ethambutol are given until susceptibility studies are performed (Horsburgh, 2015). Cure rates with 6-month short-course directy observed therapy-DOT -approach 90 percent for new infections (Raviglione, 2015). Other second-line drugs may need to be added. Drug susceptibility is performed on all irst isolates.

1	The considerable influx of women into the United States from Asia, Africa, Mexico, and Central America has been accompanied by an increased frequency of tuberculosis in pregnancy. Sackof and coworkers (2006) reported positive tuberculin test results in half of 678 foreign-born women attending prenatal clinics in New York City. Almost 60 percent were newly diagnosed. Pillay and colleagues (2004) stress the prevalence of tuberculosis in HIV-positive pregnant women. At Jackson Memorial Hospital in Miami, Schulte and associates (2002) reported that 21 percent of 207 HIV-infected pregnant women had a positive skin test result. Recall also that silent endometrial tuberculosis can cause tubal infertility (Levison, 2010; Raviglione, 2015).

1	Without therapy, active tuberculosis appears to have adverse efects on pregnancy (Mnyani, 2011). Several studies indicate that outcomes are dependent on the site of infection and gestational age at diagnosis. J ana and colleagues (1994) from India and Figueroa-Damian and Arrendondo-Garcia (1998) from Mexico City noted that active pulmonary tuberculosis was associated with increased incidences of preterm delivery, low-birthweight and growth-restricted newborns, and perinatal mortality. Others have found similar efects (EI-Messidi, 2016; Lin, 2010; Sobhy, 2017). From her review, Eferen (2007) cited twofold greater rates of low birthweight, preterm delivery, and preeclampsia. The perinatal mortality rate was increased almost tenfold. Adverse outcomes correlate with late diagnosis, incomplete or irregular treatment, and advanced pulmonary lesions. Conversely, treated tuberculosis is associated with good pregnancy outcomes (Nguyen, 2014; Taylor, 2013).

1	Extrapulmonary tuberculosis is less common. Jana and coworkers (1999) reported outcomes in 33 pregnant women with renal, intestinal, and skeletal tuberculosis, and a third had low-birthweight newborns. Llewelyn and associates (2000) reported that nine of 13 pregnant women with extrapulmonary disease had delayed diagnoses. Prevost and Fung Kee Fung (I999) reviewed 56 cases of tuberculous meningitis in which a third of mothers died. Spinal tuberculosis may cause paraplegia, but vertebral fusion may prevent it from becoming permanent (Badve, 2011; Nanda, 2002). Psoas abscess develops in 5 percent of those with spinal infections (Nigam, 2013). Other presentations include widespread intraperitoneal tuberculosis simulating ovarian carcinomatosis and degenerating leiomyoma, and hyperemesis gravidarum from tubercular meningitis (Kutiu, 2007; Moore, 2008; Sherer, 2005).

1	Two types of tests are used to detect latent or active tuberculosis. One is the time-honored tuberculin skin test (TSj) and the others are inteeron-gamma release assays IGRAs)} which are becoming preferred (Getahun, 2015; Horsburgh, 2011). IGRAs are blood tests that measure interferon-gamma release in response to antigens present in M tuberculosis, but not bacille Calmette-Guerin (BCG) vaccine (Levison, 2010). he CDC (2005b, 2010b) recommends either skin testing or IGRA testing of gravidas who are in any of the high-risk groups. For those who have received BCG vaccination, IGRA testing is used (Mazurek, 2010). For skin testing, the preferred antigen is purified protein derivative (PPD) of intermediate strength of 5 tuberculin units. If the intracutaneously applied test result is negative, no further evaluation is needed. A positive skin test result measures �5 mm in diameter and requires evaluation for active disease, including a chest radiograph.

1	Two IGRAs are available: QuantiFERON-TB Gold and T-SPOT TB tests are recommended by the CDC (2005a,b) for the same indications as skin testing. Although these tests have not been evaluated as extensively as tuberculin skin testing, Kowada (2014) concluded that the tests are cost efective. Other essential laboratory methods for detection or verification of infection-both active and latent-include microscopy, culture, nucleic acid amplification assay, and drug-susceptibility testing (Horsburgh, 2015; Raviglione, 2015).

1	Latent Infection. In nonpregnant tuberculin-positive patients with latent infection who are younger than 35 years and who have no evidence of active disease, isoniazid, 300 mg orally daily, is given for 9 months. Isoniazid has been used for decades, and it is considered safe in pregnancy (Briggs, 2015; Taylor, 2013). Compliance is a major problem, and Sackof (2006) and Cruz (2005) and their associates reported a disappointing 10-percent treatment completion. One obvious disconnect is that care for tuberculosis is given in diferent health systems than prenatal care (Zenner, 2012). hese observations are important because most recommend that isoniazid therapy be delayed until ater delivery. Because of possibly increased isoniazid-induced hepatitis risk in postpartum women, some even recommend withholding treatment until 3 to 6 months ater delivery. hat said, neither method is as efective as antepartum treatment to prevent active infection. Boggess and colleagues (2000) reported that only

1	withholding treatment until 3 to 6 months ater delivery. hat said, neither method is as efective as antepartum treatment to prevent active infection. Boggess and colleagues (2000) reported that only 42 percent of 167 tuberculin-positive asymptomatic women delivered at San Francisco General Hospital completed 6-month therapy that was not begun until the irst postpartum visit.

1	There are exceptions to delayed treatment for latent infection in pregnancy. Known recent skin-test convertors are treated antepartum because the incidence of active infection is 5 percent in the first year (Zumla, 2013). Skin-test-positive women exposed to active infection are also treated because the incidence of infection is 0.5 percent per year. Finally, HIVpositive women are treated because they have an approximate 10-percent annual risk of active disease.

1	Active Infection. Recommended initial treatment for active tuberculosis in pregnant women is a four-drug regimen with isoniazid, rifampin, ethambutol, and pyrazinamide, along with pyridoxine. For meningitis, levoBoxacin may be added (Kalita, 2014). In the irst 2-month phase, all four drugs are givenbactericidal phase. his is followed by a 4-month phase of isoniazid and rifampin-continuation phase (Raviglione, 2015; Zumla, 2013). A few reports describe MDR-TB during pregnancy, and treatment options have been reviewed (Horsburgh, 2015; Lessnau, 2003). Breastfeeding is not prohibited during antituberculous therapy.

1	Treatment of active disease is of special concern if there is antiretroviral naivete. In these circumstances, beginning concomitant therapy with antituberculosis and antiretroviral therapy can cause the immune reconstitution inlammatory syndrome IRIS) with toxic drug efects (Lai, 2016; Torok, 2011). hat said, recent studies support earlier administration of highly active antiretroviral therapy (HAART)-within 2 to 4 weeksafter beginning antituberculosis therapy (Blanc, 2011; Havlir, 2011; Karim, 2011). lso, for HIV-infected women, rifampin or rifabutin use may be contraindicated if certain protease inhibitors or nonnucleoside reverse transcriptase inhibitors are being administered. If there is resistance to rifabutin or rifampin, then pyrazinamide therapy is given. Of the secondline regimens, the aminoglycosides-streptomycin, kanamycin, amikacin, and capreomycin-are ototoxic to the fetus and are contraindicated (Briggs, 2015).

1	Neonatal Tuberculosis. Tubercular bacillemia can infect the placenta, but the fetus infrequently becomes infected-con genital tuberculosis. The term also applies to newborns who are infected by aspiration of infected secretions at delivery. Each route of infection constitutes approximately half of the cases. manifests with hepatosplenomegaly, respiratory distress, fever, and lymphadenopathy (Dewan, 2014; Osowicki, 2016). Cantwell and associates (1994) reviewed 29 cases of congenital tuberculosis reported since 1980. Only 12 of the mothers had active infection, and tuberculosis was frequently demonstrated by postpartum endometrial biopsy. Adhikari and colleagues (1997) described 11 South African postpartum women whose endometrial biopsy was culture-positive. Six of their neonates had congenital tuberculosis.

1	Neonatal infection is unlikely if the mother with active disease has been treated before delivery or if her sputum culture is negative. Because the newborn is susceptible to tuberculosis, most experts recommend isolation from the mother suspected of having active disease. If untreated, the risk of disease in the infant born to a woman with active infection is 50 percent in the irst year Qacobs, 1988).

1	his is a chronic, multisystem inflammatory disease of unknown etiology characterized by an accumulation of T-helper lymphocytes and phagocytes within noncaseating granulomas (Baughman, 2015; Celada, 2015). Predisposition to the disease is genetically determined and characterized by an exaggerated response of helper T lymphocytes to environmental triggers. Pulmonary involvement is most common, followed by skin, eyes, lymph nodes, and then all other organ systems. The prevalence of sarcoid in the United States is 20 to 60 per 100,000, with equal sex distribution. It is more than 10 times more common for blacks than for whites (Baughman, 2015). Most patients are between 20 and 40 years. Clinical presentation varies, but more than half of patients have dyspnea and a dry cough without constitutional symptoms that develop insidiously over months. Disease onset is abrupt in approximately 25 percent of patients, and 10 to 20 percent are asymptomatic at discovery.

1	Pulmonary symptoms are dominant, and more than 90 percent of patients have an abnormal chest radiograph at some point. Interstitial pneumonitis is the hallmark of pulmonary involvement, and half of afected patients develop permanent radiological changes. Lymphadenopathy, especially of the mediastinum, is present in 75 to 90 percent of cases. A fourth each have uveitis and skin involvement, the latter usually manifest as eythema nodosum. In women, sarcoid causes approximately 10 percent of cases of erythema nodosum (Mert, 2007). Impor tantly, any other organ system may be involved (Kandolin, 2015; Powe, 2015; Wallmiiller, 2012). Conirmation of the diagnosis is with biopsy-preferably a lymph node. However, because the lung may be the only obviously involved organ, tissue acquisition is often diicult.

1	The overall prognosis for sarcoidosis is good, and it resolves without treatment in 50 percent of patients. Still, quality of life is diminished (de Vries, 2007). In the other 50 percent, permanent organ dysfunction, albeit mild and nonprogressive, persists. Approximately 10 percent die because of their disease. Glucocorticoids are the most widely used treatment for symptomatic disease. Permanent organ derangement is seldom reversed by their use (Paramothayan, 2002). Thus, the deci sion to treat is based on symptoms, physical indings, chest radiograph, and pulmonary function tests. Unless respiratory symptoms are prominent, therapy is usually withheld for a several-month observation period. If inlammation does not subside, then prednisone, 1 mg/kg, is given daily and tapered to < 10 mg by 6 months (Baughman, 2015). For those with an inadequate response, immunosuppressive or cytotoxic agents and cytokine modulators can be used.

1	Because it is uncommon and frequently benign, sarcoidosis is not often seen in pregnancy. Although it seldom afects pregnancy adversely, meningitis, heart failure, and neurosarcoidosis have been described (Cardonick, 2000; Maisel, 1996; Wallmiiller, 2012). In a study of the Nationwide Inpatient Sample of 678 cases of sarcoidosis in pregnancy, incidences of preeclampsia, preterm delivery, and thromboembolism were increased (Hadid, 2015). Selroos (1990) studied 252 women with sarcoidosis in Finland, and 15 percent had sarcoidosis during pregnancy. Disease did not progress in the 26 pregnancies in women with active disease. Three aborted spontaneously, and the other 23 women were delivered at term. Agha and coworkers (1982) reported similar experiences with 35 pregnancies at the University of Michigan.

1	Active sarcoidosis is treated using the same guidelines as for the woman who is not pregnant. Severe disease warrants serial determination of pulmonary function. Symptomatic uveitis, constitutional symptoms, and pulmonary symptoms are treated with prednisone, 1 mg/kg orally per day.

1	This autosomal recessive exocrinopathy is one of the most common fatal genetic disorders in whites. Cystic ibrosis is caused by one of more than 2000 mutations in a 230-kb gene on the long arm of chromosome 7 that encodes an amino acid polypeptide (Patel, 2015; Sorscher, 2015). his peptide functions as a chloride channel and is termed the cystic ibrosis transmembrane conductance regulator (CFTR). As discussed in Chapter 14 (p. 289), phenotypes vary widely, even among homozygotes for the common 6F508 mutation (Rowntree, 2003). Approximately 10 to 20 percent of afected newborns are diagnosed shortly after birth because of meconium peritonitis (Boczar, 2015; Sorscher, 2015). Currently, the median predicted survival is 37 years, and nearly 80 percent of females with cystic ibrosis now survive to adulthood (Gillet, 2002; Patel, 2015).

1	Mutations in the chloride channel cause altered epithelial cell membrane transport of electrolytes. his afects all sites in which epithelium expresses CFTR-secretory cells. These include the sinuses, lung, pancreas, liver, and reproductive tract. Disease severity depends on which two alleles are inherited, and approximately 10 percent are disease-causing mutations (Sorscher, 2015). Homozygosity for Phe508del (�F508) is one of the most severe, and 90 percent of individuals with clinical disease carry at least one F508 allele. Exocrine gland ductal obstruction develops from thick, viscid secretions (Rowe, 2005). In the lung, submucosal glandular ducts are afected. Eccrine sweat gland abnormalities are the basis for the diagnostic sweat test, characterized by elevated sodium, potassium, and chloride levels in sweat.

1	Lung involvement is commonplace and is usually the cause of death. Bronchial gland hypertrophy with mucous plugging and small-airway obstruction leads to subsequent infection that ultimately causes chronic bronchitis and bronchiectasis. For complex and not completely explicable reasons, chronic inflammation from Pseudomonas aeruginosa occurs in more than 90 percent of patients. In a minority,aS aureus, H inluenzae, and Burkholderia cepacia are recovered (Rowe, 2005). Colonization with the last has been reported to signiy a worse prognosis, especially in pregnancy (Gillet, 2002). Acute and chronic parenchymal inlammation ultimately causes extensive fibrosis, and along with airway obstruction, ventilation-perfusion mismatch develops. Pulmonary insuiciency is the end result. Lung or heart-lung transplantation has a 5-year survival rate of only 50 to 60 percent (Sorscher, 2015). A few women have successfully undergone pregnancy following lung transplantation (Kruszka, 2002; Shaner, 2012).

1	This CFTR2 resource delineates gene variants with a clear etiological role-http://ww.cftr2.org. Women with clinical cystic fibrosis are sub fertile because of tenacious cervical mucus. Males have oligospermia or aspermia from vas deferens obstruction, and 98 percent are infertile (hmad, 2013). Despite this, the North American Cystic Fibrosis Foundation estimated that 4 percent of afected women become pregnant every year (Edenborough, 1995). he endometrium and tubes express some CFTR but are functionally normal, and the ovaries do not express the CFTR gene (Eden borough, 2001). Both intrauterine insemination and in vitro fertilization can be successful for afected women (Rodgers, 2000). Several ethical considerations regarding plans for pregnancy by these women were reviewed by Wexler and colleagues (2007). One important factor is the long-term prognosis for the mother. For male infertility, Sobczynska-Tomaszewska and associates (2006) have emphasized the importance of molecular

1	and colleagues (2007). One important factor is the long-term prognosis for the mother. For male infertility, Sobczynska-Tomaszewska and associates (2006) have emphasized the importance of molecular diagnosis.

1	he American College of Obstetricians and Gynecologists (20a17) recommends that carrier screening be ofered to all women currently pregnant or considering conception (Chap. 14, p. 289). The CDC also added cystic ibrosis to newborn screening programs (Southern, 2009) (Chap. 32, p. 614).

1	Pregnancy outcome is inversely related to severity of lung dysfunction. Advanced chronic lung disease, hypoxia, and frequent infections may prove deleterious. At least in the past, cor pulmonale was common, but even that does not preclude successful pregnancy (Cameron, 2005). In some women, pancreatic dyunction may cause poor maternal nutrition. Otherwise normal pregnancy-induced insulin resistance frequently results in gestational diabetes after midpregnancy (Hardin, 2005). In one study of 48 pregnancies, half had pancreatic insuiciency and a third required insulin (horpe-Beeston, 2013). Up to 25 percent of patients develop diabetes by age 20, and diabetes is most frequent with the Phe508del homozygous mutation (Giacobbe, 2012; Patel, 2015).

1	Cystic fibrosis per se is not afected by pregnancy (Schechter, 2013). Early reports of a deleterious efect on the course of cystic fibrosis were related to severe disease (Olson, 1997). When matched with nonpregnant women by disease severity, recent reports indicate no deleterious efects on long-term survival (Schechter, 2013). Prep regnancy counseling is imperative. Women who choose to become pregnant require close surveillance for development of superimposed infection, diabetes, and heart failure. Serial pulmonary function testing assists management and estimating prognosis. When the FEV] is at least 70 percent, women usually tolerate pregnancy well. Emphasis is placed on postural drainage, bronchodilator therapy, and infection control.

1	�-Adrenergic bronchodilators help control airway constriction. Inhaled recombinant human deoxyribonuclease I improves lung function by reducing sputum viscosity (Sorscher, 2015). Inhaled 7 -percent saline produces short-and long-term benefits (Elkins, 2006). Nutritional status is assessed and appropriate dietary counseling given. Pancreatic insuiciency requires replacement of oral pancreatic enzymes. Promising new therapy to correct CFTR protein dysfunction was recently described by Wainwright and colleagues (2015). Using a combination of lumacaftor and ivacaftor, these investigators showed that patients homozygous for the Phe508del mutation were significantly benefitted. No reports of either drug are available regarding pregnant women.

1	Infection is heralded by increasing cough and mucus production. Oral semisynthetic penicillins or cephalosporins usually suice to treat staphylococcal infections. Pseudomonas infection is problematic, and inhaled tobramycin and colistin have been used successfully to control this organism. Immediate hospitalization and aggressive therapy are warranted for serious pulmonary infections. he threshold for hospitalization with other complications is low. For labor and delivery, epidural analgesia is recommended (Deighan, 2014).

1	Earlier reports chronicled the poor maternal and perinatal outcomes of women with cystic ibrosis (Cohen, 1980; Kent, 1993). vlore recent reports describe better outcomes, but there still are serious complications. Disease severity is now quantified by pulmonary function studies, which are the best predictor of pregnancy and long-term maternal outcome. Edenborough and colleagues (2000) reported 69 pregnancies and found that if prep regnancy FEV! was <60 percent of predicted, the risk for preterm delivery, respiratory complications, and death of the mother within a few years of childbirth was substantive. Thorpe-Beeston (2013) and Fitzsimmons (1996) and their associates reported similar indings. Gillet and colleagues (2002) reported 75 pregnancies from the French Cystic Fibrosis Registry. Almost 20 percent of newborns were delivered preterm, and 30 percent had growth restriction. he one maternal death was due to Pseudomonas sepsis in a woman whose prepregnancy FEV! was 60 percent.

1	Almost 20 percent of newborns were delivered preterm, and 30 percent had growth restriction. he one maternal death was due to Pseudomonas sepsis in a woman whose prepregnancy FEV! was 60 percent. Long-term, however, 17 percent of women died, and four infants had conirmed cystic fibrosis.

1	Maternal complications are daunting. Patel and coworkers (2015) recently queried the National Inpatient Sample database and reported that the prevalence of cystic ibrosis in pregnancy had a signiicant linear increase from 2000 to 2010. They analyzed 1119 afected women in more than 12 million births and reported a litany of risks (Table 51-5). In contrast, perinatal outcomes were surprisingly good. Cystic fibrosis is a common antecedent disease leading to lung transplantation. Gyi and coworkers (2006) reviewed 10 pregnancies in such women and reported nine liveborn neonates. Maternal outcomes were less favorable. Of the three gravidas who developed rejection during pregnancy, all had progressively declining pulmonary function and died of chronic rejection by 38 months after delivery. TABLE 51-5. Odds Ratios for Maternal Complications in 11e19 Pregnant Women with Cystic Fibrosis Compared with Controls

1	TABLE 51-5. Odds Ratios for Maternal Complications in 11e19 Pregnant Women with Cystic Fibrosis Compared with Controls Carbon monoxide is a ubiquitous gas, and most nonsmoking adults have a carbon monoxyhemoglobin saturation of 1 to 3 per cent. In cigarette smokers, levels may be as high as 5 to 10 per cent. Carbon monoxide is the most frequent cause of poisoning worldwide (Stoller, 2007). Toxic levels are oten encountered in inadequately ventilated areas warmed by space heaters.

1	Carbon monoxide is particularly toxic because it is odorless and tasteless and has a high ainity for hemoglobin binding. hus, it displaces oxygen and impedes its transfer with resultant hypoxia. Besides acute sequelae including death and anoxic encephalopathy, cognitive defects develop in as many as half of patients following loss of consciousness or in those with carbon monoxide levels >25 percent (Weaver, 2002). Hypoxic brain damage has a predilection for the cerebral cortex and white matter and for the basal ganglia (Lo, 2007; Prockop, 2007). A Parkinson syndrome sometimes follows after recovery (Hemphill,a2015).

1	Through several physiological alterations, the rate of endogenous carbon monoxide production almost doubles in normal pregnancy (Longo, 1977). Although the pregnant woman is not more susceptible to carbon monoxide poisoning, the fetus does not tolerate excessive exposure (Friedman, 2015). With chronic exposure, maternal symptoms usually appear when the carboxyhemoglobin concentration is 5 to 20 percent. Symptoms include headache, weakness, dizziness, physical and visual impairment, palpitations, and nausea and vomiting. With acute exposure, concentrations of 30 to 50 percent produce symptoms of impending cardiovascular collapse. Levels > 50 percent may be fatal for the mother.

1	Because hemoglobin F has an even higher ainity for carbon monoxide, fetal carboxyhemoglobin levels are 10 to 15 percent higher than those in the mother. This may be due to facilitated difusion (Longo, 1977). Importantly, the half-life of carboxyhemoglobin is 2 hours in the mother but 7 hours in the fetus. Because carbon monoxide is bound so tightly to hemoglobin F, the fetus may be hypoxic even before maternal carbon monoxide levels are appreciably elevated. Several anomalies are associated with embryonic exposure, and anoxic encephalopathy is the primary sequela of later fetal exposure (Alehan, 2007; Aubard,a2000).

1	For all victims, treatment of carbon monoxide poisoning is supportive along with immediate administration of 100-percent inspired oxygen. Indications for hyperbaric oxygen treatment in nonpregnant individuals are unclear (Kao, 2005). Weaver and associates (2002) reported that hyperbaric oxygen treatment minimized the incidence of cognitive defects in adults at both 6 weeks and 1 year compared with that with normobaric oxygen. Hyperbaric oxygen is generally recommended in pregnancy if carbon monoxide exposure has been "significant" (Aubard, 2000; Ernst, 1998). The problem is how to deine signiicant exposure (Friedman, 2015). Although maternal carbon monoxide levels are not accurately predictive of those in the fetus, some clinicians recommend hyperbaric therapy if maternal levels exceed 15 to 20 percent. With fetal heart rate pattern evaluation, Towers and Corcoran (2009) described afected fetuses to have an elevated baseline, diminished variability, and absent accelerations and

1	15 to 20 percent. With fetal heart rate pattern evaluation, Towers and Corcoran (2009) described afected fetuses to have an elevated baseline, diminished variability, and absent accelerations and decelerations. Treatment of the afected newborn with hyperbaric oxygen is also controversial (Bar, 2007).

1	Elkharrat and colleagues (1991) reported successful hyperbaric treatments in 44 pregnant women. Silverman and Montano (1997) reported successful management of a woman whose abnormal neurological and cardiopulmonary indings abated in a parallel fashion with resolution of associated variable decelerations in fetal heart rate. Greingor and coworkers (2001) used 2.5-atm hyperbaric 100-percent oxygen for 90 minutes in a 21-week pregnant woman who was delivered of a healthy infant at term. According to the Divers Alert Network-DAN (201a6)-at Duke University, 700 chambers are located in North and Central America. Emergency consultation from the Network is available at 91a9-684-91a11a. Adhikari M, Pillay T, Pillay DG: Tuberculosis in the newborn: an emerging disease. Pediatr Infect Dis J 16: 1108, 1997 Agha FP, Vade A, Amendola A, et al: Efects of pregnancy on sarcoidosis. Surg Gynecol Obstet 155:817,t1982

1	Agha FP, Vade A, Amendola A, et al: Efects of pregnancy on sarcoidosis. Surg Gynecol Obstet 155:817,t1982 Ahmad A, Ahmed A, Patrizio P: Cystic fibrosis and fertility. Curr Opin Obstet GynecoIt25(3):167,t2013 Ahmad H, Mehta NJ, Manikal VM, et al: Pneumoystis carinii pneumonia in pregnancy. Chest 120:666, 2001 Alehan F, Erol I, Onay OS: Cerebral palsy due to nonlethal maternal carbon monoxide intoxication. Birth Defects Res A Clin Mol Teratol 79(8):614, 2007 Ali Z, Ulrik CS: Incidence and risk factors for exacerbations of asthma during pregnancy. J Asthma Allergy 6:53, 2013 American College of Obstetricians and Gynecologists: Asthma in pregnancy. Practice Bulletin No. 90, February 2008, Reairmed 2016a American College of Obstetricians and Gynecologists: Inluenza vaccination during pregnancy. Committee Opinion No. 608, September 2014, Reaffirmed 2016b

1	American College of Obstetricians and Gynecologists: Inluenza vaccination during pregnancy. Committee Opinion No. 608, September 2014, Reaffirmed 2016b American College of Obstetricians and Gynecologists: Carrier screening for genetic conditions. Committee Opinion No. 691, March 2017 Andrews W: Cesarean delivety for refractory status asthmaticus. Obstet Gynecol 121:417,t2013 Anselmi A, Ruggieri VG, Letheulle J, et al: Extracorporeal membrane oxygenation in pregnancy. J Card Surg 30(10):781,t2015 Asadi Gharabaghi M, llameh SF: Primary pulmonary cryptococcosis. BMJ Case Rep 2014:pii: bcr2014203821, 2014 Aubard Y, Magne I: Carbon monoxide poisoning in pregnancy. BJOG 107:833, 2000 Badve SA, Ghate SO, Badve MS, et al: Tuberculosis of spine with neurological deicit in advanced pregnancy: a report of three cases. Spine J 11 (1):e9, 2011 Bain E, Pierides KL, Clifton VL, et al: Interventions for managing asthma in pregnancy. Cochrane Database Syst Rev 10:CD010660, 2014

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1	Varner MW, rice MM, Anderson B, et al: Inluenza-like illness in hospitalized pregnant and postpartum women during the 2009-20120 HI N 1 pandemic. Obstet Gynecol 118(3),t2011 Wainwright CE, Elborn ]5, Ramsey BW, et al: Lumacaftor-ivacaftor in patients with cystic ibrosis homozygous for Phe508del CFTR. N Engl ] Med 3:220,t2015 Wallmiiller e, Domanovits H, Mayr FB, et al: Cardiac arrest in a 35-year-old pregnant woman with sarcoidosis. Resuscitation 83(6)eI51, 2012 Walzer PO: Pneumocystis infection. In Kasper DL, Fauci AS, Longo DL, et al (eds): Harrison's Principles of Internal Medicine, 16th ed. New York, McGraw-Hill, 2005, p 1194 Wang G, Murphy VE, Namazy ], et al: he risk of maternal and placental complications in pregnant women with asthma: a systematic review and meta-analysis. J Matern Fetal Neonatal Med 27(9):934-42,t2014 Weaver LK, Hopkins RO, Chan K], et al: Hyperbaric oxygen for acute carbon monoxide poisoning. N Englt] Med 347: ,t2002

1	Weaver LK, Hopkins RO, Chan K], et al: Hyperbaric oxygen for acute carbon monoxide poisoning. N Englt] Med 347: ,t2002 Wendel P], Ramin SM, Hamm CB, et al: Asthma treatment in pregnancy: a randomized controlled study. Am] Obstet Gynecol 175: 150, 1996 Wenzel RP, Fowler A 3rd: Acute bronchitis. N Engl ] Med 355:2125, 2006 Wexler 10, ]ohnnesson M, Edenborough FP, et al: Pregnancy and chronic progressive pulmonary disease. Amt] Respir Crit Care Med 175:330, 2007 Wise A, Polito A], Krishnan V: Respiratory physiologic changes in preg nancy. Immunol Allergy Clin North Am 26:1, 2006 Wong SF, Chow M, Leung TN, et al: Pregnancy and perinatal outcomes of women with severe acute respiratory syndrome. Am ] Obstet Gynecol 191: 292,t2004 Wunderink RG: How important is methicillin-resistant Staphylococcus aureus as a cause of community-acquired pneumonia and what is best antimicrobial therapy? Infect Dis Clin North Am 27(1):17 ,2013

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1	Zenner 0, Kruijshaar ME, Andrews N, et al: Risk of tuberculosis in pregnancy: a national, primary care-based cohort and self-controlled case series study. Amt] Respir Crit Care Med 9,t2012 Zumla A, Raviglione M, Hafner R, et al: Tuberculosis. N Engl] Med 368:745, 2013 PATHOPHYSIOLOGY .........................i. 1004 THROMBOPHILIAS ............i..i........i..... 1005 THROMBOPHILIA SCREENING . . . . . . . . . . . . . . . .... 1009 DEEP-VEIN THROMBOSIS ..................i..... 1010 LABOR AND DELIVERY . . . . . . . . . . . . . . . . . . . . . .... 1014 SUPERFICIAL VENOUS THROMBOPHLEBITIS ...i..... 1016 PULMONARY EMBOLISM ..................i..... 1016 THROMBOPROPHYLAXIS ..................i.i... 1019 he patient complains of intense and sudden precordial pain, becomes livid in appearance, and presents symptoms of proound dyspnea and eventualy of air hunger. hese embolisms, however, are not always atal, a smal proportion of the patients recovering. The treatment is purey palliative.

1	-J. Whitridge Williams (1903) During the past century, the frequency of venous thromboembolism (VTE) during the puerperium decreased remarkably as early ambulation became more widely practiced. Despite this and other advances in prevention and treatment, however, thromboembolism remains a leading cause of maternal morbidity and mortality. Indeed, thrombotic pulmonary embolism accounted for 9.2 percent of pregnancy-related deaths in the United States between 2011 and 2013 (Creanga, 2017).

1	he absolute incidence ofVTE during pregnancy is low-1 or 2 cases per 1000 pregnancies. However, the risk is approximately ive times higher than that among women who are not pregnant (Greer, 2015). Approximately equal numbers of cases are identiied antepartum and in the puerperium. Deep-vein thrombosis alone is more frequent antepartum, and pulmonary embolism is more common in the first 6 weeks postpartum (Jacobsen, 2008). During the puerperium, the estimated incidence of a thromboembolic complication is 22 events per 100,000 deliveries. Although still elevated, the risk falls to approximately 3 cases per 100,000 deliveries during the second 6-week postpartum period (Kamel, 2014).

1	Rudolf Virchow (1856) postulated that stasis, local trauma to the vessel wall, and hypercoagulability predisposed to venous thrombosis. During normal pregnancy, the risk for each of these rises. Compression of the pelvic veins and inferior vena cava by the enlarging uterus renders the lower extremity venous system particularly vulnerable to stasis. From their review, Marik and Plante (2008) cite a 50-percent reduction in venous Row velocity in the legs that lasts from the early third trimester until 6 weeks postpartum. This stasis is the most constant predisposing risk factor for venous thrombosis. Venous stasis and delivery may also contribute to endothelial cell injury. Last, as listed in the Appendix (p. 1256), the synthesis of most clotting factors is markedly enhanced during pregnancy and favors coagulation.

1	Characteristics for developing thromboembolism during pregnancy are shown in Table 52-1. he most important of these is a personal history of thrombosis. Speciically, 15 to 25 percent of all VTE cases during pregnancy are recurrent events (American College of Obstetricians and Gynecologists, 20 17b). In one study, the magnitude of other risks was estimated from 7177 VTE cases during pregnancy and 7158 events during the postpartum period (James, 2006). Calculated risks for thromboembolism were approximately doubled in women with multifetal gestation, anemia, hyperemesis, hemorrhage, and cesarean

1	TABLE 52-1. Some Risk Factors Associated with an Increased Risk for Thromboembolism delivery. he risk was even greater in pregnancies complicated by postpartum infection. Waldman and associates (2013) found that the risk of VTE was slightly higher in women with advanced maternal age and approximately doubled in women with great parity, a hypertensive disorder, cesarean delivery, or obesity. Risks were signiicantly higher among women who had a stillbirth or who underwent peripartum hysterectomy. he next most important individual risk factor is a genetically determined thrombophilia. An estimated 20 to 50 percent ofwomen who develop a venous thrombosis during pregnancy or postpartum have an identiiable underlying genetic disorder (American College ofObstetricians and Gynecologists, 2017b).

1	Several important regulatory proteins act as inhibitors in the coagulation cascade (Fig. 52-1). Normal values for many of these proteins during pregnancy are found in the Appendix (p. 1256). Inherited or acquired deficiencies of these inhibitory proteins are collectively referred to as thrombophilias. These can lead to hypercoagulability and recurrent TE (Connors, 2017). lthough these disorders are collectively present in approximately 15 percent ofwhite European populations, they are responsible for approximately 50 percent of all thromboembolic events during pregnancy (Lockwood, 2002; Pierangeli, alncludes May-Thurner syndrome (iliac vein compression syndrome).

1	Prothrombin------....Thrombin---....Coagulation Factor V Leiden mutation Factor V resistant to degradation by protein C\\\,, "0''� Inactivates factor Va PROTEINS . /" IIIII Inactivates factor Villa Thrombin binds to thrombomodulin on endothelial cells Protein C deficiencyIIIIII tProtein S deficiency -; ActivatedproteinC ProteinCII Hyperhomocysteinemia (inhibits activation of protein C) FIGURE 52-1 Inherited thrombophilias and their efect(s) on the coagulation cascade. aOdds ratio for pregnancy-associated TE compared with gravid noncarriers. Data from the American College of Obstetricians and Gynecologists, 2017c; Croles, 2017. 2011). Some aspects of the more common inherited thrombophilias are summarized in Table 52-2.

1	Data from the American College of Obstetricians and Gynecologists, 2017c; Croles, 2017. 2011). Some aspects of the more common inherited thrombophilias are summarized in Table 52-2. Patients with inherited thrombophilic disorders often have a family history of thrombosis. Inherited thrombophilias are also found in up to half of all patients who present with VTE before the age of 45 years, particularly in those whose event occurred in the absence of well-recognized risk factors. Of greatest significance is a family history of sudden death due to pulmonary embolism or a history of multiple family members requiring long-term anticoagulation therapy because of recurrent thrombosis (Anderson, 2011).

1	Synthesized in the liver, antithrombin is one of the most important inhibitors of thrombin and inactivates thrombin and factor Xa (heaume, 2016). Notably, the rate of antithrombin interaction with its target is accelerated by heparin (Anderson, 2011). Antithrombin deiciency may result from hundreds of diferent mutations that are almost always autosomal dominant. Type I deficiency results from reduced synthesis of biologically normal antithrombin, and type II deficiency is characterized by normal levels of antithrombin with reduced functional activity (Anderson, 2011). Homozygous antithrombin deficiency is lethal.

1	Antithrombin deiciency is rare-it afects approximately 1 in 500 to 5000 individuals (Ilonczai, 2015; Rheaume, 2016). It is the most thrombogenic of the heritable coagulopathies. Indeed, antithrombin deiciency is associated with a 25-to 50-fold higher relative risk of VTE in the general population and a sixfold increased risk of thromboembolic complications during pregnancy (Ilonczai, 2015). Those afected have approximatelyaa 50-percent lifetime risk ofVTE (Duhl, 2007).

1	Sabadell and associates (2010) studied the outcomes of 18 pregnancies complicated by antithrombin deiciency. Twelve of these women were treated with therapeutic doses of lowmolecular-weight heparin (LMWH), and six were not treated because antithrombin deiciency had not yet been diagnosed. Three of the untreated patients sufered a thromboembolic episode compared with none in the treated group. Untreated women also had a 50-percent risk of stillbirth and fetal-growth restriction. By comparison, none of the treated women had a stillbirth, but a fourth developed fetal-growth restriction. Similar results were reported by Ilonczai and colleagues (2015). Garda-Botella and associates (2016) described a mesenteric vein thrombosis in a pregnant woman with antithrombin deiciency. In one review of outcomes in 23 newborns with antithrombin deficiency, there were 11 cases of thrombosis and 10 infant deaths (Seguin, 1994).

1	Given such risk, afected women are treated during pregnancy with heparin regardless of whether they have had a prior thrombosis. When anticoagulation is necessarily withheld, such as during surgery or delivery, Paidas and colleagues (2016) found that treatment with recombinant human antithrombin protected against VTE development. Sharpe and associates (201a1) described successful use of antithrombin concentrate infusions plus therapeutic anticoagulation in a pregnant woman with antithrombin deficiency who developed a thrombosis during the third trimester despite therapeutic LMWH.

1	When thrombin is bound to thrombomodulin on endothelial cells of small vessels, its procoagulant activities are neutralized. his binding also activates protein C, a natural anticoagulant that in the presence of protein S controls thrombin generation, in part, by inactivating factors Va and VIlla (see Fig. 52-1). Protein C activity increases modestly but significantly throughout the first half of pregnancy, and some have speculated that this augmentation may play a role in maintaining early pregnancy through both anticoagulant and inflammatory regulatory pathways (Said, 2010b).

1	More than 160 diferent autosomal dominant mutations for the protein C gene have been described (Louis-Jacques, 2016). he prevalence of protein C deiciency is 2 to 3 per 1000, but many of these individuals do not have a thrombosis history because the phenotypic expression is highly variable (Anderson, 2011). hese prevalence estimates correspond with functional activity threshold values of 50 to 60 percent, which are used by most laboratories and which are associated with a six-to 12-fold higher risk for VTE (Lockwood, 2012).

1	his circulating aticoagulant is activated by protein C, which enhances the capacity of protein S to inactivate factors Va and VIlla (see Fig. 52-1). Protein S deiciency may be caused by more than 130 diferent mutations, with an aggregate prevalence of approximately 0.3 to 1.3 per 1000 individuals (LouisJacques, 2016). Protein S deiciency may be measured by antigenically determined free, functional, and total S levels. ll three decline substantively during normal gestation (Appendix, p. 1256). Thus, the diagnosis in pregnant women-as well as in those taking certain oral contraceptives-is diicult (Archer, 1999). If screening during pregnancy is necessary, threshold values for free protein S antigen levels in the second and third trimesters have been identiied at <30 percent and <24 percent, respectively. Among those with a positive family history, the VTE risk in pregnancy is 6 to 7 percent (American College of Obstetricians and Gynecologists, 2017c).

1	Conard and coworkers (1990) described thrombosis in five of 29 pregnant women with protein S deiciency. hey, as well as Burneo and colleagues (2002), reported maternal cerebral vein thrombosis. Neonatal homozygous protein C or S deiciency is usually associated with a fatal clinical phenotype known as purpurafulminans (Shanbhag, 2015).

1	his is the most prevalent of the known thrombophilia syndromes and is characterized by resistance of plasma to the anticoagulant efects of activated protein C. here are several mutations that create this resistance, but the most common is the factor V Leiden mutation, which was named after the city in which it was described. his missense mutation in the factor V gene results from a substitution of glutamine for arginine at position 506 in the factor V polypeptide. As a result of this mutation, activated factor V is neutralized approximately tenfold more slowly by activated protein C (see Fig. 52-1). his leads to enhanced thrombin generation (MacCallum, 2014).

1	Heterozygous inheritance of factor V Leiden is the most common heritable thrombophilia. Found in 3 to 15 percent of select European populations and 3 percent of African Americans, it is virtually absent in African blacks and Asians (Lockwood, 2012). One theory for its relatively high prevalence suggests that the heterozygous state may confer a survival advantage, possibly because of reduced bleeding with childbirth or trauma (MacCallum, 2014).

1	Women who are heterozygous for factor V Leiden account for approximately 40 percent of VTE cases during pregnancy. However, the actual risk among pregnant women who are heterozygous and who do not have a personal history or a irstdegree relative with a thrombotic episode before age 50 years is 5 to 12 events per 1000 gravidas (see Table 52-2). In contrast, this risk rises to at least 10 percent among pregnant women with a personal or family history. Pregnant women who are homozygous without a personal or family history have a 1-to 4-percent risk for VTE, whereas those with such a history have an approximately 17 -percent risk (American College of Obste tricians and Gynecologists, 2017c).

1	Diagnosis during pregnancy is performed by DNA analy sis for the mutant factor V gene. Bioassay is not used because of the normal resistance that develops after early pregnancy (Walker, 1997). Of note, activated protein C resistance can also be caused by anti phospholipid syndrome, which is described on page 1008 and also detailed in Chapter 59 (p. 1143). To assess the prognostic signiicance of maternal factor V Leiden mutation during pregnancy, Kjellberg and colleagues (2010) compared the outcomes of 491 carriers with those of 1055 controls. All three of the thromboembolic events occurred among the carriers. But, preterm birth rates, birthweights, or hypertensive complication rates did not difer between the two groups. In a prospective observational study of approximately 5000 women conducted by the Maternal-Fetal Medicine Units

1	Network, the heterozygous mutant gene incidence was 2.7 percent (Dizon-Townson, 2005). Of three pulmonary emboli and one deep-vein thrombosis cases-a rate of 0.8 per 1000 pregnancies-none were among these carriers. Moreover, in the heterozygous women, the risks of preeclampsia, placental abruption, fetal-growth restriction, or pregnancy loss were not elevated. he investigators concluded that universal prenatal screening for the Leiden mutation and prophylaxis for carriers without a prior VTE is not indicated.

1	his missense mutation in the prothrombin gene leads to excessive accumulation of prothrombin, which then may be converted to thrombin. Prothrombin levels are increased approximately 30 percent in heterozygotes and 70 percent in homozygotes (MacCallum, 2014). As with factor V Leiden, a personal or family history of VTE in a irst-degree relative before age 50 years raises the risk of VTE during pregnancy (see Table 52-2). For a heterozygous carrier with such a history, the risk exceeds 10 percent. Without such a history, heterozygous carriers of the mutation have less than a I-percent risk of VTE during pregnancy (American College of Obstetricians and Gynecologists, 2017 c).

1	Silver and coworkers (2010) tested nearly 4200 women for the prothrombin G20210A mutation. A total of 157-or 3.8 percent-of the women carried the mutation, and only one of these was homozygous. Carriers had similar rates of pregnancy loss, preeclampsia, fetal-growth restriction, and placental abruption compared with noncarriers. hree thromboembolic events occurred in women who tested negative for the mutation. Homozygous patients, or those who coinherit a G20210A mutation with a factor V Leiden mutation, have a greater thromboembolism risk than heterozygous carriers (Connors, 2017). Lim and associates (2016) have provided detailed information on pregnancy outcomes in women with such rare compound thrombophilias.

1	he most common cause of elevated homocysteine is the C667T thermolabile mutation of the 5,1 O-methylene-tetrahydrofolate reductase (MTHFR) enzyme. Inheritance is autosomal recessive. Elevated homocysteine levels may also result from deiciency of one of several enzymes involved in methionine metabolism and from correctible nutritional deficiencies of folic acid, vitamin B6, or vitamin Bl2 (Hague, 2003). During normal pregnancy, mean homocysteine plasma concentrations decline (Lopez-Quesada, 2003). hus, to make a diagnosis during pregnancy, Lockwood (2002) recommends a fasting threshold > 12 �mol/L to deine hyperhomocysteinemia.

1	In an interesting metaanalysis, Den Heijer and associates (2005) found that international studies of MTHFR polymorphisms were collectively associated with slightly greater risks for thrombosis. In contrast, studies conducted in North America collectively demonstrated no such association. The authors speculated that folic acid supplementation could explain the diference. Recall that folic acid serves as a cofactor in the remethylation reaction of homocysteine to methionine. Similarly, the American College of Chest Physicians concluded that the lack of an association with thromboembolism could relect the physiological reductions in homocysteine levels associated with pregnancy and the efects of widespread prenatal folic acid supplementation (Bates, 2012). he American College of Obstetricians and Gynecologists (2017 c) has concluded that there is insuicient evidence to support assessment of MTHFR polymorphisms or measurement of fasting homocysteine levels in the evaluation for VTE.

1	Potentially thrombophilic polymorphisms are being discovered at an ever-increasing rate. Unfortunately, information regarding the prognostic signiicance of such newly discovered mutations is limited. For example, protein Z is a vitamin K-dependent protein that serves as a cofactor in factor Xa inactivation. Studies have found that low protein Z levels are associated with an elevated thromboembolism risk in nonpregnant patients and may be implicated in the pathogenesis of poor pregnancy outcomes (lmawi, 2013). Similarly, plasminogen activator inhibitor ype I (PAl-I) is an important regulator of ibrinolysis. Certain polymorphisms in the gene promoter have been associated with slightly greater VTE risks. Although these thrombophilias may exacerbate risk among patients when coinherited with other thrombophilias, the American College of Obstetricians and Gynecologists (2017 c) has concluded that evidence to recommend screening is insuicient.

1	As an interesting aside, Galanaud and coworkers (2010) hypothesized that a paternal thrombophilia could increase the risk of maternal thromboembolism. Specifically, these investigators found that a paternal thrombophilia-the PROCR 6936G allele-afects the endothelial protein C receptor. his receptor is expressed by villous trophoblast and thus is exposed to maternal blood. Although this research is preliminary, it could help explain the pathogenesis of recurrent idiopathic thromboses in pregnant women. Some examples of acquired hypercoagulable states include anti phospholipid syndrome (APS), heparin-induced thrombocytopenia (p. 1015), and cancer.

1	Some examples of acquired hypercoagulable states include anti phospholipid syndrome (APS), heparin-induced thrombocytopenia (p. 1015), and cancer. his prothrombotic disorder can afect both the venous and arterial circulations. The deeper veins of the lower limbs and the cerebral arterial circulation are the most frequent sites of venous and arterial thrombosis, respectively (Connors, 2017; Giannakopoulos, 2013). Besides thrombosis, the other major clinical manifestations of the APS are obstetrical (Table 18-5, p. 353). Criteria include: (1) at least one otherwise unexplained fetal death at or beyond 10 weeks; (2) at least one preterm birth before 34 weeks' gestation because of eclampsia, severe preeclampsia, or placental insuiciency; or (3) at least three unexplained consecutive spontaneous abortions before 10 weeks.

1	Once one of the above clinical criteria-thrombosis or obstetrical-is met, anti phospholipid antibody testing should be performed to diagnose APS. hese patients should be tested for the presence of three factors: (1) lupus anticoagulant, (2) anticardiolipin immunoglobulin G and M (IgG and IgM) antibodies, and (3) anti-�2-glycoprotein I IgG and IgM antibodies. If any of these laboratory test results are positive, a conirmatory test is performed 12 weeks later (Connors, 2017).

1	Based on their study of 750 singleton pregnancies complicated by APS, Saccone and associates (2017) found that anticardiolipin antibody is the most common sole antiphospholipid antibody present; but anti-�rglycoprotein I is associated with the lowest live birth rate and highest incidences of preeclampsia, fetal-growth restriction, and stillbirth compared with anticardiolipin antibodies or lupus anticoagulant alone. hese investigators also observed that despite therapy with lowdose aspirin and prophylactic LMWH heparin, the chance of a liveborn neonate was only 30 percent for women with positive test results for all three antibodies.

1	The thrombosis risk rises signiicantly during pregnancy in women with APS. Indeed, up to 25 percent of thrombotic events in women with APS occur during pregnancy or in the puerperium. Looking at this a diferent way, women with APS have a 5-to 12-percent risk of thrombosis during pregnancy or the puerperium (American College of Obstetricians and Gynecologists, 2017 a). This syndrome is discussed in more detail in Chapter 59 (p. 1143). Attention has been directed toward possible relationships between inherited thrombophilias and pregnancy complications other than thromboses. Summarized in Table 52-3 are the findings of 25 studies systematically reviewed by Robertson and associates (2005) and incorporated into the recommendations of the American College of Chest Physicians (Bates, 2012). Importantly, the considerable heterogeneity and wide conidence intervals illustrate the uncertainty of these associations. Other investigations underscore the heterogeneity of results.

1	Other investigations underscore the heterogeneity of results. For example, Kahn and coworkers (2009) found no higher risk for early-onset or severe preeclampsia in women with factor V Leiden mutation, prothrombin G20210A mutation, MTHFR C677T polymorphism, or hyperhomocysteinemia. Said and associates (201 Oa) prospectively screened more than 2000 healthy nulliparous women for factor V Leiden, prothrombin Protein C deficiency 2.29 NA NA 3.05 5.15 NA (0.20-26.43) (0.24-38.51) (0.26-102.22) (0.23-151.58) Protein 5 deficiency 3.55 NA NA 20.09 2.83 2.1o1 NA (0.35-35.72) (3.70-109.1•5) (0.76-10.57) (0.47-9.34) Anticardiolipin 3.40 5.05 NA 3.30 2.73 1.42 6.91 antibodies (1.33-8.68) (1.82-14.01 ) (1.62-6.70) (1.65-4.51 ) (0.42-4.77) (2.70-1•7.68) Lupus anticoagulants 2.97 NA 14.28 2.38 1.45 NA NA (nonspecific (1.03-9.76) (4.72-43.20) (0.81-6.98) (0.70-4.61) Hyper-6.25 4.21 NA 0.98 3.49 2.40 NA homocysteinemia (1.37-28.42) (1.28-13.87) (0.1o7-5.55) (1.21-10.1•1) (0.36-15.89)

1	Hyper-6.25 4.21 NA 0.98 3.49 2.40 NA homocysteinemia (1.37-28.42) (1.28-13.87) (0.1o7-5.55) (1.21-10.1•1) (0.36-15.89) Homozygous and heterozygous carriers were grouped together; it is not possible to extract data for each state. Data are presented as odds ratio (OR [95% CI]) and are derived from Robertson, 2005. Bolded numbers are statistically significant. MTHFR = methylene tetrahydrofolate reductase variant; NA = not available. Reproduced with permission from Bates 5M, Greer iA, Middledorp 5, et al: TE, thrombophilia, antithrombotic therapy, and pregnancy. Chest 141 :e691 5,201o2.

1	Reproduced with permission from Bates 5M, Greer iA, Middledorp 5, et al: TE, thrombophilia, antithrombotic therapy, and pregnancy. Chest 141 :e691 5,201o2. gene mutation, MTHFR C677T, MTHFR A1298C, and thrombomodulin polymorphism. Women who carried the prothrombin gene mutation had a 3.6-fold greater risk of adverse pregnancy outcome, including severe preeclampsia, fetal-growth restriction, placental abruption, or stillbirth. But, none of the other polymorphisms conferred an elevated risk of these adverse outcomes. From the Stillbirth Collaborative Research Network, Silver and associates (2016) found a weak association between maternal factor V Leiden and stillbirth. here was no association between stillbirth and the other inherited thrombophilias. Based on their prospective study of 750 pregnancies complicated by stillbirth, Korteweg and colleagues (2010) concluded that routine thrombophilia testing after fetal death is inadvisable.

1	he American College of Obstetricians and Gynecologists (2017 c) notes that a deinitive causal link cannot be made between inherited thrombophilias and adverse pregnancy outcomes. Moreover, in one randomized trial, Rodger and associates (2014) found that antepartum prophylactic LMWH did not reduce a composite outcome of pregnancy loss, severe or early-onset preeclampsia, small-for-gestational age neonates, and TE in thrombophilic women. Thus, because of uncertainties in the magnitude of risk and in the beneits of prophylaxis given to prevent pregnancy complications in women with heritable thrombophilias, it remains unproven that universal screening is indicated (Louis-Jacques, 2016). In contrast, the association between APS and adverse pregnancy outcomes-including fetal loss, recurrent pregnancy loss, and preeclampsia-is much stronger.

1	Given the relatively high incidence of thrombophilia in the population and the low incidence of TE, universal screening during pregnancy is not cost efective (Carbone, 2010). hus, a selective screening strategy is required. he American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) recommend that thrombophilia screening be considered in the following clinical circumstances: (1) a personal history ofTE that was associated with a nonrecurrent risk factor such as fractures, surgery, and/or prolonged immobilization; and (2) a irst-degree relative (parent or sibling) with a history of high-risk thrombophilia or VTE before age 50 years in the absence of other risk factors.

1	The American College of Obstetricians and Gynecologists (2017 c) notes that testing for inherited thrombophilias in women who have experienced recurrent fetal loss or placental abruption is not recommended because clinical evidence that antepartum heparin prophylaxis prevents recurrence is insuicient. Similarly, testing is not recommended for women with a history of fetal-growth restriction or preeclampsia. The American College of Chest Physicians also recommends against screening women with prior pregnancy complications (Bates, TABLE 52-4. How to Test for Thrombophilias Is Testing Is Testing Reliable Reliable Is Testing During During Acute Reliable with Testing Method Pregnancy? Thrombosis? Anticoagulation? alf screening in pregnancy is necessary, cutoff values for free protein S antigen levels in the second and third trimesters been identified at less than 30% and less than 24%, respectively.

1	alf screening in pregnancy is necessary, cutoff values for free protein S antigen levels in the second and third trimesters been identified at less than 30% and less than 24%, respectively. Reproduced with permission from American College of and Gynecologists Women's Health Care Physicians: ACOG Practice Bulletin No. 138: Inherited thrombophilias in pregnancy, Obstet Gynecol. 20103 Sep;122(3):706-717. 2012). However, screening for antiphospholipid antibodies may be appropriate in women who have experienced a fetal loss or early-onset preeclampsia (Berks, 2015). Methods of screening for the more common inherited thrombophilias are shown in Table 52-4. Whenever possible, laboratory testing is performed at least 6 weeks after the thrombotic event, while the patient is not pregnant, and when she is not receiving anticoagulation or hormonal therapy. Screening for hyperhomocysteinemia is not recommended (American College of Obstetricians and Gynecologists, 2017 c).

1	During pregnancy, most venous thromboses are confined to the deep veins of the lower extremity. Approximately 70 percent of cases are located in the iliofemoral veins without involvement of the calf veins. Isolated iliac vein and calf vein thromboses occur in approximately 17 and 6 percent of cases, respectively (Chan, 2010). In contrast, in the general population, more than 80 percent of deep-vein thromboses involve calf veins, and iliofemoral or isolated iliac vein thromboses are uncommon (Huisman, 2015).

1	he signs and symptoms vary greatly and depend on the degree of occlusion and the intensity of the inlammatory response. Ginsberg and coworkers (1992) reported that 58 of 60 antepartum women-97 percent-had left leg thromboses. Blanco-Molina and coworkers (2007) reported left-leg involvement in 78 percent. Greer (2003) hypothesizes that this results from compression of the left iliac vein by the right iliac and ovarian artery, both of which cross the vein only on the left side. Yet, as described in Chapter 53 (p. 1026), the ureter is compressed more on the right side.

1	Classically, thrombosis involving the lower extremity is abrupt in onset, and there is pain and edema of the leg and thigh. The thrombus typically involves much of the deepvenous system to the iliofemoral region. Occasionally, relex arterial spasm causes a pale, cool extremity with diminished pulsations. Alternatively, there may be appreciable clot, yet little pain, heat, or swelling. Importantly, calf pain, either spontaneous or in response to squeezing or to Achilles tendon stretching-Homans sign-may be caused by a strained muscle or contusion. Between 30 and 60 percent of women with a confirmed lower-extremity acute deep-vein thrombosis have an asymptomatic pulmonary embolism (p. 1016).

1	Clinical diagnosis of deep-vein thrombosis is diicult, and in an earlier study of pregnant women, the clinical diagnosis was conirmed in only 10 percent (Hull, 1990). Another challenge is that many of the common diagnostic tests that have been investigated extensively in nonpregnant patients have not been validated appropriately in pregnancy (Huisman, 2015). Shown in Figure 52-2 is one diagnostic algorithm recommended by the American College of Chest Physicians that can be used for evaluation of pregnant women (Guyate, 2012). With a few modifications, we follow a similar evaluation at Parkland Hospital.

1	In pregnant women with suspected deep-vein thrombosis, the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017) recommend compression ultrasonography of the proximal veins as the initial diagnostic test. According to the American College of Chest Physicians, this noninvasive technique is currently the most-used irst-line test to detect deep-vein thrombosis (Guyatt, 2012). The diagnosis is based on the noncompressibility and typical echoarchitecture of a thrombosed vein. For nonpregnant patients with suspected thrombosis, the safety of withholding anticoagulation for 1 week has been established for those who have a compression ultrasound examination that is initially normal (Birdwell, 1998; Heijboer, 1993). Serial compression examinations are then performed because isolated undetected calf thromboses that ultimately extend into the proximal veins will do so within 1 to 2 weeks of presentation in approximately a fourth of patients.

1	Pregnant women with suspected Negative, but isolated iliac vein thrombosis is suspecteda Doppler ultrasound of iliac vein, or CT-or MR-imaging or venography FIGURE 52-2 Algorithm for evaluation of suspected deep-vein thrombosis in pregnancy. CT = computed tomography; MR = magnetic resonance. aSigns and symptoms include swelling of the entire leg, with or without flank, buttock, or back pain. (Data from Guyatt GH, Akl EA, Crowther M, et al: Executive summary: Antithrombotic therapy and prevention of thrombosis, 9th ed: American College ofChest Physicians evidence-based clinical practice guidelines, Chest. 2012 Feb;141(2 Suppl):7S-47S.)

1	In pregnant women, the important caveat is that normal findings with venous ultrasonography do not always exclude a pulmonary embolism. his is because the thrombosis may have already embolized or because it arose from iliac or other deeppelvic veins, which are less accessible to ultrasound evaluation (Goldhaber, 2004). As discussed, thrombosis associated with pulmonary embolism during pregnancy commonly originates in the iliac veins.

1	he results of two studies are helpful for evaluating the need for serial examinations in pregnant women suspected of having a deep-vein thrombosis but who have a negative initial compression ultrasound examination. The combined results are depicted in Figure 52-3. Chan and coworkers (20l3) studied 221 pregnant and postpartum women presenting with a suspected deep-vein thrombosis. The 205 women with a negative initial study result underwent serial testing, which was negative in all cases. Of these, one woman with normal serial testing had a pulmonary embolism 7 weeks later. Le Gal and colleagues (2012) studied 210 pregnant and postpartum women with a suspected deep-vein thrombosis. Of these, 177 women without a deep-vein thrombosis were not anticoagulated and did not undergo serial testing. Two had an objectively conirmed thrombosis diagnosed within 3 months. In sum, these preliminary data suggest that a negative single complete compression ultrasonography study may safely exclude the

1	Two had an objectively conirmed thrombosis diagnosed within 3 months. In sum, these preliminary data suggest that a negative single complete compression ultrasonography study may safely exclude the diagnosis of deep-vein thrombosis in most pregnant women.

1	This imaging technique allows excellent delineation ofanatomical detail above the inguinal ligament. Thus, in many cases, magnetic resonance (MR) imaging is immensely useful for diagnosis of iliofemoral and pelvic vein thrombosis. The venous system can also be reconstructed using MR venography (Chap. 46, p. 911). Erdman and associates (1990) reported that MR imaging was 100-percent sensitive and 90-percent speciic for detection of venographically proven deep-vein thrombosis in nonpregnant patients. Importantly, almost half of those without deep-vein thrombosis were found to have nonthrombotic conditions that included cellulitis, myositis, edema, hematomas, and supericial phlebitis.

1	halil and coworkers (2012) used MR venography to study the natural history ofpelvic vein thrombosis after vaginal delivery. Among the 30 asymptomatic patients who were all within four days of delivery, 30 percent had a deinitive thrombosis in either the iliac or ovarian veins, and another 37 percent had a suspected thrombosis. Our experiences with hundreds of postpartum MR scans do not support these findings. Thus, although the clinical significance of their findings is uncertain, it seems clear that some degree of pelvic vein intraluminal illing defect may be a normal inding.

1	These speciic ibrin degradation products are generated when fibrinolysin degrades ibrin, as occurs in thromboembolism (Chap. 41, p. 783). heir measurement is frequently incorporated into diagnostic algorithms for VTE in nonpregnant patients (Wells, 2003). Screening with the D-dimer test in pregnancy, however, is problematic for several reasons. As shown in the Appendix (p. 1256), depending on assay sensitivity, D-dimer serum levels rise with gestational age along n= 205Lost to follow-up Untreated during ortreated despite..follow-up Treat noDVT n = 177 Diagnosed with VTE within 3 mas n= 3 (0.8%) FIGURE 52-3 Findings from two studies of serial and nonserial compression ultrasound examinations in pregnant and postpartum women. CUS = compression ultrasonography. Df = deep vein thrombosis. VfE = venous thromboembolism. (Data from Chan, 2013;

1	Le Gal, 201o2.) with substantively elevated plasma fibrinogen concentrations (Murphy, 2015). Levels are also afected by multifetal gestation and cesarean delivery (Morikawa, 2011). D-Dimer concentrations can also be elevated in certain pregnancy complications such as placental abruption, preeclampsia, and sepsis syndrome. Moreover, higher levels have been observed in sickle-cell carriers and in women of Mrican and South Asian racial origin (Grossman, 2016). For all these reasons, their use during pregnancy remains uncertain, but a negative D-dimer test should be considered reassuring (Lockwood, 2012; Marik, 2008).

1	Optimal management ofVTE during pregnancy has not undergone major clinical study to provide evidence-based practices. here is, however, consensus for treatment with anticoagulation and limited activity. If thrombophilia testing is performed, it is done before anticoagulation. Heparin induces a decline in antithrombin levels, and warfarin lowers protein C and S concentrations. The results of these tests do not change treatment (Connors, 2017). Anticoagulation is initiated with either unfractionated heparin (UFH) or LMWH. lthough either type is acceptable, most recommend one of the LMWHs (Bates, 2016; Kearon, 2016). For example, the American College of Chest Physicians suggests preferential use of LMWH during pregnancy because of better bioavailability, longer plasma half-life, more predictable dose response, reduced risks of osteoporosis and thrombocytopenia, and less frequent dosing (Bates, 2012). Dosages are shown in Table 52-5.

1	During pregnancy, heparin therapy is continued, and for postpartum women, anticoagulation is begun simultaneously with warfarin. Recall that pulmonary embolism develops in s many s 60 percent of patients with untreated venous thrombosis, and anticoagulation decreases this risk to less than 5 percent. In nonpregnant patients, the mortality rate with a pulmonary embolism approximates 1 percent (Douketis, 1998; Pollack, 2011). Over several days, leg pain dissipates. Mter symptoms have abated, graded ambulation is begun. Elastic stockings are itted, and anticoagulation is continued. Recovery to this stage usually takes 7 to 10 days. Graduated compression stockings are continued for 2 years after the diagnosis to reduce the incidence of postthrombotic syndrome (Brandjes, 1997). This syndrome can include chronic leg paresthesias or pain, intractable edema, skin changes, and leg ulcers.

1	his agent should be considered for the initial treatment of thromboembolism and in situations in which delivery, surgery, or thrombolysis may be necessary (American College of Obstetricians and Gynecologists, 20 17b). U nfractionated heparin can be administered by one of two alternatives: (I) initial intravenous therapy followed by adjusted-dose subcutaneous UFH given every 12 hours; or (2) twice-daily, adjusted-dose subcutaneous UFH with doses adjusted to prolong the activated partial thromboplastin time (aPTT) into the therapeutic range 6 hours postinjection (Bates, 2012). As shown in Table 52-5, the therapeutic dose for subcutaneous UFH is usually 10,000 units or more every 12 hours. For intravenous therapy, several protocols are acceptable. In general, if UFH is used, it is initiated with a bolus intravenous dose of70 to 100 U/kg, which is 5000 to 10,000 U. This is followed by continuous intravenous infusions beginning at TABLE 52-5. Anticoagulation Regimen Definitions

1	TABLE 52-5. Anticoagulation Regimen Definitions Prophylactic LMWHa Enoxaparin,n40 mg SC once daily Dalteparin, 5,000 units SC once daily Tinzaparin, 4,500 units SC once daily Therapeutic LMWHb Enoxaparin, 1 mg/kg every 12 hours Dalteparin, 200 units/kg once daily Tinzaparin, 175 units/kg once daily Dalteparin, 100 units/kg every 12 hours May target an anti-Xa level in the therapeutic range of 0.6-1.0 units/mL for twice daily regimen; slightly higher doses may be needed for a once-daily regimen. Minidose prophylactic UFH UFH, 5,000 units SC every 12 hours Prophylactic UFH UFH, 5,000-10,000 units SC every 12 hours UFH, 5,000-7,500 units SC every 12 hours in first trimester UFH 7,500-10,000 units SC every 12 hours in the second trimester UFH, 10,000 units SC every 12 hours in the third trimester, unless the aPTI is elevated Therapeutic UFHb UFH, 10,000 units or more SC every 12 hours in doses adjusted to target aPTI in the therapeutic range (1n.5-2.5) 6 hours after injection

1	Therapeutic UFHb UFH, 10,000 units or more SC every 12 hours in doses adjusted to target aPTI in the therapeutic range (1n.5-2.5) 6 hours after injection Postpartum anticoagulation Prophylactic LMWH/UFH for 4-6 weeks or vitamin K antagonists for 4-6 weeks with a target INR of 2.0-3.0, with initial UFH or LMWH therapy overlap until the INR is 2.0 or more for Surveillance Clinical vigilance and appropriate objective investigation of women symptoms suspicious of deep-vein thrombosis or pulmonary embolism Abbreviations: aPTI, activated partial thromboplastin time; INR, international normalized ratio; LMWH, low-molecular-weight heparin; SC, subcutaneously; UFH, unfractionated heparin. aAlthough at extremes of body weight, modification of dose may be required. bAlso referred to as weight adjusted, full treatment dose. Reproduced with permission from American College of Obstetricians and Gynecologists Women's Health Care Physicians:

1	Reproduced with permission from American College of Obstetricians and Gynecologists Women's Health Care Physicians: Bulletin No. 138: Inherited thrombophilias in pregnancy, Obstet Gynecol. 2013 Sep;122(3):706-717. 1000 U Ihr or 15 to 20 U Ikg/hr. his infusion rate is titrated to achieve an aPTT 1.5 to 2.5 times control values (Brown, 2010; Linnemann, 2016). Intravenous anticoagulation is maintained for at least 5 to 7 days, after which treatment is converted to subcutaneous heparin to maintain the aPTT to at least 1.5 to 2.5 times control throughout the dosing interval. For women with lupus anticoagulant, aPTT does not accurately assess heparin anticoagulation, and thus anti-factor Xa levels are preferred.

1	The duration of full anticoagulation varies, and no studies have defined the optimal duration for pregnancy-related thromboembolism. In nonpregnant patients with TE, evidence supports a minimum treatment duration of 3 months (Kearon, 2012). For pregnant patients, the American College of Chest Physicians recommends anticoagulation throughout pregnancy and postpartum for a minimum total duration of 3 months (Bates, 2012). Lockwood (2012) recommends that full anticoagulation be continued for at least 20 weeks followed by prophylactic doses if the woman is still pregnant. Prophylactic doses of subcutaneous UFH can range from 5000 to 10,000 U every 12 hours titrated to maintain an anti-factor Xa level of 0.1 to 0.2 U/mL, measured 6 hours after the last injection. If the TE occurs during the postpartum period, Lockwood (2012) recommends a minimum of 6 months of anticoagulation treatment.

1	This is a family of derivatives of unfractionated heparin, and their molecular weights average 4000 to 5000 daltons compared with 12,000 to 16,000 daltons for conventional heparin. None of these heparins cross the placenta, and all exert their anticoagulant activity by activating antithrombin. The primary diference is their relative inhibitory activity against factor a and thrombin. Specifically, UFH has equivalent activity against factor a and thrombin, but LMWHs have greater activity against factor a than against thrombin. They lso have a more predictable anticoagulant response and fewer bleeding complications than UFH because of their better bioavailability, longer half-life, dose-independent clearance, and decreased interference with platelets (Tapson, 2008). hese LMWH compounds are cleared by the kidneys and must be used cautiously when there is renal dysunction.

1	Several studies have shown that TE is treated efectively with LMWH (Quinlan, 2004; Tapson, 2008). Using serial venograms, Breddin and associates (2001) observed that these compounds were more efective than UFH in reducing thrombus size without increasing mortality rates or major bleeding complications. Several diferent treatment regimens using adjusted-dose LMWH for treatment of acute TE are recommended by the American College of Obstetricians and Gynecologists (2017b,c) and are listed in Table 52-5.

1	LMWHs available for use in pregnancy include enoxaparin, tinzaparin, and dalteparin. Enoxaparin (Lovenox) pharmacokinetics were studied in 36 women with TE during pregnancy or immediately postpartum (Rodie, 2002). he dose was approximately 1 mg/kg given twice daily based on early pregnancy weight. Treatment was monitored by peak anti-factor Xa activity at 3 hours postinjection, with a target therapeutic range of 0.4 to 1.0 U/mL. In 33 women, enoxaparin provided satisfactory anticoagulation. In the other three women, dose reduction was necessary. None developed recurrent thromboembolism or bleeding complications. In postcesarean women with a body mass index (BMI) �35, Stephenson and associates (2016) found that weight-based dosing of enoxaparin 0.5 mg/kg twice daily more efectively achieved prophylactic peak anti-Xa levels between 0.2 to 0.6 U/mL than a ixed dose of 40 mg daily. Similar indings were reported by Overcash and colleagues (2015).

1	For tinzaparin (Innohep), a dosage of 75 to 175 U/kg/d was necessary to achieve peak anti-factor Xa levels of 0.1 to 1.0 U/mL (Smith, 2004). In studies of dalteparin (Fragmin) pharmacokinetics, conventional starting doses of dalteparin-100 U/kg every 12 hours-were likely insuicient to maintain ull anticoagulation (Barbour, 2004; Jacobsen, 2003). Thus, slightly higher doses than that shown in Table 52-5 may be required.

1	Standard prophylactic and therapeutic dosages recommended by the American College of Obstetricians and Gynecologists (2017b) for various L\1WHs are listed in Table 52-5. Whether such dosages require adjustments during the course of pregnancy is controversial (Berresheim, 2014; Cutts, 2013). Some suggest periodic measurement of anti-factor Xa levels 4 to 6 hours after an injection with dose adjustment to maintain a therapeutic level. Large studies using clinical end points that demonstrate an optimal therapeutic range or show that dose adjustments increase therapy safety or eicacy are lacking. Accordingly, the American College of Chest Physicians and others note that routine monitoring with anti-Xa levels is difficult to justiy (Bates, 2012; McDonnell, 2017).

1	Early reviews concluded that LMWHs were safe and efective (Lepercq, 2001; Sanson, 1999). Despite this, in 2002, the manufacturer of Lovenox warned that its use in pregnancy had been associated with congenital anomalies and a higher risk of hemorrhage. Ater its own extensive review, the American College of Obstetricians and Gynecologists (20 17b) concluded that these risks were rare, that their incidence was not higher than expected, and that no cause-and-efect relationship had been established. It further concluded that enoxaparin and dalteparin could be given safely during pregnancy. Other reports confirm their safety (Andersen, 2010; Bates, 2012; Galambosi, 2012).

1	N elson-Piercy and coworkers (201a1) assessed the safety of tinzaparin through a comprehensive study of 1267 treated pregnant women. There were no maternal deaths or complications from regional analgesia. Although thrombocytopenia developed in 1.8 percent, there were no cases of heparin-induced thrombocytopenia (p. 1015). he allergy incidence was 1.3 percent. Osteoporotic fractures in three women (0.2 percent) were judged to be related to tinzaparin (p. 1015). A total of 43 women (3.4 percent) required medical intervention for bleeding. Of 15 stillbirths, four were judged as possibly being related to tinzaparin use. But, none of the neonatal deaths or congenital abnormalities was attributed to tinzaparin. The authors concluded that tinzaparin during pregnancy was safe for mother and fetus. LMWHs are also safe during breastfeeding (Lim, 2010).

1	However, LMWHs should be avoided in women with renal failure. Moreover, when given within 2 hours of cesarean delivery, these agents raise the risk of wound hematoma (van Wijk, 2002).

1	Women receiving either therapeutic or prophylactic anticoagulation should be converted from LMWH to the shorter halflife UFH in the last month of pregnancy or sooner if delivery appears imminent. he purpose of conversion to UFH has less to do with any risk of maternal bleeding at the time of delivery, but rather with neuraxial blockade complicated by an epidural or spinal hematoma (Chap. 25, p. 496). The American College of Chest Physicians recommends that women scheduled for a planned delivery who are receiving twice-daily adjusteddose subcutaneous UFH or fWH discontinue their heparin 24 hours before labor induction or cesarean delivery (Bates, 2012). Patients receiving once-daily LMWH should take only 50 percent of their normal dose on the morning of the day before delivery. he American College of Obstetricians and Gynecologists (2017 c) advises that adjusted-dose subcutaneous LlvfWH or UFH can be discontinued 24 to 36 hours before an induction of labor or scheduled cesarean

1	College of Obstetricians and Gynecologists (2017 c) advises that adjusted-dose subcutaneous LlvfWH or UFH can be discontinued 24 to 36 hours before an induction of labor or scheduled cesarean delivery. he American Society of Regional Anesthesia and Pain Medicine advises withholding neuraxial blockade for 10 to 12 hours after the last prophylactic dose of LMWH or 24 hours after the last therapeutic dose (Horlocker, 2010).

1	If a woman begins labor while taking UFH, clearance can be veriied by an aPTT. Reversal of heparin with protamine sulfate is rarely required and is not indicated with a prophylactic dose of heparin. For women in whom anticoagulation therapy has temporarily been discontinued, pneumatic compression devices are recommended. • Anticoagulation with Warfarin Compounds Vitamin K antagonists are generally contraindicated because they readily cross the placenta and may cause fetal death and malformations from hemorrhages (Chap. 12, p. 247). They do not accumulate in breast milk and are thus safe during breastfeeding.

1	Postpartum venous thrombosis is usually treated with intravenous heparin and oral warfarin initiated simultaneously. The initial dose of warfarin is usually 5 to 10 mg for the first 2 days. Subsequent doses are titrated to achieve an international normalized ratio (INR) of 2 to 3. To avoid paradoxical thrombosis and skin necrosis from the early anti-protein C efect of warfarin, these women are maintained on therapeutic doses of UFH or LMWH for 5 days and until the INR is in a therapeu tic range for 2 consecutive days (American College of Obstetri cians and Gynecologists, 2017 c; Stewart, 2010). Treatment in the puerperium may require larger doses of anticoagulant. Brooks and colleagues (2002) compared antico agulation in postpartum women with that of age-matched non pregnant controls. The former required a significantly larger median total dose of warfarin-45 versus 24 mg-and a longer time-7 versus 4 days-to achieve the target INR.

1	Of newer oral anticoagulants, dabigatran (Pradaxal) inhibits thrombin. Rivaroxaban (Xarelto) and apixaban (Eliquis) inhibit factor Xa. Currently, very few reports address these newer agents during pregnancy, and thus the human reproductive risks are essentially unknown (Bates, 2012). Dabigatran crosses the human placenta (Bapat, 2014). However, it is unknown whether any of these agents are excreted in breast milk. Because of the potential for infant harm, a decision should be made to either avoid breastfeeding or use an alternative anticoagulant, such as warfarin, in postpartum women (Burnett, 2016).

1	• Complications of Anticoagulation hree significant complications associated with anticoagulation are hemorrhage, thrombocytopenia, and osteoporosis. he latter two are unique to heparin, and their risk may be reduced with LMWHs. The most serious complication is hemorrhage, which is more likely if there has been recent surgery or lacerations. Troublesome bleeding also is more likely if the heparin dosage is excessive. Unfortunately, management schemes using laboratory testing to identiY when a heparin dosage is suicient to inhibit further thrombosis, yet not cause serious hemorrhage, have been discouraging.

1	There are two types-the most common is a nonimmune, benign, reversible thrombocytopenia that develops within the first few days of therapy and resolves in approximately 5 days without therapy cessation. The second is the severe form of heparin-induced thrombocytopenia (HIT), which results from an immune reaction involving IgG antibodies directed against complexes of platelet factor 4 and heparin. The diagnosis of HIT is based on a drop in the platelet count of more than 50 percent or thrombosis beginning 5 to 10 days after the start of heparin in association with the appearance of platelet-activating HIT antibodies. The fall in platelet count in HIT occurs rapidly-over a period of 1 to 3 days-and is assessed relative to the highest platelet count after the start of heparin. he typical nadir is 40,000 to 80,000 platelets per microliter (Greinacher, 2015).

1	Although the incidence of HIT is approximately 3 to 5 percent in nonpregnant individuals, it is <0.a1 percent in obstetrical patients (Linkins, 2012). Fausett and coworkers (2001) reported no cases among 244 heparin-treated gravidas compared with 10 among 244 nonpregnant patients. Accordingly, the American College of Chest Physicians recommends against platelet count monitoring when the risk of HIT is considered to be less than 1 percent. In others, they sug (Linkins, 2012).

1	When HIT is diagnosed, heparin therapy is stopped and alternative anticoagulation initiated. Platelet transfusions are avoided (Greinacher, 2015). LMWH may not be entirely safe because it has some cross reactivity with UFH. The Ameri can College of Chest Physicians recommends danaparoid (Orgaran)-a sulfated glycosaminoglycan heparinoid (Bates, 2012; Linkins, 2012). In a review of nearly 50 pregnant women with either HIT or a skin rash, Lindhof-Last and associates tive. However, they reported two fatal maternal hemorrhages and three fetal deaths. Magnani (2010) reviewed case reports of 83 pregnant women treated with danaparoid. Although it was generally efective, two patients died related to bleeding, three patients sufered nonfatal major bleeds, and three women developed thromboembolic events unresponsive to danaparoid. The drug has been removed from the u.S. market.

1	The drug has been removed from the u.S. market. bin inhibitor (Kelton, 2013; Linkins, 2012). Successful use in pregnancy has been reported (Elsaigh, 2015; Knol, 2010). Tanimura and coworkers (2012) successfully used argatroban, and later fondaparinux, to manage HIT in a pregnant woman with hereditary antithrombin deficiency. Bone loss may develop with long-term heparin administration-usually 6 months or longer-and is more prevalent in cigarette smokers. UFH can cause osteopenia, and this is less likely with LMWHs (Deruelle, 2007). Women treated with any heparin should be encouraged to take an oral daily 1500-mg calcium supplement (Cunningham, 2005; Lockwood, 2012). In one study, Rodger and colleagues (2007) found that long-term use of dalteparin for a mean of212 days was not associated with a signiicant decline in bone mineral density.

1	he treatment of deep-vein thrombosis with heparin does not preclude pregnancy termination by careful curettage. After the products are removed without trauma to the reproductive tract, full-dose heparin can be restarted in several hours.

1	The efects of heparin on blood loss at delivery depend on several variables: (1) dose, route, and timing of administration; (2) number and depth of incisions and lacerations; (3) intensity of postpartum myometrial contractions; and (4) presence of other coagulation defects. Blood loss should not be greatly increased with vaginal delivery if the episiotomy is modest in depth, there are no lacerations, and the uterus promptly contracts. Unfortunately, such ideal circumstances do not always prevail. For example, Mueller and Lebherz (1969) described 10 women with antepartum thrombophlebitis treated with heparin. hree women who continued to receive heparin during labor and delivery bled remarkably and developed large hematomas. Thus, heparin therapy generally is stopped during labor and delivery. The American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (20a17) recommend restarting UFH or LMWH no sooner than 4 to 6 hours after vaginal delivery or 6 to

1	The American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (20a17) recommend restarting UFH or LMWH no sooner than 4 to 6 hours after vaginal delivery or 6 to 12 hours after cesarean delivery. We wait at least 24 hours to restart therapy after cesarean delivery or after vaginal delivery with significant lacerations.

1	Slow intravenous administration of protamine sulfate generally reverses the efect of heparin promptly and efectively. It should not be given in excess of the amount needed to neutralize the heparin, because it also has an anticoagulant efect. hrombosis limited strictly to the superficial veins of the saphenous system is treated with analgesia, elastic support, heat, and rest. If it does not soon subside or if deep-vein involvement is suspected, appropriate diagnostic measures are performed. Supericial vein thrombosis raises the risk of deep-vein thrombosis four-to sixfold. Heparin is given if deep-vein involvement is conirmed (Roach, 2013). Superficial thrombophlebitis is typically seen in association with varicosities or as a sequela of an indwelling intravenous catheter.

1	lthough it causes approximately 10 percent of maternal deaths, pulmonary embolism is relatively uncommon during pregnancy and the puerperium. he incidence averages 1 in 7000 pregnancies. According to Marik and Plante (200S), 70 percent of gravidas presenting with a pulmonary embolism have associated clinical evidence of deep-vein thrombosis. And recall that between 30 and 60 percent of women with a deep-vein thrombosis will have a coexisting silent pulmonary embolism. In almost 2500 nonpregnant patients with a proven pulmonary embolism, symptoms included dyspnea in S2 percent, chest pain in 49 percent, cough in 20 percent, syncope in 14 percent, and hemoptysis in 7 percent (Goldhaber, 1999). Pollack and coworkers (2011) found similar symptoms. Other predominant clinical indings typically include tachypnea, apprehension, and tachycardia. In some cases, an accentuated pulmonic closure sound, rales, and/or friction rub is heard.

1	ight axis deviation and T -wave inversion in the anterior chest leads may be evident on the electrocardiogram. In at least 40 percent, chest radiography results are normal. In others, nonspecific indings may include atelectasis, an iniltrate, cardiomegaly, or an efusion (Pollack, 2011). Vascular markings in the lung region supplied by the obstructed artery can be lost. Although most women are hypoxemic, a normal arterial blood gas analysis does not exclude pulmonary embolism. Approximatelyaa third of young patients have P02 values >SO mm Hg. hus, the alveolar-arterial oxygen tension diference is a more useful indicator of disease. More than S6 percent of patients with acute pulmonary embolism will have an alveolar-arterial diference >20 mm Hg (Lockwood, 2012). Even with massive pulmonary embolism, signs, symptoms, and laboratory data to support the diagnosis may be deceptively nonspecific.

1	his is defined as embolism causing hemodynamic instability (Tapson, 200S). Acute mechanical obstruction of the pulmonary vasculature causes increased vascular resistance and pulmonary hypertension followed by acute right ventricular dilation. In otherwise healthy patients, significant pulmonary hypertension does not develop until 60 to 75 percent of the pulmonary vascular tree is occluded (Guyton, 1954). Moreover, circulatory collapse requires 75-to SO-percent obstruction. This is depicted schematically in Figure 52-4 and emphasizes that most acutely symptomatic emboli are large and likely a saddle embolism. hese are suspected when the pulmonary artery pressure is substantively increased as estimated by echocardiography.

1	If there is evidence of right ventricular dysfunction, the mortality rate approaches 25 percent. This compares with a I-percent rate without such dysfunction (Kinane, 200S). It is important in these cases to infuse crystalloids carefully and to support blood pressure with vasopressors. As discussed on page lOIS, oxygen treatment, endotracheal intubation, and mechanical ventilation are completed preparatory to thrombolysis, filter placement, or embolectomy (T apson, 200S).

1	In most cases, recognition of a pulmonary embolism requires a high index of suspicion that prompts objective evaluation. Exposure of the mother and fetus to ionizing radiation is a concern when investigating a suspected pulmonary embolism during pregnancy. However, this concern is largely overruled by the hazards of missing a potentially fatal diagnosis. Moveover, erroneously assigning a diagnosis of pulmonary embolism to a pregnant woman is also fraught with problems. It unnecessarily exposes the mother and fetus to the risks of anticoagulation treatment and will impact delivery plans, future contraception, and thromboprophylaxis during subsequent pregnancies. herefore, investigations should aim at diagnostic certainty (Konstantinides, 2014).

1	In 2011a, the American horacic Society and the Society of Thoracic Radiology developed an algorithm-shown in Figure 52-5 for the diagnosis of pulmonary embolism during pregnancy (Leung, 201l). In addition to compression ultrasonography, which was previously discussed (p. 1010), the algorithm includes computed-tomographic pulmonary angiography (CTPA) and ventilation-perfusion scintigraphy. Multidetector computed tomography with pulmonary angiography is currently the most commonly employed technique used for pulmonary embolism diagnosis in nonpregnant patients (Bourjeily, 2012; Pollack, 2011). he technique is described further in Chapter 46 (p. 907), and an imaging example is Dia. 1.5 cm each; total area 9 cm2 Dia. 8 mm each; total area 1 3 cm2 arteries Dia. 6 mm each; = arteries Dia. 4 mm each;

1	Dia. 1.5 cm each; total area 9 cm2 Dia. 8 mm each; total area 1 3 cm2 arteries Dia. 6 mm each; = arteries Dia. 4 mm each; FIGURE 52-4 Schematic of pulmonary arterial circulation. Note that the cross-sectional area of the pulmonary trunk and the combined 2 pulmonary arteries is 9 cmo. A large saddle embolism could occlude 50 to 90 percent of the pulmonary tree, causing hemodynamic instability. As the arteries give of distal branches, the total surface area rapidly increases, that is, 13 cm2 for the combined five lobar arteries, 36 cm2 for the combined 19 segmental arteries, and more than 800 cm2 for the total 65 subsegmental arterial branches. Thus, hemodynamic instability is less likely with emboli past the lobar arteries. (Data from Singhal S, Henderson R, Horsfield K, et al: Morphometry of the human pulmonary arterial tree, Circ Res. 1973 Aug;33(2):190-197.) Stop CUS, CTPA Treat Stop

1	Stop CUS, CTPA Treat Stop FIGURE 52-5 The American Thoracic Society and Society of Thoracic Radiology diagnostic algorithm for suspected pulmonary embolism during pregnancy. CTPA = computed tomographic pulmonary angiography; CUS = compression ultrasonography; CXR = chest x-ray; PE = pulmonary embolism; V/O = ventilation/perfusion scintigraphy. (Modified with permission from Leung AN, Bull TM, jaeschke R, et al: An oficial American Thoracic Society/Society of Thoracic Radiology Clinical Practice Guideline: Evaluation of suspected pulmonary embolism in pregnancy, Am j Respir Crit Care Med. 2011 Nov 15;184(10):1200-1208.)

1	FIGURE 52-6 Axial image of the chest from a four-channel multidetector spiral computed tomographic scan performed ater administration of intravenous contrast. There is enhancement of the pulmonary artery with a large thrombus on the right (arrow) consistent with pulmonary embolism. (Reproduced with permission from Dr. Michael Landay.) shown in Figure 52-6. The estimated fetal radiation exposure averages 0.45 to 0.6 mGy. he estimated maternal breast dose is 10 to 70 mGy (Waksmonski, 2014). Bourjeily and colleagues (2012) performed a follow-up study of 318 pregnant women who had a negative CTP A performed for a suspected pulmonary embolism. All were seen 3 months following their initial presentation or at 6 weeks postpartum. None of these women were subsequently diagnosed with a thromboembolism.

1	CTP A has many advantages, but we find that the higher resolution allows detection of previously inaccessible smaller distal emboli that have uncertain clinical significance. Similar observations have been reported by others (Anderson, 2007; Hall, 2009). Also, the hyperdynamic circulation and augmented plasma volume associated with pregnancy leads to a higher number of nondiagnostic studies compared with nonpregnant patients (Ridge, 2011; Scarsbrook, 2006). his technique involves a small dose of radiotracer such as intravenously administered technetium-99m-macroaggregated albumin. There is negligible fetal and maternal breast radiation exposure-0.1 to 0.4 mGy. The scan may not provide a deinite diagnosis because many other conditions can cause perfusion defects. Examples are pneumonia or local bronchospasm. Chan and coworkers (2002) found that a fourth of ventilationperfusion scans in pregnant women were nondiagnostic. In these instances, CTPA is preferred (T romeur, 2017).

1	To compare the performance of lung scintigraphy and CTPA, Revel and colleagues (201a1) evaluated 137 pregnant women with suspected pulmonary embolism. he two modalities performed comparably and had no signiicant diferences between the proportions of positive, negative, or indeterminate results. Speciically, the proportion of indeterminate results for both approximated 20 percent. By way of comparison, about a fourth of the nonpregnant population had indeterminate studies. The investigators attributed this diference to the younger age of the pregnant patients. Similarly, one systematic review concluded that both CTP A and lung scintigraphy seem appropriate for exclusion of pulmonary embolism during pregnancy (van Mens, 2017).

1	his requires catheterization of the right side of the heart and is considered the reference test for pulmonary embolism. With newer generation multidetector CT scanners, however, the role of invasive pulmonary angiography has been questioned. his is especially true given the higher radiation exposure for the fetus (Konstantinides, 2014; Kuriakose, 2010). Other detractions are that it can be time consuming, uncomfortable, and associated with dye-induced allergy and renal failure. Indeed, the procedure-related mortality rate approximates 1 in 200 (Stein, 1992). It is reserved for confirmation when less invasive tests are equivocal. Immediate treatment for pulmonary embolism is full anticoagulation similar to that for deep-vein thrombosis as discussed on page 1012. Several complementary procedures may be indicated.

1	Immediate treatment for pulmonary embolism is full anticoagulation similar to that for deep-vein thrombosis as discussed on page 1012. Several complementary procedures may be indicated. The woman who has very recently sufered a pulmonary embolism and who must undergo cesarean delivery presents a particularly serious problem. Reversal of anticoagulation may be followed by another embolus, and surgery while ully anticoagulated frequently results in life-threatening hemorrhage or troublesome hematomas. In these cases, placement of a vena caval ilter should be considered before surgery (Marik, 2008). Moreover, in the very infrequent circumstances in which heparin therapy fails to prevent recurrent pulmonary embolism from the pelvis or legs, or when embolism develops from these sites despite heparin treatment, a vena caval ilter may also be indicated. Such filters can also be used following massive emboli in patients who are not candidates for thrombolysis (Deshpande, 2002).

1	he device is inserted through either the jugular or femoral vein and can be inserted during labor Jamjute, 2006). Routine filter placement has no added advantage to heparin given alone (Decousus, 1998). Retrievable ilters may be used as short-term protection and then removed 1 to 2 weeks later (Liu, 2012). From their systematic review, Harris and associates (2016) found that complication rates in pregnant women with vena caval filters are comparable to those in nonpregnant patients.

1	Compared with heparin, thrombolytic agents provide more rapid lysis of pulmonary clots and improvement of pulmonary hypertension (Tapson, 2008). Konstantinides and coworkers (2002) studied 256 nonpregnant patients receiving heparin for an acute submassive pulmonary embolism. hey also were randomly assigned to a placebo or the recombinant tissue plasminogen activator alteplase. hose given the placebo had a threefold greater risk of death or treatment escalation compared with those given alteplase. Agnelli and associates (2002) per formed a metaanalysis of trials involving 461 nonpregnant patients. They reported that the risk of recurrence or death and heparin compared with those given heparin alone-1 0 versus 17 percent. Importantly, however, there were ive-2 percent-fatal bleeding episodes in the thrombolysis group and none in the heparin-only group.

1	In their review, Leonhardt and colleagues (2006) identiied 28 reports of tissue plasminogen activator use during pregnancy. T en cases were for thromboembolism. Complication rates were similar to those in nonpregnant patients, and the authors concluded that such therapy should not be withheld during pregnancy if indicated. However, Akazawa and Nishida (2017) reviewed 13 cases of systemic thrombolytic therapy administered during the irst 48 hours after delivery. Blood transfusion was required in five of the eight cesarean deliveries, including three cases of hysterectomy and two cases of hematoma removal. Given the eicacy of thrombolysis and filters, surgical embolectomy is uncommonly indicated. Published experience with emergency embolectomy during pregnancy is limited to case reports (Colombier, 2015; Saeed, 2014). From their review, Ahearn and associates (2002) found that although the operative risk to the mother is reasonable, the stillbirth rate is 20 to 40 percent.

1	Most recommendations regarding thromboprophylaxis during pregnancy stem from consensus guidelines. In one review of guidelines for thromboprophylaxis in pregnancy, the authors concluded that there is a lack of overall agreement about which women should be ofered thromboprophylaxis or ofered testing for thrombophilias (Okoroh, 2012). Bates and associates (2016) also conducted a review of guidelines for obstetrically associated VTE. hey summarized that evidence-based recommendations are based largely on observational studies and extrapolated from data in nonpregnant patients. Similarly, a Cochrane review concluded that evidence is insuicient for firm recommendations regarding thromboprophylaxis during pregnancy (Bain, 2014).

1	he confusion that has ensued has provided fertile ground for litigators. Cleary-Goldman and associates (2007) surveyed 151 fellows of the American College of Obstetricians and Gynecologists and reported that intervention without a clear indication is common. Table 52-6 lists several consensus recommendations for thromboprophylaxis. In some cases, more than one option is listed, thus illustrating the confusion that currently reigns. In general, either antepartum surveillance or heparin prophylaxis is recommended for women with prior TE but without a recurrent risk factor, including no known thrombophilia. The study by Tengborn and coworkers (1989), however, suggested that such management may not be efective. hey reported out boembolic disease and were not tested for thrombophilias.

1	Despite unfractionated heparin prophylaxis, which was usually 5000 U twice daily, three of 20 women (15 percent) developed antepartum recurrence. his compared with eight of 67 women (12 percent) not given heparin. 125 pregnant women with a single prior VTE. Antepartum hep arin was not given, but anticoagulant therapy was given for 4 to 6 weeks postpartum. Six women had a recurrent venous throm bosis-three antepartum and three postpartum. here were no recurrences in the 44 women without a known thrombophilia or whose prior thrombosis was associated with a temporary risk factor. These findings imply that prophylactic heparin may not be required for these two groups of women. In contrast, and as shown in Table 52-6, women with a prior thrombosis in association with a thrombophilia or in the absence of a tempo postpartum prophylaxis (Connors, 2017).

1	De Stefano and coworkers (2006) studied 1104 nonpregnant women who had a irst-episode VTE before the age of 40 years. After excluding those with antiphospholipid antibodies, 88 women were identiied who subsequently had a total of 155 pregnancies and who were not given anti thrombotic prophylaxis. There were 19 women (22 percent) who had a subsequent pregnancy-or puerperium-related VTE. Of 20 women whose original thrombosis was associated with a transient risk factornot including pregnancy or oral contraceptive use-there were no recurrences during pregnancy, but two during the puerperium. hese data also suggest that for women with a prior VTE, anti thrombotic prophylaxis during pregnancy could be tailored according to the circumstances of the original event.

1	It is important to emphasize that VTE may recur despite antithrombotic prophylaxis. Galambosi and associates (2014) studied 270 women during 369 pregnancies who had at least one previous VTE. A total of 28 women (l0.4 percent) sufered a recurrent VTE. Twelve of these recurrences occurred early in pregnancy before the initiation of antithrombotic prophylaxis, and 16 occurred despite prophylactic use of LMWH. Our practice at Parkland Hospital for many years for women with a history of prior VTE was to administer subcutaneous UFH, 5000 to 7500 units two to three times daily. With this regimen, the recurrence of documented deep-vein thrombosis embolization was rare. Beginning approximately 10 years ago, we have successfully used 40 mg enoxaparin given subcutaneously daily for thromboprophylaxis.

1	The risk for deep-vein thrombosis and especially for fatal thromboembolism rises manyfold in women following cesarean compared with that ater vaginal delivery. When considering that a third of women giving birth in the United States yearly undergo cesarean delivery, pulmonary embolism is understandably a major cause of maternal mortality (Creanga, 2017). hat said, the "lack TABLE 52-6. Some Recommendations for Thromboprophylaxis during Pregnancy some experts." INR 2.0-3.0 x long-term therapy some experts" INR 2.0-3.0 x warfarin LMWH or 75% of anticoagulation Associated with a high-Prophylactic, NSS Postpartum Prophylactic or risk thrombophiliad intermediate-, r anticoagulationC intermediate- anticoagulation or an LMWH or UFH x INR 2.0-3.0 x affected fi rst -d eg ree 6 weeksc 6 weeks relative Associated with a low-Prophylactic or NSS Postpartum Prophylactic or risk thrombophiliae intermed iate-dose anticoagulationc or intermediate- INR 2.0-3.0 x

1	INR 2.0-3.0 x Two or more prior VTEs with or without thrombophilia 6 weeks INR 2.0-3.0 x 6 weeks Receiving long-term Therapeutic-dose Adjusted-dose Resumption of long-Resumption of anticoagulation LMWH or UFH LMWH or 75% of term anticoagulation long-term a therapeutic dose anticoagulation No prior.VTE prophylactic or intermed iate-dose a nticoag u lation. dose LMWH or intermediate-dose LMWH warfarin target LMWH or UFH INR 2.0-3.0 x mutation INR 2.0-3.0 x TABLE 52-6. Continued mutation INR 2.0-3.0 x or S deficient, INR 2.0-3.0 History of fE Prophylactic NSS Prophylactic NSS anticoagulation with a nticoag u lationC; UFH or LMWH (?plus referral to specialist9 LMWH or UFH plus low-LMWH, both given pregnancy loss r dose aspirin if prior with 75-100 mg/ stillbirth9 aAmerican College of Obstetricians and Gynecologists, 2017a, c. bAmerican College of Chest PhYSicians (Bates, 201n2). (Postpartum treatment levels should be � antepartum treatment.

1	bAmerican College of Chest PhYSicians (Bates, 201n2). (Postpartum treatment levels should be � antepartum treatment. dAntithrombin deficiency; doubly heterozygous or homozygous for prothrombin 2021 OA and factor V Leiden. eHeterozygous factor V Leiden or prothrombin 2021 OA; protein S or C deficiency. fFirst-degree relative with fE at <50 years; other major thrombotic risk factors, e.g., obesity, prolonged immobility. 9Women with antiphospholipid syndrome should not use estrogen-containing contraceptives. hTreatment is recommended if the diagnosis of antiphospholipid syndrome is based on three or more prior pregnancy losses. LMWH = low-molecular-weight heparin; NSS = not specifically stated; UFH = unfractionated heparin; fE = venous thromboembolism. Prophylactic, intermediate-, and adjusted-dose regimens are listed in Table 52-5 (p. 1013).

1	Prophylactic, intermediate-, and adjusted-dose regimens are listed in Table 52-5 (p. 1013). of high quality data" described earlier by Bates and colleagues compression devices before cesarean delivery for all women (2016) creates considerable variation in the current recommennot already receiving thromboprophylaxis. his recommendations promulgated by the American College of Obstetricians dation was based primarily on consensus and expert opinion. and Gynecologists, the Royal College of Obstetricians, and the For patients undergoing cesarean delivery with additional risk American College of Chest Physicians (Palmero la, 2016). factors for thromboembolism, both pneumatic compression

1	In 2011, the American College of Obstetricians and Gynedevices and UFH or LMH may be recommended. he Colcologists (20 17b) recommended placement of pneumatic lege stipulated that cesarean delivery in an emergency setting should not be delayed because of the time necessary to implement thromboprophylaxis. Implementation of this strategy by the Hospital Corporation of America, the largest for-profit obstetrical health care delivery system in the United States, was associated with a reduction in deaths from pulmonary embolism from 7 of 458,097 cesarean births to 1 of 465,880 cesarean births (Clark, 2011, 2014).

1	In 2016, the National Partnership for Maternal Safety published several consensus recommendations for the prevention of maternal TE (D'lton, 2016). These recommendations included expanded use of antenatal prophylaxis for women hospitalized 3 days or longer, expanded use of prophylaxis during and after vaginal delivery, and expanded use of pharmacological prophylaxis to most women after cesarean delivery. In response, Sibai and Rouse (2016) expressed concern that these new recommendations derive from sparse data of questionable applicability to obstetrical patients. They called for better quality evidence to measure the benefits, harms, and costs of increased pharmacological thromboprophylaxis. As aptly expressed by Macones (2017), "an intervention, such as increased postcesarean pharmacologic thromboprophylaxis, where there are legitimate concerns about eicacy and safety, requires a much higher degree of evidence before a national guideline is implemented." We agree with these

1	thromboprophylaxis, where there are legitimate concerns about eicacy and safety, requires a much higher degree of evidence before a national guideline is implemented." We agree with these sentiments.

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1	Revel MP, Cohen S, Sanchez 0, et al: Pulmonary embolism during pregnancy: diagnosis with lung scintigraphy or CT angiography? Radiology 258:590, 2011 Rheaume M, Weber F, Durand M, et al: Pregnancy-related venous thromboembolism risk in asymptomatic women with antithrombin deficiency: a systematic review. Obstet GynecoIt127(4):649, 2016 Ridge CA, Mhuircheartaigh IN, Dodd JD, et al: Pulmonary CT angiography protocol adapted to the hemodynamic efects of pregnancy. AJR Am J RoentgenoIt197:1058,t2011 Roach RE, Ufering WM, van Hylckama Vlieg A, et al: he risk of venous thrombosis in individuals with a history of superficial vein thrombosis and acquired venous thrombotic risk factors. Blood 122(26):4264,t2013 Robertson L, Wu 0, Langhorne P, et al: hrombophilia in pregnancy: a systematic review. Br J HaematoIt132:171, 2005

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1	Sabadell J, Casellas M, Alijotas-Reig J, et al: Inherited antithrombin deficiency and pregnancy: maternal and fetal outcomes. Eur J Obstet Gynecol Reprod Bioi 149:47, 2010 Saccone G, Berghella V, Maruotti GM, et al: Antiphospholipid antibody profile based obstetric outcomes of primary anti phospholipid syndrome: the PREGNANTS study. Am J Obstet GynecoIt216(5):525.e1, 2017 Saeed G, Moller M, Neuzner J, et al: Emergent surgical pulmonary embolectomy in a pregnant woman: case report and literature review. Tex Heart InstJ 41(2):188,t2014 Said JM, Higgins JR, Moses EK, et al: Inherited thrombophilia polymorphisms and pregnancy outcomes in nulliparous women. Obstet Gynecol 115:5, 2010a Said JM, Ignjatovic V, Monagle PT, et al: Altered reference ranges for protein C and protein S during early pregnancy: implications for the diagnosis of protein C and protein S deficiency during pregnancy. Thromb Haemost 103:984,t2010b

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1	Virchow R: Gesammelte Abhandlungen zur wissenschaftlichen Medizin. Frankfurt, Medinger Sohn & Co., 1856 Waksmonski CA: Cardiac imaging and functional assessment I pregnancy. Semin PerinatoIt38(5):240, 2014 Waldman M, Sheiner E, Vardi IS: Can we profile patients at risk for thromboembolic events after delivery: a decade of follow up. Am J Obstet Gynecol 208:S234,t2013 Walker MC, Garner PR, Keely EJ, et al: Changes in activated protein C resistance during normal pregnancy. Am J Obstet Gynecol 177: 162, 1997 Wells PS, Anderson DR, Rodger M, et al: Evaluation ofD-dimer in the diagnosis of suspected deep-vein thrombosis. N Engl J Med 349: 1227, 2003 PREGNANCY-INDUCED URINARY TRACT CHANGES.... 1025 URINARY TRACT INFECTIONS.. . . . . . . . . . . . . . . . . . .. 1026 NEPHROLITHIASIS.. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1030 PREGNANCY AFTER RENAL TRANSPLANTATION .... 1030 POLYCYSTIC KIDNEY DISEASE . . . . . . . . . . . . . . . . . .. 1031

1	GLOMERULAR DISEASES ....i......i....i...0..o...i.0...... 1032 CHRONIC KIDNEY DISEASE.. . . . . . . . . . . . . . . . . . . . .. 1034 ACUTE KIDNEY INJURY.. . . . . . . . . . . . . . . . . . . . . . . .. 1036 LOWER GENITAL TRACT LESIONS .0...0.i.......i......i.. 1037 In rare instances in patients suiringfrom pyelitis, the preg nant uterus may so compress the ureter as to cause a dam ming back of the purulent discharge, and thus give rise to a pyelonephritis. -]. Whitridge Williams (1903)

1	-]. Whitridge Williams (1903) Renal and urinary tract disorders are frequently encountered in pregnancy. Some precede pregnancy-one example being nephrolithiasis. In some women, pregnancy-induced changes may predispose to development or worsening of urinary tract disorders-an example is the markedly increased risk for pyelonephritis, described above by Williams. Finally, some renal pathology is unique to pregnancy, such as preeclampsia. With good prenatal care, however, most women with these disorders will likely have no long-term sequelae. Significant changes in both structure and function within the urinary tract during normal pregnancy are discussed in Chapter 4 (p. 65). he kidneys become larger, and dilatation of the right renal calyces and ureters can be striking Some dilatation develops before 14 weeks and likely E .

1	E . FIGURE 53-1 The 50th, 75th, and 90th percentiles for maternal renal caliceal diameters measured using sonography in 1395 pregnant women from 4 to 42 weeks' gestation. (Redrawn from Faundes A, Bricola-Filho M, Pinto e Silva JC: Dilatation of the urinary tract during pregnancy: proposal of a curve of maximal caliceal diameter by gestational age. Am J Obstet Gynecol 178: 1082, 1998.) from progesterone-induced relaxation of the muscularis. More marked dilatation is apparent beginning in midpregnancy because of more distal ureteral compression, especially on the right side (Faundes, 1998). There is also some vesicoureteral relux during pregnancy. Because of these physiological changes, the risk of upper urinary infection rises. Also, imaging studies done to evaluate urinary tract obstruction may occasionally be erroneously interpreted.

1	Evidence of functional renal hypertrophy becomes apparent very soon ater conception. Glomeruli are larger, although cell numbers do not grow (Strevens, 2003). Pregnancy-induced intrarenal vasodilatation develops, and both aferent and eferent resistances decline. This leads to greater efective renal plasma flow and glomerular filtration (Helal, 2012; Hussein, 2014). By 12 weeks' gestation, the glomerular filtration rate (GFR) is already augmented 20 percent above nonpregnant values (Hladunewich, 2004). Ultimately, plasma low and GFR rise by 40 and 65 percent, respectively. Consequently, serum concentrations of creatinine and urea drop substantively across pregnancy, and values within a nonpregnant normal range may be abnormal for pregnancy (Appendix, p. 1257). Other alterations include those related to maintaining normal acid-base homeostasis, osmoregulation, and fluid and electrolyte retention. • Assessment of Renal Function During

1	• Assessment of Renal Function During Urinalysis results are essentially unchanged during pregnancy, except for occasional glucosuria. Although protein excretion normally rises, it seldom reaches levels that are detected by usual screening methods. Higby and colleagues (1a994) reported 24-hour protein excretion in pregnancy to be 11a5 mg/d with a 95-percent confidence level of 260 mg/d. Values did not signiicantly difer by trimester (Fig. 4-14, p. 67). Albumin constitutes only a small part of total protein excretion and ranges from 5 to 30 mg/d. Airoldi and Weinstein (2007) concluded that proteinuria must exceed 300 mg/ d to be considered abnormal. Many consider 500 mg/d to be important with gestational hypertension. Investigators have correlated a urinary proteinto-creatinine ratio of :0.3 in a spot urine sample-ideally from a irst morning void-with a 24-hour protein excretion rate of :300 mg (Kuper, 2016).

1	In one study, 3 percent of 4589 nulliparas screened before 20 weeks had idiopathicihematuria, deined as 1 + or greater blood on urine dipstick (Stehman-Breen, 2002). These women had a twofold risk of developing preeclampsia. In another study of 1000 women screened during pregnancy, the incidence of dipstick hematuria was 15 percent (Brown, 2005). Most women had only trace levels of hematuria, and the falsepositive rate was 40 percent.

1	If the serum creatinine level in pregnancy persistently exceeds 0.9 mg/dL (75 LmollL), then intrinsic renal disease is suspected. In these cases, some determine the creatinine clearance as an estimate of the GFR. Of other assessment tools, sonography provides imaging of renal size, relative consistency, and elements of obstruction (see Fig. 53-1). Magnetic resonance (MR) imaging of renal masses provides excellent anatomic information (Putra, 2009). Full-sequence intravenous pyelography is not done routinely, but injection of contrast media with one or two abdominal radiographs may be indicated by the clinical situation. The usual clinical indications for cystoscopy are followed. Ureteroscopy is another available tool when indicated.

1	Although renal biopsy is relatively safely performed during pregnancy, it usually is postponed unless results may change therapy. From a review of 243 biopsies in pregnant women, the incidence of complications was 7 percent-this compares with 1 percent in postpartum women (Piccoli, 2013). Some consider biopsy for rapid deterioration of renal function with no obvious cause or for symptomatic nephrotic syndrome (Lindheimer, 2007 a). We and others have found biopsy helpful in selected cases to direct management (Chen, 2001; Piccoli, 2013). In one series, renal biopsy in 12 normal pregnant volunteers showed that five had slight to moderate glomerular endotheliosis (Strevens, 2003). Recall this is the histopathological lesion that is putatively typical of preeclampsia and is characterized by ibrin deposition within the glomerular endothelium leading to capillary occlusion. In contrast, all 27 women with protein uric hypertension had endotheliosis, and in all but one, it was moderate to

1	ibrin deposition within the glomerular endothelium leading to capillary occlusion. In contrast, all 27 women with protein uric hypertension had endotheliosis, and in all but one, it was moderate to severe.

1	In these cases, if the remaining kidney is normal, renal function becomes augmented. However, women who have donated a kidney have a higher frequency of gestational hypertension or preeclampsia in subsequent pregnancy-IaI versus 5 percent compared with non-donors (Garg, 2015). Otherwise, women with one normal kidney most often have no diiculty in pregnancy. Moreover, kidney donation does not lead to long-term adverse consequences. That said, thorough functional evaluation of the remaining kidney is essential (Ibrahim, 2009).

1	These infections are the most frequent bacterial infections complicating pregnancy. Although asymptomatic bacteriuria is the most common, symptomatic infection includes cystitis, or it may involve the renal calyces, pelvis, and parenchyma to cause pyelonephritis. Organisms that cause urinary infections are those from the normal perineal lora. Approximately 90 percent of Escherichia coli strains that cause nonobstructive pyelonephritis have adhesins such as P-and S-imbriae. These are cell-surface protein structures that enhance bacterial adherence and, thereby, virulence (Foxman, 2010; Hooton, 2012).

1	Data suggest that pregnant women have more severe sequelae from urosepsis. One possible underlying factor is the T -helper cell-Th1/h2 ratio-reversal of normal pregnancy, which is discussed in Chapter 4 (p. 59). Other perturbations of cytokine or of adhesin expression may be contributory (Chaemsaithong, 2013; Sledzinska, 2011). But even if pregnancy itself does not enhance these virulence factors, urinary stasis, vesicoureteral relux, and diabetes predispose to symptomatic upper urinary infections (Czaja, 2009). In the puerperium, several risk factors predispose to urinary infections. Bladder sensitivity to intravesical fluid tension is often diminished due to labor trauma or epidural analgesia.

1	In the puerperium, several risk factors predispose to urinary infections. Bladder sensitivity to intravesical fluid tension is often diminished due to labor trauma or epidural analgesia. Bladder sensations can also be obscured by discomfort from vaginal or perineal injury. Normal postpartum diuresis may worsen bladder overdistention, and catheterization to relieve retention often leads to urinary infection. Postpartum pyelonephritis is treated in the same manner as antepartum renal infections (McDonnold, 2012). This refers to persistent, actively multiplying bacteria within the urinary tract in asymptomatic women. he incidence during pregnancy is similar to that in nonpregnant women. It var ies from 2 to 7 percent, and it is characteristically population dependent. he highest incidence is in African-American mul tiparas with sickle-cell trait, and the lowest is in aluent white women of low parity. Asymptomatic infection is also more common in diabetics (Schneeberger, 2014).

1	Bacteriuria is typically present at the first antepartum visit. An initial positive urine culture result done as a part of prenatal care should prompt treatment. After this, fewer than 1 percent of women develop a urinary tract infection (Whalley, 1967). A clean-voided specimen containing more than 100,000 organ isms/mL is diagnostic. It may be prudent to treat when lower concentrations are identified, because pyelonephritis devel ops in some women despite colony counts of only 20,000 to 50,000 organisms/mL (Lucas, 1993).

1	Most studies indicate that if asymptomatic bacteriuria is not treated, approximately 25 percent of infected women will develop symptomatic infection during pregnancy (Smaill, 2015). In a more recent study, only 2.4 percent of treated women developed pyelonephritis (Kazemier, 2015). Eradication of bacteriuria with antimicrobial agents prevents most of these serious infections. The American Academy of Pediatrics and the American College of Obstetricians and Gynecologists (2017), as well as the U.S. Preventive Services Task Force (2008), recommend screening for bacteriuria at the first prenatal visit. Standard urine cultures may not be cost efective when the prevalence is low. Less expensive screening tests such as the leukocyte esterase/nitrite dipstick are cost efective when the prevalence is ;2 percent (Rogozinska, 2016; Rouse, 1995). Also, a dipstick culture technique has excellent positive-and negative-predictive values (Mignini, 2009). With this, a special agar-coated dipstick is

1	is ;2 percent (Rogozinska, 2016; Rouse, 1995). Also, a dipstick culture technique has excellent positive-and negative-predictive values (Mignini, 2009). With this, a special agar-coated dipstick is first placed into urine and then also serves as the culture plate. Because of a high prevalence-5 to 8 percent-at Parkland Hospital, most women are screened by traditional urine culture. Susceptibility determination is not necessary because initial treatment is empirical (Hooton, 2012).

1	In some but not all studies, covert bacteriuria has been associated with preterm or low-birthweight infants. It is even more controversial whether eradication of bacteriuria decreases these complications. Evaluating a cohort of 25,746 mother-infant pairs, Schieve and coworkers (1994) reported urinary tract infection to be associated with greater risks for low-birthweight infants, preterm delivery, pregnancy-associated hypertension, and anemia. hese findings vary from those of others (Gilstrap, 1981b; Whalley, 1967). Notably, in most studies, cohorts with asymptomatic infection are not evaluated separately from those with acute renal infection (Banhidy, 2007). One Cochrane database review noted insuicient data to answer this question (Smaill, 2015). Bacteriuria responds to empirical treatment with any of several antimicrobial regimens listed in Table

1	Bacteriuria responds to empirical treatment with any of several antimicrobial regimens listed in Table Although selection can be based on in vitro susceptibilities, in our extensive experience, empirical oral treatment for 10 days with nitrofurantoin macrocrystals, 100 mg at bedtime, is usually efective. Satisfactory results are also achieved with a 7-day oral course of nitrofurantoin, 100 mg given twice daily (Lumbiganon, 2009). Single-dose antimicrobial therapy is less successful (Widmer, 2015). The important caveat is that, regardless of regimen given, the recurrence rate is approximatey 30 percent. This may indicate covert upper tract infection and the need for longer therapy. hus, after initial therapy, periodic surveillance is necessary to prevent recurrent urinary infections (Schneeberger, 2015).

1	For recurrent bacteriuria, we have had success with nitrofurantoin, 100 mg orally at bedtime for 21 days (Lucas, 1994). For women with persistent or frequent bacteriuria recurrences, suppressive therapy for the remainder of pregnancy can be given. We routinely use nitrofurantoin, 100 mg orally at bedtime. This drug may rarely cause an acute pulmonary reaction that dissipates on its withdrawal (Boggess, 1996). TABLE 53-1. Oral Antimicrobial Agents Used for Treatment of Pregnant Women with Asymptomatic Bacteriuria Amoxicillin,n3 9 Ampicillin,n2 9 Cephalosporin, 2 9 Nitrofurantoin, 200 mg Trimethoprim-sulfamethoxazole, 320/1600 mg Amoxicillin, 500 mg three times daily Ampicillin, 250 mg four times daily Cephalosporin, 250 mg four times daily Ciprofloxacin, 250 mg twice daily Levofloxacin, 250 or 500 mg daily Nitrofurantoin, 50 to 100 mg four times daily or 100 mg twice daily Trimethoprim-sulfamethoxazole, 160/800 mg twice daily

1	Levofloxacin, 250 or 500 mg daily Nitrofurantoin, 50 to 100 mg four times daily or 100 mg twice daily Trimethoprim-sulfamethoxazole, 160/800 mg twice daily Nitrofurantoin, 100 mg four times daily for 10 days Nitrofurantoin, 100 mg twice daily for 5 to 7 days Nitrofurantoin, 100 mg at bedtime for 10 days Nitrofurantoin, 100 mg four times daily for 21 days Nitrofurantoin, 100 mg at bedtime for pregnancy remainder

1	Nitrofurantoin, 100 mg four times daily for 21 days Nitrofurantoin, 100 mg at bedtime for pregnancy remainder Lower urinary infection during pregnancy may develop without antecedent covert bacteriuria (Harris, 1981). Cystitis produces dysuria, urgency, and frequency, but with few associated systemic indings. Pyuria and bacteriuria are usually found. Microscopic hematuria is common, and occasionally there is gross hematuria from hemorrhagic cystitis. Although cystitis is usually uncomplicated, the upper urinary tract may become involved by ascending infection. Almost 40 percent of pregnant women with acute pyelonephritis have preceding symptoms of lower tract infection (Gilstrap, 1981a). Women with cystitis respond readily to any of several regimens. Most of the 3-day regimens listed in Table 53-1 are usually 90-percent efective (Fihn, 2003). Single-dose therapy is less efective, and if it is used, concomitant pyelonephritis must be confidently excluded.

1	Lower urinary tract symptoms with pyuria accompanied by a sterile urine culture may stem from urethritis caused by Chlamydia trachomatis. Mucopurulent cervicitis usually coexists, and azithromycin therapy is efective (Chap. 65, p. 1240).

1	Renal infection is one of the most frequent serious medical complications of pregnancy. Data from the 2006 Nationwide Inpatient Sample showed that nearly 29,000 pregnancy-associated hospitalizations were for acute pyelonephritis Qolley, 2012). In one hospital-system database of nearly 550,000 births, its incidence was 0.5 percent (Wing, 2014). Importantly, pyelonephritis is a leading cause of septic shock during pregnancy (Snyder, 2013). In one Parkland Hospital Obstetrical Intensive Care Unit review, 12 percent of antepartum admissions were for sepsis syndrome caused by renal infections (Zeeman, 2003). Urosepsis may be related to an increased incidence of cerebral palsy in preterm infants Qacobsson, 2002). Fortunately, afected mothers sufer no serious long-term sequelae (Raz, 2003). Renal infection develops more frequently in the second trimester, and nulliparity and young age are risks (Hill, 2005).

1	Renal infection develops more frequently in the second trimester, and nulliparity and young age are risks (Hill, 2005). Pyelonephritis is unilateral and right-sided in more than half of cases, and it is bilateral in a fourth. Fever and shaking chills usually develop rather abruptly, and patients have aching pain in one or both lumbar regions. Anorexia, nausea, and vomiting may worsen dehydration. Tenderness usually can be elicited by percussion in one or both costovertebral angles. he differential diagnosis includes, among others, labor, chorioamnionitis, adnexal torsion, appendicitis, placental abruption, or infarcted leiomyoma. Evidence of the sepsis syndrome is common (Chap. 47, p. 921).

1	If this infection is suspected, a urine sample obtained by catheterization may be preferred to avoid obscuring contamination from the lower genital tract. he urinary sediment contains many leukocytes, frequently in clumps, and numerous bacteria. Bacteremia is demonstrated in 15 to 20 percent of these women. E coli is isolated from urine or blood in 70 to 80 percent of infections, Klebsiela pneumoniae in 3 to 5 percent, Enterobacter or Proteus species in 3 to 5 percent, and gram-positive organisms, including group B Streptococcus and Staphylococcus aureus, in up to 10 percent of cases (Hill, 2005; Wing, 2000).

1	Plasma creatinine is monitored because early studies reported that 20 percent of pregnant women developed acute kidney injury . More recent findings, however, show this to be only 5 percent if aggressive fluid resuscitation is provided (Hill, 2005). Follow-up studies have demonstrated that this endotoxin-induced damage is reversible long term. s shown in Figure 53-2, varying degrees of respiratory distress syndrome from endotoxin-induced alveolar injury are manifest in up to 2 percent of women (Cunningham, 1987; Snyder, 2013; Wing, 2014).

1	Uterine activity from endotoxin is common and is related to fever severity (Graham, 1993). In one study, women with pyelonephritis averaged five contractions per hour at admission, and this decreased to two per hour within 6 hours of intravenous luid and antimicrobial administration (Millar, 2003). Notably, 3-agonist therapy for tocolysis increases the likelihood of respiratory insuiciency from permeability edema (Lamont, 2000). The incidence of pulmonary edema in women with pyelonephritis who were given 3-agonists was reported to be 8 percent-a fourfold increase over that expected (Towers, 1991) . a second-trimester pregnant woman with severe pyelonephritis. A.An extensive infiltrative process and complete obliteration of the diaphragm (white arrows) is seen. B. Improved aeration of lung fields bilaterally is noted as pleural disease resolves (arrows). c. Markedly improved visualization of the lung fields with residual platelike atelectasis and normal appearance of the diaphragm.

1	TABLE 53-2. Management of the Pregnant Woman with Acute Pyelonephritis Evaluate hemogram, serum creatinine, and electrolytes Monitor vital signs frequently, including urinary output- Establish urinary output �50 mL/hr with intravenous Obtain chest radiograph if there is dyspnea or tachypnea Change to oral antimicrobials when afebrile Discharge when afebrile 24 hours, consider antimicrobial therapy for 7 to 10 days Repeat urine culture 1 to 2 weeks after antimicrobial Modified from Lucas, 1994; Sheffield, 2005. Endotoxin-induced hemoysis is common, and approximately a third of these women with pyelonephritis develop anemia (Cox, 1991). With recovery, hemoglobin regeneration is normal, and acute infection does not afect erythropoietin production (Cavenee, 1994).

1	One scheme for management of acute pyelonephritis is shown in Table 53-2. Urine cultures are taken, but prospective trials show that blood cultures are of limited clinical utility (Gomi, 2015; Wing, 2000). We obtain blood cultures if the temperature is >39°C. Intravenous hydration to ensure adequate urinay ouput is the cornerstone of treatment. Antimicrobials are also begun promptly with the caveat that they may initially worsen endotoxemia from bacterial lysis. Surveillance for worsening sepsis syndrome includes serial monitoring of urinary output, blood pressure, pulse, temperature, and oxygen saturation. High fevers are lowered with a cooling blanket and acetaminophen. his is especially important in early pregnancy because of possible teratogenic efects from hyperthermia.

1	Antimicrobial therapy usually is empirical, and ampicillin plus gentamicin; cefazolin or ceftriaxone; or an extended-spectrum antibiotic are all 95-percent efective in randomized trials (Sanchez-Ramos, 1995; Wing, 1998,a2000). Fewer than half of E coli strains are sensitive to ampicillin in vitro, but cephalosporins and gentamicin generally have excellent activity. Serum creatinine levels are monitored if nephrotoxic drugs are given. Initial treatment at Parkland Hospital is ampicillin plus gentamicin. Some recommend suitable substitutes if bacterial studies show in vitro resistance. With any of the regimens discussed, response is usually prompt, and 95 percent of women are afebrile by 72 hours (Hill, 2005; Sheield, 2005). ter discharge, most recommend oral therapy for a total of7 to 14 days (Hooton, 2012).

1	Persistent Infection. Generally, intravenous hydration and antimicrobial therapy are followed by stepwise defervescence of approximately 1°F per day. With persistent spiking fever or lack of clinical improvement by 48 to 72 hours, urinary tract obstruction, another complication, or both are considered. In these women, renal sonography is recommended to search for obstruction, which is manifest by abnormal ureteral or pyelo caliceal dilatation (Seidman, 1998). Although most women with continuing infection have no evidence of obstruction, some are found to have calculi. Although renal sonography will detect hydronephrosis, stones are not always seen in preg nancy (Butler, 2000; Maikranz, 1987). If stones are strongly suspected despite a nondiagnostic sonographic examination, a plain abdominal radiograph will identiy nearly 90 percent. Another option is the modified one-shot intravenous pyelo injection-which usually provides adequate imaging (Butler, 2000).

1	Another option is the modified one-shot intravenous pyelo injection-which usually provides adequate imaging (Butler, 2000). In some women, MR imaging may disclose the cause of persistent infection (Spencer, 2004). Even without urinary obstruction, persistent infection can be due to an intrarenal or perinephric abscess or phlegmon (Cox, 1988; Rai, 2012). Obstruction relief is important, and one method is cystoscopic placement of a double-J ureteral stent (Rodriguez, 1988). Because these stents are usually left in place until after deliv ery, they frequently become encrusted and require replacement. We have found that percutaneous nephrostomy is preferable because the stents are more easily replaced. Finally, surgical removal of stones may be required in some women (p. 1030).

1	We have found that percutaneous nephrostomy is preferable because the stents are more easily replaced. Finally, surgical removal of stones may be required in some women (p. 1030). Outpatient Management. This is sometimes done for non pregnant women with uncomplicated pyelonephritis (Hooton, 2012). Outpatient management was described in 92 pregnant axone, two 1-g doses 24 hours apart (Wing, 1999). After this, only a third of the group was considered suitable for outpatient therapy, and these women were randomly assigned either to discharge home and oral antimicrobials or to continued hospi talization with intravenous therapy. A third of the outpatient management group was unable to adhere to the treatment regi men and required readmission. his suggests that outpatient management is applicable to very few gravidas.

1	Recurrent urinary tract infection-either covert or symptomatic-develops in 30 to 40 percent of women following completion of pyelonephritis treatment (Cunningham, 1973). Unless other measures are taken to ensure urine sterility, nitrofurantoin, 100 mg orally at bedtime given for the remainder of the pregnancy, reduces bacteriuria recurrence (Van Dorsten, 1987).

1	Vesicoureteral reflux in early childhood can cause recurrent urinary tract infections, and subsequent chronic interstitial nephritis is attributed to chronic pyelonephritis. Moreover, high-pressure sterile reflux impairs normal renal growth. Combined, this leads to patchy interstitial scarring, tubular atrophy, and loss of nephron mass and is termed relux nephropathy. In some casesespecially those with staghorn calculi-xanthogranulomatous pyelonephritis causes suppurative destruction of renal tissue. In adults, long-term complications of chronic pyelonephritis include hypertension, which may be severe (Beck, 2015; Diamond,a2012).

1	Perhaps half of women with reflux nephropathy were treated during childhood for renal infections. Many also had surgical correction of reflux as children, and these women commonly have bacteriuria when pregnant (Mor, 2003). In the other half of women with relux nephropathy, a clear history of recurrent cystitis, acute pyelonephritis, or obstructive disease is lacking. Reports describing 939 pregnancies in 379 women with reflux nephropathy indicate that impaired renal function and bilateral renal scarring were associated with increased maternal complications (EI-Khatib, 1994; Jungers, 1996; Kohler, 2003). Chronic renal disease and pregnancy outcome are discussed on page 1034.

1	Kidney stones develop in up to 9 percent of women during their lifetime with an average age of onset in the third decade (Curhan, 2015). Calcium salts make up approximately 90 percent of stones, and hyperparathyroidism should be excluded. Although calcium oxalate stones in young nonpregnant women are most common, most stones in pregnancy-65 to 75 percent-are calcium phosphate or hydroxyapatite (Ross, 2008; Tan, 2013). Patients who have a stone typically form another one every 2 to 3 years. One study found pregnancy was a risk factor for stone formation (Reinstatler, 2017). Contrary to past teachings, a low-calcium diet promotes stone formation. Thiazide diuretics diminish stone formation. In general, obstruction, infection, intractable pain, and heavy bleeding are indications for stone removal, discussed later.

1	he incidence of stone disease complicating pregnancy varies. At the low end, the incidence was 0.3 admissions per 1000 pregnancies at Parkland Hospital (Butler, 2000). In an Israeli study, the incidence in nearly 220,000 pregnancies was 0.8 per 1000 (Rosenberg, 2011). In Washington state, the incidence was 1.7 per 1000 pregnancies (Swartz, 2007). Bladder stones are rare, but recurrent infection and labor obstructed by stones have been reported (Ait Benkaddour, 2006; Ruan, 2011).

1	Data are conflicting whether women with kidney stones have an increased risk for low-birthweight and preterm newborns. In one study of 2239 women with nephrolithiasis compared with normal controls, stones were associated with a signiicantly elevated preterm delivery rate-l0.6 versus 6.4 percent (Swartz, 2007). The more recent nationwide study from Taiwan also found 20-to 40-percent increases in rates of low birthweight and pre term birth (Chung, 2013). In contrast, a study from Hungary reported that pregnancy outcomes, including preterm delivery, were similar in women with stones and normal controls (Banhidy, 2007). Comparable conclusions were drawn from the Israeli study discussed above (Rosenberg, 2011). Pregnant women may have fewer symptoms with stone passage because of urinary tract dilatation (Hendricks, 1991; Tan, 2013). hat said, more than 90 percent of pregnant women with symptomatic nephrolithiasis present with pain.

1	Gross hematuria is less common than in afected nonpregnant women. It was a presenting symptom in 23 percent of women described by Butler and associates (2000). In another study, however, only 2 percent had gross hematuria (Lewis, 2003). Sonography is usually selected to visualize stones, but many are not detected because hydronephrosis may obscure indings (Iv1cAleer, 2004). Transabdominal color Doppler sonography to detect presence or absence of ureteral "jets" of urine into the bladder may exclude obstruction (As rat, 1998).

1	If the ureter is abnormally dilated but no stone is seen, then other imaging studies are indicated. While helical computed tomography (CT) scanning is the preferred imaging method for nonpregnant individuals, the associated x-ray exposure has led some to recommend MR imaging as a second-line test in pregnancy (Masselli, 2015). Thus CT scanning is usually avoided during pregnancy if possible (Curhan, 2015; Masselli, 2015). If it is used, the slices can be tailored as needed. White and colleagues (2007) recommend unenhanced helical CT and cite an average fetal radiation dose to be 7 mGy.

1	Treatment depends on symptoms and gestational age (Semins, 2014). Intravenous hydration and analgesics are given. In up to half of women with symptomatic stones, infection will be identiied, and this is treated vigorously as described earlier (p. 1029). Although stones infrequently cause symptomatic obstruction during pregnancy, persistent pyelonephritis should prompt a search for obstruction due to nephrolithiasis. Urinary obstruction with concomitant infection is an emergency-"pus under pressure" (Curhan, 2015).

1	Approximately 65 to 80 percent of symptomatic women will improve with conservative therapy, and the stone usually passes spontaneously (Tan, 2013). Others require an invasive procedure such as ureteral stenting, ureteroscopy, percutaneous nephrostomy, transurethral laser lithotripsy, or basket extraction (Butler, 2000; Johnson, 2012; Semins, 2014). Removal by a flexible basket via cystoscopy, although used less often than in the past, is still a reasonable consideration for pregnant women. In one study, 623 various procedures were performed in 2239 symptomatic pregnant women, but less than 2 percent required surgical exploration (Swartz, 200*). Of other treatments, the need for fluoroscopy limits the utility of percutaneous nephrolithotomy (Toth, 2005). Extracorporeal shock-wave lithotripsy is contraindicated in pregnancy.

1	Following transplantation, the I-year graft survival rate is 95 percent for grafts from living donors and 89 percent for those from deceased donors. Survival rates approximately doubled between 1988 and 1996, due in large part to the introduction of cyclosporine and muromonab-CD3 (OKT3 monoclonal antibody) to prevent and treat organ rejection. Since then, mycophenolate mofetil and tacrolimus have further reduced acute rejection episodes, however, the former is considered teratogenic (Briggs, 2014). he National Transplant Pregnancy Registry reports that 23 percent of fetuses exposed to mycophenolate had birth defects (Coscia, 2010). Importantly, resumption of renal function after transplantation promptly restores fertility in reproductive-aged women (Hladunewich, 2011; Rao, 2016). But, more than half of transplant recipients in one study reported that they were not counseled regarding contraception (French, 2013).

1	Women after transplantation do better with pregnancy than those with end-stage renal disease receiving dialysis (Saliem, 2016). In one review of 2000 pregnancies in transplant recipi ents, most were treated with cyclosporine and tacrolimus, and approximately 75 percent of pregnancies resulted in a live birth (Coscia, 2010). Studies from other countries describe similar outcomes (Bramham, 2013; Wyld, 20l3). In a study from Uruguay, 62 percent of liveborns were preterm (Orihuela, 2016). Two other reports also cited a high prevalence of pre term delivery (Erman Akar, 2015; Stoumpos, 2016). Notably, the incidence of fetal malformations was not increased, except in those who took mycophenolate mofetil (Coscia, 2010).

1	The incidence of preeclampsia is increased in all transplant recipients (Brosens, 20l3). In the UK National Cohort Study, the incidence of preeclampsia was 22 percent (Bramham, 2013). From their review, Josephson and McKay (2011) cite an incidence of a third of pregnancies but question the validity of this frequency. Importantly, in some cases, rejection is difficult to distinguish from preeclampsia. hat said, the incidence of rejection episodes approximates 2 to 5 percent (Bramham, 2013; Orihuela, 2016). Viral infections-especially those by poyomavirus hominis 1, also called BK virus, are frequent. In kidney transplant recipients, this virus can cause nephropathy and graft loss, and afected patients generally have an asymptomatic decline in renal function (Wright, 2016). Gestational diabetes is also found in approximately 5 percent of transplant recipients. Both are likely related to immunosuppression therapy. Similar outcomes are reported by other investigators (Al Duraihimh, 2008;

1	is also found in approximately 5 percent of transplant recipients. Both are likely related to immunosuppression therapy. Similar outcomes are reported by other investigators (Al Duraihimh, 2008; Cruz Lemini, 2007; Ghafari, 2008).

1	Several requisites should be satisfied by renal transplantation patients before attempting pregnancy G osephson, 2011; Lopez, 2014). First, women should be in good general health for at least 1 to 2 years after transplantation. Also, renal function should be stable and without severe renal insuiciency. Thus, serum creatinine is <2 mg/dL and preferably < 1.5 mg/ dL, and proteinuria is <500 mg/d. Evidence for graft rejection should be absent for 6 months, and pyelocalyceal distention by urography should not be seen. Moreover, hypertension should be absent or well controlled. And last, women should be taking no teratogenic drugs, and drug therapy should be reduced to maintenance levels.

1	Cyclosporine, tacrolimus, prednisone, and azathioprine are given routinely to renal transplantation recipients Gain, 2004; Lopez, 2014). Cyclosporine blood levels decline during pregnancy, although this was not reported to be associated with rejection episodes (Akturk, 2015; Kim, 2015). Unfortunately, these agents are nephrotoxic and also may cause renal hypertension. In fact, they likely contribute substantively to chronic renal disease that develops in 10 to 20 percent of patients with nonrenal solid-organ transplantation (Goes, 2007). Concern persists regarding the possible late efects in ofspring subjected to immunosuppressive therapy in utero. hese include malig nancy, germ cell dysfunction, and malformations in the chil dren of the ofspring. In addition, cyclosporine is secreted in breast milk (Moretti, 2003).

1	Finally, although pregnancy-induced renal hyperiltration theoretically may impair long-term graft survival, Sturgiss and study of 34 allograft recipients followed for a mean of 15 years. Others have reported similar findings (Debska-Slizien, 2014; Stoumpos, 2016). Close surveillance is necessary. Covert bacteriuria is treated, and if it is recurrent, suppressive therapy is given for the remainder of the pregnancy. Serial hepatic enzyme concentrations and blood counts are monitored for toxic efects of azathioprine and cyclosporine. Some recommend measurement of serum cyclo sporine levels. Gestational diabetes is more common if corti costeroids are taken, and overt diabetes must be excluded with tation. Surveillance for opportunistic infections from herpesvi rus, cytomegalovirus, and toxoplasmosis is important because these infections are common. Some recommend surveillance for BK virus, however, treatment is problematic Gosephson, 2011).

1	Renal function is monitored, and the GFR usually increases 20 to 25 percent. If a signiicant rise in the serum creatinine level is detected, then its cause must be determined. Possibilities include acute rejection, cyclosporine toxicity, preeclampsia, infection, and urinary tract obstruction. Evidence of pyelonephritis or graft rejection should prompt aggressive management. Imaging studies and kidney biopsy may be indicated. The woman is carefully monitored for development or worsening of underlying hypertension, and especially superimposed preeclampsia. Management of hypertension during pregnancy is the same as for patients without a transplant.

1	Because of increased incidences of fetal-growth restriction and preterm delivery, vigilant fetal surveillance is indicated (Chaps. 42, p. 814 and 44, p. 852). Although cesarean delivery is reserved for obstetrical indications, occasionally the transplanted kidney obstructs labor. In all women with a renal transplant, the cesarean delivery rate exceeds 60 percent (Bramham, 2013; Rocha, 2013). This usually autosomally dominant systemic disease primarily afects the kidneys. The disease is found in 1 in 800 live births and causes approximately 5 to 10 percent of end-stage renal disease in the United States. Although genetically heterogeneous, almost 85 percent of cases are due to PD1 gene mutations on chromosome 16, and the other 15 percent to PD2 mutations on chromosome 4 (Zhou, 2015). Prenatal diagnosis is available if the mutation has been identiied in a family member or if linkage is established in the family.

1	Renal complications are more common in men than in women, and symptoms usually appear in the third or fourth decade. Flank pain, hematuria, proteinuria, abdominal masses, and associated calculi and infection are common. Hypertension develops in 75 percent, and progression to renal failure is a major problem. Superimposed acute kidney injury may also develop from infection or obstruction from ureteral angulation by cyst displacement. Other organs are frequently involved. Asymptomatic hepatic cysts coexist in a third of patients with polycystic kidneys. Hepatic involvement is more common and more aggressive in women, and massive polycystic liver disease is almost exclusively found in multiparous women (Zhou, 2015). Approximately 10 percent of patients with polycystic kidney disease die from rupture of an associated intracranial bery aneurysm. Up to a fourth of patients have cardiac valvular lesions that involve valve prolapse or incompetence.

1	Because of its generally late onset, adult polycystic kidney disease is uncommon in pregnancy (Banks, 2015). The prognosis for pregnancy in these women depends on the degree of associated hypertension and renal insuiciency. Urinary tract infections are common. One study compared pregnancy outcomes in 235 afected women who had 605 pregnancies with those of 108 unafected family members who had 244 pregnancies (Chapman, 1994). Composite perinatal complication rates were similar-33 versus 26 percent. However, hypertension, including preeclampsia, was significantly more frequent in women with polycystic kidneys. Pregnancy does not seem to accelerate the natural disease course (Lindheimer, 2007b). he glomerulus and its capillaries are subject to various conditions and stimuli that can lead to acute and chronic diseases. Glomerular damage can be caused by toxins or infections or from systemic diseases that include hypertension, diabetes, or systemic

1	TABLE 53-3. Patterns of Clinical Glomerulonephritis lupus erythematosus (Lewis, 2015). It may also be idiopathic. When there is capillary inflammation, the process is termed glomerulonephritis, and oten, an autoimmune process is involved. Persistent glomerulonephritis eventually leads to worsening renal function. Progression is variable and often does not manifest until chronic renal insuiciency is diagnosed. Lewis and Neilson (2015) group glomerular injuries into six syndromes based on clinical patterns (Table 53-3). Within each of these categories, there are disorders encountered in young women, and thus, these may antedate or irst appear during pregnancy.

1	Acute glomerulonephritis may result from any of several causes (see Table 53-3). The clinical presentation usually includes hypertension, hematuria, red-cell casts, pyuria, and proteinuria. Varying degrees of renal insuiciency and salt and water retention result in edema, hypertension, and circulatory congestion (Lewis, 2015). The prognosis and treatment of nephritic syndromes depends on their etiology. Some recede spontaneously or with treatment. However, in some patients, rapidy progressive glomerulonephritis leads to end-stage renal failure, whereas in others, chronic glomerulonephritis develops with slowly progressive renal disease.

1	Lupus nephritis identified before pregnancy has a 50percent chance of laring during pregnancy (Koh, 2015). IgA nephropathy, also known as Berger disease, is the most common form of acute glomerulonephritis worldwide (Wyatt, 2013). he isolated form occurs sporadically, and it may be related to Henoch-Schonlein purpura as the systemic form (Donadio, 2002). Isolated nephritis may be due to anti-glomerular basement membrane (anti-GBM) antibodies. hese may also involve the lungs to manifest as a pulmonary-renal syndrome with alveolar hemorrhage, which is termed Goodpasture syndrome (Friend, 2015; Huser, 2015). Acute nephritic syndromes during pregnancy can be diicult to diferentiate from severe preeclampsia or eclampsia (Cabiddu, Acute Nephritic Syndromes: poststreptococcal, infective endocarditis, SLE, antiglomerular basement membrane disease, IgA nephropathy (Berger disease), ANCA

1	Acute Nephritic Syndromes: poststreptococcal, infective endocarditis, SLE, antiglomerular basement membrane disease, IgA nephropathy (Berger disease), ANCA Henoch-Schonlein purpura, cryoglobulinemia, membranoproliferative and mesangioproliferative glomerulonephritis Pulmonary-Renal Syndromes: Goodpasture, ANCA vasculitis, Henoch-Schonlein purpura, cryoglobulinemia Nephrotic Syndromes: minimal change disease, focal segmental glomerulosclerosis, membranous glomerulonephritis, diabetes, amyloidosis, others Basement Membrane Syndromes: anti-GBM disease, others Glomerular Vascular Syndromes: atherosclerosis, chronic hypertension, sickle-cell disease, thrombotic microangiopathies, anti phospholipid antibody syndrome, ANCA vasculitis, others Infectious Disease-Associated Syndromes: poststreptococcal, infective endocarditis, HIV, HBV, HCV, syphilis, others

1	Infectious Disease-Associated Syndromes: poststreptococcal, infective endocarditis, HIV, HBV, HCV, syphilis, others ANCA = antineutrophilic cytoplasmic antibodies; anti-GBM = anti-glomerular basement membrane; HBV = hepatitis B virus; HCV = hepatitis C virus; HIV = human immunodeficiency virus; IgA = immunoglobulin A; SLE = systemic lupus erythematosus. Adapted from Lewis, 2015. 2016). One example is systemic lupus erythematosus with a flare during the second half of pregnancy (Bramham, 2012; Zhao, 2013). In some of these cases, renal biopsy is sometimes needed to determine etiology and direct management (Lind heimer, 2007a; Ramin, 2006). Whatever the underlying etiology, acute glomerulonephritis has profound efects on pregnancy outcome. One older study described 395 pregnancies in 238 women with primay glo merulonephritis diagnosed before pregnancy (Pacham, 1989).

1	merulonephritis, IgA glomerulonephritis, and difuse mesan gial glomerulonephritis. Although most of these women had normal renal function, half developed hypertension, a fourth were delivered preterm, and the perinatal mortality rate after 28 weeks' gestation was 80 per 1000. As expected, the worst peri natal outcomes were in women with impaired renal function, early or severe hypertension, and nephrotic-range proteinuria. women with IgA nephropathy. From their review of more than 300 such pregnancies, Lindheimer and colleagues (2000) con cluded that pregnancy outcome was related to the degree of renal insuiciency and hypertension. Liu and coworkers (2014) reached similar conclusions. Heavy proteinuria is the hallmark of the nephrotic syndromes.

1	Heavy proteinuria is the hallmark of the nephrotic syndromes. ders that cause immunological or toxin-mediated injury with glomerular capillary wall breakdown to allow excessive filtra tion of plasma proteins. In addition to heavy urine protein excretion, the syndrome is characterized by hypoalbuminemia, hypercholesterolemia, and edema. There frequently is hyper tension, and along with albumin nephrotoxicity, renal insuf iciency eventually develops.

1	Some of the more frequent causes of the nephrotic syndrome are minimal change disease (10-15 percent), focal segmental glomerulosclerosis (35 percent), membranous glomerulonephritis (30 percent), and diabetic nephropathy. In most cases, renal biopsy will disclose microscopic abnormalities that may help direct treatment (Chen, 2015; Lo, 2014). Edema is problematic, especially during pregnancy. Normal amounts of dietary protein of high biological value are encouraged. The incidence of thromboembolism is increased and varies with the severity of hypertension, proteinuria, and renal insuiciency (Stratta, 2006). Although both arterial and venous thromboses may develop, renal vein thrombosis is particularly worrisome. he value, if any, of prophylactic anticoagulation is unclear. Some cases of nephrosis from primary glomerular disease respond to glucocorticosteroids and other immunosuppressants or cytotoxic drug therapy. In most of those cases caused by infection or drugs, proteinuria

1	of nephrosis from primary glomerular disease respond to glucocorticosteroids and other immunosuppressants or cytotoxic drug therapy. In most of those cases caused by infection or drugs, proteinuria recedes when the underlying cause is corrected.

1	Maternal and perinatal outcomes in women with the nephrotic syndromes depend on its underlying cause and severity. Whenever possible, these should be ascertained, and renal biopsy may FIGURE 53-3 Massive vulvar edema in a pregnant woman with the nephrotic syndrome due to secondary syphilis. (Used with permission from Dr. George Wendel, Jr.) be indicated to determine if the etiology will respond to treat ment. Half of women with nephrotic-range proteinuria will have a rise in daily protein excretion as pregnancy progresses (Packham, 1989). In women with nephrosis cared for at Park land Hospital, we reported that two thirds had protein excre tion that exceeded 3 g/d (Stettler, 1992). At the same time, however, if these women had only mild degrees of renal dys function, they had normally augmented CFR across pregnancy (Cunningham, 1990).

1	Management of edema during pregnancy can be particularly challenging as it is intensiied by normally increasing hydrostatic pressure in the lower extremities. In some women, massive vulvar edema may develop. An example of massive vulvar edema associated with the nephrotic syndrome caused by secondary syphilis is shown in Figure 53-3. Another major problem is that up to half of these women have chronic hypertension that may require treatment. In these, as well as in previously normotensive women, preeclampsia is common and often develops early in pregnancy.

1	Most women with nephrotic syndromes who do not have severe hypertension or renal insuiciency will have successful pregnancy outcomes. Conversely, if there is renal insuiciency, moderate to severe hypertension, or both, the prognosis is much worse. In a group of such women with 65 pregnancies cared for at Parkland Hospital, complications were frequent (Stettler, 1992). Protein excretion during pregnancy averaged 4 gl d, and a third of the women had classic nephrotic syndrome. here was some degree of renal insuiciency in 75 percent, chronic hypertension in 40 percent, and persistent anemia in 25 percent. Importantly, preeclampsia developed in 60 percent, and 45 percent had preterm deliveries. Even so, after excluding abortions, 53 of 57 neonates were born alive. In another series, fetal-growth restriction was noted in a third of pregnancies in afected women (Stratta, 2006).

1	Serious long-term adverse outcomes are a risk for women identiied to have nephrotic syndromes either before or during pregnancy (Su, 2017). In our series above, at least 20 percent of women followed for 10 years progressed to end-stage renal disease (Stettler, 1992). Similarly, in another group of 15 women, by 2 years postpartum, three had died, three had developed chronic renal failure, and two had progressed to end-stage renal disease (Chen, 2001). Of predictors, serum creatinine levela> 1.4 mg/ dL and 24-hour protein excretiona> 1 g/d are associated with the shortest renal survival times following pregnancy (Imbasciati, 2007).

1	his describes a pathophysiological process that can progress to end-stage renal disease. he National Kidney Foundation describes six stages of chronic kidney disease defined by decreasing GFR. It progresses from stage O-GFR >90 mLi min/1.73 mato stage 5-GFR < 15 mLimin/1.73 ma. Several diseases can worsen renal function, and many result from one of the glomerular diseases discussed earlier. hose that most frequently lead to end-stage disease requiring dialysis and kidney transplantation and their approximate percentages include: diabetes, 35 percent; hypertension, 25 percent; glomerulonephritis, 20 percent; and polycystic kidney disease, 15 percent (Abboud, 2010; Bargman, 2015).

1	reproductive-aged women with these diseases have varying degrees of renal insuiciency, proteinuria, or both. To counsel regarding fertility and pregnancy outcome, the degree of renal functional impairment and of associated hypertension are assessed. Successful pregnancy outcome in general may be more related to these two factors than to the speciic underlying renal disorder. A general prognosis can be estimated by considering women with chronic renal disease in arbitrary categories of renal function (Davison, 2011). hese include normal or mild impairment--deined as a serum creatinine < 1.5 mg/ dL; moderate impairment-deined as a serum creatinine 1.5 to 3.0 mg/dL; and severe renal insuiciencydeined as a serum creatinine >3.0 mg/dL. Although some have suggested adopting the classification of the National Kidney Foundation, others recommend using the older categories (Davison, 2011a; Piccoli, 201 Oa, 2011). Thus, the obstetrician is ideally familiar with both.

1	Most women have relatively mild renal insuiciency, and its severity along with any underlying hypertension is prognostic of pregnancy outcome. Renal disease with comorbidities secondary to a systemic disorder-for example, diabetes or systemic lupus erythematosus-portends a worse prognosis (Davison, 2011; Koh, 2015). For all women with chronic renal disease, the incidences of hypertension and preeclampsia, preterm and growthrestricted newborns, and other problems are high (Kendrick, 2015). Despite these, the National High Blood Pressure Education Program (2000) concluded that the prognosis has substantively improved since the 1980s. his has been verified by several reviews (Hladunewich, 2016a; Nevis, 2011; Ramin, 2006).

1	Loss of renal tissue is associated with compensatory intrarenal vasodilation and hypertrophy of the surviving nephrons. he resultant hyperperfusion and hyperiltration eventually damage surviving nephrons to cause nephrosclerosis and worsening renal function. With mild renal insuiciency, pregnancy causes 0. ..,c :J,� FIGURE 53-4 Blood volume expansion in 44 normally pregnant women at term compared with 29 who had eclampsia; 10 with moderate chronic renal insufficiency (CRI)-serum creatinine 1.5 to 2.9 mg/dL; and four with severe CRI-serum creatinine �3.0 mg/dL . (Data from Cunningham, 1990; Zeeman, 2009.) greater augmentation of renal plasma low and GFR (Baylis, 2003; Helal, 2012). With progressively declining renal function, there is little, if any, augmented renal plasma low. In one study, only half of women with moderate renal insuiciency demonstrated a pregnancy-augmented GFR, and women with severe disease had no increase (Cunningham, 1990).

1	Importanty, severe chronic renal insuiciency curtails normal pregnancy-induced hypervolemia. Blood volume expansion during pregnancy is related to disease severity and correlates inversely with serum creatinine concentration. As shown in Figure 53-4, women with mild to moderate renal dysfunction have normal blood volume expansion that averages 55 percent. With severe renal insuiciency, however, volume expansion averages only 25 percent, which is similar to that seen with hemoconcentration from eclampsia. In addition, these women have variable degrees of chronic anemia due to intrinsic renal disease. Renal Disease with Preserved Function

1	In some women, although glomerular disease has not yet caused renal dysfunction, incidences of pregnancy complications are still increased. As shown in Table 53-4, these problems are less frequent than in cohorts of women with moderate and severe renal insuiciency. Two earlier studies illustrate this. In one describing 123 pregnancies in women with biopsy-proven glomerular disease, only a few of the women had renal dysfunction, yet 40 percent developed obstetrical or renal complications (Surian, 1984). In another study of 395 pregnancies in women with preexisting glomerulonephritis and minimal renal insuiciency, impaired renal function developed in 15 percent during pregnancy, and 60 percent had worsening proteinuria (Packham, 1989). Only 12 percent had antecedent chronic hypertension, however, more than half of the 395 pregnancies were complicated by hypertension. he perinatal mortality rate was 140 per 1000, but even without early-onset or severe hypertension or nephrotic-range

1	however, more than half of the 395 pregnancies were complicated by hypertension. he perinatal mortality rate was 140 per 1000, but even without early-onset or severe hypertension or nephrotic-range proteinuria, the perinatal

1	TABLE 53-4. Complications (Percent) Associated with 5000 Chronic Renal Disease During Pregnancy 4500 90th .�2500 apreeclampsia only. NS = not stated. Data from Alsuwaida, 2011; Cunningham, 1990; Farwell, 2013; Feng, 2015; Imbasciati, 2007; Maruotti, 2012; Nevis, 2011; Packham, 1989; Piccoli, 201 Oa, 2011; Stettler, 1992; Surian, 1984; Trevisan, 2004. death rate was 50 per 1000. Importantly, in 5 percent of these women, worsening renal function was permanent.

1	In women with chronic kidney disease who also have renal insuiciency, adverse outcomes are generally directly related to the degree of renal impairment. Outcomes of women with moderate versus severe renal insuiciency are usually not separated (Table 53-5). hat said, Piccoli and associates (2010a) described 91 pregnancies complicated by stage 1 chronic kidney disease. Primarily because of hypertension, 33 percent were delivered preterm, and 13 percent had fetal-growth restriction. Alsuwaida and colleagues (2011) reported similar observations. Other investigators have described pregnancies complicated by moderate or severe renal insuiciency (Cunningham, 1990; Imbasciati, 2007; Zhang, 2015). Despite a high incidence of chronic hypertension, anemia, preeclampsia, preterm delivery, (red points). (Data from Cunningham, 1990; Stettler, 1992. Growth curves are those reported by Alexander, 1996.) and fetal-growth restriction, perinatal outcomes were generally acceptable. As shown in Figure

1	(Data from Cunningham, 1990; Stettler, 1992. Growth curves are those reported by Alexander, 1996.) and fetal-growth restriction, perinatal outcomes were generally acceptable. As shown in Figure 53-5, fetal growth is frequently impaired and related to renal dysfunction severity.

1	Prenatal care is tailored for women with chronic renal disease. Frequent monitoring of blood pressure is paramount, and serum creatinine levels, protein/creatinine ratio, and 24-hour protein excretion are quantiied as indicated. Bacteriuria is treated to decrease the risk of pyelonephritis and urther nephron loss. Protein-rich diets are recommended Gim, 2016; Lindheimer, 2000). In some women with anemia from chronic renal insuiciency, a response is seen with recombinant erythropoietin, however, hypertension is a common side efect. Serial sonography is performed to follow fetal growth. he diferentiation between worsening hypertension and superimposed preeclampsia is problematic. Preliminary data indicate that the angiogenic biomarkers placental growth factor (PIGF) and its soluble antiangiogenic receptor (sFlt-l) may be useful to separate chronic from gestational hypertension. This is described in Chapter 40 (p. 716).

1	In some women, pregnancy may accelerate chronic renal disease progression by increasing hyperfiltration and glomerular pressure to worsen nephrosclerosis (Baylis, 2003; Helal, 2012). his is more likely in women with severe chronic renal insuficiency (Abe, 1991 ; Jones, 1996). For example, Jungers and associates (1995) reported few long-term pregnancy-related adverse efects in 360 women with chronic glomerulonephritis and antecedent normal renal function. However, at 1 year after pregnancy, Jones and Hayslett (1996) reported that 10 percent of such women with moderate or severe renal insuiciency had developed end-stage renal failure-stage 5 chronic kidney disease. In a study from Parkland Hospital, we found that 20 percent of pregnant women with similar insuiciency had developed end-stage renal failure by a mean of 4 years (Cunningham, 1990). Similar indings in women with a median follow-up of 3 years were described by Imbasciati and coworkers (2007). By this time, end-stage disease

1	renal failure by a mean of 4 years (Cunningham, 1990). Similar indings in women with a median follow-up of 3 years were described by Imbasciati and coworkers (2007). By this time, end-stage disease was apparent in 30 percent of women whose serum creatinine was ::l.4 mg/dL and who had proteinuriaa> 1 g/d. Chronic proteinuria is also a marker for subsequent development of renal failure. In another report from Parkland Hospital, 20 percent of women with chronic proteinuria discovered during pregnancy progressed to end-stage renal failure within several years (Stettler, 1992).

1	Signiicantly impaired renal function is accompanied by subfertility that may be corrected with chronic renal replacement therapy-either hemodialysis or peritoneal dialysis (Hladunewich, 2016b; Shahir, 2013). Not unexpectedly, these pregnancies can be complicated. In one review of 131 cases, mean fetal birthweights were higher in women who conceived while undergoing dialysis-1530 g versus 1245 g-than in women who conceived before starting dialysis (Chou, 2008). his was also true for 77 pregnancies described by J esudason and coworkers (2014). Similar outcomes from several reports are shown in Table 53-5.

1	Outcomes are similar with either hemodialysis or peritoneal dialysis. hus, for the woman already undergoing either method, it seems reasonable to continue that method with consideration for its increasing frequency. In the woman who has never been dialyzed, the threshold for initiation during pregnancy is unclear. Lindheimer and colleagues (2007a) recommend dialysis when serum creatinine levels are between 5 and 7 mg/ dL. Because it is imperative to avoid abrupt volume changes that cause hypotension, dialysis frequency may be extended to five to six times weekly (Reddy, 2007). Certain protocols emphasize attention to replacement of substances lost through dialysis Qim, 2016). Multivitamin doses are doubled, and calcium and iron salts are provided along with suicient dietary protein and calories. Chronic anemia is treated with erythropoietin. To meet pregnancy changes, extra calcium is added to the dialysate along with less bicarbonate.

1	Maternal complications are common and include severe hypertension, placental abruption, heart ailure, and sepsis. In a review of 90 pregnancies in 78 women, as well as those shown in Table 53-5, high incidences of maternal hypertension and anemia, preterm and growth-restricted infants, stillbirths, and hydramnios were reported (Piccoli, 2010b).

1	Previously termed acute renal ailure, acute kidney injury (AKI) is now used to describe suddenly impaired kidney function with retention of nitrogenous and other waste products normally excreted by the kidneys (W aikar, 2015). Severe AKI associated with pregnancy is less frequent today. For example, in a 6-year period, the overall incidence at the Mayo Clinic was 0.4 percent (Gurrieri, 2012). It is even less common for women who require dialysis-1 case per 10,000 births (Hildebrand, 2015). But, it still occasionally causes signiicant obstetrical morbidity, and women who require acute dialysis have increased maternal mortality rates (Kuklina, 2009; Van Hook, 2014). Outcomes are available from four older studies comprising a total of 266 women with renal failure (Drakeley, 2002; Nzerue, 1998; Sibai, 1990; Turney, 1989). Nearly 70 percent had preeclampsia, 50 percent had obstetrical hemorrhage, and 30 percent had a placental abruption. Almost 20 percent required dialysis, and the

1	1998; Sibai, 1990; Turney, 1989). Nearly 70 percent had preeclampsia, 50 percent had obstetrical hemorrhage, and 30 percent had a placental abruption. Almost 20 percent required dialysis, and the maternal mortality rate was 15 percent.

1	lthough obstetrical cases of AKI that require dialysis have become less prevalent, acute renal ischemia is still often associated with severe preeclampsia and hemorrhage (Gurrieri, 2012; Jim, 2017). Particularly contributory are HELLP (hemolysis, devated liver enzymes, low .latelet levels) syndrome and placental abruption (Audibert, 1996; Drakely, 2002). Septicemia is another frequent comorbidity, especially in resource-poor countries (Acharya, 2013; Srinil, 2011; Zeeman, 2003). AKI is also common in women with acute fatty liver of pregnancy (Sibai, 2007). Some degree of renal insuiciency was found in virtually all of 52 such women cared for at Parkland Hospital (Nelson, 2013). Another woman from Parkland Hospital developed AKI from dehydration caused by severe hyperemesis gravidarum at 15 weeks (Hill, 2002). Other causes include thrombotic micro angiopathies (Balofsky, 2016; Ganesan, 201a1) (Chap. 56, p. 1088).

1	In most women, AKI develops postpartum, thus management is usually not complicated by fetal considerations. An abrupt rise in serum creatinine level is most often due to renal ischemia. Oliguria is an important sign. In obstetrical cases, both prerenal and intrarenal factors are often contributory. For example, with total placental abruption, severe hypovolemia from massive hemorrhage is common, and preexistent renal ischemia from preeclampsia is often comorbid. In addition, disseminated intravascular coagulopathy may be contributory.

1	When azotemia is evident and severe oliguria persists, some form of renal replacement treatment is indicated. Hemofiltration or dialysis is initiated before marked deterioration occurs. Hemodynamic measurements are normalized. Importantly, medication doses are adjusted, and magnesium sulfate, iodinated contrast agents, aminoglycosides, and nonsteroidal antiinlammatory drugs (NSAIDs) are prominent examples (Waikar, 2015). Early dialysis appears to reduce the maternal mortality rate appreciably and may enhance the extent of renal function recovery. With time, renal function usually returns to normal or near normal. AKI in obstetrics is most often due to acute blood loss, especially that associated with preeclampsia. hus, it may often be prevented by the following means: 1.

1	AKI in obstetrics is most often due to acute blood loss, especially that associated with preeclampsia. hus, it may often be prevented by the following means: 1. Prompt and vigorous volume replacement with crystalloid solutions and blood in instances of massive hemorrhage, such as in placental abruption, placenta previa, uterine rupture, and postpartum uterine atony (Chap. 41, p. 788). 2. Delivery or termination of pregnancies complicated by severe preeclampsia or eclampsia, and careful blood transfusion ifloss is more than average (Chap. 40, p. 718). 3. Close observation for early signs of sepsis syndrome and shock in women with pyelonephritis, septic abortion, chorioamnionitis, or sepsis from other pelvic infections (Chap. 47, p. 921). 4. Avoidance of loop diuretics to treat oliguria before ensuring that blood volume and cardiac output are adequate for renal perfusion. 5.

1	4. Avoidance of loop diuretics to treat oliguria before ensuring that blood volume and cardiac output are adequate for renal perfusion. 5. Judicious use of vasoconstrictor drugs to treat hypotension, and only after it has been determined that pathological vasodilatation is the cause.

1	Irreversible ischemic renal failure caused by acute cortical necrosis is rare now in obstetrics (F rimat, 2016). Before widespread availability of dialysis, it complicated a fourth of obstetrical renal failure cases (Griinfeld, 1987; Turney, 1989). Most cases followed placental abruption, preeclampsia-eclampsia, and endotoxin-induced shock. Once common with septic abortion, this is a rare cause in this country today (Lim, 2011; Srinil, 2011). Histologically, the lesion appears to result from thrombosis of segments of the renal vascular system. he lesions may be focal, patchy, conluent, or gross. Clinically, renal cortical necrosis follows the course of AKI, and its diferentiation from acute tubular necrosis is not possible during the early phase. he prognosis depends on the extent of the necrosis. Recovery of function is variable, and stable renal insuiciency may result (Lindheimer, 2007a).

1	Rarely, bilateral ureteral compression by a very large pregnant uterus is greatly exaggerated. Resultant ureteral obstruction in turn may cause severe oliguria and azotemia. An extreme example is shown in Figure 53-6. In their series of 13 obstruction cases, Brandes and Fritsche (1991) described one woman with twins who developed anuria and a serum creatinine level of 12.2 mg/dL at 34 weeks' gestation. After amniotomy, urine flow resumed at 500 mL/hr, and her serum creatinine levels rapidly dropped to normal range. Eckford and Gingell (1991) described 10 women in whom ureteral obstruction was relieved by stenting. he stents were left in place for a mean of 15.5 weeks and removed 4 to 6 weeks postpartum. Others have reported similar experiences (Sadan, 1994; Satin, 1993).

1	FIGURE 53-6 A.Magnetic resonance image in a coronal plane of a pregnant woman with unilateral hydronephrosis caused by ureteral obstruction. The serum creatinine level was 8 mg/dL and decreased to 0.8 mg/dL ater a percutaneous nephrostomy tube was placed. B. Let kidney (arrow) and associated hydronephrosis (asterisk) are again noted in this axial plane image from the same patient. Partial ureteral obstruction may be accompanied by fluid retention and significant hypertension. When the obstructive uropathy is relieved, diuresis ensues and hypertension dissipates. In our experience, women with previous urinary tract surgery for reflux are more likely to have such obstructions.

1	Although infrequently complicating pregnancy, this type of diverticulum originates from an enlarging paraurethral gland abscess that ruptures into the urethral lumen. As infection clears, the remaining dilated diverticular sac and its ostium into the urethra persist. Urine collecting within and dribbling from the sac, pain, a palpable mass, and recurrent urinary infections may be associated findings. In general, a diverticulum is managed expectantly during pregnancy. Rarely, drainage may be necessary, or surgery required (Iyer, 2013). If additional antepartum evaluation is needed, MR imaging is preferred for its superior soft tissue resolution and ability to deine complex diverticula (Dwarkasing, 2011; Pathi, 2013) .

1	Fistulas found during pregnancy likely existed previously, but in rare cases, they form during pregnancy. In developed countries, vesicovaginal or cervicovaginal istula following a McDonald cerclage has been reported (Massengill, 2012; Zanconato, 2015). These fistulas may also form with prolonged obstructed labor that is more often seen in resource-poor countries (Cowgill, 2015). In these cases, the genital tract is compressed between the fetal head and bony pelvis. Brief pressure is not signiicant, but prolonged pressure leads to tissue necrosis with subsequent fistula formation (Wall, 2012). Vesicouterine istulas can develop after prior vaginal or cesarean delivery (DiMarco, 2006; Harfouche, 2014; Manjunatha, 2012). Rarely, vesicocervical istula can follow cesarean delivery or may form if the anterior cervical lip is compressed against the symphysis pubis (Dudderidge, 2005). Finally, an ileouterine istula from a degenerating posterior wall fibroid tumor has been described (Shehata,

1	if the anterior cervical lip is compressed against the symphysis pubis (Dudderidge, 2005). Finally, an ileouterine istula from a degenerating posterior wall fibroid tumor has been described (Shehata, 2016).

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1	Urol Clin North Am 40(1):79, 2013 Toth C, Toth G, Varga A, et al: Percutaneous nephrolithotomy in early pregnancy. Int Urol NephroI37:1, 2005 Towers CV, Kaminskas CM, Garite TJ, et al: Pulmonary injury associated with antepartum pyelonephritis: can patients at risk be identified? Am J Obstet Gynecol 164:974, 1991 Trevisan G, Ramos JG, Martins-Costa S, et al: Pregnancy in patients with chronic renal insuiciency at Hospital de Clinicas of Porto Alegre, Brazil. Ren Fail 26:29, 2004 Turney JH, Ellis CM, Parsons FM: Obstetric acute renal failure 1956-1987. BJOG 96:679, 1989 U.S. Preventive Services Task Force. Screening for asymptomatic bacteriuria in adults. Reairmation recommendation statement. 2008. Available at: http://ww. uspreven tiveservicestaskforce.org/ uspstf08/ asym ptbact/ asbactrs. htm. Accessed September 22,2016

1	Van Dorsten JP, Lenke RR, Schifrin BS: Pyelonephritis in pregnancy: the role of in-hospital management and nitrofurantoin suppression. ] Reprod Med 32:897, 1987 Van Hook JW: Acute kidney injury during pregnancy. Clin Obstet Gynecol (4):851-61,s2014 Waikar SS, Bonventre J\\1: Acute kidney injury. In Kasper DL, Fauci AS, Hauser SL, et al (eds): Harrison's Principles of Internal Medicine, 19th ed. New York, McGraw-Hill Education, 2015 Wall LL: Preventing obstetric fistulas in low-resource countries: insights from a Haddon matrix. Obstet Gynecol Surv 67(2):111,s2012 %alley PJ: Bacteriuria of pregnancy. Am J Obstet Gynecol 97:723, 1967 White WM, Zite NB, Gash J, et al: Low-dose computed tomography for the evaluation of flank pain in the pregnant population. J Endourol 21(11):1255,s2007 Widmer M, Lopez 1, Gulmezoglu AM, et al: Duration of treatment for asymptomatic bacteriuria during pregnancy. Cochrane Database Syst Rev II:CD000491,s2015

1	Widmer M, Lopez 1, Gulmezoglu AM, et al: Duration of treatment for asymptomatic bacteriuria during pregnancy. Cochrane Database Syst Rev II:CD000491,s2015 Wing DA, Fassett MJ, Getahun D: Acute pyelonephritis in pregnancy: an 18-year retrospective analysis. Am J Obstet Gynecol 210(3):219.el, 2014 Wing DA, Hendershott CM, Debuque L, et al: A randomized trial of three antibiotic regimens for the treatment of pyelonephritis in pregnancy. Am J Obstet Gynecol 92:249, 1998 Wing DA, Hendershott CM, Debuque L, et al: Outpatient treatment of acute pyelonephritis in pregnancy after 24 weeks. Obstet Gynecol 94:683, 1999 Wing DA, Park AS, DeBuque L, et al: Limited clinical utiliry of blood and urine cultures in the treatment of acute pyelonephritis during pregnancy. Am J Obstet GynecoIs182:1437, 2000 Wright A], Gill JS: Strategies to prevent BK virus infection in kidney transplant recipients. Curr Opin Infect Dis 29(4):353, 2016

1	Wright A], Gill JS: Strategies to prevent BK virus infection in kidney transplant recipients. Curr Opin Infect Dis 29(4):353, 2016 Wyatt RJ, Julian BA: IgA nephropathy. N Engl J Med 368(25):2402,s2013 Wyld ML, Clayton PA, Jesudason S, et al: Pregnancy outcomes for kidney transplant recipients. Am J Transplant 13:3173,s2013 Zanconato G, Bergamini V, Baggio S, et al: Successful pregnancy outcomes after laparoscopic cerclage in a patient with cervicovaginal fistula. Case Rep Obstet GynecoIs2015:784025, 2015 Zeeman GG, Cunningham GC, PritchardJA: he magnitude ofhemoconcentration with eclampsia. Hypertens Pregnancy 28(2):127,s2009 Zeeman GG, Wendel GO Jr, Cunningham FG: A blueprint for obstetric critical care. Am J Obstet Gynecol 188:532,2003 Zhang n, MaX, Hao L, et al: A systematic review and meta-analysis of outcomes of pregnancy in CKD and CKD outcomes in pregnancy. Clin J Soc Nephrols10(11):1964,s2015

1	Zhang n, MaX, Hao L, et al: A systematic review and meta-analysis of outcomes of pregnancy in CKD and CKD outcomes in pregnancy. Clin J Soc Nephrols10(11):1964,s2015 Zhao C, Zhao J, Huang Y, et al: New-onset systemic lupus erythematosus during pregnancy. Clin Rheumatol 32(6):815, 2013 Zhou J, Pollak MR: Polycystic kidney disease and other inherited disorders of tubule growth and development. In Kasper DL, Fauci AS, Hauser SL, et al (eds): Harrison's Principles of Internal Medicine, 19th ed. New York, McGraw-Hill Education, 2015 GENERAL CONSIDERATIONS . . . . . . . ............. 1042 UPPER GASTROINTESTINAL TRACT DISORDERS ..... 1043 HYPEREMESIS GRAVIDARUM.. . . . . . . ............. 1043 GASTROESOPHAGEAL REFLUX DISEASE ........... 1046 PEPTIC ULCER DISEASE.. . . . . . . . . . . . ............. 1047 SMALL BOWEL AND COLON DISORDERS .......... 1047 ACUTE DIARRHEA.. . . . . . . . . . . . . . . . ............. 1047 INFLAMMATORY BOWEL DISEASE ....i....i........i.... 1048

1	SMALL BOWEL AND COLON DISORDERS .......... 1047 ACUTE DIARRHEA.. . . . . . . . . . . . . . . . ............. 1047 INFLAMMATORY BOWEL DISEASE ....i....i........i.... 1048 INTESTINAL OBSTRUCTION . . . . . . . . ............. 1051 APPENDICITIS ..................i.............. 1052 The diagnosis of acute appendicitis is more dficult than at other times, as the enlarged uterus renders it almost impossible to explore the right iliac region satiactoriy.

1	The diagnosis of acute appendicitis is more dficult than at other times, as the enlarged uterus renders it almost impossible to explore the right iliac region satiactoriy. -J. Whitridge Williams (1903) hese words summarize that during normal pregnancy, the gastrointestinal tract and its appendages undergo remarkable anatomical, physiological, and functional alterations. hese changes, which are discussed in detail in Chapter 4 (p. 68), can appreciably alter clinical findings normally relied on for diagnosis and treatment of gastrointestinal disorders such as appendicitis. Moreover, as pregnancy progresses, gastrointestinal symptoms become more diicult to assess. Physical findings are often obscured by a large uterus that displaces abdominal organs and can alter the location and intensity of pain and tenderness.

1	Fiberoptic endoscopic instruments have revolutionized diagnosis and management of most gastrointestinal conditions, and these are particularly well suited for pregnancy. Endoscopy in pregnancy is associated with a slightly increased risk for preterm birth, but this is likely due to the disease itself (Ludvigsson, 2017). With endoscopy, the esophagus, stomach, duodenum, and colon can be inspected (Cappell, 2011; Savas, 2014). The proximal jejunum can also be studied, and the ampulla of Vater cannulated to perform endoscopic retrograde cholangiopancreatography-ERCP (Akcakaya, 2014; Fogel, 2014). Preliminary data suggest that postendoscopic pancreatitis following gallstone removal may have a higher incidence in pregnant women (Inamdar, 2016). Experience in pregnancy with videocapsule endoscopy for small-bowel evaluation remains limited (Storch, 2006).

1	Upper gastrointestinal endoscopy is used for management as well as diagnosis of several problems. Common bile duct exploration and drainage are used for choledocholithiasis as described in Chapter 55 (p. 1070). It is also used for sclerotherapy and for placement of percutaneous endoscopic gastrostomy (PEG) tubes. Several concise reviews have been provided (Cappell, 2011; Fogel, 2014; Gilinsky, 2006).

1	For visualization of the large bowel, lexible sigmoidoscopy can be used safely in pregnant women (Siddiqui, 2006). Coonoscopy is indispensible for viewing the entire colon and distal ileum to aid diagnosis and management of several bowel disorders. Except for the midtrimester, reports of colonoscopy during pregnancy are limited, but most results indicate that it should be performed if indicated (Cappell, 2010, 2011; De Lima, 2015). Bowel preparation is completed using polyethylene glycol electrolyte or sodium phosphate solutions. With these, serious maternal dehydration that may cause diminished uteroplacental perusion should be avoided.

1	he obvious ideal technique for gastrointestinal evaluation during pregnancy is abdominal sonography. Because computed tomography (CT) use is limited in pregnancy due to radiation exposure, magnetic resonance (MR) imaging is now commonly used to evaluate the abdomen and retroperitoneal space (handelwal, 2013). One example is magnetic resonance cholangiopancreatography-MRCP (Oto, 2009). Another is magnetic resonance enterography-MRE (Stern, 2014). These and other imaging modalities, and their safe use in pregnancy, are considered in more detail in Chapter 46.

1	Surgery is lifesaving for certain gastrointestinal conditionsperforative appendicitis being the most common example. Laparoscopic procedures have replaced traditional surgical techniques for many abdominal disorders during pregnancy. hese are shown in detail with descriptions of surgical technique in Chapter 46 (p. 903) and in Cunningham and Gilstrap 5 Operative Obstetrics, 3rd edition (Kho, 2016). Guidelines for diagnosis, treatment, and use of laparoscopy for surgical problems during pregnancy have been provided by the Society of American Gastrointestinal and Endoscopic Surgeons-SAGES (Pearl, 2017). Specialized nutritional support can be delivered enteraly, usually via nasogastric tube feedings, orparenteraly with nutrition given by venous catheter access, either peripherally or centrally.

1	Specialized nutritional support can be delivered enteraly, usually via nasogastric tube feedings, orparenteraly with nutrition given by venous catheter access, either peripherally or centrally. When possible, enteral alimentation is preferable because it has fewer serious complications (Bistrian, 2012; Stokke, 2015). In obstetrical patients, very few conditions prohibit enteral nutrition as a first efort to prevent catabolism. For extreme cases, such as recalcitrant hyperemesis gravidarum, percutaneous endoscopic gastrostomy with a jejunal port (PEG-J tube) has been described (Saha, 2009). he purpose of parenteral eeding, or hyperalimentation, is to provide nutrition when the intestinal tract must be quiescent. Central venous access is necessary for total parenteral nutrition because its hyperosmolarity requires rapid dilution in a highflow vascular system. hese solutions provide 24 to 40 kcall kg/ d, principally as a hypertonic glucose solution.

1	Not surprisingly, gastrointestinal disorders are the most common indication, and in the many studies cited, feeding duration averaged approximately 33 days. Importantly, complications of parenteral nutrition are frequent, and they may be severe (Guglielmi, 2006). n early report of 26 pregnancies described a 50-percent rate of complications, which included pneumothorax, hemothorax, and brachial plexus injury (Russo-Stieglitz, 1999). he most frequent serious complication is catheter sepsis, and Folk (2004) reported a 25-percent incidence in 27 women with hyperemesis gravidarum. Although bacterial sepsis is most common, Candia septicemia has been described (Paranyuk, 2006). he Centers for Disease Control and Prevention has updated its detailed guidelines to prevent catheter-related sepsis, and these serve to lessen the dangers of serious infections (O'Grady, 2011). Perinatal complications are

1	TABLE 54-1. Some Conditions Treated with Enteral or Parenteral Nutrition During Pregnancya aOisorders are listed alphabetically. Data from Folk, 2004; Guglielmi, 2006; Manliadevan, 2015; Ogura, 2003; Porter, 2014; Russo-Stieglitz, 1999; Saha, 2009; Spiliopoulos, 201n3 . uncommon, however, fetal subdural hematoma caused by mater nal vitamin K deiciency has been described (Sakai, 2003). Appreciable morbidity is also associated with long-term use of a peripheraly inserted central catheter (PICC). Infection is the most common serious long-term complication (Holmgren, 2008; Ogura, 2003). In a series of84 such catheters inserted in 66 preg nant women, Cape and coworkers (2014) reported a 56-percent complication rate, of which bacteremia was the most frequent.

1	Ogura, 2003). In a series of84 such catheters inserted in 66 preg nant women, Cape and coworkers (2014) reported a 56-percent complication rate, of which bacteremia was the most frequent. From a review of 48 reports of nonpregnant adults, Turcotte and associates (2006) concluded that peripherally placed catheters provided no advantages compared with centrally placed ones. Still, for short-term nutrition lasting a few weeks, it seems reasonable that PICC placement has a greater beneit-versusrisk ratio (Bistrian, 2012).

1	Mild to moderate nausea and vomiting are especially common in pregnant women until approximatel) 16 weeks' gestation (Chap. 9, p. 174). In a small but significant proportion of these, however, it is severe and unresponsive to simple dietary modiication and antiemetics. Severe unrelenting nausea and vomiting-hyperemesis gravidarum-is deined variably as being suiciently severe to produce weight loss, dehydration, ketosis, alkalosis from loss of hydrochloric acid, and hypokalemia. Acidosis develops from partial starvation. In some women, transient hepatic dysfunction develops, and biliary sludge accumulates (Matsubara, 2012). Other causes should be considered because ultimately hyperemesis gravidarum is a diagnosis of exclusion (Benson, 2013).

1	Study criteria have not been homogeneous, thus reports of population incidences vary. here does, however, appear to be an ethnic or familial predilection (Grjibovski, 2008). In population-based studies from California, Nova Scotia, and Norway, the hospitalization rate for hyperemesis gravidarum was 0.5 to 1 percent (Bailit, 2005; Fell, 2006; Vikanes, 2013). Up to 20 percent of those hospitalized in a previous pregnancy for hyperemesis will again require hospitalization (Dodds, 2006; Trogstad, 2005). In general, obese women are less likely to be hospitalized for this (Cedergren, 2008).

1	The etiopathogenesis of hyperemesis gravidarum is unknown and is likely multifactorial. It apparently is related to high or rapidly rising serum levels of pregnancy-related hormones. Putative culprits include human chorionic gonadotropin (hCG), estrogen, progesterone, leptin, placental growth hormone, prolactin, thyroxine, and adrenocortical hormones (Verberg, 2005). More recently implicated are other hormones that include ghrelins, leptin, nesfatin-1, and peptide Y (3-36) (Albayrak, 2013; Gungor, 2013).

1	Superimposed on this hormonal cornucopia are an imposing number of biological and environmental factors. Moreover, in some but not all severe cases, interrelated psychological components playaa major role (Christodoulou-Smith, 20a11a; McCarthy, 2011). Other factors that increase the risk for admission include hyperthyroidism, previous molar pregnancy, diabetes, gastrointestinal illnesses, some restrictive diets, and asthma and other allergic disorders (Fell, 2006; Mullin, 2012). n association of Helicobacter pylori infection has been proposed, but evidence is not conclusive (Goldberg, 2007). Chronic marijuana use may cause the similar cannabinoid hyperemesis syndrome (Alaniz, 2015; Andrews, 2015). And for unknown reasons-perhaps estrogenrelated-a female fetus increases the risk by 1.5-fold (Schif, 2004; Tan, 2006; Veenendaal, 2011). Finally, some but not all studies have reported an association between hyperemesis gravidarum and preterm labor, placental abruption, and preeclampsia

1	(Schif, 2004; Tan, 2006; Veenendaal, 2011). Finally, some but not all studies have reported an association between hyperemesis gravidarum and preterm labor, placental abruption, and preeclampsia (Bolin, 2013; Vandraas, 2013; Vikanes, 20l3).

1	Vomiting may be prolonged, frequent, and severe, and a list of potentially fatal complications is given in Table 54-2. Various TABLE 54-2. Some Serious and Life-Threatening Complications of Recalcitrant Hyperemesis Gravidarum Acute kidney injury-may require dialysis Depression-cause versus efect? Diaphragmatic rupture

1	TABLE 54-2. Some Serious and Life-Threatening Complications of Recalcitrant Hyperemesis Gravidarum Acute kidney injury-may require dialysis Depression-cause versus efect? Diaphragmatic rupture Esophageal rupture-Boerhaave syndrome Hypoprothrombinemia-vitamin K deficiency Hyperalimentation complications Mallory-Weiss tears-bleeding, pneumothorax, pneumomediastinum, pneumopericardium Rhabdomyolysis degrees of acute kidney injury from dehydration are encountered (Nwoko, 2012). An extreme example was a woman we cared for who required 5 days of dialysis when her serum creatinine level rose to 10.7 mg/dL (Hill, 2002). One complication from continuous retching is a Mallory-Weiss tear. Others are pneumothorax, pneumomediastinum, diaphragmatic rupture, and gastroesophageal rupture-Boerhaave syndrome (American College of 0bstetricians and Gynecologists, 2015; Chen, 2012).

1	At least two serious vitamin deiciencies have been reported with hyperemesis in pregnancy. One is Wernicke encephalopathy from thiamine deiciency that has been recognized with increasing frequency (Di Gangi, 2012; Palacios-vIarques, 2012). In two reviews, ocular signs, confusion, and ataxia were common, but only half had this triad (Chiossi, 2006; Selitsky, 2006). With this encephalopathy, an abnormal electroencephalogram (EEG) may be seen, and usually MR imaging shows findings (Vaknin, 2006; Zara, 2012). At least three maternal deaths have been described, and long-term sequelae include blindness, convulsions, and coma (Selitsky, 2006). he second is vitamin K deicieny that has been reported to cause maternal coagulopathy and fetal intracranial hemorrhage, as well as vitamin K embryopathy (Kawamura, 2008; Lane, 2015; Sakai, 2003). One algorithm for management of nausea and vomiting of pregnancy is shown in Figure Most women with mild

1	One algorithm for management of nausea and vomiting of pregnancy is shown in Figure Most women with mild Mild Dietary management; Ginger extract; Vitamin 86 plus doxylamine, diphenhydramine, or dimenhydrinate Moderate Severe Promethazine, Intravenous hydration prochlorperazine, with thiamine; trimethobenzamide, Parenteral: chlorpromazine, metoclopramide, metoclopramide, or promethazine, or ondansetron ondansetron (oral, rectal, parenteral) Intractable Enteral or parenteral nutrition ment of hyperemeSis gravidarum. TABLE 54-3. Medications for Gastric Disorders in Pregnancy At bedtime; up to 4 times daily 12.5-25 mg 1M, IV, PO, PR 1M, IV, PO, PR IV, PO 1M, IV, PO

1	TABLE 54-3. Medications for Gastric Disorders in Pregnancy At bedtime; up to 4 times daily 12.5-25 mg 1M, IV, PO, PR 1M, IV, PO, PR IV, PO 1M, IV, PO Pantoprazole (Protonix)b Lansoprazole (Prevacid)b Omeprazole (Prilosec, Zegeridr Dexlansoprazole (Dexilant)C 400 mg 4 times daily for up to 12 wks 800 mg twice daily for up to 12 wks 150 mg twice daily 20 mg twice daily up to 6 wks 40 mg daily for up to 8 wks 15 mg daily for up to 8 wks 20 mg daily for 4-8 wks 30 mg daily for up to 4 wks aFood and Drug Administration category A. bFood and Drug Administration category B. (Food and Drug Administration category C.

1	to moderate symptoms respond as outpatients to any of several irst-line antiemetic agents (Clark, 2014; Matthews, 2014). One that is becoming a mainstay is Diclegis-a combination of doxylamine (10 mg) plus pyridoxine (10 mg). It has been proven safe and efective (Briggs, 2015; Koren, 2014). The usual dose is two tablets orally at bedtime. If relief is insufficient, then additional doses, first in the morning, and then in the morning and midafternoon can be added each day to the bedtime dose. At our institution, for cost savings, we prescribe these two agents individually: Unisom (doxylamine) Y2 of a 50-mg tablet plus a 25-mg vitamin B6 tablet. The same graduated dosing is used but does not exceed three total daily doses. Ondansetron (Zofran) also does not appear to be teratogenic.

1	Ondansetron (Zofran) also does not appear to be teratogenic. It was slightly more eicacious than a combination of doxylamine and pyridoxine in a randomized trial (Oliveira, 2014; Pasternak, 2013). Its drawbacks include potential maternal efects from prolonged QT-interval and serotonin syndrome (Koren, 2014). When simple measures fail, intravenous crystalloid solutions are given to correct dehydration, ketonemia, electrolyte deicits, acid-base imbalances, and hypokalemia. No benefits are gained by infusing 5-percent dextrose along with crystalloids (Tan, 20l3). Thiamine, 100 mg, is given to prevent Wernicke encephalopathy (Giugale, 2015; Niebyl, 2010). his is usually diluted in 1 L of the selected crystalloid and infused at the maintenance rate desired for patient hydration.

1	If vomiting persists after rehydration and failed outpatient management, hospitalization is recommended (American College of Obstetricians and Gynecologists, 2015). Day care has also been shown to be efective in one randomized study (McCarthy, 2014). Intravenous hydration is continued and antiemetics such as promethazine, prochlorperazine, chlorpromazine, or metoclopramide are given parenterally (Table 54-3). The bulk of evidence is that treatment with glucocorticosteroids is not efective (Yost, 2003). Because of their putative teratogenicity, they are not routinely recommended (American College of Obstetricians and Gynecologists, 2015).

1	With persistent vomiting after hospitalization, appropriate steps should be taken to exclude possible underlying diseases as a cause of hyperemesis. That said, in one study, endoscopy did not change management in 49 women (Debby, 2008). Other potential causes of vomiting include gastroenteritis, cholecystitis, pancreatitis, hepatitis, peptic ulcer, and pyelonephritis. In addition, severe preeclampsia and fatty liver are more likely after midpregnancy. And although clinical thyrotoxicosis has been implicated as a cause of hyperemesis, it is more likely that abnormally elevated serum thyroxine levels are a surrogate for higher-than-average serum hCG levels (Sun, 2014). This is discussed further in Chapter 5 (p. 100). In our experiences, serum free thyroxine levels normalize quickly with hydration and emesis treatment.

1	With treatment, most women will have a salutary response and may be sent home with antiemetic therapy. Their readmission rate is 25 to 35 percent in most prospective studies. If associated psychiatric and social factors contribute to the illness, the woman usually improves remarkably while hospitalized (Swallow, 2004). That said, symptoms may relapse in these women, and some go on to develop posttraumatic stress syndrome (Christodoulou-Smith, 2011; McCarthy, 2011). For some women, hyperemesis can be an indication for elective termination (Poursharif, 2007).

1	In the small percentage of women who continue to have recalcitrant vomiting after intensive therapy, consideration is given for enteral nutrition (p. 1043). Stokke and associates (2015) described successful use of nasojejunal feeding for up to 41 days in 107 such women. Use of sonography to conirm correct placement of the tube has been described (Swartzlander, 2013). Percutaneous endoscopic gastrostomy with a jejunal port has also been reported (Saha, 2009; Schrag, 2007). A randomized trial failed to show any advantages from early enteral feeding (Grooten, 2017). In our experiences, only a very few women will require parenteral nutrition (Yost, 2003). In a study of 599 women, however, Peled and coworkers (2014) reported that 20 percent required central venous access to be established for nutrition.

1	Symptomatic reflux is seen in up to 15 percent of nonpregnant individuals (Kahrilas, 2015). he spectrum of sequelae includes esophagitis, stricture, Barrett esophagus, and adenocarcinoma. The main symptom of relux is heartburn, or pyrosis, which is especially common in pregnancy. Its prevalence rose from 26 percent in the irst trimester to 36 percent in the second and 51 percent in the third trimesters (Malfertheiner, 2012). The retrosternal burning sensation stems from esophagitis caused by gastroesophageal relux related to relaxation of the lower esophageal sphincter.

1	Reflux symptoms usually respond to tobacco and alcohol abstinence, small meals, head of the bed elevation, and avoidance of postprandial recumbency. So-called "trigger" foods are also avoided and usually include fatty foods, tomato-based foods, and cofee. Oral antacids are first-line therapy. If severe symptoms persist, sucralfate (Carafate) is given along with a protonpump inhibitor or an H2-receptor antagonist (see Table 54-3). Both classes are generally safe for use in pregnancy (Briggs, 2015; Mahadevan, 2006b). Of these, a 1-g sucralfate tablet is taken orally 1 hour before each of the three meals and at bedtime for up to 8 weeks. Antacids are not used within Y2 hour before or after sucralfate doses. If relief is not attained, then endoscopy should be considered. Misoprostol is contraindicated because it stimulates labor (Chap. 26, p. 508).

1	In nonpregnant patients, surgical fundoplication is performed (Kahrilas, 2015). Although the procedure is not done during pregnancy, Biertho and colleagues (2006) described 25 women who had undergone laparoscopic Nissen fundoplication before pregnancy. Only 20 percent had relux symptoms during pregnancy. he older literature is informative regarding hiatal hernias in pregnancy. Upper gastrointestinal radiographs performed in 195 women in late pregnancy showed that 20 percent of 116 multiparas and 5 percent of 79 nulliparas had a hiatal hernia (Rigler, 1935). Of 10 women studied postpartum, hernia persisted in three at 1 to 18 months.

1	he relationship of hiatal hernia with reflux esophagitis, and thus symptoms, is not clear. One study demonstrated no relationship between reflux and hernia and showed that the lower esophageal sphincter functioned efectively even when displaced intrathoracically (Cohen, 1971) . Nevertheless, during pregnancy, these hiatal hernias may cause vomiting, epigastric pain, and bleeding from ulceration. Schwentner (201a1) reported severe herniation requiring surgical repair in a woman with a 12-week gestation. Curran and coworkers (1999) described a 30-week pregnancy complicated by gastric outlet obstruction from a paraesophageal hernia.

1	These are caused by herniations of abdominal contents through either the foramen of Bochdalek or vforgagni. Fortunately, they rarely complicate pregnancy. Kurzel and associates (1988) reviewed the outcomes of 18 pregnant women with such a hernia and who developed acute obstruction. Because the maternal mortality rate was 45 percent, they recommend repair during pregnancy even if a woman is asymptomatic. Herniation has been reported in one pregnant woman from a previous traumatic diaphragmatic defect and in another who had antirelux surgery in early pregnancy (Brygger, 2013; Flick, 1999). Several case reports also describe spontaneous diaphragmatic rupture from increased intraabdominal pressure during delivery (Chen, 2012; Sharifah, 2003).

1	his is a rare motility disorder in which the lower esophageal sphincter does not relax properly with swallowing. There is also nonperistaltic contraction activity of the esophageal muscularis to cause symptoms (Kahrilas, 2015; Khudyak, 2006). The defect is caused by inflammatory destruction of the myenteric (Auerbach) plexus within smooth muscle of the lower esophagus and its sphincter. Postganglionic cholinergic neurons are unafected, thus, sphincter stimulation is unopposed. Symptoms are dysphagia, chest pain, and regurgitation. Barium swallow radiography demonstrates bird beak or ace of spades narrowing at the distal esophagus. Endoscopy is performed to exclude gastric carcinoma, and manometry is confirmatory. If dilatation of the esophagus and medical therapy does not provide relief, myotomy is considered (Torquati, 2006).

1	During pregnancy, normal relaxation of the lower esophageal sphincter in women with achalasia theoretically should not occur. Even so, in most women, pregnancy does not seem to worsen achalasia. One report of 20 afected pregnant women found no excessive relux esophagitis (Mayberry, 1987). Khudyak and coworkers (2006) reviewed 35 cases and described most women as symptom free, although esophageal dilatation was needed in a few. A maternal death was reported at 24 weeks' gestation associated with perforation of a 14-cm diameter megaesophagus (Fassina, 1995).

1	Management of achalasia includes soft diet and anticholinergic drugs. With persistent symptoms, other options include nitrates, calcium-channel antagonists, and botulinum toxin A injected locally (Hooft, 2015; Kahrilas, 2015). Balloon dila tation of the sphincter may be necessary, and 85 percent of nonpregnant patients respond to this. Satin (1a992) and Fiest (1993) and their associates reported successful use of pneu matic dilatation in pregnancy. One caveat is that esophageal peroration is a serious complication of dilatation. Spiliopoulos and colleagues (2013) described a 29-week pregnant woman with achalasia treated for 10 weeks with parenteral nutrition. Surgical correction was performed postpartum.

1	he lifetime prevalence of acid peptic disorders in women is 10 percent (Del Valle, 2015). Erosive ulcer disease involves the stomach and duodenum. Gastroduodenal ulcers may be caused by chronic gastritis from H pylori, or they develop from nonsteroidal antiinlammatory drug (NSAID) use. Neither is common in pregnancy (McKenna, 2003; Weyermann, 2003). Acid secretion is also important, and thus underlies the eicacy of antisecretory agents (Suerbaum, 2002). Gastroprotection during pregnancy probably originates from physiological changes that include reduced gastric acid secretion, decreased motility, and considerably increased mucus secretion (Hytten, 1991). Despite this, ulcer disease may be underdiagnosed because of frequent treatment for reflux esophagitis (Mehta, 2010). In the past 50 years at Parkland Hospital, during which time we have cared for more than 500,000 pregnant women, we have encountered very few who had proven ulcer disease. Perforation is rare (Goel, 2014). Before

1	years at Parkland Hospital, during which time we have cared for more than 500,000 pregnant women, we have encountered very few who had proven ulcer disease. Perforation is rare (Goel, 2014). Before appropriate therapy was commonplace, Clark (1953) studied 313 pregnancies in 11a8 women with ulcer disease and noted a clear remission during pregnancy in almost 90 percent. However, beneits were short lived. Symptoms recurred in more than half by 3 months postpartum and in almost all by 2 years.

1	The mainstay of management is eradication of H pylori and prevention of NSAID-induced disease. Antacids are usually self-prescribed, but irst-line therapy is with HTreceptor blockers or proton-pump inhibitors (Del Valle, 2015). Sucraote is the aluminum salt of sulfated sucrose that inhibits pepsin. It provides a protective coating at the ulcer base. Approximately 10 percent of the aluminum salt is absorbed, and it is considered safe for pregnant women (Briggs, 2015). With active ulcers, a search for H pylori is undertaken.

1	With active ulcers, a search for H pylori is undertaken. Diagnostic aids include the urea breath test, serological testing, or endoscopic biopsy. If any of these yield positive results, combination antimicrobial and proton-pump inhibitor therapy is indicated. Several efective oral treatment regimens do not include tetracycline and can be used during pregnancy. hese 14-day regimens include amoxicillin, 1000 mg twice daily plus clarithromycin, 250 to 500 mg twice daily, plus metronidazole, 500 mg twice daily given along with the proton-pump inhibitor omeprazole (Del Valle, 2015).

1	In some women, persistent vomiting is accompanied by worrisome upper gastrointestinal bleeding. Occasionally, a peptic ulceration is the source. However, most of these women have small linear mucosal tears near the gastroesophageal junction-Malloy-Weiss tears, described earlier. Bleeding usually responds promptly to conservative measures, including iced-saline irrigations, topical antacids, and intravenously administered HTblockers or protonpump inhibitors. Transfusions may be needed, and if bleeding persists, then endoscopy is usually indicated (O'Mahony, 2007). With sustained retching, the less common, but more serious, esophageal rupture-Boerhaave syndrome-may develop from greatly increased esophageal pressure. The small bowel has diminished motility during pregnancy.

1	The small bowel has diminished motility during pregnancy. Using a nonabsorbable carbohydrate, Lawson (1985) showed that small bowel mean transit times were 99, 125, and 137 minutes in each trimester, compared with 75 minutes when nonpregnant. In a study cited by Everson (1992), mean tran sit time for a mercury-illed balloon from the stomach to the cecum was 58 hours in term pregnant women compared with 52 hours in nonpregnant women. Muscular relaxation of the colon is accompanied by increased absorption of water and sodium that predisposes to consti pation. This complaint is reported by almost 40 percent of women at some time during pregnancy (Everson, 1992). Such symptoms are usually only mildly bothersome, and preventive measures include a high-iber diet and bulk-forming lxatives. Wald (2003) has reviewed treatment options. We have encoun impacted stool. These women almost invariably had chronically abused stimulatory laxatives.

1	he estimated monthly prevalence of diarrhea among adults is 3 to 7 percent (DuPont, 2014). Diarrhea can be classiied as acute «2 weeks), persistent (2 to 4 weeks), and chronic (>4 weeks). Most cases of acute diarrhea are caused by infectious agents, and a third result from foodborne pathogens. The large variety of viruses, bacteria, helminths, and protozoa that cause diarrhea in adults inevitably also alict pregnant women. Some of these are discussed in Chapter 64. Evaluation of acute diarrhea depends on its severity and duration. Some indications for evaluation include profuse watery diarrhea with dehydration, grossly bloody stools, fevera> 38°C, duration >48 hours without improvement, recent antimicrobial use, and diarrhea in the immunocompromised patient (Camilleri, 2015; DuPont, 2014). Cases of moderately severe diarrhea with fecal leukocytes or gross blood may best be treated with empirical antibiotics rather than evaluation. Some features of the more common acute diarrheal

1	2014). Cases of moderately severe diarrhea with fecal leukocytes or gross blood may best be treated with empirical antibiotics rather than evaluation. Some features of the more common acute diarrheal syndromes and their treatment are shown in Table 54-4.

1	The mainstay of treatment is intravenous hydration using normal saline or Ringer lactate with potassium supplementation in amounts to restore maternal blood volume and to ensure uteroplacental perfusion. Vital signs and urine output are monitored for signs of sepsis syndrome. For moderately severe nonfebrile illness without bloody diarrhea, antimobility agents such as loperamide (Imodium) may be useful. Bismuth subsalicylate (Pepto-Bismol) may also alleviate symptoms. TABLE 54-4. Etiology, Clinical Features, and Treatment of Common Acute Diarrheal Syndromes Toxin producers 1-72 hr 3-4+ 1-2+ 0-1 + 3-4+, watery 1. Staphylococcus 1. None 2. C perfringens 2. None 3. 1-2+ 1-3+, watery, 1. C difficile then bloody 1. Metronidazole 2. 3. Salmonella bloody 4. Campylobacter 5. Vibrio 5. Doxycycline Severe 1-8 days 0-1 + 3-4+ 3-4+ 1-2+, bloody 6. Shigella 6. Ciprofloxacin 7. E coli 7. Ciprofloxacin 8. Entamoeba histolytica 8. Metronidazole

1	Severe 1-8 days 0-1 + 3-4+ 3-4+ 1-2+, bloody 6. Shigella 6. Ciprofloxacin 7. E coli 7. Ciprofloxacin 8. Entamoeba histolytica 8. Metronidazole B cereus = Bacillus cereus; C difficile = Clostridium difficile; C perfringens = Clostridium perfringens; E coli = Escherichia coli. Data from Camilleri, 201 5; DuPont, 201 4.

1	B cereus = Bacillus cereus; C difficile = Clostridium difficile; C perfringens = Clostridium perfringens; E coli = Escherichia coli. Data from Camilleri, 201 5; DuPont, 201 4. Judicious use of antimicrobial agents is warranted. For moderate to severely ill women, some recommend empirical treatment with ciprofloxacin, 500 mg twice daily for 3 to 5 days. Speciic pathogens are treated as needed when identiied (see Table 54-4). Syndromes for which treatment is usually unnecessary include those caused by Escherichia coli, staphylococcal species, Bacilus cereus, and Norwalk-like virus. Severe illness caused by Salmonela spp is treated with ciprofloxacin or trimethoprim-sulfamethoxazole; by Campylobacter spp with azithromycin; by Clostridium diicile with oral metronidazole or vancomycin; and by Giardia spp and Entamoeba histoytica with metronidazole (DuPont, 2014; Rocha-Castro, 2016).

1	his anaerobic gram-positive bacillus is transmitted by the fecal-oral route. It is the most frequent nosocomial infection in the United States. In 2011, 453,000 cases of C dicile and 29,000 associated deaths were reported by the Centers for Disease Control and Prevention (CDC) (Lessa, 2015). he most important risk factor is antibiotic use, and the highest risk is with aminopenicillins, clindamycin, cephalosporins, and luoroquinolones. Other risk factors include inlammatory bowel disease, immunosuppression, advanced age, and gastrointestinal surgery. Most cases are hospital-acquired, however, community-acquired cases are becoming common (Leler, 2015). With severe colitis, the infection-related mortality rate is 5 percent.

1	Diagnosis is by enzyme immunoassay for toxins in the stool, or by DNA-based tests that identiy toxin genes. Only patients with diarrhea should be tested, and posttreatment testing is not recommended. Prevention is by soap-and-water hand washing, and infected individuals are isolated. Treatment is oral vancomycin or metronidazole. The risk of recurrence after an initial episode is 20 percent. Fecal microbial transplantation may become standard for recurrent clostridial colitis.

1	Two presumably noninfectious forms of intestinal inlammation are ulcerative colitis and Crohn disease. Diferentiation between these is important because treatment difers. That said, they both share common features, and sometimes are indistinguishable if Crohn disease involves the colon. he salient clinical and laboratory features shown in Table 54-5 permit a reasonably confident diagnostic diferentiation in most cases. he etiopathogenesis is enigmatic in both, but a genetic predisposition is suspected. Inlammation is thought to result from dysregulated mucosal immune function in response to commensal microbiota, with or without an autoimmune component (Friedman, 2015). TABLE 54-5. Some Shared and Diferentiating Characteristics of Inflammatory Bowel Disease Hereditary More than 100 disease-associated genetic loci-a third shared; Jewish predominance; familial in 5-10% of cases; Turner syndrome; immune dysregulation

1	Hereditary More than 100 disease-associated genetic loci-a third shared; Jewish predominance; familial in 5-10% of cases; Turner syndrome; immune dysregulation Other Chronic and intermittent with exacerbations and remissions; extraintestinal manifestations: arthritis, erythema nodosum, uveitis Major symptoms Diarrhea, tenesmus, rectal bleeding, cramping pain; Fibrostenotic-recurrent RLQ colicky pain; fever chronic, intermittent bladder, interenteric Bowel involvement Mucosa and submucosa of large bowel; usually begins Deep layers small and large bowel; at rectum (40% proctitis only); continuous disease commonly transmural; discontinuous involvement; strictures and fistulas RLQ = right lower quadrant; S cerevisiae = Saccharomyces cerevisiae. Data from Friedman, 201n5; Lichtenstein, 2009; Podolsky, 2002. Ulcerative Colitis and erythema nodosum. Another serious problem is that the risk of colon cancer approaches 1 percent per year. With either

1	Ulcerative Colitis and erythema nodosum. Another serious problem is that the risk of colon cancer approaches 1 percent per year. With either This is a mucosal disorder with inflammation confined to the ulcerative colitis or Crohn disease, there is also concern for possuperficial luminal layers of the colon. It typically begins at the sible increased risks for venous thromboembolism (Kappelman, rectum and extends proximally for a variable distance. In approx2011; Novacek, 2010).

1	imately 40 percent of cases, disease is conined to the rectum and rectosigmoid, but 20 percent have pancolitis. For unknown reasons, prior appendectomy protects against development of ulcerative coli tis (Friedman, 2015). Endoscopic indings include Also known as regional enteritis, Crohn ileitis, and granulomucosal granularity and friability that is interspersed with mucomatous colitis, Crohn disease has more protean manifestations sal ulcerations and a mucopurulent exudate (Fig. 54-2). than ulcerative colitis. It involves not only the bowel mucosa but

1	Major symptoms of ulcerative colitis include diarrhea, rectal also the deeper layers, and sometimes involvement is transmural bleeding, tenesmus, and abdominal cramps. The disease can be (see Fig. 54-2). Lesions can be seen throughout the entire gasacute or intermittent and is characterized by exacerbations and trointestinal tract, from the mouth to the anus, but it typically remissions. Toxic megacolon and catastrophic hemorrhage are is segmental (Friedman, 2015). Approximately 30 percent of particularly dangerous complications that may necessitate colpatients have small-bowel involvement, 25 percent have isolated ectomy. Extraintestinal maniestations include arthritis, uveitis, colonic involvement, and 40 percent have both, usually with the terminal ileum and colon involved. Perianal fistulas and abscesses develop in a third of those with colonic involvement.

1	Symptoms depend on which bowel segment(s) is involved. Thus, complaints may include lower-right-sided cramping abdominal pain, diarrhea, weight loss, lowgrade fever, and obstructive symptoms. The disease is chronic with exacerbations and remissions, and importantly, it cannot be cured medically or surgically. Approximately a third of patients require surgery within the first year ater diagnosis, and thereater, 5 percent per year. Reactive arthritis is common, and the gastrointestinal cancer risk, although not as great as. with ulcerative colitis, is increased substantially. FIGURE 54-2 Causes of colitis. A.Chronic ulcerative colitis with diffuse ulcerations and exudates. B. Crohn colitis with deep ulcers. (Reproduced with permission from Song LM Topazian M: Gastointestinal endoscopy. Kasper DL, Fauci AS, Hauser SL, et al (eds): Harrison's Principles of Internal Medicine, 19th ed. New York: McGraw-Hili Education; 201o5.)

1	Subfertility is commonly linked to chronic medical disease, but 1VIahadevan (2006a) cited a normal fertility rate for inflammatory bowel disease unless severe disease warranted surgery. Similarly, Alstead (2003) reported that decreased female fertility from active Crohn disease returned to normal with remission. For women requiring surgical resection, laparoscopic anastomosis has a higher subsequent fertility rate (Beyer-Berjot, 2013). With colectomy, however, even though fertility is improved, up to half of women will be persistently infertile (Bartels, 2012). Sexual function and fertility are only modestly afected by ileal pouch-anal anastomosis (Hor, 2016). Subfertility may also be partially due to sulfasalazine, which causes reversible sperm abnormalities (Feagins, 2009).

1	Because ulcerative colitis and Crohn disease are relatively common in young women, they are encountered with some frequency in pregnancy. In this regard, a few generalizations can be made. First, consensus supports that pregnancy does not increase the likelihood of an inlammatory bowel disease flare (.1ahadevan, 2015). Indeed, in a 10-year surveillance of women in the European Collaborative on Inflammatory Bowel Disease, the likelihood of a lare during pregnancy was decreased compared with the preconceptional rate (Riis, 2006). Although most women with quiescent disease in early pregnancy do not have relapses, when a lare develops, it may be severe. lso, active disease in early pregnancy increases the likelihood of poor pregnancy outcome, which is discussed subsequently. In general, most usual treatment regimens may be continued during pregnancy. Diagnostic evaluations should be undertaken if needed to direct management, and surgery should be performed if indicated. For women who

1	usual treatment regimens may be continued during pregnancy. Diagnostic evaluations should be undertaken if needed to direct management, and surgery should be performed if indicated. For women who successfully complete pregnancy, about half experience improvement in their health-relate.d q uali ty of life (Ananthakrishnan, 2012).

1	At first glance, it appears that adverse pregnancy outcomes are increased with inflammatory bowel disease (Boyd, 2015; Cornish, 2012; Getahun, 2014). Initially, this was attributed to the fact that most studies included women with either form of disease. Specifically, Crohn disease was noted to be linked to excessive morbidity (Dominitz, 2002; Stephansson, 2010). But, according to Reddy (2008) and others, these adverse outcomes were in women with severe disease and multiple recurrences. Indeed, in the prospective European case-control ECCO-EpiCom study of 332 pregnant women with inflammatory bowel disease, Bortoli and coworkers (2011) found similar outcomes in women with ulcerative colitis or Crohn disease compared with normally pregnant women. Importantly, perinatal mortality rates are not appreciably increased.

1	Ulcerative Colitis and Pregnancy. Ulcerative colitis does not signiicantly alter the course of pregnancy in afected women. In one review of 755 pregnancies, colitis that was quiescent at conception worsened in approximately a third of pregnancies (Fonager, 1998). In women with active disease at the time of conception, approximately 45 percent worsened, 25 percent remained unchanged, and only 25 percent improved. hese observations are similar to those previously described in an extensive review by Miller (1986) and a later report from Oron and colleagues (2012). Osteoporosis is a signiicant complication in up to a third of these women, and thus vitamin D-800 IU daily-and calcium-1200 mg daily-are given. Folic acid, 4 mg orally daily, is recommended preconceptionally and during the first trimester for neural-tube defect prevention. his high dose counteracts the antifolate actions of sulfasalazine. Flares may be caused by psychogenic stress, and reassurance is important.

1	Management for colitis for the most part mirrors that outside of pregnancy. Treatment of active colitis and maintenance therapy incorporate drugs that deliver 5-aminosalicyclic acid (5-ASA) or mesalamine. Suasalazine (Azuidine) is the prototype, and its 5-ASA moiety inhibits prostaglandin synthase in colonic mucosa. Others include olsalazine (Dipentum), balsalazide (Colazal), and delayed-release 5-ASA derivatives (Apriso, Asacol Pentasa, Lialda). Glucocorticoids are given orally, parenterally, or by enema for moderate or severe disease that does not respond to 5-ASA. However, these latter drugs are not given for maintenance therapy. Recalcitrant disease is managed with immunomodulating drugs, including azathioprine, 6-mercaptopurine, or cyclosporine, which appear relatively safe in pregnancy (Briggs, 2015; Mozafari, 2015). Importantly, methotrexate is contraindicated in pregnancy.

1	In the past, biological therapy was reserved for recalcitrant moderate to severe disease. Because of their considerable eicacy, these medications are now frequently given initialy for severe disease to prevent future complications. These agents are antibodies against tumor necrosis factor-alpha (TNF-alpha). Those approved for treatment of ulcerative colitis include inf liximab (Rem icade) , adalimumab (Hum ira), and golinumab (Simponi). These drugs are administered intravenously or subcutaneously. Several studies indicate that they are safe for use in pregnancy, although there are concerns that their discontinuance may prompt a relapse (Torres, 2015). Another worry is that they may cause immunosuppression in the neonate (Brams, 2016; Diav-Citrin, 2014; Gisbert, 20l3). Colorectal endoscopy is performed as indicated (Katz, 2002).

1	Colorectal endoscopy is performed as indicated (Katz, 2002). During pregnancy, colectomy and ostomy creation for fulminant colitis may be needed as a lifesaving measure, and it has been described during each trimester. Dozois (2006) reviewed 42 such cases and found that, in general, outcomes have been good in recent reports. Most women underwent partial or complete colectomy, but Ooi and colleagues (2003) described decompression colostomy with ileostomy in a 10-and a 16-week pregnancy. Parenteral nutrition discussed on page 1043 is occasionally necessary for women with prolonged exacerbations.

1	For women with an ileal pouch and an anal anastomosis performed before pregnancy, sexual function and fertility are improved (Cornish, 2007). Disadvantages that temporarily worsen in pregnancy include frequent bowel movements, fecal incontinence, and pouchitis. he last is an inflammatory condition of the ileoanal pouch probably due to bacterial proliferation and stasis. Pouchitis usually responds to cephalosporins or metronidazole. In one rare case, adhesions to the growing uterus led to ileal pouch perforation (Aouthmany, 2004). (Ravid, 2002). Hahnloser (2004) reviewed routes of delivery in women with 235 pregnancies before and 232 pregnancies after ileoanal pouch surgery. Functional outcomes were similar, for obstetrical indications. Postcesarean delivery ileoanal pouch obstruction has been described (Malecki, 2010).

1	To reiterate, ulcerative colitis likely has minimal adverse efects on pregnancy outcome. Modigliani (2000) reviewed to be not substantively diferent from those in the general obstetrical population. Speciically, the incidences of spontane ous abortion, preterm delivery, and stillbirth were remarkably low. In a population-based cohort study of 107 women from Washington state, perinatal outcomes, with two exceptions, were similar to those of 1308 normal pregnancies (Dominitz, 2002). One exception was an inexplicably increased incidence of congenital malformations. These authors and others also compared with that for normal controls (Mahadevan, 2015). lar outcomes in 187 gravidas with ulcerative colitis compared with normal pregnant controls (Bortoli, 2011).

1	lar outcomes in 187 gravidas with ulcerative colitis compared with normal pregnant controls (Bortoli, 2011). Crohn Disease and Pregnancy. In general, Crohn disease activity during pregnancy is related to its status around the time of conception. In a cohort study of 279 pregnancies in 186 women whose disease was inactive at conception, a fourth relapsed during pregnancy (Fonager, 1998). In 93 with active disease at conception, however, two thirds either remained active or worsened. Miller (1986) had described similar findings from his earlier review, as did Oron and associates (20a12).

1	Calcium, vitamin D, and folic acid supplementation mirror that for ulcerative colitis. For maintenance during asymptomatic periods, no regimen is universally efective. Suasalazine is efective for some, but the newer 5-ASA formulations are better tolerated. Prednisone therapy may control moderate to severe lares but is less efective for small-bowel involvement. Immunomodulators such as azathioprine, 6-mercaptopurine, and cyclosporine are used for active disease and for maintenance. These appear relatively safe during pregnancy (Briggs, 2015; Chande, 2015). As discussed in Chapter 12 (pp. 242 and 244), methotrexate, mycophenolate mofetil, and mycophenolic acid are contraindicated in pregnancy (Briggs, 2015; Food and Drug Administration, 2008).

1	As with ulcerative colitis, treatment with antitumor necrosis factor monoclonal antibodies is often used initially for active Crohn disease and maintenance (Casanova, 2013; Cominelli, 2013; Friedman, 2015). These biological compounds include inliximab, adalimumab, certolizumab (Cimzia), natalizumab (Tysabri), and vedolizumab (Enyvio). As discussed on page 1050, this class of immunomodulators is considered safe in pregnancy (Briggs, 2015; Clowse, 2015). heir discontinuance may be followed by a relapse (Torres, 2015). Endoscopy or conservative surgery is indicated for complications. Patients with small-bowel involvement are more likely to require surgery for complications that include istulas, strictures, abscesses, and intractable disease. An abdominal surgical procedure was required during 5 percent of pregnancies described by Woolfson (1990). Parenteral hyperalimentation

1	Stieglitz, 1999). hose with an ileal loop colostomy may have signiicant problems. Women with a perianal istula-unless these are rectovaginal-usually can undergo vaginal delivery without complications (Forsnes, 1999; Takahashi, 2007). As discussed, the likelihood is greater that Crohn disease is associated with adverse pregnancy outcomes compared with ulcerative colitis (Stephansson, 2010). Outcomes are probably related to disease activities. In a case-control Danish study, Norgard (2007) reported a twofold risk of preterm births. preterm delivery, low birthweight, fetal growth restriction, and cesarean delivery in 149 women with Crohn disease. Recall, however, that the prospective ECCO-EpiCom study found outcomes to be similar to those for normal pregnancies.

1	A colostomy or an ileostomy can be problematic during pregnancy because of its location (Hux, 2010). In a report of 82 pregnancies in 66 women with an ostomy, stomal dyfunction was common, but it responded to conservative management in most cases (Gopal, 1985). Surgical intervention was necessary, however, in three of six women who developed boweliobstruction and in another four with ileostomy prolapse-almost 10 percent overall. In this older study, only a third of 82 women underwent cesarean delivery, but Takahashi (2007) described six of seven cesarean deliveries in women with Crohn disease and a stoma. Although adhesions usually are involved with an obstructed ileostomy, the enlarging uterus may act to obstruct (Porter, 2014). Finally, Farouk and coworkers (2000) reported that pregnancy did not worsen long-term ostomy function. The incidence of bowel obstruction is not increased during pregnancy, although it generally is more diicult to diagnose.

1	Meyerson (1995) reported a 20-year incidence of 1 in 17,000 deliveries at two Detroit hospitals. In one study, adhesive dis ease leading to small-bowel obstruction was the second most common cause of an acute abdomen in pregnancy following appendicitis-15 versus 30 percent, respectively (Unal, 2011). Approximately half of cases are due to adhesions from previous pelvic surgery that includes cesarean delivery (Al-Sunaidi, 2006; Andolf, 2010; Lyell, 2011). Another 25 percent of bowel obstruction cases are caused by volvulus-sigmoid, cecal, or small bowel. hese have been reported in late pregnancy or early puerperium (Bade, 2014; Biswas, 2006; l Maksoud, 2015). Small-bowel obstruction has been reported in pregnancy following the currently popular Roux-en-Y gastric bypass for weight loss (Bokslag, 2014; Wax, 2013). Intussusception is occasionally encountered (Bosman, 2014; Harma, 201l). Bowel obstruction subsequent to colorectal surgery for cancer was increased threefold in women who had

1	2014; Wax, 2013). Intussusception is occasionally encountered (Bosman, 2014; Harma, 201l). Bowel obstruction subsequent to colorectal surgery for cancer was increased threefold in women who had open versus laparoscopic surgery (Haggar, 2013). Finally, Serra and colleagues (2014) described a massive ventral hernia with intestinal obstruction.

1	FIGURE 54-3 Characteristic "bent inner tube" seen with sigmoid volvulus on abdominal radiograph. (Reproduced with permission from Song LM, Topazian M: Gastointestinal endoscopy. Kasper DL, Fauci AS, Hauser SL, et al (eds): Harrison's Principles of Internal Medicine, 19th ed. New York: McGraw-Hili Education; 2015.)

1	Most cases of intestinal obstruction during pregnancy result from pressure of the growing uterus on intestinal adhesions. According to Davis and Bohon (1983), this more likely occurs around midpregnancy when the uterus becomes an abdominal organ; in the third trimester when the fetal head descends; or immediately postpartum when uterine size acutely shrinks. Perdue (1992) reported that 98 percent of afected pregnant women had either continuous or colicky abdominal pain, and 80 percent had nausea and vomiting. Abdominal tenderness was found in 70 percent, and abnormal bowel sounds noted in only 55 percent. Plain abdominal radiographs following soluble contrast showed evidence of obstruction in 90 percent of women (Fig. 54-3). Plain radiographs, however, are less accurate for diagnosing small-bowel obstruction, and we and others have found that CT and MR imaging can be diagnostic (Biswas, 2006; Essilie, 2007; McKenna, 2007). Colonoscopy can be both diagnostic and therapeutic for colonic

1	obstruction, and we and others have found that CT and MR imaging can be diagnostic (Biswas, 2006; Essilie, 2007; McKenna, 2007). Colonoscopy can be both diagnostic and therapeutic for colonic volvulus (Dray, 2012; Khan, 2012).

1	During pregnancy, mortality rates with obstruction can be excessive because of diicult and thus delayed diagnosis, reluctance to operate during pregnancy, and the need for emergency surgery (Firstenberg, 1998; Shui, 2011). In an older report of 66 pregnancies, Perdue and associates (1992) described a 6-percent maternal mortality rate and 26-percent fetal mortality rate. Two of the four women who died were in late pregnancy, and they had bowel perforation from sigmoid or cecal volvulus caused by adhesions.

1	Also known as Ogilvie syndrome, pseudo-obstruction is caused by adynamic colonic ileus. It is characterized by massive abdominal distention with cecal and right-hemicolon dilatation. Approximately 10 percent of all cases are associated with pregnancy, and its frequency has been reported as high as 1 in 1500 deliveries (Reeves, 2015). he syndrome usually develops postpartum-most commonly after cesarean delivery-but it has been reported antepartum (Tung, 2008). Rarely, the large bowel may rupture (Singh, 2005). Treatment with an intravenous infusion of neostigmine, 2 mg, usually results in prompt decompression (Song, 2015). In some cases, colonoscopic decompression is performed, and laparotomy is needed for perforation (De Giorgio, 2009; Rawlings, 2010).

1	The lifetime incidence for appendicitis ranges from 7 to 10 percent (Flum, 2015). hus, it is not surprising that an evaluation for possible appendicitis is relatively common during pregnancy. heilen and colleagues (2015) studied 171 such women during a 5-year period, but only 12 women ultimately were found to have pathologically confirmed appendicitis. After clinical and imaging evaluation, the frequency of suspected appendicitis is much lower and that of confirmed appendicitis in more than 8 million women ranged from 1 in 1000 to 1 in 5500 births (Abbasi, 2014; Hee, 1999; Mazze, 1991).

1	It is repeatedly-and appropriately-emphasized that pregnancy makes the diagnosis of appendicitis more diicult. Nausea and vomiting accompany normal pregnancy, but also, as the uterus enlarges, the appendix commonly moves upward and outward from the right lower quadrant (Baer, 1932; Erkek, 2015; Pates, 2009). Another often-stated reason for late diagnosis is that some degree of leukocytosis accompanies normal pregnancy. For these and other reasons, pregnant womenespecially those late in gestation-frequently do not have clinical findings "typical" for appendicitis. Thus, it commonly is confused with cholecystitis, labor, pyelonephritis, renal colic, placental abruption, or uterine leiomyoma degeneration.

1	Most reports indicate increasing morbidity and mortality rates with advancing gestational age. nd as the appendix is progressively delected upward by the growing uterus, omental containment of infection becomes increasingly unlikely. It is indisputable that appendiceal perforation is more common during later pregnancy (Abbasi, 2014). In the studies by Andersson (2001) and Ueberrueck (2004), the incidence of perforation was approximately 8, 12, and 20 percent in successive trimesters.

1	Persistent abdominal pain and tenderness are the most reproducible findings. Right-lower quadrant pain is the most frequent, although pain migrates upward with appendiceal displacement CMourad, 2000). For initial evaluation, sonographic abdominal imaging is reasonable in suspected appendicitis, even if to exclude an obstetrical cause of pain (Butala, 2010). hat said, graded compression sonography is diicult because of cecal displacement and uterine imposition (Pedrosa, 2009). Appendiceal computed tomography is more sensitive and accurate than FIGURE 54-4 Anterior-posterior magnetic resonance image of a periappendiceal abscess in a midtrimester pregnancy. The abscess is approximately 5 x 6 cm, and the appendiceal lumen (arow) is visible within the right-lower quadrant mass. The gravid uterus is seen to the right of this mass.

1	sonography to conirm suspected appendicitis (Katz, 2012; Raman, 2008). Specific views can be designed to diminish fetal radiation exposure (Chap. 46, p. 907). It is generally accepted that when available, MR imaging is the preferred modality for evaluation of suspected appendicitis in pregnancy (Fig. 54-4). MR imaging has high diagnostic yield and accuracy, and it also provides alternative diagnoses (Fonseca, 2014; Theilen, 2015). One metaanalysis cited positive-and negativepredictive values for MR imaging of 90 and 99.5 percent, respectively (Blumenfeld, 2011). Burke and associates (2015) reported similar indings. Using a decision-analysis model, CT and MR imaging were found to be cost efective (Kasten berg, 2013). This was verified in the clinical study of more than 7000 cases reported by Fonseca and coworkers (2014).

1	When appendicitis is suspected, treatment is prompt surgical exploration. Although diagnostic errors may lead to removal of a normal appendix, surgical evaluation is preferable to postponed intervention and generalized peritonitis (Abbasi, 2014). In earlier reports, the diagnosis was veriied in only 60 to 70 percent of pregnant women. As indicated above, however, with CT and MR imaging, these igures have improved (Blumenfeld, 2011; Theilen, 2015). Still and importantly, the accuracy of diagnosis is inversely proportional to gestational age.

1	Currently, laparoscopy is almost always used to treat suspected appendicitis during the irst two trimesters. In a report from a Swedish database of nearly 2000 laparoscopic appendectomies, perinatal outcomes were similar to those of more than 1500 open laparotomies done before 20 weeks' gestation (Reedy, 1997). Conversely, in their review, Wilasrusmee and coworkers (2012) reported a higher rate of fetal loss with laparoscopy. Authors of a more recent systematic review indicate that the level of evidence is not strong enough to demonstrate a preferred approach to appendectomy. They concede that laparoscopy may be associated with a higher risk of miscarriage (Walker, 2014). It has evolved that in many centers, laparo scopic appendectomy is also performed in most cases during the third trimester (Donkervoort, 2011). This is encouraged by the Society of American Gastrointestinal and Endoscopic

1	Surgeons (Pearl, 2017; Soper, 2011). That said, most are of the gestation should be performed only by the most experienced endoscopic surgeons (Parangi, 2007). Before exploration, intravenous antimicrobial therapy is begun, usually with a second-generation cephalosporin or third generation penicillin. Unless there is gangrene, perforation, or a periappendiceal phlegmon, antimicrobial therapy can usually be discontinued after surgery. Without generalized peritonitis, the prognosis is excellent. Seldom is cesarean delivery indicated at the time of appendectomy. Uterine contractions are common, and although some clinicians recommend tocolytic agents, we do not. De Veciana (1994) reported that tocolytic use substantially increased the risk for pulmonary-permeability edema caused by sepsis syndrome (Chap. 47, p. 917).

1	Because of European studies, some have advocated that many cases of appendicitis can be treated successfully with intravenous antimicrobials alone (Flum, 2015; J00, 2017). At this time, we discourage this practice until appropriate studies have been done with pregnant women. In one study, 6 percent of pregnant women with appendicitis were treated medically, and these gravidas had "considerably" elevated risks for septic shock, peritonitis, and venous thromboembolism compared with surgically managed cases (Abbasi, 2014).

1	Appendicitis increases the likelihood of abortion or preterm labor, especially if peritonitis has developed. In two studies, spontaneous labor after 23 weeks ensued with greater frequency following surgery for appendicitis compared with surgery for other indications (Cohen-Kerem, 2005; Mazze, 1991). In one study, the fetal loss rate was 22 percent if surgery was performed after 23 weeks' gestation. Two large population-based studies attest to the adverse outcomes from appendicitis in pregnancy. From the California Inpatient File of 3133 pregnant women undergoing surgery for suspected appendicitis, the fetal loss rate was 23 percent, and it was doubled-6 versus 11 percent-with simple versus complicated disease (McGory, 2007). A nationwide study from Taiwan found that risks for low birthweight and preterm delivery rose 1.5-to 2-fold when outcomes in 908 women with acute appendicitis were compared with those of controls (Wei, 2012).

1	Long-term complications are not common. The possible link between sepsis and neonatal neurological injury has not been veriied (Mays, 1995). Finally, appendicitis during pregnancy does not appear to be associated with subsequent infertility (Viktrup, 1998). Abbasi N, Patenaude V, Abenhaim HA: Management and outcomes of acute appendicitis in pregnancy-population-based study of over 7000 cases. BlOC 121(12): 1509,2014 Akcakaya A, Koc B, Adas G, et al: he useofERCP during pregnancy: is it safe and efective? Hepatogastroenterology 61(130):296,s2014 Alaniz \, Liss J, Metz TD, et al: Cannabinoid hyperemesis syndrome: a cause of refractory nausea and vomiting in pregnancy. Obstet Gynecol 125(6): 1484, 2015 Albayrak M, Karatas A, Demiraran Y, et al: Ghrelin, acylated ghrelin, leptin, and PYY-3 levels in hyperemesis gravidarum. J Matern Fetal Neonatal vIed 26(9):866,s2013

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1	Ueberrueck T, Koch A, Meyer L, et al: Ninety-four appendectomies for suspected acute appendicitis during pregnancy. World J Surg 28:508, 2004 Unal A, Sayherman SE, Ozel L, et al: Acute abdomen in pregnancy requiring surgical management: a 20-case series. Eur J Obstet Gynecol Reprod BioI 159(1):87,s2011 Vaknin Z, Halperin R, Schneider D, et al: Hyperemesis gravidarum and nonspeciic abnormal EEG indings. J Reprod Med 51 :623, 2006 Vandraas KF, Vikanes AV, Vangen S, et al: Hyperemesis gravidarum and birth outcomes-a population-based cohort study of 2.2 million births in the Norwegian Birth Registry. BJOG 120(13):1654,2013 Veenendaal MV, van Abeelen AF, Painter RC, et al: Consequences of hyperemesis gravidarum for ofspring: a systematic review and meta-analysis. BJOG 118(11):1302,2011 Verberg MF, Gillott JD, Fardan NA, et al: Hyperemesis gravidarum, a literature review. Hum Reprod Update ,2005

1	Verberg MF, Gillott JD, Fardan NA, et al: Hyperemesis gravidarum, a literature review. Hum Reprod Update ,2005 Vikanes AV, Stoer NC, Magnus P, et al: Hyperemesis gravidarum and pregnancy outcomes in the Norwegian mother and child cohort-a cohort study. BMC Pregnancy Childbirth 13:169,2013 Viktrup L, Hee P: Fertility and long-term complications four to nine years after appendectomy during pregnancy. Acta Obstet Gynecol Scand 77:746, 1998 Wald A: Constipation, diarrhea, and symptomatic hemorrhoids during pregnancy. Gastroenterol Clin North Am 32:309, 2003 Walker HG, I Samaraee A, Mills SJ, et al: Laparoscopic appendicectomy in pregnancy: a systematic review of the published evidence. Int J Surg 12(11):1235,s2014 Wax JR, Pinette MG, Cartin A: Roux-en-Y gastric bypass-associated bowel obstruction complicating pregnancy-an obstetrician's map to the clinical mineield. m J Obstet GynecoIs208(4):265, 2013

1	Wax JR, Pinette MG, Cartin A: Roux-en-Y gastric bypass-associated bowel obstruction complicating pregnancy-an obstetrician's map to the clinical mineield. m J Obstet GynecoIs208(4):265, 2013 Wei PL, Keller J], Liang HH, et al: Acute appendicitis and adverse pregnancy outcomes: a nationwide population-based study. J Gastrointest Surg 16(6):1204,s2012 Weyermann M, Brenner H, Adler G, et al: Helicobacter pylori infection and the occurrence and severity of gastrointestinal symptoms during pregnancy. Am ] Obstet Gynecol 189:526, 2003 Wilasrusmee CSukrat B, McEvoy M, et al: Systematic review and meta-analysis of safety laparoscopic versus open appendectomy for suspected appendicitis in pregnancy. Br J Surg 99(11):1470,s2012 Woolfson K, Cohen Z, McLeod RS: Crohn's disease and pregnancy. Dis Colon Rectum 33:869, 1990 Yost NP, McIntire DD, Wians FH Jr, et al: A randomized, placebo-controlled trial of corticosteroids for hyperemesis due to pregnancy. Obstet Gynecol 102: 1250, 2003

1	Yost NP, McIntire DD, Wians FH Jr, et al: A randomized, placebo-controlled trial of corticosteroids for hyperemesis due to pregnancy. Obstet Gynecol 102: 1250, 2003 Zara G, Codemo V, Palmieri A, et al: Neurological complications in hyperemesis gravidarum. Neurol Sci 33(1):133, 2012 CHAPTER 55 Hepatic, Biliary, and Pancreatic Disorders INTRAHEPATIC CHOLESTASIS OF PREGNANCY ...... 1059 ACUTE FATY LIVER OF PREGNANCY ..i............ 1060 VIRAL HEPATITIS .....i...i.......i........i...i.....i..... 1062 NONALCOHOLIC FATY LIVER DISEASE ...........i. 1067 CIRRHOSIS.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1067 ACETAMINOPHEN OVERDOSE HEPATOTOXICITY .... 1068 HEPATIC ADENOMA .i..................i.......i. 1069 GALLBLADDER DISORDERS . . . . . . . . . . . . . . . . . . . . . 1069 PANCREATIC DISORDERS ...........i......i.......i. 1070

1	HEPATIC ADENOMA .i..................i.......i. 1069 GALLBLADDER DISORDERS . . . . . . . . . . . . . . . . . . . . . 1069 PANCREATIC DISORDERS ...........i......i.......i. 1070 Prenancy is comparativey seldom complicated by jaundice. Notwithstanding the act that in most cases the jaundice disappears without treatment, too avorable a prognosis should not be ventured, or the reason that now and again the condition may represent the initial symptom of acute yellow atrophy of the liver. -J. Whitridge Williams (1903) Even though Williams only mentions acute hepatic fatty metamorphosis, in practice, disorders of the liver, gallbladder, and pancreas together comprise a formidable list of complications that may arise in pregnancy. Some stem from preexisting conditions and some are unique to gestation. The relationships of several of these with pregnancy can be fascinating, intriguing, and challenging.

1	Customarily, liver diseases complicating pregnancy are placed into three general categories. he first includes those specifically related to pregnancy that resolve either spontaneously or following delivery. Examples are intrahepatic cholestasis and acute fatty liver, both discussed in the next sections. Also, hepatic dysfunction from hyperemesis gravidarum may involve the liver. Mild hyperbilirubinemia with elevated serum transaminase levels is seen in up to half of afected women requiring hospitalization. However, these levels seldom exceed 200 U/L (Table 55-1). Liver biopsy may show minimal fatty changes. Hyperemesis gravidarum is discussed in detail in Chapter 54 (p. 1043). Another in this first category is hepatocellular damage with preeclampsia-the HELLP syndrome-which is characterized by hemolysis, �levated serum liver enzyme levels, and low 2latelet counts. hese changes are discussed in detail in Chapter 40 (p. 721).

1	The second category involves acute hepatic disorders that are coincidental to pregnancy, such as acute viral hepatitis. The third category includes chronic liver diseases that predate pregnancy, such as chronic hepatitis, cirrhosis, or esophageal varices. Importantly, several normal pregnancy-induced physiological changes induce appreciable liver-related clinical and laboratory manifestations (Chap. 4, p. 68, and Appendix, p. 1257). Findings such as elevated serum alkaline phosphatase levels, palmar erythema, and spider angiomas, which might suggest liver disease, are common during normal pregnancy. Metabolism is also afected, due to altered expression of the cytochrome P450 system. This alteration is mediated by higher levels of estrogen, progesterone, and other pregnancy hormones. For example, hepatic CYFIA2 expression declines, whereas that of CYF2D6 and CYF3A4 rises. Importantly, cytochrome enzymes are expressed in many organs besides the liver, most notably the

1	Hepatic, Biliary, and Pancreatic Disorders 1059 TABLE 55-1 . Clinical and Laboratory Findings with Acute Liver Diseases in Pregnancy Plat = platelets; PT = prothrombin time. placenta. The net efect is complex and likely influenced by gestational age and organ of expression (Isoherranen, 2013). Despite all of these functional changes, no major hepatic histological changes are induced by normal pregnancy.

1	• Intrahepatic Cholestasis of Pregnancy his condition has been called recurrent jaundice of pregnancy, cholestatic hepatosis, and icterus gravidarum and is characterized by pruritus, icterus, or both. It may be more common in multifetal pregnancy, and there is a significant genetic influence (Lausman, 2008; Webb, 2014). Because of this, its incidence varies by population. For example, cholestasis is infrequent in North America, with an overall incidence approximating 1 case in 500 to 1000 pregnancies. But, its rate nears 5.6 percent among Latina women in Los Angeles (Lee, 2006). Historically, indigenous women from Chile and Bolivia also have a relatively high incidence. For unknown reasons, this incidence has declined since the 1970s and is now less than 2 percent (Reyes, 2016). In other countries, for example Sweden, China, and Israel, the incidence varies from 0.25 to 1.5 percent (Glantz, 2004; Luo, 2015; Sheiner, 2006).

1	The cause of obstetrical cholestasis is unclear, but changes in various sex steroid levels are implicated. However, current research focuses on the numerous mutations in the many genes that control hepatocellular transport systems. Examples include mutations of the ABCB4 gene, which encodes multidrug resistance protein 3 (vlDR3) associated with progressive amilial intrahepatic cholestasis, and errors of the ABCBll gene, which encodes a bile-salt export pump (Anzivino, 2013; Dixon, 2014). Other potential gene products are the farnesoid X receptor and transporting ATPase encoded by A TP8Bl (Abu-Hayyeh, 2016; Davit-Spraul, 2012). Some drugs that similarly decrease canalicular transport of bile acids aggravate the disorder. We have encountered impressive cholestatic jaundice in gravidas taking azathioprine following renal transplantation.

1	Whatever the inciting cause(s), bile acids are cleared incompletely and accumulate in plasma. Hyperbilirubinemia results from retention of conjugated pigment, but total plasma concentrations rarely exceed 4 to 5 mg/ dL. Alkaline phosphatase levels are usually elevated even more than in normal pregnancy. Serum transaminase levels are normal to moderately elevated but seldom exceed 250 U/L (see Table 55-1). Liver biopsy shows mild cholestasis with bile plugs in the hepatocytes and canaliculi of the centrilobular regions, but without inflammation or necrosis. These changes disappear after delivery but often recur in subsequent pregnancies or with estrogencontaining contraceptives.

1	Pruritus develops in late pregnancy, although it occasionally manifests earlier. Constitutional symptoms are absent, and generalized pruritus shows predilection for the soles. Skin changes are limited to excoriations from scratching. Biochemical tests may be abnormal at presentation, but pruritus may precede laboratory indings by several weeks. Approximately 10 percent of women have jaundice.

1	With normal liver enzymes, the diferential diagnosis of pruritus includes other skin disorders (Table 62-1, p. 1185). Findings are unlikely to stem from preeclamptic liver disease if blood pressure elevation or proteinuria is absent. Sonography may be warranted to exclude cholelithiasis and biliary obstruction. Moreover, acute viral hepatitis is an unlikely diagnosis because of the usually low serum transaminase levels seen with cholestasis. Conversely, chronic hepatitis C is associated with a significantly increased risk of cholestasis, which may be as high as 20-fold among women who test positively for hepatitis C RNA (Marschall, 2013).

1	Pruritus may be troublesome and is thought to result from elevated serum bile salt concentrations. Antihistamines and topical emollients may provide some relie. Although cholestyramine is reported to be efective, this compound also lowers absorption of fat-soluble vitamins, which may lead to vitamin K deficiency. Fetal coagulopathy with subsequent intracranial hemorrhage and stillbirth have been reported (Matos, 1997; Sadler, 1995).

1	A recent metaanalysis suggests that ursodeoxycholic acid relieves pruritus, lowers bile acid and serum enzyme levels, and may reduce certain neonatal complications. hese include preterm birth, fetal distress, respiratory distress syndrome, and neonatal intensive care unit (NICU) admission (Bacq, 2012). Kondrackiene and associates (2005) randomly assigned 84 symptomatic women to receive either ursodeoxycholic acid ($ to 10 mg/kg/d) or cholestyramine. hey reported superior relief with ursodeoxycholic acid-67 versus 19 percent, respectively. Similarly, Glantz and coworkers (2005) found superior beneits to women randomly assigned to ursodeoxycholic acid versus dexamethasone. he American College of Obstetricians and Gynecologists (2015) has concluded that ursodeoxycholic acid relieves pruritus and improves fetal outcomes, although evidence for the latter is not compelling.

1	Earlier reports describe excessive adverse pregnancy outcomes in women with cholestatic jaundice. That said, data accrued during the past two decades are ambiguous concerning increased perinatal mortality rates and whether close fetal surveillance is preventative. Several studies also illustrate this. In one evaluation of 693 Swedish women, perinatal mortality rates were slightly increased, but only in mothers with severe disease (Glantz, 2004). Sheiner and coworkers (2006) described no diferences in perinatal outcomes in 376 afected pregnancies compared with their overall obstetrical population. However, rates of labor induction and cesarean delivery in afected women signiicantly rose. Lee and associates (2009) described two cases of sudden fetal death not predicted by nonstress testing. In another study of 101 afected women, no term fetuses died, but 87 percent of women underwent labor induction, ostensibly to avoid adverse outcomes (Rook, 2012). Nonetheless, neonatal complications

1	In another study of 101 afected women, no term fetuses died, but 87 percent of women underwent labor induction, ostensibly to avoid adverse outcomes (Rook, 2012). Nonetheless, neonatal complications developed in a third of the pregnancies, particularly respiratory distress, fetal distress, and meconium-stained amnionic luid. These problems were noted more frequently in those with higher total bile acid levels. Herrera and coworkers (2017) reported similar results. Finally, Wikstrom Shemer and colleagues (2013) reported outcomes in 5477 women with cholestasis from a database of 1,213,668 births. They described novel associations of cholestasis with preeclampsia and gestational diabetes. Although neonates were more likely to have a low 5-minute Apgar score and to be large for gestational age, the stillbirth rate was not increased. This was thought to reflect higher induction and preterm birth rates. Thus, by this time, many had now recommended early labor induction to avoid stillbirth.

1	the stillbirth rate was not increased. This was thought to reflect higher induction and preterm birth rates. Thus, by this time, many had now recommended early labor induction to avoid stillbirth. Relecting this, at Parkland Hospital, some maternal-fetal specialists ofer induction at 38 weeks, whereas others suggest 39 weeks.

1	As discussed, some evidence supports that high serum bile acid levels may contribute to fetal death. Bile acids typically remain < 10 ILmol/L throughout normal pregnancy (Egan, 2012). Elevated levels have been associated with meconium passage and stillbirth. For example, in the prior study of 693 Swedish women, stillbirths were limited to women with bile acid levels >40 ILmollL (Glantz, 2004). More recent data indicate that adverse outcomes are associated with even higher bile acid levels. For instance, Brouwers and coworkers (2015) reported high rates of spontaneous preterm birth (19 percent), meconium-stained amnionic fluid (48 percent), and perinatal death (10 percent) with bile acids levels > 100 ILmollL despite active management leading to earlier delivery. Kawakita and colleagues (2015) found a similar stillbirth link. In particular, among 233 women followed with cholestasis of pregnancy, there were four stillbirths, all of which were among women with bile acid levels > 100

1	(2015) found a similar stillbirth link. In particular, among 233 women followed with cholestasis of pregnancy, there were four stillbirths, all of which were among women with bile acid levels > 100 LmollL. Gao and associates (2014) implicated bile acids in cardiac dysfunction. Namely, in an exvivo preparation of cardiac myocytes, cholic acid lowered the beating rates in a dose-dependent manner, while increasing intracellular calcium levels. Intriguingly, studies have shown prolongations in the PR interval during fetal echocardiography among afected women (Rodriguez, 2016; Strehlow, 2010).

1	• Acute Fatty Liver of Pregnancy The most frequent cause of acute liver failure during pregnancy is acute fatty liver-also called acute aty metamorphosis or acute yellow atrophy. It is characterized by accumulation of microvesicular fat that literally "crowds out" normal hepatocytic function (Fig. Grossly, the liver is small, soft, yellow, and greasy. In its worst form, the incidence approximates 1 case in 10,000 pregnancies (Nelson, 2013). Fatty liver recurring in subsequent pregnancy is rare, but a few cases have been described (Usta, 1994). Although much has been learned about this disorder, interpretation of conflicting data has led to incomplete but interesting observations. For example, some if not most cases of maternal fatty liver are associated with recessively inherited mitochondrial abnormalities of fatty acid oxidation. These are similar to those in children with Reye-like syndromes. Several mutations have been described for the mitochondrial trifunctional

1	FIGURE 55-1 Acute fatty liver of pregnancy. Cross section of the liver from a woman who died as the result of pulmonary aspiration and respiratory failure. The liver has a greasy yellow appearance, which was present throughout the entire specimen. Inset: Electron photomicrograph of one swollen hepatocyte containing numerous microvesicular fat droplets (*). The nuclei (N) remain centered within the cell, in contrast to the case with macrovesicular fat deposition. (Used with permission from Dr. Don Wheeler.) protein enzyme complex that catalyzes the last oxidative steps in the pathway. The most common are the G1528C and E474Q mutations of the gene on chromosome 2 that codes for long-chain-3-hydroxyacyl-CoA-dehydrogenase-known as LCHAD. here are other mutations for medium-chain acyl CoA dehydrogenase-MCAD, as well as for carnitine palmito yltransferase 1 (CPT1) deficiency (Santos, 2007; Ylitalo, 2005).

1	LCHAD. here are other mutations for medium-chain acyl CoA dehydrogenase-MCAD, as well as for carnitine palmito yltransferase 1 (CPT1) deficiency (Santos, 2007; Ylitalo, 2005). Sims and coworkers (1995) observed that some homozygous LCHAD-deicient children with Reye-like syndromes had heterozygous mothers with fatty liver. This was also seen in women with a compound heterozygous fetus. Although some conclude that ony heterozygous LCHAD-deicient mothers are at risk when their fetus is homozygous, this is not always true (Baskin, 2010).

1	There is a controversial association between fatty acid 3-oxidation enzyme defects and severe preeclampsia-especially in women with HELLP syndrome (Chap. 40, p. 721). Most of these observations derive from retrospective study of mothers delivered of a child who later developed Reye-like syndrome. For example, one case-control study compared 50 mothers of children with a fatty-acid oxidation defect and 1250 mothers of matched control infants (Browning, 2006). During their pregnancy, 16 percent of mothers with an afected child developed liver problems compared with only 0.9 percent of control women. Problems included HELLP syndrome in 12 percent and fatty liver in 4 percent. Despite these indings, the clinical, biochemical, and histopathological findings are suiciently disparate to suggest that severe preeclampsia, with or without HELLP syndrome, and fatty liver are distinct syndromes (American College of Obstetricians and Gynecologists, 2015; Sibai, 2007).

1	Acute fatty liver almost always manifests late in pregnancy. Nelson and colleagues (2013) described 51 afected women at Parkland Hospital with a mean gestational age of 37 weeks (range 31.7 to 40.9). Almost 20 percent were delivered at 34 weeks' gestation or earlier. Of these 51 women, 41 percent were nulliparous, and two thirds carried a male fetus. From other data, 10 to 20 percent of cases are in women with a multifetal gestation (Fesenmeier, 2005; Vigil-De Gracia, 2011). Hepatic, Biliary, and Pancreatic Disorders 1061 Fatty liver has a clinical spectrum of severity. In the worst cases, symptoms usually develop over several days. Persistent nausea and vomiting are major complaints, and degrees of mal aise, anorexia, epigastric pain, and progressive jaundice vary.

1	Perhaps half of afected women have hypertension, protein uria, and edema, alone or in combination-signs suggestive of preeclampsia. As shown in Tables 55-1 and 55-2, degrees of moderate to severe liver dysfunction are manifest by hypofi brinogenemia, hypoalbuminemia, hypocholesterolemia, and prolonged clotting times. Serum bilirubin levels usually are < 10 mgl dL, and serum transaminase levels are modestly ele vated and usually < 1000 U/L. In almost all severe cases, profound endothelial cell activation with capillary leakage causes hemoconcentration, acute kidney injury, ascites, and sometimes pulmonary permeability edema (Bernal, 2013). With severe hemoconcentration, uteroplacental perfusion is reduced and this, along with maternal acidosis, can cause fetal death even before presentation for care. Both maternal and fetal acidemia are associated with a high incidence of fetal jeopardy and a concordantly high cesarean delivery rate.

1	Hemolysis can be severe and evidenced by leukocytosis, nucleated red cells, mild to moderate thrombocytopenia, and elevated serum levels of lactic acid dehydrogenase (LDH). Because of hemoconcentration, however, the hematocrit is often within the normal range. The peripheral blood smear demonstrates echinocytosis, and hemolysis is thought to stem from efects of hypocholesterolemia on erythrocyte membranes (Cunningham, 1985).

1	The degree of clotting dysfunction also varies and can be serious and life threatening, especially if operative delivery is undertaken. Coagulopathy is caused by diminished hepatic procoagulant synthesis, although some evidence supports increased consumption from disseminated intravascular coagulopathy. As shown in Table 55-2, hypoibrinogenemia sometimes is profound. Of 51 women with fatty liver cared for at Parkland Hospital, almost a third had a plasma fibrinogen level nadir to < 100 mg/dL (Nelson, 2014). Modest level elevations of serum D-dimers or fibrin-split products indicate an element of consumptive coagulopathy. Although usually modest, occasionally thrombocytopenia is marked (see Table 55-2). Again, among the TABLE 55-2. Laboratory Findings in 215 Women with Acute Fatty Liver of Pregnancy Series No. Fibrinogen (mg/dL) Platelets (103/.LL) Creatinine (mg/dL) AST (U/L) Pereira (1n997) 32 ND 123 (26-262) 2.7 (1n.1n-8.4) 99 (25-91n1)

1	Series No. Fibrinogen (mg/dL) Platelets (103/.LL) Creatinine (mg/dL) AST (U/L) Pereira (1n997) 32 ND 123 (26-262) 2.7 (1n.1n-8.4) 99 (25-91n1) Fesenmeier (2005) 16 ND 88 (22-226) 3.3 (0.5-8.6) 692 (122-3195) aFibrinogen and platelet values listed reflect the nadir for each patient, whereas creatinine and AST values reflect peak values for each patient. AST = aspartate transaminase; NO = not done. group from Parkland Hospital, 20 percent had platelet counts < 100,000/�L and 10 percent had platelet counts <50,000/�L (Nelson, 2014).

1	AST = aspartate transaminase; NO = not done. group from Parkland Hospital, 20 percent had platelet counts < 100,000/�L and 10 percent had platelet counts <50,000/�L (Nelson, 2014). Various liver imaging techniques have been used to confirm the diagnosis, however, none are particularly reliable. Speciically, Castro and associates (1996) reported poor sensitivity for conirmation by sonography-three of 11 patients, computed tomography (CT)-five of 10, and magnetic resonance (MR) imaging-none of ive. Similarly, in a prospective evaluation of the Swansea criteria proposed by Ch'ng and coworkers (2002), only a quarter of women had classic sonographic findings that include maternal ascites or an �chogenic hepatic appearance (Knight, 2008). Our experiences are similar (Nelson, 2013). The syndrome typically continues to worsen after diagnosis.

1	The syndrome typically continues to worsen after diagnosis. Hypoglycemia is common, and obvious hepatic encephalopathy, severe coagulopathy, and some degree of renal failure each develop in approximately half of women. Fortunately, delivery arrests liver function deterioration. We have encountered several women with a orme fruste of this disorder. Clinical involvement is relatively minor and laboratory aberrations-usually only hemolysis and a decreased plasma fibrinogen level-herald the syndrome. hus, the spectrum of liver involvement varies from milder cases that go unnoticed or are attributed to preeclampsia, to overt hepatic failure with encephalopathy.

1	Intensive supportive measures and good obstetrical care are essential. In some cases, the fetus may already be dead when the diagnosis is made, and the route of delivery is less problematic. Oten, living fetuses tolerate labor poorly. Because significant procrastination in efecting delivery may increase maternal and fetal risks, we prefer a trial oflabor induction with close fetal surveillance. Although some recommend cesarean delivery to hasten hepatic healing, this increases maternal risk when coagulopathy is severe. Nonetheless, cesarean delivery is common, and rates approach 90 percent. Transfusions with whole blood or packed red cells, along with fresh-frozen plasma, cryoprecipitate, and platelets, are usually necessary if surgery is performed or if obstetricalrlacerations complicate vaginal delivery (Chap. 41, p. 788).

1	Hepatic dysfunction resolves postpartum. It usually normalizes within a week, and in the interim, intensive medical support may be required. Two associated conditions can be seen around this time. Perhaps a fourth of women have evidence for transient diabetes insipidus. This presumably stems from elevated vasopressinase concentrations caused by diminished hepatic production of its inactivating enzyme. Finally, acute pancreatitis develops in approximately 20 percent.

1	With supportive care, recovery usually is complete. lvIaternal deaths are caused by sepsis, hemorrhage, aspiration, renal failure, pancreatitis, and gastrointestinal bleeding. Two women died in the series from Parkland Hospital. One was an encephalopathic woman who aspirated before intubation during transfer to our care. The other was in a woman with massive liver failure and nonresponsive hypotension (Nelson, 2013). In some centers, other measures have included plasma exchange and even liver transplantation (Fesenmeier, 2005; Franco, 2000; Martin, 2008).

1	Although maternal mortality rates with acute fatty liver of pregnancy have approached 75 percent in the past, the contemporaneous outlook is much better. From his review, Sibai (2007) cites an average mortality rate of 7 percent. He also cited a 70-percent preterm delivery rate and a perinatal mortality rate of 15 percent, which in the past was nearly 90 percent. At Parkland Hospital, the maternal and perinatal mortality rates during the past four decades have been 4 percent and 12 percent, respectively (Nelson, 2013).

1	Although most viral hepatitis syndromes are asymptomatic, during the past 30 years, acute symptomatic infections have become even less common in the United States (Daniels, 2009). There are at least ive distinct types of viral hepatitis: A (HA V), B (HBV), D (HDV) caused by the hepatitis B-associated delta agent, C (HCV) , and E (HEV). he clinical presentation is similar in all, and although the viruses themselves probably are not hepatotoxic, the immunological response to them causes hepatocellular necrosis (Dienstag, 2015a,b). Acute infections are most often subclinical and anicteric.

1	Acute infections are most often subclinical and anicteric. When they are clinically apparent, nausea and vomiting, headache, and malaise may precede jaundice by 1 to 2 weeks. Lowgrade fever is more common with hepatitis A. By the time jaundice develops, symptoms are usually improving. Serum transaminase levels vary, and their peaks do not correspond with disease severity (see Table 55-1). Peak levels that range from 400 to 4000 U/L are usually reached by the time jaundice develops. Serum bilirubin values typically continue to rise, despite falling serum transaminase levels, and peak at 5 to 20 mgl dL.

1	Any evidence for severe disease should prompt hospitalization. These include persistent nausea and vomiting, prolonged prothrombin time, low serum albumin level, hypoglycemia, high serum bilirubin level, or central nervous system symptoms. In most cases, however, clinical and biochemical recovery is complete within 1 to 2 months in all cases of hepatitis A, in most cases of hepatitis B, but in only a small proportion of cases of hepatitis C.

1	When patients are hospitalized, their feces, secretions, bedpans, and other articles in contact with the intestinal tract should be handled with glove-protected hands. Extra precautions, such as double gloving during delivery and surgical procedures, are recommended. Due to significant exposure of health-care personnel to hepatitis B, the Centers for Disease Control and Prevention (CDC) (2016a) recommend active and passive vaccination, described later. There is no vaccine for hepatitis C, so recommendations are for postexposure serosurveillance only.

1	Acute hepatitis has a case-fatality rate of 0.1 percent. For patients ill enough to be hospitalized, it may reach 1 percent. Most fatalities are due to fulminant hepatic necrosis, which in later pregnancy may resemble acute fatty liver. In these cases, hepatic encephalopathy is the usual presentation, and the mortality rate is 80 percent. Approximately half of patients with fulminant disease have hepatitis B infection, and co-infection with the delta agent is common. Hepatic, Biliary, and Pancreatic Disorders 1063

1	Hepatic, Biliary, and Pancreatic Disorders 1063 The CDC (20 16b) estimated that more than 4 million Americans were living with chronic viral hepatitis. Although most chroniclly infected persons are asymptomatic, approximately 20 percent develop cirrhosis within 10 to 20 years (Dienstag, 2015b). When present, symptoms are nonspecific and usually include fatigue. In some patients, cirrhosis with liver failure or bleeding varices may be the presenting inding. Indeed, asymptomatic chronic viral hepatitis as a group remains the leading cause of liver cancer and the most frequent reason for liver transplantation.

1	Chronic viral hepatitis is usually diagnosed serologically (Table 55-3). With persistently abnormal biochemical tests, liver biopsy usually discloses active inlammation, continuing necrosis, and fibrosis that may lead to cirrhosis. Chronic hepatitis is classiied by cause; by grade, defined by histological activity; and by stage, which is the degree of progression (Dienstag, 2015b). Most young women with chronic viral hepatitis either are asymptomatic or have only mild liver disease. For seropositive asymptomatic women, there usually are no problems with pregnancy. With symptomatic chronic active hepatitis, pregnancy outcome depends primarily on disease and ibrosis severity, and especially on the presence of portal hypertension. he few women whom we have managed have done well, but their long-term prognosis is poor. Accordingly, they should be counseled regarding possible liver transplantation as well as abortion and sterilization options.

1	Vaccination has reduced the incidence of hepatitis by 95 percent since 1995. In 2014, the rate was 0.4 per 100,000 individuals (Centers for Disease Control and Prevention, 2016b). his 27-nm RNA picornavirus is transmitted by the fecal-oral route, usually by ingestion of contaminated food or water. The incubation period is approximately 4 weeks. Individuals shed virus in their feces, and during the relatively brief period of viremia, their blood is also infectious. Signs and symptoms are oten nonspecific and usually mild, although jaundice develops in most patients. Symptoms usually last less than 2 months, although 10 to 15 percent of patients may remain symptomatic or relapse for up to 6 months (Dienstag, 20 15a). Early serological testing identifies IgVl anti-AV antibody, which may persist for several months. During convlescence, IgG antibody predominates, and it persists and provides subsequent immunity. here is no chronic stage of hepatitis A.

1	Management of hepatitis A in pregnancy includes a balanced diet and diminished physical activity. Women with less severe illness may be managed as outpatients. In developed countries, the efects of hepatitis A on pregnancy outcomes are not dramatic (American College of Obstetricians and Gynecologists, 2015, 2016). Both perinatal and maternal mortality rates, however, are substantively increased in resource-poor countries. Hepatitis A virus is not teratogenic, and transmission to the fetus is negligible. Preterm birth rates may be increased, and neonatal cholestasis has been reported (Urganci, 2003). Although hepatitis A RNA has been isolated in breast milk, no cases of neonatal hepatitis A have been reported secondary to breastfeeding (Daudi, 2012).

1	Preventatively, vaccination during childhood with foninactivated hepatitis viral vaccine is more than 90-percent efective. HA V vaccination is recommended by the American College of Obstetricians and Gynecologists (2016) and the Advisory Committee on Immunization (Kim, 20 15a) for highrisk adults. This category includes behavioral and occupational populations and travelers to high-risk countries. These countries are listed in the CDC (2016c) Health Information for International Travel "yellow book," which is available on the CDC website. Passive immunization for the pregnant woman recently exposed by close personal or sexual contact with a person with hepatitis A is provided by a 0.02 mLlkg dose of immune globulin (Kim, 2015a). Victor and colleagues (2007) reported that a single dose of HA V vaccine given in the usual dosage within 2 weeks of contact with an afected person was as efective as immune serum globulin to prevent hepatitis A. In both groups, HA V developed in 3 to 4

1	of HA V vaccine given in the usual dosage within 2 weeks of contact with an afected person was as efective as immune serum globulin to prevent hepatitis A. In both groups, HA V developed in 3 to 4 percent.

1	This double-stranded DNA virus is found worldwide. It is endemic in Africa, Central and Southeast Asia, China, Eastern Europe, the Middle East, and certain areas of South America, where prevalence rates reach 5 to 20 percent. The World Health Organization (WHO) (2009) estimates that more than 2 billion people worldwide are infected with HBV, and of these, 370 million have TABLE 55-3. Simplified Diagnostic Approach in Patients with Hepatitis Acute hepatitis A with + + + HAV = hepatitis A virus; HBc = hepatitis B core; HBsAg = hepatitis B surface antigen; HCV = hepatitis C virus. Compiled from the Centers for Disease Control and Prevention, 2016b; Dienstag, 2015a. chronic infection. The CDC (2016b) estimated nearly 18,100 cases of acute hepatitis B in the United States in 2014. This is a substantial decline since vaccination was introduced in the 1980s.

1	chronic infection. The CDC (2016b) estimated nearly 18,100 cases of acute hepatitis B in the United States in 2014. This is a substantial decline since vaccination was introduced in the 1980s. he hepatitis B virus is transmitted by exposure to blood or body fluids from infected individuals. In endemic countries, vertical transmission, that is, from mother to fetus or newborn, accounts for at least 35 to 50 percent of chronic HBV infections. In low-prevalence countries such as the United States, which has a prevalence <2 percent, the more frequent mode of HBV transmission is by sexual transmission or by sharing contaminated needles. HBV can be transmitted in any body luid, but exposure to virus-laden serum is the most eicient.

1	Acute hepatitis B develops after an incubation period of 30 to 180 days with a mean of 8 to 12 weeks. At least half of acute infections are asymptomatic. If symptoms are present, they are usually mild and include anorexia, nausea, vomiting, fever, abdominal pain, and jaundice. Acute HBV accounts for half of cases of fulminant hepatitis. Symptoms completely resolve within 3 to.4 months in more than 90 percent of patients.

1	Figure 55-2 details the sequence of the various HBV antigens and antibodies in acute infection. The first serological marker to be detected is the hepatitis B surface antigen (HBsAg) , often preceding the increase in transaminase levels. As HBsAg disappears, antibodies to the surface antigen develop (anti-HBs), marking complete resolution of disease. Hepatitis B core antigen is an intracellular antigen and not detectable in serum. However, anti -HBc is detectable wi thin weeks of HBsAg appearance. The hepatitis Be antigen (HBeAg) is present during times of high viral replication and often correlates with detectable HBV DNA. After acute hepatitis, approximately 90 percent of adults recover completely. The 10 percent who remain chronically infected are considered to have chronic hepatitis B. Chronic HBV infection is oten asymptomatic but may be clinically suggested by persistent anorexia, weight loss, fatigue, and

1	Chronic HBV infection is oten asymptomatic but may be clinically suggested by persistent anorexia, weight loss, fatigue, and FIGURE 55-2 Sequence of various antigens and antibodies in acute hepatitis B. ALT = alanine transaminase; anti-HBc = antibody to hepatitis B core antigen; anti-HBe = antibody to hepatitis Be antigen; anti-HBs = antibody to hepatitis B surface antigen; HBeAg = hepatitis Be antigen; HBsAg = hepatitis B surface antigen. (Reproduced with permission from Dienstag JL: Acute viral hepatitis. In Kasper DL, Fauci AS, Hauser SL, et al (eds): Harrison's Principles of Internal Medicine, 19th ed. New York, McGraw-Hili Education, 201o5).

1	hepatosplenomegaly. Extrahepatic manifestations may include arthritis, generalized vasculitis, glomerulonephritis, pericarditis, myocarditis, transverse myelitis, and peripheral neuropathy. One risk factor for chronic disease is age at acquisition. It is more than 90 percent in newborns, 50 percent in young children, and less than 10 percent in immunocompetent adults. Another risk is an immunocompromised state such as those with human immunodeiciency virus (HIV) infection, transplant recipients, or persons receiving chemotherapy. Chronically infected persons may be asymptomatic carriers or have chronic disease with or without cirrhosis. Patients with chronic disease have persistent HBsAg serum positivity. he patients with evidence of high viral replication-HBV DNA with or without HBeAg-have the highest likelihood of developing cirrhosis and hepatocellular carcinoma. The WHO considers hepatitis B to be second only to tobacco among human carcinogens. HBV DNA has been found to be the

1	the highest likelihood of developing cirrhosis and hepatocellular carcinoma. The WHO considers hepatitis B to be second only to tobacco among human carcinogens. HBV DNA has been found to be the best correlate of liver injury and disease progression risk.

1	Hepatitis B infection is not a cause of excessive maternal morbidity and mortality. It is often asymptomatic and found only on routine prenatal screening (Stewart, 2013). A review of data from the National Inpatient Sample reported a modest increase in preterm birth rates in HBV-positive mothers but no efect on fetal growth restriction or preeclampsia rates (Reddick, 2011). Others have shown similar results (Chen, 2015). Transplacental viral infection is uncommon, and Towers and associates (2001) reported that viral DNA is rarely found in amnionic luid or cord blood. Interestingly, HBV DNA has been found in the ovaries of HBV-positive pregnant women, although this may not be a significant factor in perinatal transmission Gin, 2016b). The highest HBV DNA levels were found in women who transmitted the virus to their fetuses (Dunkelberg, 2015; Society for Maternal-Fetal Medicine, 2016).

1	In the absence of HBV immunoprophylaxis, 10 to 20 percent of women positive for HBsAg transmit viral infection to their infant. This rate increases to almost 90 percent if the mother is HBsAg and HBeAg positive. Immunoprophylaxis and hepatitis B vaccine given to newborns ofHBV-infected mothers has decreased transmission dramatically and prevented approximately 90 percent of infections (Smith, 2012). But, women with high HBV viralloads-1 06 to 108 copies/ mL-or those who are HBeAg positive still have approximately a 10-percent vertical transmission rate, regardless of immunoprophylaxis (Yi, 2016).

1	The Society for Maternal-Fetal Medicine (2016) recommends antiviral therapy to decrease vertical transmission in women at highest risk because of high HBV DNA levels. Although lamivudine, a cytidine nucleoside analogue, signiicantly lowers the risk of fetal HBV infection in women with high HBV viral loads, recent data indicate that lamivudine may be less efective in the third trimester. Moreover, it is associated with the development of resistant mutations and is no longer recommended at a firstline agent. Newer drugs include the adenosine nucleoside analogue tenofovir and the thymidine analogue telbivudine. Both are associated with a lower risk of resistance than lamivudine (Ayres, 2014; Yi, 2016). T enofovir has been recommended as the irst-line agent during pregnancy by the Society for Maternal-Fetal Medicine (2016). These antiviral medications appear safe in pregnancy and are not associated with higher rates of congenital malforma tions or adverse obstetrical outcomes (Brown,

1	for Maternal-Fetal Medicine (2016). These antiviral medications appear safe in pregnancy and are not associated with higher rates of congenital malforma tions or adverse obstetrical outcomes (Brown, 2016). Hepatitis B immunoglobulin (HBIG) given antepartum to women at highest risk of transmission is also cost-efective (Fan, 2016).

1	Newborns of seropositive mothers are given HBIG very soon after birth. This is accompanied by the first of a three dose hepatitis B recombinant vaccine. Hill and colleagues the 2.4-percent transmission rate was not increased with breast feeding if vaccination was completed. Although virus is present in breast milk, the incidence of transmission is not lowered by formula feeding (Shi, 201l). he American Academy of Pedi atrics and the American College of Obstetricians and Gyne contraindication to breastfeeding.

1	For high-risk mothers who are seronegative, hepatitis B vaccine can be given during pregnancy. he eicacy is similar to that for nonpregnant adults, and overall seroconversion rates approach 95 percent after three doses (Stewart, 2013). he traditional vaccination schedule of 0, 1, and 6 months may be diicult to complete during pregnancy, and compliance rates decline after delivery. Sheield and coworkers (2011) reported that the three-dose regimen given prenatally-initially and at 1 and 4 months-resulted in seroconversion rates of 56, 77, and 90 percent, respectively. This regimen was easily completed during routine prenatal care.

1	Also called delta hepatitis, this is a defective RNA virus that is a hybrid particle with an HBsAg coat and a delta core. he virus must co-infect with hepatitis B either simultaneously or secondarily. It cannot persist in serum longer than hepatitis B virus. Transmission is similar to hepatitis B. Chronic coinfection with Brand D hepatitis is more severe and accelerated than with HBV alone, and up to 75 percent of afected patients develop cirrhosis. HDV infection is detected by the presence of anti-HDV and HDV DNA. Neonatal transmission is unusual, as neonatal HBV vaccination usually prevents delta hepatitis.

1	This is a single-stranded RNA virus, and transmission occurs via blood and body luids, although sexual transmission is ineicient. Up to a third of anti-HCV positive persons have no identifiable risk factors (Dienstag, 2015b). Screening for HCV is recommended for HIV-infected individuals, persons with injection drug use, hemodialysis patients, children born to mothers with HCV, persons exposed to HCV-positive blood or body fluids, persons with unexplained elevations in transaminase values, and recipients of blood or transplants before July 1992. Prenatal screening is recommended for high-risk women, and in the United States, seroprevalence rates reach 1 to 2.4 percent (American College of Obstetricians and Gynecologists, 2016; Arshad, 2011). It is higher in women who are infected with HIV. Santiago-Munoz and associates (2005) found that 6.3 percent of HIV-infected pregnant women at Parkland Hospital were co-infected with hepatitis B or C.

1	Hepatic, Biliary, and Pancreatic Disorders 1065 Acute HCV infection is usually asymptomatic or yields mild symptoms. Only 10 to 15 percent develop jaundice. he incubation period ranges from 15 to 160 days with a mean of 7 weeks. Transaminase levels are elevated episodically during the acute infection. Hepatitis C RNA testing is now preferred for HCV diagnosis. RNA levels may be found even before elevations of transaminase and anti-HCV levels. Speciically, anti-HCV antibody is not detected for an average of 15 weeks and in some cases up to a year (Dienstag, 20 15a).

1	Nearly 80 to 90 percent of patients with acute HCV will be chronically infected. Although most remain asymptomatic, approximately 20 to 30 percent progress to cirrhosis within 20 to 30 years. Transaminase values fluctuate, and HCV RNA levels vary over time. Liver biopsy reveals chronic disease and fibrosis in up to 50 percent, however, these indings are oten mild. Overall, the long-term prognosis for most patients is excellent. As expected, most pregnant women diagnosed with HCV have chronic disease. HCV infection was initially thought to have limited pregnancy efects. However, more recent reports have chronicled modestly increased fetal risks for low birthweight, NICU admission, preterm delivery, and mechanical ventilation (Berkley, 2008; Pergam, 2008; Reddick, 201l). In some women, high-risk behaviors associated with HCV infection.

1	he primary adverse perinatal outcome is vertical transmission of HCV infection to the fetus-infant. This is higher in mothers with viremia (Indolfi, 2014; Joshi, 2010). Airoldi and Berghella (2006) cited a rate of 1 to 3 percent in HCV-positive, RNA-negative women compared with 4 to 6 percent in those who were RNA-positive. In a report from Dublin, the vertical transmission rate in 545 HCV-infected women was 7. I-percent in RNA-positive women compared with none in those who were RNA-negative (McMenamin, 2008). Some have found an even greater risk when the mother is co-infected with HIV (Snidjewind, 2015; T ovo, 2016). Invasive prenatal diagnostic procedures have not been reported to increase transmission to the fetus. However, Rae and Sheield (2014) note that few studies have addressed this possibility, and they recommend avoiding traversing the placenta during amniocentesis. Approximately two thirds of prenatal transmission cases occur peripartum. HCV genotype, invasive prenatal

1	this possibility, and they recommend avoiding traversing the placenta during amniocentesis. Approximately two thirds of prenatal transmission cases occur peripartum. HCV genotype, invasive prenatal procedures, breastfeeding, and delivery mode are not associated with mother-to-child transmission. That said, invasive procedures such as internal electronic fetal heart rate monitoring are avoided. HCV infection is not a contraindication to breastfeeding.

1	No licensed vaccine is available for HCV prevention. The chronic HCV infection treatment has traditionally included alpha interferon (standard and pegylated), alone or in combination with ribavirin. This regimen is contraindicated in pregnancy because of the teratogenic potential of ribavirin in animals (Joshi, 2010). he initial 5-year review of the Ribavirin Pregnancy Registry found no evidence for human teratogenicity. However, the registry has enrolled fewer than half of the necessary numbers to allow a conclusive statement to be made (Roberts, 2010). The development and study of direct-acting and host-targeted antiviral drugs in the past decade shows great promise for chronic hepatitis C management (Liang, 2013; Lok, 2012; Poordad, 2013). Current interferon-free, ribavirin-free regimens are being evaluated, although no data are available for pregnant women.

1	This water-borne RNA virus usually is enterically transmitted by contaminated water supplies. Hepatitis E is probably the most common cause of acute hepatitis (Hoofnagle, 2012). It causes epidemic outbreaks in third-world countries with substantial morbidity and mortality rates. Pregnant women have a higher case-fatality rate than nonpregnant individuals. In a metaanalysis of nearly 4000 subjects from Asia and Africa, Jin and coworkers (2016a) reported maternal and fetal case-fatality rates of 21 and 34 percent, respectively. Fulminant hepatitis, although rare overall, is more common in gravidas and contributes to the increased mortality rates. An altered innate immune response to incipient hepatitis E infection during pregnancy, afecting macrophage function and toll-like receptor signaling, may be a factor in the development of fulminant hepatitis (Sehgal, 2015).

1	A recombinant HEV vaccine has been developed and licensed in China. It is >95 percent efective for 12 months after vaccination. Long-term eicacy is 87 percent, and protective titers are maintained for up to 4.5 years (Zhang, 2015). Preliminary data from inadvertently vaccinated pregnant women show no adverse maternal or fetal events (Wu, 2012). At this time, it is unclear if this Chinese-licensed vaccine is efective in other areas of the world where other genotypes predominate. Genotype 4 is most common in China, and types 2 and 3 are more common in the Americas. A Food and Drug Administration (FDA) approved vaccine is not available at this time. Hepatitis E is found worldwide, and although the highest prevalence is in east Asia, the CDC (2015) lists Mexico as a highly endemic country. Seroprevalence rates vary by age and geography, but overall seroprevalence rates of 10 percent have been reported. Durango State has the highest rate (37 percent) (Fierro, 2016).

1	Hepatitis G is the former name of an RNA flavivirus now known as HPvG or human pegivirus. This blood-borne infection of the liver, spleen, bone marrow, and mononuclear cells of the peripheral blood does not actually cause hepatitis (Chivero, 2015). It is thought to infect 750 million people worldwide, with up to two times that many with evidence of past infection. It may modulate the immune response, particularly during coinfection with HIV. Currently, no treatment aside from basic blood and body luid precautions is recommended. Vertical transmission (to the fetus/infant) and horizontal transmission (to peers) has been described (Trinks, 2014).

1	his is a generally progressive chronic hepatitis that is important to distinguish from other forms. Autoimmune hepatitis is more common in women and frequently coexists with other types of autoimmune disease, particularly autoimmune thyroid disease and Sjogren syndrome. Symptoms are typical of acute and chronic hepatitis, but one quarter may be asymptomatic. Rates of cirrhosis vary worldwide, but in western countries autoimmune hepatitis is more common and is characterized by multiple autoimmune antibodies such as antinuclear antibodies (ANA) and anti-smooth muscle antibody. Type 2 autoimmune hepatitis has an even higher prevalence in females and typically a more aggressive presentation. he incidence peaks in childhood and adolescence, before peak reproductive years. Treatment employs corticosteroids, alone or combined with azathioprine. Failure to respond to these two agents is more frequent in those with type 2 disease, and nearly all women with type 2 disease require more intensive

1	alone or combined with azathioprine. Failure to respond to these two agents is more frequent in those with type 2 disease, and nearly all women with type 2 disease require more intensive therapy that is sustained long term (Vierling, 2015). In some patients with progressive disease and cirrhosis, hepatocellular carcinoma develops. In general, autoimmune hepatitis-especially when severeincreases the risk of adverse pregnancy outcomes.

1	Westbrook and coworkers (2012) reported the outcomes of 81 pregnancies in 53 women. A third had a flare, and these were more common in those not taking medication and those with active disease in the year before conception. Maternal and fetal complications were higher among women with cirrhosis, particularly with respect to the risks of death or need for liver transplantation during the pregnancy or within 12 months postpartum. From one Swedish national database analysis, frequencies of preterm birth, low birthweight, and diabetes were higher, but not those of preeclampsia or cesarean delivery (Stokkeland, 2016). Danielsson Borssen (2016) reported stable or mild disease in 84 percent of 58 women who delivered 100 newborns. Nearly a fourth of cases were delivered before 38 weeks, and a postpartum flare developed in a third. Cirrhosis was present in 40 percent, and these women experienced more complications during pregnancy.

1	Chronic hepatitis and cirrhosis can result from iron and copper overload. Iron overload may stem from a primary cause that is generally inherited, such as hereditary hemochromatosis, or originates secondary to complications of certain hemoglobinopathies. Many of the gene mutations underlying hereditary hemochromatosis involve hepcidin and result in dysregulated iron transport (Chap. 4 p. 58). Some of these mutations are more common in certain populations originating from northern Europe (Pietrangelo, 2016; Salgia, 2015). Cardiomyopathy, diabetes, joint disease, and skin changes can coexist with liver disease. Pregnancy outcomes associated with iron overload in hereditary hemochromatosis are driven by the degree of liver dysfunction, although higher iron levels may afect birthweight (Dorak, 2009).

1	A form of neonatal hemochromatosis that does not afect the mother is now thought to be alloimmune and is called gestational aloimmune liver disease (Anastasio, 2016). With this, maternal autoantibodies cross to the fetus and mediate dysfunction of iron homeostasis, although the antigenic target of these alloantibodies remains unclear. It is associated with significant neonatal morbidity and mortality, and frequently recurs in subsequent pregnancies. In these cases, antepartum treatment with intravenous immunoglobulin (IVIG) may improve outcomes (Feldman, 2013; Roumiantsev, 2015).

1	Copper overload leading to chronic hepatitis and cirrhosis is Wilson disease. This systematic condition can also manifest with cardiomyopathy, renal disease, neuropsychiatric symp toms, and certain endocrine abnormalities. A Kayser-Fleischer ring surrounding the iris is highly speciic, but a suspected diagnosis generally requires genetic analysis. Autosomal reces sive mutations of the A TP7B gene underlie this disorder. his gene codes for the P-type ATPase involved in copper transport to ceruloplasmin and bile (Bandman, 2015). With Wilson disease, infertility may be present, but preg nancy outcomes among afected women who do conceive are inluenced by disease severity. Malik and colleagues (2013) reported four cases in pregnancy, and three had associated ges tational hypertension or preeclampsia. Maternal and neonatal outcomes were good, and the authors review chelation therapy with penicillamine and zinc sulfate in pregnancy. he American

1	College of Gastroenterology states that few data guide which of the various chelating agents is best (Tran, 2016). These include penicillamine, zinc, and trientine, and any theoretical risks are outweighed by the risks of discontinuing therapy. he latter include not only hepatic decompensation, but also injury to the placenta and fetal liver. Accordingly, the American College continue their chelation therapy, although a dose reduction of 25 to 50 percent should be considered to promote wound heal ing in the event of a surgical delivery. As a reminder, copper ions regulate the activity of proteins essential to wound repair.

1	his condition is frequently comorbid with obesity and is the most common chronic liver disease in the United States (Diehl, 2017). Its most severe form-nonacoholic steatohepatitis (NASH)-is an increasingly recognized condition that may occasionally progress to hepatic cirrhosis. Nonalcoholic fatty liver disease (NAFLD) is a macrovesicular fatty liver condition that resembles alcohol-induced liver injury but is seen without this substance abuse. Obesity, type 2 diabetes, and hyperlipidemiasyndrome X-frequently coexist (Chap. 48 p. 938). The current hypothesis suggests that these conditions may interact with other unknown etiological agents to cause multiple insults or "hits" leading to hepatic injury. For example, half of persons with type 2 diabetes have NAFLD, and insulin resistance has been postulated to act as one possible "hit" (Buzzetti, 2016). Browning and associates (2004) used MR spectroscopy to determine the prevalence ofNAFLD in Dallas County and found that approximately a

1	been postulated to act as one possible "hit" (Buzzetti, 2016). Browning and associates (2004) used MR spectroscopy to determine the prevalence ofNAFLD in Dallas County and found that approximately a third of adults were afected. This varied by ethnicity, with 45 percent of Hispanics, 33 percent of whites, and 24 percent of blacks being afected. Most people-80 percent-found to have steatosis had normal liver enzymes. In a study of obese adolescents undergoing bariatric surgery, more than a third had fatty liver without hepatitis, whereas an additional 20 percent had borderline or definite NASH (Xanthakos, 2015).

1	Liver damage follows a progressive continuum from NAFLD to NASH and then to hepatic ibrosis that may progress to cirrhosis (Goh, 2016). Still, in most persons, the disease is usually asymptomatic, and it is a frequent explanation for elevated serum transaminase levels found in blood donors and during Hepatic, Biliary, and Pancreatic Disorders 1067 other routine screening. Indeed, NAFLD is the cause of ele vated asymptomatic transaminase levels in up to 90 percent of cases in which other liver disease is ultimately excluded. It also is the most common cause of abnormal liver tests among adults in this country. Currently, weight loss along with control of diabetes and dyslipidemia is the only recommended treatment.

1	Fatty liver iniltration is probably much more common than realized in obese and diabetic gravidas. During the past decade, we encountered an increasing number of pregnant women with these disorders. Once severe liver injury, that is, acute fatty liver of pregnancy, is excluded, gravidas with fatty liver iniltra tion have no greater rates of adverse outcomes relative to liver involvement compared with pregnant women of similar weight. That said, some emerging data indicate that this condition may portend adverse pregnancy outcomes. In 110 pregnancies with NAFLD from the Swedish Medical Birth and the National

1	That said, some emerging data indicate that this condition may portend adverse pregnancy outcomes. In 110 pregnancies with NAFLD from the Swedish Medical Birth and the National Patient Registries, risks of gestational diabetes, preeclampsia, pre teI'm birth, and low-birthweight newborns were two-to threefold greater than in unafected women (Hagstrom, 2016). Yarrington and associates (2016) reported a high rate of gestational diabetes among nonobese women without liver disease, alcohol use, or diabetes, and who had elevated alanine transaminase levels in the irst trimester. s the obesity endemic worsens, adverse efects of this liver disorder on pregnancy will be clarified.

1	Irreversible chronic liver injury with extensive fibrosis and regenerative nodules is the inal common pathway for several disorders. Laennec cirrhosis from chronic alcohol exposure is the most frequent cause in the general population. But in young women-including pregnant women-most cases are caused by postnecrotic cirrhosis from chronic hepatitis Brand C. Many cases of cryptogenic cirrhosis are now known to be caused by NAFLD (Goh, 2016). Clinical manifestations of cirrhosis include jaundice, edema, coagulopathy, metabolic abnormalities, and portal hypertension with gastroesophageal varices and with splenomegaly that may cause thrombocytopenia. The incidence of deep-vein thromboembolism is increased (S0gaard, 2009). The prognosis is poor, and 75 percent have progressive disease that leads to death in 1 to 5 years. Women with symptomatic cirrhosis frequently are infertile.

1	Women with symptomatic cirrhosis frequently are infertile. hose who become pregnant generally have poor outcomes. Common complications include transient hepatic failure, variceal hemorrhage, preterm delivery, fetal growth restriction, and maternal death (Tan, 2008). Outcomes are generally worse if esophageal varices coexist. Another potentially fatal complication of cirrhosis arises from associated splenic artery aneurysms. Up to 20 percent of ruptures occur during pregnancy, and 70 percent of these rupture in the third trimester (Palatnik, 2017; Tan, 2008). In a review of 32 gravidas with aneurysm rupture, the mean aneurysm diameter was 2.25 cm, and in half of cases, the diameter was <2 cm (Ha, 2009). The 22-percent maternal mortality rate was likely related to the emergent presentation of these events. Parrish and colleagues (2015) described embolization of a 13 X 9 mm aneurysm in the third trimester leading to a splenic abscess and sepsis 3 weeks later.

1	In pregnant women, approximately half of cases of esophageal varices originate from cirrhosis or extrahepatic portal vein obstruction, which leads to portal system hypertension. Some cases of extrahepatic hypertension develop following portal vein thrombosis associated with one of the thrombophilia syndromes (Chap. 52, p. 1005). Others follow thrombosis from umbilical vein catheterization when the woman was a neonate, especially if she was born preterm.

1	With either intrahepatic or extrahepatic resistance to low, portal vein pressure rises from its normal range of 5 to 10 mm Hg, and values may exceed 30 mm Hg. Collateral circulation develops that carries portal blood to the systemic circulation. Blood drains into the gastric, intercostal, and other veins to the esophageal system, where varices develop. Bleeding is usually from varices near the gastroesophageal junction, and hemorrhage can be torrential. Bleeding during pregnancy from varices occurs in a third to half of afected women and is the major cause of maternal mortality within thisrgroup (Tan, 2008).

1	Maternal prognosis with esophageal varices largely depends on whether these rupture. Mortality rates are higher if varices are associated with cirrhosis compared with rates for varices without cirrhosis-18 versus 2 percent, respectively. Perinatal mortality rates are high in women with varices and are worse if cirrhosis caused the varices. Increased rates of neonatal demise, preterm birth, low birthweight, preeclampsia, and postpartum hemorrhage have been reported (Puljic, 2016). Treatment is the same as for nonpregnant patients. Preventatively, all patients with cirrhosis, including pregnant women, should undergo endoscopic screening for identification of variceal dilatation (Bacon, 2015). Beta-blocking drugs such as propranolol are given to reduce portal pressure and hence the bleeding risk (Bissonnette, 2015; Tran, 2016).

1	For acute bleeding and for prophylaxis, endoscopic band ligation is preferred to sclerotherapy as it avoids any potential risks of injecting sclerotherapeutic chemicals (Bissonnette, 2015; Tan, 2008). Acute medical management for bleeding varices veriied endoscopically includes the intravenous (IV) vasoconstrictors octreotide or somatostatin along with endoscopic banding. Vasopressin is less often used (Bacon, 2015). Baloon tamponade using a triple-lumen tube placed into the esophagus and stomach to compress bleeding varices can be lifesaving if endoscopy is not available. An interventional radiology procedure-tranjugular intrahepatic portosystemic stent shunting (TIPSS)-can also control bleeding from gastric varices that is unresponsive to other measures (Bissonnette, 2015; Tan, 2008). This procedure can be done electively in patients with prior variceal hemorrhage.

1	This drug is the most common cause of acute liver failure in the United States (Lee, 2013). Acetaminophen is often used during pregnancy, and overdose-either accidentally or by attempted suicide-may lead to hepatocellular necrosis and acute liver failure (Bunchorntavakul, 2013). Massive necrosis causes a cytokine storm and multiorgan dysfunction. Early symptoms of overdose are nausea, vomiting, diaphoresis, malaise, and pallor. With an acute overdose, after a latent period of 24 to 48 hours, liver failure ensues and usually begins to resolve in 5 days. In a prospective Danish study, only 35 percent of patients who were treated for fulminant hepatic failure spontaneously recovered before being listed for liver transplantation (Schmidt, 2007).

1	The antidote is N-aceycysteine, which must be given promptly. he drug is thought to increase glutathione levels, which aid metabolism of the toxic metabolite, N-acetyl-pbenzoquinoneimine. he need for treatment is based on projections of possible plasma hepatotoxic levels as a function of the time from acute ingestion. For this, many poison control centers use the nomogram established by Rumack and Matthew (1975). A plasma level is measured 4 hours after ingestion, and if the level is > 150 1g/mL, treatment is given (Smilkstein, 1988). If plasma determinations are not available, empirical treatment is given if the ingested amount exceeded 7.5 g. An oral loading dose of 140 mg/kg of N-acetylcysteine is followed by 17 maintenance doses of 70 mg/kg every 4 hours for 72 hours of total treatment time. Both the oral and an equally eicacious IV dosing regimen have been reviewed by Hodgman and Garrard (2012). he drug has been reported to reach therapeutic concentrations in the fetus (Wiest,

1	time. Both the oral and an equally eicacious IV dosing regimen have been reviewed by Hodgman and Garrard (2012). he drug has been reported to reach therapeutic concentrations in the fetus (Wiest, 2014).

1	After 14 weeks' gestation, the fetus has some cytochrome P450 activity necessary for metabolism of acetaminophen to the toxic metabolite. Riggs and colleagues (1989) reported follow-up data from the Rocky Mountain Poison and Drug Center in 60 women sufering overdose. he likelihood of maternal and fetal survival was better if the antidote was given soon after overdose. At least one 33-week fetus appears to have died as a direct result of hepatotoxicity 2 days after maternal ingestion. In another case, Crowell and associates (2008) reported a case of acetaminophen overdose at 32 weeks' gestation. The woman had taken 9.75 grams of acetaminophen approximately 1.5 hours prior to arrival. With treatment, the patient survived and went on to deliver a healthy term neonate.

1	This benign lesion of the liver is characterized in most cases by a well-delineated accumulation of normal but disordered hepatocytes that surround a central stellate scar. hese usually can be diferentiated from hepatic adenomas by magnetic resonance (vIR) or computed tomographic (CT) imaging. Except in the rare situation of unremitting pain, surgery is rarely indicated, and most women remain asymptomatic during pregnancy. In one review of 20 cases in Germany, no woman had related complications during pregnancy (Rifai, 2013). hree women showed 20-percent tumor growth; in 10 patients, the tumor decreased in size; and the remaining seven were unchanged across pregnancy. Ramirez-Fuentes and associates (2013) studied 44 lesions with MR imaging in 30 women. Of the lesions, 80 percent were unchanged in size, and most of the remainder decreased in size. They concluded that size changes were unrelated to pregnancy, combination oral contraceptive (COC) use, or menopause. Notably, this lesion

1	in size, and most of the remainder decreased in size. They concluded that size changes were unrelated to pregnancy, combination oral contraceptive (COC) use, or menopause. Notably, this lesion is not a contraindication to estrogen-containing contraceptives (Chap. 38, p. 692).

1	Hepatic, Biliary, and Pancreatic Disorders 1069 -This is an uncommon benign neoplasm but has a 5-percent risk of malignant transformation and a significant risk of rupture-associated hemorrhage, particularly in pregnancy. s just discussed, adenomas can usually be diferentiated from focal nodular hyperplasia by MR or CT imaging. Adenomas have a 9:1 predominance among women and are strongly linked with COC use. The rupture risk progresses with lesion size, and surgery is generally recommended for tumors measuring >5 cm (Agrawal, 2015). T ran and colleagues (2016) recommend sonographic surveillance of hepatic adenomas during pregnancy. In one review of 27 cases in pregnany, 23 became apparent in the third trimester .nd puerperium (Cobey, 2004). Bleeding complicated no tumors measuring <6.5 cm. However, 16 of27 women (60 percent) with an adenoma presented with tumor rupture that resulted in seven maternal deaths and six fetal deaths. Of note, 13 of 27 women presented within 2 months

1	cm. However, 16 of27 women (60 percent) with an adenoma presented with tumor rupture that resulted in seven maternal deaths and six fetal deaths. Of note, 13 of 27 women presented within 2 months postpartum, and in hal, hemorrhage heralded rupture. Wilson and coworkers (2011) described two cases of bleeding hepatic adenoma during pregnancy. One was managed by laparoscopic segmental resection, and another, which followed liver biopsy, required open surgery. The authors discourage biopsy during pregnancy for suspected hepatic adenomas, and emphasize the feasibility of resection for problematic lesions.

1	In 2013 in the United States, 5921 adult liver transplants were performed, and 34 percent of patients were women (Kim, 2015b). Currently, more than 65,000 recipients of liver transplant are living, and one literature review cited 450 pregnancies in 3026 women who had undergone transplantation (Deshpande, 2012). Although their live-birth rate of 80 percent and miscarriage rate compare favorably with those of the general population, risks of preeclampsia, cesarean delivery, and preterm birth are significantly elevated. A fourth of pregnancies were complicated by hypertension, approximately a third resulted in preterm birth, and in 10 percent, there was one or more rejection episodes (Table 55-4). Importantly, 4 percent of mothers had died within a year after delivery, but this rate is comparable to that in nonpregnant liver transplantation patients. Ghazali and associates (2016) analyzed the National Inpatient Sample database and found 2.r1 liver transplants per 100,000 deliveries.

1	to that in nonpregnant liver transplantation patients. Ghazali and associates (2016) analyzed the National Inpatient Sample database and found 2.r1 liver transplants per 100,000 deliveries. Pregnancies after liver transplant had signiicantly greater risks of maternal and fetal complications, including hypertensive disorders, gestational diabetes, and postpartum hemorrhage. Rates of preterm birth, fetal growth restriction, and congenital anomalies were also increased. Mattila and colleagues (2017) found that half of the women they cared for had maternal complications.

1	In the United States, 20 percent of women older than 40 years have gallstones. Most stones contain cholesterol, and its overse cretion into bile is thought to be a major factor in stone forma tion. The cumulative risk of all patients with silent gallstones to require surgery for symptoms or complications is 10 percent at 5 years, 15 percent at 1° years, and 18 percent at 15 years (Greenberger, 2015). For these reasons, prophylactic cholecys tectomy is not warranted for asymptomatic stones. For symp tomatic gallstone disease, nonsurgical approaches have been used and include oral bile acid therapy with ursodeoxycholic acid and extracorporeal shock wave lithotripsy. Experience with these during pregnancy is lacking.

1	Acute cholecystitis usually develops when the cystic duct is obstructed. Bacterial infection plays a role in 50 to 85 percent of cases. In more than half of patients with acute cholecystitis, a history of prior right upper quadrant pain from cholelithiasis is elicited. With acute disease, pain is accompanied by anorexia, nausea and vomiting, low-grade fever, and mild leukocytosis. As shown in Figure 55-3, sonography can help visualize stones, and both false-positive and false-negative rates range from 2 to 4 percent (Greenberger, 2015). In acute cases, medical therapy consists of IV luids, antimicrobials, analgesics, and in some instances, nasogastric suction, before surgical therapy. Laparoscopic cholecystectomy is the preferred treatment for most patients.

1	After the irst trimester, the gallbladder fasting volume and the residual volume after postprandial emptying are doubled. Incomplete emptying may result in retention of cholesterol crystals, a prerequisite for cholesterol gallstones. Maringhini and colleagues (1993) showed that the incidence of biliary sludge-which can be a forerunner to gallstones-and gallstones in pregnancy are 31 and 2 percent, respectively. Ko and colleagues (2014), however, cited a combined incidence of <5 percent. Others have identiied asymptomatic gallstones in FIGURE 55-3 This sonogram shows mUltiple hyperechoic gallstones filling an anechoic gallbladder. 2.5 to 10 percent of more than 1500 pregnant or postpartum women (Maringhini, 1993; Valdivieso, 1993).

1	2.5 to 10 percent of more than 1500 pregnant or postpartum women (Maringhini, 1993; Valdivieso, 1993). Postpartum, sludge frequently regresses, and occasionally gallstones will resorb. Still, within a year ter delivery, hospitalization for gallbladder disease remains relatively common, particularly for women managed conservatively in pregnancy. Jorge and coworkers (2015) reported that half of 53 women with symptomatic gallstones in pregnancy underwent postpartum cholecystectomy. In 80 percent of these women, recurrent symptoms developed prior to surgery, requiring readmission in half.

1	Acute cholecystitis during pregnancy or the puerperium is common and usually associated with gallstones or biliary sludge. Symptomatic cholecystitis is initially managed in a manner similar to that for nonpregnant women. In the past, most favored medical therapy. However, the recurrence rate during the same pregnancy is high, and 25 to 50 percent of women ultimately required cholecystectomy for persistent symptoms. Moreover, if cholecystitis recurs later in gestation, preterm labor is more likely and cholecystectomy is technically more diicult.

1	For these reasons, operative and endoscopic interventions are increasingly favored over conservative measures. Othman and coworkers (2012) showed that women managed conservatively had more pain, more recurrent visits to the emergency department, more hospitalizations, and a higher rate of cesarean delivery. Dhupar and associates (2010) reported more complications with conservative management of gallbladder disease compared with laparoscopic cholecystectomy in pregnancy. hese included multiple admissions, prolonged total parenteral nutrition, and unplanned labor induction for worsening gallbladder symptoms. Cholecystectomy was performed safely in all trimesters. Only one of 19 patients who underwent laparoscopic cholecystectomy had a complication, which did not require further surgery. A metaanalysis found that cholecystectomy does not increase the risk of preterm labor or of maternal or fetal mortality (Athwal, 2016). Management at Parkland Hospital has evolved to a more aggressive

1	metaanalysis found that cholecystectomy does not increase the risk of preterm labor or of maternal or fetal mortality (Athwal, 2016). Management at Parkland Hospital has evolved to a more aggressive surgical approach, especially if there is concomitant biliary pancreatitis as subsequently discussed. During the past two decades, laparoscopic cholecystectomy has evolved as the favored surgical approach and is discussed in Chapter 46 (p. 901).

1	Relief from symptomatic biliary duct gallstones during pregnancy has been greatly aided by use of endoscopic retrograde cholangiopancreatography (ERCP) (Fogel, 2014; Menees, 2006). he procedure is performed if common duct obstruction is suspected or proven. Approximately 10 percent of patients with symptomatic stone disease have common duct stones (Stinton, 2012). ERCP can be modiied in many cases so that radiation exposure from luoroscopy is avoided (Sethi, 2015).

1	Tang and associates (2009) reported results from 68 ERCP procedures performed in 65 pregnant women at Parkland Hospital. ll but two women had gallstones, and sphincterotomy was performed in all but one woman. Common duct stones were identified in half of these 65 women, and in all but one, the stones were successfully removed. A biliary stent was placed in 22 percent of cases and removed after delivery. Complications were minimal, and post-ERCP pancreatitis developed in 16 percent. Pregnancy outcomes were not diferent than for the general obstetrical population. As a less invasive approach, 1R cholangiopancreatography (MRCP) has been reported to have utility in pregnancy in small, retrospective case series. Wu and colleagues (2014) caution against its use in pregnancy, particularly for seriously ill women. Also, MRCP is not readily available, and this may lead to delayed deinitive management.

1	Ascending cholangitis can complicate acute biliary obstruction. Nearly 70 percent of afected patients develop Charcot triad-jaundice, abdominal pain, and fever. The diagnosis is aided by sonography, and treatment is broad-spectrum antibiotics and biliary drainage by ERCP (Greenberger, 2015). Acute pancreatic inlammation is triggered by factors that cause activation of pancreatic trypsinogen followed by autodigestion. It is characterized by cell-membrane disruption and proteolysis, edema, hemorrhage, and necrosis (Conwell, 2015; Fogel, 2014). Up to 10 percent of women develop necrotizing pancreatitis, which carries a mortality risk of 15 percent. This rate rises if infection develops (Cain, 2015).

1	In nonpregnant patients, acute pancreatitis is almost equally associated with gallstones and alcohol abuse. During pregnancy, however, cholelithiasis is almost always the predisposing condition. Other causes are hyperlipidemias, usually hypertriglyceridemia; hyperparathyroidism; congenital ductal anomalies; ERCP; some drugs; and rarely autoimmune pancreatitis (Cain, 2015; Ducarme, 2014). N onbiliary pancreatitis occasionally develops postoperatively, or it is associated with trauma, drugs, or some viral infections. Certain metabolic conditions, including acute fatty liver of pregnancy and familial hypertriglyceridemia, also predispose to pancreatitis (Nelson, 2013). Cases of acute and chronic pancreatitis have been linked to numerous mutations of the cystic fibrosis transmembrane conductance regulator gene (Chang, 2015).

1	The incidence of pancreatitis varies with the population studied. At Parland Hospital, with a predominant Mexican American population, acute pancreatitis complicated approximately 1 in 3300 pregnancies (Ramin, 1995). At Brigham and Women's Hospital, with a more diversely ethnic population, Hernandez and colleagues (2007) reported an incidence of 1 in 4450. In a multiple-institution :Midwestern three-state review, the incidence of acute pancreatitis was 1 in 3450 (Eddy, 2008). In contrast, from California birth certificate data, the incidence approximated only 1 case in 6000 pregnancies (Hacker, 2015).

1	Acute pancreatitis is characterized by mild to incapacitating epigastric pain, nausea and vomiting, and abdominal distention. Patients are usually distressed and have low-grade fever, tachycardia, hypotension, and abdominal tenderness. As many as 10 percent have systemic inlammatory response syndrome (SIRS), which causes endothelial activation and can lead to acute respiratory distress syndrome (Chap. 47, p. 921).

1	Of laboratory tests, serum amylase levels usually measure three times values considered the upper limit of normal. In 173 pregnant women with pancreatitis, the mean amylase value approximated 2000 lUlL, and the mean lipase value approached 3000 lUlL (Table 55-5). Importanty, the degree of enzyme elevation and disease severiy do not reliaby correlate. Indeed, by 48 to 72 hours, amylase levels may return to normal despite other evidence for continuing pancreatitis. Serum lipase activity is also increased and usually remains elevated with continued inlammation. Leukocytosis is usually found, and 25 percent of patients have hypocalcemia. Elevated serum bilirubin and aspartate transaminase levels may signiY gallstone disease. Several prognostic scoring systems have been used to classiY pancreatitis severity, but not all of these are useful in pregnancy. For instance, two of the ive Ranson criteria determined TABLE 55-5. Laboratory Values in 173 Pregnant Women with Acute Pancreatitis

1	TABLE 55-5. Laboratory Values in 173 Pregnant Women with Acute Pancreatitis Hepatic, Biliary, and Pancreatic Disorders 1071 at admission include variables speciic to nonpregnant patients. Similarly, certain criteria for the Apache II scoring system do not account for the changes in pregnancy physiology. In contrast, the Atlanta Classiication incorporates the degree of organ failure as a measure of severity and may be more applicable in pregnancy (Banks, 2013; Cain, 2015).

1	Medical treatment mirrors that for nonpregnant patients. This includes analgesics, IV hydration, and measures to decrease pancreatic secretion by interdiction of oral intake. Other than supportive therapy, antibiotics when appropriate, and targeted surgical interventions in the case of gallstone pancreatitis, no particular treatment schemes have improved outcomes. In a series by Ramin and colleagues (1995), all 43 afected pregnant women responded to conservative treatment and were hospitalized for a mean of8.5 days. Nasogastric suction does not improve outcomes of mild to moderate disease, but enteral feeding may be helpul once pain improves and associated ileus resolves. For women with more severe pancreatitis and a prolonged disease course, total enteral nutrition using nasojejunl feeding is superior to total parenteral nutrition (Cain, 2015; Conwell, 2015). If there is bacterial superinfection of necrotizing pancreatitis, sepsis, or cholangitis, then broad-spectrum antimicrobials

1	is superior to total parenteral nutrition (Cain, 2015; Conwell, 2015). If there is bacterial superinfection of necrotizing pancreatitis, sepsis, or cholangitis, then broad-spectrum antimicrobials are administered. If common duct stones are found, then ERCP is indicated (Fogel, 20 14; Tang, 2010). Cholecystectomy is considered after inflammation subsides because women with galls tonic pancreatitis carry an increased risk of recurrent pancreatitis (Cain, 2015).

1	Pregnancy outcomes are afected by acute pancreatitis severity. Eddy and associates (2008) reported a 30-percent preterm delivery rate, and 11 percent were delivered before 35 weeks' gestation. There were two pancreatitis-related maternal deaths. Importantly, almost a third of 73 women had recurrent pancreatitis during pregnancy. Of the 342 pregnancies complicated by pancreatitis in the California study, preterm delivery and fetal mortality rates were increased (Hacker, 2015). Also, preeclampsia risk was increased fourfold.

1	Few reports describe pregnancy following pancreas transplantation. Of 44 pregnancies in 73 women following pancreaskidney transplantation, outcomes are encouraging, and vaginal delivery has been described CMastrobattista, 2008). Although the incidence of hypertension, preeclampsia, preterm delivery, and fetal-growth restriction are high, there was only one perinatal death. Four rejection episodes developed during pregnancy and were treated successfully. Pancreatic islet autotransplanta tion may be performed to prevent diabetes following pancre Serum lipase 3076 36-41,824 atectomy, and at least three successful pregnancies have been 1.7 0.1-8.71 0.2-1.3 reported (J ung, 2007). Leukocytes 10,700 1000-27,200 3900-10,700 Abu-Hayyeh S, Ovadia C, Lieu T, et al: Prognostic and mechanistic potenrial of progesterone sulfates in inrrahepatic cholestasis of pregnancy and pruritus gravidarum. Hepatology ,t2016 Agrawal S, Agarwal S, Arnason T, et al: Management of hepatocellular ade

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1	Xiong HF, Liu ]Y, Guo LM, et al: Acute fatty liver of pregnancy: over six months follow-up study of twenty-ive patients. World J Gastroenterol 21(6):1927,s2015 Yarrington CD, Cantonwine DE, Seely EW, et al: The associarion of alanine aminotransferase in early pregnancy with gestational diabetes. Metab Syndr Relat Disord 14(5):254,2016 Yi P, Chen R, Huang Y, et al: Management of mother-to-child transmission of hepatitis B virus: propositions and challenges. J Clin Virol 77:32,s2016 Ylitalo K, Vanttinen T, Halmesmaki E, et al: Serious pregnancy complications in a patient with previously undiagnosed carnitine palmitoyltransferase 1 deiciency. Am] Obstet Gynecol 192:2060,2005 Zhang J, Zhang F, Huang 5], et al: Long-term eicacy of a hepatitis E vaccine. N Engl] Med 372:914,s2015 ZhangP, Kong WQ, Zhou SP, et al: Acute fatty liver of pregnancy: a retrospective analysis of 56 cases. Chin Med J (Eng!) 129(10): 1208,2016 IRON-DEFICIENCY ANEMIA.. . . . . . . . . . . . . . . . . . . . .. 1076

1	IRON-DEFICIENCY ANEMIA.. . . . . . . . . . . . . . . . . . . . .. 1076 MEGALOBLASTIC ANEMIA . . . . . . . . . . . . . . . . . . . . .. 1077 HEMOLYTIC ANEMIA ......i..........i...i.......i.....i. 1078 .APLASTIC AND HYPOPLASTIC ANEMIA.. . . . . . . . . . .. 1080 POL YCYTHEMIAS ..i......i...........i...i......i...... 1081 HEMOGLOBINOPATHIES ..i....i...i......i...i.....i..... 1081 THALASSEMIA SYNDROMES.. . . . . . . . . . . . . . . . . . . .. 1084 PLATELET DISORDERS.. . . . . . . . . . . . . . . . . . . . . . . . .. 1086 INHERITED COAGULATION DEFECTS ....i......i..... 1089 VON WILLEBRAND DISEASE.. . . . . . . . . . . . . . . . . . . .. 1090 In the later months of pregnancy there is a slight increase in the amount of hemoglobin and red corpuscles and a slight increase in the number of white corpuscles, which become markedy accentuated during the first ew days of the puerperium. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) There is virtually nothing from the irst edition of Williams' 1903 textbook that addresses the common anemias of pregnancy. Only pernicious anemia is devoted two paragraphs to say it occasionally appeared in pregnancy. Today, it is well known that pregnant women are susceptible to hematological abnormalities that may afect any woman of childbearing age. hese include chronic disorders such as hereditary anemias, immunological thrombocytopenia, and malignancies such as leukemias and lymphomas. Other disorders arise during pregnancy because of pregnancy-induced demands. Two examples are iron deiciency and megaloblastic anemias. Pregnancy may also unmask underlying hematological disorders. Finally, any hematological disease may irst arise during pregnancy. Importantly, pregnancy induces physiological changes that often confuse the diagnosis of these hematological disorders and assessment of their treatment (Chap. 4, p. 57).

1	Normal values for concentrations of many cellular elements during pregnancy are listed in the Appendix (p. 1255). The Centers for Disease Control and Prevention (1998) deined anemia in iron-supplemented pregnant women using a cutof of the 5th percentile-lrl g/dL in the irst and third trimesters, and 10.5 g/dL in the second trimester (Fig. he modest fall in hemoglobin levels and hematocrit values during pregnancy is caused by a relatively greater expansion of plasma volume compared with the increase in red cell volume. he disproportion between the rates at which plasma and erythrocytes are added to the maternal circulation is greatest during the second trimester. Late in pregnancy, plasma expansion essentially ceases, while hemoglobin mass continues to accrue.

1	he causes of more common anemias encountered in pregnancy are listed in Table 56-1r. Their frequency is dependent on multiple factors such as geography, ethniciry, socioeconomic level, nutrition, preexisting iron status, and prenatal iron supplementation (American College of Obstetricians and Gynecologists, 2017 a). In the United States, the prevalence of anemia in pregnancy is 3 to 38 percent (Centers for Disease Control and Prevention, 1989). In Latin America and the Caribbean, anemia prevalence ranges from 5 to 45 percent among women of :J taking iron supplements. (Data from the Centers for Disease Control and Prevention, 1989.) childbearing age (1ujica-Coopman, 2015). Rates are also high in Israel, China, India, South Asia, and Africa (Azulay, 2015; Kumar, 2013, Stevens, 2013). Figure 56-2 highlights the global trends in hemoglobin concentrations and anemia thresholds in pregnant and nonpregnant women.

1	Most studies of anemia during pregnancy describe large populations and deal with nutritional anemias. Anemia is associated with several adverse pregnancy outcomes including preterm birth (Kidanto, 2009; Kumar, 2013; Rukuni, 2016). Children born to iron-deicient women and without iron supplementation are reported to have lower mental development scores (Drassinower, 2016; Tran, 2014). A seemingly paradoxical inding is that healthy pregnant women with a higher hemoglobin concentration are also at greater risk for adverse perinatal outcomes (Murphy, 1986; von Tempelhof, 2008). This may result from lower than average plasma volume expansion of pregnancy concurrent with normal red cell mass accrual. Scanlon and associates (2000) studied the relationship between maternal hemoglobin levels and rates of preterm or growth-restricted newborns in 173,031 pregnancies. TABLE 56-1. Causes of Anemia During Pregnancy

1	TABLE 56-1. Causes of Anemia During Pregnancy Anemia caused by acute blood loss Anemia of inflammation or malignancy Megaloblastic anemia 0.030 ..••. Anemia cutoff for c 0.025 0 (12.0 g/dL) : ..... Anemia cutoff for 0 0.020 . (1 1.0 g/dL) 0.015 : 0.010 0.005 04.06.0 8.010.0 18.0 FIGURE 56-2 Global trends in hemoglobin concentrations in pregnant and nonpregnant women. (Reproduced with permission from Stevens GA, Finucane MM, De-Regil LM, et al: Global, regional, and national trends in haemoglobin concentration and prevalence of total and severe anaemia in children and pregnant and nonpregnant women for 1995-201o1: a systematic analysis of populationrepresentative data, Lancet Glob Health. 2013 Jul;1(l):e16-25.)

1	Women whose hemoglobin concentration was three standard deviations above the mean at 12 or 18 weeks' gestation had a 1.3-to 1.8-fold greater incidence of fetal-growth restriction. Placental weight correlates negatively with maternal hemoglobin concentration (Larsen, 2016). hese indings have led some to the illogical conclusion that withholding iron supplementation to cause iron-deiciency anemia will improve pregnancy outcomes (Ziaei, 2007). The two most common causes of anemia during pregnancy and the puerperium are iron deiciency and acute blood loss. In a study of more than 1300 women, 21 percent had thirdtrimester anemia, and 16 percent had iron-deiciency anemia (Vandevijvere, 2013). In a typical singleton gestation, the maternal need for iron averages nearly 1000 mg. Multifetal gestational requirements are considerably higher (Ru, 2016). hese amounts exceed the iron stores of most women and result in iron-deiciency anemia unless supplementation is given.

1	Iron deficiency is often manifested by an appreciable drop in hemoglobin concentration. In the third trimester, additional iron is needed to augment maternal hemoglobin and for transport to the fetus. Because the amount of iron diverted to the fetus is similar in a normal and in an iron-deicient mother, the newborn of a severely anemic mother does not sufer from irondeiciency anemia. Neonatal iron stores are related to maternal iron status and to timing of cord clamping. Classic morphological evidence of iron-deiciency anemia is erythrocyte hypochromia and microcytosis (Fig. 56-3). This may be less prominent in the pregnant woman. Serum ferritin levels are lower. And, levels of hepcidin-the master regulator of iron availability-are decreased normally in pregnancy. With iron deiciency, hepcidin levels follow those of serum ferritin (Camaschella, 2015; Koenig, 2014).

1	FIGURE 56-3 This peripheral blood smear from a women with iron-deficiency anemia contains many scattered microcytic and hypochromic red cells with characteristic central pallor. These exhibit moderate anisopoikilocytosis, namely, varying sizes and shapes including occasional elliptocytes, which can be oval or pencil-shaped. (Reproduced with permission from Werner Cl, Richardson Dl, Chang SY, et al (eds): Perioperative Considerations. In Williams Gynecology Study Guide, 3rd ed. New York, McGrawHill Education, 201o6: Photo contributor: Dr. Weina Chen.) he initial evaluation of a pregnant woman with moderate anemia includes measurements of hemoglobin, hematocrit, and red cell indices; careful examination of a peripheral blood smear; a sickle-cell preparation if the woman has African origin; and evaluation of serum iron or ferritin levels, or both (Appendix p. 1255). Serum ferritin levels normally decline during pregnancy, and levels < 10 to 15 mg/L confirm iron-deficiency anemia.

1	When pregnant women with moderate iron-deficiency anemia are given adequate iron therapy, a hematological response is detected by an elevated reticulocyte count. The rate of rise of hemoglobin concentration or hematocrit is typically slower than in nonpregnant women due to the increasing and larger blood volumes during pregnancy.

1	Routinely in pregnancy, daily oral supplementation with 30 to 60 of iron and 400 �g of folic acid is recommended (World Health Organization, 2012). A Cochrane review found that intermittent oral iron supplementation may also be appropriate (Pena-Rosas, 2015). For iron-deficiency anemia, resolution and restitution of iron stores can be accomplished with simple iron salts that provide approximately 200 mg daily of elemental iron. hese include ferrous sulfate, fumarate, or gluconate. If a woman cannot or will not take oral iron preparations, then parenteral therapy is given. Although both are administered intravenously, ferrous sucrose is safer than iron-dextran (American College of Obstetricians and Gynecologists, 2017a; Camaschella, 2015; Shi, 2015). Hemoglobin and ferritin levels show equivalent rises in women treated with either oral or parenteral iron therapy (Breymann, 2017; Daru, 20r16).

1	In early pregnancy, anemia caused by acute blood loss is common with abortion, ectopic pregnancy, and hydatidiform mole. Postpartum, anemia commonly stems from obstetrical hemorrhage. Massive hemorrhage demands immediate treatment as described in Chapter 41 (p. 788). If a moderately anemic woman-defined by a hemoglobin value of approximately 7 g/dL-is hemodynamically stable, is able to ambulate without adverse symptoms, and is not septic, then blood transfusions are not indicated. Instead, oral iron therapy is given for at least 3 months (Kraft, 2005) . • Anemia Associated with Chronic Disease Various disorders, such as chronic renal insuiciency, cancer and chemotherapy, human immunodeficiency virus (HIV) infection, and chronic inflammation result in moderate and sometimes severe anemia, usually with slightly hypochromic and microcytic erythrocytes. It is the second most common form of anemia worldwide (Weiss, 2005).

1	During pregnancy, women with chronic disorders may develop anemia for the first time. In those with preexisting anemia, it may be intensified as plasma volume expands. Causes include chronic renal insuiciency, inlammatory bowel disease, and connective-tissue disorders. Others are granulomatous infections, malignant neoplasms, rheumatoid arthritis, and chronic suppurative conditions. Chronic renal insuiciency is the most common disorder that we have encountered as a cause of this type of anemia during pregnancy. Some cases are accompanied by erythropoietin deficiency. As discussed in Chapter 53 (p. 1034), during pregnancy in women with mild chronic renal insuiciency, the degree of red cell mass expansion is inversely related to renal impairment. At the same time, plasma volume expansion usually is normal, and thus anemia is intensified (Cunningham, 1990).

1	For treatment, adequate iron stores must be ensured. Recombinant eythropoietin has been used successfully to treat anemia stemming from chronic disease (Weiss, 2005). In pregnancies complicated by chronic renal insuiciency, recombinant erythropoietin is usually considered when the hematocrit approximates 20 percent (Cyganek, 2011; Ramin, 2006). One worrisome side efect of this agent is hypertension, which is already prevalent in women with renal disease. Red cell aplasia and antierythropoietin antibodies have also reported (Casadevall, 2002; McCoy, 2008). These anemias are characterized by blood and bone-marrow abnormalities from impaired DNA synthesis. This leads to large cells with arrested nuclear maturation, whereas the cytoplasm matures more normally. Worldwide, the prevalence of megaloblastic anemia during pregnancy varies considerably. It is rare in the United States.

1	Megaloblastic anemia developing during pregnancy almost always results from folic acid deficiency. In the past, this condition was referred to as pernicious anemia of pregnancy. It usually is found in women who do not consume fresh green leay vegetables, legumes, or animal protein. As folate deficiency and anemia worsen, anorexia often becomes intense and further aggravates the dietary deiciency. Drugs and excessive ethanol ingestion either cause or contribute (Hesdorfer, 2015).

1	In nonpregnant women, the folic acid requirement is 50 to 1 00 �g/ d. During pregnancy, requirements are increased, and 400 �g/ d is recommended. he earliest biochemical evidence is low plasma folic acid concentrations (Appendix, p. 1255). Early morphological changes usually include neutrophils that are hypersegmented and newly formed erythrocytes that are macrocytic. With preexisting iron deiciency, macrocytic erythrocytes cannot be detected by measurement of the mean corpuscular volume. Careful examination of a peripheral blood smear, however, usually demonstrates some macrocytes. As the anemia becomes more intense, peripheral nucleated erythrocytes appear, and bone marrow examination discloses megaloblastic erythropoiesis. Anemia may then become severe, and thrombocytopenia, leukopenia, or both may develop. he fetus and placenta extract folate from maternal circulation so efectively that the fetus is not anemic despite severe maternal anemia.

1	For treatment, folic acid is given along with iron, and a nutritious diet is encouraged. By 4 to 7 days after beginning folic acid treatment, the reticulocyte count is increased, and leukopenia and thrombocytopenia are corrected.

1	For prevention of megaloblastic anemia, a diet should contain suicient folic acid. The role of folate deiciency in the genesis of neural-tube defects has been well studied (Chap. 13, p. 270). Since the early 1990s, nutritional experts and the American College of Obstetricians and Gynecologists (2016a) have recommended that all women of childbearing age consume at least 400 �g of folic acid daily. More folic acid is given with multifetal pregnancy, hemolytic anemia, Crohn disease, alcoholism, and inflammatory skin disorders. Women with a family history of congenital heart disease may also benefit from higher doses (Huhta, 2015). Women who previously have had infants with neural-tube defects have a lower recurrence rate if a daily 4-mg folic acid supplement is given.

1	During pregnancy, vitamin B12 levels are lower than nonpregnant values because of decreased levels of binding proteins, namely, the transcobalamins. During pregnancy, megaloblastic anemia is rare from deficiency of vitamin B12, that is, cyanocobalamin. Instead, a typical example is Addisonian pernicious anemia, which results from absent intrinsic factor that is requisite for dietary vitamin B12 absorption. his autoimmune disorder usually has its onset after age 40 years (Stabler, 2013).

1	In our limited experience, vitamin Bl2 deiciency in pregnancy is more likely encountered following gastric resection. hose who have undergone total gastrectomy require 1 000 �g of vitamin B12 given intramuscularly each month. Those with a partial gastrectomy usually do not need supplementation, but adequate serum vitamin B12 levels should be ensured (Appendix, p. 1258). Other causes of megaloblastic anemia from vitamin B12 deiciency include Crohn disease, ileal resection, some drugs, and bacterial overgrowth in the small bowel (Hesdorfer, 2015; Stabler, 2013).

1	Several conditions feature accelerated erythrocyte destruction. Damage may be stimulated by a congenital red-cell abnormality or in other cases by antibodies directed against red-cell membrane proteins. Hemolysis may be the primary disorder, and sickle-cell disease and hereditary spherocytosis are examples. In other cases, hemolysis develops secondary to an underlying condition such as systemic lupus erythematosus or preeclampsia. Microangiopathic hemolytic anemia due to malignancy has been reported in pregnancy (Happe, 2016).

1	he cause of aberrant antibody production is unknown. Typically, both the direct and indirect antiglobulin (Coombs) tests are positive. Anemias caused by these factors may be due to warm-active autoantibodies (80 to 90 percent), cold-active antibodies, or a combination. hese syndromes also may be classiied as primary (idiopathic) or secondary due to underlying diseases or other factors. Examples of the latter include lymphomas and leukemias, connective-tissue diseases, infections, chronic inlammatory diseases, and drug-induced antibodies (Provan, 2000). Cold-aglutinin disease may be induced by infectious etiologies such as Mycoplasma pneumoniae or Epstein-Barr viral mononucleosis (Dhingra, 2007). Hemolysis and positive antiglobulin test results may be the consequence of either immunoglobulin M (IgM) or immunoglobulin G (IgG) antierythrocyte antibodies. When thrombocytopenia is comorbid, it is termed Evans syndrome (Wright, 2013).

1	In pregnancy, hemolysis can be markedly accelerated. Rituximab, along with prednisone, is first-line treatment (Luzzatto, 2015). Coincidental thrombocytopenia usually corrects with therapy. Transfusion of red cells is complicated by antierythrocyte antibodies, but warming the donor cells to body temperature may decrease their destruction by cold agglutinins.

1	These hemolytic anemias must be diferentiated from other causes of autoimmune hemolysis. In most cases, hemolysis is mild, it resolves with drug withdrawal, and recurrence is prevented by avoidance of the drug. One mechanism is hemolysis induced through drug-mediated immunological injury to red cells. he drug may act as a high-ainity hapten when bound to a red-cell protein to which antidrug antibodies attach-for example, IgM anti penicillin or anticephalosporin antibodies. Some other drugs act as low-ainity hap tens and adhere to cell membrane proteins. Examples include probenecid, quinidine, rifampin, and thiopental. A more common mechanism for drug-induced hemolysis is related to a congenital erythrocyte enzymatic defect. An example is glucose-6-phosphate dehydrogenase deiciency, which is common in African-American women and discussed later (p. 1080).

1	Drug-induced hemolysis is usually chronic and mild to moderate, but occasionally acute hemolysis is severe. Garratty and coworkers (1999) described seven women with severe Coombs-positive hemolysis stimulated by cefotetan given as prophylaxis for obstetrical procedures. Alpha-methyldopa can cause similar hemolysis (Grigoriadis, 2013). Moreover, maternal hemolysis has been reported after intravenous immune globulin therapy (Rink, 2013). Withdrawal of the ofending drug frequently halts the hemolysis.

1	Unexplained severe hemolytic anemia can develop during early pregnancy, and it resolves within months postpartum. A clear immune mechanism or red cell defects are not contributory (Starksen, 1983). Because the fetus-neonate also may demonstrate transient hemolysis, an immunological cause is suspected. Maternal corticosteroid treatment is often-bur not alwaysefective (Kumar, 2001). We have cared for a woman who during each pregnancy developed intense severe hemolysis with anemia that was controlled by prednisone. Her fetuses were not afected, and in all instances, hemolysis abated spontaneously after delivery.

1	In some cases, hemolysis is induced by conditions unique to pregnancy. Mild microangiopathic hemolysis with thrombocytopenia is relatively common with severe preeclampsia and eclampsia (Cunningham, 2015; Kenny, 2015). This HELLP (hemoysis) devated fiver enzyme levels) Low .latelet count} syndrome is discussed in Chapter 40 (p. 719). Another is acure fatty liver of pregnancy, which is associated with moderate to severe hemolytic anemia (Nelson, 2013). It is discussed in Chapter 55 (p. 1060).

1	Although commonly regarded as a hemolytic anemia, this hemopoietic stem cell disorder is characterized by formation of defective platelets, granulocytes, and erythrocytes. Paroxysmal nocturnal hemoglobinuria is acquired and arises from one abnormal clone of cells, much like a neoplasm (Luzzatto, 2015). One mutated X-linked gene responsible for this condition is termed PIG-A because it codes for phosphatidylinositol glycan protein A. Resultant abnormal anchor proteins of the erythrocyte and granulocyte membrane make these cells unusually susceptible to lysis by complement (Provan, 2000). The most serious complication is thrombosis, which is heightened in the hypercoagulable state of pregnancy.

1	Chronic hemolysis has an insidious onset, and its severity ranges from mild to lethal. Hemoglobinuria develops at irregular intervals and is not necessarily nocturnal. Hemolysis may be initiated by transfusions, infections, or surgery. Almost 40 percent of patients sufer venous thromboses and may also experience renal failure, hypertension, and Budd-Chiari syndrome. Because of the thrombotic risk, prophylactic anticoagulation is recommended (Parker, 2005). The treatment of choice is eculizumab, an antibody that inhibits complement activation (Kelly, 2015). Median survival after diagnosis is 10 years, and bone marrow transplantation is the deinitive treatment.

1	During pregnancy, paroxysmal nocturnal hemoglobinuria can be serious and unpredictable. Complications have been reported in up to three fourths of afected women, and the maternal mortality rate in the past was 10 to 20 percent (De Gramont, 1987; de Guibert, 2011). Complications more often develop postpartum, and half of afected women develop venous thrombosis (Fieni, 2006; Ray, 2000). Kelly and colleagues (2015) described 75 pregnancies in 61 afected women treated with eculizumab. In half of these, the dose was increased during pregnancy. They described no maternal deaths but 4 percent stillbirths.

1	he most fulminant acquired hemolytic anemia encountered during pregnancy is caused by the exotoxin of Clostridium petingens or by group A J-hemolytic streptococcus (Chap. 47, p. 922). Endotoxin of gram-negative bacteria, that is, lipopolysaccharide, may be accompanied by hemolysis and mild-to-moderate anemia (Cox, 1991). For example, anemia often accompanies acute pyelonephritis. With normal erythropoietin production, red cell mass is restored following infection resolution as pregnancy progresses (Cavenee, 1994; Dotters-Katz, 2013).

1	he normal erythrocyte is a lexible biconcave disc that allows numerous cycles of reversible deformations. Several genes encode expression of erythrocyte structural membrane proteins or intraerythrocytic enzymes. Various mutations of these genes may result in inherited membrane defects or enzyme deiciencies that destabilize the lipid bilayer. The loss of lipids from the erythrocyte membrane causes a surface area deficiency and poorly deformable cells that undergo hemolysis. Anemia severity depends on the degree of rigidity or decreased distensibility. Erythrocyte morphology similarly is dependent on these factors, and these disorders are usually named after the most dominant red-cell shape characteristic of the disorder. hree examples are hereditary spherocytosis) pyropoikilocytosis, and ovalocytosis.

1	Hereditary Spherocytosis. Hemolytic anemias that compose this group of inherited membrane defects are among the most common hemolytic anemias found in gravidas. Mutations are usually an autosomally dominant, variably penetrant spectrin deiciency. Others are autosomally recessive or de novo gene mutations that result from deiciency of ankyrin) protein 4.2, moderate band 3, or combinations of these (Gallagher, 2010; Rencic, 2017; Yawara, 2000). The degrees of anemia and jaundice vary, and diagnosis is conirmed by identiication of spherocytes on peripheral smear and increased osmotic fragility.

1	Spherocytic anemias may be associated with a so-called crisis that is characterized by severe anemia from accelerated hemolysis, and it develops in patients with an enlarged spleen. Infection can also accelerate hemolysis or suppress erythropoiesis to worsen anemia. An example of the latter is infection with parvovirus B19 (Chap. 64, p. 1216). In severe cases, splenectomy reduces hemolysis, anemia, and jaundice.

1	Pregnancy. In general, women with inherited red-cell membrane defects do well during pregnancy. Folic acid supplementation of 4 mg daily is given orally to sustain erythropoiesis. Women with hereditary spherocytosis cared for at Parkland Hospital had hematocrits ranging from 23 to 41 volumes percent mean 31 (Maberry, 1992). Reticulocyte counts ranged from 1 to 23 percent. Among 50 pregnancies in 23 women, eight women miscarried. Four of 42 infants were born preterm, but none was growth restricted. Infection in four women intensified hemolysis, and three of these required transusions. Similar results were reported by Pajor and coworkers (1993). Because these disorders are inherited, the newborn may be afected. Celkan and Alhaj (2008) report prenatal diagnosis via cordocentesis at 18 weeks' gestation and testing for osmotic fragility. Newborns with hereditary spherocytosis may manifest hyperbilirubinemia and anemia shortly after birth.

1	n intraerythrocytic deficiency of enzymes that permit anaerobic glucose metabolism may cause hereditay nonspherocytic anemia. Most of these mutations are autosomal recessive traits. As discussed earlier (p. 1078), most episodes of severe anemia with enzyme deficiencies are induced by drugs or infections. Pyruvate kinase deficiency is associated with variable anemia and hypertensive complications (Wx, 2007). Due to recurrent transfusions in homozygous carriers, iron overload is frequent, and associated myocardial dysfunction should be monitored (Dolan, 2002). The fetus that is homozygous for this mutation may develop hydrops ttalis from anemia and heart failure (Chap. 15, p. 309).

1	Glucose-6-phosphate dehydrogenase (G6PD) deiciency is complex because there are more than 400 known enzyme variants. he most common are caused by a base substitution that leads to an amino acid replacement and a broad range of phenotypic severity (Luzzatto, 2015; Puig, 2013). In the homozygous or A variant, both X chromosomes are afected, and erythrocytes are markedly deicient in G6PD activity. Approximately 2 percent of African-American women are afected, and the heterozygous variant is found in 10 to 15 percent (Mockenhaupt, 2003). In both instances, random X-chromosome inactivation-yonization-results in variable enzyme activity.

1	During pregnancy, infections or drugs can induce hemolysis in G6PD deficiency heterozygotes or homozygotes, and severity is related to enzyme activity. Anemia is usually episodic, although some variants induce chronic nonspherocytic hemolysis. Because young erythrocytes contain more enzyme activity, anemia ultimately stabilizes and is corrected soon after the inciting cause is eliminated. Newborn screening for G6PD deiciency is not recommended by the American College of Obstetricians and Gynecologists (20 16b).

1	Aplastic anemia is a grave complication that is characterized by pancytopenia and markedly hypocellular bone marrow (Young, 2015). There are multiple etiologies, and at least one is linked to autoimmune diseases (Stalder, 2009). he inciting cause can be identified in approximately a third of cases. These include drugs and other chemicals, infection, irradiation, leukemia, immunological disorders, and inherited conditions such as Fanconi anemia and Diamond-Backon syndrome (Green, 2009; Lipton, 2009). The functional defect appears to be a marked decrease in committed marrow stem cells.

1	Hematopoietic stem-cell transplantation is optimal therapy in a young patient (Killick, 2016). Immunosuppressive therapy is given, and in some nonresponders, eltrombopag has been successful (Olnes, 2012; Townsley, 2017). Deinitive treatment is bone marrow transplantation, and approximately three fourths of patients have a good response and long-term survival (Rosenfeld, 2003). Umbilical cord blood-derived stem cells can also serve as a potential transplant source (Moise, 2005; Pinto, 2008). Previous blood transfusions and even pregnancy enhance the risk of graft rejection (Young, 2015).

1	Hypoplastic or aplastic anemia complicating pregnancy is rare. A study of 60 pregnancies complicated by aplastic anemia found that half were diagnosed during pregnancy (Bo, 2016). There are a few well-documented cases of pregnancy-induced hypoplastic anemia, and the anemia and other cytopenias improve or remit following delivery or pregnancy termination (Bourantas, 1997; Choudhry, 2002). In some cases, anemia recurred in a subsequent pregnancy.

1	Diamond-Blackon anemia is a rare form of pure red-cell hypoplasia. Approximately 40 percent of cases are familial and have autosomal dominant inheritance (Orfali, 2004). The response to glucocorticoid therapy is usually good. Continuous treatment is necessary, and most become at least partially transfusion dependent (Vlachos, 2008). In 64 pregnancies complicated by this syndrome, Faivre and associates (2006) reported that two thirds had problems related to placental vascular etiologies that included miscarriage, preeclampsia, preterm birth, fetal-growth restriction, or stillbirth.

1	Gaucher disease is an autosomally recessive lysosomal enzyme deficiency characterized by deficient activity of acid >-glucosidase. Afected women have anemia and thrombocytopenia that is usually worsened by pregnancy (Granovsky-Grisaru, 1995). Elstein and colleagues (1997) described six pregnant women whose disease improved when they were given alglucerase enzyme replacement. Imiglucerase therapy, which is human recombinant enzyme replacement therapy, has been available since 1994. European guidelines recommend treatment in pregnancy, whereas the Food and Drug Administration states it may be given with "clear indications" (Granovsky-Grisaru, 2011).

1	he major risks with hypoplastic anemia are hemorrhage and infection. Rates of preterm labor, preeclampsia, fetal-growth restriction, and stillbirth are increased (Bo, 2016). Management depends on gestational age, and supportive care includes continuous infection surveillance and prompt antimicrobial therapy. Granulocyte transfusions are given only during infections. Red cells are transfused to improve symptomatic anemia and routinely to maintain the hematocrit at or above 20 volumes percent. Platelet transfusions may be needed to control hemorrhage. Maternal mortality rates reported since 1960 have averaged nearly 50 percent, however, better outcomes have been reported more recently (Choudhry, 2002; Kwon, 2006).

1	Several reports describe successful pregnancies in women who have undergone bone marrow transplantation (BorgnaPignatti, 1996; Eliyahu, 1994). In their review, Sanders and coworkers (1996) reported 72 pregnancies in 41 women who had undergone transplantation. In the 52 pregnancies resulting in a liveborn neonate, almost half were complicated by preterm delivery or hypertension. Our experiences with a few of these women indicate that they have normal pregnancy-augmented erythropoiesis and total blood volume expansion. Excessive erythrocytosis during pregnancy is usually related to chronic hypoxia from maternal congenital cardiac disease or a chronic pulmonary disorder. Unusually heavy cigarette smoking can cause polycythemia. We have encountered otherwise healthy pregnant women who were heavy smokers, had chronic bronchitis, and had hematocrits ranging from 55 to 60 volumes percent! If polycythemia is severe, the probability of a successful pregnancy outcome is low.

1	This is a primary clonal myeloproliferative hemopoietic stemcell disorder characterized by excessive proliferation of erythroid, myeloid, and megakaryocytic precursors (Spivak, 2015; Vannucchi, 2015). Virtually all patients have either aJA2V617F or aJA2 exon 12 gene mutation (Harrison, 2009). Symptoms are related to increased blood viscosity, and thrombotic complications are common. Treatment of nonpregnant patients is with hydroxyurea or ruxolitinib (Vannucchi, 2015). Fetal loss rates are high in women with polycythemia vera, and pregnancy outcome may be improved with aspirin therapy (Griesshammer, 2006; Robinson, 2005; Teferi, 2000). Women with a history of venous thrombosis are given prophylaxis with low-molecular-weight heparin. If cytoreduction is required during pregnancy, interferon alpha may be considered (Kreher, 2014).

1	Hemoglobin A is the most common hemoglobin tetramer and consists of two .-and two 3-chains. In contrast, sickle hemoglobin (hemoglobin S) originates from a single 3-chain substitution of glutamic acid by valine, which stems from an A-for-T substitution at codon 6 of the 3-globin gene. Hemoglobinopathies that can result in clinical features of the sickle-cell syndrome include sickle-cell anemia (Hb SS); sickle-cell hemoglobin C disease (Hb SC); sickle-cell 3-thalassemia disease (either Hb SI or Hb S/B+); and sickle-cell E disease (Hb SE) (Benz, 2015). All are also associated with increased pregnancy morbidity.

1	Sickle-cell anemia results from the inheritance of the gene for S hemoglobin from each parent. In the United States, 1 of 12 African-Americans has sickle-cell trait, which results from inheritance of one gene for hemoglobin S and one for normal hemoglobin A. he computed incidence of sickle-cell anemia among frican-Americans is 1 in 576 (1112 X 1112 X 114 = 11576). But, the disease is less common in adults because of earlier mortality. Hemoglobin C originates from a single 3-chain substitution of glutamic acid by lysine, which stems from a T -forC substitution at codon 6 of the 3-globin gene. Approximately 1 in 40 African-Americans has the gene for hemoglobin C. Thus, the theoretical incidence for co inheritance of the gene for hemoglobin S and an allelic gene for hemoglobin C in an frican-American child is about 1 in 2000 (1/12 X 1140 X 114). 3-halassemia minor is approximately 1 in 40, thus S-3-thalassemia also is found in approximately 1 in 2000 (1/12 X 1140 X 114).

1	Red cells with hemoglobin S undergo sickling when they are deoxygenated, and the hemoglobin aggregates. Constant sickling and unsickling cause membrane damage, and the cell may become irreversibly sickled. Events that slow erythrocyte transit through the microcirculation include adhesion to endothelial cells, erythrocytic dehydration, and vasomotor dysregulation. Clinically, the hallmarks of sickling episodes are periods during which there is ischemia and infarction in various organs. The sickle-cell crisis produces clinical symptoms, predominately pain, which is often severe. There may be aplastic, megaloblastic, sequestration, and hemolytic crises.

1	Chronic and acute changes from sickling include bony abnormalities such as osteonecrosis of femoral and humeral heads, renal medullary damage, autosplenectomy in homozygous SS patients and splenomegaly in other variants, hepatomegaly, ventricular hypertrophy, pulmonary infarctions, pulmonary hypertension, cerebrovascular accidents, leg ulcers, and a propensity for infection and sepsis (Benz, 2015; Gladwin, 2004). Other sequelae are cerebrovascular aneurysms and sickle-cell vasculopathy (Buonanno, 2016). Pulmonary hypertension can develop and is found in 20 percent of adults with SS hemoglobin (Gladwin, 2008).

1	Good supportive care is essential to prevent mortality. Speciic therapies are evolving, and many are still experimental. One treatment is hemoglobin F induction with drugs that stimulate gamma-chain synthesis. This increases hemoglobin F, which inhibits hemoglobin S polymerization. One example is hydroyurea, which augments hemoglobin F production and reduces the number of sickling episodes (Platt, 2008). Hydroxyurea is teratogenic in animls, although a preliminary 17 -year surveillance of antenatally exposed children was reassuring (Ballas, 2009; Briggs, 2015; Italia, 2010). A randomized trial showed no benefit from treatment with prasurel, a platelet inhibitor (Henney, 2016). Treatment with crianlizumab, an antibody against P-selectin, signiicantly lowered the incidence of adverse events (Ataga, 2017).

1	Various forms of hemopoietic cell transplantation are emerging as "cures" for sicle-cell syndromes and severe thalassemias (Hsieh, 2009). Oringanje and coworkers (20l3) performed a Cochrane review and found that only observational studies have been reported. Bone marrow transplantation has 5-year survival rates that exceed 90 percent (DaIle, 20l3). Cord-blood stem-cell transplantation from related donors also shows great promise (Shenoy, 2013). Finally, successful gene therapy has been accomplished by lentiviral vector-mediated addition of a beta globin gene into stem cells (Ribeil, 2017). only after all other possible causes have been excluded. Pain TABLE 56-2. Pregnancy Morbidity with Hemoglobin SS with sickle-cell syndromes is caused by intense sequestration of sickled erythrocytes and infarction in various organs, especially Odds Ratios Oukome Hb.SS Hb.SC bone marrow. These episodes may develop acutely, especially

1	Odds Ratios Oukome Hb.SS Hb.SC bone marrow. These episodes may develop acutely, especially Preeclampsia 2-3.1 late in pregnancy, during labor and delivery, and early in the 2.0 puerperium. Stillbirth 6.5 3.2 Preterm delivery 2-2.7 1.5 Growth restriction 2.8-3.9 1.5 Data from metaanalyses by Boafor, 201n6; Oteng-Ntim, 2015.

1	Stillbirth 6.5 3.2 Preterm delivery 2-2.7 1.5 Growth restriction 2.8-3.9 1.5 Data from metaanalyses by Boafor, 201n6; Oteng-Ntim, 2015. Pregnancy is a serious burden to women with any of the major sickle hemoglobinopathies, particularly those with hemoglobin SS disease. Several large studies have defined this relationship. Villers and colleagues (2008) studied 17,952 births in women with sickle-cell syndromes. Chakravarty and associates (2008) studied 4352 pregnancies. A more recent cohort study of 1526 women was reported by Boulet and coworkers (2013). Last, a cohort study taken from more than 2 million women compared those with sickle-cell disease to normal controls (Kuo, 2016). Common obstetrical and medical complications and their relative risks from a composite of most of these studies are shown in Table 56-2.

1	Maternal morbidity common in pregnancy includes ischemic necrosis of multiple organs, especially bone marrow that causes episodes of severe pain. Pyelonephritis, pneumonia, and pulmonary complications are frequent. Although the maternal mortality rate has improved, perinatal morbidity and mortality rates remain formidable (Boga, 2016; Lesage, 2015; Yu, 2009). Perinatal outcomes include increased risks for preterm birth, fetal-growth restriction, and perinatal mortality. In nonpregnant women, morbidity and mortality rates from SC disease are appreciably lower than those from sickle-cell anemia. Indeed, fewer than half of these women have symptoms before pregnancy. In our experiences, afected gravidas sufer attacks of severe bone pain and episodes of pulmonary infarction and embolization more commonly than when they are not pregnant (Cunningham, 1983). Some adverse pregnancy outcomes are shown in Table 56-2.

1	Women with sickle-cell hemoglobinopathies require close prenatal observation. Any factor that impairs erythropoiesis or increases red cell destruction aggravates the anemia. Prenatal folic acid supplementation with 4 mg daily is needed to support rapid red blood cell turnover. One danger is that a symptomatic woman may categorically be considered to be sufering from a "sickle-cell crisis." As a result, serious obstetrical or medical problems that cause pain, anemia, or both may be overlooked. Examples are ectopic pregnancy, placental abruption, pyelonephritis, or appendicitis. Thus, a diagnosis of sickle-cell crisis should be applied

1	Guidelines for care of these women have been appropriately stressed by Rees and colleagues (2003). !vIarti-Carvajal and coworkers (2009) performed a Cochrane review and reported that no randomized trials have evaluated treatment during pregnancy. At minimum, intravenous luids are given, and opioids are administered promptly for severe pain. Oxygen via nasal cannula may decrease the intensity of sickling at the capillary level. We have found that red cell transfusions after the onset of severe pain do not dramatically improve pain intensity and may not shorten its duration. Conversely, as discussed later, prophylactic transfusions almost always prevent further vasoocclusive episodes and pain crises. Recent reports suggest benefits from epidural analgesia (Verstraete, 2012; Winder, 2011). Long term, afected women can become habituated to narcotics. his problem is highlighted by the increased rates of neonatal abstinence syndrome, which is a constellation of withdrawal symptoms (Shirel,

1	term, afected women can become habituated to narcotics. his problem is highlighted by the increased rates of neonatal abstinence syndrome, which is a constellation of withdrawal symptoms (Shirel, 2016).

1	Rates of covert bacteriuria and acute pyelonephritis are elevated substantively, and screening and treatment for bacteriuria are essential. If pyelonephritis develops, sickle cells are extremely susceptible to bacterial endotoxin, which can cause dramatic and rapid red cell destruction while simultaneously suppressing erythropoiesis. Pneumonia, especially due to Streptococcus pneumoniae, is common. he Centers for Disease Control and Prevention recommends specific vaccination for those with sickle-cell disease and all asplenic patients (Kim, 2016). hese are polyvalent pneumococcal, Haemophilus inluenzae type B, and meningococcal vaccines, and administration guidelines are found in Table 9-7 (p. 172).

1	Pulmonary complications are frequent. Of these, acute chest syndrome is characterized by pleuritic chest pain, fever, cough, lung infiltrates, and hypoxia, and usually also by bone and joint pain (Vichinsky, 2000). In addition to symptoms, radiographs show a new pulmonary infiltrate. There are four precipitants: infection, marrow emboli, thromboembolism, and atelectasis (Medof, 2005). Bacterial or viral infection causes approximately half of cases. When acute chest syndrome develops, the mean duration of hospitalization is 10.5 days. Mechanical ventilation is required in approximately 15 percent, and the mortality rate approximates 3 percent (Gladwin, 2008). At least for nonpregnant adults, some recommend rapid simple or exchange transfusions to remove the "trigger" for acute chest syndromes (Gladwin, 2008). In a study of nonpregnant patients, Turner and colleagues (2009) reported that there were no increased beneits of exchange versus simple transfusions, and the former were

1	syndromes (Gladwin, 2008). In a study of nonpregnant patients, Turner and colleagues (2009) reported that there were no increased beneits of exchange versus simple transfusions, and the former were associated with fourfold increased blood usage.

1	Women with sickle-cell disease usually have some degree of cardiac dysfunction from ventricular hypertrophy. Chronic hypertension worsens the dysfunction (Gandhi, 2000). During pregnancy, the basal hemodynamic state characterized by high cardiac output and increased blood volume is augmented (Veille, 1994). Although most women tolerate pregnancy with out problems, complications such as severe preeclampsia or ham, 1986). Heart failure caused by pulmonary hypertension must also be considered (Chakravarty, 2008). In 4352 pregnancies in women with sickle-cell syndromes, pregnancy complication rates. Compared with controls, women with sickling disorders had a 63-percent rate of nondelivery related admissions. hey had a 1.8-fold greater incidence of hypertensive disorders-19 percent; a 2.9-fold higher rate of fetal-growth restriction-6 percent; and a 1.7-fold increased cesarean delivery rate-45 percent.

1	Prophylactic Red Cell Transfusions. Chronic transfusion therapy prevents strokes in high-risk children (DeBaun, 2014). During pregnancy, the most dramatic beneit of prophylactic transfusions has been on maternal morbidity rates (Benites, 2016). In an observational 10-year prospective study at Parkland Hospital, we ofered prophylactic transfusions to all pregnant women with sickle-cell syndromes. Transfusions were given throughout pregnancy to maintain the hematocrit above 25 volumes percent and the portion of hemoglobin S <60 percent (Cunningham, 1979). Maternal morbidity was minimal, and erythropoiesis suppression was not problematic. Their outcomes were compared with historical controls who were not routinely transfused. Overall, morbidity and hospitalization rates were signiicantly reduced in the transfused group (Asma, 2015; Cunningham, 1983; Grossetti, 2009). Still, adverse perinatal outcomes are prevalent (Ngo, 2010).

1	In a multicenter trial, Koshy and coworkers (1988) randomly assigned 72 pregnant women with sickle-cell syndromes to prophylactic or indicated transfusions. They reported a signiicant decline in the incidence of painful sickle-cell crises with prophylactic transfusions but no diferences in perinatal outcomes. Because of risks inherent with blood administration, they concluded that prophylactic transfusions were not indicated. A metaanalysis of 12 studies found prophylactic transfusions improved rates of some adverse maternal and neonatal outcomes, including maternal mortality, pulmonary complications, and perinatal mortality (Malinowski, 2015).

1	Undoubtedly, morbidity from multiple transfusions is significant. Up to 10 percent of women had a delayed hemolytic transfusion reaction, and infections are major concerns. Garratty (1997) reviewed 12 studies and found alloimmunization developed in a fourth of women. Finally, in liver biopsies in these women, we found no evidence of transfusion-related iron overload, hemochromatosis, or chronic hepatitis (Yeomans, 1990). Because of what some consider marginal benefits, routine prophylactic transfusions during pregnancy remain controversial (American College of Obstetricians and Gynecologists, 2015; Okusayna, 2013). Current consensus is that their use should be individualized. Fetal Assessment. Because of the high incidence offetal-growth restriction and perinatal mortality, serial fetal assessment with sonography and antepartum surveillance is recommended (American College of Obstetricians and Gynecologists, 2015).

1	Anyaegbunam and colleagues (1991) reported nonreactive stress tests during sickling crises, which resumed reactivity with crisis resolution. They concluded that transient efects of sicklecell crisis do not compromise umbilical blood flow. Management is essentially identical to that for women with cardiac disease (Chap. 49, p. 953). Women should be kept comfortable, but not oversedated. Conduction analgesia is ideal (Camous, 2008). Compatible blood should be available. If a diicult vaginal or cesarean delivery is contemplated, and the hematocrit is <20 volumes percent, then packed erythrocyte transfusions are administered. There is no categorical contraindication to vaginal delivery, and cesarean delivery is reserved for obstetrical indications (Rogers, 2010).

1	Many clinicians do not recommend combination hormonal contraception because of potential adverse vascular and thrombotic efects. In their systematic review, however, Haddad and coworkers (2012) found that complication rates were not higher with their use in women with sickle-cell syndromes. he Centers for Disease Control and Prevention categorizes combination hormonal contraception, intrauterine devices, implants, and progestin-only contraception as having no risk or as having advantages that generally outweigh theoretical or proven risks (Curtis, 2016).

1	The frequency of sickle-cell trait among African-Americans averages 8 percent. Carriers have occasional hematuria, renal papillary necrosis, and hyposthenuria, which is urine of low specific gravity (Tsaras, 2009). And although controversial, sickle-cell trait does not appear to be associated with increased rates of abortion, perinatal mortality, low birthweight, or pregnancy-induced hypertension (Pritchard, 1973; Tita, 2007; Tuck, 1983). One unquestioned relationship is the twofold increased incidence of asymptomatic bacteriuria and urinary infection. Sickle-cell trait should not be considered a deterrent to pregnancy or to hormonal contraception. Inheritance is a concern for the fetus of a mother with sickle-cell trait whenever the father carries a gene for abnormal hemoglobins that include S, C, and D or for 3-thalassemia trait. Prenatal diagnosis is discussed in Chapter 14 (p. 290).

1	Approximately 2 percent of African-Americans are heterozygous for hemoglobin C, but even if homozygous, hemoglobin C is innocuous (Nagel, 2003). Only when coinherited with sickle-cell trait to yield hemoglobin SC is the trait problematic. Pregnancy in women with homozygous hemoglobin CC disease or C-3-thalassemia carries relatively benign associations. Table 56-3 shows our experiences from Parkland Hospital (Maberry, 1990). Other than mild-to-moderate anemia, pregnancy outcomes were not abnormal. Supplementation with folic acid and iron is indicated. Data from Maberry, 1990.

1	Although uncommon in the United States, hemoglobin E is the second most frequent hemoglobin variant worldwide. The heterozygous E trait is common in Southeast Asia. Hurst and coworkers (1983) identified homozygous hemoglobin E, hemoglobin E plus 3-thalassemia, or hemoglobin E trait in 36 percent of Cambodians and 25 percent of Laotians. Hemoglobin EE is associated with little or no anemia, hypochromia, marked microcytosis, or erythrocyte targeting. Kemthong and colleagues (2016) studied 1073 women and 2146 controls and found that hemoglobin E trait does not increase pregnancy risks other than asymptomatic bacteriuria. Conversely, doubly heterozygous E-3-thalassemia is a common cause of severe childhood anemia in Southeast Asia (DeLoughery, 2014). In a cohort study of 54 women with singleton pregnancies, Luewan and associates (2009) reported a threefold greater risk of preterm birth and fetal-growth restriction in afected women. It is unclear if hemoglobin SE disease is ominous during

1	pregnancies, Luewan and associates (2009) reported a threefold greater risk of preterm birth and fetal-growth restriction in afected women. It is unclear if hemoglobin SE disease is ominous during pregnancy.

1	• Hemoglobinopathy in the Newborn Neonates with homozygous SS, SC, and CC disease can be identiied accurately at birth by cord blood electrophoresis. The United States Preventive Services Task Force recommends that all newborns be tested for sickle-cell disease (Lin, 2007). In most states, such screening is mandated by law and performed routinely (Chap. 32, p. 614). Many tests are available to detect sickle-cell disease ante natally. Most are DNA based and use chorionic villus samples or amnionic fluid specimens (American College of Obstetricians and Gynecologists, 2015). Several mutations that encode hemoglobin S and other abnormal hemoglobins can be detected by targeted mutation analysis and polymerase chain reaction-based techniques (Chap. 13, p. 270).

1	Hundreds of mutations afect genes that control hemoglobin production. Some of these impair synthesis of one or more of the normal globin peptide chains and may result in a clinical syndrome characterized by varying degrees of inefective erythropoiesis, hemolysis, and anemia (Benz, 2015). Thalassemias are classiied according to the globin chain that is deficient. The two major forms involve impaired production or instability of a-peptide chains to cause a-thalassemia or of 3-chains to cause 3-thalassemia. hese may form from point mutations, deletions, or translocations involving the .-or non-a-globin gene (Leung, 2012). Because there are four a-globin genes, the inheritance of a-thalassemia is more complicated than for 3-thalassemia (Piel, 2014). Possible genotypes and phenotypes are shown in

1	Because there are four a-globin genes, the inheritance of a-thalassemia is more complicated than for 3-thalassemia (Piel, 2014). Possible genotypes and phenotypes are shown in Table 56-4. Clinical severity closely correlates with the degree of a-globin chains synthesis impairment. In most populations, the a-globin chain "cluster" or gene loci are doubled on chromosome 16. Similarly, ther, chains are duplicated. Thus, the normal genotype for diploid cells can be expressed as ../.. and "r /,,. here are two main groups of a-thalassemia determinants: .0-thalassemia is the deletion of both loci from one chromosome (--/..), whereas .+ -thalassemia is the loss of a single locus from one allele (-./.. heterozygote) or a loss from each allele (-./-.homozygote). here are two major phenotypes. he deletion of all four a-globin chain genes (--/--) characterizes homozygous a-thalassemia. Because a-chains are contained in fetal hemoglobin, the fetus is afected. When none of the four genes

1	TABLE 56-4. Genotypes and Phenotypes of .-Thalassemia Syndromes Normal ../.. Normal .+-Thalassemia heterozygote -'/.. } Normal; silent carrier ../-. .+-Thalassemia homozygotea -./-. } a-Thalassemia minor-mild .°-Thalassemia heterozygoteb --/.. hypochromic microcytic anemia Compound heterozygous .0/.+ --/-. Hgb H (34) with moderate-to severe hemolytic anemia Homozygous .-thalassemia --/--Hgb Bart ('4) disease, hydrops fetalis African Americans. bMore common in Asian Americans. are expressed, no a-globin chains are produced, and instead hemoglobin Bart (14) and hemoglobin H (34) are formed as abnormal tetramers that cannot transport oxygen (Chap. 7, p. 131). he relative frequency of a-thalassemia minor, hemoglobin H disease, and hemoglobin Bart disease varies remarkably among racial groups. All of these variants are encountered in Asians.

1	he relative frequency of a-thalassemia minor, hemoglobin H disease, and hemoglobin Bart disease varies remarkably among racial groups. All of these variants are encountered in Asians. In those of African descent, although a-thalassemia minor has a frequency approximating 2 percent, hemoglobin H disease is rare and hemoglobin Bart disease is unreported. This is because Asians usually have .0-thalassemia minor inherited with both gene deletions typically from the same chromosome (--/..), whereas blacks usually have .+-thalassemia minor in which one gene is deleted from each chromosome (-xl-x). Diagnosis of 3-thalassemia minor and a-thalassemia major in the fetus can be accomplished by DNA analysis using molec ular techniques (Piel, 2014). Fetal diagnosis of hemoglobin yakul, 2009; Srivorakun, 2009). Molecular genetic testing for HBAl and HBA2 identifies 90 percent of deletions and 10 percent of point mutations in afected individuals (Galanello, 2011 b).

1	Important obstetrical aspects of some a-thalassemia syndromes depend on the number of gene deletions in a given woman. he silent carrier state with one gene deletion is of no consequence. Deletion of two genes resulting in a-thalassemia minor is characterized by minimal-to-moderate hypochromic micro cytic anemia. This is due to either .r_ or .+-thalassemia trait, and thus genotypes may be -xla or --la.. Diferentiation is possible only by DNA analysis (Piel, 2014). Because no other clinical abnormalities accompany either form of a-thalassemia minor, it often goes unrecognized and is usually of no maternal consequence (Hanprasertpong, 2013). The fetus with these forms of thalassemia minor will have hemoglobin Bart at birth, but as its levels drop, it is not replaced by hemoglobin H. Red cells are hypochromic and microcytic, and the hemoglobin concentration is normal to slightly depressed.

1	Hemoglobin H disease (34) results from the compound heterozygous state for .0_ plus .+-thalassemia with deletion of three of four alpha genes (--I-x). With only one functional a-globin gene per diploid genome, the newborn will have abnormal red cells containing a mixture of hemoglobin Bart (14), hemoglobin H (34)' and hemoglobin A. The neonate appears normal but soon develops hemolytic anemia as most of the hemoglobin Bart is replaced by hemoglobin H. In adults, anemia is moderate to severe and usually worsens during pregnancy. Inheritance of all four abnormal a genes causes homozygous a-thalassemia with predominant production of hemoglobin Bart, which has an appreciably increased ainity for oxygen. This is incompatible with extended survival. Hsieh and colleagues (1989) reported that blood obtained by funipuncture from 20 hydropic fetuses contained 65 to 98 percent Bart hemoglobin. These fetuses are stillborn, or they are hydropic and usually die very soon after birth.

1	Sonographic measurement of the fetal cardiothoracic ratio at 12 to 13 weeks' gestation can be used to identiy afected fetuses (Lam, 1999; Zhen, 2015). Sonographic assessment of myocardial performance-the Tei index-in the irst half of pregnancy has been evaluated. Changes predate hydrops in afected fetuses (Luewan, 2013). Severe anemia can be detected using Doppler velocimetry of the middle cerebral artery. ment (Galanello, 201r1a). he 3-thalassemias are the consequences of impaired 3-globin chain production or a-chain instability. Genes that encode con trol of 3-globin synthesis are in the r3-gene "cluster" located on chromosome 11 (Chap. 7, p. 131). More than 150 point mutations in the 3-globin gene have been described (Weather all, 2010). In 3-thalassemia, 3-chain production is decreased, and excess a-chains precipitate to cause cell-membrane dam age. Other forms of3-thalassemias are caused by a-chain insta bility (Kihm, 2002).

1	he heterozygous trait is 3-thalassemia minor, and those Az levels. his hemoglobin is composed of two .-and two 6-globin chains, and concentrations are usually more than 3.5 percent. Hemoglobin F-composed of two .-and two I-globin chains-also usually has increased concentrations that exceed 2 percent. Some patients with heterozygous 3-thalassemia minor do not have anemia, and others have mild-to-moderate anemia characterized by hypochromia and microcytosis.

1	Homozygous 3-thalassemia-also called 3-thalassemia major or Cooley anemia-is a serious and frequently fatal disorder. Hemolysis is intense and leads to severe anemia. Many patients become transfusion dependent, and the subsequent iron load, along with abnormally increased gastrointestinal iron absorption, leads to hemochromatosis, which is fatal in many cases. Stem cell transplantation has been used to treat 3-thalassemia major Gagannath, 2014). A heterozygous form of 3-thalassemia that clinically manifests as thalassemia intermedia produces moderate anemia. During pregnancy, women with 3-thalassemia minor may have mild anemia (Charoenboon, 2016). Iron and folate supplements are given. In some women, anemia will worsen because normal plasma volume expansion may be accompanied by slightly subnormal erythropoiesis.

1	Thalassemia major and some of the other severe forms were uncommonly encountered during pregnancy before the advent of transfusion and iron chelation therapy. With such management, 63 pregnancies were reported and sufered no serious complications (Aessopos, 1999; Daskalakis, 1998). Pregnancy is considered reasonably safe if maternal cardiac function is normal. Transfusions are given throughout pregnancy to maintain the hemoglobin concentration at 10 g/dL. This is coupled with surveillance of fetal growth (American College of Obstetricians and Gynecologists, 2015; Sheiner, 2004).

1	Because 3-thalassemia major is caused by numerous mutations, prenatal diagnosis is diicult. For a given individual, targeted mutation analysis is done that requires prior identification of the familial mutation. The analysis is done using chorionic villus sampling and other techniques discussed in Chapter 14 (p. 293). Noninvasive testing of circulating fetal nucleic acids in maternal plasma for the diagnosis of 3-thalassemia has been described (Leung, 2012; Xiong, 2015).

1	Platelet abnormalities may precede pregnancy, develop during pregnancy coincidentally, or be induced by pregnancy. hrombocytopenia-deined by a platelet count < 150,000/�L-is identified in nearly 10 percent of gravidas (American College of Obstetricians and Gynecologists, 2016c). Of these cases, 75 percent are gestational thrombocytopenia, whereas 25 percent are due to other various causes. One other common cause is HELLP syndrome. Thrombocytopenia may be inherited or idiopathic, acute or chronic, and primary or associated with other disorders. Examples are shown in Table 56-5.

1	Burrows and Kelton (1993) reported that 6.6 percent of 15,471 pregnant women had a platelet count < 150,000/�L, and in 1.2 percent, it was <100,000/�L. hey further noted that almost 75 percent of 1027 women whose platelet counts were < 150,000/�L were found to have normal-variant incidental thrombocytopenia. Of the remainder, 21 percent had a hypertensive disorder of pregnancy, and 4 percent had an immunological disorder. A platelet count of <80,000/�L should trigger TABLE 56-5. Some Causes of Thrombocytopenia in Pregnancy APAS = antiphospholipid antibody syndrome; DIC = disseminated intravascular coagulopathy; HELLP = hemolysis, elevated liver enzyme levels, low platelet count; MTP = massive transfusion protocol. Data from American College of Obstetrics and Gynecologists, 2016c; Aster, 2007; Diz-Ku:ukkaya, 2016. an evaluation for etiologies other than incidental or gestational thrombocytopenia, which is unlikely to have a platelet count <50,000/�L (Gernsheimer, 2013).

1	an evaluation for etiologies other than incidental or gestational thrombocytopenia, which is unlikely to have a platelet count <50,000/�L (Gernsheimer, 2013). he physiological decline in platelet concentration seen with gestational thrombocytopenia is usually evident in the third trimester and is thought to be predominantly due to hemodilution. The normal increased splenic mass characteristic of pregnancy may also be contributory (Maymon, 2006). Most evidence shows that platelet life span is unchanged in normal pregnancy (Kenny, 2015).

1	Benard-Soulier yndrome is characterized by lack of platelet membrane glycoprotein (GPIb/IX) and causes severe dysunction. Moreover, women exposed to fetal platelets carrying this glycoprotein can develop antibodies against this fetal GPIb/IX antigen to cause lloimmune fetal thrombocytopenia (Fujimori, 1999; Peng, 1991). A systematic review of 30 pregnancies in 18 women reported a 33-percent rate of primary postpartum hemorrhage, and half of women with bleeding required blood transfusion (Peitsidis, 2010). he reviewers also described six cases of neonatal lloimmune thrombocytopenia and two perinatl deaths. Close monitoring throughout pregnancy and 6 weeks postpartum is critical due to the possibility of life-threatening hemorrhage (Prabu, 2006).

1	May-Heglin anomay is an autosomal dominant disorder characterized by thrombocytopenia, giant platelets, and leukocyte inclusions (Chatwani, 1992). Urato and Repke (1998) described such a woman who was delivered vaginally. Despite a platelet count of 16,000/�L, she did not bleed excessively. he neonate inherited the anomaly but also had no bleeding despite a platelet count of35,000/�L. A systematic review of26 studies containing 75 pregnancies in 40 women reported four cases of postpartum hemorrhage, 34 cases of neonatal thrombocytopenia, and two fetal deaths (Hussein, 2013).

1	The primary form-also termed idiopathic thrombocytopenic purpura (ITP)-is usually caused by a cluster of IgG antibodies directed against one or more platelet glycoproteins (Konkle, 2015). Antibody-coated platelets are destroyed prematurely in the reticuloendothelial system, especially the spleen. Although not proven, the disorder is probably mediated by autoantibodies directed at platelet-associated immunoglobulins-P AlgG, P AIgM, and P lA.In adults, immune thrombocytopenia most often is a chronic disease that rarely resolves spontaneously.

1	As shown in Table 56-5, secondary forms of immunemediated chronic thrombocytopenia appear in association with systemic lupus erythematosus, lymphomas, leukemias, and several systemic diseases. Approximately 2 percent of thrombocytopenic patients have positive serological tests for lupus, and in some cases, levels of anticardiolipin antibodies are high. Finally, approximately 10 percent of HIV -positive patients have associated thrombocytopenia (Scaradavou, 2002). Diagnosis and Management. Only a few adults with primary immune thrombocytopenia recover spontaneously, and for those who do not, platelet counts usually range from 10,000 to 100,000/�L (George, 2014). Evidence does not suggest that pregnancy increases the risk of relapse in women with women with active disease. hat said, it is certainly not unusual years to have recurrent thrombocytopenia during pregnancy. Although this may be from closer surveillance, hyperestrogen emia has also been implicated.

1	Although this may be from closer surveillance, hyperestrogen emia has also been implicated. Therapy is considered if the platelet count is below 30,000 to 50,000/�L (American College of Obstetricians and Gyne cologists, 20 16c). Primary treatment includes corticosteroids or intravenous immune globulin (lYlG) (Neunert, 201r1). Initially, prednisone, 1 mg/kg daily, is given to suppress the phagocytic activity of the splenic monocyte-macrophage sys tem. IYlG given in a total dose of 2 g/kg during 2 to 5 days is also efective.

1	In pregnant women with no response to corticosteroid or IVIG therapy, open or laparoscopic splenectomy may be efective. In late pregnancy, cesarean delivery may be necessary for surgical exposure. Improvement usually follows splenectomy in 1 to 3 days and peaks at approximately 8 days. Cytotoxic agents are typically avoided in pregnancy due to teratogenicity risks. Azathioprine and rituximab, however, which are used in nonpregnant persons with ITP, have been used for other conditions in pregnancy. Finally, the thrombopoietin agonist romplostim has stimulated responses in some patients (Decrooq, 2014; Imbach, 2011; Kuter, 2010).

1	Fetal and Neonatal Efects. Pregnancy complications that are increased with ITP include stillbirth, fetal loss, and preterm birth (Wyszynski, 2016). Platelet-associated IgG antibodies cross the placenta, and fetal death from hemorrhage occurs occasionally (Webert, 2003). he severely thrombocytopenic fetus is at increased risk for intracranial hemorrhage with labor and delivery, but fortunately this is unusual. Payne and associates (1997) reviewed studies of maternal ITP published since 1973. Of 601 newborns, 12 percent had severe thrombocytopenia with counts <50,000/�L. Six infants had intracranial hemorrhage, and in three, their initial platelet count was >50,000/�L. his is consistent with a study of 127 pregnancies in women with ITP in which 10 to 15 percent of neonates had transient ITP (Koyama, 2012).

1	Investigators concut that fetal and maternal platelet counts lack strong correlation (George, 2009; Hachisuga, 2014). Because of this, maternal IgG free platelet antibody levels and platelet-associated antibody levels have been evaluated to predict fetal platelet counts. Again, however, there is little concurrence with these.

1	Investigators have also examined the association between the speciic cause of thrombocytopenia and risk of a thrombocytopenic fetus. Four researched causes include gestational th ro m bocyto penia, hypertension -associa ted thro m bocyto penia, immune thrombocytopenia, and alloimmune thrombocytopenia. Burrows and Kelton (1993) reported neonatal umbilical cord platelet counts measuring <50,000/�L in 19 of 15,932 consecutive newborns (0.12 percent). Only one of756 mothers with gestational thrombocytopenia had an afected newborn. Of 1414 hypertensive women with thrombocytopenia, five neonates had thrombocytopenia. In contrast, of 46 mothers with immunological thrombocytopenia, four infants had thrombocytopenia. Alloimmune thrombocytopenia was associated with profound thrombocytopenia and cord platelet counts <20,000/�L. One of these fetuses died, and two others had intracranial hemorrhage.

1	Detection of Fetal Thrombocytopenia. Because no test accurately predicts fetal platelet counts, direct fetal blood sampling is necessary. Scott and coworkers (1983) obtained intrapartum scalp blood samples and recommended cesarean delivery for fetuses with platelet counts <50,000/�L. Dafos and colleagues (1985) reported that percutaneous umbilical cord blood sampling (PUBS) for this indication had a high complication rate (Chap. 14, p. 294). Conversely, Berry and associates (1r997) reported no complications and described poor reliability to predict severe thrombocytopenia, but noted a high negative-predictive value. Payne and coworkers (1r997) summarized six studies in which fetal blood sampling was done for platelet estimation. Of the total of 195 fetuses, severe neonatal thrombocytopenia <50,000/�L was found in 7 percent. But, serious complications from cordocentesis were noted in 4.6 percent. Because of the low incidence of severe neonatal thrombocytopenia and morbidity, fetal

1	<50,000/�L was found in 7 percent. But, serious complications from cordocentesis were noted in 4.6 percent. Because of the low incidence of severe neonatal thrombocytopenia and morbidity, fetal platelet determinations and cesarean delivery are not recommended (N eunert, 2011).

1	Alloimmune Thrombocytopenia. Disparity between maternal and fetal platelet antigens can stimulate maternal production of antiplatelet antibodies. Such platelet alloimmunization can be severe, and its pathophysiology is identical to that caused by red cell antigens (Chap. 15, p. 307).

1	Also called thrombocythemia, thrombocytosis generally is deined as persistent platelet counts >450,000/�L. Common causes of seconday or reactive thrombocytosis are iron deficiency, infection, inflammatory diseases, and malignant tumors (Deutsch, 2013). Platelet counts seldom exceed 800,000/�L in these secondary disorders, and prognosis depends on the underlying disease. On the other hand, primary or essential thrombocytosis accounts for most cases in which platelet counts exceed 1 millionl �L. It is a clonal disorder frequently due to an acquired mutation in the fAQ gene (Konkle, 2015). Thrombocytosis usually is asymptomatic, but arterial and venous thromboses may develop, and thrombosis is associated with pregnancy complications (Rabinerson, 2007; Randi, 2014). hese cases must be diferentiated from the sticky platelet syndrome, which is also associated with thromboses (Rac, 2011).

1	Normal pregnancies have been described in women whose mean platelet counts werer> 1.25 million/�L (Beard, 1991; Randi, 1994). Others report more adverse outcomes. Niittyvuopio and associates (2004) described 40 pregnancies in 16 women with essential thrombocythemia. Almost half had a spontaneous abortion, fetal demise, or preeclampsia. In 63 pregnancies in 36 women cared for at the Mayo Clinic, a third had a spontaneous miscarriage, but other pregnancy complications were uncommon (Gangat, 2009). In this observational study, aspirin therapy was associated with a signiicantly lower abortion rate than that in untreated women-1 versus 75 percent, respectively. Suggested treatments during pregnancy include aspmn, low-molecular-weight heparin, and interferon-a (Finazzi, 2012). Interferon-a therapy during pregnancy was successful in 11 women in the review by Delage and coworkers (1996). One of these women had transient blindness at midpregnancy when her platelet count was 2.3 million/�L.

1	Although not a primary platelet disorder, some degree of thrombocytopenia accompanies the thrombotic microangiopathies, which include thrombotic thrombocytopenic purpura (TTP) and hemoytic uremic syndrome (HUS). hese syndromes have an incidence of2 to 6 per million persons per year (Miller, 2004). heir similarities to HELLP syndrome allude to their obstetrical ramiications (George, 2014). Although diferent causes account for the variable findings within these syndromes, clinically, they frequently are indistinguishable. Most cases of TTP are thought to be caused by antibodies to or a plasma deficiency of ADAMTS13 (Ganesan, 201r1; Sadler, 2010). his endothelium-derived protease cleaves von Willebrand factor (vWF) to decrease its activity. Conversely, HUS is usually due to endothelial damage incited by viral or bacterial infections and is seen primarily in children (Ardissino, 2013; George, 2014).

1	With TTP, intravascular platelet aggregation stimulates a cascade of events leading to end-organ failure. There is endothelial activation and damage, but it is unclear whether this is a consequence or a cause. Elevated levels of unusually large multimers of vWF are identiied with active TTP. Various defects in the ADAMTS13 gene create difering clinical presentations of thrombotic microangiopathy (Camilleri, 2007; Moake, 2002, 2004). In another scheme, antibodies raised against ADAMTS13 neutralize its action to cleave vWF multimers during an acute episode. he end result is micro thrombi of hyaline material consisting of platelets and small amounts of ibrin within arterioles and capillaries. When suicient in number or size, these aggregates produce ischemia or infarctions in various organs.

1	hrombotic microangiopathies are characterized by thrombocytopenia, fragmentation hemolysis, and variable organ dysfunction. TTP is characterized by the pentad of thrombocytopenia, fever, neurological abnormalities, renal impairment, and hemolytic anemia. HUS typically has more profound renal involvement and fewer neurological aberrations. hrombocytopenia is usually severe, but fortunately, even with very low platelet counts, spontaneous severe hemorrhage is uncommon. Microangiopathic hemolysis is associated with moderate-to-marked anemia, and erythrocyte transfusions are frequently necessary. he blood smear is characterized by erythrocyte fragmentation with schizocytosis. Reticulocytes and nucleated red blood cells are increased, lactate dehydrogenase (LDH) levels are high, and haptoglobin concentrations are decreased. Consumptive coagulopathy, although common, is usually subtle and clinically insignificant.

1	The cornerstone of treatment is plasmapheresis with freshfrozen plasma replacement. Plasma exchange removes inhibitors and replaces the ADAMTS13 enzyme (George, 2014; Michael, 2009). Treatment with caplacizumab, the anti-vWF immunoglobulin, inhibits the interaction between ultralarge vWF multimers and platelets (Peyandi, 2016). hese treatments have remarkably improved outcomes in patients with these formerly fatal syndromes. Red cell transfusions are imperative for life-threatening anemia. Treatment is usually continued until the platelet count is > 150,OOO/�L. Unfortunately, relapses are common. Additionally, there may be long-term sequelae such as renal impairment (Dashe, 1998; Vesely, 2015). Treatment for pregnancy-associated HUS, which is complement mediated, uses the anti-C5 humanized monoclonal antibody eculizumab (Ardissino, 2013; Caiigral, 2014; Fakhouri, 2016).

1	As shown in the Appendix (p. 1256), ADAMTS13 enzyme activity decreases across pregnancy by up to 50 percent (S8nchez-Luceros, 2004). Levels drop even further with the preeclampsia syndrome. his is consonant with prevailing opinions that TTP is more commonly seen during pregnancy. he Parkland Hospital experiences were described by Dashe and coworkers (1998), who identified 11 pregnancies complicated by these syndromes among nearly 275,000 obstetrical patients-a frequency of 1 in 25,000.

1	I t seems likely that some of the disparately higher incidence in pregnancy reported by others is because of inclusion of women with severe preeclampsia and eclampsia (Hsu, 1995; Magann, 1994). Diferences that usually allow appropriate diagnosis are listed in Table 56-6. For example, moderateto-severe hemolysis is a rather constant feature of thrombotic microangiopathies. But, this is seldom severe with the preeclampsia syndrome, even when complicated by HELLP syndrome (Chap. 40, p. 719). And, although there is deposition of hyaline micro thrombi within the liver with thrombotic microangiopathy, hepatocellular necrosis with elevated serum hepatic aminotransferase levels characteristic of preeclampsia is not a common feature (Ganesan, 2011; Sadler, 2010). Importantly, whereas delivery is imperative to reverse the preeclampsia syndrome, no evidence shows that thrombotic microangiopathy is improved by delivery (Dashe, 1998; Letsky, 2000). Finally, microangiopathic syndromes are usually

1	imperative to reverse the preeclampsia syndrome, no evidence shows that thrombotic microangiopathy is improved by delivery (Dashe, 1998; Letsky, 2000). Finally, microangiopathic syndromes are usually recurrent and frequently unassociated with pregnancy. For example, seven of 11 women described by Dashe and colleagues (1998) had recurrent disease either when not pregnant or within the first trimester of a subsequent pregnancy. George (2009) reported recurrent TTP in only five of 36 subsequent pregnancies. That said, the risk for preeclampsia in these women is increased Giang,r2014).

1	Unless the diagnosis is unequivocally one of these thrombotic microangiopathies, rather than severe preeclampsia, the TABLE 56-6. Some Differential Factors between HELLP Syndrome and Thrombotic Microangiopathiesa Thrombocytopenia Mild/mod. Mod/severe ADAMTS 1 3 def. Mild/mod. Severe (AST, ALT) hemolytic uremic syndrome (HUS). ADAMTS 13 = ADAM metallopeptidase with thrombospondin type 1 motif, 13; AST = aspartate transaminase; ALT = alanine transaminase; def. = deficiency; DIC = disseminated intravascular coagulopathy; HELLP = hemolysis, elevated liver enzyme levels, low platelet count; Mod. = moderate.

1	response to pregnancy termination should be evaluated before resorting to plasmapheresis and exchange transfusion, massivedose glucocorticoid therapy, or other therapy. Unfortunately, recall that determination of ADAMTS13 enzyme activity may be diicult to interpret with HELLP syndrome (Franchini, 2007). Plasmapheresis is not indicated or preeclampsia-eclampsia complicated by hemoysis and thrombocytopenia. During the past two decades, and coincidental with plasmapheresis and plasma exchange, maternal survival rates from thrombotic micro angiopathy have improved dramatically (Dashe, 1998). Although previously fatal in up to half of mothers, with such treatment, Egerman and coworkers (1996) reported two maternal and three fetal deaths in 11 pregnancies. Hunt and associates (2013) reported that TTP accounted for 1 percent of maternal deaths in the United Kingdom from 2003 to 2008.

1	Women who are diagnosed with thrombotic micro angiopathy during pregnancy are at risk for serious long-term complications (Egerman, 1996). he Parkland experiences included a mean 9-year surveillance period (Dashe, 1998). hese women had multiple recurrences; renal disease requiring dialysis, transplantation, or both; severe chronic hypertension; and transfusion-acquired infectious diseases. Two women died remote from pregnancy-one from dialysis complications and one from transfusion-acquired HIV infection. In nonpregnant women who have recovered from thrombotic microangiopathies, persistent cognitive defects and physical disabilities have been reported (Kennedy, 2009; Lewis, 2009). Interestingly, as discussed in Chapter 40 (p. 745), these cognitive defects are very similar to those found in long-term surveillance studies of women who had eclampsia (Aukes, 2009,r2012; Wiegman, 2012).

1	Obstetrical hemorrhage may infrequently be the consequence of an inherited defect in a protein that controls coagulation. Both types of hemophilia are examples. Severity reflects plasma factor levels and is categorized as mild-levels of 6 to 30 per cent; moderate-2 to 5 percent; or severe-less than 1 percent (Arruda, 2015).

1	Hemophilia A is an X-linked recessively transmitted disorder characterized by a marked deiciency of factor VIII. It is rare among women compared with men, in whom the heterozygous state is responsible for the disease. Heterozygous women have diminished factor VIII levels, but almost invariably, the homozygous state is requisite for hemophilia A. In a few instances, it appears in women spontaneously from a newly mutated gene. Pregnancy-associated acquired hemophilia A from antibodies may result in severe bleeding-related morbidity (Tengborn, 2012). Christmas disease or hemophilia B is caused by severe deiciency of factor IX and has similar genetic and clinical features.

1	he risk of obstetrical bleeding with these is directly related to factor VIII or IX levels. Afected women have a range of activity that is determined by random X-chromosome inactivation-Iyonization-although activity is expected to average 50 percent (Letsky, 2000). Levels below 10 to 20 percent pose hemorrhage risks. If levels fall to near zero, this risk is substantial. Pregnancy does aford some protection, however, because concentrations of both these clotting factors rise appreciably during normal pregnancy (Appendix, p. 1256). Treatment with desmopressin may also stimulate factor VIII release. Risks are further reduced by avoiding lacerations, minimizing episiotomy use, and maximizing postpartum uterine contractions. Operative vaginal deliveries should be avoided. here are few published experiences during pregnancy.

1	here are few published experiences during pregnancy. Kadir and coworkers (1997) reported that 20 percent of carriers had postpartum hemorrhage. Guy and associates (I992) reviewed five pregnancies in women with hemophilia B, and in all, outcomes were favorable. They recommended factor IX administration if levels are below 10 percent. Desmopressin has been shown in selected cases to reduce obstetrical bleeding complications (Trigg, 2012). If a male fetus has hemophilia, the risk of hemorrhage increases after delivery in the neonate. his is especially true if circumcision is attempted. If a mother has hemophilia A or B, all of her sons will have the disease, and all of her daughters will be carriers. If she is a carrier, half of her sons will inherit the disease, and half of her daughters will be carriers. Prenatal diagnosis of hemophilia is possible in some families using chorionic villus biopsy (Chap. 14, p. 293).

1	Preimplantation genetic diagnosis for hemophilia was reviewed by Lavery (2009). Rarely, antibodies directed against factor VIII or IX are acquired and may lead to life-threatening hemorrhage. Patients with hemophilia more commonly develop antibodies, and their acquisition in patients without hemophilia is extraordinary. It has been identified rarely in women during the puerperium (Santoro, 2009). he prominent clinical feature is severe, protracted, repetitive hemorrhage from the reproductive tract starting a week or so after an apparently uncomplicated delivery (Gibson, 2016). he activated partial thromboplastin time is markedly prolonged. Treatment has included multiple transfusions of blood component, immunosuppressive therapy, and attempts at various surgical procedures, especially curettage and hysterectomy. A recombinant activated factor VII (NovoSeven) stops bleeding in up to 75 percent of patients with these inhibitors (Arruda, 2015; Gibson, 2016).

1	here are at least 20 heterogeneous clinical disorders involving aberrations of factor VIII complex and platelet dysfunctioncollectively termed von Willebrand disease (vWD). hese abnormalities are the most frequently inherited bleeding disorders, and their prevalence is as high as 1 to 2 percent (Arruda, 2015; Pacheco, 2010). Most von Willebrand variants are inherited as autosomal dominant traits, and types I and II are the most common. Speciically, type I accounts for 75 percent of von Willebrand variants. Type III, which is the most severe, is a recessive trait. lthough most cases of acquired vWD develop after age 50 years, some have been reported in pregnant women (Lip kind, 2005).

1	he von Wilebrand actor is a series of large plasma multimeric glycoproteins that form part of the factor VIII complex. It is essential for normal platelet adhesion to subendothelial collagen and formation of a primary hemostatic plug. It also plays a major role in stabilizing the coagulant properties of factor VIII. he procoagulant component is factor VIII, a glycoprotein synthesized by the liver. Conversely, von Willebrand precursor, which is present in platelets and plasma, is synthesized by endothelium and megakaryocytes. he von Willebrand factor antigen (vWF:Ag) is the antigenic determinant measured by immunoassays.

1	Symptomatic patients typically present with easy bruising, epistaxis, mucosal hemorrhage, and excessive bleeding with trauma, including surgery. he classic autosomal dominant forms usually cause symptoms in the heterozygous state. With vWD, laboratory features often include a prolonged bleeding time, prolonged partial thromboplastin time, decreased vWF antigen levels, decreased factor VIII immunological and coagulation-promoting activity, and inability of platelets from an afected person to react to various stimuli.

1	During normal pregnancy, maternal levels of both factor VIII and vWF antigen increase substantively (Appendix, p. 1256). Because of this, pregnant women with vWD often develop normal levels of factor VIII coagulant activity and vWF antigen, although their measured bleeding time still may be prolonged. If factor VIII activity is very low or if there is bleeding, treatment is recommended. Desmopressin by infusion transiently increases factor VIII and vWF levels (Arruda, 2015; Kujovich, 2005). With signiicant bleeding, 15 or 20 units of cryoprecipitate are transfused every 12 hours. Alternatively, factor VIII concentrates (Alfanate, Hemate-P) may be given that contain high-molecular-weight vWF multimers. Lubetsky and colleagues (1999) described continuous infusion with Hemate-P in a woman during a vaginal delivery. According to Chi and coworkers (2009), conduction analgesia can be provided safely if coagulation defects have normalized or if hemostatic agents are administered

1	a woman during a vaginal delivery. According to Chi and coworkers (2009), conduction analgesia can be provided safely if coagulation defects have normalized or if hemostatic agents are administered prophylactically.

1	Pregnancy outcomes in women with vWD are generally good, but postpartum hemorrhage is encountered in up to half of cases. In a fourth of 38 cases summarized by Conti and associates (1986), bleeding was reported with abortion, with delivery, or in the puerperium. Kadir and coworkers (1998) reported their experiences with 84 pregnancies. They described a 20-percent incidence of immediate postpartum hemorrhage and another 20-percent incidence of late hemorrhage. Most cases were associated with low vWF levels in untreated women, and none given treatment peripartum had hemorrhage. More recently, Stoof and colleagues (2015) reviewed 185 deliveries in 154 afected women and found the risk for postpartum hemorrhage to be highest in deliveries with lowest factor levels.

1	Although most patients with vWD have heterozygous variants and associated minor bleeding complications, the disease can be severe. Moreover, homozygous ofspring develop serious clotting dysfunction. Chorionic villus sampling with DNA analysis to detect the missing genes has been described. Some authorities recommend cesarean delivery to avoid trauma to a possibly afected fetus if the mother has severe disease. In general, the activity of most procoagulant factors rises across pregnancy (Appendix, p. 1256). Factor VII deiciency is a rare autosomal recessive disorder. Levels of this factor normally increase during pregnancy, but these may rise only mildly in women with factor VII deficiency (Fadel, 1989). A systematic review of 94 births found no diference in postpartum hemorrhage rates with or without prophylaxis with recombinant factor VIla (Baumann Kreuziger, 2013).

1	Factor X or Stuart-Prower actor deiciency is rare and is inherited as an autosomal recessive trait. Factor X levels typically rise by 50 percent during normal pregnancy. Konje and colleagues (1994) described a woman who had 2-percent factor activity. She was given prophylactic treatment with plasmaderived factor X, which raised her plasma levels to 37 percent. Despite this, she sufered an intrapartum placental abruption. plasma to a woman with less than I-percent factor X activity. She delivered spontaneously without incident. Beksa: and asso ciates (20r10) described a woman with severe factor X deiciency who was successfully managed with prophylactic prothrombin complex concentrate. Nance and colleagues (2012) reported on 24 pregnancies, of which 18 resulted in a healthy baby.

1	Factor I-plasma thromboplastin antecedent-diciency is inherited as an autosomal trait. It manifests as severe disease in homozygotes but only as a minor defect in heterozygotes. It is most prevalent in Ashkenazi Jews and is rarely seen in pregnancy. Musclow and coworkers (1987) reported 41 deliveries in 17 afected women, and none required transfusion. In 105 pregnancies from 33 afected women, Myers and colleagues (2007) reported an uneventful pregnancy and delivery in 70 percent. hey recommended peripartum treatment with factor XI concentrate if cesarean delivery is performed and advised against epidural analgesia unless factor XI is given. From their review, Martin-Sakes and associates (2010) found that factor XI levels and bleeding severity correlated poorly in women with severe deiciency. Wiewel-Verschueren and colleagues (2015) performed a systematic review of 27 studies with 372 women and reported that 18 percent had postpartum hemorrhage.

1	Factor II diciency is another autosomal recessive disorder that rarely complicates pregnancy. A greater incidence of thromboembolism is encountered in nonpregnant patients with this deficiency. Lao and coworkers (1991) reported an afected pregnant woman in whom placental abruption developed at 26 weeks' gestation. Factor III diciency is an autosomal recessive trait and may be associated with maternal intracranial hemorrhage (Letsky, 2000). In their review, Kadir and associates (2009) cited an increased risk of recurrent miscarriage and placental abruption. It has also been reported to cause umbilical cord bleeding (Odame, 2014). Treatment is fresh frozen plasma. Naderi and colleagues (2012) described 17 successful pregnancies in women receiving weekly prophylaxis with Factor XIII concentrate.

1	Fibrinogen abnormalities-either qualitative or quantitativealso may cause coagulation abnormalities. Autosomally inherited abnormalities usually involve the formation of a functionally defective ibrinogen-commonly referred to as dyibrinogenemia (Edwards, 2000). Familial hypoibrinogenemia and sometimes aibrinogenemia are infrequent recessive disorders. In some cases, both are found-hypodysibrinogenemia (Deering, 2003). Our experience suggests that hypoibrinogenemia represents a heterozygous autosomal dominant state. he thrombinclottable protein level in these patients typically ranges from 80 to 110 mg/ dL when nonpregnant, and this increases by 40 or 50 percent in normal pregnancy. Those pregnancy complications that give rise to acquired hypoibrinogenemia, such as placental abruption, are more common with ibrinogen deiciency. Trehan and Fergusson (1991) and Funai and coworkers (1997) described successful outcomes in two afected women in whom ibrinogen or plasma inusions were given

1	are more common with ibrinogen deiciency. Trehan and Fergusson (1991) and Funai and coworkers (1997) described successful outcomes in two afected women in whom ibrinogen or plasma inusions were given throughout pregnancy.

1	Conduction Analgesia with Bleeding Disorders Most serious bleeding disorders would logically preclude the use of epidural or spinal analgesia for labor or delivery. If the bleeding disorder is controlled, however, conduction analgesia may be considered. Chi and colleagues (2009) reviewed intrapartum outcomes in 80 pregnancies in 63 women with an inherited bleeding disorder. These included those with factor XI deiciency, hemophilia carrier status, vWD, platelet disorders, or a deiciency of factor VII, Xl, or X. Regional block was used in 41. Of these, 35 had spontaneously normalized hemostatic dysfunction, and others were given prophylactic replacement therapy. The reviewers reported no unusual complications. Singh and associates (2009) reviewed 13 women with factor XI deiciency. Nine received neuraxial analgesia without complications, but only after fresh-frozen plasma was transfused to most to correct the activated partial thromboplastin time.

1	Several important regulatory proteins act as inhibitors at strategic sites in the coagulation cascade to maintain blood fluidity. Inherited deiciencies of these inhibitory proteins are caused by gene mutations. Because they may be associated with recurrent thromboembolism, they are collectively referred to as thrombophilias. These are discussed in Chapter 52 (p. 1005) and reviewed by the American College of Obstetricians and Gynecologists (2017b). Aessopos A, Karabatsos F, Farmakis D, et al: Pregnancy in patients with welltreated 3-thalassemia: outcome for mothers and newborn infants. Am J Obstet Gynecol 180:360, 1999 American College of Obstetricians and Gynecologists: Hemoglobinopathies in pregnancy. Practice Bulletin No. 78, January 2007, Reairmed 2015 American College of Obstetricians and Gynecologists: Neural tube defects. Practice Bulletin No. 44, July 2003, Reairmed 2016a

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1	TYPES OF DIABETES. . . . . . . . . . . . . . . . . . . . . . . . . . .. 1097 PREGESTATIONAL DIABETES .....i...i...i....i.....i....1098 DIAGNOSIS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1098 FETAL EFFECTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1099 MATERNAL EFFECTS.. . . . . . . . . . . . . . . . . . . . . . . . . .. 1103 MANAGEMENT OF DIABETES IN PREGNANCY . . . . .. 1104 GESTATIONAL DIABETES. . . . . . . . . . . . . . . . . . . . . . .. 1 107 SCREENING AND DIAGNOSIS .................... 1108 MATERNAL AND FETAL EFFECTS .....i............. 1110 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1 111 Diabetes may exist bore the inception ofpregnancy, or may not appear until labour. The prognosis is generaly believed to be ominous or mother and chil, but a review of the literature shows that less than 25 percent of the mothers died from diabetic coma, while premature labour occurred in ony one third of the cases.

1	-J. Whitridge Williams (1903) In the early 1900s, overt diabetes complicating pregnancy was associated with horriic morbidity and mortality for a mother and her fetus. Although tremendously mitigated by the discovery of insulin, overt and gestational diabetes still are formidable complications of pregnancy. According to the Centers for Disease Control and Prevention (2017), the number of adults diagnosed with diabetes in the United States is 23.r1 million. And, almost a quarter of people with diabetes in the United States remain undiagnosed. tion more likely to develop type 2 diabetes, population growth within minority group at particular risk for type 2 diabetes, and a dramatic rise in obesity rates-also referred to as diabesiy. he term reflects the strong relationship of diabetes and the current obesity epidemic in the United States and underlines the critical need for diet and lifestyle interventions to change the trajectory of both.

1	here is keen interest in events that precede diabetes, and this includes the intrauterine environment. Here, early imprinting is believed to have efects later in life (Saudek, 2002). For example, in utero exposure to maternal hyperglycemia leads to fetal hyperinsulinemia, causing an increase in fetal fat cells. his leads to obesity and insulin resistance in childhood (Feig, 2002). These factors in turn lead to impaired glucose tolerance and diabetes in adulthood. This cycle of fetal exposure to diabetes leading to childhood obesity and glucose intolerance is discussed further in Chapter 48 (p. 941).

1	In nonpregnant individuals, the type of diabetes is based on its presumed etiopathogenesis and its pathophysiological manifestations. Absolute insulin deficiency, generally autoimmune in etiology, characterizes ype 1 diabetes. In contrast, insulin resistance, relative insulin deiciency, or elevated glucose production characterizes ype 2 diabetes (Table 57-1). Both types are generally preceded by a period of abnormal glucose homeostasis oten referred to as prediabetes. The terms insulin-dependent diabetes mellitus (IDDM) and noninsulin-dependent diabetes mellitus (NIDDM) are now obsolete. Pancreatic �-cell destruction can begin at any age, but type 1 diabetes is clinically apparent most often before age 30. Type 2 diabetes usually develops with advancing age but is increasingly identified in younger obese adolescents. TABLE 57-1. Etiological Classification of Diabetes Mellitus Type 1: �-Cell destruction, usually absolute insulin deficiency Immune-mediated

1	TABLE 57-1. Etiological Classification of Diabetes Mellitus Type 1: �-Cell destruction, usually absolute insulin deficiency Immune-mediated Type 2: Ranges from predominantly insulin resistance to predominantly an insulin secretory defect with insulin resistance Genetic mutations of �-cell function-MODY 1-6, others Genetic defects in insulin action Genetic syndromes-Down, Klinefelter, Tuner Diseases of the exocrine pancreas-pancreatitis, cystic fibrosis Endocrinopathies-Cushing syndrome, pheochromocytoma, others Drug or chemical induced-glucocorticosteroids, thiazides, �-adrenergic agonists, others Infections-congenital rubella, cytomegalovirus, coxsackievirus MODY = maturit I-onset diabetes of the young. Data from Powers, 21n2.

1	MODY = maturit I-onset diabetes of the young. Data from Powers, 21n2. Diabetes is the most common medical complication of pregnancy. Women can be separated into those who were known to have diabetes before pregnancy-pregestationalior overt, and those diagnosed during pregnancy-gestational diabetes. The proportion of pregnancies complicated by diabetes more than doubled between 1994 and 2008, after which rates seem to have stabilized Oovanovic, 2015). Almost 258,000-6.5 percent-of gravidas in the United States had pregnancies coexistent with some form of diabetes in 2015 (Martin, 2017). Prevalence of diabetes is highest among non-Hispanic blacks, Mexican-Americans, Puerto Rican-Americans, and Native Americans (Golden, 2012). he incidence of gestational diabetes during the past 20 years, shown in Figure 57-1, is reminiscent of similar statistics for obesity (Chap. 48, p. 936). I:u

1	I:u FIGURE 57-1 Increasing prevalence of type 2 diabetes in the United States from 1995 to 2015. (Reproduced with permission from Centers for Disease Control and Prevention, 201o7.) Until the mid-1990s, the classification by Priscilla White (1978) for diabetic pregnant women was the linchpin of management. Today, the White classiication is used less frequently but still provides simple and useful information on pregnancy risks and prognosis (Bennett, 2015). And, because most currently cited literature also contains data from these older classifications, the one previously recommended by the American College of Obstetricians and Gynecologists (1986) is provided in Table 57-2.

1	Beginning several years ago, the American College of Obstetricians and Gynecologists no longer recommended the White classification. Instead, the current focus is whether diabetes antedates pregnancy or is irst diagnosed during pregnancy. Many now recommend adoption of the classiication proposed by the American Diabetes Association (ADA), which is shown in Table 57-3.

1	he rising prevalence of type 2 diabetes, particularly in younger people, has led to an increasing number of afected pregnancies. For example, the CDC (2015) estimates that more than 5000 new cases of type 2 diabetes are diagnosed each year in youths before the age of 20 years. Feig and coworkers (2014) reported that the incidence of pregestational diabetes doubled from 7 per 1000 women in 1996 to 15 per 1000 in 2010. When considering the previously mentioned high percentage of diabetes that is undiagnosed, then many women identified to have gestational diabetes likely have type 2 diabetes previously unrecognized. In fact,rS to 10 percent of women with gestational diabetes are found to have diabetes immediately after pregnancy. Women with high plasma glucose levels, glucosuria, and ketoacidosis present no diagnostic challenge. Women with a TABLE 57-2. Classification Scheme Used from 1986 through 1994 for Diabetes

1	Women with high plasma glucose levels, glucosuria, and ketoacidosis present no diagnostic challenge. Women with a TABLE 57-2. Classification Scheme Used from 1986 through 1994 for Diabetes Age of Onset (yr) 10 to 19 10 to 19 aWhen diagnosed during pregnancy: proteinuria :500 mg/24 hr before 20 weeks' gestation.

1	random plasma glucose level >200 mg/dL plus classic signs and symptoms such as polydipsia, polyuria, and unexplained weight loss, or those with a fasting glucose levelr> 125 mg/dL are considered by the ADA (2017 a) and the World Health Organization (2013) to have overt diabetes first detected in pregnancy. Women with only minimal metabolic derangement may be more diicult to identiY. To diagnose overt diabetes in pregnancy, he International Association of Diabetes and Pregnancy Study Groups (IADPSG) Consensus Panel (2010) recognizes the threshold values found in Table 57-4 for fasting or random plasma glucose and glycosylated hemoglobin (HbA1J levels at prenatal care initiation. The ADA (2017a) and the World Health Organization (2013) now also consider a plasma glucose level >200 mg/dL measured 2 hours after a 75 g oral glucose load to be diagnostic. No consensus has been reached as to whether such testing should be universal or limited to those women classiied as high risk.

1	mg/dL measured 2 hours after a 75 g oral glucose load to be diagnostic. No consensus has been reached as to whether such testing should be universal or limited to those women classiied as high risk. Regardless, the tentative diagnosis of overt diabetes during pregnancy based on these thresholds should be conirmed postpartum. isk factors for impaired carbohydrate metabolism in pregnant women include a strong familial history of diabetes, prior delivery of a large newborn, persistent glucosuria, or unexplained fetal losses.

1	With overt diabetes, the embryo, fetus, and mother frequently experience serious complications directly attributable to diabetes. Peterson and colleagues (2015) estimate that thousands of these complications might be prevented each year by preconceptional care for improved glycemic control. The likelihood of successful outcomes with overt diabetes, however, is not simply related to glucose control. The degree of underlying cardiovascular or renal disease may be more important. Thus, advancing stages of the White classification, seen in Table 57-2, are inversely related to favorable pregnancy outcomes. Shown in Table 57-5 are data that chronicle the deleterious pregnancy outcomes with overt diabetes. hese maternal and fetal complications are described in the following sections.

1	Spontaneous Abortion. Several studies have shown that early miscarriage is associated with poor glycemic control (Chap. 18, p. 347). Up to 25 percent of diabetic gravidas have an early pregnancy loss (Galindo, 2006; Rosenn, 1994). hose whose HbA1c concentrations were > 12 percent or whose preprandial glucose concentrations were persistently > 120 mg/dL had an elevated risk. Bacon and associates (2015) reviewed 89 pregnancies in women with maturity-onset diabetes of the young (MODy), which is a monogenic form of diabetes. hese investigators found that only women with the causative glucose kinase gene (GCK) mutation were more likely to have a miscarriage. These women are characterized by hyperglycemic variability that is diicult to control. TABLE 57-3. Proposed Classification System for Diabetes in Pregnancy Gestational diabetes: diabetes d during pregnancy that is not clearly overt (type 1 or type 2) diabetes Type 1 Diabetes: Type 2 Diabetes:

1	Gestational diabetes: diabetes d during pregnancy that is not clearly overt (type 1 or type 2) diabetes Type 1 Diabetes: Type 2 Diabetes: Diabetes resulting from �-cell destruction, usually leading to Diabetes from inadequate insulin secretion in the face of a. Without vascular complications a. Without vascular complications b. With vascular complications (speciy which) b. With vascular complications (speciy which) Other types of diabetes: genetic in origin, associated with pancreatic disease, drug-induced, or chemically induced Data from American Diabetes Association, 2017a. TABLE 57-4. Diagnosis of Overt Diabetes in Pregnancya Measure of Glycemia Threshold

1	Data from American Diabetes Association, 2017a. TABLE 57-4. Diagnosis of Overt Diabetes in Pregnancya Measure of Glycemia Threshold Fasting plasma glucose At least 7.0 mmollL (126 mg/dL) Hemoglobin Ale At least 6.5% Random plasma glucose At least 11.1 mmol/L (200 mg/dL) plus confirmation aApply to women without known diabetes antedating pregnancy. The decision to perform blood testing for evaluation of glycemia all pregnant women r only on women with characteristics indicating a high risk for diabetes is based on the background frequency of abnormal glucose metabolism in the population and on local circumstances. Data from International Association of Diabetes and Pregnancy Study Groups Consensus Panel, 201n0.

1	Data from International Association of Diabetes and Pregnancy Study Groups Consensus Panel, 201n0. Preterm Delivery. Overt diabetes is an undisputed risk factor for preterm birth. Eidem and associates (2011) analyzed 1307 births in women with type 1 diabetes from the Norwegian NIedical Birth Registry. More than 26 percent were delivered preterm compared with 6.8 percent in the general obstetrical population. Moreover, almost 60 percent were indicated preterm births, that is, due to obstetrical or medical complications. In one review of more than 500,000 California births, 19 percent of women with pregestational diabetes had a preterm birth compared with 9 percent in controls (Yanit, 2012). In the Canadian study shown in Table 57-5, the incidence of preterm birth was 28 percent.

1	Malformations. he incidence of major malformations in women with type 1 diabetes is at least doubled and approximates 11 percent Qovanovic, 2015). These account for almost half of perinatal deaths in diabetic pregnancies. As shown in Table 57-6, cardiovascular malformations accounted for more than half of the anomalies. In a cohort study of more than 2 million births in Canada, the risk of an isolated cardiac defect was ivefold higher in women with type 1 diabetes (Uu, 2013). he caudal regression sequence, described in Chapter 10 (p. 196), is a rare malformation frequently associated with maternal diabetes (Garne, 2012).

1	Poorly controlled diabetes, both preconceptionally and early in pregnancy, is thought to underlie this elevated severemalformation risk. s shown in Figure 57-2, increased maternal HbA[c levels and major malformations clearly correlate. To explain this, at least three interrelated molecular chain reactions have been linked to maternal hyperglycemia (Reece, 2012). hese include alterations in cellular lipid metabolism, excess production of toxic superoxide radicals, and activation of programmed cell death. In their review of molecular mechanisms underlying diabetic embryopathy, Yang and colleagues (2015) suggest that these cellular responses to oxidative stress represent potential therapeutic targets to prevent diabetes-induced embryopathy.

1	Altered Fetal Growth. Diminished growth may result from congenitl malformations or from substrate deprivation due to advanced maternal vascular disease. That said, fetal overgrowth is more typical of pregestational diabetes. Maternal hyperglycemia prompts fetal hyperinsulinemia, and this in turn stimulates excessive somatic growth. Except for the brain, most fetal organs are afected by the macrosomia that characterizes the fetus of a diabetic woman. Newborns such as the one shown in Figure 57-3 are described as being anthropometrically diferent from other largefor-gestational age (LGA) neonates (Catalano, 2003; Durnwald, 2004). Speciically, those whose mothers are diabetic have excesyrsive fat deposition on the shoulders and trunk, which predisposes to shoulder dystocia or cesarean delivery. he incidence of macrosomia rises signiicantly when mean maternal blood glucose concentrations chronically exceed 130 mg/dL (Hay, 2012). Hammoud and coworkers (2013)

1	he incidence of macrosomia rises signiicantly when mean maternal blood glucose concentrations chronically exceed 130 mg/dL (Hay, 2012). Hammoud and coworkers (2013) TABLE 57-5. Pregnancy Outcomes of Births in Nova TABLE 57-6. Major Congenital Anomalies in 36,345 Scotia from 1988 to 2002 in Women with Neonates Born to Women with Diabetes and without Pregestational Diabetes between 2004 and 201e1 (n.=.S16) (n.=.lS0,.S98) Factor P value �pe1.DM Type.2DM GDM Organ System n = 482 n = 4166 n = 31,700 Gestational 28 9 <.001 Cardiac 38 272 1129 Preterm birth 28 5 <.001 Urinary 3 28 260 <.001 CNS 13 64 FIGURE 57-3 This 6050-g macrosomic infant was born to a woman with gestational diabetes.

1	15.8 400.) 11.7 30 25 5 3/1e9 5 2/1e7 0 <6 6-6.9 7-7.9 �8 FIGURE 57-2 The frequency of major congenital malformations in newborns ofwomen with pregestational diabetes stratified by hemoglobin Ale levels at first prenatal visit. (Data from Galindo A, Burguillo AG, Azriel S, et al: Outcome offetuses in women with pregestational diabetes mellitus, J Perinat Med. 2006;34(4):323-331.) reported that the macrosomia rates for Nordic women with type 1, type 2, or gestational diabetes were 35 percent, 28 percent, and 24 percent, respectively. As shown in 57-4, the birthweight distribution of neonates of diabetic mothers is skewed toward consistently heavier birthweights. In the study by Hammoud and colleagues (2013), fetal growth profiles from 897 sonographic examinations in 244 women with diabetes were compared with 843 examinations in 145 control women. he abdominal circumference grew disproportionately larger in the diabetic groups. Analysis of head circumference/abdominal circumference

1	were compared with 843 examinations in 145 control women. he abdominal circumference grew disproportionately larger in the diabetic groups. Analysis of head circumference/abdominal circumference (HC/AC) ratios shows that this disproportionate growth occurs mainly ..0

1	FIGURE 57-4 Distribution of birthweight standard deviations from the normal mean for gestational age in 280 newborns of diabetic mothers and in 3959 neonates of nondiabetic mothers. (Reproduced with permission from Bradley RJ, Nicolaides KH, Brudenell JM.: Are all infants ofdiabetic mothers "macrosomic"7 BMJ 1988 Dec 17;297(6663):1o583-1o584') in diabetic pregnancies that ultimately yield macrosomic newborns. hese findings comport with the observation that virtually all neonates of diabetic mothers are growth promoted, and accelerated fetal growth is particularly evident in women with poor glycemic control.

1	Unexplained Fetal Demise. Worldwide, the risk offetal death is three to four times higher in women with pregestational diabetes (Cardosi, 2013; Patel, 2015). Stillbirth without an identiiable cause is a phenomenon relatively limited to pregnancies complicated by overt diabetes. These stillbirths are "unexplained" because common factors such as obvious placental insuiciency, placental abruption, fetal-growth restriction, or oligohydramnios are not identified. hese fetuses are typically large for gestational age and die before labor, usually late in the third trimester.

1	These unexplained stillbirths are associated with poor glycemic control. Lauenborg and coworkers (2003) identiied suboptimal glycemic control in two thirds of unexplained stillbirths between 1990 and 2000. Also, fetuses ofdiabetic mothers often have elevated lactic acid levels. Salvesen and colleagues (1992, 1993) analyzed fetal blood samples and reported that mean umbilical venous blood pH was lower in diabetic pregnancies and was signiicantly related to fetal insulin levels. Such indings support the hypothesis that hyperglycemia-mediated chronic aberrations in oxygen and fetal metabolite transport may underlie these unexplained fetal deaths (Pedersen, 1977).

1	Aside from hyperglycemia alone, maternal ketoacidosis can cause fetal death. Explicable stillbirths due to placental insufficiency also occur with increased frequency in women with overt diabetes, usually in association with severe preeclampsia. In the prior California study of nearly a halfmillion singleton deliveries, the fetal death risk was sevenfold higher in women with hypertension and pregestational diabetes compared with the threefold increased risk associated with diabetes alone (Yanit, 2012). Stillbirth rates are also greater in women with advanced diabetes and vascular complications.

1	Hydramnios. Diabetic pregnancies are oten complicated by excess amnionic fluid. According to Idris and coworkers (2010), 18 percent of 314 women with pregestational diabetes were identified to have hydramnios, deined as an amnionic fluid index (AFI) >24 cm in the third trimester. And, women with elevated HbA)c values in the third trimester were more likely to have hydramnios. A likely-albeit unproven-explanation is that fetal hyperglycemia causes polyuria (Chap. 11, p. 228). In a study from Parkland Hospital, Dashe and colleagues (2000) found that the API parallels the amnionic luid glucose level among women with diabetes. Further support for this association was provided by Vink and associates (2006), who linked poor maternal glucose control to macrosomia and hydramnios.

1	Neonatal Efects. Before tests of fetal health and maturity became available, delivery before term was deliberately selected for women with diabetes to avoid unexplained stillbirth. Although this practice has been abandoned, a higher frequency of preterm delivery in women with diabetes persists. Most are indicated deliveries due to advanced diabetes with superimposed preeclampsia. hat said, Little and associates (2015) in their analysis of early-term delivery (37°/7 to 386/7 weeks) found a 13-percent reduction in such deliveries in women with diabetes between 2005 and 2011.

1	Although modern neonatal care has reduced neonatal death rates due to immaturity, neonatal morbidiy due to preterm birth continues to be a serious consequence. In one Neonatal Research Network study of 10,781 extremely preterm neonates, those born to diabetic women treated with insulin prior to pregnancy were at greater risk for necrotizing enterocolitis and late-onset sepsis than neonates of mothers without diabetes (Boghossian, 2016). Respiratory Distress Syndrome. Gestational age rather than overt diabetes is likely the most significant factor associated with respiratory distress syndrome (Chap. 33, p. 619). Indeed, in one analysis of 19,399 very-Iow-birthweight neonates delivered between 24 and 33 weeks' gestation, rates of respiratory distress syndrome in newborns of diabetic mothers were not higher compared with rates in neonates of nondiabetic mothers (Bental, 2011).

1	Hypoglycemia. Newborns of a diabetic mother experience a rapid drop in plasma glucose concentration after delivery. This is attributed to hyperplasia of the fetal �-islet cells induced by chronic maternal hyperglycemia. Low glucose concentrationsdefined as <45 mg/dL-are particularly common in newborns of women with unstable glucose concentrations during labor (Persson, 2009). Frequent blood glucose measurements in the newborn and active early feeding practices can mitigate these complications.

1	Hypocalcemia. Deined as a total serum calcium concentration <8 mg/dL in term newborns, early onset hypocalcemia is one of the potential metabolic derangements in neonates of diabetic mothers. Its cause has not been explained. Theories include aberrations in magnesium-calcium economy, asphyxia, and preterm birth. In a randomized study, 137 pregnant women with type 1 diabetes were managed with strict versus customary glucose control (DeMarini, 1994). Almost a third of neonates in the customary control group developed hypocalcemia compared with only 18 percent of those in the strict-control group.

1	Hyperbilirubinemia and Polycythemia. he pathogenesis of hyperbilirubinemia in neonates of diabetic mothers is uncertain. A major contributing factor is newborn polycythemia, which raises the bilirubin load (Chap. 33, p. 626). Polycythemia is thought to be a fetal response to relative hypoxia. According to Hay (2012), the sources of this fetal hypoxia are hyperglycemia-mediated elevations in maternal ainity for oxygen and fetal oxygen consumption. Together with insulin-like growth factors, this hypoxia leads to elevated fetal erythropoietin levels and red cell production. Fetal renal vein thrombosis is reported to result from polycythemia.

1	Cardiomyopathy. Newborns of diabetic pregnancies may have hypertrophic cardiomyopathy that primarily afects the interventricular septum (Rolo, 2011). Huang and coworkers (2013) propose that pathological ventricular hypertrophy in neonates born to women with diabetes is due to insulin excess. In severe cases, this cardiomyopathy may lead to obstructive cardiac failure. Russell and coworkers (2008) performed serial echo cardiograms on fetuses of 26 women with pregestational diabetes. In the first trimester, fetal diastolic dysfunction was already evident in some. In the third trimester, the fetal interventricular septum and right ventricular wall were thicker in fetuses of diabetic mothers. Most afected newborns are asymptomatic following birth, and hypertrophy usually resolves in the months after delivery.

1	Long-Term Cognitive Development. Intrauterine metabolic conditions have long been linked to neurodevelopment in ofspring. In a study of more than 700,000 Swedish-born men, the intelligence quotient of those whose mothers had diabetes during pregnancy averaged 1 to 2 points lower (Fraser, 2014). DeBoer and associates (2005) demonstrated impaired memory performance in infants of diabetic mothers at age 1 year. Results from the Childhood Autism Risks from Genetics and the Environment (CHARGE) study indicated that autism spectrum disorders or developmental delay were also more common in children of diabetic women (Krakowiak, 2012). Adane and colleagues (2016) conirmed a consistent relationship between maternal diabetes and diminished cognitive and language development in studies of younger children but not older children. Because interpreting efects of the intrauterine environment on neurodevelopment is confounded by postnatal factors, the link between maternal diabetes, glycemic control,

1	but not older children. Because interpreting efects of the intrauterine environment on neurodevelopment is confounded by postnatal factors, the link between maternal diabetes, glycemic control, and long-term neurocognitive outcome remains unconfirmed.

1	Inheritance of Diabetes. The risk of developing type 1 diabetes if either parent is afected is 3 to 5 percent. Type 2 diabetes has a much stronger genetic component. If both parents have type 2 diabetes, the risk of developing it approaches 40 percent. Both types of diabetes develop after a complex interplay between genetic predisposition and environmental factors. Type 1 diabetes is prompted by environmental triggers such as infection, diet, or toxins and heralded by the appearance of islet cell autoantibodies in genetically vulnerable individuals (Pociot, 2016; Rewers, 2016). Some but not all studies have shown a reduction in risk for type 1 or type 2 diabetes associated with breastfeeding (Owen, 2006; Rewers, 2016). Diabetes and pregnancy interact signiicantly such that maternal welfare can be seriously jeopardized. With the possible exception of diabetic retinopathy, however, the long-term course of diabetes is not afected by pregnancy.

1	In an analysis ofmore than 800,000 pregnancies, Jovanovic and colleagues (2015) found that 1125 mothers with type 1 diabetes were at increased risk for hypertension and respiratory complications compared with nondiabetic women. And, 10,126 mothers with type 2 diabetes had an elevated risk for depression, hypertension, infection, and cardiac or respiratory complications compared with pregnant controls. Maternal death is uncommon, but rates in women with diabetes are still higher than those in unafected gravidas. In one analysis of972 women with type 1 diabetes, the maternal mortality rate was 0.5 percent, and deaths resulted from diabetic ketoacidosis, hypoglycemia, hypertension, and infection (Leinonen, 2001).

1	Preeclampsia. Pregnancy-associated hypertension is the complication that most often forces preterm delivery in diabetic women. he incidence of chronic and gestational hypertension-and especially preeclampsia-is remarkably increased (Chap. 40, p. 713). In a systematic review and metaanalysis of 92 studies including more than 25 million pregnancies, Bartsch and associates (2016) calculated a pooled relative risk of 3.7 for preeclampsia in women with pregestational diabetes. In the study cited earlier by Yanit and colleagues (2012), preeclampsia developed three to four times more often in women with overt diabetes. Moreover, those diabetics with coexistent chronic hypertension were 12 times more likely to develop preeclampsia. As shown in Figure 57-5, women with type 1 diabetes in more advanced White classes of overt diabetes, who typically exhibit vascular complications and have preexisting nephropathy, are more likely to develop preeclampsia. This .g:-. .E )

1	.E ) FIGURE 57-5 Incidence of preeclampsia in 491 type 1 diabetic women in Sweden and the United States. (Data from Hansona, 1993; Sibaib, 2000.) rising risk with duration ofdiabetes may be related to oxidative stress, which plays a key role in the pathogenesis of diabetic complications and preeclampsia. With this in mind, the Diabetes and Preeclampsia Intervention Trial (DAPIT) randomly assigned 762 women with type 1 diabetes to antioxidant vitamin C and E supplementation or placebo in the irst half of pregnancy (McCance, 2010). Preeclampsia rates did not difer except in a few women with a low antioxidantstatus at baseline. Diabetic Nephropathy. Diabetes is the leading cause of end stage renal disease in the United States (Chap. 53, p. 1034).

1	Diabetic Nephropathy. Diabetes is the leading cause of end stage renal disease in the United States (Chap. 53, p. 1034). Clinically detectable nephropathy begins with microalbumin uria-30 to 300 mg/24 hours. his may manifest as early as 5 years after diabetes onset. Macroalbuminuria-more than 300 mg/24 hours-develops in patients destined to have end-stage renal disease. Hypertension almost invariably develops during this period, and renal failure ensues typically in the next 5 to 10 years. The incidence ofovert proteinuria is nearly 30 percent in individuals with type 1 diabetes and ranges from 4 to 20 per cent in those with type 2 diabetes (Reutens, 2013). Regression is common and, presumably from improved glucose control, the incidence ofnephropathy with type 1 diabetes has declined.

1	Approximately 5 percent of pregnant women with diabetes already have renal involvement. Approximately 40 percent of these will develop preeclampsia (Vidaef, 2008). In those with microproteinuria, this incidence may not be as high (How, 2004). However, mbia and associates (2018) reported that the rates ofpreterm delivery, birthweight <2500 g, and growth restriction were significantly higher in neonates of diabetic women with microproteinuria compared with those of diabetic gravidas without proteinuria.

1	In general, pregnancy does not appear to worsen diabetic nephropathy. In one prospective study of 43 women with diabetes, diabetic nephropathy did not progress through 12 months after delivery (Young, 2012). Most ofthese women had only mild renal impairment. Conversely, pregnancy in women with moderate-to-severe renal impairment may have accelerated progression of their disease (Vidaef, 2008). As in women with glomerulopathies, hypertension or substantial proteinuria before or during pregnancy is a major predictive factor for ultimate progression to renal failure in women with diabetic nephropathy (Chap. 53, p. 1033).

1	Diabetic Retinopathy. Retinal vasculopathy is a highly speciic complication ofboth type 1 and type 2 diabetes. In the United States, diabetic retinopathy is the most important cause ofvisual impairment in working-aged adults. he first and most common visible lesions are small microaneurysms followed by blot hemorrhages that form when erythrocytes escape from the aneurysms. hese areas leak serous fluid that creates hard exudates. Such features are termed backgroundor nonprolierative retinopathy. With increasingly severe retinopathy, the abnormal vessels of background eye disease become occluded, leading to retinal ischemia and infarctions that appear as cotton wool exuates. hese are considered preprolerative retinopathy. In response to ischemia, neovascularization begins on the retinal surface and out into the vitreous cavity. Vision is obscured when these vessels bleed. Laser photocoagulation before hemorrhage reduces the rate of visual loss progression and blindness by half. The

1	surface and out into the vitreous cavity. Vision is obscured when these vessels bleed. Laser photocoagulation before hemorrhage reduces the rate of visual loss progression and blindness by half. The procedure may be performed during pregnancy when indicated.

1	Vestgaard and coworkers (2010) reported that almost two thirds of 102 pregnant women with type 1 diabetes examined by 8 weeks' gestation had background retinal changes, proliferative retinopathy, or macular edema. A fourth of these women developed progression of retinopathy in at least one eye during pregnancy. The same group of investigators evaluated 80 type 2 diabetics and identified retinopathy, mostly mild, in 14 percent during early pregnancy. Progression was identiied in only 14 percent (Rasmussen, 2010). This complication is believed to be a rare example of a long-term adverse efect of pregnancy.

1	Other risk factors that have been associated with progression of retinopathy include hypertension, higher levels of insulin-like growth factor-I, placental growth factor, and macular edema identiied in early pregnancy (Bargiota, 2011; Huang, 2015; Mathiesen, 2012; Ringholm, 2011; Vestgaard, 2010). The American Academy of Ophthalmology (2016) recommends that pregnant women with preexisting diabetes should routinely be ofered retinal assessment after the first prenatal visit. Subsequent eye examinations depend on severity of retinopathy and level of diabetes control. Currently, most agree that laser photocoagulation and good glycemic control during pregnancy minimize the potential for deleterious efects of pregnancy.

1	Ironically, "acute" rigorous metabolic control during pregnancy has been linked to acute worsening of retinopathy. In a study of 201 women with retinopathy, almost 30 percent suffered eye disease progression during pregnancy despite intensive glucose control (11cElvy, 2001). That said, Wang and coworkers (1993) observed that although retinopathy worsened during the critical months of rigorous glucose control, long-term progression of eye disease actually slowed. Arun and Taylor (2008) found that only four women required laser photocoagulation during pregnancy, and none required laser in the next 5 years.

1	Diabetic Neuropathy. Peripheral symmetrical sensorimotor diabetic neuropathy is uncommon in pregnant women. But, a form of this, known as diabetic gastropathy, can be troublesome during pregnancy. It causes nausea and vomiting, nutritional problems, and diiculty with glucose control. Women with gastroparesis are advised that this complication is associated with a high risk of morbidity and poor perinatal outcome (Kitzmiller, 2008). Treatment with metoclopramide and D2-receptor antagonists is sometimes successful. Gastric neurostimulators have also been successfully used during pregnancy (Fuglsang, 2015). Treatment of hyperemesis gravidarum can be challenging, and we routinely provide insulin by continuous infusion for women who are admitted with this condition (Chap. 54, p. 1044).

1	Diabetic Ketoacidosis. his serious complication develops in approximately 1 percent of diabetic pregnancies and is most often encountered in women with type 1 diabetes (Hawthorne, 201r1). It is increasingly being reported in women with type 2 or even those with gestational diabetes (Bryant, 2017; Sibai, 2014). Diabetic ketoacidosis (DKA) may develop with hyperemesis gravidarum, infection, insulin noncompliance, 3-mimetic drugs given for tocolysis, and corticosteroids given to induce fetal lung maturation. DKA results from an insulin deiciency combined with an excess in counter-regulatory hormones such as glucagon. his leads to gluconeogenesis and ketone body formation. The ketone body 3-hydroxybutyrate is synthesized at a much greater rate than acetoacetate, which is preferentially detected by commonly used ketosis-detection methods. herefore, serum or plasma assays for 3-hydroxybutyrate more accurately reflect true ketone body levels.

1	Of gravidas with DA, fewer than 1 percent die, but perinatal mortality rates from a single episode of DKA may reach 35 percent (Guntupalli, 2015). Noncompliance is a prominent factor, and this and ketoacidosis were historically considered prognostically bad signs in pregnancy (Pedersen, 1974). Importantly, pregnant women usually develop ketoacidosis at lower blood glucose thresholds than when nonpregnant. In a study from Parkland Hospital, the mean glucose level for pregnant women with DKA was 380 mg/dL, and the mean HbA]c value was 10 percent (Bryant, 2017). Euglycemic ketoacidosis during pregnancy is possible but rare (Sibai, 2014). One management protocol for diabetic ketoacidosis is shown in Table 57-7. An important cornerstone of management is vigorous rehydration with crystalloid solutions of normal saline or Ringer lactate.

1	Infections. The rates of many infections are higher in diabetic pregnancies. Common ones include candidal vulvovaginitis, urinary and respiratory tract infections, and puerperal pelvic sepsis. However, in one study of more than 1250 diabetic gravidas screened before 16 weeks' gestation, rates of bacterial vaginosis or vaginal colonization with Candida or Tricho monas species were not increased (Marschalek, 2016). But, in their population-based study of almost 200,000 pregnancies, Sheiner and coworkers (2009) found a twofold greater risk of asymptomatic bacteruria in women with diabetes. Similarly, Alvarez and associates (2010) reported positive urine culture results in 25 percent of diabetic women. In a 2-year analysis of pyelonephritis at Parkland Hospital, 5 percent of women with diabetes developed pyelonephritis compared with 1.3 percent of the nondiabetic population (Hill, 2005). Fortunately, these latter infections can be minimized by screening and eradication of asymptomatic

1	developed pyelonephritis compared with 1.3 percent of the nondiabetic population (Hill, 2005). Fortunately, these latter infections can be minimized by screening and eradication of asymptomatic bacteriuria (Chap. 53, p. 1027). Finally, Johnston and colleagues (2017) reported that 16.5 percent of women with pregestational diabetes had postoperative wound complications following cesarean delivery.

1	• Management of Diabetes in Pregnancy Because of the close relationship between pregnancy complications and maternal glycemic control, eforts to achieve glucose targets are typically more aggressive during pregnancy. Management preferably should begin before pregnancy and include speciic goals during each trimester. To minimize early pregnancy loss and congenital malformations in ofspring of diabetic mothers, optimal medical care and education are recommended before conception (Chap. 8, p. 147). he National Preconception Health and Healthcare Initiative Clinical Workgroup for the CDC established values TABLE 57-7. Management of Diabetic Ketoacidosis During Pregnancy Obtain arterial blood gases to document degree of acidosis present; measure glucose, ketones, and electrolyte levels at 1-to 2-hour intervals Low-dose, intravenous Loading dose: 0.2-0.4 U/kg Maintenance: 2-10 U/hr Total replacement in first 12 hours of 4-6 L 1 L in first hour

1	Low-dose, intravenous Loading dose: 0.2-0.4 U/kg Maintenance: 2-10 U/hr Total replacement in first 12 hours of 4-6 L 1 L in first hour If initially normal or reduced, an infusion rate up to 15-20 mEq/hr may be required; if elevated, wait until levels decrease into the normal range, then add to intravenous solution in a concentration of 20-30 mEq/L Add one ampule (44 mEq) to 1 L of 0.45 normal saline if pH is <7.1 Data from Bryant, 20017; Landon, 2002; Sibai, 2014. for optimal glycemic control (Frayne, 2016). This was defined Insulin Treatment as HbA}c <6.5 percent in women with pregestational diabetes. The overtly diabetic gravida is best treated with insulin. While Unfortunately, nearly half of pregnancies in the United States oral hypoglycemic agents have been used successfully for gesare unplanned, and diabetic women frequently begin pregnancy tational diabetes (p. 11r12), these agents are not currently recwith suboptimal glucose control (Finer, 2016; Kim, 2005).

1	ommended for overt diabetes, although this is controversial The ADA (20 17b) has also deined optimal preconcep(American College of 0bstetricians and Gynecologists, 20 16b). tional glucose control using insulin. Relective values are selfMaternal glycemic control can usually be achieved with mulmonitored preprandial glucose levels of70 to 100 mg/dL, peak tiple daily insulin injections and adjustment of dietary intake. 2-hour postprandial values of 100 to 120 mg/dL, and mean he action proiles of commonly used short-and long-termdaily glucose concentrations < 110 mg/dL. In one prospective insulins are shown in Table 57-8. population-based study of 933 pregnant women with type 1 diabetes, the risk of congenital malformations was not demonstrably higher with HbA}c levels <6.9 percent compared with the risk in more than 70,000 nondiabetic controls Qensen, 2010). These investigators also found a substantial fourfold greater risk for malformations at levels > 10 percent.

1	If indicated, evaluation and treatment for diabetic complica TABLE 57-8. Action Profiles of Commonly Used Insulins Insulin Type Onset Peak (hr) Duration (hr) Lispro <15nmin 0.5-1n.5 Glulisine <15 min 0.5-1.5 3-4 tuted before pregnancy. Finally, folate, 400 jLg/d orally, is given periconceptionally and during early pregnancy to decrease the risk of neural-tube defects. Aspart <15 min 0.5-1n.5 Careful monitoring of glucose control is essential. For this reason, many clinicians hospitalize overtly diabetic women during early pregnancy to initiate an individualized glucose control program and provide education. This also provides an opportunity to assess the extent of diabetic vascular complications and precisely establish gestational age. aMinimal peak activity. NPH = neutral protamine Hagedorn; SC = subcutaneous. Data from Powers, 201 2.

1	Subcutaneous insulin infusion by a calibrated pump does not yield better pregnancy outcomes compared with multiple daily injections. But, an infusion pump is a safe alternative in appropriately selected patients (Farrar, 2016; Sibai, 2014). With the advent of sensor-augmented insulin pumps and closed-loop insulin delivery systems, improved glycemic control with either manual or computer-generated insulin adjustments based on continuous glucose monitoring is now possible. One small randomized, crossover study of 16 pregnant women compared these two technologies (Stewart, 2016). Those with automatic closed-loop systems had glucose values within target range for a higher percentage of time and had lower daily median glucose values. Moreover, their rates of hypoglycemic episodes were not increased. Roeder and colleagues (2012) noted with insulin pump use in women with type 1 diabetes that total daily insulin doses declined in the first trimester but later rose more than threefold.

1	increased. Roeder and colleagues (2012) noted with insulin pump use in women with type 1 diabetes that total daily insulin doses declined in the first trimester but later rose more than threefold. Postprandial glucose elevations prompted most of the required daily-dose increases. If a continuous-infusion insulin pump is elected, it is best started before pregnancy to avoid the hypoglycemia and ketoacidosis risk associated with the learning curve (Sibai, 2014).

1	Monitoring. Self-monitoring of capillary glucose levels using a glucometer is recommended because this involves the woman in her own care. he ADA (2017b) recommends fasting and postprandial glucose monitoring. Glucose goals recommended during pregnancy are shown in Table 57-9. Advances in noninvasive glucose monitoring will undoubtedly render intermittent capillary glucose monitoring obsolete. Subcutaneous continuous glucose monitoring devices have shown that pregnant women with diabetes experience signiicant periods of daytime hyperglycemia and nocturnal hypoglycemia that are undetected by traditional monitoring (Combs, 2012). Such glucose monitoring systems, coupled with a continuous insulin pump, ofer the potential of an "artificial pancreas" to avoid undetected hypo-or hyperglycemia during pregnancy.

1	Diet. Nutritional planning includes appropriate weight gain through carbohydrate and caloric modiications based on height, weight, and degree of glucose intolerance (merican Diabetes Association, 2017b; Bantle, 2008). he mix of carbohydrate, protein, and fat is adjusted to meet the metabolic goals and individual patient preferences. A minimum of 175 g/d of carbohydrates ideally is provided. In one analysis of more than 200 obese pregnant women with glucose intolerance, a lower carbohydrate intake, particularly late in pregnancy, was TABLE 57-9. Self-Monitored Capillary Blood Glucose Goals Fasting :;95 Premeal :;100 1-hr postprandial :;140 2-hr postprandial :;120

1	TABLE 57-9. Self-Monitored Capillary Blood Glucose Goals Fasting :;95 Premeal :;100 1-hr postprandial :;140 2-hr postprandial :;120 Hemoglobin Ale :;6% associated with lower fat mass in ofspring at birth (Renault, 2015). Allotted carbohydrates are distributed throughout the day in three small-to moderate-sized meals and two to four snacks. Weight loss is not recommended, but modest caloric restriction may be appropriate for overweight or obese women. An ideal dietary composition is 55 percent carbohydrate, 20 percent protein, and 25 percent fat, of which < 10 percent is saturated fat.

1	Hypoglycemia. Diabetes tends to be unstable in the first half of pregnancy, and the incidence of hypoglycemia peaks during the irst trimester. Chen and coworkers (2007) identified hypoglycemic events-blood glucose values <40 mg/dL-in 37 of 60 women with type 1 diabetes. A fourth of these were considered severe because the women were unable to treat their own symptoms and required assistance from another person. Caution is recommended when attempting euglycemia in women with recurrent episodes of hypoglycemia.

1	In a Cochrane database review, Middleton and colleagues (2016) determined that loose glycemic control, defined as fasting glucose valuesr> 120 mg/dL, was associated with greater risks for preeclampsia, cesarean delivery, and birthweight above the 90th percentile compared with women with tight or moderate control. Importantly, no obvious benefit were gained from very tight control, defined by fasting values <90 mg/dL, and there were more cases of hypoglycemia. hus, women with overt diabetes who have glucose values that are viewed by some as "considerably above" this 90 mg/dL threshold can expect good pregnancy outcomes.

1	Maternal serum alpha-fetoprotein determination at 16 to 20 weeks' gestation is used in association with targeted sonographic examination to detect neural-tube defects and other anomalies (Chap. 14, p. 283). hese levels may be lower in diabetic pregnancies, and interpretation is altered accordingly. Because the incidence of congenital cardiac anomalies is fivefold in mothers with diabetes, fetal echocardiography is an important part of second-trimester sonographic evaluation (Fouda, 2013). Despite advances in ultrasound technology, however, Dashe and associates (2009) cautioned that detection of fetal anomalies in obese diabetic women is more diicult than in similarly sized women without diabetes.

1	Regarding second-trimester glucose control, euglycemia with self-monitoring continues to be the goal in management. After the first-trimester instability, a stable period ensues. his is followed by a greater insulin requirement due to the elevated peripheral resistance to insulin described in Chapter 4 (p. 56). During the past several decades, the threat of late-pregnancy stillbirth in women with diabetes has prompted recommendations for various fetal surveillance programs beginning in the third trimester. Such protocols include fetal movement counting, periodic fetal heart rate monitoring, intermittent biophysical profile evaluation, and contraction stress testing (Chap. 17, p. 331). None of these techniques has been subjected to prospective randomized clinical trials, and their primary value seems related to their low false-negative rates. The American College of Obstetricians and Gynecologists (2016b) suggests initiating such testing at 32 to 34 weeks' gestation.

1	College of Obstetricians and Gynecologists (2016b) suggests initiating such testing at 32 to 34 weeks' gestation. At Parkland Hospital, women with diabetes are seen in a specialized obstetrical clinic every 2 weeks. During these visits, glycemic control records are evaluated and insulin adjusted. Women are routinely instructed to perform fetal kick counts beginning early in the third trimester. At 34 weeks, admission is ofered to all insulin-treated women. While in the hospital, they continue daily fetal movement counts and undergo fetal heart rate monitoring three times a week. Delivery is planned for 38 weeks.

1	Labor induction may be attempted when the fetus is not excessively large and the cervix is considered favorable (Chap. 26, p. 503). Little and colleagues (2015) analyzed term singleton births from 2005 to 201r1 and showed a higher percentage of diabetic women were delivered each year before 39 weeks compared with the entire cohort-37 versus 29 percent. Cesarean delivery at or near term has frequently been used to avoid traumatic birth of a large fetus in a woman with diabetes. In women with more advanced diabetes, especially those with vascular disease, the reduced likelihood of successful labor induction remote from term has also contributed to an incre�sed cesarean delivery rate. In an analysis of pregnancy outcomes of diabetic women from University of labama at Birmingham according to the White classiication, the rate of cesarean delivery and preeclampsia escalated with White class (Bennett, 2015). In another study, a HbA,c level >6.4 percent at delivery was independently

1	to the White classiication, the rate of cesarean delivery and preeclampsia escalated with White class (Bennett, 2015). In another study, a HbA,c level >6.4 percent at delivery was independently associated with urgent cesarean delivery. This suggests that tighter glycemic control during the third trimester might reduce late fetal compromise and cesarean delivery for fetal indications (Miailhe, 2013). The cesarean delivery rate for women with overt diabetes has remained at approximately 80 percent for the past 40 years at Parkland Hospital.

1	Reducing or withholding the dose of long-acting insulin to be given on the day of delivery is recommended. Regular insulin should be used to meet most or all of the insulin needs of the mother during this time, because insulin requirements typically drop markedly after delivery. We have found that continuous insulin infusion by calibrated intravenous pump is most satisfactory (Table 57-10). Throughout labor and after delivery, the woman should be adequately hydrated intravenously and given glucose in suicient amounts to maintain normoglycemia. Capillary or plasma glucose levels are checked frequently, especially during active labor, and regular insulin is administered accordingly. Often, women may require virtually no insulin for the irst 24 hours or so postpartum. Subsequently, insulin requirements may luctuate markedly during the next few days. Infection must be promptly detected and treated. When appropriate, oral agents can be restarted.

1	Counseling in the puerperium should include a discussion of birth control. Efective contraception is especially important in women with overt diabetes to allow optimal glucose control before subsequent conception. In the United States in 2010, almost 5 percent of gravidas were afected by gestational diabetes (DeSisto, 2014). W orldwide, its prevalence difers according to race, ethnicity, age, and body composition and by screening and diagnostic criteria. There continue to be several controversies pertaining to the diagnosis and treatment of gestational diabetes. A National Institutes of Health (NIH) Consensus Development Conference (2013) was convened to study this. he American College of Obstetricians and Gynecologists (2017a) has also updated its recommendations. These two authoritative sources provide an analysis of the issues surrounding the diagnosis and bolster the approach to identiying and treating women with gestational diabetes.

1	The word gestational implies that diabetes is induced by pregnancy-ostensibly because of exaggerated physiological changes in glucose metabolism (Chap. 4, p. 56). Gestational diabetes is deined as carbohydrate intolerance of variable severity with onset or first recognition during pregnancy (American College of Obstetricians and Gynecologists, 2017a). This deinition applies whether or not insulin is used for treatment and undoubtedly includes some women with previously unrecognized overt diabetes. Use of the term gestational diabetes has been encouraged to communicate the need for enhanced surveillance and to TABLE 57-1 0. Insulin Management During Labor and Delivery Usual dose of intermediate-acting insulin is given at bedtime. Morning dose of insulin is withheld. Intravenous infusion of normal saline is begun.

1	Usual dose of intermediate-acting insulin is given at bedtime. Morning dose of insulin is withheld. Intravenous infusion of normal saline is begun. Once active labor begins or glucose levels decrease to less than 70 mg/dL, the infusion is changed from saline to 5% dextrose and delivered at a rate of 100-150 cc/h (2.5 mg/kg/min) to achieve a glucose level of approximately 100 mg/dL. Glucose levels are checked hourly using a bedside meter allowing for adjustment in the insulin or glucose infusion rate. Regular (short-acting) insulin is administered by intravenous infusion at a rate of 1n.25 U/h if glucose levels exceed 100 mg/dL. Data from Coustan DR. Delivery: timing, mode, and management. In: Reece EA, Coustan DR, Gabbe SG, editors. Diabetes in women: adolescence, pregnancy, and menopause. 3rd ed. Philadelphia (PA): Lippincott Williams & Wilkins; 2004; and Jovanovic L, Peterson CM. Management of the pregnant, insulin-dependent diabetic woman. Diabetes Care 1980;3:63-8.

1	stimulate women to seek further testing postpartum. The most important perinatal correlate is excessive fetal growth, which may result in both maternal and fetal birth trauma. he likelihood of fetal death with appropriately treated gestational diabetes is not diferent from that in the general population. Importantly, more than half of women with gestational diabetes ultimately develop overt diabetes in the ensuing 20 years. And, as discussed on page 1097, evidence is mounting for long-range complications that include obesity and diabetes in their ofspring.

1	Despite almost 50 years of research, there is still no agreement regarding optimal gestational diabetes screening. The diiculty in achieving consensus is underscored by the controversy following publication of the single-step approach espoused by the International Association of Diabetes and Pregnancy Study Groups Consensus Panel (2010) and shown in Table 57-11. his strategy was greatly influenced by results of the Hypoglycemia and Pregnancy Outcomes (HAPO) Study, described later. Although the ADA (2017 a) supports this new scheme, the American College of Obstetricians and Gynecologists (2017a) continues to recommend a two-step approach to screen and diagnose gestational diabetes. Similarly, the NIH Consensus Development Conference in 2013 concluded that evidence is insuicient to adopt a one-step approach.

1	he recommended two-step approach begins with either universal or risk-based selective screening using a 50-g, I-hour oral glucose challenge test. Participants in the Fifth International TABLE 57-1 1. Threshold Values for Diagnosis of Gestational Diabetes be equaled or exceeded for the diagnosis of gestational diabetes. bin addition, 1.7% of participants in the initial cohort were unblinded because of fasting plasma glucose levels >5.8 mmol/L (105 mg/dL) or 2-hr OGTI values > 11n.1 mmollL (200 mg/dL), bringing the total to 17.8%. OGI = oral glucose tolerance test. Data from International Association of Diabetes and Pregnancy Study Groups, 2010.

1	OGI = oral glucose tolerance test. Data from International Association of Diabetes and Pregnancy Study Groups, 2010. Workshop Conferences on Gestational Diabetes endorsed use ofselective screening criteria shown in Table 57-12. Conversely, the American College of Obstetricians and Gynecologists (2017a) recommends universal screening of pregnant women using a laboratory-based blood glucose test. It is suggested that attempts to identiy the 10 percent of women who should not be screened would add unnecessary complexity. Screening should TABLE 57-12. Risk-Based Recommended Screening Strategy for Detecting GDMa GDM risk assessment: should be ascertained at the first prenatal visit Low Risk: Blood glucose testing not routinely required if all the following are present: Member of an ethnic group with a low prevalence of GDM No known diabetes in first-degree relatives No history of abnormal glucose metabolism No history of poor obstetrical outcome

1	Member of an ethnic group with a low prevalence of GDM No known diabetes in first-degree relatives No history of abnormal glucose metabolism No history of poor obstetrical outcome Average Risk: Perform blood glucose testing at 24 to 28 weeks using either: Two-step procedure: 50-g oral glucose challenge test (GCT), followed by a diagnostic 100-g OGI for those meeting the threshold value in the GCT One-step procedure: diagnostic 100-g OGTI performed on all subjects High Risk: Perform blood glucose testing as soon as feasible, using the procedures described above, if one or more of these are present: Strong family history of type 2 diabetes Previous history of GDM, impaired glucose metabolism, or glucosuria If GDM is not diagnosed, blood glucose testing should be repeated at 24 to aCriteria of the Fifth Intenational Workshop-Conference on Diabetes. GDM = gestational diabetes mellitus; OGTI = oral glucose tolerance test.

1	Reproduced with permission from Metzger BE, Coustan DR, the Organizing Committee: Summary and recommendations of the Fourth International Workshop-Conference on Gestational Diabetes Mellitus, Diabetes Care. 1998 Aug; 21 SuppIn2:B161n-B167. TABLE 57-13. Diagnosis of GDM Using Threshold women not known to have glucose intolerance earlier in preg nancy. This 50-g screening test is followed by a dianostic 100-g, Tolerance Testa,b 3-hour oral glucose tolerance test (OGT) if screening results meet or exceed a predetermined plasma glucose concentration. For the 50-g screen, the plasma glucose level is measured 1 hour ater a 50-g oral glucose load without regard to the time

1	Fasting 105 5.8 95 5.3 of day or time oflast meal. In a recent review, the pooled sensitiv1-hr 190 10.6 180 2-hr 165 9.2 155 8.6 ity for a threshold of 140 mg/dL ranged from 74 to 83 percent depending on 100-g thresholds used for diagnosis (van Leeuwen, 3-hr 145 8.0 140 2012). Sensitivity estimates for a 50-g screen threshold of 135 aThe test should be performed when the patient is fasting. bTwo or more of the venous plasma glucose concentra mg/dL improved only slightly to 78 to 85 percent. Importantly, speciicity dropped from a range of 72 to 85 percent for 140 mg/dL to 65 to 81 percent for a threshold of 135 mg/dL. Using a threshold of 130 mg/dL marginally improves sensitivity with a further decline in speciicity (Donovan, 2013). That said, in the absence of clear evidence supporting one cutof value over another, the American College of Obstetricians and Gynecolo gists (2017a) sanctions using anyrone of the three 50-g screen thresholds. At Parkland Hospital, we continue to use 140

1	cutof value over another, the American College of Obstetricians and Gynecolo gists (2017a) sanctions using anyrone of the three 50-g screen thresholds. At Parkland Hospital, we continue to use 140 mg/dL as the screening threshold to prompt the 100-g test.

1	Justiication for screening and treatment of women with gestational diabetes was strengthened by the study by Crowther and coworkers (2005). They assigned 1000 women with gestational diabetes between 24 and 34 weeks' gestation to receive dietary advice with blood glucose monitoring plus insulin therapy-the intervention group-or to undergo routine prenatal care. Women were diagnosed as having gestational diabetes if their blood glucose was > 100 mg/ dL ater an overnight fast and was between 140 and 198 mg/dL 2 hours ater ingesting a 75-g glucose solution. Women in the intervention group had a signiicantly lower risk of a composite adverse outcome that included perinatal death, shoulder dystocia, fetal bone fracture, and fetal nerve palsy. Macrosomia deined by birthweight ::4000 g complicated 10 percent of deliveries in the intervention group compared with 21 percent in the routine prenatal care group. Cesarean delivery rates were almost identical in the two study groups.

1	Slightly diferent results were reported by the MaternalFetal Medicine Units Network randomized trial of958 women (Landon, 2009). Dietary counseling plus glucose monitoring was compared with standard obstetrical care in women with mild gestational diabetes to reduce perinatal morbidity rates. Mild gestational diabetes was identiied in women with fasting glucose levels <95 mg/dL. hey reported no diferences in rates of composite morbidity that included stillbirth; neonatal hypoglycemia, hyperinsulinemia, and hyperbilirubinemia; and birth trauma. Importantly, secondary analyses demonstrated a 50-percent reduction in macrosomia, fewer cesarean deliveries, and a signiicant decrease in shoulder dystocia rate-1.5 versus 4 percent-in treated versus control women.

1	Based largely on these two landmark studies, the U.S. Preventive Sevices Task Force (2014) now recommends universal screening in low-risk women after 24 weeks' gestation. However, the Task Force concluded that evidence is insuicient to assess the balance of benefits versus harms of screening before 24 weeks. For screening, the optimal OGTT ro identiy gestational diabetes has not been agreed upon. The World Health Organization (2013) and the ADA (2017a) recommend the tions listed are met or exceeded for a positive diagnosis. (Serum glucose level. dSerum or plasma glucose level. NDDG = National Diabetes Data Group. Data from American Diabetes Association, 201n7a; Ferrara, 2002.

1	NDDG = National Diabetes Data Group. Data from American Diabetes Association, 201n7a; Ferrara, 2002. 75-g, 2-hour OGTT, but acknowledge that the diagnosis can be accomplished using the two-step strategy. In the United States, however, the 1 OO-g, 3-hour OGTT performed after an overnight fast is recommended by the American College of Obstetricians and Gynecologists (2017a). Proposed criteria for interpretation of the diagnostic 100-g OGTT are shown in Table 57-13. In a secondary analysis of the Maternal-Fetal Medicine Units Network treatment trial, Harper and colleagues (2016) showed that women diagnosed with either the National Diabetes Data Group (NDDG) or the Carpenter-Co us tan criteria beneited from treatment. However, the number needed to treat to prevent a shoulder dystocia was higher for the Carpenter-Co us tan criteria. At Parkland Hospital we continue to use the NDDG criteria for diagnosis. Criteria for the 75-g OGTT recommended are shown in Table 57-11.

1	The Hyperglycemia and Adverse Pregnancy Outcome Study

1	This was a 7 -year international epidemiological study of 23,325 pregnant women at 15 centers in nine countries (HAPO Study Cooperative Research Group, 2008). The investigation analyzed the association of various levels of glucose intolerance during the third trimester with adverse infant outcomes in women with gestational diabetes. Between 24 and 32 weeks' gestation, the general population of pregnant women underwent a 75-g OGTT after an overnight fasting. Blood glucose levels were measured fasting and then 1 and 2 hours after glucose ingestion. Caregivers were blinded to results except for women whose glucose levels exceeded values that required treatment and removal from the study. Glucose values at each of these three time posts were stratiied into seven categories (Fig. 57-6). hese values were then correlated with rates for birthweight >90th percentile (LGA) , primary cesarean delivery, neonatal hypoglycemia, and cord-serum C-peptide levels >90th percentile. Odds of each outcome

1	were then correlated with rates for birthweight >90th percentile (LGA) , primary cesarean delivery, neonatal hypoglycemia, and cord-serum C-peptide levels >90th percentile. Odds of each outcome were calculated using the lowest category-for example, fasting plasma glucose :;75 mg/dL-as the referent group. Their indings in general supported the supposition that increasing plasma glucose levels were associated with increasing adverse outcomes. Ecker and diagnosis rates but not with reduced macrosomia rates compared with a twostep approach. Remarkably, they identi � rate associated with adoption of the : IADPSG recommendations. he ADA ) (2013, 2017a) initially recommended ) adopting this new approach, however, ..J,approach described on page 1108, they now concede that data support a twostep strategy as well.

1	-1hour ; 105 106-132 133-155 156-171 172-193 194-211 ::212 Prompted by the disparate recommen -2hour ; 9091-108 109-125 126-139 140-157 158-177 ::178 dations, the NIH Consensus Development Conference on Diagnosing FIGURE 57-6 Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study. The fre quency of newborn birthweight ::90th percentile for gestational age plotted against glucose was convened. his conference included levels fasting and at 1-and 2-hr intervals following a 75-g oral glucose load. LGA = large for gestational age. (Reproduced with permission from HAPO Study Cooperative Research input from a multidisciplinary planning Group, Metzger BE, Lowe LP, et al: Hyperglycemia and adverse pregnancy outcomes, N Engl committee, a systematic evidence review J Med. 2008 May 8;358(19):1991-2002.)

1	J Med. 2008 May 8;358(19):1991-2002.) Greene (2008) concluded that it would be diicult to show that treating lesser degrees of carbohydrate intolerance would provide any meaningful improvements in clinical outcomes. We agree that changes in criteria are not justiied until clinical trials prove benefits. his position was also endorsed by the 2013 NIH Consensus Development Conference.

1	International Association of Diabetes and Pregnancy Study Group he IADPSG sponsored a workshop conference on the diagnosis and classiication of gestational diabetes in 2008. After reviewing the results of the HAPO study, a panel developed recommendations for the diagnosis and classiication of hyperglycemia during pregnancy. This panel allowed for the diagnosis of overt diabetes during pregnancy as shown in Table 57-4. It also recommended a single-step approach to the diagnosis of gestational diabetes using the 75-g, 2-hour OGTT. hresholds for fasting, 1-, and 2-hour values based on mean glucose concentrations from the entire HAPO study cohort were considered. hese glucose level thresholds were derived using an arbitrary 1.75 odds ratio of outcomes such as LGA birthweight and cord serum C-peptide levels >90th percentile. Only one of these thresholds, shown in Table 57-11, would need to be met or exceeded to make the diagnosis of gestational diabetes.

1	It is estimated that implementation of these recommendations would raise the prevalence of gestational diabetes in the United States to 17.8 percent! Said another way, the number of women with mild gestational diabetes would grow almost threefold with no evidence of treatment benefit (Cundy, 2012). Feldman and coworkers (2016) evaluated the implementation of the IADPSG paradigm in a before-ater analysis that included more than 6000 women. he new strategy was associated with a significant increase in gestational diabetes by the Agency for Healthcare Research and Quality Evidence-Based Practice Center, expert testimony, and a nonbiased panel to produce the overall report. he panel concluded that there were potential beneits to worldwide standardization. However, it found insuicient evidence to adopt a one-step diagnostic process such as the one proposed by the IADPSG. Moreover, as mentioned previously, after consideration of these indings, the American College of Obstetricians and

1	to adopt a one-step diagnostic process such as the one proposed by the IADPSG. Moreover, as mentioned previously, after consideration of these indings, the American College of Obstetricians and Gynecologists (2017a) continues to recommend a two-step screening and diagnostic approach to gestational diabetes diagnosis. he College noted no significant improvements in maternal or perinatal outcomes that would ofset the tripling of gestational diabetes incidence that would derive from the one-step approach. We applaud this decision.

1	Adverse consequences of gestational diabetes difer from those of pregestational diabetes. Unlike in women with overt diabetes, women with gestational diabetes do not appear to have fetuses with substantially higher rates of anomalies than the general obstetrical population (Sheield, 2002). In a study of more than 1 million women from the Swedish Medical Birth Registry, major malformation rates were marginally elevated in fetuses of gestational diabetics compared with those of nondiabetic controls-2.3 versus 1.8 percent (Fadl, 2010). The stillbirth rate was not greater in this study. Similarly, the stillbirth rate was not increased in an analysis by Jovanovic and associates (2015) of more than 800,000 pregnancies from 2005 through 2011. In contrast, and not unexpectedly, women with elevatedfasting glucose levels have elevated rates of unexplained stillbirths similar to those of women with overt diabetes. This increasing risk with progressive maternal hyperglycemia emphasizes the

1	glucose levels have elevated rates of unexplained stillbirths similar to those of women with overt diabetes. This increasing risk with progressive maternal hyperglycemia emphasizes the importance of identiying women with evidence of preexisting diabetes early in pregnancy (see Table 57-4). Similar to women with overt diabetes, adverse maternal efects associated with gestational diabetes include a higher frequency of hypertension and cesarean delivery.

1	he primary efect attributed to gestational diabetes is exces sive fetal size or macrosomia that is variably defined and dis cussed further in Chapter 44 (p. 857). The perinatal goal is birth trauma associated with shoulder dystocia. In a retrospec tive analysis of more than 80,000 vaginal deliveries in Chinese women, Cheng and associates (2013) calculated a 76-fold greater risk for shoulder dystocia in newborns weighing :4200 g compared with the risk in those weighing <3500 g. Impor tantly, however, the odds ratio for shoulder dystocia in women with diabetes was <2. Although gestational diabetes is certainly a risk factor, it accounts for onl) a small number of pregnancies complicated by shoulder dystocia.

1	he excessive shoulder and trunk fat that commonly characterizes the macrosomic newborn of a diabetic mother theoretically predisposes such neonates to shoulder dystocia or cesarean delivery (Durnwald, 2004; McFarland, 2000). Landon and associates (2011) identified shoulder dystocia in approximately 4 percent of women with mild gestational diabetes compared with < 1 percent of women with a 50-g glucose screen result < 120 mg/dL. In a ptospective study of fetal adipose measurements, however, Buhling and coworkers (2012) demonstrated no diferences between measurements in 630 ofspring of women with gestational diabetes and 142 without diabetes. he authors attributed this negative inding to successful treatment of gestational diabetes.

1	Extensive evidence supports that insulin-like growth factors also playra role in fetal-growth regulation (Chap. 44, p. 845). hese proinsulin-like polypeptides are produced by virtually all fetal organs and are potent stimulators of cell diferentiation and division. Luo and coworkers (2012) reported that insulinlike growth factor-1 strongly correlated with birthweight. he HAPO study investigators also reported dramatic increases in cord-serum C-peptide levels with rising maternal glucose levels following a 75-g OGTT. C-peptide levels above the 90th percentile were found in almost a third of newborns in the highest glucose categories. Other factors implicated in macrosomia include epidermal growth factor, ibroblast growth factor, platelet-derived growth factor, leptin, and adiponectin (Grissa, 2010; Loukovaara, 2004; Mazaki-Tovi, 2005).

1	Hyperinsulinemia may provoke severe hypoglycemia within minutes of birth, but only three fourths of these episodes occur in the irst 6 hours (Harris, 2012). he deinition of neonatal hypoglycemia is controversial, with recommended clinical thresholds ranging from 35 to 45 mg/dL. An NIH workshop conference on neonatal hypoglycemia supported using a threshold of 35 mg/dL in term newborns but cautioned that this practice is not strictly evidence based (Hay, 2009). Newborns described by the HAPO study (2008) had an incidence of clinical neonatal hypoglycemia that rose with increasing maternal OGTT result values deined in Figure 57-6. The frequency varied from 1 to 2 percent, but it was as high as 4.6 percent in women with fasting glucose levels : 100 mg/ dL. Similarly,

1	Cho and colleagues (2016), analyzed more than 3000 Korean women who underwent a 50-g OGTT and found that neonates born to women with a screening result :200 mg/dL were 84 times more likely to have hypoglycemia than those born to women with a result < 140 mg/dL. he risk of neonatal hypoglycemia correlates with umbilical cord C-peptide levels. But, importantly, the risk also rises with birthweight, independent of a maternal diabetes diagnosis (Mitanchez, 2014).

1	Maternal Obesity. In women with gestational diabetes, maternal body mass index (BMI) is an independent and more substantial risk factor for fetal macrosomia than is glucose intolerance (Ehrenberg, 2004; Mission, 2013). Stuebe and associates (2012) completed a secondary analysis of women with either untreated mild gestational diabetes or normal glucose tolerance testing results. They found that higher BMI levels were associated with rising birthweight, regardless of glucose levels. In one analysis of more than 600,000 pregnant women, gestational diabetes, compared with obesity or gestational weight gain, contributed the least to the population-attributable fraction of LGA neonates (Kim, 2014). he highest fraction of LGA neonates was attributable to maternal obesity plus excessive gestational weight gain. Similarly, Egan and colleagues (2014) found that excessive gestational weight gain is common in women with gestational diabetes and confers an additive risk for fetal macrosomia.

1	weight gain. Similarly, Egan and colleagues (2014) found that excessive gestational weight gain is common in women with gestational diabetes and confers an additive risk for fetal macrosomia. Weight distribution also seems to playra role because the risk of gestational diabetes is greater with maternal truncal obesity. Suresh and colleagues (2012) veriied that increased maternal abdominal subcutaneous fat thickness as measured by sonography at 18 to 22 weeks' gestation correlated with BMI and was a better predictor of gestational diabetes.

1	Women with gestational diabetes can be divided into two functional classes using fasting glucose levels. Pharmacological methods are usually recommended if diet modiication does not consistently maintain the fasting plasma glucose levels <95 mg/dL or the 2-hour postprandial plasma glucose < 120 mg/dL (American College of Obstetricians and Gynecologists, 2017 a). Whether pharmacological treatment should be used in women with lesser degrees of fasting hyperglycemia is unclear. here have been no controlled trials to identiy ideal glucose targets for fetal risk prevention. On the other hand, the HAPO study (2008) did demonstrate increased fetal risk at glucose levels below the threshold used for diagnosis of diabetes. The Fifth International Workshop Conference recommended that fasting capillary glucose levels be kept ;95 mg/ dL (Metzger, 2007).

1	In a systematic review, Hartling and colleagues (2013) concluded that treating gestational diabetes resulted in a signiicantly lower incidence of preeclampsia, shoulder dystocia, and macrosomia. For example, the calculated risk ratio was 0.50 for delivering a newborn >4000 g ater treatment. These investigators caution that the attributed risk for these outcomes is low, especially when glucose values are only moderately elevated. Importantly, they were unable to demonstrate an efect on neonatal hypoglycemia or on future metabolic outcomes in the ofspring.

1	Nutritional instructions generally include a carbohydratecontrolled diet suicient to maintain normoglycemia and avoid ketosis. On average, this includes a daily caloric intake of 30 to 35 kcallkg. Moreno-Castilla and associates (2013) randomly assigned 152 women with gestational diabetes to either a 40-or a 55-percent daily carbohydrate diet and found no diference in insulin levels and pregnancy outcomes. he American College of Obstetricians and Gynecologists (2017 a) suggests that carbohydrate intake be limited to 40 percent of total calories. The remaining calories are apportioned to give 20 percent as protein and 40 percent as fat.

1	The most appropriate dietary approach for women with gestational diabetes has not been established. One metaanalysis of trials of low-glycemic index diets found that diets higher in complex carbohydrates and dietary iber reduced the risk of macrosomia and likelihood of insulin use in women with gestational diabetes (Wei, 2016). That said, there clearly are limitations to what can be accomplished with various dietary approaches alone. Most and Langer (2012) found that insulin was efective in reducing the risk of excessive birthweight in ofspring of obese women with gestational diabetes. Casey and colleagues (20 15 b) also found that dietary treatment alone for morbidly obese women with mild gestational diabetes did not reduce neonatal fat mass or LGA birthweights.

1	Few trials have evaluated exercise speciically for women with gestational diabetes. he American College of Obstetricians and Gynecologists (2017a,b) recommends regular physical activity that incorporates aerobic and strength-conditioning exercise during pregnancy and extends this to women with gestational diabetes. Two recent metaanalyses demonstrate that structured exercise programs during pregnancy diminish weight gain during pregnancy and even reduce the risk of developing gestational diabetes (Russo, 2015; Sanabria-Martinez, 2015). Exercise during pregnancy in woman with gestational diabetes also lowers glucose levels Qovanovic-Peterson, 1989).

1	Hawkins and colleagues (2008) compared outcomes in 315 women with diet-treated gestational diabetes who used personal glucose monitors with those of 615 gestational diabetics who were also diet-treated but who underwent intermittent fasting glucose evaluation during weekly obstetrical visits. Women using daily blood-glucose self monitoring had significantly fewer macrosomic newborns. They also gained less weight after diagnosis than women evaluated during clinic visits only. hese indings support the common practice of blood-glucose self monitors for women with diet-treated gestational diabetes.

1	Postprandial surveillance for gestational diabetes has been shown to be superior to preprandial surveillance (De Veciana, 1995). At Parkland Hospital, we reviewed the impact of changing to postprandial monitoring in women with diet-treated gestational diabetes and demonstrated a signiicant reduction in maternal weight gain per week-0.63lb/week to 0.45 lb/weekin women managed with a postprandial monitoring schema. The merican College of Obstetricians and Gynecologists (2017 a) and the ADA (20 17b) recommend glucose assessment four times daily. The first check is performed fasting, and the remainder are done 1 or 2 hours ater each meal.

1	Historically, insulin has been considered standard therapy in women with gestational diabetes when target glucose levels cannot be consistently achieved through nutrition and exercise. It does not cross the placenta, and tight glycemic control can typically be achieved. Insulin therapy is typically added if fasting levels persistently exceed 95 mg/ dL in women with gestational diabetes. The American College of Obstetricians and Gynecologists (2017 a) also recommends that insulin be considered in women with I-hour postprandial levels that persistently exceed 140 mg/dL or those with 2-hour levelsr> 120 mg/dL. Importantly, all of these thresholds are extrapolated from recommendations for managing women with overt diabetes.

1	If insulin is initiated, the starting dose is typically 0.7 to 1.0 units/kg/d and is given in divided doses (American College of Obstetricians and Gynecologists, 2017a). A combination of intermediate-acting and short -acting insulin may be used, and dose adjustments are based on glucose levels at particular times of the day. At Parkland Hospital, the starting daily dose is divided so that two thirds is given in the morning before breakfast and one third in the evening before dinner. In the morning dose, one third is regular insulin and two thirds are NPH (neutral protamine Hagedorn). For the evening dose, one half is regular insulin and the other half is NPH. Insulin instruction for these women is accomplished either in a specialized outpatient clinic or during a short hospital stay. As shown in Table 57-8, insulin analogues such as insulin aspart and insulin lispro have a more rapid onset of action than regular insulin and theoretically could be helpful in postprandial glucose

1	As shown in Table 57-8, insulin analogues such as insulin aspart and insulin lispro have a more rapid onset of action than regular insulin and theoretically could be helpful in postprandial glucose management. Experience with these analogues with gestational diabetes is limited, and Singh and coworkers (2009) were unable to demonstrate a beneit compared with conventional insulins.

1	Insulin is the preferred first-line agent for persistent hyperglycemia in women with gestational diabetes. However, both the American College of Obstetricians and Gynecologists (2017 a) and the ADA (20 17b) acknowledge that several studies support the safety and eicacy of either glyburide (Micronase) or metformin (Glucophage) (Langer, 2000; Nicholson, 2009; Rowan, 2008). Balsells and colleagues (2015) performed metaanalyses of trials comparing both agents to insulin or to each other. In the seven trials comparing glyburide with insulin, glyburide was associated with higher birthweight, more macrosomia, and more frequent neonatal hypoglycemia. In the six trials comparing metformin with insulin, metformin was associated with less maternal weight gain, more ?reterm birth, and less severe neonatal hypoglycemia. On average from all trials, treatment failures occurred in 6 percent of women treated with glyburide and 34 percent of those treated with metformin. In the two studies comparing

1	hypoglycemia. On average from all trials, treatment failures occurred in 6 percent of women treated with glyburide and 34 percent of those treated with metformin. In the two studies comparing oral hypoglycemic agents with each other, metformin treatment was associated with less maternal weight gain, lower birthweight, and less macrosomia. In contrast to trials of each agent compared with insulin, treatment failure rates of both agents in these two studies were equivalent. Importantly, in a randomized trial of glyburide treatment as an adjunct to diet therapy in 395 women with mild gestational diabetes, improvements in pregnancy outcomes in women treated with glyburide.

1	comes among women treated with glyburide. First, like metfor min, glyburide crosses the placenta and reaches concentrations in the fetus that are more than two thirds of maternal lev els (Caritis, 2013). Additionally, a study of more than 9000 women with gestational diabetes treated with either insulin or glyburide showed a signiicant rise in rates of neonatal intensive care unit admission, respiratory distress, and neonatal hypogly cemia associated with glyburide use (Castillo, 2015).

1	Metformin reaches fetal serum concentrations similar to maternal levels. However, in one study of 751 women with gestational diabetes who were randomly assigned to metformin or insulin treatment, short-term perinatal adverse events did not difer between groups (Rowan, 2008). Outcomes included neonatal hypoglycemia, respiratory distress syndrome, photo therapy, birth trauma, 5-minute Apgar score .7, and preterm birth. Overall growth of ofspring at age 2 years also did not difer (Rowan, 2011). Nevertheless, the fat distribution in chil dren exposed to metformin showed a tendency toward a more favorable pattern. From a smaller randomized metformin trial, at 18 months, ofspring exposed to metformin were slightly heavier, but markers of early motor or language development did not difer compared with those in ofspring exposed to insulin (Ijas, 2015).

1	The Food and Drug Administration has not approved glyburide and metformin use for treatment of gestational diabetes. However, the American College of Obstetricians and Gynecologists (2017 a) recognizes both as reasonable choices for second-line glycemic control in women with gestational diabetes. Because long-term outcomes have not been fully studied, the committee recommends appropriate counseling, which includes disclosing the limitations in current safety data.

1	In general, for women with gestational diabetes who do not require insulin, early delivery or other interventions are seldom required. here is no consensus regarding the value or timing of antepartum fetal testing. It is typically reserved for women with pregestational diabetes because of the greater stillbirth risk. The American College of Obstetricians and Gynecologists (2017a) endorses fetal surveillance in women with gestational diabetes and poor glycemic control. At Parkland Hospital, women with gestational diabetes are routinely instructed to perform daily fetal kick counts in the third trimester (Chap. 17, p. 332). Insulin-treated women are ofered inpatient admission after 34 weeks' gestation, and antepartum monitoring is performed three times each week.

1	Women with gestational diabetes and adequate glycemic control are managed expectantly. Elective labor induction to prevent shoulder dystocia compared with spontaneous labor remains controversial. Alberico and colleagues (2017) recently described their truncated randomized trial of 425 women with tation. Although underpowered, this GINEXMAL Trial dem onstrated no clinically meaningful diference in the cesarean delivery rate between the induction and expectant management groups-12.6 versus 11.8 percent. However, with early labor induction, neonatal hyperbilirubinemia rates were signiicantly higher, and ironically, there was a nonsigniicant threefold greater shoulder dystocia rate. In a retrospective cohort study of 8392 Canadian women with gestational diabetes, Melamed 39 weeks was associated with a lower rate of cesarean delivery but with an elevated rate of neonatal intensive care unit admis sion. The American College of Obstetricians and Gynecologists 39 weeks' gestation. At Parkland

1	with a lower rate of cesarean delivery but with an elevated rate of neonatal intensive care unit admis sion. The American College of Obstetricians and Gynecologists 39 weeks' gestation. At Parkland Hospital, women with diet treated gestational diabetes are not electively induced for this indication. However, those treated with insulin are delivered at 38 weeks' gestation.

1	Elective cesarean delivery to avoid brachial plexus injuries in overgrown fetuses is another important issue. The American College of Obstetricians and Gynecologists (2017a) has concluded that data are insuicient to determine whether cesarean delivery in women with gestational diabetes whose fetuses have a sonographically estimated weight �4500 g should be performed to avoid risk of birth trauma. From their systematic review, Garabedian and coworkers (2010) estimated that as many as 588 cesarean deliveries in women with gestational diabetes and an estimated fetal weight of �4500 g would be necessary to avoid one case of permanent brachial plexus palsy. Scifres and colleagues (2015), in their retrospective analysis of 903 women with gestational diabetes who underwent so nographic evaluation within 1 month of delivery, demonstrated that sonographic estimates of fetal weight typically overdiagnosed fetuses as being LGA. Only 22 percent of women estimated to have an LGA fetus actually

1	within 1 month of delivery, demonstrated that sonographic estimates of fetal weight typically overdiagnosed fetuses as being LGA. Only 22 percent of women estimated to have an LGA fetus actually delivered an overgrown newborn. Still, the American College of Obstetricians and Gynecologists (2016a) acknowledges that prophylactic cesarean delivery may be considered in diabetic women with an estimated fetal weight �4500 g.

1	Recommendations for postpartum evaluation are based on the 50-percent likelihood of women with gestational diabetes developing overt diabetes within 20 years (O'Sullivan, 1982). The Fifth International Workshop Conference on Gestational Diabetes recommended that women diagnosed with gestational diabetes undergo postpartum evaluation with a 75-g OGTT (Metzger, 2007). These recommendations are shown in Table 57-14 along with the classification scheme of the ADA (20 17b). Eggleston and colleagues (2016) reviewed insurance claim data from 2000 to 2013 and found that only 24 percent of women with a pregnancy complicated by gestational diabetes underwent postpartum screening within a year, and less than half of those underwent a 75-g OGTT. he American College TABLE 57-14. Fifth International Workshop-Conference: Metabolic Assessments Recommended after Pregnancy with Fasting or random plasma glucose 75-g, 2-hr OGI 75-g, 2-hr OGTI 75-g, 2-hr OGTI 75-g, 2-hr OGTI

1	Fasting or random plasma glucose 75-g, 2-hr OGI 75-g, 2-hr OGTI 75-g, 2-hr OGTI 75-g, 2-hr OGTI Classification of the American Diabetes Association (2013) 2 hr <140 mg/dL 2 hr �140-199 mg/dL Hemoglobin A1c<5.7% 5.7-6.4% OGTI = oral glucose tolerance test. Detect persistent, overt diabetes Postpartum classification of glucose metabolism Assess glucose metabolism 2 hr �200 mg/dL �6.5% Data from American Diabetes Association, 2013, 2017a; Metzger, 2007. of Obstetricians and Gynecologists (2017a) recommends either a fasting glucose or the 75-g, 2-hour OGTT at 4 to 12 weeks postpartum for the diagnosis of overt diabetes. he ADA (2017a) recommends testing at least every 3 years in women with a history of gestational diabetes but normal postpartum glucose screening.

1	Women with a history of gestational diabetes are also at risk for cardiovascular complications associated with dyslipidemia, hypertension, and abdominal obesity-the metabolic syndrome (Chap. 48, p. 937). In a study of 47,909 parous women, Kessous and coworkers (2013) evaluated subsequent hospitalizations due to cardiovascular morbidity. hey found that almost 5000 women with gestational diabetes were 2.6 times more likely to be hospitalized for cardiovascular morbidity. Another study evaluated 483 women between 5 and 10 years ater being diagnosed with mild gestational diabetes (Varner, 2017). Investigators found no increased risk for developing metabolic syndrome associated with additional pregnancies. However, risk for subsequent diabetes rose almost fourfold if gestational diabetes complicated at least one subsequent pregnancy.

1	In a metaanalysis of published reports from 1973 through 2014, the pooled gestational diabetes recurrence rate was 48 percent (Schwartz, 2015). Rates in primiparas were lower (40 percent) than in multiparas (73 percent). The same group of investigators identified maternal BMI, insulin use, fetal macrosomia, and weight gain between pregnancies as additional risk factors for gestational diabetes recurrence (Schwartz, 2016). Thus, lifestyle behavioral changes that include weight control and exercise between pregnancies would seem likely to prevent gestational diabetes recurrence. Gueli and colleagues (2016) were unable to demonstrate a lower recurrence rate in women randomized to an exercise program that started before 14 weeks' gestation in a subsequent pregnancy. Conversely, Ehrlich and colleagues (2011) found that prepregnancy loss of at least two BMI units was associated with a lower subsequent risk of gestational diabetes in women who were overweight or obese in the irst pregnancy.

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1	Pociot F, Lernmark A: Genetic risk factors for type 1 diabetes. Lancet 387(10035):2331,t2016 Powers AC: Diabetes mellitus. In: Longo DL, Fauci AS, Kaspar DL, et al (eds): Harrison's Principles of Internal Medicine, 18th ed. McGraw-Hili, New York,t2012 Rasmussen L, Laugesen CS, Ringholm L, et al: Progression of diabetic retinopathy during pregnancy in women with type 2 diabetes. Diabetologia 53:1076,t2010 Reece A: Diabetes-induced birth defects: what do we know? What can we do? CUff Diab Rep 12:24,t2012 Renault KM, Carlsen EM, N0gaard K, et al: Intake of carbohydrates during pregnancy in obese women is associated with fat mass in the newborn ofspring. Am] Clin Nutr 102(6):1475, 2015 Reutens AT: Epidemiology of diabetic kidney disease. Med Clin North Am 97:1,t2013 Rewers M, Ludvigsson ]: Environmental risk factors for type 1 diabetes. Lancet 387(10035):2340,t2016

1	Reutens AT: Epidemiology of diabetic kidney disease. Med Clin North Am 97:1,t2013 Rewers M, Ludvigsson ]: Environmental risk factors for type 1 diabetes. Lancet 387(10035):2340,t2016 Ringholm L, Vestgaard M, Laugesen CS, et al: Pregnancy-induced increase in circulating IGF-1 is associated with progression of diabetic retinopathy in women with type 1 diabetes. Growth Horm IGF Res 21:25, 2011 Roeder A, Moore TR, Ramos GA: Insulin pump dosing across gestation in women with well-controlled type 1 diabetes mellitus. Amt] Obstet Gynecol 207:324.e1,t2012 Rolo LC, Nardozza LMM, Junior EA, et al: Reference curve of the fetal ventricular septum area by the STIC method: preliminary study. Arq Bras Cardiol 96(5):386, 201t1 Rosenn B, Miodovnik M, Combs CA, et al: Glycemic thresholds for spontaneous abortion and congenital malformations in insulin-dependent diabetes mellitus. Obstet Gynecol 84:515, 1994

1	Rosenn B, Miodovnik M, Combs CA, et al: Glycemic thresholds for spontaneous abortion and congenital malformations in insulin-dependent diabetes mellitus. Obstet Gynecol 84:515, 1994 Rowan ]A, Hague WM, Wanzhen G, et al: Metformin versus insulin for the treatment of gestational diabetes. N Engl] Med 358:2003, 2008 Rowan ]A, Rush EC, Obolonkin V, et al: Metformin in gestational diabetes: the ofspring follow-up (MiG TOFU): body composition at 2 years of age. Diabetes Care 34(10):2279,t2011 Russell NE, Foley M, Kinsley BT, et al: Efect of pregestational diabetes mellitus on fetal cardiac function and structure. Am] Obstet Gynecol 199:312. e1,t2008 Russo LM, Nobles e, Ertel A, et al: Physical activity interventions in pregnancy and risk of gestational diabetes mellitus: a systematic review and metaanalysis. Obstet Gynecol 125(3):576,t2015

1	Russo LM, Nobles e, Ertel A, et al: Physical activity interventions in pregnancy and risk of gestational diabetes mellitus: a systematic review and metaanalysis. Obstet Gynecol 125(3):576,t2015 Salvesen DR, Brudenell M], Nicolaides KH: Fetal polycythemia and thrombocytopenia in pregnancies complicated by maternal diabetes mellitus. Am ] Obstet Gynecol 166:1287, 1992 Salvesen DR, Brudenell M], Snijders ]M, et al: Fetal plasma erythropoietin in pregnancies complicated by maternal diabetes mellitus. Amt] Obstet Gynecol 168:88, 1993 Sanabria-Martinez G, Garda-Hermoso A, Poyatos-Le6n R, et al: Efectiveness of physical activity interventions on preventing gestational diabetes mellitus and excessive maternal weight gain: a meta-analysis. B]OG 122(9):1t167, 2015 Saudek CD: Progress and promise of diabetes research. ]AMA 287:2582, 2002 Schwartz N, Nachum Z, Green MS: Risk factors of gestational diabetes mellitus recurrence: a meta-analysis. Endocrine 53(3):662, 2016

1	Schwartz N, Nachum Z, Green MS: Risk factors of gestational diabetes mellitus recurrence: a meta-analysis. Endocrine 53(3):662, 2016 Schwartz N, Nachum Z, Green MS: The prevalence of gestational diabetes mellitus recurrence-efect of ethnicity and parity: a metaanalysis. Am ] Obstet GynecoIt213(3):31O, 2015 Scifres CM, Feghali M, Dumont T, et al: Large-for-gestational-age ultrasound diagnosis and risk for cesarean delivery in women with gestational diabetes mellitus. Obstet Gynecol 126(5):978, 2015 Sheield ]S, Buder-Koster EL, Casey BM, et al: Maternal diabetes mellitus and infant malformations. Obstet Gynecol 100:925,t2002 Sheiner E, Mazor-Drey E, Levy A: Asymptomatic bacteriuria during pregnancy. ] Matern Fetal Neonatal Med 22(5):423, 2009 Sibai BM, Caritis S, Hauth], et al: Risks of preeclampsia and adverse neonatal outcomes among women with pregestational diabetes mellitus. Amt] Obstet Gynecol 182:364, 2000

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1	U.S. Preventive Services Task Force: Gestational diabetes mellitus, screening. 2014. Available at: https:llwww.uspreventiveservicestaskforce.org/Page/ Documen t/U pdateS ummaryF inall gestational-diabetes-melli tus-screeni ng. Accessed November 11, 2017 Van Leeuwen M, Louwerse MD, Opmeer Be, et al: Glucose challenge test for detecting gestational diabetes mellitus: a systematic review. B]OG 119(4):393,t2012 Varner MW, Rice MM, Landon MB, et al: Pregnancies after the diagnosis of mild gestational diabetes mellitus and risk of cardiometabolic disorders. Obstet GynecoIt129(2):273, 2017 Vestgaard M, Ringholm L, Laugesen CS, et al: Pregnancy-induced sightthreatening diabetic retinopathy in women with type 1 diabetes. Diabet Med 27:431, 2010 Vidaef AC, Yeomans ER, Ramin SM: Pregnancy in women with renal disease. Part II: speciic underlying renal conditions. Amt] Perinatol 25:399, 2008

1	Vidaef AC, Yeomans ER, Ramin SM: Pregnancy in women with renal disease. Part II: speciic underlying renal conditions. Amt] Perinatol 25:399, 2008 Vink ]Y, Poggi SH, Ghidini A: Amniotic fluid index and birth weight: is there a relationship in diabetics with poor glycemic control? Amt] Obstet Gynecol 195:848,t2006 Wang PH, Lau], Chalmers TC: Meta-analysis of efects of intensive blood-glucose control on late complications of type 1 diabetes. Lancet 341:1306,t1993 Wei ], Heng W, Gao ]: Efects of low glycemic index diets on gestational diabetes mellitus: a meta-analysis of randomized controlled clinical trials. Medicine (Baltimore) 95(22):e3792, 2016 White P: Classiication of obstetric diabetes. Am] Obstet Gynecol 130:228, 8 World Health Organization: Diagnostic criteria and classification of hyperglycemia irst detected in pregnancy. Geneva, WHO, 2013 Yang ], Cummings EA, O'Connell C, et al: Fetal and neonatal outcomes of diabetic pregnancies. Obstet Gynecol 108:644,t2006

1	Yang ], Cummings EA, O'Connell C, et al: Fetal and neonatal outcomes of diabetic pregnancies. Obstet Gynecol 108:644,t2006 Yang P, Reece EA, Wang F, et al: Decoding the oxidative stress hypothesis in diabetic embryopathy through proapoprotic kinase signaling. Am ] Obstet Gynecol 212(5):569, 2015 Yanit KE, Snowden ]M, Cheng W, et al: he impact of chronic hypertension and pregestational diabetes on pregnancy outcomes. Am ] Obstet Gynecol 207:333,t2012 Young EC, Pires M, Marques L, et al: Efects of pregnancy on the onset and progression of diabetic nephropathy and of diabetic nephropathy on pregnancy outcomes. Diabetes Metab Syndr 5: 13 7, 2012 THYROID PHYSIOLOGY AND PREGNANCY .i......i... 1118 HYPERTHYROIDISM. . . . . . . . . . . . . . . . . . . . . . . . . . .. 1120 HYPOTHYROIDISM . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1123 SUBCLINICAL HYPOTHYROIDISM. . . . . . . . . . . . . . . .. 1124 IODINE DEFICIENCY ...i..............i.........i..... 1126

1	SUBCLINICAL HYPOTHYROIDISM. . . . . . . . . . . . . . . .. 1124 IODINE DEFICIENCY ...i..............i.........i..... 1126 CONGENITAL HYPOTHYROIDISM ..i....i.....i....i..... 1127 POSTPARTUM THYROIDITIS.. . . . . . . . . . . . . . . . . . . .. 1127 NODULAR THYROID DISEASE .....i...i....i.......i.....i1128 PARATHYROID DISEASE .....i.......i.......i......i.... 1128 ADRENAL GLAND DISORDERS.. . . . . . . . . . . . . . . . . .. 1130 PITUITARY DISORDERS .i....i.........i....i.....i....i... 1132 In a smal number of cases the thyroid gland increases markedy in size, though we are ignorant as to its signicance. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) In 1903, little was known of many endocrine disorders. Still, endocrinopathies seem particularly closely related to pregnancy because of its gestational proclivity for prodigious hormone secretion. This is best illustrated by placental lactogen in diabetes, the most common endocrinopathy encountered in pregnancy (Chap. 57, p. 1097). Pregnancy is also interrelated with some endocrinopathies that are at least partially due to autoimmune dysregulation. Clinical manifestations of this result from complex interplay among genetic, environmental, and endogenous factors that activate the immune system against targeted cells within endocrine organs. An extraordinary example of these interactions comes from studies that implicate maternal organ engraftment by fetal cells that were transferred during pregnancy. These cells later provoke antibody production, tissue destruction, and autoimmune endocrinopathies.

1	Taken in aggregate, disorders of the thyroid gland are common in young women and thus frequently encountered in pregnancy. Maternal and fetal thyroid function are intimately related, and drugs that afect the maternal thyroid also afect the fetal gland. Moreover, thyroid autoantibodies have been associated with increased rates of early pregnancy wastage. Also, uncontrolled thyrotoxicosis and untreated hypothyroidism are both associated with adverse pregnancy outcomes. Finally, evidence suggests that the severity of some autoimmune thyroid disorders may be ameliorated during pregnancy, only to be exacerbated postpartum .

1	Maternal thyroid changes are substantial, and normally altered gland structure and function are sometimes confused with thyroid abnormalities. These alterations are discussed in detail in Chapter 4 (p. 69), and normal serum hormone level values are found in the Appendix (p. 1258). First, maternal serum concentrations of thyroid binding globulin are increased concomitantly with total or bound thyroid hormone levels (Fig. 4-16, p. 70). Second, thyrotropin, also called thyroidstimulating hormone (TSH) , currently plays a central role in screening and diagnosis of many thyroid disorders. Notably, TSH receptors are cross stimulated, albeit weakly, by massive quantities of human chorionic gonadotropin (hCG) secreted 6 2.0 1.5 1 :J0l� Se3 1.0 �2 : 0.5 0.0 o 10 20 30 40

1	Se3 1.0 �2 : 0.5 0.0 o 10 20 30 40 FIGURE 58-1 Gestational age specific values for serum thyroid stimulating hormone (TSH) levels (black lines) and free thyroxine (T4) levels (blue lines). Data were derived from 17,298 women tested during pregnancy. For each color, the dark solid lines represent the 50th percentile, whereas the upper and lower light lines represent the 25th and 975th percentiles, respectively. (Data from Casey, 2005; Dashe, 2005.) by placental trophoblast. Because TSH does not cross the placenta, it has no direct fetal efects. During the irst 12 weeks of gestation, when maternal hCG serum levels are maximal, thyroid hormone secretion is stimulated. he resulting greater serum free thyroxine (T4) levels act to suppress hypothalamic thyrotropin-releasing hormone (TRH) and in turn limit pituitary TSH secretion (Fig.

1	Accordingly, TRH is undetectable in maternal serum. Conversely, in fetal serum, beginning at midpregnancy, TRH becomes detectable, but levels are static and do not increase. hroughout pregnancy, maternal thyroxine is transferred to the fetus (American College of Obstetricians and Gynecologists, 2017). Maternal thyroxine is important for normal fetal brain development, especially before the onset of fetal thyroid gland function (Bernal, 2007; Korevaar, 2016). And, even though the fetal gland begins concentrating iodine and synthesizing thyroid hormone after 12 weeks' gestation, maternal thyroxine contribution remains important. In fact, maternal sources account for 30 percent of thyroxine in fetal serum at term (horpe-Beeston, 1991). Still, developmental risks associated with maternal hypothyroidism after midpregnancy remain poorly understood (Morreale de Escobar, 2004; Sarkhail, 2016).

1	Most thyroid disorders are inextricably linked to autoantibodies against nearly 200 thyrocyte components. These antibodies variably stimulate thyroid function, block function, or cause thyroid inlammation that may lead to follicular cell destruction. Often, these efects overlap or even coexist. Thyroid-stimulating autoantibodies, also called thyroidstimulating immunoglobulins (TSls), bind to the TSH receptor and activate it, causing thyroid hyperfunction and growth. Although these antibodies are identiied in most patients with classic Graves disease, simultaneous production of thyroidstimulating blocking antibodies may blunt this efect Qameson, ;C ..EJ,50 w)0:J::0 w ...•30 ..•5 -..,20 FIGURE 58-2 Incidence in percent of antithyroid peroxidase anti bodies in 16,407 women who are normal or euthyroid, in 233 with isolated maternal hypothyroxinemia (1M H), in 598 with subclinical hypothyroidism (SCH), and in 134 with overt hypothyroidism. (Data from Casey, 2007).

1	(Data from Casey, 2007). 2015). Thyroidperoxidase (TPO) is a thyroid gland enzyme that normally functions in the production of thyroid hormones. Thyroid peroxidase antibodies, previously called thyroid microsomal autoantibodies, are directed against TPO and, as shown in Figure 58-2, have been identiied in 5 to 15 percent of all pregnant women (Abbassi-Ghanavati, 2010; Sarkhail, 2016). These antibodies have been associated in some studies with early pregnancy loss and preterm birth (Negro, 2006; Korevaar, 2013; Plowden, 2017; Thangaratinam, 201l). In another study with more than 1000 TPO antibody-positive pregnant women, the risk for preterm birth was not elevated, however, the risk for placental abruption was greater (Abbassi-Ghanavati, 2010). These women are also at high risk for postpartum thyroid dysfunction and at lifelong risk for permanent thyroid failure (Andersen, 2016; Jameson, 2015).

1	Autoimmune thyroid disease is much more common in women than in men. One intriguing explanation for this disparity is fetal-to-maternal cell traicking (Greer, 201l). Fetal cells are known to enter maternal circulation during pregnancy. When fetal lymphocytes enter maternal circulation, they can live for more than 20 years. Stem cell interchange can lead to engraftment in several maternal tissues and is termed etal microchimerism. In some cases, this may involve the thyroid gland (Bianchi, 2003; Boddy, 2015; Khosrotehrani, 2004). A high prevalence of Y-chromosome-positive cells has been identiied using luorescence in situ hybridization (FISH) in the thyroid glands of women with Hashimoto thyroiditis-60 percent, or with Graves disease-40 percent (Renne, 2004). In another study of women giving birth to a male fetus, Lepez and colleagues (201l) identiied signiicantly more circulating male mononuclear cells in those with Hashimoto thyroiditis. Ironically, such micro chimerism may have a

1	birth to a male fetus, Lepez and colleagues (201l) identiied signiicantly more circulating male mononuclear cells in those with Hashimoto thyroiditis. Ironically, such micro chimerism may have a protective role for autoimmune thyroid disorders (Cirello, 2015).

1	TABLE 58-1. Incidence of Overt Hyperthyroidism in Pregnancy Vaidya (2007)a United Kingdom 0.7% Lazarus (2007)b United Kingdom 1.7% Casey (2006r United States 0.4% Andersen (201n6r,d Denmark 0.4-0.7% aScreened irl the first trimester. bScreened at 9-15 weeks. (Screened before 20 weeks. dDiagnosed in early versus later pregnancy. The incidence of thyrotoxicosis or hyperthyroidism in pregnancy is varied and complicates between 2 and 17 per 1000 births when gestational-age appropriate TSH threshold values are used (Table

1	The incidence of thyrotoxicosis or hyperthyroidism in pregnancy is varied and complicates between 2 and 17 per 1000 births when gestational-age appropriate TSH threshold values are used (Table Because normal pregnancy simulates some clinical findings similar to thyroxine excess, clinically mild thyrotoxicosis may be diicult to diagnose. Suggestive indings include tachycardia that exceeds that usually seen with normal pregnancy, thyromegaly, exophthalmos, and failure to gain weight despite adequate food intake. Laboratory testing is confirmatory. TSH levels are markedly depressed, while serum free T4 (IT4) levels are elevated Qameson, 2015). Rarely, hyperthyroidism is caused by abnormally high serum triiodothyronine (T 3) levels-so-called Trtoxicosis.

1	he overwhelming cause of thyrotoxicosis in pregnancy is Graves disease, an organ-speciic autoimmune process associated with thyroid-stimulating TSH-receptor antibodies as previously discussed'(De Leo, 2016). Because these antibodies are specific to Graves hyperthyroidism, such assays have been proposed for diagnosis, management, and prognosis in pregnancies complicated by hyperthyroidism (Barbesino, 2013). At Parkland Hospital, these receptor antibody assays are generally reserved for cases in which fetal thyrotoxicosis is suspected. With Graves disease, during the course of pregnancy, hyperthyroid symptoms may initially worsen because ofhCG stimulation but then subsequently diminish with drops in receptor antibody titers in the second half of pregnancy (Mestman, 2012; Sarkhail, 2016). Amino and coworkers (2003) have found that levels of blocking antibodies also decline during pregnancy.

1	Treatment. Thyrotoxicosis during pregnancy can nearly always be controlled by thionamide drugs. Propylthiouracil (PTU) has been historically preferred because it partially inhibits the conversion of T4 to T3 and crosses the placenta less readily than methimazole. he latter has also been associated with a rare methimazole embryopathy, characterized by esophageal or choanal atresia as well as aplasia cutis, a congenital skin defect. yoshihara and associates (2012, 2015) analyzed outcomes in Japanese women with irst-trimester hyperthyroidism and found a twofold increased risk of major fetal malformations in pregnancies exposed to methimazole compared with either PTU or potassium iodide. Speciically, seven of nine cases with aplasia cutis and the only case of esophageal atresia were in the group of methimazole-exposed fetuses. here have also been reports of a PTU-associated embryopathy (Andersen, 2014).

1	In 2009, the Food and Drug Administration issued a safety alert on PTU-associated hepatotoxicity. his warning prompted the American Thyroid Association and the American Association of Clinical Endocrinologists (201r1) to recommend PTU therapy during the first trimester followed by methimazole beginning in the second trimester. he obvious disadvantage is that this might lead to poorly controlled thyroid function. Accordingly, at Parkland Hospital, we continue to prescribe PTU treatment throughout pregnancy. Transient leukopenia can be documented in up to 10 percent of women taing antithyroid drugs, but this does not require therapy cessation (American College of Obstetricians and Gynecologists, 2017). In approximately 0.3 percent, however, agranuloytosis develops suddenly and mandates drug discontinuance (Thomas, 2013). It is not dose related, and because of its acute onset, serial leukocyte counts during therapy are not helpul.

1	Thus, fever or sore throat develops, women are instructed to discontinue medication immediatey and report or a complete blood count. herapy may have other side efects. First, as noted, hepatotoxicity is a possibility and develops in approximately 0.1 percent of treated women. Serial measurement of hepatic enzyme levels does not prevent fulminant PTU-related hepatotoxicity. Second, approximately 20 percent of patients treated with PTU develop antineutrophil cytoplasmic antibodies NC). Despite this, only a small percentage of these subsequently develops serious vasculitis (Kimura, 2013). Finally, although thionamides have the potential to cause fetal complications, these are uncommon. In some cases, thionamides may even be therapeutic for the fetus, because TSH-receptor antibodies cross the placenta and can stimulate the fetal thyroid gland to cause thyrotoxicosis and goiter.

1	The initial thionamide dose is empirical. For nonpregnant patients, the American hyroid Association recommends that methimazole be used at an initial higher daily dose of 10 to 20 mg orally followed by a lower maintenance dose of 5 to 10 mg. If PTU is selected, a dose of 50 to 150 mg orally three times daily may be initiated depending on clinical severity (Bahn, 2011). At Parkland Hospital, we usually initially give 300 or 450 mg of PTU daily in three divided doses for pregnant women. Occasionally, daily doses of 600 mg or higher are necessary. As discussed, we generally do not transition women to methimazole during the second trimester. he goal is treatment with the lowest possible thionamide dose to maintain thyroid hormone levels slightly above or in the high normal range, while TSH levels remains suppressed (Bahn, 2011). Serum free T 4 concentrations are measured every 4 to 6 weeks.

1	Subtotal thyroidectomy can be performed after thyrotoxicosis is medically controlled. This seldom is done during pregnancy but may be appropriate for the very few women who cannot adhere to medical treatment or in whom drug therapy proves toxic (Stagnaro-Green, 2012a). Surgery is best accomplished in the second trimester. Potential drawbacks of thyroidectomy include inadvertent resection of parathyroid glands and injury to the recurrent laryngeal nerve.

1	Thyroid ablation with therapeutic radioactive iodine is contraindicated during pregnancy. The necessary doses may also cause fetal thyroid gland destruction. Thus, when radioactive iodine is given unintentionally, many clinicians recommend abortion. Any exposed fetus must be carefully evaluated, and the incidence of fetal hypothyroidism depends on gestational age and radioiodine dose (Berlin, 2001). There is no evidence that radioiodine given before pregnancy causes fetal anomalies if enough time has passed to allow radiation efects to dissipate and if the woman is euthyroid (Ayala, 1998). The International Commission on Radiological Protection has recommended that women avoid pregnancy for 6 months after radioablative therapy (Brent, 2008). Moreover, during lactation, the breast also concentrates a substantial amount of iodine. This may pose neonatal risk due to I-containing milk ingestion and maternal risk from significant breast irradiation. To limit the latter, a delay of 3 months

1	a substantial amount of iodine. This may pose neonatal risk due to I-containing milk ingestion and maternal risk from significant breast irradiation. To limit the latter, a delay of 3 months after breastfeeding cessation will more reliably ensure complete breast involution.

1	Pregnancy Outcome. Women with thyrotoxicosis have pregnancy outcomes that largely depend on whether metabolic control is achieved. For example, excess thyroxine may cause miscarriage or preterm birth (Andersen, 2014; Sheehan, 2015). In untreated women or in those who remain hyperthyroid despite therapy, incidences of preeclampsia, heart failure, and adverse perinatal outcomes are higher (Table 58-2). A prospective cohort study from China showed that women with clinical hyperthyroidism had a 12-fold greater risk of delivering an infant with hearing loss (Su, 2011). In most cases, the perinate is euthyroid. In some, however, hyper-or hypothyroidism can develop with or without a goiter (Fig. 58-3). Clinical hyperthyroidism develops in up to 1 percent of neonates born to women with Graves disease (Barbesino, 2013; Fitzpatrick, 2010). If fetal thyroid disease is

1	TABLE 58-2. Pregnancy Outcomes in Women with Overt Thyrotoxicosis aData presented as n (%). Data from Davis, 1989; Kriplani, 1994; Luewan, 201n1; Medici, 2014; Millar, 1994. FIGURE 58-3 Term hypothyroid neonate delivered of a woman with a 3-year history of thyrotoxicosis that recurred at 26 weeks' gestation. The mother was given methimazole 30 mg orally daily and was euthyroid at delivery. suspected, nomograms are available for sonographically mea sured thyroid volume (Gietka-Czernel, 2012).

1	The fetus or neonate who was exposed to excessive maternal thyroxine may have any of several clinical presentations. First, goitrous thyrotoxicosis is caused by placental transfer of thyroidstimulating immunoglobulins. Nonimmune hydrops and fetal demise have been reported with fetal thyrotoxicosis (Nachum, 2003; Stulberg, 2000). The best predictor of perinatal thyrotoxicosis is presence of thyroid-stimulating TSH-receptor antibodies in women with Graves disease (Nathan, 2014). This is especially true if their levels are more than threefold higher than the upper normal limit (Barbesino, 2013). In a study of 72 pregnant women with Graves disease, Luton and associates (2005) reported that none of the fetuses in 31 low-risk mothers had a goiter, and all were euthyroid at delivery. Low risk was defined as no requirement for antithyroid medications during the third trimester or an absence of antithyroid antibodies. Conversely, in a group of 41 women who either were taking antithyroid

1	was defined as no requirement for antithyroid medications during the third trimester or an absence of antithyroid antibodies. Conversely, in a group of 41 women who either were taking antithyroid medication at delivery or had thyroid receptor antibodies, 11 fetuses-27 percent-had sonographic evidence of a goiter at 32 weeks' gestation. Seven of these 11 fetuses were determined to be hypothyroid, and the remaining fetuses were hyperthyroid. In response to these results, the merican Thyroid Association and American Association of Clinical Endocrinologists (201r1) recommend routine evaluation of TSH-receptor antibodies between 22 and 26 weeks' gestation in women with Graves disease. he American College of Obstetricians and Gynecologists (2017), however, does not recommend such testing. If the fetus is thyrotoxic, maternal thionamide drugs are adjusted even though maternal thyroid function may be within the targeted range (Mestman, 2012). lthough usually short-lived, neonatal

1	If the fetus is thyrotoxic, maternal thionamide drugs are adjusted even though maternal thyroid function may be within the targeted range (Mestman, 2012). lthough usually short-lived, neonatal thyrotoxicosis may require short-course antithyroid drug treatment (Levy-Shraga, 2014; Nathan, 2014).

1	A second presentation is goitrous hypothyroidism caused by fetal exposure to maternally administered thionamides (see Fig. 58-3). Although there are theoretical neurological implications, reports of adverse fetal efects seem to have been exaggerated. Available data indicate that thionamides carry an extremely small risk for causing neonatal hypothyroidism (Momotani, 1997; O'Doherty, 1999). For example, in at least 239 treated thyrotoxic women shown in Table 58-1, evidence of hypothyroidism was found in only four newborns. Furthermore, at least four long-term studies report no abnormal intellectual and physical development of these children (Mestman, 1998). If maternal hypothyroidism developed, the fetus can be treated by a reduced maternal antithyroid medication dose and injections of intraamnionic thyroxine if necessary.

1	A third presentation, nongoitrous hypothyroidism, may develop from transplacental passage of maternal TSH-receptor blocking antibodies (Fitzpatrick, 2010; Gallagher, 2001). And finally,etal thyrotoxicosis after maternal thyroid gland ablation, usually with 1 radioiodine, may result from transplacental thyroid-stimulating antibodies. In one report of early fetal exposure to radioiodine, neonatal thyroid studies indicated transient hyperthyroidism from maternal transfer of stimulating antibodies (Tran, 2010).

1	Fetal Diagnosis. Evaluation of fetal thyroid function is somewhat controversial. Although the fetal thyroid volume can be measured sonographically in women taking thionamide drugs or in those with thyroid-stimulating antibodies, most investigators do not currently recommend this routinely (Cohen, 2003; Luton, 2005). Kilpatrick (2003) recommends umbilical cord blood sampling and fetal antibody testing only if the mother has previously undergone radioiodine ablation. Because fetal hyper-or hypothyroidism may cause hydrops, growth restriction, goiter, or tachycardia, fetal blood sampling may be appropriate if these are identified (Brand, 2005). he Endocrine Society clinical practice guidelines recommend umbilical cord blood sampling only when the diagnosis of fetal thyroid disease cannot be reasonably ascertained based on clinical and sonographic data (Garber, 2012). Diagnosis and treatment are considered further in Chapter 16 (p. 318).

1	Both of these are acute and life-threatening in pregnancy. hyroid storm is a hypermetabolic state and is rare in pregnancy. In contrast, pulmonary hypertension and heart failure from cardiomyopathy caused by the profound myocardial efects of thyroxine are common in pregnant women (Sheield, 2004). As shown in Table 58-2, heart failure developed in 8 percent of 90 women with uncontrolled thyrotoxicosis. In these women, cardiomyopathy is characterized by a high-output state, which may lead to a dilated cardiomyopathy (Fadel, 2000; Klein, 1998). The pregnant woman with thyrotoxicosis has minimal cardiac reserve, and decompensation is usually precipitated by preeclampsia, anemia, sepsis, or a combination of these. Fortunately, thyroxine-induced cardiomyopathy and pulmonary hypertension are frequently reversible (Sheield, 2004; Siu, 2007; Vydt, 2006).

1	Management. Treatment is similar for thyroid storm and heart failure and should be carried out in an intensive care area that may include special-care units within labor and delivery (American College of Obstetricians and Gynecologists, 2017). Shown in Figure 58-4 is our stepwise approach to medical management of thyroid storm or thyrotoxic heart failure. An hour or two ater initial thionamide administration, iodide is given to inhibit thyroidal release ofT3 and T4. It can be given intravenously as sodium iodide or orally as either saturated solution of potassium iodide (SSKI) or Lugol solution. With a history of iodine-induced anaphylaxis, lithium carbonate, 300 mg every 6 hours, is given instead. Most authorities recommend dexamethasone, 2 mg intravenously every 6 hours for four doses, to further block peripheral conversion ofT4 to T3. If a 3-blocker drug is given to control tachycardia, its efect on heart failure must be considered. Propranolol, labetalol, and esmolol have all been

1	further block peripheral conversion ofT4 to T3. If a 3-blocker drug is given to control tachycardia, its efect on heart failure must be considered. Propranolol, labetalol, and esmolol have all been used successfully. Coexisting severe preeclampsia, infection, or anemia should be aggressively managed before delivery is considered.

1	Transient biochemical features of hyperthyroidism may be observed in 2 to 15 percent of women in early pregnancy (Fitzpatrick, 2010). Many women with hyperemesis gravidarum have abnormally high serum thyroxine levels and low TSH levels (Chap. 54, p. 1045). his results from TSH-receptor stimulation from massive-but normal for pregnancyconcentrations of hCG. This transient condition is also termed gestational transient thyrotoxicosis. Even if associated with hyperemesis, antithyroid drugs are not warranted (American College of Obstetricians and Gynecologists, 2017). The degree of hCG level elevation does not correlate with thyroxine and TSH values, which become more normal by midpregnancy (Nathan, 2014; Yoshihara, 2015).

1	he prevalence of increased thyroxine levels in women with a molar pregnancy ranges between 25 and 65 percent (Hershman, 2004). As discussed, abnormally high hCG levels lead to overstimulation of the TSH receptor. Because these tumors are now usually diagnosed early, clinically apparent hyperthyroidism has become less common. With molar evacuation, serum free T4 levels usually normalize rapidly in parallel with declining hCG concentrations. This is discussed further in Chapter 20 (p. 391).

1	hird-generation TSH assays with an analytical sensitivity of 0.002 mU/mL permit identiication of subclinical thyroid disorders. These biochemically defined extremes usually represent normal biological variations but may herald the earliest stages of thyroid dysfunction. Subclinical hyperthyroidism is characterized by an abnormally low serum TSH concentration in concert with normal thyroxine hormone levels (Surks, 2004). Long-term efects of persistent subclinical thyrotoxicosis include osteoporosis, cardiovascular morbidity, and progression After 1-2 hours of thionamide therapy start iodine: if iodine anaphylaxis history, Lithium carbonate 300 mg PO every 6 hr Consider corticosteroid therapy for 24 hours: FIGURE 58-4 One management method for thyroid storm or thyrotoxic heart failure. gtt = drops; IV = intravenous; NGT = nasogastric tube; PO = orally; PTU = propylthiouracil.

1	FIGURE 58-4 One management method for thyroid storm or thyrotoxic heart failure. gtt = drops; IV = intravenous; NGT = nasogastric tube; PO = orally; PTU = propylthiouracil. to overt thyrotoxicosis or thyroid failure. Casey and Leveno (2006) reported that subclinical hyperthyroidism was found in 1.7 percent of pregnant women. Importantly, subclinical hyperthyroidism was not associated with adverse pregnancy outcomes. In separate retrospective analyses of almost 25,000 women who underwent thyroid screening throughout pregnancy, Wilson and colleagues (2012) and Tudela and coworkers (2012) also found no relationship between subclinical hyperthyroidism and preeclampsia or gestational diabetes. Treatment of subclinical hyperthyroidism is unwarranted in pregnancy because antithyroid drugs may afect the fetus. hese women may beneit from periodic surveillance, and approximately half eventually have normal TSH concentrations.

1	Overt or symptomatic hypothyroidism, as shown in Table 58-3, more likely in women in areas of endemic iodine deiciency or those with Hashimoto thyroiditis. Other indings include edema, dry skin, hair loss, and prolonged relaxation phase of deep tendon relexes. Clinical or overt hypothyroidism is conirmed when an abnormally high serum TSH level is accompanied by an abnormally low thyroxine level. Subclinical TABLE 58-3. Frequency of Overt Hypothyroidism Wang (201n1)a China 0.3% Cleary-Goldman (2008)a United States 0.3% Vaidya (2007)d United Kingdom 1.0% Casey (200S)b United States 0.2% Andersen (2016)d Denmark 1.2% has been reported to complicate between 2 and 12 per 1000 aScreened during first trimester. pregnancies. It is characterized by insidious nonspeciic clini bScreened before 20 weeks. cal indings that include fatigue, constipation, cold intolerance, (Includes those treated before pregnancy.

1	pregnancies. It is characterized by insidious nonspeciic clini bScreened before 20 weeks. cal indings that include fatigue, constipation, cold intolerance, (Includes those treated before pregnancy. muscle cramps, and weight gain. A pathologically enlarged thy dDiagnosed in early versus later pregnancy. roid gland depends on the etiology of hypothyroidism and is hypothyroidism, discussed later, is defined by an elevated serum TSH level and normal serum thyroxine concentration Qameson, 2015). Sometimes included in the spectrum of subclinical thyroid disease are asymptomatic individuals with high levels of anti-TPO or anti thyroglobulin antibodies. Autoimmune euthyroid disease represents a new investigative frontier in screening and treatment of thyroid dysfunction during pregnancy.

1	he most common cause of hypothyroidism in pregnancy is Hashimoto thyroiditis, characterized by glandular destruction from autoantibodies, particularly anti-TPO antibodies. Another cause is postablative Graves disease. Clinical identiication of hypothyroidism is especially diicult during pregnancy because many of the signs or symptoms are also common to pregnancy itself. Thyroid analyte testing should be performed on symptomatic women or those with a history of thyroid disease (American College of Obstetricians and Gynecologists, 2017). Severe hypothyroidism during pregnancy is uncommon, probably because it is often associated with infertility and higher spontaneous abortion rates (De Groot, 2012). Even women with treated hypothyroidism undergoing in vitro fertilization have a significantly lower chance of achieving pregnancy (Scoccia,r2012).

1	Treatment. The American hyroid Association and American Association of Clinical Endocrinologists (2011) recommend replacement therapy for overt hypothyroidism beginning with levothyroxine in doses of 1 to 2 ILg/kgl d or approximately 100 Lg daily. Women who are athyreotic after thyroidectomy or radioiodine therapy may require higher doses. Surveillance is with TSH levels measured at 4-to 6-week intervals, and the thyroxine dose is adjusted by 25-to 50-lLg increments until TSH values become normal. Pregnancy is associated with an increased thyroxine requirement in approximately a third of supplemented women (Abalovich, 2010; Alexander, 2004). The increased demand in pregnancy is believed to be related to augmented estrogen production (Arafah, 2001).

1	Greater thyroxine requirements begin as early as 5 weeks' gestation. In a randomized trial that provided an increased levothyroxine dose at pregnancy confirmation in 60 mothers, Yassa and coworkers (2010) found that a 29-to 43-percent increase in the weekly dose maintained serum TSH values <5.0 mUlL during the first trimester in all women. Importantly, however, this increase caused TSH suppression in more than a third of women. Significant hypothyroidism may develop early in women without thyroid reserve, such as those with a previous thyroidectomy, those with prior radioiodine ablation, or those undergoing assisted reproductive techniques (Alexander, 2004; Loh, 2009). Anticipatory 25-percent increases in thyroxine replacement at pregnancy conirmation will reduce this likelihood. All other women with hypothyroidism should instead undergo TSH testing at initiation of prenatal care.

1	Pregnancy Outcome with Overt Hypothyroidism. Observational studies, although limited, indicate that excessive adverse perinatal outcomes are associated with overt thyroxine TABLE 58-4. Pregnancy Complications in 440 Women with Hypothyroidism a,bBirthweight <2000 gr apreterm or term deliveries were the only outcomes reported by Abalovich, 2002. SU,r2011. COne infant died from syphilis. Data from Abalovich, 2002; Davis, 1988; leung, 1993; Mannistb, 2009; Su, 2011. deficiency (Table 58-4). Preterm birth rates, for example, are higher (Sheehan, 2015). With appropriate replacement therapy, however, rates of adverse efects are not increased in most reports (Bryant, 2015; Matalon, 2006; Tan, 2006). In one dissenting study, however, risks for some pregnancy complications were greater even in women taking replacement therapy (Wikner, 2008). Most experts agree that adequate hormone replacement during pregnancy minimizes the risk of adverse outcomes and most complications.

1	Fetal and Neonatal Efects. Undoubtedly, maternal and fetal thyroid abnormalities are related. In both, thyroid unction is dependent on adequate iodide intake, and its deiciency early in pregnancy can cause both maternal and fetal hypothyroidism. And, as discussed, maternal TSH-receptor-blocking antibodies can cross the placenta and cause fetal thyroid dysunction. Rovelli and colleagues (2010) evaluated 129 neonates born to women with autoimmune thyroiditis. They found that 28 percent had an elevated TSH level on the third or fourth day of life, and 47 percent of these had TPO antibodies on day 15. Still, autoantibodies were undetectable at 6 months of age. It seems paradoxical that despite these transient laboratory findings in the neonate, TPO and anti thyroglobulin antibodies have little or no efect on etalthyroid function (Fisher, 1997). Indeed, prevalence of fetal hypothyroidism in women with Hashimoto thyroiditis is estimated to be only 1 in 180,000 newborns (Brown, 1996).

1	Although common in women, the incidence of subclinical hypothyroidism varies depending on age, race, dietary iodine intake, and serum TSH thresholds used to establish the diagnosis Qameson, 2015). In two large studies totaling more than 25,000 pregnant women screened in the first half of pregnancy, subclinical hypothyroidism was identified in 2.3 percent (Casey, 2005; Cleary-Goldman, 2008). he rate of progression to overt thyroid failure is afected by TSH level, age, other disorders such as diabetes, and presence and concentration of antithyroid antibodies. nant women with subclinical hypothyroidism for 5 years and reported that in a third, TSH values became normal. In the other two thirds, those women whose TSH levels were 10 to 15 mUlL developed overt disease at a rate of 19 per 100 patient years. Those women whose TSH levels were < 10rm U IL devel oped overt hypothyroidism at a rate of 2 per 100 patient years.

1	pregnant individuals, the U.S. Preventative Services Task Force (Helfand, 2004; Karmisholt, 2008). For gravidas, in a 20-year follow-up study of 5805 women who were screened in early pregnancy, only 3 percent developed thyroid disease. Of the 224 women identified with subclinical hypothyroidism during pregnancy, 17 percent developed thy roid disease in the next 20 years, and most of these had either TPO or thyroglobulin antibodies during pregnancy (Mannisto, 2010). hus, the likelihood of progression to overt hypothy roidism during pregnancy in otherwise healthy women with subclinical hypothyroidism seems remote.

1	Earlier studies were suggestive that subclinical hypothyroidism might be associated with adverse pregnancy outcomes. In 1999, interest was heightened by two studies indicating that undiagnosed maternal thyroid hypofunction may impair fetal neuropsychological development. In one study, Pop and associates (1999) described 22 women with free T 4 levels < 10th percentile whose ofspring were at higher risk for impaired psychomotor development. In the other study, Haddow and coworkers (1999) retrospectively evaluated children born to 48 untreated women whose serum TSH values were >98th percentile. Some had diminished school performance, reading recognition, and intelligent quotient (IQ) scores. Although described as "subclinically hypothyroid," these women had an abnormally low mean serum free thyroxine level, and thus, many had overt hypothyroidism.

1	To further evaluate any adverse efects, Casey and colleagues (2005) identiied subclinical hypothyroidism in 2.3 percent of 17,298 women screened at Parkland Hospital before midpregnancy. hese women had small but signiicantly higher incidences of preterm birth, placental abruption, and neonates admitted to the intensive care nursery compared with euthyroid women. In another study of 10,990 similar women, however, ClearyGoldman and associates (2008) did not ind such associations. Other studies subsequently conirmed a link between subclinical thyroid function and adverse outcomes (Chen, 2017; Maraka, 2016). One included 24,883 women screened throughout pregnancy and showed an almost twofold greater risk of severe preeclampsia (Wilson, 2012). In an analysis of the same cohort, a consistent relationship was shown between rising TSH levels and the risk for gestational diabetes (Tudela, 2012). Finally, Nelson and colleagues (2014) found an elevated risk for diabetes and stillbirth.

1	Lazarus and colleagues (2012) reported the indings of the international multicenter Controlled Antenatal Thyroid Screening (CATS) study. his study evaluated prenatal thyroid screening and randomized treatment of both subclinical hypothyroidism and isolated maternal hypothyroxinemia. They rior in the treated pregnancies.

1	Despite these indings, the unanswered question concerned whether treatment of subclinical hypothyroidism would miti gate any or all of these reported adverse outcomes. To address this, the Maternal-Fetal Medicine Units Network screened more than 97,000 pregnant women for thyroid disorders and reported that 3.3 percent had subclinical hypothyroid ism. hese 677 women were randomly assigned to thyroxine replacement therapy or placebo. As reported by Casey and col leagues (2017), and shown in Table 58-5, maternal adverse pregnancy outcomes or cognitive development in the ofspring at 5 years did not difer between groups. Annual developmental disorder results also did not difer.

1	Screening in Pregnancy. Because of the findings in the studies from 1999 cited above, some professional organizations began to recommend routine prenatal screening and treatment for subclinical hypothyroidism. Consequent to the Lazarus study, however, clinical practice guidelines from the Endocrine Society, the American hyroid Association, and the American Association of Clinical Endocrinologists uniformly recommended screening only those at greater risk during pregnancy (De Groot, 2012; Garber, 2012). his has been and still is the recommendation of the American College of Obstetricians and Gynecologists (2017). he indings of Casey and colleagues (2017) further buttress these recommendations. Women with low serum free T4 values but a normal-range TSH level are considered to have isolated matenal hypothyroxinemia.

1	Women with low serum free T4 values but a normal-range TSH level are considered to have isolated matenal hypothyroxinemia. Its incidence in two large trials was l.3 to 2.1 percent (Casey, 2007; Cleary-Goldman, 2008). As shown in Figure 58-2, unlike in subclinical hypothyroidism, these women had a low preva lence of antithyroid antibodies. Evolution of the knowledge of this thyroid disorder was similar to that seen with subclinical hypothyroidism. Initial studies reported that ofspring of women with isolated hypothyroxinemia had neurodevelopmental diiculties (Kooistra, 2006; Pop, 1999, 2003). In another study, Casey and colleagues (2007) found no higher risks for other adverse perinatal outcomes compared with those of euthyroid women. Also, the aforementioned CATS study did not ind improved neurodevelopmental outcomes in women with isolated hypothyroxinemia who were then treated with thyroxine (Lazarus, 2012).

1	he randomized trial conducted by the Maternal-Fetal Medicine Units Network also provided data to settle this question. Casey and colleagues (2017) noted no higher rates of adverse outcomes between groups and found that early thyroxine treatment ofered no beneits (see Table 58-5). Autoantibodies to TPO and thyroglobulin have been identiied in 6 to 20 percent of reproductive-aged women (Thangaratinam, 201l). Most who test positive for such antibodies, TABLE 58-5. Pregnancy and Perinatal Outcomes According to Diagnosis and Treatment Group of Thyroid Disordersa EGA at delivery (weeks) 39.1 ± 2.5 38.9 ± 3.1 39.0 ± 2.4 38.8 ± 3.1 birth <34 weeks 9.1 % 10.9% 3.8% 2.7% Placental abruption 0.3% 1.5% 1.1% 0.8% Preeclampsia 6.5% 5.9% 3.4% 4.2% Diabetes 7.4% 6.5% 8.0% 9.2% NICU admission 8.6% 6.2% 11.8% 11.9% Birthweight < 10th centile 9.8% 8.1 % 8.8% 7.8% IQ median (25th, 75th 97 (85,105) 94 (85,107) 94(83,101) 91 (82,101) dFor all comparisons, p >0.05.

1	NICU admission 8.6% 6.2% 11.8% 11.9% Birthweight < 10th centile 9.8% 8.1 % 8.8% 7.8% IQ median (25th, 75th 97 (85,105) 94 (85,107) 94(83,101) 91 (82,101) dFor all comparisons, p >0.05. EGA = estimated gestational age; IQ = intelligence quotient; [lICU = neonatal intensive care unit. Data from Casey, 201r7.

1	EGA = estimated gestational age; IQ = intelligence quotient; [lICU = neonatal intensive care unit. Data from Casey, 201r7. however, are euthyroid. That said, such women carry a two-to ivefold increased risk for early pregnancy loss (Stagnaro-Green, 2004; hangaratinam, 2011). he presence of thyroid antibodies has also been associated with preterm birth (Stagnaro-Green, 2009). In a randomized treatment trial of 115 euthyroid women with TPO antibodies, Negro and coworkers (2006) reported that treatment with levothyroxine astoundingly reduced the preterm birth rate from 22 to 7 percent. Contrarily, AbbassiGhanavati and associates (2010) evaluated pregnancy outcomes in more than 1000 untreated women with TPO antibodies and did not ind an increased risk for preterm birth compared with the risk in 16,000 euthyroid women without antibodies. hese investigators, however, did find a threefold greater risk of placental abruption in these women.

1	As with nonpregnant subjects with TPO antibodies, these women are also at increased risk for progression of thyroid disease and postpartum thyroiditis (Jameson, 2015; StagnaroGreen, 2012a). Currently, universal screening for the thyroid autoantibodies is not recommended by any professional organization (De Groot, 2012; Stagnaro-Green, 20lla, 2012a).

1	Decreasing iodide fortification of table salt and bread products in the United States during the past 25 years has led to occasional iodide deficiency (Caldwell, 2005; Hollowell, 1998). Importantly, the most recent National Health and Nutrition Examination Survey indicated that, overall, the United States population remains iodine suicient (Caldwell, 201l). Even so, experts agree that iodine nutrition in vulnerable populations, such as pregnant women, requires continued monitoring. In 2011, the Oice of Dietary Supplements of the National Institutes of Health sponsored a workshop to prioritize iodine research. Participants emphasized the decline in median urinary iodine levels to 125 Lg/L in pregnant women and the serious potential efects on developing fetuses (Swanson, 2012).

1	Dietary iodine requirements are higher during pregnancy due to augmented thyroid hormone production, increased renal losses, and fetal iodine requirements. Adequate iodine is requisite for fetal neurological development beginning soon after conception, and abnormalities are dependent on the degree of deficiency. he World Health Organization (WHO) has estimated that 38 million children are born every year at risk of lifelong brain damage associated with iodine deiciency (Alipui, 2008).

1	Although it is doubtful that mild diciency causes intellectual impairment, supplementation does prevent fetal goiter (Stagnaro-Green, 2012b). Severe diciency, on the other hand, is frequently associated with damage typically encountered with endemic cretinism (Delange, 2001). It is presumed that moderate deiciency has intermediate and variable efects. Berbel and associates (2009) began daily supplementation in more than 300 pregnant women with moderate deiciency at three time periods-4 to 6 weeks, 12 to 14 weeks, and after delivery. They found improved neurobehavioral development scores in ofspring of women supplemented with 200 ILg potassium iodide very early in pregnancy. Similarly, Velasco and coworkers (2009) found improved Bayley Psychomotor Development scores in ofspring of women supplemented with 300 ILg of iodine daily in the irst trimester. In contrast, Murcia and colleagues (2011) identified lower psychomotor scores in 1-yearold infants whose mothers reported daily

1	women supplemented with 300 ILg of iodine daily in the irst trimester. In contrast, Murcia and colleagues (2011) identified lower psychomotor scores in 1-yearold infants whose mothers reported daily supplementation of more than 150 ILg. To address this, randomized controlled trial of iodine supplementation in mildly to moderately iodinedeficient pregnant women in India and hailand is nearing completion (Pearce, 2016).

1	Regarding daily iodine intake, the Institute of Medicine during lactation (Chap. 9, p. 168). he Endocrine Society recommends an average iodine intake of 150 �g/d in repro ductive-aged women, and this should be increased to 250 �g during pregnancy and breastfeeding (De Groot, 2012). The of iodine be added to prenatal vitamins to achieve this average daily intake (Becker, 2006). According to Leung and coworkers (2011), however, only 51 percent of the prenatal multivitamins in the United States contain iodine. It has even been suggested that because most cases of maternal hypothyroxinemia world wide are related to relative iodine deficiency, supplementation may obviate the need to consider thyroxine treatment in such women (Gyami, 2009). However, without evidence of benefit, it is hard to justiy the cost of iodine supplementation of large numbers of pregnant women in areas with mild iodine dei ciency (Pearce, 2016).

1	Importantly, experts caution against oversupplementa tion. T eng and associates (2006) contend that excessive iodine intake-deined as > 300 �g/ d-may lead to subclinical hypo thyroidism and autoimmune thyroiditis. he Endocrine Soci ety, in accordance with the WHO, advises against exceeding twice the daily recommended intake of iodine, or 500 �g/ d (De Groot, 2012; Leung, 2011). Universal newborn screening for neonatal hypothyroidism was introduced in 1974 and is now required by law in all states (Chap. 32, p. 614). This develops in approximately 1 in 3000 newborns and is one of the most preventable causes of mental retardation (LaF ranchi, 2011). Developmental disorders of the thyroid gland such as agenesis and hypoplasia account for 80 to 90 percent of these cases. he remainder is caused by hereditary defects in thyroid hormone production (Moreno, 2008).

1	Early and aggressive thyroxine replacement is critical for newborns with congenital hypothyroidism. Still, some neonates identified by screening programs who were treated promptly will exhibit cognitive deicits into adolescence (Song, 2001). herefore, in addition to timing of treatment, the severity of congenital hypothyroidism is an important factor in long-term cognitive outcomes. Olivieri and colleagues (2002) reported that 8 percent of 1420 newborns with congenital hypothyroidism also had other major congenital malformations.

1	Transient autoimmune thyroiditis is consistently found in approximately 5 to 10 percent of women during the first year after childbirth (Nathan, 2014; Stagnaro-Green, 2011b, 2012a). Postpartum thyroid dysfunction with an onset within 12 months includes hyperthyroidism, hypothyroidism, or both. The propensity for thyroiditis antedates pregnancy and is directly related to increasing serum levels of thyroid autoantibodies. Up to 50 percent of women who are thyroid-antibody positive in the irst trimester will develop postpartum thyroiditis (Stagnaro-Green, 2012a). In a Dutch study of 82 women with type 1 diabetes, postpartum thyroiditis developed in 16 percent and was threefold higher than in the general population (Gallas, 2002). Importantly, 46 percent of those identified with overt postpartum thyroiditis had TPO antibodies in the first trimester.

1	In clinical practice, postpartum thyroiditis is diagnosed infre quently because it typically develops months after delivery and causes vague and nonspeciic symptoms (Stagnaro-Green, 2004). he clinical presentation varies, and classically two clinical phases that may develop in succession are recognized.

1	The first and earliest is destruction-induced thyrotoxicosis with symptoms from excessive release of hormone from glandular disruption. The onset is abrupt, and a small, painless goiter is common. Although there may be many symptoms, only fatigue and palpitations are more frequent in thyrotoxic women com pared with normal controls. This thyrotoxic phase usually lasts only a few months. hionamides are inefective, and if symp toms are severe, a 3-blocking agent may be given. he second and usually later phase between 4 and 8 months postpartum is hypothyroidism from thyroiditis. hyromegaly and other symp toms are common and more prominent than during the thyro toxic phase. hyroxine replacement at doses of25 to 75 �g/d is typically given for 6 to 12 months.

1	Stagnaro-Green and associates (20 11 b) reported postpartum surveillance results in 4562 Italian gravidas who had been screened for thyroid disease in pregnancy. Serum TSH and anti-TPO antibody levels were measured again at 6 and 12 months. Overall, two thirds of 169 women (3.9 percent) with postpartum thyroiditis were identiied to have hypothyroidism only. The other third were diagnosed with hyperthyroidism. Only 14 percent of all women demonstrated the "classic" biphasic progression described above. These indings are consistent with data compiled from 20 other studies between 1982 and 2008 (Stagnaro-Green, 2012a).

1	Importantly, women who experience either type of postpartum thyroiditis have a 20-to 30-percent risk of eventually developing permanent hypothyroidism, and the annual progression rate is 3.6 percent (Nathan, 2014). Women at greater risk for developing hypothyroidism are those with higher titers of thyroid antibodies and higher TSH levels during the initial hypothyroid phase. Others may develop subclinical disease, but half of those with thyroiditis who are positive for TPO antibodies develop permanent hypothyroidism by 6 to 7 years (Stagnaro-Green,r2012a).

1	n association between postpartum thyroiditis and postpartum depression has been proposed but remains unresolved. Lucas and coworkers (2001) found a 1.7-percent incidence of postpartum depression at 6 months in women with thyroiditis as well as in controls. Pederson and colleagues (2007) found a signiicant correlation between abnormal scores on the Edinburgh Postnatal Depression Scale and total thyroxine values in the low normal range during pregnancy in 31 women. Similarly unsettled is the link between depression and thyroid antibodies. Kuijpens and associates (2001) reported that TPO antibodies were a marker for postpartum depression in euthyroid women. In a randomized trial, however, Harris and coworkers (2002) reported no diference in postpartum depression in 342 women with TPO antibodies who were given either levothyroxine or placebo.

1	hyroid nodules can be found in 1 to 2 percent of reproductive-aged women (Fitzpatrick, 2010). Management of a palpable thyroid nodule during pregnancy depends on gestational age and mass size. Small nodules detected by sensitive sonographic methods are more common during pregnancy in some populations. Kung and associates (2002) used high-resolution sonography and found that 15 percent of Chinese women had nodules larger than 2 mm in diameter. Almost half were multiple, and the nodules usually enlarged modestly across preg nancy and did not regress postpartum. Biopsy of thoser> 5 mmrthat persisted at 3 months usually showed nodular hyperplasia, and none were malignant. In most studies, 90 to 95 percent of solitary nodules are benign (Burch, 2016).

1	Evaluation of thyroid nodules during pregnancy should be similar to that for nonpregnant patients. As discussed in Chapter 46 (p. 908), radioiodine scanning in pregnancy is usually not recommended (American College of Obstetricians and Gynecologists, 2017). Sonographic examination reliably detects nodules > 5 mm, and their solid or cystic structure also is determined. According to the American Association of Clinical Endocrinologists, sonographic characteristics associated with malignancy include hypoechogenic pattern, irregular margins, and microcalcifications (Gharib, 2005). Fine-needle aspiration (FNA) is an excellent assessment method, and histological tumor markers and immunostaining are reliable to evaluate for malignancy (Hegedus, 2004). If the FNA biopsy shows a follicular lesion, surgery may be deferred until after delivery.

1	Evaluation of thyroid cancer involves a multidisciplinary approach (Fagin, 2016). Most thyroid carcinomas are well differentiated and pursue an indolent course. Messuti and coworkers (2014) provided evidence that persistence or recurrence of these tumors may be more common in pregnant women. When thyroid malignancy is diagnosed during the first or second trimester, thyroidectomy may be performed before the third trimester (Chap. 63, p. 1201). In women without evidence of an aggressive thyroid cancer or in those diagnosed in the third trimester, surgical treatment can be deferred to the immediate puerperium (Gharib, 2010).

1	The function of parathyroid hormone (PTH) is to maintain extracellular fluid calcium concentration. his 84-amino acid hormone acts directly on bone and kidney and indirectly on small intestine through its efects on synthesis of vitamin D (l,25-(OH)2D) to increase serum calcium (Potts, 2015). Secretion is regulated by serum ionized calcium concentration through a negative feedback system. Cacitonin is a potent parathyroid hormone that acts as a physiological parathyroid hormone antagonist. The interrelationships between these hormones, calcium metabolism, and PTH-related protein produced by fetal tissue are discussed in Chapter 4 (p. 71).

1	Of fetal demands, calcium requirements reach 300 mg/d in late pregnancy and 30 g for the entire gestation. These needs and greater renal calcium loss from augmented glomerular iltration substantively raise maternal calcium demands. Pregnancy is associated with a twofold rise in serum concentrations of 1,25-dihydroxyvitamin D, which increases gastrointestinal calcium absorption. he efectuating hormone is probably of placental and decidual origin because maternal PTH levels are low normal or decreased during pregnancy (Cooper, 2011; Molitch, 2000). Total serum calcium levels decline with serum albumin concentrations, but ionized calcium levels remain unchanged. Vargas Zapata and colleagues (2004) have suggested a role for insulin-like growth factor-1 (IGF-1) in maternal calcium homeostasis and bone turnover.

1	Hypercalcemia is caused by hyperparathyroidism or cancer in 90 percent of cases (Potts, 2015). Because many automated laboratory systems include serum calcium measurement, hyperparathyroidism has changed from being a condition deined by symptoms to one that is discovered on routine screening (Pallan, 2012). It has a reported prevalence of 2 to 3 per 1000 women, but some have estimated the rate to be as high as 14 per 1000 when asymptomatic cases are included. Almost 80 percent are caused by a solitary adenoma, and another 15 percent by hyperfunctioning of all four glands. In the remainder, a malignancy as the cause of increased serum calcium levels is usually obvious. Of note, PTH produced by tumors is not identical to the natural hormone and may not be detected by routine assays.

1	In most patients, the serum calcium level is elevated to within only 1 to 1.5 mg/dL above the upper normal limit. This may help to explain why only 20 percent of those who have abnormally elevated levels are symptomatic (Bilezikian, 2004). In a fourth, however, symptoms become apparent when the serum calcium level continues to rise. Hypercacemic crisis manifests as stupor, nausea, vomiting, weakness, fatigue, and dehydration. All women with symptomatic hyperparathyroidism should be surgically treated (Potts, 2015). Indications for parathyroidectomy include a serum calcium level 1.0 mg/dL above the upper normal range, a calculated creatinine clearance < 60 mLi min, reduced bone density, or age >50 years (Bilezikian, 2009). Those not meeting these criteria should undergo annual serum calcium and creatinine level measurement and bone density assessment every 1 to 2 years (Pallan, 2012).

1	In their review, Schnatz and Thaxton (2005) found fewer than 200 reported cases of hyperparathyroidism complicating pregnancy. As in nonpregnant patients, parathyroid adenoma is the most common etiology. Ectopic parathyroid hormone production and rare cases of parathyroid carcinoma have been reported in pregnancy (Montoro, 2000; Saad, 2014). Symptoms include hyperemesis, generalized weakness, renal calculi, and psychiatric disorders. Occasionally, pancreatitis is the presenting disorder (Cooper, 2011; Hirsch, 2015).

1	Pregnancy theoretically improves hyperparathyroidism because of signiicant calcium shunting to the fetus and augmented renal excretion (Power, 1999). When the "protective efects" of pregnancy are withdrawn, however, postpartum hypercalcemic crisis is a significant danger. his life-threatening complication can be seen with serum calcium levels greater than 14 mgl dL and is characterized by nausea, vomiting, tremors, dehydration, and mental status changes (Malekar-Raikar, 2011). ism. More recent reports, however, described lower rates of stillbirth, neonatal death, and neonatal tetany (Kovacs, 201r1). Other fetal complications include miscarriage,r· fetal-growth restriction, and low birthweight (Chamarthi, 2011). Schnatz (2005) reported a 25-percent incidence of preeclampsia.

1	Other fetal complications include miscarriage,r· fetal-growth restriction, and low birthweight (Chamarthi, 2011). Schnatz (2005) reported a 25-percent incidence of preeclampsia. Management in Pregnancy. Surgical removal of a symptomatic parathyroid adenoma is preferable. This should prevent fetal and neonatal morbidities and postpartum parathyroid crises (Kovacs, 2011). Elective neck exploration during pregnancy is usually well tolerated, even in the third trimester (Hirsch, 2015; Schnatz, 2005; Stringer, 2017). In at least two cases, a mediastinal adenoma was removed at midpregnancy (Rooney, 1998; Saad, 2014).

1	Medical management may be appropriate in asymptomatic pregnant women with mild hypercalcemia (Hirsch, 2015). If so, patients are careully monitored in the puerperium for hypercalcemic crisis. Initial medical management might include cacitonin to decrease skeletal calcium release, or oral phosphate, 1 to 1.5 g daily in divided doses, to bind excess calcium. For women with dangerously elevated serum calcium levels or those who are mentally obtunded with hypercacemic crisis, emergency treatment is instituted. Diuresis with intravenous normal saline is begun so that urine low exceeds 150 mLlhr. Furosemie is given in conventional doses to block tubular calcium reabsorption. Importantly, hypokalemia and hypomagnesemia should be prevented. Adjunctive therapy includes mithramycin, which inhibits bone resorption.

1	Neonatal Efects. Normally, cord blood calcium levels are higher than maternal levels (Chap. 7, p. 139). With maternal hyperparathyroidism, abnormally elevated maternal and thus fetal levels further suppress fetal parathyroid function. Because of this, newborn calcium levels rapidly drop after birth, and 15 to 25 percent of these neonates develop severe hypocalcemia with or without tetany (Molitch, 2000). Neonatal hypoparathyroidism caused by maternal hyperparathyroidism is usually transient and is treated with calcium and 1,25-dihydroxyvitamin D3 (calcitriol). he latter will not be efective in preterm infants, however, because the intestinal vitamin D receptor is not suiciently expressed (Kovacs, 2011) . Neonatal tetany or seizures should stimulate an evaluation for maternal hyperparathyroidism (Beattie, 2000; Ip, 2003).

1	The most common cause of hypocalcemia is hypoparathyroidism that usually follows parathyroid or thyroid surgery. Hypoparathyroidism is estimated to follow up to 7 percent of total thyroidectomies (Shoback, 2008). It is characterized by facial muscle spasms, muscle cramps, and paresthesias of the lips, tongue, fingers, and feet. This can progress to tetany and seizures (Potts, 2015). Chronically, hypocalcemic pregnant women may also have a fetus with skeletal demineralization resulting in multiple bone fractures in the neonatal period (Alikasifoglu, 2005).

1	Maternal treatment includes calcitriol, dihydrotachysterol, or large vitamin D doses of 50,000 to 150,000 U/d; calcium gluconate or calcium lactate in doses of 3 to 5 g/d; and a lowphosphate diet. Fetal risks from large doses of vitamin D have not been established. During treatment, the therapeutic challenge in women with known hypoparathyroidism is management of blood calcium levels. It is possible that the greater calcium absorption typical of pregnancy will result in lower calcium requirements or that the fetal demand for calcium will result in greater need. he goal during pregnancy is to maintain a corrected calcium level in the low normal range.

1	In most gravidas, even with their remarkably increased calcium requirements, it is uncertain whether pregnancy causes osteopenia (Kaur, 2003; To, 2003). In one study of200 pregnant women in which bone mass was measured, Kraemer and colleagues (2011) demonstrated a decline in bone density during pregnancy. Women who breastfed, carried twin pregnancies, or had a low body mass index were at higher risk of bone loss. From their review, Thomas and Weisman (2006) cite a 3-to 4-percent average reduction in bone mineral density during pregnancy. Lactation also represents a period of negative calcium balance that is corrected through maternal skeletal resorption. Feigenberg and coworkers (2008) found cortical bone mass reductions using ultrasound in young primiparas in the puerperium compared with nulligravid controls. Rarely, some women develop idiopathic osteoporosis while pregnant or lactating (Hellmeyer, 2007).

1	The most common symptom of osteoporosis is back pain in late pregnancy or postpartum. Other symptoms are hip pain, either unilateral or bilateral, and diiculty in weight bearing until the woman is nearly immobilized (Maliha, 2012). In more than half of women, no apparent reason for osteopenia is found. Some known causes include heparin (unfractionated only), prolonged bed rest, and corticosteroid therapy (Cunningham, 2005; Galambosi, 2016). In a few cases, overt hyperparathyroidism or thyrotoxicosis eventually develops. Treatment is problematic and includes calcium and vitamin D supplementation and standard pain management. Shown in Figure 58-5 is a hip radiograph from a woman FIGURE 58-5 Anteroposterior plain hip radiograph of a 25-yearold woman at 26 weeks' gestation. She complained of left hip and knee pain and progressive weakness. Transient osteoporosis of the left femur responded over 3 months to physical therapy combined with vitamin 0 and calcium supplementation.

1	treated at Parkland Hospital during the third trimester for transient osteoporosis of pregnancy. For women with pregnancy-associated osteopenia, long-term surveillance indicates that although bone density improves, these women and their ofspring may have chronic osteopenia (Carbone, 1995). Related, prenatal supplementation of normal women with cholecalciferol, 1000 IU / d, did not increase ofspring bone mineral content, although it did ensure maternal vitamin D repletion (Cooper, 2016). Pregnancy has profound efects on adrenal cortical secretion and its control or stimulation. These interrelationships were reviewed by Lekarev and New (2011) and are discussed in detail in Chapter 4 (p. 71).

1	Pheochromocytomas are chromain tumors that secrete catecholamines and usually are located in the adrenal medulla, although 10 percent are located in sympathetic ganglia. They are called the 10-percent tumor because approximately 10 percent are bilateral, 10 percent are extraadrenal, and 10 percent are malignant. These tumors can be associated with medullary thyroid carcinoma and hyperparathyroidism in some of the autosomally dominant or recessive multiple endocrine neoplasia syndromes, as well as in neurofibromatosis and von HippelLindau disease (Neumann, 2015).

1	These tumors complicate approximately 1 per 50,000 pregnancies (Quartermaine, 2017). Notably, they are found in 0.1 percent of hypertensive patients (Abdelmannan, 2011). However, they are more commonly found at autopsy but with infrequent clinical recognition. Symptoms are usually paroxysmal and manifest as hypertensive crisis, seizure disorders, or anxiety attacks. Hypertension is sustained in 60 percent of patients, but half of these also have paroxysmal crises. Other symptoms during paroxysmal attacks are headaches, profuse sweating, palpitations, chest pain, nausea and vomiting, and pallor or flushing.

1	The standard screening test is quantification of metanephrines and catecholamine metabolites in a 24-hour urine specimen (Neumann, 2015). Diagnosis is established by measurement of a 24-hour urine collection with at least two of three assays for free catecholamines, metanephrines, or vanillylmandelic acid (VMA). Determination of plasma catecholamine levels is the most sensitive test. In nonpregnant patients, adrenal localization is usually successful with either computed tomography (CT) or magnetic resonance (MR) imaging. For most cases, preferred treatment is laparoscopic adrenalectomy (Neumann, 2015).

1	These tumors are rare but result in dangerous pregnancy complications. Geelhoed (1983) provided an earlier review of 89 cases in which 43 mothers died. Maternal death was much more common if the tumor was not diagnosed antepartum-58 versus 18 percent. As seen in Table 58-6, maternal mortality rates are now lower but still formidable. In their review of 77 cases, Biggar and Lennard (2013) reported that antepartum diagnosis is the most important determinant of maternal mortality risk. That said, Salazar-Vega and colleagues (2014) described good outcomes in women diagnosed after delivery. TABLE 58-6. Outcomes of Pregnancies Complicated by Pheochromocytoma and Reported in Four Contiguous Epochs

1	TABLE 58-6. Outcomes of Pregnancies Complicated by Pheochromocytoma and Reported in Four Contiguous Epochs Diagnosis of pheochromocytoma in pregnancy is similar to that for nonpregnant patients. MR imaging is the preferred technique because it almost always locates adrenal and extraadrenal pheochromocytomas (Fig. 58-6). In many cases, the principal challenge is to diferentiate preeclampsia from the hypertensive crisis caused by pheochromocytoma. Grimbert and colleagues (1999) diagnosed two pheochromocytomas during 56 pregnancies in 30 women with von Hippel-Lindau disease. FIGURE 58-6 Coronal magnetic resonance image taken in a 32-week pregnant woman shows a right-sided pheochromocytoma (arrow) and its position relative to the liver above it.

1	FIGURE 58-6 Coronal magnetic resonance image taken in a 32-week pregnant woman shows a right-sided pheochromocytoma (arrow) and its position relative to the liver above it. Immediate control of hypertension and symptoms with an .-adrenergic blocker such as phenoxybenzamine is impera tive. he dose is 10 to 30 mg, two to four times daily. After .-blockade is achieved, 3-blockers may be given for tachycar dia. In many cases, surgical exploration and tumor removal are performed during pregnancy, preferably during the second trimester (Biggar, 2013; Dong, 2014). Successfulrlaparoscopic removal of adrenal tumors has become the norm (Miller, 2012; Zuluaga-G6mez, 2012). If diagnosed later in pregnancy, either planned cesarean delivery with tumor excision or postpartum resection is appropriate.

1	Zuluaga-G6mez, 2012). If diagnosed later in pregnancy, either planned cesarean delivery with tumor excision or postpartum resection is appropriate. Recurrent tumors are troublesome, and even with good blood pressure control, dangerous peripartum hypertension may develop. We have cared for three women in whom recur rent pheochromocytoma was identiied during pregnancy. Hypertension was managed with phenoxybenzamine in all three. Two newborns were healthy, but a third was stillborn in a mother with a massive tumor burden who was receiving phenoxybenzamine, 100 mg daily. In all three women, tumor was resected postpartum.

1	his syndrome is rare and the female:male ratio is 3: 1 (Arit, 2015). Most cases are iatrogenic from long-term corticosteroid treatment. Cushing disease refers to bilateral adrenal hyperplasia stimulated by corticotropin-producing pituitary adenomas. Corticotropin is also called adrenocorticotropic hormone (ACTH). Most adenomas are microadenomas measuring < 1 em, and half measure <5 mm. Rarely, abnormal secretion of hypothalamic corticotropin-releasing factor may cause corticotropic hyperplasia. Such hyperplasia may also be caused by nonendocrine tumors that produce polypeptides similar to either corticotropin-releasing factor or corticotropin. Less than a fourth of cases of Cushing syndrome are corticotropin independent, and most of these are caused by an adrenal adenoma. Tumors are usually bilateral, and half are malignant. Occasionally, associated androgen excess may lead to severe virilization.

1	he typical cushingoid body habitus is caused by adipose tissue deposition that characteristically results in moon acies, a builo hump, and truncal obesiy. Fatigability and weakness, hypertension, hirsutism, and amenorrhea are each encountered in 75 to 85 percent of nonpregnant patients (Hatipoglu, 2012). Personality changes, easy bruisability, and cutaneous striae are common. Up to 60 percent may have impaired glucose tolerance. Diagnosis can be diicult and is suggested by elevated plasma cortisol levels that cannot be suppressed by dexamethasone or by elevated 24-hour urine free cortisol excretion (Arit, 2015; Loriaux, 2017). Neither test is totally accurate, and each is more diicult to interpret in obese patients. Serum corticotropin levels and CT and MR imaging are used to localize pituitary and adrenal tumors or hyperplasia. Because most women have corticotropin-dependent Cushing syndrome, associated androgen excess may cause anovulation,

1	Because most women have corticotropin-dependent Cushing syndrome, associated androgen excess may cause anovulation, TABLE 58-7. Maternal and Perinatal Complications Data from Lindsay, 2005. and pregnancy is rare. In their review, Lekarev and New (2011) identiied fewer than 140 reported cases of Cushing syndrome in pregnancy. These difer compared with nonpregnant women in that half are caused by corticotropin-independent adrenal adenomas (Kamoun, 2014; Lacroix, 2015). Approximately 30 percent of cases are from a pituitary adenoma, and 10 percent from adrenal carcinomas. All reports stress diiculties in diagnosis because of pregnancy-induced increases in plasma cortisol, corticotropin, and corticotropin-releasing factor levels. Measurement of24-hour urinary free cortisol excretion is recommended, with consideration for the normal elevation seen in pregnancy.

1	Pregnancy outcomes in women with Cushing syndrome are listed in Table 58-7. Heart failure is common during pregnancy and is a major cause of maternal mortality (Buescher, 1992). Hypercortisolism in pregnancy may also cause poor wound healing, osteoporotic fracture, and psychiatric complications (Kamoun, 2014).

1	Long-term medical therapy for Cushing syndrome usually is inefective, and deinitive therapy is resection of the pituitary or adrenal adenoma or bilateral adrenalectomy for hyperplasia (Lacroix, 2015; Motivala, 2011). During pregnancy, management of hypertension in mild cases may suice until delivery. In their review, Lindsay and associates (2005) described primary medical therapy in 20 women with Cushing syndrome. Most were successfully treated with meyrapone as an interim treatment until deinitive surgery after delivery. A few cases were treated with oral ketoconazole. However, because this drug also blocks testicular steroidogenesis, treatment during pregnancy with a male fetus is worrisome. Miepristone, the norethindrone derivative used for abortion and labor induction, has shown promise for treating Cushing disease but should not be used in pregnancy for obvious reasons. If necessary, pituitary adenomas can be treated by transsphenoidal resection (Boscaro, 2001; Lindsay, 2005).

1	for treating Cushing disease but should not be used in pregnancy for obvious reasons. If necessary, pituitary adenomas can be treated by transsphenoidal resection (Boscaro, 2001; Lindsay, 2005). Unilateral adrenalectomy has been safely performed in the early third trimester and can also be curative (Abdelmannan, 2011).

1	Primary adrenocortical insuiciency is rare because more than 90 percent of total gland volume must be destroyed for symptoms to develop. Autoimmune adrenalitis is the most common cause in the developed world, but tuberculosis is a more frequent etiology in resource-poor countries (Arit, 2015; Kamoun, 2014). The incidence has been cited as being as high as 1 in 3000 births in Norway (Lekarev, 2011). In the United States, the prevalence was 1 per 10,000 to 20,000 pregnancies (Schneiderman, 2017). here is an increased incidence of concurrent Hashimoto thyroiditis, primary ovarian insuiciency, type 1 diabetes, and Graves disease. These poyglandular autoimmune syndromes also include pernicious anemia, vitiligo, alopecia, nontropical sprue, and myasthenia gravis.

1	Untreated adrenal hypofunction frequently causes infertility, but with replacement therapy, ovulation is restored. If untreated, symptoms often include weakness, fatigue, nausea and vomiting, and weight loss. Because serum cortisol levels are increased during pregnancy, the inding of a low value should prompt a cosyntropin test to document the lack of response to infused corticotropin (Salvatori, 2005).

1	In a large Swedish cohort study, 1188 women with Addison disease were compared with more than 11,000 age-matched controls who delivered between 1973 and 2006 (Bjornsdottir, 2010). Women diagnosed with adrenal insuiciency within 3 years of delivery were signiicantly more likely to deliver preterm, to deliver a low-birthweight newborn, and to undergo cesarean delivery. Others have reported similar adverse outcomes (Quartermaine, 2017). Most pregnant women with Addison disease are already taking glucocorticoid and mineralocorticoid replacement drugs. hese should be continued and women observed for evidence of either inadequate or excessive corticosteroid replacement (Lebbe, 2013). During labor, delivery,r and postpartum, or after a surgical procedure, corticosteroid replacement must be increased appreciably to approximate the normal adrenal response-the so-called stress dose. Hydrocortisone, 100 mg, is usually given intravenously every 8 hours for 48 hours. It is important that shock

1	appreciably to approximate the normal adrenal response-the so-called stress dose. Hydrocortisone, 100 mg, is usually given intravenously every 8 hours for 48 hours. It is important that shock from causes other than adrenocortical insuiciency-for example, hemorrhage or sepsis-be recognized and treated promptly.

1	Hyperaldosteronism is caused by an adrenal adenoma-Conn syndrome-in approximately 75 percent of cases. Idiopathic bilateral adrenal hyperplasia makes up the remainder, except for rare cases of adrenal carcinoma (Abdelmannan, 2011; Eschler, 2015). Findings include hypertension, hypokalemia, and muscle weakness. High serum or urine levels of aldosterone confirm the diagnosis.

1	In normal pregnancy, as discussed in Chapter 4 (p. 72), progesterone blocks aldosterone action, and thus there are very high aldosterone levels (Appendix, p. 1258). Accordingly, the diagnosis of hyperaldosteronism during pregnancy can be diicult. Since renin levels are suppressed in pregnant women with hyperaldosteronism, a plasma aldosterone-to-renin activity ratio may be helpful for diagnosis (Kamoun, 2014). Hypertension worsens as pregnancy progresses, and medical management includes potassium supplementation and antihypertensive therapy. In many cases, hypertension responds to spironolactone, but 3-blockers or calcium-channel blockers may be preferred because of the potential fetal antiandrogenic efects of spironolactone. Mascetti and coworkers (201r1) reported successful use of amiloride in a pregnant woman. Use of eplerenone, a selective aldosterone-receptor antagonist, has also been reported (Cabassi, 2012). Laparoscopic tumor resection is curative (Eschler, 2015; Miller,

1	amiloride in a pregnant woman. Use of eplerenone, a selective aldosterone-receptor antagonist, has also been reported (Cabassi, 2012). Laparoscopic tumor resection is curative (Eschler, 2015; Miller, 2012).

1	The pituitary enlarges impressively during pregnancy, predominately from lactotrophic cellular hyperplasia induced by estrogen stimulation (Chap. 4, p. 68). Several pituitary disorders can also complicate pregnancy. These adenomas are found often in nonpregnant women since the advent of widely available serum prolactin assays. Serum levels <25 pg/mL are considered normal in nonpregnant women (Nlotivala, 2011). Adenoma symptoms and indings include amenorrhea, galactorrhea, and hyperprolactinemia. Tumors are classiied arbitrarily by their size measured by CT or MR imaging. A micro adenoma is :;10 mm, and a macro adenoma is >10 mm. Treatment for microadenomas is usually with bromocriptine, a dopamine agonist and powerful prolactin inhibitor, which frequently restores ovulation. For suprasellar macroadenomas, most recommend surgical resection before pregnancy is attempted (Araujo, 2015).

1	In a pooled analysis of more than 750 pregnant women with prolactinomas, only 2.4 percent with microadenomas developed symptomatic enlargement during pregnancy (Molitch, 2015). Symptomatic enlargement of macroadenomas, however, is more frequent and was found in 21 percent of 238 pregnant women. Schlechte (2007) also reported that 15 to 35 percent of suprasellar macroadenomas have tumor enlargement that causes visual disturbances, headaches, and diabetes insipidus. Nonfunctioning adenomas can also cause symptoms of pituitary expansion in pregnancy (Lambert, 2017).

1	Pregnant women with microadenomas should be queried regularly for headaches and visual symptoms. Those with macroadenomas are followed more closely and have visual field testing during each trimester. CT or MR imaging is recommended only if symptoms develop (Fig. 58-7). Serial serum prolactin levels serve little use because of normal rises during pregnancy (Appendix, p. 1259). Symptomatic tumor enlargement should be treated immediately with a dopamine antagonist. he safety of bromocriptine in pregnancy is well established. The safety profile is less well known for cabergoline, which is increasingly used in nonpregnant women because it is better tolerated and more efective. Cabergoline is generally considered safe for use in pregnancy (Araujo, 2015; Auriemma, 2013). Lebbe and colleagues (2010) described 100 pregnancies exposed to cabergoline and found no adverse efects. Similar indings were reported in 85 exposed Japanese pregnant women (Ono, 2010). Surgery is recommended for women

1	(2010) described 100 pregnancies exposed to cabergoline and found no adverse efects. Similar indings were reported in 85 exposed Japanese pregnant women (Ono, 2010). Surgery is recommended for women with no response.

1	suprasellar mass (arrow). Note the layering of complex fluid within the mass, which was found during surgery to be hemorrhage. (Used with permission from Dr. April Baily.) his is caused by excessive growth hormone, usually from an acidophilic or a chromophobic pituitary adenoma. In normal pregnancy, pituitary growth hormone levels decrease as placental epitopes are secreted. Diagnosis is confirmed by elevated IGF-1 serum levels (Katznelson, 2014). Fewer than 100 cases of acromegaly have been reported during pregnancy (Cheng, 2012; Dias, 2013; Motivala, 2011). Pregnancy is probably rare in women with acromegaly because half are hyperprolactinemic and anovulatory. During pregnancy, afected women are at marginally greater risk for gestational diabetes and hypertension (Caron, 2010; Dias, 20l3).

1	Management is similar to that for prolactinomas, with close monitoring for symptoms of tumor enlargement. Dopamine agonist therapy is less efective than for prolactinomas. And, transsphenoidal resection, generally considered irst-line treatment outside of pregnancy, may be necessary for symptomatic tumor enlargement during pregnancy (Motivala, 2011). Guven and associates (2006) reported a case of pituitary apoplexy necessitating emergent transsphenoidal adenoma resection and cesarean delivery at 34 weeks. Successful treatment of pregnant women with the somatostatin-receptor ligand octreotide and with the GH analogue pegvisomant has also been reported (Dias, 2013; Fleseriu, 2015). The vasopressin deficiency evident in diabetes insipidus is usually due to agenesis or destruction of the neurophypophysis (Robertson, 2015). True diabetes insipidus is a rare complication of pregnancy.

1	Diabetes insipidus therapy is intranasal administration of a synthetic analogue of vasopressin, desmopressin, which is 1-deamino8-D-arginine vasopressin (DDA P). Ray (1998) reviewed 53 cases in which DDAP was used during pregnancy with no adverse sequelae. Most women require increased doses during pregnancy because of an increased metabolic clearance rate stimulated by placentl vasopressinase (Lindheimer, 1994). By this same mecha nism, subclinicaL diabetes inspidus may become symptomatic or cases of transient diabetes inspidus may be encountered during pregnancy (Bellastella, 2012; Robertson, 2015). he prevalence of vasopressinase-induced diabetes insipidus is estimated at 2 to 4 per 100,000 pregnancies (Wallia, 20l3). In our experiences, as described in Chapter 55 (p. 1062), transient secondary diabetes insipidus is more likely encoun tered with acute atty Liver of pregnancy (Nelson, 20 l3). This probably is due to altered vasopressinase clearance because of hepatic dysfunction.

1	Sheehan (1937) reported that pituitary ischemia and necrosis associated with obstetrical blood loss could result in hypopituitarism. With modern methods of hemorrhagic shock treatment, Sheehan syndrome is now seldom encountered (Feinberg, 2005; Pappachan, 2015; Robalo, 2012). Afected women may have persistent hypotension, tachycardia, hypoglycemia, and lactation failure. Because deficiencies of some or all pituitary-responsive hormones may develop after the initial insult, Sheehan syndrome can be heterogenous and may not be identified for years (Tessnow, 2010). In one cohort study of 60 women from Costa Rica with Sheehan syndrome, the average time to diagnosis was 13 years (Gei-Guardia, 2011). Because adrenal insuiciency is the most life-threatening complication, adrenal function should be immediately assessed in any woman suspected of having Sheehan syndrome. Mter glucocorticoid replacement, subsequent analyses and replacement of thyroid, gonadal, and growth hormones is considered.

1	This rare autoimmune pituitary disorder is characterized by massive iniltration by lymphocytes and plasma cells with parenchymal destruction of the gland. vIany cases are temporally linked to pregnancy (Foyouzi, 2011; Honegger, 2015; Melmed, 2015). here are varying degrees of hypopituitarism or symptoms of mass efect, including headaches and visual ield defects. A sellar mass is seen with CT or MR imaging. A mass accompanied by a modestly elevated serum prolactin level-usually < 100 pgl mL-suggests lymphocytic hypophysitis. In contrast, levels >200 pg/mL are encountered with a prolactinoma. he etiology is unknown, but nearly 30 percent have a history of coexisting autoimmune diseases including Hashimoto thyroiditis, Addison disease, type 1 diabetes, or pernicious anemia. Treatment is with glucocorticoids and pituitary hormone replacement. he disease may be self-limited, and a careful withdrawal of hormone replacement is attempted after inflammation subsides (Foyouzi, 2011; Melmed,

1	glucocorticoids and pituitary hormone replacement. he disease may be self-limited, and a careful withdrawal of hormone replacement is attempted after inflammation subsides (Foyouzi, 2011; Melmed, 2015).

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1	Salvatori R: Adrenal insuiciency. ]AMA 294:2481, 2005 Sarathi V, Lila A, Bandgar T, et al: Pheochromocytoma and pregnancy: a rare but dangerous combination. Endocr Pract 16(2):300,t2010 Sarkhail P, Mehran L, Askari S, et al: Maternal thyroid function and autoimmunity in 3 trimesters of pregnancy and their ofspring's thyroid function. Horm Metab Res 48:20,t2016 Schlechte ]A: Long-term management of prolactinomas. ] Clin Endocrinol Metab 92:2861, 2007 Schnatz PF, haxton S: Parathyroidectomy in the third trimester of pregnancy. Obstet Gynecol Surv 60:672, 2005 Schneiderman M, Czuzoj-Shulman N, Spence AR, et al: Maternal and neonatal outcomes of pregnancies in women with Addison's disease: a populationbased cohort study on 7.7 millions births. B]OG 124:1772,t2017 Scoccia B, Demir H, Kang Y, et al: In viro fertilization pregnancy rates in levothyroxine-treated women with hypothyroidism compared to women without thyroid dysfunction disorders. TIlyroid 22(6):631, 2012

1	Sheehan HL: Post-partum necrosis of the anterior pituitary. ] Pathol Bacteriol 45:189, 1937 Sheehan PM, Nankervis A, Araujo ]linior E, et al: Maternal thyroid disease and preterm birth: systematic review and meta-analysis. ] Clin Endocrinol Metab 100(11):4325, 2015 Sheield ]S, Cunningham FG: Thyrotoxicosis and heart failure that complicate pregnancy, Am ] Obstet Gynecol 190:211, 2004 Shoback 0: Hypoparathyroidism. N Engl] Med 359:391, 2008 Siu CW, Zhang XH, Yung C, et al: Hemodynamic changes in hyperthyroidismrelated pulmonary hypertension: a prospective echocardiographic study. ] Clin Endocrinol Metab 92: 1736, 2007 Song SI, Daneman D, Rovet]: he inluence of etiology and treatment factors on intellectual outcome in congenital hypothyroidism. ] Dev Behav Pediatr 6,t2001 Stagnaro-Green A: Maternal thyroid disease and preterm delivery.t] Clin Endocrinol Metab 94:21, 2009

1	Stagnaro-Green A: Maternal thyroid disease and preterm delivery.t] Clin Endocrinol Metab 94:21, 2009 Stagnaro-Green A: Overt hyperthyroidism and hypothyroidism during pregnancy. Clin Obstet Gynecol 54(3):478, 2011a Stagnaro-Green A, Glinoer 0: Thyroid autoimmunity and the risk of miscarriage. Baillieres Best Pract Res Clin Endocrinol Metab 18: , 2004 Stagnaro-Green A, Pearce E: hyroid disorders in pregnancy. Nat Rev Endocrinol 8:650, 20t12a Stagnaro-Green A, Schwartz A, Gismondi R, et al: High rate of persistent hypothyroidism in a large-scale prospective study of postpartum thyroiditis in southern Italy. ] Clin Endocrinol Metab 96(3):652, 201t1 b Stagnaro-Green A, Sullivan S, Pearch EN: Iodine supplementation during pregnancy and lactation.t]AMA 308(23):2463, 2012b Stringer KM, Gought], Gough IR: Primary hyperparathyroidism during pregnancy: management by minimally invasive surgery based on ultrasound localization. ANZ] Surg 87(10):E134, 2017

1	Stringer KM, Gought], Gough IR: Primary hyperparathyroidism during pregnancy: management by minimally invasive surgery based on ultrasound localization. ANZ] Surg 87(10):E134, 2017 Stulberg RA, Davies GAL: Maternal thyrotoxicosis and fetal nonimmune hydrops. Obstet Gynecol 95: 1036, 2000 Su PY, Huang K, Hao ]H, et al: Maternal thyroid function in the first rwenry weeks of pregnancy and subsequent fetal and infant development: a prospective population-based cohort study in China. ] Clin Endocrinol Metab 96(10):3234,t201t1 Surks MI, Ortiz E, Daniels GH, et al: Subclinical thyroid disease: scientiic review and guidelines for diagnosis and management.]AMA 291(2):228,t2004 Swanson CA, Zimmerman MB, Skeaf S, et al: Summary of an NIH workshop to identiy research needs to improve the monitoring of iodine status in the United States and to inform the DRIl-3.] Nutr 142:1175S, 2012

1	Tan TO, Cheng W, Caughey AB: Are women who are treated for hypothyroidism at risk for pregnancy complications? Am] Obstet Gynecol 194:el, 2006 T eng W, Shan Z, T eng X, et al: Efect of iodine intake on thyroid diseases in China. N Englt] Med 354:2783, 2006 Tessnow A, Wilson ]: The changing face of Sheehan's syndrome. Am] Med Sci 340(5):402, 2010 Thangaratinam S, Tan A, Knox E, et al: Association berween thyroid autoantibodies and miscarriage and preterm birth: meta-analysis of evidence. BM] 342:d2616, 2011 Thomas M, Weisman SM: Calcium supplementation during pregnancy and lactation: efects on the mother and the fetus. Am ] Obstet Gynecol 194:937,t2006 Thomas SK, Sheield ]S, Roberts SW: Thionamide-induced neutropenia and ecthyma in a pregnant patient with hyperthyroidism. Obstet Gynecol 122:940,t2013 Thorpe-Beeston ]G, Nicolaides KH, Snijders RJ, et al: Thyroid function in small for gestational age fetuses. Obstet Gynecol 77: 701, 1991

1	Thorpe-Beeston ]G, Nicolaides KH, Snijders RJ, et al: Thyroid function in small for gestational age fetuses. Obstet Gynecol 77: 701, 1991 To W, Wong MW, Leung TW: Relationship berween bone mineral density changes in pregnancy and maternal and pregnancy characteristics: a longitudinal study. Acta Obstet Gynecol Scand 82:820, 2003 Tran P, DeSimone S, Barrett M, et al: 1-131 treatment of Graves' disease in an unsuspected irst trimester pregnancy; the potential for adverse efects on the fetus and a review of the current guidelines for pregnancy screening. Int ] Pediatr EndocrinoIt2010:858359, 2010 Tudela CM, Casey BM, McIntire DD, et al: Relationship of subclinical thyroid disease to the incidence of gestational diabetes. Obstet Gynecol 119(5):983,t2012 Vaidya B, Anthony S, Bilous M, et al: Detection of thyroid dysfunction in early pregnancy: universal screening or targeted high-risk case finding? ] Clin Endocrinol Metab 92(1):203, 2007

1	Vaidya B, Anthony S, Bilous M, et al: Detection of thyroid dysfunction in early pregnancy: universal screening or targeted high-risk case finding? ] Clin Endocrinol Metab 92(1):203, 2007 Vargas Zapata CL, Donangelo CM, Woodhouse LR, et al: Calcium homeostasis during pregnancy and lactation in Brazilian women with low calcium intakes: a longitudinal study. Am] Clin Nutr 80:417,t2004 Velasco I, Carreira M, Santiago P, et al: Efect of iodine prophylaxis during pregnancy on neurocognitive development of children during the first rwo years of life. ] Clin Endocrinol Metab 94:3234, 2009 Vydt T, Verhelst ], De Keulenaer G: Cardiomyopathy and thyrotoxicosis: tachycardiomyopathy or thyrotoxic cardiomyopathy? Acta Cardiol 61:1t15, 2006 Wallia A, Bizhanova A, Huang W, et aI: Acute diabetes insipidus mediated by vasopressinase after placental abruption. ] Clin Endocrinol Metab 98:881, 20t13

1	Wallia A, Bizhanova A, Huang W, et aI: Acute diabetes insipidus mediated by vasopressinase after placental abruption. ] Clin Endocrinol Metab 98:881, 20t13 Wang W, Teng W, Shan Z, et al: he prevalence of thyroid disorders during early pregnancy in China: the beneits of universal screening in the irst trimester of pregnancy. Eur] Endocrinol 164(2):263, 2011 Wikner BN, Sparre LS, Stiller CO, et al: Maternal use of thyroid hormones in pregnancy and neonatal outcome. Acta Obstet Gynecol Scand 87(6):617, 2008 Wilson KL, Casey BM, McIntire DO, et al: Diagnosis of subclinical hypothyroidism early in pregnancy is a risk factor for the development of severe preeclampsia. Clin Thyroidol 24(5):15, 2012 Yassa L, Marqusee E, Fawcett R, et al: hyroid hormone early adjustment in pregnancy (the THERAPy) trial. ] Clin Endocrinol Metab 95(7):3234, 2010

1	Yassa L, Marqusee E, Fawcett R, et al: hyroid hormone early adjustment in pregnancy (the THERAPy) trial. ] Clin Endocrinol Metab 95(7):3234, 2010 Yoshihara A, Noh ]Y, Mukasa K, attel: Serum human chorionic gonadotropin levels and thyroid hormone levels in gestational transient thyrotoxicosis: is the serum hCG level useful for diferentiating berween active Graves' disease and GTT? Endocr] 62(6):557,t2015 Yoshihara A, Noh ]Y, Yamaguchi T, et al: Treatment of Graves disease with antithyroid drugs in the irst trimester of pregnancy and the prevalence of congenital malformation. ] Clin Endocrinol Metab 9 :2396, 2012 Zuluaga-Gomez A, Arrabal-Polo A, Arrabal-Martin M, et al: Management of pheochromocytoma during pregnancy: laparoscopic adrenalectomy. Am 8(3):E156,t2012 IMMUNE-MEDIATED CONNECTIVE TISSUE DISEASES.. 1138 SYSTEMIC LUPUS ERYTHEMATOSUS ............... 1139

1	IMMUNE-MEDIATED CONNECTIVE TISSUE DISEASES.. 1138 SYSTEMIC LUPUS ERYTHEMATOSUS ............... 1139 ANTI PHOSPHOLIPID SYNDROME ......i.......i.... 1i143 ..RHEUMATOID ARTHRITIS .....i................i.... 1146 SYSTEMIC SCLEROSIS-SCLERODERMA.. . . . . . . . . .. 1148 VASCULITIS SYNDROMES.. . . . . . . . . . . . . . . . . . . . . .. 1149 INFLAMMATORY MYOPATHIES .........i......... 1150 HEREDITARY CONNECTIVE TISSUE DISORDERS.. . . .. 1151 Owing to the great vasculariy incident to pregnancy, the various pelvic joints always show a somewhat increased motiliy. In rare instances, particulary when the pelvis is contracted in the lower portion, spontaneous rupture of the symphysis pubis or one or both sacro-iliac joints has been observe. -]. Whitridge Williams (1903)

1	-]. Whitridge Williams (1903) The principal concerns in the 1st edition of Williams Obstetrics with disorders of the joints were the obstructed pelvis caused by rickets. There is no mention of the arthritides complicating pregnancy. And of course, immune-mediated disease had not yet been elucidated.

1	Connective tissue disorders, which are also termed collagen vascular disorders, have two basic underlying causes. First are the immune-complex diseases in which connective tissue damage is caused by deposition of immune complexes. Because these are manifest by sterile inflammation-predominately of the skin, joints, blood vessels, and kidneys-they are referred to as rheumatic diseases. Many of these immune-complex diseases are more prevalent in women, for example, systemic lupus erythematosus (SLE) and rheumatoid arthritis. Second are the inherited disorders of bone, skin, cartilage, blood vessels, and basement membranes. Some examples include Marfan syndrome, osteogenesis imperfecta, and Ehlers-Danlos syndrome.

1	hese disorders can be separated into those associated with and those without autoantibody formation. The rheumatoid actor (F) is an autoantibody found in many autoimmune inflammatory conditions such as SLE, rheumatoid arthritis, systemic sclerosis (scleroderma), mixed connective tissue disease, dermatomyositis, polymyositis, and various vasculitis syndromes. he F-seronegative spondyloarthropathies are strongly associated with expression of the human leukocyte antigen B27 (HLAB27) antigen and include ankylosing spondylitis, psoriatic arthritis, Reiter disease, and other arthritis syndromes.

1	Pregnancy may mitigate activity in some of these syndromes as a result of the immunosuppression that also allows successful engraftment of fetal and placental tissues. These changes are discussed in detail in Chapters 4 (p. 58) and 5 (p. 95). One example is pregnancy-induced predominance ofT2 helper cells compared with cytokine-producing T 1 helper cells (Keeling, 2009). Pregnancy hormones also alter immune cells. Namely, estrogens upregulate and androgens down regulate T-cell response, and progesterone is immunosuppressive (Cutolo, 2006; Haupl, 2008a; Robinson, 2012).

1	In contrast, immune-mediated disease may contribute to obstetrical complications. One longitudinal cohort study found that unrecognized autoimmune systemic rheumatic disorders are associated with significant risk for preeclampsia and fetalgrowth restriction (Spinillo, 2016). In this study, the prevalence of unrecognized rheumatic arthritis was 0.4 percent, and was 0.3 percent each for SLE, Sjogren syndrome, and antiphospholipid syndrome.

1	Last, some immune-mediated diseases may either be caused or activated as a result of prior pregnancies. To explain, fetal cells and free fetal DNA are detectable in maternal blood beginning early in pregnancy (Simpson, 2013; Sitar, 2005; Waldorf, 2008). Fetal cell microchimerism is the persistence of fetal cells in the maternal circulation and in organs following pregnancy. These fetal cells may become engrafted in maternal tissues and stimulate autoantibodies. This raises the possibility that fetal cell micro chimerism leads to the predilection for autoimmune disorders among women (Adams, 2004). Evidence for this includes fetal stem cells engrafted in tissues in women with autoimmune thyroiditis and systemic sclerosis Gimenez, 2005; S;ivatsa, 2001). Such microchimerism has also been described in women with SLE and those with rheumatoid arthritis-associated HLA alleles (da Silva, 2016; Lee, 2010; Rak, 2009a). Conversely, engrafted maternal cells may provoke autoimmune conditions in a

1	in women with SLE and those with rheumatoid arthritis-associated HLA alleles (da Silva, 2016; Lee, 2010; Rak, 2009a). Conversely, engrafted maternal cells may provoke autoimmune conditions in a woman's ofspring (Ye, 2012; Stevens, 2016).

1	Lupus is a heterogeneous autoimmune disease with a complex pathogenesis that results in interactions between susceptibility genes and environmental factors (Hahn, 2015). Immune system abnormalities include overactive B lymphocytes that are responsible for autoantibody production. hese result in tissue and cellular damage when autoantibodies or immune complexes are directed at one or more cellular nuclear components (T sokos, 201r1). In addition, immunosuppression is impaired, including regulatory T-cell function (Tower, 2013). Some autoantibodies produced in patients with SLE are shown in Table Almost 90 percent of SLE cases are in women, and its prevalence in those of childbearing age approximates 1 in 500 (Lockshin, 2000). Accordingly, the disease is encountered relatively frequently during pregnancy. The 10-year survival rate is 70 to 90 percent (Tsokos, 201r1). Infection, lupus lares, endorgan failure, hypertension, stroke, and cardiovascular disease account for most deaths.

1	Genetic inluences are implicated by a higher concordance with monozygotic compared with dizygotic twins-25 versus 2 percent, respectively. Moreover, frequency in patients with one afected family member is 10 percent. he relative risk of disease rises if there is inheritance of the "autoimmunity gene" on chromosome 16 that predisposes to SLE, rheumatoid arthritis, Crohn disease, and psoriasis. Susceptibility genes such as HA-Al, -B8, -D3, -DBl, and -TET3 explain only a portion of the genetic heritability (Tsokos, 201r1; Yang, 2013). Interestingly, even maternal exposure to fetal genes elevates susceptibility to SLE development. A case-control study found that a child's HLA-DRB1 genotype increases the risk of SLE in the mother (Cruz, 2016). Furthermore, neonatal lupus erythematosus has been reported in an infant conceived via oocyte donor to a mother with autoimmune disease with circulating anti-Ro and anti-La antibodies (Chiou, 2016).

1	Lupus is notoriously variable in its presentation, course, and outcome (Table 59-2). Findings may be conined initially to one organ system, and others become involved later. Or, the disease may irst be multisystem. Frequent findings are malaise, fever, arthritis, rash, pleuropericarditis, photosensitivity, anemia, and cognitive dysfunction. At least half of patients have renal involvement. SLE is also associated with declines in attention, memory, and reasoning (Hahn, 2015; Kozora, 2008).

1	Identification of antinuclear antibodies (ANA) is the best screening test, however, a positive result is not speciic for SLE. For example, low titers are found in normal individuals, other autoimmune diseases, acute viral infections, and chronic inlammatory processes. Several drugs can also cause a positive reaction. Antibodies to double-stranded DNA (dsDNA) and to Smith (Sm) antigens are relatively speciic for SLE, whereas other antibodies are not (see Table 59-1). Although hundreds of autoantibodies have been described in SLE, only a few have been shown to participate in tissue injury (Sherer, 2004; Tsokos, 2011). Currently, micro array proiles are being developed for more accurate SLE diagnosis (Putterman, 2016). TABLE 59-1. Some Autoantibodies Produced in Patients with Systemic Lupus Erythematosus (SLE)

1	TABLE 59-1. Some Autoantibodies Produced in Patients with Systemic Lupus Erythematosus (SLE) Best screening test, multiple antibodies; a second negative test makes SLE unlikely High titers SLE-specific; may correlate with disease activity, nephritis, and vasculitis Specific for SLE Not SLE-specific, high titers associated with rheumatic syndromes Not SLE-specific; associated with sicca syndrome, predisposes to cutaneous lupus, neonatal lupus with heart block, reduced risk of nephritis Associated with anti-Ro Common in drug-induced lupus Lupus anticoagulant and anticardiolipin antibodies associated with thrombosis, fetal loss, thrombocytopenia, valvular heart disease; false-positive test for syphilis Direct Coombs test, may develop hemolysis Thrombocytopenia in 15%; poor clinical test dsDNA = double-stranded DNA; RNP = ribonucleoprotein. Data from Arbuckle, 2003; Hahn, 2015. TABLE 59-2. Some Clinical Manifestations of Systemic Lupus Erythematosus Modified from Kasper, 201r5.

1	TABLE 59-2. Some Clinical Manifestations of Systemic Lupus Erythematosus Modified from Kasper, 201r5. Anemia develops frequently, and there may be leukopenia and thrombocytopenia. Proteinuria and casts are found in the half of patients with glomerular lesions. Lupus nephritis can also cause renal insuiciency, which is more common if there are anti phospholipid antibodies (Moroni, 2004). Other laboratory indings include false-positive syphilis serology, prolonged partial thromboplastin time, and higher RF levels. Elevated serum D-dimer concentrations often follow a lare or infection, but unexplained persistent elevations are associated with a high risk for thrombosis (Wu, 2008). The diagnostic criteria for SLE are listed in Table 59-3. If any four or more of these 11 criteria are present, serially or TABLE 59-3. Clinical Criteria for Classification of Systemic Lupus Erythematosus Renal: proteinuria, casts, biopsy Neurological: seizures, psychosis, myelitis, neuropathies,

1	TABLE 59-3. Clinical Criteria for Classification of Systemic Lupus Erythematosus Renal: proteinuria, casts, biopsy Neurological: seizures, psychosis, myelitis, neuropathies, ANA = antinuclear antibodies; dsDNA = double-stranded DNA; Sm = Smith. Data from Hahn, 201r5; Hochberg, 1997. simultaneously, the diagnosis of lupus is made. Importantly, numerous drugs can induce a lupus-like syndrome. These include proton-pump inhibitors, thiazide diuretics, antifungals, chemotherapeutics, statins, and antiepileptics. Druginduced lupus is rarely associated with glomerulonephritis and usually regresses when the medication is discontinued (Laurinaviciene, 2017).

1	Of nearly 16.7 million pregnancies from 2000 to 2003 in the United States, 13,555 were complicated by lupus-an incidence of approximately 1 in 1250 pregnancies (Clowse, 2008). During pregnancy, lupus improves in a third of women, remains unchanged in a third, and worsens in the remaining third. Thus, in any given pregnancy, the clinical condition can worsen orlare without warning (Hahn, 2015; Khamashta, 1997). Petri (1998) reported a 7 -percent risk of major morbidity during pregnancy. In a cohort of 13,555 women with SLE during pregnancy, the maternal mortality and severe morbidity rate was 325 per 100,000 (Clowse, 2008). In a review of 13 studies with 17 maternal deaths attributable to SLE and lupus nephritis, all occurred in those with active disease (Ritchie, 2012). Results of a prospective cohort study of 385 women are shown in Figure

1	During the past several decades, pregnancy outcomes in women with SLE have improved remarkably. For most women with inactive or mild/moderate SLE, pregnancy outcomes are relatively favorable. Women who have conined cutaneous lupus do not usually have adverse outcomes (Hamed, 2013). However, newly diagnosed SLE during pregnancy tends to be severe (Zhao, 2013). In general, pregnancy outcome is best in women for whom: (1) lupus activity has been quiescent for at least 6 months before conception; (2) there is no lupus nephritis manifest by proteinuria or renal dysfunction; (3) anti phospholipid syndrome or lupus anticoagulant is absent; and (4) superimposed preeclampsia does not develop (Peart, 2014; Stojan, 2012; Wei, 2017; Yang, 2014). SLE, low risk (n = 129) . 10

1	SLE, low risk (n = 129) . 10 FIGURE 59-1 Frequency of adverse pregnancy outcomes. All women with systemic lupus erythematosus (SLE) in the PROMISSE study are compared with a subset of low-risk SLE patients and with control patients without SLE. (Data from Buyon, 201o5.) Active nephritis is associated with adverse pregnancy outcomes, ease remains in remission (Moroni, 2002, 2005; Stojan, 2012).

1	Of complications, women with renal disease have a high inci dence of gestational hypertension and preeclampsia. Of 80 gravidas with SLE reported by Lockshin (1989), 63 percent of women with preexisting renal disease developed preeclampsia compared with only 14 percent of those without underlying renal disease. In a review of 309 pregnancies complicated by lupus nephritis, 30 percent sufered a lare, and 40 percent of these had associated renal insuiciency (Moroni, 2005). he maternal mortality rate was 1.3 percent. These indings were corroborated in a subsequent prospective study (Moroni, 2016b). In addition, a third of the 113 pregnancies were delivered preterm (Imbasciati, 2009; Moroni, 2016a). Wagner and coworkers (2009) compared outcomes of 58 women with 90 pregnancies and found that active nephritis was linked with a signiicantly higher incidence of maternal complications-57 versus 11 percent.

1	Most recommend continuation during pregnancy of immunosuppressive therapy for nephritis. New-onset nephritis or severe renal lare is treated aggressively with intravenous corticosteroids and consideration of immunosuppressive drugs or intravenous immunoglobulin (Lazzaroni, 2016).

1	Chronic hypertension complicates up to 30 percent of pregnancies in women with SLE (Egerman, 2005). Also, as mentioned, preeclampsia is common, and superimposed preeclampsia is encountered even more often, and earlier, in those with nephritis or antiphospholipid antibodies (Bertsias, 2008). Preeclampsia and lupus nephritis share features of hypertension, proteinuria, edema, and renal function deterioration. However, the management is distinct, as lupus nephritis is treated with immunosuppression, and severe preeclampsia/eclampsia requires delivery (Lazzaroni, 2016). It may be diicult, if not impossible, to diferentiate lupus lare with nephropathy from severe preeclampsia if the kidney is the only involved organ (Petri, 2007). Central nervous system involvement with lupus may culminate in convulsions similar to those of eclampsia. One proposed schema for diferentiating the two is shown in Table 59-4. Management for preeclampsia-eclampsia is described in Chapter 40 (p. 728).

1	Lupus management consists primarily of monitoring fetal well-being and maternal clinical and laboratory status (Lateef, 2012). Pregnancy-induced thrombocytopenia and proteinuria resemble SLE disease activity, and the identiication of a lupus lare is confounded by the increased facial and palmar erythema of normal pregnancy (Lockshin, 2003). For SLE activity monitoring, various laboratory techniques have been recommended, but interpretation may be challenging. The sedimentation rate may be misleading because of pregnancy-induced hyperibrinogenemia. Serum complement levels are also normally increased in pregnancy (Appendix, p. 1259). And, although falling or low levels of complement components C3, C4, and CH50 are more likely to be associated with active disease, higher levels provide no assurance against disease activation. Our experiences and those of others suggest that clinical manifestations of disease and complement levels correlate poorly (Lockshin, 1995; Varner, 1983).

1	Serial hematological studies may detect changes in disease activity. Hemolysis is characterized by a positive Coombs test, anemia, reticulocytosis, and unconjugated hyperbilirubinemia. hrombocytopenia, leukopenia, or both may develop. According to Lockshin and Druzin (1995), chronic thrombocytopenia Data from Andreoli, 2012. in early pregnancy may be due to anti phospholipid antibodies. Later, thrombocytopenia may indicate preeclampsia.

1	Data from Andreoli, 2012. in early pregnancy may be due to anti phospholipid antibodies. Later, thrombocytopenia may indicate preeclampsia. Urine is tested frequently to detect new-onset or worsening proteinuria. The fetus is closely observed for adverse efects such as growth restriction and oligohydramnios. Many recommend screening for anti-SS-A (anti-Ro) and anti-SS-B (anti-La) antibodies, because of associated fetal complications described subsequently. Antepartum, the fetus is surveilled as outlined by the American College of Obstetricians and Gynecologists (2016a) and described in Chapter 17 (p. 331). Unless hypertension or evidence of fetal compromise or growth restriction develops, pregnancy is allowed to progress to term. Peripartum corticosteroids in "stress doses" are given to women who are taking these drugs or who recently have done so.

1	here is no cure for SLE, and complete remissions are rare. Approximately a fourth of pregnant women have mild disease, which is not life threatening, but may be disabling because of pain and fatigue. Arthralgia and serositis can be managed by nonsteroidal antiinflammatory drugs (NSAIDs). However, chronic or large intermittent dosing is avoided due to related oligohydramnios or ductus arteriosus closure (Chap. 12, p. 241). Low-dose aspirin can be used throughout gestation. Severe disease is managed with corticosteroids such as prednisone, 1 to 2 mg/kg/d orally. Mter the disease is controlled, this dose is tapered to a daily morning dose of 10 to 15 mg. Corticosteroid therapy can lead to gestational diabetes.

1	Immunosuppressive agents such as azathioprine are beneicial for active disease. These are usually reserved for lupus nephritis or disease that is corticosteroid resistant. Azathioprine has a good safety record during pregnancy (Fischer-Betz, 2013; Petri, 2007). Its recommended daily oral dose is 2 to 3 mg/kg. Teratogenic medications to be avoided include mycophenolate mofetil, methotrexate, and cyclophosphamide (Gotestam Skorpen, 2016). However, cyclophosphamide can be considered in the second or third trimester for severe disease (Lazzaroni, 2016). In some situations, mycophenolate is the only treatment that achieves disease stability. In these cases, counseling is essential regarding fetal risks described in Chapter 12 (p. 244) (Bramham, 2012).

1	Antimalarials reduced dermatitis, arthritis, and fatigue (Hahn, 2015). Although these agents cross the placenta, hydroxychloroquine is not associated with congenital malformations. Because of the long half-life of antimalarials and because discontinuing therapy can precipitate a lupus lare, most recommend their continuation during pregnancy (Borden, 2001). 'When severe disease supervenes-usually a lupus larehigh-dose glucocorticoid therapy is given. Petri (2007) recommends pulse therapy consisting of methylprednisolone, 1000 mg given intravenously over 90 minutes daily for 3 days, then a return to maintenance doses if possible. In nonpregnant subjects, antihypertensive therapy oten includes an angiotensin-converting enzyme (ACE) inhibitor or an angiotensin receptor blocker. For pregnancy, these should be changed to safer fetal options such as calcium-channel blockers, alpha methyldopa, or labetalol (Cabiddu, 2016).

1	Adverse perinatal outcome rates are signiicantly elevated in pregnancies complicated by SLE. Among these are preterm delivery, fetal-growth restriction, stillbirth, and neonatal lupus syndrome (Madazli, 2014; Phansenee, 2017). Perinatal outcome rates worsen in mothers with a lupus flare, significant proteinuria, or renal impairment and in those with chronic hypertension, preeclampsia, or both (Lazzaroni, 2016). Adverse outcomes are also more common in women with neuropsychiatric lupus (de Jesus, 2017). Reasons at least partially responsible for adverse fetal consequences include decidual vasculopathy with placental infarction and decreased perfusion (Hanly, 1988).

1	This is characterized by newborn skin lesions-lupus dermatitis; a variable number of hematological and systemic derangements; and occasionally congenital heart block (Hahn, 2015). Cutaneous manifestations can be present in 30 to 40 percent of infants and appears at 4 to 6 weeks of age (Silverman, 2010). hese are usually associated with anti-SS-A and SS-B antibodies, and approximately 40 percent of women with SLE are positive for these (Buyon, 2015). Thrombocytopenia and hepatic involvement are seen in 5 to 10 percent of afected infants.

1	In a review of outcomes in 91 infants born to women with lupus, eight of these were possibly afected-four had deinite neonatal lupus and four had possible disease (Lockshin, 1988). Cutaneous lupus, thrombocytopenia, and autoimmune hemolysis are transient and clear within a few months (Zuppa, 2017). This is not always so for congenital heart block, discussed next. In subsequent ofspring, the recurrence risk for neonatal lupus may near 25 percent Qulkunen, 1993).

1	Fetal and neonatal heart block results from difuse myocarditis and ibrosis in the region between the atrioventricular (A ) node and bundle of His. Congenital heart block develops almost exclusively in fetuses of women with antibodies to the SS-A or SS-B antigens (Buyon, 1993). Even in the presence of such antibodies, however, the incidence of fetal myocarditis is only 2 to 3 percent but rises to 20 percent with a prior afected child (Bramham, 2012; Lockshin, 1988). Fetal cardiac monitoring is performed between 18 and 26 weeks' gestation in pregnancies with either of these antibodies. The cardiac lesion is permanent, and a pacemaker is generally necessary. Long-term prognosis is poor. Of 325 infants with cardiac neonatal lupus, nearly 20 percent died, and of these, one third was stillborn (Izmirly, 2011).

1	Maternal administration of corticosteroids, plasma exchange, or intravenous immunoglobulin does not reduce the risk of congenital heart block. Maternal corticosteroid administration for treatment congenital heart block is controversial, is currently not recommended, and is discussed further in Chapter 16 (p. 316). Although this therapy to treat fetal heart block has not been studied in randomized trials, some evidence suggests that early fetal exposure to the mother's corticosteroid treatment for SLE may mitigate fetal myocarditis. Namely, Shinohara and coworkers (1999) reported no heart block in 26 neonates whose mothers received corticosteroids before 16 weeks' gestation as part of SLE maintenance therapy. By contrast, 15 of 61 neo nates with heart block were born to women in whom corticoste roid therapy was begun after 16 weeks for an SLE exacerbation.

1	There are reports that maternal treatment with hydroxychlo roquine (Plaquenil) is associated with a lower incidence of fetal heart block (Izmirly, 2012). Research in this area is ongoing.

1	The survival rate for women with SLE is 95 percent at 5 years, 90 percent at 10 years, and 78 percent at 20 years (Hahn, 2015). In general, women with lupus and chronic vascular or renal disease may limit family size because of morbidity associated with the disease and greater risks for adverse perinatal outcomes. For contraception, combination oral contraceptives did not increase the incidence oflupus lares in two large multicenter trials (Petri, 2005; Sanchez-Guerrero, 2005). Progestinonly implants and injections provide efective contraception with no known efects on lupus lares (Sammaritano, 2014). Concerns that intrauterine device (IUD) use and immunosuppressive therapy lead to greater infection rates in these patients are not evidenced-based. Notably, comorbid antiphospholipid antibodies are a contraindication to hormonal methods. Tubal sterilization may be advantageous and is performed with greatest safety postpartum or whenever SLE is quiescent.

1	his syndrome is an autoantibody-mediated acquired thrombophilia with recurrent thrombosis or pregnancy morbidity as part of its clinical constellation (Moutsopoulos, 2015). Speciically, antiphospholipid syndrome (APS) is diagnosed in women with persistently positive serum tests for antiphospholipid antibodies andwith arterial and/or venous thromboses or obstetrical morbidity. Antibodies include lupus anticoagulant, anti cardiolipin antibody, and anti-32 glycoprotein-I antibody.

1	Phospholipids are the main lipid constituents of cell and organelle membranes. There are proteins in plasma that associate noncovalently with these phospholipids. Antiphospholipid antibodies are directed against these phospholipids or phospholipid-binding proteins (Giannakopoulos, 2013; Tsokos, 201r1). his antibody group may be ofIgG, IgM, and IgA classes, alone or in combination. Antiphospholipid antibodies are most common with SLE, other connective tissue disorders, and APS. However, a small proportion of otherwise normal women and men have low levels of these antibodies.

1	The stimulus for autoantibody production is unclear, but it possibly is due to a preceding infection. he pathophysiology encountered is mediated by one or more of the following: (1) activation of various procoagulants, (2) inactivation of natural anticoagulants, (3) complement activation, and (4) inhibition of syncytiotrophoblast diferentiation (Tsokos, 2011). Clinically, these actions lead to arterial or venous thromboses or to pregnancy morbidity. Virtually every organ system may be afected. Central nervous system involvement is one of the most prominent clinical manifestations. In addition to cerebrovascular arterial and venous thrombotic events, there may be psychiatric features and even multiple sclerosis (Binder, 2010). Renovascular involvement may lead to renal failure that can be diicult to diferentiate from lupus nephritis (D'Cruz, 2009).

1	Renovascular involvement may lead to renal failure that can be diicult to diferentiate from lupus nephritis (D'Cruz, 2009). Peripheral and visceral thromboses are also a feature. For example, Ahmed and associates (2009) reported a postpartum ing a mesenteric vessel infarction. Obstetrical complications relected by fetal-growth restriction, stillbirth, preeclampsia, and preterm birth. Treatment using aspirin, anticoagulation, and close monitoring has increased live birth rates to more than 70 percent in these women (Schreiber, 2016). A small proportion of these patients develop the catastrophic drome. his is deined as a rapidly progressive thromboembolic or tissues (Schreiber, 2016). It has a high mortality rate from activation of a cytokine storm. In half of cases, a "triggering" event is identiied. As mentioned, several antibodies in APS are directed against a speciic phospholipid or phospholipid-binding protein: 1.

1	32-Gycoprotein I-also known as apolipoprotein H-is a phospholipid-binding plasma protein that inhibits prothrombinase activity within platelets and inhibits platelet aggregation (Giannakopoulos, 2013). hus, its normal action is to limit procoagulant binding and thereby prevent coagulation cascade activation. Logically, antibodies directed against this glycoprotein would reverse its anticoagulant activity and promote thrombosis. This is important from an obstetrical viewpoint because 32-glycoprotein I is expressed in high concentrations on the syncytiotrophoblast surface. Complement activation may contribute to its pathogenesis (Avalos, 2009; Tsokos, 2011). Teleologically, this seems appropriate because the decidua intuitively should be a critical area to prevent coagulation that might lead to intervillous space thrombosis. Another possibility is that 32-glycoprotein I may be involved in implantation, and this glycoprotein may result in pregnancy loss via an inlammatory mechanism

1	lead to intervillous space thrombosis. Another possibility is that 32-glycoprotein I may be involved in implantation, and this glycoprotein may result in pregnancy loss via an inlammatory mechanism (Iwasawa, 2012; Meroni, 2011).

1	2. Lupus anticoagulant (LAC) is a heterogeneous group of antibodies directed against phospholipid-binding proteins. This antibody group induces prolongation in vitro of the prothrombin, partial thromboplastin, and Russell viper venom times. hus, paradoxically, this so-called anticoagulant is actually powerfully thrombotic in vivo. 3. Anticardiolipin antibodies (ACAs) are directed against one of the many phospholipid cardiolipins found in mitochondrial membranes and platelets. Some anti phospholipid antibodies are also directed against the natural anticoagulant proteins C and S (Robertson, 2006). Another is directed against the anticoagulant protein annexin V, which is expressed in high concentrations by syncytiotrophoblast TABLE 59-5. Some Clinical Features of Antiphospholipid Syndrome Venous thrombosis-thromboembolism, thrombophlebitis, livedo reticularis

1	TABLE 59-5. Some Clinical Features of Antiphospholipid Syndrome Venous thrombosis-thromboembolism, thrombophlebitis, livedo reticularis Arterial thrombosis-stroke, transient ischemic attack, Libman-Sacks cardiac vegetations, myocardial ischemia, distal extremity and visceral thrombosis and gangrene Hematological-thrombocytopenia, autoimmune hemolytic anemia Other-neurological manifestations, migraine headaches, epilepsy; renal artery, Pregnancy-preeclampsia syndrome, recurrent miscarriage, preterm delivery, fetal-growth restriction, fetal death Data from Giannakopoulos, 2013; Moutsopoulos, 2015.

1	Pregnancy-preeclampsia syndrome, recurrent miscarriage, preterm delivery, fetal-growth restriction, fetal death Data from Giannakopoulos, 2013; Moutsopoulos, 2015. (Giannakopoulos, 2013). Testing for these other antibodies is not recommended (American College of Obstetricians and Gynecologists, 2017). However, some studies have evaluated these nonconventional antiphospholipid antibodies in women who clinically meet APS criteria but do not have the classic antibody proile. In one study, treatment of women with these nonconventional antibodies ofered some beneits, such as a lower pregnancy loss rate (Mekinian, 2016).

1	Clinical features shown in Table 59-5 provide indications for testing. By international consensus, APS is diagnosed based on laboratory and clinical criteria found in Table 18-5 (p. 353). First, one of two clinical criteria-which are vascular thrombosis or certain pregnancy morbidity-must be present. With laboratory criteria, elevated levels of LAC, ACA, and anti-3zglycoprotein I should be conirmed on two occasions 12 weeks apart. he diagnosis can be further stratified based on the number of these tests that are positive (Miyakis, 2006). Tests for LAC are nonspeciic coagulation tests. he partial thromboplastin time is generally prolonged because the anticoagulant interferes with conversion of prothrombin to thrombin in vitro. Tests considered more speciic are the dilute Russell vper venom test and the platelet neutralization procedure. here is currently disagreement as to which of these two is best for screening. If either is positive, then identiication of LAC is conirmed.

1	Branch and Khamashta (2003) recommend conservative interpretation of results based on repeated tests from a reliable laboratory that are consistent with each clinical case. Only approximately 20 percent of patients with PS have a positive LAC assay alone. Thus, ACA enzyme-linked immunosorbent assay (ELISA) testing should also be performed. Eforts have been made to standardize ACA assays, however, these remain without international standards (Adams, 2013). For each APS test, interlaboratory variation can be large, and agreement between commercial kits is poor.

1	As noted, nonspecific low levels of antiphospholipid antibodies are identified in approximately 5 percent of normal adults (Branch, 2010). When Lockwood and coworkers (1989) irst studied 737 normal pregnant women, they reported that 0.3 percent had LAC and 2.2 percent had elevated concentrations of either IgM or IgG ACAs. Subsequent investigations confirmed this, and taken together, they totaled almost 4000 normal pregnancies with an average prevalence for antiphospholipid antibodies of 4.7 percent. This is similar to that for normal nonpregnant individuals (Harris, 1991 ; Yasuda, 1995).

1	In women with high ACA levels, and especially when AC is identiied, risks for decidual vasculopathy, placental infarction, fetal-growth restriction, early-onset preeclampsia, and recurrent fetal death are increased (Saccone, 2017). Some of these women, like those with SLE, also have a high incidence of venous and arterial thromboses, cerebral thrombosis, hemolytic anemia, thrombocytopenia, and pulmonary hypertension (American College of Obstetricians and Gynecologists, 2017; Clowse, 2008). In 191 LAC-negative women with APS, women with antibodies to cardiolipin and 32-glycoprotein I had signiicantly higher miscarriage rates than if either one alone was positive (Liu, 2013). Women with higher titers tend to have more adverse outcomes (Hadar, 2017).

1	It is not precisely known how antiphospholipid antibodies cause damage, but it is likely that their actions are multifactorial. Platelets may be damaged directly by antiphospholipid antibody or indirectly by binding 32-glycoprotein I, which causes platelets to be susceptible to aggregation (Giannakopoulos, 2013). One theory proposes that phospholipid-containing endothelial cell or syncytiotrophoblast membranes may be damaged directly by the antiphospholipid antibody or indirectly by antibody binding to either 3z-glycoprotein lor annexin V (Rand, 1997, 1998). his prevents the cell membranes from protecting the syncytiotrophoblast and endothelium. This exposes the basement membrane, to which damaged platelets can adhere and form a thrombus.

1	Pierro and associates (1999) reported that antiphospholipid antibodies decreased decidual production of the vasodilating prostaglandin E2. Diminished protein C or S activity and greater prothrombin activation may also be contributory (Zangari, 1997). Evidence also supports that thrombosis with APS stems from activation of the tissue factor pathway (Amengual, 2003). Finally, uncontrolled placental complement activation by anti phospholipid antibodies may playra role in fetal loss and growth restriction (Holers, 2002). Complications from APS cannot be completely explained by thrombosis alone. Animal models suggest that efects are due to inflammation rather than thrombosis (Cohen, 2011). Some investigators hypothesize that APS-associated clotting is triggered as a "second hit" from innate inflammatory immune responses. These investigators recommend treatment with antiinlammatory agents (Meroni, 2011).

1	Overall, antiphospholipid antibodies are generally associated with higher rates of fetal wastage (Chap. 18, p. 353). In most early reports that describe these outcomes, however, women were included because they had had repeated adverse outcomes. Both antibody prevalence and miscarriage are common-recall that the incidence of antiphospholipid antibodies in the general obstetrical population is about 5 percent and early pregnancy loss approximates 20 percent. Accordingly, current data are too limited to conclude the exact risks for adverse efects of these antibodies on pregnancy outcomes. Fetal deaths, however, are more characteristic with APS than are first-trimester miscar riages (Oshiro, 1996; Roque, 2004). Moreover, women with higher titers have worse obstetrical outcomes compared with those with low titers (Hadar, 2017; Nodler, 2009).

1	When otherwise unexplained fetal deaths are examined, the data are mixed. One study measured ACA levels in 309 pregnancies with fetal death and found no diferences in their frequency compared with levels in 618 normal pregnancies (Haddow, 1991). In another of women with recurrent pregnancy loss, those with antiphospholipid antibodies had a higher rate of preterm delivery (Clark, 2007). In a case-control study of 582 stillbirths and 1547 live births, a three-to fivefold higher risk for stillbirth was found in women with elevated ACA and anti-3rglycoprotein I levels (Silver, 2013). In women with antiphospholipid antibodies, adverse outcomes are more common in the presence of: (1) all three classic anti phospholipid antibody types, (2) comorbid SLE or systemic autoimmune diseases, and (3) prior thrombosis and pregnancy morbidity. Logistic regression found the probability of pregnancy failure was 93 percent with two or more antiphospholipid antibody types but was 63 percent for those with

1	thrombosis and pregnancy morbidity. Logistic regression found the probability of pregnancy failure was 93 percent with two or more antiphospholipid antibody types but was 63 percent for those with only one (Rufatti, 2011).

1	Because of study heterogeneity, current treatment recommendations for women with antiphospholipid antibodies can be confusing. Therapy is directed at thrombosis prevention. As discussed, antiphospholipid antibodies are immunoglobulins that may be of G, M, or A classes. Those directed against the phospholipids (PL) are termed GPL, MPL, and APL, respectively. During testing, these are reported as semiquantified phospholipid binding-unit levels and expressed as negative, low-positive, medium-positive, or high-positive (American College of Obstetricians and Gynecologists, 2017). Of the three, higher titers for GPL and MPL anticardiolipin antibodies are clinically important, whereas low-positive titers are of questionable clinical signiicance.

1	As discussed in Chapter 52 (p. 1008), women with prior thromboembolic events who have antiphospholipid antibodies are at risk for recurrence in subsequent pregnancies. For these women, prophylactic anticoagulation with heparin throughout pregnancy and then for 6 weeks postpartum with either heparin or warfarin is recommended (American College of Obstetricians and Gynecologists, 2017). For those without prior thromboembolic events, recommendations for management from the American College of Obstetricians and Gynecologists (2017) and the American College of Chest Physicians (Bates, 2012) vary and are listed in Table 52-6 (p. 1020). Some acceptable schemes include close antepartum maternal observation with or without prophylactic or intermediate-dose heparin, and some form of postpartum anticoagulation for 4 to 6 weeks. Scias results with treatment with hydroxychloroquine.

1	Several trials have questioned the need for heparin for women with antibodies but no history of thrombosis (Branch, 2010). lthough this is less clear, some recommend that fetal death not attributable to other causes (Dizon-Townson, 1998; Lockshin, 1995). Some report that women with recur rent early pregnancy loss and medium-or high-positive titers of antibodies may beneit from therapy (Robertson, 2006). Described earlier (p. 1143), catastrophic antiphospholipid syndrome is treated aggressively with full anticoagulation, high-dose corticosteroids, plasma exchange, and/or intravenous immunoglobulins (Cervera, 2010; Tenti, 2016). If needed, rituximab may be added (Sukara, 2015). Due to the risk of fetal-growth abnormalities and stillbirth, serial sonographic assessment of fetal growth and antepartum testing in the third trimester is recommended by the American College of Obstetricians and Gynecologists (2016a, 2017).

1	College of Obstetricians and Gynecologists (2016a, 2017). here are other agents used to treat pregnant women with APS, but with no prior thromboembolic event. Aspirin, in doses of 60 to 80 mg orally daily, blocks conversion of arachidonic acid to thromboxane A2 while sparing prostacyclin production. This reduces synthesis of thromboxane A2, which usually causes platelet aggregation and vasoconstriction, and simultaneously spares prostacyclin, which normally has the opposite efect. There appear to be no major side efects from low-dose aspirin other than a slight risk of small-vessel bleeding during surgical procedures. Low-dose aspirin does not reduce adverse pregnancy outcomes in anti phospholipid antibody-positive women without the complete APS syndrome (Amengual, 2015). Its use is recommended for women with SLE or APS (American College of Obstetricians and Gynecologists, 20 16b).

1	Unractionated heparin is given subcutaneously in dosages of 5000 to 10,000 units every 12 hours. Some prefer lowmolecular-weight heparin, such as 40 mg enoxaparin (Lovenox) once daily (Kwak-Kim, 2013). With therapeutic dosing, measurement of heparin levels may be useful because clotting tests can be altered by LAC. he rationale for heparin therapy is to prevent venous and arterial thrombotic episodes. Heparin therapy also prevents thrombosis in the microcirculation, including the decidual-trophoblastic interface (Toglia, 1996). As discussed, heparin binds to 3rglycoprotein I, which coats the syncytiotrophoblast. This prevents binding of ACAs and anti-3rglycoprotein I antibodies to their surfaces, which likely prevents cellular damage (Tsokos, 2011). Heparin also binds to antiphospholipid antibodies in vitro and likely in vivo. Aspirin plus heparin therapy is the most eicacious regimen (de

1	Jesus, 2014). However, heparin therapy is associated with several complications that include bleeding, thrombocytopenia, TABLE 59-6. Pregnancy Outcomes (%) in 750 Women Treated for the Antiphospholipid Syndrome-the PREGNANTS Study ACA = anticardiolipin antibodies; LAC = lupus anticoagulant. aNonsevere only. Data from Saccone, 201r7. osteopenia, and osteoporosis. A description of various heparins and their adverse efects is found in Chapter 52 (p. 1012). Corticosteroids generally should not be used with primary APS-that is, without an associated connective tissue disorder. For women with SLE or those being treated for APS who develop SLE, corticosteroid therapy is indicated (Carbone, 1999). In such cases ofseconday APS seen with SLE, the dose of prednisone should be maintained at the lowest efective level to prevent lares.

1	Intravenous immunoglobulin therapy (NIG) is controversial and has usually been reserved for women with overt diseaseincluding CAPS or heparin-induced thrombocytopenia or both (Alijotas-Reig, 2013). It is used when other first-line therapies have failed, especially in the setting of preeclampsia and fetalgrowth restriction. IlG is administered by some in doses of 0.4 g/kgl d for 5 days-total dose of 2 g/kg. This is repeated monthly, or it is given as a single dose of 1 g/kg each month. Treatment is expensive, as one course costs more than $10,000. A recent literature review found no beneits from adding IlG to low-dose aspirin and low-molecular-weight heparin (Tenti, 2016). And, a Cochrane review found no improvement in the live birth rate for immunotherapy given to women with recurrent pregnancy loss (Wong, 2014). Trials are needed before application of this expensive and cumbersome therapy becomes widespread.

1	Immunosuppression with hydroxychloroquine may be beneficial with APS by reducing the risk of thrombosis and improving pregnancy outcomes in women with APS (Mekinian, 2015; Sciascia, 2016). Hydroxychloroquine is commonly used with low-dose aspirin in the treatment of women with antiphospholipid antibodies and SLE. Statins have been examined due to their protective efects on endothelium. In a small trial in 21 women with APS who developed fetal-growth restriction or preeclampsia, the addition of pravastatin to low-dose aspirin and low-molecular-weight heparin improved placental blood flow, preeclampsia features, and pregnancy outcomes (Lekou, 2016). Larger trials are needed.

1	Fetal loss is common in women with APS if untreated (Rai, 1995). Even with treatment, recurrent fetal loss rates remain at 20 to 30 percent (Branch, 2003; Empson, 2005; Ernest, 2011). Shown in Table 59-6 are pregnancy outcomes from 750 treated women with primary APS-the PREGNANTS study (Saccone, 2017). Participants were treated with low-dose aspirin and prophylactic low-molecular-weight heparin starting in the irst trimester. Importantly, some women with SLE and antiphospholipid antibodies have normal pregnancy outcomes without treatment. Also, it is emphasized that women with LAC and prior bad pregnancy outcomes have had liveborn neonates without treatment.

1	In a manner similar to neonatal lupus syndrome (p. 1142), up to 30 percent of newborns demonstrate passively acquired antiphospholipid antibodies, and thus there is concern for their adverse neonatal efects (N alii, 2017). One group found higher rates of learning disabilities in these children (Tincani, 2009). Simchen and colleagues (2009) reported a fourfold greater risk for perinatal strokes. Of 141 newborns followed in a European registry, the rate of preterm birth was 16 percent; low birthweight, 17 percent; and later behavioral abnormalities in 4 percent of the children. here were no cases of neonatal thrombosis (Motta, 2012). A 7-year study of 26 women who had APS with 36 pregnancies reported three cases of autism spectrum disorder, all associated with persistent neonatal anti �2_g1ycoprotein-1 IgG antibodies (Abisror, 2013).

1	This chronic inflammatory disease stems from immunological dysunction, and iniltrating T cells secrete ctokines to cause inflammation, polyarthritis, and systemic symptoms. he cardinal feature is inflammatory synovitis that usually involves the peripheral joints. he disease has a propensity for cartilage destruction, bony erosions, and joint deformities. Pain, aggravated by movement, is accompanied by swelling and tenderness. Extraarticular manifestations include rheumatoid nodules, vasculitis, and pleuropulmonary symptoms. Other complaints are fatigue, anorexia, and depression. he American College of Rheumatology criteria for rheumatoid arthritis diagnosis are shown in Table 59-7. A score of 6 or greater ulills the requirements for definitive diagnosis.

1	The worldwide prevalence of rheumatoid arthritis is 0.5 to 1 percent, women are afected three times more often than men, and peak onset is from 25 to 55 years (Shah, 2015). There is a genetic predisposition, and heritability is estimated at 15 to 30 percent (McInnes, 2011). Genome-wide associated studies have identified more than 30 loci involved in rheumatoid arthritis pathogenesis (Kurka, 2013). There is an association with the TABLE 59-7. Criteria for Classification of Rheumatoid Arthritis CRP = C-reactive protein; ESR = erythrocyte sedimentation rate; IP = interphalangeal joint; MCP = metacarpophalangeal joint; MTP = metatarsophalangeal joint; PIP = proximal inteLphalangeal joint; RF = rheumatoid factor. Criteria established in collaboration with the American College of Rheumatology and the European League Against Rheumatism. A scorer:::6 fulfills criteria for diagnosis. Data from Aletaha, 2010; Shah, 2015.

1	class II major histocompatibility complex molecule HLADR4 and HLA-DRB1 alleles (McInnes, 2011; Shah, 2015). Pregnancy provides a protection against rheumatoid arthritis development, and this may be related to HLA-disparate fetal microchimerism (Guthrie, 2010). Of other inluences, cigarette smoking raises the risk of rheumatoid arthritis (Papadopoulos, 2005).

1	Treatment is directed at pain relief, inlammation reduction, protection of articular structures, and preservation of function. Physical and occupational therapy and self-management instructions are essential. Until recently, aspirin and other NSAIDs were the cornerstone of therapy, but they do not retard disease progression. According to Shah and St. Clair (2015), methotrexate has become the preferred disease-modiying antirheumatic drug (DMARD). NSAIDs serve as adjunctive therapy but are important to pregnancy because methotrexate is contraindicated. Conventional NSAIDs nonspeciically inhibit both cyclooxygenase-1 (COX-I), which is an enzyme critical to normal platelet function, and COX-2, which mediates inflammatory response mechanisms. Because gastritis with acute bleeding is an unwanted side efect common to conventional NSAIDs, the more specific COX-2 inhibitors have been recommended. However, their long-term use is associated with higher risk for myocardial infarction and major

1	side efect common to conventional NSAIDs, the more specific COX-2 inhibitors have been recommended. However, their long-term use is associated with higher risk for myocardial infarction and major vascular events (Patrono, 2016).

1	In one systematic review, a higher rate of cardiac malformations was found in newborns exposed to NSAIDs in the first trimester (Adams, 2012). In addition, NSAIDs are associated with early spontaneous abortions, ductus arteriosus constriction, and neonatal pulmonary hypertension. Thus, risks versus beneits of these medications must be considered. Glucocorticoid therapy in low-to-moderate doses is given to achieve more rapid symptom control. Of these, prednisone, 7.5 mg orally daily for the first 2 years of active disease, substantively reduces progressive joint erosions (Kirwan, 1995; Shah, 201r5).

1	The American College of Rheumatology recommends several DMARDs that may reduce or prevent joint damage (Singh, 2016). Lelunomide, like methotrexate, is teratogenic (Briggs, 2015) (Chap. 12, p. 241). Sulfasalazine and hydroxychloroquine are safe for use in pregnancy (Pardett, 201r1). These, combined with COX-2 inhibitors and with relatively low-dose prednisone-7.5 to 20 mg daily-usually successfully treat flares. In one review of drug exposure, a fourth of women with rheumatoid arthritis took a DMARD within 6 months of conception (Kuriya, 2011). During pregnancy, 4 percent of 393 pregnant women were given a category D or X medication. Methotrexate was the most common 2.9 percent.

1	Biological DMARDs have revolutionized the treatment of rheumatoid arthritis. These include tumor necrosis factor alpha (TNF-.) inhibitors-infliximab, adalimumab, golimumab, certolizumab, and etanercept (Shah, 2015). Their use in pregnancy is limited, and fetal safety is a concern (Mako!, 201r1; Ojeda-Uribe, 2013). In one drug-exposure review, 13 percent of 393 women were given a biological cytokine-inhibiting DMARD-primarily etanercept (Kuriya, 2011). In another review of 300 exposures, no fetal efects were noted (Berthelot, 2009). A prospective study of 38 women found similar results (Hoxha, 2017). In 74 women exposed to adalimumab during pregnancy, no risks were identified (Burmester, 2017). There is also little known regarding pregnancy efects of anakinra, an interleukin-1 receptor antagonist, or of rituximab, an antagonist to the B-cell CD20 antigen.

1	In up to 90 percent of women with rheumatoid arthritis, their disease will improve during pregnancy (de .Man, 2008). Animal studies suggest this may be due to regulatory T-cell alterations (Munoz-Suano, 2012). Even so, some women develop disease during pregnancy, and others become worse (Nelson, 1997). A downside to this respite during pregnancy is that postpartum exacerbation is common (0stensen, 2007). This may stem from postpartum alterations in innate immunity (Haupl, 2008b). In one review, a postpartum flare was more common if women were breastfeeding (Barrett, 2000a). These same investigators followed 140 women with rheumatoid arthritis during 1 to 6 months postpartum (Barrett, 2000b). There was only a modest fall in objective disease activity, and only 16 percent had complete remission. They observed that although overall disease actually did not exacerbate postpartum, the mean number of inflamed joints rose signiicantly.

1	Some studies report a protective efect of pregnancy against developing new-onset rheumatoid arthritis. In a case-control study of 88 afected women, there was a protective efect of pregnancy in the long term, but the likelihood of new-onset rheumatoid arthritis was increased sixfold during the irst 3 postpartum months (Silman, 1992). Pikwer and colleagues (2009) reported a significant reduction in the risk of subsequent arthritis in women who breastfed longer than 12 months.

1	hese indings may reflect the interference of sex hormones with several putative processes involved in arthritis pathogenesis, including immunoregulation (Haupl, 2008a,b). First, Unger and associates (1983) reported that amelioration of rheumatoid arthritis correlated with serum levels of pregnancyassociated apha2-gycoprotein. his compound has immunosuppressive properties. Second, Nelson and coworkers (1993) noted that amelioration of disease was associated with a disparity in HLA class II antigens between mother and fetus. They s�ggested that the maternal immune response to paternal HLA antigens may play a role in pregnancy-induced remission of arthritis. In addition to monocyte activations, there also may be T -lymphocyte activation (Forger, 2008).

1	This group of diseases is the most frequent cause of chronic arthritis in children and persists into adulthood. In 76 pregnancies of 51 afected Norwegian women, pregnancy had no efects on clinical presentation, but disease activity usually became quiescent or remained so during pregnancy (0stensen, 1991). Postpartum flares were common as was discussed for rheumatoid arthritis. Joint deformities often developed in these women, and 15 of 20 cesarean deliveries were done for contracted pelves or joint prostheses. Results from a summary of 39 Polish women with juvenile rheumatoid arthritis were similar (Musiej-Nowakowska, 1999).

1	This arthritis portends few adverse pregnancy outcomes. The risk for preterm birth is increased, but later fetal development is normal (Mohamed, 2016; Rom, 2014; Wallenius, 2014). Disease severity in early pregnancy was predictive of preterm delivery and fetal-growth restriction in a cohort study (Bharti, 2015). nother study of 190 pregnancies followed from first trimester to delivery found patients with low disease activity scores in the first trimester were likely to have low disease activity or remission in the third trimester (Ince-Askan, 2017). In a study of 1807 births, Remaeus and associates (2017) reported increased incidences of preterm birth, fetal-growth restriction, and preeclampsia.

1	Primary treatment of symptomatic women during pregnancy is with aspirin and NSAIDs. These are used with appropriate concerns for irst-trimester efects, impaired hemostasis, prolonged gestation, premature ductus arteriosus closure, and persistent pulmonary circulation. Low-dose corticosteroids are also prescribed as indicated. Gold compounds have been administered in pregnancy (Almarzouqi, 2007). Immunosuppressive therapy with azathioprine, cyclophosphamide, or methotrexate is not routinely used during pregnancy. Only azathioprine is considered during early pregnancy because the other agents are teratogens (Briggs, 2015). As discussed on page 1147, DMARDs including sulfasalazine and hydroxychloroquine are acceptable for use in pregnancy.

1	If the cervical spine is involved, particular attention is warranted during pregnancy. Subluxation is common, and pregnancy, at least theoretically, predisposes to this because of joint laxity. Importantly, there are anesthesia concerns during endotracheal intubation. Following pregnancy in women with rheumatoid arthritis and its juvenile form, contraceptive counseling may include combination oral contraceptives. These are a logical choice because of their efectiveness and their potential to improve disease (Farr, 2010). That said, all methods of contraception are appropriate.

1	This is a chronic multisystem connective tissue disorder of unknown etiology. It is characterized by microvascular damage, immune system activation leading to inlammation, and excessive deposition of collagen in the skin and often in the lungs, heart, gastrointestinal tract, and kidneys. It is uncommon, displays a 5-to-1 female dominance, and typically afects those aged 30 to 50 years (Meier, 2012; Varga, 2015). This strong prevalence of scleroderma in women and its greater incidence in the years following childbirth give credence to the hypothesis that microchimerism is involved as discussed earlier (p. 1139) (Lambert, 2010). Artlett and coworkers (1998) demonstrated Y-chromosomal DNA in almost half of women with systemic sclerosis compared with only 4 percent of controls. Rak and colleagues (2009b) identiied male microchimerism in peripheral blood mononuclear cells more frequently in women with limited versus diuse scleroderma-20 versus 5 percent.

1	The hallmark is overproduction of normal collagen. In the more benign form-Limited cutaneous systemic sclerosis-progression is slow. With diuse cutaneous systemic sclerosis, skin thickening progresses rapidly, and skin fibrosis is followed by gastrointestinal tract ibrosis, especially the distal esophagus (Varga, 2015). Pulmonary interstitial fibrosis along with vascular changes may cause pulmonary hypertension, which develops in 15 percent of patients. Antinuclear antibodies are found in 95 percent of patients, and immunoincompetence often develops. Raynaud phenomenon, which includes cold-induced epi sodic digital ischemia, is seen in 95 percent of patients, and there may also be swelling of the distal extremities and face.

1	Raynaud phenomenon, which includes cold-induced epi sodic digital ischemia, is seen in 95 percent of patients, and there may also be swelling of the distal extremities and face. Half of patients have symptoms from esophageal involve ment, especially fullness and epigastric burning pain. Pulmo nary involvement is frequent and causes dyspnea. The 10-year cumulative survival rate is 70 percent in those with pulmo nary fibrosis, and pulmonary arterial hypertension is the main cause of death Goven, 2010; Varga, 2015). Women with lim ited cutaneous disease such as the CEST syndrome-falcinosis, Baynaud phenomenon, .sophageal involvement, .clerodacyy, and .elangiectasia-have milder disease.

1	Baynaud phenomenon, .sophageal involvement, .clerodacyy, and .elangiectasia-have milder disease. Overlap syndrome refers to systemic sclerosis with features of other connective tissue disorders. Mixed connective tissue disease is a term used for the syndrome involving features of SLE, systemic sclerosis, polymyositis, rheumatoid arthritis, and high titers of anti-ribonucleoprotein (RNP) antibodies (see Table 59-1). he disorder is also termed undierentiated connective tissue disease (Spinillo, 2008). Although systemic sclerosis is incurable, treatment directed improve function. Renal involvement and hypertension are often comorbid. At times, ACE inhibitors may be required for blood pressure control despite their known teratogenicity. Scleroderma renal crisis develops in up to a fourth of these patients and is characterized by obliterative vasculopathy of the renal cortical arteries. his leads to renal failure and malignant hypertension.

1	Interstitial restrictive lung disease is common and frequently becomes life threatening. Associated pulmonary hypertension is treated with bosentan or sildenail (Chap. 49, p. 960). The prevalence of scleroderma in pregnancy approximates 1 in 22,000 pregnancies (Chakravarty, 2008). These women usually have stable disease during gestation if their baseline unction is good. As perhaps expected, are aggravated by pregnancy (Steen, 1999). Dysphagia results from loss of esophageal motility due to neuromuscular dysunction. A decrease in amplitude or disappearance of peristaltic waves in the lower two thirds of the esophagus is seen using manometry. Symptomatic treatment for reflux is described in Chapter 54 (p. 1046).

1	Women with renal insuiciency and malignant hypertension have a higher incidence of superimposed preeclampsia. With rapidly worsening renal or cardiac disease, pregnancy termination should be considered. As discussed, renal crisis is life threatening and is treated with ACE inhibitors, but it does not improve with delivery (Gayed, 2007). Pulmonary hypertension usually contraindicates pregnancy.

1	Vaginal delivery may be anticipated, unless the soft tissue thickening wrought by scleroderma produces dystocia requiring cesarean delivery. Tracheal intubation for general anesthesia has special concerns because of limited ability of these women to open their mouths widely (Sobanski, 2016). Because of esophageal dysfunction, aspiration is also more likely, and epidural analgesia is preferable. Warming the delivery room and intravenous fluids, extra blankets, and socks and gloves are recommended to improve impaired circulation from Raynaud phenomenon. If corticosteroids were used frequently, stress doses of hydrocortisone are recommended (Sobanski, 2016).

1	Maternal and fetal outcomes correlate with underlying dis ease severity. In a review of 214 gravidas with systemic sclerosis, 45 percent had difuse disease. Major complications included renal crisis in three and greater rates of preterm birth (Steen, 1989, 1999). Chung and coworkers (2006) also reported elevated rates of preterm delivery, fetal-growth restriction, and perinatal mortality. A multicenter study of 109 pregnan cies from 25 centers reported higher rates of preterm delivery, fetal-growth restriction, and very-low-birthweight newborns (Taraborelli, 2012). hese are likely related to placental abnor malities that include decidual vasculopathy, acute atherosis, and infarcts (Sobanski, 2016). Scleroderma may be associated with subfertility (Bernatsky, 2008; Lambe, 2004). For women who do not choose preg nancy, several reversible contraceptive methods are acceptable.

1	Scleroderma may be associated with subfertility (Bernatsky, 2008; Lambe, 2004). For women who do not choose preg nancy, several reversible contraceptive methods are acceptable. However, hormonal agents, especially combination oral con traceptives, probably should not be used, especially in women with pulmonary, cardiac, or renal involvement. Due to the often unrelenting progression of systemic sclerosis, permanent sterilization is also considered.

1	Inlammation and damage to blood vessels may be primary or caused by another disease. Immune-complex deposition is presumed to underlie most cases (Langford, 2015). Primary types include polyarteritis nodosa, temporal or giant-cell arteritis, T akayasu arteritis, Henoch-Schonlein purpura, Behyet syndrome, and cutaneous or hypersensitivity arteritis (Goodman, 2014). Small vessel vasculitides such as granuomatosis with poyangiitis and eosinophilic granuomatosis with poyangiitis have antibodies directed against proteins in the cytoplasmic granules of leukocytes-antineutrophil ytopsmic antibodies-NA (Pagnox, 201).

1	This necrotizing vasculitis of small and medium-sized arteries is characterized clinically by myalgia, neuropathy, gastrointestinal disorders, hypertension, and renal disease (Goodman, 2014). Of cases, approximately a third is associated with hepatitis B antigenemia (Langford, 2015). Symptoms are nonspecific, and fever, weight loss, and malaise are present in more than half of cases. Diagnosis is made by biopsy, and treatment consists of high-dose prednisone plus cyclophosphamide. Vasculitis due to hepatitis B antigenemia responds to antivirals, glucocorticosteroids, and plasma exchange (Chap. 55, p. 1063). Only a few reports describe polyarteritis nodosa associated with pregnancy. Of 12 afected gravidas, polyarteritis first manifested during pregnancy in seven, and it was rapidly fatal by 6 weeks postpartum (Owen, 1989). he diagnosis was not made until autopsy in six of the seven women. Four women continued pregnancy, which resulted in one stillborn and three successful outcomes.

1	• Granulomatosis with Polyangiitis Formerly Wegener granulomatosis, this is a small-vessel necrotizing granulomatous vasculitis afecting the upper and lower respiratory tract and kidney (Pagnoux, 2016). Disease frequently includes sinusitis and nasal disease-90 percent; pulmonary infiltrates, cavities, or nodules-85 percent; glomerulonephritis-75 percent; and musculoskeletalrlesions-65 percent (Sneller, 1995). At least 90 percent have polyangiitis (Langford, 2015). It is uncommon and usually encountered after age 50. Koukoura and associates (2008) reviewed 36 cases in association with pregnancy and found a higher preterm birth rate. In another report, a second woman had disease-related pneumonitis, but pregnancy did not appear to afect disease activity (Pagnoux, 2011). Because subglottic stenosis is found in up to a fourth of patients, the anesthesia team is ideally consulted antepartum (Engel, 2011).

1	Corticosteroids are standard treatment, but azathioprine, cyclosporine, and IVIG therapy may also be used. For severe disease in the late second or third trimester, cyclophosphamide in combination with prednisolone seems acceptable. Also called pulseless disease, this is a chronic inflammatory arteritis afecting large vessels (Goodman, 2014). Unlike temporal arteritis, which develops almost exclusively after age 55, the onset ofTakayasu arteritis is almost always before age 40. It is associated with abnormal angiography of the upper aorta and its main branches and with upper extremity vascular impairment. Death usually results from congestive heart failure or cerebrovascular events. Computed tomography or magnetic resonance angiography can detect this disorder before the development of severe vascular compromise. Takayasu arteritis may respond symptomatically to corticosteroid therapy, however, it is not curative. Surgical bypass or angioplasty improves survival rates.

1	Comorbid severe renovascular hypertension, cardiac involvement, or pulmonary hypertension worsen pregnancy prognosis (Singh, 2015). Hypertension is relatively common and should be carefully controlled. Blood pressure is most accurately measured in the lower extremity. Overall, the prognosis for pregnancy is good Oohnston, 2002). A study of 58 women with Takayasu arteritis found an elevated risk of pregnancyrelated hypertension and preeclampsia but overall favorable maternal and fetal outcomes (Gudbrandsson, 2017). A study of 52 patients comparing obstetrical outcomes before and ater diagnosis reported higher rates of obstetrical complications after diagnosis. These included preeclampsia, preterm birth, and fetal-growth restriction or death (Comarmond, 2015). Involvement of the abdominal aorta portends worse perinatal outcome (Sharma, 2000). Vaginal delivery is preferred, and epidural analgesia has been advocated for labor and delivery.

1	Henoch-Schonlein purpura is uncommon after childhood. T ayabali and associates (2012) reviewed 20 pregnancies complicated by this vasculitis and described cutaneous lesions in three fourths. Approximately half had arthralgias. For Beh;et disease, Gungor and colleagues (2014) described 298 pregnancies in 94 women and found higher miscarriage rates and smaller babies compared with healthy controls. Formerly Churg-Strauss vasculitis, eosinophilic granulomatosis with polyangiitis is rare in pregnancy Oennette, 2013). Hot and associates (2007) described a pregnant woman who responded to IVIG therapy. Corradi and associates (2009) described an afected 35-yearold woman at term whose necrotizing vasculitis involved the heart, and she subsequently underwent cardiac transplantation. Edwards (2015) described one woman who developed postpartum relapses of this vasculitis in each of two pregnancies.

1	These are acquired and potentially treatable causes of skeletal muscle weakness with a prevalence of 1 in 100,000 persons (Dalakas, 2012). There are three major groups: polymyositis, dermatomyositis, and inclusion-body myositis, which all present with progressive asymmetrical muscle weakness. They have a variable association with connective tissue diseases, malignancy, drugs, systemic autoimmune disease such as Crohn disease, and viral, bacterial, and parasitic infections.

1	Poymyositis is a subacute inflammatory myopathy that is frequently associated with one of the autoimmune connective tissue disorders. Dermatomyositis manifests as a characteristic rash accompanying or preceding weakness. Laboratory findings include elevated muscle enzyme levels in serum and an abnormal electromyogram. Conirmation is by biopsy, which shows perivascular and perimysial inlammatory infiltrates, vasculitis, and muscle iber degeneration. It usually develops alone but can overlap with systemic sclerosis or mixed connective tissue disease.

1	Prevailing theories suggest that the syndromes are caused by viral infections, autoimmune disorders, or both. Importantly, approximatey 15 percent of adults who develop dermatomyositis have an associated malignant tumor. The timing of myositis and tumor appearance may be separated by several years. he most common sites of associated cancer are breast, lung, stomach, and ovary. The disease usually responds to high-dose corticosteroid therapy, immunosuppressive drugs such as azathioprine or methotrexate, or IVIG (Dalakas, 2012; Linardaki, 2011).

1	Experiences in pregnancy are garnered mostly from case series and reviews. Chen and colleagues (2015) found 17 women with polymyositis/ dermatomyositis in an Australian population-based cohort of births. These women had higher rates of hypertension (23 percent), antepartum hemorrhage (11 percent), cesarean delivery (88 percent), and preterm birth (35 percent). Another series of 60 women with dermatomyositis and 38 with polymyositis found that in 80 percent, pregnancy had no adverse efect on their disease. Similar results have been reported by others (Missumi, 2015; Pinal-Fernandez, 2014). Rosenzweig and colleagues (1989) reviewed 24 pregnancy outcomes in 18 women with primary disease. Of these, a fourth had an exacerbation in the second or third trimester. In 12 in whom disease became manifest first during pregnancy, half of the eight pregnancies resulted in perinatal death, and one woman died postpartum.

1	From their review, Doria and associates (2004) concluded that pregnancy outcome was related to dermatomyositis activity and that new-onset disease was particularly aggressive. Numerous inherited mutations involve genes that encode for structural proteins of bone, skin, cartilage, blood vessels, and basement membranes. Although connective tissues contain many complex macromolecules such as elastin and more than 30 proteoglycans, the most common constituents are ibrillar collagen types I, II, and III. Various mutations, some recessively and some dominantly inherited, result in clinical syndromes that include Marfan and Ehlers-Danlos syndromes, osteogenesis imperfecta, chondrodysplasias, and epidermolysis bulla. Of concern during pregnancy is the predilection for these disorders to result in aortic aneurysms (Schoenhof, 2013).

1	This is an autosomal dominant connective tissue disorder that has a population prevalence of 1 in 3000 to 5000 (Prockop, 2015). Marfan syndrome afects both sexes equally. The syndrome is due to abnormal fibrillin-a constituent of elastincaused by any of several diferent mutations in the FENI gene (Biggin, 2004). Located on chromosome 15q21, the FENI gene has a high mutation rate, and there are many mild, subclinical cases. A 50-percent risk of disease transmission to the ofspring exists, however, the ability to predict disease severity in progeny is limited by the lack of distinct genotype-phenotype correlation and large clinical variability. Currently, preimplantation and prenatal diagnoses are limited to the 80 percent of cases in which the mutation in the FBNI gene is known (Smok, 2014).

1	In severe disease, there is degeneration of the elastic lamina in the media of the aorta. his weakness predisposes to aortic dilation or dissecting aneurysm, which appears more commonly during pregnancy (Curry, 2014; Roman, 2016). Marfan syndrome complicating pregnancy is discussed in more detail in Chapter 49 (p. 967).

1	This disease is characterized by various connective tissue changes, including skin hyperelasticity. In the more severe types, rupture of any of several arteries can cause either stroke or bleeding. There are several disease types based on skin, joint, or other tissue involvement. Some are autosomal dominant, some recessive, and some X-linked (Solomons, 2013). Their aggregate prevalence approximates 1 in 5000 births (Prockop, 2015). Types I, II, and III are autosomally dominant, and each accounts for approximately 30 percent of cases. Type IV is uncommon but is known to predispose to preterm delivery, maternal great-vessel rupture, postpartum bleeding, and uterine rupture (Pepin, 2000). In most, the underlying molecular defect afects collagen or procollagen.

1	In general, women with Ehlers-Danlos syndrome reportedly have a higher frequency of preterm rupture of membranes, preterm delivery, and antepartum and postpartum hemorrhage (Volkov, 2006). That said, a recent cohort study of314 women reported no greater risk of adverse pregnancy outcome, including preterm birth (Sundelin, 2017). Several cases of spontaneous uterine rupture have been described (Rudd, 1983). Tissue fragility makes episiotomy repair and cesarean delivery diicult. Hurst and colleagues (2014) surveyed 1769 respondents of the Ehlers-Danlos National Foundation and found a preterm birth rate of 25 percent and infertility rate of 44 percent. A maternal and fetal death from spontaneous rupture of the right iliac artery has been reported (Esaka, 2009). Bar-Yosef and associates (2008) described a newborn with multiple congenital skull fractures and intracranial hemorrhage caused by Ehlers-Danlos type VIIC.

1	This disorder has a prevalence of 1 in 20,000 births for type I and 1 in 60,000 for type II. It is characterized by brittle bones and afected patients often have blue sclerae, hearing loss, multiple prior bone fractures, and dental abnormalities. There are up to 15 subtypes based on the causative gene and clinical picture, which ranges from mild to severe (Van Dijk, 2010). Genetic inheritance includes autosomal dominant, autosomal recessive, and sporadic patterns. Type I is the mildest form, and the typical mutation afects the COLIAI gene (Sykes, 1990). Type II is typically lethal in utero (Prockop, 2015).

1	Women with osteogenesis imperfecta, most commonly type I, may have successful pregnancies. hat said, several risks in pregnancy include fractures, complications related to scoliosis with restrictive lung disease, micrognathia, brittle teeth, an unstable cervical spine, uterine rupture, and cephalopelvic disproportion. A retrospective cohort of 295. women with osteogenesis imperfecta found greater risks of antepartum hemorrhage, abruption, fetal-growth restriction, congenital malformations, and preterm birth (Ruiter-Ligeti, 2016). It is not unusual for afected women to enter pregnancy having had 20 to 30 prior fractures. Most require minimal treatment other than management of the fractures and consideration of bisphosphonates to decrease bone loss.

1	Depending on the type of osteogenesis imperfecta, the fetus may be afected and may also sufer fractures in utero or during delivery (Chap. 10, p. 210). Prenatal diagnosis is available in many situations, if desired, and in utero stem cell therapy is being evaluated (Couzin-Frankel, 2016). Abisror N, Mekinian A, Lachassinne E, et al: Autism spectrum disorders in babies born to mothers with antiphospholipid syndrome. Semin Arthritis

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1	Yasuda M, Takakuwa K, Tokunaga A, et al: Prospective studies of the association between anticardiolipin antibody and outcome of pregnancy. Obstet Gynecol 86:555, 1995 Ye Y, van Zyl B, Varsani H, et al: Maternal microchimerism in muscle biopsies from children with juvenile dermatomyositis. Rheumatology (Oxford) 51(6):987,t2012 Zangari M, Lockwood CJ, Scher J, et al: Prothrombin activation fragment (F1.2) is increased in pregnant patients with antiphospholipid antibodies. Thromb Res 85:177, 1997 Zhao C, Zhao J, Huang Y, et al: New-onset systemic lupus erythematosus during pregnancy. Clin Rheumatol 32(6):815, 2013 Zuppa A, Riccardi R, Frezza A, et al: Neonatal lupus: follow-up in infants with anti SSA/Ro antibodies and review of the literature. Autoimmun Rev ,2017 CENTRAL NERVOUS SYSTEM IMAGING.. . . . . . . . . . .. 1156 HEADACHE ................................... 1157

1	CENTRAL NERVOUS SYSTEM IMAGING.. . . . . . . . . . .. 1156 HEADACHE ................................... 1157 SEIZURE DISORDERS.. . . . . . . . . . . . . . . . . . . . . . . . . .. 1158 CEREBROVASCULAR DISEASES . . . . . . . . . . . . . . . . .. 1160 DEMYELINATING OR DEGENERATIVE DISEASES ..... 1164 NEUROPATHIES ............................... 1166 SPINAL CORD INJURY.. . . . . . . . . . . . . . . . . . . . . . . . .. 1167 IDIOPATHIC INTRACRANIAL HYPERTENSION ....... 1168 MATERNAL VENTRICULAR SHUNTS ............... 1168 MATERNAL BRAIN DEATH ....................... 1168 Epilepsy appears to have no fect on pregnancy, though at the time of labour it may be mistaken or eclampsia by inexperienced observers. f the attacks are requent the patient should be put upon large doses of potassium bromide and treated just as at other times. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) Although several neurological diseases are relatively common in women of childbearing age, less than two pages were devoted to Diseases of the Nervous System in this textbook's first edition. In the past, some may have precluded pregnancy, however, few do so now. Most encountered during pregnancy are the same as for nonpregnant women, however a few neurological disorders may be seen more frequently in pregnant women. Examples are Bell palsy, specific types of strokes, and benign intracranial hypertension or pseudotumor cerebri. Neurovascular disorders are an important cause of maternal mortality and accounted for nearly 7 percent of maternal deaths in the United States from 2011 through 2013 (Creanga, 2017).

1	Many neurological disorders frequently precede pregnancy. fost women with chronic neurological disease who become pregnant will have successful outcomes, but some disorders have speciic risks. Conversely, other women will have newonset neurological symptoms during pregnancy, and these often must be distinguished from pregnancy complications. Psychiatric disorders can also manifest with cognitive and neuromuscular abnormalities and should be considered in the evaluation.

1	Computed tomography (CT) and magnetic resonance (MR) imaging assist in the diagnosis, classification, and management of many neurological and psychiatric disorders. As discussed in Chapter 46 (p. 907), these imaging methods can be used safely during pregnancy. CT scanning is often used when rapid diagnosis is necessary and is excellent for detecting recent hemorrhage. Because it does not use radiation, MR imaging is oten preferred and is particularly helpful to diagnose demyelinating diseases, arteriovenous malformations, congenital and developmental nervous system abnormalities, posterior fossa lesions, and spinal cord diseases. Whenever either test is done, the woman with advanced pregnancy should be positioned in a left lateral tilt with a wedge under one hip to prevent hypotension and to diminish aortic pulsations, which may degrade the image.

1	Cerebral angiography with contrast injection, usually via the femoral artery, is a valuable adjunct to the diagnosis and treatment of some cerebrovascular diseases. Fluoroscopy delivers more radiation but can be performed with abdominal shielding. Positron emission tomography (PET) and functional MR imaging (fMI) have not been evaluated for use in pregnant patients (Chiapparini, 2010).

1	In one national survey in the United States in 2012, 17 percent ofthose aged 18 to 44 years reported a severe headache or migraine within the past 3 months (Blackwell, 2014). Burch and coworkers (2015) reported that 24 percent ofnonpregnant women in this age group were similarly afected. Of pregnant women presenting with headache who received a neurological consultation, two thirds were due to primary disorders, with over 90 percent due to migraine. Of the other third due to secondary conditions, over half were due to hypertensive disorders (Robbins, 2015). Interestingly, Aegidius and associates (2009) reported a decline in the rate of all headache types during pregnancy in nulliparas, especially during the third trimester. The classification by the International Headache Society (20l3) is shown in Table

1	The classification by the International Headache Society (20l3) is shown in Table In pregnant women, primary headaches are more common than those stemming from secondary causes (Digre, 2013; Sperling, 2015). Migraine headaches are those most likely to be afected by the hormonal changes of pregnancy (Pavlovic, 2017). The incidences ofsevere headache causes in pregnancy are shown in Figure 60-1 . These are the most frequent cause of all headaches. Characteristic features include muscle tightness and mild-to-moderate pain in the back ofthe neck and head that can persist for hours. Neurological disturbances or nausea are typically absent. The pain usually responds to rest, massage, application of heat or ice, antiinlammatory medications, or mild tranquilizers. Hospital admission is seldom necessary.

1	The term migraine describes a periodic, sometimes incapacitating neurological disorder with episodic attacks ofsevere headache and autonomic nervous system dysfunction (Goadsby, 2015). The International Headache Society (20l3) classifies TABLE 60-1. Headache Classification Disorders of homeostasis Data from International Headache Society, 2013. Pituitary, 3.6% Infection, 2.1% Stroke, 2.8% Other headache, 6% (tension 3%) Migraine, 59% With aura, 37% Without aura, 24••• Chronic, 6% FIGURE 60-1 Incidences of headache causes in 140 consecutive pregnant women for whom in-hospital neurology consultation was requested. (Data from Robbins, 201o5.) three migraine types based on chronicity and on the presence or absence ofan aura. 1. Migrainewithoutaura-formerlytermedcommon migraineis characterized by a unilateral throbbing headache, nausea and vomiting, or photophobia. 2.

1	1. Migrainewithoutaura-formerlytermedcommon migraineis characterized by a unilateral throbbing headache, nausea and vomiting, or photophobia. 2. Migraine with aura-formerly termed classic migraine-has similar symptoms preceded by premonitory neurological phenomena such as visual scotoma or hallucinations. A third of patients have this type of migraine, which sometimes can be averted ifmedication is taken at the first premonitory sign. 3. Chronic migraine is defined by a migraine headache occurring at least 15 days each month for more than 3 months.

1	3. Chronic migraine is defined by a migraine headache occurring at least 15 days each month for more than 3 months. These are the most common reason for admission for headache evaluation and management. Migraines may begin in childhood, peak in adolescence, and tend to diminish in both frequency and severity with advancing years. According to Lipton and associates (2007), their annual prevalence is 17 percent in women and 6 percent in men. Another 5 percent ofwomen have probable migraine, that is, they have all criteria but one (Silberstein, 2007). Speciic polymorphisms have been identified that modulate the risk of migraines (Chen, 2015; Schiirks, 2010). hese headaches are especially common in young women and have been linked to hormone levels (Charles, 2017; Pavlovic, 2017). They are frequently encountered during pregnancy.

1	Sensory sensitivity with migraines is likely caused by monoaminergic sensory control systems in the brainstem and hypothalamus (Goadsby, 2015). his exact pathophysiology is uncertain, but they occur when neuronal dysfunction leads to decreased cortical blood How, activation ofvascular and meningeal nociceptors, and stimulation oftrigeminal sensory neurons (Brandes, 2007; D'Andrea, 2010). A predilection for the posterior circulation has been described (Kruit, 2004). Migrainesespecially those with aura in young women-are associated with increased risk for ischemic strokes. The risk is greater in those who smoke or use combination oral contraceptives.

1	The prevalence of migraine headaches in the first trimester is 2 percent (Chen, 1994). Most migraineurs have improvement during pregnancy (Kvisvik, 2011). Still, migraines-usually those with an aura-occasionally appear for the first time during pregnancy. Pregnant women with preexisting migraine symptoms may have other symptoms suggestive of a more serious disorder, and new neurological symptoms should prompt a complete evaluation (Detsky, 2006; Heaney, 2010).

1	Although conventional thinking has been that migraine headaches do not pose increased maternal or fetal risks, several recent studies have refuted this (Allais, 2010). In these, rates of preeclampsia, gestational hypertension, preterm birth, and other cardiovascular morbidities, including ischemic stroke, were increased (Grossman, 2017; Wabnitz, 2015). Bushnell and coworkers (2009) identiied an incidence of migraines during pregnancy of 185 per 100,000 deliveries. Of diagnoses associated in these gravid migraineurs, risks were signiicantly higher for stroke, 16-fold; myocardial infarction, ivefold; heart disease, twofold; venous thromboembolism, twofold; and preeclampsia/gestational hypertension, twofold.

1	Data are limited regarding non pharmacological management in pregnancy such as biofeedback techniques, acupuncture, and transcranial magnetic stimulation (Airola, 2010; Dodick, 2010). Efective medications include nonsteroidal antiinlammatory drugs (NSAIDs), and most migraine headaches respond to simple analgesics such as ibuprofen, acetaminophen, or Midrin, especially if given early. Severe migraines are vexing for the patient and her caregivers. Multitargeted drug therapy is necessary in most cases for migraine relief (Gonzalez-Hernandez, 2014). Headaches are treated aggressively with intravenous hydration and parenteral antiemetics and opioids for immediate pain relie. Although a 2-g infusion of magnesium sulfate has gained favor in the past few years, a metaanalysis reported no beneits (Choi, 2014). Ergotamine derivatives are potent vasoconstrictors and are avoided in pregnancy because of their uterotonic efects (Briggs, 2015).

1	Triptans are serotonin 5-HT I Bl20-receptor agonists that efectively relieve headaches by causing intracranial vasoconstriction (Contag, 2010). They also relieve nausea and vomiting and greatly reduce the need for analgesics. Triptans can be given as an oral tablet, injection, rectal suppository, or intranasal spray. hey are best used in combination with NSAIDs (Goadsby, 2015). The greatest experience is with sumatriptan (Imitrex), and although not studied extensively in pregnancy, it appears to be safe (Briggs, 2015; Nezvalova-Henriksen, 2010). However, in one follow-up study at 36 months of children exposed to triptans in pregnancy, Wood and colleagues (2016) found neurodevelopment diferences, including emotionality and activity problems.

1	Some women will beneit from peripheral nerve blocks, and Govindappagari and coworkers (2014) described their experiences with 13 pregnant women. For women with frequent migraine headaches, oral prophylactic therapy is warranted. Amitriptyline (Elavil), 10 to 175 mg daily; propranolol (Inderal), 40 to 120 mg daily; or metoprolol (Lopressor, Toprol), 25 to 100 mg daily, have been used with success (Contag, 2010; Goadsby, 2015; Lucas, 2009). This rare primary headache disorder is characterized by severe unilateral lancinating pain radiating to the face and orbit, lasting 15 to 180 minutes, and occurring with autonomic symptoms and agitation. Pregnancy does not afect symptom severity. Afected women should avoid tobacco and lcohol. Acute mnagement includes 100-percent oxygen therapy and sumatriptan given as a 6-mg dose subcutaneously (VanderPluym, 2016). If recurrent, prophylaxis is administered using a calcium-channel blocking agent.

1	The Centers for Disease Control and Prevention reported that the prevalence of epilepsy in adults in 2005 was 1.65 percentthus over 1 million American women of childbearing age are afected (Kobau, 2008). After headaches, seizures are the next most prevalent neurological condition encountered in pregnancy, and they complicate 1 in 200 births (Brodie, 1996; Yerby, 1994). Importantly, epilepsy accounted for 5 percent of maternal deaths in the United Kingdom for the 2011 to 2013 triennium (Knight, 2015).

1	A seizure is deined as a paroxysmal disorder of the central nervous system characterized by an abnormal neuronal discharge with or without loss of consciousness. Some identiiable causes of convulsive disorders in young adults include head trauma, alcohol-and other drug-induced withdrawals, cerebral infections, brain tumors, biochemical abnormalities, and arteriovenous malformations. A search for these is prudent with a new-onset seizure disorder in a pregnant woman. he diagnosis of idiopathic epilepsy is one of exclusion. Epilepsy encompasses diferent syndromes whose cardinal feature is a predisposition to recurrent unprovoked seizures. he International League Against Epilepsy Commission on Classiication and Terminology recently updated the following deinitions (Fisher, 2014).

1	These originate in one localized brain area and afect a correspondingly localized area of neurological unction. They are believed to result from trauma, abscess, tumor, or perinatal factors, although a speciic lesion is rarely demonstrated. Focal seizures without dyscognitive eatures start in one region of the body and progress toward other ipsilateral areas of the body, producing tonic and then clonic movements. Simple seizures can afect sensory unction or produce autonomic dysfunction or psychological changes. Cognitive function is not impaired, and recovery is rapid. Focal seizures with dyscognitive eatures are oten preceded by an aura and followed by impaired awareness manifested by sudden behavioral arrest or motionless stare. Involuntary movements such as picking motions or lip smacking are common.

1	These involve both brain hemispheres and may be preceded by an aura before an abrupt loss of consciousness. There is a strong hereditary component. In generalized tonic-clonic seizures, loss of consciousness is followed by tonic contraction of the muscles and rigid posturing, and then by clonic contractions of all extremities while the muscles gradually relax. Return to consciousness is gradual, and the patient may remain confused and disoriented for several hours. Absence seizures-also called petit mal seizures-are a form of generalized epilepsy that involve a brief loss of consciousness without muscle activity and are characterized by immediate recovery of consciousness and orientation.

1	Women with epilepsy ideally are counseled before pregnancy and relevant points are presented also in Chapter 8 (p. 148). Folic acid supplementation with 0.4 mg per day is begun at least 1 month before conception. The dose is increased to 4 mg when the woman taking antiepileptic medication becomes pregnant. hese medications are assessed and adjusted with a goal of mono therapy using the least teratogenic medication. If this is not feasible, then attempts are made to reduce the number of medications used and to use them at the lowest efective dose (Patel, 2016). Medication withdrawal should be considered if a woman is seizure free for 2 years or more.

1	The major pregnancy-related risks to women with epilepsy are fetal malformations and increased seizure rates. Seizure control is the main priority to avoid its attendant morbidity and mortality risks. Early studies described worsening seizure activity during pregnancy, however, this is less so now because of more efective drugs. Contemporary studies cite higher rates of seizure activity in only 20 to 30 percent of pregnant women (Mawer, 2010; Vajda, 2008). Women who are seizure free for at least 9 months before conception will likely remain so during pregnancy (Harden, 2009b).

1	Greater seizure frequency is often associated with decreased and thus subtherapeutic anticonvulsant serum levels, a lower seizure threshold, or both. An impressive number of pregnancyassociated alterations can result in sub therapeutic serum levels. These include nausea and vomiting, slower gastrointestinal motility, antacid use that diminishes drug absorption, pregnancy hypervolemia ofset by protein binding, induction of hepatic enzymes such as cytochrome oxidases, placental enzymes that metabolize drugs, and increased glomerular iltration that hastens drug clearance. Importantly, some women discontinue medication because of teratogenicity concerns. Finally, the seizure threshold can be afected by pregnancyrelated sleep deprivation and by hyperventilation and pain during labor.

1	Women with epilepsy have a small increased risk of pregnancy complications that include spontaneous abortion, hemorrhage, hypertensive disorders, preterm birth, fetal-growth restriction, and cesarean delivery (Harden, 2009b; Viale, 2015). Importantly, MacDonald (2015) also reports a tenfold higher maternal death rate, and, as mentioned earlier, epilepsy accounted for 5 percent of maternal deaths in the United Kingdom. Postpartum depression rates are also reportedly higher in epileptic women (Turner, 2009). Finally, children of epileptic mothers have a 10-percent risk of developing a seizure disorder. For years, it was diicult to separate efects of epilepsy from those of its therapy as the primary cause of fetal malforma tions. As discussed in Chapter 8 (p. 148), it is now believed that untreated epilepsy is not associated with an elevated fetal malformation rate (Thomas, 2008). That said, the fetus of an has an indisputably greater risk for congenital malformations.

1	Moreover, mono therapy is associated with a lower birth defect rate compared with multiagent therapy. hus, if necessary, increasing monotherapy dosage is at least initially preferable to adding another agent (Buhimschi, 2009). Specific drugs, when given alone, increase the malformation rate (Chap. 12, p. 240). Some of these are listed in Table 60-2. Phenytoin and phenobarbital increase the major malformation rate two-to threefold above baseline (Perucca, 2005; Thomas, 2008). Valproate is a particularly potent teratogen, which has a dose-dependent efect and raises the malformation risk four-to eightfold (Eadie, 2008; lein, 2014; Wyszynski, 2005). Val proate is also associated with lower cognitive performance (Kas radze, 2017). In general, with poly therapy, the risk rises with each drug added. A metaanalysis of 31 studies found lamotrig ine and levetiracetam to carry the lowest risk of malformations (Weston, 2016).

1	The American Academy of Neurology and the American Epilepsy Society have guidelines regarding treatment in pregnant women (Harden, 2009a-c). The major goal is seizure prevention. To accomplish this, treatment for nausea and vomiting is provided, seizure-provoking stimuli are avoided, and medication compliance is emphasized. The fewest necessary anticonvulsants are given at the lowest dosage efective for seizure control. Although some providers routinely monitor serum drug levels, these concentrations may be unreliable because of altered protein binding. Free or unbound drug levels, although perhaps more accurate, are not widely available. Importantly, there is no evidence that such monitoring improves seizure control (Adab, 2006). For these reasons, drug levels may be most informative if measured following seizures or if noncompliance is suspected.

1	For women taking anticonvulsant drugs, a targeted so nographic examination at midpregnancy is recommended by some to search for anomalies. Testing to assess fetal well-being is generally not indicated for women with uncomplicated epilepsy. For women desiring to breastfeed, data regarding the safety of the various anticonvulsant medications are limited. That said, no obvious deleterious efects, such as long-term cognitive issues, have been reported (Briggs, 2015; Harden, 2009c). Of birth control methods, oral contraceptive pill failure rates are higher with some of the anticonvulsant agents, especially lamotrigine. Thus, other more reliable methods should be considered (Chap. 38, p. 680). TABLE 60-2. Teratogenic Effects of Common Anticonvulsant Medications Neural-tube defects, clefts, cardiac anomalies; associated developmental delay Fetal hydantoin syndrome-craniofacial anomalies, fingernail hypoplasia, growth deficiency, developmental delay, cardiac anomalies, clefts

1	Fetal hydantoin syndrome-craniofacial anomalies, fingernail hypoplasia, growth deficiency, developmental delay, cardiac anomalies, clefts Fetal hydantoin syndrome, as above; spina bifida Clefts, cardiac anomalies, urinary tract malformations aRisk categories from Briggs, 2015; Food and Drug Administration, 2011; Harden, 2009b; Holmes, 2008; Hunt, 2008.

1	Clefts, cardiac anomalies, urinary tract malformations aRisk categories from Briggs, 2015; Food and Drug Administration, 2011; Harden, 2009b; Holmes, 2008; Hunt, 2008. Abnormalities of the cerebrovascular circulation include strokes-both ischemic and hemorrhagic, as well as anatomical anomalies, such as arteriovenous malformations and aneurysms. Cerebral ischemia is caused by reduction in blood low that lasts longer than several seconds. Early, neurological symptoms may manifest. Ater a few minutes, however, infarction often follows. Hemorrhagic stroke is caused by bleeding directly into or around the brain. It produces symptoms by its mass efect, by toxic efects of blood, or by increasing intracranial pressure. Of strokes in pregnant women, roughly half are ischemic and the other half hemorrhagic (Zofkie, 2018).

1	he current obesity endemic in this country, along with concomitant increases in rates of heart disease, hypertension, and diabetes, has increased the prevalence of strokes (Centers for Disease Control and Prevention, 2012). Women have higher lifetime risk of stroke than men and greater associated mortality rates (Martinez-Sanchez, 2011; Roger, 2012). Moreover, pregnancy increases the immediate and lifetime risk of both ischemic and hemorrhagic stroke (Jamieson, 2010; lung, 2010).

1	Stroke is relatively uncommon in pregnant women, occurring in 10 to 40 per 100,000 births, but it contributes disparately to maternal mortality rates (Lefert, 2016; Miller, 20 16; Yoshida, 2017). he incidence is rising as measured by pregnancy-related hospitalizations for stroke (Callaghan, 2008; Kukiina, 2011). Importantly, most are associated with hypertensive disorders or heart disease. Of the pregnancy-related mortality rate in the United States, 6.6 percent is due to cerebrovascular accidents, and 7.4 percent is associated with preeclampsia (Creanga, 2017). Of maternal deaths after 42 days postpartum, 9.8 percent were attributable to cerebrovascular accidents.

1	Most strokes in pregnancy manifest either during labor and delivery or in the puerperium. In a study of 2850 pregnancyrelated strokes, approximately 10 percent developed antepartum, 40 percent intrapartum, and almost 50 percent postpartum (James, 2005). In contrast, Lefert (2016) reports a timing of 45 percent antepartum, 3 percent intrapartum, and 53 percent postpartum in 145 women. Several risk factorsunrelated and related to pregnancy-have been reported from studies that included more than 10 million pregnancies. These include age; migraines, hypertension, obesity, and diabetes; cardiac disorders such as endocarditis, valvular prostheses, and patent foramen ovale; and smoking. hose related to pregnancy include hypertensive disorders, gestational diabetes, obstetrical hemorrhage, and cesarean delivery. By ar, the most common risk actors are pregnancy-associated hypertensive disorders. A third of strokes are associated with gestational hypertension, and hypertensive women compared

1	cesarean delivery. By ar, the most common risk actors are pregnancy-associated hypertensive disorders. A third of strokes are associated with gestational hypertension, and hypertensive women compared with normotensive counterparts have a three-to eightfold greater risk of stroke (Scott, 2012; Wang, 2011). Women with preeclampsia undergoing general anesthesia may be at higher risk of stroke compared with those given neuraxial anesthesia (Huang, 2010). Another risk factor for peripartum stroke is cesarean delivery, which raises the risk 1.5-fold compared with vaginal delivery (Lin, 2008).

1	Pregnancy-induced efects on cerebrovascular hemodynamics include enhanced autoregulation that maintains blood low despite changes in systemic blood pressure (van Teen, 2016). Although cerebral blood low decreases by 20 percent from midpregnancy until term, it increases signiicantly with gestational hypertension (Zeeman, 2003, 2004b). Such hyperperfusion at least intuitively would be dangerous for women with certain vascular anomalies. FIGURE 60-2 Illustrations of a brain showing various types of strokes seen in pregnancy: (1) subcortical infarction (preeclampsia), (2) hypertensive hemorrhage/ (3) aneurysm, (4) embolism or thrombosis in middle cerebral artery, (5) arteriovenous malformation, and (6) cortical vein thrombosis. Cocaine, amphetamines From Smith, 2015; Yager, 2012. Neurological Disorders 1161

1	Cocaine, amphetamines From Smith, 2015; Yager, 2012. Neurological Disorders 1161 Acute occlusion or embolization of an intracranial blood vessel causes cerebral ischemia, which may result in death of brain tissue (Fig. 60-2). he more common associated conditions and etiologies of ischemic stroke are shown in Table 60-3. A transient ischemic attack (TIA) is caused by reversible ischemia, and symptoms usually last less than 24 hours. Approximately 10 percent of these patients have a stroke by 1 year (Amarenco, 2016). Patients with a stroke usually have a sudden onset of severe headache, hemiplegia or other neurological deicits, or occasionally seizures. In contrast, focal neurological symptoms accompanied by an aura usually signiy a irst-episode migraine (Liberman, 2008).

1	Evaluation of an ischemic stroke includes echocardiography and cranial imaging with CT, MR, or angiography. Serum lipids are measured with the caveat that their values are distorted by normal pregnancy (Appendix, p. 1259). Tests to detect antiphospholipid antibodies and lupus anticoagulant are performed. These underlie up to a third of ischemic strokes in otherwise healthy young women (Chap. 59, p. 1143). Also, sickle-cell syndromes are evaluated when indicated (Buonanno, 2016). With a thorough evaluation, most causes of embolism can be identified, although treatment is not always available. Some of these include cardiacassociated embolism, vasculitis, or vasculopathy such as Moyamoya disease (Ishimori, 2006; Miyakoshi, 2009; Simolke, 1991). Outcomes of embolic strokes were reported to be favorable and similar to those of nonpregnant women (Lefert, 2016). hrombolysis for ischemic stroke during pregnancy has been reported (Tversky, 2016).

1	In reproductive-age women, a significant proportion of pregnancyrelated ischemic strokes are caused by gestational hypertension and preeclampsia (Jeng, 2004; Miller, 2016). s shown in Figure 60-2, areas of subcortical perivascular edema and petechial hemorrhage may progress to cerebral infarction (Aukes, 2007, 2009; Zeeman, 2004a). Although these are usually clinically manifest by an eclamptic convulsion, a few women will sufer a symptomatic stroke from a larger cortical infarction (Chap. 40, p. 734). Other conditions with findings similar to preeclampsia include thrombotic microangiopathies (Chap. 56, p. 1088) and the reversible cerebral vasoconstriction syndrome (Chap. 40, p. 744). The latter, also termed pospartum angiopathy, can cause extensive cerebral edema with necrosis and widespread infarction with areas of hemorrhage (Edlow, 2013; Katz, 2014; Miller, 2016).

1	These strokes usually involve the middle cerebral artery (see Fig. 60-2). he diagnosis can be made with conidence only after thrombosis and hemorrhage have been excluded and is more certain if an embolic source is identified. Hemorrhage may be more diicult to exclude because embolization and thrombosis are both followed by hemorrhagic infarction. Paradoxical embolism is an uncommon cause, even considering that more than a fourth of adults have a patent foramen ovale through which right-sided venous thromboemboli are deported (Scott, 2012). Foraminal closure may not improve outcomes in these patients, however, this procedure has been performed during pregnancy (Dark, 2011). Assorted cardioembolic causes of stroke include arrhythmias-especially atrial ibrillation, valvular lesions, mitral valve prolapse, mural thrombus, infective endocarditis, and peripartum cardiomyopathy.

1	Management of embolic stroke in pregnancy consists of supportive measures and antiplatelet therapy. Thrombolytic therapy and anticoagulation in pregnancy are controversial issues (U,r2012). Most thrombotic strokes afect older individuals and are caused by atherosclerosis, especially of the internal carotid artery. Many are preceded by one or more TIAs. hrombolytic therapy with a recombinant tissue plasminogen activator (rt-PAj is recommended. Alteplase is one of these and given within the irst 3-hour window if there is measurable neurological deficit and if neuroimaging has excluded hemorrhage. his recombinant enzyme can be used in pregnancy. A principal risk is hemorrhagic transformation of an ischemic stroke in 3 to 5 percent of treated patients (Smith, 2015; van der Worp, 2007).

1	In one study in the United States, 7 percent of cerebral venous thromboses were associated with pregnancy (Wasay, 2008). But, in the Nationwide Inpatient Sample of more than 8 million deliveries, James and associates (2005) reported that venous thrombosis caused only 2 percent of pregnancy-related strokes (Saposnik, 2011). here are numerous predisposing causes, and for gravidas, late pregnancy and the puerperium are times of greatest risk. Thrombosis of the lateral or superior sagittal venous sinus usually occurs in the puerperium and often in association with preeclampsia, sepsis, or thrombophilia (see Fig. 60-2). It is more common in patients with inherited thrombophilias or antiphospholipid antibodies (Chaps. 52, p. 1006 and 59, p. 1143). Headache is the most frequent presenting symptom, neurological deficits are common, and up to a third of patients have convulsions (Wasay, 2008). he diagnosis is made using MR venography (Saposnik, 2011).

1	Management includes anticonvulsants for seizures, and although heparinization is recommended by most, its eicacy is controversial (Saposnik, 2011; Smith, 2015). Antimicrobials are given if there is septic thrombophlebitis, and fibrinolytic therapy is reserved for those women failing systemic anticoagulation. The acute prognosis for venous thrombosis in pregnant women is better than in nonpregnant subjects, and mortality rates are less than 10 percent (McCaulley, 2011). In women with a prior cerebral venous thrombosis, one systematic review found only one recurrence in 217 pregnancies and five noncerebral venous thrombotic events in 186 pregnancies (Aguiar de Sousa, 2016). In a study of 52 women on prophylactic anticoagulation with prior cerebral venous thrombosis, there were no cases of recurrent thrombosis or bleeding, however 24 percent had late obstetrical complications (Martinelli, 2016). Recurrence Risk of Ischemic Stroke

1	Recurrence Risk of Ischemic Stroke Women with prior ischemic stroke have a low risk for recurrence during a subsequent pregnancy unless a specific, persistent cause is identiied. During a 5-year follow-up of 373 women with arterial ischemic strokes, there were 187 pregnancies in 125 women. Thirteen women had a recurrent ischemic stroke, and of these, only two were associated with pregnancy. The authors concluded that the risk of stroke recurrence is low and a previous ischemic stroke is not a contraindication to pregnancy (Lamy, 2000). In one study of 1770 nonpregnant women with anti phospholipid-related ischemic stroke, investigators reported no diference in the recurrence risk as long as preventative treatment was given with warfarin or aspirin (Levine, 2004).

1	Currently, no irm guidelines define prophylaxis in pregnant women with a stroke history (Helms, 2009). The American Heart Association stresses the importance of controlling risk factors such as hypertension and diabetes (Furie, 201l). Women with anti phospholipid syndrome or certain cardiac conditions should be considered for prophylactic anticoagulation as discussed in Chapter 49 (p. 954) and 52 (p. 1008). The two distinct categories of spontaneous intracranial bleeding are intracerebral and subarachnoid hemorrhage. The symptoms of a hemorrhagic stroke are similar to those of an ischemic stroke, and their diferentiation is only possible with CT or MR imaging (Morgenstern, 2010; Smith, 2015). Bleeding into the brain parenchyma most often is caused by spontaneous rupture of small vessels previously damaged by chronic hypertension (see Fig. 60-2). Thus, pregnancy-associated hemorrhagic strokes such as the one shown in Figure 60-3 are

1	FIGURE 60-3 A 37-year-old gravida with intrapartum eclampsia at term. A noncontrast computed tomography axial head image demonstrates a large intraparenchymal hemorrhage. oten associated with chronic hypertension and superimposed preeclampsia (Cunningham, 2005; Martin, 2005). Because of its location, this type of hemorrhage has much higher morbidity and mortality rates than does subarachnoid hemorrhage (Smith, 2015). Pressure-induced rupture causes bleeding into the putamen, thalamus, adjacent white matter, pons, and cerebellum. In the 28 women described by Martin and associates (2005), half died and most survivors had permanent disabilities. his cautions for the importance of proper management for gestational hypertension-especially systolic hypertension-to prevent cerebrovascular pathology (Chap. 40, p. 738).

1	In a study of 639 cases of pregnancy-related subarachnoid hemorrhage from the Nationwide Inpatient Sample, the incidence was 5.8 per 100,000 pregnancies, with half occurring postpartum (Bateman, 2012). A remarkably similar incidence was reported in Japanese women (Yoshida, 2017). hese bleeds are more likely caused by an underlying cerebrovascular malformation in an otherwise norml patient (see Fig. 60-2). Ruptured saccular or "berry" aneurysms cause 80 percent of all subarachnoid hemorrhages. The remaining cases are caused by a ruptured arteriovenous malformation, coagulopathy, angiopathy, venous thrombosis, infection, drug abuse, tumors, or trauma. Such cases are uncommon, and a ruptured aneurysm or angioma or bleeding from a vascular mlformation has an incidence of 1 in 75,000 pregnancies. Although this frequency is not diferent from that in the general population, the mortality rate during pregnancy is reported to be as high as 35 percent (Yoshida, 2017).

1	Intracranial Aneurysm. Approximately 1 to 2 percent of adults have this lesion (Lawton, 2017). Fortunately, only a small percentage rupture. he rate approximates 0.1 percent for aneurysms <10 mm and 1 percent for those >10 mm (Smith, 2015). Most aneurysms identified during pregnancy arise from the circle of Willis, and in 20 percent of case, there are multiple lesions. Pregnancy does not raise the risk for aneurysmal rupture. However, because of their high prevalence, they are more likely to cause subarachnoid bleeding than other etiologies (Hirsch, 2009; Tiel Groenestege, 2009). A systematic review of 44 women with 50 aneurysms in pregnancy reported that 72 percent ruptured during pregnancy, and 78 percent of these did so during the third trimester (Barbarite, 2016). his proclivity for rupture late in pregnancy was also reported by Yoshida and colleagues (2017).

1	The cardinal symptom of a subarachnoid hemorrhage from an aneurysm rupture is sudden severe headache that is accompanied by visual changes, cranial nerve abnormalities, focal neurological deicits, and altered consciousness. Patients typically have signs of meningeal irritation, nausea and vomiting, tachycardia, transient hypertension, low-grade fever, leukocytosis, and proteinuria. Prompt diagnosis and treatment may prevent potentially lethal complications. The American Heart Association recommends noncontrast cranial CT imaging as the irst diagnostic test, although MR imaging may be superior (Connolly, 2012; Smith, 2015).

1	Treatment of subarachnoid hemorrhage includes bed rest, analgesia, and sedation, with neurological monitoring and strict blood pressure control. Repair of a potentially accessible aneurysm during pregnancy depends in part on the risk of recurrent hem orrhage versus the surgical risks. At least in nonpregnant patients, the risk of subsequent bleeding with conservative treatment is 20 to 30 percent for the irst month and then 3 percent per year. The risk of rebleeding is highest within the irst 24 hours, and recurrent hemorrhage leads to death in 70 percent.

1	The risk of rebleeding is highest within the irst 24 hours, and recurrent hemorrhage leads to death in 70 percent. Early repair after the sentinel hemorrhage is done by surgi cal clipping of the aneurysm. Also, an endovascular coil can be placed using luoroscopic angiography, while attempting to limit fetal radiation exposure. Barbarite and colleagues (2016) report lower complication rates with coil embolization than clipping. For unruptured aneurysms, surgical management resulted in a third fewer complications than no treatment. For gravidas remote from term, repair without hypotensive anes thesia seems optimal. For women near term, cesarean delivery followed by aneurysm repair is a consideration, and we have successfully done this in several cases.

1	For aneurysms repaired either before or during pregnancy, mal repair. Problems arise in defining "remote," and although some recommend 2 months, the time for complete healing is unknown. For women who survive subarachnoid hemor rhage, but in whom surgical repair is not done, we agree with another way, we favor cesarean delivery.

1	Arteriovenous Malformations. hese are congenital focal abnormal conglomerations of dilated arteries and veins with sub arteriolar disorganization (see Fig. 60-2). hey lack capillaries and have resultant arteriovenous shunting. Although unclear, the risk of bleeding may rise with gestational age. When arteriovenous malformations (A VMs) bleed, half do so into the subarachnoid space, whereas half are intraparenchymal with subarachnoid extension (Smith, 2015). hey are uncommon and are estimated to occur in 0.01 percent of the general population. Of 65 identiied cases of A VM in pregnancy, 83 percent ruptured during pregnancy or postpartum, and more than 80 percent of these ruptured in the second or third trimester. Hemorrhage upon presentation is associated with poor maternal outcome (Lu, 2016).

1	Bleeding does not appear to be more likely during pregnancy. lthough these malformations are correspondingly rare during pregnancy, A VM bleeding accounted for 17 percent of hemorrhagic strokes in one study (Yoshida, 2017). At Parkland Hospital in a 33-year period during which there were about 466,000 births, 57 women had a CVA, and five of these strokes were due to a bleeding A M (Simolkie, 1991; Zokie, 2018).

1	Treatment of AVMs in nonpregnant patients is largely individualized. No consensus guides whether all accessible lesions should be resected. Factors include AVM symptoms; its anatomy and size; presence of an associated aneurysm, which is found in up to 60 percent of cases; and especially, prior A VM bleeding. Mter hemorrhage, the risk of recurrent bleeding in unrepaired lesions is 6 to 20 percent within the first year, and 2 to 4 percent per year thereater (Friedlander, 2007; Smith, 2015). The mortality rate with a bleeding AVM is 10 to 20 percent. In pregnancy, the decision to operate is usually based on neurosurgical considerations, and Friedlander (2007) recommends strong consideration for treatment if bleeding occurs. Because of the high risk of recurrent hemorrhage from an unresected or inoperable lesion, we favor cesarean delivery.

1	The demyelinating diseases are neurological disorders characterized by immune-mediated focal or patchy destruction of myelin sheaths accompanied by an inflammatory response. The degenerative diseases are multifactorial and are characterized by progressive neuronal death. In the United States, multiple sclerosis (MS) is second only to trauma as a cause of neurological disability in middle adulthood (Hauser, 2015b). The disease afects women twice as often as men, and it usually begins in the 20s and 30s. he familial recurrence rate of MS is 15 percent, and the incidence in ofspring is increased IS-fold. Studying California deliveries, Fong and colleagues (2018) reported that 0.03 percent of deliveries were complicated by MS between 2001 and 2009.

1	The demyelinating characteristic of this disorder results predominately from T cell-mediated autoimmune destruction of oligodendrocytes that synthesize myelin. here is a genetic susceptibility and likely an environmental trigger such as exposure to certain bacteria and viruses. Of these, Chlamydophila pneumoniae, human herpesvirus 6, or Epstein-Barr virus are implicated (Frohman, 2006; Goodin, 2009). There are four clinical types of MS: 1. Relapsing-remitting MS accounts for initial presentation in 85 percent of afected individuals. With it, unpredictable recurrent episodes of focal or multifocal neurological dysfunction usually are followed by full recovery. Over time, however, relapses lead to persistent deficits. 2. Secondary progressive MS disease is relapsing-remitting disease that begins to pursue a progressive downhill course after each relapse. All patients likely develop this type eventually. 3. Primary progressive MS accounts for 15 percent of cases.

1	3. Primary progressive MS accounts for 15 percent of cases. With it, disability gradually progresses from the time of initial diagnosis. 4. Progressive-relapsing MS refers to primary progressive vIS with apparent relapses. Classic findings of MS include sensory loss, visual symptoms from optic neuritis, weakness, paresthesias, and a host of other neurological symptoms. Almost 75 percent of women with isolated optic neuritis develop MS within 15 years. Clinical diagnosis is confirmed by MR imaging and cerebrospinal fluid analysis. In greater than 95 percent of cases, MR imaging shows characteristic multifocal white matter plaques that represent discrete areas of demyelination (Fig. 60-4). Their appearance and extent are less helpful for predicting treatment response. Similarly, identification of serum antibodies against myelin oligodendrocyte glycoprotein and myelin basic protein is not predictive of recurrent disease activity (Kuhle, 2007). Efects of Pregnancy

1	Efects of Pregnancy The PRegnancy In Multiple Sclerosis-PlMS-study was a European prospective multicenter study in which 254 pregnancies were described (Vukusic, 2006). Relapse risk was reduced 70 percent during pregnancy, but with a significantly greater relapse rate postpartum. This may be related to higher pregnancy-induced numbers of T-helper lymphocytes and an increased T2/Tl ratio (Airas, 2008). In a metaanalysis of women with more than 1200 pregnancies complicated by lvIS, their relapse rate was 0.4 per year before pregnancy; 0.26 per year during pregnancy; and this increased to 0.7 per year

1	FIGURE 60-4 Magnetic resonance cranial images from a woman with mUltiple sclerosis. A.T2-weighted axial image shows bright signal abnormalities in white matter, typical for multiple sclerosis. B. Sagittal T2-FLAIR image shows hyperintense areas within the corpus callosum that are representative of demyelination in multiple sclerosis. (Reproduced with permission from Hauser SL, Goodin DS: Multiple Sclerosis and other demyelinating diseases. In Kasper DL, Fauci AS, Hauser SL, et al (eds): Harrison's Principles of Internal Medicine, 19th ed. McGraw-Hili, New York, 201o5b.) after delivery (Finkelsztejn, 2011). Bove and associates (2014) reached similar conclusions after a systematic review. Factors associated with postpartum relapse include a high relapse rate before pregnancy, relapses during pregnancy, and a high MS disability score (Portaccio, 2014; Vukusic, 2006). Breastfeeding has no apparent efect on postpartum relapses (Hellwig, 2015; Portaccio, 2011).

1	Efects of Multiple Sclerosis on Pregnancy With uncomplicated disease, there are usually no adverse efects on pregnancy ourcome (Bove, 2014). Some women may become fatigued more easily, those with bladder dysfunction are predisposed to urinary infection, and women with spinal lesions at or above T 6 are at risk for autonomic dysrelexia. In one study of 449 pregnancies in afected women, the labor induction rate was higher, and second-stage labor was longer (Dahl, 2006). he greater induction rate and elective operations contributed to the overall higher cesarean delivery rate. In an analysis of 649 afected women, the mean birthweight was lower but the perinatal mortality rate was similar compared with that of controls (Dahl, 2005). Other studies have corroborated that MS does not significantly afect obstetrical and neonatal outcomes (Finkelsztejn, 2011; Fong, 2018).

1	Goals are to arrest acute or initial attacks, employ diseasemodiying agents, and provide symptomatic relief. Some treatments may need to be modiied during pregnancy. Acute or initial attacks are treated with high-dose intravenous methylprednisolone-500 to 1000 mg daily for 3 to 5 days, followed by oral prednisone for 2 weeks. Plasma exchange may be considered. Symptomatic relief can be provided by analgesics; carbamazepine, phenytoin, or amitriptyline for neurogenic pain; baclofen for spasticity; :Tadrenergic blockade for bladder neck relaxation; and cholinergic and anticholinergic drugs to stimulate or inhibit bladder contractions.

1	Several disease-modiying therapies can be used for relapsing MS or for exacerbations. Examples include in terferons 31 a (Rebi), 31 b (Betaseron), and glatiramer acetate (Copaxone), which lower relapse rates by a third (Rudick, 201l). Data concerning safety in pregnancy are limited but overall reassuring (Amato, 2010; Salminen, 2010). In clinical trials, natalizumab (Tysabri), an :4-integrin antagonist, especially when combined with interferon 31a, significantly reduced MS clinical relapse rates (Polman, 2006; Rudick, 2006). In a review of 35 pregnancies, irst-trimester drug exposure did not worsen outcomes (Hellwig, 2011). If these drugs are used in pregnancy, the neonate should be monitored for thrombocytopenia and anemia (Alroughani, 2016).

1	Fetal exposure in 89 pregnancies to ingolimod (Gilenya), another immune-modulating drug, was associated with six fetal malformations and nine spontaneous losses. Because of this and associated animal teratogenicity, its use in pregnancy is not recommended. Due to its prolonged persistence, contraception is recommended for 2 months ater drug cessation (Alroughani, 2016; Karlsson, 2014). Prevention of relapses postpartum is aforded by treatment with intravenous immunoglobulin (I I G) , given in a dose of 0.4 g/kg daily for 5 days during weeks 1, 6, and 12 (Argyriou, 2008).

1	Prevention of relapses postpartum is aforded by treatment with intravenous immunoglobulin (I I G) , given in a dose of 0.4 g/kg daily for 5 days during weeks 1, 6, and 12 (Argyriou, 2008). his adult-onset neurodegenerative disease stems from an autosomal dominant expanded CAG trinucleotide repeat within the Huntington gene on chromosome 4. It is characterized by choreoathetotic movements, progressive dementia, and psychiatric manifestations. Because the mean age of onset is 40 years, Huntington disease rarely complicates pregnancy. Prenatal diagnosis is discussed in Chapter 14 (p. 288). Prenatal screening is controversial, and because this usually is a late-onset adult disease, extensive counseling is important (Schulman, 2015).

1	approximately 1 in 7500 persons. It is more common in women, and its incidence peaks in their 20s and 30s. he eti ology is unknown, but genetic factors likely playra role. Most patients demonstrate antibodies to the acetylcholine receptor, although 10 to 20 percent are seronegative (Drachman, 2015). he latter often have antibodies to muscle-specific tyrosine kinase (MuSK), which regulates assembly of the acetylcholine receptor subunits at the neuromuscular junction (Pal, 201l).

1	he latter often have antibodies to muscle-specific tyrosine kinase (MuSK), which regulates assembly of the acetylcholine receptor subunits at the neuromuscular junction (Pal, 201l). Cardinal features of myasthenia are weakness and easy fatigability of facial, oropharyngeal, extraocular, and limb muscles. Deep tendon reflexes are preserved. Cranial muscles are involved early and disparately, and diplopia and ptosis are common. Facial muscle weakness causes diiculty in smiling, chewing, and speaking. In 85 percent of patients, the weakness becomes generalized. Other autoimmune diseases may coexist, and hypothyroidism should be excluded. The clinical course is marked by exacerbations and remissions, especially when it first becomes clinically apparent. Remissions are not always complete and are seldom permanent. Systemic diseases, concurrent infections, and even emotional upset may precipitate exacerbations, of which there are three types: 1.

1	Myasthenic crises-characterized by severe muscle weakness, inability to swallow, and respiratory muscle paralysis. 2. Rtactory crises-characterized by the same symptoms but unresponsive to the usual therapy. 3. Cholinergic crisesexcessive cholinergic medication leads to nausea, vomiting, muscle weakness, abdominal pain, and diarrhea. All three of these can be life threatening, but a refractory crisis is a medical emergency. Those with bulbar myasthenia are at particular risk because they may be unable to swallow or even ask for help.

1	Myasthenia is manageable but not curable. Oral pyridostigmine is the first-line treatment. Thymectomy is recommended but postponed until ater pregnancy (Sanders, 2016). Anticholinesterase medications improve symptoms by impeding acetylcholine degradation but seldom produce normal muscle function. Ironically, overdose is manifest by increased weness-cholinergic crisisthat may be diicult to diferentiate from myasthenic symptoms. Most of those refractory to anticholinesterase therapy respond to immunosuppressive therapy with glucocorticoids, azathioprine, or cyclosporine in pregnancy. When short-term, rapid clinical improvement is needed-such as for a surgical procedure or a myasthenic crisis-high-dose IVIG or plasma exchange is usually efective (Barth, 2011; Cortese, 2011; Sanders, 2016).

1	Because the greatest period of risk is within the first year following diagnosis, postponing pregnancy until there is sustained improvement is reasonable. Antepartum management of myasthenia includes close observation with liberal rest and prompt treatment of infections (Heaney, 2010; Kalidindi, 2007). Women in remission who become pregnant while taking corticosteroids or azathioprine should continue these. hymectomy has been successfully performed during pregnancy in refractory cases (Ip, 1986). Acute onset of myasthenia or its exacerbation demands prompt hospitalization and supportive care. Plasmapheresis and high-dose IVIG are options for emergency situations (Drachman, 2015).

1	Although pregnancy does not appear to afect the overall course of myasthenia, fatigue common to most pregnancies may be exacerbated, and the expanding uterus may compromise respiration. Maternal hypotension or hypovolemia are ideally avoided as they can trigger crises. he clinical course of myasthenia during pregnancy is unpredictable, and frequent hospitalizations are the norm. Up to a third of women have worsening myasthenia during pregnancy, and exacerbations occur equally in all three trimesters (Djelmis, 2002; Podciechowski, 2005). In women with stable disease, most will remain stable throughout pregnancy but likely worsen in the first few months postpartum (Sanders, 2016).

1	Myasthenia gravis has no significant adverse efects on pregnancy outcomes (Wen, 2009). Preeclampsia is a concern because magnesium sulfate may precipitate a severe myasthenic crisis (Hamaoui, 2009; Heaney, 2010). Although phenytoin use is also problematic in this regard, its adverse efects are less troublesome. hus, many choose it for neuroprophylaxis in women with severe preeclampsia.

1	Because smooth muscle is unafected, most women have normal labor. Oxytocin is given for the usual indications, and cesarean delivery is reserved for obstetrical indications. Narcotics may cause respiratory depression, and close observation and respiratory support are essential during labor and delivery. Curariform drugs are avoided-examples include magnesium sulfate discussed above, muscle relaxants used with general anesthesia, and aminoglycosides. Neuraxial analgesia is accomplished with amide-type local agents. Regional analgesia is preferred unless there is signiicant bulbar involvement or respiratory compromise (Almeida, 2010; Blichfeldt-Lauridsen, 2012). During second-stage labor, some women may have impaired voluntary expulsive eforts that may warrant operative vaginal delivery.

1	As discussed above, 80 percent of mothers with myasthenia gravis have anti-acetylcholine-receptor immunoglobulin G (IgG) antibodies. These and anti-MuSK antibodies cross transplacentally, and the fetus can be afected. Poor fetal swallowing may yield hydramnios (Heaney, 2010). Similarly, 10 to 20 percent of neonates manifest myasthenia symptoms (Jovandaric, 2016). Transient symptoms usually include a feeble cry, poor suckling, and respiratory distress. Symptoms usually respond to cholinesterase inhibitors and resolve within a few weeks as maternal IgG antibodies clear. Peripheral neuropathy is a general term used to describe disorders of peripheral nerve(s) from various sources. Poyneuropathies can be axonal or demyelinating as well as acute, subacute, or chronic (Amato, 2015). hese are often associated with systemic diseases such as diabetes, with drug or environmental toxin exposure, or with genetic disease.

1	Mononeuropathies are relatively common in pregnancy and signiY focal involvement of a single nerve trunk. These imply local causation such as trauma, compression, or entrapment. Traumatic pudendal, obturator, femoral, and common fibular mononeuropathies are usually caused by childbirth and are discussed in Chapter 36 (p. 661). In 75 percent of cases, this acute demyelinating polyradiculoneuropathy has clinical or serological evidence for an acute infection. Commonly associated are infections with Campylobacter jjuni, cytomegalovirus, Zika virus, and Epstein-Barr virus; surgical procedures; and immunizations (Haber, 2009; Hauser, 2015a; Pacheco, 2016). Guillain-Barre syndrome is thought to be immune-mediated from antibodies formed against nonself antigens. Demyelination causes sensory and motor conduction blockade, and remyelination yields recovery in most cases.

1	Clinical features include areflexic paralysis-usually ascending-with or without sensory disturbances. Autonomic dysfunction is common. The full syndrome develops over 1 to 3 weeks. Some manifest as chronic inlammatoy demyelinating poyneuropathy, and our experiences indicate that this may be relatively common in these young women.

1	Guillain-Barre syndrome is not more common in pregnancy, and its clinical course mirrors that for nonpregnant individuals. After an insidious onset, paresis and paralysis most oten continue to ascend to cause ventilatory weakness. Management is supportive and incorporates venous thromboembolism prophylaxis, pressure ulcer prevention, and enteral nutrition. In the worsening phase, patients are hospitalized, and a fourth requires ventilatory assistance. IVIG or plasmapheresis is beneficial if begun within 1 to 2 weeks of motor symptoms, however, neither decreases mortality rates (Cortese, 2011; Gwathmey, 2011; Pritchard, 2016). Up to 10 percent of patients deteriorate after initial improvement on therapy, and retreatment with 2 g/kg IVIG over 5 days is recommended. Although most patients recover fully within several months to a year, the mortality rate is 5 percent, mainly due to pulmonary complications and arrhythmias (Hauser, 20 15a; Pacheco, 2016).

1	FIGURE 60-5 Bell facial nerve palsy developing on the day of cesarean delivery for dichorionic twins. This woman was treated with prednisone and antiviral medication, and the palsy had almost resolved 3 weeks postpartum. his disfiguring palsy is usually a mononeuropathic acute facial paralysis that is relatively common in reproductive-aged women (Fig. 60-5). It has a female predominance, and pregnant women carry a fourfold risk compared with nonpregnant women (Cohen, 2000; Heaney, 2010). The disease is characterized by facial nerve inlammation and often is associated with reactivation of herpes simplex virus or herpes zoster virus.

1	Bell palsy usually has an abrupt and painful onset with maximum weakness by 48 hours. In some cases, hyperacusis and loss of taste accompany paralysis (Beal, 2015). Management includes supportive care with facial muscle massage and eye protection against corneal lacerations from drying. here is general consensus that prednisone, 1 mg/kg given orally daily for 5 days, will improve outcomes and shorten the recovery period (Salinas, 2016; Sullivan, 2007). It is controversial whether addition of an antiviral medication will add benefits (de Almeida, 2009; Gagyor, 2015; Quant, 2009).

1	It is unclear if pregnancy alters the prognosis for spontaneous facial palsy recovery. Gillman and associates (2002) found that only half of pregnant women recovered to a satisfactory level after 1 year, compared with approximately 80 percent of nonpregnant women and men. Some prognostic markers for incomplete recovery are bilateral palsy, recurrence in a subsequent pregnancy, greater percentage of nerve function loss, and a faster rate of loss (Cohen, 2000; Gilden, 2004). Other than a ivefold greater rate for gestational hypertension or preeclampsia, women with Bell palsy do not have increased adverse pregnancy outcomes rates (Katz, 2011; Shmorgun, 2002).

1	This syndrome results from compression of the median nerve and is the most frequent mononeuropathy in pregnancy (Padua, 2010). Symptoms include burning, numbness, or tingling along the inner half of one or both hands. Others are wrist pain and numbness extending into the forearm and sometimes into the shoulder (Katz, 2002). Symptoms are bilateral in 80 percent of gravidas, and 10 percent have evidence for severe denervation (Seror, 1998). Diferential diagnosis includes cer vical radiculopathy of C6-C7 and de Quervain tendonitis. he latter is caused by swelling of the conjoined tendons and their sheaths near the distal radius. Nerve conduction studies may be helpful for clariication (Alfonso, 2010).

1	In pregnancy, the reported incidence of carpal tunnel syn drome is 7 to 43 percent and varies greatly because the range of symptoms is marked (Meems, 2015; Padua, 2010). Symp tomatic treatment with a splint applied to a slightly lexed wrist during sleep lightens pressure and usually provides relief. Although symptoms typically are self-limited, occasionally sur gical decompression and corticosteroid injections are necessary (Keith, 2009; Shi, 2011). Symptoms may persist in more than half of patients at 1 year and in a third at 3 years (Padua, 2010).

1	According to the National Spinal Cord Injury Statistical Center (2017), there are approximately 17,000 new spinal cord injuries each year. he average age is 42 years, and males account for 80 percent of new cases. Cord injury severity determines the short-and long-term prognosis as well as that for pregnancy. For women, many have altered sexual function and transient hypothalamic pituitary hypogonadism. hat said, pregnancy is not uncommon if menstruation resumes (Bughi, 2008). In a review of nearly 2000 women in the National Spinal Cord Injury Database, 2 percent reported pregnancy in the prior 12 months (Iezzoni, 2015).

1	Gravidas with spinal cord injury have an increased frequency of pregnancy complications that include preterm and low-birth weight neonates. Recent observations in nonpregnant women note that the vaginal microbiota is altered in these women (Pires, 2016). Perhaps related, most have asymptomatic bacteriuria with sporadic symptomatic urinary infections. Bowel dysfunction causes constipation in more than half, and anemia and pressure-necrosis skin lesions are also common. Two serious and life-threatening events can complicate spinal cord injuries. First, if the cord is transected above T 10' the cough reflex is impaired, respiratory function may be compromised, and pneumonitis from covert aspiration can be serious. Pulmonary function tests are considered to assess this risk, and some women may need ventilatory support in late pregnancy or in labor.

1	Second, women with lesions above T 5-T6 are at risk for autonomic dysrlexia. With this, stimuli from structures innervated below the level of the spinal lesion lead to massive, disordered sympathetic stimulation. Abrupt catecholamine release can cause vasoconstriction with severe hypertension and symptoms that include throbbing headaches, facial flushing, sweating, bradycardia, tachycardia, arrhythmias, and respiratory distress . Dysrelexia can be precipitated by various stimuli. hese include urethral catheterization, bladder distention from urinary retention, rectal or cervical stretch during digital examinations, uterine contractions and cervical dilation, or any manipulation of other pelvic structures (American College of Obstetricians and Gynecologists, 2016; Krassioukov, 2009). In one report, 12 of 15 women at risk for autonomic dysreflexia sufered at least one episode during pregnancy (Westgren, 1993).

1	Because uterine contractions are not afected by spinal cord lesions, labor is usually easy-even precipitous, and comparatively painless. If the lesion is below T12' uterine contractions are felt normally. For lesions above T 2, the risk of out-of 1rhospital delivery is substantial and can be minimized by teaching women to palpate for uterine contractions. This is especially important because up to 20 percent of women deliver preterm (Westgren, 1993). Some recommend tocodynamometry and weekly cervical examinations beginning at 28 to 30 weeks. Another reasonable option that we frequently employ at Park land Hospital is elective hospitalization after 36 to 37 weeks' gestation (Hughes, 1991).

1	Spinal or epidural analgesia extending to TIO prevents autonomic dysrelexia and should be instituted at the start of labor. If there are severe symptoms before epidural placement, steps are taken to abolish the provoking stimulus. A parenteral antihypertensive agent such as hydralazine or labetalol is given. Labor and vaginal delivery with epidural or spinal analgesia is preferable and will minimize autonomic dysrelexia (Kuczkowski, 2006). Operative vaginal delivery is frequently necessary.

1	Also known as pseudotumor cerebri, this disorder is typified by increased intracranial pressure without hydrocephalus. The cause is unknown, but it may result from overproduction or under absorption of cerebrospinal luid (CSF). Symptoms include headache in at least 90 percent of cases, visual disturbances such as loss of a visual field or central visual acuity in 70 percent, and commonly occurring papilledema that may be sight-threatening (Evans, 2000; Heaney, 2010). Other complaints are stif neck, back pain, pulsatile tinnitus, and cranial nerve palsies. he syndrome is oten found in young women and is prevalent in those who are obese, who recently gained weight, or both (Fraser, 201r1). Along with symptoms, other criteria for diagnosis include elevated intracranial pressurer> 25 em H20, normal CSF composition, normal cranial CT or MR imaging indings, papilledema, and no evidence for systemic disease. If papilledema is not present, other criteria are required (Friedman, 2013).

1	Idiopathic intracranial hypertension is usually self-limited. Visual defects can be prevented by lowering the CSF pressure, and agents include acetazolamide to reduce fluid production, furosemide, or topiramate. Corticosteroids are now rarely used. Surgical intervention is occasionally necessary and is accomplished by either lumboperitoneal shunting of spinal fluid or optic nerve sheath fenestration. It is controversial if pregnancy is a risk factor for idiopathic intracranial hypertension. Certainly, symptoms may first appear in pregnancy, and women previously diagnosed may become symptomatic. Symptoms usually develop by midpregnancy, tend to be self-limited, and usually resolve postpartum.

1	Pregnancy does not alter management. Some recommend serial visual field testing to forestall permanent vision loss. In a report of 16 pregnant women, visual ield loss developed in four, and it became permanent in one (Huna-Baron, 2002). Visual ield loss is often coincident with the development of papilledema, for which acetazolamide is given. Lee and associates (2005) reported successful treatment of 12 pregnant women. Although outmoded for treatment of nonpregnant individuals, repeated lumbar punctures are generally successful in providing temporary relief throughout pregnancy. In some pregnant women, surgical therapy becomes necessary, and we and others have had promising results with optic nerve sheath fenestration (hambisetty, 2007).

1	Pregnancy complications are likely due to associated obesity and not to intracranial hypertension. In a review of 54 pregnancies, rates of adverse perinatal outcomes were not elevated (Katz, 1989). he route of delivery depends on obstetrical indications, and conduction analgesia is safe (Aly, 2007; Karmaniolou, 2011).

1	Pregnancies in women with previously placed ventricular shunts for obstructive hydrocephalus usually have satisfactory outcomes (Landwehr, 1994). Shunts may be ventriculoperitoneal, ventriculoatrial, or ventriculopleural. Partial obstruction of a shunt is common, especially late in pregnancy (Schiza, 2012). In one report of 17 such pregnancies, neurological complications were reported in 13 (Wisof, 1991). Findings included headaches in 60 percent, nausea and vomiting in 35 percent, lethargy in 30 percent, and ataxia or gaze paresis, each in 20 percent. Most symptoms respond to conservative management. However, if CT scanning during symptom evaluation discloses acute hydrocephaly, then the shunt is tapped or pumped several times daily. In some cases, surgical revision is necessary and may be emergently indicated (Murakami, 2010).

1	Another shunting procedure is placement of an endoscopic third ventriculostomy for hydrocephalus (de Ribaupierre, 2007). With this, a small hole is created in the loor of the third ventricle to allow CSF to low directly into lower cisterns. One report described successful results in ive pregnant women who underwent endoscopic ventriculostomy (Rifaud, 2006). In a review, however, reproductive function and miscarriage rates were found to significantly worsen in these women (Bedaiwy, 2008). Vaginal delivery is preferred in women with shunts, and unless there is a meningomyelocele, conduction analgesia is permitted. Antimicrobial prophylaxis is indicated if the peritoneal cavity is entered for cesarean delivery or tubal sterilization.

1	Brain death is rare in obstetrics. Life-support systems and parenteral alimentation for up to 15 weeks while awaiting delivery have been described (Hussein, 2006; Powner, 2003; Souza, 2006). Some women were treated with aggressive tocolysis and antimicrobial therapy. In one review of 17 women with persistent vegetative state who were given various levels of support, five women died after delivery, and most of the others remained in their vegetative state (Chiossi, 2006). With a diagnosis of brain death using the uniform Determination of Death Act definition, there are no published reports of neurological recovery (Wijdicks, 2010). Few institutional brain-death policies address pregnancy (Lewis, 2016). The ethical, inancial, and legal implications, both civil and criminal, that arise from attempting or not attempting such care are profound (Farragher, 2005; Feldman, 2000). In some women, perimortem cesarean delivery is performed as discussed in Chapter 47 (p. 931).

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1	Van Teen TR, Panerai RB, Haeri S, et al: Changes in cerebral autoregulation in the second half of pregnancy and compared to non-pregnant controls. Pregnancy Hypertens 6(4):380, 2016 Viale L, Allotey ], Cheong-See F, et al: Epilepsy in pregnancy and reproductive outcomes: a systematic review and meta-analysis. Lancet 386: 1845, 2015 Vukusic S, Confavreux C: Pregnancy and multiple sclerosis: the children of PRIMS. Clin Neurol Neurosurg 108:266, 2006 Wabnitz A, Bushnell C: Migraine, cardiovascular disease, and stroke during pregnancy: systematic review of the literature. Cephalalgia 35(2): 132, 2015 Wang IK, Chang SN, Liao CC, et al: Hypertensive disorders in pregnancy and preterm delivery and subsequent stroke in Asian women: a retrospective cohort study. Stroke 42:716, 2011 Wasay M, Bakshi R, Bobustuc G, et al: Cerebral venous thrombosis: analysis of a multicenter cohort from the United States. ] Stroke Cerebrovasc Dis 17:49, 2008

1	Wasay M, Bakshi R, Bobustuc G, et al: Cerebral venous thrombosis: analysis of a multicenter cohort from the United States. ] Stroke Cerebrovasc Dis 17:49, 2008 Wen ]C, Liu TC, Chen H, et al: No increased risk of adverse pregnancy outcomes for women with myasthenia gravis: a nationwide population-based study. Eur] NeuroI 16:889, 2009 Westgren N, Hultling C, Levi R, et al: Pregnancy and delivery in women with a trauma spinal cord injury in Sweden, 1980-1991. Obstet Gynecol 81:926, 1993 Weston ], Bromley R, Jackson CF, et al: Monotherapy treatment of epilepsy in pregnancy: congenital malformation outcomes in the child. Cochrane Database Syst Rev II:CDOI0224, 2016 Wijdicks EFM, Varelas PN, Gronseth GS, et al: Evidence-based guideline update: determining brain death in adults. Neurology 74:1911, 2010 Wisof ]H, Kratzert K], Handwerker SM, et al: Pregnancy in patients with cerebrospinal fluid shunts: report of a series and review of the literature. Neurosurgery 29:827, 1991

1	Wisof ]H, Kratzert K], Handwerker SM, et al: Pregnancy in patients with cerebrospinal fluid shunts: report of a series and review of the literature. Neurosurgery 29:827, 1991 Wood ME, Frazier ]A, Nordeng HM, et al: Longitudinal changes in neurodevelopmental outcomes between 18 and 36 months in children with prenatal trip tan exposure: findings from the Norwegian Mother and Child Cohort Study. BM] Open 6(9):eOI1971, 2016 Wyszynski DF, Nambisan M, Surve T, et al: Increased rate of major malformations in ofspring exposed to valproate during pregnancy. Neurology 64:961, 2005 Yager PH, SinghalAV, Nogueira RG: Case 31-2012: an 18-year-old man with blurred vision, dysarthria, and ataxia. N Engl] Med 367: 1450, 2012 Yerby MS: Pregnancy, teratogenesis, and epilepsy. Neurol Clin 12:749, 1994

1	Yerby MS: Pregnancy, teratogenesis, and epilepsy. Neurol Clin 12:749, 1994 Yoshida K, Takahashi ]C, Takenobu Y, et al: Strokes associated with pregnancy and puerperium. A nationwide study by the Japan Stroke Society. Stroke 48:276, 2017 Zeeman GG, Fleckenstein ]L, Twickler DM, et al: Cerebral infarction in eclampsia. Am] Obstet Gynecol 190:714, 2004a Zeeman GG, Hatab M, Twickler DM: Increased cerebral blood low in preeclampsia with magnetic resonance imaging. Am ] Obstet Gynecol 191:1425, 2004b Zeeman GG, Hatab M, Twickler DM: Maternal cerebral blood low changes in pregnancy. Am] Obstet Gynecol 189:968, 2003 Zofkie A, Cunningham FG: A 33-year single-center experience with pregnancy-associated strokes. Abstract. Presented at the 38th Annual Meeting of the Society for Maternal-Fetal Medicine. February 1-3, 2018 CHAPTER 61 . PSYCHOLOGICAL ADJUSTMENTS TO PREGNANCY .. 1173 MAJOR DEPRESSION.. . . . . . . . . . . . . . . . . . . . . . . . . .. 1175

1	CHAPTER 61 . PSYCHOLOGICAL ADJUSTMENTS TO PREGNANCY .. 1173 MAJOR DEPRESSION.. . . . . . . . . . . . . . . . . . . . . . . . . .. 1175 BIPOLAR AND RELATED DISORDERS .............. 1178 ANXIETY DISORDERS. . . . . . . . . . . . . . . . . . . . . . . . . .. 1179 SCHIZOPHRENIA SPECTRUM DISORDERS.. . . . . . . . .. 1180 EATING DISORDERS. . . . . . . . . . . . . . . . . . . . . . . . . . .. 1180 PERSONALITY DISORDERS.. . . . . . . . . . . . . . . . . . . . .. 1181 he insaniy ofpregnancy is usualy a manestation of autointoxication, and may be accompanied by melancholic or maniacal symptoms. It usualy persists throughout gestation, but disappears shorty ater labour, unless the patient has an hereditary tendency to mental derangement. -]. Whitridge Williams (1903)

1	-]. Whitridge Williams (1903) The subject of mental illness was only briely addressed by Williams in 1903, when it appears that acute puerperal psychoses were manifestations of eclampsia or sepsis. More than 100 years later, we have learned that pregnancy and the puerperium are at times suiciently stressful to provoke mental illness. Such illness may represent recurrence or exacerbation of a preexisting psychiatric disorder, or it may signal the onset of a new condition. his 25th edition of Williams Obstetrics marks only the second edition with a focused chapter dedicated to psychiatric illnesses. To emphasize the rising national interest, American College of 0bstetricians and Gynecologists President Dr. Gerald F. Joseph Jr. declared postpartum depression as an initiative in 2009.

1	Psychiatric disorders during pregnancy are associated with less prenatal care, substance use, poor obstetrical and neonatal outcomes, and higher rates of postpartum psychiatric illness (Frieder, 2008). Despite these known risks, obstetrical providers often are reluctant to confront or fail to identiy some of these mental health issues during pregnancy. For example, Lyell and colleagues (2012) found that the diagnosis of depression was not documented in nearly half of the records of depressed women. Yet, perinatal mood disorders can have far-reaching consequences beyond the immediate efect on maternal mental health and social function by adversely afecting the motherchild relationship (Weinberg, 1998).

1	lso, suicide is a primary cause of death among women during the perinatal period in the United States, and major depression is among the strongest predictors of suicidal ideation (Melville, 2010). Between 2004 and 2012, self-harm, suicide, or drug overdose was the leading cause of maternal death in Colorado (Metz, 2016). In a 10-year analysis of Washington state hospitalizations, Comtois and associates (2008) studied 355 women with a postpartum suicide attempt. Substance abuse was linked with a sixfold higher and prior psychiatric hospitalization with a 27 -fold greater risk for suicide. These rates rose further if there were multiple hospitalizations. Also of note, 54 percent of pregnancy-associated suicides involve intimatepartner conlict (Palladino, 201r1).

1	Biochemical factors and life stressors can markedly inluence mental health and mental illness during the perinatal period. Intuitively, pregnancy exacerbates some coexisting psychological disorders. Namely, an increased risk for mood disorders is linked with pregnancy-related shifts in sex steroid and monoamine neurotransmitter levels, dysfunction of the hypothalamicpituitary-adrenal axis, thyroid dysfunction, and alterations in immune response (Yonkers, 2011). These changes, coupled with familial clustering of depression cases, suggest that there may be a subgroup of women at risk for developing a unipolar major depressive disorder during pregnancy.

1	Women respond in various ways to stressors of pregnancy, and some express persistent concerns regarding fetal health, child care, lifestyle changes, or fear of childbirth pain. Anxiety, sleep disorders, and unctional impairment are common (Romero, 2014; Vythilingum, 200S). However, according to Littleton and coworkers (2007), anxiety symptoms in pregnancy are associated with psychosocial variables similar to those for nonpregnant women. he level of perceived stress is significantly higher for women whose fetus is at high risk for a malformation, for those with preterm labor or delivery, and for those with other medical complications (Alder, 2007; Ross, 2006). Hippman and colleagues (2009) screened for depression in Sl women who had an increased risk for a fetus with aneuploidy. Half of these women had a positive depression screening score, whereas only 2.4 percent of those with a normal pregnancy did so.

1	Several steps can be taken to diminish psychological stress in the event of a poor obstetrical outcome. For example, following a stillbirth, Gold (2007) encouraged parental contact with the newborn and provision of photographs and other infant memorabilia. Addressing associated sleep disorders also seems reasonable Quulia Paavonen, 2017; Romero, 2014). • The Puerperium This is a particularly stressful time for women, and risks for mental illness are increased. Up to 15 percent of women develop a nonpsychotic postpartum depressive disorder within 6 months of delivery (Tam, 2007; Yonkers, 201r1). A few have a psychotic illness following delivery, and half of these manifest a bipolar disorder. Depressive disorders are more likely in women with obstetrical complications such as severe preeclampsia or fetal-growth restriction, especially if associated with early delivery. Houston and coworkers (2015) found that expectations at delivery also increased the risk for postpartum depression.

1	Importantly, stressors beyond those directly related to the pregnancy can raise perinatal depression rates. T arney and colleagues (2015) identified spouse deployment as a factor for postpartum depression in a study at Womack Army Medical Center. But, among women with a history of bipolar disorder, these elements playra lesser role in the development of mania or depression (Yonkers, 2011). Also called pospartum blues, this is a time-limited period of heightened emotional reactivity experienced by half of women within the irst week after parturition. Prevalence estimates for the blues range from 26 to S4 percent depending on diagnostic criteria (O'Hara, 2014). his emotional state generally peaks on the fourth or ifth postpartum day and normalizes by day 10 (O'Keane, 2011).

1	he predominant mood is happiness, but afected mothers are more emotionally labile. hey also may have insomnia, weepiness, depression, anxiety, poor concentration, and irritability. Mothers may be transiently tearful for several hours and then recover completely, only to be tearful again the next day. Supportive treatment is indicated, and afected women are reassured that the dysphoria is transient and most likely due to biochemical changes. They should be monitored for development of depression and other severe psychiatric disturbances.

1	Both the American College of Obstetricians and Gynecologists (2016a) and the United States Preventative Services Task Force now recommend screening at least once during the perinatal period for depression and anxiety (Siu, 2016). Identiication of psychiatric disorders in pregnancy can be challenging because changes in behavior and mood are often attributed to pregnancy. To diferentiate these, Yonkers (2011) recommends assessment of cognitive symptoms-for example, loss of concentration. Excessive symptoms of anxiety and insomnia-even during periods of infant sleep-can also suggest postpartum depression. Speciic factors for depression are reviewed and include a prior personal or family history of depression.

1	Universal-screening programs for depression continue to evolve (Venkatesh, 2016). At Parkland Hospital, mental illness screening is generally done at the irst prenatal visit using a brief risk-based query and again postpartum using a universalapplied screening tool for postpartum depression. Questions search for psychiatric disorders, related therapy, prior or current use of psychoactive medications, and current symptoms. Women with a history of sexual, physical, or verbal abuse; substance abuse; and personality disorders are also at greater risk for depression (man, 2007; Janssen, 2012). History of neglect and abuse are especially powerful antecedents of depression in adolescent pregnancy (Meltzer-Brody, 2014). Smoking and nicotine dependence and obesity also raise rates of all mental disorders in pregnancy (Goodwin, 2007; Molyneaux, 2014). Finally, because eating disorders may be exacerbated by pregnancy, afected women are followed closely (p. l1S0).

1	Several screening instruments shown in Table 61-1 are available and have been validated for use during pregnany and the puerperium. Use of one of these screening tools is encouraged because symptom-or risk-based screening alone may be insuficient (American College of Obstetricians and Gynecologists, 2016a). Cerimele and colleagues (2013) found that obstetricians and gynecologists failed to identiY 60 percent of depressed women in clinical practice. As mentioned earlier, at Parkland Hospital, all women are screened during their irst postpartum visit using the Edinburgh Postnatal Depression Scale (EPDS). In an analysis of more than 17,000 women, 6 percent had scores that indicated either minor or major depressive symptoms, and 12 women had thoughts of self-harm (Nelson, 2013). Similarly, Kim and coworkers (2015) assessed suicidal ideation in more than 22,000 women screened using the EPDS both during pregnancy and postpartum. They found rates of depression as high as 3.4 percent during

1	and coworkers (2015) assessed suicidal ideation in more than 22,000 women screened using the EPDS both during pregnancy and postpartum. They found rates of depression as high as 3.4 percent during the puerperium. A small fraction of those with thoughts of self-harm had a credible plan, intent, and means for attempted suicide. Obviously, suicidal ideation warrants prompt psychiatric consultation for evaluation and management.

1	Screening for perinatal depression without appropriate subsequent treatment is insuicient (American College of Obstetricians and Gynecologists, 20 16a). hat said, mechanisms to ensure adequate ensuing care can be problematic. In TABLE 61 -1 . Depression Screening Toolsa Time to Complete Edinburgh Postnatal 10 <5 http://www.fresno.ucsf.ed u/ped iatricsl down loadsl edi nbu rg hsca Ie. pdf Patient Health 9 <5 http://www.integration.samhsa.gov/images/res/PHQ%20-%20Questions.pdf Center for Epidemiologic 20 5-10 http://www.perinatalweb.org/assets/cms/uploads/files/CES-D.pdf aAIl depression screening tools are also available in Spanish. bAvailable free online.

1	the study by Nelson and associates (2013) more than three fourths of the 1106 women with abnormally elevated EPDS scores did not keep their later appointment, which ofered more formal psychiatric evaluation. Barriers include diiculties with access to care, personal perception of depression, and societal stigmata (Flynn, 2010; Smith, 2008). Women referred to a behavioral health provider located at the same site as their obstetrical care are four times more likely to access treatment than those referred elsewhere (Smith, 2009). To take advantage of this, at Parkland Hospital, mental health counselors also practice at postpartum clinic sites. Other promising interventions for puerperal depression include home visits, telephonebased peer support, and interpersonal psychotherapy (Dennis, 2013; Lavender, 2013; Yonemoto, 2017). A report from Kaiser Permanente, which describes the benefits and hurdles to system-based perinatal mental health care, provides a glimpse into the possible future of

1	Lavender, 2013; Yonemoto, 2017). A report from Kaiser Permanente, which describes the benefits and hurdles to system-based perinatal mental health care, provides a glimpse into the possible future of universal perinatal screening and treatment (Avalos, 2016; Flanagan, 2016).

1	Many psychiatric disorders can be improved with counseling and psychotherapies. In some instances, psychotropic medications are needed. Treatment decisions are ideally shared between patients and their health-care providers. In particular, women taking psychotropic medication are informed of likely side efects. Many of these drugs are discussed in Chapter 12 and by the American College of Obstetricians and Gynecologists (2016b) in their Practice Bulletin No. 92. Some of these drugs are discussed subsequently.

1	Emerging information on psychiatric disorders and pregnancy outcomes suggest a link between maternal psychiatric illness and untoward outcomes such as preterm birth, low birthweight, and perinatal mortality (Grigoriadis, 2013; Steinberg, 2014; Straub, 2012; Yonkers, 2009). In a study of 16,334 deliveries, Shaw and coworkers (2014) identiied a signiicant association between posttraumatic stress disorder and spontaneous pre term delivery. Domestic abuse-another aforementioned risk factor for perinatal mood disorder-is also linked with adverse perinatal outcomes (Yost, 2005). Finally, Littleton and associates (2007) reviewed 50 studies and concluded that anxiety symptoms, which are commonly comorbid with depression, had no adverse efect on perinatal outcomes. is the most recent version by the American Psychiatric Associa tion (2013). It assists in classiying mental disorders and speci fies criteria for each diagnosis.

1	is the most recent version by the American Psychiatric Associa tion (2013). It assists in classiying mental disorders and speci fies criteria for each diagnosis. Of categories, depressive disorders are common. According to the National Institute of Mental Health (2010), the life time prevalence of depressive disorders in the United States is 21 percent. Historically, depressive disorders include major bipolar disorder with both manic and depressive episodes. It also includes dysthymia, which is chronic, mild depression. This is the most common depressive disorder, and the 12-month prevalence of major depressive episodes among U.S. women is 8.2 percent (Center for Behavioral Health Statistics and Quality, 2015). In 2011 to 2014, 16 percent of U.S. women had used an antidepressant in the prior month (Pratt, 2017). The diagnosis is made by identiying symptoms listed in Table 61-2, but very few patients manifest all of these.

1	Major depression is multifactorial and prompted by genetic and environmental factors. Families of afected individuals often also have members sufering with alcohol abuse and anxiety disorders. Provocative conditions leading to depression include life events that prompt grief reactions, substance abuse, use of certain medications, and other medical disorders. Although life events can trigger depression, genes influence the response to these events and render the distinction between genetic and environmental factors diicult. One genome-wide linkage analysis of more than 1200 mothers suggests that variation in chromosomes 1 and 9 raises susceptibility to postpartum mood symptoms (Mahon, 2009). It is unquestionable that pregnancy is a major life stressor that can precipitate or exacerbate depressive tendencies. In TABLE 61-2. Symptoms of Depressive IIlnessa Persistent sad, anxious, or "empty" feelings worthlessness, and/or helplessness Irritabi Iity, restlessness

1	TABLE 61-2. Symptoms of Depressive IIlnessa Persistent sad, anxious, or "empty" feelings worthlessness, and/or helplessness Irritabi Iity, restlessness Loss of interest in activities once pleasurable, including sex Fatigue and decreased energy Difficulty concentrating, remembering details, and making decisions Insomnia, early-morning wakefulness, or excessive sleeping Overeating or appetite loss Thoughts of suicide, suicide attempts Persistent aches or pains, headaches, cramps or digestive problems that do not ease with treatment aNot all patients experience the same symptoms. Modified with permission from National Institute of Mental Health, 2010.

1	addition, various pregnancy-induced efects are implicated. Hormones certainly afect mood, as evidenced by premenstrual Major or minor depression develops postpartum in 10 to 20 syndrome and menopausal depression. Estrogen has been percent of women (Mental Health America, 2016). Available linked to increased serotonin synthesis, decreased serotonin data indicate that unipolar major depression may be slightly breakdown, and serotonin-receptor modulation (Deecher, more prevalent during the puerperium than among women in 2008). Concordantly, women who experience postpartum the general population (Yonkers, 2011). Postpartum depressive depression often have higher predelivery serum estrogen and symptoms are associated with young maternal age, antenatal progesterone levels and experience a greater decline postpardepression, unmarried status, smoking, newborns requiring tum (Ahokas, 1999). intensive care, and those with a history of stressors during preg

1	Dennis and associates (2007) queried the Cochrane Datanancy (Ko, 2017; Silverman, 2017). Speciically, physical or base and reported that the prevalence of antenatal depression verbal abuse during pregnancy is a potent risk for postpartum averaged 11 percent. Melville and coworkers (2010) found it depression (McFarlane, 2014). Finally, serious adverse obstetriin nearly 10 percent of more than 1800 women enrolled for cal events, especially those involving the neonate, are strongly prenatal care at a single university clinic. Others have reported linked to postpartum depression (Nelson, 2013, 2015). the incidence to be much higher depending on the population Depression is frequently recurrent. Up to 70 percent of studied (Gavin, 2005; Hayes, 2012; Lee, 2007). women with previous postpartum depression have a subsequent aChapter 12 (p. 245). bChapter 60 (p. 11r60). SSRI = Selective serotonin-reuptake inhibitor. Data from Briggs, 2015; Huybrechts, 2015; Koren, 2012.

1	SSRI = Selective serotonin-reuptake inhibitor. Data from Briggs, 2015; Huybrechts, 2015; Koren, 2012. Immediate referral to psychiatrist if no response, consider antidepressant Rx; antidepressant psychotherapy beneficial for pregnant women with mood disorders. episode. Women with both prior puerperal depression and a current episode of "maternity blues" carry an inordinately high risk for major depression. Indeed, 2 to 9 months postpartum, assistance with postpartum depression was the fourth most common challenge identiied in women in the Pregnancy Risk Assessment Monitoring System-PRAMS (Kanotra, 2007).

1	Postpartum depression is generally underrecognized and undertreated. Major depression during pregnancy or after delivery can have devastating consequences for afected women, their children, and families. One of the most significant contributions to the mortality rate among new mothers is suicide, which is most frequent among women with mental illness (Koren, 2012; Palladino, 2011). If left untreated, up to 25 percent of women with postpartum depression will be depressed 1 year later. As the duration of depression increases, so too does the number of sequelae and their severity. Maternal depression during the first weeks and months after delivery can lead to insecure attachment and later behavioral problems in the child.

1	Therapy for mood disorders during pregnancy and postpartum has undergone a significant evolution during the past decade. Babbitt (2014) and Pozzi (2014) and their associates have reviewed principles of antenatal and intrapartum care of women with major mental disorders. In general, for mild and mild-moderate depression, psychological treatment options, such as cognitive behavioral therapy, are considered irst (Yonkers, 2011). Antidepressant medications together with some form of psychotherapy are indicated for moderate to severe depression during pregnancy or the puerperium (American College of Obstetricians and Gynecologists, 2016b).

1	Shown in Figure 61-1 is one algorithm regarding treatment of mood disorders. Some of these medications are listed in 1 able 61-3. For women with severe depression, a selective sero tonin-reuptake inhibitor (SSRI) is selected initially. In contrast, tricyclic antidepressants and monoamine oxidase inhibitors are infrequently selected in contemporary practice. If depressive symptoms improve during a 6-week trial, the medication is continued for a minimum of 6 months to prevent relapse (Wisner, 2002). At least 60 percent of women taking antidepressant medication before pregnancy have symptoms during pregnancy. According to Hayes and colleagues (2012), approximately three fourths of women taking antidepressants before pregnancy stopped taking them before or during early pregnancy. For those who discontinue treatment, almost 70 percent have a relapse compared with approximately 25 percent who continue therapy. If the response is suboptimal or a relapse occurs, another SSRI is substituted,

1	discontinue treatment, almost 70 percent have a relapse compared with approximately 25 percent who continue therapy. If the response is suboptimal or a relapse occurs, another SSRI is substituted, or psychiatric referral is considered.

1	Various dietary deiciencies have suggested links to perinatal depression (Yonkers, 2011). Supplements that include omega-3 fatty acids, iron, folate, ribolavin, vitamin D, calcium, and docosahexaenoic acid (DHA) have been studied (Keenan, 2014; Miller, 2013). However, evidence currently is insuicient to support use of these dietary supplements for this purpose.

1	Importantly, in a metaanalysis by Huang and coworkers (2014), women using antidepressants during pregnancy have higher rates of preterm birth and low-birthweight neonates. Nevertheless, in their review, Ray and Stowe (2014) concluded that the relative reproductive safety data are reassuring and that antidepressants remain a viable treatment option. Furthermore, recurrence sometime after medication is discontinued develops in 50 to 85 percent of women with an initial postpartum depressive episode. Women with a history of more than one depressive episode carry greater risk (American Psychiatric Association, 2000). Surveillance includes monitoring for thoughts of suicide or infanticide, emergence of psychosis, and response to therapy. For some women, the course of illness is severe enough to warrant hospitalization.

1	Fetal and Neonatal Efects of Therapy. Some known and possible fetal and neonatal efects of treatment are listed in Table 61-3. Some studies suggest that SSRIs pose an elevated teratogenic risk for fetal cardiac defects, and these have mainly focused on paroxetine (Paxil). Associations were most consistent for ventricular septal defects. The estimated risk is no greater than 1 in 200 exposed newborns (Koren, 2012). Nevertheless, the American College of Obstetricians and Gynecologists (20 16b) recommends that paroxetine be avoided in women who are either pregnant or planning pregnancy. In women exposed to paroxetine in the irst trimester, fetal echocardiography is considered. Jimenez-Solem and coworkers (2013) in their analysis of SSRIs found no association between exposure to SSRIs during pregnancy and perinatal mortality. Andersen and associates (2014) found that women discontinuing SSRI treatment in early pregnancy had a small increased risk of miscarriage, but that this was similar

1	pregnancy and perinatal mortality. Andersen and associates (2014) found that women discontinuing SSRI treatment in early pregnancy had a small increased risk of miscarriage, but that this was similar to the risk in women discontinuing SSRI treatment months before pregnancy. Taken together, these investigators concluded that treatment with SSRIs during pregnancy should not be discontinued for fear of miscarriage.

1	Of other potential efects, the risk of persistent pulmonary hypertension of the newborn rose sixfold in neonates exposed to SSRIs after 20 weeks' gestation (Chambers, 2006). his translates to an overall risk of pulmonary hypertension that would be less than 1 in 100 exposed newborns (Koren, 2012). In contrast, a population-based cohort study of 1.6 million pregnancies identiied a twofold greater rate in exposed neonates. his yields an estimated attributable risk of 2 cases per 1000 births (Kieler, 2012). In a study of more than 120,000 gravidas prescribed antidepressants, Huybrechts and coworkers (2015) found an attributed risk of 1 case per 1000 births. In sum, the maternal risk associated with discontinuing or tapering SSRI use during pregnancy must be weighed against marginally increased neonatal risks (Ornoy, 2017). Women who abruptly discontinue either serotonin-or norepinephrinereuptake inhibitor therapy typically experience some form of withdrawal.

1	Not surprisingly, up to 30 percent of exposed neonates may also exhibit withdrawal symptoms. Symptoms are similar to opioid withdrawal, but typically are less severe. Neonatal SSRI withdrawal is usually self-limited, and the newborn rarely remains in the nursery more than 5 days (Koren, 2009). Currently, convincing evidence of long-term neurobehavioral efects of fetal exposure to these medications is lacking (Koren, 2012). Grzeskowiak and coworkers (2016) found no increased risk of behavioral problems in 7 -year-old children exposed to antidepressants prior to their birth. Some psychotropic medications pass into breast milk. In most cases, however, levels are very low or undetectable. Efects may be transient irritability, sleep disturbances, and colic.

1	Electroconvulsive Therapy. This form of depression treatment is occasionally necessary during pregnancy for women with major mood disorders unresponsive to pharmacotherapy. Women undergoing electroconvulsive therapy (ECT) should be fasting for at least 6 hours. hey are given a rapid-acting antacid before the procedure, and their airway is protected to decrease the likelihood of aspiration. Ater midpregnancy, a wedge is placed under the right hip to prevent sudden maternal hypotension from aortocaval compression. Other important preparatory steps include assessment of the cervix, discontinuation of nonessential anticholinergic medication, uterine and fetal heart rate monitoring, and intravenous hydration. During the procedure, excessive hyperventilation is avoided. In most cases, maternal and fetal heart rate and maternal blood pressure and oxygen saturation remain normal throughout the procedure.

1	With proper preparation, the risks to both mother and fetus appear to be reasonable (Pinette, 2007). hat said, adverse maternal and perinatal outcomes have followed ECT. Balki and associates (2006) reported a pregnancy in which fetal brain damage likely was caused by sustained maternal hypotension associated with treatment of status epilepticus stimulated by ECT.

1	At least two extensive reviews have evaluated ECT outcomes in pregnancy. In the earlier one, Miller (1994) found 300 cases and reported complications in 10 percent. These included fetal arrhythmias, vaginal bleeding, abdominal pain, and self-limited contractions. Women not adequately prepared had increased risks for aspiration, aortocaval compression, and respiratory alkalosis. In the more recent review, Andersen and Ryan (2009) described 339 cases, undoubtedly with some homology with the earlier study. In most cases, ECT therapy was done to treat depression, and it was 78-percent efective. hey reported a 5-percent maternal ECT -related complication rate. here was a 3-percent associated perinatal complication rate, which included two fetal deaths. For all of these reasons, we agree with Richards (2007) that ECT in pregnancy is not "low risk" and that it should be reserved for women whose severe depression is resistant to intensive pharmacotherapy.

1	According to the National Institute of Mental Health (2010), the lifetime prevalence for manic-depression illness is 3.9 percent. he prevalence of bipolar disorder does not vary between gravidas and nonpregnant reproductive-aged women (Yonkers, 2011). It has a strong genetic component and has been linked to possible mutations on chromosomes 16 and 8 (Jones, 2007). The risk that monozygotic twins are both afected is 40 to 70 percent, and the risk for first-degree relatives is 5 to 10 percent (Muller-Oerlinghausen, 2002). Periods of depression last at least 2 weeks. At other times, patients are manic, in which mood is abnormally raised, expan sive, or irritable. Potential organic causes of mania include substance abuse, hyperthyroidism, and central nervous system (CNS) tumors. hese are all excluded during an acute event.

1	Importantly, pregnancy frequently prompts medication dis continuation, which poses a twofold increased risk for relapse (Viguera, 2007). Afected women are considered high risk, and as many as 20 percent of patients with manic-depression illness commit suicide. his has also been associated with adverse perinatal outcomes, for example, preterm birth (Mei-Dan, 2015). Di Florio and associates (2013) found that those women who experience pregnancy complications are more likely to exhibit periods of mania or depression. Women who tend to be manic present with exacerbations earlier in the postpartum period.

1	Typical therapy for bipolar disorder includes mood stabilizers such as lithium, valproic acid, and carbamazepine, as well as antipsychotic medications (see Table 61-3). Treatment of bipolar disorder in pregnancy is complex and is ideally managed concurrently with a psychiatrist. Decisions include risks versus benefits of using mood stabilizers, some of which are teratogenic. For example, lithium has been linked to Ebstein anomaly in exposed fetuses. More recent data, however, suggest a lower risk of cardiac malformations than previously indicated (Micromedex, 2016; Patomo, 2017) . Nevertheless, many recommend fetal echocardiography for lithium-exposed fetuses. Some limited evidence suggests that lithium in breast milk, when its elimination is impaired as in dehydration or immaturity, can adversely afect the infant (Davanzo, 2011). However, lithium use in mothers with a healthy, term fetus is considered moderately safe. A more detailed discussion of other mood stabilizers and

1	can adversely afect the infant (Davanzo, 2011). However, lithium use in mothers with a healthy, term fetus is considered moderately safe. A more detailed discussion of other mood stabilizers and antipsychotic medications side efects can be found in Chapter 12 (p. 244).

1	This severe mental disorder is usually a bipolar disorder, but it may be due to major depression (American Psychiatric Association, 2013). Its incidence is estimated to be 1 in every 1000 deliveries, and it is more common in nulliparas, especially those with obstetrical complications (Bergink, 2011; Blackmore, 2006). In most cases, illness manifests within 2 weeks of delivery. In one study of postpartum women with their first lifetime episode of psychosis, the median onset of psychiatric symptoms was 8 days after delivery, and the median duration of the episode was 40 days (Bergink, 2011). Because those with underlying psychiatric disease have a 10-to 15-fold risk for recurrence postpartum, close monitoring is imperative.

1	The most important risk for postpartum psychosis is a history of bipolar disease. These women typically exhibit symptoms within 1 to 2 days after delivery (Heron, 2007, 2008). Manic symptoms include feeling excited or elated, being active or energetic, feeling "chatty," and sufering insomnia. Afected women have signs of confusion and disorientation but may also have lucid episodes. Postpartum psychosis has a 50-percent recurrence risk in the next pregnancy. As a result, Bergink and associates (2012) rec in women with a history of postpartum psychosis. The clinical course of bipolar illness with postpartum psycho sis is comparable with that for nonpregnant women. Patients usually require hospitalization, pharmacological treatment, and long-term psychiatric care. Psychotic women may have delu sions leading to thoughts of self-harm or harm to their infants.

1	Unlike women with nonpsychotic depression, these women commit infanticide, albeit uncommonly (Kim, 2008). In most instances, women with postpartum psychosis ultimately develop relapsing, chronic psychotic manic-depression. These relatively common disorders-18 percent prevalence overall-include panic attack, panic disorder, social anxiety disorder, specific phobia, separation anxiety disorder, and generalized anxiety disorder. All are characterized by irrational fear, tension, and worry, which are accompanied by physiological changes such as trembling, nausea, hot or cold lashes, dizziness, dyspnea, insomnia, and frequent urination (Schneier, 2006). They are treated with psychotherapy and medication, including SSRIs, tricyclic antidepressants, monoamine oxide inhibitors, or others.

1	Despite the relative high prevalence in childbearing-aged women, little speciic attention has been directed to anxiety disorders in pregnancy. Most reports conclude that rates between pregnant and nonpregnant women do not difer. One recent analysis of 268 gravidas with generalized anxiety disorder demonstrated that both symptoms and severity of anxiety decline across pregnancy (Buist, 2011).

1	From their review, Ross and McLean (2006) concluded that some of the anxiety disorders may have important matemalfetal implications. Some have been linked to preterm birth, fetal-growth restriction, and poor neurobehavioral development (Van den Bergh, 2005). Children with a history of in utero exposure to maternal anxiety are felt to be at increased risk for various neuropsychiatric conditions such as attention-deicit/ hyperactivity disorder (ADHD). Hunter and coworkers (2012) analyzed infants of 60 mothers with an anxiety disorder and found that auditory sensory gating-a relection of inhibitory neurotransmission-was impaired, particularly in ofspring of untreated women. Conversely, Littleton and associates (2007) found no excessive adverse pregnancy outcomes with "anxiety symptoms." One important exception is their link with postpartum depression (Vythilingum, 2008).

1	Anxiety disorders can be efectively treated during pregnancy with psychotherapy, cognitive-behavioral therapy, or medications. vlood and anxiety disorders coexist in more than half of women identified with either diagnosis (Frieder, 2008). hus, antidepressants listed in Table 61-3 are often the first line of pharmacotherapy. Benzodiazepines are also commonly used to treat anxiety or panic disorders before and during pregnancy. Earlier casecontrol studies linked use of these CNS depressants to a possible increased risk for cleft lip and palate. A metaanalysis of more than 1 million exposed pregnancies, however, did not identiY a teratogenic risk (Enato, 2011). Benzodiazepines, especially when taken during the third trimester, can cause neonatal withdrawal syndrome, which persists for days to weeks after delivery.

1	his major form of mental illness afects 1.1 percent of adults (National Institute of Mental Health, 2016). Schizophrenia spectrum disorders are defined by abnormalities in one or more of the following domains: delusions, hallucinations, disorganized thinking, grossly disorganized or abnormal motor behavior, and negative symptoms. Brain-scanning techniques such as positron emission tomography (PET) and functional magnetic resonance imaging (MRI) show that schizophrenia is a degenerative brain disorder. Subtle anatomical abnormalities are present early in life and worsen with time.

1	Schizophrenia has a major genetic component, and there is a 50-percent concordance in monozygotic twins. If one parent has schizophrenia, the risk to ofspring is 5 to 10 percent. Some data, including a strong association between schizophrenia and the velocardiofacial syndrome, suggest that associated genes are located on chromosome 22q 11 (Murphy, 2002). But sophisticated gene mapping studies clearly show that schizophrenia is not related to a single gene or mutation. Instead, multiple DNA variants likely interact to lead to schizophrenia (Kukshal, 2012). Other putative risks for subsequent schizophrenia in an exposed fetus include maternal iron-deiciency anemia, diabetes, and acute maternal stress (Insel, 2008; Malaspina, 2008; Van Lieshout, 2008). hese remain unproven, as does the association with maternal inluenza A infection.

1	Signs of illness begin approximately at age 20 years, and commonly, work and psychosocial functioning deteriorate over time. Women have a slightly later onset than men and are less susceptible to autism and other neurodevelopmental abnormalities. hus, many investigators theorize that estrogen is protective. Afected women may become pregnant before symptoms manifest. With appropriate treatment, patients can experience a decrease or cessation of symptoms. Within 5 years from the irst signs of illness, 60 percent have social recovery, 50 percent are employed, 30 percent are mentally handicapped, and 10 percent require continued hospitalization (American Psychiatric Association, 2013).

1	vlost studies have not found adverse maternal outcomes, although researchers in a Swedish study noted increased rates of low birthweight, fetal-growth restriction, and preterm delivery (Bennedsen, 1999). In a study of more than 3000 pregnancies in schizophrenic women, Jablensky and coworkers (2005) reported that placental abruption was increased threefold and "fetal distress" -vaguely deined-was increased l.4-fold.

1	Because schizophrenia has a high recurrence if medications are discontinued, continued therapy during pregnancy is advised. Ater 40 years of use, no evidence links the conventional or "typical" antipsychotic drugs listed in Table 61-3 and adverse fetal or maternal sequelae (McKenna, 2005; Robinson, 2012; Yaeger, 2006). Because less is known about "atypical" antipsychotics, the American College of Obstetricians and Gynecologists (2016b) recommends against their routine use in pregnant and breastfeeding women. In response to adverse event reports, the Food and Drug Administration (2011) issued a safety communication alerting health-care providers concerning some antipsychotic medications. hese have been associated with neonatal extrapyramidal and withdrawal symptoms similar to the neonatal behavioral syndrome seen in those exposed to SSRIs.

1	hese include anorexia nervosa, in which the patient refuses to maintain minimally normal body weight, and bulimia nervosa, in which binge eating is usually followed by purging or excessive fasting to maintain normal body weight (Zerbe, 2008). Eating behavior disturbances largely afect adolescent females and young adults. With anorexia and bulimia, the lifetime prevalence for each is 2 to 3 percent (National Institute of Mental Health, 2016).

1	Bulik and coworkers (2009) studied pregnancy outcomes in almost 36,000 Norwegian women screened for eating disorders. Approximately 0.1 percent had anorexia nervosa, 0.85 percent had bulimia nervosa, and 5.1 percent reported a binge-eating disorder. This 6-percent pregnancy prevalence is similar to the 6-month prevalence for nonpregnant individuals (National Institute of Mental Health, 2016). he last subtype had a higher risk for large-for-gestational age neonates with a concomitantly increased cesarean delivery rate. All eating disorders begin with the desire to be slim, and women with chronic eating disorders may migrate between subtypes (Andersen, 2009).

1	Early pregnancy complication rates are increased with both eating disorders, but especially in women with bulimia nervosa (Andersen, 2009; Hofman, 2011). Generally, eating disorder symptoms improve during pregnancy, and remission rates may reach 75 percent. In contrast, typical cases of hyperemesis gravidarum may actually be a new or relapsing case of bulimia nervosa or of binge-purge type anorexia nervosa (Torgerson, 2008). As perhaps expected, anorexia is associated with low-birthweight neonates (Micali, 2007). Additional risks associated with eating disorders include poor wound healing and diiculties with breastfeeding (Andersen, 2009). At a minimum, closely monitoring gestational weight gain in women with a suspected history of an eating disorder seems prudent. Care for these women involves a multidisciplinary team that includes an obstetrician, mental health provider, and either dietician or nutritionist (American Dietetic Association, 2006).

1	Care for these women involves a multidisciplinary team that includes an obstetrician, mental health provider, and either dietician or nutritionist (American Dietetic Association, 2006). Psychological treatment is the cornerstone for treatment in women with eating disorders and frequently includes cognitivebehavioral therapy. Anorexia nervosa oten responds to motivational interactions with meal planning (Cardwell, 2013). After delivery, women with eating disorders are more prone to postpartum depression. Women with bulimia are at particular risk for disease rebound ater delivery because of body image concerns. hese disorders are characterized by the chronic use of certain coping mechanisms in an inappropriate, stereotyped, and maladaptive manner. hey are rigid and unyielding personality traits. The American Psychiatric Association (2013) recognizes three clusters of personality disorders: 1.

1	Paranoid, schizoid, and schizotypal personality disorders, which are characterized by oddness or eccentricity. 2. Histrionic, narcissistic, antisocial, and borderline disorders, which are all characterized by dramatic presentations along with self-centeredness and erratic behavior. 3. Avoidant, dependent, compulsive, and passive-aggressive personalities, which are characterized by underlying fear and anxiety. Genetic and environmental factors are important in the genesis of these disorders, whose prevalence may be as high as 20 percent. lthough management is through psychotherapy, most afected individuals do not recognize their problem, and thus only 20 percent seek help. In an observational study of 202 women with borderline personality disorder, De Genna during the most severe trajectory of their illness. hey are at increased risk for teen and unintended pregnancies.

1	Personality disorders during pregnancy are probably no diferent than in nonpregnant women. Akman and colleagues (2007) reported that avoidant, dependent, and obsessive-compulsive disorders are associated with an excessive prevalence of postpartum major depression. Magnusson and associates (2007) found a link between some personaliy traits-not disordersand excessive alcohol consumption, but not necessarily addiction or dependence. Conroy and coworkers (2010) found that a mother's ability to care for her newborn was impaired only when a personality disorder was coupled with depression. Ahokas A, Kaukorantat], Aito M: Efect of oestradiol on postpartum depres sion. Psychopharmacology 146: 108, 1999 Akman C, Uguz F, Kaya N: Postpartum-onset major depression is associated with personality disorders. Compr Psychiatry 48:343, 2007

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1	Malaspina 0, Corcoran C, Kleinhaus KR, et al: Acute maternal stress in pregnancy and schizophrenia in ofspring: a cohort prospective. BMC Psychiatry 8:71,t2008 McFarlane ], Maddoux ], Cesario S, et al: Efect of abuse during pregnancy on maternal and child safety and functioning for 24 months after delivery. Obstet GynecoIt123(4):839, 2014 McKenna K, Koren G, Tetelbaum M, et al: Pregnancy outcome of women using atypical antipsychotic drugs: a prospective comparative study. ] Clin Psychiatry 66:444, 2005 Mei-Dan E, Ray ]G, Vigod SN, et al: Perinatal outcomes among women with bipolar disorder: a population-based cohort study. Am ] Obstet Gynecol 212(3):367.el,t2015 Meltzer-Brody S: Treating perinatal depression: risks and stigma. Obstet Gynecol 124(4):653,t2014 Melville ]L, Gavin A, Guo Y, et al: Depressive disorders during pregnancy. Obstet GynecoIt116:1064, 2010

1	Melville ]L, Gavin A, Guo Y, et al: Depressive disorders during pregnancy. Obstet GynecoIt116:1064, 2010 Mental Health America: Postpartum disorders. 2016. Available at: http:// ww.mentalhealthamerica.net/conditions/postpartum-disorders. Accessed July 12, 2016 Metz TO, Rovner P, Allshouse A, et al: Maternal deaths from suicide and drug overdose in Colorado. Am] Obstet Gynecol 214(1):S126, 2016 Micali N, Simonof E, Treasure]: Risk of major adverse perinatal outcomes in women with eating disorders. Brt] Psychiatry 190:255, 2007 Micromedex: Lithium. Available at: http://ww.micromedexsolutions.com/ micromedex2/li brarian/PF Defaul tActionld/ evidencexpert. Doln tegratedSearch. Accessed June 29, 2016 Miller B], Murray L, Beckman MM, et al: Dietary supplements for preventing postnatal depression. Cochrane Database Syst Rev 10:CD009104, 2013 Miller L]: Use of electroconvulsive therapy during pregnancy. Hosp Community Psychiatry 45:444, 1994

1	Molyneaux E, Poston L, Ashurst-Williams S, et al: Obesity and mental disorders during pregnancy and postpartum: a systematic review and metaanalysis. Obstet GynecoIt123(4):857, 2014 Muller-Oerlinghausen B, Berghofer A, Bauer JvI: Bipolar disorder. Lancet 359:241, 2002 Murphy KC: Schizophrenia and velocardiofacial syndrome. Lancet 359(9304): 426, 2002 National Institute of Mental Health: Spotlight on postpartum depression. 2010. Available at: http://ww.nimh.nih.gov/about/directorl20 1 0/ spotlight-on-postpartum-depression.shtml. Accessed] uly 12, 2016 National Institute of Mental Health: he numbers count: mental disorders in America. NIH Publication No. 06-4584, 2016 Nelson DB, Doty M, McIntire DO, et al: Rates and precipitating factors for postpartum depression following screening in consecutive births. ] Matern Fetal Neonatal Med 11:1,t2015

1	Nelson DB, Doty M, McIntire DO, et al: Rates and precipitating factors for postpartum depression following screening in consecutive births. ] Matern Fetal Neonatal Med 11:1,t2015 Nelson DB, Freeman MP, Johnson NL, et al: A prospective study of postpartum depression in 17648 parturients. ] Matern Fetal Neonatal Med 26(12):1156,t2013 O'Hara MW, Wisner L: Perinatal mental illness: definition, description and aetiology. Best Pract Res Clin Obstet Gynaecol 28(1):3, 2014 O'Keane V, Lightman S, Patrick K, et al: Changes in the maternal hypothalamicpituitary-adrenal axis during the early puerperium may be related to the postpartum blues. ] Neuroendocrinol 23(11): 1149, 2011 Ornoy A, Koren G: Selective serotonin reuptake inhibitors during pregnancy: do we have now more definite answers related to prenatal exposure. Birth Defects Res 109(12):898, 2017

1	Ornoy A, Koren G: Selective serotonin reuptake inhibitors during pregnancy: do we have now more definite answers related to prenatal exposure. Birth Defects Res 109(12):898, 2017 Palladino CL, Singh V, Campbell H, et al: Homicide and suicide during the perinatal period: indings from the National Violent Death Reporting System. Obstet Gynecol 118(5):1056,t2011 Patorno E, Huybrechts KF, Bateman BT, et al: Lithium use in pregnancy and the risk of cardiac malformations. N Engl J Med 376(23):2245, 2017 Pinette MG, Santarpio C, Wax JR, et al: Electroconvulsive therapy in pregnancy. Obstet Gynecol 110:465, 2007 Pozzi A, Yee LM, Brown K, et al: Pregnancy in the severely mentally ill patient as an opportuniry for global coordination of care. Am J Obstet Gynecol 210:32, 2014 Pratt LA, Brody OJ, Gu Q: antidepressant use among persons aged 12 and over: United States, 2011-2014. NCHS Data Brief 283: 1,2017

1	Pratt LA, Brody OJ, Gu Q: antidepressant use among persons aged 12 and over: United States, 2011-2014. NCHS Data Brief 283: 1,2017 Ray 5, Stowe ZN: he use of antidepressant medication in pregnancy. Best Pract Res Clin Obstet GynaecoIt28(l):71, 2014 Richards OS: Is electroconvulsive therapy in pregnancy safe? Obstet Gynecol 11t0:45t1,t2007 Robinson GE: Treatment of schizophrenia in pregnancy and postpartum. J Popul her Clin PharmacoIt19(3):e380, 2012 Romero R, Badr MS: A role for sleep disorders in pregnancy complications: challenges and opportunities. Am J Obstet Gynecol 210:3, 2014 Ross LE, McLean LM: Anxiery disorders during pregnancy and the postpartum period: a systematic review. J Clin Psychiatry 67:1285,t2006 Schneier FR: Clinical practice. Social anxiery disorder. N Engl J Med 355(10): 1029,t2006 Shaw JG, Asch SM, Kimerling R, et al: Posttraumatic stress disorder and risk of spontaneous preterm birth. Obstet Gynecol 124(6): 1111, 2014

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1	Steinberg JR, McCulloch CE, Adler NE: Abortion and mental health: findings from the National Comorbidiry Survey Replication. Obstet Gynecol 123:263,t2014 Straub H, Adams M, Kim JJ, et al: Antenatal depressive symptoms increase the likelihood of pre term birth. Am J Obstet GynecoIt207:329.el, 2012 Tam H, Chung T: Psychosomatic disorders in pregnancy. CUlT Opin Obstet Gynecol 19: 126, 2007 Tarney CM, Berry-Caban C, Jain RB, et al: Association of spouse deployment on pregnancy outcomes in a U.S. military population. Obstet Gynecol 126(3):569,t2015 Torgerson L, Von Holle A, Reichborn-Kjennerud T, et al: Nausea and vomiting of pregnancy in women with bulimia nervosa and eating disorders not otherwise specified. Int J Eat Disord 41t:722, 2008 Van den Bergh BR, Mulder EJ, Mennes M, et al: Antenatal maternal anxiery and stress and the neurobehavioural development of the fetus and child: links and possible mechanisms. A review. Neurosci Biobehav Rev 29(2):237, 2005

1	Van Lieshout RJ, Voruganti LP: Diabetes mellitus during pregnancy and increased risk of schizophrenia in ofspring: a review of the evidence and putative mechanisms. J Psychiatry Neurosci 33(5):395, 2008 Venkatesh KK, Kaimal A, Nadel H, et al: Implementation of universal screening for depression during pregnancy: feasibiliry and impact on obstetric care. 215(4):517.e1,t2016 Viguera AC, Whitfield T, Baldessarini RJ, et al: Risk of recurrence in women with bipolar disorder during pregnancy: prospective study of mood stabilizer discontinuation. Am J Psychiatry 164(12): 1817,t2007 Vythilingum B: Anxiery disorders in pregnancy. Curr Psychiatry Rep 10(4): 331,t2008 Weinberg MK, Tronick EZ: The impact of maternal illness on infant development. J Clin Psychiatry 59:53, 1998 Wisner KL, Parry BL, Piontek CM: Postpartum depression. N Engl J Med 347:t194,t2002

1	Weinberg MK, Tronick EZ: The impact of maternal illness on infant development. J Clin Psychiatry 59:53, 1998 Wisner KL, Parry BL, Piontek CM: Postpartum depression. N Engl J Med 347:t194,t2002 Yaeger 0, Smith HG, Altshuler LL: Arypical antipsychotics in the treatment of schizophrenia during pregnancy and the postpartum. Am J Psychiatry 163:2064,t2006 Yonemoto N, Dowswell T, Nagai 5, et al: Schedules for home visits in early postpartum period. Cochrane Database Syst Rev 8:CD009326, 2017 Yonkers A, Vigod 5, Ross LE: Diagnosis, pathophysiology, and management of mood disorders in pregnant and postpartum women. Obstet Gynecol 117:961,201t1 Yonkers A, Wisner KL, Stewart DE, et al: he management of depression during pregnancy: a report from the American Psychiatric Association and the American College of Obstetricians and Gynecologists. Obstet Gynecol 114:703,t2009

1	Yost NP, Bloom SL, McIntire DD, et al: A prospective observational study of domestic violence during pregnancy. Obstet Gynecol 106(1):61, 2005 Zerbe KJ, Rosenberg J: Eating disorders. Clinical Updates in Women's Health Care. American College of Obstetricians and Gynecologists, Vol VU, No.t1, January 2008 PREGNANCY-SPECIFIC DERMATOSES .i...i.....i....i... 1184 SPECIFIC TO PREGNANCY.. . . . . . . . . . . . . . . . . . . . . .. 1186 DERMATOLOGICAL TREATMENT.. . . . . . . . . . . . . . . .. 1188 Herpes Gestationis-This disease, more requenty known as dermatitis herpetormis is an inlammatory supeicialy seated multorm herpetiorm eruption, which is characterized by erythematous, vesicular, pustular, and bulous lesions. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) Four dermatoses considered unique to pregnancy include intrahepatic cholestasis of pregnancy, pruritic urticarial papules and plaques of pregnancy (PUPPP), atopic eruption of pregnancy (AEP), and pemphigoid gestationis (PG). Descriptions of these are given in Table 62-1. As a group, these are diagnosed in up to 5 percent of pregnancies (Chander, 2011). Their gross appearance may be similar to each other or to other skin disorders, and pruritus is a common feature of all four. Only intrahepatic cholestasis and pemphigoid gestationis have been linked with adverse fetal outcomes. • Intrahepatic Cholestasis of Pregnancy

1	• Intrahepatic Cholestasis of Pregnancy Previously termed pruritus gravidarum, this condition is found in 0.5 percent of pregnancies (Wikstrom Shemer, 2013). In contrast to the other pregnancy-speciic dermatoses, intrahepatic cholestasis of pregnancy generally has no primary skin lesions. Rarely, a rash precedes pruritus, which is usually associated with abnormally elevated serum bile acid levels and mildly increased hepatic aminotransferase levels (Chao, 2011). Adverse fetal efects have been linked to this condition, and it is discussed in detail in Chapter 55 (p. 1059). his rare autoimmune bullous disease is notable for its maternal and fetal efects. Initially, pruritic papules and urticarial plaques form and are then followed in most cases after 1 to 2 weeks by vesicles or bullae. Lesions are frequently distributed periumbilically, often develop on other skin surfaces, but spare mucous membranes, scalp, and face (Fig. 62-1).

1	Previously termed hepes gestationis, pemphigoid gestationis is not related to the herpesvirus. Instead, maternal immunoglobulin G (IgG) antibodies target collagen VII found in the basement membrane of skin and amnionic epithelium (Kelly, 1988; Shimanovich, 2002). Collagen VII is also termed bullous pemphigoid 180 (BP 180). Autoantibody binding to collagen VII activates complement to promote eosinophil chemotaxis to the antigenantibody complexes. Eosinophilic degranulation damages the dermal-epidermal junction and leads to blistering (Engineer, 2000). In most cases, PG develops during a irst pregnancy. It may rarely be associated with gestational trophoblastic disease (Matsumoto, 2013; Takatsuka, 2012). Most subsequent pregnancies are also afected, usually earlier and more severely (Tani, 2015). Whites have a higher incidence, and other autoimmune diseases are frequent in afected women (Shornick, 1984, 1992).

1	PG usually begins during the second or third trimester, but postpartum onset or exacerbation is common (Lawley, 1978). The disease course is often marked by antepartum flares and remissions. And, especially in cases with early-onset and blistering, PG has an association with preterm birth and fetalgrowth restriction (Al-Saif, 2016; Chi, 2009). One theory for TABLE 62-1 . Pregnancy-specific Dermatoses Cholestasis of Common No primary lesions, secondary Increased perinatal Anti pru ritics, pregnancy excoriations from scratching morbidity cholestyramine, plaques of on abdomen, thighs, buttocks, pregnancy (PUPPP) especially within striae, but Atopic eruptions of None Anti pru ritics, Eczema of pregnancy Common Dry, red scaly patches on emollients, topicalextremity flexures, neck, face corticosteroids, oral Prurigo of pregnancy Common 1-5 mm pruritic red papules on steroids if severe extensor surfaces, trunk Pruritic folliculitis of Rare Small red papules, sterile

1	Pruritic folliculitis of Rare Small red papules, sterile Pemphigoid Rare Erythematous pruritic papules, Preterm birth, fetal gestationis plaques, vesicles, and bullae; growth restriction, abdomen often with umbilical transient neonatal involvement, extremities lesions this is mild placental insuiciency that stems from IgG and resolution is protracted, and disease may be exacerbated during complement deposition along the amnionic basement memmenses or by oral contraceptives (Semkova, 2009). brane (Huilaja, 2013). Accordingly, antepartum surveillance of afected pregnancies is reasonable.

1	In 5 to 10 percent of cases, IgG antibodies passively transfer Before bullae form, these lesions may resemble pruritic urtifrom the mother to cause similar skin lesions in the newborn carial papules and plaques of pregnancy. Other diagnoses (Erickson, 2002). hese eruptions in the neonate require only include pustular psoriasis, dermatitis herpetiformis, erythema wound care and clear spontaneously within a few weeks as the multiforme, linear 19A bullous dermatosis, urticaria, allergic passively acquired IgG levels decline. Slowly following contact dermatitis, bullous pemphigoid, and atopic eruption maternal lesions resolve without scarring, and most women are of pregnancy (LipozenCic, 2012). Drug-induced blistering disease-free after 6 months (Jenkins, 1999). In some, however, syndromes must also be excluded as some are life-threatening,

1	FIGURE 62-1 Pemphigoid gestation is. A. Abdominal plaques classically involve the umbilicus. B. Blistered lesions on the wrist and forearm. (Used with permission from Dr. Kara Ehlers.) for example, Stevens-Johnson syndrome and toxic epidermal necrolysis (Stern, 2012). Skin biopsy and serum antibody assays are informative. Immunofluorescent staining of a skin punch biopsy sample is the gold standard, and C3 complement and sometimes IgC are seen deposited along the basement membrane between the epidermis and the dermis (Katz, 1976). Also, in many cases, circulating IgC antibodies against collagen V1I can be detected in maternal serum (Powell, 2005; Sitaru, 2004).

1	Pruritus can be severe. Early in its course, topical highpotency corticosteroids and oral antihistamines may be efective. Oral prednisone, 0.5 to 1 mg/kg daily gradually tapered to a maintenance dose, may be needed for relief and also for inhibition of new lesions. Plasmapheresis, high-dose intravenous immunoglobulin (IVIC) therapy, or cyclosporine has been used in intractable cases (Huilaja, 2015; Ko, 2014; Van de Wiel, 1980).

1	Plaques of Pregnancy his relatively common pregnancy-speciic dermatosis is characterized by its benign efects on pregnancy and by intensely pruritic 1-to 2-mm erythematous papules that coalesce to form urticarial plaques. Also known as poymorphic eruption of pregnancy, PUPPP usually appears late in pregnancy (Rudolph, 2005). Rarely, postpartum onset has been described (Park, 2013). he rash afects the abdomen and proximal thighs in 97 percent of women (Fig. 62-2). Lesions often initially form within striae but show periumbilical sparing. The face, palms, and soles also are rarely involved (High, 2005). It is more frequent in white and nulliparous women, those with multifetal gestation, and those carrying a male fetus (Regnier, 2008). PUPPP seldom recurs in subsequent pregnancies (Ahmadi, 2005). Its cause is unknown, but an autoimmune basis is not implicated (Lawley, 1979).

1	PUPPP may be compared with several skin eruptions. Some include contact dermatitis, drug eruption, viral exanthem, insect bites, scabies infestation, pityriasis rosea, and the other pregnancy-speciic dermatoses. It also may appear similar to early PC that has not yet blistered. In unclear cases, skin biopsy and negative serum collagen V1I antibody levels help to differentiate the two. Pruritus will usually respond to treatment with oral antihistamines, skin emollients, and topical corticosteroids. A few women will need systemic corticosteroids to relieve severe itching (Scheinfeld, 2008). FIGURE 62-2 Pruritic urticarial papules and plaques of pregnancy (PUPPP) shows sma" papules on the buttock and proximal thigh and within abdominal striae. PUPPP usually resolves within several days following delivery and leaves no scarring. In 15 to 20 percent of women, however, symptoms persist for 2 to 4 weeks postpartum (Vaughan Jones, 1999).

1	PUPPP usually resolves within several days following delivery and leaves no scarring. In 15 to 20 percent of women, however, symptoms persist for 2 to 4 weeks postpartum (Vaughan Jones, 1999). • Atopic Eruption of Pregnancy his umbrella term encompasses three conditions previously considered separate: eczema in pregnancy, prurigo of pregnancy, and pruritic folliculitis of pregnancy (Ambros-Rudolph, 2006). Two thirds of women with atopic eruption have widespread eczematous changes, whereas the other third have papular lesions (American Academy of Dermatology, 201r1). As a group, these pose no risk to the fetus. Diagnosis is greatly aided by a history of atopy and by rash characteristics.

1	Eczema in pregnany has the appearance of traditional eczema but with a pregnancy onset. It is the most common pregnancy-specific dermatosis, and afected skin shows dry, thickened, scaly, red patches involving extremity lexures, nipples, neck, and face. In contrast, prurigo of pregnancy, also known as prurigo gestationis) is characterized by 5-to 10-mm, itchy, erythematous papules or nodules commonly found on the extensor surfaces and trunk. Last, pruritic olliculitis of pregnancy is rare and notable for small, erythematous follicular papules and sterile pustules predominantly on the trunk. Onset for all is during the second or third trimester, although eczema in pregnancy may develop earlier than the other two. All lesions commonly resolve with delivery, but may persist for up to 3 months postpartum. Recurrence with subsequent pregnancies is variable but common.

1	Diagnosis is one of exclusion. Serum bile acid levels are elevated but not greater than concentrations expected for normal pregnancy, and aminotransferase levels are normal. Serology specific for PC is negative. Many women with eczema of pregnancy have elevated serum IgE levels, which are not seen with the two other AEP dermatoses (Ambros-Rudolph, 201r1). For all three manifestations, skin lesions and pruritus are usually controlled with low-or moderate-potency topical corticosteroids and oral antihistamines. For severe eczema, second-line agents include short-course ultrapotent topical corticosteroids. In some cases, however, an oral corticosteroid, narrow-band ultraviolet B, or cyclosporine is required (Lehrhof, 2013). pregnancy. Some of those chronic conditions are considered here.

1	his common chronic dermatosis is unpredictably afected by pregnancy and, if necessary, is treated with benzoyl peroxide alone or coupled with either topical erythromycin or topical clindamycin (Zaenglein, 2016). In these combinations, benzoyl peroxide minimizes Propionibacterium acnes drug resistance. Azelaic acid is another comedolytic agent, which is category B. Topical salicylic acid is category C, but amounts in over-the-counter products are considered safe (Murase, 2014). Topical retinoids, which include tretinoin and adapalene, also appear safe, are category C drugs, but are probably best avoided during pregnancy, especially during the irst trimester (Kaplan, 2015; Panchaud, 2012). Topical tazarotene is contraindicated. For more severe cases, oral antibiotics that include erythromycin, azithromycin, cephalexin, or amoxicillin may be coupled with benzoyl peroxide. Systemic antibiotics are ideally delayed until the second trimester, and therapy duration limited to 4 to 6 weeks

1	azithromycin, cephalexin, or amoxicillin may be coupled with benzoyl peroxide. Systemic antibiotics are ideally delayed until the second trimester, and therapy duration limited to 4 to 6 weeks (Chien, 2016).

1	his chronic dermatosis also has a variable course during pregnancy, however, postpartum lares are common (Oumeish, 2006). Emollients alone are given initially, and low-or moderate-potency topical corticosteroids can be added. In resistant cases, restrained use of high-potency or ultrapotent corticosteroids appears safe in the second and third trimesters. Ultraviolet B phototherapy can be used as a second-line option. Last, cyclosporine, systemic corticosteroids, or tumor necrosis factor (TNF)-a antagonists that include adalimumab, etanercept, and inliximab are third-tier agents for pregnancy (Bae, 2012). Overall, data do not support an increased risk of adverse pregnancy outcomes with psoriasis (Bobotsis, 2016). With severe disease, a small increased risk for low-birthweight neonates was found by some (Lima, 2012; Yang, 2011). Also, in general, psoriatic patients have higher associated rates of depression (Bandoli, 2017; Cohen, 2016).

1	Psoriasis is most commonly of the chronic plaque variety. In contrast, with generalized pustuar psoriasis of pregnancy, severe systemic symptoms may develop. Formerly called impetigo herpetiformis, this rare pustular form has erythematous, sometimes pruritic plaques ringed by sterile pustules that enlarge and then crust (Fig. 62-3). Lesions initially involve intertriginous areas but may spread to the torso, extremities, and oral mucosa. Comorbid constitutional symptoms are common. Laboratory testing may reveal hypocalcemia, elevated erythrocte sedimentation rate, leukocytosis, and hypoalbuminemia (Lehrhof, 2013). Extensive lesions can lead to sepsis from secondary infection and to massive fluid loss with hypovolemia and placental insuiciency. First-line treatment is with oral prednisone, cyclosporine, infliximab, topical corticosteroids, or topical calcipotriene (Robinson, 2012). Phototherapy is a second-line option. For secondary infections, intravenous antibiotics are added (Huang,

1	cyclosporine, infliximab, topical corticosteroids, or topical calcipotriene (Robinson, 2012). Phototherapy is a second-line option. For secondary infections, intravenous antibiotics are added (Huang, 2011). Pustular psoriasis typically resolves quickly in the puerperium, but recurrences have been reported in subsequent pregnancies and with menstruation or oral contraceptive use (Roth, 2011).

1	his skin condition represents inflammation of subcutaneous fat associated with numerous disorders, including pregnancy. Other triggers are infections, sarcoidosis, drugs, Beh:et syndrome, inlammatory bowel disease, or a malignancy (Mert, 2007; Papagrigoraki, 2010). Characteristically, 1-to 6-cm tender, red, warm nodules and plaques develop rapidly on the extensor surface of the legs and arms. Within a few days, lesions flatten and undergo the color evolution of a bruise-from dark red and purple to yellow green. Constitutional symptoms may also be present. Initial evaluation and treatment focuses on the underlying etiology. Symptoms spontaneously resolve in 1 to 6 weeks without scarring but may leave residual hyperpigmentation (Acosta, 2013). his lesion is frequently seen in pregnancy (Fig. 62-4). Poorly named, pyogenic granuloma is actually a lobular capillary hemangioma commonly forming on the mouth or hand in response to low-grade local irritation or traumatic injury. hey grow

1	FIGURE 62-3 Generalized pustular psoriasis of pregnancy displays FIGURE 62-4 Pyogenic granuloma is characterized grossly by a erythematous, sometimes pruritic plaques ringed by sterile pustules lobulated red growth on a pedunculated or sessile base. With minithat enlarge and then form a scaling crust. (Used with permission mal trauma, these vascular lesions bleed easily. (Used with permisfrom Dr. Paul Slocum.) sion from Dr. Abel Moron.) quickly and bleed with minimal provocation. Active bleeding can be controlled with pressure and application of a silver nitrate stick or Monsel paste (ferric subsulfate). These growths often resolve within months postpartum. But with a symptomatic antepartum growth, a persistent postpartum lesion, or with an unclear diagnosis, excision can be done using suture and scalpel, electrosurgical curettage, laser photocoagulation, or cryotherapy. Oral lesions are best referred to oral health-care specialists.

1	These lesions are typiied by benign cutaneous neuroibromas, cafe-au-lait spots, axillary and inguinal freckling, benign nodules of the iris (Lisch nodules), and optic nerve gliomas. Neuroibromas may increase in size and number during pregnancy (Cesaretti, 2013; Dugof, 1996). With the more common neuroibromatosis type 1, higher rates of preclampsia and preterm birth complicate pregnancy (Leppavirta, 2017; Terry, 2013). With neurofibromatosis type 2, some evidence suggests a risk for preeclampsia (Terry, 2015). Prenatal genetic diagnosis is available for both types (Merker, 2015; Spits, 2007). Rosacea ulminans is also known as pyoderma aciale. It is rare and characterized by facial pustules and coalescing draining sinuses. Topical or oral antimicrobials are primary treatment, although surgical drainage and corticosteroids have also been used (F uentelsaz, 2011; Jarrett, 2010).

1	Hidradenitis suppurativa is said to improve with pregnancy, but in our experiences, it is not appreciably changed. Twice daily topically applied I-percent clindamycin gel for 12 weeks aims to prevent new lesions. This can be supplemented by 7-to 10-day courses of oral amoxicillin plus clavulanic acid or oral clindamycin to reduce lesion progression (Margesson, 2014). Other options are reviewed by Perng and coworkers (2017). Other skin conditions that are discussed elsewhere in this book include hirsutism and melanoma (Chap. 63, p. 1203), cutaneous lupus (Chap. 59, p. 1140), hyperpigmentation (Chap. 4, p. 53), and skin lesions seen with infections (Chaps. 64 and 65).

1	Local skin care, oral antihistamines, and topical corticosteroids are commonly used for many dermatoses. Oral antihistamines are given for pruritus. Suitable options include irst-generation agents such s diphenhydramine (Benadryl), 25 to 50 mg every 6 hours, or chlorpheniramine (Chlor-Trimeton), 4 mg every 6 hours. Second-generation agents-Ioratadine (Claritin) 10 mg daily or cetirizine (Zyrtec) 5 or 10 mg daily-may produce less sedation and are also pregnancy category B.

1	Hundreds of topical corticosteroid preparations are available, and in the United States, these are categorized by potency into seven groups. For initial treatment of dermatological disorders, low-or moderate-potency agents are preferred. Low-potency agents include those in groups 6 and 7, such as I-percent hydrocortisone or 0.05-percent desonide (DesOwen). Moderate-potency drugs are in groups 5, 4, and 3-such as O.I-percent triamcinolone acetonide (Aristocort) or O.I-percent mometasone furoate (Elocon). High-potency medications are in group 2, such as 0.05-percent betamethasone dipropionate (Diprolene). Ultrapotent agents in group 1, such as 0.05-percent clobetasol propionate (Temovate), are best reserved for refractory disorders and used for only 2 to 4 weeks on small surface areas.

1	Mild and moderate strengths are not associated with adverse pregnancy outcomes, whereas high-and ultrapotent agents pose a small risk for fetal-growth restriction with large cumulative doses (Chi, 2013, 2015). Even then, this risk is less than that with systemic corticosteroids. Importantly, with any topical agent, factors that increase systemic absorption include a large surface area treated, compromised epidermal barrier, occlusive dressings, prolonged treatment duration, and coadministration of topical agents that increase absorption.

1	The list of other agents used for dermatological conditions is extensive. For use in pregnancy and lactation, Murase (2014) and Butler (2014) have compiled tables and evidencebased descriptions of most. Notable therapeutic agents to avoid during pregnancy include methotrexate, psoralen plus ultraviolet A, mycophenolate mofetil, podophyllin, and systemic retinoids. hese are discussed in more detail in Chapter 12. Bacterial infections are a potential secondary complication of skin disorders and are treated promptly with oral antimicrobial agents with gram-positive coverage. Acosta A, Haver MC, Kelly B: Etiology and therapeutic management of erythema nodosum during pregnancy: an update. Am J Clin Dermatol 14(3):215,t2013 Ahmadi S, Powell FC: Pruritic urticarial papules and plaques of pregnancy: current status. Australas J Dermatol 46(2):53, 2005

1	Ahmadi S, Powell FC: Pruritic urticarial papules and plaques of pregnancy: current status. Australas J Dermatol 46(2):53, 2005 Al-Saif F, Elisa A, Al-Homidy A, et al: Retrospective analysis of pemphigoid gestationis in 32 Saudi patients-clinicopathological features and a literature review. J Reprod Immunol 11t6:42,t20t16 Ambros-Rudolph CM: Dermatoses of pregnancy-clues to diagnosis, fetal risk and therapy. Ann Dermatol23(3):265, 2011 Ambros-Rudolph CM, Mullegger R, Vaughan-Jones SA, et al: The specific dermatoses of pregnancy revisited and reclassified: results of a retrospective two-center study on 505 pregnant patients. J Am Acad Dermatol 54:395, 2006 American Academy of Dermatology: Learning module: dermatoses in pregnancy. 2011t. Available at: https://www.aad.org/education/basic-dermcurricul um/ suggested -order-of-modules/ dermatoses-in -p regnancy. Accessed May 17, 2016

1	Bae YS, Van Voorhees AS, Hsu S, et al: Review of treatment options for psoriasis in pregnant or lactating women: from the Medical Board of the National Psoriasis Foundation. J Am Acad Dermatol67(3):459, 2012 Bandoli G, Chambers CD: Autoimmune conditions and comorbid depression in pregnancy: examining the risk of preterm birth and preeclampsia. J Perinatolt37(lO):1082,t2017 Bobotsis R, Gulliver P, Monaghan K, et al: Psoriasis and adverse pregnancy outcomes: a systematic review of observational studies. Br J Dermatol 175(3):464,t2016 Butler DC, Heller MM, Murase JE: Safety of dermatologic medications in pregnancy and lactation: Part II. Lactation. J Am Acad Dermatol 70(3):417.e1, 2014 Cesaretti C, Melloni G, Quagliarini D: Neuroibromatosis type 1 and pregnancy: maternal complications and attitudes about prenatal diagnosis. m J Med Genet A 161A (2):386, 2013

1	Cesaretti C, Melloni G, Quagliarini D: Neuroibromatosis type 1 and pregnancy: maternal complications and attitudes about prenatal diagnosis. m J Med Genet A 161A (2):386, 2013 Chander R, Garg T, Kakkar S, et al: Speciic pregnancy dermatoses in 1430 females from Northern India. J Dermatol Case Rep 5(4):69, 201t1 Chao TT, Sheield JS: Primary dermatologic indings with early-onset intrahepatic cholestasis of pregnancy. Obstet Gynecol 117:456, 2011 Chi CC, Wang SH, Charles-Holmes R, et al: Pemphigoid gestationis: early onset and blister formations are associated with adverse pregnancy outcomes. Br] DermatoIt160(6):1222, 2009 Chi CC, Wang SH, Mayon-White R: Pregnancy outcomes after maternal exposure to topical corticosteroids: a UK population-based cohort study. ]AMA DermatoIt149(11):1274, 2013 Chi CC, Wang SH, Wojnarowska F, et al: Safety of topical corticosteroids in pregnancy. Cochrane Database Syst Rev 10:CD007346, 2015

1	Chi CC, Wang SH, Wojnarowska F, et al: Safety of topical corticosteroids in pregnancy. Cochrane Database Syst Rev 10:CD007346, 2015 Chien AL, Qi], Rainer B, et al: Treatment of acne in pregnancy.] Am Board Fam Med 29(2):254,t2016 Cohen BE, Martires K], Ho RS: Psoriasis and the risk of depression in the US population: national health and nutrition examination survey 2009-2012. ]AMA Dermatol 3,t2016 Dugof L, Sujansky E: Neuroibromatosis type 1 and pregnancy. Am ] Med Genet 66(1):7, 1996 Engineer L, Bhol K, Ahmed AR: Pemphigoid gestationis: a review. Am ] Obstet GynecoIt183(2):483, 2000 Erickson NI, Ellis L: Neonatal rash due to herpes gestation is. N Englt] Med 347(9):660, 2002 Fuentelsaz V, Ara M, Corredera C, et al: Rosacea fulminans in pregnancy: successful treatment with azithromycin. Clin Exp DermaroI36(6):674, 2011

1	High WA, Hoang MP, Miller MD: Pruritic urticarial papules and plaques of pregnancy with unusual and extensive palmoplantar involvement. Obstet Gynecol 105:1261,t2005 Huang H, Chen P, Liang CC, et al: Impetigo herpetiform is with gestational hypertension: a case report and literature review. Dermatology 222(3):221, 2011 Huilaja L, Makikallio K, Hannula-]ouppi K, et al: Cyclosporine treatment in severe gestational pemphigoid. Acta Derm Venereol 95(5):593, 2015 Huilaja L, Makikallio K, Sormunen R, et al: Gestational pemphigoid: placental morphology and function. Acta Derm Venereol 93(1):33, 2013 Jarrett R, Gonsalves R, Anstey AV: Difering obstetric outcomes of rosacea fulminans in pregnancy: report of three cases with review of pathogenesis and management. Clin Exp Dermatol 35(8):888, 2010 Jenkins E, Hern S, Black MM: Clinical features and management of 87 patients with pemphigoid gestationis. Clin Exp DermatoI24(4):255, 1999

1	Jenkins E, Hern S, Black MM: Clinical features and management of 87 patients with pemphigoid gestationis. Clin Exp DermatoI24(4):255, 1999 Kaplan YC, Ozsarfati ], Etwel F, et al: Pregnancy outcomes following irsttrimester exposure to topical retinoids: a systematic review and meta-analysis. Br] DermatoIt173(5):1132, 2015 Katz SI, Hertz KC, Yaoita H: Herpes gestationis. Immunopathology and characterization of the HG factor.t] Clin Invest 57(6):1434,t1976 Kelly SE, Bhogal BS, Wojnarowska F, et al: Expression of a pemphigoid gestationisrelated antigen by human placenta. Br] DermatoIt118:605, 1988 Ko B], Whang KU: Intravenous immunoglobulin therapy for persistent pemphigoid gestationis with steroid induced iatrogenic Cushing's syndrome. Ann DermatoIt26(5):661, 2014 Lawley T], Hertz KC, Wade TR, et al: Pruritic urticarial papules and plaques of pregnancy. ]AMA 241 (16): 1696, 1979

1	Lawley T], Hertz KC, Wade TR, et al: Pruritic urticarial papules and plaques of pregnancy. ]AMA 241 (16): 1696, 1979 Lawley T], Stingl G, Katz SI: Fetal and maternal risk factors in herpes gestationis. Arch DermatoIt114(4):552, 1978 Lehrhof S, Pomeranz MK: Specific dermatoses of pregnancy and their treatment. Dermatol her 26(4):274,t2013 Leppavirtat], Kallionpaa A, Uusitalo E, et al: he pregnancy in neuroibromatosis 1: A retrospective register-based total population study. Amt] Med Genet A 173(10):2641,t2017 Lima XT, ]anakiraman V, Hughes MD, et al: The impact of psoriasis on pregnancy outcomes.t] Invest Dermatol 132(1):85,t2012 Lipozencic ], Ljubojevic S, Bukvic-Mokos Z: Pemphigoid gestationis. Clin DermatoIt30(1):51,t2012 Margesson L], Danby FW: Hidradenitis suppurativa. Best Pract Res Clin Obstet GynaecoIt28(7):1013, 2014

1	Margesson L], Danby FW: Hidradenitis suppurativa. Best Pract Res Clin Obstet GynaecoIt28(7):1013, 2014 Matsumoto N, Osada M, Kaneko K, et al: Pemphigoid gestation is after sponta neous expulsion of a massive complete hydatidiform mole. Case Rep Obstet 268,t2013 Merker VL, Murphy TP, Hughes ]B, et al: Outcomes of preimplantation genetic diagnosis in neuroibromatosis type 1. Ferti! Steriltl03(3):76t1, 20t15 Men A, Kumbasar H, Ozaras R, et al: Erythema nodosum: an evaluation of 100 cases. Clin Exp Rheumatol 25(4):563, 200 Murase ]E, Heller MM, Buder DC: Safety of dermatologic medications in pregnancy and lactation: part r. Pregnancy.] Am Acad Dermatol 70(3):40 l.e1, 2014 Oumeish OY, AI-Fouzan A W: Miscellaneous diseases afected by pregnancy. Clin Dermatol 24: 113, 2006 Panchaud A, Csajka C, Merlob P, et al: Pregnancy outcome following exposure to topical retinoids: a multicenter prospective study. ] Clin Pharmacol 52(12):1844,t2012

1	Clin Dermatol 24: 113, 2006 Panchaud A, Csajka C, Merlob P, et al: Pregnancy outcome following exposure to topical retinoids: a multicenter prospective study. ] Clin Pharmacol 52(12):1844,t2012 Papagrigoraki A, Gisondi P, Rosina P, et al: Erythema nodosum: etiological factors and relapses in a retrospective cohort study. Eur ] Dermatol 3,t2010 Park SY, Kim ]H, Lee WS: Pruritic urticarial papules and plaques of pregnancy with unique distribution developing in postpartum period. Ann Dermatol 25(4):506,t2013 Perng P, Zampella]G, Okoye GA: Management of hidradenitis suppurativa in pregnancy.] Am Acad Dermatol 76(5):979, 2017 Powell M, Sakuma-Oyama Y, Oyama N, et al: Usefulness of BP180 NC16a enzyme-linked immunosorbent assay in the serodiagnosis of pemphigoid gestationis and in diferentiating between pemphigoid gestationis and pruritic urticarial papules and plaques of pregnancy. Arch Dermatol 141(6): 705, 2005

1	Regnier S, Fermand V, Levy P, et al: A case-control study of polymorphic eruption of pregnancy. ] Am Acad Dermatol 58 (1):63, 2008 Robinson A, Van Voorhees AS, Hsu S, et al: Treatment of pustular psoriasis: from the Medical Board of the National Psoriasis Foundation. ] Am Acad Dermatol 67(2):279, 2012 Roth MM: Pregnancy dermatoses: diagnosis, management, and controversies. Amt] Clin Dermatol 12(1):25,t2011 Rudolph CM, AI-Fares S, Vaughan-Jones SA, et al: Polymorphic eruption of pregnancy: clinicopathology and potential trigger factors in 181 patients. Br ] Dermatol 154:54,t2005 Scheinfeld N: Pruritic urticarial papules and plaques of pregnancy wholly abated with one week twice daily application of fluticasone propionate lotion: a case report and review of the literature. Dermatol Online 14(11):4,2008

1	Segal 0, Holcberg G, Sapir 0, et al: Neuroibromatosis in pregnancy. Maternal and perinatal outcome. Eur] Obstet Gynecol Reprod Bioi 84(1):59, 1999 Semkova K, Black M: Pemphigoid gestationis: current insights into pathogenesis and treatment. Eur] Obstet Gynecol Reprod Bioi 145(2):138, 2009 Shimanovich 1, Skrobek C, Rose C, et al: Pemphigoid gestationis with predominant involvement of oral mucous membranes and IgA autoantibodies targeting the C-terminus ofBP180.] Am Acad DermaroI47:780, 2002 Shornick]K, Black MM: Fetal risks in herpes gestationis. ] Am Acad Dermatol 26:63, 1992 Shornick ]K, Meek T], Nesbitt LT, et al: Herpes gestationis in blacks. Arch DermatoIt120(4):511,t1984 Sitaru C, Powell], Messer G, et al: Immunoblotting and enzyme-linked immunosorbent assay for the diagnosis of pemphigoid gestationis. Obstet Gynecol 103(4):757,t2004 Spits C, De Rycke M, Van Ranst N, et al: Preimplantation genetic diagnosis for cancer predisposition syndromes. Prenat Diagn 27(5):44 ,t2007

1	Spits C, De Rycke M, Van Ranst N, et al: Preimplantation genetic diagnosis for cancer predisposition syndromes. Prenat Diagn 27(5):44 ,t2007 Stern RS: Exanthematous drug eruptions. N Engl] Med 366:2492, 2012 Takatsuka Y, Komine \1, Ohtsuki M: Pemphigoid gestation is with a complete hydatidiform mole.] DermatoI39(5):474, 2012 Tani N, Kimura Y, Koga H, et al: Clinical and immunological proiles of 25 patients with pemphigoid gestationis. Br] DermatoIt172(1):120, 2015 Terry AR, Barker FG 2nd, Lefert L, et al: Neuroibromatosis type 1 and pregnancy complications: a population-based study. Am ] Obstet Gynecol 209(1) :46.e1, 2013 Terry AR, Merker L, Barker FG 2nd, et al: Pregnancy complications in women with rare tumor suppressor syndromes afecting central and peripheral nervous system. Amt] Obstet GynecoIt213(1):108, 2015 Van de Wiel A, Hart HC, Flinterman], et al: Plasma exchange in herpes gestationis. BM] 281: 1041, 1980

1	Van de Wiel A, Hart HC, Flinterman], et al: Plasma exchange in herpes gestationis. BM] 281: 1041, 1980 Vaughan Jones SA, Hern S, Nelson-Piercy C, et al: A prospective study of 200 women with dermatoses of pregnancy correlating clinical indings with hormonal and immunopathological proiles. Br J Dermatol 1, 1999 iksrr:im Shemer E, Marschall HU, Ludvigsson ]F, et al: Intrahepatic cholestasis of pregnancy and associated adverse pregnancy and fetal outcomes: a 12-year population-based cohort study. B]OG ,t2012 Yang YW, Chen CS, Chen H, et al: Psoriasis and pregnancy outcomes: a nationwide population-based study.t] Am Acad Dermatol 64(1):71, 201t1 Zaenglein AL, Pathy AL, Schlosser B], et al: Guidelines of care for the manage ment of acne vulgaris.] Am Acad Dermatol 74(5):945, 2016 CANCER THERAPY IN PREGNANCY .i........i....... 1191 REPRODUCTIVE TRACT NEOPLASMS.. . . . . . . . . . . . .. 1192

1	CANCER THERAPY IN PREGNANCY .i........i....... 1191 REPRODUCTIVE TRACT NEOPLASMS.. . . . . . . . . . . . .. 1192 BREAST CARCINOMA. . . . . . . . . . . . . . . . . . . . . . . . . .. 1200 THYROID CANCER.. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1201 LYMPHOMAS.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1202 MALIGNANT MELANOMA. . . . . . . . . . . . . . . . . . . . . .. 1203 GASTROINTESTINAL TRACT . . . . . . . . . . . .. 1204 MISCELLANEOUS TUMORS. . . . . . . . . . . . . . . . . . . . .. 1204 of the more frequent ones are shown in Figure 63-1. Breast cancer is found in 1 in 5000 pregnancies, thyroid-1 in 7000, and cervical-1 in 8500 (Smith, 2003). These, along with lymphoma and melanoma, account for 65 percent of malignancy cases in pregnancy (Eibye, 2013). For some cancers-ovary, endometrium, and breast-evidence suggests that high parity is protective (Hognas, 2014).

1	During pregnancy, cancer management poses unique problems related to fetal concerns, and treatment must be individualized. Considerations include the type and stage of malignancy, the desire for pregnancy continuation, and inherent risks associated with modiying or delaying cancer treatment. Any excessive enlargement of the abdomen or the appearance of pressure symptoms should always lead one to make a carul examination, and in not a ew cases a tumour will be ound occupying the pelvic caviy. In rare instances malignant tumours of the rectum may so obstruct the pelvic canal as to render caesarean section imperative. -J. Whitridge Williams (1903) Any neoplasm can complicate pregnancy, and as written by Williams, physical examination often suggests the diagnosis. Current imaging also allows a greater number of these to be identiied antepartum. Most encountered neoplasms are benign, and uterine leiomyomas and ovarian cysts are the most frequent.

1	Cancer has an incidence approximating 1 per 1000 pregnancies (Parazzini, 2017; Salani, 2014). One third are diagnosed prenatally, and the others within 12 months of delivery. Some FIGURE 63-1 Proportion of malignancies during pregnancy and within 12 months of delivery in 4.85 million women from the California Cancer Registry. GI = gastrointestinal. (Data from Smith, 2003.) diagnosis, staging, or therapy. Fortunately, most procedures that do not interfere with the reproductive tract are well toler ated by both mother and fetus (Chap. 46, p. 901). Although many operations have classically been deferred until after 12 to 14 weeks' gestation to minimize miscarriage risks, this probably is not necessary. We are of the opinion that surgery should be performed regardless of gestational age if maternal well-being is imperiled.

1	Both pregnancy and malignancy are risk factors for venous thromboembolism (VTE). In one study, Bleau and coworkers (2016) reported a higher risk ofVTE in gravidas with myeloid leukemia, Hodgkin disease, cervical cancer, and ovarian cancer compared with that in pregnant women without a malignancy. The risk was not increased in those with brain or thyroid cancer, melanoma, or lymphoid leukemia. That said, current guidelines ing surgeries for cancer. hus, depending on the complexity of the planned procedure, it seems reasonable to use prophylactic low-molecular-weight heparin combined with elastic stockings and/or intermittent pneumatic compression as described in Chapter 52 (p. 1019).

1	Sonography is a preferred imaging tool during pregnancy. Even so, according to the American College of Obstetricians and Gynecologists (2017a), most diagnostic radiographic procedures deliver very low x-ray doses and should not be delayed if they would directly afect therapy (Chap. 46, p. 906). Magnetic resonance (MR) imaging can safely be performed in any trimester, but delay until after the irst trimester may lower potential risks. Gadolinium should not be used in the irst trimester and should be used later in pregnancy only when the beneits overwhelmingly outweigh the risks (American College of Radiology, 2016; Kanal, 2013). Computed tomography (CT) is less often selected due to ionizing radiation, and procedure-related doses are listed in Chapter 46 (p. 907). Accordingly, CT is used in pregnancy most often to evaluate acute concerns that include pulmonary embolism, bowel or renal obstruction, and acute neurological events. To enhance CT, oral and intravenous contrasts may be

1	in pregnancy most often to evaluate acute concerns that include pulmonary embolism, bowel or renal obstruction, and acute neurological events. To enhance CT, oral and intravenous contrasts may be added. hese lack known fetal harm, and postpartum breastfeeding need not be interrupted. Last, some radioisotopes are relatively safe in pregnancy and are listed in Table 46-8 (p. 909).

1	herapeutic radiation often results in signiicant fetal exposure depending on the dose, tumor location, field size, and gestational age. Potential adverse efects include fetal malformation, intellectual disability, growth restriction, sterility, and carcinogenesis (Brent, 1999; Stovall, 1995). In the 2 weeks following fertilization, exposure typically leads to chromosomal damage and embryonic death. he next most susceptible period is during organogenesis in weeks 2 through 8, and exposure can cause malformation. These might develop above a threshold dose of 0.1 to 0.2 Gy. During weeks 8 through 25, the fetal central nervous system is especially vulnerable. The threshold dose for intellectual disability at 8 to 15 weeks' gestation approximates 0.06 Gy, and at 16 to 25 weeks it is about 0.25 Gy (Kal, 2005; Otake, 1996). After 25 weeks' gestation, susceptibility is less, although no gestational age is considered safe for therapeutic radiation exposure. hus, radiotherapy to the maternal

1	Gy (Kal, 2005; Otake, 1996). After 25 weeks' gestation, susceptibility is less, although no gestational age is considered safe for therapeutic radiation exposure. hus, radiotherapy to the maternal abdomen is contraindicated. With some head and neck cancers, however, radiotherapy to supradiaphragmatic areas can be used relatively safely with abdominal shielding of the fetus (Amant, 2015a).

1	Various antineoplastic drugs may be given for primary treatment or for adjunctive therapy. Although chemotherapy often improves long-term maternal outcomes, many are reluctant to employ it during pregnancy. Concerns for the fetus include malformations, growth restriction, intellectual disability, and the risk of future childhood malignancies. Risks are dependent primarily on fetal age at exposure, and most agents are potentially detrimental in the irst trimester during organogenesis. Indeed, in one review, 14 percent of major malformations were attributable to irst-trimester exposure to cytotoxic drugs (National Toxicology Program, 2013).

1	Ater the first trimester, most antineoplastic drugs are without immediate obvious adverse fetal sequelae (Abdel-Hady, 2012; Vercruysse, 2016). Similarly, late mutagenic efects appear limited (Amant, 2015b; Cardonick, 2015). Although not always practicable, some recommend that chemotherapy be withheld in the 3 weeks before expected delivery because neutropenia or pancytopenia might cause undue risk for maternal infection or hemorrhage. Another concern is that neonatal hepatic and renal clearance of chemotherapy metabolites is limited (Ko, 2011). For these reasons, most cytotoxic chemotherapy agents are contraindicated with breastfeeding (Pistilli, 2013).

1	Drugs designed to stimulate hemopoiesis are commonly used with cancer treatments. Some of these include the granulocyte colony-stimulating factors filgrastim (Neupogen) and pegilgrastim (Neulasta). If required in pregnancy, limited data support the safety of these agents (Boxer, 2015). Red blood cells can be stimulated by erythropoietin alfa (Procrit), which from case reports also appears safe in pregnancy (Sienas, 2013). However, maternal hypertension is a known potential risk. he two main types of targeted therapy are monoclonal antibodies and small molecule inhibitors. Both block the actions of specific enzymes, proteins, or other molecules involved in cancer cell growth. hese drugs are designed to treat an everexpanding list of cancers, and some are described in later discussions of specific tumors. Most of these compounds are labeled by the Food and Drug Administration (FDA) as class D, and data are limited regarding their pregnancy or breastfeeding efects.

1	D, and data are limited regarding their pregnancy or breastfeeding efects. Many of these drugs target tyrosine kinase, an important enzyme that regulates signaling pathways involved with cell division, diferentiation, and apoptosis. With irst-trimester use, embryo toxicity or teratogenicity has been attributed to these. Thus, this speciic group of targeted agents is considered for use in pregnancy only if the potential beneit to the pregnant woman justifies the potential risk to the fetus (Lodish, 2013). Of other agents, the monoclonal antibody trastuzumab (Herceptin) inhibits the human epidermal growth factor receptor type 2 (HER2), which some breast cancers express. Although not teratogenic, its use in the second and third trimesters is associated with oligohydramnios, which appears to be reversible upon stopping the drug (Sarno, 2013; Zagouri, 2013b). Because of sparse available data, other HER2 inhibitors are best avoided in pregnancy (Lambertini, 2015).

1	Fertility may be diminished after chemotherapy or radiotherapy. Counseling ideally takes place before cancer treatment, and guidelines for this have been developed (American Society for Reproductive Medicine, 2013a; Lambertini, 2016; Loren, 2013; Peccatori, 2013). Prior to therapy, cryopreservation of embryos or of oocytes is a recognized fertility-preserving option (American Society for Reproductive Medicine, 2013b,c). Surgical transposition of the ovaries can be considered if pelvic radiation is planned. For this, ovaries and their intact primary blood supply are carried out of the pelvis and ixed to the lateral abdominal wall at a site 3 to 4 cm above the level of the umbilicus. In one review, functional preservation was reported in 65 to 94 percent, depending on radiotherapy type (Gubbala, 2014). Also, such transposition requires transabdominal egg retrieval if later in vitro fertilization is planned (American Society for Reproductive Medicine, 20 13a).

1	Ovarian suppression with gonadotropin-releasing hormone agonists is not beneficial, according to a recent review (Elgindy, 2015). At this time, cryopreservation of ovarian tissue is considered investigational. These methods currently are limited to referral centers. In counseling cancer survivors, evidence suggests that exposure to most radiotherapy or chemotherapy agents in childhood or adulthood does not signiicantly increase the risk of congenital anomalies or genetic disease in their ofspring (Haggar, 2014; Signorello, 2012; Stensheim, 2013; Winther, 2012). In those treated as children with chemotherapy, studies also do not show a consistent link to adverse obstetrical outcomes (Melin, 2015; Reulen, 2009). Data are limited regarding those with cancer treated as an adult, and some studies have shown slightly higher rates of preterm birth and cesarean delivery (Haggar, 2014; Stensheim, 2013).

1	Notably, prior abdominopelvic radiation more convincingly afects neonatal outcomes. Adverse efects include elevated rates of abortion, low birthweight, stillbirth, and preterm birth (Signorello, 2006, 2010; Winther, 2008). Radiation may lower reproductive potential by creating reduced uterine volume, a thinned endometrium, and impaired uterine blood flow (Critchley, 1992; Larsen, 2004). Greater efects are seen with direct uterine radiation and with radiotherapy at younger ages (Teh,r2014). Importantly, many cancer survivors conceive by assisted reproductive technology, which by itself has associated obstetrical risks. his is discussed in Chapter 8 (p. 151).

1	Importantly, many cancer survivors conceive by assisted reproductive technology, which by itself has associated obstetrical risks. his is discussed in Chapter 8 (p. 151). Tumors infrequently metastasize to the placenta. As described in Chapter 6 (p. 116), the most common types are malignant melanomas, leukemias, lymphomas, and breast cancer (AlAdnani, 2007). Placentas from pregnancies in these and all other women with a malignancy should be sent for histological evaluation. Because tumor cells are usually confined within the intervillous spaces, fetal metastases are infrequent. Benign neoplasms are common and include leiomyomas, ovarian neoplasms, and endocervical polyps. Cancer in these organs may also complicate pregnancy, and of these, cervical neoplasia makes up the majority (Fig. 63-2).

1	These are overgrowths of endocervical stroma covered by epithelium. hey typically appear as single, red, elongated leshy masses of variable size that extend outward from the endocervical canal. Usually benign, they can bleed and can be a source of Pap test results describing aypical glandular cels of undetermined signiicance-AGUS. With removal and histological Ovary 23% Uterus, vulva, FIGURE 63-2 Frequency of reproductive-tract malignancies in 844 pregnant women. (Data from Haas, 1984; Lutz, 1977; Smith, 2003.) evaluation of these polyps, dysplasia is diagnosed in up to 0.5 percent, and there is malignant transformation in up to 0.1 percent (Esim Buyukbayrak, 2011; Long, 2013).

1	Few formal data guide management in pregnancy. Small asymptomatic lesions may be left alone to slough during delivery or puerperal remodeling. Removal and histological evaluation is reasonable if malignancy is suspected or if bleeding is trouble some. For those with a slender stalk, the polyp is grasped with ring forceps and twisted repeatedly about its base to strangulate feeding vessels. With repeated twisting, the base narrows and avulses. Monsel paste, which is ferric subsulfate, can be applied with pressure to the stalk stub for hemostasis. A thick-pedicle polyp may sometimes warrant surgical ligation and excision.

1	Pregnancy provides an opportune time to screen for cervical intraepithelial neoplasia (CIN), especially in women without regular access to health care. With Pap test screening, preg nancy status is noted on the requisition form because interpre changes. Some of these changes include presence of decidual cells and less often, Arias-Stella reaction. he latter gives an appearance of endocervical gland hyperplasia, which can make diferentiating this from truly atypical glandular cells diicult.

1	Screening guidelines also applicable in pregnant women were updated in 2012 by the American Society for Colposcopy and Cervical Pathology (ASCCP). hese include: (1) no screening until age 21, (2) cytology alone every 3 years in those aged 21 to 29 years, and (3) in those older than 30, human papillomavirus (HPV) and cytology co-testing every 5 years, or cytology alone every 3 years (Massad, 2013). High-risk conditions for cervical neoplasia include human immunodeficiency virus (HIV) infection, other immunocompromised states, and in utero diethylstilbestrol (DES) exposure. For women with HIV infection, initiation of cervical cancer screening with cytology alone begins within the first year after HIV diagnosis (American College of Obstetricians and Gynecologists, 20 16b).

1	Human Papillomaviru5. This virus infects cervical epithelia. In most instances, the infection clears, but in a smaller number, the virus may promote benign, premalignant, or cancerous neoplastic growth. he prevalence of HPV infection in pregnant women approximates 15 percent (Hong, 2013; Liu, 2014). here are more than 100 serotypes, and several are associated with high-grade intraepithelial lesions and invasive cancer. he most prominent of these are serotypes 16 and 18. Cervical cancer screening that combines both cytology and testing for high-risk HPV serotypes is termed co-testing and is suitable for women 30 years and older. Notably, as a new paradigm for screening, primary HPV testing alone can be considered an appropriate sole method for women older than 25 years (Huh, 2015). With this, identiication of serotypes 16 or 18 prompts colposcopic evaluation.

1	Serotypes HPV 6 and 11 are linked with benign maternal genital warts. Congenital HPV infection from vertical transmission-mother to fetus or newborn-beyond transient skin colonization is rare. Still, conjunctival, laryngeal, vulvar, or perianal warts present at birth in the neonate or that develop within 1 to 3 years of birth are most likely due to perinatal exposure to these maternal HPV serotypes. his is described further in Chapter 65 (p. 1245). Importantly, cesarean delivery does not lower the risk of neonatal laryngeal papillomatosis. has prompted development of three approved vaccines. These are not administered during pregnancy, are compatible with breastfeeding, and are discussed in Chapter 65 (p. 1245).

1	has prompted development of three approved vaccines. These are not administered during pregnancy, are compatible with breastfeeding, and are discussed in Chapter 65 (p. 1245). Abnormal Cytology and Histology. The incidence of abnor mal cervical cytology during pregnancy is at least as high as that reported for nonpregnant women. Abnormal cytological indings and their suggested management according to consen sus guidelines are summarized in Table 63-1. Many of these cytological abnormalities should prompt colposcopy, and the main goal during pregnancy is exclusion of invasive cancer.

1	Accordingly, lesions suspicious for high-grade disease or cancer should undergo biopsy. Unsatisfactory colposcopic evaluation is less common during pregnancy because the transformation zone is better exposed due to cervical eversion. With insui cient visualization of the zone, colposcopy is repeated in 6 to 8 weeks. During this time, the squamocolumnar junction usually will further evert to permit satisfactory examination.

1	Women with histologically confirmed CIN during pregnancy may be allowed to deliver vaginally, with further evaluation planned after delivery. For those with CIN 1, the recommended management is reevaluation postpartum. For those with CIN 2 or 3 in which invasive disease has been excluded, deferring reevaluation until at least 6 weeks postpartum is acceptable. Alternatively, repeat colposcopic and cytological evaluations are performed at intervals no more frequent than 12 weeks. Repeat biopsy is recommended only if appearance of the lesion worsens or if cytology suggests invasive cancer (Massad,r2013).

1	Regression of a CIN lesion is common during pregnancy or postpartum. In a study of 1079 pregnant women with cervical dysplasia in which biopsy correlated with colposcopic findings, 61 percent of lesions reverted to normal postpartum (Fader, 2010). In another study, Yost and colleagues (1999) reported postpartum lesion regression in 70 percent of women with CIN 2 or 3. And, although 7 percent of women had CIN 2 lesions that progressed to CIN 3, no lesion progressed to invasive carcinoma. In another study of 77 women with carcinoma in situ (CIS) diagnosed during pregnancy, a third had postpartum regression of their lesion, two thirds had persistent CIS, and only two women had microinvasive cancer on cone biopsy after delivery (Ackermann, 2006). Adenocarcinoma in situ (AIS) is managed similarly to CIN 3 (Dunton, 2008). hus, unless invasive cancer is identified, treatment of AIS is not recommended until 6 weeks postpartum.

1	Adenocarcinoma in situ (AIS) is managed similarly to CIN 3 (Dunton, 2008). hus, unless invasive cancer is identified, treatment of AIS is not recommended until 6 weeks postpartum. Cervical Conization. If invasive epithelial lesions are suspected, conization is indicated and may be done with loop electrosurgical excisional procedure (LEEP) or by cold-knife conization. However, the epithelium and underlying stroma within the endocervical canal cannot be extensively excised without the risk of membrane rupture. Logically, residual disease is common. Of 376 cone biopsies during pregnancy, Hacker and aWomen aged 30+ years. bEndocervical curettage and endometrial sampling are contraindicated in pregnancy. adenoCA = adenocarcinoma; AGC = atypical glandular cells; AIS = adenocarcinoma in situ; ASC-H = atypical squamous cells, cannot exclude high-grade squamous intraepithelialrlesion; ASC-US = atypical squamous cells of undetermined significance; HPV = human papillomavirus;

1	HSIL = high-grade squamous intraepithelialrlesion; LSIL = low7grade squamous intraepithelial lesion; NILM = negative for intraepithelialrlesion or malignancy; SCCA = squamous cell carcinoma. Adapted from American Society for Colposcopy and Cervical Pathology (ASCCP) 2012 Consensus Guidelines; Massad, 20lr3. Table summarized and used with permission from Dr. Claudia L. Werner. associates (1982) found residual neoplasia in 43 percent of subsequent specimens. In addition, nearly 10 percent of 180 pregnant women required transfusion after conization (Averette, 1970). hus, if possible, conization is avoided in pregnancy because of its higher risks for abortion, membrane rupture, hemorrhage, and preterm delivery.

1	Women with CIN treated bore pregnancy may also encounter pregnancy complications. First, cicatricial cervical stenosis is uncommon but may follow conization, LEEP, or laser surgery. Cervical stenosis almost always yields during labor. A so-called conglutinated cervix may undergo almost complete intrapartum efacement without dilation, and the presenting part is separated from the vagina by only a thin layer of cervical tissue. Spontaneous dilation usually promptly follows firm pressure with a ingertip, although instrumented dilation or cruciate incisions may be required. Second, preconceptional cold-knife conization is associated with cervical insuiciency and preterm birth. That said, the relationship between preterm birth and LEEP continues to be debated (Castanon, 2012; Conner, 2014; Stout, 2015; Werner, 2010). he size of tissue excised seems to be directly related to adverse outcomes (Weinmann, 2017).

1	he incidence of invasive cervical carcinoma has dramatically declined in the United States as a result of PAP testing (American College of Obstetricians and Gynecologists, 2016b). This cancer is found in approximately 1 in 8500 pregnancies (Bigelow, 2017; Pettersson, 2010). The diagnosis is conirmed with biopsies taken during colposcopy, with conization, or from a grossly abnormal lesion. Of the histological types, squamous cell carcinomas account for 75 percent of all cervical cancers, whereas adenocarcinomas compose the remainder. Cancers may appear as exophytic or endophytic growth; as a polypoid mass, papillary tissue, or barrel-shaped cervix; or as focal ulceration or necrosis. A watery, purulent, foul, or bloody discharge may also be present. Biopsy with Tischler forceps is warranted for suspicious lesions. Abnormal tumor vessels may cause heavier than expected biopsy-site bleeding, which is usually controlled by Monsel paste and pressure.

1	Cervical cancer is staged clinically, and 70 to 75 percent of cases that are diagnosed in pregnancy are stage I (Bigelow, 2017; Morice, 2012). Physiological pregnancy changes may impede accurate staging, and the extent of cancer is more likely to be underestimated in pregnant women. Specifically, induration of the broad ligament base, which characterizes tumor spread beyond the cervix, may be less prominent due to cervical, paracervical, and parametrial pregnancy-induced softening. Staging in pregnancy typically incorporates indings from pelvic examination and from renal sonography, chest radiography, cystoscopy, proctoscopy, and perhaps cone biopsy. Although MR imaging is not formally considered for clinical staging, it can be used without gadolinium contrast to ascertain involvement of the urinaty tract and lymph nodes (Fig. 63-3). FIGURE 63-3 Sagittal T2-weighted magnetic resonance image of a gravid uterus at 32 weeks' gestation with a large cervical carcinoma (arrows).

1	FIGURE 63-3 Sagittal T2-weighted magnetic resonance image of a gravid uterus at 32 weeks' gestation with a large cervical carcinoma (arrows). Management and Prognosis. Cervical cancer treatment in pregnant women is individualized, and factors include the clinical stage, fetal age, and individual desire to continue pregnancy. Stage IA1 is termed microinvasive disease and describes lesions with deepest invasion ;3 mm and widest lateral extension ;7 mm (FIGO Committee on Gynecologic Oncology, 2009). If diagnosed by cone biopsy, then treatment follows guidelines similar to those for intraepithelial disease. In general, continuation of pregnancy and vaginal delivery are considered safe, and definitive therapy is reserved until 6 weeks postpartum.

1	In contrast, invasive cancer demands relatively prompt therapy. During the first half of pregnancy, immediate treatment is advised by most, but this depends on the decision whether to continue pregnancy. During the latter half of pregnancy, most agree that pregnancy can safely be continued until fetal lung maturity is attained (Greer, 1989). In two studies with a total of 40 women past 20 weeks' gestation with either stage I or stage IIA carcinoma, delayed treatment was considered reasonable in women without bulky lesions (Takushi, 2002; van Vliet, 1998). Another option is to complete staging using laparoscopic lymphadenectomy and to delay treatment if metastases are excluded (louini, 2008; Favero, 2010). In a metaanalysis, neoadj uvant chemotherapy, that is, prior to surgery, with platinum derivatives was found to be promising for treatment in pregnancy (Zagouri, 2013a).

1	Although surgical therapy and radiation are equally efective, radical hysterectomy plus pelvic lymphadenectomy is the preferred treatment for invasive cervical cancer in most young women with stage I and early stage IIA lesions. Disadvantageously, radiotherapy for cervical cancer destroys ovarian and possibly sexual function, and frequently causes intestinal and urinary tract injury. In 49 women with pregnancy-associated stage IB cancer, a 30-percent severe complication rate accompanied radiotherapy compared with that of only 7 percent with radical surgery (Nisker, 1983). With surgery before 20 weeks' gestation, radical hysterectomy is usually performed with the fetus in situ. In later pregnancy, however, hysterotomy is oten performed first.

1	In later pregnancy, however, hysterotomy is oten performed first. Although less commonly selected during pregnancy, other cers. U ngGr and colleagues (2006) performed abdominal radical in five pregnant women. Yahata and associates (2008) treated four women at 16 to 23 weeks for stage IA1 adenocarcinoma with laser conization, and all delivered at term. Van Calsteren and coworkers (2008) reported similar success. For more advanced-stage cancer, radiotherapy is given. External beam radiation in early in pregnancy typically leads to spontaneous abortion. If miscarriage does not ensue, curettage is performed. During the second trimester, spontaneous abortion up to a fourth of cases. his is selected because labor induction or dilation and evacuation may pose serious hemorrhage risks. Pregnancy does not have a negative efect on the prognosis of cervical cancer, and survival outcomes are similar for preg nant and nonpregnant women (Amant, 2014; Mogos, 2013).

1	Pregnancy does not have a negative efect on the prognosis of cervical cancer, and survival outcomes are similar for preg nant and nonpregnant women (Amant, 2014; Mogos, 2013). In a case-control study of 44 women with pregnancy-associated cervical cancer, the overall 5-year survival rate approximated (van de' Vange, 1995). Delivery. Any adverse prognostic efects that vaginal delivery through a cancerous cervix might have are unknown. For this reason, the mode of delivery is controversial, especially for small, early-stage lesions. In some cases of bulky or friable tumors, significant hemorrhage from the cancer may complicate vaginal delivery. Also, recurrences have been reported in the episiotomy scar, which result from tumor cells apparently "seeding" the episiotomy (Goldman, 2003). Thus, most favor cesarean delivery.

1	Pregnancy ater Radical Trachelectomy. There is growing experience with pregnancy in women who have undergone fertility-sparing radical trachelectomy for stage IB 1 and IB2 cervical cancer before conception. During the typically vaginal procedure, the cervix is amputated at the level of the internal os, and a permanent-suture cerclage is placed around the isthmus for support in future pregnancies. The uterine isthmus is then reconstructed to the vagina. Because of the permanent cerclage, a classical cesarean incision is required for delivery. Shepherd and colleagues (2006) presented outcomes for 123 such women cared for at their institution. Of 63 women who attempted pregnancy, 19 had 28 live births. All underwent classical cesarean delivery, and one fourth delivered before 32 weeks. Similar findings were reported by Kim (2012) and Park (2014) and their coworkers. Also known as myomas and somewhat erroneously called ibrois, uterine leiomyomas are common benign smooth-muscle tumors.

1	Also known as myomas and somewhat erroneously called ibrois, uterine leiomyomas are common benign smooth-muscle tumors. heir incidence during pregnancy approximates 2 percent, and the cited range depends on the frequency of routine sonography and population characteristics (Qidwai, 2006; Stout, 2010). In one study of 4271 women, the irst-trimester leiomyoma prevalence was highest in black women-18 percent-and lowest in whites-8 percent (Laughlin, 2009).

1	Leiomyomas vary in location and may develop as submucous, subserosal, or intramural growths. Less often, these develop in the cervix or broad ligament. Some become parasitic and their blood supply is derived from adjacent structures such as the highly vascularized omentum. In one rare manifestation-leiomyomatosis peritonealis disseminata-numerous, small, benign subperitoneal smooth-muscle tumors appear similar to carcinomatosis. The tumors are likely caused by estrogen stimulation of multicentric subcoelomic mesenchymal cells to become smooth-muscle cells (Bulun, 2015). hese growths often regress after pregnancy. he stimulatory efects of pregnancy progesterone on myoma growth are unpredictable and can be impressive. hese tumors respond diferently in individual women and may grow, regress, or remain unchanged in size during pregnancy (Laughlin, 2009; Neiger, 2006).

1	Especially during pregnancy, myomas can be conused with other adnexal masses, and sonographic imaging is indispensable (Fig. 63-4). In women in whom sonographic indings are unclear, MR imaging performed ater the first trimester may be necessary. Once diagnosed, leiomyomas do not require surveillance with serial sonography unless associated complications are anticipated. Symptoms. Most leiomyomas are asymptomatic, but acute or chronic pain or pressure may develop. Large myomas more oten require admission for pain (Dogan, 2016). For chronic pain secondary to large tumor size, nonnarcotic analgesic drugs usually suice. More acutely, some myomas can outgrow their blood supply and hemorrhagic infarction follows, which is termed red

1	FIGURE 63-4 Sonogram of a pregnant uterus with a large uterine leiomyoma. The heterogeneous mass (arrows) lies beside the fetus (seen in cross section) and has the classic appearance of a leiomyoma in pregnancy. The placenta is located anteriorly, and the mass originates from the posterior lower uterine segment and occupies more than half of the total uterine volume. or carneous degeneration. Clinically, there is acute focal abdominal pain and tenderness, and sometimes a low-grade fever and leukocytosis. As such, tumor degeneration may be diicult to diferentiate from appendicitis, placental abruption, ureteral stone, or pyelonephritis. Sonographic imaging can be helpful, but close observation is requisite because an infarcted myoma is essentially a diagnosis of exclusion. In some women, preterm labor is stimulated by associated inlammation.

1	Treatment of a degenerated myoma is analgesic medications, and symptoms usually abate within a few days. In severe cases, close observation may be needed to exclude a septic cause. Although surgery is rarely necessary during pregnancy, myomectomy in highly selected cases has resulted in good outcomes. Of 23 reported cases, women were 14 to 20 weeks' gestation, and in almost half, surgery was performed because of pain (Celik, 2002; De Carolis, 2001). In some, an intramural leiomyoma was in contact with the implantation site. Except for one loss immediately following surgery at 19 weeks, most underwent cesarean delivery later, at term. Occasionlly, a pedunculated subserosal myoma will undergo torsion with subsequent painul necrosis. Laparoscopy or laparotomy can be used to ligate the stalk and resect the necrotic tumor. That said, we believe that surgery should be limited to tumors with a discrete pedicle that can be easily clamped and ligated.

1	Pregnancy Complications. Myomas are associated with several complications that include preterm labor, placental abruption, fetal malpresentation, obstructed labor, cesarean delivery, and postpartum hemorrhage. In a review of pregnancy outcomes in 2065 women with leiomyomas, Coronado and colleagues (2000) reported that placental abruption and breech presentation were each increased fourfold; irst-trimester bleeding and dysfunctional labor, twofold; and cesarean delivery, sixfold. Salvador and associates (2002) reported an eightfold higher second-trimester abortion risk in these women.

1	Factors most important in determining morbidity in pregnancy are leiomyoma number, size, and location (Ciavattini, 2015; Jenabi, 2018; Lam, 2014). If the placenta is adjacent to or implanted over a leiomyoma, rates of abortion, preterm labor, placental abruption, and postpartum hemorrhage are all increased. Retroplacental myomas are also associated with fetal-growth restriction (Knight, 2016) . Tumors in the cervix or lower uterine segment may obstruct labor, as did the one shown in Figure 63-5. Despite these complications, Qidwai and associates (2006) reported a 70-percent vaginal delivery rate in women in whom myomas measured : 10 cm. hese data argue against empirical cesarean delivery for leiomyomas, and we allow a trial of labor unless myomas clearly obstruct the birth canal. If cesarean delivery is indicated, uterine malrotation should be excluded prior to hysterotomy. Myomas are generally let alone unless they cause recalcitrant bleeding. n important caveat is that cesarean

1	delivery is indicated, uterine malrotation should be excluded prior to hysterotomy. Myomas are generally let alone unless they cause recalcitrant bleeding. n important caveat is that cesarean hysterectomy may be technically diicult because of lateral ureteral displacement by the masses.

1	Bleeding due to myomas may develop during pregnancy from any of several factors. Especially common is bleeding with miscarriage, preterm labor, placenta previa, and placental abruption. Much less often, bleeding may result from a submucous myoma that has prolapsed from the uterus and into the cervix or vagina. In this unusual circumstance, although heavy or persistent bleeding may require earlier intervention, the stalk, if accessible, can be ligated vaginally near term to avoid tumor avulsion during delivery. FIGURE 63-5 Cesarean delivery performed because of a large leiomyoma in the lower uterine segment. A classical vertical uterine incision, seen to the left of the myoma, was required for delivery of the fetus. Fortunately, myomas rarely become infected (Genta, 2001).

1	Fortunately, myomas rarely become infected (Genta, 2001). When infection develops, it usually is postpartum, especially if the tumor is located immediately adjacent to the implantation site (Lin, 2002). hey also may become infected with an associated septic abortion and myoma perforation by a sound, dilator, or curette. Fertility Considerations. Despite the relatively high prevalence of myomas in young women, it is not clear whether they diminish fertility, other than by possibly causing miscarriage. In a review of 11 studies, Pritts (2001) concluded that submucous myomas did signiicantly afect fertility. He also found that hysteroscopic myomectomy improved infertility and early miscarriage rates in these women. If truly implicated in infertility, myomas in other locations may require laparoscopy or laparotomy for excision.

1	Some of these methods of treatment for infertility may afect subsequent pregnancies. For example, after myomectomy, the gravid uterus can rupture either before or during labor (American College of Obstetricians and Gynecologists, 20 16a). Management is individualized, and review of the prior operative report is prudent. If resection resulted in a defect into or immediately adjacent to the endometrial cavity, then cesarean delivery is usually done before labor begins. Although less efective than surgery, uterine artery embolization of myomas has also been used to treat infertility or symptoms (Mara, 2008). Women so treated have higher rates of miscarriage, cesarean delivery, and postpartum hemorrhage (Homer, 2010). he Society of Interventional Radiology considers myoma embolization relatively contraindicated in women who plan future pregnancies (Stokes, 2010).

1	Finally, outside the United States, ulipristal-a selective progesterone-receptor modulator-can be used for myoma regression. Successful subsequent pregnancies without tumor regrowth have been reported (Luycx, 2014). Occasionally, endometriosis can develop after delivery from otomy scars (Bumpers, 2002). Here, they form a palpable mass and can cause cyclic localized pain. Endometriomas within an ovary are discussed in the next section. Adenomyosis is traditionally found in late reproductive life and beyond. Its acquisition may be at least partially related to disruption of the endometrial-myometrial border during sharp curettage for abortion (Curtis, 2002). In a case-control study, associated rates of second-trimester abortion, preeclampsia, fetal malposition, and preterm delivery.

1	Endometrial carcinoma is an estrogen-dependent neoplasia also usually found in women older than 40 years. hus, it is seen only rarely with pregnancy. Of 27 cases that were identi fied during pregnancy or within the irst 4 months postpartum, nuna, 2009). These are usually early-stage, well-diferentiated adenocarcinomas for which treatment consists primarily of tomy. Much less commonly, to preserve future fertility, curet tage with or without postprocedural progestational therapy has been used for the rare patient with cancer identified in a miscar riage curettage specimen (Schammel, 1998).

1	Many more studies describe a conservative approach for well-selected nonpregnant women diagnosed with endometrial cancer who wish to preserve fertility. One study followed 13 women treated with progestins for early-stage, well-diferentiated adenocarcinoma who then later conceived after apparent remission (Godieb, 2003). Nine had liveborn neonates, and four of six women with a recurrence responded to another course of therapy. Similar outcomes were described in 12 women by Niwa (2005) and in 21 women by Signorelli (2009), each with their coworkers. Despite these acceptable pregnancy rates, recurrences and death have been reported, and conservative management is not considered standard (Erkanli, 2010).

1	Ovarian masses found during pregnancy are relatively common. Among studies, incidences vary depending on the frequency of prenatal sonography, the ovarian size threshold used to define a clinically signiicant "mass," and whether the study site is tertiary or primary care. Thus, the incidence of ovarian masses not surprisingly ranges from 1 in 100 to 2000 pregnancies (Whitecar, 1999; Zan etta, 2003). Of ovarian malignancies, the absolute incidence in the California Cancer Registry was 1 in 19,000 pregnancies (Smith, 2003). The most frequent types of ovarian masses are corpus luteum cysts, endometriomas, benign cystadenomas, and mature cystic teratomas. Because pregnant women are usually young, malignant tumors and those of low malignant potential are proportionately uncommon. Our experiences from Parkland Hospital are similar to those of Leiserowitz and associates (2006), who

1	FIGURE 63-6 Sonographic characteristics of common adnexal masses in pregnancy. A. A simple anechoic cyst with smooth walls is characteristic of a physiological corpus luteum cyst or benign cystadenoma. B. Cystic structure with difuse internal low-level echoes sug gestive of an endometrioma or hemorrhagic corpus luteum. C. Mature cystic teratoma appears as an adnexal cyst (marked by calipers) with accentuated lines and dots that represent hair in both longitudinal and transverse planes. At the central inferior aspect of this cyst, a mural nodule-Rokitansky protuberance-is seen. These typically rounded protuberances range in size from 1 to 4 cm, they are predominantly hyperechoic, and they create an acute angle with the cyst wall. Although not seen here, fat-fluid levels are often identified with cystic teratomas. (Used with permission from Dr. Elysia Moschos.) found that 1 percent of9375 ovarian masses were frankly malignant and that another 1 percent were of low malignant potential.

1	with cystic teratomas. (Used with permission from Dr. Elysia Moschos.) found that 1 percent of9375 ovarian masses were frankly malignant and that another 1 percent were of low malignant potential. In surgically excised masses, rates of malignancy are higher, vary from 4 to 13 percent, and probably reRect a greater preoperative concern for cancer (Hofman, 2007; Sherard, 2003).

1	Most ovarian masses are asymptomatic in pregnant women. Some cause pressure or chronic pain, and acute abdominal pain may be due to torsion, rupture, or hemorrhage. Seldom is blood loss signiicant enough to cause hypovolemia. Many ovarian masses are detected during routine prenatal sonography or during imaging done for other indications, including evaluation of symptoms. The typical sonographic appearance of these masses is shown in Figure 63-6. In some instances, MR imaging can be used to evaluate complicated anatomy.

1	Cancer antigen 125 (CA125) serves as a tumor marker, and levels are frequently elevated with ovarian malignancy. Importantly, concentrations of CA125 in early pregnancy and early puerperium are normally elevated, possibly from the decidua (slam , 2000; Spitzer, 1998). As shown in the Appendix (p. 1257), from the second trimester until term, levels are not normally higher than those in the nonpregnant woman (Szecsi, 2014). With severe preeclampsia, however, levels are abnormally elevated (Karaman, 2014). Other tumor markers that are not useul for diagnosis or posttreatment surveillance in pregnancy include human chorionic gonadotropin (hCG) , alpha-fetoprotein, inhibins A and B, and the multimarker OVAl test (Liu, 2011).

1	The two most common are torsion and hemorrhage. Torsion usually causes acute constant or episodic lower abdominal pain that frequently is accompanied by nausea and vomiting. Sonography often aids the diagnosis. With color Doppler, presence of an ovarian mass with absent Row strongly correlates with torsion. However, minimal or early twisting may compromise only venous Row, thus leaving the arterial supply intact. If torsion is suspected, laparoscopy or laparotomy is warranted. Contrary to prior teaching, adnexectomy is generally unnecessary to avoid clot release, thus, most recommend attempts at untwlstmg (McGovern, 1999; Zweizig, 1993). With a salvageable ovary, within minutes, congestion is relieved, and ovarian volume and cyanosis diminish. If cyanosis persists, however, then removal of the infarcted adnexum is typically indicated.

1	If the adnexum is healthy, there are options. First, neoplasms are resected. However, ovarian cystectomy in an ischemic, edematous ovary may be technically diicult, and adnexectomy may be necessary. Second, unilateral or bilateral oophoropexy has been described to minimize the risk of repeated torsion (Djavadian, 2004; Germain, 1996). Techniques described include shortening of the utero ovarian ligament or fixing the utero ovarian ligament to the posterior uterus, the lateral pelvic wall, or the round ligament (Fuchs, 2010; Weitzman, 2008).

1	he most common cause of ovarian hemorrhage follows rupture of a corpus luteum cyst. If the diagnosis is certain and symptoms abate, then observation and surveillance is usually suicient. Concern for ongoing bleeding will typically prompt surgical evaluation. If the corpus luteum is removed before 10 weeks' gestation, progestational support is recommended to maintain the pregnancy. Suitable regimens include: (1) micronized progesterone (Prometrium) 200 or 300 mg orally once daily; (2) 8-percent progesterone vaginal gel (Crinone), one premeasured applicator vaginally daily plus micronized progesterone 100 or 200 mg orally once daily; or (3) intramuscular 17 -hydroxyprogesterone caproate, 150 mg. The first two regimens are given until 10 completed weeks. For the last, if given between 8 and 10 weeks' gestation, only one injection is required immediately after surgery. If the corpus luteum is excised between 6 and 8 weeks' gestation, then two additional doses should be given 1 and 2 weeks

1	weeks' gestation, only one injection is required immediately after surgery. If the corpus luteum is excised between 6 and 8 weeks' gestation, then two additional doses should be given 1 and 2 weeks after the irst.

1	Because most of these are incidental findings, management considerations include whether resection is necessary and its timing. A cystic benign-appearing mass that is <5 cm often requires no additional antepartum surveillance. Early in pregnancy, this is likely a corpus luteum cyst, which typically resolves by the early second trimester. For cysts ::10 cm, because of the substan tial risk of malignancy, torsion, or labor obstruction, surgical removal is reasonable. Tumors between 5 and 10 cm should be carefully evaluated by sonography along with color Doppler and possibly MR imaging. If they have a simple cystic appear ance, these cysts can be managed expectantly with sonographic surveillance (Schmeler, 2005; Zan etta, 2003). Resection is done if cysts grow, begin to display malignant qualities, or become symptomatic. hose with classic indings of endometrioma or cesarean for obstetrical indications.

1	On the other hand, if sonographic characteristics suggest cancer-thick septa, nodules, papillary excrescences, or solid components-immediate resection is indicated (Caspi, 2000). In one review of 563 masses, approximately half were simple, and the other half complex (Webb, 2015). Among simple masses, 1 percent were malignant, and of complex masses, 9 percent were cancerous. Approximately 1 in 1000 pregnant women undergoes surgical exploration for an adnexal mass (Boulay, 1998). In general, we plan resection at 14 to 20 weeks' gestation because most masses that will regress will have done so by this time. As outlined in Chapter 46 (p. 901), laparoscopic removal is ideal (Naqvi, 2015; Sisodia, 2015). Importantly, in any instance in which cancer is strongly suspected, the American College of Obstetricians and Gynecologists (2017b) recommends consultation with a gynecologic oncologist.

1	Pregnancy Luteoma. One group of ovarian masses results directly from the stimulating efects of various pregnancy hormones on ovarian stroma. These include pregnancy luteoma, hyperreactio luteinalis, and ovarian hyperstimulation syndrome. Of these, pregnancy luteoma is a rare, benign ovarian neoplasm that arises from luteinized stromal cells and classically causes elevated testosterone levels (Hakim, 2016; Irving, 2011). Up to 25 percent of afected women will be virilized, and of these afected women, nearly half of their female fetuses will have some degree of virilization. However, most mothers and their fetuses are unafected because the placenta rapidly converts testosterone to estrogen (KanovG, 2011).

1	In typical cases, an adnexal mass along with maternal virilization will prompt sonography and measurement of testosterone and CA125 levels. Luteomas range in size from microscopic to >20 cm. hey appear as solid tumors, may be multiple or bilateral, and may be complex because of internal hemorrhage (Choi, 2000). Concerns for malignancy may be further investigated with MR imaging (Kao, 2005; Tannus, 2009). Total testosterone levels are increased, but notably, levels in normal pregnancy can be substantially elevated (Appendix, p. 1259). Diferential diagnoses include granulosa cell tumors, thecomas, Sertoli-Leydig cell tumors, Leydig cell tumors, stromal hyperthecosis, and hyperreactio luteinalis.

1	Generally, luteomas do not require surgical intervention unless there is torsion, rupture, or hemorrhage (Masarie, 2010). hese tumors spontaneously regress during the irst few months postpartum, and androgen levels drop precipitously during the irst 2 weeks following delivery (Wang, 2005). Lactation may be delayed a week or so by hyperandrogenemia (Dahl, 2008). Recurrence in subsequent pregnancies is rare. Hyperreactio Luteinalis. In this condition, one or both ovaries develop multiple, large theca-lutein cysts, typically after the first trimester. Cysts are caused by luteinization of the follicular theca interna layer, and most are in response to stimulation by excep tionally high hCG levels (Russell, 2009). For this reason, they are more common with gestational trophoblastic disease, twins, fetal hydrops, and other conditions with increased placental mass. Maternal virilization may develop, but fetal virilization has not been reported (KanovG, 2011; Malinowski, 2015).

1	s reported by Baxi and coworkers (2014), these ovarian tumors appear sonographically to have a "spoke wheel" pattern (Fig. 20-3, p. 391). If the diagnosis is confident, and unless com plicated by torsion or hemorrhage, surgical intervention is not required. hese masses resolve ater delivery. Few data allow pre diction of risk in a subsequent pregnancy, but in one case report, a woman had hyperreactio with three pregnancies (Bishop, 2016).

1	Ovarian Hyperstimulation Syndrome. This is typified by multiple ovarian follicular cysts accompanied by increased capillary permeability. It most often is a complication of ovulationinduction therapy for infertility, although it rarely may develop in an otherwise normal pregnancy. It has also been reported with a partial molar pregnancy (Suzuki, 2014). Its etiopathogenesis is thought to involve hCG stimulation of vascular endothelial growth factor (VEGF) expression in granulosa-lutein cells (Soares, 2008). This causes greater that can lead to ascites, pleural or pericardial efusion, hypovolemia with acute kidney injury, and hypercoagulability. Serious complications are renal dysfunction, adult respiratory distress syndrome, ovarian rupture with hemorrhage, and VTE. Unlike hyperreactio lutealis, virilization is absent (Suzuki, 2004).

1	Detailed guidelines for management are outlined by the American Society for Reproductive Medicine (2016). Treatment is primarily supportive with attention to maintaining vascular volume and thromboprophylaxis. In severe cases, paracentesis can be helpful. Malignancies of the ovary are the leading cause of death from genital-tract cancers in all women (American Cancer Society, 2017). Still, it is uncommon in young women, and the incidence of ovarian malignancy ranges from 1 in 20,000 to 1 in 50,000 births (Eibye, 2013; Palmer, 2009). Fortunately, 75 percent of these found in pregnancy are early-stage cancers that carry a 5-year survival rate between 70 and 90 percent (Brewer, 2011). The types of malignancy are also markedly diferent in pregnant women compared with those in older women. In gravidas, these are, in decreasing order of frequency, germ cell and sex cord-stromal tumors, low-malignant-potential tumors, and epithelial tumors (Morice, 2012).

1	Pregnancy apparently does not alter the prognosis of most ovarian malignancies. Management is similar to that for nonpregnant women, with the usual proviso that it may be modiied depending on gestational age. Thus, if frozen section histopathological analysis veriies malignancy, surgical staging is done with careful inspection of all accessible peritoneal and visceral surfaces (Giuntoli, 2006). Peritoneal washings are taken for cytology, biopsies are obtained from the diaphragmatic surface and peritoneum, omentectomy is completed, and pelvic and infrarenal paraaortic lymph nodes are sampled, if accessible.

1	If there is advanced disease, bilateral adnexectomy and omentectomy will decrease most tumor burden. In early pregnancy, hysterectomy and aggressive surgical debulking procedures may be elected. In other cases, minimal debulking as described in the previous paragraph is done and the operation terminated. In some cases of aggressive or large-volume disease, chemotherapy can be given during pregnancy while awaiting pulmonary maturation. Monitoring maternal CA125 serum levels during chemotherapy is not accurate in pregnancy (Aslam, 2000; Morice, 2012).

1	Paratubal and paroovarian cysts are either distended remnants of the paramesonephric ducts or are mesothelil inclusion cysts. Although most measure 53 cm, they occasionally attain worrisome dimensions. Their reported incidence is inluenced by size, but one autopsy series in nonpregnant women cited this to be 5 percent (Dorum, 2005). The most common parmesonephric cyst is the hydatid of Morgagni, which is pedunculated and typically dangles from one of the imbria. hese cysts infrequently cause complications and are most oten identiied at the time of cesarean delivery or puerperal sterilization. In these instances, they can simply be excised or drained by creating a large window in the cyst wll. Neoplastic paraovarian cysts are rare, sonographically and histologically resemble tumors of ovarian origin, and rarely are of borderline potential or frankly malignant (Korbin, 1998).

1	Preinvasive disease in young women-vulvar intraepithelial neoplasia (YIN) and vaginal intraepithelial neoplasia (VAIN)are seen more often than invasive disease and are commonly associated with HPV infection. As with cervical neoplasia, these premalignant conditions are treated after delivery. Cancer of the vulva or vagina is generally a malignancy of older women, and thus, these are rarely associated with pregnancy. Even so, any suspicious lesions should be biopsied. Treatment is individualized according to the clinical stage and depth of invasion. In a review of 23 cases, investigators concluded that radical surgery for stage I disease was feasible during pregnancy-including in the last trimester (Heller, 2000).

1	We and others question the necessity of resection in late pregnany because deinitive therapy can oten be delayed due to these cancers' typically slow progression (Anderson, 2001). It appears that vaginal delivery is not contraindicated if vulvar and inguinal incisions are well healed. Vulvar sarcoma, vulvar melanoma, and vaginal malignancies are rare in pregnancy and are the subjects of case reports (lexander, 2004; Kuller, 1990; Matsuo, 2009).

1	Breast cancer rates rise most acutely between ages 40 and 80 years. However, because of its overall high frequency, breast cancer is relatively common even in younger women and is the most frequent cancer found in gravidas. From the Nationwide Inpatient Sample of 11.8 million births, the incidence approximated 1 in 15,000 (Maor, 2017). And, as more women choose to delay childbearing, the frequency of associated breast cancer is certain to grow. Postponed childbearing was considered partially responsible for the increase in pregnancy-associated breast cancer in Sweden and Denmark (Andersson, 2015; Eibye, 2013).

1	Some studies suggest that women with a family history of breast cancer-especially those with BRCAl and BRC2 breast cancer gene mutations-are more likely to develop breast malignancy during pregnancy (Wohlfahrt, 2002). However, it may be that it is parity that modiies this risk. Namely, parous women older than 40 years with these mutations have a signiicantly lower cancer risk than nulliparas with these mutated genes (Andrieu, 2006; Antoniou, 2006). Women with BRAl and BRC2 gene mutations who undergo induced abortion or those who breastfeed do not have an increased breast cancer risk (Friedman, 2006). Moreover, Jernstrom and associates (2004) found that breastfeeding conveyed a protective efect against this cancer in those with BRCAl gene mutation, but not in those with BRCA2 mutations. Of other congenital factors, it is controversial whether DES exposure raises breast cancer risks (Hoover, 2011; Titus-Ernstof, 2006).

1	More than 90 percent of gravidas with breast cancer have a palpable mass, and greater than 80 percent of cases are selfreported (Brewer, 2011). In pregnancy, clinical assessment, diagnostic procedures, and treatment of women with breast tumors are often slightly delayed (Berry, 1999). his can be partially attributed to pregnancy-induced breast tissue that obscures masses.

1	The evaluation of pregnant women with a breast mass does not difer from that for nonpregnant women (Loibl, 2015). hus, any suspicious breast mass should be pursued to diagnosis. Pragmatically, a palpable discrete mass can be biopsied or excised. If imaging is desirable to distinguish between a solid mass and a cystic lesion, sonography has high sensitivity and specificity (Navrozoglou, 2008). Mammography is appropriate if indicated, and the fetal radiation risk is negligible-0.04 mGywith appropriate shielding (rishna, 2013). But, because breast tissue is denser in pregnancy, mammography has a false-negative rate of 35 to 40 percent (Woo, 2003). If the decision to biopsy is uncertain, then MR imaging may be used. With such techniques, masses can usually be described as either solid or cystic.

1	Cystic breast lesions are simple, complicated, or complex (Berg, 2003). Simple cysts do not require special management or monitoring, but they may be aspirated if symptomatic. Complicated cysts show internal echoes during sonography, and they sometimes are indistinguishable from solid masses. These are typically aspirated, and if the sonographic abnormality does not resolve completely, a core-needle biopsy is usually performed. Complex cysts have septa or intracystic masses seen sonographically. Because some breast cancer may form complex cysts, excision is usually recommended.

1	For solid breast masses, evaluation is with the triple test, that is, clinical examination, imaging, and core needle biopsy. If all three suggest a benign lesion or if all three suggest a breast cancer, the test is said to be concordant. A concordant benign triple test is �99-percent accurate, and breast lumps in this category can be followed by clinical examination alone. Fortunately, most masses in pregnancy have these three reassuring features. In contrast, if any of the three assessments suggests malignancy, the mass should be excised. Once breast cancer is diagnosed, a limited search of the most common metastatic sites is completed. For most women, this includes a chest radiograph, liver sonography, and skeletal MR imaging (Becker, 2016; Krishna, 2013).

1	Treatment of breast cancer is multidisciplinary and includes an obstetrician, breast surgeon, and medical oncologist. Ini tially, desires for pregnancy continuation are addressed, and data indicate that pregnancy interruption does not influence the course or prognosis of breast cancer (Cardonick, 2010). With pregnancy continuation, treatment in general mirrors that for nonpregnant women. Important caveats are that chemotherapy and surgery are postponed to the second trimester, and adjuvant radiotherapy is withheld until after delivery (Brewer, 2011).

1	Surgical treatment may be definitive. In the absence of metastatic disease, either a wide excision or a modified or total mastectomy-each with axillary node staging-can be performed (Rosenkranz, 2006). Staging by sentinel lymph node biopsy and lymphoscintigraphy with technetium-99m is safe. Breast reconstruction, if desired, is typically delayed until after delivery (Viswanathan, 2011). hat said, Caragacianu and coworkers (2016) described good results in 10 pregnant women who underwent immediate reconstruction after mastectomy.

1	Chemotherapy is usually given with both positive-and negative-node breast cancers. In premenopausal women, survival rates with this approach are improved, even if lymph nodes are cancer free. For node-positive disease, multiagent chemotherapy is begun if delivery is not anticipated within several weeks. Cyclophosphamide, doxorubicin, and cisplatin are currently used (Euhus, 2016). If an anthracycline-based agent such as doxorubicin is used, pretherapy maternal echocardiography is performed because of associated cardiotoxicity (Brewer, 2011). Good maternal and perinatal results have been reported (Berry, 1999; Hahn, 2006). Immunotherapy for breast cancers is now commonplace.

1	Immunotherapy for breast cancers is now commonplace. T rastuzumab (Herceptin) is a monoclonal antibody to the HER2/neu receptor, which is found in approximately a third of invasive breast cancers (Hudis, 2007). The drug is not recommended in pregnancy. This is because HER21 neu is strongly expressed in fetal renal epithelium, and trastuzumab has been linked with miscarriage, fetal renal failure and related oligohydramnios, and preterm birth (Amant, 2010; Azim, 2010). he efects of pregnancy on the course of breast cancer and its prognosis are complex. Breast cancer is more aggressive in younger women, but whether it is more aggressive during pregnancy in these same women is debatable (Azim, 2014). Clinically, most studies indicate little diference in overall survival rates with pregnancy-associated breast cancer compared with similarly aged and staged nonpregnant women (Beadle, 2009).

1	Other reports note worse overall survival rates with pregnancy associated breast cancer (Rodriguez, 2008). hese investigators do conclude, however, that later disease stages are more preva lent in pregnant women. Indeed, breast cancer is usually found at a more advanced stage in pregnant women, and thus overall prognosis is dimin ished (Andersson, 2015). The aggregate of studies published after 1990 indicate that up to 60 percent of pregnant women have concomitant axillary node involvement at diagnosis. And although, stage for stage, the 5-year survival rate is compara ble in pregnant and nonpregnant women, the more advanced stages that are typical of pregnant women worsen their progno sis (Kuo, 2017; Zemlickis, 1992).

1	After breast cancer treatment, chemotherapy will render some women infertile, and options for childbearing are limited (Kim, 2011). For those who become pregnant, long-term maternal survival rates are not adversely afected (Averette, 1999; Velentgas, 1999). One metaanalysis of 10 studies found that for women with early breast cancer, pregnancy that occurs 10 months after diagnosis may, in fact, confer a survival benefit (Valachis, 2010). Data do not indicate that breastfeeding adversely the course.

1	In women successfully treated for breast cancer, recurrence is a concern. Because recurrences are more common soon after treatment, it seems reasonable to delay conception for 2 to 3 years. Hormonal contraceptive methods are contraindicated, and a copper-containing intrauterine device is an excellent long-acting reversible method for many. That said, women who conceive do not appear to have diminished survival rates (Ives, 2006). Notably, women treated with tamoxifen are at risk for several months after its discontinuation to have a newborn with congenital anomalies. This drug has an extremely long half-life, and thus delaying conception is recommended for at least 2 months after tamoxifen completion (Braems, 2011).

1	Palpable thyroid nodules are detected in 4 to 7 percent of the population, and approximately 10 percent are malignant (Burman, 2015). Clinical nodules are typically evaluated with sonography and measurement of serum thyroid-stimulating hormone (TSH) and free thyroxine levels. Fine-needle aspiration is indicated for a suspicious nodule (lexander, 2017; Gharib, 2016). With a diagnosis of thyroid malignancy, pregnancy termination is not necessary. Primary therapy is thyroidectomy performed ideally during the second trimester. Postoperatively, replacement thyroxine is given. Most thyroid cancers are well diferentiated and follow an indolent course. Thus, delayed surgical treatment does not usually alter outcome (Yazbeck, 2012; Yu, 2016).

1	In some types of thyroid cancer, radioiodine is used for primary or postoperative treatment. This is contraindicated in both pregnancy and lactation for several reasons. First, transplacental 1311 is avidly trapped by the fetal thyroid gland to cause hypothyroidism. Second, during lactation, the breast also concentrates a substantial amount of iodide. This may pose neonatal risk due to radioiodine-contaminated milk ingestion and maternal risk from significant breast irradiation. To limit maternal exposure, a delay of 3 months between lactation and thyroid ablation will more reliably ensure complete breast involution (Sisson, 2011). In women with thyroid cancer who ultimately receive 131 I doses, pregnancy should be avoided for 6 months to 1 year. This time ensures thyroid function stability and permits confirmation of cancer remission (Abalovich, 2007).

1	This lymphoma is probably B-cell derived and is cytologically distinguished from other lymphomas by Reed-Sternberg cells. Of cancers in pregnancy, lymphomas are common, and gestational rates are rising because of delayed childbearing (Horowitz, 2016). In pregnant women, Hodgkin lymphomas are more frequent than non-Hodgkin lymphomas. In one populationbased review of 7.9 million births from the Nationwide Inpatient Sample, El-Messidi and colleagues (2015) reported its incidence to be 1 in 12,400. In more than 70 percent of Hodgkin disease cases, lymph nodes painlessly enlarge at sites above the diaphragm, that is, in the axillary, cervical, or submandibular chains. Approximately one third of patients have symptoms such as fever, night sweats, malaise, weight loss, and pruritus. Diagnosis is by histological examination of involved nodes (Longo, 2015).

1	The Ann Arbor staging system, shown in Table 63-2, is applied to Hodgkin and other lymphomas. For staging, pregnancy limits the use of some radiographic studies, but at minimum, chest radiography, abdominal imaging with sonography or MR imaging, and bone marrow biopsy are completed (Williams, 2001). MR imaging is excellent for evaluating thoracic and abdominal paraaortic lymph nodes (Brenner, 2012). Staging laparotomy is seldom done today (Longo, 2015). The current trend for nonpregnant individuals is to administer chemotherapy for all stages of Hodgkin disease. In pregnancy, for early-stage disease in the first trimester, options include observation until after 12 weeks' gestation, single-agent vinblastine until the second trimester, pregnancy termination followed by multiagent chemotherapy, or radiotherapy alone for isolated neck or axillary sites (EI-Hemaidi, 2012; Eyre, 2015).

1	For advanced-stage disease, chemotherapy is recommended regardless of gestational age. Before 20 weeks, therapeutic abortion is a consideration, but if termination is unacceptable, then treatment with vinblastine followed by multi agent therapy in the second trimester can be used (Eyre, 2015). For most advancedstage disease after the irst trimester, cycles of doxorubicin, bleomycin, vinblastine, and dacarbazine are given, and radiotherapy can be added postpartum (Cohen, 2011). In general, postponement of therapy until fetal maturity is achieved seems justiiable only when the diagnosis is made late in pregnancy. Women with Hodgkin lymphoma have a higher incidence of VTE (El-Messidi, 2015; Horowitz, 2016). lso, in our experiences, pregnant women with Hodgkin disease-even after they arer" cured" -are inordinately susceptible to infection and sepsis. Active antineoplastic therapy only increases this vulnerability.

1	The overall prognosis with Hodgkin lymphoma is good, and survival rates exceed 70 percent. Pregnancy does not adversely afect the cancer course or pregnancy outcomes in women with this lymphoma. Speciically, neither chemotherapy after the first trimester nor mediastinal and neck irradiation has adverse fetal efects (Brenner, 2012; El-v1essidi, 2015; Pinnix, 2016). For women with disease in remission, pregnancy does not stimulate a relapse (Weibull, 2016).

1	lthough usually B-cell tumors, non-Hodgkin lymphomas can also be T-cell or natural-killer-cell neoplasms. heir biology, classification, and treatment are complex (Longo, 2015; O'Gara, 2009). hey are associated with viral infections, and indeed, their incidence has risen sharply at least pardy because 5 to 10 percent of HIV-infected persons develop lymphoma. Other associated viruses include Epstein-Barr virus, hepatitis C virus, and human herpes virus 8. Some of these lymphomas are aggressive, and survival rates vary with the type of cell line involved (Longo, 2015).

1	Non-Hodgkin lymphomas are infrequent during pregnancy (Brenner, 2012; Pinnix, 2016). They are also staged according to the Ann Arbor system. If diagnosed in the irst trimester, pregnancy termination followed by multiagent chemotherapy is recommended for all but indolent or very early disease. These less aggressive forms may either be observed or be temporized with focal supradiaphragmatic radiotherapy and then full treatment in the second trimester. If one of these lymphomas is diagnosed after the irst trimester, chemotherapy and immunotherapy TABLE 63-2. Ann Arbor Staging System for Hodgkin and Other Lymphomas I Involvement in a single lymph node region or lymphoid site-e.g., spleen or thymus II Involvement of two or more lymph node groups on the same side of the diaphragm-the mediastinum is a single site III Involvement of lymph nodes on both sides of the diaphragm 1. Limited to spleen or splenic hilar, celiac, or portal nodes 2.

1	III Involvement of lymph nodes on both sides of the diaphragm 1. Limited to spleen or splenic hilar, celiac, or portal nodes 2. Includes paraaortic, iliac, or nodes plus those in III IV Extralymphatic involvement-e.g., liver or bone marrow Substage A = no symptoms; substage B = fever, sweats, or weight loss; substage E = extralymphatic involvement excluding liver and bone marrow. with rituximab are given (Cohen, 2011; Rizack, 2009). In one follow-up of 55 individuals at 6 to 29 years after exposure to chemotherapy in utero during maternal lymphoma treatment, no congenital, neurological, or psychological abnormalities were noted (Aviles, 2001). Burkitt lymphoma is an aggressive B-cell tumor associ ated with Epstein-Barr virus infection. Prognosis is poor, and treatment is given with multiagent chemotherapy. In a review of 19 women whose pregnancies were complicated by this lymphoma, 17 died within a year of diagnosis (Barnes, 1998).

1	In general, these malignancies arise either from lymphoid tissues-lymphoblastic or lymphocytic leukemias, or from bone marrow-myeloid leukemias. hey can be acute or chronic. Although adult leukemias are more prevalent after age 40, they still are among the most common malignancies of young women (see Fig. 63-1). Leukemia was diagnosed in 1 in 40,000 pregnancies reported to the California Cancer Registry (Smith, 2003). In a review of 72 pregnancies complicated by leukemia from 1975 until 1988, 44 had acute myelogenous leukemia; 20 had acute lymphocytic leukemia; and eight had one of the chronic leukemias (Caligiuri, 1989). Acute leukemias almost always cause marked peripheral blood count abnormalities, and often the white blood cell count is elevated with readily recognizable circulating blast cells. The diagnosis is made from bone marrow biopsy.

1	With current multiagent chemotherapy, remission during pregnancy is common, compared with an almost 100-percent mortality rate before 1970. Pregnancy termination does not further improve the prognosis, however, abortion is a consideration in early pregnancy to avoid potential chemotherapy teratogenesis. One example of the latter is treatment of acute promyelocytic leukemia with all-trans-retinoic acid, also known as tretinoin (Carradice, 2002; Sanz, 2015). This potent teratogen causes retinoic acid syndrome (Chap. 12, p. 245). In another example, acute myeloid leukemia is treated with tyrosine kinase inhibitors, another teratogen group (Palani, 2015). In other cases, pregnancy termination before viability may simpliY management of an acutely ill woman.

1	Other than these caveats, treatment of gravidas with leukemia is similar to that for nonpregnant women. Acute myeloid leukemia is treated without delay (Ali, 2015). After induction chemotherapy, postremission maintenance therapy is mandatory to prevent a relapse, which is then usually treated with stem-cell transplantation. If allogeneic stem-cell transplantation is indicated, early delivery is considered. With some chronic leukemias, it may be possible to delay therapy until after delivery (Fey, 2008). As with lymphoma, infection and hemorrhage are signiicant complications that should be anticipated in women with active disease.

1	Most descriptions of leukemia treatment in pregnancy are single cases or small series (Routledge, 2016; Sanz, 2015). In an earlier review of 58 cases, 75 percent were diagnosed after the irst trimester (Reynoso, 1987). Half were acute myelogenous leukemia, which had a remission rate of 75 percent with chemotherapy. Only 40 percent of these pregnancies resulted in liveborn neonates (Caligiuri, 1989). and its pigment-producing melanocytes. Melanomas should be suspected in pigmented lesions that show changes in contour, surface elevation, discoloration, bleeding, or ulceration, which should prompt biopsy (Richtig, 2017). They are most com in women of childbearing age.

1	In some population studies, melanoma is the most frequent malignancy complicating pregnancy (Andersson, 2015; Ban nister-Tyrrell, 2015). Still, the reported incidence ranges widely from 0.03 to 2.8 per 1000 live births (Eibye, 2013; Smith, 2003). One explanation is that many are treated as outpatients and thus are not entered into tumor registries. As noted earlier (p. 1192), malignant melanoma is one of the tumors that is known to metastasize to the placenta and fetus. Placental evaluation for metastases should be performed after delivery. Staging is clinical. Stage I is a melanoma with no palpable lymph nodes; in stage II, lymph nodes are palpable; and in stage III, there are distant metastases. For patients with stage

1	Staging is clinical. Stage I is a melanoma with no palpable lymph nodes; in stage II, lymph nodes are palpable; and in stage III, there are distant metastases. For patients with stage I, tumor thickness is the single most important predictor of survival. he Clark classiication includes ive levels of involve ment by depth into the epidermis, dermis, and subcutaneous fat. he Breslow scale measures tumor thickness and size, in addition to depth of invasion.

1	Primary surgical treatment for melanoma is determined by the stage and includes wide local resection, sometimes with extensive regional lymph node dissection. Schwartz and associates (2003) recommend sentinel lymph node mapping and biopsy using 99mT c-sulfur colloid, which has a calculated fetal dose of 0.014 mSv or 0.014 mGy. Routine regional node dissection reportedly improves survival rates in nonpregnant patients with microscopic metastases (Cascinelli, 1998). For pregnant patients, an algorithm has been proposed that begins with resection of the primary tumor under local anesthesia but postpones sentinel lymph node biopsy until after delivery (Broer, 2012). lthough prophylactic chemotherapy or immunotherapy is usually avoided during pregnancy, it may be given if indicated by tumor stage and maternal prognosis. In most cases of distant metastatic melanoma, treatment is at best palliative. Currently, the role of estrogen receptor-� in melanoma progression is under investigation,

1	stage and maternal prognosis. In most cases of distant metastatic melanoma, treatment is at best palliative. Currently, the role of estrogen receptor-� in melanoma progression is under investigation, and it may be a target for future therapeutic intervention (de Giorgi, 2011).

1	Stage-for-stage, survival is equivalent between pregnant and nonpregnant women (Driscoll, 2016; Johansson, 2014). In one study, half of pregnant women had stage III or IV lesions (de Haan, 2017). Therapeutic abortion does not improve maternal survival rates. Clinical stage is the strongest determinant of survival, and women with deep cutaneous invasion or regional node involvement have the worst prognosis. Approximately 60 percent of recurrences will manifest within 2 years, and 90 percent by 5 years. Thus, most recommend that pregnancy be avoided for 3 to 5 years after surgical resection. Interim contraception can include combination oral contraceptives, as they do not appear to have adverse cancer efects (Gandini, 2011). Related, subsequent pregnancies in women with localized melanoma do not lower cancer survival rates (Driscoll, 2009).

1	Carcinomas of the colon and rectum are the third most frequent in women of all age groups in the United States (American Cancer Society, 2016). heir incidence in pregnancy is rising because of delayed childbearing (Rogers, 2016) . Even so, colorectal tumors are uncommon before age 40. Smith and colleagues (2003) reported an approximate incidence of 1 per 150,000 deliveries in the California Cancer Registry. he incidence approximated 1 per 35,000 births in a Danish Registry (Eibye, 2013). Most-80 percent-of colorectal carcinomas in pregnant women arise from the rectum. In one review, only 41 cases in pregnancy were colon cancers above the peritoneal reflection (Chan, 1999).

1	he most frequent symptoms of colorectal cancer are abdominal pain, distention, nausea, constipation, and rectal bleeding. If symptoms of colon disease persist, digital rectal examination, stool tests for occult blood, and lexible sigmoidoscopy or colonoscopy are done. Some malignancies of the gastrointestinal tract are discovered because of metastases to the ovary. Kruckenberg tumors are tumor-laden ovaries from another primary, often gastrointestinal, and have a bleak prognosis (GliSic, 2006; Kodama, 2016).

1	Treatment of colon cancer in pregnant women mirrors the same general guidelines as for nonpregnant women. Without evidence of metastatic disease, surgical resection is preferred, but most gravidas have advanced lesions (AI-Ibrahim, 2014). During the first half of pregnancy, hysterectomy is not necessary to perform colon or rectal resection, and thus, therapeutic abortion is not mandated. During later pregnancy, therapy may be delayed until fetal maturation, however, bowel hemorrhage, obstruction, or perforation may force surgical intervention (Minter, 2005).

1	Gastric cancer is rarely associated with pregnancy, and most reported cases are from Japan. Hirabayashi and coworkers (1987) reviewed outcomes in 60 pregnant women during a 70-year period from 1916 to 1985. Delayed diagnosis during pregnancy is common, and the prognosis is consistently poor (Lee, 2009). Esophageal cancer has similar symptoms, but it is rare (Sahin, 2015). It is axiomatic that persistent unexplained upper gastrointestinal symptoms should be evaluated by endoscopy.

1	Various other neoplasms have been reported in pregnancy and are usually the subject of case reports. Examples include carcinoid tumors, which are usually of gastrointestinal origin (Durkin, 1983). Both pancreatic and hepatocellular cancer are rare during pregnancy (Kakoza, 2009; Marinoni, 2006; Papoutsis, 2012; Perera, 2011). Another report described a massive intrahepatic cholangiocarcinoma masquerading as HELLP syndrome (Bladerston, 1998). Except for thyroid cancer, malignant tumors of the head and neck are rare (Cheng, 2015). Lung cancer is also uncommon (Boussios, 2013). Central nervous system neoplasms had a reported frequency of 1 in 10,000 to 28,000 births (Eibye, 2013; Smith, 2003). Bladder and urachal duct carcinoma are rarely coincident with pregnancy (McNally, 2013; Yeaton-Massey, 2013). Last, bone tumors have been described (Kathiresan, 2011).

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1	MATERNAL AND FETAL.IMMUNOLOGY .......i.... 1209 VIRAL INFECTIONS ...i.....i............i...... 1210 BACTERIAL INFECTIONS .............i.......... 1220 PROTOZOAL INFECTIONS .i.i....i.............. 1225 BIOTERRORISM . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 1228 According to many authorities, inluena exerts a very pernicious influence upon pregnancy. t would appear that the fects of inluena must vary with the severiy of the epidemic, and more particuary with the frequeny of pneumonic com plications. As a rule, any septic condition oers a worse prognosis in prenany. Several instances have been reported of transmission of the oending bacteria to the oetus. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) Infections have historically been a major cause of maternal and fetal morbidity and mortality worldwide, and they remain so in the 21st century. he unique maternal-fetal vascular connection in some cases serves to protect the fetus from infectious agents, whereas in other instances it provides a conduit for their transmission to the fetus. Maternal serological status, gestational age at the time infection is acquired, the mode of acquisition, and the immunological status of both the mother and her fetus all inluence disease outcome.

1	Even after intensive study, many of the maternal immunological adaptations to pregnancy are not well elucidated. It is known that pregnancy is associated with an increase in the CD4+ T cells that secrete h2-type cytokines-for example interleukins (Fragiadakis, 2016). Th1-type cytokine production-for example, interferon gamma and interleukin 2-appears to be somewhat suppressed, leading to a h2 bias in pregnancy. This bias afects the ability to rapidly eliminate certain intracellular pathogens during pregnancy, although the clinical implications of this suppression are unknown (Kourtis, 2014; Svensson-Arvelund, 2014). Importantly, the h2 humoral immune response remains intact. It also appears that human leukocyte antigen (HA)-C expressed by extravillous trophoblasts elicits responses from decidual natural killer (dNK) and decidual CD8+ T cells (Crespo, 2017).

1	In describing infections, horizontal transmission is the spread of an infectious agent from one individual to another. Vertical transmission refers to passage from the mother to her fetus of an infectious agent through the placenta, during labor or delivery, or by breastfeeding. Thus, preterm rupture of membranes, prolonged labor, and obstetrical manipulations may enhance the risk of neonatal infection (Centers for Disease Control and Prevention, 2010). Table 64-1 details speciic infections by mode and timing of acquisition. A inal term, the seconday attack rate, is the probability that infection develops in a susceptible individual following known contact with an infectious person.

1	The active immunological capacity of the fetus and neonate is compromised compared with that of older children and adults. That said, fetal cell-mediated and humoral immunity begin to develop by 9 to 15 weeks' gestation (Warner, 2010). The primary fetal response to infection is immunoglobulin M (Ig.1). Passive immunity is provided by IgG transferred across the placenta. By 16 weeks, this transfer begins to rise rapidly, and by 26 weeks, fetal concentrations are equivalent to those of the mother. After birth, breastfeeding is protective against some infections, although this protection begins to decline at TABLE 4-1 . Specific Causes of Some Fetal and Neonatal Infections Viruses: varicella-zoster, coxsackie, human parvovirus B19, rubella, CMV, HIV, Zika Bacteria: Listeria, syphilis, Borrelia Protozoa: toxoplasmosis, malaria group B streptococcus, coliforms Viruses: HIV

1	Bacteria: Listeria, syphilis, Borrelia Protozoa: toxoplasmosis, malaria group B streptococcus, coliforms Viruses: HIV Bacteria: gonorrhea, chlamydia, group B streptococcus, tuberculosis, mycoplasmas Viruses: HSV, HPV, HIV, hepatitis B, hepatitis C, Zika External contamination Bacteria: staphylococcus, coliforms Viruses: HSV, varicella zoster Human transmission: staphylococcus, HSV Respirators and catheters: staphylococcus, coliforms CMV = cytomegalovirus; HIV = human immunodeficiency virus; HPV = human papillomavirus; HSV = herpes simplex virus. 2 months of age. Current World Health Organization (2013) recommendations are to exclusively breastfeed for the first 6 months of life with partial breastfeeding until 2 years of age.

1	2 months of age. Current World Health Organization (2013) recommendations are to exclusively breastfeed for the first 6 months of life with partial breastfeeding until 2 years of age. Neonatal infection, especially in its early stages, may be dificult to diagnose because these newborns often fail to express classic clinical signs. If the fetus was infected in utero, there may be depression and acidosis at birth for no apparent reason. The neonate may suck poorly, vomit, or show abdominal distention. Respiratory insuiciency can develop, which may present similarly to idiopathic respiratory distress syndrome. he neonate may be lethargic or jittery. he response to sepsis may be hypothermia rather than hyperthermia, and the total leukocyte and neutrophil counts may be depressed.

1	Several viruses cause severe maternal infections, and some can also cause devastating fetal infections. Of these, cytomegalovirus (CMV) is a ubiquitous DNA herpes virus that eventually infects most humans. CMV is also the most common perinatal infection in the developed world. Speciically, some evidence of fetal infection is found in 0.2 to 2.2 percent of all neonates (American College of Obstetricians and Gynecologists, 2017). The virus is secreted into all body luids, and person-to-person contact with viral-laden saliva, semen, urine, blood, and nasopharyngeal and cervical secretions can transmit infection. The fetus may become infected by transplacental viremia, or the neonate is infected at delivery or during breastfeeding. vloreover, acquisition continues to accrue. Day-care centers, for example, are a frequent source. Revello and coworkers (2008) reported that amniocentesis in women whose blood is positive for CMV DNA does not result in iatrogenic fetal transmission.

1	Up to 85 percent of women from lower socioeconomic backgrounds are seropositive by the time of pregnancy, whereas only half of women in higher income groups are immune. Following primary CMV infection, and in a manner similar to other herpesvirus infections, the virus becomes latent with periodic reactivation characterized by viral shedding. This occurs despite high serum levels of anti-CMV IgG antibody. These antibodies do not prevent maternal recurrence, reactivation, or reinfection, nor do they totally mitigate fetal or neonatal infection.

1	Women who are seronegative before pregnancy, but who develop primary CMV infection during pregnancy, are at greatest risk to have an infected fetus. It is estimated that 25 percent of congenital CMV infections in the United States are from primary maternal infection (Wang, 2011). Most CMV infections are clinically silent, but they can be detected by seroconversion, and this may be as high as 1 to 7 percent annually (Hyde, 2010). Conversely, diagnosis of CMV nonprimary infection is a challenge (Picone, 2017).

1	Pregnancy does not increase the risk or severity of maternal CMV infection. Most infections are asymptomatic, but 10 to 15 percent of infected adults have a mononucleosis-like syndrome characterized by fever, pharyngitis, lymphadenopathy, and polyarthritis. Immunocompromised women may develop myocarditis, pneumonitis, hepatitis, retinitis, gastroenteritis, or meningoencephalitis. Nigro and associates (2003) reported that most women in a cohort with primary infection had elevated serum aminotransferases or lymphocytosis. Reactivation disease usually is asymptomatic, although viral shedding is common.

1	Transmission rates for primary infection are 30 to 36 percent in the first trimester, 34 to 40 percent in the second, and 40 to 72 percent in the third trimester (American College of Obstetricians and Gynecologists, 2017; Picone, 2017). In contrast, recurrent maternal infection infects the fetus in only 0.15 to 1 percent of cases. Naturally acquired immunity during pregnancy results in a 70-percent risk reduction of congenital CMV infection in future pregnancies (Fowler, 2003; LeruezVille, 2017). However, as noted earlier, maternal immunity does not prevent recurrences, and maternal antibodies do not prevent fetal infection (Ross, 2011) .

1	Newborns with apparent sequelae of in-utero-acquired CMV infection are described as having symptomatic CMV inection. Congenital infection is a syndrome that may include growth restriction, microcephaly, intracranial calcifications, chorioretinitis, mental and motor retardation, sensorineural deficits, hepatosplenomegaly, jaundice, hemolytic anemia, and thrombocytopenic purpura (Cheeran, 2009). An example of periventricular calcifications is shown in Figure 64-1. Of the estimated 40,000 infected neonates born each year, only 5 to 10 percent demonstrate this syndrome (Fowler, 1992). hus, most FIGURE 64-1 Coronal view of cranial sonogram from a neonate with congenital cytomegalovirus infection showing multiple periventricular calcifications.

1	FIGURE 64-1 Coronal view of cranial sonogram from a neonate with congenital cytomegalovirus infection showing multiple periventricular calcifications. infected infants are asymptomatic at birth, but some develop late-onset sequelae. Complications may include hearing loss, neurological deicits, chorioretinitis, psychomotor retardation, and learning disabilities. Infections in dichorionic twins most 1ikely are nonconcordant (Egana-U grinovic, 2016).

1	Routine prenatal CMV serological screening is currently not recommended by the Society for Maternal-Fetal Medicine (2016). An algorithm for management is shown in Figure 64-2. Pregnant women should be tested for CMV if they present with a mononucleosis-like illness or if congenital infection is suspected based on abnormal sonographic indings. Primary infection is diagnosed using CMV-speciic IgG testing of paired acute and convalescent sera. CMV IgM does not accurately relect timing of seroconversion because IgM antibody levels may be elevated for more than a year (Stagno, 1985). Moreover, CMV IgM may be found with reactivation disease or reinfection with a new strain. Thus, specific CMV IgG avidity testing is valuable Abnormal Maternal CMV Screening CMV IgG: positive CMV IgM: positive CMV-specific IgG and IgM by EIA, IgG avidity by EIA, and CMV-specific IgM by immunoblot CMV IgG: negative CMV IgM: negative CMV IgG: positive IgG avidity index: high

1	CMV-specific IgG and IgM by EIA, IgG avidity by EIA, and CMV-specific IgM by immunoblot CMV IgG: negative CMV IgM: negative CMV IgG: positive IgG avidity index: high CMV IgM: negative in conirming primary CMV infection. High anti-CMV IgG avidity indicates primary maternal infection >6 months before testing (Kanengisser-Pines, 2009). Finally, viral culture may be useful, although a minimum of 21 days is required before findings are considered negative.

1	Several fetal abnormalities associated with CMV infection may be seen with sonography, computed tomography, or magnetic resonance imaging. In some cases, they are found at the time of routine prenatal sonographic screening, but in others they are part of a specific evaluation in women with CMV infection. Findings include microcephaly, ventriculomegaly, and cerebral calciications; ascites, hepatomegaly, splenomegaly, and hyperechoic bowel; hydrops; and oligohydramnios (Society for Maternal-Fetal Medicine, 2016). Abnormal sonographic findings seen in combination with positive findings in fetal blood or amnionic fluid are predictive of an approximate 75-percent risk of symptomatic congenital infection (Enders, 2001).

1	CMV nucleic acid amplification testing (NAA T) of amnionic luid is considered the gold standard for the diagnosis of fetal infection. Sensitivities range from 70 to 99 percent and depend on amniocentesis timing. Sensitivity is highest when amniocentesis is performed at least 6 weeks after maternal infection and after 21 weeks' gestation (Azam, 2001; Guerra, 2000). A negative result from amnionic luid polymerase chain reaction (PCR) testing does not exclude fetal infection and may need to be repeated if suspicion for fetal infection is high.

1	The management of the immunocompetent pregnant woman with primary or recurrent CMV is limited to symptomatic treatment. If recent primary CMV infection is conirmed, amnionic fluid analysis should be ofered. Counseling regarding fetal outcome depends on the gestational age during which primary infection is documented. Despite the high infection rate with primary infection in the first half of pregnancy, most fetuses develop normally. However, pregnancy termination may be an option for some.

1	Currently, no proven treatments are available for CMV infection (Society for Maternal-Fetal Medicine, 2016). Leruez-Ville and associates (2016) recently reported that oral treatment with valacyclovir, 8 g daily, apparently mitigated adverse outcomes in eight of 11 afected fetuses treated beginning at median of 25.9 weeks' gestation. Kimberlin and colleagues (2015) previously showed that intravenous valganciclovir administered for 6 weeks to neonates with symptomatic central nevous system (CNS) disease prevented hearing deterioration at 6 months and possibly later. Passive immunization with CMV-specific hyperimmune globulin may lower the risk of congenital CMV infection when given to pregnant women with primary disease (Nigro, 2005, 2012; Visentin, 2012). he Maternal-Fetal Medicine Units Network currently is conducting a randomized trial designed to address this.

1	here is no CMV vaccine, although several clinical trials are underway (Arvin, 2004; Schleiss, 2016). Prevention of congenital infection relies on avoiding maternal primary infection, especially in early pregnancy. Basic measures such as good hygiene and hand washing have been promoted, particularly for women with toddlers in day-care settings (Fowler, 2000). CMV may be sexually transmitted among infected partners, but no data address the eicacy of preventive strategies.

1	CMV may be sexually transmitted among infected partners, but no data address the eicacy of preventive strategies. Varicella-zoster virus (VZV) is a double-stranded DNA herpesvirus acquired predominately during childhood, and 90 percent of adults have serological evidence of immunity (Whitley, 2015). The incidence of adult varicella declined by 82 percent after the introduction of varicella vaccination, and this has resulted in a drop in maternal and fetal varicella rates (American College of Obstetricians and Gynecologists, 2017). In the United States between 2003 and 2010, the incidence of maternal varicella among 7.7 million pregnancy admissions was 1.21 per 10,000 (Zhang, 2015).

1	Primary infection-varicela or chickenpox-is transmitted by direct contact with an infected individual, although respiratory transmission has been reported. The incubation period is 10 to 21 days, and a nonimmune woman has a 60-to 95-percent risk of becoming infected after exposure (Whitley, 2015). Primary varicella presents with a 1-to 2-day flulike prodrome, which is followed by pruritic vesicular lesions that crust after 3 to 7 days. Infection tends to be more severe in adults (Marin, 2007). Afected patients are then contagious from 1 day before the onset of the rash until the lesions become crusted.

1	Mortality is predominately due to VZV pneumonia, which is thought to be more severe during adulthood and particularly in pregnancy. Although the incidence was once thought to be higher, only 2 to 5 percent of infected pregnant women develop pneumonitis (Marin, 2007; Zhang, 2015). Risk factors for VZV pneumonia include smoking and having more than 100 cutaneous lesions. Maternal mortality rates with pneumonia have decreased to 1 to 2 percent (Chandra, 1998). Symptoms of VZV pneumonia usually appear 3 to 5 days into the course of illness. Fever, tachypnea, dry cough, dyspnea, and pleuritic pain are characteristic. Nodular iniltrates are similar to other viral pneumonias (Chap. 51, p. 994). Although resolution of pneumonitis parallels that of skin lesions, fever and compromised pulmonary function may persist for weeks.

1	If primary varicella is reactivated years later, it causes herpes zoster or shingles (Whirley, 2015). This presents as a unilateral dermatomal vesicular eruption associated with severe pain. Zoster does not appear to be more frequent or severe in pregnant women. Congenital varicella syndrome rarely develops in cases of maternal herpes zoster (hn, 2016; Enders, 1994). Zoster is contagious if blisters are broken, although less so than with primary varicella.

1	In women with varicella during the irst half of pregnancy, the fetus may develop congenital varicella syndrome. Some features include chorioretinitis, microphthalmia, cerebral cortical atrophy, growth restriction, hydronephrosis, limb hypoplasia, and cicatricial skin lesions as shown in Figure 64-3 (Ahn, 2016; Auriti, 2009). Enders and coworkers (1994) evaluated 13 3 pregnant women with varicella. When maternal infection developed before 13 weeks, only two of 472 pregnancies-O.4 and scarring in a fetus infected during the first trimester by vari cella. (Reproduced with permission from Paryani SG, Arvin AM:

1	Intrauterine infection with varicella zoster virus after maternal varicella, N Engl J Med. 1986 Jun 12;314(24):1542-1546.) percent-had neonates with congenital varicella syndrome. he highest risk was between 13 and 20 weeks, during which time seven of 351 exposed fetuses-2 percent-had evidence of congenital varicella. Mter 20 weeks' gestation, the researchers found no clinical evidence of congenital infection. Ahn and colleagues (2016) recently described similar findings. hat said, sporadic reports have described CNS abnormalities and skin lesions in fetuses who developed congenital varicella in weeks 21 to 28 of gestation (Lamont, 2011 a; Marin, 2007).

1	If the fetus or neonate is exposed to active infection just before or during delivery, and therefore before maternal antibody has been formed, the newborn faces a serious threat. Attack rates range from 25 to 50 percent, and mortality rates approach 30 percent. In some instances, neonates develop disseminated visceral and CNS disease, which is commonly fatal. For this reason, varicella-zoster immune globulin (VZIG) should be administered to neonates born to mothers who have clinical evidence of varicella 5 days before and up to 2 days ater delivery.

1	Maternal varicella is usually diagnosed clinically. Infection may be confirmed by NAA T of vesicular fluid, which is very sensitive. The virus may also be isolated by scraping the vesicle base during primary infection and performing a Tzanck smear, tissue culture, or direct luorescent antibody testing. Congenital varicella may be diagnosed using NAA T analysis of amnionic luid, although a positive result does not correlate well with the development of congenital infection (Mendelson, 2006). A detailed anatomical sonographic evaluation performed at least 5 weeks after maternal infection may disclose abnormalities, but the sensitivity is low (Mandelbrot, 2012).

1	Maternal Viral Exposure. Several aspects of maternal VZV exposure and infection in pregnancy afect management. Exposed gravidas with a negative history for chickenpox should undergo VZV serological testing. At least 70 percent of these women will be seropositive, and thus immune. Exposed pregnant women who are susceptible (seronegative) should be given varicella-zoster immune globulin (VariZIG). Although best given within 96 hours of exposure, its use is approved for up to 10 days to prevent or attenuate varicella infection (Centers for Disease Control and Prevention, 2012, 2013d). Passive immunization appears to be highly efective Qespersen, 2016). In women with known history of varicella, VariZI G is not indicated. Maternal Infection. Any patient dianosed with primay varicella inection or herpes zoster should be isolated rom pregnant women.

1	Maternal Infection. Any patient dianosed with primay varicella inection or herpes zoster should be isolated rom pregnant women. Because VZV pneumonia often presents with few symptoms, a chest radiograph is recommended by many. Most women require only supportive care, but those who require intravenous (IV) luids and especially those with pneumonia are hospitalized. IV acyclovir therapy is given to women requiring hospital ization-500 mg/mror 10 to 15 mg/kg every 8 hours. Vaccination. An attenuated live-virus vaccine is recommended for nonpregnant adolescents and adults with no history of vari cella. Two doses of Varivax are given 4 to 8 weeks apart, and the seroconversion rate is 98 percent (Marin, 2007). Importantly, vaccine-induced immunity diminishes over time, and the (Chaves, 2007).

1	The vaccine is not recommended or pregnant women or or vaccine dose. That said, a registry of more than 1000 vaccine exposed pregnancies reports no cases of congenital varicella syn drome or other associated congenital malformations (Marin, 2014; Wilson, 2008). he attenuated vaccine virus is not secreted in breast milk. hus, postpartum vaccination should not be delayed because of breastfeeding (American College of Obstetricians and Gynecologists, 2016c). hese respiratory infections are caused by members of the family Orthomyxoviridae. Inluenza A and B form one genus of these RNA viruses, and both cause epidemic human disease (Cohen, 2015b). Inluenza A viruses are subclassiied further by hemagglutinin (H) and neuraminidase (N) surface antigens. Inluenza outbreaks occur annually, and the most recent epidemic was in 2016 to 2017 caused by an inluenza A/H3N2 strain (Shang, 2016).

1	Fever, dry cough, and systemic symptoms characterize this infection, which usually is not life-threatening in otherwise healthy adults. However, pregnant women appear to be more susceptible to serious complications, particularly pulmonary involvement (Cohen, 2015b; Mertz, 2017; Rasmussen, 2012). Severe infection has a maternal mortality rate of 1 percent (Duryea, 2015). And from 2009 to 2010, widespread inluenza A infection afected pregnant women and caused 12 percent of pregnancy-related deaths (Callaghan, 2015). No firm evidence links inluenza A virus and congenital malformations (Irving, 2000; Zerbo, 2017). Conversely, TABLE 64-2. Outpatient Influenza A and B Virus Testing Methods aNasopharyngeal or throat swab. RT-PCR = reverse transcription-polymerase chain reaction. Data from Centers for Disease Control and Prevention, 201r7e.

1	RT-PCR = reverse transcription-polymerase chain reaction. Data from Centers for Disease Control and Prevention, 201r7e. Lynberg and colleagues (1994) reported higher rates of neuraltube defects in neonates born to women with inluenza early in pregnancy. his was possibly associated with hyperthermia. Viremia is infrequent, and transplacental passage is rare (Rasmussen, 2012). Stillbirth, preterm delivery, and irst-trimester abortion have all been reported, but usually correlate with the severity of maternal infection (Centers for Disease Control and Prevention, 2011; Fell, 2017; vleijer, 2015).

1	Inluenza may be detected in nasopharyngeal swabs using viral antigen rapid detection assays (Table 64-2). Reverse transcriptase-polymerase chain reaction (RT-PCR) is the more sensitive and specific test, although not widely available (Cohen, 2015b). In contrast, rapid influenza diagnostic tests (RIDTs) are least indicative, with sensitivities of 40 to 70 percent. Decisions to administer antiviral medications or inluenza treatment or chemoprophylaxis should be based on clinical symptoms and epidemiological actors. Specifically, the start of therapy should not be delayed pending testing results (Centers for Disease Control and Prevention, 2017 e).

1	Two classes of antiviral medications are currently available. Neuraminidase inhibitors are highly efective for the treatment of early inluenza A and B. hese include oseltamivir (Tamilu), which is taken orally for treatment and for chemoprophylaxis; zanamivir (Relenza), which is inhaled for treatment; andperamivir (Rapivab), which is administered intravenously. he adamantanes include amantadine and rimantadine, which were used for years for treatment and chemoprophylaxis of inluenza A. In 2005, inluenza A resistance to adamantine was reported to exceed 90 percent in the United States. hus, its use is not currently recommended. It is possible that these drugs may again be efective for subsequently mutated strains. Patterns of resistance are available at cdc.gov/flu.

1	Experience with all of these antiviral agents in pregnant women is limited (Beau, 2014; Beigi, 2014; Dunstan, 2014). hey are Food and Drug Administration category C drugs and thus used when potential benefits outweigh risks. At Parkland Hospital, we start oral oseltamivir treatment within 48 hours of symptom onset-75 mg twice daily for 5 days. Earlyadministration may reduce length of hospital stays (Meijer, 2015; Oboho, 2016). Prophylaxis with oseltamivir, 75 mg orally once daily for 7 days, is also recommended for signiicant exposures. Antibacterial medications are added when a secondary bacterial pneumonia is suspected (Chap. 51, p. 993).

1	Efective vaccines are formulated annually. Vaccination against inluenza throughout the influenza season, but optimally in October or November, is recommended by the Centers for Disease Control and Prevention (CDC) (2013a) and the American College of Obstetricians and Gynecologists (2016b) for all women who will be pregnant during the influenza season. his is especially important for those afected by chronic medical disorders such as diabetes, heart disease, asthma, or human immunodeficiency virus (HIV) infection. Inactivated vaccine prevents clinical illness in 70 to 90 percent of healthy adults. Importantly, there is no evidence of teratogenicity or other adverse maternal or fetal events (Chambers, 2016; Fell, 2017; Kharbanda, 2017; Polyzos, 2015; Sukumaran, 2015). Moreover, for mothers vaccinated during pregnancy, several studies found lower rates of influenza in their infants up to 6 months of age (Nunes, 2017; Steinhof, 2012; Zaman, 2008). Immunogenicity of the trivalent

1	mothers vaccinated during pregnancy, several studies found lower rates of influenza in their infants up to 6 months of age (Nunes, 2017; Steinhof, 2012; Zaman, 2008). Immunogenicity of the trivalent inactivated seasonal inluenza vaccine in pregnant women is similar to that in the nonpregnant individual. A live attenuated influenza virus vaccine is available for intranasal use but is not recommended for pregnant women (Cohen, 2015b).

1	his uncommon adult infection is caused by an RNA paramyxovirus. Because of childhood immunization, up to 90 percent of adults are seropositive (Rubin, 2012). The virus primarily infects the salivary glands but also may involve the gonads, meninges, pancreas, and other organs. It is transmitted by direct contact with respiratory secretions, saliva, or through fomites. Most transmission occurs before and within 5 days of parotitis onset, and droplet isolation is recommended during this time (Kutty, 2010). Treatment is symptomatic, and mumps during pregnancy is no more severe than in nonpregnant adults. Women who develop mumps in the first trimester may have a greater risk of spontaneous abortion. Infection in pregnancy is not associated with congenital malformations, and fetal infection is rare (McLean, 2013).

1	he live attenuated J eryl-Lynn vaccine strain is part of the MMR vaccine-measles, mumps, and rubella. This vaccine is contraindicated in pregnancy according to the CDC (McLean, 2013). No malformations attributable to MMR vaccination in pregnancy have been reported, but pregnancy should be avoided for 30 da) s after mumps vaccination. The vaccine may be given to susceptible women postpartum, and breastfeeding is not a contraindication.

1	his is a highly contagious RNA virus of the family Paramyxoviridae that only infects humans. In endemic areas, annual outbreaks of measles, also called rubeola, occur in late winter and early spring, transmission is primarily by respiratory droplets, and the secondary attack rate among contacts exceeds 90 per cent (Rainwater-Lovett, 2015). Resurgences in measles have been linked to clusters of vaccine-eligible but unvaccinated individuals (Fiebelkorn, 2010; Phadke, 2016). Fever, coryza, conjunctivitis, and cough are typical symptoms. he charac teristic erythematous maculopapular rash develops on the face and neck and then spreads to the back, trunk, and extremities.

1	Koplik spots are small white lesions with surrounding erythema found within the oral cavity. Immediate or delayed neurologi cal sequelae of measles may manifest in several forms, making diagnosis diicult (Buchanan, 2012; Chiu, 2016). Diagnosis of acute infection is most commonly performed by serological evidence of IgM antibodies, although RT-PCR tests are avail able. Treatment is supportive. Pregnant women without evidence of measles immunity should be administered passive immunoprophylaxis with immune globulin, 400 mg/kg intravenously (Centers for Dis ease Control and Prevention, 2017d). Active vaccination is not performed during pregnancy, however, susceptible women can be vaccinated routinely postpartum, and breastfeeding is not contraindicated (Ohji, 2009). he virus does not appear to be teratogenic (Siegel, 1973).

1	he virus does not appear to be teratogenic (Siegel, 1973). However, rates of spontaneous abortion, pre term delivery, and low-birthweight neonates are increased with maternal measles (Rasmussen, 2015). If a woman develops measles shortly before birth, risk of serious infection developing in the neonate is considerable, especially in a preterm neonate. his RNA togavirus causes rubella, also called German measles, which is of minor importance in the absence of pregnancy. Rubella inection in the irst trimester, however, poses signicant risk or abortion and severe congenital maormations. T ransmission occurs via nasopharyngeal secretions, and the transmission rate is 80 percent to susceptible individuals. he peak incidence is late winter and spring in endemic areas (Lambert, 2015).

1	Maternal rubella is usually a mild febrile illness with a generalized maculopapular rash beginning on the face and spreading to the trunk and extremities. hat said, 25 to 50 percent of infections are asymptomatic. Other symptoms may include arthralgias or arthritis, head and neck lymphadenopathy, and conjunctivitis. he incubation period is 12 to 23 days. Viremia usually precedes clinical signs by about a week, and adults are infectious during viremia and through 7 days after the rash appears. Up to half of maternal infections are subclinical despite viremia that may cause devastating fetal infection (McLean, 2013).

1	Rubella virus may be isolated from the urine, blood, nasopharynx, and cerebrospinal fluid for up to 2 weeks after rash onset. The diagnosis is usually made, however, with serological analysis. In one study, 6 percent of nonimmune women seroconverted to rubella virus during pregnancy (Hutton, 2014). Specific IgM antibody can be detected using enzyme-linked immunoassay for 4 to 5 days after onset of clinical disease, but antibody can persist for up to 6 weeks after appearance of the rash. Importantly, rubella virus reinfection can give rise to transient low levels of IgM. With this, fetal infection can rarely occur, but no adverse fetal efects have been described. Serum IgG antibody titers peak 1 to 2 weeks after rash onset. his rapid antibody response may complicate serodiagnosis unless samples are initially collected within a few days after the onset of the rash. If, for example, the first specimen was obtained 10 days after the rash, detection of IgG antibodies would fail to

1	samples are initially collected within a few days after the onset of the rash. If, for example, the first specimen was obtained 10 days after the rash, detection of IgG antibodies would fail to diferentiate between very recent disease and preexisting immunity to rubella. IgG avidity testing is performed concomitant with the serological tests above. High-avidity IgG antibodies indicate an infection at least 2 months in the past.

1	The rubella virus is one of the most complete teratogens, and efects of fetal infection are worst during organogenesis (Adams Waldorf, 2013). Pregnant women with rubella and a rash during the first 12 weeks of gestation have an afected fetus with congenital infection in up to 90 percent of cases (Miller, 1982). At 13 to 14 weeks' gestation, this incidence is 50 percent, and by the end of the second trimester, it is 25 percent. Defects are rare after 20 weeks' gestation. Features of congenital rubella syndrome amenable to prenatal diagnosis are cardiac septal defects, pulmonary stenosis, microcephaly, cataracts, microphthalmia, and hepatosplenomegaly (Yazigi, 2017). Other abnormalities include sensorineural deafness, intellectual disability, neonatal purpura, and radiolucent bone disease. Neonates born with congenital rubela may shed the virus or many months and thus be a threat to other inants and to susceptible adults who contact them. Reports of delayed morbidities associated with

1	Neonates born with congenital rubela may shed the virus or many months and thus be a threat to other inants and to susceptible adults who contact them. Reports of delayed morbidities associated with congenital rubella syndrome may include a rare, progressive panencephalitis, insulin-dependent diabetes mellitus, and thyroid disorders (Sever, 1985; Webster, 1998).

1	here is no speciic treatment for rubella. Droplet precautions for 7 days after the onset of the rash are recommended. Postexposure passive immunization with polyclonal immunoglobulin may be of benefit if given within 5 days of exposure (Young, 2015). Although large epidemics of rubella have virtually disappeared in the United States because of immunization, up to 10 percent of women in the United States are susceptible. Cluster outbreaks during the 1990s mainly involved persons born outside the United States, as congenital rubella is still common in developing nations (Centers for Disease Control and Prevention, 20 13f) . To eradicate rubella and prevent congenital rubella syndrome completely, a comprehensive approach is recommended for immunizing the adult population (Grant, 2015).

1	MMR vaccine should be ofered to nonpregnant women of childbearing age who do not have evidence of immunity whenever they make contact with the health-care system. Vaccination of all susceptible hospital personnel who might be exposed to patients with rubella or who might have contact with pregnant women is important. Rubella vaccination should be avoided 1 month before or during pregnancy because the vaccine contains attenuated live virus. No observed evidence links the vaccine and induced malformations, although the overall theoretical risk is up to 2.6 percent (McLean, 2013; Swamy, 2015). MMR vaccination is not an indication for pregnancy termination. Prenatal serological screening for rubella is indicated for all pregnant women. Women found to be nonimmune are ofered the MMR vaccine postpartum.

1	Prenatal serological screening for rubella is indicated for all pregnant women. Women found to be nonimmune are ofered the MMR vaccine postpartum. More than 200 antigenically distinct respiratory viruses cause the cominon cold, pharyngitis, laryngitis, bronchitis, and pneumonia. Rhinovirus, coronavirus, and adenovirus are major causes of the common cold. The RNA-containing rhinovirus and coronavirus usually produce a trivial, self-limited illness characterized by rhinorrhea, sneezing, and congestion. he DNA-containing adenovirus is more likely to produce cough and lower respiratory tract involvement, including pneumonia.

1	The potential teratogenic efects of respiratory viruses are controversial. In a case-control study using data from the Finnish Register of Congenital vlalformations, 393 gravidas with a common cold had a four-to ivefold greater risk of fetal anencephaly (Kurppa, 1991). In another population study of California births from 1989 to 1991, low attributable risks for neural-tube defects were associated with many illnesses in early pregnancy (Shaw, 1998). Adams and colleagues (2012) performed amnionic fluid viral PCR studies in 1191 women undergoing amniocentesis for fetal karyotyping. Viral PCR was positive in 6.5 percent, with adenovirus being the virus most frequently identified. There was an association with fetalgrowth restriction, nonimmune hydrops, foot/hand abnormalities, and neural-tube defects. Adenoviral infection is a known cause of childhood myocarditis. T owbin (1994) and Forsnes (1998) and their associates used PCR tests to identiy and link adenovirus to fetal myocarditis and

1	defects. Adenoviral infection is a known cause of childhood myocarditis. T owbin (1994) and Forsnes (1998) and their associates used PCR tests to identiy and link adenovirus to fetal myocarditis and nonimmune hydrops.

1	hese RNA viruses are members of the family Bunyaviridae. They are associated with a rodent reservoir, and transmission involves inhalation of virus excreted in rodent urine and feces. Outbreaks of hantaviruses including Sin Nombre virus and Seoul virus have been reported in the United States, the most recent in early 2017 (Centers for Disease Control and Prevention, 2017b). Hantaviruses are a heterogenous group of viruses with low and variable rates of transplacental transmission. Howard and associates (1999) reported the Hantavirus pulmonay syndrome to cause maternal death, fetal demise, and preterm birth. hey found no evidence of vertical transmission of the causative Sin N ombre virus.

1	hese viruses are a major subgroup of RNA picornaviruses that include coxsackievirus, poliovirus, and echovirus. They are trophic for intestinal epithelium but can also cause widespread maternal, fetal, and neonatal infections that may include the CNS, skin, heart, and lungs. Most maternal infections are subclinical yet can be fatal to the fetus-neonate (Tassin, 2014). Hepatitis A is an enterovirus that is discussed in Chapter 55 (p. 1063). Coxsackievirus infections with group A and B are usually asymptomatic. Symptomatic infections-usually with group B-include aseptic meningitis, polio-like illness, hand foot and mouth disease, rashes, respiratory disease, pleuritis, pericarditis, and myocarditis. No treatment or vaccination is available (Cohen, 2015a). Coxsackievirus may be transmitted by maternal secretions to the fetus at delivery in up to half of mothers who seroconverted during pregnancy (Modlin, 1988). Transplacental passage has also been reported (Ornoy, 2006).

1	Congenital malformation rates may be slightly increased in fetuses of pregnant women who had serological evidence of coxsackievirus (Brown, 1972). Viremia can cause fetal hepatitis, skin lesions, myocarditis, and encephalomyelitis, all of which may be fatal. Some have reported higher rates of cardiac anomalies and of low-birthweight, preterm, and small-for-gestational-age newborns (Chen, 2010; Koro'lkova, 1989). Maternal-fetal infection has been associated with massive perivillous ibrin deposition and fetal death (Yu, 2015). Finally, a rare association between maternal coxsackievirus infection and insulin-dependent diabetes in ofspring has been described (Viskari, 2012).

1	Polio viruses cause highly contagious infections that are subclinical or mild. he virus is trophic for the CNS, and it can cause paralytic poliomyelitis (Cohen, 2015a). Siegel (1955) demonstrated that pregnant women not only were more susceptible to polio but also had a higher death rate. Perinatal transmission has been observed, especially when maternal infection developed in the third trimester (Bates, 1955). Inactivated subcutaneous polio vaccine is recommended for susceptible pregnant women who must travel to endemic areas or are placed in other high-risk situations. Live oral polio vaccine has been used for mass vaccination during pregnancy without harmful fetal efects (Harjulehto, 1989).

1	This B 19 virus causes erythema inectiosum, orith disease. It is a small, single-stranded DNA virus that replicates in rapidly proliferating cells such as erythroblast precursors (Brown, 2015). his can lead to anemia, which is its primary fetal efect. Only individuals with the erythrocyte globoside membrane P antigen are susceptible. In women with severe hemolytic anemia-for example, sickle-cell disease-parvovirus infection may cause an aplastic crisis.

1	The main mode of parvovirus transmission is respiratory or hand-to-mouth contact, and the infection is common in spring months. The maternal infection rate is highest in women with school-aged children and in day-care workers, but not in schoolteachers. An infected person develops viremia 4 to 14 days after exposure, and an otherwise immunocompetent individual is no longer infectious at the onset of the rash. By adulthood, only 40 percent of women are susceptible. The annual seroconversion rate is 1 to 2 percent but is >10 percent during epidemic periods (Brown, 2015). The secondary attack rate approaches 50 percent.

1	In 20 to 30 percent of adults, infection is asymptomatic. Fever, headache, and flulike symptoms may begin in the last few days of the viremic phase. Several days later, a bright red rash with erythroderma afects the face and gives a slapped-cheek appearance. The rash becomes lacelike and spreads to the trunk and extremities. Adults often have milder rashes and develop symmetrical polyarthralgia that may persist several weeks. Mayama and associates (2014) described a pregnant woman in whom B 19 infection was associated with hemophagocytic lymphohistiocytosis. No evidence suggests that parvovirus infection is altered by pregnancy. With recovery, IgM antibody is generated 7 to 10 days postinfection, and production persists for 3 to 4 months. Several days after IgM is produced, IgG antibody is detectable and persists for life with natural immunity (American College of Obstetricians and Gynecologists, 2017).

1	here is vertical transmission to the fetus in up to a third of maternal parvovirus infections (de long, 2011; Lamont, 20 11 b). Fetal infection has been associated with abortion, nonimmune hydrops, and stillbirth (Lassen, 2012; Mace, 2014; McClure, 2009). According to the American College of Obstetricians and Gynecologists (2017), the rate of fetal loss with serologically proven parvovirus infection is 8 to 17 percent before 20 weeks' gestation, and 2 to 6 percent after midpregnancy. Currently, no data support evaluating asymptomatic mothers and stillborn fetuses for parvovirus infection.

1	Hydrops develops in only approximately 1 percent of fetuses of women infected with parvovirus (American College of Obstetricians and Gynecologists, 2017; Pasquini, 2016; Puccetti, 2012). Still, it is the most frequent infectious agent of nonimmune hydrops in autopsied fetuses (Rogers, 1999). Hydrops usually stems from infection in the irst half of gestation. In one report, more than 80 percent of hydrops cases were found in the second trimester, with a mean gestational age of 22 to 23 weeks (Yaegashi, 2000). At least 85 percent of cases of fetal infection developed within 10 weeks of maternal infection, and the mean interval was 6 to 7 weeks. he critical period for maternal infection leading to fetal hydrops was estimated to be between 13 and 16 weeks' gestation, which coincided with the period in which fetal hepatic hemopoiesis is greatest.

1	An algorithm for diagnosis of maternal parvoviral infection is illustrated in Figure 64 4. Diagnosis is generally made by maternal serological testing for speciic IgG and IgM antibodies (Bonvicini, 2011; Brown, 2015). Viral DNA may be detectable by PCR in maternal serum during the prodrome and persist for months to years after infection. Fetal infection is diagnosed by detection of B 19 viral DNA in amnionic luid or IgM antibodies in fetal serum obtained by cordocentesis (de long, 2011; Weifenbach, 2012). Fetal and maternal viral loads do not predict fetal morbidity and mortality (de Haan, 2007).

1	Most cases of parvovirus-associated hydrops develop in the first 10 weeks after infection. Thus, serial sonography every 2 weeks should be performed in women with recent infection (see Fig. 64-4). As discussed in Chapter 10 (p. 214), middle cerebral artery (MCA) Doppler interrogation can also be used to predict fetal anemia (Chauvet, 2011). Fetal blood sampling is warranted with hydrops to assess the degree of fetal anemia. Comorbid fetal myocarditis may induce hydrops with lesser degrees of anemia. Depending on gestational age, fetal transfusion for hydrops may improve outcome in some cases (Enders, 2004). Mortal ity rates as high as 30 percent have been reported in hydropic fetuses without transfusions. With transfusion, 94 percent of hydrops cases resolve within 6 to 12 weeks, and the overall mortality rate is <10 percent. Most fetuses require only one transfusion because hemopoiesis resumes as infection resolves.

1	Concurrent fetal thrombocytopenia worsens the prognosis (Melamed,r2015). Reports describing neurodevelopmental outcomes in fetuses transfused for B 19 infection-induced anemia are conlicting. In one review of 24 transfused hydropic fetuses, abnormal neurodevelopment was noted in ive of 16 survivors-32 percent-at 6 months to 8 years (Nagel, 2007). Outcomes were not related to severity of fetal anemia or acidemia, and these investigators hypothesized that the infection itself induced cerebral damage. In another study of 28 children treated with intrauterine transfusion, 11 percent had neurodevelopmental impairment during evaluation at a median age of 5 years (de long, 2012). Conversely, Dembinski (2003) found no signiicant neurodevelopmental delay despite severe fetal anemia.

1	Currently, no parvovirus vaccine is available, and no evidence suggests that antiviral treatment prevents maternal or fetal infection. Decisions to avoid higher-risk work settings are complex and require assessment of exposure risks. Pregnant women should be counseled that risks for infection approximate 5 percent for casual, infrequent contact; 20 percent for intense, prolonged work exposure such as for teachers; and 50 percent for close, frequent interaction such as in the home. Workers at day-care centers and schools need not avoid infected children because infectivity is greatest before clinical illness. Finally, infected children do not require isolation.

1	This mosquito-borne RNA flavivirus is a human neuropathogen. It has become the most common cause of arthropod-borne viral encephalitis in the United States (Centers for Disease Control and Prevention, 2017f; Krow-Lucal, 2017). West Nile viral infections are typically acquired through mosquito bites in late summer or perhaps through blood transfusion. The incubation period is 2 to 14 days, and most persons have mild or no symptoms. Fewer than 1 percent of infected adults develop meningoencephalitis or acute laccid paralysis (Granwehr, 2004). Presenting symptoms may include fever, mental status changes, muscle weakness, and coma (Stewart, 2013). Diagnosis of West Nile infection is based on clinical symptoms and the detection of viral IgG and IgM in serum and IgM in cerebrospinal fluid. There is no known efective antiviral treatment, and management is supportive. he primary strategy for preventing exposure in pregnancy is the use of insect

1	Clinical disease: exposure or infection Sonographic evidence of fetal infection: hydrops fetalis, hepatomegaly, splenomegaly, placentomegaly, elevated FIGURE 64-4 Algorithm for evaluation and management of human parvovirus B 19 infection in pregnancy. eBe = complete blood count; IgG = immunoglobulin G; IgM = immunoglobulin M; MeA = middle cerebral artery; peR = polymerase chain reaction; RNA = ribonucleic acid. repellant containing ,N-diethyl-m-toluamide (DEET). This is infections initially reported to the West Nile Virus Pregnancy considered safe for use among pregnant women (Wylie, 2016). Registry, there were four miscarriages, two elective abortions, Avoiding outdoor activity and stagnant water and wearing proand 72 live births, 6 percent of which were preterm (O'Leary, tective clothing are also recommended. 2006). Three of these 72 newborns were shown to have West

1	Adverse efects of West Nile viremia on pregnancy are Nile infection, and it could not be established conclusively that unclear. Animal data suggest that embryos are susceptible, and infection was acquired congenitally. Of three major malformaa case report of human fetal infection at 27 weeks' gestation tions possiby associated with viral infection, none was deinidescribed chorioretinitis and severe temporal and occipital lobe tively conirmed. Similar conclusions were reached by Pridjian leukomalacia (Alpert, 2003; Julander, 2006). In 77 maternal and colleagues (2016), who analyzed data from the CDC West Nile Virus Registry. Transmission of West Nile virus through breastfeeding is rare . hese are single-stranded RNA viruses that are prevalent world wide. In 2002, an especially virulent strain of coronavirus

1	hese are single-stranded RNA viruses that are prevalent world wide. In 2002, an especially virulent strain of coronavirus China. It rapidly spread throughout Asia, Europe, and North and South America. he case-fatality rate approached 10 percent in the nonpregnant population and was as high as 25 percent in pregnant women (Lam, 2004; Wong, 2004). Although no addi tional cases have been conirmed since 2004, the CDC (2013b) now lists SARS-Co V as a "select agent" that has the potential to pose a severe threat to public health and safety. Another novel regional coronavirus with a high case-fatality coronavirus eMERS-Co ) (Arabi, 2017). Although experience with MERS-Co V is sparse in pregnancy, infection has been reported to cause maternal and perinatal deaths (Assiri, 2016). A member of the RNA Filoviridae family, the Ebola virus is transmitted by direct person-to-person contact (Kuhn, 2015).

1	A member of the RNA Filoviridae family, the Ebola virus is transmitted by direct person-to-person contact (Kuhn, 2015). Infection produces a severe hemorrhagic fever with pronounced thy. Treatment is supportive, and the mortality rate approaches 50 percent. Data are few concerning Ebola viral infection in pregnancy (Beigi, 2017; Money, 2015; Oduyebo, 2015). The CDC concludes that pregnant women are at increased risk for severe illness and death a amieson, 2014). hat said, no evidence suggests that pregnant women are more susceptible to Ebola virus infection. One report described trophoblast infection (Muehlenbachs, 2017). This RNA virus of the Flavivirdae family has recently been recognized as the first major mosquito-borne teratogen (Rasmussen, 2016). Although Zika virus is primaril) transmitted by mosquito bite, sexual transmission is also possible, and the virus may be detected in body fluids for months following acute infection (Hills, 2016; Joguet, 2017; Paz-Bailey, 2017).

1	Reminiscent of the rubella epidemic in the 1960s, in adults Zika infection may be asymptomatic or cause mild symptoms of rash, fever, headache, arthralgia, and conjunctivitis lasting a few days. Virus is typically detectable in blood around the time of symptom onset and may persist days to months in pregnant women (Driggers, 2016; Meaney-Delman, 2016). Serum IgM antibodies typically become detectable within the first two weeks after symptom onset and remain a median of four months (Oduyebo, 2017). Rarely, Guillain-Barre syndrome may develop following infection (da Silva, 2017; Parra, 2016).

1	The fetus can be severely infected whether or not the mother is symptomatic. Honein and coworkers (2017) describe a fetus with congenital Zika infection. Findings shown include a thin cerebral cortex, increased extraaxial space (, dilated ventricles (F,), and absent cavum septum pellucidum. (Reproduced with permission from Driggers RW, Ho (Y, Korhonen EM, et al: Zika virus infection with prolonged maternal viremia and fetal brain abnor malities, N Engl J Med. 2016 Jun 2;374(22):2142-2151.) a 6-percent overall fetal infection rate. In one report of 134 women with positive RT-PCR results, fetal mortality was 7 percent (Brasil, 2016). Among live births, the rate of fetal birth defects ranges from 5 percent-among women with possible Zika infection-to 15 percent among pregnant women with laboratory-confirmed infection in the irst trimester (Reynolds, 2017). In the most severely afected fetuses, a congenital Zika syndrome has been described that includes microcephaly, lissencephaly,

1	laboratory-confirmed infection in the irst trimester (Reynolds, 2017). In the most severely afected fetuses, a congenital Zika syndrome has been described that includes microcephaly, lissencephaly, ventriculomegaly, intracranial calcifications, ocular abnormalities, and congenital contractures (Honein, 2017; Moore, 2017; Soares de Oliveira-Szejnfeld, 2016). Sonographic findings from a Zika-infected fetus are shown in 64-5.

1	Diagnosis of this infection in pregnant women is made with detection of Zika virus RNA in blood or urine or by serological testing. Detection of Zika virus RNA by PCR confirms infection. Serological assays for Zika IgM antibodies may cross react with other flaviviruses. Thus, a positive assay result is followed by another assay containing virus-specific neutralizing antibodies (Oduyebo, 2017) . Testing recommendations and interpretation have evolved for pregnant women who are symptomatic and those who are asymptomatic but have ongoing exposure risk. his risk includes living in or traveling to an area with active local transmission. Large-scale screening programs have been described to identiy women at high risk for travelassociated Zika infection (Adhikari, 2017).

1	Currently, no specific treatment or vaccine is available for Zika infection, although several vaccine candidates are in development (Beigi, 2017; World Health Organization, 2017). Prophylaxis includes protective netting and insect spray to control the vector mosquito and avoidance of sexual contact with partners recently exposed. he CDC has established a pregnancy hotline (770-488-7100) and U.S. Zika Pregnanc) Registry (ZikaPregnancy@cdc.gov) for clinicians with concerns related to management of women with Zika infection or exposure.

1	Registry (ZikaPregnancy@cdc.gov) for clinicians with concerns related to management of women with Zika infection or exposure. Infections caused by Streptococcus pyogenes are important in pregnant women. his organism is the most frequent bacterial cause of acute pharyngitis and is associated with several systemic and cutaneous infections. S pyogenes produces numerous toxins and enzymes responsible for its local and systemic toxicity. Pyrogenic exotoxin-producing strains are usually associated with severe disease (Shinar, 2016; Wessels, 2015). In most cases, streptococcal pharyngitis, scarlet fever, and erysipelas are not life threatening. Treatment, usually with penicillin, is similar in pregnant and nonpregnant women.

1	In the United States, S pyogenes infrequently causes puerperal infection. Still, it remains the most common cause of severe maternal postpartum infection and death worldwide, and the incidence of these infections is rising (Deutscher, 2011; Hamilton, 2013; Wessels, 2015). Puerperal infections are discussed in detail in Chapter 37. he early 1990s saw the emergence of streptococcal toxic shock syndrome, manifested by hypotension, fever, and evidence of multiorgan failure with associated bacteremia. Group A puerperal sepsis is seriously complicated in 20 percent of cases (Shinar, 2016). The case-fatality rate approximates 30 percent, and morbidity and mortality rates are improved with early recognition. Treatment includes clindamycin plus penicillin therapy and often surgical debridement (Chapter 47, p. 924). No vaccine for group A streptococcus is commercially available.

1	Streptococcus agalactiae is a group B organism that can be found to colonize the gastrointestinal and genitourinary tract in 10 to 25 percent of pregnant women (Kwatra, 2016). Throughout pregnancy, group B Streptococcus (GBS) is isolated in a transient, intermittent, or chronic fashion. Although the organism is most likely always present in these same women, their isolation is not always homologous. he spectrum of maternal and fetal G BS efects ranges from asymptomatic colonization to septicemia. S agalactiae has been implicated in adverse pregnancy outcomes that include preterm labor, prematurely ruptured membranes, clinical and subclinical chorioamnionitis, and fetal infections (Randis, 2014). GBS can also cause maternal bacteriuria, pyelonephritis, osteomyelitis, postpartum mastitis, and puerperal infections. It remains the leading infectious cause of morbidity and mortality among infants in the United States (Centers for Disease Control and Prevention, 2010; Schrag, 2016).

1	Neonatal sepsis has received the most attention due to its devastating consequences and available efective preventative measures. Infection <7 days after birth is deined as eary-onset disease and is seen in 0.21/1000 live births (Centers for Disease Control and Prevention, 2015). Many investigators use a threshold of <72 hours of life as most compatible with intrapartum acquisition of disease (Stoll, 2011). We and others have also encountered unexpected intrapartum stillbirths from GBS infections (Nan, 2015). Tudela and associates (2012) reported that newborns with early-onset CBS infection often had clinical evidence of fetal infection during labor or at delivery.

1	In many neonates, septicemia involves signs of serious illness that usually develop within 6 to 12 hours of birth. These include respiratory distress, apnea, and hypotension. At the outset, therefore, neonatal infection must be diferentiated from respiratory distress syndrome caused by insuicient surfactant production (Chap. 34, p. 636). he mortality rate with early-onset disease has declined to approximately 4 percent, and preterm newborns are disparately afected. Late-onset disease caused by GBS is noted in 0.32 per 1000 live births and usually manifests as meningitis 1 week to 3 months after birth (Centers for Disease Control and Prevention, 2015). he mortality rate, although appreciable, is less for late-onset meningitis than for early-onset sepsis. Unfortunately, it is not uncommon for surviving infants of both early-and late-onset disease to exhibit devastating neurological sequelae.

1	As GBS neonatal infections evolved beginning in the 1970s and before widespread intrapartum chemoprophylaxis, rates of early-onset sepsis ranged from 2 to 3 per 1000 live births. By 2010, these outcomes led to a policy of universal rectovaginal culture screening for GBS at 35 to 37 weeks' gestation followed by intrapartum antibiotic prophylaxis for women identified to be carriers. hese outcomes stimulated development of expanded laboratory identiication criteria for GBS; updated algorithms for screening and intrapartum chemoprophylaxis for women with preterm prematurely ruptured membranes, preterm labor, or penicillin allergy; and described new dosing for penicillin G chemoprophylaxis. Following these changes, the incidence of early-onset G BS neonatal sepsis decreased to 0.21 cases per 1000 live births by 2015 (Centers for Disease Control and Prevention, 2015).

1	hus, during the past three decades, several strategies have been proposed to prevent perinatal acquisition of GBS infections. These strategies have not been compared in randomized trials and are either culture-based or risk-based guidelines (Ohlsson, 2014). hese methods have been adopted in the United States, but not all European countries have guidelines (Di Renzo, 2015).

1	Culture-Based Prevention. he CDC (2010) GBS guidelines recommend a culture-based approach, which was also adopted by the American College of Obstetricians and Gynecologists (20 16e). Shown in Figure 64-6, this strategy is designed to identiy women who should be given intrapartum antimicrobial prophylaxis. Women are screened for GBS colonization at 35 to 37 weeks' gestation, and intrapartum antimicrobials are given to women with rectovaginal GBS-positive cultures. Selective enrichment broth followed by subculture improves detection. In addition, more rapid techniques such as DNA probes and NAATs are being developed (Helali, 2012). A previous sibling with GBS invasive disease and identiication of GBS Vaginal and rectal GBS screening cultures at 35-37 weeks' gestation for ALL pregnant women (unless patient had GBS bacteriuria during the current pregnancy or a previous infant with invasive GBS disease) Previous infant with invasive GBS disease

1	Previous infant with invasive GBS disease Positive GBS screening culture during current pregnancy (unless a planned cesarean delivery, in the absence of labor or amniotic membrane rupture, is performed) Unknown GBS status (culture not done, incomplete, or results unknown) and any of the following: Previous pregnancy with a positive GBS screening culture (unless a culture was also positive during the current pregnancy) Planned cesarean delivery performed in the absence of labor or membrane rupture (regardless of maternal GBS culture status) Negative vaginal and rectal GBS screening culture in late gestation during the current pregnancy, regardless of intrapartum risk factors Intrapartum temperature � 1 OOAoF (�38.0°C)

1	Negative vaginal and rectal GBS screening culture in late gestation during the current pregnancy, regardless of intrapartum risk factors Intrapartum temperature � 1 OOAoF (�38.0°C) FIGURE 64-6 Indications for intrapartum prophylaxis to prevent perinatal group B streptococcal (GBS) disease under a universal prenatal screening strategy based on combined vaginal and rectal cultures obtained at 35 to 37 weeks' gestation. (From Centers for Disease Control and Prevention, 201o0.) bacteriuria in the current pregnancy are also considered indications for prophylaxis.

1	Risk-Based Prevention. This approach is recommended for women in labor and whose GBS culture results are not known. It relies on risk factors associated with intrapartum GBS transmission. Intrapartum chemoprophylaxis is given to women who have any of the following: delivery <37 weeks, ruptured membranes � 18 hours, or intrapartum temperature � 100.4°F (�38.0°C). Women with GBS during the current pregnancy and women with a prior infant with invasive early-onset GBS disease are also given chemoprophylaxis.

1	At Parkland Hospital in 1995-and prior to consensus guidelines-we adopted and continue to use the risk-based approach for intrapartum treatment of women at high risk. Importantly, in addition, all term neonates who were not given intrapartum prophylaxis were treated in the delivery room with aqueous penicillin G, 50,000 to 60,000 units intramuscularly. Rates of early-onset GBS sepsis decreased to 0.4 to 0.66 per 1000 live births (Staford, 2012; Wendel, 2002). Non-GBS early-onset sepsis decreased from 0.66 to 0.24 per 1000 live births (Staford, 2012). Thus, this approach has results similar to those reported by the CDC (2010) for culture-based prevention. Serotype-specific capsular antibody concentrations clinically correlate with GBS neonatal disease. Antibody-producing vaccines have been tested, but none are clinically available (Donders, 2016; Kobayashi, 2016; Madhi, 2016).

1	Preventive antimicrobials administered 4 or more hours before delivery are highly efective (Fairlie, 2013). Regardless of screening method, penicillin remains the first-line agent TABLE 64-3. Regimens for Intrapartum Antimicrobial Prophylaxis for Perinatal GBS Disease Recommended Penicillin G, 5 million units IV initial dose, then 2.5 to 3.0 million units IV every 4 hours until delivery Alternative Ampicillin, 2 g IV initial dose, then 1 9 IV every 4 hours or 2 g every Patients not at high risk for anaphylaxis Cefazolin, 2 9 IV initial dose, then 1 9 IV every 8 hours until delivery Patients at high risk for anaphylaxis and with GBS Clindamycin, 900 mg IV every 8 hours until delivery susceptible to clindamycin Patients at high risk for anaphylaxis and with GBS Vancomycin, 1 9 IV every 12 hours until delivery resistant to clindamycin or susceptibility unknown

1	GBS = group B Streptococcus; IV = intravenous. Data from the Verani, 2010 for prophylaxis, and ampicillin is an acceptable alternative Women undergoing cesarean delivery before labor onset with (T able 64-3). Women with a penicillin allergy and no hisintact membranes do not need intrapartum GBS chemoprophytory of anaphylaxis are given cefazolin (Briody, 2016). hose laxis, regardless of GBS colonization status or gestational age. at high risk for anaphylaxis should have antimicrobial susceptibility testing performed to exclude clindamycin resistance. Clindamycin-sensitive but erythromycin-resistant isolates • Methicillin-Resistant Staphylococcus aureus should have a D-zone test performed to assess for inducible Staphylococcus aureus is a pyogenic gram-positive organism and clindamycin resistance. If clindamycin resistance is conirmed, is considered the most virulent of the staphylococcal species. vancomycin should be administered. Eythromycin is no longer It primarily colonizes the

1	If clindamycin resistance is conirmed, is considered the most virulent of the staphylococcal species. vancomycin should be administered. Eythromycin is no longer It primarily colonizes the nares, skin, genital tissues, and oroused or penicilin-alergic patients. pharynx. Approximately 20 percent of normal individuals are

1	Further recommendations for management of spontanepersistent carriers, 30 to 60 percent are intermittent carriers, ous preterm labor, threatened preterm delivery, or preterm and 20 to 50 percent are noncarriers (Gorwitz, 2008). Coloprematurely ruptured membranes are shown in Figure 64-7. nization is considered the greatest risk factor for infection Onset of labor orrupture of membranes at < 37 weeks' gestation with significant risk for imminent preterm delivery rectal GBS culture -...for�48hours prophylaxis

1	Onset of labor orrupture of membranes at < 37 weeks' gestation with significant risk for imminent preterm delivery rectal GBS culture -...for�48hours prophylaxis FIGURE 64-7 Sample algorithm for prophylaxis for women with group B streptococcal (GBS) disease and threatened preterm delivery. This algorithm is not an exclusive course of management, and variations that incorporate individual circumstances or institutional preferences may be appropriate. IV = intravenous. (Adapted from Centers for Disease Control and Prevention, 201o6a.) (Marzec, 2016; Sheield, 2013). Methicillin-resistant 5 aureus (MRSA) colonizes only 2 percent of adults but is a signiicant contributor to the health-care burden (Gorwitz, 2008). MRSA infections are associated with increased cost and higher mortality rates compared with those by methicillin-sensitive 5 aureus (NISSA) (Beigi, 2009; Butterly, 2010).

1	Community-acquired MRSA (CA-MRSA) is diagnosed when identified in an outpatient setting or within 48 hours of hospitalization in a person without traditional risk factors. Such risk factors include prior MRSA infection, hospitalization, dialysis or surgery within the past year, and indwelling catheters or devices (Dantes, 2013). Hospital-associated MRSA (HA-MRSA) infections are nosocomial. Most cases of \1RSA in pregnant women are CA-MRSA.

1	Anovaginal colonization with 5 aureus is identified in 10 to 25 percent of obstetrical patients (Top, 2010). Skin and soft tissue infections are the most common presentation of MRSA in pregnant women (Fig. 64-8). Mastitis and breast abscesses have been reported in up to a fourth of cases of MRSA complicating pregnancy (Laibl, 2005; Lee, 2010). Perineal abscesses, wound infections at sites such as abdominal and episiotomy incisions, and chorioamnionitis are also associated with MRSA (Pimentel, 2009; hurman, 2008). Finally, osteomyelitis has been reported (Nguyen, 2015; Tanamai, 2016). A rise in CA-MRSA infections has been reported in neonatal intensive care units and newborn nurseries. In these settings, infection is frequently associated with maternal and health-care worker skin infections and infected breast milk. Vertical transmission is rare (Jimenez-Truque, 2012; Pinter, 2009).

1	he Infectious Diseases Society of America has published guidelines for the treatment of MRSA infections (Liu, 2011). Uncomplicated supericial infections are primarily managed by drainage and local wound care. Although historically FIGURE 64-8 This antepartum patient presented with multiple small microabscesses for which culture identified methicillinresistant Staphylococcus aureus. (Used with permission from Dr. Stephan Shivvers.) de-emphasized, recent evidence suggests benefit from antibi otic therapy in addition to incision and drainage of smaller abscesses (Daum, 2017; Forcade, 2012). Severe superficial infections, especially those that fail to respond to local care or those in patients with medical comorbidities, are treated with MRSA-appropriate antibiotics. Purulent cellulitis should be treated empirically for CA-MRSA until culture results are available. Most CA-MRSA strains are sensitive to trimethoprim-sul famethoxazole and clindamycin (\1iller, 2015; T alan, 2016).

1	Most CA-MRSA strains are sensitive to trimethoprim-sul famethoxazole and clindamycin (\1iller, 2015; T alan, 2016). for mono therapy. Linezolid, although efective against MRSA, is expensive, and there is little information regarding its use in pregnancy. Doxycycline, minocycline, and tetracycline, although efective for MRSA infections, should not be used in pregnancy. Vancomycin remains the irst-line therapy for inpatient serious MRSA infections.

1	he control and prevention of HA-NIRSA and CA-MRSA rely on appropriate hand hygiene and prevention of skin-toskin contact or contact with wound dressings. Decolonization should be considered only in cases in which a patient develops recurrent supericial infections despite optimal hygiene measures or if ongoing transmission occurs among household or close contacts (Liu, 2011). Decolonization measures include nasal treatment with mupirocin, chlorhexidine gluconate baths, and oral rifampin therapy if previous measures have failed. Routine decolonization is not efective in the general obstetrical population. For women with culture-proven CA-MRSA infection during pregnancy, we add single-dose vancomycin to routine beta-Iactam perioperative prophylaxis for cesarean deliveries and higher-order perineal lacerations. Breastfeeding in these women is not prohibited, but optimal hygiene and attention to minor skin breaks is encouraged.

1	Listeria monocytogenes is an uncommon but probably underdiagnosed cause of neonatal sepsis (Kylat, 2016). This facultative intracellular gram-positive bacillus can be isolated from the feces of 1 to 5 percent of adults. Nearly all cases oflisteriosis are thought to be foodborne. Outbreaks have been caused by raw vegetables, coleslaw, apple cider, melons, milk, fresh Mexicanstyle cheese, smoked ish, and processed foods such as pate, hummus, wieners, and sliced deli meats (Centers for Disease Control and Prevention, 20 13e).

1	Listerial infections are more common in pregnant women, immunocompromised patients, and the very old or young. he incidence of such infections in pregnancy is estimated to be up to 100 times that in the general population (Kourtis, 2014; Rouse, 2016). In 1651 cases reported in 2009 to 2011, the CDC found that 14 percent were in pregnant women (Silk, 2013). It is unclear why pregnant women still account for a significant number of these reported cases. One hypothesis is that pregnant women are susceptible because of decreased cellmediated immunity (Baud, 2011).

1	Listeriosis during pregnancy may be asymptomatic or may cause a febrile illness that is confused with influenza, pyelonephritis, or meningitis (Centers for Disease Control and Prevention, 2013e). he diagnosis usually is not apparent until blood cultures are reported as positive. Occult or clinical infection also may stimulate labor. Discolored, brownish, or meconiumstained amnionic fluid is common with fetal infection, even in preterm gestations. VIaternalrlisteriosis causes fetal infection that characteristically produces disseminated granulomatous lesions with microabscesses (Fig. 64-9). Chorioamnionitis is common, and placental lesions include multiple, well-demarcated macroabscesses. Early-and late-onset neonatal infections are similar to group B streptococcal sepsis. In a review of 222 cases, infection resulted in abortion or stillbirth in 20 percent, and neonatal sepsis developed in 68 percent of surviving newborns (Mylonakis, 2002). In one large, prospective cohort study, 24

1	222 cases, infection resulted in abortion or stillbirth in 20 percent, and neonatal sepsis developed in 68 percent of surviving newborns (Mylonakis, 2002). In one large, prospective cohort study, 24 percent of mothers experienced fetal loss, but none after 29 weeks' gestation (Charlier, 2017). However, a neonatal-case fatality rate of 21 percent has been reported (Sapuan, 2017).

1	FIGURE 64-9 The pale placenta (A)and stillborn infant (B) resulted from maternal listeriosis. Treatment with ampicillin plus gentamicin is usually recommended because of synergism against Listeria species (Rouse, 2016). T rimethoprim-sulfamethoxazole can be given to penicillin-allergic women. Maternal treatment in most cases is also efective for fetal infection (Chan, 2013). No vaccine is available. Prevention is by washing raw vegetables, cooking all raw food, and avoiding the implicated foods listed previously (American College of Obstetricians and Gynecologists, 2016d).

1	Infections from Salmonella species continue to be a major cause of foodborne illness (Peques, 2012). Six serotypes, including Salmonella subtypes yphimurium and enteritidis, account for most cases in the United States. Nontyphoid Salmonella gastroenteritis is contracted through contaminated food. Symptoms that include nonbloody diarrhea, abdominal pain, fever, chills, nausea, and vomiting begin 6 to 48 hours after exposure. Diagnosis is made by stool studies (Chap. 54, p. 1048). Intravenous crystalloid solutions are given for rehydration. ntimicrobials are not given in uncomplicated infections because they do not commonly shorten illness and may prolong the convalescent carrier state. If gastroenteritis is complicated by bacteremia, antimicrobials are given as discussed below. Rare case reports have linked Salmonella bacteremia with abortion (Coughlin, 2002).

1	Typhoid fever caused by Salmonela yphi remains a global health problem, although it is uncommon in the United States. Infection is spread by oral ingestion of contaminated food, water, or milk. In pregnant women, the disease is more likely to be encountered during epidemics or in those with HIV infection (Hedriana, 1995). In former years, antepartum typhoid fever resulted in abortion, preterm labor, and maternal or fetal death (Dildy, 1990). Fluoroquinolones and third-generation cephalosporins are the preferred treatment. For enteric (typhoid) fever, antimicrobial susceptibility testing is important because of the development of drug-resistant strains (Crump, 2015). Typhoid vaccines appear to exert no harmful efects when administered to pregnant women and are given in an epidemic or before travel to endemic areas.

1	Bacillary dysentery caused by Shigela is a relatively common, highly contagious cause of inlammatory exudative diarrhea in adults. Shigellosis is more common in children attending daycare centers and is transmitted via the fecal-oral route. Clinical manifestations range from mild diarrhea to severe dysentery, bloody stools, abdominal cramping, tenesmus, fever, and systemic toxicity. Although shigellosis may be self-limited, careful attention to treatment of dehydration is essential in severe cases. We have cared for pregnant women in whom secretory diarrhea exceeded lO•Ll day! Antimicrobial therapy is imperative, and efective treatment during pregnancy includes fluoroquinolones, ceftriaxone, or azithromycin. Antimicrobial resistance is rapidly emerging, and antibiotic susceptibility testing can help guide appropriate therapy (Centers for Disease Control and Prevention, 2016). Shigellosis can stimulate uterine contractions and cause preterm birth (Parisot, 2016).

1	Also known as leprosy, this chronic infection is caused by Mycobacterium leprae and is rare in this country. Diagnosis is confirmed by PCR. Multidrug therapy with dapsone, rifampin, and clofazimine is recommended for treatment and is generally safe during pregnancy (Gimovsky, 2013; Ozturk, 2017). Duncan (1980) reported an excessive incidence oflow-birthweight newborns among infected women. The placenta is not involved, and neonatal infection apparently is acquired from skin-to-skin or droplet transmission (Duncan, 1984). Vertical transmission is common in untreated mothers (Moschella, 2004).

1	Caused by the spirochete Borrelia burgdoeri, Lyme disease is the most commonly reported vectorborne illness in the United States (Centers for Disease Control and Prevention, 2017c). Lyme borreliosis follows tick bites of the genus Ixodes. There are three stages (Steere, 2015). Early infection-stage I-causes a distinctive local skin lesion, erythema migrans, which may be accompanied by a flulike syndrome and regional adenopathy. If untreated, disseminated infection-stage 2-follows in days to weeks. Multisystem involvement is frequent, but skin lesions, arthralgia, myalgia, carditis, and meningitis predominate. If still untreated after several weeks to months, late or persistent infection-stage 3-manifests in perhaps half of patients. Native immunity is acquired, and the disease enters a chronic phase in about 10 percent. Some patients remain asymptomatic, but others in the chronic phase develop various skin, joint, or neurological manifestations (Shapiro, 2014).

1	Clinical diagnosis is important because serological and PCR testing has many pitfalls (Steere, 2015). IgM and IgG serological testing is recommended in early infection and is followed by Western blotting for conirmation. Ideally, acute and convalescent serological evaluation is completed if possible, however, false-positive and -negative rates are high. Optimal treatment of Lyme disease was published by the Infectious Diseases Society of America (Sanchez, 2016). For early infection, treatment with doxycycline, amoxicillin, or cefuroxime is recommended for 14 days, although doxycycline is usually avoided in pregnancy. A 14-to 28-day course of IV ceftriaxone, cefotaxime, or penicillin G is given for complicated early infections that include meningitis, carditis, or disseminated infections. Chronic arthritis and post-Lyme disease syndrome are treated with prolonged oral or IV regimens, however, symptoms respond poorly to treatment (Steere, 2015).

1	No vaccine is commercially available. Avoiding areas with endemic Lyme disease and improving tick control in those areas is the most efective prevention. Self-examination with removal of unengorged ticks within 36 hours of attachment reduces infection risk (Hayes, 2003). For tick bites recognized within 72 hours, a single 200-mg oral dose of doxycycline may reduce infection development. Several reports describe Lyme disease in pregnancy, although large series are lacking. Transplacental transmission has been confirmed, but no congenital efects of maternal borreliosis have been conclusively identiied (Shapiro, 2014; Walsh, 2006). Prompt treatment of maternal early infection should prevent most adverse pregnancy outcomes (Mylonas, 201l). Diagnosis and management of tuberculosis during pregnancy is discussed in detail in Chapter 51 (p. 995).

1	Diagnosis and management of tuberculosis during pregnancy is discussed in detail in Chapter 51 (p. 995). The obligate intracellular parasite Toxoplasma gondii has a life cycle with two distinct stages (Kim, 2015). he eline stage takes place in the cat-the deinitive host-and its prey. Unsporu lated oocysts are excreted in feces. In the non eline stage, tissue cysts containing bradyzoites or oocysts are ingested by the inter mediate host, including humans. Gastric acid digests the cysts to release bradyzoites, which infect small-intestinal epithelium. Here, they are transformed into rapidly dividing tachyzoites, which can infect all cells within the host mammal. Humoral and cell-mediated immune defenses eliminate most of these, but tissue cysts develop. Their lifelong persistence is the chronic form of toxoplasmosis.

1	Human infection is acquired by eating raw or undercooked meat infected with tissue cysts or by contact with oocysts from cat feces in contaminated litter, soil, or water. Prior infection is conirmed by serological testing, and its prevalence depends on geographic locale and parasite genotype. In the United States, seroprevalence in persons aged 10 to 19 years is 5 to 30 per cent, and this can exceed 60 percent in those older than 50 (Kim, 2015). Thus, a significant segment of pregnant women in this country are susceptible to infection. The incidence of prenatal infection resulting in birth of a newborn with con genital toxoplasmosis varies from 0.8 per 10,000 live births in the United States to 10 per 10,000 in France (Cook, 2000). Between 400 and 4000 cases of congenital toxoplasmosis are diagnosed annually in the United States Oones, 2014).

1	Between 400 and 4000 cases of congenital toxoplasmosis are diagnosed annually in the United States Oones, 2014). Most acute maternal infections are subclinical and are detected only by prenatal or newborn serological screening. In some cases, maternal symptoms may include fatigue, fever, headache, muscle pain, and sometimes a maculopapular rash and posterior cervical lymphadenopathy. In immunocompetent adults, initial infection confers immunity, and prep regnancy infection nearly eliminates any risk of vertical transmission. Infection in immu nocompromised women, however, may be severe, and reactiva tion may cause encephalitis, retinochoroiditis, or mass lesions. Maternal infection is associated with a fourfold increased pre term delivery rate before 37 weeks (Freeman, 2005).

1	Maternal infection is associated with a fourfold increased pre term delivery rate before 37 weeks (Freeman, 2005). The incidence and severity of fetal toxoplasmosis depend on gestational age at the time of maternal infection. Risks for fetal infection rise with gestational age. A metaanalysis estimated the risk to be 15 percent at 13 weeks, 44 percent at 26 weeks, and 71 percent at 36 weeks (SYROCOT Study Group, 2007). Conversely, the severity of fetal infection is much greater in early pregnancy, and these fetuses are much more likely to have clinical findings of infection (American College of Obstetricians and Gynecologists, 2017).

1	Importantly, most infected fetuses are born without obvious stigmata of toxoplasmosis. Clinically afected neonates usually have generalized disease expressed as low birthweight, hepatosplenomegaly, jaundice, and anemia. Some primarily have neurological disease with intracranial calciications and with hydrocephaly or microcephaly (Dhombres, 2017). Many eventually develop chorioretinitis and exhibit learning disabilities. This classic triad-chorioretinitis, intracranial calcifications, and hydrocephalus-is often accompanied by convulsions. Infected neonates with clinical signs are at risk for long-term complications (Abdoli, 2014; Wallon, 2014).

1	With IgG antibody conirmed before pregnancy, there is no risk for a congenitally infected fetus. he American College of Obstetricians and Gynecologists (2017) does not recommend prenatal screening for toxoplasmosis in areas of low prevalence, including the United States. Screening should be performed in immunocompromised pregnant women, including those with HIV infection. In areas of high toxoplasmosis prevalence-for example, France and Austria-routine screening has resulted in diminished congenital disease (Kim, 2015; Wallon, 2013).

1	Pregnant women with suspected toxoplasmosis should be tested. he parasite is rarely detected in tissue or body fluids. Antitoxoplasma IgG develops within 2 to 3 weeks after infection, peaks at 1 to 2 months, and usually persists for lifesometimes in high titers. Although IgM antibodies appear by 10 days after infection and usually become negative within 3 to 4 months, they may remain detectable for years. hus, IgM antibodies are not used alone to diagnose acute toxoplasmosis (Dhakal, 2015). Best results are obtained with the Toxoplasma Serologic Proile performed at the Palo Alto Medical Foundation Research Institute (ww.toxolab@pamf.org). Toxoplasma IgG avidity increases with time. hus, if a high-avidity IgG result is found, infection in the preceding 3 to 5 months is excluded. Multiple tests are available that allow high avidity results to conirm latent infection with a 100-percent positivepredictive value (Villard, 2013).

1	Congenital toxoplasmosis is suspected when sonography reveals findings such as hydrocephaly, intracranial or hepatic calciications, ascites, placental thickening, hyperechoic bowel, and growth restriction. Prenatal diagnosis of congenital toxoplasmosis is performed using PCR ampliication of toxoplasma DNA in amnionic luid (Filisetti, 2015; Montoya, 2008). he sensitivity of PCR varies with gestational age and is lowest before 18 weeks (Romand, 2001). No randomized clinical trials have assessed the benefit and eicacy of treatment to decrease the risk for congenital infection. A systematic review of data from 1438 treated pregnancies found weak evidence for early treatment to reduce congenital toxoplasmosis risks (SYROCOT Study Group, 2007). Treatment has been associated with a reduction in rates of serious neurological sequelae and neonatal demise (Cortina-Borja, 2010).

1	Prenatal treatment is based on two regimens-spiramycin alone or a pyrimethamine-sulfonamide combination given with folinic acid (American College of Obstetricians and Gynecologists, 2017). These two regimens have also been used consecutively (Hotop, 2012). Little evidence supports the use of a speciic regimen (Montazeri, 2017; Valentini, 2015). hat said, most experts will use spiramycin in women with acute infection early in pregnancy to reduce vertical transmission. Because it does not cross the placenta, spiramycin may not be used to treat fetal infection. Pyrimethamine-sulfadiazine with folinic acid is selected for maternal infection after 18 weeks' gestation or if fetal infection is suspected.

1	There is no vaccine for toxoplasmosis, so avoidance of infection is necessary if congenital infection is to be prevented. Eforts include: (1) cooking meat to safe temperatures; (2) peeling or thoroughly washing fruits and vegetables; (3) cleaning all food preparation surfaces and utensils that have contacted raw meat, poultry, seafood, or unwashed fruits and vegetables; (4) wearing gloves when changing cat litter, or else delegating this duty; and (5) avoiding feeding cats raw or undercooked meat and keeping cats indoors. Although these preventive steps are recommended, no data support their efectiveness (American College of Obstetricians and Gynecologists, 2017; Di Mario, 2015).

1	his protozoan infection remains a global health crisis and causes 2000 deaths per day worldwide (White, 2015). Malaria has been efectively eradicated in Europe and in most of North America, and worldwide mortality rates have fallen more than 25 percent. In the United States, most cases of malaria are imported-some in returning military personnel (Mace, 2017). Transmitted by infected Anopheles mosquitoes, six species of Plasmodium cause human disease-alciparum, vivax, two species of ovale, malariae, and knowlesi.

1	Pregnant women have increased susceptibility to malarial infections (Kourtis, 2014). Antibodies to the parasite surface antigen VAR2CSA mediate placental accumulation of infected erythrocytes and lead to the harmful efects of malaria (Mayor, 2015). hrough this mechanism, some immunity accrues with parity and is called pregnancy-specic antimalarial immuniy. Ironically, malaria treatment dampens this immunity, and resurgence in pregnancy has been documented in Mozambique (Mayor, 2015).

1	Clinical findings are fever, chills, and flulike symptoms including headaches, myalgia, and malaise, which may occur at intervals. Symptoms are less severe with recurrences. Malaria may be associated with anemia and jaundice, and aciparum infections may cause kidney failure, coma, and death. That said, many otherwise healthy but infected adults in endemic areas are asymptomatic because of partial immunity. Pregnant women, although often asymptomatic, are said to be more likely to develop traditional symptoms (Desai, 2007) . infections during pregnancy-whether symptomatic or asymptomatic-are associated with higher rates of perinatal morbidity and mortality (Menendez, 2007; Nosten, 2007). Adverse outcomes include stillbirth, preterm birth, low birthweight, and maternal anemia. The latter two are documented most frequently (Machado Filho, 2014; McClure, 2013). Maternal infection is associated with a 14-percent rate of low-birthweight newborns worldwide (Eisele, 2012). These

1	FIGURE 64-10 Photomicrograph of placental malaria. A.Multiple infected red blood cells (long black arrow) are seen in the intervillous space of this placenta. Multiple villi cut in cross section are shown, and three are highlighted (short arrows). B. Increased magnification of image (A). Multiple infected erythrocytes are seen, and two are identified (arrows). adverse perinatal outcomes correlate with high levels of placental parasitemia (Rogerson, 2007). The latter occurs when parasitized erythrocytes, monocytes, and macrophages accumulate in the vascular areas of the placenta (Fig. 64-10). Infections with P aciparum are the worst, and early infection raises the risk for abortion. The incidence of malaria increases signiicantly in the latter two trimesters and postpartum (Diagne, 2000). Despite this, congenital malaria occurs in <5 percent of neonates born to infected mothers.

1	Identiication of parasites by microscopical evaluation of a thick and thin blood smear remains the gold standard for diagnosis. In women with low parasite densities, however, the sensitivity of microscopy is poor. Malaria-speciic antigens are now being used for rapid diagnostic testing. Not only is their sensitivity still an issue in pregnancy, but these tests are not routinely available (Kashif, 2013; White, 2015). For treatment, the most frequently used antimalarial drugs are not contraindicated in pregnancy. The World Health Organization recommends that all infected patients living in or traveling from endemic areas be treated with an artemisinin-based regimen for uncomplicated falciparum malaria (Taming, 2016). The CDC (2013c) recommends using atovaquone options are not available or tolerated.

1	nosed with uncomplicated malaria caused by P vivax, malariae, ovale, and chloroquine-sensitive P aciparum should be treated with chloroquine or hydroxychloroquine. For women infected with multidrug-resistant P aciparum, one irst-line agent for nonpregnant persons is artemether-Iumefantrine. Another primary option is artesunate plus meRoquine or artesunate plus dihydroartemisinin-piperaquine (White, 2015). he PREGACT Study Group (2016) recently compared four artemisinin-based drugs in 3428 pregnant women with falciparum malaria and reported no serious maternal or perinatal adverse efects. Second-line treatment regimens are artesunate; quinine plus either tetracycline, doxycycline, or clindamycin; or atovaquone-proguanii. Chloroquine-resistant P vivax should be treated with meRoquine. Chloroquine-sensitive P vivax or P ovale should be treated with chloroquine throughout pregnancy and then primaquine postpartum. Resistance to all the antimalarial drugs has been reported, including the

1	P vivax or P ovale should be treated with chloroquine throughout pregnancy and then primaquine postpartum. Resistance to all the antimalarial drugs has been reported, including the recently added artemisinin-based compounds.

1	Treatment regimens for uncomplicated and severe malarial infections in pregnancy are detailed at: ww.cdc.gov/malaria/ diagnosis_treatment. he CDC also maintains a malaria hotline for treatment recommendations (855-856-4713). Malaria control and prevention relies on chemoprophylaxis when traveling to or living in endemic areas. Vector control is also important. Insecticide-treated netting, pyrethroid insecticides, and D EET -based insect repellent lower malarial rates in endemic areas. hese are well tolerated in pregnancy (Menendez, 2007). If travel is necessary, chemoprophylaxis is recommended.

1	Chloroquine and hydroxychloroquine prophylaxis is safe and well tolerated in pregnancy. Prophylaxis lowers placental infection rates from 20 percent to 4 percent in asymptomatic infected women in areas without chloroquine resistance (Cot, 1992). For travelers to areas with chloroquine-resistant P aciparum, meRoquine prophylaxis is recommended (Freedman, 2016). One evaluation compared prophylaxis during pregnancy with either sulfadoxine-pyrimethamine or dihydroartemisinin-piperaquine and found the latter to be more efective (Kakuru, 2016). Primaquine and doxycycline are contraindicated in pregnancy, and data are insuicient for atovaquone/proguanil use. The latest chemoprophylaxis regimens for pregnancy can be obtained from the CDC Travelers' Health website at: ww.cdc.gov/malaria/ travelers/drugs.htmi. The CDC also publishes Health Inormation or Intenational Travel (The Yellow Book) at: ww.cdc. gov/yellowbook. For women living in endemic areas, intermittent preventive treatment was

1	The CDC also publishes Health Inormation or Intenational Travel (The Yellow Book) at: ww.cdc. gov/yellowbook. For women living in endemic areas, intermittent preventive treatment was found to be superior to intermittent screening with treatmet (Desai, 2015).

1	Approximately 10 percent of the world population is infected with Entamoeba histo6ltica, and most are asymptomatic (Andrade, 201r5). Amebic dysentelY, however, may take a fulminant course during pregnancy, with fever, abdominal pain, and bloody stools. Prognosis is worse if complicated by a hepatic abscess. Diagnosis is made by identiying E histoytica cysts or trophozoites within a stool sample. herapy is similar to that for the nonpregnant woman, and metronidazole or tinidazole are the preferred drugs for amebic colitis and invasive disease. Noninvasive infections may be treated with iodoquinol or paromomycin. Disseminated fungal infection-usually pneumonitis-during pregnancy is uncommon with coccidiomycosis, blastomycosis, cryptococcosis, or histoplasmosis. heir identification and management are considered in Chapter 51 (p. 995).

1	Pregnant travelers face general medical, obstetrical, and potentially hazardous destination risks. Several sources provide travel information (Freedman, 2016). he International Federation for Tropical Medicine has comprehensive information available at ww.iftm-hp.org, and the International Society of Travel Medicine publishes information at www.istm.org/ bodyofknowledge. lso, 1he Yelow Book, mentioned earlier, by the CDC has extensive travel information regarding pregnancy and breastfeeding.

1	The concept ofbioterrorism involves the deliberate release ofbacteria, viruses, or other infectious agents to cause illness or death. hese natural agents are often altered to increase their infectivity or their resistance to medical therapy. Health-care providers should be alert for signiicant increases in the number of persons with febrile illnesses accompanied by respiratory symptoms or with rashes not easily associated with common illnesses. Clinicians are urged to contact their state health department or the CDC for current information and recommendations. The merican College of Obstetricians and Gynecologists (2016a) has addressed disaster preparedness for obstetricians. It provides both general considerations and recommendations for hospital readiness and obstetrics-specific issues.

1	he variola virus causes smallpox and is considered a serious weapon. The virus is highly transmissible and carries an overall 30-percent case-fatality rate. The last case of smallpox in the United States was reported in 1949, and worldwide it was reported in Somalia in 1977. Nishiura (2006) has reviewed the severe perinatal and maternal morbidity and mortality caused by smallpox. The case-fatality rate of smallpox in pregnancy is 61 percent if the pregnant woman is unvaccinated. Rates of stillbirth, abortion, preterm labor and delivery, and neonatal demise rise significantly in pregnancies complicated by this infection.

1	Because the smallpox vaccine currently available is made with live vaccinia virus, pregnancy should be delayed for 4 weeks in recipients. It is generally not given to pregnant women because of the risk of fetal vaccinia, a rare but serious complication. Inadvertent smallpox vaccination during pregnancy has not, however, been convincingly associated with fetal malformations or preterm birth (Badell, 2015). Moreover, no cases of fetal vaccinia have been reported with second-generation smallpox vaccine exposure. The Smallpox Vaccine in Pregnancy Registry remains active, and vaccinated women are still being enrolled: DOD .NHRC-birthregistry@mail.mil.

1	Bacillus anthracis is a gram-positive, spore-forming, aerobic bacterium. It can cause three main types of clinical anthrax: inhalational, cutaneous, and gastrointestinal (Centers for Disease Control and Prevention, 2017a). The bioterrorist anthrax attacks of 2001 involved inhalational anthrax (Inglesby, 2002). Spores are inhaled and deposited in the alveoli. They are engulfed by macrophages and germinate in mediastinal lymph nodes. The incubation period is usually less than 1 week but may be as long as 2 months. Within 1 to 5 days of symptom onset, the second stage is heralded by the abrupt onset of severe respiratory distress and high fevers. Mediastinitis and hemorrhagic thoracic lymphadenitis are common. Chest radiographs show a widened mediastinum. Case-fatality rates with inhalational anthrax are high, even with aggressive antibiotic and supportive therapy (Holty, 2006).

1	Anthrax afecting pregnant women and its treatment were reviewed by Meaney-Delman and coworkers (2012, 2013). They reported data on 20 pregnant and postpartum women. The overall mortality rate was 80 percent, with a 60-percent fetal or neonatal loss rate. Of note, most cases were published before the advent of antibiotics. Regimens for postexposure anthrax prophylaxis are given for 2 months. The CDC recommend that asymptomatic pregnant and lactating women with documented exposure to B anthracis be given postexposure prophylaxis with ciproloxacin, 500 mg orally twice daily for 60 days (Hendricks, 2014; Meaney-Delman, 2013). Amoxicillin, 500 mg orally three times daily, can be substituted if the strain is proven sensitive. In the case of ciproloxacin allergy and either penicillin allergy or resistance, doxycycline, 100 mg orally twice daily, is given for 60 days. Risks from anthrax far outweigh any fetal risks from doxycycline (lvleaney-Delman, 2013).

1	The anthrax vaccine is an inactivated, cell-free product that requires three injections over 28 days. Vaccination is generally avoided in pregnancy because safety data are limited. Inadvertent vaccination of pregnant women with the vaccine has not been linked with a significant increase in fetal malformation or miscarriage rates (Conlin, 2015; Ryan, 2008). The anthrax vaccine is an essential adjunct to postexposure antimicrobial prophylaxis, even in pregnancy. Other category A bioterrorism agents include Francisella tularemis-tularemia, Clostridium botulinum-botulism, Yersinia pestis-plague, and viral hemorrhagic fevers-for example, Ebola, Marburg, Lassa, and Machupo. The guidelines for these biological agents are evolving and are detailed at the CDC Bioterrorism website: emergency.cdc.gov/bioterrorism/index.asp. Abdoli A, Dalimi A, Arbabi M, et al: Neuropsychiatric manifestations of latent toxoplasmosis on mothers and their ofspring. J Matern Fetal Neonatal Med 27(13):1368,t2014

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1	SYPHILIS.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1235 GONORRHEA.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1239 CHLAMYDIAL.INFECTIONS . . . . . . . . . . . . . . . . . . . . .. 1240 HERPES SIMPLEX VIRUS . . . . . . . . . . . . . . . . . . . . . . .. 1241 HUMAN PAPILLOMAVIRUS ............i.......i...i...i. 1244 VAGINITIS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1245 HUMAN IMMUNODEFICIENCY VIRUS .....i....i...i... 1247 Syphilis is one of the most important complications of preg nancy, as it is one of the most frequent causes of abortion or premature labour. Syphilis is the most common cause of oetal death in the later months of pregnancy, and may be maternal or paternal in origin. -J. Whitridge Williams (1903)

1	-J. Whitridge Williams (1903) Syphilis and gonorrhea were prominently mentioned in the irst edition of this book, with special consideration for their harmful efects on fetal development. Although Williams confined his discussion to these two infections, today, sexually transmitted diseases (STDs) include chlamydial and trichomonal infections and viral STDs such as hepatitis B, human immunodeiciency virus (HIV), herpes simplex virus (HSV), and human papillomavirus (HPV) infections. In some form, all can be injurious to the mother or fetus and thus should be aggressively sought and treated. In many instances, recommended therapies are provided in guidelines from the Centers for Disease Control and Prevention (CDC) and listed throughout the chapter.

1	Vertical transmISSIOn refers to passage from the mother to her fetus of an infectious agent through the placenta, during labor or delivery, or by breastfeeding. Treatment of most STDs is clearly associated with improved pregnancy outcome and prevention of perinatal morbidity. Logically, education, screening, treatment, and prevention are essential components of prenatal care.

1	Despite the availability of adequate therapy for decades, syphilis remains a major issue for both mother and fetus. From 2001 through 2015, the primary and secondary syphilis rates have risen almost yearly (Centers for Disease Control and Prevention, 2016c). In the United States in 2015, the combined rate for both of these among women was 1.8 cases per 100,000 persons (de Voux, 2017). For congenital syphilis, after a nadir in 2012, rates have also risen yearly to reach 12.4 cases per 100,000 live births in 2015. Of risks, higher congenital syphilis rates are linked to inadequate prenatal care, black or Hispanic race, and lack of treatment (Su, 2016). Similarly, syphilis remains a signiicant global health problem, with many countries reporting high numbers of new infections (Newman, 2015; World Health Organization, 2012).

1	Syphilis is caused by the spirochetal bacterium Treponema pallidum. Minute abrasions on the vaginal mucosa provide an entry portal, and cervical eversion, hyperemia, and friability raise transmission risk. Spirochetes replicate and then disseminate through lymphatic channels within hours to days. The incubation period is 3 to 4 weeks depending on host factors and inoculum size. he early stages of syphilis include primary, secondary, and early latent syphilis. hese are associated with high spirochete loads, and partner transmission rates approximate 30 to 60 percent (Garnett, 1997; Singh, 1999). In late-stage disease, transmission rates decline because of smaller inoculum sizes. FIGURE 65-1 Primary syphilis. Photograph of a chancre with a raised, firm border and smooth, red base.

1	FIGURE 65-1 Primary syphilis. Photograph of a chancre with a raised, firm border and smooth, red base. Maternal syphilis can cause fetal infection by several routes. Spirochetes readily cross the placenta to cause congenital infection. Although transplacental transmission is the most common route, neonatal infection may follow after contact with spirochetes through lesions at delivery or across the placental membranes. Fetal infection develops in >50 percent of untreated early syphilis cases and in 10 percent of late latent disease (Fiumara, 1975; Hollier, 2001). This is staged according to clinical features and disease duration. 1. Primary syphilis is diagnosed by its characteristic chancre, which develops at the inoculation site. This solitary, painless

1	This is staged according to clinical features and disease duration. 1. Primary syphilis is diagnosed by its characteristic chancre, which develops at the inoculation site. This solitary, painless FIGURE 65-3 Condyloma lata. (Reproduced with permission from Horsager R, Roberts S, Roger V, et al (eds): Williams Obstetrics 24th Edition Study Guide, New York, McGraw Hill Education, 2014; Photo contributor: Dr. Jonathan Willms.) lesion typically has a raised, firm border and a red, smooth ulcerated base without signiicant pus (Fig. 65-1). Nonsuppurative lymphadenopathy may develop. A chancre will usually resolve spontaneously in 2 to 8 weeks, even if untreated. Multiple lesions, if found, are predominantly in HIV-1 coinfected women.

1	2. Secondary syphilis stems from dissemination of spirochetes to afect multiple organ systems. Manifestations develop 4 to 10 weeks after the chancre appears and include dermatological abnormalities in up to 90 percent of women. A difuse macular rash, plantar and palmar targetlike lesions, patchy alopecia, and mucous patches may be seen (Fig. 65-2). Condylomata lata are flesh-colored papules and nodules found on the perineum and perianal area (Fig. 65-3). These papules are teeming with spirochetes and are highly infectious. Most women with secondary syphilis also express constitutional symptoms such as fever, malaise, headache, and myalgias. FIGURE 65-2 Secondary syphilis. A.Target lesions on the palms. B. Mucous patches around the nose and mouth. (Used with permission from Dr. Devin Macias.) Hepatitis, nephropathy, ocular changes, anterior uveitis, and periostitis can also develop. 3.

1	Hepatitis, nephropathy, ocular changes, anterior uveitis, and periostitis can also develop. 3. Latent syphilis develops when primary or secondary syphilis is not treated but clinical manifestations still resolve. It is identified instead by serological testing. Eary latent syphilis is subclinical disease acquired within the preceding 12 months. Disease diagnosed beyond 12 months is either late latent syphilis or latent syphilis of unknown duration. 4. Tertiary syphilis is a slowl) progressive disease afecting any organ system but is rarely seen in reproductive-aged women.

1	4. Tertiary syphilis is a slowl) progressive disease afecting any organ system but is rarely seen in reproductive-aged women. Without screening and treatment, approximately 70 percent of infected women will have an adverse pregnancy outcome (Hawkes, 201l). Maternal infection can lead to preterm labor, fetal death, fetal-growth restriction, or fetal infection (Gomez, 2013). Because of immune incompetence prior to midpregnancy, the fetus generally does not manifest the immunological inlammatory response characteristic of clinical disease before this time (Silverstein, 1962). Once fetal syphilis develops, however, it manifests as a continuum. Fetal hepatic abnormalities are followed by anemia and thrombocytopenia, then ascites and hydrops (Hollier, 2001). Stillbirth remains a major complication (Lawn, 2016; Su, 2016). he newborn may have jaundice with petechiae or purpuric skin lesions, lymphadenopathy, rhinitis, pneumonia, myocarditis, nephrosis, or long-bone involvement (Fig. 65-4).

1	With syphilitic infection, the placenta becomes large and pale (see Fig. 65-4). Microscopically, villi lose their characteristic arborization and become thicker and clubbed. Sheield and colleagues (2002c) described these villi in more than 60 percent of syphilitic placentas. Blood vessels markedly diminish in number, and in advanced cases, they almost entirely disappear as a result of endarteritis and stromal cell proliferation. Likely related, Lucas and coworkers (1991) demonstrated increased vascular resistance in uterine and umbilical arteries of infected pregnancies. he cord may also show evidence of infection. In a study of 25 untreated women, Schwartz and associates (1995) reported that necrotizing funisitis was present in a third.

1	The United States Preventative Services Task Force recom mends that clinicians screen all pregnant women for syphilis to prevent congenital infection (Wolf, 2009). Testing is ideally performed at the irst prenatal visit. In populations with a high prevalence of syphilis, serological testing is repeated in the third trimester and again at delivery (Workowski, 2015). Treponema pallidum cannot be cultured from clinical specimens. However, direct diagnosis of early-stage disease from lesion exudate, tissue, or body luid can be completed by darkield microscopic examination, by polymerase chain reaction (PCR), or by direct fluorescent antibody tests for T pallidum (DFA-TP) (Tsang, 2015). hese methods are not widely available and are less sensitive for blood specimens (Grange, 2012; HenaoMartinez, 2014). hus, in practice, diagnoses are mainly derived from clinical indings coupled with serological blood testing.

1	Serological testing is used for diagnostic and for screening purposes. here are two types. If the irst of these is positive, then the second type is also performed. his combination identifies infection and clariies disease stage. Traditionally, the first type is nontreponemal testing, and either the Venereal Disease Research Laboratory (VDRL) or the rapid plasma reagin (RPR) is selected. Both tests measure patient immunoglobulin M and G (IgM and IgG) antibodies formed against cardiolipin that is released from damaged host cells and possibly also from treponemes. Notably, these same antibodies can also be produced in response to other acute events that include recent vaccination, febrile illness, and pregnancy itself or in response to chronic conditions such as intravenous drug abuse, systemic lupus erythematosus, aging, leprosy, or cancer. As such, these all serve as potential sources of false-positive results (Larsen, 1995). Conversely, seroconversion occurs at around 3 weeks, but

1	lupus erythematosus, aging, leprosy, or cancer. As such, these all serve as potential sources of false-positive results (Larsen, 1995). Conversely, seroconversion occurs at around 3 weeks, but can take up to 6 weeks (Peeling, 2004). Thus, women with very early primary syphilis can have initially false-negative serological test results.

1	FIGURE 65-4 Congenital syphilis. A. Fetogram of a stillborn infant infected with syphilis showing the "moth-eaten" appearance of the femurs (arrow). B. Enlarged hydropic placenta of a syphilis-infected neonate.

1	With positive nontreponemal test results, findings are quantified and expressed as titers. Because titers reflect disease activity, they increase during early syphilis and often exceed levels of 1 :32 in secondary syphilis. Following treatment of primary and secondary syphilis, serological testing at 3 to 6 months usually confirms a fourfold drop in VDRL or RPR titers (Rac, 2014a). Because VDRL titers do not correspond directly to RPR titers, consistent use of the same test for surveillance is recommended. hose with treatment failure or reinfection may lack this expected decline. Importantly, some successfully treated patients may still exhibit persistently low-level positive titers, which are referred to as "serofast." his state is more likely in older individuals, those with lower initial nontreponemal antibody titers, and those with later stages of syphilis (Seia, 2015). The second type of serological testing is trep0 nemal-specic.

1	The second type of serological testing is trep0 nemal-specic. It seeks patient antibodies formed specifically against T palidum. he antibodies detected by treponemal assays appear up to a few weeks earlier than those detected by nontreponemal tests (Levett, 2015). Tests includes the fluorescent treponemalantibody absorption tests (FTA-ABS), the T palidum passive particle agglutination (TP-PA) test, and various immunoassays (Association of Public Health Laboratories, 2015). O/note, these treponemal-speciic tests generaly remain positive throughout lie.

1	Each of the serological tests has limitations including falsepositive and -negative results. Traditionally, nontreponemal tests have been used for screening in the United States, and results are then conirmed by a specific treponemal test. Within the past several years, some laboratories have implemented a reverse screening algorithm, namely, screening first with a treponemalspecific test (Binnicker, 2012; Centers for Disease Control and Prevention, 2011). Both approaches are efective if there is a program for appropriate screening, follow-up, and treatment.

1	In contrast to these tests, rapid "point-of-care" (POC) syphilis screening of blood or serum samples is being developed (Singh, 2015; Tucker, 2010). hese may be best used for women with limited prenatal care. Most tests are treponemalspecific, and positive POC results can then be confirmed by a laboratory nontreponemal test. In hard-to-reach populations, some countries immediately treat women with positive POC results. This practice, however, risks overtreating previously cured women who still have residual persistent treponemal antibodies. his limitation may be overcome by newer POC dual tests, which simultaneously assess nontreponemal and treponemal antibodies (Causer, 2015).

1	Following maternal diagnosis, sonographic evaluation is performed for fetuses >20 weeks' gestation to search for signs of congenital syphilis. Rac and associates (20 14b) noted that 31 percent of infected women diagnosed at � 18 weeks' gestation had abnormal fetal sonographic indings. Hepatomegaly, placental thickening, hydramnios, ascites, hydrops fetalis, and elevated middle cerebral artery Doppler velocimetry measurements are indicative of fetal infection. Before 20 weeks, treatment is highly successful, and sonographic findings are rare (Nathan, 1997).

1	For fetuses of viable age with sonographic findings, antepartum fetal heart rate monitoring prior to treatment is recommended. Spontaneous late decelerations or a nonreactive tracing likely reflects an extremely ill fetus that may poorly tolerate a Jarisch-Herxheimer reaction, described next. In this extreme case, consultation with a neonatologist regarding a plan of delaying treatment, pursuing delivery, and then treating in the nursery is a consideration (Wendel, 2002). Syphilis therapy during pregnancy is given to eradicate maternal infection and to prevent or treat congenital syphilis. Parenteral penicillin G remains the preferred treatment for all stages of syphilis during pregnancy (Table 65-1). During pregnancy, authorities recommend that a second dose of benzathine penicillin G be given 1 week after the initial dose. Such treatment is also given for women with concomitant HIV infection (Workowski, 2015).

1	Benzathine penicillin G is highly efective for early maternal infection. In a study of 340 pregnant women so treated, Alexander and associates (1999) reported six cases-1.8 percent-of congenital syphilis. Four of these six neonates were from a group of75 women with secondary syphilis. The other two were identified in those delivered from a group of 102 women with early latent syphilis. Congenital syphilis was generally confined to neonates of women treated ater 26 weeks and is likely related to the duration and severity of fetal infection. Sheield and coworkers (2002b) reported that high maternal serological titers, preterm delivery, and delivery shortly ater antepartum therapy are all risks for failure of maternal treatment to prevent neonatal infection.

1	here are no proven alternatives to penicillin therapy during pregnancy. Erythromycin and azithromycin may be curative for the mother, but because of limited transplacental passage, these drugs do not prevent all congenital disease (Berman, 2004; Wendel, 1988; Zhou, 2007). Moreover, in several countries, TABLE 65-1. Recommended Treatment for Pregnant Women with Syphilis G, 2.4 million units as a single injection-some recommend a second More than l-year durationb Benzathine penicillin G, 2.4 million units intramuscularly weekly for three doses aprimary, secondary, early latent syphilis of less than duration. bLatent syphilis of unknown or more than 1-year duration; tertiary syphilis. Missed doses are not acceptable for pregnant women, and those who miss any dose of therapy must repeat the full course of therapy. Data from Workowski, 201r5.

1	Data from Workowski, 201r5. macrolide-resistant strains of T pallidum are now prevalent (Stamm, 2015). Cephalosporins may prove useful, but data are limited (Liang, 2016). Tetracyclines, including doxycycline, are efective but generally not recommended during pregnancy, because of the risk for fetal deciduous-teeth discoloration. All women with syphilis are ofered counseling and testing for HIV and other STDs. Following syphilis treatment, serological testing to detect treatment failures is done at 3 to 6 months and usually confirms a fourfold drop in VDRL or RPR titers. During pregnancy, serological titers can be checked monthly in women at high risk for reinfection (Workowski, 2015).

1	In some instances, a woman may present without symptoms but describes recent sexual contact with a person who has been diagnosed with syphilis. She should be evaluated clinically and serologically. If her partner is diagnosed and their sexual contact occurred within the preceding 90 days, the gravida is treated presumptively for early syphilis, even if serological test results are negative. his accounts for early infection but before seroconversion. If contact was earlier than 90 days ago, treatment is based on serological results (Workwoski, 2015). Women with a history of penicillin allergy should have either an oral stepwise penicillin-dose challenge or skin testing performed to confirm the risk of immunoglobulin E (IgE)mediated anaphylaxis. If conirmed, penicillin desensitization, shown in Table 65-2, is recommended and then followed by benzathine penicillin G treatment (Wendel, 1985). TABLE 65-2. Penicillin Allergy-Oral Desensitization

1	TABLE 65-2. Penicillin Allergy-Oral Desensitization Protocol for Patients with a Positive Skin Test Penicillin V Amountb Cumulative Dosea (units/mL) mL Units Dose (units) 1 1000 0.1 100 100 2 1000 0.2 200 300 3 ·1000 0.4 400 700 4 1000 O.S 800 1500 5 1000 1.6 1600 3100 6 1000 3.2 3200 6300 7 1000 6.4 6400 12,700 8 10,000 1.2 12,000 24,700 9 10,000 2.4 24,000 4S,700 10 10,000 4.S 4S,OOO 96,700 11 SO,OOO 1.0 80,000 176,700 12 80,000 2.0 160,000 336,700 13 SO,OOO 4.0 320,000 656,700 14 SO,OOO 8.0 640,000 1,296,700 alnterval between doses: 15 Elapsed time: 3 hours and 45 minutes. Cumulative dose: 1.3 million units. Observation period: 30 minutes before parenteral adminis tration of penicillin. bThe specific amount of drug was diluted in approximately 30 mL of water and administered orally. From Wendel, 1985, with permission.

1	Distinct from allergy, a Jarisch-Herxheimer reaction develops following penicillin treatment in most women with primary syphilis and approximately half with secondary infection. Uterine contractions, mild maternal temperature elevation, decreased fetal movement, and fetal heart rate decelerations are findings. Reaction treatment is supportive with antipyretics as needed, hydration, and oxygen supplementation (Klein, 1990). In a study of 50 gravidas who received benzathine penicillin for syphilis, Myles and associates (1998) reported a 40-percent incidence of Jarisch-Herxheimer reactions. Of the 31 women monitored electronically, 42 percent developed regular uterine contractions, and 39 percent developed variable decelerations. All contractions resolved within 24 hours of therapy. Accordingly, for fetuses of viable age, some recommend administering the irst dose of antibiotic in labor and delivery and with continuous fetal monitoring for at least 24 hours (Rae, 2017). Others recommend

1	for fetuses of viable age, some recommend administering the irst dose of antibiotic in labor and delivery and with continuous fetal monitoring for at least 24 hours (Rae, 2017). Others recommend this only if sonographic signs of fetal syphilis, described earlier, are found (Duf, 2014; Wendel, 2002). If this second plan is elected, patients are counseled on reaction signs and encouraged to seek evaluation if they develop.

1	Of notiiable STDs, infections caused by Neisseria gonorrhoeae are the second most common. The incidence of gonorrhea in the United States has continued to rise since 2009, and in 2015, the rate was 124 cases per 100,000 persons (Centers for Disease Control and Prevention, 2016c). he highest rates in women of any ethnicity were in those aged 15 to 24 years. In pregnant women, its prevalence approximates 0.6 percent (Blatt, 2012). In most pregnant women, infection is limited to the lower genital tract-the cervix, urethra, and periurethral and vestibular glands. Acute salpingitis is rare in pregnancy. But, pregnant women account for a disproportionate number of disseminated gonococcal infections (Bleich, 2012).

1	Gonococcal infection can have deleterious efects in any trimester. Untreated gonococcal cervicitis is associated with septic abortion as well as infection after voluntary abortion (Burkman, 1976). Preterm delivery, prematurely ruptured membranes, chorioamnionitis, and postpartum infection are more frequent in women with gonococcal infection (lger, 1988; Johnson, 2011). Vertical transmission of gonorrhea is predominantly due to fetal contact with vaginal infection during birth. he predominant sequela is gonococcal ophthalmia neonatorum, which can lead to corneal scarring, ocular perforation, and blindness. Transmission rates are high and approximate 40 percent (Laga, 1986). Accordingly, as discussed in Chapter 32 (p. 613), ocular prophylaxis is provided to newborns (Mabry-Hernandez, 2010).

1	Pregnant women who live in high-prevalence areas or who are at risk for gonorrhea should undergo irst-trimester screening. Risk factors include age �25 years; prior gonococcal infection; other STDs; prostitution; new or multiple sexual partners; drug abuse; black, Hispanic, or American Indian or laska Native ethnicity; and inconsistent condom use (American Academy of Pediatrics, 2017). For women who test positive, screening for syphilis, chlamydial infection, and HIV should precede treatment, if possible. Gonococcal infection is a marker for concomitant chlamydial infection. Thus, if chlamydial testing is unavailable, presumptive chlamydial therapy is given to women treated for gonorrhea.

1	Screening for gonorrhea in women is by culture or nucleic acid ampliication tests (NAATs). NAATs have replaced culture in most laboratories, and kits are available for specific collection from the vagina, en do cervix, or urine. Of these, vaginal or cervical samples are preferred, as urine collection may detect up to 10 percent fewer infections (Papp, 2014). If used, the initial urine stream, not midstream, is collected. NAATs are also recommended for diagnosis of rectal or pharyngeal disease, but participating laboratories must be CLIA (Clinical Laboratory Improvement Amendments) compliant with required test modifications. Culture is also available for these anatomical sites. Rapid POC tests for gonorrhea, although available, do not yet reach the sensitivity or specificity of culture or NAAT and have not been rigorously studied in pregnant women (Herbst de Cortina, 2016).

1	Gonorrhea treatment has evolved during the past decade due to the ability of N gonorrhoeae to rapidly develop antimicrobial resistance. The current treatment for uncomplicated gonococcal infection during pregnancy is 250 mg of cetriaxone intramuscularly plus 1 g of azithromycin orally (Workowski, 2015). he latter provides another drug with a diferent mechanism of action against N gonorrhoeae and treats chlamydial co-infections. Patients are instructed to abstain from sexual intercourse for 7 days ater they and their sexual partners have completed treatment. u an alternative regimen, a single, 400-mg oral dose of cefixime plus 1 g of azithromycin should be reserved for situations that preclude cetriaxone treatment. With cephalosporin allergy, a 240-mg intramuscular dose of gentamicin can be coupled with a 2-g oral azithromycin dose. Repeat testing is recommended in the third trimester for any woman treated for gonorrhea in the first trimester and for any uninfected woman who is at high

1	coupled with a 2-g oral azithromycin dose. Repeat testing is recommended in the third trimester for any woman treated for gonorrhea in the first trimester and for any uninfected woman who is at high risk for gonococcal infection (American Academy of Pediatrics, 2017).

1	Treatment is recommended for sexual contacts. Expedited therapy, discussed on page 1241, is a less-desirable option due to the now-preferred injectable regimen.

1	Gonococcal bacteremia may cause disseminated infections that manifest as petechial or pustular skin lesions, arthralgias, or septic arthritis. For treatment of septic arthritis, the CDC recommends ceftriaxone, 1 g intramuscularly or intravenously (IV) every 24 hours plus a single 1-g oral dose of azithromycin (Workowski, 2015). Treatment is continued for 24 to 48 hours ater clinical improvement, and therapy is then changed to an oral agent to complete 1 week of therapy. Prompt recognition and antimicrobial treatment will usually yield favorable outcomes in pregnancy (Bleich, 2012). Meningitis and endocarditis rarely complicate pregnancy, but they may be fatal (Bataskov, 1991; Burgis, 2006). For gonococcal endocarditis, ceftriaxone 1 to 2 g IV every 12 hours should be continued for at least 4 weeks, and for meningitis, 10 to 14 days. A single 1-g oral dose of azithromycin is also provided for chlamydial co-infection (Workowski, 2015).

1	Chlamydia trachomatis is an obligate intracellular bacterium that has several serotypes, including those that cause lymphogranuloma venereum. The most commonly encountered strains are those that attach only to columnar or transitional cell epithelium and cause cervical infection. It is the most common reportable STD in the United States, and the overall chlamydial infection rate among women was 646 cases per 100,000 females in 2015 (Centers for Disease Control and Prevention, 2016c). Most pregnant women have asymptomatic infection, but a third have urethral syndrome, urethritis, or Bartholin gland infection (Peipert, 2003). Mucopurulent cervicitis may be due to chlamydial or gonococcal infection or both. Other chlamydial infections not usually seen in pregnancy are endometritis, salpingitis, reactive arthritis, and Reiter syndrome.

1	The role of chlamydial infection in pregnancy complications remains controversial. A few studies have reported a direct association between C trachomatis and miscarriage, whereas most show no correlation (Baud, 2011; Coste, 1991; Paukku, 1999). It is disputed whether untreated cervical infection increases the risk of preterm delivery, preterm ruptured membranes, low birthweight, or perinatal mortality (Andrews, 2000, 2006; BIas, 2007; Johnson, 2011; Moodley, 2017; Silva, 2011). Chlamydial infection has not been associated with a greater risk of chorioamnionitis or with peripartum pelvic infection (Berman, 1987; Gibbs, 1987). However, delayed postpartum uterine infection has been described by Hoyme and associates (1986). The syndrome, which develops 2 to 3 weeks postpartum, is distinct from early postpartum metritis. It is characterized by vaginal bleeding or discharge, low-grade fever, and uterine tenderness.

1	Infection poses a higher risk to the newborn than to the mother. Vertical transmission leads to infection in 8 to 44 percent of neonates delivered vaginally from afected women (Rosenman, 2003). Of neonatal infections, conjunctivitis is the most common (Chap. 32, p. 613). Perinatal transmission to newborns can also cause pneumonia. Currently, the U.S. Preventive Services Task Force (LeFevre, 2014) as well as the American Academy of Pediatrics and American College of Obstetricians and Gynecologists (2017) recommend chlamydia screening for all women at the irst prenatal visit. he College further suggests testing in the third trimester for those treated in the irst trimester; all women aged ;25 years; and those aged �25 years with behavioral factors, which mirror those for women at risk for gonorrhea. In one review of repeat chlamydial infections among women, the reinfection rate was 14 percent, and most recurred within the first 8 to 10 months (Hosenfeld, 2009).

1	Diagnosis is made predominantly by culture or NAAT. Cultures are more expensive and less accurate than newer NAA T s (Greer, 2008). Of samples for NAAT, vaginal or cervical TABLE 65-3. Oral Treatment of Chlamydia trachomatis Infections During Pregnancy Erythromycin ethylsuccinate, 400 mg four times daily for 14 d Data from Workowski, 2015. samples are preferred, as urine collection may detect up to 10 infections (Papp, Wiesenfeld, 2017). Roberts and associates (2011) evaluated NAA T of urine speci mens compared with cervical secretions in more than 2000 pregnant women and found them to be equivalent. As with gonorrhea, the irst portion of the urine stream is collected.

1	Currently recommended treatment regimens for chlamydial infections are shown in Table 65-3. Azithromycin is irst-line treatment and is safe and efective in pregnancy. The luoroquinolones and doxycycline are usually avoided in pregnancy, and erythromycin estolate is contraindicated because of drug-related hepatotoxicity. Chlamydial testing is repeated 3 to 4 weeks after therapy completion and ,gain 3 months after treatment. In high-risk individuals, third-trimester rescreening is recommended (Workowski, 2015).

1	To prevent STD transmission, guidelines for expedited partner therapy (EPT) have been created by the Centers for Disease Control and Prevention (2006a) and are endorsed by the American College of Obstetricians and Gynecologists (2015). With EPT, a prescription is provided to the diagnosed patient for their partner. It is delivered by the patient to their partner without medical assessment of the partner or professional counseling. EPT ideally does not replace traditional strategies, such as standard patient referral with screening for other STDs. EPT is acceptable for treatment of sexual contacts with chlamydial infection. In light of new guidelines that recommend injectable ceftriaxone, EPT for gonorrhea is less desirable unless the partner will otherwise not seek treatment (Centers for Disease Control and Prevention, 2016a). Fewer data are available to assess this strategy for trichomoniasis (Kissinger, 2006; Schwebke, 2010). EPT is not recommended for syphilis (Workowski,r2015).

1	Although sanctioned by the CDC, EPT is not legal in several states. \{oreover, the risk of litigation in the event of adverse outcomes may be elevated when a practice has uncertain legal status or is outside formally accepted community practice standards (Centers for Disease Control and Prevention, 2006a). The legal status of EPT in each of the 50 states can be found at: http://ww.cdc.gov/std/ept/legal!default.htm.

1	L], L2, and L3 serovars of C trachomatis cause ymphogranuloma venereum (LG). he primary genital infection is transient, is seldom recognized, and is not linked with vertical transmission to the fetus. It can be confused with chancroid. Classically, matted inguinal adenitis may develop on either side of the inguinal ligament to give rise to the "groove sign." At times, these nodes may suppurate. Ultimately, the lymphatics of the lower genital tract and perirectal tissues may be involved. Here, sclerosis and fibrosis can cause vulvar elephantiasis and severe rectal stricture. Fistula formation involving the rectum, perineum, and vulva may also evolve. For treatment during pregnancy, erythromycin base, 500 mg orally four times daily, is given for 21 days (Workowski, 2015). Some authorities instead use szithromycin, 1 g orally weekly for 21 days, although data regarding eicacy are scarce.

1	his virus poses a disproportionately higher risk to the newborn than to the mother. hus, strategies in pregnancy aim to curb rates of vertical transmission.

1	Two types of herpes simplex viruses are distinguished based on immunological diferences. Yet, the two viruses have signiicant DNA sequence homology, and thereby, prior infection with one type attenuates a primary infection with the other. Type 2 HSV is recovered almost exclusively from the genital tract and is usually transmitted by sexual contact. Type 1 is responsible for most nongenital infections and typically is acquired in childhood. However, more than half of new cases of genital herpes in adolescents and young adults are now caused by HSV-1 infection (Bernstein, 2013). his rise in the prevalence of HSV-1 genital disease is thought to stem from an increase in oral-genital sexual practices. Another explanation is that HSV-1 acquisition has declined in childhood as a result of improved living conditions and hygiene (Bradley, 2014; Xu, 2007). Without prior exposure, this renders young people without HSV-l antibodies susceptible to genital acquisition of HSV-l or -2.

1	Genital herpes simplex virus afects an estimated 50 million adolescents and adults (Workowski, 2015). Most women are unaware of their infection, but HSV-2 seroprevalence among non-Hispanic white females in the United States was 15.3 from 2007 to 2010 and among black females, it was 53 percent (Fanfair, 2014; Schulte, 2014). In one study of nearly 16,000 pregnant women from 2000 to 2010, the overall seroprevalence of HSV-2 was 16 percent, and for HSV-l, it was 66 percent (Delaney, 2014). Seronegative pregnant women have a 4 to 5 percent risk to acquire HSV-l or -2 during pregnancy (Brown, 1997; Kulhanjian, 1992). For those who are HSV-l seropositive, acquisition risk for HSV-2 approximates 2 percent (Brown, 1997).

1	Once transmitted by contact, HSV-l or -2 replicates at the entry site. Following mucocutaneous infection, the virus moves retrograde along sensory nerves. It then remains latent in cranial nerves or dorsal spinal ganglia, but recurrences are common. HSV infections may be categorized into three groups.

1	First episode primary inection describes the case in which HSV-l or 2 is isolated from a lesion in the absence of HSV-l or -2 serological antibodies. The typical incubation period of 6 to 8 days (range 1 to 26 days) may be followed by a papular eruption with itching or tingling, which then becomes painful and vesicular. Multiple vulvar and perineal lesions mayror may not coalesce, and then ulcerate (Fig. 65-5). Associated inguinal adenopathy can be severe. Many women do not present with typical lesions. Instead, a pruritic or painful abraded area or knife-cut may be found. Cervical involvement is common, although it may be inapparent clinically. Transient systemic influenza-like symptoms are frequent and are presumably caused by viremia. Some cases are severe enough to require hospitalization. Hepatitis, encephalitis, or pneumonia infrequently develop, and disseminated disease is rare. After 2 to 4 weeks, all signs and symptoms of infection disappear. Instead of these classic

1	Hepatitis, encephalitis, or pneumonia infrequently develop, and disseminated disease is rare. After 2 to 4 weeks, all signs and symptoms of infection disappear. Instead of these classic symptoms, the percentage of asymptomatic primary HSV-2 genital infections may be as high as 90 percent (Fanfair, 2013).

1	FIGURE 65-5 First-episode primary genital herpes simplex virus infection. Vesicles and knife-cut lesions are indicated by arrows. Small ulcers rim the anus. Similar lesions can typically be seen on the vulva. First episode nonprimay inection is diagnosed when one HSV type is isolated from a lesion in a woman who has only the other serological HSV-type antibody present. In general, compared with primary infection, nonprimary infections are characterized by fewer lesions, less pain, fewer systemic manifestations, and briefer duration of lesions and viral shedding. This is likely because of some immunity from cross-reacting antibodies, for example, from childhood-acquired HSV-l infection.

1	Recurrent disease is characterized by isolation of HSV-l or -2 from the genital tract in women with the same serotype antibodies. During the latency period, in which viral particles reside in nerve ganglia, reactivation is common and mediated through poorly understood stimuli. The resulting lesions generally are fewer in number, are less tender, and shed virus for a shorter period than those of primary infection. Typically, they recur at the same sites. Genital disease recurrences are more frequently caused by HSV-2 compared with HSV-l. Recurrences are most frequent in the irst year after initial infection, and rates slowly decline subsequently (Benedetti, 1999). Gravidas with a known prior history of genital HSV often experience recurrences (Sheield, 2006).

1	Asymptomatic viral shedding is defined by the absence of clinical findings. Most infected women shed virus intermittently over time, and most HSV transmission to a partner occurs during these periods of asymptomatic viral shedding. he virus can be passed to the fetus/neonate by three routes: peripartum in 85 percent, (2) postnatal in 10 percent, or intrauterine in 5 percent Qames, 2015). As discussed in Chapter 18 (p. 347), evidence does not suggest an obvious link between HSV infection and miscarriage (Zhou, 2015).

1	Peripartum transmission is by far the more frequent route of infection, and the fetus is exposed to virus shed from the cervix or lower genital tract. HSV-l or -2 invades the uterus following membrane rupture or is transmitted by contact at delivery. The newborn is mainly infected, but rare cases of maternal endometritis have .been described (Hollier, 1997; McGill, 2012). Neonatal manifestations vary. First, infection may be localized to the skin, eye, or mouth-SEM disease-in approximately 40 percent of cases. Second, central nervous system disease with encephalitis is seen in 30 percent. Last, disseminated disease with involvement of multiple major organs is found in 32 percent. Localized infection is usually associated with a good outcome. Conversely, even with acyclovir treatment, disseminated infection has a mortality rate of nearly 30 percent (Corey, 2009; Kimberlin, 2011). Of disseminated or cerebral infection survivors, serious developmental and central nervous system morbidity

1	infection has a mortality rate of nearly 30 percent (Corey, 2009; Kimberlin, 2011). Of disseminated or cerebral infection survivors, serious developmental and central nervous system morbidity is seen in 20 to 50 percent.

1	The neonatal infection rate is 0.5 to 1 per 10,000 births in the United States (Flagg, 2011; Mahnert, 2007). Most infected newborns are born to mothers with no reported history ofHSV infection (Gardella, 2010). The risk of neonatal infection cor relates with the presence of HSV in the genital tract, the HSV type, invasive obstetrical procedures, and stage of maternal infection (Brown, 2005, 2007). For example, neonates born to women who acquire genital HSV near the time of delivery have a 30-to 50-percent risk of infection. This is attributed to higher viral loads and the lack of transplacental protective antibodies (Brown, 1997,r2000). Women with recurrent HSV have less than a I-percent risk of neonatal infection (Pasternak, 2010; Prober, 1987). Postpartum transmission is uncommon and passed to the newborn by contact with an infected mother, family member, or health-care worker. The clinical presentation mirrors that with peripartum transmission.

1	In utero transmission ofHSV-l or HSV-2 is rare and is part of the TORCH (roxoplasmosis, Qther, rubella, �ytomegalovi rus, herpes virus) collection of infections. Intrauterine HSV infection classically leads to disease involving the skin (blisters, scarring), the central nervous system (hydranencephaly, micro cephaly, intracranial calcification), or the eyes (chorioretini tis, microphthalmia) (Hutto, 1987). Bone and viscera can be involved (Marquez, 2011). If seen sonographically, indings should prompt viral serological testing as described next. PCR analysis of an amniocentesis sample is another potential tool (Diguet, 2006). Several organizations recommend against routine serological HSV screening in asymptomatic gravidas (American College of Obstetricians and Gynecologists, 20 16b; Workowski, 2015;

1	Several organizations recommend against routine serological HSV screening in asymptomatic gravidas (American College of Obstetricians and Gynecologists, 20 16b; Workowski, 2015; U.S. Preventive Services Task Force, 2016). However, for those with a clinically suspicious lesion, a diagnosis should be conirmed by laboratory testing. Available HSV tests are either virological or type-speciic serological tests.

1	Direct virological tests are can be performed on a specimen from the mucocutaneous lesion. PCR or culture of the sample is a testing option. Of the two, PCR assays are more sensitive, the results generally are available in 1 to 2 days, and specimen handling is easier. In contrast, for viral culture, the sensitivity of HSV isolation is relatively low as vesicular lesions ulcerate and then crust. Also, results sometimes are not available for 7 to 14 days (Strick, 2006). Regardless of the test performed, HSV viral types should be diferentiated (LeGof, 2014). Importantly, a negative culture or PCR result does not exclude infection. In contrast, false-positive results are rare.

1	Serological assays are available to detect antibodies produced against speciic HSV glycoproteins, Glrand G2. These proteins evoke type-speciic antibody responses to HSV-l and HSV-2 infection, respectively, and they reliably diferentiate the two. IgG antibodies develop 1 to 2 weeks after a primary infection and then persist. This permits conirmation of clinical infection and identiication of asymptomatic carriers. Providers should request type-speciic glycoprotein G-based assays when serology is being performed. Sensitivity approaches 90 to 100 percent, and speciicity is 99 to 100 percent (Wald, 2002). IgM antibody detection is not a useful test.

1	In nonpregnant patients, antiviral therapy with acyclovir, vala cyclovir, or famciclovir is used to treat irst-episode genital herpes. Oral or parenteral preparations attenuate clinical infec tion and viral shedding duration. Suppressive therapy is also an option to limit recurrent infections and to reduce heterosexual transmission (Corey, 2004). In pregnant women, acyclovir is safe (Briggs, 2015). Through 1999, the manufacturers of acyclovir and valacyclovir maintained a registry of outcomes following exposure to these drugs during pregnancy. More than 700 neonates exposed dur ing the irst trimester were evaluated, and there were no adverse efects attributable to acyclovir (Stone, 2004). At this time, data are insuicient regarding famciclovir exposure, although a pregnancy registry is being maintained (1-888-669-6682).

1	For a primary outbreak during pregnancy, women may be given antiviral therapy to attenuate and decrease the duration of symptoms and viral shedding (Table 65-4). Women with HIV co-infection may require a longer duration of treatment. Those with severe or disseminated HSV are given IV acyclovir, 5 to 10 mg/kg every 8 hours for 2 to 7 days until clinically improved. This is followed by oral antiviral drugs to complete at least 10 days of total therapy (W orkowski, 2015). For intense discomfort, oral analgesics and topical anesthetics may provide some relief, and comorbid urinary retention is treated with an indwelling bladder catheter. For recurrent HSV infections during pregnancy, antiviral treatment is provided mainly for symptom relief (see Table 65-4) . Although uncommon, acyclovir resistance has been reported, predominantly with HSV-2 and in immunocompromised patients (Andrei,r2013).

1	During pregnancy, amniocentesis, percutaneous cord blood sampling, or transabdominal chorionic villus sampling may be performed even with active genital lesions. With active lesions, however, internal electronic monitoring during labor is not recommended. T ranscervical procedures may best be delayed until lesions have resolved (American College of Obstetricians and Gynecologists, 20 16b).

1	To diminish vertical transmission risks, cesarean delivery is indicated for women with active genital lesions or prodromal symptoms (American College of Obstetricians and Gynecologists, 20 16b). Several studies have shown that acyclovir or valacyclovir suppression initiated at 36 weeks' gestation for gravidas with recurrences during pregnancy lowers the number of HSV outbreaks at term. The goal is to decrease the need for cesarean delivery (Hollier, 2008). his suppressive therapy will also decrease viral shedding (Scott, 2002; Sheield, 2006; Watts, 2003). One systematic review evaluated acyclovir prophylaxis given from 36 weeks to delivery to women with HSV recurrence during pregnancy. Sheield and colleagues (2003) found that suppressive therapy was associated with signiicantly lower rates of clinical HSV recurrence, cesarean deliveries for HSV recurrences, total HSV detection, and asymptomatic shedding. Subsequent studies using valacyclovir suppression have shown similar results

1	rates of clinical HSV recurrence, cesarean deliveries for HSV recurrences, total HSV detection, and asymptomatic shedding. Subsequent studies using valacyclovir suppression have shown similar results (Andrews, 2006; Sheield, 2006). Because of these studies, the

1	TABLE 65-4. Oral Antiviral Medications for Herpesvirus Infection in Pregnancya Acyclovir, 400 mg three times daily for 7-10 d Valacyclovir, 1 9 twice daily for 7-1r0 days Acyclovir, 400 mg three times daily for 5 days Acyclovir, 800 mg twice daily for 5 days Acyclovir, 800 mg three times daily for 2 d Valacyclovir, 500 mg twice daily for 3 days Valacyclovir, 1 9 once daily for 5 days Acyclovir,r400 mg three times daily from 36 weeks until delivery Valacyclovir, 500 mg twice daily from 36 weeks until delivery Famciclovir not preferred during pregnancy due to fewer safety data. Data from Workowski, 2015.

1	Valacyclovir, 500 mg twice daily from 36 weeks until delivery Famciclovir not preferred during pregnancy due to fewer safety data. Data from Workowski, 2015. American College of Obstetricians and Gynecologists (2016b) recommends viral therapy at or beyond 36 weeks for women who had primary genital herpes infection or active recurrent genital herpes during pregnancy. It is unclear whether suppression is needed for women with outbreaks before but not during pregnancy. Notably, despite maternal antiviral suppression, several cases of atypical neonatal herpes infection have been reported (Pinninti, 2012).

1	On presentation for delivery, a woman with a history of HSV should be questioned regarding prodromal symptoms such as vulvar burning or itching. A careful examination of the vulva, vagina, and cervix is performed, and women without genitalrlesions may proceed with labor and delivery. Use of a fetal scalp electrode can raise the transmission risk. But, electrode placement is reasonable if needed in the absence of active lesions (American College of Obstetricians and Gynecologists, 2016b). Suspicious lesions should be cultured or PCR tested. Cesarean delivery is indicated for women with genital lesions or prodromal symptoms. It is not recommended for women with a history of HSV infection but no active genital disease at the time of delivery. Moreover, an active lesion in a nongenital area is not an indication for cesarean delivery. Instead, an occlusive dressing is placed, and vaginal delivery is allowed.

1	With preterm ruptured membranes, no evidence suggests that external lesions cause ascending fetal infection. Major and associates (2003) described expectant management of preterm premature membrane rupture in 29 women at gestational ages <31 weeks. There were no cases of neonatal HSV, and the maximum infection risk was calculated to be 10 percent. Antiviral treatment is recommended. For women with a clinical recurrence at delivery, there is not an absolute duration of membrane rupture beyond which the fetus would not beneit from cesarean delivery (merican College of Obstetricians and Gynecologists, 2016d).

1	Women with active HSV may breastfeed if there are no active breast lesions. Strict hand washing is essential. Val acyclovir and acyclovir may be used for symptomatic maternal lesions during breastfeeding, as drug concentrations in breast milk are low. One study found the acyclovir concentration to be only 2 percent of that used for therapeutic dosing of the neonate (Sheield, 2002a).

1	Haemophilus ducryi can cause painul, nonindurated genital ulcers termed sot chancres. At times, these are accompanied by painul suppurative inguinal lymphadenopathy. Although common in some developing countries, only 11 cases were reported in the United States in 2015 (Centers for Disease Control and Prevention, 20 16c). Appropriate media are not widely accessible, and no Food and Drug Administration (FDA)-cleared PCR test is yet available. Instead, painul genital ulcer(s) and negative screening for syphilis or HSV leads to a presumptive diagnosis. Treatment in pregnancy is azithromycin, 1 g orally as a single dose; erythromycin base, 500 mg orally three times daily for 7 days; or cetriaxone, 250 mg in a single intramuscular dose (Workowski, 2015).

1	This is a common STD, and more than 40 types infect the genital tract. In the United States from 2005 to 2006, the overall HPV prevalence was 40 percent in females aged 14 to 59 years (Liu, 2016). Prevalence is highest in younger women, and some of this seroprevalence now relects HPV vaccination in this age group (Brouwer, 2015). Most reproductive-aged women become infected within a few years of becoming sexually active, and most infections are asymptomatic and transient. High-risk types are those with the most oncogenic potential. Of these, HPV types 16 and 18 are oten associated with dysplasia (Chap. 63, p. 1193). Mucocutaneous external genital warts termed condyloma acuminata are usually caused by types 6 and 11r.

1	For unknown reasons, genital warts frequently increase in number and size during pregnancy. hese lesions may sometimes grow to fill the vagina or cover the perineum, thus making vaginal delivery or episiotomy diicult. Maternal HPV infection does not appear to be related to preterm labor (Subramaniam, 2016). Genital wart eradication during pregnancy is usually not neces sary unless they are symptomatic. herapy is directed toward ity to the mother and fetus. Several agents are available, but no deinitive evidence supports superiority of one over another (Workowski, 2015). Response to treatment during pregnancy may be incomplete, but lesions frequently improve or regress rapidly following delivery. Trichloroacetic or bichloracetic acid, 80-to 90-percent solu tion, applied topically weekly, is an efective regimen for external warts. Some prefer cyotherapy, laser ablation, or surgical excision.

1	Trichloroacetic or bichloracetic acid, 80-to 90-percent solu tion, applied topically weekly, is an efective regimen for external warts. Some prefer cyotherapy, laser ablation, or surgical excision. Agents not recommended in pregnancy because of concerns for maternal and fetal safety include podophyllin resin, podoilox solution or gel, imiquimod cream, and sinecatechins. There are three vaccines available for long-term prevention.

1	Gardasil (HPV 4) is a quadrivalent vaccine against HPV types 6,11, 16, and 18. his is being replaced by Gardasil9 (HPV9), a nonavalent vaccine that protects against all the types in HPV 4 plus types 31,r33,r45, 52, and 58. Cervarix (HPV2) is a bivalent vaccine against HPVs 16 and 18. One of these vaccines is selected and given as a three-dose series on a schedule of 0, 1-2, and 6 months for those aged 15 to 26 years. A two-dose regimen, given at 0 and again at 6 to 12 months, is now recommended for girls aged 9 to 14 years (Meites, 2016). Vaccines are licensed for females aged 9 to 26 years, and the target age is 11 to 12 years. he vaccines are not recommended for pregnant women, however, inadvertent exposures do occur. No adverse pregnancy outcomes are associated with the vaccines (Moreira, 2016; Panagiotou, 2015; Vichnin, 2015). If a woman is found to be pregnant after starting the vaccination series, the remaining doses are delayed and given after delivery (American College of

1	2016; Panagiotou, 2015; Vichnin, 2015). If a woman is found to be pregnant after starting the vaccination series, the remaining doses are delayed and given after delivery (American College of Obstetricians and Gynecologists, 2017a). Women who are breastfeeding may receive the vaccine.

1	Vertical transmission rates ofHPV to the newborn are minimal. Juvenile-onset recurrent respiratory papillomatosis QoRRP) is a rare, benign neoplasm of the larynx. It can cause hoarseness and respiratory distress in children and is most often caused by HPV 6 or 11. Risks for infection are maternal genital HPV infection and longer labors (Niyibizi, 2014). Many newborns are likely exposed to HPV, but few develop JoRRP (Silverberg, 2003; Smith, 2004; Tenti, 1999). For example, the national incidence of JoRRP in 2006 in the United States ranged from 0.5 to 1 per 100,000 children (Marsico, 2014). The beneit of cesarean delivery to decrease transmission risk is unknown, and thus it is currently not recommended solely to prevent HPV transmission (Workowski, 2015). HPV vaccination may ultimately decrease JoRRP rates in the uture (Matys, 2012).

1	Pregnant women frequently develop increased vaginal discharge. his may be a physiological discharge, described in Chapter 4 (p. 51), but should be diferentiated from symptomatic vaginitis, which is also common in pregnancy. Fortunately, vaginitis is prevented in part by normal vaginal lora. To better understand this, studies of the composition and function of the normal vaginal microflora are currently underway with the Vaginal Human Microbiome Project (Huang, 2014). Not an infection in the ordinary sense, bacterial vaginosis (BY) is a maldistribution of normal vaginal flora. With BV, num bers of lactobacilli are decreased, and anaerobic bacteria spe cies are overrepresented. hese anaerobes include Gardnerela, Prevotela, Mobiluncus, and Bacteroides species; Atopobium vagi nae; and BV-associated bacteria, provisionally named BVAB 1, BVAB2, and BVAB3. hese last three are newly recognized bacteria found in women with BV (Fredricks, 2005).

1	BVAB2, and BVAB3. hese last three are newly recognized bacteria found in women with BV (Fredricks, 2005). Molecular ribosomal RNA gene sequencing techniques have greatly aided this understanding of the vaginal lora, also called the vaginal m icro biota. Five types of vaginal microbiota exist, referred to as communiy state ypes (eSTs). And, a woman can be categorized to one of these ive CSTs based on her vaginal microbiota composition (Ravel, 2011). Researchers have begun to quantiy the risk of BV by these CST groups. Speciically, CST s I, II, III, and V are rich in lactobacilli. In contrast, CST IV is a heterogeneous microbiota of strict anaerobes and is associated with BV. CSTs vary racially, and CST IV is also the most common in asymptomatic, healthy black women (Fettweis, 2014). Pregnancy-related changes in vaginal micro biota are also being defined and may hold keys to adverse BV-related pregnancy outcomes, discussed subsequently (Romero, 2014).

1	Of childbearing-aged women in the United States, nearly 30 percent have BV. In black women, the prevalence approximates 50 percent (llsworth, 2007). Most women are asymptomatic, but a foul, thin vaginal discharge is a typical complaint. Associated risk factors are douching, multiple partners, smoking, and altered host immunity (Desseauve, 2012; Koumans, 2007; Murphy, 2016) .

1	For clinical diagnosis of BV, three of the four following criteria are present: (1) vaginal pH >4.5; (2) a thin, milky, noninlammatory vaginal discharge; (3) > 20 percent clue cells seen microscopically; and (4) a fishy odor ater addition of 10-percent potassium hydroxide to vaginal secretion samples (Amsel, 1983). The last is described as a positive "whif test." Likewise, alkalinity of seminal fluid and blood are responsible for foul-odor complaints ater intercourse and with menses in afected women. Clue cells are vaginal epithelial cells containing many attached bacteria, which create a poorly deined stippled cellular border (Fig. 65-6). he higher vaginal pH stems from diminished acid production by lactobacilli. Similarly, Trichomonas vaginalis infection is also associated with anaerobic overgrowth and resultant elaborated amines. hus, women diagnosed with BV should have no microscopic evidence of trichomoniasis (see Fig. 65-6).

1	FIGURE 65-6 A. Bacterial vaginosis. Microscopy reveals several squamous cells heavily studded with bacteria. Clue cells are covered to the extent that cell borders are blurred and nuclei are not visible (arrows). B. Trichomonads (arrows). (Reproduced with permission from McCord E, Rahn DO, Hoffman BL: Gynecologic infection. In Hoffman BL, Schorge JO, Bradshaw KD, et al (eds): Williams Gynecology, 3rd ed. New York, McGraw Hill Education, 2016. Photo contributors: Lauri Campagna and Mercedes Pineda, WHNP.) The Nugent score, used primarily in research studies rather than clinical practice, is a system employed for diagnosing BV (Nugent, 1991). During microscopic examination of a gramstained vaginal discharge smear, scores are calculated by assessing bacteria staining and morphology.

1	Several adverse pregnancy-related health outcomes associated with BV are preterm birth, premature rupture of the membranes, and postpartum endometritis (Hillier, 1995; Leitich, 2003; Watts, 1990). It also increases susceptibility to STDs, including HIV (Atashili, 2008; Brotman, 2010). For women at low risk for preterm birth, however, treatment of BV does not reduce preterm birth rates (Brocklehurst, 2013; Carey, 2000). For high-risk women, evidence is conlicting. Currently, the American College of Obstetricians and Gynecologists (2016c), CDC, and U.S. Preventive Services Task Force do not recommend routine BV screening of asymptomatic gravidas-at either high or low risk for pre term delivery-to prevent preterm birth (Nygren, 2008; Workowski, 2015).

1	Treatment is reserved for symptomatic women. Preferred drugs are metronidazole, 500 mg twice daily orally for 7 days; metronidazole 0.75-percent gel, one applicator intravaginally, daily for 5 days; or clindamycin 2-percent cream, one applicator intravaginally nightly for 7 days. Alternatives are clindamycin, 300 mg orally twice daily for 7 days, or 100-mg clindamycin ovules placed intravaginally nightly for 3 days (Workowski, 2015). It is still debated whether BV is a sexually transmitted infection. But, treatment of a male partner does not appear to lower recurrence rates (Amaya-Guio, 2016).

1	Vaginitis caused by Trichomonas vaginalis is common, and its prevalence in the United States approximates 3 percent in nonpregnant and pregnant women (llsworth, 2009; Satterwhite, 2013). The prevalence is higher in those older than 30 years compared with younger women. Risks include black race, douching, and greater number of lifetime sexual partners (Sutton, 2007). Among women, frequent sites of infection include the urethra, endocervix, and vagina. Symptomatic vaginitis is characterized by yellow purulent discharge, pruritus, vulvovaginal erythema, and colpitis macularis, which is often termed a "strawberry cervix" and reflects a patchy, maculoerythematous ectocervix (W0Iner-Hanssen, 1989).

1	Trichomonads are flagellated, pear-shaped, motile organisms that are somewhat larger than leukocytes. These parasites can readily be seen microscopically moving briskly in a sample mixed on a slide with saline. Prompt inspection of vaginal secretions is advantageous because trichomonads slow with cooling. At times, T vaginalis may be found incidentally on a Pap test slide. Both of these microscopic slide tests have low diagnostic sensitivity that approximates only 60 percent (Krieger, 1988; Wiese, 2000). And, Pap tests can yield false-positive results. Thus, Pap test trichomonad indings warrant wet-prep microscopy or other diagnostic confirmation (American College of Obstetricians and Gynecologists, 2017 c). Of other tests, culture is expensive, lengthy, and sensitivities are 75 to 95 percent (Association of Public Health Laboratories, 2016; Huppert, 2007). Laboratory NAA T analysis of a vaginal, endocervical, or urine sample is available, is completed in minutes to hours, and ofers

1	(Association of Public Health Laboratories, 2016; Huppert, 2007). Laboratory NAA T analysis of a vaginal, endocervical, or urine sample is available, is completed in minutes to hours, and ofers superior sensitivity of95 to 100 percent (Schwebke, 2011; Van Der Pol, 2014). Rapid POC testing is also available but may sacriice sensitivity for speed. The OSOi Trichomonas Rapid Test provides results in 10 minutes, is suitable for oice use, and has sensitivities of 88 to 98 percent (Herbst de Cortina, 2016).

1	Metronidazole, administered orally in a single 2-g dose, is efective in eradicating T vaginalis. For those with HIV infection, treatment instead with metronidazole 500 mg orally twice daily for 7 days improves eicacy. Because of the high rate of reinfection among women treated for trichomoniasis, retesting for T vaginalis is recommended for all sexually active women within 3 months following initial treatment (Workowski, 2015). Metronidazole, an FDA category B drug, is not teratogenic or fetotoxic, but has displayed some tumorigenicity in animal stud ies (Briggs, 2015; Czeizel, 1998). For this reason, the manufac turer recommends against its use during the irst trimester (Pfizer, 2016). Fewer data are available for tinidazole, which is a category

1	C drug, and thus metronidazole is preferred. vletronidazole and tinidazole have similar chemical structures, and those allergic to metronidazole may also react to tinidazole. For allergic patients, metronidazole desensitization is efective, and one scheme is out lined in the study by Helms and coworkers (2008). With post partum breastfeeding, feeds are delayed for 24 hours following the mation. For tinidazole, the delay is 72 hours.

1	Perinatal transmission of trichomoniasis by direct contact in the birth canal is rare but may lead to neonatal respiratory or genital infection (Bruins, 2013; Trintis, 2010). Some studies have linked trichomonal infection with preterm birth. A few other studies implicate this infection with preterm premature rupture of membranes and small-for-gestational age newborns (Silver, 2014). However, treatment did not lower preterm birth rates in a randomized study by Klebanof and colleagues (2001). Also, in this study, but not one by Mann and coworkers (2009), treatment for trichomoniasis was associated instead with a higher preterm birth rate.

1	In sum, treatment for symptomatic women is reasonable and outlined above. For most asymptomatic women during pregnancy, screening is not recommended. However, for pregnant women with HIV infection, screening at the first prenatal visit and prompt treatment are encouraged. his is because T vaginalis infection in pregnant women with HIV may be a risk factor for vertical HIVtransmission (Gumbo, 2010; Workowski, 2015). Candida albicans or other candidal species can be identified by culture from the vagina during pregnancy in approximately 20 percent of women. A link between candidiasis and preterm birth is not robust (Cotch, 1998; Kiss, 2004; Roberts, 2015). hus, asymptomatic colonization requires no treatment. he organism, however, can create an extremely profuse, irritating discharge.

1	For symptoms, efective treatment is a 100-mg miconazole vaginal suppository or a 2-percent butoconazole, I-percent clotrimazole, 2-percent miconazole, or 0.4-percent terconazole cream, any of which is used daily for 7 days. A shorter, 3-day regimen is daily 2-percent clotrimazole, 4-percent miconazole, or 0.8-percent tioconazole cream, or daily 200-mg miconazole or 80-mg terconazole suppository (Workowski, 2015). In some women, infection is likely to recur and require repeated treatment during pregnancy. In these cases, symptomatic infection usually subsides after pregnancy (Sobel, 2007). For treatment, the American College of Obstetricians and Gynecologists (2017 c) and the CDC recommend topical rather than oral azoles for symptoms. As discussed urther in Chapter 12 (p. 241), oral fluconazole is generally not considered teratogenic, but in 2016, the FDA released a safety alert regarding a possible link with miscarriage (M0Igaard-Nielsen, 2016).

1	Causative agents of acquired immunodeficiency syndrome (AIDS) are RNA retroviruses termed human immunodiciency viruses, HIV-J and HIV-2. Most cases worldwide are caused by HIV-l infection. Sexual intercourse is the major mode of transmission. he virus also is passed by blood, and infected by breast milk. he primary determinant of transmission is the plasma HIV-l viral load. For sexual transmission, the viral HIV envelope binds to mucosal dendritic cells. These cells then present the viral par ticle to specific T lymphocytes. These lymphocytes are defined phenotypically by their cluster of dierentiation 4 (CD4) glyco protein surface antigens. The CD4 site serves as a receptor for the virus. Once infected, CD4 T lymphocytes may die, and the common denominator of clinical illness with AIDS is pro found immunodeficiency that gives rise to various opportunis tic infections and neoplasms.

1	In the United States, the CDC (2016c) estimated that more than 1.2 million individuals were infected in 2013, and new cases numbered more than 39,000. Approximately 8500 women with HIV give birth annually in the United States. However, the estimated number of perinatally acquired HIV cases has decreased dramatically, and the perinatal transmission rate in 2013 was 1.8 percent (Centers for Disease Control and Prevention, 2016b, 2017). his is predominantly due to the implementation of prenatal HIV testing and antiretroviral therapy (ART) for the woman and then her neonate.

1	he incubation period from exposure to clinical disease averages 3 to 6 weeks. Acute HIV infection is similar to many other viral syndromes and usually lasts less than 10 days. Common symptoms, if any, include fever, fatigue, rash, headache, lymphadenopathy, pharyngitis, myalgias, nausea, and diarrhea. After symptoms abate, the level of viremia usually decreases to a set point, and patients with the highest viral burden at this time progress more rapidly to AIDS and death (Fauci, 2007). According to the CDC, AIDS is defined by a CD4 T-cell count <200 cells/lLL, by CD4 T-cells comprising < 14 percent of alirlymphocytes, or one of several AIDS-defining illnesses (Schneider, 2008; Selik, 2014). Route of infection, the pathogenicity of the infecting viral strain, the initial viral inoculum, and the immunological status of the host all afect the rapidity of progression.

1	he CDC (2006b) and the American College of Obstetricians and Gynecologists (2016e) recommend prenatal HIV screening using an opt-out approach. his means that a woman is notiied that HIV testing is included in a comprehensive set of antenatal tests, but that testing may be declined. Women are given information regarding HIV but are not required to sign a specific consent. hrough the use of such opt-out strategies, HIV testing rates have increased. Specific state laws concerning screening vary and can be found at: ww.cdc.gov/hiv/policies/ law / states/ testing.h tml.

1	Repeat testing during the third trimester, preferably before 36 weeks' gestation, is considered for all pregnant women. Retesting is recommended for those at risk for acquiring HIV or for women in high-risk areas, namely, those with rates of HIV infection of 1 per 1000 pregnant women screened (Workowski, 2015). At-risk factors include injection drug use, prostitution, a suspected or known HIV-infected sexual partner, multiple sexual partners, or a diagnosis of another STD (American College of Obstetricians and Gynecologists, 20 16e).

1	he initial laboratory screening test for HIV is an antigen/ antibody combination immunoassay that detects antibodies against HIV-1 and HIV-2 and detects HIV-1 p24 antigen (Centers for Disease Control and Prevention, 2014). Antibody can be detected in most patients within 1 month of infection, and thus, antibody serotesting may not exclude early infection. Instead, for acute primary HIV infection, identiication of viral p24 core antigen or viral RNA is possible. No further testing is required for specimens that are negative on the initial immunoassay unless a known exposure to HIV has occurred.

1	As shown in Figure 65-7, specimens with a "reactive" (that is, a positive) antigen/antibody combination immunoassay result should be tested with an antibody immunoassay that diferentiates HIV-1 antibodies from HIV-2 antibodies. The HIV-1/ HIV-2 antibody diferentiation immunoassay is resulted as positive or negative for HN-1 antibodies, for HN-2 antibodies, or for HIV antibodies, undiferentiated. If these two serial immunoassays are discordant, an HIV -1 NAA T -qualitative or quantitative HIV RNA test-is performed (Centers for Disease Control and Prevention, 2014).

1	Women with undocumented HIV status at delivery should have a fourth-generation HIV antigen/antibody combination screening test performed on a blood sample. A negative screening test result does not need confirmation. However, in cases of recent HIV exposure, consideration is given to peripartum interventions to reduce perinatal transmission despite negative HIV testing. Repeat interval testing is recommended to exclude very early infection not identified with the initial screen. With a positive fourth-generation HIV testing result, peripartum and neonatal interventions to reduce perinatal transmission are initiated. This includes avoidance of breastfeeding, although breast milk may be stored until confirmatory test results are available. Interventions can be discontinued if confirmatory testing is negative. To confirm a positive result from any initial HIV test, the laboratory testing algorithm in Figure 65-7 should be used and begins with the antigen/antibody combination immunoassay.

1	Viral burden and neonatal infection rates are directly related. In one cohort, neonatal infection was 1 percent with < 400 copies/mL, and it was 23 percent when maternal viral RNA levels were >30,000 copies/mL (Cooper, 2002). Among 2615 infants born to mothers taking ART before conception and during pregnancy, there were no cases of vertical transmission at delivery (Mandelbrot, 2015). Transmis sion of HIV infection, however, has been observed at all HIV RNA levels, including .r assays. Transplacental HIV transmission Negative for HIV-1 and HIV-2 can occur early, and the virus has even antibodies and p24 antigen HIV-1/HIV-2 antibody differentiation immunoassay abortion (Lewis, 1990). Kourtis and col leagues (2001) estimated that 20 percent of vertical transmission occurs before 36

1	HIV-1/HIV-2 antibody differentiation immunoassay abortion (Lewis, 1990). Kourtis and col leagues (2001) estimated that 20 percent of vertical transmission occurs before 36 HIV-1 (+) HIV-1 (-) HIV-1 (+) weeks' gestation, 50 percent in the days HIV-2 (-) HIV-2 (+) HIV-2 (+) before delivery, and 30 percent intrapar tum. Transmission rates for breastfeeding may be as high as 30 to 40 percent and HIV-1 NAAT are associated with increasing HIV viral burden (Kourtis, 2006, 2007; Slyker, 2012). In nonpregnant individuals, con mission are linked. Evidence also supports that vertical transmission rates may be FIGURE 65-7 Algorithm for HIV testing. In the pathway colored light-blue, for specimens increased by comorbid STDs (Schulte, that are reactive on the initial antigen/antibody combination immunoassay and nonreac tive or indeterminate on the HIV-1/HIV-2 antibody diferentiation immunoassay, a nucleic 2001; Watts, 2012).

1	acid amplification test (NAAT) is implemented. A positive HIV-1 NAAT result and nonreac for acute HIV-1 infection. A positive HIV-1 NAAT result and indeterminate HIV-1 /HIV-2 • Antepartum Care antibody diferentiation immunoassay result indicate the presence of HIV-1 con Pregnant women with HIV infection need firmed by HIV-1 NAAT. A negative HIV-1 NAAT result and nonreactive or indeterminate special attention and are seen in consultation on the initial antigen/antibody combination immunoassay. (Reproduced with permission by physicians with special interest in this from Centers for Disease Control and Prevention, 2014.) field. An additional resource is the National Perinatal HIV Hotline (1-888-448-8765), which is a federally unded service that provides free antepartum, intrapartum, or postpartum consultation to providers. At Parkland Hospital, an HIVinfected pregnant woman is initially assessed with the following:

1	Standard prenatal laboratory surveys that include serum creatinine, complete blood count, and bacteriuria screening Plasma HIV RNA quantification-"viralrload" -CD4 T-cell count, and antiretroviral resistance testing HSV-1 and 2, cytomegalovirus, toxoplasmosis, and hepatitis Brand C serological screening Tuberculosis testing with puriied protein derivative (PPD) skin testing, or interferon-gamma release assay Evaluation of need for pneumococcal, hepatitis A, hepatitis B, T dap, and influenza vaccines Sonographic evaluation to establish gestational age.

1	Evaluation of need for pneumococcal, hepatitis A, hepatitis B, T dap, and influenza vaccines Sonographic evaluation to establish gestational age. During pregnancy, the risk of HIV transmission does not appear to be increased with amniocentesis or other invasive diagnostic procedures in women receiving efective ART resulting in viral suppression (Floridia, 2017). For women not receiving ART, the risk rises approximately twofold (Mandelbrot, 1996). If amniocentesis is performed, eforts are taken to avoid passing through the placenta (Panel on Treatment of HI V-Infected Pregnant Women and Prevention of Perinatal Transmission, 2016).

1	In overview, the ideal strategy to suppress viral load and minimize vertical HIV transmission includes: (1) preconceptional ART, (2) antepartum ART, (3) intrapartum continuation of the antepartum oral ART regimen plus IV zidovudine, and (4) newborn ART prophylxis. ART is recommended for all HIVinfected pregnant women, and it should be initiated as early in pregnancy as possible. Treatment reduces the risk of perinatal transmission regardless of CD4 T-cell count or HIV RNA level. Adherence is essential because the risk of viral drug resistance is lessened. As for nonpregnant adults, pregnant women are treated with at least three antiviral agents. The Panel on Treatment of HI V-Infected Pregnant Women and Prevention of Perinatal Transmission (2016) has issued guidelines for four diferent scenarios during pregnancy (Table 65-5). he following paragraphs summarize these recommendations.

1	First, women already taking ART at pregnancy onset are encouraged to continue the regimen if viral suppression is adequate. Didanosine, stavudine, and full-dose ritonavir, which difers from ritonavir-boosted agents, are exceptions due to pregnancy toxicity but not teratogenicity. Second, women who have never received antiretroviral therapy-antiretroviral nai've-are given ART regardless of trimester. In general, the starting regimen comprises two nucleoside reverse transcriptase inhibitors plus either a ritonavir-boosted protease inhibitor or an integrase inhibitor.

1	hird, women who have previously received antiretroviral therapy but are currently not taking medications should undergo HIV resistance testing because prior ART use raises their risk of drug resistance. Typically, ART is initiated prior to receiving results of these drug-resistance tests. In this case, initial ART selection should factor results of prior resistance testing, if available; prior ART regimen; and current ART preg nancy guidelines, that is, those for ART-naive women. Drug resistance testing may then modiy the initial regimen. For these three categories of women taking antepartum ART, therapy surveillance is outlined in Table 65-5. Most patients with adequate viral response have at least a I-log viral load decline within 1 to 4 weeks after starting therapy. For those who fail to achieve this decline, options include review of drug resistance study results, confirmation of regimen compli ance, and ART modiication.

1	During labor and delivery, oral medications can be taken with sips of water. Additionally, IV zidovudine is given to women with an HIV RNA viral load > 1000 copies/ mL or who have an unknown viral load near delivery. At Parkland Hospital, we administer intrapartum IV zidovudine to all HIV-positive women, regardless of viral load. A 2 mg/kg load delivery. In this instance, for gravidas already taking antepar tum oral zidovudine, their oral dose can be held, and IV drug is instead administered. HIV-infected women undergoing a scheduled cesarean delivery are given IV zidovudine as a load ing dose followed by 2 more hours of continuous maintenance therapy-a total of 3 hours of infused zidovudine. who are taking no medications. hese women are given IV zid ovudine intrapartum as just described.

1	who are taking no medications. hese women are given IV zid ovudine intrapartum as just described. During labor, artificial membrane rupture, fetal scalp electrode placement, episiotomy, and operative vaginal delivery are reserved for clear obstetrical indications (Mandelbrot, 1996; Peters, 2016). Labor augmentation is used when needed to shorten the interval to delivery to further lower the transmission risk. Delayed cord clamping in preterm neonates is acceptable. Neuraxial analgesia is suitable. Postpartum hemorrhage is best managed with oxytocin and prostaglandin analogues. Methylergonovine (Methergine) and other ergot alkaloids adversey interact with reverse transcriptase and protease inhibitors to cause severe vasoconstriction.

1	In some cases, cesarean delivery lowers HIV prenatal transmission (European Mode of Delivery Collaboration, 1999; International Perinatal HIV Group, 1999). The American College of Obstetricians and Gynecologists (2017b) recommends that scheduled cesarean delivery be discussed and recommended for HIV-infected women with HIV-1 RNA loads > 1000 copies/mL. Scheduled delivery is recommended at 38 weeks' gestation in these women to avert spontaneous labor.

1	For women with HIV RNA levels �1000 copies/mL, data are insuicient to predict similar beneits, and scheduled cesarean delivery is unlikely to confer additional risk reduction for women already taking ART and achieving viral suppression (Briand, 2013; Jamieson, 2007; Read, 2005). Vaginal delivery in this group may be elected. However, if cesarean delivery is instead chosen for a well-counseled woman in this group, it should be performed at 39 weeks. Similarly, cesarean delivery performed for obstetrical indications in this lower-viral-Ioad group should be done at 39 weeks when possible. TABLE 65-5. Recommendations for HIV Antiviral Drug Use During Pregnancy Antepartum care See antepartum screening test list (p. 1249)

1	TABLE 65-5. Recommendations for HIV Antiviral Drug Use During Pregnancy Antepartum care See antepartum screening test list (p. 1249) ART should be initiated as early as possible For those with HIV RNA levels >500-'1000 copies/mL, order HIV antiretroviral drug resistance testing but do not delay ART initiation awaiting results Repeat HIV RNA levels 2-4 weeks after initiating (or changing) ART drugs; monthly until RNA levels are undetectable; then at least every 3 mo; and finally at 34-36 weeks' gestation for delivery planning CD4+ count should be monitored at the initial visit and every 3-6 mo

1	CD4+ count should be monitored at the initial visit and every 3-6 mo Intrapartum care If HIV RNA levelr> 1 000 copies/mL or is unknown before labor or ROM, plan cesarean delivery at 38 weeks' gestation If HIV RNA levelsr> 1000 or is unknown but labor or ROM has ensued, benefits of cesarean delivery are unclear and labor plans are individualized If HIV RNA levelr:;1 000 copies/mL, vaginal delivery is permitted; cesarean delivery not routinely recommended Start IV ZDV if HIV RNA levelr> 1 000 copies/mL near delivery or is unknown. Dosing is 2 mg/kg IV load over 1 hr, then 1 mg/kg/hr until delivery. IV ZDV should begin 3 hr before scheduled cesarean delivery Those taking oral antepartum ART should take this during labor with sips of water

1	IV ZDV should begin 3 hr before scheduled cesarean delivery Those taking oral antepartum ART should take this during labor with sips of water ART = antiretroviral therapy; NRTI = nucleoside reverse transcriptase inhibitor; ROM = rupture of membranes; ZDV = zidovudine. Adapted from the Panel on Treatment of HIV-Infected Pregnant Women and Prevention of Perinatal Transmission, 2016. Department of Health and Human Services.

1	healthy woman with HIV has no signiicant efect on disease Vertical transmission is increased by breastfeeding, and it genprogression (Calvert, 2015). Linkage to general HIV care posterally is not recommended for HIV-positive women in the partum is critical to maintain viral suppression (Swain, 2016). United States, where formula is readily available (Read, 2003). For women who do not have HIV infection, but whose In nutritionally deprived countries, where infectious disease partner is seropositive, current guidance supports the use of and malnutrition are primary causes of infant death, the World highly active antiretroviral therapy with viral suppression in the Health Organization (2016) recommends exclusive breastfeeding infected partner (treatment as prevention), and consideration during the first 6 to 12 months. of antiretroviral preexposure prophylaxis (PreP) for the HIV he Panel on Treatment of HI V-Infected Pregnant Women negative partner. The well-counseled couple can consider

1	the first 6 to 12 months. of antiretroviral preexposure prophylaxis (PreP) for the HIV he Panel on Treatment of HI V-Infected Pregnant Women negative partner. The well-counseled couple can consider periand Prevention of Perinatal Transmission (2016) strongly recovulatory condomless intercourse, or uterine insemination or ommends that ART regimens not be discontinued postpartum in vitro fertilization after sperm washing for assisted conception but continued lifelong for the advantages of viral suppression. (Brooks, 2017; Kawwass, 2017). Ideally, all those planning pregnancy should be receiving ART If, instead, pregnancy is undesired, efective contraception is and have a plasma viral load below detectable levels before discussed (Chap. 38, p. 680). Counseling also includes education conception. As one benefit, interpregnancy viral load suppresfor decreasing high-risk sexual behaviors to prevent transmission sion is associated with less vertical transmission in a subsequent and to

1	As one benefit, interpregnancy viral load suppresfor decreasing high-risk sexual behaviors to prevent transmission sion is associated with less vertical transmission in a subsequent and to decrease other STD acquisition. Similarly, women with pregnancy (French, 2014; Mandelbrot, 2015; Stewart, 2014; HIV have unique gynecological issues, such as genital neoplasia, Townsend, 2014). Reassuringly, for those seeking subsequent which require special attention (American College of Obstetripregnancy, when ART is available, repeated pregnancy in a cians and Gynecologists, 2016a; Werner, 2016).

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1	Xu F, Lee FK, Morrow A,et al: Seroprevalence of herpes simplex virus type 1 in children in the United States. J Pediatr 151(4):374,t2007 Zhou P, Qian Y, Xu J, et al: Occurrence of congenital syphilis after matenal treatment with azithromycin during pregnancy. Sex Transm Dis 34:472, 2007 Zhou Y, Bian G, Zhou Q, et al: Detection of cytomegalovirus, human parvovirus B 19, and herpes simplex virus-112 in women with irst-trimester spontaneous abortions. J Med Virol 87(10): 1749,t2015 I. SERUM AND BLOOD CONSTITUENTS ............... 1255 II. MATERNAL ECHOCARDIOGRAPHIC MEASUREMENTS .. 1261 III. FETAL SONOGRAPHIC MEASUREMENTS ...r........ 1262 APPENDIX I. Serum and Blood Constituents Erythropoietinb (U/L) 4-27 12-25 8-67 14-222 7,10,47 Ferritinb (ng/mL) 10-150d 6-130 2-230 0-116 � lQ 3�r42,r45,r4�r62, 70 Folate, red blood cell 150-450 137-589 94-828 109-663 45,r46,r72 (ng/mL) Folate, serum (ng/mL) 5.4-18.0 2.6-15.0 0.8-24.0 1.4-20.7 7,r43,r45,r46,r53,r58,r72

1	Folate, red blood cell 150-450 137-589 94-828 109-663 45,r46,r72 (ng/mL) Folate, serum (ng/mL) 5.4-18.0 2.6-15.0 0.8-24.0 1.4-20.7 7,r43,r45,r46,r53,r58,r72 Hemoglobinb (g/dL) 12-15.8d 11.6-13.9 9.7-14.8 9.5-15.0 10,45,47,58,r62 HematocritiJ (%) 35.4-44.4 31.0-41.0 30.0-39.0 28.0-40.0 6, 7, 10,42,45 58,66 Iron, total binding 251-406 278-403 Not reported 359-609 62 capacity (TIBC)b (.Lg/dL) Iron, serum.b (Lg/dL) 41-141 72-143 44-178 30-193 10,62 Mean corpuscular volume 79-93 81r-96 82-97 81r-99 6,r42,r45,r58 Platelet (x109 /L) 165-41r5 174-39r1 155-409 146-429 4,r6,r16,42,r45 Mean platelet volume 6.4-11.0 7.7-10.3 7.8-10.2 8.2-10.4 42 Red blood cell count 4.00-5.20 3.42-4.55 2.81-4.49 2.71-4.43 6,42,45,58 Red cell distribution <14.5 12.5-14.1 13.4-13.6 12.7-15.3 42 White blood cell count 3.5-9.1 5.7-13.6 5.6-14.8 5.9-16.9 6,9,42,45,r58 Neutrophils (x 103/mm3) 1.4-4.6 3.6-10.1 3.8-12.3 3.9-13.1 4,6,9,42 Lymphocytes (x103/mm3) 0.7-4.6 1.1-3.6 0.9-3.9 1.0-3.6 4,6,9,42

1	Neutrophils (x 103/mm3) 1.4-4.6 3.6-10.1 3.8-12.3 3.9-13.1 4,6,9,42 Lymphocytes (x103/mm3) 0.7-4.6 1.1-3.6 0.9-3.9 1.0-3.6 4,6,9,42 Monocytes (x103/mm3) 0.1-0.7 0.1-1.1 0.1-1.1 0.1-1.4 6,9,42 Eosinophils (x 103/mm3) 0-0.6 0-0.6 0-0.6 0-0.6 6, 9 Basophils (x103/mm3) 0-0.2 0-0.1 0-0.1 0-0.1 6,r9 200-400c 254-344 220-441 288-530 39,r42 Transferrin, saturation 22-46b Not reported 10-44 5-37 Transferrin, saturation 22-46b Not reported 18-92 9-98 Creatinine (mg/dL) 0.5-0.9d 0.4-0.7 0.4-0.8 0.4-0.9 39,r42,r45 Gamma-glutamyl 9-58 2-23 4-22 3-26 5,r42,r39,r70 transpeptidase (GGT) Lactate dehydrogenase 115-221 78-433 80-447 82-524 42,29,39,r70 Magnesium (mg/dL) 1.5-2.3 1r.6-2.2 1.5-2.2 1.1r-2.2 3,r26,r29,r39,r42,r48,r63 Osmolality (mOsm/kg H2O) 275-295 275-280 276-289 278-280 17,r63 Phosphate (mg/dL) 2.5-4.3 3.1r-4.6 2.5-4.6 2.8-4.6 3,r26,r33,r39,r42 Potassium (mEq/L) 3.5-5.0 3.6-5.0 3.3-5.0 3.3-5.1 20,r26,r29,r39,r42,r63,r66

1	Phosphate (mg/dL) 2.5-4.3 3.1r-4.6 2.5-4.6 2.8-4.6 3,r26,r33,r39,r42 Potassium (mEq/L) 3.5-5.0 3.6-5.0 3.3-5.0 3.3-5.1 20,r26,r29,r39,r42,r63,r66 Protein, total (g/dL) 6.7-8.6 6.2-7.6 5.7-6.9 5.6-6.7 26,r29,r42 Sodium (mEq/L) 136-146 133-148 129-148 130-148 17,26,29,39,42,63,66 Urea nitrogen (mg/dL) 7-20 7-1r2 3-1r3 3-1r1 20,r39,r42 Uric acid (mg/dL) 2.5-5.6d 2.0-4.2 2.4-4.9 3.1r-6.3 17,r39,r42 Aldosterone (ng/dL) 2-9 6-104 9-104 15-101 21,r34,69 Angiotensin-converting 9-67 1-38 1-36 1-39 20, 54 Cortisol (19/dL) 0-25 7-1r9 10-42 12-50 42,r69 4-6 4-7 48,r49,r59 Thyroxine, total (T 4) 5.4-1r1.7 6.5-10.r1 7.5-1r0.3 6.3-9.7 29,r42 C-reactive protein (CRP) 0.2-3.0 Not reported 0.4-20.3 0.4-8.1 28 Dehydroepiandrosterone 1.3-6.8e 2.0-16.5 0.9-7.8 0.8-6.5 52 sulfate (DHEAS) Estradiol (pg/mU <20-443dJ 188--2497 1278-7192 6137-3460 1.3,52 Progesterone (ng/mL) <1-20d 8-48 99-342 13,n52 Prolactin (ng/mL) 0-20d 36-2n13 11n0-330 137-372 3,n13,n38,n49

1	Estradiol (pg/mU <20-443dJ 188--2497 1278-7192 6137-3460 1.3,52 Progesterone (ng/mL) <1-20d 8-48 99-342 13,n52 Prolactin (ng/mL) 0-20d 36-2n13 11n0-330 137-372 3,n13,n38,n49 Sex hormone binding 18-114d 39-131 214-717 216-724 1,52 Testosterone (ng/dL) 6-86d 25.7-211.4 34.3-242.9 62.9-308.6 17-Hydroxyprogesterone 0.6-1 O.6d.e 5.2-28.5 5.2-28.5 15.5-84 52 P02 (mm Hg) 90-100 93-100 90-98 92-107 23,67 pH 7.38-7.42 7.36-7.52 7.40-7.52 7.41r-7.53 23, 26 7.39-7.45 aUnless otherwise specified, all normal reference values are from the seventeenth edition of Harrison's Principles of Internal Medicine (37). bRange includes references with and without iron supplementation. (Reference values are from Laboratory Reference Handbook, Pathology Department, Parkland Hospital, 2005. dNormal reference range is specific range for females.

1	(Reference values are from Laboratory Reference Handbook, Pathology Department, Parkland Hospital, 2005. dNormal reference range is specific range for females. =Reference values are from the 15th edition of Harrison's Principles of Intenal Medicine (1r2). fRange is for premenopausal females and varies by menstrual cycle phase. 9Reference values are from Cerneca et al: Coagulation and fibrinolysis changes in normal pregnancy increased levels of procoagulants and reduced levels of inhibitors during pregnancy induce a hypercoagulable state, combined with a reactive fibrinolysis (1r5). hReference values are from Cerneca et al and Choi et al: Tissue plasminogen activator levels change with plasma fibrinogen concentrations during pregnancy (1r5, 16). 'Reference values are from Mannucci et al: Changes in health and disease of the metalloprotease that cleaves von Willebrand factor (44A).

1	'Reference values are from Mannucci et al: Changes in health and disease of the metalloprotease that cleaves von Willebrand factor (44A). jReference values are from Bacq et al: Liver function tests in normal pregnancy: a prospective study of 102 pregnant women and 102 matched controls (5). kReference values are from et al: Creatine kinase and its MB isoenzyme in the third trimester and the peripartum period (41). 'Reference values are from Dunlop: Serial changes in renal haemodynamics during normal human pregnancy (1r9). Appendix courtesy of Dr. Mina Abbassi-Ghanavati and Dr. Laura G. Greer. APPENDIX II. Maternal Echocardiographic Measurements IVSd (mm) 7.3±1.0 7.4 ± 1.1 7.8 ± 1.2 7.1 ± 0.9 LVEDD (mm) 45-47.8 47-48.9 47-49.6 46-48.8 LVESD (mm) 28-30 29-30.1 30-30.8 28-30.6 PWd 6.3 ± 0.7 6.6 ± 0.7 6.9 ± 1.0 6.1 ± 0.6 RVT 0.26-0.36 0.27-0.37 0.28-0.38 0.25-0.35 PW thickening (%) 66 ± 16 72 ± 16 74 ± 16 71 ± 14 VCFC (circ/sec) 1.15-0.3 1.18-0.16 1.18-0.12 1.18-0.·12

1	PWd 6.3 ± 0.7 6.6 ± 0.7 6.9 ± 1.0 6.1 ± 0.6 RVT 0.26-0.36 0.27-0.37 0.28-0.38 0.25-0.35 PW thickening (%) 66 ± 16 72 ± 16 74 ± 16 71 ± 14 VCFC (circ/sec) 1.15-0.3 1.18-0.16 1.18-0.12 1.18-0.·12 Mitral E wave (m/sec) 0.85 ± 0.13 0.84 ± 0.16 0.77 ± 0.15 0.77 ± 0.11 Mitral A wave (m/sec) 0.5 ± 0.09 0.5 ± 0.1 0.55 ± 0.1 0.46 ± 0.1 Values are ranges or means ± SD. Circ = circumference; d = diastolic; ESS = end-systolic wall stress; FS = fractional shortening; IVRT = isovolumic relaxation time; IVSd = interventricular septum-diastole; LV = let ventricle; LVEDD = left ventricular end-diastolic dimension; LVESD = let ventricular end-systolic dimension; PW = posterior wall; RWT = relative wall thickening; SW = septal wall; VCFC = rate-adjusted mean velocity of circumferential fiber thickening. Data from Savu (62A) and Vitarelli (71 A). APPENDIX III. Fetal Sonographic Measurements

1	Data from Savu (62A) and Vitarelli (71 A). APPENDIX III. Fetal Sonographic Measurements TABLE 111-1. Mean Gestational Sac Diameter and Crown-Rump Length and Corresponding Menstrual Age 32 4.6 3 34 4.9 5 36 5.1 6 38 5.4 8 10 0.2 42 6.0 12 OJ5 44 6J 14 0.5 46 6.6 16 0.7 48 6.9 18 0.9 50 7.1 1r.0 52 7.4 22 1.2 54 7.7 24 1.4 56 8.0 58 8.3 27 1.8 60 8.6 29 2.0 8.9 31 2.2 64 9.1 33 2.4 66 9.4 2.6 2.9 70 10.0 3.1 74 10.6 3.7 76 10.9 4.0 78 11.1 47 4.2 80 11.4 49 4.6 12.0 53 5.4 Data from Nyberg, 1992; Hadlock, 1992; 1975; Daya, 1991r. TABLE 111-2. Mean Gestational Age Percentiles Corresponding to Crown-Rump Length (CRL) Measurements TABLE 111-3. Fetal Weight Percentiles According to Gestational Age Adapted with permission from Hadlock, 1991n. TABLE 111-4. Smoothed Birth Weight Percentiles for Twins with Dichorionic Placentation GA = gestational age. Reproduced with permission from Ananth, 1998.

1	TABLE 111-4. Smoothed Birth Weight Percentiles for Twins with Dichorionic Placentation GA = gestational age. Reproduced with permission from Ananth, 1998. TABLE 111-5. Smoothed Birth Weight Percentiles for Twins with Monochorionic Placentation Reproduced with permission from Ananth, 1998. TABLE 111-6. Fetal Thoracic Circumference Measurements (cm) According to Gestational Age

1	Age (wk) No. 2.5 5 10 25 50 75 90 95 97.5 16 6 6.4 7.0 8.0 9.1 10.3 11.3 11.9 12.4 8.9 10.0 11.2 12.2 12.8 13.3 22 6.8 8.2 8.8 9.8 1 '1.0 12.1 13.1 13.7 14.2 19 21 8.6 9.7 10.7 11.9 13.0 14.0 14.6 15.1 20 20 9.6 10.0 10.6 11.7 12.8 13.9 15.0 15.5 16.0 30 10.4 11.0 11.6 12.6 13.7 14.8 15.8 16.4 16.9 18 11.3 11.9 12.5 13.5 14.6 15.7 16.7 17.3 17.8 12.2 12.8 13.4 14.4 15.5 16.6 17.6 18.2 18.8 27 13.2 13.7 14.3 15.3 16.4 17.5 18.5 19.1 19.7 20 14.1 14.6 15.2 16.2 17.3 18.4 19.4 20.0 20.6 25 15.0 15.5 16.1 17.1 18.2 19.3 21n.0 21n.5 27 24 15.9 16.4 17.0 18.0 19.1 20.2 21.3 21.9 22.4 24 16.8 17.3 18.9 20.0 21.2 22.2 22.8 23.3 24 17.7 18.2 18.8 19.8 21.0 22.1 23.1 23.7 24.2 18.6 19.1 19.7 21n.9 23.0 24.0 24.6 25.1 24 19.5 20.0 20.6 21.6 22.8 23.9 24.9 25.5 26.0 20.9 32 28 21n.5 22.6 23.7 24.8 25.8 26.4 26.9 21.3 21.8 22.5 23.5 24.6 25.7 27.3 27.8 28.2 28.722.2 22.8 23.4 24.4 25.5 26.6 27.6 20 23.1 23.7 24.3 25.3 26.4 27.5 28.5 29.1 29.6 23 24.0 24.6 25.2 26.2 27.3 28.4 29.4 30.0 30.6 37 22

1	26.4 26.9 21.3 21.8 22.5 23.5 24.6 25.7 27.3 27.8 28.2 28.722.2 22.8 23.4 24.4 25.5 26.6 27.6 20 23.1 23.7 24.3 25.3 26.4 27.5 28.5 29.1 29.6 23 24.0 24.6 25.2 26.2 27.3 28.4 29.4 30.0 30.6 37 22 24.8 25.5 26.1 27.1 28.2 29.3 30.3 30.9 31.5 25.9 26.4 27.0 28.0 29.1 30.2 31.2 31.9 32.4 39 7 26.8 27.3 27.9 30.0 31.1 32.2 32.8 33.3 6 27.7 28.2 28.8 29.8 30.9 32.1 33.1 33.7 34.2

1	Reproduced with permission from Chitkara, 1987. Reproduced with permission from Jeanty, 1983, TABLE 111-8. Ocular Parameters According to Gestational Age Adapted with permission from Romero R, 1988. TABLE 111-9. Transverse Cerebellar Diameter Measurements According to Gestational Age Age (wk) 10 25 50 75.. 90 23 21.5 28 28 29 40.5 43 38 40 40 48.5 52 55 Adapted with permission from Goldstein, 1987.

1	Age (wk) 10 25 50 75.. 90 23 21.5 28 28 29 40.5 43 38 40 40 48.5 52 55 Adapted with permission from Goldstein, 1987. TABLE 111-10. Reference Values for Umbilical Artery Doppler Indices 23 0.63 2.71 0.73 3.73 0.83 24 0.62 2.64 0.72 3.59 0.82 25 0.61 2.57 0.71 3.46 0.81 5.28 26 0.60 2.50 0.70 0.80 5.01 27 0.59 244 0.69 3.22 0.79 4.76 28 0.58 2.38 0.68 3.1n2 0.78 29 0.57 2.32 0.67 3.02 0.77 30 0.56 2.26 0.66 2.93 0.76 4.'14 31 0.55 2.21 0.65 2.84 0.75 32 0.54 2.1n6 0.64 2.76 0.74 3.81 0.53 2.1n1 0.63 2.68 0.73 3.66 0.52 2.07 0.62 2.61 0.72 3.53 0.51 2.03 0.61 2.54 0.71 340 36 0.50 1n.98 0.60 2.47 0.70 3.29 37 049 1n.94 0.59 2.41 0.69 3.18 38 0.47 1.90 0.57 2.35 0.67 3.08 39 0.46 1n.87 0.56 2.30 0.66 2.98 40 0n45 1.83 0.55 2.24 0.65 2.89 0.44 1.80 0.54 2.1n9 0.64 2.81 0.43 1.76 0.53 2.14 0.63 2.73 GA = gestational age. Adapted with permission from Kofnas AD, 1992. 1. Acromite MT, Mantzoros CS, Leach E, et al: Androgens in preeclampsia. mJ Obstet Gynecol 180:60, 1999 2.

1	GA = gestational age. Adapted with permission from Kofnas AD, 1992. 1. Acromite MT, Mantzoros CS, Leach E, et al: Androgens in preeclampsia. mJ Obstet Gynecol 180:60, 1999 2. Alvarez S1, Castanon SG, Ruata MLC, et al: Updating of normal levels of copper, zinc and selenium in serum of pregnant women. J Trace Elem Med BioI 21(Sl):49, 200 2A. Ananth CV, Vintzileos, Shen-Schwarz S, et al: Standards of birth weight in rwin gestations. Obstet Gynecol 91 :917, 1998 3. Ardawi MSM, Nasrat HAN, BA'Aqueel HS: Calcium-regulating hormones and parathyroid hormone-related peptide in normal human pregnancy and postpartum: a longitudinal study. Eur J Endocrinol 13 :402, 1997 3A. Aslam N, Ong C, Woelfer B, et al: Serum CA ] 25 at 11-14 weeks of gestation in women with morphologically normal ovaries. BJOG 107(5): 689, 2000 4. Aziz Karim S, Khurshid M, Rizvi JH, et al: Platelets and leucocyte counts in pregnancy. J Pak Med Assoc 42:86, 1992 5.

1	Aziz Karim S, Khurshid M, Rizvi JH, et al: Platelets and leucocyte counts in pregnancy. J Pak Med Assoc 42:86, 1992 5. Bacq Y, Zarka 0, Brechot JF, et al: Liver function tests in normal pregnancy: a prospective study of 102 pregnant women and 102 matched controls. Hepatology 23: 1 030, 1996 6. Balloch AJ, Cauchi MN: Reference ranges for haematology parameters in pregnancy derived from patient populations. Clin Lab HaematoIt15:7, 1993 7. Beguin Y, Lipscei G, Thourmsin H, et al: Blunted erythropoietin production and decreased erythropoiesis in early pregnancy. Blood 78(1):89, 1991 8. Belo L, Cas lake M, Gafney 0, et al: Changes in LDL size and HDL concentration in normal and preeclamptic pregnancies. Atherosclerosis 162:425,t2002 9. Belo L, Santos-Silva A, Rocha S, et al: Fluctuations in C-reactive protein concentration and neurrophil activation during normal human pregnancy. Eur J Obstet Gynecol Reprod BioI 123:46, 2005 10.

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1	Larrea F, Mendez 1, Parra A: Serum pattern of diferent molecular forms of prolactin during normal human pregnancy. Hum Reprod 8: 1617, 1993 39. Larsson A, Palm M, Hansson L-O, et al: Reference values for clinical chemistry tests during normal pregnancy. BJOG 115:874, 2008 39A. Lattuada A, Rossi E, Calzarossa C, et al: Mild to moderate reduction of a von Willebrand factor cleaving protease (ADMTS-13) in pregnant women with HELLP microangiopathic syndrome. Haematologica 88(9): 1029,t2003 39B. Leek AE, Ruoss CF, Kitau MG, et al: Maternal plasma alphafetoprotein levels in the second half of normal pregnancy: relationship to fetal weight, and maternal age and parity. BJOG 82:669, 1975 40. Lefkowitz ]B, Clarke SH, Barbour LA: Comparison of protein S functional and antigenic assays in normal pregnancy. Am J Obstet Gynecol 175:657,t1996 41.

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1	L6pez-Quesada E, Vilaseca MA, Lailla JM: Plasma total homocysteine in uncomplicated pregnancy and in preeclampsia. Eur J Obstet Gynecol Reprod BioI 108:45, 2003 44. Louro MO, Cocho ]A, Tutor ]e Assessment of copper status in pregnancy by means of determining the speciic oxidase activity of ceruloplasmin. Clin Chim Acta 312:123,t2001 44A. Mannucci PM, Canciani MT, Forza I, et al: Changes in health and disease of the metalloprotease that cleaves von Willebrand factor. Blood 98(9): 2730,t2001 45. Milman N, Bergholt T, Byg KE, et al: Reference intervals for haematological variables during normal pregnancy and postpartum in 434 healthy Danish women. Eur J Haematol 79:39, 2007 46. Milman N, Byg KE, Hvas vI, et al: Erythrocyte folate, plasma folate and plasma homocysteine during normal pregnancy and postpartum: a longitudinal study comprising 404 Danish women. Eur J Haematol 76:200, 2006 47.

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1	The surface of our planet is populated by living things—curious, intricately organized chemical factories that take in matter from their surroundings and use these raw materials to generate copies of themselves. These living organisms appear extraordinarily diverse. What could be more different than a tiger and a piece of seaweed, or a bacterium and a tree? Yet our ancestors, knowing nothing of cells or DNA, saw that all these things had something in common. They called that something “life,” marveled at it, struggled to define it, and despaired of explaining what it was or how it worked in terms that relate to nonliving matter.

1	The discoveries of the past century have not diminished the marvel—quite the contrary. But they have removed the central mystery regarding the nature of life. We can now see that all living things are made of cells: small, membrane-enclosed units filled with a concentrated aqueous solution of chemicals and endowed with the extraordinary ability to create copies of themselves by growing and then dividing in two.

1	Because cells are the fundamental units of life, it is to cell biology—the study of the structure, function, and behavior of cells—that we must look for answers to the questions of what life is and how it works. With a deeper understanding of cells and their evolution, we can begin to tackle the grand historical problems of life on Earth: its mysterious origins, its stunning diversity, and its invasion of every conceivable habitat. Indeed, as emphasized long ago by the pioneering cell biologist E. B. Wilson, “the key to every biological problem must finally be sought in the cell; for every living organism is, or at some time has been, a cell.”

1	Despite their apparent diversity, living things are fundamentally similar inside. The whole of biology is thus a counterpoint between two themes: astonishing variety in individual particulars; astonishing constancy in fundamental mechanisms. In this first chapter, we begin by outlining the universal features common to all life on our planet. We then survey, briefly, the diversity of cells. And we see how, thanks to the common molecular code in which the specifications for all living organisms are written, it is possible to read, measure, and decipher these specifications to help us achieve a coherent understanding of all the forms of life, from the smallest to the greatest. The unIveRsAl FeATuRes oF cells on eARTh The dIveRsITy oF Genomes And The TRee oF lIFe The unIveRsAl FeATuRes oF cells on eARTh

1	The unIveRsAl FeATuRes oF cells on eARTh It is estimated that there are more than 10 million—perhaps 100 million—living species on Earth today. Each species is different, and each reproduces itself faithfully, yielding progeny that belong to the same species: the parent organism hands down information specifying, in extraordinary detail, the characteristics that the offspring shall have. This phenomenon of heredity is central to the definition of life: it distinguishes life from other processes, such as the growth of a crystal, or the burning of a candle, or the formation of waves on water, in which orderly structures are generated but without the same type of link between the peculiarities of parents and the peculiarities of offspring. Like the candle flame, the living organism must consume free energy to create and maintain its organization. But life employs the free energy to drive a hugely complex system of chemical processes that are specified by hereditary information.

1	Most living organisms are single cells. Others, such as ourselves, are vast multicellular cities in which groups of cells perform specialized functions linked by intricate systems of communication. But even for the aggregate of more than 1013 cells that form a human body, the whole organism has been generated by cell divisions from a single cell. The single cell, therefore, is the vehicle for all of the hereditary information that defines each species (Figure 1–1). This cell includes the machinery to gather raw materials from the environment and to construct from them a new cell in its own image, complete with a new copy of its hereditary information. Each and every cell is truly amazing. All cells store Their hereditary Information in the same linear chemical code: dnA

1	All cells store Their hereditary Information in the same linear chemical code: dnA Computers have made us familiar with the concept of information as a measurable quantity—a million bytes (to record a few hundred pages of text or an image from a digital camera), 600 million bytes for the music on a CD, and so on. Computers have also made us well aware that the same information can be recorded in many different physical forms: the discs and tapes that we used 20 years ago for our electronic archives have become unreadable on present-day machines. Living

1	Figure 1–1 The hereditary information in the fertilized egg cell determines the nature of the whole multicellular organism. Although their starting cells look superficially similar, as indicated: a sea urchin egg gives rise to a sea urchin (A and B). A mouse egg gives rise to a mouse (c and d). An egg of the seaweed Fucus gives rise to a Fucus seaweed (e and F). (A, courtesy of david mcclay; B, courtesy of m. Gibbs, oxford scientific Films; c, courtesy of Patricia calarco, from G. martin, Science 209:768–776, 1980. With permission from AAAs; d, courtesy of o. newman, oxford scientific Films; e and F, courtesy of colin Brownlee.) (A) building block of DNA (D) double-stranded DNA (C) templated polymerization of new strand cells, like computers, store information, and it is estimated that they have been evolving and diversifying for over 3.5 billion years. It is scarcely to be expected that they would all store their information in the same form, or that the archives of one type of

1	they have been evolving and diversifying for over 3.5 billion years. It is scarcely to be expected that they would all store their information in the same form, or that the archives of one type of cell should be readable by the information-handling machinery of another. And yet it is so. All living cells on Earth store their hereditary information in the form of double-stranded molecules of DNA—long, unbranched, paired polymer chains, formed always of the same four types of monomers. These monomers, chemical compounds known as nucleotides, have nicknames drawn from a four-letter alphabet—A, T, C, G—and they are strung together in a long linear sequence that encodes the genetic information, just as the sequence of 1s and 0s encodes the information in a computer file. We can take a piece of DNA from a human cell and insert it into a bacterium, or a piece of bacterial DNA and insert it into a human cell, and the information will be successfully read, interpreted, and copied. Using

1	of DNA from a human cell and insert it into a bacterium, or a piece of bacterial DNA and insert it into a human cell, and the information will be successfully read, interpreted, and copied. Using chemical methods, scientists have learned how to read out the complete sequence of monomers in any DNA molecule—extending for many millions of nucleotides—and thereby decipher all of the hereditary information that each organism contains.

1	The mechanisms that make life possible depend on the structure of the double-stranded DNA molecule. Each monomer in a single DNA strand—that is, each nucleotide—consists of two parts: a sugar (deoxyribose) with a phosphate group attached to it, and a base, which may be either adenine (A), guanine (G), cytosine (C), or thymine (T) (Figure 1–2). Each sugar is linked to the next via the phosphate group, creating a polymer chain composed of a repetitive sugar-phosphate backbone with a series of bases protruding from it. The DNA polymer is extended by adding monomers at one end. For a single isolated strand, these monomers can, in principle, be added in any order, because each one links to the next in the same way, through the part of the molecule that is the same for all of them. In the living cell, however, DNA is not synthesized as a free strand in isolation, but on a template formed by a preexisting DNA strand. The bases protruding from the Figure 1–2 DNA and its building blocks.

1	(A) dnA is made from simple subunits, called nucleotides, each consisting of a sugar-phosphate molecule with a nitrogen-containing side group, or base, attached to it. The bases are of four types (adenine, guanine, cytosine, and thymine), corresponding to four distinct nucleotides, labeled A, G, c, and T. (B) A single strand of dnA consists of nucleotides joined together by sugar-phosphate linkages. note that the individual sugar-phosphate units are asymmetric, giving the backbone of the strand a definite directionality, or polarity. This directionality guides the molecular processes by which the information in dnA is interpreted and copied in cells: the information is always “read” in a consistent order, just as written english text is read from left to right. (c) Through templated polymerization, the sequence of nucleotides in an existing dnA strand controls the sequence in which nucleotides are joined together in a new dnA strand; T in one strand pairs with A in the other, and G in

1	the sequence of nucleotides in an existing dnA strand controls the sequence in which nucleotides are joined together in a new dnA strand; T in one strand pairs with A in the other, and G in one strand with c in the other. The new strand has a nucleotide sequence complementary to that of the old strand, and a backbone with opposite directionality: corresponding to the GTAA... of the original strand, it has ...TTAc. (d) A normal dnA molecule consists of two such complementary strands. The nucleotides within each strand are linked by strong (covalent) chemical bonds; the complementary nucleotides on opposite strands are held together more weakly, by hydrogen bonds. (e) The two strands twist around each other to form a double helix—a robust structure that can accommodate any sequence of nucleotides without altering its basic structure (see movie 4.1).

1	existing strand bind to bases of the strand being synthesized, according to a strict rule defined by the complementary structures of the bases: A binds to T, and C binds to G. This base-pairing holds fresh monomers in place and thereby controls the selection of which one of the four monomers shall be added to the growing strand next. In this way, a double-stranded structure is created, consisting of two exactly complementary sequences of As, Cs, Ts, and Gs. The two strands twist around each other, forming a DNA double helix (Figure 1–2E).

1	The bonds between the base pairs are weak compared with the sugar-phosphate links, and this allows the two DNA strands to be pulled apart without breakage of their backbones. Each strand then can serve as a template, in the way just described, for the synthesis of a fresh DNA strand complementary to itself—a fresh copy, that is, of the hereditary information (Figure 1–3). In different types of cells, this process of DNA replication occurs at different rates, with different controls to start it or stop it, and different auxiliary molecules to help it along. But the basics are universal: DNA is the information store for heredity, and templated polymerization is the way in which this information is copied throughout the living world. All cells Transcribe Portions of Their hereditary Information into the same Intermediary Form: RnA

1	All cells Transcribe Portions of Their hereditary Information into the same Intermediary Form: RnA To carry out its information-bearing function, DNA must do more than copy itself. It must also express its information, by letting the information guide the synthesis of other molecules in the cell. This expression occurs by a mechanism that is the same in all living organisms, leading first and foremost to the production of two other key classes of polymers: RNAs and proteins. The process (discussed in detail in Chapters 6 and 7) begins with a templated polymerization called transcription, in which segments of the DNA sequence are used as templates for the synthesis of shorter molecules of the closely related polymer ribonucleic acid, or RNA. Later, in the more complex process of translation, many of these RNA molecules direct the synthesis of polymers of a radically different chemical class—the proteins (Figure 1–4).

1	In RNA, the backbone is formed of a slightly different sugar from that of DNA— ribose instead of deoxyribose—and one of the four bases is slightly different—uracil (U) in place of thymine (T). But the other three bases—A, C, and G—are the same, and all four bases pair with their complementary counterparts in DNA—the A, U, C, and G of RNA with the T, A, G, and C of DNA. During transcription, the RNA monomers are lined up and selected for polymerization on a template strand of DNA, just as DNA monomers are selected during replication. The outcome is a polymer molecule whose sequence of nucleotides faithfully represents a portion of the cell’s genetic information, even though it is written in a slightly different alphabet—consisting of RNA monomers instead of DNA monomers.

1	The same segment of DNA can be used repeatedly to guide the synthesis of many identical RNA molecules. Thus, whereas the cell’s archive of genetic information in the form of DNA is fixed and sacrosanct, these RNA transcripts are Figure 1–3 The copying of genetic information by DNA replication. In this process, the two strands of a dnA double helix are pulled apart, and each serves as a template for synthesis of a new complementary strand. Figure 1–4 From DNA to protein. Genetic information is read out and put to use through a two-step process. First, in transcription, segments of the dnA sequence are used to guide the synthesis of molecules of RnA. Then, in translation, the RnA molecules are used to guide the synthesis of molecules of protein.

1	strand used as a template to direct RNA synthesis many identical RNA transcripts mass-produced and disposable (Figure 1–5). As we shall see, these transcripts function as intermediates in the transfer of genetic information. Most notably, they serve as messenger RNA (mRNA) molecules that guide the synthesis of proteins according to the genetic instructions stored in the DNA.

1	RNA molecules have distinctive structures that can also give them other specialized chemical capabilities. Being single-stranded, their backbone is flexible, so that the polymer chain can bend back on itself to allow one part of the molecule to form weak bonds with another part of the same molecule. This occurs when segments of the sequence are locally complementary: a ...GGGG... segment, for example, will tend to associate with a ...CCCC... segment. These types of internal associations can cause an RNA chain to fold up into a specific shape that is dictated by its sequence (Figure 1–6). The shape of the RNA molecule, in turn, may enable it to recognize other molecules by binding to them selectively—and even, in certain cases, to catalyze chemical changes in the molecules that are bound. In fact, some chemical reactions catalyzed by RNA molecules are crucial for several of the most ancient and fundamental processes in living cells, and it has been suggested that an extensive catalysis

1	fact, some chemical reactions catalyzed by RNA molecules are crucial for several of the most ancient and fundamental processes in living cells, and it has been suggested that an extensive catalysis by RNA played a central part in the early evolution of life (discussed in Chapter 6).

1	All cells use Proteins as catalysts Protein molecules, like DNA and RNA molecules, are long unbranched polymer chains, formed by stringing together monomeric building blocks drawn from a standard repertoire that is the same for all living cells. Like DNA and RNA, proteins carry information in the form of a linear sequence of symbols, in the same way as a human message written in an alphabetic script. There are many different protein molecules in each cell, and—leaving out the water—they form most of the cell’s mass. Figure 1–5 How genetic information is broadcast for use inside the cell.

1	Figure 1–5 How genetic information is broadcast for use inside the cell. each cell contains a fixed set of dnA molecules—its archive of genetic information. A given segment of this dnA guides the synthesis of many identical RnA transcripts, which serve as working copies of the information stored in the archive. many different sets of RnA molecules can be made by transcribing different parts of a cell’s dnA sequences, allowing different types of cells to use the same information store differently.

1	Figure 1–6 The conformation of an RNA molecule. (A) nucleotide pairing between different regions of the same RnA polymer chain causes the molecule to adopt a distinctive shape. (B) The three-dimensional structure of an actual RnA molecule produced by hepatitis delta virus; this RnA can catalyze RnA strand cleavage. The blue ribbon represents the sugar-phosphate backbone and the bars represent base pairs (see movie 6.1). (B, based on A.R. Ferré-d’Amaré, k. Zhou, and J.A. doudna, Nature 395:567–574, 1998. With permission from macmillan Publishers ltd.) Figure 1–7 How a protein molecule acts as a catalyst for a chemical reaction. (A) In a protein molecule, the polymer chain folds up into a specific shape defined by its amino acid sequence. A groove in the surface of this particular folded molecule, the enzyme lysozyme, forms a catalytic site.

1	(B) A polysaccharide molecule (red)—a polymer chain of sugar monomers—binds to the catalytic site of lysozyme and is broken apart, as a result of a covalent bond-breaking reaction catalyzed by the amino acids lining the groove (see movie 3.9). (PdB code: 1lyd.)

1	The monomers of protein, the amino acids, are quite different from those of DNA and RNA, and there are 20 types instead of 4. Each amino acid is built around the same core structure through which it can be linked in a standard way to any other amino acid in the set; attached to this core is a side group that gives each amino acid a distinctive chemical character. Each of the protein molecules is a polypeptide, created by joining its amino acids in a particular sequence. Through billions of years of evolution, this sequence has been selected to give the protein a useful function. Thus, by folding into a precise three-dimensional form with reactive sites on its surface (Figure 1–7A), these amino-acid polymers can bind with high specificity to other molecules and can act as enzymes to catalyze reactions that make or break covalent bonds. In this way they direct the vast majority of chemical processes in the cell (Figure 1–7B).

1	Proteins have many other functions as well—maintaining structures, generating movements, sensing signals, and so on—each protein molecule performing a specific function according to its own genetically specified sequence of amino acids. Proteins, above all, are the main molecules that put the cell’s genetic information into action. Thus, polynucleotides specify the amino acid sequences of proteins. Proteins, in turn, catalyze many chemical reactions, including those by which new DNA molecules are synthesized. From the most fundamental point of view, a living cell is a self-replicating collection of catalysts that takes in food, processes this food to derive both the building blocks and energy needed to make more catalysts, and discards the materials left over as waste (Figure 1–8A). A feedback loop that connects proteins and polynucleotides forms the basis for this autocatalytic, self-reproducing behavior of living organisms (Figure 1–8B).

1	All cells Translate RnA into Protein in the same Way How the information in DNA specifies the production of proteins was a complete mystery in the 1950s when the double-stranded structure of DNA was first revealed as the basis of heredity. But in the intervening years, scientists have discovered the elegant mechanisms involved. The translation of genetic information from the 4-letter alphabet of polynucleotides into the 20-letter alphabet of proteins is a complex process. The rules of this translation seem in some respects neat and rational but in other respects strangely arbitrary, given that they are (with minor exceptions) identical in all living things. These arbitrary features, it is thought, reflect frozen accidents in the early history of life. They stem from the chance properties of the earliest organisms that were passed on by heredity and have become so deeply embedded in the constitution of all living cells that they cannot be changed without disastrous effects.

1	It turns out that the information in the sequence of a messenger RNA molecule is read out in groups of three nucleotides at a time: each triplet of nucleotides, or codon, specifies (codes for) a single amino acid in a corresponding protein. Since the number of distinct triplets that can be formed from four nucleotides is 43, there are 64 possible codons, all of which occur in nature. However, there are only 20 naturally occurring amino acids. That means there are necessarily many cases in which several codons correspond to the same amino acid. This genetic code is read out by a special class of small RNA molecules, the transfer RNAs (tRNAs). Each type of tRNA becomes attached at one end to a specific amino acid, and displays at its other end a specific sequence of three nucleotides—an anticodon— that enables it to recognize, through base-pairing, a particular codon or subset of codons in mRNA. The intricate chemistry that enables these tRNAs to translate a specific sequence of A, C,

1	that enables it to recognize, through base-pairing, a particular codon or subset of codons in mRNA. The intricate chemistry that enables these tRNAs to translate a specific sequence of A, C, G, and U nucleotides in an mRNA molecule into a specific sequence of amino acids in a protein molecule occurs on the ribosome, a large multimolecular machine composed of both protein and ribosomal RNA. All of these processes are described in detail in Chapter 6.

1	DNA molecules as a rule are very large, containing the specifications for thousands of proteins. Special sequences in the DNA serve as punctuation, defining where the information for each protein begins and ends. And individual segments of the long DNA sequence are transcribed into separate mRNA molecules, coding for different proteins. Each such DNA segment represents one gene. A complication is that RNA molecules transcribed from the same DNA segment can often be processed in more than one way, so as to give rise to a set of alternative versions of a protein, especially in more complex cells such as those of plants and animals. In addition, some DNA segments—a smaller number—are transcribed into RNA molecules that are not translated but have catalytic, regulatory, or structural functions; such DNA segments also count as genes. A gene therefore is defined as the segment of DNA sequence corresponding to a single protein or set of alternative protein variants or to a single catalytic,

1	such DNA segments also count as genes. A gene therefore is defined as the segment of DNA sequence corresponding to a single protein or set of alternative protein variants or to a single catalytic, regulatory, or structural RNA molecule.

1	In all cells, the expression of individual genes is regulated: instead of manufacturing its full repertoire of possible proteins at full tilt all the time, the cell adjusts the rate of transcription and translation of different genes independently, according to need. Stretches of regulatory DNA are interspersed among the segments that code for protein, and these noncoding regions bind to special protein molecules that control the local rate of transcription. The quantity and organization of the regulatory DNA vary widely from one class of organisms to another, but the basic strategy is universal. In this way, the genome of the cell—that is, the totality of its genetic information as embodied in its complete DNA sequence— dictates not only the nature of the cell’s proteins, but also when and where they are to be made.

1	Figure 1–8 Life as an autocatalytic process. (A) The cell as a self-replicating collection of catalysts. (B) Polynucleotides (the nucleic acids dnA and RnA, which are nucleotide polymers) provide the sequence information, while proteins (amino acid polymers) provide most of the catalytic functions that serve—through a complex set of chemical reactions—to bring about the synthesis of more polynucleotides and proteins of the same types. A living cell is a dynamic chemical system, operating far from chemical equilibrium. For a cell to grow or to make a new cell in its own image, it must take in free energy from the environment, as well as raw materials, to drive the necessary synthetic reactions. This consumption of free energy is fundamental to life. When it stops, a cell decays toward chemical equilibrium and soon dies.

1	Genetic information is also fundamental to life, and free energy is required for the propagation of this information. For example, to specify one bit of information—that is, one yes/no choice between two equally probable alternatives— costs a defined amount of free energy that can be calculated. The quantitative relationship involves some deep reasoning and depends on a precise definition of the term “free energy,” as explained in Chapter 2. The basic idea, however, is not difficult to understand intuitively.

1	Picture the molecules in a cell as a swarm of objects endowed with thermal energy, moving around violently at random, buffeted by collisions with one another. To specify genetic information—in the form of a DNA sequence, for example—molecules from this wild crowd must be captured, arranged in a specific order defined by some preexisting template, and linked together in a fixed relationship. The bonds that hold the molecules in their proper places on the template and join them together must be strong enough to resist the disordering effect of thermal motion. The process is driven forward by consumption of free energy, which is needed to ensure that the correct bonds are made, and made robustly. In the simplest case, the molecules can be compared with spring-loaded traps, ready to snap into a more stable, lower-energy attached state when they meet their proper partners; as they snap together into the bonded arrangement, their available stored energy—their free energy—like the energy of

1	a more stable, lower-energy attached state when they meet their proper partners; as they snap together into the bonded arrangement, their available stored energy—their free energy—like the energy of the spring in the trap, is released and dissipated as heat. In a cell, the chemical processes underlying information transfer are more complex, but the same basic principle applies: free energy has to be spent on the creation of order.

1	To replicate its genetic information faithfully, and indeed to make all its complex molecules according to the correct specifications, the cell therefore requires free energy, which has to be imported somehow from the surroundings. As we shall see in Chapter 2, the free energy required by animal cells is derived from chemical bonds in food molecules that the animals eat, while plants get their free energy from sunlight. All cells Function as Biochemical Factories dealing with the same Basic molecular Building Blocks

1	All cells Function as Biochemical Factories dealing with the same Basic molecular Building Blocks Because all cells make DNA, RNA, and protein, all cells have to contain and manipulate a similar collection of small molecules, including simple sugars, nucleotides, and amino acids, as well as other substances that are universally required. All cells, for example, require the phosphorylated nucleotide ATP (adenosine triphosphate), not only as a building block for the synthesis of DNA and RNA, but also as a carrier of the free energy that is needed to drive a huge number of chemical reactions in the cell.

1	Although all cells function as biochemical factories of a broadly similar type, many of the details of their small-molecule transactions differ. Some organisms, such as plants, require only the simplest of nutrients and harness the energy of sunlight to make all their own small organic molecules. Other organisms, such as animals, feed on living things and must obtain many of their organic molecules ready-made. We return to this point later. Another universal feature is that each cell is enclosed by a membrane—the plasma membrane. This container acts as a selective barrier that enables the cell to concentrate nutrients gathered from its environment and retain the products it

1	Figure 1–9 Formation of a membrane by amphiphilic phospholipid molecules. Phospholipids have a hydrophilic (water-loving, phosphate) head group and a hydrophobic (water-avoiding, hydrocarbon) tail. At an interface between oil and water, they arrange themselves as a single sheet with their head groups facing the water and their tail groups facing the oil. But when immersed in water, they aggregate to form bilayers enclosing aqueous compartments, as indicated. synthesizes for its own use, while excreting its waste products. Without a plasma membrane, the cell could not maintain its integrity as a coordinated chemical system.

1	The molecules that form a membrane have the simple physicochemical property of being amphiphilic—that is, consisting of one part that is hydrophobic (water-insoluble) and another part that is hydrophilic (water-soluble). Such molecules placed in water aggregate spontaneously, arranging their hydrophobic portions to be as much in contact with one another as possible to hide them from the water, while keeping their hydrophilic portions exposed. Amphiphilic molecules of appropriate shape, such as the phospholipid molecules that comprise most of the plasma membrane, spontaneously aggregate in water to create a bilayer that forms small closed vesicles (Figure 1–9). The phenomenon can be demonstrated in a test tube by simply mixing phospholipids and water together; under appropriate conditions, small vesicles form whose aqueous contents are isolated from the external medium.

1	Although the chemical details vary, the hydrophobic tails of the predominant membrane molecules in all cells are hydrocarbon polymers (–CH2–CH2–CH2–), and their spontaneous assembly into a bilayered vesicle is but one of many examples of an important general principle: cells produce molecules whose chemical properties cause them to self-assemble into the structures that a cell needs.

1	The cell boundary cannot be totally impermeable. If a cell is to grow and reproduce, it must be able to import raw materials and export waste across its plasma membrane. All cells therefore have specialized proteins embedded in their membrane that transport specific molecules from one side to the other. Some of these membrane transport proteins, like some of the proteins that catalyze the fundamental small-molecule reactions inside the cell, have been so well preserved over the course of evolution that we can recognize the family resemblances between them in comparisons of even the most distantly related groups of living organisms.

1	The transport proteins in the membrane largely determine which molecules enter the cell, and the catalytic proteins inside the cell determine the reactions that those molecules undergo. Thus, by specifying the proteins that the cell is to manufacture, the genetic information recorded in the DNA sequence dictates the entire chemistry of the cell; and not only its chemistry, but also its form and its behavior, for these too are chiefly constructed and controlled by the cell’s proteins. A living cell can exist with Fewer Than 500 Genes

1	A living cell can exist with Fewer Than 500 Genes The basic principles of biological information transfer are simple enough, but how complex are real living cells? In particular, what are the minimum requirements? We can get a rough indication by considering a species that has one of the smallest known genomes—the bacterium Mycoplasma genitalium (Figure 1–10). This organism lives as a parasite in mammals, and its environment provides it with many of its small molecules ready-made. Nevertheless, it still has to make all the large molecules—DNA, RNAs, and proteins—required for the basic processes of heredity. It has about 530 genes, about 400 of which are essential. Its genome of 580,070 nucleotide pairs represents 145,018 bytes of information—about as much as it takes to record the text of one chapter of this book. Cell biology may be complicated, but it is not impossibly so.

1	The minimum number of genes for a viable cell in today’s environments is probably not less than 300, although there are only about 60 genes in the core set that is shared by all living species.

1	The individual cell is the minimal self-reproducing unit of living matter, and it consists of a self-replicating collection of catalysts. Central to this reproduction is the transmission of genetic information to progeny cells. Every cell on our planet stores its genetic information in the same chemical form—as double-stranded DNA. The cell replicates its information by separating the paired DNA strands and using each as a template for polymerization to make a new DNA strand with a complementary sequence of nucleotides. The same strategy of templated polymerization is used to transcribe portions of the information from DNA into molecules of the closely related polymer, RNA. These RNA molecules in turn guide the synthesis of protein molecules by the more complex machinery of translation, involving a large multi-molecular machine, the ribosome. Proteins are the principal catalysts for almost all the chemical reactions in the cell; their other functions include the selective import and

1	involving a large multi-molecular machine, the ribosome. Proteins are the principal catalysts for almost all the chemical reactions in the cell; their other functions include the selective import and export of small molecules across the plasma membrane that forms the cell’s boundary. The specific function of each protein depends on its amino acid sequence, which is specified by the nucleotide sequence of a corresponding segment of the DNA—the gene that codes for that protein. In this way, the genome of the cell determines its chemistry; and the chemistry of every living cell is fundamentally similar, because it must provide for the synthesis of DNA, RNA, and protein. The simplest known cells can survive with about 400 genes.

1	The dIveRsITy oF Genomes And The TRee oF lIFe The success of living organisms based on DNA, RNA, and protein has been spectacular. Life has populated the oceans, covered the land, infiltrated the Earth’s crust, and molded the surface of our planet. Our oxygen-rich atmosphere, the deposits of coal and oil, the layers of iron ores, the cliffs of chalk and limestone and marble—all these are products, directly or indirectly, of past biological activity on Earth.

1	Living things are not confined to the familiar temperate realm of land, water, and sunlight inhabited by plants and plant-eating animals. They can be found in the darkest depths of the ocean, in hot volcanic mud, in pools beneath the frozen surface of the Antarctic, and buried kilometers deep in the Earth’s crust. The creatures that live in these extreme environments are generally unfamiliar, not only because they are inaccessible, but also because they are mostly microscopic. In more homely habitats, too, most organisms are too small for us to see without special equipment: they tend to go unnoticed, unless they cause a disease or rot the timbers of our houses. Yet microorganisms make up most of the total mass of living matter on our planet. Only recently, through new methods of molecular analysis and specifically through the analysis of DNA sequences, have we begun to get a picture of life on Earth that is not grossly distorted by our biased perspective as large animals living on

1	analysis and specifically through the analysis of DNA sequences, have we begun to get a picture of life on Earth that is not grossly distorted by our biased perspective as large animals living on dry land.

1	In this section, we consider the diversity of organisms and the relationships among them. Because the genetic information for every organism is written in the universal language of DNA sequences, and the DNA sequence of any given organism can be readily obtained by standard biochemical techniques, it is now possible to characterize, catalog, and compare any set of living organisms with reference to these sequences. From such comparisons we can estimate the place of each organism in the family tree of living species—the “tree of life.” But before describing what this approach reveals, we need first to consider the routes by which cells in different environments obtain the matter and energy they require to survive and proliferate, and the ways in which some classes of organisms depend on others for their basic chemical needs. cells can Be Powered by a variety of Free-energy sources

1	cells can Be Powered by a variety of Free-energy sources Living organisms obtain their free energy in different ways. Some, such as animals, fungi, and the many different bacteria that live in the human gut, get it by feeding on other living things or the organic chemicals they produce; such organisms 0.2 µm Figure 1–10 Mycoplasma genitalium. scanning electron micrograph showing the irregular shape of this small bacterium, reflecting the lack of any rigid cell wall.

1	Figure 1–10 Mycoplasma genitalium. scanning electron micrograph showing the irregular shape of this small bacterium, reflecting the lack of any rigid cell wall. cross section (transmission electron micrograph) of a Mycoplasma cell. of the 530 genes of Mycoplasma genitalium, 43 code for transfer, ribosomal, and other non-messenger RnAs. Functions are known, or can be guessed, for 339 of the genes coding for protein: of these, 154 are involved in replication, transcription, translation, and related processes involving dnA, RnA, and protein; 98 in the membrane and surface structures of the cell; 46 in the transport of nutrients and other molecules across the membrane; 71 in energy conversion and the synthesis and degradation of small molecules; and 12 in the regulation of cell division and other processes. note that these categories are partly overlapping, so that some genes feature twice. (A, from s.

1	Razin et al., Infect. Immun. 30:538–546, 1980. With permission from the American society for microbiology; B, courtesy of Roger cole, in medical microbiology, 4th ed. [s. Baron ed.]. Galveston: university of Texas medical Branch, 1996.) are called organotrophic (from the Greek word trophe, meaning “food”). Others derive their energy directly from the nonliving world. These primary energy converters fall into two classes: those that harvest the energy of sunlight, and those that capture their energy from energy-rich systems of inorganic chemicals in the environment (chemical systems that are far from chemical equilibrium). Organisms of the former class are called phototrophic (feeding on sunlight); those of the latter are called lithotrophic (feeding on rock). Organotrophic organisms could not exist without these primary energy converters, which are the most plentiful form of life.

1	Phototrophic organisms include many types of bacteria, as well as algae and plants, on which we—and virtually all the living things that we ordinarily see around us—depend. Phototrophic organisms have changed the whole chemistry of our environment: the oxygen in the Earth’s atmosphere is a by-product of their biosynthetic activities. Lithotrophic organisms are not such an obvious feature of our world, because they are microscopic and mostly live in habitats that humans do not frequent— deep in the ocean, buried in the Earth’s crust, or in various other inhospitable environments. But they are a major part of the living world, and they are especially important in any consideration of the history of life on Earth.

1	Some lithotrophs get energy from aerobic reactions, which use molecular oxygen from the environment; since atmospheric O2 is ultimately the product of living organisms, these aerobic lithotrophs are, in a sense, feeding on the products of past life. There are, however, other lithotrophs that live anaerobically, in places where little or no molecular oxygen is present. These are circumstances similar to those that existed in the early days of life on Earth, before oxygen had accumulated. The most dramatic of these sites are the hot hydrothermal vents on the floor of the Pacific and Atlantic Oceans. They are located where the ocean floor is spreading as new portions of the Earth’s crust form by a gradual upwelling of material from the Earth’s interior (Figure 1–11). Downward-percolating seawater is heated and driven back upward as a submarine geyser, carrying with it a current of chemicals from the hot rocks below. A typical cocktail might include H2S, H2, CO,

1	Mn2+, Fe2+, Ni2+, CH2, NH4 , and phosphorus-containing compounds. A dense SEA dark cloud of hot, mineral-rich water 350°C percolation contour of seawater Figure 1–11 The geology of a hot hydrothermal vent in the ocean floor. As indicated, water percolates down toward the hot molten rock upwelling from the earth’s interior and is heated and driven back upward, carrying minerals leached from the hot rock. A temperature gradient is set up, from more than 350°c near the core of the vent, down to 2–3°c in the surrounding ocean. minerals precipitate from the water as it cools, forming a chimney. different classes of organisms, thriving at different temperatures, live in different neighborhoods of the chimney. A typical chimney might be a few meters tall, spewing out hot, mineral-rich water at a flow rate of 1–2 m/sec.

1	multicellular animals, e.g., tube worms population of microbes lives in the neighborhood of the vent, thriving on this austere diet and harvesting free energy from reactions between the available chemicals. Other organisms—clams, mussels, and giant marine worms—in turn live off the microbes at the vent, forming an entire ecosystem analogous to the world of plants and animals that we belong to, but powered by geochemical energy instead of light (Figure 1–12).

1	To make a living cell requires matter, as well as free energy. DNA, RNA, and protein are composed of just six elements: hydrogen, carbon, nitrogen, oxygen, sulfur, and phosphorus. These are all plentiful in the nonliving environment, in the Earth’s rocks, water, and atmosphere. But they are not present in chemical forms that allow easy incorporation into biological molecules. Atmospheric N2 and CO2, in particular, are extremely unreactive. A large amount of free energy is required to drive the reactions that use these inorganic molecules to make the organic compounds needed for further biosynthesis—that is, to fix nitrogen and carbon dioxide, so as to make N and C available to living organisms. Many types of living cells lack the biochemical machinery to achieve this fixation; they instead rely on other classes of cells to do the job for them. We animals depend on plants for our supplies of organic carbon and nitrogen compounds. Plants in turn, although they can fix carbon dioxide

1	rely on other classes of cells to do the job for them. We animals depend on plants for our supplies of organic carbon and nitrogen compounds. Plants in turn, although they can fix carbon dioxide from the atmosphere, lack the ability to fix atmospheric nitrogen; they depend in part on nitrogen-fixing bacteria to supply their need for nitrogen compounds. Plants of the pea family, for example, harbor symbiotic nitrogen-fixing bacteria in nodules in their roots.

1	Living cells therefore differ widely in some of the most basic aspects of their biochemistry. Not surprisingly, cells with complementary needs and capabilities have developed close associations. Some of these associations, as we see below, have evolved to the point where the partners have lost their separate identities altogether: they have joined forces to form a single composite cell. The Greatest Biochemical diversity exists Among Prokaryotic cells From simple microscopy, it has long been clear that living organisms can be classified on the basis of cell structure into two groups: the eukaryotes and the

1	Figure 1–12 Organisms living at a depth of 2500 meters near a vent in the ocean floor. close to the vent, at temperatures up to about 120°c, various lithotrophic species of bacteria and archaea (archaebacteria) live, directly fueled by geochemical energy. A little further away, where the temperature is lower, various invertebrate animals live by feeding on these microorganisms. most remarkable are these giant (2 meter) tube worms, Riftia pachyptila, which, rather than feed on the lithotrophic cells, live in symbiosis with them: specialized organs in the worms harbor huge numbers of symbiotic sulfur-oxidizing bacteria. These bacteria harness geochemical energy and supply nourishment to their hosts, which have no mouth, gut, or anus. The tube worms are thought to have evolved from more conventional animals, and to have become secondarily adapted to life at hydrothermal vents. (courtesy of monika Bright, university of vienna, Austria.) rod-shaped cells the smallest cells e.g.,

1	more conventional animals, and to have become secondarily adapted to life at hydrothermal vents. (courtesy of monika Bright, university of vienna, Austria.) rod-shaped cells the smallest cells e.g., Escherichia coli, e.g., Mycoplasma, Vibrio cholerae Spiroplasma prokaryotes. Eukaryotes keep their DNA in a distinct membrane-enclosed intracellular compartment called the nucleus. (The name is from the Greek, meaning “truly nucleated,” from the words eu, “well” or “truly,” and karyon, “kernel” or “nucleus.”) Prokaryotes have no distinct nuclear compartment to house their DNA. Plants, fungi, and animals are eukaryotes; bacteria are prokaryotes, as are archaea—a separate class of prokaryotic cells, discussed below.

1	Most prokaryotic cells are small and simple in outward appearance (Figure 1–13), and they live mostly as independent individuals or in loosely organized communities, rather than as multicellular organisms. They are typically spherical or rod-shaped and measure a few micrometers in linear dimension. They often have a tough protective coat, called a cell wall, beneath which a plasma membrane encloses a single cytoplasmic compartment containing DNA, RNA, proteins, and the many small molecules needed for life. In the electron microscope, this cell interior appears as a matrix of varying texture without any discernible organized internal structure (Figure 1–14).

1	Prokaryotic cells live in an enormous variety of ecological niches, and they are astonishingly varied in their biochemical capabilities—far more so than eukaryotic cells. Organotrophic species can utilize virtually any type of organic molecule as food, from sugars and amino acids to hydrocarbons and methane gas. Photo-trophic species (Figure 1–15) harvest light energy in a variety of ways, some of them generating oxygen as a by-product, others not. Lithotrophic species can feed on a plain diet of inorganic nutrients, getting their carbon from CO2, and relying on H2S to fuel their energy needs (Figure 1–16)—or on H2, or Fe2+, or elemental sulfur, or any of a host of other chemicals that occur in the environment.

1	Figure 1–14 The structure of a bacterium. (A) The bacterium Vibrio cholerae, showing its simple internal organization. like many other species, Vibrio has a helical appendage at one end—a flagellum—that rotates as a propeller to drive the cell forward. It can infect the human small intestine to cause cholera; the severe diarrhea that accompanies this disease kills more than 100,000 people a year. (B) An electron micrograph of a longitudinal section through the widely studied bacterium Escherichia coli (E. coli). The cell’s dnA is concentrated in the lightly stained region. Part of our normal intestinal flora, E. coli is related to Vibrio, and it has many flagella distributed over its surface that are not visible in this section. (B, courtesy of e. kellenberger.) spherical cells e.g., Streptococcus spiral cells e.g., Treponema pallidum

1	Figure 1–13 Shapes and sizes of some bacteria. Although most are small, as shown, measuring a few micrometers in linear dimension, there are also some giant species. An extreme example (not shown) is the cigar-shaped bacterium Epulopiscium fishelsoni, which lives in the gut of a surgeonfish and can be up to 600 μm long. Much of this world of microscopic organisms is virtually unexplored. Traditional methods of bacteriology have given us an acquaintance with those species that can be isolated and cultured in the laboratory. But DNA sequence analysis of the populations of bacteria and archaea in samples from natural habitats—such as soil or ocean water, or even the human mouth—has opened our eyes to the fact that most species cannot be cultured by standard laboratory techniques. According to one estimate, at least 99% of prokaryotic species remain to be characterized. Detected only by their DNA, it has not yet been possible to grow the vast majority of them in laboratories.

1	The Tree of life has Three Primary Branches: Bacteria, Archaea, and eukaryotes The classification of living things has traditionally depended on comparisons of their outward appearances: we can see that a fish has eyes, jaws, backbone, brain, and so on, just as we do, and that a worm does not; that a rosebush is cousin to an apple tree, but is less similar to a grass. As Darwin showed, we can readily interpret such close family resemblances in terms of evolution from common ancestors, and we can find the remains of many of these ancestors preserved in the fossil record. In this way, it has been possible to begin to draw a family tree of living organisms, showing the various lines of descent, as well as branch points in the history, where the ancestors of one group of species became different from those of another.

1	When the disparities between organisms become very great, however, these methods begin to fail. How do we decide whether a fungus is closer kin to a plant or to an animal? When it comes to prokaryotes, the task becomes harder still: one microscopic rod or sphere looks much like another. Microbiologists have therefore sought to classify prokaryotes in terms of their biochemistry and nutritional requirements. But this approach also has its pitfalls. Amid the bewildering variety of biochemical behaviors, it is difficult to know which differences truly reflect differences of evolutionary history.

1	Genome analysis has now given us a simpler, more direct, and much more powerful way to determine evolutionary relationships. The complete DNA sequence of an organism defines its nature with almost perfect precision and in exhaustive detail. Moreover, this specification is in a digital form—a string of let-ters—that can be entered straightforwardly into a computer and compared with the corresponding information for any other living thing. Because DNA is subject to random changes that accumulate over long periods of time (as we shall see shortly), the number of differences between the DNA sequences of two organisms can provide a direct, objective, quantitative indication of the evolutionary distance between them.

1	This approach has shown that the organisms that were traditionally classed together as “bacteria” can be as widely divergent in their evolutionary origins as is any prokaryote from any eukaryote. It is now clear that the prokaryotes comprise two distinct groups that diverged early in the history of life on Earth, before the eukaryotes diverged as a separate group. The two groups of prokaryotes are called the bacteria (or eubacteria) and the archaea (or archaebacteria). Detailed genome analyses have recently revealed that the first eukayotic cell formed after a Figure 1–15 The phototrophic bacterium Anabaena cylindrica viewed in the light microscope. The cells of this species form long, multicellular filaments. most of the cells (labeled v) perform photosynthesis, while others become specialized for nitrogen fixation (labeled h) or develop into resistant spores (labeled s). (courtesy of dave G. Adams.) Figure 1–16 A lithotrophic bacterium.

1	Figure 1–16 A lithotrophic bacterium. Beggiatoa, which lives in sulfurous environments, gets its energy by oxidizing h2s and can fix carbon even in the dark. note the yellow deposits of sulfur inside the cells. (courtesy of Ralph W. Wolfe.) ARCHAEA BACTERIA EUKARYOTES common ancestor cellAquifex Thermotoga cyanobacteria Bacillus E. coli Aeropyrum Sulfolobus Haloferax Methano-thermobacter Methanococcus Paramecium Dictyostelium Euglena Trypanosoma maize yeast human 1 change/10 nucleotides frst eukaryote Giardia Trichomonas particular type of ancient archaeal cell engulfed an ancient bacterium (see Figure 12–3). Thus, the living world today is considered to consist of three major divisions or domains: bacteria, archaea, and eukaryotes (Figure 1–17).

1	Archaea are often found inhabiting environments that we humans avoid, such as bogs, sewage treatment plants, ocean depths, salt brines, and hot acid springs, although they are also widespread in less extreme and more homely environments, from soils and lakes to the stomachs of cattle. In outward appearance they are not easily distinguished from bacteria. At a molecular level, archaea seem to resemble eukaryotes more closely in their machinery for handling genetic information (replication, transcription, and translation), but bacteria more closely in their apparatus for metabolism and energy conversion. We discuss below how this might be explained.

1	Both in the storage and in the copying of genetic information, random accidents and errors occur, altering the nucleotide sequence—that is, creating mutations. Therefore, when a cell divides, its two daughters are often not quite identical to one another or to their parent. On rare occasions, the error may represent a change for the better; more probably, it will cause no significant difference in the cell’s prospects. But in many cases, the error will cause serious damage—for example, by disrupting the coding sequence for a key protein. Changes due to mistakes of the first type will tend to be perpetuated, because the altered cell has an increased likelihood of reproducing itself. Changes due to mistakes of the second type—selectively neutral changes—may be perpetuated or not: in the competition for limited resources, it is a matter of chance whether the altered cell or its cousins will succeed. But changes that cause serious damage lead nowhere: the cell that suffers them dies,

1	competition for limited resources, it is a matter of chance whether the altered cell or its cousins will succeed. But changes that cause serious damage lead nowhere: the cell that suffers them dies, leaving no progeny. Through endless repetition of this cycle of error and trial—of mutation and natural selection—organisms evolve: their genetic specifications change, giving them new ways to exploit the environment more effectively, to survive in competition with others, and to reproduce successfully.

1	Some parts of the genome will change more easily than others in the course of evolution. A segment of DNA that does not code for protein and has no significant regulatory role is free to change at a rate limited only by the frequency of random errors. In contrast, a gene that codes for a highly optimized essential protein or RNA molecule cannot alter so easily: when mistakes occur, the faulty cells are almost always eliminated. Genes of this latter sort are therefore highly conserved. Through 3.5 billion years or more of evolutionary history, many features of the genome have changed beyond all recognition, but the most highly conserved genes remain perfectly recognizable in all living species.

1	Figure 1–17 The three major divisions (domains) of the living world. note that the word bacteria was originally used to refer to prokaryotes in general, but more recently has been redefined to refer to eubacteria specifically. The tree shown here is based on comparisons of the nucleotide sequence of a ribosomal RnA (rRnA) subunit in the different species, and the distances in the diagram represent estimates of the numbers of evolutionary changes that have occurred in this molecule in each lineage (see Figure 1–18). The parts of the tree shrouded in gray cloud represent uncertainties about details of the true pattern of species divergence in the course of evolution: comparisons of nucleotide or amino acid sequences of molecules other than rRnA, as well as other arguments, can lead to somewhat different trees. As indicated, the nucleus of the eukaryotic cell is now thought to have emerged from a sub-branch within the archaea, so that in the beginning the tree of life had only two

1	somewhat different trees. As indicated, the nucleus of the eukaryotic cell is now thought to have emerged from a sub-branch within the archaea, so that in the beginning the tree of life had only two branches—bacteria and archaea.

1	GTTCCGGGGGGAGTATGGTTGCAAAGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAACCTCACCC human GCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTTAAAGGAATTGGCGGGGGAGCACTACAACGGGTGGAGCCTGCGGTTTAATTGGATTCAACGCCGGGCATCTTACCA Methanococcus ACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGC.ACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCT E. coli GTTCCGGGGGGAGTATGGTTGCAAAGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAACCTCACCC human

1	These latter genes are the ones we must examine if we wish to trace family relationships between the most distantly related organisms in the tree of life. The initial studies that led to the classification of the living world into the three domains of bacteria, archaea, and eukaryotes were based chiefly on analysis of one of the rRNA components of the ribosome. Because the translation of RNA into protein is fundamental to all living cells, this component of the ribosome has been very well conserved since early in the history of life on Earth (Figure 1–18). Natural selection has generally favored those prokaryotic cells that can reproduce the fastest by taking up raw materials from their environment and replicating themselves most efficiently, at the maximal rate permitted by the available food supplies. Small size implies a large ratio of surface area to volume, thereby helping to maximize the uptake of nutrients across the plasma membrane and boosting a cell’s reproductive rate.

1	Presumably for these reasons, most prokaryotic cells carry very little superfluous baggage; their genomes are small, with genes packed closely together and minimal quantities of regulatory DNA between them. The small genome size has made it easy to use modern DNA sequencing techniques to determine complete genome sequences. We now have this information for thousands of species of bacteria and archaea, as well as for hundreds of species of eukaryotes. Most bacterial and archaeal genomes contain between 106 and 107 nucleotide pairs, encoding 1000–6000 genes.

1	A complete DNA sequence reveals both the genes an organism possesses and the genes it lacks. When we compare the three domains of the living world, we can begin to see which genes are common to all of them and must therefore have been present in the cell that was ancestral to all present-day living things, and which genes are peculiar to a single branch in the tree of life. To explain the findings, however, we need to consider a little more closely how new genes arise and genomes evolve. The raw material of evolution is the DNA sequence that already exists: there is no natural mechanism for making long stretches of new random sequence. In this sense, no gene is ever entirely new. Innovation can, however, occur in several ways (Figure 1–19): 1. Intragenic mutation: an existing gene can be randomly modified by changes in its DNA sequence, through various types of error that occur mainly in the process of DNA replication. 2.

1	Intragenic mutation: an existing gene can be randomly modified by changes in its DNA sequence, through various types of error that occur mainly in the process of DNA replication. 2. Gene duplication: an existing gene can be accidentally duplicated so as to create a pair of initially identical genes within a single cell; these two genes may then diverge in the course of evolution. 3. DNA segment shuffling: two or more existing genes can break and rejoin to make a hybrid gene consisting of DNA segments that originally belonged to separate genes. 4. Horizontal (intercellular) transfer: a piece of DNA can be transferred from the genome of one cell to that of another—even to that of another species. This process is in contrast with the usual vertical transfer of genetic information from parent to progeny. Each of these types of change leaves a characteristic trace in the DNA sequence of the organism, and there is clear evidence that all four processes have frequently

1	Each of these types of change leaves a characteristic trace in the DNA sequence of the organism, and there is clear evidence that all four processes have frequently Figure 1–18 Genetic information conserved since the days of the last common ancestor of all living things.

1	Figure 1–18 Genetic information conserved since the days of the last common ancestor of all living things. A part of the gene for the smaller of the two main rRnA components of the ribosome is shown. (The complete molecule is about 1500–1900 nucleotides long, depending on species.) corresponding segments of nucleotide sequence from an archaean (Methanococcus jannaschii), a bacterium (Escherichia coli), and a eukaryote (Homo sapiens) are aligned. sites where the nucleotides are identical between species are indicated by a vertical line; the human sequence is repeated at the bottom of the alignment so that all three two-way comparisons can be seen. A dot halfway along the E. coli sequence denotes a site where a nucleotide has been either deleted from the bacterial lineage in the course of evolution or inserted in the other two lineages. note that the sequences from these three organisms, representative of the three domains of the living world, still retain unmistakable similarities.

1	occurred. In later chapters, we discuss the underlying mechanisms, but for the present we focus on the consequences. Gene duplications Give Rise to Families of Related Genes Within a single cell

1	Gene duplications Give Rise to Families of Related Genes Within a single cell A cell duplicates its entire genome each time it divides into two daughter cells. However, accidents occasionally result in the inappropriate duplication of just part of the genome, with retention of original and duplicate segments in a single cell. Once a gene has been duplicated in this way, one of the two gene copies is free to mutate and become specialized to perform a different function within the same cell. Repeated rounds of this process of duplication and divergence, over many millions of years, have enabled one gene to give rise to a family of genes that may all be found within a single genome. Analysis of the DNA sequence of prokaryotic genomes reveals many examples of such gene families: in the bacterium Bacillus subtilis, for example, 47% of the genes have one or more obvious relatives (Figure 1–20).

1	When genes duplicate and diverge in this way, the individuals of one species become endowed with multiple variants of a primordial gene. This evolutionary process has to be distinguished from the genetic divergence that occurs when one species of organism splits into two separate lines of descent at a branch point in the family tree—when the human line of descent became separate from that of chimpanzees, for example. There, the genes gradually become different in the course of evolution, but they are likely to continue to have corresponding functions in the two sister species. Genes that are related by descent in this way—that is, genes in two separate species that derive from the same ancestral gene in the last common ancestor of those two species—are called orthologs. Related genes that have resulted from a gene duplication event within a single genome—and

1	Figure 1–19 Four modes of genetic innovation and their effects on the DNA sequence of an organism. A special form of horizontal transfer occurs when two different types of cells enter into a permanent symbiotic association. Genes from one of the cells then may be transferred to the genome of the other, as we shall see below when we discuss mitochondria and chloroplasts. 283 genes in families with 38–77 gene members 764 genes in families with 4–19 gene members 2126 genes with no family relationship273 genes in families with 3 gene members 568 genes in families with 2 gene members are likely to have diverged in their function—are called paralogs. Genes that are related by descent in either way are called homologs, a general term used to cover both types of relationship (Figure 1–21). Genes can Be Transferred Between organisms, Both in the laboratory and in nature

1	Prokaryotes provide good examples of the horizontal transfer of genes from one species of cell to another. The most obvious tell-tale signs are sequences recognizable as being derived from viruses, those infecting bacteria being called bacteriophages (Figure 1–22). Viruses are small packets of genetic material that have evolved as parasites on the reproductive and biosynthetic machinery of host cells. Although not themselves living cells, they often serve as vectors for gene transfer. A virus will replicate in one cell, emerge from it with a protective wrapping, and then enter and infect another cell, which may be of the same or a different species. Often, the infected cell will be killed by the massive proliferation of virus particles inside it; but sometimes, the viral DNA, instead of directly generating these particles, may persist in its host for many cell generations as a relatively innocuous passenger, either as a separate intracellular fragment of DNA, known as a plasmid, or as

1	generating these particles, may persist in its host for many cell generations as a relatively innocuous passenger, either as a separate intracellular fragment of DNA, known as a plasmid, or as a sequence inserted into the cell’s regular genome. In their travels, viruses can accidentally pick up fragments of DNA from the genome of one host cell and ferry them into another cell. Such transfers of genetic material are very common in prokaryotes.

1	Horizontal transfers of genes between eukaryotic cells of different species are very rare, and they do not seem to have played a significant part in eukaryote evolution (although massive transfers from bacterial to eukaryotic genomes have occurred in the evolution of mitochondria and chloroplasts, as we discuss below). genes GA and GB are orthologs genes G1 and G2 are paralogs Figure 1–20 Families of evolutionarily related genes in the genome of Bacillus subtilis. The largest gene family in this bacterium consists of 77 genes coding for varieties of ABc transporters—a class of membrane transport proteins found in all three domains of the living world. (Adapted from F. kunst et al., Nature 390:249–256, 1997. With permission from macmillan Publishers ltd.) Figure 1–21 Paralogous genes and orthologous genes: two types of gene homology based on different evolutionary pathways. (A) orthologs. (B) Paralogs.

1	In contrast, horizontal gene transfers occur much more frequently between different species of prokaryotes. Many prokaryotes have a remarkable capacity to take up even nonviral DNA molecules from their surroundings and thereby capture the genetic information these molecules carry. By this route, or by virus-mediated transfer, bacteria and archaea in the wild can acquire genes from neighboring cells relatively easily. Genes that confer resistance to an antibiotic or an ability to produce a toxin, for example, can be transferred from species to species and provide the recipient bacterium with a selective advantage. In this way, new and sometimes dangerous strains of bacteria have been observed to evolve in the bacterial ecosystems that inhabit hospitals or the various niches in the human body. For example, horizontal gene transfer is responsible for the spread, over the past 40 years, of penicillin-resistant strains of Neisseria gonorrhoeae, the bacterium that causes gonorrhea. On a

1	body. For example, horizontal gene transfer is responsible for the spread, over the past 40 years, of penicillin-resistant strains of Neisseria gonorrhoeae, the bacterium that causes gonorrhea. On a longer time scale, the results can be even more profound; it has been estimated that at least 18% of all of the genes in the present-day genome of E. coli have been acquired by horizontal transfer from another species within the past 100 million years.

1	sex Results in horizontal exchanges of Genetic Information Within a species Horizontal gene transfer among prokaryotes has a parallel in a phenomenon familiar to us all: sex. In addition to the usual vertical transfer of genetic material from parent to offspring, sexual reproduction causes a large-scale horizontal transfer of genetic information between two initially separate cell lineages—those of the father and the mother. A key feature of sex, of course, is that the genetic exchange normally occurs only between individuals of the same species. But no matter whether they occur within a species or between species, horizontal gene Figure 1–22 The viral transfer of DNA into a cell. (A) An electron micrograph of particles of a bacterial virus, the T4 bacteriophage. The head of this virus contains the viral dnA; the tail contains the apparatus for injecting the dnA into a host bacterium. (B) A cross section of an

1	E. coli bacterium with a T4 bacteriophage latched onto its surface. The large dark objects inside the bacterium are the heads of new T4 particles in the course of assembly. When they are mature, the bacterium will burst open to release them. (c–e) The process of dnA injection into the bacterium, as visualized in unstained, frozen samples by cryoelectron microscopy. (c) Attachment begins. Attached state during dnA injection.

1	Attached state during dnA injection. virus head has emptied all of its dnA into the bacterium. (A, courtesy of James Paulson; B, courtesy of Jonathan king and erika hartwig from G. karp, cell and molecular Biology, 2nd ed. new york: John Wiley & sons, 1999. With permission from John Wiley & sons; c–e, courtesy of Ian molineux, university of Texas at Austin and Jun liu, university of Texas health science center, houston.) transfers leave a characteristic imprint: they result in individuals who are related more closely to one set of relatives with respect to some genes, and more closely to another set of relatives with respect to others. By comparing the DNA sequences of individual human genomes, an intelligent visitor from outer space could deduce that humans reproduce sexually, even if it knew nothing about human behavior.

1	Sexual reproduction is widespread (although not universal), especially among eukaryotes. Even bacteria indulge from time to time in controlled sexual exchanges of DNA with other members of their own species. Natural selection has clearly favored organisms that can reproduce sexually, although evolutionary theorists dispute precisely what that selective advantage is. The Function of a Gene can often Be deduced from Its sequence

1	The Function of a Gene can often Be deduced from Its sequence Family relationships among genes are important not just for their historical interest, but because they simplify the task of deciphering gene functions. Once the sequence of a newly discovered gene has been determined, a scientist can tap a few keys on a computer to search the entire database of known gene sequences for genes related to it. In many cases, the function of one or more of these homo-logs will have been already determined experimentally. Since gene sequence determines gene function, one can frequently make a good guess at the function of the new gene: it is likely to be similar to that of the already known homologs. In this way, it is possible to decipher a great deal of the biology of an organism simply by analyzing the DNA sequence of its genome and using the information we already have about the functions of genes in other organisms that have been more intensively studied.

1	more Than 200 Gene Families Are common to All Three Primary Branches of the Tree of life

1	Given the complete genome sequences of representative organisms from all three domains—archaea, bacteria, and eukaryotes—we can search systematically for homologies that span this enormous evolutionary divide. In this way we can begin to take stock of the common inheritance of all living things. There are considerable difficulties in this enterprise. For example, individual species have often lost some of the ancestral genes; other genes have almost certainly been acquired by horizontal transfer from another species and therefore are not truly ancestral, even though shared. In fact, genome comparisons strongly suggest that both lineage-specific gene loss and horizontal gene transfer, in some cases between evolutionarily distant species, have been major factors of evolution, at least among prokaryotes. Finally, in the course of 2 or 3 billion years, some genes that were initially shared will have changed beyond recognition through mutation.

1	Because of all these vagaries of the evolutionary process, it seems that only a small proportion of ancestral gene families has been universally retained in a recognizable form. Thus, out of 4873 protein-coding gene families defined by comparing the genomes of 50 species of bacteria, 13 archaea, and 3 unicellular eukaryotes, only 63 are truly ubiquitous (that is, represented in all the genomes analyzed). The great majority of these universal families include components of the translation and transcription systems. This is not likely to be a realistic approximation of an ancestral gene set. A better—though still crude—idea of the latter can be obtained by tallying the gene families that have representatives in multiple, but not necessarily all, species from all three major domains. Such an analysis reveals 264 ancient conserved families. Each family can be assigned a function (at least in terms of general biochemical activity, but usually with more precision). As shown in Table 1–1,

1	an analysis reveals 264 ancient conserved families. Each family can be assigned a function (at least in terms of general biochemical activity, but usually with more precision). As shown in Table 1–1, the largest number of shared gene families are involved in translation and in amino acid metabolism and transport. However, this set of highly conserved gene families represents only a very rough sketch of the common inheritance of all modern life. A more precise reconstruction of the gene complement of the last universal common ancestor will hopefully become feasible with further genome sequencing and more sophisticated forms of comparative analysis.

1	mutations Reveal the Functions of Genes Without additional information, no amount of gazing at genome sequences will reveal the functions of genes. We may recognize that gene B is like gene A, but how do we discover the function of gene A in the first place? And even if we know the function of gene A, how do we test whether the function of gene B is truly the same as the sequence similarity suggests? How do we connect the world of abstract genetic information with the world of real living organisms?

1	The analysis of gene functions depends on two complementary approaches: genetics and biochemistry. Genetics starts with the study of mutants: we either find or make an organism in which a gene is altered, and then examine the effects on the organism’s structure and performance (Figure 1–23). Biochemistry more directly examines the functions of molecules: here we extract molecules from an organism and then study their chemical activities. By combining genetics and biochemistry, it is possible to find those molecules whose production depends on a given gene. At the same time, careful studies of the performance of the mutant organism show us what role those molecules have in the operation of the organism as a whole. Thus, genetics and biochemistry used in combination with cell biology provide the best way to relate genes and molecules to the structure and function of an organism.

1	In recent years, DNA sequence information and the powerful tools of molecular biology have accelerated progress. From sequence comparisons, we can often identify particular subregions within a gene that have been preserved nearly unchanged over the course of evolution. These conserved subregions are likely to be the most important parts of the gene in terms of function. We can test their individual contributions to the activity of the gene product by creating in the laboratory mutations of specific sites within the gene, or by constructing artificial hybrid genes that combine part of one gene with part of another. Organisms can be engineered to make either the RNA or the protein specified by the gene in large quantities to facilitate biochemical analysis. Specialists in molecular structure can determine the three-dimensional conformation of the gene product, revealing the exact position of every atom in it. Biochemists can determine how each of the

1	Figure 1–23 A mutant phenotype reflecting the function of a gene. A normal yeast (of the species Schizosaccharomyces pombe) is compared with a mutant in which a change in a single gene has converted the cell from a cigar shape (left) to a T shape (right). The mutant gene therefore has a function in the control of cell shape. But how, in molecular terms, does the gene product perform that function? That is a harder question, and it needs biochemical analysis to answer it. (courtesy of kenneth sawin and Paul nurse.) parts of the genetically specified molecule contributes to its chemical behavior. Cell biologists can analyze the behavior of cells that are engineered to express a mutant version of the gene.

1	There is, however, no one simple recipe for discovering a gene’s function, and no simple standard universal format for describing it. We may discover, for example, that the product of a given gene catalyzes a certain chemical reaction, and yet have no idea how or why that reaction is important to the organism. The functional characterization of each new family of gene products, unlike the description of the gene sequences, presents a fresh challenge to the biologist’s ingenuity. Moreover, we will never fully understand the function of a gene until we learn its role in the life of the organism as a whole. To make ultimate sense of gene functions, therefore, we have to study whole organisms, not just molecules or cells. molecular Biology Began with a spotlight on E. coli

1	molecular Biology Began with a spotlight on E. coli Because living organisms are so complex, the more we learn about any particular species, the more attractive it becomes as an object for further study. Each discovery raises new questions and provides new tools with which to tackle general questions in the context of the chosen organism. For this reason, large communities of biologists have become dedicated to studying different aspects of the same model organism.

1	In the early days of molecular biology, the spotlight focused intensely on just one species: the Escherichia coli, or E. coli, bacterium (see Figures 1–13 and 1–14). This small, rod-shaped bacterial cell normally lives in the gut of humans and other vertebrates, but it can be grown easily in a simple nutrient broth in a culture bottle. It adapts to variable chemical conditions and reproduces rapidly, and it can evolve by mutation and selection at a remarkable speed. As with other bacteria, different strains of E. coli, though classified as members of a single species, differ genetically to a much greater degree than do different varieties of a sexually reproducing organism such as a plant or animal. One E. coli strain may possess many hundreds of genes that are absent from another, and the two strains could have as little as 50% of their genes in common. The standard laboratory strain

1	E. coli K-12 has a genome of approximately 4.6 million nucleotide pairs, contained in a single circular molecule of DNA that codes for about 4300 different kinds of proteins (Figure 1–24). In molecular terms, we know more about E. coli than about any other living organism. Most of our understanding of the fundamental mechanisms of life— for example, how cells replicate their DNA, or how they decode the instructions represented in the DNA to direct the synthesis of specific proteins—initially came from studies of E. coli. The basic genetic mechanisms have turned out to be highly conserved throughout evolution: these mechanisms are essentially the same in our own cells as in E. coli.

1	Prokaryotes (cells without a distinct nucleus) are biochemically the most diverse organisms and include species that can obtain all their energy and nutrients from inorganic chemical sources, such as the reactive mixtures of minerals released at hydrothermal vents on the ocean floor—the sort of diet that may have nourished the first living cells 3.5 billion years ago. DNA sequence comparisons reveal the family relationships of living organisms and show that the prokaryotes fall into two groups that diverged early in the course of evolution: the bacteria (or eubacteria) and the archaea. Together with the eukaryotes (cells with a membrane-enclosed nucleus), these constitute the three primary branches of the tree of life.

1	Most bacteria and archaea are small unicellular organisms with compact genomes comprising 1000–6000 genes. Many of the genes within a single organism show strong family resemblances in their DNA sequences, implying that they originated from the same ancestral gene through gene duplication and divergence. Family resemblances (homologies) are also clear when gene sequences are compared between different species, and more than 200 gene families have been so highly (A) 4,639,221 nucleotide pairs Escherichia coli K-12 origin of replication terminus of replication (B) Figure 1–24 The genome of E. coli. (A) A cluster of E. coli cells. (B) A diagram of the genome of

1	E. coli strain k-12. The diagram is circular because the dnA of E. coli, like that of other prokaryotes, forms a single, closed loop. Protein-coding genes are shown as yellow or orange bars, depending on the dnA strand from which they are transcribed; genes encoding only RnA molecules are indicated by green arrows. some genes are transcribed from one strand of the dnA double helix (in a clockwise direction in this diagram), others from the other strand (counterclockwise). (A, courtesy of dr. Tony Brain and david Parker/Photo Researchers; B, adapted from F.R. Blattner et al., Science 277:1453–1462, 1997.) conserved that they can be recognized as common to most species from all three domains of the living world. Thus, given the DNA sequence of a newly discovered gene, it is often possible to deduce the gene’s function from the known function of a homologous gene in an intensively studied model organism, such as the bacterium E. coli.

1	E. coli. Eukaryotic cells, in general, are bigger and more elaborate than prokaryotic cells, and their genomes are bigger and more elaborate, too. The greater size is accompanied by radical differences in cell structure and function. Moreover, many classes of eukaryotic cells form multicellular organisms that attain levels of complexity unmatched by any prokaryote.

1	Because they are so complex, eukaryotes confront molecular biologists with a special set of challenges that will concern us in the rest of this book. Increasingly, biologists attempt to meet these challenges through the analysis and manipulation of the genetic information within cells and organisms. It is therefore important at the outset to know something of the special features of the eukaryotic genome. We begin by briefly discussing how eukaryotic cells are organized, how this reflects their way of life, and how their genomes differ from those of prokaryotes. This leads us to an outline of the strategy by which cell biologists, by exploiting genetic and biochemical information, are attempting to discover how eukaryotic organisms work. eukaryotic cells may have originated as Predators

1	By definition, eukaryotic cells keep their DNA in an internal compartment called the nucleus. The nuclear envelope, a double layer of membrane, surrounds the nucleus and separates the DNA from the cytoplasm. Eukaryotes also have other features that set them apart from prokaryotes (Figure 1–25). Their cells are, typically, 10 times bigger in linear dimension and 1000 times larger in volume. They have an elaborate cytoskeleton—a system of protein filaments crisscrossing the cytoplasm and forming, together with the many proteins that attach to them, a system of girders, ropes, and motors that gives the cell mechanical strength, controls its shape, and drives and guides its movements (Movie 1.1). And the nuclear envelope is only one part of a set of internal membranes, each structurally similar to the plasma membrane and enclosing different types of spaces inside the cell, many of them involved in digestion and secretion. Lacking the tough cell wall of most bacteria, animal cells and the

1	to the plasma membrane and enclosing different types of spaces inside the cell, many of them involved in digestion and secretion. Lacking the tough cell wall of most bacteria, animal cells and the free-living eukaryotic cells called protozoa can change their shape rapidly and engulf other cells and small objects by phagocytosis (Figure 1–26).

1	How all of the unique properties of eukaryotic cells evolved, and in what sequence, is still a mystery. One plausible view, however, is that they are all reflections of the way of life of a primordial cell that was a predator, living by capturing other cells and eating them (Figure 1–27). Such a way of life requires a large cell with a flexible plasma membrane, as well as an elaborate cytoskeleton to support Figure 1–25 The major features of eukaryotic cells. The drawing depicts a typical animal cell, but almost all the same components are found in plants and fungi as well as in single-celled eukaryotes such as yeasts and protozoa. Plant cells contain chloroplasts in addition to the components shown here, and their plasma membrane is surrounded by a tough external wall formed of cellulose.

1	and move this membrane. It may also require that the cell’s long, fragile DNA molecules be sequestered in a separate nuclear compartment, to protect the genome from damage by the movements of the cytoskeleton.

1	A predatory way of life helps to explain another feature of eukaryotic cells. All such cells contain (or at one time did contain) mitochondria (Figure 1–28). These small bodies in the cytoplasm, enclosed by a double layer of membrane, take up oxygen and harness energy from the oxidation of food molecules—such as sugars—to produce most of the ATP that powers the cell’s activities. Mitochondria are similar in size to small bacteria, and, like bacteria, they have their own genome in the form of a circular DNA molecule, their own ribosomes that differ from those elsewhere in the eukaryotic cell, and their own transfer RNAs. It is now generally accepted that mitochondria originated from free-living oxygen-metabolizing (aerobic) bacteria that were engulfed by an ancestral cell that could otherwise make no such use of oxygen (that is, was anaerobic). Escaping digestion, these bacteria evolved in symbiosis with the engulfing cell and its progeny, receiving

1	Figure 1–27 A single-celled eukaryote that eats other cells. (A) Didinium is a carnivorous protozoan, belonging to the group known as ciliates. It has a globular body, about 150 μm in diameter, encircled by two fringes of cilia—sinuous, whiplike appendages that beat continually; its front end is flattened except for a single protrusion, rather like a snout. (B) A Didinium engulfing its prey. Didinium normally swims around in the water at high speed by means of the synchronous beating of its cilia. When it encounters a suitable prey (yellow), usually another type of protozoan, it releases numerous small paralyzing darts from its snout region. Then, the Didinium attaches to and devours the other cell by phagocytosis, inverting like a hollow ball to engulf its victim, which can be almost as large as itself. (courtesy of d. Barlow.)

1	Figure 1–26 Phagocytosis. This series of stills from a movie shows a human white blood cell (a neutrophil) engulfing a red blood cell (artificially colored red) that has been treated with an antibody that marks it for destruction (see movie 13.5). (courtesy of stephen e. malawista and Anne de Boisfleury chevance.) shelter and nourishment in return for the power generation they performed for their hosts. This partnership between a primitive anaerobic predator cell and an aerobic bacterial cell is thought to have been established about 1.5 billion years ago, when the Earth’s atmosphere first became rich in oxygen. As indicated in Figure 1–29, recent genomic analyses suggest that the first eukaryotic cells formed after an archaeal cell engulfed an aerobic bacterium. This would explain why all eukaryotic cells today, including those that live as strict anaerobes show clear evidence that they once contained mitochondria.

1	Many eukaryotic cells—specifically, those of plants and algae—also contain another class of small membrane-enclosed organelles somewhat similar to mitochondria—the chloroplasts (Figure 1–30). Chloroplasts perform photosynthesis, using the energy of sunlight to synthesize carbohydrates from atmospheric carbon dioxide and water, and deliver the products to the host cell as food. Like mitochondria, chloroplasts have their own genome. They almost certainly originated as symbiotic photosynthetic bacteria, acquired by eukaryotic cells that already possessed mitochondria (Figure 1–31).

1	A eukaryotic cell equipped with chloroplasts has no need to chase after other cells as prey; it is nourished by the captive chloroplasts it has inherited from its ancestors. Correspondingly, plant cells, although they possess the cytoskeletal equipment for movement, have lost the ability to change shape rapidly and to engulf other cells by phagocytosis. Instead, they create around themselves a tough, protective cell wall. If the first eukaryotic cells were predators on other organisms, we can view plant cells as cells that have made the transition from hunting to farming. Fungi represent yet another eukaryotic way of life. Fungal cells, like animal cells, possess mitochondria but not chloroplasts; but in contrast with animal cells and protozoa, they have a tough outer wall that limits their ability to move rapidly Figure 1–28 A mitochondrion. (A) A cross section, as seen in the electron microscope.

1	Figure 1–28 A mitochondrion. (A) A cross section, as seen in the electron microscope. (B) A drawing of a mitochondrion with part of it cut away to show the three-dimensional structure (Movie 1.2). (c) A schematic eukaryotic cell, with the interior space of a mitochondrion, containing the mitochondrial dnA and ribosomes, colored. note the smooth outer membrane and the convoluted inner membrane, which houses the proteins that generate ATP from the oxidation of food molecules. (A, courtesy of daniel s. Friend.) Figure 1–29 The origin of mitochondria. archaeon) is thought to have engulfed the bacterial ancestor of mitochondria, initiating a symbiotic relationship. clear evidence of a dual bacterial and archaeal inheritance can be discerned today in the genomes of all eukaryotes.

1	or to swallow up other cells. Fungi, it seems, have turned from hunters into scavengers: other cells secrete nutrient molecules or release them upon death, and fungi feed on these leavings—performing whatever digestion is necessary extracellularly, by secreting digestive enzymes to the exterior.

1	The genetic information of eukaryotic cells has a hybrid origin—from the ancestral anaerobic archaeal cell, and from the bacteria that it adopted as symbionts. Most of this information is stored in the nucleus, but a small amount remains inside the mitochondria and, for plant and algal cells, in the chloroplasts. When mitochondrial DNA and the chloroplast DNA are separated from the nuclear DNA and individually analyzed and sequenced, the mitochondrial and chloroplast genomes are found to be degenerate, cut-down versions of the corresponding bacterial genomes. In a human cell, for example, the mitochondrial genome consists of only 16,569 nucleotide pairs, and codes for only 13 proteins, 2 ribosomal RNA components, and 22 transfer RNAs.

1	Figure 1–30 Chloroplasts. These organelles capture the energy of sunlight in plant cells and some single-celled eukaryotes. (A) A single cell isolated from a leaf of a flowering plant, seen in the light microscope, showing the green chloroplasts (Movie 1.3 and see movie 14.9). (B) A drawing of one of the chloroplasts, showing the highly folded system of internal membranes containing the chlorophyll molecules by which light is absorbed. (A, courtesy of Preeti dahiya.) early eukaryotic cell eukaryotic cell capable of photosynthesis

1	Many of the genes that are missing from the mitochondria and chloroplasts have not been lost; instead, they have moved from the symbiont genome into the DNA of the host cell nucleus. The nuclear DNA of humans contains many genes coding for proteins that serve essential functions inside the mitochondria; in plants, the nuclear DNA also contains many genes specifying proteins required in chloroplasts. In both cases, the DNA sequences of these nuclear genes show clear evidence of their origin from the bacterial ancestor of the respective organelle.

1	Natural selection has evidently favored mitochondria with small genomes. By contrast, the nuclear genomes of most eukaryotes seem to have been free to enlarge. Perhaps the eukaryotic way of life has made large size an advantage: predators typically need to be bigger than their prey, and cell size generally increases in proportion to genome size. Whatever the reason, aided by a massive accumulation of DNA segments derived from parasitic transposable elements (discussed in Chapter 5), the genomes of most eukaryotes have become orders of magnitude larger than those of bacteria and archaea (Figure 1–32). The freedom to be extravagant with DNA has had profound implications. Eukaryotes not only have more genes than prokaryotes; they also have vastly more DNA that does not code for protein. The human genome contains 1000 times as many nucleotide pairs as the genome of a typical bacterium, perhaps 10 times as Figure 1–31 The origin of chloroplasts.

1	Figure 1–31 The origin of chloroplasts. An early eukaryotic cell, already possessing mitochondria, engulfed a photosynthetic bacterium (a cyanobacterium) and retained it in symbiosis. Present-day chloroplasts are thought to trace their ancestry back to a single species of cyanobacterium that was adopted as an internal symbiont (an endosymbiont) over a billion years ago. Figure 1–32 Genome sizes compared. Genome size is measured in nucleotide pairs of dnA per haploid genome, that is, per single copy of the genome. (The cells of sexually reproducing organisms such as ourselves are generally diploid: they contain two copies of the genome, one inherited from the mother, the other from the father.) closely related organisms can vary widely in the quantity of dnA in their genomes, even though they contain similar numbers of functionally distinct genes. (data from

1	W.h. li, molecular evolution, pp. 380–383. sunderland, mA: sinauer, 1997.) many genes, and a great deal more noncoding DNA (~98.5% of the genome for a human does not code for proteins, as opposed to 11% of the genome for the bacterium E. coli). The estimated genome sizes and gene numbers for some eukaryotes are compiled for easy comparison with E. coli in Table 1–2; we shall discuss how each of these eukaryotes serves as a model organism shortly.

1	Much of our noncoding DNA is almost certainly dispensable junk, retained like a mass of old papers because, when there is little pressure to keep an archive small, it is easier to retain everything than to sort out the valuable information and discard the rest. Certain exceptional eukaryotic species, such as the puffer fish, bear witness to the profligacy of their relatives; they have somehow managed to rid themselves of large quantities of noncoding DNA. Yet they appear similar in structure, behavior, and fitness to related species that have vastly more such DNA (see Figure 4–71).

1	Even in compact eukaryotic genomes such as that of puffer fish, there is more noncoding DNA than coding DNA, and at least some of the noncoding DNA certainly has important functions. In particular, it regulates the expression of adjacent genes. With this regulatory DNA, eukaryotes have evolved distinctive ways of controlling when and where a gene is brought into play. This sophisticated gene regulation is crucial for the formation of complex multicellular organisms. The Genome defines the Program of multicellular development The cells in an individual animal or plant are extraordinarily varied. Fat cells, skin cells, bone cells, nerve cells—they seem as dissimilar as any cells could be (Figure 1–33). Yet all these cell types are the descendants of a single fertilized egg cell, and all (with minor exceptions) contain identical copies of the genome of the species.

1	The differences result from the way in which the cells make selective use of their genetic instructions according to the cues they get from their surroundings in the developing embryo. The DNA is not just a shopping list specifying the molecules that every cell must have, and the cell is not an assembly of all the items on the list. Rather, the cell behaves as a multipurpose machine, with sensors to receive environmental signals and with highly developed abilities to call different sets of genes into action according to the sequences of signals to which the cell has been exposed. The genome in each cell is big enough to accommodate the Figure 1–33 Cell types can vary enormously in size and shape. An animal nerve cell is compared here with a neutrophil, a type of white blood cell. Both are drawn to scale. information that specifies an entire multicellular organism, but in any individual cell only part of that information is used.

1	information that specifies an entire multicellular organism, but in any individual cell only part of that information is used. A large number of genes in the eukaryotic genome code for proteins that regulate the activities of other genes. Most of these transcription regulators act by binding, directly or indirectly, to the regulatory DNA adjacent to the genes that are to be controlled, or by interfering with the abilities of other proteins to do so. The expanded genome of eukaryotes therefore not only specifies the hardware of the cell, but also stores the software that controls how that hardware is used (Figure 1–34).

1	Cells do not just passively receive signals; rather, they actively exchange signals with their neighbors. Thus, in a developing multicellular organism, the same control system governs each cell, but with different consequences depending on the messages exchanged. The outcome, astonishingly, is a precisely patterned array of cells in different states, each displaying a character appropriate to its position in the multicellular structure. many eukaryotes live as solitary cells

1	many eukaryotes live as solitary cells Many species of eukaryotic cells lead a solitary life—some as hunters (the protozoa), some as photosynthesizers (the unicellular algae), some as scavengers (the unicellular fungi, or yeasts). Figure 1–35 conveys something of the astonishing variety of the single-celled eukaryotes. The anatomy of protozoa, especially, is often elaborate and includes such structures as sensory bristles, photoreceptors, sinuously beating cilia, leglike appendages, mouth parts, stinging darts, and musclelike contractile bundles. Although they are single cells, protozoa can be as intricate, as versatile, and as complex in their behavior as many multicellular organisms (see Figure 1–27, Movie 1.4, and Movie 1.5).

1	In terms of their ancestry and DNA sequences, the unicellular eukaryotes are far more diverse than the multicellular animals, plants, and fungi, which arose as three comparatively late branches of the eukaryotic pedigree (see Figure 1–17). As with prokaryotes, humans have tended to neglect them because they are microscopic. Only now, with the help of genome analysis, are we beginning to understand their positions in the tree of life, and to put into context the glimpses these strange creatures can offer us of our distant evolutionary past. A yeast serves as a minimal model eukaryote The molecular and genetic complexity of eukaryotes is daunting. Even more than for prokaryotes, biologists need to concentrate their limited resources on a few selected model organisms to unravel this complexity. Figure 1–34 Genetic control of the program of multicellular development.

1	Figure 1–34 Genetic control of the program of multicellular development. The role of a regulatory gene is demonstrated in the snapdragon Antirrhinum. In this example, a mutation in a single gene coding for a regulatory protein causes leafy shoots to develop in place of flowers: because a regulatory protein has been changed, the cells adopt characters that would be appropriate to a different location in the normal plant. The mutant is on the left, the normal plant on the right. (courtesy of enrico coen and Rosemary carpenter.) To analyze the internal workings of the eukaryotic cell without the additional problems of multicellular development, it makes sense to use a species that is unicellular and as simple as possible. The popular choice for this role of minimal model eukaryote has been the yeast Saccharomyces cerevisiae (Figure 1–36)—the same species that is used by brewers of beer and bakers of bread.

1	S. cerevisiae is a small, single-celled member of the kingdom of fungi and thus, according to modern views, is at least as closely related to animals as it is to plants. It is robust and easy to grow in a simple nutrient medium. Like other fungi, it has a tough cell wall, is relatively immobile, and possesses mitochondria but not chloroplasts. When nutrients are plentiful, it grows and divides almost as rapidly as a bacterium. It can reproduce either vegetatively (that is, by simple cell division), or sexually: two yeast cells that are haploid (possessing a single copy of the genome) can fuse to create a cell that is diploid (containing a double genome); and the diploid cell can undergo meiosis (a reduction division) to produce cells that are once again haploid (Figure 1–37). In contrast with higher plants and animals, the yeast can divide indefinitely in either the haploid or the diploid state, and the process leading from one state to the other can be induced at will by changing the

1	higher plants and animals, the yeast can divide indefinitely in either the haploid or the diploid state, and the process leading from one state to the other can be induced at will by changing the growth conditions.

1	In addition to these features, the yeast has a further property that makes it a convenient organism for genetic studies: its genome, by eukaryotic standards, is exceptionally small. Nevertheless, it suffices for all the basic tasks that every eukaryotic cell must perform. Mutants are available for essentially every gene, Figure 1–35 An assortment of protozoa: a small sample of an extremely diverse class of organisms. The drawings are done to different scales, but in each case the scale bar represents 10 μm. The organisms in (A), (c), and (G) are ciliates; is a heliozoan; (d) is an amoeba; is a dinoflagellate; and (F) is a euglenoid. (From m.A. sleigh, Biology of Protozoa. cambridge, uk: cambridge university Press, 1973.)

1	Figure 1–36 The yeast Saccharomyces cerevisiae. (A) A scanning electron micrograph of a cluster of the cells. This species is also known as budding yeast; it proliferates by forming a protrusion or bud that enlarges and then separates from the rest of the original cell. many cells with buds are visible in this micrograph. (B) A transmission electron micrograph of a cross section of a yeast cell, showing its nucleus, mitochondrion, and thick cell wall. (A, courtesy of Ira herskowitz and eric schabatach.) Figure 1–37 The reproductive cycles of the yeast S. cerevisiae.

1	Figure 1–37 The reproductive cycles of the yeast S. cerevisiae. depending on environmental conditions and on details of the genotype, cells of this species can exist in either a diploid (2n) state, with a double chromosome set, or a haploid (n) state, with a single chromosome set. The diploid form can either proliferate by ordinary cell-division cycles or undergo meiosis to produce haploid cells. The haploid form can either proliferate by ordinary cell-division cycles or undergo sexual fusion with another haploid cell to become diploid. meiosis is triggered by starvation and gives rise to spores—haploid cells in a dormant state, resistant to harsh environmental conditions.

1	and studies on yeasts (using both S. cerevisiae and other species) have provided a key to many crucial processes, including the eukaryotic cell-division cycle—the critical chain of events by which the nucleus and all the other components of a cell are duplicated and parceled out to create two daughter cells from one. The control system that governs this process has been so well conserved over the course of evolution that many of its components can function interchangeably in yeast and human cells: if a mutant yeast lacking an essential yeast cell-division-cycle gene is supplied with a copy of the homologous cell-division-cycle gene from a human, the yeast is cured of its defect and becomes able to divide normally. The expression levels of All the Genes of An organism can Be monitored simultaneously

1	The expression levels of All the Genes of An organism can Be monitored simultaneously The complete genome sequence of S. cerevisiae, determined in 1997, consists of approximately 13,117,000 nucleotide pairs, including the small contribution (78,520 nucleotide pairs) of the mitochondrial DNA. This total is only about 2.5 times as much DNA as there is in E. coli, and it codes for only 1.5 times as many distinct proteins (about 6600 in all). The way of life of S. cerevisiae is similar in many ways to that of a bacterium, and it seems that this yeast has likewise been subject to selection pressures that have kept its genome compact.

1	Knowledge of the complete genome sequence of any organism—be it a yeast or a human—opens up new perspectives on the workings of the cell: things that once seemed impossibly complex now seem within our grasp. Using techniques described in Chapter 8, it is now possible, for example, to monitor, simultaneously, the amount of mRNA transcript that is produced from every gene in the yeast genome under any chosen conditions, and to see how this whole pattern of gene activity changes when conditions change. The analysis can be repeated with mRNA prepared from mutant cells lacking a chosen gene—any gene that we care to test. In principle, this approach provides a way to reveal the entire system of control relationships that govern gene expression—not only in yeast cells, but in any organism whose genome sequence is known. Arabidopsis has Been chosen out of 300,000 species As a model Plant

1	Arabidopsis has Been chosen out of 300,000 species As a model Plant The large multicellular organisms that we see around us—the flowers and trees and animals—seem fantastically varied, but they are much closer to one another in their evolutionary origins, and more similar in their basic cell biology, than the great host of microscopic single-celled organisms. Thus, while bacteria and archaea are separated by perhaps 3.5 billion years of evolution, vertebrates and insects are separated by about 700 million years, fish and mammals by about 450 million years, and the different species of flowering plants by only about 150 million years.

1	Because of the close evolutionary relationship between all flowering plants, we can, once again, get insight into the cell and molecular biology of this whole class of organisms by focusing on just one or a few species for detailed analysis. Out of the several hundred thousand species of flowering plants on Earth today, molecular biologists have chosen to concentrate their efforts on a small weed, the common Thale cress Arabidopsis thaliana (Figure 1–38), which can be grown indoors in large numbers and produces thousands of offspring per plant after 8–10 weeks. Arabidopsis has a total genome size of approximately 220 million nucleotide pairs, about 17 times the size of yeast’s (see Table 1–2). The World of Animal cells Is Represented By a Worm, a Fly, a Fish, a mouse, and a human

1	The World of Animal cells Is Represented By a Worm, a Fly, a Fish, a mouse, and a human Multicellular animals account for the majority of all named species of living organisms, and for the largest part of the biological research effort. Five species have emerged as the foremost model organisms for molecular genetic studies. In order of increasing size, they are the nematode worm Caenorhabditis elegans, the fly Drosophila melanogaster, the zebrafish Danio rerio, the mouse Mus musculus, and the human, Homo sapiens. Each has had its genome sequenced.

1	Caenorhabditis elegans (Figure 1–39) is a small, harmless relative of the eel-worm that attacks crops. With a life cycle of only a few days, an ability to survive in a freezer indefinitely in a state of suspended animation, a simple body plan, and an unusual life cycle that is well suited for genetic studies (described in Chapter 21), it is an ideal model organism. C. elegans develops with clockwork precision from a fertilized egg cell into an adult worm with exactly 959 body cells (plus a variable number of egg and sperm cells)—an unusual degree of regularity for an animal. We now have a minutely detailed description of the sequence of events by which this occurs, as the cells divide, move, and change their character according to strict and predictable rules. The genome of 130 million nucleotide pairs codes for about 21,000 proteins, and many mutants and other tools are available for the testing of gene functions. Although the worm has a body plan very different from our own, the

1	nucleotide pairs codes for about 21,000 proteins, and many mutants and other tools are available for the testing of gene functions. Although the worm has a body plan very different from our own, the conservation of biological mechanisms has been sufficient for the worm to be a model for many of the developmental and cell-biological processes that occur in the human body. Thus, for example, studies of the worm have been critical for helping us to understand the programs of cell division and cell death that determine the number of cells in the body—a topic of great importance for both developmental biology and cancer research.

1	studies in Drosophila Provide a key to vertebrate development The fruit fly Drosophila melanogaster (Figure 1–40) has been used as a model genetic organism for longer than any other; in fact, the foundations of classical genetics were built to a large extent on studies of this insect. Over 80 years ago, it provided, for example, definitive proof that genes—the abstract units of hereditary information—are carried on chromosomes, concrete physical objects whose behavior had been closely followed in the eukaryotic cell with the light microscope, but whose function was at first unknown. The proof depended on one of the many features that make Drosophila peculiarly convenient for genetics—the giant chromosomes, with characteristic banded appearance, that are visible in Figure 1–38 Arabidopsis thaliana, the plant chosen as the primary model for studying plant molecular genetics. (courtesy of Toni hayden and the John Innes Foundation.) 0.2 mm

1	Figure 1–38 Arabidopsis thaliana, the plant chosen as the primary model for studying plant molecular genetics. (courtesy of Toni hayden and the John Innes Foundation.) 0.2 mm Figure 1–39 Caenorhabditis elegans, the first multicellular organism to have its complete genome sequence determined. This small nematode, about 1 mm long, lives in the soil. most individuals are hermaphrodites, producing both eggs and sperm. (courtesy of maria Gallegos, university of Wisconsin, madison.) some of its cells (Figure 1–41). Specific changes in the hereditary information, manifest in families of mutant flies, were found to correlate exactly with the loss or alteration of specific giant-chromosome bands.

1	In more recent times, Drosophila, more than any other organism, has shown us how to trace the chain of cause and effect from the genetic instructions encoded in the chromosomal DNA to the structure of the adult multicellular body. Drosophila mutants with body parts strangely misplaced or mispatterned provided the key to the identification and characterization of the genes required to make a properly structured body, with gut, limbs, eyes, and all the other parts in their correct places. Once these Drosophila genes were sequenced, the genomes of vertebrates could be scanned for homologs. These were found, and their functions in vertebrates were then tested by analyzing mice in which the genes had been mutated. The results, as we see later in the book, reveal an astonishing degree of similarity in the molecular mechanisms that govern insect and vertebrate development (discussed in Chapter 21).

1	The majority of all named species of living organisms are insects. Even if Drosophila had nothing in common with vertebrates, but only with insects, it would still be an important model organism. But if understanding the molecular genetics of vertebrates is the goal, why not simply tackle the problem head-on? Why sidle up to it obliquely, through studies in Drosophila?

1	Drosophila requires only 9 days to progress from a fertilized egg to an adult; it is vastly easier and cheaper to breed than any vertebrate, and its genome is much smaller—about 200 million nucleotide pairs, compared with 3200 million for a human. This genome codes for about 15,000 proteins, and mutants can now be obtained for essentially any gene. But there is also another, deeper reason why genetic mechanisms that are hard to discover in a vertebrate are often readily revealed in the fly. This relates, as we now explain, to the frequency of gene duplication, which is substantially greater in vertebrate genomes than in the fly genome and has probably been crucial in making vertebrates the complex and subtle creatures that they are. The vertebrate Genome Is a Product of Repeated duplications

1	The vertebrate Genome Is a Product of Repeated duplications Almost every gene in the vertebrate genome has paralogs—other genes in the same genome that are unmistakably related and must have arisen by gene duplication. In many cases, a whole cluster of genes is closely related to similar clusters present elsewhere in the genome, suggesting that genes have been duplicated in linked groups rather than as isolated individuals. According to one hypothesis, at an early stage in the evolution of the vertebrates, the entire genome underwent duplication twice in succession, giving rise to four copies of every gene. The precise course of vertebrate genome evolution remains uncertain, because many further evolutionary changes have occurred since these ancient events. Figure 1–40 Drosophila melanogaster.

1	The precise course of vertebrate genome evolution remains uncertain, because many further evolutionary changes have occurred since these ancient events. Figure 1–40 Drosophila melanogaster. molecular genetic studies on this fly have provided the main key to understanding how all animals develop from a fertilized egg into an adult. (From e.B. lewis, Science 221:cover, 1983. With permission from AAAs.) Figure 1–41 Giant chromosomes from salivary gland cells of Drosophila.

1	Figure 1–41 Giant chromosomes from salivary gland cells of Drosophila. Because many rounds of dnA replication have occurred without an intervening cell division, each of the chromosomes in these unusual cells contains over 1000 identical dnA molecules, all aligned in register. This makes them easy to see in the light microscope, where they display a characteristic and reproducible banding pattern. specific bands can be identified as the locations of specific genes: a mutant fly with a region of the banding pattern missing shows a phenotype reflecting loss of the genes in that region. Genes that are being transcribed at a high rate correspond to bands with a “puffed” appearance. The bands stained dark brown in the micrograph are sites where a particular regulatory protein is bound to the dnA. (courtesy of B. Zink and R. Paro, from R. Paro, Trends Genet. 6:416–421, 1990. With permission from elsevier.)

1	R. Paro, Trends Genet. 6:416–421, 1990. With permission from elsevier.) Genes that were once identical have diverged; many of the gene copies have been lost through disruptive mutations; some have undergone further rounds of local duplication; and the genome, in each branch of the vertebrate family tree, has suffered repeated rearrangements, breaking up most of the original gene orderings. Comparison of the gene order in two related organisms, such as the human and the mouse, reveals that—on the time scale of vertebrate evolution—chromosomes frequently fuse and fragment to move large blocks of DNA sequence around. Indeed, it is possible, as discussed in Chapter 4, that the present state of affairs is the result of many separate duplications of fragments of the genome, rather than duplications of the genome as a whole.

1	There is, however, no doubt that such whole-genome duplications do occur from time to time in evolution, for we can see recent instances in which duplicated chromosome sets are still clearly identifiable as such. The frog genus Xenopus, for example, comprises a set of closely similar species related to one another by repeated duplications or triplications of the whole genome. Among these frogs are X. tropicalis, with an ordinary diploid genome; the common laboratory species X. laevis, with a duplicated genome and twice as much DNA per cell; and X. ruwenzoriensis, with a sixfold reduplication of the original genome and six times as much DNA per cell (108 chromosomes, compared with 36 in X. laevis, for example). These species are estimated to have diverged from one another within the past 120 million years (Figure 1–42). The Frog and the Zebrafish Provide Accessible models for vertebrate development

1	The Frog and the Zebrafish Provide Accessible models for vertebrate development Frogs have long been used to study the early steps of embryonic development in vertebrates, because their eggs are big, easy to manipulate, and fertilized outside of the animal, so that the subsequent development of the early embryo is easily followed (Figure 1–43). Xenopus laevis, in particular, continues to be an important model organism, even though it is poorly suited for genetic analysis (Movie 1.6 and see Movie 21.1).

1	The zebrafish Danio rerio has similar advantages, but without this drawback. Its genome is compact—only half as big as that of a mouse or a human—and it has a generation time of only about three months. Many mutants are known, and genetic engineering is relatively easy. The zebrafish has the added virtue that it is transparent for the first two weeks of its life, so that one can watch the behavior of individual cells in the living organism (see Movie 21.2). All this has made it an increasingly important model vertebrate (Figure 1–44). The mouse Is the Predominant mammalian model organism

1	The mouse Is the Predominant mammalian model organism Mammals have typically two times as many genes as Drosophila, a genome that is 16 times larger, and millions or billions of times as many cells in their adult bodies. In terms of genome size and function, cell biology, and molecular mechanisms, mammals are nevertheless a highly uniform group of organisms. Even anatomically, the differences among mammals are chiefly a matter of size and proportions; it is hard to think of a human body part that does not have a counterpart in elephants and mice, and vice versa. Evolution plays freely with quantitative features, but it does not readily change the logic of the structure.

1	Figure 1–42 Two species of the frog genus Xenopus. X. tropicalis, above, has an ordinary diploid genome; X. laevis, below, has twice as much dnA per cell. From the banding patterns of their chromosomes and the arrangement of genes along them, as well as from comparisons of gene sequences, it is clear that the large-genome species have evolved through duplications of the whole genome. These duplications are thought to have occurred in the aftermath of matings between frogs of slightly divergent Xenopus species. (courtesy of e. Amaya, m. offield, and R. Grainger, Trends Genet. 14:253– 255, 1998. With permission from elsevier.) Figure 1–43 Stages in the normal development of a frog. These drawings show the development of a Rana pipiens tadpole from a fertilized egg. The entire process takes place outside of the mother, making the mechanisms tail bud involved readily accessible for experimental studies. (From W. shumway, Anat. Rec. 78:139–147, 1940.)

1	For a more exact measure of how closely mammalian species resemble one another genetically, we can compare the nucleotide sequences of corresponding (orthologous) genes, or the amino acid sequences of the proteins that these genes encode. The results for individual genes and proteins vary widely. But typically, if we line up the amino acid sequence of a human protein with that of the orthologous protein from, say, an elephant, about 85% of the amino acids are identical. A similar comparison between human and bird shows an amino acid identity of about 70%—twice as many differences, because the bird and the mammalian lineages have had twice as long to diverge as those of the elephant and the human (Figure 1–45).

1	The mouse, being small, hardy, and a rapid breeder, has become the foremost model organism for experimental studies of vertebrate molecular genetics. Many naturally occurring mutations are known, often mimicking the effects of corresponding mutations in humans (Figure 1–46). Methods have been developed, moreover, to test the function of any chosen mouse gene, or of any noncoding portion of the mouse genome, by artificially creating mutations in it, as we explain later in the book. Just one made-to-order mutant mouse can provide a wealth of information for the cell biologist. It reveals the effects of the chosen mutation in a host of different contexts, simultaneously testing the action of the gene in all the different kinds of cells in the body that could in principle be affected. As humans, we have a special interest in the human genome. We want to know the full set of parts from which we are made, and to discover how they work. But even

1	As humans, we have a special interest in the human genome. We want to know the full set of parts from which we are made, and to discover how they work. But even Figure 1–44 Zebrafish as a model for studies of vertebrate development. These small, hardy tropical fish are convenient for genetic studies. Additionally, they have transparent embryos that develop outside of the mother, so that one can clearly observe cells moving and changing their character in the living organism throughout its development. (A) Adult fish. (B) An embryo 24 hours after fertilization. (A, with permission from steve Baskauf; B, from m. Rhinn et al., Neural Dev. 4:12, 2009.)

1	Figure 1–45 Times of divergence of different vertebrates. The scale on the left shows the estimated date and geological era of the last common ancestor of each specified pair of animals. each time estimate is based on comparisons of the amino acid sequences of orthologous proteins; the longer the animals of a pair have had to evolve independently, the smaller the percentage of amino acids that remain identical. The time scale has been calibrated to match the fossil evidence showing that the last common ancestor of mammals and birds lived 310 million years ago.

1	The figures on the right give data on protein—the α chain of hemoglobin. note that although there is a clear general trend of increasing divergence with increasing time for this protein, there are irregularities that are thought to reflect the action of changes of hemoglobin sequence when the organisms experienced special physiological demands. some proteins, subject to stricter functional constraints, evolve much more slowly than hemoglobin, time in millions of years others as much as five times faster. All this gives rise to substantial uncertainties in estimates of divergence times, and some experts believe that the major groups of mammals diverged from one another as if you were a mouse, preoccupied with the molecular biology of mice, humans would be attractive as model genetic organisms, because of one special property: through medical examinations and self-reporting, we catalog our own genetic (and other) disorders. The human population is enormous, consisting today of some 7

1	because of one special property: through medical examinations and self-reporting, we catalog our own genetic (and other) disorders. The human population is enormous, consisting today of some 7 billion individuals, and this self-documenting property means that a huge database of information exists on human mutations. The human genome sequence of more than 3 billion nucleotide pairs has been determined for thousands of different people, making it easier than ever before to identify at a molecular level the precise genetic change responsible for any given human mutant phenotype.

1	By drawing together the insights from humans, mice, fish, flies, worms, yeasts, plants, and bacteria—using gene sequence similarities to map out the correspondences between one model organism and another—we are enriching our understanding of them all. much as 60 million years more recently than shown here. (Adapted from s. kumar and s.B. hedges, Nature 392:917–920, 1998. With permission from macmillan Publishers ltd.) Figure 1–46 Human and mouse: similar genes and similar development. The human baby and the mouse shown here have similar white patches on their foreheads because both have mutations in the same gene (called Kit), required for the development and maintenance of pigment cells. (courtesy of R.A. Fleischman.)

1	What precisely do we mean when we speak of the human genome? Whose genome? On average, any two people taken at random differ in about one or two in every 1000 nucleotide pairs in their DNA sequence. The genome of the human species is, properly speaking, a very complex thing, embracing the entire pool of variant genes found in the human population. Knowledge of this variation is helping us to understand, for example, why some people are prone to one disease, others to another; why some respond well to a drug, others badly. It is also providing clues to our history—the population movements and minglings of our ancestors, the infections they suffered, the diets they ate. All these things have left traces in the variant forms of genes that survive today in the human communities that populate the globe. To understand cells and organisms Will Require mathematics, computers, and Quantitative Information

1	To understand cells and organisms Will Require mathematics, computers, and Quantitative Information Empowered by knowledge of complete genome sequences, we can list the genes, proteins, and RNA molecules in a cell, and we have methods that allow us to begin to depict the complex web of interactions between them. But how are we to turn all this information into an understanding of how cells work? Even for a single cell type belonging to a single species of organism, the current deluge of data seems overwhelming. The sort of informal reasoning on which biologists usually rely seems totally inadequate in the face of such complexity.

1	In fact, the difficulty is more than just a matter of information overload. Biological systems are, for example, full of feedback loops, and the behavior of even the simplest of systems with feedback is remarkably difficult to predict by intuition alone (Figure 1–47); small changes in parameters can cause radical changes in outcome. To go from a circuit diagram to a prediction of the behavior of the system, we need detailed quantitative information, and to draw deductions from that information we need mathematics and computers.

1	Such tools for quantitative reasoning are essential, but they are not all-powerful. You might think that, knowing how each protein influences each other protein, and how the expression of each gene is regulated by the products of others, we should soon be able to calculate how the cell as a whole will behave, just as an astronomer can calculate the orbits of the planets, or a chemical engineer can calculate the flows through a chemical plant. But any attempt to perform this feat for anything close to an entire living cell rapidly reveals the limits of our present knowledge. The information we have, plentiful as it is, is full of gaps and uncertainties. Moreover, it is largely qualitative rather than quantitative. Most often, cell biologists studying the cell’s control systems sum up their knowledge in simple schematic diagrams—this book is full of them—rather than in numbers, graphs, and differential equations.

1	To progress from qualitative descriptions and intuitive reasoning to quantitative descriptions and mathematical deduction is one of the biggest challenges for contemporary cell biology. So far, the challenge has been met only for a few very simple fragments of the machinery of living cells—subsystems involving a handful of different proteins, or two or three cross-regulatory genes, where theory and experiment go closely hand in hand. We discuss some of these examples later in the book and devote the entire final section of Chapter 8 to the role of quantitation in cell biology.

1	Knowledge and understanding bring the power to intervene—with humans, to avoid or prevent disease; with plants, to create better crops; with bacteria, to turn them to our own uses. All these biological enterprises are linked, because the genetic information of all living organisms is written in the same language. The new-found ability of molecular biologists to read and decipher this language has already begun to transform our relationship to the living world. The account of cell biology in the subsequent chapters will, we hope, equip the reader to understand, and possibly to contribute to, the great scientific adventure of the twenty-first century.

1	Figure 1–47 A very simple regulatory circuit—a single gene regulating its own expression by the binding of its protein product to its own regulatory DNA. simple schematic diagrams such as this are found throughout this book. They are often used to summarize what we know, but they leave many questions unanswered. When the protein binds, does it inhibit or stimulate transcription from the gene? how steeply does the transcription rate depend on the protein concentration? how long, on average, does a molecule of the protein remain bound to the dnA? how long does it take to make each molecule of mRnA or protein, and how quickly does each type of molecule get degraded? As explained in chapter 8, mathematical modeling shows that we need quantitative answers to all these and other questions before we can predict the behavior of even this single-gene system. For different parameter values, the system may settle to a unique steady state; or it may behave as a switch, capable of existing in one

1	we can predict the behavior of even this single-gene system. For different parameter values, the system may settle to a unique steady state; or it may behave as a switch, capable of existing in one or another of a set of alternative states; or it may oscillate; or it may show large random fluctuations.

1	Eukaryotic cells, by definition, keep their DNA in a separate membrane-enclosed compartment, the nucleus. They have, in addition, a cytoskeleton for support and movement, elaborate intracellular compartments for digestion and secretion, the capacity (in many species) to engulf other cells, and a metabolism that depends on the oxidation of organic molecules by mitochondria. These properties suggest that eukaryotes may have originated as predators on other cells. Mitochondria—and, in plants, chloroplasts—contain their own genetic material, and they evidently evolved from bacteria that were taken up into the cytoplasm of ancient cells and survived as symbionts.

1	Eukaryotic cells typically have 3–30 times as many genes as prokaryotes, and often thousands of times more noncoding DNA. The noncoding DNA allows for great complexity in the regulation of gene expression, as required for the construction of complex multicellular organisms. Many eukaryotes are, however, unicellular—among them the yeast Saccharomyces cerevisiae, which serves as a simple model organism for eukaryotic cell biology, revealing the molecular basis of many fundamental processes that have been strikingly conserved during a billion years of evolution. A small number of other organisms have also been chosen for intensive study: a worm, a fly, a fish, and the mouse serve as “model organisms” for multicellular animals; and a small milkweed serves as a model for plants.

1	Powerful new technologies such as genome sequencing are producing striking advances in our knowledge of human beings, and they are helping to advance our understanding of human health and disease. But living systems are incredibly complex, and mammalian genomes contain multiple closely related homologs of most genes. This genetic redundancy has allowed diversification and specialization of genes for new purposes, but it also makes biological mechanisms harder to decipher. For this reason, simpler model organisms have played a key part in revealing universal genetic mechanisms of animal development, and research using these systems remains critical for driving scientific and medical advances. Which statements are true? explain why or why not.

1	Which statements are true? explain why or why not. What new approaches might provide a clearer view of the anaerobic archaeon that is thought to have formed the nucleus of the first eukaryotic cell? how did its symbiosis with an aerobic bacterium lead to the mitochondrion? somewhere on earth, are there cells not yet identified that can fill in the details of how eukaryotic cells originated? dnA sequencing has revealed a rich and previously undiscovered world of microbial cells, the vast majority of which fail to grow in a laboratory. how might these cells be made more accessible for detailed study? What new model cells or organisms should be developed for scientists to study? Why might a concerted focus on these models speed progress toward understanding a critical aspect of cell function that is poorly understood? how did the first cell membranes arise?

1	how did the first cell membranes arise? 1–1 Each member of the human hemoglobin gene illustrated in Figure Q1–1. Only one in a million computfamily, which consists of seven genes arranged in two clus-er-generated “random” codes is more error-resistant than ters on different chromosomes, is an ortholog to all of the the natural genetic code. Does the extraordinary mutation other members. resistance of the genetic code argue in favor of its origin as a frozen accident or as a result of natural selection? Explain 1–2 Horizontal gene transfer is more prevalent in sin- your reasoning. gle-celled organisms than in multicellular organisms. 1–3 Most of the DNA sequences in a bacterial genome code for proteins, whereas most of the DNA sequences in the human genome do not. Discuss the following problems.

1	1–3 Most of the DNA sequences in a bacterial genome code for proteins, whereas most of the DNA sequences in the human genome do not. Discuss the following problems. 1–4 Since it was deciphered four decades ago, some have claimed that the genetic code must be a frozen acci number of codes (thousands) dent, while others have argued that it was shaped by nat susceptibility to mutation ural selection. A striking feature of the genetic code is its inherent resistance to the effects of mutation. For example, Figure Q1–1 susceptibility to mutation of the natural code shown a change in the third position of a codon often specifies the relative to that of millions of computer-generated alternative genetic codes (Problem 1–4). susceptibility measures the average change in same amino acid or one with similar chemical properties. amino acid properties caused by random mutations in a genetic code. The natural code resists mutation more effectively (is less

1	amino acid properties caused by random mutations in a genetic code. The natural code resists mutation more effectively (is less A small value indicates that mutations tend to cause minor changes.susceptible to error) than most other possible versions, as (data courtesy of steve Freeland.) 1–5 You have begun to characterize a sample obtained from the depths of the oceans on Europa, one of Jupiter’s moons. Much to your surprise, the sample contains a life-form that grows well in a rich broth. Your preliminary analysis shows that it is cellular and contains DNA, RNA, and protein. When you show your results to a colleague, she suggests that your sample was contaminated with an organism from Earth. What approaches might you try to distinguish between contamination and a novel cellular life-form based on DNA, RNA, and protein?

1	1–6 It is not so difficult to imagine what it means to feed on the organic molecules that living things produce. That is, after all, what we do. But what does it mean to “feed” on sunlight, as phototrophs do? Or, even stranger, to “feed” on rocks, as lithotrophs do? Where is the “food,” for example, in the mixture of chemicals (H2S, H2, CO, Mn+, Fe2+, Ni2+, CH4, and NH4+) that spews from a hydrothermal vent? 1–7 How many possible different trees (branching patterns) can in theory be drawn to display the evolution of bacteria, archaea, and eukaryotes, assuming that they all arose from a common ancestor? 1–8 The genes for ribosomal RNA are highly conserved (relatively few sequence changes) in all organisms on Earth; thus, they have evolved very slowly over time. Were ribosomal RNA genes “born” perfect?

1	1–9 Genes participating in informational processes such as replication, transcription, and translation are transferred between species much less often than are genes involved in metabolism. The basis for this inequality is unclear at present, but one suggestion is that it relates to the underlying complexity of the two types of processes. Informational processes tend to involve large aggregates of different gene products, whereas metabolic reactions are usually catalyzed by enzymes composed of a single protein. Why would the complexity of the underlying process—informational or metabolic—have any effect on the rate of horizontal gene transfer?

1	1–10 Animal cells have neither cell walls nor chloroplasts, whereas plant cells have both. Fungal cells are somewhere in between; they have cell walls but lack chloroplasts. Are fungal cells more likely to be animal cells that gained the ability to make cell walls, or plant cells that lost their chloroplasts? This question represented a difficult issue for early investigators who sought to assign evolutionary relationships based solely on cell characteristics and morphology. How do you suppose that this question was eventually decided?

1	Figure Q1–2 Phylogenetic tree for hemoglobin genes from a variety of species (Problem 1–11). The legumes are highlighted in green. The lengths of lines that connect the present-day species represent the evolutionary distances that separate them. Barley ChlamydomonasParameciumNematode Clam Insect Earthworm Goldfsh Frog Salamander Cobra Chicken Rabbit Whale Cat Human Cow LotusAlfalfaBeanVERTEBRATES INVERTEBRATES PROTOZOA PLANTS 1–11 When plant hemoglobin genes were first discovered in legumes, it was so surprising to find a gene typical of animal blood that it was hypothesized that the plant gene arose by horizontal transfer from an animal. Many more hemoglobin genes have now been sequenced, and a phylogenetic tree based on some of these sequences is shown in Figure Q1–2. A. Does this tree support or refute the hypothesis that the plant hemoglobins arose by horizontal gene transfer?

1	A. Does this tree support or refute the hypothesis that the plant hemoglobins arose by horizontal gene transfer? b. Supposing that the plant hemoglobin genes were originally derived from a parasitic nematode, for example, what would you expect the phylogenetic tree to look like? 1–12 Rates of evolution appear to vary in different lineages. For example, the rate of evolution in the rat lineage is significantly higher than in the human lineage. These rate differences are apparent whether one looks at changes in nucleotide sequences that encode proteins and are subject to selective pressure or at changes in noncoding nucleotide sequences, which are not under obvious selection pressure. Can you offer one or more possible explanations for the slower rate of evolutionary change in the human lineage versus the rat lineage? Alberts B, Bray d, hopkin k et al. (2014) essential cell Biology, 4th ed. new york: Garland science.

1	Alberts B, Bray d, hopkin k et al. (2014) essential cell Biology, 4th ed. new york: Garland science. Barton nh, Briggs deG, eisen JA et al. (2007) evolution. cold spring harbor, ny: cold spring harbor laboratory Press. darwin c (1859) on the origin of species. london: murray. Graur d & li W-h (1999) Fundamentals of molecular evolution, 2nd ed. sunderland, mA: sinauer Associates. madigan mT, martinko Jm, stahl d et al. (2010) Brock Biology of microorganisms, 13th ed. menlo Park, cA: Benjamin-cummings. margulis l & chapman mJ (2009) kingdoms and domains: An Illustrated Guide to the Phyla of life on earth, 1st ed. san diego: Academic Press. moore JA (1993) science As a Way of knowing. cambridge, mA: harvard university Press. moore JA (1972) heredity and development, 2nd ed. new york: oxford university Press. (Free download at www.nap.edu) yang Z (2014) molecular evolution: A statistical Approach. oxford: oxford university Press. The Universal Features of Cells On earth

1	The Universal Features of Cells On earth Andersson sGe (2006) The bacterial world gets smaller. Science 314, 259–260. Brenner s, Jacob F & meselson m (1961) An unstable intermediate carrying information from genes to ribosomes for protein synthesis. Nature 190, 576–581. deamer d & szostak JW eds. (2010) The origins of life (cold spring harbor Perspectives in Biology). ny: cold spring harbor laboratory Press. Gibson dG, Benders GA, Andrews-Pfannkoch c et al. (2008) complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215–1220. Glass JI, Assad-Garcia n, Alperovich n et al. (2006) essential genes of a minimal bacterium. Proc. Natl Acad. Sci. USA 103, 425–430. harris Jk, kelley sT, spiegelman GB et al. (2003) The genetic core of the universal ancestor. Genome Res. 13, 407–413. koonin ev (2005) orthologs, paralogs, and evolutionary genomics. Annu. Rev. Genet. 39, 309–338.

1	koonin ev (2005) orthologs, paralogs, and evolutionary genomics. Annu. Rev. Genet. 39, 309–338. noller h (2005) RnA structure: reading the ribosome. Science 309, 1508–1514. Rinke c, schwientek P, sczyrba A et al. (2013) Insights into the phylogeny and coding potential of microbial dark matter. Nature 499, 431–437. Watson Jd & crick Fhc (1953) molecular structure of nucleic acids. A structure for deoxyribose nucleic acid. Nature 171, 737–738. The Diversity of Genomes and the Tree of Life Blattner FR, Plunkett G, Bloch cA et al. (1997) The complete genome sequence of Escherichia coli k-12. Science 277, 1453–1474. Boucher y, douady cJ, Papke RT et al. (2003) lateral gene transfer and the origins of prokaryotic groups. Annu. Rev. Genet. 37, 283–328. cavicchioli R (2010) Archaea–timeline of the third domain. Nat. Rev. Microbiol. 9, 51–61.

1	cavicchioli R (2010) Archaea–timeline of the third domain. Nat. Rev. Microbiol. 9, 51–61. choudhuri s (2014) Bioinformatics for Beginners: Genes, Genomes, molecular evolution, databases and Analytical Tools, 1st ed. san diego: Academic Press. dixon B (1997) Power unseen: how microbes Rule the World. oxford:oxford university Press. handelsman J (2004) metagenomics: applications of genomics to uncultured microorganisms. Microbiol. Mol. Biol. Rev. 68, 669–685. kerr RA (1997) life goes to extremes in the deep earth—and elsewhere? Science 276, 703–704. lee TI, Rinaldi nJ, Robert F et al. (2002) Transcriptional regulatory networks in Saccharomyces cerevisiae. Science 298, 799–804. olsen GJ & Woese cR (1997) Archaeal genomics: an overview. Cell 89:991–994. Williams TA, Foster PG, cox cJ & embley Tm (2013) An archaeal origin of eukaryotes supports only two primary domains of life. Nature 504, 231–235. Woese c (1998) The universal ancestor. Proc. Natl Acad. Sci. USA 95, 6854–6859.

1	Woese c (1998) The universal ancestor. Proc. Natl Acad. Sci. USA 95, 6854–6859. Adams md, celniker se, holt RA et al. (2000) The genome sequence of Drosophila melanogaster. Science 287, 2185–2195. Amborella Genome Project (2013) The Amborella genome and the evolution of flowering plants. Science 342, 1241089. Andersson sG, Zomorodipour A, Andersson Jo et al. (1998) The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396, 133–140. The Arabidopsis Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815. carroll sB, Grenier Jk & Weatherbee sd (2005) From dnA to diversity: molecular Genetics and the evolution of Animal design, 2nd ed. maldon, mA: Blackwell science. de duve c (2007) The origin of eukaryotes: a reappraisal. Nat. Rev. Genet. 8, 395–403. delsuc F, Brinkmann h & Philippe h (2005) Phylogenomics and the reconstruction of the tree of life. Nat. Rev. Genet. 6, 361–375.

1	delsuc F, Brinkmann h & Philippe h (2005) Phylogenomics and the reconstruction of the tree of life. Nat. Rev. Genet. 6, 361–375. deRisi Jl, Iyer vR & Brown Po (1997) exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278, 680–686. Gabriel sB, schaffner sF, nguyen h et al. (2002) The structure of haplotype blocks in the human genome. Science 296, 2225–2229. Goffeau A, Barrell BG, Bussey h et al. (1996) life with 6000 genes. Science 274, 546–567. International human Genome sequencing consortium (2001) Initial sequencing and analysis of the human genome. Nature 409, 860–921. keeling PJ & koonin ev eds. (2014) The origin and evolution of eukaryotes (cold spring harbor Perspectives in Biology). ny: cold spring harbor laboratory Press. lander es (2011) Initial impact of the sequencing of the human genome. Nature 470, 187–197. lynch m & conery Js (2000) The evolutionary fate and consequences of duplicate genes. Science 290, 1151–1155.

1	lynch m & conery Js (2000) The evolutionary fate and consequences of duplicate genes. Science 290, 1151–1155. national center for Biotechnology Information. http://www.ncbi.nlm.nih.gov/ owens k & king mc (1999) Genomic views of human history. Science 286, 451–453. Palmer Jd & delwiche cF (1996) second-hand chloroplasts and the case of the disappearing nucleus. Proc. Natl Acad. Sci. USA 93, 7432–7435. Reed FA & Tishkoff sA (2006) African human diversity, origins and migrations. Curr. Opin. Genet. Dev. 16, 597–605. Rine J (2014) A future of the model organism model. Mol. Biol. Cell 25, 549–553. Rubin Gm, yandell md, Wortman JR et al. (2000) comparative genomics of the eukaryotes. Science 287, 2204–2215. shen y, yue F, mccleary d et al. (2012) A map of the cis-regulatory sequences in the mouse genome. Nature 488, 116–120. The C. elegans sequencing consortium (1998) Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282, 2012–2018.

1	The C. elegans sequencing consortium (1998) Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282, 2012–2018. Tinsley Rc & kobel hR eds. (1996) The Biology of Xenopus. oxford: clarendon Press. Tyson JJ, chen kc & novak B (2003) sniffers, buzzers, toggles and blinkers: dynamics of regulatory and signaling pathways in the cell. Curr. Opin. Cell Biol. 15, 221–231. venter Jc, Adams md, myers eW et al (2001) The sequence of the human genome. Science 291, 1304–1351. It is at first sight difficult to accept the idea that living creatures are merely chemical systems. Their incredible diversity of form, their seemingly purposeful behavior, and their ability to grow and reproduce all seem to set them apart from the world of solids, liquids, and gases that chemistry normally describes. Indeed, until the nineteenth century animals were believed to contain a Vital Force—an “animus”—that was responsible for their distinctive properties.

1	We now know that there is nothing in living organisms that disobeys chemical or physical laws. However, the chemistry of life is indeed special. First, it is based overwhelmingly on carbon compounds, the study of which is known as organic chemistry. Second, cells are 70% water, and life depends largely on chemical reactions that take place in aqueous solution. Third, and most important, cell chemistry is enormously complex: even the simplest cell is vastly more complicated in its chemistry than any other chemical system known. In particular, although cells contain a variety of small carbon-containing molecules, most of the carbon atoms present are incorporated into enormous polymeric molecules—chains of chemical subunits linked end-to-end. It is the unique properties of these macromolecules that enable cells and organisms to grow and reproduce—as well as to do all the other things that are characteristic of life. The ChemiCal ComponenTs of a Cell

1	The ChemiCal ComponenTs of a Cell Living organisms are made of only a small selection of the 92 naturally occurring elements, four of which—carbon (C), hydrogen (H), nitrogen (N), and oxygen (O)—make up 96.5% of an organism’s weight (Figure 2–1). The atoms of these elements are linked together by covalent bonds to form molecules (see Panel 2–1, pp. 90–91). Because covalent bonds are typically 100 times stronger than the thermal energies within a cell, they resist being pulled apart by thermal motions, and they are normally broken only during specific chemical reactions with other atoms and molecules. Two different molecules can be held together by noncovalent bonds, The ChemiCal ComponenTs of a Cell CaTalYsis anD The Use of eneRGY BY Cells

1	The ChemiCal ComponenTs of a Cell CaTalYsis anD The Use of eneRGY BY Cells Figure 2–1 The main elements in cells, highlighted in the periodic table. When ordered by their atomic number and arranged in this manner, elements fall into vertical columns that show similar properties. atoms in the same vertical column must gain (or lose) the same number of electrons to attain a filled outer shell, and they thus behave similarly in bond or ion formation. Thus, for example, mg and Ca tend to give away the two electrons in their outer shells. C, n, and o occur in the same horizontal row, and tend to complete their second shells by sharing electrons.

1	The four elements highlighted in red constitute 99% of the total number of atoms present in the human body. an additional seven elements, highlighted in blue, together represent about 0.9% of the total. The elements shown in green are required in trace amounts by humans. it remains unclear whether those elements shown in yellow are essential in humans. The chemistry of life, it seems, is therefore predominantly the chemistry of lighter elements. The atomic weights shown here are those of the most common isotope of each element. 1000 10,000 kJ which are much weaker (Figure 2–2). We shall see later that noncovalent bonds are important in the many situations where molecules have to associate and dissociate readily to carry out their biological functions. Water is held Together by hydrogen Bonds

1	Water is held Together by hydrogen Bonds The reactions inside a cell occur in an aqueous environment. Life on Earth began in the ocean, and the conditions in that primeval environment put a permanent stamp on the chemistry of living things. Life therefore hinges on the chemical properties of water, which are reviewed in Panel 2–2, pp. 92–93.

1	In each water molecule (H2O) the two H atoms are linked to the O atom by covalent bonds. The two bonds are highly polar because the O is strongly attractive for electrons, whereas the H is only weakly attractive. Consequently, there is an unequal distribution of electrons in a water molecule, with a preponderance of positive charge on the two H atoms and of negative charge on the O. When a positively charged region of one water molecule (that is, one of its H atoms) approaches a negatively charged region (that is, the O) of a second water molecule, the electrical attraction between them can result in a hydrogen bond. These bonds are much weaker than covalent bonds and are easily broken by the random thermal motions that reflect the heat energy of the molecules. Thus, each bond lasts only a short time. But the combined effect of many weak bonds can be profound. For example, each water molecule can form hydrogen bonds through its two H atoms to two other water molecules, producing a

1	a short time. But the combined effect of many weak bonds can be profound. For example, each water molecule can form hydrogen bonds through its two H atoms to two other water molecules, producing a network in which hydrogen bonds are being continually broken and formed. It is only because of the hydrogen bonds that link water molecules together that water is a liquid at room temperature—with a high boiling point and high surface tension—rather than a gas.

1	Molecules, such as alcohols, that contain polar bonds and that can form hydrogen bonds with water dissolve readily in water. Molecules carrying charges (ions) likewise interact favorably with water. Such molecules are termed hydrophilic, meaning that they are water-loving. Many of the molecules in the aqueous environment of a cell necessarily fall into this category, including sugars, DNA, RNA, and most proteins. Hydrophobic (water-hating) molecules, by contrast, are uncharged and form few or no hydrogen bonds, and so do not dissolve in water. Hydrocarbons are an important example. In these molecules all of the H atoms are covalently linked to C atoms by a largely nonpolar bond; thus they cannot form effective hydrogen bonds to other molecules (see Panel 2–1, p. 90). This makes the hydrocarbon as a whole hydrophobic—a property that is exploited in cells, whose membranes are constructed from molecules that have long hydrocarbon tails, as we see in Chapter 10.

1	four Types of noncovalent attractions help Bring molecules Together in Cells Much of biology depends on the specific binding of different molecules caused by three types of noncovalent bonds: electrostatic attractions (ionic bonds), hydrogen bonds, and van der Waals attractions; and on a fourth factor that can push molecules together: the hydrophobic force. The properties of the four types of noncovalent attractions are presented in Panel 2–3 (pp. 94–95). Although each

1	Figure 2–2 Some energies important for cells. a crucial property of any bond— covalent or noncovalent—is its strength. Bond strength is measured by the amount of energy that must be supplied to break it, expressed in units of either kilojoules per mole (kJ/mole) or kilocalories per mole (kcal/mole). Thus if 100 kJ of energy must be supplied to break 6 × 1023 bonds of a specific type (that is, 1 mole of these bonds), then the strength of that bond is 100 kJ/mole. note that, in this diagram, energies are compared on a logarithmic scale. Typical strengths and lengths of the main classes of chemical bonds are given in Table 2–1.

1	one joule (J) is the amount of energy required to move an object a distance of one meter against a force of one newton. This measure of energy is derived from the si units (système internationale d’Unités) universally employed by physical scientists. a second unit of energy, often used by cell biologists, is the kilocalorie (kcal); one calorie is the amount of energy needed to raise the temperature of 1 gram of water by 1°C. one kJ is equal to 0.239 kcal (1 kcal = 4.18 kJ). Figure 2–3 Schematic indicating how two macromolecules with complementary surfaces can bind tightly to one another through noncovalent interactions. noncovalent chemical bonds have less than 1/20 the strength of a covalent bond. They are able to produce tight binding only when many of them are formed simultaneously. although only electrostatic attractions are illustrated here, in reality all four noncovalent forces often contribute to holding two macromolecules together (Movie 2.1).

1	individual noncovalent attraction would be much too weak to be effective in the face of thermal motions, their energies can sum to create a strong force between two separate molecules. Thus sets of noncovalent attractions often allow the complementary surfaces of two macromolecules to hold those two macromolecules together (Figure 2–3). Table 2–1 compares noncovalent bond strengths to that of a typical covalent bond, both in the presence and in the absence of water. Note that, by forming competing interactions with the involved molecules, water greatly reduces the strength of both electrostatic attractions and hydrogen bonds.

1	The structure of a typical hydrogen bond is illustrated in Figure 2–4. This bond represents a special form of polar interaction in which an electropositive hydrogen atom is shared by two electronegative atoms. Its hydrogen can be viewed as a proton that has partially dissociated from a donor atom, allowing it to be shared by a second acceptor atom. Unlike a typical electrostatic interaction, this bond is highly directional—being strongest when a straight line can be drawn between all three of the involved atoms.

1	The fourth effect that often brings molecules together in water is not, strictly speaking, a bond at all. However, a very important hydrophobic force is caused by a pushing of nonpolar surfaces out of the hydrogen-bonded water network, where they would otherwise physically interfere with the highly favorable interactions between water molecules. Bringing any two nonpolar surfaces together reduces their contact with water; in this sense, the force is nonspecific. Nevertheless, we shall see in Chapter 3 that hydrophobic forces are central to the proper folding of protein molecules.

1	One of the simplest kinds of chemical reaction, and one that has profound significance in cells, takes place when a molecule containing a highly polar covalent bond between a hydrogen and another atom dissolves in water. The hydrogen atom in such a molecule has given up its electron almost entirely to the companion atom, and so exists as an almost naked positively charged hydrogen nucleus—in *an ionic bond is an electrostatic attraction between two fully charged atoms. **Values in parentheses are kcal/mole. 1 kJ = 0.239 kcal and 1 kcal = 4.18 kJ. (A) hydrogen bond ~0.3 nm long covalent bond ~0.1 nm long Figure 2–4 Hydrogen bonds. (a) Ball-andstick model of a typical hydrogen bond. The distance between the hydrogen and the oxygen atom here is less than the sum of their van der Waals radii, indicating a partial sharing of electrons. (B) The most common hydrogen bonds in cells.

1	H2O H2O molecule to H3O+ OH– the other hydronium hydroxyl(B) ion ion other words, a proton (H+). When the polar molecule becomes surrounded by water molecules, the proton will be attracted to the partial negative charge on the O atom of an adjacent water molecule. This proton can easily dissociate from its original partner and associate instead with the oxygen atom of the water molecule, generating a hydronium ion (H3O+) (Figure 2–5A). The reverse reaction also takes place very readily, so in the aqueous solution protons are constantly flitting to and fro between one molecule and another.

1	Substances that release protons when they dissolve in water, thus forming H3O+, are termed acids. The higher the concentration of H3O+, the more acidic the solution. H3O+ is present even in pure water, at a concentration of 10–7 M, as a result of the movement of protons from one water molecule to another (Figure 2–5B). By convention, the H3O+ concentration is usually referred to as the H+ concentration, even though most protons in an aqueous solution are present as H3O+. To avoid the use of unwieldy numbers, the concentration of H3O+ is expressed using a logarithmic scale called the pH scale. Pure water has a pH of 7.0 and is said to be neutral—that is, neither acidic (pH <7) nor basic (pH >7).

1	Acids are characterized as being strong or weak, depending on how readily they give up their protons to water. Strong acids, such as hydrochloric acid (HCl), lose their protons quickly. Acetic acid, on the other hand, is a weak acid because it holds on to its proton more tightly when dissolved in water. Many of the acids important in the cell—such as molecules containing a carboxyl (COOH) group— are weak acids (see Panel 2–2, pp. 92–93). Because the proton of a hydronium ion can be passed readily to many types of molecules in cells, altering their character, the concentration of H3O+ inside a cell (the acidity) must be closely regulated. Acids—especially weak acids—will give up their protons more readily if the concentration of H3O+ in solution is low and will tend to receive them back if the concentration in solution is high.

1	The opposite of an acid is a base. Any molecule capable of accepting a proton from a water molecule is called a base. Sodium hydroxide (NaOH) is basic (the term alkaline is also used) because it dissociates readily in aqueous solution to form Na+ ions and OH– ions. Because of this property, NaOH is called a strong base. More important in living cells, however, are the weak bases—those that have a weak tendency to reversibly accept a proton from water. Many biologically important molecules contain an amino (NH2) group. This group is a weak base that can generate OH– by taking a proton from water: –NH2 + H2O →–NH3+ + OH– (see Panel 2–2, pp. 92–93).

1	Because an OH– ion combines with a H3O+ ion to form two water molecules, an increase in the OH– concentration forces a decrease in the concentration of H3O+, and vice versa. A pure solution of water contains an equal concentration (10–7 M) of both ions, rendering it neutral. The interior of a cell is also kept close to neutrality by the presence of buffers: weak acids and bases that can release or take up protons near pH 7, keeping the environment of the cell relatively constant under a variety of conditions. Figure 2–5 Protons readily move in aqueous solutions. (a) The reaction that takes place when a molecule of acetic acid dissolves in water. at ph 7, nearly all of the acetic acid is present as acetate ion. (B) Water molecules are continuously exchanging protons with each other to form hydronium and hydroxyl ions. These ions in turn rapidly recombine to form water molecules. a Cell is formed from Carbon Compounds

1	a Cell is formed from Carbon Compounds Having reviewed the ways atoms combine into molecules and how these molecules behave in an aqueous environment, we now examine the main classes of small molecules found in cells. We shall see that a few categories of molecules, formed from a handful of different elements, give rise to all the extraordinary richness of form and behavior shown by living things.

1	If we disregard water and inorganic ions such as potassium, nearly all the molecules in a cell are based on carbon. Carbon is outstanding among all the elements in its ability to form large molecules; silicon is a poor second. Because carbon is small and has four electrons and four vacancies in its outermost shell, a carbon atom can form four covalent bonds with other atoms. Most important, one carbon atom can join to other carbon atoms through highly stable covalent C–C bonds to form chains and rings and hence generate large and complex molecules with no obvious upper limit to their size. The carbon compounds made by cells are called organic molecules. In contrast, all other molecules, including water, are said to be inorganic.

1	Certain combinations of atoms, such as the methyl (–CH3), hydroxyl (–OH), carboxyl (–COOH), carbonyl (–C=O), phosphate (–PO32–), sulfhydryl (–SH), and amino (–NH2) groups, occur repeatedly in the molecules made by cells. Each such chemical group has distinct chemical and physical properties that influence the behavior of the molecule in which the group occurs. The most common chemical groups and some of their properties are summarized in Panel 2–1, pp. 90–91. Cells Contain four major families of small organic molecules

1	Cells Contain four major families of small organic molecules The small organic molecules of the cell are carbon-based compounds that have molecular weights in the range of 100–1000 and contain up to 30 or so carbon atoms. They are usually found free in solution and have many different fates. Some are used as monomer subunits to construct giant polymeric macromolecules— proteins, nucleic acids, and large polysaccharides. Others act as energy sources and are broken down and transformed into other small molecules in a maze of intracellular metabolic pathways. Many small molecules have more than one role in the cell—for example, acting both as a potential subunit for a macromolecule and as an energy source. Small organic molecules are much less abundant than the organic macromolecules, accounting for only about one-tenth of the total mass of organic matter in a cell. As a rough guess, there may be a thousand different kinds of these small molecules in a typical cell.

1	All organic molecules are synthesized from and are broken down into the same set of simple compounds. As a consequence, the compounds in a cell are chemically related and most can be classified into a few distinct families. Broadly speaking, cells contain four major families of small organic molecules: the sugars, the fatty acids, the nucleotides, and the amino acids (Figure 2–6). Although many compounds present in cells do not fit into these categories, these four families of small organic molecules, together with the macromolecules made by linking them into long chains, account for a large fraction of the cell mass. Amino acids and the proteins that they form will be the subject of Chapter 3. A summary of the structures and properties of the remaining three families— sugars, fatty acids, and nucleotides—is presented in Panels 2–4, 2–5, and 2–6, respectively (see pages 96–101). The Chemistry of Cells is Dominated by macromolecules with Remarkable properties

1	The Chemistry of Cells is Dominated by macromolecules with Remarkable properties By weight, macromolecules are the most abundant carbon-containing molecules in a living cell (Figure 2–7). They are the principal building blocks from which a cell is constructed and also the components that confer the most distinctive properties of living things. The macromolecules in cells are polymers that are constructed by covalently linking small organic molecules (called monomers) into of the cell of the cell SUGARS FATTY ACIDS POLYSACCHARIDES AMINO ACIDS NUCLEOTIDES PROTEINS FATS, LIPIDS, MEMBRANES NUCLEIC ACIDS

1	SUGARS FATTY ACIDS POLYSACCHARIDES AMINO ACIDS NUCLEOTIDES PROTEINS FATS, LIPIDS, MEMBRANES NUCLEIC ACIDS Figure 2–6 The four main families of small organic molecules in cells. These small molecules form the monomeric building blocks, or subunits, for most of the macromolecules and other assemblies of the cell. some, such as the sugars and the fatty acids, are also energy sources. Their structures are outlined here and shown in more detail in the panels at the end of this chapter and in Chapter 3. long chains (Figure 2–8). They have remarkable properties that could not have been predicted from their simple constituents.

1	long chains (Figure 2–8). They have remarkable properties that could not have been predicted from their simple constituents. Proteins are abundant and spectacularly versatile, performing thousands of distinct functions in cells. Many proteins serve as enzymes, the catalysts that facilitate the many covalent bond-making and bond-breaking reactions that the cell needs. Enzymes catalyze all of the reactions whereby cells extract energy from food molecules, for example, and an enzyme called ribulose bisphosphate carboxylase helps to convert CO2 to sugars in photosynthetic organisms, producing most of the organic matter needed for life on Earth. Other proteins are used to build structural components, such as tubulin, a protein that self-assembles to make the cell’s long microtubules, or histones, proteins that compact the DNA in chromosomes. Yet other proteins act as molecular motors to produce force and

1	Figure 2–7 The distribution of molecules in cells. The approximate composition of a bacterial cell is shown by weight. The composition of an animal cell is similar, even though its volume is roughly 1000 times greater. note that macromolecules dominate. The major inorganic ions include na+, K+, mg2+, Ca2+, and Cl–. movement, as for myosin in muscle. Proteins perform many other functions, and we shall examine the molecular basis for many of them later in this book.

1	Although the chemical reactions for adding subunits to each polymer are different in detail for proteins, nucleic acids, and polysaccharides, they share important features. Each polymer grows by the addition of a monomer onto the end of a growing chain in a condensation reaction, in which one molecule of water is lost with each subunit added (Figure 2–9). The stepwise polymerization of monomers into a long chain is a simple way to manufacture a large, complex molecule, since the subunits are added by the same reaction performed over and over again by the same set of enzymes. Apart from some of the polysaccharides, most macromolecules are made from a limited set of monomers that are slightly different from one another—for example, the 20 different amino acids from which proteins are made. It is critical to life that the polymer chain is not assembled at random from these subunits; instead the subunits are added in a precise order, or sequence. The elaborate mechanisms that allow

1	made. It is critical to life that the polymer chain is not assembled at random from these subunits; instead the subunits are added in a precise order, or sequence. The elaborate mechanisms that allow enzymes to accomplish this task are described in detail in Chapters 5 and 6.

1	noncovalent Bonds specify Both the precise shape of a macromolecule and its Binding to other molecules Most of the covalent bonds in a macromolecule allow rotation of the atoms they join, giving the polymer chain great flexibility. In principle, this allows a macromolecule to adopt an almost unlimited number of shapes, or conformations, as random thermal energy causes the polymer chain to writhe and rotate. However, the shapes of most biological macromolecules are highly constrained because of the many weak noncovalent bonds that form between different parts of the same molecule. If these noncovalent bonds are formed in sufficient numbers, the polymer chain can strongly prefer one particular conformation, determined by the linear sequence of monomers in its chain. Most protein molecules and many of the small RNA molecules found in cells fold tightly into a highly preferred conformation in this way (Figure 2–10).

1	The four types of noncovalent interactions important in biological molecules were presented earlier, and they are discussed further in Panel 2–3 (pp. 94–95). In addition to folding biological macromolecules into unique shapes, they can also add up to create a strong attraction between two different molecules (see Figure 2–3). This form of molecular interaction provides for great specificity, inasmuch as the close multipoint contacts required for strong binding make it possible for a macromolecule to select out—through binding—just one of the many thousands of other types of molecules present inside a cell. Moreover, because the strength of the binding depends on the number of noncovalent bonds that are formed, interactions of almost any affinity are possible—allowing rapid dissociation where appropriate.

1	As we discuss next, binding of this type underlies all biological catalysis, making it possible for proteins to function as enzymes. In addition, noncovalent interactions allow macromolecules to be used as building blocks for the formation of Figure 2–8 Three families of macromolecules. each is a polymer formed from small molecules (called monomers) linked together by covalent bonds. Figure 2–9 Condensation and hydrolysis as opposite reactions. The macromolecules of the cell are polymers that are formed from subunits (or monomers) by a condensation reaction, and they are broken down by hydrolysis. The condensation reactions are all energetically unfavorable; thus polymer formation requires an energy input, as will be described in the text.

1	Figure 2–10 The folding of proteins and RNa molecules into a particularly stable three-dimensional shape, or conformation. if the noncovalent bonds maintaining the stable conformation are disrupted, the molecule becomes a flexible chain that loses its biological activity. larger structures, thereby forming intricate machines with multiple moving parts that perform such complex tasks as DNA replication and protein synthesis (Figure 2–11).

1	Living organisms are autonomous, self-propagating chemical systems. They are formed from a distinctive and restricted set of small carbon-based molecules that are essentially the same for every living species. Each of these small molecules is composed of a small set of atoms linked to each other in a precise configuration through covalent bonds. The main categories are sugars, fatty acids, amino acids, and nucleotides. Sugars are a primary source of chemical energy for cells and can be incorporated into polysaccharides for energy storage. Fatty acids are also important for energy storage, but their most critical function is in the formation of cell membranes. Long chains of amino acids form the remarkably diverse and versatile macromolecules known as proteins. Nucleotides play a central part in energy transfer, while also serving as the subunits for the informational macromolecules, RNA and DNA.

1	Most of the dry mass of a cell consists of macromolecules that have been produced as linear polymers of amino acids (proteins) or nucleotides (DNA and RNA), covalently linked to each other in an exact order. Most of the protein molecules and many of the RNAs fold into a unique conformation that is determined by their sequence of subunits. This folding process creates unique surfaces, and it depends on a large set of weak attractions produced by noncovalent forces between atoms. e.g., sugars, amino acids, and nucleotides e.g., globular proteins and RNA e.g., ribosome Figure 2–11 Small molecules become covalently linked to form macromolecules, which in turn assemble through noncovalent interactions to form large complexes. small molecules, proteins, and a ribosome are drawn approximately to scale. Ribosomes are a central part of the machinery that the cell uses to make proteins: each ribosome is formed as a complex of about 90 macromolecules (protein and Rna molecules).

1	These forces are of four types: electrostatic attractions, hydrogen bonds, van der Waals attractions, and an interaction between nonpolar groups caused by their hydrophobic expulsion from water. The same set of weak forces governs the specific binding of other molecules to macromolecules, making possible the myriad associations between biological molecules that produce the structure and the chemistry of a cell. CaTalYsis anD The Use of eneRGY BY Cells

1	CaTalYsis anD The Use of eneRGY BY Cells One property of living things above all makes them seem almost miraculously different from nonliving matter: they create and maintain order, in a universe that is tending always to greater disorder (Figure 2–12). To create this order, the cells in a living organism must perform a never-ending stream of chemical reactions. In some of these reactions, small organic molecules—amino acids, sugars, nucleotides, and lipids—are being taken apart or modified to supply the many other small molecules that the cell requires. In other reactions, small molecules are being used to construct an enormously diverse range of proteins, nucleic acids, and other macromolecules that endow living systems with all of their most distinctive properties. Each cell can be viewed as a tiny chemical factory, performing many millions of reactions every second. Cell metabolism is organized by enzymes

1	Cell metabolism is organized by enzymes The chemical reactions that a cell carries out would normally occur only at much higher temperatures than those existing inside cells. For this reason, each reaction requires a specific boost in chemical reactivity. This requirement is crucial, because it allows the cell to control its chemistry. The control is exerted through specialized biological catalysts. These are almost always proteins called enzymes, although RNA catalysts also exist, called ribozymes. Each enzyme accelerates, or catalyzes, just one of the many possible kinds of reactions that a particular molecule might undergo. Enzyme-catalyzed reactions are connected in series, so that the product of one reaction becomes the starting material, or substrate, for the next (Figure 2–13). Long linear reaction pathways are in turn linked to one another, forming a maze of interconnected reactions that enable the cell to survive, grow, and reproduce.

1	Two opposing streams of chemical reactions occur in cells: (1) the catabolic pathways break down foodstuffs into smaller molecules, thereby generating both a useful form of energy for the cell and some of the small molecules that the cell needs as building blocks, and (2) the anabolic, or biosynthetic, pathways use the 20 nm 50 nm 10 µm 0.5 mm 20 mm

1	Figure 2–12 biological structures are highly ordered. Well-defined, ornate, and beautiful spatial patterns can be found at every level of organization in living organisms. in order of increasing size: (a) protein molecules in the coat of a virus (a parasite that, although not technically alive, contains the same types of molecules as those found in living cells); (B) the regular array of microtubules seen in a cross section of a sperm tail; (C) surface contours of a pollen grain (a single cell); (D) cross section of a fern stem, showing the patterned arrangement of cells; and (e) a spiral arrangement of leaves in a succulent plant. (a, courtesy of Robert Grant, stéphane Crainic, and James m. hogle; B, courtesy of lewis Tilney; C, courtesy of Colin macfarlane and Chris Jeffree; D, courtesy of Jim haseloff.)

1	Figure 2–13 How a set of enzyme-catalyzed reactions generates a metabolic pathway. each enzyme catalyzes a particular chemical reaction, leaving the enzyme unchanged. in this example, a set of enzymes acting in series converts molecule a to molecule f, forming a metabolic pathway. (for a diagram of many of the reactions in a human cell, abbreviated as shown, see figure 2–63.) small molecules and the energy harnessed by catabolism to drive the synthesis of the many other molecules that form the cell. Together these two sets of reactions constitute the metabolism of the cell (Figure 2–14). The details of cell metabolism form the traditional subject of biochemistry and most of them need not concern us here. But the general principles by which cells obtain energy from their environment and use it to create order are central to cell biology. We begin with a discussion of why a constant input of energy is needed to sustain all living things.

1	Biological order is made possible by the Release of heat energy from Cells The universal tendency of things to become disordered is a fundamental law of physics—the second law of thermodynamics—which states that in the universe, or in any isolated system (a collection of matter that is completely isolated from the rest of the universe), the degree of disorder always increases. This law has such profound implications for life that we will restate it in several ways.

1	For example, we can present the second law in terms of probability by stating that systems will change spontaneously toward those arrangements that have the greatest probability. If we consider a box of 100 coins all lying heads up, a series of accidents that disturbs the box will tend to move the arrangement toward a mixture of 50 heads and 50 tails. The reason is simple: there is a huge number of possible arrangements of the individual coins in the mixture that can achieve the 50–50 result, but only one possible arrangement that keeps all of the coins oriented heads up. Because the 50–50 mixture is therefore the most probable, we say that it is more “disordered.” For the same reason, it is a common experience that one’s living space will become increasingly disordered without intentional effort: the movement toward disorder is a spontaneous process, requiring a periodic effort to reverse it (Figure 2–15).

1	The amount of disorder in a system can be quantified and expressed as the entropy of the system: the greater the disorder, the greater the entropy. Thus, another way to express the second law of thermodynamics is to say that systems will change spontaneously toward arrangements with greater entropy.

1	Living cells—by surviving, growing, and forming complex organisms—are generating order and thus might appear to defy the second law of thermodynamics. How is this possible? The answer is that a cell is not an isolated system: it takes in energy from its environment in the form of food, or as photons from the sun (or even, as in some chemosynthetic bacteria, from inorganic molecules alone). It then uses this energy to generate order within itself. In the course of the chemical reactions that generate order, the cell converts part of the energy it uses into heat. The heat is discharged into the cell’s environment and disorders the surroundings. As a result, the total entropy—that of the cell plus its surroundings—increases, as demanded by the second law of thermodynamics.

1	To understand the principles governing these energy conversions, think of a cell surrounded by a sea of matter representing the rest of the universe. As the cell lives and grows, it creates internal order. But it constantly releases heat energy as it synthesizes molecules and assembles them into cell structures. Heat is energy in its most disordered form—the random jostling of molecules. When food the many molecules molecules that form the cell Figure 2–14 Schematic representation of the relationship between catabolic and anabolic pathways in metabolism. as suggested in this diagram, a major portion of the energy stored in the chemical bonds of food molecules is dissipated as heat. in addition, the mass of food required by any organism that derives all of its energy from catabolism is much greater than the mass of the molecules that it can produce by anabolism.

1	the cell releases heat to the sea, it increases the intensity of molecular motions there (thermal motion)—thereby increasing the randomness, or disorder, of the sea. The second law of thermodynamics is satisfied because the increase in the amount of order inside the cell is always more than compensated for by an even greater decrease in order (increase in entropy) in the surrounding sea of matter (Figure 2–16).

1	Where does the heat that the cell releases come from? Here we encounter another important law of thermodynamics. The first law of thermodynamics states that energy can be converted from one form to another, but that it cannot be created or destroyed. Figure 2–17 illustrates some interconversions between different forms of energy. The amount of energy in different forms will change as a result of the chemical reactions inside the cell, but the first law tells us that the total amount of energy must always be the same. For example, an animal cell takes in foodstuffs and converts some of the energy present in the chemical bonds between the atoms of these food molecules (chemical-bond energy) into the random thermal motion of molecules (heat energy).

1	The cell cannot derive any benefit from the heat energy it releases unless the heat-generating reactions inside the cell are directly linked to the processes that generate molecular order. It is the tight coupling of heat production to an increase sea of matter cell Figure 2–15 an everyday illustration of the spontaneous drive toward disorder. Reversing this tendency toward disorder requires an intentional effort and an input of energy: it is not spontaneous. in fact, from the second law of thermodynamics, we can be certain that the human intervention required will release enough heat to the environment to more than compensate for the reordering of the items in this room.

1	Figure 2–16 a simple thermodynamic analysis of a living cell. in the diagram on the left, the molecules of both the cell and the rest of the universe (the sea of matter) are depicted in a relatively disordered state. in the diagram on the right, the cell has taken in energy from food molecules and released heat through reactions that order the molecules the cell contains. The heat released increases the disorder in the environment around the cell (depicted by jagged arrows and distorted molecules, indicating increased molecular motions caused by heat). as a result, the second law of thermodynamics—which states that the amount of disorder in the universe must always increase—is satisfied as the cell grows and divides. for a detailed discussion, see panel 2–7 (pp. 102–103).

1	heat is released to pull of the foor 1 potential energy due to position kinetic energy heat energy two hydrogen oxygen gas rapid vibrations and heat dispersed to gas molecules molecule rotations of two newly surroundings formed water molecules in order that distinguishes the metabolism of a cell from the wasteful burning of fuel in a fire. Later, we illustrate how this coupling occurs. For now, it is sufficient to recognize that a direct linkage of the “controlled burning” of food molecules to the generation of biological order is required for cells to create and maintain an island of order in a universe tending toward chaos. Cells obtain energy by the oxidation of organic molecules

1	Cells obtain energy by the oxidation of organic molecules All animal and plant cells are powered by energy stored in the chemical bonds of organic molecules, whether they are sugars that a plant has photosynthesized as food for itself or the mixture of large and small molecules that an animal has eaten. Organisms must extract this energy in usable form to live, grow, and reproduce. In both plants and animals, energy is extracted from food molecules by a process of gradual oxidation, or controlled burning. The Earth’s atmosphere contains a great deal of oxygen, and in the presence of oxygen the most energetically stable form of carbon is CO2 and that of hydrogen Figure 2–17 Some interconversions between different forms of energy.

1	Figure 2–17 Some interconversions between different forms of energy. all energy forms are, in principle, interconvertible. in all these processes the total amount of energy is conserved. Thus, for example, from the height and weight of the brick in (1), we can predict exactly how much heat will be released when it hits the floor. in (2), note that the large amount of chemical-bond energy released when water is formed is initially converted to very rapid thermal motions in the two new water molecules; but collisions with other molecules almost instantaneously spread this kinetic energy evenly throughout the surroundings (heat transfer), making the new molecules indistinguishable from all the rest. is H2O. A cell is therefore able to obtain energy from sugars or other organic molecules by allowing their carbon and hydrogen atoms to combine with oxygen to produce CO2 and H2O, respectively—a process called aerobic respiration.

1	Photosynthesis (discussed in detail in Chapter 14) and respiration are complementary processes (Figure 2–18). This means that the transactions between plants and animals are not all one way. Plants, animals, and microorganisms have existed together on this planet for so long that many of them have become an essential part of the others’ environments. The oxygen released by photosynthesis is consumed in the combustion of organic molecules during aerobic respiration. And some of the CO2 molecules that are fixed today into organic molecules by photosynthesis in a green leaf were yesterday released into the atmosphere by the respiration of an animal—or by the respiration of a fungus or bacterium decomposing dead organic matter. We therefore see that carbon utilization forms a huge cycle that involves the biosphere (all of the living organisms on Earth) as a whole (Figure 2–19). Similarly, atoms of nitrogen, phosphorus, and sulfur move between the living and nonliving worlds in cycles that

1	the biosphere (all of the living organisms on Earth) as a whole (Figure 2–19). Similarly, atoms of nitrogen, phosphorus, and sulfur move between the living and nonliving worlds in cycles that involve plants, animals, fungi, and bacteria.

1	The cell does not oxidize organic molecules in one step, as occurs when organic material is burned in a fire. Through the use of enzyme catalysts, metabolism takes these molecules through a large number of reactions that only rarely involve the direct addition of oxygen. Before we consider some of these reactions and their purpose, we discuss what is meant by the process of oxidation.

1	Figure 2–18 Photosynthesis and respiration as complementary processes in the living world. photosynthesis converts the electromagnetic energy in sunlight into chemical-bond energy in sugars and other organic molecules. plants, algae, and cyanobacteria obtain the carbon atoms that they need for this purpose from atmospheric Co2 and the hydrogen from water, releasing o2 gas as a by-product. The organic molecules produced by photosynthesis in turn serve as food for other organisms. many of these organisms carry out aerobic respiration, a process that uses o2 to form Co2 from the same carbon atoms that had been taken up as Co2 and converted into sugars by photosynthesis. in the process, the organisms that respire obtain the chemical-bond energy that they need to survive.

1	The first cells on the earth are thought to have been capable of neither photosynthesis nor respiration (discussed in Chapter 14). however, photosynthesis must have preceded respiration on the earth, since there is strong evidence that billions of years of photosynthesis were required before o2 had been released in sufficient quantity to create an atmosphere rich in this gas. (The earth’s atmosphere currently contains 20% o2.) HUMUS AND DISSOLVED ORGANIC MATTER SEDIMENTS AND FOSSIL FUELS RESPIRATION PHOTOSYNTHESIS FOOD CHAIN CO2 IN ATMOSPHERE AND WATER PLANTS, ALGAE, BACTERIA ANIMALS Figure 2–19 The carbon cycle. individual carbon atoms are incorporated into organic molecules of the living world by the photosynthetic activity of bacteria, algae, and plants. They pass to animals, microorganisms, and organic material in soil and oceans in cyclic paths. Co2 is restored to the atmosphere when organic molecules are oxidized by cells or burned by humans as fuels.

1	Chapter 2: Cell Chemistry and Bioenergetics enzymes lower the activation-energy Barriers That Block Chemical Reactions

1	Consider the reaction paper + O2→ smoke + ashes + heat + CO2 + H2O Once ignited, the paper burns readily, releasing to the atmosphere both energy as heat and water and carbon dioxide as gases. The reaction is irreversible, since the smoke and ashes never spontaneously retrieve these entities from the heated atmosphere and reconstitute themselves into paper. When the paper burns, its chemical energy is dissipated as heat—not lost from the universe, since energy can never be created or destroyed, but irretrievably dispersed in the chaotic random thermal motions of molecules. At the same time, the atoms and molecules of the paper become dispersed and disordered. In the language of thermodynamics, there has been a loss of free energy; that is, of energy that can be harnessed to do work or drive chemical reactions. This loss reflects a reduction of orderliness in the way the energy and molecules were stored in the paper. We shall discuss free energy in more detail shortly, but the general

1	chemical reactions. This loss reflects a reduction of orderliness in the way the energy and molecules were stored in the paper. We shall discuss free energy in more detail shortly, but the general principle is clear enough intuitively: chemical reactions proceed spontaneously only in the direction that leads to a loss of free energy. In other words, the spontaneous direction for any reaction is the direction that goes “downhill,” where a “downhill” reaction is one that is energetically favorable. Although the most energetically favorable form of carbon under ordinary conditions is CO2, and that of hydrogen is H2O, a living organism does not disappear in a puff of smoke, and the paper book in your hands does not burst into flames. This is because the molecules both in the living organism and in the book are in a relatively stable state, and they cannot be changed to a state of lower energy without an input of energy: in other words, a molecule requires activation energy—a kick over an

1	in the book are in a relatively stable state, and they cannot be changed to a state of lower energy without an input of energy: in other words, a molecule requires activation energy—a kick over an energy barrier—before it can undergo a chemical reaction that leaves it in a more stable state (Figure 2–21). In the case of a burning book, the activation energy can be provided by the heat of a lighted match. For the molecules in the watery solution inside a cell, the kick is delivered by an unusually energetic random collision with surrounding molecules—collisions that become more violent as the temperature is raised. The chemistry in a living cell is tightly controlled, because the kick over energy barriers is greatly aided by a specialized class of proteins—the enzymes. Each enzyme binds tightly to one or more molecules, called substrates, and holds them in a way that greatly reduces the activation energy of a particular chemical reaction that the bound substrates can undergo. A

1	tightly to one or more molecules, called substrates, and holds them in a way that greatly reduces the activation energy of a particular chemical reaction that the bound substrates can undergo. A substance that can lower the activation energy of a reaction is termed a catalyst; catalysts increase the rate of chemical reactions because they allow a much larger proportion of the random collisions with surrounding molecules to kick the substrates over the energy barrier, as illustrated in Figure 2–22. Enzymes are among the most effective catalysts

1	Figure 2–21 The important principle of activation energy. (a) Compound Y (a reactant) is in a relatively stable state, and energy is required to convert it to compound X (a product), even though X is at a lower overall energy level than Y. This conversion will not take place, therefore, unless compound Y can acquire enough activation energy (energy a minus energy b) from its surroundings to undergo the reaction that converts it into compound X. This energy may be provided by means of an unusually energetic collision with other molecules. for the reverse reaction, X → Y, the activation energy will be d more rarely. activation energies are always positive; note, however, that the total energy change for the energetically known: some are capable of speeding up reactions by factors of 1014 or more. Enzymes thereby allow reactions that would not otherwise occur to proceed rapidly at normal temperatures.

1	An enzyme cannot change the equilibrium point for a reaction. The reason is simple: when an enzyme (or any catalyst) lowers the activation energy for the reaction Y → X, of necessity it also lowers the activation energy for the reaction X → Y by exactly the same amount (see Figure 2–21). The forward and backward reactions will therefore be accelerated by the same factor by an enzyme, and the equilibrium point for the reaction will be unchanged (Figure 2–23). Thus no matter how much an enzyme speeds up a reaction, it cannot change its direction.

1	Despite the above limitation, enzymes steer all of the reactions in cells through specific reaction paths. This is because enzymes are both highly selective and very precise, usually catalyzing only one particular reaction. In other words, each enzyme selectively lowers the activation energy of only one of the several possible chemical reactions that its bound substrate molecules could undergo. In this way, sets of enzymes can direct each of the many different molecules in a cell along a particular reaction pathway (Figure 2–24). The success of living organisms is attributable to a cell’s ability to make enzymes of many types, each with precisely specified properties. Each enzyme

1	The success of living organisms is attributable to a cell’s ability to make enzymes of many types, each with precisely specified properties. Each enzyme Figure 2–23 enzymes cannot change the equilibrium point for reactions. enzymes, like all catalysts, speed up the forward and backward rates of a reaction by the same factor. Therefore, for both the catalyzed and the uncatalyzed reactions shown here, the number of molecules undergoing the transition X → Y is equal to the number of molecules undergoing the transition Y → X when the ratio of Y molecules to X molecules is 3 to 1. in other words, the two reactions reach equilibrium at exactly the same point.

1	Figure 2–22 lowering the activation energy greatly increases the probability of a reaction. at any given instant, a population of identical substrate molecules will have a range of energies, distributed as shown on the graph. The varying energies come from collisions with surrounding molecules, which make the substrate molecules jiggle, vibrate, and spin. for a molecule to undergo a chemical reaction, the energy of the molecule must exceed the activation-energy barrier for that reaction (dashed lines). for most biological reactions, this almost never happens without enzyme catalysis. even with enzyme catalysis, the substrate molecules must experience a particularly energetic collision to react (red shaded area). Raising the temperature will also increase the number of molecules with sufficient energy to overcome the activation energy needed for a reaction; but in marked contrast to enzyme catalysis, this effect is nonselective, speeding up all reactions (Movie 2.2).

1	Figure 2–24 Directing substrate molecules through a specific reaction pathway by enzyme catalysis. a substrate molecule in a cell (green ball) is converted into a different molecule (blue ball) by means of a series of enzyme-catalyzed reactions. as indicated (yellow box), several reactions are energetically favorable at each step, but only one is catalyzed by each enzyme. sets of enzymes thereby determine the exact reaction pathway that is followed by each molecule inside the cell. has a unique shape containing an active site, a pocket or groove in the enzyme into which only particular substrates will fit (Figure 2–25). Like all other catalysts, enzyme molecules themselves remain unchanged after participating in a reaction and therefore can function over and over again. In Chapter 3, we discuss further how enzymes work. how enzymes find Their substrates: The enormous Rapidity of molecular motions

1	how enzymes find Their substrates: The enormous Rapidity of molecular motions An enzyme will often catalyze the reaction of thousands of substrate molecules every second. This means that it must be able to bind a new substrate molecule in a fraction of a millisecond. But both enzymes and their substrates are present in relatively small numbers in a cell. How do they find each other so fast? Rapid binding is possible because the motions caused by heat energy are enormously fast at the molecular level. These molecular motions can be classified broadly into three kinds: (1) the movement of a molecule from one place to another (translational motion), (2) the rapid back-and-forth movement of covalently linked atoms with respect to one another (vibrations), and (3) rotations. All of these motions help to bring the surfaces of interacting molecules together.

1	The rates of molecular motions can be measured by a variety of spectroscopic techniques. A large globular protein is constantly tumbling, rotating about its axis about a million times per second. Molecules are also in constant translational motion, which causes them to explore the space inside the cell very efficiently by wandering through it—a process called diffusion. In this way, every molecule in a cell collides with a huge number of other molecules each second. As the molecules in a liquid collide and bounce off one another, an individual molecule moves first one way and then another, its path constituting a random walk (Figure 2–26). In such a walk, the average net distance that each molecule travels (as the “crow flies”) from its starting point is proportional to the square root of the time involved: that is, if it takes a molecule 1 second on average to travel 1 μm, it takes 4 seconds to travel 2 μm, 100 seconds to travel 10 μm, and so on.

1	The inside of a cell is very crowded (Figure 2–27). Nevertheless, experiments in which fluorescent dyes and other labeled molecules are injected into cells show that small organic molecules diffuse through the watery gel of the cytosol nearly Figure 2–25 How enzymes work. each enzyme has an active site to which one or more substrate molecules bind, forming an enzyme–substrate complex. a reaction occurs at the active site, producing an enzyme–product complex. The product is then released, allowing the enzyme to bind further substrate molecules. as rapidly as they do through water. A small organic molecule, for example, takes only about one-fifth of a second on average to diffuse a distance of 10 μm. Diffusion is therefore an efficient way for small molecules to move the limited distances in the cell (a typical animal cell is 15 μm in diameter).

1	Since enzymes move more slowly than substrates in cells, we can think of them as sitting still. The rate of encounter of each enzyme molecule with its substrate will depend on the concentration of the substrate molecule. For example, some abundant substrates are present at a concentration of 0.5 mM. Since pure water is 55.5 M, there is only about one such substrate molecule in the cell for every 105 water molecules. Nevertheless, the active site on an enzyme molecule that binds this substrate will be bombarded by about 500,000 random collisions with the substrate molecule per second. (For a substrate concentration tenfold lower, the number of collisions drops to 50,000 per second, and so on.) A random collision between the active site of an enzyme and the matching surface of its substrate molecule often leads immediately to the formation of an enzyme–substrate complex. A reaction in which a covalent bond is broken or formed can now occur extremely rapidly. When one appreciates how

1	molecule often leads immediately to the formation of an enzyme–substrate complex. A reaction in which a covalent bond is broken or formed can now occur extremely rapidly. When one appreciates how quickly molecules move and react, the observed rates of enzymatic catalysis do not seem so amazing.

1	Two molecules that are held together by noncovalent bonds can also dissociate. The multiple weak noncovalent bonds that they form with each other will persist until random thermal motion causes the two molecules to separate. In general, the stronger the binding of the enzyme and substrate, the slower their rate of dissociation. In contrast, whenever two colliding molecules have poorly matching surfaces, they form few noncovalent bonds and the total energy of association will be negligible compared with that of thermal motion. In this case, the two molecules dissociate as rapidly as they come together, preventing incorrect and unwanted associations between mismatched molecules, such as between an enzyme and the wrong substrate. The free-energy Change for a Reaction, ∆G, Determines Whether it Can occur spontaneously

1	The free-energy Change for a Reaction, ∆G, Determines Whether it Can occur spontaneously Although enzymes speed up reactions, they cannot by themselves force energetically unfavorable reactions to occur. In terms of a water analogy, enzymes by themselves cannot make water run uphill. Cells, however, must do just that in order to grow and divide: they must build highly ordered and energy-rich molecules from small and simple ones. We shall see that this is done through enzymes that directly couple energetically favorable reactions, which release energy and produce heat, to energetically unfavorable reactions, which produce biological order.

1	What do cell biologists mean by the term “energetically favorable,” and how can this be quantified? According to the second law of thermodynamics the universe tends toward maximum disorder (largest entropy or greatest probability). Thus, a chemical reaction can proceed spontaneously only if it results in a net increase in the disorder of the universe (see Figure 2–16). This disorder of the universe can be expressed most conveniently in terms of the free energy of a system, a concept we touched on earlier. Free energy, G, is an expression of the energy available to do work—for example, the work of driving chemical reactions. The value of G is of interest only when a system undergoes a change, denoted ∆G (delta G). The change in G is critical because, as explained in Panel 2–7 (pp. 102–103), ∆G is a direct measure of the

1	Figure 2–27 The structure of the cytoplasm. The drawing is approximately to scale and emphasizes the crowding in the cytoplasm. only the macromolecules are shown: Rnas are shown in blue, ribosomes in green, and proteins in red. enzymes and other macromolecules diffuse relatively slowly in the cytoplasm, in part because they interact with many other macromolecules; small molecules, by contrast, diffuse nearly as rapidly as they do in water (Movie 2.4). (adapted from D.s. Goodsell, Trends Biochem. Sci. 16:203–206, 1991. With permission from elsevier.) Figure 2–26 a random walk. molecules in solution move in a random fashion as a result of the continual buffeting they receive in collisions with other molecules. This movement allows small molecules to diffuse rapidly from one part of the cell to another, as described in the text (Movie 2.3).

1	amount of disorder created in the universe when a reaction takes place. Energetically favorable reactions, by definition, are those that decrease free energy; in other words, they have a negative ∆G and disorder the universe (Figure 2–28). An example of an energetically favorable reaction on a macroscopic scale is the “reaction” by which a compressed spring relaxes to an expanded state, releasing its stored elastic energy as heat to its surroundings; an example on a microscopic scale is salt dissolving in water. Conversely, energetically unfavorable reactions with a positive ∆G—such as the joining of two amino acids to form a peptide bond—by themselves create order in the universe. Therefore, these reactions can take place only if they are coupled to a second reaction with a negative ∆G so large that the ∆G of the overall process is negative (Figure 2–29). The Concentration of Reactants influences the free-energy Change and a Reaction’s Direction

1	The Concentration of Reactants influences the free-energy Change and a Reaction’s Direction As we have just described, a reaction Y ↔ X will go in the direction Y →X when the associated free-energy change, ∆G, is negative, just as a tensed spring left to itself will relax and lose its stored energy to its surroundings as heat. For a chemical reaction, however, ∆G depends not only on the energy stored in each individual molecule, but also on the concentrations of the molecules in the reaction mixture. Remember that ∆G reflects the degree to which a reaction creates a more disordered—in other words, a more probable—state of the universe. Recalling our coin analogy, it is very likely that a coin will flip from a head to a tail orientation if a jiggling box contains 90 heads and 10 tails, but this is a less probable event if the box has 10 heads and 90 tails.

1	The same is true for a chemical reaction. For a reversible reaction Y ↔ X, a large excess of Y over X will tend to drive the reaction in the direction Y → X. Therefore, as the ratio of Y to X increases, the ∆G becomes more negative for the transition Y → X (and more positive for the transition X → Y). The amount of concentration difference that is needed to compensate for a given decrease in chemical-bond energy (and accompanying heat release) is not intuitively obvious. In the late nineteenth century, the relationship was determined through a thermodynamic analysis that makes it possible to separate the concentration-dependent and the concentration-independent parts of the free-energy change, as we describe next. The standard free-energy Change, ∆G°, makes it possible to Compare the energetics of Different Reactions

1	The standard free-energy Change, ∆G°, makes it possible to Compare the energetics of Different Reactions Because ∆G depends on the concentrations of the molecules in the reaction mixture at any given time, it is not a particularly useful value for comparing the relative energies of different types of reactions. To place reactions on a comparable basis, we need to turn to the standard free-energy change of a reaction, ∆G°. The ∆G° is the change in free energy under a standard condition, defined as that where the concentrations of all the reactants are set to the same fixed value of 1 mole/liter. Defined in this way, ∆G° depends only on the intrinsic characters of the reacting molecules.

1	For the simple reaction Y → X at 37°C, ∆G° is related to ∆G as follows: where ∆G is in kilojoules per mole, [Y] and [X] denote the concentrations of Y and X in moles/liter, ln is the natural logarithm, and RT is the product of the gas constant, R, and the absolute temperature, T. At 37°C, RT = 2.58 J mole–1. (A mole is 6 × 1023 molecules of a substance.) A large body of thermodynamic data has been collected that has made it possible to determine the standard free-energy change, ∆G°, for the important metabolic reactions of a cell. Given these ∆G° values, combined with additional information about metabolite concentrations and reaction pathways, it is possible to quantitatively predict the course of most biological reactions. The free energy of Y is greater than the free energy of X. Therefore ˜G < 0, and the disorderENERGETICALLY of the universe increasesFAVORABLE during the reactionREACTION Y X. If the reaction X Y occurred, ˜G would be > 0, and theENERGETICALLY universe would

1	X. If the reaction X Y occurred, ˜G would be > 0, and theENERGETICALLY universe would REACTION ordered. this reaction can occur only if it is coupled to a second, energetically favorable reaction Figure 2–28 The distinction between energetically favorable and energetically unfavorable reactions. the energetically unfavorable reaction X Y is driven by the energetically favorable reaction C D, because the net free-energy change for the pair of coupled reactions is less than zero Figure 2–29 How reaction coupling is used to drive energetically unfavorable reactions. FOR THE ENERGETICALLY FAVORABLE REACTION Y °X, Figure 2–30 Chemical equilibrium. When a reaction reaches equilibrium, the forward and backward fluxes of reacting molecules are equal and opposite.

1	FOR THE ENERGETICALLY FAVORABLE REACTION Y °X, Figure 2–30 Chemical equilibrium. When a reaction reaches equilibrium, the forward and backward fluxes of reacting molecules are equal and opposite. when X and Y are at equal concentrations, [Y] = [X], the formation of X is energetically favored. In other words, the ˜G of Y °X is negative and the ˜G of X °Y is positive. But because of thermal bombardments, there will always be some X converting to Y. THUS, FOR EACH INDIVIDUAL MOLECULE, conversion of Y to X will occur often. Conversion of X to Y will occur less often than the transition Y °X, because it requires a more energetic collision. Therefore the ratio of X to Y molecules will increase with time

1	Conversion of X to Y will occur less often than the transition Y °X, because it requires a more energetic collision. Therefore the ratio of X to Y molecules will increase with time EVENTUALLY, there will be a large enough excess of X over Y to just compensate for the slow rate of X °Y, such that the number of Y molecules being converted to X molecules each second is exactly equal to the number of X molecules being converted to Y molecules each second. At this point, the reaction will be at equilibrium. AT EQUILIBRIUM, there is no net change in the ratio of Y to X, and the ˜G for both forward and backward reactions is zero. The equilibrium Constant and ∆G° are Readily Derived from each other

1	The equilibrium Constant and ∆G° are Readily Derived from each other Inspection of the above equation reveals that the ∆G equals the value of ∆G° when the concentrations of Y and X are equal. But as any favorable reaction proceeds, the concentrations of the products will increase as the concentration of the substrates decreases. This change in relative concentrations will cause [X]/[Y] to become increasingly large, making the initially favorable ∆G less and less negative (the logarithm of a number x is positive for x > 1, negative for x < 1, and zero for x =1). Eventually, when ∆G = 0, a chemical equilibrium will be attained; here there is no net change in free energy to drive the reaction in either direction, inasmuch as the concentration effect just balances the push given to the reaction by ∆G°. As a result, the ratio of product to substrate reaches a constant value at chemical equilibrium (Figure 2–30).

1	We can define the equilibrium constant, K, for the reaction Y →X as where [X] is the concentration of the product and [Y] is the concentration of the reactant at equilibrium. Remembering that ∆G = ∆G° + RT ln [X]/[Y], and that ∆G = 0 at equilibrium, we see that At 37°C, where RT = 2.58, the equilibrium equation is therefore: ∆G° = –2.58 ln K

1	At 37°C, where RT = 2.58, the equilibrium equation is therefore: ∆G° = –2.58 ln K Converting this equation from the natural logarithm (ln) to the more commonly used base 10 logarithm (log), we get ∆G° = –5.94 log K The above equation reveals how the equilibrium ratio of X to Y (expressed as the equilibrium constant, K) depends on the intrinsic character of the molecules, (as expressed in the value of ∆G° in kilojoules per mole). Note that for every 5.94 kJ/mole difference in free energy at 37°C, the equilibrium constant changes by a factor of 10 (Table 2–2). Thus, the more energetically favorable a reaction, the more product will accumulate if the reaction proceeds to equilibrium. More generally, for a reaction that has multiple reactants and products, such as A + B → C + D,

1	The concentrations of the two reactants and the two products are multiplied because the rate of the forward reaction depends on the collision of A and B and the rate of the backward reaction depends on the collision of C and D. Thus, at 37°C, ∆G° = –5.94 log where ∆G° is in kilojoules per mole, and [A], [B], [C], and [D] denote the concentrations of the reactants and products in moles/liter. The free-energy Changes of Coupled Reactions are additive We have pointed out that unfavorable reactions can be coupled to favorable ones to drive the unfavorable ones forward (see Figure 2–29). In thermodynamic terms, this is possible because the overall free-energy change for a set of coupled reactions is the sum of the free-energy changes in each of its component steps. Consider, as a simple example, two sequential reactions

1	X →Y and Y →Z whose ∆G° values are +5 and –13 kJ/mole, respectively. If these two reactions occur sequentially, the ∆G° for the coupled reaction will be –8 kJ/mole. This means that, with appropriate conditions, the unfavorable reaction X → Y can be driven by the favorable reaction Y → Z, provided that this second reaction follows the first. For example, several of the reactions in the long pathway that converts sugars into CO2 and H2O have positive ∆G° values. But the pathway nevertheless proceeds because the total ∆G° for the series of sequential reactions has a large negative value. Forming a sequential pathway is not adequate for many purposes. Often the desired pathway is simply X → Y, without further conversion of Y to some other product. Fortunately, there are other more general ways of using enzymes to couple reactions together. These often involve the activated carrier molecules that we discuss next. activated Carrier molecules are essential for Biosynthesis

1	activated Carrier molecules are essential for Biosynthesis The energy released by the oxidation of food molecules must be stored temporarily before it can be channeled into the construction of the many other molecules needed by the cell. In most cases, the energy is stored as chemical-bond energy in a small set of activated “carrier molecules,” which contain one or more energy-rich covalent bonds. These molecules diffuse rapidly throughout the cell and thereby carry their bond energy from sites of energy generation to the sites where the energy will be used for biosynthesis and other cell activities (Figure 2–31).

1	The activated carriers store energy in an easily exchangeable form, either as a readily transferable chemical group or as electrons held at a high energy level, and they can serve a dual role as a source of both energy and chemical groups in biosynthetic reactions. For historical reasons, these molecules are also sometimes referred to as coenzymes. The most important of the activated carrier molecules are ATP and two molecules that are closely related to each other, NADH and NADPH. Cells use such activated carrier molecules like money to pay for reactions that otherwise could not take place. The formation of an activated Carrier is Coupled to an energetically favorable Reaction

1	Coupling mechanisms require enzymes and are fundamental to all the energy transactions of the cell. The nature of a coupled reaction is illustrated by a mechanical analogy in Figure 2–32, in which an energetically favorable chemical reaction is represented by rocks falling from a cliff. The energy of falling rocks would normally be entirely wasted in the form of heat generated by friction when the rocks hit the ground (see the falling-brick diagram in Figure 2–17). By careful design, however, part of this energy could be used instead to drive a paddle wheel that lifts a bucket of water (Figure 2–32B). Because the rocks can now reach the ground only after moving the paddle wheel, we say that the energetically favorable reaction of rock falling has been directly coupled to the energetically unfavorable reaction of lifting the bucket of water. Note that because part of the energy is used to do work in Figure 2–32B, the rocks hit the ground with less velocity than in Figure 2–32A, and

1	unfavorable reaction of lifting the bucket of water. Note that because part of the energy is used to do work in Figure 2–32B, the rocks hit the ground with less velocity than in Figure 2–32A, and correspondingly less energy is dissipated as heat.

1	Similar processes occur in cells, where enzymes play the role of the paddle wheel. By mechanisms that we discuss later in this chapter, enzymes couple an Figure 2–31 energy transfer and the role of activated carriers in metabolism. By serving as energy shuttles, activated carrier molecules perform their function as go-betweens that link the breakdown of food molecules and the release of energy (catabolism) to the energy-requiring biosynthesis of small and large organic molecules (anabolism).

1	Figure 2–32 a mechanical model illustrating the principle of coupled chemical reactions. The spontaneous reaction shown in (a) could serve as an analogy for the direct oxidation of glucose to Co2 and h2o, which produces heat only. in (B), the same reaction is coupled to a second reaction; this second reaction is analogous to the synthesis of activated carrier molecules. The energy produced in (B) is in a more useful form than in (a) and can be used to drive a variety of otherwise energetically unfavorable reactions (C).

1	energetically favorable reaction, such as the oxidation of foodstuffs, to an energetically unfavorable reaction, such as the generation of an activated carrier molecule. In this example, the amount of heat released by the oxidation reaction is reduced by exactly the amount of energy stored in the energy-rich covalent bonds of the activated carrier molecule. And the activated carrier molecule picks up a packet of energy of a size sufficient to power a chemical reaction elsewhere in the cell. aTp is the most Widely Used activated Carrier molecule

1	aTp is the most Widely Used activated Carrier molecule The most important and versatile of the activated carriers in cells is ATP (adenosine triphosphate). Just as the energy stored in the raised bucket of water in Figure 2–32B can drive a wide variety of hydraulic machines, ATP is a convenient and versatile store, or currency, of energy used to drive a variety of chemical reactions in cells. ATP is synthesized in an energetically unfavorable phosphorylation reaction in which a phosphate group is added to ADP (adenosine diphosphate). When required, ATP gives up its energy packet through its energetically favorable hydrolysis to ADP and inorganic phosphate (Figure 2–33). The regenerated ADP is then available to be used for another round of the phosphorylation reaction that forms ATP.

1	The energetically favorable reaction of ATP hydrolysis is coupled to many otherwise unfavorable reactions through which other molecules are synthesized. Many of these coupled reactions involve the transfer of the terminal phosphate in ATP to another molecule, as illustrated by the phosphorylation reaction in Figure 2–34. As the most abundant activated carrier in cells, ATP is the principle energy currency. To give just two examples, it supplies energy for many of the pumps that transport substances into and out of the cell (discussed in Chapter 11), and it powers the molecular motors that enable muscle cells to contract and nerve cells to transport materials from one end of their long axons to another (discussed in Chapter 16). energy stored in aTp is often harnessed to Join Two molecules Together

1	energy stored in aTp is often harnessed to Join Two molecules Together We have previously discussed one way in which an energetically favorable reaction can be coupled to an energetically unfavorable reaction, X → Y, so as to enable it to occur. In that scheme, a second enzyme catalyzes the energetically favorable reaction Y → Z, pulling all of the X to Y in the process. But when the required product is Y and not Z, this mechanism is not useful.

1	Figure 2–33 The hydrolysis of aTP to aDP and inorganic phosphate. The two outermost phosphates in aTp are held to the rest of the molecule by high-energy phosphoanhydride bonds and are readily transferred. as indicated, water can be added to aTp to form aDp and inorganic phosphate (pi). hydrolysis of the terminal phosphate of aTp yields between 46 and 54 kJ/mole of usable energy, depending on the intracellular conditions. The large negative ΔG of this reaction arises from several factors: release of the terminal phosphate group removes an unfavorable repulsion between adjacent negative charges, and the inorganic phosphate ion (pi) released is stabilized by resonance and by favorable hydrogen-bond formation with water.

1	Figure 2–34 an example of a phosphate transfer reaction. Because an energy-rich phosphoanhydride bond in aTp is converted to a phosphoester bond, this reaction is energetically favorable, having a large negative ΔG. Reactions of this type are involved in the synthesis of phospholipids and in the initial steps of reactions that catabolize sugars. A typical biosynthetic reaction is one in which two molecules, A and B, are joined together to produce A–B in the energetically unfavorable condensation reaction There is an indirect pathway that allows A–H and B–OH to form A–B, in which a coupling to ATP hydrolysis makes the reaction go. Here, energy from ATP hydrolysis is first used to convert B–OH to a higher-energy intermediate compound, which then reacts directly with A–H to give A–B. The simplest possible mechanism involves the transfer of a phosphate from ATP to B–OH to make B–O–PO3, in which case the reaction pathway contains only two steps: 1. 2.

1	2. A–H + B–O–PO3 →A–B + Pi Net result: B–OH + ATP + A–H →A–B + ADP + Pi The condensation reaction, which by itself is energetically unfavorable, is forced to occur by being directly coupled to ATP hydrolysis in an enzyme-catalyzed reaction pathway (Figure 2–35A). A biosynthetic reaction of exactly this type synthesizes the amino acid glutamine (Figure 2–35B). We will see shortly that similar (but more complex) mechanisms are also used to produce nearly all of the large molecules of the cell.

1	Figure 2–35 an example of an energetically unfavorable biosynthetic reaction driven by aTP hydrolysis. (a) schematic illustration of the formation of a–B in the condensation reaction described in the text. (B) The biosynthesis of the common amino acid glutamine from glutamic acid and ammonia. Glutamic acid is first converted to a high-energy phosphorylated intermediate (corresponding to the compound B–o–po3 described in the text), which then reacts with ammonia (corresponding to a–h) to form glutamine. in this example, both steps occur on the surface of the same enzyme, glutamine synthetase. The high-energy bonds are shaded red; here, as elsewhere throughout the book, the symbol pi = hpo42–, and a yellow “circled p” = po3 . naDh and naDph are important electron Carriers

1	naDh and naDph are important electron Carriers Other important activated carrier molecules participate in oxidation–reduction reactions and are commonly part of coupled reactions in cells. These activated carriers are specialized to carry electrons held at a high energy level (sometimes called “high-energy” electrons) and hydrogen atoms. The most important of these electron carriers are NAD+ (nicotinamide adenine dinucleotide) and the closely related molecule NADP+ (nicotinamide adenine dinucleotide phosphate). Each picks up a “packet of energy” corresponding to two electrons plus a proton (H+), and they are thereby converted to NADH (reduced nicotinamide adenine dinucleotide) and NADPH (reduced nicotinamide adenine dinucleotide phosphate), respectively (Figure 2–36). These molecules can therefore be regarded as carriers of hydride ions (the H+ plus two electrons, or H–).

1	Like ATP, NADPH is an activated carrier that participates in many important biosynthetic reactions that would otherwise be energetically unfavorable. The NADPH is produced according to the general scheme shown in Figure 2–36A. During a special set of energy-yielding catabolic reactions, two hydrogen atoms are removed from a substrate molecule. Both electrons but just one proton (that is, a hydride ion, H–) are added to the nicotinamide ring of NADP+ to form NADPH; the second proton (H+) is released into solution. This is a typical oxidation–reduction reaction, in which the substrate is oxidized and NADP+ is reduced. NADPH readily gives up the hydride ion it carries in a subsequent oxidation–reduction reaction, because the nicotinamide ring can achieve a more stable arrangement of electrons without it. In this subsequent reaction, which Figure 2–36 NaDPH, an important carrier of electrons.

1	Figure 2–36 NaDPH, an important carrier of electrons. HC (a) naDph is produced in reactions of the general type shown on the left, in which two hydrogen atoms are removed from a substrate. The oxidized form of the carrier molecule, naDp+, receives one hydrogen atom plus an electron (a hydride ion); the proton (h+) from the other h atom is released into solution. Because naDph holds its hydride ion in a high-energy linkage, the hydride ion can easily be transferred to other molecules, as shown on the right. (B) and (C) The structures of naDp+ and naDph. The part of the naDp+ molecule known as the nicotinamide ring accepts the hydride ion, h–, forming naDph. The molecules naD+ and naDh are identical in structure to naDp+ and naDph, respectively, except that they lack the indicated phosphate group.

1	this phosphate group is missing in NAD+ and NADH regenerates NADP+, it is the NADPH that is oxidized and the substrate that is reduced. The NADPH is an effective donor of its hydride ion to other molecules for the same reason that ATP readily transfers a phosphate: in both cases the transfer is accompanied by a large negative free-energy change. One example of the use of NADPH in biosynthesis is shown in Figure 2–37. The extra phosphate group on NADPH has no effect on the electron-transfer properties of NADPH compared with NADH, being far away from the region involved in electron transfer (see Figure 2–36C). It does, however, give a molecule of NADPH a slightly different shape from that of NADH, making it possible for NADPH and NADH to bind as substrates to completely different sets of enzymes. Thus, the two types of carriers are used to transfer electrons (or hydride ions) between two different sets of molecules.

1	Why should there be this division of labor? The answer lies in the need to regulate two sets of electron-transfer reactions independently. NADPH operates chiefly with enzymes that catalyze anabolic reactions, supplying the high-energy electrons needed to synthesize energy-rich biological molecules. NADH, by contrast, has a special role as an intermediate in the catabolic system of reactions that generate ATP through the oxidation of food molecules, as we will discuss shortly. The genesis of NADH from NAD+, and of NADPH from NADP+, occur by different pathways and are independently regulated, so that the cell can adjust the supply of electrons for these two contrasting purposes. Inside the cell the ratio of NAD+ to NADH is kept high, whereas the ratio of NADP+ to NADPH is kept low. This provides plenty of NAD+ to act as an oxidizing agent and plenty of NADPH to act as a reducing agent (Figure 2–37B)—as required for their special roles in catabolism and anabolism, respectively.

1	There are many other activated Carrier molecules in Cells Other activated carriers also pick up and carry a chemical group in an easily transferred, high-energy linkage. For example, coenzyme A carries a readily transferable Figure 2–37 NaDPH as a reducing agent. (a) The final stage in the biosynthetic route leading to cholesterol. as in many other biosynthetic reactions, the reduction of the C=C bond is achieved by the transfer of a hydride ion from the carrier molecule naDph, plus a proton (h+) from the solution. (B) Keeping naDph levels high and naDh levels low alters their affinities for electrons (see panel 14–1, p. 765). This causes naDph to be a much stronger electron donor (reducing agent) than naDh, and naD+ therefore to be a much better electron acceptor (oxidizing agent) than naDp+, as indicated.

1	acetyl group in a thioester linkage, and in this activated form is known as acetyl CoA (acetyl coenzyme A). Acetyl CoA (Figure 2–38) is used to add two carbon units in the biosynthesis of larger molecules.

1	In acetyl CoA, as in other carrier molecules, the transferable group makes up only a small part of the molecule. The rest consists of a large organic portion that serves as a convenient “handle,” facilitating the recognition of the carrier molecule by specific enzymes. As with acetyl CoA, this handle portion very often contains a nucleotide (usually adenosine), a curious fact that may be a relic from an early stage of evolution. It is currently thought that the main catalysts for early life-forms—before DNA or proteins—were RNA molecules (or their close relatives), as described in Chapter 6. It is tempting to speculate that many of the carrier molecules that we find today originated in this earlier RNA world, where their nucleotide portions could have been useful for binding them to RNA enzymes (ribozymes).

1	Thus, ATP transfers phosphate, NADPH transfers electrons and hydrogen, and acetyl CoA transfers two-carbon acetyl groups. FADH2 (reduced flavin adenine dinucleotide) is used like NADH in electron and proton transfers (Figure 2–39). The reactions of other activated carrier molecules involve the transfer of a methyl, carboxyl, or glucose group for biosyntheses (Table 2–3). These activated carriers Figure 2–38 The structure of the important activated carrier molecule acetyl Coa. a ball-and-stick model is shown above the structure. The sulfur atom (yellow) forms a thioester bond to acetate. Because this is a high-energy linkage, releasing a large amount of free energy when it is hydrolyzed, the acetate molecule can be readily transferred to other molecules. Figure 2–39 FaDH2 is a carrier of hydrogens and high-energy electrons, like NaDH and NaDPH. (a) structure of faDh2, with its hydrogen-carrying atoms highlighted in yellow. (B) The formation of faDh2 from faD.

1	are generated in reactions that are coupled to ATP hydrolysis, as in the example in Figure 2–40. Therefore, the energy that enables their groups to be used for biosynthesis ultimately comes from the catabolic reactions that generate ATP. Similar processes occur in the synthesis of the very large molecules of the cell—the nucleic acids, proteins, and polysaccharides—that we discuss next. The synthesis of Biological polymers is Driven by aTp hydrolysis

1	The synthesis of Biological polymers is Driven by aTp hydrolysis As discussed previously, the macromolecules of the cell constitute most of its dry mass (see Figure 2–7). These molecules are made from subunits (or monomers) that are linked together in a condensation reaction, in which the constituents of a water molecule (OH plus H) are removed from the two reactants. Consequently, the reverse reaction—the breakdown of all three types of polymers—occurs by the enzyme-catalyzed addition of water (hydrolysis). This hydrolysis reaction is energetically favorable, whereas the biosynthetic reactions require an energy input (see Figure 2–9).

1	The nucleic acids (DNA and RNA), proteins, and polysaccharides are all polymers that are produced by the repeated addition of a monomer onto one end of a growing chain. The synthesis reactions for these three types of macromolecules are outlined in Figure 2–41. As indicated, the condensation step in each case depends on energy from nucleoside triphosphate hydrolysis. And yet, except for the nucleic acids, there are no phosphate groups left in the final product molecules. How are the reactions that release the energy of ATP hydrolysis coupled to polymer synthesis? For each type of macromolecule, an enzyme-catalyzed pathway exists which resembles that discussed previously for the synthesis of the amino acid glutamine (see Figure 2–35). The principle is exactly the same, in that the –OH group that will

1	Figure 2–40 a carboxyl group-transfer reaction using an activated carrier molecule. Carboxylated biotin is used by the enzyme pyruvate carboxylase to transfer a carboxyl group in the production of oxaloacetate, a molecule needed for the citric acid cycle. The acceptor molecule for this group-transfer reaction is pyruvate. other enzymes use biotin, a B-complex vitamin, to transfer carboxyl groups to other acceptor molecules. note that synthesis of carboxylated biotin requires energy that is derived from aTp—a general feature of many activated carriers.

1	Figure 2–41 The synthesis of polysaccharides, proteins, and nucleic acids. synthesis of each kind of biological polymer involves the loss of water in a condensation reaction. not shown is the consumption of high-energy nucleoside triphosphates that is required to activate each monomer before its addition. in contrast, the reverse reaction—the breakdown CH2OHOHOOHOHCH2OHOHOOHOHCH2OHOOHOHOOCH2OHOOHOHCH2OHOOHOHOOCH2OHOOHOHOHOOH(A)POLYSACCHARIDESglucoseglycogenglycogenH2O energy from nucleoside triphosphate hydrolysis HCROCNHHCRCOOHHHRHCCNOOHHCROCNHHCRCRHCCNOOHHOprotein amino acid (C) PROTEINS A OCH2OHOOOO_POC OCH2OHOHOHOPOG OOHOHCH2O_A OCH2OHOOOO_POC OCH2OHOOPOG OOHOHCH2O_H2O (B) NUCLEIC ACIDS RNA nucleotide H2O energy from nucleoside triphosphate hydrolysis energy from nucleoside triphosphate hydrolysis RNA protein of all three types of polymers—occurs by the simple addition of water (hydrolysis).

1	be removed in the condensation reaction is first activated by becoming involved in a high-energy linkage to a second molecule. However, the actual mechanisms used to link ATP hydrolysis to the synthesis of proteins and polysaccharides are more complex than that used for glutamine synthesis, since a series of high-energy intermediates is required to generate the final high-energy bond that is broken during the condensation step (discussed in Chapter 6 for protein synthesis).

1	Each activated carrier has limits in its ability to drive a biosynthetic reaction. The ∆G for the hydrolysis of ATP to ADP and inorganic phosphate (Pi) depends on the concentrations of all of the reactants, but under the usual conditions in a cell it is between –46 and –54 kJ/mole. In principle, this hydrolysis reaction could drive an unfavorable reaction with a ∆G of, perhaps, +40 kJ/mole, provided that a suitable reaction path is available. For some biosynthetic reactions, however, even –50 kJ/mole does not provide enough of a driving force. In these cases, the path of ATP hydrolysis can be altered so that it initially produces AMP and pyrophosphate (PPi), which is itself then hydrolyzed in a subsequent step (Figure 2–42). The whole process makes available a total free-energy change of about –100 kJ/ mole. An important type of biosynthetic reaction that is driven in this way is the synthesis of nucleic acids (polynucleotides) from nucleoside triphosphates, as illustrated on the

1	of about –100 kJ/ mole. An important type of biosynthetic reaction that is driven in this way is the synthesis of nucleic acids (polynucleotides) from nucleoside triphosphates, as illustrated on the right side of Figure 2–43.

1	Note that the repetitive condensation reactions that produce macromolecules can be oriented in one of two ways, giving rise to either the head polymerization or the tail polymerization of monomers. In so-called head polymerization, the reactive bond required for the condensation reaction is carried on the end of the Figure 2–43 Synthesis of a polynucleotide, RNa or DNa, is a multistep process driven by aTP hydrolysis. in the first step, a nucleoside monophosphate is activated by the sequential transfer of the terminal phosphate groups from two aTp molecules. The high-energy intermediate formed—a nucleoside triphosphate—exists free in solution until it reacts with the growing end of an Rna or a Dna chain with release of pyrophosphate. hydrolysis of the latter to inorganic phosphate is highly favorable and helps to drive the overall reaction in the direction of polynucleotide synthesis. for details, see Chapter 5.

1	Figure 2–42 an alternative pathway of aTP hydrolysis, in which pyrophosphate is first formed and then hydrolyzed. This route releases about twice as much free energy (approximately –100 kJ/mole) as the reaction shown earlier in figure 2–33, and it forms amp instead of aDp. (a) in the two successive hydrolysis reactions, oxygen atoms from the participating water molecules are retained in the products, as indicated, whereas the hydrogen atoms dissociate to form free hydrogen ions (h+, not shown). (B) summary of overall reaction. HEAD POLYMERIZATION (e.g., PROTEINS, FATTY ACIDS) TAIL POLYMERIZATION (e.g., DNA, RNA, POLYSACCHARIDES) growing polymer, and it must therefore be regenerated each time that a monomer is added. In this case, each monomer brings with it the reactive bond that will be used in adding the next monomer in the series. In tail polymerization, the reactive bond carried by each monomer is instead used immediately for its own addition (Figure 2–44).

1	We shall see in later chapters that both of these types of polymerization are used. The synthesis of polynucleotides and some simple polysaccharides occurs by tail polymerization, for example, whereas the synthesis of proteins occurs by a head polymerization process.

1	Living cells need to create and maintain order within themselves to survive and grow. This is thermodynamically possible only because of a continual input of energy, part of which must be released from the cells to their environment as heat that disorders the surroundings. The only chemical reactions possible are those that increase the total amount of disorder in the universe. The free-energy change for a reaction, ∆G, measures this disorder, and it must be less than zero for a reaction to proceed spontaneously. This ∆G depends both on the intrinsic properties of the reacting molecules and their concentrations, and it can be calculated from these concentrations if either the equilibrium constant (K) for the reaction or its standard free-energy change, ∆G°, is known.

1	The energy needed for life comes ultimately from the electromagnetic radiation of the sun, which drives the formation of organic molecules in photosynthetic organisms such as green plants. Animals obtain their energy by eating organic molecules and oxidizing them in a series of enzyme-catalyzed reactions that are coupled to the formation of ATP—a common currency of energy in all cells.

1	To make possible the continual generation of order in cells, energetically favorable reactions, such as the hydrolysis of ATP, are coupled to energetically unfavorable reactions. In the biosynthesis of macromolecules, ATP is used to form reactive phosphorylated intermediates. Because the energetically unfavorable reaction of biosynthesis now becomes energetically favorable, ATP hydrolysis is said to drive the reaction. Polymeric molecules such as proteins, nucleic acids, and polysaccharides are assembled from small activated precursor molecules by repetitive condensation reactions that are driven in this way. Other reactive molecules, called either activated carriers or coenzymes, transfer other chemical groups in the course of biosynthesis: NADPH transfers hydrogen as a proton plus two electrons (a hydride ion), for example, whereas acetyl CoA transfers an acetyl group.

1	The constant supply of energy that cells need to generate and maintain the biological order that keeps them alive comes from the chemical-bond energy in food molecules. The proteins, lipids, and polysaccharides that make up most of the food we eat must be broken down into smaller molecules before our cells can use them—either Figure 2–44 The orientation of the active intermediates in the repetitive condensation reactions that form biological polymers. The head growth of polymers is compared with its alternative, tail growth. as indicated, these two mechanisms are used to produce different types of biological macromolecules.

1	as a source of energy or as building blocks for other molecules. Enzymatic digestion breaks down the large polymeric molecules in food into their monomer subunits—proteins into amino acids, polysaccharides into sugars, and fats into fatty acids and glycerol. After digestion, the small organic molecules derived from food enter the cytosol of cells, where their gradual oxidation begins. Sugars are particularly important fuel molecules, and they are oxidized in small controlled steps to carbon dioxide (CO2) and water (Figure 2–45). In this section, we trace the major steps in the breakdown, or catabolism, of sugars and show how they produce ATP, NADH, and other activated carrier molecules in animal cells. A very similar pathway also operates in plants, fungi, and many bacteria. As we shall see, the oxidation of fatty acids is equally important for cells. Other molecules, such as proteins, can also serve as energy sources when they are funneled through appropriate enzymatic pathways.

1	Glycolysis is a Central aTp-producing pathway The major process for oxidizing sugars is the sequence of reactions known as glycolysis—from the Greek glukus, “sweet,” and lusis, “rupture.” Glycolysis produces ATP without the involvement of molecular oxygen (O2 gas). It occurs in the cytosol of most cells, including many anaerobic microorganisms. Glycolysis probably evolved early in the history of life, before photosynthetic organisms introduced oxygen into the atmosphere. During glycolysis, a glucose molecule with six carbon atoms is converted into two molecules of pyruvate, each of which contains three carbon atoms. For each glucose molecule, two molecules of ATP are hydrolyzed to provide energy to drive the early steps, but four molecules of ATP are produced in the later steps. At the end of glycolysis, there is consequently a net gain of two molecules of ATP for each glucose molecule broken down. Two molecules of the activated carrier NADH are also produced.

1	The glycolytic pathway is outlined in Figure 2–46 and shown in more detail in Panel 2–8 (pp. 104–105) and Movie 2.5. Glycolysis involves a sequence of 10 separate reactions, each producing a different sugar intermediate and each catalyzed by a different enzyme. Like most enzymes, these have names ending in ase—such as isomerase and dehydrogenase—to indicate the type of reaction they catalyze. Although no molecular oxygen is used in glycolysis, oxidation occurs, in that electrons are removed by NAD+ (producing NADH) from some of the carbons derived from the glucose molecule. The stepwise nature of the process releases the energy of oxidation in small packets, so that much of it can be stored in activated carrier molecules rather than all of it being released as heat (see Figure 2–45). Thus, some of the energy released by oxidation drives the direct synthesis of ATP molecules from ADP and Pi, and some remains with the electrons in the electron carrier NADH.

1	Figure 2–45 Schematic representation of the controlled stepwise oxidation of sugar in a cell, compared with ordinary burning. (a) if the sugar were oxidized to Co2 and h2o in a single step, it would release an amount of energy much larger than could be captured for useful purposes. (B) in the cell, enzymes catalyze oxidation via a series of small steps in which free energy is transferred in conveniently sized packets to carrier molecules—most often aTp and naDh. at each step, an enzyme controls the reaction by reducing the activation-energy barrier that has to be surmounted before the specific reaction can occur. The total free energy released is exactly the same in (a) and (B). large activation energy overcome by the heat from a fre all free energy is released as heat; none is stored

1	large activation energy overcome by the heat from a fre all free energy is released as heat; none is stored Two molecules of NADH are formed per molecule of glucose in the course of glycolysis. In aerobic organisms, these NADH molecules donate their electrons to the electron-transport chain described in Chapter 14, and the NAD+ formed from the NADH is used again for glycolysis (see step 6 in Panel 2–8, pp. 104–105). fermentations produce aTp in the absence of oxygen For most animal and plant cells, glycolysis is only a prelude to the final stage of the breakdown of food molecules. In these cells, the pyruvate formed by glycolysis is rapidly transported into the mitochondria, where it is converted into CO2 plus acetyl CoA, whose acetyl group is then completely oxidized to CO2 and H2O.

1	In contrast, for many anaerobic organisms—which do not utilize molecular oxygen and can grow and divide without it—glycolysis is the principal source of the cell’s ATP. Certain animal tissues, such as skeletal muscle, can also continue to function when molecular oxygen is limited. In these anaerobic conditions, the pyruvate and the NADH electrons stay in the cytosol. The pyruvate is converted into products excreted from the cell—for example, into ethanol and CO2 in the yeasts used in brewing and breadmaking, or into lactate in muscle. In this process, the NADH gives up its electrons and is converted back into NAD+. This regeneration of NAD+ is required to maintain the reactions of glycolysis (Figure 2–47). Energy-yielding pathways like these, in which organic molecules both donate and accept electrons (and which are often, as in these cases, anaerobic), are called one molecule of glucose

1	Energy-yielding pathways like these, in which organic molecules both donate and accept electrons (and which are often, as in these cases, anaerobic), are called one molecule of glucose OH to be fructose 1,6 six-carbon sugar to two three-carbon sugarsSTEP 5 two molecules of glyceraldehyde Figure 2–46 an outline of glycolysis. each of the 10 steps shown is catalyzed by a different enzyme. note that step 4 cleaves a six-carbon sugar into two three-carbon sugars, so that the number of molecules at every stage after this doubles. as indicated, step 6 begins the energy-generation phase of glycolysis. Because two molecules of aTp are hydrolyzed in the early, energy-investment phase, glycolysis results in the net synthesis of 2 aTp and 2 naDh molecules per molecule of glucose (see also panel 2–8). Figure 2–47 Two pathways for the anaerobic breakdown of pyruvate.

1	Figure 2–47 Two pathways for the anaerobic breakdown of pyruvate. (a) When there is inadequate oxygen, for example, in a muscle cell undergoing vigorous contraction, the pyruvate produced by glycolysis is converted to lactate as shown. This reaction regenerates the naD+ consumed in step 6 of glycolysis, but the whole pathway yields much less energy overall than complete oxidation. (B) in some organisms that can grow anaerobically, such as yeasts, pyruvate is C regeneration C converted via acetaldehyde into carbon dioxide and ethanol. again, this pathway CO HCOH regenerates naD+ from naDh, as required to enable glycolysis to continue. Both (a) and (B) are examples of fermentations.

1	fermentations. Studies of the commercially important fermentations carried out by yeasts inspired much of early biochemistry. Work in the nineteenth century led in 1896 to the then startling recognition that these processes could be studied outside living organisms, in cell extracts. This revolutionary discovery eventually made it possible to dissect out and study each of the individual reactions in the fermentation process. The piecing together of the complete glycolytic pathway in the 1930s was a major triumph of biochemistry, and it was quickly followed by the recognition of the central role of ATP in cell processes. Glycolysis illustrates how enzymes Couple oxidation to energy storage

1	Glycolysis illustrates how enzymes Couple oxidation to energy storage The formation of ATP during glycolysis provides a particularly clear demonstration of how enzymes couple energetically unfavorable reactions with favorable ones, thereby driving the many chemical reactions that make life possible. Two central reactions in glycolysis (steps 6 and 7) convert the three-carbon sugar intermediate glyceraldehyde 3-phosphate (an aldehyde) into 3-phosphoglycerate (a carboxylic acid; see Panel 2–8, pp. 104–105), thus oxidizing an aldehyde group to a carboxylic acid group. The overall reaction releases enough free energy to convert a molecule of ADP to ATP and to transfer two electrons (and a proton) from the aldehyde to NAD+ to form NADH, while still liberating enough heat to the environment to make the overall reaction energetically favorable (∆G° for the overall reaction is –12.5 kJ/mole).

1	Figure 2–48 outlines this remarkable feat of energy harvesting. The chemical reactions are precisely guided by two enzymes to which the sugar intermediates A short-lived covalent bond is formed between glyceraldehyde 3-phosphate and the –SH group of a cysteine side chain of the enzyme glyceraldehyde 3-phosphate dehydrogenase. The enzyme also binds noncovalently to NAD+. Glyceraldehyde 3-phosphate is oxidized as the enzyme removes a hydrogen atom (yellow) and transfers it, along with an electron, to NAD+, forming NADH (see Figure 2–37). Part of the energy + H+ released by the oxidation of the aldehyde is thus stored in NADH, and part is stored in the high- glyceraldehyde 3-phosphate to the CO enzyme. A molecule of inorganic phosphatehigh-energy inorganic displaces the high-energy thioesterphosphate phosphate bond to create 1,3-bisphospho-glycerate, which contains a high-energy phosphate bond. 1,3-bisphosphoglycerate

1	1,3-bisphosphoglycerate The high-energy phosphate group is transferred to ADP to form ATP. The oxidation of an aldehyde to a carboxylic acid releases energy, much of which is captured in the activated carriers ATP and NADH.

1	The oxidation of an aldehyde to a carboxylic acid releases energy, much of which is captured in the activated carriers ATP and NADH. Figure 2–48 energy storage in steps 6 and 7 of glycolysis. (a) in step 6, the enzyme glyceraldehyde 3-phosphate dehydrogenase couples the energetically favorable oxidation of an aldehyde to the energetically unfavorable formation of a high-energy phosphate bond. at the same time, it enables energy to be stored in naDh. The formation of the high-energy phosphate bond is driven by the oxidation reaction, and the enzyme thereby acts like the “paddle wheel” coupler in figure 2–32B. in step 7, the newly formed high-energy phosphate bond in 1,3-bisphosphoglycerate is transferred to aDp, forming a molecule of aTp and leaving a free carboxylic acid group on the oxidized sugar. The part of the molecule that undergoes a change is shaded in blue; the rest of the molecule remains unchanged throughout all these reactions.

1	(B) summary of the overall chemical change produced by reactions 6 and 7. are tightly bound. As detailed in Figure 2–48, the first enzyme (glyceraldehyde 3-phosphate dehydrogenase) forms a short-lived covalent bond to the aldehyde through a reactive –SH group on the enzyme, and catalyzes its oxidation by NAD+ in this attached state. The reactive enzyme–substrate bond is then displaced by an inorganic phosphate ion to produce a high-energy phosphate intermediate, which is released from the enzyme. This intermediate binds to the second enzyme (phosphoglycerate kinase), which catalyzes the energetically favorable transfer of the high-energy phosphate just created to ADP, forming ATP and completing the process of oxidizing an aldehyde to a carboxylic acid. Note that the C–H bond oxidation energy in step 6 drives the formation of both NADH and a high-energy phosphate bond. The breakage of the high-energy bond then drives ATP formation.

1	We have shown this particular oxidation process in some detail because it provides a clear example of enzyme-mediated energy storage through coupled reactions (Figure 2–49). Steps 6 and 7 are the only reactions in glycolysis that create a high-energy phosphate linkage directly from inorganic phosphate. As such, they account for the net yield of two ATP molecules and two NADH molecules per molecule of glucose (see Panel 2–8, pp. 104–105). As we have just seen, ATP can be formed readily from ADP when a reaction intermediate is formed with a phosphate bond of higher energy than the terminal phosphate bond in ATP. Phosphate bonds can be ordered in energy by comparing the standard free-energy change (∆G°) for the breakage of each bond by hydrolysis. Figure 2–50 compares the high-energy phosphoanhydride bonds in ATP with the energy of some other phosphate bonds, several of which are generated during glycolysis.

1	All organisms need to maintain a high ATP/ADP ratio to maintain biological order in their cells. Yet animals have only periodic access to food, and plants need to survive overnight without sunlight, when they are unable to produce sugar from photosynthesis. For this reason, both plants and animals convert sugars and fats to special forms for storage (Figure 2–51). To compensate for long periods of fasting, animals store fatty acids as fat droplets composed of water-insoluble triacylglycerols (also called triglycerides). The triacylglycerols in animals are mostly stored in the cytoplasm of specialized fat cells called adipocytes. For shorter-term storage, sugar is stored as glucose 1,3-bisphosphoglycerate formation of hydrolysis of high-energy bond high-energy bond TOTAL ENERGY CHANGE for step 6 followed by step 7 is a favorable –12.5 kJ/mole

1	TOTAL ENERGY CHANGE for step 6 followed by step 7 is a favorable –12.5 kJ/mole Figure 2–49 Schematic view of the coupled reactions that form NaDH and aTP in steps 6 and 7 of glycolysis. The C–h bond oxidation energy drives the formation of both naDh and a high-energy phosphate bond. The breakage of the high-energy bond then drives aTp formation. bond to carbon anhydride bond to phosphate type of phosphate bond –61.9 kJ (see Panel 2–8, pp. 104–105) for example, 1,3-bisphosphoglycerate –49.0 kJ (see Panel 2–8) –40 creatine phosphate (activated carrier that –43.0 kJ stores energy in muscle) for example, ATP when hydrolyzed –30.6 kJ to ADP –20 for example, glucose 6-phosphate –17.5 kJ (see Panel 2–8) specifc examples showing the standard free-energy change (˜G°) for hydrolysis of phosphate bond

1	Figure 2–50 Phosphate bonds have different energies. examples of different types of phosphate bonds with their sites of hydrolysis are shown in the molecules depicted on the left. Those starting with a gray carbon atom show only part of a molecule. examples of molecules containing such bonds are given on the right, with the standard free-energy change for hydrolysis in kilojoules. The transfer of a phosphate group from one molecule to another is energetically favorable if the free-energy change (ΔG) for hydrolysis of the phosphate bond of the first molecule is more negative than that for hydrolysis of the phosphate bond in the second. Thus, under standard conditions, a phosphate group is readily transferred from 1,3-bisphosphoglycerate to aDp to form aTp. (standard conditions often do not pertain to living cells, where the relative concentrations of reactants and products will influence the actual change in free energy.) The hydrolysis reaction can be viewed as the transfer of the

1	not pertain to living cells, where the relative concentrations of reactants and products will influence the actual change in free energy.) The hydrolysis reaction can be viewed as the transfer of the phosphate group to water.

1	subunits in the large branched polysaccharide glycogen, which is present as small granules in the cytoplasm of many cells, including liver and muscle. The synthesis and degradation of glycogen are rapidly regulated according to need. When cells need more ATP than they can generate from the food molecules taken in from the bloodstream, they break down glycogen in a reaction that produces glucose 1-phosphate, which is rapidly converted to glucose 6-phosphate for glycolysis (Figure 2–52). Quantitatively, fat is far more important than glycogen as an energy store for animals, presumably because it provides for more efficient storage. The oxidation of a gram of fat releases about twice as much energy as the oxidation of a gram of glycogen. Moreover, glycogen differs from fat in binding a great deal of water, producing a sixfold difference in the actual mass of glycogen required to store the same amount of energy as fat. An average adult human stores enough glycogen

1	Figure 2–51 The storage of sugars and fats in animal and plant cells. (a) The structures of starch and glycogen, the storage form of sugars in plants and animals, respectively. Both are storage polymers of the sugar glucose and differ only in the frequency of branch points. There are many more branches in glycogen than in starch. (B) an electron micrograph of glycogen granules in the cytoplasm of a liver cell. (C) a thin section of a chloroplast from a plant cell, showing the starch granules and lipid (fat droplets) that have accumulated as a result of the biosyntheses occurring there. (D) fat droplets (stained red) beginning to accumulate in developing fat cells of an animal. (B, courtesy of Robert fletterick and Daniel s. friend; C, courtesy of K. plaskitt; D, courtesy of Ronald m. evans and peter Totonoz.) for only about a day of normal activities, but enough fat to last for nearly a month. If our main fuel reservoir had to be carried as glycogen instead of fat, body weight would

1	and peter Totonoz.) for only about a day of normal activities, but enough fat to last for nearly a month. If our main fuel reservoir had to be carried as glycogen instead of fat, body weight would increase by an average of about 60 pounds.

1	The sugar and ATP needed by plant cells are largely produced in separate organelles: sugars in chloroplasts (the organelles specialized for photosynthesis), 2 Figure 2–52 How sugars are produced from glycogen. Glucose subunits are released from glycogen by the enzyme glycogen phosphorylase. This produces glucose 1-phosphate, which is rapidly converted to glucose 6-phosphate for OH glycolysis. Figure 2–53 Some plant seeds that serve as important foods for humans.

1	Corn, nuts, and peas all contain rich stores of starch and fat that provide the young plant embryo in the seed with energy and building blocks for biosynthesis. (Courtesy of the John innes foundation.) and ATP in mitochondria. Although plants produce abundant amounts of both ATP and NADPH in their chloroplasts, this organelle is isolated from the rest of its plant cell by a membrane that is impermeable to both types of activated carrier molecules. Moreover, the plant contains many cells—such as those in the roots— that lack chloroplasts and therefore cannot produce their own sugars. Thus, sugars are exported from chloroplasts to the mitochondria present in all cells of the plant. Most of the ATP needed for general plant cell metabolism is synthesized in these mitochondria, using exactly the same pathways for the oxidative breakdown of sugars as in nonphotosynthetic organisms; this ATP is then passed to the rest of the cell (see Figure 14–42).

1	During periods of excess photosynthetic capacity during the day, chloroplasts convert some of the sugars that they make into fats and into starch, a polymer of glucose analogous to the glycogen of animals. The fats in plants are triacyl-glycerols (triglycerides), just like the fats in animals, and differ only in the types of fatty acids that predominate. Fat and starch are both stored inside the chloroplast until needed for energy-yielding oxidation during periods of darkness (see Figure 2–51C). The embryos inside plant seeds must live on stored sources of energy for a prolonged period, until they germinate and produce leaves that can harvest the energy in sunlight. For this reason plant seeds often contain especially large amounts of fats and starch—which makes them a major food source for animals, including ourselves (Figure 2–53).

1	After a meal, most of the energy that an animal needs is derived from sugars obtained from food. Excess sugars, if any, are used to replenish depleted glycogen stores, or to synthesize fats as a food store. But soon the fat stored in adipose tissue is called into play, and by the morning after an overnight fast, fatty acid oxidation generates most of the ATP we need. Low glucose levels in the blood trigger the breakdown of fats for energy production. As illustrated in Figure 2–54, the triacylglycerols stored in fat droplets in adipocytes are hydrolyzed to produce fatty acids and glycerol, and the fatty acids released are transferred to cells in the body through the bloodstream. While animals readily convert sugars to fats, they cannot convert fatty acids to sugars. Instead, the fatty acids are oxidized directly. sugars and fats are Both Degraded to acetyl Coa in mitochondria

1	sugars and fats are Both Degraded to acetyl Coa in mitochondria In aerobic metabolism, the pyruvate that was produced by glycolysis from sugars in the cytosol is transported into the mitochondria of eukaryotic cells. There, it is rapidly decarboxylated by a giant complex of three enzymes, called the pyruvate dehydrogenase complex. The products of pyruvate decarboxylation are a molecule of CO2 (a waste product), a molecule of NADH, and acetyl CoA (see Panel 2–9). The fatty acids imported from the bloodstream are moved into mitochondria, where all of their oxidation takes place (Figure 2–55). Each molecule of fatty acid (as the activated molecule fatty acyl CoA) is broken down completely by a cycle of reactions that trims two carbons at a time from its carboxyl end, generating one molecule of acetyl CoA for each turn of the cycle. A molecule of NADH and a molecule of FADH2 are also produced in this process (Figure 2–56).

1	Sugars and fats are the major energy sources for most nonphotosynthetic organisms, including humans. However, most of the useful energy that can be extracted from the oxidation of both types of foodstuffs remains stored in the acetyl CoA molecules that are produced by the two types of reactions just described. The citric acid cycle of reactions, in which the acetyl group (–COCH3) in acetyl CoA is oxidized to CO2 and H2O, is therefore central to the energy metabolism of aerobic organisms. In eukaryotes, these reactions all take place in mitochondria. We should therefore not be surprised to discover that the mitochondrion is the place where most of the ATP is produced in animal cells. In contrast, aerobic bacteria carry out all of their reactions, including the citric acid cycle, in a single compartment, the cytosol. The Citric acid Cycle Generates naDh by oxidizing acetyl Groups to Co2

1	The Citric acid Cycle Generates naDh by oxidizing acetyl Groups to Co2 In the nineteenth century, biologists noticed that in the absence of air cells produce lactic acid (for example, in muscle) or ethanol (for example, in yeast), while in its presence they consume O2 and produce CO2 and H2O. Efforts to define the pathways of aerobic metabolism eventually focused on the oxidation of pyruvate and led in 1937 to the discovery of the citric acid cycle, also known as the

1	Figure 2–54 How stored fats are mobilized for energy production in animals. low glucose levels in the blood trigger the hydrolysis of the triacylglycerol molecules in fat droplets to free fatty acids and glycerol. These fatty acids enter the bloodstream, where they bind to the abundant blood protein, serum albumin. special fatty acid transporters in the plasma membrane of cells that oxidize fatty acids, such as muscle cells, then pass these fatty acids into the cytosol, from which they are moved into mitochondria for energy production.

1	Figure 2–55 Pathways for the production of acetyl Coa from sugars and fats. The mitochondrion in eukaryotic cells is where acetyl Coa is produced from both types of major food molecules. it is therefore the place where most of the cell’s oxidation reactions occur and where most of its aTp is made. amino acids (not shown) can also enter the mitochondria, to be converted there into acetyl Coa or another intermediate of the citric acid cycle. The structure and function of mitochondria are discussed in detail in Chapter 14.

1	tricarboxylic acid cycle or the Krebs cycle. The citric acid cycle accounts for about two-thirds of the total oxidation of carbon compounds in most cells, and its major end products are CO2 and high-energy electrons in the form of NADH. The CO2 is released as a waste product, while the high-energy electrons from NADH are passed to a membrane-bound electron-transport chain (discussed in Chapter 14), eventually combining with O2 to produce H2O. The citric acid cycle itself does not use gaseous O2 (it uses oxygen atoms from H2O). But the cycle does require O2 in subsequent reactions to keep it going. This is because there is no other efficient way for the NADH to get rid of its electrons and thus regenerate the NAD+ that is needed.

1	The citric acid cycle takes place inside mitochondria in eukaryotic cells. It results in the complete oxidation of the carbon atoms of the acetyl groups in acetyl CoA, converting them into CO2. But the acetyl group is not oxidized directly. Instead, this group is transferred from acetyl CoA to a larger, four-carbon molecule, oxaloacetate, to form the six-carbon tricarboxylic acid, citric acid, for which the subsequent cycle of reactions is named. The citric acid molecule is then gradually oxidized, allowing the energy of this oxidation to be harnessed to produce energy-rich activated carrier molecules. The chain of eight reactions forms a cycle because at the end the oxaloacetate is regenerated and enters a new turn of the cycle, as shown in outline in Figure 2–57.

1	We have thus far discussed only one of the three types of activated carrier molecules that are produced by the citric acid cycle; NADH, the reduced form of the NAD+/NADH electron carrier system (see Figure 2–36). In addition to three molecules of NADH, each turn of the cycle also produces one molecule of FADH2 (reduced flavin adenine dinucleotide) from FAD (see Figure 2–39), and one molecule of the ribonucleoside triphosphate GTP from GDP. The structure of GTP is illustrated in Figure 2–58. GTP is a close relative of ATP, and the transfer of its terminal phosphate group to ADP produces one ATP molecule in each cycle. As we discuss shortly, the energy that is stored in the readily transferred electrons of NADH and FADH2 will be utilized subsequently for ATP production through the rest of hydrocarbon tail fatty acyl CoA shortened by two carbons

1	Figure 2–56 The oxidation of fatty acids to acetyl Coa. (a) electron micrograph of a lipid droplet in the cytoplasm. (B) The structure of fats. fats are triacylglycerols. The glycerol portion, to which three fatty acids are linked through ester bonds, is shown in blue. fats are insoluble in water and form large lipid droplets in the specialized fat cells (adipocytes) in which they are stored. (C) The fatty acid oxidation cycle. The cycle is catalyzed by a series of four enzymes in mitochondria. each turn of the cycle shortens the fatty acid chain by two carbons (shown in red) and generates one molecule of acetyl Coa and one molecule each of naDh and faDh2. (a, courtesy of Daniel s. friend.)

1	NET RESULT: ONE TURN OF THE CYCLE PRODUCES THREE NADH, ONE GTP, AND ONE FADH2 MOLECULE, AND RELEASES TWO MOLECULES OF CO2 process of oxidative phosphorylation, the only step in the oxidative catabolism of foodstuffs that directly requires gaseous oxygen (O2) from the atmosphere. Panel 2–9 (pp. 106–107) and Movie 2.6 present the complete citric acid cycle. Water, rather than molecular oxygen, supplies the extra oxygen atoms required to make CO2 from the acetyl groups entering the citric acid cycle. As illustrated in the panel, three molecules of water are split in each cycle, and the oxygen atoms of some of them are ultimately used to make CO2.

1	In addition to pyruvate and fatty acids, some amino acids pass from the cytosol into mitochondria, where they are also converted into acetyl CoA or one of the other intermediates of the citric acid cycle. Thus, in the eukaryotic cell, the mitochondrion is the center toward which all energy-yielding processes lead, whether they begin with sugars, fats, or proteins. Both the citric acid cycle and glycolysis also function as starting points for important biosynthetic reactions by producing vital carbon-containing intermediates, such as oxaloacetate and α-ketoglutarate. Some of these substances produced by catabolism are transferred back from the mitochondria to the cytosol, where they serve in anabolic reactions as precursors for the synthesis of many essential molecules, such as amino acids (Figure 2–59). electron Transport Drives the synthesis of the majority of the aTp in most Cells

1	Most chemical energy is released in the last stage in the degradation of a food molecule. In this final process, NADH and FADH2 transfer the electrons that they gained when oxidizing food-derived organic molecules to the electron-transport chain, which is embedded in the inner membrane of the mitochondrion (see Figure 14–10). As the electrons pass along this long chain of specialized electron acceptor and donor molecules, they fall to successively lower energy states. The energy that the electrons release in this process pumps H+ ions (protons) across the membrane—from the innermost mitochondrial compartment (the matrix) to the intermembrane space (and then to the cytosol)—generating a gradient of H+ ions (Figure 2–60). This gradient serves as a major source of energy for cells, being tapped like a battery to drive a variety of energy-requiring reactions. The most prominent of these reactions is the generation of ATP by the phosphorylation of ADP.

1	Figure 2–57 Simple overview of the citric acid cycle. The reaction of acetyl Coa with oxaloacetate starts the cycle by producing citrate (citric acid). in each turn of the cycle, two molecules of Co2 are produced as waste products, plus three molecules of naDh, one molecule of GTp, and one molecule of faDh2. The number of carbon atoms in each intermediate is shown in a yellow box. for details, see panel 2–9 (pp. 106–107). O Figure 2–58 The structure of GTP. GTp and GDp are close relatives of aTp and aDp, respectively.

1	O Figure 2–58 The structure of GTP. GTp and GDp are close relatives of aTp and aDp, respectively. At the end of this series of electron transfers, the electrons are passed to molecules of oxygen gas (O2) that have diffused into the mitochondrion, which simultaneously combine with protons (H+) from the surrounding solution to produce water. The electrons have now reached a low energy level, and all the available energy has been extracted from the oxidized food molecule. This process, termed oxidative phosphorylation (Figure 2–61), also occurs in the plasma membrane of bacteria. As one of the most remarkable achievements of cell evolution, it is a central topic of Chapter 14. In total, the complete oxidation of a molecule of glucose to H2O and CO2 is used by the cell to produce about 30 molecules of ATP. In contrast, only 2 molecules of ATP are produced per molecule of glucose by glycolysis alone. amino acids and nucleotides are part of the nitrogen Cycle

1	amino acids and nucleotides are part of the nitrogen Cycle So far we have concentrated mainly on carbohydrate metabolism and have not yet considered the metabolism of nitrogen or sulfur. These two elements are important constituents of biological macromolecules. Nitrogen and sulfur atoms pass Figure 2–59 Glycolysis and the citric acid cycle provide the precursors needed to synthesize many important biological molecules. The amino acids, nucleotides, lipids, sugars, and other molecules—shown here as products—in turn serve as the precursors for the many macromolecules of the cell. each black arrow in this diagram denotes a single enzyme-catalyzed reaction; the red arrows generally represent pathways with many steps that are required to produce the indicated products. from compound to compound and between organisms and their environment in a series of reversible cycles.

1	from compound to compound and between organisms and their environment in a series of reversible cycles. Although molecular nitrogen is abundant in the Earth’s atmosphere, nitrogen is chemically unreactive as a gas. Only a few living species are able to incorporate it into organic molecules, a process called nitrogen fixation. Nitrogen fixation occurs in certain microorganisms and by some geophysical processes, such as lightning discharge. It is essential to the biosphere as a whole, for without it life could not exist on this planet. Only a small fraction of the nitrogenous compounds in today’s organisms, however, is due to fresh products of nitrogen fixation from the atmosphere. Most organic nitrogen has been in circulation for some time, passing from one living organism to another. Thus, present-day nitrogen-fixing reactions can be said to perform a “topping-up” function for the total nitrogen supply.

1	Vertebrates receive virtually all of their nitrogen from their dietary intake of proteins and nucleic acids. In the body, these macromolecules are broken down to amino acids and the components of nucleotides, and the nitrogen they contain is used to produce new proteins and nucleic acids—or other molecules. About half of the 20 amino acids found in proteins are essential amino acids for vertebrates (Figure 2–62), which means that they cannot be synthesized from other ingredients of the diet. The other amino acids can be so synthesized, using a variety of raw materials, including intermediates of the citric acid cycle. The essential amino acids are made by plants and other organisms, usually by long and energetically expensive pathways that have been lost in the course of vertebrate evolution.

1	The nucleotides needed to make RNA and DNA can be synthesized using specialized biosynthetic pathways. All of the nitrogens in the purine and pyrimidine bases (as well as some of the carbons) are derived from the plentiful amino acids glutamine, aspartic acid, and glycine, whereas the ribose and deoxyribose sugars are derived from glucose. There are no “essential nucleotides” that must be provided in the diet. Amino acids not used in biosynthesis can be oxidized to generate metabolic energy. Most of their carbon and hydrogen atoms eventually form CO2 or H2O, whereas their nitrogen atoms are shuttled through various forms and eventually appear as urea, which is excreted. Each amino acid is processed differently, and a whole constellation of enzymatic reactions exists for their catabolism.

1	Sulfur is abundant on Earth in its most oxidized form, sulfate (SO42–). To be useful for life, sulfate must be reduced to sulfide (S2–), the oxidation state of sulfur required for the synthesis of essential biological molecules, including the amino acids methionine and cysteine, coenzyme A (see Figure 2–39), and the iron-sulfur centers essential for electron transport (see Figure 14–16). The sulfur-reduction process begins in bacteria, fungi, and plants, where a special group of enzymes use ATP and reducing power to create a sulfate assimilation pathway. Humans and other animals cannot reduce sulfate and must therefore acquire the sulfur they need for their metabolism in the food that they eat.

1	Figure 2–60 The generation of an H+ gradient across a membrane by electron-transport reactions. an electron held in a high-energy state (derived, for example, from the oxidation of a metabolite) is passed sequentially by carriers a, B, and C to a lower energy state. in this diagram, carrier B is arranged in the membrane in such a way that it takes up h+ from one side and releases it to the other as the electron passes. The result is an h+ gradient. as discussed in Chapter 14, this gradient is an important form of energy that is harnessed by other membrane proteins to drive the formation of aTp (for an actual example, see figure 14–21).

1	Figure 2–61 The final stages of oxidation of food molecules. molecules of naDh and faDh2 (faDh2 is not shown) are produced by the citric acid cycle. These activated carriers donate high-energy electrons that are eventually used to reduce oxygen gas to water. a major portion of the energy released during the transfer of these electrons along an electron-transfer chain in the mitochondrial inner membrane (or in the plasma membrane of bacteria) is harnessed to drive the synthesis of aTp— hence the name oxidative phosphorylation (discussed in Chapter 14). metabolism is highly organized and Regulated One gets a sense of the intricacy of a cell as a chemical machine from the relation of glycolysis and the citric acid cycle to the other metabolic pathways sketched out in Figure 2–63. This chart represents only some of the enzymatic pathways in a human cell. It is obvious that our discussion of cell metabolism has dealt with only a tiny fraction of the broad field of cell chemistry.

1	All these reactions occur in a cell that is less than 0.1 mm in diameter, and each requires a different enzyme. As is clear from Figure 2–63, the same molecule can often be part of many different pathways. Pyruvate, for example, is a substrate for half a dozen or more different enzymes, each of which modifies it chemically in a different way. One enzyme converts pyruvate to acetyl CoA, another to oxaloacetate; a third enzyme changes pyruvate to the amino acid alanine, a fourth to lactate, and so on. All of these different pathways compete for the same pyruvate molecule, and similar competitions for thousands of other small molecules go on at the same time.

1	The situation is further complicated in a multicellular organism. Different cell types will in general require somewhat different sets of enzymes. And different tissues make distinct contributions to the chemistry of the organism as a whole. In addition to differences in specialized products such as hormones or antibodies, there are significant differences in the “common” metabolic pathways among various types of cells in the same organism. Although virtually all cells contain the enzymes of glycolysis, the citric acid cycle, lipid synthesis and breakdown, and amino acid metabolism, the levels of these processes required in different tissues are not the same. For example, nerve cells, which are probably the most fastidious cells in the body, maintain almost no reserves of glycogen or fatty acids and rely almost entirely on a constant Figure 2–62 The nine essential amino acids. These cannot be synthesized by human cells and so must be supplied in the diet.

1	Figure 2–63 Glycolysis and the citric acid cycle are at the center of an elaborate set of metabolic pathways in human cells. some 2000 metabolic reactions are shown schematically with the reactions of glycolysis and the citric acid cycle in red. many other reactions either lead into these two central pathways—delivering small molecules to be catabolized with production of energy—or they lead outward and thereby supply carbon compounds for the purpose of biosynthesis. (adapted with permission from Kanehisa laboratories.) supply of glucose from the bloodstream. In contrast, liver cells supply glucose to WhaT We Don’T KnoW actively contracting muscle cells and recycle the lactic acid produced by muscle cells back into glucose. All types of cells have their distinctive metabolic traits, and • Did chemiosmosis precede they cooperate extensively in the normal state, as well as in response to stress and fermentation as the source of starvation. One might think that the whole system would

1	• Did chemiosmosis precede they cooperate extensively in the normal state, as well as in response to stress and fermentation as the source of starvation. One might think that the whole system would need to be so finely bal-biological energy, or did some form of anced that any minor upset, such as a temporary change in dietary intake, would fermentation come first, as had been be disastrous. assumed for many years? In fact, the metabolic balance of a cell is amazingly stable. Whenever the balance is perturbed, the cell reacts so as to restore the initial state. The cell can adapt • What is the minimum number of and continue to function during starvation or disease. Mutations of many kinds components required to make a living can damage or even eliminate particular reaction pathways, and yet—provided cell from scratch? how might we find out?

1	that certain minimum requirements are met—the cell survives. It does so because an elaborate network of control mechanisms regulates and coordinates the rates of all of its reactions. These controls rest, ultimately, on the remarkable abilities • are other life chemistries possible of proteins to change their shape and their chemistry in response to changes in besides the single one known on earth (and described in this chapter)? When their immediate environment. The principles that underlie how large molecules screening for life on other planets, such as proteins are built and the chemistry behind their regulation will be our what type of chemical signatures next concern. should we search for?

1	should we search for? • is the shared chemistry inside all Glucose and other food molecules are broken down by controlled stepwise oxidation living cells a clue for deciphering the to provide chemical energy in the form of ATP and NADH. There are three main sets environment on earth where the first of reactions that act in series, the products of each being the starting material for the cells originated? for example, what next: glycolysis (which occurs in the cytosol), the citric acid cycle (in the mitochon-might we conclude from the universally shared high K+/na+ ratio, neutral ph, drial matrix), and oxidative phosphorylation (on the inner mitochondrial mem and central role of phosphates?

1	brane). The intermediate products of glycolysis and the citric acid cycle are used both as sources of metabolic energy and to produce many of the small molecules used as the raw materials for biosynthesis. Cells store sugar molecules as glycogen in animals and starch in plants; both plants and animals also use fats extensively as a food store. These storage materials in turn serve as a major source of food for humans, along with the proteins that comprise the majority of the dry mass of most of the cells in the foods we eat. Which statements are true? explain why or why not. Discuss the following problems. 2–1 A 10–8 M solution of HCl has a pH of 8. 2–8 The organic chemistry of living cells is said to be special for two reasons: it occurs in an aqueous environ2–2 Most of the interactions between macromolecules ment and it accomplishes some very complex reactions. could be mediated just as well by covalent bonds as by

1	But do you suppose it is really all that much different from noncovalent bonds. the organic chemistry carried out in the top laboratories in the world? Why or why not? 2–3 Animals and plants use oxidation to extract energy from food molecules. 2–9 The molecular weight of ethanol (CH3CH2OH) is 46 and its density is 0.789 g/cm3. 2–4 If an oxidation occurs in a reaction, it must be a. What is the molarity of ethanol in beer that is 5% accompanied by a reduction. ethanol by volume? [Alcohol content of beer varies from about 4% (lite beer) to 8% (stout beer).] 2–5 Linking the energetically unfavorable reaction A b. The legal limit for a driver’s blood alcohol content → B to a second, favorable reaction B → C will shift the varies, but 80 mg of ethanol per 100 mL of blood (usually equilibrium constant for the first reaction.

1	referred to as a blood alcohol level of 0.08) is typical. What 2–6 The criterion for whether a reaction proceeds is the molarity of ethanol in a person at this legal limit? spontaneously is ΔG not ΔG°, because ΔG takes into C. How many 12-oz (355-mL) bottles of 5% beer could account the concentrations of the substrates and products. a 70-kg person drink and remain under the legal limit? A 70-kg person contains about 40 liters of water. Ignore the 2–7 The oxygen consumed during the oxidation of glu-metabolism of ethanol, and assume that the water content cose in animal cells is returned as CO2 to the atmosphere. of the person remains constant. activity (% of maximum) D. Ethanol is metabolized at a constant rate of about 120 mg per hour per kg body weight, regardless of its concentration. If a 70-kg person were at twice the legal limit (160 mg/100 mL), how long would it take for their blood alcohol level to fall below the legal limit?

1	2–10 A histidine side chain is known to play an important role in the catalytic mechanism of an enzyme; however, it is not clear whether histidine is required in its protonated (charged) or unprotonated (uncharged) state. To answer this question you measure enzyme activity over a range of pH, with the results shown in Figure Q2–1. Which form of histidine is required for enzyme activity? activity as a function of ph (problem 2–10). 2–11 The three molecules in Figure Q2–2 contain the seven most common reactive groups in biology. Most molecules in the cell are built from these functional groups. Indicate and name the functional groups in these molecules. O Figure Q2–2 Three molecules that illustrate the seven most common functional groups in biology (problem 2–11). 1,3-Bisphosphoglycerate and O pyruvate are intermediates in glycolysis and cysteine is an amino acid.

1	O pyruvate are intermediates in glycolysis and cysteine is an amino acid. 1,3-bisphosphoglycerate pyruvate cysteine 2–12 “Diffusion” sounds slow—and over everyday distances it is—but on the scale of a cell it is very fast. The average instantaneous velocity of a particle in solution—that is, the velocity between the very frequent collisions—is where k = 1.38 × 10–16 g cm2/K sec2, T = temperature in K (37°C is 310 K), and m = mass in g/molecule. Calculate the instantaneous velocity of a water molecule (molecular mass = 18 daltons), a glucose molecule (molecular mass = 180 daltons), and a myoglobin molecule (molecular mass = 15,000 daltons) at 37°C. Just for fun, convert these numbers into kilometers/hour. Before you do any calculations, try to guess whether the molecules are moving at a slow crawl (<1 km/hr), an easy walk (5 km/hr), or a record-setting sprint (40 km/hr).

1	Before you do any calculations, try to guess whether the molecules are moving at a slow crawl (<1 km/hr), an easy walk (5 km/hr), or a record-setting sprint (40 km/hr). 2–13 Polymerization of tubulin subunits into microtubules occurs with an increase in the orderliness of the subunits. Yet tubulin polymerization occurs with an increase in entropy (decrease in order). How can that be? 2–14 A 70-kg adult human (154 lb) could meet his or her entire energy needs for one day by eating 3 moles of glucose (540 g). (We do not recommend this.) Each molecule of glucose generates 30 molecules of ATP when it is oxidized to CO2. The concentration of ATP is maintained in cells at about 2 mM, and a 70-kg adult has about 25 liters of intracellular fluid. Given that the ATP concentration remains constant in cells, calculate how many times per day, on average, each ATP molecule in the body is hydrolyzed and resynthesized.

1	2–15 Assuming that there are 5 × 1013 cells in the human body and that ATP is turning over at a rate of 109 ATP molecules per minute in each cell, how many watts is the human body consuming? (A watt is a joule per second.) Assume that hydrolysis of ATP yields 50 kJ/mole. 2–16 Does a Snickers™ candy bar (65 g, 1360 kJ) provide enough energy to climb from Zermatt (elevation 1660 m) to the top of the Matterhorn (4478 m, Figure Q2–3), or might you need to stop at HÖrnli Hut (3260 m) to eat another one? Imagine that you and your gear have a mass of 75 kg, and that all of your work is done against gravity (that is, you are just climbing straight up). Remember from your introductory physics course that where g is acceleration due to gravity (9.8 m/sec2). One joule is 1 kg m2/sec2. What assumptions made here will greatly underestimate how much candy you need?

1	What assumptions made here will greatly underestimate how much candy you need? Figure Q2–3 The matterhorn (problem 2–16). (Courtesy of Zermatt Tourism.) 2–17 In the absence of oxygen, cells consume glucose at a high, steady rate. When oxygen is added, glucose consumption drops precipitously and is then maintained at the lower rate. Why is glucose consumed at a high rate in the absence of oxygen and at a low rate in its presence? PaNel 2–1: Chemical bonds and Groups Commonly encountered in biological Molecules Carbon has a unique role in the cell because of its ability to form strong covalent bonds with other branched trees ringscarbon atoms. Thus carbon atoms can join to form: also written as also written as also written as

1	A covalent bond forms when two atoms come very close together and share one or more of their electrons. In a single Carbon and hydrogen combinebond, one electron from each of the two atoms is shared; in together to make stablea double bond, a total of four electrons are shared. compounds (or chemical groups)Each atom forms a fxed number of covalent bonds in a called hydrocarbons. These aredefned spatial arrangement. For example, carbon forms four nonpolar, do not formsingle bonds arranged tetrahedrally, whereas nitrogen forms hydrogen bonds, and arethree single bonds and oxygen forms two single bonds arranged generally insoluble in water. as shown below. cannot rotate freely HH around the bond axis. This restriction is a Double bonds exist and have a different spatial arrangement. major infuence on the three-dimensional shape of many macromolecules. methane methyl group

1	This restriction is a Double bonds exist and have a different spatial arrangement. major infuence on the three-dimensional shape of many macromolecules. methane methyl group The carbon chain can include double Alternating double bonds in a ring bonds. If these are on alternate carbon can generate a very stable structure. CH2 atoms, the bonding electrons move within the molecule, stabilizing the structure by a phenomenon called CH2 resonance. HH H HH H the truth is somewhere between H2C these two structures HH often written as part of the hydrocarbon “tail” of a fatty acid molecule Many biological compounds contain a carbon Amines and amides are two important examples ofbonded to an oxygen. For example, compounds containing a carbon linked to a nitrogen. alcohol H Amines in water combine with an H+ ion to become The –OH is called a positively charged. hydroxyl group. Amides are formed by combining an acid and an

1	alcohol H Amines in water combine with an H+ ion to become The –OH is called a positively charged. hydroxyl group. Amides are formed by combining an acid and an The C—O is called aamine. Unlike amines, amides are uncharged in water. carbonyl group. ketone C An example is the peptide bond that joins amino acids in a protein. carboxylic acid O The –COOH is called a C carboxyl group. In water H this loses an H+ ion to OH Nitrogen also occurs in several ring compounds, including become –COO . important constituents of nucleic acids: purines and pyrimidines. esters Esters are formed by a condensation reaction between an acid and an alcohol. SULFHYDRYL GROUP The C SH is called a sulfhydryl group. In the amino acid cysteine, the sulfhydryl group may exist in the reduced form, C SH or more rarely in an oxidized, cross-bridging form, CSSC

1	SULFHYDRYL GROUP The C SH is called a sulfhydryl group. In the amino acid cysteine, the sulfhydryl group may exist in the reduced form, C SH or more rarely in an oxidized, cross-bridging form, CSSC PHOSPHATES Inorganic phosphate is a stable ion formed from Phosphate esters can form between a phosphate and a free hydroxyl group. phosphoric acid, H3PO4. It is also written as Pi . Phosphate groups are often attached to proteins in this way. also _ __ written as HOPO C OHHOPO C OPO H2O The combination of a phosphate and a carboxyl group, or two or more phosphate groups, gives an acid anhydride. Because compounds of this kind are easily hydrolysed in the cell, they are sometimes said to contain a “high-energy” bond. _ also written as __ also written as molecules such as ATP PaNel 2–2: Water and Its Influence on the behavior of biological Molecules

1	_ also written as __ also written as molecules such as ATP PaNel 2–2: Water and Its Influence on the behavior of biological Molecules Two atoms, connected by a covalent bond, may exert different attractions for Molecules of water join together transiently the electrons of the bond. In such cases the bond is polar, with one end in a hydrogen-bonded lattice. Even at 37oC, slightly negatively charged (˜–) and the other slightly positively charged (˜+). 15% of the water molecules are joined to four others in a short-lived assembly known as a “fickering cluster.”

1	Although a water molecule has an overall neutral charge (having the same number of electrons and protons), the electrons are asymmetrically distributed, which makes the molecule polar. The oxygen nucleus draws electrons away The cohesive nature of water is from the hydrogen nuclei, leaving these nuclei with a small net positive charge. responsible for many of its unusual The excess of electron density on the oxygen atom creates weakly negative properties, such as high surface tension, regions at the other two corners of an imaginary tetrahedron. specifc heat, and heat of vaporization. Because they are polarized, two ˜+ adjacent H2O molecules can form H H hydrogen bond a linkage known as a hydrogen H 0.17 nm bond. Hydrogen bonds have only about 1/20 the strength O H O 2˜+ of a covalent bond. H H 0.10 nmHydrogen bonds are strongest when hydrogen bond covalent bondthe three atoms lie in a straight line. ˜+

1	H H 0.10 nmHydrogen bonds are strongest when hydrogen bond covalent bondthe three atoms lie in a straight line. ˜+ Substances that dissolve readily in water are termed hydrophilic. They are Molecules that contain a preponderance composed of ions or polar molecules that attract water molecules through of nonpolar bonds are usually insoluble in electrical charge effects. Water molecules surround each ion or polar molecule water and are termed hydrophobic. This is on the surface of a solid substance and carry it into solution. true, especially, of hydrocarbons, which H contain many C–H bonds. Water molecules are not attracted to such molecules and so O have little tendency to surround them and H OH carry them into solution. Ionic substances such as sodium chloride H dissolve because water molecules are

1	O have little tendency to surround them and H OH carry them into solution. Ionic substances such as sodium chloride H dissolve because water molecules are C attracted to the positive (Na+) or negative Polar substances such as urea HO (Cl_) charge of each ion. dissolve because their molecules HH HH form hydrogen bonds with the OO HH surrounding water molecules. Many substances, such as household sugar, dissolve in water. That is, their molecules separate from each other, each becoming surrounded by water molecules. When a substance dissolves in a liquid, the mixture is termed a solution. The dissolved substance (in this case sugar dissolves sugar) is the solute, and the liquid that does the dissolving (in this case water) is the solvent. Water is an excellent solvent for many substances because of its polar bonds.

1	Many of the acids important in the cell are only partially dissociated, and they are therefore weak acids—for example, the carboxyl group (–COOH), which dissociates to give a hydrogen ion in solution. (weak acid) Note that this is a reversible reaction. The acidity of a solution is defned by the concentration of H+ ions it possesses. For convenience we use the pH scale, where H+ conc. moles/liter Positively charged hydrogen ions (H+) can spontaneously move from one water molecule to another, thereby creating two ionic species. hydronium ion (water acting as a weak base) often written as: H2O hydroxyl ion (water acting as a weak acid) Since the process is rapidly reversible, hydrogen ions are continually shuttling between water molecules. Pure water contains a steady-state concentration of hydrogen ions and hydroxyl ions (both 10–7 M).

1	Substances that reduce the number of hydrogen ions in solution are called bases. Some bases, such as ammonia, combine directly with hydrogen ions. Other bases, such as sodium hydroxide, reduce the number of H+ ions indirectly, by making OH– ions that then combine directly with H+ ions to make H2O. Many bases found in cells are partially associated with H+ ions and are termed weak bases. This is true of compounds that contain an amino group (–NH2), which has a weak tendency to reversibly accept an H+ ion from water, increasing the quantity of free OH– ions. PaNel 2–3: The Principal Types of Weak Noncovalent bonds that Hold Macromolecules Together Organic molecules can interact with other molecules through three types of short-range attractive forces known as noncovalent bonds: van der Waals attractions, electrostatic attractions, and hydrogen bonds. The repulsion of hydrophobic groups from water is also important for the folding of biological macromolecules.

1	Weak noncovalent chemical bonds have less than 1/20 the strength of a strong covalent bond. They are strong enough to provide tight binding only when many of them are formed simultaneously. As already described for water (see Panel 2–2), hydrogen bondsform when a hydrogen atom is (usually oxygen or nitrogen). Hydrogen bonds are strongest when the three atoms are in a straight line: Examples in macromolecules: together. These stabilize the structure of folded proteins. Two bases, G and C, are hydrogen-bonded in a DNA double helix. If two atoms are too close together they repel each other very strongly. For this reason, an atom can often be treated as a sphere with a fxed radius. The characteristic “size” for each atom is specifed by a unique van der Waals radius. The contact distance between any two noncovalently bonded atoms is the sum of their van der Waals radii. 0.12 nm 0.2 nm 0.15 nm 0.14 nm radius radius radius radius

1	0.12 nm 0.2 nm 0.15 nm 0.14 nm radius radius radius radius At very short distances any two atoms show a weak bonding interaction due to their fuctuating electrical charges. The two atoms will be attracted to each other in this way until the distance between their nuclei is approximately equal to the sum of their van der Waals radii. Although they are individually very weak, van der Waals attractions can become important when two macromolecular surfaces ft very close together, because many atoms are involved. Note that when two atoms form a covalent bond, the centers of the two atoms (the two atomic nuclei) are much closer together than the sum of the two van der Waals radii. Thus, 0.4 nm 0.15 nm 0.13 nm Any molecules that can form hydrogen bonds to each other can alternatively form hydrogen bonds to water molecules. Because of this competition with water molecules, the hydrogen bonds formed between two molecules dissolved in water are relatively weak.

1	HYDROPHOBIC FORCES Water forces hydrophobic groups together, because doing so minimizes their disruptive effects on the hydrogen-bonded water network. Hydrophobic groups held H together in this way are sometimes said to be held together by “hydrophobic bonds,” even though the apparent attraction is actually caused by a repulsion from the water. Attractive forces occur both between fully charged groups (ionic bond) and between the partially charged groups on polar molecules. The force of attraction between the two charges, ˜+ and ˜–, falls off rapidly as the distance between the charges increases. In the absence of water, electrostatic forces are very strong. They are responsible for the strength of such minerals as marble and agate, and for crystal formation in common table salt, NaCl. a crystal of salt, NaCl Charged groups are shielded by their interactions with water molecules. Electrostatic attractions are therefore quite weak in water.

1	a crystal of salt, NaCl Charged groups are shielded by their interactions with water molecules. Electrostatic attractions are therefore quite weak in water. Similarly, ions in solution can cluster around charged groups and further weaken these attractions. Despite being weakened by water and salt, electrostatic attractions are very important in biological systems. For example, an enzyme that binds a positively charged substrate will often have a negatively charged amino acid side chain at the appropriate place. PaNel 2–4: an Outline of Some of the Types of Sugars Commonly Found in Cells Monosaccharides usually have the general formula (CH2O) n, where n can be 3, 4, 5, 6, 7, or 8, and have two or more hydroxyl groups. They either contain an aldehyde group ( ) and are called aldoses, or a ketone group ( ) and are called ketoses. MONOSACCHARIDESC O C O H

1	In aqueous solution, the aldehyde or ketone group of a sugar Many monosaccharides differ only in the spatial arrangementmolecule tends to react with a hydroxyl group of the same of atoms—that is, they are isomers. For example, glucose,molecule, thereby closing the molecule into a ring. galactose, and mannose have the same formula (C6H12O6) but differ in the arrangement of groups around one or two carbon atoms. chemical properties of the sugars. But they are recognized by enzymes and other proteins and therefore can have major 5CH2OH has a number. biological effects.

1	˜ AND ° LINKS The hydroxyl group on the carbon that carries the aldehyde or ketone can rapidly change from one position to the other. These two positions are called ˜ and °. As soon as one sugar is linked to another, the ˜ or ° form is frozen. OH O OH O ° hydroxyl ˜ hydroxyl CH2OH NH2 H O OH OH HO glucosamine CH2OH O OH OH HO CH3 O NH C H N-acetylglucosamine C O OH OH OH HO OH glucuronic acid O CH2OH HO O OH OH CH2OH OH HOCH2 HO CH2OH HO O OH OH OH CH2OH OH HOCH2 HO H O + sucrose ˜glucose °fructoseDISACCHARIDES The carbon that carries the aldehyde or the ketone can react with any hydroxyl group on a second sugar molecule to form a disaccharide. The linkage is called a glycosidic bond. Three common disaccharides are maltose (glucose + glucose) lactose (galactose + glucose) sucrose (glucose + fructose) The reaction forming sucrose is shown here. H2O O O O OLIGOSACCHARIDES AND POLYSACCHARIDES Large linear and branched molecules can be made from simple repeating sugar subunits. Short

1	+ fructose) The reaction forming sucrose is shown here. H2O O O O OLIGOSACCHARIDES AND POLYSACCHARIDES Large linear and branched molecules can be made from simple repeating sugar subunits. Short chains are called oligosaccharides, while long chains are called polysaccharides. Glycogen, for example, is a polysaccharide made entirely of glucose units joined together. branch points glycogen CH2OH NH O CH2OH O CH2OH O OH O HO OH HO O OH NH O O HO CH3 O In many cases a sugar sequence is nonrepetitive. Many different molecules are possible. Such complex oligosaccharides are usually linked to proteins or to lipids, as is this oligosaccharide, which is part of a cell-surface molecule that defnes a particular blood group. COMPLEX OLIGOSACCHARIDES C O CH3 C O CH3 SUGAR DERIVATIVES The hydroxyl groups of a simple monosaccharide such as glucose can be replaced by other groups. For example, 97

1	Fatty acids are stored as an energy reserve (fats and oils) through an ester linkage to glycerol to form triacylglycerols, also known as triglycerides. H2COCOHCOCOH2COCOH2COHHCOHH2COHglycerol TRIACYLGLYCEROLS These are carboxylic acids with long hydrocarbon tails. COOHCH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3COOHCH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2COOHCH2CH2CH2CH2CH2CH2CH2CHCHCH2CH2CH2CH2CH2CH2CH2CH3CH3stearic acid (C18) palmitic acid (C16) oleic acid (C18) COMMON FATTY ACIDS OOCstearic acid This double bond is rigid and creates a kink in the chain. The rest of the chain is free to rotate about the other C–C bonds. –OCARBOXYL GROUP O_OCOOCNOCCHIf free, the carboxyl group of a fatty acid will be ionized. But more usually it is linked to other groups to form either esters or amides. PHOSPHOLIPIDS CH2CHCH2PO_OOOhydrophobic fatty acid tails general structure of a phospholipid –OOColeic acid space-flling model carbon skeleton hydrophilic head choline In phospholipids, two

1	amides. PHOSPHOLIPIDS CH2CHCH2PO_OOOhydrophobic fatty acid tails general structure of a phospholipid –OOColeic acid space-flling model carbon skeleton hydrophilic head choline In phospholipids, two of the –OH groups in glycerol are linked to fatty acids, while the third –OH group is linked to phosphoric acid. The phosphate is further linked to one of a variety of small polar groups, such as choline. space-flling model of the phospholipid phosphatidylcholine Hundreds of different kinds of fatty acids exist. Some have one or more double bonds in their hydrocarbon tail and are said to be unsaturated. Fatty acids with no double bonds are saturated. Phospholipids are the major constituents of cell membranes. UNSATURATED SATURATED 98 PaNel 2–5: Fatty acids and Other lipids

1	Fatty acids have a hydrophilic head and a hydrophobic tail. In water they can form a surface flm or form small micelles. Their derivatives can form larger aggregates held together by hydrophobic forces: Triacylglycerols (triglycerides) can form large spherical fat droplets in the cell cytoplasm. Phospholipids and glycolipids form self-sealing lipid bilayers that are the basis for all cell membranes. 200 nm or more micelle long-chain polymers of isoprene O OTHER LIPIDS Lipids are defned as the water-insoluble molecules in cells that are soluble in organic solvents. Two other common types of lipids are steroids and polyisoprenoids. Both are made from isoprene units. CH3CCHCH2CH2isoprene STEROIDS Steroids have a common multiple-ring structure.

1	STEROIDS Steroids have a common multiple-ring structure. GLYCOLIPIDS Like phospholipids, these compounds are composed of a hydrophobic region, containing two long hydrocarbon tails and a polar region, which contains one or more sugars and, unlike phospholipids, no phosphate. CCCH2HNHOHHOCOsugar a simple glycolipid Hdolichol phosphate—used to carry activated sugars in the membrane-associated synthesis of glycoproteins and some polysaccharides galactose BASES The bases are nitrogen-containing ring compounds, either pyrimidines or purines. CCCHCNHNHOOCCHCNHNONH2H3CCCHCNHNHOOHCHCUCTuracilcytosinethymineN N 1 2 3 4 5 6 N N 1 2 3 4 5 6N N 7 8 9 O N N H C C C C N NH NH2 HC N N H C C C CH N N HC NH2 adenine guanine A G PYRIMIDINE PURINE 100 PaNel 2–6: a Survey of the Nucleotides

1	NUCLEOTIDESA nucleotide consists of a nitrogen-containing base, a fve-carbon sugar, and one or more phosphate groups. HHONH2OCH2OHOHBASE PHOSPHATE SUGAR Nucleotides are the subunits of the nucleic acids.The phosphate makes a nucleotide negatively charged. PHOSPHATES The phosphates are normally joined to the C5 hydroxyl of the ribose or deoxyribose sugar (designated 5'). Mono-, di-, and triphosphates are common. The base is linked to the same carbon (C1) used in sugar–sugar bonds. SUGARS Each numbered carbon on the sugar of a nucleotide is followed by a prime mark; therefore, one speaks of the “5-prime carbon,” etc. OH OH O H H HOCH2 OH H H OH H O H H HOCH2 OH H H PENTOSE a fve-carbon sugar O 4’ 3’ 2’ 1’ C 5’ two kinds are used ˜-D-ribose used in ribonucleic acid ˜-D-2-deoxyribose used in deoxyribonucleic acid

1	NOMENCLATURE A nucleoside or nucleotide is named according to its nitrogenous base. BASE adenine guanine cytosine uracil thymine NUCLEOSIDE adenosine guanosine cytidine uridine thymidine ABBR. A G C U T Single-letter abbreviations are used variously as shorthand for (1) the base alone, (2) the nucleoside, or (3) the whole nucleotide— the context will usually make clear which of the three entities is meant. When the context is not suffcient, we will add the terms “base”, “nucleoside”, “nucleotide”, or—as in the examples below—use the full 3-letter nucleotide code. AMP dAMP UDP ATP = adenosine monophosphate = deoxyadenosine monophosphate = uridine diphosphate = adenosine triphosphate sugar base sugar base BASE + SUGAR = NUCLEOSIDE BASE + SUGAR + PHOSPHATE = NUCLEOTIDE NUCLEIC ACIDS Nucleotides are joined together by a phosphodiester linkage between 5’ and 3’ carbon atoms to form nucleic acids. The linear sequence of nucleotides in a nucleic acid chain is commonly abbreviated by a

1	are joined together by a phosphodiester linkage between 5’ and 3’ carbon atoms to form nucleic acids. The linear sequence of nucleotides in a nucleic acid chain is commonly abbreviated by a one-letter code, such as A—G—C—T—T—A—C—A, with the 5’ end of the chain at the left. OOHsugar base CH2OO––OPO+OOHsugarbaseCH2H2OOO––OPOOsugarbaseCH2OO––OPOOsugarbase CH2PO–OOO5’OH3’3’ end of chain 3’5’phosphodiester linkage 5’ end of chain example: DNA OO––OPOO–POOOO–PONUCLEOTIDES HAVE MANY OTHER FUNCTIONS OCH2NNNNNH2OHOH1 They carry chemical energy in their easily hydrolyzed phosphoanhydride bonds. OOO–POCH2NNNNNH2OH2 They combine with other groups to form coenzymes. OO–POOCCCCNCCCNCCHSOOHHHHHHHHHHHHHHOCH3example: coenzyme A (CoA) CH33 They are used as specifc signaling molecules in the cell. OOO–POCH2NNNNNH2OOHexample: cyclic AMP (cAMP) phosphoanhydride bonds example: ATP (or ) OPO–O–OATPP proceed in cells only because they are coupled to very favorable reactions that drive them. The question of

1	cyclic AMP (cAMP) phosphoanhydride bonds example: ATP (or ) OPO–O–OATPP proceed in cells only because they are coupled to very favorable reactions that drive them. The question of whether a reaction dynamics—that are required for understanding what free energy is and why it is so important to cells.

1	ENERGY RELEASED BY CHANGES IN CHEMICAL BONDING IS CONVERTED INTO HEAT of molecular collisions that heat up frst the walls of the box and then the outside world (represented by the sea in our example). In the end, the system returns to its initial temperature, by which time all the chemical-bond energy released in the box has been converted into heat energy and transferred out of the box to the surroundings. According to the frst law, the change in the energy in the box (˜Ebox, which we shall denote as ˜E) must be equal and opposite to the amount of heat energy transferred, which we shall designate as h: that is, ˜E = _h. Thus, the energy in the box (E) decreases when heat leaves the system. E also can change during a reaction as a result of work being done on the outside world. For example, suppose that there is a small increase in the volume (˜V) of the box during a reaction. Since the walls of the box must push against the constant pressure (P) in the surroundings in order to

1	example, suppose that there is a small increase in the volume (˜V) of the box during a reaction. Since the walls of the box must push against the constant pressure (P) in the surroundings in order to expand, this does work on the outside world and requires energy. The energy used is P(˜V), which according to the frst law must decrease the energy in the box (E) by the same amount. In most reactions, chemical-bond energy is converted into both work and heat. Enthalpy (H) is a composite function that includes both of these (H = E + PV). To be rigorous, it is the change in enthalpy (˜H) in an enclosed system, and not the change in energy, that is equal to the heat transferred to the outside world during a reaction. Reactions in which H decreases release heat to the surroundings and are said to be “exothermic,” while reactions in which H increases absorb heat from the surroundings and are said to be “endothermic.” Thus, _h = ˜H. However, the volume change is negligible in most biological

1	to be “exothermic,” while reactions in which H increases absorb heat from the surroundings and are said to be “endothermic.” Thus, _h = ˜H. However, the volume change is negligible in most biological reactions, so to a good approximation An enclosed system is defned as a collection of molecules that does not exchange matter with the rest of the universe (for example, the “cell in a box” shown above). Any such system will contain molecules with a total energy E. This energy will be distributed in a variety of ways: some as the translational energy of the molecules, some as their vibrational and rotational energies, but most as the bonding energies between the individual atoms that make up the molecules. Suppose that a reaction occurs in the system. The frst law of thermodynamics places a constraint on what types of reactions are possible: it states that “in any process, the total energy of the universe remains constant.” For example, suppose that reaction A B occurs somewhere in the

1	constraint on what types of reactions are possible: it states that “in any process, the total energy of the universe remains constant.” For example, suppose that reaction A B occurs somewhere in the box and releases a great deal of chemical-bond energy. This energy will initially increase the intensity of molecular motions (translational, vibrational, and rotational) in the system, which is equivalent to raising its temperature. However, these increased motions will soon be transferred out of the system by a series _h = ˜H = ˜E BOX CELL SEA UNIVERSE ~ THE SECOND LAW OF THERMODYNAMICS Consider a container in which 1000 coins are all lying heads up. If the container is shaken vigorously, subjecting the coins to the types of random motions that all molecules experience due to their frequent collisions with other molecules, one will end up with about half the coins oriented heads down. The reason for this reorientation is that there is only a single way in which the original orderly state

1	with other molecules, one will end up with about half the coins oriented heads down. The reason for this reorientation is that there is only a single way in which the original orderly state of the coins can be reinstated (every coin must lie heads up), whereas there are many different ways (about 10298) to achieve a disorderly state in which there is an equal mixture of heads and tails; in fact, there are more ways to achieve a 50-50 state than to achieve any other state. Each state has a probability of occurrence that is proportional to the number of ways it can be realized. The second law of thermo-dynamics states that “systems will change spontaneously from states of lower probability to states of higher probability.” Since states of lower probability are more “ordered” than states of high probability, the second law can be restated: “the universe constantly changes so as to become more disordered.”

1	THE ENTROPY, S The second law (but not the frst law) allows one to predict the direction of a particular reaction. But to make it useful for this purpose, one needs a convenient measure of the probability or, equivalently, the degree of disorder of a state. The entropy (S) is such a measure. It is a logarithmic function of the probability such that the change in entropy (˜S) that occurs when the reaction A

1	B converts one mole of A into one mole of B is where pA and pB are the probabilities of the two states A and B, R is the gas constant (8.31 J K–1 mole_1), and ˜S is measured in entropy units (eu). In our initial example of 1000 coins, the relative probability of all heads (state A) versus half heads and half tails (state B) is equal to the ratio of the number of different ways that the two results can be obtained. One can calculate that pA = 1 and pB = 1000!(500! x 500!) = 10299. Therefore, the entropy change for the reorientation of the coins when their container is vigorously shaken and an equal mixture of heads and tails is obtained is R In (10298), or about 1370 eu per mole of such containers (6 x 1023 containers). We see that, because ˜S defned above is positive for the transition from state A to state B (pB /pA > 1), reactions with a large increase in S (that is, for which ˜S > 0) are favored and will occur spontaneously.

1	As discussed in Chapter 2, heat energy causes the random commotion of molecules. Because the transfer of heat from an enclosed system to its surroundings increases the number of different arrangements that the molecules in the outside world can have, it increases their entropy. It can be shown that the release of a fxed quantity of heat energy has a greater disordering effect at low temperature than at high temperature, and that the value of ˜S for the surroundings, as defned above (˜Ssea), is precisely equal to h, the amount of heat transferred to the surroundings from the system, divided by the absolute temperature (T ):

1	THE GIBBS FREE ENERGY, G When dealing with an enclosed biological system, one would like to have a simple way of predicting whether a given reaction will or will not occur spontaneously in the system. We have seen that the crucial question is whether the entropy change for the universe is positive or negative when that reaction occurs. In our idealized system, the cell in a box, there are two separate components to the entropy change of the universe—the entropy change for the system enclosed in the box and the entropy change for the surrounding “sea”—and both must be added together before any prediction can be made. For example, it is possible for a reaction to absorb heat and thereby decrease the entropy of the sea (˜Ssea < 0) and at the same time to cause such a large degree of disordering inside the box (˜Sbox > 0) that the total ˜Suniverse = ˜Ssea + ˜Sbox is greater than 0. In this case, the reaction will occur spontaneously, even though the sea gives up heat to the box during the

1	inside the box (˜Sbox > 0) that the total ˜Suniverse = ˜Ssea + ˜Sbox is greater than 0. In this case, the reaction will occur spontaneously, even though the sea gives up heat to the box during the reaction. An example of such a reaction is the dissolving of sodium chloride in a beaker containing water (the “box”), which is a spontaneous process even though the temperature of the water drops as the salt goes into solution. Chemists have found it useful to defne a number of new “composite functions” that describe combinations of physical properties of a system. The properties that can be combined include the temperature (T), pressure (P), volume (V), energy (E), and entropy (S). The enthalpy (H) is one such composite function. But by far the most useful composite function for biologists is the Gibbs free energy, G. It serves as an accounting device that allows one to deduce the entropy change of the universe resulting from a chemical reaction in the box, while avoiding any separate

1	is the Gibbs free energy, G. It serves as an accounting device that allows one to deduce the entropy change of the universe resulting from a chemical reaction in the box, while avoiding any separate consideration of the entropy change in the sea. The defnition of G is where, for a box of volume V, H is the enthalpy described above (E + PV), T is the absolute temperature, and S is the entropy. Each of these quantities applies to the inside of the box only. The change in free energy during a reaction in the box (the G of the products minus the G of the starting materials) is denoted as ˜G and, as we shall now demonstrate, it is a direct measure of the amount of disorder that is created in the universe when the reaction occurs. G = H _ TS At constant temperature the change in free energy (˜G) during a reaction equals ˜H _ T˜S. Remembering that ˜H = _h, the heat absorbed from the sea, we have _˜G = _˜H + T˜S _˜G = h + T˜S, so _˜G/T = h/T + ˜S But h/T is equal to the entropy change of the

1	during a reaction equals ˜H _ T˜S. Remembering that ˜H = _h, the heat absorbed from the sea, we have _˜G = _˜H + T˜S _˜G = h + T˜S, so _˜G/T = h/T + ˜S But h/T is equal to the entropy change of the sea (˜Ssea), and the ˜S in the above equation is ˜Sbox. Therefore _˜G/T = ˜Ssea + ˜Sbox = ˜Suniverse We conclude that the free-energy change is a direct measure of the entropy change of the universe. A reaction will proceed in the direction that causes the change in the free energy (˜G) to be less than zero, because in this case there will be a positive entropy change in the universe when the reaction occurs. For a complex set of coupled reactions involving many different molecules, the total free-energy change can be com-puted simply by adding up the free energies of all the different molecular species after the reaction and comparing this value with the sum of free energies before the reaction; for common substances the required free-energy values can be found from published tables. In

1	species after the reaction and comparing this value with the sum of free energies before the reaction; for common substances the required free-energy values can be found from published tables. In this way, one can predict the direction of a reaction and thereby readily check the feasibility of any proposed mechanism. Thus, for example, from the observed values for the magnitude of the electrochemical proton gradient across the inner mitochondrial membrane and the ˜G for ATP hydrolysis inside the mitochondrion, one can be certain that ATP synthase requires the passage of more than one proton for each molecule of ATP that it synthesizes. The value of ˜G for a reaction is a direct measure of how far the reaction is from equilibrium. The large negative value for ATP hydrolysis in a cell merely refects the fact that cells keep the ATP hydrolysis reaction as much as 10 orders of magnitude away from equilibrium. If a reaction reaches equilibrium, ˜G = 0, the reaction then proceeds at

1	merely refects the fact that cells keep the ATP hydrolysis reaction as much as 10 orders of magnitude away from equilibrium. If a reaction reaches equilibrium, ˜G = 0, the reaction then proceeds at precisely equal rates in the forward and backward direction. For ATP hydrolysis, equilibrium is reached when the vast majority of the ATP has been hydrolyzed, as occurs in a dead cell.

1	CH2OHOOHOHOHHOglucoseCH2OH+OOHOHOHHOglucose 6-phosphate + + + hexokinase CH2OOOHOHOHHOglucose 6-phosphate fructose 6-phosphate (ring form) (ring form) (open-chain form) 1 1 2 2 3 4 5 6 6 OHCCHOHCHOHCHOHCHOHCH2O3 4 5 (open-chain form) 1 1 2 2 6 CCHOHCHOHCHOHCH2OCH2OH334455phosphoglucoseisomeraseOH2CCH2OHOHOOHOH6+H+++phosphofructokinaseOH2CCH2OHOHOOHOHOH2COHOOHOHGlucose is phosphorylated by ATP to form a sugar phosphate. The negative charge of the phosphate prevents passage of the sugar phosphate through the plasma membrane, trapping glucose inside the cell. The six-carbon sugar is cleaved to produce two three-carbon molecules. Only the glyceraldehyde 3-phosphate can proceed immediately through glycolysis. A readily reversible rearrangement of the chemical structure (isomerization) moves the carbonyl oxygen from carbon 1 to carbon 2, forming a ketose from an aldose sugar. (See Panel 2–3, pp. 70–71.) The new hydroxyl group on carbon 1 is phosphorylated by ATP, in preparation for the

1	the carbonyl oxygen from carbon 1 to carbon 2, forming a ketose from an aldose sugar. (See Panel 2–3, pp. 70–71.) The new hydroxyl group on carbon 1 is phosphorylated by ATP, in preparation for the formation of two three-carbon sugar phosphates. The entry of sugars into glycolysis is controlled at this step, through regulation of the enzyme phosphofructokinase. fructose 6-phosphate fructose 1,6-bisphosphate + (ring form) OHCCHOHaldolase (open-chain form) CCHOHCHOHCHOHCH2OCH2OOCCHOHHCH2OCH2OOOH2CCH2OOHOOHOHfructose 1,6-bisphosphate dihydroxyacetone phosphate glyceraldehyde 3-phosphate OFor each step, the part of the molecule that undergoes a change is shadowed in blue, and the name of the enzyme that catalyzes the reaction is in a yellow box. ATPATPADPADPP P P P P PP P P P P P P P CH2OStep 1 Step 2 Step 3 Step 4 104 PaNel 2–8: Details of the 10 Steps of Glycolysis + + enolase phosphoglycerate mutase + OO–CCHOHCH2O3-phosphoglycerate OO–CCHOCH2OH2-phosphoglycerate

1	P P P P P P P CH2OStep 1 Step 2 Step 3 Step 4 104 PaNel 2–8: Details of the 10 Steps of Glycolysis + + enolase phosphoglycerate mutase + OO–CCHOHCH2O3-phosphoglycerate OO–CCHOCH2OH2-phosphoglycerate OO–CCHOCH2OH2-phosphoglycerate OO–CCOCH2H2Ophosphoenolpyruvate OO–CCOCH2phosphoenolpyruvate OO–CCOCH3pyruvate + phosphoglycerate kinaseOCCHOHCH2O1,3-bisphosphoglycerate + OO–CCHOHCH2O3-phosphoglycerate The two molecules of glyceraldehyde 3-phosphate are oxidized. The energy-generation phase of glycolysis begins, as NADH and a new high-energy anhydride linkage to phosphate are formed (see Figure 13–5). H++ ++ glyceraldehyde 3-phosphate dehydrogenaseOHCCHOHCH2Oglyceraldehyde 3-phosphate OOCCHOHCH2O1,3-bisphosphoglycerate H++ The transfer to ADP of the high-energy phosphate group that was generated in step 6 forms ATP. The remaining phosphate ester linkage in 3-phosphoglycerate, which has a relatively low free energy of hydrolysis, is moved from carbon 3 to carbon 2 to form

1	that was generated in step 6 forms ATP. The remaining phosphate ester linkage in 3-phosphoglycerate, which has a relatively low free energy of hydrolysis, is moved from carbon 3 to carbon 2 to form 2-phosphoglycerate. The removal of water from 2-phosphoglycerate creates a high-energy enol phosphate linkage. The transfer to ADP of the high-energy phosphate group that was generated in step 9 forms ATP, completing glycolysis. + pyruvate kinase 1 2 3 ATPADPATPADPNADHNAD+Pi P P P P P P P P P P P OStep 6 Step 7 Step 8 Step 9 Step 10 105

1	In addition to the pyruvate, the net products are two molecules glucose two molecules of ATP and two molecules of NADH. of pyruvate Step 2 An isomerization COO–

1	After the enzyme removes a proton from the CH3 group on acetyl CoA, the negatively charged CH2 – forms a bond to a carbonyl carbon of oxaloacetate. The subsequent loss by hydrolysis of the coenzyme A (HS–CoA) drives the reaction strongly forward. acetyl CoA S-citryl-CoA intermediate citrateoxaloacetate COO–COO–OOCCSCoAcitrate synthase CH3CH2H2OOCSCoACH2COO–COO–CHOCH2CH2COO–COO–COO–CCH2CoAHSH+CoAHO+++Details of these eight steps are shown below. In this part of the panel, for each step, the part of the molecule that undergoes a change is shadowed in blue, and the name of the enzyme that catalyzes the reaction is in a yellow box. CH2COO–COO–COO–CHOCH2H2OH2OH2OCH2CO2CO2CO2COO–COO–COO–HCCHHOCH2COO–COO–COCH2CH2COO–COO–CH2CHCOO–COO–CHHOHCCOO–COO–CH2COO–COCH2SCoAOCH3SCoAacetyl CoA coenzyme A HSCoAHSHSCoAHSCoACH2OCCOO–COO–CH2OOCCOO–COO–COO–CH2CH3Cnext cycle + Step 1 Step 2 Step 3 Step 4 Step 6 Step 7 Step 8 Step 5 citrate (6C) isocitrate (6C) succinyl CoA (4C)succinate (4C) fumarate (4C)

1	HSCoAHSHSCoAHSCoACH2OCCOO–COO–CH2OOCCOO–COO–COO–CH2CH3Cnext cycle + Step 1 Step 2 Step 3 Step 4 Step 6 Step 7 Step 8 Step 5 citrate (6C) isocitrate (6C) succinyl CoA (4C)succinate (4C) fumarate (4C) malate (4C) oxaloacetate (4C) oxaloacetate (4C) pyruvate ˜-ketoglutarate (5C) + H+ + H+ + H+ + H+ (2C) CITRIC ACID CYCLE Overview of the complete citric acid cycle. The two carbons from acetyl CoA that enter this turn of the cycle (shadowed in red ) will be converted to CO2 in subsequent turns of the cycle: it is the two carbons shadowed in blue that are converted to CO2 in this cycle. CGTPGDPNADHNADHNADHNAD+NAD+NAD+Pi FADFADH2Step 1 106 PaNel 2–9: The Complete Citric acid Cycle NADHNAD+ reaction, in which water is aconitaseadded back, moves the hydroxyl group from one carbon atom to its neighbor.

1	In the frst of four oxidation steps in the cycle, the carbon carrying the hydroxyl group is converted to a carbonyl group. The immediate product is unstable, losing CO2 while still bound to the enzyme. The ˜-ketoglutarate dehydrogenase complex closely resembles the large enzyme complex that converts pyruvate to acetyl CoA, the pyruvate dehydrogenase complex in Figure 13–10. It likewise catalyzes an oxidation that produces NADH, CO2, and a high-energy thioester bond to coenzyme A (CoA). A phosphate molecule from solution displaces the CoA, forming a high-energy phosphate linkage to succinate. This phosphate is then passed to GDP to form GTP. (In bacteria and plants, ATP is formed instead.) In the third oxidation step in the cycle, FAD accepts two hydrogen atoms from succinate. The addition of water to fumarate places a hydroxyl group next to a carbonyl carbon. COO–COO–HOHHHHCCCisocitrate isocitrate dehydrogenase CO2COO–COO–COO–HHHOCCCoxalosuccinate intermediate COO–+ H+

1	The addition of water to fumarate places a hydroxyl group next to a carbonyl carbon. COO–COO–HOHHHHCCCisocitrate isocitrate dehydrogenase CO2COO–COO–COO–HHHOCCCoxalosuccinate intermediate COO–+ H+ H+COO–COO–HHHHOCCC˜-ketoglutarate succinate dehydrogenase COO–COO–HHHHCCsuccinate COO–COO–HHCCfumarate fumarase COO–COO–HOHHHCCmalate COO–COO–HHCCfumarate H2O˜-ketoglutarate dehydrogenase complex CO2 + H+ COO–COO–HHHHOCCC˜-ketoglutarate COO–HHHHOCCCsuccinyl-CoA HSCoASCoA+ H2OCOO–HHHHOCCCsuccinyl-CoA SCoACOO–COO–HHHHCCsuccinate HSCoA+ succinyl-CoA synthetase GTPGDPFADNADHFADH2NAD+NADHNAD+Pi Step 3 Step 4 Step 5 Step 6 Step 7 107

1	Step 8 In the last of four malate dehydrogenaseoxidation steps in the cycle, the carbon carrying the hydroxyl group is converted to a carbonyl group, regenerating the oxaloacetate + H+ needed for step 1. COO– Berg Jm, Tymoczko Jl & stryer l (2011) Biochemistry, 7th ed. new York: Wh freeman. Garrett Rh & Grisham Cm (2012) Biochemistry, 5th ed. philadelphia: Thomson Brooks/Cole. moran la, horton hR, scrimgeour G & perry m (2011) principles of Biochemistry, 5th ed. Upper saddle River, nJ: prentice hall. nelson Dl & Cox mm (2012) lehninger principles of Biochemistry, 6th ed. new York: Worth. van holde Ke, Johnson WC & ho ps (2005) principles of physical Biochemistry, 2nd ed. Upper saddle River, nJ: prentice hall. Van Vranken D & Weiss G (2013) introduction to Bioorganic Chemistry and Chemical Biology. new York: Garland science. Voet D, Voet JG & pratt Cm (2012) fundamentals of Biochemistry, 4th ed. new York: Wiley.

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1	eisenberg D & Kauzmann W (1969) The structure and properties of Water. oxford: oxford University press. franks f (1993) Water. Cambridge: Royal society of Chemistry. henderson lJ (1927) The fitness of the environment, 1958 ed. Boston, ma: Beacon. neidhardt fC, ingraham Jl & schaechter m (1990) physiology of the Bacterial Cell: a molecular approach. sunderland, ma: sinauer. phillips R & milo R (2009) a feeling for the numbers in biology. Proc. Natl Acad. Sci. USA 106, 21465–21471. skinner Jl (2010) following the motions of water molecules in aqueous solutions. Science 328, 985–986. Taylor me & Drickamer K (2011) introduction to Glycobiology, 3rd ed. new York: oxford University press. Vance De & Vance Je (2008) Biochemistry of lipids, lipoproteins and membranes, 5th ed. amsterdam: elsevier. Catalysis and the Use of energy by Cells atkins pW (1994) The second law: energy, Chaos and form. new York: scientific american Books.

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1	fell D (1997) Understanding the Control of metabolism. london: portland press. fothergill-Gilmore la (1986) The evolution of the glycolytic pathway. Trends Biochem. Sci. 11, 47–51. friedmann hC (2004) from Butyribacterium to E. coli: an essay on unity in biochemistry. Perspect. Biol. Med. 47, 47–66. heinrich R, meléndez-hevia e, montero f et al. (1999) The structural design of glycolysis: an evolutionary approach. Biochem. Soc. Trans. 27, 294–298. huynen ma, Dandekar T & Bork p (1999) Variation and evolution of the citric-acid cycle: a genomic perspective. Trends Microbiol. 7, 281–291. Koonin eV (2014) The origins of cellular life. Antonie van Leeuwenhoek 106, 27–41. Kornberg hl (2000) Krebs and his trinity of cycles. Nat. Rev. Mol. Cell Biol. 1, 225–228. Krebs ha (1970) The history of the tricarboxylic acid cycle. Perspect. Biol. Med. 14, 154–170. Krebs ha & martin a (1981) Reminiscences and Reflections. oxford/ new York: Clarendon press/oxford University press.

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1	When we look at a cell through a microscope or analyze its electrical or biochemical activity, we are, in essence, observing proteins. Proteins constitute most of a cell’s dry mass. They are not only the cell’s building blocks; they also execute the majority of the cell’s functions. Thus, proteins that are enzymes provide the intricate molecular surfaces inside a cell that catalyze its many chemical reactions. Proteins embedded in the plasma membrane form channels and pumps that control the passage of small molecules into and out of the cell. Other proteins carry messages from one cell to another, or act as signal integrators that relay sets of signals inward from the plasma membrane to the cell nucleus. Yet others serve as tiny molecular machines with moving parts: kinesin, for example, propels organelles through the cytoplasm; topoisomerase can untangle knotted DNA molecules. Other specialized proteins act as antibodies, toxins, hormones, antifreeze molecules, elastic fibers, ropes,

1	organelles through the cytoplasm; topoisomerase can untangle knotted DNA molecules. Other specialized proteins act as antibodies, toxins, hormones, antifreeze molecules, elastic fibers, ropes, or sources of luminescence. Before we can hope to understand how genes work, how muscles contract, how nerves conduct electricity, how embryos develop, or how our bodies function, we must attain a deep understanding of proteins.

1	From a chemical point of view, proteins are by far the most structurally complex and functionally sophisticated molecules known. This is perhaps not surprising, once we realize that the structure and chemistry of each protein has been developed and fine-tuned over billions of years of evolutionary history. The theoretical calculations of population geneticists reveal that, over evolutionary time periods, a surprisingly small selective advantage is enough to cause a randomly altered protein sequence to spread through a population of organisms. Yet, even to experts, the remarkable versatility of proteins can seem truly amazing. In this section, we consider how the location of each amino acid in the long string of amino acids that forms a protein determines its three-dimensional shape. Later in the chapter, we use this understanding of protein structure at the atomic level to describe how the precise shape of each protein molecule determines its function in a cell.

1	The Shape of a Protein Is Specified by Its Amino Acid Sequence There are 20 different of amino acids in proteins that are coded for directly in an organism’s DNA, each with different chemical properties. A protein molecule is made from a long unbranched chain of these amino acids, each linked to its neighbor through a covalent peptide bond. Proteins are therefore also known as polypeptides. Each type of protein has a unique sequence of amino acids, and there are many thousands of different proteins in a cell. The repeating sequence of atoms along the core of the polypeptide chain is referred to as the polypeptide backbone. Attached to this repetitive chain are those portions of the amino acids that are not involved in making a peptide bond and that give each amino acid its unique properties: the 20 different amino acid side chains (Figure 3–1). Some of these side chains are nonpolar and hydrophobic Figure 3–1 The components of a protein.

1	Figure 3–1 The components of a protein. A protein consists of a polypeptide backbone with attached side chains. Each type of protein differs in its sequence and number of amino acids; therefore, it is the sequence of the chemically different side chains that makes each protein distinct. The two ends of a polypeptide chain are chemically different: the end carrying the free amino group (NH3+, also written NH2) is the amino terminus, or N-terminus, and that carrying the free carboxyl group (COO–, also written COOH) is the carboxyl terminus or C-terminus. The amino acid sequence of a protein is always presented in the N-to-C direction, reading from left to right. (“water-fearing”), others are negatively or positively charged, some readily form covalent bonds, and so on. Panel 3–1 (pp. 112–113) shows their atomic structures and Figure 3–2 lists their abbreviations.

1	As discussed in Chapter 2, atoms behave almost as if they were hard spheres with a definite radius (their van der Waals radius). The requirement that no two atoms overlap plus other constraints limit the possible bond angles in a polypeptide chain (Figure 3–3), severely restricting the possible three-dimensional arrangements (or conformations) of atoms. Nevertheless, a long flexible chain such as a protein can still fold in an enormous number of ways.

1	The folding of a protein chain is also determined by many different sets of weak noncovalent bonds that form between one part of the chain and another. These involve atoms in the polypeptide backbone, as well as atoms in the amino acid side chains. There are three types of these weak bonds: hydrogen bonds, electrostatic attractions, and van der Waals attractions, as explained in Chapter 2 (see p. 44). Individual noncovalent bonds are 30–300 times weaker than the typical covalent bonds that create biological molecules. But many weak bonds acting in parallel can hold two regions of a polypeptide chain tightly together. In this way, the combined strength of large numbers of such noncovalent bonds determines the stability of each folded shape (Figure 3–4).

1	Figure 3–2 The 20 amino acids commonly found in proteins. Each amino acid has a three-letter and a one-letter abbreviation. There are equal numbers of polar and nonpolar side chains; however, some side chains listed here as polar are large enough to have some nonpolar properties (for example, Tyr, Thr, Arg, Lys). For atomic structures, see Panel 3–1 (pp. 112–113).

1	Figure 3–3 Steric limitations on the bond angles in a polypeptide –180 chain. (A) Each amino acid contributes three bonds (red) to the backbone –180 0 +180 of the chain. The peptide bond is planar (gray shading) and does not permit rotation. By contrast, rotation can occur about the Cα–C bond, whose angle of rotation is called psi (ψ), and about the N–Cα bond, whose angle of left-handed rotation is called phi (ϕ). By convention, an R group is often used to denote helix an amino acid side chain (purplecircles). (B) The conformation of the main-chain atoms in a protein is determined by one pair of ϕ and ψ angles for each amino acid; because of steric collisions between atoms within each amino acid, most of the possible pairs of ϕ and ψ angles do not occur. In this so-called Ramachandran plot, each dot represents an observed pair of angles in a protein. The three differently shaded clusters of dots reflect three different “secondary structures” repeatedly found in proteins, as will be

1	each dot represents an observed pair of angles in a protein. The three differently shaded clusters of dots reflect three different “secondary structures” repeatedly found in proteins, as will be described in the text. (B, from J. Richardson, Adv. Prot. Chem. 34:174–175, 1981. © Academic Press.)

1	A fourth weak force—a hydrophobic clustering force—also has a central role in determining the shape of a protein. As described in Chapter 2, hydrophobic molecules, including the nonpolar side chains of particular amino acids, tend to be forced together in an aqueous environment in order to minimize their disruptive effect on the hydrogen-bonded network of water molecules (see Panel 2–2, pp. 92–93). Therefore, an important factor governing the folding of any protein is Figure 3–4 Three types of noncovalent bonds help proteins fold. Although a single one of these bonds is quite weak, many of them act together to create a strong bonding arrangement, as in the example shown. As in the previous figure, R is used as a general designation for an amino acid side chain. 112 Panel 3–1: The 20 amino acids Found in Proteins THE AMINO ACID OPTICAL ISOMERS The ˜-carbon atom is asymmetric, which allows for two mirror images (or stereo-)

1	112 Panel 3–1: The 20 amino acids Found in Proteins THE AMINO ACID OPTICAL ISOMERS The ˜-carbon atom is asymmetric, which allows for two mirror images (or stereo-) The general formula of an amino acid is isomers, L and D. ˜-carbon atom R is commonly one of 20 different side chains. At pH 7 both the amino and carboxyl groups are ionized. Proteins consist exclusively of L-amino acids. Amino acids are commonly joined together by an amide linkage, Peptide bond: The four atoms in each gray box form a rigid called a peptide bond. planar unit. There is no rotation around the C–N bond. aminoor carboxylor Proteins are long polymers N-terminus HO CH2 HH C-terminus of amino acids linked by + peptide bonds, and they are always written with the CH2H O CH N-terminus toward the left. The sequence of this tripeptide C is histidine-cysteine-valine. HN CH These two single bonds allow rotation, so that long chains of amino acids are very fexible.

1	The sequence of this tripeptide C is histidine-cysteine-valine. HN CH These two single bonds allow rotation, so that long chains of amino acids are very fexible. The common amino acids (Lys, or K) (Arg, or R) (His, or H) are grouped according to whether their side chains are CH2 This group is CH2 very basic CH2 because its NH These nitrogens have a and one-letter abbreviations. NH3 is stabilized by C relatively weak affnity for an resonance. +H2N NH2 H+ and are only partly positive Thus: alanine = Ala = A at neutral pH. alanine valine aspartic acid glutamic acid (Ala, or A) (Val, or V) (Asp, or D) (Glu, or E) C O O– (Leu, or L) asparagine glutamine proline phenylalanine (Asn, or N) (Gln, or Q) (Pro, or P) (Phe, or F) methionine tryptophan (Met, or M) (Trp, or W) Although the amide N is not charged at neutral pH, it is polar. (Ser, or S) (Thr, or T) (Tyr, or Y) (Gly, or G) (Cys, or C) The –OH group is polar. in proteins.

1	The –OH group is polar. in proteins. polar side chain on the hydrophobic core region outside of the molecule contains nonpolar can form hydrogen side chains bonds to water the distribution of its polar and nonpolar amino acids. The nonpolar (hydrophobic) side chains in a protein—belonging to such amino acids as phenylalanine, leucine, valine, and tryptophan—tend to cluster in the interior of the molecule (just as hydrophobic oil droplets coalesce in water to form one large droplet). This enables them to avoid contact with the water that surrounds them inside a cell. In contrast, polar groups—such as those belonging to arginine, glutamine, and histidine—tend to arrange themselves near the outside of the molecule, where they can form hydrogen bonds with water and with other polar molecules (Figure 3–5). Polar amino acids buried within the protein are usually hydrogen-bonded to other polar amino acids or to the polypeptide backbone. Proteins Fold into a Conformation of Lowest Energy

1	Proteins Fold into a Conformation of Lowest Energy As a result of all of these interactions, most proteins have a particular three-dimensional structure, which is determined by the order of the amino acids in its chain. The final folded structure, or conformation, of any polypeptide chain is generally the one that minimizes its free energy. Biologists have studied protein folding in a test tube using highly purified proteins. Treatment with certain solvents, which disrupt the noncovalent interactions holding the folded chain together, unfolds, or denatures, a protein. This treatment converts the protein into a flexible polypeptide chain that has lost its natural shape. When the denaturing solvent is removed, the protein often refolds spontaneously, or renatures, into its original conformation. This indicates that the amino acid sequence contains all of the information needed for specifying the three-dimensional shape of a protein, a critical point for understanding cell biology.

1	Most proteins fold up into a single stable conformation. However, this conformation changes slightly when the protein interacts with other molecules in the cell. This change in shape is often crucial to the function of the protein, as we see later. Although a protein chain can fold into its correct conformation without outside help, in a living cell special proteins called molecular chaperones often assist in protein folding. Molecular chaperones bind to partly folded polypeptide chains and help them progress along the most energetically favorable folding pathway. In the crowded conditions of the cytoplasm, chaperones are required to prevent the temporarily exposed hydrophobic regions in newly synthesized protein chains from associating with each other to form protein aggregates (see p. 355). However, the final three-dimensional shape of the protein is still specified by its amino acid sequence: chaperones simply make reaching the folded state more reliable.

1	Figure 3–5 How a protein folds into a compact conformation. The polar amino acid side chains tend to lie on the outside of the protein, where they can interact with water; the nonpolar amino acid side chains are buried on the inside forming a tightly packed hydrophobic core of atoms that are hidden from water. In this schematic drawing, the protein contains only about 35 amino acids. Figure 3–6 Four representations describing the structure of a small protein domain. Constructed from a string of 100 amino acids, the SH2 domain is part of many different proteins (see, for example, Figure 3–61). Here, the structure of the SH2 domain is displayed as (A) a polypeptide backbone model, (B) a ribbon model, (C) a wire model that includes the amino acid side chains, and (D) a space-filling model (Movie 3.1). These images are colored in a way that allows the polypeptide chain to be followed from its N-terminus (purple) to its C-terminus (red) (PDB code: 1SHA).

1	Proteins come in a wide variety of shapes, and most are between 50 and 2000 amino acids long. Large proteins usually consist of several distinct protein domains—structural units that fold more or less independently of each other, as we discuss below. The structure of even a small domain is complex, and for clarity, several different representations are conventionally used, each of which emphasizes distinct features. As an example, Figure 3–6 presents four representations of a protein domain called SH2, a structure present in many different proteins in eukaryotic cells and involved in cell signaling (see Figure 15–46). Descriptions of protein structures are aided by the fact that proteins are built up from combinations of several common structural motifs, as we discuss next. The α Helix and the β Sheet Are Common Folding Patterns

1	When we compare the three-dimensional structures of many different protein molecules, it becomes clear that, although the overall conformation of each protein is unique, two regular folding patterns are often found within them. Both patterns were discovered more than 60 years ago from studies of hair and silk. The first folding pattern to be discovered, called the α helix, was found in the protein α-keratin, which is abundant in skin and its derivatives—such as hair, nails, and horns. Within a year of the discovery of the α helix, a second folded structure, called a β sheet, was found in the protein fibroin, the major constituent of silk. These two patterns are particularly common because they result from hydrogen-bonding between the N–H and C=O groups in the polypeptide backbone, without involving the side chains of the amino acids. Thus, although incompatible with some amino acid side chains, many different amino acid sequences can form them. In each case, the protein chain adopts a

1	involving the side chains of the amino acids. Thus, although incompatible with some amino acid side chains, many different amino acid sequences can form them. In each case, the protein chain adopts a regular, repeating conformation. Figure 3–7 illustrates the detailed structures of these two important conformations, which in ribbon models of proteins are represented by a helical ribbon and by a set of aligned arrows, respectively.

1	0.54 nm R R R R R R R R R R RR R R R H-bond hydrogen amino acid side chain nitrogen carbon carbon peptide bond oxygen (C) (D) 0.7 nm

1	Figure 3–7 The regular conformation of the polypeptide backbone in the α helix and the β sheet. The α helix is shown in (A) and (B). The N–H of every peptide bond is hydrogen-bonded to the C=O of a neighboring peptide bond located four peptide bonds away in the same chain. Note that all of the N–H groups point up in this diagram and that all of the C=O groups point down (toward the C-terminus); this gives a polarity to the helix, with the C-terminus having a partial negative and the N-terminus a partial positive charge (Movie 3.2). The β sheet is shown in (C) and (D). In this example, adjacent peptide chains run in opposite (antiparallel) directions. Hydrogen-bonding between peptide bonds in different strands holds the individual polypeptide chains (strands) together in a β sheet, and the amino acid side chains in each strand alternately project above and below the plane of the sheet (Movie 3.3). (A) and (C) show all the atoms in the polypeptide backbone, but the amino acid side

1	the amino acid side chains in each strand alternately project above and below the plane of the sheet (Movie 3.3). (A) and (C) show all the atoms in the polypeptide backbone, but the amino acid side chains are truncated and denoted by R. In contrast, (B) and (D) show only the carbon and nitrogen backbone atoms.

1	The cores of many proteins contain extensive regions of β sheet. As shown in Figure 3–8, these β sheets can form either from neighboring segments of the polypeptide backbone that run in the same orientation (parallel chains) or from a polypeptide backbone that folds back and forth upon itself, with each section of the chain running in the direction opposite to that of its immediate neighbors (antiparallel chains). Both types of β sheet produce a very rigid structure, held together by hydrogen bonds that connect the peptide bonds in neighboring chains (see Figure 3–7C).

1	An α helix is generated when a single polypeptide chain twists around on itself to form a rigid cylinder. A hydrogen bond forms between every fourth peptide bond, linking the C=O of one peptide bond to the N–H of another (see Figure 3–7A). This gives rise to a regular helix with a complete turn every 3.6 amino acids. The SH2 protein domain illustrated in Figure 3–6 contains two α helices, as well as a three-stranded antiparallel β sheet. Regions of α helix are abundant in proteins located in cell membranes, such as transport proteins and receptors. As we discuss in Chapter 10, those portions of a transmembrane protein that cross the lipid bilayer usually cross as α helices composed largely of amino acids with nonpolar side chains. The polypeptide backbone, which is hydrophilic, is hydrogen-bonded to itself in the α helix and shielded from the hydrophobic lipid environment of the membrane by its protruding nonpolar side chains (see also Figure 3–75A).

1	In other proteins, α helices wrap around each other to form a particularly stable structure, known as a coiled-coil. This structure can form when the two (or in some cases, three or four) α helices have most of their nonpolar (hydrophobic) side chains on one side, so that they can twist around each other with these side chains facing inward (Figure 3–9). Long rodlike coiled-coils provide the structural framework for many elongated proteins. Examples are α-keratin, which forms the intracellular fibers that reinforce the outer layer of the skin and its appendages, and the myosin molecules responsible for muscle contraction.

1	Even a small protein molecule is built from thousands of atoms linked together by precisely oriented covalent and noncovalent bonds. Biologists are aided in visualizing these extremely complicated structures by various graphic and computer-based three-dimensional displays. The student resource site that accompanies this book contains computer-generated images of selected proteins, displayed and rotated on the screen in a variety of formats. Scientists distinguish four levels of organization in the structure of a protein. The amino acid sequence is known as the primary structure. Stretches of polypeptide chain that form α helices and β sheets constitute the protein’s secondary structure. The full three-dimensional organization of a polypeptide chain is sometimes referred to as the tertiary structure, and if a particular protein molecule is formed as a complex of more than one polypeptide chain, the complete structure is designated as the quaternary structure.

1	Studies of the conformation, function, and evolution of proteins have also revealed the central importance of a unit of organization distinct from these four. This is the protein domain, a substructure produced by any contiguous part of a polypeptide chain that can fold independently of the rest of the protein into a compact, stable structure. A domain usually contains between 40 and 350 amino acids, and it is the modular unit from which many larger proteins are constructed. The different domains of a protein are often associated with different functions. Figure 3–10 shows an example—the Src protein kinase, which functions in signaling pathways inside vertebrate cells (Src is pronounced “sarc”). This protein stripe of hydrophobic helices wrap around each other to minimize exposure of hydrophobic amino acid side chains to aqueous environment 0.5 nm

1	Figure 3–8 Two types of β sheet structures. (A) An antiparallel β sheet (see Figure 3–7C). (B) A parallel β sheet. Both of these structures are common in proteins.

1	Figure 3–9 a coiled-coil. (A) A single α helix, with successive amino acid side chains labeled in a sevenfold sequence, “abcdefg” (from bottom to top). Amino acids “a” and “d” in such a sequence lie close together on the cylinder surface, forming a “stripe” (green) that winds slowly around the α helix. Proteins that form coiled-coils typically have nonpolar amino acids at positions “a” and “d.” Consequently, as shown in (B), the two α helices can wrap around each other with the nonpolar side chains of one α helix interacting with the nonpolar side chains of the other. (C) The atomic structure of a coiled-coil determined by x-ray crystallography. The alpha helical backbone is shown in red and the nonpolar side chains in green, while the more hydrophilic amino acid side chains, shown in gray, are left exposed to the aqueous environment (Movie 3.4). (PDB code: 3NMD.) is considered to have three domains: the SH2 and SH3 domains have regulatory roles, while the C-terminal domain is

1	in gray, are left exposed to the aqueous environment (Movie 3.4). (PDB code: 3NMD.) is considered to have three domains: the SH2 and SH3 domains have regulatory roles, while the C-terminal domain is responsible for the kinase catalytic activity. Later in the chapter, we shall return to this protein, in order to explain how proteins can form molecular switches that transmit information throughout cells.

1	Figure 3–11 presents ribbon models of three differently organized protein domains. As these examples illustrate, the central core of a domain can be constructed from α helices, from β sheets, or from various combinations of these two fundamental folding elements. The smallest protein molecules contain only a single domain, whereas larger proteins can contain several dozen domains, often connected to each other by short, relatively unstructured lengths of polypeptide chain that can act as flexible hinges between domains. Few of the Many Possible Polypeptide Chains Will Be Useful to Cells

1	Few of the Many Possible Polypeptide Chains Will Be Useful to Cells Since each of the 20 amino acids is chemically distinct and each can, in principle, occur at any position in a protein chain, there are 20 × 20 × 20 × 20 = 160,000 different possible polypeptide chains four amino acids long, or 20n different possible polypeptide chains n amino acids long. For a typical protein length of about 300 amino acids, a cell could theoretically make more than 10390 (20300) different polypeptide chains. This is such an enormous number that to produce just one molecule of each kind would require many more atoms than exist in the universe. Only a very small fraction of this vast set of conceivable polypeptide chains would adopt a stable three-dimensional conformation—by some estimates, less

1	Only a very small fraction of this vast set of conceivable polypeptide chains would adopt a stable three-dimensional conformation—by some estimates, less Figure 3–10 a protein formed from multiple domains. In the Src protein shown, a C-terminal domain with two lobes (yellow and orange) forms a protein kinase enzyme, while the SH2 and SH3 domains perform regulatory functions. (A) A ribbon model, with ATP substrate in red. (B) A space-filling model, with ATP substrate in red. Note that the site that binds ATP is positioned at the interface of the two lobes that form the kinase. The structure of the SH2 domain was illustrated in Figure 3–6. (PDB code: 2SRC.)

1	Figure 3–11 Ribbon models of three different protein domains. (A) Cytochrome b562, a single-domain protein involved in electron transport in mitochondria. This protein is composed almost entirely of α helices. (B) The NAD-binding domain of the enzyme lactic dehydrogenase, which is composed of a mixture of α helices and parallel β sheets. (C) The variable domain of an immunoglobulin (antibody) light chain, composed of a sandwich of two antiparallel β sheets. In these examples, the α helices are shown in green, while strands organized as β sheets are denoted by red arrows. Note how the polypeptide chain generally traverses back and forth across the entire domain, making sharp turns only at the protein surface (Movie 3.5). It is the protruding loop regions (yellow) that often form the binding sites for other molecules. (Adapted from drawings courtesy of Jane Richardson.) than one in a billion. And yet the majority of proteins present in cells do adopt unique and stable conformations.

1	sites for other molecules. (Adapted from drawings courtesy of Jane Richardson.) than one in a billion. And yet the majority of proteins present in cells do adopt unique and stable conformations. How is this possible? The answer lies in natural selection. A protein with an unpredictably variable structure and biochemical activity is unlikely to help the survival of a cell that contains it. Such proteins would therefore have been eliminated by natural selection through the enormously long trial-and-error process that underlies biological evolution.

1	Because evolution has selected for protein function in living organisms, the amino acid sequence of most present-day proteins is such that a single conformation is stable. In addition, this conformation has its chemical properties finely tuned to enable the protein to perform a particular catalytic or structural function in the cell. Proteins are so precisely built that the change of even a few atoms in one amino acid can sometimes disrupt the structure of the whole molecule so severely that all function is lost. And, as discussed later in this chapter, when certain rare protein misfolding accidents occur, the results can be disastrous for the organisms that contain them.

1	Once a protein had evolved that folded up into a stable conformation with useful properties, its structure could be modified during evolution to enable it to perform new functions. This process has been greatly accelerated by genetic mechanisms that occasionally duplicate genes, allowing one gene copy to evolve independently to perform a new function (discussed in Chapter 4). This type of event has occurred very often in the past; as a result, many present-day proteins can be grouped into protein families, each family member having an amino acid sequence and a three-dimensional conformation that resemble those of the other family members.

1	Consider, for example, the serine proteases, a large family of protein-cleaving (proteolytic) enzymes that includes the digestive enzymes chymotrypsin, trypsin, and elastase, and several proteases involved in blood clotting. When the protease portions of any two of these enzymes are compared, parts of their amino acid sequences are found to match. The similarity of their three-dimensional conformations is even more striking: most of the detailed twists and turns in their polypeptide chains, which are several hundred amino acids long, are virtually identical (Figure 3–12). The many different serine proteases nevertheless have distinct enzymatic activities, each cleaving different proteins or the peptide bonds between different types of amino acids. Each therefore performs a distinct function in an organism. The story we have told for the serine proteases could be repeated for hundreds of other protein families. In general, the structure of the different members of a

1	The story we have told for the serine proteases could be repeated for hundreds of other protein families. In general, the structure of the different members of a Figure 3–12 a comparison of the conformations of two serine proteases. The backbone conformations of elastase and chymotrypsin. Although only those amino acids in the polypeptide chain shaded in green are the same in the two proteins, the two conformations are very similar nearly everywhere. The active site of each enzyme is circled in red; this is where the peptide bonds of the proteins that serve as substrates are bound and cleaved by hydrolysis. The serine proteases derive their name from the amino acid serine, whose side chain is part of the active site of each enzyme and directly participates in the cleavage reaction. The two dots on the right side of the chymotrypsin molecule mark the new ends created when this enzyme cuts its own backbone.

1	Figure 3–13 a comparison of a class of Dna-binding domains, called homeodomains, in a pair of proteins from two organisms separated by more than a billion years of evolution. (A) A ribbon model of the structure common to both proteins. (B) A trace of the α-carbon positions. The three-dimensional structures shown were determined by x-ray crystallography for the yeast α2 protein (green) and the Drosophila engrailed protein (red). (C) A comparison of amino acid sequences for the region of the proteins shown in (A) and (B). Black dots mark sites with identical amino acids. Orange dots indicate the position of a three-amino-acid insert in the α2 protein. (Adapted from C. Wolberger et al., Cell 67:517–528, 1991. With permission from Elsevier.) protein family has been more highly conserved than has the amino acid sequence. In many cases, the amino acid sequences have diverged so far that we cannot be certain of a family relationship between two proteins without determining their

1	than has the amino acid sequence. In many cases, the amino acid sequences have diverged so far that we cannot be certain of a family relationship between two proteins without determining their three-dimensional structures. The yeast α2 protein and the Drosophila engrailed protein, for example, are both gene regulatory proteins in the homeodomain family (discussed in Chapter 7). Because they are identical in only 17 of their 60 amino acid residues, their relationship became certain only by comparing their three-dimensional structures (Figure 3–13). Many similar examples show that two proteins with more than 25% identity in their amino acid sequences usually share the same overall structure.

1	The various members of a large protein family often have distinct functions. Some of the amino acid changes that make family members different were no doubt selected in the course of evolution because they resulted in useful changes in biological activity, giving the individual family members the different functional properties they have today. But many other amino acid changes are effectively “neutral,” having neither a beneficial nor a damaging effect on the basic structure and function of the protein. In addition, since mutation is a random process, there must also have been many deleterious changes that altered the three-dimensional structure of these proteins sufficiently to harm them. Such faulty proteins would have been lost whenever the individual organisms making them were at enough of a disadvantage to be eliminated by natural selection.

1	Protein families are readily recognized when the genome of any organism is sequenced; for example, the determination of the DNA sequence for the entire human genome has revealed that we contain about 21,000 protein-coding genes. (Note, however, that as a result of alternative RNA splicing, human cells can produce much more than 21,000 different proteins, as will be explained in Chapter 6.) Through sequence comparisons, we can assign the products of at least 40% of our protein-coding genes to known protein structures, belonging to more than 500 different protein families. Most of the proteins in each family have evolved to perform somewhat different functions, as for the enzymes elastase and chymotrypsin illustrated previously in Figure 3–12. As explained in Chapter 1 (see Figure 1–21), these are sometimes called paralogs to distinguish them from the many corresponding proteins in different organisms (orthologs, such as mouse and human elastase).

1	As described in Chapter 8, because of the powerful techniques of x-ray crystallography and nuclear magnetic resonance (NMR), we now know the three-dimensional shapes, or conformations, of more than 100,000 proteins. By carefully comparing the conformations of these proteins, structural biologists (that is, experts on the structure of biological molecules) have concluded that there are a limited number of ways in which protein domains fold up in nature—maybe as few as 2000, if we consider all organisms. For most of these so-called protein folds, representative structures have been determined.

1	The present database of known protein sequences contains more than twenty million entries, and it is growing very rapidly as more and more genomes are sequenced—revealing huge numbers of new genes that encode proteins. The encoded polypeptides range widely in size, from 6 amino acids to a gigantic protein of 33,000 amino acids. Protein comparisons are important because related structures often imply related functions. Many years of experimentation can be saved by discovering that a new protein has an amino acid sequence similarity with a protein of known function. Such sequence relationships, for example, first indicated that certain genes that cause mammalian cells to become cancerous encode protein kinases (discussed in Chapter 20).

1	As previously stated, most proteins are composed of a series of protein domains, in which different regions of the polypeptide chain fold independently to form compact structures. Such multidomain proteins are believed to have originated from the accidental joining of the DNA sequences that encode each domain, creating a new gene. In an evolutionary process called domain shuffling, many large proteins have evolved through the joining of preexisting domains in new combinations (Figure 3–14). Novel binding surfaces have often been created at the juxtaposition of domains, and many of the functional sites where proteins bind to small molecules are found to be located there. A subset of protein domains has been especially mobile during evolution; these seem to have particularly versatile structures and are sometimes referred to as protein modules. The structure of one, the SH2 domain, was illustrated in Figure 3–6. Three other abundant protein domains are illustrated in Figure 3–15.

1	Each of the domains shown has a stable core structure formed from strands of β sheets, from which less-ordered loops of polypeptide chain protrude. The loops are ideally situated to form binding sites for other molecules, as most clearly demonstrated for the immunoglobulin fold, which forms the basis for antibody molecules. Such β-sheet-based domains may have achieved their evolutionary success because they provide a convenient framework for the generation of new binding sites for ligands, requiring only small changes to their protruding loops (see Figure 3–42).

1	Figure 3–14 Domain shuffling. An extensive shuffling of blocks of protein sequence (protein domains) has occurred during protein evolution. Those portions of a protein denoted by the same shape and color in this diagram are evolutionarily related. Serine proteases like chymotrypsin are formed from two domains (brown). In the three other proteases shown, which are highly regulated and more specialized, these two protease domains are connected to one or more domains that are similar to domains found in epidermal growth factor (EGF; green), to a calcium-binding protein (yellow), or to a “kringle” domain (blue). Chymotrypsin is illustrated in Figure 3–12. Figure 3–15 The three-dimensional structures of three commonly used protein domains. In these ribbon diagrams, β-sheet strands are shown as arrows, and the Nand C-termini are indicated by red spheres. Many more such “modules” exist in nature. (Adapted from

1	M. Baron, D.G. Norman and I.D. Campbell, Trends Biochem. Sci. 16:13–17, 1991, with permission from Elsevier, and D.J. Leahy et al., Science 258:987–991, 1992, with permission from AAAS.) Figure 3–16 an extended structure formed from a series of protein domains. Four fibronectin type 3 domains (see Figure 3–15) from the extracellular matrix molecule fibronectin are illustrated in (A) ribbon and (B) space-filling models. (Adapted from D.J. Leahy, I. Aukhil and H.P. Erickson, Cell 84:155–164, 1996. With permission from Elsevier.)

1	A second feature of these protein domains that explains their utility is the ease with which they can be integrated into other proteins. Two of the three domains illustrated in Figure 3–15 have their Nand C-terminal ends at opposite poles of the domain. When the DNA encoding such a domain undergoes tandem duplication, which is not unusual in the evolution of genomes (discussed in Chapter 4), the duplicated domains with this “in-line” arrangement can be readily linked in series to form extended structures—either with themselves or with other in-line domains (Figure 3–16). Stiff extended structures composed of a series of domains are especially common in extracellular matrix molecules and in the extracellular portions of cell-surface receptor proteins. Other frequently used domains, including the kringle domain illustrated in Figure 3–15 and the SH2 domain, are of a “plug-in” type, with their Nand C-termini close together. After genomic rearrangements, such domains are usually

1	including the kringle domain illustrated in Figure 3–15 and the SH2 domain, are of a “plug-in” type, with their Nand C-termini close together. After genomic rearrangements, such domains are usually accommodated as an insertion into a loop region of a second protein.

1	A comparison of the relative frequency of domain utilization in different eukaryotes reveals that, for many common domains, such as protein kinases, this frequency is similar in organisms as diverse as yeast, plants, worms, flies, and humans. But there are some notable exceptions, such as the Major Histocompatibility Complex (MHC) antigen-recognition domain (see Figure 24–36) that is present in 57 copies in humans, but absent in the other four organisms just mentioned. Domains such as these have specialized functions that are not shared with the other eukaryotes; they are assumed to have been strongly selected for during recent evolution to produce the multiple copies observed. Similarly, the SH2 domain shows an unusual increase in its numbers in higher eukaryotes; such domains might be assumed to be especially useful for multicellularity. Certain Pairs of Domains Are Found Together in Many Proteins

1	Certain Pairs of Domains Are Found Together in Many Proteins We can construct a large table displaying domain usage for each organism whose genome sequence is known. For example, the human genome contains the DNA sequences for about 1000 immunoglobulin domains, 500 protein kinase domains, 250 DNA-binding homeodomains, 300 SH3 domains, and 120 SH2 domains. In addition, we find that more than two-thirds of all proteins consist of two or more domains, and that the same pairs of domains occur repeatedly in the same relative arrangement in a protein. Although half of all domain families are common to archaea, bacteria, and eukaryotes, only about 5% of the two-domain combinations are similarly shared. This pattern suggests that most proteins containing especially useful two-domain combinations arose through domain shuffling relatively late in evolution. The Human Genome Encodes a Complex Set of Proteins, Revealing That Much Remains Unknown

1	The Human Genome Encodes a Complex Set of Proteins, Revealing That Much Remains Unknown The result of sequencing the human genome has been surprising, because it reveals that our chromosomes contain only about 21,000 protein-coding genes. Based on this number alone, we would appear to be no more complex than the tiny mustard weed, Arabidopsis, and only about 1.3-fold more complex than a nematode worm. The genome sequences also reveal that vertebrates have inherited nearly all of their protein domains from invertebrates—with only 7% of identified human domains being vertebrate-specific. Each of our proteins is on average more complicated, however (Figure 3–17). Domain shuffling during vertebrate evolution has given rise to many novel

1	Each of our proteins is on average more complicated, however (Figure 3–17). Domain shuffling during vertebrate evolution has given rise to many novel Figure 3–17 Domain structure of a group of evolutionarily related proteins that are thought to have a similar function. In general, there is a tendency for the proteins in more complex organisms, such as humans, to contain additional domains—as is the case for the DNA-binding protein compared here. combinations of protein domains, with the result that there are nearly twice as many combinations of domains found in human proteins as in a worm or a fly. Thus, for example, the trypsinlike serine protease domain is linked to at least 18 other types of protein domains in human proteins, whereas it is found covalently joined to only 5 different domains in the worm. This extra variety in our proteins greatly increases the range of protein–protein interactions possible (see Figure 3–79), but how it contributes to making us human is not known.

1	The complexity of living organisms is staggering, and it is quite sobering to note that we currently lack even the tiniest hint of what the function might be for more than 10,000 of the proteins that have thus far been identified through examining the human genome. There are certainly enormous challenges ahead for the next generation of cell biologists, with no shortage of fascinating mysteries to solve.

1	The same weak noncovalent bonds that enable a protein chain to fold into a specific conformation also allow proteins to bind to each other to produce larger structures in the cell. Any region of a protein’s surface that can interact with another molecule through sets of noncovalent bonds is called a binding site. A protein can contain binding sites for various large and small molecules. If a binding site recognizes the surface of a second protein, the tight binding of two folded polypeptide chains at this site creates a larger protein molecule with a precisely defined geometry. Each polypeptide chain in such a protein is called a protein subunit.

1	In the simplest case, two identical folded polypeptide chains bind to each other in a “head-to-head” arrangement, forming a symmetric complex of two protein subunits (a dimer) held together by interactions between two identical binding sites. The Cro repressor protein—a viral gene regulatory protein that binds to DNA to turn specific viral genes off in an infected bacterial cell—provides an example (Figure 3–18). Cells contain many other types of symmetric protein complexes, formed from multiple copies of a single polypeptide chain (for example, see Figure 3–20 below). Many of the proteins in cells contain two or more types of polypeptide chains. Hemoglobin, the protein that carries oxygen in red blood cells, contains two identical α-globin subunits and two identical β-globin subunits, symmetrically arranged (Figure 3–19). Such multisubunit proteins are very common in cells, and they can be very large (Movie 3.6).

1	Most of the proteins that we have discussed so far are globular proteins, in which the polypeptide chain folds up into a compact shape like a ball with an irregular surface. Some of these protein molecules can nevertheless assemble to form filaments that may span the entire length of a cell. Most simply, a long chain of identical protein molecules can be constructed if each molecule has a binding Figure 3–18 Two identical protein subunits binding together to form a symmetric protein dimer. The Cro repressor protein from bacteriophage lambda binds to DNA to turn off a specific subset of viral genes. Its two identical subunits bind head-to-head, held together by a combination of hydrophobic forces (blue) and a set of hydrogen bonds (yellow region). (Adapted from D.H. Ohlendorf, D.E. Tronrud and B.W. Matthews, J. Mol. Biol. 280:129–136, 1998. With permission from Academic Press.)

1	D.E. Tronrud and B.W. Matthews, J. Mol. Biol. 280:129–136, 1998. With permission from Academic Press.) Figure 3–19 a protein formed as a symmetric assembly using two each of two different subunits. Hemoglobin is an abundant protein in red blood cells that contains two copies of α-globin (green) and two copies of β-globin (blue). Each of these four polypeptide chains contains a heme molecule (red), which is the site that binds oxygen (O2). Thus, each molecule of hemoglobin in the blood carries four molecules of oxygen. (PDB code: 2DHB.) Figure 3–20 Protein assemblies. (A) A protein with just one binding site can (A) form a dimer with another identical protein. (B) Identical proteins with two free assembled different binding sites often form a long helical filament. (C) If the two binding subunits structures sites are disposed appropriately in relation to each other, the protein subunits may form a closed ring instead of a helix. (For an example of A, see

1	Figure 3–18; for an example of B, see Figure 3–21; for examples of C, see Figures 5–14 and 14–31.) site site complementary to another region of the surface of the same molecule (Figure 3–20). An actin filament, for example, is a long helical structure produced from many molecules of the protein actin (Figure 3–21). Actin is a globular protein that is very abundant in eukaryotic cells, where it forms one of the major filament systems of the cytoskeleton (discussed in Chapter 16).

1	We will encounter many helical structures in this book. Why is a helix such a common structure in biology? As we have seen, biological structures are often formed by linking similar subunits into long, repetitive chains. If all the subunits are identical, the neighboring subunits in the chain can often fit together in only one way, adjusting their relative positions to minimize the free energy of the contact between them. As a result, each subunit is positioned in exactly the same way in relation to the next, so that subunit 3 fits onto subunit 2 in the same way that subunit 2 fits onto subunit 1, and so on. Because it is very rare for subunits to join up in a straight line, this arrangement generally results in a helix—a regular structure that resembles a spiral staircase, as illustrated in Figure 3–22. Depending on the twist of the staircase, a helix is said to be either right-handed or left-handed (see Figure 3–22E). Handedness is not affected by turning the helix upside down, but

1	in Figure 3–22. Depending on the twist of the staircase, a helix is said to be either right-handed or left-handed (see Figure 3–22E). Handedness is not affected by turning the helix upside down, but it is reversed if the helix is reflected in the mirror.

1	The observation that helices occur commonly in biological structures holds true whether the subunits are small molecules linked together by covalent bonds (for example, the amino acids in an α helix) or large protein molecules that are linked by noncovalent forces (for example, the actin molecules in actin filaments). This is not surprising. A helix is an unexceptional structure, and it is generated simply by placing many similar subunits next to each other, each in the same strictly repeated relationship to the one before—that is, with a fixed rotation followed by a fixed translation along the helix axis, as in a spiral staircase. Many Protein Molecules Have Elongated, Fibrous Shapes

1	Many Protein Molecules Have Elongated, Fibrous Shapes Enzymes tend to be globular proteins: even though many are large and complicated, with multiple subunits, most have an overall rounded shape. In Figure 3–21, we saw that a globular protein can also associate to form long filaments. But there are also functions that require each individual protein molecule to span a large distance. These proteins generally have a relatively simple, elongated three-dimensional structure and are commonly referred to as fibrous proteins.

1	One large family of intracellular fibrous proteins consists of α-keratin, introduced when we presented the α helix, and its relatives. Keratin filaments are extremely stable and are the main component in long-lived structures such as hair, horn, and nails. An α-keratin molecule is a dimer of two identical subunits, with the long α helices of each subunit forming a coiled-coil (see Figure 3–9). The coiled-coil regions are capped at each end by globular domains containing binding sites. This enables this class of protein to assemble into ropelike intermediate filaments—an important component of the cytoskeleton that creates the cell’s internal structural framework (see Figure 16–67).

1	Fibrous proteins are especially abundant outside the cell, where they are a main component of the gel-like extracellular matrix that helps to bind collections of cells together to form tissues. Cells secrete extracellular matrix proteins into their surroundings, where they often assemble into sheets or long fibrils. Collagen is the most abundant of these proteins in animal tissues. A collagen molecule consists of three long polypeptide chains, each containing the nonpolar amino Figure 3–21 actin filaments. (A) Transmission electron micrographs of negatively stained actin filaments. (B) The helical arrangement of actin molecules in an actin filament. (A, courtesy of Roger Craig.) Figure 3–22 Some properties of a helix.

1	(A–D) A helix forms when a series of subunits bind to each other in a regular way. At the bottom, each of these helices is viewed from directly above the helix and seen to have two (A), three (B), and six (C and D) subunits per helical turn. Note that the helix in (D) has a wider path than that in (C), but the same number of subunits per turn. (E) As discussed in the text, a helix can be either right-handed or left-handed. As a reference, it is useful to remember that standard metal screws, which insert when turned clockwise, are right-handed. Note that a helix retains the same handedness when it is turned upside down. (PDB code: 2DHB.) acid glycine at every third position. This regular structure allows the chains to wind around one another to generate a long regular triple helix (Figure 3–23A). Many collagen molecules then bind to one another side-by-side and end-toend to create long overlapping arrays—thereby generating the extremely tough collagen fibrils that give connective

1	3–23A). Many collagen molecules then bind to one another side-by-side and end-toend to create long overlapping arrays—thereby generating the extremely tough collagen fibrils that give connective tissues their tensile strength, as described in Chapter 19.

1	Proteins Contain a Surprisingly Large Amount of Intrinsically Disordered Polypeptide Chain It has been well known for a long time that, in complete contrast to collagen, another abundant protein in the extracellular matrix, elastin, is formed as a highly disordered polypeptide. This disorder is essential for elastin’s function. Its relatively loose and unstructured polypeptide chains are covalently cross-linked to short section of 50 nm collagen fbril collagen molecule 300 nm × 1.5 nm 1.5 nm Figure 3–23 Collagen and elastin. (A) Collagen is a triple helix formed by three extended protein chains that wrap around one another (bottom). Many rodlike collagen molecules are cross-linked together in the extracellular space to form unextendable collagen fibrils (top) that have the tensile strength of steel. The striping on the collagen fibril is caused by the regular repeating arrangement of the collagen molecules within the fibril.

1	(B) Elastin polypeptide chains are cross-linked together in the extracellular space to form rubberlike, elastic fibers. Each elastin molecule uncoils into a more extended conformation when the fiber is stretched and recoils spontaneously as soon as the stretching force is relaxed. The cross-linking in the extracellular space mentioned creates covalent linkages between lysine side chains, but the chemistry is different for collagen and elastin. produce a rubberlike, elastic meshwork that can be reversibly pulled from one conformation to another, as illustrated in Figure 3–23B. The elastic fibers that result enable skin and other tissues, such as arteries and lungs, to stretch and recoil without tearing.

1	Intrinsically disordered regions of proteins are frequent in nature, and they have important functions in the interior of cells. As we have already seen, proteins often have loops of polypeptide chain that protrude from the core region of a protein domain to bind other molecules. Some of these loops remain largely unstructured until they bind to a target molecule, adopting a specific folded conformation only when this other molecule is bound. Many proteins were also known to have intrinsically disordered tails at one or the other end of a structured domain (see, for example, the histones in Figure 4–24). But the extent of such disordered structure only became clear when genomes were sequenced. This allowed bioinformatic methods to be used to analyze the amino acid sequences that genes encode, searching for disordered regions based on their unusually low hydrophobicity and relatively high net charge. Combining these results with other data, it is now thought that perhaps a quarter of

1	searching for disordered regions based on their unusually low hydrophobicity and relatively high net charge. Combining these results with other data, it is now thought that perhaps a quarter of all eukaryotic proteins can adopt structures that are mostly disordered, fluctuating rapidly between many different conformations. Many such intrinsically disordered regions contain repeated sequences of amino acids. What do these disordered regions do?

1	Some known functions are illustrated in Figure 3–24. One predominant function is to form specific binding sites for other protein molecules that are of high specificity, but readily altered by protein phosphorylation, protein dephosphorylation, or any of the other covalent modifications that are triggered by cell signaling events (Figure 3–24A and B). We shall see, for example, that the eukaryotic RNA polymerase enzyme that produces mRNAs contains a long, unstructured C-terminal tail that is covalently modified as its RNA synthesis proceeds, thereby attracting specific other proteins to the transcription complex at different times (see Figure 6–22). And this unstructured tail interacts with a different type of low complexity domain when the RNA polymerase is recruited to the specific sites on the DNA where it begins synthesis.

1	As illustrated in Figure 3–24C, an unstructured region can also serve as a “tether” to hold two protein domains in close proximity to facilitate their interaction. For example, it is this tethering function that allows substrates to move between active sites in large multienzyme complexes (see Figure 3–54). A similar tethering function allows large scaffold proteins with multiple protein-binding sites to concentrate sets of interacting proteins, both increasing reaction rates and confining their reaction to a particular site in a cell (see Figure 3–78).

1	Like elastin, other proteins have a function that directly requires that they remain largely unstructured. Thus, large numbers of disordered protein chains in close proximity can create micro-regions of gel-like consistency inside the cell that restrict diffusion. For example, the abundant nucleoporins that coat the inner surface of the nuclear pore complex form a random coil meshwork (Figure 3–24) that is critical for selective nuclear transport (see Figure 12–8).

1	Figure 3–24 Some important functions for intrinsically disordered protein sequences. (A) Unstructured regions of polypeptide chain often form binding sites for other proteins. Although these binding events are of high specificity, they are often of low affinity due to the free-energy cost of folding the normally unfolded partner (and they are thus readily reversible). (B) Unstructured regions can be easily modified covalently to change their binding preferences, and they are therefore frequently involved in cell signaling processes. In this schematic, multiple sites of protein phosphorylation are indicated. (C) Unstructured regions frequently create “tethers” that hold interacting protein domains in close proximity. (D) A dense network of unstructured proteins can form a diffusion barrier, as the nucleoporins do for the nuclear pore.

1	Figure 3–25 Disulfide bonds. Covalent disulfide bonds form between adjacent cysteine side chains. These cross-linkages can join either two parts of the same polypeptide chain or two different polypeptide chains. Since the energy required to break one covalent bond is much larger than the energy required to break even a whole set of noncovalent bonds (see Table 2–1, p. 45), a disulfide bond can have a major stabilizing effect on a protein (Movie 3.7).

1	Many protein molecules are either attached to the outside of a cell’s plasma membrane or secreted as part of the extracellular matrix. All such proteins are directly exposed to extracellular conditions. To help maintain their structures, the polypeptide chains in such proteins are often stabilized by covalent cross-linkages. These linkages can either tie together two amino acids in the same protein, or connect different polypeptide chains in a multisubunit protein. Although many other types exist, the most common cross-linkages in proteins are covalent sulfur– sulfur bonds. These disulfide bonds (also called S–S bonds) form as cells prepare newly synthesized proteins for export. As described in Chapter 12, their formation is catalyzed in the endoplasmic reticulum by an enzyme that links together two pairs of –SH groups of cysteine side chains that are adjacent in the folded protein (Figure 3–25). Disulfide bonds do not change the conformation of a protein but instead act as atomic

1	together two pairs of –SH groups of cysteine side chains that are adjacent in the folded protein (Figure 3–25). Disulfide bonds do not change the conformation of a protein but instead act as atomic staples to reinforce its most favored conformation. For example, lysozyme—an enzyme in tears that dissolves bacterial cell walls—retains its antibacterial activity for a long time because it is stabilized by such cross-linkages.

1	Disulfide bonds generally fail to form in the cytosol, where a high concentration of reducing agents converts S–S bonds back to cysteine –SH groups. Apparently, proteins do not require this type of reinforcement in the relatively mild environment inside the cell. Protein Molecules Often Serve as Subunits for the Assembly of Large Structures The same principles that enable a protein molecule to associate with itself to form rings or a long filament also operate to generate much larger structures formed from a set of different macromolecules, such as enzyme complexes, ribosomes, viruses, and membranes. These large objects are not made as single, giant, covalently linked molecules. Instead they are formed by the noncovalent assembly of many separately manufactured molecules, which serve as the subunits of the final structure. The use of smaller subunits to build larger structures has several advantages: 1.

1	The use of smaller subunits to build larger structures has several advantages: 1. A large structure built from one or a few repeating smaller subunits requires only a small amount of genetic information. 2. Both assembly and disassembly can be readily controlled reversible processes, because the subunits associate through multiple bonds of relatively low energy. 3. Errors in the synthesis of the structure can be more easily avoided, since correction mechanisms can operate during the course of assembly to exclude malformed subunits.

1	3. Errors in the synthesis of the structure can be more easily avoided, since correction mechanisms can operate during the course of assembly to exclude malformed subunits. Some protein subunits assemble into flat sheets in which the subunits are arranged in hexagonal patterns. Specialized membrane proteins are sometimes arranged this way in lipid bilayers. With a slight change in the geometry of the individual subunits, a hexagonal sheet can be converted into a tube (Figure 3–26) or, with more changes, into a hollow sphere. Protein tubes and spheres that bind specific RNA and DNA molecules in their interior form the coats of viruses.

1	The formation of closed structures, such as rings, tubes, or spheres, provides additional stability because it increases the number of bonds between the protein subunits. Moreover, because such a structure is created by mutually dependent, cooperative interactions between subunits, a relatively small change that affects each subunit individually can cause the structure to assemble or disassemble. These principles are dramatically illustrated in the protein coat or capsid of many simple viruses, which takes the form of a hollow sphere based on an icosahedron (Figure 3–27). Capsids are often made of hundreds of identical protein subunits that enclose and protect the viral nucleic acid (Figure 3–28). The protein in such a capsid must have a particularly adaptable structure: not only must it make several different kinds of contacts to create the sphere, it must also change this arrangement to let the nucleic acid out to initiate viral replication once the virus has entered a cell.

1	Many Structures in Cells Are Capable of Self-Assembly The information for forming many of the complex assemblies of macromolecules in cells must be contained in the subunits themselves, because purified subunits can spontaneously assemble into the final structure under the appropriate conditions. The first large macromolecular aggregate shown to be capable of self-assembly from its component parts was tobacco mosaic virus (TMV ). This virus is a long rod in which a cylinder of protein is arranged around a helical RNA core (Figure 3–29). If the dissociated RNA and protein subunits are mixed together in solution, they recombine to form fully active viral particles. The assembly process is unexpectedly complex and includes the formation of double rings of protein, which serve as intermediates that add to the growing viral coat.

1	Another complex macromolecular aggregate that can reassemble from its component parts is the bacterial ribosome. This structure is composed of about 55 different protein molecules and 3 different rRNA molecules. Incubating a mixture of the individual components under appropriate conditions in a test tube causes them to spontaneously re-form the original structure. Most importantly, such reconstituted ribosomes are able to catalyze protein synthesis. As might be expected, the reassembly of ribosomes follows a specific pathway: after certain proteins have bound to the RNA, this complex is then recognized by other proteins, and so on, until the structure is complete. It is still not clear how some of the more elaborate self-assembly processes are regulated. Many structures in the cell, for example, seem to have a precisely defined length that is many times greater than that of their component macromolecules. How such length determination is achieved is in many cases a mystery. In

1	Figure 3–26 Single protein subunits form protein assemblies that feature multiple protein–protein contacts. Hexagonally packed globular protein subunits are shown here forming either flat sheets or tubes. Generally, such large structures are not considered to be single “molecules.” Instead, like the actin filament described previously, they are viewed as assemblies formed of many different molecules.

1	Figure 3–27 The protein capsid of a virus. The structure of the simian virus SV40 capsid has been determined by x-ray crystallography and, as for the capsids of many other viruses, it is known in atomic detail. (Courtesy of Robert Grant, Stephan Crainic, and James M. Hogle.) capsid protein intact virus monomer particle shown as (90 dimers) the simplest case, a long core protein or other macromolecule provides a scaffold that determines the extent of the final assembly. This is the mechanism that determines the length of the TMV particle, where the RNA chain provides the core. Similarly, a core protein interacting with actin is thought to determine the length of the thin filaments in muscle.

1	Figure 3–28 The structure of a spherical virus. In viruses, many copies of a single protein subunit often pack together to create a spherical shell (a capsid). This capsid encloses the viral genome, composed of either RNA or DNA (see also Figure 3–27). For geometric reasons, no more than 60 identical subunits can pack together in a precisely symmetric way. If slight irregularities are allowed, however, more subunits can be used to produce a larger capsid that retains icosahedral symmetry. The tomato bushy stunt virus (TBSV) shown here, for example, is a spherical virus about 33 nm in diameter formed from 180 identical copies of a 386-amino-acid capsid protein plus an RNA genome of 4500 nucleotides. To construct such a large capsid, the protein must be able to fit into three somewhat different environments. This requires three slightly different conformations, each of which is differently colored in the virus particle shown here. The postulated pathway of assembly is shown; the precise

1	environments. This requires three slightly different conformations, each of which is differently colored in the virus particle shown here. The postulated pathway of assembly is shown; the precise three-dimensional structure has been determined by x-ray diffraction. (Courtesy of Steve Harrison.)

1	Figure 3–29 The structure of tobacco mosaic virus (TMV). (A) An electron micrograph of the viral particle, which consists of a single long RNA molecule enclosed in a cylindrical protein coat composed of identical protein subunits. (B) A model showing part of the structure of TMV. A single-stranded RNA molecule of 6395 nucleotides is packaged in a helical coat constructed from 2130 copies of a coat protein 158 amino acids long. Fully infective viral particles can self-assemble in a test tube from purified RNA and protein molecules. (A, courtesy of Robley Williams; B, courtesy of Richard J. Feldmann.)

1	Figure 3–30 Proteolytic cleavage in insulin assembly. The polypeptide hormone insulin cannot spontaneously re-form efficiently if its disulfide bonds are disrupted. It is synthesized as a larger protein (proinsulin) that is cleaved by a proteolytic enzyme after the protein chain has folded into a specific shape. Excision of part of the proinsulin polypeptide chain removes some of the information needed for the protein to fold spontaneously into its normal conformation. Once insulin has been denatured and its two polypeptide chains have separated, its ability to reassemble is lost. Assembly Factors Often Aid the Formation of Complex Biological Structures

1	Assembly Factors Often Aid the Formation of Complex Biological Structures Not all cellular structures held together by noncovalent bonds self-assemble. A cilium, or a myofibril of a muscle cell, for example, cannot form spontaneously from a solution of its component macromolecules. In these cases, part of the assembly information is provided by special enzymes and other proteins that perform the function of templates, serving as assembly factors that guide construction but take no part in the final assembled structure.

1	Even relatively simple structures may lack some of the ingredients necessary for their own assembly. In the formation of certain bacterial viruses, for example, the head, which is composed of many copies of a single protein subunit, is assembled on a temporary scaffold composed of a second protein that is produced by the virus. Because the second protein is absent from the final viral particle, the head structure cannot spontaneously reassemble once it has been taken apart. Other examples are known in which proteolytic cleavage is an essential and irreversible step in the normal assembly process. This is even the case for some small protein assemblies, including the structural protein collagen and the hormone insulin (Figure 3–30). From these relatively simple examples, it seems certain that the assembly of a structure as complex as a cilium will involve a temporal and spatial ordering that is imparted by numerous other components.

1	A special class of protein structures, utilized for some normal cell functions, can also contribute to human diseases when not controlled. These are self-propagating, stable β-sheet aggregates called amyloid fibrils. These fibrils are built from a series of identical polypeptide chains that become layered one over the other to create a continuous stack of β sheets, with the β strands oriented perpendicular to the fibril axis to form a cross-beta filament (Figure 3–31). Typically, hundreds of monomers will aggregate to form an unbranched fibrous structure that is several micrometers long and 5 to 15 nm in width. A surprisingly large fraction of proteins have the potential to form such structures, because the short segment of the polypeptide chain that forms the spine of the fibril can have a variety of different sequences and follow one of several different paths (Figure 3–32). However, very few proteins will actually form this structure inside cells.

1	In normal humans, the quality control mechanisms governing proteins gradually decline with age, occasionally permitting normal proteins to form pathological aggregates. The protein aggregates may be released from dead cells and accumulate as amyloid in the extracellular matrix. In extreme cases, the accumulation of such amyloid fibrils in the cell interior can kill the cells and damage tissues. Because the brain is composed of a highly organized collection of nerve cells that cannot regenerate, the brain is especially vulnerable to this sort of cumulative damage. Thus, although amyloid fibrils may form in different tissues, and are known to cause pathologies in several sites in the body, the most severe amyloid pathologies are neurodegenerative diseases. For example, the abnormal formation of highly stable amyloid fibrils is thought to play a central causative role in both Alzheimer’s and Parkinson’s diseases.

1	Prion diseases are a special type of these pathologies. They have attained special notoriety because, unlike Parkinson’s or Alzheimer’s, prion diseases can spread from one organism to another, providing that the second organism eats a S S S S S S SH SH SH SH SH SH specifc folding stabilized by disulfde bonds connecting peptide removed, leaving complete two-chain insulin molecule reduction irreversibly separates the two chains

1	Figure 3–31 Detailed structure of the core of an amyloid fibril. Illustrated here is the cross-beta spine of the amyloid fibril that is formed by a peptide of seven amino acids from the protein Sup35, an extensively studied yeast prion. Consisting of the sequence glycine-asparagine-asparagine-glutamine-glutamine-asparaginetyrosine (GNNQQNY), its structure was determined by X-ray crystallography. Although the cross-beta spines of other amyloids are similar, being composed of two long β sheets held together by a “steric zipper,” different detailed structures are observed depending on the short peptide sequence involved. (A) One half of the spine is illustrated. Here, a standard parallel β-sheet structure (see p. 116) is held together by a set of hydrogen bonds between two side chains plus hydrogen bonds between two backbone atoms, as illustrated (oxygen atoms red and nitrogen atoms blue). Note that in this example, the adjacent peptides are exactly in register. Although only five

1	plus hydrogen bonds between two backbone atoms, as illustrated (oxygen atoms red and nitrogen atoms blue). Note that in this example, the adjacent peptides are exactly in register. Although only five layers are shown (each layer depicted as an arrow), the actual structure would extend for many tens of thousands of layers in the plane of the paper. (B) The complete cross-beta spine. A second, identical β-sheet is paired with the first one to form a two-sheet motif that runs the entire length of the fibril. (C) View of the complete spine in (B) from the top. The closely interdigitated side chains form a tight, water-free junction known as a steric zipper. (Courtesy of David Eisenberg and Michael Sawaya, UCLA; based on R. Nelson et al., Nature 435:773–778, 2005. With permission from Macmillan Publishers Ltd.) tissue containing the protein aggregate. A set of closely related diseases—scrapie in sheep, Creutzfeldt–Jakob disease (CJD) in humans, Kuru in humans, and bovine spongiform

1	Publishers Ltd.) tissue containing the protein aggregate. A set of closely related diseases—scrapie in sheep, Creutzfeldt–Jakob disease (CJD) in humans, Kuru in humans, and bovine spongiform encephalopathy (BSE) in cattle—are caused by a misfolded, aggregated form of a particular protein called PrP (for prion protein). PrP is normally located on the outer surface of the plasma membrane, most prominently in neurons, and it has the unfortunate property of forming amyloid fibrils that are “infectious” because they convert normally folded molecules of PrP to the same pathological form (Figure 3–33). This property creates a positive feedback loop that propagates the abnormal form of PrP, called PrP*, and allows the pathological conformation to spread rapidly from cell to cell in the brain, eventually causing death. It can be dangerous to eat the tissues of animals that contain PrP*, as witnessed by the spread of BSE (commonly referred to as “mad cow disease”) from cattle to humans.

1	eventually causing death. It can be dangerous to eat the tissues of animals that contain PrP*, as witnessed by the spread of BSE (commonly referred to as “mad cow disease”) from cattle to humans. Fortunately, in the absence of PrP*, PrP is extraordinarily difficult to convert to its abnormal form.

1	A closely related “protein-only inheritance” has been observed in yeast cells. The ability to study infectious proteins in yeast has clarified another remarkable feature of prions. These protein molecules can form several distinctively different types of amyloid fibrils from the same polypeptide chain. Moreover, each type of aggregate can be infectious, forcing normal protein molecules to adopt the same type of abnormal structure. Thus, several different “strains” of infectious particles can arise from the same polypeptide chain.

1	Figure 3–32 The structure of an amyloid fibril. (A) Schematic diagram of the structure of a amyloid fibril that is formed by the aggregation of a protein. Only the cross-beta spine of an amyloid fibril resembles the structure shown in Figure 3–31. (B) A cut-away view of a structure proposed for the amyloid fibril that can be formed in a test tube by the enzyme ribonuclease A, illustrating how the core of the fibril—formed by a short segment— relates to the rest of the structure. Electron micrograph of amyloid fibrils. (A, from L. Esposito, C. Pedone and L. Vitagliano, Proc. Natl Acad. Sci. USA 103:11533–11538, 2006; B, from S. Sambashivan et al., Nature 437:266–269, 2005; C, courtesy of David Eisenberg.) Figure 3–33 The special protein aggregates that cause prion diseases.

1	Figure 3–33 The special protein aggregates that cause prion diseases. (A) Schematic illustration of the type of conformational change in the PrP protein (prion protein) that produces material for an amyloid fibril. (B) The self-infectious nature of the protein aggregation that is central to prion diseases. PrP is highly unusual because the misfolded version of the protein, called PrP*, induces the normal PrP protein it contacts to change its conformation, as shown.

1	Amyloid fibrils were initially studied because they cause disease. But the same type of structure is now known to be exploited by cells for useful purposes. Eukaryotic cells, for example, store many different peptide and protein hormones that they will secrete in specialized “secretory granules,” which package a high concentration of their cargo in dense cores with a regular structure (see Figure 13–65). We now know that these structured cores consist of amyloid fibrils, which in this case have a structure that causes them to dissolve to release soluble cargo after being secreted by exocytosis to the cell exterior (Figure 3–34A). Many bacteria use the amyloid structure in a very different way, secreting proteins that form long amyloid fibrils projecting from the cell exterior that help to bind bacterial neighbors into biofilms (Figure 3–34B). Because these biofilms help bacteria to survive in adverse environments (including in humans treated with antibiotics), new drugs that

1	help to bind bacterial neighbors into biofilms (Figure 3–34B). Because these biofilms help bacteria to survive in adverse environments (including in humans treated with antibiotics), new drugs that specifically disrupt the fibrous networks formed by bacterial amyloids have promise for treating human infections.

1	Until recently, those amyloids with useful functions were thought to be either confined to the interior of specialized vesicles or expressed on the exterior of cells, as in Figure 3–34. However, new experiments reveal that a large set of low complexity domains can form amyloid fibers that have functional roles in both the cell nucleus and the cell cytoplasm. These domains are normally unstructured and consist of stretches of amino acid sequence that can span hundreds of amino acids, while containing only a small subset of the 20 different amino acids. In contrast to the disease-associated amyloid in Figure 3–33, these newly discovered structures are held together by weaker noncovalent bonds and readily dissociate in response to signals—hence their name reversible amyloids. Many proteins with such domains also contain a different set of domains that bind to specific other protein or RNA molecules. Thus, their controlled aggregation

1	Many proteins with such domains also contain a different set of domains that bind to specific other protein or RNA molecules. Thus, their controlled aggregation Golgi amyloid template subunit cisterna fbril subunit of fbril (A) prion protein can adopt an abnormal, misfolded form normal Prp abnormal prion form protein of PrP protein (Prp*) (B) misfolded protein can induce formation of protein aggregates the conversion of more PrP to misfolded form creates a stable amyloid fbril protein aggregate in form of amyloid fbril

1	Figure 3–34 Two normal functions for amyloid fibrils. (A) In eukaryotic cells, protein cargo can be packed very densely in secretory vesicles and stored until signals cause a release of this cargo by exocytosis. For example, proteins and peptide hormones of the endocrine system, such as glucagon and calcitonin, are efficiently stored as short amyloid fibrils, which dissociate when they reach the cell exterior. (B) Bacteria produce amyloid fibrils on their surface by secreting the precursor proteins; these fibrils then create biofilms that link together, and help to protect, large numbers of individual bacteria. soluble protein with green fuorescent tag hnRNPA2 t/2 = 10.1 minSOLUBLE PROTEIN REPLACED BY BUFFER hnRNPA1 t/2 = 3.6 min 0.5 1 2 3 5 101520304560dissociation of green protein from time after washinggel is measured by fuorescence (A) microscope as a function of time (B)

1	Figure 3–35 Measuring the association between “reversible amyloids.” (A) Experimental setup. The fiber-forming domains from proteins that contain a low-complexity domain were produced in large quantities by cloning the DNA sequence that encodes them into an E. coli plasmid so as to allow overproduction of that domain (see p. 483). After these protein domains were purified by affinity chromatography, a tiny droplet of concentrated solution of one of the domains (here the FUS low-complexity domain) was deposited onto a microscope dish and allowed to gel. The gel was then soaked in a dilute solution of a fluorescently labeled low-complexity domain from the same or a different protein, making the gel fluorescent. After replacing the dilute protein solution with buffer, the relative strength of binding of the various domains to each other could then be measured by the decay of fluorescence, as indicated. (B) Results. The low-complexity domain from the FUS protein binds more tightly to

1	of binding of the various domains to each other could then be measured by the decay of fluorescence, as indicated. (B) Results. The low-complexity domain from the FUS protein binds more tightly to itself than it does to the low-complexity domains from the proteins hnRNPA1 or hnRNPA2. A separate experiment reveals that these three different RNA binding proteins associate by forming mixed amyloid fibrils. (Adapted from M.Kato et al., Cell 149: 753-767, 2012).

1	in the cell can form a hydrogel that pulls these and other molecules into punctate structures called intracellular bodies, or granules. Specific mRNAs can be sequestered in such granules, where they are stored until made available by a controlled disassembly of the core amyloid structure that holds them together.

1	Consider the FUS protein, an essential nuclear protein with roles in the transcription, processing, and transport of specific mRNA molecules. Over 80 percent of its C-terminal domain of two hundred amino acids is composed of only four amino acids: glycine, serine, glutamine, and tyrosine. This low complexity domain is attached to several other domains that bind to RNA molecules. At high enough concentrations in a test tube, it forms a hydrogel that will associate with either itself or with the low complexity domains from other proteins. As illustrated by the experiment in Figure 3–35, although different low complexity domains bind to each other, homotypic interactions appear to be of greatest affinity (thus, the FUS low complexity domain binds most tightly to itself). Further experiments reveal that that both the homotypic and the heterotypic bindings are mediated through a β-sheet core structure forming amyloid fibrils, and that these structures bind to other types of repeat

1	reveal that that both the homotypic and the heterotypic bindings are mediated through a β-sheet core structure forming amyloid fibrils, and that these structures bind to other types of repeat sequences in the manner indicated in Figure 3–36. Many of these interactions appear to be controlled by the phosphorylation of serine side chains in the one or both of the interacting partners. However, a great deal remains to be learned concerning these newly discovered structures and the varied roles that they play in the cell biology of eukaryotic cells.

1	protein with low-complexity domain weak cross-beta spine binding site for other proteins with repeated sequences or for RNA molecules Figure 3–36 One type of complex that is formed by reversible amyloids. The structure shown is based on the observed interaction of RNA polymerase with a low-complexity domain of a protein that regulates DNA transcription. (Adapted from I. Kwon et al., Cell 155:1049–1060, 2013.) A protein molecule’s amino acid sequence determines its three-dimensional conformation. Noncovalent interactions between different parts of the polypeptide chain stabilize its folded structure. The amino acids with hydrophobic side chains tend to cluster in the interior of the molecule, and local hydrogen-bond interactions between neighboring peptide bonds give rise to α helices and β sheets.

1	Regions of amino acid sequence known as domains are the modular units from which many proteins are constructed. Such domains generally contain 40–350 amino acids, often folded into a globular shape. Small proteins typically consist of only a single domain, while large proteins are formed from multiple domains linked together by various lengths of polypeptide chain, some of which can be relatively disordered. As proteins have evolved, domains have been modified and combined with other domains to construct large numbers of new proteins.

1	Proteins are brought together into larger structures by the same noncovalent forces that determine protein folding. Proteins with binding sites for their own surface can assemble into dimers, closed rings, spherical shells, or helical polymers. The amyloid fibril is a long unbranched structure assembled through a repeating aggregate of β sheets. Although some mixtures of proteins and nucleic acids can assemble spontaneously into complex structures in a test tube, not all structures in the cell are capable of spontaneous reassembly after they have been dissociated into their component parts, because many biological assembly processes involve assembly factors that are not present in the final structure.

1	We have seen that each type of protein consists of a precise sequence of amino acids that allows it to fold up into a particular three-dimensional shape, or conformation. But proteins are not rigid lumps of material. They often have precisely engineered moving parts whose mechanical actions are coupled to chemical events. It is this coupling of chemistry and movement that gives proteins the extraordinary capabilities that underlie the dynamic processes in living cells. In this section, we explain how proteins bind to other selected molecules and how a protein’s activity depends on such binding. We show that the ability to bind to other molecules enables proteins to act as catalysts, signal receptors, switches, motors, or tiny pumps. The examples we discuss in this chapter by no means exhaust the vast functional repertoire of proteins. You will encounter the specialized functions of many other proteins elsewhere in this book, based on similar principles.

1	All Proteins Bind to Other Molecules A protein molecule’s physical interaction with other molecules determines its biological properties. Thus, antibodies attach to viruses or bacteria to mark them for destruction, the enzyme hexokinase binds glucose and ATP so as to catalyze a reaction between them, actin molecules bind to each other to assemble into actin filaments, and so on. Indeed, all proteins stick, or bind, to other molecules. In some cases, this binding is very tight; in others it is weak and short-lived. But the binding always shows great specificity, in the sense that each protein molecule can usually bind just one or a few molecules out of the many thousands of different types it encounters. The substance that is bound by the protein—whether it is an ion, a small molecule, or a macromolecule such as another protein—is referred to as a ligand for that protein (from the Latin word ligare, meaning “to bind”).

1	The ability of a protein to bind selectively and with high affinity to a ligand depends on the formation of a set of weak noncovalent bonds—hydrogen bonds, electrostatic attractions, and van der Waals attractions—plus favorable hydrophobic interactions (see Panel 2–3, pp. 94–95). Because each individual bond is weak, effective binding occurs only when many of these bonds form simultaneously. Such binding is possible only if the surface contours of the ligand molecule fit very closely to the protein, matching it like a hand in a glove (Figure 3–37).

1	The region of a protein that associates with a ligand, known as the ligand’s binding site, usually consists of a cavity in the protein surface formed by a particular arrangement of amino acids. These amino acids can belong to different portions of the polypeptide chain that are brought together when the protein folds (Figure 3–38). Separate regions of the protein surface generally provide binding sites for different ligands, allowing the protein’s activity to be regulated, as we shall see later. And other parts of the protein act as a handle to position the protein in the cell—an example is the SH2 domain discussed previously, which often moves a protein containing it to particular intracellular sites in response to signals.

1	Although the atoms buried in the interior of the protein have no direct contact with the ligand, they form the framework that gives the surface its contours and its chemical and mechanical properties. Even small changes to the amino acids in the interior of a protein molecule can change its three-dimensional shape enough to destroy a binding site on the surface. The Surface Conformation of a Protein Determines Its Chemistry The impressive chemical capabilities of proteins often require that the chemical groups on their surface interact in ways that enhance the chemical reactivity of one or more amino acid side chains. These interactions fall into two main categories. First, the interaction of neighboring parts of the polypeptide chain may restrict the access of water molecules to that protein’s ligand-binding sites. Because water molecules readily form hydrogen bonds that can compete with ligands for sites

1	Figure 3–37 The selective binding of a protein to another molecule. Many weak bonds are needed to enable a protein to bind tightly to a second molecule, or ligand. A ligand must therefore fit precisely into a protein’s binding site, like a hand into a glove, so that a large number of noncovalent bonds form between the protein and the ligand. (A) Schematic; (B) space-filling model. (PDB code: 1G6N.) Figure 3–38 The binding site of a protein. (A) The folding of the polypeptide chain typically creates a crevice or cavity on the protein surface. This crevice contains a set of amino acid side chains disposed in such a way that they can form noncovalent bonds only with certain ligands. (B) A close-up of an actual binding site, showing the hydrogen bonds and electrostatic interactions formed between a protein and its ligand. In this example, a molecule of cyclic AMP is the bound ligand.

1	on the protein surface, a ligand will form tighter hydrogen bonds (and electrostatic interactions) with a protein if water molecules are kept away. It might be hard to imagine a mechanism that would exclude a molecule as small as water from a protein surface without affecting the access of the ligand itself. However, because of the strong tendency of water molecules to form water–water hydrogen bonds, water molecules exist in a large hydrogen-bonded network (see Panel 2–2, pp. 92–93). In effect, a protein can keep a ligand-binding site dry, increasing that site's reactivity, because it is energetically unfavorable for individual water molecules to break away from this network—as they must do to reach into a crevice on a protein’s surface.

1	Second, the clustering of neighboring polar amino acid side chains can alter their reactivity. If protein folding forces together a number of negatively charged side chains against their mutual repulsion, for example, the affinity of the site for a positively charged ion is greatly increased. In addition, when amino acid side chains interact with one another through hydrogen bonds, normally unreactive groups (such as the –CH2OH on the serine shown in Figure 3–39) can become reactive, enabling them to be used to make or break selected covalent bonds. The surface of each protein molecule therefore has a unique chemical reactivity that depends not only on which amino acid side chains are exposed, but also on their exact orientation relative to one another. For this reason, two slightly different conformations of the same protein molecule can differ greatly in their chemistry.

1	As we have described previously, genome sequences allow us to group many of the domains in proteins into families that show clear evidence of their evolution from a common ancestor. The three-dimensional structures of members of the same domain family are remarkably similar. For example, even when the amino acid sequence identity falls to 25%, the backbone atoms in a domain can follow a common protein fold within 0.2 nanometers (2 Å).

1	We can use a method called evolutionary tracing to identify those sites in a protein domain that are the most crucial to the domain’s function. Those sites that bind to other molecules are the most likely to be maintained, unchanged as organisms evolve. Thus, in this method, those amino acids that are unchanged, or nearly unchanged, in all of the known protein family members are mapped onto a model of the three-dimensional structure of one family member. When this is done, the most invariant positions often form one or more clusters on the protein surface, as illustrated in Figure 3–40A for the SH2 domain described previously (see Figure 3–6). These clusters generally correspond to ligand-binding sites.

1	The SH2 domain functions to link two proteins together. It binds the protein containing it to a second protein that contains a phosphorylated tyrosine side chain in a specific amino acid sequence context, as shown in Figure 3–40B. The amino acids located at the binding site for the phosphorylated polypeptide have been the slowest to change during the long evolutionary process that produced Figure 3–39 an unusually reactive amino acid at the active site of an enzyme. This example is the “catalytic triad” Asp-His-Ser found in chymotrypsin, elastase, and other serine proteases (see Figure 3–12). The aspartic acid side chain (Asp) induces the histidine (His) to remove the proton from a particular serine (Ser). This activates the serine and enables it to form a covalent bond with an enzyme substrate, hydrolyzing a peptide bond. The many convolutions of the polypeptide chain are omitted here.

1	the large SH2 family of peptide recognition domains. Mutation is a random process; survival is not. Thus, natural selection (random mutation followed by nonrandom survival) produces the sequence conservation by preferentially eliminating organisms whose SH2 domains become altered in a way that inactivates the SH2 binding site, destroying SH2 function. Genome sequencing has revealed huge numbers of proteins whose functions are unknown. Once a three-dimensional structure has been determined for one member of a protein family, evolutionary tracing allows biologists to determine binding sites for the members of that family, providing a useful start in deciphering protein function. Proteins Bind to Other Proteins Through Several Types of Interfaces

1	Proteins Bind to Other Proteins Through Several Types of Interfaces Proteins can bind to other proteins in multiple ways. In many cases, a portion of the surface of one protein contacts an extended loop of polypeptide chain (a “string”) on a second protein (Figure 3–41A). Such a surface–string interaction, for example, allows the SH2 domain to recognize a phosphorylated polypeptide loop on a second protein, as just described, and it also enables a protein kinase to recognize the proteins that it will phosphorylate (see below). A second type of protein–protein interface forms when two α helices, one from each protein, pair together to form a coiled-coil (Figure 3–41B). This type of protein interface is found in several families of gene regulatory proteins, as discussed in Chapter 7.

1	The most common way for proteins to interact, however, is by the precise matching of one rigid surface with that of another (Figure 3–41C). Such interactions can be very tight, since a large number of weak bonds can form between two surfaces that match well. For the same reason, such surface–surface interactions can be extremely specific, enabling a protein to select just one partner from the many thousands of different proteins found in a cell. Figure 3–40 The evolutionary trace method applied to the SH2 domain.

1	Figure 3–40 The evolutionary trace method applied to the SH2 domain. Front and back views of a space-filling model of the SH2 domain, with evolutionarily conserved amino acids on the protein surface colored yellow, and those more toward the protein interior colored red. (B) The structure of one specific SH2 domain with its bound polypeptide. Here, those amino acids located within 0.4 nm of the bound ligand are colored blue. The two key amino acids of the ligand are yellow, and the others are purple. Note the high degree of correspondence between and (B). (Adapted from O. Lichtarge, H.R. Bourne and F.E. Cohen, J. Mol. Biol. 257:342–358, 1996. With permission from Elsevier; PDB codes: 1SPR, 1SPS.)

1	H.R. Bourne and F.E. Cohen, J. Mol. Biol. 257:342–358, 1996. With permission from Elsevier; PDB codes: 1SPR, 1SPS.) Figure 3–41 Three ways in which two proteins can bind to each other. Only the interacting parts of the two proteins are shown. (A) A rigid surface on one protein can bind to an extended loop of polypeptide chain (a “string”) on a second protein. (B) Two α helices can bind together to form a coiled-coil. (C) Two complementary rigid surfaces often link two proteins together. Binding interactions can also involve the pairing of β strands (see, for example, Figure 3–18).

1	(C) Two complementary rigid surfaces often link two proteins together. Binding interactions can also involve the pairing of β strands (see, for example, Figure 3–18). hypervariable loops variable domain of light chain (VL) (B) constant domain of light chain (CL) disulfde bond COOH NH2 S S S S SS SSSSSSSSVL CL VH CH1CH1 CH2 CH3 Figure 3–42 an antibody molecule. A typical antibody molecule is Y-shaped and has two identical binding sites for its antigen, one on each arm of the Y. As explained in Chapter 24, the protein is composed of four polypeptide chains (two identical heavy chains and two identical and smaller light chains) held together by disulfide bonds. Each chain is made All proteins must bind to particular ligands to carry out their various functions. The antibody family is notable for its capacity for tight, highly selective binding (discussed in detail in Chapter 24).

1	Antibodies, or immunoglobulins, are proteins produced by the immune system in response to foreign molecules, such as those on the surface of an invading microorganism. Each antibody binds tightly to a particular target molecule, thereby either inactivating the target molecule directly or marking it for destruction. An antibody recognizes its target (called an antigen) with remarkable specificity. Because there are potentially billions of different antigens that humans might encounter, we have to be able to produce billions of different antibodies.

1	Antibodies are Y-shaped molecules with two identical binding sites that are complementary to a small portion of the surface of the antigen molecule. A detailed examination of the antigen-binding sites of antibodies reveals that they are formed from several loops of polypeptide chain that protrude from the ends of a pair of closely juxtaposed protein domains (Figure 3–42). Different antibodies generate an enormous diversity of antigen-binding sites by changing only the length and amino acid sequence of these loops, without altering the basic protein structure. Loops of this kind are ideal for grasping other molecules. They allow a large number of chemical groups to surround a ligand so that the protein can link to it with many weak bonds. For this reason, loops often form the ligand-binding sites in proteins. The Equilibrium Constant Measures Binding Strength

1	The Equilibrium Constant Measures Binding Strength Molecules in the cell encounter each other very frequently because of their continual random thermal movements. Colliding molecules with poorly matching surfaces form few noncovalent bonds with one another, and the two molecules dissociate as rapidly as they come together. At the other extreme, when many noncovalent bonds form between two colliding molecules, the association can persist for a very long time (Figure 3–43). Strong interactions occur in cells whenever a biological function requires that molecules remain associated for a long time—for example, when a group of RNA and protein molecules come together to make a subcellular structure such as a ribosome.

1	up of several different immunoglobulin domains, here shaded either blue or gray. The antigen-binding site is formed where a heavy-chain variable domain (VH) and a light-chain variable domain (VL) come close together. These are the domains that differ most in their sequence and structure in different antibodies. At the end of each of the two arms of the antibody molecule, these two domains form loops that bind to the antigen (see Movie 24.5). the surfaces of molecules A and B, and A and C, are a poor match and are capable of forming only a few weak bonds; thermal motion rapidly breaks them apart molecule A randomly encounters other molecules (B, C, and D) the surfaces of molecules A and D match well and therefore can form enough weak bonds to withstand thermal jolting; they therefore

1	We can measure the strength with which any two molecules bind to each other. As an example, consider a population of identical antibody molecules that suddenly encounters a population of ligands diffusing in the fluid surrounding them. At frequent intervals, one of the ligand molecules will bump into the binding site of an antibody and form an antibody–ligand complex. The population of antibody–ligand complexes will therefore increase, but not without limit: over time, a second process, in which individual complexes break apart because of thermally induced motion, will become increasingly important. Eventually, any population of antibody molecules and ligands will reach a steady state, or equilibrium, in which the number of binding (association) events per second is precisely equal to the number of “unbinding” (dissociation) events (see Figure 2–30).

1	From the concentrations of the ligand, antibody, and antibody–ligand complex at equilibrium, we can calculate a convenient measure of the strength of bind-ing—the equilibrium constant (K)—(Figure 3–44A). This constant was described in detail in Chapter 2, where its connection to free energy differences was derived (see p. 62). The equilibrium constant for a reaction in which two molecules (A and B) bind to each other to form a complex (AB) has units of liters/mole, and half of the binding sites will be occupied by ligand when that ligand’s concentration (in moles/liter) reaches a value that is equal to 1/K. This equilibrium constant is larger the greater the binding strength, and it is a direct measure of the free-energy difference between the bound and free states (Figure 3–44B). Even a change stay bound to each other Figure 3–43 How noncovalent bonds mediate interactions between macromolecules (see Movie 2.1).

1	Figure 3–44 Relating standard free-energy difference (ΔG°) to the equilibrium constant (K). (A) The equilibrium between molecules A and B and the complex AB is maintained by a balance between the two opposing reactions shown in panels 1 and 2. Molecules A and B must collide if they are to react, and the association rate is therefore proportional to the product of their individual concentrations [A] × [B]. (Square brackets indicate concentration.) As shown in panel 3, the ratio of the rate constants for the association and the dissociation reactions is equal to the equilibrium constant (K) for the reaction (see also p. 63). (B) The equilibrium constant in panel 3 is that for the reaction A + B ↔ AB, and the larger its value, the stronger the binding between A and B. Note that for every 5.91 kJ/mole decrease in standard free energy, the equilibrium constant increases by a factor of 10 at 37°C.

1	The equilibrium constant here has units of liters/mole; for simple binding interactions it is also called the affinity constant or association constant, denoted Ka. The reciprocal of Ka is called the dissociation 1 2 3 dissociation AB A + B dissociation rate = dissociation rate constant ×concentration of AB dissociation rate = koff [AB] association ABA + B association rate = association rate constant ×concentration of A association rate = kon [A] [B] ×concentration of B AT EQUILIBRIUM: association rate = dissociation rate kon [A] [B] = koff [AB] [A][B] koff [AB] kon = = K = equilibrium constant equilibrium constant [A][B] [AB] = K(liters/mole) 1 10 102 103 104 105 106 107 108 109 1010 standard free-energy difference of AB minus free energy of A + B (kJ/mole) 0 –5.9 –11.9 –17.8 –23.7 –29.7 –35.6 –41.5 –47.4 –53.4 –59.4 The relationship between standard free-energy differences (°G°) and equilibrium constants (37°C) (A) (B) constant, Kd (in units of moles/liter).

1	of a few noncovalent bonds can have a striking effect on a binding interaction, as shown by the example in Figure 3–45. (Note that the equilibrium constant, as defined here, is also known as the association or affinity constant, Ka.) We have used the case of an antibody binding to its ligand to illustrate the effect of binding strength on the equilibrium state, but the same principles apply to any molecule and its ligand. Many proteins are enzymes, which, as we now discuss, first bind to their ligands and then catalyze the breakage or formation of covalent bonds in these molecules.

1	Many proteins can perform their function simply by binding to another molecule. An actin molecule, for example, need only associate with other actin molecules to form a filament. There are other proteins, however, for which ligand binding is only a necessary first step in their function. This is the case for the large and very important class of proteins called enzymes. As described in Chapter 2, enzymes are remarkable molecules that cause the chemical transformations that make and break covalent bonds in cells. They bind to one or more ligands, called substrates, and convert them into one or more chemically modified products, doing this over and over again with amazing rapidity. Enzymes speed up reactions, often by a factor of a million or more, without themselves being changed—that is, they act as catalysts that permit cells to make or break covalent bonds in a controlled way. It is the catalysis of organized sets of chemical reactions by enzymes that creates and maintains the cell,

1	act as catalysts that permit cells to make or break covalent bonds in a controlled way. It is the catalysis of organized sets of chemical reactions by enzymes that creates and maintains the cell, making life possible.

1	We can group enzymes into functional classes that perform similar chemical reactions (Table 3–1). Each type of enzyme within such a class is highly specific,

1	Figure 3–45 Small changes in the number of weak bonds can have drastic effects on a binding interaction. This example illustrates the dramatic effect of the presence or absence of a few weak noncovalent bonds in a biological context. Consider 1000 molecules of A and 1000 molecules of B in a eukaryotic cell. The concentration of both will be about 10–9 M. If the equilibrium constant (K ) for A + B AB is 1010, then one can calculate that at equilibrium there will be If the equilibrium constant is a little weaker at 108, which represents a loss of 11.9 kilojoule/mole of binding energy from the example above, or 2–3 fewer hydrogen bonds, then there will be 270 A molecules 270 B molecules 730 AB molecules 915 A molecules 915 B molecules 85 AB molecules 0.5Vmax catalyzing only a single type of reaction. Thus, hexokinase adds a phosphate group to D-glucose but ignores its optical isomer L-glucose; the blood-clotting enzyme thrombin cuts one type of blood protein between a particular arginine

1	Thus, hexokinase adds a phosphate group to D-glucose but ignores its optical isomer L-glucose; the blood-clotting enzyme thrombin cuts one type of blood protein between a particular arginine and its adjacent glycine and nowhere else, and so on. As discussed in detail in Chapter 2, enzymes work in teams, with the product of one enzyme becoming the substrate for the next. The result is an elaborate network of metabolic pathways that provides the cell with energy and generates the many large and small molecules that the cell needs (see Figure 2–63).

1	Substrate Binding Is the First Step in Enzyme Catalysis

1	For a protein that catalyzes a chemical reaction (an enzyme), the binding of each substrate molecule to the protein is an essential prelude. In the simplest case, if we denote the enzyme by E, the substrate by S, and the product by P, the basic reaction path is E + S → ES →EP → E + P. There is a limit to the amount of substrate that a single enzyme molecule can process in a given time. Although an increase in the concentration of substrate increases the rate at which product is formed, this rate eventually reaches a maximum value (Figure 3–46). At that point the enzyme molecule is saturated with substrate, and the rate of reaction (Vmax) depends only on how rapidly the enzyme can process the substrate molecule. This maximum rate divided by the enzyme concentration is called the turnover number. Turnover numbers are often about 1000 substrate molecules processed per second per enzyme molecule, although turnover numbers between 1 and 10,000 are known.

1	The other kinetic parameter frequently used to characterize an enzyme is its Km, the concentration of substrate that allows the reaction to proceed at one-half its maximum rate (0.5 Vmax) (see Figure 3–46). A low Km value means that the enzyme reaches its maximum catalytic rate at a low concentration of substrate and generally indicates that the enzyme binds to its substrate very tightly, whereas a high Km value corresponds to weak binding. The methods used to characterize enzymes in this way are explained in Panel 3–2 (pp. 142–143).

1	Enzymes achieve extremely high rates of chemical reaction—rates that are far higher than for any synthetic catalysts. There are several reasons for this efficiency. First, when two molecules need to react, the enzyme greatly increases the local concentration of both of these substrate molecules at the catalytic site, holding them in the correct orientation for the reaction that is to follow. More importantly, however, some of the binding energy contributes directly to the catalysis. Substrate molecules must pass through a series of intermediate states of altered geometry and electron distribution before they form the ultimate products of the reaction. The free energy required to attain the most unstable intermediate state, called the transition state, is known as the activation energy for the reaction, and it is the major determinant of the reaction rate. Enzymes have a much higher affinity for the transition state of the substrate than they have for the stable form.

1	rate of reaction Figure 3–46 enzyme kinetics. The rate of an enzyme reaction (V) increases as the substrate concentration increases until a maximum value (Vmax) is reached. At this point all substrate-binding sites on the enzyme molecules are fully occupied, and the rate of reaction is limited by the rate of the catalytic process on the enzyme surface. For most enzymes, the concentration of substrate at which the reaction rate is half-maximal (Km) is a measure of how tightly the substrate is bound, with a large value of Km corresponding to weak binding.

1	WHY ANALYZE THE KINETICS OF ENZYMES? Enzymes are the most selective and powerful catalysts known. An understanding of their detailed mechanisms provides a critical tool for the discovery of new drugs, for the large-scale industrial synthesis of useful chemicals, and for appreciating the chemistry of cells and organisms. A detailed study of the rates of the chemical reactions that are catalyzed by a purifed enzyme—more specifcally how these rates change with changes in conditions such as the concentrations of substrates, products, inhibitors, and regulatory ligands—allows biochemists to fgure out exactly how each enzyme works. For example, this is the way that the ATP-producing reactions of glycolysis, shown previously in Figure 2–48, were deciphered—allowing us to appreciate the rationale for this critical enzymatic pathway. In this Panel, we introduce the important feld of enzyme kinetics, which has been indispensable for deriving much of the detailed knowledge that we now have about

1	for this critical enzymatic pathway. In this Panel, we introduce the important feld of enzyme kinetics, which has been indispensable for deriving much of the detailed knowledge that we now have about cell chemistry. 142 Panel 3–2: Some of the Methods Used to Study enzymes

1	STEADY-STATE ENZYME KINETICS Many enzymes have only one substrate, which they bind and then process to produce products according to the scheme outlined in Figure 3–50A. In this case, the reaction is written as Here we have assumed that the reverse reaction, in which E + P recombine to form EP and then ES, occurs so rarely that we can ignore it. In this case, EP need not be represented, and we can express the rate of the reaction—known as its velocity, V, as where [ES] is the concentration of the enzyme–substrate complex, and kcat is the turnover number, a rate constant that has a value equal to the number of substrate molecules processed per enzyme molecule each second. But how does the value of [ES] relate to the concentrations that we know directly, which are the total concentration of the enzyme, [Eo], and the concentration of the substrate, [S]? When enzyme and substrate are frst mixed, the concentration [ES] will rise rapidly from zero to a so-called steady-state level, as

1	of the enzyme, [Eo], and the concentration of the substrate, [S]? When enzyme and substrate are frst mixed, the concentration [ES] will rise rapidly from zero to a so-called steady-state level, as illustrated below. E + S E + P ES k1 k–1 kcat V = kcat [ES]

1	At this steady state, [ES] is nearly constant, so that or, since the concentration of the free enzyme, [E], is equal to [Eo] – [ES], Rearranging, and defning the constant Km as we get or, remembering that V = kcat [ES], we obtain the famous Michaelis–Menten equation As [S] is increased to higher and higher levels, essentially all of the enzyme will be bound to substrate at steady state; at this point, a maximum rate of reaction, Vmax , will be reached where V = Vmax = kcat [Eo]. Thus, it is convenient to rewrite the Michaelis–Menten equation as rate of ES formation k1 [E][S] rate of ES breakdown k–1 [ES] + kcat [ES] = k1 k–1 + kcat [ES] = [E][S] = [Eo] – [ES] [S] k1 k–1 + kcat k1 k–1 + kcat Km + [S] kcat [Eo][S] [ES] = [Eo][S] Km + [S] V = Km + [S] Vmax [S]V = THE DOUBLE-RECIPROCAL PLOT A typical plot of V versus [S] for an enzyme that follows Michaelis–Menten kinetics is shown below. From this plot, neither the value of Vmax nor of Km is immediately clear.

1	THE SIGNIFICANCE OF Km, kcat, and kcat /Km As described in the text, Km is an approximate measure of substrate affnity for the enzyme: it is numerically equal to the concentration of [S] at V = 0.5 Vmax. In general, a lower value of Km means tighter substrate binding. In fact, for those cases where kcat is much smaller than k–1, the Km will be equal to Kd, the dissociation constant for substrate binding to the enzyme (Kd = 1/Ka; see Figure 3–44). We have seen that k cat is the turnover number for the enzyme. At very low substrate concentrations, where 20 0 0 2 4 6 8 40 60 80 [S] mmole/liter To obtain Vmax and Km from such data, a double-reciprocal [S] << Km, most of the enzyme is free. Thus we can think of [E] = [Eo], so that the Michaelis–Menten equation becomes V = kcat/Km [E][S]. Thus, the ratio kcat/Km is equivalent to the rate constant for the reaction between free enzyme and free substrate. A comparison of kcat/Km for the same enzyme with different substrates, or for two enzymes

1	the ratio kcat/Km is equivalent to the rate constant for the reaction between free enzyme and free substrate. A comparison of kcat/Km for the same enzyme with different substrates, or for two enzymes with their different substrates, is widely used as a measure of enzyme effectiveness. For simplicity, in this Panel we have discussed enzymes that have only one substrate, such as the lysozyme enzyme described in the text (see p. 144). Most enzymes have two substrates, one of which is often an active carrier

1	SOME ENZYMES ARE DIFFUSION LIMITED The values of kcat, Km, and kcat /Km for some selected molecule—such as NADH or ATP. A similar, but more complex, analysis is used to determine the kinetics of such enzymes—allowing the order of substrate binding and the presence of covalent intermediates along the pathway to be revealed.

1	plot is often used, in which the Michaelis–Menten equation has merely been rearranged, so that 1/V can be plotted versus 1/ [S]. 1/V= + 1/ Vmax Km Vmax [S] 1 123468 1 Vmax 0.01 0.02 0.03 0.04 slope = KM / Vmax 1/V (second/µmole) enzymes are given below: Because an enzyme and its substrate must collide before they can react, kcat /Km has a maximum possible value that is limited by collision rates. If every collision forms an enzyme–substrate complex, one can calculate from diffusion theory that kcat /Km will be between 108 and 109 sec–1M–1, in the case where all subsequent steps proceed immediately. – 0.25 0 0.25 0.5 0.75 1.0– 0.5 fumarase fumarate 8x102 5x10–6 1.6x108 catalase H2O2 4x107 1 4x107 acetylcholinesterase acetylcholine 1.4x104 9x10–5 1.6x108 enzyme substrate kcat (sec–1) kcat/Km (sec–1M–1) Km (M)

1	Thus, it is claimed that enzymes like acetylcholinesterase and1 liter/mmole fumarase are “perfect enzymes,” each enzyme having evolved to the point where nearly every collision with its substrate converts the substrate to a product. [S] Km Figure 3–47 enzymatic acceleration of chemical reactions by decreasing the activation energy. There is a single transition state in this example. However, often both the uncatalyzed reaction (A) and the enzyme-catalyzed reaction (B) go through a series of transition states. In that case, it is the transition state with the highest energy (ST and EST) that determines the activation energy and limits the rate of the reaction. (S = substrate; P = product of the reaction; ES = enzyme–substrate complex; EP = enzyme– product complex.) Because this tight binding greatly lowers the energy of the transition state, the enzyme greatly accelerates a particular reaction by lowering the activation energy that is required (Figure 3–47).

1	Because this tight binding greatly lowers the energy of the transition state, the enzyme greatly accelerates a particular reaction by lowering the activation energy that is required (Figure 3–47). Figure 3–48 compares the spontaneous reaction rates and the corresponding enzyme-catalyzed rates for five enzymes. Rate accelerations range from 109 to 1023. Enzymes not only bind tightly to a transition state, they also contain precisely positioned atoms that alter the electron distributions in the atoms that participate directly in the making and breaking of covalent bonds. Peptide bonds, for example, can be hydrolyzed in the absence of an enzyme by exposing a polypeptide to either a strong acid or a strong base. Enzymes are unique, however, in being able to use acid and base catalysis simultaneously, because the rigid framework of the protein constrains the acidic and basic residues and prevents them from combining with each other, as they would do in solution (Figure 3–49).

1	The fit between an enzyme and its substrate needs to be precise. A small change introduced by genetic engineering in the active site of an enzyme can therefore have a profound effect. Replacing a glutamic acid with an aspartic acid in one enzyme, for example, shifts the position of the catalytic carboxylate ion by only 1 Å (about the radius of a hydrogen atom); yet this is enough to decrease the activity of the enzyme a thousandfold.

1	To demonstrate how enzymes catalyze chemical reactions, we examine an enzyme that acts as a natural antibiotic in egg white, saliva, tears, and other secretions. Lysozyme catalyzes the cutting of polysaccharide chains in the cell walls of bacteria. The bacterial cell is under pressure from osmotic forces, and cutting even a small number of these chains causes the cell wall to rupture and the cell to burst. A relatively small and stable protein that can be easily isolated in large quantities, lysozyme was the first enzyme to have its structure worked out in atomic detail by x-ray crystallography (in the mid-1960s).

1	The reaction that lysozyme catalyzes is a hydrolysis: it adds a molecule of water to a single bond between two adjacent sugar groups in the polysaccharide chain, thereby causing the bond to break (see Figure 2–9). The reaction is energetically favorable because the free energy of the severed polysaccharide chain is lower progress of reaction activation energy for catalyzed reaction Figure 3–48 The rate accelerations caused by five different enzymes.

1	(Adapted from A. Radzicka and R. Wolfenden, Science 267:90–93, 1995.) catalysis. (A) The start of the uncatalyzed reaction that hydrolyzes a peptide bond, with blue shading used to indicate electron distribution in the water and carbonyl bonds. (B) An acid likes to donate a proton (H+) to other atoms. By pairing with the carbonyl oxygen, an acid causes electrons to move away from the carbonyl carbon, making this atom much more attractive to the electronegative oxygen of an attacking water molecule. (C) A base likes to take up H+. By pairing with a hydrogen of the attacking water molecule, a base causes base catalyses electrons to move toward the water oxygen, making it a better attacking group for the carbonyl carbon. (D) By having appropriately than the free energy of the intact chain. However, there is an energy barrier to the positioned atoms on its surface, an enzyme reaction, and a colliding water molecule can break a bond linking two sugars only if the polysaccharide molecule

1	However, there is an energy barrier to the positioned atoms on its surface, an enzyme reaction, and a colliding water molecule can break a bond linking two sugars only if the polysaccharide molecule is distorted into a particular shape—the transition state—in which the atoms around the bond have an altered geometry and electron distribution. Because of this requirement, random collisions must supply a very large activation energy for the reaction to take place. In an aqueous solution at room temperature, the energy of collisions almost never exceeds the activation energy. The pure polysaccharide can therefore remain for years in water without being hydrolyzed to any detectable degree.

1	This situation changes drastically when the polysaccharide binds to lysozyme. The active site of lysozyme, because its substrate is a polymer, is a long groove that holds six linked sugars at the same time. As soon as the polysaccharide binds to form an enzyme–substrate complex, the enzyme cuts the polysaccharide by adding a water molecule across one of its sugar–sugar bonds. The product chains are then quickly released, freeing the enzyme for further cycles of reaction (Figure 3–50).

1	An impressive increase in hydrolysis rate is possible because conditions are created in the microenvironment of the lysozyme active site that greatly reduce the activation energy necessary for the hydrolysis to take place. In particular, lysozyme distorts one of the two sugars connected by the bond to be broken from its normal, most stable conformation. The bond to be broken is also held close to two amino acids with acidic side chains (a glutamic acid and an aspartic acid) that participate directly in the reaction. Figure 3–51 shows the three central steps in this enzymatically catalyzed reaction, which occurs millions of times faster than uncatalyzed hydrolysis.

1	Other enzymes use similar mechanisms to lower activation energies and speed up the reactions they catalyze. In reactions involving two or more reactants, the active site also acts like a template, or mold, that brings the substrates together in the proper orientation for a reaction to occur between them (Figure 3–52A). As we saw for lysozyme, the active site of an enzyme contains precisely positioned can perform both acid catalysis and base catalysis at the same time.

1	Figure 3–50 The reaction catalyzed by lysozyme. (A) The enzyme lysozyme (E) catalyzes the cutting of a polysaccharide chain, which is its substrate (S). The enzyme first binds to the chain to form an enzyme–substrate complex (ES) and then catalyzes the cleavage of a specific covalent bond in the backbone of the polysaccharide, forming an enzyme–product complex (EP) that rapidly dissociates. Release of the severed chain (the products P) leaves the enzyme free to act on another substrate molecule. (B) A space-filling model of the lysozyme molecule bound to a short length of polysaccharide chain before cleavage (Movie 3.8). (B, courtesy of Richard J. Feldmann; PDB code: 3AB6.) This substrate is an oligosaccharide of six sugars, The fnal products are an oligosaccharide of four sugars labeled A through F. Only sugars D and E are shown in detail. (left) and a disaccharide (right), produced by hydrolysis.

1	In the enzyme–substrate complex (ES), the The Asp52 has formed a covalent bond between enzyme forces sugar D into a strained the enzyme and the C1 carbon atom of sugar D. conformation. The Glu35 in the enzyme is The Glu35 then polarizes a water molecule (red ), positioned to serve as an acid that attacks the so that its oxygen can readily attack the C1 adjacent sugar–sugar bond by donating a proton carbon atom and displace Asp52. (H+) to sugar E; Asp52 is poised to attack the C1 carbon atom.

1	atoms that speed up a reaction by using charged groups to alter the distribution of electrons in the substrates (Figure 3–52B). And as we have also seen, when a substrate binds to an enzyme, bonds in the substrate are often distorted, changing the substrate shape. These changes, along with mechanical forces, drive a substrate toward a particular transition state (Figure 3–52C). Finally, like lysozyme, many enzymes participate intimately in the reaction by transiently forming a covalent bond between the substrate and a side chain of the enzyme. Subsequent steps in the reaction restore the side chain to its original state, so that the enzyme remains unchanged after the reaction (see also Figure 2–48). Tightly Bound Small Molecules Add Extra Functions to Proteins

1	Tightly Bound Small Molecules Add Extra Functions to Proteins Although we have emphasized the versatility of enzymes—and proteins in general—as chains of amino acids that perform remarkable functions, there are many instances in which the amino acids by themselves are not enough. Just as humans The reaction of the water molecule (red) completes the hydrolysis and returns the enzyme to its initial state, forming the fnal enzyme– product complex (EP).

1	The reaction of the water molecule (red) completes the hydrolysis and returns the enzyme to its initial state, forming the fnal enzyme– product complex (EP). Figure 3–51 events at the active site of lysozyme. The top left and top right drawings show the free substrate and the free products, respectively, whereas the other three drawings show the sequential events at the enzyme active site. Note the change in the conformation of sugar D in the enzyme–substrate complex; this shape change stabilizes the oxocarbenium ion-like transition states required for formation and hydrolysis of the covalent intermediate shown in the middle panel. It is also possible that a carbonium ion intermediate forms in step 2, but the covalent intermediate shown in the middle panel has been detected with a synthetic substrate (Movie 3.9). (See D.J. Vocadlo et al., Nature 412:835–838, 2001.)

1	Figure 3–52 Some general strategies of enzyme catalysis. (A) Holding substrates (A) enzyme binds to two (B) binding of substrate (C) enzyme strains the together in a precise alignment. (B) Charge substrate molecules and to enzyme rearranges bound substrate stabilization of reaction intermediates. orients them precisely to electrons in the substrate, molecule, forcing it encourage a reaction to creating partial negative toward a transition (C) Applying forces that distort bonds in the occur between them and positive charges state to favor a reaction substrate to increase the rate of a particular that favor a reaction reaction.

1	employ tools to enhance and extend the capabilities of their hands, enzymes and other proteins often use small nonprotein molecules to perform functions that would be difficult or impossible to do with amino acids alone. Thus, enzymes frequently have a small molecule or metal atom tightly associated with their active site that assists with their catalytic function. Carboxypeptidase, for example, an enzyme that cuts polypeptide chains, carries a tightly bound zinc ion in its active site. During the cleavage of a peptide bond by carboxypeptidase, the zinc ion forms a transient bond with one of the substrate atoms, thereby assisting the hydrolysis reaction. In other enzymes, a small organic molecule serves a similar purpose. Such organic molecules are often referred to as coenzymes. An example is biotin, which is found in enzymes that transfer a carboxylate group (–COO–) from one molecule to another (see Figure 2–40). Biotin participates in these reactions by forming a transient covalent

1	is biotin, which is found in enzymes that transfer a carboxylate group (–COO–) from one molecule to another (see Figure 2–40). Biotin participates in these reactions by forming a transient covalent bond to the –COO– group to be transferred, being better suited to this function than any of the amino acids used to make proteins. Because it cannot be synthesized by humans, and must therefore be supplied in small quantities in our diet, biotin is a vitamin. Many other coenzymes are either vitamins or derivatives of vitamins (Table 3–2).

1	Other proteins also frequently require specific small-molecule adjuncts to function properly. Thus, the signal receptor protein rhodopsin, which is made by the photoreceptor cells in the retina, detects light by means of a small molecule, retinal, embedded in the protein (Figure 3–53A). Retinal, which is derived from vitamin A, changes its shape when it absorbs a photon of light, and this change causes the protein to trigger a cascade of enzymatic reactions that eventually lead to an electrical signal being carried to the brain. Figure 3–53 Retinal and heme. (A) The structure of retinal, the light-sensitive molecule attached to rhodopsin in the eye. The structure shown isomerizes when it absorbs light. (B) The structure of a heme group. The carbon-containing heme ring is red and the iron atom at its center is orange. A heme group is tightly bound to each of the four polypeptide chains in hemoglobin, the oxygen-carrying protein whose structure is shown in Figure 3–19.

1	Another example of a protein with a nonprotein portion is hemoglobin (see Figure 3–19). Each molecule of hemoglobin carries four heme groups, ring-shaped molecules each with a single central iron atom (Figure 3–53B). Heme gives hemoglobin (and blood) its red color. By binding reversibly to oxygen gas through its iron atom, heme enables hemoglobin to pick up oxygen in the lungs and release it in the tissues. Sometimes these small molecules are attached covalently and permanently to their protein, thereby becoming an integral part of the protein molecule itself. We shall see in Chapter 10 that proteins are often anchored to cell membranes through covalently attached lipid molecules. And membrane proteins exposed on the surface of the cell, as well as proteins secreted outside the cell, are often modified by the covalent addition of sugars and oligosaccharides. Multienzyme Complexes Help to Increase the Rate of Cell Metabolism

1	Multienzyme Complexes Help to Increase the Rate of Cell Metabolism The efficiency of enzymes in accelerating chemical reactions is crucial to the maintenance of life. Cells, in effect, must race against the unavoidable processes of decay, which—if left unattended—cause macromolecules to run downhill toward greater and greater disorder. If the rates of desirable reactions were not greater than the rates of competing side reactions, a cell would soon die. We can get some idea of the rate at which cell metabolism proceeds by measuring the rate of ATP utilization. A typical mammalian cell “turns over” (i.e., hydrolyzes and restores by phosphorylation) its entire ATP pool once every 1 or 2 minutes. For each cell, this turnover represents the utilization of roughly 107 molecules of ATP per second (or, for the human body, about 1 gram of ATP every minute).

1	The rates of reactions in cells are rapid because enzyme catalysis is so effective. Some enzymes have become so efficient that there is no possibility of further useful improvement. The factor that limits the reaction rate is no longer the enzyme’s intrinsic speed of action; rather, it is the frequency with which the enzyme collides with its substrate. Such a reaction is said to be diffusion-limited (see Panel 3–2, pp. 142–143).

1	The amount of product produced by an enzyme will depend on the concentration of both the enzyme and its substrate. If a sequence of reactions is to occur extremely rapidly, each metabolic intermediate and enzyme involved must be present in high concentration. However, given the enormous number of different reactions performed by a cell, there are limits to the concentrations that can be achieved. In fact, most metabolites are present in micromolar (10–6 M) concentrations, and most enzyme concentrations are much lower. How is it possible, therefore, to maintain very fast metabolic rates?

1	The answer lies in the spatial organization of cell components. The cell can increase reaction rates without raising substrate concentrations by bringing the various enzymes involved in a reaction sequence together to form a large protein assembly known as a multienzyme complex (Figure 3–54). Because this assembly is organized in a way that allows the product of enzyme A to be passed directly to enzyme B, and so on, diffusion rates need not be limiting, even when the concentrations of the substrates in the cell as a whole are very low. It is perhaps not surprising, therefore, that such enzyme complexes are very common, and they are involved in nearly all aspects of metabolism—including the central genetic processes of DNA, RNA, and protein synthesis. In fact, few enzymes in eukaryotic cells diffuse freely in solution; instead, most seem to have evolved binding sites that concentrate them with other proteins of related function in particular regions of the cell, thereby increasing the

1	diffuse freely in solution; instead, most seem to have evolved binding sites that concentrate them with other proteins of related function in particular regions of the cell, thereby increasing the rate and efficiency of the reactions that they catalyze (see p. 331).

1	Eukaryotic cells have yet another way of increasing the rate of metabolic reactions: using their intracellular membrane systems. These membranes can segregate particular substrates and the enzymes that act on them into the same membrane-enclosed compartment, such as the endoplasmic reticulum or the cell nucleus. If, for example, a compartment occupies a total of 10% of the volume of TE2 2 2 1 1 4 4 5 21 4 5 3 3 3 enzyme domains (C) (E) (D) etc.5 nm C acyl carrier domain termination domain (TE) PYRUVATE DEHYDROGENASE COMPLEX FATTY ACID SYNTHASE 3 1 20 nm

1	Figure 3–54 How unstructured regions of polypeptide chain serving as tethers allow reaction intermediates to be passed from one active site to another in large multienzyme complexes. (A–C) The fatty acid synthase in mammals. (A) The location of seven protein domains with different activities in this 270 kilodalton protein. The numbers refer to the order in which each enzyme domain must function to complete each two-carbon addition step. After multiple cycles of two-carbon addition, the termination domain releases the final product once the desired length of fatty acid has been synthesized. (B) The structure of the dimeric enzyme, with the location of the five active sites in one monomer indicated. (C) How a flexible tether allows the substrate that remains linked to the acyl carrier domain (red) to be passed from one active site to another in each monomer, sequentially elongating and modifying the bound fatty acid intermediate (yellow). The five steps are repeated until the final

1	(red) to be passed from one active site to another in each monomer, sequentially elongating and modifying the bound fatty acid intermediate (yellow). The five steps are repeated until the final length of fatty acid chain has been synthesized. (Only steps 1 through 4 are illustrated here.) (D) Multiple tethered subunits in the giant pyruvate dehydrogenase complex (9500 kilodaltons, larger than a ribosome) that catalyzes the conversion of pyruvate to acetyl CoA. As in (C), a covalently bound substrate held on a flexible tether (red balls with yellow substrate) is serially passed through active sites on subunits (here labeled 1 through 3) to produce the final products. Here, subunit 1 catalyzes the decarboxylation of pyruvate accompanied by the reductive acetylation of a lipoyl group linked to one of the red balls. Subunit 2 transfers this acetyl group to CoA, forming acetyl CoA, and subunit 3 reoxidizes the lipoyl group to prepare it for the next cycle. Only one-tenth of the subunits

1	to one of the red balls. Subunit 2 transfers this acetyl group to CoA, forming acetyl CoA, and subunit 3 reoxidizes the lipoyl group to prepare it for the next cycle. Only one-tenth of the subunits labeled 1 and 3, attached to the core formed by subunit 2, are illustrated here. This important reaction takes place in the mammalian mitochondrion, as part of the pathway that oxidizes sugars to CO2 and H2O (see page 82). (A–C, adapted from T. Maier et al., Quart. Rev. Biophys. 43:373–422, 2010; D, from J.L.S. Milne et al., J. Biol. Chem. 281:4364–4370, 2006.) the cell, the concentration of reactants in that compartment may be increased by 10 times compared with a cell with the same number of enzyme and substrate molecules, but no compartmentalization. Reactions limited by the speed of diffusion can thereby be speeded up by a factor of 10.

1	The Cell Regulates the Catalytic Activities of Its Enzymes A living cell contains thousands of enzymes, many of which operate at the same time and in the same small volume of the cytosol. By their catalytic action, these enzymes generate a complex web of metabolic pathways, each composed of chains of chemical reactions in which the product of one enzyme becomes the substrate of the next. In this maze of pathways, there are many branch points (nodes) where different enzymes compete for the same substrate. The system is complex (see Figure 2–63), and elaborate controls are required to regulate when and how rapidly each reaction occurs.

1	Regulation occurs at many levels. At one level, the cell controls how many molecules of each enzyme it makes by regulating the expression of the gene that encodes that enzyme (discussed in Chapter 7). The cell also controls enzymatic activities by confining sets of enzymes to particular subcellular compartments, whether by enclosing them in a distinct membrane-bounded compartment (discussed in Chapters 12 and 14) or by concentrating them on a protein scaffold (see Figure 3–77). As will be explained later in this chapter, enzymes are also covalently modified to control their activity. The rate of protein destruction by targeted proteolysis represents yet another important regulatory mechanism (see Figure 6–86). But the most general process that adjusts reaction rates operates through a direct, reversible change in the activity of an enzyme in response to the specific small molecules that it binds.

1	The most common type of control occurs when an enzyme binds a molecule that is not a substrate to a special regulatory site outside the active site, thereby altering the rate at which the enzyme converts its substrates to products. For example, in feedback inhibition, a product produced late in a reaction pathway inhibits an enzyme that acts earlier in the pathway. Thus, whenever large quantities of the final product begin to accumulate, this product binds to the enzyme and slows down its catalytic action, thereby limiting the further entry of substrates into that reaction pathway (Figure 3–55). Where pathways branch or intersect, there are usually multiple points of control by different final products, each of which works to regulate its own synthesis (Figure 3–56). Feedback inhibition can work almost instantaneously, and it is rapidly reversed when the level of the product falls.

1	Figure 3–55 Feedback inhibition of a single biosynthetic pathway. The end product Z inhibits the first enzyme that is unique to its synthesis and thereby controls its own level in the cell. This is an example of negative regulation. Figure 3–56 Multiple feedback inhibition. In this example, which shows the biosynthetic pathways for four different amino acids in bacteria, the red lines indicate positions at which products feed back to inhibit enzymes. Each amino acid controls the first enzyme specific to its own synthesis, thereby controlling its own levels and avoiding a wasteful, or even dangerous, buildup of intermediates. The products can also separately inhibit the initial set of reactions common to all the syntheses; in this case, three different enzymes catalyze the initial reaction, each inhibited by a different product.

1	Feedback inhibition is negative regulation: it prevents an enzyme from acting. Enzymes can also be subject to positive regulation, in which a regulatory molecule stimulates the enzyme’s activity rather than shutting the enzyme down. Positive regulation occurs when a product in one branch of the metabolic network stimulates the activity of an enzyme in another pathway. As one example, the accumulation of ADP activates several enzymes involved in the oxidation of sugar molecules, thereby stimulating the cell to convert more ADP to ATP.

1	A striking feature of both positive and negative feedback regulation is that the regulatory molecule often has a shape totally different from the shape of the substrate of the enzyme. This is why the effect on a protein is termed allostery (from the Greek words allos, meaning “other,” and stereos, meaning “solid” or “three-dimensional”). As biologists learned more about feedback regulation, they recognized that the enzymes involved must have at least two different binding sites on their surface—an active site that recognizes the substrates, and a regulatory site that recognizes a regulatory molecule. These two sites must somehow communicate so that the catalytic events at the active site can be influenced by the binding of the regulatory molecule at its separate site on the protein’s surface.

1	The interaction between separated sites on a protein molecule is now known to depend on a conformational change in the protein: binding at one of the sites causes a shift from one folded shape to a slightly different folded shape. During feedback inhibition, for example, the binding of an inhibitor at one site on the protein causes the protein to shift to a conformation that incapacitates its active site located elsewhere in the protein.

1	It is thought that most protein molecules are allosteric. They can adopt two or more slightly different conformations, and a shift from one to another caused by the binding of a ligand can alter their activity. This is true not only for enzymes but also for many other proteins, including receptors, structural proteins, and motor proteins. In all instances of allosteric regulation, each conformation of the protein has somewhat different surface contours, and the protein’s binding sites for ligands are altered when the protein changes shape. Moreover, as we discuss next, each ligand will stabilize the conformation that it binds to most strongly, and thus—at high enough concentrations—will tend to “switch” the protein toward the conformation that the ligand prefers.

1	The effects of ligand binding on a protein follow from a fundamental chemical principle known as linkage. Suppose, for example, that a protein that binds glucose also binds another molecule, X, at a distant site on the protein’s surface. If the binding site for X changes shape as part of the conformational change in the protein induced by glucose binding, the binding sites for X and for glucose are said to be coupled. Whenever two ligands prefer to bind to the same conformation of an allosteric protein, it follows from basic thermodynamic principles that each ligand must increase the affinity of the protein for the other. For example, if the shift of a protein to a conformation that binds glucose best also causes the binding site for X to fit X better, then the protein will bind glucose more tightly when X is present than when X is absent. In other words, X will positively regulate the protein’s binding of glucose (Figure 3–57).

1	Conversely, linkage operates in a negative way if two ligands prefer to bind to different conformations of the same protein. In this case, the binding of the first ligand discourages the binding of the second ligand. Thus, if a shape change caused by glucose binding decreases the affinity of a protein for molecule X, the binding of X must also decrease the protein’s affinity for glucose (Figure 3–58). The linkage relationship is quantitatively reciprocal, so that, for example, if glucose has a very large effect on the binding of X, X has a very large effect on the binding of glucose.

1	The relationships shown in Figures 3–57 and 3–58 apply to all proteins, and they underlie all of cell biology. The principle seems so obvious in retrospect that we now take it for granted. But the discovery of linkage in studies of a few enzymes in the 1950s, followed by an extensive analysis of allosteric mechanisms in proteins in the early 1960s, had a revolutionary effect on our understanding of biology. Since molecule X in these examples binds at a site on the enzyme that is distinct from the site where catalysis occurs, it need not have any chemical relationship to the substrate that binds at the active site. Moreover, as we have just seen, for enzymes that are regulated in this way, molecule X can either turn the enzyme on (positive regulation) or turn it off (negative regulation). By such a mechanism, allosteric proteins serve as general switches that, in principle, can allow one molecule in a cell to affect the fate of any other.

1	A single-subunit enzyme that is regulated by negative feedback can at most decrease from 90% to about 10% activity in response to a 100-fold increase in the concentration of an inhibitory ligand that it binds (Figure 3–59, red line). Responses of this type are apparently not sharp enough for optimal cell regulation, and most enzymes that are turned on or off by ligand binding consist of symmetric assemblies of identical subunits. With this arrangement, the binding of a molecule of ligand to a single site on one subunit can promote an allosteric change in the entire assembly that helps the neighboring subunits bind the same ligand. As a result, a cooperative allosteric transition occurs (Figure 3–59, blue line), allowing

1	Figure 3–57 Positive regulation caused by conformational coupling between two separate binding sites. In this example, both glucose and molecule X bind best to the closed conformation of a protein with two domains. Because both glucose and molecule X drive the protein toward its closed conformation, each ligand helps the other to bind. Glucose and molecule X are therefore said to bind cooperatively to the protein. Figure 3–58 negative regulation caused by conformational coupling between two separate binding sites. The scheme here resembles that in the previous figure, but here molecule X prefers the open conformation, while glucose prefers the closed conformation. Because glucose and molecule X drive the protein toward opposite conformations (closed and open, respectively), the presence of either ligand interferes with the binding of the other.

1	a relatively small change in ligand concentration in the cell to switch the whole assembly from an almost fully active to an almost fully inactive conformation (or vice versa). The principles involved in a cooperative “all-or-none” transition are the same for all proteins, whether or not they are enzymes. Thus, for example, they are critical for the efficient uptake and release of O2 by hemoglobin in our blood. But they are perhaps easiest to visualize for an enzyme that forms a symmetric dimer. In the example shown in Figure 3–60, the first molecule of an inhibitory ligand binds with great difficulty since its binding disrupts an energetically favorable interaction between the two identical monomers in the dimer. A second molecule of inhibitory ligand now binds more easily, however, because its binding restores the energetically favorable monomer–monomer contacts of a symmetric dimer (this also completely inactivates the enzyme).

1	As an alternative to this induced fit model for a cooperative allosteric transition, we can view such a symmetric enzyme as having only two possible conformations, corresponding to the “enzyme on” and “enzyme off” structures in Figure 3–60. In this view, ligand binding perturbs an all-or-none equilibrium between these two states, thereby changing the proportion of active molecules. Both models represent true and useful concepts. Proteins are regulated by more than the reversible binding of other molecules. A second method that eukaryotic cells use extensively to regulate a protein’s function is the covalent addition of a smaller molecule to one or more of its amino acid side chains. The most common such regulatory modification in higher eukaryotes is the addition of a phosphate group. We shall therefore use protein phosphorylation to illustrate some of the general principles involved in the control of protein function through the modification of amino acid side chains.

1	A phosphorylation event can affect the protein that is modified in three important ways. First, because each phosphate group carries two negative charges, the enzyme-catalyzed addition of a phosphate group to a protein can cause a major conformational change in the protein by, for example, attracting a cluster of positively charged amino acid side chains. This can, in turn, affect the binding of ligands elsewhere on the protein surface, dramatically changing the

1	Figure 3–60 a cooperative allosteric transition in an enzyme composed of two identical subunits. This diagram illustrates how the conformation of one subunit can influence that of its neighbor. The binding of a single molecule of an inhibitory ligand (yellow) to one subunit of the enzyme occurs with difficulty because it changes the conformation of this subunit and thereby disrupts the symmetry of the enzyme. Once this conformational change has occurred, however, the energy gained by restoring the symmetric pairing interaction between the two subunits makes it especially easy for the second subunit to bind the inhibitory ligand and undergo the same conformational change. Because the binding of the first molecule of ligand increases the affinity with which the other subunit binds the same ligand, the response of the enzyme to changes in the concentration of the ligand is much steeper than the response of an enzyme with only one subunit (see Figure 3–59 and Movie 3.10).

1	Figure 3–59 enzyme activity versus the concentration of inhibitory ligand for single-subunit and multisubunit allosteric enzymes. For an enzyme with a single subunit (red line), a drop from 90% enzyme activity to 10% activity (indicated by the two dots on the curve) requires a 100-fold increase in the concentration of inhibitor. The enzyme activity is calculated from the simple equilibrium relationship K = [IP]/[I][P], where P is active protein, I is inhibitor, and IP is the inactive protein bound to inhibitor. An identical curve applies to any simple binding interaction between two molecules, A and B. In contrast, a multisubunit allosteric enzyme can respond in a switchlike manner to a change in ligand concentration: the steep response is caused by a cooperative binding of the ligand molecules, as explained in Figure 3–60. Here, the green line represents the idealized result expected for the cooperative binding of two inhibitory ligand molecules to an allosteric enzyme with two

1	molecules, as explained in Figure 3–60. Here, the green line represents the idealized result expected for the cooperative binding of two inhibitory ligand molecules to an allosteric enzyme with two subunits, and the blue line shows the idealized response of an enzyme with four subunits. As indicated by the two dots on each of these curves, the more complex enzymes drop from 90% to 10% activity over a much narrower range of inhibitor concentration than does the enzyme composed of a single subunit.

1	protein’s activity. When a second enzyme removes the phosphate group, the protein returns to its original conformation and restores its initial activity. Second, an attached phosphate group can form part of a structure that the binding sites of other proteins recognize. As previously discussed, the SH2 domain binds to a short peptide sequence containing a phosphorylated tyrosine side chain (see Figure 3–40B). More than ten other common domains provide binding sites for attaching their protein to phosphorylated peptides in other protein molecules, each recognizing a phosphorylated amino acid side chain in a different protein context. Third, the addition of a phosphate group can mask a binding site that otherwise holds two proteins together, and thereby disrupt protein–protein interactions. As a result, protein phosphorylation and dephosphorylation very often drive the regulated assembly and disassembly of protein complexes (see, for example, Figure 15–11).

1	Reversible protein phosphorylation controls the activity, structure, and cellular localization of both enzymes and many other types of proteins in eukaryotic cells. In fact, this regulation is so extensive that more than one-third of the 10,000 or so proteins in a typical mammalian cell are thought to be phosphorylated at any given time—many with more than one phosphate. As might be expected, the addition and removal of phosphate groups from specific proteins often occur in response to signals that specify some change in a cell’s state. For example, the complicated series of events that takes place as a eukaryotic cell divides is largely timed in this way (discussed in Chapter 17), and many of the signals mediating cell–cell interactions are relayed from the plasma membrane to the nucleus by a cascade of protein phosphorylation events (discussed in Chapter 15). A Eukaryotic Cell Contains a Large Collection of Protein Kinases and Protein Phosphatases

1	Protein phosphorylation involves the enzyme-catalyzed transfer of the terminal phosphate group of an ATP molecule to the hydroxyl group on a serine, threonine, or tyrosine side chain of the protein (Figure 3–61). A protein kinase catalyzes this reaction, and the reaction is essentially unidirectional because of the large amount of free energy released when the phosphate–phosphate bond in ATP is broken to produce ADP (discussed in Chapter 2). A protein phosphatase catalyzes the reverse reaction of phosphate removal, or dephosphorylation. Cells contain hundreds of different protein kinases, each responsible for phosphorylating a different protein or set of proteins. There are also many different protein phosphatases; some are highly specific and remove phosphate groups from only one or a few proteins, whereas others act on a broad range of proteins and are targeted to specific substrates by regulatory subunits. The state of phosphorylation of a protein at any moment, and thus its

1	a few proteins, whereas others act on a broad range of proteins and are targeted to specific substrates by regulatory subunits. The state of phosphorylation of a protein at any moment, and thus its activity, depends on the relative activities of the protein kinases and phosphatases that modify it.

1	The protein kinases that phosphorylate proteins in eukaryotic cells belong to a very large family of enzymes that share a catalytic (kinase) sequence of about 290 amino acids. The various family members contain different amino acid sequences on either end of the kinase sequence (for example, see Figure 3–10), and often have short amino acid sequences inserted into loops within it. Some of these additional amino acid sequences enable each kinase to recognize the specific set of proteins it phosphorylates, or to bind to structures that localize it in specific regions of the cell. Other parts of the protein regulate the activity of each kinase, so it can be turned on and off in response to different specific signals, as described below.

1	By comparing the number of amino acid sequence differences between the various members of a protein family, we can construct an “evolutionary tree” that is thought to reflect the pattern of gene duplication and divergence that gave rise to the family. Figure 3–62 shows an evolutionary tree of protein kinases. Kinases with related functions are often located on nearby branches of the tree: the protein kinases involved in cell signaling that phosphorylate tyrosine side chains, for example, are all clustered in the top left corner of the tree. The other kinases shown

1	Figure 3–61 Protein phosphorylation. Many thousands of proteins in a typical eukaryotic cell are modified by the covalent addition of a phosphate group. (A) The general reaction transfers a phosphate group from ATP to an amino acid side chain of the target protein by a protein kinase. Removal of the phosphate group is catalyzed by a second enzyme, a protein phosphatase. In this example, the phosphate is added to a serine side chain; in other cases, the phosphate is instead linked to the –OH group of a threonine or a tyrosine in the protein. (B) The phosphorylation of a protein by a protein kinase can either increase or decrease the protein’s activity, depending on the site of phosphorylation and the structure of the protein. phosphorylate either a serine or a threonine side chain, and many are organized into clusters that seem to reflect their function—in transmembrane signal transduction, intracellular signal amplification, cell-cycle control, and so on.

1	As a result of the combined activities of protein kinases and protein phosphatases, the phosphate groups on proteins are continually turning over—being added and then rapidly removed. Such phosphorylation cycles may seem wasteful, but they are important in allowing the phosphorylated proteins to switch rapidly from one state to another: the more rapid the cycle, the faster a population of protein molecules can change its state of phosphorylation in response to a sudden change in its phosphorylation rate (see Figure 15–14). The energy required to drive this phosphorylation cycle is derived from the free energy of ATP hydrolysis, one molecule of which is consumed for each phosphorylation event. The Regulation of the Src Protein Kinase Reveals How a Protein Can Function as a Microprocessor

1	The Regulation of the Src Protein Kinase Reveals How a Protein Can Function as a Microprocessor The hundreds of different protein kinases in a eukaryotic cell are organized into complex networks of signaling pathways that help to coordinate the cell’s activities, drive the cell cycle, and relay signals into the cell from the cell’s environment. Many of the extracellular signals involved need to be both integrated and amplified by the cell. Individual protein kinases (and other signaling proteins) serve as input–output devices, or “microprocessors,” in the integration process. An important part of the input to these signal-processing proteins comes from the control that is exerted by phosphates added and removed from them by protein kinases and protein phosphatases, respectively.

1	The Src family of protein kinases (see Figure 3–10) exhibits such behavior. The Src protein (pronounced “sarc” and named for the type of tumor, a sarcoma, that its deregulation can cause) was the first tyrosine kinase to be discovered. It is now known to be part of a subfamily of nine very similar protein kinases, which are found only in multicellular animals. As indicated by the evolutionary tree in Figure 3–62, sequence comparisons suggest that tyrosine kinases as a group were a relatively late innovation that branched off from the serine/threonine kinases, with the Src subfamily being only one subgroup of the tyrosine kinases created in this way. The Src protein and its relatives contain a short N-terminal region that becomes covalently linked to a strongly hydrophobic fatty acid, which anchors the kinase at the cytoplasmic face of the plasma membrane. Next along the linear sequence of

1	Figure 3–62 an evolutionary tree of selected protein kinases. A higher eukaryotic cell contains hundreds of such enzymes, and the human genome codes for more than 500. Note that only some of these, those discussed in this book, are shown. Figure 3–64 The activation of a Src-type protein kinase by two sequential events. As described in the text, the requirement for multiple upstream events to trigger these processes allows the kinase to serve as a signal integrator (Movie 3.11). (Adapted from S.C. Harrison et al., Cell 112:737–740, 2003. With permission from Elsevier.) Figure 3–65 How a Src-type protein kinase acts as a signal-integrating device. A disruption of the inhibitory interaction illustrated for the SH3 domain (green) occurs when its binding to the indicated orange linker region is replaced with its higher-affinity binding to an activating ligand.

1	change that inactivates the protein. The three-dimensional structure of a prototypical member of this family, the monomeric GTPase called Ras, is shown in Figure 3–67. The Ras protein has an important role in cell signaling (discussed in Chapter 15). In its GTP-bound form, it is active and stimulates a cascade of protein phosphorylations in the cell. Most of the time, however, the protein is in its inactive, GDP-bound form. It becomes active when it exchanges its GDP for a GTP molecule in response to extracellular signals, such as growth factors, that bind to receptors in the plasma membrane (see Figure 15–47). Regulatory Proteins GAP and GEF Control the Activity of GTP-Binding Proteins by Determining Whether GTP or GDP Is Bound

1	GTP-binding proteins are controlled by regulatory proteins that determine whether GTP or GDP is bound, just as phosphorylated proteins are turned on and off by protein kinases and protein phosphatases. Thus, Ras is inactivated by a GTPase-activating protein (GAP), which binds to the Ras protein and induces Ras to hydrolyze its bound GTP molecule to GDP—which remains tightly bound— and inorganic phosphate (Pi), which is rapidly released. The Ras protein stays in its inactive, GDP-bound conformation until it encounters a guanine nucleotide exchange factor (GEF), which binds to GDP-Ras and causes Ras to release its GDP. Because the empty nucleotide-binding site is immediately filled by a GTP molecule (GTP is present in large excess over GDP in cells), the GEF activates Ras by indirectly adding back the phosphate removed by GTP hydrolysis. Thus, in a sense, the roles of GAP and GEF are analogous to those of a protein phosphatase and a protein kinase, respectively (Figure 3–68).

1	Proteins Can Be Regulated by the Covalent Addition of Other Proteins

1	Cells contain a special family of small proteins whose members are covalently attached to many other proteins to determine the activity or fate of the second protein. In each case, the carboxyl end of the small protein becomes linked to the amino group of a lysine side chain of a “target” protein through an isopeptide bond. The first such protein discovered, and the most abundantly used, is ubiquitin (Figure 3–69A). Ubiquitin can be covalently attached to target proteins in a variety of ways, each of which has a different meaning for cells. The major form of ubiquitin addition produces polyubiquitin chains in which—once the first ubiquitin molecule is attached to the target—each subsequent ubiquitin molecule links to Lys48 of the previous ubiquitin, creating a chain of Lys48-linked ubiquitins that are attached to a single lysine side chain of the target protein. This form of polyubiquitin directs the target protein to the interior of a proteasome, where it is digested to small

1	ubiquitins that are attached to a single lysine side chain of the target protein. This form of polyubiquitin directs the target protein to the interior of a proteasome, where it is digested to small peptides (see Figure 6–84). In other circumstances, only single molecules of ubiquitin are added to proteins. In addition, some target proteins are has this binding been disrupted? has this phosphate been added? has this phosphate been removed? P P

1	Src-type protein kinase activity turns on fully only if the answers to all of the above questions are yes Figure 3–66 GTP-binding proteins as molecular switches. The activity of a GTP-binding protein (also called a GTPase) generally requires the presence of a tightly bound GTP molecule (switch “on”). Hydrolysis of this GTP molecule by the GTP-binding protein produces GDP and inorganic phosphate (Pi), and it causes the protein to convert to a different, usually inactive, conformation (switch “off”). Resetting the switch requires that the tightly bound GDP dissociates. This is a slow step that is greatly accelerated by specific signals; once the GDP has dissociated, a molecule of GTP is quickly rebound. modified with a different type of polyubiquitin chain. These modifications have different functional consequences for the protein that is targeted (Figure 3–69B).

1	modified with a different type of polyubiquitin chain. These modifications have different functional consequences for the protein that is targeted (Figure 3–69B). Related structures are created when a different member of the ubiquitin family, such as SUMO (small ubiquitin-related modifier), is covalently attached to a lysine side chain of target proteins. Not surprisingly, all such modifications are reversible. Cells contain sets of ubiquitylating and deubiquitylating (and sumoylating and desumoylating) enzymes that manipulate these covalent adducts, thereby playing roles analogous to the protein kinases and phosphatases that add and remove phosphates from protein side chains. An Elaborate Ubiquitin-Conjugating System Is Used to Mark Proteins

1	An Elaborate Ubiquitin-Conjugating System Is Used to Mark Proteins How do cells select target proteins for ubiquitin addition? As an initial step, the carboxyl end of ubiquitin needs to be activated. This activation is accomplished when a protein called a ubiquitin-activating enzyme (E1) uses ATP hydrolysis energy to attach ubiquitin to itself through a high-energy covalent bond (a thioester). E1 then passes this activated ubiquitin to one of a set of ubiquitin-conjugating (E2) enzymes, each of which acts in conjunction with a set of accessory (E3) proteins called ubiquitin ligases. There are roughly 30 structurally similar but distinct E2 enzymes in mammals, and hundreds of different E3 proteins that form complexes with specific E2 enzymes.

1	Figure 3–70 illustrates the process used to mark proteins for proteasomal degradation. [Similar mechanisms are used to attach ubiquitin (and SUMO) to other types of target proteins.] Here, the ubiquitin ligase binds to specific degradation signals, called degrons, in protein substrates, thereby helping E2 to form a polyubiquitin chain linked to a lysine of the substrate protein. This polyubiquitin chain on a target protein will then be recognized by a specific receptor in the proteasome, causing the target protein to be destroyed. Distinct ubiquitin ligases recognize different degradation signals, thereby targeting distinct subsets of intracellular proteins for destruction, often in response to specific signals (see Figure 6–86).

1	Figure 3–67 The structure of the Ras protein in its GTP-bound form. This monomeric GTPase illustrates the structure of a GTP-binding domain, which is present in a large family of GTP-binding proteins. The red regions change their conformation when the GTP molecule is hydrolyzed to GDP and inorganic phosphate by the protein; the GDP remains bound to the protein, while the inorganic phosphate is released. The special role of the “switch helix” in proteins related to Ras is explained in the text (see Figure 3–72 and Movie 15.7). Figure 3–68 a comparison of two major intracellular signaling mechanisms in eukaryotic cells. In both cases, a signaling protein is activated by the addition of a phosphate group and inactivated by the removal of this phosphate. Note that the addition of a phosphate to a protein can also be inhibitory. (Adapted from E.R. Kantrowitz and W.N. Lipscomb, Trends Biochem. Sci. 15:53–59, 1990.)

1	Protein Complexes with Interchangeable Parts Make Efficient Use of Genetic Information

1	The SCF ubiquitin ligase is a protein complex that binds different “target proteins” at different times in the cell cycle, covalently adding polyubiquitin polypeptide chains to these targets. Its C-shaped structure is formed from five protein subunits, the largest of which serves as a scaffold on which the rest of the complex is built. The structure underlies a remarkable mechanism (Figure 3–71). At one end of the C is an E2 ubiquitin-conjugating enzyme. At the other end is a substrate-binding arm, a subunit known as an F-box protein. These two subunits are separated by a gap of about 5 nm. When this protein complex is activated, the F-box protein binds to a specific site on a target protein, positioning the protein in the gap so that some of its lysine side chains contact the ubiquitin-conjugating enzyme. The enzyme can then catalyze repeated additions of ubiquitin polypeptide to these lysines (see Figure 3–71C), producing polyubiquitin chains that mark the target proteins for rapid

1	enzyme. The enzyme can then catalyze repeated additions of ubiquitin polypeptide to these lysines (see Figure 3–71C), producing polyubiquitin chains that mark the target proteins for rapid destruction in a proteasome.

1	Figure 3–69 The marking of proteins by ubiquitin. (A) The three-dimensional structure of ubiquitin, a small protein of 76 amino acids. A family of special enzymes couples its carboxyl end to the amino group of a lysine side chain in a target protein molecule, forming an isopeptide bond. (B) Some modification patterns that have specific meanings to the cell. Note that the two types of polyubiquitylation differ in the way the ubiquitin molecules are linked together. Linkage through Lys48 signifies degradation by the proteasome (see Figure 6–84), whereas that through Lys63 has other meanings. Ubiquitin markings are “read” by proteins that specifically recognize each type of modification.

1	Figure 3–70 The marking of proteins with ubiquitin. (A) The C-terminus of ubiquitin is initially activated by being linked via a high-energy thioester bond to a cysteine side chain on the E1 protein. This reaction requires ATP, and it proceeds via a covalent AMP-ubiquitin intermediate. The activated ubiquitin on E1, also known as the ubiquitin-activating enzyme, is then transferred to the cysteine on an E2 molecule. (B) The addition of a polyubiquitin chain to a target protein. In a mammalian cell, there are several hundred distinct E2–E3 complexes. The E2s are called ubiquitinconjugating enzymes. The E3s are referred to as ubiquitin ligases. (Adapted from D.R. Knighton et al., Science 253:407–414, 1991.) two of many polyubiquitylated possible protein targeted substrate-binding for destruction arms

1	In this manner, specific proteins are targeted for rapid destruction in response to specific signals, thereby helping to drive the cell cycle (discussed in Chapter 17). The timing of the destruction often involves creating a specific pattern of phosphorylation on the target protein that is required for its recognition by the F-box subunit. It also requires the activation of an SCF ubiquitin ligase that carries the appropriate substrate-binding arm. Many of these arms (the F-box subunits) are interchangeable in the protein complex (see Figure 3–71B), and there are more than 70 human genes that encode them.

1	As emphasized previously, once a successful protein has evolved, its genetic information tends to be duplicated to produce a family of related proteins. Thus, for example, not only are there many F-box proteins—making possible the recognition of different sets of target proteins—but there is also a family of scaffolds (known as cullins) that give rise to a family of SCF-like ubiquitin ligases. A protein machine like the SCF ubiquitin ligase, with its interchangeable parts, makes economical use of the genetic information in cells. It also creates opportunities for “rapid” evolution, inasmuch as new functions can evolve for the entire complex simply by producing an alternative version of one of its subunits. Ubiquitin ligases form a diverse family of protein complexes. Some of these complexes are far larger and more complicated than SCF, but their underlying enzymatic function remains the same (Figure 3–71D).

1	Detailed structures obtained for one of the GTP-binding protein family members, the EF-Tu protein, provide a good example of how allosteric changes in protein conformations can produce large movements by amplifying a small, local conformational change. As will be discussed in Chapter 6, EF-Tu is an abundant molecule that serves as an elongation factor (hence the EF) in protein synthesis, loading each aminoacyl-tRNA molecule onto the ribosome. EF-Tu contains a Ras-like domain (see Figure 3–67), and the tRNA molecule forms a tight complex with its GTP-bound form. This tRNA molecule can transfer its amino acid to the growing

1	Figure 3–71 The structure and mode of action of an SCF ubiquitin ligase. (A) The structure of the five-protein ubiquitin ligase complex that includes an E2 ubiquitinconjugating enzyme. Four proteins form the E3 portion. The protein denoted here as adaptor protein 1 is the Rbx1/Hrt1 protein, adaptor protein 2 is the Skp1 protein, and the cullin is the Cul1 protein. One of the many different F-box proteins completes the complex. (B) Comparison of the same complex with two different substrate-binding arms, the F-box proteins Skp2 (top) and β-trCP1 (bottom), respectively. (C) The binding and ubiquitylation of a target protein by the SCF ubiquitin ligase. If, as indicated, a chain of ubiquitin molecules is added to the same lysine of the target protein, that protein is marked for rapid destruction by the proteasome. (D) Comparison of SCF (bottom) with a low-resolution electron microscopy structure of a ubiquitin ligase called the anaphase-promoting complex (APC/C; top) at the same scale.

1	by the proteasome. (D) Comparison of SCF (bottom) with a low-resolution electron microscopy structure of a ubiquitin ligase called the anaphase-promoting complex (APC/C; top) at the same scale. The APC/C is a large, 15-protein complex. As discussed in Chapter 17, its ubiquitylations control the late stages of mitosis. It is distantly related to SCF and contains a cullin subunit (green) that lies along the side of the complex at right, only partly visible in this view. E2 proteins are not shown here, but their binding sites are indicated in orange, along with substrate-binding sites in purple. (A and B, adapted from G. Wu et al., Mol. Cell 11:1445–1456, 2003. With permission from Elsevier; D, adapted from

1	P. da Fonseca et al., Nature 470:274–278, 2011. With permission from Macmillan Publishers Ltd.)

1	Figure 3–72 The large conformational change in eF-Tu caused by GTP hydrolysis. (A and B) The three-dimensional structure of EF-Tu with GTP bound. The domain at the top has a structure similar to the Ras protein, and its red α helix is the switch helix, which moves after GTP hydrolysis. (C) The change in the conformation of the switch helix in domain 1 allows domains 2 and 3 to rotate as a single unit by about 90 degrees toward the viewer, which releases the tRNA that was bound to this structure (see also Figure 3–73). (A, adapted from H. Berchtold et al., Nature 365:126–132, 1993. With permission from Macmillan Publishers Ltd. B, courtesy of Mathias Sprinzl and Rolf Hilgenfeld. PDB code: 1EFT.) (B) domain 1 P P P GTP-binding site switch helix domain 3 domain 2 PP bound GDP release of tRNA GTP hydrolysis site of tRNA binding G G HOOC NH2 switch helix GTP (A) polypeptide chain only after the GTP bound to EF-Tu is hydrolyzed, dissociating the EF-Tu. Since this GTP hydrolysis is triggered

1	GTP hydrolysis site of tRNA binding G G HOOC NH2 switch helix GTP (A) polypeptide chain only after the GTP bound to EF-Tu is hydrolyzed, dissociating the EF-Tu. Since this GTP hydrolysis is triggered by a proper fit of the tRNA to the mRNA molecule on the ribosome, the EF-Tu serves as a factor that discriminates between correct and incorrect mRNA–tRNA pairings (see Figure 6–65).

1	By comparing the three-dimensional structure of EF-Tu in its GTP-bound and GDP-bound forms, we can see how the repositioning of the tRNA occurs. The dissociation of the inorganic phosphate group (Pi), which follows the reaction GTP →GDP + Pi, causes a shift of a few tenths of a nanometer at the GTP-binding site, just as it does in the Ras protein. This tiny movement, equivalent to a few times the diameter of a hydrogen atom, causes a conformational change to propagate along a crucial piece of αhelix, called the switch helix, in the Ras-like domain of the protein. The switch helix seems to serve as a latch that adheres to a specific site in another domain of the molecule, holding the protein in a “shut” conformation. The conformational change triggered by GTP hydrolysis causes the switch helix to detach, allowing separate domains of the protein to swing apart, through a distance of about 4 nm (Figure 3–72). This releases the bound tRNA molecule, allowing its attached amino acid to be

1	helix to detach, allowing separate domains of the protein to swing apart, through a distance of about 4 nm (Figure 3–72). This releases the bound tRNA molecule, allowing its attached amino acid to be used (Figure 3–73).

1	Notice in this example how cells have exploited a simple chemical change that occurs on the surface of a small protein domain to create a movement 50 times larger. Dramatic shape changes of this type also cause the very large movements that occur in motor proteins, as we discuss next.

1	We have seen that conformational changes in proteins have a central role in enzyme regulation and cell signaling. We now discuss proteins whose major function is to move other molecules. These motor proteins generate the forces responsible for muscle contraction and the crawling and swimming of cells. Motor proteins also power smaller-scale intracellular movements: they help to move chromosomes to opposite ends of the cell during mitosis (discussed in Chapter 17), to move organelles along molecular tracks within the cell (discussed in Chapter 16), and to move enzymes along a DNA strand during the synthesis of a new DNA molecule (discussed in Chapter 5). All these fundamental processes depend on proteins with moving parts that operate as force-generating machines.

1	How do these machines work? In other words, how do cells use shape changes in proteins to generate directed movements? If, for example, a protein is required to walk along a narrow thread such as a DNA molecule, it can do this by undergoing a series of conformational changes, such as those shown in Figure 3–74. But with nothing to drive these changes in an orderly sequence, they are perfectly

1	Figure 3–73 an aminoacyl tRna molecule bound to eF-Tu. Note how the bound protein blocks the use of the tRNA-linked amino acid (green) for protein synthesis until GTP hydrolysis triggers the conformational changes shown in Figure 3–72C, dissociating the protein-tRNA complex. EF-Tu is a bacterial protein; however, a very similar protein exists in eukaryotes, where it is called EF-1 (Movie 3.12). (Coordinates determined by P. Nissen et al., Science 270:1464–1472, 1995. PDB code: 1B23.) 162 Chapter 3: Proteins reversible, and the protein can only wander randomly back and forth along the thread. We can look at this situation in another way. Since the directional movement of a protein does work, the laws of thermodynamics (discussed in Chapter 2) demand that such movement use free energy from some other source (otherwise the protein could be used to make a perpetual motion machine). Therefore, without an input of energy, the protein molecule can only wander aimlessly.

1	How can the cell make such a series of conformational changes unidirectional? To force the entire cycle to proceed in one direction, it is enough to make any one of the changes in shape irreversible. Most proteins that are able to walk in one direction for long distances achieve this motion by coupling one of the conformational changes to the hydrolysis of an ATP molecule that is tightly bound to the protein. The mechanism is similar to the one just discussed that drives allosteric protein shape changes by GTP hydrolysis. Because ATP (or GTP) hydrolysis releases a great deal of free energy, it is very unlikely that the nucleotide-binding protein will undergo the reverse shape change needed for moving backward— since this would require that it also reverse the ATP hydrolysis by adding a phosphate molecule to ADP to form ATP.

1	In the model shown in Figure 3–75A, ATP binding shifts a motor protein from conformation 1 to conformation 2. The bound ATP is then hydrolyzed to produce ADP and inorganic phosphate (Pi), causing a change from conformation 2 to conformation 3. Finally, the release of the bound ADP and Pi drives the protein back to conformation 1. Because the energy provided by ATP hydrolysis drives the transition 2 → 3, this series of conformational changes is effectively irreversible. Thus, the entire cycle goes in only one direction, causing the protein molecule to walk continuously to the right in this example. Many motor proteins generate directional movement through the use of a similar unidirectional ratchet, including the muscle motor protein myosin, direction of Figure 3–74 an allosteric “walking” protein. Although its three different conformations allow it to wander randomly back and forth while bound to a thread or a filament, the protein cannot move uniformly in a single direction.

1	Figure 3–75 How a protein can walk in one direction. (A) An allosteric motor protein driven by ATP hydrolysis. The transition between three different conformations includes a step driven by the hydrolysis of a bound ATP molecule, creating a “unidirectional ratchet” that makes the entire cycle essentially irreversible. By repeated cycles, the protein therefore moves continuously to the right along the thread. (B) Direct visualization of a walking myosin motor protein by high-speed atomic force microscopy; the elapsed time between steps was less than 0.5 sec (see Movie 16.3). (B, modified from

1	N. Kodera et al., Nature 468:72–76, 2010. With permission from Macmillan Publishers Ltd.) which walks along actin filaments (Figure 3–75B), and the kinesin proteins that walk along microtubules (both discussed in Chapter 16). These movements can be rapid: some of the motor proteins involved in DNA replication (the DNA helicases) propel themselves along a DNA strand at rates as high as 1000 nucleotides per second. Membrane-Bound Transporters Harness Energy to Pump Molecules Through Membranes We have thus far seen how proteins that undergo allosteric shape changes can act as microprocessors (Src family kinases), as assembly factors (EF-Tu), and as generators of mechanical force and motion (motor proteins). Allosteric proteins can also harness energy derived from ATP hydrolysis, ion gradients, or electron-transport processes to pump specific ions or small molecules across a membrane. We consider one example here that will be discussed in more detail in Chapter 11.

1	The ABC transporters (ATP-binding cassette transporters) constitute an important class of membrane-bound pump proteins. In humans, at least 48 different genes encode them. These transporters mostly function to export hydrophobic molecules from the cytoplasm, serving to remove toxic molecules at the mucosal surface of the intestinal tract, for example, or at the blood–brain barrier. The study of ABC transporters is of intense interest in clinical medicine, because the overproduction of proteins in this class contributes to the resistance of tumor cells to chemotherapeutic drugs. In bacteria, the same types of proteins primarily function to import essential nutrients into the cell.

1	A typical ABC transporter contains a pair of membrane-spanning subunits linked to a pair of ATP-binding subunits located just below the plasma membrane. As in other examples we have discussed, the hydrolysis of the bound ATP molecules drives conformational changes in the protein, transmitting forces that cause the membrane-spanning subunits to move their bound molecules across the lipid bilayer (Figure 3–76). Humans have invented many different types of mechanical pumps, and it should not be surprising that cells also contain membrane-bound pumps that

1	Humans have invented many different types of mechanical pumps, and it should not be surprising that cells also contain membrane-bound pumps that Figure 3–76 The abC (aTP-binding cassette) transporter, a protein machine that pumps molecules through a membrane. (A) How this large family of transporters pumps molecules into the cell in bacteria. As indicated, the binding of two molecules of ATP causes the two ATP-binding domains to clamp together tightly, opening a channel to the cell exterior. The binding of a substrate molecule to the extracellular face of the protein complex then triggers ATP hydrolysis followed by ADP release, which opens the cytoplasmic gate; the pump is then reset for another cycle.

1	(B) As discussed in Chapter 11, in eukaryotes an opposite process occurs, causing selected substrate molecules to be pumped out of the cell. (C) The structure of a bacterial ABC transporter (see Movie 11.5). (C, from R.J. Dawson and K.P. Locher, Nature 443:180–185, 2006. With permission from Macmillan Publishers Ltd; PDB code: 2HYD). function in other ways. Among the most notable are the rotary pumps that couple the hydrolysis of ATP to the transport of H+ ions (protons). These pumps resemble miniature turbines, and they are used to acidify the interior of lysosomes and other eukaryotic organelles. Like other ion pumps that create ion gradients, they can function in reverse to catalyze the reaction ADP + Pi → ATP, if the gradient across their membrane of the ion that they transport is steep enough.

1	One such pump, the ATP synthase, harnesses a gradient of proton concentration produced by electron-transport processes to produce most of the ATP used in the living world. This ubiquitous pump has a central role in energy conversion, and we shall discuss its three-dimensional structure and mechanism in Chapter 14. Proteins Often Form Large Complexes That Function as Protein Machines

1	Large proteins formed from many domains are able to perform more elaborate functions than small, single-domain proteins. But large protein assemblies formed from many protein molecules linked together by noncovalent bonds perform the most impressive tasks. Now that it is possible to reconstruct most biological processes in cell-free systems in the laboratory, it is clear that each of the central processes in a cell—such as DNA replication, protein synthesis, vesicle budding, or transmembrane signaling—is catalyzed by a highly coordinated, linked set of 10 or more proteins. In most such protein machines, an energetically favorable reaction such as the hydrolysis of bound nucleoside triphosphates (ATP or GTP) drives an ordered series of conformational changes in one or more of the individual protein subunits, enabling the ensemble of proteins to move coordinately. In this way, each enzyme can be moved directly into position, as the machine catalyzes successive reactions in a series

1	protein subunits, enabling the ensemble of proteins to move coordinately. In this way, each enzyme can be moved directly into position, as the machine catalyzes successive reactions in a series (Figure 3–77). This is what occurs, for example, in protein synthesis on a ribosome (discussed in Chapter 6)—or in DNA replication, where a large multiprotein complex moves rapidly along the DNA (discussed in Chapter 5).

1	Cells have evolved protein machines for the same reason that humans have invented mechanical and electronic machines. For accomplishing almost any task, manipulations that are spatially and temporally coordinated through linked processes are much more efficient than the use of many separate tools. Scaffolds Concentrate Sets of Interacting Proteins

1	Scaffolds Concentrate Sets of Interacting Proteins As scientists have learned more of the details of cell biology, they have recognized an increasing degree of sophistication in cell chemistry. Thus, not only do we now know that protein machines play a predominant role, but it has also become clear that they are very often localized to specific sites in the cell, being assembled and activated only where and when they are needed. As one example, when extracellular signaling molecules bind to receptor proteins in the plasma membrane, the activated receptors often recruit a set of other proteins to the inside surface of the plasma membrane to form a large protein complex that passes the signal on (discussed in Chapter 15).

1	The mechanisms frequently involve scaffold proteins. These are proteins with binding sites for multiple other proteins, and they serve both to link together specific sets of interacting proteins and to position them at specific locations inside a cell. At one extreme are rigid scaffolds, such as the cullin in SCF ubiquitin ligase (see Figure 3–71). At the other extreme are the large, flexible scaffold proteins that often underlie regions of specialized plasma membrane. These include the Figure 3–77 How “protein machines” carry out complex functions.

1	Figure 3–77 How “protein machines” carry out complex functions. These machines are made of individual proteins that collaborate to perform a specific task (Movie 3.13). The movement of these proteins is often coordinated by the hydrolysis of a bound nucleotide such as ATP or GTP. Directional allosteric conformational changes of proteins that are driven in this way often occur in a large protein assembly in which the activities of several different protein molecules are coordinated by such movements within the complex.

1	Discs-large protein (Dlg), a protein of about 900 amino acids that is concentrated in special regions beneath the plasma membrane in epithelial cells and at synapses. Dlg contains binding sites for at least seven other proteins, interspersed with regions of more flexible polypeptide chain. An ancient protein, conserved in organisms as diverse as sponges, worms, flies, and humans, Dlg derives its name from the mutant phenotype of the organism in which it was first discovered; the cells in the imaginal discs of a Drosophila embryo with a mutation in the Dlg gene fail to stop proliferating when they should, and they produce unusually large discs whose epithelial cells can form tumors.

1	Although incompletely studied, Dlg and a large number of similar scaffold proteins are thought to function like the protein that is schematically illustrated in Figure 3–78. By binding a specific set of interacting proteins, these scaffolds can enhance the rate of critical reactions, while also confining them to the particular region of the cell that contains the scaffold. For similar reasons, cells also make extensive use of scaffold RNA molecules, as discussed in Chapter 7. Many Proteins Are Controlled by Covalent Modifications That Direct Them to Specific Sites Inside the Cell We have thus far described only a few ways in which proteins are post-translationally modified. A large number of other such modifications also occur, more than 200 distinct types being known. To give a sense of the variety, Table 3–3 presents

1	Figure 3–78 How the proximity created by scaffold proteins can greatly speed reactions in a cell. In this example, long unstructured regions of polypeptide chain in a large scaffold protein connect a series of structured domains that bind a set of reacting proteins. The unstructured regions serve as flexible “tethers” that greatly speed reaction rates by causing a rapid, random collision of all of the proteins that are bound to the scaffold. (For specific examples of protein tethering, see Figure 3–54 and Figure 16–18; for scaffold RNA molecules, see Figure 7–49B.) a few of the modifying groups with known regulatory roles. As in phosphate and ubiquitin additions described previously, these groups are added and then removed from proteins according to the needs of the cell.

1	A large number of proteins are now known to be modified on more than one amino acid side chain, with different regulatory events producing a different pattern of such modifications. A striking example is the protein p53, which plays a central part in controlling a cell’s response to adverse circumstances (see Figure 17–62). Through one of four different types of molecular additions, this protein can be modified at 20 different sites. Because an enormous number of different combinations of these 20 modifications are possible, the protein’s behavior can in principle be altered in a huge number of ways. Such modifications will often create a site on the modified protein that binds it to a scaffold protein in a specific region of the cell, thereby connecting it—via the scaffold—to the other proteins required for a reaction at that site.

1	One can view each protein’s set of covalent modifications as a combinatorial regulatory code. Specific modifying groups are added to or removed from a protein in response to signals, and the code then alters protein behavior—changing the activity or stability of the protein, its binding partners, and/or its specific location within the cell (Figure 3–79). As a result, the cell is able to respond rapidly and with great versatility to changes in its condition or environment. A Complex Network of Protein Interactions Underlies Cell Function

1	A Complex Network of Protein Interactions Underlies Cell Function There are many challenges facing cell biologists in this information-rich era when a large number of complete genome sequences are known. One is the need to dissect and reconstruct each one of the thousands of protein machines that exist in an organism such as ourselves. To understand these remarkable protein complexes, each will need to be reconstituted from its purified protein parts, so that we can study its detailed mode of operation under controlled conditions in a test tube, free from all other cell components. This alone is a massive task. But we now know that each of these subcomponents of a cell also interacts with other sets of macromolecules, creating a large network of protein–protein and protein–nucleic acid interactions throughout the cell. To understand the cell, therefore, we will need to analyze most of these other interactions as well.

1	Figure 3–79 Multisite protein modification and its effects. (A) A protein that carries a post-translational addition to more than one of its amino acid side chains can be considered to carry a combinatorial regulatory code. Multisite modifications are added to (and removed from) a protein through signaling networks, and the resulting combinatorial regulatory code on the protein is read to alter its behavior in the cell. (B) The pattern of some covalent modifications to the protein p53. We can gain some idea of the complexity of intracellular protein networks from a particularly well-studied example described in Chapter 16: the many dozens of proteins that interact with the actin cytoskeleton to control actin filament behavior (see Panel 16–3, p. 905).

1	The extent of such protein–protein interactions can also be estimated more generally. An enormous amount of valuable information is now freely available in protein databases on the Internet: tens of thousands of three-dimensional protein structures plus tens of millions of protein sequences derived from the nucleotide sequences of genes. Scientists have been developing new methods for mining this great resource to increase our understanding of cells. In particular, computer-based bioinformatics tools are being combined with robotics and other technologies to allow thousands of proteins to be investigated in a single set of experiments. Proteomics is a term that is often used to describe such research focused on the analysis of large sets of proteins, analogous to the term genomics describing the large-scale analysis of DNA sequences and genes.

1	A biochemical method based on affinity tagging and mass spectroscopy has proven especially powerful for determining the direct binding interactions between the many different proteins in a cell (discussed in Chapter 8). The results are being tabulated and organized in Internet databases. This allows a cell biologist studying a small set of proteins to readily discover which other proteins in the same cell are likely to bind to, and thus interact with, that set of proteins. When displayed graphically as a protein interaction map, each protein is represented by a box or dot in a two-dimensional network, with a straight line connecting those proteins that have been found to bind to each other.

1	When hundreds or thousands of proteins are displayed on the same map, the network diagram becomes bewilderingly complicated, serving to illustrate the enormous challenges that face scientists attempting to understand the cell (Figure 3–80). Much more useful are small subsections of these maps, centered on a few proteins of interest.

1	We have previously described the structure and mode of action of the SCF ubiquitin ligase, using it to illustrate how protein complexes are constructed from interchangeable parts (see Figure 3–71). Figure 3–81 shows a network of protein– protein interactions for the five proteins that form this protein complex in a yeast cell. Four of the subunits of this ligase are located at the bottom right of this figure. The remaining subunit, the F-box protein that serves as its substrate-binding arm, appears as a set of 15 different gene products that bind to adaptor protein 2 (the Skp1 protein). Along the top and left of the figure are sets of additional protein interactions marked with yellow and green shading: as indicated, these protein sets function at the origin of DNA replication, in cell cycle regulation, in methionine synthesis, in the kinetochore, and in vacuolar H+-ATPase assembly. We shall use this figure to explain how such protein interaction maps are used, and what they do and do

1	regulation, in methionine synthesis, in the kinetochore, and in vacuolar H+-ATPase assembly. We shall use this figure to explain how such protein interaction maps are used, and what they do and do not mean.

1	1. Protein interaction maps are useful for identifying the likely function of previously uncharacterized proteins. Examples are the products of the genes that have thus far only been inferred to exist from the yeast genome sequence, which are the three proteins in the figure that lack a simple three-letter abbreviation (white letters beginning with Y). The three in this diagram are F-box proteins that bind to Skp1; these are therefore likely to function as part of the ubiquitin ligase, serving as substrate-binding arms that recognize different target proteins. However, as we discuss next, neither assignment can be considered certain without additional data. 2.

1	2. Protein interaction networks need to be interpreted with caution because, as a result of evolution making efficient use of each organism’s genetic information, the same protein can be used as part of different protein complexes that have different types of functions. Thus, although protein A binds to protein B and protein B binds to protein C, proteins A and C need not function in the same process. For example, we know from detailed biochemical studies that the functions of Skp1 in the kinetochore and in Figure 3–80 a network of protein-binding interactions in a yeast cell. Each line connecting a pair of dots (proteins) indicates a protein–protein interaction. (From A. Guimerá and M. Sales–Pardo, Mol. Syst. Biol. 2:42, 2006. With permission from Macmillan Publishers Ltd.) Figure 3–81 a map of some protein–protein interactions of the SCF ubiquitin ligase and other proteins in the yeast

1	Figure 3–81 a map of some protein–protein interactions of the SCF ubiquitin ligase and other proteins in the yeast S. cerevisiae. The symbols and/or colors used for the five proteins of the ligase are those in Figure 3–71. Note that 15 different F-box proteins are shown (purple); those with white lettering (beginning with Y) are known from the genome sequence as open reading frames. For additional details, see text. (Courtesy of Peter Bowers and David Eisenberg, UCLA-DOE Institute for Genomics and Proteomics, UCLA.) vacuolar H+-ATPase assembly (yellow shading) are separate from its function in the SCF ubiquitin ligase. In fact, only the remaining three functions of Skp1 illustrated in the diagram—methionine synthesis, cell cycle regulation, and origin of replication (green shading)—involve ubiquitylation.

1	3. In cross-species comparisons, those proteins displaying similar patterns of interactions in the two protein interaction maps are likely to have the same function in the cell. Thus, as scientists generate more and more highly detailed maps for multiple organisms, the results will become increasingly useful for inferring protein function. These map comparisons will be a particularly powerful tool for deciphering the functions of human proteins, because a vast amount of direct information about protein function can be obtained from genetic engineering, mutational, and genetic analyses in experimental organisms—such as yeast, worms, and flies—that are not feasible in humans.

1	What does the future hold? There are likely to be on the order of 10,000 different proteins in a typical human cell, each of which interacts with 5 to 10 different partners. Despite the enormous progress made in recent years, we cannot yet claim to understand even the simplest known cells, such as the small Mycoplasma bacterium formed from only about 500 gene products (see Figure 1–10). How then can we hope to understand a human? Clearly, a great deal of new biochemistry will be essential, in which each protein in a particular interacting set is purified so that its chemistry and interactions can be dissected in a test tube. But in addition, more powerful ways of analyzing networks will be needed based on mathematical and computational tools not yet invented, as we shall emphasize in Chapter 8. Clearly, there are many wonderful challenges that remain for future generations of cell biologists.

1	Proteins can form enormously sophisticated chemical devices, whose functions largely depend on the detailed chemical properties of their surfaces. Binding sites for ligands are formed as surface cavities in which precisely positioned amino acid side chains are brought together by protein folding. In this way, normally unreactive amino acid side chains can be activated to make and break covalent bonds. Enzymes are catalytic proteins that greatly speed up reaction rates by binding the high-energy transition states for a specific reaction path; they also can perform acid catalysis and base catalysis simultaneously. The rates of enzyme reactions are often so fast that they are limited only by diffusion. Rates can be further increased only if enzymes that act sequentially on a substrate are joined into a single multienzyme complex, or if the enzymes and their substrates are attached to protein scaffolds, or otherwise confined to the same part of the cell.

1	Proteins reversibly change their shape when ligands bind to their surface. The allosteric changes in protein conformation produced by one ligand affect the binding of a second ligand, and this linkage between two ligand-binding sites provides a crucial mechanism for regulating cell processes. Metabolic pathways, for example, are controlled by feedback regulation: some small molecules inhibit and other small molecules activate enzymes early in a pathway. Enzymes controlled in this way generally form symmetric assemblies, allowing cooperative conformational changes to create a steep response to changes in the concentrations of the ligands that regulate them.

1	The expenditure of chemical energy can drive unidirectional changes in protein shape. By coupling allosteric shape changes to ATP hydrolysis, for example, proteins can do useful work, such as generating a mechanical force or moving for long distances in a single direction. The three-dimensional structures of proteins have revealed how a small local change caused by nucleoside triphosphate hydrolysis is amplified to create major changes elsewhere in the protein. By such means, these proteins can serve as input–output devices that transmit information, as assembly factors, as motors, or as membrane-bound pumps. Highly efficient protein machines are formed by incorporating many different protein molecules into larger assemblies that coordinate the allosteric movements of the individual components. Such machines perform most of the important reactions in cells.

1	Proteins are subjected to many reversible, post-translational modifications, such as the covalent addition of a phosphate or an acetyl group to a specific amino acid side chain. The addition of these modifying groups is used to regulate the activity of a protein, changing its conformation, its binding to other proteins, and its location inside the cell. A typical protein in a cell will interact with more than five different partners. Through proteomics, biologists can analyze thousands of proteins in one set of experiments. One important result is the production of detailed protein interaction maps, which aim at describing all of the binding interactions between the thousands of distinct proteins in a cell. However, understanding these networks will require new biochemistry, through which small sets of interacting proteins can be purified and their chemistry dissected in detail. In addition, new computational techniques will be required to deal with the enormous complexity.

1	What are the functions of the surprisingly large amount of unfolded polypeptide chain found in proteins? How many types of protein functions remain to be discovered? What are the most promising approaches for discovering them? When will scientists be able to take any amino acid sequence and accurately predict both that protein’s three-dimensional conformations and its chemical properties? What breakthroughs will be needed to accomplish this important goal? Are there ways to reveal the detailed workings of a protein machine that do not require the purification of each of its component parts in large amounts, so that the machine’s functions can be reconstituted and dissected using chemical techniques in a test tube? What are the roles of the dozens of different types of covalent modifications of proteins that have been found in addition to those listed in Table 3–3? Which ones are critical for cell function and why?

1	Why is amyloid toxic to cells and how does it contribute to neurodegenerative diseases such as Alzheimer’s disease? Which statements are true? explain why or why not. 3–1 Each strand in a β sheet is a helix with two amino acids per turn. 3–2 Intrinsically disordered regions of proteins can be identified using bioinformatic methods to search genes for encoded amino acid sequences that possess high hydrophobicity and low net charge. 3–3 Loops of polypeptide that protrude from the surface of a protein often form the binding sites for other molecules. 3–4 An enzyme reaches a maximum rate at high substrate concentration because it has a fixed number of active sites where substrate binds. 3–5 Higher concentrations of enzyme give rise to a higher turnover number. 3–6 Enzymes that undergo cooperative allosteric transitions invariably consist of symmetric assemblies of multiple subunits.

1	3–6 Enzymes that undergo cooperative allosteric transitions invariably consist of symmetric assemblies of multiple subunits. 3–7 Continual addition and removal of phosphates by protein kinases and protein phosphatases is wasteful of energy—since their combined action consumes ATP— but it is a necessary consequence of effective regulation by phosphorylation. Discuss the following problems.

1	Discuss the following problems. 3–8 Consider the following statement. “To produce one molecule of each possible kind of polypeptide chain, 300 amino acids in length, would require more atoms than exist in the universe.” Given the size of the universe, do you suppose this statement could possibly be correct? Since counting atoms is a tricky business, consider the problem from the standpoint of mass. The mass of the observable universe is estimated to be about 1080 grams, give or take an order of magnitude or so. Assuming that the average One such kelch repeat domain is shown in Figure Q3–1. Would you classify this domain as an “in-line” or “plug-in” type domain? ˜7 Figure Q3–1 The kelch repeat domain of D. dendroides (Problem 3–10). The seven individual β propellers are color coded and labeled. The Nand C-termini are indicated by N and C.

1	˜7 Figure Q3–1 The kelch repeat domain of D. dendroides (Problem 3–10). The seven individual β propellers are color coded and labeled. The Nand C-termini are indicated by N and C. 3–11 Titin, which has a molecular weight of about 3 × 106, is the largest polypeptide yet described. Titin molecules extend from muscle thick filaments to the Z disc; they are thought to act as springs to keep the thick filaments centered in the sarcomere. Titin is composed of a large number of repeated immunoglobulin (Ig) sequences of 89 amino acids, each of which is folded into a domain about 4 nm in length (Figure Q3–2A).

1	You suspect that the springlike behavior of titin is caused by the sequential unfolding (and refolding) of individual Ig domains. You test this hypothesis using the atomic force microscope, which allows you to pick up one end of a protein molecule and pull with an accurately measured force. For a fragment of titin containing seven repeats of the Ig domain, this experiment gives the sawtooth force-versus-extension curve shown in Figure Q3–2B. If the experiment is repeated in a solution of 8 M urea (a protein denaturant), the peaks disappear and the measured extension becomes much longer for a given force. If the experiment is repeated after the protein has been cross-linked by treatment with glutaraldehyde, once again the peaks disappear but the extension becomes much smaller for a given force.

1	mass of an amino acid is 110 daltons, what would be the mass of one molecule of each possible kind of polypeptide chain 300 amino acids in length? Is this greater than the mass of the universe? homologous proteins is to search the database using a short signature sequence indicative of the particular protein function. Why is it better to search with a short sequence than with a long sequence? Do you not have more chances 0 for a “hit” in the database with a long sequence? 3–10 The so-called kelch motif consists of a four-stranded β sheet, which forms what is known as a β pro- Figure Q3–2 Springlike behavior of titin (Problem 3–11). (A) The peller. It is usually found to be repeated four to seven times, structure of an individual Ig domain. (B) Force in piconewtons versus forming a kelch repeat domain in a multidomain protein. extension in nanometers obtained by atomic force microscopy. a.

1	a. Are the data consistent with your hypothesis that titin’s springlike behavior is due to the sequential unfolding of individual Ig domains? Explain your reasoning. b. Is the extension for each putative domain-unfolding event the magnitude you would expect? (In an extended polypeptide chain, amino acids are spaced at intervals of 0.34 nm.) C. Why is each successive peak in Figure Q3–2B a little higher than the one before? D. Why does the force collapse so abruptly after each peak?

1	C. Why is each successive peak in Figure Q3–2B a little higher than the one before? D. Why does the force collapse so abruptly after each peak? 3–12 Rous sarcoma virus (RSV) carries an oncogene called Src, which encodes a continuously active protein tyrosine kinase that leads to unchecked cell proliferation. Normally, Src carries an attached fatty acid (myristoylate) group that allows it to bind to the cytoplasmic side of the plasma membrane. A mutant version of Src that does not allow attachment of myristoylate does not bind to the membrane. Infection of cells with RSV encoding either the normal or the mutant form of Src leads to the same high level of protein tyrosine kinase activity, but the mutant Src does not cause cell proliferation. a.

1	a. Assuming that the normal Src is all bound to the plasma membrane and that the mutant Src is distributed throughout the cytoplasm, calculate their relative concentrations in the neighborhood of the plasma membrane. For the purposes of this calculation, assume that the cell is a sphere with a radius (r) of 10 μm and that the mutant Src is distributed throughout the cell, whereas the normal Src is confined to a 4-nm-thick layer immediately beneath the membrane. [For this problem, assume that the membrane has no thickness. The volume of a sphere is (4/3)πr3.] b. The target (X) for phosphorylation by Src resides in the membrane. Explain why the mutant Src does not cause cell proliferation.

1	3–13 An antibody binds to another protein with an equilibrium constant, K, of 5 × 109 M–1. When it binds to a second, related protein, it forms three fewer hydrogen bonds, reducing its binding affinity by 11.9 kJ/mole. What is the K for its binding to the second protein? (Free-energy change is related to the equilibrium constant by the equation ΔG° = –2.3 RT log K, where R is 8.3 × 10–3 kJ/(mole K) and T is 310 K.) 3–14 The protein SmpB binds to a special species of tRNA, tmRNA, to eliminate the incomplete proteins made from truncated mRNAs in bacteria. If the binding of SmpB to tmRNA is plotted as fraction tmRNA bound versus SmpB concentration, one obtains a symmetrical S-shaped curve as shown in Figure Q3–3. This curve is a visual display of a very useful relationship between Kd and concentration, which has broad applicability. The general expression for fraction of ligand bound is derived from the equation for Kd(Kd = [Pr][L]/[Pr–L]) by substituting ([L]TOT – [L]) for [Pr–L] and

1	which has broad applicability. The general expression for fraction of ligand bound is derived from the equation for Kd(Kd = [Pr][L]/[Pr–L]) by substituting ([L]TOT – [L]) for [Pr–L] and rearranging. Because the total concentration of ligand ([L]TOT) is equal to the free ligand ([L]) plus bound ligand ([Pr–L]), 1.0

1	Figure Q3–3 Fraction of tmRNA bound versus SmpB concentration (Problem 3–14). concentration of SmpB (M) For SmpB and tmRNA, the fraction bound = [SmpB– tmRNA]/[tmRNA]TOT = [SmpB]/([SmpB] + Kd). Using this relationship, calculate the fraction of tmRNA bound for SmpB concentrations equal to 104 Kd, 103 Kd, 102 Kd, 101 Kd, Kd, 10–1 Kd, 10–2 Kd, 10–3 Kd, and 10–4 Kd. 3–15 Many enzymes obey simple Michaelis–Menten kinetics, which are summarized by the equation where Vmax = maximum velocity, [S] = concentration of substrate, and Km = the Michaelis constant. It is instructive to plug a few values of [S] into the equation to see how rate is affected. What are the rates for [S] equal to zero, equal to Km, and equal to infinite concentration? 3–16 The enzyme hexokinase adds a phosphate to D-glucose but ignores its mirror image, L-glucose. Suppose that you were able to synthesize hexokinase entirely from D-amino acids, which are the mirror image of the normal L-amino acids. a.

1	a. Assuming that the “D” enzyme would fold to a stable conformation, what relationship would you expect it to bear to the normal “L” enzyme? b. Do you suppose the “D” enzyme would add a phosphate to L-glucose, and ignore D-glucose? 3–17 How do you suppose that a molecule of hemoglobin is able to bind oxygen efficiently in the lungs, and yet release it efficiently in the tissues? 3–18 Synthesis of the purine nucleotides AMP and GMP proceeds by a branched pathway starting with ribose 5-phosphate (R5P), as shown schematically in Figure Q3–4. Using the principles of feedback inhibition, propose a regulatory strategy for this pathway that ensures an adequate supply of both AMP and GMP and minimizes the buildup of the intermediates (A–I) when supplies of AMP and GMP are adequate. 0 0.25 0.5 0.75 Figure Q3–4 Schematic diagram of the metabolic pathway for synthesis of AMP and GMP from R5P (Problem 3–18).

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1	Fersht AR (1999) Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding. New York: WH Freeman. Haucke V, Neher E & Sigrist SJ (2011) Protein scaffolds in the coupling of synaptic exocytosis and endocytosis. Nat. Rev. Neurosci. 12, 127–138. Hua Z & Vierstra RD (2011) The cullin-RING ubiquitin-protein ligases. Annu. Rev. Plant Biol. 62, 299–334. Hunter T (2012) Why nature chose phosphate to modify proteins. Philos. Trans. R. Soc. Lond. B Biol. Sci. 367, 2513–2516. Johnson LN & Lewis RJ (2001) Structural basis for control by phosphorylation. Chem. Rev. 101, 2209–2242. Kantrowitz ER & Lipscomb WN (1988) Escherichia coli aspartate transcarbamylase: the relation between structure and function. Science 241, 669–674. Kerscher O, Felberbaum R & Hochstrasser M (2006) Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell Dev. Biol. 22, 159–180.

1	Kerscher O, Felberbaum R & Hochstrasser M (2006) Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell Dev. Biol. 22, 159–180. Kim E & Sheng M (2004) PDZ domain proteins of synapses. Nat. Rev. Neurosci. 5, 771–781. Koshland DE Jr (1984) Control of enzyme activity and metabolic pathways. Trends Biochem. Sci. 9, 155–159. Krogan NJ, Cagney G, Yu H et al. (2006) Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440, 637–643. Lichtarge O, Bourne HR & Cohen FE (1996) An evolutionary trace method defines binding surfaces common to protein families. J. Mol. Biol. 257, 342–358. Maier T, Leibundgut M & Ban N (2008) The crystal structure of a mammalian fatty acid synthase. Science 321, 1315–1322. Monod J, Changeux JP & Jacob F (1963) Allosteric proteins and cellular control systems. J. Mol. Biol. 6, 306–329.

1	Monod J, Changeux JP & Jacob F (1963) Allosteric proteins and cellular control systems. J. Mol. Biol. 6, 306–329. Perutz M (1990) Mechanisms of Cooperativity and Allosteric Regulation in Proteins. Cambridge: Cambridge University Press. Radzicka A & Wolfenden R (1995) A proficient enzyme. Science 267, 90–93. Schramm VL (2011) Enzymatic transition states, transition-state analogs, dynamics, thermodynamics, and lifetimes. Annu. Rev. Biochem. 80, 703–732. Scott JD & Pawson T (2009) Cell signaling in space and time: where proteins come together and when they’re apart. Science 326, 1220–1224. Taylor SS, Keshwani MM, Steichen JM & Kornev AP (2012) Evolution of the eukaryotic protein kinases as dynamic molecular switches. Philos. Trans. R. Soc. Lond. B Biol. Sci. 367, 2517–2528. Vale RD & Milligan RA (2000) The way things move: looking under the hood of molecular motor proteins. Science 288, 88–95.

1	Vale RD & Milligan RA (2000) The way things move: looking under the hood of molecular motor proteins. Science 288, 88–95. Wilson MZ & Gitai Z (2013) Beyond the cytoskeleton: mesoscale assemblies and their function in spatial organization. Curr. Opin. Microbiol. 16, 177–183. DNA, Chromosomes, and genomes Life depends on the ability of cells to store, retrieve, and translate the genetic instructions required to make and maintain a living organism. This hereditary information is passed on from a cell to its daughter cells at cell division, and from one generation of an organism to the next through the organism’s reproductive cells. The instructions are stored within every living cell as its genes, the information-containing elements that determine the characteristics of a species as a whole and of the individuals within it.

1	As soon as genetics emerged as a science at the beginning of the twentieth century, scientists became intrigued by the chemical structure of genes. The information in genes is copied and transmitted from cell to daughter cell millions of times during the life of a multicellular organism, and it survives the process essentially unchanged. What form of molecule could be capable of such accurate and almost unlimited replication and also be able to exert precise control, directing multicellular development as well as the daily life of every cell? What kind of instructions does the genetic information contain? And how can the enormous amount of information required for the development and maintenance of an organism fit within the tiny space of a cell?

1	The answers to several of these questions began to emerge in the 1940s. At this time researchers discovered, from studies in simple fungi, that genetic information consists largely of instructions for making proteins. Proteins are phenomenally versatile macromolecules that perform most cell functions. As we saw in Chapter 3, they serve as building blocks for cell structures and form the enzymes that catalyze most of the cell’s chemical reactions. They also regulate gene expression (Chapter 7), and they enable cells to communicate with each other (Chapter 15) and to move (Chapter 16). The properties and functions of cells and organisms are determined to a great extent by the proteins that they are able to make.

1	Painstaking observations of cells and embryos in the late nineteenth century had led to the recognition that the hereditary information is carried on chromosomes—threadlike structures in the nucleus of a eukaryotic cell that become visible by light microscopy as the cell begins to divide (Figure 4–1). Later, when biochemical analysis became possible, chromosomes were found to consist of deoxyribonucleic acid (DNA) and protein, with both being present in roughly the same amounts. For many decades, the DNA was thought to be merely a structural

1	Figure 4–1 Chromosomes in cells. (A) Two adjacent plant cells photographed through a light microscope. The DNA has been stained with a fluorescent dye (DAPI) that binds to it. The DNA is present in chromosomes, which become visible as distinct structures in the light microscope only when they become compact, sausage-shaped structures in preparation for cell division, as shown on the left. The cell on the right, which is not dividing, contains identical chromosomes, but they cannot be clearly distinguished at this phase in the cell’s life cycle, because they are in a more extended conformation. (b) Schematic diagram of the outlines of the two cells along with their chromosomes. (A, courtesy of Peter Shaw.) element. However, the other crucial advance made in the 1940s was the identification of DNA as the likely carrier of genetic information. This breakthrough in our understanding of cells came from studies of inheritance in bacteria (Figure 4–2). But still, as the 1950s began, both how

1	of DNA as the likely carrier of genetic information. This breakthrough in our understanding of cells came from studies of inheritance in bacteria (Figure 4–2). But still, as the 1950s began, both how proteins could be specified by instructions in the DNA and how this information might be copied for transmission from cell to cell seemed completely mysterious. The puzzle was suddenly solved in 1953, when James Watson and Francis Crick derived the mechanism from their model of DNA structure. As outlined in Chapter 1, the determination of the double-helical structure of DNA immediately solved the problem of how the information in this molecule might be copied, or replicated. It also provided the first clues as to how a molecule of DNA might use the sequence of its subunits to encode the instructions for making proteins. Today, the fact that DNA is the genetic material is so fundamental to biological thought that it is difficult to appreciate the enormous intellectual gap that was filled

1	for making proteins. Today, the fact that DNA is the genetic material is so fundamental to biological thought that it is difficult to appreciate the enormous intellectual gap that was filled by this breakthrough discovery.

1	We begin this chapter by describing the structure of DNA. We see how, despite its chemical simplicity, the structure and chemical properties of DNA make it ideally suited as the raw material of genes. We then consider how the many proteins in chromosomes arrange and package this DNA. The packing has to be done in an orderly fashion so that the chromosomes can be replicated and apportioned correctly between the two daughter cells at each cell division. And it must also allow access to chromosomal DNA, both for the enzymes that repair DNA damage and for the specialized proteins that direct the expression of its many genes. In the past two decades, there has been a revolution in our ability to determine the exact order of subunits in DNA molecules. As a result, we now know the RNA protein DNA lipid carbohydratelive R strain cells grown in presence of either heat-killed S strain cells or cell-free molecules tested for transformation of R strain cellsextract of S strain cells

1	CONCLUSION: Molecules that can CONCLUSION: The molecule that carry heritable information are carries the heritable information present in S strain cells. is DNA. Figure 4–2 The first experimental demonstration that DNA is the genetic material. These experiments, carried out in the 1920s (A) and 1940s (b), showed that adding purified DNA to a bacterium changed the bacterium’s properties and that this change was faithfully passed on to subsequent generations. Two closely related strains of the bacterium Streptococcus pneumoniae differ from each other in both their appearance under the microscope and their pathogenicity. One strain appears smooth (S) and causes death when injected into mice, and the other appears rough (R) and is nonlethal. An initial experiment shows that some substance present in the S strain can change (or transform) the R strain into the S strain and that this change is inherited by subsequent generations of bacteria.

1	This experiment, in which the R strain has been incubated with various classes of biological molecules purified from the S strain, identifies the active substance as DNA. sequence of the 3.2 billion nucleotide pairs that provide the information for producing a human adult from a fertilized egg, as well as having the DNA sequences for thousands of other organisms. Detailed analyses of these sequences are providing exciting insights into the process of evolution, and it is with this subject that the chapter ends.

1	This is the first of four chapters that deal with basic genetic mechanisms—the ways in which the cell maintains, replicates, and expresses the genetic information carried in its DNA. In the next chapter (Chapter 5), we shall discuss the mechanisms by which the cell accurately replicates and repairs DNA; we also describe how DNA sequences can be rearranged through the process of genetic recombination. Gene expression—the process through which the information encoded in DNA is interpreted by the cell to guide the synthesis of proteins—is the main topic of Chapter 6. In Chapter 7, we describe how this gene expression is controlled by the cell to ensure that each of the many thousands of proteins and RNA molecules encrypted in its DNA is manufactured only at the proper time and place in the life of a cell.

1	Biologists in the 1940s had difficulty in conceiving how DNA could be the genetic material. The molecule seemed too simple: a long polymer composed of only four types of nucleotide subunits, which resemble one another chemically. Early in the 1950s, DNA was examined by x-ray diffraction analysis, a technique for determining the three-dimensional atomic structure of a molecule (discussed in Chapter 8). The early x-ray diffraction results indicated that DNA was composed of two strands of the polymer wound into a helix. The observation that DNA was double-stranded provided one of the major clues that led to the Watson–Crick model for DNA structure that, as soon as it was proposed in 1953, made DNA’s potential for replication and information storage apparent. A DNA molecule Consists of Two Complementary Chains of Nucleotides

1	A deoxyribonucleic acid (DNA) molecule consists of two long polynucleotide chains composed of four types of nucleotide subunits. Each of these chains is known as a DNA chain, or a DNA strand. The chains run antiparallel to each other, and hydrogen bonds between the base portions of the nucleotides hold the two chains together (Figure 4–3). As we saw in Chapter 2 (Panel 2–6, pp. 100–101), nucleotides are composed of a five-carbon sugar to which are attached one or more phosphate groups and a nitrogen-containing base. In the case of the nucleotides in DNA, the sugar is deoxyribose attached to a single phosphate group (hence the name deoxyribonucleic acid), and the base may be either adenine (A), cytosine (C), guanine (G), or thymine (T). The nucleotides are covalently linked together in a chain through the sugars and phosphates, which thus form a “backbone” of alternating sugar–phosphate–sugar–phosphate. Because only the base differs in each of the four types of nucleotide subunit, each

1	through the sugars and phosphates, which thus form a “backbone” of alternating sugar–phosphate–sugar–phosphate. Because only the base differs in each of the four types of nucleotide subunit, each polynucleotide chain in DNA is analogous to a sugar-phosphate necklace (the backbone), from which hang the four types of beads (the bases A, C, G, and T). These same symbols (A, C, G, and T) are commonly used to denote either the four bases or the four entire nucleotides—that is, the bases with their attached sugar and phosphate groups.

1	The way in which the nucleotides are linked together gives a DNA strand a chemical polarity. If we think of each sugar as a block with a protruding knob (the 5ʹ phosphate) on one side and a hole (the 3ʹ hydroxyl) on the other (see Figure 4–3), each completed chain, formed by interlocking knobs with holes, will have all of its subunits lined up in the same orientation. Moreover, the two ends of the chain will be easily distinguishable, as one has a hole (the 3ʹ hydroxyl) and the other a knob (the 5ʹ phosphate) at its terminus. This polarity in a DNA chain is indicated by referring to one end as the 3ʹ end and the other as the 5ʹ end, names derived from the orientation of the deoxyribose sugar. With respect to DNA’s building blocks of DNA DNA strand information-carrying capacity, the chain of nucleotides in a DNA strand, being both directional and linear, can be read in much the same way as the letters on this page.

1	The three-dimensional structure of DNA—the DNA double helix—arises from the chemical and structural features of its two polynucleotide chains. Because these two chains are held together by hydrogen-bonding between the bases on the different strands, all the bases are on the inside of the double helix, and the sugar-phosphate backbones are on the outside (see Figure 4–3). In each case, a bulkier two-ring base (a purine; see Panel 2–6, pp. 100–101) is paired with a single-ring base (a pyrimidine): A always pairs with T, and G with C (Figure 4–4). This complementary base-pairing enables the base pairs to be packed in the energetically most favorable arrangement in the interior of the double helix. In this arrangement, each base pair is of similar width, thus holding the sugar-phosphate backbones a constant distance apart along the DNA molecule. To maximize the efficiency of base-pair packing, the two sugar-phosphate backbones wind around each other to form a right-handed double helix,

1	a constant distance apart along the DNA molecule. To maximize the efficiency of base-pair packing, the two sugar-phosphate backbones wind around each other to form a right-handed double helix, with one complete turn every ten base pairs (Figure 4–5).

1	The members of each base pair can fit together within the double helix only if the two strands of the helix are antiparallel—that is, only if the polarity of one strand is oriented opposite to that of the other strand (see Figures 4–3 and 4–4). A consequence of DNA’s structure and base-pairing requirements is that each strand of a DNA molecule contains a sequence of nucleotides that is exactly complementary to the nucleotide sequence of its partner strand. Figure 4–3 DNA and its building blocks.

1	Figure 4–3 DNA and its building blocks. DNA is made of four types of nucleotides, which are linked covalently into a polynucleotide chain (a DNA strand) with a sugar-phosphate backbone from which the bases (A, C, g, and T) extend. A DNA molecule is composed of two antiparallel DNA strands held together by hydrogen bonds between the paired bases. The arrowheads at the ends of the DNA strands indicate the polarities of the two strands. In the diagram at the bottom left of the figure, the DNA molecule is shown straightened out; in reality, it is twisted into a double helix, as shown on the right. For details, see Figure 4–5 and Movie 4.1. The Structure of DNA Provides a mechanism for Heredity

1	The Structure of DNA Provides a mechanism for Heredity The discovery of the structure of DNA immediately suggested answers to the two most fundamental questions about heredity. First, how could the information to specify an organism be carried in a chemical form? And second, how could this information be duplicated and copied from generation to generation? The answer to the first question came from the realization that DNA is a linear polymer of four different kinds of monomer, strung out in a defined sequence like the letters of a document written in an alphabetic script. The answer to the second question came from the double-stranded nature of the structure: because each strand of DNA contains a sequence of nucleotides that is exactly complementary to the nucleotide sequence of its partner strand, each strand can act as a template, or mold, for the synthesis of a new complementary strand. In other words, if we designate the two DNA strands as S and Sʹ, strand 0.34 nm

1	Figure 4–4 Complementary base pairs in the DNA double helix. The shapes and chemical structures of the bases allow hydrogen bonds to form efficiently only between A and T and between g and C, because atoms that are able to form hydrogen bonds (see Panel 2–3, pp. 94–95) can then be brought close together without distorting the double helix. As indicated, two hydrogen bonds form between A and T, while three form between g and C. The bases can pair in this way only if the two polynucleotide chains that contain them are antiparallel to each other. Figure 4–5 The DNA double helix. A space-filling model of 1.5 turns of the DNA double helix. Each turn of DNA is made up of 10.4 nucleotide pairs, and the center-to-center distance between adjacent nucleotide pairs is 0.34 nm. The coiling of the two strands around each other creates two grooves in the double helix: the wider groove is called the major groove, and the smaller the minor groove, as indicated.

1	A short section of the double helix viewed from its side, showing four base pairs. The nucleotides are linked together covalently by phosphodiester bonds that join the 3ʹ-hydroxyl (–OH) group of one sugar to the 5ʹ-hydroxyl group of the next sugar. Thus, each polynucleotide strand has a chemical polarity; that is, its two ends are chemically different. The 5ʹ end of the DNA polymer is by convention often illustrated carrying a phosphate group, while the 3ʹ end is shown with a hydroxyl. S can serve as a template for making a new strand Sʹ, while strand Sʹ can serve as a template for making a new strand S (Figure 4–6). Thus, the genetic information in DNA can be accurately copied by the beautifully simple process in which strand S separates from strand Sʹ, and each separated strand then serves as a template for the production of a new complementary partner strand that is identical to its former partner.

1	The ability of each strand of a DNA molecule to act as a template for producing a complementary strand enables a cell to copy, or replicate, its genome before passing it on to its descendants. We shall describe the elegant machinery that the cell uses to perform this task in Chapter 5. Organisms differ from one another because their respective DNA molecules have different nucleotide sequences and, consequently, carry different biological messages. But how is the nucleotide alphabet used to make messages, and what do they spell out?

1	As discussed above, it was known well before the structure of DNA was determined that genes contain the instructions for producing proteins. If genes are made of DNA, the DNA must therefore somehow encode proteins (Figure 4–7). As discussed in Chapter 3, the properties of a protein, which are responsible for its biological function, are determined by its three-dimensional structure. This structure is determined in turn by the linear sequence of the amino acids of which it is composed. The linear sequence of nucleotides in a gene must therefore somehow spell out the linear sequence of amino acids in a protein. The exact correspondence between the four-letter nucleotide alphabet of DNA and the twenty-letter amino acid alphabet of proteins—the genetic code—is not at all obvious from the DNA structure, and it took over a decade after the discovery of the double helix before it was worked out. In Chapter 6, we will describe this code in detail in the course of elaborating the process of

1	structure, and it took over a decade after the discovery of the double helix before it was worked out. In Chapter 6, we will describe this code in detail in the course of elaborating the process of gene expression, through which a cell converts the nucleotide sequence of a gene first into the nucleotide sequence of an RNA molecule, and then into the amino acid sequence of a protein.

1	The complete store of information in an organism’s DNA is called its genome, and it specifies all the RNA molecules and proteins that the organism will ever synthesize. (The term genome is also used to describe the DNA that carries this information.) The amount of information contained in genomes is staggering. The nucleotide sequence of a very small human gene, written out in the four-letter nucleotide alphabet, occupies a quarter of a page of text (Figure 4–8), while the complete sequence of nucleotides in the human genome would fill more than a thousand books the size of this one. In addition to other critical information, it includes roughly 21,000 protein-coding genes, which (through alternative splicing; see p. 415) give rise to a much greater number of distinct proteins. In Eukaryotes, DNA Is Enclosed in a Cell Nucleus

1	In Eukaryotes, DNA Is Enclosed in a Cell Nucleus As described in Chapter 1, nearly all the DNA in a eukaryotic cell is sequestered in a nucleus, which in many cells occupies about 10% of the total cell volume. This compartment is delimited by a nuclear envelope formed by two concentric lipid Figure 4–6 DNA as a template for its own duplication. because the nucleotide A successfully pairs only with T, and g pairs with C, each strand of DNA can act as a template to specify the sequence of nucleotides in its complementary strand. In this way, double-helical DNA can be copied precisely, with each parental DNA helix producing two identical daughter DNA helices. Figure 4–7 The relationship between genetic information carried in DNA and proteins. (Discussed in Chapter 1.)

1	Figure 4–7 The nucleotide sequence of the human β-globin gene. by convention, a nucleotide sequence is written from its 5ʹ end to its 3ʹ end, and it should be read from left to right in successive lines down the page as though it were normal English text. This gene carries the information for the amino acid sequence of one of the two types of subunits of the hemoglobin molecule; a different gene, the α-globin gene, carries the information for the other. (Hemoglobin, the protein that carries oxygen in the blood, has four subunits, two of each type.) Only one of the two strands of the DNA double helix containing the β-globin gene is shown; the other strand has the exact complementary sequence. The DNA sequences highlighted in yellow show the three regions of the gene that specify the amino acid sequence for the β-globin protein. we shall see in Chapter 6 how the cell splices these three sequences together at the level of messenger RNA in order to synthesize a full-length β-globin

1	amino acid sequence for the β-globin protein. we shall see in Chapter 6 how the cell splices these three sequences together at the level of messenger RNA in order to synthesize a full-length β-globin protein.

1	bilayer membranes (Figure 4–9). These membranes are punctured at intervals by large nuclear pores, through which molecules move between the nucleus and the cytosol. The nuclear envelope is directly connected to the extensive system of intracellular membranes called the endoplasmic reticulum, which extend out from it into the cytoplasm. And it is mechanically supported by a network of intermediate filaments called the nuclear lamina—a thin feltlike mesh just beneath the inner nuclear membrane (see Figure 4–9B). The nuclear envelope allows the many proteins that act on DNA to be concentrated where they are needed in the cell, and, as we see in subsequent chapters, it also keeps nuclear and cytosolic enzymes separate, a feature that is crucial for the proper functioning of eukaryotic cells.

1	Genetic information is carried in the linear sequence of nucleotides in DNA. Each molecule of DNA is a double helix formed from two complementary antiparallel strands of nucleotides held together by hydrogen bonds between G-C and A-T base pairs. Duplication of the genetic information occurs by the use of one DNA strand as a template for the formation of a complementary strand. The genetic information stored in an organism’s DNA contains the instructions for all the RNA molecules and proteins that the organism will ever synthesize and is said to comprise its genome. In eukaryotes, DNA is contained in the cell nucleus, a large membrane-bound compartment.

1	The most important function of DNA is to carry genes, the information that specifies all the RNA molecules and proteins that make up an organism—including information about when, in what types of cells, and in what quantity each RNA molecule and protein is to be made. The nuclear DNA of eukaryotes is divided up into chromosomes, and in this section we see how genes are typically arranged on each chromosome. In addition, we describe the specialized DNA sequences that are required for a chromosome to be accurately duplicated as a separate entity and passed on from one generation to the next.

1	We also confront the serious challenge of DNA packaging. If the double helices comprising all 46 chromosomes in a human cell could be laid end to end, they would reach approximately 2 meters; yet the nucleus, which contains the DNA, is only about 6 μm in diameter. This is geometrically equivalent to packing 40 km (24 miles) of extremely fine thread into a tennis ball. The complex task of packaging DNA is accomplished by specialized proteins that bind to the DNA and fold it, generating a series of organized coils and loops that provide increasingly higher levels of organization, and prevent the DNA from becoming an unmanageable tangle. Amazingly, although the DNA is very tightly compacted, it nevertheless remains accessible to the many enzymes in the cell that replicate it, repair it, and use its genes to produce RNA molecules and proteins.

1	endoplasmic reticulum nuclear pore outer nuclear membrane inner nuclear membrane nuclear envelope nuclear lamina microtubule centrosome nucleolus DNA and associated proteins (chromatin), plus many RNA and protein molecules 2 µm (A) (B) nucleolus nuclear envelope heterochromatinheterochromatin

1	Figure 4–9 A cross-sectional view of a typical cell nucleus. (A) Electron micrograph of a thin section through the nucleus of a human fibroblast. (b) Schematic drawing, showing that the nuclear envelope consists of two membranes, the outer one being continuous with the endoplasmic reticulum (ER) membrane (see also Figure 12–7). The space inside the endoplasmic reticulum (the ER lumen) is colored yellow; it is continuous with the space between the two nuclear membranes. The lipid bilayers of the inner and outer nuclear membranes are connected at each nuclear pore. A sheetlike network of intermediate filaments (brown) inside the nucleus forms the nuclear lamina (brown), providing mechanical support for the nuclear envelope (for details, see Chapter 12). The dark-staining heterochromatin contains specially condensed regions of DNA that will be discussed later. (A, courtesy of E.g. Jordan and J. mcgovern.) Eukaryotic DNA Is Packaged into a Set of Chromosomes

1	Eukaryotic DNA Is Packaged into a Set of Chromosomes Each chromosome in a eukaryotic cell consists of a single, enormously long linear DNA molecule along with the proteins that fold and pack the fine DNA thread into a more compact structure. In addition to the proteins involved in packaging, chromosomes are also associated with many other proteins (as well as numerous RNA molecules). These are required for the processes of gene expression, DNA replication, and DNA repair. The complex of DNA and tightly bound protein is called chromatin (from the Greek chroma, “color,” because of its staining properties).

1	Bacteria lack a special nuclear compartment, and they generally carry their genes on a single DNA molecule, which is often circular (see Figure 1–24). This DNA is also associated with proteins that package and condense it, but they are different from the proteins that perform these functions in eukaryotes. Although the bacterial DNA with its attendant proteins is often called the bacterial “chromosome,” it does not have the same structure as eukaryotic chromosomes, and less is known about how the bacterial DNA is packaged. Therefore, our discussion of chromosome structure will focus almost entirely on eukaryotic chromosomes.

1	With the exception of the gametes (eggs and sperm) and a few highly specialized cell types that cannot multiply and either lack DNA altogether (for example, red blood cells) or have replicated their DNA without completing cell division (for example, megakaryocytes), each human cell nucleus contains two copies of each chromosome, one inherited from the mother and one from the father. The maternal and paternal chromosomes of a pair are called homologous chromosomes (homologs). The only nonhomologous chromosome pairs are the sex chromosomes in males, where a Y chromosome is inherited from the father and an X chromosome from the mother. Thus, each human cell contains a total of 46 chromosomes—22 pairs common to both males and females, plus two so-called sex chromosomes (X and Y in males, two Xs in females). These human chromosomes can be readily distinguished by “painting” each one a different color using a technique based on DNA hybridization (Figure 4–10). In this method (described in

1	Xs in females). These human chromosomes can be readily distinguished by “painting” each one a different color using a technique based on DNA hybridization (Figure 4–10). In this method (described in detail in Chapter 8), a short strand of nucleic acid tagged with a fluorescent dye serves as a “probe” that picks out its complementary DNA sequence, lighting up the target chromosome at any site where it binds. Chromosome painting is most frequently done at the stage in the cell cycle called mitosis, when chromosomes are especially compacted and easy to visualize (see below).

1	Another more traditional way to distinguish one chromosome from another is to stain them with dyes that reveal a striking and reproducible pattern of bands along each mitotic chromosome (Figure 4–11). These banding patterns presumably reflect variations in chromatin structure, but their basis is not well understood. Nevertheless, the pattern of bands on each type of chromosome is unique, and it provided the initial means to identify and number each human chromosome reliably.

1	Figure 4–10 The complete set of human chromosomes. These chromosomes, from a female, were isolated from a cell undergoing nuclear division (mitosis) and are therefore highly compacted. Each chromosome has been “painted” a different color to permit its unambiguous identification under the fluorescence microscope, using a technique called “spectral karyotyping.” Chromosome painting can be performed by exposing the chromosomes to a large collection of DNA molecules whose sequence matches known DNA sequences from the human genome. The set of sequences matching each chromosome is coupled to a different combination of fluorescent dyes. DNA molecules derived from chromosome 1 are labeled with one specific dye combination, those from chromosome 2 with another, and so on. because the labeled DNA can form base pairs, or hybridize, only to the chromosome from which it was derived, each chromosome becomes labeled with a different combination of dyes. For such experiments, the chromosomes are

1	can form base pairs, or hybridize, only to the chromosome from which it was derived, each chromosome becomes labeled with a different combination of dyes. For such experiments, the chromosomes are subjected to treatments that separate the two strands of double-helical DNA in a way that permits base-pairing with the single-stranded labeled DNA, but keeps the overall chromosome structure relatively intact. (A) The chromosomes visualized as they originally spilled from the lysed cell.

1	The same chromosomes artificially lined up in their numerical order. This arrangement of the full chromosome set is called a karyotype. (Adapted from N. mcNeil and T. Ried, Expert Rev. Mol. Med. 2:1–14, 2000. with permission from Cambridge University Press.)

1	N. mcNeil and T. Ried, Expert Rev. Mol. Med. 2:1–14, 2000. with permission from Cambridge University Press.) Figure 4–11 The banding patterns of human chromosomes. Chromosomes 1–22 are numbered in approximate order of size. A typical human cell contains two of each of these chromosomes, plus two sex chromosomes—two X chromosomes in a female, one X and one Y chromosome in a male. The chromosomes used to make these maps were stained at an early stage in mitosis, when the chromosomes are incompletely compacted. The horizontal red line represents the position of the centromere (see Figure 4–19), which appears as a constriction on mitotic chromosomes. The red knobs on chromosomes 13, 14, 15, 21, and 22 indicate the positions of genes that code for the large ribosomal RNAs (discussed in Chapter 6). These banding patterns are obtained by staining chromosomes with giemsa stain, and they can be observed under the light microscope. (Adapted from

1	U. Francke, Cytogenet. Cell Genet. 31:24– 32, 1981. with permission from the author.) Figure 4–12 Aberrant human chromosomes. (A) Two normal human chromosomes, 4 and 6. (b) In an individual carrying a balanced chromosomal translocation, the DNA double helix in one chromosome has crossed over with the DNA double helix in the other chromosome due to an abnormal recombination event. The chromosome painting technique used on the chromosomes in each of the sets allows the identification of even short pieces of chromosomes that have become translocated, a frequent event in cancer cells. (Courtesy of Zhenya Tang and the NIgmS Human genetic Cell Repository at the Coriell Institute for medical Research: gm21880.)

1	The display of the 46 human chromosomes at mitosis is called the human karyotype. If parts of chromosomes are lost or are switched between chromosomes, these changes can be detected either by changes in the banding patterns or—with greater sensitivity—by changes in the pattern of chromosome painting (Figure 4–12). Cytogeneticists use these alterations to detect inherited chromosome abnormalities and to reveal the chromosome rearrangements that occur in cancer cells as they progress to malignancy (discussed in Chapter 20). Chromosomes Contain long Strings of genes

1	Chromosomes Contain long Strings of genes Chromosomes carry genes—the functional units of heredity. A gene is often defined as a segment of DNA that contains the instructions for making a particular protein (or a set of closely related proteins), but this definition is too narrow. Genes that code for protein are indeed the majority, and most of the genes with clear-cut mutant phenotypes fall under this heading. In addition, however, there are many “RNA genes”—segments of DNA that generate a functionally significant RNA molecule, instead of a protein, as their final product. We shall say more about the RNA genes and their products later.

1	As might be expected, some correlation exists between the complexity of an organism and the number of genes in its genome (see Table 1–2, p. 29). For example, some simple bacteria have only 500 genes, compared to about 30,000 for humans. Bacteria, archaea, and some single-celled eukaryotes, such as yeast, have concise genomes, consisting of little more than strings of closely packed genes. However, the genomes of multicellular plants and animals, as well as many other eukaryotes, contain, in addition to genes, a large quantity of interspersed DNA whose function is poorly understood (Figure 4–13). Some of this additional DNA is crucial for the proper control of gene expression, and this may in part explain why there is so much of it in multicellular organisms, whose genes have to be switched on and off according to complicated rules during development (discussed in Chapters 7 and 21).

1	Differences in the amount of DNA interspersed between genes, far more than differences in numbers of genes, account for the astonishing variations in genome size that we see when we compare one species with another (see Figure 1–32). For example, the human genome is 200 times larger than that of the yeast Saccharomyces cerevisiae, but 30 times smaller than that of some plants and amphibians and 200 times smaller than that of a species of amoeba. Moreover, because of differences in the amount of noncoding DNA, the genomes of closely related organisms (bony fish, for example) can vary several hundredfold in their DNA content, even though they contain roughly the same number of genes. Whatever the excess

1	Figure 4–13 The arrangement of genes in the genome of S. cerevisiae compared to humans. (A) S. cerevisiae is a budding yeast widely used for brewing and baking. The genome of this single-celled eukaryote is distributed over 16 chromosomes. A small region of one chromosome has been arbitrarily selected to show its high density of genes. (b) A region of the human genome of equal length to the yeast segment in (A). The human genes are much less densely packed and the amount of interspersed DNA sequence is far greater. Not shown in this sample of human DNA is the fact that most human genes are much longer than yeast genes (see Figure 4–15). DNA may do, it seems clear that it is not a great handicap for a eukaryotic cell to carry a large amount of it.

1	DNA may do, it seems clear that it is not a great handicap for a eukaryotic cell to carry a large amount of it. How the genome is divided into chromosomes also differs from one eukaryotic species to the next. For example, while the cells of humans have 46 chromosomes, those of some small deer have only 6, while those of the common carp contain over 100. Even closely related species with similar genome sizes can have very different numbers and sizes of chromosomes (Figure 4–14). Thus, there is no simple relationship between chromosome number, complexity of the organism, and total genome size. Rather, the genomes and chromosomes of modern-day species have each been shaped by a unique history of seemingly random genetic events, acted on by poorly understood selection pressures over long evolutionary times. The Nucleotide Sequence of the Human genome Shows How Our genes Are Arranged

1	The Nucleotide Sequence of the Human genome Shows How Our genes Are Arranged With the publication of the full DNA sequence of the human genome in 2004, it became possible to see in detail how the genes are arranged along each of our chromosomes (Figure 4–15). It will be many decades before the information contained in the human genome sequence is fully analyzed, but it has already stimulated new experiments that have had major effects on the content of every chapter in this book. (A) human chromosome 22 in its mitotic conformation, composed of two double-stranded DNA molecules, each 48 ×106 nucleotide pairs long 10% of chromosome arm ~40 genes (B) ×10 1% of chromosome arm containing 4 genes (C) one gene of 3.4 ×104 nucleotide pairs (D) exon intron

1	Figure 4–14 Two closely related species of deer with very different chromosome numbers. In the evolution of the Indian muntjac, initially separate chromosomes fused, without having a major effect on the animal. These two species contain a similar number of genes. (Chinese muntjac photo courtesy of Deborah Carreno, Natural wonders Photography.)

1	Figure 4–15 The organization of genes on a human chromosome. (A) Chromosome 22, one of the smallest human chromosomes, contains 48 × 106 nucleotide pairs and makes up approximately 1.5% of the human genome. most of the left arm of chromosome 22 consists of short repeated sequences of DNA that are packaged in a particularly compact form of chromatin (heterochromatin) discussed later in this chapter. (b) A tenfold expansion of a portion of chromosome 22, with about 40 genes indicated. Those in dark brown are known genes and those in red are predicted genes. (C) An expanded portion of (b) showing four genes. (D) The intron–exon arrangement of a typical gene is shown after a further tenfold expansion. Each exon (red) codes for a portion of the protein, while the DNA sequence of the introns (gray) is relatively unimportant, as discussed in detail in Chapter 6.

1	The human genome (3.2 × 109 nucleotide pairs) is the totality of genetic information belonging to our species. Almost all of this genome is distributed over the 22 different autosomes and 2 sex chromosomes (see Figures 4–10 and 4–11) found within the nucleus. A minute fraction of the human genome (16,569 nucleotide pairs—in multiple copies per cell) is found in the mitochondria (introduced in Chapter 1, and discussed in detail in Chapter 14). The term human genome sequence refers to the complete nucleotide sequence of DNA in the 24 nuclear chromosomes and the mitochondria. being diploid, a human somatic cell nucleus contains roughly twice the haploid amount of DNA, or 6.4 × 109 nucleotide pairs, when not duplicating its chromosomes in preparation for division. (Adapted from International Human genome Sequencing Consortium, Nature 409:860–921, 2001. with permission from macmillan Publishers ltd.)

1	The first striking feature of the human genome is how little of it (only a few percent) codes for proteins (Table 4–1 and Figure 4–16). It is also notable that nearly half of the chromosomal DNA is made up of mobile pieces of DNA that have gradually inserted themselves in the chromosomes over evolutionary time, multiplying like parasites in the genome (see Figure 4–62). We discuss these transposable elements in detail in later chapters.

1	A second notable feature of the human genome is the large average gene size—about 27,000 nucleotide pairs. As discussed above, a typical gene carries in its linear sequence of nucleotides the information for the linear sequence of the amino acids of a protein. Only about 1300 nucleotide pairs are required to encode a protein of average size (about 430 amino acids in humans). Most of the remaining sequence in a gene consists of long stretches of noncoding DNA that interrupt the relatively short segments of DNA that code for protein. As will be discussed in detail in Chapter 6, the coding sequences are called exons; the intervening (noncoding) sequences in genes are called introns (see Figure 4–15 and Table 4–1). The majority of human genes thus consist of a long string of alternating exons and introns, with most of the gene consisting of introns. In contrast, the majority of genes from organisms with concise genomes lack introns. This accounts for the much smaller size of their genes

1	and introns, with most of the gene consisting of introns. In contrast, the majority of genes from organisms with concise genomes lack introns. This accounts for the much smaller size of their genes (about one-twentieth that of human genes), as well as for the much higher fraction of coding DNA in their chromosomes.

1	Figure 4–16 Scale of the human genome. If drawn with a 1 mm space between each nucleotide pair, as in (A), the human genome would extend 3200 km (approximately 2000 miles), far enough to stretch across the center of Africa, the site of our human origins (red line in b). At this scale, there would be, on average, a protein-coding gene every 150 m. An average gene would extend for 30 m, but the coding sequences in this gene would add up to only just over a meter.

1	In addition to introns and exons, each gene is associated with regulatory DNA sequences, which are responsible for ensuring that the gene is turned on or off at the proper time, expressed at the appropriate level, and only in the proper type of cell. In humans, the regulatory sequences for a typical gene are spread out over tens of thousands of nucleotide pairs. As would be expected, these regulatory sequences are much more compressed in organisms with concise genomes. We discuss how regulatory DNA sequences work in Chapter 7.

1	Research in the last decade has surprised biologists with the discovery that, in addition to 21,000 protein-coding genes, the human genome contains many thousands of genes that encode RNA molecules that do not produce proteins, but instead have a variety of other important functions. What is thus far known about these molecules will be presented in Chapters 6 and 7. Last, but not least, the nucleotide sequence of the human genome has revealed that the archive of information needed to produce a human seems to be in an alarming state of chaos. As one commentator described our genome, “In some ways it may resemble your garage/bedroom/refrigerator/life: highly individualistic, but unkempt; little evidence of organization; much accumulated clutter (referred to by the uninitiated as ‘junk’); virtually nothing ever discarded; and the few patently valuable items indiscriminately, apparently carelessly, scattered throughout.” We shall discuss how this is thought to have come about in the final

1	nothing ever discarded; and the few patently valuable items indiscriminately, apparently carelessly, scattered throughout.” We shall discuss how this is thought to have come about in the final sections of this chapter entitled “How Genomes Evolve.”

1	Each DNA molecule That Forms a linear Chromosome must Contain a Centromere, Two Telomeres, and Replication Origins

1	To form a functional chromosome, a DNA molecule must be able to do more than simply carry genes: it must be able to replicate, and the replicated copies must be separated and reliably partitioned into daughter cells at each cell division. This process occurs through an ordered series of stages, collectively known as the cell cycle, which provides for a temporal separation between the duplication of chromosomes and their segregation into two daughter cells. The cell cycle is briefly summarized in Figure 4–17, and it is discussed in detail in Chapter 17. Briefly, during a long interphase, genes are expressed and chromosomes are replicated, with the two replicas remaining together as a pair of sister chromatids. Throughout this time, the chromosomes are extended and much of their chromatin exists as long threads in the nucleus so that individual chromosomes cannot be easily distinguished. It is only during a much briefer period of mitosis that each chromosome condenses so that its two

1	exists as long threads in the nucleus so that individual chromosomes cannot be easily distinguished. It is only during a much briefer period of mitosis that each chromosome condenses so that its two sister chromatids can be separated and distributed to the two daughter nuclei. The highly condensed chromosomes in a dividing cell are known as mitotic chromosomes (Figure 4–18). This is the form in which chromosomes are most easily visualized; in fact, the images of chromosomes shown so far in the chapter are of chromosomes in mitosis.

1	Each chromosome operates as a distinct structural unit: for a copy to be passed on to each daughter cell at division, each chromosome must be able to replicate, and the newly replicated copies must subsequently be separated and partitioned

1	Figure 4–17 A simplified view of the eukaryotic cell cycle. During interphase, the cell is actively expressing its genes and is therefore synthesizing proteins. Also, during interphase and before cell division, the DNA is replicated and each chromosome is duplicated to produce two closely paired sister DNA molecules (called sister chromatids). A cell with only one type of chromosome, present in maternal and paternal copies, is illustrated here. Once DNA replication is complete, the cell can enter M phase, when mitosis occurs and the nucleus is divided into two daughter nuclei. During this stage, the chromosomes condense, the nuclear envelope breaks down, and the mitotic spindle forms from microtubules and other proteins. The condensed mitotic chromosomes are captured by the mitotic spindle, and one complete set of chromosomes is then pulled to each end of the cell by separating the members of each sister-chromatid pair. A nuclear envelope re-forms around each chromosome set, and in

1	and one complete set of chromosomes is then pulled to each end of the cell by separating the members of each sister-chromatid pair. A nuclear envelope re-forms around each chromosome set, and in the final step of m phase, the cell divides to produce two daughter cells. most of the time in the cell cycle is spent in interphase; m phase is brief in comparison, occupying only about an hour in many mammalian cells.

1	nuclear envelope surrounding the nucleus INTERPHASE M PHASE INTERPHASE correctly into the two daughter cells. These basic functions are controlled by three types of specialized nucleotide sequences in the DNA, each of which binds specific proteins that guide the machinery that replicates and segregates chromosomes (Figure 4–19). Experiments in yeasts, whose chromosomes are relatively small and easy to manipulate, have identified the minimal DNA sequence elements responsible for each of these functions. One type of nucleotide sequence acts as a DNA replication origin, the location at which duplication of the DNA begins. Eukaryotic chromosomes contain many origins of replication to ensure that the entire chromosome can be replicated rapidly, as discussed in detail in Chapter 5.

1	After DNA replication, the two sister chromatids that form each chromosome remain attached to one another and, as the cell cycle proceeds, are condensed further to produce mitotic chromosomes. The presence of a second specialized DNA sequence, called a centromere, allows one copy of each duplicated and condensed chromosome to be pulled into each daughter cell when a cell divides. A protein complex called a kinetochore forms at the centromere and attaches the duplicated chromosomes to the mitotic spindle, allowing them to be pulled apart (discussed in Chapter 17).

1	The third specialized DNA sequence forms telomeres, the ends of a chromosome. Telomeres contain repeated nucleotide sequences that enable the ends of chromosomes to be efficiently replicated. Telomeres also perform another function: the repeated telomere DNA sequences, together with the regions adjoining them, form structures that protect the end of the chromosome from being mistaken by the cell for a broken DNA molecule in need of repair. We discuss both this type of repair and the structure and function of telomeres in Chapter 5.

1	In yeast cells, the three types of sequences required to propagate a chromosome are relatively short (typically less than 1000 base pairs each) and therefore use only a tiny fraction of the information-carrying capacity of a chromosome. Although telomere sequences are fairly simple and short in all eukaryotes, the DNA sequences that form centromeres and replication origins in more complex organisms are much longer than their yeast counterparts. For example, experiments suggest that a human centromere can contain up to a million nucleotide pairs and that it may not require a stretch of DNA with a defined nucleotide sequence. Instead, as we shall discuss later in this chapter, a human centromere is thought to consist of a large, regularly repeating protein–nucleic acid structure that can be inherited when a chromosome replicates. Figure 4–18 A mitotic chromosome.

1	Figure 4–18 A mitotic chromosome. A mitotic chromosome is a condensed duplicated chromosome in which the two new chromosomes, called sister chromatids, are still linked together (see Figure 4–17). The constricted region indicates the position of the centromere. (Courtesy of Terry D. Allen.) Figure 4–19 The three DNA sequences required to produce a eukaryotic chromosome that can be replicated and then segregated accurately at mitosis.

1	Figure 4–19 The three DNA sequences required to produce a eukaryotic chromosome that can be replicated and then segregated accurately at mitosis. Each chromosome has multiple origins of replication, one centromere, and two telomeres. Shown here is the sequence of events that a typical chromosome follows during the cell cycle. The DNA replicates in interphase, beginning at the origins of replication and proceeding bidirectionally from the origins across the chromosome. In m phase, the centromere attaches the duplicated chromosomes to the mitotic spindle so that a copy of the entire genome is distributed to each daughter cell during mitosis; the special structure that attaches the centromere to the spindle is a protein complex called the kinetochore (dark green). The centromere also helps to hold the duplicated chromosomes together until they are ready to be moved apart. The telomeres form special caps at each chromosome end.

1	All eukaryotic organisms have special ways of packaging DNA into chromosomes. For example, if the 48 million nucleotide pairs of DNA in human chromosome 22 could be laid out as one long perfect double helix, the molecule would extend for about 1.5 cm if stretched out end to end. But chromosome 22 measures only about 2 μm in length in mitosis (see Figures 4–10 and 4–11), representing an endto-end compaction ratio of over 7000-fold. This remarkable feat of compression is performed by proteins that successively coil and fold the DNA into higher and higher levels of organization. Although much less condensed than mitotic chromosomes, the DNA of human interphase chromosomes is still tightly packed.

1	In reading these sections it is important to keep in mind that chromosome structure is dynamic. We have seen that each chromosome condenses to an extreme degree in the M phase of the cell cycle. Much less visible, but of enormous interest and importance, specific regions of interphase chromosomes decondense to allow access to specific DNA sequences for gene expression, DNA repair, and replication—and then recondense when these processes are completed. The packaging of chromosomes is therefore accomplished in a way that allows rapid localized, on-demand access to the DNA. In the next sections, we discuss the specialized proteins that make this type of packaging possible. Nucleosomes Are a basic Unit of Eukaryotic Chromosome Structure

1	Nucleosomes Are a basic Unit of Eukaryotic Chromosome Structure The proteins that bind to the DNA to form eukaryotic chromosomes are traditionally divided into two classes: the histones and the non-histone chromosomal proteins, each contributing about the same mass to a chromosome as the DNA. The complex of both classes of protein with the nuclear DNA of eukaryotic cells is known as chromatin (Figure 4–20).

1	Histones are responsible for the first and most basic level of chromosome packing, the nucleosome, a protein–DNA complex discovered in 1974. When interphase nuclei are broken open very gently and their contents examined under the electron microscope, most of the chromatin appears to be in the form of a fiber with a diameter of about 30 nm (Figure 4–21A). If this chromatin is subjected to treatments that cause it to unfold partially, it can be seen under the electron microscope as a series of “beads on a string” (Figure 4–21B). The string is DNA, and each bead is a “nucleosome core particle” that consists of DNA wound around a histone core (Movie 4.2).

1	The structural organization of nucleosomes was determined after first isolating them from unfolded chromatin by digestion with particular enzymes (called nucleases) that break down DNA by cutting between the nucleosomes. After digestion for a short period, the exposed DNA between the nucleosome core particles, the linker DNA, is degraded. Each individual nucleosome core particle consists of a complex of eight histone proteins—two molecules each of histones H2A, Figure 4–20 Chromatin. As illustrated, chromatin consists of DNA bound to both histone and non-histone proteins. The mass of histone protein present is about equal to the total mass of non-histone protein, but—as schematically indicated here—the latter class is composed of an enormous number of different species. In total, a chromosome is about one-third DNA and two-thirds protein by mass.

1	H2B, H3, and H4—and double-stranded DNA that is 147 nucleotide pairs long. The histone octamer forms a protein core around which the double-stranded DNA is wound (Figure 4–22). The region of linker DNA that separates each nucleosome core particle from the next can vary in length from a few nucleotide pairs up to about 80. (The term nucleosome technically refers to a nucleosome core particle plus one of its adjacent DNA linkers, but it is often used synonymously with nucleosome core particle.) On average, therefore, nucleosomes repeat at intervals of about 200 nucleotide pairs. For example, a diploid human cell with 6.4 × 109 nucleotide pairs contains approximately 30 million nucleosomes. The formation of nucleosomes converts a DNA molecule into a chromatin thread about one-third of its initial length. The Structure of the Nucleosome Core Particle Reveals How DNA Is Packaged

1	The Structure of the Nucleosome Core Particle Reveals How DNA Is Packaged The high-resolution structure of a nucleosome core particle, solved in 1997, revealed a disc-shaped histone core around which the DNA was tightly wrapped in a left-handed coil of 1.7 turns (Figure 4–23). All four of the histones that make up the core of the nucleosome are relatively small proteins (102–135 amino acids), and they share a structural motif, known as the histone fold, formed from three α helices connected by two loops (Figure 4–24). In assembling a nucleosome, the histone folds first bind to each other to form H3–H4 and H2A–H2B dimers, and the H3–H4 dimers combine to form tetramers. An H3–H4 tetramer then further combines with two H2A–H2B dimers to form the compact octamer core, around which the DNA is wound.

1	The interface between DNA and histone is extensive: 142 hydrogen bonds are formed between DNA and the histone core in each nucleosome. Nearly half of these bonds form between the amino acid backbone of the histones and the sugar-phosphate backbone of the DNA. Numerous hydrophobic interactions and salt linkages also hold DNA and protein together in the nucleosome. More than one-fifth of the amino acids in each of the core histones are either lysine or arginine (two amino acids with basic side chains), and their positive charges can effectively

1	Figure 4–22 Structural organization of the nucleosome. A nucleosome contains a protein core made of eight histone molecules. In biochemical experiments, the nucleosome core particle can be released from isolated chromatin by digestion of the linker DNA with a nuclease, an enzyme that breaks down DNA. (The nuclease can degrade the exposed linker DNA but cannot attack the DNA wound tightly around the nucleosome core.) After dissociation of the isolated nucleosome into its protein core and DNA, the length of the DNA that was wound around the core can be determined. This length of 147 nucleotide pairs is sufficient to wrap 1.7 times around the histone core.

1	Figure 4–21 Nucleosomes as seen in the electron microscope. (A) Chromatin isolated directly from an interphase nucleus appears in the electron microscope as a thread about 30 nm thick. (b) This electron micrograph shows a length of chromatin that has been experimentally unpacked, or decondensed, after isolation to show the nucleosomes. (A, courtesy of barbara Hamkalo; b, courtesy of victoria Foe.) core histones linker DNA of nucleosome “beads-on-a-string” nucleosome includes form of chromatin ~200 nucleotide pairs of DNA

1	Figure 4–23 The structure of a nucleosome core particle, as determined by x-ray diffraction analyses of crystals. Each histone is colored according to the scheme in Figure 4–22, with the DNA double helix in light gray. (Adapted from k. luger et al., Nature 389:251–260, 1997. with permission from macmillan Publishers ltd.) neutralize the negatively charged DNA backbone. These numerous interactions explain in part why DNA of virtually any sequence can be bound on a histone octamer core. The path of the DNA around the histone core is not smooth; rather, several kinks are seen in the DNA, as expected from the nonuniform surface of the

1	Figure 4–24 The overall structural organization of the core histones. (A) Each of the core histones contains an N-terminal tail, which is subject to several forms of covalent modification, and a histone fold region, as indicated. (b) The structure of the histone fold, which is formed by all four of the core histones. (C) Histones 2A and 2b form a dimer through an interaction known as the “handshake.” Histones H3 and H4 form a dimer through the same type of interaction. (D) The final histone octamer on DNA. Note that all eight N-terminal tails of the histones protrude from the disc-shaped core structure. Their conformations are highly flexible, and they serve as binding sites for sets of other proteins.

1	core. The bending requires a substantial compression of the minor groove of the DNA helix. Certain dinucleotides in the minor groove are especially easy to compress, and some nucleotide sequences bind the nucleosome more tightly than others (Figure 4–25). This probably explains some striking, but unusual, cases of very precise positioning of nucleosomes along a stretch of DNA. However, the sequence preference of nucleosomes must be weak enough to allow other factors to dominate, inasmuch as nucleosomes can occupy any one of a number of positions relative to the DNA sequence in most chromosomal regions. In addition to its histone fold, each of the core histones has an N-terminal amino acid “tail,” which extends out from the DNA–histone core (see Figure 4–24D). These histone tails are subject to several different types of covalent modifications that in turn control critical aspects of chromatin structure and function, as we shall discuss shortly.

1	As a reflection of their fundamental role in DNA function through controlling chromatin structure, the histones are among the most highly conserved eukaryotic proteins. For example, the amino acid sequence of histone H4 from a pea differs from that of a cow at only 2 of the 102 positions. This strong evolutionary conservation suggests that the functions of histones involve nearly all of their amino acids, so that a change in any position is deleterious to the cell. But in addition to this remarkable conservation, eukaryotic organisms also produce smaller amounts of specialized variant core histones that differ in amino acid sequence from the main ones. As discussed later, these variants, combined with the surprisingly large number of covalent modifications that can be added to the histones in nucleosomes, give rise to a variety of chromatin structures in cells.

1	Nucleosomes Have a Dynamic Structure, and Are Frequently Subjected to Changes Catalyzed by ATP-Dependent Chromatin Remodeling Complexes For many years biologists thought that, once formed in a particular position on DNA, a nucleosome would remain fixed in place because of the very tight association between its core histones and DNA. If true, this would pose problems for genetic readout mechanisms, which in principle require easy access to many specific DNA sequences. It would also hinder the rapid passage of the DNA transcription and replication machinery through chromatin. But kinetic experiments show that the DNA in an isolated nucleosome unwraps from each end at a rate of about four times per second, remaining exposed for 10 to 50 milliseconds before the partially unwrapped structure recloses. Thus, most of the DNA in an isolated nucleosome is in principle available for binding other proteins.

1	For the chromatin in a cell, a further loosening of DNA–histone contacts is clearly required, because eukaryotic cells contain a large variety of ATP-dependent chromatin remodeling complexes. These complexes include a subunit that hydrolyzes ATP (an ATPase evolutionarily related to the DNA helicases discussed in Chapter 5). This subunit binds both to the protein core of the nucleosome and to the double-stranded DNA that winds around it. By using the energy of ATP hydrolysis to move this DNA relative to the core, the protein complex changes the structure of a nucleosome temporarily, making the DNA less tightly bound to the histone core. Through repeated cycles of ATP hydrolysis that pull the nucleosome core along the DNA double helix, the remodeling complexes can catalyze nucleosome sliding. In this way, they can reposition nucleosomes to expose specific regions of DNA, thereby making them available to other proteins in the cell (Figure 4–26). In addition, by cooperating with a variety

1	In this way, they can reposition nucleosomes to expose specific regions of DNA, thereby making them available to other proteins in the cell (Figure 4–26). In addition, by cooperating with a variety of other proteins that bind to histones and serve as histone chaperones, some remodeling complexes are able to remove either all or part of the nucleosome core from a nucleosome—catalyzing either an exchange of its H2A–H2B histones, or the complete removal of the octameric core from the DNA (Figure 4–27). As a result of such processes, measurements reveal that a typical nucleosome is replaced on the DNA every one or two hours inside the cell.

1	DNA of nucleosome histone core of nucleosome (histone octamer) AA, TT, and TA dinucleotides preferred here (minor groove inside) (minor groove outside) Figure 4–25 The bending of DNA in a nucleosome. The DNA helix makes 1.7 tight turns around the histone octamer. This diagram illustrates how the minor groove is compressed on the inside of the turn. Owing to structural features of the DNA molecule, the indicated dinucleotides are preferentially accommodated in such a narrow minor groove, which helps to explain why certain DNA sequences will bind more tightly than others to the nucleosome core.

1	Cells contain dozens of different ATP-dependent chromatin remodeling complexes that are specialized for different roles. Most are large protein complexes that can contain 10 or more subunits, some of which bind to specific modifications on histones (see Figure 4–26C). The activity of these complexes is carefully controlled by the cell. As genes are turned on and off, chromatin remodeling complexes are brought to specific regions of DNA where they act locally to influence chromatin structure (discussed in Chapter 7; see also Figure 4–40, below).

1	Although some DNA sequences bind more tightly than others to the nucleosome core (see Figure 4–25), the most important influence on nucleosome positioning appears to be the presence of other tightly bound proteins on the DNA. Some bound proteins favor the formation of a nucleosome adjacent to them. Others create obstacles that force the nucleosomes to move elsewhere. The exact positions of nucleosomes along a stretch of DNA therefore depend mainly on the presence and nature of other proteins bound to the DNA. And due to the presence of ATP-dependent chromatin remodeling complexes, the arrangement of nucleosomes on DNA can be highly dynamic, changing rapidly according to the needs of the cell.

1	Although enormously long strings of nucleosomes form on the chromosomal DNA, chromatin in a living cell probably rarely adopts the extended “beads-on-astring” form. Instead, the nucleosomes are packed on top of one another, generating arrays in which the DNA is even more highly condensed. Thus, when nuclei are very gently lysed onto an electron microscope grid, much of the chromatin is seen to be in the form of a fiber with a diameter of about 30 nm, which is considerably wider than chromatin in the “beads-on-a-string” form (see Figure 4–21).

1	Figure 4–26 The nucleosome sliding catalyzed by ATP-dependent chromatin remodeling complexes. (A) Using the energy of ATP hydrolysis, the remodeling complex is thought to push on the DNA of its bound nucleosome and loosen its attachment to the nucleosome core. Each cycle of ATP binding, ATP hydrolysis, and release of the ADP and Pi products thereby moves the DNA with respect to the histone octamer in the direction of the arrow in this diagram. It requires many such cycles to produce the nucleosome sliding shown.

1	(b) The structure of a nucleosome-bound dimer of the two identical ATPase subunits (green) that slide nucleosomes back and forth in the ISw1 family of chromatin remodeling complexes. (C) The structure of a large chromatin remodeling complex, showing how it is thought to wrap around a nucleosome. modeled in green is the yeast RSC complex, which contains 15 subunits— including an ATPase and at least four subunits with domains that recognize specific covalently modified histones. (b, from l.R. Racki et al., Nature 462:1016–1021, 2009. with permission from macmillan Publishers ltd; C, adapted from A.E. leschziner et al., Proc. Natl Acad. Sci. USA 104:4913–4918, 2007.) 192 Chapter 4: DNA, Chromosomes, and genomes

1	A.E. leschziner et al., Proc. Natl Acad. Sci. USA 104:4913–4918, 2007.) 192 Chapter 4: DNA, Chromosomes, and genomes How nucleosomes are organized into condensed arrays is unclear. The structure of a tetranucleosome (a complex of four nucleosomes) obtained by x-ray crystallography and high-resolution electron microscopy of reconstituted chromatin have been used to support a zigzag model for the stacking of nucleosomes in a 30-nm fiber (Figure 4–28). But cryoelectron microscopy of carefully prepared nuclei suggests that most regions of chromatin are less regularly structured.

1	What causes nucleosomes to stack so tightly on each other? Nucleosome-tonucleosome linkages that involve histone tails, most notably the H4 tail, constitute one important factor (Figure 4–29). Another important factor is an additional histone that is often present in a 1-to-1 ratio with nucleosome cores, known as Figure 4–28 A zigzag model for the 30histone H1. This so-called linker histone is larger than the individual core histones nm chromatin fiber. (A) The conformation of two of the four nucleosomes in a and it has been considerably less well conserved during evolution. A single his tetranucleosome, from a structure tone H1 molecule binds to each nucleosome, contacting both DNA and protein, determined by x-ray crystallography. and changing the path of the DNA as it exits from the nucleosome. This change in (b) Schematic of the entire tetranucleosome; the exit path of DNA is thought to help compact nucleosomal DNA (Figure 4–30). the fourth nucleosome is not visible, being stacked

1	This change in (b) Schematic of the entire tetranucleosome; the exit path of DNA is thought to help compact nucleosomal DNA (Figure 4–30). the fourth nucleosome is not visible, being stacked on the bottom nucleosome and behind it in this diagram. (C) Diagrammatic illustration of a possible zigzag structure that could account for the 30-nm chromatin fiber. (A, PDb code: 1Zbb; C, adapted from C.l. woodcock, Nat. Struct. Mol. Biol. 12:639–640, 2005. with permission from macmillan Publishers ltd.)

1	Most eukaryotic organisms make several histone H1 proteins of related but quite distinct amino acid sequences. The presence of many other DNA-binding proteins, as well as proteins that bind directly to histones, is certain to add important additional features to any array of nucleosomes.

1	A gene is a nucleotide sequence in a DNA molecule that acts as a functional unit for the production of a protein, a structural RNA, or a catalytic or regulatory RNA molecule. In eukaryotes, protein-coding genes are usually composed of a string of alternating introns and exons associated with regulatory regions of DNA. A chromosome is formed from a single, enormously long DNA molecule that contains a linear array of many genes, bound to a large set of proteins. The human genome contains 3.2 × 109 DNA nucleotide pairs, divided between 22 different autosomes (present in two copies each) and 2 sex chromosomes. Only a small percentage of this DNA codes for proteins or functional RNA molecules. A chromosomal DNA molecule also contains three other types of important nucleotide sequences: replication origins and telomeres allow the DNA molecule to be efficiently replicated, while a centromere attaches the sister DNA molecules to the mitotic spindle, ensuring their accurate segregation to

1	origins and telomeres allow the DNA molecule to be efficiently replicated, while a centromere attaches the sister DNA molecules to the mitotic spindle, ensuring their accurate segregation to daughter cells during the M phase of the cell cycle.

1	The DNA in eukaryotes is tightly bound to an equal mass of histones, which form repeated arrays of DNA–protein particles called nucleosomes. The nucleosome is composed of an octameric core of histone proteins around which the DNA double helix is wrapped. Nucleosomes are spaced at intervals of about 200 nucleotide pairs, and they are usually packed together (with the aid of histone H1 molecules) into quasi-regular arrays to form a 30-nm chromatin fiber. Even though compact, the structure of chromatin must be highly dynamic to allow access to the DNA. There is some spontaneous DNA unwrapping and rewrapping in the nucleosome itself; however, the general strategy for reversibly changing local chromatin structure features ATP-driven chromatin remodeling complexes. Cells contain a large set of such complexes, which are targeted to specific regions of chromatin at appropriate times. The remodeling complexes collaborate with histone chaperones to allow nucleosome cores to be repositioned,

1	of such complexes, which are targeted to specific regions of chromatin at appropriate times. The remodeling complexes collaborate with histone chaperones to allow nucleosome cores to be repositioned, reconstituted with different histones, or completely removed to expose the underlying DNA.

1	Figure 4–29 A model for the role played by histone tails in the compaction of chromatin. (A) A schematic diagram shows the approximate exit points of the eight histone tails, one from each histone protein, that extend from each nucleosome. The actual structure is shown to its right. In the high-resolution structure of the nucleosome, the tails are largely unstructured, suggesting that they are highly flexible. (b) As indicated, the histone tails are thought to be involved in interactions between nucleosomes that help to pack them together. (A, PDb code: 1k X5.)

1	Figure 4–30 How the linker histone binds to the nucleosome. The position and structure of histone H1 is shown. The H1 core region constrains an additional 20 nucleotide pairs of DNA where it exits from the nucleosome core and is important for compacting chromatin. (A) Schematic, and (b) structure inferred for a single nucleosome from a structure determined by high-resolution electron microscopy of a reconstituted chromatin fiber (C). (b and C, adapted from F. Song et al., Science 344:376–380, 2014.)

1	Having described how DNA is packaged into nucleosomes to create a chromatin fiber, we now turn to the mechanisms that create different chromatin structures in different regions of a cell’s genome. Mechanisms of this type have a variety of important functions in cells. Most strikingly, certain types of chromatin structure can be inherited; that is, the structure can be directly passed down from a cell to its descendants. Because the cell memory that results is based on an inherited chromatin structure rather than on a change in DNA sequence, this is a form of epigenetic inheritance. The prefix epi is Greek for “on”; this is appropriate, because epigenetics represents a form of inheritance that is superimposed on the genetic inheritance based on DNA.

1	In Chapter 7, we shall introduce the many different ways in which the expression of genes is regulated. There we discuss epigenetic inheritance in detail and present several different mechanisms that can produce it. Here, we are concerned with only one, that based on chromatin structure. We begin this section by reviewing the observations that first demonstrated that chromatin structures can be inherited. We then describe some of the chemistry that makes this possible— the covalent modification of histones in nucleosomes. These modifications have many functions, inasmuch as they serve as recognition sites for protein domains that link specific protein complexes to different regions of chromatin. Histones thereby have effects on gene expression, as well as on many other DNA-linked processes. Through such mechanisms, chromatin structure plays an important role in the development, growth, and maintenance of all eukaryotic organisms, including ourselves.

1	Light-microscope studies in the 1930s distinguished two types of chromatin in the interphase nuclei of many higher eukaryotic cells: a highly condensed form, called heterochromatin, and all the rest, which is less condensed, called euchromatin. Heterochromatin represents an especially compact form of chromatin (see Figure 4–9), and we are finally beginning to understand its molecular properties. It is highly concentrated in certain specialized regions, most notably at the centromeres and telomeres introduced previously (see Figure 4–19), but it is also present at many other locations along chromosomes—locations that can vary according to the physiological state of the cell. In a typical mammalian cell, more than 10% of the genome is packaged in this way.

1	The DNA in heterochromatin typically contains few genes, and when euchromatic regions are converted to a heterochromatic state, their genes are generally switched off as a result. However, we know now that the term heterochromatin encompasses several distinct modes of chromatin compaction that have different implications for gene expression. Thus, heterochromatin should not be thought of as simply encapsulating “dead” DNA, but rather as a descriptor for compact chromatin domains that share the common feature of being unusually resistant to gene expression. The Heterochromatic State Is Self-Propagating

1	The Heterochromatic State Is Self-Propagating Through chromosome breakage and rejoining, whether brought about by a natural genetic accident or by experimental artifice, a piece of chromosome that is normally euchromatic can be translocated into the neighborhood of heterochromatin. Remarkably, this often causes silencing—inactivation—of the normally active genes. This phenomenon is referred to as a position effect. It reflects a spreading of the heterochromatic state into the originally euchromatic region, and it has provided important clues to the mechanisms that create and maintain heterochromatin. First recognized in Drosophila, position effects have now been observed in many eukaryotes, including yeasts, plants, and humans. early in the developing embryo, heterochromatin forms and spreads into neighboring euchromatin to different extents in different cells clone of cells with clone of cells with clone of cells with gene 1 inactive genes 1, 2, and 3 inactive no genes inactivated

1	Figure 4–31 The cause of position effect variegation in Drosophila. (A) Heterochromatin (green) is normally prevented from spreading into adjacent regions of euchromatin (red) by barrier DNA sequences, which we shall discuss shortly. In flies that inherit certain chromosomal rearrangements, however, this barrier is no longer present. (b) During the early development of such flies, heterochromatin can spread into neighboring chromosomal DNA, proceeding for different distances in different cells. This spreading soon stops, but the established pattern of heterochromatin is subsequently inherited, so that large clones of progeny cells are produced that have the same neighboring genes condensed into heterochromatin and thereby inactivated (hence the “variegated” appearance of some of these flies; see Figure 4–32). Although “spreading” is used to describe the formation of new heterochromatin close to previously existing heterochromatin, the term may not be wholly accurate. There is evidence

1	see Figure 4–32). Although “spreading” is used to describe the formation of new heterochromatin close to previously existing heterochromatin, the term may not be wholly accurate. There is evidence that during expansion, the condensation of DNA into heterochromatin can “skip over” some regions of chromatin, sparing the genes that lie within them from repressive effects.

1	In chromosome breakage-and-rejoining events of the sort just described, the zone of silencing, where euchromatin is converted to a heterochromatic state, spreads for different distances in different early cells in the fly embryo. Remarkably, these differences then are perpetuated for the rest of the animal’s life: in each cell, once the heterochromatic condition is established on a piece of chromatin, it tends to be stably inherited by all of that cell’s progeny (Figure 4–31). This remarkable phenomenon, called position effect variegation, was first recognized through a detailed genetic analysis of the mottled loss of red pigment in the fly eye (Figure 4–32). It shares features with the extensive spread of heterochromatin that inactivates one of the two X chromosomes in female mammals. There too, a random process acts in each cell of the early embryo to dictate which X chromosome will be inactivated, and that same X chromosome then remains inactive in all the cell’s progeny, creating

1	too, a random process acts in each cell of the early embryo to dictate which X chromosome will be inactivated, and that same X chromosome then remains inactive in all the cell’s progeny, creating a mosaic of different clones of cells in the adult body (see Figure 7–50).

1	These observations, taken together, point to a fundamental strategy of heterochromatin formation: heterochromatin begets more heterochromatin. This positive feedback can operate both in space, causing the heterochromatic state to spread along the chromosome, and in time, across cell generations, propagating the heterochromatic state of the parent cell to its daughters. The challenge is to explain the molecular mechanisms that underlie this remarkable behavior.

1	Figure 4–32 The discovery of position effects on gene expression. The White gene in the fruit fly Drosophila controls eye pigment production and is named after the mutation that first identified it. wild-type flies with a normal White gene (White+) have normal pigment production, which gives them red eyes, but if the White gene is mutated and inactivated, the mutant flies (White–) make no pigment and have white eyes. In flies in which a normal White gene has been moved near a region of heterochromatin, the eyes are mottled, with both red and white patches. The white patches represent cell lineages in which the White gene has been silenced by the effects of the heterochromatin. In contrast, the red patches represent cell lineages in which the White gene is expressed. Early in development, when the heterochromatin is first formed, it spreads into neighboring euchromatin to different extents in different embryonic cells (see Figure 4–31). The presence of large patches of red and white

1	when the heterochromatin is first formed, it spreads into neighboring euchromatin to different extents in different embryonic cells (see Figure 4–31). The presence of large patches of red and white cells reveals that the state of transcriptional activity, as determined by the packaging of this gene into chromatin in those ancestor cells, is inherited by all daughter cells.

1	Figure 4–33 Some prominent types of covalent amino acid side-chain (B) SERINE PHOSPHORYLATION modifications found on nucleosomal histones. (A) Three different levels of lysine methylation are shown; each can be recognized by a different binding protein and thus each can have a different significance for the cell. Note that acetylation removes the plus charge on lysine, and that, most importantly, an acetylated lysine cannot be methylated, and vice versa. (b) Serine phosphorylation adds a negative charge to a histone. modifications of histones not shown here include the monoor dimethylation of an arginine, the phosphorylation of a threonine, the addition of ADP-ribose to a glutamic acid, and the addition of a ubiquityl, sumoyl, or biotin group to a lysine. OPO As a first step, one can carry out a search for the molecules that are involved.

1	As a first step, one can carry out a search for the molecules that are involved. This has been done by means of genetic screens, in which large numbers of mutants are generated, after which one picks out those that show an abnormality of the process in question. Extensive genetic screens in Drosophila, fungi, and mice have identified more than 100 genes whose products either enhance or suppress the spread of heterochromatin and its stable inheritance—in other words, genes that serve as either enhancers or suppressors of position effect variegation. Many of these genes turn out to code for non-histone chromosomal proteins that interact with histones and are involved in modifying or maintaining chromatin structure. We shall discuss how they work in the sections that follow. The Core Histones Are Covalently modified at many Different Sites

1	The Core Histones Are Covalently modified at many Different Sites The amino acid side chains of the four histones in the nucleosome core are subjected to a remarkable variety of covalent modifications, including the acetylation of lysines, the mono-, di-, and trimethylation of lysines, and the phosphorylation of serines (Figure 4–33). A large number of these side-chain modifications occur on the eight relatively unstructured N-terminal “histone tails” that protrude from the nucleosome (Figure 4–34). However, there are also more than 20 specific side-chain modifications on the nucleosome’s globular core.

1	All of the above types of modifications are reversible, with one enzyme serving to create a particular type of modification, and another to remove it. These enzymes are highly specific. Thus, for example, acetyl groups are added to specific lysines by a set of different histone acetyl transferases (HATs) and removed by a set of histone deacetylase complexes (HDACs). Likewise, methyl groups are added to lysine side chains by a set of different histone methyl transferases and removed by a set of histone demethylases. Each enzyme is recruited to specific sites on the chromatin at defined times in each cell’s life history. For the most part, the initial recruitment depends on transcription regulator proteins (sometimes called “transcription factors”). As we shall explain in Chapter 7, these proteins recognize and bind to specific DNA sequences in the chromosomes. They are produced at KEY:

1	Figure 4–34 The covalent modification of core histone tails. (A) The structure of the nucleosome highlighting the location of the first 30 amino acids in each of its eight N-terminal histone tails (green). These tails are unstructured and highly mobile, and thus will change their conformation depending on other bound proteins. (b) well-documented modifications of the four histone core proteins are indicated. Although only a single symbol is used here for methylation (m), each lysine (k) or arginine (R) can be methylated in several different ways. Note also that some positions (e.g., lysine 9 of H3) can be modified either by methylation or by acetylation, but not both. most of the modifications shown add a relatively small molecule onto the histone tails; the exception is ubiquitin, a 76-amino-acid protein also used for other cell processes (see Figure 3–69). Not shown are more than 20 possible modifications located in the globular core of the histones. (A, PDb: 1kX5; b, adapted from

1	protein also used for other cell processes (see Figure 3–69). Not shown are more than 20 possible modifications located in the globular core of the histones. (A, PDb: 1kX5; b, adapted from H. Santos-Rosa and

1	C. Caldas, Eur. J. Cancer 41:2381–2402, 2005. with permission from Elsevier.) different times and places in the life of an organism, thereby determining where and when the chromatin-modifying enzymes will act. In this way, the DNA sequence ultimately determines how histones are modified. But in at least some cases, the covalent modifications on nucleosomes can persist long after the transcription regulator proteins that first induced them have disappeared, thereby providing the cell with a memory of its developmental history. Most remarkably, as in the related phenomenon of position effect variegation discussed above, this memory can be transmitted from one cell generation to the next.

1	Very different patterns of covalent modification are found on different groups of nucleosomes, depending both on their exact position in the genome and on the history of the cell. The modifications of the histones are carefully controlled, and they have important consequences. The acetylation of lysines on the N-terminal tails loosens chromatin structure, in part because adding an acetyl group to lysine removes its positive charge, thereby reducing the affinity of the tails for adjacent nucleosomes. However, the most profound effects of the histone modifications lie in their ability to recruit specific other proteins to the modified stretch of chromatin. Trimethylation of one specific lysine on the histone H3 tail, for instance, attracts the heterochromatin-specific protein HP1 and contributes to the establishment and spread of heterochromatin. More generally, the recruited proteins act with the modified histones to determine how and when genes will be expressed, as well as other

1	to the establishment and spread of heterochromatin. More generally, the recruited proteins act with the modified histones to determine how and when genes will be expressed, as well as other chromosome functions. In this way, the precise structure of each domain of chromatin governs the readout of the genetic information that it contains, and thereby the structure and function of the eukaryotic cell.

1	H3.3 gene expression,H2AZ chromosome segregation transcriptional repression,macroH2A X-chromosome inactivation Chromatin Acquires Additional variety Through the Site-Specific Insertion of a Small Set of Histone variants In addition to the four highly conserved standard core histones, eukaryotes also contain a few variant histones that can also assemble into nucleosomes. These histones are present in much smaller amounts than the major histones, and they have been less well conserved over long evolutionary times. Variants are known for each of the core histones with the exception of H4; some examples are shown in Figure 4–35.

1	The major histones are synthesized primarily during the S phase of the cell cycle and assembled into nucleosomes on the daughter DNA helices just behind the replication fork (see Figure 5–32). In contrast, most histone variants are synthesized throughout interphase. They are often inserted into already-formed chromatin, which requires a histone-exchange process catalyzed by the ATP-dependent chromatin remodeling complexes discussed previously. These remodeling complexes contain subunits that cause them to bind both to specific sites on chromatin and to histone chaperones that carry a particular variant. As a result, each histone variant is inserted into chromatin in a highly selective manner (see Figure 4–27). Covalent modifications and Histone variants Act in Concert to Control Chromosome Functions

1	Covalent modifications and Histone variants Act in Concert to Control Chromosome Functions The number of possible distinct markings on an individual nucleosome is in principle enormous, and this potential for diversity is still greater when we allow for nucleosomes that contain histone variants. However, the histone modifications are known to occur in coordinated sets. More than 15 such sets can be identified in mammalian cells. However, it is not yet clear how many different types of chromatin are functionally important in cells.

1	Some combinations are known to have a specific meaning for the cell in the sense that they determine how and when the DNA packaged in the nucleosomes is to be accessed or manipulated—a fact that led to the idea of a “histone code.” For example, one type of marking signals that a stretch of chromatin has been newly replicated, another signals that the DNA in that chromatin has been damaged and needs repair, while others signal when and how gene expression should take place. Various regulatory proteins contain small domains that bind to specific marks, recognizing, for example, a trimethylated lysine 4 on histone H3 (Figure 4–36). These domains are often linked together as modules in a single large

1	Figure 4–35 The structure of some histone variants compared with the major histone that they replace. The histone variants are inserted into nucleosomes at specific sites on chromosomes by ATP-dependent chromatin remodeling enzymes that act in concert with histone chaperones (see Figure 4–27). The CENP-A (Centromere Protein-A) variant of histone H3 is discussed later in this chapter (see Figure 4–42); other variants are discussed in Chapter 7. The sequences in each variant that are colored differently (compared to the major histone above it) denote regions with an amino acid sequence different from this major histone. (Adapted from k. Sarma and D. Reinberg, Nat. Rev. Mol. Cell Biol. 6:139–149, 2005. with permission from macmillan Publishers ltd.)

1	Figure 4–36 How a mark on a nucleosome is read. The figure shows the structure of a protein module (called an INg PHD domain) that specifically recognizes histone H3 trimethylated on lysine 4. (A) A trimethyl group. (b) Space-filling model of an INg PHD domain bound to a histone tail (green, with the trimethyl group highlighted in yellow). (C) A ribbon model showing how the N-terminal six amino acids in the H3 tail are recognized. The red lines represent hydrogen bonds. This is one of a family of PHD domains that recognize methylated lysines on histones; different members of the family bind tightly to lysines located at different positions, and they can discriminate between a mono-, di-, and trimethylated lysine. In a similar way, other small protein modules recognize specific histone side chains that have been marked with acetyl groups, phosphate groups, and so on. (Adapted from P.v. Peña et al., Nature 442:100–103, 2006. with permission from macmillan Publishers ltd.) protein or

1	side chains that have been marked with acetyl groups, phosphate groups, and so on. (Adapted from P.v. Peña et al., Nature 442:100–103, 2006. with permission from macmillan Publishers ltd.) protein or protein complex, which thereby recognizes a specific combination of histone modifications (Figure 4–37). The result is a reader complex that allows particular combinations of markings on chromatin to attract additional proteins, so as to execute an appropriate biological function at the right time (Figure 4–38).

1	The marks on nucleosomes due to covalent additions to histones are dynamic, being constantly removed and added at rates that depend on their chromosomal locations. Because the histone tails extend outward from the nucleosome core and are likely to be accessible even when chromatin is condensed, they would seem to provide an especially suitable format for creating marks that can be readily altered as a cell’s needs change. Although much remains to be learned about the meaning of the different histone modifications, a few well-studied examples of the information that can be encoded in the histone H3 tail are listed in Figure 4–39. A Complex of Reader and writer Proteins Can Spread Specific The phenomenon of position effect variegation described previously requires that some modified forms of chromatin have the ability to spread for substantial distances along a chromosomal DNA molecule (see Figure 4–31). How is this possible?

1	The enzymes that add or remove modifications to histones in nucleosomes are part of multisubunit complexes. They can initially be brought to a particular region of chromatin by one of the sequence-specific DNA-binding proteins (transcription regulators) discussed in Chapters 6 and 7 (for a specific example,

1	Figure 4–37 Recognition of a specific combination of marks on a nucleosome. In the example shown, two adjacent domains that are part of the NURF (Nucleosome Remodeling Factor) chromatin remodeling complex bind to the nucleosome, with the PHD domain (red) recognizing a methylated H3 lysine 4 and another domain (a bromodomain, blue) recognizing an acetylated H4 lysine 16. These two histone marks constitute a unique histone modification pattern that occurs in subsets of nucleosomes in human cells. Here the two histone tails are indicated by green dotted lines, and only half of one nucleosome is shown. (Adapted from A.J. Ruthenburg et al., Cell 145:692–706, 2011. with permission from Elsevier.) protein modules scaffold binding to specifc protein histone modifcations on nucleosome attachment to other components in nucleus, leading to gene expression, gene silencing, or other biological function see Figure 7–20). But after a modifying enzyme “writes” its mark on one or a few neighboring

1	to other components in nucleus, leading to gene expression, gene silencing, or other biological function see Figure 7–20). But after a modifying enzyme “writes” its mark on one or a few neighboring nucleosomes, events that resemble a chain reaction can ensue. In such a case, the “writer enzyme” works in concert with a “reader protein” located in the same protein complex. The reader protein contains a module that recognizes the mark and binds tightly to the newly modified nucleosome (see Figure heterochromatin formation, gene silencingK 9

1	Figure 4–38 Schematic diagram showing how a particular combination of histone modifications can be recognized by a reader complex. A large protein complex that contains a series of protein modules, each of which recognizes a specific histone mark, is schematically illustrated (green). This “reader complex” will bind tightly only to a region of chromatin that contains several of the different histone marks that it recognizes. Therefore, only a specific combination of marks will cause the complex to bind to chromatin and attract the additional protein complexes (purple) needed to catalyze a biological function.

1	Figure 4–39 Some specific meanings of histone modifications. (A) The modifications on the histone H3 N-terminal tail are shown, repeated from Figure 4–34. (b) The H3 tail can be marked by different sets of modifications that act in combination to convey a specific meaning. Only a small number of the meanings are known, including the three examples shown. Not illustrated is the fact that, as just implied (see Figure 4–38), reading a histone mark generally involves the joint recognition of marks at other sites on the nucleosome along with the indicated H3 tail recognition. In addition, specific levels of methylation (mono-, di-, or trimethyl groups) are generally required. Thus, for example, the trimethylation of lysine 9 attracts the heterochromatin-specific protein HP1, which induces a spreading wave of further lysine 9 trimethylation followed by further HP1 binding, according to the general scheme that will be illustrated shortly (see Figure 4–40). Also important in this process,

1	spreading wave of further lysine 9 trimethylation followed by further HP1 binding, according to the general scheme that will be illustrated shortly (see Figure 4–40). Also important in this process, however, is a synergistic trimethylation of the histone H4 N-terminal tail on lysine 20.

1	4–36), activating an attached writer enzyme and positioning it near an adjacent nucleosome. Through many such read–write cycles, the reader protein can carry the writer enzyme along the DNA—spreading the mark in a hand-over-hand manner along the chromosome (Figure 4–40). In reality, the process is more complicated than the scheme just described. Both readers and writers are part of a protein complex that is likely to contain multiple readers and writers, and to require multiple marks on the nucleosome to spread. Moreover, many of these reader–writer complexes also contain an ATP-dependent chromatin remodeling protein (see Figure 4–26C), and the reader, writer, and remodeling proteins can work in concert to either decondense or condense long stretches of chromatin as the reader moves progressively along the nucleosome-packaged DNA.

1	A similar process is used to remove histone modifications from specific regions of the DNA; in this case, an “eraser enzyme,” such as a histone demethylase or his-tone deacetylase, is recruited to the complex. As for the writer complex in Figure 4–40, sequence-specific DNA-binding proteins (transcription regulators) direct where such modifications occur (discussed in Chapter 7). Some idea of the complexity of the above processes can be derived from the results of genetic screens for genes that either enhance or suppress the spreading and stability of heterochromatin, as manifest in effects on position effect variegation in Drosophila (see Figure 4–32). As pointed out previously, more than 100 such genes are known, and most of them are likely to code for subunits in one or more reader–writer–remodeling protein complexes.

1	Figure 4–40 How the recruitment of a reader–writer complex can spread chromatin changes along a chromosome. The writer is an enzyme that creates a specific modification on one or more of the four nucleosomal histones. After its recruitment to a specific site on a chromosome by a transcription regulatory protein, the writer collaborates with a reader protein to spread its mark from nucleosome to nucleosome by means of the indicated reader–writer complex. For this mechanism to work, the reader must recognize the same histone modification mark that the writer produces; its binding to that mark can be shown to activate the writer. In this schematic example, a spreading wave of chromatin condensation is thereby induced. Not shown are the additional proteins involved, including an ATP-dependent chromatin remodeling complex required to reposition the modified nucleosomes. barrier DNA Sequences block the Spread of Reader–writer Complexes and thereby Separate Neighboring Chromatin Domains

1	barrier DNA Sequences block the Spread of Reader–writer Complexes and thereby Separate Neighboring Chromatin Domains The above mechanism for spreading chromatin structures raises a potential problem. Inasmuch as each chromosome contains one continuous, very long DNA molecule, what prevents a cacophony of confusing cross-talk between adjacent chromatin domains of different structure and function? Early studies of position effect variegation had suggested an answer: certain DNA sequences mark the boundaries of chromatin domains and separate one such domain from another (see Figure 4–31). Several such barrier sequences have now been identified and characterized through the use of genetic engineering techniques that allow specific DNA segments to be deleted from, or inserted in, chromosomes.

1	For example, in cells that are destined to give rise to red blood cells, a sequence called HS4 normally separates the active chromatin domain that contains the human β-globin locus from an adjacent region of silenced, condensed chromatin. If this sequence is deleted, the β-globin locus is invaded by condensed chromatin. This chromatin silences the genes it covers, and it spreads to a different extent in different cells, causing position effect variegation similar to that observed in Drosophila. As described in Chapter 7, the consequences are dire: the globin genes are poorly expressed, and individuals who carry such a deletion have a severe form of anemia.

1	In genetic engineering experiments, the HS4 sequence is often added to both ends of a gene that is to be inserted into a mammalian genome, in order to protect that gene from the silencing caused by spreading heterochromatin. Analysis of this barrier sequence reveals that it contains a cluster of binding sites for histone acetylase enzymes. Since the acetylation of a lysine side chain is incompatible with the methylation of the same side chain, and specific lysine methylations are required to spread heterochromatin, histone acetylases are logical candidates for the formation of DNA barriers to spreading (Figure 4–41). However, several other types of chromatin modifications are known that can also protect genes from silencing. Figure 4–41 Some mechanisms of barrier action. These models are derived from experimental analyses of barrier action, and a combination of several of them may function at any one site.

1	Figure 4–41 Some mechanisms of barrier action. These models are derived from experimental analyses of barrier action, and a combination of several of them may function at any one site. The tethering of a region of chromatin to a large fixed site, such as the nuclear pore complex illustrated here, can form a barrier that stops the spread of heterochromatin. The tight binding of barrier proteins to a group of nucleosomes can make this chromatin resistant to heterochromatin spreading. (C) by recruiting a group of highly active histone-modifying enzymes, barriers can erase the histone marks that are required for heterochromatin to spread. For example, a potent acetylation of lysine 9 on histone H3 will compete with lysine 9 methylation, thereby preventing the binding of the HP1 protein needed to form a major form of heterochromatin. (based on A.g. west and P. Fraser, Hum. Mol. Genet. 14:R101–R111, 2005. with permission from Oxford University Press.)

1	A.g. west and P. Fraser, Hum. Mol. Genet. 14:R101–R111, 2005. with permission from Oxford University Press.) The Chromatin in Centromeres Reveals How Histone variants Can Create Special Structures

1	Nucleosomes carrying histone variants have a distinctive character and are thought to be able to produce marks in chromatin that are unusually long-lasting. An important example is seen in the formation and inheritance of the specialized chromatin structure at the centromere, the region of each chromosome required for attachment to the mitotic spindle and orderly segregation of the duplicated copies of the genome into daughter cells each time a cell divides. In many complex organisms, including humans, each centromere is embedded in a stretch of special centromeric chromatin that persists throughout interphase, even though the centromere-mediated attachment to the spindle and movement of DNA occur only during mitosis. This chromatin contains a centromere-specific variant H3 histone, known as CENP-A (Centromere Protein-A; see Figure 4–35), plus additional proteins that pack the nucleosomes into particularly dense arrangements and form the kinetochore, the special structure required for

1	as CENP-A (Centromere Protein-A; see Figure 4–35), plus additional proteins that pack the nucleosomes into particularly dense arrangements and form the kinetochore, the special structure required for attachment of the mitotic spindle (see Figure 4–19).

1	A specific DNA sequence of approximately 125 nucleotide pairs is sufficient to serve as a centromere in the yeast S. cerevisiae. Despite its small size, more than a dozen different proteins assemble on this DNA sequence; the proteins include the CENP-A histone H3 variant, which, along with the three other core histones, forms a centromere-specific nucleosome. The additional proteins at the yeast centromere attach this nucleosome to a single microtubule from the yeast mitotic spindle (Figure 4–42).

1	The centromeres in more complex organisms are considerably larger than those in budding yeasts. For example, fly and human centromeres extend over hundreds of thousands of nucleotide pairs and, while they contain CENP-A, they do not seem to contain a centromere-specific DNA sequence. These centromeres largely consist of short, repeated DNA sequences, known as alpha satellite DNA in humans. But the same repeat sequences are also found at other (non-centromeric) positions on chromosomes, indicating that they are not sufficient to direct centromere formation. Most strikingly, in some unusual cases, new human centromeres (called neocentromeres) have been observed to form spontaneously on fragmented chromosomes. Some of these new positions were originally euchromatic and lack alpha satellite DNA altogether (Figure 4–43). It seems that centromeres in complex organisms are defined by an assembly of proteins, rather than by a specific DNA sequence.

1	Inactivation of some centromeres and genesis of others de novo seem to have played an essential part in evolution. Different species, even when quite closely Figure 4–42 A model for the structure of a simple centromere. (A) In the yeast Saccharomyces cerevisiae, a special centromeric DNA sequence assembles a single nucleosome in which two copies of an H3 variant histone (called CENP-A in most organisms) replace the normal H3. (b) How peptide sequences unique to this variant histone (see Figure 4–35) help to assemble additional proteins, some of which form a kinetochore. The yeast kinetochore is unusual in capturing only a single microtubule; humans have much larger centromeres and form kinetochores that can capture 20 or more microtubules (see Figure 4–43). The kinetochore is discussed in detail in Chapter 17. (Adapted from A. Joglekar et al., Nat. Cell Biol. 8:581–585, 2006. with permission from macmillan Publishers ltd.)

1	Figure 4–43 evidence for the plasticity of human centromere formation. (A) A series of A-T-rich alpha satellite DNA sequences is repeated many thousands of times at each human centromere (red), and is surrounded by pericentric heterochromatin (brown). However, due to an ancient chromosome breakage-and-rejoining event, some human chromosomes contain two blocks of alpha satellite DNA, each of which presumably functioned as a centromere in its original chromosome. Usually, chromosomes with two functional centromeres are not stably propagated because they attach improperly to the spindle and are broken apart during mitosis. In chromosomes that do survive, however, one of the centromeres has somehow become inactivated, even though it contains all the necessary DNA sequences. This allows the chromosome to be stably propagated. (b) In a small fraction (1/2000) of human births, extra chromosomes are observed in cells of the offspring. Some of these extra chromosomes, which have formed from a

1	to be stably propagated. (b) In a small fraction (1/2000) of human births, extra chromosomes are observed in cells of the offspring. Some of these extra chromosomes, which have formed from a breakage event, lack alpha satellite DNA altogether, yet new centromeres (neocentromeres) have arisen from what was originally euchromatic DNA.

1	The complexity of centromeric chromatin is not illustrated in these diagrams. The alpha satellite DNA that forms centromeric chromatin in humans is packaged into alternating blocks of chromatin. One block is formed from a long string of nucleosomes containing the CENP-A H3 variant histone; the other block contains nucleosomes that are specially marked with dimethyl lysine 4 on the normal H3 histone. Each block is more than a thousand nucleosomes long. This centromeric chromatin is flanked by pericentric heterochromatin, as shown. The pericentric chromatin contains methylated lysine 9 on its H3 histones, along with HP1 protein, and it is an example of “classical” heterochromatin (see Figure 4–39).

1	related, often have different numbers of chromosomes; see Figure 4–14 for an extreme example. As we shall discuss below, detailed genome comparisons show that in many cases the changes in chromosome numbers have arisen through chromosome breakage-and-rejoining events, creating novel chromosomes, some of which must initially have contained abnormal numbers of centromeres—either more than one, or none at all. Yet stable inheritance requires that each chromosome should contain one centromere, and one only. It seems that surplus centromeres must have been inactivated, and/or new centromeres created, so as to allow the rearranged chromosome sets to be stably maintained. The changes in centromere activity just discussed, once established, need to be perpetuated through subsequent cell generations. What could be the mechanism of this type of epigenetic inheritance?

1	It has been proposed that de novo centromere formation requires an initial seeding event, involving the formation of a specialized DNA–protein structure that contains nucleosomes formed with the CENP-A variant of histone H3. In humans, this seeding event happens more readily on arrays of alpha satellite DNA than on other DNA sequences. The H3–H4 tetramers from each nucleosome on the parental DNA helix are directly inherited by the sister DNA helices at a replication fork (see Figure 5–32). Therefore, once a set of CENP-A-containing nucleosomes has been assembled on a stretch of DNA, it is easy to understand how a new centromere could be generated in the same place on both daughter chromosomes following each round of cell division. One need only assume that the presence of the CENP-A histone in an inherited nucleosome selectively recruits more CENP-A histone to its newly formed neighbors.

1	There are some striking similarities between the formation and maintenance of centromeres and the formation and maintenance of some other regions of heterochromatin. In particular, the entire centromere forms as an all-or-none entity, suggesting that the creation of centromeric chromatin is a highly cooperative process, spreading out from an initial seed in a manner reminiscent of the phenomenon of position effect variegation that we discussed earlier. In both cases, a particular chromatin structure, once formed, seems to be directly inherited on the DNA following each round of chromosome replication. A cooperative recruitment of proteins, along with the action of reader–writer complexes, can thus not only account for the spreading of specific forms of chromatin in space along the chromosome, but also for its propagation across cell generations—from parent cell to daughter cell (Figure 4–44).

1	Experiments with Frog Embryos Suggest that both Activating and Repressive Chromatin Structures Can be Inherited Epigenetically Epigenetic inheritance plays a central part in the creation of multicellular organisms. Their differentiated cell types become established during development, and persist thereafter even through repeated cell-division cycles. The daughters of a liver cell persist as liver cells, those of an epidermal cell as epidermal cells, and so on, even though they all contain the same genome; and this is because distinctive patterns of gene expression are passed on faithfully from parent cell to daughter cell. Chromatin structure has a role in this epigenetic transmission of information from one cell generation to the next.

1	One type of evidence comes from studies in which the nucleus of a cell from a frog or tadpole is transplanted into a frog egg whose own nucleus has been removed (an enucleated egg). In a classic set of experiments performed in 1968, it was shown that a nucleus taken from a differentiated donor cell can be reprogrammed in this way to support development of a whole new tadpole (see Figure 7–2). But this reprogramming occurs only with difficulty, and it becomes less and less efficient as nuclei from older animals are used. Thus, for example, less than 2% of the enucleated eggs injected with a nucleus from a tadpole epithelial cell developed to the swimming tadpole stage, compared with 35% when the donor nuclei were taken instead from an early (gastrula-stage) embryo. With new experimental tools, the cause of this resistance to reprogramming can now be traced. It arises, at least in part, because specific chromatin structures in the original differentiated nucleus tend to persist and be

1	the cause of this resistance to reprogramming can now be traced. It arises, at least in part, because specific chromatin structures in the original differentiated nucleus tend to persist and be transmitted through the many cell-division cycles required for embryonic development. In experiments with Xenopus embryos, specific forms of either repressive or active chromatin structures could be demonstrated to persist through as many as 24 cell divisions, causing the misplaced expression of genes. Figure 4–45 briefly describes one such experiment,

1	Figure 4–44 How the packaging of DNA in chromatin can be inherited following chromosome replication. In this model, some of the specialized chromatin components are distributed to each sister chromosome after DNA duplication, along with the specially marked nucleosomes that they bind. After DNA replication, the inherited nucleosomes that are specially modified, acting in concert with the inherited chromatin components, change the pattern of histone modification on the newly formed nucleosomes nearby. This creates new binding sites for the same chromatin components, which then assemble to complete the structure. The latter process is likely to involve reader– writer–remodeling complexes operating in a manner similar to that previously illustrated in Figure 4–40.

1	inject normal no injection inject mutant H3.3 mRNA (control) H3.3 mRNA cells analyzed for MyoD expression and for H3.3 histone on MyoD promoter focused on chromatin containing the histone variant, H3.3. We shall return to these phenomena in the final section of Chapter 22, where we discuss stem cells and the ways in which one cell type can be converted into another. Although a great deal remains to be learned about the functions of different chromatin structures, the packaging of DNA into nucleosomes was probably crucial for the evolution of eukaryotes like ourselves. To form a complex multicellular organism, the cells in different lineages must specialize by changing the accessibility and activity of many hundreds of genes. As described in Chapter 21, this process depends on cell memory: each cell holds a record of its past developmental history in the regulatory circuits that control its many genes. That record, it seems, is partly stored in the structure of the chromatin.

1	Although bacteria also have cell memory mechanisms, the complexity of the memory circuits in higher eukaryotes is unparalleled. Strategies based on local variations in chromatin structure, unique to eukaryotes, can enable individual genes, once they are switched on or switched off, to stay in that state until some new factor acts to reverse it. At one extreme are structures like centromeric chromatin that, once established, are stably inherited from one cell generation to the next. Likewise, the major “classical” type of heterochromatin, which contains long arrays of the HP1 protein (see Figure 4–39), can persist stably throughout life. In contrast, a form of condensed chromatin that is created by the Polycomb group of proteins serves to silence genes that must be kept inactive in some conditions, but are active in others. The latter mechanism governs the expression of a large number of genes that encode transcription regulators important in early embryonic development, as discussed

1	but are active in others. The latter mechanism governs the expression of a large number of genes that encode transcription regulators important in early embryonic development, as discussed in Chapter 21. There are many other variant forms of chromatin, some with much shorter lifetimes, often less than the division time of the cell. We shall say more about the variety of chromatin types in the next section.

1	Figure 4–45 evidence for the inheritance of a gene-activating chromatin state.

1	The well-characterized MyoD gene encodes a master transcription regulatory protein for muscle, myoD (see p. 399). This gene is normally turned on in the indicated region of the young embryo where somites form. when a nucleus from this region is injected into an enucleated egg as shown, many of the progeny cell nuclei abnormally express the myoD protein in non-muscle regions of the “nuclear transplant embryo” that forms. This abnormal expression can be attributed to maintenance of the MyoD promoter region in its active chromatin state through the many cycles of cell division that produce the blastula-stage embryo—a so-called “epigenetic memory” that persists in this case in the absence of transcription. The active chromatin surrounding the MyoD promoter contains the variant histone H3.3 (see Figure 4–35) in a lys4 methylated form. As indicated, an overproduction of this histone caused by injecting excess mRNA encoding the normal H3.3 protein increases both H3.3 occupancy on the MyoD

1	(see Figure 4–35) in a lys4 methylated form. As indicated, an overproduction of this histone caused by injecting excess mRNA encoding the normal H3.3 protein increases both H3.3 occupancy on the MyoD promoter and the epigenetic myoD production, whereas injection of an mRNA producing a mutant form of H3.3 that cannot be methylated at lys4 reduces the epigenetic myoD production. Such experiments provide evidence that an inherited chromatin state underlies the epigenetic memory observed. (Adapted from R.k. Ng and J.b. gurdon, Nat. Cell Biol. 10:102–109, 2008. with permission from macmillan Publishers ltd.)

1	In the chromosomes of eukaryotes, DNA is uniformly assembled into nucleosomes, but a variety of different chromatin structures is possible. This variety is based on a large set of reversible covalent modifications of the four histones in the nucleosome core. These modifications include the mono-, di-, and trimethylation of many different lysine side chains, an important reaction that is incompatible with the acetylation that can occur on the same lysines. Specific combinations of the modifications mark many nucleosomes, governing their interactions with other proteins. These marks are read when protein modules that are part of a larger protein complex bind to the modified nucleosomes in a region of chromatin. These reader proteins then attract additional proteins that perform various functions.

1	Some reader protein complexes contain a histone-modifying enzyme, such as a histone lysine methylase, that “writes” the same mark that the reader recognizes. A reader–writer–remodeling complex of this type can spread a specific form of chromatin along a chromosome. In particular, large regions of condensed heterochromatin are thought to be formed in this way. Heterochromatin is commonly found around centromeres and near telomeres, but it is also present at many other positions in chromosomes. The tight packaging of DNA into heterochromatin usually silences the genes within it.

1	The phenomenon of position effect variegation provides strong evidence for the inheritance of condensed states of chromatin from one cell generation to the next. A similar mechanism appears to be responsible for maintaining the specialized chromatin at centromeres. More generally, the ability to propagate specific chromatin structures across cell generations makes possible an epigenetic cell memory process that plays a role in maintaining the set of different cell states required by complex multicellular organisms.

1	Having discussed the DNA and protein molecules from which the chromatin fiber is made, we now turn to the organization of the chromosome on a more global scale and the way in which its various domains are arranged in space. As a 30-nm fiber, a typical human chromosome would still be 0.1 cm in length and able to span the nucleus more than 100 times. Clearly, there must be a still higher level of folding, even in interphase chromosomes. Although the molecular details are still largely a mystery, this higher-order packaging almost certainly involves the folding of the chromatin into a series of loops and coils. This chromatin packing is fluid, frequently changing in response to the needs of the cell.

1	We begin this section by describing some unusual interphase chromosomes that can be easily visualized. Exceptional though they are, these special cases reveal features that are thought to be representative of all interphase chromosomes. Moreover, they provide ways to investigate some fundamental aspects of chromatin structure that we have touched on in the previous section. Next, we describe how a typical interphase chromosome is arranged in the mammalian cell nucleus. Finally, we shall discuss the additional tenfold compaction that chromosomes undergo in the passage from interphase to mitosis. Chromosomes Are Folded into large loops of Chromatin

1	Chromosomes Are Folded into large loops of Chromatin Insight into the structure of the chromosomes in interphase cells has come from studies of the stiff and enormously extended chromosomes in growing amphibian oocytes (immature eggs). These very unusual lampbrush chromosomes (the largest chromosomes known), paired in preparation for meiosis, are clearly visible even in the light microscope, where they are seen to be organized into a series of large chromatin loops emanating from a linear chromosomal axis (Figure 4–46 and Figure 4–47). In these chromosomes, a given loop always contains the same DNA sequence that remains extended in the same manner as the oocyte grows. These chromosomes are producing large amounts of RNA for the oocyte, and most of the genes Figure 4–46 A model for the chromatin domains in a lampbrush chromosome.

1	Figure 4–46 A model for the chromatin domains in a lampbrush chromosome. Shown is a small portion of one pair of sister chromatids. Here, two identical DNA double helices are aligned side by side, packaged into different types of chromatin. The set of lampbrush chromosomes in many amphibians contains a total of about 10,000 loops resembling those shown here. The rest of the DNA in each chromosome (the great majority) remains highly condensed. Four copies of each loop are present in the cell, since each lampbrush chromosome consists of two aligned sets of paired chromatids. This four-stranded structure is characteristic of this stage of development of the oocyte, which has arrested at the diplotene stage of meiosis; see Figure 17–56. present in the DNA loops are being actively expressed. The majority of the DNA, however, is not in loops but remains highly condensed on the chromosome axis, where genes are generally not expressed.

1	It is thought that the interphase chromosomes of all eukaryotes are similarly arranged in loops. Although these loops are normally too small and fragile to be easily observed in a light microscope, other methods can be used to infer their presence. For example, modern DNA technologies have made it possible to assess the frequency with which any two loci along an interphase chromosome are held together, thus revealing likely candidates for the sites on chromatin that form the bases of loop structures (Figure 4–48). These experiments and others suggest that the DNA in human chromosomes is likely to be organized into loops of various lengths. A typical loop might contain between 50,000 and 200,000 nucleotide pairs of DNA, although loops of a million nucleotide pairs have also been suggested (Figure 4–49).

1	Further insight has come from another unusual class of cells—the polytene cells of flies, such as the fruit fly Drosophila. Some types of cells, in many organisms, grow abnormally large through multiple cycles of DNA synthesis without cell division. Such cells, containing increased numbers of standard chromosomes, are said to be polyploid. In the salivary glands of fly larvae, this process is taken to an extreme degree, creating huge cells that contain hundreds or thousands of copies of the Figure 4–47 lampbrush chromosomes.

1	(A) A light micrograph of lampbrush chromosomes in an amphibian oocyte. Early in oocyte differentiation, each chromosome replicates to begin meiosis, and the homologous replicated chromosomes pair to form this highly extended structure containing a total of four replicated DNA double helices, or chromatids. The lampbrush chromosome stage persists for months or years, while the oocyte builds up a supply of materials required for its ultimate development into a new individual. (b) An enlarged region of a similar chromosome, stained with a fluorescent reagent that makes the loops active in RNA synthesis clearly visible. (Courtesy of Joseph g. gall.) genome. Moreover, in this case, all the copies of each chromosome are aligned side by side in exact register, like drinking straws in a box, to create giant polytene chromosomes. These allow features to be detected that are thought to be shared with ordinary interphase chromosomes, but are normally hard to see.

1	When polytene chromosomes from a fly’s salivary glands are viewed in the light microscope, distinct alternating dark bands and light interbands are visible (Figure 4–50), each formed from a thousand identical DNA sequences arranged side by side in register. About 95% of the DNA in polytene chromosomes is in bands, and 5% is in interbands. A very thin band can contain 3000 nucleotide pairs, while a thick band may contain 200,000 nucleotide pairs in each of its chromatin strands. The chromatin in each band appears dark because the DNA is more condensed than the DNA in interbands; it may also contain a higher concentration of proteins (Figure 4–51). This banding pattern seems to reflect the same sort of organization detected in the amphibian lampbrush chromosomes described earlier.

1	There are approximately 3700 bands and 3700 interbands in the complete set of Drosophila polytene chromosomes. The bands can be recognized by their different thicknesses and spacings, and each one has been given a number to generate a chromosome “map” that has been indexed to the finished genome sequence of this fly. The Drosophila polytene chromosomes provide a good starting point for examining how chromatin is organized on a large scale. In the previous section, we saw that there are many forms of chromatin, each of which contains nucleosomes with a different combination of modified histones. Specific sets of non-histone proteins assemble on these nucleosomes to affect biological function in different ways. Recruitment of some of these non-histone proteins can spread for long distances along the DNA, imparting a similar chromatin structure to broad tracts DNA product is obtained only if proteins hold the two DNA sequences close together in the cell

1	Figure 4–48 A method for determining the position of loops in interphase chromosomes. In this technique, known as the chromosome conformation capture (3C) method, cells are treated with formaldehyde to create the indicated covalent DNA–protein and DNA–DNA cross-links. The DNA is then treated with an enzyme (a restriction nuclease) that chops the DNA into many pieces, cutting at strictly defined nucleotide sequences and forming sets of identical “cohesive ends” (see Figure 8–28). The cohesive ends can be made to join through their complementary base-pairing. Importantly, prior to the ligation step shown, the DNA is diluted so that the fragments that have been kept in close proximity to each other (through cross-linking) are the ones most likely to join. Finally, the cross-links are reversed and the newly ligated fragments of DNA are identified and quantified by PCR (the polymerase chain reaction, described in Chapter 8). From the results, combined with DNA sequence information, one can

1	and the newly ligated fragments of DNA are identified and quantified by PCR (the polymerase chain reaction, described in Chapter 8). From the results, combined with DNA sequence information, one can derive models for the interphase conformation of chromosomes.

1	Figure 4–49 A model for the organization of an interphase chromosome. A section of an interphase chromosome is shown folded into a series of looped domains, each containing perhaps 50,000–200,000 or more nucleotide pairs of double-helical DNA condensed into a chromatin fiber. The chromatin in each individual loop is further condensed through poorly understood folding processes that are reversed when the cell requires direct access to the DNA packaged in the loop. Neither the composition of the postulated chromosomal axis nor how the folded chromatin fiber is anchored to it is clear. However, in mitotic chromosomes, the bases of the chromosomal loops are enriched both in condensins (discussed below) and in DNA topoisomerase II enzymes (discussed in Chapter 5), two proteins that may form much of the axis at metaphase.

1	normal mitotic right arm of chromosomes at chromosome 2 same scale region where two homologous chromosomes are separated left arm of chromosome 2 left arm of chromocenter chromosome 3 20 µm right arm of chromosome 3 of the genome (see Figure 4–40). Such regions, where all of the chromatin has a similar structure, are separated from neighboring domains by barrier proteins (see Figure 4–41). At low resolution, the interphase chromosome can therefore be considered as a mosaic of chromatin structures, each containing particular nucleosome modifications associated with a particular set of non-histone proteins. Polytene chromosomes allow us to see details of this mosaic of domains in the light microscope, as well as to observe some of the changes associated with gene expression. There Are multiple Forms of Chromatin

1	There Are multiple Forms of Chromatin By staining Drosophila polytene chromosomes with antibodies, or by using a more recent technique called ChIP (chromatin immunoprecipitation) analysis (see Chapter 8), the locations of the histone and non-histone proteins in chromatin can be mapped across the entire DNA sequence of an organism’s genome. Such an analysis in Drosophila has thus far localized more than 50 different chromatin proteins and histone modifications. The results suggest that three major types of repressive chromatin predominate in this organism, along with two major types of chromatin on actively transcribed genes, and that each type is associated with a different complex of non-histone proteins. Thus, classical heterochromatin contains more than six such proteins, including heterochromatin protein 1 (HP1),

1	Figure 4–50 The entire set of polytene chromosomes in one Drosophila salivary cell. In this drawing of a light micrograph, the giant chromosomes have been spread out for viewing by squashing them against a microscope slide. Drosophila has four chromosomes, and there are four different chromosome pairs present. but each chromosome is tightly paired with its homolog (so that each pair appears as a single structure), which is not true in most nuclei (except in meiosis). Each chromosome has undergone multiple rounds of replication, and the homologs and all their duplicates have remained in exact register with each other, resulting in huge chromatin cables many DNA strands thick.

1	The four polytene chromosomes are normally linked together by heterochromatic regions near their centromeres that aggregate to create a single large chromocenter (pink region). In this preparation, however, the chromocenter has been split into two halves by the squashing procedure used. (Adapted from T.S. Painter, J. Hered. 25:465–476, 1934. with permission from Oxford University Press.) Figure 4–51 Micrographs of polytene chromosomes from Drosophila salivary glands. (A) light micrograph of a portion of a chromosome. The DNA has been stained with a fluorescent dye, but a reverse image is presented here that renders the DNA black rather than white; the bands are clearly seen to be regions of increased DNA concentration. This chromosome has been processed by a high-pressure treatment so as to show its distinct pattern of bands and interbands more clearly.

1	(b) An electron micrograph of a small section of a Drosophila polytene chromosome seen in thin section. bands of very different thickness can be readily distinguished, separated by interbands, which contain less condensed chromatin. (A, adapted from D.v. Novikov, I. kireev and A.S. belmont, Nat. Methods 4:483– 485, 2007. with permission from macmillan Publishers ltd; b, courtesy of veikko Sorsa.) Figure 4–52 RNA synthesis in polytene chromosome puffs.

1	Figure 4–52 RNA synthesis in polytene chromosome puffs. An autoradiograph of a single puff in a polytene chromosome from the salivary glands of the freshwater midge Chironomus tentans. As outlined in Chapter 1 and described in detail in Chapter 6, the first step in gene expression is the synthesis of an RNA molecule using the DNA as a template. The decondensed portion of the chromosome is undergoing RNA synthesis and has become labeled with 3H-uridine, an RNA precursor molecule that is incorporated into growing RNA chains. (Courtesy of José bonner.) whereas the so-called Polycomb form of heterochromatin contains a similar number of proteins of a different set (PcG proteins). In addition to the five major chromatin types, other more minor forms of chromatin appear to be present, each of which may be differently regulated and have distinct roles in the cell.

1	The set of proteins bound as part of the chromatin at a given locus varies depending on the cell type and its stage of development. These variations make the accessibility of specific genes different in different tissues, helping to generate the cell diversification that accompanies embryonic development (described in Chapter 21). Chromatin loops Decondense when the genes within Them Are Expressed When an insect progresses from one developmental stage to another, distinctive chromosome puffs arise and old puffs recede in its polytene chromosomes as new genes become expressed and old ones are turned off (Figure 4–52). From inspection of each puff when it is relatively small and the banding pattern is still discernible, it seems that most puffs arise from the decondensation of a single chromosome band.

1	The individual chromatin fibers that make up a puff can be visualized with an electron microscope. In favorable cases, loops are seen, much like those observed in amphibian lampbrush chromosomes. When genes in the loop are not expressed, the loop assumes a thickened structure, possibly that of a folded 30-nm fiber, but when gene expression is occurring, the loop becomes more extended. In electron micrographs, the chromatin located on either side of the decondensed loop appears considerably more compact, suggesting that a loop constitutes a distinct functional domain of chromatin structure.

1	Observations in human cells also suggest that highly folded loops of chromatin expand to occupy an increased volume when a gene within them is expressed. For example, quiescent chromosome regions from 0.4 to 2 million nucleotide pairs in length appear as compact dots in an interphase nucleus when visualized by fluorescence microscopy. However, the same DNA is seen to occupy a larger territory when its genes are expressed, with elongated, punctate structures replacing the original dot. New ways of visualizing individual chromosomes have shown that each of the 46 interphase chromosomes in a human cell tends to occupy its own discrete ter- ritory within the nucleus: that is, the chromosomes are not extensively entangled with one another (Figure 4–53). However, pictures such as these present only an average view of the DNA in each chromosome. Experiments that specifically localize the heterochromatic regions of a chromosome reveal that they are often

1	Figure 4–53 Simultaneous visualization of the chromosome territories for all of the human chromosomes in a single interphase nucleus. Here, a mixture of DNA probes for each chromosome has been labeled so as to fluoresce with a different spectra; this allows DNA–DNA hybridization to be used to detect each chromosome, as in Figure 4–10. Three-dimensional reconstructions were then produced. below the micrograph, each chromosome is identified in a schematic of the actual image. Note that homologous chromosomes (e.g., the two copies of chromosome 9) are not in general co-located. (From m.R. Speicher and N.P. Carter, Nat. Rev. Genet. 6:782–792, 2005. with permission from macmillan Publishers ltd.)

1	Figure 4–54 The distribution of gene-rich regions of the human genome in an interphase nucleus. gene-rich regions have been visualized with a fluorescent probe that hybridizes to the Alu interspersed repeat, which is present in more than a million copies in the human genome (see page 292). For unknown reasons, these sequences cluster in chromosomal regions rich in genes. In this representation, regions enriched for the Alu sequence are green, regions depleted for these sequences are red, while the average regions are yellow. The gene-rich regions are seen to be largely absent in the DNA near the nuclear envelope. (From A. bolzer et al., PLoS Biol. 3:826–842, 2005.) closely associated with the nuclear lamina, regardless of the chromosome examined. And DNA probes that preferentially stain gene-rich regions of human chromosomes produce a striking picture of the interphase nucleus that presumably reflects different average positions for active and inactive genes (Figure 4–54).

1	How is most of the chromatin in each interphase chromosome condensed when its genes are not being expressed? A powerful extension of the chromosome conformation capture method described previously (see Figure 4–48), which exploits a high-throughput DNA sequencing technology called massive parallel sequencing (see Panel 8–1, pp. 478–481), allows the connections between all of the different one-megabase (1 Mb) segments of the human genome to be mapped in human interphase chromosomes. The results reveal that most regions of our chromosomes are folded into a conformation referred to as a fractal globule: a knot-free arrangement that facilitates maximally dense packing while, at the same time, preserving the ability of the chromatin fiber to unfold and fold (Figure 4–55). Chromatin Can move to Specific Sites within the Nucleus to Alter gene Expression

1	Chromatin Can move to Specific Sites within the Nucleus to Alter gene Expression A variety of different types of experiments has led to the conclusion that the position of a gene in the interior of the nucleus changes when it becomes highly expressed. Thus, a region that becomes very actively transcribed is sometimes found to extend out of its chromosome territory, as if in an extended loop (Figure 4–56). We will see in Chapter 6 that the initiation of transcription—the first step in gene expression—requires the assembly of over 100 proteins, and it makes sense that this would be facilitated in regions of the nucleus enriched in these proteins. More generally, it is clear that the nucleus is very heterogeneous, with functionally different regions to which portions of chromosomes can move as they are subjected to different biochemical processes—such as when their gene expression changes. It is this issue that we discuss next.

1	Figure 4–55 A fractal globule model for interphase chromatin. An extension of the 3C method in Figure 4–48, called Hi-C, was used to measure the extent to which each of the three thousand 1 mb segments in the human genome was located adjacent to any other of these segments. The results support the type of model shown. In the enlarged fractal globule illustrated, a region of 5 million base pairs is seen to fold in a way that keeps regions that are neighbors along the one-dimensional DNA helix as neighbors in three dimensions; this gives rise to monochromatic blocks in this representation that are obvious both on the surface and in cross section. The fractal globule is a knot-free conformation of the DNA that permits dense packing, yet retains an ability to easily fold and unfold any genomic locus. (Adapted from E. lieberman-Aiden et al., Science 326:289–293, 2009. with permission from AAAS.)

1	E. lieberman-Aiden et al., Science 326:289–293, 2009. with permission from AAAS.) Figure 4–56 An effect of high levels of gene expression on the intranuclear location of chromatin. (A) Fluorescence micrographs of human nuclei showing how the position of a gene changes when it becomes highly transcribed. The region of the chromosome adjacent to the gene (red) is seen to leave its chromosomal territory (green) only when it is highly active. (b) Schematic representation of a large loop of chromatin that expands when the gene is on, and contracts when the gene is off. Other genes that are less actively expressed can be shown by the same methods to remain inside their chromosomal territory when transcribed. (From J.R. Chubb and w.A. bickmore, Cell 112:403–406, 2003. with permission from Elsevier.) Networks of macromolecules Form a Set of Distinct biochemical Environments inside the Nucleus

1	Networks of macromolecules Form a Set of Distinct biochemical Environments inside the Nucleus In Chapter 6, we shall describe the function of a variety of subcompartments that are present within the nucleus. The largest and most obvious of these is the nucleolus, a structure well known to microscopists even in the nineteenth century (see Figure 4–9). The nucleolus is the cell’s site of ribosome subunit formation, as well as the place where many other specialized reactions occur (see Figure 6–42): it consists of a network of RNAs and proteins concentrated around ribosomal RNA genes that are being actively transcribed. In eukaryotes, the genome contains multiple copies of the ribosomal RNA genes, and although they are typically clustered together in a single nucleolus, they are often located on several separate chromosomes.

1	A variety of less obvious organelles are also present inside the nucleus. For example, spherical structures called Cajal bodies and interchromatin granule clusters are present in most plant and animal cells (Figure 4–57). Like the nucleolus, these organelles are composed of selected protein and RNA molecules that bind together to create networks that are highly permeable to other protein and RNA molecules in the surrounding nucleoplasm.

1	Structures such as these can create distinct biochemical environments by immobilizing select groups of macromolecules, as can other networks of proteins and RNA molecules associated with nuclear pores and with the nuclear envelope. In principle, this allows other molecules that enter these spaces to be processed with great efficiency through complex reaction pathways. Highly permeable, 1 µm fibrous networks of this sort can thereby impart many of the kinetic advantages of compartmentalization (see p. 164) to reactions that take place in subregions of the nucleus (Figure 4–58A). However, unlike the membrane-bound compartments in nuclear subcompartments. The large the cytoplasm (discussed in Chapter 12), these nuclear subcompartments—lack sphere here is a Cajal body. The smaller ing a lipid bilayer membrane—can neither concentrate nor exclude specific small darker sphere is an interchromatin granule molecules. cluster, also known as a speckle (see also Figure 6–46). These “subnuclear

1	The cell has a remarkable ability to construct distinct environments to per- organelles” are from the nucleus of a form complex biochemical tasks efficiently. Those that we have mentioned in the Xenopus oocyte. (From k.E. Handwerger nucleus facilitate various aspects of gene expression, and will be further discussed and J.g. gall, Trends Cell Biol. 16:19–26, in Chapter 6. These subcompartments, including the nucleolus, appear to form 2006. with permission from Elsevier.) only as needed, and they create a high local concentration of the many different enzymes and RNA molecules needed for a particular process. In an analogous way, when DNA is damaged by irradiation, the set of enzymes needed to carry out DNA repair are observed to congregate in discrete foci inside the nucleus, creating “repair factories” (see Figure 5–52). And nuclei often contain hundreds of discrete foci representing factories for DNA or RNA synthesis (see Figure 6–47).

1	It seems likely that all of these entities make use of the type of tethering illustrated in Figure 4–58B, where long flexible lengths of polypeptide chain and/or long noncoding RNA molecules are interspersed with specific binding sites that concentrate the multiple proteins and other molecules that are needed to catalyze a particular process. Not surprisingly, tethers are similarly used to help to speed biological processes in the cytoplasm, increasing specific reaction rates there (for example, see Figure 16–18).

1	Is there also an intranuclear framework, analogous to the cytoskeleton, on which chromosomes and other components of the nucleus are organized? The nuclear matrix, or scaffold, has been defined as the insoluble material left in the nucleus after a series of biochemical extraction steps. Many of the proteins and RNA molecules that form this insoluble material are likely to be derived from the fibrous subcompartments of the nucleus just discussed, while others may be proteins that help to form the base of chromosomal loops or to attach chromosomes to other structures in the nucleus.

1	Having discussed the dynamic structure of interphase chromosomes, we now turn to mitotic chromosomes. The chromosomes from nearly all eukaryotic cells become readily visible by light microscopy during mitosis, when they coil up to form highly condensed structures. This condensation reduces the length of a typical interphase chromosome only about tenfold, but it produces a dramatic change in chromosome appearance. Figure 4–59 depicts a typical mitotic chromosome at the metaphase stage of mitosis (for the stages of mitosis, see Figure 17–3). The two DNA molecules produced by DNA replication during interphase of the cell-division cycle are separately folded to produce two sister chromosomes, or sister chromatids, held together at their centromeres, as mentioned earlier. These chromosomes are normally covered with a variety of molecules, including large amounts of RNA–protein

1	Figure 4–58 effective compartmentalization without a bilayer membrane. (A) Schematic illustration of the organization of a spherical subnuclear organelle (left) and of a postulated similarly organized subcompartment just beneath the nuclear envelope (right). In both cases, RNAs and/or proteins (gray) associate to form highly porous, gel-like structures that contain binding sites for other specific proteins and RNA molecules (colored objects). (b) How the tethering of a selected set of proteins and RNA molecules to long flexible polymer chains, as in (A), can create “staging areas” that greatly speed the rates of reactions in subcompartments of the nucleus. The reactions catalyzed will depend on the particular macromolecules that are localized by the tethering. The same strategy for accelerating complex sets of reactions is also employed in subcompartments elsewhere in the cell (see also Figure 3–78).

1	Figure 4–59 A typical mitotic chromosome at metaphase. Each sister chromatid contains one of two identical sister DNA molecules generated earlier in the cell cycle by DNA replication (see also Figure 17–21).

1	Figure 4–60 A scanning electron micrograph of a region near one end of a typical mitotic chromosome. Each knoblike projection is believed to represent the tip of a separate looped domain. Note that the two identical paired chromatids (drawn in Figure 4–59) can be clearly distinguished. (From m.P. marsden and U.k. laemmli, Cell 17:849–858, 1979. with permission from Elsevier.) complexes. Once this covering has been stripped away, each chromatid can be seen in electron micrographs to be organized into loops of chromatin emanating from a central scaffolding (Figure 4–60). Experiments using DNA hybridization to detect specific DNA sequences demonstrate that the order of visible features along a mitotic chromosome at least roughly reflects the order of genes along the DNA molecule. Mitotic chromosome condensation can thus be thought of as the final level in the hierarchy of chromosome packaging (Figure 4–61).

1	The compaction of chromosomes during mitosis is a highly organized and dynamic process that serves at least two important purposes. First, when condensation is complete (in metaphase), sister chromatids have been disentangled from each other and lie side by side. Thus, the sister chromatids can easily separate when the mitotic apparatus begins pulling them apart. Second, the compaction of chromosomes protects the relatively fragile DNA molecules from being broken as they are pulled to separate daughter cells.

1	The condensation of interphase chromosomes into mitotic chromosomes begins in early M phase, and it is intimately connected with the progression of the cell cycle. During M phase, gene expression shuts down, and specific modifications are made to histones that help to reorganize the chromatin as it compacts. Two classes of ring-shaped proteins, called cohesins and condensins, aid this compaction. How they help to produce the two separately folded chromatids of a mitotic chromosome will be discussed in Chapter 17, along with the details of the cell cycle. short region of 11 nm form of chromatin “beads-on-a-string” chromatin fber of packed 30 nm NET RESULT: EACH DNA MOLECULE HAS BEEN PACKAGED INTO A MITOTIC CHROMOSOME THAT IS 10,000-FOLD SHORTER THAN ITS FULLY EXTENDED LENGTH 0.1 µm Figure 4–61 Chromatin packing. This model shows some of the many levels of chromatin packing postulated to give rise to the highly condensed mitotic chromosome.

1	Figure 4–61 Chromatin packing. This model shows some of the many levels of chromatin packing postulated to give rise to the highly condensed mitotic chromosome. Chromosomes are generally decondensed during interphase, so that the details of their structure are difficult to visualize. Notable exceptions are the specialized lampbrush chromosomes of vertebrate oocytes and the polytene chromosomes in the giant secretory cells of insects. Studies of these two types of interphase chromosomes suggest that each long DNA molecule in a chromosome is divided into a large number of discrete domains organized as loops of chromatin that are compacted by further folding. When genes contained in a loop are expressed, the loop unfolds and allows the cell’s machinery access to the DNA.

1	Interphase chromosomes occupy discrete territories in the cell nucleus; that is, they are not extensively intertwined. Euchromatin makes up most of interphase chromosomes and, when not being transcribed, it probably exists as tightly folded fibers of compacted nucleosomes. However, euchromatin is interrupted by stretches of heterochromatin, in which the nucleosomes are subjected to additional packing that usually renders the DNA resistant to gene expression. Heterochromatin exists in several forms, some of which are found in large blocks in and around centromeres and near telomeres. But heterochromatin is also present at many other positions on chromosomes, where it can serve to help regulate developmentally important genes.

1	The interior of the nucleus is highly dynamic, with heterochromatin often positioned near the nuclear envelope and loops of chromatin moving away from their chromosome territory when genes are very highly expressed. This reflects the existence of nuclear subcompartments, where different sets of biochemical reactions are facilitated by an increased concentration of selected proteins and RNAs. The components involved in forming a subcompartment can self-assemble into discrete organelles such as nucleoli or Cajal bodies; they can also be tethered to fixed structures such as the nuclear envelope.

1	During mitosis, gene expression shuts down and all chromosomes adopt a highly condensed conformation in a process that begins early in M phase to package the two DNA molecules of each replicated chromosome as two separately folded chromatids. The condensation is accompanied by histone modifications that facilitate chromatin packing, but satisfactory completion of this orderly process, which reduces the end-to-end distance of each DNA molecule from its interphase length by an additional factor of ten, requires additional proteins.

1	In this final section of the chapter, we provide an overview of some of the ways that genes and genomes have evolved over time to produce the vast diversity of modern-day life-forms on our planet. The sequencing of the genomes of thousands of organisms is revolutionizing our view of the process of evolution, uncovering an astonishing wealth of information about not only family relationships among organisms, but also about the molecular mechanisms by which evolution has proceeded.

1	It is perhaps not surprising that genes with similar functions can be found in a diverse range of living things. But the great revelation of the past 30 years has been the extent to which the actual nucleotide sequences of many genes have been conserved. Homologous genes—that is, genes that are similar in both their nucleotide sequence and function because of a common ancestry—can often be recognized across vast phylogenetic distances. Unmistakable homologs of many human genes are present in organisms as diverse as nematode worms, fruit flies, yeasts, and even bacteria. In many cases, the resemblance is so close that, for example, the protein-coding portion of a yeast gene can be substituted with its human homolog—even though humans and yeast are separated by more than a billion years of evolutionary history.

1	As emphasized in Chapter 3, the recognition of sequence similarity has become a major tool for inferring gene and protein function. Although a sequence match does not guarantee similarity in function, it has proved to be an excellent clue. Thus, it is often possible to predict the function of genes in humans for which no biochemical or genetic information is available simply by comparing their nucleotide sequences with the sequences of genes that have been characterized in other more readily studied organisms. In general, the sequences of individual genes are much more tightly conserved than is overall genome structure. Features of genome organization such as genome size, number of chromosomes, order of genes along chromosomes, abundance and size of introns, and amount of repetitive DNA are found to differ greatly when comparing distant organisms, as does the number of genes that each organism contains.

1	A first obstacle in interpreting the sequence of the 3.2 billion nucleotide pairs in the human genome is the fact that much of it is probably functionally unimportant. The regions of the genome that code for the amino acid sequences of proteins (the exons) are typically found in short segments (average size about 145 nucleotide pairs), small islands in a sea of DNA whose exact nucleotide sequence is thought to be mostly of little consequence. This arrangement makes it difficult to identify all the exons in a stretch of DNA, and it is often hard too to determine exactly where a gene begins and ends.

1	One very important approach to deciphering our genome is to search for DNA sequences that are closely similar between different species, on the principle that DNA sequences that have a function are much more likely to be conserved than those without a function. For example, humans and mice are thought to have diverged from a common mammalian ancestor about 80 × 106 years ago, which is long enough for the majority of nucleotides in their genomes to have been changed by random mutational events. Consequently, the only regions that will have remained closely similar in the two genomes are those in which mutations would have impaired function and put the animals carrying them at a disadvantage, resulting in their elimination from the population by natural selection. Such closely similar pieces of DNA sequence are known as conserved regions. In addition to revealing those DNA sequences that encode functionally important exons and RNA molecules, these conserved regions will include

1	pieces of DNA sequence are known as conserved regions. In addition to revealing those DNA sequences that encode functionally important exons and RNA molecules, these conserved regions will include regulatory DNA sequences as well as DNA sequences with functions that are not yet known. In contrast, most nonconserved regions will reflect DNA whose sequence is much less likely to be critical for function.

1	The power of this method can be increased by including in such comparisons the genomes of large numbers of species whose genomes have been sequenced, such as rat, chicken, fish, dog, and chimpanzee, as well as mouse and human. By revealing in this way the results of a very long natural “experiment,” lasting for hundreds of millions of years, such comparative DNA sequencing studies have highlighted the most interesting regions in our genome. The comparisons reveal that roughly 5% of the human genome consists of “multispecies conserved sequences.” To our great surprise, only about one-third of these sequences code for proteins (see Table 4–1, p. 184). Many of the remaining conserved sequences consist of DNA containing clusters of protein-binding sites that are involved in gene regulation, while others produce RNA molecules that are not translated into protein but are important for other known purposes. But, even in the most intensively studied species, the function of the majority of

1	while others produce RNA molecules that are not translated into protein but are important for other known purposes. But, even in the most intensively studied species, the function of the majority of these highly conserved sequences remains unknown. This remarkable discovery has led scientists to conclude that we understand much less about the cell biology of vertebrates than we had thought. Certainly, there are enormous opportunities for new discoveries, and we should expect many more surprises ahead.

1	genome Alterations Are Caused by Failures of the Normal mechanisms for Copying and maintaining DNA, as well as by Transposable DNA Elements

1	Evolution depends on accidents and mistakes followed by nonrandom survival. Most of the genetic changes that occur result simply from failures in the normal mechanisms by which genomes are copied or repaired when damaged, although the movement of transposable DNA elements (discussed below) also plays an important part. As we will explain in Chapter 5, the mechanisms that maintain DNA sequences are remarkably precise—but they are not perfect. DNA sequences are inherited with such extraordinary fidelity that typically, along a given line of descent, only about one nucleotide pair in a thousand is randomly changed in the germ line every million years. Even so, in a population of 10,000 diploid individuals, every possible nucleotide substitution will have been “tried out” on about 20 occasions in the course of a million years—a short span of time in relation to the evolution of species.

1	Errors in DNA replication, DNA recombination, or DNA repair can lead either to simple local changes in DNA sequence—so-called point mutations such as the substitution of one base pair for another—or to large-scale genome rearrangements such as deletions, duplications, inversions, and translocations of DNA from one chromosome to another. In addition to these failures of the genetic machinery, genomes contain mobile DNA elements that are an important source of genomic change (see Table 5–3, p. 267). These transposable DNA elements (transposons) are parasitic DNA sequences that can spread within the genomes they colonize. In the process, they often disrupt the function or alter the regulation of existing genes. On occasion, they have created altogether novel genes through fusions between transposon sequences and segments of existing genes. Over long periods of evolutionary time, DNA transposition events have profoundly affected genomes, so much so that nearly half of the DNA in the human

1	sequences and segments of existing genes. Over long periods of evolutionary time, DNA transposition events have profoundly affected genomes, so much so that nearly half of the DNA in the human genome consists of recognizable relics of past transposition events (Figure 4–62). Even more of our genome is thought to have been derived from transpositions that occurred so long ago (>108 years) that the sequences can no longer be traced to transposons.

1	The genome Sequences of Two Species Differ in Proportion to the length of Time Since They Have Separately Evolved The differences between the genomes of species alive today have accumulated over more than 3 billion years. Although we lack a direct record of changes over time, scientists can reconstruct the process of genome evolution from detailed comparisons of the genomes of contemporary organisms. The basic organizing framework for comparative genomics is the phylogenetic tree. A simple example is the tree describing the divergence of humans from the great apes (Figure 4–63). The primary support for this tree comes from comparisons of gene or protein sequences. For example, comparisons between the sequences of human genes or proteins and those of the great apes typically reveal the fewest differences between human and chimpanzee and the most between human and orangutan.

1	For closely related organisms such as humans and chimpanzees, it is relatively easy to reconstruct the gene sequences of the extinct, last common ancestor of the two species (Figure 4–64). The close similarity between human and chimpanzee genes is mainly due to the short time that has been available for the accumulation of mutations in the two diverging lineages, rather than to functional constraints TRANSPOSONS nonrepetitive DNA that is

1	Figure 4–62 A representation of the nucleotide sequence content of the sequenced human genome. The lINEs (long interspersed nuclear elements), SINEs (short interspersed nuclear elements), retroviral-like elements, and DNA-only transposons are mobile genetic elements that have multiplied in our genome by replicating themselves and inserting the new copies in different positions. These mobile genetic elements are discussed in Chapter 5 (see Table 5–3, p. 267). Simple sequence repeats are short nucleotide sequences (less than 14 nucleotide pairs) that are repeated again and again for long stretches. Segmental duplications are large blocks of DNA sequence (1000–200,000 nucleotide pairs) that are present at two or more locations in the genome. The most highly repeated blocks of DNA in heterochromatin have not yet been completely sequenced; therefore about 10% of human DNA sequences are not represented in this diagram. (Data courtesy of E. margulies.) showing the relationship between 1.5

1	have not yet been completely sequenced; therefore about 10% of human DNA sequences are not represented in this diagram. (Data courtesy of E. margulies.) showing the relationship between 1.5 humans and the great apes based on nucleotide sequence data. As indicated, the sequences of the genomes of all four millions of years before present species are estimated to differ from the sequence of the genome of their last common ancestor by a little over 1.5%. because changes occur independently on both diverging lineages, pairwise comparisons reveal twice the sequence divergence from the last common ancestor. For example, human–orangutan comparisons typically show sequence 1.0 0.5 0.0 divergences of a little over 3%, while that have kept the sequences the same. Evidence for this view comes from the observation that the human and chimpanzee genomes are nearly identical even where there is no functional constraint on the nucleotide sequence—such as in the third position of “synonymous” codons

1	the observation that the human and chimpanzee genomes are nearly identical even where there is no functional constraint on the nucleotide sequence—such as in the third position of “synonymous” codons (codons specifying the same amino acid but differing in their third nucleotide).

1	For much less closely related organisms, such as humans and chickens (which have evolved separately for about 300 million years), the sequence conservation found in genes is almost entirely due to purifying selection (that is, selection that eliminates individuals carrying mutations that interfere with important genetic functions), rather than to an inadequate time for mutations to occur. Phylogenetic Trees Constructed from a Comparison of DNA Sequences Trace the Relationships of All Organisms Phylogenetic trees based on molecular sequence data can be compared with the fossil record, and we get our best view of evolution by integrating the two approaches. The fossil record remains essential as a source of absolute dates, human–chimpanzee comparisons show divergences of approximately 1.2%. (modified from F.C. Chen and w.H. li, Am. J. Hum. Genet. 68:444–456, 2001.)

1	Figure 4–64 Tracing the ancestral sequence from a sequence comparison of the coding regions of human and chimpanzee leptin genes. Reading left to right and top to bottom, a continuous gene is illustrated. leptin is a hormone that regulates food intake and energy utilization in response to the adequacy of 300-nucleotide segment of a leptin-coding fat reserves. As indicated by the codons boxed in green, only 5 nucleotides (of 120 441 total) differ between the two species. moreover, in only one of the five positions does the difference in nucleotide lead to a difference in the encoded amino acid. For each of the five variant nucleotide positions, the corresponding sequence in the gorilla is also indicated. In two cases, sequence, while in three cases it agrees with the chimpanzee sequence.

1	the gorilla sequence agrees with the human what was the sequence of the leptin gene in the last common ancestor? The most economical assumption is that evolution minimum number of mutations consistent with the data. Thus, it seems likely that chimp TACCAACAGATCCTCACCAGTATGCCTTCCAGAAACATGATCCAAATATCCAACGACCTG protein Y Q Q I L T S M P S R N M I Q I S N D L the leptin sequence of the last common has followed a pathway requiring the ancestor was the same as the human and chimpanzee sequences when they agree; when they disagree, the gorilla sequence would be used as a tiebreaker. For convenience, only the first 300 nucleotides of the leptin-coding sequences are given. The remaining 141 are identical between humans and chimpanzees.

1	Figure 4–65 The very different rates of evolution of exons and introns, as illustrated by comparing a portion of the mouse and human leptin genes. Positions where the sequences differ by a single nucleotide substitution are boxed in green, and positions that differ by the addition or deletion of nucleotides are boxed in yellow. Note that, thanks to purifying selection, the coding sequence of the exon is much more conserved than is the adjacent intron sequence. based on radioisotope decay in the rock formations in which fossils are found. Because the fossil record has many gaps, however, precise divergence times between species are difficult to establish, even for species that leave good fossils with distinctive morphology.

1	Phylogenetic trees whose timing has been calibrated according to the fossil record suggest that changes in the sequences of particular genes or proteins tend to occur at a nearly constant rate, although rates that differ from the norm by as much as twofold are observed in particular lineages. This provides us with a molecular clock for evolution—or rather a set of molecular clocks corresponding to different categories of DNA sequence. As in the example in Figure 4–65, the clock runs most rapidly and regularly in sequences that are not subject to purifying selection. These include portions of introns that lack splicing or regulatory signals, the third position in synonymous codons, and genes that have been irreversibly inactivated by mutation (the so-called pseudogenes). The clock runs most slowly for sequences that are subject to strong functional constraints—for example, the amino acid sequences of proteins that engage in specific interactions with large numbers of other proteins and

1	slowly for sequences that are subject to strong functional constraints—for example, the amino acid sequences of proteins that engage in specific interactions with large numbers of other proteins and whose structure is therefore highly constrained, or the nucleotide sequences that encode the RNA subunits of the ribosome, on which all protein synthesis depends.

1	Occasionally, rapid change is seen in a previously highly conserved sequence. As discussed later in this chapter, such episodes are especially interesting because they are thought to reflect periods of strong positive selection for mutations that have conferred a selective advantage in the particular lineage where the rapid change occurred. The pace at which molecular clocks run during evolution is determined not only by the degree of purifying selection, but also by the mutation rate. Most notably, in animals, although not in plants, clocks based on functionally unconstrained mitochondrial DNA sequences run much faster than clocks based on functionally unconstrained nuclear sequences, because the mutation rate in animal mitochondria is exceptionally high.

1	Categories of DNA for which the clock runs fast are most informative for recent evolutionary events; the mitochondrial DNA clock has been used, for example, to chronicle the divergence of the Neanderthal lineage from that of modern Homo sapiens. To study more ancient evolutionary events, one must examine DNA for which the clock runs more slowly; thus the divergence of the major branches of the tree of life—bacteria, archaea, and eukaryotes—has been deduced from study of the sequences specifying ribosomal RNA.

1	In general, molecular clocks, appropriately chosen, have a finer time resolution than the fossil record, and they are a more reliable guide to the detailed structure of phylogenetic trees than are classical methods of tree construction, which are based on family resemblances in anatomy and embryonic development. For example, the precise family tree of great apes and humans was not settled until sufficient molecular sequence data accumulated in the 1980s to produce the pedigree shown previously in Figure 4–63. And with huge amounts of DNA sequence now determined from a wide variety of mammals, much better estimates of our relationship to them are being obtained (Figure 4–66). A Comparison of Human and mouse Chromosomes Shows How the Structures of genomes Diverge

1	A Comparison of Human and mouse Chromosomes Shows How the Structures of genomes Diverge As would be expected, the human and chimpanzee genomes are much more alike than are the human and mouse genomes, even though all three genomes are roughly the same size and contain nearly identical sets of genes. Mouse and human lineages have had approximately 80 million years to diverge through accumulated mutations, versus 6 million years for humans and chimpanzees. In addition, as indicated in Figure 4–66, rodent lineages (represented by the rat and the mouse) have unusually fast molecular clocks, and have diverged from the human lineage more rapidly than otherwise expected.

1	While the way that the genome is organized into chromosomes is almost identical between humans and chimpanzees, this organization has diverged greatly between humans and mice. According to rough estimates, a total of about 180 breakage-and-rejoining events have occurred in the human and mouse lineages since these two species last shared a common ancestor. In the process, although the number of chromosomes is similar in the two species (23 per haploid genome in the human versus 20 in the mouse), their overall structures differ greatly. Nonetheless, even after the extensive genomic shuffling, there are many large blocks of DNA in which the gene order is the same in the human and the mouse. These stretches of conserved gene order in chromosomes are referred to as regions of synteny. Figure 4–67 illustrates how segments of the different mouse chromosomes map onto the human chromosome set. For much more distantly related vertebrates, such as chicken and human, the number of

1	Figure 4–67 illustrates how segments of the different mouse chromosomes map onto the human chromosome set. For much more distantly related vertebrates, such as chicken and human, the number of breakage-and-rejoining events has been much greater and the regions of synteny are much shorter; in addition, they are often hard to discern because of the divergence of the DNA sequences that they contain.

1	An unexpected conclusion from a detailed comparison of the complete mouse and human genome sequences, confirmed by subsequent comparisons between the genomes of other vertebrates, is that small blocks of DNA sequence are being deleted from and added to genomes at a surprisingly rapid rate. Thus, if we assume that our common ancestor had a genome of human size (about 3.2 billion nucleotide pairs), mice would have lost a total of about 45% of that genome from accumulated deletions during the past 80 million years, while humans would have lost about 25%. However, substantial sequence gains from many small chromosome duplications and from the multiplication of transposons have compensated for these deletions. As a result, our genome size is thought to be practically unchanged from that of the last common ancestor of humans and mice, while the mouse genome is smaller by only about 0.3 billion nucleotides.

1	Figure 4–66 A phylogenetic tree showing the evolutionary relationships of some present-day mammals. The length of each line is proportional to the number of “neutral substitutions”—that is, nucleotide changes at sites where there is assumed to be no purifying selection. (Adapted from g.m. Cooper et al., Genome Res. 15:901–913, 2005. with permission from Cold Spring Harbor laboratory Press.) Good evidence for the loss of DNA sequences in small blocks during evolution can be obtained from a detailed comparison of regions of synteny in the human and mouse genomes. The comparative shrinkage of the mouse genome can be clearly seen from such comparisons, with the net loss of sequences scattered throughout the long stretches of DNA that are otherwise homologous (Figure 4–68).

1	DNA is added to genomes both by the spontaneous duplication of chromosomal segments that are typically tens of thousands of nucleotide pairs long (as will be discussed shortly) and by insertion of new copies of active transposons. Most transposition events are duplicative, because the original copy of the transposon stays where it was when a copy inserts at the new site; see, for example, Figure 5–63. Comparison of the DNA sequences derived from transposons in the human and the mouse readily reveals some of the sequence additions (Figure 4–69). It remains a mystery why all mammals have maintained genome sizes of roughly 3 billion nucleotide pairs that contain nearly identical sets of genes, even though only approximately 150 million nucleotide pairs appear to be under sequence-specific functional constraints. The Size of a vertebrate genome Reflects the Relative Rates of DNA Addition and DNA loss in a lineage

1	The Size of a vertebrate genome Reflects the Relative Rates of DNA Addition and DNA loss in a lineage In more distantly related vertebrates, genome size can vary considerably, apparently without a drastic effect on the organism or its number of genes. Thus, the chicken genome, at one billion nucleotide pairs, is only about one-third the size 200,000 bases Figure 4–67 Synteny between human and mouse chromosomes. In this diagram, the human chromosome set is shown above, with each part of each chromosome colored according to the mouse chromosome with which it is syntenic. The color coding used for each mouse chromosome is shown below. Heterochromatic highly repetitive regions (such as centromeres) that are difficult to sequence cannot be mapped in this way; these are colored black. (Adapted from E.E. Eichler and D. Sankoff, Science 301:793–797, 2003. with permission from AAAS.) Figure 4–68 Comparison of a syntenic portion of mouse and human genomes.

1	About 90% of the two genomes can be aligned in this way. Note that while there is an identical order of the matched index sequences (red marks), there has been a net loss of DNA in the mouse lineage that is interspersed throughout the entire region. This type of net loss is typical for all such regions, and it accounts for the fact that the mouse genome contains 14% less DNA than does the human genome. (Adapted from mouse genome Sequencing Consortium, Nature 420:520–562, 2002. with permission from macmillan Publishers ltd.) 10,000 nucleotide pairs of the mammalian genome. An extreme example is the puffer fish, Fugu rubripes (Figure 4–70), which has a tiny genome for a vertebrate (0.4 billion nucleotide pairs compared to 1 billion or more for many other fish). The small size of the Fugu genome is largely due to the small size of its introns. Specifically, Fugu introns, as well as other noncoding segments of the Fugu genome, lack the repetitive DNA that makes up a large portion of the

1	is largely due to the small size of its introns. Specifically, Fugu introns, as well as other noncoding segments of the Fugu genome, lack the repetitive DNA that makes up a large portion of the genomes of most well-studied vertebrates. Nevertheless, the positions of the Fugu introns between the exons of each gene are almost the same as in mammalian genomes (Figure 4–71).

1	While initially a mystery, we now have a simple explanation for such large differences in genome size between similar organisms: because all vertebrates experience a continuous process of DNA loss and DNA addition, the size of a genome merely depends on the balance between these opposing processes acting over millions of years. Suppose, for example, that in the lineage leading to Fugu, the rate of DNA addition happened to slow greatly. Over long periods of time, this would result in a major “cleansing” from this fish genome of those DNA sequences whose loss could be tolerated. The result is an unusually compact genome, relatively free of junk and clutter, but retaining through purifying selection the vertebrate DNA sequences that are functionally important. This makes Fugu, with its 400 million nucleotide pairs of DNA, a valuable resource for genome research aimed at understanding humans. we Can Infer the Sequence of Some Ancient genomes

1	The genomes of ancestral organisms can be inferred, but most can never be directly observed. DNA is very stable compared with most organic molecules, but it is not perfectly stable, and its progressive degradation, even under the best circumstances, means that it is virtually impossible to extract sequence information from fossils that are more than a million years old. Although a modern organism such as the horseshoe crab looks remarkably similar to fossil ancestors that lived 200 million years ago, there is every reason to believe that the horse-shoe-crab genome has been changing during all that time in much the same way as in other evolutionary lineages, and at a similar rate. Selection must have maintained key functional properties of the horseshoe-crab genome to account for the morphological stability of the lineage. However, comparisons between different present-day organisms show that the fraction of the genome subject to purifying selection is small; hence, it is fair to

1	morphological stability of the lineage. However, comparisons between different present-day organisms show that the fraction of the genome subject to purifying selection is small; hence, it is fair to assume that the genome of the modern horseshoe crab, while preserving features critical for function, must differ greatly from that of its extinct ancestors, known to us only through the fossil record.

1	It is possible to get direct sequence information by examining DNA samples from ancient materials if these are not too old. In recent years, technical advances have allowed DNA sequencing from exceptionally well-preserved bone fragments that date from more than 100,000 years ago. Although any DNA this old will be imperfectly preserved, a sequence of the Neanderthal genome has been reconstructed from many millions of short DNA sequences, revealing—among other things—that our human ancestors interbred with Neanderthals in Europe and

1	Figure 4–69 A comparison of the β-globin gene cluster in the human and mouse genomes, showing the locations of transposable elements. This stretch of the human genome contains five functional β-globin-like genes (orange); the comparable region from the mouse genome has only four. The positions of the human Alu sequences are indicated by green circles, and the human L1 sequences by red circles. The mouse genome contains different but related transposable elements: the positions of b1 elements (which are related to the human Alu sequences) are indicated by blue triangles, and the positions of the mouse L1 elements (which are related to the human L1 sequences) are indicated by orange triangles. The absence of transposable elements from the globin structural genes can be attributed to purifying selection, which would have eliminated any insertion that compromised gene function. (Courtesy of Ross Hardison and webb miller.)

1	Figure 4–70 The puffer fish, Fugu rubripes. (Courtesy of byrappa venkatesh.) 0.0 100.0 180.0 thousands of nucleotide pairs that modern humans have inherited specific genes from them (Figure 4–72). The average difference in DNA sequence between humans and Neanderthals shows that our two lineages diverged somewhere between 270,000 and 440,000 years ago, well before the time that humans are believed to have migrated out of Africa.

1	But what about deciphering the genomes of much older ancestors, those for which no useful DNA samples can be isolated? For organisms that are as closely related as human and chimpanzee, we saw that this may not be difficult: reference to the gorilla sequence can be used to sort out which of the few sequence differences between human and chimpanzee are inherited from our common ancestor some 6 million years ago (see Figure 4–64). And for an ancestor that has produced a large number of different organisms alive today, the DNA sequences of many species can be compared simultaneously to unscramble much of the ancestral sequence, allowing scientists to derive DNA sequences much farther back in time. For example, from the genome sequences currently being obtained for dozens of modern placental mammals, it should be possible to infer much of the genome sequence of their 100 million-year-old common ancestor—the precursor of species as diverse as dog, mouse, rabbit, armadillo, and human (see

1	mammals, it should be possible to infer much of the genome sequence of their 100 million-year-old common ancestor—the precursor of species as diverse as dog, mouse, rabbit, armadillo, and human (see Figure 4–66).

1	multispecies Sequence Comparisons Identify Conserved DNA Sequences of Unknown Function The mass of DNA sequence now in databases (hundreds of billions of nucleotide pairs) provides a rich resource that scientists can mine for many purposes. This information can be used not only to unscramble the evolutionary pathways that have led to modern organisms, but also to provide insights into how cells and organisms function. Perhaps the most remarkable discovery in this realm comes from the observation that a striking amount of DNA sequence that does not code for protein has been conserved during mammalian evolution (see Table 4–1, p. 184). This is most clearly revealed when we align and compare DNA synteny

1	Figure 4–71 Comparison of the genomic sequences of the human and Fugu genes encoding the protein huntingtin. both genes (indicated in red) contain 67 short exons that align in 1:1 correspondence to one another; these exons are connected by curved lines. The human gene is 7.5 times larger than the Fugu gene (180,000 versus 24,000 nucleotide pairs). The size difference is entirely due to larger introns in the human gene. The larger size of the human introns is due in part to the presence of retrotransposons (discussed in Chapter 5), whose positions are represented by green vertical lines; the Fugu introns lack retrotransposons. In humans, mutation of the huntingtin gene causes Huntington’s disease, an inherited neurodegenerative disorder. (Adapted from S. baxendale et al., Nat. Genet. 10:67–76, 1995. with permission from macmillan Publishers ltd.)

1	Figure 4–72 The Neanderthals. (A) map of Europe showing the location of the cave in Croatia where most of the bones used to isolate the DNA used to derive the Neanderthal genome sequence were discovered. (b) Photograph of the vindija cave. (C) Photograph of the 38,000-yearold bones from vindija. more recent studies have succeeded in extracting DNA sequence information from hominid remains that are considerably older (see movie 8.3). (b, courtesy of Johannes krause; C, from R.E. green et al., Science 328: 710–722, 2010. Reprinted with permission from AAAS.) cave in Vindija, Croatia blocks from many different species, thereby identifying large numbers of so-called multispecies conserved sequences: some of these code for protein, but most of them do not (Figure 4–73).

1	Most of the noncoding conserved sequences discovered in this way turn out to be relatively short, containing between 50 and 200 nucleotide pairs. Among the most mysterious are the so-called “ultraconserved” noncoding sequences, exemplified by more than 5000 DNA segments over 100 nucleotides long that are exactly the same in human, mouse, and rat. Most have undergone little or no change since mammalian and bird ancestors diverged about 300 million years ago. The strict conservation implies that even though the sequences do not encode proteins, each nevertheless has an important function maintained by purifying selection. The puzzle is to unravel what those functions are.

1	Many of the conserved sequences that do not code for protein are now known to produce untranslated RNA molecules, such as the thousands of long noncoding RNAs (lncRNAs) that are thought to have important functions in regulating gene transcription. As we shall also see in Chapter 7, others are short regions of DNA scattered throughout the genome that directly bind proteins involved in gene regulation. But it is uncertain how much of the conserved noncoding DNA can be accounted for in these ways, and the function of most of it remains a mystery. This enigma highlights how much more we need to learn about the fundamental biological mechanisms that operate in animals and other complex organisms, and its solution is certain to have profound consequences for medicine.

1	How can cell biologists tackle the mystery of noncoding conserved DNA? Traditionally, attempts to determine the function of a puzzling DNA sequence begin by looking at the consequences of its experimental disruption. But many DNA sequences that are crucial for an organism in the wild can be expected to have no noticeable effect on its phenotype under laboratory conditions: what is required for a mouse to survive in a laboratory cage is very much less than what is required 190,000 nucleotide pairs 100 nucleotide pairs 10,000 nucleotide pairs Figure 4–73 The detection of multispecies conserved sequences.

1	In this example, genome sequences for each of the organisms shown have been compared with the indicated region of the human CFTR (cystic fibrosis transmembrane conductance regulator) gene; this region contains one exon plus a large amount of intronic DNA. For each organism, the percent identity with human for each 25-nucleotide block is plotted in green. In addition, a computational algorithm has been used to detect the sequences within this region that are most highly conserved when the sequences from all of the organisms are taken into account. besides the exon (dark blue on the line at the top of the figure), the positions of three other blocks of multispecies conserved sequences are indicated (pale blue). The function of most such sequences in the human genome is not known. (Courtesy of Eric D. green.) for it to succeed in nature. Moreover, calculations based on population genetics reveal that just a tiny selective advantage—less than a 0.1% difference in survival—can be enough to

1	Eric D. green.) for it to succeed in nature. Moreover, calculations based on population genetics reveal that just a tiny selective advantage—less than a 0.1% difference in survival—can be enough to strongly favor retaining a particular DNA sequence over evolutionary time spans. One should therefore not be surprised to find that many DNA sequences that are ultraconserved can be deleted from the mouse genome without any noticeable effect on that mouse in a laboratory.

1	A second important approach for discovering the function of a mysterious noncoding DNA sequence uses biochemical techniques to identify proteins or RNA molecules that bind to it—and/or to any RNA molecules that it produces. Most of this task still lies before us, but a start has been made (see p. 435).

1	Given genome sequence information, we can tackle another intriguing question: What alterations in our DNA have made humans so different from other animals—or for that matter, what makes any individual species so different from its relatives? For example, as soon as both the human and the chimpanzee genome sequences became available, scientists began searching for DNA sequence changes that might account for the striking differences between us and chimpanzees. With 3.2 billion nucleotide pairs to compare in the two species, this might seem an impossible task. But the job was made much easier by confining the search to 35,000 clearly defined multispecies conserved sequences (a total of about 5 million nucleotide pairs), representing parts of the genome that are most likely to be functionally important. Though these sequences are conserved strongly, they are not conserved perfectly, and when the version in one species is compared with that in another they are generally found to have

1	important. Though these sequences are conserved strongly, they are not conserved perfectly, and when the version in one species is compared with that in another they are generally found to have drifted apart by a small amount corresponding simply to the time elapsed since the last common ancestor. In a small proportion of cases, however, one sees signs of a sudden evolutionary spurt. For example, some DNA sequences that have been highly conserved in other mammalian species are found to have accumulated nucleotide changes exceptionally rapidly during the 6 million years of human evolution since we diverged from the chimpanzees. These human accelerated regions (HARs) are thought to reflect functions that have been especially important in making us different in some useful way.

1	About 50 such sites were identified in one study, one-fourth of which were located near genes associated with neural development. The sequence exhibiting the most rapid change (18 changes between human and chimpanzee, compared to only two changes between chimpanzee and chicken) was examined further and found to encode a 118-nucleotide noncoding RNA molecule, HAR1F (human accelerated region 1F), that is produced in the human cerebral cortex at a critical time during brain development. The function of this HAR1F RNA is not yet known, but findings of this type are stimulating research studies that may shed light on crucial features of the human brain.

1	A related approach in the search for the important mutations that contributed to human evolution likewise begins with DNA sequences that have been conserved during mammalian evolution, but rather than screening for accelerated changes in individual nucleotides, it focuses instead on chromosome sites that have experienced deletions in the 6 million years since our lineage diverged from that of chimpanzees. More than 500 such sequences—conserved among other species but deleted in humans—have been discovered. Each deletion removes an average of 95 nucleotides of DNA sequence. Only one of these deletions affects a protein-coding region: the rest are thought to alter regions that affect how nearby genes are expressed, an expectation that has been experimentally confirmed in a few cases. A large proportion of the presumed regulatory regions identified in this way lie near genes that affect neural function and/or near genes involved in steroid signaling, suggesting that changes in the

1	A large proportion of the presumed regulatory regions identified in this way lie near genes that affect neural function and/or near genes involved in steroid signaling, suggesting that changes in the nervous system and in immune or reproductive functions have played an especially important role in human evolution.

1	mutations in the DNA Sequences That Control gene Expression Have Driven many of the Evolutionary Changes in vertebrates

1	The vast hoard of genomic sequence data now being accumulated can be explored in many other ways to reveal events that happened even hundreds of millions of years ago. For example, one can attempt to trace the origins of the regulatory elements in DNA that have played critical parts in vertebrate evolution. One such study began with the identification of nearly 3 million noncoding sequences, averaging 28 base pairs in length, that have been conserved in recent vertebrate evolution while being absent in more ancient ancestors. Each of these special non-coding sequences is likely to represent a functional innovation peculiar to a particular branch of the vertebrate family tree, and most of them are thought to consist of regulatory DNA that governs the expression of a neighboring gene. Given full genome sequences, one can identify the genes that lie closest and thus appear most likely to have fallen under the sway of these novel regulatory elements. By comparing many different species,

1	full genome sequences, one can identify the genes that lie closest and thus appear most likely to have fallen under the sway of these novel regulatory elements. By comparing many different species, with known divergence times, one can also estimate when each such regulatory element came into existence as a conserved feature. The findings suggest remarkable evolutionary differences between the various functional classes of genes (Figure 4–74). Conserved regulatory elements that originated early in vertebrate evolution—that is, more than about 300 million years ago, which is when the mammalian lineage split from the lineage leading to birds and reptiles—seem to be mostly associated with genes that code for transcription regulator proteins and for proteins with roles in organizing embryonic development. Then came an era when the regulatory DNA innovations arose next to genes coding for receptors for extracellular signals. Finally, over the course of the past 100 million years, the

1	development. Then came an era when the regulatory DNA innovations arose next to genes coding for receptors for extracellular signals. Finally, over the course of the past 100 million years, the regulatory innovations seem to have been concentrated in the neighborhood of genes coding for proteins (such as protein kinases) that function to modify other proteins post-translationally.

1	Many questions remain to be answered about these phenomena and what they mean. One possible interpretation is that the logic—the circuit diagram—of the gene regulatory network in vertebrates was established early, and that more recent evolutionary change has mainly occurred through the tuning of quantitative parameters. This could help to explain why, among the mammals, for example, the basic body plan—the topology of the tissues and organs—has been largely conserved. gene Duplication Also Provides an Important Source of genetic Novelty During Evolution Evolution depends on the creation of new genes, as well as on the modification of those that already exist. How does this occur? When we compare organisms that seem very different—a primate with a rodent, for example, or a mouse with a fish—we rarely encounter genes in the one species that have no homolog in the reception of extracellular signals HUMAN 500 400 300 200 100 0 millions of years before present

1	Figure 4–74 The types of changes in gene regulation inferred to have predominated during the evolution of our vertebrate ancestors. To produce the information summarized in this plot, wherever possible the type of gene regulated by each conserved noncoding sequence was inferred from the identity of its closest protein-coding gene. The fixation time for each conserved sequence was then used to derive the conclusions shown. (based on C.b. lowe et al., Science 333:1019–1024, 2011. with permission from AAAS.) other. Genes without homologous counterparts are relatively scarce even when we compare such divergent organisms as a mammal and a worm. On the other hand, we frequently find gene families that have different numbers of members in different species. To create such families, genes have been repeatedly duplicated, and the copies have then diverged to take on new functions that often vary from one species to another.

1	Gene duplication occurs at high rates in all evolutionary lineages, contributing to the vigorous process of DNA addition discussed previously. In a detailed study of spontaneous duplications in yeast, duplications of 50,000 to 250,000 nucleotide pairs were commonly observed, most of which were tandemly repeated. These appeared to result from DNA replication errors that led to the inexact repair of double-strand chromosome breaks. A comparison of the human and chimpanzee genomes reveals that, since the time that these two organisms diverged, such segmental duplications have added about 5 million nucleotide pairs to each genome every million years, with an average duplication size being about 50,000 nucleotide pairs (although there are some duplications five times larger). In fact, if one counts nucleotides, duplication events have created more differences between our two species than have single-nucleotide substitutions.

1	What is the fate of newly duplicated genes? In most cases, there is presumed to be little or no selection—at least initially—to maintain the duplicated state since either copy can provide an equivalent function. Hence, many duplication events are likely to be followed by loss-of-function mutations in one or the other gene. This cycle would functionally restore the one-gene state that preceded the duplication. Indeed, there are many examples in contemporary genomes where one copy of a duplicated gene can be seen to have become irreversibly inactivated by multiple mutations. Over time, the sequence similarity between such a pseudogene and the functional gene whose duplication produced it would be expected to be eroded by the accumulation of many mutations in the pseudogene—the homologous relationship eventually becoming undetectable.

1	An alternative fate for gene duplications is for both copies to remain functional, while diverging in their sequence and pattern of expression, thus taking on different roles. This process of “duplication and divergence” almost certainly explains the presence of large families of genes with related functions in biologically complex organisms, and it is thought to play a critical role in the evolution of increased biological complexity. An examination of many different eukaryotic genomes suggests that the probability that any particular gene will undergo a duplication event that spreads to most or all individuals in a species is approximately 1 percent every million years.

1	Whole-genome duplications offer particularly dramatic examples of the duplication–divergence cycle. A whole-genome duplication can occur quite simply: all that is required is one round of genome replication in a germ-line cell lineage without a corresponding cell division. Initially, the chromosome number simply doubles. Such abrupt increases in the ploidy of an organism are common, particularly in fungi and plants. After a whole-genome duplication, all genes exist as duplicate copies. However, unless the duplication event occurred so recently that there has been little time for subsequent alterations in genome structure, the results of a series of segmental duplications—occurring at different times— are hard to distinguish from the end product of a whole-genome duplication. In mammals, for example, the role of whole-genome duplications versus a series of piecemeal duplications of DNA segments is quite uncertain. Nevertheless, it is clear that a great deal of gene duplication has

1	for example, the role of whole-genome duplications versus a series of piecemeal duplications of DNA segments is quite uncertain. Nevertheless, it is clear that a great deal of gene duplication has occurred in the distant past.

1	Analysis of the genome of the zebrafish, in which at least one whole-genome duplication is thought to have occurred hundreds of millions of years ago, has cast some light on the process of gene duplication and divergence. Although many duplicates of zebrafish genes appear to have been lost by mutation, a significant fraction—perhaps as many as 30–50%—have diverged functionally while both Figure 4–75 A comparison of the structure of one-chain and four-chain globins. The four-chain globin shown is hemoglobin, which is a complex of two α-globin and two β-globin chains. The one-chain globin present in some primitive vertebrates represents an intermediate in the evolution of the four-chain globin. with oxygen bound it exists as a monomer; without oxygen it dimerizes.

1	copies have remained active. In many cases, the most obvious functional difference between the duplicated genes is that they are expressed in different tissues or at different stages of development. One attractive theory to explain such an end result imagines that different, mildly deleterious mutations occur quickly in both copies of a duplicated gene set. For example, one copy might lose expression in a particular tissue as a result of a regulatory mutation, while the other copy loses expression in a second tissue. Following such an occurrence, both gene copies would be required to provide the full range of functions that were once supplied by a single gene; hence, both copies would now be protected from loss through inactivating mutations. Over a longer period, each copy could then undergo further changes through which it could acquire new, specialized features. The Evolution of the globin gene Family Shows How DNA Duplications Contribute to the Evolution of Organisms

1	The Evolution of the globin gene Family Shows How DNA Duplications Contribute to the Evolution of Organisms The globin gene family provides an especially good example of how DNA duplication generates new proteins, because its evolutionary history has been worked out particularly well. The unmistakable similarities in amino acid sequence and structure among the present-day globins indicate that they all must derive from a common ancestral gene, even though some are now encoded by widely separated genes in the mammalian genome.

1	We can reconstruct some of the past events that produced the various types of oxygen-carrying hemoglobin molecules by considering the different forms of the protein in organisms at different positions on the tree of life. A molecule like hemoglobin was necessary to allow multicellular animals to grow to a large size, since large animals cannot simply rely on the diffusion of oxygen through the body surface to oxygenate their tissues adequately. But oxygen plays a vital part in the life of nearly all living organisms, and oxygen-binding proteins homologous to hemoglobin can be recognized even in plants, fungi, and bacteria. In animals, the most primitive oxygen-carrying molecule is a globin polypeptide chain of about 150 amino acids that is found in many marine worms, insects, and primitive fish.

1	The hemoglobin molecule in more complex vertebrates, however, is composed of two kinds of globin chains. It appears that about 500 million years ago, during the continuing evolution of fish, a series of gene mutations and duplications occurred. These events established two slightly different globin genes in the genome of each individual, coding for αand β-globin chains that associate to form a hemoglobin molecule consisting of two α chains and two β chains (Figure 4–75). The four oxygen-binding sites in the α2β2 molecule interact, allowing a cooperative allosteric change in the molecule as it binds and releases oxygen, which enables hemoglobin to take up and release oxygen more efficiently than the single-chain version.

1	Still later, during the evolution of mammals, the β-chain gene apparently underwent duplication and mutation to give rise to a second β-like chain that millions of years ago is synthesized specifically in the fetus. The resulting hemoglobin molecule has a higher affinity for oxygen than adult hemoglobin and thus helps in the transfer of oxygen from the mother to the fetus. The gene for the new β-like chain subsequently duplicated and mutated again to produce two new genes, ε and γ, the ε chain being produced earlier in development (to form α2ε2) than the fetal γ chain, which forms α2γ2. A duplication of the adult β-chain gene occurred still later, during primate evolution, to give rise to a δ-globin gene and thus to a minor form of hemoglobin (α2δ2) that is found only in adult primates (Figure 4–76).

1	Each of these duplicated genes has been modified by point mutations that affect the properties of the final hemoglobin molecule, as well as by changes in regulatory regions that determine the timing and level of expression of the gene. Figure 4–76 An evolutionary scheme for the globin chains that carry oxygen in the blood of animals. The scheme emphasizes the β-like globin gene family. A relatively recent gene duplication of the γ-chain gene produced γg and γA, which are fetal β-like chains of identical function. The location of the globin genes in the human genome is shown at the top of the figure. As a result, each globin is made in different amounts at different times of human development.

1	As a result, each globin is made in different amounts at different times of human development. The history of these gene duplications is reflected in the arrangement of hemoglobin genes in the genome. In the human genome, the genes that arose from the original β gene are arranged as a series of homologous DNA sequences located within 50,000 nucleotide pairs of one another on a single chromosome. A similar cluster of human α-globin genes is located on a separate chromosome. Not only other mammals, but birds too have their αand β-globin gene clusters on separate chromosomes. In the frog Xenopus, however, they are together, suggesting that a chromosome translocation event in the lineage of birds and mammals separated the two gene clusters about 300 million years ago, soon after our ancestors diverged from amphibians (see Figure 4–76).

1	There are several duplicated globin DNA sequences in the αand β-globin gene clusters that are not functional genes but pseudogenes. These have a close sequence similarity to the functional genes but have been disabled by mutations that prevent their expression as functional proteins. The existence of such pseudogenes makes it clear that, as expected, not every DNA duplication leads to a new functional gene. genes Encoding New Proteins Can be Created by the Recombination of Exons

1	genes Encoding New Proteins Can be Created by the Recombination of Exons The role of DNA duplication in evolution is not confined to the expansion of gene families. It can also act on a smaller scale to create single genes by stringing together short duplicated segments of DNA. The proteins encoded by genes generated in this way can be recognized by the presence of repeating similar protein domains, which are covalently linked to one another in series. The immunoglobulins (Figure 4–77), for example, as well as most fibrous proteins (such as collagens) are encoded by genes that have evolved by repeated duplications of a primordial DNA sequence.

1	In genes that have evolved in this way, as well as in many other genes, each separate exon often encodes an individual protein folding unit, or domain. It is believed that the organization of DNA coding sequences as a series of such exons separated by long introns has greatly facilitated the evolution of new proteins. The duplications necessary to form a single gene coding for a protein with repeating domains, for example, can easily occur by breaking and rejoining the DNA anywhere in the long introns on either side of an exon; without introns there would be only a few sites in the original gene at which a recombinational exchange between DNA molecules could duplicate the domain and not disrupt it. By enabling the duplication to occur by recombination at many potential sites rather than just a few, introns increase the probability of a favorable duplication event.

1	More generally, we know from genome sequences that the various parts of genes—both their individual exons and their regulatory elements—have served as modular elements that have been duplicated and moved about the genome to create the great diversity of living things. Thus, for example, many present-day proteins are formed as a patchwork of domains from different origins, reflecting their complex evolutionary history (see Figure 3–17). Neutral mutations Often Spread to become Fixed in a Population, with a Probability That Depends on Population Size In comparisons between two species that have diverged from one another by millions of years, it makes little difference which individuals from each species are Figure 4–77 Schematic view of an antibody (immunoglobulin) molecule.

1	Figure 4–77 Schematic view of an antibody (immunoglobulin) molecule. This molecule is a complex of two identical heavy chains and two identical light chains. Each heavy chain contains four similar, covalently linked domains, while each light chain contains two such domains. Each domain is encoded by a separate exon, and all of the exons are thought to have evolved by the serial duplication of a single ancestral exon.

1	compared. For example, typical human and chimpanzee DNA sequences differ from one another by about 1%. In contrast, when the same region of the genome is sampled from two randomly chosen humans, the differences are typically about 0.1%. For more distantly related organisms, the interspecies differences outshine intraspecies variation even more dramatically. However, each “fixed difference” between the human and the chimpanzee (in other words, each difference that is now characteristic of all or nearly all individuals of each species) started out as a new mutation in a single individual. If the size of the interbreeding population in which the mutation occurred is N, the initial allele frequency for a new mutation would be 1/(2N) for a diploid organism. How does such a rare mutation become fixed in the population, and hence become a characteristic of the species rather than of a few scattered individuals?

1	The answer to this question depends on the functional consequences of the mutation. If the mutation has a significantly deleterious effect, it will simply be eliminated by purifying selection and will not become fixed. (In the most extreme case, the individual carrying the mutation will die without producing progeny.) Conversely, the rare mutations that confer a major reproductive advantage on individuals who inherit them can spread rapidly in the population. Because humans reproduce sexually and genetic recombination occurs each time a gamete is formed (discussed in Chapter 5), the genome of each individual who has inherited the mutation will be a unique recombinational mosaic of segments inherited from a large number of ancestors. The selected mutation along with a modest amount of neighboring sequence—ultimately inherited from the individual in which the mutation occurred—will simply be one piece of this huge mosaic.

1	The great majority of mutations that are not harmful are not beneficial either. These selectively neutral mutations can also spread and become fixed in a population, and they make a large contribution to evolutionary change in genomes. For example, as we saw earlier, they account for most of the DNA sequence differences between apes and humans. The spread of neutral mutations is not as rapid as the spread of the rare strongly advantageous mutations. It depends on a random variation in the number of mutation-bearing progeny produced by each mutation-bearing individual, causing changes in the relative frequency of the mutant allele in the population. Through a sort of “random walk” process, the mutant allele may eventually become extinct, or it may become commonplace. This can be modeled mathematically for an idealized interbreeding population, on the assumption of constant population size and random mating, as well as selective neutrality for the mutations. While neither of the first

1	for an idealized interbreeding population, on the assumption of constant population size and random mating, as well as selective neutrality for the mutations. While neither of the first two assumptions is a good description of human population history, study of this idealized case reveals the general principles in a clear and simple way.

1	When a new neutral mutation occurs in a population of constant size N that is undergoing random mating, the probability that it will ultimately become fixed is approximately 1/(2N). This is because there are 2N copies of the gene in the diploid population, and each of them has an equal chance of becoming the predominant version in the long run. For those mutations that do become fixed, the mathematics shows that the average time to fixation is approximately 4N generations. Detailed analyses of data on human genetic variation have suggested an ancestral population size of approximately 10,000 at the time when the current pattern of genetic variation was largely established. With a population that has reached this size, the probability that a new, selectively neutral mutation would become fixed is small (1/20,000), while the average time to fixation would be on the order of 800,000 years (assuming a 20-year generation time). Thus, while we know that the human population has grown

1	fixed is small (1/20,000), while the average time to fixation would be on the order of 800,000 years (assuming a 20-year generation time). Thus, while we know that the human population has grown enormously since the development of agriculture approximately 15,000 years ago, most of the present-day set of common human genetic variants reflects the mixture of variants that was already present long before this time, when the human population was still small.

1	Similar arguments explain another phenomenon with important practical implications for genetic counseling. In an isolated community descended from a small group of founders, such as the people of Iceland or the Jews of Eastern individual with rare allele Europe, genetic variants that are rare in the human population as a whole can often be present at a high frequency, even if those variants are mildly deleterious (Figure 4–78). A great Deal Can be learned from Analyses of the variation Among Humans Even though the common variant gene alleles among modern humans originate from variants present in a comparatively tiny group of ancestors, the total number of variants now encountered, including those that are individually rare, is very large. New neutral mutations are constantly occurring and accumulating, even though no single one of them has had enough time to become fixed in the vast modern human population.

1	From detailed comparisons of the DNA sequences of a large number of modern humans located around the globe, scientists can estimate how many generations have elapsed since the origin of a particular neutral mutation. From such data, it has been possible to map the routes of ancient human migrations. For example, by combining this type of genetic analysis with archaeological findings, scientists have been able to deduce the most probable routes that our ancestors took when they left Africa 60,000 to 80,000 years ago (Figure 4–79).

1	We have been focusing on mutations that affect a single gene, but these are not the only source of variation. Another source, perhaps even more important but missed for many years, lies in the many duplications and deletions of large blocks of human DNA. When one compares any individual human with the standard reference genome in the database, one will generally find roughly 100 differences involving gain or loss of long sequence blocks, totaling perhaps 3 million nucleotide pairs. Some of these copy number variations (CNVs) will be very common, presumably reflecting relatively ancient origins, while others will be present in only a small minority of people (Figure 4–80). On average, nearly half of the CNVs contain known genes. CNVs have been implicated in many human traits, including color blindness, infertility, hypertension, and a wide variety of disease susceptibilities. In retrospect, this type of variation is not surprising, given the prominent role of DNA addition and DNA loss

1	blindness, infertility, hypertension, and a wide variety of disease susceptibilities. In retrospect, this type of variation is not surprising, given the prominent role of DNA addition and DNA loss in vertebrate evolution.

1	The intraspecies variations that have been most extensively characterized, however, are single-nucleotide polymorphisms (SNPs). These are simply points in the genome sequence where one large fraction of the human population has one nucleotide, while another substantial fraction has another. To qualify as Figure 4–78 How founder effects determine the set of genetic variants in a population of individuals belonging to the same species. This example illustrates how a rare allele (red) can become established in an isolated population, even though the mutation that produced it has no selective advantage—or is mildly deleterious.

1	Figure 4–79 Tracing the course of human history by analyses of genome sequences. The map shows the routes of the earliest successful human migrations. Dotted lines indicate two alternative routes that our ancestors are thought to have taken out of Africa. DNA sequence comparisons suggest that modern Europeans descended from a small ancestral population that existed about 30,000 to 50,000 years ago. In agreement, archaeological findings suggest that the ancestors of modern native Australians (solid red arrows)—and of modern European and middle Eastern populations—reached their destinations about 45,000 years ago. Even more recent studies, comparing the genome sequences of living humans with those of Neanderthals and another extinct population from southern Siberia (the Denisovans), suggest that our exit from Africa was a bit more convoluted, while also revealing that a number of our ancestors interbred with these hominid neighbors as they made their way across the globe. (modified from

1	P. Forster and S. matsumura, Science 308:965–966, 2005.) density of known genes a polymorphism, the variants must be common enough to give a reasonably high probability that the genomes of two randomly chosen individuals will differ at the given site; a probability of 1% is commonly chosen as the cutoff. Two human genomes sampled from the modern world population at random will differ at approximately 2.5 × 106 such sites (1 per 1300 nucleotide pairs). As will be described in the overview of genetics in Chapter 8, SNPs in the human genome can be extremely useful for genetic mapping analyses, in which one attempts to associate specific traits (phenotypes) with specific DNA sequences for medical or scientific purposes (see p. 493). But while useful as genetic markers, there is good evidence that most of these SNPs have little or no effect on human fitness. This is as expected, since deleterious variants will have been selected against during human evolution and, unlike SNPs, should

1	that most of these SNPs have little or no effect on human fitness. This is as expected, since deleterious variants will have been selected against during human evolution and, unlike SNPs, should therefore be rare.

1	Against the background of ordinary SNPs inherited from our prehistoric ancestors, certain sequences with exceptionally high mutation rates stand out. A dramatic example is provided by CA repeats, which are ubiquitous in the human genome and in the genomes of other eukaryotes. Sequences with the motif (CA)n are replicated with relatively low fidelity because of a slippage that occurs between the template and the newly synthesized strands during DNA replication; hence, the precise value of n can vary over a considerable range from one genome to the next. These repeats make ideal DNA-based genetic markers, since most humans are heterozygous, having inherited one repeat length (n) from their mother and a different repeat length from their father. While the value of n changes sufficiently rarely that most parent–child transmissions propagate CA repeats faithfully, the changes are sufficiently frequent to maintain high levels of heterozygosity in the human population. These and some other

1	that most parent–child transmissions propagate CA repeats faithfully, the changes are sufficiently frequent to maintain high levels of heterozygosity in the human population. These and some other simple repeats that display exceptionally high variability therefore provide the basis for identifying individuals by DNA analysis in crime investigations, paternity suits, and other forensic applications (see Figure 8–39).

1	While most of the SNPs and CNVs in the human genome sequence are thought to have little or no effect on phenotype, a subset of the genome sequence variations must be responsible for the heritable aspects of human individuality. We know that even a single nucleotide change that alters one amino acid in a protein can cause a serious disease, as for example in sickle-cell anemia, which is caused by such a mutation in hemoglobin (Movie 4.3). We also know that gene dosage—a doubling or halving of the copy number of some genes—can have a profound effect on development by altering the level of gene product, as can changes in regulatory DNA sequences. There is therefore every reason to suppose that some of the many differences between any two human beings will have substantial Figure 4–80 Detection of copy number variations on human chromosome 17.

1	when 100 individuals were tested by a DNA microarray analysis that detects the copy number of DNA sequences throughout the entire length of this chromosome, the indicated distributions of DNA additions (green bars) and DNA losses (red bars) were observed compared with an arbitrary human reference sequence. The shortest red and green bars represent a single occurrence among the 200 chromosomes examined, whereas the longer bars indicate that the addition or loss was correspondingly more frequent. The results show preferred regions where the variations occur, and these tend to be in or near regions that already contain blocks of segmental duplications. many of the changes include known genes. (Adapted from J.l. Freeman et al., Genome Res. 16:949–961, 2006. with permission from Cold Spring Harbor laboratory Press.) effects on human health, physiology, behavior, and physique. A major challenge in human genetics is to recognize those relatively few variations that are functionally important

1	laboratory Press.) effects on human health, physiology, behavior, and physique. A major challenge in human genetics is to recognize those relatively few variations that are functionally important against a large background of variation that is neutral and of no consequence.

1	Comparisons of the nucleotide sequences of present-day genomes have revolutionized our understanding of gene and genome evolution. Because of the extremely high fidelity of DNA replication and DNA repair processes, random errors in maintaining the nucleotide sequences in genomes occur so rarely that only about one nucleotide in a thousand is altered in every million years in any particular eukaryotic line of descent. Not surprisingly, therefore, a comparison of human and chimpanzee chromosomes—which are separated by about 6 million years of evolution— reveals very few changes. Not only are our genes essentially the same, but their order on each chromosome is almost identical. Although a substantial number of segmental duplications and segmental deletions have occurred in the past 6 million years, even the positions of the transposable elements that make up a major portion of our noncoding DNA are mostly unchanged.

1	When one compares the genomes of two more distantly related organisms—such as a human and a mouse, separated by about 80 million years—one finds many more changes. Now the effects of natural selection can be clearly seen: through purifying selection, essential nucleotide sequences—both in regulatory regions and in coding sequences (exons)—have been highly conserved. In contrast, nonessential sequences (for example, much of the DNA in introns) have been altered to such an extent that one can no longer see any family resemblance.

1	Because of purifying selection, the comparison of the genome sequences of multiple related species is an especially powerful way to find DNA sequences with important functions. Although about 5% of the human genome has been conserved as a result of purifying selection, the function of the majority of this DNA (tens of thousands of multispecies conserved sequences) remains mysterious. Future experiments characterizing its functions should teach us many new lessons about vertebrate biology.

1	Other sequence comparisons show that a great deal of the genetic complexity of present-day organisms is due to the expansion of ancient gene families. DNA duplication followed by sequence divergence has clearly been a major source of genetic novelty during evolution. On a more recent time scale, the genomes of any two humans will differ from each other both because of nucleotide substitutions (single-nucleotide polymorphisms, or SNPs) and because of inherited DNA gains and DNA losses that cause copy number variations (CNVs). Understanding the effects of these differences will improve both medicine and our understanding of human biology. Which statements are true? explain why or why not. How many different types of chromatin structure are important for cells? How is each of these structures established and maintained, and which ones tend to be inherited following DNA replication?

1	How many different types of chromatin structure are important for cells? How is each of these structures established and maintained, and which ones tend to be inherited following DNA replication? why are there so many different chromatin remodeling complexes in cells? what are their essential roles, and how do they get loaded onto chromatin at specific places and at specific times? How do chromosomal loops form during interphase, and what happens to these loops in condensed mitotic chromosomes? what genetic changes made us uniquely human? what further aspects of our recent evolutionary development can be reconstructed by sequencing DNA from remains of ancient hominids? How much of the enormous complexity that we find in cell biology is unnecessary, having evolved by random drift? 4–1 Human females have 23 different chromosomes, served DNA sequences facilitates the search for function-whereas human males have 24. ally important regions.

1	4–1 Human females have 23 different chromosomes, served DNA sequences facilitates the search for function-whereas human males have 24. ally important regions. 4–2 The four core histones are relatively small proteins 4–5 Gene duplication and divergence is thought to with a very high proportion of positively charged amino have played a critical role in the evolution of increased bioacids; the positive charge helps the histones bind tightly to logical complexity. DNA, regardless of its nucleotide sequence. Discuss the following problems. 4–3 Nucleosomes bind DNA so tightly that they cannot 4–6 DNA isolated from the bacterial virus M13 con-move from the positions where they are first assembled. tains 25% A, 33% T, 22% C, and 20% G. Do these results 4–4 In a comparison between the DNAs of related strike you as peculiar? Why or why not? How might you organisms such as humans and mice, identifying the con-explain these values?

1	Figure Q4–1 Three nucleotides from the interior of a single strand of DNA (Problem 4–7). Arrows O at the ends of the DNA strand indicate that the structure continues in both directions. 4–7 A segment of DNA from the interior of a single strand is shown in O Figure Q4–1. What is the polarity of this –O P O DNA from top to bottom? O on a molar basis. What are the mole percents of A, G, and T? in the human lineage (Figure Q4–2). Draw the intermediate chromosome that resulted from the first inversion and explicitly indicate the segments O included in each inversion. (Problem 4–9). Differently colored blocks indicate segments of the chromosomes that are homologous in DNA sequence.

1	(Problem 4–9). Differently colored blocks indicate segments of the chromosomes that are homologous in DNA sequence. 4–10 Assuming that the 30-nm chromatin fiber contains about 20 nucleosomes (200 bp/nucleosome) per 50 nm of length, calculate the degree of compaction of DNA associated with this level of chromatin structure. What fraction of the 10,000-fold condensation that occurs at mitosis does this level of DNA packing represent? 4–11 In contrast to histone acetylation, which always correlates with gene activation, histone methylation can lead to either transcriptional activation or repression. How do you suppose that the same modification—methylation—can mediate different biological outcomes?

1	4–12 Why is a chromosome with two centromeres (a dicentric chromosome) unstable? Would a backup centromere not be a good thing for a chromosome, giving it two chances to form a kinetochore and attach to microtubules during mitosis? Would that not help to ensure that the chromosome did not get left behind at mitosis? 4–13 Look at the two yeast colonies in Figure Q4–3. Each of these colonies contains about 100,000 cells descended from a single yeast cell, originally somewhere in the middle of the clump. A white colony arises when the Ade2 gene is expressed from its normal chromosomal location. When the Ade2 gene is moved to a location near a telomere, it is packed into heterochromatin and inactivated in most cells, giving rise to colonies that are mostly red. In these largely red colonies, white sectors fan out from the middle of the colony. In both the red and white sectors, the Ade2 white colony of yeast cells red colony of yeast cells with white sectors

1	Figure Q4–3 Position effect on expression of the yeast Ade2 gene (Problem 4–13). The Ade2 gene codes for one of the enzymes of adenosine biosynthesis, and the absence of the Ade2 gene product leads to the accumulation of a red pigment. Therefore a colony of cells that express Ade2 is white, and one composed of cells in which the Ade2 gene is not expressed is red. gene is still located near telomeres. Explain why white sectors have formed near the rim of the red colony. Based on the patterns observed, what can you conclude about the propagation of the transcriptional state of the Ade2 gene from mother to daughter cells in this experiment?

1	4–14 Mobile pieces of DNA—transposable elements— that insert themselves into chromosomes and accumulate during evolution make up more than 40% of the human genome. Transposable elements of four types—long interspersed nuclear elements (LINEs), short interspersed nuclear elements (SINEs), long terminal repeat (LTR) retrotransposons, and DNA transposons—are inserted more-or-less randomly throughout the human genome. These elements are conspicuously rare at the four homeobox gene clusters, HoxA, HoxB, HoxC, and HoxD, as illustrated for HoxD in Figure Q4–4, along with an equivalent region of chromosome 22, which lacks a Hox cluster. Each Hox cluster is about 100 kb in length and contains 9 to 11 genes, whose differential expression along the anteroposterior axis of the developing embryo establishes the basic body plan for humans (and for other animals). Why do you suppose that transposable elements are so rare in the Hox clusters?

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1	lieberman-Aiden E, van berkum Nl, williams l et al. (2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293. maeshima k & laemmli Uk (2003) A two-step scaffolding model for mitotic chromosome assembly. Dev. Cell 4, 467–480. moser SC & Swedlow JR (2011) How to be a mitotic chromosome. Chromosome Res. 19, 307–319. Nizami ZF, Deryusheva S & gall Jg (2010) Cajal bodies and histone locus bodies in Drosophila and Xenopus. Cold Spring Harb. Symp. Quant. Biol. 75, 313–320. Zhimulev IF (1997) Polytene chromosomes, heterochromatin, and position effect variegation. Adv. Genet. 37, 1–566. batzer mA & Deininger Pl (2002) Alu repeats and human genomic diversity. Nat. Rev. Genet. 3, 370–379. Feuk l, Carson AR & Scherer S (2006) Structural variation in the human genome. Nat. Rev. Genet. 7, 85–97. green RE, krause J, briggs Aw et al. (2010) A draft sequence of the Neandertal genome. Science 328, 710–722.

1	green RE, krause J, briggs Aw et al. (2010) A draft sequence of the Neandertal genome. Science 328, 710–722. International Human genome Sequencing Consortium (2001) Initial sequencing and analysis of the human genome. Nature 409, 860–921. International Human genome Sequencing Consortium (2004) Finishing the euchromatic sequence of the human genome. Nature 431, 931–945. kellis m, wold b, Snyder mP et al. (2014) Defining functional DNA elements in the human genome. Proc. Natl Acad. Sci. USA 111, 6131–6138. lander ES (2011) Initial impact of the sequencing of the human genome. Nature 470, 187–197. lee C & Scherer Sw (2010) The clinical context of copy number variation in the human genome. Expert Rev. Mol. Med. 12, e8. mouse genome Sequencing Consortium (2002) Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520–562.

1	mouse genome Sequencing Consortium (2002) Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520–562. Pollard kS, Salama SR, lambert N et al. (2006) An RNA gene expressed during cortical development evolved rapidly in humans. Nature 443, 167–172. DNA Replication, Repair, and Recombination

1	DNA Replication, Repair, and Recombination The ability of cells to maintain a high degree of order in a chaotic universe depends upon the accurate duplication of vast quantities of genetic information carried in chemical form as DNA. This process, called DNA replication, must occur before a cell can produce two genetically identical daughter cells. Maintaining order also requires the continued surveillance and repair of this genetic information, because DNA inside cells is repeatedly damaged by chemicals and radiation from the environment, as well as by thermal accidents and reactive molecules generated inside the cell. In this chapter, we describe the protein machines that replicate and repair the cell’s DNA. These machines catalyze some of the most rapid and accurate processes that take place within cells, and their mechanisms illustrate the elegance and efficiency of cell chemistry.

1	While the short-term survival of a cell can depend on preventing changes in its DNA, the long-term survival of a species requires that DNA sequences be changeable over many generations to permit evolutionary adaptation to changing circumstances. We shall see that despite the great efforts that cells make to protect their DNA, occasional changes in DNA sequences do occur. Over time, these changes provide the genetic variation upon which selection pressures act during the evolution of organisms. We begin this chapter with a brief discussion of the changes that occur in DNA as it is passed down from generation to generation. Next, we discuss the cell mechanisms—DNA replication and DNA repair—that are responsible for minimizing these changes. Finally, we consider some of the most intriguing pathways that alter DNA sequences—in particular, those of DNA recombination including the movement within chromosomes of special DNA sequences called transposable elements.

1	Although, as just pointed out, occasional genetic changes enhance the long-term survival of a species through evolution, the survival of the individual demands a high degree of genetic stability. Only rarely do the cell’s DNA-maintenance processes fail, resulting in permanent change in the DNA. Such a change is called a mutation, and it can destroy an organism if it occurs in a vital position in the DNA sequence.

1	The mutation rate, the rate at which changes occur in DNA sequences, can be determined directly from experiments carried out with a bacterium such as Escherichia coli—a resident of our intestinal tract and a commonly used laboratory organism (see Figure 1–24). Under laboratory conditions, E. coli divides about once every 30 minutes, and a single cell can generate a very large population— several billion—in less than a day. In such a population, it is possible to detect the small fraction of bacteria that have suffered a damaging mutation in a particular gene, if that gene is not required for the bacterium’s survival. For example, the mutation rate of a gene specifically required for cells to use the sugar lactose as an energy source can be determined by growing the cells in the presence of a different sugar, such as glucose, and testing them subsequently to see how many have lost the ability to survive on a lactose diet. The fraction of damaged genes underestimates the actual mutation

1	a different sugar, such as glucose, and testing them subsequently to see how many have lost the ability to survive on a lactose diet. The fraction of damaged genes underestimates the actual mutation rate because many mutations are silent (for example, those that change a codon but not the amino acid it specifies, or those that change an amino acid without affecting the activity of the protein coded for by the gene). After correcting for these silent mutations, one finds that a single gene that encodes an average-sized protein (~103 coding nucleotide pairs) accumulates a mutation (not necessarily one that would inactivate the protein) approximately once in about 106 bacterial cell generations. Stated differently, bacteria display a mutation rate of about three nucleotide changes per 1010 nucleotides per cell generation.

1	Recently, it has become possible to measure the germ-line mutation rate directly in more complex, sexually reproducing organisms such as humans. In this case, the complete genomes from a family—parents and offspring—were directly sequenced, and a careful comparison revealed that approximately 70 new single-nucleotide mutations arose in the germ lines of each offspring. Normalized to the size of the human genome, the mutation rate is one nucleotide change per 108 nucleotides per human generation. This is a slight underestimate because some mutations will be lethal and will therefore be absent from progeny; however, because relatively little of the human genome carries critical information, this consideration has only a small effect on the true mutation rate. It is estimated that approximately 100 cell divisions occur in the germ line from the time of conception to the time of production of the eggs and sperm that go on to make the next generation. Thus, the human mutation rate,

1	100 cell divisions occur in the germ line from the time of conception to the time of production of the eggs and sperm that go on to make the next generation. Thus, the human mutation rate, expressed in terms of cell divisions (instead of human generations), is approximately 1 mutation/1010 nucleotides/ cell division.

1	Although E. coli and humans differ greatly in their modes of reproduction and in their generation times, when the mutation rates of each are normalized to a single round of DNA replication, they are both extremely low and within a factor of three of each other. We shall see later in the chapter that the basic mechanisms that ensure these low rates of mutation have been conserved since the very early history of cells on Earth. Low Mutation Rates Are Necessary for Life as We Know It

1	Low Mutation Rates Are Necessary for Life as We Know It Since many mutations are deleterious, no species can afford to allow them to accumulate at a high rate in its germ cells. Although the observed mutation frequency is low, it is nevertheless thought to limit the number of essential proteins that any organism can depend upon to perhaps 30,000. More than this, and the probability that at least one critical component will suffer a damaging mutation becomes catastrophically high. By an extension of the same argument, a mutation frequency tenfold higher would limit an organism to about 3000 essential genes. In this case, evolution would have been limited to organisms considerably less complex than a fruit fly.

1	The cells of a sexually reproducing animal or plant are of two types: germ cells and somatic cells. The germ cells transmit genetic information from parent to offspring; the somatic cells form the body of the organism (Figure 5–1). We have seen that germ cells must be protected against high rates of mutation to maintain the species. However, the somatic cells of multicellular organisms must also be protected from genetic change to properly maintain the organized structure of the body. Nucleotide changes in somatic cells can give rise to variant cells, some of which, through “local” natural selection, proliferate rapidly at the expense of the rest of the organism. In an extreme case, the result is the uncontrolled cell proliferation that we know as cancer, a disease that causes (in Europe and North America) more than 20% of human deaths each year. These deaths are due largely to an accumulation of changes in the DNA sequences of somatic cells, as discussed in Chapter 20. A significant

1	and North America) more than 20% of human deaths each year. These deaths are due largely to an accumulation of changes in the DNA sequences of somatic cells, as discussed in Chapter 20. A significant increase in the mutation frequency would presumably cause a disastrous increase in the incidence of cancer by accelerating the rate at which somatic-cell variants arise. Thus, both for the perpetuation of a species with a large number of genes (germ-cell stability) and for the prevention of cancer resulting from mutations in somatic cells (somatic-cell stability), multicellular organisms like ourselves depend on the remarkably high fidelity with which their DNA sequences are replicated and maintained.

1	In all cells, DNA sequences are maintained and replicated with high fidelity. The mutation rate, approximately one nucleotide change per 1010 nucleotides each time the DNA is replicated, is roughly the same for organisms as different as bacteria and humans. Because of this remarkable accuracy, the sequence of the human genome (approximately 3.2 × 109 nucleotide pairs) is unchanged or changed by only a few nucleotides each time a typical human cell divides. This allows most humans to pass accurate genetic instructions from one generation to the next, and also to avoid the changes in somatic cells that lead to cancer. All organisms duplicate their DNA with extraordinary accuracy before each cell division. In this section, we explore how an elaborate “replication machine” achieves this accuracy, while duplicating DNA at rates as high as 1000 nucleotides per second.

1	As introduced in Chapter 1, DNA templating is the mechanism the cell uses to copy the nucleotide sequence of one DNA strand into a complementary DNA sequence (Figure 5–2). This process requires the separation of the DNA helix into two template strands, and entails the recognition of each nucleotide in the DNA template strands by a free (unpolymerized) complementary nucleotide. The separation of Figure 5–1 Germ-line cells and somatic cells carry out fundamentally different functions. In sexually reproducing organisms, the germ-line cells (red) propagate genetic information into the next generation. Somatic cells (blue), which form the body of the organism, are necessary for the survival of germ-line cells but do not themselves leave any progeny. Figure 5–2 The DNA double helix acts as a template for its own duplication.

1	Figure 5–2 The DNA double helix acts as a template for its own duplication. Because the nucleotide A will pair successfully only with T, and G only with C, each strand of DNA can serve as a template to specify the sequence of nucleotides in its complementary strand by DNA base-pairing. In this way, a double-helical DNA molecule can be copied precisely. the DNA helix exposes the hydrogen-bond donor and acceptor groups on each DNA base for base-pairing with the appropriate incoming free nucleotide, aligning it for its enzyme-catalyzed polymerization into a new DNA chain. The first nucleotide-polymerizing enzyme, DNA polymerase, was discovered in 1957. The free nucleotides that serve as substrates for this enzyme were found to be deoxyribonucleoside triphosphates, and their polymerization into DNA required a single-strand DNA template. Figure 5–3 and Figure 5–4 illustrate the stepwise mechanism of this reaction. The DNA Replication Fork Is Asymmetrical

1	The DNA Replication Fork Is Asymmetrical During DNA replication inside a cell, each of the two original DNA strands serves as a template for the formation of an entire new strand. Because each of the two daughters of a dividing cell inherits a new DNA double helix containing one original and one new strand (Figure 5–5), the DNA double helix is said to be replicated “semiconservatively.” How is this feat accomplished? 3˜ end of strand 5˜ end of strand Figure 5–3 The chemistry of DNA synthesis. The addition of a deoxyribonucleotide to the 3ʹ end of a polynucleotide chain (the primer strand) is the fundamental reaction by which DNA is synthesized. As shown, base-pairing between an incoming deoxyribonucleoside triphosphate and an existing strand of DNA (the template strand) guides the formation of the new strand of DNA and causes it to have a complementary nucleotide sequence. The way in which complementary nucleotides base-pair is shown in Figure 4–4. direction of chain growth

1	Figure 5–4 DNA synthesis catalyzed by DNA polymerase. (A) DNA polymerase catalyzes the stepwise addition of a deoxyribonucleotide to the 3ʹ-OH end of a polynucleotide chain, the growing primer strand that is paired to an existing template strand. The newly synthesized DNA strand therefore polymerizes in the 5ʹ-to-3ʹ direction as shown also in the previous figure. Because each incoming deoxyribonucleoside triphosphate must pair with the template strand to be recognized by the DNA polymerase, this strand determines which of the four possible deoxyribonucleotides (A, C, G, or T) will be added. The reaction is driven by a large, favorable free-energy change, caused by the release of pyrophosphate and its subsequent hydrolysis to two molecules of inorganic phosphate. (B) Structure of DNA polymerase complexed wth DNA (orange), as determined by x-ray crystallography (Movie 5.1). The template DNA strand is the longer strand and the newly synthesized DNA is the shorter. (C) Schematic diagram

1	complexed wth DNA (orange), as determined by x-ray crystallography (Movie 5.1). The template DNA strand is the longer strand and the newly synthesized DNA is the shorter. (C) Schematic diagram of DNA polymerase, based on the structure in (B). The proper base-pair geometry of a correct incoming deoxyribonucleoside triphosphate causes the polymerase to tighten around the base pair, thereby initiating the nucleotide addition reaction (middle diagram (C)). Dissociation of pyrophosphate relaxes the polymerase, allowing translocation of the DNA by one nucleotide so the active site of the polymerase is ready to receive the next deoxyribonucleoside triphosphate.

1	Analyses carried out in the early 1960s on whole replicating chromosomes revealed a localized region of replication that moves progressively along the parental DNA double helix. Because of its Y-shaped structure, this active region is called a replication fork (Figure 5–6). At the replication fork, a multienzyme complex that contains the DNA polymerase synthesizes the DNA of both new daughter strands.

1	Initially, the simplest mechanism of DNA replication seemed to be the continuous growth of both new strands, nucleotide by nucleotide, at the replication fork as it moves from one end of a DNA molecule to the other. But because of the antiparallel orientation of the two DNA strands in the DNA double helix (see Figure 5–2), this mechanism would require one daughter strand to polymerize in the 5ʹ-to-3ʹ direction and the other in the 3ʹ-to-5ʹ direction. Such a replication fork would require two distinct types of DNA polymerase enzymes. However, as attractive as this model might be, the DNA polymerases at replication forks can synthesize only in the 5ʹ-to-3ʹ direction.

1	How, then, can a DNA strand grow in the 3ʹ-to-5ʹ direction? The answer was first suggested by the results of an experiment performed in the late 1960s. Researchers added highly radioactive 3H-thymidine to dividing bacteria for a few seconds, so that only the most recently replicated DNA—that just behind the replication fork—became radiolabeled. This experiment revealed the transient existence of pieces of DNA that were 1000–2000 nucleotides long, now commonly known as Okazaki fragments, at the growing replication fork. (Similar replication Figure 5–5 The semiconservative nature of DNA replication. In a round of replication, each of the two strands of DNA is used as a template for the formation of a complementary DNA strand. The original strands therefore remain intact through many cell generations.

1	intermediates were later found in eukaryotes, where they are only 100–200 nucleotides long.) The Okazaki fragments were shown to be polymerized only in the 5ʹ-to-3ʹ chain direction and to be joined together after their synthesis to create long DNA chains. A replication fork therefore has an asymmetric structure (Figure 5–7). The DNA daughter strand that is synthesized continuously is known as the leading strand. Its synthesis slightly precedes the synthesis of the daughter strand that is synthesized discontinuously, known as the lagging strand. For the lagging strand, the direction of nucleotide polymerization is opposite to the overall direction of DNA chain growth. The synthesis of this strand by a discontinuous “backstitching” mechanism means that DNA replication requires only the 5ʹ-to-3ʹ type of DNA polymerase. The High Fidelity of DNA Replication Requires Several Proofreading Mechanisms

1	The High Fidelity of DNA Replication Requires Several Proofreading Mechanisms As discussed above, the fidelity of copying DNA during replication is such that only about one mistake occurs for every 1010 nucleotides copied. This fidelity is much higher than one would expect from the accuracy of complementary base-pairing. The standard complementary base pairs (see Figure 4–4) are not the only ones possible. For example, with small changes in helix geometry, two hydrogen bonds can form between G and T in DNA. In addition, rare tautomeric forms of the four DNA bases occur transiently in ratios of 1 part to 104 or 105. These forms mispair without a change in helix geometry: the rare tautomeric form of C pairs with A instead of G, for example.

1	If the DNA polymerase did nothing special when a mispairing occurred between an incoming deoxyribonucleoside triphosphate and the DNA template, the wrong nucleotide would often be incorporated into the new DNA chain, producing frequent mutations. The high fidelity of DNA replication, however, depends not only on the initial base-pairing but also on several “proofreading” mechanisms that act sequentially to correct any initial mispairings that might have occurred. Figure 5–6 Two replication forks moving in opposite directions on a circular chromosome. An active zone of DNA replication moves progressively along a replicating DNA molecule, creating a Y-shaped DNA structure known as a replication fork: the two arms of each Y are the two daughter DNA molecules, and the stem of the Y is the parental DNA helix. In this diagram, parental strands are orange; newly synthesized strands are red. 1 µm (Micrograph courtesy of Jerome Vinograd.)

1	Figure 5–7 The structure of a DNA replication fork. Left, replication fork with newly synthesized DNA in red and arrows indicating the 5ʹ-to-3ʹ direction of DNA synthesis. Because both daughter DNA strands are polymerized in the 5ʹ-to-3ʹ direction, the DNA synthesized on the lagging strand must be made initially as a series of short DNA molecules, called Okazaki fragments, named after the scientist who discovered them. Right, the same fork a short time later. On the lagging strand, the Okazaki fragments are synthesized sequentially, with those nearest the fork being the most recently made.

1	DNA polymerase performs the first proofreading step just before a new nucleotide is covalently added to the growing chain. Our knowledge of this mechanism comes from studies of several different DNA polymerases, including one produced by a bacterial virus, T7, that replicates inside E. coli. The correct nucleotide has a higher affinity for the moving polymerase than does the incorrect nucleotide, because the correct pairing is more energetically favorable. Moreover, after nucleotide binding, but before the nucleotide is covalently added to the grow-AAAAAAAAA ing chain, the enzyme must undergo a conformational change in which its “grip” tightens around the active site (see Figure 5–4). Because this change occurs more readily with correct than incorrect base-pairing, it allows the polymerase to “double-check” the exact base-pair geometry before it catalyzes the addition of the nucleotide. Incorrectly paired nucleotides are harder to add and therefore more likely to diffuse away before

1	to “double-check” the exact base-pair geometry before it catalyzes the addition of the nucleotide. Incorrectly paired nucleotides are harder to add and therefore more likely to diffuse away before the polymerase can mistakenly add them.

1	The next error-correcting reaction, known as exonucleolytic proofreading, takes place immediately after those rare instances in which an incorrect nucle otide is covalently added to the growing chain. DNA polymerase enzymes are unpaired or mispaired residues at the primer terminus, continuing until enough nucleotides have been removed to regenerate a correctly base-paired 3ʹ-OH ter minus that can prime DNA synthesis. In this way, DNA polymerase functions as a attached to DNA polymerase “self-correcting” enzyme that removes its own polymerization errors as it moves chews back to create a base- along the DNA (Figure 5–8 and Figure 5–9). paired 3˜-OH end on the primer

1	paired 3˜-OH end on the primer The self-correcting properties of the DNA polymerase depend on its require ment for a perfectly base-paired primer terminus, and it is apparently not possible for such an enzyme to start synthesis de novo, without an existing primer. By contrast, the RNA polymerase enzymes involved in gene transcription do not need such an efficient exonucleolytic proofreading mechanism: errors in making RNA are not passed on to the next generation, and the occasional defective RNA molecule that is produced has no long-term significance. RNA polymerases are thus able to start new polynucleotide chains without a primer. DNA polymerase resumes the process of adding nucleotides to the base-paired 3˜-OH end of the primer strand

1	DNA polymerase resumes the process of adding nucleotides to the base-paired 3˜-OH end of the primer strand Figure 5–8 Exonucleolytic proofreading by DNA polymerase during DNA replication. In this example, a C is accidentally incorporated at the growing 3ʹ-OH end of a DNA chain. The part of DNA polymerase that removes the misincorporated nucleotide is a specialized member of a large class of enzymes, known as exonucleases, that cleave nucleotides one at a time from the ends of polynucleotides. Figure 5–9 Editing by DNA polymerase. DNA polymerase complexed with the DNA template in the polymerizing mode (left) and the editing mode (right). The catalytic sites for the exonucleolytic (E) and the polymerization (P) reactions are indicated. In the editing mode, the newly synthesized DNA transiently unpairs from the template and enters the editing site where the most recently added nucleotide is catalytically removed.

1	There is an error frequency of about one mistake for every 104 polymerization events both in RNA synthesis and in the separate process of translating mRNA sequences into protein sequences. This error rate is over 100,000 times greater than that in DNA replication, where, as we have seen, a series of proofreading processes makes the process unusually accurate (Table 5–1). Only DNA Replication in the 5ʹ-to-3ʹ Direction Allows Efficient Error Correction

1	Only DNA Replication in the 5ʹ-to-3ʹ Direction Allows Efficient Error Correction The need for accuracy probably explains why DNA replication occurs only in the 5ʹ-to-3ʹ direction. If there were a DNA polymerase that added deoxyribonucleoside triphosphates in the 3ʹ-to-5ʹ direction, the growing 5ʹ end of the chain, rather than the incoming mononucleotide, would have to provide the activating triphosphate needed for the covalent linkage. In this case, the mistakes in polymerization could not be simply hydrolyzed away, because the bare 5ʹ end of the chain thus created would immediately terminate DNA synthesis (see Figure 5–3). It is therefore possible to correct a mismatched base only if it has been added to the 3ʹ end of a DNA chain. Although the backstitching mechanism for DNA replication seems complex, it preserves the 5ʹ-to-3ʹ direction of polymerization that is required for exonucleolytic proofreading.

1	Despite these safeguards against DNA replication errors, DNA polymerases occasionally make mistakes. However, as we shall see later, cells have yet another Figure 5–10 RNA primer synthesis. A schematic view of the reaction catalyzed by DNA primase, the enzyme that synthesizes the short RNA primers made on the lagging strand using DNA as a template. Unlike DNA polymerase, this enzyme can start a new polynucleotide chain by joining two nucleoside triphosphates together. The primase synthesizes a short polynucleotide in the 5ʹ-to-3ʹ direction and then stops, making the 3ʹ end of this primer available for the DNA polymerase. chance to correct these errors by a process called strand-directed mismatch repair. Before discussing this mechanism, however, we describe the other types of proteins that function at the replication fork. A Special Nucleotide-Polymerizing Enzyme Synthesizes Short RNA Primer Molecules on the Lagging Strand

1	A Special Nucleotide-Polymerizing Enzyme Synthesizes Short RNA Primer Molecules on the Lagging Strand For the leading strand, a primer is needed only at the start of replication: once a replication fork is established, the DNA polymerase is continuously presented with a base-paired chain end on which to add new nucleotides. On the lagging side of the fork, however, each time the DNA polymerase completes a short DNA Okazaki fragment (which takes a few seconds), it must start synthesizing a completely new fragment at a site further along the template strand (see Figure 5–7). A special mechanism produces the base-paired primer strand required by the DNA polymerase molecules. The mechanism depends on an enzyme called DNA primase, which uses ribonucleoside triphosphates to synthesize short RNA primers on the lagging strand (Figure 5–10). In eukaryotes, these primers are about 10 nucleotides long and are made at intervals of 100–200 nucleotides on the lagging strand.

1	The chemical structure of RNA was introduced in Chapter 1 and is described in detail in Chapter 6. Here, we note only that RNA is very similar in structure to DNA. A strand of RNA can form base pairs with a strand of DNA, generating a DNA–RNA hybrid double helix if the two nucleotide sequences are complementary. Thus, the same templating principle used for DNA synthesis guides the synthesis of RNA primers. Because an RNA primer contains a properly base-paired nucleotide with a 3ʹ-OH group at one end, it can be elongated by the DNA polymerase at this end to begin an Okazaki fragment. The synthesis of each Okazaki fragment ends when this DNA polymerase runs into the RNA primer attached to the 5ʹ end of the previous fragment. To produce a continuous DNA chain from the many DNA fragments made on the lagging strand, a special DNA repair system acts quickly to erase the old RNA primer and replace it with DNA. An enzyme called DNA ligase then joins the 3ʹend of the new DNA fragment to the

1	on the lagging strand, a special DNA repair system acts quickly to erase the old RNA primer and replace it with DNA. An enzyme called DNA ligase then joins the 3ʹend of the new DNA fragment to the 5ʹend of the previous one to complete the process (Figure 5–11 and Figure 5–12).

1	Why might an erasable RNA primer be preferred to a DNA primer that would not need to be erased? The argument that a self-correcting polymerase cannot start chains de novo also implies the converse: an enzyme that starts chains anew cannot be efficient at self-correction. Thus, any enzyme that primes the synthesis of Okazaki fragments will of necessity make a relatively inaccurate copy (at least one error in 105). Even if the copies retained in the final product constituted as little as 5% of the total genome (for example, 10 nucleotides per 200-nucleotide DNA fragment), the resulting increase in the overall mutation rate would be enormous. It therefore seems likely that the use of RNA rather than DNA for priming brings a powerful advantage to the cell: the ribonucleotides in the primer automatically mark these sequences as “suspect copy” to be efficiently removed and replaced.

1	Figure 5–11 The synthesis of one of many DNA fragments on the lagging strand. In eukaryotes, RNA primers are made at intervals spaced by about 200 nucleotides on the lagging strand, and each RNA primer is approximately 10 nucleotides long. This primer is erased by a special DNA repair enzyme (an RNAse H) that recognizes an RNA strand in an RNA/DNA helix and fragments it; this leaves gaps that are filled in by DNA polymerase and DNA ligase. DNA polymerase adds to new RNA primer to start new Okazaki fragment sealing by DNA ligase joins new Okazaki fragment to the growing chain Special Proteins Help to Open Up the DNA Double Helix in Front of the Replication Fork

1	Special Proteins Help to Open Up the DNA Double Helix in Front of the Replication Fork For DNA synthesis to proceed, the DNA double helix must be opened up (“melted”) ahead of the replication fork so that the incoming deoxyribonucleoside triphosphates can form base pairs with the template strands. However, the DNA double helix is very stable under physiological conditions; the base pairs are locked in place so strongly that it requires temperatures approaching that of boiling water to separate the two strands in a test tube. For this reason, two additional types of replication proteins—DNA helicases and single-strand DNA-binding proteins—are needed to open the double helix and provide the appropriate single-strand DNA templates for the DNA polymerase to copy.

1	DNA helicases were first isolated as proteins that hydrolyze ATP when they are bound to single strands of DNA. As described in Chapter 3, the hydrolysis of ATP can change the shape of a protein molecule in a cyclical manner that allows the protein to perform mechanical work. DNA helicases use this principle to propel themselves rapidly along a DNA single strand. When they encounter a region of double helix, they continue to move along their strand, thereby prying apart the helix at rates of up to 1000 nucleotide pairs per second (Figure 5–13 and Figure 5–14). The two strands of DNA have opposite polarities, and, in principle, a helicase

1	The two strands of DNA have opposite polarities, and, in principle, a helicase Figure 5–12 The reaction catalyzed by DNA ligase. This enzyme seals a broken phosphodiester bond. As shown, DNA ligase uses a molecule of ATP to activate the 5ʹ end at the nick (step 1) before forming the new bond (step 2). In this way, the energetically unfavorable nick-sealing reaction is driven by being coupled to the energetically favorable process of ATP hydrolysis. could unwind the DNA double helix by moving in the 5ʹ-to-3ʹ direction along one 5˜ 3˜ strand or in the 3ʹ-to-5ʹ direction along the other. In fact, both types of DNA helicase exist. In the best-understood replication systems in bacteria, a helicase moving 5ʹ to 3ʹ along the lagging-strand template appears to have the predominant role, for reasons that will become clear shortly.

1	Single-strand DNA-binding (SSB) proteins, also called helix-destabilizing proteins, bind tightly and cooperatively to exposed single-strand DNA without covering the bases, which therefore remain available as templates. These proteins are unable to open a long DNA helix directly, but they aid helicases by stabilizing the unwound, single-strand conformation. In addition, through cooperative binding, they coat and straighten out the regions of single-strand DNA, which occur routinely in the lagging-strand template, thereby preventing the formation of the short hairpin helices that readily form in single-strand DNA (Figure 5–15 and Figure 5–16). If not removed, these hairpin helices can impede the DNA synthesis catalyzed by DNA polymerase. A Sliding Ring Holds a Moving DNA Polymerase Onto the DNA

1	A Sliding Ring Holds a Moving DNA Polymerase Onto the DNA On their own, most DNA polymerase molecules will synthesize only a short string of nucleotides before falling off the DNA template. The tendency to dissociate quickly from a DNA molecule allows a DNA polymerase molecule that has just Figure 5–13 An assay for DNA helicase enzymes. A short DNA fragment is annealed to a long DNA single strand to form a region of DNA double helix. The double helix is melted as the helicase runs along the DNA single strand, releasing the short DNA fragment in a reaction that requires the presence of both the helicase protein and ATP. The rapid stepwise movement of the helicase is powered by its ATP hydrolysis (shown schematically in Figure 3–75A). As indicated, many DNA helicases are composed of six subunits.

1	Figure 5–14 The structure of a DNA helicase. (A) Diagram of the protein 5˜ as a hexameric ring drawn to scale with a replication fork. (B) Detailed structure of the bacteriophage T7 replicative helicase, as determined by x-ray diffraction. Six identical subunits bind and hydrolyze ATP in an ordered fashion to propel this molecule, like a rotary engine, along a DNA single strand that passes through the central hole. Red indicates bound ATP molecules in the structure (Movie 5.2). (PDB code: 1E0J.) finished synthesizing one Okazaki fragment on the lagging strand to be recycled quickly, so as to begin the synthesis of the next Okazaki fragment on the same strand. This rapid dissociation, however, would make it difficult for the polymerase to synthesize the long DNA strands produced at a replication fork were it not for an accessory protein (called PCNA in eukaryotes) that functions as a regulated sliding clamp. This clamp keeps the polymerase firmly on the DNA when it is moving, but

1	replication fork were it not for an accessory protein (called PCNA in eukaryotes) that functions as a regulated sliding clamp. This clamp keeps the polymerase firmly on the DNA when it is moving, but releases it as soon as the polymerase runs into a double-strand region of DNA.

1	How can a sliding clamp prevent the polymerase from dissociating without at the same time impeding the polymerase’s rapid movement along the DNA molecule? The three-dimensional structure of the clamp protein, determined by x-ray diffraction, revealed it to be a large ring around the DNA double helix. One face of the ring binds to the back of the DNA polymerase, and the whole ring slides freely along the DNA as the polymerase moves. The assembly of the clamp around the DNA requires ATP hydrolysis by a special protein complex, the clamp loader, which hydrolyzes ATP as it loads the clamp on to a primer–template junction (Figure 5–17).

1	On the leading-strand template, the moving DNA polymerase is tightly bound to the clamp, and the two remain associated for a very long time. The DNA polymerase on the lagging-strand template also makes use of the clamp, but each time the polymerase reaches the 5ʹ end of the preceding Okazaki fragment, the polymerase releases itself from the clamp and dissociates from the template. This polymerase molecule then associates with a new clamp that is assembled on the RNA primer of the next Okazaki fragment. single-stranded region of DNA template with short regions of base-paired “hairpins”

1	single-stranded region of DNA template with short regions of base-paired “hairpins” Figure 5–15 The effect of single-strand DNA-binding proteins (SSb proteins) on the structure of single-strand DNA. Because each protein molecule prefers to bind next to a previously bound molecule, long rows of this protein form on a DNA single strand. This cooperative binding straightens out the DNA template and facilitates the DNA polymerization process. The “hairpin helices” shown in the bare, single-strand DNA result from a chance matching of short regions of complementary nucleotide sequence; they are similar to the short helices that typically form in RNA molecules (see Figure 1–6).

1	Figure 5–16 Human single-strand binding protein bound to DNA. (A) Front view of the two DNA-binding domains of the protein (called RPA) which cover a total of eight nucleotides. Note that the DNA bases remain exposed in this protein–DNA complex. (B) Diagram showing the three-dimensional structure, with the DNA strand (orange) viewed end-on. (PDB code: 1JMC.)

1	Figure 5–17 The regulated sliding clamp that holds DNA polymerase on the DNA. (A) The structure of the clamp protein from E. coli, as determined by x-ray crystallography, with a DNA helix added to indicate how the protein fits around DNA (Movie 5.3). (B) Schematic illustration showing how the clamp (with red and yellow subunits) is loaded onto DNA to serve as a tether for a moving DNA polymerase molecule. The structure of the clamp loader (dark green) resembles a screw nut, (A) (B) sliding clamp clamp loader 5˜ATPADPPi ATPATP+ DNA + DNA polymerase 3˜5˜5˜3˜3˜RECYCLING OF RELEASED CLAMP LOADER ATP BINDING TO CLAMP LOADER OPENS SLIDING CLAMP DNA ENGAGED IN CLAMP ATP HYDROLYSIS LOCKS SLIDING CLAMP AROUND DNA AND RELEASES CLAMP LOADER DNA POLYMERASE BINDS TO SLIDING CLAMP + with its threads matching the grooves of double-stranded DNA. The loader binds to a free clamp molecule, forcing a gap in its ring of subunits so that this ring is able to slip around DNA. The clamp loader, thanks to

1	matching the grooves of double-stranded DNA. The loader binds to a free clamp molecule, forcing a gap in its ring of subunits so that this ring is able to slip around DNA. The clamp loader, thanks to its screw-nut structure, recognises the region of DNA that is double-stranded and latches onto it, tightening around the complex of a template strand with a freshly synthesized elongating (primer) strand. It carries the clamp along this double-stranded region until it encounters the 3ʹ end of the primer, at which point the loader hydrolyzes ATP and releases the clamp, allowing it to close around the DNA and bind to DNA polymerase. In the simplified reaction shown here, the clamp loader dissociates into solution once the clamp has been assembled. At a true replication fork, the clamp loader remains close to the polymerase so that, on the lagging strand, it is ready to assemble a new clamp at the start of each new Okazaki fragment (see Figure 5–18). (A, from X.P. Kong et al., Cell

1	remains close to the polymerase so that, on the lagging strand, it is ready to assemble a new clamp at the start of each new Okazaki fragment (see Figure 5–18). (A, from X.P. Kong et al., Cell 69:425–437, 1992. With permission from Elsevier; B, adapted from B.A. Kelch et al., Science 334:1675–1680, 2011. With permission from AAAS. PDB code: 3BEP.)

1	The Proteins at a Replication Fork Cooperate to Form a Replication Machine Although we have discussed DNA replication as though it were performed by a mix of proteins all acting independently, in reality most of the proteins are held together in a large and orderly multienzyme complex that rapidly synthesizes DNA. This complex can be likened to a tiny sewing machine composed of protein parts and powered by nucleoside triphosphate hydrolysis. Like a sewing machine, the replication complex probably remains stationary with respect to its immediate surroundings; the DNA can be thought of as a long strip of cloth being rapidly threaded through it. Although the replication complex has been most intensively studied in E. coli and several of its viruses, a very similar complex also operates in eukaryotes, as we see below.

1	Figure 5–18 summarizes the functions of the subunits of the replication machine. At the front of the replication fork, DNA helicase opens the DNA helix. Two DNA polymerase molecules work at the fork, one on the leading strand and one on the lagging strand. Whereas the DNA polymerase molecule on the leading strand can operate in a continuous fashion, the DNA polymerase molecule on the lagging strand must restart at short intervals, using a short RNA primer made by a DNA primase molecule. The close association of all these protein components increases the efficiency of replication and is made possible by a folding back of the lagging strand as shown in Figure 5–18A. This arrangement also facilitates the loading of the polymerase clamp each time that an Okazaki fragment is synthesized: the clamp loader and the lagging-strand DNA polymerase molecule are kept in place as a part of the protein machine even when they detach from their DNA template. The replication proteins are thus linked

1	clamp loader and the lagging-strand DNA polymerase molecule are kept in place as a part of the protein machine even when they detach from their DNA template. The replication proteins are thus linked together into a single large

1	Figure 5–18 A bacterial replication fork. (A) This schematic diagram shows a current view of the arrangement of replication proteins at a replication fork when DNA is being synthesized. The lagging-strand DNA is folded to bring the lagging-strand DNA polymerase molecule into a complex with the leading-strand DNA polymerase molecule. This folding also brings the 3ʹ end of each completed Okazaki fragment close to the start site for the next Okazaki fragment. Because the lagging-strand DNA polymerase molecule remains bound to the rest of the replication proteins, it can be reused to synthesize successive Okazaki fragments. In this diagram, it is about to let go of its completed DNA fragment and move to the RNA primer that is just being synthesized. Additional proteins (not shown) help to hold the different protein components of the fork together, enabling them to function as a well-coordinated protein machine (Movie 5.4 and Movie 5.5). (B) An electron micrograph showing the replication

1	the different protein components of the fork together, enabling them to function as a well-coordinated protein machine (Movie 5.4 and Movie 5.5). (B) An electron micrograph showing the replication machine from the bacteriophage T4 as it moves along a template synthesizing DNA behind it. (C) An interpretation of the micrograph is given in the sketch: note especially the DNA loop on the lagging strand. Apparently, the replication proteins became partly detached from the very front of the replication fork during the preparation of this sample for electron microscopy. (B, courtesy of Jack Griffith; see P.D. Chastain et al., J. Biol. Chem. 278:21276–21285, 2003.) unit (total molecular mass >106 daltons), enabling DNA to be synthesized on both sides of the replication fork in a coordinated and efficient manner.

1	On the lagging strand, the DNA replication machine leaves behind a series of unsealed Okazaki fragments, which still contain the RNA that primed their synthesis at their 5ʹ ends. As discussed earlier, this RNA is removed and the resulting gap is filled in by DNA repair enzymes that operate behind the replication fork (see Figure 5–11). A Strand-Directed Mismatch Repair System Removes Replication Errors That Escape from the Replication Machine

1	A Strand-Directed Mismatch Repair System Removes Replication Errors That Escape from the Replication Machine As stated previously, bacteria such as E. coli are capable of dividing once every 30 minutes, making it relatively easy to screen large populations to find a rare mutant cell that is altered in a specific process. One interesting class of mutants consists of those with alterations in so-called mutator genes, which greatly increase the rate of spontaneous mutation. Not surprisingly, one such mutant makes a defective form of the 3ʹ-to-5ʹ proofreading exonuclease that is a part of the DNA polymerase enzyme (see Figures 5–8 and 5–9). The mutant DNA polymerase no longer proofreads effectively, and many replication errors that would otherwise have been removed accumulate in the DNA.

1	The study of other E. coli mutants exhibiting abnormally high mutation rates has uncovered a proofreading system that removes replication errors made by the polymerase that have been missed by the proofreading exonuclease. This strand-directed mismatch repair system detects the potential for distortion in the DNA helix from the misfit between noncomplementary base pairs. If the proofreading system simply recognized a mismatch in newly replicated DNA and randomly corrected one of the two mismatched nucleotides, it would mistakenly “correct” the original template strand to match the error exactly half the time, thereby failing to lower the overall error rate. To be effective, such a proofreading system must be able to distinguish and remove the mismatched nucleotide only on the newly synthesized strand, where the replication error occurred.

1	The strand-distinction mechanism used by the mismatch proofreading system in E. coli depends on the methylation of selected A residues in the DNA. Methyl groups are added to all A residues in the sequence GATC, but not until some time after the A has been incorporated into a newly synthesized DNA chain. As a result, the only GATC sequences that have not yet been methylated are in the new strands just behind a replication fork. The recognition of these unmethylated GATCs allows the new DNA strands to be transiently distinguished from old ones, as required if their mismatches are to be selectively removed. The three-step process involves recognition of a newly synthesized strand, excision of the portion containing the mismatch, and resynthesis of the excised segment using the old strand as a template. This strand-directed mismatch repair system reduces the number of errors made during DNA replication by an additional factor of 100 to 1000 (see Table 5–1, p. 244).

1	A similar mismatch proofreading system functions in eukaryotic cells but uses a different strategy to distinguish the new strand from the old (Figure 5–19). Newly synthesized lagging-strand DNA transiently contains nicks (before they are sealed by DNA ligase) and such nicks (also called single-strand breaks) provide the signal that directs the mismatch proofreading system to the appropriate strand. This strategy also requires that the newly synthesized DNA on the leading strand be transiently nicked; how this occurs is uncertain.

1	The importance of mismatch proofreading in humans is seen in individuals who inherit one defective copy of a mismatch repair gene (along with a functional gene on the other copy of the chromosome). These people have a marked predisposition for certain types of cancers. For example, in a type of colon cancer called hereditary nonpolyposis colon cancer (HNPCC), spontaneous mutation of the one functional gene produces a clone of somatic cells that, because they are deficient in mismatch proofreading, accumulate mutations unusually rapidly. Most cancers arise in cells that have accumulated multiple mutations (see pp. 1096–1097), and cells deficient in mismatch proofreading therefore have a greatly enhanced chance of becoming cancerous. Fortunately, most of us inherit two good copies of each gene that encodes a mismatch proofreading protein; this protects us, because it is highly unlikely for both copies to become mutated in the same cell.

1	As a replication fork moves along double-strand DNA, it creates what has been called the “winding problem.” The two parental strands, which are wound around each other, must be unwound and separated for replication to occur. For every 10 nucleotide pairs replicated at the fork, one complete turn of the parental double helix must be unwound. In principle, this unwinding can be achieved by rapidly rotating the entire chromosome ahead of a moving fork; however, this is energetically highly unfavorable (particularly for long chromosomes) and, instead, the DNA in front of a replication fork becomes overwound (Figure 5–20). The over-winding, in turn, is continually relieved by proteins known as DNA topoisomerases. A DNA topoisomerase can be viewed as a reversible nuclease that adds itself covalently to a DNA backbone phosphate, thereby breaking a phosphodiester bond in a DNA strand. This reaction is reversible, and the phosphodiester bond re-forms as the protein leaves.

1	One type of topoisomerase, called topoisomerase I, produces a transient sin-gle-strand break; this break in the phosphodiester backbone allows the two sections of DNA helix on either side of the nick to rotate freely relative to each other, using the phosphodiester bond in the strand opposite the nick as a swivel point (Figure 5–21). Any tension in the DNA helix will drive this rotation in the direction that relieves the tension. As a result, DNA replication can occur with the rotation of only a short length of helix—the part just ahead of the fork. Because the covalent linkage that joins the DNA topoisomerase protein to a DNA phosphate retains rotate, torsional stress will build up

1	Figure 5–19 Strand-directed mismatch repair. (A) The two proteins shown are present in both bacteria and eukaryotic cells: MutS binds specifically to a mismatched base pair, while MutL scans the nearby DNA for a nick. Once MutL finds a nick, it triggers the degradation of the nicked strand all the way back through the mismatch. Because nicks are largely confined to newly replicated strands in eukaryotes, replication errors are selectively removed. In bacteria, an additional protein in the complex (MutH) nicks unmethylated (and therefore newly replicated) GATC sequences, thereby beginning the process illustrated here. In eukaryotes, MutL contains a DNA nicking activity that aids in the removal of the damaged strand.

1	(B) The structure of the MutS protein bound to a DNA mismatch. This protein is a dimer, which grips the DNA double helix as shown, kinking the DNA at the mismatched base pair. It seems that the MutS protein scans the DNA for mismatches by testing for sites that can be readily kinked, which are those with an abnormal base pair. (PDB code: 1EWQ.) Figure 5–20 The “winding problem” that arises during DNA replication.

1	Figure 5–20 The “winding problem” that arises during DNA replication. (A) For a bacterial replication fork moving at 500 nucleotides per second, the parental DNA helix ahead of the fork must rotate at 50 revolutions per second. (B) If the ends of the DNA double helix remain fixed (or difficult to rotate), tension builds up in front of the replication fork as it becomes overwound. Some of this tension can be taken up by supercoiling, whereby the DNA double helix twists around itself (see Figure 6–19). However, if the tension continues to build up, the replication fork will eventually stop because further unwinding requires more energy than the helicase can provide. Note that in (A), the dotted line represents about 20 turns of DNA. one end of the DNA double helix cannot rotate relative to the other end topoisomerase with tyrosine at the active site

1	one end of the DNA double helix cannot rotate relative to the other end topoisomerase with tyrosine at the active site CH2 OH CH2 OH DNA topoisomerase covalently attaches to a DNA phosphate, thereby breaking a phosphodiester linkage in one DNA strand the two ends of the DNA double helix can now rotate relative to each other, relieving accumulated strain Figure 5–21 The reversible DNA nicking reaction catalyzed by a eukaryotic DNA topoisomerase I enzyme. As indicated, these enzymes transiently form a single covalent bond with DNA; this allows free rotation of the DNA around the covalent backbone bonds linked to the blue phosphate.

1	CH2HO the original phosphodiester bond energy is stored in the phosphotyrosine linkage, making the reaction reversible CH2 OH spontaneous re-formation of the phosphodiester bond regenerates both the DNA helix and the DNA topoisomerase the energy of the cleaved phosphodiester bond, resealing is rapid and does not require additional energy input. In this respect, the rejoining mechanism differs from that catalyzed by the enzyme DNA ligase, discussed previously (see Figure 5–12). A second type of DNA topoisomerase, topoisomerase II, forms a covalent linkage to both strands of the DNA helix at the same time, making a transient

1	A second type of DNA topoisomerase, topoisomerase II, forms a covalent linkage to both strands of the DNA helix at the same time, making a transient Figure 5–22 The DNA-helix-passing reaction catalyzed by DNA topoisomerase II. Unlike type I topoisomerases, type II enzymes hydrolyze ATP (red), which is needed to release and reset the enzyme after each cycle. Type II topoisomerases are largely confined to proliferating cells in eukaryotes; partly for that reason, they have been effective targets for anticancer drugs. Some of these drugs inhibit topoisomerase II at the third step in the figure and thereby produce high levels of double-strand breaks that kill rapidly dividing cells. The small yellow circles represent the phosphates in the DNA backbone that become covalently bonded to the topoisomerase (see Figure 5–21).

1	double-strand break in the helix. These enzymes are activated by sites on chromosomes where two double helices cross over each other such as those generated by supercoiling in front of a replication fork (see Figure 5–20). Once a topoisomerase II molecule binds to such a crossing site, the protein uses ATP hydrolysis to perform the following set of reactions efficiently: (1) it breaks one double helix reversibly to create a DNA “gate”; (2) it causes the second, nearby double helix to pass through this opening; and (3) it then reseals the break and dissociates from the DNA. At crossover points generated by supercoiling, passage of the double helix through the gate occurs in the direction that will reduce supercoiling. In this way, type II topoisomerases can relieve the overwinding tension generated in front of a replication fork. Their reaction mechanism also allows type II DNA topoisomerases to efficiently separate two interlocked DNA circles (Figure 5–22).

1	Topoisomerase II also prevents the severe DNA tangling problems that would otherwise arise during DNA replication. This role is nicely illustrated by mutant yeast cells that produce, in place of the normal topoisomerase II, a version that is inactive above 37°C. When the mutant cells are warmed to this temperature, their daughter chromosomes remain intertwined after DNA replication and are unable to separate. The enormous usefulness of topoisomerase II for untangling chromosomes can readily be appreciated by anyone who has struggled to remove a tangle from a fishing line without the aid of scissors.

1	Much of what we know about DNA replication was first derived from studies of purified bacterial and bacteriophage multienzyme systems capable of DNA replication in vitro. The development of these systems in the 1970s was greatly facilitated by the prior isolation of mutants in a variety of replication genes; these mutants were exploited to identify and purify the corresponding replication proteins. The first mammalian replication system that accurately replicated DNA in vitro was described in the mid-1980s, and mutations in genes encoding nearly all of the replication components have now been isolated and analyzed in the yeast Saccharomyces cerevisiae. As a result, much is known about the detailed enzymology of DNA replication in eukaryotes, and it is clear that the fundamental features of DNA replication—including replication-fork geometry and the use of a multi-protein replication machine—have been conserved during the long evolutionary process that separated bacteria from

1	of DNA replication—including replication-fork geometry and the use of a multi-protein replication machine—have been conserved during the long evolutionary process that separated bacteria from eukaryotes.

1	There are more protein components in eukaryotic replication machines than there are in the bacterial analogs, even though the basic functions are the same. Thus, for example, the eukaryotic single-strand binding (SSB) protein is formed from three subunits, whereas only a single subunit is found in bacteria. Similarly, the eukaryotic DNA primase is incorporated into a multisubunit enzyme that also contains a polymerase called DNA polymerase α-primase. This protein complex begins each Okazaki fragment on the lagging strand with RNA and then extends the RNA primer with a short length of DNA. At this point, the two main eukaryotic replicative DNA polymerases, Polδand Polε, come into play: Polδcompletes each Okazaki fragment on the lagging strand and Polε extends the leading strand. The increased complexity of eukaryotic replication machinery probably reflects reversal of the covalent attachment of the topoisomerase restores an intact orange double helix more elaborate controls. For

1	increased complexity of eukaryotic replication machinery probably reflects reversal of the covalent attachment of the topoisomerase restores an intact orange double helix more elaborate controls. For example, the orderly maintenance of different cell types and tissues in animals and plants requires that DNA replication be tightly regulated. Moreover, eukaryotic DNA replication must be coordinated with the elaborate process of mitosis, as we discuss in Chapter 17.

1	As we see in the next section, the eukaryotic replication machinery has the added complication of having to replicate through nucleosomes, the repeating structural unit of chromosomes discussed in Chapter 4. Nucleosomes are spaced at intervals of about 200 nucleotide pairs along the DNA, which, as we will see, explains why new Okazaki fragments are synthesized on the lagging strand at intervals of 100–200 nucleotides in eukaryotes, instead of 1000–2000 nucleotides as in bacteria. Nucleosomes may also act as barriers that slow down the movement of DNA polymerase molecules, which may be why eukaryotic replication forks move only about one-tenth as fast as bacterial replication forks.

1	DNA replication takes place at a Y-shaped structure called a replication fork. A self-correcting DNA polymerase enzyme catalyzes nucleotide polymerization in a 5ʹ-to-3ʹ direction, copying a DNA template strand with remarkable fidelity. Since the two strands of a DNA double helix are antiparallel, this 5ʹ-to-3ʹ DNA synthesis can take place continuously on only one of the strands at a replication fork (the leading strand). On the lagging strand, short DNA fragments must be made by a “backstitching” process. Because the self-correcting DNA polymerase cannot start a new chain, these lagging-strand DNA fragments are primed by short RNA primer molecules that are subsequently erased and replaced with DNA.

1	DNA replication requires the cooperation of many proteins. These include (1) DNA polymerase and DNA primase to catalyze nucleoside triphosphate polymerization; (2) DNA helicases and single-strand DNA-binding (SSB) proteins to help in opening up the DNA helix so that it can be copied; (3) DNA ligase and an enzyme that degrades RNA primers to seal together the discontinuously synthesized lagging-strand DNA fragments; and (4) DNA topoisomerases to help to relieve helical winding and DNA tangling problems. Many of these proteins associate with each other at a replication fork to form a highly efficient “replication machine,” through which the activities and spatial movements of the individual components are coordinated.

1	We have seen how a set of replication proteins rapidly and accurately generates two daughter DNA double helices behind a replication fork. But how is this replication machinery assembled in the first place, and how are replication forks created on an intact, double-strand DNA molecule? In this section, we discuss how cells initiate DNA replication and how they carefully regulate this process to ensure that it takes place not only at the proper positions on the chromosome but also at the appropriate time in the life of the cell. We also discuss a few of the special problems that the replication machinery in eukaryotic cells must overcome. These include the need to replicate the enormously long DNA molecules found in eukaryotic chromosomes, as well as the difficulty of copying DNA molecules that are tightly complexed with histones in nucleosomes.

1	As discussed previously, the DNA double helix is normally very stable: the two DNA strands are locked together firmly by many hydrogen bonds formed between the bases on each strand. To begin DNA replication, the double helix must first be opened up and the two strands separated to expose unpaired bases. As we shall see, the process of DNA replication is begun by special initiator proteins that bind to double-strand DNA and pry the two strands apart, breaking the hydrogen bonds between the bases. leading strand of fork 1 of fork 2 leading strand lagging strand of fork 1 of fork 2 Figure 5–23 A replication bubble formed by replication-fork initiation. This diagram outlines the major steps in the initiation of replication forks at replication origins. The structure formed at the last step, in which both strands of the parental DNA helix have been separated from each other and serve as templates for DNA synthesis, is called a replication bubble.

1	Figure 5–24 DNA replication of a bacterial genome. It takes E. coli about 30 minutes to duplicate its genome of 4.6 × 106 nucleotide pairs. For simplicity, no Okazaki fragments are shown on the lagging strand. What happens as the two replication forks approach each other and collide at the end of the replication cycle is not well understood, although the replication machines are disassembled as part of the process.

1	The positions at which the DNA helix is first opened are called replication origins (Figure 5–23). In simple cells like those of bacteria or yeast, origins are specified by DNA sequences several hundred nucleotide pairs in length. This DNA contains both short sequences that attract initiator proteins and stretches of DNA that are especially easy to open. We saw in Figure 4–4 that an A-T base pair is held together by fewer hydrogen bonds than a G-C base pair. Therefore, DNA rich in A-T base pairs is relatively easy to pull apart, and regions of DNA enriched in A-T base pairs are typically found at replication origins.

1	Although the basic process of replication-fork initiation depicted in Figure 5–23 is fundamentally the same for bacteria and eukaryotes, the detailed way in which this process is performed and regulated differs between these two groups of organisms. We first consider the simpler and better-understood case in bacteria and then turn to the more complex situation found in yeasts, mammals, and other eukaryotes. Bacterial Chromosomes Typically Have a Single Origin of DNA Replication

1	The genome of E. coli is contained in a single circular DNA molecule of 4.6 × 106 nucleotide pairs. DNA replication begins at a single origin of replication, and the two replication forks assembled there proceed (at approximately 1000 nucleotides per second) in opposite directions until they meet up roughly halfway around the chromosome (Figure 5–24). The only point at which E. coli can control DNA replication is initiation: once the forks have been assembled at the origin, they synthesize DNA at relatively constant speed until replication is finished. Therefore, it is not surprising that the initiation of DNA replication is highly regulated. The process begins when initiator proteins (in their ATP-bound state) bind in multiple copies to specific DNA sites located at the replication origin, wrapping the DNA around the proteins to form a large protein–DNA complex that destabilizes the adjacent double helix. This complex then attracts two DNA helicases, each bound to a helicase loader,

1	wrapping the DNA around the proteins to form a large protein–DNA complex that destabilizes the adjacent double helix. This complex then attracts two DNA helicases, each bound to a helicase loader, and these are placed around adjacent DNA single strands whose bases have been exposed by the assembly of the initiator protein–DNA complex. The helicase loader is analogous to the clamp loader we encountered above; it has the additional job of keeping the helicase in an inactive form until it is properly loaded onto a nascent replication fork. Once the helicases are loaded, the loaders dissociate and the helicases begin to unwind DNA, exposing enough single-strand DNA for DNA primase to synthesize the first RNA primers (Figure 5–25). This quickly leads to the assembly of remaining proteins to create two replication forks, with replication machines that move, with respect to the replication origin, in opposite directions. They continue to synthesize DNA until all of the DNA template

1	to create two replication forks, with replication machines that move, with respect to the replication origin, in opposite directions. They continue to synthesize DNA until all of the DNA template downstream of each fork has been replicated.

1	In E. coli, the interaction of the initiator protein with the replication origin is carefully regulated, with initiation occurring only when sufficient nutrients are available for the bacterium to complete an entire round of replication. Initiation is also controlled to ensure that only one round of DNA replication occurs for each cell division. After replication is initiated, the initiator protein is inactivated by hydrolysis of its bound ATP molecule, and the origin of replication experiences a “refractory period.” The refractory period is caused by a delay in the methylation of newly incorporated A nucleotides in the origin (Figure 5–26). Initiation cannot occur again until the A’s are methylated and the initiator protein is restored to its ATP-bound state. Eukaryotic Chromosomes Contain Multiple Origins of Replication

1	Eukaryotic Chromosomes Contain Multiple Origins of Replication We have seen how two replication forks begin at a single replication origin in bacteria and proceed in opposite directions, moving away from the origin until all of the DNA in the single circular chromosome is replicated. The bacterial genome is sufficiently small for these two replication forks to duplicate the genome in about 30 minutes. Because of the much greater size of most eukaryotic chromosomes, a different strategy is required to allow their replication in a timely manner.

1	A method for determining the general pattern of eukaryotic chromosome replication was developed in the early 1960s. Human cells growing in culture are labeled for a short time with 3H-thymidine so that the DNA synthesized during this period becomes highly radioactive. The cells are then gently lysed, and the DNA is stretched on the surface of a glass slide coated with a photographic emulsion. Development of the emulsion reveals the pattern of labeled DNA through a technique known as autoradiography. The time allotted for radioactive labeling is chosen to allow each replication fork to move several micrometers along the DNA, so that the replicated DNA can be detected in the light microscope as lines of silver grains, even though the DNA molecule itself is too thin to be visible.

1	Figure 5–25 The proteins that initiate DNA replication in bacteria. The mechanism shown was established by studies in vitro with mixtures of highly purified proteins. For E. coli DNA replication, the major initiator protein, the helicase, and the primase are the dnaA, dnaB, and dnaG proteins, respectively. In the first step, several molecules of the initiator protein bind to specific DNA sequences at the replication origin and destabilize the double helix by forming a compact structure in which the DNA is tightly wrapped around the protein. Next, two helicases are brought in by helicaseloading proteins (the dnaC proteins), which inhibit the helicases until they are properly loaded at the replication origin. Helicase-loading proteins prevent the replicative DNA helices from inappropriately entering other single-strand stretches of DNA in the bacterial genome. Aided by single-strand binding protein (not shown), the loaded helicases open up the DNA, thereby enabling primases to enter and

1	entering other single-strand stretches of DNA in the bacterial genome. Aided by single-strand binding protein (not shown), the loaded helicases open up the DNA, thereby enabling primases to enter and synthesize initial primers. In subsequent steps, two complete replication forks are assembled at the origin and move off in opposite directions. The initiator proteins are displaced as the left-hand fork moves through them (not shown).

1	fully methylated hemimethylated origins are origin resistant to initiation initiation occurs if suffcient origins become fully resources are available to complete methylated, making them a round of DNA replication again competent for initiation In this way, both the rate and the direction of replication-fork movement can be determined (Figure 5–27). From the rate at which tracks of replicated DNA increase in length with increasing labeling time, the eukaryotic replication forks are estimated to travel at about 50 nucleotides per second. This is approximately twentyfold slower than the rate at which bacterial replication forks move, possibly reflecting the increased difficulty of replicating DNA that is packaged tightly in chromatin.

1	An average-size human chromosome contains a single linear DNA molecule of about 150 million nucleotide pairs. It would take 0.02 seconds/nucleotide × 150 × 106 nucleotides = 3.0 × 106 seconds (about 35 days) to replicate such a DNA molecule from end to end with a single replication fork moving at a rate of 50 nucleotides per second. As expected, therefore, the autoradiographic experiments just described reveal that many forks, belonging to separate replication bubbles, are moving simultaneously on each eukaryotic chromosome.

1	Much faster and more sophisticated methods now exist for monitoring DNA replication initiation and tracking the movement of DNA replication forks across whole genomes. One approach uses DNA microarrays—grids the size of a postage stamp studded with hundreds of thousands of fragments of known DNA sequence. As we will see in detail in Chapter 8, each different DNA fragment is placed at a unique position on the microarray, and whole genomes can thereby be represented in an orderly manner. If a DNA sample from a group of replicating cells is broken up and hybridized to a microarray representing that organism’s genome, the amount of each DNA sequence can be determined. Because a segment of a genome that has been replicated will contain twice as much DNA as an unreplicated segment, replication-fork initiation and fork movement can be accurately monitored across an entire genome (Figure 5–28).

1	Experiments of this type have shown the following: (1) Approximately 30,000– 50,000 origins of replication are used each time a human cell divides. (2) The human genome has many more (perhaps tenfold more) potential origins than this, and different cell types use different sets of origins. This may allow a cell to coordinate its active origins with other features of its chromosomes such as which Figure 5–26 Methylation of the E. coli replication origin creates a refractory period for DNA initiation. DNA methylation occurs at GATC sequences, 11 of which are found in the origin of replication (spanning approximately 250 nucleotide pairs). In its hemimethylated state, the origin of replication is bound by an inhibitor protein (Seq A, not shown), which blocks the ability of the initiator proteins to unwind the origin DNA. Eventually (about 15 minutes after replication is initiated), the hemimethylated origins become fully methylated by a DNA methylase enzyme; Seq A then dissociates.

1	A single enzyme, the Dam methylase, is responsible for methylating all E. coli GATC sequences. A lag in methylation after the replication of GATC sequences is also used by the E. coli mismatch proofreading system to distinguish the newly synthesized DNA strand from the parental DNA strand; in that case, the relevant GATC sequences are scattered throughout the chromosome, and they are not bound by Seq A. Figure 5–27 The experiments that demonstrated the pattern in which replication forks are formed and move on eukaryotic chromosomes. The new DNA made in human cells in culture was labeled briefly with a pulse of highly radioactive thymidine (3H-thymidine).

1	(A) In this experiment, the cells were lysed, and the DNA was stretched out on a glass slide that was subsequently covered with a photographic emulsion. After several months, the emulsion was developed, revealing a line of silver grains over the radioactive DNA. The brown DNA in this figure is shown only to help with the interpretation of the autoradiograph; the unlabeled DNA is invisible in such experiments. (B) This experiment was the same except that a further incubation in unlabeled medium allowed additional DNA, with a lower level of radioactivity, to be replicated. The pairs of dark tracks in (B) were found to have silver grains tapering off in opposite directions, demonstrating bidirectional fork movement from a central replication origin where a replication bubble forms (see Figure 5–23). A replication fork is thought to stop only when it encounters a replication fork moving in the opposite direction or when it reaches the end of the chromosome; in this way, all the DNA is

1	5–23). A replication fork is thought to stop only when it encounters a replication fork moving in the opposite direction or when it reaches the end of the chromosome; in this way, all the DNA is eventually replicated.

1	258 Chapter 5: DNA Replication, Repair, and Recombination culture of cells arrested before DNA replication begins allow replication to begin fragment DNA, separate strands, and fuorescently label genes are being expressed. The excess origins also provide “backups” in case a primary origin fails. (3) As in bacteria, replication forks are formed in pairs and create a replication bubble as they move in opposite directions away from a common point of origin, stopping only when they collide head-on with a replication fork moving in the opposite direction or when they reach a chromosome end. In this way, many replication forks operate independently on each chromosome and yet form two complete daughter DNA helices. In Eukaryotes, DNA Replication Takes Place During Only One Part of the Cell Cycle

1	In Eukaryotes, DNA Replication Takes Place During Only One Part of the Cell Cycle When growing rapidly, bacteria replicate their DNA nearly continuously. In contrast, DNA replication in most eukaryotic cells occurs only during a specific part of the cell-division cycle, called the DNA synthesis phase or S phase (Figure 5–29). In a mammalian cell, the S phase typically lasts for about 8 hours; in simpler eukaryotic cells such as yeasts, the S phase can be as short as 40 minutes. By its end, each chromosome has been replicated to produce two complete copies, which remain joined together at their centromeres until the M phase (M for mitosis), which soon follows. In Chapter 17, we describe the control system that runs the cell cycle, and we explain why entry into each phase of the cycle requires the cell to have successfully completed the previous phase. In the following sections, we explore how chromosome replication is coordinated within the S phase of the cell cycle.

1	In the following sections, we explore how chromosome replication is coordinated within the S phase of the cell cycle. Different Regions on the Same Chromosome Replicate at Distinct Times in S Phase In mammalian cells, the replication of DNA in the region between one replication origin and the next should normally require only about an hour to complete, given the rate at which a replication fork moves and the largest distances measured between replication origins. Yet S phase usually lasts for about 8 hours in a mammalian cell. This implies that the replication origins are not all activated simultaneously; indeed, replication origins are activated in clusters of about 50 adjacent replication origins, each of which is replicated during only a small part of the total S-phase interval.

1	Figure 5–28 Use of DNA microarrays to monitor the formation and progress of replication forks. For this experiment, a population of cells is synchronized so that they all begin replication at the same time. DNA is collected and hybridized to the microarray; DNA that has been replicated once gives a hybridization signal (dark green squares) twice as high as that of unreplicated DNA (light green squares). The spots on these microarrays represent consecutive sequences along a segment of a chromosome arranged left to right, top to bottom. Only 81 spots are shown here, but the actual arrays contain hundreds of thousands of sequences that span an entire genome. As can be seen, replication begins at an origin and proceeds bidirectionally. For simplicity, only one origin is shown here. In human cells, replication begins at 30,000–50,000 origins located throughout the genome. Using this approach it is possible to observe the formation and progress of every replication fork across a genome.

1	Figure 5–29 The four successive phases of a standard eukaryotic cell cycle. During the G1, S, and G2 phases, the cell grows continuously. During M phase growth stops, the nucleus divides, and the cell divides in two. DNA replication is confined to the part of the cell cycle known as S phase. G1 is the gap between M phase and S phase; G2 is the gap between S phase and M phase. It seems that the order in which replication origins are activated depends, in part, on the chromatin structure in which the origins reside. We saw in Chapter 4 that heterochromatin is a particularly condensed state of chromatin, while euchromatin, where most transcription occurs, has a less condensed conformation. Heterochromatin tends to be replicated very late in S phase, suggesting that the timing of replication is related to the packing of the DNA in chromatin.

1	Once initiated, however, replication forks seem to move at comparable rates throughout S phase, so the extent of chromosome condensation seems to influence the time at which replication forks are initiated, rather than their speed once formed. A Large Multisubunit Complex Binds to Eukaryotic Origins of Replication Having seen that a eukaryotic chromosome is replicated using many origins of replication, each of which “fires” at a characteristic time in S phase of the cell cycle, we turn to the nature of these origins of replication. We saw earlier in this chapter that replication origins have been precisely defined in bacteria as specific DNA sequences that attract initiator proteins, which then assemble the DNA replication machinery. We shall see that this is the case for the single-cell budding yeast S. cerevisiae, but it appears not to be strictly true for most other eukaryotes.

1	For budding yeast, the location of every origin of replication on each chromosome has been determined. The particular chromosome shown in Figure 5–30— chromosome III from S. cerevisiae—is one of the smallest chromosomes known, with a length less than 1/100 that of a typical human chromosome. Its major origins are spaced an average of 30,000 nucleotide pairs apart, but only a subset of these origins is used by a given cell. Nonetheless, this chromosome can be replicated in about 15 minutes.

1	The minimal DNA sequence required for directing DNA replication initiation in S. cerevisiae has been determined by taking a segment of DNA that spans an origin of replication and testing smaller and smaller DNA fragments for their ability to function as origins. Most DNA sequences that can serve as an origin of replication are found to contain (1) a binding site for a large, multisubunit initiator protein called ORC, for origin recognition complex; (2) a stretch of DNA that is rich in As and Ts and therefore easy to melt; and (3) at least one binding site for proteins that facilitate ORC binding, probably by adjusting chromatin structure.

1	In bacteria, once the initiator protein is properly bound to the single origin of replication, the assembly of the replication forks seems to follow more or less automatically. In eukaryotes, the situation is significantly different because of a profound problem eukaryotes have in replicating chromosomes: with so many places to begin replication, how is the process regulated to ensure that all the DNA is copied once and only once?

1	The answer lies in the sequential manner in which the replicative helicase is first loaded onto origins and is then activated to initiate DNA replication. This matter is discussed in detail in Chapter 17, where we consider the machinery that underlies the cell-division cycle. In brief, during G1 phase, the replicative helicases are loaded onto DNA next to ORC to create a prereplicative complex. Then, upon passage from G1 phase to S phase, specialized protein kinases come into play to activate the helicases. The resulting opening of the double helix allows the loading of the remaining replication proteins, including the DNA polymerases. origins of replication telomere centromere telomere

1	origins of replication telomere centromere telomere Figure 5–30 The origins of DNA replication on chromosome III of the yeast S. cerevisiae. This chromosome, one of the smallest eukaryotic chromosomes known, carries a total of 180 genes. As indicated, it contains 18 replication origins, although they are used with different frequencies. Those in red are typically used in less than 10% of cell divisions, while those in green are used about 90% of the time.

1	Figure 5–31 DNA replication initiation in eukaryotes. This mechanism ensures that each origin of replication is activated only once per cell cycle. An origin of replication can be used only if a prereplicative complex forms there in G1 phase. At the beginning of S phase, specialized kinases phosphorylate Mcm and ORC, activating the former and inactivating the latter. A new prereplicative complex cannot form at the origin until the cell progresses to the next G1 phase, when the bound ORC has been dephosphorylated. Note that the eukaryotic Mcm helicase moves along the leading-strand template, whereas the bacterial helicase moves along the lagging-strand template (see Figure 5–25). As the forks begin to move, ORC is displaced, and new ORCs rapidly bind to the newly replicated origins.

1	The protein kinases that trigger DNA replication simultaneously prevent assembly of new prereplicative complexes until the next M phase resets the entire cycle (for details, see pp. 974–975). They do this, in part, by phosphorylating ORC, rendering it unable to accept new helicases. This strategy provides a single window of opportunity for prereplicative complexes to form (G1 phase, when kinase activity is low) and a second window for them to be activated and subsequently disassembled (S phase, when kinase activity is high). Because these two phases of the cell cycle are mutually exclusive and occur in a prescribed order, each origin of replication can fire once and only once during each cell cycle. Features of the Human Genome That Specify Origins of Replication Remain to Be Discovered

1	Features of the Human Genome That Specify Origins of Replication Remain to Be Discovered Compared with the situation in budding yeast, the determinants of replication origins in other eukaryotes have been difficult to discover. It has been possible to identify specific human DNA sequences, each several thousand nucleotide pairs in length, that are sufficient to serve as replication origins. These origins continue to function when moved to a different chromosomal region by recombinant DNA methods, as long as they are placed in a region where the chromatin is relatively uncondensed. However, comparisons of such DNA sequences have not revealed specific DNA sequences that mark origins of replication.

1	Despite this, a human ORC that is very similar to the yeast ORC binds to origins of replication and initiates DNA replication in humans. Many of the other proteins that function in the initiation process in yeast likewise have central roles in humans. It therefore seems likely that the yeast and human initiation mechanisms are similar in outline, but chromatin structure, transcriptional activity, or some property of the genome other than a specific DNA sequence has the central role in attracting ORC and specifying mammalian origins of replication. These ideas could also help to explain how a given mammalian cell chooses which of the many possible origins to use when it replicates its genome and how this choice could differ from cell to cell. Clearly, we have a great deal to discover about the fundamental process of DNA replication initiation. New Nucleosomes Are Assembled Behind the Replication Fork

1	Several additional aspects of DNA replication are specific to eukaryotes. As discussed in Chapter 4, eukaryotic chromosomes are composed of roughly equal mixtures of DNA and protein. Chromosome duplication therefore requires not only the replication of DNA, but also the synthesis and assembly of new chromosomal proteins onto the DNA behind each replication fork. Although we are far from understanding this process in detail, we are beginning to learn how the fundamental unit of chromatin packaging, the nucleosome, is duplicated. The cell requires a large amount of new histone protein, approximately equal in mass to the newly synthesized DNA, to make the new nucleosomes in each cell cycle. For this reason, most eukaryotic organisms possess multiple copies of the gene for each histone. Vertebrate cells, for example, have about 20 repeated gene sets, most containing the genes that encode all five histones (H1, H2A, H2B, H3, and H4).

1	Unlike most proteins, which are made continuously, histones are synthesized mainly in S phase, when the level of histone mRNA increases about fiftyfold as a result of both increased transcription and decreased mRNA degradation. The major histone mRNAs are degraded within minutes when DNA synthesis stops at the end of S phase. The mechanism depends on special properties of the 3ʹends of these mRNAs, as discussed in Chapter 7. In contrast, the histone proteins themselves are remarkably stable and may survive for the entire life of a cell. The tight linkage between DNA synthesis and histone synthesis appears to reflect a feedback mechanism that monitors the level of free histone to ensure that the amount of histone made exactly matches the amount of new DNA synthesized.

1	As a replication fork advances, it must pass through the parental nucleosomes. In the cell, efficient replication requires chromatin remodeling complexes (discussed in Chapter 4) to destabilize the DNA–histone interfaces. Aided by such complexes, replication forks can transit even highly condensed chromatin efficiently.

1	As a replication fork passes through chromatin, the histones are transiently displaced leaving about 600 nucleotide pairs of non-nucleosomal DNA in its wake. The reestablishment of nucleosomes behind a moving fork occurs in an intriguing way. When a nucleosome is traversed by a replication fork, the histone octamer appears to be broken into an H3-H4 tetramer and two H2A-H2B dimers (discussed in Chapter 4). The H3-H4 tetramer remains loosely associated with DNA and is distributed at random to one or the other daughter duplex, but the H2A-H2B dimers are released completely from DNA. Freshly made H3-H4 tetramers are added to the newly synthesized DNA to fill in the “spaces,” and H2A-H2B dimers—half of which are old and half new—are then added at random to complete the nucleosomes (Figure 5–32). The formation of new nucleosomes behind a replication fork has an important consequence for the process of DNA replication itself. As DNA polymerase δ discontinuously synthesizes the lagging

1	5–32). The formation of new nucleosomes behind a replication fork has an important consequence for the process of DNA replication itself. As DNA polymerase δ discontinuously synthesizes the lagging strand (see pp. 253–254), the length of each Okazaki fragment is determined by the point at which DNA polymerase δ is blocked by a newly formed nucleosome. This tight coupling between nucleosome duplication and DNA replication explains why the length of Okazaki fragments in eukaryotes (~200 nucleotides) is approximately the same as the nucleosome repeat length.

1	262 Chapter 5: DNA Replication, Repair, and Recombination H2A-H2B dimer displaced in front of replication fork The orderly and rapid addition of new H3-H4 tetramers and H2A-H2B dimers behind a replication fork requires histone chaperones (also called chromatin assembly factors). These multisubunit complexes bind the highly basic histones and release them for assembly only in the appropriate context. The histone chaperones, along with their cargoes, are directed to newly replicated DNA through a specific interaction with the eukaryotic sliding clamp called PCNA (see Figure 5–32B). These clamps are left behind moving replication forks and remain on the DNA long enough for the histone chaperones to complete their tasks. Telomerase Replicates the Ends of Chromosomes

1	Telomerase Replicates the Ends of Chromosomes We saw earlier that synthesis of the lagging strand at a replication fork must occur discontinuously through a backstitching mechanism that produces short DNA fragments. This mechanism encounters a special problem when the replication fork reaches an end of a linear chromosome. The final RNA primer synthesized on the lagging-strand template cannot be replaced by DNA because there is no 3ʹ-OH end available for the repair polymerase. Without a mechanism to deal with this problem, DNA would be lost from the ends of all chromosomes each time a cell divides.

1	Bacteria solve this “end-replication” problem by having circular DNA molecules as chromosomes (see Figure 5–24). Eukaryotes solve it in a different way: they have specialized nucleotide sequences at the ends of their chromosomes that are incorporated into structures called telomeres (discussed in Chapter 4). Telomeres contain many tandem repeats of a short sequence that is similar in organisms as diverse as protozoa, fungi, plants, and mammals. In humans, the sequence of the repeat unit is GGGTTA, and it is repeated roughly a thousand times at each telomere.

1	Telomere DNA sequences are recognized by sequence-specific DNA-binding proteins that attract an enzyme, called telomerase, that replenishes these sequences each time a cell divides. Telomerase recognizes the tip of an existing telomere DNA repeat sequence and elongates it in the 5ʹ-to-3ʹ direction, using an RNA template that is a component of the enzyme itself to synthesize new copies of the repeat (Figure 5–33). The enzymatic portion of telomerase resembles other reverse transcriptases, proteins that synthesize DNA using an RNA template, although, in this case, the telomerase RNA also contributes functional groups to make the catalysis more efficient. After extension of the parental DNA strand by telomerase, replication of the lagging strand at the chromosome end can be completed by the conventional DNA polymerases, using these extensions as a template to synthesize the complementary strand (Figure 5–34).

1	Figure 5–32 Formation of nucleosomes behind a replication fork. Parental H3-H4 tetramers are distributed at random to the daughter DNA molecules, with roughly equal numbers inherited by each daughter. In contrast, H2A-H2B dimers are released from the DNA as the replication fork passes. This release begins just in front of the replication fork and is facilitated by chromatin remodeling complexes that move with the fork. Histone chaperones (NAP1 and CAF1) restore the full complement of histones to daughter molecules using both parental and newly synthesized histones. Although some daughter nucleosomes contain only parental histones or only newly synthesized histones, most are hybrids of old and new. For simplicity, the DNA double helix shown as a single red line. (Adapted from J.D. Watson et al., Molecular Biology of the Gene, 5th ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2004.) remainder of telomerase proteintelomerase RNA region of telomerase RNA used as

1	Watson et al., Molecular Biology of the Gene, 5th ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2004.) remainder of telomerase proteintelomerase RNA region of telomerase RNA used as template “palm“—active site of telomerase protein rest of synthesized chromosome telomere DNA

1	Telomeres Are Packaged Into Specialized Structures That Protect the Ends of Chromosomes The ends of chromosomes present cells with an additional problem. As we will see in the next part of this chapter, when a chromosome is accidently broken, the break is rapidly repaired (see Figure 5–45). Telomeres must clearly be distinguished from these accidental breaks; otherwise the cell will attempt to “repair” telomeres, causing chromosome fusions and other genetic abnormalities. Telomeres have several features to prevent this from happening.

1	A specialized nuclease chews back the 5ʹend of a telomere leaving a protruding single-strand end. This protruding end—in combination with the GGGTTA repeats in telomeres—attracts a group of proteins that form a protective chromosome cap known as shelterin. In particular, shelterin “hides” telomeres from the cell’s damage detectors that continually monitor DNA. When human telomeres are artificially cross-linked and viewed by electron microscopy, structures known as “t-loops” are observed in which the protruding end of the telomere loops back and tucks itself into the duplex DNA of the telomere repeat sequence (Figure 5–35). It is believed that t-loops are regulated by shelterin and provide additional protection for the ends of chromosomes. incomplete, newly synthesized lagging strand direction of AACCCC telomere 5˜ synthesisTELOMERASE EXTENDS 3˜ END telomerase with bound RNA template

1	incomplete, newly synthesized lagging strand direction of AACCCC telomere 5˜ synthesisTELOMERASE EXTENDS 3˜ END telomerase with bound RNA template Figure 5–33 Structure of a portion of telomerase. Telomerase is a large protein– RNA complex. The RNA (blue) contains a templating sequence for synthesizing new DNA telomere repeats. The synthesis reaction itself is carried out by the reverse transcriptase domain of the protein, shown in green. A reverse transcriptase is a special form of polymerase enzyme that uses an RNA template to make a DNA strand; telomerase is unique in carrying its own RNA template with it. Telomerase also has several additional protein domains (not shown) that are needed to assemble the enzyme at the ends of chromosomes. (Modified from J. Lingner and T.R. Cech, Curr. Opin. Genet. Dev. 8:226–232, 1998. With permission from Elsevier.)

1	Figure 5–34 Telomere replication. Shown here are the reactions that synthesize the repeating sequences that form the ends of the chromosomes (telomeres) of diverse eukaryotic organisms. The 3ʹ end of the parental DNA strand is extended by RNA-templated DNA synthesis; this allows the incomplete daughter DNA strand that is paired with it to be extended in its 5ʹ direction. This incomplete, lagging strand is presumed to be completed by DNA polymerase α, which carries a DNA primase as one of its subunits (Movie 5.6). The telomere sequence illustrated is that of the ciliate Tetrahymena, in which these reactions were first discovered. Because the processes that grow and shrink each telomere sequence are only approximately balanced, a chromosome end contains a variable number of telomeric repeats. Not surprisingly, many cells have homeostatic mechanisms that maintain the number of these repeats within a limited range (Figure 5–36).

1	In most of the dividing somatic cells of humans, however, telomeres gradually shorten, and it has been proposed that this provides a counting mechanism that helps prevent the unlimited proliferation of wayward cells in adult tissues. In its simplest form, this idea holds that our somatic cells start off in the embryo with a full complement of telomeric repeats. These are then eroded to different extents in different cell types. Some stem cells, notably those in tissues that must be replenished at a high rate throughout life—bone marrow or gut lining, for example— retain full telomerase activity. However, in many other types of cells, the level of telomerase is turned down so that the enzyme cannot quite keep up with chromosome duplication. Such cells lose 100–200 nucleotides from each telomere every time they divide. After many cell generations, the descendant cells will inherit chromosomes that lack telomere function, and, as a result of this defect, activate a DNA-damage response

1	every time they divide. After many cell generations, the descendant cells will inherit chromosomes that lack telomere function, and, as a result of this defect, activate a DNA-damage response causing them to withdraw permanently from the cell cycle and cease dividing—a process called replicative cell senescence (discussed in Chapter 17). In theory, such a mechanism could provide a safeguard against the uncontrolled cell proliferation of abnormal cells in somatic tissues, thereby helping to protect us from cancer.

1	fraction of chromosome ends Figure 5–35 A t-loop at the end of a mammalian chromosome. (A) Electron micrograph of the DNA at the end of an interphase human chromosome. The chromosome was fixed, deproteinated, and artificially thickened before viewing. The loop seen here is approximately 15,000 nucleotide pairs in length. (B) Structure of a t-loop. The insertion of the single-strand 3ʹ end into the duplex repeats is carried out, and the structure maintained, by specialized proteins. (From J.D. Griffith et al., Cell 97:503–514, 1999. With permission from Elsevier.)

1	Figure 5–36 A demonstration that yeast cells control the length of their telomeres. In this experiment, the telomere at one end of a particular chromosome is artificially made either longer (left) or shorter (right) than average. After many cell divisions, the chromosome recovers, showing an average telomere length and a length distribution that is typical of the other chromosomes in the yeast cell. A similar feedback mechanism for controlling telomere length has been proposed for the germ-line cells of animals.

1	The idea that telomere length acts as a “measuring stick” to count cell divisions and thereby regulate the lifetime of the cell lineage has been tested in several ways. For certain types of human cells grown in tissue culture, the experimental results support such a theory. Human fibroblasts normally proliferate for about 60 cell divisions in culture before undergoing replicative cell senescence. Like most other somatic cells in humans, fibroblasts produce only low levels of telomerase, and their telomeres gradually shorten each time they divide. When telomerase is provided to the fibroblasts by inserting an active telomerase gene, telomere length is maintained and many of the cells now continue to proliferate indefinitely.

1	It has been proposed that this type of control on cell proliferation may contribute to the aging of animals like ourselves. These ideas have been tested by producing transgenic mice that lack telomerase entirely. The telomeres in mouse chromosomes are about five times longer than human telomeres, and the mice must therefore be bred through three or more generations before their telomeres have shrunk to the normal human length. It is therefore perhaps not surprising that the first generations of mice develop normally. However, the mice in later generations develop progressively more defects in some of their highly proliferative tissues. In addition, these mice show signs of premature aging and have a pronounced tendency to develop tumors. In these and other respects these mice resemble humans with the genetic disease dyskeratosis congenita. Individuals afflicted with this disease carry one functional and one nonfunctional copy of the telomerase RNA gene; they have prematurely shortened

1	with the genetic disease dyskeratosis congenita. Individuals afflicted with this disease carry one functional and one nonfunctional copy of the telomerase RNA gene; they have prematurely shortened telomeres and typically die of progressive bone marrow failure. They also develop lung scarring and liver cirrhosis and show abnormalities in various epidermal structures including skin, hair follicles, and nails.

1	The above observations demonstrate that controlling cell proliferation by telomere shortening poses a risk to an organism, because not all of the cells that begin losing the ends of their chromosomes will stop dividing. Some apparently become genetically unstable, but continue to divide, giving rise to variant cells that can lead to cancer. Clearly, the use of telomere shortening as a regulating mechanism is not foolproof and, like many mechanisms in the cell, seems to strike a balance between benefit and risk. The proteins that initiate DNA replication bind to DNA sequences at a replication origin to catalyze the formation of a replication bubble with two outward-moving replication forks. The process begins when an initiator protein–DNA complex is formed that subsequently loads a DNA helicase onto the DNA template. Other proteins are then added to form the multienzyme “replication machine” that catalyzes DNA synthesis at each replication fork.

1	In bacteria and some simple eukaryotes, replication origins are specified by specific DNA sequences that are only several hundred nucleotide pairs long. In other eukaryotes, such as humans, the sequences needed to specify an origin of DNA replication seem to be less well defined, and the origin can span several thousand nucleotide pairs.

1	Bacteria typically have a single origin of replication in a circular chromosome. With fork speeds of up to 1000 nucleotides per second, they can replicate their genome in less than an hour. Eukaryotic DNA replication takes place in only one part of the cell cycle, the S phase. The replication fork in eukaryotes moves about 10 times more slowly than the bacterial replication fork, and the much longer eukaryotic chromosomes each require many replication origins to complete their replication in an S phase, which typically lasts for 8 hours in human cells. The different replication origins in these eukaryotic chromosomes are activated in a sequence, determined in part by the structure of the chromatin, with the most condensed regions of chromatin typically beginning their replication last. After the replication fork has passed, chromatin structure is re-formed by the addition of new histones to the old histones that are directly inherited by each daughter DNA molecule.

1	Eukaryotes solve the problem of replicating the ends of their linear chromosomes with a specialized end structure, the telomere, maintained by a special nucleotide polymerizing enzyme called telomerase. Telomerase extends one of the DNA strands at the end of a chromosome by using an RNA template that is an integral part of the enzyme itself, producing a highly repeated DNA sequence that typically extends for thousands of nucleotide pairs at each chromosome end. Telomeres have specialized structures that distinguish them from broken ends of chromosomes, ensuring that they are not mistakenly repaired.

1	Maintaining the genetic stability that an organism needs for its survival requires not only an extremely accurate mechanism for replicating DNA, but also mechanisms for repairing the many accidental lesions that DNA continually suffers. Most such spontaneous changes in DNA are temporary because they are immediately corrected by a set of processes that are collectively called DNA repair. Of the tens of thousands of random changes created every day in the DNA of a human cell by heat, metabolic accidents, radiation of various sorts, and exposure to substances in the environment, only a few (less than 0.02%) accumulate as permanent mutations in the DNA sequence. The rest are eliminated with remarkable efficiency by DNA repair.

1	The importance of DNA repair is evident from the large investment that cells make in the enzymes that carry it out: several percent of the coding capacity of most genomes is devoted solely to DNA repair functions. The importance of DNA repair is also demonstrated by the increased rate of mutation that follows the inactivation of a DNA repair gene. Many DNA repair proteins and the genes that encode them—which we now know operate in a wide range of organisms, including humans—were originally identified in bacteria by the isolation and characterization of mutants that displayed an increased mutation rate or an increased sensitivity to DNA-damaging agents.

1	Recent studies of the consequences of a diminished capacity for DNA repair in humans have linked many human diseases with decreased repair (Table 5–2). Thus, we saw previously that defects in a human gene whose product normally functions to repair the mismatched base pairs resulting from DNA replication errors can lead to an inherited predisposition to cancers of the colon and some other organs, reflecting an increased mutation rate. In another human disease, xeroderma pigmentosum (XP), the afflicted individuals have an extreme sensitivity to ultraviolet radiation because they are unable to repair certain DNA photo-products. This repair defect results in an increased mutation rate that leads to serious skin lesions and an increased susceptibility to skin cancers. Finally, mutations in the Brca1 and Brca2 genes compromise a type of DNA repair known as homologous recombination and are a cause of hereditary breast and ovarian cancer.

1	Without DNA Repair, Spontaneous DNA Damage Would Rapidly Change DNA Sequences Although DNA is a highly stable material—as required for the storage of genetic information—it is a complex organic molecule that is susceptible, even under normal cell conditions, to spontaneous changes that would lead to mutations if left unrepaired (Figure 5–37 and see Table 5–3). For example, the DNA of each Figure 5–37 A summary of spontaneous alterations that require DNA repair.

1	The sites on each nucleotide modified by spontaneous oxidative damage (red arrows), hydrolytic attack (blue arrows), and methylation (green arrows) are shown, with the width of each arrow indicating the relative frequency of each event (see Table 5–3). (After T. Lindahl, Nature 362:709–715, 1993. With permission from Macmillan Publishers Ltd.) human cell loses about 18,000 purine bases (adenine and guanine) every day because their N-glycosyl linkages to deoxyribose hydrolyze, a spontaneous reaction called depurination. Similarly, a spontaneous deamination of cytosine to uracil in DNA occurs at a rate of about 100 bases per cell per day (Figure 5–38). DNA bases are also occasionally damaged by an encounter with reactive metabolites produced in the cell, including reactive forms of oxygen and the high-energy methyl donor S-adenosylmethionine, or by exposure to chemicals in the environment. Likewise, ultraviolet radiation from the sun can produce a covalent linkage between two adjacent

1	and the high-energy methyl donor S-adenosylmethionine, or by exposure to chemicals in the environment. Likewise, ultraviolet radiation from the sun can produce a covalent linkage between two adjacent pyrimidine bases in DNA to form, for example, thymine dimers (Figure 5–39). If left uncorrected when the DNA is replicated, most of these changes would be expected to lead either to the deletion of one or more base pairs or to a base-pair substitution in the daughter DNA chain (Figure 5–40). The mutations would then be propagated throughout subsequent cell generations. Such a high rate of random changes in the DNA sequence would have disastrous consequences.

1	The DNA Double Helix Is Readily Repaired The double-helical structure of DNA is ideally suited for repair because it carries two separate copies of all the genetic information—one in each of its two strands. Thus, when one strand is damaged, the complementary strand retains an intact copy of the same information, and this copy is generally used to restore the correct nucleotide sequences to the damaged strand.

1	An indication of the importance of a double-strand helix to the safe storage of genetic information is that all cells use it; only a few small viruses use single-strand DNA or RNA as their genetic material. The types of repair processes described in this section cannot operate on such nucleic acids, and once damaged, the chance of a permanent nucleotide change occurring in these single-strand genomes of viruses is thus very high. It seems that only organisms with tiny genomes (and therefore tiny targets for DNA damage) can afford to encode their genetic information in any molecule other than a DNA double helix.

1	Figure 5–38 Depurination and deamination. These reactions are two of the most frequent spontaneous chemical reactions that create serious DNA damage in cells. Depurination can release guanine (shown here), as well as adenine, from DNA. The major type of deamination reaction converts cytosine to an altered DNA base, uracil (shown here), but deamination occurs on other bases as well. These reactions normally take place in double-helical DNA; for convenience, only one strand is shown. Figure 5–39 The most common type of thymine dimer. This type of damage occurs in the DNA of cells exposed to ultraviolet irradiation (as in sunlight). A similar dimer will form between any two neighboring pyrimidine bases (C or T residues) in DNA.

1	Cells have multiple pathways to repair their DNA using different enzymes that act upon different kinds of lesions. Figure 5–41 shows two of the most common pathways. In both, the damage is excised, the original DNA sequence is restored by a DNA polymerase that uses the undamaged strand as its template, and a remaining break in the double helix is sealed by DNA ligase (see Figure 5–12).

1	The two pathways differ in the way in which they remove the damage from DNA. The first pathway, called base excision repair, involves a battery of enzymes called DNA glycosylases, each of which can recognize a specific type of altered base in DNA and catalyze its hydrolytic removal. There are at least six types of these enzymes, including those that remove deaminated Cs, deaminated As, different types of alkylated or oxidized bases, bases with opened rings, and bases in which a carbon–carbon double bond has been accidentally converted to a carbon–carbon single bond. How is an altered base detected within the context of the double helix? A key step is an enzyme-mediated “flipping-out” of the altered nucleotide from the helix, which allows the DNA glycosylase to probe all faces of the base for damage (Figure 5–42). It is thought that these enzymes travel along DNA using base-flipping to evaluate the status of each base. Once an enzyme finds the damaged base that it recognizes, it

1	base for damage (Figure 5–42). It is thought that these enzymes travel along DNA using base-flipping to evaluate the status of each base. Once an enzyme finds the damaged base that it recognizes, it removes that base from its sugar.

1	The “missing tooth” created by DNA glycosylase action is recognized by an enzyme called AP endonuclease (AP for apurinic or apyrimidinic, endo to signify that the nuclease cleaves within the polynucleotide chain), which cuts the phosphodiester backbone, after which the resulting gap is repaired (see Figure 5–41A). Depurination, which is by far the most frequent type of damage suffered by DNA, also leaves a deoxyribose sugar with a missing base. Depurinations are directly repaired beginning with AP endonuclease, following the bottom half of the pathway in Figure 5–41A. a G has been changed to an A

1	a G has been changed to an A Figure 5–40 How chemical modifications of nucleotides produce mutations. (A) Deamination of cytosine, if uncorrected, results in the substitution of one base for another when the DNA is replicated. As shown in Figure 5–38, deamination of cytosine produces uracil. Uracil differs from cytosine in its base-pairing properties and preferentially base-pairs with adenine. The DNA replication machinery therefore adds an adenine when it encounters a uracil on the template strand. (B) Depurination can lead to the loss of a nucleotide pair. When the replication machinery encounters a missing purine on the template strand, it may skip to the next complete nucleotide as illustrated here, thus producing a nucleotide deletion in the newly synthesized strand. Many other types of DNA damage (see Figure 5–37), if left uncorrected, also produce mutations when the DNA is replicated. G C T A T C C DNA helix with missing base C G A G T A G G

1	G C T A T C C DNA helix with missing base C G A G T A G G DNA helix with single-nucleotide gap C G A G T A G G DNA POLYMERASE ADDS NEW NUCLEOTIDE, DNA LIGASE SEALS NICK G C T C A T C C DNA helix with 12nucleotide gap

1	Figure 5– 41 A comparison of two major DNA repair pathways. (A) Base excision repair. This pathway starts with a DNA glycosylase. Here, the enzyme uracil DNA glycosylase removes an accidentally deaminated cytosine in DNA. After the action of this glycosylase (or another DNA glycosylase that recognizes a different kind of damage), the sugar phosphate with the missing base is cut out by the sequential action of AP endonuclease and a phosphodiesterase. (These same enzymes begin the repair of depurinated sites directly.) The gap of a single nucleotide is then filled by DNA polymerase and DNA ligase. The net result is that the U that was created by accidental deamination is restored to a C. AP endonuclease is so-named because it recognizes any site in the DNA helix that contains a deoxyribose sugar with a missing base; such sites can arise either by the loss of a purine (apurinic sites) or by the loss of a pyrimidine (apyrimidinic sites). (B) Nucleotide excision repair. In bacteria, after

1	sugar with a missing base; such sites can arise either by the loss of a purine (apurinic sites) or by the loss of a pyrimidine (apyrimidinic sites). (B) Nucleotide excision repair. In bacteria, after a multienzyme complex has recognized a lesion such as a pyrimidine dimer (see Figure 5–39), one cut is made on each side of the lesion, and an associated DNA helicase then removes the entire portion of the damaged strand. The excision repair machinery in bacteria leaves the gap of 12 nucleotides shown. In humans, once the damaged DNA is recognized, a helicase is recruited to unwind the DNA duplex locally. Next, the excision nuclease enters and cleaves on either side of the damage, leaving a gap of about 30 nucleotides. The nucleotide excision repair machinery in both bacteria and humans can recognize and repair many different types of DNA damage.

1	The second major repair pathway is called nucleotide excision repair. This mechanism can repair the damage caused by almost any large change in the structure of the DNA double helix. Such “bulky lesions” include those created by the covalent reaction of DNA bases with large hydrocarbons (such as the carcinogen benzopyrene, found in tobacco smoke, coal tar, and diesel exhaust), as well as the various pyrimidine dimers (T-T, T-C, and C-C) caused by sunlight. In this pathway, a large multienzyme complex scans the DNA for a distortion in the double helix, rather than for a specific base change. Once it finds a lesion, it cleaves the phosphodiester backbone of the abnormal strand on both sides of the distortion, and a DNA helicase peels away the single-strand oligonucleotide containing the lesion. The large gap produced in the DNA helix is then repaired by DNA polymerase and DNA ligase (see Figure 5–41B).

1	An alternative to base and nucleotide excision repair processes is direct chemical reversal of DNA damage, and this strategy is selectively employed for the rapid removal of certain highly mutagenic or cytotoxic lesions. For example, the alkylation lesion O6-methylguanine has its methyl group removed by direct transfer to a cysteine residue in the repair protein itself, which is destroyed in the reaction. In another example, methyl groups in the alkylation lesions 1-methyladenine and 3-methylcytosine are “burnt off” by an iron-dependent demethylase, with release of formaldehyde from the methylated DNA and regeneration of the native base. Coupling Nucleotide Excision Repair to Transcription Ensures That the Cell’s Most Important DNA Is Efficiently Repaired

1	All of a cell’s DNA is under constant surveillance for damage, and the repair mechanisms we have described act on all parts of the genome. However, cells have a way of directing DNA repair to the DNA sequences that are most urgently needed. They do this by linking RNA polymerase, the enzyme that transcribes DNA into RNA as the first step in gene expression, to the nucleotide excision repair pathway. As discussed above, this repair system can correct many different types of DNA damage. RNA polymerase stalls at DNA lesions and, through the use of coupling proteins, directs the excision repair machinery to these sites. In bacteria, where genes are relatively short, the stalled RNA polymerase can be dissociated from the DNA; the DNA is repaired, and the gene is transcribed again from the beginning. In eukaryotes, where genes can be enormously long, a more complex reaction is used to “back up” the RNA polymerase, repair the damage, and then restart the polymerase.

1	The importance of transcription-coupled excision repair is seen in people with Cockayne syndrome, which is caused by a defect in this coupling. These individuals suffer from growth retardation, skeletal abnormalities, progressive neural retardation, and severe sensitivity to sunlight. Most of these problems are thought to arise from RNA polymerase molecules that become permanently stalled at sites of DNA damage that lie in important genes. The Chemistry of the DNA Bases Facilitates Damage Detection

1	The Chemistry of the DNA Bases Facilitates Damage Detection The DNA double helix seems optimal for repair. As noted above, it contains a backup copy of all genetic information. Equally importantly, the nature of the four bases in DNA makes the distinction between undamaged and damaged bases very clear. For example, every possible deamination event in DNA yields an “unnatural” base, which can be directly recognized and removed by a specific DNA glycosylase. Hypoxanthine, for example, is the simplest purine base capable of pairing specifically with C, but hypoxanthine is the direct deamination product of A (Figure 5–43A). The addition of a second amino group to hypoxanthine

1	Figure 5–42 The recognition of an unusual nucleotide in DNA by base-flipping. The DNA glycosylase family of enzymes recognizes specific inappropriate bases in the conformation shown. Each of these enzymes cleaves the glycosyl bond that connects a particular recognized base (yellow) to the backbone sugar, removing it from the DNA. (A) Stick model; (B) space-filling model.

1	Figure 5–43 The deamination of DNA nucleotides. In each case, the oxygen atom that is added in this reaction with water is colored red. (A) The spontaneous deamination products of A and G are recognizable as unnatural when they occur in DNA and thus are readily found and repaired. The deamination of C to U was also illustrated in Figure 5–38; T has no amino group to remove. (B) About 3% of the C nucleotides in vertebrate DNAs are methylated to help in controlling gene expression (discussed in Chapter 7). When these 5-methyl C nucleotides are accidentally deaminated, they form the natural nucleotide T. However, this T will be paired with a G on the opposite strand, forming a mismatched base pair. produces G, which cannot be formed from A by spontaneous deamination, and whose deamination product (xanthine) is likewise unique.

1	produces G, which cannot be formed from A by spontaneous deamination, and whose deamination product (xanthine) is likewise unique. As discussed in Chapter 6, RNA is thought, on an evolutionary time scale, to have served as the genetic material before DNA, and it seems likely that the genetic code was initially carried in the four nucleotides A, C, G, and U. This raises the question of why the U in RNA was replaced in DNA by T (which is 5-methyl U). We have seen that the spontaneous deamination of C converts it to U, but that this event is rendered relatively harmless by uracil DNA glycosylase. However, if DNA contained U as a natural base, the repair system would not be able to distinguish a deaminated C from a naturally occurring U.

1	A special situation occurs in vertebrate DNA, in which selected C nucleotides are methylated at specific CG sequences that are associated with inactive genes (discussed in Chapter 7). The accidental deamination of these methylated C nucleotides produces the natural nucleotide T (Figure 5–43B) in a mismatched base pair with a G on the opposite DNA strand. To help in repairing deaminated methylated C nucleotides, a special DNA glycosylase recognizes a mismatched base pair involving T in the sequence T-G and removes the T. This DNA repair mechanism must be relatively ineffective, however, because methylated C nucleotides are exceptionally common sites for mutations in vertebrate DNA. It is striking that, even though only about 3% of the C nucleotides in human DNA are methylated, mutations in these methylated nucleotides account for about one-third of the single-base mutations that have been observed in inherited human diseases.

1	If a cell’s DNA suffers heavy damage, the repair mechanisms that we have discussed are often insufficient to cope with it. In these cases, a different strategy is called into play, one that entails some risk to the cell. The highly accurate replicative DNA polymerases stall when they encounter damaged DNA, and in emergencies cells employ versatile, but less accurate, backup polymerases, known as translesion polymerases, to replicate through the DNA damage.

1	Human cells have seven translesion polymerases, some of which can recognize a specific type of DNA damage and correctly add the nucleotide required to restore the initial sequence. Others make only “good guesses,” especially when the template base has been extensively damaged. These enzymes are not as accurate as the normal replicative polymerases when they copy a normal DNA sequence. For one thing, the translesion polymerases lack exonucleolytic proofreading activity; in addition, many are much less discriminating than the replicative polymerase in choosing which nucleotide to incorporate initially. Presumably for this reason, each such translesion polymerase is given a chance to add only one or a few nucleotides before the highly accurate replicative polymerase resumes DNA synthesis.

1	Despite their usefulness in allowing heavily damaged DNA to be replicated, these translesion polymerases do, as noted above, pose risks to the cell. They are probably responsible for most of the base-substitution and single-nucleotide deletion mutations that accumulate in genomes; although they generally produce mutations when copying damaged DNA (see Figure 5–40), they probably also create mutations—at a low level—on undamaged DNA. Clearly, it is important for the cell to tightly regulate these polymerases, releasing them only at sites of DNA damage. Exactly how this happens for each translesion polymerase remains to be discovered, but a conceptual model is given in Figure 5–44. The principle of this model applies to many of the DNA repair processes discussed in this chapter: because the enzymes that carry out these reactions are potentially dangerous to the genome, they must be brought into play only at sites of damage.

1	An especially dangerous type of DNA damage occurs when both strands of the double helix are broken, leaving no intact template strand to enable accurate Figure 5–44 Translesion DNA polymerases can use damaged templates. According to this model, a replicative polymerase stalled at a site of DNA damage is recognized by the cell as needing rescue. Specialized enzymes covalently modify the sliding clamp (typically, it is ubiquitylated—see Figure 3–69) which releases the replicative DNA polymerase and, together with damaged DNA, attracts a translesion polymerase specific to that type of damage. Once the damaged DNA is bypassed, the covalent modification of the clamp is removed, the translesion polymerase dissociates, and the replicative polymerase is brought back into play.

1	removal of covalent modifcations, reloading of replicative DNA polymerase, DNA synthesis continues repair. Ionizing radiation, replication errors, oxidizing agents, and other metabolites produced in the cell cause breaks of this type. If these lesions were left unrepaired, they would quickly lead to the breakdown of chromosomes into smaller fragments and to loss of genes when the cell divides. However, two distinct mechanisms have evolved to deal with this type of damage (Figure 5–45). The simplest to understand is nonhomologous end joining, in which the broken ends are simply brought together and rejoined by DNA ligation, generally with the loss of nucleotides at the site of joining (Figure 5–46). This end-joining mechanism, which can be seen as a “quick and dirty” solution to the repair of double-strand breaks, is common in mammalian somatic cells. Although a change in the DNA sequence (a mutation) results at the site of breakage, so little of the mammalian genome is essential for

1	of double-strand breaks, is common in mammalian somatic cells. Although a change in the DNA sequence (a mutation) results at the site of breakage, so little of the mammalian genome is essential for life that this mechanism is apparently an acceptable solution to the problem of rejoining broken chromosomes. By the time a human reaches the age of 70, the typical somatic cell contains over 2000 such “scars,” distributed throughout its genome, representing places where DNA has been inaccurately repaired by nonhomologous end joining. But nonhomologous end joining presents another danger: because there seems to be no mechanism to ensure that two ends being joined were originally next to each other in the genome, nonhomologous end joining can occasionally generate rearrangements in which one broken chromosome becomes covalently attached to another. This can result in chromosomes with two centromeres and chromosomes lacking centromeres altogether; both processing of processing of 5˜ endsDNA

1	chromosome becomes covalently attached to another. This can result in chromosomes with two centromeres and chromosomes lacking centromeres altogether; both processing of processing of 5˜ endsDNA ends by nuclease deletion of DNA sequence damage repaired accurately using sister chromatid as the template types of aberrant chromosomes are missegregated during cell division. As previously discussed, the specialized structure of telomeres prevents the natural ends of chromosomes from being mistaken for broken DNA and “repaired” in this way.

1	A much more accurate type of double-strand break repair occurs in newly replicated DNA (Figure 5–45B). Here, the DNA is repaired using the sister chromatid as a template. This reaction is an example of homologous recombination, and we consider its mechanism later in this chapter. Most organisms employ both non-homologous end joining and homologous recombination to repair double-strand breaks in DNA. Nonhomologous end joining predominates in humans; homologous recombination is used only during and shortly after DNA replication (in S and G2 phases), when sister chromatids are available to serve as templates. repaired DNA has generally suffered a deletion of nucleotides (A)

1	repaired DNA has generally suffered a deletion of nucleotides (A) Figure 5–45 Two ways to repair double-strand breaks. (A) Nonhomologous end joining alters the original DNA sequence when repairing a broken chromosome. The initial degradation of the broken DNA ends is important because the nucleotides at the site of the initial break are often damaged and cannot be ligated. Nonhomologous end joining usually takes place when cells have not yet duplicated their DNA. (B) Repairing double-strand breaks by homologous recombination is more difficult to accomplish but restores the original DNA sequence. It typically takes place after the DNA has been duplicated (when a duplex template is available) but before the cell has divided. Details of the homologous recombination pathway are presented in the following section (see Figure 5–48).

1	Figure 5–46 Nonhomologous end joining. (A) A central role is played by the Ku protein, a heterodimer that grasps the broken chromosome ends. The additional proteins shown are needed to hold the broken ends together while they are processed and eventually joined covalently. (B) Three-dimensional structure of a Ku heterodimer bound to the end of a duplex DNA fragment. The Ku protein is also essential for V(D)J joining, a specific recombination process through which antibody and T cell receptor diversity is generated in developing B and T cells (discussed in Chapter 24). V(D)J joining and nonhomologous end joining show many similarities in mechanism but the former relies on specific double-strand breaks produced deliberately by the cell. (B, from J.R. Walker, R.A. Corpina, and J. Goldberg, Nature 412:607–614, 2001. With permission from Macmillan Publishers Ltd.) DNA Damage Delays Progression of the Cell Cycle

1	DNA Damage Delays Progression of the Cell Cycle We have just seen that cells contain multiple enzyme systems that can recognize and repair many types of DNA damage (Movie 5.7). Because of the importance of maintaining intact, undamaged DNA from generation to generation, eukaryotic cells have an additional mechanism that maximizes the effectiveness of their DNA repair enzymes: they delay progression of the cell cycle until DNA repair is complete. As discussed in detail in Chapter 17, the orderly progression of the cell cycle is stopped if damaged DNA is detected, and it restarts when the damage has been repaired. Thus, in mammalian cells, the presence of DNA damage can block entry from G1 into S phase, it can slow S phase once it has begun, and it can block the transition from G2 phase to M phase. These delays facilitate DNA repair by providing the time needed for the repair to reach completion.

1	DNA damage also results in an increased synthesis of some DNA repair enzymes. This response depends on special signaling proteins that sense DNA damage and up-regulate the appropriate DNA repair enzymes. The importance of this mechanism is revealed by the phenotype of humans who are born with defects in the gene that encodes the ATM protein. These individuals have the disease ataxia telangiectasia (AT ), the symptoms of which include neurodegeneration, a predisposition to cancer, and genome instability. The ATM protein is a large kinase needed to generate the intracellular signals that sound the alarm in response to many types of spontaneous DNA damage (see Figure 17–62), and individuals with defects in this protein therefore suffer from the effects of unrepaired DNA lesions.

1	Genetic information can be stored stably in DNA sequences only because a large set of DNA repair enzymes continuously scan the DNA and replace any damaged nucleotides. Most types of DNA repair depend on the presence of a separate copy of the genetic information in each of the two strands of the DNA double helix. An accidental lesion on one strand can therefore be cut out by a repair enzyme and a corrected strand resynthesized by reference to the information in the undamaged strand.

1	Most of the damage to DNA bases is excised by one of two major DNA repair pathways. In base excision repair, the altered base is removed by a DNA glycosylase enzyme, followed by excision of the resulting sugar phosphate. In nucleotide excision repair, a small section of the DNA strand surrounding the damage is removed from the DNA double helix as an oligonucleotide. In both cases, the gap left in the DNA helix is filled in by the sequential action of DNA polymerase and DNA ligase, using the undamaged DNA strand as the template. Some types of DNA damage can be repaired by a different strategy—the direct chemical reversal of the damage— which is carried out by specialized repair proteins. When DNA damage is excessive, a special class of inaccurate DNA polymerases, called translesion polymerases, is used to bypass the damage, allowing the cell to survive but sometimes creating permanent mutations at the sites of damage.

1	Other critical repair systems—based on either nonhomologous end joining or homologous recombination—reseal the accidental double-strand breaks that occur in the DNA helix. In most cells, an elevated level of DNA damage causes a delay in the cell cycle, which ensures that DNA damage is repaired before a cell divides.

1	In the two preceding sections, we discussed the mechanisms that allow the DNA sequences in cells to be maintained from generation to generation with very little change. In this section, we further explore one of the DNA repair mechanisms, a diverse set of reactions known collectively as homologous recombination. The key feature of homologous recombination (also known as general recombination) is an exchange of DNA strands between a pair of homologous duplex DNA sequences, that is, segments of double helix that are very similar or identical in nucleotide sequence. This exchange allows one stretch of duplex DNA to act as a template to restore lost or damaged information on a second stretch of duplex DNA. Because the template for repair is not limited to the strand complementary to that containing the damage, homologous recombination can repair many types of DNA damage. It is, for example, the main way to accurately repair double-strand breaks, as introduced in the previous section (see

1	the damage, homologous recombination can repair many types of DNA damage. It is, for example, the main way to accurately repair double-strand breaks, as introduced in the previous section (see Figure 5–45B). Double-strand breaks can result from radiation and reactive chemicals, but most of the time they arise from DNA replication forks that become stalled or broken independently of any such external cause. Homologous recombination accurately corrects these accidents and, because they occur during nearly every round of DNA replication, this repair mechanism is essential for every proliferating cell. Homologous recombination is perhaps the most versatile DNA repair mechanism available to the cell; the “all-purpose” nature of recombinational repair probably explains why its mechanism and the proteins that carry it out have been conserved in virtually all cells on Earth.

1	Additionally, we shall see that homologous recombination plays a special role in sexually reproducing organisms. During meiosis, a key step in gamete (sperm and egg) production, it catalyzes the orderly exchange of bits of genetic information between corresponding (homologous) maternal and paternal chromosomes to create new combinations of DNA sequences in the chromosomes passed to the offspring.

1	The current view of homologous recombination as a critical DNA repair mechanism in all cells evolved slowly from its original discovery as a key component in the specialized process of meiosis in plants and animals. The subsequent recognition that homologous recombination also occurs in unicellular organisms made it much more amenable to molecular analyses. Thus, most of what we know about the biochemistry of genetic recombination was originally derived from studies of bacteria, especially of E. coli and its viruses, as well as from experiments with simple eukaryotes such as yeasts. For these organisms with short generation times and relatively small genomes, it was possible to isolate a large set of mutants with defects in their recombination processes. The protein altered in each mutant was then identified and, ultimately, studied biochemically. Close relatives of these proteins have been found in more complex eukaryotes including flies, mice, and humans, and more recently, it has

1	was then identified and, ultimately, studied biochemically. Close relatives of these proteins have been found in more complex eukaryotes including flies, mice, and humans, and more recently, it has been possible to directly analyze homologous recombination in these species as well. These studies reveal that the fundamental processes that catalyze homologous recombination are common to all cells.

1	The hallmark of homologous recombination is that it takes place only between DNA duplexes that have extensive regions of sequence similarity (homology). Not surprisingly, base-pairing underlies this requirement, and two DNA duplexes that are undergoing homologous recombination “sample” each other’s DNA sequence by engaging in extensive base-pairing between a single strand from one DNA duplex and the complementary single strand from the other. The match need not be perfect, but it must be very close for homologous recombination to succeed.

1	In its simplest form, this type of base-pairing interaction can be mimicked in a test tube by allowing a DNA double helix to re-form from its separated single strands. This process, called DNA renaturation or hybridization, occurs when a rare random collision juxtaposes complementary nucleotide sequences on two matching DNA single strands, allowing the formation of a short stretch of double helix between them. This relatively slow helix-nucleation step is followed by a very rapid “zippering” step, as the region of double helix is extended to maximize the number of base-pairing interactions (Figure 5–47).

1	DNA hybridization can create a region of DNA double helix consisting of strands that originate from two different duplex DNA molecules as long as they are complementary, or nearly so. As we will see shortly, the formation of such a hybrid molecule, known as a heteroduplex, is an essential feature of homologous recombination. DNA hybridization and heteroduplex formation is also the basis for many of the methods used to study cells, and we will discuss these uses in Chapter 8. The DNA in a living cell is almost all in the stable double-helical form, so the reaction depicted in Figure 5–47 rarely occurs in vivo. Instead, as we shall see, homologous recombination is brought about through a carefully controlled set of reactions that allow two DNA duplexes to sample each other’s sequences without fully dissociating into single strands.

1	We saw in the previous section that nonhomologous end-joining occurs without a template and usually leaves a mutation at the site at which a double-strand break is repaired. In contrast, homologous recombination can repair double-strand breaks accurately, without any loss or alteration of nucleotides at the site of repair. For homologous recombination to do this repair job, the broken DNA has to be brought into proximity with homologous but unbroken DNA, which can serve as a template for repair. For this reason, homologous recombination often occurs just after DNA replication, when the two daughter DNA molecules lie close together and one can serve as a template for repair of the other. As we shall see, the process of DNA replication itself creates a special risk of accidents requiring this sort of repair.

1	The simplest pathway through which homologous recombination can repair double-strand breaks is shown in Figure 5–48. In essence, the broken DNA duplex and the template duplex carry out a “strand dance” so that one of the damaged strands can use the complementary strand of the intact DNA duplex as a template for repair. First, the ends of the broken DNA are chewed back, or “resected,” by specialized nucleases to produce overhanging, single-strand 3ʹ ends. The next step is strand exchange (also called strand invasion), during which one of the single-strand 3ʹ ends from the damaged DNA molecule worms its way into the template duplex and searches it for homologous sequences through base-pairing. We describe this remarkable reaction in detail in the next section. Once stable base-pairing is established (which completes the strand exchange step), an accurate DNA polymerase extends the invading strand by using the information provided by the undamaged template molecule, thus restoring the

1	established (which completes the strand exchange step), an accurate DNA polymerase extends the invading strand by using the information provided by the undamaged template molecule, thus restoring the damaged DNA. The last steps—strand displacement, further repair synthesis, and ligation—restore the two original DNA double helices and complete the repair process. Homologous recombination resembles other DNA repair reactions in that a

1	Figure 5–47 DNA hybridization. DNA double helices can re-form from their separated strands in a reaction that depends on the random collision of two complementary DNA strands. The vast majority of such collisions are not productive, as shown on the left, but a few result in a short region where complementary base pairs have formed (helix nucleation). A rapid zippering then leads to the formation of a complete double helix. Through this trial-and-error process, a DNA strand will find its complementary partner even in the midst of millions of nonmatching DNA strands. DNA polymerase utilizes a pristine template to restore damaged DNA. However, instead of using the partner complementary strand as a template, as occurs in most DNA repair pathways, homologous recombination exploits a complementary strand from a separate DNA duplex. Strand Exchange Is Carried Out by the RecA/Rad51 Protein

1	Strand Exchange Is Carried Out by the RecA/Rad51 Protein Of all the steps of homologous recombination, strand exchange is the most difficult to imagine. How does the invading single strand rapidly sample a DNA duplex for homology? Once the homology is found, how does the exchange occur? How is the inherent stability of the template double helix overcome? The answers to these questions came from biochemical and structural studies of the protein that carries out these feats, called RecA in E. coli and Rad51 in virtually all eukaryotic organisms. To catalyze strand exchange, RecA first binds cooperatively to the invading single strand, forming a protein–DNA filament that forces the DNA into an unusual configuration: groups of three consecutive nucleotides are held as though they were in a conventional DNA double helix but, between adjacent triplets, the DNA backbone is untwisted and stretched out (Figure 5–49). This unusual protein–DNA filament then binds to duplex DNA

1	Figure 5–48 Mechanism of double-strand break repair by homologous recombination. This is the preferred method for repairing DNA double-strand breaks that arise shortly after the DNA has been replicated, while the daughter DNA molecules are still held close together. In general, homologous recombination can be regarded as a flexible series of reactions, with the exact pathway differing from one case to the next. For example, the length of the repair “patch” can vary considerably depending on the extent of 5ʹ processing and new DNA synthesis, indicated in green.

1	in a way that stretches the duplex, destabilizing it and making it easy to pull the strands apart. The invading single strand then can sample the sequence of the duplex by conventional base-pairing. This sampling occurs in triplet nucleotide blocks: if a triplet match is found, the adjacent triplet is sampled, and so on. In this way, mismatches quickly lead to dissociation and only an extended stretch of base-pairing (at least 15 nucleotides) stabilizes the invading strand and leads to strand exchange.

1	RecA hydrolyzes ATP, and the steps described above require that each RecA monomer along the filament be in the ATP-bound state. However, the searching itself does not require ATP hydrolysis; instead, the process occurs by simple molecular collision, allowing many potential sequences to be rapidly sampled. Once the strand-exchange reaction is completed, however, ATP hydrolysis is necessary to disassemble RecA from the complex of DNA molecules. At this point, repair DNA polymerases and DNA ligase can complete the repair process, as shown in Figure 5–48.

1	Although accurately repairing double-strand breaks, which can arise from radiation or chemical reactions, is a crucial function of homologous recombination, perhaps its most important role is in rescuing stalled or broken DNA replication forks. Many types of events can cause a replication fork to break, and here we consider just one example: a single-strand nick or gap in the parental DNA helix just ahead of a replication fork. When the fork reaches this lesion, it falls apart—resulting in one broken and one intact daughter chromosome. The broken fork can be flawlessly repaired (Figure 5–50) using the same basic homologous recombination reactions we discussed above for the repair of double-strand breaks. With slight modifications, the set of reactions depicted in Figures 5–48 and 5–50— known collectively as homologous recombination—can accurately repair many different types of DNA damage. Cells Carefully Regulate the Use of Homologous Recombination in DNA Repair

1	Cells Carefully Regulate the Use of Homologous Recombination in DNA Repair Although homologous recombination neatly solves the problem of accurately repairing double-strand breaks and other types of DNA damage, it does present

1	Figure 5–49 Strand invasion catalyzed by the RecA protein. Our understanding of this reaction is based in part on structures determined by x-ray diffraction studies of RecA bound to singleand double-strand DNA. These DNA structures (shown without the RecA protein) are on the left side of the diagram. Starting at the top, ATP-bound RecA associates with single-strand DNA, holding it in an elongated form where groups of three bases are separated from each other by a stretched and twisted backbone. In the next step, the RecA-bound single strand then binds to duplex DNA, destabilizing it and allowing the single strand to sample its sequence through base-pairing, three bases at a time. If no match is found, the RecA-bound single strand of DNA rapidly dissociates and begins a new search. If an extensive match is found, the structure is disassembled through ATP hydrolysis, resulting in the dissociation of RecA and the exchange of one single strand of DNA for another, thereby forming a

1	If an extensive match is found, the structure is disassembled through ATP hydrolysis, resulting in the dissociation of RecA and the exchange of one single strand of DNA for another, thereby forming a heteroduplex. (PDB code: 3CMX.)

1	Figure 5–50 Repair of a broken replication fork by homologous recombination. When a moving replication fork encounters a single-strand break, it will collapse, but can be repaired by homologous recombination. The process uses many of the same reactions shown in Figure 5–48 and proceeds through the same basic steps. Green strands represent the new DNA synthesis that takes place after the replication fork has broken. This pathway allows the fork to move past the site that was nicked on the original template by using the undamaged duplex as a template to synthesize DNA. (Adapted from M.M. Cox, Proc. Natl Acad. Sci. USA 98:8173–8180, 2001. With permission from National Academy of Sciences.) some dangers to the cell as it sometimes “repairs” damage using the wrong bit of the genome as the template. For example, sometimes a broken human chromosome is “repaired” using the homolog from the other parent instead of the sister chromatid as the template. Because maternal and paternal chromosomes

1	For example, sometimes a broken human chromosome is “repaired” using the homolog from the other parent instead of the sister chromatid as the template. Because maternal and paternal chromosomes differ in DNA sequence at many positions along their lengths, this type of repair can convert the sequence of the repaired DNA from the maternal to the paternal sequence or vice versa. The result of this type of errant recombination is known as loss of heterozygosity. It can have severe consequences if the homolog used for repair contains a deleterious mutation, because the recombination event destroys the “good” copy. Loss of heterozygosity, although rare, is a critical step in the formation of many cancers (discussed in Chapter 20).

1	Cells go to great lengths to minimize the risk of mishaps of these types; indeed, nearly every step of homologous recombination is carefully regulated. For example, the first step, processing of the broken ends, is coordinated with the cell cycle: the nuclease enzymes that carry out this process are activated (in part, by phosphorylation) only in the S and G2 phases of the cell cycle, when a daughter duplex (either as a partially replicated chromosome or a fully replicated sister chromatid) can serve as a template for repair (see Figure 5–50). The close proximity of the two daughter chromosomes disfavors the use of other genome sequences in the repair process.

1	The loading of RecA or Rad52 onto the processed DNA ends and the subsequent strand-exchange reaction are also tightly controlled. Although these proteins alone can carry out these steps in vitro, a series of accessory proteins, including Rad52, is needed in eukaryotic cells to ensure that homologous recombination is efficient and accurate (Figure 5–51). There are many such accessory proteins, and exactly how they coordinate and control homologous recombination remains a mystery. We do know that the enzymes that catalyze recombinational repair are made at relatively high levels in eukaryotes and are dispersed throughout the nucleus in an inactive form. In response to DNA damage, they rapidly converge on the sites of DNA damage, become activated, and form “repair factories” where many lesions are apparently brought together and repaired (Figure 5–52).

1	In Chapter 20, we shall see that both too much and too little homologous recombination can lead to cancer in humans, the former through repair using the “wrong” template (as described above) and the latter through an increased mutation rate caused by inefficient DNA repair. Clearly, a delicate balance has evolved that keeps this process in check on undamaged DNA, while still allowing it to act efficiently and rapidly on DNA lesions as soon as they arise. Not surprisingly, mutations in the components that carry out and regulate homologous recombination are responsible for several inherited forms of cancer. Two of these, the Brca1 and Brca2 proteins, were first discovered because

1	Figure 5–51 Structure of a portion of the Rad52 protein. This doughnutshaped structure is composed of 11 subunits. Single-strand DNA has been modeled into the deep groove running along the protein surface. Rad52 helps load Rad51 onto single-strand DNA to form the nucleoprotein filament that carries out strand exchange. Rad52 also acts later to re-form the double helix and complete the homologous recombination reaction. (From

1	M.R. Singleton et al., Proc. Natl Acad. Sci. USA 99:13492–13497, 2002. With permission from National Academy of Sciences.) mutations in their genes lead to a greatly increased frequency of breast cancer. Because these mutations cause inefficient repair by homologous recombination, accumulation of DNA damage can, in a small proportion of cells, give rise to a cancer. Brca1 regulates an early step in broken-end processing; without it, such ends are not processed correctly for homologous recombination and instead are repaired inaccurately by the nonhomologous end-joining pathway (see Figure 5–45). Brca2 binds to the Rad51 protein, preventing its polymerization on DNA, and thereby maintaining it in an inactive form until it is needed. Normally, upon DNA damage, Brca2 helps to bring Rad51 protein rapidly to sites of damage and, once in place, to release it in its active form onto single-strand DNA.

1	We have seen that homologous recombination comprises a group of reactions— including broken-end processing, strand exchange, limited DNA synthesis, and ligation—to exchange DNA sequences between two double helices of similar nucleotide sequence. Having discussed its role in accurately repairing damaged DNA, we now turn to homologous recombination as a means to generate DNA molecules that carry novel combinations of genes as a result of the deliberate exchange of material between different chromosomes. Although this occasionally occurs by accident in mitotic cells (and is often detrimental), it is a frequent and necessary part of meiosis, which occurs in sexually reproducing organisms such as fungi, plants, and animals.

1	Here, homologous recombination occurs as an integral part of the process whereby chromosomes are parceled out to germ cells (sperm and eggs in animals). We discuss the process of meiosis in detail in Chapter 17; in the following sections, we discuss how homologous recombination during meiosis produces chromosome crossing-over and gene conversion, resulting in hybrid chromosomes that contain genetic information from both the maternal and paternal homologs (Figure 5–53). Crossing-over and gene conversion are both generated by homologous recombination mechanisms that, at their core, resemble those used to repair double-strand breaks. Meiotic Recombination Begins with a Programmed Double-Strand Break

1	Meiotic Recombination Begins with a Programmed Double-Strand Break Homologous recombination in meiosis starts with a bold stroke: a specialized protein (called Spo11 in budding yeast) breaks both strands of the DNA double helix in one of the recombining chromosomes (Figure 5–54). Like a topoisomerase, Spo11, after catalyzing this reaction, remains covalently bound to the broken site of gene site of conversion crossover

1	Figure 5–52 Experiment demonstrating the rapid localization of repair proteins to DNA double-strand breaks. Human fibroblasts were x-irradiated to produce DNA double-strand breaks. Before the x-rays struck the cells, they were passed through a microscopic grid with x-ray-absorbing “bars” spaced 1 μm apart. This produced a striped pattern of DNA damage, allowing a comparison of damaged and undamaged DNA in the same nucleus. (A) Total DNA in a fibroblast nucleus stained with the dye DAPI. (B) Sites of new DNA synthesis due to repair of DNA damage, indicated by incorporation of BudR (a thymidine analog) and subsequent staining with fluorescently labeled antibodies to BudR (green).

1	(C) Localization of the Mre11 complex to damaged DNA as visualized by antibodies against the Mre11 subunit (red). Mre11 is a nuclease that processes damaged DNA in preparation for homologous recombination (see Figure 5–48). (A), (B), and (C) were processed 30 minutes after x-irradiation. (From B.E. Nelms et al., Science 280:590– 592, 1998. With permission from AAAS.) Figure 5–53 Chromosome crossing-over occurs in meiosis. Meiosis is the process by which a diploid cell gives rise to four haploid germ cells, as described in detail in Chapter 17. Meiosis produces germ cells in which the paternal and maternal genetic information (red and blue) has been reassorted through chromosome crossovers. In addition, many short regions of gene conversion occur, as indicated.

1	Figure 5–54 Homologous recombination during meiosis can generate chromosome crossovers. Once the meiosis-specific protein Spo11 and the Mre11 complex break the duplex DNA and process the ends, homologous recombination can proceed along alternative pathways. One (right side of figure) closely resembles the double-strand break repair reaction shown in Figure 5–48 and results in chromosomes that have been “repaired” but have not crossed over. The other (left side with strand breaks as shown by the blue arrows) proceeds through a double Holliday junction and produces two chromosomes that have crossed over. During meiosis, homologous recombination takes place between maternal and paternal chromosome homologs when they are held tightly together (see Figure 17–54). DNA (see Figure 5–21). A specialized nuclease then rapidly degrades the ends bound by Spo11, removing the protein along with the DNA and leaving protruding 3ʹ single-strand ends.

1	DNA (see Figure 5–21). A specialized nuclease then rapidly degrades the ends bound by Spo11, removing the protein along with the DNA and leaving protruding 3ʹ single-strand ends. At this point, many of the recombination reactions resemble those described above for the repair of double-strand breaks; indeed, some of the same proteins are used for both processes. However, several meiosis-specific proteins direct them to perform their tasks somewhat differently, resulting in the distinctive outcomes observed for meiosis. Another important difference is that, in meiosis, recombination occurs preferentially between maternal and paternal chromosomal homo-logs rather than between the newly replicated, identical DNA duplexes that pair in double-strand break repair. In the sections that follow, we describe in more detail those aspects of homologous recombination that are especially important for meiosis.

1	Of special importance in meiosis is an intermediate known as a Holliday junction or cross-strand exchange (Figure 5–55). Each Holliday junction can adopt multiple conformations and a special set of recombination proteins binds to, and thereby stabilizes, the open, symmetric isomer. Specialized proteins that bind to Holliday junctions can catalyze a reaction known as branch migration (Figure 5–56), whereby DNA is spooled through the Holliday junction by continually breaking and re-forming base pairs (Figure 5–57). In this way, the Holliday junction proteins use ATP hydrolysis to expand the region of heteroduplex DNA initially created by the strand-exchange reaction. In meiosis, heteroduplex regions often “migrate” thousands of nucleotides from the original site of the double-strand break. As shown in Figure 5–54, Holliday junctions usually occur in pairs, known as double Holliday junctions.

1	As shown in Figure 5–54, there are two basic outcomes of homologous recombination during meiosis. In humans, approximately 90% of the double-strand breaks produced during meiosis are resolved as non-crossovers (see right side of Figure 5–54). Here, the two original DNA duplexes separate from each other in a form unaltered except for a region of heteroduplex that formed near the site of the original double-strand break. This set of reactions resembles that described above for the repair of double-strand breaks (see Figure 5–48). The other outcome is more profound: a double Holliday junction is formed and is cleaved by specialized enzymes to create a crossover (see left side of Figure 5–54). The two original portions of each chromosome upstream and downstream

1	Figure 5–55 A Holliday junction. The initially formed structure (A) is usually drawn with two strands crossing, as in Figure 5–54. An isomerization of the Holliday junction produces an open, symmetrical structure that is bound by specialized proteins. These proteins “move” the Holliday junctions by a coordinated set of branch-migration reactions (see Figure 5–57 and Movie 5.8). (D) Structure of the Holliday junction in the open form depicted in (B). The Holliday junction is named for the scientist who first proposed its formation. (PDB code: 1DCW.) direction of branch migration

1	Figure 5–56 Simplified view of branch migration. In branch migration, base pairs are continually broken and formed as the branch point moves. Although branch migration can happen spontaneously on naked DNA molecules, the process is inefficient and the branch moves back and forth at random. In the cell, branch migration is carried out using specialized proteins and ATP hydrolysis to ensure that, as shown, the branch moves rapidly and in one direction. As shown in Figure 5–57, branch migrations often occur at Holliday junctions, where two branch-migration reactions are coupled.

1	Figure 5–57 Enzyme-catalyzed branch movement at a Holliday junction by branch migration. In E. coli, a tetramer of the RuvA protein (green) and two hexamers of the RuvB protein (yellow) bind to the open form of the junction. The RuvB protein, which resembles the hexameric helicases used in DNA replication (Figure 5–14), uses the energy of ATP hydrolysis to spool DNA rapidly through the Holliday junction, extending the heteroduplex region as shown. The RuvA protein coordinates this movement, threading the DNA strands to avoid tangling. (PDB codes: 1IXR, 1C7Y.) from the two Holliday junctions are thereby swapped, creating two chromosomes that have crossed over.

1	How does the cell decide which Spo11-induced double-strand breaks to resolve as crossovers? The answer is not yet known, but we know the decision is an important one. The relatively few crossovers that do form are distributed along chromosomes in such a way that a crossover in one position inhibits crossing-over in neighboring regions. Termed crossover control, this fascinating but poorly understood regulatory mechanism ensures the roughly even distribution of crossover points along chromosomes. It also ensures that each chromosome— no matter how small—undergoes at least one crossover every meiosis. For many organisms, roughly two crossovers per chromosome occur during each meiosis, one on each arm. As discussed in detail in Chapter 17, these crossovers play an important mechanical role in the proper segregation of chromosomes during meiosis.

1	Whether a meiotic recombination event is resolved as a crossover or a non-crossover, the recombination machinery leaves behind a heteroduplex region where a strand with the DNA sequence of the paternal homolog is base-paired with a strand from the maternal homolog (Figure 5–58). These heteroduplex regions can tolerate a small percentage of mismatched base pairs, and because of branch migration, they often extend for thousands of nucleotide pairs. The many non-crossover events that occur in meiosis thereby produce scattered sites in the germ cells where short DNA sequences from one homolog have been pasted into the other homolog. Heteroduplex regions mark sites of potential gene conversion—where the four haploid chromosomes produced by meiosis contain three copies of a DNA sequence from one homolog and only one copy of this sequence from the other homolog (see Figure 5–53), as explained next.

1	Figure 5–58 Heteroduplexes formed during meiosis. Heteroduplex DNA is present at sites of recombination that are resolved either as crossovers or non-crossovers. Because the DNA sequences of maternal and paternal chromosomes differ at many positions along their lengths, heteroduplexes often contain a small number of base-pair mismatches.

1	Figure 5–59 Gene conversion caused by mismatch correction. In this process, heteroduplex DNA is formed at the sites of homologous recombination between maternal and paternal chromosomes. If the maternal and paternal DNA sequences are slightly different, the heteroduplex region will include some mismatched base pairs, which may then be corrected by the DNA mismatch repair machinery (see Figure 5–19). Such repair can “erase” nucleotide sequences on either the paternal or the maternal strand. The consequence of this mismatch repair is gene conversion, detected as a deviation from the segregation of equal copies of maternal and paternal alleles that normally occurs in meiosis.

1	In sexually reproducing organisms, it is a fundamental law of genetics that—aside from mitochondrial DNA, which is inherited only through the mother—each parent makes an equal genetic contribution to an offspring. One complete set of nuclear genes is inherited from the father and one complete set is inherited from the mother. Underlying this law is the accurate parceling out of chromosomes to the germ cells (eggs and sperm) that takes place during meiosis. Thus, when a diploid cell in a parent undergoes meiosis to produce four haploid germ cells, exactly half of the genes distributed among these four cells should be maternal (genes inherited from the mother of this parent) and the other half paternal (genes inherited from the father of this parent). In some organisms (fungi, for example), it is possible to recover and analyze all four of the haploid gametes produced from a single cell by meiosis. Studies in such organisms have revealed rare cases in which the parceling out of genes

1	it is possible to recover and analyze all four of the haploid gametes produced from a single cell by meiosis. Studies in such organisms have revealed rare cases in which the parceling out of genes violates the standard genetic rules. Occasionally, for example, meiosis yields three copies of the maternal version of a gene and only one copy of the paternal allele. Alternative versions of the same gene are called alleles, and it is the divergence from their expected distribution during meiosis that is known as gene conversion. Genetic studies show that only small sections of DNA typically undergo gene conversion, and in many cases only a part of a gene is changed.

1	Several pathways in the cell can lead to gene conversion, but one of the most important arises from a particular consequence of recombination during meiosis. We have seen that both crossovers and non-crossovers produce heteroduplex regions of DNA. If the two strands that make up a heteroduplex region do not have identical nucleotide sequences, mismatched base pairs are formed, and these are often repaired by the cell’s mismatch repair system (see Figure 5–19). However, the mismatch repair system cannot distinguish between the paternal and maternal strands and will randomly choose the strand to be used as a template. As a consequence, one allele will be lost and the other duplicated (Figure 5–59), resulting in net “conversion” of one allele to the other. Thus, gene conversion, originally regarded as a mysterious deviation from the rules of genetics, can be seen as a straightforward consequence of the mechanisms of homologous recombination.

1	Homologous recombination describes a flexible set of reactions resulting in the exchange of DNA sequences between a pair of identical or nearly identical duplex DNA molecules. In all cells, this process is essential for the error-free repair of chromosome damage, particularly double-strand breaks and broken or stalled replication forks. Homologous recombination is also responsible for the crossing-over of chromosomes that occurs during meiosis. Homologous recombination takes place through a variety of pathways, but they have in common a strand-exchange step whereby a single strand from one DNA duplex invades a second duplex and base-pairs with one strand while displacing the other. This reaction, catalyzed by the RecA/Rad51 family of proteins, can only occur if the invading strand can form a short stretch of consecutive nucleotide pairs with one of the strands of the duplex. This requirement ensures that homologous recombination occurs only between identical or very similar DNA

1	form a short stretch of consecutive nucleotide pairs with one of the strands of the duplex. This requirement ensures that homologous recombination occurs only between identical or very similar DNA sequences.

1	DNA SYNTHESIS FILLS GAP, CREATING AN EXTRA COPY OF THE RED ALLELE OF When used as a repair mechanism, homologous recombination occurs between a damaged DNA molecule and its recently duplicated sister molecule, with the undamaged duplex acting as a template to repair the damaged copy flawlessly. In meiosis, homologous recombination is initiated by deliberate, carefully regulated double-strand breaks and occurs preferentially between the homologous chromosomes rather than the newly replicated sister chromatids. The outcome can be either two chromosomes that have crossed over (that is, chromosomes in which the DNA on either side of the site of DNA pairing originates from two different homo-logs) or two non-crossover chromosomes. In the latter case, the two chromosomes that result are identical to the original two homologs, except for relatively minor DNA sequence changes at the site of recombination.

1	We have seen that homologous recombination can result in the exchange of DNA sequences between chromosomes. However, the order of genes on the interacting chromosomes typically remains the same following homologous recombination, inasmuch as the recombining sequences must be very similar for the process to occur. In this section, we describe two very different types of recombination—transposition (also called transpositional recombination) and conservative site-specific recombination—that do not require substantial regions of DNA homology. These two types of recombination reactions can alter gene order along a chromosome and can cause unusual types of mutations that introduce whole blocks of DNA sequence into the genome.

1	Transposition and conservative site-specific recombination are largely dedicated to moving a wide variety of specialized segments of DNA—collectively termed mobile genetic elements—from one position in a genome to another. We will see that mobile genetic elements can range in size from a few hundred to tens of thousands of nucleotide pairs, and each typically carries a unique set of genes. Often, one of these genes encodes a specialized enzyme that catalyzes the movement of only that element, thereby making this type of recombination possible.

1	Virtually all cells contain mobile genetic elements (known informally as “jumping genes”). As explained in Chapter 4, over evolutionary time scales, they have had a profound effect on the shaping of modern genomes. For example, nearly half of the human genome can be traced to these elements (see Figure 4–62). Over time, random mutation has altered their nucleotide sequences, and, as a result, only a few of the many copies of these elements in our DNA are still active and capable of movement. The remainder are molecular fossils whose existence provides striking clues to our evolutionary history.

1	Mobile genetic elements are often considered to be molecular parasites (they are also termed “selfish DNA”) that persist because cells cannot get rid of them; they certainly have come close to overrunning our own genome. However, mobile DNA elements can provide benefits to the cell. For example, the genes they carry are sometimes advantageous, as in the case of antibiotic resistance in bacterial cells, discussed below. The movement of mobile genetic elements also produces many of the genetic variants upon which evolution depends, because, in addition to moving themselves, mobile genetic elements occasionally rearrange neighboring sequences of the host genome. Thus, spontaneous mutations observed in Drosophila, humans, and other organisms are often due to the movement of mobile genetic elements. While many of these mutations will be deleterious to the organism, some will be advantageous and may spread throughout the population. It is almost certain that much of the variety of life we

1	While many of these mutations will be deleterious to the organism, some will be advantageous and may spread throughout the population. It is almost certain that much of the variety of life we see around us originally arose from the movement of mobile genetic elements.

1	In this section, we introduce mobile genetic elements and describe the mechanisms that enable them to move around a genome. We shall see that some of these elements move through transposition mechanisms and others through conservative site-specific recombination. We begin with transposition, as there are many more known examples of this type of movement. Through Transposition, Mobile Genetic Elements Can Insert Into Any DNA Sequence

1	Through Transposition, Mobile Genetic Elements Can Insert Into Any DNA Sequence Mobile elements that move by way of transposition are called transposons, or transposable elements. In transposition, a specific enzyme, usually encoded by the transposon itself and typically called a transposase, acts on specific DNA sequences at each end of the transposon, causing it to insert into a new target DNA site. Most transposons are only modestly selective in choosing their target site, and they can therefore insert themselves into many different locations in a genome. In particular, there is no general requirement for sequence similarity between the ends of the element and the target sequence. Most transposons move only rarely. In bacteria, where it is possible to measure the frequency accurately, transposons typically move once every 105 cell divisions. More frequent movement would probably destroy the host cell’s genome.

1	On the basis of their structure and transposition mechanism, transposons can be grouped into three large classes: DNA-only transposons, retroviral-like retrotransposons, and nonretroviral retrotransposons. The differences among them are briefly outlined in Table 5–4, and each class will be discussed in turn.

1	DNA-only transposons, so named because they exist only as DNA during their movement, predominate in bacteria, and they are largely responsible for the spread of antibiotic resistance in bacterial strains. When antibiotics like penicillin and streptomycin first became widely available in the 1950s, most bacteria that caused human disease were susceptible to them. Now, the situation is different— antibiotics such as penicillin (and its modern derivatives) are no longer effective against many modern bacterial strains, including those causing gonorrhea and bacterial pneumonia. The spread of antibiotic resistance is due largely to genes that encode antibiotic-inactivating enzymes that are carried on transposons (Figure 5–60). Although these mobile elements can transpose only within cells that already carry them, they can be moved from one cell to another through other mechanisms known collectively as horizontal gene transfer (see Figure 1–19). Once introduced into a new cell, a transposon

1	already carry them, they can be moved from one cell to another through other mechanisms known collectively as horizontal gene transfer (see Figure 1–19). Once introduced into a new cell, a transposon can insert itself into the genome and be faithfully passed on to all progeny cells through the normal processes of DNA replication and cell division.

1	DNA-only transposons can relocate from a donor site to a target site by cutand-paste transposition (Figure 5–61). Here, the transposon is literally excised from one spot on a genome and inserted into another. This reaction produces a short duplication of the target DNA sequence at the insertion site; these direct repeat sequences that flank the transposon serve as convenient records of prior transposition events. Such “signatures” often provide valuable clues in identifying transposons in genome sequences.

1	When a cut-and-paste DNA-only transposon is excised from its original location, it leaves behind a “hole” in the chromosome. This lesion can be perfectly healed by recombinational double-strand break repair (see Figure 5–48), provided that the chromosome has just been replicated and an identical copy of the damaged host sequence is available. Alternatively, a nonhomologous end-joining reaction can reseal the break; in this case, the DNA sequence that originally flanked the transposon is altered, producing a mutation at the chromosomal site from which the transposon was excised (see Figure 5–45). Remarkably, the same mechanism used to excise cut-and-paste transposons from DNA has been found to operate in developing immune systems of short direct repeats of target DNA sequences in chromosome B

1	Figure 5–60 Three of the many DNA-only transposons found in bacteria. Each of these mobile DNA elements contains a gene that encodes a transposase, an enzyme that carries out the DNA breakage and joining reactions needed for the element to move. Each transposon also carries short DNA sequences (indicated in red) that are recognized only by the transposase encoded by that element and are necessary for movement of the element. In addition, two of the three mobile elements shown carry genes that encode enzymes that inactivate the antibiotics ampicillin (AmpR)—a penicillin derivative—and tetracycline (TetR). The transposable element Tn10, shown in the bottom diagram, is thought to have evolved from the chance landing of two much shorter mobile elements on either side of a tetracycline-resistance gene.

1	Figure 5–61 Cut-and-paste transposition. DNA-only transposons can be recognized in chromosomes by the “inverted repeat DNA sequences” (red) present at their ends. These sequences, which can be as short as 20 nucleotides, are all that is necessary for the DNA between them to be transposed by the particular transposase enzyme associated with the element. The cut-and-paste movement of a DNA-only transposable element from one chromosomal site to another begins when the transposase brings the two inverted DNA sequences together, forming a DNA loop. Insertion into the target chromosome, also catalyzed by the transposase, occurs at a random site through the creation of staggered breaks in the target chromosome (purple arrowheads). Following the transposition reaction, the single-strand gaps created by the staggered breaks are repaired by DNA polymerase and ligase (black). As a result, the insertion site is marked by a short direct repeat of the target DNA sequence, as shown. Although the

1	by the staggered breaks are repaired by DNA polymerase and ligase (black). As a result, the insertion site is marked by a short direct repeat of the target DNA sequence, as shown. Although the break in the donor chromosome (green) is repaired, this process often alters the DNA sequence, causing a mutation at the original site of the excised transposable element (not shown).

1	vertebrates, catalyzing the DNA rearrangements that produce antibody and T cell receptor diversity. Known as V(D)J recombination, this process will be discussed in Chapter 24. Found only in vertebrates, V(D)J recombination is a relatively recent evolutionary novelty, but it is believed to be derived from the much more ancient cut-and-paste transposons. Some Viruses Use a Transposition Mechanism to Move Themselves Into Host-Cell Chromosomes

1	Some Viruses Use a Transposition Mechanism to Move Themselves Into Host-Cell Chromosomes Certain viruses are considered mobile genetic elements because they use transposition mechanisms to integrate their genomes into that of their host cell. However, unlike transposons, these viruses encode proteins that package their genetic information into virus particles that can infect other cells. Many of the viruses that insert themselves into a host chromosome do so by employing one of the first two mechanisms listed in Table 5–4; namely, by behaving like DNA-only transposons or like retroviral-like retrotransposons. Indeed, much of our knowledge of these mechanisms has come from studies of particular viruses that employ them.

1	Transposition has a key role in the life cycle of many viruses. Most notable are the retroviruses, which include the human AIDS virus, HIV. Outside the cell, a retrovirus exists as a single-strand RNA genome packed into a protein shell or capsid along with a virus-encoded reverse transcriptase enzyme. During the infection process, the viral RNA enters a cell and is converted to a double-strand DNA molecule by the action of this crucial enzyme, which is able to polymerize DNA on either an RNA or a DNA template (Figure 5–62). The term retrovirus refers to the virus’s ability to reverse the usual flow of genetic information, which normally is from DNA to RNA (see Figure 1–4). Once the reverse transcriptase has produced a double-strand DNA molecule, specific sequences near its two ends are recognized by a virus-encoded

1	Once the reverse transcriptase has produced a double-strand DNA molecule, specific sequences near its two ends are recognized by a virus-encoded Figure 5–62 The life cycle of a retrovirus. The retrovirus genome consists of an RNA molecule (blue) that is typically between 7000 and 12,000 nucleotides in length. It is packaged inside a protein capsid, which is surrounded by a lipid-based envelope that contains virus-encoded envelope proteins (green). Inside an infected cell, the enzyme reverse transcriptase (red circle) first makes a DNA copy of the viral RNA molecule and then a second DNA strand, generating a double-strand DNA copy of the RNA genome. The integration of this DNA double helix into the host chromosome is then catalyzed by a virus-encoded integrase enzyme. This integration is required for the synthesis of new viral RNA molecules by the host-cell RNA polymerase, the enzyme that transcribes DNA into RNA (discussed in Chapter 6).

1	transposase called integrase. Integrase then inserts the viral DNA into the chromosome by a mechanism similar to that used by the cut-and-paste DNA-only transposons (see Figure 5–61). Retroviral-like Retrotransposons Resemble Retroviruses, but Lack a Protein Coat

1	A large family of transposons called retroviral-like retrotransposons (see Table 5–4) move themselves in and out of chromosomes by a mechanism that is similar to that used by retroviruses. These elements are present in organisms as diverse as yeasts, flies, and mammals; unlike viruses, they have no intrinsic ability to leave their resident cell but are passed along to all descendants of that cell through the normal processes of DNA replication and cell division. The first step in their transposition is the transcription of the entire transposon, producing an RNA copy of the element that is typically several thousand nucleotides long. This transcript, which is translated as a messenger RNA by the host cell, encodes a reverse transcriptase enzyme. This enzyme makes a double-strand DNA copy of the RNA molecule via an RNA–DNA hybrid intermediate, precisely mirroring the early stages of infection by a retrovirus (see Figure 5–62). Like a retrovirus, the linear, double-strand DNA molecule

1	of the RNA molecule via an RNA–DNA hybrid intermediate, precisely mirroring the early stages of infection by a retrovirus (see Figure 5–62). Like a retrovirus, the linear, double-strand DNA molecule then integrates into a site on the chromosome using an integrase enzyme that is also encoded by the element. The structure and mechanisms of these integrases closely resemble those of the transposases of DNA-only transposons.

1	A Large Fraction of the Human Genome Is Composed of Nonretroviral Retrotransposons A significant fraction of many vertebrate chromosomes is made up of repeated DNA sequences. In human chromosomes, these repeats are mostly mutated and truncated versions of nonretroviral retrotransposons, the third major type of transposon (see Table 5–4). Although most of these transposons in the human genome are immobile, a few retain the ability to move. Relatively recent movements of the L1 element (sometimes referred to as a LINE or long interspersed nuclear element) have been identified, some of which result in human disease; for example, a particular type of hemophilia results from an L1 insertion into the gene encoding the blood-clotting protein Factor VIII (see Figure 6–24). binds to L1 RNA

1	binds to L1 RNA Nonretroviral retrotransposons are found in many organisms and move via a 5˜ distinct mechanism that requires a complex of an endonuclease and a reverse transcriptase. As illustrated in Figure 5–63, the RNA and reverse transcriptase CLEAVAGE OF FIRST STRAND have a much more direct role in the recombination event than they do in the ret- roviral-like retrotransposons described above.

1	Inspection of the human genome sequence reveals that the bulk of nonretro ber of the SINE (short interspersed nuclear element) family—do not carry their viral retrotransposons—for example, the many copies of the Alu element, a mem-target DNA own endonuclease or reverse transcriptase genes. Nonetheless, they have suc cessfully amplified themselves to become major constituents of our genome, pre- sumably by pirating enzymes encoded by other transposons. Together the LINEs and SINEs make up over 30% of the human genome (see Figure 4–62); there are 500,000 copies of the former and over a million of the latter. Figure 5–63 Transposition by a nonretroviral retrotransposon.

1	Figure 5–63 Transposition by a nonretroviral retrotransposon. Transposition of the L1 element (red) begins when an endonuclease attached to the L1 reverse transcriptase (green) and the L1 RNA (blue) nick the target DNA at the point at which insertion will occur. This cleavage releases a 3ʹ-OH DNA end in the target DNA, which is then used as a primer for the reverse transcription step shown. This generates a single-strand DNA copy of the 3˜ element that is directly linked to the target DNA. In subsequent reactions, further processing of the single-strand DNA copy results in the generation of a new double-strand DNA copy of the L1 element that is inserted at the site of the initial nick.

1	We have described several types of transposable elements: (1) DNA-only transposons, the movement of which is based on DNA breaking and joining reactions; (2) retroviral-like retrotransposons, which also move via DNA breakage and joining, but where RNA has a key role as a template to generate the DNA recombination substrate; and (3) nonretroviral retrotransposons, in which an RNA copy of the element is central to the incorporation of the element into the target DNA, acting as a direct template for a DNA target-primed reverse transcription event.

1	Intriguingly, different types of transposons predominate in different organisms. For example, the vast majority of bacterial transposons are DNA-only types, with a few related to the nonretroviral retrotransposons also present. In yeasts, the main mobile elements are retroviral-like retrotransposons. In Drosophila, DNA-based, retroviral, and nonretroviral transposons are all found. Finally, the human genome contains all three types of transposon, but as discussed below, their evolutionary histories are strikingly different. Genome Sequences Reveal the Approximate Times at Which Transposable Elements Have Moved

1	The nucleotide sequence of the human genome provides a rich fossil record of the activity of transposons over evolutionary time spans. By carefully comparing the nucleotide sequences of the approximately 3 million transposable element remnants in the human genome, it has been possible to broadly reconstruct the movements of transposons in our ancestors’ genomes over the past several hundred million years. For example, the DNA-only transposons appear to have been very active well before the divergence of humans and Old World monkeys (25–35 million years ago), but because they gradually accumulated inactivating mutations, they have been dormant in the human lineage since that time. Likewise, although our genome is littered with relics of retroviral-like retrotransposons, none appear to be active today. Only a single family of retroviral-like retrotransposons is believed to have transposed in the human genome since the divergence of human and chimpanzee approximately 6 million years ago.

1	today. Only a single family of retroviral-like retrotransposons is believed to have transposed in the human genome since the divergence of human and chimpanzee approximately 6 million years ago. The nonretroviral retrotransposons are also ancient, but in contrast to other types, some are still moving in our genome, as mentioned previously. For example, it is estimated that de novo movement of an Alu element is seen once in every 100–200 human births. The movement of nonretroviral retrotransposons is responsible for a small but significant fraction of new human mutations—perhaps two mutations out of every thousand.

1	The situation in mice is significantly different. Although the mouse and human genomes contain roughly the same density of the three types of transposons, both types of retrotransposons are still actively transposing in the mouse genome, being responsible for approximately 10% of new mutations. Although we are only beginning to understand how the movements of transposons have shaped the genomes of present-day mammals, it has been proposed that bursts in transposition activity could have been responsible for critical speciation events during the radiation of the mammalian lineages from a common ancestor, a process that began approximately 170 million years ago. At present, we can only wonder how many of our uniquely human qualities arose from the past activity of the many mobile genetic elements whose remnants are found today scattered throughout our chromosomes.

1	A different kind of recombination mechanism, known as conservative site-specific recombination, rearranges other types of mobile DNA elements. In this pathway, breakage and joining occur at two special sites, one on each participating DNA Figure 5–64 Two types of DNA rearrangement produced by conservative site-specific recombination. The only difference between the reactions in (A) and (B) is the relative orientation of the two short DNA sites (indicated by arrows) at which a site-specific recombination event occurs. Through an integration reaction, a circular DNA molecule can become incorporated into a second DNA molecule; by the reverse reaction (excision), it can exit to re-form the original DNA circle. Many bacterial viruses move in and out of their host chromosomes in this way.

1	Conservative site-specific recombination can also invert a specific segment of DNA in a chromosome. A well-studied example of DNA inversion through site-specific recombination occurs in the bacterium Salmonella typhimurium, an organism that is a major cause of food poisoning in humans; as described in the following section, the inversion of a DNA segment changes the type of flagellum that is produced by the bacterium. molecule. Depending on the positions and relative orientations of the two recombination sites, DNA integration, DNA excision, or DNA inversion can occur (Figure 5–64). Conservative site-specific recombination is carried out by specialized enzymes that break and rejoin two DNA double helices at specific sequences on each DNA molecule. The same enzyme system that joins two DNA molecules can often take them apart again, precisely restoring the sequence of the two original DNA molecules (see Figure 5–64A).

1	Conservative site-specific recombination is often used by DNA viruses to move their genomes in and out of the genomes of their host cells. When integrated into its host genome, the viral DNA is replicated along with the host DNA and is faithfully passed on to all descendent cells. If the host cell suffers damage (for example, by UV irradiation), the virus can reverse the site-specific recombination reaction, excise its genome, and package it into a virus particle. In this way, many viruses can replicate themselves passively as a component of the host genome, but can also “leave the sinking ship” by excising their genomes and packaging them in a protective coat until a new, healthy host cell is encountered.

1	Several features distinguish conservative site-specific recombination from transposition. First, conservative site-specific recombination requires specialized DNA sequences on both the donor and recipient DNA (hence the term site-specific). These sequences contain recognition sites for the particular recombinase that will catalyze the rearrangement. In contrast, transposition requires only that the transposon have a specialized sequence; for most transposons, the recipient DNA can be of any sequence. Second, the reaction mechanisms are fundamentally different. The recombinases that catalyze conservative site-specific recombination resemble topoisomerases in the sense that they form transient high-energy covalent bonds with the DNA and use this energy to complete the DNA rearrangements (see Figure 5–21). Thus, all the phosphate bonds that are broken during a recombination event are restored upon its completion (hence the term conservative). Transposition, in contrast, does not proceed

1	Figure 5–21). Thus, all the phosphate bonds that are broken during a recombination event are restored upon its completion (hence the term conservative). Transposition, in contrast, does not proceed through a covalently joined protein–DNA intermediate, and this process leaves gaps in the DNA that must be repaired by DNA polymerases.

1	Conservative Site-Specific Recombination Can Be Used to Turn Genes On or Off

1	Many bacteria use conservative site-specific recombination to control the expression of particular genes. A well-studied example occurs in Salmonella bacteria and is known as phase variation. The switch in gene expression results from the occasional inversion of a specific 1000-nucleotide-pair piece of DNA, brought about by a conservative site-specific recombinase encoded in the Salmonella genome. This change alters the expression of the cell-surface protein flagellin, for which the bacterium has two different genes (Figure 5–65). The DNA inversion changes the orientation of a promoter (a DNA sequence that directs transcription of a gene) that is located within the inverted DNA segment. With the promoter in one orientation, the bacteria synthesize one type of flagellin; with the promoter in the other orientation, they synthesize the other type. The recombination reaction is reversible, allowing bacterial populations to switch back and forth between the two types of flagellin.

1	in the other orientation, they synthesize the other type. The recombination reaction is reversible, allowing bacterial populations to switch back and forth between the two types of flagellin. Inversions occur only rarely, and because such changes in the genome will be copied faithfully during all subsequent replication cycles, entire clones of bacteria will have one type of flagellin or the other.

1	Phase variation helps protect the bacterial population against the immune response of its vertebrate host. If the host makes antibodies against one type of flagellin, a few bacteria whose flagellin has been altered by gene inversion will still be able to survive and multiply.

1	Like many of the mechanisms used by cells and viruses, site-specific recombination has been put to work by scientists to study a wide variety of problems. To decipher the roles of specific genes and proteins in complex multicellular organisms, genetic engineering techniques are used to produce worms, flies, and mice carrying a gene encoding a site-specific recombination enzyme plus a carefully designed target DNA with the DNA sites that this enzyme recognizes. At an appropriate time, the gene encoding the enzyme can be activated to rearrange the target DNA sequence. Such a rearrangement is widely used to delete a specific gene in a particular tissue of a multicellular organism (Figure 5–66). It is particularly useful when the gene of interest plays a key role in the early development of many tissues, and a complete deletion of the gene from the germ line would cause death Figure 5–65 Switching gene expression by DNA inversion in bacteria.

1	Figure 5–65 Switching gene expression by DNA inversion in bacteria. Alternating transcription of two flagellin genes in a Salmonella bacterium is caused by a conservative site-specific recombination event that inverts a small DNA segment containing a promoter. In one orientation, the promoter activates transcription of the H2 flagellin gene as well as that of a repressor protein that blocks the expression of the H1 flagellin gene. Promoters and repressors are described in detail in Chapter 7; here we note simply that a promoter is needed to express a gene into protein and that a repressor blocks this from happening.

1	When the promoter is inverted, it no longer turns on H2 or the repressor, and the H1 gene, which is thereby released from repression, is expressed instead. The inversion reaction requires specific DNA sequences (red) and a recombinase enzyme that is encoded in the invertible DNA segment. This site-specific recombination mechanism is activated only rarely (about once in every 105 cell divisions). Therefore, the production of one or the other flagellin tends to be faithfully inherited in each clone of cells. IN SPECIFIC TISSUE (e.g., LIVER) Cre recombinase gene gene of interest gene of interest deleted from chromosome and lost as liver cells divide IN OTHER TISSUES, THE GENE OF INTEREST IS EXPRESSED NORMALLY Cre recombinase gene gene of interest mRNA(e.g., promoter active only in liver) protein of interest

1	Figure 5–66 How a conservative site-specific recombination enzyme from bacteria is used to delete specific genes from particular mouse tissues. This approach requires the insertion of two specially engineered DNA molecules into the animal’s germ line. The first contains the gene for a recombinase (in this case, the Cre recombinase from the bacteriophage P1) under the control of a tissue-specific promoter, which ensures that the recombinase is expressed only in that tissue. The second DNA molecule contains the gene of interest flanked by recognition sites (in this case, LoxP sites) for the recombinase. The mouse is engineered so that this is the only copy of this gene. Therefore, if the recombinase is expressed only in the liver, the gene of interest will be deleted there, and only there. The reaction that excises the gene is the same as that shown in Figure 5–64A. As described in Chapter 7, many tissue-specific promoters are known; moreover, many of these promoters are active only at

1	The reaction that excises the gene is the same as that shown in Figure 5–64A. As described in Chapter 7, many tissue-specific promoters are known; moreover, many of these promoters are active only at specific times in development. Thus, it is possible to study the effect of deleting specific genes at different times during the development of each tissue.

1	• How does DNA replication contend with all the other processes that occur simultaneously on chromosomes, very early in development. The same strategy can also be used to inappropriately including DNA repair and gene express any specific gene in a tissue of interest; here, the triggered deletion joins transcription? a strong transcriptional promoter to the gene of interest. With this tool one can in principle determine the influence of any protein in any desired tissue of an intact • What is the basis for the low animal. frequency of errors in DNA replication observed in all cells? Is this the best that cells can do given the speed of replication and the limits of molecular The genomes of nearly all organisms contain mobile genetic elements that can move diffusion? Was this mutation rate from one position in the genome to another by either transpositional or conserva-selected in evolution to provide genetic variation?

1	tive site-specific recombination processes. In most cases, this movement is random and happens at a very low frequency. Mobile genetic elements include transposons, which move within a single cell (and its descendants), plus those viruses whose genomes can integrate into the genomes of their host cells. ways of repairing it. Are there still There are three classes of transposons: the DNA-only transposons, the retrovi other, undiscovered ways that cells ral-like retrotransposons, and the nonretroviral retrotransposons. All but the last have for repairing DNA? have close relatives among the viruses. Although viruses and transposable elements can be viewed as parasites, many of the new arrangements of DNA sequences that • Do the many “dead” transposons their site-specific recombination events produce have played an important part in in the human genome provide any creating the genetic variation crucial for the evolution of cells and organisms. benefits to humans?

1	Which statements are true? Explain why or why not. 5–1 The different cells in your body rarely have genomes with the identical nucleotide sequence. 5–2 In E. coli, where the replication fork travels at 500 nucleotide pairs per second, the DNA ahead of the fork— in the absence of topoisomerase—would have to rotate at nearly 3000 revolutions per minute. 5–3 In a replication bubble, the same parental DNA strand serves as the template strand for leading-strand synthesis in one replication fork and as the template for lagging-strand synthesis in the other fork. 5–4 When bidirectional replication forks from adjacent origins meet, a leading strand always runs into a lagging strand. 5–5 DNA repair mechanisms all depend on the existence of two copies of the genetic information, one in each of the two homologous chromosomes. Discuss the following problems.

1	5–5 DNA repair mechanisms all depend on the existence of two copies of the genetic information, one in each of the two homologous chromosomes. Discuss the following problems. 5–6 To determine the reproducibility of mutation frequency measurements, you do the following experiment. You inoculate each of 10 cultures with a single E. coli bacterium, allow the cultures to grow until each contains 106 cells, and then measure the number of cells in each culture that carry a mutation in your gene of interest. You were so surprised by the initial results that you repeated the experiment to confirm them. Both sets of results display the same extreme variability, as shown in Table Q5–1. Assuming that the rate of mutation is constant, why do you suppose there is so much variation in the frequencies of mutant cells in different cultures?

1	5–7 DNA repair enzymes preferentially repair mismatched bases on the newly synthesized DNA strand, using the old DNA strand as a template. If mismatches were instead repaired without regard for which strand served as template, would mismatch repair reduce replication errors? Would such a mismatch repair system result in fewer mutations, more mutations, or the same number of mutations as there would have been without any repair at all? Explain your answers. 5–8 Discuss the following statement: “Primase is a sloppy enzyme that makes many mistakes. Eventually, the RNA primers it makes are replaced with DNA made by a polymerase with higher fidelity. This is wasteful. It would be more energy-efficient if a DNA polymerase made an accurate copy in the first place.” 5–9 If DNA polymerase requires a perfectly paired primer in order to add the next nucleotide, how is it that any mismatched nucleotides “escape” this requirement and become substrates for mismatch repair enzymes?

1	5–10 The laboratory you joined is studying the life cycle of an animal virus that uses circular, double-strand DNA as its genome. Your project is to define the location of the origin(s) of replication and to determine whether replication proceeds in one or both directions away from an origin (unidirectional or bidirectional replication). To accomplish your goal, you broke open cells infected with the virus, isolated replicating viral genomes, cleaved them with a restriction nuclease that cuts the genome at only one site to produce a linear molecule from the circle, and examined the resulting molecules in the electron microscope. Some of the molecules you observed are illustrated schematically in Figure Q5–1. (Note that it is impossible to distinguish the orientation of one DNA molecule relative to another in the electron microscope.)

1	You must present your conclusions to the rest of the lab tomorrow. How will you answer the two questions your advisor posed for you? Is there a single, unique origin of replication or several origins? Is replication unidirectional or bidirectional? Figure Q5–1 Parental and replicating forms of an animal virus (Problem 5–10). 5–11 You are investigating DNA synthesis in tissue-culture cells, using 3H-thymidine to radioactively label the replication forks. By breaking open the cells in a way that allows some of the DNA strands to be stretched out, very long DNA strands can be isolated intact and examined. You overlay the DNA with a photographic emulsion, and expose it for 3 to 6 months, a procedure known as auto-radiography. Because the emulsion is sensitive to radioactive emissions, the 3H-labeled DNA shows up as tracks of silver grains. Because the stretching collapses replication

1	Figure Q5–2 Autoradiographic investigation of DNA replication in cultured cells (Problem 5–11). (A) Addition of 3H-labeled thymidine immediately after release from the synchronizing block. (B) Addition of 3H-labeled thymidine 30 minutes after release from the synchronizing block. bubbles, the daughter duplexes lie side by side and cannot be distinguished from each other.

1	bubbles, the daughter duplexes lie side by side and cannot be distinguished from each other. You pretreat the cells to synchronize them at the beginning of S phase. In the first experiment, you release the synchronizing block and add 3H-thymidine immediately. After 30 minutes, you wash the cells and change the medium so that the total concentration of thymidine is the same as it was, but only one-third of it is radioactive. After an additional 15 minutes, you prepare DNA for autoradiography. The results of this experiment are shown in Figure Q5–2A. In the second experiment, you release the synchronizing block and then wait 30 minutes before adding 3H-thymidine. After 30 minutes in the presence of 3H-thymidine, you once again change the medium to reduce the concentration of radioactive thymidine and incubate the cells for an additional 15 minutes. The results of the second experiment are shown in Figure Q5–2B.

1	A. Explain why, in both experiments, some regions of the tracks are dense with silver grains (dark), whereas others are less dense (light). b. In the first experiment, each track has a central dark section with light sections at each end. In the second experiment, the dark section of each track has a light section at only one end. Explain the reason for this difference. C. Estimate the rate of fork movement (μm/min) in these experiments. Do the estimates from the two experiments agree? Can you use this information to gauge how long it would take to replicate the entire genome?

1	5–12 If you compare the frequency of the sixteen possible dinucleotide sequences in the E. coli and human genomes, there are no striking differences except for one dinucleotide, 5ʹ-CG-3ʹ. The frequency of CG dinucleotides in the human genome is significantly lower than in E. coli and significantly lower than expected by chance. Why do you suppose that CG dinucleotides are underrepresented in the human genome? 5–13 With age, somatic cells are thought to accumulate genomic “scars” as a result of the inaccurate repair of double-strand breaks by nonhomologous end joining (NHEJ).

1	Estimates based on the frequency of breaks in primary human fibroblasts suggest that by age 70, each human somatic cell may carry some 2000 NHEJ-induced mutations due to inaccurate repair. If these mutations were distributed randomly around the genome, how many pro-tein-coding genes would you expect to be affected? Would you expect cell function to be compromised? Why or why not? (Assume that 2% of the genome—1.5% protein-coding and 0.5% regulatory—is crucial information.) 5–14 Draw the structure of the double Holliday junction that would result from strand invasion by both ends of the broken duplex into the intact homologous duplex shown in Figure Q5–3. Label the left end of each strand in the Holliday junction 5ʹor 3ʹ so that the relationship to the parental and recombinant duplexes is clear. Indicate how DNA synthesis would be used to fill in any single-strand gaps in your double Holliday junction. (Problem 5–14).

1	(Problem 5–14). 5–15 In addition to correcting DNA mismatches, the mismatch repair system functions to prevent homologous recombination from taking place between similar but not identical sequences. Why would recombination between similar, but nonidentical sequences pose a problem for human cells? 5–16 Cre recombinase is a site-specific enzyme that catalyzes recombination between two LoxP DNA sites. Cre recombinase pairs two LoxP sites in the same orientation, breaks both duplexes at the same point in each LoxP site, and joins the ends with new partners so that each LoxP site is regenerated, as shown schematically in Figure Q5–4A. Based on this mechanism, predict the arrangement of sequences that will be generated by Cre-mediated site-specific recombination for each of the two DNAs shown in Figure Q5–4B.

1	Figure Q5–4 Cre recombinase-mediated site-specific recombination (Problem 5–16). (A) Schematic representation of Cre/LoxP site-specific recombination. The LoxP sequences in the DNA are represented by triangles that are colored so that the site-specific recombination event can be followed more readily. In reality their DNA sequences are identical. (B) DNA substrates containing two arrangements of LoxP sites. Brown TA (2007) Genomes 3. New York: Garland Science. Friedberg EC, Walker GC, Siede W et al. (2005) DNA Repair and Mutagenesis. Washington, DC: ASM Press. Haber JE (2013) Genome Stability: DNA Repair and Recombination. New York: Garland Science. Hartwell L, Hood L, Goldberg ML et al. (2010) Genetics: from Genes to Genomes. Boston: McGraw Hill. Stent GS (1971) Molecular Genetics: An Introductory Narrative. San Francisco: WH Freeman. Watson J, Baker T, Bell S et al. (2013) Molecular Biology of the Gene, 7th ed. Menlo Park, CA: Benjamin Cummings.

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1	Szostak JW, Orr-Weaver TK, Rothstein RJ et al. (1983) The double-strand break repair model for recombination. Cell 33, 25–35. West SC (2003) Molecular views of recombination proteins and their control. Nat. Rev. Mol. Cell Biol. 4(6), 435–445. Yeeles JY, Poli J, Marians KJ et al. (2013) Rescuing stalled or damaged replication forks. Cold Spring Harb. Perspect. Biol. 5, a012815. Zickler D & Kleckner N (1999) Meiotic chromosomes: integrating structure and function. Annu. Rev. Genet. 33, 603–754. Comfort NC (2001) From controlling elements to transposons: Barbara McClintock and the Nobel Prize. Trends Biochem. Sci. 26, 454–457. Grindley ND, Whiteson KL & Rice PA (2006) Mechanisms of site-specific recombination. Annu. Rev. Biochem. 75, 567–605. Huang, CR, Burns KH & Boeke JD (2012) Active transposition in genomes. Annu. Rev. Genet. 46, 651–675. Varmus H (1988) Retroviruses. Science 240, 1427–1435. How Cells Read the Genome: From DNA to Protein

1	Varmus H (1988) Retroviruses. Science 240, 1427–1435. How Cells Read the Genome: From DNA to Protein Since the structure of DNA was discovered in the early 1950s, progress in cell and molecular biology has been astounding. We now know the complete genome sequences for thousands of different organisms, revealing fascinating details of their biochemistry as well as important clues as to how these organisms evolved. Complete genome sequences have also been obtained for thousands of individual humans, as well as for a few of our now-extinct relatives, such as the Neanderthals. Knowing the maximum amount of information that is required to produce a complex organism like ourselves puts constraints on the biochemical and structural features of cells and makes it clear that biology is not infinitely complex.

1	As discussed in Chapter 1, the DNA in genomes does not direct protein synthesis itself, but instead uses RNA as an intermediary. When the cell needs a particular protein, the nucleotide sequence of the appropriate portion of the immensely long DNA molecule in a chromosome is first copied into RNA (a process called transcription). It is these RNA copies of segments of the DNA that are used directly as templates to direct the synthesis of the protein (a process called translation). The flow of genetic information in cells is therefore from DNA to RNA to protein (Figure 6–1). All cells, from bacteria to humans, express their genetic information in this way—a principle so fundamental that it is termed the central dogma of molecular biology. Despite the universality of the central dogma of molecular biology, there are important variations between organisms in the way in which information flows from DNA to protein. Principal among these is that RNA transcripts in eukaryotic cells are

1	of molecular biology, there are important variations between organisms in the way in which information flows from DNA to protein. Principal among these is that RNA transcripts in eukaryotic cells are subject to a series of processing steps in the nucleus, including RNA splicing, before they are permitted to exit from the nucleus and be translated into protein. As we discuss in this chapter, these processing steps can critically change the “meaning” of an RNA molecule and are therefore crucial for understanding how eukaryotic cells read their genome.

1	Although we focus on the production of the proteins encoded by the genome in this chapter, we see that for many genes, RNA is the final product. Like proteins, some of these RNAs fold into precise three-dimensional structures that have structural and catalytic roles in the cell. Other RNAs, as we discuss in the next chapter, act primarily as regulators of gene expression. But the roles of many non-coding RNAs are not yet known.

1	One might have predicted that the information present in genomes would be arranged in an orderly fashion, resembling a dictionary or a telephone directory. But it turns out that the genomes of most multicellular organisms are surprisingly disorderly, reflecting their chaotic evolutionary histories. The genes in these organisms largely consist of a long string of alternating short exons and long introns, as discussed in Chapter 4 (see Figure 4–15D). Moreover, small bits of DNA sequence that code for protein are interspersed with large blocks of seemingly meaningless DNA. Some sections of the genome contain many genes and others lack genes altogether. Proteins that work closely with one another in the cell often have their genes located on different chromosomes, and adjacent genes typically encode proteins that have little to do with each other in the cell. Decoding genomes is therefore no simple matter. Even with the aid of powerful computers, it is difficult for researchers to locate

1	encode proteins that have little to do with each other in the cell. Decoding genomes is therefore no simple matter. Even with the aid of powerful computers, it is difficult for researchers to locate definitively the beginning and end of genes, much less to decipher when and where each gene is expressed in the life of the

1	Figure 6–1 The pathway from DNA to protein. The flow of genetic information from DNA to RNA (transcription) and from RNA to protein (translation) occurs in all living cells. human X chromosome: 155 million nucleotide base pairs (~5% of genome) total length of this section = 1.25 million nucleotide pairs

1	Figure 6–2 Schematic depiction of a small portion of the human X chromosome. As summarized in the key, the known protein-coding genes (starting with Abcd1 and ending with F8) are shown in dark gray, with coding regions (exons) indicated by bars that extend above and below the central line. Noncoding RNAs with known functions are indicated by purple diamonds. Yellow triangles indicate positions within protein-coding regions where the Neanderthal genome sequences codes for a different amino acid than the human genome. The stretch of yellow triangles in the Txtl1 gene appear to have been positively selected for since the divergence of Homo sapiens from Neanderthals some 200,000 years ago. Note that most of the proteins are identical between us and our extinct relative. The blue histogram indicates the extent to which portions of the human genome are conserved with other vertebrate species. It is likely that additional genes, currently unrecognized, also lie within this portion of the

1	indicates the extent to which portions of the human genome are conserved with other vertebrate species. It is likely that additional genes, currently unrecognized, also lie within this portion of the human genome.

1	Genes whose mutation causes an inherited human condition are indicated by red brackets. The Abcd1 gene codes for a protein that imports fatty acids into the peroxisome; mutations in the gene cause demylination of nerves which can result in cognition and movement disorders. Incontinentia pigmenti is a disease of the skin, hair, nails, teeth, and eyes. Hemophilia A is a bleeding disorder caused by mutations in the Factor VIII gene, which codes for a blood-clotting protein. Because males have only a single copy of the X chromosome, most of the conditions shown here affect only males; females that inherit one of these defective genes are often asymptomatic because a functional protein is made from their other X chromosome. (Courtesy of Alex Williams, obtained from the University of California, Genome Browser, http://genome.ucsc.edu) organism. Yet the cells in our body do this automatically, thousands of times a second.

1	The problems that cells face in decoding genomes can be appreciated by considering a tiny portion of the human genome (Figure 6–2). The region illustrated represents less than 1/2000th of our genome and includes at least 48 genes that encode proteins and 6 genes for noncoding RNAs. When we consider the entire human genome, we can only marvel at the capacity of our cells to rapidly and accurately handle such large amounts of information.

1	In this chapter, we explain how cells decode and use the information in their genomes. Much has been learned about how the genetic instructions written in an alphabet of just four “letters”—the four different nucleotides in DNA—direct the formation of a bacterium, a fruit fly, or a human. Nevertheless, we still have a great deal to discover about how the information stored in an organism’s genome produces even the simplest unicellular bacterium with 500 genes, let alone how it directs the development of a human with approximately 30,000 genes. An enormous amount of ignorance remains; many fascinating challenges therefore await the next generation of cell biologists.

1	Transcription and translation are the means by which cells read out, or express, the genetic instructions in their genes. Because many identical RNA copies can be made from the same gene, and each RNA molecule can direct the synthesis of many identical protein molecules, cells can synthesize a large amount of protein from a gene when necessary. But genes can be transcribed and translated with different efficiencies, allowing the cell to make vast quantities of some proteins and tiny amounts of others (Figure 6–3). Moreover, as we see in the next chapter, Figure 6–3 Genes can be expressed with different efficiencies. In this example, gene A is transcribed much more efficiently than gene B and each RNA molecule that it produces is also translated more frequently. This causes the amount of protein A in the cell to be much greater than that of protein B.

1	a cell can change (or regulate) the expression of each of its genes according to its needs—most commonly by controlling the production of its RNA. The first step a cell takes in reading out a needed part of its genetic instructions is to copy a particular portion of its DNA nucleotide sequence—a gene—into an RNA nucleotide sequence (Figure 6–4). The information in RNA, although copied into another chemical form, is still written in essentially the same language as it is in DNA—the language of a nucleotide sequence. Hence the name given to producing RNA molecules on DNA is transcription.

1	Like DNA, RNA is a linear polymer made of four different types of nucleotide subunits linked together by phosphodiester bonds (see Figure 6–4). It differs from DNA chemically in two respects: (1) the nucleotides in RNA are ribonucleotides— that is, they contain the sugar ribose (hence the name ribonucleic acid) rather than deoxyribose; (2) although, like DNA, RNA contains the bases adenine (A), guanine (G), and cytosine (C), it contains the base uracil (U) instead of the thymine (T) in DNA (Figure 6–5). Since U, like T, can base-pair by hydrogen-bonding with A (Figure 6–6), the complementary base-pairing properties described for DNA in Chapters 4 and 5 apply also to RNA (in RNA, G pairs with C, and A pairs with U). We also find other types of base pairs in RNA: for example, G occasionally pairs with U.

1	Although these chemical differences are slight, DNA and RNA differ quite dramatically in overall structure. Whereas DNA always occurs in cells as a double-stranded helix, RNA is single-stranded. An RNA chain can therefore fold up into a particular shape, just as a polypeptide chain folds up to form the final shape of a protein (Figure 6–7). As we see later in this chapter, the ability to fold into complex three-dimensional shapes allows some RNA molecules to have precise structural and catalytic functions. Transcription Produces RNA Complementary to One Strand of DNA

1	Transcription Produces RNA Complementary to One Strand of DNA The RNA in a cell is made by DNA transcription, a process that has certain similarities to the process of DNA replication discussed in Chapter 5. Transcription begins with the opening and unwinding of a small portion of the DNA double helix to expose the bases on each DNA strand. One of the two strands of the DNA double helix then acts as a template for the synthesis of an RNA molecule. As in DNA replication, the nucleotide sequence of the RNA chain is determined by the complementary base-pairing between incoming nucleotides and the DNA Figure 6–4 A short length of RNA. The phosphodiester chemical linkage between nucleotides in RNA is the same as that in DNA.

1	Figure 6–4 A short length of RNA. The phosphodiester chemical linkage between nucleotides in RNA is the same as that in DNA. Figure 6–5 The chemical structure of RNA. (A) RNA contains the sugar ribose, which differs from deoxyribose, the sugar used in DNA, by the presence of an additional –OH group. (B) RNA contains the base uracil, which differs from thymine, the equivalent base in DNA, by the absence of a –CH3 group. Figure 6–6 Uracil forms base pairs with adenine. The absence of a methyl group in U has no effect on base-pairing; thus, U-A base pairs closely resemble T-A base pairs (see Figure 4–4).

1	Figure 6–6 Uracil forms base pairs with adenine. The absence of a methyl group in U has no effect on base-pairing; thus, U-A base pairs closely resemble T-A base pairs (see Figure 4–4). template. When a good match is made (A with T, U with A, G with C, and C with G), the incoming ribonucleotide is covalently linked to the growing RNA chain in an enzymatically catalyzed reaction. The RNA chain produced by transcription— the transcript—is therefore elongated one nucleotide at a time, and it has a nucleotide sequence that is exactly complementary to the strand of DNA used as the template (Figure 6–8).

1	Transcription, however, differs from DNA replication in several crucial ways. Unlike a newly formed DNA strand, the RNA strand does not remain hydrogen-bonded to the DNA template strand. Instead, just behind the region where the ribonucleotides are being added, the RNA chain is displaced and the DNA helix re-forms. Thus, the RNA molecules produced by transcription are released from the DNA template as single strands. In addition, because they are copied from only a limited region of the DNA, RNA molecules are much shorter than DNA molecules. A DNA molecule in a human chromosome can be up to 250 million nucleotide-pairs long; in contrast, most RNAs are no more than a few thousand nucleotides long, and many are considerably shorter.

1	The enzymes that perform transcription are called RNA polymerases. Like the DNA polymerase that catalyzes DNA replication (discussed in Chapter 5), RNA polymerases catalyze the formation of the phosphodiester bonds that link the nucleotides together to form a linear chain. The RNA polymerase moves stepwise along the DNA, unwinding the DNA helix just ahead of the active site for polymerization to expose a new region of the template strand for complementary Figure 6–7 RNA can fold into specific structures. RNA is largely single-stranded, but it often contains short stretches of nucleotides that can form conventional base pairs with complementary sequences found elsewhere on the same molecule. These interactions, along with additional “nonconventional” base-pair interactions, allow an RNA molecule to fold into a three-dimensional structure that is determined by its sequence of nucleotides (Movie 6.1).

1	Diagram of a folded RNA structure showing only conventional base-pair interactions. (B) Structure with both conventional (red) and nonconventional (green) base-pair interactions. Structure of an actual RNA, one that catalyzes its own splicing (see p. 324). Each conventional base-pair interaction is indicated by a “rung” in the double helix. Bases in other configurations are indicated by broken rungs. base-pairing. In this way, the growing RNA chain is extended by one nucleotide at a time in the 5ʹ-to-3ʹdirection (Figure 6–9). The substrates are ribonucleoside triphosphates (ATP, CTP, UTP, and GTP); as in DNA replication, the hydrolysis of high-energy bonds provides the energy needed to drive the reaction forward (see Figure 5–4 and Movie 6.2).

1	The almost immediate release of the RNA strand from the DNA as it is synthesized means that many RNA copies can be made from the same gene in a relatively short time, with the synthesis of additional RNA molecules being started before the previous RNA molecules are completed (Figure 6–10). When RNA polymerase molecules follow hard on each other’s heels in this way, each moving at about 50 nucleotides per second, over a thousand transcripts can be synthesized in an hour from a single gene.

1	Although RNA polymerase catalyzes essentially the same chemical reaction as DNA polymerase, there are some important differences between the activities of the two enzymes. First, and most obviously, RNA polymerase catalyzes the linkage of ribonucleotides, not deoxyribonucleotides. Second, unlike the DNA polymerases involved in DNA replication, RNA polymerases can start an RNA chain without a primer. This difference is thought possible because transcription need not be as accurate as DNA replication (see Table 5–1, p. 244). RNA polymerases make about one mistake for every 104 nucleotides copied into RNA (compared with an error rate for direct copying by DNA polymerase of about one in 107 nucleotides); and the consequences of an error in RNA transcription are much less significant as RNA does not permanently store genetic information in cells. Finally, unlike DNA polymerases, which make their products in segments that are later stitched together, RNA polymerases are absolutely

1	as RNA does not permanently store genetic information in cells. Finally, unlike DNA polymerases, which make their products in segments that are later stitched together, RNA polymerases are absolutely processive; that is, the same RNA polymerase that begins an RNA molecule must finish it without dissociating from the DNA template.

1	Although not nearly as accurate as the DNA polymerases that replicate DNA, RNA polymerases nonetheless have a modest proofreading mechanism. If an incorrect ribonucleotide is added to the growing RNA chain, the polymerase can back up, and the active site of the enzyme can perform an excision reaction that resembles the reverse of the polymerization reaction, except that a water molecule replaces the pyrophosphate and a nucleoside monophosphate is released. Given that DNA and RNA polymerases both carry out template-dependent nucleotide polymerization, it might be expected that the two types of enzymes would be structurally related. However, x-ray crystallographic studies reveal that, other than containing a critical Mg2+ ion at the catalytic site, the two enzymes are quite different. Template-dependent nucleotide-polymerizing enzymes seem to have arisen at least twice during the early evolution of cells. One lineage led to the short region of DNA/RNA helix 5˜5˜ direction of

1	Figure 6–8 DNA transcription produces a single-stranded RNA molecule that is complementary to one strand of the DNA double helix. Note that the sequence of bases in the RNA molecule produced is the same as the sequence of bases in the non-template DNA strand, except that a U replaces every T base in the DNA.

1	Figure 6–9 DNA is transcribed by the enzyme RNA polymerase. The RNA polymerase (pale blue) moves stepwise along the DNA, unwinding the DNA helix at its active site indicated by the Mg2+ (red), which is required for catalysis. As it progresses, the polymerase adds nucleotides one by one to the RNA chain at the polymerization site, using an exposed DNA strand as a template. The RNA transcript is thus a complementary copy of one of the two DNA strands. A short region of DNA/RNA helix (approximately nine nucleotide pairs in length) is formed only transiently, and a “window” of DNA/RNA helix therefore moves along the DNA with the polymerase as the DNA double helix reforms behind it. The incoming nucleotides are in the form of ribonucleoside triphosphates (ATP, UTP, CTP, and GTP), and the energy stored in their phosphate– phosphate bonds provides the driving force for the polymerization reaction (see Figure 5–4). The figure, based on an x-ray crystallographic structure, shows a cutaway view

1	stored in their phosphate– phosphate bonds provides the driving force for the polymerization reaction (see Figure 5–4). The figure, based on an x-ray crystallographic structure, shows a cutaway view of the polymerase: the part facing the viewer has been sliced away to reveal the interior (Movie 6.3). (Adapted from P. Cramer et al., Science 288:640–649, 2000; PDB code: 1HQM.) modern DNA polymerases and reverse transcriptases discussed in Chapter 5, as well as to a few RNA polymerases from viruses. The other lineage formed all of the modern RNA polymerases that we discuss in this chapter.

1	Cells Produce Different Categories of RNA Molecules

1	The majority of genes carried in a cell’s DNA specify the amino acid sequence of proteins; the RNA molecules that are copied from these genes (which ultimately direct the synthesis of proteins) are called messenger RNA (mRNA) molecules. The final product of other genes, however, is the RNA molecule itself. These RNAs are known as noncoding RNAs because they do not code for protein. In a well-studied, single-celled eukaryote, the yeast Saccharomyces cerevisiae, over 1200 genes (more than 15% of the total) produce RNA as their final product. Humans may produce on the order of ten thousand noncoding RNAs. These RNAs, like proteins, serve as enzymatic, structural, and regulatory components for a wide variety of processes in the cell. In Chapter 5, we encountered one of them as the template carried by the enzyme telomerase. Although many of the noncoding RNAs are still mysterious, we shall see in this chapter that small nuclear RNA (snRNA) molecules direct the splicing of pre-mRNA to form

1	by the enzyme telomerase. Although many of the noncoding RNAs are still mysterious, we shall see in this chapter that small nuclear RNA (snRNA) molecules direct the splicing of pre-mRNA to form mRNA, that ribosomal RNA (rRNA) molecules form the core of ribosomes, and that transfer RNA (tRNA) molecules form the adaptors that select amino acids and hold them in place on a ribosome for incorporation into protein. In Chapter 7, we shall see that microRNA (miRNA) molecules and small interfering RNA (siRNA) molecules serve as key regulators of eukaryotic gene expression, and that piwi-interacting RNAs (piRNAs) protect animal germ lines from transposons; we also discuss the long noncoding RNAs (lncRNAs), a diverse set of RNAs whose functions are just being discovered (Table 6–1).

1	Figure 6–10 Transcription of two genes as observed under the electron microscope. The micrograph shows many molecules of RNA polymerase simultaneously transcribing each of two adjacent genes. Molecules of RNA polymerase are visible as a series of dots along the DNA with the newly synthesized transcripts (fine threads) attached to them. The RNA molecules (ribosomal RNAs) shown in this example are not translated into protein but are instead used directly as components of ribosomes, the machines on which translation takes place. The particles at the 5ʹ end (the free end) of each rRNA transcript are believed to reflect the beginnings of ribosome assembly. From the relative lengths of the newly synthesized transcripts, it can be deduced that the RNA polymerase molecules are transcribing from left to right. (Courtesy of Ulrich Scheer.)

1	Each transcribed segment of DNA is called a transcription unit. In eukaryotes, a transcription unit typically carries the information of just one gene, and therefore codes for either a single RNA molecule or a single protein (or group of related proteins if the initial RNA transcript is spliced in more than one way to produce different mRNAs). In bacteria, a set of adjacent genes is often transcribed as a unit; the resulting mRNA molecule therefore carries the information for several distinct proteins. Overall, RNA makes up a few percent of a cell’s dry weight, whereas proteins comprise about 50%. Most of the RNA in cells is rRNA; mRNA comprises only 3–5% of the total RNA in a typical mammalian cell. The mRNA population is made up of tens of thousands of different species, and there are on average only 10–15 molecules of each species of mRNA present in each cell. Signals Encoded in DNA Tell RNA Polymerase Where to Start and Stop

1	Signals Encoded in DNA Tell RNA Polymerase Where to Start and Stop To transcribe a gene accurately, RNA polymerase must recognize where on the genome to start and where to finish. The way in which RNA polymerases perform these tasks differs somewhat between bacteria and eukaryotes. Because the processes in bacteria are simpler, we discuss them first.

1	The initiation of transcription is an especially important step in gene expression because it is the main point at which the cell regulates which proteins are to be produced and at what rate. The bacterial RNA polymerase core enzyme is a multisubunit complex that synthesizes RNA using the DNA template as a guide. An additional subunit called sigma (σ) factor associates with the core enzyme and assists it in reading the signals in the DNA that tell it where to begin transcribing (Figure 6–11). Together, σ factor and core enzyme are known as the RNA polymerase holoenzyme; this complex adheres only weakly to bacterial DNA when the two collide, and a holoenzyme typically slides rapidly along the long DNA molecule and then dissociates. However, when the polymerase holoenzyme slides into a special sequence of nucleotides indicating the starting point for RNA synthesis called a promoter, the polymerase binds tightly, because its σ factor makes specific contacts with the edges of bases

1	a special sequence of nucleotides indicating the starting point for RNA synthesis called a promoter, the polymerase binds tightly, because its σ factor makes specific contacts with the edges of bases exposed on the outside of the DNA double helix (step 1 in Figure 6–11A).

1	The tightly bound RNA polymerase holoenzyme at a promoter opens up the double helix to expose a short stretch of nucleotides on each strand (step 2 in Figure 6–11A). The region of unpaired DNA (about 10 nucleotides) is called the transcription bubble and it is stabilized by the binding of σ factor to the unpaired bases on one of the exposed strands. The other exposed DNA strand then acts as a template for complementary base-pairing with incoming ribonucleotides, two of which are joined together by the polymerase to begin an RNA chain (step 3 in Figure 6–11A). The first ten or so nucleotides of RNA are synthesized using a “scrunching” mechanism, in which RNA polymerase remains bound to the promoter and pulls the upstream DNA into its active site, thereby expanding the transcription bubble. This process creates considerable stress and the short RNAs are often released, thereby relieving the stress and forcing the polymerase, which remains in place, to begin synthesis over again.

1	bubble. This process creates considerable stress and the short RNAs are often released, thereby relieving the stress and forcing the polymerase, which remains in place, to begin synthesis over again. Eventually this process of abortive initiation is overcome and the stress generated by scrunching helps the core enzyme to break free of its interactions with the promoter DNA (step 4 in Figure 6–11A) and discard the σ factor (step 5 in Figure 6–11A). At this point, the polymerase begins to move down the DNA, synthesizing RNA, in a stepwise fashion: the polymerase moves forward one base pair for every nucleotide added. During this process, the transcription bubble continually expands at the front of the polymerase and contracts at its rear. Chain elongation continues (at a speed of approximately 50 nucleotides/sec for bacterial RNA polymerases) until the enzyme encounters a second signal, the terminator (step 6 in Figure 6–11A), where the polymerase halts and releases both the newly made

1	50 nucleotides/sec for bacterial RNA polymerases) until the enzyme encounters a second signal, the terminator (step 6 in Figure 6–11A), where the polymerase halts and releases both the newly made RNA molecule and the DNA template (step 7 in Figure 6–11A). The free polymerase core enzyme then reassociates with a free σ factor to form a holoenzyme that can begin the process of transcription again (step 8 in Figure 6–11A).

1	The process of transcription initiation is complicated and requires that the RNA polymerase holoenzyme and the DNA undergo a series of conformational changes. We can view these changes as opening up and positioning the DNA in the active site followed by a successive tightening of the enzyme around the DNA and RNA to ensure that it does not dissociate before it has finished transcribing a gene. If an RNA polymerase does dissociate prematurely, it must start over again at the promoter.

1	How do the termination signals in the DNA stop the elongating polymerase? For most bacterial genes, a termination signal consists of a string of A-T nucleotide pairs preceded by a twofold symmetric DNA sequence, which, when transcribed into RNA, folds into a “hairpin” structure through Watson–Crick base-pairing (see Figure 6–11A). As the polymerase transcribes across a terminator, the formation of the hairpin helps to disengage the RNA transcript from the active site (step 7 in Figure 6–11A). The process of termination provides an example of a common theme in this chapter: the folding of RNA into specific structures affects many steps in decoding the genome.

1	As we have just seen, the processes of transcription initiation and termination involve a complicated series of structural transitions in protein, DNA, and RNA molecules. The signals encoded in DNA that specify these transitions are often difficult for researchers to recognize. Indeed, a comparison of many different bacterial promoters reveals a surprising degree of variation. Nevertheless, they all contain related sequences, reflecting aspects of the DNA that are recognized directly

1	Figure 6–11 The transcription cycle of bacterial RNA polymerase. (A) In step 1, the RNA polymerase holoenzyme (polymerase core enzyme plus σ factor) assembles and then locates a promoter DNA sequence (see Figure 6–12). The polymerase opens (unwinds) the DNA at the position at which transcription is to begin (step 2) and begins transcribing (step 3). This initial RNA synthesis (abortive initiation) is relatively inefficient as short, unproductive transcripts are often released. However, once RNA polymerase has managed to synthesize about 10 nucleotides of RNA, it breaks its interactions with the promoter DNA (step 4) and eventually releases σ factor—as the polymerase tightens around the DNA and shifts to the elongation mode of RNA synthesis, moving along the DNA (step 5). During the elongation mode, transcription is highly processive, with the polymerase leaving the DNA template and releasing the newly transcribed RNA only when it encounters a termination signal (steps 6 and 7).

1	elongation mode, transcription is highly processive, with the polymerase leaving the DNA template and releasing the newly transcribed RNA only when it encounters a termination signal (steps 6 and 7). Termination signals are typically encoded in DNA, and many function by forming an RNA hairpin-like structure that destabilizes the polymerase’s hold on the RNA.

1	In bacteria, all RNA molecules are synthesized by a single type of RNA polymerase, and the cycle depicted in the figure therefore applies to the production of mRNAs as well as structural and catalytic RNAs. (B) Two-dimensional image of an elongating bacterial RNA polymerase, as determined by atomic force microscopy (see Figure 9–33). (C) Interpretation of the image in (B). (Adapted from K.M. Herbert et al., Annu. Rev. Biochem. 77:149–176, 2008.) frequency of nucleotide in each position (%) sequence and sequence logo for the major class of E. coli promoters.

1	(A) On the basis of a comparison of 300 promoters, the frequencies of each of the four nucleotides at each position in the promoter are given. The consensus sequence, shown below the graph, reflects the most common nucleotide found at each position in the collection of promoters. These promoters are characterized by two hexameric DNA sequences—the –35 sequence and the –10 sequence, named for their approximate location relative to the start point of transcription (designated +1). The sequence of nucleotides between the –35 and –10 hexamers shows no significant similarities among promoters.

1	For convenience, the nucleotide sequence of a single strand of DNA is shown; in reality, promoters are double-stranded by the σ factor. These common features are often summarized in the form of a consensus sequence (Figure 6–12). A consensus nucleotide sequence is derived by comparing many sequences with the same basic function and tallying up the most common nucleotides found at each position. It therefore serves as a summary or “average” of a large number of individual nucleotide sequences. A more accurate way of displaying the range of DNA sequences recognized by a protein is through the use of a sequence logo, which reveals the relative frequencies of each nucleotide at each position (Figure 6–12C).

1	The DNA sequences of individual bacterial promoters differ in ways that determine their strength (the number of initiation events per unit time of the promoter). Evolutionary processes have fine-tuned each to initiate as often as necessary and have thereby created a wide spectrum of promoter strengths. Promoters for genes that code for abundant proteins are much stronger than those associated with genes that encode rare proteins, and the nucleotide sequences of their promoters are responsible for these differences. Like bacterial promoters, transcription terminators also have a wide range of sequences, with the potential to form a simple hairpin RNA structure being the most important common feature. Since an almost unlimited number of nucleotide sequences have this potential, terminator sequences are even more heterogeneous than promoter sequences.

1	We have discussed bacterial promoters and terminators in some detail to illustrate an important point regarding the analysis of genome sequences. Although we know a great deal about bacterial promoters and terminators and can construct consensus sequences that summarize their most salient features, their variation in nucleotide sequence makes it difficult to definitively locate them simply DNA. The nucleotides shown in the figure are recognized by σ factor, a subunit of the RNA polymerase holoenzyme.

1	DNA. The nucleotides shown in the figure are recognized by σ factor, a subunit of the RNA polymerase holoenzyme. (B) The distribution of spacing between the –35 and –10 hexamers found in E. coli promoters. (C) A sequence logo displaying the same information as in panel (A). Here, the height of each letter is proportional to the frequency at which that base occurs at that position across a wide variety of promoter sequences. The total height of all the letters at each position is proportional to the information content (expressed in bits) at that position. For example, the total information content of a position that can tolerate several different bases is small (see the last three bases of the –35 sequences), but statistically greater than random.

1	DNA of E. coli chromosome gene a by analysis of the nucleotide sequence of a genome. It is even more difficult to locate analogous sequences in eukaryotic genomes, due in part to the excess DNA carried in these genomes. Often we need additional information, some of it from direct experimentation, to locate and accurately interpret the short DNA signals in genomes. As shown in Figure 6–11, promoter sequences are asymmetric, ensuring that RNA polymerase can bind in only one orientation. Because the polymerase can synthesize RNA only in the 5ʹ-to-3ʹ direction, the promoter orientation specifies the strand to be used as a template. Genome sequences reveal that the DNA strand that is used as the template for RNA synthesis varies from gene to gene, depending on the orientation of the promoter (Figure 6–13). Having considered transcription in bacteria, we now turn to the situation in eukaryotes, where the synthesis of RNA molecules is a much more elaborate affair.

1	Having considered transcription in bacteria, we now turn to the situation in eukaryotes, where the synthesis of RNA molecules is a much more elaborate affair. In contrast to bacteria, which contain a single type of RNA polymerase, eukaryotic nuclei have three: RNA polymerase I, RNA polymerase II, and RNA polymerase III. The three polymerases are structurally similar to one another and share some common subunits, but they transcribe different categories of genes (Table 6–2). RNA polymerases I and III transcribe the genes encoding transfer RNA, ribosomal RNA, and various small RNAs. RNA polymerase II transcribes most genes, including all those that encode proteins, and our subsequent discussion therefore focuses on this enzyme. Eukaryotic RNA polymerase II has many structural similarities to bacterial RNA polymerase (Figure 6–14). But there are several important differences in the way in which the bacterial and eukaryotic enzymes function, two of which concern us immediately. 1.

1	1. While bacterial RNA polymerase requires only a single transcriptioninitiation factor (σ) to begin transcription, eukaryotic RNA polymerases require many such factors, collectively called the general transcription factors. 2. Eukaryotic transcription initiation must take place on DNA that is packaged into nucleosomes and higher-order forms of chromatin structure (described in Chapter 4), features that are absent from bacterial chromosomes.

1	Figure 6–13 Directions of transcription along a short portion of a bacterial chromosome. Some genes are transcribed using one DNA strand as a template, while others are transcribed using the other DNA strand. The direction of transcription is determined by the promoter at the beginning of each gene (green arrowheads). This diagram shows approximately 0.2% (9000 base pairs) of the E. coli chromosome. The genes transcribed from left to right use the bottom DNA strand as the template; those transcribed from right to left use the top strand as the template. RNA Polymerase II Requires a Set of General Transcription Factors

1	RNA Polymerase II Requires a Set of General Transcription Factors The general transcription factors help to position eukaryotic RNA polymerase correctly at the promoter, aid in pulling apart the two strands of DNA to allow transcription to begin, and release RNA polymerase from the promoter to start its elongation mode. The proteins are “general” because they are needed at nearly all promoters used by RNA polymerase II. They consist of a set of interacting proteins denoted arbitrarily as TFIIA, TFIIB, TFIIC, TFIID, and so on (TFII standing for “transcription factor for polymerase II).” In a broad sense, the eukaryotic general transcription factors carry out functions equivalent to those of the σ factor in bacteria; indeed, portions of TFIIF have the same three-dimensional structure as the equivalent portions of σ.

1	Figure 6–15 illustrates how the general transcription factors assemble at promoters used by RNA polymerase II, and Table 6–3 summarizes their activities. The assembly process begins when TFIID binds to a short double-helical DNA sequence primarily composed of T and A nucleotides. For this reason, this sequence is known as the TATA sequence, or TATA box, and the subunit of TFIID that recognizes it is called TBP (for TATA-binding protein). The TATA box is typically located 25 nucleotides upstream from the transcription start site. It is not the only DNA sequence that signals the start of transcription (Figure 6–16), but for most polymerase II promoters it is the most important. The binding of TFIID

1	Figure 6–15 Initiation of transcription of a eukaryotic gene by RNA polymerase II. To begin transcription, RNA polymerase requires several general transcription factors. (A) The promoter contains a DNA sequence called the TATA box, which is located 25 nucleotides away from the site at which transcription is initiated. (B) Through its subunit TBP, TFIID recognizes and binds the TATA box, which then enables the adjacent binding of TFIIB (C). For simplicity the DNA distortion produced by the binding of TFIID (see Figure 6–17) is not shown. (D) The rest of the general transcription factors, as well as the RNA polymerase itself, assemble at the promoter. (E) TFIIH then uses energy from ATP hydrolysis to pry apart the DNA double helix at the transcription start point, locally exposing the template strand. TFIIH also phosphorylates RNA polymerase II, changing its conformation so that the polymerase is released from the general factors and can begin the elongation phase of transcription. As

1	strand. TFIIH also phosphorylates RNA polymerase II, changing its conformation so that the polymerase is released from the general factors and can begin the elongation phase of transcription. As shown, the site of phosphorylation is a long C-terminal polypeptide tail, also called the C-terminal domain (CTD), that extends from the polymerase molecule. The assembly scheme shown in the figure was deduced from experiments performed in vitro, and the exact order in which the general transcription factors assemble on promoters probably varies from gene to gene in vivo. The general transcription factors are highly conserved; some of those from human cells can be replaced in biochemical experiments by the corresponding factors from simple yeasts.

1	Figure 6–14 Structural similarity between a bacterial RNA polymerase and a eukaryotic RNA polymerase II. Regions of the two RNA polymerases that have similar structures are indicated in green. The eukaryotic polymerase is larger than the bacterial enzyme (12 subunits instead of 5), and some of the additional regions are shown in gray. The blue spheres represent Zn atoms that serve as structural components of the polymerases, and the red sphere represents the Mg atom present at the active site, where polymerization takes place. The RNA polymerases in all modern-day cells (bacteria, archaea, and eukaryotes) are closely related, indicating that the basic features of the enzyme were in place before the divergence of the three major branches of life. (Courtesy of

1	P. Cramer and R. Kornberg.) start of transcription TATA box (D) UTP, ATP CTP, GTP FACTOR RELEASE causes a large distortion in the DNA of the TATA box (Figure 6–17). This distortion is thought to serve as a physical landmark for the location of an active promoter in the midst of a very large genome, and it brings DNA sequences on both sides of the distortion closer together to allow for subsequent protein assembly steps. Other factors then assemble, along with RNA polymerase II, to form a complete transcription initiation complex (see Figure 6–15). The most complicated of the general transcription factors is TFIIH. Consisting of nine subunits, it is nearly as large as RNA polymerase II itself and, as we shall see shortly, performs several enzymatic steps needed for the initiation of transcription.

1	After forming a transcription initiation complex on the promoter DNA, RNA polymerase II must gain access to the template strand at the transcription start point. TFIIH, which contains a DNA helicase as one of its subunits, makes this step possible by hydrolyzing ATP and unwinding the DNA, thereby exposing the template strand. Next, RNA polymerase II, like the bacterial polymerase, remains at the promoter synthesizing short lengths of RNA until it undergoes a series of conformational changes that allow it to move away from the promoter and enter the elongation phase of transcription. A key step in this transition is the addition of phosphate groups to the “tail” of the RNA polymerase (known as the CTD or C-terminal domain). In humans, the CTD consists of 52 tandem repeats of a subunit of TFIID C/T C/T A N T/A C/T C/T TFIID

1	Figure 6–16 Consensus sequences found in the vicinity of eukaryotic RNA polymerase II start points. The name given to each consensus sequence (first column) and the general transcription factor that recognizes it (last column) are indicated. N indicates any nucleotide, and two nucleotides separated by a slash indicate an equal probability of either nucleotide at the indicated position. In reality, each consensus sequence is a shorthand representation of a histogram similar to that of Figure 6–12. For most RNA polymerase II transcription start points, only two or three of the four sequences are present. For example, many polymerase II promoters have a TATA box sequence, but those that do not typically have a “strong” INR sequence. Although most of the DNA sequences that influence transcription initiation are located upstream of the transcription start point, a few, such as the DPE shown in the figure, are located in the transcribed region.

1	seven-amino-acid sequence, which extend from the RNA polymerase core structure. During transcription initiation, the serine located at the fifth position in the repeat sequence (Ser5) is phosphorylated by TFIIH, which contains a protein kinase in one of its subunits (see Figure 6–15D and E). The polymerase can then disengage from the cluster of general transcription factors. During this process, it undergoes a series of conformational changes that tighten its interaction with DNA, and it acquires new proteins that allow it to transcribe for long distances, in some cases for many hours, without dissociating from DNA.

1	Once the polymerase II has begun elongating the RNA transcript, most of the general transcription factors are released from the DNA so that they are available to initiate another round of transcription with a new RNA polymerase molecule. As we see shortly, the phosphorylation of the tail of RNA polymerase II has an additional function: it causes components of the RNA-processing machinery to load onto the polymerase and thus be positioned to modify the newly transcribed RNA as it emerges from the polymerase. Polymerase II Also Requires Activator, Mediator, and Chromatin-Modifying Proteins

1	Studies of RNA polymerase II and its general transcription factors acting on DNA templates in purified in vitro systems established the model for transcription initiation just described. However, as discussed in Chapter 4, DNA in eukaryotic cells is packaged into nucleosomes, which are further arranged in higher-order chromatin structures. As a result, transcription initiation in a eukaryotic cell is more complex and requires more proteins than it does on purified DNA. First, gene regulatory proteins known as transcriptional activators must bind to specific sequences in DNA (called enhancers) and help to attract RNA polymerase II to the start point of transcription (Figure 6–18). We discuss the role of these activators in Chapter 7, because they are one of the main ways in which cells regulate expression of their genes. Here we simply note that their presence on DNA is required for transcription initiation in a eukaryotic cell. Second, eukaryotic transcription initiation in vivo

1	regulate expression of their genes. Here we simply note that their presence on DNA is required for transcription initiation in a eukaryotic cell. Second, eukaryotic transcription initiation in vivo requires the presence of a large protein complex known as Mediator, which allows the activator proteins to communicate properly with the polymerase II and with the general transcription factors. Finally, transcription initiation in a eukaryotic cell typically requires the recruitment of chromatin-modifying enzymes, including chromatin remodeling complexes and

1	Figure 6–17 Three-dimensional structure of TbP (TATA-binding protein) bound to DNA. The TBP is the subunit of the general transcription factor TFIID that is responsible for recognizing and binding to the TATA box sequence in the DNA (red). The unique DNA bending caused by TBP—kinks in the double helix separated by partly unwound DNA—is thought to serve as a landmark that helps to attract the other general transcription factors (Movie 6.4). TBP is a single polypeptide chain that is folded into two very similar domains (blue and green). (Adapted from J.L. Kim et al., Nature 365:520–527, 1993. With permission from Macmillan Publishers Ltd.) start of (binding site for GENERAL TRANSCRIPTION FACTORS, RNA POLYMERASE, MEDIATOR, CHROMATIN REMODELING COMPLEXES, AND HISTONE-MODIFYING ENZYMES

1	GENERAL TRANSCRIPTION FACTORS, RNA POLYMERASE, MEDIATOR, CHROMATIN REMODELING COMPLEXES, AND HISTONE-MODIFYING ENZYMES RNA polymerase bound to histone-modifying enzymes. As discussed in Chapter 4, both types of enzymes can increase access to the DNA in chromatin, and by doing so they facilitate the assembly of the transcription initiation machinery onto DNA. As illustrated in Figure 6–18, many proteins (well over 100 individual subunits) must assemble at the start point of transcription to initiate transcription in a eukaryotic cell. The order of assembly of these proteins does not seem to follow a prescribed pathway; rather, the order differs from gene to gene. Indeed, some of these different protein complexes may be brought to DNA as preformed subassemblies.

1	To begin transcribing, RNA polymerase II must be released from this large complex of proteins. In addition to the steps described in Figure 6–14, this release often requires the in situ proteolysis of the activator protein. We shall return to some of these issues, including the role of chromatin remodeling complexes and histone-modifying enzymes, in Chapter 7, where we discuss how eukaryotic cells regulate the process of transcription initiation.

1	Once RNA polymerase has initiated transcription, it moves jerkily, pausing at some DNA sequences and rapidly transcribing through others. Elongating RNA polymerases, both bacterial and eukaryotic, are associated with a series of elongation factors, proteins that decrease the likelihood that RNA polymerase will dissociate before it reaches the end of a gene. These factors typically associate with RNA polymerase shortly after initiation and help the polymerase move through the wide variety of different DNA sequences that are found in genes. Eukaryotic RNA polymerases must also contend with chromatin structure as they move along a DNA template, and they are typically aided by ATP-dependent chromatin remodeling complexes that either move with the polymerase or may simply seek out and rescue the occasional stalled polymerase. In addition, histone chaperones help by partially disassembling nucleosomes in front of a moving RNA polymerase and assembling them behind.

1	As RNA polymerase moves along a gene, some of the enzymes bound to it modify the histones, leaving behind a record of where the polymerase has been. Although it is not clear exactly how the cell uses this information, it may aid in Figure 6–18 Transcription initiation by RNA polymerase II in a eukaryotic cell.

1	Transcription initiation in vivo requires the presence of transcription activator proteins. As described in Chapter 7, these proteins bind to specific short sequences in DNA. Although only one is shown here, a typical eukaryotic gene utilizes many transcription activator proteins, which in combination determine its rate and pattern of transcription. Sometimes acting from a distance of several thousand nucleotide pairs (indicated by the dashed DNA molecule), these proteins help RNA polymerase, the general transcription factors, and Mediator all to assemble at the promoter. In addition, activators attract ATP-dependent chromatin remodeling complexes and histone-modifying enzymes. One of the main roles of Mediator is to coordinate the assembly of all these proteins at the promoter so that transcription can begin. As discussed in Chapter 4, the “default” state of chromatin is a condensed fiber (see Figure 4–28), and this is likely to be the form of DNA upon which most transcription is

1	transcription can begin. As discussed in Chapter 4, the “default” state of chromatin is a condensed fiber (see Figure 4–28), and this is likely to be the form of DNA upon which most transcription is initiated. For simplicity, the chromatin is not shown in this figure.

1	transcribing a gene over and over again once it has become active for the first time. It may also be used to coordinate transcription elongation with the processing of RNA as it emerges from RNA polymerase, a topic we discuss later in this chapter.

1	There is yet another barrier to elongating RNA polymerases, both bacterial and eukaryotic, one that also applies to DNA polymerases, as discussed in Chapter 5 (see Figure 5–20). To describe this issue in more detail, we need first to consider a subtle property inherent in the DNA double helix called DNA supercoiling. DNA supercoiling is the name given to a conformation that DNA adopts in response to superhelical tension; alternatively, creating loops or coils in a double-helical DNA molecule can create such tension. Figure 6–19 illustrates why. There are approximately 10 nucleotide pairs for every helical turn in a DNA double helix. If we imagine a helix whose two ends are fixed with respect to each other (as they are in a DNA circle, such as a bacterial chromosome, or in a tightly clamped loop, as is thought to exist in eukaryotic chromosomes), one large DNA supercoil will form to compensate for each 10 nucleotide pairs that are opened (unwound). The formation of this supercoil is

1	loop, as is thought to exist in eukaryotic chromosomes), one large DNA supercoil will form to compensate for each 10 nucleotide pairs that are opened (unwound). The formation of this supercoil is energetically favorable because it restores a normal helical twist to the base-paired regions that remain, which would otherwise need to be overwound because of the fixed ends.

1	RNA polymerase creates superhelical tension as it moves along a stretch of DNA that is anchored at its ends (see Figure 6–19C). As long as the polymerase is not free to rotate rapidly (and such rotation is unlikely given the size of RNA polymerases and their attached transcripts), a moving polymerase generates positive superhelical tension in the DNA in front of it and negative helical tension behind it. For eukaryotes, this situation is thought to provide a bonus: although the positive superhelical tension ahead of the polymerase makes the DNA helix DNA with free end DNA with fxed ends Figure 6–19 Superhelical tension in DNA causes DNA supercoiling. (A) For a DNA molecule with one free end (or a nick in one strand that serves as a swivel), the DNA double helix rotates by one turn for every 10 nucleotide pairs opened.

1	(B) If rotation is prevented, superhelical tension is introduced into the DNA by helix opening. In the example shown, the DNA helix contains 10 helical turns, one of which is opened. One way of accommodating the tension created would be to increase the helical twist from 10 to 11 nucleotide pairs per turn in the double helix that remains. The DNA helix, however, resists such a deformation in a springlike fashion, preferring to relieve the superhelical tension by bending into supercoiled loops. As a result, one DNA supercoil forms in the DNA double helix for every 10 nucleotide pairs opened. The supercoil formed in this case is a positive supercoil. (C) Supercoiling of DNA is induced by a protein tracking through the DNA double helix. The two ends of the DNA shown here are unable to rotate freely relative to each other, and the protein molecule is assumed also to be prevented from rotating freely as it moves. Under these conditions, the movement of the protein causes an excess of

1	freely relative to each other, and the protein molecule is assumed also to be prevented from rotating freely as it moves. Under these conditions, the movement of the protein causes an excess of helical turns to accumulate in the DNA helix ahead of the protein and a deficit of helical turns to arise in the DNA behind the protein, as shown.

1	more difficult to open, the tension should facilitate the partial unwrapping of the DNA in nucleosomes, inasmuch as the release of DNA from the histone core helps to relax this tension.

1	Any protein that propels itself alone along a DNA strand of a double helix, such as a DNA helicase or an RNA polymerase, tends to generate superhelical tension. In eukaryotes, DNA topoisomerase enzymes rapidly remove this superhelical tension (see pp. 251–253). But in bacteria a specialized topoisomerase called DNA gyrase uses the energy of ATP hydrolysis to pump supercoils continuously into the DNA, thereby maintaining the DNA under constant tension. These are negative supercoils, having the opposite handedness from the positive supercoils that form when a region of DNA helix opens (see Figure 6–19B). Whenever a region of helix opens, it removes these negative supercoils from bacterial DNA, reducing the superhelical tension. DNA gyrase therefore makes the opening of the DNA helix in bacteria energetically favorable compared with helix opening in DNA that is not supercoiled. For this reason, it facilitates those genetic processes in bacteria, such as the initiation of transcription by

1	energetically favorable compared with helix opening in DNA that is not supercoiled. For this reason, it facilitates those genetic processes in bacteria, such as the initiation of transcription by bacterial RNA polymerase, that require helix opening (see Figure 6–11).

1	Transcription Elongation in Eukaryotes Is Tightly Coupled to RNA Processing We have seen that bacterial mRNAs are synthesized by the RNA polymerase starting and stopping at specific spots on the genome. The situation in eukaryotes is substantially different. In particular, transcription is only the first of several steps needed to produce a mature mRNA molecule. Other critical steps are the covalent modification of the ends of the RNA and the removal of intron sequences that are discarded from the middle of the RNA transcript by the process of RNA splicing (Figure 6–20). Both ends of eukaryotic mRNAs are modified: by capping on the 5ʹ end and by polyadenylation of the 3ʹ end (Figure 6–21). These special ends allow the cell to assess whether both ends of an mRNA molecule are present (and if the message is therefore intact) before it exports the RNA from the nucleus and translates it

1	Figure 6–20 Comparison of the steps leading from gene to protein in eukaryotes and bacteria. The final level of a protein in the cell depends on the efficiency of each step and on the rates of degradation of the RNA and protein molecules. (A) In eukaryotic cells, the mRNA molecule resulting from transcription contains both coding (exon) and noncoding (intron) sequences. Before it can be translated into protein, the two ends of the RNA are modified, the introns are removed by an enzymatically catalyzed RNA splicing reaction, and the resulting mRNA is transported from the nucleus to the cytoplasm. For convenience, the steps in this figure are depicted as occurring one at a time; in reality, many occur concurrently. For example, the RNA cap is added and splicing begins before transcription has been completed. Because of the coupling between transcription and RNA processing, intact primary transcripts—the full-length RNAs that would, in theory, be produced if no processing had

1	has been completed. Because of the coupling between transcription and RNA processing, intact primary transcripts—the full-length RNAs that would, in theory, be produced if no processing had occurred—are found only rarely. (B) In prokaryotes, the production of mRNA is much simpler. The 5ʹ end of an mRNA molecule is produced by the initiation of transcription, and the 3ʹ end is produced by the termination of transcription. Since prokaryotic cells lack a nucleus, transcription and translation take place in a common compartment, and the translation of a bacterial mRNA often begins before its synthesis has been completed.

1	prokaryotic mRNA 5˜ end of coding noncoding 7-methylguanosine primary transcript sequence sequence

1	Figure 6–21 A comparison of the structures of prokaryotic and eukaryotic mRNA molecules. (A) The 5ʹ and 3ʹ ends of a bacterial mRNA are the unmodified ends of the chain synthesized by the RNA polymerase, which initiates and terminates transcription at those points, respectively. The corresponding ends of a eukaryotic mRNA are formed by adding a 5ʹ cap and by cleavage of the pre-mRNA transcript near the 3ʹ end and the addition of a poly-A tail, respectively. The figure also illustrates another difference between the prokaryotic and eukaryotic mRNAs: bacterial mRNAs can contain the instructions for several different proteins, whereas eukaryotic mRNAs nearly always contain the information for only a single protein. (B) The structure of the cap at the 5ʹ end of eukaryotic mRNA molecules. Note the unusual 5ʹ-to-5ʹ linkage of the 7-methyl G to the remainder of the RNA. Many eukaryotic mRNAs carry an additional modification: methylation of the 2ʹ-hydroxyl group of the ribose sugar at the 5ʹ

1	the unusual 5ʹ-to-5ʹ linkage of the 7-methyl G to the remainder of the RNA. Many eukaryotic mRNAs carry an additional modification: methylation of the 2ʹ-hydroxyl group of the ribose sugar at the 5ʹ end of the primary transcript (see Figure 6–23).

1	into protein. RNA splicing joins together the different portions of a protein-coding sequence, and it provides eukaryotes with the ability to synthesize several different proteins from the same gene.

1	A simple strategy has evolved to couple all of the above RNA processing steps to transcription elongation. As discussed previously, a key step in transcription initiation by RNA polymerase II is the phosphorylation of the RNA polymerase II tail, also called the CTD (C-terminal domain). This phosphorylation, which proceeds gradually as the RNA polymerase initiates transcription and moves along the DNA, not only helps dissociate the RNA polymerase II from other proteins present at the start point of transcription, but also allows a new set of proteins to associate with the RNA polymerase tail that function in transcription elongation and RNA processing. As discussed next, some of these processing proteins are thought to “hop” from the polymerase tail onto the nascent RNA molecule to begin processing it as it emerges from the RNA polymerase. Thus, we can view RNA polymerase II in its elongation mode as an RNA factory that not only moves along the DNA synthesizing an RNA molecule, but

1	processing it as it emerges from the RNA polymerase. Thus, we can view RNA polymerase II in its elongation mode as an RNA factory that not only moves along the DNA synthesizing an RNA molecule, but also processes the RNA that it produces (Figure 6–22). Fully extended, the CTD is nearly 10 times longer than the remainder of RNA polymerase. As a flexible protein domain, it serves as a scaffold or tether, holding a variety of proteins close by so that they can rapidly act when needed. This strategy, which greatly speeds up the overall rate of a series of consecutive reactions, is one that is commonly utilized in the cell (see Figures 4–58 and 16–18).

1	RNA Capping Is the First Modification of Eukaryotic Pre-mRNAs As soon as RNA polymerase II has produced about 25 nucleotides of RNA, the 5ʹ end of the new RNA molecule is modified by addition of a cap that consists of a modified guanine nucleotide (see Figure 6–21B). Three enzymes, acting in succession, perform the capping reaction: one (a phosphatase) removes a phosphate from the 5ʹ end of the nascent RNA, another (a guanyl transferase) adds a GMP in

1	Figure 6–22 eukaryotic RNA polymerase II as an “RNA factory.” As the polymerase transcribes DNA into RNA, it carries RNA-processing proteins on its tail that are transferred to the nascent RNA at the appropriate time. The tail contains 52 tandem repeats of a seven-amino-acid sequence, and there are two serines in each repeat. The capping proteins first bind to the RNA polymerase tail when it is phosphorylated on Ser5 of the heptad repeat late in the process of transcription initiation (see Figure 6–15). This strategy ensures that the RNA molecule is efficiently capped as soon as its 5ʹ end emerges from the RNA polymerase. As the polymerase continues transcribing, its tail is extensively phosphorylated on the Ser2 positions by a kinase associated with the elongating polymerase and is eventually dephosphorylated at Ser5 positions. These further modifications attract splicing and 3ʹ-end processing proteins to the moving polymerase, positioning them to act on the newly synthesized RNA as

1	dephosphorylated at Ser5 positions. These further modifications attract splicing and 3ʹ-end processing proteins to the moving polymerase, positioning them to act on the newly synthesized RNA as it emerges from the RNA polymerase. There are many RNA-processing enzymes, and not all travel with the polymerase. For RNA splicing, for example, the tail carries only a few critical components; once transferred to an RNA molecule, they serve as a nucleation site for the remaining components.

1	When RNA polymerase II finishes transcribing a gene, it is released from DNA, soluble phosphatases remove the phosphates on its tail, and it can reinitiate transcription. Only the fully dephosphorylated form of RNA polymerase II is competent to begin RNA synthesis at a promoter. a reverse linkage (5ʹ to 5ʹ instead of 5ʹ to 3ʹ), and a third (a methyl transferase) adds a methyl group to the guanosine (Figure 6–23). Because all three enzymes bind to the RNA polymerase tail phosphorylated at the Ser5 position—the modification added by TFIIH during transcription initiation— they are poised to modify the 5ʹ end of the nascent transcript as soon as it emerges from the polymerase.

1	The 5ʹ-methyl cap signifies the 5ʹ end of eukaryotic mRNAs, and this landmark helps the cell to distinguish mRNAs from the other types of RNA molecules present in the cell. For example, RNA polymerases I and III produce uncapped RNAs during transcription, in part because these polymerases lack a CTD. In the nucleus, the cap binds a protein complex called CBC (cap-binding complex), which, as we discuss in subsequent sections, helps a future mRNA be further processed and exported. The 5ʹ-methyl cap also has an important role in the translation of mRNAs in the cytosol, as we discuss later in the chapter. 5˜ end of nascent RNA transcript

1	5˜ end of nascent RNA transcript As discussed in Chapter 4, the protein-coding sequences of eukaryotic genes are typically interrupted by noncoding intervening sequences (introns). Discovered 5˜ 3˜ in 1977, this feature of eukaryotic genes came as a surprise to scientists, who had been, until that time, familiar only with bacterial genes, which typically consist of a continuous stretch of coding DNA that is directly transcribed into mRNA. In marked contrast, eukaryotic genes were found to be broken up into small pieces of coding sequence (expressed sequences or exons) interspersed with much longer intervening sequences or introns; thus, the coding portion of a eukaryotic gene is often only a small fraction of the length of the gene (Figure 6–24).

1	Both intron and exon sequences are transcribed into RNA. The intron sequences are removed from the newly synthesized RNA through the process of RNA splicing. The vast majority of RNA splicing that takes place in cells func- tions in the production of mRNA, and our discussion of splicing focuses on this so-called precursor-mRNA (or pre-mRNA) splicing. Only after 5ʹand 3ʹ-end pro- add methyl group to base cessing and splicing have taken place is such RNA termed mRNA.

1	Figure 6–23 The reactions that cap the 5ʹ end of each RNA molecule synthesized by RNA polymerase II. The final cap contains a novel 5ʹ-to-5ʹ add methyl group to ribose linkage between the positively charged 7-methyl G residue and the 5ʹ end of the RNA transcript (see Figure 6–21B). The letter N represents any one of the four ribonucleotides, although the nucleotide that starts an RNA chain is usually a purine (an A or a G). (After A.J. Shatkin, BioEssays 7:275–277, 1987. With permission from Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.) CH3 exons 2000 200,000 nucleotide pairs

1	Each splicing event removes one intron, proceeding through two sequential phosphoryl-transfer reactions known as transesterifications; these join two exons together while removing the intron between them as a “lariat” (Figure 6–25). The machinery that catalyzes pre-mRNA splicing is complex, consisting of five addi-tional RNA molecules and several hundred proteins, and it hydrolyzes many ATP molecules per splicing event. This complexity ensures that splicing is accurate, while at the same time being flexible enough to deal with the enormous variety of introns found in a typical eukaryotic cell. It may seem wasteful to remove large numbers of introns by RNA splicing. In attempting to explain why it occurs, scientists have pointed out that the exon– intron arrangement would seem to facilitate the emergence of new and useful proteins over evolutionary time scales. Thus, the presence of numerous introns in DNA allows genetic recombination to readily combine the exons of different genes,

1	the emergence of new and useful proteins over evolutionary time scales. Thus, the presence of numerous introns in DNA allows genetic recombination to readily combine the exons of different genes, enabling genes for new proteins to evolve more easily by the combination of parts of preexisting genes. The observation, described in Chapter 3, that many proteins in present-day cells resemble patchworks composed from a common set of protein domains, supports this idea (see pp. 121–122). RNA splicing also has a present-day advantage. The transcripts of many eukaryotic genes (estimated at 95% of genes in humans) are spliced in more than one way, thereby allowing the same gene to produce a corresponding set of dif-ferent proteins (Figure 6–26). Rather than being the wasteful process it may have seemed at first sight, RNA splicing enables eukaryotes to increase the coding potential of their genomes. We shall return to this idea again in this chapter and the next, but we first need to describe

1	at first sight, RNA splicing enables eukaryotes to increase the coding potential of their genomes. We shall return to this idea again in this chapter and the next, but we first need to describe the cellular machinery that performs this remarkable task. O(A) (B) 5˜5˜AOH AHO 3˜5˜exon sequence intron sequence 3˜exon 2˜

1	Figure 6–24 Structure of two human genes showing the arrangement of exons and introns. (A) The relatively small β-globin gene, which encodes a subunit of the oxygen-carrying protein hemoglobin, contains 3 exons (see also Figure 4–7). (B) The much larger Factor VIII gene contains 26 exons; it codes for a protein (Factor VIII) that functions in the blood-clotting pathway. The most prevalent form of hemophilia results from mutations in this gene.

1	Figure 6–25 The pre-mRNA splicing reaction. (A) In the first step, a specific adenine nucleotide in the intron sequence (indicated in red ) attacks the 5ʹ splice site and cuts the sugar-phosphate backbone of the RNA at this point. The cut 5ʹ end of the intron becomes covalently linked to the adenine nucleotide, as shown in detail in (B), thereby creating a loop in the RNA molecule. The released free 3ʹ-OH end of the exon sequence then reacts with the start of the next exon sequence, joining the two exons together and releasing the intron sequence in the shape of a lariat. The two exon sequences thereby become joined into a continuous coding sequence. The released intron sequence is eventually broken down into single nucleotides, which 5˜ 3˜ are recycled. TRANSCRIPTION, SPLICING, AND 3˜ CLEAVAGE/POLYADENYLATION

1	TRANSCRIPTION, SPLICING, AND 3˜ CLEAVAGE/POLYADENYLATION The mechanism of pre-mRNA splicing shown in Figure 6–24 requires that the splicing machinery recognize three portions of the precursor RNA molecule: the 5ʹ splice site, the 3ʹ splice site, and the branch point in the intron sequence that forms the base of the excised lariat. Not surprisingly, each site has a consensus nucleotide sequence that is similar from intron to intron and provides the cell with cues for where splicing is to take place (Figure 6–27). However, these consensus sequences are relatively short and can accommodate extensive sequence variability; as we shall see shortly, the cell incorporates additional types of information to ultimately choose exactly where, on each RNA molecule, splicing is to take place.

1	The high variability of the splicing consensus sequences presents a special challenge for scientists attempting to decipher genome sequences. Introns range in size from about 10 nucleotides to over 100,000 nucleotides, and choosing the precise borders of each intron is a difficult task even with the aid of powerful computers. The possibility of alternative splicing compounds the problem of predicting protein sequences solely from a genome sequence. This difficulty is one of the main barriers to identifying all of the genes in a complete genome sequence, and it is one of the primary reasons why we know only the approximate number of different proteins produced by the human genome. RNA Splicing Is Performed by the Spliceosome

1	RNA Splicing Is Performed by the Spliceosome Unlike the other steps of mRNA production we have discussed, key steps in RNA splicing are performed by RNA molecules rather than proteins. Specialized RNA molecules recognize the nucleotide sequences that specify where splicing is to occur and also catalyze the chemistry of splicing. These RNA molecules are relatively short (less than 200 nucleotides each), and there are five of them, U1, U2, U4, U5, and U6. Known as snRNAs (small nuclear RNAs), each is complexed with at least seven protein subunits to form an snRNP (small nuclear ribonucleoprotein). –°–°–°AG GURAGU –°– –°– °YURAC – ....°– YYYYYYYYNCAG G –°–°–° 5˜3˜ portion of a primary transcript exon 1 intron exon 2 portion of mRNA exon 1 exon 2 Figure 6–26 Alternative splicing of the α-tropomyosin gene from rat.

1	Figure 6–26 Alternative splicing of the α-tropomyosin gene from rat. α-Tropomyosin is a coiled-coil protein (see Figure 3–9) that carries out several tasks, most notably the regulation of contraction in muscle cells. The primary transcript can be spliced in different ways, as indicated in the figure, to produce distinct mRNAs, which then give rise to variant proteins. Some of the splicing patterns are specific for certain types of cells. For example, the α-tropomyosin made in striated muscle is different from that made from the same gene in smooth muscle. The arrowheads in the top part of the figure mark the sites where cleavage and poly-A addition form the 3ʹ ends of the mature mRNAs.

1	Figure 6–27 The consensus nucleotide sequences in an RNA molecule that signal the beginning and the end of most introns in humans. The three blocks of nucleotide sequences shown are required to remove an intron sequence. Here A, G, U, and C are the standard RNA nucleotides; R stands for purines (A or G); and Y stands for pyrimidines (C or U). The A highlighted in red forms the branch point of the lariat produced by splicing (see Figure 6–25). Only the GU at the start of the intron and the AG at its end are invariant nucleotides in the splicing consensus sequences. Several different nucleotides can occupy the remaining positions, although the indicated nucleotides are preferred. The distances along the RNA between the three splicing consensus sequences are highly variable; however, the distance between the branch point and 3ʹ splice junction is typically much shorter than that between the 5ʹ splice junction and the branch point.

1	These snRNPs form the core of the spliceosome, the large assembly of RNA and protein molecules that performs pre-mRNA splicing in the cell. During the splicing reaction, recognition of the 5ʹ splice junction, the branch-point site, and the 3ʹsplice junction is performed largely through base-pairing between the snRNAs and the consensus RNA sequences in the pre-mRNA substrate. The spliceosome is a complex and dynamic machine. When studied in vitro, a few components of the spliceosome assemble on pre-mRNA and, as the splicing reaction proceeds, new components enter and those that have already performed their tasks are jettisoned (Figure 6–28). However, many scientists believe that, inside the cell, the spliceosome is a preexisting, loose assembly of all the components—capturing, splicing, and releasing RNA as a coordinated unit, and undergoing extensive rearrangements each time a splice is made. intron portion of a 5˜ 3˜ pre-mRNA transcript

1	intron portion of a 5˜ 3˜ pre-mRNA transcript U1, U4 excised intron sequence in the form of a lariat 3˜ (intron RNA will be degraded in the nucleus; snRNPs will exon 1 exon 2 The U1 snRNP forms base pairs with the 5˜ splice junction (see Figure 6–29) and the BBP (branch-point binding protein) and U2AF (U2 auxilliary factor) recognize the branch-point site. The U2 snRNP displaces BBP and U2AF and forms base pairs with the branch-point site consensus sequence. The U4/U6•U5 “triple” snRNP enters the reaction. In this triple snRNP, the U4 and U6 snRNAs are held frmly together by base-pair interactions. Subsequent rearrangements break apart the U4/U6 base pairs, allowing U6 to displace U1 at the 5˜ splice junction (see Figure 6–29). This creates the active site that catalyzes the frst phosphoryl-transferase reaction. Additional RNA–RNA rearrangements create the active site for the second phosphoryl-transferase reaction, which then completes the splice (see Figure 6–25A).

1	Additional RNA–RNA rearrangements create the active site for the second phosphoryl-transferase reaction, which then completes the splice (see Figure 6–25A). Figure 6–28 The pre-mRNA splicing mechanism. RNA splicing is catalyzed by an assembly of snRNPs (shown as colored circles) plus other proteins (most of which are not shown), which together constitute the spliceosome. The spliceosome recognizes the splicing signals on a pre-mRNA molecule, brings the two ends of the intron together, and provides the enzymatic activity for the two reaction steps required (see Figure 6–25A and Movie 6.5). As indicated, a set of proteins called the exon junction complex (EJC) remains on the spliced mRNA molecule; its subsequent role will be discussed shortly. The Spliceosome Uses ATP Hydrolysis to Produce a Complex Series of RNA–RNA Rearrangements

1	The Spliceosome Uses ATP Hydrolysis to Produce a Complex Series of RNA–RNA Rearrangements ATP hydrolysis is not required for the chemistry of RNA splicing per se since the two transesterification reactions preserve the high-energy phosphate bonds. However, extensive ATP hydrolysis is required for the assembly and rearrangements of the spliceosome. Some of the additional proteins that make up the spliceosome use the energy of ATP hydrolysis to break existing RNA–RNA interactions to allow the formation of new ones. Each successful splice requires approximately 200 proteins, if we include those that form the snRNPs.

1	What is the purpose of these rearrangements? First, they allow the splicing signals on the pre-RNA to be examined by snRNPs several times during the course of splicing. For example, the U1 snRNP initially recognizes the 5ʹ splice site through conventional base-pairing; as splicing proceeds, these base pairs are broken (using the energy of ATP hydrolysis) and U1 is replaced by U6 (Figure 6–29). This type of RNA–RNA rearrangement (in which the formation of one RNA–RNA interaction requires the disruption of another) occurs several times during splicing and allows the spliceosomes to check and recheck the splicing signals, thereby increasing the overall accuracy of splicing. Second, the rearrangements that take place in the spliceosome create the active sites for the two transesterification reactions. These two active sites are created, one after the other, and only after the splicing signals on the pre-mRNA have been checked several times. This orderly progression ensures that splicing

1	reactions. These two active sites are created, one after the other, and only after the splicing signals on the pre-mRNA have been checked several times. This orderly progression ensures that splicing accidents occur only rarely.

1	One of the most surprising features of the spliceosome is the nature of the catalytic sites: they are formed by both protein and RNA molecules, although the RNA molecules catalyze the actual chemistry of splicing. In the last section of this chapter, we discuss in general terms the structural and chemical properties of RNA molecules that allow them to act as catalysts.

1	Once the splicing chemistry is completed, the snRNPs remain bound to the lariat. The disassembly of these snRNPs from the lariat (and from each other) requires another series of RNA–RNA rearrangements that require ATP hydrolysis, thereby returning the snRNAs to their original configuration so that they can be used again in a new reaction. At the completion of a splice, the spliceosome directs a set of proteins to bind to the mRNA near the position formerly occupied by the intron. Called the exon junction complex (EJC), these proteins mark the site of a successful splicing event and, as we shall see later in this chapter, influence the subsequent fate of the mRNA. Other Properties of Pre-mRNA and Its Synthesis Help to Explain the Choice of Proper Splice Sites

1	Other Properties of Pre-mRNA and Its Synthesis Help to Explain the Choice of Proper Splice Sites As we have seen, intron sequences vary enormously in size, with some being in excess of 100,000 nucleotides. If splice-site selection were determined solely by the snRNPs acting on a preformed, protein-free RNA molecule, we would expect frequent splicing mistakes—such as exon skipping and the use of “cryptic” splice sites (Figure 6–30). The fidelity mechanisms built into the spliceosome to suppress errors, however, are supplemented by two additional strategies that further increase the accuracy of splicing. The first is a simple consequence of splicing being coupled to transcription. As transcription proceeds, the phosphorylated tail of RNA polymerase carries several components of the spliceosome (see Figure Figure 6–29 One of the many rearrangements that take place in the spliceosome during pre-mRNA splicing.

1	Figure 6–29 One of the many rearrangements that take place in the spliceosome during pre-mRNA splicing. This example comes from the yeast Saccharomyces cerevisiae, in which the nucleotide sequences involved are slightly different from those in human cells. The exchange of U1 snRNP for U6 snRNP occurs just before the first phosphoryltransfer reaction (see Figure 6–28). This exchange requires the 5ʹ splice site to be read by two different snRNPs, thereby increasing the accuracy of 5ʹ splice-site selection by the spliceosome. portion of exon 2 exon 1 exon 3 exon 1 6–22), and these components are transferred directly from the polymerase to the

1	portion of exon 2 exon 1 exon 3 exon 1 6–22), and these components are transferred directly from the polymerase to the RNA as the RNA emerges from the polymerase. This strategy helps the cell keep track of introns and exons: for example, the snRNPs that assemble at a 5ʹ splice site are initially presented only with the single 3ʹ splice site that emerges next from the polymerase; the potential sites further downstream have not yet been synthe sized. The coordination of transcription with splicing is especially important in preventing inappropriate exon skipping.

1	A strategy called “exon definition” also helps cells choose the appropriate splice sites. Exon size tends to be much more uniform than intron size, averaging about 150 nucleotide pairs across a wide variety of eukaryotic organisms (Figure 6–31). Through exon definition, the splicing machinery can seek out the relatively homogeneously sized exon sequences. As RNA synthesis proceeds, a group of additional components (most notably SR proteins, so-named because they con help to mark off each 3ʹand 5ʹ splice site, starting at the 5ʹ end of the RNA (Figure 6–32). These proteins, in turn, recruit U1 snRNA, which marks the downstream exon boundary, and U2 snRNA, which specifies the upstream one. By specifically marking the exons in this way and thereby taking advantage of the relatively uni form size of exons, the cell increases the accuracy with which it deposits the initial splicing components on the nascent RNA and thereby avoids “near miss” splice sites. How the SR proteins discriminate

1	size of exons, the cell increases the accuracy with which it deposits the initial splicing components on the nascent RNA and thereby avoids “near miss” splice sites. How the SR proteins discriminate exon sequences from intron sequences is not understood in detail; however, it is known that some of the SR proteins bind preferentially to specific RNA sequences in exons, termed splicing enhancers.

1	In principle, since any one of several different codons can be used to code for a given amino acid, there is freedom to evolve the exon nucleotide sequence so as to form a binding site for an SR protein, without necessarily affecting the amino acid sequence that the exon specifies. Both the marking of exon and intron boundaries and the assembly of the spli ceosome begin on an RNA molecule while it is still being elongated by RNA poly merase at its 3ʹ end. However, the actual chemistry of splicing can take place later. This delay means that intron sequences are not necessarily removed from a pre mRNA molecule in the order in which they occur along the RNA chain. Figure 6–30 Two types of splicing errors. (A) Exon skipping. (B) Cryptic splice-site selection. Cryptic splicing signals are nucleotide sequences of RNA that closely resemble true splicing signals and are sometimes mistakenly used by the spliceosome.

1	Figure 6–31 Variation in intron and exon lengths in the human, worm, and fly genomes. (A) Size distribution of exons. (B) Size distribution of introns. Note that exon length is much more uniform than intron length. (Adapted from International Human Genome Sequencing Consortium, Nature 409:860–921, 2001. With permission from Macmillan Publishers Ltd.) percentage of introns percentage of exons 100 200 300 400 500 600 700 800 900 1000 <100 100–2000 2000–5000 5000–30,000 >30,000 exon length (nucleotide pairs) intron length (nucleotide pairs)

1	Although it may seem at first counterintuitive, the way a gene is packaged into chromatin can affect how the RNA transcript of that gene is ultimately spliced. Nucleosomes tend to be positioned over exons (which are, on average, close to the length of DNA in a nucleosome), and it has been proposed that these act as “speed bumps,” allowing the proteins responsible for exon definition to assemble on the RNA as it emerges from the polymerase. In addition, changes in chromatin structure are used to alter splicing patterns. There are two ways this can happen. First, because splicing and transcription are coupled, the rate at which RNA polymerase moves along DNA can affect RNA splicing. For example, if polymerase is moving slowly, exon skipping (see Figure 6–30A) is minimized: assembly of the initial spliceosome may be complete before an alternative choice of splice site even emerges from the RNA polymerase. The nucleosomes in condensed chromatin can cause polymerase to pause; the pattern

1	initial spliceosome may be complete before an alternative choice of splice site even emerges from the RNA polymerase. The nucleosomes in condensed chromatin can cause polymerase to pause; the pattern of pauses in turn affects the extent of RNA exposed at any given time to the splicing machinery.

1	There is a second and more direct way that chromatin structure can affect RNA splicing. Although the details are not yet understood, specific histone modifications attract components of the spliceosome, and, because the chromatin being transcribed is in close association with the nascent RNA, these splicing components can easily be transferred to the emerging RNA. In this way, certain types of histone modifications can affect the final pattern of splicing.

1	We have seen that the choice of splice sites depends on such features of the premRNA transcript as the strength of the three signals on the RNA (the 5ʹand 3ʹ splice junctions and the branch point) for the splicing machinery, the co-transcriptional assembly of the spliceosome, chromatin structure, and the “bookkeeping” that underlies exon definition. We do not know exactly how accurate splicing normally is because, as we see later, there are several quality control systems that rapidly destroy mRNAs whose splicing goes awry. However, we do know that, compared with other steps in gene expression, splicing is unusually flexible.

1	Thus, for example, a mutation in a nucleotide sequence critical for splicing of a particular intron does not necessarily prevent splicing of that intron altogether. Instead, the mutation typically creates a new pattern of splicing (Figure 6–33). Most commonly, an exon is simply skipped (Figure 6–33B). In other cases, the mutation causes a cryptic splice junction to be efficiently used (Figure 6–33C). Apparently, the splicing machinery has evolved to pick out the best possible pattern of splice junctions, and if the optimal one is damaged by mutation, it will seek out the next best pattern, and so on. This inherent plasticity in the process of RNA splicing suggests that changes in splicing patterns caused by random mutations have been important in the evolution of genes and organisms. It also means that mutations that affect splicing can be severely detrimental to the organism: in addition to the β thalassemia, example presented in Figure 6–33, aberrant

1	Figure 6–32 The exon definition hypothesis. According to this idea, SR proteins bind to each exon sequence in the pre-mRNA and thereby help to guide the snRNPs to the proper intron/ exon boundaries. This demarcation of exons by the SR proteins occurs cotranscriptionally, beginning at the CBC (cap-binding complex) at the 5ʹ end. It has been proposed that a group of proteins known as the heterogeneous nuclear ribonucleoproteins (hnRNPs) may preferentially associate with intron sequences, further helping the spliceosome distinguish introns from exons. (Adapted from R. Reed, Curr. Opin. Cell Biol. 12:340–345, 2000. With permission from Elsevier.) splicing plays important roles in the development of cystic fibrosis, frontotemporal dementia, Parkinson’s disease, retinitis pigmentosa, spinal muscular atrophy, myotonic dystrophy, premature aging, and cancer. It has been estimated that of the many point mutations that cause inherited human diseases, 10% produce aberrant splicing of the gene

1	atrophy, myotonic dystrophy, premature aging, and cancer. It has been estimated that of the many point mutations that cause inherited human diseases, 10% produce aberrant splicing of the gene containing the mutation.

1	The plasticity of RNA splicing also means that the cell can easily regulate the pattern of RNA splicing. Earlier in this section we saw that alternative splicing can give rise to different proteins from the same gene and that this is a common strategy to enhance the coding potential of genomes. Some examples of alternative splicing are constitutive; that is, the alternatively spliced mRNAs are produced continuously by cells of an organism. However, in many cases, the cell regulates the splicing patterns so that different forms of the protein are produced at different times and in different tissues (see Figure 6–26). In Chapter 7, we return to this issue to discuss some specific examples of regulated RNA splicing.

1	When the spliceosome was first discovered, it puzzled molecular biologists. Why do RNA molecules instead of proteins perform important roles in splice-site recognition and in the chemistry of splicing? Why is a lariat intermediate used rather than the apparently simpler alternative of bringing the 5ʹand 3ʹ splice sites together in a single step, followed by their direct cleavage and rejoining? The answers to these questions reflect the way in which the spliceosome has evolved.

1	As discussed briefly in Chapter 1 (and in more detail in the final section of this chapter), it is likely that early cells used RNA molecules rather than proteins as their major catalysts and that they stored their genetic information in RNA rather than in DNA sequences. RNA-catalyzed splicing reactions presumably had critical roles in these early cells. As evidence, some self-splicing RNA introns (that is, intron sequences in RNA whose splicing out can occur in the absence of proteins or any other RNA molecules) remain today—for example, in the nuclear rRNA genes of the ciliate Tetrahymena, in a few bacteriophage T4 genes, and in some mitochondrial and chloroplast genes. In these cases, the RNA molecule folds into a specific three-dimensional structure that brings the intron/exon junctions together and catalyzes the two transesterification reactions. A self-splicing intron sequence can be identified in a test tube by incubating a pure RNA molecule that contains the intron sequence

1	together and catalyzes the two transesterification reactions. A self-splicing intron sequence can be identified in a test tube by incubating a pure RNA molecule that contains the intron sequence and observing the splicing reaction. Because the basic chemistry of some self-splicing reactions is so similar to pre-mRNA splicing, it has been proposed that the much more involved process of pre-mRNA splicing evolved from a simpler, ancestral form of RNA self-splicing.

1	RNA-Processing Enzymes Generate the 3ʹ End of Eukaryotic mRNAs We have seen that the 5ʹ end of the pre-mRNA produced by RNA polymerase II is capped almost as soon as it emerges from the RNA polymerase. Then, as the polymerase continues its movement along a gene, the spliceosome assembles on the RNA and delineates the intron and exon boundaries. The long C-terminal tail of the RNA polymerase coordinates these processes by transferring capping and splicing components directly to the RNA as it emerges from the enzyme. In this section, we shall see that, as RNA polymerase II reaches the end of a gene, a similar mechanism ensures that the 3ʹ end of the pre-mRNA is appropriately processed.

1	The position of the 3ʹ end of each mRNA molecule is specified by signals encoded in the genome (Figure 6–34). These signals are transcribed into RNA as the RNA polymerase II moves through them, and they are then recognized (as RNA) by a series of RNA-binding proteins and RNA-processing enzymes (Figure 6–35). Two multisubunit proteins, called CstF (cleavage stimulation factor) and CPSF (cleavage and polyadenylation specificity factor), are of special importance. intron sequences normal mRNA is formed from three exons A SINGLE-NUCLEOTIDE CHANGE THAT DESTROYS A NORMAL SPLICE SITE, THEREBY CAUSING EXON SKIPPING mRNA with exon 2 missing A SINGLE-NUCLEOTIDE CHANGE THAT DESTROYS A NORMAL SPLICE SITE, THEREBY ACTIVATING A CRYPTIC SPLICE SITE mRNA with extended exon 3 mRNA with extra exon inserted between exon 2 and exon 3 Figure 6–33 Abnormal processing of the β-globin primary RNA transcript in humans with the disease β thalassemia.

1	Figure 6–33 Abnormal processing of the β-globin primary RNA transcript in humans with the disease β thalassemia. In the examples shown, the disease (a severe anemia due to aberrant hemoglobin synthesis) is caused by splice-site mutations found in the genomes of affected patients. The dark blue boxes represent the three normal exon sequences; the red lines connect the 5ʹ and 3ʹ splice sites that are used. In (B), (C), and (D), the light blue boxes depict new nucleotide sequences included in the final mRNA molecule as a result of the mutation denoted by the black arrowhead. Note that when a mutation leaves a normal splice site without a partner, an exon is skipped (B) or one or more abnormal cryptic splice sites nearby is used as the partner site (C). [Adapted in part from S.H. Orkin, in The Molecular Basis of Blood Diseases (G. Stamatoyannopoulos et al., eds.), pp. 106–126. Philadelphia: Saunders, 1987.] degraded in the nucleus

1	Both of these proteins travel with the RNA polymerase tail and are transferred to the 3ʹ-end processing sequence on an RNA molecule as it emerges from the RNA polymerase.

1	Once CstF and CPSF bind to their recognition sequences on the emerging RNA molecule, additional proteins assemble with them to create the 3ʹend of the mRNA. First, the RNA is cleaved from the polymerase (see Figure 6–35). Next an enzyme called poly-A polymerase (PAP) adds, one at a time, approximately 200 A nucleotides to the 3ʹ end produced by the cleavage. The nucleotide precursor for these additions is ATP, and the same type of 5ʹ-to-3ʹ bonds are formed as in conventional RNA synthesis. But unlike other RNA polymerases, poly-A polymerase does not require a template; hence the poly-A tail of eukaryotic mRNAs is not directly encoded in the genome. As the poly-A tail is synthesized, proteins called poly-A-binding proteins assemble onto it and, by a poorly understood mechanism, help determine the final length of the tail.

1	After the 3ʹ-end of a eukaryotic pre-mRNA molecule has been cleaved, the RNA polymerase II continues to transcribe, in some cases for hundreds of nucleotides. Once 3ʹ-end cleavage has occurred, the newly synthesized RNA that emerges from the polymerases lacks a 5ʹ cap; this unprotected RNA is rapidly degraded by a 5ʹ →3ʹ exonuclease carried along on the polymerase tail. Apparently, it is this continued RNA degradation that eventually causes the RNA polymerase to release its grip on the template and terminate transcription. Mature Eukaryotic mRNAs Are Selectively Exported from the Nucleus

1	Mature Eukaryotic mRNAs Are Selectively Exported from the Nucleus Eukaryotic pre-mRNA synthesis and processing take place in an orderly fashion within the cell nucleus. But of the pre-mRNA that is synthesized, only a small fraction—the mature mRNA—is of further use to the cell. Most of the rest—excised introns, broken RNAs, and aberrantly processed pre-mRNAs—is not only useless but potentially dangerous. How does the cell distinguish between the relatively rare mature mRNA molecules it wishes to keep and the overwhelming amount of debris created by RNA processing?

1	The answer is that, as an RNA molecule is processed, it loses certain proteins and acquires others. For example, we have seen that acquisition of cap-binding complexes, exon junction complexes, and poly-A-binding proteins mark the completion of capping, splicing, and poly-A addition, respectively. A properly completed mRNA molecule is also distinguished by the proteins it lacks. For example, the presence of an snRNP protein would signify incomplete or aberrant splicing. Only when the proteins present on an mRNA molecule collectively signify that processing was successfully completed is the mRNA exported from the nucleus into the cytosol, where it can be translated into protein. Improperly processed mRNAs

1	Figure 6–35 Some of the major steps in generating the 3ʹ end of a eukaryotic mRNA. This process is much more complicated than the analogous process in bacteria, where the RNA polymerase simply stops at a termination signal and releases both the 3ʹ end of its transcript and the DNA template (see Figure 6–11). Figure 6–34 Consensus nucleotide sequences that direct cleavage and polyadenylation to form the 3ʹ end of a eukaryotic mRNA. These sequences are encoded in the genome, and specific proteins recognize them—as RNA—after they are transcribed. As shown in Figure 6–35, the hexamer AAUAAA is bound by CPSF and the GU-rich element beyond the cleavage site is bound by CstF; the CA sequence is bound by a third protein factor required for the cleavage step. Like other consensus nucleotide sequences discussed in this chapter (see Figure 6–12), the sequences shown in the figure represent a variety of individual cleavage and polyadenylation signals.

1	mature 3˜ end of an mRNA molecule and other RNA debris (excised intron sequences, for example) are retained in the nucleus, where they are eventually degraded by the nuclear exosome, a large protein complex whose interior is rich in 3ʹ-to-5ʹ RNA exonucleases (Figure 6–36). Eukaryotic cells thus export only useful RNA molecules to the cytoplasm, while debris is disposed of in the nucleus.

1	Of all the proteins that assemble on pre-mRNA molecules as they emerge from transcribing RNA polymerases, the most abundant are the hnRNPs (heterogeneous nuclear ribonuclear proteins). Some of these proteins (there are approximately 30 different ones in humans) unwind the hairpin helices in the RNA so that splicing and other signals on the RNA can be read more easily. Others preferentially package the RNA contained in the very long intron sequences typical in complex organisms (see Figure 6–31) and these may play an important role in distinguishing mature mRNA from the debris left over from RNA processing.

1	Successfully processed mRNAs are guided through the nuclear pore complexes (NPCs)—aqueous channels in the nuclear membrane that directly connect the nucleoplasm and cytosol (Figure 6–37). Small molecules (less than 60,000 daltons) can diffuse freely through these channels. However, most of the macromolecules in cells, including mRNAs complexed with proteins, are far too large to pass through the channels without a special process. The cell uses energy to actively transport such macromolecules in both directions through the nuclear pore complexes.

1	As explained in detail in Chapter 12, macromolecules are moved through nuclear pore complexes by nuclear transport receptors, which, depending on the identity of the macromolecule, escort it from the nucleus to the cytoplasm or vice versa. For mRNA export to occur, a specific nuclear transport receptor must be loaded onto the mRNA, a step that, in many organisms, takes place in concert with 3ʹ cleavage and polyadenylation. Once it helps to move an RNA molecule through the nuclear pore complex, the transport receptor dissociates from the mRNA, re-enters the nucleus, and is then used to export a new mRNA molecule.

1	The export of mRNA–protein complexes from the nucleus can be readily observed with the electron microscope for the unusually abundant mRNA of the insect Balbiani Ring genes. As these genes are transcribed, the newly formed RNA is seen to be packaged by proteins, including hnRNPs, SR proteins, and components of the spliceosome. This protein–RNA complex undergoes a series of structural transitions, probably reflecting RNA processing events, culminating in a curved fiber (see Figure 6–37). This curved fiber moves through the nucleoplasm and enters the nuclear pore complex (with its 5ʹ cap proceeding first), and it then undergoes another series of structural transitions as it moves through the pore. These and other observations reveal that the pre-mRNA–protein and mRNA–protein complexes are dynamic structures that gain and lose numerous specific proteins during RNA synthesis, processing, and export (Figure 6–38).

1	The analysis just described has been complemented by new methods that allow researchers to track the fate of more typical mRNA molecules, which can RNA as it emerges from RNA polymerase

1	RNA as it emerges from RNA polymerase Figure 6–36 Structure of the core of human RNA exosome. RNA is fed into one end of the central pore and is degraded by RNAses that associate with the other end. Nine different protein subunits (each represented by a different color) make up this large ring structure. Eukaryotic cells have both a nuclear exosome and a cytoplasmic exosome; both forms include the core exosome shown here and additional subunits (including specialized RNAses) that differentiate the two forms. The nuclear exosome degrades aberrant RNAs before they are exported to the cytosol. It also processes certain types of RNA (for example, the ribosomal RNAs) to produce their final form. The cytoplasmic form of the exosome is responsible for degrading mRNAs in the cytosol, and is thus crucial in determining the lifetime of each mRNA molecule. (PDB code: 2NN6.)

1	Figure 6–37 Transport of a large mRNA molecule through the nuclear pore complex. (A) The maturation of an mRNA molecule as it is synthesized by RNA polymerase and packaged by a variety of nuclear proteins. This drawing of an unusually large and abundant insect RNA, called the Balbiani Ring mRNA, is based on electron microscope micrographs such as that shown in (B). (A, adapted from B. Daneholt, Cell 88:585–588, 1997. With permission from Elsevier; B, from B.J. Stevens and H. Swift, J. Cell Biol. 31:55–77, 1966. With permission from The Rockefeller University Press.) be fluorescently labeled and observed individually. A typical RNA molecule is released from its site of transcription and spends several minutes diffusing to a nuclear pore complex. During this time it is likely that RNA processing events continue and that the RNA sheds previously bound proteins and acquires new ones. Once it arrives at the entrance to the pore, the “export-ready” mRNA hovers for several seconds, during

1	events continue and that the RNA sheds previously bound proteins and acquires new ones. Once it arrives at the entrance to the pore, the “export-ready” mRNA hovers for several seconds, during which time the completion of processing may occur, and then is transported through the pore very rapidly, in tens of milliseconds. Some mRNA–protein complexes are very large, and how they move through the nuclear pore complexes so rapidly remains a mystery.

1	Some of the proteins deposited on the mRNA while it is still in the nucleus can affect the fate of the RNA after it is transported to the cytosol. Thus, the stability of an mRNA in the cytosol, the efficiency with which it is translated into protein, and its ultimate destination in the cytosol can all be determined by proteins acquired in the nucleus that remain bound to the RNA after it leaves the nucleus. But before discussing what happens to mRNAs in the cytosol, we briefly consider how the synthesis and processing of some noncoding RNA molecules occurs. There are many types of noncoding RNAs produced by cells (see Table 6–1, p. 305), but here we focus on the rRNAs, which are critically important for the translation of mRNAs into protein. Noncoding RNAs Are Also Synthesized and Processed in the Nucleus

1	Noncoding RNAs Are Also Synthesized and Processed in the Nucleus Only a few percent of the dry weight of a mammalian cell is RNA; of that, only about 3–5% is mRNA. The bulk of the RNA in cells performs structural and catalytic functions (see Table 6–1). The most abundant RNAs in cells are the ribosomal RNAs (rRNAs), constituting approximately 80% of the RNA in rapidly dividing cells. As discussed later in this chapter, these RNAs form the core of the ribosome. Unlike bacteria—in which a single RNA polymerase synthesizes all RNAs in the cell—eukaryotes have a separate, specialized polymerase, RNA polymerase I, that is dedicated to producing rRNAs. RNA polymerase I is similar structurally to the RNA polymerase II discussed previously; however, the absence of a C-terminal tail in polymerase I helps to explain why its transcripts are neither capped nor polyadenylated.

1	Because multiple rounds of translation of each mRNA molecule can provide an enormous amplification in the production of protein molecules, many of the proteins that are very abundant in a cell can be synthesized from genes that are present in a single copy per haploid genome (see Figure 6–3). In contrast, the RNA components of the ribosome are final gene products, and a growing mammalian cell must synthesize approximately 10 million copies of each type of ribosomal RNA in each cell generation to construct its 10 million ribosomes. The cell can produce adequate quantities of ribosomal RNAs only because it contains multiple copies of the rRNA genes that code for ribosomal RNAs (rRNAs). Even E. coli needs seven copies of its rRNA genes to meet the cell’s need for ribosomes. Human cells contain about 200 rRNA gene copies per haploid genome, spread Figure 6–38 Schematic illustration of an export-ready mRNA molecule and its transport through the nuclear pore.

1	Figure 6–38 Schematic illustration of an export-ready mRNA molecule and its transport through the nuclear pore. As indicated, some proteins travel with the mRNA as it moves through the pore, whereas others remain in the nucleus. The nuclear export receptor for mRNAs is a complex of proteins that binds to an mRNA molecule once it has been correctly spliced and polyadenylated. After the mRNA has been exported to the cytosol, this export receptor dissociates from the mRNA and is re-imported into the nucleus, where it can be used again. The final check indicated here, called nonsense-mediated decay, will be described later in the chapter. out in small clusters on five different chromosomes (see Figure 4–11), while cells of the frog Xenopus contain about 600 rRNA gene copies per haploid genome in a single cluster on one chromosome (Figure 6–39).

1	There are four types of eukaryotic rRNAs, each present in one copy per ribosome. Three of the four rRNAs (18S, 5.8S, and 28S) are made by chemically modifying and cleaving a single large precursor rRNA (Figure 6–40); the fourth (5S RNA) is synthesized from a separate cluster of genes by a different polymerase, RNA polymerase III, and does not require chemical modification.

1	Extensive chemical modifications occur in the 13,000-nucleotide-long precursor rRNA before the rRNAs are cleaved out of it and assembled into ribosomes. These include about 100 methylations of the 2ʹ-OH positions on nucleotide sugars and 100 isomerizations of uridine nucleotides to pseudouridine (Figure 6–41A). The functions of these modifications are not understood in detail, but they probably aid in the folding and assembly of the final rRNAs, or subtly alter the function of ribosomes. Each modification is made at a specific position in the precursor rRNA, specified by “guide RNAs,” which position themselves on the precursor rRNA through base-pairing and thereby bring an RNA-modifying enzyme to the appropriate position (Figure 6–41B). Other guide RNAs promote cleavage of the precursor rRNAs into the mature rRNAs, probably by causing conformational changes in the precursor rRNA that expose these sites to nucleases. All of these guide RNAs are members of a large class of RNAs called

1	rRNAs into the mature rRNAs, probably by causing conformational changes in the precursor rRNA that expose these sites to nucleases. All of these guide RNAs are members of a large class of RNAs called small nucleolar RNAs (or snoRNAs), so named because these RNAs perform their functions in a sub-compartment of the nucleus called the nucleolus. Many snoRNAs are encoded in 13,000 nucleotides degraded regions of nucleotide sequence

1	CLEAVAGE 5.8S rRNA 28S rRNA 5S rRNA made elsewhere incorporated into large ribosomal subunit Figure 6–39 Transcription from tandemly arranged rRNA genes, as seen in the electron microscope. The pattern of alternating transcribed gene and nontranscribed spacer is readily seen. A higher-magnification view of rRNA genes is shown in Figure 6–10. (From V.E. Foe, Cold Spring Harb. Symp. Quant. Biol. 42:723–740, 1978. With permission from Cold Spring Harbor Laboratory Press.) Figure 6–40 The chemical modification and nucleolytic processing of a eukaryotic 45S precursor rRNA molecule into three separate ribosomal RNAs.

1	Figure 6–40 The chemical modification and nucleolytic processing of a eukaryotic 45S precursor rRNA molecule into three separate ribosomal RNAs. Two types of chemical modifications (color-coded as indicated in Figure 6–41) are made to the precursor rRNA before it is cleaved. Nearly half of the nucleotide sequences in this precursor rRNA are discarded and degraded in the nucleus by the exosome. The rRNAs are named according to their “S” values, which refer to their rate of sedimentation in an ultracentrifuge. The larger the S value, the larger the rRNA. the introns of other genes, especially those encoding ribosomal proteins. They are synthesized by RNA polymerase II and processed from excised intron sequences. The Nucleolus Is a Ribosome-Producing Factory

1	The Nucleolus Is a Ribosome-Producing Factory The nucleolus is the most obvious structure seen in the nucleus of a eukaryotic cell when viewed in the light microscope. It was so closely scrutinized by early cytologists that an 1898 review could list some 700 references. We now know that the nucleolus is the site for the processing of rRNAs and their assembly into ribosome subunits. Unlike many of the major organelles in the cell, the nucleolus is not bound by a membrane (Figure 6–42); instead, it is a huge aggregate Figure 6–41 Modifications of the precursor rRNA by guide RNAs. (A) Two prominent covalent modifications made to rRNA; the differences from the initially incorporated nucleotide are indicated by red atoms. Pseudouridine is an isomer of uridine; the base has been “rotated,” and is attached to the red C rather than to the red N of the sugar (compare to Figure 6–5B).

1	(B) As indicated, snoRNAs determine the sites of modification by base-pairing to complementary sequences on the precursor rRNA. The snoRNAs are bound to proteins, and the complexes are called snoRNPs (small nucleolar ribonucleoproteins). snoRNPs contain both the guide sequences and the enzymes that modify the rRNA. Figure 6–42 electron micrograph of a thin section of a nucleolus in a human fibroblast, showing its three distinct zones. (A) View of entire nucleus. Higher-power view of the nucleolus. It is believed that transcription of the rRNA genes takes place between the fibrillar center and the dense fibrillar component and that processing of the rRNAs and their assembly into the two subunits of the ribosome proceeds outward from the dense fibrillar component to the surrounding granular components. (Courtesy of E.G. Jordan and J. McGovern.)

1	J. McGovern.) Figure 6–43 Changes in the appearance of the nucleolus in a human cell during the cell cycle. Only the cell nucleus is represented in this diagram. In most eukaryotic cells, the nuclear envelope breaks down during mitosis, as indicated by the dashed circles. of macromolecules, including the rRNA genes themselves, precursor rRNAs, mature rRNAs, rRNA-processing enzymes, snoRNPs, a large set of assembly factors (including ATPases, GTPases, protein kinases, and RNA helicases), ribosomal proteins, and partly assembled ribosomes. The close association of all these components allows the assembly of ribosomes to occur rapidly and smoothly.

1	Various types of RNA molecules play a central part in the chemistry and structure of the nucleolus, suggesting that it may have evolved from an ancient structure present in cells dominated by RNA catalysis. In present-day cells, the rRNA genes have an important role in forming the nucleolus. In a diploid human cell, the rRNA genes are distributed into 10 clusters, located near the tips of five different chromosome pairs (see Figure 4–11). During interphase, these 10 chromosomes contribute DNA loops (containing the rRNA genes) to the nucleolus; in

1	M phase, when the chromosomes condense, the nucleolus fragments and then disappears. Then, in the telophase part of mitosis, as chromosomes return to their semi-dispersed state, the tips of the 10 chromosomes reform small nucleoli, which progressively coalesce into a single nucleolus (Figure 6–43 and Figure 6–44). As might be expected, the size of the nucleolus reflects the number of ribosomes that the cell is producing. Its size therefore varies greatly in different cells and can change in a single cell, occupying 25% of the total nuclear volume in cells that are making unusually large amounts of protein. Ribosome assembly is a complex process, the most important features of which are outlined in Figure 6–45. In addition to its central role in ribosome biogenesis, the nucleolus is the site where other noncoding RNAs are produced and other

1	RNA–protein complexes are assembled. For example, the U6 snRNP, which func tions in pre-mRNA splicing (see Figure 6–28), is composed of one RNA molecule and at least seven proteins. The U6 snRNA is chemically modified by snoRNAs in the nucleolus before its final assembly there into the U6 snRNP. Other important RNA–protein complexes, including telomerase (encountered in Chapter 5) and the signal-recognition particle (which we discuss in Chapter 12), are assembled at the nucleolus. Finally, the tRNAs (transfer RNAs) that carry the amino acids for protein synthesis are processed there as well; like the rRNA genes, the genes encoding tRNAs are clustered in the nucleolus. Thus, the nucleolus can be thought of as a large factory at which different noncoding RNAs are transcribed, processed, and assembled with proteins to form a large variety of ribonucleoprotein complexes.

1	Figure 6–44 Nucleolar fusion. These light micrographs of human fibroblasts grown in culture show various stages of nucleolar fusion. After mitosis, each of the 10 human chromosomes that carry a cluster of rRNA genes begins to form a tiny nucleolus, but these rapidly coalesce as they grow to form the single large nucleolus typical of many interphase cells. (Courtesy of E.G. Jordan and J. McGovern.) The Nucleus Contains a Variety of Subnuclear Aggregates

1	Although the nucleolus is the most prominent structure in the nucleus, several other nuclear bodies have been observed and studied (Figure 6–46). These include Cajal bodies (named for the scientist who first described them in 1906) and interchromatin granule clusters (also called “speckles”). Like the nucleolus, these other nuclear structures lack membranes and are highly dynamic depending on the needs of the cell. Their assembly is likely mediated by the association of low complexity protein domains, as described in Chapter 3 (see Figure 3–36). Their appearance is the result of the tight association of protein and RNA components involved in the synthesis, assembly, and storage of macromolecules involved in gene expression. Cajal bodies are sites where the snRNPs and snoRNPs undergo their final maturation steps, and where the snRNPs are recycled and their RNAs are “reset” after the rearrangements that occur during splicing (see p. 321). In contrast, the interchromatin granule clusters

1	final maturation steps, and where the snRNPs are recycled and their RNAs are “reset” after the rearrangements that occur during splicing (see p. 321). In contrast, the interchromatin granule clusters have been proposed to be stockpiles of fully mature snRNPs and other RNA processing components that are ready to be used in the production of mRNA.

1	Scientists have had difficulties in working out the function of these small sub-nuclear structures, in part because their appearances can change dramatically as cells traverse the cell cycle or respond to changes in their environment. Moreover, Figure 6–45 The function of the nucleolus in ribosome and other ribonucleoprotein synthesis. The 45S precursor rRNA is packaged in a large ribonucleoprotein particle containing many ribosomal proteins imported from the cytoplasm. While this particle remains at the nucleolus, selected components are added and others discarded as it is processed into immature large and small ribosomal subunits. The two ribosomal subunits attain their final functional form only after each is individually transported through the nuclear pores into the cytoplasm. Other ribonucleoprotein complexes, including telomerase shown here, are also assembled in the nucleolus.

1	Figure 6–46 Visualization of some prominent nuclear bodies. The protein fibrillarin (red), a component of several snoRNPs, is present at both nucleoli and Cajal bodies; the latter are indicated by the arrows. The Cajal bodies (but not the nucleoli) are also highlighted by staining one of their main components, the protein coilin; the superposition of the snoRNP and coilin stains appears pink. Interchromatin granule clusters (green) have been revealed by using antibodies against a protein involved in pre-mRNA splicing. DNA is stained blue by the dye DAPI. (From J.R. Swedlow and A.I. Lamond, Gen. Biol. 2:1–7, 2001. With permission from BioMed Central. Micrograph courtesy of Judith Sleeman.) disrupting a particular type of nuclear body often has little effect on cell viability. It seems that the main function of these aggregates is to bring components together at high concentration in order to speed up their assembly. For example, it is estimated that assembly of the U4/U6 snRNP (see

1	the main function of these aggregates is to bring components together at high concentration in order to speed up their assembly. For example, it is estimated that assembly of the U4/U6 snRNP (see Figure 6–28) occurs ten times more rapidly in Cajal bodies than would be the case if the same number of components were dispersed throughout the nucleus. Consequently, Cajal bodies appear dispensible in many types of cells but are absolutely required in situations where cells must proliferate rapidly, such as in early vertebrate development. Here, protein synthesis (which depends on RNA splicing) must be especially rapid, and delays can be lethal.

1	Given the prominence of nuclear bodies in RNA processing, it might be expected that pre-mRNA splicing would occur in a particular location in the nucleus, as it requires numerous RNA and protein components. However, as we have seen, the assembly of splicing components on pre-mRNA is co-transcriptional; thus, splicing must occur at many locations along chromosomes. Although a typical mammalian cell may be expressing on the order of 15,000 genes, transcription and RNA splicing takes place in only several thousand sites in the nucleus. These sites are highly dynamic and probably result from the association of transcription and splicing components to create small factories, the name given to specific aggregates containing a high local concentration of selected components that create biochemical assembly lines (Figure 6–47). Interchromatin tail of RNA polymerase

1	Figure 6–47 A model for an mRNA production factory. mRNA production is made more efficient in the nucleus by an aggregation of the many components needed for transcription and pre-mRNA processing, thereby producing a specialized biochemical factory. In (A), a postulated scaffold protein holds various components in the proximity of a transcribing RNA polymerase. Other key components are bound directly to the RNA polymerase tail, which likewise serves as a scaffold (see Figure 6–22), but for simplicity these are not shown here. In (B), a large number of such scaffolds have been brought together to form an aggregate that is highly enriched in the many components needed for the synthesis and processing of pre-mRNAs. Such a scaffold model can account for the several thousand sites of active RNA transcription and processing typically observed in the nucleus of a mammalian cell, each of which has a diameter of roughly 100nm and is estimated to contain, on average, about 10 RNA polymerase II

1	transcription and processing typically observed in the nucleus of a mammalian cell, each of which has a diameter of roughly 100nm and is estimated to contain, on average, about 10 RNA polymerase II molecules in addition to many other proteins. (C) Here, mRNA production factories and DNA replication factories have been visualized in the same mammalian cell by briefly incorporating differently modified nucleotides into each nucleic acid and detecting the RNA and DNA produced using antibodies, one (green) detecting the newly synthesized DNA and the other (red) detecting the newly synthesized RNA. (C, from D.G. Wansink et al., J. Cell Sci. 107:1449–1456, 1994. With permission from The Company of Biologists.) granule clusters—which contain stockpiles of RNA processing components—are often observed next to these sites of transcription, as though poised to replenish supplies. We can thus view the nucleus as organized into subdomains, with snRNPs, snoRNPs, and other nuclear components moving

1	next to these sites of transcription, as though poised to replenish supplies. We can thus view the nucleus as organized into subdomains, with snRNPs, snoRNPs, and other nuclear components moving among them in an orderly fashion according to the needs of the cell.

1	Before the synthesis of a particular protein can begin, the corresponding mRNA molecule must be produced by transcription. Bacteria contain a single type of RNA polymerase (the enzyme that carries out the transcription of DNA into RNA). An mRNA molecule is produced after this enzyme initiates transcription at a promoter, synthesizes the RNA by chain elongation, stops transcription at a terminator, and releases both the DNA template and the completed mRNA molecule. In eukaryotic cells, the process of transcription is much more complex, and there are three RNA polymerases—polymerase I, II, and III—that are related evolutionarily to one another and to the bacterial polymerase.

1	RNA polymerase II synthesizes eukaryotic mRNA. This enzyme requires a set of additional proteins, both the general transcription factors, and specific transcriptional activator proteins, to initiate transcription on a DNA template. It requires still more proteins (including chromatin remodeling complexes and histone-modifying enzymes) to initiate transcription on its chromatin templates inside the cell.

1	During the elongation phase of transcription, the nascent RNA undergoes three types of processing events: a special nucleotide is added to its 5ʹ end (capping), intron sequences are removed from the middle of the RNA molecule (splicing), and the 3ʹ end of the RNA is generated (cleavage and polyadenylation). Each of these processes is initiated by proteins that travel along with RNA polymerase II by binding to sites on its long, extended C-terminal tail. Splicing is unusual in that many of its key steps are carried out by specialized RNA molecules rather than proteins. Only properly processed mRNAs are passed through nuclear pore complexes into the cytosol, where they are translated into protein. For many genes, RNA, rather than protein, is the final product. In eukaryotes, these genes are usually transcribed by either RNA polymerase I or RNA polymerase

1	For many genes, RNA, rather than protein, is the final product. In eukaryotes, these genes are usually transcribed by either RNA polymerase I or RNA polymerase III. RNA polymerase I makes the ribosomal RNAs. After their synthesis as a large precursor, the rRNAs are chemically modified, cleaved, and assembled into the two ribosomal subunits in the nucleolus—a distinct subnuclear structure that also helps to process some smaller RNA–protein complexes in the cell. Additional subnuclear structures (including Cajal bodies and interchromatin granule clusters) are sites where components involved in RNA processing are assembled, stored, and recycled. The high concentration of components in such “factories” ensures that the processes being catalyzed are rapid and efficient.

1	In the preceding section, we have seen that the final product of some genes is an RNA molecule itself, such as the RNAs present in the snRNPs and in ribosomes. However, most genes in a cell produce mRNA molecules that serve as intermediaries on the pathway to proteins. In this section, we examine how the cell converts the information carried in an mRNA molecule into a protein molecule. This feat of translation was a strong focus of attention for biologists in the late 1950s, when it was posed as the “coding problem”: how is the information in a linear sequence of nucleotides in RNA translated into the linear sequence of a chemically quite different set of units—the amino acids in proteins? This fascinating question stimulated great excitement. Here was a cryptogram set up by nature that, after more than 3 billion years of evolution, could finally be solved by one of the products of evolution—human beings. And indeed, not only was the code cracked step by step, but in the year 2000 the

1	more than 3 billion years of evolution, could finally be solved by one of the products of evolution—human beings. And indeed, not only was the code cracked step by step, but in the year 2000 the structure of the elaborate machinery by which cells read this code—the ribosome—was finally revealed in atomic detail.

1	An mRNA Sequence Is Decoded in Sets of Three Nucleotides Once an mRNA has been produced by transcription and processing, the information present in its nucleotide sequence is used to synthesize a protein. Transcription is simple to understand as a means of information transfer: since DNA and RNA are chemically and structurally similar, the DNA can act as a direct template for the synthesis of RNA by complementary base-pairing. As the term transcription signifies, it is as if a message written out by hand is being converted, say, into a typewritten text. The language itself and the form of the message do not change, and the symbols used are closely related.

1	In contrast, the conversion of the information in RNA into protein represents a translation of the information into another language that uses quite different symbols. Moreover, since there are only 4 different nucleotides in mRNA and 20 different types of amino acids in a protein, this translation cannot be accounted for by a direct one-to-one correspondence between a nucleotide in RNA and an amino acid in protein. The nucleotide sequence of a gene, through the intermediary of mRNA, is instead translated into the amino acid sequence of a protein by rules that are known as the genetic code. This code was deciphered in the early 1960s.

1	The sequence of nucleotides in the mRNA molecule is read in consecutive groups of three. RNA is a linear polymer of four different nucleotides, so there are 4 × 4 × 4 = 64 possible combinations of three nucleotides: the triplets AAA, AUA, AUG, and so on. However, only 20 different amino acids are commonly found in proteins. Either some nucleotide triplets are never used, or the code is redundant and some amino acids are specified by more than one triplet. The second possibility is, in fact, the correct one, as shown by the completely deciphered genetic code in Figure 6–48. Each group of three consecutive nucleotides in RNA is called a codon, and each codon specifies either one amino acid or a stop to the translation process.

1	This genetic code is used universally in all present-day organisms. Although a few slight differences in the code have been found, these are chiefly in the DNA of mitochondria. Mitochondria have their own transcription and protein-synthesis systems that operate quite independently from those of the rest of the cell, and it is understandable that their tiny genomes have been able to accommodate minor changes to the code (discussed in Chapter 14). In principle, an RNA sequence can be translated in any one of three different reading frames, depending on where the decoding process begins (Figure 6–49). However, only one of the three possible reading frames in an mRNA encodes the required protein. We see later how a special punctuation signal at the beginning of each RNA message sets the correct reading frame at the start of protein synthesis. tRNA Molecules Match Amino Acids to Codons in mRNA

1	tRNA Molecules Match Amino Acids to Codons in mRNA The codons in an mRNA molecule do not directly recognize the amino acids they specify: the group of three nucleotides does not, for example, bind directly to the amino acid. Rather, the translation of mRNA into protein depends on adaptor molecules that can recognize and bind both to the codon and, at another site on their surface, to the amino acid. These adaptors consist of a set of small RNA molecules known as transfer RNAs (tRNAs), each about 80 nucleotides in length.

1	Figure 6–48 The genetic code. The standard one-letter abbreviation for each amino acid is presented below its three-letter abbreviation (see Panel 3–1, pp. 112– 113, for the full name of each amino acid and its structure). By convention, codons are always written with the 5ʹ-terminal nucleotide to the left. Note that most amino acids are represented by more than one codon, and that there are some regularities in the set of codons that specifies each amino acid: codons for the same amino acid tend to contain the same nucleotides at the first and second positions, and vary at the third position. Three codons do not specify any amino acid but act as termination sites (stop codons), signaling the end of the protein-coding sequence. One codon—AUG—acts both as an initiation codon, signaling the start of a protein-coding message, and also as the codon that specifies methionine. Figure 6–49 The three possible reading frames in protein synthesis.

1	Figure 6–49 The three possible reading frames in protein synthesis. In the process of translating a nucleotide sequence (blue) into an amino acid sequence (red), the sequence of nucleotides in an mRNA molecule is read from the 5ʹ end to the 3ʹ end in consecutive sets of three nucleotides. In principle, therefore, the same RNA sequence can specify three completely different amino acid sequences, depending on the reading frame. In reality, however, only one of these reading frames contains the actual message.

1	Figure 6–50 A tRNA molecule. A tRNA specific for the amino acid phenylalanine (Phe) is depicted in various ways. (A) The cloverleaf structure showing the complementary base-pairing (red lines) that creates the double-helical regions of the molecule. The anticodon is the sequence of three nucleotides that base-pairs with a codon in mRNA. The amino acid matching the codon/anticodon pair is attached at the 3ʹ end of the tRNA. tRNAs contain some unusual bases, which are produced by chemical modification after the tRNA has been synthesized. For example, the bases denoted ψ (pseudouridine—see Figure 6–41) and D (dihydrouridine—see Figure 6–53) are derived from uracil. (B and C) Views of the L-shaped molecule, based on x-ray diffraction analysis. Although this diagram shows the tRNA for the amino acid phenylalanine, all other tRNAs have similar structures. (D) The tRNA icon we use in this book. (E) The linear nucleotide sequence of the molecule, color-coded to match (A), (B), and (C).

1	We saw earlier in this chapter that RNA molecules can fold into precise three-dimensional structures, and the tRNA molecules provide a striking example. Four short segments of the folded tRNA are double-helical, producing a molecule that looks like a cloverleaf when drawn schematically (Figure 6–50). For example, a 5ʹ-GCUC-3ʹ sequence in one part of a polynucleotide chain can form a relatively strong association with a 5ʹ-GAGC-3ʹ sequence in another region of the same molecule. The cloverleaf undergoes further folding to form a compact L-shaped structure that is held together by additional hydrogen bonds between different regions of the molecule (see Figure 6–50B and C).

1	Two regions of unpaired nucleotides situated at either end of the L-shaped molecule are crucial to the function of tRNA in protein synthesis. One of these regions forms the anticodon, a set of three consecutive nucleotides that pairs with the complementary codon in an mRNA molecule. The other is a short single-stranded region at the 3ʹend of the molecule; this is the site where the amino acid that matches the codon is attached to the tRNA.

1	We saw above that the genetic code is redundant; that is, several different codons can specify a single amino acid. This redundancy implies either that there is more than one tRNA for many of the amino acids or that some tRNA molecules can base-pair with more than one codon. In fact, both situations occur. Some amino acids have more than one tRNA and some tRNAs are constructed so that they require accurate base-pairing only at the first two positions of the codon and can tolerate a mismatch (or wobble) at the third position (Figure 6–51). This wobble base-pairing explains why so many of the alternative codons for an amino acid differ only in their third nucleotide (see Figure 6–48). In bacteria, wobble base-pairings make it possible to fit the 20 amino acids to their 61 codons with as

1	Figure 6–51 Wobble base-pairing between codons and anticodons. If the nucleotide listed in the first column is present at the third, or wobble, position of the codon, it can base-pair with any of the nucleotides listed in the second column. Thus, for example, when inosine (I) is present in the wobble position of the tRNA anticodon, the tRNA can recognize any one of three different codons in bacteria and either of two codons in eukaryotes. The inosine in tRNAs is formed from the deamination of adenosine (see Figure 6–53), a chemical modification that takes place after the tRNA has been synthesized. The nonstandard base pairs, including those made with inosine, are generally weaker than conventional base pairs. Codon–anticodon base-pairing is more stringent at positions 1 and 2 of the codon, where only conventional base pairs are permitted. The differences in wobble base-pairing interactions between bacteria and eukaryotes presumably result from subtle structural differences between

1	where only conventional base pairs are permitted. The differences in wobble base-pairing interactions between bacteria and eukaryotes presumably result from subtle structural differences between bacterial and eukaryotic ribosomes, the molecular machines that perform protein synthesis. (Adapted from C. Guthrie and

1	J. Abelson, in The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression, pp. 487–528. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 1982.) few as 31 kinds of tRNA molecules. The exact number of different kinds of tRNAs, however, differs from one species to the next. For example, humans have nearly 500 tRNA genes, and among them 48 different anticodons are represented. tRNAs Are Covalently Modified Before They Exit from the Nucleus

1	tRNAs Are Covalently Modified Before They Exit from the Nucleus Like most other eukaryotic RNAs, tRNAs are covalently modified before they are allowed to exit from the nucleus. Eukaryotic tRNAs are synthesized by RNA polymerase III. Both bacterial and eukaryotic tRNAs are typically synthesized as larger precursor tRNAs, which are then trimmed to produce the mature tRNA. In addition, some tRNA precursors (from both bacteria and eukaryotes) contain introns that must be spliced out. This splicing reaction differs chemically from pre-mRNA splicing; rather than generating a lariat intermediate, tRNA splicing uses a cutand-paste mechanism that is catalyzed by proteins (Figure 6–52). Trimming and splicing both require the precursor tRNA to be correctly folded in its cloverleaf configuration. Because misfolded tRNA precursors will not be processed properly, the trimming and splicing reactions serve as quality-control steps in the generation of tRNAs.

1	All tRNAs are modified chemically—nearly 1 in 10 nucleotides in each mature tRNA molecule is an altered version of a standard G, U, C, or A ribonucleotide. Over 50 different types of tRNA modifications are known; a few are shown in Figure 6–53. Some of the modified nucleotides—most notably inosine, produced by the deamination of adenosine—affect the conformation and base-pairing of the anticodon and thereby facilitate the recognition of the appropriate mRNA codon by the tRNA molecule (see Figure 6–51). Others affect the accuracy with which the tRNA is attached to the correct amino acid. Specific Enzymes Couple Each Amino Acid to Its Appropriate tRNA Molecule

1	We have seen that, to read the genetic code in DNA, cells make a series of different tRNAs. We now consider how each tRNA molecule becomes linked to the one amino acid in 20 that is its appropriate partner. Recognition and attachment of the correct amino acid depends on enzymes called aminoacyl-tRNA synthetases, which covalently couple each amino acid to its appropriate set of tRNA molecules (Figure 6–54 and Figure 6–55). Most cells have a different synthetase enzyme for each amino acid (that is, 20 synthetases in all); one attaches glycine to all tRNAs that recognize codons for glycine, another attaches alanine to all tRNAs that recognize codons for alanine, and so on. Many bacteria, however, have fewer than 20 synthetases, and the same synthetase enzyme is responsible for coupling more than one amino acid to the appropriate tRNAs. In these cases, a single synthetase places the identical amino acid on two different types of tRNAs, only one of which wobble codon possible base

1	more than one amino acid to the appropriate tRNAs. In these cases, a single synthetase places the identical amino acid on two different types of tRNAs, only one of which wobble codon possible base anticodon bases U A, G, or I C G or I A U or I G C or U wobble codon possible base anticodon bases U A, G, or I C G or I AU GC

1	Figure 6–52 Structure of a tRNA-splicing endonuclease docked to a precursor tRNA. The endonuclease (a four-subunit enzyme) removes the tRNA intron (dark blue, bottom). A second enzyme, a multifunctional tRNA ligase (not shown), then joins the two tRNA halves together. (Courtesy of Hong Li, Christopher Trotta, and John Abelson; PDB code: 2A9L.) ribose HHNNNNNCH3CH3ribose HNNOHHHHribose HNNSOHHtwo methyl groups added to G (N,N-dimethyl G) two hydrogens added to U (dihydro U) ribose HNNNNHHOdeamination of A (inosine) sulfur replaces oxygen in U (4-thiouridine) P P P P has an anticodon that matches the amino acid. A second enzyme then chemically modifies each “incorrectly” attached amino acid so that it now corresponds to the anticodon displayed by its covalently linked tRNA.

1	The synthetase-catalyzed reaction that attaches the amino acid to the 3ʹend of the tRNA is one of many reactions coupled to the energy-releasing hydrolysis of ATP (see pp. 64–65), and it produces a high-energy bond between the tRNA and the amino acid. The energy of this bond is used at a later stage in protein synthesis to link the amino acid covalently to the growing polypeptide chain. The aminoacyl-tRNA synthetase enzymes and the tRNAs are equally important in the decoding process (Figure 6–56). This was established by an experiment in

1	Figure 6–54 Amino acid activation by synthetase enzymes. An amino acid is activated for protein synthesis by an aminoacyl-tRNA synthetase enzyme in two steps. As indicated, the energy of ATP hydrolysis is used to attach each amino acid to its tRNA molecule in a high-energy linkage. The amino acid is first activated through the linkage of its carboxyl group directly to AMP, forming an adenylated amino acid; the linkage of the AMP, normally an unfavorable reaction, is driven by the hydrolysis of the ATP molecule that donates the AMP. Without leaving the synthetase enzyme, the AMP-linked carboxyl group on the amino acid is then transferred to a hydroxyl group on the sugar at the 3ʹ end of the tRNA molecule. This transfer joins the amino acid by an activated ester linkage to the tRNA and forms the final aminoacyl-tRNA molecule. The synthetase enzyme is not shown in this diagram. Figure 6–53 A few of the unusual nucleotides found in tRNA molecules.

1	Figure 6–53 A few of the unusual nucleotides found in tRNA molecules. These nucleotides are produced by covalent modification of a normal nucleotide after it has been incorporated into an RNA chain. Two other types of modified nucleotides are shown in Figure 6–41. In most tRNA molecules, about 10% of the nucleotides are modified (see Figure 6–50). As shown in Figure 6–51, inosine is sometimes present at the wobble position in the tRNA anticodon.

1	which one amino acid (cysteine) was chemically converted into a different amino acid (alanine) after it already had been attached to its specific tRNA. When such “hybrid” aminoacyl-tRNA molecules were used for protein synthesis in a cell-free system, the wrong amino acid was inserted at every point in the protein chain where that tRNA was used. Although, as we shall see, cells have several quality control mechanisms to avoid this type of mishap, the experiment did establish that the genetic code is translated by two sets of adaptors that act sequentially. Each matches one molecular surface to another with great specificity, and it is their combined action that associates each sequence of three nucleotides in the mRNA molecule—that is, each codon—with its particular amino acid.

1	Several mechanisms working together ensure that an aminoacyl-tRNA synthetase links the correct amino acid to each tRNA. Most synthetase enzymes select the correct amino acid by a two-step mechanism. The correct amino acid has the highest affinity for the active-site pocket of its synthetase and is therefore favored over the other 19; in particular, amino acids larger than the correct one are excluded from the active site. However, accurate discrimination between two similar amino acids, such as isoleucine and valine (which differ by only a methyl linkage of amino acid to tRNA Figure 6–55 The structure of the aminoacyl-tRNA linkage. The carboxyl end of the amino acid forms an ester bond to ribose. Because the hydrolysis of this ester bond is associated with a large favorable change in free energy, an amino acid held in this way is said to be activated. Schematic drawing of the structure. The amino acid is linked to the nucleotide at the 3ʹ end of the tRNA (see Figure 6–50).

1	Schematic drawing of the structure. The amino acid is linked to the nucleotide at the 3ʹ end of the tRNA (see Figure 6–50). Actual structure corresponding to the boxed region in (A). There are two major classes of synthetase enzymes: one links the amino acid directly to the 3ʹ-OH group of the ribose, and the other links it initially to the 2ʹ-OH group. In the latter case, a subsequent transesterification reaction shifts the amino acid to the 3ʹ position. As in Figure 6–54, the “R group” indicates the side chain of the amino acid.

1	Figure 6–56 The genetic code is translated by means of two adaptors that act one after another. The first adaptor is the aminoacyl-tRNA synthetase, which couples a particular amino acid to its corresponding tRNA; the second adaptor is the tRNA molecule itself, whose anticodon forms base pairs with the appropriate codon on the mRNA. An error in either step would cause the wrong amino acid to be incorporated into a protein chain (Movie 6.6). In the sequence of events shown, the amino acid tryptophan (Trp) is selected by the codon UGG on the mRNA.

1	group), is very difficult to achieve in a single step. A second discrimination step occurs after the amino acid has been covalently linked to AMP (see Figure 6–54): when tRNA binds, the synthetase tries to force the adenylated amino acid into a second editing pocket in the enzyme. The precise dimensions of this pocket exclude the correct amino acid, while allowing access by closely related amino acids. In the editing pocket, an amino acid is removed from the AMP (or from the tRNA itself if the aminoacyl-tRNA bond has already formed) by hydrolysis. This hydrolytic editing, which is analogous to the exonucleolytic proofreading by DNA polymerases, increases the overall accuracy of tRNA charging to approximately one mistake in 40,000 couplings (Figure 6–57).

1	The tRNA synthetase must also recognize the correct set of tRNAs, and extensive structural and chemical complementarity between the synthetase and the tRNA allows the synthetase to probe various features of the tRNA (Figure 6–58). Most tRNA synthetases directly recognize the matching tRNA anticodon; these synthetases contain three adjacent nucleotide-binding pockets, each of which is complementary in shape and charge to a nucleotide in the anticodon. For other synthetases, the nucleotide sequence of the amino acid-accepting arm (acceptor stem) is the key recognition determinant. In most cases, however, the synthetase “reads” the nucleotides at several different positions on the tRNA. Amino Acids Are Added to the C-terminal End of a Growing Polypeptide Chain

1	Amino Acids Are Added to the C-terminal End of a Growing Polypeptide Chain Having seen that each amino acid is first coupled to specific tRNA molecules, we now turn to the mechanism that joins these amino acids together to form proteins. The fundamental reaction of protein synthesis is the formation of a peptide bond between the carboxyl group at the end of a growing polypeptide chain and a free amino group on an incoming amino acid. Consequently, a protein is synthesized stepwise from its N-terminal end to its C-terminal end. Throughout the entire process, the growing carboxyl end of the polypeptide chain remains activated by its covalent attachment to a tRNA molecule (forming a peptidyl-tRNA). Each Figure 6–57 Hydrolytic editing. Aminoacyl tRNA synthetases correct their own coupling errors through hydrolytic editing of incorrectly attached amino acids. As described in the text, the correct amino acid is rejected by the editing site.

1	The error-correction process performed by DNA polymerase has similarities; however, it differs because the removal process depends strongly on a mispairing with the template (see Figure 5–8). (P, polymerization site; E, editing site.) Figure 6–58 The recognition of a tRNA molecule by its aminoacyl-tRNA synthetase. For this tRNA (tRNAGln), specific nucleotides in both the anticodon (dark blue) and the amino acid-accepting arm (green) allow the correct tRNA to be recognized by the synthetase enzyme (yellow-green). A bound ATP molecule is yellow. (Courtesy of Tom Steitz; PDB code: 1QRS.) peptidyl-tRNA attached to C-terminus of the growing polypeptide chain new peptidyl-tRNA molecule attached to C-terminus of the growing polypeptide chain

1	Figure 6–59 The incorporation of an amino acid into a protein. A polypeptide chain grows by the stepwise addition of amino acids to its C-terminal end. The formation of each peptide bond is energetically favorable because the growing C-terminus has been activated by the covalent attachment of a tRNA molecule. The peptidyl-tRNA linkage that activates the growing end is regenerated during each addition. The amino acid side chains have been abbreviated as R1, R2, R3, and R4; as a reference point, all of the atoms in the second amino acid in the polypeptide chain are shaded gray. The figure shows the addition of the fourth amino acid (red) to the growing chain.

1	addition disrupts this high-energy covalent linkage, but immediately replaces it with an identical linkage on the most recently added amino acid (Figure 6–59). In this way, each amino acid added carries with it the activation energy for the addition of the next amino acid rather than the energy for its own addition—an example of the “head growth” type of polymerization described in Figure 2–44. The RNA Message Is Decoded in Ribosomes

1	The RNA Message Is Decoded in Ribosomes The synthesis of proteins is guided by information carried by mRNA molecules. To maintain the correct reading frame and to ensure accuracy (about 1 mistake every 10,000 amino acids), protein synthesis is performed in the ribosome, a complex catalytic machine made from more than 50 different proteins (the ribosomal proteins) and several RNA molecules, the ribosomal RNAs (rRNAs). A typical eukaryotic cell contains millions of ribosomes in its cytoplasm (Figure 6–60). The large and small ribosome subunits are assembled at the nucleolus, where newly transcribed and modified rRNAs associate with the ribosomal proteins that have been transported into the nucleus after their synthesis in the cytoplasm. These two ribosomal subunits are then exported to the cytoplasm, where they join together to synthesize proteins.

1	Figure 6–60 Ribosomes in the cytoplasm of a eukaryotic cell. This electron micrograph shows a thin section of a small region of cytoplasm. The ribosomes appear as black dots (red arrows). Some are free in the cytosol; others are attached to membranes of the endoplasmic reticulum. 400 nm (Courtesy of Daniel S. Friend.) 70S MW 2,800,000 30S (small) subunit MW 900,000 16S rRNA MW 4,200,000 60S (large) subunit 40S (small) subunit MW 1,400,000 28S rRNA 5.8S rRNA 18S rRNA MW 2,500,000 MW 1,600,000

1	MW 1,400,000 28S rRNA 5.8S rRNA 18S rRNA MW 2,500,000 MW 1,600,000 Figure 6–61 A comparison of bacterial and eukaryotic ribosomes. Despite differences in the number and size of their rRNA and protein components, both bacterial and eukaryotic ribosomes have nearly the same structure and they function similarly. Although the 18S and 28S rRNAs of the eukaryotic ribosome contain many nucleotides not present in their bacterial counterparts, these nucleotides are present as multiple insertions that form extra domains and leave the basic structure of the rRNA largely unchanged.

1	Eukaryotic and bacterial ribosomes have similar structures and functions, being composed of one large and one small subunit that fit together to form a complete ribosome with a mass of several million daltons (Figure 6–61). The small subunit provides the framework on which the tRNAs are accurately matched to the codons of the mRNA, while the large subunit catalyzes the formation of the peptide bonds that link the amino acids together into a polypeptide chain (see Figure 6–58).

1	When not actively synthesizing proteins, the two subunits of the ribosome are separate. They join together on an mRNA molecule, usually near its 5ʹ end, to initiate the synthesis of a protein. The mRNA is then pulled through the ribosome, three nucleotides at a time. As its codons enter the core of the ribosome, the mRNA nucleotide sequence is translated into an amino acid sequence using the tRNAs as adaptors to add each amino acid in the correct sequence to the growing end of the polypeptide chain. When a stop codon is encountered, the ribosome releases the finished protein, and its two subunits separate again. These subunits can then be used to start the synthesis of another protein on another mRNA molecule. Ribosomes operate with remarkable efficiency: in one second, a eukaryotic ribosome adds 2 amino acids to a polypeptide chain; the ribosomes of bacterial cells operate even faster, at a rate of about 20 amino acids per second.

1	To choreograph the many coordinated movements required for efficient translation, a ribosome contains four binding sites for RNA molecules: one is for the mRNA and three (called the A site, the P site, and the E site) are for tRNAs (Figure 6–62). A tRNA molecule is held tightly at the A and P sites only if its anticodon

1	Figure 6–62 The RNA-binding sites in the ribosome. Each ribosome has one binding site for mRNA and three binding sites for tRNA: the A, P, and E sites (short for aminoacyl-tRNA, peptidyl-tRNA, and exit, respectively). (A) A bacterial ribosome viewed with the small subunit in the front (dark green) and the large subunit in the back (light green). Both the rRNAs and the ribosomal proteins are illustrated. tRNAs are shown bound in the E site (red), the P site (orange), and the A site (yellow). Although all three tRNA sites are shown occupied here, during the process of protein synthesis not more than two of these sites are thought to contain tRNA molecules at any one time (see Figure 6–64). (B) Large and small ribosomal subunits arranged as though the ribosome in (A) were opened like a book. (C) The ribosome in (A) rotated through 90° and viewed with the large subunit on top and small subunit on the bottom. (D) Schematic representation of a ribosome [in the same orientation as (C)],

1	(C) The ribosome in (A) rotated through 90° and viewed with the large subunit on top and small subunit on the bottom. (D) Schematic representation of a ribosome [in the same orientation as (C)], which will be used in subsequent figures. (A, B, and C, adapted from M.M. Yusupov et al., Science 292:883–896, 2001. With permission from AAAS; courtesy of Albion Baucom and Harry Noller.) forms base pairs with a complementary codon (allowing for wobble) on the mRNA molecule that is threaded through the ribosome (Figure 6–63). The A and P sites are close enough together for their two tRNA molecules to be forced to form base pairs with adjacent codons on the mRNA molecule. This feature of the ribosome maintains the correct reading frame on the mRNA.

1	Once protein synthesis has been initiated, each new amino acid is added to the elongating chain in a cycle of reactions containing four major steps: tRNA binding (step 1), peptide bond formation (step 2), large subunit translocation (step 3), and small subunit translocation (step 4). As a result of the two translocation steps, the entire ribosome moves three nucleotides along the mRNA and is positioned to start the next cycle. Figure 6–64 illustrates this four-step process, beginning at a point at which three amino acids have already been linked together and there is a tRNA molecule in the P site on the ribosome, covalently joined to the C-terminal end of the short polypeptide. In step 1, a tRNA carrying the next amino acid in the chain binds to the ribosomal A site by forming base pairs with the mRNA codon positioned there, so that the P site and the A site contain adjacent bound tRNAs. In step 2, the carboxyl end of the polypeptide chain is released from the tRNA at the P site (by

1	with the mRNA codon positioned there, so that the P site and the A site contain adjacent bound tRNAs. In step 2, the carboxyl end of the polypeptide chain is released from the tRNA at the P site (by breakage of the high-energy bond between the tRNA and its amino acid) and joined to the free amino group of the amino acid linked to the tRNA at the A site, forming a new peptide bond. This central reaction of protein synthesis is catalyzed by a peptidyl transferase contained in the large ribosomal subunit. In step 3, the large subunit moves relative to the mRNA held by the small subunit, thereby shifting the acceptor stems of the two tRNAs to the E and P sites of the large subunit. In step 4, another series of conformational changes moves the small subunit and its bound mRNA exactly three nucleotides, ejecting the spent tRNA from the E site and resetting the ribosome so it is ready to receive the next aminoacyl-tRNA. Step 1 is then repeated with a new incoming aminoacyl-tRNA, and so on.

1	This four-step cycle is repeated each time an amino acid is added to the polypeptide chain, as the chain grows from its amino to its carboxyl end. The basic cycle of polypeptide elongation shown in outline in Figure 6–64 has an additional feature that makes translation especially efficient and accurate. Two elongation factors enter and leave the ribosome during each cycle, each hydrolyzing GTP to GDP and undergoing conformational changes in the process. These factors are called EF-Tu and EF-G in bacteria, and EF1 and EF2 in eukaryotes. Under some conditions in vitro, ribosomes can be forced to synthesize proteins

1	Figure 6–64 Translating an mRNA molecule. Each amino acid added to the growing end of a polypeptide chain is selected by complementary base-pairing between the anticodon on its attached tRNA molecule and the next codon on the mRNA chain. Because only one of the many types of tRNA molecules in a cell can base-pair with each codon, the codon determines the specific amino acid to be added to the growing polypeptide chain. The four-step cycle shown is repeated over and over during the synthesis of a protein. In step 1, an aminoacyl-tRNA molecule binds to a vacant A site on the ribosome. In step 2, a new peptide bond is formed. In step 3, the large subunit translocates relative to the small subunit, leaving the two tRNAs in hybrid sites: P on the large subunit and A on the small, for one; E on the large subunit and P on the small, for the other. In step 4, the small subunit translocates carrying its mRNA a distance of three nucleotides through the ribosome. This “resets” the ribosome with

1	the large subunit and P on the small, for the other. In step 4, the small subunit translocates carrying its mRNA a distance of three nucleotides through the ribosome. This “resets” the ribosome with a fully empty A site, ready for the next aminoacyl-tRNA molecule to bind. As indicated, the mRNA is translated in the 5ʹ-to-3ʹ direction, and the N-terminal end of a protein is made first, with each cycle adding one amino acid to the C-terminus of the polypeptide chain (Movie 6.7 and Movie 6.8).

1	Figure 6–63 The path of mRNA (blue) through the small ribosomal subunit. The orientation is the same as that in the right-hand panel of Figure 6–62B. (Courtesy of Harry F. Noller, based on data in G.Z. Yusupova et al., Cell 106:233–241, 2001. With permission from Elsevier.) Figure 6–65 Detailed view of the translation cycle. The outline of translation presented in Figure 6–64 has been expanded to show the roles of the two elongation factors EF-Tu and EF-G, which drive translation in the forward direction. As explained in the text, EF-Tu provides opportunities for proofreading of the codon–anticodon match. In this way, incorrectly paired tRNAs are selectively rejected, and the accuracy of translation is improved. The binding of a molecule of EF-G to the ribosome and the subsequent hydrolysis of GTP lead to a rearrangement of the ribosome structure, moving the mRNA being decoded exactly three nucleotides through it (Movie 6.9).

1	without the aid of these elongation factors and GTP hydrolysis, but this synthesis is very slow, inefficient, and inaccurate. Coupling the GTP hydrolysis-driven changes in the elongation factors to transitions between different states of the ribosome speeds up protein synthesis enormously. The cycles of elongation factor association, GTP hydrolysis, and dissociation also ensure that all such changes occur in the “forward” direction, helping translation to proceed efficiently (Figure 6–65).

1	In addition to moving translation forward, EF-Tu increases its accuracy. As we discussed in Chapter 3, EF-Tu can simultaneously bind GTP and aminoacyl-tR-NAs (see Figures 3–72 and 3–73), and it is in this form that the initial codon–anticodon interaction occurs in the A site of the ribosome. Because of the free-energy change associated with base-pair formation, a correct codon–anticodon match will bind more tightly than an incorrect interaction. However, this difference in affinity is relatively modest and cannot by itself account for the high accuracy of translation.

1	To increase the accuracy of this binding reaction, the ribosome and EF-Tu work together in the following ways. First, the 16s rRNA in the small subunit of the ribosome assesses the “correctness” of the codon–anticodon match by folding around it and probing its molecular details (Figure 6–66). When a correct match is found, the rRNA closes tightly around the codon–anticodon pair, causing a conformational change in the ribosome that triggers GTP hydrolysis by EF-Tu. Only when GTP is hydrolyzed does EF-Tu release its grip on the aminoacyl-tRNA and allow it to be used in protein synthesis. Incorrect codon–anticodon matches do not readily trigger this conformational change, and these errant tRNAs mostly fall off the ribosome before they can be used in protein synthesis. Proofreading, however, does not end here.

1	After GTP is hydrolyzed and EF-Tu dissociates from the ribosome, there is a second opportunity for the ribosome to prevent an incorrect amino acid from being added to the growing chain. There is a short time delay as the amino acid carried by the tRNA moves into position on the ribosome. This time delay is shorter for correct than incorrect codon–anticodon pairs. Moreover, incorrectly matched tRNAs dissociate more rapidly than those correctly bound because their interaction with the codon is weaker. Thus, most incorrectly bound tRNA molecules (as well as a significant number of correctly bound molecules) will leave the ribosome without being used for protein synthesis. The two proofreading steps, acting in series, are largely responsible for the 99.99% accuracy of the ribosome in translating RNA into protein.

1	Even if the wrong amino acid slips through the proofreading steps just described and is incorporated onto the growing polypeptide chain, there is still one more opportunity for the ribosome to detect the error and provide a solution, albeit one that is not, strictly speaking, proofreading. An incorrect codonanticodon interaction in the P site of the ribosome (which would occur after the misincorporation) causes an increased rate of misreading in the A site. Successive rounds of amino acid misincorporation eventually lead to premature termination of the protein by release factors, which are described below. Normally, these release factors act when translation of a protein is complete; here, they act early. Although this mechanism does not correct the original error, it releases the flawed protein for degradation, ensuring that no additional peptide synthesis is wasted on it.

1	Figure 6–66 Recognition of correct codon–anticodon matches by the small-subunit rRNA of the ribosome. Shown here is the interaction between a nucleotide of the small-subunit rRNA and the first nucleotide pair of a correctly paired codon–anticodon. Similar interactions form between other nucleotides of the rRNA and the second and third positions of codon– anticodon pair. The small-subunit rRNA can form this network of hydrogen bonds only when an anticodon is correctly matched to a codon. As explained in the text, this codon–anticodon monitoring by the small-subunit rRNA increases the accuracy of protein synthesis. (From J.M. Ogle et al., Science 292:897–902, 2001. With permission from AAAS.) Many Biological Processes Overcome the Inherent Limitations of Complementary Base-Pairing

1	Many Biological Processes Overcome the Inherent Limitations of Complementary Base-Pairing We have seen in this and the previous chapter that DNA replication, repair, transcription, and translation all rely on complementary base-pairing—G with C, and A with T (or U). However, if only the difference in hydrogen bonding is considered, a correct versus incorrect match should differ in affinity only by a factor of 10to 100-fold. These processes have an accuracy much higher than can be accounted for by this difference. Although the mechanisms used to “squeeze out” additional specificity from complementary base-pairing differ from one process to the next, two principles exemplified by the ribosome appear to be general.

1	The first is induced fit. We have seen that, before an amino acid is added to a growing polypeptide chain, the ribosome folds around the codon–anticodon interaction, and only when the match is correct is this folding completed and the reaction allowed to proceed. Thus, the codon–anticodon interaction is thereby checked twice—once by the initial complementary base-pairing and a second time by the folding of the ribosome, which depends on the correctness of the match. This same principle of induced fit is seen in transcription by RNA polymerase; here, an incoming nucleoside triphosphate initially forms a base pair with the template; at this point the enzyme folds around the base pair (thereby assessing its correctness) and, in doing so, creates the active site of the enzyme. The enzyme then covalently adds the nucleotide to the growing chain. Because their geometry is “wrong,” incorrect base pairs block this induced fit, and they are therefore likely to dissociate before being

1	then covalently adds the nucleotide to the growing chain. Because their geometry is “wrong,” incorrect base pairs block this induced fit, and they are therefore likely to dissociate before being incorporated into the growing chain.

1	A second principle used to increase the specificity of complementary base-pairing is called kinetic proofreading. We have seen that after the initial codon‒anticodon pairing and conformational change of the ribosome, GTP is hydrolyzed. This creates an irreversible step and starts the clock on a time delay during which the aminoacyl-tRNA moves into the proper position for catalysis. During this delay, those incorrect codon–anticodon pairs that have somehow slipped through the induced-fit scrutiny have a higher likelihood of dissociating than correct pairs. There are two reasons for this: (1) the interaction of the wrong tRNA with the codon is weaker, and (2) the delay is longer for incorrect than correct matches.

1	In its most general form, kinetic proofreading refers to a time delay that begins with an irreversible step such as ATP or GTP hydrolysis, during which an incorrect substrate is more likely to dissociate than a correct one. In this case, kinetic proofreading thus increases the specificity of complementary base-pairing above what is possible from simple thermodynamic associations alone. The increase in specificity produced by kinetic proofreading comes at an energetic cost in the form of ATP or GTP hydrolysis. Kinetic proofreading is believed to operate in many biological processes, but its role is understood particularly well for translation. Accuracy in Translation Requires an Expenditure of Free Energy

1	Accuracy in Translation Requires an Expenditure of Free Energy Translation by the ribosome is a compromise between the opposing constraints of accuracy and speed. We have seen, for example, that the accuracy of translation (1 mistake per 104 amino acids joined) requires time delays each time a new amino acid is added to a growing polypeptide chain, producing an overall speed of translation of 20 amino acids incorporated per second in bacteria. Mutant bacteria with a specific alteration in the small ribosomal subunit have longer delays and translate mRNA into protein with an accuracy considerably higher than this; however, protein synthesis is so slow in these mutants that the bacteria are barely able to survive.

1	We have also seen that attaining the observed accuracy of protein synthesis requires the expenditure of a great deal of free energy; this is expected, since, as discussed in Chapter 2, there is a price to be paid for any increase in order in the cell. In most cells, protein synthesis consumes more energy than any other biosynthetic process. At least four high-energy phosphate bonds are split to make each new peptide bond: two are consumed in charging a tRNA molecule with an amino acid (see Figure 6–54), and two more drive steps in the cycle of reactions occurring on the ribosome during protein synthesis itself (see Figure 6–65). In addition, extra energy is consumed each time that an incorrect amino acid linkage is hydrolyzed by a tRNA synthetase (see Figure 6–57) and each time that an incorrect tRNA enters the ribosome, triggers GTP hydrolysis, and is rejected (see Figure 6–65). To be effective, any proofreading mechanism must also allow an appreciable fraction of correct

1	that an incorrect tRNA enters the ribosome, triggers GTP hydrolysis, and is rejected (see Figure 6–65). To be effective, any proofreading mechanism must also allow an appreciable fraction of correct interactions to be removed; for this reason, proofreading is even more costly in energy than it might at first seem.

1	The Ribosome Is a Ribozyme The ribosome is a large complex composed of two-thirds RNA and one-third protein. The determination, in 2000, of the entire three-dimensional conformation of its large and small subunits is a major triumph of modern structural biology. The findings confirm earlier evidence that rRNAs—and not proteins—are responsible for the ribosome’s overall structure, its ability to position tRNAs on the mRNA, and its catalytic activity in forming covalent peptide bonds. The ribosomal RNAs are folded into highly compact, precise three-dimensional structures that form the compact core of the ribosome and determine its overall shape (Figure 6–67).

1	In marked contrast to the central positions of the rRNAs, the ribosomal proteins are generally located on the surface and fill in the gaps and crevices of the folded RNA (Figure 6–68). Some of these proteins send out extended regions of polypeptide chain that penetrate short distances into holes in the RNA core (Figure 6–69). The main role of the ribosomal proteins seems to be to stabilize the

1	Figure 6–67 Structure of the rRNAs in the large subunit of a bacterial ribosome, as determined by x-ray crystallography. (A) Three-dimensional conformations of the large-subunit rRNAs (5S and 23S) as they appear in the ribosome. One of the protein subunits of the ribosome (L1) is also shown as a reference point, since it forms a characteristic protrusion on the ribosome. (B) Schematic diagram of the secondary structure of the 23S rRNA, showing the extensive network of base-pairing. The structure has been divided into six “domains” whose colors correspond to those in (A). The secondary-structure diagram is highly schematized to represent as much of the structure as possible in two dimensions. To do this, several discontinuities in the RNA chain have been introduced, although in reality the 23S rRNA is a single RNA molecule. For example, the base of Domain III is continuous with the base of Domain IV even though a gap appears in the diagram. (Adapted from N. Ban et al., Science

1	the 23S rRNA is a single RNA molecule. For example, the base of Domain III is continuous with the base of Domain IV even though a gap appears in the diagram. (Adapted from N. Ban et al., Science 289:905–920, 2000. With permission from AAAS.)

1	RNA core, while permitting the changes in rRNA conformation that are necessary for this RNA to catalyze efficient protein synthesis. The proteins also aid in the initial assembly of the rRNAs that make up the core of the ribosome.

1	Not only are the A, P, and E binding sites for tRNAs formed primarily by ribosomal RNAs, but the catalytic site for peptide bond formation is also formed by RNA, as the nearest amino acid is located more than 1.8 nm away. This discovery came as a surprise to biologists because, unlike proteins, RNA does not contain easily ionizable functional groups that can be used to catalyze sophisticated reactions like peptide bond formation. Moreover, metal ions, which are often used by RNA molecules to catalyze chemical reactions (as discussed later in the chapter), were not observed at the active site of the ribosome. Instead, it is believed that the 23S rRNA forms a highly structured pocket that, through a network of hydrogen bonds, precisely orients the two reactants (the growing peptide chain and an aminoacyl-tRNA) and thereby greatly accelerates their covalent joining. An additional surprise came from the discovery that the tRNA in the P site contributes an important OH group to the active

1	an aminoacyl-tRNA) and thereby greatly accelerates their covalent joining. An additional surprise came from the discovery that the tRNA in the P site contributes an important OH group to the active site and participates directly in the catalysis. This mechanism may ensure that catalysis occurs only when the P site tRNA is properly positioned in the ribosome.

1	RNA molecules that possess catalytic activity are known as ribozymes. We saw earlier in this chapter that some ribozymes function in self-splicing reactions. In the final section of this chapter, we consider what the ability of RNA molecules to function as catalysts might mean for the early evolution of living cells. For now, we merely note that there is good reason to suspect that RNA rather than protein molecules served as the first catalysts for living cells. If so, the ribosome, with its RNA core, may be a relic of an earlier time in life’s history—when protein synthesis evolved in cells that were run almost entirely by ribozymes. Nucleotide Sequences in mRNA Signal Where to Start Protein Synthesis

1	Nucleotide Sequences in mRNA Signal Where to Start Protein Synthesis The initiation and termination of translation share features of the translation elongation cycle described above. The site at which protein synthesis begins on the mRNA is especially crucial, since it sets the reading frame for the whole length of the message. An error of one nucleotide either way at this stage would cause every subsequent codon in the message to be misread, resulting in a nonfunctional protein with a garbled sequence of amino acids. The initiation step is also important because for most genes it is the last point at which the cell can decide whether the mRNA is to be translated to produce a protein. The rate of this step is thus one determinant of the rate at which any particular protein will be synthesized. We shall see in Chapter 7 how regulation of this step occurs.

1	The translation of an mRNA begins with the codon AUG, and a special tRNA is required to start translation. This initiator tRNA always carries the amino acid methionine (in bacteria, a modified form of methionine—formylmethionine—is used), with the result that all newly made proteins have methionine as the first amino acid at their N-terminus, the end of a protein that is synthesized first. (This methionine is usually removed later by a specific protease.) The initiator tRNA is specially recognized by initiation factors because it has a nucleotide sequence distinct from that of the tRNA that normally carries methionine.

1	In eukaryotes, the initiator tRNA–methionine complex (Met–tRNAi) is first loaded into the small ribosomal subunit along with additional proteins called eukaryotic initiation factors, or eIFs. Of all the aminoacyl-tRNAs in the cell, only the methionine-charged initiator tRNA is capable of tightly binding the small ribosome subunit without the complete ribosome being present, and unlike other tRNAs it binds directly to the P site (Figure 6–70). Next, the small ribosomal subunit binds to the 5ʹ end of an mRNA molecule, which is recognized by virtue of its 5ʹ cap that has previously bound two initiation factors, eIF4E and eIF4G (see Figure 6–38). The small ribosomal subunit then moves forward (5ʹ to 3ʹ) along the mRNA, searching for the first AUG; additional initiation factors that act as ATP-powered

1	Figure 6–68 location of the protein components of the bacterial large ribosomal subunit. The rRNAs (5S and 23S) are shown in blue and the proteins of the large subunit in green. This view is toward the outside of the ribosome; the interface with the small subunit is on the opposite face. (PDB code: 1FFK.) Figure 6–69 Structure of the l15 protein in the large subunit of the bacterial ribosome. The globular domain of the protein lies on the surface of the ribosome and an extended region penetrates deeply into the RNA core of the ribosome. The L15 protein is shown in green and a portion of the ribosomal RNA core is shown in blue. (From D. Klein, P.B. Moore and T.A. Steitz, J. Mol. Biol. 340:141–177, 2004. With permission from Academic Press. PDB code: 1S72.)

1	T.A. Steitz, J. Mol. Biol. 340:141–177, 2004. With permission from Academic Press. PDB code: 1S72.) Figure 6–70 The initiation of protein synthesis in eukaryotes. Only three eIF2 of the many translation initiation factors required for this process are shown. Efficient translation initiation also requires the poly-A tail of the mRNA bound by poly-A-binding proteins, which, in turn, interact with eIF4G (see Figure 6–38). In this way, the translation apparatus ascertains that both ends of the mRNA are intact before initiating protein synthesis. Although only one GTP-hydrolysis event is shown in the figure, a second is known to occur subunit with just before the large and small ribosomal subunits join. In the last two steps initiator tRNA shown in the figure, the ribosome has begun the standard elongation cycle, bound to P site depicted in Figure 6–64. helicases facilitate this movement. In 90% of mRNAs, translation begins at the first

1	helicases facilitate this movement. In 90% of mRNAs, translation begins at the first AUG encountered by the small subunit. At this point, the initiation factors dis sociate, allowing the large ribosomal subunit to assemble with the complex and complete the ribosome. The initiator tRNA remains at the P site, leaving the A site vacant. Protein synthesis is therefore ready to begin (see Figure 6–70). The nucleotides immediately surrounding the start site in eukaryotic mRNAs influence the efficiency of AUG recognition during the above scanning process. If this recognition site differs substantially from the consensus recognition sequence (5ʹ-ACCAUGG-3ʹ), scanning ribosomal subunits will sometimes ignore the first

1	AUG codon in the mRNA and skip to the second or third AUG codon instead. Cells frequently use this phenomenon, known as “leaky scanning,” to produce two or more proteins, differing in their N-termini, from the same mRNA molecule. This mechanism allows some genes to produce the same protein with and without a signal sequence attached at its N-terminus, for example, so that the protein is directed to two different compartments in the cell. rial mRNAs have no 5ʹ caps to signal the ribosome where to begin searching for the start of translation. Instead, each bacterial mRNA contains a specific ribo- some-binding site (called the Shine–Dalgarno sequence, named after its discov-DISSOCIATE erers) that is located a few nucleotides upstream of the AUG at which translation

1	The mechanism for selecting a start codon in bacteria is different. Bacte is to begin. This nucleotide sequence, with the consensus 5ʹ-AGGAGGU-3ʹ, forms base pairs with the 16S rRNA of the small ribosomal subunit to position the initiating AUG codon in the ribosome. A set of translation initiation factors orchestrates this interaction, as well as the subsequent assembly of the large ribosomal subunit to complete the ribosome. Unlike a eukaryotic ribosome, a bacterial ribosome can readily assemble directly on a start codon that lies in the interior of an mRNA molecule, so long as a ribosome-binding site precedes it by several nucleotides. As a result, bac-AMINOACYL terial mRNAs are often polycistronic—that is, they encode several different pro teins, each of which is translated from the same mRNA molecule (Figure 6–71). In contrast, a eukaryotic mRNA generally encodes only a single protein, or more accurately, a single set of closely related proteins.

1	Stop Codons Mark the End of Translation The end of the protein-coding message is signaled by the presence of one of three stop codons (UAA, UAG, or UGA) (see Figure 6–48). These are not recognized by a tRNA and do not specify an amino acid, but instead signal to the ribosome to stop translation. Proteins known as release factors bind to any ribosome with a stop codon positioned in the A site, forcing the peptidyl transferase in the ribosome to catalyze the addition of a water molecule instead of an amino acid to the peptidyl-tRNA (Figure 6–72). This reaction frees the carboxyl end of the growing polypeptide chain from its attachment to a tRNA molecule, and since only this etc.

1	attachment normally holds the growing polypeptide to the ribosome, the com pleted protein chain is immediately released into the cytoplasm. The ribosome then releases its bound mRNA molecule and separates into the large and small subunits. These subunits can then assemble on this or another mRNA molecule to begin a new round of protein synthesis. Figure 6–71 Structure of a typical bacterial mRNA molecule. Unlike eukaryotic ribosomes, which typically require a capped 5ʹ end on the mRNA, prokaryotic ribosomes initiate translation at ribosome-binding sites (Shine–Dalgarno sequences), which can be located anywhere along an mRNA molecule. This property of their ribosomes permits bacteria to synthesize more than one type of protein from a single mRNA molecule.

1	During translation, the nascent polypeptide moves through a large, water-filled tunnel (approximately 10 nm × 1.5 nm) in the large subunit of the ribosome. The walls of this tunnel, made primarily of 23S rRNA, are a patchwork of tiny hydrophobic surfaces embedded in a more extensive hydrophilic surface. This structure is not complementary to any peptide, and thus provides a “Teflon” coating through which a polypeptide chain can easily slide. The dimensions of the tunnel suggest that nascent proteins are largely unstructured as they pass through the ribosome, although some α-helical regions of the protein can form before leaving the ribosome tunnel. As it leaves the ribosome, a newly synthesized protein must fold into its proper three-dimensional conformation to be useful to the cell. Later in this chapter we discuss how this folding occurs. First, however, we describe several additional aspects of the translation process itself.

1	The synthesis of most protein molecules takes between 20 seconds and several minutes. During this very short period, however, it is usual for multiple initiations to take place on each mRNA molecule being translated. As soon as the preceding ribosome has translated enough of the nucleotide sequence to move out of the way, the 5ʹ end of the mRNA is threaded into a new ribosome. The mRNA molecules being translated are therefore usually found in the form of polyribosomes (or polysomes): large cytoplasmic assemblies made up of several ribosomes spaced as close as 80 nucleotides apart along a single mRNA molecule (Figure 6–73). These multiple initiations allow the cell to make many more protein molecules in a given time than would be possible if each protein had to be completed before the next could start.

1	Both bacteria and eukaryotes use polysomes, and both employ additional strategies to speed up the overall rate of protein synthesis. Because bacterial mRNA does not need to be processed and is accessible to ribosomes while it is being made, ribosomes can attach to the free end of a bacterial mRNA molecule and start translating it even before the transcription of that RNA is complete, following closely behind the RNA polymerase as it moves along DNA. In eukaryotes, as we have seen, the 5ʹand 3ʹ ends of the mRNA interact (see Figure 6–73A); therefore, as soon as a ribosome dissociates, its two subunits are in an optimal position to reinitiate translation on the same mRNA molecule. There Are Minor Variations in the Standard Genetic Code

1	There Are Minor Variations in the Standard Genetic Code As discussed in Chapter 1, the genetic code (shown in Figure 6–48) applies to all three major branches of life, providing important evidence for the common ancestry of all life on Earth. Although rare, there are exceptions to this code. For example, Candida albicans, the most prevalent human fungal pathogen, translates the codon CUG as serine, whereas nearly all other organisms translate it as leucine. Mitochondria (which have their own genomes and encode much of their translational apparatus) often deviate from the standard code. For example, in mammalian mitochondria AUA is translated as methionine, whereas in the

1	Figure 6–72 The final phase of protein synthesis. The binding of a release factor to an A site bearing a stop codon terminates translation. The completed polypeptide is released and, in a series of reactions that requires additional proteins and GTP hydrolysis (not shown), the ribosome dissociates into its two separate subunits. messenger RNA Figure 6–73 A polyribosome. (A) Schematic drawing showing how a series of ribosomes can simultaneously translate the same eukaryotic mRNA molecule. (B) Electron micrograph of a polyribosome from a eukaryotic cell (Movie 6.10). (B, courtesy of John Heuser.) cytosol of the cell it is translated as isoleucine (see Table 14–3, p. 805). This type of deviation in the genetic code is “hardwired” into the organisms or the organelles in which it occurs.

1	A different type of variation, sometimes called translation recoding, occurs in many cells. In this case, other nucleotide sequence information present in an mRNA can change the meaning of the genetic code at a particular site in the mRNA molecule. The standard code allows cells to manufacture proteins using only 20 amino acids. However, bacteria, archaea, and eukaryotes have available to them a twenty-first amino acid that can be incorporated directly into a growing polypeptide chain through translation recoding. Selenocysteine, which is essential for the efficient function of a variety of enzymes, contains a selenium atom in place of the sulfur atom of cysteine. Selenocysteine is enzymatically produced from a serine attached to a special tRNA molecule that base-pairs with the UGA codon, a codon normally used to signal a translation stop. The mRNAs for proteins in which selenocysteine is to be inserted at a UGA codon carry an additional nearby nucleotide sequence in the mRNA that

1	a codon normally used to signal a translation stop. The mRNAs for proteins in which selenocysteine is to be inserted at a UGA codon carry an additional nearby nucleotide sequence in the mRNA that triggers this recoding event (Figure 6–74).

1	added to growing seryl-tRNA converted to selenocysteine selenocysteine tRNA signal that the preceding UGA encodes selenocysteine Figure 6–74 Incorporation of selenocysteine into a growing polypeptide chain. A specialized tRNA is charged with serine by the normal seryltRNA synthetase, and the serine is subsequently converted enzymatically to selenocysteine. A specific RNA structure in the mRNA (a stem and loop structure with a particular nucleotide sequence) signals that selenocysteine is to be inserted at the neighboring UGA codon. As indicated, this event requires the participation of a selenocysteine-specific translation factor. After the addition of selenocysteine, translation continues until a conventional stop codon is encountered. Inhibitors of Prokaryotic Protein Synthesis Are Useful as Antibiotics

1	Many of the most effective antibiotics used in modern medicine are compounds made by fungi that inhibit bacterial protein synthesis. Fungi and bacteria compete for many of the same environmental niches, and millions of years of coevolution have resulted in fungi producing potent bacterial inhibitors. Some of these drugs exploit the structural and functional differences between bacterial and eukaryotic ribosomes so as to interfere preferentially with the function of bacterial ribosomes. Thus, humans can take high dosages of some of these compounds without undue toxicity. Many antibiotics lodge in pockets in the ribosomal RNAs and simply interfere with the smooth operation of the ribosome; others block specific parts of the ribosome such as the exit channel (Figure 6–75). Table 6–4 lists some common antibiotics of this kind along with several other inhibitors of protein synthesis, some of which act on eukaryotic cells and therefore cannot be used as antibiotics.

1	Because they block specific steps in the processes that lead from DNA to protein, many of the compounds listed in Table 6–4 are useful for cell biological studies. Among the most commonly used drugs in such investigations are chloramphenicol, cycloheximide, and puromycin, all of which specifically inhibit protein synthesis. In a eukaryotic cell, for example, chloramphenicol inhibits protein synthesis on ribosomes only in mitochondria (and in chloroplasts in plants), presumably reflecting the prokaryotic origins of these organelles (discussed in Chapter 14). Cycloheximide, in contrast, affects only ribosomes in the cytosol. Puromycin is especially interesting because it is a structural analog of a tRNA molecule linked to an amino acid and is therefore another example of molecular mimicry; the ribosome mistakes it for an authentic amino acid and covalently incorporates it at the C-terminus of the growing peptide chain, thereby causing the premature termination and release of the

1	the ribosome mistakes it for an authentic amino acid and covalently incorporates it at the C-terminus of the growing peptide chain, thereby causing the premature termination and release of the polypeptide. As might be expected, puromycin inhibits protein synthesis in both prokaryotes and eukaryotes.

1	Quality Control Mechanisms Act to Prevent Translation of Damaged mRNAs In eukaryotes, mRNA production involves both transcription and a series of elaborate RNA processing steps; as we have seen, these take place in the nucleus, segregated from ribosomes, and only when the processing is complete are the mRNAs transported to the cytosol to be translated (see Figure 6–38). However, this scheme is not foolproof, and some incorrectly processed mRNAs are inadvertently sent to the cytosol. In addition, mRNAs that were flawless when they left the nucleus can become broken or otherwise damaged in the cytosol. The danger of

1	Figure 6–75 binding sites for antibiotics on the bacterial ribosome. The small (left) and large (right) subunits of the ribosome are arranged as though the ribosome has been opened like a book. Antibiotic binding sites are marked with colored spheres, and the bound tRNA molecules are shown in purple (see Figure 6–62). Most of the antibiotics shown bind directly to pockets formed by the ribosomal RNA molecules. Hygromycin B induces errors in translation, spectinomycin blocks the translocation of the peptidyl-tRNA from the A site to the P site, and streptogramin B prevents elongation of nascent peptides. Table 6–4 lists the inhibitory mechanisms of the other antibiotics shown in the figure. (Adapted from J. Poehlsgaard and S. Douthwaite, Nat. Rev. Microbiol. 3:870–881, 2005. With permission from Macmillan Publishers Ltd.) translating damaged or incompletely processed mRNAs (which would produce truncated or otherwise aberrant proteins) is apparently so great that the cell has several

1	from Macmillan Publishers Ltd.) translating damaged or incompletely processed mRNAs (which would produce truncated or otherwise aberrant proteins) is apparently so great that the cell has several backup measures to prevent this from happening. To avoid translating broken mRNAs, for example, the 5ʹ cap and the poly-A tail are both recognized by the translation-initiation machinery before translation begins (see Figure 6–70).

1	The most powerful mRNA surveillance system, called nonsense-mediated mRNA decay, eliminates defective mRNAs before they move away from the nucleus. This mechanism is brought into play when the cell determines that an mRNA molecule has a nonsense (stop) codon (UAA, UAG, or UGA) in the “wrong” place. This situation is likely to arise in an mRNA molecule that has been improperly spliced, because aberrant splicing will usually result in the random introduction of a nonsense codon into the reading frame of the mRNA—especially in organisms, such as humans, that have a large average intron size (see Figure 6–31B).

1	The nonsense-mediated mRNA decay mechanism begins as an mRNA molecule is being transported from the nucleus to the cytosol. As its 5ʹ end emerges from a nuclear pore, the mRNA is met by a ribosome, which begins to translate it. As translation proceeds, the exon junction complexes (EJCs) that are bound to the mRNA at each splice site are displaced by the moving ribosome. The normal stop codon will lie within the last exon, so by the time the ribosome reaches it and stalls, no more EJCs will be bound to the mRNA. In this case, the mRNA “passes inspection” and is released to the cytosol where it can be translated in earnest (Figure 6–76). However, if the ribosome reaches a stop codon earlier, when EJCs remain bound, the mRNA molecule is rapidly degraded. In this way, the first round of translation allows the cell to test the fitness of each mRNA molecule as it exits the nucleus.

1	Nonsense-mediated decay may have been especially important in evolution, allowing eukaryotic cells to more easily explore new genes formed by DNA rearrangements, mutations, or alternative patterns of splicing—by selecting only those mRNAs for translation that can produce a full-length protein. Nonsense-mediated decay is also important in cells of the developing immune system, where the extensive DNA rearrangements that occur (see Figure 24–28) often generate mRNA SURVIVES, EFFICIENT TRANSLATION Upf TRIGGERS mRNA DEGRADATION

1	Figure 6–76 Nonsense-mediated mRNA decay. As shown on the right, the failure to correctly splice a pre-mRNA often introduces a premature stop codon into the reading frame for the protein. These abnormal mRNAs are destroyed by the nonsense-mediated decay mechanism. To activate this mechanism, an mRNA molecule, bearing exon junction complexes (EJCs) to mark successfully completed splices, is first met by a ribosome that performs a “test” round of translation. As the mRNA passes through the tight channel of the ribosome, the EJCs are stripped off, and successful mRNAs are released to undergo multiple rounds of translation (left side). However, if an in-frame stop codon is encountered before the final EJC is reached (right side), the mRNA undergoes nonsense-mediated decay, which is triggered by the Upf proteins (green) that bind to each EJC. Note that this mechanism ensures that nonsense-mediated decay is triggered only when the premature stop codon is in the same reading frame as that of

1	the Upf proteins (green) that bind to each EJC. Note that this mechanism ensures that nonsense-mediated decay is triggered only when the premature stop codon is in the same reading frame as that of the normal protein. (Adapted from J. Lykke-Andersen et al., Cell 103:1121–1131, 2000. With permission from Elsevier.) premature termination codons. The surveillance system degrades the mRNAs produced from such rearranged genes, thereby avoiding the potential toxic effects of truncated proteins.

1	The nonsense-mediated surveillance pathway also plays an important role in mitigating the symptoms of many inherited human diseases. As we have seen, inherited diseases are usually caused by mutations that spoil the function of a key protein, such as hemoglobin or one of the blood-clotting factors. Approximately one-third of all genetic disorders in humans result from nonsense mutations or mutations (such as frameshift mutations or splice-site mutations) that place nonsense mutations into the gene’s reading frame. In individuals that carry one mutant and one functional gene, nonsense-mediated decay eliminates the aberrant mRNA and thereby prevents a potentially toxic protein from being made. Without this safeguard, individuals with one functional and one mutant “disease gene” would likely suffer much more severe symptoms. Some Proteins Begin to Fold While Still Being Synthesized

1	Some Proteins Begin to Fold While Still Being Synthesized The process of gene expression is not over when the genetic code has been used to create the sequence of amino acids that constitutes a protein. To be useful to the cell, this new polypeptide chain must fold up into its unique three-dimensional conformation, bind any small-molecule cofactors required for its activity, be appropriately modified by protein kinases or other protein-modifying enzymes, and assemble correctly with the other protein subunits with which it functions (Figure 6–77). The information needed for all of the steps listed above is ultimately contained in the sequence of amino acids that the ribosome produces when it translates an mRNA molecule into a polypeptide chain. As discussed in Chapter 3, when a

1	Figure 6–77 Steps in the creation of a functional protein. As indicated, translation of an mRNA sequence into an amino acid sequence on the ribosome is not the end of the process of forming a protein. To function, the completed polypeptide chain must fold correctly into its three-dimensional conformation, bind any cofactors required, and assemble with its partner protein chains, if any. Noncovalent bond formation drives these changes. As indicated, many proteins also require covalent modifications of selected amino acids. Although the most frequent modifications are protein glycosylation and protein phosphorylation, over 200 different types of covalent modifications are known (see pp. 165–166).

1	protein folds into a compact structure, it buries most of its hydrophobic residues in an interior core. In addition, large numbers of noncovalent interactions form between various parts of the molecule. It is the sum of all of these energetically favorable arrangements that determines the final folding pattern of the polypeptide chain—as the conformation of lowest free energy (see pp. 114–115).

1	Through many millions of years of evolution, the amino acid sequence of each protein has been selected not only for the conformation that it adopts but also for an ability to fold rapidly. For some proteins, this folding begins immediately, as the protein chain emerges from the ribosome, starting from the N-terminal end. In these cases, as each protein domain emerges from the ribosome, within a few seconds it forms a compact structure that contains most of the final secondary features (α helices and β sheets) aligned in roughly the right conformation (Figure 6–78). For some protein domains, this unusually dynamic and flexible state, called a molten globule, is the starting point for a relatively slow process in which many side-chain adjustments occur that eventually form the correct tertiary structure. It takes several minutes to synthesize a protein of average size, and for some proteins much of the folding process is complete by the time the ribosome releases the C-terminal end of a

1	structure. It takes several minutes to synthesize a protein of average size, and for some proteins much of the folding process is complete by the time the ribosome releases the C-terminal end of a protein (Figure 6–79).

1	Molecular Chaperones Help Guide the Folding of Most Proteins Most proteins probably do not fold correctly during their synthesis and require a special class of proteins called molecular chaperones to do so. Molecular chaperones are useful for cells because there are many different folding paths available to an unfolded or partially folded protein. Without chaperones, some of these pathways would not lead to the correctly folded (and most stable) form because the protein would become “kinetically trapped” in structures that are off-pathway. Some of these off-pathway configurations would aggregate and be left as irreversible dead ends of nonfunctional (and potentially dangerous) structures. covalent modifcation by glycosylation, phosphorylation, acetylation etc. binding to other protein subunits

1	covalent modifcation by glycosylation, phosphorylation, acetylation etc. binding to other protein subunits Figure 6–78 The structure of a molten globule. (A) A molten globule form of cytochrome b562 is more open and less highly ordered than the final folded form of the protein, shown in (B). Note that the molten globule contains most of the secondary structure of the final form, although the ends of the α helices are unraveled and one of the helices is only partly formed. (Courtesy of Joshua Wand, from Y. Feng et al., Nat. Struct. Biol. 1:30– 35, 1994. With permission from Macmillan Publishers Ltd.) Figure 6–79 Co-translational protein folding. A growing polypeptide chain is shown acquiring its secondary and tertiary structure as it emerges from a ribosome. The N-terminal domain folds first, while the C-terminal domain is still being synthesized. This protein has not achieved its final conformation at the time it is released from the ribosome. (Modified from A.N. Fedorov and

1	T.O. Baldwin, J. Biol. Chem. 272:32715–32718, 1997.) Molecular chaperones specifically recognize incorrect, off-pathway configurations by their exposure of hydrophobic surfaces, which in correctly folded proteins are typically buried in the interior. The binding of these exposed hydrophobic surfaces to each other is what causes off-pathway conformations to irreversibly aggregate. We saw in Chapter 3 that in some cases of inherited human diseases, aggregates do form and can cause severe symptoms and even death. Chaperones prevent this from happening in normal proteins by binding to the exposed hydrophobic surfaces using hydrophobic surfaces of their own. As we shall see shortly, there are several types of chaperones; once bound to an incorrectly folded protein, they ultimately release it in a way that gives the protein another chance to fold correctly. Cells Utilize Several Types of Chaperones

1	Cells Utilize Several Types of Chaperones Many molecular chaperones are called heat-shock proteins (designated hsp), because they are synthesized in dramatically increased amounts after a brief exposure of cells to an elevated temperature (for example, 42°C for cells that normally live at 37°C). This reflects the operation of a feedback system that responds to an increase in misfolded proteins (such as those produced by elevated temperatures) by boosting the synthesis of the chaperones that help these proteins refold. There are several major families of molecular chaperones, including the hsp60 and hsp70 proteins. Different members of these families function in different organelles. Thus, as discussed in Chapter 12, mitochondria contain their own hsp60 and hsp70 molecules that are distinct from those that function in the cytosol; and a special hsp70 (called BIP) helps to fold proteins in the endoplasmic reticulum.

1	The hsp60 and hsp70 proteins each work with their own small set of associated proteins when they help other proteins to fold. These hsps share an affinity for the exposed hydrophobic patches on incompletely folded proteins, and they hydrolyze ATP, often binding and releasing their protein substrate with each cycle of ATP hydrolysis. In other respects, the two types of hsp proteins function differently. The hsp70 machinery acts early in the life of many proteins (often before the protein leaves the ribosome), with each monomer of hsp70 binding to a string of about four or five hydrophobic amino acids (Figure 6–80). On binding ATP, hsp70 releases the protein into solution allowing it a chance to re-fold. In contrast, hsp60-like proteins form a large barrel-shaped structure that acts after a protein has been fully synthesized. This type of chaperone, sometimes called a chaperonin, forms an “isolation chamber” for the folding process (Figure 6–81).

1	To enter a chamber, a substrate protein is first captured via the hydrophobic entrance to the chamber. The protein is then released into the interior of the chamber, which is lined with hydrophilic surfaces, and the chamber is sealed with a lid, a step requiring ATP. Here, the substrate is allowed to fold into its final conformation in isolation, where there are no other proteins with which to aggregate. When ATP is hydrolyzed, the lid pops off, and the substrate protein, whether folded or not, is released from the chamber.

1	The chaperones shown in Figures 6–80 and 6–81 often need many cycles of ATP hydrolysis to fold a single polypeptide chain correctly. This energy is used to perform mechanical movements of the hsp60 and hsp70 “machines,” converting them from binding forms to releasing forms. Just as we saw for transcription, splicing, and translation, the expenditure of free energy can be used by cells to improve the accuracy of a biological process. In the case of protein folding, ATP hydrolysis allows chaperones to recognize a wide variety of misfolded structures, to halt any further misfolding, and to recommence the folding of a protein in an orderly way.

1	Figure 6–80 The hsp70 family of molecular chaperones. These proteins act early, recognizing a small stretch of hydrophobic amino acids on a protein’s surface. Aided by a set of smaller hsp40 proteins (not shown), ATP-bound hsp70 molecules grasp their target protein and then hydrolyze ATP to ADP, undergoing conformational changes that cause the hsp70 molecules to associate even more tightly with the target. After the hsp40 dissociates, the rapid rebinding of ATP induces the dissociation of the hsp70 protein after ADP release. Repeated cycles of hsp binding and release help the target protein to refold.

1	Figure 6–81 The structure and function of the hsp60 family of molecular chaperones. (A) A misfolded protein is initially captured by hydrophobic interactions with the exposed surface of the opening. The initial binding often helps to unfold a misfolded protein. The subsequent binding of ATP and a cap releases the substrate protein into an enclosed space, where it has a new opportunity to fold. After about 10 seconds, ATP hydrolysis occurs, weakening the binding of the cap. Subsequent binding of additional ATP molecules ejects the cap, and the protein is released. As indicated, only half of the symmetric barrel operates on a client protein at any one time. This type of molecular chaperone is also known as a chaperonin; it is designated as hsp60 in mitochondria, TCP1 in the cytosol of vertebrate cells, and GroEL in bacteria. (B) The structure of GroEL bound to its GroES cap, as determined by x-ray crystallography. On the left is shown the outside of the barrel-like structure, and on the

1	cells, and GroEL in bacteria. (B) The structure of GroEL bound to its GroES cap, as determined by x-ray crystallography. On the left is shown the outside of the barrel-like structure, and on the right a cross section through its center. (B, adapted from B. Bukau and A.L. Horwich, Cell 92:351–366, 1998. With permission from Elsevier.) 10 nm

1	Although our discussion focuses on only two types of chaperones, the cell has a variety of others. The enormous diversity of proteins in cells presumably requires a wide range of chaperones with versatile surveillance and correction capabilities. If radioactive amino acids are added to cells for a brief period, the newly synthesized proteins can be followed as they mature into their final functional forms. This type of experiment demonstrates that the hsp70 proteins act first, beginning when a protein is still being synthesized on a ribosome, and the hsp60-like proteins act only later to help fold completed proteins. We have seen that the cell distinguishes misfolded proteins, which require additional rounds of ATP-catalyzed refolding, from those with correct structures through the recognition of hydrophobic surfaces.

1	Usually, if a protein has a sizable exposed patch of hydrophobic amino acids on its surface, it is abnormal: it has either failed to fold correctly after leaving the ribosome, suffered an accident that partly unfolded it at a later time, or failed to find its normal partner subunit in a larger protein complex. Such a protein is not merely useless to the cell, it can be dangerous. Proteins that rapidly fold correctly on their own do not display such patterns and generally bypass the chaperones. For the others, the chaperones can carry out “protein repair” by giving them additional chances to fold while, at the same time, preventing their aggregation.

1	Figure 6–82 outlines all of the quality-control choices that a cell makes for a difficult-to-fold, newly synthesized protein. As indicated, when attempts to refold a protein fail, an additional mechanism is called into play that completely destroys the protein by proteolysis. This proteolytic pathway begins with the recognition of an abnormal hydrophobic patch on a protein’s surface, and it ends with the delivery of the entire protein to a protein-destruction machine, a complex protease known as the proteasome. As described next, this process depends on an elaborate protein-marking system that also carries out other central functions in the cell by destroying selected normal proteins. The Proteasome Is a Compartmentalized Protease with Sequestered Active Sites

1	The Proteasome Is a Compartmentalized Protease with Sequestered Active Sites The proteolytic machinery and the chaperones compete with one another to recognize a misfolded protein. If a newly synthesized protein folds rapidly, at most only a small fraction of it is degraded. In contrast, a slowly folding protein is vulnerable to the proteolytic machinery for a longer time, and many more of its molecules may be destroyed before the remainder attain the proper folded state. Due to mutations or to errors in transcription, RNA splicing, and translation, some proteins never fold properly, and it is particularly important that the cell destroy these potentially harmful proteins. The apparatus that deliberately destroys aberrant proteins is the proteasome, an abundant ATP-dependent protease that constitutes nearly 1% of cell protein.

1	Figure 6–82 The processes that monitor protein quality following protein synthesis. A newly synthesized protein sometimes folds correctly and assembles on its own with its partner proteins, in which case the quality control mechanisms leave it alone. Incompletely folded proteins are helped to properly fold by molecular chaperones: first by a family of hsp70 proteins, and then, in some cases, by hsp60-like proteins. For both types of chaperones, the substrate proteins are recognized by an abnormally exposed patch of hydrophobic amino acids on their surface. These “protein-rescue” processes compete with another mechanism that, upon recognizing an abnormally exposed hydrophobic patch, marks the protein for destruction by the proteasome. The combined activity of all of these processes is needed to prevent massive protein aggregation in a cell, which can occur when many hydrophobic regions on proteins clump together nonspecifically.

1	Figure 6–83 The proteasome. (A) A cutaway view of the structure of the central 20S cylinder, as determined by x-ray crystallography, with the active sites of the proteases indicated by red dots. (B) The entire proteasome, in which the central cylinder (yellow) is supplemented by a 19S cap (blue) at each end. The complex cap (also called the regulatory particle) selectively binds proteins that have been marked by ubiquitin for destruction; it then uses ATP hydrolysis to unfold their polypeptide chains and feed them through a narrow channel (see Figure 6–85) into the inner chamber of the 20S cylinder for digestion to short peptides. (B, from W. Baumeister et al., Cell 92:367–380, 1998. With permission from Elsevier.)

1	W. Baumeister et al., Cell 92:367–380, 1998. With permission from Elsevier.) Present in many copies dispersed throughout the cytosol and the nucleus, the proteasome also destroys aberrant proteins that have entered the endoplasmic reticulum (ER). In the latter case, an ER-based surveillance system detects proteins that have failed either to fold or to be assembled properly after they enter the ER, and retrotranslocates them back to the cytosol for degradation by the proteasome (discussed in Chapter 12).

1	Each proteasome consists of a central hollow cylinder (the 20S core proteasome) formed from multiple protein subunits that assemble as a stack of four heptameric rings (Figure 6–83). Some of the subunits are proteases whose active sites face the cylinder’s inner chamber, thus preventing them from running rampant through the cell. Each end of the cylinder is normally associated with a large protein complex (the 19S cap) that contains a six-subunit protein ring through which target proteins are threaded into the proteasome core, where they are degraded (Figure 6–84). The threading reaction, driven by ATP hydrolysis, unfolds the target proteins as they move through the cap, exposing them to the proteases lining the proteasome core (Figure 6–85). The proteins that make up the ring structure in the proteasome cap belong to a large class of protein “unfoldases” known as AAA proteins. Many of them function as hexamers, and they share mechanistic features with the ATP-dependent DNA helicases

1	the proteasome cap belong to a large class of protein “unfoldases” known as AAA proteins. Many of them function as hexamers, and they share mechanistic features with the ATP-dependent DNA helicases that unwind DNA (see Figure 5–14).

1	(A) (B) ubiquitin target protein with receptor polyubiquitin chain

1	Figure 6–84 Processive protein digestion by the proteasome. (A) The proteasome cap recognizes proteins marked by a polyubiquitin chain (see Figure 3–70), and subsequently translocates them into the proteasome core, where they are digested. At an early stage, the ubiquitin is cleaved from the substrate protein and is recycled. Translocation into the core of the proteasome is mediated by a ring of ATPases that unfold the substrate protein as it is threaded through the ring and into the proteasome core. This unfoldase ring is depicted in Figure 6–85). (B) Detailed structure of the proteasome cap. The cap includes a ubiquitin receptor, which holds a ubiquitylated protein in place while it begins to be pulled into the proteasome core, and a ubiquitin hydrolase, which cleaves ubiquitin from the doomed protein. (A, from S. Prakash and A. Matouschek, Trends Biochem. Sci. 29:593–600, 2004. With permission from Elsevier. B, adapted from G.C. Lander et al., Nature 482:186–191, 2012.)

1	A crucial property of the proteasome, and one reason for the complexity of its design, is the processivity of its mechanism: in contrast to a “simple” protease that cleaves a substrate’s polypeptide chain just once before dissociating, the proteasome keeps the entire substrate bound until all of it is converted into short peptides.

1	One would expect that a machine as efficient as the proteasome would be tightly regulated; in particular, it must be able to distinguish abnormal proteins from those that are properly folded. The 19S cap of the proteasome acts as a gate at the entrance to the inner proteolytic core, and only those proteins marked for destruction are threaded through the cap. The destruction “mark” is the covalent attachment of the small protein ubiquitin. As we saw in Chapter 3, ubiquitin modification of proteins is used for many purposes in the cell. The particular type of ubiquitin linkage that concerns us here is a chain of ubiquitin molecules linked together at lysine 48 (see Figure 3–69); this is the distinguishing feature of the ubiquitin tag that marks a protein for destruction in the proteasome.

1	A special set of E3 molecules (see Figure 3–70B) is responsible for the ubiquitylation of denatured or otherwise misfolded proteins, as well as proteins containing oxidized or other abnormal amino acids. Abnormal proteins tend to display on their surface hydrophobic amino acid sequences or conformational motifs that are recognized as degradation signals by these E3 molecules; these sequences are buried and therefore inaccessible in the normal, properly folded version. However, a proteolytic pathway that recognizes and destroys abnormal proteins must be able to distinguish completed proteins that have “wrong” conformations from the many growing polypeptides on ribosomes (as well as polypeptides just released from ribosomes) that have not yet achieved their normal folded conformation. This is not a trivial problem; in the course of carrying out its main job, the ubiquitin–proteasome system probably destroys many nascent and newly formed protein molecules, not because these proteins are

1	is not a trivial problem; in the course of carrying out its main job, the ubiquitin–proteasome system probably destroys many nascent and newly formed protein molecules, not because these proteins are abnormal as such, but because they have transiently exposed degradation signals that are buried in their mature (folded) state.

1	One function of intracellular proteolytic mechanisms is to recognize and eliminate misfolded or otherwise abnormal proteins, as just described. Indeed, every protein in the cell eventually accumulates damage and is probably degraded by the proteasome. Yet another function of these proteolytic pathways is to confer short lifetimes on specific normal proteins whose concentrations must change promptly with alterations in the state of a cell. Some of these short-lived proteins are degraded rapidly at all times, while many others are conditionally short-lived; that is, they are metabolically stable under some conditions, but become unstable upon a change in the cell’s state. For example, mitotic cyclins are long-lived throughout the cell cycle until their sudden degradation at the end of mitosis, as explained in Chapter 17.

1	Figure 6–85 A hexameric protein unfoldase. (A) The proteasome cap includes proteins (orange) that recognize and hydrolyze ubiquitin and a hexameric ring (blue) through which ubiquitylated proteins are threaded. The hexameric ring is formed from six subunits, each belonging to the AAA family of proteins. (B) Model for the ATP-dependent unfoldase activity of AAA proteins. The ATP-bound form of a hexameric ring of AAA proteins binds a folded substrate protein that is held in place by its ubiquitin tag. A conformational change, driven by ATP hydrolysis, pulls the substrate into the central core and strains the ring structure. At this point, the substrate protein, which is being tugged upon, can partially unfold and enter further into the pore or it can maintain its structure and partially withdraw. Very stable protein substrates may require hundreds of cycles of ATP hydrolysis and dissociation before they are successfully pulled through the AAA protein ring. Once unfolded (and

1	withdraw. Very stable protein substrates may require hundreds of cycles of ATP hydrolysis and dissociation before they are successfully pulled through the AAA protein ring. Once unfolded (and de-ubiquitylated), the substrate protein moves relatively quickly through the pore by successive rounds of ATP hydrolysis. (A, adapted from G.C. Lander et al., Nature 482:186–191, 2012; B, adapted from

1	R.T. Sauer et al., Cell 119:9–18, 2004. With permission from Elsevier.) N phosphorylation unmasking by creation of destabilizing by protein kinase protein dissociation N-terminus How is such a regulated destruction of a protein controlled? Several general mechanisms are illustrated in Figure 6–86. Specific examples of each mechanism are discussed in later chapters. In one general class of mechanism (Figure 6–86A), the activity of a ubiquitin ligase is turned on either by E3 phosphorylation or by an allosteric transition in an E3 protein caused by its binding to a specific small or large molecule. For example, the anaphase-promoting complex (APC) is a multi-subunit ubiquitin ligase that is activated by a cell-cycle-timed subunit addition at mitosis. The activated APC then causes the degradation of mitotic cyclins and several other regulators of the metaphase–anaphase transition (see Figure 17–15A).

1	Alternatively, in response either to intracellular signals or to signals from the environment, a degradation signal can be created in a protein, causing its rapid ubiquitylation and destruction by the proteasome (Figure 6–86B). One common way to create such a signal is to phosphorylate a specific site on a protein that unmasks a normally hidden degradation signal. Another way to unmask such a signal is by the regulated dissociation of a protein subunit. Finally, powerful degradation signals can be created by cleaving a single peptide bond, provided that this cleavage creates a new N-terminus that is recognized by a specific E3 protein as a “destabilizing” N-terminal residue. This E3 protein recognizes only certain amino acids at the N-terminus of a protein; thus not all protein-cleavage events will lead to degradation of the C-terminal fragment produced.

1	In humans, nearly 80% of proteins are acetylated on their N-terminal residue, and we now know that this modification is recognized by a specific E3 enzyme, Figure 6–86 Two general ways of inducing the degradation of a specific protein. (A) Activation of a specific E3 molecule creates a new ubiquitin ligase. Eukaryotic cells have many different E3 molecules, each activated by a different signal. (B) Creation of an exposed degradation signal in the protein to be degraded. This signal binds a ubiquitin ligase, causing the addition of a polyubiquitin chain to a nearby lysine on the target protein. All six pathways shown are known to be used by cells to induce the movement of selected proteins into the proteasome.

1	which directs the ubiquitylation of the protein and sends it to the proteasome for degradation. Thus, the majority of human proteins carry their own signals for destruction. It has been proposed that when a protein is properly folded (and, before that, when it is in contact with a chaperone), this acetylated N-terminus is buried and therefore inaccessible to the E3 enzyme. According to this idea, as a protein ages and becomes damaged (or if it fails to fold correctly from the start), this destruction signal becomes exposed, and the protein is destroyed. There Are Many Steps From DNA to Protein

1	There Are Many Steps From DNA to Protein We have seen so far in this chapter that many different types of chemical reactions are required to produce a properly folded protein from the information contained in a gene (Figure 6–87). The final level of a properly folded protein in a cell therefore depends upon the efficiency with which each of the many steps is performed. We also now know that the cell devotes enormous resources to selectively degrading proteins, particularly those that fail to fold properly or accumulate damage as they age. It is the balance between the rates of synthesis and degradation that determines the final amount of every protein in the cell.

1	In the following chapter, we shall see that cells have the ability to change the levels of their proteins according to their needs. In principle, any or all of the steps in Figure 6–87 could be regulated for each individual protein. As we shall see in Chapter 7, there are examples of regulation at each step from gene to protein. 5˜3˜DNA introns exons INITIATION OF TRANSCRIPTION CLEAVAGE, POLYADENYLATION, AND TERMINATION EXPORT CAPPING, ELONGATION SPLICING AAAA mRNA NUCLEUS CYTOSOL cap poly-A tail Figure 6–87 The production of a protein by a eukaryotic cell. The final level of each protein in a eukaryotic cell depends upon the efficiency of each step depicted.

1	Figure 6–87 The production of a protein by a eukaryotic cell. The final level of each protein in a eukaryotic cell depends upon the efficiency of each step depicted. The translation of the nucleotide sequence of an mRNA molecule into protein takes place in the cytosol on a large ribonucleoprotein assembly called a ribosome. Each amino acid used for protein synthesis is first attached to a tRNA molecule that recognizes, by complementary base-pair interactions, a particular set of three nucleotides (codons) in the mRNA. As an mRNA is threaded through a ribosome, its sequence of nucleotides is then read from one end to the other in sets of three according to the genetic code.

1	To initiate translation, a small ribosomal subunit binds to the mRNA molecule at a start codon (AUG) that is recognized by a unique initiator tRNA molecule. A large ribosomal subunit then binds to complete the ribosome and begin protein synthesis. During this phase, aminoacyl-tRNAs—each bearing a specific amino acid—bind sequentially to the appropriate codons in mRNA through complementary base-pairing between tRNA anticodons and mRNA codons. Each amino acid is added to the C-terminal end of the growing polypeptide in four sequential steps: aminoacyl-tRNA binding, followed by peptide bond formation, followed by two ribosome translocation steps. Elongation factors use GTP hydrolysis both to drive these reactions forward and to improve the accuracy of amino acid selection. The mRNA molecule progresses codon by codon through the ribosome in the 5ʹ-to-3ʹ direction until it reaches one of three stop codons. A release factor then binds to the ribosome, terminating translation and releasing

1	progresses codon by codon through the ribosome in the 5ʹ-to-3ʹ direction until it reaches one of three stop codons. A release factor then binds to the ribosome, terminating translation and releasing the completed polypeptide.

1	Eukaryotic and bacterial ribosomes are closely related, despite differences in the number and size of their rRNA and protein components. The rRNA has the dominant role in translation, determining the overall structure of the ribosome, forming the binding sites for the tRNAs, matching the tRNAs to codons in the mRNA, and creating the active site of the peptidyl transferase enzyme that links amino acids together during translation.

1	In the final steps of protein synthesis, two distinct types of molecular chaperones guide the folding of polypeptide chains. These chaperones, known as hsp60 and hsp70, recognize exposed hydrophobic patches on proteins and serve to prevent the protein aggregation that would otherwise compete with the folding of newly synthesized proteins into their correct three-dimensional conformations. This pro-tein-folding process must also compete with an elaborate quality control mechanism that destroys proteins with abnormally exposed hydrophobic patches. In this case, ubiquitin is covalently added to a misfolded protein by a ubiquitin ligase, and the resulting polyubiquitin chain is recognized by the cap on a proteasome that unfolds the protein and threads it into the interior of the proteasome for proteolytic degradation. A closely related proteolytic mechanism, based on special degradation signals recognized by ubiquitin ligases, is used to determine the lifetimes of many normally folded

1	for proteolytic degradation. A closely related proteolytic mechanism, based on special degradation signals recognized by ubiquitin ligases, is used to determine the lifetimes of many normally folded proteins as well as to remove selected proteins from the cell in response to specific signals.

1	We have seen that the expression of hereditary information requires extraordinarily complex machinery and proceeds from DNA to protein through an RNA intermediate. This machinery presents a central paradox: if nucleic acids are required to synthesize proteins and proteins are required, in turn, to synthesize nucleic acids, how did such a system of interdependent components ever arise? One view is that an RNA world existed on Earth before modern cells arose (Figure 6–88). According to this hypothesis, RNA both stored genetic information and catalyzed the chemical reactions in primitive cells. Only later in evolutionary time did DNA take over as the genetic material and proteins become the major catalysts and structural components of cells. If this idea is correct, then the transition out of the RNA world was never complete; as we have seen in this chapter, RNA still catalyzes several fundamental reactions in modern-day cells, which can be viewed as molecular fossils from an earlier

1	of the RNA world was never complete; as we have seen in this chapter, RNA still catalyzes several fundamental reactions in modern-day cells, which can be viewed as molecular fossils from an earlier world.

1	formed with DNA Figure 6–88 Time line for the universe, suggesting the early existence of an RNA world of living systems. The RNA world hypothesis relies on the fact that, among present-day biological molecules, RNA is unique in being able to act as both a carrier of genetic information and as a ribozyme to catalyze chemical reactions. In this section, we discuss these properties of RNA and how they may have been especially important in early cells.

1	We have seen in this chapter that RNA can carry genetic information in mRNAs, and we saw in Chapter 5 that the genomes of some viruses are composed solely of RNA. We have also seen that complementary base-pairing and other types of hydrogen bonds can occur between nucleotides in the same chain of RNA, causing an RNA molecule to fold up in a unique way determined by its nucleotide sequence (see, for example, Figures 6–50 and 6–67). Comparisons of many RNA structures have revealed conserved motifs, short structural elements that are used over and over again as parts of larger structures (Figure 6–89).

1	Protein catalysts require a surface with unique contours and chemical properties on which a given set of substrates can react (discussed in Chapter 3). In exactly the same way, an RNA molecule with an appropriately folded shape can serve as a catalyst (Figure 6–90). Like some proteins, many of these ribozymes work by positioning metal ions at their active sites. This feature gives them a wider range of catalytic activities than provided by the limited chemical groups of a polynucleotide chain. Much of our inference about the RNA world has come from experiments in which large pools of RNA molecules of random nucleotide sequences are generated in the laboratory. Those rare RNA molecules with a property specified by the experimenter are then selected out and studied (Figure 6–91). Such experiments have created RNAs that can catalyze a wide variety of biochemical reactions (Table 6–5), with reaction rate enhancements only a few orders of magnitude lower than

1	Figure 6–89 Some common elements of RNA structure. Conventional, complementary base-pairing interactions are indicated by red “rungs” in double-helical portions of the RNA. Figure 6–90 A ribozyme. This simple RNA molecule catalyzes the cleavage of a second RNA at a specific site. This ribozyme is found embedded in larger RNA genomes—called viroids—which infect plants. The cleavage, which occurs in nature at a distant location on the same RNA molecule that contains the ribozyme, is a step in the replication of the viroid genome. Although not shown in the figure, the reaction requires a magnesium ion positioned at the active site. (Adapted from T.R. Cech and O.C. Uhlenbeck, Nature 372:39–40, 1994. With permission from Macmillan Publishers Ltd.)

1	Figure 6–91 In vitro selection of a synthetic ribozyme. Beginning with a large pool of nucleic acid molecules synthesized in the laboratory, those rare RNA molecules that possess a specified catalytic activity can be isolated and studied. Although a specific example (that of an autophosphorylating ribozyme) is shown, variations of this procedure have been used to generate many of the ribozymes listed in Table 6–5. During the autophosphorylation step, the RNA molecules are kept sufficiently dilute to prevent the “cross”phosphorylation of additional RNA molecules. In reality, several repetitions of this procedure are necessary to select the very rare RNA molecules with this catalytic activity. Thus, the material initially eluted from the column is converted back into DNA, amplified many fold (using reverse transcriptase and PCR, as explained in Chapter 8), transcribed back into RNA, and subjected to repeated rounds of selection. (Adapted from J.R. Lorsch and

1	J.W. Szostak, Nature 371:31–36, 1994. With permission from Macmillan Publishers Ltd.) those of the “fastest” protein enzymes. Given these findings, it is not clear why protein catalysts greatly outnumber ribozymes in modern cells. Experiments have shown, however, that RNA molecules may have more difficulty than proteins in binding to flexible, hydrophobic substrates. In any case, the availability of 20 types of amino acids presumably provides proteins with a greater number of catalytic strategies. RNA molecules have one property that contrasts with those of polypeptides: they can directly guide the formation of copies of their own sequence. This capacity depends on complementary base-pairing of their nucleotide subunits, which enables one RNA to act as a template for the formation of another. As we have seen in this and the preceding chapter, these complementary templating mechanisms lie at the heart of DNA replication and transcription in modern-day cells.

1	large pool of double-stranded DNA molecules, each with a different, randomly generated nucleotide sequence large pool of single-stranded RNA molecules, each with a different, randomly generated nucleotide sequence only the rare RNA molecules able to phosphorylate themselves incorporate sulfur molecules that fail to bind to the rare RNA molecules that can catalyze their own phosphorylation using ATP as a substrate But the efficient synthesis of RNA by such complementary templating mechanisms requires catalysts to promote the polymerization reaction: without catalysts, polymer formation is slow, error-prone, and inefficient.

1	Because RNA has all the properties required of a molecule that could catalyze a variety of chemical reactions, including those that lead to its own synthesis (Figure 6–92), it has been proposed that RNAs served long ago as the catalysts for template-dependent RNA synthesis. Although self-replicating systems of RNA molecules have not been found in nature, scientists have made significant progress toward constructing them in the laboratory. While such demonstrations would not prove that self-replicating RNA molecules were central to the origin of life on Earth, they would establish that such a scenario is plausible. How Did Protein Synthesis Evolve?

1	How Did Protein Synthesis Evolve? The molecular processes underlying protein synthesis in present-day cells seem inextricably complex. Although we understand most of them, they do not make conceptual sense in the way that DNA transcription, DNA repair, and DNA replication do. It is especially difficult to imagine how protein synthesis evolved because it is now performed by a complex interlocking system of protein and RNA molecules; obviously the proteins could not have existed until an early version of the translation apparatus was already in place. As attractive as the RNA world idea is for envisioning early life, it does not explain how the modern-day system of protein synthesis arose. Although we can only speculate on the origins of the genetic code, several experimental observations have provided plausible scenarios.

1	In modern cells, some short peptides (such as antibiotics) are synthesized without the ribosome; peptide synthetase enzymes assemble these peptides, with their proper sequence of amino acids, without mRNAs to guide their synthesis. It is plausible that this noncoded, primitive version of protein synthesis first developed in the RNA world, where it would have been catalyzed by RNA molecules. This idea presents no conceptual difficulties because, as we have seen, rRNA catalyzes peptide bond formation in present-day cells. However, it leaves unexplained how the genetic code—which lies at the core of protein synthesis in today’s cells— might have arisen. We know that ribozymes created in the laboratory can perform specific aminoacylation reactions; that is, they can match specific amino acids to specific tRNAs. It is therefore possible that tRNA-like adaptors, each matched to a specific amino acid, could have arisen in the RNA world, marking the beginnings of a genetic code.

1	Once coded protein synthesis evolved, the transition to a protein-dominated world could proceed, with proteins eventually taking over the majority of catalytic and structural tasks because of their greater versatility, with 20 rather than 4 different subunits. Although these ideas are highly speculative, they are consistent with the known properties of RNA and protein molecules. All Present-Day Cells Use DNA as Their Hereditary Material If the evolutionary speculations embodied in the RNA world hypothesis are correct, early cells would have differed fundamentally from the cells we know today in having their hereditary information stored in RNA rather than in DNA (Figure 6–93). Evidence that RNA arose before DNA in evolution can be found

1	Figure 6–92 An RNA molecule that can catalyze its own synthesis. This hypothetical process would require catalysis both of the production of a second RNA strand of complementary nucleotide sequence (not shown) and the use of this second RNA molecule as a template to form many molecules of RNA with the original sequence. The red rays represent the active site of this hypothetical RNA enzyme. Figure 6–93 The hypothesis that RNA preceded DNA and proteins in evolution. In the earliest cells, RNA molecules (or their close analogs) would have had combined genetic, structural, and catalytic functions. In present-day cells, DNA is the repository of genetic information, and proteins perform the vast majority of catalytic functions in cells. RNA primarily functions today as a go-between in protein synthesis, although it remains a catalyst for a small number of crucial reactions.

1	366 Chapter 6: How Cells Read the Genome: From DNA to Protein in the chemical differences between them. Ribose, like glucose and other sim-WHAT WE DON’T KNOW ple carbohydrates, can be formed from formaldehyde (HCHO), a simple chemical which is readily produced in laboratory experiments that attempt to simulate • How did the present relationships conditions on the primitive Earth. The sugar deoxyribose is harder to make, and in present-day cells it is produced from ribose in a reaction catalyzed by a pro- evolve? How did the genetic code tein enzyme, suggesting that ribose pre-dates deoxyribose in cells. Presumably, originate?

1	DNA appeared on the scene later, but then proved more suitable than RNA as a permanent repository of genetic information. In particular, the deoxyribose in • The information carried in genomes its sugar-phosphate backbone makes chains of DNA chemically more stable than specifies the sequences of all proteins chains of RNA, so that much greater lengths of DNA can be maintained without and RNA molecules in the cell, breakage. and it determines when and where these molecules are synthesized. The other differences between RNA and DNA—the double-helical structure of DNA and the use of thymine rather than uracil—further enhance DNA stability by making the many unavoidable accidents that occur to the molecule much easier discovered? to repair, as discussed in detail in Chapter 5 (pp. 271–273). • Cells go to great length to correct mistakes in the processes of DNA replication, transcription, splicing, and translation. Are there analogous

1	• Cells go to great length to correct mistakes in the processes of DNA replication, transcription, splicing, and translation. Are there analogous From our knowledge of present-day organisms and the molecules they contain, it strategies to correct mistakes in the seems likely that the development of the distinctive autocatalytic mechanisms fun- selection of which genes are to be damental to living systems began with the evolution of families of RNA molecules expressed in a given cell type? Could that could catalyze their own replication. DNA is likely to have been a late addition: the great complexity of transcription as the accumulation of protein catalysts allowed more efficient and complex cells to initiation in animals and plants reflect evolve, the DNA double helix replaced RNA as a more stable molecule for storing the such a strategy? increased amounts of genetic information required by such cells. Which statements are true? explain why or why not.

1	Which statements are true? explain why or why not. 6–1 The consequences of errors in transcription are less severe than those of errors in DNA replication. 6–2 Since introns are largely genetic “junk,” they do not Figure Q6–1 Supercoils around a moving RNA polymerase (Problem have to be removed precisely from the primary transcript 6–6). during RNA splicing. 6–3 Wobble pairing occurs between the first position 6–7 You have attached an RNA polymerase molecule in the codon and the third position in the anticodon. to a glass slide and have allowed it to initiate transcription on a template DNA that is tethered to a magnetic bead as 6–4 During protein synthesis, the thermodynamics of shown in Figure Q6–2. If the DNA with its attached mag-base-pairing between tRNAs and mRNAs sets the upper netic bead moves relative to the RNA polymerase as indilimit for the accuracy with which protein molecules are cated in the figure, in which direction will the bead rotate? made.

1	made. 6–5 Protein enzymes are thought to greatly outnum-Figure Q6–2 System for measuring the rotation of DNA ber ribozymes in modern cells because they can catalyze magnet a much greater variety of reactions and all of them have (Problem 6–7). The magnet faster rates than any ribozyme. holds the bead upright (but doesn’t interfere with its Discuss the following problems. bead rotation), and the attached tiny fluorescent beads allow 6–6 In which direction along the template must the the direction of motion to DNA be visualized under the RNA polymerase in Figure Q6–1 be moving to have gen- microscope. RNA polymerase is erated the supercoiled structures that are shown? Would you expect supercoils to be generated if the RNA poly-RNA the glass slide. merase were free to rotate about the axis of the DNA as it progressed along the template? glass slide

1	merase were free to rotate about the axis of the DNA as it progressed along the template? glass slide Figure Q6–3 Alternatively spliced mRNAs from the human α-tropomyosin gene (Problem 6–8). (A) Exons in the human α-tropomyosin gene. The locations and relative sizes of exons are shown by the blue and red rectangles, with alternative exons in red. (B) Splicing patterns for four α-tropomyosin mRNAs. Splicing is indicated by lines connecting the exons that are included in the mRNA.

1	(B) Splicing patterns for four α-tropomyosin mRNAs. Splicing is indicated by lines connecting the exons that are included in the mRNA. 6–8 The human α-tropomyosin gene is alternatively spliced to produce different forms of α-tropomyosin mRNA in different cell types (Figure Q6–3). For all forms of the mRNA, the protein sequences encoded by exon 1 are the same, as are the protein sequences encoded by exon 10. Exons 2 and 3 are alternative exons used in different mRNAs, as are exons 7 and 8. Which of the following statements about exons 2 and 3 is the most accurate? Is that statement also the most accurate one for exons 7 and 8? Explain your answers. A. Exons 2 and 3 must have the same number of nucleotides. b. Exons 2 and 3 must each contain an integral number of codons (that is, the number of nucleotides divided by 3 must be an integer). C. Exons 2 and 3 must each contain a number of nucleotides that when divided by 3 leaves the same remainder (that is, 0, 1, or 2).

1	C. Exons 2 and 3 must each contain a number of nucleotides that when divided by 3 leaves the same remainder (that is, 0, 1, or 2). 6–9 After treating cells with a chemical mutagen, you isolate two mutants. One carries alanine and the other carries methionine at a site in the protein that normally contains valine (Figure Q6–4). After treating these two mutants again with the mutagen, you isolate mutants from each that now carry threonine at the site of the original valine (Figure Q6–4). Assuming that all mutations involve single-nucleotide changes, deduce the codons that are used for valine, methionine, threonine, and alanine at the affected site. Would you expect to be able to isolate valineto-threonine mutants in one step? 6–10 Which of the following mutational changes would you predict to be the most deleterious to gene function? Explain your answers.

1	6–10 Which of the following mutational changes would you predict to be the most deleterious to gene function? Explain your answers. frst Ala second Figure Q6–4 Two rounds of treatment treatment mutagenesis and the altered amino acids at a single position in Val Thr a protein (Problem 6–9). 1. Insertion of a single nucleotide near the end of the coding sequence. 2. Removal of a single nucleotide near the beginning of the coding sequence. 3. Deletion of three consecutive nucleotides in the middle of the coding sequence. 4. Substitution of one nucleotide for another in the middle of the coding sequence. 6–11 Prokaryotes and eukaryotes both protect against the dangers of translating broken mRNAs. What dangers do partial mRNAs pose for the cell?

1	6–11 Prokaryotes and eukaryotes both protect against the dangers of translating broken mRNAs. What dangers do partial mRNAs pose for the cell? 6–12 Both hsp60-like and hsp70 molecular chaperones share an affinity for exposed hydrophobic patches on proteins, using them as indicators of incomplete folding. Why do you suppose hydrophobic patches serve as critical signals for the folding status of a protein? 6–13 Most proteins require molecular chaperones to assist in their correct folding. How do you suppose the chaperones themselves manage to fold correctly? 6–14 What is so special about RNA that it is hypothesized to be an evolutionary precursor to DNA and protein? What is it about DNA that makes it a better material than RNA for storage of genetic information?

1	6–15 If an RNA molecule could form a hairpin with a symmetric internal loop, as shown in Figure Q6–5, could the complement of this RNA form a similar structure? If so, would there be any regions of the two structures that are identical? Which ones? Figure Q6–5 An RNA hairpin with 5’-G-C-A C-C-G a symmetric internal loop (Problem U 6–15). 6–16 Imagine a warm pond on the primordial Earth. Chance processes have just assembled a single copy of an RNA molecule with a catalytic site that can carry out RNA replication. This RNA molecule folds into a structure that is capable of linking nucleotides according to instructions in an RNA template. Given an adequate supply of nucleotides, will this single RNA molecule be able to use itself as a template to catalyze its own replication? Why or why not? Atkins JF, Gesteland RF & Cech TR (eds) (2011) The RNA Worlds: From Life’s Origins to Diversity in Gene Regulation. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

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1	Kruger K, Grabowski P, Zaug P et al. (1982) Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening seuence of Tetrahymena. Cell 31, 147–157. Orgel L (2000) Origin of life. A simpler nucleic acid. Science 290, 1306–1307. Robertson MP& Joyce GF (2012) The origins of the RNA world. Cold Spring Harb. Perspect. Biol. 4, a003608. Control of Gene expression 7 An organism’s DNA encodes all of the RNA and protein molecules required to construct its cells. Yet a complete description of the DNA sequence of an organism—be it the few million nucleotides of a bacterium or the few billion nucleotides of a human—no more enables us to reconstruct the organism than a list of English words enables us to reconstruct a play by Shakespeare. In both cases, the problem is to know how the elements in the DNA sequence or the words on the list are used. Under what conditions is each gene product made, and, once made, what does it do?

1	In this chapter, we focus on the first half of this problem—the rules and mechanisms that enable a subset of genes to be selectively expressed in each cell. These mechanisms operate at many levels, and we shall discuss each level in turn. But first we present some of the basic principles involved. The different cell types in a multicellular organism differ dramatically in both structure and function. If we compare a mammalian neuron with a liver cell, for example, the differences are so extreme that it is difficult to imagine that the two cells contain the same genome (Figure 7–1). For this reason, and because cell differentiation often seemed irreversible, biologists originally suspected that genes might be selectively lost when a cell differentiates. We now know, however, that cell differentiation generally occurs without changes in the nucleotide sequence of a cell’s genome. the Different Cell types of a Multicellular Organism Contain the Same DnA

1	the Different Cell types of a Multicellular Organism Contain the Same DnA The cell types in a multicellular organism become different from one another because they synthesize and accumulate different sets of RNA and protein molecules. The initial evidence that they do this without altering the sequence of their DNA came from a classic set of experiments in frogs. When the nucleus of a fully differentiated frog cell is injected into a frog egg whose nucleus has been removed, the injected donor nucleus is capable of directing the recipient egg to

1	Figure 7–1 A neuron and a liver cell share the same genome. the long branches of this neuron from the retina enable it to receive electrical signals from many other neurons and convey them to neighboring neurons. the liver cell, which is drawn to the same scale, is involved in many metabolic processes, including digestion and the detoxification of alcohol and other drugs. Both of these mammalian cells contain the same genome, but they express different sets of rnAs and proteins. (neuron adapted from S. ramón y Cajal, histologie du Systeme nerveux de l’homme et de vertebres, 1909–1911. paris: Maloine; reprinted, Madrid: C.S.i.C, 1972.) 370 Chapter 7: Control of Gene expression produce a normal tadpole (Figure 7–2A). The tadpole contains a full range of differentiated cells that derived their DNA sequences from the nucleus of the original donor cell. Thus, the differentiated donor cell cannot have lost any important DNA sequences. A similar conclusion came from experiments performed

1	their DNA sequences from the nucleus of the original donor cell. Thus, the differentiated donor cell cannot have lost any important DNA sequences. A similar conclusion came from experiments performed with plants. When differentiated pieces of plant tissue are placed in culture and then dissociated into single cells, often one of these individual cells can regenerate an entire adult plant (Figure 7–2B). And the same principle has been more recently demonstrated in mammals that include sheep, cattle, pigs, goats, dogs, and mice (Figure 7–2C).

1	Most recently, detailed DNA sequencing has confirmed the conclusion that the changes in gene expression that underlie the development of multicellular organisms do not generally involve changes in the DNA sequence of the genome. Different Cell types Synthesize Different Sets of rnAs and proteins As a first step in understanding cell differentiation, we would like to know how many differences there are between any one cell type and another. Although we

1	Figure 7–2 Differentiated cells contain all the genetic instructions necessary to direct the formation of a complete organism. (A) the nucleus of a skin cell from an adult frog transplanted into an enucleated egg can give rise to an entire tadpole. the broken arrow indicates that, to give the transplanted genome time to adjust to an embryonic environment, a further transfer step is required in which one of the nuclei is taken from an early embryo that begins to develop and is put back into a second enucleated egg. (B) in many types of plants, differentiated cells retain the ability to “de-differentiate,” so that a single cell can form a clone of progeny cells that later give rise to an entire plant. (C) A nucleus removed from a differentiated cell from an adult cow and introduced into an enucleated egg from a different cow can give rise to a calf. Different calves produced from the same differentiated cell donor are all clones of the donor and are therefore genetically identical. (A,

1	enucleated egg from a different cow can give rise to a calf. Different calves produced from the same differentiated cell donor are all clones of the donor and are therefore genetically identical. (A, modified from

1	J.B. Gurdon, Sci. Am. 219:24–35, 1968.) unfertilized MEIOTIC SPINDLE egg cell AND ASSOCIATED CHROMOSOMES REMOVED no. of reads no. of reads (B) start of transcription exons introns CELL LINE still do not have an exact answer to this fundamental question, we can make several general statements. 1. Many processes are common to all cells, and any two cells in a single organism therefore have many gene products in common. These include the structural proteins of chromosomes, RNA and DNA polymerases, DNA repair enzymes, ribosomal proteins and RNAs, the enzymes that catalyze the central reactions of metabolism, and many of the proteins that form the cytoskeleton such as actin (Figure 7–3A). 2.

1	2. Some RNAs and proteins are abundant in the specialized cells in which they function and cannot be detected elsewhere, even by sensitive tests. Hemoglobin, for example, is expressed specifically in red blood cells, where it carries oxygen, and the enzyme tyrosine aminotransferase (which breaks down tyrosine in food) is expressed in liver but not in most other tissues (Figure 7–3B). 3.

1	3. Studies of the number of different RNAs suggest that, at any one time, a typical human cell expresses 30–60% of its approximately 30,000 genes at some level. There are about 21,000 protein-coding genes and a roughly estimated 9000 noncoding RNA genes in humans. When the patterns of RNA expression in different human cell lines are compared, the level of expression of almost every gene is found to vary from one cell type to another. A few of these differences are striking, like those of hemoglobin and tyrosine aminotransferase noted above, but most are much more subtle. But even those genes that are expressed in all cell types usually vary in their level of expression from one cell type to the next. 4.

1	4. Although there are striking differences in coding RNAs (mRNAs) in specialized cell types, they underestimate the full range of differences in the final pattern of protein production. As we discuss in this chapter, there are many steps after RNA production at which gene expression can be regulated. And, as we saw in Chapter 3, proteins are often covalently modified after they are synthesized. The radical differences in gene expression between cell types are therefore most fully revealed through methods that directly display the levels of proteins along with their post-translational modifications (Figure 7–4).

1	Figure 7–3 Differences in RNA levels for two human genes in seven different tissues. to obtain the rnA data by the technique known as RNA-seq (see p. 447), rnA was collected from human cell lines grown in culture, derived from each of the seven indicated tissues. Millions of “sequence reads” were obtained and mapped across the human genome by matching rnA sequences to the DnA sequence of the genome. At each position along the genome, the height of the colored trace is proportional to the number of sequence reads that match the genome sequence at that point. As seen in the figure, the exon sequences in transcribed genes are present at high levels, reflecting their presence in mature mrnAs. intron sequences are present at much lower levels and reflect pre-mrnA molecules that have not yet been spliced plus intron sequences that have been spliced out but not yet degraded. (A) the gene coding for “all-purpose” actin, a major component of the cytoskeleton. note that the left-hand end of the

1	spliced plus intron sequences that have been spliced out but not yet degraded. (A) the gene coding for “all-purpose” actin, a major component of the cytoskeleton. note that the left-hand end of the mature β-actin mrnA is not translated into protein. As explained later in this chapter, many mrnAs have 5ʹ untranslated regions that regulate their translation into protein. (B) the same type of data displayed for the enzyme tyrosine aminotransferase, which is highly expressed in liver cells but not in the other cell types tested. (information for both panels from the University of California, Santa Cruz, Genome Browser (http://genome.ucsc.edu), which provides this type of information for every human gene. See also S. Djebali et al., Nature 489:101–108, 2012.) external Signals Can Cause a Cell to Change the expression of its Genes

1	Although the specialized cells in a multicellular organism have characteristic patterns of gene expression, each cell is capable of altering its pattern of gene expression in response to extracellular cues. If a liver cell is exposed to a glucocorticoid hormone, for example, the production of a set of proteins is dramatically increased. Released in the body during periods of starvation or intense exercise, glucocorticoids signal the liver to increase the production of energy from amino acids and other small molecules; the set of proteins whose production is induced includes the enzyme tyrosine aminotransferase, mentioned above. When the hormone is no longer present, the production of these proteins drops to its normal, unstimulated level in liver cells.

1	Other cell types respond to glucocorticoids differently. Fat cells, for example, reduce the production of tyrosine aminotransferase, while some other cell types do not respond to glucocorticoids at all. These examples illustrate a general feature of cell specialization: different cell types often respond very differently to the same extracellular signal. Other features of the gene expression pattern do not change and give each cell type its permanently distinctive character. Gene expression Can Be regulated at Many of the Steps in the pathway from DnA to rnA to protein

1	If differences among the various cell types of an organism depend on the particular genes that the cells express, at what level is the control of gene expression exercised? As we saw in the previous chapter, there are many steps in the pathway leading from DNA to protein. We now know that all of them can in principle be regulated. Thus a cell can control the proteins it makes by (1) controlling when and how often a given gene is transcribed (transcriptional control), (2) controlling the splicing and processing of RNA transcripts (RNA processing control), (3) selecting which completed mRNAs are exported from the nucleus to the cytosol and determining where in the cytosol they are localized (RNA transport and localization control), (4) selecting which mRNAs in the cytoplasm are translated by ribosomes (translational control), (5) selectively destabilizing certain mRNA molecules in the cytoplasm (mRNA degradation control), or (6) selectively activating, inactivating, degrading, or

1	by ribosomes (translational control), (5) selectively destabilizing certain mRNA molecules in the cytoplasm (mRNA degradation control), or (6) selectively activating, inactivating, degrading, or localizing specific protein molecules after they have been made (protein activity control) (Figure 7–5).

1	For most genes, transcriptional controls are paramount. This makes sense because, of all the possible control points illustrated in Figure 7–5, only transcriptional control ensures that the cell will not synthesize superfluous intermediates. In the following sections, we discuss the DNA and protein components that perform this function by regulating the initiation of gene transcription. We shall then return to the additional ways of regulating gene expression.

1	Figure 7–4 Differences in the proteins expressed by two human tissues, (A) brain and (B) liver. in each panel, the proteins are displayed using two-dimensional polyacrylamide-gel electrophoresis (see pp. 452–454). the proteins have been separated by molecular weight (top to bottom) and isoelectric point, the ph at which the protein has no net charge (right to left). the protein spots artificially colored red are common to both samples; those in blue are specific to that tissue. the differences between the two tissue samples vastly outweigh their similarities: even for proteins that are shared between the two tissues, their relative abundances are usually different. note that this technique separates proteins by both size and charge; therefore a protein that has several different phosphorylation states will appear as a series of horizontal spots (see upper right-hand portion of right panel). Only a small portion of the complete protein spectrum is shown for each sample.

1	Methods based on mass spectrometry (see pp. 455–457) provide much more detailed information, including the identity of each protein, the position of each modification, and the nature of the modification. (Courtesy of tim Myers and leigh Anderson, large Scale Biology Corporation.) The genome of a cell contains in its DNA sequence the information to make many thousands of different protein and RNA molecules. A cell typically expresses only a fraction of its genes, and the different types of cells in multicellular organisms arise because different sets of genes are expressed. Moreover, cells can change the pattern of genes they express in response to changes in their environment, such as signals from other cells. Although all of the steps involved in expressing a gene can in principle be regulated, for most genes the initiation of RNA transcription provides the most important point of control.

1	How does a cell determine which of its thousands of genes to transcribe? Perhaps the most important concept, one that applies to all species on Earth, is based on a group of proteins known as transcription regulators. These proteins recognize specific sequences of DNA (typically 5–10 nucleotide pairs in length) that are often called cis-regulatory sequences, because they must be on the same chromosome (that is, in cis) to the genes they control. Transcription regulators bind to these sequences, which are dispersed throughout genomes, and this binding puts into motion a series of reactions that ultimately specify which genes are to be transcribed and at what rate. Approximately 10% of the protein-coding genes of most organisms are devoted to transcription regulators, making them one of the largest classes of proteins in the cell. In most cases, a given transcription regulator recognizes its own cis-regulatory sequence, which is different from those recognized by all the other

1	of the largest classes of proteins in the cell. In most cases, a given transcription regulator recognizes its own cis-regulatory sequence, which is different from those recognized by all the other regulators in the cell.

1	Transcription of each gene is, in turn, controlled by its own collection of cis-regulatory sequences. These typically lie near the gene, often in the intergenic region directly upstream from the transcription start point of the gene. Although a few genes are controlled by a single cis-regulatory sequence that is recognized by a single transcription regulator, the majority have complex arrangements of cis-regulatory sequences, each of which is recognized by a different transcription regulator. It is therefore the positions, identity, and arrangement of cis-regulatory sequences—which are an important part of the information embedded in the genome—that ultimately determine the time and place that each gene is transcribed. We begin our discussion by describing how transcription regulators recognize cis-regulatory sequences. the Sequence of nucleotides in the DnA Double helix Can Be read by proteins

1	We begin our discussion by describing how transcription regulators recognize cis-regulatory sequences. the Sequence of nucleotides in the DnA Double helix Can Be read by proteins As discussed in Chapter 4, the DNA in a chromosome consists of a very long double helix that has both a major and a minor groove (Figure 7–6). Transcription regulators must recognize short, specific cis-regulatory sequences within Figure 7–5 Six steps at which eukaryotic gene expression can be controlled. Controls that operate at steps 1 through 5 are discussed in this chapter. Step 6, the regulation of protein activity, occurs largely through covalent post-translational modifications including phosphorylation, acetylation, and ubiquitylation (see table 3–3, p. 165). Step 6 was introduced in Chapter 3 and is subsequently discussed in many chapters throughout the book.

1	Figure 7–6 Double-helical structure of DNA. A space-filling model of DnA showing the major and minor grooves on the outside of the double helix (see Movie 4.1). the atoms are colored as follows: carbon, dark blue; nitrogen, light blue; hydrogen, white; oxygen, red; phosphorus, yellow.

1	this structure. When first discovered in the 1960s, it was thought that these proteins might require direct access to the interior of the double helix to distinguish between one DNA sequence and another. It is now clear, however, that the outside of the double helix is studded with DNA sequence information that transcription regulators recognize: the edge of each base pair presents a distinctive pattern of hydrogen-bond donors, hydrogen-bond acceptors, and hydrophobic patches in both the major and minor grooves (Figure 7–7). Because the major groove is wider and displays more molecular features than does the minor groove, nearly all transcription regulators make the majority of their contacts with the major groove—as we shall see.

1	Molecular recognition in biology generally relies on an exact fit between the surfaces of two molecules, and the study of transcription regulators has provided some of the clearest examples of this principle. A transcription regulator recognizes a specific cis-regulatory sequence because the surface of the protein is extensively complementary to the special surface features of the double helix that displays that sequence. Each transcription regulator makes a series of contacts with the DNA, involving hydrogen bonds, ionic bonds, and hydrophobic interactions. Although each individual contact is weak, the 20 or so contacts that are typically formed at the protein–DNA interface add together to ensure that the interaction is both highly specific and very strong (Figure 7–8). In fact, DNA–protein

1	Figure 7–7 How the different base pairs in DNA can be recognized from their edges without the need to open the double helix. the four possible configurations of base pairs are shown, with potential hydrogen-bond donors indicated in blue, potential hydrogen-bond acceptors in red, and hydrogen bonds of the base pairs themselves as a series of short, parallel red lines. Methyl groups, which form hydrophobic protuberances, are shown in yellow, and hydrogen atoms that are attached to carbons, and are therefore unavailable for hydrogen-bonding, are white. from the major groove, each of the four base-pair configurations projects a unique pattern of features. (from C. Branden and J. tooze, introduction to protein Structure, 2nd ed. new York: Garland publishing, 1999.) outer limit of sugar-phosphate of DNAbackbone on outside of double helix interactions include some of the tightest and most specific molecular interactions known in biology.

1	Although each example of protein–DNA recognition is unique in detail, x-ray crystallographic and nuclear magnetic resonance (NMR) spectroscopic studies of hundreds of transcription regulators have revealed that many of them contain one or another of a small set of DNA-binding structural motifs (Panel 7–1). These motifs generally use either α helices or β sheets to bind to the major groove of DNA. The amino acid side chains that extend from these protein motifs make the specific contacts with the DNA. Thus, a given structural motif can be used to recognize many different cis-regulatory sequences depending on the specific side chains present. Dimerization of transcription regulators increases their Affinity and Specificity for DnA

1	Dimerization of transcription regulators increases their Affinity and Specificity for DnA A monomer of a typical transcription regulator recognizes about 6–8 nucleotide pairs of DNA. However, sequence-specific DNA-binding proteins do not bind tightly to a single DNA sequence and reject all others; rather, they recognize a range of closely related sequences, with the affinity of the protein for the DNA varying according to how closely the DNA matches the optimal sequence. Hence, cis-regulatory sequences are often depicted as “logos” which display the range of sequences recognized by a particular transcription regulator (Figure 7–9A and B). In Chapter 6, we saw this same representation at work for the binding of RNA polymerase to promoters (see Figure 6–12).

1	The DNA sequence recognized by a monomer does not contain sufficient information to be picked out from the background of such sequences that would occur at random all over the genome. For example, an exact six-nucleotide DNA sequence would be expected to occur by chance approximately once every 4096 nucleotides (46), and the range of six-nucleotide sequences described by a typical logo would be expected to occur by chance much more often, perhaps every 1000 nucleotides. Clearly, for a bacterial genome of 4.6 × 106 nucleotide pairs, not to mention a mammalian genome of 3 × 109 nucleotide pairs, this is insufficient information to accurately control the transcription of individual genes. Additional contributions to DNA-binding specificity must therefore be present. Many transcription regulators form dimers, with both monomers making nearly identical contacts with DNA (Figure 7–9C). This arrangement doubles the length of the cis-regulatory sequence recognized and greatly increases both

1	form dimers, with both monomers making nearly identical contacts with DNA (Figure 7–9C). This arrangement doubles the length of the cis-regulatory sequence recognized and greatly increases both the affinity and the specificity of transcription regulator binding. Because the DNA sequence

1	Figure 7–8 The binding of a transcription regulator to a specific DNA sequence. On the left, a single contact is shown between a transcription regulator and DnA; such contacts allow the protein to “read” the DnA sequence. On the right, the complete set of contacts between a transcription regulator (a member of the homeodomain family—see panel 7–1) and its cis-regulatory sequence is shown. the DnA-binding portion of the protein is 60 amino acids long. Although the interactions in the major groove are the most important, the protein is also seen to contact both the minor groove and phosphates in the sugar–phosphate DnA backbone. (See C. wolberger et al., Cell 67:517–528, 1991.) tryptophan repressor lambda Cro lambda repressor fragment CAP fragment DNA DNA 3.4 nm (A) Arg C 3 1 2 N 2 Ser Arg Asn 3 1 (B) Originally identifed in bacterial transcription regulators, this motif has since been found in many hundreds of DNA-binding proteins from both eukaryotes and prokaryotes. It is constructed

1	3 1 (B) Originally identifed in bacterial transcription regulators, this motif has since been found in many hundreds of DNA-binding proteins from both eukaryotes and prokaryotes. It is constructed from two ˜ helices (blue and red) connected by a short extended chain of amino acids, which constitutes the “turn.” The two helices are held at a fxed angle, primarily through interactions between the two helices. The more C-terminal helix (in red) is called the recognition helix because it fts into the major groove of DNA; its amino acid side chains, which differ from protein to protein, play an important part in recognizing the specifc DNA sequence to which the protein binds. All of the proteins shown here bind DNA as dimers in which the two copies of the recognition helix (in red) are separated by exactly one turn of the DNA helix (3.4 nm); thus both recognition helices of the dimer can ft into the major groove of DNA. Not long after the frst transcription regulators were discovered in

1	by exactly one turn of the DNA helix (3.4 nm); thus both recognition helices of the dimer can ft into the major groove of DNA. Not long after the frst transcription regulators were discovered in bacteria, genetic analyses of the fruit fy Drosophila led to the characterization of an important class of genes, the homeotic selector genes, that play a critical part in orchestrating fy development (discussed in Chapter 21). It was later shown that these genes coded for transcription regulators that bound DNA through a structural motif named the homeodomain. Two different views of the same structure are shown. (A) The homeodomain is folded into three ˜ helices, which are packed tightly together by hydrophobic interactions. The part containing helices 2 and 3 closely resembles the helix–turn–helix motif. (B) The recognition helix (helix 3, red) forms important contacts with the major groove of DNA. The asparagine (Asn) of helix 3, for example, contacts an adenine, as shown in Figure 7–8. A

1	motif. (B) The recognition helix (helix 3, red) forms important contacts with the major groove of DNA. The asparagine (Asn) of helix 3, for example, contacts an adenine, as shown in Figure 7–8. A fexible arm attached to helix 1 forms contacts with nucleotide pairs in the minor groove. The leucine zipper motif is named because of the way the two ˜helices, one from each monomer, are joined together to form a short coiled-coil. These proteins bind DNA as dimers where the two long ˜helices are held together by interactions between hydrophobic amino acid side chains (often on leucines) that extend from one side of each helix. Just beyond the dimerization interface, the two ˜helices separate from each other to form a Y-shaped structure, which allows their side chains to contact the major groove of DNA. The dimer thus grips the double helix like a clothespin on a clothesline. recognition helix recognition helix DNA dimerization interface HELIX–TURN–HELIX PROTEINS HOMEODOMAIN PROTEINS LEUCINE

1	of DNA. The dimer thus grips the double helix like a clothespin on a clothesline. recognition helix recognition helix DNA dimerization interface HELIX–TURN–HELIX PROTEINS HOMEODOMAIN PROTEINS LEUCINE ZIPPER PROTEINS 376 PANEL 7–1: Common Structural Motifs in Transcription Regulators

1	In the other DNA-binding motifs displayed in this panel, ˜ helices are the primary mechanism used to recognize specifc DNA sequences. In one large group of transcription regulators, however, a two-stranded ° sheet, with amino acid side chains extending from the sheet toward the DNA, reads the information on the surface of the major groove. As in the case of a recognition ˜ helix, this °-sheet motif can be used to recognize many different DNA sequences; the exact DNA sequence recognized depends on the sequence of amino acids that make up the ° sheet. Shown is a transcription regulator that binds two molecules of S-adenosyl methionine (red). On the left is a dimer of the protein; on the right is a simplifed diagram showing just the two-stranded ° sheet bound to the major groove of DNA. This group of DNA-binding motifs includes one or more zinc atoms as structural components. All such zinc-coordinated DNA-binding motifs are called zinc fngers, referring to their appearance in early

1	This group of DNA-binding motifs includes one or more zinc atoms as structural components. All such zinc-coordinated DNA-binding motifs are called zinc fngers, referring to their appearance in early schematic drawings (left). They fall into several distinct structural groups, only one of which we consider here. It has a simple structure, in which the zinc atom holds an ˜ helix and a ° sheet together (middle). This type of zinc fnger is often found in clusters with the ˜ helix of each fnger contacting the major groove of the DNA, forming a nearly continuous stretch of ˜ helices along that groove. In this way, a strong and specifc DNA–protein interaction is built up through a repeating basic structural unit. Three such fngers are shown on the right.N C C N C His Cys Zn His N Cys CCGLEHHZnKYKNQRVRRSFKEVLSASCOOHNH2COOHNH2ZnZnZnDNADNA° SHEET DNA RECOGNITION PROTEINS ZINC FINGER PROTEINS 377

1	Related to the leucine zipper, the helix–loop–helix motif consists of a short ˜ helix connected by a loop (red) to a second, longer ˜ helix. The fexibility of the loop allows one helix to fold back and park against the other thereby forming the dimerization surface. As shown, this two-helix structure binds both to DNA and to the two-helix structure of a second protein to create either a homodimer or a heterodimer. Two ˜helices that extend from the dimerization interface make specifc contacts with the major groove of DNA. CCNNDNAloopHELIX–LOOP–HELIX PROTEINS recognized by the protein has increased from approximately 6 nucleotide pairs to 12 nucleotide pairs, there are many fewer random occurrences of matching sequences.

1	Heterodimers are often formed from two different transcription regulators. Transcription regulators may form heterodimers with more than one partner protein; in this way, the same transcription regulator can be “reused” to create several distinct DNA-binding specificities (see Figure 7–9C). transcription regulators Bind Cooperatively to DnA In the simplest case, the collection of noncovalent bonds that holds the above dimers or heterodimers together is so extensive that these structures form obligatorily, and never fall apart. In this case, the unit of binding is the dimer or heterodimer, and the binding curve for the transcription regulator (the fraction of DNA bound as a function of protein concentration) has a standard exponential shape (Figure 7–10A).

1	In many cases, however, the dimers and heterodimers are held together very weakly; they exist predominantly as monomers in solution, and yet dimers are observed on the appropriate DNA sequence. Here, the proteins are said to bind to DNA cooperatively, and the curve describing their binding is sigmoidal in shape (Figure 7–10B). Cooperative binding means that, over a range of concentrations of the transcription regulator, binding is more of an all-or-none phenomenon than Figure 7–9 Transcription regulators and cis-regulatory sequences. (A) Depiction of the cis-regulatory sequence for nanog, a homeodomain family member that is a key regulator in embryonic stem cells. this “logo” form (see figure 6–12) shows that the protein can recognize a collection of closely related DnA sequences and gives the preferred nucleotide pair at each position. Cis-regulatory sequences are “read” as double-stranded DnA, but only one strand typically is shown in a logo.

1	(B) representation of the cis-regulatory sequence as a colored box. (C) Many transcription regulators form dimers (homodimers) and heterodimers. in the example shown, three different DnAbinding specificities are formed from two transcription regulators. Figure 7–10 Occupancy of a cisregulatory sequence by a transcription regulator. (A) noncooperative binding by a stable heterodimer. (B) Cooperative binding by components of a heterodimer that are predominantly monomers in solution. the shape of the curve differs from that of (A) because the fraction of protein in a form competent to bind DnA (the heterodimer) increases with increasing protein concentration. for noncooperative binding; that is, at most protein concentrations, the cis-regulatory sequence is either nearly empty or nearly fully occupied and rarely is somewhere in between. A discussion of the mathematics behind cooperative binding is given in Chapter 8 (see Figure 8–79A).

1	nucleosome Structure promotes Cooperative Binding of transcription regulators As we have just seen, cooperative binding of transcription regulators to DNA often occurs because the monomers have only a weak affinity for each other. However, there is a second, indirect mechanism for cooperative binding, one that arises from the nucleosome structure of eukaryotic chromosomes.

1	In general, transcription regulators bind to DNA in nucleosomes with lower affinity than they do to naked DNA. There are two reasons for this difference. First, the surface of the cis-regulatory sequence recognized by the transcription regulator may be facing inward on the nucleosome, toward the histone core, and therefore not be readily available to the regulatory protein. Second, even if the face of the cis-regulatory sequence is exposed on the outside of the nucleosome, many transcription regulators subtly alter the conformation of the DNA when they bind, and these changes are generally opposed by the tight wrapping of the DNA around the histone core. For example, many transcription regulators induce a bend or kink in the DNA when they bind.

1	We saw in Chapter 4 that nucleosome remodeling can alter the structure of the nucleosome, allowing transcription regulators access to the DNA. Even without remodeling, however, transcription regulators can still gain limited access to DNA in a nucleosome. The DNA at the end of a nucleosome “breathes,” transiently exposing the DNA and allowing regulators to bind. This breathing happens at a much lower rate in the middle of the nucleosome; therefore, the positions where the DNA exits the nucleosome are much easier to occupy (Figure 7–11).

1	These properties of the nucleosome promote cooperative DNA binding by transcription regulators. If a regulatory protein enters the DNA of a nucleosome and prevents the DNA from tightly rewrapping around the nucleosome core, it will increase the affinity of a second transcription regulator for a nearby cis-regulatory sequence. If the two transcription regulators also interact with each other (as described above), the cooperative effect is even greater. In some cases, the combined action of the regulatory proteins can eventually displace the histone core of the nucleosome altogether. Figure 7–11 How nucleosomes effect the binding of transcription regulators.

1	Figure 7–11 How nucleosomes effect the binding of transcription regulators. “breathing” cis-regulatory sequence this open form occurs about 1/20th of the time (A) (B) compared to its affnity for naked DNA, a typical transcription regulator will bind with 20 times lower affnity if its cis-regulatory sequence is located near the end of a nucleosome a typical transcription regulator will bind with roughly 200-fold less affnity + + transcription regulatorhistone core + + + (C) if its cis-regulatory sequence is located in the middle of a nucleosome (D) one transcription regulator can destabilize the nucleosome, facilitating binding of another

1	The cooperation among transcription regulators can become much greater when nucleosome remodeling complexes are involved. If one transcription regulator binds its cis-regulatory sequence and attracts a chromatin remodeling complex, the localized action of the remodeling complex can allow a second transcription regulator to efficiently bind nearby. Moreover, we have discussed how transcription regulators can work together in pairs; in reality, larger numbers often cooperate by repeated use of the same principles. A highly cooperative binding of transcription regulators to DNA probably explains why many sites in eukaryotic genomes that are bound by transcription regulators are “nucleosome free.”

1	Transcription regulators recognize short stretches of double-helical DNA of defined sequence called cis-regulatory sequences, and thereby determine which of the thousands of genes in a cell will be transcribed. Approximately 10% of the protein-coding genes in most organisms produce transcription regulators, and they control many features of cells. Although each of these transcription regulators has unique features, most bind to DNA as homodimers or heterodimers and recognize DNA through one of a small number of structural motifs. Transcription regulators typically work in groups and bind DNA cooperatively, a feature that has several underlying mechanisms, some of which exploit the packaging of DNA in nucleosomes.

1	Having seen how transcription regulators bind to cis-regulatory sequences embedded in the genome, we can now discuss how, once bound, these proteins influence the transcription of genes. The situation in bacteria is simpler than in eukaryotes (for one thing, chromatin structure is not an issue), and we therefore discuss it first. Following this, we turn to the more complex situation in eukaryotes. the tryptophan repressor Switches Genes Off

1	the tryptophan repressor Switches Genes Off The genome of the bacterium E. coli consists of a single, circular DNA molecule of about 4.6 × 106 nucleotide pairs. This DNA encodes approximately 4300 proteins, although only a fraction of these are made at any one time. Bacteria regulate the expression of many of their genes according to the food sources that are available in the environment. For example, in E. coli, five genes code for enzymes that manufacture the amino acid tryptophan. These genes are arranged in a cluster on the chromosome and are transcribed from a single promoter as one long mRNA molecule; such coordinately transcribed clusters are called operons (Figure 7–12). Although operons are common in bacteria, they are rare in eukaryotes, where genes are typically transcribed and regulated individually (see Figure 7–3).

1	When tryptophan concentrations are low, the operon is transcribed; the resulting mRNA is translated to produce a full set of biosynthetic enzymes, which work in tandem to synthesize tryptophan from much simpler molecules. When tryptophan is abundant, however—for example, when the bacterium is in the gut of a mammal that has just eaten a protein-rich meal—the amino acid is imported into the cell and shuts down production of the enzymes, which are no longer needed. E. coli chromosome series of enzymes required for tryptophan biosynthesis

1	E. coli chromosome series of enzymes required for tryptophan biosynthesis Figure 7–12 A cluster of bacterial genes can be transcribed from a single promoter. each of these five genes encodes a different enzyme, and all of these enzymes are needed to synthesize the amino acid tryptophan from simpler molecules. the genes are transcribed as a single mrnA molecule, a feature that allows their expression to be coordinated. Clusters of genes transcribed as a single mrnA molecule are common in bacteria. each of these clusters is called an operon because its expression is controlled by a cis-regulatory sequence called the operator (green), situated within the promoter. (in this and subsequent figures, the yellow blocks in the promoter represent DnA sequences that bind rnA polymerase; see figure 6–12). start of transcription

1	start of transcription We now understand exactly how this repression of the tryptophan operon comes about. Within the operon’s promoter is a cis-regulatory sequence that is recognized by a transcription regulator. When this regulator binds to this sequence, it blocks access of RNA polymerase to the promoter, thereby preventing transcription of the operon (and thus production of the tryptophan-producing enzymes). The transcription regulator is known as the tryptophan repressor and its cis-regulatory sequence is called the tryptophan operator. These components are controlled in a simple way: the repressor can bind to DNA only if it has also bound several molecules of tryptophan (Figure 7–13).

1	The tryptophan repressor is an allosteric protein, and the binding of tryptophan causes a subtle change in its three-dimensional structure so that the protein can bind to the operator sequence. Whenever the concentration of free tryptophan in the bacterium drops, tryptophan dissociates from the repressor, the repressor no longer binds to DNA, and the tryptophan operon is transcribed. The repressor is thus a simple device that switches production of a set of biosynthetic enzymes on and off according to the availability of the end product of the pathway that the enzymes catalyze. The tryptophan repressor protein itself is always present in the cell. The gene that encodes it is continuously transcribed at a low level, so that a small amount of the repressor protein is always being made. Thus the bacterium can respond very rapidly to a rise or fall in tryptophan concentration.

1	The tryptophan repressor, as its name suggests, is a transcriptional repressor protein: in its active form, it switches genes off, or represses them. Some bacterial transcription regulators do the opposite: they switch genes on, or activate them. These transcriptional activator proteins work on promoters that—in contrast to the promoter for the tryptophan operon—are only marginally able to bind and position RNA polymerase on their own. However, these poorly functioning promoters can be made fully functional by activator proteins that bind to nearby cis-regulatory sequences and contact the RNA polymerase to help it initiate transcription (Figure 7–14).

1	Figure 7–13 Genes can be switched off by repressor proteins. if the concentration of tryptophan inside a bacterium is low (left), rnA polymerase (blue) binds to the promoter and transcribes the five genes of the tryptophan operon. however, if the concentration of tryptophan is high (right), the repressor protein (dark green) becomes active and binds to the operator (light green), where it blocks the binding of rnA polymerase to the promoter. whenever the concentration of intracellular tryptophan drops, the repressor falls off the DnA, allowing the polymerase to again transcribe the operon. Although not shown in the figure, the repressor is a stable dimer.

1	Figure 7–14 Genes can be switched on by activator proteins. An activator protein binds to its cis-regulatory sequence on the DnA and interacts with the rnA polymerase to help it initiate transcription. without the activator, the promoter fails to initiate transcription efficiently. in bacteria, the binding of the activator to DnA is often controlled by the interaction of a metabolite or other small molecule (red triangle) with the activator protein. the Lac operon works in this manner, as we discuss shortly.

1	DNA-bound activator proteins can increase the rate of transcription initiation as much as 1000-fold, a value consistent with a relatively weak and nonspecific interaction between the transcription regulator and RNA polymerase. For example, a 1000-fold change in the affinity of RNA polymerase for its promoter corresponds to a change in ∆G of ≈18 kJ/mole, which could be accounted for by just a few weak, noncovalent bonds. Thus, many activator proteins work simply by providing a few favorable interactions that help to attract RNA polymerase to the promoter. To provide this assistance, however, the activator protein must be bound to its cis-regulatory sequence, and this sequence must be positioned, with respect to the promoter, so that the favorable interactions can occur.

1	Like the tryptophan repressor, activator proteins often have to interact with a second molecule to be able to bind DNA. For example, the bacterial activator protein CAP has to bind cyclic AMP (cAMP) before it can bind to DNA. Genes activated by CAP are switched on in response to an increase in intracellular cAMP concentration, which rises when glucose, the bacterium’s preferred carbon source, is no longer available; as a result, CAP drives the production of enzymes that allow the bacterium to digest other sugars. An Activator and a repressor Control the Lac Operon

1	In many instances, the activity of a single promoter is controlled by several different transcription regulators. The Lac operon in E. coli, for example, is controlled by both the Lac repressor and the CAP activator that we just discussed. The Lac operon encodes proteins required to import and digest the disaccharide lactose. In the absence of glucose, the bacterium makes cAMP, which activates CAP to switch on genes that allow the cell to utilize alternative sources of carbon— including lactose. It would be wasteful, however, for CAP to induce expression of the Lac operon if lactose itself were not present. Thus the Lac repressor shuts off the operon in the absence of lactose. This arrangement enables the control region of the Lac operon to integrate two different signals, so that the operon is highly expressed only when two conditions are met: glucose must be absent and lactose must be present (Figure 7–15). This genetic circuit thus behaves much like

1	RNA-cis-regulatory polymerase-start of transcription sequence binding site for CAP (promoter) Figure 7–15 The Lac operon is controlled by two transcription regulators, the Lac repressor and CAP. LacZ, the first gene of the operon, encodes the enzyme β-galactosidase, which breaks down lactose to galactose and glucose. when lactose is absent, the lac repressor binds to a cisregulatory sequence, called the Lac operator, and shuts off expression of the operon (Movie 7.4). Addition of lactose increases the intracellular concentration of a related compound, allolactose; allolactose binds to the lac repressor, causing it to undergo a conformational change that releases its grip on the operator DnA (not shown). when glucose is absent, cyclic AMp (red triangle) is produced by the cell, and CAp binds to DnA.

1	a switch that carries out a logic operation in a computer. When lactose is present AND glucose is absent, the cell executes the appropriate program—in this case, transcription of the genes that permit the uptake and utilization of lactose. All transcription regulators, whether they are repressors or activators, must be bound to DNA to exert their effects. In this way, each regulatory protein acts selectively, controlling only those genes that bear a cis-regulatory sequence recognized by it. The logic of the Lac operon first attracted the attention of biologists more than 50 years ago. The way it works was uncovered by a combination of genetics and biochemistry, providing some of the first insights into how transcription is controlled in any organism.

1	We have seen that transcription activators help RNA polymerase initiate transcription and repressors hinder it. However, the two types of proteins are very similar to one another. For example, to occupy their cis-regulatory sequences, both the tryptophan repressor and the CAP activator protein must bind a small molecule; moreover, they both recognize their cis-regulatory sequences using the same structural motif (the helix–turn–helix shown in Panel 7–1). Indeed, some proteins (for example, the CAP protein) can act as both a repressor and an activator, depending on the exact placement of their cis-regulatory sequence relative to the promoter: for some genes, the CAP cis-regulatory sequence overlaps the promoter, and CAP binding thereby prevents the assembly of RNA polymerase at the promoter.

1	Most bacteria have small, compact genomes, and the cis-regulatory sequences that control the transcription of a gene are typically located very near to the start point of transcription. But there are some exceptions to this generalization— cis-regulatory sequences can be located hundreds and even thousands of nucleotide pairs from the bacterial genes they control (Figure 7–16). In these cases, the intervening DNA is looped out, allowing a protein bound at a distant site along the DNA to contact RNA polymerase. Here, the DNA acts as a tether, enormously increasing the probability that the proteins will collide, compared with the situation where one protein is bound to DNA and the other is free in solution. We will see shortly that, although it is the exception in bacteria, DNA looping occurs in the regulation of nearly every eukaryotic gene.

1	A possible explanation for this difference is based on evolutionary considerations. It has been proposed that the compact, simple genetic switches found in bacteria evolved in response to large population sizes where competition for growth put selective pressure on bacteria to maintain small genome sizes. In contrast, there appears to have been little selective pressure to “streamline” the genomes of multicellular organisms. Figure 7–16 Transcriptional activation at a distance. (A) the ntrC protein is a bacterial transcription regulator that activates transcription by directly contacting rnA polymerase. (B) the interaction of ntrC and rnA polymerase, with the intervening DnA looped out, can be seen in the electron microscope. (B, courtesy of harrison echols and Sydney Kustu.)

1	When compared to the situation in bacteria, transcription regulation in eukaryotes involves many more proteins and much longer stretches of DNA. It often seems bewilderingly complex. Yet many of the same principles apply. As in bacteria, the time and place that each gene is to be transcribed is specified by its cis-regulatory sequences, which are “read” by the transcription regulators that bind to them. Once bound to DNA, positive transcription regulators (activators) help RNA polymerase begin transcribing genes, and negative regulators (repressors) block this from happening. In bacteria, as we have seen, most of the interactions between DNA-bound transcription regulators and RNA polymerases (whether they activate or repress transcription) are direct. In contrast, these interactions are almost always indirect in eukaryotes: many intermediate proteins, including the his-tones, act between the DNA-bound transcription regulator and RNA polymerase. Moreover, in multicellular organisms, it

1	always indirect in eukaryotes: many intermediate proteins, including the his-tones, act between the DNA-bound transcription regulator and RNA polymerase. Moreover, in multicellular organisms, it is common for dozens of transcription regulators to control a single gene, with cis-regulatory sequences spread over tens of thousands of nucleotide pairs. DNA looping allows the DNA-bound regulatory proteins to interact with each other and ultimately with RNA polymerase at the promoter. Finally, because nearly all of the DNA in eukaryotic organisms is compacted by nucleosomes and higher-order structures, transcription initiation in eukaryotes must overcome this inherent block.

1	In the next sections, we discuss these features of transcription initiation in eukaryotes, emphasizing how they provide extra levels of control not found in bacteria. A eukaryotic Gene Control region Consists of a promoter plus Many cis-regulatory Sequences In eukaryotes, RNA polymerase II transcribes all the protein-coding genes and many noncoding RNA genes, as we saw in Chapter 6. This polymerase requires five general transcription factors (27 subunits in toto; see Table 6–3, p. 311), in contrast to bacterial RNA polymerase, which needs only a single general transcription factor (the σ subunit). As we have seen, the stepwise assembly of the general transcription factors at a eukaryotic promoter provides, in principle, multiple steps at which the cell can speed up or slow down the rate of transcription initiation in response to transcription regulators.

1	Because the many cis-regulatory sequences that control the expression of a typical gene are often spread over long stretches of DNA, we use the term gene control region to describe the whole expanse of DNA involved in regulating and initiating transcription of a eukaryotic gene. This includes the promoter, where the general transcription factors and the polymerase assemble, plus all of the cis-regulatory sequences to which transcription regulators bind to control the rate of the assembly processes at the promoter (Figure 7–17). In animals and plants, it is not unusual to find the regulatory sequences of a gene dotted over stretches of DNA as large as 100,000 nucleotide pairs. Some of this DNA is transcribed (but not translated), and we discuss these long noncoding RNAs (lncRNAs) later in this chapter. For now, we can regard much of this DNA as “spacer” sequences that transcription regulators do not directly recognize. It is important to keep in mind that, like other regions of

1	in this chapter. For now, we can regard much of this DNA as “spacer” sequences that transcription regulators do not directly recognize. It is important to keep in mind that, like other regions of eukaryotic chromosomes, most of the DNA in gene control regions is packaged into nucleosomes and higher-order forms of chromatin, thereby compacting its overall length and altering its properties.

1	In this chapter, we shall loosely use the term gene to refer to a segment of DNA that is transcribed into a functional RNA molecule, one that either codes for a protein or has a different role in the cell (see Table 6–1, p. 305). However, the classical view of a gene includes the gene control region as well, since mutations in it can produce an altered phenotype. Alternative RNA splicing further complicates the definition of a gene—a point we shall return to later.

1	In contrast to the small number of general transcription factors, which are abundant proteins that assemble on the promoters of all genes transcribed by the gene control region for gene X TATA TATA promoter “spacer” DNA coactivatorstranscription regulators general transcription factors RNA polymerase II gene X cis-regulatory sequence gene X Mediator Figure 7–17 The gene control region for a typical eukaryotic gene. the promoter is the DnA sequence where the general transcription factors and the polymerase assemble (see figure 6–15). the cis-regulatory sequences are binding sites for transcription regulators, whose presence on the DnA affects the rate of transcription initiation. these sequences can be located adjacent to the promoter, far upstream of it, or even within introns or entirely downstream of the gene. the broken stretches of DnA signify that the length of DnA between the cis-regulatory sequences and the start of transcription

1	RNA polymerase II, there are thousands of different transcription regulators devoted to turning individual genes on and off. In eukaryotes, operons—sets of genes transcribed as a unit—are rare, and, instead, each gene is regulated individually. Not surprisingly, the regulation of each gene is different in detail from that of every other gene, and it is difficult to formulate simple rules for gene regulation that apply in every case. We can, however, make some generalizations about how transcription regulators, once bound to gene control regions on DNA, set in motion the series of events that lead to gene activation or repression.

1	In bacteria, we saw that proteins such as the tryptophan repressor, the Lac repressor, and the CAP protein bind to DNA on their own and directly affect RNA polymerase at the promoter. Eukaryotic transcription regulators, in contrast, usually assemble in groups at their cis-regulatory sequences. Often two or more regulators bind cooperatively, as discussed earlier in the chapter. In addition, a broad class of multisubunit proteins termed coactivators and co-repressors assemble on DNA with them. Typically, these coactivators and co-repressors do not recognize specific DNA sequences themselves; they are brought to those sequences by the transcription regulators. Often the protein–protein interactions between transcription regulators and between regulators and coactivators are too weak for them to assemble in solution; however, the appropriate combination of cis-regulatory sequences can “crystallize” the assembly of these complexes on DNA (Figure 7–18).

1	As their names imply, coactivators are typically involved in activating transcription and co-repressors in repressing it. In the following sections, we will see that coactivators and co-repressors can act in a variety of different ways to influence transcription after they have been localized on the genome by transcription regulators.

1	As shown in Figure 7–18, an individual transcription regulator can often participate in more than one type of regulatory complex. A protein might function, varies, sometimes reaching tens of thousands of nucleotide pairs in length. the TATA box is a DnA recognition sequence for the general transcription factor tfiiD. As shown in the lower panel, DnA looping allows transcription regulators bound at any of these positions to interact with the proteins that assemble at the promoter. Many transcription regulators act through Mediator (described in Chapter 6), while some interact with the general transcription factors and rnA polymerase directly. transcription regulators also act by recruiting proteins that alter the chromatin structure of the promoter (not shown, but discussed below).

1	whereas Mediator and the general transcription factors are the same for all rnA polymerase ii-transcribed genes, the transcription regulators and the locations of their binding sites relative to the promoter differ for each gene. for example, in one case as part of a complex that activates transcription and in another case as part of a complex that represses transcription. Thus, individual eukaryotic transcription regulators function as regulatory parts that are used to build complexes whose function depends on the final assembly of all of the individual components. Each eukaryotic gene is therefore regulated by a “committee” of proteins, all of which must be present to express the gene at its proper level. Activator proteins promote the Assembly of rnA polymerase at the Start point of transcription

1	Activator proteins promote the Assembly of rnA polymerase at the Start point of transcription The cis-regulatory sequences to which eukaryotic transcription activator proteins bind were originally called enhancers because their presence “enhanced” the rate of transcription initiation. It came as a surprise when it was discovered that these sequences could be found tens of thousands of nucleotide pairs away from the promoter; as we have seen, DNA looping, which was not widely appreciated at the time, can now explain this initially puzzling observation. Once bound to DNA, how do assemblies of activator proteins increase the rate of transcription initiation? At most genes, mechanisms work in concert. Their function is both to attract and position RNA polymerase II at the promoter and to release it so that transcription can begin.

1	Some activator proteins bind directly to one or more of the general transcription factors, accelerating their assembly on a promoter that has been brought in proximity—through DNA looping—to that activator. Most transcription activators, however, attract coactivators that then perform the biochemical tasks needed to initiate transcription. One of the most prevalent coactivators is the large Mediator protein complex, composed of more than 30 subunits. About the same size as RNA polymerase itself, Mediator serves as a bridge between DNA-bound transcription activators, RNA polymerase, and the general transcription factors, facilitating their assembly at the promoter (see Figure 7–17). eukaryotic transcription Activators Direct the Modification of local Chromatin Structure

1	eukaryotic transcription Activators Direct the Modification of local Chromatin Structure The eukaryotic general transcription factors and RNA polymerase are unable, on their own, to assemble on a promoter that is packaged in nucleosomes. Thus, in addition to directing the assembly of the transcription machinery at the promoter, eukaryotic transcription activators promote transcription by triggering changes to the chromatin structure of the promoters, making the underlying DNA more accessible. The most important ways of locally altering chromatin are through covalent histone modifications, nucleosome remodeling, nucleosome removal, and his-tone replacement (discussed in Chapter 4). Eukaryotic transcription activators use all four of these mechanisms: thus they attract coactivators that include his-tone modification enzymes, ATP-dependent chromatin remodeling complexes, and histone chaperones, each of which can alter the chromatin structure of

1	Figure 7–18 Eukaryotic transcription regulators assemble into complexes on DNA. (A) Seven transcription regulators are shown. the nature and function of the complex they form depend on the specific cis-regulatory sequences that seed their assembly. (B) Some assembled complexes activate gene transcription, while another represses transcription. note that the light green and dark green proteins are shared by both activating and repressing complexes. proteins that do not themselves bind DnA but assemble on other DnA-bound transcription regulators are termed coactivators or co-repressors. in some cases (lower right), rnA molecules are found in these assemblies. As described later in this chapter, these rnAs often act as scaffolds to hold a group of proteins together.

1	promoters (Figure 7–19). These local alterations in chromatin structure provide greater access to DNA, thereby facilitating the assembly of the general transcription factors at the promoter. In addition, some histone modifications specifically attract these proteins to the promoter. These mechanisms often work together during transcription initiation (Figure 7–20). Finally, as discussed earlier in this chapter, the local chromatin changes directed by one transcriptional regulator can allow the binding of additional regulators. By repeated use of this principle, large assemblies of proteins can form on control regions of genes to regulate their transcription.

1	The alterations of chromatin structure that occur during transcription initiation can persist for different lengths of time. In some cases, as soon as the transcription regulator dissociates from DNA, the chromatin modifications are rapidly reversed, restoring the gene to its pre-activated state. This rapid reversal is especially important for genes that the cell must quickly switch on and off in response to external signals. In other cases, the altered chromatin structure persists, even after the transcription regulator that directed its establishment has dissociated from DNA. In principle, this memory can extend into the next cell generation because, as discussed in Chapter 4, chromatin structure can be self-renewing (see Figure 4–44). The fact that different histone modifications persist for different times provides the cell with a mechanism that makes possible both longerand shorter-term memory of gene expression patterns.

1	A special type of chromatin modification occurs as RNA polymerase II transcribes through a gene. The histones just ahead of the polymerase can be acetylated by enzymes carried by the polymerase, removed by histone chaperones, and deposited behind the moving polymerase. These histones are then rapidly deacetylated and methylated, also by complexes that are carried by the polymerase, leaving behind nucleosomes that are especially resistant to transcription. This remarkable process seems to prevent spurious transcription reinitiation Figure 7–19 Eukaryotic transcription activator proteins direct local alterations in chromatin structure. nucleosome remodeling, nucleosome removal, histone replacement, and certain types of histone modifications favor transcription initiation (see figure 4–39). these alterations increase the accessibility of DnA and facilitate the binding of rnA polymerase and the general transcription factors.

1	Figure 7–20 Successive histone modifications during transcription initiation. in this example, taken from the human interferon gene promoter, a transcription activator binds to DnA packaged into chromatin and attracts a histone acetyl transferase that acetylates lysine 9 of histone h3 and lysine 8 of histone h4. then a histone kinase, also attracted by the transcription activator, phosphorylates serine 10 of histone h3 but it can only do so after lysine 9 has been acetylated. this serine modification signals the histone acetyl transferase to acetylate position K14 of histone h3. next, the general transcription factor tfiiD and a chromatin remodeling complex bind to the chromatin to promote the subsequent steps of transcription initiation. tfiiD and the remodeling complex both recognize acetylated histone tails through a bromodomain, a protein domain specialized to read this particular mark on histones; a bromodomain is carried in a subunit of each protein complex.

1	the histone acetyl transferase, the histone kinase, and the chromatin remodeling complex are all coactivators. the order of events shown applies to a specific promoter; at other genes, the steps may occur in a different order or individual steps may be omitted altogether. (Adapted from t. Agalioti, G. Chen and D. thanos, Cell 111:381–392, 2002. with permission from elsevier.) behind a moving polymerase, which, in essence, must clear a path through chromatin as it transcribes. Later in this chapter, when we discuss RNA interference, the potential dangers to the cell of such inappropriate transcription will become especially obvious. The modification of nucleosomes behind a moving RNA polymerase also plays an important role in RNA splicing (see p. 323).

1	In some cases, transcription initiation requires that a DNA-bound transcription activator releases RNA polymerase from the promoter so as to allow it to begin transcribing the gene. In other cases, the RNA polymerase halts after transcribing about 50 nucleotides of RNA, and further elongation requires a transcription activator bound behind it (Figure 7–21). These paused polymerases are common in humans, where a significant fraction of genes that are not being transcribed have a paused polymerase located just downstream from the promoter.

1	The release of RNA polymerase can occur in several ways. In some cases, the activator brings in a chromatin remodeling complex that removes a nucleosome block to the elongating RNA polymerase. In other cases, the activator communicates with RNA polymerase (typically through a coactivator), signaling it to move ahead. Finally, as we saw in Chapter 6, RNA polymerase requires elongation factors to effectively transcribe through chromatin. In some cases, the key step in gene activation is the loading of these factors onto RNA polymerase, which can be directed by DNA-bound transcription activators. Once loaded, these factors allow the polymerase to move through blocks imposed by chromatin structure and begin transcribing the gene in earnest. Having RNA polymerase already poised on a promoter in the beginning stages of transcription bypasses the step of assembling many components at the promoter, which is often slow. This mechanism can therefore allow cells to begin transcribing a gene as a

1	the beginning stages of transcription bypasses the step of assembling many components at the promoter, which is often slow. This mechanism can therefore allow cells to begin transcribing a gene as a rapid response to an extracellular signal.

1	We have seen that complexes of transcription activators and coactivators assemble cooperatively on DNA. We have also seen that these assemblies can promote different steps in transcription initiation. In general, where several factors work together to enhance a reaction rate, the joint effect is not merely the sum of the enhancements that each factor alone contributes, but the product. If, for example, factor A lowers the free-energy barrier for a reaction by a certain amount and thereby speeds up the reaction 100-fold, and factor B, by acting on another aspect of the reaction, does likewise, then A and B acting in parallel will lower the barrier

1	Figure 7–21 Transcription activators can act at different steps. in addition to (A) promoting binding of additional transcription regulators and (B) assembling rnA polymerase at promoters, transcription activators are often needed (C) to release already assembled rnA polymerases from promoters or (D) to release rnA polymerase molecules that become stalled after transcribing about 50 nucleotides of rnA. the activities shown in figure 7–19 can affect each of these four steps. by a double amount and speed up the reaction 10,000-fold. Even if A and B work simply by attracting the same protein, the affinity of that protein for the reaction site increases multiplicatively. Thus, transcription activators often exhibit transcriptional synergy, where several DNA-bound activator proteins working together produce a transcription rate that is much higher than the sum of their transcription rates working alone (Figure 7–22).

1	An important point is that a transcription activator protein must be bound to DNA to influence transcription of its target gene. And the rate of transcription of a gene ultimately depends upon the spectrum of regulatory proteins bound upstream and downstream of its transcription start site, along with the coactivator proteins they bring to DNA. Although the “default” state of eukaryotic DNA packaged into nucleosomes is resistant to transcription, eukaryotes nonetheless use transcription regulators to Figure 7–22 Transcriptional synergy. this experiment compares the rate of transcription produced by three experimentally constructed regulatory regions in a eukaryotic cell and reveals transcriptional synergy, a greater than additive effect of multiple activators working together. for simplicity, coactivators have been omitted from the diagram.

1	Such transcriptional synergy is not only observed between different transcription activators from the same organism; it is also seen between activator proteins from different eukaryotic species when they are experimentally introduced into the same cell. this last observation reflects the high degree of conservation of the machinery responsible for eukaryotic transcription initiation. recruitment of chromatin masking the activation surface for activator for repressor binding site recruitment of with the general transcription factors recruitment of TATA histone methyl transferase histone methylation proteins that bind to methylated histones

1	Figure 7–23 Six ways in which eukaryotic repressor proteins can operate. (A) Activator proteins and repressor proteins compete for binding to the same regulatory DnA sequence. (B) Both proteins bind DnA, but the repressor prevents the activator from carrying out its functions. (C) the repressor blocks assembly of the general transcription factors. (D) the repressor recruits a chromatin remodeling complex, which returns the nucleosomal state of the promoter region to its pre-transcriptional form. (e) the repressor attracts a histone deacetylase to the promoter. As we have seen, histone acetylation can stimulate transcription initiation (see figure 7–20), and the repressor simply reverses this modification. (f) the repressor attracts a histone methyl transferase, which modifies certain positions on histones by attaching methyl groups; the methylated histones, in turn, are bound by proteins that maintain the chromatin in a transcriptionally silent form.

1	repress the transcription of genes. These transcription repressors can both depress the rate of transcription below the default value and rapidly shut off genes that were previously activated. We saw in Chapter 4 that large regions of the genome can be shut down by the packaging of DNA into especially resistant forms of chromatin. However, eukaryotic genes are rarely organized along the genome according to function, and this strategy is not generally applicable for shutting off a set of genes that work together. Instead, most eukaryotic repressors work on a geneby-gene basis. Unlike bacterial repressors, eukaryotic repressors do not directly compete with the RNA polymerase for access to the DNA; rather, they use a variety of other mechanisms, some of which are illustrated in Figure 7–23. Although all of these mechanisms ultimately block transcription by RNA polymerase, eukaryotic transcription repressors typically act by bringing co-repressors to DNA. Like transcription activation,

1	Although all of these mechanisms ultimately block transcription by RNA polymerase, eukaryotic transcription repressors typically act by bringing co-repressors to DNA. Like transcription activation, transcription repression can act through more than one mechanism at a given target gene, thereby ensuring especially efficient repression.

1	Gene repression is especially important to animals and plants whose growth depends on elaborate and complex developmental programs. Misexpression of a single gene at a critical time can have disastrous consequences for the individual. For this reason, many of the genes encoding the most important developmental regulatory proteins are kept tightly repressed when they are not needed. sequence domain of actively Figure 7–24 Schematic diagram summarizing the properties of insulators and barrier sequences. (A) insulators directionally block the action of cis-regulatory sequences, whereas barrier sequences prevent the spread of heterochromatin. how barrier sequences likely function is depicted in figure 4–41. (B) insulator-binding proteins (purple) hold chromatin in loops, thereby favoring “correct” cis-regulatory sequence–gene associations. thus, gene B is properly regulated, and gene B’s cisregulatory sequences are prevented from influencing the transcription of gene A.

1	We have seen that all genes have control regions, which dictate at which times, under what conditions, and in what tissues the gene will be expressed. We have also seen that eukaryotic transcription regulators can act across very long stretches of DNA, with the intervening DNA looped out. How, then, are control regions of different genes kept from interfering with one another? For example, what keeps a transcription regulator bound on the control region of one gene from looping in the wrong direction and inappropriately influencing the transcription of an adjacent gene?

1	To avoid such cross-talk, several types of DNA elements compartmentalize the genome into discrete regulatory domains. In Chapter 4, we discussed barrier sequences that prevent the spread of heterochromatin into genes that need to be expressed. A second type of DNA element, called an insulator, prevents cis-regulatory sequences from running amok and activating inappropriate genes (Figure 7–24). Insulators function by forming loops of chromatin, an effect mediated by specialized proteins that bind them (see Figures 4–48 and 7–24B). The loops hold a gene and its control region in rough proximity and help to prevent the control region from “spilling over” to adjacent genes. Importantly, these loops can be in different in different cell types, depending on the particular proteins and chromatin structures that are present.

1	The distribution of insulators and barrier sequences in a genome is thought to divide it into independent domains of gene regulation and chromatin structure (see pp. 207–208). Aspects of this organization can be visualized by staining whole chromosomes for the specialized proteins that bind these DNA elements (Figure 7–25). Figure 7–25 Localization of a Drosophila insulator-binding protein on polytene chromosomes.

1	Figure 7–25 Localization of a Drosophila insulator-binding protein on polytene chromosomes. A polytene chromosome (discussed in Chapter 4) was stained with propidium iodide (red) to show its banding patterns, with bands appearing bright red and interbands as dark gaps in the pattern (top). the positions on this polytene chromosome that are bound by a particular insulator protein are stained bright green using antibodies directed against the protein (bottom). this protein is preferentially localized to interband regions, reflecting its role in organizing chromosomes into structural, as well as functional, domains. for convenience, these two micrographs of the same polytene chromosome are arranged as mirror images. (Courtesy of Uli laemmli, from K. Zhao et al., Cell 81:879–889, 1995. with permission from elsevier.)

1	Although chromosomes are organized into orderly domains that discourage control regions from acting indiscriminately, there are special circumstances where a control region located on one chromosome has been found to activate a gene located on a different chromosome. Although there is much we do not understand about this mechanism, it indicates the extreme versatility of transcriptional regulation strategies.

1	Transcription regulators switch the transcription of individual genes on and off in cells. In prokaryotes, these proteins typically bind to specific DNA sequences close to the RNA polymerase start site and, depending on the nature of the regulatory protein and the precise location of its binding site relative to the start site, either activate or repress transcription of the gene. The flexibility of the DNA helix, however, also allows proteins bound at distant sites to affect the RNA polymerase at the promoter by the looping out of the intervening DNA. The regulation of higher eukaryotic genes is much more complex, commensurate with a larger genome size and the large variety of cell types that are formed. A single eukaryotic gene is typically controlled by many transcription regulators bound to sequences that can be tens or even hundreds of thousands of nucleotide pairs from the promoter that directs transcription of the gene. Eukaryotic activators and repressors act by a wide variety

1	to sequences that can be tens or even hundreds of thousands of nucleotide pairs from the promoter that directs transcription of the gene. Eukaryotic activators and repressors act by a wide variety of mechanisms—generally altering chromatin structure and controlling the assembly of the general transcription factors and RNA polymerase at the promoter. They do this by attracting coactivators and co-repressors, protein complexes that perform the necessary biochemical reactions. The time and place that each gene is transcribed, as well as its rates of transcription under different conditions, are determined by the particular spectrum of transcription regulators that bind to the regulatory region of the gene.

1	Although all cells must be able to switch genes on and off in response to changes in their environments, the cells of multicellular organisms have evolved this capacity to an extreme degree. In particular, once a cell in a multicellular organism becomes committed to differentiate into a specific cell type, the cell maintains this choice through many subsequent cell generations, which means that it remembers the changes in gene expression involved in the choice. This phenomenon of cell memory is a prerequisite for the creation of organized tissues and for the maintenance of stably differentiated cell types. In contrast, other changes in gene expression in eukaryotes, as well as most such changes in bacteria, are only transient. The tryptophan repressor, for example, switches off the tryptophan genes in bacteria only in the presence of tryptophan; as soon as tryptophan is removed from the medium, the genes are switched back on, and the descendants of the cell will have no memory that

1	genes in bacteria only in the presence of tryptophan; as soon as tryptophan is removed from the medium, the genes are switched back on, and the descendants of the cell will have no memory that their ancestors had been exposed to tryptophan.

1	In this section, we shall examine not only cell memory mechanisms, but also how gene regulatory devices can be combined to create the “logic circuits” through which cells integrate signals and remember events in their past. We begin by considering one such complex gene control region in detail. We have seen that transcription regulators can be positioned at multiple sites along long stretches of DNA and that these proteins can bring into play coactivators and co-repressors. Here, we discuss how the numerous transcription regulators that are bound to the control region of a gene can cause the gene to be transcribed at the proper place and time.

1	Consider the Drosophila Even-skipped (Eve) gene, whose expression plays an important part in the development of the Drosophila embryo. If this gene is inactivated by mutation, many parts of the embryo fail to form, and the embryo dies early in development. As discussed in Chapter 21, at the stage of development when Eve begins to be expressed, the embryo is a single giant cell containing multiple nuclei in a common cytoplasm. This cytoplasm contains a mixture of transcription regulators that are distributed unevenly along the length of the embryo, thus providing positional information that distinguishes one part of the embryo from another (Figure 7–26). Although the nuclei are initially identical, they rapidly begin to express different genes because they are exposed to different transcription regulators. For example, the nuclei near the anterior end of the developing embryo are exposed to a set of transcription regulators that is distinct from the set that influences nuclei at the

1	regulators. For example, the nuclei near the anterior end of the developing embryo are exposed to a set of transcription regulators that is distinct from the set that influences nuclei at the middle or at the posterior end of the embryo.

1	The regulatory DNA sequences that control the Eve gene have evolved to “read” the concentrations of transcription regulators at each position along the length of the embryo, and they cause the Eve gene to be expressed in seven precisely positioned stripes, each initially five to six nuclei wide (Figure 7–27). How is this remarkable feat of information processing carried out? Although there is still much to learn, several general principles have emerged from studies of Eve and other genes that are similarly regulated.

1	The regulatory region of the Eve gene is very large (approximately 20,000 nucleotide pairs). It is formed from a series of relatively simple regulatory modules, each of which contains multiple cis-regulatory sequences and is responsible for specifying a particular stripe of Eve expression along the embryo. This modular organization of the Eve gene control region was revealed by experiments in which a particular regulatory module (say, that specifying stripe 2) is removed from its normal setting upstream of the Eve gene, placed in front of a reporter gene, and reintroduced into the Drosophila genome. When developing embryos derived from flies carrying this genetic construct are examined, the reporter gene is found to be expressed in precisely the position of stripe 2 (Figure 7–28). Similar experiments reveal the existence of other regulatory modules, each of which specifies other stripes.

1	Figure 7–27 The seven stripes of the protein encoded by the Even-skipped (Eve) gene in a developing Drosophila embryo. two and one-half hours after fertilization, the egg was fixed and stained with antibodies that recognize the eve protein (green) and antibodies that recognize the Giant protein (red). where eve and Giant proteins are both present, the staining appears yellow. At this stage in development, the egg contains approximately 4000 nuclei. the eve and Giant proteins are both located in the nuclei, and the eve stripes are about four nuclei wide. the pattern for the Giant protein is also shown in figure 7–26. (Courtesy of Michael levine.)

1	Figure 7–26 The nonuniform distribution of transcription regulators in an early Drosophila embryo. At this stage, the embryo is a syncytium; that is, multiple nuclei are contained in a common cytoplasm. Although not shown in these drawings, all of these proteins are concentrated in the nuclei. how such differences are established is discussed in Chapter 21. start of segment INSERT start of transcription

1	start of segment INSERT start of transcription Figure 7–28 Experiment demonstrating the modular construction of the Eve gene regulatory region. (A) A 480-nucleotidepair section of the Eve regulatory region was removed and (B) inserted upstream of a test promoter that directs the synthesis of the enzyme β-galactosidase (the product of the E. coli LacZ gene—see figure 7–15). (C, D) when this artificial construct was reintroduced into the genome of Drosophila embryos, the embryos (D) expressed β-galactosidase (detectable by histochemical staining) precisely in the position of the second of the seven Eve stripes (C). β-Galactosidase is simple to detect and thus provides a convenient way to monitor the expression specified by a gene control region. As used here, β-galactosidase is said to serve as a reporter, since it “reports” the activity of a gene control region. (C and D, courtesy of Stephen Small and Michael levine.) the Drosophila Eve Gene is regulated by Combinatorial Controls

1	A detailed study of the stripe 2 regulatory module has provided insights into how it reads and interprets positional information. The module contains recognition sequences for two transcription regulators (Bicoid and Hunchback) that activate Eve transcription and for two transcription regulators (Krüppel and Giant) that repress it (Figure 7–29). The relative concentrations of these four proteins determine whether the protein complexes that form at the stripe 2 module activate transcription of the Eve gene. Figure 7–30 shows the distributions of the four transcription regulators across the region of a Drosophila embryo where stripe 2 forms. It is thought that either of the two repressor proteins, when bound to the DNA, will turn off the stripe 2 module, whereas both Bicoid and Hunchback must bind for this module’s maximal activation. This simple regulatory scheme suffices to turn on the stripe 2 module (and therefore the expression of the Eve gene) only in those nuclei located where

1	must bind for this module’s maximal activation. This simple regulatory scheme suffices to turn on the stripe 2 module (and therefore the expression of the Eve gene) only in those nuclei located where the levels of both Bicoid and Hunchback are high and both Krüppel and Giant are absent—a combination that occurs in only one region of the early embryo. It is not known exactly how these four transcription regulators interact with coactivators and co-repressors to specify the final level of transcription across the stripe, but the outcome very likely relies on competition between activators and repressors that act by the mechanisms outlined in Figures 7–17, 7–19, and 7–23.

1	The stripe 2 element is autonomous, inasmuch as it specifies stripe 2 when isolated from its normal context (see Figure 7–28). The other stripe regulatory modules are thought to be constructed similarly, reading positional information provided by other combinations of transcription regulators. The entire Eve gene control region binds more than 20 different transcription regulators. Seven combinations of regulators—one combination for each stripe—specify Eve expression, while many other combinations (all those found in the interstripe regions of

1	Figure 7–29 The Eve stripe 2 unit. the segment of the Eve gene control region identified in figure 7–28 contains cisregulatory sequences for four transcription regulators. it is known from genetic experiments that these four regulatory proteins are responsible for the proper expression of Eve in stripe 2. flies that are deficient in the two gene activators Bicoid and hunchback, for example, fail to efficiently express Eve in stripe 2. in flies deficient in either of the two gene repressors, Giant and Krüppel, stripe 2 expands and covers an abnormally broad region of the embryo. As indicated, in some cases the binding sites for the transcription regulators overlap, and the proteins can compete for binding to the DnA. for example, binding of Krüppel and binding of Bicoid to the site at the far right is mutually exclusive.

1	the embryo) keep the stripe elements silent. A large and complex control region is thereby built from a series of smaller modules, each of which consists of a unique arrangement of short cis-regulatory sequences recognized by specific transcription regulators. The Eve gene itself encodes a transcription regulator, which, after its pattern of expression is set up in seven stripes, controls the expression of other Drosophila genes. As development proceeds, the embryo is thus subdivided into finer and finer regions that eventually give rise to the different body parts of the adult fly, as discussed in Chapter 21.

1	Eve exemplifies the complex control regions found in plants and animals. As this example shows, control regions can respond to many different inputs, integrate this information, and produce a complex spatial and temporal output as development proceeds. However, exactly how all these mechanisms work together to produce the final output is understood only in broad outline (Figure 7–31).

1	The above example from Drosophila clearly illustrates the power of combinatorial control, but this case is unusual in that the nuclei are exposed directly to positional cues in the form of concentrations of transcription regulators. In embryos of most other organisms and in all adults, individual nuclei are in separate cells, and extracellular information (including positional cues) must be passed across the plasma membrane so as to generate signals in the cytosol that cause different transcription regulators to become active in different cell types. Some of the different mechanisms that are known to be used to activate transcription regulators are diagrammed in Figure 7–32, and in Chapter 15, we discuss how extracellular signals trigger these changes. strongly activating assembly neutral assembly of

1	strongly activating assembly neutral assembly of Figure 7–30 Distribution of the transcription regulators responsible for ensuring that Eve is expressed in stripe 2. the distributions of these proteins were visualized by staining a developing Drosophila embryo with antibodies directed against each of the four proteins. the expression of Eve in stripe 2 occurs only at the position where the two activators (Bicoid and hunchback) are present and the two repressors (Giant and Krüppel) are absent. in fly embryos that lack Krüppel, for example, stripe 2 expands posteriorly. likewise, stripe 2 expands posteriorly if the DnA-binding sites for Krüppel in the stripe 2 module are inactivated by mutation (see also figures 7–26 and 7–27).

1	Figure 7–31 The integration of multiple inputs at a promoter. Multiple sets of transcription regulators, coactivators, and co-repressors can work together to influence transcription initiation at a promoter, as they do in the Eve stripe 2 module illustrated in figure 7–29. it is not yet understood in detail how the cell achieves integration of multiple inputs, but it is likely that the final transcriptional activity of the gene results from a competition between activators and repressors that act by the mechanisms summarized in figures 7–17, 7–19, and 7–23.

1	We have seen that transcription regulators can act in combination to control the expression of an individual gene. It is also generally true that each transcription regulator in an organism contributes to the control of many genes. This point is illustrated schematically in Figure 7–33, which shows how combinatorial gene control makes it possible to generate a great deal of biological complexity even with relatively few transcription regulators. Due to combinatorial control, a given transcription regulator does not necessarily have a single, simply definable function as commander of a particular battery of genes or specifier of a particular cell type. Rather, transcription regulators can be likened to the words of a language: they are used with different meanings in a variety of contexts and rarely alone; it is the well-chosen combination that conveys the information that specifies a gene regulatory event.

1	Combinatorial gene control causes the effect of adding a new transcription regulator to a cell to depend on that cell’s past history, since it is this history that determines which transcription regulators are already present. Thus, during development, a cell can accumulate a series of transcription regulators that need not initially alter gene expression. The addition of the final members of the requisite combination of transcription regulators will complete the regulatory message, and can lead to large changes in gene expression.

1	The importance of combinations of transcription regulators for the specification of cell types is most easily demonstrated by their ability—when expressed artificially—to convert one type of cell to another. Thus, the artificial expression of three neuron-specific transcription regulators in liver cells can convert the liver cells into functional nerve cells (Figure 7–34). In some cases, expression of even a single transcription regulator is sufficient to convert one cell type to another. For example, when the gene encoding the transcription regulator MyoD is artificially introduced into fibroblasts cultured from skin connective tissue, the fibroblasts form muscle-like cells. As discussed in Chapter 22, fibroblasts, which are derived from the same broad class of embryonic cells as muscle cells, have already accumulated many of the other necessary transcription regulators required for the

1	Figure 7–32 Some ways in which the activity of transcription regulators is controlled inside eukaryotic cells. (A) the protein is synthesized only when needed and is rapidly degraded by proteolysis so that it does not accumulate. (B) Activation by ligand binding. (C) Activation by covalent modification. phosphorylation is shown here, but many other modifications are possible (see table 3–3, p. 165). formation of a complex between a DnA-binding protein and a separate protein with a transcription-activating domain. Unmasking of an activation domain by the phosphorylation of an inhibitor protein. Stimulation of nuclear entry by removal of an inhibitory protein that otherwise keeps the regulatory protein from entering the nucleus. (G) release of a transcription regulator from a membrane bilayer by regulated proteolysis.

1	combinatorial control of the muscle-specific genes, and the addition of MyoD completes the unique combination required to direct the cells to become muscle. An even more striking example is seen by artificially expressing, early in development, a single Drosophila transcription regulator (Eyeless) in groups of cells Figure 7–34 A small set of transcription regulators can convert one differentiated cell type into another. in this experiment, (A) liver cells grown in culture were converted into (B) neuronal cells via the artificial expression of three nerve-specific transcription regulators. Both types of cells express an artificial red fluorescent protein, which is used to visualize them. this conversion involves the activation of many nerve-specific genes as well as the repression of many liver-specific genes. (from S. Marro et al., Cell Stem Cell 9:374–382, 2011. with permission from elsevier.)

1	Figure 7–33 The importance of combinatorial gene control for development. Combinations of a few transcription regulators can generate many cell types during development. in this simple, idealized scheme, a “decision” to make one of a pair of different transcription regulators (shown as numbered circles) is made after each cell division. Sensing its relative position in the embryo, the daughter cell toward the left side of the embryo is always induced to synthesize the even-numbered protein of each pair, while the daughter cell toward the right side of the embryo is induced to synthesize the odd-numbered protein. the production of each transcription regulator is assumed to be self-perpetuating once it has become initiated (see figure 7–39). in this way, through cell memory, the final combinatorial specification is built up step by step. in this purely hypothetical example, five different transcription regulators have created eight final cell types (G–n).

1	fy with Eyeless gene artifcially that would normally go on to form leg parts. Here, this abnormal gene expression change causes eye-like structures to develop in the legs (Figure 7–35). Specialized Cell types Can Be experimentally reprogrammed to Become pluripotent Stem Cells

1	Specialized Cell types Can Be experimentally reprogrammed to Become pluripotent Stem Cells Manipulation of transcription regulators can also coax various differentiated cells to de-differentiate into pluripotent stem cells that are capable of giving rise to the different cell types in the body, much like the embryonic stem (ES) cells discussed in Chapter 22. When three specific transcription regulators are artificially expressed in cultured mouse fibroblasts, a number of cells become induced pluripotent stem cells (iPS cells)—cells that look and behave like the pluripotent ES cells that are derived from embryos (Figure 7–36). This approach has been adapted to produce iPS cells from a variety of specialized cell types, including cells taken from humans. Such human iPS cells can then be directed to generate a population of differentiated cells for use in the study or treatment of disease, as we discuss in Chapter 22.

1	Although it was once thought that cell differentiation was irreversible, it is now clear that by manipulating combinations of master transcription regulators, cell types and differentiation pathways can be readily altered. Combinations of Master transcription regulators Specify Cell types by Controlling the expression of Many Genes As we saw in the introduction of this chapter, different cell types of multicellular organisms differ enormously in the proteins and RNAs they express. For example, only muscle cells express special types of actin and myosin that form the contractile Figure 7–35 Expression of the Drosophila Eyeless gene in precursor cells of the leg triggers the development of an eye on the leg. (A) Simplified diagrams showing the result when a fruit fly larva contains either the normally expressed Eyeless gene (left) or an Eyeless gene that is additionally expressed artificially in cells that normally give rise to leg tissue (right).

1	(B) photograph of an abnormal leg that contains a misplaced eye (see also figure 21–2). the transcription regulator was named eyeless because its inactivation in otherwise normal flies causes the loss of eyes. (B, courtesy of walter Gehring.) Figure 7–36 A combination of transcription regulators can induce a differentiated cell to de-differentiate into a pluripotent cell. the artificial expression of a set of three genes, each of which encodes a transcription regulator, can reprogram a fibroblast into a pluripotent cell with embryonic stem (eS)-cell-like properties. like eS cells, such induced pluripotent stem (ipS) cells can proliferate indefinitely in culture and can be stimulated by appropriate extracellular signal molecules to differentiate into almost any cell type found in the body. transcription regulators such as Oct4, Sox2, and Klf4 are often called master transcription regulators because their expression is sufficient to trigger a change in cell identity.

1	apparatus, while nerve cells must make and assemble all the proteins needed to form dendrites and synapses. We have seen that these patterns of cell-type-specific expression are orchestrated by a combination of master transcription regulators. In many cases, these proteins bind directly to cis-regulatory sequences of the genes particular to that cell type. Thus, MyoD binds directly to cis-regulatory sequences located in the control regions of the muscle-specific genes. In other cases, the master regulators control the expression of “downstream” transcription regulators which, in turn, bind to the control regions of other cell-type-specific genes and control their synthesis.

1	The specification of a particular cell type typically involves changes in the expression of several thousand genes. Genes whose protein products are required in the cell type are expressed at high levels, while those not needed are typically down-regulated. As might be imagined, the pattern of binding between the master regulators and all of the regulated genes can be extremely elaborate (Figure 7–37). When we consider that many of these regulated genes have control regions that span tens of thousands of nucleotide pairs, commensurate with the Eve example discussed above, we can begin to appreciate the enormous complexity of cell-type specification. An outstanding question in biology is how the information in a genome is used to specify a multicellular organism. Although we have the general outline of the answer, we are far from understanding how a single cell type is completely specified, let alone a whole organism. Specialized Cells Must rapidly turn Sets of Genes On and Off

1	Although they generally maintain their identities, specialized cells must constantly respond to changes in their environment. Among the most important changes are signals from other cells that coordinate the behavior of the whole organism. Many of these signals induce transient changes in gene transcription, and we discuss the nature of these signals in detail in Chapter 15. Here, we consider how specialized cell types rapidly and decisively switch groups of genes on and off in response to their environment. Even though control of gene expression is combinatorial, the effect of a single transcription regulator can still be decisive in switching any particular gene on or off, simply by completing the combination needed to maximally activate or repress that gene. This situation is analogous to dialing in the final number of a combination lock: the lock will spring open with only this simple addition if all of the other numbers have been previously entered.

1	Figure 7–37 A portion of the transcription network specifying embryonic stem cells. (A) the three master transcription regulators in figure 7–36 are shown as large circles. Genes whose cis-regulatory sequences are bound by each regulator in embryonic stem cells are indicated by a small dot (representing the gene) connected by a thin line (representing the binding reaction). note that many of the target genes are bound by more than one of the regulators. (B) the master regulators control their own expression. As shown here, the three transcriptional regulators bind to their own control regions (indicated by feedback loops), as well as those of the other master regulators (indicated by straight arrows). (Courtesy of trevor Sorrells, based on data from J. Kim et al., Cell 132:1049– 1061, 2008.) Moreover, the same number can complete the combination for many different locks. Likewise, the addition of a particular protein can turn on many different genes.

1	An example is the rapid control of gene expression by the human glucocorticoid receptor protein. To bind to its cis-regulatory sequences in the genome, this transcription regulator must first form a complex with a molecule of a glucocorticoid steroid hormone, such as cortisol (see Figure 15–64). The body releases this hormone during times of starvation and intense physical activity, and among its other activities, it stimulates liver cells to increase the production of glucose from amino acids and other small molecules. To respond in this way, liver cells increase the expression of many different genes that code for metabolic enzymes, such as tyrosine aminotransferase, as we discussed earlier in this chapter (see Figure 7–3). Although these genes all have different and complex control regions, their maximal expression depends on the binding of the hormone–glucocorticoid receptor complex to its cis-regulatory sequence, which is present in the control region of each gene. When the body

1	their maximal expression depends on the binding of the hormone–glucocorticoid receptor complex to its cis-regulatory sequence, which is present in the control region of each gene. When the body has recovered and the hormone is no longer present, the expression of each of these genes drops to its normal level in the liver. In this way, a single transcription regulator can rapidly control the expression of many different genes (Figure 7–38).

1	The effects of the glucocorticoid receptor are not confined to cells of the liver. In other cell types, activation of this transcription regulator by hormone also causes changes in the expression levels of many genes; the genes affected, however, are usually different from those affected in liver cells. As we have seen, each cell type has an individualized set of transcription regulators, and because of combinatorial control, these critically influence the action of the glucocorticoid receptor. Because the receptor is able to assemble with many different sets of celltype-specific transcription regulators, switching it on with hormone produces a different spectrum of effects in each cell type.

1	Once a cell has become differentiated into a particular cell type, it will generally remain differentiated, and all its progeny cells will remain that same cell type. Some highly specialized cells, including skeletal muscle cells and neurons, never divide again once they have differentiated—that is, they are terminally differentiated (as discussed in Chapter 17). But many other differentiated cells—such as receptor in absence of glucocorticoid hormone

1	Figure 7–38 A single transcription regulator can coordinate the expression of many different genes. the action of the glucocorticoid receptor is illustrated schematically. On the left is a series of genes, each of which has various transcription regulators bound to its regulatory region. however, these bound proteins are not sufficient on their own to fully activate transcription. On the right is shown the effect of adding an additional transcription regulator—the glucocorticoid receptor in a complex with glucocorticoid hormone—that has a cisregulatory sequence in the control region of each gene. the glucocorticoid receptor completes the combination of transcription regulators required for maximal initiation of transcription, and the genes are now switched on as a set. when the hormone is no longer present, the glucocorticoid receptor dissociates from DnA and the genes return to their pre-stimulated levels.

1	fibroblasts, smooth muscle cells, and liver cells—will divide many times in the life of an individual. When they do, these specialized cell types give rise only to cells like themselves: smooth muscle cells do not give rise to liver cells, nor liver cells to fibroblasts. For a proliferating cell to maintain its identity—a property called cell memory—the patterns of gene expression responsible for that identity must be remembered and passed on to its daughter cells through subsequent cell divisions. Thus, in the model we discussed in Figure 7–33, the production of each transcription regulator, once begun, has to be continued in the daughter cells of each cell division. How is such perpetuation accomplished?

1	Cells have several ways of ensuring that their daughters “remember” what kind of cells they are. One of the simplest and most important is through a positive feedback loop, where a master cell-type transcription regulator activates transcription of its own gene, in addition to that of other cell-type-specific genes. Each time a cell divides, the regulator is distributed to both daughter cells, where it continues to stimulate the positive feedback loop, making more of itself each division. Positive feedback is crucial for establishing “self-sustaining” circuits of gene expression that allow a cell to commit to a particular fate—and then to transmit that information to its progeny (Figure 7–39).

1	As was previously shown in Figure 7–37B, the master regulators needed to maintain the pluripotency of iPS cells bind to cis-regulatory sequences in their own control regions, providing examples of the type of positive feedback loop. In addition, most of these pluripotent cell regulators also activate transcription of other master regulators, resulting in a complex series of indirect feedback loops. For example, if A activates B, and B activates A, this forms a positive feedback loop where A activates its own expression, albeit indirectly. The series of direct and indirect feedback loops observed in the iPS circuit is typical of other specialized cell circuits. Such a network structure strengthens cell memory, increasing the probability that a particular pattern of gene expression is transmitted through successive generations. For example, if the level of A drops below the critical threshold to stimulate its own synthesis, regulator B can rescue it. By successive application of this

1	through successive generations. For example, if the level of A drops below the critical threshold to stimulate its own synthesis, regulator B can rescue it. By successive application of this mechanism, a complex series of positive feedback loops among multiple transcription regulators can stably maintain a differentiated state through many cell divisions.

1	Figure 7–39 A positive feedback loop can create cell memory. protein A is a master transcription regulator that activates the transcription of its own gene—as well as other cell-type-specific genes (not shown). All of the descendants of the original cell will therefore “remember” that the progenitor cell had experienced a transient signal that initiated the production of protein A.

1	Positive feedback loops formed by transcription regulators are probably the most prevalent way of ensuring that daughter cells remember what kind of cells they are meant to be, and they are found in all species on Earth. For example, many bacteria and single-cell eukaryotes form different types of cells, and positive feedback loops lie at the heart of mechanisms that maintain their cell types through many rounds of cell division. Plants and animals also make extensive use of transcription feedback loops; as we shall discuss later in the chapter, they have additional, more specialized mechanisms for making cell memory even stronger. But first, we briefly consider how combinations of transcription regulators and cis-regulatory sequences can be combined to create useful logic devices for the cell. transcription Circuits Allow the Cell to Carry Out logic Operations

1	Simple gene regulatory switches can be combined to create all sorts of control devices, just as simple electronic switching elements in a computer can be linked to perform different types of operations. An analysis of gene regulatory circuits reveals that certain simple types of arrangements (called network motifs) are found over and over again in cells from widely different species. For example, positive and negative feedback loops are common in all cells (Figure 7–40). Whereas the former provides a simple memory device, the latter is often used to keep the expression of a gene close to a standard level despite the variations in biochemical conditions inside a cell. Suppose, for example, that a transcription repressor protein binds to the regulatory region of its own gene and exerts a strong negative feedback, such that transcription falls to a very low rate when the concentration of the repressor protein is above some critical value (determined by its affinity for its DNA binding

1	strong negative feedback, such that transcription falls to a very low rate when the concentration of the repressor protein is above some critical value (determined by its affinity for its DNA binding site). The concentration of the protein can then be held close to the critical value, since any circumstance that causes a fall below that value can lead to a steep increase in synthesis, and any that causes a rise above that value will lead to synthesis being switched off. Such adjustments will, however, take time, so that an abrupt change of conditions will cause a disturbance of gene expression that is strong but transient. If there is a delay in the feedback loop, the result may be spontaneous oscillations in the expression of the gene (see Figure 15–18). The different types of behavior produced by a feedback loop will depend on the details of the system; for example, how tightly the transcription regulator binds to its cis-regulatory sequence, its rate of synthesis, and its rate of

1	produced by a feedback loop will depend on the details of the system; for example, how tightly the transcription regulator binds to its cis-regulatory sequence, its rate of synthesis, and its rate of decay. We discuss these issues in quantitative terms and in more detail in Chapter 8.

1	With two or more transcription regulators, the possible range of circuit behaviors becomes more complex. Some bacterial viruses contain a common type of two-gene circuit that can flip-flop between expression of one gene and expression of the other. Another common circuit arrangement is called a feed-forward loop; such a loop can serve as a filter, responding to input signals that are prolonged but disregarding those that are brief (Figure 7–41). These various network motifs resemble miniature logic devices, and they can process information in surprisingly sophisticated ways. The simple types of devices just illustrated are found to be interwoven in eukaryotic cells, creating exceedingly complex circuits (Figure 7–42). Each cell in a developing multicellular organism is equipped with similarly complex control

1	Figure 7–40 Common types of network motifs in transcription circuits. A and B represent transcription regulators, arrows indicate positive transcription control, while lines with bars depict negative transcription control. in the feed-forward loop, A and B represent transcription regulators that both activate the transcription of target gene Z (see also figure 8–86). machinery, and it must, in effect, use its intricate system of interlocking transcription switches to compute how it should behave at each time point in response to the many different past and present inputs received. We are only beginning to understand how to study such complex intracellular control networks. Indeed, without new approaches, coupled with quantitative information that is far more precise and complete than we now possess, it will be impossible to predict the behavior of a system such as that shown in Figure 7–42. As explained in Chapter 8, a circuit diagram by itself is not enough.

1	Figure 7–41 How a feed-forward loop can measure the duration of a signal. in this theoretical example, transcription regulators A and B are both required for transcription of Z, and A becomes active only when an input signal is present. if the input signal to A is brief, A does not stay active long enough for B to accumulate, and the Z gene is not transcribed. (C) if the signal to A persists, B accumulates, A remains active, and Z is transcribed. this arrangement allows the cell to ignore rapid fluctuations of the input signal and respond only to persistent levels. this strategy could be used, for example, to distinguish between random noise and a true signal.

1	the behavior shown here was computed for one particular set of parameter values describing the quantitative properties of A, B, and the product of Z, along with their syntheses. with different values of these parameters, feed-forward loops can in principle perform other types of “calculations.” Many feed-forward loops have been discovered in cells, and theoretical analysis helps researchers to discern—and subsequently test—the different ways in which they may function (see figure 8–86). (Adapted from S.S. Shen-Orr et al., Nat. Genet. 31:64–68, 2002. with permission from Macmillan publishers ltd.)

1	S.S. Shen-Orr et al., Nat. Genet. 31:64–68, 2002. with permission from Macmillan publishers ltd.) Figure 7–42 The exceedingly complex gene circuit that specifies a portion of the developing sea urchin embryo. each colored small box represents a different gene. those in yellow code for transcription regulators and those in green and blue code for proteins that give cells of the mesoderm and endoderm, respectively, their specialized characteristics. Genes depicted in gray are largely active in the mother and provide the egg with cues needed for proper development. As in figure 7–40, arrows depict instances in which a transcription regulator activates the transcription of another gene. lines ending in bars indicate examples of gene repression. (from i.S. peter and e.h. Davidson, Nature 474:635–639, 2011. with permission from Macmillan publishers ltd.)

1	The many types of cells in animals and plants are created largely through mechanisms that cause different sets of genes to be transcribed in different cells. The transcription of any particular gene is generally controlled by a combination of transcription regulators. Each type of cell in a higher eukaryotic organism contains a specific set of transcription regulators that ensures the expression of only those genes appropriate to that type of cell. A given transcription regulator may be active in a variety of circumstances and is typically involved in the regulation of many different genes.

1	Since specialized animal cells can maintain their unique character through many cell-division cycles, and even when grown in culture, the gene regulatory mechanisms involved in creating them must be stable once established and heritable when the cell divides. These features reflect the cell’s memory of its developmental history. Direct or indirect positive feedback loops, which enable transcription regulators to perpetuate their own synthesis, provide the simplest mechanism for cell memory. Transcription circuits also provide the cell with the means to carry out other types of logic operations. Simple transcription circuits combined into large regulatory networks drive highly sophisticated programs of embryonic development that will require new approaches to decipher.

1	Thus far in this chapter, we have emphasized the regulation of gene transcription by proteins that associate either directly or indirectly with DNA. However, DNA itself can be covalently modified, and certain types of chromatin states appear to be inherited. In this section, we shall see how these phenomena also provide opportunities for the regulation of gene expression. At the end of this section, we discuss how, in mice and humans, an entire chromosome can be transcription-ally inactivated using such mechanisms, and how this state can be maintained through many cell divisions. patterns of DnA Methylation Can Be inherited when vertebrate Cells Divide

1	In vertebrate cells, the methylation of cytosine provides a mechanism through which gene expression patterns can be passed on to progeny cells. The methylated form of cytosine, 5-methyl cytosine (5-methyl C), has the same relation to cytosine that thymine has to uracil, and the modification likewise has no effect on base-pairing (Figure 7–43). DNA methylation in vertebrate DNA occurs on cytosine (C) nucleotides largely in the sequence CG, which is base-paired to exactly the same sequence (in opposite orientation) on the other strand of the DNA helix. Consequently, a simple mechanism permits the existing pattern of DNA methylation to be inherited directly by the daughter DNA strands. An enzyme called maintenance methyl transferase acts preferentially on those CG sequences that are base-paired with a CG sequence that is already methylated. As a result, the pattern of DNA methylation on the parental DNA strand serves as a template for the methylation of the daughter DNA strand, causing

1	with a CG sequence that is already methylated. As a result, the pattern of DNA methylation on the parental DNA strand serves as a template for the methylation of the daughter DNA strand, causing this pattern to be inherited directly following DNA replication (Figure 7–44).

1	Although DNA methylation patterns can be maintained in differentiated cells by the mechanism shown in Figure 7–44, methylation patterns are dynamic during mammalian development. Shortly after fertilization, there is a genome-wide wave of demethylation, when the vast majority of methyl groups are lost from the DNA. This demethylation may occur either by suppression of maintenance DNA methyl transferase activity, resulting in the passive loss of methyl groups during each round of DNA replication, or by demethylating enzymes (discussed below). Later in development, new methylation patterns are established by several de novo DNA methyl transferases that are directed to DNA by sequence Figure 7–43 Formation of 5-methyl cytosine occurs by methylation of a cytosine base in the DNA double helix.

1	Figure 7–43 Formation of 5-methyl cytosine occurs by methylation of a cytosine base in the DNA double helix. in vertebrates, this event is largely confined to selected cytosine (C) nucleotides located in the sequence CG. CG sequences are sometimes denoted as CpG sequences, where the p indicates a phosphate linkage to distinguish it from a CG base pair. in this chapter, we will continue to use the simpler nomenclature CG to indicate this dinucleotide. Figure 7–44 How DNA methylation patterns are faithfully inherited. in vertebrate DnA, a large fraction of the cytosine nucleotides in the sequence CG is methylated (see figure 7–43). Because of the existence of a methyl-directed methylating enzyme (the maintenance methyl transferase), once a pattern of DnA methylation is established, that pattern of methylation is inherited in the progeny DnA, as shown.

1	specific DNA-binding proteins. Once the new patterns of methylation are established, they can be propagated through rounds of DNA replication by the maintenance methyl transferases. DNA methylation has several uses in the vertebrate cell. A very important role is to work in conjunction with other gene expression control mechanisms to establish a particularly efficient form of gene repression. This combination of mechanisms ensures that unneeded eukaryotic genes can be repressed to very high degrees. For example, the rate at which a vertebrate gene is transcribed can vary 106-fold between one tissue and another. The unexpressed vertebrate genes are much less “leaky” in terms of transcription than bacterial genes, in which the largest known differences in transcription rates between expressed and unexpressed gene states are about 1000-fold.

1	DNA methylation helps to repress transcription in several ways. The methyl groups on methylated cytosines lie in the major groove of DNA and interfere directly with the binding of proteins (transcription regulators as well as the general transcription factors) required for transcription initiation. In addition, the cell contains a repertoire of proteins that bind specifically to methylated DNA.The best characterized of these associate with histone modifying enzymes, leading to a repressive chromatin state where chromatin structure and DNA methylation act synergistically (Figure 7–45). One reflection of the importance of DNA methylation to humans is the widespread involvement of “incorrect” DNA methylation patterns in cancer progression (discussed in Chapter 20). CG-rich islands Are Associated with Many Genes in Mammals

1	CG-rich islands Are Associated with Many Genes in Mammals Because of the way in which DNA repair enzymes work, methylated C nucleotides in the vertebrate genome tend to be eliminated in the course of evolution. Accidental deamination of an unmethylated C gives rise to U (see Figure 5–38), which is not normally present in DNA and thus is recognized easily by the DNA repair enzyme uracil DNA glycosylase, excised, and then replaced with a C (as discussed in Chapter 5). But accidental deamination of a 5-methyl C cannot be repaired in this way, for the deamination product is a T and so is indistinguishable from the other, nonmutant T nucleotides in the DNA. Although a special repair system exists to remove these mutant T nucleotides, many of the deaminations escape detection, so that those C nucleotides in the genome that are methylated tend to mutate to T over evolutionary time. 406 Chapter 7: Control of Gene expression

1	406 Chapter 7: Control of Gene expression Figure 7–45 Multiple mechanisms contribute to stable gene repression.

1	in this schematic example, histone reader and writer proteins (discussed in Chapter 4), under the direction of transcription regulators, establish a repressive form of chromatin. A de novo DnA methylase is attracted by the histone reader and methylates nearby cytosines in DnA, which are, in turn, bound by DnA methyl-binding proteins. During DnA replication, some of the modified (blue dot) histones will be inherited by one daughter chromosome, some by the other, and in each daughter they can induce reconstruction of the same pattern of chromatin modifications (discussed in Chapter 4). At the same time, the mechanism shown in figure 7–44 will cause both daughter chromosomes to inherit the same methylation pattern. in these cases where DnA methylation stimulates the activity of the histone writer, the two inheritance mechanisms will be mutually reinforcing. this scheme can account for the inheritance by daughter cells of both the histone and the DnA modifications. it can also explain the

1	the two inheritance mechanisms will be mutually reinforcing. this scheme can account for the inheritance by daughter cells of both the histone and the DnA modifications. it can also explain the tendency of some chromatin modifications to spread along a chromosome (see figure 4–44).

1	During the course of evolution, more than three out of every four CGs have been lost in this way, leaving vertebrates with a remarkable deficiency of this dinucleotide. The CG sequences that remain are very unevenly distributed in the genome; they are present at 10 times their average density in selected regions, called CG islands, which average 1000 nucleotide pairs in length. The human genome contains roughly 20,000 CG islands and they usually include promoters of genes. For example, 60% of human protein-coding genes have promoters embedded in CG islands and these include virtually all the promoters of the so-called housekeeping genes—those genes that code for the many proteins that are essential for cell viability and are therefore expressed in nearly all cells (Figure 7–46). Over evolutionary timescales, the CG islands were spared the accelerated mutation rate of bulk CG sequences because they remained unmethylated in the germ line (Figure 7–47).

1	CG islands also remain unmethylated in most somatic tissues whether or not the associated gene is expressed. The unmethylated state is maintained by sequence-specific DNA-binding proteins, many of whose cis-regulatory sequences contain a CG. By binding to these sequences, which are spread across CG islands, they protect the DNA from methyl transferases. These proteins also recruit DNA demethylases, which convert 5-methyl C to hydroxy-methyl C, which 10,000 nucleotide pairs is later replaced by C either through DNA repair (see Figure 5–41A) or, passively, through multiple rounds of DNA replication. Unmethylated CG islands have several properties that make them particularly suitable for promoters. For example, some of the same proteins that bind to CG islands and protect them from methylation recruit histone modifying enzymes that make the islands particularly “promoter friendly.” As a result, RNA polymerase is often found bound to promoters within CG islands, even when the associated

1	recruit histone modifying enzymes that make the islands particularly “promoter friendly.” As a result, RNA polymerase is often found bound to promoters within CG islands, even when the associated gene is not being actively transcribed. At unmethylated CG islands, the balance between polymerase and nucleosome assembly is thus tipped toward the former. Additional steps are needed to “push” the bound polymerase into transcribing the adjacent gene, and these are directed by transcription regulators that bind to cis-regulatory sequences of DNA (often well upstream from the CG islands). These regulators serve to release the polymerase with the appropriate elongation factors (see Figure 7–21C and D).

1	Genomic imprinting is Based on DnA Methylation Mammalian cells are diploid, containing one set of genes inherited from the father and one set from the mother. The expression of a small minority of genes depends on whether they have been inherited from the mother or the father: when the paternally inherited gene copy is active, the maternally inherited gene copy is silent, or vice versa. This phenomenon is called genomic imprinting. Roughly 300 genes are imprinted in humans. Because only one copy of an imprinted gene is expressed, imprinting can “unmask” mutations that would normally be covered by the other, functional copy. For example, Angelman syndrome, a disorder of the nervous system in humans that causes reduced mental ability and severe speech impairment, results from a gene deletion on one chromosomal homolog and the silencing, by imprinting, of the intact gene on the other homolog.

1	The insulin-like growth factor-2 (Igf 2) gene in the mouse provides a well-studied example of imprinting. Mice that do not express Igf 2 at all are born half the size of normal mice. However, only the paternal copy of Igf 2 is transcribed, and only this gene copy matters for the phenotype. As a result, mice with a mutated paternally derived Igf 2 gene are stunted, while mice with a mutated maternally derived Igf 2 gene are normal.

1	Figure 7–47 A mechanism to explain both the marked overall deficiency of CG sequences and their clustering into CG islands in vertebrate genomes. White lines mark the location of CG dinucleotides in the DnA sequences, while red circles indicate the presence of a methyl group on the CG dinucleotide. CG sequences that lie in regulatory sequences of genes that are transcribed in germ cells are unmethylated and therefore tend to be retained in evolution. Methylated CG sequences, on the other hand, tend to be lost through deamination of 5-methyl C to t, unless the CG sequence is critical for survival.

1	Figure 7–46 The CG islands surrounding the promoter in three mammalian housekeeping genes. the yellow boxes show the extent of each island. As for most genes in mammals, the exons (dark red) are very short relative to the introns (light red). (Adapted from A.p. Bird, Trends Genet. 3:342–347, 1987. with permission from elsevier.) methylation of most CG sequences in germ line many millions of years of evolution mRNA mRNA BOTH PARENTS EXPRESS THE SAME ALLELE OF GENE Aimprinted allele of gene Aexpressed allele of gene Asomatic cell somatic cell REMOVAL OF IMPRINTING IN GERM CELLS, FOLLOWED BY MEIOSIS FEMALE IMPRINT ESTABLISHED MALE IMPRINT ESTABLISHED EGGS SPERM OFFSPRING DIFFER IN THE ALLELE OF GENE ATHAT IS EXPRESSED mRNA mRNA chromosome inherited from father

1	Figure 7–48 Imprinting in the mouse. the top portion of the figure shows a pair of homologous chromosomes in the somatic cells of two adult mice, one male and one female. in this example, both mice have inherited the top homolog from their father and the bottom homolog from their mother, and the paternal copy of a gene subject to imprinting (indicated in orange) is methylated, preventing its expression. the maternally derived copy of the same gene (yellow) is expressed. the remainder of the figure shows the outcome of a cross between these two mice. During germ-cell formation, but before meiosis, the imprints are erased and then, much later in germ-cell development, they are reimposed in a sex-specific pattern (middle portion of figure). in eggs produced from the female, neither allele of the A gene is methylated. in sperm from the male, both alleles of gene A are methylated. Shown at the bottom of the figure are two of the possible imprinting patterns inherited by the progeny mice;

1	the A gene is methylated. in sperm from the male, both alleles of gene A are methylated. Shown at the bottom of the figure are two of the possible imprinting patterns inherited by the progeny mice; the mouse on the left has the same imprinting pattern as each of the parents, whereas the mouse on the right has the opposite pattern. if the two alleles of gene A are distinct, these different imprinting patterns can cause phenotypic differences in the progeny mice, even though they carry exactly the same DnA sequences of the two A gene alleles. imprinting provides an important exception to classical genetic behavior, and several hundred mouse genes are thought to be affected in this way. however, the majority of mouse genes are not imprinted, and therefore the rules of Mendelian inheritance apply to most of the mouse genome.

1	In the early embryo, genes subject to imprinting are marked by methylation according to whether they were derived from a sperm or an egg chromosome. In this way, DNA methylation is used as a mark to distinguish two copies of a gene that may be otherwise identical (Figure 7–48). Because imprinted genes are somehow protected from the wave of demethylation that takes place shortly after fertilization (see pp. 404–405), this mark enables somatic cells to “remember” the parental origin of each of the two copies of the gene and to regulate their expression accordingly. In most cases, the methyl imprint silences nearby gene expression. In some cases, however, it can activate expression of a gene. In the case of Igf 2, for example, methylation of an insulator element on the paternally derived chromosome blocks its function and allows distant cis-regulatory sequences to activate transcription of the Igf 2 gene. On the maternally derived chromosome, the insulator is not methylated and the Igf 2

1	blocks its function and allows distant cis-regulatory sequences to activate transcription of the Igf 2 gene. On the maternally derived chromosome, the insulator is not methylated and the Igf 2 gene is therefore not transcribed (Figure 7–49A).

1	Other cases of imprinting involve long noncoding RNAs, which are defined as RNA molecules over 200 nucleotides in length that do not code for proteins. We discuss lncRNAs broadly at the end of this chapter; here, we focus on the role of a specific lncRNA in imprinting. In the case of the Kcnq1 gene, which codes for a voltage-gated calcium channel needed for proper heart function, the lncRNA is made from the paternal allele (which is unmethylated) but it is not released by the RNA polymerase, remaining instead at its site of synthesis on the DNA template. This RNA in turn recruits histone-modifying and DNA-methylating enzymes that direct the formation of repressive chromatin, which silences the protein-coding gene associated on the paternally derived chromosome (Figure 7–49B). The maternally derived gene, on the other hand, is immune to these effects because the specific methylation present from imprinting blocks the synthesis of the lncRNA but allows transcription of the

1	The maternally derived gene, on the other hand, is immune to these effects because the specific methylation present from imprinting blocks the synthesis of the lncRNA but allows transcription of the protein-coding gene. Like Igf 2, the specificity of Kcnq1 imprinting arises from the inherited methylation patterns; the difference lies in the way these patterns bring about differential expression of the imprinted gene.

1	Why imprinting should exist at all is a mystery. In vertebrates, it is restricted to placental mammals, and many of the imprinted genes are involved in fetal development. One idea is that imprinting reflects a middle ground in the evolutionary struggle between males to produce larger offspring and females to limit offspring size. Whatever its purpose might be, imprinting provides startling evidence that features of DNA other than its sequence of nucleotides can be inherited. We have seen that DNA methylation and certain types of chromatin structure can be heritable, preserving patterns of gene expression across cell generations. Perhaps the most striking example of this effect occurs in mammals, in which an alteration in the chromatin structure of an entire chromosome can modulate the levels of expression of most genes on that chromosome. Figure 7–49 Mechanisms of imprinting.

1	Figure 7–49 Mechanisms of imprinting. On chromosomes inherited from the female, a protein called CtCf binds to an insulator (see figure 7–24), blocking communication between cis-regulatory sequences (green) and the Igf2 gene (orange). Igf2 is therefore not expressed from the maternally inherited chromosome. Because of imprinting, the insulator on the male-derived chromosome is methylated (red circles); this inactivates the insulator by blocking the binding of the CtCf protein, and allows the cis-regulatory sequences to activate transcription of the Igf2 gene. in other examples of imprinting, methylation simply blocks gene expression by interfering with the binding of proteins required for a gene’s transcription.

1	imprinting of the mouse Kcnq1 gene. On the maternally derived chromosome, synthesis of the lncrnA is blocked by methylation of the DnA (red circles), and the Kcnq1 gene is expressed. On the paternally derived chromosome, the lncrnA is synthesized, remains in place, and by directing alterations in chromatin structure blocks expression of the Kcnq1 gene. Although shown as directly binding to lncrnA, the histone-modifying enzymes are likely to be recruited indirectly, through additional proteins.

1	Males and females differ in their sex chromosomes. Females have two X chromosomes, whereas males have one X and one Y chromosome. As a result, female cells contain twice as many copies of X-chromosome genes as do male cells. In mammals, the X and Y sex chromosomes differ radically in gene content: the X chromosome is large and contains more than a thousand genes, whereas the Y chromosome is small and contains less than 100 genes. Mammals have evolved a dosage compensation mechanism to equalize the dosage of X-chromosome gene products between males and females. The correct ratio of X chromosome to auto-some (non-sex chromosome) gene products is carefully controlled, and mutations that interfere with this dosage compensation are generally lethal.

1	Mammals achieve dosage compensation by the transcriptional inactivation of one of the two X chromosomes in female somatic cells, a process known as X-inactivation. As a result of X-inactivation, two X chromosomes can coexist within the same nucleus, exposed to the same diffusible transcription regulators, yet differ entirely in their expression.

1	Early in the development of a female embryo, when it consists of a few hundred cells, one of the two X chromosomes in each cell becomes highly condensed into a type of heterochromatin. The initial choice of which X chromosome to inactivate, the maternally inherited one (Xm) or the paternally inherited one (Xp), is random. Once either Xp or Xm has been inactivated, it remains silent throughout all subsequent cell divisions of that cell and its progeny, indicating that the inactive state is faithfully maintained through many cycles of DNA replication and mitosis. Because X-inactivation is random and takes place after several hundred cells have already formed in the embryo, every female is a mosaic of clonal groups of cells in which either Xp or Xm is silenced (Figure 7–50). These clonal groups are

1	Xp Xm Xp Xm Xp Xm CONDENSATION OF A RANDOMLY SELECTED X CHROMOSOME DIRECT INHERITANCE OF THE PATTERN OF CHROMOSOME CONDENSATION DIRECT INHERITANCE OF THE PATTERN OF CHROMOSOME CONDENSATION Figure 7–50 X-inactivation. the clonal inheritance in female mammals of a only Xm active in this clone only Xp active in this clone condensed, inactive X chromosome.

1	Figure 7–51 Photoreceptor cells in the retina of a female mouse showing patterns of X-chromosome inactivation. Using genetic engineering techniques (described in Chapter 8), the germ line of a mouse was modified so that one copy of the X chromosome (if active) makes a green fluorescent protein and the other a red fluorescent protein. Both proteins concentrate in the nucleus and, in the field of cells shown here, it is clear that only one of the two X chromosomes is active in each cell. (from h. wu et al., Neuron 81:103–119, 2014. with permission from elsevier.) distributed in small clusters in the adult animal because sister cells tend to remain close together during later stages of development (Figure 7–51). For example, X-chromosome inactivation causes the orange and black “tortoiseshell” coat coloration of some female cats. In these cats, one X chromosome carries a gene that produces orange hair color, and the other X chromosome carries an allele of the same gene that results in

1	coat coloration of some female cats. In these cats, one X chromosome carries a gene that produces orange hair color, and the other X chromosome carries an allele of the same gene that results in black hair color; it is the random X-inactivation that produces patches of cells of two distinctive colors. In contrast, male cats of this genetic stock are either solid orange or solid black, depending on which X chromosome they inherit from their mothers. Although X-chromosome inactivation is maintained over thousands of cell divisions, it is reversed during germ-cell formation, so that all haploid oocytes contain an active X chromosome and can express X-linked gene products.

1	How is an entire chromosome transcriptionally inactivated? X-chromosome inactivation is initiated and spreads from a single site near the middle of the X chromosome, the X-inactivation center (XIC). Within the XIC is a transcribed 20,000-nucleotide lncRNA (called Xist), which is expressed solely from the inactive X chromosome. Xist RNA spreads from the XIC over the entire chromosome and directs gene silencing. Although we do not know exactly how this is accomplished, it likely involves recruitment of histone-modifying enzymes and other proteins to form a repressive form of chromatin analogous to that of Figure 7–45. Curiously, about 10% of the genes on the X chromosome (including Xist itself) escape this silencing and remain active.

1	The spread of Xist RNA along the X chromosome does not proceed linearly along the DNA. Rather, starting at its site of synthesis, it is first handed off across the base of the DNA loops that make up the chromosome; these shortcuts explain how Xist can spread rapidly, by a “hand-over-hand” mechanism, along the X chromosome once the inactivation process begins (Figure 7–52). It also helps to explain why the inactivation does not spread to the other, active X chromosome.

1	Imprinting and X-chromosome inactivation are examples of monoallelic gene expression, where in a diploid genome, only one of the two copies of a gene is expressed. In addition to the approximately 1000 genes on the X chromosome and the 300 or so genes that are imprinted, there are another 1000–2000 human genes that exhibit monoallelic expression. Like X-chromosome inactivation (but unlike imprinting), the choice of which copy of the gene is to be expressed and which is to be silenced often appears random. Yet once the choice is made, it can persist for many cell divisions. Because the choice is often made relatively late in development, cells of the same tissue in the same individual can express different copies of a given gene. In other words, somatic tissues are often mosaics, where different clones of cells have subtly different patterns of gene expression. The mechanisms responsible for this type of monoallelic expression are not known in detail, and its general purpose—if any—is

1	clones of cells have subtly different patterns of gene expression. The mechanisms responsible for this type of monoallelic expression are not known in detail, and its general purpose—if any—is poorly understood. Several different mechanisms may contribute to such epigenetic inheritance, as we explain next.

1	epigenetic Mechanisms ensure that Stable patterns of Gene expression Can Be transmitted to Daughter Cells

1	As we have seen, once a cell in an organism differentiates into a particular cell type, it generally remains specialized in that way; if it divides, its daughters inherit the same specialized character. Perhaps the simplest way for a cell to remember transcription of Xist RNA Xist RNA spreads Xist RNA binds histone-modifying from one X chromosome “hand-over-hand” and continues to spread its identity is through a positive feedback loop in which a key transcription regulator activates, either directly or indirectly, the transcription of its own gene (see Figure 7–39). Interlocking positive feedback loops of the type shown in Figure 7–37 provide greater stability by buffering the circuit against fluctuations in the level of any one transcription regulator. Because transcription regulators are synthesized in the cytosol and diffuse throughout the nucleus, feedback loops based on this mechanism will affect both copies of a gene in a diploid cell. However, as discussed in this section, the

1	synthesized in the cytosol and diffuse throughout the nucleus, feedback loops based on this mechanism will affect both copies of a gene in a diploid cell. However, as discussed in this section, the expression pattern of a gene on one chromosome can differ from the copy of the same gene on the other chromosome (as in X-chromosome inactivation or in imprinting), and such differences can also be inherited through many cell divisions.

1	The ability of a daughter cell to retain a memory of the gene expression patterns that were present in the parent cell is an example of epigenetic inheritance: a heritable alteration in a cell or organism’s phenotype that does not result from changes in the nucleotide sequence of DNA (discussed in Chapter 4). (Unfortunately, the term epigenetic is sometimes also used to refer to all covalent modifications to histones and DNA, whether or not they are self-propagating; many of these modifications are erased each time a cell divides and do not generate cell memory.) In Figure 7–53, we contrast two self-propagating epigenetic mechanisms that work in cis, affecting only one chromosomal copy with two self-propagating mechanisms that work in trans, affecting both chromosomal copies of a gene. Cells can combine these mechanisms to ensure that patterns of gene expression are maintained and inherited accurately and reliably—over a period of up to a hundred years or more, in our own case.

1	We can get some idea of the prevalence of epigenetic changes by comparing identical twins. Their genomes have the same sequence of nucleotides, and, obviously, many features of identical twins—such as their appearance—are strongly determined by the genome sequences they inherit. When their gene expression, histone modification, and DNA methylation patterns are compared, however, many differences are observed. Because these differences are roughly correlated not only with age but also with the time that the twins have spent apart from each other, it has been proposed that some of these differences are heritable from cell to cell and are the result of environmental factors. Although these studies are in early stages, the idea that environmental events can be permanently registered as epigenetic changes in our cells is a fascinating one that presents an important challenge to the next generation of biological scientists.

1	Figure 7–52 Mammalian X-chromosome inactivation. X-chromosome inactivation begins with the synthesis of Xist (X-inactivation specific transcript) rnA from the XiC (X-inactivation center) locus and moves outward to the chromosome ends. According to the model depicted here, the long (≈20,000 nucleotides) Xist rnA has many low-affinity binding sites for the structural components of chromosomes and spreads by releasing its hold on one portion of the chromosome while grasping another. the continued synthesis of Xist from the center of the chromosome drives it to the ends. As shown, Xist rnA does not move linearly along the chromosomal DnA, but, instead, moves first across the base of chromosome loops. it has been proposed that the portions of chromosomal DnA at the tips of long loops contain the 10% of genes that escape X-chromosome inactivation.

1	Eukaryotic cells can use inherited forms of DNA methylation and inherited states of chromatin condensation as additional mechanisms for generating cell memory of gene expression patterns. An especially dramatic case that involves chromatin condensation is the inactivation of an entire X chromosome in female mammals. DNA methylation underlies the phenomenon in mammals of genomic imprinting, in which the expression of a gene depends on whether it was inherited from the mother or the father.

1	In principle, every step required for the process of gene expression can be controlled. Indeed, one can find examples of each type of regulation, and many genes are regulated by multiple mechanisms. As we have seen, controls on the initiation of gene transcription are a critical form of regulation for all genes. But other controls can act later in the pathway from DNA to protein to modulate the amount of gene product that is made—and in some cases, to determine the exact amino acid sequence of the protein product. These post-transcriptional controls, which operate after RNA polymerase has bound to the gene’s promoter and has begun RNA synthesis, are crucial for the regulation of many genes. In the following sections, we consider the varieties of post-transcriptional regulation in temporal order, according to the sequence of events that an RNA molecule might experience after its transcription has begun (Figure 7–54).

1	Figure 7–53 Four distinct mechanisms that can produce an epigenetic form of inheritance in an organism. (A) epigenetic mechanisms that act in cis. As discussed in this chapter, a maintenance methylase can propagate specific patterns of cytosine methylation (see figure 7–44). As discussed in Chapter 4, a histone modifying enzyme that replicates the same modification that attracts it to chromatin can result in the modification being self-propagating (see figure 4–44). (B) epigenetic mechanisms that act in trans. positive feedback loops, formed by transcriptional regulators are found in all species and are probably the most common form of cell memory. As discussed in Chapter 3, some proteins can form self-propagating prions (figure 3–33). if these proteins are involved in gene expression, they can transmit patterns of gene expression to daughter cells.

1	Figure 7–54 Post-transcriptional controls of gene expression. the final synthesis rate of a protein can, in principle, be controlled at any of the steps listed in capital letters. in addition, rnA splicing, rnA editing, and translation recoding can also alter the sequence of amino acids in a protein, making it possible for the cell to produce more than one protein variant from the same gene. Only a few of the steps depicted here are likely to be critical for the regulation of any one particular protein. transcription Attenuation Causes the premature termination of Some rnA Molecules

1	transcription Attenuation Causes the premature termination of Some rnA Molecules It has long been known that the expression of some genes is inhibited by premature termination of transcription, a phenomenon called transcription attenuation. In some of these cases, the nascent RNA chain adopts a structure that causes it to interact with the RNA polymerase in such a way as to abort its transcription. When the gene product is required, regulatory proteins bind to the nascent RNA chain and remove the attenuation, allowing the transcription of a complete RNA molecule.

1	A well-studied example of transcription attenuation occurs during the life cycle of HIV, the human immunodeficiency virus that is the causative agent of acquired immune deficiency syndrome, or AIDS. Once the HIV genome has been integrated into the host genome, the viral DNA is transcribed by the cell’s RNA polymerase II (see Figure 5–62). However, this polymerase usually terminates transcription after synthesizing transcripts of several hundred nucleotides and therefore fails to efficiently transcribe the entire viral genome. When conditions for viral growth are optimal, a virus-encoded protein called Tat, which binds to a specific stem-loop structure in the nascent RNA that contains a “bulged base,” prevents this premature termination (see Figure 6–89). Once bound to this specific RNA structure (called TAR), Tat assembles several host-cell proteins that allow the RNA polymerase to continue transcribing. The normal role of at least some of these proteins is to prevent pausing and

1	structure (called TAR), Tat assembles several host-cell proteins that allow the RNA polymerase to continue transcribing. The normal role of at least some of these proteins is to prevent pausing and premature termination by RNA polymerase when it transcribes normal cell genes. Thus, a normal cell mechanism has apparently been highjacked by HIV to permit transcription of its genome to be controlled by a single viral protein.

1	riboswitches probably represent Ancient forms of Gene Control In Chapter 6, we discussed the idea that, before modern cells arose on Earth, RNA played the role of both DNA and proteins, both storing hereditary information and catalyzing chemical reactions (see pp. 362–366). The discovery of riboswitches shows that RNA can also form control devices. Riboswitches are short sequences of RNA that change their conformation on binding small molecules, such as metabolites. Each riboswitch recognizes a specific small molecule and the resulting conformational change is used to regulate gene expression. Riboswitches are often located near the 5ʹ end of mRNAs, and they fold while the mRNA is being synthesized, blocking or permitting progress of the RNA polymerase according to whether the regulatory small molecule is bound (Figure 7–55).

1	Riboswitches are particularly common in bacteria, in which they sense key small metabolites in the cell and adjust gene expression accordingly. Perhaps their most remarkable feature is the high specificity and affinity with which each recognizes only the appropriate small molecule; in many cases, every chemical feature of the small molecule is read by the RNA (Figure 7–55C). Moreover, the binding affinities observed are as tight as those typically observed between small molecules and proteins.

1	Figure 7–55 A riboswitch that responds to guanine. (A) in this example from bacteria, the riboswitch controls expression of the purine biosynthetic genes. when guanine levels in cells are low, an elongating rnA polymerase transcribes the purine biosynthetic genes, and the enzymes needed for guanine synthesis are therefore expressed. (B) when guanine is abundant, it binds the riboswitch, causing it to undergo a conformational change that forces the rnA polymerase to terminate transcription (see figure 6–11). (C) Guanine (red) bound to the riboswitch. Only those nucleotides that form the guanine-binding pocket are shown. Many other riboswitches exist, including those that recognize S-adenosylmethionine, coenzyme B12, flavin mononucleotide, adenine, lysine, and glycine. (Adapted from M. Mandal and r.r. Breaker, Nat. Rev. Mol. Cell Biol. 5:451–463, 2004. with permission from Macmillan publishers ltd; and C.K. vanderpool and S. Gottesman, Mol. Microbiol. 54:1076–1089, 2004. with permission

1	and r.r. Breaker, Nat. Rev. Mol. Cell Biol. 5:451–463, 2004. with permission from Macmillan publishers ltd; and C.K. vanderpool and S. Gottesman, Mol. Microbiol. 54:1076–1089, 2004. with permission from Blackwell publishing.)

1	Figure 7–56 Five patterns of alternative RNA splicing. in each case, a single type of rnA transcript is spliced in two alternative ways to produce two distinct mrnAs (1 and 2). the dark blue boxes mark exon sequences 1 that are retained in both mrnAs. the light blue boxes mark possible exon 2 sequences that are included in only one of the mrnAs. the boxes are joined by red lines to indicate where intron sequences (yellow) are removed. (Adapted from h. Keren et al. Nat. Rev. Genet. 11:345–355, 2010. with permission from Macmillan publishers ltd.)

1	Riboswitches are perhaps the most economical examples of gene control devices, inasmuch as they bypass the need for regulatory proteins altogether. In the example shown in Figure 7–55, the riboswitch controls transcription elongation, but they can also regulate other steps in gene expression, as we shall see later 1 in this chapter. Clearly, highly sophisticated gene control devices can be made from short sequences of RNA, a fact that supports the hypothesis of an early “RNA world.” 1 Alternative rnA Splicing Can produce Different forms of a protein 2 from the Same Gene

1	As discussed in Chapter 6 (see Figure 6–26), RNA splicing shortens the transcripts of many eukaryotic genes by removing the intron sequences from the mRNA pre-1 cursor. We also saw that a cell can splice an RNA transcript differently and thereby 2 make different polypeptide chains from the same gene—a process called alternative RNA splicing (Figure 7–56). A substantial proportion of animal genes (estimated at 90% in humans) produce multiple proteins in this way.

1	When different splicing possibilities exist at several positions in the transcript, a single gene can produce dozens of different proteins. In one extreme case, a Drosophila gene may produce as many as 38,000 different proteins from a single gene through alternative splicing (Figure 7–57), although only a fraction of these forms have thus far been experimentally observed. Considering that the Drosophila genome has approximately 14,000 identified genes, it is clear that the protein complexity of an organism can greatly exceed the number of its genes. This example also illustrates the perils in equating gene number with an organism’s complexity. For example, alternative splicing is rare in single-celled budding yeasts one out of 38,016 possible splicing patterns

1	Figure 7–57 Alternative splicing of RNA transcripts of the Drosophila Dscam gene. DSCAM proteins have several different functions. in cells of the fly immune system, they mediate the phagocytosis of bacterial pathogens. in cells of the nervous system, DSCAM proteins are needed for proper wiring of neurons. the final mrnA contains 24 exons, four of which (denoted A, B, C, and D) are present in the Dscam gene as arrays of alternative exons. each rnA contains 1 of 12 alternatives for exon A (red), 1 of 48 alternatives for exon B (green), 1 of 33 alternatives for exon C (blue), and 1 of 2 alternatives for exon D (yellow). this figure shows only one of the many possible splicing patterns (indicated by the red line and by the mature mrnA below it). each variant DSCAM protein would fold into roughly the same structure (predominantly a series of extracellular immunoglobulin-like domains linked to a membrane-spanning region; see figure 24–48), but the amino acid sequence of the domains vary

1	the same structure (predominantly a series of extracellular immunoglobulin-like domains linked to a membrane-spanning region; see figure 24–48), but the amino acid sequence of the domains vary according to the splicing pattern. the diversity of DSCAM variants contributes to the plasticity of the immune system as well as the formation of complex neural circuits; we take up the specific role of the DSCAM variants in more detail when we describe the development of the nervous system in Chapter 21. (Adapted from D.l. Black, Cell 103:367–370, 2000. with permission from elsevier.) but very common in flies. Budding yeast has ≈6200 genes, only about 300 of which are subject to splicing, and nearly all of these have only a single intron. To say that flies have only 2–3 times as many genes as yeasts greatly underestimates the difference in complexity of these two genomes.

1	In some cases, alternative RNA splicing occurs because there is an intron sequence ambiguity: the standard spliceosome mechanism for removing intron sequences (discussed in Chapter 6) is unable to distinguish clearly between two or more alternative pairings of 5ʹ and 3ʹ splice sites, so that different choices are made by chance on different individual transcripts. Where such constitutive alternative splicing occurs, several versions of the protein encoded by the gene are made in all cells in which the gene is expressed.

1	In many cases, however, alternative RNA splicing is regulated. In the simplest examples, regulated splicing is used to switch from the production of a nonfunctional protein to the production of a functional one (or the other way around). The transposase that catalyzes the transposition of the Drosophila P element, for example, is produced in a functional form in germ cells and a nonfunctional form in somatic cells of the fly, allowing the P element to spread throughout the genome of the fly without causing damage in somatic cells (see Figure 5–61). The difference in transposon activity has been traced to the presence of an intron sequence in the transposase RNA that is removed only in germ cells.

1	In addition to enabling switching from the production of a functional protein to the production of a nonfunctional one (or vice versa), the regulation of RNA splicing can generate different versions of a protein in different cell types, according to the needs of the cell. Tropomyosin, for example, is produced in specialized forms in different types of cells (see Figure 6–26). Cell-type-specific forms of many other proteins are produced in the same way. RNA splicing can be regulated either negatively, by a regulatory molecule that prevents the splicing machinery from gaining access to a particular splice site on the RNA, or positively, by a regulatory molecule that helps direct the splicing machinery to an otherwise overlooked splice site (Figure 7–58).

1	Because of the plasticity of RNA splicing, the blocking of a “strong” splicing site will often expose a “weak” site and result in a different pattern of splicing. Thus, the splicing of a pre-mRNA molecule can be thought of as a delicate balance between competing splice sites—a balance that can easily be tipped by effects on splicing of regulatory proteins. the Definition of a Gene has Been Modified Since the Discovery of Alternative rnA Splicing The discovery that eukaryotic genes usually contain introns and that their coding sequences can be assembled in more than one way raised new questions about the definition of a gene. A gene was first clearly defined in molecular terms in the early 1940s from work on the biochemical genetics of the fungus Neurospora. Figure 7–58 Negative and positive control of alternative RNA splicing.

1	Figure 7–58 Negative and positive control of alternative RNA splicing. in negative control, a repressor protein binds to a specific sequence in the premrnA transcript and blocks access of the splicing machinery to a splice junction. this often results in the use of a secondary splice site, thereby producing an altered pattern of splicing (see figure 7–56). in positive control, the splicing machinery is unable to remove a particular intron sequence efficiently without assistance from an activator protein. Because rnA is flexible, the nucleotide sequences that bind these activators can be located many nucleotide pairs from the splice junctions they control, and they are often called splicing enhancers, by analogy with the transcriptional enhancers mentioned earlier in this chapter.

1	Until then, a gene had been defined operationally as a region of the genome that segregates as a single unit during meiosis and gives rise to a definable phenotypic trait, such as a red or a white eye in Drosophila or a round or wrinkled seed in peas. The work on Neurospora showed that most genes correspond to a region of the genome that directs the synthesis of a single enzyme. This led to the hypothesis that one gene encodes one polypeptide chain. The hypothesis proved fruitful for subsequent research; as more was learned about the mechanism of gene expression in the 1960s, a gene became identified as that stretch of DNA that was transcribed into the RNA coding for a single polypeptide chain (or a single structural RNA such as a tRNA or an rRNA molecule). The discovery of split genes and introns in the late 1970s could be readily accommodated by the original definition of a gene, provided that a single polypeptide chain was specified by the RNA transcribed from any one DNA sequence.

1	introns in the late 1970s could be readily accommodated by the original definition of a gene, provided that a single polypeptide chain was specified by the RNA transcribed from any one DNA sequence. But it is now clear that many DNA sequences in higher eukaryotic cells can produce a set of distinct (but related) proteins by means of alternative RNA splicing. How, then, is a gene to be defined?

1	In those relatively rare cases in which a single transcription unit produces two very different eukaryotic proteins, the two proteins are considered to be produced by distinct genes that overlap on the chromosome. It seems unnecessarily complex, however, to consider most of the protein variants produced by alternative RNA splicing as being derived from overlapping genes. A more sensible alternative is to modify the original definition to count a DNA sequence that is transcribed as a single unit and encodes one set of closely related polypeptide chains (protein isoforms) as a single protein-coding gene. This definition also accommodates those DNA sequences that encode protein variants produced by post-transcriptional processes other than RNA splicing, such as transcript cleavage and RNA editing (discussed below). A Change in the Site of rnA transcript Cleavage and poly-A Addition Can Change the C-terminus of a protein

1	We saw in Chapter 6 that the 3ʹ end of a eukaryotic mRNA molecule is not formed by the termination of RNA synthesis by the RNA polymerase, as it is in bacteria. Instead, it results from an RNA cleavage reaction that is catalyzed by additional proteins while the transcript is elongating (see Figure 6–34). A cell can control the site of this cleavage so as to change the C-terminus of the resultant protein. In the simplest cases, one protein variant is simply a truncated version of the other; in many other cases, however, the alternative cleavage and polyadenylation sites lie within intron sequences and the pattern of splicing is thereby altered. This process can produce two closely related proteins differing only in the amino acid sequences at their C-terminal ends. Close analysis of RNAs produced from the human genome in a variety of cell types (see Figure 7–3) indicate that as many as 50% of human protein-coding genes produce mRNA species that differ at their site of polyadenylation.

1	A well-studied example of regulated polyadenylation is the switch from the synthesis of membrane-bound to secreted antibody molecules that occurs during the development of B lymphocytes (see Figure 24–22). Early in the life history of a B lymphocyte, the antibody it produces is anchored in the plasma membrane, where it serves as a receptor for antigen. Antigen stimulation causes B lymphocytes to multiply and to begin secreting their antibody. The secreted form of the antibody is identical to the membrane-bound form except at the extreme C-terminus. In this part of the protein, the membrane-bound form has a long string of hydrophobic amino acids that traverses the lipid bilayer of the membrane, whereas the secreted form has a much shorter string of hydrophilic amino acids. The switch from membrane-bound to secreted antibody is generated through a change in the site of RNA cleavage and polyadenylation, as shown in Figure 7–59.

1	The change is caused by an increase in the concentration of a subunit of a protein (CstF) that promotes RNA cleavage (see Figure 6–34). The first cleavage/ poly-A addition site that a transcribing RNA polymerase encounters is suboptimal and is usually skipped in unstimulated B lymphocytes, leading to production of the longer RNA transcript. When activated to produce antibodies, the B lymphocyte increases its CstF concentration; as a result, cleavage now occurs at the suboptimal site, and the shorter transcript is produced. In this way, a change in concentration of a general RNA-processing factor has a dramatic effect on the expression of a particular gene. rnA editing Can Change the Meaning of the rnA Message

1	rnA editing Can Change the Meaning of the rnA Message The molecular mechanisms used by cells are a continual source of surprises. An example is the process of RNA editing, which alters the nucleotide sequences of RNA transcripts once they are synthesized and thereby changes the coded message they carry. We saw in Chapter 6 that tRNA and rRNA molecules are chemically modified after they are synthesized: here we focus on changes to mRNAs.

1	In animals, two principal types of mRNA editing occur: the deamination of adenine to produce inosine (A-to-I editing) and, less frequently, the deamination of cytosine to produce uracil (C-to-U editing), as shown in Figure 5–43. Because these chemical modifications alter the pairing properties of the bases (I pairs with C, and U pairs with A), they can have profound effects on the meaning of the RNA. If the edit occurs in a coding region, it can change the amino acid sequence of the protein or produce a truncated protein by creating a premature stop codon. Edits that occur outside coding sequences can affect the pattern of pre-mRNA splicing, the transport of mRNA from the nucleus to the cytosol, the efficiency with which the RNA is translated, or the base-pairing between microRNAs (miRNAs) and their mRNA targets, a form of regulation that will be discussed later in the chapter.

1	The process of A-to-I editing is particularly prevalent in humans, where it occurs in approximately 1000 genes. Enzymes called ADARs (adenosine deaminases acting on RNA) perform this type of editing; these enzymes recognize a double-stranded RNA structure that is formed through base-pairing between the site to be edited and a complementary sequence located elsewhere on the same RNA molecule, typically in an intron (Figure 7–60). The structure of the double-stranded RNA specifies whether the mRNA is to be edited, and if so, where the edit should be made. An especially important example of A-to-I editing takes place in the mRNA that codes for a transmitter-gated ion channel in the brain. A single edit changes a glutamine to an arginine; the affected amino acid lies on the inner wall of the channel, and the editing change alters the Ca2+ permeability of the channel. Mutant mice that cannot make this edit are prone to epileptic seizures and die during or shortly after weaning, showing

1	channel, and the editing change alters the Ca2+ permeability of the channel. Mutant mice that cannot make this edit are prone to epileptic seizures and die during or shortly after weaning, showing that editing of the ion channel RNA is normally crucial for proper brain development.

1	C-to-U editing, which is carried out by a different set of enzymes, is also crucial in mammals. For example, in certain cells of the gut, the mRNA for apolipoprotein B undergoes a C-to-U edit that creates a premature stop codon and therefore

1	Figure 7–59 Regulation of the site of RNA cleavage and poly-A addition determines whether an antibody molecule is secreted or remains membrane-bound. in unstimulated B lymphocytes (left), a long rnA transcript is produced, and the intron sequence (yellow) near its 3ʹ end is removed by rnA splicing to provide an mrnA molecule that codes for a membrane-bound antibody molecule. Only a portion of the antibody gene is shown in the figure; the actual gene and its mrnA would extend further to the left of the diagram. After antigen stimulation (right), the rnA transcript is cleaved and polyadenylated upstream from the intron’s 3′ splice site. As a result, some of the intron sequence remains as a coding sequence in the short transcript and specifies the hydrophilic C-terminal portion of the secreted antibody molecule (brown). (Adapted from D. Di Giammartino et al., Mol. Cell 43:853–866, 2011. with permission from elsevier.)

1	Figure 7–60 Mechanism of A-to-I RNA editing in mammals. typically, a sequence complementary to the position of the edit is present in an intron, and the resulting double-stranded rnA structure attracts an A-to-i editing enzyme (ADAr). in the case illustrated, the edit is made in an exon; in most cases, however, this occurs in noncoding portions of the mrnA. editing by ADAr takes place in the nucleus, before the pre-mrnA has been fully processed. Mice and humans have two ADAr genes: ADR1 is expressed in many tissues and is required in the liver for proper red blood cell development; ADR2 is expressed only in the brain, where it is required for proper brain development.

1	no editing editing, CAA UAA produces a shorter form of the protein. In cells of the liver, the editing enzyme is not expressed, and the full-length apolipoprotein B is produced. The two protein isoforms have different properties, and each plays a role in lipid metabolism that is specific to the organ that produces it (Figure 7–61).

1	Why RNA editing exists at all is a mystery. One idea is that it arose in evolution to correct “mistakes” in the genome. Another is that it arose as a somewhat slapdash way for the cell to produce subtly different proteins from the same gene. A third possibility is that RNA editing originally evolved as a defense mechanism against retroviruses and retrotransposons and was later adapted by the cell to change the meanings of certain mRNAs. Indeed, RNA editing still plays important roles in cell defense. Some retroviruses, including HIV, are extensively edited after they infect cells. This hyperediting creates many harmful mutations in the viral RNA genome and also causes viral mRNAs to be retained in the nucleus, where they are eventually degraded. Although some modern retroviruses protect themselves against this defense mechanism, RNA editing presumably helps to hold many viruses in check. rnA transport from the nucleus Can Be regulated

1	rnA transport from the nucleus Can Be regulated It has been estimated that in mammals only about one-twentieth of the total mass of RNA synthesized ever leaves the nucleus. We saw in Chapter 6 that most mammalian RNA molecules undergo extensive processing and that the “leftover” RNA fragments (excised introns and RNA sequences 3ʹ to the cleavage/poly-A site) are degraded in the nucleus. Incompletely processed and otherwise damaged RNAs are also eventually degraded as part of the quality control system for RNA production. As described in Chapter 6, the export of RNA molecules from the nucleus is delayed until processing has been completed. However, mechanisms that deliberately override this control point can be used to regulate gene expression. This strategy forms the basis for one of the best-understood examples of regulated nuclear transport of mRNA, which occurs in the human AIDS virus, HIV.

1	As we saw in Chapter 5, HIV, once inside the cell, directs the formation of a double-stranded DNA copy of its genome, which is then inserted into the genome of the host (see Figure 5–62). Once inserted, the viral DNA can be transcribed as one long RNA molecule by the host cell’s RNA polymerase II. This transcript is then spliced in many different ways to produce over 30 different species of mRNA, which in turn are translated into a variety of different proteins (Figure 7–62). In order to make progeny virus, entire, unspliced viral transcripts must be exported from the nucleus to the cytosol, where they are packaged into viral capsids and serve as the viral genome. This large transcript, as well as alternatively spliced HIV mRNAs that the virus needs to move to the cytoplasm for protein synthesis, still carries complete introns. The host cell’s normal block to the nuclear export of unspliced RNAs therefore presents a special problem for HIV.

1	Figure 7–61 C-to-U RNA editing produces a truncated form of apolipoprotein B. integrated into host genome1,000 nucleotide pairs The block is overcome in an ingenious way. The virus encodes a protein (called Rev) that binds to a specific RNA sequence (called the Rev responsive element, RRE) located within a viral intron. The Rev protein interacts with a nuclear export receptor (Crm1), which directs the movement of viral RNAs through nuclear pores into the cytosol despite the presence of intron sequences. We discuss in detail the way in which export receptors function in Chapter 12.

1	The regulation of nuclear export by Rev has several important consequences for HIV growth and pathogenesis. In addition to ensuring the nuclear export of specific unspliced RNAs, it divides the viral infection into an early phase (in which Rev is translated from a fully spliced RNA and all of the intron-containing viral RNAs are retained in the nucleus and degraded) and a late phase (in which unspliced RNAs are exported due to Rev function). This timing helps the virus replicate by providing the gene products in roughly the order in which they are needed (Figure 7–63). Regulation by Rev and by Tat, the HIV protein that counteracts premature transcription termination (see p. 414), allows the virus to achieve latency, a condition in which the HIV genome has become integrated into the host-cell genome but the production of viral proteins has temporarily ceased.

1	Figure 7–62 The compact genome of HIV, the human AIDS virus. the positions of the nine hiv genes are shown in green. the red double line indicates a DnA copy of the viral genome that has become integrated into the host DnA (gray). note that the coding regions of many genes overlap, and that those of Tat and Rev are split by introns. the blue line at the bottom of the figure represents the pre-mrnA transcript of the viral DnA and shows the locations of all the possible splice sites (arrows). there are many alternative ways of splicing the viral transcript; for example, the Env mrnAs retain the intron that has been spliced out of the Tat and Rev mrnAs. the rev response element (rre) is indicated by a blue ball and stick. it is a 234-nucleotide-long stretch of rnA that folds into a defined structure; rev recognizes a particular hairpin within this larger structure.

1	the Gag gene codes for a protein that is cleaved into several smaller proteins that form the viral capsid. the Pol gene codes for a protein that is cleaved to produce reverse transcriptase (which transcribes rnA into DnA), as well as the integrase involved in integrating the viral genome (as double-stranded DnA) into the host genome. the Env gene codes for the envelope proteins (see figure 5–62). tat, rev, vif, vpr, vpu, and nef are small proteins with a variety of functions. for example, rev regulates nuclear export (see figure 7–63) and tat regulates the elongation of transcription across the integrated viral genome (see p. 414).

1	Figure 7–63 Regulation of nuclear export by the HIV Rev protein. (A) early in hiv infection, only the fully spliced rnAs (which contain the coding sequences for rev, tat, and nef) are exported from the nucleus and translated. (B) Once sufficient rev protein has accumulated and been transported into the nucleus, unspliced viral rnAs can be exported from the nucleus. Many of these rnAs are translated into protein, and the full-length transcripts are packaged into new viral particles. If, after its initial entry into a host cell, conditions become unfavorable for viral transcription and replication, Rev and Tat are made at levels too low to promote transcription and export of unspliced RNA. This situation stalls the viral growth cycle until conditions improve, whereupon Rev and Tat levels increase, and the virus enters the replication cycle. Some mrnAs Are localized to Specific regions of the Cytosol

1	Once a newly made eukaryotic mRNA molecule has passed through a nuclear pore and entered the cytosol, it is typically met by ribosomes, which translate it into a polypeptide chain (see Figure 6–8). Once the first round of translation “passes” the nonsense-mediated decay test (see Figure 6–76), the mRNA is usually translated in earnest. If the mRNA encodes a protein that is destined to be secreted or expressed on the cell surface, a signal sequence at the protein’s N-terminus will direct it to the endoplasmic reticulum (ER). In this case, as discussed in Chapter 12, components of the cell’s protein-sorting apparatus recognize the signal sequence as soon as it emerges from the ribosome and direct the entire complex of ribosome, mRNA, and nascent protein to the membrane of the ER, where the remainder of the polypeptide chain is synthesized. In other cases, free ribosomes in the cytosol synthesize the entire protein, and signals in the completed polypeptide chain may then direct the

1	the remainder of the polypeptide chain is synthesized. In other cases, free ribosomes in the cytosol synthesize the entire protein, and signals in the completed polypeptide chain may then direct the protein to other sites in the cell.

1	Many mRNAs are themselves directed to specific intracellular locations before their efficient translation begins, allowing the cell to position its mRNAs close to the sites where the encoded protein is needed. RNA localization has been observed in many organisms, including unicellular fungi, plants, and animals, and it is likely to be a common mechanism that cells use to concentrate high-level production of proteins at specific sites. This strategy also provides the cell with other advantages. For example, it allows the establishment of asymmetries in the cytosol of the cell, a key step in many stages of development. Localized mRNA, coupled with translational control, also allows the cell to regulate gene expression independently in different regions. This feature is particularly important in large, highly polarized cells such as neurons, where it plays a central role in synaptic function.

1	Several mechanisms for mRNA localization have been discovered (Figure 7–64), all of which require specific signals in the mRNA itself. These signals are usually concentrated in the 3ʹ untranslated region (UTR), the region of RNA that

1	Figure 7–64 Mechanisms for the localization of mRNAs. the mrnA to be localized leaves the nucleus through nuclear pores (top). Some localized mrnAs (left diagram) travel to their destination by associating with cytoskeletal motors, which use the energy of Atp hydrolysis to move the mrnAs unidirectionally along filaments in the cytoskeleton (red) (see Chapter 16). At their destination, the mrnAs are held in place by anchor proteins (black). Other mrnAs randomly diffuse through the cytosol and are simply trapped by anchor proteins and at their sites of localization (center diagram). Some mrnAs (right diagram) are degraded in the cytosol unless they have bound, through random diffusion, a localized protein complex that anchors and protects the mrnA from degradation (black). each mechanism requires specific signals on the mrnA, which are typically located in the 3ʹ Utr. Additional components can block the translation of the mrnA until it is properly localized. (Adapted from h.D. lipshitz

1	specific signals on the mrnA, which are typically located in the 3ʹ Utr. Additional components can block the translation of the mrnA until it is properly localized. (Adapted from h.D. lipshitz and C.A. Smibert, Curr. Opin. Genet. Dev. 10:476–488, 2000. with permission from elsevier.)

1	Figure 7–65 An experiment demonstrating the importance of the 3ʹ UTR in localizing mRNAs to specific regions of the cytoplasm. for this experiment, two different fluorescently labeled rnAs were prepared by transcribing DnA in vitro in the presence of fluorescently labeled derivatives of Utp. One rnA (labeled with a red fluorochrome) contains the coding region for the Drosophila hairy protein and includes the adjacent 3ʹ Utr (see figure 6–21). the other rnA (labeled green) contains the hairy coding region with the 3ʹ Utr deleted. the two rnAs were mixed and injected into a Drosophila embryo at a stage of development when multiple nuclei reside in a common cytoplasm (see figure 7–26). when the fluorescent rnAs were visualized 10 minutes later, the full-length hairy rnA (red) was localized to the apical side of nuclei (blue) but the transcript missing the 3ʹ Utr (green) failed to localize. hairy is one of many transcriptional regulators that specify positional information in the

1	to the apical side of nuclei (blue) but the transcript missing the 3ʹ Utr (green) failed to localize. hairy is one of many transcriptional regulators that specify positional information in the developing Drosophila embryo (discussed in Chapter 21), and the localization of its mrnA (shown in this experiment to depend on its 3ʹ Utr) is critical for proper fly development. (Courtesy of Simon Bullock and David ish-horowicz.) extends from the stop codon that terminates protein synthesis to the start of the poly-A tail (Figure 7–65). The mRNA localization is usually coupled with translational controls to ensure that the mRNA remains quiescent until it has been moved into place.

1	The Drosophila egg exhibits an especially striking example of mRNA localization. The mRNA encoding the Bicoid transcription regulator is localized by attachment to the cytoskeleton at the anterior tip of the developing egg. When fertilization triggers the translation of this mRNA, it generates a gradient of the Bicoid protein that plays a crucial part in directing the development of the anterior part of the embryo (see Figure 7–26). Many mRNAs in somatic cells are also localized in a similar way. The mRNA that encodes actin, for example, is localized to the actin-filament-rich cell cortex in mammalian fibroblasts by means of a 3ʹ UTR signal.

1	We saw in Chapter 6 that mRNA molecules exit from the nucleus bearing numerous markings in the form of RNA modifications (the 5ʹ cap and the 3ʹ poly-A tail) and bound proteins (exon-junction complexes, for example) that signify the successful completion of the different pre-mRNA processing steps. As just described, the 3ʹ UTR of an mRNA can be thought of as a “zip code” that directs mRNAs to different places in the cell. Below, we will also see that mRNAs carry information specifying their average lifetime in the cytosol and the efficiency with which they are translated into protein. In a broad sense, the untranslated regions of eukaryotic mRNAs resemble the transcriptional control regions of genes: their nucleotide sequences contain information specifying the way the RNA is to be used, and proteins interpret this information by binding specifically to these sequences. Thus, over and above the specification of the amino acid sequences of proteins, mRNA molecules are rich with

1	used, and proteins interpret this information by binding specifically to these sequences. Thus, over and above the specification of the amino acid sequences of proteins, mRNA molecules are rich with information.

1	the 5ʹ and 3ʹ Untranslated regions of mrnAs Control their translation Once an mRNA has been synthesized, one of the most common ways of regulating the levels of its protein product is to control the step that initiates translation. Even though the details of translation initiation differ between eukaryotes and bacteria (as we saw in Chapter 6), they each use some of the same basic regulatory strategies. In bacterial mRNAs, a conserved stretch of nucleotides, the Shine–Dalgarno sequence, is always found a few nucleotides upstream of the initiating AUG codon. In bacteria, translational control mechanisms are carried out by proteins or by RNA molecules, and they generally involve either exposing or blocking the Shine– Dalgarno sequence (Figure 7–66).

1	Eukaryotic mRNAs do not contain such a sequence. Instead, as discussed in Chapter 6, the selection of an AUG codon as a translation start site is largely determined by its proximity to the cap at the 5ʹ end of the mRNA molecule, which is the site at which the small ribosomal subunit binds to the mRNA and begins scanning for an initiating AUG codon. In eukaryotes, translational repressors can bind to the 5ʹ end of the mRNA and thereby inhibit translation initiation. Other repressors recognize nucleotide sequences in the 3ʹ UTR of specific mRNAs and decrease translation initiation by interfering with the communication between the 5ʹ cap and 3ʹ poly-A tail, a step required for efficient translation (see Figure 6–70). A particularly important type of translational control in eukaryotes relies on small RNAs (termed microRNAs or miRNAs) that bind to mRNAs and reduce protein output, as described later in this chapter.

1	the phosphorylation of an initiation factor regulates protein Synthesis Globally Eukaryotic cells decrease their overall rate of protein synthesis in response to a variety of situations, including deprivation of growth factors or nutrients, infection by viruses, and sudden increases in temperature. Much of this decrease is caused by the phosphorylation of the translation initiation factor eIF2 by specific protein kinases that respond to the changes in conditions.

1	The normal function of eIF2 was outlined in Chapter 6. It forms a complex with GTP and mediates the binding of the methionyl initiator tRNA to the small ribosomal subunit, which then binds to the 5ʹ end of the mRNA and begins scanning along the mRNA. When an AUG codon is recognized, the eIF2 protein hydrolyzes the bound GTP to GDP, causing a conformational change in the protein and releasing it from the small ribosomal subunit. The large ribosomal subunit then joins the small one to form a complete ribosome that begins protein synthesis. Figure 7–66 Mechanisms of translational control. Although these examples are from bacteria, many of the same principles operate in eukaryotes.

1	(A) Sequence-specific rnA-binding proteins repress translation of specific mrnAs by blocking access of the ribosome to the Shine–Dalgarno sequence (orange). for example, some ribosomal proteins repress translation of their own rnA. this mechanism allows the cell to maintain correctly balanced quantities of the various components needed to form ribosomes. (B) An rnA “thermosensor” permits efficient translation initiation only at elevated temperatures at which the stem-loop structure has been melted. An example occurs in the human pathogen Listeria monocytogenes, in which the translation of its virulence genes increases at 37°C, the temperature of the host. (C) Binding of a small molecule to a riboswitch causes a major rearrangement of the rnA forming a different set of stem-loop structures. in the bound structure, the Shine–Dalgarno sequence (orange) is sequestered and translation initiation is thereby blocked. in many bacteria, S-adenosylmethionine acts in this manner to block

1	in the bound structure, the Shine–Dalgarno sequence (orange) is sequestered and translation initiation is thereby blocked. in many bacteria, S-adenosylmethionine acts in this manner to block production of the enzymes that synthesize it. (D) An “antisense” rnA produced elsewhere from the genome base-pairs with a specific mrnA and blocks its translation. Many bacteria regulate expression of iron-storage proteins in this way.

1	Because eIF2 binds very tightly to GDP, a guanine nucleotide exchange factor (see p. 157), designated eIF2B, is required to cause GDP release so that a new GTP molecule can bind and eIF2 can be reused (Figure 7–67A). The reuse of eIF2 is inhibited when it is phosphorylated—the phosphorylated eIF2 binds to eIF2B unusually tightly, inactivating eIF2B. There is more eIF2 than eIF2B in cells, and even a fraction of phosphorylated eIF2 can trap nearly all of the eIF2B. This prevents the reuse of the nonphosphorylated eIF2 and greatly slows protein synthesis (Figure 7–67B). Regulation of the level of active eIF2 is especially important in mammalian cells; eIF2 is part of the mechanism that allows cells to enter a nonproliferating, resting state (called G0) in which the rate of total protein synthesis is reduced to about one-fifth the rate in proliferating cells. initiation at AUG Codons Upstream of the translation Start Can regulate eukaryotic translation initiation

1	We saw in Chapter 6 that eukaryotic translation typically begins at the first AUG downstream of the 5ʹend of the mRNA, which is the first AUG encountered by a scanning small ribosomal subunit. But the nucleotides immediately surrounding the AUG also influence the efficiency of translation initiation. If the recognition site is poor enough, scanning ribosomal subunits will sometimes ignore the first AUG codon in the mRNA and skip to the second or third AUG codon instead. This phenomenon, known as “leaky scanning,” is a strategy frequently used to produce two or more closely related proteins, differing only in their N-termini, from the same mRNA. A particularly important use of this mechanism is the production of the same protein with and without a signal sequence attached at its N-terminus. This allows the protein to be directed to two different locations in the cell (for example, to both mitochondria and the cytosol). Cells can regulate the relative abundance of the protein isoforms

1	This allows the protein to be directed to two different locations in the cell (for example, to both mitochondria and the cytosol). Cells can regulate the relative abundance of the protein isoforms produced by leaky scanning; for example, a cell-type-specific increase in the abundance of the initiation factor eIF4F favors the use of the AUG closest to the 5ʹ end of the mRNA.

1	Another type of control found in eukaryotes uses one or more short open reading frames—short stretches of DNA that begin with a start codon (ATG) and end with a stop codon, with no stop codons in between—that lie between the 5ʹend of the mRNA and the beginning of the gene. Often, the amino acid sequences coded by these upstream open reading frames (uORFs) are not important; rather, the uORFs serve a purely regulatory function. An uORF present on an mRNA molecule will generally decrease translation of the downstream gene by trapping a guanine nucleotide exchange factor, eIF2B IN ABSENCE OF ACTIVE eIF2B, THE REMAINING UNBOUND eIF2 REMAINS IN ITS Figure 7–67 The eIF2 cycle. (A) the INACTIVE, GDP-recycling of used eif2 by a guanine BOUND FORM nucleotide exchange factor (eif2B). (B) eif2 (B) INACTIVE COMPLEX rates by tying up eif2B.

1	scanning ribosome initiation complex and causing the ribosome to translate the uORF and dissociate from the mRNA before it reaches the bona fide protein-coding sequence. When the activity of a general translation factor (such as the eIF2 discussed above) is reduced, one might expect that the translation of all mRNAs would be reduced equally. Contrary to this expectation, however, the phosphorylation of eIF2 can have selective effects, even enhancing the translation of specific mRNAs that contain uORFs. This can enable cells, for example, to adapt to starvation for specific nutrients by shutting down the synthesis of all proteins except those that are required for synthesis of the missing nutrients. The details of this mechanism have been worked out for a specific yeast mRNA that encodes a protein called Gcn4, a transcription regulator that activates many genes that encode proteins that are important for amino acid synthesis.

1	The Gcn4 mRNA contains several short uORFs, and when amino acids are abundant, ribosomes translate the uORFs and generally dissociate before they reach the Gcn4 coding region. A global decrease in eIF2 activity brought about by amino acid starvation makes it more likely that a scanning small ribosomal subunit will move across the uORFs (without translating them) before it acquires a molecule of eIF2 (see Figure 6–70). Such a ribosomal subunit is then free to initiate translation on the actual Gcn4 sequences. The increased level of this transcription regulator increases production of the amino acid biosynthetic enzymes.

1	Although approximately 90% of eukaryotic mRNAs are translated beginning with the first AUG downstream from the 5ʹ cap, certain AUGs, as we saw in the previous section, can be skipped over during the scanning process. In this section, we discuss yet another way that cells can initiate translation at positions distant from the 5ʹ end of the mRNA, using a specialized type of RNA sequence called an internal ribosome entry site (IRES). In some cases, two distinct protein-coding sequences are carried in tandem on the same eukaryotic mRNA; translation of the first occurs by the usual scanning mechanism, and translation of the second occurs through an IRES. IRESs are typically several hundred nucleotides in length and fold into specific structures that bind many, but not all, of the same proteins that are used to initiate normal 5ʹ cap-dependent translation (Figure 7–68). In fact, different IRESs require different subsets of initiation factors. However, all of them bypass the need for a 5ʹ

1	that are used to initiate normal 5ʹ cap-dependent translation (Figure 7–68). In fact, different IRESs require different subsets of initiation factors. However, all of them bypass the need for a 5ʹ cap structure and the translation initiation factor that recognizes it, eIF4E.

1	Figure 7–68 Two mechanisms of translation initiation. (A) the normal, cap-dependent mechanism requires a set of initiation factors whose assembly on the mrnA is stimulated by the presence of a 5ʹ cap and a poly-A tail (see also figure 6–70). (B) the ireS-dependent mechanism, seen mainly in viruses, requires only a subset of the normal translation initiating factors, and these assemble directly on the folded ireS. (Adapted from A. Sachs, Cell 101:243–245, 2000. with permission from elsevier.)

1	Some viruses use IRESs as part of a strategy to get their own mRNA molecules translated while blocking normal 5ʹ cap-dependent translation of host mRNAs. On infection, these viruses produce a protease (encoded in the viral genome) that cleaves the host-cell translation factor eIF4G, rendering it unable to bind to eIF4E, the cap-binding complex. This shuts down most of the host cell’s translation and effectively diverts the translation machinery to the IRES sequences present on the viral mRNAs. (The truncated eIF4G remains competent to initiate translation at these internal sites.) The many ways in which viruses manipulate their host’s protein-synthesis machinery for their own advantage continue to surprise cell biologists. Studying this “arms race” between humans and pathogens has led to many fundamental insights into the workings of the cell, and we revisit this topic in more detail in Chapter 23.

1	Most mRNAs in a bacterial cell are very unstable, having half-lives of less than a couple of minutes. Exonucleases, which degrade in the 3ʹ-to-5ʹ direction, are usually responsible for the rapid destruction of these mRNAs. Because its mRNAs are both rapidly synthesized and rapidly degraded, a bacterium can adapt quickly to environmental changes. As a general rule, the mRNAs in eukaryotic cells are more stable. Some, such as that encoding β globin, have half-lives of more than 10 hours, but most have considerably shorter half-lives, typically less than 30 minutes. The mRNAs that code for proteins such as growth factors and transcription regulators, whose production rates need to change rapidly in cells, have especially short half-lives.

1	We saw in Chapter 6 that the cell has several mechanisms that rapidly destroy incorrectly processed RNAs; here, we consider the fate of the typical “normal” eukaryotic mRNA. Two general mechanisms exist for eventually destroying each mRNA that is made by the cell. Both begin with the gradual shortening of the poly-A tail by an exonuclease, a process that starts as soon as the mRNA reaches the cytosol. In a broad sense, this poly-A shortening acts as a timer that counts down the lifetime of each mRNA. Once the poly-A tail is reduced to a critical length (about 25 nucleotides in humans), the two pathways diverge. In one, the 5ʹ cap is removed (a process called decapping) and the “exposed” mRNA is rapidly degraded from its 5ʹ end. In the other, the mRNA continues to be degraded from the 3ʹ end, through the poly-A tail, into the coding sequences (Figure 7–69).

1	Nearly all mRNAs are subject to both types of decay, and the specific sequences of each mRNA determine how fast each step occurs and therefore how long each mRNA will persist in the cell and be able to produce protein. The 3ʹ UTR sequences are especially important in controlling mRNA lifetimes, and they often carry binding sites for specific proteins that increase or decrease the rate of poly-A shortening, decapping, or 3ʹ-to-5ʹ degradation. The half-life of an mRNA is also affected by how efficiently it is translated. Poly-A shortening and decapping compete directly with the machinery that translates the mRNA; therefore, any factors that affect the translation efficiency of an mRNA will tend to have the opposite effect on its degradation (Figure 7–70). Although poly-A shortening controls the half-life of most eukaryotic mRNAs, some mRNAs can be degraded by a specialized mechanism that bypasses this step altogether. In these cases, specific nucleases cleave the mRNA internally,

1	Figure 7–69 Two mechanisms of eukaryotic mRNA decay. A critical threshold of poly-A tail length induces rapid 3ʹ-to-5ʹ degradation, which may be triggered by the loss of the poly-Abinding proteins. As shown in figure 7–70, a deadenylase associates with both the 3ʹ poly-A tail and the 5ʹ cap, and this connection may be involved in signaling decapping after poly-A shortening. Although 5ʹ-to-3ʹ and 3ʹ-to-5ʹ degradation are shown here on separate rnA molecules, these two processes can occur together on the same molecule. (Adapted from C.A. Beelman and r. parker, Cell 81:179–183, 1995. with permission from elsevier.)

1	Figure 7–70 The competition between effectively decapping one end and removing the poly-A tail from the other so that both halves are rapidly degraded. The mRNAs that are destroyed in this way carry specific nucleotide sequences, often in the 3ʹ UTRs, that serve as recognition sequences for these endonucleases. This strategy makes it especially simple to tightly regulate the stability of these mRNAs by blocking or exposing the endonuclease site in response to extracellular signals. For example, the addition of iron to cells decreases the stability of the mRNA that encodes the receptor protein that binds the iron-transporting protein transferrin, causing less of this receptor to be made. This effect is mediated by the iron-sensitive RNA-binding protein aconitase. Aconitase can bind to the 3ʹ UTR of the transferrin receptor mRNA and increase receptor production by blocking endonucleolytic cleavage of the mRNA. On the addition of iron, aconitase is released from the mRNA, exposing the

1	3ʹ UTR of the transferrin receptor mRNA and increase receptor production by blocking endonucleolytic cleavage of the mRNA. On the addition of iron, aconitase is released from the mRNA, exposing the cleavage site and thereby decreasing the stability of the mRNA (Figure 7–71).

1	regulation of mrnA Stability involves p-bodies and Stress Granules We saw in Chapters 3 and 6 that large aggregates of proteins and nucleic acids that work together are often held in proximity by loose, low-affinity connections (see Figure 3–36). In this way, they function as “organelles” even though they are not surrounded by membranes. Many of the events discussed in the previous mRNA translation and mRNA decay. the same two features of an mrnA molecule—its 5ʹ cap and the 3ʹ poly-A decay (see figure 7–69). the deadenylase that shortens the poly-A tail in the 3ʹ-to5ʹ direction associates with the 5ʹ cap. As described in Chapter 6 (see figure 6–70), the translation initiation machinery also associates with both the 5ʹ cap and the poly-A tail. (Adapted from M. Gao et al., Mol. Cell 5:479–488, 2000. with permission from elsevier.)

1	Figure 7–71 Two post-translational controls mediated by iron. (A) During iron starvation, the binding of aconitase to the 5ʹ Utr of the ferritin mrnA blocks translation initiation; its binding to the 3ʹ Utr of the transferrin receptor mrnA blocks an endonuclease cleavage site and thereby stabilizes the mrnA.

1	(B) in response to an increase in iron concentration in the cytosol, a cell increases its synthesis of ferritin in order to bind the extra iron and decreases its synthesis of transferrin receptors in order to import less iron across the plasma membrane. Both responses are mediated by the same iron-responsive regulatory protein, aconitase, which recognizes common features in a stem-loop structure in the mrnAs encoding ferritin and the transferrin receptor. Aconitase dissociates from the mrnA when it binds iron. But because the transferrin receptor and ferritin are regulated by different types of mechanisms, their levels respond oppositely to iron concentrations even though they are regulated by the same iron-responsive regulatory protein. section—including decapping and RNA degradation—take place in aggregates known as Processingor P-bodies, which are present in the cytosol (Figure 7–72).

1	Although many mRNAs are eventually degraded in P-bodies, some remain intact and are later returned to the pool of translating mRNAs. To be “rescued” in this way, mRNAs move from P-bodies to another type of aggregate known as a stress granule, which contains translation initiation factors, poly-A-binding protein, and small ribosomal subunits. Translation itself does not occur in stress granules, but mRNAs can become “translation-ready” as the proteins bound to them in P-bodies are replaced with those in stress granules. The movement of mRNAs between active translation, P-bodies, and stress granules can be seen as an mRNA cycle (Figure 7–73) where the competition between translation and mRNA degradation is carefully controlled. Thus, when translation initiation is blocked (by starvation, drugs, or genetic manipulation), stress granules enlarge as more and more nontranslated mRNAs are moved directly into them for storage. Clearly, once a cell has made the large investment in producing a

1	or genetic manipulation), stress granules enlarge as more and more nontranslated mRNAs are moved directly into them for storage. Clearly, once a cell has made the large investment in producing a properly processed mRNA molecule, it carefully controls its subsequent fate.

1	Many steps in the pathway from RNA to protein are regulated by cells in order to control gene expression. Most genes are regulated at multiple levels, in addition to being controlled at the initiation stage of transcription. The regulatory mechanisms include (1) attenuation of the RNA transcript by its premature termination, (2) alternative RNA splice-site selection, (3) control of 3ʹ-end formation by cleavage and poly-A addition, (4) RNA editing, (5) control of transport from the nucleus to the cytosol, (6) localization of mRNAs to particular parts of the cell, (7) control of translation initiation, and (8) regulated mRNA degradation. Most of these control processes require the recognition of specific sequences or structures in the RNA molecule being regulated, a task performed by either regulatory proteins or regulatory RNA molecules. Figure 7–72 Visualization of P-bodies.

1	Figure 7–72 Visualization of P-bodies. human cells were stained with antibodies to a component of the mrnA decapping enzyme Dcp1a (left panels) and to the Argonaute protein (middle panels). As described later in this chapter, Argonaute is a key component of rnA interference pathways. the merged image (right panels) shows that the two proteins co-localize to p-bodies in the cytoplasm. (Adapted from J. liu et al., Nat. Cell Biol. 7:719–723, 2005. with permission from Macmillan publishers ltd.) Figure 7–73 Possible fates of an mRNA molecule. An mrnA molecule released from the nucleus can be actively translated (center), stored in stress granules (right), or degraded in p-bodies (left). As the needs of the cell change, mrnAs can be shuffled from one pool to the next, as indicated by the arrows.

1	In the previous chapter, we introduced the central dogma, according to which the flow of genetic information proceeds from DNA through RNA to protein (Figure 6–1). But we have seen throughout this book that RNA molecules perform many critical tasks in the cell besides serving as intermediate carriers of genetic information. Among these noncoding RNAs are the rRNA and tRNA molecules, which are responsible for reading the genetic code and synthesizing proteins. The RNA molecule in telomerase serves as a template for the replication of chromosome ends, snoRNAs modify ribosomal RNA, and snRNAs carry out the major events of RNA splicing. And we saw in the previous section that Xist RNA has an important role in inactivating one copy of the X chromosome in females.

1	A series of recent discoveries has revealed that noncoding RNAs are even more prevalent than previously imagined. We now know that such RNAs play widespread roles in regulating gene expression and in protecting the genome from viruses and transposable elements. These newly discovered RNAs are the subject of this section.

1	We begin our discussion with a group of short RNAs that carry out RNA interference or RNAi. Here, short single-stranded RNAs (20–30 nucleotides) serve as guide RNAs that selectively reorganize and bind—through base-pairing—other RNAs in the cell. When the target is a mature mRNA, the small noncoding RNAs can inhibit its translation or even catalyze its destruction. If the target RNA molecule is in the process of being transcribed, the small noncoding RNA can bind to it and direct the formation of certain types of repressive chromatin on its attached DNA template (Figure 7–74). Three classes of small noncoding RNAs work in this way—microRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs)—and we discuss them in turn in the next sections. Although they differ in the way the short pieces of single-stranded RNA are generated, all three types of short RNAs locate their targets through RNA–RNA base-pairing, and they generally cause reductions in gene

1	differ in the way the short pieces of single-stranded RNA are generated, all three types of short RNAs locate their targets through RNA–RNA base-pairing, and they generally cause reductions in gene expression.

1	Over 1000 different microRNAs (miRNAs) are produced from the human genome, and these appear to regulate at least one-third of all human protein-coding genes. Once made, miRNAs base-pair with specific mRNAs and fine-tune their translation and stability. The miRNA precursors are synthesized by RNA polymerase II and are capped and polyadenylated. They then undergo a special type of processing, after which the miRNA (typically 23 nucleotides in length) is assembled with a set of proteins to form an RNA-induced silencing complex or RISC. Once formed, the RISC seeks out its target mRNAs by searching for complementary nucleotide translational repression formation of heterochromatin cleavage of of target RNA is being transcribed

1	Figure 7–74 RNA interference in eukaryotes. Single-stranded interfering rnAs are generated from double-stranded rnA. they locate target rnAs through base-pairing and, at this point, several fates are possible, as shown. As described in the text, there are several types of rnA interference; the way the double-stranded rnA is produced and processed and the ultimate fate of the target rnA depends on the particular system. sequences (Figure 7–75). This search is greatly facilitated by the Argonaute protein, a component of RISC, which holds the 5ʹ region of the miRNA so that it is optimally positioned for base-pairing to another RNA molecule (Figure 7–76). In animals, the extent of base-pairing is typically at least seven nucleotide pairs, and this pairing most often occurs in the 3ʹ UTR of the target mRNA.

1	Once an mRNA has been bound by an miRNA, several outcomes are possible. If the base-pairing is extensive (which is unusual in humans but common in many plants), the mRNA is cleaved (sliced) by the Argonaute protein, effectively removing the mRNA’s poly-A tail and exposing it to exonucleases (see Figure 7–69). Following cleavage of the mRNA, the RISC with its associated miRNA is released, and it can seek out additional mRNAs (see Figure 7–75). Thus, a single miRNA can act catalytically to destroy many complementary mRNAs. These miRNAs can be thus thought of as guide sequences that bring destructive nucleases into contact with specific mRNAs.

1	If the base-pairing between the miRNA and the mRNA is less extensive (as observed for most human miRNAs), Argonaute does not slice the mRNA; rather, translation of the mRNA is repressed and the mRNA is shuttled to P-bodies (see Figure 7–73) where, sequestered from ribosomes, it eventually undergoes poly-A tail shortening, decapping, and degradation.

1	Several features make miRNAs especially useful regulators of gene expression. First, a single miRNA can regulate a whole set of different mRNAs, so long as the mRNAs carry a common short sequence in their UTRs. This situation is common in humans, where a single miRNA can control hundreds of different mRNAs. Second, regulation by miRNAs can be combinatorial. When the base-pairing between the miRNA and mRNA fails to trigger cleavage, additional miRNAs binding to the same mRNA lead to further reductions in its translation. As discussed earlier for transcription regulators, combinatorial control greatly expands the possibilities available to the cell by linking gene expression to a combination of different regulators rather than a single regulator. Third, an miRNA occupies relatively little

1	Figure 7–75 miRNA processing and mechanism of action. the precursor mirnA, through complementarity between one part of its sequence and another, forms a double-stranded structure. this rnA is cropped while still in the nucleus and then exported to the cytosol, where it is further cleaved by the Dicer enzyme to form the mirnA proper. Argonaute, in conjunction with other components of riSC, initially associates with both strands of the mirnA and then cleaves and discards one of them. the other strand guides riSC to specific mrnAs through base-pairing. if the rnA–rnA match is extensive, as is commonly seen in plants, Argonaute cleaves the target mrnA, causing its rapid degradation. in mammals, the mirnA–mrnA match often does not extend beyond a short seven-nucleotide “seed” region near the 5ʹ end of the mirnA. this less extensive base-pairing leads to inhibition of translation, mrnA destabilization, and transfer of the mrnA to p-bodies, where it is eventually degraded.

1	3˜ OH (end of RNA) active site, showing 2 Mg2+ atoms needed for “slicing” space in the genome when compared with a protein. Indeed, their small size is one reason that miRNAs were discovered only recently. Although we are only beginning to appreciate the full impact of miRNAs, it is clear that they represent an important part of the cell’s equipment for regulating the expression of genes. We discuss specific examples of miRNAs that have key roles in development in Chapter 21. rnA interference is Also Used as a Cell Defense Mechanism

1	rnA interference is Also Used as a Cell Defense Mechanism Many of the proteins that participate in the miRNA regulatory mechanisms just described also serve a second function as a defense mechanism: they orchestrate the degradation of foreign RNA molecules, specifically those that occur in double-stranded form. Many transposable elements and viruses produce double-stranded RNA, at least transiently, in their life cycles, and RNA interference helps to keep these potentially dangerous invaders in check. As we shall see, this form of RNAi also provides scientists with a powerful experimental technique to turn off the expression of individual genes.

1	The presence of double-stranded RNA in the cell triggers RNAi by attracting a protein complex containing Dicer, the same nuclease that processes miR-NAs (see Figure 7–75). This protein cleaves the double-stranded RNA into small fragments (approximately 23 nucleotide pairs) called small interfering RNAs (siRNAs). These double-stranded siRNAs are then bound by Argonaute and other components of RISC. As we saw above for miRNAs, one strand of the duplex RNA is then cleaved by Argonaute and discarded. The single-stranded siRNA molecule that remains directs RISC back to complementary RNA molecules produced by the virus or transposable element. Because the match is usually exact, Argonaute cleaves these molecules, leading to their rapid destruction.

1	Each time RISC cleaves a new RNA molecule, the RISC is released; thus, as we saw for miRNAs, a single RNA molecule can act catalytically to destroy many complementary RNAs. Some organisms employ an additional mechanism that amplifies the RNAi response even further. In these organisms, RNA-dependent RNA polymerases use siRNAs as primers to produce additional copies of double-strand RNAs which are then cleaved into siRNAs. This amplification ensures that, once initiated, RNA interference can continue even after all the initiating double-stranded RNA has been degraded or diluted out. For example, it permits progeny cells to continue carrying out the specific RNA interference that was provoked in the parent cells.

1	In some organisms, the RNA interference activity can be spread by the transfer of RNA fragments from cell to cell. This is particularly important in plants (whose cells are linked by fine connecting channels, as discussed in Chapter 19), because it allows an entire plant to become resistant to an RNA virus after only a few of its cells have been infected. In a broad sense, the RNAi response resembles certain aspects of the animal immune system; in both, an invading organism elicits a customized response, and—through amplification of the “attack” molecules—the host becomes systemically protected.

1	Figure 7–76 Human Argonaute protein carrying an miRNA. the protein is folded into four structural domains, each indicated by a different color. the mirnA is held in an extended form that is optimal for forming rnA–rnA base pairs. the active site of Argonaute that “slices” a target rnA, when it is extensively base-paired with the mirnA, is indicated in red. Many Argonaute proteins (three out of the four human proteins, for example) lack the catalytic site and therefore bind target rnAs without slicing them. (Adapted from C.D. Kuhn and l. Joshua-tor, Trends Biochem. Sci. 38:263–271, 2013. with permission from Cell press.)

1	We have seen that although miRNAs and siRNAs are generated in slightly different ways, they rely on the same proteins and seek out their targets in a fundamentally similar manner. Because siRNAs are found in widespread species, they are believed to be the most ancient form of RNA interference, with miRNAs being a later refinement. These siRNA-mediated defense mechanisms are crucial for plants, worms, and insects. In mammals, a protein-based system (described in Chapter 24) has largely taken over the task of fighting off viruses.

1	The siRNA interference pathway just described does not necessarily stop with the destruction of target RNA molecules. In some cases, the RNA interference machinery can also selectively shut off synthesis of the target RNAs. For this to occur, the short siRNAs produced by the Dicer protein are assembled with a group of proteins (including Argonaute) to form the RITS (RNA-induced transcriptional silencing) complex. Using single-stranded siRNA as a guide sequence, this complex binds complementary RNA transcripts as they emerge from a transcribing RNA polymerase II (Figure 7–77). Positioned on the genome in this way, the RITS complex attracts proteins that covalently modify nearby histones and eventually direct the formation of heterochromatin to prevent further transcription initiation. In some cases, an RNA-dependent RNA polymerase and a Dicer enzyme are also recruited by the RITS complex to continually generate additional siRNAs in situ. This positive feedback loop ensures continued

1	some cases, an RNA-dependent RNA polymerase and a Dicer enzyme are also recruited by the RITS complex to continually generate additional siRNAs in situ. This positive feedback loop ensures continued repression of the target gene even after the initiating siRNA molecules have disappeared.

1	RNAi-directed heterochromatin formation is an important cell defense mechanism that limits the spread of transposable elements in genomes by maintaining their DNA sequences in a transcriptionally silent form. However, this same mechanism is also used in some normal processes in the cell. For example, in many organisms, the RNA interference machinery maintains the heterochromatin formed around centromeres. Centromeric DNA sequences are transcribed in both directions, producing complementary RNA transcripts that can base-pair to form double-stranded RNA. This double-stranded RNA triggers the RNA Figure 7–77 RNA interference directed by siRNAs. in many organisms, double-stranded rnA can trigger both the destruction of complementary mrnAs (left) and transcriptional silencing (right). the change in chromatin structure induced by the bound ritS (rnAinduced transcriptional silencing) complex resembles that in figure 7–45.

1	interference pathway and stimulates formation of the heterochromatin that surrounds centromeres, which is necessary for the centromeres to segregate chromosomes accurately during mitosis. pirnAs protect the Germ line from transposable elements

1	pirnAs protect the Germ line from transposable elements A third system of RNA interference relies on piRNAs (piwi-interacting RNAs, named for Piwi, a class of proteins related to Argonaute). piRNAs are made specifically in the germ line, where they block the movement of transposable elements. Found in many organisms, including humans, genes coding for piRNAs consist largely of sequence fragments of transposable elements. These clusters of fragments are transcribed and broken up into short, single-stranded piRNAs. The processing differs from that for miRNAs and siRNAs (for one thing, the Dicer enzyme is not involved), and the resulting piRNAs are slightly longer than miRNAs and siRNAs; moreover, they are complexed with Piwi rather than Argonaute proteins. Once formed, the piRNAs seek out RNA targets by base-pairing and, much like siRNAs, transcriptionally silence intact transposon genes and destroy any RNA (including mRNAs) produced by them.

1	Many mysteries surround piRNAs. Over a million piRNA species are coded in the genomes of many mammals and expressed in the testes, yet only a small fraction seem to be directed against the transposons present in those genomes. Are the piRNAs remnants of past invaders? Do they cover so much “sequence space” that they are broadly protective for any foreign DNA? Another curious feature of piRNAs is that many of them (particularly if base-pairing does not have to be perfect) should, in principle, attack the normal mRNAs made by the organism, yet they do not. It has been proposed that these large numbers of piRNAs may form a system to distinguish “self” RNAs from “foreign” RNAs and attack only the latter. If this is the case, there must be a special way for the cell to spare its own RNAs. One idea is that RNAs produced in the previous generation of an organism are somehow registered and set aside from piRNA attack in subsequent generations. Whether or not this mechanism truly exists, and,

1	idea is that RNAs produced in the previous generation of an organism are somehow registered and set aside from piRNA attack in subsequent generations. Whether or not this mechanism truly exists, and, if so, how it might work, are questions that demonstrate our incomplete understanding of the full implications of RNA interference.

1	Although it likely arose as a defense mechanism against viruses and transposable elements, RNA interference, as we have seen, has become thoroughly integrated into many aspects of normal cell biology, ranging from the control of gene expression to the structure of chromosomes. It has also been developed by scientists into a powerful experimental tool that allows almost any gene to be inactivated by evoking an RNAi response to it. This technique, which can be readily carried out in cultured cells and, in many cases, whole animals and plants, has made possible new genetic approaches in cell and molecular biology. We shall discuss it in detail in the following chapter where we cover modern genetic methods used to study cells (see pp. 499–501). RNAi also has potential in treating human disease. Since many human disorders result from the misexpression of genes, the ability to turn these genes off by experimentally introducing complementary siRNA molecules holds great medical promise.

1	Since many human disorders result from the misexpression of genes, the ability to turn these genes off by experimentally introducing complementary siRNA molecules holds great medical promise. Although the mechanism of RNA interference was discovered a few decades ago, we are still being surprised by its mechanistic details and by its broad biological implications.

1	Bacteria Use Small noncoding rnAs to protect themselves from viruses

1	Bacteria make up the vast majority of the Earth’s biomass and, not surprisingly, viruses that infect bacteria greatly outnumber plant and animal viruses. These viruses generally have DNA genomes. A recent discovery revealed that many species of bacteria (and almost all species of archaebacteria) use a repository of small noncoding RNA molecules to seek out and destroy the DNA of the invading viruses. Many features of this defense mechanism, known as the CRISPR system, resemble those we saw above for miRNAs and siRNAs, but there are two important differences. First, when bacteria and archaea are first infected by a virus, they have a mechanism that causes short fragments of that viral DNA to become integrated into their genomes. These serve as “vaccinations,” in the sense that they become the templates for producing small noncoding RNAs known as crRNAs (CRISPR RNAs) that will thereafter destroy the virus should it reinfect the descendants of the original cell. This aspect of the CRISPR

1	templates for producing small noncoding RNAs known as crRNAs (CRISPR RNAs) that will thereafter destroy the virus should it reinfect the descendants of the original cell. This aspect of the CRISPR system is similar in principle to adaptive immunity in mammals, in that the cell carries a record of past exposures that is used to protect against future exposures. The second distinguishing feature of the CRISPR system is that these crRNAs then become associated with special proteins that allow them to seek out and destroy double-stranded DNA molecules, rather than single-stranded RNA molecules.

1	Although many details of CRISPR-mediated immunity remain to be discovered, we can outline the general process in three steps (Figure 7–78). In the first, viral DNA sequences are integrated into special regions of the bacterial genome known as CRISPR (clustered regularly interspersed short palindromic repeat) loci, named for the peculiar structure that first drew the attention of scientists. In its simplest form, a CRISPR locus consists of several hundred repeats of a host DNA sequence interspersed with a large collection of sequences (typically 25–70 nucleotide pairs each) that has been derived from prior exposures to viruses and other foreign DNA. The newest viral sequence is always integrated at the 5ʹend of the CRISPR locus, the end that is transcribed first. Each locus, therefore, carries a temporal record of prior infections. Many bacterial and archaeal species carry several large CRISPR loci in their genomes and are thus immune to a wide range of viruses.

1	In the second step, the CRISPR locus is transcribed to produce a long RNA molecule, which is then processed into the much shorter (approximately 30 nucleotides) crRNAs. In the third step, crRNAs complexed with Cas (CRISPR-associated) proteins seek out complementary viral DNA sequences and direct their destruction by nucleases. Although structurally dissimilar, Cas proteins are analogous to the Argonaute and Piwi proteins discussed above: they hold small single-stranded RNAs in an extended configuration that is optimized, in this case, for seeking and forming complementary base pairs with DNA.

1	We still have much to learn about CRISPR-based immunity in bacteria and archaebacteria. The mechanism through which viral sequences are first identified and integrated into the host genome is poorly understood, as is the way that the crRNAs find their complementary sequences in double-stranded DNA. Moreover, in different species of bacteria and archaebacteria, crRNAs are processed in different ways, and in some cases, the crRNAs can attack viral RNAs as well as DNAs. We shall see in the following chapter that bacterial CRISPR systems have already been artificially “moved” into plants and animals, where they have become very powerful experimental tools for manipulating genomes.

1	Figure 7–78 CRISPR-mediated immunity in bacteria and archaebacteria. After infection by a virus (left panel), a small bit of DnA from the viral genome is inserted into the CriSpr locus. for this to happen, a small fraction of infected cells must survive the initial viral infection. the surviving cells, or more generally their descendants, transcribe the CriSpr locus and process the transcript into crrnAs (middle panel). Upon reinfection with a virus that the population has already been “vaccinated” against, the incoming viral DnA is destroyed by a complementary crrnA (right panel). for a CriSpr system to be effective, the crrnAs must not destroy the CriSpr locus itself, even though the crrnAs are complementary in sequence to it. in many species, in order for crrnAs to attack an invading DnA molecule, there must be additional short nucleotide sequences that are carried by the target molecule. Because these sequences, known as pAMs (protospacer adjacent motifs), lie outside the crrnA

1	DnA molecule, there must be additional short nucleotide sequences that are carried by the target molecule. Because these sequences, known as pAMs (protospacer adjacent motifs), lie outside the crrnA sequences, the host CriSpr locus is spared (see figure 8–55).

1	long noncoding rnAs have Diverse functions in the Cell

1	In this and the preceding chapters, we have seen that noncoding RNA molecules have many functions in the cell. Yet, as is the case with proteins, there remain many noncoding RNAs whose function is still unknown. Many RNAs of unknown function belong to a group known as long noncoding RNA (lncRNA). These are arbitrarily defined as RNAs longer than 200 nucleotides that do not code for protein. As methods have improved for determining the nucleotide sequences of all the RNA molecules produced by a cell line or tissue, the sheer number of lncRNAs (an estimated 8000 for the human genome, for example) came as a surprise to scientists. Most lncRNAs are transcribed by RNA polymerase II and have 5ʹ caps and poly-A tails, and, in many cases, they are spliced. It has been difficult to annotate lncRNAs because low levels of RNA are now known to be made from 75% of the human genome. Most of these RNAs are thought to result from the background “noise” of transcription and RNA processing. According

1	because low levels of RNA are now known to be made from 75% of the human genome. Most of these RNAs are thought to result from the background “noise” of transcription and RNA processing. According to this idea, such nonfunctional RNAs provide no fitness advantage or disadvantage to the organism and are a tolerated by-product of the complex patterns of gene expression that need to be produced in multicellular organisms. For these reasons, it is difficult to estimate the number of lncRNAs that are likely to have a function in the cell and to distinguish them from the background transcription.

1	We have already encountered a few lncRNAs, including the RNA in telomerase (see Figure 5–33), Xist RNA (see Figure 7–52), and an RNA involved in imprinting (see Figure 7–49). Other lncRNAs have been implicated in controlling the enzymatic activity of proteins, inactivating transcription regulators, affecting splicing patterns, and blocking translation of certain mRNAs.

1	In terms of biological function, lncRNA should be considered a catch-all phrase encompassing a great diversity of functions. Nevertheless, there are two unifying features of lncRNAs that can account for their many roles in the cell. The first is that lncRNAs can function as scaffold RNA molecules, holding together groups of proteins to coordinate their functions (Figure 7–79A). We have already seen an example in telomerase, where the RNA molecule holds together and organizes protein components. These RNA-based scaffolds are analogous to protein scaffolds we discussed in Chapter 3 (see Figure 3–78) and Chapter 6 (see Figure 6–47). RNA molecules are well suited to act as scaffolds: small bits of RNA sequence, often those portions that form stem-loop structures, can serve as binding sites for proteins, and these can be strung together with random sequences of RNA in between. This property may be one reason that lncRNAs show relatively little primary-sequence conservation across species.

1	The second key feature of lncRNAs is their ability to serve as guide sequences, binding to specific RNA or DNA target molecules through base-pairing. By doing so, they bring proteins that are bound to them into close proximity with the DNA Figure 7–79 Roles of long noncoding RNA (lncRNA). (A) lncrnAs can serve as scaffolds, bringing together proteins that function in the same process. As described in Chapter 6, rnAs can fold into specific three-dimensional structures that are often recognized by proteins. (B) in addition to serving as scaffolds, lncrnAs can, through formation of complementary base pairs, localize proteins to specific sequences on rnA or DnA molecules. (C) in some cases, lncrnAs act only in cis, for example, when the rnA is held in place by rnA polymerase (top). Other lncrnAs, however, diffuse from their sites of synthesis and therefore act in trans.

1	(A) IncRNA IncRNA IncRNA RNA DNA RNA polymerase controls transcription of genes on same chromosome controls transcription of genes on other chromosomes, chromosome A chromosome A chromosome B ACTS IN CISACTS IN TRANS and RNA sequences (Figure 7–79B). This behavior is similar to that of snoRNAs (see Figure 6–41), crRNAs (see Figure 7–78), and miRNAs (see Figure 7–75), all of which act in this way to guide protein enzymes to specific nucleic acid sequences.

1	In some cases, lncRNAs work simply by base-pairing, without bringing in enzymes or other proteins. For example, a number of lncRNA genes are embedded in protein-coding genes, but they are transcribed in the “wrong direction.” These antisense RNAs can form complementary base pairs with the mRNA (transcribed in the “correct” direction) and block its translation into protein (see Figure 7–66D). Other antisense lncRNAs base-pair with pre-mRNAs as they are synthesized and change the pattern of RNA splicing by masking splice-site sequences. Still others act as “sponges,” base-pairing with miRNAs and thereby reducing their effects.

1	Finally, we note that some lncRNAs can act only in cis; that is, they affect only the chromosome from which they are transcribed. This readily occurs when the transcribed RNA has not yet been released from RNA polymerases (Figure 7–79C). Many lncRNAs, however, diffuse from their site of synthesis and act in trans. Although the best understood lncRNAs work in the nucleus, many are found in the cytosol. The functions—if any—of the great majority of these cytosolic lncRNAs remain undiscovered.

1	RNA molecules have many uses in the cell besides carrying the information needed to specify the order of amino acids during protein synthesis. Although we have encountered noncoding RNAs in other chapters (tRNAs, rRNAs, snoRNAs, for example), the sheer number of noncoding RNAs produced by cells has surprised scientists. One well understood use of noncoding RNAs occurs in RNA interference, where guide RNAs (miRNAs, siRNAs, piRNAs) base-pair with mRNAs. RNA interference can cause mRNAs to be either destroyed or translationally repressed. It can also cause specific genes to be packaged into heterochromatin suppressing their transcription. In bacteria and archaebacteria, RNA interference is used as an adaptive immune response to destroy viruses that infect them. A large family of large noncoding RNAs (lncRNAs) has recently been discovered. Although the function of most of these RNAs is unknown, some serve as RNA scaffolds to bring specific proteins and how is the final rate of

1	noncoding RNAs (lncRNAs) has recently been discovered. Although the function of most of these RNAs is unknown, some serve as RNA scaffolds to bring specific proteins and how is the final rate of transcription of a gene specified by the hundreds of proteins that assemble on its control regions? will we ever be able to predict this rate from inspection of the DnA sequences of control regions?

1	how does the collection of cisregulatory sequences embedded in a genome orchestrate the developmental program of a multicellular organism? how much of the human genome sequence is functional, and why is the remainder retained? which of the thousands of unstudied noncoding rnAs have functions in the cell, and what are these functions? were introns present in early cells (and subsequently lost in some organisms), or did they arise at later times? RNA molecules together to speed up needed reactions. Which statements are true? Explain why or why not. 7–1 In terms of the way it interacts with DNA, the helix–loop–helix motif is more closely related to the leucine zipper motif than it is to the helix–turn–helix motif. 7–2 Once cells have differentiated to their final specialized forms, they never again alter expression of their genes.

1	7–2 Once cells have differentiated to their final specialized forms, they never again alter expression of their genes. 7–3 CG islands are thought to have arisen during evolution because they were associated with portions of the genome that remained unmethylated in the germ line. 7–4 In most differentiated tissues, daughter cells retain a memory of gene expression patterns that were present in the parent cell through mechanisms that do not involve changes in the sequence of their genomic DNA. Discuss the following problems. 7–5 A small portion of a two-dimensional display of proteins from human brain is shown in Figure Q7–1. These proteins were separated on the basis of size in one dimension and electrical charge (isoelectric point) in the other. Not all protein spots on such displays are products

1	Figure Q7–1 two-dimensional separation of proteins from the human brain (problem 7–5). the proteins were displayed using two-dimensional gel electrophoresis. Only a small portion of the protein spectrum is shown. (Courtesy of tim Myers and leigh Anderson, large Scale Biology Corporation.) of different genes; some represent modified forms of a protein that migrate to different positions. Pick out a couple of sets of spots that could represent proteins that differ by the number of phosphates they carry. Explain the basis for your selection.

1	7–6 Comparisons of the patterns of mRNA levels across different human cell types show that the level of expression of almost every active gene is different. The patterns of mRNA abundance are so characteristic of cell type that they can be used to determine the tissue of origin of cancer cells, even though the cells may have metastasized to different parts of the body. By definition, however, cancer cells are different from their noncancerous precursor cells. How do you suppose then that patterns of mRNA expression might be used to determine the tissue source of a human cancer? 7–7 What are the two fundamental components of a genetic switch?

1	7–7 What are the two fundamental components of a genetic switch? 7–8 The nucleus of a eukaryotic cell is much larger than a bacterium, and it contains much more DNA. As a consequence, a transcription regulator in a eukaryotic cell must be able to select its specific binding site from among many more unrelated sequences than does a transcription regulator in a bacterium. Does this present any special problems for eukaryotic gene regulation? Consider the following situation. Assume that the eukaryotic nucleus and the bacterial cell each have a single copy of the same DNA binding site. In addition, assume that the nucleus is 500 times the volume of the bacterium, and has 500 times as much DNA. If the concentration of the transcription regulator that binds the site were the same in the nucleus and in the bacterium, would the regulator occupy its binding site equally as well in the eukaryotic nucleus as it does in the bacterium? Explain your answer.

1	7–9 Some transcription regulators bind to DNA and cause the double helix to bend at a sharp angle. Such “bending proteins” can affect the initiation of transcription without directly contacting any other protein. Can you devise a plausible explanation for how such proteins might work to modulate transcription? Draw a diagram that illustrates your explanation. 7–10 How is it that protein–protein interactions that are too weak to cause proteins to assemble in solution can nevertheless allow the same proteins to assemble into complexes on DNA?

1	7–10 How is it that protein–protein interactions that are too weak to cause proteins to assemble in solution can nevertheless allow the same proteins to assemble into complexes on DNA? 7–11 Imagine the two situations shown in Figure Q7–2. In cell 1, a transient signal induces the synthesis of protein A, which is a transcription activator that turns on many genes including its own. In cell 2, a transient signal induces the synthesis of protein R, which is a transcription repressor that turns off many genes including its own. In which, if either, of these situations will the descendants of the original cell “remember” that the progenitor cell had experienced the transient signal? Explain your reasoning. transcription turns on transcription activator protein activator of activator mRNA turns on its own transcription Figure Q7–2 Gene regulatory circuits and cell memory (problem 7–11).

1	transcription turns on transcription activator protein activator of activator mRNA turns on its own transcription Figure Q7–2 Gene regulatory circuits and cell memory (problem 7–11). (A) induction of synthesis of transcription activator A by a transient signal. (B) induction of synthesis of transcription repressor r by a transient signal.

1	(A) induction of synthesis of transcription activator A by a transient signal. (B) induction of synthesis of transcription repressor r by a transient signal. 7–12 Examine the two pedigrees shown in Figure Q7–3. One results from deletion of a maternally imprinted autosomal gene. The other pedigree results from deletion of a paternally imprinted autosomal gene. In both pedigrees, affected individuals (red symbols) are heterozygous for the deletion. These individuals are affected because one copy of the chromosome carries an imprinted, inactive gene, while the other carries a deletion of the gene. Dotted yellow symbols indicate individuals that carry the deleted locus, but do not display the mutant phenotype. Which pedigree is based on paternal imprinting and which on maternal imprinting? Explain your answer.

1	Figure Q7–3 pedigrees reflecting maternal and paternal imprinting (problem 7–12). in one pedigree, the gene is paternally imprinted; in the other, it is maternally imprinted. in generations 3 and 4, only one of the two parents in the indicated matings is shown; the other parent is a normal individual from outside this pedigree. Affected individuals are represented by red circles for females and red squares for males. Dotted yellow symbols indicate individuals that carry the deletion but do not display the phenotype. 7–13 If you insert a β-galactosidase gene lacking its own transcription control region into a cluster of piRNA genes in Drosophila, you find that β-galactosidase expression from a normal copy elsewhere in the genome is strongly inhibited in the fly’s germ cells. If the inactive β-galactosidase gene is inserted outside the piRNA gene cluster, the normal gene is properly expressed. What do you suppose is the basis for this observation? How would you test your hypothesis?

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1	wiedenheft B, Sternberg Sh & Doudna JA (2012) rnA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338. Analyzing Cells, Molecules, and Systems Progress in science is often driven by advances in technology. The entire field of cell biology, for example, came into being when optical craftsmen learned to grind small lenses of sufficiently high quality to observe cells and their substructures. Innovations in lens grinding, rather than any conceptual or philosophical advance, allowed Hooke and van Leeuwenhoek to discover a previously unseen cellular world, where tiny creatures tumble and twirl in a small droplet of water (Figure 8–1).

1	The twenty-first century is a particularly exciting time for biology. New methods for analyzing cells, proteins, DNA, and RNA are fueling an information explosion and allowing scientists to study cells and their macromolecules in previously unimagined ways. We now have access to the sequences of many billions of nucleotides, providing the complete molecular blueprints for hundreds of organ-isms—from microbes and mustard weeds to worms, flies, mice, dogs, chimpanzees, and humans. And powerful new techniques are helping us to decipher that information, allowing us not only to compile huge, detailed catalogs of genes and proteins but also to begin to unravel how these components work together to form functional cells and organisms. The long-range goal is nothing short of obtaining a complete understanding of what takes place inside a cell as it responds to its environment and interacts with its neighbors.

1	In this chapter, we present some of the principal methods used to study cells and their molecular components. We consider how to separate cells of different types from tissues, how to grow cells outside the body, and how to disrupt cells and isolate their organelles and constituent macromolecules in pure form. We also present the techniques used to determine protein structure, function, and interactions, and we discuss the breakthroughs in DNA technology that continue to revolutionize our understanding of cell function. We end the chapter with an overview of some of the mathematical approaches that are helping us deal with the enormous complexity of cells. By considering cells as dynamic systems with many moving parts, mathematical approaches can reveal hidden insights into how the many components of cells work together to produce the special qualities of life.

1	Although the organelles and large molecules in a cell can be visualized with microscopes, understanding how these components function requires a detailed biochemical analysis. Most biochemical procedures require that large numbers of cells be physically disrupted to gain access to their components. If the sample is a piece of tissue, composed of different types of cells, heterogeneous cell populations will be mixed together. To obtain as much information as possible about the cells in a tissue, biologists have developed ways of dissociating cells from tissues and separating them according to type. These manipulations result in a relatively homogeneous population of cells that can then be analyzed—either directly or after their number has been greatly increased by allowing the cells to proliferate in culture.

1	Intact tissues provide the most realistic source of material, as they represent the actual cells found within the body. The first step in isolating individual cells is to disrupt the extracellular matrix and cell–cell junctions that hold the cells together. For this purpose, a tissue sample is typically treated with proteolytic enzymes (such as trypsin and collagenase) to digest proteins in the extracellular matrix and with agents (such as ethylenediaminetetraacetic acid, or EDTA) that bind, or chelate, the Ca2+ on which cell–cell adhesion depends. The tissue can then be teased apart into single cells by gentle agitation.

1	For some biochemical preparations, the protein of interest can be obtained in sufficient quantity without having to separate the tissue or organ into cell types. Examples include the preparation of histones from calf thymus, actin from rabbit muscle, or tubulin from cow brain. In other cases, obtaining the desired protein requires enrichment for a specific cell type of interest. Several approaches are used to separate the different cell types from a mixed cell suspension. One of the most sophisticated cell-separation techniques uses an antibody coupled to a fluorescent dye to label specific cells. An antibody is chosen that specifically binds to the surface of only one cell type in the tissue. The labeled cells can then be separated from the unlabeled ones in a fluorescence-activated cell sorter. In this remarkable machine, individual cells traveling single file in a fine stream pass through a laser beam, and the fluorescence of each cell is rapidly measured. A vibrating nozzle

1	cell sorter. In this remarkable machine, individual cells traveling single file in a fine stream pass through a laser beam, and the fluorescence of each cell is rapidly measured. A vibrating nozzle generates tiny droplets, most containing either one cell or no cells. The droplets containing a single cell are automatically given a positive or a negative charge at the moment of formation, depending on whether the cell they contain is fluorescent; they are then deflected by a strong electric field into an appropriate container. Occasional clumps of cells, detected by their increased light scattering, are left uncharged and are discarded into a waste container. Such machines can accurately select 1 fluorescent cell from a pool of 1000 unlabeled cells and sort several thousand cells each second (Figure 8–2).

1	Although molecules can be extracted from whole tissues, this is often not the most convenient or useful source of material. The complexity of intact tissues and organs is an inherent disadvantage when trying to purify particular molecules. Cells grown in culture provide a more homogeneous population of cells from which to extract material, and they are also much more convenient to work with in the laboratory. Given appropriate surroundings, most plant and animal cells can live, multiply, and even express differentiated properties in a culture dish. The cells can be watched continuously under the microscope or analyzed biochemically, and the effects of adding or removing specific molecules, such as hormones or growth factors, can be systematically explored.

1	Experiments performed on cultured cells are sometimes said to be carried out in vitro (literally, “in glass”) to contrast them with experiments using intact organisms, which are said to be carried out in vivo (literally, “in the living organism”). Figure 8–1 Microscopic life. A sample of “diverse animalcules” seen by van Leeuwenhoek using his simple microscope. (A) Bacteria seen in material he excavated from between his teeth. Those in fig. B he described as “swimming first forward and then backwards” (1692). (B) The eukaryotic green alga Volvox (1700). (Courtesy of the John Innes Foundation.) These terms can be confusing, however, because they are often used in a very different sense by biochemists. In the biochemistry lab, in vitro refers to reactions carried out in a test tube in the absence of living cells, whereas in vivo refers to any reaction taking place inside a living cell, even if that cell is growing in culture.

1	Tissue culture began in 1907 with an experiment designed to settle a controversy in neurobiology. The hypothesis under examination was known as the neuronal doctrine, which states that each nerve fiber is the outgrowth of a single nerve cell and not the product of the fusion of many cells. To test this contention, small pieces of spinal cord were placed on clotted tissue fluid in a warm, moist chamber and observed at regular intervals under the microscope. After a day or so, individual nerve cells could be seen extending long, thin filaments (axons) into the clot. Thus, the neuronal doctrine received strong support, and the foundation was laid for the cell-culture revolution.

1	These original experiments on nerve fibers used cultures of small tissue fragments called explants. Today, cultures are more commonly made from suspensions of cells dissociated from tissues. Unlike bacteria, most tissue cells are not adapted to living suspended in fluid and require a solid surface on which to grow and divide. For cell cultures, this support is usually provided by the surface of a plastic culture dish. Cells vary in their requirements, however, and many do not proliferate or differentiate unless the culture dish is coated with materials that cells like to adhere to, such as polylysine or extracellular matrix components.

1	Cultures prepared directly from the tissues of an organism are called primary cultures. These can be made with or without an initial fractionation step to separate different cell types. In most cases, cells in primary cultures can be removed from the culture dish and recultured repeatedly in so-called secondary cultures; in this way, they can be repeatedly subcultured (passaged) for weeks or months. Such cells often display many of the differentiated properties appropriate to their Figure 8–2 A fluorescence-activated cell sorter. A cell passing through the laser beam is monitored for fluorescence. Droplets containing single cells are given a negative or positive charge, depending on whether the cell is fluorescent or not. The droplets are then deflected by an electric field into collection tubes according to their charge. Note that the cell concentration must be adjusted so that most droplets contain no cells and flow to a waste container together with any cell clumps.

1	Figure 8–3 Light micrographs of cells in culture. (A) Mouse fibroblasts. (B) Chick myoblasts fusing to form multinucleate muscle cells. (C) Purified rat retinal ganglion nerve cells. (D) Tobacco cells in liquid culture. (A, courtesy of Daniel Zicha; B, courtesy of Rosalind Zalin; C, from A. Meyer-Franke et al., Neuron 15:805–819, 1995. With permission from Elsevier; D, courtesy of Gethin Roberts.) origin (Figure 8–3): fibroblasts continue to secrete collagen; cells derived from embryonic skeletal muscle fuse to form muscle fibers that contract spontaneously in the culture dish; nerve cells extend axons that are electrically excitable and make synapses with other nerve cells; and epithelial cells form extensive sheets with many of the properties of an intact epithelium. Because these properties are maintained in culture, they are accessible to study in ways that are often not possible in intact tissues.

1	Cell culture is not limited to animal cells. When a piece of plant tissue is cul-50 µm tured in a sterile medium containing nutrients and appropriate growth regulators, many of the cells are stimulated to proliferate indefinitely in a disorganized manner, producing a mass of relatively undifferentiated cells called a callus. If the nutrients and growth regulators are carefully manipulated, one can induce the formation of a shoot and then root apical meristems within the callus, and, in many species, regenerate a whole new plant. Similar to animal cells, callus cultures can be mechanically dissociated into single cells, which will grow and divide as a suspension culture (see Figure 8–3D). Eukaryotic Cell Lines Are a Widely Used Source of Homogeneous Cells

1	The cell cultures obtained by disrupting tissues tend to suffer from a problem— eventually the cells die. Most vertebrate cells stop dividing after a finite number of cell divisions in culture, a process called replicative cell senescence (discussed in Chapter 17). Normal human fibroblasts, for example, typically divide only 25–40 times in culture before they stop. In these cells, the limited proliferation capacity reflects a progressive shortening and uncapping of the cell’s telomeres, the repetitive DNA sequences and associated proteins that cap the ends of each chromosome (discussed in Chapter 5). Human somatic cells in the body have turned off production of the enzyme, called telomerase, that normally maintains the telomeres, which is why their telomeres shorten with each cell division. Human fibroblasts can often be coaxed to proliferate indefinitely by providing them with the gene that encodes the catalytic subunit of telomerase; in this case, they can be propagated as an

1	Human fibroblasts can often be coaxed to proliferate indefinitely by providing them with the gene that encodes the catalytic subunit of telomerase; in this case, they can be propagated as an “immortalized” cell line.

1	Some human cells, however, cannot be immortalized by this trick. Although their telomeres remain long, they still stop dividing after a limited number of divisions because culture conditions cause excessive mitogenic stimulation, which activates a poorly understood protective mechanism (discussed in Chapter 17) that stops cell division—a process sometimes called “culture shock.” To immortalize these cells, one has to do more than introduce telomerase. One must also inactivate the protective mechanisms, which can be done by introducing certain cancer-promoting oncogenes (discussed in Chapter 20). Unlike human cells, most rodent cells do not turn off production of telomerase and therefore their telomeres do not shorten with each cell division. Therefore, if culture shock can be avoided, some rodent cell types will divide indefinitely in culture. In addition, rodent cells often undergo spontaneous genetic changes in culture that inactivate their protective mechanisms, thereby producing

1	rodent cell types will divide indefinitely in culture. In addition, rodent cells often undergo spontaneous genetic changes in culture that inactivate their protective mechanisms, thereby producing immortalized cell lines.

1	Cell lines can often be most easily generated from cancer cells, but these cultures—referred to as transformed cell lines—differ from those prepared from normal cells in several ways. Transformed cell lines often grow without attaching to a surface, for example, and they can proliferate to a much higher density in a culture dish. Similar properties can be induced experimentally in normal cells by transforming them with a tumor-inducing virus or chemical. The resulting transformed cell lines can usually cause tumors if injected into a susceptible animal.

1	Transformed and nontransformed cell lines are extremely useful in cell research as sources of very large numbers of cells of a uniform type, especially since they can be stored in liquid nitrogen at –196°C for an indefinite period and retain their viability when thawed. It is important to keep in mind, however, that cell lines nearly always differ in important ways from their normal progenitors in the tissues from which they were derived. Some widely used cell lines are listed in Table 8–1. Different lines have different advantages; for example, the PtK epithelial cell lines derived from the rat 444 Chapter 8: Analyzing Cells, Molecules, and Systems kangaroo, unlike many other cell types, remain flat during mitosis, allowing the mitotic apparatus to be readily observed in action.

1	As we see throughout this book, antibodies are particularly useful tools for cell biology. Their great specificity allows precise visualization of selected proteins among the many thousands that each cell typically produces. Antibodies are often produced by inoculating animals with the protein of interest and subsequently isolating the antibodies specific to that protein from the serum of the animal. However, only limited quantities of antibodies can be obtained from a single inoculated animal, and the antibodies produced will be a heterogeneous mixture of antibodies that recognize a variety of different antigenic sites on a macromolecule that differs from animal to animal. Moreover, antibodies specific for the antigen will constitute only a fraction of the antibodies found in the serum. An alternative technology, which allows the production of an unlimited quantity of identical antibodies and greatly increases the specificity and convenience of antibody-based methods, is the

1	serum. An alternative technology, which allows the production of an unlimited quantity of identical antibodies and greatly increases the specificity and convenience of antibody-based methods, is the production of monoclonal antibodies by hybridoma cell lines.

1	This technology, developed in 1975, revolutionized the production of antibodies for use as tools in cell biology, as well as for the diagnosis and treatment of certain diseases, including rheumatoid arthritis and cancer. The procedure requires hybrid cell technology (Figure 8–4), and it involves propagating a clone of cells from a single antibody-secreting B lymphocyte to obtain a homogeneous preparation of antibodies in large quantities. B lymphocytes normally have a limited life-span in culture, but individual antibody-producing B lymphocytes from an immunized mouse, when fused with cells derived from a transformed B lymphocyte cell line, can give rise to hybrids that have both the ability to make a particular antibody and the ability to multiply indefinitely in culture. These hybridomas are propagated as individual clones, each of which provides a permanent and stable source of a single type of monoclonal antibody. Each type of monoclonal antibody recognizes a single type of

1	are propagated as individual clones, each of which provides a permanent and stable source of a single type of monoclonal antibody. Each type of monoclonal antibody recognizes a single type of antigenic site—for example, a particular cluster of five or six amino acid side chains on the surface of a protein. Their uniform specificity makes monoclonal antibodies much more useful than conventional antisera for many purposes.

1	An important advantage of the hybridoma technique is that monoclonal antibodies can be made against molecules that constitute only a minor component of a complex mixture. In an ordinary antiserum made against such a mixture, the proportion of antibody molecules that recognize the minor component would be too small to be useful. But if the B lymphocytes that produce the various components of this antiserum are made into hybridomas, it becomes possible to screen individual hybridoma clones from the large mixture to select one that produces the desired type of monoclonal antibody and to propagate the selected hybridoma SUSPENSION OF TWO CELL three clones of hybrid cellsTYPES CENTRIFUGED WITH A FUSING AGENT ADDED SELECTIVE MEDIUM FORMATION OF PROLIFERATE. THESE HETEROKARYONS, BECOME HYBRID WHICH ARE CELLS, WHICH ARE THEN CULTURED THEN CLONED

1	FORMATION OF PROLIFERATE. THESE HETEROKARYONS, BECOME HYBRID WHICH ARE CELLS, WHICH ARE THEN CULTURED THEN CLONED Figure 8–4 The production of hybrid cells. It is possible to fuse one cell with another to form a heterokaryon, a combined cell with two separate nuclei. Typically, a suspension of cells is treated with certain inactivated viruses or with polyethylene glycol, each of which alters the plasma membranes of cells in a way that induces them to fuse. Eventually, a heterokaryon proceeds to mitosis and produces a hybrid cell in which the two separate nuclear envelopes have been disassembled, allowing all the chromosomes to be brought together in a single large nucleus. Such hybrid cells can give rise to immortal hybrid cell lines. If one of the parent cells was from a tumor cell line, the hybrid cell is called a hybridoma.

1	indefinitely so as to produce that antibody in unlimited quantities. In principle, therefore, a monoclonal antibody can be made against any protein in a biological sample. Once an antibody has been made, it can be used to localize the protein in cells and tissues, to follow its movement, and to purify the protein to study its structure and function.

1	Tissues can be dissociated into their component cells, from which individual cell types can be purified and used for biochemical analysis or for the establishment of cell cultures. Many animal and plant cells survive and proliferate in a culture dish if they are provided with a suitable culture medium containing nutrients and appropriate signal molecules. Although many animal cells stop dividing after a finite number of cell divisions, cells that have been immortalized through spontaneous mutations or genetic manipulation can be maintained indefinitely as cell lines. Hybridoma cells are widely employed to produce unlimited quantities of uniform monoclonal antibodies, which are used to detect and purify cell proteins, as well as to diagnose and treat diseases.

1	The challenge of isolating a single type of protein from the thousands of other proteins present in a cell is a formidable one, but must be overcome in order to study protein function in vitro. As we shall see later in this chapter, recombinant DNA technology can enormously simplify this task by “tricking” cells into producing large quantities of a given protein, thereby making its purification much easier. Whether the source of the protein is an engineered cell or a natural tissue, a purification procedure usually starts with subcellular fractionation to reduce the complexity of the material, and is then followed by purification steps of increasing specificity.

1	To purify a protein, it must first be extracted from inside the cell. Cells can be broken up in various ways: they can be subjected to osmotic shock or ultrasonic vibration, forced through a small orifice, or ground up in a blender. These procedures break many of the membranes of the cell (including the plasma membrane and endoplasmic reticulum) into fragments that immediately reseal to form small closed vesicles. If carefully carried out, however, the disruption procedures leave organelles such as nuclei, mitochondria, the Golgi apparatus, lysosomes, and peroxisomes largely intact. The suspension of cells is thereby reduced to a thick slurry (called a homogenate or extract) that contains a variety of membrane-enclosed organelles, each with a distinctive size, charge, and density. Provided that the homogenization medium has been carefully chosen (by trial and error for each organelle), the various components—including the vesicles derived from the endoplasmic reticulum, called

1	Provided that the homogenization medium has been carefully chosen (by trial and error for each organelle), the various components—including the vesicles derived from the endoplasmic reticulum, called microsomes—retain most of their original biochemical properties.

1	The different components of the homogenate must then be separated. Such cell fractionations became possible only after the commercial development in the early 1940s of an instrument known as the preparative ultracentrifuge, which rotates extracts of broken cells at high speeds (Figure 8–5). This treatment separates cell components by size and density: in general, the largest objects experience the largest centrifugal force and move the most rapidly. At relatively low speed, large components such as nuclei sediment to form a pellet at the bottom of the centrifuge tube; at slightly higher speed, a pellet of mitochondria is deposited; and at even higher speeds and with longer periods of centrifugation, first the small closed vesicles and then the ribosomes can be collected (Figure 8–6). All of these fractions are impure, but many of the contaminants can be removed by resuspending the pellet and repeating the centrifugation procedure several times.

1	Figure 8–5 The preparative ultracentrifuge. (A) The sample is contained in tubes that are inserted into a ring of angled cylindrical holes in a metal rotor. Rapid rotation of the rotor generates enormous centrifugal forces, which cause particles in the sample to sediment against the bottom sides of the sample tubes, as shown here. The vacuum reduces friction, preventing heating of the rotor and allowing the refrigeration system to maintain the sample at 4°C. (B) Some fractionation methods require a different type of rotor called a swinging-bucket rotor. In this case, the sample tubes are placed in metal tubes on hinges that allow the tubes to swing outward when the rotor spins. Sample tubes are therefore horizontal during spinning, and samples are sedimented toward the bottom, not the sides, of the tube, providing better separation of differently sized components (see Figures 8–6 and 8–7).

1	Centrifugation is the first step in most fractionations, but it separates only components that differ greatly in size. A finer degree of separation can be achieved by layering the homogenate in a thin band on top of a salt solution that fills a centrifuge tube. When centrifuged, the various components in the mixture move as a series of distinct bands through the solution, each at a different rate, in a process called velocity sedimentation (Figure 8–7A). For the procedure to work effectively, the bands must be protected from convective mixing, which would normally occur whenever a denser solution (for example, one containing organelles) finds itself on top of a lighter one (the salt solution). This is achieved by augmenting the solution in the tube with a shallow gradient of sucrose prepared by a special mixing device. The resulting density gradient—with the dense end at the bottom of the tube—keeps each region of the solution denser than any solution above it, and it thereby prevents

1	by a special mixing device. The resulting density gradient—with the dense end at the bottom of the tube—keeps each region of the solution denser than any solution above it, and it thereby prevents convective mixing from distorting the separation.

1	When sedimented through sucrose gradients, different cell components separate into distinct bands that can be collected individually. The relative rate at which each component sediments depends primarily on its size and shape—normally being described in terms of its sedimentation coefficient, or S value. Present-day ultracentrifuges rotate at speeds of up to 80,000 rpm and produce forces as high as 500,000 times gravity. These enormous forces drive even small macromolecules, such as tRNA molecules and simple enzymes, to sediment at an appreciable rate and allow them to be separated from one another by size. The ultracentrifuge is also used to separate cell components on the basis of their buoyant density, independently of their size and shape. In this case, the

1	Figure 8–6 Cell fractionation by centrifugation. Repeated centrifugation at progressively higher speeds will fractionate homogenates of cells into their components. In general, the smaller the subcellular component, the greater the centrifugal force required to sediment it. Typical values for the various centrifugation steps referred to in the figure are: low speed: 1000 times gravity for 10 minutes medium speed: 20,000 times gravity for 20 minutes high speed: 80,000 times gravity for 1 hour very high speed: 150,000 times gravity for 3 hours steep(e.g., 5–20%) sucrose gradient (e.g., 20–70%) sample is sedimented through a steep density gradient that contains a very high concentration of sucrose or cesium chloride. Each cell component begins to move down the gradient as in Figure 8–7A, but it eventually reaches a position where the density of the solution is equal to its own density. At this point, the component floats and can move no farther. A series of distinct bands is thereby

1	but it eventually reaches a position where the density of the solution is equal to its own density. At this point, the component floats and can move no farther. A series of distinct bands is thereby produced in the centrifuge tube, with the bands closest to the bottom of the tube containing the components of highest buoyant density (Figure 8–7B). This method, called equilibrium sedimentation, is so sensitive that it can separate macromolecules that have incorporated heavy isotopes, such as 13C or 15N, from the same macromolecules that contain the lighter, common isotopes (12C or 14N). In fact, the cesium-chloride method was developed in 1957 to separate the labeled from the unlabeled DNA produced after exposure of a growing population of bacteria to nucleotide precursors containing 15N; this classic experiment provided direct evidence for the semiconservative replication of DNA (see Figure 5–5).

1	Cell Extracts Provide Accessible Systems to Study Cell Functions Studies of organelles and other large subcellular components isolated in the ultracentrifuge have contributed enormously to our understanding of the functions of different cell components. Experiments on mitochondria and chloroplasts purified by centrifugation, for example, demonstrated the central function of these organelles in converting energy into forms that the cell can use. Similarly, resealed vesicles formed from fragments of rough and smooth endoplasmic reticulum

1	Figure 8–7 Comparison of velocity sedimentation and equilibrium sedimentation. (A) In velocity sedimentation, subcellular components sediment at different speeds according to their size and shape when layered over a solution containing sucrose. To stabilize the sedimenting bands against convective mixing caused by small differences in temperature or solute concentration, the tube contains a continuous shallow gradient of sucrose, which increases in concentration toward the bottom of the tube (typically from 5% to 20% sucrose). After centrifugation, the different components can be collected individually, most simply by puncturing the plastic centrifuge tube with a needle and collecting drops from the bottom, as illustrated here.

1	(B) In equilibrium sedimentation, subcellular components move up or down when centrifuged in a gradient until they reach a position where their density matches their surroundings. Although a sucrose gradient is shown here, denser gradients, which are especially useful for protein and nucleic acid separation, can be formed from cesium chloride. The final bands, at equilibrium, can be collected as in (A). (microsomes) have been separated from each other and analyzed as functional models of these compartments of the intact cell. Similarly, highly concentrated cell extracts, especially extracts of Xenopus laevis (African clawed frog) oocytes, have played a critical role in the study of such complex and highly organized processes as the cell-division cycle, the separation of chromosomes on the mitotic spindle, and the vesicular-transport steps involved in the movement of proteins from the endoplasmic reticulum through the Golgi apparatus to the plasma membrane.

1	Cell extracts also provide, in principle, the starting material for the complete separation of all of the individual macromolecular components of the cell. We now consider how this separation is achieved, focusing on proteins. Proteins are most often fractionated by column chromatography, in which a mixture of proteins in solution is passed through a column containing a porous solid matrix. Different proteins are retarded to different extents by their interaction with the matrix, and they can be collected separately as they flow out of the bottom of the column (Figure 8–8). Depending on the choice of matrix, proteins can be separated according to their charge (ion-exchange chromatography), their hydrophobicity (hydrophobic chromatography), their size (gel-filtration chromatography), or their ability to bind to particular small molecules or to other macromolecules (affinity chromatography).

1	Many types of matrices are available. Ion-exchange columns (Figure 8–9A) are packed with small beads that carry either a positive or a negative charge, so that proteins are fractionated according to the arrangement of charges on their surface. Hydrophobic columns are packed with beads from which hydrophobic side chains protrude, selectively retarding proteins with exposed hydrophobic regions. Gel-filtration columns (Figure 8–9B), which separate proteins according to their size, are packed with tiny porous beads: molecules that are small enough to enter the pores linger inside successive beads as they pass down the column, while larger molecules remain in the solution flowing between the beads and therefore move more rapidly, emerging from the column first. Besides providing applied to the top of applied reservoir of solvent

1	Figure 8–8 The separation of molecules by column chromatography. The sample, a solution containing a mixture of different molecules, is applied to the top of a cylindrical glass or plastic column filled with a permeable solid matrix, such as cellulose. A large amount of solvent is then passed slowly through the column and collected in separate tubes as it emerges from the bottom. Because various components of the sample travel at different rates through the column, they are fractionated into different tubes. a means of separating molecules, gel-filtration chromatography is a convenient way to estimate their size.

1	a means of separating molecules, gel-filtration chromatography is a convenient way to estimate their size. Affinity chromatography (Figure 8–9C) takes advantage of the biologically important binding interactions that occur on protein surfaces. If a substrate molecule is covalently coupled to an inert matrix such as a polysaccharide bead, the enzyme that operates on that substrate will often be specifically retained by the matrix and can then be eluted (washed out) in nearly pure form. Likewise, short DNA oligonucleotides of a specifically designed sequence can be immobilized in this way and used to purify DNA-binding proteins that normally recognize this sequence of nucleotides in chromosomes. Alternatively, specific antibodies can be coupled to a matrix to purify protein molecules recognized by the antibodies. Because of the great specificity of all such affinity columns, 1000to 10,000-fold purifications can sometimes be achieved in a single pass.

1	If one starts with a complex mixture of proteins, a single passage through an ion-exchange or a gel-filtration column does not produce very highly purified fractions, since these methods individually increase the proportion of a given protein in the mixture no more than twentyfold. Because most individual proteins represent less than 1/1000 of the total cell protein, it is usually necessary to use several different types of columns in succession to attain sufficient purity, with affinity chromatography being the most efficient (Figure 8–10).

1	Inhomogeneities in the matrices (such as cellulose), which cause an uneven flow of solvent through the column, limit the resolution of conventional column chromatography. Special chromatography resins (usually silica-based) composed of tiny spheres (3–10 μm in diameter) can be packed with a special apparatus to form a uniform column bed. Such high-performance liquid chromatography (HPLC) columns attain a high degree of resolution. In HPLC, the solutes equilibrate very rapidly with the interior of the tiny spheres, and so solutes with different affinities for the matrix are efficiently separated from one another even at very fast flow rates. HPLC is therefore the method of choice for separating many proteins and small molecules.

1	Immunoprecipitation is a useful variation on the theme of affinity chromatography. Specific antibodies that recognize the protein to be purified are attached to small agarose beads. Rather than being packed into a column, as in affinity chromatography, a small quantity of the antibody-coated beads is simply added to a protein extract in a test tube and mixed in suspension for a short period of time— thereby allowing the antibodies to bind the desired protein. The beads are then collected by low-speed centrifugation, and the unbound proteins in the supernatant are discarded. This method is commonly used to purify small amounts of enzymes from cell extracts for analysis of enzymatic activity or for studies of associated proteins. bead with covalently attached substrate

1	Figure 8–9 Three types of matrices used for chromatography. (A) In ion-exchange chromatography, the insoluble matrix carries ionic charges that retard the movement of molecules of opposite charge. Matrices used for separating proteins include diethylaminoethylcellulose (DEAE-cellulose), which is positively charged, and carboxymethylcellulose (CM-cellulose) and phosphocellulose, which are negatively charged. Analogous matrices based on agarose or other polymers are also frequently used. The strength of the association between the dissolved molecules and the ion-exchange matrix depends on both the ionic strength and the pH of the solution that is passing down the column, which may therefore be varied systematically (as in Figure 8–10) to achieve an effective separation. (B) In gel-filtration chromatography, the small beads that form the matrix are inert but porous. Molecules that are small enough to penetrate into the matrix beads are thereby delayed and travel more slowly through the

1	chromatography, the small beads that form the matrix are inert but porous. Molecules that are small enough to penetrate into the matrix beads are thereby delayed and travel more slowly through the column than larger molecules that cannot penetrate. Beads of cross-linked polysaccharide (dextran, agarose, or acrylamide) are available commercially in a wide range of pore sizes, making them suitable for the fractionation of molecules of various molecular mass, from less than 500 daltons to more than 5 × 106 daltons.

1	(C) Affinity chromatography uses an insoluble matrix that is covalently linked to a specific ligand, such as an antibody molecule or an enzyme substrate, that will bind a specific protein. Enzyme molecules that bind to immobilized substrates on such columns can be eluted with a concentrated solution of the free form of the substrate molecule, while molecules that bind to immobilized antibodies can be eluted by dissociating the antibody–antigen complex with concentrated salt solutions or solutions of high or low pH. High degrees of purification can be achieved in a single pass through an affinity column. pool these fractions and apply them to the next column below fraction number pool these fractions and apply them to the next column below pool these fractions, which now contain the highly purifed protein Genetically Engineered Tags Provide an Easy Way to Purify Proteins

1	Using the recombinant DNA methods discussed in subsequent sections, any gene can be modified to produce its protein with a special recognition tag attached to it, so as to make subsequent purification of the protein simple and rapid. Often the recognition tag is itself an antigenic determinant, or epitope, which can be recognized by a highly specific antibody. The antibody can then be used to purify the protein by affinity chromatography or immunoprecipitation (Figure 8–11). Other types of tags are specifically designed for protein purification. For example, a repeated sequence of the amino acid histidine binds to certain metal ions, including nickel and copper. If genetic engineering techniques are used to attach a short string of histidines to one end of a protein, the slightly modified protein can be retained selectively on an affinity column containing immobilized nickel ions. Metal affinity chromatography can thereby be used to purify the modified protein from a complex molecular

1	protein can be retained selectively on an affinity column containing immobilized nickel ions. Metal affinity chromatography can thereby be used to purify the modified protein from a complex molecular mixture.

1	Figure 8–10 Protein purification by chromatography. Typical results obtained when three different chromatographic steps are used in succession to purify a protein. In this example, a homogenate of cells was first fractionated by allowing it to percolate through an ion-exchange resin packed into a column (A). The column was washed to remove all unbound contaminants, and the bound proteins were then eluted by pouring a solution containing a gradually increasing concentration of salt onto the top of the column. Proteins with the lowest affinity for the ion-exchange resin passed directly through the column and were collected in the earliest fractions eluted from the bottom of the column. The remaining proteins were eluted in sequence according to their affinity for the resin—those proteins binding most tightly to the resin requiring the highest concentration of salt to remove them. The protein of interest was eluted in several fractions and was detected by its enzymatic activity. The

1	binding most tightly to the resin requiring the highest concentration of salt to remove them. The protein of interest was eluted in several fractions and was detected by its enzymatic activity. The fractions with activity were pooled and then applied to a gel-filtration column (B). The elution position of the still-impure protein was again determined by its enzymatic activity, and the active fractions were pooled and purified to homogeneity on an affinity column (C) that contained an immobilized substrate of the enzyme.

1	gene for protein of interest rapid purifcation of tagged protein and any associated proteins Figure 8–11 epitope tagging for the purification of proteins. Using standard genetic engineering techniques, a short peptide tag can be added to a protein of interest. If the tag is itself an antigenic determinant, or epitope, it can be targeted by an appropriate antibody, which can be used to purify the protein by immunoprecipitation or affinity chromatography. In other cases, an entire protein is used as the recognition tag. When cells are engineered to synthesize the small enzyme glutathione S-transferase (GST) attached to a protein of interest, the resulting fusion protein can be purified from the other contents of the cell with an affinity column containing glutathione, a substrate molecule that binds specifically and tightly to GST.

1	As a further refinement of purification methods using recognition tags, an amino acid sequence that forms a cleavage site for a highly specific proteolytic enzyme can be engineered between the protein of choice and the recognition tag. Because the amino acid sequences at the cleavage site are very rarely found by chance in proteins, the tag can later be cleaved off without destroying the purified protein.

1	This type of specific cleavage is used in an especially powerful purification methodology known as tandem affinity purification tagging (TAP-tagging). Here, one end of a protein is engineered to contain two recognition tags that are separated by a protease cleavage site. The tag on the very end of the construct is chosen to bind irreversibly to an affinity column, allowing the column to be washed extensively to remove all contaminating proteins. Protease cleavage then releases the protein, which is then further purified using the second tag. Because this two-step strategy provides an especially high degree of protein purification with relatively little effort, it is used extensively in cell biology. Thus, for example, a set of approximately 6000 yeast strains, each with a different gene fused to DNA that encodes a TAP-tag, has been constructed to allow any yeast protein to be rapidly purified. Purified Cell-free Systems Are Required for the Precise Dissection of Molecular Functions

1	Purified Cell-free Systems Are Required for the Precise Dissection of Molecular Functions Purified cell-free systems provide a means of studying biological processes free from all of the complex side reactions that occur in a living cell. To make this possible, cell homogenates are fractionated with the aim of purifying each of the individual macromolecules that are needed to catalyze a biological process of interest. For example, the experiments to decipher the mechanisms of protein synthesis began with a cell homogenate that could translate RNA molecules to produce proteins. Fractionation of this homogenate, step by step, produced in turn the ribosomes, tRNAs, and various enzymes that together constitute the protein-synthetic machinery. Once individual pure components were available, each could be added or withheld separately to define its exact role in the overall process.

1	A major goal for cell biologists is the reconstitution of every biological process in a purified cell-free system. Only in this way can we define all of the components needed for the process and control their concentrations, which is required to work out their precise mechanism of action. Although much remains to be done, a great deal of what we know today about the molecular biology of the cell has been discovered by studies in such cell-free systems. They have been used, for example, to decipher the molecular details of DNA replication and DNA transcription, RNA splicing, protein translation, muscle contraction, and particle transport along microtubules, and many other processes that occur in cells.

1	Populations of cells can be analyzed biochemically by disrupting them and fractionating their contents, allowing functional cell-free systems to be developed. Highly purified cell-free systems are needed for determining the molecular details of complex cell processes, and the development of such systems requires extensive purification of all the proteins and other components involved. The proteins in soluble cell extracts can be purified by column chromatography; depending on the type of column matrix, biologically active proteins can be separated on the basis of their molecular weight, hydrophobicity, charge characteristics, or affinity for other molecules. In a typical purification, the sample is passed through several different columns in turn, with the enriched fractions obtained from one column being applied to the next. Recombinant DNA techniques (described later) allow special recognition tags to be attached to proteins, thereby greatly simplifying their purification.

1	Proteins perform most cellular processes: they catalyze metabolic reactions, use nucleotide hydrolysis to do mechanical work, and serve as the major structural elements of the cell. The great variety of protein structures and functions has stimulated the development of a multitude of techniques to study them.

1	Proteins usually possess a net positive or negative charge, depending on the mixture of charged amino acids they contain. An electric field applied to a solution containing a protein molecule causes the protein to migrate at a rate that depends on its net charge and on its size and shape. The most popular application of this property is SDS polyacrylamide-gel electrophoresis (SDS-PAGE). It uses a highly cross-linked gel of polyacrylamide as the inert matrix through which the proteins migrate. The gel is prepared by polymerization of monomers; the pore size of the gel can be adjusted so that it is small enough to retard the migration of the protein molecules of interest. The proteins are dissolved in a solution that includes a powerful negatively charged detergent, sodium dodecyl sulfate, or SDS (Figure 8–12). Because this detergent binds to hydrophobic regions of the protein molecules, causing them to unfold into extended polypeptide chains, the individual protein molecules are

1	or SDS (Figure 8–12). Because this detergent binds to hydrophobic regions of the protein molecules, causing them to unfold into extended polypeptide chains, the individual protein molecules are released from their associations with other proteins or lipid molecules and rendered freely soluble in the detergent solution. In addition, a reducing agent such as β-mercaptoethanol (see Figure 8–12) is usually added to break any S–S linkages in the proteins, so that all of the constituent polypeptides in multisubunit proteins can be analyzed separately.

1	What happens when a mixture of SDS-solubilized proteins is run through a slab of polyacrylamide gel? Each protein molecule binds large numbers of the negatively charged detergent molecules, which mask the protein’s intrinsic charge and cause it to migrate toward the positive electrode when a voltage is applied. Proteins of the same size tend to move through the gel with similar speeds because (1) their native structure is completely unfolded by the SDS, so that their shapes are the same, and (2) they bind the same amount of SDS and therefore have the same amount of negative charge. Larger proteins, with more charge, are subjected to larger electrical forces but also to a larger drag. In free solution, the two effects would cancel out, but, in the mesh of the polyacrylamide gel, which acts as a molecular sieve, large proteins are retarded much more than small ones. As a result, a complex mixture of proteins is fractionated into a series of discrete protein bands arranged in order of

1	as a molecular sieve, large proteins are retarded much more than small ones. As a result, a complex mixture of proteins is fractionated into a series of discrete protein bands arranged in order of molecular weight (Figure 8–13). The major proteins are readily detected by staining the proteins in the gel with a dye such as Coomassie blue. Even minor proteins are seen in gels treated with a silver stain, so that as little as 10 ng of protein can be detected in a band. For some purposes, specific proteins can also be labeled with a radioactive isotope tag; exposure of the gel to film results in an autoradiograph on which the labeled proteins are visible (see Figure 8–16).

1	SDS-PAGE is widely used because it can separate all types of proteins, including those that are normally insoluble in water—such as the many proteins in membranes. And because the method separates polypeptides by size, it provides information about the molecular weight and the subunit composition of proteins. Figure 8–14 presents a photograph of a gel that has been used to analyze each of the successive stages in the purification of a protein. Because different proteins can have similar sizes, shapes, masses, and overall charges, most separation techniques such as SDS polyacrylamide-gel electrophoresis or ion-exchange chromatography cannot typically separate all the proteins Figure 8–12 The detergent sodium dodecyl sulfate (SDS) and the reducing agent β-mercaptoethanol. These two chemicals are used to solubilize proteins for SDS polyacrylamide-gel electrophoresis. The SDS is shown here in its ionized form.

1	sample loaded onto gel protein with two by pipette subunits, A and B, joined by a disulfde single-subunit bridge protein

1	Figure 8–13 SDS polyacrylamide-gel electrophoresis (SDS-PAGe). (A) An electrophoresis apparatus. (B) Individual polypeptide chains form a complex with negatively charged molecules of sodium dodecyl sulfate (SDS) and therefore migrate as a negatively charged SDS–protein complex through a porous gel of polyacrylamide. Because the speed of migration under these conditions is greater the smaller the polypeptide, this technique can be used to determine the approximate molecular weight of a polypeptide chain as A well as the subunit composition of a protein. If the protein contains a large amount of carbohydrate, however, it will move anomalously on the gel and its apparent molecular weight estimated by SDS-PAGE will be misleading. Other modifications, such as phosphorylation, can also cause small changes in a protein’s migration in the gel. slab of polyacrylamide gel in a cell or even in an organelle. In contrast, two-dimensional gel electrophoresis, which combines two different separation

1	changes in a protein’s migration in the gel. slab of polyacrylamide gel in a cell or even in an organelle. In contrast, two-dimensional gel electrophoresis, which combines two different separation procedures, can resolve up to 2000 proteins in the form of a two-dimensional protein map.

1	In the first step, the proteins are separated by their intrinsic charges. The sample is dissolved in a small volume of a solution containing a nonionic (uncharged) detergent, together with β-mercaptoethanol and the denaturing reagent urea. This solution solubilizes, denatures, and dissociates all the polypeptide chains but leaves their intrinsic charge unchanged. The polypeptide chains are then separated in a pH gradient by a procedure called isoelectric focusing, which takes advantage of the variation in the net charge on a protein molecule with the pH of its surrounding solution. Every protein has a characteristic isoelectric point, the pH at which the protein has no net charge and therefore does not migrate in an electric field. In isoelectric focusing, proteins are separated electrophoretically in a narrow tube of polyacrylamide gel in which a gradient of pH is established by a mixture of special buffers. Each protein moves to a position in the gradient that

1	Figure 8–14 Analysis of protein samples by SDS polyacrylamide-gel electrophoresis. The photograph shows a Coomassie-stained gel that has been used to detect the proteins present at successive stages in the purification of an enzyme. The leftmost lane (lane 1) contains the complex mixture of proteins in the starting cell extract, and each succeeding lane analyzes the proteins obtained after a chromatographic fractionation of the protein sample analyzed in the previous lane (see Figure 8–10). The same total amount of protein (10 μg) was loaded onto the gel at the top of each lane. Individual proteins normally appear as sharp, dye-stained bands; a band broadens, however, when it contains a large amount of protein. (From T. Formosa and B.M. Alberts, J. Biol. Chem. 261:6107–6118, 1986.) mass(daltons)100,00040,00015,000 Figure 8–15 Separation of protein molecules by isoelectric focusing. at low pH,

1	T. Formosa and B.M. Alberts, J. Biol. Chem. 261:6107–6118, 1986.) mass(daltons)100,00040,00015,000 Figure 8–15 Separation of protein molecules by isoelectric focusing. at low pH, At low pH (high H+ concentration), the charged the protein carboxylic acid groups of proteins tend to at the isoelectric example, –NH3+), giving most proteins point, the protein a net positive charge. At high pH, the and therefore no carboxylic acid groups are negatively at high pH, charged (–COO–) and the basic groups the electric feld; tend to be uncharged (for example, –NH2), for the protein giving most proteins a net negative charge. shown, thethe protein

1	shown, thethe protein At its isoelectric pH, a protein has no net is 6.5 charge since the positive and negative charges balance. Thus, when a tube containing a fixed pH gradient is subjected to a strong electric field in the appropriate direction, each protein species migrates until it forms a sharp band at its isoelectric corresponds to its isoelectric point and remains there (Figure 8–15). This is the pH, as shown. first dimension of two-dimensional polyacrylamide-gel electrophoresis.

1	In the second step, the narrow tube gel containing the separated proteins is again subjected to electrophoresis but in a direction that is at a right angle to the direction used in the first step. This time SDS is added, and the proteins separate according to their size, as in one-dimensional SDS-PAGE: the original tube gel is soaked in SDS and then placed along the top edge of an SDS polyacrylamide-gel slab, through which each polypeptide chain migrates to form a discrete spot. This is the second dimension of two-dimensional polyacrylamide-gel electrophoresis. The only proteins left unresolved are those that have both identical sizes and identical isoelectric points, a relatively rare situation. Even trace amounts of each polypeptide chain can be detected on the gel by various staining procedures—or by autoradiography if the protein sample was initially labeled with a radioisotope (Figure 8–16). The technique has such great resolving power that it can distinguish between two proteins

1	by autoradiography if the protein sample was initially labeled with a radioisotope (Figure 8–16). The technique has such great resolving power that it can distinguish between two proteins that differ in only a single charged amino acid, or a single negatively charged phosphorylation site.

1	Specific Proteins Can Be Detected by Blotting with Antibodies A specific protein can be identified after its fractionation on a polyacrylamide gel by exposing all the proteins present on the gel to a specific antibody that has been labeled with a radioactive isotope or a fluorescent dye. This procedure is normally carried out after transferring all of the separated proteins present in the 100 50 25SDS migration (mol. mass in kilodaltons) Figure 8–16 Two-dimensional polyacrylamide-gel electrophoresis.

1	All the proteins in an E. coli bacterial cell are separated in this gel, in which each spot corresponds to a different polypeptide chain. The proteins were first separated on the basis of their isoelectric points by isoelectric focusing in the horizontal dimension. They were then further fractionated according to their molecular mass by electrophoresis from top to bottom in the presence of SDS. Note that different proteins are present in very different amounts. The bacteria were fed with a mixture of radioisotope-labeled amino acids so that all of their proteins were radioactive and could be detected by autoradiography. (Courtesy of Patrick O’Farrell.) gel onto a sheet of nitrocellulose paper or nylon membrane. Placing the membrane over the gel and driving the proteins out of the gel with a strong electric current transfers the protein onto the membrane. The membrane is then soaked in a solution of labeled antibody to reveal the protein of interest. This method of detecting proteins

1	a strong electric current transfers the protein onto the membrane. The membrane is then soaked in a solution of labeled antibody to reveal the protein of interest. This method of detecting proteins is called Western blotting, or immunoblotting (Figure 8–17). Sensitive Western-blotting methods can detect very small amounts of a specific protein (1 nanogram or less) in a total cell extract or some other heterogeneous protein mixture. The method can be very useful when assessing the amounts of a specific protein in the cell or when measuring changes in those amounts under various conditions.

1	Hydrodynamic Measurements Reveal the Size and Shape of a Protein Complex

1	Most proteins in a cell act as part of larger complexes, and knowledge of the size and shape of these complexes often leads to insights regarding their function. This information can be obtained in several important ways. Sometimes, a complex can be directly visualized using electron microscopy, as described in Chapter 9. A complementary approach relies on the hydrodynamic properties of a complex; that is, its behavior as it moves through a liquid medium. Usually, two separate measurements are made. One measure is the velocity of a complex as it moves under the influence of a centrifugal field produced by an ultracentrifuge (see Figure 8–7A). The sedimentation coefficient (or S value) obtained depends on both the size and the shape of the complex and does not, by itself, convey especially useful information. However, once a second hydrodynamic measurement is performed—by charting the migration of a complex through a gel-filtration chromatography column (see Figure 8–9B)—both the

1	useful information. However, once a second hydrodynamic measurement is performed—by charting the migration of a complex through a gel-filtration chromatography column (see Figure 8–9B)—both the approximate shape of a complex and its molecular weight can be calculated.

1	Molecular weight can also be determined more directly by using an analytical ultracentrifuge, a complex device that allows protein absorbance measurements to be made on a sample while it is subjected to centrifugal forces. In this approach, the sample is centrifuged until it reaches equilibrium, where the centrifugal force on a protein complex exactly balances its tendency to diffuse away. Because this balancing point is dependent on a complex’s molecular weight but not on its particular shape, the molecular weight can be directly calculated.

1	A frequent problem in cell biology and biochemistry is the identification of a protein or collection of proteins that has been obtained by one of the purification procedures discussed in the preceding pages. Because the genome sequences of most experimental organisms are now known, catalogs of all the proteins produced in those organisms are available. The task of identifying an unknown protein (or collection of unknown proteins) thus reduces to matching some of the amino acid

1	Figure 8–17 Western blotting. All the proteins from dividing tobacco cells in culture were first separated by two-dimensional polyacrylamide-gel electrophoresis. In (A), the positions of the proteins are revealed by a sensitive protein stain. In (B), the separated proteins on an identical gel were then transferred to a sheet of nitrocellulose and exposed to an antibody that recognizes only those proteins that are phosphorylated on threonine residues during mitosis. The positions of the few proteins that are recognized by this antibody are revealed by an enzyme-linked second antibody. (From J.A. Traas et al., Plant J. 2:723–732, 1992. With permission from Blackwell Publishing.)

1	Figure 8–18 The mass spectrometer. (A) Mass spectrometers used in biology contain an ion source that generates gaseous peptides or other molecules under conditions that render most molecules positively charged. The two major types of ion source are MALDI and electrospray, as described in the text. Ions are accelerated into a mass analyzer, which separates the ions on the basis of their mass and charge by one of three major methods: 1. Time-of-flight (TOF) analyzers determine the massto-charge ratio of each ion in the mixture from the rate at which it travels from the ion source to the detector. 2. Quadropole mass filters contain a long chamber lined by four electrodes that produce oscillating electric fields that govern the trajectory of ions; by varying the properties of the electric field over a wide range, a spectrum of ions with specific mass-to-charge ratios is allowed to pass through the chamber to the detector, while other ions are discarded. 3. Ion traps contain

1	electric field over a wide range, a spectrum of ions with specific mass-to-charge ratios is allowed to pass through the chamber to the detector, while other ions are discarded. 3. Ion traps contain doughnut-shaped electrodes producing a three-dimensional electric field that traps all ions in a circular chamber; the properties of the electric field can be varied over a wide range to eject a spectrum of specific ions to a detector. (B) Tandem mass spectrometry typically involves two mass analyzers separated by a collision chamber containing an inert, high-energy gas. The electric field of the first mass analyzer is adjusted to select a specific peptide ion, called a precursor ion, which is then directed to the collision chamber. Collision of the peptide with gas molecules causes random peptide fragmentation, primarily at peptide bonds, resulting in a highly complex mixture of fragments containing one or more amino acids from throughout the original peptide. The second mass analyzer is

1	fragmentation, primarily at peptide bonds, resulting in a highly complex mixture of fragments containing one or more amino acids from throughout the original peptide. The second mass analyzer is then used to measure the masses of the fragments (called product or daughter ions). With computer assistance, the pattern of fragments can be used to deduce the amino acid sequence of the original peptide.

1	sequences present in the unknown sample with known cataloged genes. This task is now performed almost exclusively by using mass spectrometry in conjunction with computer searches of databases. Charged particles have very precise dynamics when subjected to electrical and magnetic fields in a vacuum. Mass spectrometry exploits this principle to separate ions according to their mass-to-charge (m/z) ratio. It is an enormously sensitive technique. It requires very little material and is capable of determining the precise mass of intact proteins and of peptides derived from them by enzymatic or chemical cleavage. Masses can be obtained with great accuracy, often with an error of less than one part in a million.

1	Mass spectrometry is performed using complex instruments with three major components (Figure 8–18A). The first is the ion source, which transforms tiny amounts of a peptide sample into a gas containing individual charged peptide molecules. These ions are accelerated by an electric field into the second component, the mass analyzer, where electric or magnetic fields are used to separate the ions on the basis of their mass-to-charge ratios. Finally, the separated ions collide with a detector, which generates a mass spectrum containing a series of peaks representing the masses of the molecules in the sample.

1	There are many different types of mass spectrometer, varying mainly in the nature of their ion sources and mass analyzers. One of the most common ion sources depends on a technique called matrix-assisted laser desorption ionization (MALDI). In this approach, the proteins in the sample are first cleaved into short peptides by a protease such as trypsin. These peptides are mixed with an organic acid and then dried onto a metal or ceramic slide. A brief laser burst is directed toward the sample, producing a gaseous puff of ionized peptides, each carrying one or more positive charges. In many cases, the MALDI ion source is coupled to a mass analyzer called a time-of-flight (TOF) analyzer, which is a long chamber through which the ionized peptides are accelerated by an electric field toward a detector. Their mass and charge determine the time it takes them to reach the detector: large peptides move more slowly, and more highly charged molecules move more quickly. By analyzing those ionized

1	Their mass and charge determine the time it takes them to reach the detector: large peptides move more slowly, and more highly charged molecules move more quickly. By analyzing those ionized peptides that bear a single charge, the precise masses of peptides present in the original sample can be determined. This information is then used to search genomic databases, in which the masses of all proteins and of all their predicted peptide fragments have been tabulated from the genomic sequences of the organism. An unambiguous match to a particular open reading frame can often be made by knowing the mass of only a few peptides derived from a given protein.

1	By employing two mass analyzers in tandem (an arrangement known as MS/ MS; Figure 8–18B), it is possible to directly determine the amino acid sequences of individual peptides in a complex mixture. The MALDI-TOF instrument described above is not ideal for this method. Instead, MS/MS typically involves an electro-spray ion source, which produces a continuous thin stream of peptides that are ionized and accelerated into the first mass analyzer. The mass analyzer is typically either a quadropole or ion trap, which employs large electrodes to produce oscillating electric fields inside the chamber containing the ions. These instruments act as mass filters: the electric field is adjusted over a broad range to select a single peptide ion and discard all the others in the peptide mixture. In tandem mass spectrometry, this single ion is then exposed to an inert, high-energy gas, which collides with the peptide, resulting in fragmentation, primarily at peptide bonds. The second mass analyzer

1	mass spectrometry, this single ion is then exposed to an inert, high-energy gas, which collides with the peptide, resulting in fragmentation, primarily at peptide bonds. The second mass analyzer then determines the masses of the peptide fragments, which can be used by computational methods to determine the amino acid sequence of the original peptide and thereby identify the protein from which it came.

1	Tandem mass spectrometry is also useful for detecting and precisely mapping post-translational modifications of proteins, such as phosphorylations or acetylations. Because these modifications impart a characteristic mass increase to an amino acid, they are easily detected during the analysis of peptide fragments in the second mass analyzer, and the precise site of the modification can often be deduced from the spectrum of peptide fragments.

1	A powerful, “two-dimensional” mass spectrometry technique can be used to determine all of the proteins present in an organelle or another complex mixture of proteins. First, the mixture of proteins present is digested with trypsin to produce short peptides. Next, these peptides are separated by automated high-performance liquid chromatography (LC). Every peptide fraction from the chromatographic column is injected directly into an electrospray ion source on a tandem mass spectrometer (MS/MS), providing the amino acid sequence and post-translational modifications for every peptide in the mixture. This method, often called LC-MS/MS, is used to identify hundreds or thousands of proteins in complex protein mixtures from specific organelles or from whole cells. It can also be used to map all of the phosphorylation sites in the cell, or all of the proteins targeted by other post-translational modifications such as acetylation or ubiquitylation.

1	Sets of Interacting Proteins Can Be Identified by Biochemical Methods Because most proteins in the cell function as part of complexes with other proteins, an important way to begin to characterize the biological role of an unknown protein is to identify all of the other proteins to which it specifically binds. A key method for identifying proteins that bind to one another tightly is coimmunoprecipitation. A specific target protein is immunoprecipitated from a cell lysate using specific antibodies coupled to beads, as described earlier. If the target protein is associated tightly enough with another protein when it is captured by the antibody, the partner precipitates as well and can be identified by mass spectrometry. This method is useful for identifying proteins that are part of a complex inside cells, including those that interact only transiently—for example, when extracellular signal molecules stimulate cells (discussed in Chapter 15).

1	In addition to capturing protein complexes on columns or in test tubes, researchers are developing high-density protein arrays to investigate protein interactions. These arrays, which contain thousands of different proteins or antibodies spotted onto glass slides or immobilized in tiny wells, allow one to examine the biochemical activities and binding profiles of a large number of proteins at once. For example, if one incubates a fluorescently labeled protein with arrays containing thousands of immobilized proteins, the spots that remain fluorescent after extensive washing each contain a protein that specifically binds the labeled protein.

1	Once two proteins—or a protein and a small molecule—are known to associate, it becomes important to characterize their interaction in more detail. Proteins can associate with each other more or less permanently (like the subunits of RNA polymerase or the proteasome), or engage in transient encounters that may last only a few milliseconds (like a protein kinase and its substrate). To understand how a protein functions inside a cell, we need to determine how tightly it binds to other proteins, how rapidly it dissociates from them, and how covalent modifications, small molecules, or other proteins influence these interactions.

1	As we discussed in Chapter 3 (see Figure 3–44), the extent to which two proteins interact is determined by the rates at which they associate and dissociate. These rates depend, respectively, on the association rate constant (kon) and dissociation rate constant (koff). The kinetic rate constant koff is a particularly useful number because it provides valuable information about how long two proteins remain bound to one another. The ratio of the two kinetic constants (kon/koff) yields another very useful number called the equilibrium constant (K, also known as Keq or Ka), the inverse of which is the more commonly used dissociation constant Kd. The equilibrium constant is useful as a general indicator of the affinity of the interaction, and it can be used to estimate the amount of bound complex at different concentrations of the two protein partners—thereby providing insights into the importance of the interaction at the protein concentrations found inside the cell.

1	A wide range of methods can be used to determine binding constants for a two-protein complex. In a simple equilibrium binding experiment, two proteins are mixed at a range of concentrations, allowed to reach equilibrium, and the amount of bound complex is measured; half of the protein complex will be bound at a concentration that is equal to Kd. Equilibrium experiments often involve the use of radioactive or fluorescent tags on one of the protein partners, coupled with biochemical or optical methods for measuring the amount of bound protein. In a more complex kinetic binding experiment, the kinetic rate constants are determined using rapid methods that allow real-time measurement of the formation of a bound complex over time (to determine kon) or the dissociation of a bound complex over time (to determine koff).

1	Optical techniques provide particularly rapid, convenient, and accurate binding measurements, and in some cases the proteins do not even need to be labeled. Certain amino acids (tryptophan, for example) exhibit weak fluorescence that can be detected with sensitive fluorimeters. In many cases, the fluorescence intensity, or the emission spectrum of fluorescent amino acids located in a protein–protein interface, will change when two proteins associate. When this change can be detected by fluorimetry, it provides a simple and sensitive measure of protein binding that is useful in both equilibrium and kinetic binding experiments. A related but more widely useful optical binding technique is based on fluorescence anisotropy, a change in the polarized light that is emitted by a fluorescently tagged protein in the bound and free states (Figure 8–19).

1	Figure 8–19 Measurement of binding with fluorescence anisotropy. This method depends on a fluorescently tagged protein that is illuminated with polarized light at the appropriate wavelength for excitation; a fluorimeter is used to measure the intensity and polarization of the emitted light. If the fluorescent protein is fixed in position and therefore does not rotate during the brief period between excitation and emission, then the emitted light will be polarized at the same angle as the excitation light. This directional effect is called fluorescence anisotropy. Protein molecules in solution rotate or tumble rapidly, however, so that there is a decrease in the amount of anisotropic fluorescence. Larger molecules tumble at a slower rate and therefore have higher fluorescence anisotropy. (A) To measure the binding between a small molecule and a large receptor protein, the smaller molecule is labeled with a fluorophore. In the absence of its binding partner, the molecule tumbles

1	(A) To measure the binding between a small molecule and a large receptor protein, the smaller molecule is labeled with a fluorophore. In the absence of its binding partner, the molecule tumbles rapidly, resulting in low fluorescence anisotropy (top). When the small molecule binds to its larger partner, however, it tumbles less rapidly, resulting in an increase in fluorescence anisotropy (bottom). (B) In the equilibrium binding experiment shown here, a small, fluorescent peptide ligand was present at a low concentration, and the amount of fluorescence anisotropy (in millipolarization units, mP) was measured after incubation with various concentrations of a larger protein receptor for the ligand. From the hyperbolic curve that fits the data, it can be seen that 50% binding occurred at about 10 μM, which is equal to the dissociation constant Kd for the binding interaction.

1	Another optical method for probing protein interactions uses green fluorescent protein (discussed in detail below) and its derivatives of different colors. In this application, two proteins of interest are each labeled with a different fluorescent protein, such that the emission spectrum of one fluorescent protein overlaps the absorption spectrum of the second. If the two proteins come very close to each other (within about 1–5 nm), the energy of the absorbed light is transferred from one fluorescent protein to the other. The energy transfer, called fluorescence resonance energy transfer (FRET), is determined by illuminating the first fluorescent protein and measuring emission from the second (see Figure 9–26). When combined with fluorescence microscopy, this method can be used to characterize protein–protein interactions at specific locations inside living cells (discussed in Chapter 9).

1	Small chemical inhibitors of specific proteins have contributed a great deal to the development of cell biology. For example, the microtubule inhibitor colchicine is routinely used to test whether microtubules are required for a given biological process; it also led to the first purification of tubulin several decades ago. In the past, these small molecules were usually natural products; that is, they were synthesized by living creatures. Although natural products have been extraordinarily useful in science and medicine (see, for example, Table 6–4, p. 352), they act on a limited number of biological processes. However, the recent development of methods to synthesize hundreds of thousands of small molecules and to carry out large-scale automated screens holds the promise of identifying chemical inhibitors for virtually any biological process. In such approaches, large collections of small chemical compounds are simultaneously tested, either on living cells or in cell-free assays. Once

1	inhibitors for virtually any biological process. In such approaches, large collections of small chemical compounds are simultaneously tested, either on living cells or in cell-free assays. Once an inhibitor is identified, it can be used as a probe to identify, through affinity chromatography or other means, the protein to which the inhibitor binds. This general strategy, sometimes called chemical biology, has successfully identified inhibitors of many proteins that carry out key processes in cell biology. An inhibitor of a kinesin protein that functions in mitosis, for

1	Figure 8–20 Small-molecule inhibitors for manipulating living cells. (A) Chemical structure of monastrol, a kinesin inhibitor identified in a large-scale screen for small molecules that disrupt mitosis. (B) Normal mitotic spindle seen in an untreated cell. The microtubules are stained green and chromosomes blue. (C) Monopolar spindle that forms in cells treated with monastrol, which inhibits a kinesin protein required for separation of the spindle poles in early mitosis. (B and C, from T.U. Mayer et al., Science 286:971–974, 1999. With permission from AAAS.) example, was identified by this method (Figure 8–20). Chemical inhibitors give the cell biologist great control over the timing of inhibition, as drugs can be rapidly added to or removed from cells, allowing protein function to be switched on or off quickly.

1	The main technique that has been used to discover the three-dimensional structure of molecules, including proteins, at atomic resolution is x-ray crystallography. X-rays, like light, are a form of electromagnetic radiation, but they have a much shorter wavelength, typically around 0.1 nm (the diameter of a hydrogen 5 µm atom). If a narrow beam of parallel x-rays is directed at a sample of a pure protein, most of the x-rays pass straight through it. A small fraction, however, are scattered by the atoms in the sample. If the sample is a well-ordered crystal, the scattered waves reinforce one another at certain points and appear as diffraction spots when recorded by a suitable detector (Figure 8–21).

1	The position and intensity of each spot in the x-ray diffraction pattern contain information about the locations of the atoms in the crystal that gave rise to it. Deducing the three-dimensional structure of a large molecule from the diffraction pattern of its crystal is a complex task and was not achieved for a protein molecule until 1960. But in recent years x-ray diffraction analysis has become increasingly automated, and now the slowest step is likely to be the generation of suitable protein crystals. This step requires large amounts of very pure protein and often involves years of trial and error to discover the proper crystallization conditions; the pace has greatly accelerated with the use of recombinant DNA techniques to produce pure proteins and robotic techniques to test large numbers of crystallization conditions.

1	Analysis of the resulting diffraction pattern produces a complex three-dimensional electron-density map. Interpreting this map—translating its contours into a three-dimensional structure—is a complicated procedure that requires knowledge of the amino acid sequence of the protein. Largely by trial and error, the sequence and the electron-density map are correlated by computer to give the best possible fit. The reliability of the final atomic model depends on the resolution of the original crystallographic data: 0.5 nm resolution might produce a low-resolution map of the polypeptide backbone, whereas a resolution of 0.15 nm allows all of the non-hydrogen atoms in the molecule to be reliably positioned.

1	A complete atomic model is often too complex to appreciate directly, but simplified versions that show a protein’s essential structural features can be readily derived from it (see Panel 3–2, pp. 142–143). The three-dimensional structures of tens of thousands of different proteins have been determined by x-ray crystallography or by NMR spectroscopy (see page 461)—enough to allow the grouping of common structures into families (Movie 8.1). These structures or protein folds often seem to be more conserved in evolution than are the amino acid sequences that form them (see Figure 3–13).

1	X-ray crystallographic techniques can also be applied to the study of macromolecular complexes. The method was used, for example, to determine the structure of the ribosome, a large and complex machine made of several RNAs and more than 50 proteins (see Figure 6–62). The determination required the use of a synchrotron, a radiation source that generates x-rays with the intensity needed to analyze the crystals of such large macromolecular complexes.

1	beam of x-rays x-ray source protein crystal diffracted beams x-ray diffraction pattern obtained from the protein crystal beam stop (A) (B) (C)Figure 8–21 X-ray crystallography. (A) A narrow beam of x-rays is directed at a well-ordered crystal (B). Shown here is a protein crystal of ribulose bisphosphate carboxylase, an enzyme with a central role in CO2 fixation during photosynthesis. The atoms in the crystal scatter some of the beam, and the scattered waves reinforce one another at certain points and appear as a pattern of diffraction spots (C). This diffraction pattern, together with the amino acid sequence of the protein, can be used to produce an atomic model (D). The complete atomic model is hard to interpret, but this simplified version, derived from the x-ray diffraction data, shows the protein’s structural features clearly (α helices, green; β strands, red). The components pictured in A to D are not shown to scale. (B, courtesy of C. Branden; C, courtesy of J. Hajdu and I.

1	the protein’s structural features clearly (α helices, green; β strands, red). The components pictured in A to D are not shown to scale. (B, courtesy of C. Branden; C, courtesy of J. Hajdu and I. Andersson; D, adapted from original provided by B. Furugren.)

1	NMR Can Be Used to Determine Protein Structure in Solution Nuclear magnetic resonance (NMR) spectroscopy has been widely used for many years to analyze the structure of small molecules, small proteins, or protein domains. Unlike x-ray crystallography, NMR does not depend on having a crystalline sample. It simply requires a small volume of concentrated protein solution that is placed in a strong magnetic field; indeed, it is the main technique that yields detailed evidence about the three-dimensional structure of molecules in solution.

1	Certain atomic nuclei, particularly hydrogen nuclei, have a magnetic moment or spin: that is, they have an intrinsic magnetization, like a bar magnet. The spin aligns along the strong magnetic field, but it can be changed to a misaligned, excited state in response to applied radiofrequency (RF) pulses of electromagnetic radiation. When the excited hydrogen nuclei return to their aligned state, they emit RF radiation, which can be measured and displayed as a spectrum. The nature of the emitted radiation depends on the environment of each hydrogen nucleus, and if one nucleus is excited, it influences the absorption and emission of radiation by other nuclei that lie close to it. It is consequently possible, by an ingenious elaboration of the basic NMR technique known as two-dimensional NMR, to distinguish the signals from hydrogen nuclei in different amino acid residues, and to identify and measure the small shifts in these signals that occur when these hydrogen nuclei lie close enough

1	to distinguish the signals from hydrogen nuclei in different amino acid residues, and to identify and measure the small shifts in these signals that occur when these hydrogen nuclei lie close enough together to interact. Because the size of such a shift reveals the distance between the interacting pair of hydrogen atoms, NMR can provide information about the distances between the parts of the protein molecule. By combining this information with a knowledge of the amino acid

1	Figure 8–22 NMR spectroscopy. An example of the data from an NMR machine. This two-dimensional NMR spectrum is derived from the C-terminal domain of the enzyme cellulase. The spots represent interactions between hydrogen atoms that are near neighbors in the protein and hence reflect the distance that separates them. Complex computing methods, in conjunction with the known amino acid sequence, enable possible compatible structures to be derived. Ten structures of the enzyme, which all satisfy the distance constraints equally well, are shown superimposed on one another, giving a good indication of the probable three-dimensional structure. (Courtesy of P. Kraulis.) sequence, it is possible in principle to compute the three-dimensional structure of the protein (Figure 8–22).

1	P. Kraulis.) sequence, it is possible in principle to compute the three-dimensional structure of the protein (Figure 8–22). For technical reasons, the structure of small proteins of about 20,000 daltons or less can be most readily determined by NMR spectroscopy. Resolution decreases as the size of a macromolecule increases. But recent technical advances have now pushed the limit to about 100,000 daltons, thereby making the majority of proteins accessible for structural analysis by NMR. Because NMR studies are performed in solution, this method also offers a convenient means of monitoring changes in protein structure, for example during protein folding or when the protein binds to another molecule. NMR is also used widely to investigate molecules other than proteins and is valuable, for example, as a method to determine the three-dimensional structures of RNA molecules and the complex carbohydrate side chains of glycoproteins.

1	A third major method for the determination of protein structure, and particularly the structure of large protein complexes, is single-particle analysis by electron microscopy. We discuss this approach in Chapter 9. Having discussed methods for purifying and analyzing proteins, we now turn to a common situation in cell and molecular biology: an investigator has identified a gene important for a biological process but has no direct knowledge of the biochemical properties of its protein product.

1	Thanks to the proliferation of protein and nucleic acid sequences that are cataloged in genome databases, the function of a gene—and its encoded protein— can often be predicted by simply comparing its sequence with those of previously characterized genes. Because amino acid sequence determines protein structure, and structure dictates biochemical function, proteins that share a similar amino acid sequence usually have the same structure and usually perform similar biochemical functions, even when they are found in distantly related organisms. In modern cell biology, the study of a newly discovered protein usually begins with a search for previously characterized proteins that are similar in their amino acid sequences.

1	Searching a collection of known sequences for similar genes or proteins is typically done over the Internet, and it simply involves selecting a database and entering the desired sequence. A sequence-alignment program—the most popular is BLAST—scans the database for similar sequences by sliding the submitted sequence along the archived sequences until a cluster of residues falls into full or partial alignment (Figure 8–23). Such comparisons can predict the functions of individual proteins, families of proteins, or even most of the protein complement of a newly sequenced organism.

1	As was explained in Chapter 3, many proteins that adopt the same conformation and have related functions are too distantly related to be identified from a comparison of their amino acid sequences alone (see Figure 3–13). Thus, an ability to reliably predict the three-dimensional structure of a protein from its amino acid sequence would improve our ability to infer protein function from the sequence information in genomic databases. In recent years, major progress has been made in predicting the precise structure of a protein. These predictions are based, in part, on our knowledge of the thousands of protein structures that have already been determined by x-ray crystallography and NMR spectroscopy and, in part, on computations using our knowledge of the physical forces acting on the atoms. However, it remains a substantial and important challenge to predict the structures of proteins that are large or have multiple domains, or to predict structures at the very high levels of resolution

1	However, it remains a substantial and important challenge to predict the structures of proteins that are large or have multiple domains, or to predict structures at the very high levels of resolution needed to assist in computer-based drug discovery.

1	While finding related sequences and structures for a new protein will provide many clues about its function, it is usually necessary to test these insights through direct experimentation. However, the clues generated from sequence comparisons typically point the investigator in the correct experimental direction, and their use has therefore become one of the most important strategies in modern cell biology. Many methods exist for identifying proteins and analyzing their biochemical properties, structures, and interactions with other proteins. Small-molecule inhibitors allow the functions of proteins they act upon to be studied in living cells. Because proteins with similar structures often have similar functions, the biochemical activity of a protein can often be predicted by searching databases for previously characterized proteins that are similar in their amino acid sequences.

1	Until the early 1970s, DNA was the most difficult biological molecule for the biochemist to analyze. Enormously long and chemically monotonous, the string of nucleotides that forms the genetic material of an organism could be examined only indirectly, by protein sequencing or by genetic analysis. Today, the situation has changed entirely. From being the most difficult macromolecule of the cell to Figure 8–23 Results of a bLAST search.

1	Sequence databases can be searched to find similar amino acid or nucleic acid sequences. Here, a search for proteins similar to the human cell-cycle regulatory protein Cdc2 (Query) locates maize Cdc2 (Sbjct), which is 68% identical to human Cdc2 in its amino acid sequence. The alignment begins at residue 57 of the Query protein, suggesting that the human protein has an N-terminal region that is absent from the maize protein. The green blocks indicate differences in sequence, and the yellow bar summarizes the similarities: when the two amino acid sequences are identical, the residue is shown; similar amino acid substitutions are indicated by a plus sign (+). Only one small gap has been introduced—indicated by the red arrow at position 194 in the Query sequence—to align the two sequences maximally. The alignment score (Score), which is expressed in two different types of units, takes into account penalties for substitutions and gaps; the higher the alignment score, the better the match.

1	The alignment score (Score), which is expressed in two different types of units, takes into account penalties for substitutions and gaps; the higher the alignment score, the better the match. The significance of the alignment is reflected in the Expectation (E) value, which specifies how often a match this good would be expected to occur by chance. The lower the E value, the more significant the match; the extremely low value here (e–111) indicates certain significance. E values much higher than 0.1 are unlikely to reflect true relatedness. For example, an E value of 0.1 means there is a 1 in 10 likelihood that such a match would arise solely by chance.

1	analyze, DNA has become the easiest. It is now possible to determine the entire nucleotide sequence of a bacterial or fungal genome in a matter of hours, and the sequence of an individual human genome in less than a day. Once the nucleotide sequence of a genome is known, any individual gene can be easily isolated, and large quantities of the gene product (be it RNA or protein) can be made either by introducing the gene into bacteria or animal cells and coaxing these cells to over-express the foreign gene or by synthesizing the gene product in vitro. In this way, proteins and RNA molecules that might be present in only tiny amounts in living cells can be produced in large quantities for biochemical and structural analyses. And this approach can also be used to produce large quantities of human proteins (such as insulin, or interferon, or blood-clotting proteins) for use as human pharmaceuticals. As we will see later in this chapter, it is also possible for scientists to alter an

1	of human proteins (such as insulin, or interferon, or blood-clotting proteins) for use as human pharmaceuticals. As we will see later in this chapter, it is also possible for scientists to alter an isolated gene and transfer it back into the germ line of an animal or plant, so as to become a functional and heritable part of the organism’s genome. In this way, the biological roles of any gene can be assessed by observing—in the whole organism—the results of modifying it.

1	The ability to manipulate DNA with precision in a test tube or an organism, known as recombinant DNA technology has had a dramatic impact on all aspects of cell and molecular biology, allowing us to routinely study cells and their macromolecules in ways that were unimaginable even twenty years ago. Central to the technology are the following manipulations: 1. Cleavage of DNA at specific sites by restriction nucleases, which greatly facilitates the isolation and manipulation of individual pieces of a genome. 2. DNA ligation, which makes it possible to seamlessly join together DNA molecules from widely different sources. 3. DNA cloning (through the use of either cloning vectors or the polymerase chain reaction) in which a portion of a genome (often an individual gene) is “purified” away from the remainder of the genome by repeatedly copying it to generate many billions of identical molecules. 4.

1	4. Nucleic acid hybridization, which makes it possible to identify any specific sequence of DNA or RNA with great accuracy and sensitivity based on its ability to selectively bind a complementary nucleic acid sequence. 5. DNA synthesis, which makes it possible to chemically synthesize DNA molecules with any sequence of nucleotides, whether or not the sequence occurs in nature. 6. Rapid determination of the sequence of nucleotides of any DNA or RNA molecule. In the following sections, we describe each of these basic techniques which, together, have revolutionized the study of cell and molecular biology.

1	Unlike a protein, a gene does not exist as a discrete entity in cells, but rather as a small region of a much longer DNA molecule. Although the DNA molecules in a cell can be randomly broken into small pieces by mechanical force, a fragment containing a single gene in a mammalian genome would still be only one among a hundred thousand or more DNA fragments, indistinguishable in their average size. How could such a gene be separated from all the others? Because all DNA molecules consist of an approximately equal mixture of the same four nucleotides, they cannot be readily separated, as proteins can, on the basis of their different charges and biochemical properties. The solution to this problem began to emerge with the discovery of restriction nucleases. These enzymes, which are purified from bacteria, cut the DNA double helix at specific sites defined by the local nucleotide sequence, thereby cleaving a long, double-stranded DNA molecule into fragments of strictly defined sizes.

1	Like many of the tools of recombinant DNA technology, restriction nucleases were discovered by researchers trying to understand an intriguing biological phenomenon. It had been observed that certain bacteria always degraded “foreign” DNA that was introduced into them experimentally. A search for the mechanism responsible revealed a then unanticipated class of bacterial nucleases that cleave DNA at specific nucleotide sequences. The bacterium’s own DNA is protected from cleavage by methylation of these same sequences, thereby protecting a bacterium’s own genome from being overrun by foreign DNA. Because these enzymes restrict the transfer of DNA into bacteria, they were called restriction nucleases. The pursuit of this seemingly arcane biological puzzle set off the development of technologies that have forever changed the way cell and molecular biologists study living things.

1	Different bacterial species produce different restriction nucleases, each cutting at a different, specific nucleotide sequence (Figure 8–24). Because these target sequences are short—generally four to eight nucleotide pairs—many sites of cleavage will occur, purely by chance, in any long DNA molecule. The reason restriction nucleases are so useful in the laboratory is that each enzyme will always cut a particular DNA molecule at the same sites. Thus for a given sample of DNA (which contains many identical molecules), a particular restriction nuclease will reliably generate the same set of DNA fragments.

1	The size of the resulting fragments depends on the length of the target sequences of the restriction nucleases. As shown in Figure 8–24, the enzyme HaeIII cuts at a sequence of four nucleotide pairs; a sequence this long would be expected to occur purely by chance approximately once every 256 nucleotide pairs (1 in 44). In comparison, a restriction nuclease with a target sequence that is eight nucleotides long would be expected to cleave DNA on average once every 65,536 nucleotide pairs (1 in 48). This difference in sequence selectivity makes it possible to cleave a long DNA molecule into the fragment sizes that are most suitable for a given application. Gel Electrophoresis Separates DNA Molecules of Different Sizes

1	The same types of gel-electrophoresis methods that have proved so useful in the analysis of proteins (see Figure 8–13) can be applied to DNA molecules. The procedure is actually simpler than for proteins: because each nucleotide in a nucleic acid molecule already carries a single negative charge (on the phosphate group), there is no need to add the negatively charged detergent SDS that is required to make protein molecules move uniformly toward the positive electrode. Larger DNA fragments will migrate more slowly because their progress is impeded to a greater extent by the gel matrix. Over several hours, the DNA fragments become spread out across the gel according to size, forming a ladder of discrete bands, each composed of a collection of DNA molecules of identical length (Figure 8–25A and B). To separate DNA molecules longer than 500 nucleotide pairs, the gel is made of a diluted solution of agarose (a polysaccharide isolated from seaweed). For DNA fragments less than 500

1	8–25A and B). To separate DNA molecules longer than 500 nucleotide pairs, the gel is made of a diluted solution of agarose (a polysaccharide isolated from seaweed). For DNA fragments less than 500 nucleotides long, specially designed polyacrylamide gels allow the separation of molecules that differ in length by as little as a single nucleotide (see Figure 8–25C).

1	Figure 8–24 Restriction nucleases cleave DNA at specific nucleotide sequences.

1	Like the sequence-specific DNA-binding proteins we encountered in Chapter 7 (see Figure 7–8), restriction enzymes often work as dimers, and the DNA sequence they recognize and cleave is often symmetrical around a central point. Here, both strands of the DNA double helix are cut at specific points within the target sequence (orange). Some enzymes, such as HaeIII, cut straight across the double helix and leave two blunt-ended DNA molecules; with others, such as EcoRI and HindIII, the cuts on each strand are staggered. These staggered cuts generate “sticky ends”—short, single-stranded overhangs that help the cut DNA molecules join back together through complementary base-pairing. This rejoining of DNA molecules becomes important for DNA cloning, as we discuss below. Restriction nucleases are usually obtained from bacteria, and their names reflect their origins: for example, the enzyme EcoRI comes from Escherichia coli. There are currently hundreds of different restriction enzymes

1	are usually obtained from bacteria, and their names reflect their origins: for example, the enzyme EcoRI comes from Escherichia coli. There are currently hundreds of different restriction enzymes available; they can be ordered from companies that commercially produce them.

1	A variation of agarose-gel electrophoresis, called pulsed-field gel electrophoresis, makes it possible to separate extremely long DNA molecules, even those found in whole chromosomes. Ordinary gel electrophoresis fails to separate very large DNA molecules because the steady electric field stretches them out so that they travel end-first through the gel in snakelike configurations at a rate that is independent of their length. In pulsed-field gel electrophoresis, by contrast, the direction of the electric field changes periodically, which forces the molecules to reorient before continuing to move snakelike through the gel. This re-orientation takes much more time for larger molecules, so that longer molecules move more slowly than shorter ones. As a consequence, entire bacterial or yeast chromosomes separate into discrete bands in pulsed-field gels and so can be sorted and identified on the basis of their size (Figure 8–25D). Although a typical mammalian chromosome of 108 nucleotide

1	chromosomes separate into discrete bands in pulsed-field gels and so can be sorted and identified on the basis of their size (Figure 8–25D). Although a typical mammalian chromosome of 108 nucleotide pairs is still too long to be sorted even in this way, large segments of these chromosomes are readily separated and identified if the chromosomal DNA is first cut with a restriction nuclease selected to recognize sequences that occur only rarely.

1	The DNA bands on agarose or polyacrylamide gels are invisible unless the DNA is labeled or stained in some way. A particularly sensitive method of staining DNA is to soak the gel in the dye ethidium bromide, which fluoresces under ultraviolet light when it is bound to DNA (see Figure 8–25B and D). Even more sensitive detection methods incorporate a radioisotope or chemical marker into the DNA molecules before electrophoresis, as we next describe.

1	Figure 8–25 DNA molecules can be separated by size using gel electrophoresis. (A) Schematic illustration comparing the results of cutting the same DNA molecule (in this case, the genome of a virus that infects wasps) with two different restriction nucleases, EcoRI (middle) and HindIII (right). The fragments are then separated by gel electrophoresis using a gel matrix of agarose. Because larger fragments migrate more slowly than smaller ones, the lowermost bands on the gel contain the smallest DNA fragments. The sizes of the fragments can be estimated by comparing them to a set of DNA fragments of known sizes (left). (B) Photograph of an actual agarose gel showing DNA “bands” that have been stained with ethidium bromide.

1	A polyacrylamide gel with small pores was used to separate short DNA molecules that differ by only a single nucleotide. Shown here are the results of a dideoxy sequencing reaction, explained later in this chapter. From left to right, the bands in the four lanes were produced by adding G, A, T, and C chain-terminating nucleotides (see Panel 8–1). The DNA molecules were labeled with 32P, and the image shown was produced by laying a piece of photographic film over the gel and allowing the 32P to expose the film, producing the dark bands observed when the film was developed.

1	The technique of pulsed-field agarose-gel electrophoresis was used to separate the 16 different chromosomes of the yeast species Saccharomyces cerevisiae, which range in size from 220,000 to 2.5 million nucleotide pairs. The DNA was stained as in (B). DNA molecules as large as 107 nucleotide pairs can be separated in this way. (B, from U. Albrecht et al., J. Gen. Virol. 75:3353-3363, 1994; C, courtesy of Leander Lauffer and Peter Walter; D, from D. Vollrath and R.W. Davis, Nucleic Acids Res. 15:7865–7876, 1987. With permission from Oxford University Press.) 2.5 4 million 15 7 950,000 6.5 4.3 14 10 11 610,000 2.3 2 3 6 1 220,000 (A) slab of agarose gel (B) (C) (D) Purified DNA Molecules Can Be Specifically Labeled with Radioisotopes or Chemical Markers in vitro

1	The DNA polymerases that synthesize and repair DNA (discussed in Chapter 5) have become important tools in experimentally manipulating DNA. Because they synthesize sequences complementary to an existing DNA molecule, they are often used in the test tube to create exact copies of existing DNA molecules. The copies can include specially modified nucleotides (Figure 8–26). To synthesize DNA in this way, the DNA polymerase is presented with a template and a pool of nucleotide precursors that contain the modification. As long as the polymerase can use these precursors, it automatically makes new, modified molecules that match the sequence of the template. Modified DNA molecules have many uses. DNA labeled with the radioisotope 32P can be detected following gel electrophoresis by placing the gel next to a piece of photographic film (see Figure 8–25C). The 32P atoms emit β particles which expose the film, producing a visible record of every band on the gel. Alternatively, the gel can be

1	gel next to a piece of photographic film (see Figure 8–25C). The 32P atoms emit β particles which expose the film, producing a visible record of every band on the gel. Alternatively, the gel can be scanned by a detector that measures the β emissions directly. Other types of modified DNA, such as that labeled by digoxigenin (see Figure 8–26B), are useful for visualizing DNA molecules in whole cells, a topic we discuss later in this chapter.

1	Any DNA fragment can be cloned. In molecular biology, the term DNA cloning is used in two senses. It literally refers to the act of making many identical copies (typically billions) of a DNA molecule—the amplification of a particular DNA sequence. However, the term also describes the isolation of a particular stretch of DNA (often a particular gene) from the rest of the cell’s genome; the same term purifed fragment of duplex DNA DNA polymerase incorporates 32P nucleotides, resulting in a population of radiolabeled DNA molecules that contain sequences from both strands

1	Figure 8–26 Methods for labeling DNA molecules in vitro. (A) A purified DNA polymerase enzyme can incorporate radiolabeled nucleotides as it synthesizes new DNA molecules. In this way, radiolabeled versions of any DNA sequence can be prepared in the laboratory. (B) The method in (A) is also used to produce nonradioactive DNA molecules that carry a specific chemical marker that can be detected with an appropriate antibody. The base on the nucleoside triphosphate shown is an analog of thymine, in which the methyl group on T has been replaced by a spacer arm linked to the plant steroid digoxigenin. An anti-digoxigenin antibody coupled to a visible marker such as a fluorescent dye is then used to visualize the DNA. Other chemical labels, such as biotin, can be attached to nucleotides and used in the same way. The only requirements are that the modified nucleotides properly base-pair and appear “normal” to the DNA polymerase.

1	circular, is used because this isolation is usually accomplished by making many identical copies of only the DNA of interest. We note that elsewhere in the book, cloning, particularly when used in the context of developmental biology, can also refer to the generation of many genetically identical cells starting from a single cell or even to the generation of genetically identical organisms (see, for example, Figure 7–2). In all cases, cloning refers to the act of making many identical copies, and in this section, we use the term to refer to methods designed to generate many identical copies of a defined segment of nucleic acid.

1	DNA cloning can be accomplished in several ways. One of the simplest involves inserting a particular fragment of DNA into the purified DNA genome of a self-replicating genetic element—usually a plasmid. The plasmid vectors most widely used for gene cloning are small, circular molecules of double-stranded DNA derived from plasmids that occur naturally in bacterial cells. They generally account for only a minor fraction of the total host bacterial cell DNA, but owing to their small size, they can easily be separated from the much larger chromosomal DNA molecules, which precipitate as a pellet upon centrifugation. For use as cloning vectors, the purified plasmid DNA circles are first cut with a restriction nuclease to create linear DNA molecules. The DNA to be cloned is added to the cut plasmid and then covalently joined using the enzyme DNA ligase (Figure 8–27 and Figure 8–28). As discussed in Chapter 5, this enzyme is used by the cell to stitch together the Okazaki fragments produced

1	and then covalently joined using the enzyme DNA ligase (Figure 8–27 and Figure 8–28). As discussed in Chapter 5, this enzyme is used by the cell to stitch together the Okazaki fragments produced during DNA replication. The recombinant DNA circle is introduced back into bacterial cells that have been made transiently permeable to DNA. As the cells grow and divide, doubling in number every 30 minutes, the recombinant plasmids also replicate to produce an

1	Figure 8–27 The insertion of a DNA fragment into a bacterial plasmid with the enzyme DNA ligase. The plasmid is cut open with a restriction nuclease (in this case, one that produces staggered ends) and is mixed with the DNA fragment to be cloned (which has been prepared with the same restriction nuclease). DNA ligase and ATP are added. The staggered ends base-pair, and DNA ligase seals the nicks in the DNA backbone, producing a complete recombinant DNA molecule. In the accompanying micrographs, the inserted DNA is colored red. (Micrographs courtesy of Huntington Potter and David Dressler.) Figure 8–28 DNA ligase can join together any two DNA fragments in vitro to produce recombinant DNA molecules. ATP provides the energy necessary to reseal the sugar-phosphate backbone of DNA (see Figure 5–12).

1	DNA ligase can readily join two DNA fragments produced by the same restriction nuclease, in this case EcoRI. Note that the staggered ends produced by this enzyme enable the ends of the two fragments to base-pair correctly with each other, greatly facilitating their rejoining. DNA ligase can also be used to join DNA fragments produced by different restriction nucleases—for example, EcoRI and HaeIII. In this case, before the fragments undergo ligation, DNA polymerase plus a mixture of deoxyribonucleoside triphosphates (dNTPs) are used to fill in the staggered cut produced by EcoRI. Each DNA fragment shown in the figure is oriented so that its 5ʹ ends are at the left end of the upper strand and the right end of the lower strand, as indicated.

1	cell culture produces hundreds of millions of new bacteria enormous number of copies of DNA circles containing the foreign DNA (Figure 8–29). Once the cells are lysed and the plasmid DNA isolated, the cloned DNA fragment can be readily recovered by cutting it out of the plasmid DNA with the same restriction nuclease that was used to insert it, and then separating it from the plasmid DNA by gel electrophoresis. Together, these steps allow the amplification and purification of any segment of DNA from the genome of any organism.

1	A particularly useful plasmid vector is based on the naturally occurring F plasmid of E. coli. Unlike smaller bacterial plasmids, the F plasmid—and its engineered derivative, the bacterial artificial chromosome (BAC)—is present in only one or two copies per E. coli cell. The fact that BACs are kept in such low numbers means that they can stably maintain very long DNA sequences, up to 1 million nucleotide pairs in length. With only a few BACs present per bacterium, it is less likely that the cloned DNA fragments will become scrambled by recombination with sequences carried on other copies of the plasmid. Because of their stability, ability to accept large DNA inserts, and ease of handling, BACs are now the preferred vector for handling large fragments of foreign DNA. As we will see below, BACs were instrumental in determining the complete nucleotide sequence of the human genome.

1	Often it is useful to break up a genome into much smaller fragments and clone every fragment, separately, using a plasmid vector. This approach is useful because it allows scientists to work with easily managed, discrete pieces of a genome instead of whole, unwieldy chromosomes.

1	This strategy involves cleaving genomic DNA into small pieces using a restriction nuclease (or, in some cases, by mechanically shearing the DNA) and ligating the entire collection of DNA fragments into plasmid vectors, using conditions that favor the insertion of a single DNA fragment into each plasmid molecule. These recombinant plasmids are then introduced into E. coli at a concentration that ensures that no more than one plasmid molecule is taken up by each bacterium. The collection of cloned plasmid molecules is known as a DNA library. Because the DNA fragments were derived directly from the chromosomal DNA of the organism of interest, the resulting collection—called a genomic library—will represent the entire genome of that organism (Figure 8–30), spread out over tens of thousands of individual bacterial colonies.

1	An alternative strategy, one that enriches for protein-coding genes, is to begin the cloning process by selecting only those DNA sequences that are transcribed into mRNA and thus correspond to protein-encoding genes. This is done by extracting the mRNA from cells and then making a DNA copy of each mRNA Figure 8–30 Human genomic libraries containing DNA fragments that represent the whole human genome can be constructed using restriction nucleases and DNA ligase. Such a genomic library consists of a set of bacteria, each carrying a different fragment of human DNA. For simplicity, only the colored DNA fragments are shown in the library; in reality, all of the different gray fragments will also be represented.

1	Figure 8–29 A DNA fragment can be replicated inside a bacterial cell. To clone a particular fragment of DNA, it is first inserted into a plasmid vector, as shown in Figure 8–27. The resulting recombinant plasmid DNA is then introduced into a bacterium, where it is replicated many millions of times as the bacterium multiplies. For simplicity, the genome of the bacterial cell is not shown.

1	millions of genomic DNA fragments molecule present—a so-called complementary DNA, or cDNA. The copying reaction is catalyzed by the reverse transcriptase enzyme of retroviruses, which synthesizes a complementary DNA chain on an RNA template. The single-stranded cDNA molecules synthesized by the reverse transcriptase are converted by DNA polymerase into double-stranded cDNA molecules, and these molecules are inserted into a plasmid or virus vector and cloned (Figure 8–31). Each clone obtained in this way is called a cDNA clone, and the entire collection of clones derived from one mRNA preparation constitutes a cDNA library.

1	Figure 8–32 illustrates some important differences between genomic DNA clones and cDNA clones. Genomic clones represent a random sample of all of the DNA sequences in an organism—both coding and noncoding—and, with very rare exceptions, are the same regardless of the cell type used to prepare them. By contrast, cDNA clones contain only those regions of the genome that have been transcribed into mRNA. Because the cells of different tissues produce distinct sets of mRNA molecules, a distinct cDNA library is obtained for each type of cell used to prepare the library. tissue (e.g., brain) double-stranded cDNA copy of original mRNA

1	Figure 8–31 The synthesis of cDNA. Total mRNA is extracted from a particular tissue, and the enzyme reverse transcriptase (see Figure 5–62) is used to produce DNA copies (cDNA) of the mRNA molecules. For simplicity, the copying of just one of these mRNAs into cDNA is illustrated. A short oligonucleotide complementary to the poly-A tail at the 3ʹ end of the mRNA (discussed in Chapter 6) is first hybridized to the RNA to act as a primer for the reverse transcriptase, which then copies the RNA into a complementary DNA chain, thereby forming a DNA–RNA hybrid helix. Treating the DNA–RNA hybrid with a specialized nuclease (RNAse H) that attacks only the RNA produces nicks and gaps in the RNA strand. DNA polymerase then copies the remaining single-stranded cDNA into double-stranded cDNA. Because DNA polymerase can synthesize through the bound RNA molecules, the RNA fragment that is base-paired to the 3ʹ end of the first DNA strand usually acts as the primer for the second strand synthesis,

1	DNA polymerase can synthesize through the bound RNA molecules, the RNA fragment that is base-paired to the 3ʹ end of the first DNA strand usually acts as the primer for the second strand synthesis, as shown. Any remaining RNA is eventually degraded during subsequent cloning steps. As a result, the nucleotide sequences at the extreme 5ʹ ends of the original mRNA molecules are often absent from cDNA libraries.

1	Genomic libraries are especially useful in determining the nucleotide sequences of a whole genome. For example, to determine the nucleotide sequence of the human genome, it was broken up into roughly 100,000-nucleotide-pair pieces, each of which was inserted into a BAC plasmid and amplified in E. coli. The resulting genomic library consisted of tens of thousands of bacterial colonies, each containing a different human DNA insert. The nucleotide sequence of each insert was determined separately and the sequence of the entire genome was stitched together from the pieces. The most important advantage of cDNA clones, over genomic clones, is that they contain the uninterrupted coding sequence of a gene. When the aim of the cloning, for example, is to produce the protein in large quantities by expressing the cloned gene in a bacterial or yeast cell, it is more preferable to start with cDNA.

1	Genomic and cDNA libraries are inexhaustible resources, which are widely shared among investigators. Today, many such libraries are also available from commercial sources. Because the identity of each insert in a library is often known (through sequencing the insert), it is often possible to order a particular region of a chromosome (or, in the case of cDNA, a complete, intron-less protein-coding gene) and have it delivered by mail. Cloning DNA by using bacteria revolutionized the study of genomes and is still in wide use today. However, there is an even simpler way to clone DNA, one that can be carried out entirely in vitro. We discuss this approach, called the polymerase chain reaction, below. However, first we need to review a fundamental, far-reaching property of DNA and RNA called hybridization. Figure 8–32 The differences between cDNA clones and genomic DNA clones derived from the same region of DNA.

1	Figure 8–32 The differences between cDNA clones and genomic DNA clones derived from the same region of DNA. In this example, gene A is infrequently transcribed, whereas gene B is frequently transcribed, and both genes contain introns (orange). In the genomic DNA library, both the introns and the nontranscribed DNA (gray) are included in the clones, and most clones contain, at most, only part of the coding sequence of a gene (red). In the cDNA clones, the intron sequences (yellow) have been removed by RNA splicing during the formation of the mRNA (blue), and a continuous coding sequence is therefore present in each clone. Because gene B is transcribed more frequently than gene A in the cells from which the cDNA library was made, it is represented much more frequently than A in the cDNA library. In contrast, A and B are represented equally in the genomic DNA library. Hybridization Provides a Powerful, But Simple Way to Detect Specific Nucleotide Sequences

1	Hybridization Provides a Powerful, But Simple Way to Detect Specific Nucleotide Sequences Under normal conditions, the two strands of a DNA double helix are held together by hydrogen bonds between the complementary base pairs (see Figure 4–3). But these relatively weak, noncovalent bonds can be fairly easily broken. Such DNA denaturation will release the two strands from each other, but does not break the covalent bonds that link together the nucleotides within each strand. Perhaps the simplest way to achieve this separation involves heating the DNA to around 90°C. When the conditions are reversed—by slowly lowering the temperature— the complementary strands will readily come back together to re-form a double helix. This hybridization, or DNA renaturation, is driven by the re-formation of the hydrogen bonds between complementary base pairs (Figure 8–33). We saw in Chapter 5 that DNA hybridization underlies the crucial process of homologous recombination (see Figure 5–47).

1	This fundamental capacity of a single-stranded nucleic acid molecule, either DNA or RNA, to form a double helix with a single-stranded molecule of a complementary sequence provides a powerful and sensitive technique for detecting specific nucleotide sequences. Today, one simply designs a short, single-stranded DNA molecule (often called a DNA probe) that is complementary to the nucleotide sequence of interest. Because the nucleotide sequences of so many genomes are known—and are stored in publicly accessible databases—designing a probe to hybridize anywhere in a genome is straightforward. Probes are single-stranded, typically 30 nucleotides in length, and are usually synthesized chemically by a commercial service for pennies per nucleotide. A DNA sequence of 30 nucleotides will occur by chance only once every 1 × 1018 nucleotides (430); so, even in the human genome of 3 × 109 nucleotide pairs, a DNA probe designed to match a particular 30-nucleotide sequence will be highly unlikely to

1	only once every 1 × 1018 nucleotides (430); so, even in the human genome of 3 × 109 nucleotide pairs, a DNA probe designed to match a particular 30-nucleotide sequence will be highly unlikely to hybridize—by chance— anywhere else on the genome. This, of course, presumes that the sequence complementary to the probe does not occur multiple times in the genome, a condition that can be checked beforehand by scanning the genomic sequence in silico (using a computer) and designing probes that match only one spot. The hybridization conditions can be set so that even a single mismatch will prevent hybridization to “near-miss” sequences. The exquisite specificity of nucleic acid hybridization can be easily appreciated by the in situ (Latin for “in place”) hybridization experiment shown in Figure 8–34. As we will see throughout this chapter, nucleic acid

1	Figure 8–34 In situ hybridization can be used to locate genes on isolated chromosomes. Here, six different DNA probes have been used to mark the locations of their complementary nucleotide sequences on human Chromosome 5, isolated from a mitotic cell in metaphase (see Figure 4–59 and Panel 17–1, pp. 980–981). The DNA probes have been labeled with different chemical groups (see Figure 8–26B) and are detected using fluorescent antibodies specific for those groups. The chromosomal DNA has been partially denatured to allow the probes to base-pair with their complementary sequences. Both the maternal and paternal copies of Chromosome 5 are shown, aligned side by side. Each probe produces two dots on each chromosome because chromosomes undergoing mitosis have already replicated their DNA; therefore, each chromosome contains two identical DNA helices. The technique employed here is nicknamed FISH, for fluorescence in situ hybridization. (Courtesy of David C. Ward.)

1	Figure 8–33 A molecule of DNA can undergo denaturation and renaturation (hybridization). For two single-stranded molecules to hybridize, they must have complementary nucleotide sequences that allow base-pairing. In this example, the red and orange strands are complementary to each other, and the blue and green strands are complementary to each other. Although denaturation by heating is shown, DNA can also be renatured after being denatured by alkali treatment. hybridization has many uses in modern cell and molecular biology; one of the most powerful is in the cloning of DNA by the polymerase chain reaction, as we next discuss.

1	hybridization has many uses in modern cell and molecular biology; one of the most powerful is in the cloning of DNA by the polymerase chain reaction, as we next discuss. Genomic and cDNA libraries were once the only route to cloning genes and they are still used for cloning very large genes and for sequencing whole genomes. However, a powerful and versatile method for amplifying DNA, known as the polymerase chain reaction (PCR), provides a more rapid and straightforward approach to DNA cloning, particularly in organisms whose complete genome sequence is known. Today, since genome sequences are abundant, most cloning is carried out by PCR.

1	Invented in the 1980s, PCR revolutionized the way that DNA and RNA are analyzed. The technique can amplify any nucleotide sequence selectively and is performed entirely in a test tube. Eliminating the need for bacteria makes PCR convenient and rapid—billions of copies of a nucleotide can be generated in a matter of hours. Starting with an entire genome, PCR allows DNA from a specified region—selected by the experimenter—to be greatly amplified, effectively “purifying” this DNA away from the remainder of the genome, which remains unamplified. Because of its power to greatly amplify nucleic acids, PCR is remarkably sensitive: the method can be used to detect the trace amounts of DNA in a drop of blood left at a crime scene or in a few copies of a viral genome in a patient’s blood sample.

1	The success of PCR depends both on the selectivity of DNA hybridization and on the ability of DNA polymerase to copy a DNA template faithfully through repeated rounds of replication in vitro. As discussed in Chapter 5, this enzyme adds nucleotides to the 3ʹ end of a growing strand of DNA (see Figure 5–4). To copy DNA, the polymerase requires a primer—a short nucleotide sequence that provides a 3ʹ end from which synthesis can begin. For PCR, the primers are designed by the experimenter, synthesized chemically, and, by hybridizing to genomic DNA, “tell” the polymerase which part of the genome to copy. As discussed in the previous section, DNA primers (in essence, the same type of molecules as DNA probes but without a radioactive or fluorescent label) can be designed to uniquely locate any position on a genome.

1	PCR is an iterative process in which the cycle of amplification is repeated dozens of times. At the start of each cycle, the two strands of the double-stranded DNA template are separated and a different primer is annealed to each. These primers mark the right and left boundaries of the DNA to be amplified. DNA polymerase is then allowed to replicate each strand independently (Figure 8–35). In

1	Figure 8–35 A pair of primers directs the synthesis of a desired segment of DNA in a test tube. Each cycle of PCR includes three steps: (1) The double-stranded DNA is heated briefly to separate the two strands. (2) The DNA is exposed to a large excess of a pair of specific primers— designed to bracket the region of DNA to be amplified—and the sample is cooled to allow the primers to hybridize to complementary sequences in the two DNA strands. (3) This mixture is incubated with DNA polymerase and the four deoxyribonucleoside triphosphates so that DNA can be synthesized, starting from the two primers. To amplify the DNA, the cycle is repeated many times by reheating the sample to separate the newly synthesized DNA strands (see Figure 8–36).

1	The technique depends on the use of a special DNA polymerase isolated from a thermophilic bacterium; this polymerase is stable at much higher temperatures than eukaryotic DNA polymerases, so it is not denatured by the heat treatment shown in step 1. The enzyme therefore does not have to be added again after each cycle. products of frst cycle

1	Figure 8–36 PCR uses repeated rounds of strand separation, hybridization, and synthesis to amplify DNA. As the procedure outlined in Figure 8–35 is repeated, all the newly synthesized fragments serve as templates in their turn. Because the polymerase and the primers remain in the sample after the first cycle, PCR involves simply heating and then cooling the same sample, in the same test tube, again and again. Each cycle doubles the amount of DNA synthesized in the previous cycle, so that within a few cycles, the predominant DNA is identical to the sequence bracketed by and including the two primers in the original template. In the example illustrated here, three cycles of reaction produce 16 DNA chains, 8 of which (boxed in yellow) correspond exactly to one or the other strand of the original bracketed sequence. After four more cycles, 240 of the 256 DNA chains will correspond exactly to the original sequence, and after several more cycles, essentially all of the DNA strands will be

1	bracketed sequence. After four more cycles, 240 of the 256 DNA chains will correspond exactly to the original sequence, and after several more cycles, essentially all of the DNA strands will be this length. Typically, 20–30 cycles are carried out to effectively clone a region of DNA starting from genomic DNA; the rest of the genome remains unamplified, and its concentration is therefore negligible compared with that of the amplified region (Movie 8.2).

1	subsequent cycles, all the newly synthesized DNA molecules produced by the polymerase serve as templates for the next round of replication (Figure 8–36). Through this iterative amplification process, many copies of the original sequence can be made—billions after about 20 to 30 cycles. PCR is now the method of choice for cloning relatively short DNA fragments (say, under 10,000 nucleotide pairs). Each cycle takes only about five minutes, and automation of the whole procedure enables cell-free cloning of a DNA fragment in a few hours. The original template for PCR can be either DNA or RNA, so this method can be used to obtain either a genomic clone (complete with introns and exons) or a cDNA copy of an mRNA (Figure 8–37).

1	The PCR method is extraordinarily sensitive; it can detect a single DNA molecule in a sample if at least part of the sequence of that molecule is known. Trace amounts of RNA can be analyzed in the same way by first transcribing them into DNA with reverse transcriptase. For these reasons, PCR is frequently employed for uses that go beyond simple cloning. For example, it can be used to detect invading pathogens at very early stages of infection. In this case, short sequences complementary to a segment of the infectious agent’s genome are used as primers and following many cycles of amplification, even a few copies of an invading bacterial or viral genome in a patient’s sample can be detected (Figure 8–38). For many infections, PCR has replaced the use of antibodies against microbial molecules to isolate total DNA isolate total mRNA DNA segment to be cloned mRNA sequence to be cloned ADD FIRST PRIMER, REVERSE TRANSCRIPTASE, AND DEOXYRIBONUCLEOSIDE TRIPHOSPHATES SEPARATE STRANDS AND ADD

1	to isolate total DNA isolate total mRNA DNA segment to be cloned mRNA sequence to be cloned ADD FIRST PRIMER, REVERSE TRANSCRIPTASE, AND DEOXYRIBONUCLEOSIDE TRIPHOSPHATES SEPARATE STRANDS AND ADD SECOND PRIMER PCR AMPLIFICATION WITH BOTH PRIMERS PRESENTPCR AMPLIFICATION mRNA DNA SEPARATE STRANDS AND ADD PRIMERS genomic clones (A) cDNA clones (B) chromosomal DNA detect the presence of the invader. It is also used to verify the authenticity of a food source—for example, whether a sample of beef actually came from a cow.

1	Finally, PCR is now widely used in forensics. The method’s extreme sensitivity allows forensic investigators to isolate DNA from minute traces of human blood or other tissue to obtain a DNA fingerprint of the person who left the sample behind. rare HIV particle control, usingin plasma of blood frominfected person Figure 8–38 PCR can be used to detect the presence of a viral genome in a sample of blood. Because of its ability to amplify enormously the signal from a single molecule of nucleic acid, PCR is an extraordinarily sensitive method for detecting trace amounts of virus in a sample of blood or tissue, without the need to purify the virus. For HIV, the virus that causes AIDS, the genome is a single-stranded molecule of RNA, as illustrated here. In addition to HIV, many other viruses that infect humans are now detected in this way. Figure 8–37 PCR can be used to obtain either genomic or cDNA clones.

1	Figure 8–37 PCR can be used to obtain either genomic or cDNA clones. (A) To use PCR to clone a segment of chromosomal DNA, total genomic DNA is first purified from cells. PCR primers that flank the stretch of DNA to be cloned are added, and many cycles of PCR are completed (see Figure 8–36). Because only the DNA between (and including) the primers is amplified, PCR provides a way to obtain selectively any short stretch of chromosomal DNA in an effectively pure form. (B) To use PCR to obtain a cDNA clone of a gene, total mRNA is first purified from cells. The first primer is added to the population of mRNAs, and reverse transcriptase is used to make a DNA strand complementary to the specific RNA sequence of interest. The second primer is then added, and the DNA molecule is amplified through many cycles of PCR.

1	Figure 8–39 PCR is used in forensic science to distinguish one individual from another. The DNA sequences analyzed are short tandem repeats (STRs) composed of sequences such as CACACA… or GTGTGT… STRs are found in various positions (loci) in the human genome. The number of repeats in each STR locus is highly variable in the population, ranging from 4 to 40 in different individuals. Because of the variability in these sequences, individuals will usually inherit a different number of repeats at each STR locus from their mother and from their father; two unrelated individuals, therefore, rarely contain the same pair of sequences at a given STR locus. (A) PCR using primers that recognize unique sequences on either side of one particular STR locus produces a pair of bands of amplified DNA from each individual, one band representing the maternal STR variant and the other representing the paternal STR variant. The length of the amplified DNA, and thus its position after gel electrophoresis,

1	each individual, one band representing the maternal STR variant and the other representing the paternal STR variant. The length of the amplified DNA, and thus its position after gel electrophoresis, will depend on the exact number of repeats at the locus. (B) In the schematic example shown here, the same three STR loci are analyzed in samples from three suspects (individuals A, B, and C), producing six bands for each individual. Although different people can have several bands in common, the overall pattern is quite distinctive for each person. The band pattern can therefore serve as a DNA fingerprint to identify an individual nearly uniquely. The fourth lane (F) contains the products of the same PCR amplifications carried out on a hypothetical forensic DNA sample, which could have been obtained from a single hair or a tiny spot of blood left at a crime scene.

1	The more loci that are examined, the more confident one can be about the results. When examining the variability at 5–10 different STR loci, the odds that two random individuals would share the same fingerprint by chance are approximately one in 10 billion. In the case shown here, individuals A and C can be eliminated from inquiries, while B is a clear suspect. A similar approach is used routinely in paternity testing. With the possible exception of identical twins, the genome of each human differs in DNA sequence from that of every other person on Earth. Using primer pairs targeted at genome sequences that are known to be highly variable in the human population, PCR makes it possible to generate a distinctive DNA fingerprint for any individual (Figure 8–39). Such forensic analyses can be used not only to help identify those who have done wrong, but also—equally important—to exonerate those who have been wrongfully accused.

1	Most current methods of manipulating DNA, RNA, and proteins rely on prior knowledge of the nucleotide sequence of the genome of interest. But how were these sequences determined in the first place? And how are new DNA and RNA molecules sequenced today? In the late 1970s, researchers developed several strategies for determining, simply and quickly, the nucleotide sequence of any purified DNA fragment. The one that became the most widely used is called dideoxy sequencing or Sanger sequencing (Panel 8–1). This method was used to determine the nucleotide sequence of many genomes, including those of E. coli, fruit flies, nematode worms, mice, and humans. Today, cheaper and faster methods are routinely used to sequence DNA, and even more efficient strategies are being developed (see Panel 8–1). The original “reference” sequence of the human genome, completed in 2003, cost over $1 billion and required many scientists from around the world working together for 13 years. The enormous progress

1	The original “reference” sequence of the human genome, completed in 2003, cost over $1 billion and required many scientists from around the world working together for 13 years. The enormous progress made in the past decade makes it possible for a single person to complete the sequence of an individual human genome in less than a day.

1	The methods summarized in Panel 8–1 for rapidly sequencing DNA can also be applied to RNA. Although methods are being developed to sequence RNA directly, it is most commonly carried out by converting the RNA to complementary DNA (using reverse transcriptase) and using one of the methods described for DNA sequencing. It is important to keep in mind that although genomes remain the same from cell to cell and from tissue to tissue, the RNA produced from the genome can vary enormously. We will see later in this chapter that sequencing the entire repertoire of RNA from a cell or tissue (known as deep RNA sequencing, or RNA-seq) is a powerful way to understand how the information present in the genome is used by different cells under different circumstances. In the next section, we shall see how RNA-seq has also become a valuable tool for annotating genomes. To Be Useful, Genome Sequences Must Be Annotated

1	To Be Useful, Genome Sequences Must Be Annotated Long strings of nucleotides, at first glance, reveal nothing about how this genetic information directs the development of a living organism—or even what types of DNA, protein, and RNA molecules are produced by a genome. The process of genome annotation attempts to mark out all the genes (both protein-coding and noncoding) in a genome and ascribe a role to each. It also seeks to understand more subtle types of genome information, such as the cis-regulatory sequences that specify the time and place that a given gene is expressed and whether its mRNA undergoes alternative splicing to produce different protein isotypes. Clearly, this is a daunting task, and we are far short of completing it for any form of life, even the simplest bacterium. For many organisms, we know the approximate number of genes, and, for very simple organisms, we understand the functions of about half their genes.

1	In this section, we discuss broadly how genes are identified in genome sequences and what clues we can discern about their roles from simply inspecting their sequences. Later in the chapter, we turn to the more difficult problem of experimentally determining gene function. How does one begin to make sense of a genome sequence? The first step is usually to translate in silico the entire genome into protein. There are six different reading frames for any piece of double-stranded DNA (three on each strand). We saw in Chapter 6 that a random sequence of nucleotides, read in frame, will PANeL 8–1: DNA Sequencing Methods

1	OOCH25˜OH3˜3˜3˜OH allows strand extension at 3˜ end normal deoxyribonucleoside triphosphate (dNTP) OOCH25˜3˜ H prevents strand extension at 3˜end chain-terminating dideoxyribonucleoside triphosphate (ddNTP) basebase PPP PPP ATGTCAGTCCAG 3˜RESULT TACAGTCAGGTC 5˜GCAT ATG GCAT ATGTCAG GCAT ATGTCAGTCCAG G sequence of DNA primer sequence read from gel 3˜ CGTATACAGTCAGGTC 5˜3˜ CGTATACAGTCAGGTC 5˜5˜ GCAT 3˜GCAT A GCAT ATGTCA GCAT ATGTCAGTCCA GCAT AT GCAT ATGT GCAT ATGTCAGT GCAT ATGTC GCAT ATGTCAGTC GCAT ATGTCAGTCC A T C G A C C T G A C T G T A A T C G 3˜5˜ADD LABELED DNA PRIMER single-stranded DNA fragment to be sequenced T T T TTT T T T T A A A A AA AA GG GGG GG CC C C C CC C CADD DNA POLYMERASE AND DIVIDE INTO 4 SEPARATE TUBES ADD EXCESS AMOUNTS OF NORMAL dNTPs sequence of original DNA strand ADD SMALL AMOUNT OF ONE CHAIN-TERMINATING ddNTP TO EACH TUBE TT C T A T AG T G T C A CC T AAATA G C TT GG C G T AATC A T GG T(B) (A) GCATATG GCATA GCATATGT GCATAT GCATATGTC mixture of DNA

1	DNA strand ADD SMALL AMOUNT OF ONE CHAIN-TERMINATING ddNTP TO EACH TUBE TT C T A T AG T G T C A CC T AAATA G C TT GG C G T AATC A T GG T(B) (A) GCATATG GCATA GCATATGT GCATAT GCATATGTC mixture of DNA products, each containing a chain-terminating ddNTP labeled with a different fuorescent marker PRODUCTS LOADED ONTO CAPILLARY GEL electrophoresis size-separated products are read in sequence Dideoxy sequencing, or Sanger sequencing (named after the scientist who invented it), uses DNA polymerase, along with special chain-terminating nucleotides called dideoxyribonucleoside triphosphates (left), to make partial copies of the DNA fragment to be sequenced. These ddNTPs are derivatives of the normal deoxyribonucleoside triphosphates that lack the 3˜ hydroxyl group. When incorporated into a growing DNA strand, they block further elongation of that strand. DNA SEQUENCING MANUAL DIDEOXY SEQUENCING AUTOMATED DIDEOXY SEQUENCING To determine the complete sequence of a single-stranded fragment of DNA

1	DNA strand, they block further elongation of that strand. DNA SEQUENCING MANUAL DIDEOXY SEQUENCING AUTOMATED DIDEOXY SEQUENCING To determine the complete sequence of a single-stranded fragment of DNA (gray), the DNA is frst hybridized with a short DNA primer (orange) that is labeled with a fuorescent dye or radioisotope. DNA polymerase and an excess of all four normal deoxyribonucleoside triphosphates (blue A, C, G, or T) are added to the primed DNA, which is then divided into four reaction tubes. Each of these tubes receives a small amount of a single chain-terminating dideoxyribonucleoside triphosphate (red A, C, G, or T). Because these will be incorporated only occasionally, each reaction produces a set of DNA copies that terminate at different points in the sequence. The products of these four reactions are separated by electrophoresis in four parallel lanes of a polyacrylamide gel (labeled here A, T, C, and G). In each lane, the bands represent fragments that have terminated at a

1	four reactions are separated by electrophoresis in four parallel lanes of a polyacrylamide gel (labeled here A, T, C, and G). In each lane, the bands represent fragments that have terminated at a given nucleotide but at different positions in the DNA. By reading off the bands in order, starting at the bottom of the gel and reading across all lanes, the DNA sequence of the newly synthesized strand can be determined (see Figure 8–25C). The sequence, which is given in the green arrow to the right of the gel, is complementary to the sequence of the original gray single-stranded DNA. Fully automated machines can run dideoxy sequencing reactions. (A) The automated method uses an excess amount of normal dNTPs plus a mixture of four different chain-terminating ddNTPs, each of which is labeled with a fuorescent tag of a different color. The reaction products are loaded onto a long, thin capillary gel and separated by electrophoresis. A camera (not shown) reads the color of each band as it

1	with a fuorescent tag of a different color. The reaction products are loaded onto a long, thin capillary gel and separated by electrophoresis. A camera (not shown) reads the color of each band as it moves through the gel and feeds the data to a computer that assembles the sequence. (B) A tiny part of the data from such an automated sequencing run. Each colored peak represents a nucleotide in the DNA sequence. 5˜ GCAT 3˜ CGTA 479SEQUENCING WHOLE GENOMES Shotgun sequencing: To determine the nucleotide sequence of a whole genome, the genomic DNA is frst fragmented into small pieces and a genomic library is constructed, typically using plasmids and bacteria (see Figure 8–30). In shotgun sequencing, the nucleotide sequence of tens of thousands of individual clones is determined; the full genome sequence is then reconstructed by stitching together (in silico) the nucleotide sequence of each clone, using the overlaps between clones as a guide. The shotgun method works well for small

1	genome sequence is then reconstructed by stitching together (in silico) the nucleotide sequence of each clone, using the overlaps between clones as a guide. The shotgun method works well for small genomes (such as those of viruses and bacteria) that lack repetitive DNA. SECOND-GENERATION SEQUENCING TECHNOLOGIES The dideoxy method made it possible to sequence the genomes of humans and most of the other organisms discussed in this book. But newer methods, developed since 2005, have made genome sequencing even more rapid—and very much cheaper. With these so-called second-generation sequencing methods, the cost of sequencing DNA has decreased dramatically. Not surprisingly, the number of genomes that have been sequenced has increased enormously. These rapid methods allow multiple genomes to be sequenced in parallel in a matter of weeks, enabling investigators to examine thousands of individual human genomes, catalog the variation in nucleotide sequences from people around the world, and

1	be sequenced in parallel in a matter of weeks, enabling investigators to examine thousands of individual human genomes, catalog the variation in nucleotide sequences from people around the world, and uncover the mutations that increase the risk of various diseases, from cancer to autism. These methods have also made it possible to determine the genome sequence of extinct species, including Neanderthal man and the wooly mammoth. By sequencing genomes from many closely related species, they have also made it possible to understand the molecular basis of key evolutionary events in the tree of life, such as the “inventions” of multicellularity, vision, and language. The ability to rapidly sequence DNA has had major impacts on all branches of biology and medicine; it is almost impossible to imagine where we would be without it. BAC clones: Most plant and animal genomes are large (often over 109 nucleotide pairs) and contain extensive amounts of repetitive DNA spread throughout the genome.

1	where we would be without it. BAC clones: Most plant and animal genomes are large (often over 109 nucleotide pairs) and contain extensive amounts of repetitive DNA spread throughout the genome. Because a nucleotide sequence of a fragment of repetitive DNA will “overlap” every instance of the repeated DNA, it is diffcult, if not impossible, to assemble the fragments into a unique order solely by the shotgun method. To circumvent this problem, the human genome was frst broken down into very large DNA fragments (each approximately 100,000 nucleotide pairs) and cloned into BACs (see p. 469). The order of the BACs along a chromosome was determined by comparing the pattern of restriction enzyme cleavage sites in a given BAC clone with that of the whole genome. In this way, a given BAC clone can be mapped, say, to the left arm of human Chromosome 3. Once a collection of BAC clones was Thousands of genomes from individual humans have now been sequenced and it is not necessary to painstakingly

1	be mapped, say, to the left arm of human Chromosome 3. Once a collection of BAC clones was Thousands of genomes from individual humans have now been sequenced and it is not necessary to painstakingly reconstruct the order of DNA sequence “reads” each time; they are simply assembled using the order determined from the original human genome sequencing project. For this reason, resequencing, the term applied when the genome of a species is sequenced again (even though it may be from a different individual), is far easier than the original sequencing. sequence one strand of fragments GTTCAGCATTG------GCCATTAGTTCA---GCCATTAGTTCAGCATTG---original sequence reconstructed based on sequence overlap sequences of two fragments AA D B BA B C E C restriction pattern for individual BAC clones restriction map of one segment of human genome cleavage sites for restriction nucleases A, B, C, D, and E obtained that spanned the entire genome, each individual BAC was sequenced by the shotgun method. At the

1	map of one segment of human genome cleavage sites for restriction nucleases A, B, C, D, and E obtained that spanned the entire genome, each individual BAC was sequenced by the shotgun method. At the end, the sequences of all the BAC inserts were stitched together using the knowledge of the position of each BAC insert in the human genome. In all, approximately 30,000 BAC clones were sequenced to complete the human genome. random fragmentation multiple copies of genome 479

1	ILLUMINA® SEQUENCING Several second-generation sequencing methods are now in wide use, and we will discuss two of the most common. Both rely on the construction of libraries of DNA fragments that represent—in toto—the DNA of the genome. Instead of using bacterial cells to generate these libraries, as we saw in Figure 8–30), they are made using PCR amplifcation of billions of DNA fragments, each attached to a solid support. The amplifcation is carried out so that the PCR-generated copies, instead of foating away in solution, remain bound in proximity to the original DNA fragment. This process generates clusters of DNA fragments, where each cluster contains about 1000 identical copies of a small bit of the genome. These clusters—a billion of which can ft in a single slide or plate—are then sequenced at the same time; that is, in parallel. One method, known as Illumina sequencing, is based on the dideoxy method described above, but it incorporates several innovations. Here, each

1	then sequenced at the same time; that is, in parallel. One method, known as Illumina sequencing, is based on the dideoxy method described above, but it incorporates several innovations. Here, each nucleotide is attached to a removable fuorescent molecule (a different color for each of the four bases) as well as a special chain-terminating chemical adduct: instead of a 3’-OH group, as in conventional dideoxy sequencing, the nucleotides carry a chemical group that blocks elongation by DNA polymerase but which can be removed chemically. Sequencing is then carried out as follows: the four fuorescently labeled nucleotides along with DNA polymerase are added to billions of DNA clusters immobilized on a slide. Only the appropriate nucleotide (that is complementary to the next nucleotide in the template) is covalently incorporated at each cluster; the unincorporated nucleotides are washed away, and a high-resolution digital camera takes an image that registers which of the four nucleotides

1	template) is covalently incorporated at each cluster; the unincorporated nucleotides are washed away, and a high-resolution digital camera takes an image that registers which of the four nucleotides was added to the chain at each cluster. The fuorescent label and the 3˜-OH blocking group are then removed enzymatically, washed away, and the process is repeated many times. In this way, billions of sequencing reactions are carried out simultaneously. By keeping track of the color changes occuring at each cluster, the DNA sequence represented by each spot can be read. Although each individual sequence read is relatively short (approximately 200 nucleotides), the billions that are carried out simultaneously can produce several human genomes worth of sequence in about a day. Principle behind Illumina sequencing. This reaction is carried out stepwise, on billions of DNA clusters at once. The method relies on a color digital camera that rapidly scans all the DNA clusters after each round of

1	sequencing. This reaction is carried out stepwise, on billions of DNA clusters at once. The method relies on a color digital camera that rapidly scans all the DNA clusters after each round of modifed nucleotide incorporation. The DNA sequence of each cluster is then determined by the sequence of color changes it undergoes as the elongation reaction proceeds stepwise. Each round of modifed nucleotide incorporation, image acquisition, and removal of the 3˜block and the fuorescent group takes less than an hour. Each cluster on the slide contains many copies of different, random bits of a genome; in preparing the clusters, a DNA sequence (specifed by the experimenter) is joined to each copy in every cluster, and a primer complementary to this sequence is used to begin the elongation reaction by DNA polymerase. 100 µm A slide showing individual clusters of PCR-generated DNA molecules. Each cluster carries about 1000 identical DNA molecules; the four colors are produced by incorporation of

1	by DNA polymerase. 100 µm A slide showing individual clusters of PCR-generated DNA molecules. Each cluster carries about 1000 identical DNA molecules; the four colors are produced by incorporation of C, G, A, or T, each of which has a different color fuorophore. The image has been taken just after a fuorescent nucleotide has been incorporated into each growing DNA chain. (From Illumina Sequencing Overview, 2013.) PPP PP fuor 3˜ block 5˜3˜ block incorporation into growing chain by DNA polymerase P 5˜free 3˜ end P DNA template OH fuor removed block removed next cycle no fuor photo taken 480

1	ION TORRENT™ SEQUENCING THE FUTURE OF DNA SEQUENCING Another widely used strategy for rapid DNA sequencing is called the ion torrent method. Here, a genome is fragmented, and the individual fragments are attached to microscopic beads. Using PCR, each DNA fragment is then amplifed so that copies of it eventually coat the bead to which it was initially attached. This process produces a library of billions of individual beads, each covered with identical copies of a particular DNA fragment. Like eggs in a carton, the beads are placed into individual wells on an array that can hold a billion beads in a square inch. Beginning with a primer, DNA synthesis is then initiated on each bead. A hydrogen ion (H+) is released (along with pyrophosphate) each time a nucleotide is incorporated into a growing DNA chain (see Figure 5–3), and the ion torrent method is based on this simple fact. Each of the four nucleotides is washed in, one at a time, over the array of beads; when a nucleotide is

1	a growing DNA chain (see Figure 5–3), and the ion torrent method is based on this simple fact. Each of the four nucleotides is washed in, one at a time, over the array of beads; when a nucleotide is incorporated in the DNA of a given bead, the release of an H+ ion changes the pH, which is registered by a semiconductor chip placed beneath the array of wells. In this way, the DNA sequence on a given bead can be read from the pattern of pH changes observed as nucleotides are washed over them. Like a high-resolution sensor in a digital camera, the ion torrent semiconductor chip can register enormous amounts of information and can thus keep track of billions of parallel sequencing reactions. Using this technology it is currently possible, using a single chip, to determine the nucleotide sequences of several human genomes in just a few hours. PPP PP 5˜DNA template A OH OH H+ pH change detected volts 1 µm voltage-sensing chip well bead coated with DNA template A DNA sequencing by the ion

1	of several human genomes in just a few hours. PPP PP 5˜DNA template A OH OH H+ pH change detected volts 1 µm voltage-sensing chip well bead coated with DNA template A DNA sequencing by the ion torrent method. Beads, each coated with a DNA molecule that has been amplifed many times, are placed in wells along with primers and DNA polymerase. As nucleotides are sequentially washed over the beads, those incorporated by the polymerase cause a pH change. In the example shown, an A is incorporated; thus, the template must have a T in this position. As the four nucleotides are sequentially washed over the beads, the sequence of the DNA on each bead can be “read” by the pattern of pH fuctuations. Billions of beads are monitored at once by a voltage-sensitive semiconductor chip placed below the array of beads. Even newer, potentially faster, methods of sequencing DNA are being developed. Some of these “third-generation” technologies bypass the DNA amplifcation steps altogether and determine the

1	of beads. Even newer, potentially faster, methods of sequencing DNA are being developed. Some of these “third-generation” technologies bypass the DNA amplifcation steps altogether and determine the sequence of single molecules of DNA. In one technique, a DNA molecule is pushed through a tiny channel, like a thread through the eye of a needle. As the DNA molecule moves through the pore, it generates electrical currents that depend on its sequence of nucleotides; the pattern of currents can then be used to deduce the nucleotide sequence. Other methods visualize single DNA molecules using electron or atomic force microscopy; the nucleotide sequence is read from the small differences in the “appearance” of the DNA as it is scanned. Finally, another method is based on immobilizing a single DNA polymerase molecule (with a template) and measuring the “dwell” time of each of the four nucleotides, which are labeled with different removable fuorescent dyes. Nucleotides that reside longer on the

1	polymerase molecule (with a template) and measuring the “dwell” time of each of the four nucleotides, which are labeled with different removable fuorescent dyes. Nucleotides that reside longer on the polymerase (before their dye is removed) are those incorporated by the polymerase. Although the two methods we have described in detail (Illumina and ion torrent) are now used extensively, it is likely that faster and cheaper methods will continue to be developed. 2001 2003 2005 2007 2009 2011 2013 2015 100,000,000 10,000,000 1,000,000 100,000 10,000 1000 100 cost in dollars years Shown here are the costs of sequencing a human genome, which was $100 million in 2001 and about a thousand dollars by the end of 2014. (Data from the National Human Genome Research Initiative.) 481 (A) reading direction for sequence of top DNA strand frames –3 reading direction for sequence of bottom DNA strand 500 base pairs contain a stop codon about every 20 amino acids; protein-coding regions will, in

1	direction for sequence of top DNA strand frames –3 reading direction for sequence of bottom DNA strand 500 base pairs contain a stop codon about every 20 amino acids; protein-coding regions will, in contrast, usually contain much longer stretches without stop codons (Figure 8–40). Known as open reading frames (ORFs), these usually signify bona fide protein-coding genes. This assignment is often “double-checked” by comparing the ORF amino acid sequence to the many databases of documented proteins from other species. If a match is found, even as imperfect one, it is very likely that the ORF will code for a functional protein (see Figure 8–23).

1	This strategy works very well for compact genomes, where intron sequences are rare and ORFs often extend for many hundreds of amino acids. However, in many animals and plants, the average exon size is 150–200 nucleotide pairs (see Figure 6–31) and additional information is usually required to unambiguously locate all the exons of a gene. Although it is possible to search genomes for splicing signals and other features that help to identify exons (codon bias, for example), one of the most powerful methods is simply to sequence the total RNA produced from the genome in living cells. As can be seen in Figure 7–3, this RNA-seq information, when mapped onto the genome sequence, can be used to accurately locate all the introns and exons of even complex genes. By sequencing total RNA from different cell types, it is also possible to identify cases of alternative splicing (see Figure 6–26).

1	RNA-seq also identifies noncoding RNAs produced by a genome. Although the function of some of them can be readily recognized (tRNAs or snoRNAs, for example), many have unknown functions and still others probably have no function at all (discussed in Chapter 7, pp. 429–436). The existence of the many noncoding RNAs and our relative ignorance of their function is the main reason that we know only the approximate number of genes in the human genome.

1	But even for protein-coding genes that have been unambiguously identified, we still have much to learn. Thousands of genomes have been sequenced, and we know from comparative genomics that many organisms share the same basic set of proteins. However, the functions of a very large number of identified proteins remain unknown. Depending on the organism, approximately one-third of the proteins encoded by a sequenced genome do not clearly resemble any protein that has been studied biochemically. This observation underscores a limitation of the emerging field of genomics: although comparative analysis of genomes reveals a great deal of information about the relationships between genes and organisms, it often does not provide immediate information about how these genes function, or what roles they have in the physiology of an organism. Comparison of the full Figure 8–40 Finding the regions in a DNA sequence that encode a protein.

1	(A) Any region of the DNA sequence can, in principle, code for six different amino acid sequences, because any one of three different reading frames can be used to interpret the nucleotide sequence on each strand. Note that a nucleotide sequence is always read in the 5ʹ-to-3ʹ direction and encodes a polypeptide from the N-terminus to the C-terminus. For a random nucleotide sequence read in a particular frame, a stop signal for protein synthesis is encountered, on average, about once every 20 amino acids. In this sample sequence of 48 base pairs, each such signal (stop codon) is colored blue, and only reading frame 2 lacks a stop signal. (B) Search of a 1700-base-pair DNA sequence for a possible protein-encoding sequence. The information is displayed as in (A), with each stop signal for protein synthesis denoted by a blue line. In addition, all of the regions between possible start and stop signals for protein synthesis (see pp. 347–349) are displayed as red bars. Only reading frame 1

1	synthesis denoted by a blue line. In addition, all of the regions between possible start and stop signals for protein synthesis (see pp. 347–349) are displayed as red bars. Only reading frame 1 actually encodes a protein, which is 475 amino acid residues long.

1	gene complement of several thermophilic bacteria, for example, does not reveal why these bacteria thrive at temperatures exceeding 70°C. And examination of the genome of the incredibly radioresistant bacterium Deinococcus radiodurans does not explain how this organism can survive a blast of radiation that can shatter glass. Further biochemical and genetic studies, like those described in the other sections of this chapter, are required to determine how genes, and the proteins they produce, function in the context of living organisms. DNA Cloning Allows Any Protein to be Produced in Large Amounts

1	In the last section, we saw how protein-coding genes can be identified in genome sequences. Using the genetic code (and provided the intron and exon boundaries are known), the amino acid sequence of any protein coded in a genome can be deduced. As was discussed earlier, this sequence can often provide an important clue to the protein’s function if found to be similar to the amino acid sequence of a protein that has already been studied (see Figure 8–23). Although this strategy is often successful, it typically provides only the likely biochemical function of the protein; for example, whether the protein resembles a kinase or a protease. It usually remains for the experimenter to verify (or refute) this assignment and, most importantly, to discover the protein’s biological function in the whole organism; that is, to what attributes of the organism does the kinase or the protease contribute and in what molecular pathways does it function? Nowadays, most new proteins are “discovered”

1	whole organism; that is, to what attributes of the organism does the kinase or the protease contribute and in what molecular pathways does it function? Nowadays, most new proteins are “discovered” through genome sequencing, and it often remains a great challenge to ascertain their functions.

1	An important approach in determining gene function is to alter the gene (or in some cases, its expression pattern), to put the altered copy back into the germ line of the organism, and to deduce the function of the normal gene by the changes caused by its alteration. Various techniques to implement this strategy are discussed in the next section of this chapter. But it is equally important to study the biochemical and structural properties of a gene product, as outlined in the first part of this chapter. One of the most important contributions of DNA cloning to cell and molecular biology is the ability to produce any protein, even the rare ones, in nearly unlimited amounts—as long as the gene coding for it is known. Such high-level production is usually carried out in living cells using expression vectors (Figure 8–41). These are generally plasmids that have been designed to produce a large amount of stable mRNA that can be efficiently translated into protein when the plasmid is

1	expression vectors (Figure 8–41). These are generally plasmids that have been designed to produce a large amount of stable mRNA that can be efficiently translated into protein when the plasmid is introduced into bacterial, yeast, insect, or mammalian cells. To prevent the high level of the foreign protein from interfering with the cell’s growth, the expression vector is often designed to delay the synthesis of the foreign mRNA and protein until shortly before the cells are harvested and lysed (Figure 8–42).

1	Because the desired protein made from an expression vector is produced inside a cell, it must be purified away from the host-cell proteins by chromatography following cell lysis; but because it is such a plentiful species in the cell (often 1–10% of the total cell protein), the purification is usually easy to accomplish in only a few steps. As we saw in the first part of this chapter, many expression

1	Figure 8–41 Production of large amounts of a protein from a protein-coding DNA sequence cloned into an expression vector and introduced into cells. A plasmid vector has been engineered to contain a highly active promoter, which causes unusually large amounts of mRNA to be produced from an adjacent protein-coding gene inserted into the plasmid vector. Depending on the characteristics of the cloning vector, the plasmid is introduced into bacterial, yeast, insect, or mammalian cells, where the inserted gene is efficiently transcribed and translated into protein. If the gene to be overexpressed has no introns (typical for genes from bacteria, archaea, and simple eukaryotes), it can simply be cloned from genomic DNA by PCR. For cloned animal and plant genes, it is often more convenient to obtain the gene as cDNA, either from a cDNA library (see Figure 8–32) or cloned directly by PCR from RNA isolated from the organism (see Figure 8–37). Alternatively, the DNA coding for the protein can be

1	the gene as cDNA, either from a cDNA library (see Figure 8–32) or cloned directly by PCR from RNA isolated from the organism (see Figure 8–37). Alternatively, the DNA coding for the protein can be made by chemical synthesis (see p. 472).

1	vectors have been designed to add a molecular tag—a cluster of histidine residues or a small marker protein—to the expressed protein to facilitate easy purification by affinity chromatography (see Figure 8–11). A variety of expression vectors is available, each engineered to function in the type of cell in which the protein is to be made. This technology is also used to make large amounts of many medically useful proteins, including hormones (such as insulin and growth factors) used as human pharmaceuticals, and viral coat proteins for use in vaccines. Expression vectors also allow scientists to produce many proteins of biological interest in large enough amounts for detailed structural studies. Nearly all three-dimensional protein structures depicted in this book are of proteins produced in this way. Recombinant DNA techniques thus allow scientists to move with ease from protein to gene, and vice versa, so that the functions of both can be explored on multiple fronts (Figure 8–43).

1	DNA cloning allows a copy of any specific part of a DNA or RNA sequence to be selected from the millions of other sequences in a cell and produced in unlimited amounts in pure form. DNA sequences can be amplified after breaking up chromosomal DNA and inserting the resulting DNA fragments into the chromosome of a self-replicating genetic element such as a plasmid. The resulting “genomic DNA library” is housed in millions of bacterial cells, each carrying a different cloned DNA fragment. Individual cells from this library that are allowed to proliferate produce large amounts of a single cloned DNA fragment. Bypassing cloning vectors and bacterial cells altogether, the polymerase chain reaction (PCR) allows DNA cloning to be performed directly with a DNA polymerase and DNA primers—provided that the DNA sequence of interest is already known.

1	The procedures used to obtain DNA clones that correspond in sequence to mRNA molecules are the same, except that a DNA copy of the mRNA sequence, called cDNA, is first made. Unlike genomic DNA clones, cDNA clones lack intron sequences, making them the clones of choice for analyzing the protein product of a gene. Nucleic acid hybridization reactions provide a sensitive means of detecting any nucleotide sequence of interest. The enormous specificity of this hybridization reaction allows any single-stranded sequence of nucleotides to be labeled with a radioisotope or chemical and used as a probe to find a complementary partner strand, even in a cell or cell extract that contains millions of different DNA and RNA sequences. DNA hybridization also makes it possible to use PCR to amplify any section of any genome once its sequence is known. direction of electrophoresis

1	direction of electrophoresis Figure 8–42 Production of large amounts of a protein by using a plasmid expression vector. In this example, an expression vector that overproduces a DNA helicase has been introduced into bacteria. In this expression vector, transcription from this coding sequence is under the control of a viral promoter that becomes active only at a temperature of 37°C or higher. The total cell protein, either from bacteria grown at 25°C (no helicase protein made) or after a shift of the same bacteria to 42°C for up to 2 hours (helicase protein has become the most abundant protein species in the lysate), has been analyzed by SDS polyacrylamide-gel electrophoresis. (Courtesy of Jack Barry.) sequence of a peptide fragment E. coli or otherPROTEIN host cell to produce protein DNA primers for PCR insert protein-coding region of gene into expression vector (from cDNA clone)

1	E. coli or otherPROTEIN host cell to produce protein DNA primers for PCR insert protein-coding region of gene into expression vector (from cDNA clone) Figure 8–43 Recombinant DNA techniques make it possible to move experimentally from gene to protein and from protein to gene. If a gene has been identified (right), its protein-coding sequence can be inserted into an expression vector to produce large quantities of the protein (see Figure 8–41), which can then be studied biochemically or structurally. If a protein has been purified based on its biochemical properties, mass spectrometry (see Figure 8–18) can be used to obtain a partial amino acid sequence, which is used to search a genome sequence for the corresponding nucleotide sequence. The complete gene can then be cloned by PCR from a sequenced genome (see Figure 8–37). The gene can also be manipulated and introduced into cells or organisms to study its function, a topic covered in the next section of this chapter.

1	The nucleotide sequence of any genome can be determined rapidly and simply by using highly automated techniques based on several different strategies. Comparison of the genome sequences of different organisms allows us to trace the evolutionary relationships among genes and organisms, and it has proved valuable for discovering new genes and predicting their functions. Taken together, these techniques for analyzing and manipulating DNA have made it possible to sequence, identify, and isolate genes from any organism of interest. Related technologies allow scientists to produce the protein products of these genes in the large quantities needed for detailed analyses of their structure and function, as well as for medical purposes.

1	Ultimately, one wishes to determine how genes—and the proteins they encode— function in the intact organism. Although it may seem counterintuitive, one of the most direct ways to find out what a gene does is to see what happens to the organism when that gene is missing. Studying mutant organisms that have acquired changes or deletions in their nucleotide sequences is a time-honored practice in biology and forms the basis of the important field of genetics. Because mutations can disrupt cell processes, mutants often hold the key to understanding gene function. In the classical genetic approach, one begins by isolating mutants that have an interesting or unusual appearance: fruit flies with white eyes or curly wings, for example. Working backward from the phenotype—the appearance or behavior of the individual—one then determines the organism’s genotype, the form of the gene responsible for that characteristic (Panel 8–2).

1	Today, with numerous genome sequences available, the exploration of gene function often begins with a DNA sequence. Here, the challenge is to translate sequence into function. One approach, discussed earlier in the chapter, is to search databases for well-characterized proteins that have similar amino acid sequences to the protein encoded by a new gene. From there, the protein (or for noncoding genes, the RNA molecule) can be overexpressed and purified and the methods described in the first part of this chapter can be employed to study its three-dimensional structure and its biochemical properties. But to determine directly a gene’s function in a cell or organism, the most effective approach involves studying mutants that either lack the gene or express an altered version of it. Determining which cell processes have been disrupted or compromised in such mutants will usually shed light on a gene’s biological role.

1	In this section, we describe several approaches to determining a gene’s function, starting either from an individual with an interesting phenotype or from a DNA sequence. We begin with the classical genetic approach, which starts with a genetic screen for isolating mutants of interest and then proceeds toward identification of the gene or genes responsible for the observed phenotype. We then describe the set of techniques that are collectively called reverse genetics, in which one begins with a gene or gene sequence and attempts to determine its function. This approach often involves some intelligent guesswork—searching for similar sequences in other organisms or determining when and where a gene is expressed—as well as generating mutant organisms and characterizing their phenotype.

1	Before the advent of gene cloning technology, most genes were identified by the abnormalities produced when the gene was mutated. Indeed, the very concept of the gene was deduced from the heritability of such abnormalities. This classical genetic approach—identifying the genes responsible for mutant phenotypes—is most easily performed in organisms that reproduce rapidly and are amenable to genetic manipulation, such as bacteria, yeasts, nematode worms, and fruit flies. Although spontaneous mutants can sometimes be found by examining extremely PANeL 8–2: Review of Classical Genetics GENES AND PHENOTYPES Gene: a functional unit of inheritance, usually corresponding to the segment of DNA coding for a single protein. Genome: all of an organism’s DNA sequences. locus:the site of the gene in the genome Wild-type: the normal, Mutant: differing from the alleles:alternative forms of a gene naturally occurring type wild-type because of a genetic change (a mutation)

1	Wild-type: the normal, Mutant: differing from the alleles:alternative forms of a gene naturally occurring type wild-type because of a genetic change (a mutation) GENOTYPE: the specifc set of alleles forming the genome of an individual PHENOTYPE: the visible character of the individual allele A is dominant (relative to a); allele a is recessive (relative to A) In the example above, the phenotype of the heterozygote is the same as that of one of the homozygotes; in cases where it is different from both, the two alleles are said to be co-dominant. a chromosome at the beginning of the cell cycle, in G1 phase; the single long bar centromere represents one long double helix of DNA a chromosome near the end of the cell cycle, in metaphase; it is duplicated and condensed, consisting of mother father two identical sister chromatids (each containing one DNA double helix) joined at the centromere.

1	“p” arm “q” arm A normal diploid chromosome set, as seen in a metaphase spread, prepared pair of autosomes by bursting open a cell at metaphase and staining the scattered maternal 1 chromosomes. In the example shown schematically here, there are three pairs of autosomes (chromosomes both parents, regardless of sex) and two sex chromosomes—an X from the mother and a Y from the father. The maternal 2 numbers and types of sex determination are variable from one class of organisms to another, as is the X number of pairs of autosomes. For simplicity, the cycle is shown for only sex chromosomes one chromosome/chromosome pair. MEIOSIS AND GENETIC RECOMBINATION The greater the distance chromosome, the greater is the chance that they will be

1	MEIOSIS AND GENETIC RECOMBINATION The greater the distance chromosome, the greater is the chance that they will be RECOMBINATION site of crossing-over occurring at a site between them. If two genes are thus reassorted in x% of gametes, they are said to be separated on a chromosome by a genetic map AB distance of x map units (or genotype ab haploid gametes (eggs or sperm) x centimorgans). POINT MUTATION: maps to a single site in the genome, corresponding to a single nucleotide pair or a very small part of a single gene INVERSION: inverts a segment of a chromosome lethal mutation: causes the developing organism to die prematurely. conditional mutation: produces its phenotypic effect only under certain conditions, called the restrictive conditions. Under other conditions—the permissive conditions—the effect is not seen. For a temperature-sensitive mutation, the restrictive condition typically is high temperature, while the permissive condition is low temperature.

1	loss-of-function mutation: either reduces or abolishes the activity of the gene. These are the most common class of mutations. Loss-of-function mutations are usually recessive—the organism can usually function normally as long as it retains at least one normal copy of the affected gene. null mutation: a loss-of-function mutation that completely abolishes the activity of the gene. TWO GENES OR ONE? Given two mutations that produce the same phenotype, how can we tell whether they are mutations in the same gene? If the mutations are recessive (as they most often are), the answer can be found by a complementation test. COMPLEMENTATION: MUTATIONS IN TWO DIFFERENT GENES DELETION: deletes a segment of a chromosome

1	COMPLEMENTATION: MUTATIONS IN TWO DIFFERENT GENES DELETION: deletes a segment of a chromosome TRANSLOCATION: breaks off a segment from one chromosome and attaches it to another gain-of-function mutation: increases the activity of the gene or makes it active in inappropriate circumstances; these mutations are usually dominant. dominant-negative mutation: dominant-acting mutation that blocks gene activity, causing a loss-of-function phenotype even in the presence of a normal copy of the gene. This phenomenon occurs when the mutant gene product interferes with the function of the normal gene product. suppressor mutation: suppresses the phenotypic effect of another mutation, so that the double mutant seems normal. An intragenic suppressor mutation lies within the gene affected by the frst mutation; an extragenic suppressor mutation lies in a second gene—often one whose product interacts directly with the product of the frst.

1	In the simplest type of complementation test, an individual who is homozygous for one mutation is mated with an individual who is homozygous for the other. The phenotype of the offspring gives the answer to the question. NONCOMPLEMENTATION: TWO INDEPENDENT MUTATIONS IN THE SAME GENE hybrid offspring shows normal phenotype: hybrid offspring shows mutant phenotype: one normal copy of each gene is present no normal copies of the mutated gene are present large populations—thousands or tens of thousands of individual organisms—isolating mutant individuals is much more efficient if one generates mutations with chemicals or radiation that damage DNA. By treating organisms with such mutagens, very large numbers of mutant individuals can be created quickly and then screened for a particular defect of interest, as we discuss shortly.

1	An alternative approach to chemical or radiation mutagenesis is called insertional mutagenesis. This method relies on the fact that exogenous DNA inserted randomly into the genome can produce mutations if the inserted fragment interrupts a gene or its regulatory sequences. The inserted DNA, whose sequence is known, then serves as a molecular tag that aids in the subsequent identification and cloning of the disrupted gene (Figure 8–44). In Drosophila, the use of the transposable P element to inactivate genes has revolutionized the study of gene function in the fly. Transposable elements (see Table 5–4, p. 288) have also been used to generate mutations in bacteria, yeast, mice, and the flowering plant Arabidopsis. Genetic Screens Identify Mutants with Specific Abnormalities

1	Genetic Screens Identify Mutants with Specific Abnormalities Once a collection of mutants in a model organism such as yeast or fly has been produced, one generally must examine thousands of individuals to find the altered phenotype of interest. Such a search is called a genetic screen, and the larger the genome, the less likely it is that any particular gene will be mutated. Therefore, the larger the genome of an organism, the bigger the screening task becomes. The phenotype being screened for can be simple or complex. Simple phenotypes are easiest to detect: one can screen many organisms rapidly, for example, for mutations that make it impossible for the organism to survive in the absence of a particular amino acid or nutrient.

1	More complex phenotypes, such as defects in learning or behavior, may require more elaborate screens (Figure 8–45). But even genetic screens that are used to dissect complex physiological systems can be simple in design, which permits the simultaneous examination of large numbers of mutants. As an example, one particularly elegant screen was designed to search for genes involved in visual processing in zebrafish. The basis of this screen, which monitors the fishes’ response to motion, is a change in behavior. Wild-type fish tend to swim in the direction of a perceived motion, whereas mutants with defects in their visual processing systems swim in random directions—a behavior that is easily detected. One mutant discovered in this screen is called lakritz, which is missing 80% of the retinal ganglion cells that help to relay visual signals from the eye to the brain. As the cellular organization of the zebrafish retina is similar to that of all vertebrates, the study of such mutants

1	ganglion cells that help to relay visual signals from the eye to the brain. As the cellular organization of the zebrafish retina is similar to that of all vertebrates, the study of such mutants should also provide insights into visual processing in humans.

1	Because defects in genes that are required for fundamental cell processes— RNA synthesis and processing or cell-cycle control, for example—are usually lethal, the functions of these genes are often studied in individuals with Figure 8–44 Insertional mutant of the snapdragon, Antirrhinum. A mutation in a single gene coding for a regulatory protein causes leafy shoots (left) to develop in place of flowers, which occur in the normal plant (right). The mutation causes cells to adopt a character that would be appropriate to a different part of the normal plant, so instead of a flower, the cells produce a leafy shoot. (Courtesy of Enrico Coen and Rosemary Carpenter.)

1	Figure 8–45 A behavioral phenotype detected in a genetic screen. (A) Wild-type C. elegans engage in social feeding. The worms migrate around until they encounter their neighbors and commence feeding on bacteria. (B) Mutant animals feed by themselves. (Courtesy of Cornelia Bargmann, Cell 94: cover, 1998. With permission from Elsevier.) mutant cells proliferate and form a colony at the permissive temperature mutant cells fail to proliferate and form a colony at the nonpermissive conditional mutations. The mutant individuals function normally as long as “permissive” conditions prevail, but demonstrate abnormal gene function when subjected to “nonpermissive” (restrictive) conditions. In organisms with temperature-sensitive mutations, for example, the abnormality can be switched on and off experimentally simply by changing the ambient temperature; thus, a cell containing a temperature-sensitive mutation in a gene essential for survival will die at a nonpermissive temperature but

1	off experimentally simply by changing the ambient temperature; thus, a cell containing a temperature-sensitive mutation in a gene essential for survival will die at a nonpermissive temperature but proliferate normally at the permissive temperature (Figure 8–46). The temperature-sensitive gene in such a mutant usually contains a point mutation that causes a subtle change in its protein product; for example, the mutant protein may function normally at low temperatures but unfold at higher temperatures.

1	Temperature-sensitive mutations were crucial to find the bacterial genes that encode the proteins required for DNA replication. The mutants were identified by screening populations of mutagen-treated bacteria for cells that stop making DNA when they are warmed from 30°C to 42°C. These mutants were later used to identify and characterize the corresponding DNA replication proteins (discussed in Chapter 5). Similarly, screens for temperature-sensitive mutations led to the identification of many proteins involved in regulating the cell cycle, as well as many proteins involved in moving proteins through the secretory pathway in yeast. Related screening approaches demonstrated the function of enzymes involved in the principal metabolic pathways of bacteria and yeast (discussed in Chapter 2) and identified many of the gene products responsible for the orderly development of the Drosophila embryo (discussed in Chapter 21). Mutations Can Cause Loss or Gain of Protein Function

1	Mutations Can Cause Loss or Gain of Protein Function Gene mutations are generally classed as “loss of function” or “gain of function.” A loss-of-function mutation results in a gene product that either does not work or works too little; thus, it can reveal the normal function of the gene. A gain-of-function mutation results in a gene product that works too much, works at the wrong time or place, or works in a new way (Figure 8–47).

1	An important early step in the genetic analysis of any mutant cell or organism is to determine whether the mutation causes a loss or a gain of function. A standard test is to determine whether the mutation is dominant or recessive. A dominant mutation is one that still causes the mutant phenotype in the presence of a single copy of the wild-type gene. A recessive mutation is one that is no longer able to cause the mutant phenotype in the presence of a single wild-type copy of the gene. Although cases have been described in which a loss-of-function mutation is dominant or a gain-of-function mutation is recessive, in the vast majority of cases, recessive mutations are loss of function and dominant mutations are gain Figure 8–46 Screening for temperature-sensitive bacterial or yeast mutants.

1	Figure 8–46 Screening for temperature-sensitive bacterial or yeast mutants. Mutagenized cells are plated out at the permissive temperature. They divide and form colonies, which are transferred to two identical Petri dishes by replica plating. One of these plates is incubated at the permissive temperature, the other at the nonpermissive temperature. Cells containing a temperature-sensitive mutation in a gene essential for proliferation can divide at the normal, permissive temperature but fail to divide at the elevated, nonpermissive temperature. Temperature-sensitive mutations of this type were especially useful for identifying genes needed for DNA replication, an essential process. Figure 8–47 Gene mutations that affect their protein product in different ways.

1	Figure 8–47 Gene mutations that affect their protein product in different ways. In this example, the wild-type protein has a specific cell function denoted by the red rays. Mutations that eliminate this function or inactivate it at higher temperatures are shown. The conditional mutant protein carries an amino acid substitution (red) that prevents its proper folding at 37ºC, but allows the protein to fold and function normally at 25ºC. Such temperature-sensitive conditional mutations are especially useful for studying essential genes; the organism can be grown under the permissive condition and then be moved to the nonpermissive condition to study the consequences of losing the gene product.

1	of function. It is easy to determine if a mutation is dominant or recessive. One simply mates a mutant with a wild type to obtain diploid cells or organisms. The progeny from the mating will be heterozygous for the mutation. If the mutant phenotype is no longer observed, one can conclude that the mutation is recessive and is very likely to be a loss-of-function mutation (see Panel 8–2). Complementation Tests Reveal Whether Two Mutations Are in the Same Gene or Different Genes

1	A large-scale genetic screen can turn up many different mutations that show the same phenotype. These defects might lie in different genes that function in the same process, or they might represent different mutations in the same gene. Alternative forms of the same gene are known as alleles. The most common difference between alleles is a substitution of a single nucleotide pair, but different alleles can also bear deletions, substitutions, and duplications. How can we tell, then, whether two mutations that produce the same phenotype occur in the same gene or in different genes? If the mutations are recessive—if, for example, they represent a loss of function of a particular gene—a complementation test can be used to ascertain whether the mutations fall in the same gene or in different genes. To test complementation in a diploid organism, an individual that is homozygous for one mutation—that is, it possesses two identical alleles of the mutant gene in question—is mated with an

1	genes. To test complementation in a diploid organism, an individual that is homozygous for one mutation—that is, it possesses two identical alleles of the mutant gene in question—is mated with an individual that is homozygous for the other mutation. If the two mutations are in the same gene, the offspring show the mutant phenotype, because they still will have no normal copies of the gene in question (Figure 8–48). If, in contrast, the mutations fall in different genes, the resulting offspring show a normal phenotype, because they retain one normal copy (and one mutant copy) of each gene; the mutations thereby complement one another and restore a normal phenotype. Complementation testing of mutants identified during genetic screens has revealed, for example, that 5 different genes are required for yeast to digest the sugar galactose, 20 genes are needed for E. coli to build a functional flagellum, 48 genes are involved in assembling bacteriophage T4 viral particles, and hundreds of

1	for yeast to digest the sugar galactose, 20 genes are needed for E. coli to build a functional flagellum, 48 genes are involved in assembling bacteriophage T4 viral particles, and hundreds of genes are involved in the development of an adult nematode worm from a fertilized egg.

1	Once a set of genes involved in a particular biological process has been identified, the next step is often to determine in which order the genes function. Gene order is perhaps easiest to explain for metabolic pathways, where, for example, enzyme A is necessary to produce the substrate for enzyme B. In this case, we would say that the gene encoding enzyme A acts before (upstream of) the gene encoding enzyme B in the pathway. Similarly, where one protein regulates the activity of another protein, we would say that the former gene acts before the latter. Gene order can, in many cases, be determined purely by genetic analysis without any knowledge of the mechanism of action of the gene products involved. Suppose we have a biosynthetic process consisting of a sequence of steps, such that performance of step B is conditional on completion of the preceding step A; and suppose gene A is required for step A, and gene B is required for step

1	B. Then a null mutation (a mutation that abolishes function) in gene A will arrest the process at step A, regardless of whether gene B is functional or not, whereas a null mutation in gene B will cause arrest at step B only if gene A is still active. In such a case, gene A is said to be epistatic to gene B. By comparing the phenotypes of the different combinations of mutations, we can therefore discover the order in which the genes act. This type of analysis is called epistasis analysis. As an example, the pathway of protein secretion in yeast has been analyzed in this way. Different mutations in this pathway cause proteins to accumulate aberrantly in the endoplasmic reticulum (ER) or in the Golgi apparatus. When a yeast cell is engineered to carry both a mutation that blocks protein processing in the ER and a mutation that blocks processing in the Golgi apparatus, proteins accumulate in

1	Figure 8–48 A complementation test can reveal that mutations in two different genes are responsible for the same abnormal phenotype. When an albino (white) bird from one strain is bred with an albino from a different strain, the resulting offspring (bottom) have normal coloration. This restoration of the wild-type plumage indicates that the two white breeds lack color because of recessive mutations in different genes. (From W. Bateson, Mendel’s Principles of Heredity, 1st ed. Cambridge, UK: Cambridge University Press, 1913.) the ER. This indicates that proteins must pass through the ER before being sent to the Golgi before secretion (Figure 8–49). Strictly speaking, an epistasis analysis can only provide information about gene order in a pathway when both mutations are null alleles. When the mutations retain partial function, their epistasis interactions can be difficult to interpret.

1	Sometimes, a double mutant will show a new or more severe phenotype than either single mutant alone. This type of genetic interaction is called a synthetic phenotype, and if the phenotype is death of the organism, it is called synthetic lethality. In most cases, a synthetic phenotype indicates that the two genes act in two different parallel pathways, either of which is capable of mediating the same cell process. Thus, when both pathways are disrupted in the double mutant, the process fails altogether, and the synthetic phenotype is observed.

1	Once a collection of mutant organisms with interesting phenotypes has been obtained, the next task is to identify the gene or genes responsible for the altered phenotype. If the phenotype has been produced by insertional mutagenesis, locating the disrupted gene is fairly simple. DNA fragments containing the insertion (a transposon or a retrovirus, for example) are amplified by PCR, and the nucleotide sequence of the flanking DNA is determined. The gene affected by the insertion can then be identified by a computer-aided search of the complete genome sequence of the organism.

1	If a DNA-damaging chemical was used to generate the mutations, identifying the inactivated gene is often more laborious, but there are several powerful strategies available. If the genome size of the organism is small (for example, for bacteria or simple eukaryotes), it is possible to simply determine the genome sequence of the mutant organism and identify the affected gene by comparison with the wild-type sequence. Because of the continuous accumulation of neutral mutations, there will probably be differences between the two genome sequences in addition to the mutation responsible for the phenotype. One way of proving that a mutation is causative is to introduce the putative mutation back into a normal organism and determine whether or not it causes the mutant phenotype. We will discuss how this is accomplished later in the chapter.

1	Genetic screens in model experimental organisms have been spectacularly successful in identifying genes and relating them to various phenotypes, including many that are conserved between these organisms and humans. But how can we study humans directly? They do not reproduce rapidly, cannot be treated with mutagens, and, if they have a defect in an essential process such as DNA replication, would die long before birth. Despite their limitations compared to model organisms, humans are becoming increasingly attractive subjects for genetic studies. Because the human

1	Despite their limitations compared to model organisms, humans are becoming increasingly attractive subjects for genetic studies. Because the human Figure 8–49 Using genetics to determine the order of function of genes. In normal cells, secretory proteins are loaded into vesicles, which fuse with the plasma membrane to secrete their contents into the extracellular medium. Two mutants, A and B, fail to secrete proteins. In mutant A, secretory proteins accumulate in the ER. In mutant B, secretory proteins accumulate in the Golgi. In the double mutant AB, proteins accumulate in the ER; this indicates that the gene defective in mutant A acts before the gene defective in mutant B in the secretory pathway.

1	population is so large, spontaneous, nonlethal mutations have arisen in all human genes—many times over. A substantial proportion of these remain in the genomes of present-day humans. The most deleterious of these mutations are discovered when the mutant individuals call attention to themselves by seeking medical help. With the recent advances that have enabled the sequencing of entire human genomes cheaply and quickly, we can now identify such mutations and study their evolution and inheritance in ways that were impossible even a few years ago. By comparing the sequences of thousands of human genomes from all around the world, we can begin to identify directly the DNA differences that distinguish one individual from another. These differences hold clues to our evolutionary origins and can be used to explore the roots of disease. Linked Blocks of Polymorphisms Have Been Passed Down from Our Ancestors

1	Linked Blocks of Polymorphisms Have Been Passed Down from Our Ancestors When we compare the sequences of multiple human genomes, we find that any two individuals will differ in roughly 1 nucleotide pair in 1000. Most of these variations are common and relatively harmless. When two sequence variants coexist in the population and both are common, the variants are called polymorphisms. The majority of polymorphisms are due to the substitution of a single nucleotide, called single-nucleotide polymorphisms or SNPs (Figure 8–50). The rest are due largely to insertions or deletions—called indels when the change is small, or copy number variations (CNVs) when it is large. Although these common variants can be found throughout the genome, they are not scattered randomly—or even independently. Instead, they tend to travel in groups called haplotype blocks—combinations of polymorphisms that are inherited as a unit.

1	To understand why such haplotype blocks exist, we need to consider our evolutionary history. It is thought that modern humans expanded from a relatively small population—perhaps around 10,000 individuals—that existed in Africa about 60,000 years ago. Among that small group of our ancestors, some individuals will have carried one set of genetic variants, others a different set. The chromosomes of a present-day human represent a shuffled combination of chromosome segments from different members of this small ancestral group of people. Because only about two thousand generations separate us from them, large segments of these ancestral chromosomes have passed from parent to child, unbroken by the crossover events that occur during meiosis. As described in Chapter 5, only a few crossovers occur between each set of homologous chromosomes during each meiosis (see Figure 5–53).

1	As a result, certain sets of DNA sequences—and their associated polymorphisms—have been inherited in linked groups, with little genetic rearrangement across the generations. These are the haplotype blocks. Like genes that exist in different allelic forms, haplotype blocks also come in a limited number of variants that are common in the human population, each representing a combination of DNA polymorphisms passed down from a particular ancestor long ago. Figure 8–50 Single-nucleotide polymorphisms (SNPs) are sites in the genome where two or more alternative choices of a nucleotide are common in the population. Most such variations in the human genome occur at locations where they do not significantly affect a gene’s function. Polymorphisms Can Aid the Search for Mutations Associated with Disease

1	Mutations that give rise, in a reproducible way, to rare but clearly defined abnormalities, such as albinism, hemophilia, or congenital deafness, can often be identified by studies of affected families. Such single-gene, or monogenic, disorders are often referred to as Mendelian because their pattern of inheritance is easy to track. Moreover, individuals who inherit the causative mutation will exhibit the abnormality irrespective of environmental factors such as diet or exercise. But for many common diseases, the genetic roots are more complex. Instead of a single allele of a single gene, such disorders stem from a combination of contributions from multiple genes. And often, environmental factors have strong influences on the severity of the disorder. For these multigenic conditions, such as diabetes or arthritis, population studies are often helpful in tracking down the genes that increase the risk of getting the disease.

1	In population studies, investigators collect DNA samples from a large number of people who have the disease and compare them to samples from a group of people who do not have the disease. They look for variants—SNPs, for example— that are more common among the people who have the disease. Because DNA sequences that are close together on a chromosome tend to be inherited together, the presence of such SNPs could indicate that an allele that increases the risk of the disease might lie nearby (Figure 8–51). Although, in principle, the disease could be caused by the SNP itself, the culprit is much more likely to be a change that is merely linked to the SNP as part of a haplotype block.

1	Such genome-wide association studies have been used to search for genes that predispose individuals to common diseases, including diabetes, coronary artery disease, rheumatoid arthritis, and even depression. For many of these conditions, the DNA polymorphisms identified increase the risk of disease only slightly. Moreover, environmental factors (diet, exercise, for example) play an important role in the onset and severity of the disease. Nonetheless, the identification of genes affected by these polymorphisms is leading to a mechanistic understanding of some of our most common disorders. Genomics Is Accelerating the Discovery of Rare Mutations That Predispose Us to Serious Disease

1	Genomics Is Accelerating the Discovery of Rare Mutations That Predispose Us to Serious Disease The genetic variants that have thus far allowed us to identify some of the genes that increase our risk of disease are common ones. They arose long ago in our evolutionary past and are now present, in one form or another, in a substantial portion (1% or more) of the population. Such polymorphisms are thought to account

1	Figure 8–51 Genes that affect the risk of developing a common disease can often be tracked down through linkage to SNPs. Here, the patterns of SNPs are compared between two sets of individuals—a set of healthy controls and a set affected by a particular common disease. A segment of a typical chromosome is shown. For most polymorphic sites in this segment, it is a random matter whether an individual has one SNP variant (red vertical bars) or another (blue vertical bars); this same randomness is seen both for the control group and for the affected individuals. However, in the part of the chromosome that is shaded in darker gray, a bias is seen: most normal individuals have the blue SNP variants, whereas most affected individuals have the red SNP variants. This suggests that this region contains, or is close to, a gene that is genetically linked to these red SNP variants and that predisposes individuals to the disease. Using carefully selected controls and thousands of affected

1	contains, or is close to, a gene that is genetically linked to these red SNP variants and that predisposes individuals to the disease. Using carefully selected controls and thousands of affected individuals, this approach can help track down disease-related genes, even when they confer only a slight increase in the risk of developing the disease.

1	for about 90% of the differences between one person’s genome and another’s. But when we try to tie these common variants to differences in disease susceptibility or other heritable traits, such as height, we find that they do not have as much predictive power as we had anticipated: thus, for example, most confer relatively small increases—less than twofold—in the risk of developing a common disease.

1	In contrast to polymorphisms, rare DNA variants—those much less frequent in humans than SNPs—can have large effects on the risk of developing some common diseases. For example, a number of different loss-of-function mutations, each individually rare, have been found to increase greatly the predisposition to autism and schizophrenia. Many of these are de novo mutations, which arose spontaneously in the germ-line cells of one or the other parent. The fact that these mutations arise spontaneously with some frequency could help explain why these common disorders—each observed in about 1% of the population—remain with us, even though the affected individuals leave few or no descendants. These rare mutations may arise in any one of hundreds of different genes, which could explain much of the clinical variability of autism and schizophrenia. Because they are kept rare by natural selection, most such variants with a large effect on risk would be missed by genome-wide association studies.

1	Now that DNA sequencing has become fast and inexpensive, the most efficient and cost-effective way to identify these rare, large-effect mutations is by sequencing the genomes of affected individuals, along with those of their parents and siblings as controls. Reverse Genetics Begins with a Known Gene and Determines Which Cell Processes Require Its Function

1	As we have seen, classical genetics starts with a mutant phenotype (or, in the case of humans, a range of characteristics) and identifies the mutations, and consequently the genes, responsible for it. Recombinant DNA technology has made possible a different type of genetic approach, one that is used widely in a variety of genetically tractable species. Instead of beginning with a mutant organism and using it to identify a gene and its protein, an investigator can start with a particular gene and proceed to make mutations in it, creating mutant cells or organisms so as to analyze the gene’s function. Because this approach reverses the traditional direction of genetic discovery—proceeding from genes to mutations, rather than vice versa—it is commonly referred to as reverse genetics. And because the genome of the organism is deliberately altered in a particular way, this approach is also called genome engineering or genome editing. We shall see in this chapter that this approach can be

1	the genome of the organism is deliberately altered in a particular way, this approach is also called genome engineering or genome editing. We shall see in this chapter that this approach can be scaled upward so that whole collections of organisms can be created, each of which has a different gene altered.

1	There are several ways a gene of interest can be altered. In the simplest, the gene can simply be deleted from the genome, although in a diploid organism, this requires that both copies—one on each chromosome homolog—be deleted. Although somewhat counterintuitive, one of the best ways to discover the function of a gene is by observing the effects of not having it. Such “gene knockouts” are especially useful if the gene is not essential. Through reverse genetics, the gene in question (even if it is essential) can also be replaced by one that is expressed in the wrong tissue or at the wrong time in development; this type of manipulation often provides important clues to the gene’s normal function. For example, a gene of interest can be modified to be expressed at will by the experimenter (Figure 8–52). Finally, genes can also be engineered so that they are expressed normally in most cell types and tissues but deleted in certain cell types or tissues selected by the experimenter (see

1	(Figure 8–52). Finally, genes can also be engineered so that they are expressed normally in most cell types and tissues but deleted in certain cell types or tissues selected by the experimenter (see Figure 5–66). This approach is especially useful when a gene has different roles in different tissues.

1	It is also possible to make subtler changes to a gene. It is sometimes useful to make slight changes in a protein’s structure so that one can begin to dissect which portions of a protein are important for its function. The activity of an enzyme, for example, can be studied by changing a single amino acid in its active site. It is also possible, through genome engineering, to create new types of proteins in an

1	Figure 8–52 engineered genes can be turned on and off with small molecules. Here, the DNA-binding portion of a bacterial protein (the tetracycline, Tet, repressor) has been fused to a portion of a mammalian transcriptional activator and expressed in cultured mammalian cells. The engineered gene X, present in place of the normal gene, has its usual gene control region replaced by cis-regulatory sequences recognized by the tetracycline repressor. In the absence of doxycycline (a particularly stable version of tetracycline), the engineered gene is expressed; in the presence of doxycycline, the gene is turned off because the drug causes the tetracycline repressor to dissociate from the DNA. This strategy can also be used in mice by incorporating the engineered genes into the germ line. In many tissues, the gene can be turned on and off simply by adding or removing doxycycline from the animal’s water. If the tetracycline repressor construct is placed under the control of a tissue-specific

1	tissues, the gene can be turned on and off simply by adding or removing doxycycline from the animal’s water. If the tetracycline repressor construct is placed under the control of a tissue-specific gene control region, the engineered gene will be turned on and off only in that tissue.

1	animal. For example, a gene can be fused to the gene for a fluorescent protein. When this altered gene is introduced into the genome, the protein can be tracked in the living organism by monitoring its fluorescence.

1	Altered genes can be created in several ways. Perhaps the simplest is to chemically synthesize the DNA that makes up the gene. In this way, the investigator can specify any type of variant of the normal gene. It is also possible to construct altered genes using recombinant DNA technology, as described earlier in this chapter. Once obtained, altered genes can be introduced into cells in a variety of ways. DNA can be microinjected into mammalian cells with a glass micropipette or introduced by a virus that has been engineered to carry foreign genes. In plant cells, genes are frequently introduced by a technique called particle bombardment: DNA samples are painted onto tiny gold beads and then literally shot through the cell wall with a specially modified gun. Electroporation is the method of choice for introducing DNA into bacteria and some other cells. In this technique, a brief electric shock renders the cell membrane temporarily permeable, allowing foreign DNA to enter the cytoplasm.

1	To be most useful to experimenters, the altered gene, once it is introduced into a cell, must recombine with the cell’s genome so that the normal gene is replaced. In simple organisms such as bacteria and yeasts, this process occurs with high frequency using the cell’s own homologous recombination machinery, as described in Chapter 5. In more complex organisms that have elaborate developmental programs, the procedure is more complicated because the altered gene must be introduced into the germ line, as we next describe.

1	Animals and plants that have been genetically engineered by gene deletion or gene replacement are called transgenic organisms, and any foreign or modified genes that are added are called transgenes. We discuss transgenic plants later in this chapter and, for now, concentrate our discussion on transgenic mice, as enormous progress has been made in this area. If a DNA molecule carrying a mutated mouse gene is transferred into a mouse cell, it often inserts into the chromosomes at random, but methods have been developed to direct the mutant gene to replace the normal gene by homologous recombination. By exploiting these “gene targeting” events, any specific gene can be altered or inactivated in a mouse cell by a direct gene replacement. In the case in which both copies of the gene of interest are completely inactivated or deleted, the resulting animal is called a “knockout” mouse. The technique is summarized in Figure 8–53.

1	TRANSGENIC MOUSE WITH ONE COPY OF TARGET GENE REPLACED BY ALTERED GENE IN GERM LINE SOMATIC CELLS OF OFFSPRING TESTED FOR PRESENCE OF ALTERED GENE, AND SELECTED MICE BRED TO TEST FOR GENE IN GERM-LINE CELLS BIRTH MATE AND HARVEST EARLY EMBRYOS HYBRID EARLY EMBRYO PARTLY FORMED FROM ES CELLS INTRODUCE HYBRID EARLY EMBRYO INTO PSEUDOPREGNANT MOUSE isolated early embryo in culture altered version of target gene constructed by genetic engineering INTRODUCE A DNA FRAGMENT CONTAINING ALTERED GENE INTO MANY CELLS TEST FOR THE RARE COLONY IN WHICH THE DNA FRAGMENT HAS REPLACED ONE COPY OF THE NORMAL GENE LET EACH CELL PROLIFERATE TO FORM A COLONY ES cells with one copy of target gene replaced by mutant gene INJECT ES CELLS INTO EARLY EMBRYO

1	The ability to prepare transgenic mice lacking a known normal gene has been a major advance, and the technique has been used to determine the functions of many mouse genes (Figure 8–54). If the gene functions in early development, a knockout mouse will usually die before it reaches adulthood. These lethal defects can be carefully analyzed to help determine the function of the missing gene. As described in Chapter 5, an especially useful type of transgenic animal takes advantage of a site-specific recombination system to excise—and thus disable— the target gene in a particular place or at a particular time (see Figure 5–66). In this case, the target gene in embryonic stem (ES) cells is replaced by a fully functional version of the gene that is flanked by a pair of the short DNA sequences, called lox sites, that are recognized by the Cre recombinase protein. The transgenic mice that result are phenotypically normal. They are then mated with transgenic mice that express the Cre

1	called lox sites, that are recognized by the Cre recombinase protein. The transgenic mice that result are phenotypically normal. They are then mated with transgenic mice that express the Cre recombinase gene under the control of an inducible promoter. In

1	Figure 8–53 Summary of the procedures used for making gene replacements in mice. In the first step (A), an altered version of the gene is introduced into cultured ES (embryonic stem) cells. These cells are described in detail in Chapter 22. Only a few ES cells will have their corresponding normal genes replaced by the altered gene through a homologous recombination event. These cells can be identified by PCR and cultured to produce many descendants, each of which carries an altered gene in place of one of its two normal corresponding genes. In the next step of the procedure (B), these altered ES cells are injected into a very early mouse embryo; the cells are incorporated into the growing embryo, and a mouse produced by such an embryo will contain some somatic cells (indicated by orange) that carry the altered gene. Some of these mice will also contain germ-line cells that contain the altered gene; when bred with a normal mouse, some of the progeny of these mice will contain one copy

1	that carry the altered gene. Some of these mice will also contain germ-line cells that contain the altered gene; when bred with a normal mouse, some of the progeny of these mice will contain one copy of the altered gene in all of their cells.

1	The mice with the transgene in their germ line are then bred to produce both a male and a female animal, each heterozygous for the gene replacement (that is, they have one normal and one mutant copy of the gene). When these two mice are mated (not shown), one-fourth of their progeny will be homozygous for the altered gene. the specific cells or tissues in which Cre is switched on, it catalyzes recombination between the lox sequences—excising a target gene and eliminating its activity (see Figure 22–5). The Bacterial CRISPR System Has Been Adapted to Edit Genomes in a Wide Variety of Species One of the difficulties in making transgenic mice by the procedure just described is that the introduced DNA molecule (bearing the experimentally altered gene) often inserts at random in the genome, and many ES cells must therefore be screened individually to find one that has the “correct” gene replacement.

1	Creative use of the CRISPR system, discovered in bacteria as a defense against viruses, has largely solved this problem. As described in Chapter 7, the CRISPR system uses a guide RNA sequence to target (through complementary base-pairing) double-stranded DNA, which it then cleaves (see Figure 7–78). The gene coding for the key component of this system, the bacterial Cas9 protein, has been transferred into a variety of organisms, where it greatly simplifies the process of making transgenic organisms (Figure 8–55A and B). The basic strategy is as follows: Cas9 protein is expressed in ES cells along with a guide RNA designed by the experimenter to target a particular location on the genome. The Cas9 and guide RNA associate, the complex is brought to the matching sequence on the genome, and the Cas9 protein makes a double-strand break. As we saw in Chapter 5, double-strand breaks are often repaired by homologous recombination; here, the template chosen by the cell to repair the damage is

1	Cas9 protein makes a double-strand break. As we saw in Chapter 5, double-strand breaks are often repaired by homologous recombination; here, the template chosen by the cell to repair the damage is often the altered gene, which is introduced to ES cells by the experimenter. In this way, the normal gene can be selectively damaged by the CRISPR system and replaced at high efficiency by the experimentally altered gene.

1	The CRISPR system has a variety of other uses. Its particular power lies with its ability to target Cas9 to thousands of different positions across a genome through the simple rules of complementary base-pairing. Thus, if a catalytically inactive Cas9 protein is fused to a transcription activator or repressor, it is possible, in principle, to turn any gene on or off (Figure 8–55C and D).

1	Figure 8–54 Transgenic mice engineered to express a mutant DNA helicase show premature aging. The helicase, encoded by the Xpd gene, is involved in both transcription and DNA repair. Compared with a wild-type mouse of the same age (A), a transgenic mouse that expresses a defective version of Xpd (B) exhibits many of the symptoms of premature aging, including osteoporosis, emaciation, early graying, infertility, and reduced life-span. The mutation in Xpd used here impairs the activity of the helicase and mimics a mutation that in humans causes trichothiodystrophy, a disorder characterized by brittle hair, skeletal abnormalities, and a very reduced life expectancy. These results indicate that an accumulation of DNA damage can contribute to the aging process in both humans and mice. (From J. de Boer et al., Science 296:1276–1279, 2002. With permission from AAAS.)

1	Figure 8–55 Use of CRISPR to study gene function in a wide variety of species. (A) The Cas9 protein (artificially expressed in the species of interest) binds to a guide RNA, designed by the experimenter and also expressed. The portion of RNA in light blue is needed for associations with Cas9; that in dark blue is specified by the experimenter to match a position on the genome. The only other requirement is that the adjacent genome sequence includes a short PAM (protospacer adjacent motif) that is needed for Cas9 to cleave. As described in Chapter 7, this sequence is how the CRISPR system in bacteria distinguishes its own genome from that of invading viruses. (B) When directed to make double-strand breaks, the CRISPR system greatly improves the ability to replace an endogenous gene with an experimentally altered gene since the altered gene is used to “repair” the double-strand break (C, D). By using a mutant form of Cas9 that can no longer cleave DNA, Cas9 can be used to activate a

1	an experimentally altered gene since the altered gene is used to “repair” the double-strand break (C, D). By using a mutant form of Cas9 that can no longer cleave DNA, Cas9 can be used to activate a normally dormant gene (C) or turn off an actively expressed gene (D). (Adapted from P. Mali et al., Nat. Methods 10:957– 963, 2013. With permission from Macmillan Publishers Ltd.)

1	The CRISPR system has several advantages over other strategies for experimentally manipulating gene expression. First, it is relatively easy for the experimenter to design the guide RNA: it simply follows standard base pairing convention. Second, the gene to be controlled does not have to be modified; the CRISPR strategy exploits DNA sequences already present in the genome. Third, numerous genes can be controlled simultaneously. Cas9 has to be expressed only once, but many guide RNAs can be expressed in the same cell; this strategy allows the experimenter to turn on or off a whole set of genes at once.

1	The export of the CRISPR system from bacteria to virtually all other experimental organisms (including mice, zebrafish, worms, flies, rice, and wheat) has revolutionized the study of gene function. Like the earlier discovery of restriction enzymes, this breakthrough came from scientists studying a fascinating phenomenon in bacteria without—at first—realizing the enormous impact these discoveries would have on all aspects of biology. Large Collections of Engineered Mutations Provide a Tool for Examining the Function of Every Gene in an Organism

1	Extensive collaborative efforts have produced comprehensive libraries of mutations in a variety of model organisms, including S. cerevisiae, C. elegans, Drosophila, Arabidopsis, and even the mouse. The ultimate aim in each case is to produce a collection of mutant strains in which every gene in the organism has been systematically deleted or altered in such a way that it can be conditionally disrupted. Collections of this type provide an invaluable resource for investigating gene function on a genomic scale. For example, a large collection of mutant organisms can be screened for a particular phenotype. Like the classic genetic approaches described earlier, this is one of the most powerful ways to identify the genes responsible for a particular phenotype. Unlike the classical genetic approach, however, the set of mutants is “pre-engineered,” so that there is no need to rely on chance events such as spontaneous mutations or transposon insertions. In addition, each of the individual

1	however, the set of mutants is “pre-engineered,” so that there is no need to rely on chance events such as spontaneous mutations or transposon insertions. In addition, each of the individual mutations within the collection is often engineered to contain a distinct molecular “barcode”—in the form of a unique DNA sequence—designed to make identification of the altered gene rapid and routine (Figure 8–56).

1	Figure 8–56 Making barcoded collections of mutant organisms. A deletion construct for use in yeast contains DNA sequences (red) homologous to each end of a target gene x, a selectable marker gene (blue), and a unique “barcode” sequence approximately 20 nucleotide pairs in length (green). This DNA is introduced into yeast cells, where it readily replaces the target gene by homologous recombination. Cells that carry a successful gene replacement are identified by expression of the selectable marker gene, typically a gene that provides resistance to a drug. By using a collection of such constructs, each specific for one gene, a library of yeast mutants was constructed containing a mutant for every gene. Essential genes cannot be studied this way, as their deletion from the genome causes the cells to die. In this case, the target gene is replaced by a version of the gene that can be regulated by the experimenter (see Figure 8–52). The gene can then be turned off and the effect of this can

1	to die. In this case, the target gene is replaced by a version of the gene that can be regulated by the experimenter (see Figure 8–52). The gene can then be turned off and the effect of this can be monitored before the cells die.

1	Figure 8–57 Genome-wide screens for fitness using a large pool of barcoded yeast deletion mutants. A large pool of yeast mutants, each with a different gene deleted and present in equal amounts, is grown under conditions selected by the experimenter. Some mutants (blue) grow normally, but others show reduced growth (orange and green) or no growth at all (red). The fitness of each mutant is experimentally determined in the following way. After the growth phase is completed, genomic DNA (isolated from the mixture of strains) is purified and the relative abundance of each mutant is determined by quantifying the level of the DNA barcode matched to each deletion. This can be done by sequencing the pooled genomic DNA or hybridizing it to microarrays (see Figure 8–64) that contain DNA oligonucleotides complementary to each barcode. In this way, the contribution of every gene to growth under the specified condition can be rapidly ascertained. This type of study has revealed that of the

1	complementary to each barcode. In this way, the contribution of every gene to growth under the specified condition can be rapidly ascertained. This type of study has revealed that of the approximately 6000 coding genes in yeast, only about 1000 are essential under standard growth conditions.

1	In S. cerevisiae, the task of generating a complete set of 6000 mutants, each missing only one gene, was accomplished several years ago. Because each mutant strain has an individual barcode sequence embedded in its genome, a large mixture of engineered strains can be grown under various selective test conditions— such as nutritional deprivation, a temperature shift, or the presence of various drugs—and the cells that survive can be rapidly identified by the unique sequence tags present in their genomes. By assessing how well each mutant in the mixture fares, one can begin to discern which genes are essential, useful, or irrelevant for growth under the various conditions (Figure 8–57).

1	The insights generated by examining mutant libraries can be considerable. For example, studies of an extensive collection of mutants in Mycoplasma genitalium—the organism with the smallest known genome—have identified the minimum complement of genes essential for cellular life. Growth under laboratory conditions requires about three-quarters of the 480 protein-coding genes in M. genitalium. Approximately 100 of these essential genes are of unknown function, which suggests that a surprising number of the basic molecular mechanisms that underlie life have yet to be discovered.

1	Collections of mutant organisms are also available for many animal and plant species. For example, it is possible to “order,” by phone or email from a consortium of investigators, a deletion or insertion mutant for almost all coding genes in Drosophila. Likewise, a nearly complete set of mutants exists for the “model” plant Arabidopsis. And the adaptation of the CRISPR system for use in mice means that, in the near future, we can expect to be able to turn on or off—at will—each gene in the mouse genome. Although we are still ignorant about the function of most genes in most organisms, these technologies allow an exploration of gene function on a scale that was unimaginable a decade ago. RNA Interference Is a Simple and Rapid Way to Test Gene Function

1	RNA Interference Is a Simple and Rapid Way to Test Gene Function Although knocking out (or conditionally expressing) a gene in an organism and studying the consequences is the most powerful approach for understanding the functions of the gene, RNA interference (RNAi, for short), is an alternative, particularly convenient approach. As discussed in Chapter 7, this method exploits a natural mechanism used in many plants, animals, and fungi to protect themselves against viruses and transposable elements. The technique introduces into a cell or organism a double-stranded RNA molecule whose nucleotide sequence matches that of part of the gene to be inactivated. After the RNA is processed, it hybridizes with the target-gene RNA (either mRNA or noncoding RNA) and reduces its expression by the mechanisms shown in Figure 7–75.

1	RNAi is frequently used to inactivate genes in Drosophila and mammalian cell culture lines. Indeed, a set of 15,000 Drosophila RNAi molecules (one for every coding gene) allows researchers, in several months, to test the role of every fly gene in any process that can be monitored using cultured cells. RNAi has also been widely used to study gene function in whole organisms, including the nematode pool of barcoded yeast mutants, each deleted for a different gene E. coli, expressing double-stranded RNA, eaten by worm

1	E. coli, expressing double-stranded RNA, eaten by worm Figure 8–58 Gene function can be tested by RNA interference. (A) Double-stranded RNA (dsRNA) can be introduced into C. elegans by (1) feeding the worms E. coli that express the dsRNA or (2) injecting the dsRNA directly into the animal’s gut. (B) In a wild-type worm embryo, the egg and sperm pronuclei (red arrowheads) come together in the posterior half of the embryo shortly after fertilization. (C) In an embryo in which a particular gene has been inactivated by RNAi, the pronuclei fail to migrate. This experiment revealed an important but previously unknown function of this gene in embryonic development. (B and C, from P. Gönczy et al., Nature 408:331–336, 2000. With permission from Macmillan Publishers Ltd.)

1	C. elegans. When working with worms, introducing the double-stranded RNA is quite simple: the RNA can be injected directly into the intestine of the animal, or the worm can be fed with E. coli engineered to produce the RNA (Figure 8–58). The RNA is amplified (see p. 431) and distributed throughout the body of the worm, where it inhibits expression of the target gene in different tissue types. RNAi is being used to help in assigning functions to the entire complement of worm genes (Figure 8–59).

1	A related technique has also been applied to mice. In this case, the RNAi molecules are not injected or fed to the mouse; rather, recombinant DNA techniques are used to make transgenic animals that express the RNAi under the control of an inducible promoter. Often this is a specially designed RNA that can fold back on itself and, through base-pairing, produce a double-stranded region that is recognized by the RNAi machinery. In the simplest cases, the process inactivates only the genes that exactly match the RNAi sequence. Depending on the inducible promoter used, the RNAi can be produced only in a specified tissue or only at a particular time in development, allowing the functions of the target genes to be analyzed in elaborate detail. RNAi has made reverse genetics simple and efficient in many organisms, but it has several potential limitations compared with true genetic knockouts. For C. elegans each well contains WORMS INGEST E. coli; RESULTING PHENOTYPES RECORDED AND ANALYZED

1	C. elegans each well contains WORMS INGEST E. coli; RESULTING PHENOTYPES RECORDED AND ANALYZED E. coli expressing a different dsRNA ADD TO WELLS IN PLATE 96-well plate

1	Figure 8–59 RNA interference provides a convenient method for conducting genome-wide genetic screens. In this experiment, each well in this 96-well plate is filled with E. coli that produce a different double-stranded RNA. Each interfering RNA matches the nucleotide sequence of a single C. elegans gene, thereby inactivating it. About 10 worms are added to each well, where they ingest the genetically modified bacteria. The plate is incubated for several days, which gives the RNAs time to inactivate their target genes—and the worms time to grow, mate, and produce offspring. The plate is then examined in a microscope, which can be controlled robotically, to screen for genes that affect the worms’ ability to survive, reproduce, develop, and behave. Shown here are normal worms alongside worms that show an impaired ability to reproduce due to inactivation of a particular “fertility” gene. (From B. Lehner et al., Nat. Genet. 38:896–903, 2006. With permission from Macmillan Publishers Ltd.)

1	that show an impaired ability to reproduce due to inactivation of a particular “fertility” gene. (From B. Lehner et al., Nat. Genet. 38:896–903, 2006. With permission from Macmillan Publishers Ltd.) unknown reasons, RNAi does not efficiently inactivate all genes. Moreover, within whole organisms, certain tissues may be resistant to the action of RNAi (for example, neurons in nematodes). Another problem arises because many organisms contain large gene families, the members of which exhibit sequence similarity. RNAi therefore sometimes produces “off-target” effects, inactivating related genes in addition to the targeted gene. One strategy to avoid such problems is to use multiple small RNA molecules matched to different regions of the same gene. Ultimately, the results of any RNAi experiment must be viewed as a strong clue to, but not necessarily a proof of, normal gene function.

1	In the preceding section, we discussed how genetic approaches can be used to assess a gene’s function in cultured cells or, even better, in the intact organism. Although this information is crucial to understanding gene function, it does not generally reveal the molecular mechanisms through which the gene product works in the cell. For example, genetics on its own rarely tells us all the places in the organism where the gene is expressed, or how its expression is controlled. It does not necessarily reveal whether the gene acts in the nucleus, the cytosol, on the cell surface, or in one of the numerous other compartments of the cell. And it does not reveal how a gene product might change its location or its expression pattern when the external environment of the cell changes. Key insights into gene function can be obtained by simply observing when and where a gene is expressed. A variety of approaches, most involving some form of genetic engineering, can easily provide this critical

1	into gene function can be obtained by simply observing when and where a gene is expressed. A variety of approaches, most involving some form of genetic engineering, can easily provide this critical information.

1	As discussed in detail in Chapter 7, cis-regulatory DNA sequences, located upstream or downstream of the coding region, control gene transcription. These regulatory sequences, which determine precisely when and where the gene is expressed, can be easily studied by placing a reporter gene under their control and introducing these recombinant DNA molecules into cells (Figure 8–60). In this way, the normal expression pattern of a gene can be determined, as well as 123 start site for RNA pattern of normal gene X DNA sequences that determine the expression of gene X cells 123 pattern of reporter gene Y expression CONCLUSIONS —cis-regulatory sequence 3 normally turns on gene X in cell B —cis-regulatory sequence 2 normally turns on gene X in cells D, E, and F —cis-regulatory sequence 1 normally turns off gene X in cell D

1	Figure 8–60 Using a reporter protein to determine the pattern of a gene’s expression. (A) In this example, the coding sequence for protein X is replaced by the coding sequence for reporter protein Y. The expression patterns for X and Y are the same. (B) Various fragments of DNA containing candidate cis-regulatory sequences are added in combinations to produce test DNA molecules encoding reporter gene Y. These recombinant DNA molecules are then tested for expression after introducing them into a variety of different types of mammalian cells. The results are summarized in (C). For experiments in eukaryotic cells, two commonly used reporter proteins are the enzyme β-galactosidase (β-gal) (see Figure 7–28) and green fluorescent protein (GFP) (see Figure 9–22). the contribution of individual cis-regulatory sequences in establishing this pattern (see also Figure 7–29).

1	the contribution of individual cis-regulatory sequences in establishing this pattern (see also Figure 7–29). Reporter genes also allow any protein to be tracked over time in living cells. Here, the reporter gene typically encodes a fluorescent protein, often green fluorescent protein (GFP), the molecule that gives luminescent jellyfish their greenish glow. The GFP is simply attached—in the coding frame—to the protein-coding gene of interest. The resulting GFP fusion protein often behaves in the same way the normal protein does and its location can be monitored by fluorescence microscopy, a topic that is discussed in the next chapter (see Figure 9–25). GFP fusion has become a standard strategy for tracking not only the location but also the movement of specific proteins in living cells. In addition, the use of multiple GFP variants that fluoresce at different wavelengths can provide insights into how different cells interact in a living tissue (Figure 8–61).

1	In situ Hybridization Can Reveal the Location of mRNAs and Noncoding RNAs

1	It is also possible to directly observe the time and place that an RNA product of a gene is expressed using in situ hybridization. For protein-coding genes, this strategy often provides the same general information as the reporter gene approaches described above; however, it is crucial for genes whose final product is RNA rather than protein. We encountered in situ hybridization earlier in the chapter (see Figure 8–34); it relies on the basic principles of nucleic acid hybridization. Typically, tissues are gently fixed so that their RNA is retained in an exposed form that can hybridize with a labeled complementary DNA or RNA probe. In this way, the patterns of differential gene expression can be observed in tissues, and the location of specific RNAs can be determined (Figure 8–62). An advantage of in situ hybridization over other approaches is that genetic engineering is not required. Thus, it is often simpler and faster and can be used for genetically intractable species.

1	Expression of Individual Genes Can Be Measured Using Quantitative RT-PCR Although reporter genes and in situ hybridization accurately reveal patterns of gene expression, they are not the most powerful methods for quantifying amounts of individual RNAs in cells. We have seen that RNA sequencing can provide information about the relative abundance of different RNA molecules (see Figure 7–3). Here, the number of “sequence reads” (short bits of nucleotide sequence) is proportional to the abundance of the RNA species. But this method is limited to RNAs

1	Figure 8–61 GFPs that fluoresce at different wavelengths help reveal the connections that individual neurons make within the brain. This image shows differently colored neurons in one region of a mouse brain. The neurons randomly express different combinations of differently colored GFPs (see Figure 9–13), making it possible to distinguish and trace many individual neurons within a population. These images were obtained by genetically engineering the genes for four different fluorescent proteins, each flanked by loxP sites of recombination (see Figure 5–66), and integrating them into the mouse germ line. When crossed to a mouse that produced the Cre recombinase in neuronal cells, the fluorescent protein genes were randomly excised, producing neurons that express many different combinations of the four fluorescent proteins. Over 100 combinations of fluorescent protein can be produced, allowing scientists to distinguish one neuron from the next. The stunning appearance of these labeled

1	of the four fluorescent proteins. Over 100 combinations of fluorescent protein can be produced, allowing scientists to distinguish one neuron from the next. The stunning appearance of these labeled neurons has earned these animals the colorful nickname “brainbow mice.” (From J. Livet et al., Nature 450:56–62, 2007. With permission from Macmillan Publishers Ltd.)

1	Figure 8–62 In situ hybridization to mRNAs has been used to generate an atlas of gene expression in the mouse brain. This computer-generated image shows the expression of several different mRNAs specific to an area of the brain associated with learning and memory. Similar maps of expression patterns of all known genes in the mouse brain are compiled in the brain atlas project, which is available online. (From M. Hawrylycz et al., PLoS Comput. Biol. 7:e1001065, 2011.) Figure 8–63 RNA levels can be measured by quantitative RT-PCR. The fluorescence measured is generated by a dye that fluoresces only when bound to the double-stranded DNA products of the RT-PCR (see Figure 8–36). The red sample has a higher concentration of the mRNA being measured than does the blue sample, since it requires fewer PCR cycles to reach the same half-maximal concentration of double-stranded DNA. Based on this difference, the relative amounts of the mRNA in the two samples can be precisely determined.

1	that are expressed at reasonably high levels, and it is difficult to quantify (or even identify) rare RNAs. A more accurate method is based on the principles of PCR (Figure 8–63). Called quantitative RT-PCR (reverse transcription–polymerase chain reaction), this method begins with the total population of RNA molecules purified from a tissue or a cell culture. It is important that no DNA be present in the preparation; it must be purified away or enzymatically degraded. Two DNA primers that specifically match the mRNA of interest are added, along with reverse transcriptase, DNA polymerase, and the four deoxyribonucleoside triphosphates needed for DNA synthesis. The first round of synthesis is the reverse transcription of the RNA into DNA using one of the primers. Next, a series of heating and cooling cycles allows the amplification of that DNA strand by PCR (see Figure 8–36). The quantitative part of this method relies on a direct relationship between the rate at which the PCR product

1	and cooling cycles allows the amplification of that DNA strand by PCR (see Figure 8–36). The quantitative part of this method relies on a direct relationship between the rate at which the PCR product is generated and the original concentration of the mRNA species of interest. By adding chemical dyes to the PCR that fluoresce only when bound to double-stranded DNA, a simple fluorescence measurement can be used to track the progress of the reaction and thereby accurately deduce the starting concentration of the mRNA that is amplified. Although it seems complicated, this quantitative RT-PCR technique is relatively fast and simple to perform in the laboratory; it is currently the method of choice for accurately quantifying mRNA levels from any given gene.

1	Analysis of mRNAs by Microarray or RNA-seq Provides a Snapshot of Gene Expression As discussed in Chapter 7, a cell expresses only a subset of the many thousands of genes available in its genome; moreover, this subset differs from one cell type to another or, in the same cell, from one environment to the next. One way to determine which genes are being expressed by a population of cells or a tissue is to analyze which mRNAs are being produced.

1	The first tool that allowed investigators to analyze simultaneously the thousands of different RNAs produced by cells or tissues was the DNA microarray. Developed in the 1990s, DNA microarrays are glass microscope slides that contain hundreds of thousands of DNA fragments, each of which serves as a probe for the mRNA produced by a specific gene. Such microarrays allow investigators to monitor the expression of every gene in a genome in a single experiment. To do the analysis, mRNAs are extracted from cells or tissues and converted to cDNAs (see Figure 8–31). The cDNAs are fluorescently labeled and allowed to hybridize to the fragments bound to the microarray. An automated fluorescence microscope then determines which mRNAs were present in the original sample based on the array positions to which the cDNAs are bound (Figure 8–64).

1	Although microarrays are relatively inexpensive and easy to use, they suffer from one obvious drawback: the sequences of the mRNA samples to be analyzed must be known in advance and represented by a corresponding probe on the array. With the development of improved sequencing technologies, investigators increasingly use RNA-seq, discussed earlier, as a more direct approach for cataloging the RNAs produced by a cell. For example, this approach can readily detect alternative RNA splicing, RNA editing, and the many noncoding RNAs produced from a complex genome. DNA microarrays and RNA-seq analysis have been used to examine everything from the changes in gene expression that make strawberries ripen to the gene expression “signatures” of different types of human cancer cells; or from changes time (number of PCR cycles)

1	Figure 8–64 DNA microarrays are used to analyze the production of mRNA from mRNA from thousands of different mRNAs in a single experiment. In this example, sample 1 sample 2 mRNA is collected from two different cell samples—for example, cells treated with a hormone and untreated cells of the same type—to allow for a direct comparison of the specific genes expressed under both conditions. convert to cDNA, convert to cDNA,

1	The mRNAs are converted to cDNAs that are labeled with a red fluorescent labeled with red labeled with green dye for one sample and a green fluorescent dye for the other. The labeled samples are mixed and then allowed to hybridize to the microarray. Each microscopic spot on the microarray is a 50-nucleotide DNA molecule of defined sequence made by chemical synthesis and spotted on the array. The DNA sequence represented by each spot is different, and the hundreds of thousands of such spots are designed to span the sequence of the genome. The DNA sequence of each spot is kept track of by computer. After incubation, the array is washed and the fluorescence scanned. Only a small proportion of the microarray, representing 676 genes, is shown. Red spots indicate that the gene in sample 1 is expressed at a higher level than the corresponding gene in sample 2, and the green spots indicate the opposite. Yellow spots reveal genes that are expressed at about equal levels in both cell samples.

1	at a higher level than the corresponding gene in sample 2, and the green spots indicate the opposite. Yellow spots reveal genes that are expressed at about equal levels in both cell samples. The intensity of the fluorescence provides an estimate of how much RNA is present from a gene. Dark spots indicate little or no expression of the gene whose probe is located at that position in the array.

1	that occur as cells progress through the cell cycle to those made in response to sudden shifts in temperature. Indeed, because these approaches allow the simultaneous monitoring of large numbers of RNAs, they can detect subtle changes in a cell, changes that might not be manifested in its outward appearance or behavior.

1	Comprehensive studies of gene expression also provide information that is useful for predicting gene function. Earlier in this chapter, we discussed how identifying a protein’s interaction partners can yield clues about that protein’s function. A similar principle holds true for genes: information about a gene’s function can be deduced by identifying genes that share its expression pattern. Using an approach called cluster analysis, one can identify sets of genes that are coordinately regulated. Genes that are turned on or turned off together under different circumstances are likely to work in concert in the cell: they may encode proteins that are part of the same multiprotein machine, or proteins that are involved in a complex coordinated activity, such as DNA replication or RNA splicing. Characterizing a gene whose function is unknown by grouping it with known genes that share its transcriptional behavior is sometimes called “guilt by association.” Cluster analyses have been used to

1	Characterizing a gene whose function is unknown by grouping it with known genes that share its transcriptional behavior is sometimes called “guilt by association.” Cluster analyses have been used to analyze the gene expression profiles that underlie many interesting biological processes, including wound healing in humans (Figure 8–65).

1	Figure 8–65 Using cluster analysis to identify sets of genes that are coordinately regulated. Genes that have the same expression pattern are likely to be involved in common pathways or processes. To perform a cluster analysis, RNA-seq or microarray data are obtained from cell samples exposed to a variety of different conditions, and genes that show coordinate changes in their expression pattern are grouped together. In this experiment, human fibroblasts were deprived of serum for 48 hours; serum was then added back to the cultures at time 0 and the cells were harvested for microarray analysis at different time points. Of the 8600 genes depicted here (each represented by a thin, vertical line), just over 300 showed threefold or greater variation in their expression patterns in response to serum reintroduction. Here, red indicates an increase in expression; green is a decrease in expression. On the basis of the results of many other experiments, the 8600 genes have been grouped in

1	to serum reintroduction. Here, red indicates an increase in expression; green is a decrease in expression. On the basis of the results of many other experiments, the 8600 genes have been grouped in clusters based on similar patterns of expression. The results of this analysis show that genes involved in wound healing are turned on in response to serum, while genes involved in regulating cell-cycle progression and cholesterol biosynthesis are shut down. (From M.B. Eisen et al., Proc. Natl Acad. Sci. USA 94:14863–14868, 1998. With permission from National Academy of Sciences.)

1	Figure 8–66 Chromatin immunoprecipitation. This method allows the identification of all the sites in a genome that a transcription regulator occupies in vivo. The identities of the precipitated, amplified DNA fragments are determined by DNA sequencing. Genome-wide Chromatin Immunoprecipitation Identifies Sites on the Genome Occupied by Transcription Regulators

1	We have discussed several strategies to measure the levels of individual RNAs in a cell and to monitor changes in their levels in response to external signals. But this information does not tell us how such changes are brought about. We saw in Chapter 7 that transcription regulators, by binding to cis-regulatory sequences in DNA, are responsible for establishing and changing patterns of transcription. Typically, these proteins do not occupy all of their potential cis-regulatory sequences in the genome under all conditions. For example, in some cell types, the regulatory protein may not be expressed, or it may be present but lack an obligatory partner protein, or it may be excluded from the nucleus until an appropriate signal is received from the cell’s environment. Even if the protein is present in the nucleus and is competent to bind DNA, other transcription regulators or components of chromatin can occupy overlapping DNA sequences and thereby occlude some of its cis-regulatory

1	is present in the nucleus and is competent to bind DNA, other transcription regulators or components of chromatin can occupy overlapping DNA sequences and thereby occlude some of its cis-regulatory sequences in the genome.

1	Chromatin immunoprecipitation provides a way to experimentally determine all the cis-regulatory sequences in a genome that are occupied by a given transcription regulator under a particular set of conditions (Figure 8–66). In this approach, proteins are covalently cross-linked to DNA in living cells, the cells are broken open, and the DNA is mechanically sheared into small fragments. Antibodies directed against a given transcription regulator are then used to purify the DNA that became covalently cross-linked to that protein in the cell. This DNA is then sequenced using the rapid methods discussed earlier; the precise location of each precipitated DNA fragment along the genome is determined by comparing its DNA sequence to that of the whole genome sequence (Figure 8–67). In this way, all of the sites occupied by the transcription regulator in the cell sample can be mapped across the cell’s genome (see Figure 7–37). In combination with microarray or RNA-seq information, chromatin

1	all of the sites occupied by the transcription regulator in the cell sample can be mapped across the cell’s genome (see Figure 7–37). In combination with microarray or RNA-seq information, chromatin immunoprecipitation can identify the key transcriptional regulator responsible for specifying a particular pattern of gene expression.

1	Chromatin immunoprecipitation can also be used to deduce the cis-regulatory sequences recognized by a given transcription regulator. Here, all the DNA sequences precipitated by the regulator are lined up (by computer) and features in common are tabulated to produce the spectrum of cis-regulatory sequences recognized by the protein (see Figure 7–9A). Chromatin immunoprecipitation is also used routinely to identify the positions along a genome that are bound by the various types of modified histones discussed in Chapter 4. In this case, antibodies specific to the particular histone modification are employed (see Figure 8–67). A variation of the technique can also be used to map positions of chromosomes that are in physical proximity (see Figure 4–48). Ribosome Profiling Reveals Which mRNAs Are Being Translated in the Cell

1	Ribosome Profiling Reveals Which mRNAs Are Being Translated in the Cell In preceding sections, we discussed several ways that RNA levels in the cell can be monitored. But for mRNAs, this represents only one step in gene expression, and we are often more interested in the final level of the protein produced by the gene. As described in the first part of this chapter, mass-spectroscopy methods can be used to monitor the levels of all proteins in the cell, including modified forms of the proteins. However, if we want to understand how synthesis of proteins is controlled by the cell, we need to consider the translation step of gene expression.

1	An approach called ribosome profiling provides an instantaneous map of the position of ribosomes on each mRNA in the cell and thereby identifies those many other DNA fragments that comprise the rest of the genome mRNAs that are being actively translated. To accomplish this, total RNA from a cell line or tissue is exposed to RNAses under conditions where only those RNA sequences covered by ribosomes are spared. The protected RNAs are released from ribosomes, converted to DNA, and the nucleotide sequence of each is determined (Figure 8–68). When these sequences are mapped on the genome, the position of ribosomes across each mRNA species can be ascertained.

1	Ribosome profiling has revealed many cases where mRNAs are abundant but are not translated until the cell receives an external signal. It has also shown that many open reading frames (ORFs) that were too short to be annotated as genes are actively translated and probably encode functional, albeit very small, proteins (Figure 8–69). Finally, ribosome profiling has revealed the ways that cells rapidly and globally change their translation patterns in response to sudden changes in temperature, nutrient availability, or chemical stress.

1	We have seen that nucleic acid methodologies developed in the past 40 years have completely changed the way that cell and molecular biology is studied. But they have also had a profound effect on our day-to-day lives. Many human pharmaceuticals in routine use (insulin, human growth hormone, blood-clotting factors, and interferon, for example) are based on cloning human genes and expressing the encoded proteins in large amounts. As DNA sequencing continues to drop in cost, more and more individuals will elect to have their genome sequenced; this information can be used to predict susceptibility to diseases (often with the option of minimizing this possibility by appropriate behavior) or to predict the way an individual will respond to a given drug. The genomes of tumor cells from an individual can be sequenced to determine the best type of anticancer treatment. And mutations that cause or greatly increase the risk of disease continue to be identified at an unprecedented pace. Using the

1	can be sequenced to determine the best type of anticancer treatment. And mutations that cause or greatly increase the risk of disease continue to be identified at an unprecedented pace. Using the recombinant DNA technologies discussed in this chapter, these mutations can then be introduced into animals, such as mice, that can be studied in the laboratory. The resulting transgenic animals, which often mimic some of the phenotypic abnormalities associated with the condition in patients, can be used to explore the cellular and molecular basis of the disease and to screen for drugs that could potentially be used therapeutically in humans.

1	number of sequence reads

1	Figure 8–67 Results of several chromatin immunoprecipitations showing proteins bound to the control region that control expression of the Oct4 gene. In this series of chromatin immunoprecipitation experiments, antibodies directed against a transcription regulator (first three panels) or a particular histone modification (fourth panel) were used to precipitate bound, cross-linked DNA. Precipitated DNA was sequenced, and the positions across the genome were mapped. (Only the small part of the mouse genome containing the Oct4 gene is shown.) The results show that, in the embryonic stem cells analyzed in these experiments, Oct4 binds upstream of its own gene and that Sox2 and Nanog are bound in close proximity. Oct4, Sox2, and Nanog are key regulators in embryonic stem cells (discussed in Chapter 22) and this experiment reveals the position on the genome through which they exert their effects on Oct4 expression. In the fourth panel, the positions of a histone modification associated with

1	22) and this experiment reveals the position on the genome through which they exert their effects on Oct4 expression. In the fourth panel, the positions of a histone modification associated with actively transcribed genes is shown (see Figure 4–39). Finally, the bottom panel shows the RNA produced from the Oct4 gene under the same conditions used for the chromatin immunoprecipitations. Note that the introns and exons are relatively easy to identify from these RNA-seq data.

1	Figure 8–68 Ribosome profiling. RNA AUG UGA AAAAAAAAAAAA is purified from cells and digested with an RNAse to leave only those portions AUG of the mRNAs that are protected by a bound ribosome. These short pieces remove ribosomes convert RNA to DNA and sequence Although we tend to think of recombinant DNA research in terms of animal biology, these techniques have also had a profound impact on the study of plants. In fact, certain features of plants make them especially amenable to recombinant DNA methods.

1	When a piece of plant tissue is cultured in a sterile medium containing nutrients and appropriate growth regulators, some of the cells are stimulated to proliferate indefinitely in a disorganized manner, producing a mass of relatively undifferentiated cells called a callus. If the nutrients and growth regulators are carefully manipulated, one can induce the formation of a shoot within the callus, and in many species a whole new plant can be regenerated from such shoots. In a number of plants—including tobacco, petunia, carrot, potato, and Arabidopsis—a single cell from such a callus (known as a totipotent cell) can be grown into a small clump of cells from which a whole plant can be regenerated (see Figure 7–2B). Just as mutant mice can be derived by the genetic manipulation of embryonic stem 200 ORF discovered through ribosome profling nucleotide pairs codes for a protein of 20 amino acids of protected RNA (approximately 20 nucleotides in length) are converted to DNA and sequenced.

1	stem 200 ORF discovered through ribosome profling nucleotide pairs codes for a protein of 20 amino acids of protected RNA (approximately 20 nucleotides in length) are converted to DNA and sequenced. The resulting information is displayed as the number of sequence reads along each position of the genome. In the diagram here, the data for only one gene, whose mRNA is being efficiently translated, are shown. Ribosome profiling provides this type of information for every mRNA produced by the cell.

1	Figure 8–69 Ribosome profiling can identify new genes. This experiment shows the discovery of a previously unrecognized gene—one that encodes a protein of only 20 amino acids. At the top is shown a portion of a viral genome with two previously annotated genes. Below are the results of a ribosome profiling experiment, displayed across the same section of the genome, after the virus was infected into human cells. The results show that the left-hand gene is not expressed under these conditions, the right-hand gene is expressed at low levels, and a previously unrecognized gene that lies between them position along genome is expressed at high levels. 508 Chapter 8: Analyzing Cells, Molecules, and Systems leaf discs incubated with genetically engineereddiscs removed from cells in culture, so transgenic plants can be created from plant cells transfected with DNA in culture (Figure 8–70).

1	The ability to produce transgenic plants has greatly accelerated progress in many areas of plant cell biology. It has played an important part, for example, in isolating receptors for growth regulators and in analyzing the mechanisms of morphogenesis and of gene expression in plants. These techniques have also opened up many new possibilities in agriculture that could benefit both the farmer and the consumer. They have made it possible, for example, to modify the ratio of lipid, starch, and protein in seeds, to impart pest and virus resistance to plants, and to create modified plants that tolerate extreme habitats such as salt marshes or water-stressed soil. One variety of rice has been genetically engineered to produce β-carotene, the precursor of vitamin A. Were it to replace conventional rice, this “golden rice”—so-called because of its faint yellow color—could help to alleviate severe vitamin A deficiency, which causes blindness in hundreds of thousands of children in the

1	rice, this “golden rice”—so-called because of its faint yellow color—could help to alleviate severe vitamin A deficiency, which causes blindness in hundreds of thousands of children in the developing world each year.

1	Genetics and genetic engineering provide powerful tools for understanding the function of individual genes in cells and organisms. In the classical genetic approach, random mutagenesis is coupled with screening to identify mutants that are deficient in a particular biological process. These mutants are then used to locate and study the genes responsible for that process.

1	Gene function can also be ascertained by reverse genetic techniques. DNA engineering methods can be used to alter genes and to re-insert them into a cell’s chromosomes so that they become a permanent part of the genome. If the cell used for this gene transfer is a fertilized egg (for an animal) or a totipotent plant cell in culture, transgenic organisms can be produced that express the mutant gene and pass it on to their progeny. Especially important for cell and molecular biology is the ability to alter cells and organisms in highly specific ways—allowing one to discern the effect on the cell or the organism of a designed change in a single protein or RNA molecule. For example, genomes can be altered so that the expression of any gene can be switched on or off by the experimenter.

1	Figure 8–70 Transgenic plants can be made using recombinant DNA techniques optimized for plants. A disc is cut out of a leaf and incubated in a culture of Agrobacterium that carries a recombinant plasmid with both a selectable marker and a desired genetically engineered gene. The wounded plant cells at the edge of the disc release substances that attract the bacteria, which inject their DNA into the plant cells. Only those plant cells that take up the appropriate DNA and express the selectable marker gene survive and proliferate and form a callus. The manipulation of growth factors supplied to the callus induces it to form shoots, which subsequently root and grow into adult plants carrying the engineered gene.

1	Many of these methods are being expanded to investigate gene function on a genome-wide scale. The generation of mutant libraries in which every gene in an organism has been systematically deleted, disrupted, or made controllable by the experimenter provides invaluable tools for exploring the role of each gene in the elaborate molecular collaboration that gives rise to life. Technologies such as RNA-seq and DNA microarrays can monitor the expression of tens of thousands of genes simultaneously, providing detailed, comprehensive snapshots of the dynamic patterns of gene expression that underlie complex cell processes.

1	Quantitative experiments combined with mathematical theory mark the beginning of modern science. Galileo, Kepler, Newton, and their contemporaries did more than set out some rules of mechanics and offer an explanation of the movements of the planets around the Sun: they showed how a quantitative mathematical approach could provide a depth and precision of understanding, at least for physical systems, that had never before been dreamed to be possible. What is it that gives mathematics this almost magical power to explain the natural world, and why has mathematics played so much more important a part in physical sciences than in biology? What do biologists need to know about mathematics?

1	Mathematics can be viewed as a tool for deriving logical consequences from propositions. It differs from ordinary intuitive reasoning in its insistence on rigorous, accurate logic and the precise treatment of quantitative information. If the initial propositions are correct, then the deductions drawn from them by mathematics will be true. The surprising power of mathematics comes from the length of the chains of reasoning that rigorous logic and mathematical arguments make possible, and from the unexpectedness of the conclusions that can be reached, often revealing connections that one would not otherwise have guessed at. Reversing the argument, mathematics provides a way to test experimental hypotheses: if mathematical reasoning from a given hypothesis leads to a prediction that is not true, then the hypothesis is not true.

1	Clearly, mathematics is not much use unless we can frame our ideas—our initial hypotheses—about the given system in a precise, quantitative form. A mathematical edifice raised on a rickety or—even worse—a vague or overcomplicated set of propositions is likely to lead us astray. For mathematics to be useful, we must focus our analysis on simple subsystems in which we can pick out key quantitative parameters and frame well-defined hypotheses. This approach has been used with great success in physics for centuries, but it has been less common in biology. But times are changing, and more and more it is becoming possible for biologists to exploit the power of quantitative mathematical analysis.

1	In this final section of our methods chapter, we do not attempt to teach readers every way in which mathematics can be fruitfully applied to biological problems. Rather, we simply aim to give a sense of what mathematics and quantitative approaches can do for us in modern biology. We focus primarily on the important principles that mathematics teaches us about the dynamics of molecular interactions, and how mathematics can unveil surprising and useful features of complex systems containing feedback. We will illustrate these principles using the regulation of gene expression by transcription regulators like those discussed in Chapter 7. The same principles apply to the post-transcriptional regulatory systems that govern cell signaling (Chapter 15), cell-cycle control (Chapter 17), and essentially all cell processes.

1	Cell function and regulation depend on transient interactions among thousands of different macromolecules in the cell. We often summarize these interactions in this book with schematic cartoons. These diagrams are useful, but a complete picture requires a deeper, more quantitative level of understanding. To meaningfully assess the biological impact of any interaction in the cell, we need to know in precise terms how the molecules interact, how they catalyze reactions, and, most importantly, how the behaviors of the molecules change over time. If a cartoon shows that protein A activates protein B, for example, we cannot judge the importance of this relationship without quantitative details about the concentrations, affinities, and kinetic behaviors of proteins A and B.

1	Let us begin by defining two different types of regulatory interaction in our cartoons: one designating inhibition and the other designating activation. If the protein product of gene X is a transcription repressor that inhibits the expression of gene Z, we depict the relationship as a red bar-headed line ( ) drawn between genes X and Z (Figure 8–71). If the protein product of gene Y is a transcription activator that induces the expression of gene Z, then a green arrow ( ) is drawn between genes Y and Z.

1	The regulation of one gene’s expression by another is more complicated than a single arrow connecting them, and a complete understanding of this regulation requires that we tease apart the underlying biochemical processes. Figure 8–72A sketches some of the biochemical steps in the activation of gene expression by a transcription activator. A gene encoding the activator, designated as gene A, will produce its product, protein A, via an RNA intermediate. This protein A will then bind to pX, the regulatory promoter of gene X, to form the complex A:pX. Once the A:pX complex forms, it stimulates the production of an RNA transcript that is subsequently translated to produce protein X.

1	We will focus here on the binding interaction that lies at the heart of this regulatory system: the interaction between protein A and the promoter pX. Any molecule of protein A that is bound to pX can also dissociate from it. The steps represented by the green activation arrow in Figure 8–72A include both the binding of A to pX and the dissociation of the complex A:pX to re-form A and pX, as illustrated A:pX rate of complex formation = k [A][pX] rate of complex dissociation = koff [A:pX] at steady state: on [A][pX] = koff [A:pX] GENE X GENE Y Figure 8–71 Diagrams that summarize biochemical relationships. Here, a simple cartoon indicates that gene X represses gene Z (left) whereas gene Y activates gene Z (right).

1	Figure 8–72 A simple transcriptional interaction. (A) Genes A and X each produce a protein, with the product of gene A serving as a transcription activator to stimulate expression of gene X. As indicated by the green arrow, stimulation depends in part on the binding of protein A to the promoter region of gene X, designated as pX. (B) The binding of protein A to the gene promoter is determined by the concentrations of the two binding partners (denoted as [A] and [pX], in units of mol/liter, or M), the association rate constant kon (in units of sec–1 M–1), and the dissociation rate constant koff (in units of sec–1). (C) At steady state, the rates of association and dissociation are equal, and the concentration of the bound complex is determined by Equation 8–1, in which the two rate constants are combined in the equilibrium constant K. (D) Equation 8–2 can be derived to calculate the steady-state concentration of bound complex at a known total concentration of the promoter [pXT]. (E)

1	are combined in the equilibrium constant K. (D) Equation 8–2 can be derived to calculate the steady-state concentration of bound complex at a known total concentration of the promoter [pXT]. (E) Rearrangement of Equation 8–2 yields Equation 8–3, which allows calculation of the fraction of promoter pX that is occupied by protein A.

1	by the notation in Figure 8–72B. This reaction notation is more informative than the diagrams in our figures, but has its own limitations. Suppose that the concentration of A increases by a factor of ten as a response to an environmental input. If A increases, we intuitively know that A:pX should increase too, but we cannot determine the amount of the increase without additional information. We need to know the affinity of the binding interaction and the concentrations of the components. With this information in hand, we can rigorously derive the answer.

1	As discussed earlier and in Chapter 3 (see Figure 3–44), we know that the formation of a complex between two binding partners, such as A and pX, depends on a rate constant kon, which describes how many productive collisions occur per unit time per protein at a given concentration of pX. The rate of complex formation equals the product of this rate constant kon and the concentrations of A and pX (see Figure 8–72B). Complex dissociation occurs at a rate koff multiplied by the concentration of the complex. The rate constant koff can differ by orders of magnitude for different DNA sequences because it depends on the strength of the non-covalent bonds formed between A and pX.

1	We are primarily interested in understanding the amount of bound promoter complex at equilibrium or steady state, where the rate of complex formation equals the rate of complex dissociation. Under these conditions, the concentration of the promoter complex is specified by a simple equation that combines the two rate constants into a single equilibrium constant K = kon/koff (Equation 8–1; Figure 8–72C). K is sometimes called the association constant, Ka. The larger this constant K, the stronger the interaction between A and pX (see Figure 3–44). The reciprocal of K is the dissociation constant, Kd.

1	To calculate the steady-state concentration of promoter complex using Equation 8–1, we need to account for another complication: both A and pX exist in two forms—free in solution and bound to each other. In most cases, we know the total concentration of pX and not the free or bound concentrations, so we must find a way to use the total concentration in our calculations. To do this, we first specify that the total concentration of pX ([pXT ]) is the sum of the concentrations of free ([pX]) and bound ([A:pX]) forms (Figure 8–72D). This leads to a new equation that allows us to use [pXT ] to calculate the steady-state concentration of the promoter complex ([A:pX]) (Equation 8–2, Figure 8–72D).

1	Protein A also exists in two forms: free ([A]) and bound to pX ([A:pX]). In a cell, there are typically one or two copies of pX (assuming there is only one gene X per haploid genome) and multiple copies of A. As a result, we can safely assume that from the viewpoint of A, [A:pX] is negligible relative to the total [AT ]. This means that [A] ≈ [AT ], and we can just plug in the values of total [AT ] in Equation 8–2 without incurring appreciable error in the calculation of [A:pX]. Now, we are ready to determine the effects of increasing the concentration of

1	Now, we are ready to determine the effects of increasing the concentration of A. Suppose that K = 108 M–1, which is a typical value for many such interactions. The starting concentration of A is [AT ] = 10–9 M, and [pXT ] = 10–10 M (assuming there is one copy of gene X in a haploid yeast cell, for example, with a volume of around 2 × 10–14 L). Using Equation 8–2, we find that a tenfold increase in the concentration of A causes the amount of promoter complex [A:pX] to increase 5.5fold, from 0.09 × 10–10 M to 0.5 × 10–10 M at steady state. The effects of a tenfold increase in the concentration of A will vary dramatically depending on its starting concentration relative to the equilibrium constant. Only through this mathematical approach can we achieve a thorough understanding of what these effects will be and what impact they will have on the biological response.

1	To assess the biological impact of a change in transcription activator levels, it is also important in many cases to determine the fraction of the target gene promoter that is bound by the activator, since this number will be directly proportional to the activity of the gene’s promoter. In our case, we can calculate the fraction of the gene X promoter, pX, that has protein A bound to it by rearranging Equation 8–2 (Equation 8–3, Figure 8–72E). This fraction can be viewed as the probability that promoter pX is occupied, averaged over time. It is also equal to the average occupancy across a large population of cells at any instant in time. When there is no protein A present, pX is always free, the bound fraction is zero, and transcription is off. When [A] = 1/K, the promoter pX has a 50% chance of being occupied. When [A] greatly exceeds 1/K, the bound fraction is almost equal to one, meaning that pX is fully occupied and transcription is maximal.

1	The most important and basic insights for which we, as biologists, depend on mathematics concern the behavior of regulatory systems over time. This is the central theme of dynamics, and it was for the solution of problems in dynamics that the techniques of calculus were developed, by Newton and Leibniz, in the seventeenth century. Briefly, the general problem is this: if we are given the rates of change of a set of variables that characterize the system at any instant, how can we compute its future state? The problem becomes especially interesting, and the predictions often remarkable, when the rates of change themselves depend on the values of the state variables, as in systems with feedback.

1	Let us return to Equation 8–2 (Figure 8–72D), which tells us that when [A] changes, [A:pX] at steady state will also change to a new concentration that we can calculate with precision. However, [A:pX] does not change instantaneously to this value. If we hope to understand the behavior of this system in detail, we must also ask how long it takes [A:pX] to get to its new steady-state value inside the cell. Equation 8–2 cannot answer this question. We need calculus.

1	The most common strategy for solving this problem is to use ordinary differential equations. The equations that describe biochemical reactions have a simple premise: the rate of change in the concentration of any molecular species X (that is, d[X]/dt) is given by the balance of the rate of its appearance with that of its disappearance. For our example, the rate of change in the concentration of the bound promoter complex, [A:pX], is determined by the rates of complex assembly and disassembly. We can incorporate these rates into the differential equation shown in Figure 8–73A (Equation 8–4). When [A] changes, Equation 8–4 can be solved to generate the concentration of [A:pX] as a function of time. Notice that when kon [A][pX] = koff [A:pX], then d[A:pX]/dt = 0 and [A:pX] stops changing. At this point, the system has reached the steady state.

1	Calculation of all [A:pX] values as a function of time, using Equation 8–4, allows us to determine the rate at which [A:pX] reaches its steady-state value. Because this value is attained asymptotically, it is often most useful to compare the times needed to get to 50, 90, or 99 percent of this new steady state. The simplest way to determine these values is to solve Equation 8–4 with a method called numerical integration, which involves plugging in values for all of the parameters (kon, koff, etc.) and then using a computer to determine the values of [A:pX] over time, starting from given initial concentrations of [A] and [pX]. For kon = 0.5 × 107 sec–1 M–1, koff = 0.5 × 10–1 sec–1 (K = 108 M–1 as above), and [pXT ] = 10–10 M, it takes [A:pX] about 5, 20, and 40 seconds to reach 50, 90, and 99 percent of the new steady-state value following a sudden tenfold change in [A] (Figure 8–73B). Thus, a sudden jump in [A] does not have instantaneous effects, as we might have assumed from looking

1	99 percent of the new steady-state value following a sudden tenfold change in [A] (Figure 8–73B). Thus, a sudden jump in [A] does not have instantaneous effects, as we might have assumed from looking at the cartoon in Figure 8–72A.

1	Differential equations therefore allow us to understand the transient dynamics of biochemical reactions. This tool is critical for achieving a deep understanding of cell behavior, in part because it allows us to determine the dependence of the dynamics inside cells on parameters that are specific to the particular molecules Figure 8–73 Using differential equations to study the dynamics and steady-state behavior of a biological system.

1	Figure 8–73 Using differential equations to study the dynamics and steady-state behavior of a biological system. (A) Equation 8–4 is an ordinary differential equation for calculating the rate of change in the formation of bound promoter complex in response to a change in other components. (B) Formation of [A:pX] after a tenfold increase in [A], as determined by solving Equation 8–4. In blue is the solution corresponding to kon = 0.5 × 107 sec–1 M–1 and koff = 0.5 × 10–1 sec–1. In this case, it takes [A:pX ] about 5, 20, and 40 seconds to reach 50, 90, and 99 percent of the new steady-state value. For the red curve, the kon and koff values are doubled, and the system reaches the same steady state more rapidly.

1	[A:pX] (multiples of initial value) involved. For example, if we double the values of both kon and koff, then Equa tion 8–1 (Figure 8–72C) indicates that the steady-state value of [A:pX] does not change. However, the time it takes to reach 50% of this steady state after a ten-fold 4.5 3.5 2.5 d[A:pX] = rate of complex formation – rate of complex dissociation 1.5 d[A:pX] 0.5 on [A][pX] – koff [A:pX] protein production rate = ˜.m change in [A] in our example changes from about 5 seconds to 2 seconds (see Figure 8–73B). These insights are not accessible from either cartoons or equilibrium equations. This is an unusually simple example; mathematical descriptions such as differential equations become more indispensible for understanding biological interactions as the number of interactions increases. Both Promoter Activity and Protein Degradation Affect the Rate of Change of Protein Concentration

1	Both Promoter Activity and Protein Degradation Affect the Rate of Change of Protein Concentration To understand our gene regulatory system further, we also need to describe the dynamics of protein X production in response to changes in the amount of transcription activator protein A. Here again, we use an ordinary differential equation for the rate of change of protein X concentration—determined by the balance of the rate of production of protein X through expression of gene X and the protein’s rate of degradation.

1	Let us begin with the rate of protein X production, which is determined primarily by the occupancy of the promoter of gene X by protein A. The binding and dissociation of a transcription regulator at a promoter generally occurs on a much faster time scale than transcription initiation, causing many binding and unbinding events to occur before transcription proceeds. As a result, we can assume that the binding reaction is at equilibrium on the time scale of transcription, and we can calculate promoter occupancy by protein A using the equilibrium equation discussed earlier (Equation 8–3, Figure 8–72E). To determine transcription rate, we simply multiply the occupied promoter fraction by a transcription rate constant, β, that represents the binding of RNA polymerase and the subsequent steps that lead to production of mRNA and protein (Figure 8–74A). If each mRNA molecule produces, on average, m molecules of protein product, then we can determine protein production rate by multiplying the

1	lead to production of mRNA and protein (Figure 8–74A). If each mRNA molecule produces, on average, m molecules of protein product, then we can determine protein production rate by multiplying the transcription rate by m (Figure 8–74A).

1	Now let us consider the factors that influence protein X degradation and its dilution due to cell growth. Degradation generally results in an exponential decline in protein levels, and the average time required for a specific protein to be 1.0 0.5 Figure 8–74 effect of protein lifetime on the timing of the response.

1	Figure 8–74 effect of protein lifetime on the timing of the response. (A) Equations for calculation of the rates of gene X transcription, protein X production, and protein X degradation, as explained in the text. (B) Equation 8–5 is an ordinary differential equation for calculating the rate of change in protein X in response to changes in other components. (C) When the rate of change in protein X is zero (steady state), its concentration can be calculated with Equation 8–6, revealing a direct relationship with protein lifetime (τ). (D) The solution of Equation 8–5 specifies the concentration of protein X over time as it approaches its steady-state concentration. (E) Response time depends on protein lifetime. As described in the text, the time that it takes a protein to reach a new steady state is greater when the protein is more stable. Here, the blue line corresponds to a protein with a lifetime that is 2.5-fold shorter than the lifetime of the protein in red.

1	degraded is defined as its mean lifetime, τ. In our current example, the rate of degradation of protein X depends on its mean lifetime τX, which takes into account active degradation as well as its dilution as the cell grows. The degradation rate depends on the concentration of protein X and is calculated by dividing this concentration by the lifetime (Figure 8–74A).

1	With equations for rates of production and degradation in hand, we can now generate a differential equation to determine the rate of change of protein X as a function of time (Equation 8–5, Figure 8–74B). This equation can be solved by the numerical methods mentioned earlier. According to the solution of this equation, when transcription begins, the concentration of protein X rises to a steady-state level at which the concentration of X is not changing anymore; that is, its rate of change is zero. When this occurs, rearrangement of Equation 8–5 yields an equation that can be used to determine the steady-state value of X, [Xst] (Equation 8–6, Figure 8–74C). An important concept emerges from the mathematics: the steady-state concentration of a gene product is directly proportional to its lifetime. If lifetime doubles, protein concentration doubles as well. The Time Required to Reach Steady State Depends on Protein Lifetime

1	We can see from Equation 8–6 (see Figure 8–74C) that when the concentration of protein A rises, protein X increases to a new steady-state value, [Xst]. But this cannot happen instantaneously. Instead, X changes dynamically according to the solution of its differential rate equation (Equation 8–5). The solution of this equation reveals that the concentration of X over time is related to its steady-state concentration according to the equation in Figure 8–74D. Once again, mathematics uncovers a simple but important concept that is not intuitively obvious: following a sudden increase in [A], [X] rises to a new steady state at an exponential rate that is inversely related to its lifetime; the faster X is degraded, the less time it takes it to reach its new steady-state value (Figure 8–74E). The faster response time comes at a higher metabolic cost, however, since proteins with a rapid response time must be produced and degraded at a high rate. For proteins that are not rapidly turned

1	The faster response time comes at a higher metabolic cost, however, since proteins with a rapid response time must be produced and degraded at a high rate. For proteins that are not rapidly turned over, the response time is very long, and protein concentration is determined primarily by the dilution that results from cell growth and division.

1	Positive control is not the only mechanism that cells use to regulate the expression of their genes. As we discussed in Chapter 7, cells also actively shut off genes, often by employing transcription repressor proteins that bind to specific sites on target genes, thereby blocking access to RNA polymerase. We can analyze the function of these repressors by the same quantitative methods described above for transcription activators. If a repressor protein R binds to the regulatory region of gene X and represses its transcription, then the fraction of gene binding sites occupied by the repressor is specified by the same equation we used earlier for the transcription activator (Figure 8–75A). In this case, however, it is only when the DNA is free that RNA polymerase can bind to the promoter and transcribe the gene. Thus, the quantity of interest is the unbound fraction, which can be viewed as the probability that the site is free, averaged over multiple binding and unbinding events. When

1	transcribe the gene. Thus, the quantity of interest is the unbound fraction, which can be viewed as the probability that the site is free, averaged over multiple binding and unbinding events. When the repressor concentration is zero, the unbound fraction is 1 and the promoter is fully active; when the repressor concentration greatly exceeds 1/K, the unbound fraction approaches zero. Figures 8–75B and C compare these relationships for a transcription activator and a transcription repressor.

1	We can create a differential equation that provides the rate of change in protein X when repressor concentrations change (Equation 8–7, Figure 8–75D). As in the case of the transcription activator, the steady-state concentration of protein X increases as its lifetime increases, but it decreases as the concentration of the transcription repressor increases. 1.0 Thus far, we have considered simple regulatory systems of just a few components. In most of the complex regulatory systems that govern cell behaviors, multiple

1	Figure 8–75 How promoter occupancy depends on the binding affinity of a transcription regulator protein. (A) The fraction of a binding site that is occupied by a transcription repressor R is determined by an equation that is similar to the one we used for a transcription activator (see Figure 8–72E), except that in the case of a repressor we are interested primarily in the unbound fraction. (B) For a transcription activator A, half of the promoters are occupied when [A] = 1/KA. Gene activity is proportional to this bound fraction. (C) For a transcription repressor R, gene activity is proportional to the unbound fraction of promoters. As indicated, this fraction is reduced to half of its maximal value when [R]=1/KR. (D) As in the case of the transcription activator A (see Figure 8–74), 0.5 0.5 we can derive equations to assess the timing of protein X production as a function of repressor concentrations.

1	modules are linked to produce larger circuits that we call network motifs, which can produce surprisingly complex and biologically useful responses whose properties become apparent only through mathematical analysis. A particularly common and important network motif is the negative feedback loop, which can have dramatically different functions depending on how it is structured.

1	We take as a first example a network motif consisting of two linked modules (Figure 8–76A). Here, an input signal initiates the transcription of gene A, which produces a transcription activator protein A. This activates gene R, which synthesizes a transcription repressor protein R. Protein R in turn binds to the promoter of gene A to inhibit its expression. This cyclical organization creates a negative feedback loop that one can intuitively understand as a mechanism to prevent proteins from accumulating to high levels. But what can we learn about negative feedback loops, and their value in biology, by using mathematics to model them?

1	The negative feedback loop in Figure 8–76A can be modeled using Equation 8–7 (see Figure 8–75D) for the repression of gene A and Equation 8–5 (see Figure 8–74B) for the activation of gene R. Thus, for proteins A and R, we use the set of differential equations (Equation set 8–8) shown in Figure 8–76B. The two equations in this set are coupled, which means that they must be solved together to describe d[A] ˜A.mA [A] = ˜R.mR – 1 0.5 0 fraction steady-stateprotein level

1	Figure 8–76 A simple negative feedback motif. (A) Gene A negatively regulates its own expression by activating gene R. The product of gene R is a transcription repressor that inhibits gene A. (B) Equation set 8–8 can be solved to determine the dynamics of system components over time. (C) A system with negative feedback (blue) reaches its steady state faster than a system with no feedback (red). The plots indicate the levels of protein A, expressed as a fraction of the steady-state level. The blue line reflects the solution of Equation set 8–8, which includes negative feedback of gene A by the repressor R. The red line represents the solution when the rate of synthesis of A was set to a constant value that is unaffected by the repressor R. (C) time the behavior of A and R over time for any value of the input. As before, we plug in values for the parameters (βR, τR, etc.) and then use a computer to determine the values of [A] and [R] as a function of time after a sudden input activates

1	value of the input. As before, we plug in values for the parameters (βR, τR, etc.) and then use a computer to determine the values of [A] and [R] as a function of time after a sudden input activates gene A.

1	The results reveal several important properties of negative feedback. First, rather surprisingly, negative feedback increases the speed of the response to the activating input. As shown in Figure 8–76C, the system with negative feedback reaches its new steady state faster than the system with no feedback. Second, negative feedback is useful for protecting cells from perturbations that continuously arise in the cell’s internal environment—due either to random variations in the birth and death of molecules or to fluctuations in environmental variables such as temperature and nutritional supplies. Let us imagine, for example, that βA, the transcription rate constant for gene A, fluctuates by 25% of its value and ask whether and how much the levels of protein R are affected. The results, shown in Figure 8–77, reveal that a change in βA causes a smaller change in the steady-state value of R when the network has negative feedback.

1	A beautiful thing happens when a negative feedback loop contains some delay mechanism that slows the feedback signal through the loop: rather than generating a new stable state as in a rapid negative feedback loop, a delayed loop generates pulses, or oscillations, in the levels of its components. This can be seen, for example, if the number of components in a negative feedback loop increases, which leads to delays in the amount of time required for the cycle of signals to be completed. Figure 8–78 compares the behavior of two network motifs—one with a three-stage and one with a five-stage negative feedback loop. Using the same kinetic parameters at each stage in the two loops, one finds that stable oscillations arise in the longer loop, while in the shorter loop the same parameters lead to relatively rapid convergence to a stable steady state.

1	Changes in the parameters of a delayed negative feedback loop—binding affinities, transcription rates, or protein stabilities, for example—can change the amplitude and period of the oscillations, providing a remarkably versatile mechanism for generating all sorts of oscillators that can be used for various purposes in the cell. Indeed, many naturally occurring oscillators, including the calcium oscillators described in Chapter 15 and the cell-cycle network described in Chapter 17, use delayed negative feedback as the basis for biologically important oscillations. Not all of the oscillations observed in cells are thought to have a function, however. Oscillations become inevitable in a highly complex, multicomponent biochemical pathway like glycolysis, due simply to the large number of feedback loops that appear to be required for its regulation.

1	We have focused thus far on the binding of a single transcription regulator to a single site in a gene promoter. Many promoters, however, contain multiple adjacent binding sites for the same transcription regulator, and it is not uncommon for these regulators to interact with each other on the DNA to form dimers or larger oligomers. These interactions can result in a cooperative form of DNA binding,

1	Figure 8–77 The effect of fluctuations in kinetic rate constants on a system with negative feedback compared to one without feedback. The plot at left represents the levels of protein R after a sudden activating stimulus, according to the regulatory scheme in Figure 8–76A and determined by the solution of Equation set 8–8 (see Figure 8–76B). A perturbation was induced by changing βA from 4 M/min (red line) to 3 M/min (blue line). The plot at right shows the results when negative feedback was removed. The system with negative feedback deviates less from its normal operation as β changes than does the system with no feedback. Notice that, as in Figure 8–76C, the system with negative feedback also reaches its steady state more rapidly.

1	such that DNA-binding affinity increases at higher concentrations of the transcription regulator. Cooperativity produces a steeper transcriptional response to increasing regulator concentration than the response that can be generated by the binding of a monomeric protein to a single site. A steep transcriptional response of this sort, when present in conjunction with positive feedback, is an important ingredient for producing systems with the ability to switch between different discrete phenotypic states. To begin to understand how this occurs, we need to modify our equations to include cooperativity.

1	Cooperative binding events can produce steep S-shaped (or sigmoidal) relationships between the concentration of regulatory protein and the amount bound on the DNA (see Figure 15–16). In this case, a number called the Hill coefficient (h) describes the degree of cooperativity, and we can include this coefficient in our equations for calculating the bound fraction of promoter (Figure 8–79A). As the Hill coefficient increases, the dependence of binding on protein concentration becomes steeper (Figure 8–79B). In principle, the Hill coefficient is similar to the number of molecules that must come together to generate a reaction. In practice, however, cooperativity is rarely complete, and the Hill coefficient does not reach Figure 8–78 Oscillations arising from delayed negative feedback.

1	Figure 8–78 Oscillations arising from delayed negative feedback. A transcriptional circuit with three components (A, B) is less likely to oscillate than a transcriptional circuit with five components (C, D). The X (light blue), Y (dark blue), and Z (brown) here represent transcription regulatory proteins. For the simulations in (B) and (D), the system was initiated from random initial conditions for X, Y, and Z. Oscillations are produced by a delay induced as the signal propagates through the loop.

1	Figure 8–79 How the cooperative binding of transcription regulatory proteins affects the fraction of promoters bound. (A) Cooperativity is incorporated into our mathematical models by including a Hill coefficient (h) in the equations used previously to determine the fraction of bound promoter (see Figures 8–72E and 8–75A). When h is 1, the equations shown here become identical to the equations used previously, and there is no cooperativity. (B) The left panel depicts a cooperatively bound transcription activator and the right panel depicts a cooperatively 0.5 bound transcription repressor. Recall from Figure 8–75B that gene activity is proportional to bound activator (left panel) or unbound repressor (right panel). 1/KR Note that the plots get steeper as the Hill (B) concentration of protein A concentration of protein R coefficient increases.

1	1/KR Note that the plots get steeper as the Hill (B) concentration of protein A concentration of protein R coefficient increases. We turn now to positive feedback and its very important consequences. First and foremost, positive feedback can make a system bistable, enabling it to persist in either of two (or more) alternative steady states. The idea is simple and can be conveyed by drawing an analogy with a candle, which can exist either in a burning state or in an unlit state. The burning state is maintained by positive feedback: the heat generated by burning keeps the flame alight. The unlit state is maintained by the absence of this feedback signal: so long as sufficient heat has never been applied, the candle will stay unlit.

1	For the biological system, as for the candle, bistability has an important corollary: it means that the system has a memory, such that its present state depends on its history. If we start with the system in an Off state and gradually rack up the concentration of the activator protein, there will come a point where autostimulation becomes self-sustaining (the candle lights), and the system moves rapidly to an On state. If we now intervene to decrease the level of activator, there will come a point where the same thing happens in reverse, and the system moves rapidly back to an Off state. But the transition points for switching on and switching off are different, and so the current state of the system depends on the route by which it has been taken in the past—a phenomenon called hysteresis.

1	A simple case of positive feedback can be seen in a regulatory system in which a transcription regulator activates (directly or indirectly) its own expression, as in Figure 8–80A. Positive feedback can also arise in a circuit with many intervening repressors or activators, so long as the net overall effect of the interactions is activation (Figure 8–80B and C).

1	To illustrate how positive feedback can generate stable states, let us focus on a simple positive feedback loop containing two repressors, X and Y, each of which inhibits expression of the other (Figure 8–81A). As we saw with Equation set 8–8 (Figure 8–76B) earlier, we can create differential equations describing the rate of change of [X] and [Y] (Equation set 8–9, Figure 8–81B). We can further modify these equations to include cooperativity by adding Hill coefficients. As we did earlier, we can then create equations for calculating the concentrations of [X] and [Y] when the system reaches a steady state (that is, when (d[X]/dt ) = 0 and (d[Y]/dt ) = 0; Equations 8–10 and 8–11, Figure 8–81C).

1	Equations 8–10 and 8–11 can be used to carry out an intriguing mathematical procedure called a nullcline analysis. These equations define the relationships between the concentration of X at steady state, [Xst], and the concentration of Y at steady state, [Yst], which must be simultaneously satisfied. We can plug in different values for [Yst] in Equation 8–10, and calculate the corresponding [Xst] for each of these values. We can then graph [Xst] as a function of [Yst]. Next, we repeat the process by varying [Xst] in Equation 8–11 to graph the resulting [Yst]. The intersections of these two graphs determine the theoretically possible steady states of the system. For systems in which the Hill coefficients hX and hY are much larger than 1, the lines in the two graphs intersect at three locations (Figure 8–81D). In other systems that have the same arrangement of regulators but different parameters, there might only be one intersection, indicating the presence of only a single

1	Figure 8–80 Positive feedback of a gene onto itself through serially connected interactions. A sequence of activators and repressors of any length can be connected to produce a positive feedback loop, as long as the overall sign is positive. Because the negative of a negative is positive, not only circuit (A) and (B) but also circuit (C) create positive feedback. = ˜X.mX – dt 1 + (KY[Y])hY ˜X = ˜Y.mY – GENE Y dt 1 + (KX[X])hX ˜Y (B) concentration of X = ˜X.mX.˜X = ˜Y.mY.˜Y

1	Figure 8–81 A graphical nullcline analysis. (A) X inhibits Y and Y inhibits X, resulting in a positive feedback loop. (B) Equation set 8–9 can be used to determine the rate of change in the concentrations of proteins X and Y. (C) Equations 8–10 and 8–11 provide the concentrations of proteins X and Y, respectively, when these concentrations reach concentration of X a steady state. (D, E) Blue curves (called nullclines) are plots of [Xst]calculated from Equation 8–10 over a range of concentrations of [Yst]. Red curves indicate values of [Yst] calculated from Equation 8–11 over a range of concentrations of [Xst]. At an intersection of the two lines, both [X] and [Y] are at steady state. For plot (D), the binding of both proteins to their target gene promoters was cooperative (hX and hY much larger than 1), resulting in the presence of multiple intersections of the nullclines––suggesting that the system can assume multiple discrete steady states. In plot (E), the binding of protein X to

1	larger than 1), resulting in the presence of multiple intersections of the nullclines––suggesting that the system can assume multiple discrete steady states. In plot (E), the binding of protein X to the promoter of gene Y was not cooperative (hX close to 1), resulting in only one nullcline intersection and thus just one likely steady state.

1	steady state. For example, when there is a low cooperativity of protein X binding to the promoter of gene Y (that is, a small Hill coefficient, hX, in Equation 8–11), the plot of [Y] is less curved (Figure 8–81E), and it is less likely that there will be multiple intersections of the two curves.

1	We emphasized earlier that positive feedback typically generates a bistable system with two stable steady states. Why does the system modeled in Figure 8–81D have three? This conundrum can be explained by solving the reaction rate equations (Equation set 8–9, Figure 8–81B) for various different starting conditions of [X] and [Y], determining all values of [X] and [Y] as a function of time. Starting Figure 8–82 Analysis of the stability of a system’s steady states. (A) The dotted with each set of initial concentrations of [X] and [Y], these calculations produce lines are the nullclines for the system shown a so-called trajectory of points, each indicated by a curved green line on Figure in Figure 8–81. Also shown are dynamic 8–82A. A fascinating pattern emerges: each trajectory moves across the plot and trajectories (green) that show the changes settles in one of two steady states, but never in the third (middle steady state). over time in [X] and [Y], starting at a variety We conclude

1	the plot and trajectories (green) that show the changes settles in one of two steady states, but never in the third (middle steady state). over time in [X] and [Y], starting at a variety We conclude that the middle steady state is unstable because it cannot “attract” of different initial concentrations (determined by solution of Equation set 8–9; see Figure any trajectories. The system therefore has only two stable steady states. Thus, the 8–81B). By plotting [X] versus [Y] at each number of stable steady states in a system need not be equal to the total number time point, we find that, although there are of its theoretically possible steady states. In fact, stable steady states are usually three possible steady states in this system, separated by unstable ones, as in our example. the dynamic trajectories converge on only Once this system adopts a fate by settling in one of the two steady states, does two of them. The middle steady state is avoided: it is unstable, being unable to it

1	trajectories converge on only Once this system adopts a fate by settling in one of the two steady states, does two of them. The middle steady state is avoided: it is unstable, being unable to it have the ability to switch to the other state? The numerical solution of Equation attract any trajectories. (B) Imagine that the system is at the upper-left steady state and experiences a perturbation (black arrows), such as a random fluctuation in the production rates of X and/or Y. If the perturbation is small (arrow 1), the system will return to the same steady state. On the other hand, a perturbation that drives the system beyond the unstable (middle) steady state (arrow 2) causes it to switch to the lower-right steady state. The set of perturbations that a system can withstand without switching from one steady state to the other is known as the region of concentration of X concentration of X attraction of that steady state.

1	set 8–9 can again provide an answer. In Figure 8–82B, we show the solution of this equation set for two perturbations from the upper-left steady state. For a small perturbation, the system returns to its original steady state. But the larger perturbation causes the system to switch to the alternate steady state. Thus, this system can be switched from one stable steady state to the other by subjecting it to an input (or a perturbation) that is large enough to make the other steady state more attractive. More generally, every stable steady state has a corresponding region of attraction, which can be intuitively thought of as the range of perturbations (of [X] or [Y] in this example) for which the dynamic trajectories converge back to that particular steady state, rather than switch to the other one.

1	The concept of a region of attraction has interesting implications for the heritability of transcriptional states and the transition rates between them. If the region of attraction around one steady state is large, for example, then most cells in the population will assume this particular state. Furthermore, this state is likely to be inherited by daughter cells, since minor perturbations, like those ensuing from an asymmetric distribution of molecules during cell division, will rarely be sufficient to induce switching to the other steady state. We should expect that the use of positive feedback, coupled to cooperativity, will quite often be associated with systems requiring stable cell memory. Robustness Is an Important Characteristic of Biological Networks

1	Biological regulatory systems are exposed to frequent and sometimes extreme variations in external conditions or the concentrations or activities of key components. The ability of these systems to function normally in the face of such perturbations is called robustness. If we understand a complex system to the extent that we can reproduce its behavior with a computational model, then the robustness of the system can be assessed by determining how well its normal function persists following changes in various parameters, such as rate constants and component concentrations. We have already seen, for example, how the presence of negative feedback reduces the sensitivity of the steady state to changes in the values of the system’s parameters (see Figure 8–77). Considerations of robustness also apply to dynamic behaviors. Thus, for example, when discussing negative feedback, we described how the behavior of a system tends to become more oscillatory as the number of components that

1	also apply to dynamic behaviors. Thus, for example, when discussing negative feedback, we described how the behavior of a system tends to become more oscillatory as the number of components that constitute the feedback loop increases. If we use different values of the parameters in models derived for systems like those in Figure 8–78, we find that the system with the longer loop tends to exhibit stable oscillations within a much broader range of parameters, indicating that this system provides a more robust oscillator. We can perform similar calculations to determine the ability of different systems to achieve robust bistability arising from positive feedback. Thus, one benefit of computational models is that they allow us to probe the robustness of biological networks in a systematic and rigorous way.

1	Two Transcription Regulators That Bind to the Same Gene Promoter Can Exert Combinatorial Control Thus far, we have discussed how one transcription regulator can modulate the expression level of a gene. Most genes, however, are controlled by more than one type of transcription regulator, providing combinatorial control that allows two or more inputs to influence the expression of one gene. We can use computational methods to unveil some of the important regulatory features of combinatorial control systems.

1	Consider a gene whose promoter contains binding sites for two regulatory proteins, A and R, which bind to their individual sites independently. There are four possible binding configurations (Figure 8–83A). Suppose that A is a transcription activator, R is a transcription repressor, and the gene is only active when A is bound and R is not bound. We learned earlier that the probability that A is bound and the probability that R is not bound can be determined by the equations in Figure 8–84A. The product of these two probabilities gives us the probability of gene activation.

1	This example illustrates an AND NOT logic function (A and not R) (see Figure 8–83A). Maximal activation of this gene is accomplished when [A] is high and [R] is zero. However, intermediate levels of gene activation are also possible depending on the levels of A and R and also on the binding affinities of [A] and [R] for their respective sites (that is, KA and KR). When KA » KR, even a small concentration of [A] is capable of overcoming repression by R. Conversely, if KA « KR, then much more [A] is needed to activate the gene (Figure 8–84B and C).

1	Many other logic functions can govern combinatorial gene regulation. For example, an AND logic gate results when two activators, A1 and A2, are both required for a gene to be transcribed (Figures 8–83B and 8–84D). In E. coli cells, the AraJ gene controls some aspects of arabinose sugar metabolism: its expression requires two transcription regulators, one activated by arabinose and the other activated by the small molecule cAMP (Figure 8–84E). KA[A]fraction of A bound = fraction of R not bound = P(A,R) = . = concentration of R concentration of R concentration of A concentration of A

1	KA[A]fraction of A bound = fraction of R not bound = P(A,R) = . = concentration of R concentration of R concentration of A concentration of A Figure 8–83 Combinatorial control of gene expression. There are many ways in which gene expression can be controlled by two transcription regulators. To define precisely the relationship between the two inputs and the gene expression output, a regulatory circuit is often described as a specific type of logic gate, a term borrowed from electronic circuit design. A simple example is the OR logic gate (not shown here), in which a gene is controlled by two transcription activators, and one or the other can activate gene expression. (A) In a system with an activator A and repressor R, if transcription is turned on only when A is bound and R is not, then the result is an AND NOT logic gate. We saw an example of this logic in Chapter 7 (Figure 7–15).

1	(B) An AND gate results when two transcription activators, A1 and A2, are both required to turn on a gene. Figure 8–84 How the quantitative output of a gene depends on both its combinatorial logic and the affinities of transcription regulators. (A) In a combinatorial gene regulatory system like that illustrated in Figure 8–83A, the fraction of promoters bound by activator A and not bound by repressor R are each determined as shown here. The product of these probabilities provides the probability, P(A, R), that a gene promoter is active. (B–E) In these four panels, red indicates high gene expression and blue indicates low gene expression. (B) and (C) depict gene expression from the system described in panel (A). The two panels demonstrate how the system behaves when the relative concentration of A1 concentration of A2 arabinose (mM) S. Kaplan et al., Mol. Cell 29:786–792, 2008.) 0.02 1.3 43 affinities of the two transcription regulators change as indicated above each panel.

1	(D) Gene expression in a case where the gene turns on only at high levels of both activating inputs (A1 and A2), as shown in Figure 8–83B. (E) Experimental data showing measured expression of a gene in E. coli that is combinatorially regulated by two inputs: arabinose and cAMP. Note the close resemblance to panel (D). (E, adapted from rate of protein synthesis rate of protein synthesis

1	Imagine that a sudden input signal immediately activates a transcription activator A and that the same input signal induces the much slower synthesis of a transcription repressor protein R that acts on the same gene X. If A and R control gene expression by an AND NOT logic function like that described above, our intuition tells us that this system should be able to generate a pulse of transcription: when A is activated (and R is absent), the transcription of gene X will begin and cause an increase in the concentration of protein X, but then transcription will shut off when the concentration of R increases to a sufficiently high value.

1	Arrangements of this type are common in the cell. In E. coli, for example, galactose metabolic genes are positively regulated by the catabolite activator protein (CAP), which is activated at high levels of cAMP. The same genes are repressed by the GalS repressor protein, which is encoded by a gene whose transcription is likewise activated by CAP. Thus, an increase in input (cAMP) activates A (CAP), and transcription of the galactose genes begins. But activation of A also causes a subsequent buildup of R (GalS), which causes the same genes to be repressed after a delay. This results in an incoherent feed-forward motif (Figure 8–85A).

1	The response of the incoherent feed-forward motif will vary, depending on the parameters of the system. Suppose, for example, that the transcription activator protein A binds more weakly to the gene regulatory region than does the transcription repressor protein R (KA < KR). In this case, there will be a transient burst of protein synthesized by the affected gene (gene X) in response to a sudden activating input (Figure 8–85B). In contrast, the output will be more sustained if KA is much larger than KR, because the repression will be too weak to overcome the gene activation (Figure 8–85C). Other properties of this network, such as the dependence of the amplitude of the pulse on the various rate constants in the system, can be explored with the same computational tools. Thus, our intuitive guess about how this system would behave was only partially correct; even the simplest of networks depends on precise interaction strengths, demonstrating yet again why mathematics is needed to

1	guess about how this system would behave was only partially correct; even the simplest of networks depends on precise interaction strengths, demonstrating yet again why mathematics is needed to complement cartoon drawings.

1	In the bacterium E. coli, the sugar arabinose is only consumed when the preferred sugar, glucose, is scarce. The strategy that cells use to assess the presence of arabinose and absence of glucose involves a feed-forward arrangement that is different from the one just described. In this case, depletion of glucose causes an increase of cAMP, which is sensed by the CAP transcription activator protein, as described previously. In this case, however, CAP also induces the synthesis of a second transcription activator, AraC. Both activator proteins are necessary to activate arabinose metabolic genes (the AND logic function in Figure 8–83B). Figure 8–85 How an incoherent feed-forward motif can generate a brief pulse of gene activation in response to a sustained input. (A) Diagram of an incoherent feed-forward motif in which the transcription activator A and the repressor R control the expression of gene X using the AND NOT logic of Figure 8–83A.

1	(B) When KA « KR, this motif generates a pulse of protein X expression, such that the output goes back down even if the input remains high. (C) When KA » KR, the same motif responds to a sustained input by generating a sustained output.

1	This arrangement, known as a coherent feed-forward motif, has the interesting characteristics illustrated in Figure 8–86. Imagine that two activators, A1 and A2, are both required to initiate transcription of a gene. The input to the network activates A1 directly, but only activates A2 through this A1 activation. Thus, for a protein to be synthesized from this gene, long-term inputs are required that allow both A1 and A2 to be produced in active form. Brief input pulses are either ignored or produce small outputs. The requirement for a long input is important if assurances about a signal are needed before a costly cellular program is triggered. For example, glucose is the sugar on which E. coli cells grow best. Before cells trigger arabinose metabolism in the example above, it might be beneficial to be sure that glucose has been depleted (a sustained CAP pulse), rather than inducing the arabinose program during a transient glucose fluctuation.

1	The Same Network Can Behave Differently in Different Cells Due to Stochastic Effects Up to this point, we have assumed that all cells in a population produce identical behaviors if they contain the same network. It is important, however, to account for the fact that cells often show considerable individuality in their responses. Consider a situation in which a single mother cell divides into two daughter cells of equal volume. If the mother cell has only one molecule of a given protein, then only one daughter will inherit it. The daughters, though genetically identical, are already different. This variability is most pronounced for molecules that are present in small numbers. Nevertheless, even when there are many copies of a particular protein (or RNA), it is very unlikely that both daughter cells will end up with exactly the same number of molecules.

1	This is just one illustration of a universal feature of cells: their behaviors are often stochastic, meaning that they display variability in their protein content and therefore exhibit variations in phenotypes. In addition to the asymmetric partitioning of molecules following cell division, variability can originate from many chemical reactions. Imagine, for example, that our mother cell contains a simple gene regulatory circuit with a positive feedback loop like that shown in Figure 8–80B. Even if both daughter cells receive a copy of this circuit, including one copy of the initial transcription activator protein, there will be variability in the time required for promoter binding—and it will be statistically nearly impossible for the genes in the two daughter cells to become activated at precisely the same time. If the system is bistable and poised near a switching point, then variability in the response might flip the switch in only one daughter cell. Two daughter cells that were

1	at precisely the same time. If the system is bistable and poised near a switching point, then variability in the response might flip the switch in only one daughter cell. Two daughter cells that were born identical can thereby acquire, by chance, a dramatic difference in phenotype.

1	More generally, isogenic populations of cells grown in the same environment display diversity in size, shape, cell-cycle position, and gene expression. These differences arise because biochemical reactions require probabilistic collisions Figure 8–86 How a coherent feed-forward motif responds to various inputs. (A) Diagram of a coherent feed-forward motif in which the transcription activators A1 and A2 together activate expression of gene X using the AND logic of Figure 8–83B. (B) The response to a brief input can be either weak (as shown) or nonexistent. This allows the motif to ignore random fluctuations in the concentration of signaling molecules. (C) A prolonged input produces a strong response that can turn off rapidly.

1	between randomly moving molecules, with each event resulting in changes in the number of molecular species by integer amounts. The amplified effect of fluctuations in a molecular reactant, or the compounded effects of fluctuations across many molecular reactants, often accumulates as an observable phenotype. This can endow a cell with individuality and generate non-genetic cell-to-cell variability in a population.

1	Non-genetic variability can be studied in the laboratory by single-cell measurements of fluorescent proteins expressed from genes under the control of a specific promoter. Live cells can be mounted on a slide and viewed through a fluorescence microscope, revealing the striking variability in protein expression levels (Figure 8–87). Another approach is to use flow cytometry, which works by streaming a dilute suspension of cells past an illuminator and measuring the fluorescence of individual cells as they flow past the detector (see Figure 8–2). Fluorescence values can be used to build histograms that reveal the variability in a process across a population of cells, with a broad histogram indicating higher variability. Several Computational Approaches Can Be Used to Model the Reactions in Cells

1	We have focused primarily on the use of ordinary differential equations to model the dynamics of simple regulatory circuits. These models are called deterministic, because they do not incorporate stochastic variability and will always produce the same result from a specific set of parameters. As we have seen, such models can provide useful insights, particularly in the detailed mechanistic analysis of small regulatory circuits. However, other types of computational approaches are also needed to comprehend the great complexity of cell behavior. Stochastic models, for example, attempt to account for the very important problem of random variability in molecular networks. These models do not provide deterministic predictions about the behavior of molecules; instead, they incorporate random variation into molecule numbers and interactions, and the purpose of these models is to obtain a better understanding of the probability that a system will exist in a certain state over time.

1	Numerous other modeling strategies have been or are being developed. Boolean networks are used for the qualitative analysis of complex gene regulatory networks containing large numbers of interacting components. In these models, each molecule is a node that can exist in either the active or inactive state, thereby affecting the state of the nodes it is linked to. Models of this sort provide insights into the flow of information through a network, and they were useful in helping us understand the complex gene regulatory network that controls the early development of the sea urchin (see Figure 7–43). Boolean networks therefore reduce complex networks to a highly simplified (and potentially inaccurate) form. At the other extreme are agent-based simulations, in which thousands of molecules (or “agents”) in a system are modeled individually, and their probable behaviors and interactions with each other over time are calculated on the basis of predicted physical and chemical behaviors,

1	(or “agents”) in a system are modeled individually, and their probable behaviors and interactions with each other over time are calculated on the basis of predicted physical and chemical behaviors, often while taking stochastic variation into account. Agent-based approaches are computationally demanding but have the potential to generate highly lifelike simulations of real biological systems.

1	Statistical Methods Are Critical For the Analysis of Biological Data Dynamics, differential equations, and theoretical modeling are not the be-all and end-all of mathematics. Other branches of the subject are no less important for biologists. Statistics—the mathematics of probabilistic processes and noisy datasets—is an inescapable part of every biologist’s life. This is true in two main ways. First, imperfect measurement devices and other errors generate experimental noise in our data. Second, all cell-biological processes depend on the stochastic behavior of individual molecules, as we just discussed, and this results in biological noise in our results. How, in the face of all this noise, do we come to conclusions about the truth of hypotheses? The answer is statistical analysis, which shows how to move from one level of description to

1	Figure 8–87 Different levels of gene expression in individual cells within a population of E. coli bacteria. For this experiment, two different reporter proteins (one fluorescing green, the other red), controlled by a copy of the same promoter, have been introduced into all of the bacteria. Some cells express only one gene copy, and so appear either red or green, while others express both gene copies, and so appear yellow. This experiment reveals variable levels of fluorescence, indicating variable levels of gene expression within an apparently uniform population of cells. (From M.B. Elowitz et al., Science 297:1183–1186, 2002. With permission from AAAS.) another: from a set of erratic individual data points to a simpler description of the WHAT WE DON’T KNOW key features of the data.

1	Statistics teaches us that the more times we repeat our measurements, the bet • Many of the tools that revolutionized ter and more refined the conclusions we can draw from them. Given many repeti-DNA technology were discovered by tions, it becomes possible to describe our data in terms of variables that summa-scientists studying basic biological rize the features that matter: the mean value of the measured variable, taken over problems that had no obvious the set of data points; the magnitude of the noise (the standard deviation of the applications. What are the best set of data points); the likely error in our estimate of the mean value (the standard strategies to ensure that such crucially important technologies will continue to error of the mean); and, for specialists, the details of the probability distribution be discovered?

1	describing the likelihood that an individual measurement will yield a given value. For all these things, statistics provides recipes and quantitative formulas that biol • As the cost of DNA sequencing ogists must understand if they are to make rigorous conclusions on the basis of decreases and the amount of variable results.

1	sequence data accumulates, how are we going to keep track of and Summary meaningfully analyze this vast amount of information? What new questions Quantitative mathematical analysis can provide a powerful extra dimension in our will this information allow us to answer? understanding of cell regulation and function. Cell regulatory systems often depend • Can we develop tools to analyze on macromolecular interactions, and mathematical analysis of the dynamics of each of the post-transcriptional these interactions can unveil important insights into the importance of binding modifications on the proteins in living affinities and protein stability in the generation of transcriptional or other signals. cells, so as to follow all of their Regulatory systems often employ network motifs that generate useful behaviors: changes in real time?

1	cells, so as to follow all of their Regulatory systems often employ network motifs that generate useful behaviors: changes in real time? a rapid negative feedback loop dampens the response to input signals; a delayed negative feedback loop creates a biochemical oscillator; positive feedback yields a models to accurately describe the systems that generate transient signal pulses or respond only to sustained inputs. enormous complexity of cellular The dynamic behavior of these network motifs can be dissected in detail with deter-networks and to predict undiscovered ministic and stochastic mathematical modeling. components and mechanisms? Which statements are true? explain why or why not. Discuss the following problems.

1	Which statements are true? explain why or why not. Discuss the following problems. 8–1 Because a monoclonal antibody recognizes a spe-8–7 A common step in the isolation of cells from a cific antigenic site (epitope), it binds only to the specific sample of animal tissue is to treat the tissue with trypsin, protein against which it was made. collagenase, and EDTA. Why is such a treatment nece ssary, and what does each component accomplish? And 8–2 Given the inexorable march of technology, it why does this treatment not kill the cells?

1	seems inevitable that the sensitivity of detection of molecules will ultimately be pushed beyond the yoctomole 8–8 Tropomyosin, at 93 kd, sediments at 2.6S, whereas level (10–24 mole). the 65-kd protein, hemoglobin, sediments at 4.3S. (The sedimentation coefficient S is a linear measure of the rate 8–3 If each cycle of PCR doubles the amount of DNA of sedimentation.) These two proteins are drawn to scale synthesized in the previous cycle, then 10 cycles will give a in Figure Q8–1. How is it that the bigger protein sediments 103-fold amplification, 20 cycles will give a 106-fold amplimore slowly than the smaller one? Can you think of an fication, and 30 cycles will give a 109-fold amplification. analogy from everyday experience that might help you 8–4 To judge the biological importance of an interac-with this problem? tion between protein A and protein B, we need to know quantitative details about their concentrations, affinities, and kinetic behaviors.

1	8–5 The rate of change in the concentration of any molecular species X is given by the balance between its rate of appearance and its rate of disappearance. 8–6 After a sudden increase in transcription, a protein with a slow rate of degradation will reach a new steady state level more quickly than a protein with a rapid rate of degradation. tropomyosinhemoglobinFigure Q8–1 Scale models of tropomyosin and hemoglobin (Problem 8–8). 8–9 Hybridoma technology allows one to generate monoclonal antibodies to virtually any protein. Why is it, then, that genetically tagging proteins with epitopes is such a commonly used technique, especially since an epitope tag has the potential to interfere with the function of the protein?

1	8–10 How many copies of a protein need to be present in a cell in order for it to be visible as a band on an SDS gel? Assume that you can load 100 μg of cell extract onto a gel and that you can detect 10 ng in a single band by silver staining the gel. The concentration of protein in cells is about 200 mg/mL, and a typical mammalian cell has a volume of about 1000 μm3 and a typical bacterium a volume of about 1 μm3. Given these parameters, calculate the number of copies of a 120-kd protein that would need to be present in a mammalian cell and in a bacterium in order to give a detectable band on a gel. You might try an order-of-magnitude guess before you make the calculations.

1	8–11 You have isolated the proteins from two adjacent spots after two-dimensional polyacrylamide-gel electrophoresis and digested them with trypsin. When the masses of the peptides were measured by MALDI-TOF mass spectrometry, the peptides from the two proteins were found to be identical except for one (Figure Q8–2). For this peptide, the mass-to-charge (m/z) values differed by 80, a value that does not correspond to a difference in amino acid sequence. (For example, glutamic acid instead of valine at one position would give an m/z difference of around 30.) Can you suggest a possible difference between the two peptides that might account for the observed m/z difference? Masses of peptides measured by MALDI-TOF mass spectrometry (Problem 8–11). Only the numbered peaks differ between the two protein samples.

1	Masses of peptides measured by MALDI-TOF mass spectrometry (Problem 8–11). Only the numbered peaks differ between the two protein samples. 8–12 You want to amplify the DNA between the two stretches of sequence shown in Figure Q8–3. Of the listed primers, choose the pair that will allow you to amplify the DNA by PCR. 8–13 In the very first round of PCR using genomic DNA, the DNA primers prime synthesis that terminates only when the cycle ends (or when a random end of DNA is encountered). Yet, by the end of 20 to 30 cycles—a typical amplification—the only visible product is defined precisely by the ends of the DNA primers. In what cycle is a double-stranded fragment of the correct size first generated? DNA to be amplifed Figure Q8–3 DNA to be amplified and potential PCR primers (Problem 8–12). 8–14 Explain the difference between a gain-of-function mutation and a dominant-negative mutation. Why are both these types of mutation usually dominant?

1	8–14 Explain the difference between a gain-of-function mutation and a dominant-negative mutation. Why are both these types of mutation usually dominant? 8–15 Discuss the following statement: “We would have no idea today of the importance of insulin as a regulatory hormone if its absence were not associated with the human disease diabetes. It is the dramatic consequences of its absence that focused early efforts on the identification of insulin and the study of its normal role in physiology.” 8–16 You have just gotten back the results from an RNA-seq analysis of mRNAs from liver. You had anticipated counting the number of reads of each mRNA to determine the relative abundance of different mRNAs. But you are puzzled because many of the mRNAs have given you results like those shown in Figure Q8–4. How is it that different parts of an mRNA can be represented at different levels?

1	8–17 Examine the network motifs in Figure Q8–5. Decide which ones are negative feedback loops and which are positive. Explain your reasoning. 8–18 Imagine that a random perturbation positions a bistable system precisely at the boundary between two stable states (at the orange dot in Figure Q8–6). How would the system respond? Figure Q8–4 RNA-seq reads for a liver mRNA (Problem 8–16). The exon structure of the mRNA is indicated, with protein-coding segments indicated in light blue and untranslated regions in dark blue. The numbers of sequencing reads are indicated by the heights of the vertical lines above the mRNA. Figure Q8–5 Network motifs composed of transcription activators and repressors (Problem 8–17).

1	8–19 Detailed analysis of the regulatory region of the Lac operon has revealed surprising complexity. Instead of a single binding site for the Lac repressor, as might be expected, there are three sites termed operators: O1, O2, and O3, arrayed along the DNA as shown in Figure Q8–7. To probe the functions of these three sites, you make a series of constructs in which various combinations of operator sites are present. You examine their ability to repress expression of β-galactosidase, using either tetrameric (wild type) or dimeric (mutant) forms of the Lac repressor. The dimeric form of the repressor can bind to a single operator (with the same affinity as the tetramer) with each monomer binding to half the site. The tetramer, the form normally expressed in cells, can bind to two sites simultaneously. When you measure repression of β-galactosidase expression, you find the results shown in Figure Q8–7, with higher numbers indicating more effective repression. concentration of X

1	concentration of X Figure Q8–6 Perturbations of a bistable system (Problem 8–18). As shown by the green lines, after perturbation 1 the system returns to its original stable state (green dot at left), and after perturbation 2, the system moves to the other stable state (green dot at right). Perturbation 3 moves the system to the precise boundary between the two stable states (orange dot). A. Which single operator site is the most important for repression? How can you tell? b. Do combinations of operator sites (Figure Q8–7, constructs 1, 2, 3, and 5) substantially increase repression by the dimeric repressor? Do combinations of operator sites substantially increase repression by the tetrameric repressor? If the two repressors behave differently, offer an explanation for the difference. C. The wild-type repressor binds O3 very weakly when it is by itself on a segment of DNA. However, if O1 is included on the same segment of DNA, the repressor binds O3 quite well. How can that be?

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1	Understanding the structural organization of cells is essential for learning how they function. In this chapter, we briefly describe some of the principal microscopy methods used to study cells. Optical microscopy will be our starting point because cell biology began with the light microscope, and it is still an indispensible tool. The development of methods for the specific labeling and imaging of individual cellular constituents and the reconstruction of their threedimensional architecture has meant that, far from falling into disuse, optical microscopy continues to increase in importance. One advantage of optical microscopy is that light is relatively nondestructive. By tagging specific cell components with fluorescent probes, such as intrinsically fluorescent proteins, we can watch their movement, dynamics, and interactions in living cells. Although conventional optical microscopy is limited in resolution by the wavelength of visible light, new methods cleverly bypass this

1	their movement, dynamics, and interactions in living cells. Although conventional optical microscopy is limited in resolution by the wavelength of visible light, new methods cleverly bypass this limitation and allow the position of even single molecules to be mapped. By using a beam of electrons instead of visible light, electron microscopy can image the interior of cells, and their macromolecular components, at almost atomic resolution, and in three dimensions.

1	This chapter is intended as a companion, rather than an introduction, to the chapters that follow; readers may wish to refer back to it as they encounter applications of microscopy to basic biological problems in the later pages of the book. Looking at CeLLs in the Light MiCrosCope A typical animal cell is 10–20 μm in diameter, which is about onefifth the size of the smallest object that we can normally see with the naked eye. Only after good light microscopes became available in the early part of the nineteenth century did Schleiden and Schwann propose that all plant and animal tissues were aggregates of individual cells. Their proposal in 1838, known as the cell doctrine, marks the formal birth of cell biology.

1	Animal cells are not only tiny, but they are also colorless and translucent. The discovery of their main internal features, therefore, depended on the development, in the late nineteenth century, of a variety of stains that provided sufficient contrast to make those features visible. Similarly, the far more powerful electron microscope introduced in the early 1940s required the development of new techniques for preserving and staining cells before the full complexities of their internal fine structure could begin to emerge. To this day, microscopy often relies as much on techniques for preparing the specimen as on the performance of the microscope itself. In the following discussions, we therefore consider both instruments and specimen preparation, beginning with the light microscope.

1	The images in Figure 9–1 illustrate a stepwise progression from a thumb to a cluster of atoms. Each successive image represents a tenfold increase in magnification. The naked eye can see features in the first two panels, the light microscope allows us to see details corresponding to about the fourth or fifth panel, and the electron microscope takes us to about the seventh or eighth panel. Figure 9–2 shows the sizes of various cellular and subcellular structures and the ranges of size that different types of microscopes can visualize. Looking at CeLLs in the Light MiCrosCope Looking at CeLLs anD MoLeCULes in the eLeCtron MiCrosCope 20 mm 2 mm 0.2 mm 20 µm 2 µm 0.2 µm 20 nm 2 nm 0.2 nm the Light Microscope Can resolve Details 0.2 μm apart

1	Looking at CeLLs anD MoLeCULes in the eLeCtron MiCrosCope 20 mm 2 mm 0.2 mm 20 µm 2 µm 0.2 µm 20 nm 2 nm 0.2 nm the Light Microscope Can resolve Details 0.2 μm apart For well over 100 years, all microscopes were constrained by a fundamental limitation: that a given type of radiation cannot be used to probe structural details much smaller than its own wavelength. A limit to the resolution of a light microscope was therefore set by the wavelength of visible light, which ranges from about 0.4 μm (for violet) to 0.7 μm (for deep red). In practical terms, bacteria and mitochondria, which are about 500 nm (0.5 μm) wide, are generally the smallest objects whose shape we can clearly discern in the light microscope; details smaller than this are obscured by effects resulting from the wavelike nature of light. To understand why this occurs, we must follow the behavior of a beam of light as it passes through the lenses of a microscope (Figure 9–3).

1	Because of its wave nature, light does not follow the idealized straight ray paths that geometrical optics predicts. Instead, light waves travel through an optical system by many slightly different routes, like ripples in water, so that they Figure 9–1 A sense of scale between living cells and atoms. each diagram shows an image magnified by a factor of ten in an imaginary progression from a thumb, through skin cells, to a ribosome, to a cluster of atoms forming part of one of the many protein molecules in our body. atomic details of biological macromolecules, as shown in the last two panels, are usually beyond the power of the electron microscope. While color has been used here in all the panels, it is not a feature of objects much smaller than the wavelength of light, so the last five panels should really be in black and white. limit of limit of super-conventional 0.1 nm(1 A)

1	limit of limit of super-conventional 0.1 nm(1 A) Figure 9–2 Resolving power. sizes of cells and their components are drawn on a logarithmic scale, indicating the range of objects that can be readily resolved by the naked eye and in the light and electron microscopes. note that new superresolution microscopy techniques, discussed in detail later, allow an improvement in resolution by an order of magnitude compared with conventional light microscopy.

1	interfere with one another and cause optical diffraction effects. If two trains of waves reaching the same point by different paths are precisely in phase, with crest matching crest and trough matching trough, they will reinforce each other so as to increase brightness. In contrast, if the trains of waves are out of phase, they will interfere with each other in such a way as to cancel each other partly or entirely (Figure 9–4). The interaction of light with an object changes the phase relationships of the light waves in a way that produces complex interference effects. At high magnification, for example, the shadow of an edge that is evenly illuminated with light of uniform wavelength appears as a set of parallel lines (Figure 9–5), whereas that of a circular spot appears as a set of concentric rings. For the same reason, a single point seen through a microscope appears as a blurred disc, and two point objects close together give overlapping images and may merge into one.

1	the following units of length are commonly employed in microscopy: μm (micrometer) = 10–6 m nm (nanometer) = 10–9 m Å (Ångström unit) = 10–10 m Figure 9–3 A light microscope. (a) Diagram showing the light path in a compound microscope. Light is focused on the specimen by lenses in the condenser. a combination of objective lenses, tube lenses, and eyepiece lenses is arranged to focus an image of the illuminated specimen in the eye. (B) a modern research light microscope. (B, courtesy of Carl Zeiss Microscopy, gmbh.) Although no amount of refinement of the lenses can overcome the diffraction limit imposed by the wavelike nature of light, other ways of cleverly bypassing this limit have emerged, creating socalled superresolution imaging techniques that can even detect the position of single molecules.

1	The limiting separation at which two objects appear distinct—the socalled limit of resolution—depends on both the wavelength of the light and the numerical aperture of the lens system used. The numerical aperture affects the lightgathering ability of the lens and is related both to the angle of the cone of light that can enter it and to the refractive index of the medium the lens is operating in; the wider the microscope opens its eye, so to speak, the more sharply it can see (Figure 9–6). The refractive index is the ratio of the speed of light in a vacuum to the speed of light in a particular transparent medium. For example, for water this is 1.33, meaning that light travels 1.33 times slower in water than in a vacuum. Under the best conditions, with violet light (wavelength = 0.4 μm) and a numerical aperture of 1.4, the basic light microscope can theoretically achieve a limit of resolution of about 0.2 μm, or 200 nm. Some microscope makers at the end of the nineteenth century

1	μm) and a numerical aperture of 1.4, the basic light microscope can theoretically achieve a limit of resolution of about 0.2 μm, or 200 nm. Some microscope makers at the end of the nineteenth century achieved this resolution, but it is routinely matched in contemporary, factoryproduced microscopes. Although it is possible to enlarge an image as much as we want—for example, by projecting it onto a screen—it is not possible, in a conventional light microscope, to resolve two objects in the light microscope that are separated by less than about 0.2 μm; they will appear as a single object. It is important, however, to distinguish between resolution and detection. If a small object, below the resolution limit, itself emits light, then we may still be able to see or detect it. Thus, we can see a single fluorescently labeled microtubule even though it is about ten times thinner than the resolution limit of the light microscope. Diffraction effects, however, will cause it to appear blurred

1	a single fluorescently labeled microtubule even though it is about ten times thinner than the resolution limit of the light microscope. Diffraction effects, however, will cause it to appear blurred and at least 0.2 μm thick (see Figure 9–16). In a similar way, we can see the stars in the night sky, even though their diameters are far below the angular resolution of our unaided eyes: they all appear as similar, slightly blurred points of light, differing only in their color and brightness.

1	photon noise Creates additional Limits to resolution When Light Levels are Low Any image, whether produced by an electron microscope or by an optical microscope, is made by particles—electrons or photons—striking a detector of some sort. But these particles are governed by quantum mechanics, so the numbers reaching the detector are predictable only in a statistical sense. Finite samples, collected by imaging for a limited period of time (that is, by taking a snapshot), will show random variation: successive snapshots of the same scene will not be exactly identical. Moreover, every detection method has some level of background signal or noise, adding to the statistical uncertainty. With bright illumination, corresponding to very large numbers of photons or electrons, the features of the imaged

1	Figure 9–4 Interference between light waves. When two light waves combine in phase, the amplitude of the resultant wave is larger and the brightness is increased. two light waves that are out of phase cancel each other partly and produce a wave whose amplitude, and therefore brightness, is decreased. Figure 9–5 Images of an edge and of a point of light. (a) the interference effects, or fringes, seen at high magnification when light of a specific wavelength passes the edge of a solid object placed between the light source and the observer. (B) the image of a point source of light. Diffraction spreads this out into a complex, circular pattern, whose width depends on the numerical aperture of the optical system: the smaller the aperture, the bigger (more blurred) the diffracted image. two point sources can be just resolved when the center of the image of one lies on the first dark ring in the image of the other: this is used to define the limit of resolution.

1	Figure 9–6 Numerical aperture. the path of light rays passing through a transparent specimen in a microscope illustrates the concept of numerical aperture and its relation to the limit of resolution. the objective lens collects a cone of light rays to create an image the condenser lens focuses a cone of light rays onto each point of the specimen RESOLUTION: the resolving power of the microscope depends on the width of the cone of illumination and therefore on both the condenser and the objective lens. It is calculated using the formula 0.61 ˛ where: ˜ = half the angular width of the cone of rays collected by the objective lens from a typical point in the central region of the specimen (since the maximum width is 180o, sin ˜ has a maximum value of 1) n = the refractive index of the medium (usually air or oil) separating the specimen from the objective and condenser lenses ˛ = the wavelength of light used (for white light a fgure of 0.53 µm is commonly assumed)

1	NUMERICAL APERTURE: n sin ˜ in the aperture, the greater the resolution and the equation above is called the numerical aperture brighter the image (brightness is important in of the lens and is a function of its light-fuorescence microscopy). However, this advancollecting ability. For dry lenses this cannot be tage does necessitate very short working distances more than 1, but for oil-immersion lenses it can and a very small depth of feld. be as high as 1.4. The higher the numerical specimen are accurately determined based on the distribution of these particles at the detector. However, with smaller numbers of particles, the structural details of the specimen are obscured by the statistical fluctuations in the numbers of particles detected in each region, which give the image a speckled appearance and limit its precision. The term noise describes this random variability. Living Cells are seen Clearly in a phase-Contrast or a Differential-interference-Contrast Microscope

1	Living Cells are seen Clearly in a phase-Contrast or a Differential-interference-Contrast Microscope There are many ways in which contrast in a specimen can be generated (Figure 9–7A). While fixing and staining a specimen can generate contrast through color, microscopists have always been challenged by the possibility that some components of the cell may be lost or distorted during specimen preparation. The only certain way to avoid the problem is to examine cells while they are alive, without fixing or freezing. For this purpose, light microscopes with special optical systems are especially useful.

1	In the normal bright-field microscope, light passing through a cell in culture forms the image directly. Another system, dark-field microscopy, exploits the fact that light rays can be scattered in all directions by small objects in their path. If oblique lighting from the condenser is arranged, which does not directly enter the objective, focused but unstained objects in a living cell can scatter the rays, some of which then enter the objective to create a bright image against a black background (Figure 9–7B).

1	When light passes through a living cell, the phase of the light wave is changed according to the cell’s refractive index: a relatively thick or dense part of the cell, such as a nucleus, slows the light passing through it. The phase of the light, consequently, is shifted relative to light that has passed through an adjacent thinner region of the cytoplasm (Figure 9–7C). The phase-contrast microscope and, in a more complex way, the differential-interference-contrast microscope increase these phase differences so that the waves are more nearly out of phase, producing amplitude differences when the sets of waves recombine, thereby creating an image of the cell’s structure. Both types of light microscopy are widely used to visualize living cells (see Movie 17.2). Figure 9–8 compares images of the same cell obtained by four kinds of light microscopy.

1	Figure 9–7 Contrast in light microscopy. (a) the stained portion of the cell will absorb light of some wavelengths, which depends on the stain, but will allow other wavelengths to pass through it. a colored image of the cell is thereby obtained that is visible in the normal bright-field light microscope. (B) in the dark-field microscope, oblique rays of light focused on the specimen do not enter the objective lens, but light that is scattered by components in the living cell can be collected to produce a bright image on a dark background. (C) Light passing through the unstained living cell experiences very little change in amplitude, and the structural details cannot be seen even if the image is highly magnified. the phase of the light, however, is altered by its passage through either thicker or denser parts of the cell, and small phase differences can be made visible by exploiting interference effects using a phase-contrast or a differential-interference-contrast microscope.

1	Phasecontrast, differentialinterferencecontrast, and darkfield microscopy make it possible to watch the movements involved in such processes as mitosis and cell migration. Since many cellular motions are too slow to be seen in real time, it is often helpful to make timelapse movies in which the camera records successive frames separated by a short time delay, so that when the resulting picture series is played at normal speed, events appear greatly speeded up.

1	In recent years, electronic, or digital, imaging systems, and the associated technology of image processing, have had a major impact on light microscopy. Certain practical limitations of microscopes relating to imperfections in the optical system have been largely overcome. Electronic imaging systems have also circumvented two fundamental limitations of the human eye: the eye cannot see well in extremely dim light, and it cannot perceive small differences in light intensity against a bright background. To increase our ability to observe cells in these difficult conditions, we can attach a sensitive digital camera to a microscope. These cameras detect light by means of chargecoupled devices (CCDs), or high sensitivity complementary metaloxide semiconductor (CMOS) sensors, similar to those found in digital cameras. Such image sensors are 10 times more sensitive than the human eye and can detect 100 times more intensity levels. It is therefore possible to observe cells for long periods

1	found in digital cameras. Such image sensors are 10 times more sensitive than the human eye and can detect 100 times more intensity levels. It is therefore possible to observe cells for long periods at very low light levels, thereby avoiding the damaging effects of prolonged bright light (and heat). Such lowlight cameras are especially important for viewing fluorescent molecules in living cells, as explained below.

1	Because images produced by digital cameras are in electronic form, they can be processed in various ways to extract latent information. Such image processing makes it possible to compensate for several optical faults in microscopes. Moreover, by digital image processing, contrast can be greatly enhanced to overcome Figure 9–8 Four types of light microscopy. Four images are shown of the same fibroblast cell in culture. all images can be obtained with most modern microscopes by interchanging optical components. (a) Bright-field microscopy, in which light is transmitted straight through the specimen. (B) phase-contrast microscopy, in which phase alterations of light transmitted through the specimen are translated into brightness changes. (C) Differential-interference-contrast microscopy, which highlights edges where there is a steep change of refractive index. (D) Dark-field microscopy, in which the specimen is lit from the side and only the scattered light is seen.

1	the eye’s limitations in detecting small differences in light intensity, and background irregularities in the optical system can be digitally subtracted. This procedure reveals small transparent objects that were previously impossible to distinguish from the background. intact tissues are Usually Fixed and sectioned Before Microscopy Because most tissue samples are too thick for their individual cells to be examined directly at high resolution, they are often cut into very thin transparent slices, or sections. To preserve the cells within the tissue they must be treated with a fixative. Common fixatives include glutaraldehyde, which forms covalent bonds with the free amino groups of proteins, crosslinking them so they are stabilized and locked into position.

1	Because tissues are generally soft and fragile, even after fixation, they need to be either frozen or embedded in a supporting medium before being sectioned. The usual embedding media are waxes or resins. In liquid form, these media both permeate and surround the fixed tissue; they can then be hardened (by cooling or by polymerization) to form a solid block, which is readily sectioned with a microtome. This is a machine with a sharp blade, usually of steel or glass, which operates like a meatslicer (Figure 9–9). The sections (typically 0.5–10 μm thick) are then laid flat on the surface of a glass microscope slide.

1	There is little in the contents of most cells (which are 70% water by weight) to impede the passage of light rays. Thus, most cells in their natural state, even if fixed and sectioned, are almost invisible in an ordinary light microscope. We have seen that cellular components can be made visible by techniques such as phasecontrast and differentialinterferencecontrast microscopy, but these methods tell us almost nothing about the underlying chemistry. There are three main approaches to working with thin tissue sections that reveal differences in types of molecules that are present. First, and traditionally, sections can be stained with organic dyes that have some specific affinity for particular subcellular components. The dye hematoxylin, for example, has an affinity for negatively charged molecules and therefore reveals the distribution of DNA, RNA, and acidic proteins in a cell (Figure 9–10). The chemical basis for the specificity of many dyes, however, is not known.

1	movement of microtome arm ribbon of ribbon of sections on glass slide, stained and mounted under a glass cover slip Figure 9–9 Making tissue sections. this illustration shows how an embedded tissue is sectioned with a microtome in preparation for examination in the light microscope. Second, sectioned tissues can be used to visualize specific patterns of differential gene expression. In situ hybridization, discussed earlier (see Figure 8–34), reveals the cellular distribution and abundance of specific expressed RNA molecules in sectioned material or in whole mounts of small organisms or organs. This is particularly effective when used in conjunction with fluorescent probes (Figure 9–11). A third and very sensitive approach, generally and widely applicable for localizing proteins of interest, also depends on using fluorescent probes and markers, as we explain next.

1	A third and very sensitive approach, generally and widely applicable for localizing proteins of interest, also depends on using fluorescent probes and markers, as we explain next. Fluorescent molecules absorb light at one wavelength and emit it at another, longer wavelength (Figure 9–12A). If we illuminate such a molecule at its absorbing wavelength and then view it through a filter that allows only light of the emitted wavelength to pass, it will glow against a dark background. Because the background is dark, even a minute amount of the glowing fluorescent dye can be detected. In contrast, the same number of molecules of a nonfluorescent stain, viewed conventionally, would be practically indiscernible because the absorption of light by molecules in the stain would result in only the faintest tinge of color in the light transmitted through that part of the specimen.

1	The fluorescent dyes used for staining cells are visualized with a fluorescence microscope. This microscope is similar to an ordinary light microscope except that the illuminating light, from a very powerful source, is passed through two sets of filters—one to filter the light before it reaches the specimen and one to Figure 9–10 Staining of cell components. (a) this section of cells in the urine-collecting ducts of the kidney was stained with hematoxylin and eosin, two dyes commonly used in histology. each duct is made of closely packed cells (with nuclei stained red) that form a ring. the ring is surrounded by extracellular matrix, stained purple. (B) this section of a young plant root is stained with two dyes, safranin and fast green. the fast green stains the cellulosic cell walls while the safranin stains the lignified xylem cell walls bright red. (a, from p.r. Wheater et al., Functional histology, 2nd ed. London: Churchill Livingstone, 1987; B, courtesy of stephen grace.)

1	Figure 9–11 RNA in situ hybridization.

1	as described in Chapter 8 (see Figure 8–62), it is possible to visualize the distribution of different rnas in tissues using in situ hybridization. here, the transcription pattern of five different genes involved in patterning the early fly embryo is revealed in a single embryo. each rna probe has been fluorescently labeled in a different way, some directly and some indirectly; the resulting images are displayed each in a different color (“false-colored”) and combined to give an image where different color combinations represent different sets of genes expressed. the genes whose expression pattern is revealed here are wingless (yellow), engrailed (blue), short gastrulation (red), intermediate neuroblasts defective (green), and muscle specific homeobox (purple). (From D. kosman et al., Science 305:846, 2004. With permission from aaas.) energy of orbital electronin fuorophore emission of photon 3 second barrier flter: cuts out unwanted fuorescent signals, passing the specifc green

1	Science 305:846, 2004. With permission from aaas.) energy of orbital electronin fuorophore emission of photon 3 second barrier flter: cuts out unwanted fuorescent signals, passing the specifc green fuorescein emission between 520 and 560 nm 2 beam-splitting mirror: refects light below 510 nm but transmits light above 510 nm

1	Figure 9–12 Fluorescence and the fluorescence microscope. (a) an orbital electron of a fluorochrome molecule can be raised to an excited state following the absorption of a photon. Fluorescence occurs when the electron returns to its ground state and emits a photon of light at a longer wavelength. too much exposure to light, or too bright a light, can also destroy the fluorochrome molecule, in a process called photobleaching. (B) in the fluorescence microscope, a filter set consists of two barrier filters (1 and 3) and a dichroic (beam-splitting) mirror (2). this example shows the filter set for detection of the fluorescent molecule fluorescein. high-numerical-aperture objective lenses are especially important in this type of microscopy because, for a given magnification, the brightness of the fluorescent image is proportional to the fourth power of the numerical aperture Figure 9–14 Different fluorescent probes can be visualized in the same cell. in this composite micrograph of a

1	of the fluorescent image is proportional to the fourth power of the numerical aperture Figure 9–14 Different fluorescent probes can be visualized in the same cell. in this composite micrograph of a cell in mitosis, three different fluorescent probes have been used to label three different cellular components (Movie 9.1). the spindle microtubules are revealed with a green fluorescent antibody, centromeres with a red fluorescent antibody, and the Dna of the condensed chromosomes with the blue fluorescent dye Dapi. (Courtesy of kevin F. sullivan.) (see Figure 9–13) but, like many organic fluorochromes, they fade fairly rapidly when continuously illuminated. More stable fluorochromes have been developed based on inorganic chemistry. Tiny crystals of semiconductor material, called nanoparticles, or quantum dots, can be excited to fluoresce by a broad spectrum of blue light. Their emitted light has a color that depends on the exact size of the nanocrystal, between 2 and 10 nm in diameter,

1	or quantum dots, can be excited to fluoresce by a broad spectrum of blue light. Their emitted light has a color that depends on the exact size of the nanocrystal, between 2 and 10 nm in diameter, and additionally the fluorescence fades only slowly with time (Figure 9–15). These nanoparticles, when coupled to other probes such as antibodies, are therefore ideal for tracking molecules over time. If introduced into a living cell, in an embryo for example, the progeny of that cell can be followed many days later by their fluorescence, allowing cell lineages to be tracked.

1	Figure 9–15 Fluorescent nanoparticles or quantum dots. (a) Quantum dots are tiny particles of cadmium selenide, a semiconductor, with a coating to make them water-soluble. they can be coupled to protein molecules such as antibodies or streptavidin and, when introduced into a cell, will bind to a target protein of interest. Different-sized quantum dots emit light of different colors—the larger the dot, the longer the wavelength—but they are all excited by the same blue light. Quantum dots can keep shining for weeks, unlike most fluorescent organic dyes. (B) in this cell, microtubules are labeled (green) with an organic fluorescent dye (alexa 488), while a nuclear protein is stained (red) with quantum dots bound to streptavidin. on continuous exposure to strong blue light, the fluorescent dye fades quickly while the quantum dots continue to shine. (C) in this cell, the labeling pattern is reversed; a nuclear protein is labeled (green) with an organic fluorescent dye (alexa 488), while

1	dye fades quickly while the quantum dots continue to shine. (C) in this cell, the labeling pattern is reversed; a nuclear protein is labeled (green) with an organic fluorescent dye (alexa 488), while microtubules are labeled (red) with quantum dots. again, the quantum dots far outlast the fluorescent dye. (B and C, from L. Medintz et al., Nat. Mater. 4:435–446, 2005. With permission from Macmillan publishers Ltd.)

1	Later in the chapter, additional fluorescence microscopy methods will be discussed that can be used to monitor changes in the concentration and location of specific molecules inside living cells. antibodies Can Be Used to Detect specific Molecules

1	antibodies Can Be Used to Detect specific Molecules Antibodies are proteins produced by the vertebrate immune system as a defense against infection (discussed in Chapter 24). They are unique among proteins in that they are made in billions of different forms, each with a different binding site that recognizes a specific target molecule (or antigen). The precise antigen specificity of antibodies makes them powerful tools for the cell biologist. When labeled with fluorescent dyes, antibodies are invaluable for locating specific molecules in cells by fluorescence microscopy (Figure 9–16); labeled with electrondense particles such as colloidal gold spheres, they are used for similar purposes in the electron microscope (discussed below). The antibodies employed in microscopy are commonly either purified from antiserum so as to remove all nonspecific antibodies, or they are specific monoclonal antibodies that only recognize the target molecule.

1	When we use antibodies as probes to detect and assay specific molecules in cells, we frequently use chemical methods to amplify the fluorescent signal they produce. For example, although a marker molecule such as a fluorescent dye can be linked directly to an antibody—the primary antibody—a stronger signal is achieved by using an unlabeled primary antibody and then detecting it with a group of labeled secondary antibodies that bind to it (Figure 9–17). This process is called indirect immunocytochemistry. Some amplification methods use an enzyme as a marker molecule attached to the secondary antibody. The enzyme alkaline phosphatase, for example, in the presence of appropriate chemicals, produces inorganic phosphate that in turn primary antibody: secondary antibodies: Figure 9–16 Immunofluorescence.

1	Figure 9–16 Immunofluorescence. a transmission electron micrograph of the periphery of a cultured epithelial cell showing the distribution of microtubules and other filaments. (B) the same area stained with fluorescent antibodies against tubulin, the protein that assembles to form microtubules, using the technique of indirect immunocytochemistry (see Figure 9–17). Red arrows indicate individual microtubules that are readily recognizable in both images. note that, because of diffraction effects, the microtubules in the light microscope appear 0.2 μm wide rather than their true width of 0.025 μm. (From M. osborn, r. Webster and k. Weber, J. Cell Biol. 77:r27–r34, 1978. With permission from the rockefeller University press.) Figure 9–17 Indirect immunocytochemistry.

1	Weber, J. Cell Biol. 77:r27–r34, 1978. With permission from the rockefeller University press.) Figure 9–17 Indirect immunocytochemistry. this detection method is very sensitive because many molecules of the secondary antibody recognize each primary antibody. the secondary antibody is covalently coupled to a marker molecule that makes it readily detectable. Commonly used marker molecules include fluorescent probes (for fluorescence microscopy), the enzyme horseradish peroxidase (for either conventional light microscopy or electron microscopy), colloidal gold spheres (for electron microscopy), and the enzymes alkaline phosphatase or peroxidase (for biochemical detection).

1	leads to the local formation of a colored precipitate. This reveals the location of the secondary antibody and hence the location of the antibody–antigen complex. Since each enzyme molecule acts catalytically to generate many thousands of molecules of product, even tiny amounts of antigen can be detected. Although the enzyme amplification makes enzymelinked methods sensitive, diffusion of the colored precipitate away from the enzyme limits the spatial resolution of this method for microscopy, and fluorescent labels are usually used for the most sensitive and precise optical localization. imaging of Complex three-Dimensional objects is possible with the optical Microscope

1	imaging of Complex three-Dimensional objects is possible with the optical Microscope For ordinary light microscopy, as we have seen, a tissue has to be sliced into thin sections to be examined; the thinner the section, the crisper the image. Since information about the third dimension is lost upon sectioning, how, then, can we get a picture of the threedimensional architecture of a cell or tissue, and how can we view the microscopic structure of a specimen that, for one reason or another, cannot first be sliced into sections? Although an optical microscope is focused on a particular focal plane within a threedimensional specimen, all the other parts of the specimen, above and below the plane of focus, are also illuminated and the light originating from these regions contributes to the image as “outoffocus” blur. This can make it very hard to interpret the image in detail and can lead to fine image structure being obscured by the outoffocus light.

1	Two distinct but complementary approaches solve this problem: one is computational, the other optical. These threedimensional microscopic imaging methods make it possible to focus on a chosen plane in a thick specimen while rejecting the light that comes from outoffocus regions above and below that plane. Thus one sees a crisp, thin optical section. From a series of such optical sections taken at different depths and stored in a computer, a threedimensional image can be reconstructed. The methods do for the microscopist what the computed tomography (CT) scanner does (by different means) for the radiologist investigating a human body: both machines give detailed sectional views of the interior of an intact structure.

1	The computational approach is often called image deconvolution. To understand how it works, remember that the wavelike nature of light means that the microscope lens system produces a small blurred disc as the image of a point light source (see Figure 9–5), with increased blurring if the point source lies above or below the focal plane. This blurred image of a point source is called the point spread function (see Figure 9–36). An image of a complex object can then be thought of as being built up by replacing each point of the specimen by a corresponding blurred disc, resulting in an image that is blurred overall. For deconvolution, we first obtain a series of (blurred) images, usually with a cooled CCD camera or more recently a CMOS camera, focusing the microscope in turn on a series of focal planes—in effect, a (blurred) threedimensional image. Digital processing of the stack of digital images then removes as much of the blur as possible. In essence, the computer program uses the

1	focal planes—in effect, a (blurred) threedimensional image. Digital processing of the stack of digital images then removes as much of the blur as possible. In essence, the computer program uses the measured point spread function of a point source of light from that microscope to determine what the effect of the blurring would have been on the image, and then applies an equivalent “deblurring” (deconvolution), turning the blurred threedimensional image into a series of clean optical sections, albeit still constrained by the diffraction limit. Figure 9–18 shows an example.

1	the Confocal Microscope produces optical sections by excluding out-of-Focus Light The confocal microscope achieves a result similar to that of deconvolution, but does so by manipulating the light before it is measured; it is an analog technique rather than a digital one. The optical details of the confocal microscope are complex, but the basic idea is simple, as illustrated in Figure 9–19, and the results are Figure 9–18 Image deconvolution. a light micrograph of the large polytene chromosomes from Drosophila, stained with a fluorescent Dna-binding dye. the same field of view after image deconvolution clearly reveals the banding pattern on the chromosomes. each band is about 0.25 μm thick, approaching the diffraction limit of the light microscope. (Courtesy of the John sedat Laboratory.)

1	Figure 9–19 The confocal fluorescence microscope. (a) this simplified diagram shows that the basic arrangement of optical components is similar to that of the in Figure 9–12, except that a laser is used to illuminate a small pinhole whose image is focused at a single point in the three-dimensional (3-D) specimen. (B) emitted fluorescence from this focal point in the specimen is focused at a second (confocal) pinhole. (C) emitted light from elsewhere in the specimen is not focused at the pinhole and therefore does not contribute to the final image. By scanning the beam of light across the specimen, a very sharp two-dimensional image of the exact plane of focus is built up that is not significantly point of focus fuorescent specimen is illuminated with a focused point of light from a pinhole far superior to those obtained by conventional light microscopy (Figure 9–20A and B).

1	The confocal microscope is generally used with fluorescence optics (see Figure 9–12), but instead of illuminating the whole specimen at once, in the usual way, the optical system at any instant focuses a spot of light onto a single point at a specific depth in the specimen. This requires a source of pinpoint illumination that is usually supplied by a laser whose light has been passed through a pinhole. The fluorescence emitted from the illuminated material is collected at a suitable light detector and used to generate an image. A pinhole aperture is placed in front of the detector, at a position that is confocal with the illuminating pinhole—that is, precisely where the rays emitted from the illuminated point in the specimen come to a focus. Thus, the light from this point in the specimen converges on this aperture and enters the detector.

1	By contrast, the light from regions out of the plane of focus of the spotlight is also out of focus at the pinhole aperture and is therefore largely excluded from the detector (see Figure 9–19). To build up a twodimensional image, data from each point in the plane of focus are collected sequentially by scanning across the field from left to right in a regular pattern of pixels and are displayed on a computer screen. Although not shown in Figure 9–19, the scanning is usually done by deflecting the beam with an oscillating mirror placed between the dichroic mirror and the objective lens in such a way that the illuminating spotlight and the degraded by light from other regions of the specimen. Figure 9–20 Confocal fluorescence microscopy produces clear optical sections and three-dimensional data sets. the first two micrographs are of the same intact gastrula-stage Drosophila embryo, which has been stained with a fluorescent probe for actin filaments.

1	(a) the conventional, unprocessed image is blurred by the presence of fluorescent structures above and below the plane of focus. (B) in the confocal image, this out-of-focus information is removed, resulting in a crisp optical section of the cells in the embryo. (C) a three-dimensional reconstruction of an object can be assembled from a stack of such optical sections. in this case, the complex branching structure of the mitochondrial compartment in a single live yeast cell is shown. (a and B, courtesy of richard Warn and peter shaw; C, courtesy of stefan hell.)

1	Figure 9–21 Multiphoton imaging. infrared laser light causes less damage to living cells than visible light and can also penetrate further, allowing microscopists to peer deeper into living tissues. the two-photon effect, in which a fluorochrome can be excited by two coincident infrared photons instead of a single high-energy photon, allows us to see nearly 0.5 mm inside the cortex of a live mouse brain. a dye, whose fluorescence changes with the calcium concentration, reveals active synapses (yellow) on the dendritic spines (red) that change as a function of time; in this case, there is a day between each image. (Courtesy of thomas oertner and karel svoboda.) confocal pinhole at the detector remain strictly in register. Variations in design now allow the rapid collection of data at video rates.

1	The confocal microscope has been used to resolve the structure of numerous complex threedimensional objects (Figure 9–20C) including the networks of cytoskeletal fibers in the cytoplasm and the arrangements of chromosomes and genes in the nucleus. The relative merits of deconvolution methods and confocal microscopy for threedimensional optical microscopy depend on the specimen being imaged. Confocal microscopes tend to be better for thicker specimens with high levels of outoffocus light. They are also generally easier to use than deconvolution systems and the final optical sections can be seen quickly. In contrast, the cooled CCD or CMOS cameras used for deconvolution systems are extremely efficient at collecting small amounts of light, and they can be used to make detailed threedimensional images from specimens that are too weakly stained or too easily damaged by the bright light used for confocal microscopy.

1	Both methods, however, have another drawback; neither is good at coping with very thick specimens. Deconvolution methods quickly become ineffective any deeper than about 40 μm into a specimen, while confocal microscopes can only obtain images up to a depth of about 150 μm. Special microscopes can now take advantage of the way in which fluorescent molecules are excited, to probe even deeper into a specimen. Fluorescent molecules are usually excited by a single highenergy photon, of shorter wavelength than the emitted light, but they can in addition be excited by the absorption of two (or more) photons of lower energy, as long as they both arrive within a femtosecond or so of each other. The use of this longerwavelength excitation has some important advantages. In addition to reducing background noise, red or nearinfrared light can penetrate deeper into a specimen. Multiphoton microscopes, constructed to take advantage of this two-photon effect, can obtain sharp images, sometimes even

1	noise, red or nearinfrared light can penetrate deeper into a specimen. Multiphoton microscopes, constructed to take advantage of this two-photon effect, can obtain sharp images, sometimes even at a depth of 250 μm within a specimen. This is particularly valuable for studies of living tissues, notably in imaging the dynamic activity of synapses and neurons just below the surface of living brains (Figure 9–21).

1	Even the most stable cell structures must be assembled, disassembled, and reorganized during the cell’s life cycle. Other structures, often enormous on the molecular scale, rapidly change, move, and reorganize themselves as the cell conducts its internal affairs and responds to its environment. Complex, highly organized pieces of molecular machinery move components around the cell, controlling traffic into and out of the nucleus, from one organelle to another, and into and out of the cell itself.

1	Various techniques have been developed to visualize the specific components involved in such dynamic phenomena. Many of these methods use fluorescent proteins, and they require a tradeoff between structural preservation and efficient labeling. All of the fluorescent molecules discussed so far are made outside the cell and then artificially introduced into it. But use of genes coding for protein molecules that are themselves inherently fluorescent also enables the creation of organisms and cell lines that make their own visible tags and labels, without the introduction of foreign molecules. These cellular exhibitionists display their inner workings in glowing fluorescent color.

1	Foremost among the fluorescent proteins used for these purposes by cell biologists is the green fluorescent protein (GFP), isolated from the jellyfish Aequorea victoria. This protein is encoded by a single gene, which can be cloned and introduced into cells of other species. The freshly translated protein is not fluorescent, but within an hour or so (less for some alleles of the gene, more for others) it undergoes a selfcatalyzed posttranslational modification to generate an efficient fluorochrome, shielded within the interior of a barrellike protein, which will now fluoresce when illuminated appropriately with blue light (Figure 9–22). Extensive sitedirected mutagenesis performed on the original gene sequence has resulted in multiple variants that can be used effectively in organisms ranging from animals and plants to fungi and microbes. The fluorescence efficiency has also been improved, and variants have been generated with altered absorption and emission spectra from the

1	ranging from animals and plants to fungi and microbes. The fluorescence efficiency has also been improved, and variants have been generated with altered absorption and emission spectra from the blue–green, like blue fluorescent protein or BFP, to the far visible red. Other, related fluorescent proteins have since been discovered (for example, in corals) that also extend the range into the red region of the spectrum, like red fluorescent protein or RFP.

1	One of the simplest uses of GFP is as a reporter molecule, a fluorescent probe to monitor gene expression. A transgenic organism can be made with the GFPcoding sequence placed under the transcriptional control of the promoter belonging to a gene of interest, giving a directly visible readout of the gene’s expression pattern in the living organism (Figure 9–23). In another application, a peptide location signal can be added to the GFP to direct it to a particular cell compartment, such as the endoplasmic reticulum or a mitochondrion, lighting up these organelles so they can be observed in the living state (see Figure 12–31).

1	The GFP DNA coding sequence can also be inserted at the beginning or end of the gene for another protein, yielding a chimeric product consisting of that protein with a GFP domain attached. In many cases, this GFP fusion protein behaves in the same way as the original protein, directly revealing its location and activities by means of its genetically encoded fluorescence (Figure 9–24). It is often possible to prove that the GFP fusion protein is functionally equivalent to the untagged protein, for example by using it to rescue a mutant lacking that protein. GFP tagging is the clearest and most unequivocal way of showing the distribution and dynamics of a protein in a living organism (Figure 9–25 and see Movie 16.8).

1	Fluorescent proteins are now exploited not just to see where in a cell a particular protein is located, but also to uncover its kinetic properties and to find out whether it might interact with other molecules. We now describe three techniques in which fluorescent proteins are used in this way. First, interactions between one protein and another can be monitored by fluorescence resonance energy transfer, also called Förster resonance energy

1	First, interactions between one protein and another can be monitored by fluorescence resonance energy transfer, also called Förster resonance energy Figure 9–23 Green fluorescent protein (GFP) as a reporter. For this experiment, carried out in the fruit fly, the gFp gene was joined (using recombinant Dna techniques) to a fly promoter that is active only in a specialized set of neurons. this image of a live fly embryo was captured by a fluorescence microscope and shows approximately 20 neurons, each with long projections (axons and dendrites) that communicate with other (nonfluorescent) cells. these neurons are located just under the surface of the animal and allow it to sense its immediate environment. (From W.B. grueber et al., Curr. Biol. 13:618–626, 2003. With permission from elsevier.)

1	Figure 9–22 Green fluorescent protein (GFP). the structure of gFp, shown here schematically, highlights the eleven β strands that form the staves of a barrel. Buried within the barrel is the active chromophore (dark green) that is formed post-translationally from the protruding side chains of three amino acid residues. (From M. ormö et al., Science 273:1392–1395, 1996. With permission from aaas.)

1	Figure 9–24 GFP-tagged proteins. this living cell from a tobacco plant is expressing high levels of green fluorescent protein, fused to a protein that is targeted to mitochondria, which accordingly appear green. the mitochondria are seen to cluster around the chloroplasts, whose chlorophyll autofluorescence marks them out in red. (Courtesy of olivier grandjean.) transfer, both abbreviated FRET. In this technique, two molecules of interest are each labeled with a different fluorochrome, chosen so that the emission spectrum of one fluorochrome, the donor, overlaps with the absorption spectrum of the other, the acceptor. If the two proteins bind so as to bring their fluorochromes into very close proximity (closer than about 5 nm), one fluorochrome, when excited, can transfer energy from the absorbed light directly (by resonance, nonradiatively) to the other. Thus, when the complex is illuminated at the excitation wavelength of the first fluorochrome, fluorescent light is produced at the

1	absorbed light directly (by resonance, nonradiatively) to the other. Thus, when the complex is illuminated at the excitation wavelength of the first fluorochrome, fluorescent light is produced at the emission wavelength of the second. This method can be used with two different spectral variants of GFP as fluorochromes to monitor processes such as the interaction of signaling molecules with their receptors, or proteins in macromolecular complexes at specific locations inside living cells (Figure 9–26). The FRET can be measured by quantifying the reduction of the donor fluorescence in the presence of the acceptor.

1	A second example of a fluorescencetagging technique that allows detailed observations of proteins within cells involves synthesizing an inactive form of the fluorescent molecule of interest, introducing it into the cell, and then activating it suddenly at a chosen site in the cell by focusing a spot of light on it. This process is referred to as photoactivation. Many inactive photosensitive precursors of this type, often called caged molecules, have been made based on a variety of fluorescent molecules. A microscope can be used to focus a strong pulse of light from a laser on any tiny region of the cell, so that the experimenter can control exactly where and when the fluorescent molecule is photoactivated. The technique allows us to follow complex and rapid intracellular processes, such as the actions of signaling molecules or the movements of cytoskeletal proteins.

1	When a photoactivatable fluorescent tag is attached to a purified protein, it is important that the modified protein remain biologically active: labeling with a caged fluorescent dye adds a bulky group to the surface of a protein, which can easily change the protein’s properties. A satisfactory labeling protocol is usually found by trial and error. Once a biologically active labeled protein has been produced, it needs to be introduced into the living cell where its behavior can be followed. Tubulin labeled with caged fluorescein, for example, can be injected into a dividing cell, where it is incorporated into microtubules of the mitotic spindle. When a small region of the spindle is illuminated with a laser, the labeled tubulin

1	Figure 9–25 Dynamics of GFP tagging. this sequence of micrographs shows a set of three-dimensional images of a living nucleus taken over the course of 135 minutes. tobacco cells have been stably transformed with gFp fused to a spliceosomal protein that is concentrated in small nuclear bodies called Cajal bodies (see Figure 6–46). the fluorescent Cajal bodies, easily visible in a living cell with confocal microscopy, are dynamic structures that move around within the nucleus. (Courtesy of kurt Boudonck, Liam Dolan, and peter shaw.) becomes fluorescent, so that its movement along the spindle microtubules can be readily followed (Figure 9–27).

1	A further development in photoactivation is the discovery that the genes encoding GFP and related fluorescent proteins can be engineered to produce protein variants, usually with one or more amino acid changes, that fluoresce only weakly under normal excitation conditions, but can be induced to fluoresce either more strongly or with a color shift (for example, from green to red) by activating them with a strong pulse of light at a different wavelength. In principle, the microscopist can then follow the local in vivo behavior of any protein that can be expressed as a fusion with one of these GFP variants. These genetically encoded, photoactivatable fluorescent proteins allow the lifetime and behavior of any protein to be studied independently of other newly synthesized proteins (Figure 9–28).

1	A third way to exploit GFP fused to a protein of interest is known as fluorescence recovery after photobleaching (FRAP). Here, one uses a strong focused beam of light from a laser to extinguish the GFP fluorescence in a specified region of the cell, after which one can analyze the way in which remaining unbleached fluorescent protein molecules move into the bleached area as a function of time.

1	Figure 9–26 Fluorescence resonance energy transfer (FRET). to determine whether (and when) two proteins interact inside a cell, the proteins are first produced as fusion proteins attached to different color variants of green fluorescent protein (gFp). (a) in this example, protein X is coupled to a blue fluorescent protein, which is excited by violet light (370–440 nm) and emits blue light (440–480 nm); protein Y is coupled to a green fluorescent protein, which is excited by blue light (440–480 nm) and emits green light (510 nm). (B) if protein X and Y do not interact, illuminating the sample with violet light yields fluorescence from the blue fluorescent protein only. (C) When protein X and protein Y interact, the resonance transfer of energy, Fret, can now occur. illuminating the sample with violet light excites the blue fluorescent protein, which transfers its energy to the green fluorescent protein, resulting in an emission of green light. the fluorochromes must be quite close

1	with violet light excites the blue fluorescent protein, which transfers its energy to the green fluorescent protein, resulting in an emission of green light. the fluorochromes must be quite close together—within about 1–5 nm of one another—for Fret to occur. Because not every molecule of protein X and protein Y is bound at all times, some blue light may still be detected. But as the two proteins begin to interact, emission from the donor blue fluorescent protein falls as the emission from the acceptor gFp rises.

1	Figure 9–27 Determining microtubule flux in the mitotic spindle with caged fluorescein linked to tubulin. a metaphase spindle formed in vitro from an extract of Xenopus eggs has incorporated three fluorescent markers: rhodamine-labeled tubulin (red) to mark all the microtubules, a blue Dna-binding dye that labels the chromosomes, and caged-fluorescein-labeled tubulin, which is also incorporated into all the microtubules but is invisible because it is nonfluorescent until activated by ultraviolet (UV) light.

1	a beam of UV light activates, or “uncages,” the caged-fluorescein-labeled tubulin locally, mainly just to the left side of the metaphase plate. over the next few minutes—after 1.5 minutes in (C) and after 2.5 minutes in (D)—the uncagedfluorescein–tubulin signal moves toward the left spindle pole, indicating that tubulin is continuously moving poleward even though the spindle (visualized by the red rhodamine-labeled tubulin fluorescence) remains largely unchanged. (From k.e. sawin and t.J. Mitchison, J. Cell Biol. 112:941–954, 1991. With permission from the rockefeller University press.) 546 Chapter 9: Visualizing Cells This technique is usually carried out with a confocal microscope and, like photoactivation, can deliver valuable quantitative data about a protein’s kinetic parameters, such as diffusion coefficients, active transport rates, or binding and dissociation rates from other proteins (Figure 9–29).

1	One way to study the chemistry of a single living cell is to insert the tip of a fine, glass, ionsensitive microelectrode directly into the cell interior through the plasma membrane. This technique is used to measure the intracellular concentrations of common inorganic ions, such as H+, Na+, K+, Cl–, and Ca2+. However, ionsensitive microelectrodes reveal the ion concentration only at one point in a cell, and for an ion present at a very low concentration, such as Ca2+, their responses are slow and somewhat erratic. Thus, these microelectrodes are not ideally suited to record the rapid and transient changes in the concentration of cytosolic Ca2+ that have an important role in allowing cells to respond to extracellular signals. Such changes can be analyzed with ion-sensitive indicators, whose light emission reflects the local concentration of the ion. Some of these indicators are luminescent (emitting light spontaneously), while others are fluorescent (emitting light on exposure to

1	whose light emission reflects the local concentration of the ion. Some of these indicators are luminescent (emitting light spontaneously), while others are fluorescent (emitting light on exposure to light).

1	Figure 9–28 Photoactivation. photoactivation is the light-induced activation of an inert molecule to an active state. in this experiment, shown schematically in (a), a photoactivatable variant of gFp is expressed in a cultured animal cell. (B) Before activation (time 0 sec), little or no gFp fluorescence is detected in the selected region (red circle) when excited by blue light at 488 nm. after activation of the gFp with an ultraviolet laser pulse at 413 nm, it rapidly fluoresces brightly in the selected region (green). the movement of gFp, as it diffuses out of this region, can be measured. since only the photoactivated proteins are fluorescent within the cell, the trafficking, turnover, and degradative pathways of proteins can be monitored. (B, from J. Lippincott-schwartz and g.h. patterson, Science 300:87–91, 2003.)

1	Figure 9–29 Fluorescence recovery after photobleaching (FRAP). a strong focused pulse of laser light will extinguish, or bleach, the fluorescence of gFp. By selectively photobleaching a set of fluorescently tagged protein molecules within a defined region of a cell, the microscopist can monitor recovery over time, as the remaining fluorescent molecules move into the bleached region (see Movie 10.6). (a) the experiment shown uses monkey cells in culture that express galactosyltransferase, an enzyme that constantly recycles between the golgi apparatus and the endoplasmic reticulum (er). the golgi apparatus in one of the two cells is selectively photobleached, while the production of new fluorescent protein is blocked by treating the cells with cycloheximide. the recovery, resulting from fluorescent enzyme molecules moving from the er to the golgi, can then be followed over a period of time. (B) schematic diagram of the experiment shown in (a). (a, from J. Lippincott-schwartz,

1	from fluorescent enzyme molecules moving from the er to the golgi, can then be followed over a period of time. (B) schematic diagram of the experiment shown in (a). (a, from J. Lippincott-schwartz, Histochem. Cell Biol. 116:97–107, 2001. With permission from springer-Verlag.) only molecules in the evanescent feld fuoresce

1	Figure 9–32 TIRF microscopy allows the detection of single fluorescent molecules. (a) tirF microscopy uses excitatory laser light to illuminate the cover-slip surface at the critical angle at which all the light is reflected by the glass–water interface. some electromagnetic energy extends a short distance across the interface as an evanescent wave that excites just those molecules that are attached to the cover slip or are very close to its surface. (B) tirF microscopy is used here to image individual myosin–gFp molecules (green dots) attached to nonfluorescent actin filaments (C), which are invisible but stuck to the surface of the cover slip. (Courtesy of Dmitry Cherny and Clive r. Bagshaw.) particular molecule of interest. This problem can be solved by the use of a special optical technique called total internal reflection fluorescence (TIRF) microscopy. In a TIRF microscope, laser light shines onto the coverslip surface at the precise critical angle at which total internal

1	technique called total internal reflection fluorescence (TIRF) microscopy. In a TIRF microscope, laser light shines onto the coverslip surface at the precise critical angle at which total internal reflection occurs (Figure 9–32A). Because of total internal reflection, the light does not enter the sample, and the majority of fluorescent molecules are not, therefore, illuminated. However, electromagnetic energy does extend, as an evanescent field, for a very short distance beyond the surface of the cover slip and into the specimen, allowing just those molecules in the layer closest to the surface to become excited. When these molecules fluoresce, their emitted light is no longer competing with outoffocus light from the overlying molecules, and can now be detected. TIRF has allowed several dramatic experiments, for instance imaging of single motor proteins moving along microtubules or single actin filaments forming and branching. At present, the technique is restricted to a thin layer

1	dramatic experiments, for instance imaging of single motor proteins moving along microtubules or single actin filaments forming and branching. At present, the technique is restricted to a thin layer within only 100–200 nm of the cell surface (Figure 9–32B and C).

1	individual Molecules Can Be touched, imaged, and Moved Using atomic Force Microscopy

1	While TIRF allows single molecules to be visualized under certain conditions, it is strictly a passive observation method. In order to probe molecular function, it is ultimately useful to be able to manipulate individual molecules themselves, and atomic force microscopy (AFM) provides a method to do just that. In an AFM device, an extremely small and sharply pointed tip, often of silicon or silicon nitride, is made using nanofabrication methods similar to those used in the semiconductor industry. The tip of the AFM probe is attached to a springy cantilever arm mounted on a highly precise positioning system that allows it to be moved over very small distances. In addition to this precise movement capability, the AFM device is able to collect information about a variety of forces that it encounters—including electrostatic, van der Waals, and mechanical forces—which are felt by its tip as it moves close to or touches the surface (Figure 9–33A). When AFM was first developed, it was

1	it encounters—including electrostatic, van der Waals, and mechanical forces—which are felt by its tip as it moves close to or touches the surface (Figure 9–33A). When AFM was first developed, it was intended as an imaging technology to measure molecularscale (B) (C) 1000 800 600 400 200 0 0 100 200 300 extension (nm) laser photodiode (detector) detector and feedback electronics springy cantilever arm AFM tip stretching molecule attached to substrate e.g., mica (A) 28 nm force (pN)

1	Figure 9–33 Single molecules can be imaged and manipulated by atomic force microscopy. (a) schematic diagram of the key components of an atomic force microscope (aFM), showing the force-sensing tip attached to one end of a single protein molecule, as in the experiment described in (D). (B) and (C) an aFM in imaging mode created these images of a single heteroduplex Dna molecule with a Muts protein dimer (larger white regions) bound near its center, at the point of a mismatched base pair. Muts is the first protein that binds to Dna when the mismatch repair process is initiated (see Figure 5–19). the smaller white dots are single streptavidin molecules, used to label the two ends of each Dna molecule. (D) titin is an enormous protein molecule that provides muscle with its passive elasticity (see Figure 16–34). the extensibility of this protein can be directly tested using a short, artificially produced protein that contains eight repeated immunoglobulin (ig) domains from one region of

1	(see Figure 16–34). the extensibility of this protein can be directly tested using a short, artificially produced protein that contains eight repeated immunoglobulin (ig) domains from one region of the titin protein. in this experiment, the tip of the aFM is used to pick up, and progressively stretch, a single molecule until it eventually ruptures. as force is applied, each ig domain suddenly begins to unfold, and the force needed in each case (about 200 pn) can be recorded. the region of the force–extension curve shaded green records the sequential unfolding event for each of the eight protein domains. (B and C, from Y. Jiang and p.e. Marszalek, EMBO J. 30:2881–2893, 2011. reprinted with permission of John Wiley & sons; D, adapted from W.a. Linke et al., J. Struct. Biol. 137:194–205, 2002. With permission from elsevier.) features on a surface. When used in this mode, the probe is scanned over the surface, moving up and down as necessary to maintain a constant interaction force with

1	With permission from elsevier.) features on a surface. When used in this mode, the probe is scanned over the surface, moving up and down as necessary to maintain a constant interaction force with the surface, thus revealing any objects such as proteins or other molecules that might be present on the otherwise flat surface (Figure 9–33B and C). AFM is not limited to simply imaging surfaces, however, and can also be used to pick up and move single molecules that adsorb strongly to the tip. Using this technology, the mechanical properties of individual protein molecules can be measured in detail. For example, AFM has been used to unfold a single protein molecule in order to measure the energetics of domain folding (Figure 9–33 D).

1	The variations on light microscopy we have described so far are all constrained by the classic diffraction limit to resolution described earlier; that is, to about 200 nm (see Figure 9–6). Yet many cellular structures—from nuclear pores to nucleosomes and clathrincoated pits—are much smaller than this and so are unresolvable by conventional light microscopy. Several approaches, however, are now available that bypass the limit imposed by the diffraction of light, and successfully allow objects as small as 20 nm to be imaged and clearly resolved: a remarkable, orderofmagnitude improvement.

1	The first of these socalled superresolution approaches, structured illumination microscopy (SIM), is a fluorescence imaging method with a resolution of about 100 nm, or twice the resolution of conventional brightfield and confocal microscopy. SIM overcomes the diffraction limit by using a grated or structured pattern of light to illuminate the sample. The microscope’s physical setup and operation is quite complex, but the general principle can be thought of as similar to creating a moiré pattern, an interference pattern created by overlaying two grids with different angles or mesh sizes (Figure 9–34). In a similar way to creating a moiré pattern, the illuminating grid and the sample features combine into an interference pattern, from which the original highresolution contributions to the image of features beyond the classical resolution limit can be calculated. Illumination by a grid means that the parts of the sample in the dark stripes of the grid are not illuminated and therefore

1	image of features beyond the classical resolution limit can be calculated. Illumination by a grid means that the parts of the sample in the dark stripes of the grid are not illuminated and therefore not imaged, so the imaging is repeated several times (usually three) after translating the grid through a fraction of the grid spacing between each image. As the interference effect is strongest for image components close to the direction of the grid bars, the whole process is repeated with the grid pattern rotated through a series of angles to obtain an equivalent enhancement in all directions. Finally, mathematically combining all these separate images by computer creates an enhanced superresolution image. SIM is versatile because it can be used with any fluorescent dye or protein, and combining SIM images captured at consecutive focal planes can create threedimensional data sets (Figure 9–35).

1	Figure 9–34 Structured illumination microscopy. the principle, illustrated here, is to illuminate a sample with patterned light and measure the moiré pattern. shown are (a) the pattern from an unknown structure and (B) a known pattern. (C) When these are combined, the resulting moiré pattern contains more information than is easily seen in (a), the original pattern. if the known pattern (B) has higher spatial frequencies, then better resolution will result. however, because the spatial patterns that can be created optically are also diffraction-limited, siM can only improve the resolution by about a factor of two. (From B.o. Leung and k.C. Chou, Appl. Spectrosc. 65:967–980, 2011.)

1	Figure 9–35 Structured illumination microscopy can be used to create three-dimensional data. these three-dimensional projections of the meiotic chromosomes at pachytene in a maize cell show the paired lateral elements of the synaptonemal complexes. (a) the chromosome set has been stained with a fluorescent antibody to cohesin and is viewed here by conventional fluorescence microscopy. Because the distance between the two lateral elements is about 200 nm, the diffraction limit, the two lateral elements that make up each complex are not resolved. (B) in the three-dimensional siM image, the improved resolution enables each lateral element, about 100 nm across, to be clearly resolved, and the two chromosomes can clearly be seen to coil around each other. (C) Because the complete three-dimensional data set for the whole nucleus is available, the path of each separate pair of chromosomes can be traced and artificially assigned a different color. (Courtesy of C.J. rachel Wang, peter Carlton

1	data set for the whole nucleus is available, the path of each separate pair of chromosomes can be traced and artificially assigned a different color. (Courtesy of C.J. rachel Wang, peter Carlton and Zacheus Cande.) approximates a gaussian distribution, whose width at half-maximum height under ideal conditions is about 200 nm. (C) two point sources that are about 200 nm apart

1	To get around the diffraction limit, the other two superresolutiontechniques exploit aspects of the point spread function, a property of the optical system mentioned earlier. The point spread function is the distribution of light intensity within the threedimensional, blurred image that is formed when a single point source of light is brought to a focus with a lens. Instead of being identical to the point source, the image has an intensity distribution that is approximately described by a Gaussian distribution, which in turn determines the resolution of the lens system (Figure 9–36). Two points that are closer than the width at halfmaximum height of this distribution will become hard to resolve because their images overlap too much (see Figure 9–36C).

1	In fluorescence microscopy, the excitation light is focused to a spot on the specimen by the objective lens, which then captures the photons emitted by any fluorescent molecule that the beam has raised from a ground state to an excited state. Because the excitation spot is blurred according to the point spread function, fluorescent molecules that are closer than about 200 nm will be imaged as a single blurred spot. One approach to increasing the resolution is to switch all the fluorescent molecules at the periphery of the blurry excitation spot back to their ground state, or to a state where they no longer fluoresce in the normal way, leaving only those at the very center to be recorded. This can be done in practice by adding a second, very bright laser beam that wraps around the excitation beam like a torus. The wavelength and intensity of this second beam are adjusted so as to switch the fluorescent molecules off everywhere except at the very center of the point spread function, a

1	beam like a torus. The wavelength and intensity of this second beam are adjusted so as to switch the fluorescent molecules off everywhere except at the very center of the point spread function, a region that can be as small as 20 nm across (Figure 9–37). The fluorescent probes used must be in a special class that is photoswitchable: their emission can be reversibly switched on and off with lights of different wavelengths. As the specimen is scanned with this arrangement of lasers, fluorescent molecules are switched on and off, and the small point spread function at each location is recorded. The diffraction limit is breached because the technique ensures that similar but very closely spaced molecules are in one of two different states, either fluorescing or dark. This approach is called STED (stimulated emission depletion microscopy) and various microscopes using versions of the general method are now in wide use. Resolutions of 20 nm have been achieved in biological specimens, and

1	STED (stimulated emission depletion microscopy) and various microscopes using versions of the general method are now in wide use. Resolutions of 20 nm have been achieved in biological specimens, and even higher resolution attained with nonbiological specimens (see Figure 9–37).

1	If a single fluorescent molecule is imaged, it appears as a circular blurry disc, but if sufficient photons have contributed to this image, the precise mathematical center of the disclike image can be determined very accurately, often to within a few nanometers. But the problem with a specimen that contains a large number

1	Figure 9–37 Superresolution microscopy can be achieved by reducing the size of the point spread function. (a) the size of a normal focused beam of excitatory light. (B) an extremely strong superimposed laser beam, at a different wavelength and in the shape of a torus, depletes emitted fluorescence everywhere in the specimen except right in the center of the beam, reducing the effective width of the point spread function (C). as the specimen is scanned, this small point spread function can then build up a crisp image in a process called steD (stimulated emission depletion microscopy). (D) synaptic vesicles in live cultured neurons, fluorescently labeled and imaged by ordinary confocal microscopy, with a resolution of 260 nm. (e) the same vesicles imaged by steD, with a resolution of 60 nm, which allows single vesicles to be resolved. (F) Fluorescently labeled replication factories in the nucleus of a cultured cell, imaged by ordinary confocal microscopy. (g) the same replication

1	60 nm, which allows single vesicles to be resolved. (F) Fluorescently labeled replication factories in the nucleus of a cultured cell, imaged by ordinary confocal microscopy. (g) the same replication factories imaged by steD. single, discrete replication sites can be resolved by steD that cannot be seen in the confocal image. (a, B, and C, from g. Donnert et al., Proc. Natl Acad. Sci. USA 103:11440–11445, 2006. With permission from national academy of sciences; D and e, from V. Westphal et al., Science 320:246– 249, 2008. With permission from aaas; F and g, from Z. Cseresnyes,

1	U. schwarz and C.M. green, BMC Cell Biol. 10:88, 2009.) of adjacent fluorescent molecules, as we saw earlier, is that they each contribute blurry, overlapping point spread functions to the image, making the exact position of any one molecule impossible to resolve. Another way round this limitation is to arrange for only a very few, clearly separated molecules to actively fluoresce at any one moment. The exact position of each of these can then be computed, before subsequent sets of molecules are examined.

1	In practice, this can be achieved by using lasers to sequentially switch on a sparse subset of fluorescent molecules in a specimen containing photoactivatable or photoswitchable fluorescent labels. Labels are activated, for example, by illumination with nearultraviolet light, which modifies a small subset of molecules so that they fluoresce when exposed to an excitation beam at another wavelength. These are then imaged before bleaching quenches their fluorescence and a new subset is activated. Each molecule emits a few thousand photons in response to the excitation before switching off, and the switching process can be repeated hundreds or even thousands of times, allowing the exact coordinates of a very large set of single molecules to be determined. The full set can be combined and digitally displayed as an image in which the computed location of each individual molecule is exactly marked (Figure 9–38). This class of methods has been variously termed photoactivated localization

1	digitally displayed as an image in which the computed location of each individual molecule is exactly marked (Figure 9–38). This class of methods has been variously termed photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM).

1	By switching the fluorophores off and on sequentially in different regions of the specimen as a function of time, all the superresolution imaging methods described above allow the resolution of molecules that are much closer together than the 200 nm diffraction limit. In STED, the locations of the molecules are determined by using optical methods to define exactly where their fluorescence will be on or off. In PALM and STORM, individual fluorescent molecules are switched on and off at random over a period of time, allowing their positions to be accurately determined. PALM and STORM techniques have depended on the 100 photons 1000 photons 10,000 photons successive cycles of activation and bleaching allow well-separated single fuorescent molecules to be detected the exact center of each fuorescent molecule is determined and its position added to the map (C)a super-resolution image of the fuorescent structure is built up as the positions of successive small 1 µmgroups of molecules are

1	molecule is determined and its position added to the map (C)a super-resolution image of the fuorescent structure is built up as the positions of successive small 1 µmgroups of molecules are added to the map

1	Figure 9–38 Single fluorescent molecules can be located with great accuracy. (a) Determining the exact mathematical center of the blurred image of a single fluorescent molecule becomes more accurate the more photons contribute to the final image. the point spread function described in the text dictates that the size of the molecular image is about 200 nm across, but in very bright specimens, the position of its center can be pinpointed to within a nanometer. (B) in this imaginary specimen, sparse subsets of fluorescent molecules are individually switched on briefly and then bleached. the exact positions of all these well-spaced molecules can be gradually built up into an image at superresolution. (C) in this portion of a cell, the microtubules have been fluorescently labeled and imaged at the top in a tirF microscope (see Figure 9–32) and below, at superresolution, in a paLM microscope. the diameter of the microtubules in the lower panel now resembles their true size, about 25 nm,

1	the top in a tirF microscope (see Figure 9–32) and below, at superresolution, in a paLM microscope. the diameter of the microtubules in the lower panel now resembles their true size, about 25 nm, rather than the 250 nm in the blurred image at the top. (a, from a.L. Mcevoy et al., BMC Biol. 8:106, 2010; C, courtesy of Carl Zeiss Ltd.) development of novel fluorescent probes that exhibit the appropriate switching behavior. All these methods are now being extended to incorporate multicolor imaging, threedimensional imaging (Figure 9–39), and livecell imaging in real time. Ending the long reign of the diffraction limit has certainly reinvigorated light microscopy and its place in cell biology research.

1	Many light-microscope techniques are available for observing cells. Cells that have been fixed and stained can be studied in a conventional light microscope, whereas antibodies coupled to fluorescent dyes can be used to locate specific molecules in cells in a fluorescence microscope. Living cells can be seen with phase-contrast, differential-interference-contrast, dark-field, or bright-field microscopes. All forms of light microscopy are facilitated by digital image-processing techniques, which enhance sensitivity and refine the image. Confocal microscopy and image deconvolution both provide thin optical sections and can be used to reconstruct three-dimensional images.

1	Techniques are now available for detecting, measuring, and following almost any desired molecule in a living cell. Fluorescent indicator dyes can be introduced to measure the concentrations of specific ions in individual cells or in different parts of a cell. Virtually any protein of interest can be genetically engineered as a fluorescent fusion protein, and then imaged in living cells by fluorescence microscopy. The dynamic behavior and interactions of many molecules can be followed in living cells by variations on the use of fluorescent protein tags, in some cases at the level of single molecules. Various superresolution techniques can circumvent the diffraction limit and resolve molecules separated by distances as small as 20 nm. Looking at CeLLs anD MoLeCULes in the eLeCtron MiCrosCope

1	Looking at CeLLs anD MoLeCULes in the eLeCtron MiCrosCope Light microscopy is limited in the fineness of detail that it can reveal. Microscopes using other types of radiation—in particular, electron microscopes—can resolve much smaller structures than is possible with visible light. This higher resolution comes at a cost: specimen preparation for electron microscopy is complex and it is harder to be sure that what we see in the image corresponds precisely to the original living structure. It is possible, however, to use very rapid freezing to preserve structures faithfully for electron microscopy. Digital image analysis can be used to reconstruct threedimensional objects by combining information either from many individual particles or from multiple tilted views of a single object. Together, these approaches extend the resolution and scope of electron microscopy to the point at which we can faithfully image the structures of individual macromolecules and the complexes they form.

1	the electron Microscope resolves the Fine structure of the Cell

1	The formal relationship between the diffraction limit to resolution and the wavelength of the illuminating radiation (see Figure 9–6) holds true for any form of radiation, whether it is a beam of light or a beam of electrons. With electrons, however, the limit of resolution is very small. The wavelength of an electron decreases as its velocity increases. In an electron microscope with an accelerating voltage of 100,000 V, the wavelength of an electron is 0.004 nm. In theory, the resolution of such a microscope should be about 0.002 nm, which is 100,000 times that of the light microscope. Because the aberrations of an electron lens are considerably harder to correct than those of a glass lens, however, the practical resolving power of modern electron microscopes is, even with careful image processing to correct for lens aberrations, about 0.05 nm (0.5 Å) (Figure 9–40). This is because only the very center of the electron lenses can be used, and the effective numerical aperture is tiny.

1	processing to correct for lens aberrations, about 0.05 nm (0.5 Å) (Figure 9–40). This is because only the very center of the electron lenses can be used, and the effective numerical aperture is tiny. Furthermore, problems of specimen preparation, contrast, and radiation damage have generally limited the normal effective resolution for biological objects to 1 nm (10 Å). This is nonetheless about 200 times better than the resolution of the light microscope. Moreover, the performance of electron microscopes is improved by electron illumination sources called field emission guns. These very bright and coherent sources substantially improve the resolution achieved.

1	In overall design, the transmission electron microscope (TEM) is similar to a light microscope, although it is much larger and “upside down” (Figure 9–41). 0.14 nm Figure 9–40 The resolution of the electron microscope. this transmission electron micrograph of a monolayer of graphene resolves the individual carbon atoms as bright spots in a hexagonal lattice. graphene is a single isolated atomic plane of graphite and forms the basis of carbon nanotubes. the distance between adjacent bonded carbon atoms is 0.14 nm (1.4 Å). such resolution can only be obtained in a specially built transmission electron microscope in which all lens aberrations are carefully corrected, and with optimal specimens; it cannot be achieved with most conventional biological specimens. (From a. Dato et al., Chem. Commun. 40:6095–6097, 2009. With permission from the royal society of Chemistry.)

1	The source of illumination is a filament or cathode that emits electrons at the top of a cylindrical column about 2 m high. Since electrons are scattered by collisions with air molecules, air must first be pumped out of the column to create a vacuum. The electrons are then accelerated from the filament by a nearby anode and allowed to pass through a tiny hole to form an electron beam that travels down the column. Magnetic coils placed at intervals along the column focus the electron beam, just as glass lenses focus the light in a light microscope. The specimen is put into the vacuum, through an airlock, into the path of the electron beam. As in light microscopy, the specimen is usually stained—in this case, with electron-dense material. Some of the electrons passing through the specimen are scattered by structures stained with the electrondense material; the remainder are focused to form an image, in a manner analogous to the way an image is formed in a light microscope. The image can

1	scattered by structures stained with the electrondense material; the remainder are focused to form an image, in a manner analogous to the way an image is formed in a light microscope. The image can be observed on a phosphorescent screen or recorded with a highresolution digital camera. Because the scattered electrons are lost from the beam, the dense regions of the specimen show up in the image as areas of reduced electron flux, which look dark.

1	In the early days of its application to biological materials, the electron microscope revealed many previously unimagined structures in cells. But before these discoveries could be made, electron microscopists had to develop new procedures for embedding, cutting, and staining tissues.

1	Since the specimen is exposed to a very high vacuum in the electron microscope, living tissue is usually killed and preserved by fixation—first with glutaraldehyde, which covalently crosslinks protein molecules to their neighbors, and then with osmium tetroxide, which binds to and stabilizes lipid bilayers as well as proteins (Figure 9–42). Because electrons have very limited penetrating power, the fixed tissues normally have to be cut into extremely thin sections (25–100 nm thick, about 1/200 the thickness of a single cell) before they are viewed. This is achieved by dehydrating the specimen, permeating it with a monomeric resin that polymerizes to form a solid block of plastic, then cutting the block with a fine glass or diamond knife on a special microtome. The resulting thin sections, free of water and other volatile solvents, are supported on a small metal grid for viewing in the microscope (Figure 9–43).

1	Figure 9–41 The principal features of a light microscope and a transmission electron microscope. these drawings emphasize the similarities of overall design. Whereas the lenses in the light microscope are made of glass, those in the electron microscope are magnetic coils. the electron microscope requires that the specimen be placed in a vacuum. the inset shows a transmission electron microscope in use. (photograph courtesy of JeoL Ltd.) Figure 9–42 Two common chemical fixatives used for electron microscopy. the two reactive aldehyde groups of glutaraldehyde enable it to cross-link various types of molecules, forming covalent bonds between them. osmium tetroxide forms cross-linked complexes with many organic compounds, and in the process becomes reduced. this reaction is especially useful for fixing cell membranes, since the C=C double bonds present in many fatty acids react with osmium tetroxide.

1	The steps required to prepare biological material for electron microscopy are challenging. How can we be sure that the image of the fixed, dehydrated, resinembedded specimen bears any relation to the delicate, aqueous biological system present in the living cell? The best current approaches to this problem depend on rapid freezing. If an aqueous system is cooled fast enough and to a low enough temperature, the water and other components in it do not have time to rearrange themselves or crystallize into ice. Instead, the water is supercooled into a rigid but noncrystalline state—a “glass”—called vitreous ice. This state can be achieved by slamming the specimen onto a polished copper block cooled by liquid helium, by plunging it into or spraying it with a jet of a coolant such as liquid propane, or by cooling it at high pressure.

1	Some rapidly frozen specimens can be examined directly in the electron microscope using a special cooled specimen holder. In other cases, the frozen block can be fractured to reveal interior cell surfaces, or the surrounding ice can be sublimed away to expose external surfaces. However, we often want to examine thin sections. A compromise is therefore to rapidfreeze the tissue, replace the water with organic solvents, embed the tissue in plastic resin, and finally cut sections and stain. Although technically still difficult, this approach stabilizes and preserves the tissue in a condition very close to its original living state (Figure 9–44).

1	Image clarity in an electron micrograph depends upon having a range of contrasting electron densities within the specimen. Electron density in turn depends on the atomic number of the atoms that are present: the higher the atomic number, the more electrons are scattered and the darker that part of the image. Biological tissues are composed mainly of atoms of very low atomic number (primarily carbon, oxygen, nitrogen, and hydrogen). To make them visible, tissues are usually impregnated (before or after sectioning) with the salts of heavy metals such as uranium, lead, and osmium. The degree of impregnation, or “staining,” with these salts will vary for different cell constituents. Lipids, for example, tend to stain darkly after osmium fixation, revealing the location of cell membranes. We have seen how antibodies can be used in conjunction with fluorescence microscopy to localize specific macromolecules. An analogous method—immunogold copper grid covered with carbon and/or plastic flm

1	Figure 9–43 The metal grid that supports the thin sections of a specimen in a transmission electron microscope. Figure 9–44 Thin section of a cell. this thin section is of a yeast cell that has been very rapidly frozen and the vitreous ice replaced by organic solvents and then by plastic resin. the nucleus, mitochondria, cell wall, golgi stacks, and ribosomes can all be readily seen in a state that is presumed to be as lifelike as possible. (Courtesy of andrew staehelin.) 0.5 µm electron microscopy—can be used in the electron microscope. The usual procedure is to incubate a thin section first with a specific primary antibody, and then with a secondary antibody to which a colloidal gold particle has been attached. The gold particle is electrondense and can be seen as a black dot in the electron microscope (Figure 9–45). Different antibodies can be conjugated to different sized gold particles so multiple proteins can be localized in a single sample.

1	A complication for immunogold labeling is that the antibodies and colloidal gold particles do not penetrate into the resin used for embedding; therefore, they detect antigens only at the surface of the section. This means that the method’s sensitivity is low, since antigen molecules in the deeper parts of the section are not detected. Furthermore, we may get a false impression regarding which structures contain the antigen and which do not. One solution is to label the specimen before embedding it in plastic, when cells and tissues are still fully accessible to labeling reagents. Extremely small gold particles, about 1 nm in diameter, work best for this procedure. Such small gold particles are usually not easily visible in the final sections, so additional silver or gold is nucleated around the tiny 1 nm gold particles in a chemical process very much like photographic development. Different Views of a single object Can Be Combined to give a three-Dimensional reconstruction

1	Different Views of a single object Can Be Combined to give a three-Dimensional reconstruction Thin sections often fail to convey the threedimensional arrangement of cellular components viewed in a TEM, and the image can be very misleading: a linear structure such as a microtubule may appear in section as a pointlike object, for example, and a section through protruding parts of a single irregularly shaped solid body may give the appearance of two or more separate objects (Figure 9–46). The third dimension can be reconstructed from serial sections, but this is a lengthy and tedious process. Even thin sections, however, have a significant depth compared with the resolution of the electron microscope, so the TEM image can also be misleading in an opposite way, through the superimposition of objects that lie at different depths.

1	Figure 9–45 Localizing proteins in electron microscopy. immunogold electron microscopy is used here to find the specific location of four different protein components within the spindle pole body of yeast. at the top is a thin section of a yeast mitotic spindle showing the spindle microtubules that cross the nucleus and connect at each end to spindle pole bodies embedded in the nuclear envelope. a diagram of the components of a single spindle pole body is shown below. on separate sections, antibodies against four different proteins of the spindle pole body are used, together with colloidal gold particles (black dots), to reveal where within the complex structure each protein is located. (Courtesy of John kilmartin.)

1	Because of the large depth of field of electron microscopes, all the parts of the threedimensional specimen are in focus, and the resulting image is a projection (a superimposition of layers) of the structure along the viewing direction. The lost information in the third dimension can be recovered if we have views of the same specimen from many different directions. The computational methods for this technique are widely used in medical CT scans. In a CT scan, the imaging equipment is moved around the patient to generate the different views. In electron-microscope (EM) tomography, the specimen holder is tilted in the microscope, which achieves the same result. In this way, we can arrive at a threedimensional reconstruction, in a chosen standard orientation, by combining different views of a single object. Each individual view will be very noisy but by combining them in three dimensions and taking an average, the noise can be largely eliminated. Starting with thick plastic sections of

1	a single object. Each individual view will be very noisy but by combining them in three dimensions and taking an average, the noise can be largely eliminated. Starting with thick plastic sections of embedded material, threedimensional reconstructions, or tomograms, are used extensively to describe the detailed anatomy of specific regions of the cell, such as the Golgi apparatus (Figure 9–47) or the cytoskeleton. Increasingly, microscopists are also applying EM tomography to unstained frozen, hydrated sections, and even to rapidly frozen whole cells or organelles (Figure 9–48). Electron microscopy now provides a robust bridge between the scale of the single molecule and that of the whole cell.

1	images of surfaces Can Be obtained by scanning electron Microscopy A scanning electron microscope (SEM) directly produces an image of the threedimensional structure of the surface of a specimen. The SEM is usually smaller, simpler, and cheaper than a transmission electron microscope. Whereas the TEM uses the electrons that have passed through the specimen to form an Figure 9–46 A three-dimensional reconstruction from serial sections. single thin sections in the electron microscope sometimes give misleading impressions. in this example, most sections through a cell containing a branched mitochondrion seem to contain two or three separate mitochondria (compare Figure 9–44). sections 4 and 7, moreover, might be interpreted as showing a mitochondrion in the process of dividing. the true three-dimensional shape can be reconstructed from a complete set of serial sections.

1	Figure 9–47 Electron-microscope (EM) tomography. samples that have been rapidly frozen, and then freeze-substituted and embedded in plastic, preserve their structure in a condition that is very close to their original living state (Movie 9.2). this example shows the three-dimensional structure of the golgi apparatus from a rat kidney cell. several thick sections (250 nm) of the cell were tilted in a high-voltage electron microscope, along two different axes, and about 160 different views recorded. the digital data allow individual thin slices of the complete three-dimensional data set, or tomogram, to be viewed; for example, the serial slices, each only 4 nm thick, are shown in (a) and (B). Very little changes from one slice to the next, but using the full data set, and manually color-coding the membranes (B), one can obtain a full three-dimensional reconstruction, at a resolution of about 7 nm, of the complete golgi complex and its associated vesicles (C). (From M.s. Ladinsky et

1	the membranes (B), one can obtain a full three-dimensional reconstruction, at a resolution of about 7 nm, of the complete golgi complex and its associated vesicles (C). (From M.s. Ladinsky et al., J.Cell Biol. 144:1135–1149, 1999. With permission from the authors.)

1	Looking at CeLLs anD MoLeCULes in the eLeCtron MiCrosCope

1	Figure 9–48 Combining cryoelectronmicroscope tomography and single-particle reconstruction. small, unfixed, rapidly frozen specimens can be examined while still frozen. in this example, the small nuclei of the amoeba Dictyostelium were gently isolated and then very rapidly frozen before a series of angled views were recorded with the aid of a tilting microscope stage. these digital views are combined by eM tomographs to produce a three-dimensional tomogram. two thin digital slices (10 nm) through this tomogram show (a) top views and (B) side views of individual nuclear pores (white arrows). (C) in the three-dimensional model, a surface rendering of the pores (blue) is seen embedded in the nuclear envelope (yellow). From a series of tomograms it was possible to extract data sets for nearly 300 separate nuclear pores, whose structures could then be averaged using the techniques of single-particle reconstruction. the surface-rendered view of one of these reconstructed pores is shown (D)

1	300 separate nuclear pores, whose structures could then be averaged using the techniques of single-particle reconstruction. the surface-rendered view of one of these reconstructed pores is shown (D) from the nuclear face and (e) in (E) 50 nm cross section (compare with Figure 12–8). the pore complex is colored blue and the nuclear basket brown. (From M. Beck image, the SEM uses electrons that are scattered or emitted from the specimen’s et al., Science 306:1387–1390, 2004. With permission from aaas.) surface. The specimen to be examined is fixed, dried, and coated with a thin layer of heavy metal. Alternatively, it can be rapidly frozen, and then transferred to a cooled specimen stage for direct examination in the microscope. Often an entire plant part or small animal can be put into the microscope with very little preparation (Figure 9–49). The specimen is scanned with a very narrow beam of electrons. The quantity of electrons scattered or emitted as this primary beam bombards each

1	microscope with very little preparation (Figure 9–49). The specimen is scanned with a very narrow beam of electrons. The quantity of electrons scattered or emitted as this primary beam bombards each successive point of the metallic surface is measured and used to control the intensity of a second beam, which moves in synchrony with the primary beam and forms an image on a computer screen. Eventually a highly enlarged image of the surface as a whole is built up (Figure 9–50).

1	The SEM technique provides great depth of field; moreover, since the amount of electron scattering depends on the angle of the surface relative to the beam, the image has highlights and shadows that give it a threedimensional appearance (see Figure 9–49 and Figure 9–51). Only surface features can be examined, however, and in most forms of SEM, the resolution attainable is not very high (about 10 nm, with an effective magnification of up to 20,000 times). As a result, the technique is usually used to study whole cells and tissues rather than subcellular organelles (see Movie 21.3). Veryhighresolution SEMs have, however, been developed with a bright coherentfield emission gun as the electron source. This type of SEM can produce images that rival the resolution possible with a TEM (Figure 9–52). negative staining and Cryoelectron Microscopy Both allow Macromolecules to Be Viewed at high resolution

1	If they are shadowed with a heavy metal to provide contrast, isolated macromolecules such as DNA or large proteins can be visualized readily in the electron microscope, but negative staining allows finer detail to be seen. In this technique, the molecules are supported on a thin film of carbon and mixed with a Figure 9–49 A developing wheat flower, solution of a heavymetal salt such as uranyl acetate. After the sample has dried, or spike. this delicate flower spike was rapidly frozen, coated with a thin metal film, a very thin film of metal salt covers the carbon film everywhere except where it and examined in the frozen state with an has been excluded by the presence of an adsorbed macromolecule. Because the seM. this low-magnification micrograph macromolecule allows electrons to pass through it much more readily than does demonstrates the large depth of focus of the surrounding heavymetal stain, a reverse or negative image of the molecule is an seM. (Courtesy of kim Findlay.)

1	through it much more readily than does demonstrates the large depth of focus of the surrounding heavymetal stain, a reverse or negative image of the molecule is an seM. (Courtesy of kim Findlay.) created. Negative staining is especially useful for viewing large macromolecular aggregates such as viruses or ribosomes, and for seeing the subunit structure of protein filaments (Figure 9–53).

1	Shadowing and negative staining can provide highcontrast surface views of small macromolecular assemblies, but the size of the smallest metal particles in the shadow or stain used limits the resolution of both techniques. An alternative that allows us to visualize directly at high resolution even the interior features of threedimensional structures such as viruses and organelles is cryoelectron microscopy, in which rapid freezing to form vitreous ice is again the key. A very thin (about 100 nm) film of an aqueous suspension of virus or purified macromolecular complex is prepared on a microscope grid and is then rapidly frozen by

1	Figure 9–50 The scanning electron microscope. in an seM, the specimen is scanned by a beam of electrons brought to a focus on the specimen by the electromagnetic coils that act as lenses. the detector measures the quantity of electrons scattered or emitted as the beam bombards each successive point on the surface of the specimen and controls the intensity of successive points in an image built up on a screen. the seM creates striking images of three-dimensional objects with great depth of focus and a resolution between 3 nm and 20 nm depending on the instrument. (photograph courtesy of andrew Davies.)

1	Figure 9–51 Scanning electron microscopy. (a) a scanning electron micrograph of the stereocilia projecting from a hair cell in the inner ear of a bullfrog. For comparison, the same structure is shown by (B) differential-interferencecontrast light microscopy (Movie 9.3) and (C) thin-section transmission electron microscopy. (Courtesy of richard Jacobs and James hudspeth.) being plunged into a coolant. A special sample holder keeps this hydrated specimen at –160°C in the vacuum of the microscope, where it can be viewed directly without fixation, staining, or drying. Unlike negative staining, in which what we see is the envelope of stain exclusion around the particle, hydrated cryoelectron microscopy produces an image from the macromolecular structure itself. However, the contrast in this image is very low, and to extract the maximum amount of structural information, special imageprocessing techniques must be used, as we describe next.

1	Multiple images Can Be Combined to increase resolution As we saw earlier (p. 532), noise is important in light microscopy at low light levels, but it is a particularly severe problem for electron microscopy of unstained macromolecules. A protein molecule can tolerate a dose of only a few tens of electrons per square nanometer without damage, and this dose is orders of magnitude below what is needed to define an image at atomic resolution.

1	The solution is to obtain images of many identical molecules—perhaps tens of thousands of individual images—and combine them to produce an averaged image, revealing structural details that are hidden by the noise in the original images. This procedure is called single-particle reconstruction. Before combining all the individual images, however, they must be aligned with each other. Sometimes it is possible to induce proteins and complexes to form crystalline arrays, in which each molecule is held in the same orientation in a regular lattice. In this case, the alignment problem is easily solved, and several protein structures have been determined at atomic resolution by this type of electron crystallography. In principle, however, crystalline arrays are not absolutely required. With the help of a computer, the digital images of randomly distributed and unaligned molecules can be processed and combined to yield highresolution reconstructions (see Movie 13.1). Although structures that

1	help of a computer, the digital images of randomly distributed and unaligned molecules can be processed and combined to yield highresolution reconstructions (see Movie 13.1). Although structures that have some intrinsic symmetry make the task of alignment easier and more accurate, this technique has also been used for objects like ribosomes, with no symmetry. Figure 9–54 shows the structure of

1	Figure 9–52 The nuclear pore. rapidly frozen nuclear envelopes were imaged in a high-resolution seM, equipped with a field emission gun as the electron source. these views of each side of a nuclear pore represent the limit of resolution of the seM (compare with Figure 12–8). (Courtesy of Martin goldberg and terry allen.) We know in detail about many cell processes, such as Dna replication and transcription and rna translation, but will we ever be able to visualize such rapid molecular processes in action in cells? Will we ever be able to image intracellular structures at the resolution of the electron microscope in living cells? how can we improve crystallization and single-particle cryoelectron microscopy techniques to obtain high-resolution structures of all important membrane channels and transporters? What new concepts might these structures reveal?

1	Figure 9–53 Negatively stained actin filaments. in this transmission electron micrograph, each filament is about 8 nm in diameter and is seen, on close inspection, to be composed of a helical chain of globular actin molecules. (Courtesy of roger Craig.) the protein capsid inside a human immunodeficiency virus (HIV) that has been determined at high resolution by the combination of many particles, multiple views, and molecular modeling.

1	A resolution of 0.3 nm has been achieved by electron microscopy—enough to begin to see the internal atomic arrangements in a protein and to rival xray crystallography in resolution. Although electron microscopy is unlikely to supersede xray crystallography (discussed in Chapter 8) as a method for macromolecular structure determination, it has some very clear advantages. First, it does not absolutely require crystalline specimens. Second, it can deal with extremely large complexes—structures that may be too large or too variable to crystallize satisfactorily. Third, it allows the rapid analysis of different conformations of protein complexes.

1	The analysis of large and complex macromolecular structures is helped considerably if the atomic structure of one or more of the subunits is known, for example from xray crystallography. Molecular models can then be mathematically “fitted” into the envelope of the structure determined at lower resolution using the electron microscope (see Figures 16–16D and 16–46). Figure 9–55 shows the structure of a ribosome with the location of a bound release factor displayed in this way (see also Figure 6–72).

1	Discovering the detailed structure of membranes and organelles requires the higher resolution attainable in a transmission electron microscope. Specific macromolecules can be localized after being labeled with colloidal gold linked to antibodies. Three-dimensional views of the surfaces of cells and tissues are obtained by scanning electron microscopy. The shapes of isolated molecules can be readily determined by electron microscopy techniques involving fast freezing or negative staining. Electron tomography and single-particle reconstruction use computational manipulations of data obtained from multiple images and multiple viewing angles to produce detailed reconstructions of macromolecules and molecular complexes. The resolution obtained with these methods means that atomic structures of individual

1	Figure 9–54 Single-particle reconstruction. the structure of a complete human immunodeficiency virus (hiV) capsid has been determined by a combination of cryoelectron microscopy, protein structure determination, and modeling. (a) a single 4 nm slice from an eM tomographic model (see also Figure 9–48) of an intact hiV particle with its membrane outer envelope and its internal, irregularly shaped protein capsid that houses its rna genome. (B) electron microscopy of capsid subunits that have self-assembled into a helical tube can be used to derive an electron-density map at a resolution of 8 nm, in which details of the hexamers are clearly visible. (C) Using the known atomic coordinates of a single subunit of the hexamer, the structure has been modeled into the electron-density map from (B). (D) a molecular reconstruction of the entire hiV capsid, based on the detailed structures shown in (a) and (C). this capsid contains 216 hexamers (blue) and 12 pentamers (yellow). (adapted from g.

1	(D) a molecular reconstruction of the entire hiV capsid, based on the detailed structures shown in (a) and (C). this capsid contains 216 hexamers (blue) and 12 pentamers (yellow). (adapted from g. Zhao et al., Nature 497:643–646, 2013. With permission from Macmillan publishers Ltd. C, pDB code: 3J34.)

1	Figure 9–55 Single-particle reconstruction and molecular model fitting. Bacterial ribosomes, with and without the release factor required for peptide release from the ribosome, were used to derive high-resolution, three-dimensional cryoelectron microscopy maps at a resolution of better than 1 nm. images of nearly 20,000 separate ribosomes preserved in ice were used to produce single-particle reconstructions.

1	(a) the 30s ribosomal subunit (yellow) and the 50s subunit (blue) can be distinguished from the additional electron density that can be attributed to the release factor rF2 (purple). (B) the known molecular structure of rF2 modeled into the electron density from (a). (From U.B.s. rawat et al., Nature 421:87–90, 2003. With permission from Macmillan publishers Ltd.) macromolecules can often be “fitted” to the images derived by electron microscopy. In this way, the TEM is increasingly able to bridge the gap between structures discovered by x-ray crystallography and those discovered with the light microscope. Figure Q9–1 paths of light rays through dry and oil-immersion lenses (problem 9–3). the red circle at the origin of the light rays is the specimen. 9–4 Figure Q9–2 shows a diagram of the human eye. The refractive indices of the components in the light path are: cornea 1.38, aqueous humor 1.33, crystalline lens 1.41, and Which statements are true? Explain why or why not.

1	Which statements are true? Explain why or why not. 9–1 Because the DNA double helix is only 2 nm wide— well below the limit of resolution of the light microscope— it is impossible to see chromosomes in living cells without special stains. 9–2 A fluorescent molecule, having absorbed a single photon of light at one wavelength, always emits it at a longer wavelength. Discuss the following problems. 9–3 The diagrams in Figure Q9–1 show the paths of light rays passing through a specimen with a dry lens and with an oilimmersion lens. Offer an explanation for why oilimmersion lenses should give better resolution. Air, glass, and oil have refractive indices of 1.00, 1.51, and 1.51, 9–5 Why do humans see so poorly under water? And why do goggles help? 9–6 Explain the difference between resolution and magnification.

1	9–6 Explain the difference between resolution and magnification. 9–7 Antibodies that bind to specific proteins are important tools for defining the locations of molecules in cells. The sensitivity of the primary antibody—the antibody that reacts with the target molecule—is often enhanced by using labeled secondary antibodies that bind to it. What are the advantages and disadvantages of using secondary antibodies that carry fluorescent tags versus those that carry bound enzymes? 9–8 Figure Q9–3 shows a series of modified fluorescent proteins that emit light in a range of colors. How do you suppose the exact same chromophore can fluoresce at so many different wavelengths?

1	Figure Q9–3 a rainbow of colors produced by modified fluorescent proteins (problem 9–8). (Courtesy of nathan shaner, paul steinbach and roger tsien.) 9–9 Consider a fluorescent detector designed to report the cellular location of active protein tyrosine kinases. A blue (cyan) fluorescent protein (CFP) and a yellow fluorescent protein (YFP) were fused to either end of a hybrid protein domain. The hybrid protein segment consisted of a substrate peptide recognized by the Abl protein tyrosine kinase and a phosphotyrosinebinding domain (Figure Q9–4A). Stimulation of the CFP domain does not cause emission by the YFP domain when the domains are 1.3 (problem 9–4). binding protein retina 1.0 vitreous humor 1.38. Where does the main refraction—the main focusing—occur? What role do you suppose the lens 1.2 plays? 1.1 iris of the human eye

1	1.1 iris of the human eye Figure Q9–4 Fluorescent reporter protein designed to detect tyrosine phosphorylation (problem 9–9). (a) Domain structure of reporter protein. Four domains are indicated: CFp, YFp, tyrosine kinase substrate peptide, and a phosphotyrosine-binding domain. (B) Fret assay. YFp/CFp is normalized to 1.0 at time zero. the reporter was incubated in the presence (or absence) of abl and atp for the indicated times. Arrow indicates time of addition of a tyrosine phosphatase. (From a.Y. ting, k.h. kain, r.L. klemke and r.Y. tsien, Proc. Natl Acad. Sci. USA 98:15003–15008, 2001. With permission from national academy of sciences.) separated. When the CFP and YFP domains are brought close together, however, fluorescence resonance energy transfer (FRET) allows excitation of CFP to stimulate emission by YFP. FRET shows up experimentally as an increase in the ratio of emission at 526 nm versus 476 nm (YFP/ CFP) when CFP is excited by 434 nm light.

1	Incubation of the reporter protein with Abl protein tyrosine kinase in the presence of ATP gave an increase in YFP/CFP emission (Figure Q9–4B). In the absence of ATP or the Abl protein, no FRET occurred. FRET was also eliminated by addition of a tyrosine phosphatase (Figure Q9–4B). Describe as best you can how the reporter protein detects active Abl protein tyrosine kinase. Celis Je, Carter n, simons k et al. (eds) (2005) Cell Biology: a Laboratory handbook, 3rd ed. san Diego: academic press. (Volume 3 of this four-volume set covers the practicalities of most of the current light and electron imaging methods that are used in cell biology.) pawley Bp (ed) (2006) handbook of Biological Confocal Microscopy, 3rd ed. new York: springer science. Wayne r (2014) Light and Video Microscopy. san Diego: academic press.

1	Wayne r (2014) Light and Video Microscopy. san Diego: academic press. Looking at Cells in the Light Microscope adams MC, salmon WC, gupton sL et al. (2003) a high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells. Methods 29, 29–41. agard Da, hiraoka Y, shaw p & sedat JW (1989) Fluorescence microscopy in three dimensions. in Methods in Cell Biology, Vol. 30: Fluorescence Microscopy of Living Cells in Culture, part B (DL taylor, Y-L Wang eds). san Diego: academic press. Burnette Dt, sengupta p, Dai Y et al. (2011) Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules. Proc. Natl Acad. Sci. USA 108, 21081–21086. Chalfie M, tu Y, euskirchen g et al. (1994) green fluorescent protein as a marker for gene expression. Science 263, 802–805.

1	Chalfie M, tu Y, euskirchen g et al. (1994) green fluorescent protein as a marker for gene expression. Science 263, 802–805. giepmans Bn, adams sr, ellisman Mh & tsien rY (2006) the fluorescent toolbox for assessing protein location and function. Science 312, 217–224. harlow e & Lane D (1998) Using antibodies: a Laboratory Manual. Cold spring harbor, nY: Cold spring harbor Laboratory press. hell s (2009) Microscopy and its focal switch. Nat. Methods 6, 24–32. huang B, Babcock h & Zhuang X (2010) Breaking the diffraction barrier: super-resolution imaging of cells. Cell 143, 1047–1058. huang B, Bates M & Zhuang X (2009) super-resolution fluorescence microscopy. Annu. Rev. Biochem. 78, 993–1016. Jaiswai Jk & simon sM (2004) potentials and pitfalls of fluorescent quantum dots for biological imaging. Trends Cell Biol. 14, 497–504.

1	Jaiswai Jk & simon sM (2004) potentials and pitfalls of fluorescent quantum dots for biological imaging. Trends Cell Biol. 14, 497–504. klar ta, Jakobs s, Dyba M et al. (2000) Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97, 8206–8210. Lippincott-schwartz J & patterson gh (2003) Development and use of fluorescent protein markers in living cells. Science 300, 87–91. Lippincott-schwartz J, altan-Bonnet n & patterson g (2003) photobleaching and photoactivation: following protein dynamics in living cells. Nat. Cell Biol. 5(suppl), s7–s14. Mcevoy aL, greenfield D, Bates M & Liphardt J (2010) Q&a: single-molecule localization microscopy for biological imaging. BMC Biol. 8, 106. Minsky M (1988) Memoir on inventing the confocal scanning microscope. Scanning 10, 128–138. Miyawaki a, sawano a & kogure t (2003) Lighting up cells: labelling proteins with fluorophores. Nat. Cell Biol. 5(suppl), s1–s7.

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1	shaner nC, steinbach pa & tsien rY (2005) a guide to choosing fluorescent proteins. Nat. Methods 2, 905–909. sluder g & Wolf De (2007) Digital Microscopy, 3rd ed: Methods in Cell Biology, Vol 81. san Diego: academic press. stephens DJ & allan VJ (2003) Light microscopy techniques for live cell imaging. Science 300, 82–86. tsien rY (2008) Constructing and exploiting the fluorescent protein paintbox. nobel prize Lecture. www.nobelprize.org White Jg, amos WB & Fordham M (1987) an evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. J. Cell Biol. 105, 41–48. Zernike F (1955) how i discovered phase contrast. Science 121, 345–349. Looking at Cells and Molecules in the Electron Microscope allen tD & goldberg MW (1993) high-resolution seM in cell biology. Trends Cell Biol. 3, 205–208. Baumeister W (2002) electron tomography: towards visualizing the molecular organization of the cytoplasm. Curr. Opin. Struct. Biol. 12, 679–684.

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1	Cell membranes are crucial to the life of the cell. The plasma membrane encloses the cell, defines its boundaries, and maintains the essential differences between the cytosol and the extracellular environment. Inside eukaryotic cells, the membranes of the nucleus, endoplasmic reticulum, Golgi apparatus, mitochondria, and other membrane-enclosed organelles maintain the characteristic differences between the contents of each organelle and the cytosol. Ion gradients across membranes, established by the activities of specialized membrane proteins, can be used to synthesize ATP, to drive the transport of selected solutes across the membrane, or, as in nerve and muscle cells, to produce and transmit electrical signals. In all cells, the plasma membrane also contains proteins that act as sensors of external signals, allowing the cell to change its behavior in response to environmental cues, including signals from other cells; these protein sensors, or receptors, transfer information—rather

1	of external signals, allowing the cell to change its behavior in response to environmental cues, including signals from other cells; these protein sensors, or receptors, transfer information—rather than molecules—across the membrane.

1	Despite their differing functions, all biological membranes have a common general structure: each is a very thin film of lipid and protein molecules, held together mainly by noncovalent interactions (Figure 10–1). Cell membranes the lIpID BIlAYer MeMBrAne prOteInS lipid molecule (B) protein molecule lipid bilayer (5 nm) (A) Figure 10–1 Two views of a cell membrane. (A) An electron micrograph of a segment of the plasma membrane of a human red blood cell seen in cross section, showing its bilayer structure. (B) A three-dimensional schematic view of a cell membrane and the general disposition of its lipid and protein constituents. (A, courtesy of Daniel S. Friend.) are dynamic, fluid structures, and most of their molecules move about in the plane of the membrane. The lipid molecules are arranged as a continuous double layer about 5 nm thick. This lipid bilayer provides the basic fluid structure of the membrane and serves as a relatively impermeable barrier to the passage of most

1	are arranged as a continuous double layer about 5 nm thick. This lipid bilayer provides the basic fluid structure of the membrane and serves as a relatively impermeable barrier to the passage of most water-soluble molecules. Most membrane proteins span the lipid bilayer and mediate nearly all of the other functions of the membrane, including the transport of specific molecules across it, and the catalysis of membrane-associated reactions such as ATP synthesis. In the plasma membrane, some transmembrane proteins serve as structural links that connect the cytoskeleton through the lipid bilayer to either the extracellular matrix or an adjacent cell, while others serve as receptors to detect and transduce chemical signals in the cell’s environment. It takes many kinds of membrane proteins to enable a cell to function and interact with its environment, and it is estimated that about 30% of the proteins encoded in an animal’s genome are membrane proteins.

1	In this chapter, we consider the structure and organization of the two main constituents of biological membranes—the lipids and the proteins. Although we focus mainly on the plasma membrane, most concepts discussed apply to the various internal membranes of eukaryotic cells as well. The functions of cell membranes are considered in later chapters: their role in energy conversion and ATP synthesis, for example, is discussed in Chapter 14; their role in the transmembrane transport of small molecules in Chapter 11; and their roles in cell signaling and cell adhesion in Chapters 15 and 19, respectively. In Chapters 12 and 13, we discuss the internal membranes of the cell and the protein traffic through and between them. the lIpID BIlAYer

1	the lIpID BIlAYer The lipid bilayer provides the basic structure for all cell membranes. It is easily seen by electron microscopy, and its bilayer structure is attributable exclusively to the special properties of the lipid molecules, which assemble spontaneously into bilayers even under simple artificial conditions. In this section, we discuss the different types of lipid molecules found in cell membranes and the general properties of lipid bilayers. phosphoglycerides, Sphingolipids, and Sterols Are the Major lipids in cell Membranes

1	phosphoglycerides, Sphingolipids, and Sterols Are the Major lipids in cell Membranes Lipid molecules constitute about 50% of the mass of most animal cell membranes, nearly all of the remainder being protein. There are approximately 5 × 106 lipid molecules in a 1 μm × 1 μm area of lipid bilayer, or about 109 lipid molecules in the plasma membrane of a small animal cell. All of the lipid molecules in cell membranes are amphiphilic—that is, they have a hydrophilic (“water-loving”) or polar end and a hydrophobic (“water-fearing”) or nonpolar end.

1	The most abundant membrane lipids are the phospholipids. These have a polar head group containing a phosphate group and two hydrophobic hydrocarbon tails. In animal, plant, and bacterial cells, the tails are usually fatty acids, and they can differ in length (they normally contain between 14 and 24 carbon atoms). One tail typically has one or more cis-double bonds (that is, it is unsaturated), while the other tail does not (that is, it is saturated). As shown in Figure 10–2, each cis-double bond creates a kink in the tail. Differences in the length and saturation of the fatty acid tails influence how phospholipid molecules pack against one another, thereby affecting the fluidity of the membrane, as we discuss later.

1	The main phospholipids in most animal cell membranes are the phosphoglycerides, which have a three-carbon glycerol backbone (see Figure 10–2). Two long-chain fatty acids are linked through ester bonds to adjacent carbon atoms of the glycerol, and the third carbon atom of the glycerol is attached to a phosphate group, which in turn is linked to one of several types of head group. By combining several different fatty acids and head groups, cells make many different phosphoglycerides. Phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine are the most abundant ones in mammalian cell membranes (Figure 10–3A–C).

1	Another important class of phospholipids are the sphingolipids, which are built from sphingosine rather than glycerol (Figure10–3D–E). Sphingosine is a long acyl chain with an amino group (NH2) and two hydroxyl groups (OH) at one end. In sphingomyelin, the most common sphingolipid, a fatty acid tail is attached to the amino group, and a phosphocholine group is attached to the terminal hydroxyl group. Together, the phospholipids phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin constitute more than half the mass of lipid in most mammalian cell membranes (see Table 10–1, p. 571). Figure 10–2 The parts of a typical phospholipid molecule. this example is a phosphatidylcholine, represented (A) schematically, (B) by a formula, (c) as a space-filling model (Movie 10.1), and hydrophilicheadhydrophobictails(D) (D) as a symbol.

1	Figure 10–3 Four major phospholipids in mammalian plasma membranes. Different head groups are represented by different colors in the symbols. the lipid molecules shown in (A–c) are phosphoglycerides, which are derived from glycerol. the molecule in (D) is sphingomyelin, which is derived from sphingosine (e) and is therefore a sphingolipid. note that only phosphatidylserine carries a net negative charge, the importance of which we discuss later; the other three are electrically neutral at physiological ph, carrying one positive and one negative charge. Figure 10–4 The structure of cholesterol. polar head group cholesterol is represented (A) by a formula, (B) by a schematic drawing, and (c) as a space-filling model. on their shape, they can do this in either of two ways: they can form spherical micelles, with the tails inward, or they can form double-layered sheets, or bilayers, with the hydrophobic tails sandwiched between the hydrophilic head groups (Figure 10–7).

1	The same forces that drive phospholipids to form bilayers also provide a self-sealing property. A small tear in the bilayer creates a free edge with water; because this is energetically unfavorable, the lipids tend to rearrange spontaneously to eliminate the free edge. (In eukaryotic plasma membranes, the fusion of intracellular vesicles repairs larger tears.) The prohibition of free edges has a profound consequence: the only way for a bilayer to avoid having edges is by closing in on itself and forming a sealed compartment (Figure 10–8). This remarkable

1	In addition to phospholipids, the lipid bilayers in many cell membranes contain glycolipids and cholesterol. Glycolipids resemble sphingolipids, but, instead of a phosphate-linked head group, they have sugars attached. We discuss glycolipids later. Eukaryotic plasma membranes contain especially large amounts of cholesterol—up to one molecule for every phospholipid molecule. Cholesterol is a sterol. It contains a rigid ring structure, to which is attached a single polar hydroxyl group and a short nonpolar hydrocarbon chain (Figure 10–4). The cholesterol molecules orient themselves in the bilayer with their hydroxyl group close to the polar head groups of adjacent phospholipid molecules (Figure 10–5).

1	The shape and amphiphilic nature of the phospholipid molecules cause them to form bilayers spontaneously in aqueous environments. As discussed in Chapter 2, hydrophilic molecules dissolve readily in water because they contain charged groups or uncharged polar groups that can form either favorable electrostatic interactions or hydrogen bonds with water molecules (Figure 10–6A). Hydrophobic molecules, by contrast, are insoluble in water because all, or almost all, of their atoms are uncharged and nonpolar and therefore cannot form energetically favorable interactions with water molecules. If dispersed in water, they force the adjacent water molecules to reorganize into icelike cages that surround the hydrophobic molecule (Figure 10–6B). Because these cage structures are more ordered than the surrounding water, their formation increases the free energy. This free-energy cost is minimized, however, if the hydrophobic molecules (or the hydrophobic portions of amphiphilic molecules) cluster

1	surrounding water, their formation increases the free energy. This free-energy cost is minimized, however, if the hydrophobic molecules (or the hydrophobic portions of amphiphilic molecules) cluster together so that the smallest number of water molecules is affected. 3

1	When amphiphilic molecules are exposed to an aqueous environment, they behave as you would expect from the above discussion. They spontaneously aggregate to bury their hydrophobic tails in the interior, where they are shielded 2 from the water, and they expose their hydrophilic heads to water. Depending Figure 10–5 Cholesterol in a lipid bilayer. Schematic drawing (to scale) of a cholesterol molecule interacting with two phospholipid molecules in one monolayer of a lipid bilayer. behavior, fundamental to the creation of a living cell, follows directly from the shape and amphiphilic nature of the phospholipid molecule. A lipid bilayer also has other characteristics that make it an ideal structure for cell membranes. One of the most important of these is its fluidity, which is crucial to many membrane functions (Movie 10.2). the lipid Bilayer Is a two-dimensional Fluid

1	the lipid Bilayer Is a two-dimensional Fluid Around 1970, researchers first recognized that individual lipid molecules are able to diffuse freely within the plane of a lipid bilayer. The initial demonstration came from studies of synthetic (artificial) lipid bilayers, which can be made in the form of spherical vesicles, called liposomes (Figure 10–9); or in the form of planar bilayers formed across a hole in a partition between two aqueous compartments or on a solid support. Various techniques have been used to measure the motion of individual lipid molecules and their components. One can construct a lipid molecule, for example, with a fluorescent dye or a small gold particle attached to its polar head group and follow the diffusion of even individual molecules in a membrane. Alternatively, one can modify a lipid head group to carry a “spin label,” such as a nitroxide shape of packing of molecule molecules in water

1	Figure 10–7 Packing arrangements of amphiphilic molecules in an aqueous environment. (A) these molecules spontaneously form micelles or bilayers in water, depending on their shape. cone-shaped amphiphilic molecules (above) form micelles, whereas cylinder-shaped amphiphilic molecules such as phospholipids (below) form bilayers. (B) A micelle and a lipid bilayer seen in cross section. note that micelles of amphiphilic molecules are thought to be much more irregular than drawn here (see Figure 10–26c). Figure 10–6 How hydrophilic and hydrophobic molecules interact differently with water. (A) Because acetone is polar, it can form hydrogen bonds (red) and favorable electrostatic interactions (yellow) with water molecules, which are also polar. thus, acetone readily dissolves in water.

1	(B) By contrast, 2-methyl propane is entirely hydrophobic. Because it cannot form favorable interactions with water, it forces adjacent water molecules to reorganize into icelike cage structures, which increases the free energy. this compound is therefore virtually insoluble in water. the symbol δ– indicates a partial negative charge, and δ+ indicates a partial positive charge. polar atoms are shown in color and nonpolar groups are shown in gray. planar phospholipid bilayer with edges exposed to water Figure 10–8 The spontaneous closure of a phospholipid bilayer to form a sealed compartment. the closed structure is stable because it avoids the exposure of the hydrophobic hydrocarbon tails to water, which would be energetically unfavorable.

1	Figure 10–9 Liposomes. (A) An electron micrograph of unfixed, unstained, synthetic phospholipid vesicles—liposomes—in water, which have been rapidly frozen at liquid-nitrogen temperature. (B) A drawing of a small spherical liposome seen in cross section. liposomes are commonly used as model membranes in experimental studies, especially to study incorporated membrane proteins. (A, from p. Frederik and D. hubert, Methods Enzymol. 391:431–448, 2005. With permission from elsevier.) group (=N–O); this contains an unpaired electron whose spin creates a paramagnetic signal that can be detected by electron spin resonance (ESR) spectroscopy, the principles of which are similar to those of nuclear magnetic resonance (NMR), discussed in Chapter 8. The motion and orientation of a spin-labeled lipid in a bilayer can be deduced from the ESR spectrum. Such studies show that phospholipid molecules in synthetic bilayers very rarely migrate from the monolayer (also called a leaflet) on one side to that

1	a bilayer can be deduced from the ESR spectrum. Such studies show that phospholipid molecules in synthetic bilayers very rarely migrate from the monolayer (also called a leaflet) on one side to that on the other. This process, known as “flip-flop,” occurs on a time scale of hours for any individual molecule, although cholesterol is an exception to this rule and can flip-flop rapidly. In contrast, lipid molecules rapidly exchange places with their neighbors within a monolayer (~107 times per second). This gives rise to a rapid lateral diffusion, with a diffusion coefficient (D) of about 10–8 cm2/sec, which means that an average lipid molecule diffuses the length of a large bacterial cell (~2 μm) in about 1 second. These studies have also shown that individual lipid molecules rotate very rapidly about their long axis and have flexible hydrocarbon chains. Computer simulations show that lipid molecules in synthetic bilayers are very disordered, presenting an irregular surface of variously

1	about their long axis and have flexible hydrocarbon chains. Computer simulations show that lipid molecules in synthetic bilayers are very disordered, presenting an irregular surface of variously spaced and oriented head groups to the water phase on either side of the bilayer (Figure 10–10).

1	Similar mobility studies on labeled lipid molecules in isolated biological membranes and in living cells give results similar to those in synthetic bilayers. They demonstrate that the lipid component of a biological membrane is a two-dimensional liquid in which the constituent molecules are free to move laterally. As in synthetic bilayers, individual phospholipid molecules are normally confined to their own monolayer. This confinement creates a problem for their synthesis. Phospholipid molecules are manufactured in only one monolayer of a membrane, mainly in the cytosolic monolayer of the endoplasmic reticulum membrane. If none of these newly made molecules could migrate reasonably promptly to the noncytosolic monolayer, new lipid bilayer could not be made. The problem is solved by a special class of membrane proteins called phospholipid translocators, or flippases, which catalyze the rapid flip-flop of phospholipids from one mono-layer to the other, as discussed in Chapter 12.

1	Despite the fluidity of the lipid bilayer, liposomes do not fuse spontaneously with one another when suspended in water. Fusion does not occur because the polar lipid head groups bind water molecules that need to be displaced for the bilayers of two different liposomes to fuse. The hydration shell that keeps liposomes apart also insulates the many internal membranes in a eukaryotic cell and prevents their uncontrolled fusion, thereby maintaining the compartmental integrity of membrane-enclosed organelles. All cell membrane fusion events

1	Figure 10–10 The mobility of phospholipid molecules in an artificial lipid bilayer. Starting with a model of 100 phosphatidylcholine molecules arranged in a regular bilayer, a computer calculated the position of every atom after 300 picoseconds of simulated time. From these theoretical calculations, a model of the lipid bilayer emerges that accounts for almost all of the measurable properties of a synthetic lipid bilayer, including its thickness, number of lipid molecules per membrane area, depth of water penetration, and unevenness of the two surfaces. note that the tails in one monolayer can interact with those in the other monolayer, if the tails are long enough. (B) the different motions of a lipid molecule in a bilayer. (A, based on S.W. chiu et al., Biophys. J. 69:1230– 1245, 1995. With permission from the Biophysical Society.) are catalyzed by tightly regulated fusion proteins, which force appropriate membranes into tight proximity, squeezing out the water layer that keeps the

1	With permission from the Biophysical Society.) are catalyzed by tightly regulated fusion proteins, which force appropriate membranes into tight proximity, squeezing out the water layer that keeps the bilayers apart, as we discuss in Chapter 13.

1	the Fluidity of a lipid Bilayer Depends on Its composition The fluidity of cell membranes has to be precisely regulated. Certain membrane transport processes and enzyme activities, for example, cease when the bilayer viscosity is experimentally increased beyond a threshold level.

1	The fluidity of a lipid bilayer depends on both its composition and its temperature, as is readily demonstrated in studies of synthetic lipid bilayers. A synthetic bilayer made from a single type of phospholipid changes from a liquid state to a two-dimensional rigid crystalline (or gel) state at a characteristic temperature. This change of state is called a phase transition, and the temperature at which it occurs is lower (that is, the membrane becomes more difficult to freeze) if the hydrocarbon chains are short or have double bonds. A shorter chain length reduces the tendency of the hydrocarbon tails to interact with one another, in both the same and opposite monolayer, and cis-double bonds produce kinks in the chains that make them more difficult to pack together, so that the membrane remains fluid at lower temperatures (Figure 10–11). Bacteria, yeasts, and other organisms whose temperature fluctuates with that of their environment adjust the fatty acid composition of their

1	remains fluid at lower temperatures (Figure 10–11). Bacteria, yeasts, and other organisms whose temperature fluctuates with that of their environment adjust the fatty acid composition of their membrane lipids to maintain a relatively constant fluidity. As the temperature falls, for instance, the cells of those organisms synthesize fatty acids with more cis-double bonds, thereby avoiding the decrease in bilayer fluidity that would otherwise result from the temperature drop.

1	Cholesterol modulates the properties of lipid bilayers. When mixed with phospholipids, it enhances the permeability-barrier properties of the lipid bilayer. Cholesterol inserts into the bilayer with its hydroxyl group close to the polar head groups of the phospholipids, so that its rigid, platelike steroid rings interact with— and partly immobilize—those regions of the hydrocarbon chains closest to the polar head groups (see Figure 10–5 and Movie 10.3). By decreasing the mobility of the first few CH2 groups of the chains of the phospholipid molecules, cholesterol makes the lipid bilayer less deformable in this region and thereby decreases the permeability of the bilayer to small water-soluble molecules. Although cholesterol tightens the packing of the lipids in a bilayer, it does not make membranes any less fluid. At the high concentrations found in most eukaryotic plasma membranes, cholesterol also prevents the hydrocarbon chains from coming together and crystallizing.

1	Table 10–1 compares the lipid compositions of several biological membranes. Note that bacterial plasma membranes are often composed of one main type of phospholipid and contain no cholesterol. In archaea, lipids usually contain unsaturated saturated hydrocarbon chains hydrocarbon chains with cis-double bonds Figure 10–11 The influence of cisdouble bonds in hydrocarbon chains. the double bonds make it more difficult to pack the chains together, thereby making the lipid bilayer more difficult to freeze. In addition, because the hydrocarbon chains of unsaturated lipids are more spread apart, lipid bilayers containing them are thinner than bilayers formed exclusively from saturated lipids.

1	20–25-carbon-long prenyl chains instead of fatty acids; prenyl and fatty acid chains are similarly hydrophobic and flexible (see Figure 10–20F); in thermophilic archaea, the longest lipid chains span both leaflets, making the membrane particularly stable to heat. Thus, lipid bilayers can be built from molecules with similar features but different molecular designs. The plasma membranes of most eukaryotic cells are more varied than those of prokaryotes and archaea, not only in containing large amounts of cholesterol but also in containing a mixture of different phospholipids.

1	Analysis of membrane lipids by mass spectrometry has revealed that the lipid composition of a typical eukaryotic cell membrane is much more complex than originally thought. These membranes contain a bewildering variety of perhaps 500–2000 different lipid species with even the simple plasma membrane of a red blood cell containing well over 150. While some of this complexity reflects the combinatorial variation in head groups, hydrocarbon chain lengths, and desaturation of the major phospholipid classes, some membranes also contain many structurally distinct minor lipids, at least some of which have important functions. The inositol phospholipids, for example, are present in small quantities in animal cell membranes and have crucial functions in guiding membrane traffic and in cell signaling (discussed in Chapters 13 and 15, respectively). Their local synthesis and destruction are regulated by a large number of enzymes, which create both small intracellular signaling molecules and lipid

1	(discussed in Chapters 13 and 15, respectively). Their local synthesis and destruction are regulated by a large number of enzymes, which create both small intracellular signaling molecules and lipid docking sites on membranes that recruit specific proteins from the cytosol, as we discuss later.

1	Despite their Fluidity, lipid Bilayers can Form Domains of Different compositions Because a lipid bilayer is a two-dimensional fluid, we might expect most types of lipid molecules in it to be well mixed and randomly distributed in their own monolayer. The van der Waals attractive forces between neighboring hydrocarbon tails are not selective enough to hold groups of phospholipid molecules together. With certain lipid mixtures in artificial bilayers, however, one can observe phase segregations in which specific lipids come together in separate domains (Figure 10–12).

1	There has been a long debate among cell biologists about whether the lipid molecules in the plasma membrane of living cells similarly segregate into specialized domains, called lipid rafts. Although many lipids and membrane proteins are not distributed uniformly, large-scale lipid phase segregations are rarely seen in living cell membranes. Instead, specific membrane proteins and lipids are seen to concentrate in a more temporary, dynamic fashion facilitated by protein– protein interactions that allow the transient formation of specialized membrane regions (Figure 10–13). Such clusters can be tiny nanoclusters on a scale of a few molecules, or larger assemblies that can be seen with electron microscopy, such as the caveolae involved in endocytosis (discussed in Chapter 13). The tendency of mixtures of lipids to undergo phase partitioning, as seen in artificial bilayers (see Figure 10–12), may help create rafts in living cell membranes—organizing and concentrating membrane proteins

1	of mixtures of lipids to undergo phase partitioning, as seen in artificial bilayers (see Figure 10–12), may help create rafts in living cell membranes—organizing and concentrating membrane proteins either for transport in membrane vesicles

1	Figure 10–12 Lateral phase separation in artificial lipid bilayers. (A) giant liposomes produced from a 1:1 mixture of phosphatidylcholine and sphingomyelin form uniform bilayers. (B) By contrast, liposomes produced from a 1:1:1 mixture of phosphatidylcholine, sphingomyelin, and cholesterol form bilayers with two separate phases. the liposomes are stained with trace concentrations of a fluorescent dye that preferentially partitions into one of the two phases. the average size of the domains formed in these giant artificial liposomes is much larger than that expected in cell membranes, where “lipid rafts” (see text) may be as small as a few nanometers in diameter. (A, from n. Kahya et al., J. Struct. Biol. 147:77–89, 2004. With permission from elsevier; B, courtesy of petra Schwille.) (discussed in Chapter 13) or for working together in protein assemblies, such as when they convert extracellular signals into intracellular ones (discussed in Chapter 15).

1	Most cells store an excess of lipids in lipid droplets, from where they can be retrieved as building blocks for membrane synthesis or as a food source. Fat cells, or adipocytes, are specialized for lipid storage. They contain a giant lipid droplet that fills up most of their cytoplasm. Most other cells have many smaller lipid droplets, the number and size varying with the cell’s metabolic state. Fatty acids can be liberated from lipid droplets on demand and exported to other cells through the bloodstream. Lipid droplets store neutral lipids, such as triacylglycerols and cholesterol esters, which are synthesized from fatty acids and cholesterol by enzymes in the endoplasmic reticulum membrane. Because these lipids do not contain hydrophilic head groups, they are exclusively hydrophobic molecules, and therefore aggregate into three-dimensional droplets rather than into bilayers.

1	Lipid droplets are unique organelles in that they are surrounded by a single monolayer of phospholipids, which contains a large variety of proteins. Some of the proteins are enzymes involved in lipid metabolism, but the functions of most are unknown. Lipid droplets form rapidly when cells are exposed to high concentrations of fatty acids. They are thought to form from discrete regions of the endoplasmic reticulum membrane where many enzymes of lipid metabolism are concentrated. Figure 10–14 shows one model of how lipid droplets may form and acquire their surrounding monolayer of phospholipids and proteins. the Asymmetry of the lipid Bilayer Is Functionally Important The lipid compositions of the two monolayers of the lipid bilayer in many membranes are strikingly different. In the human red blood cell (erythrocyte) membrane, for example, almost all of the phospholipid molecules that have cho-line—(CH3)3N+CH2CH2OH—in their head group (phosphatidylcholine and

1	Figure 10–13 a model of a raft domain. Weak protein–protein, protein–lipid, and lipid–lipid interactions reinforce one another to partition the interacting components into raft domains. cholesterol, sphingolipids, glycolipids, glycosylphosphatidylinositol (gpI)-anchored proteins, and some transmembrane proteins are enriched in these domains. note that because of their composition, raft domains have an increased membrane thickness.We discuss glycolipids, gpI-anchored proteins, and oligosaccharide linkers later. (Adapted from D. lingwood and K. Simons, Science 327:46–50, 2010.)

1	Figure 10–14 a model for the formation of lipid droplets. neutral lipids are deposited between the two monolayers of the endoplasmic reticulum membrane. there, they aggregate into a three-dimensional droplet, which buds and pinches off from the endoplasmic reticulum membrane as a unique organelle, surrounded by a single monolayer of phospholipids and associated proteins. (Adapted from S. Martin and r.g. parton, Nat. Rev. Mol. Cell Biol. 7:373–378, 2006. With permission from Macmillan publishers ltd.) sphingomyelin) are in the outer monolayer, whereas almost all that contain a terminal primary amino group (phosphatidylethanolamine and phosphatidylserine) are in the inner monolayer (Figure 10–15). Because the negatively charged phosphatidylserine is located in the inner monolayer, there is a significant difference in charge between the two halves of the bilayer. We discuss in Chapter 12 how membrane-bound phospholipid translocators generate and maintain lipid asymmetry.

1	Lipid asymmetry is functionally important, especially in converting extracellular signals into intracellular ones (discussed in Chapter 15). Many cytosolic proteins bind to specific lipid head groups found in the cytosolic monolayer of the lipid bilayer. The enzyme protein kinase C (PKC), for example, which is activated in response to various extracellular signals, binds to the cytosolic face of the plasma membrane, where phosphatidylserine is concentrated, and requires this negatively charged phospholipid for its activity.

1	In other cases, specific lipid head groups must first be modified to create pro-tein-binding sites at a particular time and place. One example is phosphatidylinositol (PI), one of the minor phospholipids that are concentrated in the cytosolic monolayer of cell membranes (see Figure 13–10A–C). Various lipid kinases can add phosphate groups at distinct positions on the inositol ring, creating binding sites that recruit specific proteins from the cytosol to the membrane. An important example of such a lipid kinase is phosphoinositide 3-kinase (PI 3-kinase), which is activated in response to extracellular signals and helps to recruit specific intracellular signaling proteins to the cytosolic face of the plasma membrane (see Figure 15–53). Similar lipid kinases phosphorylate inositol phospholipids in intracellular membranes and thereby help to recruit proteins that guide membrane transport.

1	Phospholipids in the plasma membrane are used in yet another way to convert extracellular signals into intracellular ones. The plasma membrane contains various phospholipases that are activated by extracellular signals to cleave specific phospholipid molecules, generating fragments of these molecules that act as short-lived intracellular mediators. Phospholipase C, for example, cleaves an inositol phospholipid in the cytosolic monolayer of the plasma membrane to generate two fragments, one of which remains in the membrane and helps activate protein kinase C, while the other is released into the cytosol and stimulates the release of Ca2+ from the endoplasmic reticulum (see Figure 15–28).

1	Animals exploit the phospholipid asymmetry of their plasma membranes to distinguish between live and dead cells. When animal cells undergo apoptosis (a form of programmed cell death, discussed in Chapter 18), phosphatidylserine, which is normally confined to the cytosolic (or inner) monolayer of the plasma membrane lipid bilayer, rapidly translocates to the extracellular (or outer) mono-layer. The phosphatidylserine exposed on the cell surface signals neighboring cells, such as macrophages, to phagocytose the dead cell and digest it. The trans-location of the phosphatidylserine in apoptotic cells is thought to occur by two mechanisms: 1. The phospholipid translocator that normally transports this lipid from the outer monolayer to the inner monolayer is inactivated. 2. A “scramblase” that transfers phospholipids nonspecifically in both directions between the two monolayers is activated.

1	2. A “scramblase” that transfers phospholipids nonspecifically in both directions between the two monolayers is activated. Figure 10–15 The asymmetrical distribution of phospholipids and glycolipids in the lipid bilayer of human red blood cells. the colors used for the phospholipid head groups are those introduced in Figure 10–3. In addition, glycolipids are drawn with hexagonal polar head groups (blue). cholesterol (not shown) is distributed roughly equally in both monolayers. glycolipids Are Found on the Surface of All eukaryotic plasma Membranes

1	Sugar-containing lipid molecules called glycolipids have the most extreme asymmetry in their membrane distribution: these molecules, whether in the plasma membrane or in intracellular membranes, are found exclusively in the monolayer facing away from the cytosol. In animal cells, they are made from sphingosine, just like sphingomyelin (see Figure 10–3). These intriguing molecules tend to self-associate, partly through hydrogen bonds between their sugars and partly through van der Waals forces between their long and straight hydrocarbon chains, which causes them to partition preferentially into lipid raft phases (see Figure 10–13). The asymmetric distribution of glycolipids in the bilayer results from the addition of sugar groups to the lipid molecules in the lumen of the Golgi apparatus. Thus, the compartment in which they are manufactured is topologically equivalent to the exterior of the cell (discussed in Chapter 12). As they are delivered to the plasma membrane, the sugar groups

1	Thus, the compartment in which they are manufactured is topologically equivalent to the exterior of the cell (discussed in Chapter 12). As they are delivered to the plasma membrane, the sugar groups are exposed at the cell surface (see Figure 10–15), where they have important roles in interactions of the cell with its surroundings.

1	Glycolipids probably occur in all eukaryotic cell plasma membranes, where they generally constitute about 5% of the lipid molecules in the outer monolayer. They are also found in some intracellular membranes. The most complex of the glycolipids, the gangliosides, contain oligosaccharides with one or more sialic acid moieties, which give gangliosides a net negative charge (Figure 10–16). The most abundant of the more than 40 different gangliosides that have been identified are in the plasma membrane of nerve cells, where gangliosides constitute 5–10% of the total lipid mass; they are also found in much smaller quantities in other cell types.

1	Hints as to the functions of glycolipids come from their localization. In the plasma membrane of epithelial cells, for example, glycolipids are confined to the exposed apical surface, where they may help to protect the membrane against the harsh conditions frequently found there (such as low pH and high concentrations of degradative enzymes). Charged glycolipids, such as gangliosides, may be important because of their electrical effects: their presence alters the electrical field across the membrane and the concentrations of ions—especially Ca2+—at the membrane surface. Glycolipids also function in cell-recognition processes, Figure 10–16 Glycolipid molecules.

1	Figure 10–16 Glycolipid molecules. (A) galactocerebroside is called a neutral glycolipid because the sugar that forms its head group is uncharged. (B) A ganglioside always contains one or more negatively charged sialic acid moiety. there are various types of sialic acid; in human cells, it is mostly N-acetylneuraminic acid, or nAnA), whose structure is shown in (c). Whereas in bacteria and plants almost all glycolipids are derived from glycerol, as are most phospholipids, in animal cells almost all glycolipids are based on sphingosine, as is the case for sphingomyelin (see Figure 10–3). gal = galactose; glc = glucose, galnAc = N-acetylgalactosamine; these three sugars are uncharged.

1	in which membrane-bound carbohydrate-binding proteins (lectins) bind to the sugar groups on both glycolipids and glycoproteins in the process of cell–cell adhesion (discussed in Chapter 19). Mutant mice that are deficient in all of their complex gangliosides show abnormalities in the nervous system, including axonal degeneration and reduced myelination.

1	Some glycolipids provide entry points for certain bacterial toxins and viruses. The ganglioside GM1 (see Figure 10–16), for example, acts as a cell-surface receptor for the bacterial toxin that causes the debilitating diarrhea of cholera. Cholera toxin binds to and enters only those cells that have GM1 on their surface, including intestinal epithelial cells. Its entry into a cell leads to a prolonged increase in the concentration of intracellular cyclic AMP (discussed in Chapter 15), which in turn causes a large efflux of Cl–, leading to the secretion of Na+, K+, HCO3–, and water into the intestine. Polyomaviruses also enter the cell after binding initially to gangliosides.

1	Biological membranes consist of a continuous double layer of lipid molecules in which membrane proteins are embedded. This lipid bilayer is fluid, with individual lipid molecules able to diffuse rapidly within their own monolayer. The membrane lipid molecules are amphiphilic. When placed in water, they assemble spontaneously into bilayers, which form sealed compartments.

1	Although cell membranes can contain hundreds of different lipid species, the plasma membrane in animal cells contains three major classes—phospholipids, cholesterol, and glycolipids. Because of their different backbone structure, phospholipids fall into two subclasses—phosphoglycerides and sphingolipids. The lipid compositions of the inner and outer monolayers are different, reflecting the different functions of the two faces of a cell membrane. Different mixtures of lipids are found in the membranes of cells of different types, as well as in the various membranes of a single eukaryotic cell. Inositol phospholipids are a minor class of phospholipids, which in the cytosolic leaflet of the plasma membrane lipid bilayer play an important part in cell signaling: in response to extracellular signals, specific lipid kinases phosphorylate the head groups of these lipids to form docking sites for cytosolic signaling proteins, whereas specific phospholipases cleave certain inositol

1	signals, specific lipid kinases phosphorylate the head groups of these lipids to form docking sites for cytosolic signaling proteins, whereas specific phospholipases cleave certain inositol phospholipids to generate small intracellular signaling molecules.

1	Although the lipid bilayer provides the basic structure of biological membranes, the membrane proteins perform most of the membrane’s specific tasks and therefore give each type of cell membrane its characteristic functional properties. Accordingly, the amounts and types of proteins in a membrane are highly variable. In the myelin membrane, which serves mainly as electrical insulation for nerve-cell axons, less than 25% of the membrane mass is protein. By contrast, in the membranes involved in ATP production (such as the internal membranes of mitochondria and chloroplasts), approximately 75% is protein. A typical plasma membrane is somewhere in between, with protein accounting for about half of its mass. Because lipid molecules are small compared with protein molecules, however, there are always many more lipid molecules than protein molecules in cell membranes—about 50 lipid molecules for each protein molecule in cell membranes that are 50% protein by mass. Membrane proteins vary

1	are always many more lipid molecules than protein molecules in cell membranes—about 50 lipid molecules for each protein molecule in cell membranes that are 50% protein by mass. Membrane proteins vary widely in structure and in the way they associate with the lipid bilayer, which reflects their diverse functions.

1	Membrane proteins can Be Associated with the lipid Bilayer in Various Ways Figure 10–17 shows the different ways in which proteins can associate with the membrane. Like their lipid neighbors, membrane proteins are amphiphilic, having hydrophobic and hydrophilic regions. Many membrane proteins extend through the lipid bilayer, and hence are called transmembrane proteins, with part of their mass on either side (Figure 10–17, examples 1, 2, and 3). Their hydrophobic regions pass through the membrane and interact with the hydrophobic tails of the lipid molecules in the interior of the bilayer, where they are sequestered away from water. Their hydrophilic regions are exposed to water on either side of the membrane. The covalent attachment of a fatty acid chain that inserts into the cytosolic monolayer of the lipid bilayer increases the hydrophobicity of some of these transmembrane proteins (see Figure 10–17, example 1).

1	Other membrane proteins are located entirely in the cytosol and are attached to the cytosolic monolayer of the lipid bilayer, either by an amphiphilic α helix exposed on the surface of the protein (Figure 10–17, example 4) or by one or more covalently attached lipid chains (Figure 10–17, example 5). Yet other membrane proteins are entirely exposed at the external cell surface, being attached to the lipid bilayer only by a covalent linkage (via a specific oligosaccharide) to a lipid anchor in the outer monolayer of the plasma membrane (Figure 10–17, example 6).

1	The lipid-linked proteins in example 5 in Figure 10–17 are made as soluble proteins in the cytosol and are subsequently anchored to the membrane by the covalent attachment of the lipid group. The proteins in example 6, however, are made as single-pass membrane proteins in the endoplasmic reticulum (ER). While still in the ER, the transmembrane segment of the protein is cleaved off and a glycosylphosphatidylinositol (GPI) anchor is added, leaving the protein bound to the noncytosolic surface of the ER membrane solely by this anchor (discussed in Chapter 12); transport vesicles eventually deliver the protein to the plasma membrane (discussed in Chapter 13).

1	By contrast to these examples, membrane-associated proteins do not extend into the hydrophobic interior of the lipid bilayer at all; they are instead bound to either face of the membrane by noncovalent interactions with other membrane proteins (Figure 10–17, examples 7 and 8). Many of the proteins of this type can be released from the membrane by relatively gentle extraction procedures, such as exposure to solutions of very high or low ionic strength or of extreme pH, which interfere with protein–protein interactions but leave the lipid bilayer intact; these proteins are often referred to as peripheral membrane proteins. Transmembrane proteins and many proteins held in the bilayer by lipid groups or hydrophobic polypeptide regions that insert into the hydrophobic core of the lipid bilayer cannot be released in these ways. lipid Anchors control the Membrane localization of Some Signaling proteins

1	lipid Anchors control the Membrane localization of Some Signaling proteins How a membrane protein is associated with the lipid bilayer reflects the function of the protein. Only transmembrane proteins can function on both sides of Figure 10–17 Various ways in which proteins associate with the lipid bilayer. Most membrane proteins are thought to extend across the bilayer as (1) a single α helix, (2) as multiple α helices, or (3) as a rolled-up β sheet (a β barrel). Some of these “single-pass” and “multipass” proteins have a covalently attached fatty acid chain inserted in the cytosolic lipid monolayer (1). Other membrane proteins are exposed at only one side of the membrane. (4) Some of these are anchored to the cytosolic surface by an amphiphilic α helix that partitions into the cytosolic monolayer of the lipid bilayer through the hydrophobic face of the helix.

1	(5) Others are attached to the bilayer solely by a covalently bound lipid chain—either a fatty acid chain or a prenyl group (see Figure 10–18)—in the cytosolic monolayer or, (6) via an oligosaccharide linker, to phosphatidylinositol in the noncytosolic monolayer—called a gpI anchor. (7, 8) Finally, membrane-associated proteins are attached to the membrane only by noncovalent interactions with other membrane proteins. the way in which the structure in (5) is formed is illustrated in Figure 10–18, while the way in which the gpI anchor shown in (6) is formed is illustrated in Figure 12–52. the details of how membrane proteins become associated with the lipid bilayer are discussed in chapter 12.

1	the bilayer or transport molecules across it. Cell-surface receptors, for example, are usually transmembrane proteins that bind signal molecules in the extracellular space and generate different intracellular signals on the opposite side of the plasma membrane. To transfer small hydrophilic molecules across a membrane, a membrane transport protein must provide a path for the molecules to cross the hydrophobic permeability barrier of the lipid bilayer; the molecular architecture of multipass transmembrane proteins (Figure 10–17, examples 2 and 3) is ideally suited for this task, as we discuss in Chapter 11.

1	Proteins that function on only one side of the lipid bilayer, by contrast, are often associated exclusively with either the lipid monolayer or a protein domain on that side. Some intracellular signaling proteins, for example, that help relay extracellular signals into the cell interior are bound to the cytosolic half of the plasma membrane by one or more covalently attached lipid groups, which can be fatty acid chains or prenyl groups (Figure 10–18). In some cases, myristic acid, a saturated 14-carbon fatty acid, is added to the N-terminal amino group of the protein during its synthesis on a ribosome. All members of the Src family of cytoplasmic protein tyrosine kinases (discussed in Chapter 15) are myristoylated in this way. Membrane attachment through a single lipid anchor is not very strong, however, and a second lipid group is often added to anchor proteins more firmly to a membrane. For most Src kinases, the second lipid modification is the attachment of palmitic acid, a

1	very strong, however, and a second lipid group is often added to anchor proteins more firmly to a membrane. For most Src kinases, the second lipid modification is the attachment of palmitic acid, a saturated 16-carbon fatty acid, to a cysteine side chain of the protein. This modification occurs in response to an extracellular signal and helps recruit the kinases to the plasma membrane. When the signaling pathway is turned off, the palmitic acid is removed, allowing the kinase to return to the cytosol. Other intracellular signaling proteins, such as the Ras family small GTPases (discussed in Chapter 15), use a combination of prenyl group and palmitic acid attachment to recruit the proteins to the plasma membrane.

1	Many proteins attach to membranes transiently. Some are classical peripheral membrane proteins that associate with membranes by regulated protein–protein interactions. Others undergo a transition from soluble to membrane protein by a conformational change that exposes a hydrophobic peptide or covalently attached lipid anchor. Many of the small GTPases of the Rab protein family that regulate intracellular membrane traffic (discussed in Chapter 13), for example, switch depending on the nucleotide that is bound to the protein. In their GDP-bound state they are soluble and free in the cytosol, whereas in their GTP-bound state their lipid anchor is exposed and tethers them to membranes. They are

1	Figure 10–18 Membrane protein attachment by a fatty acid chain or a prenyl group. the covalent attachment of either type of lipid can help localize a water-soluble protein to a membrane after its synthesis in the cytosol. (A) A fatty acid chain (myristic acid) is attached via an amide linkage to an n-terminal glycine.

1	(B) A fatty acid chain (palmitic acid) is attached via a thioester linkage to a cysteine. (c) A prenyl chain (either farnesyl or a longer geranylgeranyl chain) is attached via a thioether linkage to a cysteine residue that is initially located four residues from the protein’s c-terminus. After prenylation, the terminal three amino acids are cleaved off, and the new c-terminus is methylated before insertion of the anchor into the membrane (not shown). the structures of the lipid anchors are shown below: (D) a myristoyl anchor (derived from a 14-carbon saturated fatty acid chain), (e) a palmitoyl anchor (a 16-carbon saturated fatty acid chain), and (F) a farnesyl anchor (a 15-carbon unsaturated hydrocarbon chain).

1	Figure 10–19 a segment of a membrane-spanning polypeptide chain crossing the lipid bilayer as an α helix. Only the α-carbon backbone of the polypeptide chain is shown, with the hydrophobic amino acids in green and yellow. the polypeptide segment shown is part of the bacterial photosynthetic reaction center, the structure of which was determined by x-ray diffraction. (Based on data from J. Deisenhofer et al., Nature 318:618– 624, 1985, and h. Michel et al., EMBO J. 5:1149–1158, 1986.) membrane proteins at one moment and soluble proteins at the next. Such highly dynamic interactions greatly expand the repertoire of membrane functions. In Most transmembrane proteins, the polypeptide chain crosses the lipid Bilayer in an α-helical conformation

1	A transmembrane protein always has a unique orientation in the membrane. This reflects both the asymmetric manner in which it is inserted into the lipid bilayer in the ER during its biosynthesis (discussed in Chapter 12) and the different functions of its cytosolic and noncytosolic domains. These domains are separated by the membrane-spanning segments of the polypeptide chain, which contact the hydrophobic environment of the lipid bilayer and are composed largely of amino acids with nonpolar side chains. Because the peptide bonds themselves are polar and because water is absent, all peptide bonds in the bilayer are driven to form hydrogen bonds with one another. The hydrogen-bonding between peptide bonds is maximized if the polypeptide chain forms a regular α helix as it crosses the bilayer, and this is how most membrane-spanning segments of polypeptide chains traverse the bilayer (Figure 10–19).

1	In single-pass transmembrane proteins, the polypeptide chain crosses only once (see Figure 10–17, example 1), whereas in multipass transmembrane proteins, the polypeptide chain crosses multiple times (see Figure 10–17, example 2). An alternative way for the peptide bonds in the lipid bilayer to satisfy their hydrogen-bonding requirements is for multiple transmembrane strands of a polypeptide chain to be arranged as a βsheet that is rolled up into a cylinder (a so-called β barrel; see Figure 10–17, example 3). This protein architecture is seen in the Figure 10–20 Using hydropathy plots to porin proteins that we discuss later.

1	Progress in the x-ray crystallography of membrane proteins has enabled the determination of the three-dimensional structure of many of them. The structures confirm that it is often possible to predict from the protein’s amino acid sequence which parts of the polypeptide chain extend across the lipid bilayer. Segments containing about 20–30 amino acids, with a high degree of hydrophobicity, are long enough to span a lipid bilayer as an α helix, and they can often be identified in hydropathy plots (Figure 10–20). From such plots, it is estimated that about 30% 0 50 100 0 100 200 Rev. Biochem. 53:595–624, 1984. With amino acid number amino acid number permission from Annual reviews.) localize potential α-helical membrane-spanning segments in a polypeptide chain. the free energy needed to transfer successive segments of a polypeptide chain from a nonpolar solvent to water is calculated from the amino acid composition of each segment using data obtained from model compounds. this

1	to transfer successive segments of a polypeptide chain from a nonpolar solvent to water is calculated from the amino acid composition of each segment using data obtained from model compounds. this calculation is made for segments of a fixed size (usually around 10–20 amino acids), beginning with each successive amino acid in the chain. the “hydropathy index” of the segment is plotted on the Y axis as a function of its location in the chain. A positive value indicates that free energy is required for transfer to water (i.e., the segment is hydrophobic), and the value assigned is an index of the amount of energy needed. peaks in the hydropathy index appear at the positions of hydrophobic segments in the amino acid sequence. (A and B) hydropathy plots for two membrane proteins that are discussed later in this chapter. glycophorin (A) has a single membrane-spanning α helix, and one corresponding peak in the hydropathy plot. Bacteriorhodopsin (B) has seven membrane-spanning α helices and

1	later in this chapter. glycophorin (A) has a single membrane-spanning α helix, and one corresponding peak in the hydropathy plot. Bacteriorhodopsin (B) has seven membrane-spanning α helices and seven corresponding peaks in the hydropathy plot. (A, adapted from D. eisenberg, Annu.

1	of an organism’s proteins are transmembrane proteins, emphasizing their importance. Hydropathy plots cannot identify the membrane-spanning segments of a β barrel, as 10 amino acids or fewer are sufficient to traverse a lipid bilayer as an extended β strand and only every other amino acid side chain is hydrophobic.

1	The strong drive to maximize hydrogen-bonding in the absence of water means that a polypeptide chain that enters the lipid bilayer is likely to pass entirely through it before changing direction, since chain bending requires a loss of regular hydrogen-bonding interactions. But multipass transmembrane proteins can also contain regions that fold into the membrane from either side, squeezing into spaces between transmembrane α helices without contacting the hydrophobic core of the lipid bilayer. Because such regions interact only with other polypeptide regions, they do not need to maximize hydrogen-bonding; they can therefore have a variety of secondary structures, including helices that extend only part way across the lipid bilayer (Figure 10–21). Such regions are important for the function of some membrane proteins, including water channel and ion channel proteins, in which the regions contribute to the walls of the pores traversing the membrane and confer substrate specificity on the

1	of some membrane proteins, including water channel and ion channel proteins, in which the regions contribute to the walls of the pores traversing the membrane and confer substrate specificity on the channels, as we discuss in Chapter 11. These regions cannot be identified in hydropathy plots and are only revealed by x-ray crystallography or electron crystallography (a technique similar to x-ray diffraction but performed on two-dimensional arrays of proteins) of the protein’s three-dimensional structure.

1	transmembrane α helices Often Interact with One Another

1	The transmembrane α helices of many single-pass membrane proteins do not contribute to the folding of the protein domains on either side of the membrane. As a consequence, it is often possible to engineer cells to produce just the cytosolic or extracellular domains of these proteins as water-soluble molecules. This approach has been invaluable for studying the structure and function of these domains, especially the domains of transmembrane receptor proteins (discussed in Chapter 15). A transmembrane α helix, even in a single-pass membrane protein, however, often does more than just anchor the protein to the lipid bilayer. Many single-pass membrane proteins form homoor heterodimers that are held together by noncovalent, but strong and highly specific, interactions between the two transmembrane α helices; the sequence of the hydrophobic amino acids of these helices contains the information that directs the protein–protein interaction.

1	Similarly, the transmembrane α helices in multipass membrane proteins occupy specific positions in the folded protein structure that are determined by interactions between the neighboring helices. These interactions are crucial for the structure and function of the many channels and transporters that move molecules across cell membranes.

1	In these proteins, neighboring transmembrane helices in the folded structure of the protein shield many of the other transmembrane helices from the membrane lipids. Why, then, are these shielded helices nevertheless composed primarily of hydrophobic amino acids? The answer lies in the way in which multi-pass proteins are integrated into the membrane during their biosynthesis. As we discuss in Chapter 12, transmembrane α helices are inserted into the lipid bilayer sequentially by a protein translocator. After leaving the translocator, each helix is transiently surrounded by lipids, which requires that the helix be hydrophobic. It is only as the protein folds up into its final structure that contacts are made between adjacent helices, and protein–protein contacts replace some of the protein–lipid contacts (Figure 10–22).

1	Multipass membrane proteins that have their transmembrane segments arranged as β barrels rather than as α helices are comparatively rigid and therefore tend to form crystals readily when isolated. Thus, some of them were among the first Figure 10–21 Two short α helices in the aquaporin water channel, each of which spans only halfway through the lipid bilayer. In the plasma membrane, four monomers, one of which is shown here, form a tetramer. each monomer has a hydrophilic pore at its center, which allows water molecules to cross the membrane in single file (see Figure 11–20 and Movie 11.6). the two short colored helices are buried at an interface formed by protein–protein interactions. the mechanism by which the channel allows the passage of water molecules is discussed in more detail in chapter 11.

1	multipass membrane protein structures to be determined by x-ray crystallography. The number of β strands in a β barrel varies widely, from as few as 8 strands to as many as 22 (Figure 10–23).

1	β-barrel proteins are abundant in the outer membranes of bacteria, mitochondria, and chloroplasts. Some are pore-forming proteins, which create water-filled channels that allow selected small hydrophilic molecules to cross the membrane. The porins are well-studied examples (example 3 in Figure 10–23C). Many porin barrels are formed from a 16-strand, antiparallel β sheet rolled up into a cylindrical structure. Polar amino acid side chains line the aqueous channel on the inside, while nonpolar side chains project from the outside of the barrel to interact with the hydrophobic core of the lipid bilayer. Loops of the polypeptide chain often protrude into the lumen of the channel, narrowing it so that only certain solutes can pass. Some porins are therefore highly selective: maltoporin, for example, preferentially allows maltose and maltose oligomers to cross the outer membrane of E. coli.

1	The FepA protein is a more complex example of a β barrel transport protein (Figure 10–23D). It transports iron ions across the bacterial outer membrane. It is constructed from 22 β strands, and a large globular domain completely fills the inside of the barrel. Iron ions bind to this domain, which by an unknown mechanism moves or changes its conformation to transfer the iron across the membrane. Not all β-barrel proteins are transport proteins. Some form smaller barrels that are completely filled by amino acid side chains that project into the center of the barrel. These proteins function as receptors or enzymes (Figure 10–23A and B); the barrel serves as a rigid anchor, which holds the protein in the membrane and orients the cytosolic loops that form binding sites for specific intracellular molecules. Most multipass membrane proteins in eukaryotic cells and in the bacterial plasma membrane are constructed from transmembrane α helices. The helices

1	Most multipass membrane proteins in eukaryotic cells and in the bacterial plasma membrane are constructed from transmembrane α helices. The helices Figure 10–22 Steps in the folding of a multipass transmembrane protein. When a newly synthesized transmembrane α helix is released into the lipid bilayer, it is initially surrounded by lipid molecules. As the protein folds, contacts between the helices displace some of the lipid molecules surrounding the helices. Figure 10–23 β barrels formed from different numbers of β strands.

1	Figure 10–23 β barrels formed from different numbers of β strands. (A) the E. coli OmpA protein serves as a receptor for a bacterial virus. (B) the E. coli OMplA protein is an enzyme (a lipase) that hydrolyzes lipid molecules. the amino acids that catalyze the enzymatic reaction (shown in red) protrude from the outside surface of the barrel. (c) A porin from the bacterium Rhodobacter capsulatus forms a water-filled pore across the outer membrane. the diameter of the channel is restricted by loops (shown in blue) that protrude into the channel. (D) the E. coli FepA protein transports iron ions. the inside of the barrel is completely filled by a globular protein domain (shown in blue) that contains an iron-binding site (not shown).

1	Figure 10–24 a single-pass transmembrane protein. note that the polypeptide chain traverses the lipid bilayer as a right-handed α helix and that the oligosaccharide chains and disulfide bonds are all on the noncytosolic surface of the membrane. the sulfhydryl groups in the cytosolic domain of the protein do not normally form disulfide bonds because the reducing environment in the cytosol maintains these groups in their reduced (–Sh) form. can slide against each other, allowing conformational changes in the protein that can open and shut ion channels, transport solutes, or transduce extracellular signals into intracellular ones. In β-barrel proteins, by contrast, hydrogen bonds bind each β strand rigidly to its neighbors, making conformational changes within the wall of the barrel unlikely.

1	Most transmembrane proteins in animal cells are glycosylated. As in glycolipids, the sugar residues are added in the lumen of the ER and the Golgi apparatus (discussed in Chapters 12 and 13). For this reason, the oligosaccharide chains are always present on the noncytosolic side of the membrane. Another important difference between proteins (or parts of proteins) on the two sides of the membrane results from the reducing environment of the cytosol. This environment decreases the likelihood that intrachain or interchain disulfide (S–S) bonds will form between cysteines on the cytosolic side of membranes. These bonds form on the noncytosolic side, where they can help stabilize either the folded structure of the polypeptide chain or its association with other polypeptide chains (Figure 10–24).

1	Because the extracellular part of most plasma membrane proteins are glycosylated, carbohydrates extensively coat the surface of all eukaryotic cells. These carbohydrates occur as oligosaccharide chains covalently bound to membrane proteins (glycoproteins) and lipids (glycolipids). They also occur as the polysaccharide chains of integral membrane proteoglycan molecules. Proteoglycans, which consist of long polysaccharide chains linked covalently to a protein core, are found mainly outside the cell, as part of the extracellular matrix (discussed in Chapter 19). But, for some proteoglycans, the protein core either extends across the lipid bilayer or is attached to the bilayer by a glycosylphosphatidylinositol (GPI) anchor.

1	The terms cell coat or glycocalyx are sometimes used to describe the carbohydrate-rich zone on the cell surface. This carbohydrate layer can be visualized by various stains, such as ruthenium red (Figure 10–25A), as well as by its affinity for carbohydrate-binding proteins called lectins, which can be labeled with a fluorescent dye or some other visible marker. Although most of the sugar groups are attached to intrinsic plasma membrane molecules, the carbohydrate layer also contains both glycoproteins and proteoglycans that have been secreted into the extracellular space and then adsorbed onto the cell surface (Figure 10–25B). Many of these adsorbed macromolecules are components of the extracellular matrix, so that the boundary between the plasma membrane and the extracellular matrix is often not sharply defined. One of the many functions of the carbohydrate layer is to protect cells against mechanical and chemical damage; it also keeps various other cells at a distance, preventing

1	often not sharply defined. One of the many functions of the carbohydrate layer is to protect cells against mechanical and chemical damage; it also keeps various other cells at a distance, preventing unwanted cell–cell interactions.

1	The oligosaccharide side chains of glycoproteins and glycolipids are enormously diverse in their arrangement of sugars. Although they usually contain fewer than 15 sugars, the chains are often branched, and the sugars can be bonded together by various kinds of covalent linkages—unlike the amino acids in a polypeptide chain, which are all linked by identical peptide bonds. Even three sugars can be put together to form hundreds of different trisaccharides. Both the diversity and the exposed position of the oligosaccharides on the cell surface make them especially well suited to function in specific cell-recognition processes. As we discuss in Chapter 19, plasma-membrane-bound lectins that recognize specific oligosaccharides on cell-surface glycolipids and glycoproteins mediate a variety of transient cell–cell adhesion processes, including those occurring in lymphocyte recirculation and inflammatory responses (see Figure 19–28).

1	In general, only agents that disrupt hydrophobic associations and destroy the lipid bilayer can solubilize membrane proteins. The most useful of these for the membrane biochemist are detergents, which are small amphiphilic molecules of variable structure (Movie 10.4). Detergents are much more soluble in water than lipids. Their polar (hydrophilic) ends can be either charged (ionic), as in sodium dodecyl sulfate (SDS), or uncharged (nonionic), as in octylglucoside and Triton (Figure 10–26A). At low concentration, detergents are monomeric in solution, but when their concentration is increased above a threshold, called the critical micelle concentration (CMC), they aggregate to form micelles (Figure 10–26B–D). Above the CMC, detergent molecules rapidly diffuse in and out of micelles, keeping the concentration of monomer in the solution constant, no matter how many micelles are present. Both the CMC and the average number of detergent molecules in a micelle are characteristic properties

1	the concentration of monomer in the solution constant, no matter how many micelles are present. Both the CMC and the average number of detergent molecules in a micelle are characteristic properties of each detergent, but they also depend on the temperature, pH, and salt concentration. Detergent solutions are therefore complex systems and are difficult to study.

1	When mixed with membranes, the hydrophobic ends of detergents bind to the hydrophobic regions of the membrane proteins, where they displace lipid molecules with a collar of detergent molecules. Since the other end of the detergent Figure 10–25 The carbohydrate layer on the cell surface. (A) this electron micrograph of the surface of a lymphocyte stained with ruthenium red emphasizes the thick carbohydrate-rich layer surrounding the cell. (B) the carbohydrate layer is made up of the oligosaccharide side chains of membrane glycolipids and membrane glycoproteins and the polysaccharide chains on membrane proteoglycans. In addition, adsorbed glycoproteins, and adsorbed proteoglycans (not shown), contribute to the carbohydrate layer in many cells. note that all of the carbohydrate is on the extracellular surface of the membrane. (A, courtesy of Audrey M. glauert and g.M.W. cook.)

1	Figure 10–26 The structure and function of detergents. (A) three commonly used detergents are sodium dodecyl sulfate (SDS), an anionic detergent, and triton X-100 and β-octylglucoside, two nonionic detergents. triton X-100 is a mixture of compounds in which the region in brackets is repeated between 9 and 10 times. the hydrophobic portion of each detergent is shown in yellow, and the hydrophilic portion is shown in orange. (B) At low concentration, detergent molecules are monomeric in solution. As their concentration is increased beyond the critical micelle concentration (cMc), some of the detergent molecules form micelles. note that the concentration of detergent monomer stays constant above the cMc. (c) Because they have both polar and nonpolar ends, detergent molecules are amphiphilic; and because they are cone-shaped, they form micelles rather than bilayers (see Figure 10–7). Detergent micelles are thought to have irregular shapes, and, due to packing constraints, the hydrophobic

1	and because they are cone-shaped, they form micelles rather than bilayers (see Figure 10–7). Detergent micelles are thought to have irregular shapes, and, due to packing constraints, the hydrophobic tails are partially exposed to water. (D) the space-filling model shows the structure of a micelle composed of 20 β-octylglucoside molecules, predicted by molecular dynamics calculations. the head groups are shown in red and the hydrophobic tails in gray. (B, adapted from g. gunnarsson, B. Jönsson and h. Wennerström, J. Phys. Chem. 84:3114–3121, 1980; c, from S. Bogusz, r.M. Venable and r.W. pastor, J. Phys. Chem. B 104:5462–5470, 2000.) molecule is polar, this binding tends to bring the membrane proteins into solution as detergent–protein complexes (Figure 10–27). Usually, some lipid molecules also remain attached to the protein.

1	Strong ionic detergents, such as SDS, can solubilize even the most hydrophobic membrane proteins. This allows the proteins to be analyzed by SDS polyacrylamide-gel electrophoresis (discussed in Chapter 8), a procedure that has revolutionized the study of proteins. Such strong detergents, however, unfold (denature) proteins by binding to their internal “hydrophobic cores,” thereby rendering the proteins inactive and unusable for functional studies. Nonetheless, proteins can be readily separated and purified in their SDS-denatured form. In some cases, removal of the SDS allows the purified protein to renature, with recovery of functional activity.

1	Many membrane proteins can be solubilized and then purified in an active form by the use of mild detergents. These detergents cover the hydrophobic regions on membrane-spanning segments that become exposed after lipid removal but do not unfold the protein. If the detergent concentration of a solution of solubilized membrane proteins is reduced (by dilution, for example), membrane proteins do not remain soluble. In the presence of an excess of phospholipid molecules in such a solution, however, membrane proteins incorporate into small liposomes that form spontaneously. In this way, functionally active membrane protein systems can be reconstituted from purified components, providing a powerful means of analyzing the activities of membrane transporters, ion channels, signaling receptors, and so on (Figure 10–28). Such functional reconstitution, for example, provided proof for the hypothesis that the enzymes that make ADDITION OF PHOSPHOLIPIDS (mixed with

1	ADDITION OF PHOSPHOLIPIDS (mixed with Figure 10–27 Solubilizing a membrane protein with a mild nonionic detergent. the detergent disrupts the lipid bilayer and brings the protein into solution as protein–lipid–detergent complexes. the phospholipids in the membrane are also solubilized by the detergent, as lipid-detergent micelles. Figure 10–28 The use of mild nonionic detergents for solubilizing, purifying, and reconstituting functional membrane protein systems. In this example, functional na+-K+ pump molecules are purified and incorporated into phospholipid vesicles. this pump is present in the plasma membrane of most animal cells, where it uses the energy of Atp hydrolysis to pump na+ out of the cell and K+ in, as discussed in chapter 11. ATP (ATP synthases) use H+ gradients in mitochondrial, chloroplast, and bacterial membranes to produce ATP.

1	Membrane proteins can also be reconstituted from detergent solution into nanodiscs, which are small, uniformly sized patches of membrane that are surrounded by a belt of protein, which covers the exposed edge of the bilayer to keep the patch in solution (Figure 10–29). The belt is derived from high-density lipoproteins (HDL), which keep lipids soluble for transport in the blood. In nanodiscs the membrane protein of interest can be studied in its native lipid environment and is experimentally accessible from both sides of the bilayer, which is useful, for example, for ligand-binding experiments. Proteins contained in nanodiscs can also be analyzed by single particle electron microscopy techniques to determine their structure. By this rapidly improving technique (discussed in Chapter 9), the structure of a membrane protein can be determined to high resolution without a requirement of the protein of interest to crystallize into a regular lattice, which is often hard to achieve for

1	9), the structure of a membrane protein can be determined to high resolution without a requirement of the protein of interest to crystallize into a regular lattice, which is often hard to achieve for membrane proteins.

1	Detergents have also played a crucial part in the purification and crystallization of membrane proteins. The development of new detergents and new expression systems that produce large quantities of membrane proteins from cDNA clones has led to a rapid increase in the number of three-dimensional structures of membrane proteins and protein complexes that are known, although they are still few compared to the known structures of water-soluble proteins and protein complexes. Bacteriorhodopsin Is a light-driven proton (h+) pump that traverses the lipid Bilayer as Seven α helices

1	Bacteriorhodopsin Is a light-driven proton (h+) pump that traverses the lipid Bilayer as Seven α helices In Chapter 11, we consider how multipass transmembrane proteins mediate the selective transport of small hydrophilic molecules across cell membranes. But a detailed understanding of how such a membrane transport protein works requires precise information about its three-dimensional structure in the bilayer. Bacteriorhodopsin was the first membrane transport protein whose structure was determined, and it has remained the prototype of many multipass membrane proteins with a similar structure.

1	The “purple membrane” of the archaeon Halobacterium salinarum is a specialized patch in the plasma membrane that contains a single species of protein molecule, bacteriorhodopsin (Figure 10–30A). The protein functions as a light-activated H+ pump that transfers H+ out of the archaeal cell. Because the bacteriorhodopsin molecules are tightly packed and arranged as a planar two-dimensional crystal (FIgure 10–30B and C), it was possible to determine their three-dimensional structure by combining electron microscopy and electron diffraction analysis—a procedure called electron crystallography, which we

1	Figure 10–29 Model of a membrane protein reconstituted into a nanodisc. When detergent is removed from a solution containing a multipass membrane protein, lipids, and a protein subunit of the high-density lipoprotein (hDl), the membrane protein becomes embedded in a small patch of lipid bilayer, which is surrounded by a belt of the hDl protein. In such nanodiscs, the hydrophobic edges of the bilayer patch are shielded by the protein belt, which renders the assembly water-soluble. mentioned earlier. This method has provided the first structural views of many membrane proteins that were found to be difficult to crystallize from detergent solutions. For bacteriorhodopsin, the structure was later confirmed and extended to very high resolution by x-ray crystallography.

1	Each bacteriorhodopsin molecule is folded into seven closely packed trans-membrane α helices and contains a single light-absorbing group, or chromophore (in this case, retinal), which gives the protein its purple color. Retinal is vitamin A in its aldehyde form and is identical to the chromophore found in rhodopsin of the photoreceptor cells of the vertebrate eye (discussed in Chapter 15). Retinal is covalently linked to a lysine side chain of the bacteriorhodopsin protein. When activated by a single photon of light, the excited chromophore changes its shape and causes a series of small conformational changes in the protein, resulting in the transfer of one H+ from the inside to the outside of the cell (Figure 10–31A). In bright light, each bacteriorhodopsin molecule can pump several hundred protons per second. The light-driven proton transfer establishes an H+ gradient across the plasma membrane, which in turn drives the production of ATP by a second protein in the cell’s plasma

1	hundred protons per second. The light-driven proton transfer establishes an H+ gradient across the plasma membrane, which in turn drives the production of ATP by a second protein in the cell’s plasma membrane. The energy stored in the H+ gradient also drives other energy-requiring processes in the cell. Thus, bacteriorhodopsin converts solar energy into a H+ gradient, which provides energy to the archaeal cell.

1	The high-resolution crystal structure of bacteriorhodopsin reveals many lipid molecules bound in specific places on the protein surface (Figure 10–31B). hydrophobic core of lipid bilayer (3 nm) Figure 10–30 Patches of purple membrane, which contain bacteriorhodopsin in the archaeon Halobacterium salinarum. (A) these archaea live in saltwater pools, where they are exposed to sunlight. they have evolved a variety of light-activated proteins, including bacteriorhodopsin, which is a light-activated h+ pump in the plasma membrane. (B) the bacteriorhodopsin molecules in the purple membrane patches are tightly packed into two-dimensional crystalline arrays. (c) Details of the molecular surface visualized by atomic force microscopy. With this technique, individual bacteriorhodopsin molecules can be seen. (D) Outline of the approximate location of the bacteriorhodopsin monomer and the individual α helices in the image shown in (c). (B–c, courtesy of Dieter Oesterhelt; D, pDB code: 2BrD.)

1	Figure 10–31 The three-dimensional structure of a bacteriorhodopsin molecule. (Movie 10.5) (A) the polypeptide chain crosses the lipid bilayer seven times as α helices. the location of the retinal chromophore (purple) and the probable pathway taken by h+ during the light-activated pumping cycle are shown. the first and key step is the passing of an h+ from the chromophore to the side chain of aspartic acid 85 (red, located next to the chromophore) that occurs upon absorption of a photon by the chromophore. Subsequently, other h+ transfers—in the numerical order indicated and utilizing the hydrophilic amino acid side chains that line a path through the membrane—complete the pumping cycle and return the enzyme to its starting state. color code: glutamic acid (orange), aspartic acid (red), arginine (blue). (B) the high-resolution crystal structure of bacteriorhodopsin shows many lipid molecules (yellow with red head groups) that are tightly bound to specific places on the surface of the

1	(blue). (B) the high-resolution crystal structure of bacteriorhodopsin shows many lipid molecules (yellow with red head groups) that are tightly bound to specific places on the surface of the protein. (A, adapted from h. luecke et al., Science 286:255–261, 1999. With permission from AAAS; B, from h. luecke et al., J. Mol. Biol. 291:899–911, 1999. With permission from Academic press.)

1	Interactions with specific lipids are thought to help stabilize many membrane proteins, which work best and sometimes crystallize more readily if some of the lipids remain bound during detergent extraction, or if specific lipids are added back to the proteins in detergent solutions. The specificity of these lipid–protein interactions helps explain why eukaryotic membranes contain such a variety of lipids, with head groups that differ in size, shape, and charge. We can think of the membrane lipids as constituting a two-dimensional solvent for the proteins in the membrane, just as water constitutes a three-dimensional solvent for proteins in an aqueous solution: some membrane proteins can function only in the presence of specific lipid head groups, just as many enzymes in aqueous solution require a particular ion for activity.

1	Bacteriorhodopsin is a member of a large superfamily of membrane proteins with similar structures but different functions. For example, rhodopsin in rod cells of the vertebrate retina and many cell-surface receptor proteins that bind extracellular signal molecules are also built from seven transmembrane α helices. These proteins function as signal transducers rather than as transporters: each responds to an extracellular signal by activating a GTP-binding protein (G protein) inside the cell and they are therefore called G-protein-coupled receptors (GPCRs), as we discuss in Chapter 15 (see Figure 15–6B). Although the structures of bacteriorhodopsins and GPCRs are strikingly similar, they show no sequence similarity and thus probably belong to two evolutionarily distant branches of an ancient protein family. A related class of membrane proteins, the channelrhodopsins that green algae use to detect light, form ion channels when they absorb a photon. When engineered so that they are

1	ancient protein family. A related class of membrane proteins, the channelrhodopsins that green algae use to detect light, form ion channels when they absorb a photon. When engineered so that they are expressed in animal brains, these proteins have become invaluable tools in neurobiology because they allow specific neurons to be stimulated experimentally by shining light on them, as we discuss in Chapter 11 (Figure 11–32).

1	Membrane proteins Often Function as large complexes Many membrane proteins function as part of multicomponent complexes, several of which have been studied by x-ray crystallography. One is a bacterial photosynthetic reaction center, which was the first membrane protein complex to be crystallized and analyzed by x-ray diffraction. In Chapter 14, we discuss how such photosynthetic complexes function to capture light energy and use it to pump H+ across the membrane. Many of the membrane protein complexes involved in photosynthesis, proton pumping, and electron transport are even larger than the photosynthetic reaction center. The enormous photosystem II complex from cyanobacteria, for example, contains 19 protein subunits and well over 60 transmembrane helices (see Figure 14–49). Membrane proteins are often arranged in large complexes, not only for harvesting various forms of energy, but also for transducing extracellular signals into intracellular ones (discussed in Chapter 15).

1	Many Membrane proteins Diffuse in the plane of the Membrane Like most membrane lipids, membrane proteins do not tumble (flip-flop) across the lipid bilayer, but they do rotate about an axis perpendicular to the plane of the bilayer (rotational diffusion). In addition, many membrane proteins are able to move laterally within the membrane (lateral diffusion). An experiment in which mouse cells were artificially fused with human cells to produce hybrid cells (heterokaryons) provided the first direct evidence that some plasma membrane proteins are mobile in the plane of the membrane. Two differently labeled antibodies were used to distinguish selected mouse and human plasma membrane proteins. Although at first the mouse and human proteins were confined to their own halves of the newly formed heterokaryon, the two sets of proteins diffused and mixed over the entire cell surface in about half an hour (Figure 10–32).

1	The lateral diffusion rates of membrane proteins can be measured by using the technique of fluorescence recovery after photobleaching (FRAP). The method usually involves marking the membrane protein of interest with a specific fluorescent group. This can be done either with a fluorescent ligand such as a fluorophore-labeled antibody that binds to the protein or with recombinant DNA technology to express the protein fused to a fluorescent protein such as green fluorescent protein (GFP) (discussed in Chapter 9). The fluorescent group is then bleached in a small area of membrane by a laser beam, and the time taken for adjacent membrane proteins carrying unbleached ligand or GFP to diffuse into the bleached area is measured (Figure 10–33). From FRAP measurements, we can estimate the diffusion coefficient for the marked cell-surface protein. The values of the diffusion coefficients for different membrane proteins in different cells are highly variable, because interactions with other

1	coefficient for the marked cell-surface protein. The values of the diffusion coefficients for different membrane proteins in different cells are highly variable, because interactions with other proteins impede the diffusion of the proteins to varying degrees. Measurements of proteins that are minimally impeded in this way indicate that cell membranes have a viscosity comparable to that of olive oil.

1	One drawback to the FRAP technique is that it monitors the movement of large populations of molecules in a relatively large area of membrane; one cannot follow individual protein molecules. If a protein fails to migrate into a bleached area, for example, one cannot tell whether the molecule is truly immobile or just restricted in its movement to a very small region of membrane—perhaps by cytoskeletal proteins. Single-particle tracking techniques overcome this problem by labeling individual membrane molecules with antibodies coupled to fluorescent dyes or tiny gold particles and tracking their movement by video microscopy. Using single-particle tracking, one can record the diffusion path of a single membrane protein molecule over time. Results from all of these techniques indicate that plasma membrane proteins differ widely in their diffusion characteristics, as we now discuss.

1	Figure 10–32 an experiment demonstrating the diffusion of proteins in the plasma membrane of mouse– human hybrid cells. In this experiment, a mouse and a human cell were fused to create a hybrid cell, which was then stained with two fluorescently labeled antibodies. One antibody (labeled with a green dye) detects mouse plasma membrane proteins, the other antibody (labeled with a red dye) detects human plasma membrane proteins. When cells were stained immediately after fusion, mouse and human plasma membrane proteins are still found in the membrane domains originating from the mouse and human cell, respectively. After a short time, however, the plasma membrane proteins diffuse over the entire cell surface and completely intermix. (From l.D. Frye and M. edidine, J. Cell Sci. 7:319–335, 1970. With permission from the company of Biologists.)

1	Figure 10–33 Measuring the rate of lateral diffusion of a membrane protein by fluorescence recovery after photobleaching. A specific protein of interest can be expressed as a fusion protein with green fluorescent protein (gFp), which is intrinsically fluorescent. the fluorescent molecules are bleached in a small area using a laser beam. the fluorescence intensity recovers as the bleached molecules diffuse away and unbleached molecules diffuse into the irradiated area (shown here in side and top views). the diffusion coefficient is calculated from a graph of the rate of recovery: the greater the diffusion coefficient of the membrane protein, the faster the recovery (Movie 10.6). cells can confine proteins and lipids to Specific Domains Within a Membrane

1	cells can confine proteins and lipids to Specific Domains Within a Membrane The recognition that biological membranes are two-dimensional fluids was a major advance in understanding membrane structure and function. It has become clear, however, that the picture of a membrane as a lipid sea in which all proteins float freely is greatly oversimplified. Most cells confine membrane proteins to specific regions in a continuous lipid bilayer. We have already discussed how bacteriorhodopsin molecules in the purple membrane of Halobacterium assemble into large two-dimensional crystals, in which individual protein molecules are relatively fixed in relationship to one another (see Figure 10–30). ATP synthase complexes in the inner mitochondrial membrane also associate into long double rows, as we discuss in Chapter 14 (see Figure 14–32). Large aggregates of this kind diffuse very slowly.

1	In epithelial cells, such as those that line the gut or the tubules of the kidney, certain plasma membrane enzymes and transport proteins are confined to the apical surface of the cells, whereas others are confined to the basal and lateral surfaces (Figure 10–34). This asymmetric distribution of membrane proteins is often essential for the function of the epithelium, as we discuss in Chapter 11 (see Figure 11–11). The lipid compositions of these two membrane domains are also different, demonstrating that epithelial cells can prevent the diffusion of lipid as well as protein molecules between the domains. The barriers set up by a specific type of intercellular junction (called a tight junction, discussed in Chapter 19; see Figure 19–18) maintain the separation of both protein and lipid molecules. Clearly, the membrane proteins that form these intercellular junctions cannot be allowed to diffuse laterally in the interacting membranes.

1	A cell can also create membrane domains without using intercellular junctions. As we already discussed, regulated protein–protein interactions in membranes are thought to create nanoscale raft domains that function in signaling and membrane trafficking. A more extreme example is seen in the mammalian spermatozoon, a single cell that consists of several structurally and functionally distinct parts covered by a continuous plasma membrane. When a sperm cell is examined by immunofluorescence microscopy with a variety of antibodies, each of which reacts with a specific cell-surface molecule, the plasma membrane is found to consist of at least three distinct domains (Figure 10–35). Some of the membrane molecules are able to diffuse freely within the confines of their own domain. The molecular nature of the “fence” that prevents the molecules from Figure 10–34 How membrane molecules can be restricted to a particular membrane domain.

1	Figure 10–34 How membrane molecules can be restricted to a particular membrane domain. In this drawing of an epithelial cell, protein A (in the apical domain of the plasma membrane) and protein B (in the basal and lateral domains) can diffuse laterally in their own domains but are prevented from entering the other domain, at least partly by the specialized cell–cell junction called a tight junction. lipid molecules in the outer (extracellular) monolayer of the plasma membrane are likewise unable to diffuse between the two domains; lipids in the inner (cytosolic) monolayer, however, are able to do so (not shown). the basal lamina is a thin mat of extracellular matrix that separates epithelial sheets from other tissues (discussed in chapter 19).

1	leaving their domain is not known. Many other cells have similar membrane fences that confine membrane protein diffusion to certain membrane domains. The plasma membrane of nerve cells, for example, contains a domain enclosing the cell body and dendrites, and another enclosing the axon; it is thought that a belt of actin filaments tightly associated with the plasma membrane at the cell-body–axon junction forms part of the barrier. Figure 10–36 shows four common ways of immobilizing specific membrane proteins through protein–protein interactions. the cortical cytoskeleton gives Membranes Mechanical Strength and restricts Membrane protein Diffusion

1	As shown in Figure 10–36B and C, a common way in which a cell restricts the lateral mobility of specific membrane proteins is to tether them to macromolecular assemblies on either side of the membrane. The characteristic biconcave shape of a red blood cell (Figure 10–37), for example, results from interactions of its plasma membrane proteins with an underlying cytoskeleton, which consists mainly of a meshwork of the filamentous protein spectrin. Spectrin is a long, thin, flexible rod about 100 nm in length. As the principal component of the red cell cytoskeleton, it maintains the structural integrity and shape of the plasma membrane, which is the red cell’s only membrane, as the cell has no nucleus or other organelles. The spectrin cytoskeleton is riveted to the membrane through various membrane proteins. The final result is a deformable, netlike meshwork that covers the entire cytosolic surface of the red cell membrane (Figure 10–38). This spectrin-based cytoskeleton enables the red

1	proteins. The final result is a deformable, netlike meshwork that covers the entire cytosolic surface of the red cell membrane (Figure 10–38). This spectrin-based cytoskeleton enables the red cell to withstand the stress on its membrane as it is forced through narrow capillaries. Mice and humans with genetic abnormalities in spectrin are anemic and have red cells that are spherical (instead of concave) and fragile; the severity of the anemia increases with the degree of spectrin deficiency.

1	Figure 10–35 Three domains in the plasma membrane of a guinea pig sperm. (A) A drawing of a guinea pig sperm. (B–D) In the three pairs of micrographs, phase-contrast micrographs are on the left, and the same cell is shown with cell-surface immunofluorescence staining on the right. Different monoclonal antibodies selectively label cell-surface molecules on (B) the anterior head, (c) the posterior head, and (D) the tail. (Micrographs courtesy of Selena carroll and Diana Myles.) Figure 10–36 Four ways of restricting the lateral mobility of specific plasma membrane proteins. (A) the proteins can self-assemble into large aggregates (as seen for bacteriorhodopsin in the purple membrane of Halobacterium salinarum); they can be tethered by interactions with assemblies of macromolecules (B) outside or (c) inside the cell; or (D) they can interact with proteins on the surface of another cell.

1	junctional complex spectrin dimer actin ankyrin band 3 glycophorin band 4.1 100 nm ˜ chain H2N HOOC ° chain fexible link between domains 106-amino-acid-long repeating domain NH2spectrin spectrin tetramer plasma membrane An analogous but much more elaborate and highly dynamic cytoskeletal network exists beneath the plasma membrane of most other cells in our body. This network, which constitutes the cortex of the cell, is rich in actin filaments, which are attached to the plasma membrane in numerous ways. The dynamic remodeling of the cortical actin network provides a driving force for many essential cell functions, including cell movement, endocytosis, and the formation of transient, mobile plasma membrane structures such as filopodia and lamellopodia band 4.1

1	Figure 10–38 The spectrin-based cytoskeleton on the cytosolic side of the human red blood cell plasma membrane. (A) the arrangement shown in the drawing has been deduced mainly from studies on the interactions of purified proteins in vitro. Spectrin heterodimers (enlarged in the drawing on the right) are linked together into a netlike meshwork by “junctional complexes” (enlarged in the drawing on the left). each spectrin heterodimer consists of two antiparallel, loosely intertwined, flexible polypeptide chains called α and β. the two spectrin chains are attached noncovalently to each other at multiple points, including at both ends. Both the α and β chains are composed largely of repeating domains. two spectrin heterodimers join end-to-end to form tetramers.

1	the junctional complexes are composed of short actin filaments (containing 13 actin monomers) and these proteins—band 4.1, adducin, and a tropomyosin molecule that probably determines the length of the actin filaments. the cytoskeleton is linked to the membrane through two transmembrane proteins—a multipass protein called band 3 and a single-pass protein called glycophorin. the spectrin tetramers bind to some band 3 proteins via ankyrin molecules, and to glycophorin and band 3 (not shown) via band 4.1 proteins.

1	(B) the electron micrograph shows the cytoskeleton on the cytosolic side of a red blood cell membrane after fixation and negative staining. the spectrin meshwork has been purposely stretched out to allow the details of its structure to be seen. In a normal cell, the meshwork shown would be much more crowded and occupy only about one-tenth of this area. (B, courtesy of t. Byers and D. Branton, Proc. Natl Acad. Sci. USA 82:6153–6157, 1985. With permission from the national Academy of Sciences.) Figure 10–37 a scanning electron micrograph of human red blood cells. the cells have a biconcave shape and lack a nucleus and other organelles (Movie 10.7). (courtesy of Bernadette chailley.) discussed in Chapter 16. The cortex of nucleated cells also contains proteins that are structurally homologous to spectrin and the other components of the red cell cytoskeleton. We discuss the cortical cytoskeleton in nucleated cells and its interactions with the plasma membrane in Chapter 16.

1	The cortical cytoskeletal network restricts diffusion of not only the plasma membrane proteins that are directly anchored to it. Because the cytoskeletal filaments are often closely apposed to the cytosolic surface of the plasma membrane, they can form mechanical barriers that obstruct the free diffusion of proteins in the membrane. These barriers partition the membrane into small domains, or corrals (Figure 10–39A), which can be either permanent, as in the sperm (see Figure 10–35), or transient. The barriers can be detected when the diffusion of individual membrane proteins is followed by high-speed, single-particle tracking. The proteins diffuse rapidly but are confined within an individual corral (Figure 10–39B); occasionally, however, thermal motions cause a few cortical filaments to detach transiently from the membrane, allowing the protein to escape into an adjacent corral.

1	The extent to which a transmembrane protein is confined within a corral depends on its association with other proteins and the size of its cytoplasmic domain; proteins with a large cytosolic domain will have a harder time passing through cytoskeletal barriers. When a cell-surface receptor binds its extracellular signal molecules, for example, large protein complexes build up on the cytosolic domain of the receptor, making it more difficult for the receptor to escape from its corral. It is thought that corralling helps concentrate such signaling complexes, increasing the speed and efficiency of the signaling process (discussed in Chapter 15).

1	Cell membranes assume many different shapes, as illustrated by the elaborate and varied structures of cell-surface protrusions and membrane-enclosed organelles in eukaryotic cells. Flat sheets, narrow tubules, round vesicles, fenestrated sheets, and pitta bread-shaped cisternae are all part of the repertoire: often, a variety of shapes will be present in different regions of the same continuous bilayer. Membrane shape is controlled dynamically, as many essential cell processes— including vesicle budding, cell movement, and cell division—require elaborate transient membrane deformations. In many cases, membrane shape is influenced by dynamic pushing and pulling forces exerted by cytoskeletal or extracellular structures, as we discuss in Chapters 13 and 16). A crucial part in producing these deformations is played by membrane-bending proteins, which control local membrane curvature. Often, cytoskeletal dynamics and membrane-bending-protein forces work together. Membrane-bending proteins

1	deformations is played by membrane-bending proteins, which control local membrane curvature. Often, cytoskeletal dynamics and membrane-bending-protein forces work together. Membrane-bending proteins attach to specific membrane regions as needed and act by one or more of three principal mechanisms: 1. Some insert hydrophobic protein domains or attached lipid anchors into one of the leaflets of a lipid bilayer. Increasing the area of only one leaflet

1	Figure 10–39 Corralling plasma membrane proteins by cortical cytoskeletal filaments. (A) the filaments are thought to provide diffusion barriers that divide the membrane into small domains, or corrals. (B) high-speed, single-particle tracking was used to follow the path of single fluorescently labeled membrane protein of one type over time. the trace shows that the individual protein molecules (the movement of each shown in a different color) diffuse within a tightly delimited membrane domain and only infrequently escape into a neighboring domain. (Adapted from A. Kusumi et al., Annu. Rev. Biophys. Biomol. Struct. 34:351–378, 2005. With permission from Annual reviews.)

1	Figure 10–40 Three ways in which membrane-bending proteins shape membranes. lipid bilayers are blue and proteins are green. (A) Bilayer without protein bound. (B) A hydrophobic region of the protein can insert as a wedge into one monolayer to pry lipid head groups apart. Such regions can either be amphiphilic helices as shown or hydrophobic hairpins. (c) the curved surface of the protein can bind to lipid head groups and deform the membrane or stabilize its curvature. (D) A protein can bind to and cluster lipids that have large head groups and thereby bend the membrane. (Adapted from W.A. prinz and J.e. hinshaw, Crit. Rev. Biochem. Mol. Biol. 44:278–291, 2009.) causes the membrane to bend (Figure 10–40B). The proteins that shape the convoluted network of narrow ER tubules are thought to work in this way. 2.

1	2. Some membrane-bending proteins form rigid scaffolds that deform the membrane or stabilize an already bent membrane (Figure 10–40C). The coat proteins that shape the budding vesicles in intracellular transport fall into this class. 3. Some membrane-bending proteins cause particular membrane lipids to cluster together, thereby inducing membrane curvature. The ability of a lipid to induce positive or negative membrane curvature is determined by the relative cross-sectional areas of its head group and its hydrocarbon tails. For example, the large head group of phosphoinositides make these lipid molecules wedge-shaped, and their accumulation in a domain of one leaflet of a bilayer therefore induces positive curvature (Figure 10–40D). By contrast, phospholipases that remove lipid head groups produce inversely shaped lipid molecules that induce negative curvature.

1	Often, different membrane-bending proteins collaborate to achieve a particular curvature, as in shaping a budding transport vesicle, as we discuss in Chapter 13.

1	Whereas the lipid bilayer determines the basic structure of biological membranes, proteins are responsible for most membrane functions, serving as specific receptors, enzymes, transporters, and so on. Transmembrane proteins extend across the lipid bilayer. Some of these membrane proteins are single-pass proteins, in which the polypeptide chain crosses the bilayer as a single α helix. Others are multipass proteins, in which the polypeptide chain crosses the bilayer multiple times—either as a series of α helices or as a β sheet rolled up into the shape of a barrel. All proteins responsible for the transport of ions and other small water-soluble molecules through the membrane are multipass proteins. Some membrane proteins do not span the bilayer but instead are attached to either side of the membrane: some are attached to the cytosolic side by an amphipathic a helix on the protein surface or by the covalent attachment of one or more lipid chains, others are attached to the noncytosolic

1	membrane: some are attached to the cytosolic side by an amphipathic a helix on the protein surface or by the covalent attachment of one or more lipid chains, others are attached to the noncytosolic side by a GPI anchor. Some membrane-associated proteins are bound by noncovalent interactions with transmembrane proteins. In the plasma membrane of all eukaryotic cells, most of the proteins exposed on the cell surface and some of the lipid molecules in the outer lipid monolayer have oligosaccharide chains covalently attached to them. Like the lipid molecules in the bilayer, many membrane proteins are able to diffuse rapidly in the plane of the membrane. However, cells have ways of immobilizing specific membrane proteins, as well as ways of confining both membrane protein and lipid molecules to particular domains in a continuous lipid bilayer. The dynamic association of membrane-bending proteins confers on membranes their characteristic three-dimensional shapes.

1	given the highly complex lipid composition of cell membranes, what are the variations within different organelle membranes in an animal cell? What are the functional consequences of these differences, and what are the roles of the minor lipid species? Is the biophysical tendency of lipids to partition into separate phases within a lipid bilayer functionally utilized in cell membranes? If so, how is it regulated and what membrane functions does it control? how commonly do specific lipid molecules associate with membrane proteins to regulate their function? given that the structure of only a tiny fraction of all membrane proteins has been determined, what new principles of membrane protein structure remain to be discovered? Which statements are true? explain why or why not. 10–1 Although lipid molecules are free to diffuse in the plane of the bilayer, they cannot flip-flop across the bilayer unless enzyme catalysts called phospholipid translocators are present in the membrane.

1	10–2 Whereas all the carbohydrate in the plasma membrane faces outward on the external surface of the cell, all the carbohydrate on internal membranes faces toward the cytosol. 10–3 Although membrane domains with different protein compositions are well known, there are at present no examples of membrane domains that differ in lipid composition. Discuss the following problems. 10–4 When a lipid bilayer is torn, why does it not seal itself by forming a “hemi-micelle” cap at the edges, as shown in Figure Q10–1? seal with hemi-micelle cap Figure Q10–1 A torn lipid bilayer sealed with a hypothetical “hemimicelle” cap (problem 10–4). 10–5 Margarine is made from vegetable oil by a chemical process. Do you suppose this process converts saturated fatty acids to unsaturated ones, or vice versa? Explain your answer.

1	10–7 Monomeric single-pass transmembrane proteins span a membrane with a single α helix that has characteristic chemical properties in the region of the bilayer. Which of the three 20-amino-acid sequences listed below is the most likely candidate for such a transmembrane segment? Explain the reasons for your choice. (See back of book for one-letter amino acid code; FAMILY VW is a convenient mnemonic for hydrophobic amino acids.) 10–6 If a lipid raft is typically 70 nm in diameter and each lipid molecule has a diameter of 0.5 nm, about how many lipid molecules would there be in a lipid raft composed entirely of lipid? At a ratio of 50 lipid molecules per protein molecule (50% protein by mass), how many proteins would be in a typical raft? (Neglect the loss of lipid from the raft that would be required to accommodate the protein.) 10–8 You are studying the binding of proteins to the cytoplasmic face of cultured neuroblastoma cells and have found a method that gives a good yield of

1	would be required to accommodate the protein.) 10–8 You are studying the binding of proteins to the cytoplasmic face of cultured neuroblastoma cells and have found a method that gives a good yield of inside-out vesicles from the plasma membrane. Unfortunately, your preparations are contaminated with variable amounts of right-side-out vesicles. Nothing you have tried avoids this problem. A friend suggests that you pass your vesicles over an affinity column made of lectin coupled to solid beads. What is the point of your friend’s suggestion?

1	10–9 Glycophorin, a protein in the plasma membrane of the red blood cell, normally exists as a homodimer that is held together entirely by interactions between its trans-membrane domains. Since transmembrane domains are hydrophobic, how is it that they can associate with one another so specifically? 10–10 Three mechanisms by which membrane-binding proteins bend a membrane are illustrated in Figure Q10–2A, B, and C. As shown, each of these cytosolic membrane-bending proteins would induce an invagination of the plasma membrane. Could similar kinds of cytosolic proteins induce a protrusion of the plasma membrane (Figure Q10–2D)? Which ones? Explain how they might work.

1	Figure Q10–2 Bending of the plasma membrane by cytosolic proteins (problem 10–10). (A) Insertion of a protein “finger” into the cytosolic leaflet of the membrane. (B) Binding of lipids to the curved surface of a membrane-binding protein. (c) Binding of membrane proteins to membrane lipids with large head groups. (D) A segment of the plasma membrane showing a protrusion. Bretscher MS (1973) Membrane structure: some general principles. Science 181, 622–629. edidin M (2003) lipids on the frontier: a century of cell-membrane bilayers. Nat. Rev. Mol. Cell Biol. 4, 414–418. goñi FM (2014) the basic structure and dynamics of cell membranes: an update of the Singer-nicolson model. Biochim. Biophys. Acta 1838, 1467–1476. lipowsky r & Sackmann e (eds) (1995) Structure and Dynamics of Membranes. Amsterdam: elsevier. Singer SJ & nicolson gl (1972) the fluid mosaic model of the structure of cell membranes. Science 175, 720–731.

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1	Ichikawa S & hirabayashi Y (1998) glucosylceramide synthase and glycosphingolipid synthesis. Trends Cell Biol. 8, 198–202. Klose c, Surma MA & Simons K (2013) Organellar lipidomics— background and perspectives. Curr. Opin. Cell Biol. 25, 406–413. Kornberg rD & Mcconnell hM (1971) lateral diffusion of phospholipids in a vesicle membrane. Proc. Natl Acad. Sci. USA 68, 2564–2568. lingwood D & Simons K (2010) lipid rafts as a membrane-organizing principle. Science 327, 46–50. Mansilla Mc, cybulski le, Albanesi D & de Mendoza D (2004) control of membrane lipid fluidity by molecular thermosensors. J. Bacteriol. 186, 6681–6688. Maxfield Fr & van Meer g (2010) cholesterol, the central lipid of mammalian cells. Curr. Opin. Cell Biol. 22, 422–429. Mcconnell hM & radhakrishnan A (2003) condensed complexes of cholesterol and phospholipids. Biochim. Biophys. Acta 1610, 159–173. pomorski t & Menon AK (2006) lipid flippases and their biological functions. Cell. Mol. Life Sci. 63, 2908–2921.

1	pomorski t & Menon AK (2006) lipid flippases and their biological functions. Cell. Mol. Life Sci. 63, 2908–2921. rothman Je & lenard J (1977) Membrane asymmetry. Science 195, 743–753. Walther tc & Farese rV Jr (2012) lipid droplets and cellular lipid metabolism. Annu. Rev. Biochem. 81, 687–714. Bennett V & Baines AJ (2001) Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues. Physiol. Rev. 81, 1353–1392. Bijlmakers MJ & Marsh M (2003) the on-off story of protein palmitoylation. Trends Cell Biol. 13, 32–42. Branden c & tooze J (1999) Introduction to protein Structure, 2nd ed. new York: garland Science. Bretscher MS & raff Mc (1975) Mammalian plasma membranes. Nature 258, 43–49. Buchanan SK (1999) Beta-barrel proteins from bacterial outer membranes: structure, function and refolding. Curr. Opin. Struct. Biol. 9, 455–461.

1	Buchanan SK (1999) Beta-barrel proteins from bacterial outer membranes: structure, function and refolding. Curr. Opin. Struct. Biol. 9, 455–461. chen Y, lagerholm Bc, Yang B & Jacobson K (2006) Methods to measure the lateral diffusion of membrane lipids and proteins. Methods 39, 147–153. curran Ar & engelman DM (2003) Sequence motifs, polar interactions and conformational changes in helical membrane proteins. Curr. Opin. Struct. Biol. 13, 412–417. Deisenhofer J & Michel h (1991) Structures of bacterial photosynthetic reaction centers. Annu. Rev. Cell Biol. 7, 1–23. Drickamer K & taylor Me (1993) Biology of animal lectins. Annu. Rev. Cell Biol. 9, 237–264. Drickamer K & taylor Me (1998) evolving views of protein glycosylation. Trends Biochem. Sci. 23, 321–324. Frye lD & edidin M (1970) the rapid intermixing of cell surface antigens after formation of mouse-human heterokaryons. J. Cell Sci. 7, 319–335.

1	Frye lD & edidin M (1970) the rapid intermixing of cell surface antigens after formation of mouse-human heterokaryons. J. Cell Sci. 7, 319–335. helenius A & Simons K (1975) Solubilization of membranes by detergents. Biochim. Biophys. Acta 415, 29–79. henderson r & Unwin pn (1975) three-dimensional model of purple membrane obtained by electron microscopy. Nature 257, 28–32. Kyte J & Doolittle rF (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132. lee Ag (2003) lipid-protein interactions in biological membranes: a structural perspective. Biochim. Biophys. Acta 1612, 1–40. Marchesi Vt, Furthmayr h & tomita M (1976) the red cell membrane. Annu. Rev. Biochem. 45, 667–698. nakada c, ritchie K, Oba Y et al. (2003) Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization. Nat. Cell Biol. 5, 626–632.

1	nakada c, ritchie K, Oba Y et al. (2003) Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization. Nat. Cell Biol. 5, 626–632. Oesterhelt D (1998) the structure and mechanism of the family of retinal proteins from halophilic archaea. Curr. Opin. Struct. Biol. 8, 489–500. popot J-l (2010) Amphipols, nanodiscs, and fluorinated surfactants: three nonconventional approaches to studying membrane proteins in aqueous solution. Annu. Rev. Biochem. 79, 737–775. prinz WA & hinshaw Je (2009) Membrane-bending proteins. Crit. Rev. Biochem. Mol. Biol. 44, 278–291. rao M & Mayor S (2014) Active organization of membrane constituents in living cells. Curr. Opin. Cell Biol. 29, 126–132. reig n & van der goot Fg (2006) About lipids and toxins. FEBS Lett. 580, 5572–5579. Sharon n & lis h (2004) history of lectins: from hemagglutinins to biological recognition molecules. Glycobiology 14, 53r–62r.

1	Sharon n & lis h (2004) history of lectins: from hemagglutinins to biological recognition molecules. Glycobiology 14, 53r–62r. Sheetz Mp (2001) cell control by membrane-cytoskeleton adhesion. Nat. Rev. Mol. Cell Biol. 2, 392–396. Shibata Y, hu J, Kozlov MM & rapoport tA (2009) Mechanisms shaping the membranes of cellular organelles. Annu. Rev. Cell Dev. Biol. 25, 329–354. Steck tl (1974) the organization of proteins in the human red blood cell membrane. A review. J. Cell Biol. 62, 1–19. Subramaniam S (1999) the structure of bacteriorhodopsin: an emerging consensus. Curr. Opin. Struct. Biol. 9, 462–468. Viel A & Branton D (1996) Spectrin: on the path from structure to function. Curr. Opin. Cell Biol. 8, 49–55. Vinothkumar Kr & henderson r (2010) Structures of membrane proteins. Q. Rev. Biophys. 43, 65–158. von heijne g (2011) Membrane proteins: from bench to bits. Biochem. Soc. Trans. 39, 747–750.

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1	Because of its hydrophobic interior, the lipid bilayer of cell membranes restricts the passage of most polar molecules. This barrier function allows the cell to maintain concentrations of solutes in its cytosol that differ from those in the extracellular fluid and in each of the intracellular membrane-enclosed compartments. To benefit from this barrier, however, cells have had to evolve ways of transferring specific water-soluble molecules and ions across their membranes in order to ingest essential nutrients, excrete metabolic waste products, and regulate intracellular ion concentrations. Cells use specialized membrane transport proteins to accomplish this goal. The importance of such small molecule transport is reflected in the large number of genes in all organisms that code for the transmembrane transport proteins involved, which make up 15–30% of the membrane proteins in all cells. Some mammalian cells, such as nerve and kidney cells, devote up to two-thirds of their total

1	the transmembrane transport proteins involved, which make up 15–30% of the membrane proteins in all cells. Some mammalian cells, such as nerve and kidney cells, devote up to two-thirds of their total metabolic energy consumption to such transport processes.

1	Cells can also transfer macromolecules and even large particles across their membranes, but the mechanisms involved in most of these cases differ from those used for transferring small molecules, and they are discussed in Chapters 12 and 13.

1	We begin this chapter by describing some general principles of how small water-soluble molecules traverse cell membranes. We then consider, in turn, the two main classes of membrane proteins that mediate this transmembrane traffic: transporters, which undergo sequential conformational changes to transport specific small molecules across membranes, and channels, which form narrow pores, allowing passive transmembrane movement, primarily of water and small inorganic ions. Transporters can be coupled to a source of energy to catalyze active transport, which together with selective passive permeability, creates large differences in the composition of the cytosol compared with that of either the extracellular fluid (Table 11–1) or the fluid within membrane-enclosed organelles. By generating inorganic ion-concentration differences across the lipid bilayer, cell membranes can store potential energy in the form of electrochemical gradients, which drive various transport processes, convey

1	inorganic ion-concentration differences across the lipid bilayer, cell membranes can store potential energy in the form of electrochemical gradients, which drive various transport processes, convey electrical signals in electrically excitable cells, and (in mitochondria, chloroplasts, and bacteria) make most of the cell’s ATP. We focus our discussion mainly on transport across the plasma membrane, but similar mechanisms operate across the other membranes of the eukaryotic cell, as discussed in later chapters.

1	In the last part of the chapter, we concentrate mainly on the functions of ion channels in neurons (nerve cells). In these cells, channel proteins perform at their highest level of sophistication, enabling networks of neurons to carry out all the astonishing feats your brain is capable of. We begin this section by describing the permeability properties of protein-free, synthetic lipid bilayers. We then introduce some of the terms used to describe the various forms of membrane transport and some strategies for characterizing the proteins and processes involved. Protein-Free Lipid Bilayers Are Impermeable to Ions

1	Protein-Free Lipid Bilayers Are Impermeable to Ions Given enough time, virtually any molecule will diffuse across a protein-free lipid bilayer down its concentration gradient. The rate of diffusion, however, varies enormously, depending partly on the size of the molecule but mostly on its relative hydrophobicity (solubility in oil). In general, the smaller the molecule and the more hydrophobic, or nonpolar, it is, the more easily it will diffuse across a lipid bilayer. Small nonpolar molecules, such as O2 and CO2, readily dissolve in lipid bilayers and therefore diffuse rapidly across them. Small uncharged polar molecules, such as water or urea, also diffuse across a bilayer, albeit much more slowly (Figure 11–1 and see Movie 10.3). By contrast, lipid bilayers are essentially impermeable to charged molecules (ions), no matter how small: the charge and high degree of hydration of such molecules prevents them from entering the hydrocarbon phase of the bilayer (Figure 11–2).

1	There Are Two Main Classes of Membrane Transport Proteins: Transporters and Channels

1	Like synthetic lipid bilayers, cell membranes allow small nonpolar molecules to permeate by diffusion. Cell membranes, however, also have to allow the passage of various polar molecules, such as ions, sugars, amino acids, nucleotides, water, and many cell metabolites that cross synthetic lipid bilayers only very slowly. Special membrane transport proteins transfer such solutes across cell membranes. These proteins occur in many forms and in all types of biological membranes. Each protein often transports only a specific molecular species or sometimes a class of molecules (such as ions, sugars, or amino acids). Studies in the 1950s found that bacteria with a single-gene mutation were unable to transport sugars across their plasma membrane, thereby demonstrating the specificity of membrane transport proteins. We now know that humans with similar mutations suffer from various inherited diseases that hinder the transport of a specific solute or solute class in the kidney, intestine, or

1	transport proteins. We now know that humans with similar mutations suffer from various inherited diseases that hinder the transport of a specific solute or solute class in the kidney, intestine, or other cell type. Individuals with the inherited disease cystinuria, for example, cannot transport certain amino acids (including cystine, the disulfide-linked dimer of cysteine) from either the urine or the intestine into the

1	Figure 11–1 The relative permeability of a synthetic lipid bilayer to different classes of molecules. The smaller the molecule and, more importantly, the less strongly it associates with water, the more rapidly the molecule diffuses across the bilayer. Figure 11–2 Permeability coefficients for the passage of various molecules through synthetic lipid bilayers. The rate of flow of a solute across the bilayer is directly proportional to the difference in its concentration on the two sides of the membrane. Multiplying this concentration difference (in mol/cm3) by the permeability coefficient (in cm/sec) gives the flow of solute in moles per second per square centimeter of bilayer. A concentration difference of tryptophan of 10–4 mol/cm3 (10–4 mol / 10–3 L = 0.1 M), for example, would cause a flow of 10–4 mol/cm3 × 10–7 cm/sec = 10–11 mol/sec through 1 cm2 of bilayer, or 6 × 104 molecules/sec through 1 μm2 of bilayer.

1	blood; the resulting accumulation of cystine in the urine leads to the formation of cystine stones in the kidneys. All membrane transport proteins that have been studied in detail are multi-pass transmembrane proteins—that is, their polypeptide chains traverse the lipid bilayer multiple times. By forming a protein-lined pathway across the membrane, these proteins enable specific hydrophilic solutes to cross the membrane without coming into direct contact with the hydrophobic interior of the lipid bilayer.

1	Transporters and channels are the two major classes of membrane transport proteins (Figure 11–3). Transporters (also called carriers, or permeases) bind the specific solute to be transported and undergo a series of conformational changes that alternately expose solute-binding sites on one side of the membrane and then on the other to transfer the solute across it. Channels, by contrast, interact with the solute to be transported much more weakly. They form continuous pores that extend across the lipid bilayer. When open, these pores allow specific solutes (such as inorganic ions of appropriate size and charge and in some cases small molecules, including water, glycerol, and ammonia) to pass through them and thereby cross the membrane. Not surprisingly, transport through channels occurs at a much faster rate than transport mediated by transporters. Although water can slowly diffuse across synthetic lipid bilayers, cells use dedicated channel proteins (called water channels, or

1	at a much faster rate than transport mediated by transporters. Although water can slowly diffuse across synthetic lipid bilayers, cells use dedicated channel proteins (called water channels, or aquaporins) that greatly increase the permeability of their membranes to water, as we discuss later.

1	Active Transport Is Mediated by Transporters Coupled to an Energy Source

1	All channels and many transporters allow solutes to cross the membrane only passively (“downhill”), a process called passive transport. In the case of transport of a single uncharged molecule, the difference in the concentration on the two sides of the membrane—its concentration gradient—drives passive transport and determines its direction (Figure 11–4A). If the solute carries a net charge, however, both its concentration gradient and the electrical potential difference across the membrane, the membrane potential, influence its transport. The concentration gradient and the electrical gradient combine to form a net driving force, the electrochemical gradient, for each charged solute (Figure 11–4B). We discuss electrochemical gradients in more detail later and in Chapter 14. In fact, almost all plasma membranes have an electrical potential (i.e., a voltage) across them, with the inside usually negative with respect to the outside. This potential favors the entry of positively charged

1	all plasma membranes have an electrical potential (i.e., a voltage) across them, with the inside usually negative with respect to the outside. This potential favors the entry of positively charged ions into the cell but opposes the entry of negatively charged ions (see Figure 11–4B); it also opposes the efflux of positively charged ions.

1	Figure 11–3 Transporters and channel proteins. (A) A transporter alternates between two conformations, so that the solute-binding site is sequentially accessible on one side of the bilayer and then on the other. (B) In contrast, a channel protein forms a pore across the bilayer through which specific solutes can passively diffuse. concentration electrochemical gradient with gradient (with no a membrane potential As shown in Figure 11–4A, in addition to passive transport, cells need to be able to actively pump certain solutes across the membrane “uphill,” against their electrochemical gradients. Such active transport is mediated by transporters whose pumping activity is directional because it is tightly coupled to a source of metabolic energy, such as an ion gradient or ATP hydrolysis, as discussed later. Transmembrane movement of small molecules mediated by transporters can be either active or passive, whereas that mediated by channels is always passive (see Figure 11–4A).

1	Lipid bilayers are virtually impermeable to most polar molecules. To transport small water-soluble molecules into or out of cells or intracellular membrane-enclosed compartments, cell membranes contain various membrane transport proteins, each of which is responsible for transferring a particular solute or class of solutes across the membrane. There are two classes of membrane transport proteins—transporters and channels. Both form protein pathways across the lipid bilayer. Whereas transmembrane movement mediated by transporters can be either active or passive, solute flow through channel proteins is always passive. Both active and passive ion transport is influenced by the ion’s concentration gradient and the membrane potential—that is, its electrochemical gradient.

1	The process by which a transporter transfers a solute molecule across the lipid bilayer resembles an enzyme–substrate reaction, and in many ways transporters behave like enzymes. By contrast to ordinary enzyme–substrate reactions, however, the transporter does not modify the transported solute but instead delivers it unchanged to the other side of the membrane. Each type of transporter has one or more specific binding sites for its solute (substrate). It transfers the solute across the lipid bilayer by undergoing reversible

1	Each type of transporter has one or more specific binding sites for its solute (substrate). It transfers the solute across the lipid bilayer by undergoing reversible Figure 11–4 Different forms of membrane transport and the influence of the membrane. Passive transport down a concentration gradient (or an electrochemical gradient—see B below) occurs spontaneously, by diffusion, either through the lipid bilayer directly or through channels or passive transporters. By contrast, active transport requires an input of metabolic energy and is always mediated by transporters that pump the solute against its concentration or electrochemical gradient. (B) The electrochemical gradient of a charged solute (an ion) affects its transport. This gradient combines the membrane potential and the concentration gradient of the solute. The electrical and chemical gradients can work additively to increase the driving force on an ion across the membrane (middle) or can work against each other (right).

1	Figure 11–5 a model of how a conformational change in a transporter mediates the passive movement of a solute. The transporter is shown in three conformational states: in the outward- open state, the binding sites for solute are exposed on the outside; in the occluded state, the same sites are not accessible from either side; and in the inward-open state, the sites are exposed on the inside. The transitions between the states occur randomly. They are completely reversible and do not depend on whether the solute-binding site is occupied. Therefore, if the solute concentration is higher on the outside of the bilayer, more solute binds to the transporter in the outward-open conformation than in the inward-open conformation, and there is a net transport of solute down its concentration gradient (or, if the solute is an ion, down its electrochemical gradient).

1	conformational changes that alternately expose the solute-binding site first on one side of the membrane and then on the other—but never on both sides at the same time. The transition occurs through an intermediate state in which the solute is inaccessible, or occluded, from either side of the membrane (Figure 11–5). When the transporter is saturated (that is, when all solute-binding sites are occupied), the rate of transport is maximal. This rate, referred to as Vmax (V for velocity), is characteristic of the specific carrier. Vmax measures the rate at which the carrier can flip between its conformational states. In addition, each transporter has a characteristic affinity for its solute, reflected in the Km of the reaction, which is equal to the concentration of solute when the transport rate is half its maximum value (Figure 11–6). As with enzymes, the binding of solute can be blocked by either competitive inhibitors (which compete for the same binding site and may or may not be

1	rate is half its maximum value (Figure 11–6). As with enzymes, the binding of solute can be blocked by either competitive inhibitors (which compete for the same binding site and may or may not be transported) or noncompetitive inhibitors (which bind elsewhere and alter the structure of the transporter).

1	As we discuss shortly, it requires only a relatively minor modification of the model shown in Figure 11–5 to link a transporter to a source of energy in order to pump a solute uphill against its electrochemical gradient. Cells carry out such active transport in three main ways (Figure 11–7): 1. Coupled transporters harness the energy stored in concentration gradients to couple the uphill transport of one solute across the membrane to the downhill transport of another. 2. ATP-driven pumps couple uphill transport to the hydrolysis of ATP. 3. Lightor redox-driven pumps, which are known in bacteria, archaea, mitochondria, and chloroplasts, couple uphill transport to an input of energy from light, as with bacteriorhodopsin (discussed in Chapter 10), or from a rate of transport redox reaction, as with cytochrome c oxidase (discussed in Chapter 14).

1	Amino acid sequence and three-dimensional structure comparisons suggest that, in many cases, there are strong similarities in structure between transporters that mediate active transport and those that mediate passive transport. Some bacterial transporters, for example, that use the energy stored in the H+ gradient across the plasma membrane to drive the active uptake of various sugars are structurally similar to the transporters that mediate passive glucose transport into most animal cells. This suggests an evolutionary relationship between various transporters. Given the importance of small metabolites and sugars as energy sources, it is not surprising that the superfamily of transporters is an ancient one.

1	We begin our discussion of active membrane transport by considering a class of coupled transporters that are driven by ion concentration gradients. These proteins have a crucial role in the transport of small metabolites across membranes in all cells. We then discuss ATP-driven pumps, including the Na+-K+ pump that is found in the plasma membrane of most animal cells. Examples of the third class of active transport—lightor redox-driven pumps—are discussed in Chapter 14. Some transporters simply passively mediate the movement of a single solute from one side of the membrane to the other at a rate determined by their Vmax and

1	Some transporters simply passively mediate the movement of a single solute from one side of the membrane to the other at a rate determined by their Vmax and Figure 11–6 The kinetics of simple diffusion compared with transporter-mediated diffusion. Whereas the rate of diffusion and channel-mediated transport is directly proportional to the solute concentration (within the physical limits imposed by total surface area or total channels available), the rate of transporter-mediated diffusion reaches a maximum (Vmax) when the transporter is saturated. The solute concentration when the transport rate is at half its maximal value approximates the binding constant (Km) of the transporter for the solute and is analogous to the Km of an enzyme for its substrate. The graph applies to a transporter moving a single solute; the kinetics of coupled transport of two or more solutes is more complex and exhibits cooperative behavior.

1	Km; they are called uniporters. Others function as coupled transporters, in which the transfer of one solute strictly depends on the transport of a second. Coupled transport involves either the simultaneous transfer of a second solute in the same direction, performed by symporters (also called co-transporters), or the transfer of a second solute in the opposite direction, performed by antiporters (also called exchangers) (Figure 11–8).

1	The tight coupling between the transfer of two solutes allows the coupled transporters to harvest the energy stored in the electrochemical gradient of one solute, typically an inorganic ion, to transport the other. In this way, the free energy released during the movement of an inorganic ion down an electrochemical gradient is used as the driving force to pump other solutes uphill, against their electrochemical gradient. This strategy can work in either direction; some coupled transporters function as symporters, others as antiporters. In the plasma membrane of animal cells, Na+ is the usual co-transported ion because its electrochemical gradient provides a large driving force for the active transport of a second molecule. The Na+ that enters the cell during coupled transport is subsequently pumped out by an ATP-driven Na+-K+ pump in the plasma membrane (as we discuss later), which, by maintaining the Na+ gradient, indirectly drives the coupled transport. Such ion-driven coupled

1	pumped out by an ATP-driven Na+-K+ pump in the plasma membrane (as we discuss later), which, by maintaining the Na+ gradient, indirectly drives the coupled transport. Such ion-driven coupled transporters as just described are said to mediate secondary active transport. In contrast, ATP-driven pumps are said to mediate primary active transport because in these the free energy of ATP hydrolysis is used to directly drive the transport of a solute against its concentration gradient.

1	Intestinal and kidney epithelial cells contain a variety of symporters that are driven by the Na+ gradient across the plasma membrane. Each Na+-driven symporter is specific for importing a small group of related sugars or amino acids into the cell. Because the Na+ tends to move into the cell down its electrochemical gradient, the sugar or amino acid is, in a sense, “dragged” into the cell with it. The greater the electrochemical gradient for Na+, the more solute is pumped Figure 11–7 Three ways of driving active transport. The actively transported molecule is shown in orange, and the energy source is shown in red. Redox driven active transport is discussed in Chapter 14 (see Figures 14–18 and 14–19). Figure 11–8 This schematic diagram shows transporters functioning as uniporters, symporters, and antiporters coupled transport (Movie 11.1).

1	Figure 11–9 Mechanism of glucose transport fueled by a Na+ gradient. As in the model shown in Figure 11–5, the transporter alternates between inward-open and outward-open states via an occluded intermediate state. Binding of Na+ and glucose is cooperative—that is, the binding of either solute increases the protein’s affinity for the other. Since the Na+ concentration is much higher in the extracellular space than in the cytosol, glucose is more likely to bind to the transporter in the outward-facing state. The transition to the occluded state occurs only when both Na+ and glucose are bound; their precise interactions in the solute-binding sites slightly stabilize the occluded state and thereby make this transition energetically favorable. Stochastic fluctuations caused by thermal energy drive the transporter randomly into the inward-open or outward-open conformation. If it opens outwardly, nothing is achieved, and the process starts all over. However, whenever it opens inwardly,

1	drive the transporter randomly into the inward-open or outward-open conformation. If it opens outwardly, nothing is achieved, and the process starts all over. However, whenever it opens inwardly, Na+ dissociates quickly in the low-Na+-concentration environment of the cytosol. Glucose dissociation is likewise enhanced when Na+ is lost, because of cooperativity in binding of the two solutes. The overall result is the net transport of both Na+ and glucose into the cell. Because the occluded state is not formed when only one of the solutes is bound, the transporter switches conformation only when it is fully occupied or fully empty, thereby assuring strict coupling of the transport of Na+ and glucose.

1	into the cell (Figure 11–9). Neurotransmitters (released by nerve cells to signal at synapses—as we discuss later) are taken up again by Na+ symporters after their release. These neurotransmitter transporters are important drug targets: stimulants, such as cocaine and antidepressants, inhibit them and thereby prolong signaling by the neurotransmitters, which are not cleared efficiently.

1	Despite their great variety, transporters share structural features that can explain how they function and how they evolved. Transporters are typically built from bundles of 10 or more α helices that span the membrane. Soluteand ion-binding sites are located midway through the membrane, where some helices are broken or distorted and amino acid side chains and polypeptide backbone atoms form ionand solute-binding sites. In the inward-open and outward-open conformations, these binding sites are accessible by passageways from one side of the membrane but not the other. In switching between the two conformations, the transporter protein transiently adopts an occluded conformation, in which both passageways are closed; this prevents the driving ion and the transported solute from crossing the membrane unaccompanied, which would deplete the cell’s energy store to no purpose. Because only transporters with both types of binding sites appropriately filled change their conformation, tight

1	the membrane unaccompanied, which would deplete the cell’s energy store to no purpose. Because only transporters with both types of binding sites appropriately filled change their conformation, tight coupling between ion and solute transport is assured.

1	Like enzymes, transporters can work in the reverse direction if ion and solute gradients are appropriately adjusted experimentally. This chemical symmetry is mirrored in their physical structure. Crystallographic analyses have revealed that transporters are built from inverted repeats: the packing of the transmembrane α helices in one half of the helix bundle is structurally similar to the packing in the other half, but the two halves are inverted in the membrane relative to each other. Transporters are therefore said to be pseudosymmetric, and the passageways that open and close on either side of the membrane have closely similar geometries, allowing alternating access to the ionand solute-binding sites in the center (Figure 11–10). It is thought that the two halves evolved by gene duplication of a smaller ancestor protein.

1	Some other types of important membrane transport proteins are also built from inverted repeats. Examples even include channel proteins such as the aquaporin water channel (discussed later) and the Sec61 channel through which nascent polypeptides move into the endoplasmic reticulum (discussed in Chapter 12). It is thought that these channels evolved from coupled transporters, in which the gating functions were lost, allowing them to open toward both sides of the membrane simultaneously to provide a continuous path across the membrane. In bacteria, yeasts, and plants, as well as in many membrane-enclosed organelles of animal cells, most ion-driven active transport systems depend on H+ rather than Na+ gradients, reflecting the predominance of H+ pumps in these membranes. An electrochemical H+ gradient across the bacterial plasma membrane, for example, drives the inward active transport of many sugars and amino acids. Transporters in the Plasma Membrane Regulate Cytosolic pH

1	Transporters in the Plasma Membrane Regulate Cytosolic pH Most proteins operate optimally at a particular pH. Lysosomal enzymes, for example, function best at the low pH (~5) found in lysosomes, whereas cytosolic enzymes function best at the close-to-neutral pH (~7.2) found in the cytosol. It is therefore crucial that cells control the pH of their intracellular compartments.

1	Most cells have one or more types of Na+-driven antiporters in their plasma membrane that help to maintain the cytosolic pH at about 7.2. These transporters use the energy stored in the Na+ gradient to pump out excess H+, which either leaks in or is produced in the cell by acid-forming reactions. Two mechanisms are used: either H+ is directly transported out of the cell or HCO3 is brought into the cell to neutralize H+ in the cytosol (according to the reaction HCO3– + H+ →H2O + CO2). One of the antiporters that uses the first mechanism is a Na+–H+ exchanger, which couples an influx of Na+ to an efflux of H+. Another, which uses a combination of the two mechanisms, is a Na+-driven Cl––HCO3 – exchanger that couples an influx of Na+ and HCO3 to an efflux of Cl– and H+ (so that NaHCO3 comes – in and HCl goes out). The Na+-driven Cl––HCO3 exchanger is twice as effective as the Na+–H+ exchanger: it pumps out one H+ and neutralizes another for each Na+ that enters the cell. If HCO3– is

1	– in and HCl goes out). The Na+-driven Cl––HCO3 exchanger is twice as effective as the Na+–H+ exchanger: it pumps out one H+ and neutralizes another for each Na+ that enters the cell. If HCO3– is available, as is usually the case, this antiporter is the most important transporter regulating the cytosolic pH. The pH inside the cell regulates both exchangers; when the pH in the cytosol falls, both exchangers increase their activity.

1	A Na+-independent Cl––HCO3 exchanger adjusts the cytosolic pH in the reverse direction. Like the Na+-dependent transporters, pH regulates the Na+-in– dependent Cl––HCO3 exchanger, but the exchanger’s activity increases as the – cytosol becomes too alkaline. The movement of HCO3 in this case is normally out of the cell, down its electrochemical gradient, which decreases the pH of the

1	Figure 11–10 Transporters are built from inverted repeats. (A) LeuT, a bacterial leucine/Na+ symporter related to human neurotransmitter transporters, such as the serotonin transporter, is shown. The core of the transporter is built from two bundles, each composed of five α helices (blue and yellow). The helices shown in gray differ among members of this transporter family and are thought to play regulatory roles, which are specific to a particular transporter. (B) Both core helix bundles are packed in a similar arrangement (shown as a hand, with the broken helix as the thumb), but the second bundle is inverted with respect to the first. The transporter’s structural pseudosymmetry reflects its functional symmetry: the transporter can work in either direction, depending on the direction of the ion gradient. (Adapted from K.R. Vinothkumar and R. Henderson, Q.

1	K.R. Vinothkumar and R. Henderson, Q. Rev. Biophys. 43:65–158, 2010. With permission from Cambridge University Press. PDB code: 3F3E.) cytosol. A Na+-independent Cl––HCO3 exchanger in the membrane of red blood cells (called band 3 protein—see Figure 10–38) facilitates the quick discharge of CO2 (as HCO3–) as the cells pass through capillaries in the lung. The intracellular pH is not entirely regulated by transporters in the plasma membrane: ATP-driven H+ pumps are used to control the pH of many intracellular compartments. As discussed in Chapter 13, H+ pumps maintain the low pH in lysosomes, as well as in endosomes and secretory vesicles. These H+ pumps use the energy of ATP hydrolysis to pump H+ into these organelles from the cytosol. An Asymmetric Distribution of Transporters in Epithelial Cells Underlies the Transcellular Transport of Solutes

1	An Asymmetric Distribution of Transporters in Epithelial Cells Underlies the Transcellular Transport of Solutes In epithelial cells, such as those that absorb nutrients from the gut, transporters are distributed nonuniformly in the plasma membrane and thereby contribute to the transcellular transport of absorbed solutes. By the actions of the transporters in these cells, solutes are moved across the epithelial cell layer into the extracellular fluid from where they pass into the blood. As shown in Figure 11–11, Na+-linked symporters located in the apical (absorptive) domain of the plasma membrane actively transport nutrients into the cell, building up substantial concentration gradients for these solutes across the plasma membrane. Uniporters in the basal and lateral (basolateral) domains allow the nutrients to leave the cell passively down these concentration gradients.

1	In many of these epithelial cells, the plasma membrane area is greatly increased by the formation of thousands of microvilli, which extend as thin, fingerlike projections from the apical surface of each cell. Such microvilli can increase the total absorptive area of a cell as much as 25-fold, thereby enhancing its transport capabilities. As we have seen, ion gradients have a crucial role in driving many essential transport processes in cells. Ion pumps that use the energy of ATP hydrolysis establish and maintain these gradients, as we discuss next. transporter mediating passive transport of glucose

1	Figure 11–11 Transcellular transport. The transcellular transport of glucose across an intestinal epithelial cell depends on the nonuniform distribution of transporters in the cell’s plasma membrane. The process shown here results in the transport of glucose from the intestinal lumen to the extracellular fluid (from where it passes into the blood). Glucose is pumped into the cell through the apical domain of the membrane by a Na+-powered glucose symporter. Glucose passes out of the cell (down its concentration gradient) by passive movement through a glucose uniporter in the basal and lateral membrane domains. The Na+ gradient driving the glucose symport is maintained by the Na+-K+ pump in the basal and lateral plasma membrane domains, which keeps the internal concentration of Na+ low (Movie 11.2). Adjacent cells are connected by impermeable tight junctions, which have a dual function in the transport process illustrated: they prevent solutes from crossing the epithelium between cells,

1	11.2). Adjacent cells are connected by impermeable tight junctions, which have a dual function in the transport process illustrated: they prevent solutes from crossing the epithelium between cells, allowing a concentration gradient of glucose to be maintained across the cell sheet (see Figure 19–18). They also serve as diffusion barriers (fences) within the plasma membrane, which help confine the various transporters to their respective membrane domains (see Figure 10–34).

1	There Are Three Classes of ATP-Driven Pumps ATP-driven pumps are often called transport ATPases because they hydrolyze ATP to ADP and phosphate and use the energy released to pump ions or other solutes across a membrane. There are three principal classes of ATP-driven pumps (Figure 11–12), and representatives of each are found in all prokaryotic and eukaryotic cells. 1. P-type pumps are structurally and functionally related multipass trans-membrane proteins. They are called “P-type” because they phosphorylate themselves during the pumping cycle. This class includes many of the ion pumps that are responsible for setting up and maintaining gradients of Na+, K+, H+, and Ca2+ across cell membranes. 2. ABC transporters (ATP-Binding Cassette transporters) differ structurally from P-type ATPases and primarily pump small molecules across cell membranes. 3.

1	2. ABC transporters (ATP-Binding Cassette transporters) differ structurally from P-type ATPases and primarily pump small molecules across cell membranes. 3. V-type pumps are turbine-like protein machines, constructed from multiple different subunits. The V-type proton pump transfers H+ into organelles such as lysosomes, synaptic vesicles, and plant or yeast vacuoles (V = vacuolar), to acidify the interior of these organelles (see Figure 13–37).

1	Structurally related to the V-type pumps is a distinct family of F-type ATPases, more commonly called ATP synthases because they normally work in reverse: instead of using ATP hydrolysis to drive H+ transport, they use the H+ gradient across the membrane to drive the synthesis of ATP from ADP and phosphate (see Figure 14–30). ATP synthases are found in the plasma membrane of bacteria, the inner membrane of mitochondria, and the thylakoid membrane of chloroplasts. The H+ gradient is generated either during the electron-transport steps of oxidative phosphorylation (in aerobic bacteria and mitochondria), during photosynthesis (in chloroplasts), or by the light-driven H+ pump (bacteriorhodopsin) in Halobacterium. We discuss some of these proteins in detail in Chapter 14. For the remainder of this section, we focus on P-type pumps and ABC transporters. A P-type ATPase Pumps Ca2+ into the Sarcoplasmic Reticulum in Muscle Cells

1	For the remainder of this section, we focus on P-type pumps and ABC transporters. A P-type ATPase Pumps Ca2+ into the Sarcoplasmic Reticulum in Muscle Cells Eukaryotic cells maintain very low concentrations of free Ca2+ in their cytosol (~10–7 M) in the face of a very much higher extracellular Ca2+ concentration (~10–3 M). Therefore, even a small influx of Ca2+ significantly increases the concentration of free Ca2+ in the cytosol, and the flow of Ca2+ down its steep concentration gradient in response to extracellular signals is one means of transmitting these signals rapidly across the plasma membrane (discussed in Chapter 15). It is thus

1	Figure 11–12 Three types of aTP-driven pumps. Like any enzyme, all ATP-driven pumps can work in either direction, depending on the electrochemical gradients of their solutes and the ATP/ADP ratio. When the ATP/ADP ratio is high, they hydrolyze ATP; when the ATP/ADP ratio is low, they can synthesize ATP. The F-type ATPase in mitochondria normally works in this “reverse” mode to make most of the cell’s ATP. important that the cell maintains a steep Ca2+ gradient across its plasma membrane. Ca2+ transporters that actively pump Ca2+ out of the cell help maintain the gradient. One of these is a P-type Ca2+ ATPase; the other is an antiporter (called a Na+–Ca2+ exchanger) that is driven by the Na+ electrochemical gradient (discussed in Chapter 15).

1	The Ca2+ pump, or Ca2+ ATPase, in the sarcoplasmic reticulum (SR) membrane of skeletal muscle cells is a well-understood P-type transport ATPase. The SR is a specialized type of endoplasmic reticulum that forms a network of tubular sacs in the muscle cell cytoplasm, and it serves as an intracellular store of Ca2+. When an action potential depolarizes the muscle cell plasma membrane, Ca2+ is released into the cytosol from the SR through Ca2+-release channels, stimulating the muscle to contract (discussed in Chapters 15 and 16). The Ca2+ pump, which accounts for about 90% of the membrane protein of the SR, moves Ca2+ from the cytosol back into the SR. The endoplasmic reticulum of nonmuscle cells contains a similar Ca2+ pump, but in smaller quantities.

1	Enzymatic studies and analyses of the three-dimensional structures of transport intermediates of the SR Ca2+ pump and related pumps have revealed the molecular mechanism of P-type transport ATPases in great detail. They all have similar structures, containing 10 transmembrane α helices connected to three cytosolic domains (Figure 11–13). In the Ca2+ pump, amino acid side chains protruding from the transmembrane helices form two centrally positioned binding sites for Ca2+. As shown in Figure 11–14, in the pump’s ATP-bound nonphosphorylated state, these binding sites are accessible only from the cytosolic side of the SR membrane. Ca2+ binding triggers a series of conformational changes that close the passageway to the cytosol and activate a phosphotransfer reaction in which the terminal phosphate of the ATP is transferred to an aspartate that is highly conserved among all P-type ATPases. The ADP then dissociates and is replaced with a fresh ATP, causing another conformational change

1	phosphate of the ATP is transferred to an aspartate that is highly conserved among all P-type ATPases. The ADP then dissociates and is replaced with a fresh ATP, causing another conformational change that opens a passageway to the SR lumen through which the two Ca2+ ions exit. They are replaced by two H+ ions and a water molecule that stabilize the empty Ca2+-binding sites and close the passageway to the SR lumen. Hydrolysis of the labile phosphoryl-aspartate bond returns the pump to the initial conformation, and the cycle starts again. The transient self-phosphorylation of the pump during its cycle is an essential characteristic of all P-type pumps.

1	The Plasma Membrane Na+-K+ Pump Establishes Na+ and K+ Gradients Across the Plasma Membrane The concentration of K+ is typically 10–30 times higher inside cells than outside, whereas the reverse is true of Na+ (see Table 11–1, p. 598). A Na+-K+ pump, or Na+K+ ATPase, found in the plasma membrane of virtually all animal cells maintains

1	Figure 11–13 The structure of the sarcoplasmic reticulum Ca2+ pump. The ribbon model (left), derived from x-ray crystallographic analyses, shows the pump in its phosphorylated, ATP-bound state. The three globular cytosolic domains of the pump—the nucleotide-binding domain (dark green), the activator domain (blue), and the phosphorylation domain (red), also shown schematically on the right—change conformation dramatically during the pumping cycle. These changes in turn alter the arrangement of the transmembrane helices, which allows the Ca2+ to be released from its binding cavity into the SR lumen (Movie 11.3). (PDB code: 3B9B.) these concentration differences. Like the Ca2+ pump, the Na+-K+ pump belongs to the family of P-type ATPases and operates as an ATP-driven antiporter, actively pumping Na+ out of the cell against its steep electrochemical gradient and pumping K+ in (Figure 11–15).

1	We mentioned earlier that the Na+ gradient produced by the Na+-K+ pump drives the transport of most nutrients into animal cells and also has a crucial role in regulating cytosolic pH. A typical animal cell devotes almost one-third of its energy to fueling this pump, and the pump consumes even more energy in nerve cells and in cells that are dedicated to transport processes, such as those forming kidney tubules. Since the Na+-K+ pump drives three positively charged ions out of the cell for every two it pumps in, it is electrogenic: it drives a net electric current across the membrane, tending to create an electrical potential, with the cell’s inside being negative relative to the outside. This electrogenic effect of the pump, however, seldom directly contributes more than 10% to the membrane potential. The remaining 90%, as we discuss later, depends only indirectly on the Na+-K+ pump. Figure 11–14 The pumping cycle of the sarcoplasmic reticulum Ca2+ pump.

1	Figure 11–14 The pumping cycle of the sarcoplasmic reticulum Ca2+ pump. Ion pumping proceeds by a series of stepwise conformational changes in which movements of the pump’s three cytosolic domains [the nucleotide-binding domain (N), the phosphorylation domain (P), and the activator domain (A)] are mechanically coupled to movements of the transmembrane α helices. Helix movement opens and closes passageways through which Ca2+ enters from the cytosol and binds to the two centrally located Ca2+ binding sites. The two Ca2+ then exit into the SR lumen and are replaced by two H+, which are transported in the opposite direction. The Ca2+-dependent phosphorylation and H+-dependent dephosphorylation of aspartic acid are universally conserved steps in the reaction cycle of all P-type pumps: they cause the conformational transitions to occur in an orderly manner, enabling the proteins to do useful work. (Adapted from C. Toyoshima et al., Nature 432:361–368, 2004 and

1	J.V. Møller et al., Q. Rev. Biophys. 43:501– 566, 2010.) Figure 11–15 The function of the Na+-K+ pump. This P-type ATPase actively pumps Na+ out of and K+ into a cell against their electrochemical gradients. It is structurally closely related to the Ca2+ ATPase but differs in its selectivity for ions: for every molecule of ATP hydrolyzed by the pump, three Na+ are pumped out and two K+ are pumped in. As in the Ca2+ pump, an aspartate is phosphorylated and dephosphorylated during the pumping cycle (Movie 11.4). ABC Transporters Constitute the Largest Family of Membrane Transport Proteins

1	The last type of transport ATPase that we discuss is the family of the ABC transporters, so named because each member contains two highly conserved ATPase domains, or ATP-Binding “Cassettes,” on the cytosolic side of the membrane. ATP binding brings together the two ATPase domains, and ATP hydrolysis leads to their dissociation (Figure 11–16). These movements of the cytosolic domains are transmitted to the transmembrane segments, driving cycles of conformational changes that alternately expose solute-binding sites on one side of the membrane and then on the other, as we have seen for other transporters. In this way, ABC transporters harvest the energy released upon ATP binding and hydrolysis to drive transport of solutes across the bilayer. The transport is directional toward inside or toward outside, depending on the particular conformational change in the solute binding site that is linked to ATP hydrolysis (see Figure 11–16).

1	ABC transporters constitute the largest family of membrane transport proteins and are of great clinical importance. The first of these proteins to be characterized was found in bacteria. We have already mentioned that the plasma membranes of all bacteria contain transporters that use the H+ gradient across the membrane to actively transport a variety of nutrients into the cell. In addition, bacteria use ABC transporters to import certain small molecules. In bacteria such as E. coli that have double membranes (Figure 11–17), the ABC transporters are located in the inner membrane, and an auxiliary mechanism operates to capture the nutrients and deliver them to the transporters (Figure 11–18).

1	In E. coli, 78 genes (an amazing 5% of the bacterium’s genes) encode ABC transporters, and animal genomes encode an even larger number. Although each transporter is thought to be specific for a particular molecule or class of molecules, the variety of substrates transported by this superfamily is great and includes inorganic ions, amino acids, monoand polysaccharides, peptides, lipids, drugs, and, in some cases, even proteins that can be larger than the transporter itself.

1	Figure 11–16 Small-molecule transport by typical abC transporters. ABC transporters consist of multiple domains. Typically, two hydrophobic domains, each built of six membrane-spanning α helices, together form the translocation pathway and provide substrate specificity. Two ATPase domains protrude into the cytosol. In some cases, the two halves of the transporter are formed by a single polypeptide, whereas in other cases they are formed by two or more separate polypeptides that assemble into a similar structure. Without ATP bound, the transporter exposes a substrate-binding site on one side of the membrane. ATP binding induces a conformational change that exposes the substrate-binding site on the opposite side; ATP hydrolysis followed by ADP dissociation returns the transporter to its original conformation. Most individual ABC transporters are unidirectional. (A) Both importing and exporting ABC transporters are found in bacteria; an ABC importer is shown in this cartoon. The crystal

1	conformation. Most individual ABC transporters are unidirectional. (A) Both importing and exporting ABC transporters are found in bacteria; an ABC importer is shown in this cartoon. The crystal structure of a bacterial ABC transporter is shown in Figure 3–76.

1	(B) In eukaryotes, most ABC transporters export substances—either from the cytosol to the extracellular space or from the cytosol to a membrane-bound intracellular compartment such as the endoplasmic reticulum—or from the mitochondrial matrix to the cytosol.

1	The first eukaryotic ABC transporters identified were discovered because of their ability to pump hydrophobic drugs out of the cytosol. One of these transporters is the multidrug resistance (MDR) protein, also called P-glycoprotein. It is present at elevated levels in many human cancer cells and makes the cells simultaneously resistant to a variety of chemically unrelated cytotoxic drugs that are widely used in cancer chemotherapy. Treatment with any one of these drugs can result in the selective survival and overgrowth of those cancer cells that express an especially large amount of the MDR transporter. These cells pump drugs out of the cell very efficiently and are therefore relatively resistant to the drugs’ toxic effects (Movie 11.5). Selection for cancer cells with resistance to one drug can thereby lead to resistance to a wide variety of anticancer drugs. Some studies indicate that up to 40% of human cancers develop multidrug resistance, making it a major hurdle in the battle

1	drug can thereby lead to resistance to a wide variety of anticancer drugs. Some studies indicate that up to 40% of human cancers develop multidrug resistance, making it a major hurdle in the battle against cancer.

1	A related and equally sinister phenomenon occurs in the protist Plasmodium falciparum, which causes malaria. More than 200 million people are infected worldwide with this parasite, which remains a major cause of human death, killing almost a million people every year. The development of resistance to the antimalarial drug chloroquine has hampered the control of malaria. The resistant P. falciparum have amplified a gene encoding an ABC transporter that pumps out the chloroquine.

1	Figure 11–17 a small section of the double membrane of an E. coli bacterium. The inner membrane is the cell’s plasma membrane. Between the inner and outer membranes is a highly porous, rigid peptidoglycan layer, composed of protein and polysaccharide that constitute the bacterial cell wall. It is attached to lipoprotein molecules in the outer membrane and fills the periplasmic space (only a little of the peptidoglycan layer is shown). This space also contains a variety of soluble protein molecules. The dashed threads (shown in green) at the top represent the polysaccharide chains of the special lipopolysaccharide molecules that form the external monolayer of the outer membrane; for clarity, only a few of these chains are shown. Bacteria with double membranes are called Gram-negative because they do not retain the dark blue dye used in Gram staining. Bacteria with single membranes (but thicker peptidoglycan cell walls), such as staphylococci and streptococci, retain the blue dye and

1	they do not retain the dark blue dye used in Gram staining. Bacteria with single membranes (but thicker peptidoglycan cell walls), such as staphylococci and streptococci, retain the blue dye and are therefore called Gram-positive; their single membrane is analogous to the inner (plasma) membrane of Gram-negative bacteria.

1	Figure 11–18 The auxiliary transport system associated with transport aTPases in bacteria with double membranes. The solute diffuses through channel proteins (porins) in the outer membrane and binds to a periplasmic substrate-binding protein that delivers it to the ABC transporter, which pumps it across the plasma membrane. The peptidoglycan is omitted for simplicity; its porous structure allows the substrate-binding proteins and water-soluble solutes to move through it by diffusion.

1	In most vertebrate cells, an ABC transporter in the endoplasmic reticulum (ER) membrane (named transporter associated with antigen processing, or TAP transporter) actively pumps a wide variety of peptides from the cytosol into the ER lumen. These peptides are produced by protein degradation in proteasomes (discussed in Chapter 6). They are carried from the ER to the cell surface, where they are displayed for scrutiny by cytotoxic T lymphocytes, which kill the cell if the peptides are derived from a virus or other microorganism lurking in the cytosol of an infected cell (discussed in Chapter 24).

1	Yet another member of the ABC transporter family is the cystic fibrosis trans-membrane conductance regulator protein (CFTR), which was discovered through studies of the common genetic disease cystic fibrosis. This disease is caused by a mutation in the gene encoding CFTR, a Cl– transport protein in the plasma membrane of epithelial cells. CFTR regulates ion concentrations in the extracellular fluid, especially in the lung. One in 27 Caucasians carries a gene encoding a mutant form of this protein; in 1 in 2900, both copies of the gene are mutated, causing the disease. In contrast to other ABC transporters, ATP binding and hydrolysis in the CFTR protein do not drive the transport process. Instead, they control the opening and closing of a continuous channel, which provides a passive conduit for Cl– to move down its electrochemical gradient. Thus, some ABC proteins can function as transporters and others as gated channels.

1	Transporters bind specific solutes and transfer them across the lipid bilayer by undergoing conformational changes that alternately expose the solute-binding site on one side of the membrane and then on the other. Some transporters move a single solute “downhill,” whereas others can act as pumps to move a solute “uphill” against its electrochemical gradient, using energy provided by ATP hydrolysis, by a downhill flow of another solute (such as Na+ or H+), or by light to drive the requisite series of conformational changes in an orderly manner. Transporters belong to a small number of protein families. Each family evolved from a common ancestral protein, and its members all operate by a similar mechanism. The family of P-type transport ATPases, which includes Ca2+ and Na+-K+ pumps, is an important example; each of these ATPases sequentially phosphorylates and dephosphorylates itself during the pumping cycle. The superfamily of ABC transporters is the largest family of membrane

1	is an important example; each of these ATPases sequentially phosphorylates and dephosphorylates itself during the pumping cycle. The superfamily of ABC transporters is the largest family of membrane transport proteins and is especially important clinically. It includes proteins that are responsible for cystic fibrosis, for drug resistance in both cancer cells and malaria-causing parasites, and for pumping pathogen-derived peptides into the ER for cytotoxic lymphocytes to reorganize on the surface of infected cells.

1	Unlike transporters, channels form pores across membranes. One class of channel proteins found in virtually all animals forms gap junctions between adjacent cells; each plasma membrane contributes equally to the formation of the channel, which connects the cytoplasm of the two cells. These channels are discussed in Chapter 19 and will not be considered further here. Both gap junctions and porins, the channels in the outer membranes of bacteria, mitochondria, and chloroplasts (discussed in Chapter 10), have relatively large and permissive pores, and it would be disastrous if they directly connected the inside of a cell to an extracellular space. Indeed, many bacterial toxins do exactly that to kill other cells (discussed in Chapter 24).

1	In contrast, most channels in the plasma membrane of animal and plant cells that connect the cytosol to the cell exterior necessarily have narrow, highly selective pores that can open and close rapidly. Because these proteins are concerned specifically with inorganic ion transport, they are referred to as ion channels. For transport efficiency, ion channels have an advantage over transporters, in that they can pass up to 100 million ions through one open channel each second—a rate 105 times greater than the fastest rate of transport mediated by any known transporter. As discussed earlier, however, channels cannot be coupled to an energy source to perform active transport, so the transport they mediate is always passive (downhill). Thus, the function of ion channels is to allow specific inorganic ions—primarily Na+, K+, Ca2+, or Cl–—to diffuse rapidly down their electrochemical gradients across the lipid bilayer. In this section, we will see that the ability to control ion fluxes

1	inorganic ions—primarily Na+, K+, Ca2+, or Cl–—to diffuse rapidly down their electrochemical gradients across the lipid bilayer. In this section, we will see that the ability to control ion fluxes through these channels is essential for many cell functions. Nerve cells (neurons), in particular, have made a specialty of using ion channels, and we will consider how they use many different ion channels to receive, conduct, and transmit signals. Before we discuss ion channels, however, we briefly consider the aquaporin water channels that we mentioned earlier.

1	Aquaporins Are Permeable to Water But Impermeable to Ions

1	Because cells are mostly water (typically ~70% by weight), water movement across cell membranes is fundamentally important for life. Cells also contain a high concentration of solutes, including numerous negatively charged organic molecules that are confined inside the cell (the so-called fixed anions) and their accompanying cations that are required for charge balance. This creates an osmotic gradient, which mostly is balanced by an opposite osmotic gradient due to a high concentration of inorganic ions—chiefly Na+ and Cl–—in the extracellular fluid. The small remaining osmotic force tends to “pull” water into the cell, causing it to swell until the forces are balanced. Because all biological membranes are moderately permeable to water (see Figure 11–2), cell volume equilibrates in minutes or less in response to an osmotic gradient. For most animal cells, however, osmosis has only a minor role in regulating cell volume. This is because most of the cytoplasm is in a gel-like state and

1	or less in response to an osmotic gradient. For most animal cells, however, osmosis has only a minor role in regulating cell volume. This is because most of the cytoplasm is in a gel-like state and resists large changes in its volume in response to changes in osmolarity.

1	In addition to the direct diffusion of water across the lipid bilayer, some prokaryotic and eukaryotic cells have water channels, or aquaporins, embedded in their plasma membrane to allow water to move more rapidly. Aquaporins are particularly abundant in animal cells that must transport water at high rates, such as the epithelial cells of the kidney or exocrine cells that must transport or secrete large volumes of fluids, respectively (Figure 11–19).

1	Aquaporins must solve a problem that is opposite to that facing ion channels. To avoid disrupting ion gradients across membranes, they have to allow the rapid passage of water molecules while completely blocking the passage of ions. The three-dimensional structure of an aquaporin reveals how it achieves this remarkable selectivity. The channels have a narrow pore that allows water molecules to traverse the membrane in single file, following the path of carbonyl oxygens that line one side of the pore (Figure 11–20A and B). Hydrophobic amino acids line the other side of the pore. The pore is too narrow for any hydrated ion to enter, and the energy cost of dehydrating an ion would be enormous because the hydrophobic wall of the pore cannot interact with a dehydrated ion to compensate for the loss of water. This design readily explains why the aquaporins cannot conduct K+,

1	Figure 11–19 The role of aquaporins in fluid secretion. Cells lining the ducts of exocrine glands (as found, for example, in the pancreas and liver, and in mammary, sweat, and salivary glands) secrete large volumes of body fluids. These cells are organized into epithelial sheets in which their apical plasma membrane faces the lumen of the duct. Ion pumps and channels situated in the basolateral and apical plasma membrane move ions (mostly Na+ and Cl–) into the ductal lumen, creating an osmotic gradient between the surrounding tissue and the duct. Water molecules rapidly follow the osmotic gradient through aquaporins that are present in high concentrations in both the apical and basolateral membranes.

1	Na+, Ca2+, or Cl– ions. These channels are also impermeable to H+, which is mainly present in cells as H3O+. These hydronium ions diffuse through water extremely rapidly, using a molecular relay mechanism that requires the making and breaking of hydrogen bonds between adjacent water molecules (Figure 11–20C). Aquaporins contain two strategically placed asparagines, which bind to the oxygen atom of the central water molecule in the line of water molecules traversing the pore, imposing a bipolarity on the entire column of water molecules (Figure 11–20C and D). This makes it impossible for the “making and breaking” sequence of hydrogen bonds (shown in Figure 11–20C) to get past the central asparagine-bonded water molecule. Because both valences of this central oxygen are unavailable for hydrogen-bonding, the central water molecule cannot participate in an H+ relay, and the pore is therefore impermeable to H+. We now turn to ion channels, the subject of the rest of the chapter.

1	We now turn to ion channels, the subject of the rest of the chapter. Two important properties distinguish ion channels from aqueous pores. First, they show ion selectivity, permitting some inorganic ions to pass, but not others. This suggests that their pores must be narrow enough in places to force permeating ions into intimate contact with the walls of the channel so that only ions of appropriate size and charge can pass. The permeating ions have to shed most or all of their associated water molecules to pass, often in single file, through the narrowest part of the channel, which is called the selectivity filter; this limits their rate of passage (Figure 11–21). Thus, as the ion concentration increases, the flux of the ion through a channel increases proportionally but then levels off (saturates) at a maximum rate.

1	Figure 11–20 The structure of aquaporins. (A) A ribbon diagram of an aquaporin monomer. In the membrane, aquaporins form tetramers, with each monomer containing an aqueous pore in its center (not shown). Each individual aquaporin channel passes about 109 water molecules per second. (B) A longitudinal cross section through one aquaporin monomer, in the plane of the central pore. One face of the pore is lined with hydrophilic amino acids, which provide transient hydrogen bonds to water molecules; these bonds help line up the transiting water molecules in a single row and orient them as they traverse the pore. (C and D) A model explaining why aquaporins are impermeable to H+.

1	(C) In water, H+ diffuses extremely rapidly by being relayed from one water molecule to the next. (D) Carbonyl groups (C=O) lining the hydrophilic face of the pore align water molecules, and two strategically placed asparagines in the center help tether a central water molecule such that both valences on its oxygen are occupied. This arrangement bipolarizes the entire line of water molecules, with each water molecule acting as a hydrogen-bond acceptor from its inner neighbor (Movie 11.6). (A and B, adapted from R.M. Stroud et al., Curr. Opin. Struct. Biol. 13:424–431, 2003. With permission from Elsevier.)

1	Figure 11–21 a typical ion channel, which fluctuates between closed and open conformations. The ion channel shown here in cross section forms a pore across the lipid bilayer only in the “open” conformational state. The pore narrows to atomic dimensions in one region (the selectivity filter), where the ion selectivity of the channel is largely determined. Another region of the channel forms the gate. Figure 11–22 The gating of ion channels. This schematic drawing shows several kinds of stimuli that open ion channels. Mechanically gated channels often have cytoplasmic extensions (not shown) that link the channel to the cytoskeleton.

1	The second important distinction between ion channels and aqueous pores is that ion channels are not continuously open. Instead, they are gated, which allows them to open briefly and then close again. Moreover, with prolonged (chemical or electrical) stimulation, most ion channels go into a closed “desensitized,” or “inactivated,” state, in which they are refractory to further opening until the stimulus has been removed, as we discuss later. In most cases, the gate opens in response to a specific stimulus. As shown in Figure 11–22, the main types of stimuli that are known to cause ion channels to open are a change in the voltage across the membrane (voltage-gated channels), a mechanical stress (mechanically gated channels), or the binding of a ligand (ligand-gated channels). The ligand can be either an extracellular mediator—specifically, a neurotransmitter (transmitter-gated channels)—or an intracellular mediator such as an ion (ion-gated channels) or a nucleotide (nucleotide-gated

1	be either an extracellular mediator—specifically, a neurotransmitter (transmitter-gated channels)—or an intracellular mediator such as an ion (ion-gated channels) or a nucleotide (nucleotide-gated channels). In addition, protein phosphorylation and dephosphorylation regulates the activity of many ion channels; this type of channel regulation is discussed, together with nucleotide-gated ion channels, in Chapter 15.

1	More than 100 types of ion channels have been identified thus far, and new ones are still being discovered, each characterized by the ions it conducts, the mechanism by which it is gated, and its abundance and localization in the cell and in specific cells. Ion channels are responsible for the electrical excitability of muscle cells, and they mediate most forms of electrical signaling in the nervous system. A single neuron typically contains 10 or more kinds of ion channels, located in different domains of its plasma membrane. But ion channels are not restricted to electrically excitable cells. They are present in all animal cells and are found in plant cells and microorganisms: they propagate the leaf-closing response of the mimosa plant, for example (Movie 11.7), and allow the single-celled Paramecium to reverse direction after a collision.

1	Ion channels that are permeable mainly to K+ are found in the plasma membrane of almost all cells. An important subset of K+ channels opens even in an unstimulated or “resting” cell, and hence these are called K+ leak channels. Although this term applies to many different K+ channels, depending on the cell type, they serve a common purpose: by making the plasma membrane much more permeable to K+ than to other ions, they have a crucial role in maintaining the membrane potential across all plasma membranes, as we discuss next. The Membrane Potential in Animal Cells Depends Mainly on K+ Leak Channels and the K+ Gradient Across the Plasma Membrane

1	The Membrane Potential in Animal Cells Depends Mainly on K+ Leak Channels and the K+ Gradient Across the Plasma Membrane A membrane potential arises when there is a difference in the electrical charge on the two sides of a membrane, due to a slight excess of positive ions over negative ones on one side and a slight deficit on the other. Such charge differences can result both from active electrogenic pumping (see p. 608) and from passive ion diffusion. As we discuss in Chapter 14, electrogenic H+ pumps in the mitochondrial inner membrane generate most of the membrane potential across this membrane. Electrogenic pumps also generate most of the electrical potential across the plasma membrane in plants and fungi. In typical animal cells, however, passive ion movements make the largest contribution to the electrical potential across the plasma membrane.

1	As explained earlier, due to the action of the Na+-K+ pump, there is little Na+ inside the cell, and other intracellular inorganic cations have to be plentiful enough to balance the charge carried by the cell’s fixed anions—the negatively charged organic molecules that are confined inside the cell. This balancing role is performed largely by K+, which is actively pumped into the cell by the Na+K+ pump and can also move freely in or out through the K+ leak channels in the plasma membrane. Because of the presence of these channels, K+ comes almost to equilibrium, where an electrical force exerted by an excess of negative charges attracting K+ into the cell balances the tendency of K+ to leak out down its concentration gradient. The membrane potential (of the plasma membrane) is the manifestation of this electrical force, and we can calculate its equilibrium value from the steepness of the K+ concentration gradient. The following argument may help to make this clear.

1	Suppose that initially there is no voltage gradient across the plasma membrane (the membrane potential is zero) but the concentration of K+ is high inside the cell and low outside. K+ will tend to leave the cell through the K+ leak channels, driven by its concentration gradient. As K+ begins to move out, each ion leaves behind an unbalanced negative charge, thereby creating an electrical field, or membrane potential, which will tend to oppose the further efflux of K+. The net efflux of K+ halts when the membrane potential reaches a value at which this electrical driving force on K+ exactly balances the effect of its concentration gradient—that is, when the electrochemical gradient for K+ is zero. Although Cl– ions also equilibrate across the membrane, the membrane potential keeps most of these ions out of the cell because their charge is negative.

1	The equilibrium condition, in which there is no net flow of ions across the plasma membrane, defines the resting membrane potential for this idealized cell. A simple but very important formula, the Nernst equation, quantifies the equilibrium condition and, as explained in Panel 11–1, makes it possible to calculate the theoretical resting membrane potential if we know the ratio of internal and external ion concentrations. As the plasma membrane of a real cell is not exclusively permeable to K+ and Cl–, however, the actual resting membrane potential is usually not exactly equal to that predicted by the Nernst equation for K+ or Cl–. The Resting Potential Decays Only Slowly When the Na+-K+ Pump Is Stopped

1	The Resting Potential Decays Only Slowly When the Na+-K+ Pump Is Stopped Movement of only a minute number of inorganic ions across the plasma membrane through ion channels suffices to set up the membrane potential. Thus, we can think of the membrane potential as arising from movements of charge that leave ion concentrations practically unaffected and result in only a very slight discrepancy in the number of positive and negative ions on the two sides of the membrane (Figure 11–23). Moreover, these movements of charge are generally rapid, taking only a few milliseconds or less. Consider the change in the membrane potential in a real cell after the sudden inactivation of the Na+-K+ pump. A slight drop in the membrane potential occurs immediately. This is because the pump is electrogenic and, when active, makes a

1	The fow of any inorganic ion through a membrane channel is driven by the electrochemical gradient for that ion. This gradient represents the combination of two infuences: the voltage gradient and the concentration gradient of the ion across the membrane. When these two infuences just balance each other, the electrochemical gradient for the ion is zero, and there is no net fow of the ion through the channel. The voltage gradient (membrane potential) at which this equilibrium is reached is called the equilibrium potential for the ion. It can be calculated from an equation that will be derived below, called the Nernst equation. The Nernst equation is V = RT In Co zF Ci V = the equilibrium potential in volts (internal Co and Ci = outside and inside concentrations of the ion, respectively R = the gas constant (8.3 J mol–1 K–1) T = the absolute temperature (K) F = Faraday’s constant (9.6 × 104 J V–1 mol–1) z = the valence (charge) of the ion In = logarithm to the base e

1	R = the gas constant (8.3 J mol–1 K–1) T = the absolute temperature (K) F = Faraday’s constant (9.6 × 104 J V–1 mol–1) z = the valence (charge) of the ion In = logarithm to the base e The Nernst equation is derived as follows: A molecule in solution (a solute) tends to move from a region of high concentration to a region of low concentration simply due to the random movement of molecules, which results in their equilibrium. Consequently, movement down a concentration gradient is accompanied by a favorable free-energy change (°G < 0), whereas movement up a concentration gradient is accompanied by an unfavorable free-energy change (°G > 0). (Free energy is introduced in Chapter 2 and discussed in the context of redox reactions in Panel 14–1, p. 765.) The free-energy change per mole of solute moved across the plasma membrane (°Gconc) is equal to –RT In Co / Ci.

1	The free-energy change per mole of solute moved across the plasma membrane (°Gconc) is equal to –RT In Co / Ci. If the solute is an ion, moving it into a cell across a membrane whose inside is at a voltage V relative to the outside will cause an additional free-energy change (per mole of solute moved) of °Gvolt = zFV. At the point where the concentration and voltage gradients just balance, and the ion distribution is at equilibrium across the membrane. Thus, and, therefore, or, using the constant that converts natural logarithms to base 10, V = 2.3 log10 For a univalent cation, 2.3 = 58 mV at 20oC and 61.5 mV at 37oC. Thus, for such an ion at 37ºC, V = + 61.5 mV for Co/ Ci = 10, whereas V = 0 for Co / Ci= 1. The K+ equilibrium potential (VK), for example, is 61.5 log10([K+]o / [K+]i) millivolts (–89 mV for a typical cell, where [K+]o = 5 mM and [K+]i = 140 mM). At VK, there is no net fow of K+ across the membrane.

1	At VK, there is no net fow of K+ across the membrane. Similarly, when the membrane potential has a value of 61.5 log10([Na+]o /[Na+]i), the Na+ equilibrium potential (VNa), there is no net fow of Na+. For any particular membrane potential, VM, the net force tending to drive a particular type of ion out of the cell, is proportional to the difference between VM and the equilibrium potential for the ion: hence, for K+ it is VM – VK and for Na+ it is VM – VNa.

1	When there is a voltage gradient across the membrane, the ions responsible for it—the positive ions on one side and the negative ions on the other—are concentrated in thin layers on either side of the membrane because of the attraction between positive and negative electric charges. The number of ions that go to form the layer of charge adjacent to the membrane is minute compared with the total number inside the cell. For example, the movement of 6000 Na+ ions across 1 µm2 of membrane will carry suffcient charge to shift the membrane potential by about 100 mV. Because there are about 3 × 107 Na+ ions in a typical cell (1 µm3 of bulk cytoplasm), such a movement of charge will generally have a negligible effect on the ion concentration gradients across the membrane.

1	exact balance of charges on each side of the a few of the positive ions (red) cross the membrane; membrane potential = 0 membrane from right to left, leaving their small direct contribution to the membrane potential by pumping out three Na+ for every two K+ that it pumps in (see Figure 11–15). However, switching off the pump does not abolish the major component of the resting potential, which is generated by the K+ equilibrium mechanism just described. This component of the membrane potential persists as long as the Na+ concentration inside the cell stays low and the K+ ion concentration high—typically for many minutes. But the plasma membrane is somewhat permeable to all small ions, including Na+. Therefore, without the Na+-K+ pump, the ion gradients set up by the pump will eventually run down, and the membrane potential established by diffusion through the K+ leak channels will fall as well. As Na+ enters, the cell eventually comes to a new resting state where Na+, K+, and Cl– are

1	down, and the membrane potential established by diffusion through the K+ leak channels will fall as well. As Na+ enters, the cell eventually comes to a new resting state where Na+, K+, and Cl– are all at equilibrium across the membrane. The membrane potential in this state is much less than it was in the normal cell with an active Na+-K+ pump.

1	The resting potential of an animal cell varies between –20 mV and –120 mV, depending on the organism and cell type. Although the K+ gradient always has a major influence on this potential, the gradients of other ions (and the disequilibrating effects of ion pumps) also have a significant effect: the more permeable the membrane for a given ion, the more strongly the membrane potential tends to be driven toward the equilibrium value for that ion. Consequently, changes in a membrane’s permeability to ions can cause significant changes in the membrane potential. This is one of the key principles relating the electrical excitability of cells to the activities of ion channels. To understand how ion channels select their ions and how they open and close, we need to know their atomic structure. The first ion channel to be crystallized and studied by x-ray diffraction was a bacterial K+ channel. The details of its structure revolutionized our understanding of ion channels.

1	The Three-Dimensional Structure of a Bacterial K+ Channel Shows How an Ion Channel Can Work Scientists were puzzled by the remarkable ability of ion channels to combine exquisite ion selectivity with a high conductance. K+ leak channels, for example, conduct K+ 10,000-fold faster than Na+, yet the two ions are both featureless spheres and have similar diameters (0.133 nm and 0.095 nm, respectively). A single amino acid substitution in the pore of an animal cell K+ channel can result in a loss of ion selectivity and cell death. We cannot explain the normal K+ selectivity by pore size, because Na+ is smaller than K+. Moreover, the high conductance rate is incompatible with the channel’s having selective, high-affinity K+-binding sites, as the binding of K+ ions to such sites would greatly slow their passage.

1	The puzzle was solved when the structure of a bacterial K+ channel was determined by x-ray crystallography. The channel is made from four identical trans-membrane subunits, which together form a central pore through the membrane. Each subunit contributes two transmembrane α helices, which are tilted outward in the membrane and together form a cone, with its wide end facing the outside of

1	Figure 11–23 The ionic basis of a membrane potential. A small flow of inorganic ions through an ion channel carries sufficient charge to cause a large change in the membrane potential. The ions that give rise to the membrane potential lie in a thin (< 1 nm) surface layer close to the membrane, held there by their electrical attraction to their oppositely charged counterparts (counterions) on the other side of the membrane. For a typical cell, 1 microcoulomb of charge (6 × 1012 monovalent ions) per square centimeter of membrane, transferred from one side of the membrane to the other, changes the membrane potential by roughly 1 V. This means, for example, that in a spherical cell of diameter 10 μm, the number of K+ ions that have to flow out to alter the membrane potential by 100 mV is only about 1/100,000 of the total number of K+ ions in the cytosol. This amount is so minute that the intracellular K+ concentration remains virtually unchanged.

1	618 Chapter 11: Membrane Transport of Small Molecules and the Electrical Properties of Membranes

1	Figure 11–24 The structure of a bacterial K+ channel. (A) The transmembrane α helices from only two of the four identical subunits are shown. From the cytosolic side, the pore (schematically shaded in blue) opens up into a vestibule in the middle of the membrane. The pore vestibule facilitates transport by allowing the K+ ions to remain hydrated even though they are more than halfway across the membrane. The narrow selectivity filter of the pore links the vestibule to the outside of the cell. Carbonyl oxygens line the walls of the selectivity filter and form transient binding sites for dehydrated K+ ions. Two K+ ions occupy different sites in the selectivity filter, while a third K+ ion is located in the center of the vestibule, where it is stabilized by electrical interactions with the more negatively charged ends of the pore helices. The ends of the four short “pore helices” (only two of which are shown) point precisely toward the center of the vestibule, thereby guiding K+ ions

1	more negatively charged ends of the pore helices. The ends of the four short “pore helices” (only two of which are shown) point precisely toward the center of the vestibule, thereby guiding K+ ions into the selectivity filter (Movie 11.8). (B) Peptide bonds have an electric dipole, with more negative charge accumulated at the oxygen of the C=O bond and at the nitrogen of the N–H bond. In an α helix, hydrogen bonds (red) align the dipoles. As a consequence, every α helix has an electric dipole along its axis, resulting from summation of the dipoles of the individual peptide bonds, with a more negatively charged C-terminal end (δ–) and a more positively charged N-terminal end (δ+). (A, adapted from D.A. Doyle et al., Science 280:69–77, 1998.) the cell where K+ ions exit from the channel (Figure 11–24). The polypeptide chain that connects the two transmembrane helices forms a short αhelix (the pore helix) and a crucial loop that protrudes into the wide section of the cone to form the

1	(Figure 11–24). The polypeptide chain that connects the two transmembrane helices forms a short αhelix (the pore helix) and a crucial loop that protrudes into the wide section of the cone to form the selectivity filter. The selectivity loops from the four subunits form a short, rigid, narrow pore, which is lined by the carbonyl oxygen atoms of their polypeptide backbones. Because the selectivity loops of all known K+ channels have similar amino acid sequences, it is likely that they form a closely similar structure.

1	The structure of the selectivity filter explains the ion selectivity of the channel. A K+ ion must lose almost all of its bound water molecules to enter the filter, where it interacts instead with the carbonyl oxygens lining the filter; the oxygens are rigidly spaced at the exact distance to accommodate a K+ ion. A Na+ ion, in contrast, cannot enter the filter because the carbonyl oxygens are too far away from the smaller Na+ ion to compensate for the energy expense associated with the loss of water molecules required for entry (Figure 11–25).

1	Structural studies of K+ channels and other ion channels have also indicated some general principles of how these channels open and close. The gating involves movement of the helices in the membrane so that they either obstruct or open the path for ion movement. Depending on the particular type of channel, helices tilt, rotate, or bend during gating. The structure of a closed K+ channel shows that by tilting the inner helices, the pore constricts like a diaphragm at its cytosolic end (Figure 11–26). Bulky hydrophobic amino acid side chains block the small opening that remains, preventing the entry of ions. Many other ion channels operate on similar principles: the channel’s gating helices are allosterically coupled to domains that form the ion-conducting pathway; and a conformational change in the gate—in response, say, to ligand binding or altered membrane potential—brings about conformational change in the conducting pathway, either opening it or blocking it off.

1	All organisms, from single-cell bacteria to multicellular animals and plants, must sense and respond to mechanical forces in their external environment (such as sound, touch, pressure, shear forces, and gravity) and in their internal environment (such as osmotic pressure and membrane bending). Numerous proteins are known to be capable of responding to such mechanical forces, and a large subset of those proteins has been identified as possible mechanosensitive channels, but very few of the candidate proteins have been shown directly to be mechanically activated ion channels. One reason for this dearth in our knowledge is that most such channels are extremely rare. Auditory hair cells in the human cochlea, for example, contain extraordinarily sensitive mechanically gated ion channels, but each of the approximately 15,000 individual hair cells is thought to have a total of only 50–100 of them (Movie 11.9). Additional difficulties arise because the gating mechanisms of many

1	but each of the approximately 15,000 individual hair cells is thought to have a total of only 50–100 of them (Movie 11.9). Additional difficulties arise because the gating mechanisms of many mechanosensitive channel types require the channels to be embedded in complex architectures that require attachment to the extracellular matrix or to the cytoskeleton and are difficult to reconstitute in the test tube. The study of mechanosensitive receptors is a field of active investigation.

1	A well-studied class of mechanosensitive channels is found in the bacterial plasma membrane. These channels open in response to mechanical stretching of the lipid bilayer in which they are embedded. When a bacterium experiences a low-ionic-strength external environment (hypotonic conditions), such as

1	Figure 11–25 K+ specificity of the selectivity filter in a K+ channel. The drawings show K+ and Na+ ions (A) in the vestibule and (B) in the selectivity filter of the pore, viewed in cross section. In the vestibule, the ions are hydrated. In the selectivity filter, they have lost their water, and the carbonyl oxygens are placed to accommodate a dehydrated K+ ion. The dehydration of the K+ ion requires energy, which is precisely balanced by the energy regained by the interaction of the ion with all of the carbonyl oxygens that serve as surrogate water molecules. Because the Na+ ion is too small to interact with the oxygens, it can enter the selectivity filter only at a great energetic expense. The filter therefore selects K+ ions with high specificity. (A, adapted from Y. Zhou et al., Nature 414:43–48, 2001. With permission from Macmillan Publishers Ltd.)

1	Figure 11–26 a model for the gating of a bacterial K+ channel. The channel is viewed in cross section. To adopt the closed conformation, the four inner transmembrane helices that line the pore on the cytosolic side of the selectivity filter (see Figure 11–24) rearrange to close the cytosolic entrance to the channel. (Adapted from E. Perozo et al., Science 285:73–78, 1999.) (A) CLOSED (B) OPEN Figure 11–27 The structure of mechanosensitive channels. The crystal structures of MscS in its (A) closed and (B) open conformation are shown. The side views (lower panels) show the entire protein, including the large intracellular domain. The face views (upper panels) show the transmembrane domains only. The open structure occupies more area in the lipid bilayer and is energetically favored when a membrane is stretched. This may explain why the MscS channel opens as pressure builds up inside the cell. (PDB codes: 2OAU, 2VV5.) rainwater, the cell swells as water seeps in due to an increase in the

1	is stretched. This may explain why the MscS channel opens as pressure builds up inside the cell. (PDB codes: 2OAU, 2VV5.) rainwater, the cell swells as water seeps in due to an increase in the osmotic pressure. If the pressure rises to dangerous levels, the cell opens mechanosensitive channels that allow small molecules to leak out. Bacteria that are experimentally placed in fresh water can rapidly lose more than 95% of their small molecules in this manner, including amino acids, sugars, and potassium ions. However, they keep their macromolecules safely inside and thus can recover quickly after environmental conditions return to normal.

1	Mechanical gating has been demonstrated using biophysical techniques in which force is exerted on pure lipid bilayers containing the bacterial mechanosensitive channels; for example, by applying suction with a micropipette. Such measurements demonstrate that the cell has several different channels that open at different levels of pressure. The mechanosensitive channel of small conductance, called the MscS channel, opens at low and moderate pressures (Figure 11–27). It is composed of seven identical subunits, which in the open state form a pore about 1.3 nm in diameter—just big enough to pass ions and small molecules. Large cytoplasmic domains limit the size of molecules that can reach the pore. The mechanosensitive channel of large conductance, called the MscL channel, opens to over 3 nm in diameter when the pressure gets so high that the cell might burst. The Function of a Neuron Depends on Its Elongated Structure

1	The Function of a Neuron Depends on Its Elongated Structure The cells that make most sophisticated use of channels are neurons. Before discussing how they do so, we digress briefly to describe how a typical neuron is organized. The fundamental task of a neuron, or nerve cell, is to receive, conduct, and transmit signals. To perform these functions, neurons are often extremely elongated. In humans, for example, a single neuron extending from the spinal cord to a muscle in the foot may be as long as 1 meter. Every neuron consists of a cell body (containing the nucleus) with a number of thin processes radiating outward from it. Usually one long axon conducts signals away from the cell body toward distant

1	Figure 11–28 a typical vertebrate neuron. The arrows indicate the direction in which signals are conveyed. The single axon conducts signals away from the cell body, while the multiple dendrites (and the cell body) receive signals from the axons of other neurons. The axon terminals end on the dendrites or cell body of other neurons or on other cell types, such as muscle or gland cells. targets, and several shorter, branching dendrites extend from the cell body like antennae, providing an enlarged surface area to receive signals from the axons of other neurons (Figure 11–28), although the cell body itself also receives such signals. A typical axon divides at its far end into many branches, passing on its message to many target cells simultaneously. Likewise, the extent of branching of the dendrites can be very great—in some cases sufficient to receive as many as 100,000 inputs on a single neuron.

1	Despite the varied significance of the signals carried by different classes of neurons, the form of the signal is always the same, consisting of changes in the electrical potential across the neuron’s plasma membrane. The signal spreads because an electrical disturbance produced in one part of the membrane spreads to other parts, although the disturbance becomes weaker with increasing distance from its source, unless the neuron expends energy to amplify it as it travels. Over short distances, this attenuation is unimportant; in fact, many small neurons conduct their signals passively, without amplification. For long-distance communication, however, such passive spread is inadequate. Thus, larger neurons employ an active signaling mechanism, which is one of their most striking features. An electrical stimulus that exceeds a certain threshold strength triggers an explosion of electrical activity that propagates rapidly along the neuron’s plasma membrane and is sustained by automatic

1	An electrical stimulus that exceeds a certain threshold strength triggers an explosion of electrical activity that propagates rapidly along the neuron’s plasma membrane and is sustained by automatic amplification all along the way. This traveling wave of electrical excitation, known as an action potential, or nerve impulse, can carry a message without attenuation from one end of a neuron to the other at speeds of 100 meters per second or more. Action potentials are the direct consequence of the properties of voltage-gated cation channels, as we now discuss.

1	The plasma membrane of all electrically excitable cells—not only neurons, but also muscle, endocrine, and egg cells—contains voltage-gated cation channels, which are responsible for generating the action potentials. An action potential is triggered by a depolarization of the plasma membrane—that is, by a shift in the membrane potential to a less negative value inside. (We shall see later how the action of a neurotransmitter causes depolarization.) In nerve and skeletal muscle cells, a stimulus that causes sufficient depolarization promptly opens the voltage-gated Na+ channels, allowing a small amount of Na+ to enter the cell down its electrochemical gradient. The influx of positive charge depolarizes the membrane further, thereby opening more Na+ channels, which admit more Na+ ions, causing still further depolarization. This self-amplification process (an example of positive feedback, discussed in Chapters 8 and 15) continues until, within a fraction of a millisecond, the electrical

1	still further depolarization. This self-amplification process (an example of positive feedback, discussed in Chapters 8 and 15) continues until, within a fraction of a millisecond, the electrical potential in the local region of membrane has shifted from its resting value of about –70 mV (in squid giant axon; about –40 mV in human) to almost as far as the Na+ equilibrium potential of about +50 mV (see

1	Panel 11–1, p. 616). At this point, when the net electrochemical driving force for the flow of Na+ is almost zero, the cell would come to a new resting state, with all of its Na+ channels permanently open, if the open conformation of the channel were stable. Two mechanisms act in concert to save the cell from such a permanent electrical spasm: the Na+ channels automatically inactivate and voltage-gated K+ channels open to restore the membrane potential to its initial negative value. The Na+ channel is built from a single polypeptide chain that contains four structurally very similar domains. It is thought that these domains evolved by gene duplication followed by fusion into a single large gene (Figure 11–29A). In bacteria, in fact, the Na+ channel is a tetramer of four identical polypeptide chains, supporting this evolutionary idea.

1	Each domain contributes to the central channel, which is very similar to the K+ channel. Each domain also contains a voltage sensor that is characterized by an unusual transmembrane helix, S4, that contains many positively charged amino acids. As the membrane depolarizes, the S4 helices experience an electrostatic pulling force that attracts them to the now negatively charged extracellular side of the plasma membrane. The resulting conformational change opens the channel. The structure of a bacterial voltage-gated Na+ channel provides insights how the structural elements are arranged in the membrane (Figure 11–29B and C).

1	The Na+ channels also have an automatic inactivating mechanism, which causes the channels to reclose rapidly even though the membrane is still depolarized (see Figure 11–30). The Na+ channels remain in this inactivated state, unable to reopen, until after the membrane potential has returned to its initial negative value. The time necessary for a sufficient number of Na+ channels to recover from inactivation to support a new action potential, termed the refractory period, limits

1	Figure 11–29 Structural models of voltage-gated Na+ channels. (A) The channel in animal cells is built from a single polypeptide chain that contains four homologous domains. Each domain contains two transmembrane α helices (green) that surround the central ion-conducting pore. They are separated by sequences (blue) that form the selectivity filter. Four α additional helices (gray and red) in each domain constitute the voltage sensor. The S4 helices (red) are unique in that they contain an abundance of positively charged arginines. An inactivation gate that is part of a flexible loop connecting the third and fourth domains acts as a plug that obstructs the pore in the channel’s inactivated state, as shown in Figure 11–30. (B) Side and top views of a homologous bacterial channel protein showing its arrangement within the membrane. (C) A cross section of the pore domain of the channel shown in (B) shows lateral portals, through which the central cavity is accessible from the hydrophobic

1	its arrangement within the membrane. (C) A cross section of the pore domain of the channel shown in (B) shows lateral portals, through which the central cavity is accessible from the hydrophobic core of the lipid bilayer. In the crystals, lipid acyl chains were found to intrude into the pore. These lateral portals are large enough to allow entry of small, hydrophobic, pore-blocking drugs that are commonly used as anesthetics and block ion conductance. (PDB code: 3RVZ.)

1	Figure 11–30 Na+ channels and an action potential. (A) An action potential is triggered by a brief pulse of current, which (B) partially depolarizes the membrane, as shown in the plot of membrane potential versus time. The green curve shows how the membrane potential would have simply relaxed back to the resting value after the initial depolarizing stimulus if there had been no voltage-gated Na+ channels in the membrane. The red curve shows the course of the action potential that is caused by the opening and subsequent inactivation of voltage-gated Na+ channels. The states of the Na+ channels are indicated in (B). The membrane cannot fire a second action potential until the Na+ channels have returned from the inactivated to the closed conformation; until then, the membrane is refractory to stimulation. (C) The three states of the Na+ channel. When the membrane is at rest (highly polarized), the closed conformation of the channel has the lowest free energy and is therefore most stable;

1	stimulation. (C) The three states of the Na+ channel. When the membrane is at rest (highly polarized), the closed conformation of the channel has the lowest free energy and is therefore most stable; when the membrane is depolarized, the energy of the open conformation is lower, so the channel has a high probability of opening. But the free energy of the inactivated conformation is lower still; therefore, after a randomly variable period spent in the open state, the channel becomes inactivated. Thus, the open conformation corresponds to a metastable state that can exist only transiently when the membrane depolarizes (Movie 11.10).

1	the repetitive firing rate of a neuron. The cycle from initial stimulus to the return to the original resting state takes a few milliseconds or less. The Na+ channel can therefore exist in three distinct states—closed, open, and inactivated—which contribute to the rise and fall of the action potential (Figure 11–30). This description of an action potential applies only to a small patch of plasma membrane. The self-amplifying depolarization of the patch, however, is sufficient to depolarize neighboring regions of membrane, which then go through the same cycle. In this way, the action potential sweeps like a wave from the initial site of depolarization over the entire plasma membrane, as shown in Figure 11–31. The Use of Channelrhodopsins Has Revolutionized the Study of Neural Circuits

1	The Use of Channelrhodopsins Has Revolutionized the Study of Neural Circuits Channelrhodopsins are photosensitive ion channels that open in response to light. They evolved as sensory receptors in photosynthetic green algae to allow the algae to swim toward light. The structure of channelrhodopsin closely resembles that of bacteriorhodopsin (see Figure 10–31). It contains a covalently bound retinal group that absorbs light and undergoes an isomerization reaction, which triggers a conformational change in the protein, opening an ion channel in the plasma membrane. In contrast to bacteriorhodopsin, which is a light-driven proton pump, channelrhodopsin is a light-driven cation channel.

1	Using genetic engineering techniques, channelrhodopsin can be expressed in virtually any cell type in vertebrates and invertebrates. Researchers first introduced the gene into cultured neurons and showed that flashing light could now activate the channelrhodopsin and induce the neurons to fire action potentials. Because the frequency of the light flashes determined the frequency of the action potentials, one can control the frequency of neuronal firing with millisecond precision. Next, neurobiologists used the approach to activate specific neurons in the brain of experimental animals. Using a tiny fiber optic cable implanted near the axon at time = 0 (triggering of action potential)

1	Figure 11–31 The propagation of an action potential along an axon. (A) The voltages that would be recorded from a set of intracellular electrodes placed at intervals along the axon. (B) The changes in the Na+ channels and the current flows (curved red arrows) that give rise to a traveling action potential. The region of the axon with a depolarized membrane is shaded in blue. Note that once an action potential has started to progress, it has to continue in the same direction, traveling only away from the site of depolarization, because Na+-channel inactivation prevents the depolarization from spreading backward.

1	relevant brain region, they could flash light to specifically activate the channelrhodopsin-containing neurons to fire action potentials. One group of researchers expressed channelrhodopsin in a subset of mouse neurons thought to be involved in aggression: when these cells were activated by light, the mouse immediately attacked anything in its environment—including other mice or even an inflated rubber glove (Figure 11–32); when the light was switched off, the neurons fell silent and the mouse’s behavior returned to normal. Since these pioneering studies, researchers have engineered additional light-responsive ion channels and transporters, including some that can rapidly Figure 11–32 Optogenetic control of aggression neurons in a living mouse.

1	A gene encoding channelrhodopsin was introduced into a subpopulation of neurons in the hypothalamus of a mouse. When the neurons were exposed to flashing blue light using a tiny, implanted fiber optic cable, the channelrhodopsin channels opened, depolarizing and activating the cells. When the light was switched on, the mouse immediately became aggressive and attacked the inflated rubber glove; when the light was switched off, its behavior immediately returned to normal (Movie 11.11). (From D. Lin et al., Nature 470:221–226, 2011. With permission from Macmillan Publishers Ltd.) inactivate specific neurons. It is therefore now possible to transiently activate or inhibit specific neurons in the brains of awake animals with remarkable spatial and temporal precision. In this way, the rapidly expanding new field of optogenetics is revolutionizing neurobiology, allowing neuroscientists to analyze the neurons and circuits underlying even the most complex behaviors in experimental animals,

1	expanding new field of optogenetics is revolutionizing neurobiology, allowing neuroscientists to analyze the neurons and circuits underlying even the most complex behaviors in experimental animals, including nonhuman primates.

1	Myelination Increases the Speed and Efficiency of Action Potential Propagation in Nerve Cells The axons of many vertebrate neurons are insulated by a myelin sheath, which greatly increases the rate at which an axon can conduct an action potential. The importance of myelination is dramatically demonstrated by the demyelinating disease multiple sclerosis, in which the immune system destroys myelin sheaths in some regions of the central nervous system; in the affected regions, nerve impulse propagation greatly slows or even fails, often with devastating neurological consequences.

1	Myelin is formed by specialized non-neuronal supporting cells called glial cells. Schwann cells are the glial cells that myelinate axons in peripheral nerves, and oligodendrocytes do so in the central nervous system. These myelinating glial cells wrap layer upon layer of their own plasma membrane in a tight spiral around the axon (Figure 11–33A and B), thereby insulating the axonal membrane so that little current can leak across it. The myelin sheath is interrupted at regularly spaced nodes of Ranvier, where almost all the Na+ channels in the axon are concentrated (Figure 11–33C). This arrangement allows an action potential to propagate along a myelinated axon by jumping from node to node, a process called saltatory conduction. This type of conduction has two main advantages: action potentials travel very much faster, and metabolic energy is conserved because the active excitation is confined to the small regions of axonal plasma membrane at nodes of Ranvier.

1	Figure 11–33 Myelination.

1	(A) A myelinated axon from a peripheral nerve. Each Schwann cell wraps its plasma membrane concentrically around the axon to form a segment of myelin sheath about 1 mm long. For clarity, the membrane layers of the myelin are shown less compacted than they are in reality (see part B). (B) An electron micrograph of a nerve in the leg of a young rat. Two Schwann cells can be seen: one near the bottom is just beginning to myelinate its axon; the one above it has formed an almost mature myelin sheath. (C) Fluorescence micrograph and diagram of individual myelinated axons teased apart in a rat optic nerve, showing the confinement of the voltage-gated Na+ channels (green) in the axonal membrane at the node of Ranvier. A protein called Caspr (red) marks the junctions where the myelinating glial cell plasma membrane tightly abuts the axon on either side of the node. Voltage-gated K+ channels (blue) localize to regions in the axon plasma membrane well away from the node. (B, from Cedric S.

1	cell plasma membrane tightly abuts the axon on either side of the node. Voltage-gated K+ channels (blue) localize to regions in the axon plasma membrane well away from the node. (B, from Cedric S. Raine, in Myelin [P. Morell, ed.]. New York: Plenum, 1976; C, from M.N. Rasband and P. Shrager, J. Physiol. 525:63–73, 2000. With permission from Blackwell Publishing.)

1	Neuron and skeletal muscle cell plasma membranes contain many thousands of voltage-gated Na+ channels, and the current crossing the membrane is the sum of the currents flowing through all of these. An intracellular microelectrode can record this aggregate current, as shown in Figure 11–31A. Remarkably, however, it is also possible to record current flowing through individual channels. Patch-clamp recording, developed in the 1970s and 1980s, revolutionized the study of ion channels and made it possible to examine transport through a single channel in a small patch of membrane covering the mouth of a micropipette (Figure 11–34). With this simple but powerful technique, one can study the detailed properties of ion channels in all sorts of cell types. This work led to the discovery that even cells that are not electrically excitable usually have a variety of ion channels in their plasma membrane. Many of these cells, such as yeasts, are too small to be investigated by the traditional

1	cells that are not electrically excitable usually have a variety of ion channels in their plasma membrane. Many of these cells, such as yeasts, are too small to be investigated by the traditional electrophysiologist’s method of impalement with an intracellular microelectrode.

1	Patch-clamp recording indicates that individual ion channels open in an allor-nothing fashion. For example, a voltage-gated Na+ channel opens and closes at random, but when open, the channel always has the same large conductance, allowing more than 1000 ions to pass per millisecond (Figure 11–35). Therefore, the aggregate current crossing the membrane of an entire cell does not indicate the degree to which a typical individual channel is open but rather the total number of channels in its membrane that are open at any one time.

1	Some simple physical principles allow us to refine our understanding of voltage-gating from the perspective of a single Na+ channel. The interior of the resting neuron or muscle cell is at an electrical potential about 40–100 mV more negative than the external medium. Although this potential difference seems small, it exists across a plasma membrane only about 5 nm thick, so that the resulting voltage gradient is about 100,000 V/cm. Charged proteins in the membrane such as Na+ channels are thus subjected to a very large electrical field that can profoundly affect their conformation. Each conformation can “flip” to another conformation if given a sufficient jolt by the random thermal movements of the surroundings, and it is the relative stability of the closed, open, and inactivated conformations against flipping that is altered by changes in the membrane potential (see Figure 11–30C).

1	Na+ channels are not the only kind of voltage-gated cation channel that can generate an action potential. The action potentials in some muscle, egg, and endocrine cells, for example, depend on voltage-gated Ca2+ channels rather than on Na+ channels. pull micropipette away from cellglass to detach themicropipette patch of

1	Figure 11–34 The technique of patch-clamp recording. Because of the extremely tight seal between the micropipette and the membrane, current can enter or leave the micropipette only by passing through the ion channels in the patch of membrane covering its tip. The term clamp is used because an electronic device is employed to maintain, or “clamp,” the membrane potential at a set value while recording the ionic current through individual channels. The current through these channels can be recorded with the patch still attached to the rest of the cell, as in (A), or detached, as in (B). The advantage of the detached patch is that it is easy to alter the composition of the solution on either side of the membrane to test the effect of various solutes on channel behavior. A detached patch can also be produced with the opposite orientation, so that the cytoplasmic surface of the membrane faces the inside of the pipette.

1	There is a surprising amount of structural and functional diversity within each of the different classes of voltage-gated cation channels, generated both by multiple genes and by the alternative splicing of RNA transcripts produced from the same gene. Nonetheless, the amino acid sequences of the known voltage-gated Na+, K+, and Ca2+ channels show striking similarities, demonstrating that they all belong to a large superfamily of evolutionarily and structurally related proteins and share many of the design principles. Whereas the single-celled yeast S. cerevisiae contains a single gene that codes for a voltage-gated K+ channel, the genome of the worm C. elegans contains 68 genes that encode different but related K+ channels. This complexity indicates that even a simple nervous system made up of only 302 neurons uses a large number of different ion channels to compute its responses.

1	Humans who inherit mutant genes encoding ion channels can suffer from a variety of nerve, muscle, brain, or heart diseases, depending in which cells the channel encoded by the mutant gene normally functions. Mutations in genes that encode voltage-gated Na+ channels in skeletal muscle cells, for example, can cause myotonia, a condition in which there is a delay in muscle relaxation after voluntary contraction, causing painful muscle spasms. In some cases, this occurs because the abnormal channels fail to inactivate normally; as a result, Na+ entry persists after an action potential finishes and repeatedly reinitiates membrane depolarization and muscle contraction. Similarly, mutations that affect Na+ or K+ channels in the brain can cause epilepsy, in which excessive synchronized firing of large groups of neurons causes epileptic seizures (convulsions, or fits).

1	The particular combination of ion channels conducting Na+, K+, and Ca2+ that are expressed in a neuron largely determines how the cell fires repetitive sequences of action potentials. Some nerve cells can repeat action potentials up to 300 times per second; other neurons fire short bursts of action potentials separated by periods of silence; while others rarely fire more than one action potential at a time. There is a remarkable diversity of neurons in the brain.

1	It is estimated that the human brain contains about 1011 neurons and 1014 synaptic connections. To make matters more complex, neural circuitry is continuously sculpted in response to experience, modified as we learn and store memories, and irreversibly altered by the gradual loss of neurons and their connections as we age. How can a system so complex be subject to such change and yet continue to function stably? One emerging theory suggests that individual neurons are self-tuning devices, constantly adjusting the expression of ion channels and neurotransmitter receptors in order to maintain a stable function. How might this work?

1	Neurons can be categorized into functionally different types, based in part on their propensity to fire action potentials and their pattern of firing. For example, some neurons fire action potentials at high frequencies, while others fire rarely. The firing properties of each neuron type are determined to a large extent by the ion channels that the cell expresses. The number of ion channels in a neuron’s membrane is not fixed: as conditions change, a neuron can modify the numbers of depolarizing (Na+ and Ca2+) and hyperpolarizing (K+) channels and keep their proportions adjusted so as to maintain its characteristic firing behavior—a remarkable example of homeostatic control. The molecular mechanisms involved remain an important mystery. Neuronal signals are transmitted from cell to cell at specialized sites of contact known as synapses. The usual mechanism of transmission is indirect. The cells are

1	Figure 11–35 Patch-clamp measurements for a single voltage-gated Na+ channel. A tiny patch of plasma membrane was detached from an embryonic rat muscle cell, as in Figure 11–34. (A) The membrane was depolarized by an abrupt shift of potential from –90 to about –40 mV. (B) Three current records from three experiments performed on the same patch of membrane. Each major current step in (B) represents the opening and closing of a single channel. A comparison of the three records shows that, whereas the durations of channel opening and closing vary greatly, the rate at which current flows through an open channel (its conductance) is practically constant. The minor fluctuations in the current records arise largely from electrical noise in the recording apparatus. Current flowing into the cell, measured in picoamperes (pA), is shown as a downward deflection of the curve. By convention, the electrical potential on the outside of the cell is defined as zero.

1	(C) The sum of the currents measured in 144 repetitions of the same experiment. This aggregate current is equivalent to the usual Na+ current that would be observed flowing through a relatively large region of membrane containing 144 channels. A comparison of (B) and (C) reveals that the time course of the aggregate current reflects the probability that any individual channel will be in the open state; this probability decreases with time as the channels in the depolarized membrane adopt their inactivated conformation. (Data from J. Patlak and R. Horn, J. Gen. Physiol. 79:333–351, 1982. With permission from The Rockefeller University Press.) nerve terminal of presynaptic cell neurotransmitter in synaptic vesicle

1	Figure 11–36 a chemical synapse. (A) When an action potential reaches the nerve terminal in a presynaptic cell, it stimulates the terminal to release its neurotransmitter. The neurotransmitter molecules are contained in synaptic vesicles and are released to the cell exterior when the vesicles fuse with the plasma membrane of the nerve terminal. The released neurotransmitter binds to and opens the transmitter-gated ion channels concentrated in the plasma membrane of the postsynaptic target cell at the synapse. The resulting ion flows alter the membrane potential of the postsynaptic membrane, thereby transmitting a signal from the excited nerve (B) (Movie 11.12). (B) A thin-section electron micrograph of two nerve terminal synapses on a dendrite of a postsynaptic cell. (B, courtesy of Cedric Raine.) electrically isolated from one another, the presynaptic cell being separated from the postsynaptic cell by a narrow synaptic cleft. When an action potential arrives at the presynaptic site,

1	Raine.) electrically isolated from one another, the presynaptic cell being separated from the postsynaptic cell by a narrow synaptic cleft. When an action potential arrives at the presynaptic site, the depolarization of the membrane opens voltage-gated Ca2+ channels that are clustered in the presynaptic membrane. Ca2+ influx triggers the release into the cleft of small signal molecules known as neurotransmitters, which are stored in membrane-enclosed synaptic vesicles and released by exocytosis (discussed in Chapter 13). The neurotransmitter diffuses rapidly across the synaptic cleft and provokes an electrical change in the postsynaptic cell by binding to and opening transmitter-gated ion channels (Figure 11–36). After the neurotransmitter has been secreted, it is rapidly removed: it is either destroyed by specific enzymes in the synaptic cleft or taken up by the presynaptic nerve terminal or by surrounding glial cells. Reuptake is mediated by a variety of Na+-dependent

1	it is either destroyed by specific enzymes in the synaptic cleft or taken up by the presynaptic nerve terminal or by surrounding glial cells. Reuptake is mediated by a variety of Na+-dependent neurotransmitter symporters (see Figure 11–8); in this way, neurotransmitters are recycled, allowing cells to keep up with high rates of release. Rapid removal ensures both spatial and temporal precision of signaling at a synapse. It decreases the chances that the neurotransmitter will influence neighboring cells, and it clears the synaptic cleft before the next pulse of neurotransmitter is released, so that the timing of repeated, rapid signaling events can be accurately communicated to the postsynaptic cell. As we shall see, signaling via such chemical synapses is far more versatile and adaptable than direct electrical coupling via gap junctions at electrical synapses (discussed in Chapter 19), which are also used by neurons but to a much smaller extent.

1	Transmitter-gated ion channels, also called ionotropic receptors, are built for rapidly converting extracellular chemical signals into electrical signals at chemical synapses. The channels are concentrated in a specialized region of the postsynaptic plasma membrane at the synapse and open transiently in response to the binding of neurotransmitter molecules, thereby producing a brief permeability change in the membrane (see Figure 11–36A). Unlike the voltage-gated channels responsible for action potentials, transmitter-gated channels are relatively insensitive to the membrane potential and therefore cannot by themselves produce a self-amplifying excitation. Instead, they produce local permeability increases, and hence changes of membrane potential, that are graded according to the amount of neurotransmitter released at the synapse and how long it persists there. Only if the summation of small depolarizations at this site opens sufficient numbers of nearby voltage-gated cation channels

1	neurotransmitter released at the synapse and how long it persists there. Only if the summation of small depolarizations at this site opens sufficient numbers of nearby voltage-gated cation channels can an action potential be triggered. This may require the opening of transmitter-gated ion channels at numerous synapses in close proximity on the target nerve cell.

1	Transmitter-gated ion channels differ from one another in several important ways. First, as receptors, they have highly selective binding sites for the neurotransmitter that is released from the presynaptic nerve terminal. Second, as channels, they are selective in the type of ions that they let pass across the plasma membrane; this determines the nature of the postsynaptic response. Excitatory neurotransmitters open cation channels, causing an influx of Na+, and in many cases Ca2+, that depolarizes the postsynaptic membrane toward the threshold potential for firing an action potential. Inhibitory neurotransmitters, by contrast, open either Cl– channels or K+ channels, and this suppresses firing by making it harder for excitatory neurotransmitters to depolarize the postsynaptic membrane. Many transmitters can be either excitatory or inhibitory, depending on where they are released, what receptors they bind to, and the ionic conditions that they encounter. Acetylcholine, for example,

1	Many transmitters can be either excitatory or inhibitory, depending on where they are released, what receptors they bind to, and the ionic conditions that they encounter. Acetylcholine, for example, can either excite or inhibit, depending on the type of acetylcholine receptors it binds to. Usually, however, acetylcholine, glutamate, and serotonin are used as excitatory transmitters, and γ-aminobutyric acid (GABA) and glycine are used as inhibitory transmitters. Glutamate, for instance, mediates most of the excitatory signaling in the vertebrate brain.

1	We have already discussed how the opening of Na+ or Ca2+ channels depolarizes a membrane. The opening of K+ channels has the opposite effect because the K+ concentration gradient is in the opposite direction—high concentration inside the cell, low outside. Opening K+ channels tends to keep the cell close to the equilibrium potential for K+, which, as we discussed earlier, is normally close to the resting membrane potential because at rest K+ channels are the main type of channel that is open. When additional K+ channels open, it becomes harder to drive the cell away from the resting state. We can understand the effect of opening Cl– channels similarly. The concentration of Cl– is much higher outside the cell than inside (see Table 11–1, p. 598), but the membrane potential opposes its influx. In fact, for many neurons, the equilibrium potential for Cl– is close to the resting potential—or even more negative. For this reason, opening Cl– channels tends to buffer the membrane potential;

1	In fact, for many neurons, the equilibrium potential for Cl– is close to the resting potential—or even more negative. For this reason, opening Cl– channels tends to buffer the membrane potential; as the membrane starts to depolarize, more negatively charged Cl– ions enter the cell and counteract the depolarization. Thus, the opening of Cl– channels makes it more difficult to depolarize the membrane and hence to excite the cell. Some powerful toxins act by blocking the action of inhibitory neurotransmitters: strychnine, for example, binds to glycine receptors and prevents their inhibitory action, causing muscle spasms, convulsions, and death.

1	However, not all chemical signaling in the nervous system operates through these ionotropic ligand-gated ion channels. In fact, most neurotransmitter molecules that are secreted by nerve terminals, including a large variety of neuropeptides, bind to metabotropic receptors, which regulate ion channels only indirectly through the action of small intracellular signal molecules (discussed in Chapter 15). All neurotransmitter receptors fall into one or other of these two major classes—ionotropic or metabotropic—on the basis of their signaling mechanisms: 1. Ionotropic receptors are ion channels and feature at fast chemical synapses. Acetylcholine, glycine, glutamate, and GABA all act on transmitter-gated ion channels, mediating excitatory or inhibitory signaling that is generally immediate, simple, and brief. 2.

1	2. Metabotropic receptors are G-protein-coupled receptors (discussed in Chapter 15) that bind to all other neurotransmitters (and, confusingly, also acetylcholine, glutamate, and GABA). Signaling mediated by ligand-binding to metabotropic receptors tends to be far slower and more complex than that at ionotropic receptors, and longer-lasting in its consequences. The Acetylcholine Receptors at the Neuromuscular Junction Are Excitatory Transmitter-Gated Cation Channels

1	The Acetylcholine Receptors at the Neuromuscular Junction Are Excitatory Transmitter-Gated Cation Channels A well-studied example of a transmitter-gated ion channel is the acetylcholine receptor of skeletal muscle cells. This channel is opened transiently by acetylcholine released from the nerve terminal at a neuromuscular junction—the specialized chemical synapse between a motor neuron and a skeletal muscle cell (Figure 11–37). This synapse has been intensively investigated because it is readily accessible to electrophysiological study, unlike most of the synapses in the central nervous system, that is, the brain and spinal cord in vertebrates. Moreover, the acetylcholine receptors are densely packed in the muscle cell plasma membrane at a neuromuscular junction (about 20,000 such receptors per μm2), with relatively few receptors elsewhere in the same membrane.

1	The receptors are composed of five transmembrane polypeptides, two of one kind and three others, encoded by four separate genes (Figure 11–38A). The four genes are strikingly similar in sequence, implying that they evolved from a single ancestral gene. The two identical polypeptides in the pentamer each contribute one acetylcholine-binding site. When two acetylcholine molecules bind to the pentameric complex, they induce a conformational change that opens the channel. With ligand bound, the channel still flickers between open and closed states, but now it has a 90% probability of being open. This state continues—with acetylcholine binding and unbinding—until hydrolysis of the free acetylcholine by the enzyme acetylcholinesterase lowers its concentration at the neuromuscular junction sufficiently. Once freed of its bound neurotransmitter, the acetylcholine receptor reverts to its initial resting state. If the presence of acetylcholine persists for a prolonged time as a result of

1	sufficiently. Once freed of its bound neurotransmitter, the acetylcholine receptor reverts to its initial resting state. If the presence of acetylcholine persists for a prolonged time as a result of excessive nerve stimulation, the channel inactivates. Normally, the acetylcholine is rapidly hydrolyzed and the channel closes within about 1 millisecond, well before significant desensitization occurs. Desensitization would occur after about 20 milliseconds in the continued presence of acetylcholine.

1	The five subunits of the acetylcholine receptor are arranged in a ring, forming a water-filled transmembrane channel that consists of a narrow pore through the lipid bilayer, which widens into vestibules at both ends. Acetylcholine binding opens the channel by causing the helices that line the pore to rotate outward, thus disrupting a ring of hydrophobic amino acids that blocks ion flow in the closed state. Clusters of negatively charged amino acids at either end of the pore help to exclude negative ions and encourage any positive ion of diameter less than 0.65 nm to pass through (Figure 11–38B). The normal through-traffic consists chiefly of Na+ and K+, together with some Ca2+. Thus, unlike voltage-gated cation channels, such as the K+ channel discussed earlier, there is little selectivity among cations, and the relative contributions of the different cations to the current through the channel depend chiefly on their concentrations and on the electrochemical driving forces. When the

1	among cations, and the relative contributions of the different cations to the current through the channel depend chiefly on their concentrations and on the electrochemical driving forces. When the muscle cell membrane is at its resting potential, the net driving force for K+ is near zero, since the voltage gradient nearly balances the K+ concentration gradient across the membrane (see Panel 11–1, p. 616). For Na+, in contrast, the voltage gradient and the concentration gradient both act in the same direction to drive the ion into the cell. (The same is true for Ca2+, but the

1	Figure 11–37 a low-magnification scanning electron micrograph of a neuromuscular junction in a frog. The termination of a single axon on a skeletal muscle cell is shown. (From J. Desaki and Y. Uehara, J. Neurocytol. 10:101–110, 1981. With permission from Kluwer Academic Publishers.) extracellular concentration of Ca2+ is so much lower than that of Na+ that Ca2+ makes only a small contribution to the total inward current.) Therefore, the opening of the acetylcholine-receptor channels leads to a large net influx of Na+ (a peak rate of about 30,000 ions per channel each millisecond). This influx causes a membrane depolarization that signals the muscle to contract, as discussed below. Neurons Contain Many Types of Transmitter-Gated Channels

1	Neurons Contain Many Types of Transmitter-Gated Channels The ion channels that open directly in response to the neurotransmitters acetylcholine, serotonin, GABA, and glycine contain subunits that are structurally similar and probably form transmembrane pores in the same way as the ionotropic acetylcholine receptor, even though they have distinct neurotransmitter-binding specificities and ion selectivities. These channels are all built from homologous polypeptide subunits, which assemble as a pentamer. Glutamate-gated ion channels are an exception, in that they are constructed from a distinct family of subunits and form tetramers resembling the K+ channels discussed earlier (see Figure 11–24A).

1	For each class of transmitter-gated ion channel, there are alternative forms of each type of subunit, which may be encoded by distinct genes or else generated by alternative RNA splicing of a single gene product. The subunits assemble in different combinations to form an extremely diverse set of distinct channel subtypes, with different ligand affinities, different channel conductances, different rates of opening and closing, and different sensitivities to drugs and toxins. Some vertebrate neurons, for example, have acetylcholine-gated ion channels that differ from those of muscle cells in that they are formed from two subunits of one type and three of another; but there are at least nine genes coding for different versions of the first type of subunit and at least three coding for different versions of the second. Subsets of such neurons performing different functions in the brain express different combinations of the genes for these subunits. In principle, and already to some extent

1	versions of the second. Subsets of such neurons performing different functions in the brain express different combinations of the genes for these subunits. In principle, and already to some extent in practice, it is possible to design drugs targeted against these narrowly defined subsets, thereby specifically influencing particular brain functions.

1	Transmitter-gated ion channels have for a long time been important drug targets. A surgeon, for example, can relax muscles for the duration of an operation

1	Figure 11–38 a model for the structure of the skeletal muscle acetylcholine receptor. (A) Five homologous subunits (α, α, β, γ, δ) combine to form a transmembrane pore. Both of the α subunits contribute an acetylcholinebinding site nestled between adjoining subunits. (B) The pore is lined by a ring of five transmembrane α helices, one contributed by each subunit (just the two α subunits are shown). In its closed conformation, the pore is occluded by the hydrophobic side chains of five leucines (green), one from each α helix, which form a gate near the middle of the lipid bilayer. When acetylcholine binds to both α subunits, the channel undergoes a conformational change that opens the gate by an outward rotation of the helices containing the occluding leucines. Negatively charged side chains (indicated by the “–“ signs) at either end of the pore ensure that only positively charged ions pass through the channel. (PDB code: 2BG9.) by blocking the acetylcholine receptors on skeletal

1	chains (indicated by the “–“ signs) at either end of the pore ensure that only positively charged ions pass through the channel. (PDB code: 2BG9.) by blocking the acetylcholine receptors on skeletal muscle cells with curare, a plant-derived drug that was originally used by South American Indians to make poison arrows. Most drugs used to treat insomnia, anxiety, depression, and schizophrenia exert their effects at chemical synapses, and many of these act by binding to transmitter-gated channels. Barbiturates, tranquilizers such as Valium, and sleeping pills such as Ambien, for example, bind to GABA receptors, potentiating the inhibitory action of GABA by allowing lower concentrations of this neurotransmitter to open Cl– channels. Our increasing understanding of the molecular biology of ion channels should allow us to design a new generation of psychoactive drugs that will act still more selectively to alleviate the miseries of mental illness.

1	In addition to ion channels, many other components of the synaptic signaling machinery are potential targets for psychoactive drugs. As mentioned earlier, after release into the synaptic cleft, many neurotransmitters are cleared by reuptake mechanisms mediated by Na+-driven symports. Inhibiting such transporters prolongs the effect of the neurotransmitter, thereby strengthening synaptic transmission. Many antidepressant drugs, including Prozac, inhibit the reuptake of serotonin; others inhibit the reuptake of both serotonin and norepinephrine. Ion channels are the basic molecular units from which neuronal devices for signaling and computation are built. To provide a glimpse of how sophisticated these devices can be, we consider several examples that demonstrate how the coordinated activities of groups of ion channels allow you to move, feel, and remember. Neuromuscular Transmission Involves the Sequential Activation of Five Different Sets of Ion Channels

1	Neuromuscular Transmission Involves the Sequential Activation of Five Different Sets of Ion Channels The following process, in which a nerve impulse stimulates a muscle cell to contract, illustrates the importance of ion channels to electrically excitable cells. This apparently simple response requires the sequential activation of at least five different sets of ion channels, all within a few milliseconds (Figure 11–39). 1. The process is initiated when a nerve impulse reaches the nerve terminal and depolarizes the plasma membrane of the terminal. The depolarization transiently opens voltage-gated Ca2+ channels in this presynaptic membrane. As the Ca2+ concentration outside cells is more than 1000 times Figure 11–39 The system of ion channels at a neuromuscular junction. These gated ion channels are essential for the stimulation of muscle contraction by a nerve impulse. The various channels are numbered in the sequence in which they are activated, as described in the text.

1	greater than the free Ca2+ concentration inside, Ca2+ flows into the nerve terminal. The increase in Ca2+ concentration in the cytosol of the nerve terminal triggers the local release of acetylcholine by exocytosis into the synaptic cleft. 2. The released acetylcholine binds to acetylcholine receptors in the muscle cell plasma membrane, transiently opening the cation channels associated with them. The resulting influx of Na+ causes a local membrane depolarization. 3. The local depolarization opens voltage-gated Na+ channels in this membrane, allowing more Na+ to enter, which further depolarizes the membrane. This, in turn, opens neighboring voltage-gated Na+ channels and results in a self-propagating depolarization (an action potential) that spreads to involve the entire plasma membrane (see Figure 11–31). 4.

1	4. The generalized depolarization of the muscle cell plasma membrane activates voltage-gated Ca2+ channels in the transverse tubules (T tubules— discussed in Chapter 16) of this membrane. 5. This in turn causes Ca2+-release channels in an adjacent region of the sarcoplasmic reticulum (SR) membrane to open transiently and release Ca2+ stored in the SR into the cytosol. The T-tubule and SR membranes are closely apposed with the two types of channel joined together in a specialized structure, in which activation of the voltage-sensitive Ca2+ channel in the T-tubule plasma membrane causes a channel conformational change that is mechanically transmitted to the Ca2+-release channel in the SR membrane, opening it and allowing Ca2+ to flow from the SR lumen into the cytoplasm (see Figure 16–35). The sudden increase in the cytosolic Ca2+ concentration causes the myofibrils in the muscle cell to contract.

1	Whereas the initiation of muscle contraction by a motor neuron is complex, an even more sophisticated interplay of ion channels is required for a neuron to integrate a large number of input signals at its synapses and compute an appropriate output, as we now discuss. In the central nervous system, a single neuron can receive inputs from thousands of other neurons, and it can in turn form synapses with many thousands of other cells. Several thousand nerve terminals, for example, make synapses on an average motor neuron in the spinal cord, almost completely covering its cell body and dendrites (Figure 11–40). Some of these synapses transmit signals from the brain or spinal cord; others bring sensory information from muscles or from the skin. The motor neuron must combine the information received from all these sources and react, either by firing action potentials along its axon or by remaining quiet.

1	Of the many synapses on a neuron, some tend to excite it, while others inhibit it. Neurotransmitter released at an excitatory synapse causes a small depolarization in the postsynaptic membrane called an excitatory postsynaptic potential (excitatory PSP), whereas neurotransmitter released at an inhibitory synapse generally causes a small hyperpolarization called an inhibitory PSP. The plasma membrane of the dendrites and cell body of most neurons contains a relatively low density of voltage-gated Na+ channels, and so an individual excitatory PSP is generally too small to trigger an action potential. Instead, each incoming signal initiates a local PSP, which decreases with distance from the site of the synapse. If signals arrive simultaneously at several synapses in the same region of the dendritic tree, the total PSP in that neighborhood will be roughly the sum of the individual PSPs, with inhibitory PSPs making a negative contribution to the total. The PSPs from each neighborhood

1	dendritic tree, the total PSP in that neighborhood will be roughly the sum of the individual PSPs, with inhibitory PSPs making a negative contribution to the total. The PSPs from each neighborhood spread passively and converge on the cell body. For long-distance transmission, the combined magnitude of the PSP is then translated, or encoded, into the frequency of firing of action potentials: the greater the stimulation (depolarization), the higher the frequency of action potentials.

1	0.1 mm Neuronal Computation Requires a Combination of at Least Three Kinds of K+ Channels The intensity of stimulation that a neuron receives is encoded by that neuron into action potential frequency for long-distance transmission. The encoding takes place at a specialized region of the axonal membrane known as the initial segment, or axon hillock, at the junction of the axon and the cell body (see Figure 11–40). This membrane is rich in voltage-gated Na+ channels; but it also contains at least four other classes of ion channels—three selective for K+ and one selective for Ca2+—all of which contribute to the axon hillock’s encoding function. The three varieties of K+ channels have different properties; we shall refer to them as delayed, rapidly inactivating, and Ca2+-activated K+ channels.

1	To understand the need for multiple types of channels, consider first what would happen if the only voltage-gated ion channels present in the nerve cell were the Na+ channels. Below a certain threshold level of synaptic stimulation, the depolarization of the initial-segment membrane would be insufficient to trigger an action potential. With gradually increasing stimulation, the threshold would be crossed, the Na+ channels would open, and an action potential would fire. The action potential would be terminated by inactivation of the Na+ channels. Before another action potential could fire, these channels would have to recover from their inactivation. But that would require a return of the membrane voltage to a very negative value, which would not occur as long as the strong depolarizing stimulus (from PSPs) was maintained. An additional channel type is needed, therefore, to repolarize the membrane after each action potential to prepare the cell to fire again.

1	The delayed K+ channels perform this task, as discussed previously in relation to the propagation of the action potential (see Figure 11–31). They are voltage-gated, but because of their slower kinetics they open only during the falling phase of the action potential, when the Na+ channels are inactive. Their opening permits an efflux of K+ that drives the membrane back toward the K+ equilibrium potential, which is so negative that the Na+ channels rapidly recover from their inactivated state. Repolarization of the membrane also closes the delayed K+ channels. The initial segment is now reset so that the depolarizing stimulus from

1	Figure 11–40 a motor neuron in the spinal cord. (A) Many thousands of nerve terminals synapse on the cell body and dendrites. These deliver signals from other parts of the organism to control the firing of action potentials along the single axon of this large cell. (B) Fluorescence micrograph showing a nerve cell body and its dendrites stained with a fluorescent antibody that recognizes a cytoskeletal protein (green) that is not present in axons. Thousands of axon terminals (red) from other nerve cells (not visible) make synapses on the cell body and dendrites; the terminals are stained with a fluorescent antibody that recognizes a protein in synaptic vesicles. (B, courtesy of Olaf Mundigl and Pietro de Camilli.) frequency of fring(action potentials per second) magnitude of combined PSP synaptic inputs can fire another action potential. In this way, sustained stimulation of the dendrites and cell body leads to repetitive firing of the axon.

1	Repetitive firing in itself, however, is not enough. The frequency of firing has to reflect the intensity of stimulation, and a simple system of Na+ channels and delayed K+ channels is inadequate for this purpose. Below a certain threshold level of steady stimulation, the cell will not fire at all; above that threshold level, it will abruptly begin to fire at a relatively rapid rate. The rapidly inactivating K+ channels solve the problem. These, too, are voltage-gated and open when the membrane is depolarized, but their specific voltage sensitivity and kinetics of inactivation are such that they act to reduce the rate of firing at levels of stimulation that are only just above the threshold required for firing. Thus, they remove the discontinuity in the relationship between the firing rate and the intensity of stimulation. The result is a firing rate that is proportional to the strength of the depolarizing stimulus over a very broad range (Figure 11–41).

1	The process of encoding is usually further modulated by the two other types of ion channels in the initial segment that were mentioned earlier—voltage-gated Ca2+ channels and Ca2+-activated K+ channels. They act together to decrease the response of the cell to an unchanging, prolonged stimulation—a process called adaptation. These Ca2+ channels are similar to the Ca2+ channels that mediate the release of neurotransmitter from presynaptic axon terminals; they open when an action potential fires, transiently allowing Ca2+ into the axon cytosol at the initial segment.

1	The Ca2+-activated K+ channel opens in response to a raised concentration of Ca2+ at the channel’s cytoplasmic face (Figure 11–42). Prolonged, strong depolarizing stimuli will trigger a long train of action potentials, each of which permits a brief influx of Ca2+ through the voltage-gated Ca2+ channels, so that local cytosolic Ca2+ concentration gradually builds up to a level high enough to open the Ca2+-activated K+ channels. Because the resulting increased permeability of the membrane to K+ makes the membrane harder to depolarize, the delay between one action potential and the next is increased. In this way, a neuron that is stimulated continuously for a prolonged period becomes gradually less responsive to the constant stimulus.

1	Such adaptation, which can also occur by other mechanisms, allows a neuron—indeed, the nervous system generally—to react sensitively to change, even against a high background level of steady stimulation. It is one of the computational strategies that help us, for example, to feel a light touch on the shoulder and yet ignore the constant pressure of our clothing. We discuss adaptation as a general feature in cell signaling processes in more detail in Chapter 15. Other neurons do different computations, reacting to their synaptic inputs in myriad ways, reflecting the different assortments of ion channels in their membrane. There are several hundred genes that code for ion channels in the human genome, with over 150 encoding voltage-gated channels alone. Further complexity is introduced by alternative splicing of RNA transcripts and assembling channel subunits in different combinations. Moreover, ion channels are selectively

1	Figure 11–41 The magnitude of the combined postsynaptic potential (PSP) is reflected in the frequency of firing of action potentials. The mix of excitatory and inhibitory PSPs produces a combined PSP at the initial segment. A comparison of (A) and (B) shows how the firing frequency of an axon increases with an increase in the combined PSP, while (C) summarizes the general relationship.

1	localized to different sites in the plasma membrane of a neuron. Some K+ and Ca2+ channels are concentrated in the dendrites and participate in processing the input that a neuron receives. As we have seen, other ion channels are located at the axon’s initial segment, where they control action potential firing; and some ligand-gated channels are distributed over the cell body and, depending on their ligand occupancy, modulate the cell’s general sensitivity to synaptic inputs. The multiplicity of ion channels and their locations evidently allows each of the many types of neurons to tune the electrical behavior to the particular tasks they perform.

1	One of the crucial properties of the nervous system is its ability to learn and remember. This property depends in part on the ability of individual synapses to strengthen or weaken depending on their use—a process called synaptic plasticity. We end this chapter by considering a remarkable type of ion channel that has a special role in some forms of synaptic plasticity. It is located at many excitatory synapses in the central nervous system, where it is gated by both voltage and the excitatory neurotransmitter glutamate. It is also the site of action of the psychoactive drug phencyclidine, or angel dust. Long-Term Potentiation (LTP) in the Mammalian Hippocampus Depends on Ca2+ Entry Through NMDA-Receptor Channels

1	Practically all animals can learn, but mammals seem to learn exceptionally well (or so we like to think). In a mammal’s brain, the region called the hippocampus has a special role in learning. When it is destroyed on both sides of the brain, the ability to form new memories is largely lost, although previous long-established memories remain. Some synapses in the hippocampus show a striking form of synaptic plasticity with repeated use: whereas occasional single action potentials in the presynaptic cells leave no lasting trace, a short burst of repetitive firing causes long-term potentiation (LTP), such that subsequent single action potentials in the presynaptic cells evoke a greatly enhanced response in the postsynaptic cells. The effect lasts hours, days, or weeks, according to the number and intensity of the bursts of repetitive firing. Only the synapses that were activated exhibit LTP; synapses that have remained quiet on the same postsynaptic cell are not affected. However, while

1	and intensity of the bursts of repetitive firing. Only the synapses that were activated exhibit LTP; synapses that have remained quiet on the same postsynaptic cell are not affected. However, while the cell is receiving a burst of repetitive stimulation via one set of synapses, if a single action potential is delivered at another synapse on its surface, that latter synapse also will undergo LTP, even though a single action potential delivered there at another time would leave no such lasting trace.

1	The underlying rule in such events seems to be that LTP occurs on any occasion when a presynaptic cell fires (once or more) at a time when the postsynaptic membrane is strongly depolarized (either through recent repetitive firing of the same presynaptic cell or by other means). This rule reflects the behavior of a particular class of ion channels in the postsynaptic membrane. Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system, and glutamate-gated ion channels are the most common of all transmitter-gated channels in the brain. In the hippocampus, as elsewhere, most of the depolarizing current responsible for excitatory PSPs is carried by glutamate-gated ion channels called AMPA receptors, which operate in the standard way (Figure 11–43). But the current has, in addition, a second and more intriguing component, which is mediated by a separate subclass of glutamate-gated ion channels known as NMDA receptors, so named because they are selectively

1	has, in addition, a second and more intriguing component, which is mediated by a separate subclass of glutamate-gated ion channels known as NMDA receptors, so named because they are selectively activated by the artificial glutamate analog N-methyl-D-aspartate. The NMDA-receptor channels are doubly gated, opening only when two conditions are satisfied simultaneously: glutamate must be bound to the receptor, and the membrane must be strongly depolarized. The second condition is required for releasing the Mg2+ that normally blocks the resting channel. This means that NMDA receptors are normally activated only when AMPA receptors are activated as well and depolarize the membrane. The NMDA receptors are critical for LTP. When they are selectively blocked with a specific inhibitor or inactivated genetically, LTP does not occur, even though ordinary synaptic transmission continues, indicating the importance of NMDA receptors

1	Figure 11–42 Structure of a Ca2+activated K+ channel. The channel contains four identical subunits (which are shown in different colors for clarity). It is both voltageand Ca2+-gated. The structure shown is a composite of the cytosolic and membrane portions of the channel that were separately crystallized. (PDB codes: 2R99, 1LNQ.) Figure 11–43 The structure of the aMPa receptor. This ionotropic glutamate receptor (named after the glutamate analog α-Amino 3-hydroxy 5-Methyl 4-isoxazole Propionic Acid) is the most common mediator of fast, excitatory synaptic transmission in the central nervous system (CNS). (PDB code: 3KG2.) AMPA-receptor channels, depolarization removes Mg2+ block from NMDA-allowing Na+ infux receptor channel, which that depolarizes (with glutamate bound) the postsynaptic allows Ca2+ to enter the membrane postsynaptic cell for LTP induction. Such animals exhibit specific deficits in their learning abilities but behave almost normally otherwise.

1	How do NMDA receptors mediate LTP? The answer is that these channels, when open, are highly permeable to Ca2+, which acts as an intracellular signal in the postsynaptic cell, triggering a cascade of changes that are responsible for LTP. Thus, LTP is prevented when Ca2+ levels are held artificially low in the postsynaptic cell by injecting the Ca2+ chelator EGTA into it, and LTP can be induced by artificially raising intracellular Ca2+ levels in the cell. Among the long-term changes that increase the sensitivity of the postsynaptic cell to glutamate is the insertion of new AMPA receptors into the plasma membrane (Figure 11–44). In some forms of LTP, changes occur in the presynaptic cell as well, so that it releases more glutamate than normal when it is activated subsequently.

1	If synapses were capable only of LTP they would quickly become saturated, and thus be of limited value as an information-storage device. In fact, they also exhibit long-term depression (LTD), with the long-term effect of reducing the number of AMPA receptors in the post-synaptic membrane. This feat is accomplished by degrading AMPA receptors after their selective endocytosis. Surprisingly, LTD also requires NMDA receptor activation and a rise in Ca2+. How does Ca2+ trigger opposite effects at the same synapse? It turns out that this bidirectional control of synaptic strength depends on the magnitude of the rise in Ca2+: high Ca2+ levels activate protein kinases and LTP, whereas modest Ca2+ levels activate protein phosphatases and LTD.

1	There is evidence that NMDA receptors have an important role in synaptic plasticity and learning in other parts of the brain, as well as in the hippocampus. Moreover, they have a crucial role in adjusting the anatomical pattern of synaptic connections in the light of experience during the development of the nervous system. Thus, neurotransmitters released at synapses, besides relaying transient electrical signals, can also alter concentrations of intracellular mediators that bring about lasting changes in the efficacy of synaptic transmission. However, it is still uncertain how these changes endure for weeks, months, or a lifetime in the face of the normal turnover of cell constituents.

1	Ion channels form aqueous pores across the lipid bilayer and allow inorganic ions of appropriate size and charge to cross the membrane down their electrochemical gradients at rates about 1000 times greater than those achieved by any known transporter. The channels are “gated” and usually open transiently in response to a specific perturbation in the membrane, such as a change in membrane potential (voltage-gated channels), or the binding of a neurotransmitter to the channel (transmitter-gated channels). K+-selective leak channels have an important role in determining the resting membrane potential across the plasma membrane in most animal cells. increased Ca2+ in the cytosol induces postsynaptic cell to insert new AMPA receptors in the plasma membrane, increasing the cell's sensitivity to glutamate

1	increased Ca2+ in the cytosol induces postsynaptic cell to insert new AMPA receptors in the plasma membrane, increasing the cell's sensitivity to glutamate Figure 11–44 The signaling events in longterm potentiation. Although not shown, transmission-enhancing changes can also occur in the presynaptic nerve terminals in LTP, which may be induced by retrograde signals from the postsynaptic cell. Voltage-gated cation channels are responsible for the amplification and propaga-WHAT WE DON’T KNOW tion of action potentials in electrically excitable cells, such as neurons and skeletal muscle cells. Transmitter-gated ion channels convert chemical signals to electrical How do individual neurons establish signals at chemical synapses. Excitatory neurotransmitters, such as acetylcholine and maintain their characteristic and glutamate, open transmitter-gated cation channels and thereby depolarize the intrinsic firing properties?

1	postsynaptic membrane toward the threshold level for firing an action potential. Inhibitory neurotransmitters, such as GABA and glycine, open transmitter-gated

1	Even organisms with very simple Cl– or K+ channels and thereby suppress firing by keeping the postsynaptic mem-nervous systems have dozens of brane polarized. A subclass of glutamate-gated ion channels, called NMDA-recep-different K+ channels. Why is it tor channels, is highly permeable to Ca2+, which can trigger the long-term changes important to have so many? in synapse efficacy (synaptic plasticity) such as LTP and LTD that are thought to be involved in some forms of learning and memory. • Why do cells that are not electrically Ion channels work together in complex ways to control the behavior of electri-active contain voltage-gated ion cally excitable cells. A typical neuron, for example, receives thousands of excitatory channels? and inhibitory inputs, which combine by spatial and temporal summation to produce a combined postsynaptic potential (PSP) at the initial segment of its axon. The • How are memories stored for so magnitude of the PSP is translated into the rate of firing

1	summation to produce a combined postsynaptic potential (PSP) at the initial segment of its axon. The • How are memories stored for so magnitude of the PSP is translated into the rate of firing of action potentials by a many years in the human brain? mixture of cation channels in the initial segment membrane.

1	Which statements are true? explain why or why not. 11–1 Transport by transporters can be either active or b. Does the linked action of these two pumps cause passive, whereas transport by channels is always passive. imbalances in either the K+ concentration or the membrane potential? Why or why not? 11–2 Transporters saturate at high concentrations of 11–7 Microvilli increase the surface area of intestinal the transported molecule when all their binding sites are cells, providing more efficient absorption of nutrients.

1	occupied; channels, on the other hand, do not bind the Microvilli are shown in profile and cross section in Figure ions they transport and thus the flux of ions through a Q11–1. From the dimensions given in the figure, estimate channel does not saturate. the increase in surface area that microvilli provide (for 11–3 The membrane potential arises from movements the portion of the plasma membrane in contact with the of charge that leave ion concentrations practically unaf-lumen of the gut) relative to the corresponding surface of a fected, causing only a very slight discrepancy in the num-cell with a “flat” plasma membrane. ber of positive and negative ions on the two sides of the membrane. Discuss the following problems. 11–4 Order Ca2+, CO2, ethanol, glucose, RNA, and H2O according to their ability to diffuse through a lipid bilayer, beginning with the one that crosses the bilayer most readily. Explain your order.

1	11–4 Order Ca2+, CO2, ethanol, glucose, RNA, and H2O according to their ability to diffuse through a lipid bilayer, beginning with the one that crosses the bilayer most readily. Explain your order. 11–5 How is it possible for some molecules to be at 1 µm 0.1 µm equilibrium across a biological membrane and yet not be at the same concentration on both sides?

1	Figure Q11–1 Microvilli of intestinal epithelial cells in profile and cross section (Problem 11–7). (Left panel, from Rippel Electron Microscope 11–6 Ion transporters are “linked” together—not physi-Facility, Dartmouth College; right panel, from David Burgess.) cally, but as a consequence of their actions. For example, cells can raise their intracellular pH, when it becomes too acidic, by exchanging external Na+ for internal H+, using 11–8 According to Newton’s laws of motion, an ion a Na+–H+ antiporter. The change in internal Na+ is then exposed to an electric field in a vacuum would experience redressed using the Na+-K+ pump. a constant acceleration from the electric driving force, just a. Can these two transporters, operating together, as a falling body in a vacuum constantly accelerates due to normalize both the H+ and the Na+ concentrations inside gravity. In water, however, an ion moves at constant velocthe cell? ity in an electric field. Why do you suppose that is? 21.4 nm

1	21.4 nm Figure Q11–2 A “ball” tethered by a “chain” to a voltage-gated K+ channel (Problem 11–9). 11–9 In a subset of voltage-gated K+ channels, the N-terminus of each subunit acts like a tethered ball that occludes the cytoplasmic end of the pore soon after it opens, thereby inactivating the channel. This “ball-andchain” model for the rapid inactivation of voltage-gated K+ channels has been elegantly supported for the shaker K+ channel from Drosophila melanogaster. (The shaker K+ channel in Drosophila is named after a mutant form that causes excitable behavior—even anesthetized flies keep twitching.) Deletion of the N-terminal amino acids from the normal shaker channel gives rise to a channel that opens in response to membrane depolarization, but stays open instead of rapidly closing as the normal channel does. A peptide (MAAVAGLYGLGEDRQHRKKQ) that corresponds to the deleted N-terminus can inactivate the open channel at 100 µM.

1	Is the concentration of free peptide (100 µM) that is required to inactivate the defective K+ channel anywhere near the local concentration of the tethered ball on a normal channel? Assume that the tethered ball can explore a hemisphere [volume = (2/3)πr3] with a radius of 21.4 nm, which is the length of the polypeptide “chain” (Figure Q11–2). Calculate the concentration for one ball in this hemisphere. How does that value compare with the concentration of free peptide needed to inactivate the channel? 11–10 The giant axon of the squid (Figure Q11–3) occupies a unique position in the history of our understanding of cell membrane potentials and nerve action. When an electrode is stuck into an intact giant axon, the membrane potential registers –70 mV. When the axon, suspended in a bath of seawater, is stimulated to conduct a nerve impulse, the membrane potential changes transiently from –70 mV to +40 mV.

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1	Hodgkin AL & Huxley AF (1952) Currents carried by sodium and potassium ions through the membrane of the giant axon of Loligo. J. Physiol. 116, 449–472. Jessell TM & Kandel ER (1993) Synaptic transmission: a bidirectional and self-modifiable form of cell–cell communication. Cell 72(Suppl), 1–30. Julius D (2013) TRP channels and pain. Annu. Rev. Cell Dev. Biol. 29, 355–384. Katz B (1966) Nerve, Muscle and Synapse. New York: McGraw-Hill. King LS, Kozono D & Agre P (2004) From structure to disease: the evolving tale of aquaporin biology. Nat. Rev. Mol. Cell Biol. 5, 687–698. Liao M, Cao E, Julius D & Cheng Y (2014) Single particle electron cryo-microscopy of a mammalian ion channel. Curr. Opin. Struct. Biol. 27, 1–7. MacKinnon R (2003) Potassium channels. FEBS Lett. 555, 62–65. Miesenböck G (2011) Optogenetic control of cells and circuits. Annu. Rev. Cell Dev. Biol. 27, 731–758. Moss SJ & Smart TG (2001) Constructing inhibitory synapses. Nat. Rev. Neurosci. 2, 240–250.

1	Miesenböck G (2011) Optogenetic control of cells and circuits. Annu. Rev. Cell Dev. Biol. 27, 731–758. Moss SJ & Smart TG (2001) Constructing inhibitory synapses. Nat. Rev. Neurosci. 2, 240–250. Neher E & Sakmann B (1992) The patch clamp technique. Sci. Am. 266, 44–51. Nicholls JG, Fuchs PA, Martin AR & Wallace BG (2000) From Neuron to Brain, 4th ed. Sunderland, MA: Sinauer. Numa S (1987) A molecular view of neurotransmitter receptors and ionic channels. Harvey Lect. 83, 121–165. Payandeh J, Scheuer T, Zheng N & Catterall WA (2011) The crystal structure of a voltage-gated sodium channel. Nature 475, 353–358. Scannevin RH & Huganir RL (2000) Postsynaptic organization and regulation of excitatory synapses. Nat. Rev. Neurosci. 1, 133–141. Snyder SH (1996) Drugs and the Brain. New York: WH Freeman/ Scientific American Books. Sobolevsky AI, Rosconi MP & Gouaux E (2009) X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor. Nature 462, 745–756.

1	Sobolevsky AI, Rosconi MP & Gouaux E (2009) X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor. Nature 462, 745–756. Stevens CF (2004) Presynaptic function. Curr. Opin. Neurobiol. 14, 341–345. Verkman AS (2013) Aquaporins. Curr. Biol. 23, R52–R55. Unlike a bacterium, which generally consists of a single intracellular compartment surrounded by a plasma membrane, a eukaryotic cell is elaborately subdivided into functionally distinct, membrane-enclosed compartments. Each compartment, or organelle, contains its own characteristic set of enzymes and other specialized molecules, and complex distribution systems transport specific products from one compartment to another. To understand the eukaryotic cell, it is essential to know how the cell creates and maintains these compartments, what occurs in each of them, and how molecules move between them.

1	Proteins confer upon each compartment its characteristic structural and functional properties. They catalyze the reactions that occur there and selectively transport small molecules into and out of the compartment. For membrane-enclosed organelles in the cytoplasm, proteins also serve as organelle-specific surface markers that direct new deliveries of proteins and lipids to the appropriate organelle. An animal cell contains about 10 billion (1010) protein molecules of perhaps 10,000 kinds, and the synthesis of almost all of them begins in the cytosol, the space of the cytoplasm outside the membrane-enclosed organelles. Each newly synthesized protein is then delivered specifically to the organelle that requires it. The intracellular transport of proteins is the central theme of both this chapter and the next. By tracing the protein traffic from one compartment to another, one can begin to make sense of the otherwise bewildering maze of intracellular membranes.

1	The ComparTmenTalizaTion of Cells In this brief overview of the compartments of the cell and the relationships between them, we organize the organelles conceptually into a small number of discrete families, discuss how proteins are directed to specific organelles, and explain how proteins cross organelle membranes. all eukaryotic Cells have the same Basic set of membrane-enclosed organelles

1	Many vital biochemical processes take place in membranes or on their surfaces. Membrane-bound enzymes, for example, catalyze lipid metabolism; and oxidative phosphorylation and photosynthesis both require a membrane to couple the transport of H+ to the synthesis of ATP. In addition to providing increased membrane area to host biochemical reactions, intracellular membrane systems form enclosed compartments that are separate from the cytosol, thus creating functionally specialized aqueous spaces within the cell. In these spaces, subsets of molecules (proteins, reactants, ions) are concentrated to optimize the biochemical reactions in which they participate. Because the lipid bilayer of cell membranes is impermeable to most hydrophilic molecules, the membrane of an organelle must contain membrane transport proteins to import and export specific metabolites. Each organelle membrane must also have a mechanism for importing, and incorporating into the organelle, the specific proteins that

1	transport proteins to import and export specific metabolites. Each organelle membrane must also have a mechanism for importing, and incorporating into the organelle, the specific proteins that make the organelle unique.

1	The ComparTmenTalizaTion of Cells The TransporT of moleCUles BeTWeen The nUCleUs anD The CYTosol The TransporT of proTeins inTo miToChonDria anD ChloroplasTs The enDoplasmiC reTiCUlUm 642 Chapter 12: intracellular Compartments and protein sorting endoplasmic reticulum with membrane-bound polyribosomes Figure 12–1 illustrates the major intracellular compartments common to eukaryotic cells. The nucleus contains the genome (aside from mitochondrial and chloroplast DNA), and it is the principal site of DNA and RNA synthesis. The surrounding cytoplasm consists of the cytosol and the cytoplasmic organelles suspended in it. The cytosol constitutes a little more than half the total volume of the cell, and it is the main site of protein synthesis and degradation. It also performs most of the cell’s intermediary metabolism—that is, the many reactions that degrade some small molecules and synthesize others to provide the building blocks for macromolecules (discussed in Chapter 2).

1	About half the total area of membrane in a eukaryotic cell encloses the labyrinthine spaces of the endoplasmic reticulum (ER). The rough ER has many ribosomes bound to its cytosolic surface. Ribosomes are organelles that are not membrane-enclosed; they synthesize both soluble and integral membrane proteins, most of which are destined either for secretion to the cell exterior or for other organelles. We shall see that, whereas proteins are transported into other membrane-enclosed organelles only after their synthesis is complete, they are transported into the ER as they are synthesized. This explains why the ER membrane is unique in having ribosomes tethered to it. The ER also produces most of the lipid for the rest of the cell and functions as a store for Ca2+ ions. Regions of the ER that lack bound ribosomes are called smooth ER. The ER sends many of its proteins and lipids to the Golgi apparatus, which often consists of organized stacks of disc-like compartments called Golgi

1	ER that lack bound ribosomes are called smooth ER. The ER sends many of its proteins and lipids to the Golgi apparatus, which often consists of organized stacks of disc-like compartments called Golgi cisternae. The Golgi apparatus receives lipids and proteins from the ER and dispatches them to various destinations, usually covalently modifying them en route.

1	Mitochondria and chloroplasts generate most of the ATP that cells use to drive reactions requiring an input of free energy; chloroplasts are a specialized version of plastids (present in plants, algae, and some protozoa), which can also have other functions, such as the storage of food or pigment molecules. Lysosomes contain digestive enzymes that degrade defunct intracellular organelles, as well as macromolecules and particles taken in from outside the cell by endocytosis. On the way to lysosomes, endocytosed material must first pass through a series of organelles called endosomes. Finally, peroxisomes are small vesicular compartments that contain enzymes used in various oxidative reactions. In general, each membrane-enclosed organelle performs the same set of basic functions in all cell types. But to serve the specialized functions of cells, these organelles vary in abundance and can have additional properties that differ from cell type to cell type.

1	On average, the membrane-enclosed compartments together occupy nearly half the volume of a cell (Table 12–1), and a large amount of intracellular membrane is required to make them. In liver and pancreatic cells, for example, the Figure 12–1 The major intracellular compartments of an animal cell. The cytosol (gray), endoplasmic reticulum, Golgi apparatus, nucleus, mitochondrion, endosome, lysosome, and peroxisome are distinct compartments isolated from the rest of the cell by at least one selectively permeable membrane (see movie 9.2). endoplasmic reticulum has a total membrane surface area that is, respectively, 25 times and 12 times that of the plasma membrane (Table 12–2). The membrane-enclosed organelles are packed tightly in the cytoplasm, and, in terms of area and mass, the plasma membrane is only a minor membrane in most eukaryotic cells (Figure 12–2).

1	The abundance and shape of membrane-enclosed organelles are regulated to meet the needs of the cell. This is particularly apparent in cells that are highly specialized and therefore disproportionately rely on specific organelles. Plasma cells, for example, which secrete their own weight every day in antibody molecules into the bloodstream, contain vastly amplified amounts of rough ER, which is found in large, flat sheets. Cells that specialize in lipid synthesis also expand their ER, but in this case the organelle forms a network of convoluted tubules. Moreover, membrane-enclosed organelles are often found in characteristic positions in the cytoplasm. In most cells, for example, the Golgi apparatus is located close to the nucleus, whereas the network of ER tubules extends from the nucleus throughout the entire cytosol. These characteristic distributions depend on interactions of the organelles with the cytoskeleton. The localization of both the ER and the Golgi apparatus, for

1	nucleus throughout the entire cytosol. These characteristic distributions depend on interactions of the organelles with the cytoskeleton. The localization of both the ER and the Golgi apparatus, for instance, depends on an intact microtubule array; if the microtubules are experimentally depolymerized with a drug, the Golgi apparatus fragments and disperses throughout the cell, and the ER network collapses toward the cell center (discussed in Chapter 16). The size, shape, composition, and location are all important and regulated features of these organelles that ultimately contribute to the organelle’s function.

1	evolutionary origins may help explain the Topological relationships of organelles

1	To understand the relationships between the compartments of the cell, it is helpful to consider how they might have evolved. The precursors of the first eukaryotic cells are thought to have been relatively simple cells that—like most bacterial and archaeal cells—have a plasma membrane but no internal membranes. The plasma membrane in such cells provides all membrane-dependent functions, including the pumping of ions, ATP synthesis, protein secretion, and lipid synthesis. Typical present-day eukaryotic cells are 10–30 times larger in linear dimension and 1000–10,000 times greater in volume than a typical bacterium such as E. coli. The profusion of internal membranes can be regarded, in part, as an adaptation to this increase in size: the eukaryotic cell has a much smaller ratio of surface area to volume, and its plasma membrane therefore presumably has too small an area to sustain the many vital functions that membranes perform. The extensive internal membrane systems of a eukaryotic

1	area to volume, and its plasma membrane therefore presumably has too small an area to sustain the many vital functions that membranes perform. The extensive internal membrane systems of a eukaryotic cell alleviate this problem.

1	The evolution of internal membranes evidently went hand-in-hand with the specialization of membrane function. A hypothetical scheme for how the first eukaryotic cells, with a nucleus and ER, might have evolved by the invagination and pinching off of the plasma membrane of an ancestral cell is illustrated in Figure 12–3.This process would create membrane-enclosed organelles with an interior or lumen that is topologically equivalent to the exterior of the cell. We shall see that this topological relationship holds for all of the organelles involved in the secretory and endocytic pathways, including the ER, Golgi apparatus, endosomes, lysosomes, and peroxisomes. We can therefore think of all of these organelles as members of the same topologically equivalent compartment. As we discuss in detail in the next chapter, their interiors communicate extensively with one another and with the outside of the cell via transport vesicles, which bud off from one organelle and fuse with another

1	in detail in the next chapter, their interiors communicate extensively with one another and with the outside of the cell via transport vesicles, which bud off from one organelle and fuse with another (Figure 12–4).

1	As described in Chapter 14, mitochondria and plastids differ from the other membrane-enclosed organelles because they contain their own genomes. The nature of these genomes, and the close resemblance of the proteins in these organelles to those in some present-day bacteria, strongly suggest that mitochondria and plastids evolved from bacteria that were engulfed by other cells with which they initially lived in symbiosis (see Figures 1–29 and 1–31): the inner membrane of mitochondria and plastids presumably corresponds to the original plasma membrane of the bacterium, while the lumen of these organelles evolved from the bacterial cytosol. Like the bacteria from which they were derived, both mitochondria and plastids are enclosed by a double membrane and they remain isolated from the extensive vesicular traffic that connects the interiors of most of the other membrane-enclosed organelles to each other and to the outside of the cell.

1	Figure 12–2 An electron micrograph of part of a liver cell seen in cross section. examples of most of the major intracellular organelles are indicated. (Courtesy of Daniel s. friend.) ARCHAEAL AND BACTERIAL) WALL IN ANCIENT GREATLY INCREASES ANAEROBIC ARCHAEON HORIZONTAL GENE FACILITATES HORIZONTAL TRANSFERS, SPEEDING GENE TRANSFERS EVOLUTIONARY PROCESSES The evolutionary schemes just described group the intracellular compartments in eukaryotic cells into four distinct families: (1) the nucleus and the cytosol, which communicate with each other through nuclear pore complexes and are thus topologically continuous (although functionally distinct); (2) all organelles that function in the secretory and endocytic pathways—including the ER, Golgi apparatus, endosomes, and lysosomes, the numerous classes of transport intermediates such as transport vesicles that move between them, and peroxisomes; (3) the mitochondria; and (4) the plastids (in plants only).

1	The synthesis of all proteins begins on ribosomes in the cytosol, except for the few that are synthesized on the ribosomes of mitochondria and plastids. Their subsequent fate depends on their amino acid sequence, which can contain sorting signals that direct their delivery to locations outside the cytosol or to organelle surfaces. Some proteins do not have a sorting signal and consequently remain in the cytosol as permanent residents. Many others, however, have specific sorting signals that direct their transport from the cytosol into the nucleus, the ER, mitochondria, plastids, or peroxisomes; sorting signals can also direct the transport of proteins from the ER to other destinations in the cell.

1	Figure 12–3 One suggested pathway for the evolution of the eukaryotic cell and its internal membranes as discussed in Chapter 1, there is evidence that the nuclear genome of a eukaryotic cell evolved from an ancient archeaon. for example, clear homologs of actin, tubulin, histones, and the nuclear Dna replication system are found in archaea, but not in bacteria. Thus, it is now thought that the first eukaryotic cells arose when an ancient anaerobic archaeon joined forces with an aerobic bacterium roughly 1.6 billion years ago. as indicated, the nuclear envelope may have originated from an invagination of the plasma membrane of this ancient archaeon—an invagination that protected its chromosome while still allowing access of the Dna to the cytosol (as required for Dna to direct protein synthesis). This envelope may have later pinched off completely from the plasma membrane, so as to produce a separate nuclear compartment surrounded by a double membrane. Because this double membrane is

1	This envelope may have later pinched off completely from the plasma membrane, so as to produce a separate nuclear compartment surrounded by a double membrane. Because this double membrane is penetrated by nuclear pore complexes, the nuclear compartment is topologically equivalent to the cytosol. in contrast, the lumen of the er is continuous with the space between the inner and outer nuclear membranes, and it is topologically equivalent to the extracellular space (see figure 12–4). (adapted from J. martijn and

1	T.J.G. ettema, Biochem. Soc. Trans. 41: 451–457, 2013.) To understand the general principles by which sorting signals operate, it is important to distinguish three fundamentally different ways by which proteins move from one compartment to another. These three mechanisms are described below, and the transport steps at which they operate are outlined in Figure 12–5. We discuss the first two mechanisms (gated transport and transmembrane transport) in this chapter, and the third (vesicular transport, green arrows in Figure 12–5) in Chapter 13. 1. In gated transport, proteins and RNA molecules move between the cytosol and the nucleus through nuclear pore complexes in the nuclear envelope. The nuclear pore complexes function as selective gates that support the active transport of specific macromolecules and macromolecular assemblies between the two topologically equivalent spaces, although they also allow free diffusion of smaller molecules. 2.

1	2. In protein translocation, transmembrane protein translocators directly transport specific proteins across a membrane from the cytosol into a space that is topologically distinct. The transported protein molecule usually must unfold to snake through the translocator. The initial transport of selected proteins from the cytosol into the ER lumen or mitochondria, for example, occurs in this way. Integral membrane proteins often use the same translocators but translocate only partially across the membrane, so that the protein becomes embedded in the lipid bilayer. 3.

1	3. In vesicular transport, membrane-enclosed transport intermediates— which may be small, spherical transport vesicles or larger, irregularly shaped organelle fragments—ferry proteins from one topologically equivalent compartment to another. The transport vesicles and fragments become loaded with a cargo of molecules derived from the lumen of one compartment as they bud and pinch off from its membrane; they discharge their cargo into a second compartment by fusing with the membrane enclosing that compartment (Figure 12–6). The transfer of soluble proteins from the ER to the Golgi apparatus, for example, occurs in this way. Because the

1	Figure 12–5 A simplified “roadmap” of protein traffic within a eukaryotic cell. proteins can move from one compartment to another by gated transport (red), protein translocation (blue), or vesicular transport (green). The sorting signals that direct a given protein’s movement through the system, and thereby determine its eventual location in the cell, are contained in each protein’s amino acid sequence. The journey begins with the synthesis of a protein on a ribosome in the cytosol and, for many proteins, terminates when the protein reaches its final destination. other proteins shuttle back and forth between the nucleus and cytosol. at each intermediate station (boxes), a decision is made as to whether the protein is to be retained in that compartment or transported further. a sorting signal may direct either retention in or exit from a compartment.

1	We shall refer to this figure often as a guide in this chapter and the next, highlighting in color the particular pathway being discussed.

1	Figure 12–4 Topologically equivalent compartments in the secretory and endocytic pathways in a eukaryotic cell. Compartments are said to be topologically equivalent if they can communicate with one another, in the sense that molecules can get from one to the other without having to cross a membrane. Topologically equivalent spaces are shown in red. (a) molecules can be carried from one compartment to another topologically equivalent compartment by vesicles that bud from one and fuse with the other. (B) in principle, cycles of membrane budding and fusion permit the lumen of any of the organelles shown to communicate with any other and with the cell exterior by means of transport vesicles. Blue arrows indicate the extensive outbound and inbound vesicular traffic (discussed in Chapter 13). some organelles, most notably mitochondria and (in plant cells) plastids, do not take part in this communication and are isolated from the vesicular traffic between organelles shown here.

1	= gated transport = transmembrane transport = vesicular transport KEY: Figure 12–6 Vesicle budding and fusion during vesicular transport. Transport vesicles bud from one compartment (donor) and fuse with another topologically equivalent (target) compartment. in the process, soluble components (red dots) are transferred from lumen to lumen. note that membrane is also transferred and that the original orientation of both proteins and lipids in the donor compartment membrane is preserved in the target compartment membrane. Thus, membrane proteins retain their asymmetric orientation, with the same domains always facing the cytosol. transported proteins do not cross a membrane, vesicular transport can move proteins only between compartments that are topologically equiva lent (see Figure 12–4).

1	transported proteins do not cross a membrane, vesicular transport can move proteins only between compartments that are topologically equiva lent (see Figure 12–4). Each mode of protein transfer is usually guided by sorting signals in the transported protein, which are recognized by complementary sorting receptors. If a large protein is to be imported into the nucleus, for example, it must possess a sorting signal that receptor proteins recognize to guide it through the nuclear pore complex. If a protein is to be transferred directly across a membrane, it must possess a sorting signal that the translocator recognizes. Likewise, if a protein is to be loaded into a certain type of vesicle or retained in certain organelles, a complementary receptor in the appropriate membrane must recognize its sorting signal. signal sequences and sorting receptors Direct proteins to the Correct Cell address

1	signal sequences and sorting receptors Direct proteins to the Correct Cell address Most protein sorting signals involved in transmembrane transport reside in a stretch of amino acid sequence, typically 15–60 residues long. Such signal sequences are often found at the N-terminus of the polypeptide chain, and in many cases specialized signal peptidases remove the signal sequence from the finished protein once the sorting process is complete. Signal sequences can also be internal stretches of amino acids, which remain part of the protein. Such signals are used in gated transport into the nucleus. Sorting signals can also be composed of multiple internal amino acid sequences that form a specific three-dimensional arrangement of atoms on the protein’s surface; such signal patches are sometimes used for nuclear import and in vesicular transport.

1	Each signal sequence specifies a particular destination in the cell. Proteins destined for initial transfer to the ER usually have a signal sequence at their Nterminus that characteristically includes a sequence composed of about 5–10 hydrophobic amino acids. Many of these proteins will in turn pass from the ER to the Golgi apparatus, but those with a specific signal sequence of four amino acids at their C-terminus are recognized as ER residents and are returned to the ER. Proteins destined for mitochondria have signal sequences of yet another type, in which positively charged amino acids alternate with hydrophobic ones. Finally, many proteins destined for peroxisomes have a signal sequence of three characteristic amino acids at their C-terminus.

1	Table 12–3 presents some specific signal sequences. Experiments in which the peptide is transferred from one protein to another by genetic engineering techniques have demonstrated the importance of each of these signal sequences for protein targeting. Placing the N-terminal ER signal sequence at the beginning of a cytosolic protein, for example, redirects the protein to the ER; removing or mutating the signal sequence of an ER protein causes its retention in the cytosol. Signal sequences are therefore both necessary and sufficient for protein targeting. Even though their amino acid sequences can vary greatly, the signal sequences of proteins having the same destination are functionally interchangeable; physical properties, such as hydrophobicity, often seem to be more important in the signal-recognition process than the exact amino acid sequence.

1	Signal sequences are recognized by complementary sorting receptors that guide proteins to their appropriate destination, where the receptors unload their cargo. The receptors function catalytically: after completing one round of targeting, they return to their point of origin to be reused. Most sorting receptors budding vesicle with contents selected for recognize classes of proteins rather than an individual protein species. They can therefore be viewed as public transportation systems, dedicated to delivering numerous different components to their correct location in the cell. most organelles Cannot Be Constructed De Novo: They require information in the organelle itself

1	When a cell reproduces by division, it has to duplicate its organelles, in addition to its chromosomes. In general, cells do this by incorporating new molecules into the existing organelles, thereby enlarging them; the enlarged organelles then divide and are distributed to the two daughter cells. Thus, each daughter cell inherits a complete set of specialized cell membranes from its mother. This inheritance is essential because a cell could not make such membranes from scratch. If the ER were completely removed from a cell, for example, how could the cell reconstruct it? As we discuss later, the membrane proteins that define the ER and perform many of its functions are themselves products of the ER. A new ER could not be made without an existing ER or, at least, a membrane that specifically contains the protein translocators required to import selected proteins into the ER from the cytosol (including the ER-specific translocators themselves). The same is true for mitochondria and

1	contains the protein translocators required to import selected proteins into the ER from the cytosol (including the ER-specific translocators themselves). The same is true for mitochondria and plastids.

1	Thus, it seems that the information required to construct an organelle does not reside exclusively in the DNA that specifies the organelle’s proteins. Information in the form of at least one distinct protein that preexists in the organelle membrane is also required, and this information is passed from parent cell to daughter cells in the form of the organelle itself. Presumably, such information is essential for the propagation of the cell’s compartmental organization, just as the information in DNA is essential for the propagation of the cell’s nucleotide and amino acid sequences.

1	As we discuss in more detail in Chapter 13, however, the ER buds off a constant stream of transport vesicles that incorporate only a subset of ER proteins and therefore have a composition different from the ER itself. Similarly, the plasma membrane constantly buds off various types of specialized endocytic vesicles. Thus, some organelles can form from other organelles and do not have to be inherited at cell division. Eukaryotic cells contain intracellular membrane-enclosed organelles that make up nearly half the cell’s total volume. The main ones present in all eukaryotic cells are the endoplasmic reticulum, Golgi apparatus, nucleus, mitochondria, lysosomes, endosomes, and peroxisomes; plant cells also contain plastids such as chloroplasts. These organelles contain distinct sets of proteins, which mediate each organelle’s unique function.

1	Each newly synthesized organelle protein must find its way from a ribosome in the cytosol, where the protein is made, to the organelle where it functions. It does so by following a specific pathway, guided by sorting signals in its amino acid sequence that function as either signal sequences or signal patches. Sorting signals are recognized by complementary sorting receptors, which deliver the protein to the appropriate target organelle. Proteins that function in the cytosol do not contain sorting signals and therefore remain there after they are synthesized. During cell division, organelles such as the ER and mitochondria are distributed to each daughter cell. These organelles contain information that is required for their construction, and so they cannot be made de novo. The TransporT of moleCUles BeTWeen The nUCleUs anD The CYTosol The nuclear envelope encloses the DNA and defines the nuclear compartment.

1	This envelope consists of two concentric membranes, which are penetrated by nuclear pore complexes (Figure 12–7). Although the inner and outer nuclear membranes are continuous, they maintain distinct protein compositions. The inner nuclear membrane contains proteins that act as binding sites for chromosomes and for the nuclear lamina, a protein meshwork that provides structural support for the nuclear envelope; the lamina also acts as an anchoring site for chromosomes and the cytoplasmic cytoskeleton (via protein complexes that span the nuclear envelope). The inner membrane is surrounded by the outer nuclear membrane, which is continuous with the membrane of the ER. Like the ER membrane (discussed later), the outer nuclear membrane is studded with ribosomes engaged in protein synthesis. The proteins made on these ribosomes are transported into the space between the inner and outer nuclear membranes (the perinuclear space), which is continuous with the ER lumen (see Figure 12–7).

1	Bidirectional traffic occurs continuously between the cytosol and the nucleus. The many proteins that function in the nucleus—including histones, DNA polymerases, RNA polymerases, transcriptional regulators, and RNA-processing proteins—are selectively imported into the nuclear compartment from the cytosol, where they are made. At the same time, almost all RNAs—including mRNAs, rRNAs, tRNAs, miRNAs, and snRNAs—are synthesized in the nuclear compartment and then exported to the cytosol. Like the import process, the export process is selective; mRNAs, for example, are exported only after they have been properly modified by RNA-processing reactions in the nucleus. In some cases, the transport process is complex. Ribosomal proteins, for instance, are made in the cytosol and imported into the nucleus, where they assemble with newly made ribosomal RNA into particles. The particles are then exported to the cytosol, where they assemble into ribosomes. Each of these steps requires selective

1	nucleus, where they assemble with newly made ribosomal RNA into particles. The particles are then exported to the cytosol, where they assemble into ribosomes. Each of these steps requires selective transport across the nuclear envelope.

1	nuclear pore Complexes perforate the nuclear envelope

1	Large and elaborate nuclear pore complexes (NPCs) perforate the nuclear envelope in all eukaryotes. Each NPC is composed of a set of approximately 30 different proteins, or nucleoporins. Reflecting the high degree of internal symmetry, each nucleoporin is present in multiple copies, resulting in 500–1000 protein molecules in the fully assembled NPC, with an estimated mass of 66 million daltons in yeast and 125 million daltons in vertebrates (Figure 12–8). Most nucleoporins are composed of repetitive protein domains of only a few different types, which have evolved through extensive gene duplication. Some of the scaffold nucleoporins (see Figure 12–8) are structurally related to vesicle coat protein complexes, such as clathrin and COPII coatomer (discussed in Chapter 13), which shape transport vesicles; and one protein is used as a common building block in both NPCs and vesicle coats. These similarities suggest a common evolutionary origin for NPCs and vesicle coats: they may derive

1	vesicles; and one protein is used as a common building block in both NPCs and vesicle coats. These similarities suggest a common evolutionary origin for NPCs and vesicle coats: they may derive from an early membrane-bending protein module that helped shape the elaborate membrane systems of eukaryotic cells, and in present-day cells stabilize the sharp membrane bends required to form a nuclear pore.

1	The nuclear envelope of a typical mammalian cell contains 3000–4000 NPCs, although that number varies widely, from a few hundred in glial cells to almost 20,000 in Purkinje neurons. The total traffic that passes through each NPC is enormous: each NPC can transport up to 1000 macromolecules per second and can transport in both directions at the same time. How it coordinates the bidirectional flow of macromolecules to avoid congestion and head-on collisions is not known.

1	Each NPC contains aqueous passages, through which small water-soluble molecules can diffuse passively. Researchers have determined the effective size of these passages by injecting labeled water-soluble molecules of different sizes into the cytosol and then measuring their rate of diffusion into the nucleus. Small molecules (5000 daltons or less) diffuse in so fast that we can consider the nuclear envelope freely permeable to them. Large proteins, however, diffuse in much more slowly, and the larger a protein, the more slowly it passes through the NPC. Proteins larger than 60,000 daltons cannot enter by passive diffusion. This size cut-off to free diffusion is thought to result from the NPC structure (see Figure 12–8). The channel nucleoporins with extensive unstructured regions form a disordered tangle (much like a kelp bed in the ocean) that restricts the diffusion of large macromolecules while allowing smaller molecules to pass.

1	Because many cell proteins are too large to diffuse passively through the NPCs, the nuclear compartment and the cytosol can maintain different protein compositions. Mature cytosolic ribosomes, for example, are about 30 nm in diameter and thus cannot diffuse through the NPC, confining protein synthesis to the cytosol. But how does the nucleus export newly made ribosomal subunits or import large molecules, such as DNA polymerases and RNA polymerases, which have subunit molecular masses of 100,000–200,000 daltons? As we discuss next, these and most other transported protein and RNA molecules bind to specific receptor proteins that actively ferry large molecules through NPCs. Even small proteins like histones frequently use receptor-mediated mechanisms to cross the NPC, thereby increasing transport efficiency. nuclear localization signals Direct nuclear proteins to the nucleus

1	nuclear localization signals Direct nuclear proteins to the nucleus When proteins are experimentally extracted from the nucleus and reintroduced into the cytosol, even the very large ones reaccumulate efficiently in the nucleus. Sorting signals called nuclear localization signals (NLSs) are responsible for the selectivity of this active nuclear import process. The signals have been precisely defined by using recombinant DNA technology for numerous nuclear proteins, as well as for proteins that enter the nucleus only transiently (Figure 12–9). In many nuclear proteins, the signals consist of one or two short sequences that are rich in the positively charged amino acids lysine and arginine (see Table 12–3, p. 648), with the precise sequence varying for different proteins. Other nuclear proteins contain different signals, some of which are not yet characterized.

1	Nuclear localization signals can be located almost anywhere in the amino acid sequence and are thought to form loops or patches on the protein surface. Many function even when linked as short peptides to lysine side chains on the surface of a cytosolic protein, suggesting that the precise location of the signal within the amino acid sequence of a nuclear protein is not important. Moreover, as long as one of the protein subunits of a multicomponent complex displays a nuclear localization signal, the entire complex will be imported into the nucleus. Figure 12–7 The nuclear envelope. The double-membrane envelope is penetrated by pores in which nuclear pore complexes (not shown) are positioned. The outer nuclear membrane is continuous with the endoplasmic reticulum (er). The ribosomes that are normally bound to the cytosolic surface of the er membrane and outer nuclear membrane are not shown. The nuclear lamina is a fibrous protein meshwork underlying the inner membrane.

1	One can visualize the transport of nuclear proteins through NPCs by coating gold particles with a nuclear localization signal, injecting the particles into the cytosol, and then following their fate by electron microscopy (Figure 12–10). The particles bind to the tentaclelike fibrils that extend from the scaffold nucleoporins at the rim of the NPC into the cytosol, and then proceed through the center of the NPC. Presumably, the unstructured regions of the nucleoporins that form a diffusion barrier for large molecules (mentioned earlier) are pushed away to allow the coated gold particles to squeeze through. Macromolecular transport across NPCs differs fundamentally from the transport of proteins across the membranes of other organelles, in that it occurs (B) 0.1 µm inner nuclear membrane nuclear envelope outer nuclear membrane 50 nm scaffold nucleoporins membrane ring proteins channel nucleoporinsnuclear basket disordered region of channel nucleoporins cytosolic fbrils 0.1 µm 0.1 µm

1	Figure 12–8 The arrangement of NPCs in the nuclear envelope. (a) in a vertebrate npC, nucleoporins are arranged with striking eightfold rotational symmetry. in addition, immunoelectron microscopic studies show that the proteins that make up the central portion of the npC are oriented symmetrically across the nuclear envelope, so that the nuclear and cytosolic sides look identical. The eightfold rotational and twofold transverse symmetry explains how such a huge structure can be formed from only about 30 different proteins: many of the nucleoporins are present in 8, 16, or 32 copies. Based on their approximate localization in the central portion of the npC, nucleoporins can be classified into (1) transmembrane ring proteins that span the nuclear envelope and anchor the npC to the envelope; (2) scaffold nucleoporins that form layered ring structures. some scaffold nucleoporins are membrane-bending proteins that stabilize the sharp membrane curvature where the nuclear envelope is

1	(2) scaffold nucleoporins that form layered ring structures. some scaffold nucleoporins are membrane-bending proteins that stabilize the sharp membrane curvature where the nuclear envelope is penetrated; and (3) channel nucleoporins that line a central pore. in addition to folded domains that anchor the proteins in specific places, many channel nucleoporins contain extensive unstructured regions, where the polypeptide chains are intrinsically disordered. The central pore is filled with a tangled mesh of these disordered domains that blocks the passive diffusion of large macromolecules. The disordered regions contain a large number of phenylalanine-glycine (fG) repeats. fibrils protrude from both the cytosolic and the nuclear sides of the npC. By contrast to the twofold transverse symmetry of the npC core, the fibrils facing the cytosol and nucleus are different: on the nuclear side, the fibrils converge at their distal end to form a basketlike structure. The precise arrangement of

1	of the npC core, the fibrils facing the cytosol and nucleus are different: on the nuclear side, the fibrils converge at their distal end to form a basketlike structure. The precise arrangement of individual nucleoporins in the assembled npC is still a matter of intense debate, because atomic resolution analyses have been hindered by the sheer size and flexible nature of the npC, and by difficulties in purifying sufficient amounts of homogeneous material. a combination of electron microscopy, computational analyses, and crystal structures of nucleoporin subcomplexes has been used to develop the current models of the npC architecture. (B) a scanning electron micrograph of the nuclear side of the nuclear envelope of an oocyte (see also figure 9–52). (C) an electron micrograph showing a side view of two npCs (brackets); note that the inner and outer nuclear membranes are continuous at the edges of the pore. (D) an electron micrograph showing face-on views of negatively stained npCs. The

1	view of two npCs (brackets); note that the inner and outer nuclear membranes are continuous at the edges of the pore. (D) an electron micrograph showing face-on views of negatively stained npCs. The membrane has been removed by detergent extraction. note that some of the npCs contain material in their center, which is thought to be trapped macromolecules in transit through these npCs. (a, adapted from a. hoelz, e.W. Debler and G. Blobel, Annu. Rev. Biochem. 80:613– 643, 2011. With permission from annual reviews; B, from m.W. Goldberg and T.D. allen, J. Cell Biol. 119:1429–1440, 1992. With permission from The rockefeller University press; C, courtesy of Werner franke and Ulrich scheer; D, courtesy of ron milligan.) through a large, expandable, aqueous pore, rather than through a protein transporter spanning one or more lipid bilayers. For this reason, fully folded nuclear proteins can be transported into the nucleus through an NPC, and newly formed ribosomal subunits are transported

1	transporter spanning one or more lipid bilayers. For this reason, fully folded nuclear proteins can be transported into the nucleus through an NPC, and newly formed ribosomal subunits are transported out of the nucleus as an assembled particle. By contrast, proteins have to be extensively unfolded to be transported into most other organelles, as we discuss later.

1	nuclear import receptors Bind to Both nuclear localization signals and npC proteins To initiate nuclear import, most nuclear localization signals must be recognized by nuclear import receptors, sometimes called importins, most of which are encoded by a family of related genes. Each family member encodes a receptor protein that can bind and transport the subset of cargo proteins containing the appropriate nuclear localization signal (Figure 12–11A). Nuclear import receptors do not always bind to nuclear proteins directly. Additional adaptor proteins can form a bridge between the import receptors and the nuclear localization signals on the proteins to be transported (Figure 12–11B). Some adaptor proteins are structurally related to nuclear import receptors, suggesting a common evolutionary origin. By using a variety of import receptors and adaptors, cells are able to recognize the broad repertoire of nuclear localization signals that are displayed on nuclear proteins.

1	The import receptors are soluble cytosolic proteins that bind both to the nuclear localization signal on the cargo protein and to the phenylalanine-glycine (FG) repeats in the unstructured domains of the channel nucleoporins that line the central pore. FG-repeats are also found in the cytoplasmic and nuclear fibrils. FG-repeats in the unstructured tangle of the pore are thought to do double duty. They interact weakly, which gives the protein tangle gel-like properties that impose a permeability barrier to large macromolecules, and they serve as docking sites for nuclear import receptors. FG-repeats line the path through the NPCs taken by the import receptors and their bound cargo proteins. According to one model of nuclear transport, the receptor–cargo complexes move along the transport path by repeatedly binding, dissociating, and then re-binding to adjacent FG-repeat sequences. In this way, the complexes may hop from one nucleoporin to another to traverse the tangled interior of the

1	by repeatedly binding, dissociating, and then re-binding to adjacent FG-repeat sequences. In this way, the complexes may hop from one nucleoporin to another to traverse the tangled interior of the NPC in a random walk. As import receptors bind to FG-repeats during this journey, they would disrupt interaction between the repeats and locally dissolve the gel phase of the protein tangle that fills the pore, allowing the passage of the receptor–cargo complex. Once inside the nucleus, the import receptors dissociate from their cargo and return to the cytosol. As we will see, this dissociation only occurs on the nuclear side of the NPC and thereby confers directionality to the import process.

1	nuclear export Works like nuclear import, But in reverse The nuclear export of large molecules, such as new ribosomal subunits and RNA molecules, occurs through NPCs and also depends on a selective transport Figure 12–9 The function of a nuclear localization signal. immunofluorescence micrographs showing the cell location of sV40 virus T-antigen containing or lacking a short sequence that serves as a nuclear localization signal. (a) The normal T-antigen protein contains the lysine-rich sequence indicated and is imported to its site of action in the nucleus, as indicated by immunofluorescence staining with antibodies against the T-antigen. T-antigen with an altered nuclear localization signal (a threonine replacing a lysine) remains in the cytosol. (from D. Kalderon, B. roberts, W. richardson and a. smith, Cell 39:499–509, 1984. With permission from elsevier.)

1	Figure 12–10 Visualizing active import through NPCs. This series of electron micrographs shows colloidal gold spheres (arrowheads) coated with peptides containing nuclear localization signals entering the nucleus through npCs. The gold particles were injected into the cytosol of living cells, which then were fixed and prepared for electron microscopy at various times after injection. (a) Gold particles are first seen in proximity to the cytosolic fibrils of the npCs. (B, C) They are then seen at the center of the npCs, exclusively on the cytosolic face. (D) They then appear on the nuclear face. These gold particles have much larger diameters than the diffusion channels in the npC and are imported by active transport. (from n. panté and U. aebi, Science 273:1729–1732, 1996. With permission from aaas.) system. The transport system relies on nuclear export signals on the macromolecules to be exported, as well as on complementary nuclear export receptors, or exportins. These receptors

1	from aaas.) system. The transport system relies on nuclear export signals on the macromolecules to be exported, as well as on complementary nuclear export receptors, or exportins. These receptors bind to both the export signal and NPC proteins to guide their cargo through the NPC to the cytosol.

1	Many nuclear export receptors are structurally related to nuclear import receptors, and they are encoded by the same gene family of nuclear transport receptors, or karyopherins. In yeast, there are 14 genes encoding karyopherins; in animal cells, the number is significantly larger. It is often not possible to tell from their amino acid sequence alone whether a particular family member works as a nuclear import or nuclear export receptor. As might be expected, therefore, the import and export transport systems work in similar ways but in opposite directions: the import receptors bind their cargo molecules in the cytosol, release them in the nucleus, and are then exported to the cytosol for reuse, while the export receptors function in the opposite fashion. The ran GTpase imposes Directionality on Transport Through npCs

1	The ran GTpase imposes Directionality on Transport Through npCs The import of nuclear proteins through NPCs concentrates specific proteins in the nucleus and thereby increases order in the cell. The cell fuels this ordering process by harnessing energy stored in concentration gradients of the GTP-bound form of the monomeric GTPase Ran, which is required for both nuclear import and export.

1	Like other GTPases, Ran is a molecular switch that can exist in two conformational states, depending on whether GDP or GTP is bound (discussed in Chapter 3). Two Ran-specific regulatory proteins trigger the conversion between the two states: a cytosolic GTPase-activating protein (GAP) triggers GTP hydrolysis and thus converts Ran-GTP to Ran-GDP, and a nuclear guanine exchange factor (GEF) promotes the exchange of GDP for GTP and thus converts Ran-GDP to Ran-GTP. Because Ran-GAP is located in the cytosol and Ran-GEF is located in the nucleus where it is anchored to chromatin, the cytosol contains mainly Ran-GDP, and the nucleus contains mainly Ran-GTP (Figure 12–12).

1	Figure 12–11 Nuclear import receptors (importins). (a) Different nuclear import receptors bind different nuclear localization signals and thereby different cargo proteins. (B) Cargo protein 4 requires an adaptor protein to bind to its nuclear import receptor. The adaptors are structurally related to nuclear import receptors and recognize nuclear localization signals on cargo proteins. They also contain a nuclear localization signal that binds them to an import receptor, but this signal only becomes exposed when they are loaded with a cargo protein. Figure 12–12 The compartmentalization of Ran-GDP and Ran-GTP. localization of ran-GDp in the cytosol and ran-GTp in the nucleus results from the localization of two ran regulatory proteins: ran GTpaseactivating protein (ran-Gap) is located in the cytosol, and ran guanine nucleotide exchange factor (ran-Gef) binds to chromatin and is therefore located in the nucleus.

1	ran-GDp is imported into the nucleus by its own import receptor, which is specific for the GDp-bound conformation of ran. The ran-GDp receptor is structurally unrelated to the main family of nuclear transport receptors. however, it also binds to fG-repeats in npC channel nucleoporins. 654 Chapter 12: intracellular Compartments and protein sorting

1	654 Chapter 12: intracellular Compartments and protein sorting This gradient of the two conformational forms of Ran drives nuclear transport in the appropriate direction. Docking of nuclear import receptors to FG-repeats on the cytosolic side of the NPC, for example, occurs whether or not these receptors are loaded with appropriate cargo. Import receptors, facilitated by FG-repeat binding, then enter the channel. If they reach the nuclear side of the pore complex, Ran-GTP binds to them, and, if the receptors arrive loaded with cargo molecules, the Ran-GTP binding causes the receptors to release their cargo (Figure 12–13A). Because the Ran-GDP in the cytosol does not bind to import (or export) receptors, unloading occurs only on the nuclear side of the NPC. In this way, the nuclear localization of Ran-GTP creates the directionality of the import process.

1	Having discharged its cargo in the nucleus, the empty import receptor with Ran-GTP bound is transported back through the pore complex to the cytosol. There, Ran-GAP triggers Ran-GTP to hydrolyze its bound GTP, thereby converting it to Ran-GDP, which dissociates from the receptor. The receptor is then ready for another cycle of nuclear import. Nuclear export occurs by a similar mechanism, except that Ran-GTP in the nucleus promotes cargo binding to the export receptor, rather than promoting cargo dissociation. Once the export receptor moves through the pore to the cytosol, it encounters Ran-GAP, which induces the receptor to hydrolyze its GTP to GDP. As a result, the export receptor releases both its cargo and Ran-GDP in the cytosol. Free export receptors are then returned to the nucleus to complete the cycle (Figure 12–13B). Transport Through npCs Can Be regulated by Controlling access to the Transport machinery

1	Transport Through npCs Can Be regulated by Controlling access to the Transport machinery Some proteins contain both nuclear localization signals and nuclear export signals. These proteins continually shuttle back and forth between the nucleus and the cytosol. The relative rates of their import and export determine the steady-state localization of such shuttling proteins: if the rate of import exceeds the rate of export, a protein will be located mainly in the nucleus; conversely, if the rate of export exceeds the rate of import, a protein will be located mainly in the cytosol. Thus, changing the rate of import, export, or both, can change the location of a protein. Figure 12–13 How GTP hydrolysis by Ran in the cytosol provides directionality to nuclear transport.

1	Figure 12–13 How GTP hydrolysis by Ran in the cytosol provides directionality to nuclear transport. movement through the npC of loaded nuclear transport receptors occurs along the fG-repeats displayed by certain npC proteins. The differential localization of ran-GTp in the nucleus and ran-GDp in the cytosol provides directionality (red arrows) to both nuclear import (a) and nuclear export (B). ran-Gap stimulates the hydrolysis of GTp to produce ran-GDp on the cytosolic side of the npC (see figure 12–12).

1	Some shuttling proteins move continuously into and out of the nucleus. In other cases, however, the transport is stringently controlled. As discussed in Chapter 7, cells control the activity of some transcription regulators by keeping them out of the nucleus until they are needed there (Figure 12–14). In many cases, cells control transport by regulating nuclear localization and export signals—turning them on or off, often by phosphorylation of amino acids close to the signal sequences (Figure 12–15).

1	Other transcription regulators are bound to inhibitory cytosolic proteins that either anchor them in the cytosol (through interactions with the cytoskeleton or specific organelles) or mask their nuclear localization signals so that they cannot interact with nuclear import receptors. An appropriate stimulus releases the gene regulatory protein from its cytosolic anchor or mask, and it is then transported into the nucleus. One important example is the latent gene regulatory protein that controls the expression of proteins involved in cholesterol metabolism. The protein is made and stored in an inactive form as a transmembrane protein in the ER. When a cell is deprived of cholesterol, the protein is transported from the ER to the Golgi apparatus where it encounters specific proteases that cleave off the cytosolic domain, releasing it into the cytosol. This domain is then imported into the nucleus, where it activates the transcription of genes required for both cholesterol uptake and

1	cleave off the cytosolic domain, releasing it into the cytosol. This domain is then imported into the nucleus, where it activates the transcription of genes required for both cholesterol uptake and synthesis (Figure 12–16).

1	As we discuss in detail in Chapter 6, cells control the export of RNAs from the nucleus in a similar way. snRNAs, miRNAs, and tRNAs bind to the same family of nuclear export receptors just discussed, and they use the same Ran-GTP gradient to fuel the transport process. By contrast, the export of mRNAs out of the nucleus uses a different mechanism. mRNAs are exported as large assemblies, which can be as large as 100 million daltons (see Figure 6–37) and can contain hundreds of proteins of a few dozen different types. These mRNA ribonucleoprotein complexes (mRNPs) first dock at the nuclear side of the NPC, where they are extensively remodeled. Although Ran-GTP is indirectly involved in the export (because it imports the proteins that bind to the mRNA molecules), the translocation across the NPC is thought to be driven by ATP hydrolysis. How export directionality is assured is unclear. It is likely that the many accessory proteins tethered to the NPC’s nuclear and cytoplasmic fibrils

1	the NPC is thought to be driven by ATP hydrolysis. How export directionality is assured is unclear. It is likely that the many accessory proteins tethered to the NPC’s nuclear and cytoplasmic fibrils have important roles in remodeling the mRNPs as they pass through the pores, in particular stripping away nuclear proteins as the mRNPs exit on the cytosolic side of the NPC, thereby ensuring that transport is unidirectional. Upon entry into the cytosol, these nuclear mRNP

1	Figure 12–14 The control of nuclear transport in the early Drosophila embryo. The embryo at this stage is a syncytium, shown here in cross section, with many nuclei in a common cytoplasm, arranged around the periphery, just beneath the plasma membrane. The transcription regulatory protein Dorsal is produced uniformly throughout the peripheral cytoplasm, but it can act only when inside the nuclei. The Dorsal protein has been stained with an enzyme-coupled antibody that yields a brown product, revealing that Dorsal is excluded from the nuclei at the dorsal side (top) of the embryo but is concentrated in the nuclei toward the ventral side (bottom) of the embryo. The regulated traffic of Dorsal into the nuclei controls the differential development between the back and belly of the animal. (Courtesy of siegfried roth.)

1	Figure 12–15 The control of nuclear import during T cell activation. The nuclear factor of activated T cells (nf-aT) is a transcription regulatory protein that, in the resting T cell, is found in the cytosol in a phosphorylated state. When T cells are activated by foreign antigen (discussed in Chapter 24), the intracellular Ca2+ concentration increases. in high Ca2+, the protein phosphatase calcineurin binds to nf-aT and dephosphorylates it. The dephosphorylation exposes nuclear import signals and blocks a nuclear export signal. The complex of nf-aT and calcineurin is therefore imported into the nucleus, where nf-aT activates the transcription of numerous genes required for T cell activation.

1	The response shuts off when Ca2+ levels decrease, releasing nf-aT from calcineurin. rephosphorylation of nf-aT inactivates the nuclear import signals and re-exposes the nuclear export signal, causing nf-aT to relocate to the cytosol. some of the most potent immunosuppressive drugs, including cyclosporin a and fK506, inhibit the ability of calcineurin to dephosphorylate nf-aT and thereby block the nuclear accumulation of nf-aT and T cell activation (Movie 12.1). During mitosis the nuclear envelope Disassembles

1	During mitosis the nuclear envelope Disassembles The nuclear lamina, located on the nuclear side of the inner nuclear membrane, is a meshwork of interconnected protein subunits called nuclear lamins. The lamins are a special class of intermediate filament proteins (discussed in Chapter 16) that polymerize into a two-dimensional lattice (Figure 12–17). The nuclear lamina gives shape and stability to the nuclear envelope, to which it is anchored by attachment to both the NPCs and transmembrane proteins of the inner nuclear membrane. The lamina also interacts directly with chromatin, which itself interacts with transmembrane proteins of the inner nuclear membrane. Together with the lamina, these inner membrane proteins provide structural links between the DNA and the nuclear envelope.

1	When a nucleus is dismantled during mitosis, the NPCs and nuclear lamina disassemble and the nuclear envelope fragments. The dismantling process is at least partly a consequence of direct phosphorylation of nucleoporins and lamins by the cyclin-dependent protein kinase (Cdk) that is activated at the onset of mitosis (discussed in Chapter 17). During this process, some NPC proteins become bound to nuclear import receptors, which play an important part in the reassembly of NPCs at the end of mitosis. Nuclear envelope membrane proteins—no longer tethered to the pore complexes, lamina, or chromatin—disperse throughout the ER membrane. The dynein motor protein, which moves along microtubules (discussed in Chapter 16), actively participates in tearing the nuclear envelope off the chromatin. Together, these processes break down the barriers that normally separate the nucleus and cytosol, and the nuclear proteins that are not bound to membranes or chromosomes intermix completely with the

1	Together, these processes break down the barriers that normally separate the nucleus and cytosol, and the nuclear proteins that are not bound to membranes or chromosomes intermix completely with the proteins of the cytosol (Figure 12–18).

1	Later in mitosis, the nuclear envelope reassembles on the surface of the daughter chromosomes. In addition to its crucial role in nuclear transport, the Ran GTPase also acts as a positional marker for chromatin during cell division, when the nuclear and cytosolic components intermix. Because Ran-GEF remains bound to chromatin when the nuclear envelope breaks down, Ran molecules close to chromatin are mainly in their GTP-bound conformation. By contrast, Ran molecules further away have a high likelihood of encountering Ran-GAP, which is distributed throughout the cytosol; these Ran molecules are mainly in their GDP-bound conformation. As a result, the chromosomes in mitotic cells are surrounded by a cloud of Ran-GTP. Ran-GTP releases the NPC proteins in proximity to the chromosomes from nuclear import receptors. The free NPC proteins attach

1	Figure 12–16 Feedback regulation of cholesterol biosynthesis. sreBp (sterol response element binding protein), a latent transcription regulator that controls expression of cholesterol biosynthetic enzymes, is initially synthesized as an er membrane protein. it is anchored in the er if there is sufficient cholesterol in the membrane by interaction with another er membrane protein, called sCap (sreBp cleavage activation protein), which binds cholesterol. if the cholesterol binding site on sCap is empty (at low cholesterol concentrations), sCap changes conformation and is packaged together with sreBp into transport vesicles, which deliver their cargo to the Golgi apparatus, where two Golgi-resident proteases cleave sreBp to free its cytosolic domain from the membrane. The cytosolic domain then moves into the nucleus, where it binds to the promoters of genes that encode proteins involved in cholesterol biosynthesis and activates their transcription. in this way, more cholesterol is made

1	moves into the nucleus, where it binds to the promoters of genes that encode proteins involved in cholesterol biosynthesis and activates their transcription. in this way, more cholesterol is made when its concentration falls below a threshold.

1	Figure 12–17 The nuclear lamina. an electron micrograph of a portion of the nuclear lamina in a Xenopus oocyte prepared by freeze-drying and metal shadowing. The lamina is formed by a regular lattice of specialized intermediate filaments. lamins are only present in metazoan cells. other, yet-unknown proteins may serve similar functions in species that lack lamins. (Courtesy of Ueli aebi.) complex inner nuclear membrane outer nuclear membranenuclear envelope PHOSPHORYLATION OF LAMINS AND NPC PROTEINS DEPHOSPHORYLATION OF LAMINS FUSION OF ENVELOPED CHROMOSOMES FUSION OF NUCLEAR ENVELOPE FRAGMENTS nuclear envelope fragmentduplicated chromosome daughter chromosome phosphorylated lamins INTERPHASE NUCLEUS EARLY TELOPHASE PROPHASE LATE TELOPHASE nuclear pore complex proteins P P P P P P P P P P P P P P P P Figure 12–18 The breakdown and re-formation of the nuclear envelope and lamina during mitosis. phosphorylation of the lamins triggers the disassembly to the chromosome surface, where they

1	P P P P P P Figure 12–18 The breakdown and re-formation of the nuclear envelope and lamina during mitosis. phosphorylation of the lamins triggers the disassembly to the chromosome surface, where they assemble into new NPCs. At the same time, inner nuclear membrane proteins and dephosphorylated lamins bind again to chromatin. ER membranes wrap around groups of chromosomes until they form a sealed nuclear envelope (Movie 12.2). During this process, the NPCs start actively re-importing proteins that contain nuclear localization signals. Because the nuclear envelope is initially closely applied to the surface of the chromosomes, the newly formed nucleus excludes all proteins except those initially bound to the mitotic chromosomes and those that are selectively imported through NPCs. In this way, all other large proteins, including ribosomes, are kept out of the newly assembled nucleus.

1	As we discuss in Chapter 17, the cloud of Ran-GTP surrounding chromatin is also important in assembling the mitotic spindle in a dividing cell. The nuclear envelope consists of an inner and an outer nuclear membrane that are continuous with each other and with the ER membrane, and the space between the inner and outer nuclear membrane is continuous with the ER lumen. RNA molecules, which are made in the nucleus, and ribosomal subunits, which are assembled there, are exported to the cytosol; in contrast, all the proteins that function in the nucleus are synthesized in the cytosol and are then imported. The extensive traffic of materials between the nucleus and cytosol occurs through nuclear pore complexes (NPCs), which provide a direct passageway across the nuclear envelope. Small molecules diffuse passively through the NPCs, but large macromolecules have to be actively transported.

1	Proteins containing nuclear localization signals are actively transported into the nucleus through NPCs, while proteins containing nuclear export signals are transported out of the nucleus to the cytosol. Some proteins, including the nuclear of the nuclear lamina, which initiates the nuclear envelope to break up. Dephosphorylation of the lamins reverses the process. an analogous phosphorylation and dephosphorylation cycle occurs for some nucleoporins and proteins of the inner nuclear membrane, and some of these dephosphorylations are also involved in the reassembly process. as indicated, the nuclear envelope initially re-forms around individual decondensing daughter chromosomes. eventually, as decondensation progresses, these structures fuse to form a single complete nucleus. mitotic breakdown of the nuclear envelope occurs in all metazoan cells. however, in many other species, such as yeasts, the nuclear envelope remains intact during mitosis, and the nucleus divides by fission.

1	import and export receptors, continually shuttle between the cytosol and nucleus. The monomeric GTPase Ran provides both the free energy and the directionality for nuclear transport. Cells regulate the transport of nuclear proteins and RNA molecules through the NPCs by controlling the access of these molecules to the transport machinery. Newly transcribed messenger RNA and ribosomal RNA are exported from the nucleus as parts of large ribonucleoprotein complexes. Because nuclear localization signals are not removed, nuclear proteins can be imported repeatedly, as is required each time that the nucleus reassembles after mitosis. The TransporT of proTeins inTo miToChonDria anD ChloroplasTs

1	The TransporT of proTeins inTo miToChonDria anD ChloroplasTs Mitochondria and chloroplasts (a specialized form of plastids in green algae and plant cells) are double-membrane-enclosed organelles. They specialize in ATP synthesis, using energy derived from electron transport and oxidative phosphorylation in mitochondria and from photosynthesis in chloroplasts (discussed in Chapter 14). Although both organelles contain their own DNA, ribosomes, and other components required for protein synthesis, most of their proteins are encoded in the cell nucleus and imported from the cytosol. Each imported protein must reach the particular organelle subcompartment in which it functions.

1	There are different subcompartments in mitochondria (Figure 12–19A): the internal matrix space and the intermembrane space, which is continuous with the cristae space. These compartments are formed by the two concentric mitochondrial membranes: the inner membrane, which encloses the matrix space and forms extensive invaginations called cristae, and the outer membrane, which is in contact with the cytosol. Protein complexes provide boundaries at the junctions where the cristae invaginate and divide the inner membrane into two domains: one inner membrane domain surrounds the cristae space, and the other domain abuts the outer membrane. Chloroplasts also have an outer and inner membrane, which enclose an intermembrane space, and the stroma, which is the chloroplast equivalent of the mitochondrial matrix space (Figure 12–19B). They have an additional subcompartment, the thylakoid space, which is surrounded by the thylakoid membrane. The thylakoid membrane derives from the inner membrane

1	matrix space (Figure 12–19B). They have an additional subcompartment, the thylakoid space, which is surrounded by the thylakoid membrane. The thylakoid membrane derives from the inner membrane during plastid development and is pinched off to become discontinuous with it. Each of the subcompartments in mitochondria and chloroplasts contains a distinct set of proteins.

1	New mitochondria and chloroplasts are produced by the growth of preexisting organelles, followed by fission (discussed in Chapter 14). The growth depends mainly on the import of proteins from the cytosol. The imported proteins must be transported across a number of membranes in succession and end up in the appropriate place. The process of protein movement across membranes is called protein translocation. This section explains how it occurs. Figure 12–19 The subcompartments of mitochondria and chloroplasts. in contrast to the cristae of mitochondria (a), the thylakoids of chloroplasts (B) are not connected to the inner membrane and therefore form a sealed compartment with a separate internal space.

1	Proteins imported into mitochondria are usually taken up from the cytosol within seconds or minutes of their release from ribosomes. Thus, in contrast to protein translocation into the ER, which often takes place simultaneously with translation by a ribosome docked on the rough ER membrane (described later), mitochondrial proteins are first fully synthesized as mitochondrial precursor proteins in the cytosol and then translocated into mitochondria by a post-translational mechanism. One or more signal sequences direct all mitochondrial precursor proteins to their appropriate mitochondrial subcompartment. Many proteins entering the matrix space contain a signal sequence at their N-terminus that a signal peptidase rapidly removes after import. Other imported proteins, including all outer membrane and many inner membrane and intermembrane space proteins, have internal signal sequences that are not removed. The signal sequences are both necessary and sufficient for the import and correct

1	and many inner membrane and intermembrane space proteins, have internal signal sequences that are not removed. The signal sequences are both necessary and sufficient for the import and correct localization of the proteins: when genetic engineering techniques are used to link these signals to a cytosolic protein, the signals direct the protein to the correct mitochondrial subcompartment.

1	The signal sequences that direct precursor proteins into the mitochondrial matrix space are best understood. They all form an amphiphilic α helix, in which positively charged residues cluster on one side of the helix, while uncharged hydrophobic residues cluster on the opposite side. Specific receptor proteins that initiate protein translocation recognize this configuration rather than the precise amino acid sequence of the signal sequence (Figure 12–20). Multisubunit protein complexes that function as protein translocators mediate protein movement across mitochondrial membranes. The TOM complex transfers proteins across the outer membrane, and two TIM complexes (TIM23 and TIM22) transfer proteins across the inner membrane (Figure 12–21). These complexes contain some components that act as receptors for mitochondrial precursor proteins, and other components that form the translocation channels.

1	The TOM complex is required for the import of all nucleus-encoded mitochondrial proteins. It initially transports their signal sequences into the intermembrane space and helps to insert transmembrane proteins into the outer membrane. β-barrel proteins, which are particularly abundant in the outer membrane, are then passed on to an additional translocator, the SAM complex, which helps them to fold properly in the outer membrane. The TIM23 complex transports some soluble proteins into the matrix space and helps to insert transmembrane proteins into the inner membrane. The TIM22 complex mediates the insertion of a subclass of inner membrane proteins, including the transporter that moves ADP, ATP, and phosphate in and out of mitochondria. Yet another protein translocator in the inner mitochondrial membrane, the OXA complex, mediates the insertion of Figure 12–20 A signal sequence for mitochondrial protein import.

1	Cytochrome oxidase is a large multiprotein complex located in the inner mitochondrial membrane, where it functions as the terminal enzyme in the electron-transport chain (discussed in Chapter 14). (a) The first 18 amino acids of the precursor to subunit iV of this enzyme serve as a signal sequence for import of the subunit into the mitochondrion. (B) When the signal sequence is folded as an α helix, the positively charged amino acids (red) are clustered on one face of the helix, while the nonpolar ones (green) are clustered primarily on the opposite face. Uncharged polar amino acids are shaded orange; nitrogen atoms on the side chains of arg and Gln are colored blue. signal sequences that direct proteins into the matrix space always have the potential to form such an amphiphilic α helix, which is recognized by specific receptor proteins on the mitochondrial surface. (C) The structure of a signal sequence (of alcohol dehydrogenase, another mitochondrial matrix enzyme), bound to an

1	which is recognized by specific receptor proteins on the mitochondrial surface. (C) The structure of a signal sequence (of alcohol dehydrogenase, another mitochondrial matrix enzyme), bound to an import receptor (gray), as determined by nuclear magnetic resonance. The amphiphilic α helix binds with its hydrophobic face to a hydrophobic groove in the receptor (pDB code: 1om2).

1	660 Chapter 12: intracellular Compartments and protein sorting those inner membrane proteins that are synthesized within mitochondria. It also helps to insert some imported inner membrane proteins that are initially transported into the matrix space by the other complexes. mitochondrial precursor proteins are imported as Unfolded polypeptide Chains We have learned almost everything we know about the molecular mechanism of protein import into mitochondria from analyses of cell-free, reconstituted trans-location systems, in which purified mitochondria in a test tube import radiolabeled mitochondrial precursor proteins. By changing the conditions in the test tube, it is possible to establish the biochemical requirements for the import.

1	Mitochondrial precursor proteins do not fold into their native structures after they are synthesized; instead, they remain unfolded in the cytosol through interactions with other proteins. Some of these interacting proteins are general chaperone proteins of the hsp70 family (discussed in Chapter 6), whereas others are dedicated to mitochondrial precursor proteins and bind directly to their signal sequences. All the interacting proteins help to prevent the precursor proteins from aggregating or folding up spontaneously before they engage with the TOM complex in the outer mitochondrial membrane. As a first step in the import process, the import receptors of the TOM complex bind the signal sequence of the mitochondrial precursor protein. The interacting proteins are then stripped off, and the unfolded polypeptide chain is fed—signal sequence first—into the trans-location channel.

1	In principle, a protein could reach the mitochondrial matrix space by either crossing the two membranes all at once or crossing one at a time. One can distinguish between these possibilities by cooling a cell-free mitochondrial import system to arrest the proteins at an intermediate step in the translocation process. The result is that the arrested proteins no longer contain their N-terminal signal sequence, indicating that the N-terminus must be in the matrix space where the signal peptidase is located, but the bulk of the protein can still be attacked from outside the mitochondria by externally added proteolytic enzymes. Clearly, the precursor proteins can pass through both mitochondrial membranes at once to enter the matrix space (Figure 12–22). The TOM complex first transports the signal sequence across the outer membrane to the intermembrane space, where it binds to a TIM complex, opening the channel in the complex. The polypeptide chain is then either translocated into the

1	signal sequence across the outer membrane to the intermembrane space, where it binds to a TIM complex, opening the channel in the complex. The polypeptide chain is then either translocated into the matrix space or inserted into the inner membrane.

1	Figure 12–21 The protein translocators in the mitochondrial membranes. The Tom, Tim, sam, and oXa complexes are multimeric membrane protein assemblies that catalyze protein translocation across mitochondrial membranes. The protein components of the Tim22 and Tim23 complexes that line the import channel are structurally related, suggesting a common evolutionary origin of both Tim complexes. on the matrix side, the Tim23 complex is bound to a multimeric protein complex containing mitochondrial hsp70, which acts as an import aTpase, using aTp hydrolysis to pull proteins through the pore. in animal cells, subtle variations exist in the subunit composition of the translocator complexes to adapt the mitochondrial import machinery to the particular needs of specialized cell types. sam = sorting and assembly machinery; oXa = cytochrome oXidase activity; Tim = Translocator of the inner mitochondrial membrane; Tom = Translocator of the outer membrane.

1	mitochondria. The n-terminal signal sequence of the mitochondrial precursor protein is recognized by receptors of the Tom complex. The protein is then translocated through the Tim23 complex so that it transiently spans both mitochondrial membranes (Movie 12.3). The signal sequence is cleaved off by a signal peptidase in the matrix space to form the mature protein. The free signal sequence is then rapidly degraded (not shown). Although the TOM and TIM complexes usually work together to translocate precursor proteins across both membranes at the same time, they can work independently. In isolated outer membranes, for example, the TOM complex can translocate the signal sequence of precursor proteins across the membrane. Similarly, if the outer membrane is experimentally disrupted in isolated mitochondria, the exposed TIM23 complex can efficiently import precursor proteins into the matrix space. aTp hydrolysis and a membrane potential Drive protein import into the matrix space

1	aTp hydrolysis and a membrane potential Drive protein import into the matrix space Directional transport requires energy, which in most biological systems is supplied by ATP hydrolysis. ATP hydrolysis fuels mitochondrial protein import at two discrete sites, one outside the mitochondria and one in the matrix space. In addition, protein import requires another energy source, which is the membrane potential across the inner mitochondrial membrane (Figure 12–23). The first requirement for energy occurs at the initial stage of the translocation process, when the unfolded precursor protein, associated with chaperone proteins, interacts with the import receptors of the TOM complex. As discussed in Chapter 6, the binding and release of newly synthesized polypeptides from the chaperone proteins requires ATP hydrolysis.

1	Figure 12–23 The role of energy in protein import into the mitochondrial matrix space. (1) Bound cytosolic hsp70 chaperone is released from the precursor protein in a step that depends on aTp hydrolysis. after initial insertion of the signal sequence and of adjacent portions of the polypeptide chain into the Tom complex translocation channel, the signal sequence interacts with a Tim complex. (2) The signal sequence is then translocated into the matrix space in a process that requires the energy in the membrane potential across the inner membrane. (3) Mitochondrial hsp70, which is part of an import aTpase complex, binds to regions of the polypeptide chain as they become exposed in the matrix space, pulling the protein through the translocation channel, using the energy of aTp hydrolysis. 662 Chapter 12: intracellular Compartments and protein sorting

1	662 Chapter 12: intracellular Compartments and protein sorting Once the signal sequence has passed through the TOM complex and is bound to a TIM complex, further translocation through the TIM translocation channel requires the membrane potential, which is the electrical component of the electrochemical H+ gradient across the inner membrane (see Figure 11–4). Pumping of H+ from the matrix space to the intermembrane space, driven by electron transport processes in the inner membrane (discussed in Chapter 14), maintains the electrochemical gradient. The energy in the electrochemical H+ gradient across the inner membrane therefore not only powers most of the cell’s ATP synthesis, but it also drives the translocation of the positively charged signal sequences through the TIM complexes by electrophoresis.

1	Mitochondrial hsp70 also plays a crucial part in the import process. Mitochondria containing mutant forms of the protein fail to import precursor proteins. The mitochondrial hsp70 is part of a multisubunit protein assembly that is bound to the matrix side of the TIM23 complex and acts as a motor to pull the precursor protein into the matrix space. Like its cytosolic cousin, mitochondrial hsp70 has a high affinity for unfolded polypeptide chains, and it binds tightly to an imported protein chain as soon as the chain emerges from the TIM translocator in the matrix space. The hsp70 then undergoes a conformational change and releases the protein chain in an ATP-dependent step, exerting a ratcheting/pulling force on the protein being imported. This energy-driven cycle of binding and subsequent release provides the final driving force needed to complete protein import after a protein has initially inserted into the TIM23 complex (see Figure 12–23).

1	After the initial interaction with mitochondrial hsp70, many imported matrix proteins are passed on to another chaperone protein, mitochondrial hsp60. As discussed in Chapter 6, hsp60 helps the unfolded polypeptide chain to fold by binding and releasing it through cycles of ATP hydrolysis. Bacteria and mitochondria Use similar mechanisms to insert porins into their outer membrane

1	Bacteria and mitochondria Use similar mechanisms to insert porins into their outer membrane The outer mitochondrial membrane, like the outer membrane of Gram-negative bacteria (see Figure 11–17), contains abundant pore-forming β-barrel proteins called porins, and it is thus freely permeable to inorganic ions and metabolites (but not to most proteins). In contrast to other outer membrane proteins, which are anchored in the membrane through transmembrane α-helical regions, the TOM complex cannot integrate porins into the lipid bilayer. Instead, porins are first transported unfolded into the intermembrane space, where they transiently bind specialized chaperone proteins, which keep the porins from aggregating (Figure 12–24A). They then bind to the SAM complex in the outer membrane, which both inserts them into the outer membrane and helps them fold properly.

1	One of the central subunits of the SAM complex is homologous to a bacterial outer membrane protein that helps insert β-barrel proteins into the bacterial outer Figure 12–24 Integration of porins into the outer mitochondrial and bacterial membranes. (a) after translocation through the Tom complex in the outer mitochondrial membrane, β-barrel proteins bind to chaperones in the intermembrane space. The sam complex then inserts the unfolded polypeptide chain into the outer membrane and helps the chain fold. (B) a structurally related Bam complex in the outer membrane of Gram-negative bacteria catalyzes β-barrel protein insertion and folding (see figure 11–17). membrane from the periplasmic space (the equivalent of the intermembrane space in mitochondria) (Figure 12–24B). This conserved pathway for inserting β-barrel proteins further underscores the endosymbiotic origin of mitochondria. Transport into the inner mitochondrial membrane and intermembrane space occurs Via several routes

1	Transport into the inner mitochondrial membrane and intermembrane space occurs Via several routes The same mechanism that transports proteins into the matrix space using the TOM and TIM23 translocators (see Figure 12–22) also mediates the initial translocation of many proteins that are destined for the inner mitochondrial membrane or the intermembrane space. In the most common translocation route, only the N-terminal signal sequence of the transported protein actually enters the matrix space (Figure 12–25A). A hydrophobic amino acid sequence, strategically placed after the N-terminal signal sequence, acts as a stop-transfer sequence, preventing protease intermembrane reduced protein to TRANSLOCATION ACROSScleavage space protein be imported SH SH SH SHSHSH S S S SH S S S S S SH RECOGNITION AND OXIDATION BY Mia40 REOXIDATION BY RESPIRATORY CHAIN OUTER MEMBRANE REDUCTION OF Mia40, OXIDATION OF IMPORTED PROTEINMia40 TOM complex

1	Figure 12–25 Protein import from the cytosol into the inner mitochondrial membrane and intermembrane space. (a) The n-terminal signal sequence (red) initiates import into the matrix space (see figure 12–22). a hydrophobic sequence (blue) that follows the matrix-targeting signal sequence binds to the Tim23 translocator (orange) in the inner membrane and stops translocation. The remainder of the protein is then pulled into the intermembrane space through the Tom translocator in the outer membrane, and the hydrophobic sequence is released into the inner membrane anchoring the protein there. (B) a second route for protein integration into the inner membrane first delivers the protein completely into the matrix space. Cleavage of the signal sequence (red) used for the initial translocation unmasks an adjacent hydrophobic signal sequence (blue) at the new n-terminus. This signal then directs the protein into the inner membrane, using the same oXa-dependent pathway that inserts proteins that

1	an adjacent hydrophobic signal sequence (blue) at the new n-terminus. This signal then directs the protein into the inner membrane, using the same oXa-dependent pathway that inserts proteins that are encoded by the mitochondrial genome and translated in the matrix space. (C) some soluble proteins of the intermembrane space also use the pathways shown in (a) and (B) before they are released into the intermembrane space by a second signal peptidase, which has its active site in the intermembrane space and removes the hydrophobic signal sequence. (D) some soluble intermembrane-space proteins become oxidized by the mia40 protein (mia = mitochondrial intermembrane space assembly) during import. mia40 forms a covalent intermediate through an intermolecular disulfide bond, which helps pull the transported protein through the Tom complex. mia40 becomes reduced in the process, and then is reoxidized by the electron transport chain, so that it can catalyze the next round of import. (e)

1	the transported protein through the Tom complex. mia40 becomes reduced in the process, and then is reoxidized by the electron transport chain, so that it can catalyze the next round of import. (e) multipass inner membrane proteins that function as metabolite transporters contain internal signal sequences and snake through the Tom complex as a loop. They then bind to the chaperones in the intermembrane space, which guide the proteins to the Tim22 complex. The Tim22 complex is specialized for the insertion of multipass inner membrane proteins.

1	further translocation across the inner membrane. The remainder of the protein then crosses the outer membrane through the TOM complex into the intermembrane space; the signal sequence is cleaved off in the matrix, and the hydrophobic sequence, released from TIM23, remains anchored in the inner membrane.

1	In another transport route to the inner membrane or intermembrane space, the TIM23 complex initially translocates the entire protein into the matrix space (Figure 12–25B). A matrix signal peptidase then removes the N-terminal signal sequence, exposing a hydrophobic sequence at the new N-terminus. This signal sequence guides the protein to the OXA complex, which inserts the protein into the inner membrane. As mentioned earlier, the OXA complex is primarily used to insert proteins that are encoded and translated in the mitochondrion into the inner membrane, and only a few imported proteins use this pathway. Translocators that are closely related to the OXA complex are found in the plasma membrane of bacteria and in the thylakoid membrane of chloroplasts, where they insert membrane proteins by a similar mechanism.

1	Many proteins that use these pathways to the inner membrane remain anchored there through their hydrophobic signal sequence (see Figure 12–25A,B). Others, however, are released into the intermembrane space by a protease that removes the membrane anchor (Figure 12–25C). Many of these cleaved proteins remain attached to the outer surface of the inner membrane as peripheral subunits of protein complexes that also contain transmembrane proteins.

1	Certain intermembrane-space proteins that contain cysteine motifs are imported by a yet different route. These proteins form a transient covalent disulfide bond to the Mia40 protein (Figure 12–25D). The imported proteins are then released in an oxidized form containing intrachain disulfide bonds. Mia40 becomes reduced in the process, and is then reoxidized by passing electrons to the electron transport chain in the inner mitochondrial membrane. In this way, the energy stored in the redox potential in the mitochondrial electron transport chain is tapped to drive protein import.

1	Mitochondria are the principal sites of ATP synthesis in the cell, but they also contain many metabolic enzymes, such as those of the citric acid cycle. Thus, in addition to proteins, mitochondria must also transport small metabolites across their membranes. While the outer membrane contains porins, which make the membrane freely permeable to such small molecules, the inner membrane does not. Instead, a family of metabolite-specific transporters transfers a vast number of small molecules across the inner membrane. In yeast cells, these transporters comprise a family of 35 different proteins, the most abundant of which transport ATP, ADP, and phosphate. These are multipass transmembrane proteins, which do not have cleavable signal sequences at their N-termini but instead contain internal signal sequences. They cross the TOM complex in the outer membrane, and intermembrane-space chaperones guide them to the TIM22 complex, which inserts them into the inner membrane by a process that

1	signal sequences. They cross the TOM complex in the outer membrane, and intermembrane-space chaperones guide them to the TIM22 complex, which inserts them into the inner membrane by a process that requires the membrane potential, but not mitochondrial hsp70 or ATP (Figure 12–25E). An energetically favorable partitioning of the hydrophobic transmembrane regions into the inner membrane is likely to drive this process.

1	Two signal sequences Direct proteins to the Thylakoid membrane in Chloroplasts Protein transport into chloroplasts resembles transport into mitochondria. Both processes occur post-translationally, use separate translocation complexes in each membrane, require energy, and use amphiphilic N-terminal signal sequences that are removed after use. With the exception of some of the chaperone molecules, however, the protein components that form the translocation complexes differ. Moreover, whereas mitochondria harness the electrochemical H+ gradient across their inner membrane to drive transport, chloroplasts, which have an electrochemical H+ gradient across their thylakoid membrane but not their inner membrane, use GTP and ATP hydrolysis to power import across their double membrane. The functional similarities may thus result from convergent evolution, reflecting the common requirements for translocation across a double membrane.

1	Although the signal sequences for import into chloroplasts superficially resemble those for import into mitochondria, the same plant cells have both mitochondria and chloroplasts, so proteins must partition appropriately between the two organelles. In plants, for example, a bacterial enzyme can be directed specifically to mitochondria if it is experimentally joined to an N-terminal signal sequence of a mitochondrial protein; the same enzyme joined to an N-terminal signal sequence of a chloroplast protein ends up in chloroplasts. Thus, the import receptors on each organelle distinguish between the different signal sequences.

1	Chloroplasts have an extra membrane-enclosed compartment, the thylakoid. Many chloroplast proteins, including the protein subunits of the photosynthetic system and of the ATP synthase (discussed in Chapter 14), are located in the thylakoid membrane. Like the precursors of some mitochondrial proteins, the precursors of these proteins are translocated from the cytosol to their final destination in two steps. First, they pass across the double membrane into the matrix space (called the stroma in chloroplasts), and then they either integrate into the thylakoid membrane or translocate into the thylakoid space (Figure 12–26A). The precursors of these proteins have a hydrophobic thylakoid signal sequence following the N-terminal chloroplast signal sequence. After the N-terminal signal sequence has been used to import the protein into the stroma, a stromal signal peptidase removes it, unmasking the thylakoid signal sequence that initiates transport

1	Figure 12–26 Translocation of chloroplast precursor proteins into the thylakoid space. (a) The precursor protein contains an n-terminal chloroplast signal sequence (red), followed immediately by a thylakoid signal sequence (brown). The chloroplast signal sequence initiates translocation into the stroma by a mechanism similar to that used for the translocation of mitochondrial precursor proteins into the matrix space, although the translocator complexes, ToC and TiC, are different. The signal sequence is then cleaved off, unmasking the thylakoid signal sequence, which initiates translocation across the thylakoid membrane.

1	(B) Translocation into the thylakoid space or thylakoid membrane can occur by any one of at least four routes: (1) a Sec pathway, so called because it uses components that are homologs of sec proteins, which mediate protein translocation across the bacterial plasma membrane (discussed later); (2) an SRP-like pathway, so called because it uses a chloroplast homolog of the signal-recognition particle, or srp (discussed later); (3) a TAT (twin arginine translocation) pathway, so called because two arginines are critical in the signal sequences that direct proteins into this pathway, which depends on the h+ gradient across the thylakoid membrane; and (4) a spontaneous insertion pathway that seems not to require any protein translocator. across the thylakoid membrane. There are at least four routes by which proteins cross or become integrated into the thylakoid membrane, distinguished by their need for different stromal chaperones and energy sources (Figure 12–26B).

1	Although mitochondria and chloroplasts have their own genetic systems, they produce only a small proportion of their own proteins. Instead, the two organelles import most of their proteins from the cytosol, using similar mechanisms. In both cases, proteins are transported in an unfolded state across both outer and inner membranes simultaneously into the matrix space or stroma. Both ATP hydrolysis and a membrane potential across the inner membrane drive translocation into mitochondria, whereas GTP and ATP hydrolysis drive translocation into chloroplasts. Chaperone proteins of the cytosolic hsp70 family maintain the precursor proteins in an unfolded state, and a second set of hsp70 proteins in the matrix space or stroma pulls the polypeptide chain into the organelle. Only proteins that contain a specific signal sequence are translocated. The signal sequence can either be located at the N-terminus and cleaved off after import or be internal and retained. Transport into the inner membrane

1	a specific signal sequence are translocated. The signal sequence can either be located at the N-terminus and cleaved off after import or be internal and retained. Transport into the inner membrane sometimes uses a second, hydrophobic signal sequence that is unmasked when the first signal sequence is removed. In chloroplasts, import from the stroma into the thylakoid can occur by several routes, distinguished by the chaperones and energy source used.

1	Peroxisomes differ from mitochondria and chloroplasts in many ways. Most notably, they are surrounded by only a single membrane, and they do not contain DNA or ribosomes. Thus, because peroxisomes lack a genome, all of their proteins are encoded in the nucleus. Peroxisomes acquire most of these proteins by selective import from the cytosol, although some of them enter the peroxisome membrane via the ER. Because we do not discuss peroxisomes elsewhere, we shall digress to consider some of the functions of this diverse family of organelles, before discussing their biosynthesis. Virtually all eukaryotic cells have peroxisomes. They contain oxidative enzymes, such as catalase and urate oxidase, at such high concentrations that, in some cells, the peroxisomes stand out in electron micrographs because of the presence of a crystalloid protein core (Figure 12–27).

1	Like mitochondria, peroxisomes are major sites of oxygen utilization. One hypothesis is that peroxisomes are a vestige of an ancient organelle that performed all the oxygen metabolism in the primitive ancestors of eukaryotic cells. When the oxygen produced by photosynthetic bacteria first accumulated in the atmosphere, it would have been highly toxic to most cells. Peroxisomes might have lowered the intracellular concentration of oxygen, while also exploiting its chemical reactivity to perform useful oxidation reactions. According to this view, the later development of mitochondria rendered peroxisomes largely obsolete because many of the same biochemical reactions—which had formerly been carried out in peroxisomes without producing energy—were now coupled to ATP formation by means of oxidative phosphorylation. The oxidation reactions performed by peroxisomes in present-day cells could therefore partly be those whose functions were not taken over by mitochondria.

1	peroxisomes Use molecular oxygen and hydrogen peroxide to perform oxidation reactions Peroxisomes are so named because they usually contain one or more enzymes that use molecular oxygen to remove hydrogen atoms from specific organic substrates (designated here as R) in an oxidation reaction that produces hydrogen peroxide (H2O2): Catalase uses the H2O2 generated by other enzymes in the organelle to oxidize a variety of other substrates—including formic acid, formaldehyde, and alcohol— by the “peroxidation” reaction: H2O2 + R′H2 → R′ + 2H2O. This type of oxidation reaction is particularly important in liver and kidney cells, where the peroxisomes detoxify various harmful molecules that enter the bloodstream. About 25% of the ethanol we drink is oxidized to acetaldehyde in this way. In addition, when excess H2O2 accumulates in the cell, catalase converts it to H2O through the reaction

1	A major function of the oxidation reactions performed in peroxisomes is the breakdown of fatty acid molecules. The process, called β oxidation, shortens the alkyl chains of fatty acids sequentially in blocks of two carbon atoms at a time, thereby converting the fatty acids to acetyl CoA. The peroxisomes then export the acetyl CoA to the cytosol for use in biosynthetic reactions. In mammalian cells, β oxidation occurs in both mitochondria and peroxisomes; in yeast and plant cells, however, this essential reaction occurs exclusively in peroxisomes. An essential biosynthetic function of animal peroxisomes is to catalyze the first reactions in the formation of plasmalogens, which are the most abundant class of phospholipids in myelin (Figure 12–28). Plasmalogen deficiencies cause profound abnormalities in the myelination of nerve-cell axons, which is one reason why many peroxisomal disorders lead to neurological disease.

1	Peroxisomes are unusually diverse organelles, and even in the various cell types of a single organism they may contain different sets of enzymes. They also adapt remarkably to changing conditions. Yeasts grown on sugar, for example, have few small peroxisomes. But when some yeasts are grown on methanol, numerous large peroxisomes are formed that oxidize methanol; and when grown on fatty acids, they develop numerous large peroxisomes that break down fatty acids to acetyl CoA by β oxidation.

1	Peroxisomes are also important in plants. Two types of plant peroxisomes have been studied extensively. One is present in leaves, where it participates in photo-respiration (discussed in Chapter 14) (Figure 12–29A). The other type of peroxisome is present in germinating seeds, where it converts the fatty acids stored in seed lipids into the sugars needed for the growth of the young plant. Because this conversion of fats to sugars is accomplished by a series of reactions known as the glyoxylate cycle, these peroxisomes are also called glyoxysomes (Figure 12–29B). In the glyoxylate cycle, two molecules of acetyl CoA produced by fatty acid breakdown in the peroxisome are used to make succinic acid, which then leaves the peroxisome and is converted into glucose in the cytosol. The glyoxylate cycle does not occur in animal cells, and animals are therefore unable to convert the fatty acids in fats into carbohydrates. a short signal sequence Directs the import of proteins into peroxisomes

1	A specific sequence of three amino acids (Ser–Lys–Leu) located at the C-terminus of many peroxisomal proteins functions as an import signal (see Table 12–3, p. 648). Other peroxisomal proteins contain a signal sequence near the N-terminus. If either sequence is attached to a cytosolic protein, the protein is imported into peroxisomes. The import signals are first recognized by soluble receptor proteins in the cytosol. Numerous distinct proteins, called peroxins, participate in the import process, which is driven by ATP hydrolysis. A complex of at least six different peroxins forms a protein translocator in the peroxisome membrane. Even oligomeric proteins do not have to unfold to be imported. To allow the passage of such compactly folded cargo molecules, the pore formed by the transporter is thought to be dynamic in its dimensions, adapting in size to the particular cargo molecules to be transported. In this respect, the mechanism differs from that used by mitochondria and

1	transporter is thought to be dynamic in its dimensions, adapting in size to the particular cargo molecules to be transported. In this respect, the mechanism differs from that used by mitochondria and chloroplasts. One soluble import receptor, the peroxin Pex5 recognizes the C-terminal peroxisomal import signal. It accompanies its cargo all the way into peroxisomes and, after cargo release, cycles back to the cytosol. After

1	Figure 12–27 An electron micrograph of three peroxisomes in a rat liver cell. The paracrystalline, electron-dense inclusions are composed primarily of the enzyme urate oxidase. (Courtesy of Daniel s. friend.) Figure 12–28 The structure of a plasmalogen. plasmalogens are very abundant in the myelin sheaths that insulate the axons of nerve cells. They make up some 80–90% of the myelin membrane phospholipids. in addition to an ethanolamine head group and a long-chain fatty acid attached to the same glycerol phosphate backbone used for phospholipids, plasmalogens contain an unusual fatty alcohol that is attached through an ether linkage (bottom left).

1	Figure 12–29 Electron micrographs of two types of peroxisomes found in plant cells. (a) a peroxisome with a paracrystalline core in a tobacco leaf mesophyll cell. its close association with chloroplasts is thought to facilitate the exchange of materials between these organelles during photorespiration. The vacuole in plant cells is equivalent to the lysosome in animal cells. (B) peroxisomes in a fat-storing cotyledon cell of a tomato seed 4 days after germination. here the peroxisomes (glyoxysomes) are associated with the lipid droplets that store fat, reflecting their central role in fat mobilization and gluconeogenesis during seed germination. (a, from s.e. frederick and e.h. newcomb, J. Cell Biol. 43:343–353, 1969. With permission from The rockefeller press; B, from W.p. Wergin, p.J. Gruber and e.h. newcomb, J. Ultrastruct. Res. 30:533–557, 1970. With permission from academic press.) delivering its cargo to the peroxisome lumen, Pex5 undergoes ubiquitylation. This modification is

1	Gruber and e.h. newcomb, J. Ultrastruct. Res. 30:533–557, 1970. With permission from academic press.) delivering its cargo to the peroxisome lumen, Pex5 undergoes ubiquitylation. This modification is required to release Pex5 back into the cytosol, where the ubiquitin is removed. An ATPase composed of Pex1 and Pex6 harnesses the energy of ATP hydrolysis to help release Pex5 from peroxisomes.

1	The importance of this import process and of peroxisomes is demonstrated by the inherited human disease Zellweger syndrome, in which a defect in importing proteins into peroxisomes leads to a profound peroxisomal deficiency. These individuals, whose cells contain “empty” peroxisomes, have severe abnormalities in their brain, liver, and kidneys, and they die soon after birth. A mutation in the gene encoding peroxin Pex5 causes one form of the disease. A defect in Pex7, the receptor for the N-terminal import signal, causes a milder peroxisomal disease. It has long been debated whether new peroxisomes arise from preexisting ones by organelle growth and fission—as mentioned earlier for mitochondria and plastids—or whether they derive as a specialized compartment from the endoplasmic reticulum (ER). Aspects of both views are true (Figure 12–30). Most peroxisomal membrane proteins are made in the cytosol and insert into the membrane of

1	Figure 12–30 A model that explains how peroxisomes proliferate and how new peroxisomes arise. peroxisomal precursor vesicles bud from the er. at least two peroxisomal membrane proteins, pex3 and pex15, follow this route. The machinery that drives the budding reaction and that selects only peroxisomal proteins for packaging into these vesicles depends on pex19 and other cytosolic proteins that are still unknown. peroxisomal precursor vesicles may then fuse with one another or with preexisting peroxisomes. The peroxisome membrane contains import receptors and protein translocators that are required for the import of peroxisomal proteins made on cytosolic ribosomes, including new copies of the import receptors and translocator components. presumably, the lipids required for growth are also imported, although some may derive directly from the er in the membrane of peroxisomal precursor vesicles.

1	preexisting peroxisomes, but others are first integrated into the ER membrane, where they are packaged into specialized peroxisomal precursor vesicles. New precursor vesicles may then fuse with one another and begin importing additional peroxisomal proteins, using their own protein import machinery to grow into mature peroxisomes, which can undergo cycles of growth and fission.

1	Peroxisomes are specialized for carrying out oxidation reactions using molecular oxygen. They generate hydrogen peroxide, which they employ for oxidative purposes—and contain catalase to destroy the excess. Like mitochondria and plastids, peroxisomes are self-replicating organelles. Because they do not contain DNA or ribosomes, however, all of their proteins are encoded in the cell nucleus. Some of these proteins are conveyed to peroxisomes via peroxisomal precursor vesicles that bud from the ER, but most are synthesized in the cytosol and directly imported. A specific sequence of three amino acids near the C-terminus of many of the latter proteins functions as a peroxisomal import signal. The mechanism of protein import differs from that of mitochondria and chloroplasts, in that even oligomeric proteins are imported from the cytosol without unfolding. The enDoplasmiC reTiCUlUm

1	The enDoplasmiC reTiCUlUm All eukaryotic cells have an endoplasmic reticulum (ER). Its membrane typically constitutes more than half of the total membrane of an average animal cell (see Table 12–2, p. 643). The ER is organized into a netlike labyrinth of branching tubules and flattened sacs that extends throughout the cytosol (Figure 12–31 and Movie 12.4). The tubules and sacs interconnect, and their membrane is continuous with the outer nuclear membrane; the compartment that they enclose therefore is also continuous with the space between the inner and outer nuclear membranes. Thus, the ER and nuclear membranes form a continuous sheet enclosing a single internal space, called the ER lumen or the ER cisternal space, which often occupies more than 10% of the total cell volume (see Table 12–1, p. 643).

1	As mentioned at the beginning of this chapter, the ER has a central role in both lipid and protein biosynthesis, and it also serves as an intracellular Ca2+ store that is used in many cell signaling responses (discussed in Chapter 15). The ER membrane is the site of production of all the transmembrane proteins and lipids for most of the cell’s organelles, including the ER itself, the Golgi apparatus, lysosomes, endosomes, secretory vesicles, and the plasma membrane. The ER membrane is also the site at which most of the lipids for mitochondrial and peroxisomal membranes are made. In addition, almost all of the proteins that will be secreted to the cell exterior—plus those destined for the lumen of the ER, Golgi apparatus, or lysosomes—are initially delivered to the ER lumen.

1	Figure 12–31 Fluorescent micrographs of the endoplasmic reticulum. (a) an animal cell in tissue culture that was genetically engineered to express an er membrane protein fused to a fluorescent protein. The er extends as a network of tubules and sheets throughout the entire cytosol, so that all regions of the cytosol are close to some portion of the er membrane. The outer nuclear membrane, which is continuous with the er, is also stained. (B) part of an er network in a living plant cell that was genetically engineered to express a fluorescent protein in the er. (a, courtesy of patrick Chitwood and Gia Voeltz; B, courtesy of petra Boevink and Chris hawes.) The er is structurally and functionally Diverse

1	The er is structurally and functionally Diverse While the various functions of the ER are essential to every cell, their relative importance varies greatly between individual cell types. To meet different functional demands, distinct regions of the ER become highly specialized. We observe such functional specialization as dramatic changes in ER structure, and different cell types can therefore possess characteristically different types of ER membrane. One of the most remarkable ER specializations is the rough ER.

1	Mammalian cells begin to import most proteins into the ER before complete synthesis of the polypeptide chain—that is, import is a co-translational process (Figure 12–32A). In contrast, the import of proteins into mitochondria, chloroplasts, nuclei, and peroxisomes is a post-translational process (Figure 12–32B). In co-translational transport, the ribosome that is synthesizing the protein is attached directly to the ER membrane, enabling one end of the protein to be translocated into the ER while the rest of the polypeptide chain is being synthesized. These membrane-bound ribosomes coat the surface of the ER, creating regions termed rough endoplasmic reticulum, or rough ER; regions of ER that lack bound ribosomes are called smooth endoplasmic reticulum, or smooth ER (Figure 12–33).

1	Most cells have scanty regions of smooth ER, and the ER is often partly smooth and partly rough. Areas of smooth ER from which transport vesicles carrying newly synthesized proteins and lipids bud off for transport to the Golgi apparatus are called transitional ER. In certain specialized cells, the smooth ER is abundant and has additional functions. It is prominent, for example, in cells that specialize in lipid metabolism, such as cells that synthesize steroid hormones from cholesterol; the expanded smooth ER accommodates the enzymes that make cholesterol and modify it to form the hormones (see Figure 12–33B).

1	The main cell type in the liver, the hepatocyte, also has a substantial amount of smooth ER. It is the principal site of production of lipoprotein particles, which carry lipids via the bloodstream to other parts of the body. The enzymes that synthesize the lipid components of the particles are located in the membrane of the smooth ER, which also contains enzymes that catalyze a series of reactions to detoxify both lipid-soluble drugs and various harmful compounds produced by metabolism. The most extensively studied of these detoxification reactions are carried out by the cytochrome P450 family of enzymes, which catalyze a series of reactions in which water-insoluble drugs or metabolites that would otherwise accumulate to toxic levels in cell membranes are rendered sufficiently water-soluble to leave the cell and be excreted in the urine. Because the rough ER alone cannot house enough of these and other necessary enzymes, a substantial portion of the membrane in a hepatocyte normally

1	to leave the cell and be excreted in the urine. Because the rough ER alone cannot house enough of these and other necessary enzymes, a substantial portion of the membrane in a hepatocyte normally consists of smooth ER (see Table 12–2).

1	Another crucially important function of the ER in most eukaryotic cells is to sequester Ca2+ from the cytosol. The release of Ca2+ into the cytosol from the ER, and its subsequent reuptake, occurs in many rapid responses to extracellular Figure 12–32 Co-translational and post-translational protein translocation. (a) ribosomes bind to the er membrane during co-translational translocation. (B) By contrast, cytosolic ribosomes complete the synthesis of a protein and release it prior to post-translational translocation. in both cases, the protein is directed to the er by an er signal sequence (red and orange). 200 nm 0.5 µm

1	200 nm 0.5 µm Figure 12–33 The rough and smooth ER. (a) an electron micrograph of the rough er in a pancreatic exocrine cell that makes and secretes large amounts of digestive enzymes every day. The cytosol is filled with closely packed sheets of er membrane that is studded with ribosomes. at the top left is a portion of the nucleus and its nuclear envelope; note that the outer nuclear membrane, which is continuous with the er, is also studded with ribosomes. (B) abundant smooth er in a steroid-hormone-secreting cell. This electron micrograph is of a testosterone-secreting leydig cell in the human testis.

1	(C) a three-dimensional reconstruction of a region of smooth er and rough er in a liver cell. The rough er forms oriented stacks of flattened cisternae, each having a lumenal space 20–30 nm wide. The smooth er membrane is connected to these cisternae and forms a fine network of tubules 30–60 nm in diameter. The er lumen is colored green. (D) a tomographic reconstruction of a portion of the er network in a yeast cell. membrane-bound ribosomes (tiny dark spheres) are seen in both flat sheets and tubular regions of irregular diameter, demonstrating that the ribosomes bind to er membranes of different curvature in these cells. (a, courtesy of lelio orci; B, courtesy of Daniel s. friend; C, after r.V. Krstic´, Ultrastructure of the mammalian Cell. new York: springer-Verlag, 1979; D, from m. West et al., J. Cell Biol. 193:333–346, 2011. With permission from rockefeller University press.) signals, as discussed in Chapter 15. A Ca2+ pump transports Ca2+ from the cytosol into the ER lumen. A

1	et al., J. Cell Biol. 193:333–346, 2011. With permission from rockefeller University press.) signals, as discussed in Chapter 15. A Ca2+ pump transports Ca2+ from the cytosol into the ER lumen. A high concentration of Ca2+-binding proteins in the ER facilitates Ca2+ storage. In some cell types, and perhaps in most, specific regions of the ER are specialized for Ca2+ storage. Muscle cells have an abundant, modified smooth ER called the sarcoplasmic reticulum. The release and reuptake of Ca2+ by the sarcoplasmic reticulum trigger myofibril contraction and relaxation, respectively, during each round of muscle contraction (discussed in Chapter 16).

1	To study the functions and biochemistry of the ER, it is necessary to isolate it. This may seem to be a hopeless task because the ER is intricately interleaved with other components of the cytoplasm. Fortunately, when tissues or cells are disrupted by homogenization, the ER breaks into fragments, which reseal to form small (~100–200 nm in diameter) closed vesicles called microsomes. Microsomes are relatively easy to purify. To the biochemist, microsomes represent small authentic versions of the ER, still capable of protein translocation, protein glycosylation (discussed later), Ca2+ uptake and release, and lipid synthesis. Microsomes derived from rough ER are studded with ribosomes and are called rough microsomes. The ribosomes are always found on the outside surface, so the interior of the microsome is biochemically equivalent to the lumen of the ER (Figure 12–34A).

1	Many vesicles similar in size to rough microsomes, but lacking attached ribosomes, are also found in cell homogenates. Such smooth microsomes are derived in part from smooth portions of the ER and in part from vesiculated fragments of the plasma membrane, Golgi apparatus, endosomes, and mitochondria (the ratio depending on the tissue). Thus, whereas rough microsomes are clearly derived from rough portions of ER, it is not easy to separate smooth microsomes derived from different organelles. The smooth microsomes prepared from liver or muscle cells are an exception. Because of the unusually large quantities of smooth ER or sarcoplasmic reticulum, respectively, most of the smooth microsomes in homogenates of these tissues are derived from the smooth ER or sarcoplasmic reticulum. The ribosomes attached to rough microsomes make them more dense than smooth microsomes. As a result, we can use equilibrium centrifugation to separate the rough and smooth microsomes (Figure 12–34B). Microsomes

1	attached to rough microsomes make them more dense than smooth microsomes. As a result, we can use equilibrium centrifugation to separate the rough and smooth microsomes (Figure 12–34B). Microsomes have been invaluable in elucidating the molecular aspects of ER function, as we discuss next.

1	signal sequences Were first Discovered in proteins imported into the rough er The ER captures selected proteins from the cytosol as they are being synthesized. These proteins are of two types: transmembrane proteins, which are only partly translocated across the ER membrane and become embedded in it, and water-soluble proteins, which are fully translocated across the ER membrane and are released into the ER lumen. Some of the transmembrane proteins function in the ER, but many are destined to reside in the plasma membrane or the membrane of another organelle. The water-soluble proteins are destined either for secretion or for residence in the lumen of the ER or of another organelle. All of these proteins, regardless of their subsequent fate, are directed to the ER membrane by an ER signal sequence, which initiates their translocation by a common mechanism.

1	Signal sequences (and the signal sequence strategy of protein sorting) were first discovered in the early 1970s in secreted proteins that are translocated across the ER membrane as a first step toward their eventual discharge from the cell. In the key experiment, the mRNA encoding a secreted protein was translated by ribosomes in vitro. When microsomes were omitted from this cell-free system, the protein synthesized was slightly larger than the normal secreted protein. In the presence of microsomes derived from the rough ER, however, a protein of the correct size was produced. According to the signal hypothesis, the size difference reflects the initial presence of a signal sequence that directs the secreted protein

1	Figure 12–34 The isolation of purified rough and smooth microsomes from the ER. (a) a thin section electron micrograph of the purified rough er fraction shows an abundance of ribosome-studded vesicles. (B) When sedimented to equilibrium through a gradient of sucrose, the two types of microsomes separate from each other on the basis of their different densities. note that the smooth fraction will also contain non-er-derived material. (a, courtesy of George palade.) Figure 12–35 The signal hypothesis.

1	Figure 12–35 The signal hypothesis. a simplified view of protein translocation across the er membrane, as originally proposed. When the er signal sequence emerges from the ribosome, it directs the ribosome to a translocator on the er membrane that forms a pore in the membrane through which the polypeptide is translocated. a signal peptidase is closely associated with the translocator and clips off the signal sequence during translation, and the mature protein is released into the lumen of the er immediately after its synthesis is completed. The translocator is closed until the ribosome has bound, so that the permeability barrier of the er membrane is maintained at all times.

1	to the ER membrane and is then cleaved off by a signal peptidase in the ER membrane before the polypeptide chain has been completed (Figure 12–35). Cell-free systems in which proteins are imported into microsomes have provided powerful procedures for identifying, purifying, and studying the various components of the molecular machinery responsible for the ER import process. a signal-recognition particle (srp) Directs the er signal sequence to a specific receptor in the rough er membrane

1	a signal-recognition particle (srp) Directs the er signal sequence to a specific receptor in the rough er membrane The ER signal sequence is guided to the ER membrane by at least two components: a signal-recognition particle (SRP), which cycles between the ER membrane and the cytosol and binds to the signal sequence, and an SRP receptor in the ER membrane. The SRP is a large complex; in animal cells, it consists of six different polypeptide chains bound to a single small RNA molecule. While the SRP and SRP receptor have fewer subunits in bacteria, homologs are present in all cells, indicating that this protein-targeting mechanism arose early in evolution and has been conserved.

1	ER signal sequences vary greatly in amino acid sequence, but each has eight or more nonpolar amino acids at its center (see Table 12–3, p. 648). How can the SRP bind specifically to so many different sequences? The answer has come from the crystal structure of the SRP protein, which shows that the signal-sequence-binding site is a large hydrophobic pocket lined by methionines. Because methionines have unbranched, flexible side chains, the pocket is sufficiently plastic to accommodate hydrophobic signal sequences of different sequences, sizes, and shapes.

1	The SRP is a rodlike structure, which wraps around the large ribosomal subunit, with one end binding to the ER signal sequence as it emerges from the ribosome as part of the newly made polypeptide chain; the other end blocks the elongation factor binding site at the interface between the large and small ribosomal subunits (Figure 12–36). This block halts protein synthesis as soon as the signal peptide has emerged from the ribosome. The transient pause presumably gives the ribosome enough time to bind to the ER membrane before completion of the polypeptide chain, thereby ensuring that the protein is not released into the cytosol. This safety

1	Figure 12–36 The signal-recognition particle (SRP). (a) a mammalian srp is a rodlike ribonucleoprotein complex containing six protein subunits (brown) and one rna molecule (blue). The srp rna forms a backbone that links the protein domain containing the signal-sequencebinding pocket to the domain responsible for pausing translation. Crystal structures of various srp pieces from different species are assembled here into a composite model to approximate the structure of a complete srp. (B) The three-dimensional outline of the srp bound to a ribosome was determined by cryoelectron microscopy. srp binds to the large ribosomal subunit so that its signal-sequence-binding pocket is positioned near the growing polypeptide chain exit site, and its translational pause domain is positioned at the interface between the ribosomal subunits, where it interferes with elongation factor binding. (C) as a signal sequence emerges from the ribosome and binds to the srp, a conformational change in the srp

1	between the ribosomal subunits, where it interferes with elongation factor binding. (C) as a signal sequence emerges from the ribosome and binds to the srp, a conformational change in the srp exposes a binding site for the srp receptor. (B, adapted from m. halic et al., Nature 427:808–814, 2004. With permission from macmillan publishers ltd.) device may be especially important for secreted and lysosomal hydrolases, which could wreak havoc in the cytosol; cells that secrete large amounts of hydrolases, however, take the added precaution of having high concentrations of hydrolase inhibitors in their cytosol. The pause also ensures that large portions of a protein that could fold into a compact structure are not made before reaching the translocator in the ER membrane. Thus, in contrast to the post-translational import of proteins into mitochondria and chloroplasts, chaperone proteins are not required to keep the protein unfolded.

1	When a signal sequence binds, SRP exposes a binding site for the SRP receptor (see Figure 12–36B,C), which is a transmembrane protein complex in the rough ER membrane. The binding of the SRP to its receptor brings the SRP–ribosome complex to an unoccupied protein translocator in the same membrane. The SRP and SRP receptor are then released, and the translocator transfers the growing polypeptide chain across the membrane (Figure 12–37). This co-translational transfer process creates two spatially separate populations of ribosomes in the cytosol. Membrane-bound ribosomes, attached to the

1	Figure 12–37 How ER signal sequences and SRP direct ribosomes to the ER membrane. The srp and its receptor act in concert. The srp binds to both the exposed er signal sequence and the ribosome, thereby inducing a pause in translation. The srp receptor in the er membrane, which in animal cells is composed of two different polypeptide chains, binds the srp–ribosome complex and directs it to the translocator. in a poorly understood reaction, the srp and srp receptor are then released, leaving the ribosome bound to the translocator in the er membrane. The translocator then inserts the polypeptide chain into the membrane and transfers it across the lipid bilayer. Because one of the srp proteins and both chains of the srp receptor contain GTp-binding domains, it is thought that conformational changes that occur during cycles of GTp binding and hydrolysis (discussed in Chapter 15) ensure that srp release occurs only after the ribosome has become properly engaged with the translocator in the

1	that occur during cycles of GTp binding and hydrolysis (discussed in Chapter 15) ensure that srp release occurs only after the ribosome has become properly engaged with the translocator in the er membrane. The translocator is closed until the ribosome has bound, so that the permeability barrier of the er membrane is maintained at all times.

1	cytosolic side of the ER membrane, are engaged in the synthesis of proteins that are being concurrently translocated into the ER. Free ribosomes, unattached to any membrane, synthesize all other proteins encoded by the nuclear genome. Membrane-bound and free ribosomes are structurally and functionally identical. They differ only in the proteins they are making at any given time.

1	Since many ribosomes can bind to a single mRNA molecule, a polyribosome is usually formed. If the mRNA encodes a protein with an ER signal sequence, the polyribosome becomes attached to the ER membrane, directed there by the signal sequences on multiple growing polypeptide chains. The individual ribosomes associated with such an mRNA molecule can return to the cytosol when they finish translation and intermix with the pool of free ribosomes. The mRNA itself, however, remains attached to the ER membrane by a changing population of ribosomes, each transiently held at the membrane by the translocator (Figure 12–38). The polypeptide Chain passes Through an aqueous Channel in the Translocator

1	The polypeptide Chain passes Through an aqueous Channel in the Translocator It had long been debated whether polypeptide chains are transferred across the ER membrane in direct contact with the lipid bilayer or through a channel in a protein translocator. The debate ended with the identification of the translocator, which was shown to form a water-filled channel in the membrane through common pool of ribosomal subunits in cytosol mRNA encoding a protein targeted to ER remains membrane-bound

1	Figure 12–38 Free and membrane-bound polyribosomes. (a) a common pool of ribosomes synthesizes the proteins that stay in the cytosol and those that are transported into the er. The er signal sequence on a newly formed polypeptide chain binds to srp, which directs the translating ribosome to the er membrane. The mrna molecule remains permanently bound to the er as part of a polyribosome, while the ribosomes that move along it are recycled; at the end of each round of protein synthesis, the ribosomal subunits are released and rejoin the common pool in the cytosol. (B) a thin section electron micrograph of polyribosomes attached to the er membrane. The plane of section in some places cuts through the er roughly parallel to the membrane, giving a face-on view of the rosettelike pattern of the polyribosomes. (B, courtesy of George palade.)

1	Figure 12–39 Structure of the Sec61 complex. (a) a side view (left) and a top view (right, seen from the cytosol) of the structure of the sec61 complex of the archaeon Methanococcus jannaschii. The sec61α subunit has an inverted repeat structure (see figure 11–10) and is shown in blue and beige to indicate this pseudo-symmetry; the two smaller β and γ subunits are shown in gray. in the side view, some helices in front have been omitted to make the inside of the pore visible. The yellow short helix is thought to form a plug that seals the pore when the translocator is closed. To open, the complex rearranges itself to move the plug helix out of the way, as indicated by the red arrow. a ring of hydrophobic amino acid side chains is thought to form a tight-fitting diaphragm around translocating polypeptide chain to prevent leaks of other molecules across the membrane. The pore of the sec61 complex can also open sideways at a lateral seam. (B) models of the closed and open states of the

1	polypeptide chain to prevent leaks of other molecules across the membrane. The pore of the sec61 complex can also open sideways at a lateral seam. (B) models of the closed and open states of the translocator are shown in top view, illustrating how a signal sequence (or a transmembrane segment) could be released into the lipid bilayer after opening of the seam. (pDB codes: 1rh5 and 1rhz.) which the polypeptide chain passes. The core of the translocator, called the Sec61 complex, is built from three subunits that are highly conserved from bacteria to eukaryotic cells. The structure of the Sec61 complex suggests that α helices contributed by the largest subunit surround a central channel through which the polypeptide chain traverses the membrane (Figure 12–39). The channel is gated by a short α helix that is thought to keep the translocator closed when it is idle and to move aside when it is engaged in passing a polypeptide chain. According to this view, the pore is a dynamic gated

1	short α helix that is thought to keep the translocator closed when it is idle and to move aside when it is engaged in passing a polypeptide chain. According to this view, the pore is a dynamic gated channel that opens only transiently when a polypeptide chain traverses the membrane. In an idle translocator, it is important to keep the channel closed, so that the membrane remains impermeable to ions, such as Ca2+, which otherwise would leak out of the ER. As a polypeptide chain is translocating, a ring of hydrophobic amino acid side chains is thought to provide a flexible seal to prevent ion leaks.

1	The structure of the Sec61 complex suggests that the pore can also open along a seam on its side. Indeed, some structures of the translocator show it locked in an open-seam conformation. This opening allows a translocating peptide chain lateral access into the hydrophobic core of the membrane, a process that is important both for the release of a cleaved signal peptide into the membrane (see Figure 12–35) and for the integration of transmembrane proteins into the bilayer, as we discuss later.

1	Figure 12–40 A ribosome (green) bound to the ER protein translocator (blue). (a) a side-view reconstruction of the complex from electron microscopic images. (B) a view of the translocator seen from the er lumen. The translocator contains sec61, accessory proteins, and detergent used in the preparation. Domains of accessory proteins extend across the membrane and form the lumenal bulge. (C) a schematic drawing of a membrane-bound ribosome attached to the translocator, indicating the location of the tunnel in the large ribosomal subunit through which the growing polypeptide chain exits from the ribosome. The mrna (not shown) would be located between the small and large ribosomal subunits. (adapted from J.f. ménétret et al., J. Mol. Biol. 348:445–457, 2005. With permission from academic press.)

1	In eukaryotic cells, four Sec61 complexes form a large translocator assembly that can be visualized on ER-bound ribosomes after detergent solubilization of the ER membrane (Figure 12–40). It is likely that this assembly includes other membrane complexes that associate with the translocator, such as enzymes that modify the growing polypeptide chain, including oligosaccharide transferase and the signal peptidase. The assembly of a translocator with these accessory components is called the translocon. Translocation across the er membrane Does not always require ongoing polypeptide Chain elongation As we have seen, translocation of proteins into mitochondria, chloroplasts, and peroxisomes occurs post-translationally, after the protein has been made and released into the cytosol, whereas translocation across the ER membrane usually occurs during translation (co-translationally). This explains why ribosomes are bound to the ER but not to other organelles.

1	Some completely synthesized proteins, however, are imported into the ER, demonstrating that translocation does not always require ongoing translation. Post-translational protein translocation is especially common across the yeast ER membrane and the bacterial plasma membrane (which is thought to be evolutionarily related to the ER). To function in post-translational translocation, the ER translocator needs accessory proteins that feed the polypeptide chain into the pore and drive translocation (Figure 12–41). In bacteria, a translocation motor protein, the SecA ATPase, attaches to the cytosolic side of the translocator, where it undergoes cyclic conformational changes driven by ATP hydrolysis. Each time an ATP is hydrolyzed, a portion of the SecA protein inserts into the pore of the translocator, pushing a short segment of the passenger protein with it. As a result of this ratchet mechanism, the SecA ATPase progressively pushes the polypeptide chain of the transported protein across

1	pushing a short segment of the passenger protein with it. As a result of this ratchet mechanism, the SecA ATPase progressively pushes the polypeptide chain of the transported protein across the membrane.

1	Eukaryotic cells use a different set of accessory proteins that associate with the Sec61 complex. These proteins span the ER membrane and use a small domain on the lumenal side of the ER membrane to deposit an hsp70-like chaperone protein (called BiP, for binding protein) onto the polypeptide chain as it emerges from the pore into the ER lumen. ATP-dependent cycles of BiP binding and release drive unidirectional translocation, as described earlier for the mitochondrial hsp70 proteins that pull proteins across mitochondrial membranes. Proteins that are transported into the ER by a post-translational mechanism are first released into the cytosol, where they bind to chaperone proteins to prevent folding, as discussed earlier for proteins destined for mitochondria and chloroplasts. in single-pass Transmembrane proteins, a single internal er signal sequence remains in the lipid Bilayer as a membrane-spanning α helix

1	in single-pass Transmembrane proteins, a single internal er signal sequence remains in the lipid Bilayer as a membrane-spanning α helix The ER signal sequence in the growing polypeptide chain is thought to trigger the opening of the pore in the Sec61 protein translocator: after the signal sequence is released from the SRP and the growing chain has reached a sufficient length, the CYTOSOL ER LUMEN CYTOSOL EXTRACELLULAR SPACE SecA ATPase SecY complex Sec62,63,71,72 complex BiP SRP SRP receptor Sec61 complex ATPADPER signal sequence Pi+ bacterial inner (plasma) membrane Figure 12–41 Three ways in which protein translocation can be driven through structurally similar translocators.

1	(a) Co-translational translocation. The ribosome is brought to the membrane by the srp and srp receptor and then engages with the sec61 protein translocator. The growing polypeptide chain is threaded across the membrane as it is made. no additional energy is needed, as the only path available to the growing chain is to cross the membrane. (B) post-translational translocation in eukaryotic cells requires an additional complex composed of sec62, sec63, sec71, and sec72 proteins, which is attached to the sec61 translocator and deposits Bip molecules onto the translocating chain as it emerges from the translocator in the lumen of the er. aTp-driven cycles of Bip binding and release pull the protein into the lumen, a mechanism that closely resembles the mechanism of mitochondrial import in figure 12–23. (C) post-translational translocation in bacteria. The completed polypeptide chain is fed from the cytosolic side into the bacterial homolog of the sec61 complex (called the secY complex in

1	12–23. (C) post-translational translocation in bacteria. The completed polypeptide chain is fed from the cytosolic side into the bacterial homolog of the sec61 complex (called the secY complex in bacteria) in the plasma membrane by the seca aTpase. aTp hydrolysis-driven conformational changes drive a pistonlike motion in seca, each cycle pushing about 20 amino acids of the protein chain through the pore of the translocator. The sec pathway used for protein translocation across the thylakoid membrane in chloroplasts uses a similar mechanism (see figure 12–26B).

1	Whereas the sec61 translocator, srp, and srp receptor are found in all organisms, seca is found exclusively in bacteria, and the sec62, 63, 71, 72 complex is found exclusively in eukaryotic cells. (adapted from p. Walter and a.e. Johnson, Annu. Rev. Cell Biol. 10:87–119, 1994. With permission from annual reviews.) signal sequence binds to a specific site inside the pore itself, thereby opening the pore. An ER signal sequence is therefore recognized twice: first by an SRP in the cytosol and then by a binding site in the pore of the protein translocator, where it serves as a start-transfer signal (or start-transfer peptide) that opens the pore (for example, see Figure 12–35 for how this works for a soluble protein). Dual recognition may help ensure that only appropriate proteins enter the lumen of the ER.

1	While bound in the translocation pore, a signal sequence is in contact not only with the Sec61 complex, which forms the walls of the pore, but also, along the lateral seam, with the hydrophobic core of the lipid bilayer. This was shown in chemical cross-linking experiments in which the signal sequence and the hydrocarbon chains of lipids were covalently linked together. When the nascent polypeptide chain grew long enough, the ER signal peptidase cleaved off the signal sequence and released it from the pore into the membrane, where it was rapidly degraded to amino acids by other proteases in the ER membrane. To release the signal sequence into the membrane, the translocator opens laterally along the seam (see Figures 12–35 and 12–39). The translocator is therefore gated in two directions: it opens to form a pore across the membrane to let the hydrophilic portions of proteins cross the lipid bilayer, and it opens laterally within the membrane to let hydrophobic portions of proteins

1	it opens to form a pore across the membrane to let the hydrophilic portions of proteins cross the lipid bilayer, and it opens laterally within the membrane to let hydrophobic portions of proteins partition into the lipid bilayer. Lateral gating of the pore is an essential step during the integration of transmembrane proteins.

1	The integration of membrane proteins requires that some parts of the polypeptide chain be translocated across the lipid bilayer whereas others are not. Despite this additional complexity, all modes of insertion of membrane proteins are simply variants of the sequence of events just described for transferring a soluble protein into the lumen of the ER. We begin by describing the three ways in which single-pass transmembrane proteins (see Figure 10–17) become inserted into the ER membrane.

1	In the simplest case, an N-terminal signal sequence initiates translocation, just as for a soluble protein, but an additional hydrophobic segment in the polypeptide chain stops the transfer process before the entire polypeptide chain is trans-located. This stop-transfer signal anchors the protein in the membrane after the ER signal sequence (the start-transfer signal) has been cleaved off and released from the translocator (Figure 12–42). The lateral gating mechanism transfers the stop-transfer sequence into the bilayer, where it remains as a single α-helical membrane-spanning segment, with the N-terminus of the protein on the lumenal side of the membrane and the C-terminus on the cytosolic side.

1	In the other two cases, the signal sequence is internal, rather than at the N-terminal end of the protein. As for an N-terminal ER signal sequence, the SRP binds to an internal signal sequence by recognizing its hydrophobic α-helical features. The SRP brings the ribosome making the protein to the ER membrane, and the ER signal sequence then serves as a start-transfer signal that initiates the protein’s translocation. After release from the translocator, the internal start-transfer sequence remains in the lipid bilayer as a single membrane-spanning α helix.

1	Internal start-transfer sequences can bind to the translocation apparatus in either of two orientations; this in turn determines which protein segment (the one preceding or the one following the start-transfer sequence) is moved across the membrane into the ER lumen. In one case, the resulting membrane protein has its C-terminus on the lumenal side (pathway A in Figure 12–43), while in the other, it has its N-terminus on the lumenal side (pathway B in Figure 12–43). The orientation of the start-transfer sequence depends on the distribution of nearby charged amino acids, as described in the figure legend. Combinations of start-Transfer and stop-Transfer signals Determine the Topology of multipass Transmembrane proteins

1	Combinations of start-Transfer and stop-Transfer signals Determine the Topology of multipass Transmembrane proteins In multipass transmembrane proteins, the polypeptide chain passes back and forth repeatedly across the lipid bilayer as hydrophobic α helices (see Figure 10–17). It is thought that an internal signal sequence serves as a start-transfer signal in these proteins to initiate translocation, which continues until the translocator encounters a stop-transfer sequence; in double-pass transmembrane proteins, for example, the polypeptide can then be released into the bilayer

1	Figure 12–42 How a single-pass transmembrane protein with a cleaved ER signal sequence is integrated into the ER membrane. in this protein, the co-translational translocation process is initiated by an n-terminal er signal sequence (red) that functions as a start-transfer signal, opening the translocator as in figure 12–35. in addition to this start-transfer sequence, however, the protein also contains a stop-transfer sequence (orange); when this sequence enters the translocator and interacts with a binding site within the pore, the translocator opens at the seam and discharges the protein laterally into the lipid bilayer, where the stop-transfer sequence remains to anchor the protein in the membrane. (in this figure and the two figures that follow, the ribosomes have been omitted for clarity.) (Figure 12–44). In more complex multipass proteins, in which many hydrophobic α helices span the bilayer, a second start-transfer sequence reinitiates translocation further down the polypeptide

1	(Figure 12–44). In more complex multipass proteins, in which many hydrophobic α helices span the bilayer, a second start-transfer sequence reinitiates translocation further down the polypeptide chain until the next stop-transfer sequence causes polypeptide release, and so on for subsequent start-transfer and stop-transfer sequences (Figure 12–45 and Movie 12.5).

1	Hydrophobic start-transfer and stop-transfer signal sequences both act to fix the topology of the protein in the membrane by locking themselves into the membrane as membrane-spanning α helices; and they can do this in either orientation. Whether a given hydrophobic signal sequence functions as a start-transfer or stop-transfer sequence must depend on its location in a polypeptide chain, since its function can be switched by changing its location in the protein by using recombinant DNA techniques. Thus, the distinction between start-transfer and stop-transfer sequences results mostly from their relative order in the growing polypeptide chain. It seems that the SRP begins scanning an unfolded polypeptide chain for hydrophobic segments at its N-terminus and proceeds toward the C-terminus, in the direction that the protein is synthesized. By recognizing the first appropriate hydrophobic segment to emerge from the ribosome, the SRP sets the “reading frame” for membrane integration: after

1	the direction that the protein is synthesized. By recognizing the first appropriate hydrophobic segment to emerge from the ribosome, the SRP sets the “reading frame” for membrane integration: after the SRP initiates trans-location, the translocator recognizes the next appropriate hydrophobic segment in the direction of transfer as a stop-transfer sequence, causing the region of the polypeptide chain in between to be threaded across the membrane. A similar

1	Figure 12–43 Integration of a single-pass transmembrane protein with an internal signal sequence into the ER membrane. an internal er signal sequence that functions as a start-transfer signal can bind to the translocator in one of two ways, leading to a membrane protein that has either its C-terminus (pathway a) or its n-terminus (pathway

1	B) in the er lumen. proteins are directed into either pathway by features in the polypeptide chain flanking the internal start-transfer sequence: if there are more positively charged amino acids immediately preceding the hydrophobic core of the start-transfer sequence than there are following it, the membrane protein is inserted into the translocator in the orientation shown in pathway a, whereas if there are more positively charged amino acids immediately following the hydrophobic core of the start-transfer sequence than there are preceding it, the membrane protein is inserted into the translocator in the orientation shown in pathway B. Because translocation cannot start before a start-transfer sequence appears outside the ribosome, translocation of the n-terminal portion of the protein shown in (B) can occur only after this portion has been fully synthesized.

1	note that there are two ways to insert a single-pass membrane-spanning protein whose n-terminus is located in the er lumen: that shown in figure 12–42 and that shown here in (B). scanning process continues until all of the hydrophobic regions in the protein have been inserted into the membrane as transmembrane α helices.

1	scanning process continues until all of the hydrophobic regions in the protein have been inserted into the membrane as transmembrane α helices. Because membrane proteins are always inserted from the cytosolic side of the ER in this programmed manner, all copies of the same polypeptide chain will have the same orientation in the lipid bilayer. This generates an asymmetrical ER membrane in which the protein domains exposed on one side are different from those exposed on the other side. This asymmetry is maintained during the many membrane budding and fusion events that transport the proteins made in the ER to other cell membranes (discussed in Chapter 13). Thus, the way in which a newly synthesized protein is inserted into the ER membrane determines the orientation of the protein in all of the other membranes as well.

1	When proteins are extracted with detergent from a membrane and then reconstituted into artificial lipid vesicles, a random mixture of right-side-out and inside-out protein orientations usually results. Thus, the protein asymmetry observed in cell membranes seems not to be an inherent property of the proteins, but instead results solely from the process by which proteins are inserted into the ER membrane from the cytosol. Figure 12–44 Integration of a double-pass transmembrane protein with an internal signal sequence into the ER membrane. in this protein, an internal er signal sequence acts as a start-transfer signal (as in figure 12–43) and initiates the transfer of the C-terminal part of the protein. at some point after a stop-transfer sequence has entered the translocator, the translocator discharges the sequence laterally into the membrane.

1	Figure 12–45 The insertion of the multipass membrane protein rhodopsin into the ER membrane. rhodopsin is the light-sensitive protein in rod photoreceptor cells in the mammalian retina (discussed in Chapter 15). (a) a hydropathy plot (see figure 10–20) identifies seven short hydrophobic regions in rhodopsin. (B) The hydrophobic region nearest the n-terminus serves as a start-transfer sequence that causes the preceding n-terminal portion of the protein to pass across the er membrane. subsequent hydrophobic sequences function in alternation as start-transfer and stop-transfer sequences. The green arrows indicate the paired start and stop signals inserted into the translocator. (C) The final integrated rhodopsin has its n-terminus located in the er lumen and its C-terminus located in the cytosol. The blue hexagons represent covalently attached oligosaccharides. er Tail-anchored proteins are integrated into the er membrane by a special mechanism

1	Many important membrane proteins are anchored in the membrane by a C-terminal transmembrane, hydrophobic α helix. These ER tail-anchored proteins include a large number of SNARE protein subunits that guide vesicular traffic (discussed in Chapter 13). When such a tail-anchored protein inserts into the ER membrane from the cytosol, only a few amino acids that follow the transmembrane αhelix on its C-terminal side are translocated into the ER lumen, while most of the protein remains in the cytosol. Because of the unique position of the transmembrane α helix in the protein sequence, translation terminates while the C-terminal amino acids that will form the transmembrane α helix have not yet emerged from the ribosome exit tunnel. Recognition by SRP is therefore not possible. It was long thought that these proteins are released from the ribosome and the hydrophobic C-terminal tail spontaneously partitions into the ER membrane. Such a mechanism could not explain, however, why ER

1	long thought that these proteins are released from the ribosome and the hydrophobic C-terminal tail spontaneously partitions into the ER membrane. Such a mechanism could not explain, however, why ER tail-anchored proteins insert into the ER membrane selectively and not also into all other membranes in the cell. It is now clear that a specialized targeting machinery is involved that is fueled by ATP hydrolysis (Figure 12–46). Although the components and details differ, this post-translational targeting mechanism is conceptually similar to SRP-dependent protein targeting (see Figure 12–37).

1	Not all tail-anchored proteins are inserted into the ER, however. Some proteins contain a C-terminal membrane anchor that contains additional sorting information that directs the protein to mitochondria or peroxisomes. How these proteins are sorted there remains unknown. Translocated polypeptide Chains fold and assemble in the lumen of the rough er Many of the proteins in the lumen of the ER are in transit, en route to other destinations; others, however, normally reside there and are present at high concentrations. These ER resident proteins contain an ER retention signal of four amino acids at their C-terminus that is responsible for retaining the protein in the ER (see Table 12–3. p. 648; discussed in Chapter 13). Some of these proteins function as catalysts that help the many proteins that are translocated into the ER lumen to fold and assemble correctly.

1	One important ER resident protein is protein disulfide isomerase (PDI), which catalyzes the oxidation of free sulfhydryl (SH) groups on cysteines to form disulfide (S–S) bonds. Almost all cysteines in protein domains exposed to either the extracellular space or the lumen of organelles in the secretory and endocytic pathways are disulfide-bonded. By contrast, disulfide bonds form only very rarely in domains exposed to the cytosol, because of the reducing environment there.

1	Figure 12–46 The insertion mechanism for tail-anchored proteins. in this post-translational pathway for the insertion of tail-anchored er membrane proteins, a soluble pre-targeting complex captures the hydrophobic C-terminal α helix after it emerges from the ribosomal exit tunnel and loads it onto the Get3 aTpase. The resulting complex is targeted to the er membrane by interaction with the Get1– Get2 receptor complex that functions as a membrane protein insertion machine. after Get3 hydrolyzes its bound aTp, the tail-anchored protein is released from the receptor and inserted into the er membrane. aDp release and renewed aTp binding recycles Get3 back to the cytosol.

1	Another ER resident protein is the chaperone protein BiP. We have already discussed how BiP pulls proteins post-translationally into the ER through the Sec61 ER translocator. Like other chaperones (discussed in Chapter 13), BiP recognizes incorrectly folded proteins, as well as protein subunits that have not yet assembled into their final oligomeric complexes. It does so by binding to exposed amino acid sequences that would normally be buried in the interior of correctly folded or assembled polypeptide chains. An example of a BiP-binding site is a stretch of alternating hydrophobic and hydrophilic amino acids that would normally be buried in a β sheet with its hydrophobic side oriented towards the hydrophobic core of the folded protein. The bound BiP both prevents the protein from aggregating and helps keep it in the ER (and thus out of the Golgi apparatus and later parts of the secretory pathway). Like some other members of the hsp70 family of chaperone proteins, which bind unfolded

1	and helps keep it in the ER (and thus out of the Golgi apparatus and later parts of the secretory pathway). Like some other members of the hsp70 family of chaperone proteins, which bind unfolded proteins and facilitate their import into mitochondria and chloroplasts, BiP hydrolyzes ATP to shuttle between highand low-affinity binding states, which allow it to hold on to and let go of its substrate proteins in a dynamic cycle.

1	most proteins synthesized in the rough er are Glycosylated by the addition of a Common N-linked oligosaccharide The covalent addition of oligosaccharides to proteins is one of the major biosynthetic functions of the ER. About half of the soluble and membrane-bound proteins that are processed in the ER—including those destined for transport to the Golgi apparatus, lysosomes, plasma membrane, or extracellular space—are glycoproteins that are modified in this way. Many proteins in the cytosol and nucleus are also glycosylated, but not with oligosaccharides: they carry a much simpler sugar modification, in which a single N-acetylglucosamine group is added to a serine or threonine of the protein.

1	During the most common form of protein glycosylation in the ER, a preformed precursor oligosaccharide (composed of N-acetylglucosamine, mannose, and glucose, and containing a total of 14 sugars) is transferred en bloc to proteins. Because this oligosaccharide is transferred to the side-chain NH2 group of an asparagine in the protein, it is said to be N-linked or asparagine-linked (Figure 12–47A). The transfer is catalyzed by a membrane-bound enzyme complex, an protein with N-glycosylation site

1	Figure 12–47 N-linked protein glycosylation in the rough ER. (a) almost as soon as a polypeptide chain enters the er lumen, it is glycosylated on target asparagine amino acids. The precursor oligosaccharide (shown in color) is attached only to asparagines in the sequences asnX-ser and asn-X-Thr (where X is any amino acid except proline). These sequences occur much less frequently in glycoproteins than in nonglycosylated cytosolic proteins. evidently there has been selective pressure against these sequences during protein evolution, presumably because glycosylation at too many sites would interfere with protein folding. The five sugars in the gray box form the core region of this oligosaccharide. for many glycoproteins, only the core sugars survive the extensive oligosaccharide trimming that takes place in the Golgi apparatus. (B) The precursor oligosaccharide is transferred from a dolichol lipid anchor to the asparagine as an intact unit in a reaction catalyzed by a transmembrane

1	that takes place in the Golgi apparatus. (B) The precursor oligosaccharide is transferred from a dolichol lipid anchor to the asparagine as an intact unit in a reaction catalyzed by a transmembrane oligosaccharyl transferase enzyme. one copy of this enzyme is associated with each protein translocator in the er membrane. (The translocator is not shown.) oligosaccharyl transferase contains 13 transmembrane α helices and a large er lumenal domain that contains its substrate-binding sites. The asparagine binds a tunnel that penetrates the enzyme interior. There, the amino group of the asparagine is twisted out of the plane that stabilizes the otherwise poorly reactive amide bond, activating it for reaction with the dolichol–oligosaccharide. The structure shown is of a prokaryotic homolog that closely resembles the catalytic subunit of the eukaryotic oligosaccharyl transferase. (pDB code: 3rCe.) oligosaccharyl transferase, which has its active site exposed on the lumenal side of the ER

1	closely resembles the catalytic subunit of the eukaryotic oligosaccharyl transferase. (pDB code: 3rCe.) oligosaccharyl transferase, which has its active site exposed on the lumenal side of the ER membrane; this explains why cytosolic proteins are not glycosylated in this way. A special lipid molecule called dolichol anchors the precursor oligosaccharide in the ER membrane. The precursor oligosaccharide is transferred to the target asparagine in a single enzymatic step immediately after that amino acid has reached the ER lumen during protein translocation. The precursor oligosaccharide is linked to the dolichol lipid by a high-energy pyrophosphate bond, which provides the activation energy that drives the glycosylation reaction (Figure 12–47B). One copy of oligosaccharyl transferase is associated with each protein translocator, allowing it to scan and glycosylate the incoming polypeptide chains efficiently.

1	The precursor oligosaccharide is built up sugar by sugar on the membrane-bound dolichol lipid and is then transferred to a protein. The sugars are first activated in the cytosol by the formation of nucleotide (UDP or GDP)-sugar intermediates, which then donate their sugar (directly or indirectly) to the lipid in an orderly sequence. Part way through this process, the lipid-linked oligosaccharide is flipped, with the help of a transporter, from the cytosolic to the lumenal side of the ER membrane (Figure 12–48).

1	All of the diversity of the N-linked oligosaccharide structures on mature glycoproteins results from the later modification of the original precursor oligosaccharide. While still in the ER, three glucoses (see Figure 12–47) and one mannose are quickly removed from the oligosaccharides of most glycoproteins. We shall return to the importance of glucose trimming shortly. This oligosaccharide “trimming,” or “processing,” continues in the Golgi apparatus, as we discuss in Chapter 13. The N-linked oligosaccharides are by far the most common oligosaccharides, being found on 90% of all glycoproteins. Less frequently, oligosaccharides are linked to the hydroxyl group on the side chain of a serine, threonine, or hydroxy-lysine amino acid. A first sugar of these O-linked oligosaccharides is added in the ER and the oligosaccharide is then further extended in the Golgi apparatus (see Figure 13–32). lipid bilayer of ER membrane

1	Figure 12–48 Synthesis of the lipid-linked precursor oligosaccharide in the rough ER membrane. The oligosaccharide is assembled sugar by sugar onto the carrier lipid dolichol (a polyisoprenoid; see panel 2–5, pp. 98–99). Dolichol is long and very hydrophobic: its 22 five-carbon units can span the thickness of a lipid bilayer more than three times, so that the attached oligosaccharide is firmly anchored in the membrane. The first sugar is linked to dolichol by a pyrophosphate bridge. This high-energy bond activates the oligosaccharide for its eventual transfer from the lipid to an asparagine side chain of a growing polypeptide on the lumenal side of the rough er. as indicated, the synthesis of the oligosaccharide starts on the cytosolic side of the er membrane and continues on the lumenal face after the (man)5(Glcnac)2 lipid intermediate is flipped across the bilayer by a transporter (which is not shown). all the subsequent glycosyl transfer reactions on the lumenal side of the er

1	face after the (man)5(Glcnac)2 lipid intermediate is flipped across the bilayer by a transporter (which is not shown). all the subsequent glycosyl transfer reactions on the lumenal side of the er involve transfers from dolichol-p-glucose and dolichol-pmannose; these activated, lipid-linked monosaccharides are synthesized from dolichol phosphate and UDp-glucose or GDp-mannose (as appropriate) on the cytosolic side of the er and are then flipped across the er membrane. Glcnac = N-acetylglucosamine; man = mannose; Glc = glucose.

1	oligosaccharides are Used as Tags to mark the state of protein folding

1	It has long been debated why glycosylation is such a common modification of proteins that enter the ER. One particularly puzzling observation has been that some proteins require N-linked glycosylation for proper folding in the ER, yet the precise location of the oligosaccharides attached to the protein’s surface does not seem to matter. A clue to the role of glycosylation in protein folding came from studies of two ER chaperone proteins, which are called calnexin and calreticulin because they require Ca2+ for their activities. These chaperones are carbohydrate-binding proteins, or lectins, which bind to oligosaccharides on incompletely folded proteins and retain them in the ER. Like other chaperones, they prevent incompletely folded proteins from irreversibly aggregating. Both calnexin and calreticulin also promote the association of incompletely folded proteins with another ER chaperone, which binds to cysteines that have not yet formed disulfide bonds.

1	Calnexin and calreticulin recognize N-linked oligosaccharides that contain a single terminal glucose, and they therefore bind proteins only after two of the three glucoses on the precursor oligosaccharide have been removed during glucose trimming by ER glucosidases. When the third glucose has been removed, the glycoprotein dissociates from its chaperone and can leave the ER. How, then, do calnexin and calreticulin distinguish properly folded from incompletely folded proteins? The answer lies in yet another ER enzyme, a glucosyl transferase that keeps adding a glucose to those oligosaccharides that have lost their last glucose. It adds the glucose, however, only to oligosaccharides that are attached to unfolded proteins. Thus, an unfolded protein undergoes continuous cycles of glucose trimming (by glucosidase) and glucose addition (by glucosyl transferase), maintaining an affinity for calnexin and calreticulin until it has achieved its fully folded state (Figure 12–49).

1	improperly folded proteins are exported from the er and Degraded in the Cytosol Despite all the help from chaperones, many protein molecules (more than 80% for some proteins) translocated into the ER fail to achieve their properly folded or oligomeric state. Such proteins are exported from the ER back into the cytosol, where they are degraded in proteasomes (discussed in Chapter 6). In many ways, the mechanism of retrotranslocation is similar to other post-translational Figure 12–49 The role of N-linked glycosylation in ER protein folding.

1	The er-membrane-bound chaperone protein calnexin binds to incompletely folded proteins containing one terminal glucose on N-linked oligosaccharides, trapping the protein in the er. removal of the terminal glucose by a glucosidase releases the protein from calnexin. a glucosyl transferase is the crucial enzyme that determines whether the protein is folded properly or not: if the protein is still incompletely folded, the enzyme transfers a new glucose from UDp-glucose to the N-linked oligosaccharide, renewing the protein’s affinity for calnexin and retaining it in the er. The cycle repeats until the protein has folded completely. Calreticulin functions similarly, except that it is a soluble er resident protein. another er chaperone, erp57 (not shown), collaborates with calnexin and calreticulin in retaining an incompletely folded protein in the er. erp57 recognizes free sulfhydryl groups, which are a sign of incomplete disulfide bond formation.

1	modes of translocation. For example, like translocation into mitochondria or chloroplasts, chaperone proteins are necessary to keep the polypeptide chain in an unfolded state prior to and during translocation. Similarly, a source of energy is required to provide directionality to the transport and to pull the protein into the cytosol. Finally, a translocator is necessary.

1	Selecting proteins from the ER for degradation is a challenging process: mis-folded proteins or unassembled protein subunits should be degraded, but folding intermediates of newly made proteins should not. Help in making this distinction comes from the N-linked oligosaccharides, which serve as timers that measure how long a protein has spent in the ER. The slow trimming of a particular man-nose on the core oligosaccharide tree by an enzyme (a mannosidase) in the ER creates a new oligosaccharide structure that ER-lumenal lectins of the retrotranslocation apparatus recognize. Proteins that fold and exit from the ER faster than the mannosidase can remove its target mannose therefore escape degradation.

1	In addition to the lectins in the ER that recognize the oligosaccharides, chaperones and protein disulfide isomerases (enzymes mentioned earlier that catalyze the formation and breakage of S–S bonds) associate with the proteins that must be degraded. The chaperones prevent the unfolded proteins from aggregating, and the disulfide isomerases break disulfide bonds that may have formed incorrectly, so that a linear polypeptide chain can be translocated back into the cytosol.

1	Multiple translocator complexes move different proteins from the ER membrane or lumen into the cytosol. A common feature is that they each contain an E3 ubiquitin ligase enzyme, which attaches polyubiquitin tags to the unfolded proteins as they emerge into the cytosol, marking them for destruction. Fueled by the energy derived from ATP hydrolysis, a hexomeric ATPase of the family of AAA-ATPases (see Figure 6–85) pulls the unfolded protein through the translocator into the cytosol. An N-glycanase removes its oligosaccharide chains en bloc. Guided by its ubiquitin tag, the deglycosylated polypeptide is rapidly fed into proteasomes, where it is degraded (Figure 12–50). misfolded proteins in the er activate an Unfolded protein response

1	misfolded proteins in the er activate an Unfolded protein response Cells carefully monitor the amount of misfolded protein in various compartments. An accumulation of misfolded proteins in the cytosol, for example, triggers a heat-shock response (discussed in Chapter 6), which stimulates the transcription of genes encoding cytosolic chaperones that help to refold the proteins. Similarly, an accumulation of misfolded proteins in the ER triggers an unfolded protein response, which includes an increased transcription of genes encoding proteins involved in retrotranslocation and protein degradation in the cytosol, ER chaperones, and many other proteins that help to increase the protein-folding capacity of the ER. Figure 12–50 The export and degradation of misfolded ER proteins.

1	Figure 12–50 The export and degradation of misfolded ER proteins. misfolded soluble proteins in the er lumen are recognized and targeted to a translocator complex in the er membrane. They first interact in the er lumen with chaperones, disulfide isomerases, and lectins. They are then exported into the cytosol through the translocator. in the cytosol, they are ubiquitylated, deglycosylated, and degraded in proteasomes. misfolded membrane proteins follow a similar pathway but use a different translocator. How do misfolded proteins in the ER signal to the nucleus? There are three parallel pathways that execute the unfolded protein response (Figure 12–51A). The first pathway, which was initially discovered in yeast cells, is particularly remarkable. Misfolded proteins in the ER activate a transmembrane protein kinase in the ER, called IRE1, which causes the kinase to oligomerize and phosphorylate itself. (Some cell-surface receptor kinases in the plasma membrane are activated in a

1	Figure 12–51 The unfolded protein response. (a) By three parallel intracellular signaling pathways, the accumulation of misfolded proteins in the er lumen signals to the nucleus to activate the transcription of genes that encode proteins that help the cell cope with misfolded proteins in the er. (B) regulated rna splicing is a key regulatory switch in pathway 1 of the unfolded protein response (Movie 12.6).

1	1 MISFOLDED PROTEINS IN ER SIGNAL THE NEED FOR MORE ER CHAPERONES. THEY BIND TO AND ACTIVATE A TRANSMEMBRANE KINASE 2 ACTIVATED KINASE UNMASKS AN ENDORIBONUCLEASE ACTIVITY 3 ENDORIBONUCLEASE CUTS SPECIFIC RNA MOLECULES AT TWO POSITIONS, REMOVING AN INTRON 6 TRANSCRIPTION REGULATOR ENTERS NUCLEUS AND ACTIVATES GENES ENCODING ER CHAPERONES 7 CHAPERONES ARE MADE IN ER, WHERE THEY HELP FOLD PROTEINS 4 TWO EXONS ARE LIGATED TO FORM AN ACTIVE mRNA 5 mRNA IS TRANSLATED TO MAKE A TRANSCRIPTION REGULATOR similar way, as discussed in Chapter 15.) The oligomerization and autophosphorylation of IRE1 activates an endoribonuclease domain in the cytosolic portion of the same molecule, which cleaves a specific cytosolic mRNA molecule at two positions, excising an intron. (This is a unique exception to the rule that introns are spliced out while the RNA is still in the nucleus.) The separated exons are then joined by an RNA ligase, generating a spliced mRNA, which is translated to produce an active

1	the rule that introns are spliced out while the RNA is still in the nucleus.) The separated exons are then joined by an RNA ligase, generating a spliced mRNA, which is translated to produce an active transcription regulatory protein. This protein activates the transcription of genes encoding the proteins that help mediate the unfolded protein response (Figure 12–51B).

1	Misfolded proteins also activate a second transmembrane kinase in the ER, PERK, which inhibits a translation initiation factor by phosphorylating it, thereby reducing the production of new proteins throughout the cell. One consequence of the reduction in protein synthesis is to reduce the flux of proteins into the ER, thereby reducing the load of proteins that need to be folded there. Some proteins, however, are preferentially translated when translation initiation factors are scarce (discussed in Chapter 7, p. 424), and one of these is a transcription regulator that helps activate the transcription of the genes encoding proteins active in the unfolded protein response.

1	Finally, a third transcription regulator, ATF6, is initially synthesized as a trans-membrane ER protein. Because it is embedded in the ER membrane, it cannot activate the transcription of genes in the nucleus. When misfolded proteins accumulate in the ER, however, the ATF6 protein is transported to the Golgi apparatus, where it encounters proteases that cleave off its cytosolic domain, which can now migrate to the nucleus and help activate the transcription of genes encoding proteins involved in the unfolded protein response. (This mechanism is similar to that described in Figure 12–16 for activation of the transcription regulator that controls cholesterol biosynthesis.) The relative importance of each of these three pathways in the unfolded protein response differs in different cell types, enabling each cell type to tailor the unfolded protein response to its particular needs.

1	As discussed in Chapter 10, several cytosolic enzymes catalyze the covalent addition of a single fatty acid chain or prenyl group to selected proteins. The attached lipids help direct and attach these proteins to cell membranes. A related process is catalyzed by ER enzymes that covalently attach a glycosylphosphatidylinositol (GPI) anchor to the C-terminus of some membrane proteins destined for the plasma membrane. This linkage forms in the lumen of the ER, where, at the same time, the transmembrane segment of the protein is cleaved off (Figure 12–52). A large number of plasma membrane proteins are modified in this way. Since they are attached to the exterior of the plasma membrane only by their GPI anchors,

1	Figure 12–52 The attachment of a GPI anchor to a protein in the ER. Gpianchored proteins are targeted to the er membrane by an n-terminal signal sequence (not shown), which is removed (see figure 12–42). immediately after the completion of protein synthesis, the precursor protein remains anchored in the er membrane by a hydrophobic C-terminal sequence of 15–20 amino acids; the rest of the protein is in the er lumen. Within less than a minute, an enzyme in the er cuts the protein free from its membrane-bound C-terminus and simultaneously attaches the new C-terminus to an amino group on a preassembled Gpi intermediate. The sugar chain contains an inositol attached to the lipid from which the Gpi anchor derives its name. it is followed by a glucosamine and three mannoses. The terminal mannose links to a phosphoethanolamine that provides the amino group to attach the protein. The signal that specifies this modification is contained within the hydrophobic C-terminal sequence and a few

1	links to a phosphoethanolamine that provides the amino group to attach the protein. The signal that specifies this modification is contained within the hydrophobic C-terminal sequence and a few amino acids adjacent to it on the lumenal side of the er membrane; if this signal is added to other proteins, they too become modified in this way. Because of the covalently linked lipid anchor, the protein remains membrane-bound, with all of its amino acids exposed initially on the lumenal side of the er and eventually on the exterior of the plasma membrane.

1	lipid bilayer of ER phosphatidic they can in principle be released from cells in soluble form in response to signals Figure 12–53 The synthesis of phosphatidylcholine. as illustrated, this that activate a specific phospholipase in the plasma membrane. Trypanosome phospholipid is synthesized from glycerol parasites, for example, use this mechanism to shed their coat of GPI-anchored 3-phosphate, cytidine-diphosphocholine surface proteins when attacked by the immune system. GPI anchors may also be (CDp-choline), and fatty acids delivered used to direct plasma membrane proteins into lipid rafts and thus segregate the to the er by a cytosolic fatty acid binding proteins from other membrane proteins (see Figure 10–13). protein. The er assembles most lipid Bilayers

1	The ER membrane is the site of synthesis of nearly all of the cell’s major classes of lipids, including both phospholipids and cholesterol, required for the production of new cell membranes. The major phospholipid made is phosphatidylcholine, which can be formed in three steps from choline, two fatty acids, and glycerol phosphate (Figure 12–53). Each step is catalyzed by enzymes in the ER membrane, which have their active sites facing the cytosol, where all of the required metabolites are found. Thus, phospholipid synthesis occurs exclusively in the cytosolic leaflet of the ER membrane. Because fatty acids are not soluble in water, they are shepherded from their sites of synthesis to the ER by a fatty acid binding protein in the cytosol. After arrival in the ER membrane and activation with CoA, acyl transferases successively add two fatty acids to glycerol phosphate to produce phosphatidic acid. Phosphatidic acid is sufficiently water-insoluble to remain in the lipid bilayer; it

1	with CoA, acyl transferases successively add two fatty acids to glycerol phosphate to produce phosphatidic acid. Phosphatidic acid is sufficiently water-insoluble to remain in the lipid bilayer; it cannot be extracted from the bilayer by the fatty acid binding proteins. It is therefore this first step that enlarges the ER lipid bilayer. The later steps determine the head group of a newly formed lipid molecule and therefore the chemical nature of the bilayer, but they do not result in net membrane growth. The two other major membrane phospholipids—phosphatidylethanolamine and phosphatidylserine (see Figure 10–3)—as well as the minor phospholipid phosphatidylinositol (PI), are all synthesized in this way.

1	Because phospholipid synthesis takes place in the cytosolic leaflet of the ER lipid bilayer, there needs to be a mechanism that transfers some of the newly formed phospholipid molecules to the lumenal leaflet of the bilayer. In synthetic lipid bilayers, lipids do not “flip-flop” in this way (see Figure 10–10). In the ER, however, phospholipids equilibrate across the membrane within minutes, which is almost 100,000 times faster than can be accounted for by spontaneous “flipflop.” This rapid trans-bilayer movement is mediated by a poorly characterized asymmetric lipid bilayer of plasma membrane phospholipid translocator called a scramblase, which nonselectively equilibrates phospholipids between the two leaflets of the lipid bilayer (Figure 12–54). Thus, the different types of phospholipids are thought to be equally distributed between the two leaflets of the ER membrane.

1	The plasma membrane contains a different type of phospholipid translocator that belongs to the family of P-type pumps (discussed in Chapter 11). These flip-pases specifically recognize those phospholipids that contain free amino groups in their head groups (phosphatidylserine and phosphatidylethanolamine—see Figure 10–3) and transfers them from the extracellular to the cytosolic leaflet, using the energy of ATP hydrolysis. The plasma membrane therefore has a highly asymmetric phospholipid composition, which is actively maintained by the flip-pases (see Figure 10–15). The plasma membrane also contains a scramblase but, in contrast to the ER scramblase, which is always active, the plasma membrane enzyme is regulated and only activated in some situations, such as in apoptosis and in activated platelets, where it acts to abolish the lipid bilayer asymmetry; the resulting exposure of phosphatidylserine on the surface of apoptotic cells serves as a signal for phagocytic cells to ingest and

1	platelets, where it acts to abolish the lipid bilayer asymmetry; the resulting exposure of phosphatidylserine on the surface of apoptotic cells serves as a signal for phagocytic cells to ingest and degrade the dead cell.

1	The ER also produces cholesterol and ceramide (Figure 12–55). Ceramide is made by condensing the amino acid serine with a fatty acid to form the amino alcohol sphingosine (see Figure 10–3); a second fatty acid is then covalently added to form ceramide. The ceramide is exported to the Golgi apparatus, where it serves as a precursor for the synthesis of two types of lipids: oligosaccharide chains are added to form glycosphingolipids (glycolipids; see Figure 10–16), and phosphocholine head groups are transferred from phosphatidylcholine to other ceramide molecules to form sphingomyelin (discussed in Chapter 10). Thus, both glycolipids and sphingomyelin are produced relatively late in the process of membrane synthesis. Because they are produced by enzymes that have their active sites exposed to the Golgi lumen, they are found exclusively in the noncytosolic leaflet of the lipid bilayers that contain them. Figure 12–54 The role of phospholipid translocators in lipid bilayer synthesis.

1	Figure 12–54 The role of phospholipid translocators in lipid bilayer synthesis. (a) Because new lipid molecules are added only to the cytosolic half of the er membrane bilayer and lipid molecules do not flip spontaneously from one monolayer to the other, a transmembrane phospholipid translocator (called a scramblase) is required to transfer lipid molecules from the cytosolic half to the lumenal half so that the membrane grows as a bilayer. The scramblase is not specific for particular phospholipid head groups and therefore equilibrates the different phospholipids between the two monolayers. (B) fueled by aTp hydrolysis, a head-group-specific flippase in the plasma membrane actively flips phosphatidylserine and phosphatidylethanolamine directionally from the extracellular to the cytosolic leaflet, creating the characteristically asymmetric lipid bilayer of the plasma membrane of animal cells (see figure 10–15). Figure 12–55 The structure of ceramide.

1	Figure 12–55 The structure of ceramide. As discussed in Chapter 13, the plasma membrane and the membranes of the Golgi apparatus, lysosomes, and endosomes all form part of a membrane system that communicates with the ER by means of transport vesicles, which transfer both proteins and lipids. Mitochondria and plastids, however, do not belong to this system, and they therefore require different mechanisms to import proteins and lipids for growth. We have already seen that they import most of their proteins from the cytosol. Although mitochondria modify some of the lipids they import, they do not synthesize lipids de novo; instead, their lipids have to be imported from the ER, either directly or indirectly by way of other cell membranes. In either case, special mechanisms are required for the transfer.

1	The details of how lipid distribution between different membranes is catalyzed and regulated are not known. Water-soluble carrier proteins called phospholipid exchange proteins (or phospholipid transfer proteins) are thought to transfer individual phospholipid molecules between membranes, functioning much like fatty acid binding proteins that shepherd fatty acids through the cytosol (see Figure 12–54). In addition, mitochondria are often seen in close juxtaposition to ER membranes in electron micrographs, and specific junction complexes have been identified that hold the ER and outer mitochondrial membrane in close proximity. It is thought that these junction complexes provide specific contact-dependent lipid transfer mechanisms that operate between these adjacent membranes.

1	The extensive ER network serves as a factory for the production of almost all of the cell’s lipids. In addition, a major portion of the cell’s protein synthesis occurs on the cytosolic surface of the rough ER: virtually all proteins destined for secretion or for the ER itself, the Golgi apparatus, the lysosomes, the endosomes, and the plasma membrane are first imported into the ER from the cytosol. In the ER lumen, the proteins fold and oligomerize, disulfide bonds are formed, and N-linked oligosaccharides are added. The pattern of N-linked glycosylation is used to indicate the extent of protein folding, so that proteins leave the ER only when they are properly folded. Proteins that do not fold or oligomerize correctly are translocated back into the cytosol, where they are deglycosylated, polyubiquitylated, and degraded in proteasomes. If misfolded proteins accumulate in excess in the ER, they trigger an unfolded protein response, which activates appropriate genes in the nucleus to

1	polyubiquitylated, and degraded in proteasomes. If misfolded proteins accumulate in excess in the ER, they trigger an unfolded protein response, which activates appropriate genes in the nucleus to help the ER cope.

1	Only proteins that carry a special ER signal sequence are imported into the ER. The signal sequence is recognized by a signal-recognition particle (SRP), which binds both the growing polypeptide chain and the ribosome and directs them to a receptor protein on the cytosolic surface of the rough ER membrane. This binding to the ER membrane initiates the translocation process that threads a loop of polypeptide chain across the ER membrane through the hydrophilic pore of a protein translocator.

1	Soluble proteins—destined for the ER lumen, for secretion, or for transfer to the lumen of other organelles—pass completely into the ER lumen. Transmembrane proteins destined for the ER or for other cell membranes are translocated part way across the ER membrane and remain anchored there by one or more membrane-spanning α-helical segments in their polypeptide chains. These hydrophobic portions of the protein can act either as start-transfer or stop-transfer signals during the translocation process. When a polypeptide contains multiple, alternating start-transfer and stop-transfer signals, it will pass back and forth across the bilayer multiple times as a multipass transmembrane protein. The asymmetry of protein insertion and glycosylation in the ER establishes the sidedness of the membranes of all the other organelles that the ER supplies with membrane proteins. how do nuclear import receptors negotiate the tangled gel-like interior of a nuclear pore complex so efficiently?

1	how do nuclear import receptors negotiate the tangled gel-like interior of a nuclear pore complex so efficiently? is the nuclear pore complex a rigid structure or can it expand and contract, depending on the cargo transported? sequence comparisons show that signal sequences for an individual protein such as insulin are quite conserved across species, much more so than would be expected from our current understanding that all that matters for their function are general structural features such as hydrophobicity. What other functions might signal sequences have that could account for their evolutionary sequence conservation? how are polyribosomes on the endoplasmic reticulum membrane arranged so that the next initiating ribosome will find an unoccupied translocator? Why does the signal-recognition particle have an indispensable rna subunit? Which statements are true? Explain why or why not.

1	Why does the signal-recognition particle have an indispensable rna subunit? Which statements are true? Explain why or why not. 12–1 Like the lumen of the ER, the interior of the nucleus is topologically equivalent to the outside of the cell. 12–2 ER-bound and free ribosomes, which are structurally and functionally identical, differ only in the proteins they happen to be making at a particular time. 12–3 To avoid the inevitable collisions that would occur if two-way traffic through a single pore were allowed, nuclear pore complexes are specialized so that some mediate import while others mediate export. 12–4 Peroxisomes are found in only a few specialized types of eukaryotic cell. Discuss the following problems. 12–5 What is the fate of a protein with no sorting signal?

1	12–4 Peroxisomes are found in only a few specialized types of eukaryotic cell. Discuss the following problems. 12–5 What is the fate of a protein with no sorting signal? 12–6 The rough ER is the site of synthesis of many classes of membrane proteins. Some of these proteins remain in the ER, whereas others are sorted to compartments such as the Golgi apparatus, lysosomes, and the plasma membrane. One measure of the difficulty of the sorting problem is the degree of “purification” that must be achieved during transport from the ER. Are proteins bound for the plasma membrane common or rare among all ER membrane proteins?

1	A few simple considerations allow one to answer this question. In a typical growing cell that is dividing once every 24 hours, the equivalent of one new plasma membrane must transit the ER every day. If the ER membrane is 20 times the area of a plasma membrane, what is the ratio of plasma membrane proteins to other membrane proteins in the ER? (Assume that all proteins on their way to the plasma membrane remain in the ER for 30 minutes on average before exiting, and that the ratio of proteins to lipids in the ER and plasma membranes is the same.) 12–7 Before nuclear pore complexes were well understood, it was unclear whether nuclear proteins diffused passively into the nucleus and accumulated there by binding to residents of the nucleus such as chromosomes, or whether they were actively imported and accumulated regardless of their affinity for nuclear components.

1	A classic experiment that addressed this problem used several forms of radioactive nucleoplasmin, which is a large pentameric protein involved in chromatin assembly. In this experiment, either the intact protein or the nucleoplasmin heads, tails, or heads with a single tail were injected into the cytoplasm of a frog oocyte or into the nucleus (Figure Q12–1). All forms of nucleoplasmin, except heads, accumulated in the nucleus when injected into the cytoplasm, and all forms were retained in the nucleus when injected there. A. What portion of the nucleoplasmin molecule is responsible for localization in the nucleus? nucleoplasmin nuclear cytoplasmic Figure Q12–1 Cellular preparation injection injection location of injected nucleoplasmin components (problem 12–7). schematic diagrams of autoradiographs show the cytoplasm and nucleus with the location of nucleoplasmin indicated by the red areas.

1	B. How do these experiments distinguish between active transport, in which a nuclear localization signal triggers transport by the nuclear pore complex, and passive diffusion, in which a binding site for a nuclear component allows accumulation in the nucleus? 12–8 Assuming that 32 million histone octamers are required to package the human genome, how many his-tone molecules must be transported per second per nuclear pore complex in cells whose nuclei contain 3000 nuclear pores and are dividing once per day?

1	12–9 The nuclear pore complex (NPC) creates a barrier to the free exchange of molecules between the nucleus and cytosol, but in a way that remains mysterious. In yeast, for example, the central pore of the NPC has a diameter of 35 nm and is 30 nm long, which is somewhat smaller than its vertebrate counterpart. Even so, it is large enough to accommodate virtually all components of the cytosol. Yet the pore allows passive diffusion of molecules only up to about 40 kd; entry of anything larger requires help from a nuclear import receptor. Selective permeability is controlled by protein components of the NPC that have unstructured, polar tails extending into the central pore. These tails are characterized by periodic repeats of the hydrophobic amino acids phenylalanine (F) and glycine (G).

1	At high enough concentration (~50 mM), the FG-repeat domains of these proteins can form a gel, with a meshwork of interactions between the hydrophobic FG repeats (Figure Q12–2A). These gels allow passive diffusion of small molecules, but they prevent entry of larger proteins such as the fluorescent protein mCherry fused to maltose binding protein (MBP) (Figure Q12–2B). (The fusion to MBP makes mCherry too large to enter the nucleus by passive diffusion.) However, if the nuclear import receptor, importin, is fused to a similar protein, MBP-GFP, the importin-MBP-GFP fusion readily enters the gel (Figure Q12–2B).

1	Figure Q12–2 fG-repeat gel and influx of proteins into the nucleus (problem 12–9). (a) Cartoon of the meshwork (gel) formed by pairwise interactions between hydrophobic fG repeats. for fG-repeats separated by 17 amino acids, as is typical, the mesh formed by extended amino acid side chains would correspond to about 4 nm on a side, which would be large enough to account for the characteristic passive diffusion of proteins through nuclear pores. (B) Diffusion of mBp-mCherry and importin-mBp-Gfp into a gel of fG-repeats. in each group, the solution is shown at left and the gel at right. The bright areas indicate regions that contain the fluorescent proteins.

1	A. FG-repeats only form gels in vitro at relatively high concentration (50 mM). Is this concentration reasonable for FG repeats in the NPC core? In yeast, there are about 5000 FG-repeats in each NPC. Given the dimensions of the yeast nuclear pore (35 nm diameter and 30 nm length), calculate the concentration of FG-repeats in the cylindrical volume of the pore. Is this concentration comparable to the one used in vitro?

1	B. A second question is whether the diffusion of importin-MBP-GFP through the FG-repeat gel is fast enough to account for the efficient flow of materials between the nucleus and cytosol. From experiments of the type shown in Figure Q12–2B, the diffusion coefficient (D) of importin-MBP-GFP through the FG-repeat gel was determined to be about 0.1 μm2/s. The equation for diffusion is t = x2/2D, where t is time and x is distance. Calculate the time it would take importin-MBP-GFP to diffuse through a yeast nuclear pore (30 nm) if the pore consisted of a gel of FG-repeats. Does this time seem fast enough for the needs of a eukaryotic cell?

1	12–10 Components of the TIM complexes, the multi-subunit protein translocators in the mitochondrial inner membrane, are much less abundant than those of the TOM complex. They were initially identified using a genetic trick. The yeast Ura3 gene, whose product is an enzyme that is normally located in the cytosol where it is essential for synthesis of uracil, was modified so that the protein carried an import signal for the mitochondrial matrix. A population of cells carrying the modified Ura3 gene in place of the normal gene was then grown in the absence of uracil. Most cells died, but the rare cells that grew were shown to be defective for mitochondrial import. Explain how this selection identifies cells with defects in components required for import into the mitochondrial matrix. Why don’t normal cells with the modified Ura3 gene grow in the absence of uracil? Why do cells that are defective for mitochondrial import grow in the absence of uracil?

1	12–11 If the enzyme dihydrofolate reductase (DHFR), which is normally located in the cytosol, is engineered to carry a mitochondrial targeting sequence at its N-terminus, it is efficiently imported into mitochondria. If the modified DHFR is first incubated with methotrexate, which binds tightly to the active site, the enzyme remains in the cytosol. How do you suppose that the binding of methotrexate interferes with mitochondrial import? 12–12 Why do mitochondria need a special translocator to import proteins across the outer membrane, when the membrane already has large pores formed by porins? 12–13 Examine the multipass transmembrane protein shown in Figure Q12–3. What would you predict would be the effect of converting the first hydrophobic trans-membrane segment to a hydrophilic segment? Sketch the arrangement of the modified protein in the ER membrane.

1	Figure Q12–3 arrangement of a multipass transmembrane protein in the er membrane (problem 12–13). Blue hexagons represent covalently attached oligosaccharides. The positions of positively and negatively charged amino acids flanking the second transmembrane segment are shown. 12–14 All new phospholipids are added to the cytosolic leaflet of the ER membrane, yet the ER membrane has a symmetrical distribution of different phospholipids in its two leaflets. By contrast, the plasma membrane, which receives all its membrane components ultimately from the ER, has a very asymmetrical distribution of phospholipids in the two leaflets of its lipid bilayer. How is the symmetry generated in the ER membrane, and how is the asymmetry generated and maintained in the plasma membrane? palade G (1975) intracellular aspects of the process of protein synthesis. Science 189, 347–358. The Compartmentalization of Cells

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1	prakash s & matouschek a (2004) protein unfolding in the cell. Trends Biochem. Sci. 29, 593–600. schleiff e & Becker T (2011) Common ground for protein translocation: access control for mitochondria and chloroplasts. Nat. Rev. Mol. Cell Biol. 12, 48–59. Dimitrov l, lam sK & schekman r (2013) The role of the endoplasmic reticulum in peroxisome biogenesis. Cold Spring Harb. Perspect. Biol. 5, a013243. fujiki Y, Yagita Y & matsuzaki T (2012) peroxisome biogenesis disorders. Biochim. Biophys. Acta 1822, 1337–1342. schliebs W, Girzalsky W& erdmann r (2010) peroxisomal protein import and eraD: variations on a common theme. Nat. Rev. Mol. Cell Biol. 11, 885–890. Tabak hf, Braakman i & van der zand a (2013) peroxisome formation and maintenance are dependent on the endoplasmic reticulum. Annu. Rev. Biochem. 82, 723–744.

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1	Gething mJ (1999) role and regulation of the er chaperone Bip. Semin. Cell Dev. Biol. 10, 465–472. Görlich D, prehn s, hartmann e et al. (1992) a mammalian homolog of seC61p and seCYp is associated with ribosomes and nascent polypeptides during translocation. Cell 71, 489–503. hegde rs & ploegh hl (2010) Quality and quantity control at the endoplasmic reticulum. Curr. Opin. Cell Biol. 22, 437–446. hegde rs & Keenan rJ (2011) Tail-anchored membrane protein insertion into the endoplasmic reticulum. Nat. Rev. Mol. Cell Biol. 12, 787–798. levine T & loewen C (2006) inter-organelle membrane contact sites: through a glass, darkly. Curr. Opin. Cell Biol. 18, 371–378. lópez-marqués rl, holthuis JCm & pomorski TG (2011) pumping lipids with p4-aTpases. Biol. Chem. 392, 67–76. mamathambika Bs & Bardwell JC (2008) Disulfide-linked protein folding pathways. Annu. Rev. Cell Dev. Biol. 24, 211–235.

1	mamathambika Bs & Bardwell JC (2008) Disulfide-linked protein folding pathways. Annu. Rev. Cell Dev. Biol. 24, 211–235. marciniak sJ & ron D (2006) endoplasmic reticulum stress signaling in disease. Physiol. Rev. 86, 1133–1149. milstein C, Brownlee GG, harrison Tm & mathews mB (1972) a possible precursor of immunoglobulin light chains. Nat. New Biol. 239, 117–120. park e & rapoport Ta (2012) mechanisms of sec61/secY-mediated protein translocation across membranes. Annu. Rev. Biophys. 41, 21–40. römisch K (2005) endoplasmic reticulum-associated degradation. Annu. Rev. Cell Dev. Biol. 21, 435–456. rowland aa & Voeltz GK (2012) endoplasmic reticulum-mitochondria contacts: function of the junction. Nat. Rev. Mol. Cell Biol. 13, 607–625. Trombetta es & parodi aJ (2003) Quality control and protein folding in the secretory pathway. Annu. Rev. Cell Dev. Biol. 19, 649–676.

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1	Every cell must eat, communicate with the world around it, and quickly respond to changes in its environment. To help accomplish these tasks, cells continually adjust the composition of their plasma membrane and internal compartments in rapid response to need. They use an elaborate internal membrane system to add and remove cell-surface proteins, such as receptors, ion channels, and transporters (Figure 13–1). Through the process of exocytosis, the secretory pathway delivers newly synthesized proteins, carbohydrates, and lipids either to the plasma membrane or the extracellular space. By the converse process of endocytosis, cells remove plasma membrane components and deliver them to internal compartments called endosomes, from where they can be recycled to the same or different regions of the plasma membrane or be delivered to lysosomes for degradation. Cells also use endocytosis to capture important nutrients, such as vitamins, cholesterol, and iron; these are taken up together with

1	the plasma membrane or be delivered to lysosomes for degradation. Cells also use endocytosis to capture important nutrients, such as vitamins, cholesterol, and iron; these are taken up together with the macromolecules to which they bind and are then moved on to endosomes and lysosomes, from where they can be transported into the cytosol for use in various biosynthetic processes.

1	The interior space, or lumen, of each membrane-enclosed compartment along the secretory and endocytic pathways is equivalent to the lumen of most other membrane-enclosed compartments and to the cell exterior, in the sense that proteins can travel in this space without having to cross a membrane as they are passed from one compartment to another by means of numerous membrane-enclosed transport containers. These containers are formed from the donor compartment and are either small, spherical vesicles, larger irregular vesicles, or tubules. We shall use the term transport vesicle to apply to all forms of these containers.

1	Within a eukaryotic cell, transport vesicles continually bud off from one membrane and fuse with another, carrying membrane components and soluble lumenal molecules, which are referred to as cargo (Figure 13–2). This vesicular traffic flows along highly organized, directional routes, which allow the cell to secrete, eat, and remodel its plasma membrane and organelles. The secretory pathway leads outward from the endoplasmic reticulum (ER) toward the Golgi apparatus and cell surface, with a side route leading to lysosomes, while the endocytic pathway leads inward from the plasma membrane. In each case, retrieval pathways The Molecular MechanIsMs of MeMbrane TransporT and The MaInTenance of coMparTMenTal dIversITy TransporT froM The er Through The golgI apparaTus TransporT froM The Trans golgI neTwork To lysosoMes TransporT InTo The cell froM The plasMa MeMbrane: endocyTosIs TransporT froM The Trans golgI neTwork To The cell exTerIor: exocyTosIs

1	TransporT froM The Trans golgI neTwork To lysosoMes TransporT InTo The cell froM The plasMa MeMbrane: endocyTosIs TransporT froM The Trans golgI neTwork To The cell exTerIor: exocyTosIs Figure 13–1 Exocytosis and endocytosis. (a) In exocytosis, a transport vesicle fuses with the plasma membrane. Its content is released into the extracellular space, while the vesicle membrane (red) becomes continuous with the plasma membrane. (b) In endocytosis, a plasma membrane patch (red) is internalized, forming a transport vesicle. Its content derives from the extracellular space. 696 Chapter 13: Intracellular Membrane Traffic balance the flow of membrane between compartments in the opposite direction, Figure 13–2 Vesicle transport. Transport vesicles bud off from one compartment bringing membrane and selected proteins back to the compartment of origin and fuse with another. as they do so, they (Figure 13–3).

1	carry material as cargo from the lumen To perform its function, each transport vesicle that buds from a compartment (the space within a membrane-enclosed must be selective. It must take up only the appropriate molecules and must fuse compartment) and membrane of the donor only with the appropriate target membrane. A vesicle carrying cargo from the ER compartment to the lumen and membrane of the target compartment, as shown. to the Golgi apparatus, for example, must exclude most proteins that are to stay in the ER, and it must fuse only with the Golgi apparatus and not with any other organelle. We begin this chapter by considering the molecular mechanisms of budding and fusion that underlie all vesicle transport. We then discuss the fundamental problem of how, in the face of this transport, the cell maintains the molecular and

1	Figure 13–3 A “road-map” of the secretory and endocytic pathways. (a) In this schematic roadmap, which was introduced in chapter 12, the endocytic and secretory pathways are illustrated with green and red arrows, respectively. In addition, bluearrows denote retrieval pathways for the backflow of selected components. (b) The compartments of the eukaryotic cell involved in vesicle transport. The lumen of each membrane-enclosed compartment is topologically equivalent to the outside of the cell. all compartments shown communicate with one another and the outside of the cell by means of transport vesicles. In the secretory pathway (red arrows), protein molecules are transported from the er to the plasma membrane or (via endosomes) to lysosomes. In the endocytic pathway (green arrows), molecules are ingested in endocytic vesicles derived from the plasma membrane and delivered to early endosomes and then (via late endosomes) to lysosomes. Many endocytosed molecules are retrieved from early

1	are ingested in endocytic vesicles derived from the plasma membrane and delivered to early endosomes and then (via late endosomes) to lysosomes. Many endocytosed molecules are retrieved from early endosomes and returned (some via recycling endosomes) to the cell surface for reuse; similarly, some molecules are retrieved from the early and late endosomes and returned to the golgi apparatus, and some are retrieved from the golgi apparatus and returned to the er. all of these retrieval pathways are shown with blue arrows,as in part (a).

1	functional differences between its compartments. Finally, we consider the function of the Golgi apparatus, lysosomes, secretory vesicles, and endosomes, as we trace the pathways that connect these organelles. The Molecular MechanIsMs of MeMbrane TransporT and The MaInTenance of coMparTMenTal dIversITy

1	The Molecular MechanIsMs of MeMbrane TransporT and The MaInTenance of coMparTMenTal dIversITy Vesicle transport mediates a continuous exchange of components between the ten or more chemically distinct, membrane-enclosed compartments that collectively comprise the secretory and endocytic pathways. With this massive exchange, how can each compartment maintain its special identity? To answer this question, we must first consider what defines the character of a compartment. Above all, it is the composition of the enclosing membrane: molecular markers displayed on the cytosolic surface of the membrane serve as guidance cues for incoming traffic to ensure that transport vesicles fuse only with the correct compartment. Many of these membrane markers, however, are found on more than one compartment, and it is the specific combination of marker molecules that gives each compartment its molecular address.

1	How are these membrane markers kept at high concentration on one compartment and at low concentration on another? To answer this question, we need to consider how patches of membrane, enriched or depleted in specific membrane components, bud off from one compartment and transfer to another. We begin by discussing how cells segregate proteins into separate membrane domains by assembling a special protein coat on the membrane’s cytosolic face. We consider how coats form, what they are made of, and how they are used to extract specific cargo components from a membrane and compartment lumen for delivery to another compartment. Finally, we discuss how transport vesicles dock at the appropriate target membrane and then fuse with it to deliver their cargo. There are various Types of coated vesicles

1	There are various Types of coated vesicles Most transport vesicles form from specialized, coated regions of membranes. They bud off as coated vesicles, which have a distinctive cage of proteins covering their cytosolic surface. Before the vesicles fuse with a target membrane, they discard their coat, as is required for the two cytosolic membrane surfaces to interact directly and fuse. The coat performs two main functions that are reflected in a common two-layered structure. First, an inner coat layer concentrates specific membrane proteins in a specialized patch, which then gives rise to the vesicle membrane. In this way, the inner layer selects the appropriate membrane molecules for transport. Second, an outer coat layer assembles into a curved, basketlike lattice that deforms the membrane patch and thereby shapes the vesicle.

1	There are three well-characterized types of coated vesicles, distinguished by their major coat proteins: clathrin-coated, COPI-coated, and COPII-coated (Figure 13–4). Each type is used for different transport steps. Clathrin-coated vesicles, for example, mediate transport from the Golgi apparatus and from the plasma membrane, whereas COPIand COPII-coated vesicles most commonly mediate transport from the ER and from the Golgi cisternae (Figure 13–5). There is, however, much more variety in coated vesicles and their functions than this short list suggests. As we discuss below, there are several types of clathrin-coated vesicles, each specialized for a different transport step, and the COPIand COPII-coated vesicles may be similarly diverse. The assembly of a clathrin coat drives vesicle formation

1	The assembly of a clathrin coat drives vesicle formation Clathrin-coated vesicles, the first coated vesicles to be identified, transport material from the plasma membrane and between endosomal and Golgi compartments. COPI-coated vesicles and COPII-coated vesicles transport material early in the secretory pathway: COPI-coated vesicles bud from Golgi compartments, and COPII-coated vesicles bud from the ER (see Figure 13–5). We discuss clathrin-coated vesicles first, as they provide a good example of how vesicles form.

1	The major protein component of clathrin-coated vesicles is clathrin itself, which forms the outer layer of the coat. Each clathrin subunit consists of three large and three small polypeptide chains that together form a three-legged structure called a triskelion (Figure 13–6A,B). Clathrin triskelions assemble into a basketlike framework of hexagons and pentagons to form coated pits (buds) on the cytosolic surface of membranes (Figure 13–7). Under appropriate conditions, isolated triskelions spontaneously self-assemble into typical polyhedral cages in a test tube, even in the absence of the membrane vesicles that these baskets normally enclose (Figure 13–6C,D). Thus, the clathrin triskelions determine the geometry of the clathrin cage (Figure 13–6E).

1	Adaptor proteins, another major coat component in clathrin-coated vesicles, form a discrete inner layer of the coat, positioned between the clathrin cage and the membrane. They bind the clathrin coat to the membrane and trap various transmembrane proteins, including transmembrane receptors that capture soluble cargo molecules inside the vesicle—so-called cargo receptors. In this way, the adaptor proteins select a specific set of transmembrane proteins, together with the soluble proteins that interact with them, and package them into each newly formed clathrin-coated transport vesicle (Figure 13–8). KEY: Figure 13–4 Electron micrographs of clathrin-coated, COPI-coated, and COPII-coated vesicles. all are shown in electron micrographs at the same scale. (a clathrin-coated vesicles. (b) copIcoated vesicles and golgi cisternae (red arrows) from a cell-free system in which copI-coated vesicles bud in the test tube. copII-coated vesicles. (a and b, from l.

1	copII-coated vesicles. (a and b, from l. orci, b. glick and J. rothman, Cell 46:171–184, 1986. with permission from elsevier; c, courtesy of charles barlowe and lelio orci.) Figure 13–5 Use of different coats for different steps in vesicle traffic. different coat proteins select different cargo and shape the transport vesicles that mediate the various steps in the secretory and endocytic pathways. when the same coats function in different places in the cell, they usually incorporate different coat protein subunits that modify their properties (not shown). Many differentiated cells have additional pathways beside those shown here, including a sorting pathway from the trans golgi network to the apical surface of epithelial cells and a specialized recycling pathway for proteins of synaptic vesicles in the nerve terminals of neurons (see figure 11–36). The arrows are colored as in figure 13–3.

1	Figure 13–6 The structure of a clathrin coat. (a) electron micrograph of a clathrin triskelion shadowed with platinum.

1	each triskelion is composed of three clathrin heavy chains and three clathrin light chains, as shown in the diagram. (c and a cryoelectron micrograph taken of a clathrin coat composed of 36 triskelions organized in a network of 12 pentagons and 6 hexagons, with some heavy chains (c) and light chains (d) highlighted (Movie 13.1). The light chains link to the actin cytoskeleton, which helps generate force for membrane budding and vesicle movement, and their phosphorylation regulates clathrin coat assembly. The interwoven legs of the clathrin triskelions form an outer shell from which the n-terminal domains of the triskelions protrude inward. These domains bind to the adaptor proteins shown in figure 13–8. The coat shown was assembled biochemically from pure clathrin triskelions and is too small to enclose a membrane vesicle. (e) Images of clathrincoated vesicles isolated from bovine brain. The clathrin coats are constructed in a similar but less regular way, from pentagons, a larger

1	small to enclose a membrane vesicle. (e) Images of clathrincoated vesicles isolated from bovine brain. The clathrin coats are constructed in a similar but less regular way, from pentagons, a larger number of hexagons, and sometime heptagons, resembling the architecture of deformed soccer balls. The structures were determined by cryoelectron microscopy and tomographic reconstruction. (a, from e. ungewickell and d. branton, nature 289:420–422, 1981; c and d, from a. fotin et al., nature 432:573–579, 2004. all with permission from Macmillan publishers ltd; e, from y. cheng et al., J. Mol. Biol. 365:892–899, 2007. with permission from elsevier.)

1	There are several types of adaptor proteins. The best characterized have four different protein subunits; others are single-chain proteins. Each type of adaptor protein is specific for a different set of cargo receptors. Clathrin-coated vesicles budding from different membranes use different adaptor proteins and thus package different receptors and cargo molecules. The assembly of adaptor proteins on the membrane is tightly controlled, in part by the cooperative interaction of the adaptor proteins with other components of the coat. The adaptor protein AP2 serves as a well-understood example. When it binds to a specific phosphorylated phosphatidylinositol lipid (a phosphoinositide), it alters its conformation, exposing binding sites for cargo receptors in the membrane. The simultaneous binding to the cargo receptors and lipid head groups greatly enhances the binding of AP2 to the membrane (Figure 13–9).

1	Because several requirements must be met simultaneously to stably bind AP2 proteins to a membrane, the proteins act as coincidence detectors that only assemble at the right time and place. Upon binding, they induce membrane curvature, which makes the binding of additional AP2 proteins in its proximity more likely. The cooperative assembly of the AP2 coat layer then is further amplified by clathrin binding, which leads to the formation and budding of a transport vesicle. Adaptor proteins found in other coats also bind to phosphoinositides, which not only have a major role in directing when and where coats assemble in the cell, but also are used much more widely as molecular markers of compartment identity. This helps to control vesicular traffic, as we now discuss.

1	Figure 13–7 Clathrin-coated pits and vesicles. This rapid-freeze, deep- on the inner surface of the plasma membrane of cultured fibroblasts. The cells were rapidly frozen in liquid helium, fractured, and deep-etched to expose the cytoplasmic surface of the plasma membrane. (courtesy of John heuser.) 0.2 µm

1	Although inositol phospholipids typically comprise less than 10% of the total phospholipids in a membrane, they have important regulatory functions. They can undergo rapid cycles of phosphorylation and dephosphorylation at the 3ʹ, 4ʹ, and 5ʹ positions of their inositol sugar head groups to produce various types of phosphoinositides (phosphatidylinositol phosphates, or PIPs). The interconversion of phosphatidylinositol (PI) and PIPs is highly compartmentalized: different organelles in the endocytic and secretory pathways have distinct sets of PI and PIP kinases and PIP phosphatases (Figure 13–10). The distribution, regulation, and local balance of these enzymes determine the steady-state distribution of each PIP species. As a consequence, the distribution of PIPs varies from organelle to organelle, and often within a continuous membrane from one region to another, thereby defining specialized membrane domains.

1	Many proteins involved at different steps in vesicle transport contain domains that bind with high specificity to the head groups of particular PIPs, distinguishing one phosphorylated form from another (see Figure 13–10 E and F). Local control of the PI and PIP kinases and PIP phosphatases can therefore be used to rapidly control the binding of proteins to a membrane or membrane domain. The production of a particular type of PIP recruits proteins containing matching PIP-binding domains. The PIP-binding proteins then help regulate vesicle formation and other steps in the control of vesicle traffic (Figure 13–11). The same strategy is widely used to recruit specific intracellular signaling proteins to the plasma membrane in response to extracellular signals (discussed in Chapter 15). phosphoinositide PI(4,5)P2 endocytosis signals AP2 open AP2 locked ˜2 µ2 ˛2 ˝

1	phosphoinositide PI(4,5)P2 endocytosis signals AP2 open AP2 locked ˜2 µ2 ˛2 ˝ Figure 13–8 The assembly and disassembly of a clathrin coat. The assembly of the coat introduces curvature into the membrane, which leads in turn to the formation of a coated bud (called a coated pit if it is in the plasma membrane). The adaptor proteins bind both clathrin triskelions and membrane-bound cargo receptors, thereby mediating the selective recruitment of both membrane and soluble cargo molecules into the vesicle. other membrane-bending and fission proteins are recruited to the neck of the budding vesicle, where sharp membrane curvature is introduced. The coat is rapidly lost shortly after the vesicle buds off.

1	Figure 13–9 Lipid-induced conformation switching of AP2. The ap2 adaptor protein complex has four subunits (α, β2, μ2, and σ2). upon interaction with the phosphoinositide pI(4,5)p2 (see figure 13–10) in the cytosolic leaflet of the plasma membrane, ap2 rearranges so that binding sites for cargo receptors become exposed. each ap2 complex binds four pI(4,5)p2 molecules (for clarity, only one is shown). In the open ap2 complex, the μ2 and σ2 subunits bind the cytosolic tails of cargo receptors that display the appropriate endocytosis signals. These signals consist of short amino acid sequence motifs. when ap2 binds tightly to the membrane, it induces curvature, which favors the binding of additional ap2 complexes in the vicinity. OHOHHOHOHOPOOOOOOCCOO_CH2CH2CH123456123456PIPI(3,4)P2PI(3,5)P2PI(3)PPI(3,4)P2PIPI(4)PPI(5)PPI(4,5)P2PI(3,4,5)P3(A)(C)(E)(F)(B)(D)PPPP Membrane-bending proteins help deform the Membrane during vesicle formation

1	The forces generated by clathrin coat assembly alone are not sufficient to shape and pinch off a vesicle from the membrane. Other membrane-bending and force-generating proteins participate at every stage of the process. Membrane-bending proteins that contain crescent-shaped domains, called BAR domains, bind to and impose their shape on the underlying membrane via electrostatic interactions with the lipid head groups (Figure 13–12; also see Figure 10–40). Such BAR-domain proteins are thought to help AP2 nucleate clathrin-mediated endocytosis by shaping the plasma membrane to allow a clathrin-coated bud to form. Some of these proteins also contain amphiphilic helices that induce membrane curvature after being inserted as wedges into the cytoplasmic leaflet of the membrane. Other BAR-domain proteins are important in shaping the neck of a budding vesicle, where stabilization of sharp membrane bends is essential. Finally, the clathrin machinery nucleates the local assembly of actin

1	proteins are important in shaping the neck of a budding vesicle, where stabilization of sharp membrane bends is essential. Finally, the clathrin machinery nucleates the local assembly of actin filaments that introduce tension to help pinch off and propel the forming vesicle away from the membrane.

1	cytoplasmic proteins regulate the pinching-off and uncoating of coated vesicles As a clathrin-coated bud grows, soluble cytoplasmic proteins, including dynamin, assemble at the neck of each bud (Figure 13–13). Dynamin contains a PI(4,5) P2-binding domain, which tethers the protein to the membrane, and a GTPase domain, which regulates the rate at which vesicles pinch off from the membrane.

1	Figure 13–11 The intracellular location of phosphoinositides. different types of pIps are located in different membranes and membrane domains, where they are often associated with specific vesicle transport events. The membrane of secretory vesicles, for example, contains pI(4)p. when the vesicles fuse with the plasma membrane, a pI 5-kinase that is localized there converts the pI(4)p into pI(4,5)p2. The pI(4,5)p2, in turn, helps recruit adaptor proteins, which initiate the formation of a clathrin-coated pit, as the first step in clathrin-mediated endocytosis. once the clathrin-coated vesicle buds off from the plasma membrane, a pI(5)p phosphatase hydrolyzes pI(4,5)p2, which weakens the binding of the adaptor proteins, promoting vesicle uncoating. we discuss phagocytosis and the distinction between regulated and constitutive exocytosis later in the chapter. (Modified from M.a. de Matteis and a. godi, nat. Cell Biol. 6:487–492, 2004. with permission from Macmillan publishers ltd.)

1	M.a. de Matteis and a. godi, nat. Cell Biol. 6:487–492, 2004. with permission from Macmillan publishers ltd.) Figure 13–10 Phosphatidylinositol (PI) and phosphoinositides (PIPs). (a, b) The structure of pI shows the free hydroxyl groups in the inositol sugar that can in principle be modified.

1	(a, b) The structure of pI shows the free hydroxyl groups in the inositol sugar that can in principle be modified. (c) phosphorylation of one, two, or three of the hydroxyl groups on pI by pI and pIp kinases produces a variety of pIp species. They are named according to the ring position (in parentheses) and the number of phosphate groups (subscript) added to pI. pI(3,4)p2 is shown. (d) animal cells have several pI and pIp kinases and a similar number of pIp phosphatases, which are localized to different organelles, where they are regulated to catalyze the production of particular pIps. The red and green arrows show the kinase and phosphatase reactions, respectively. (e, f) phosphoinositide head groups are recognized by protein domains that discriminate between the different forms. In this way, select groups of proteins containing such domains are recruited to regions of membrane in which these phosphoinositides are present. pI(3)p and pI(4,5)p2 are shown. (d, modified from

1	M.a. de Matteis and a. godi, nat. Cell Biol. 6:487–492, 2004. with permission from Macmillan publishers ltd.) constitutive exocytosis regulated exocytosis endocytosis phagocytosis KEY: PI(3)P PI(4)P PI(4,5)P2 PI(3,5)P2 PI(3,4,5)P3 702 Chapter 13: Intracellular Membrane Traffic BAR domain dimer Figure 13–12 The structure of BAR domains. bar-domain proteins are diverse and enable many membrane-bending processes in the cell. bar domains are built from coiled coils that dimerize into modules with a positively charged inner + surface, which preferentially interacts with negatively charged lipid head groups to bend membranes. local membrane deformations caused by bar-domain proteins facilitate the binding of additional bar-domain proteins, thereby generating a positive feedback cycle for curvature propagation. Individual bar-domain

1	The pinching-off process brings the two noncytosolic leaflets of the membrane proteins contain a distinctive curvature and into close proximity and fuses them, sealing off the forming vesicle (see Figure often have additional features that adapt them to their specific tasks: some have 13–2). To perform this task, dynamin recruits other proteins to the neck of the bud. Together with dynamin, they help bend the patch of membrane—by directly distorting the bilayer structure, or by changing its lipid composition through the others are flanked by pIp-binding domains recruitment of lipid-modifying enzymes, or by both mechanisms. that direct them to membranes enriched in Once released from the membrane, the vesicle rapidly loses its clathrin coat. A cognate phosphoinositides. PIP phosphatase that is co-packaged into clathrin-coated vesicles depletes PI(4,5) P2 from the membrane, which weakens the binding of the adaptor proteins. In addition, an hsp70 chaperone protein (see Figure 6–80)

1	that is co-packaged into clathrin-coated vesicles depletes PI(4,5) P2 from the membrane, which weakens the binding of the adaptor proteins. In addition, an hsp70 chaperone protein (see Figure 6–80) functions as an uncoating ATPase, using the energy of ATP hydrolysis to peel off the clathrin coat. Auxilin, another vesicle protein, is thought to activate the ATPase. The release of the coat, however, must not happen prematurely, so additional control mechanisms must somehow prevent the clathrin from being removed before it has formed a complete vesicle (discussed below).

1	GTPase domain of dynamin

1	Figure 13–13 The role of dynamin in pinching off clathrin-coated vesicles. (a) Multiple dynamin molecules assemble into a spiral around the neck of the forming bud. The dynamin spiral is thought to recruit other proteins to the bud neck, which, together with dynamin, destabilize the interacting lipid bilayers so that the noncytoplasmic leaflets flow together. The newly formed vesicle then pinches off from the membrane. specific mutations in dynamin can either enhance or block the pinching-off process. (b) dynamin was discovered as the protein defective in the shibire mutant of Drosophila. These mutant flies become paralyzed because clathrin-mediated endocytosis stops, and the synaptic vesicle membrane fails to recycle, blocking neurotransmitter release. deeply invaginated clathrin-coated pits form in the nerve endings of the fly’s nerve cells, with a belt of mutant dynamin assembled around the neck, as shown in this thin-section electron micrograph. The pinching-off process fails

1	pits form in the nerve endings of the fly’s nerve cells, with a belt of mutant dynamin assembled around the neck, as shown in this thin-section electron micrograph. The pinching-off process fails because the required membrane fusion does not take place. (c, d) a model of how conformational changes in the gTpase domains of membrane-assembled dynamin may power a conformational change that constricts the neck of the bud. a single dynamin molecule is shown in orange in d. (b, from J.h. koenig and k. Ikeda, J. neurosci. 9:3844–3860, 1989. with permission from the society of neuroscience; c and d, adapted from M.g.J. ford, s. Jenni and J. nunnari, nature 477:561–566, 2011. with permission from Macmillan publishers.)

1	To balance the vesicle traffic to and from a compartment, coat proteins must assemble only when and where they are needed. While local production of PIPs plays a major part in regulating the assembly of clathrin coats on the plasma membrane and Golgi apparatus, cells superimpose additional ways of regulating coat formation. Coat-recruitment GTPases, for example, control the assembly of clathrin coats on endosomes and the COPI and COPII coats on Golgi and ER membranes.

1	Many steps in vesicle transport depend on a variety of GTP-binding proteins that control both the spatial and temporal aspects of vesicle formation and fusion. As discussed in Chapter 3, GTP-binding proteins regulate most processes in eukaryotic cells. They act as molecular switches, which flip between an active state with GTP bound and an inactive state with GDP bound. Two classes of proteins regulate the flipping: guanine nucleotide exchange factors (GEFs) activate the proteins by catalyzing the exchange of GDP for GTP, and GTPase-activating proteins (GAPs) inactivate the proteins by triggering the hydrolysis of the bound GTP to GDP (see Figures 3–68 and 15–7). Although both monomeric GTP-binding proteins (monomeric GTPases) and trimeric GTP-binding proteins (G proteins) have important roles in vesicle transport, the roles of the monomeric GTPases are better understood, and we focus on them here.

1	Coat-recruitment GTPases are members of a family of monomeric GTPases. They include the ARF proteins, which are responsible for the assembly of both COPI and clathrin coats assembly at Golgi membranes, and the Sar1 protein, which is responsible for the assembly of COPII coats at the ER membrane. Coat-recruitment GTPases are usually found in high concentration in the cytosol in an inactive, GDP-bound state. When a COPII-coated vesicle is to bud from the ER membrane, for example, a specific Sar1-GEF embedded in the ER membrane binds to cytosolic Sar1, causing the Sar1 to release its GDP and bind GTP in its place. (Recall that GTP is present in much higher concentration in the cytosol than GDP and therefore will spontaneously bind after GDP is released.) In its GTP-bound state, the Sar1 protein exposes an amphiphilic helix, which inserts into the cytoplasmic leaflet of the lipid bilayer of the ER membrane. The tightly bound Sar1 now recruits adaptor coat protein subunits to the ER

1	protein exposes an amphiphilic helix, which inserts into the cytoplasmic leaflet of the lipid bilayer of the ER membrane. The tightly bound Sar1 now recruits adaptor coat protein subunits to the ER membrane to initiate budding (Figure 13–14). Other GEFs and coat-recruitment GTPases operate in a similar way on other membranes.

1	The coat-recruitment GTPases also have a role in coat disassembly. The hydrolysis of bound GTP to GDP causes the GTPase to change its conformation so that its hydrophobic tail pops out of the membrane, causing the vesicle’s coat to disassemble. Although it is not known what triggers the GTP hydrolysis, it has been proposed that the GTPases work like timers, which hydrolyze GTP at slow but predictable rates, to ensure that vesicle formation is synchronized with the requirements of the moment. COPII coats accelerate GTP hydrolysis by Sar1, and a fully formed vesicle will be produced only when bud formation occurs faster than the timed disassembly process; otherwise, disassembly will be triggered before a vesicle pinches off, and the process will have to start again, perhaps at a more appropriate time and place. Once a vesicle pinches off, GTP hydrolysis releases Sar1, but the sealed coat is sufficiently stabilized through many cooperative interactions, including binding to the cargo

1	time and place. Once a vesicle pinches off, GTP hydrolysis releases Sar1, but the sealed coat is sufficiently stabilized through many cooperative interactions, including binding to the cargo receptors in the membrane, that it may stay on the vesicle until the vesicle docks at a target membrane. There, a kinase phosphorylates the coat proteins, which completes coat disassembly and readies the vesicle for fusion.

1	Clathrinand COPI-coated vesicles, by contrast, shed their coat soon after they pinch off. For COPI vesicles, the curvature of the vesicle membrane serves as a trigger to begin uncoating. An ARF-GAP is recruited to the COPI coat as it assembles. It interacts with the membrane, and senses the lipid packing density. It becomes activated when the curvature of the membrane approaches that of a transport vesicle. It then inactivates ARF, causing the coat to disassemble. inactive, soluble Sar1-GDP (A) amphiphilic helix active, membrane-bound Sar1-GTP donor membrane (ER) Sar1-GEF GTPGTPGDPGDPCYTOSOL ER LUMEN not all Transport vesicles are spherical

1	Although vesicle-budding is similar at various locations in the cell, each cell membrane poses its own special challenges. The plasma membrane, for example, is comparatively flat and stiff, owing to its cholesterol-rich lipid composition and underlying actin-rich cortex. Thus, the coordinated action of clathrin coats and membrane-bending proteins has to produce sufficient force to introduce curvature, especially at the neck of the bud where sharp bends are required for the pinching-off processes. In contrast, vesicle-budding from many intracellular membranes occurs preferentially at regions where the membranes are already curved, such as the rims of the Golgi cisternae or ends of membrane tubules. In these places, the primary function of the coats is to capture the appropriate cargo proteins rather than to deform the membrane.

1	Transport vesicles also occur in various sizes and shapes. Diverse COPII vesicles are required for the transport of large cargo molecules. Collagen, for example, is assembled in the ER as 300-nm-long, stiff procollagen rods that then are secreted from the cell where they are cleaved by proteases to collagen, which is embedded into the extracellular matrix (discussed in Chapter 19). Procollagen rods do not fit into the 60–80 nm COPII vesicles normally observed. To circumvent this problem, the procollagen cargo molecules bind to transmembrane packaging proteins in the ER, which control the assembly of the COPII coat components (Figure 13–15). These events drive the local assembly of much larger COPII vesicles that accommodate the oversized cargo. Human mutations in genes encoding such packaging proteins result in collagen defects with severe consequences, such as skeletal abnormalities and other developmental defects. Similar mechanisms must regulate the sizes of vesicles required to

1	proteins result in collagen defects with severe consequences, such as skeletal abnormalities and other developmental defects. Similar mechanisms must regulate the sizes of vesicles required to secrete other large macromolecular complexes, including the lipoprotein particles that transport lipids out of cells.

1	Figure 13–14 Formation of a COPII-coated vesicle. (a) Inactive, soluble sar1-gdp binds to a sar1-gef in the er membrane, causing the sar1 to release its gdp and bind gTp. a gTp-triggered conformational change in sar1 exposes an amphiphilic helix, which inserts into the cytoplasmic leaflet of the er membrane, initiating membrane bending (which is not shown). (b) gTp-bound sar1 binds to a complex of two copII adaptor coat proteins, called sec23 and sec24, which form the inner coat. sec24 has several different binding sites for the cytosolic tails of cargo receptors. The entire surface of the complex that attaches to the membrane is gently curved, matching the diameter of copII-coated vesicles.

1	(c) a complex of two additional copII coat proteins, called sec13 and sec 31, forms the outer shell of the coat. like clathrin, they can assemble on their own into symmetrical cages with appropriate dimensions to enclose a copII-coated vesicle. (d) Membrane-bound, active sar1-gTp recruits copII adaptor proteins to the membrane. They select certain transmembrane proteins and cause the membrane to deform. The adaptor proteins then recruit the outer coat proteins which help form a bud. a subsequent membrane fusion event pinches off the coated vesicle. other coated vesicles are thought to form in a similar way. (c, modified from s.M. stagg et al., nature 439:234–238, 2006. with permission from Macmillan publishers ltd.)

1	Many other vesicle budding events likewise involve variations of common mechanisms. When living cells are genetically engineered to express fluorescent membrane components, the endosomes and trans Golgi network are seen in a fluorescence microscope to continually send out long tubules. Coat proteins assemble onto the membrane tubules and help recruit specific cargo. The tubules then either regress or pinch off with the help of dynamin-like proteins to form transport vesicles of different sizes and shapes. Tubules have a higher surface-to-volume ratio than the larger organelles from which they form. They are therefore relatively enriched in membrane proteins compared with soluble cargo proteins. As we discuss later, this property of tubules is an important feature for sorting proteins in endosomes. rab proteins guide Transport vesicles to Their Target Membrane

1	rab proteins guide Transport vesicles to Their Target Membrane To ensure an orderly flow of vesicle traffic, transport vesicles must be highly accurate in recognizing the correct target membrane with which to fuse. Because of the diversity and crowding of membrane systems in the cytoplasm, a vesicle is likely to encounter many potential target membranes before it finds the correct one. Specificity in targeting is ensured because all transport vesicles display surface markers that identify them according to their origin and type of cargo, and target membranes display complementary receptors that recognize the appropriate markers. This crucial process occurs in two steps. First, Rab proteins and Rab effectors direct the vesicle to specific spots on the correct target membrane. Second, SNARE proteins and SNARE regulators mediate the fusion of the lipid bilayers.

1	Rab proteins play a central part in the specificity of vesicle transport. Like the coat-recruitment GTPases discussed earlier (see Figure 13–14), Rab proteins are also monomeric GTPases. With over 60 known members, the Rab subfamily is the largest of the monomeric GTPase subfamilies. Each Rab protein is associated with one or more membrane-enclosed organelles of the secretory or endocytic pathways, and each of these organelles has at least one Rab protein on its cytosolic surface (Table 13–1). Their highly selective distribution on these membrane systems makes Rab proteins ideal molecular markers for identifying each membrane type and guiding vesicle traffic between them. Rab proteins can function on transport vesicles, on target membranes, or both.

1	Like the coat-recruitment GTPases, Rab proteins cycle between a membrane and the cytosol and regulate the reversible assembly of protein complexes on the membrane. In their GDP-bound state, they are inactive and bound to another protein (Rab-GDP dissociation inhibitor, or GDI) that keeps them soluble in the Figure 13–15 Packaging of procollagen into large tubular COPII-coated vesicles.

1	The cartoons show models for two copII coat assembly modes. The models are based on cryoelectron tomography images of reconstituted copII vesicles. on a spherical membrane (left), the sec23/24 inner coat proteins assemble in patches that anchor the sec13/31 outer coat protein cage. The sec13/31 rods assemble a cage of triangles, squares, and pentagons. when procollagen needs to be packaged (right), special packaging proteins sense the cargo and modify the coat assembly process. This interaction recruits the copII inner coat protein sec24 and locally enhances the rate with which sar1 cycles on and off the membrane (not shown). In addition, a monoubiquitin is added to the sec31 protein, changing the assembly properties of the outer cage. sec23/24 proteins arrange in larger arrays and sec13/31 arrange in a regular lattice of diamond shapes. as the result, a large tubular vesicle is formed that can accommodate the large cargo molecules. The packaging proteins are not part of the budding

1	arrange in a regular lattice of diamond shapes. as the result, a large tubular vesicle is formed that can accommodate the large cargo molecules. The packaging proteins are not part of the budding vesicle but remain in the er. (Modified from g. Zanetti et al., eLife 2:e00951, 2013.) cytosol; in their GTP-bound state, they are active and tightly associated with the membrane of an organelle or transport vesicle. Membrane-bound Rab-GEFs activate Rab proteins on both transport vesicle and target membranes; for some membrane fusion events, activated Rab molecules are required on both sides of the reaction. Once in the GTP-bound state and membrane-bound through a now-exposed lipid anchor, Rab proteins bind to other proteins, called Rab effectors, which are the downstream mediators of vesicle transport, membrane tethering, and membrane fusion (Figure 13–16). The rate of GTP hydrolysis sets the concentration of active Rab and, consequently, the concentration of its effectors on the membrane.

1	In contrast to the highly conserved structure of Rab proteins, the structures and functions of Rab effectors vary greatly, and the same Rab proteins can often bind to many different effectors. Some Rab effectors are motor proteins that propel vesicles along actin filaments or microtubules to their target membrane. Others are tethering proteins, some of which have long, threadlike domains that serve as “fishing lines” that can extend to link two membranes more than 200 nm apart; other tethering proteins are large protein complexes that link two membranes that are closer together and interact with a wide variety of other proteins that facilitate the membrane fusion step. The tethering complex that docks COPII-coated

1	Figure 13–16 Tethering of a transport vesicle to a target membrane. rab effector proteins interact with active rab proteins (rab-gTps, yellow) located on the target membrane, vesicle membrane, or both, to establish the first connection between the two membranes that are going to fuse. In the example shown here, the rab effector is a filamentous tethering protein (dark green). next, snare proteins on the two membranes (red and blue) pair, docking the vesicle to the target membrane and catalyzing the fusion of the two apposed lipid bilayers. during docking and fusion, a rab-gap (not shown) induces the rab protein to hydrolyze its bound gTp to gdp, causing the rab to dissociate from the membrane and return to the cytosol as rab-gdp, where it is bound by a gdI protein that keeps the rab soluble and inactive.

1	vesicles, for example, contains a protein kinase that phosphorylates the coat proteins to complete the uncoating process. Coupling uncoating to vesicle delivery helps to ensure directionality of the transport process and fusion with the proper membrane. Rab effectors can also interact with SNAREs to couple membrane tethering to fusion (see Figure 13–16). The assembly of Rab proteins and their effectors on a membrane is cooperative and results in the formation of large, specialized membrane patches. Rab5, for example, assembles on endosomes and mediates the capture of endocytic vesicles arriving from the plasma membrane. The experimental depletion of Rab5 causes disappearance of the entire endosomal and lysosomal membrane system, highlighting the crucial role of Rab proteins in organelle biogenesis and maintenance.

1	A Rab5 domain concentrates tethering proteins that catch incoming vesicles. Its assembly on endosomal membranes begins when a Rab5-GDP/GDI complex encounters a Rab-GEF. GDI is released and Rab5-GDP is converted to Rab5-GTP. Active Rab5-GTP becomes anchored to the membrane and recruits more Rab5GEF to the endosome, thereby stimulating the recruitment of more Rab5 to the same site. In addition, active Rab5 activates a PI 3-kinase, which locally converts PI to PI(3)P, which in turn binds some of the Rab effectors including tethering proteins and stabilizes their local membrane attachment (Figure 13–17). This type of positive feedback greatly amplifies the assembly process and helps to establish functionally distinct membrane domains within a continuous membrane.

1	The endosomal membrane provides a striking example of how different Rab proteins and their effectors help to create multiple specialized membrane domains, each fulfilling a particular set of functions. Thus, while the Rab5 membrane domain receives incoming endocytic vesicles from the plasma membrane, distinct Rab11 and Rab4 domains in the same membrane organize the budding of recycling vesicles that return proteins from the endosome to the plasma membrane. rab cascades can change the Identity of an organelle

1	A Rab domain can be disassembled and replaced by a different Rab domain, changing the identity of an organelle. Such ordered recruitment of sequentially acting Rab proteins is called a Rab cascade. Over time, for example, Rab5 domains are replaced by Rab7 domains on endosomal membranes. This converts an early endosome, marked by Rab5, into a late endosome, marked by Rab7. Because the set of Rab effectors recruited by Rab7 is different from that recruited by Rab5, this change reprograms the compartment: as we discuss later, it alters the membrane dynamics, including the incoming and outgoing traffic, and repositions the organelle away from the plasma membrane toward the cell interior. All of the cargo contained in the early endosome that has not been recycled to the plasma membrane is now part of a late endosome. This process is also referred to as endosome maturation. The self-amplifying nature of the Rab domains renders the process of endosome maturation unidirectional and

1	is now part of a late endosome. This process is also referred to as endosome maturation. The self-amplifying nature of the Rab domains renders the process of endosome maturation unidirectional and irreversible (Figure 13–18).

1	Figure 13–17 The formation of a Rab5 domain on the endosome membrane. a rab5-gef on the endosome membrane binds a rab5 protein and induces it to exchange gdp for gTp. gdI is lost and gTp binding alters the conformation of the rab protein, exposing an amphiphilic helix and a covalently attached lipid group, which together anchor the rab5-gTp to the membrane. active rab5 activates pI 3-kinase, which converts pI into pI(3) p. pI(3)p and active rab5 together bind a variety of rab effector proteins that contain pI(3)p-binding sites, including filamentous tethering proteins that catch incoming clathrin-coated endocytic vesicles from the plasma membrane. with the help of another effector, active rab5 also recruits more rab5-gef, further enhancing the assembly of the rab5 domain on the membrane.

1	controlled cycles of gTp hydrolysis and gdp–gTp exchange dynamically regulate the size and activity of such rab domains. unlike snares, which are integral membrane proteins, the gdp/ gTp cycle, coupled to the membrane/ cytosol translocation cycle, endows the rab machinery with the ability to undergo assembly and disassembly on the membrane. (adapted from M. Zerial and h. Mcbride, nat. rev. Mol. Cell Biol. 2:107–117, 2001. with permission from Macmillan publishers ltd.)

1	Once a transport vesicle has been tethered to its target membrane, it unloads its cargo by membrane fusion. Membrane fusion requires bringing the lipid bilayers of two membranes to within 1.5 nm of each other so that they can merge. When the membranes are in such close apposition, lipids can flow from one bilayer to the other. For this close approach, water must be displaced from the hydrophilic surface of the membrane—a process that is highly energetically unfavorable and requires specialized fusion proteins that overcome this energy barrier. We have already discussed the role of dynamin in a related task during the pinching-off of clathrin-coated vesicles (see Figure 13–13).

1	The SNARE proteins (also called SNAREs, for short) catalyze the membrane fusion reactions in vesicle transport. There are at least 35 different SNAREs in an animal cell, each associated with a particular organelle in the secretory or endocytic pathways. These transmembrane proteins exist as complementary sets, with v-SNAREs usually found on vesicle membranes and t-SNAREs usually found on target membranes (see Figure 13–16). A v-SNARE is a single polypeptide chain, whereas a t-SNARE is usually composed of three proteins. The v-SNAREs and t-SNAREs have characteristic helical domains, and when a v-SNARE interacts with a t-SNARE, the helical domains of one wrap around the helical domains of the other to form a very stable four-helix bundle. The resulting trans-SNARE complex locks the two membranes together. Biochemical membrane fusion assays with all different SNARE combinations show that vand t-SNARE pairing is highly specific. The SNAREs thus provide an additional layer of specificity

1	together. Biochemical membrane fusion assays with all different SNARE combinations show that vand t-SNARE pairing is highly specific. The SNAREs thus provide an additional layer of specificity in the transport process by helping to ensure that vesicles fuse only with the correct target membrane.

1	The trans-SNARE complexes catalyze membrane fusion by using the energy that is freed when the interacting helices wrap around each other to pull the membrane faces together, simultaneously squeezing out water molecules from the interface (Figure 13–19). When liposomes containing purified v-SNAREs are mixed with liposomes containing complementary t-SNAREs, their membranes fuse, albeit slowly. In the cell, other proteins recruited to the fusion site, presumably Rab effectors, cooperate with SNAREs to accelerate fusion. Fusion does not always follow immediately after v-SNAREs and t-SNAREs pair. As we discuss later, in the process of regulated exocytosis, fusion is delayed until secretion is triggered by a specific extracellular signal.

1	Rab proteins, which can regulate the availability of SNARE proteins, exert an additional layer of control. t-SNAREs in target membranes are often associated with inhibitory proteins that must be released before the t-SNARE can function. Rab proteins and their effectors trigger the release of such SNARE inhibitory Figure 13–19 A model for how SNARE proteins may catalyze membrane fusion. bilayer fusion occurs in multiple steps. a tight pairing between vand t-snares forces lipid bilayers into close apposition and expels water molecules from the interface. lipid molecules in the two interacting (cytosolic) leaflets of the bilayers then flow between the membranes to form a connecting stalk. lipids of the two noncytosolic leaflets then contact each other, forming a new bilayer, which widens the fusion zone (hemifusion, or half-fusion). rupture of the new bilayer completes the fusion reaction.

1	Figure 13–18 A model for a generic Rab cascade. The local activation of a raba-gef leads to assembly of a raba domain on the membrane. active raba recruits its effector proteins, one of which is a gef for rabb. The rabb-gef then recruits rabb to the membrane, which in turn begins to recruit its effectors, among them a gap for raba. The raba-gap activates raba gTp hydrolysis leading to the inactivation of the raba and the disassembly of the raba domain as the rabb domain grows. In this way, the raba domain is irreversibly replaced by the rabb domain. In principle, this sequence can continued by the recruitment of a next gef by rabb. (adapted from a.h. hutagalung and p.J. novick, Physiol. rev. 91:119–149, 2011. with permission from The american physiological society.) proteins. In this way, SNARE proteins are concentrated and activated in the correct location on the membrane, where tethering proteins capture incoming vesicles. Rab proteins thus speed up the process by which appropriate

1	SNARE proteins are concentrated and activated in the correct location on the membrane, where tethering proteins capture incoming vesicles. Rab proteins thus speed up the process by which appropriate SNARE proteins in two membranes find each other.

1	For vesicle transport to operate normally, transport vesicles must incorporate the appropriate SNARE and Rab proteins. Not surprisingly, therefore, many transport vesicles will form only if they incorporate the appropriate complement of SNARE and Rab proteins in their membrane. How this crucial control process operates during vesicle budding remains a mystery. Interacting snares need to be pried apart before They can function again

1	Most SNARE proteins in cells have already participated in multiple rounds of vesicle transport and are sometimes present in a membrane as stable complexes with partner SNAREs. The complexes have to disassemble before the SNAREs can mediate new rounds of transport. A crucial protein called NSF cycles between membranes and the cytosol and catalyzes the disassembly process. NSF is a hexameric ATPase of the family of AAA-ATPases (see Figure 6–85) that uses the energy of ATP hydrolysis to unravel the intimate interactions between the helical domains of paired SNARE proteins (Figure 13–20). The requirement for NSF-mediated reactivation of SNAREs by SNARE complex disassembly helps prevent membranes from fusing indiscriminately: if the t-SNAREs in a target membrane were always active, any membrane containing an appropriate v-SNARE might fuse whenever the two membranes made contact. It is not known how the activity of NSF is controlled so that the SNARE machinery is activated at the right time

1	containing an appropriate v-SNARE might fuse whenever the two membranes made contact. It is not known how the activity of NSF is controlled so that the SNARE machinery is activated at the right time and place. It is also not known how v-SNAREs are selectively retrieved and returned to their compartment of origin so that they can be reused in newly formed transport vesicles.

1	Membrane fusion is important in other processes beside vesicle transport. The plasma membranes of a sperm and an egg fuse during fertilization, and myoblasts fuse with one another during the development of multinucleate muscle fibers (discussed in Chapter 22). Likewise, the ER network and mitochondria fuse and fragment in a dynamic way (discussed in Chapters 12 and 14). All cell membrane fusions require special proteins and are tightly regulated to ensure that only appropriate membranes fuse. The controls are crucial for maintaining both the identity of cells and the individuality of each type of intracellular compartment.

1	The membrane fusions catalyzed by viral fusion proteins are well understood. These proteins have a crucial role in permitting the entry of enveloped viruses (which have a lipid-bilayer-based membrane coat) into the cells that they infect (discussed in Chapters 5 and 23). For example, viruses such as the human immunodeficiency virus (HIV), which causes AIDS, bind to cell-surface receptors and then fuse with the plasma membrane of the target cell (Figure 13–21). This fusion event allows the viral nucleic acid inside the nucleocapsid to enter the cytosol, where it replicates. Other viruses, such as the influenza virus, first enter the cell by receptor-mediated endocytosis (discussed later) and are delivered to endosomes; the low pH in endosomes activates a fusion protein in the viral envelope that catalyzes the fusion of the viral and endosomal membranes, releasing the viral nucleic acid into the cytosol. Viral fusion proteins and SNAREs promote lipid bilayer fusion in similar ways.

1	Figure 13–20 Dissociation of SNARE pairs by NSF after a membrane fusion cycle. after a v-snare and t-snare have mediated the fusion of a transport vesicle with a target membrane, nsf binds to the snare complex and, with the help of accessory proteins, hydrolyzes aTp to pry the snares apart. Directed and selective transport of particular membrane components from one membrane-enclosed compartment to another in a eukaryotic cell maintains the differences between those compartments. Transport vesicles, which can be spherical, tubular, or irregularly shaped, bud from specialized coated regions of the donor membrane. The assembly of the coat helps to collect specific membrane and soluble cargo molecules for transport and to drive the formation of the vesicle.

1	There are various types of coated vesicles. The best characterized are clathrin-coated vesicles, which mediate transport from the plasma membrane and the trans Golgi network, and COPIand COPII-coated vesicles, which mediate transport between Golgi cisternae and between the ER and the Golgi apparatus, respectively. Coats have a common two-layered structure: an inner layer formed of adaptor proteins links the outer layer (or cage) to the vesicle membrane and also traps specific cargo molecules for packaging into the vesicle. The coat is shed before the vesicle fuses with its appropriate target membrane.

1	Local synthesis of specific phosphoinositides creates binding sites that trigger clathrin coat assembly and vesicle budding. In addition, monomeric GTPases help regulate various steps in vesicle transport, including both vesicle budding and docking. The coat-recruitment GTPases, including Sar1 and the ARF proteins, regulate coat assembly and disassembly. A large family of Rab proteins functions as vesicle-targeting GTPases. Rab proteins are recruited to both, forming transport vesicles and target membranes. The assembly and disassembly of Rab proteins and their effectors in specialized membrane domains are dynamically controlled by GTP binding and hydrolysis. Active Rab proteins recruit Rab effectors, such as motor proteins, which transport vesicles along actin filaments or microtubules, and filamentous tethering proteins, which help ensure that the vesicles deliver their contents only to the appropriate target membrane. Complementary v-SNARE proteins on transport vesicles and t-SNARE

1	filamentous tethering proteins, which help ensure that the vesicles deliver their contents only to the appropriate target membrane. Complementary v-SNARE proteins on transport vesicles and t-SNARE proteins on the target membrane form stable trans-SNARE complexes, which force the two membranes into close apposition so that their lipid bilayers can fuse.

1	TransporT froM The er Through The golgI apparaTus As discussed in Chapter 12, newly synthesized proteins cross the ER membrane from the cytosol to enter the secretory pathway. During their subsequent transport, from the ER to the Golgi apparatus and from the Golgi apparatus to the cell surface and elsewhere, these proteins are successively modified as they pass through a series of compartments. Transfer from one compartment to the next involves a delicate balance between forward and backward (retrieval) transport pathways. Some transport vesicles select cargo molecules and move them to the next compartment in the pathway, while others retrieve escaped proteins and return them to a previous compartment where they normally function. Thus, the pathway from the ER to the cell surface consists of many sorting steps, which continuously select membrane and soluble lumenal proteins for packaging and transport.

1	Figure 13–21 The entry of enveloped viruses into cells. electron micrographs showing how hIv enters a cell by fusing its membrane with the plasma membrane of the cell. (from b.s. stein et al., Cell 49:659–668, 1987. with permission from elsevier.)

1	In this section, we focus mainly on the Golgi apparatus (also called the Golgi complex). It is a major site of carbohydrate synthesis, as well as a sorting and dispatching station for products of the ER. The cell makes many polysaccharides in the Golgi apparatus, including the pectin and hemicellulose of the cell wall in plants and most of the glycosaminoglycans of the extracellular matrix in animals (discussed in Chapter 19). The Golgi apparatus also lies on the exit route from the ER, and a large proportion of the carbohydrates that it makes are attached as oligosaccharide side chains to the many proteins and lipids that the ER sends to it. A subset of these oligosaccharide groups serve as tags to direct specific proteins into vesicles that then transport them to lysosomes. But most proteins and lipids, once they have acquired their appropriate oligosaccharides in the Golgi apparatus, are recognized in other ways for targeting into the transport vesicles going to other destinations.

1	proteins leave the er in copII-coated Transport vesicles To initiate their journey along the secretory pathway, proteins that have entered the ER and are destined for the Golgi apparatus or beyond are first packaged into COPII-coated transport vesicles. These vesicles bud from specialized regions of the ER called ER exit sites, whose membrane lacks bound ribosomes. Most animal cells have ER exit sites dispersed throughout the ER network.

1	Entry into vesicles that leave the ER can be a selective process or can happen by default. Many membrane proteins are actively recruited into such vesicles, where they become concentrated. These cargo membrane proteins display exit (transport) signals on their cytosolic surface that adaptor proteins of the inner COPII coat recognize (Figure 13–22); some of these components act as cargo receptors and are recycled back to the ER after they have delivered their cargo to the Golgi apparatus. Soluble cargo proteins in the ER lumen, by contrast, have exit signals that attach them to transmembrane cargo receptors. Proteins without exit signals can also enter transport vesicles, including protein molecules that normally function in the ER (so-called ER resident proteins), some of which slowly leak out of the ER and are delivered to the Golgi apparatus. Different cargo proteins enter the transport vesicles with substantially different rates and efficiencies, which may result from differences

1	out of the ER and are delivered to the Golgi apparatus. Different cargo proteins enter the transport vesicles with substantially different rates and efficiencies, which may result from differences in their folding and oligomerization efficiencies and kinetics, as well as the factors already discussed. The exit step from the ER is a major checkpoint at which quality control is exerted on the proteins that a cell secretes or displays on its surface, as we discussed in Chapter 12.

1	The exit signals that direct soluble proteins out of the ER for transport to the Golgi apparatus and beyond are not well understood. Some transmembrane proteins that serve as cargo receptors for packaging some secretory proteins into COPII-coated vesicles are lectins that bind to oligosaccharides on the secreted

1	Figure 13–22 The recruitment of membrane and soluble cargo molecules into ER transport vesicles. Membrane proteins are packaged into budding transport vesicles through interactions of exit signals on their cytosolic tails with adaptor proteins of the inner copII coat. some of these membrane proteins function as cargo receptors, binding soluble proteins in the er lumen and helping to package them into vesicles. other proteins may enter the vesicle by bulk flow. a typical 50 nm transport vesicle contains about 200 membrane proteins, which can be of many different types. as indicated, unfolded or incompletely assembled proteins are bound to chaperones and transiently retained in the er compartment.

1	proteins. One such lectin, for example, binds to mannose on two secreted blood-clotting factors (Factor V and Factor VIII), thereby packaging the proteins into transport vesicles in the ER; its role in protein transport was identified because humans who lack it owing to an inherited mutation have lowered serum levels of Factors V and VIII, and they therefore bleed excessively. only proteins That are properly folded and assembled can leave the er

1	To exit from the ER, proteins must be properly folded and, if they are subunits of multiprotein complexes, they need to be completely assembled. Those that are misfolded or incompletely assembled transiently remain in the ER, where they are bound to chaperone proteins (discussed in Chapter 6) such as BiP or calnexin. The chaperones may cover up the exit signals or somehow anchor the proteins in the ER. Such failed proteins are eventually transported back into the cytosol, where they are degraded by proteasomes (discussed in Chapters 6 and 12). This quality-control step prevents the onward transport of misfolded or misassembled proteins that could potentially interfere with the functions of normal proteins. Such failures are surprisingly common. More than 90% of the newly synthesized subunits of the T cell receptor (discussed in Chapter 24) and of the acetylcholine receptor (discussed in Chapter 11), for example, are normally degraded without ever reaching the cell surface where they

1	of the T cell receptor (discussed in Chapter 24) and of the acetylcholine receptor (discussed in Chapter 11), for example, are normally degraded without ever reaching the cell surface where they function. Thus, cells must make a large excess of some protein molecules to produce a select few that fold, assemble, and function properly.

1	Sometimes, however, there are drawbacks to the stringent quality-control mechanism. The predominant mutations that cause cystic fibrosis, a common inherited disease, result in the production of a slightly misfolded form of a plasma membrane protein important for Cl– transport. Although the mutant protein would function normally if it reached the plasma membrane, it is retained in the ER and then is degraded by cytosolic proteasomes. This devastating disease thus results not because the mutation inactivates the protein but because the active protein is discarded before it reaches the plasma membrane. vesicular Tubular clusters Mediate Transport from the er to the golgi apparatus

1	vesicular Tubular clusters Mediate Transport from the er to the golgi apparatus After transport vesicles have budded from ER exit sites and have shed their coat, they begin to fuse with one another. The fusion of membranes from the same compartment is called homotypic fusion, to distinguish it from heterotypic fusion, in which a membrane from one compartment fuses with the membrane of a different compartment. As with heterotypic fusion, homotypic fusion requires a set of matching SNAREs. In this case, however, the interaction is symmetrical, with both membranes contributing v-SNAREs and t-SNAREs (Figure 13–23). The structures formed when ER-derived vesicles fuse with one another are called vesicular tubular clusters, because they have a convoluted appearance in

1	The structures formed when ER-derived vesicles fuse with one another are called vesicular tubular clusters, because they have a convoluted appearance in Figure 13–23 Homotypic membrane fusion. In step 1, nsf pries apart identical pairs of v-snares and t-snares in both membranes (see figure 13–20). In steps 2 and 3, the separated matching snares on adjacent identical membranes interact, which leads to membrane fusion and the formation of one continuous compartment. subsequently, the compartment grows by further homotypic fusion with vesicles from the same kind of membrane, displaying matching snares. homotypic fusion occurs when er-derived transport vesicles fuse with one another, but also when endosomes fuse to generate larger endosomes. rab proteins help regulate the extent of homotypic fusion and hence the size of a cell’s compartments (not shown).

1	Figure 13–24 Vesicular tubular clusters. (a) an electron micrograph of vesicular tubular clusters forming around an exit site. Many of the vesicle-like structures seen in the micrograph are cross sections of tubules that extend above and below the plane of this thin section and are interconnected. (b) vesicular tubular clusters move along microtubules to carry proteins from the ER er to the golgi apparatus. copI-coated vesicles mediate the budding of vesicles that return to the (A) 0.2 µm er from these clusters (and from the golgi apparatus). (a, courtesy of william balch.) the electron microscope (Figure 13–24A). These clusters constitute a compartment that is separate from the ER and lacks many of the proteins that function in the ER. They are generated continually and function as transport containers that bring material from the ER to the Golgi apparatus. The clusters move quickly along microtubules to the Golgi apparatus with which they fuse (Figure 13–24B and Movie 13.2).

1	As soon as vesicular tubular clusters form, they begin to bud off transport vesicles of their own. Unlike the COPII-coated vesicles that bud from the ER, these vesicles are COPI-coated (see Figure 13–24A). COPI-coated vesicles are unique in that the components that make up the inner and outer coat layers are recruited as a preassembled complex, called coatomer. They function as a retrieval pathway, carrying back ER resident proteins that have escaped, as well as proteins such as cargo receptors and SNAREs that participated in the ER budding and vesicle fusion reactions. This retrieval process demonstrates the exquisite control mechanisms that regulate coat assembly reactions. The COPI coat assembly begins only seconds after the COPII coats have been shed, and remains a mystery how this switch in coat assembly is controlled.

1	The retrieval (or retrograde) transport continues as the vesicular tubular clusters move toward the Golgi apparatus. Thus, the clusters continuously mature, gradually changing their composition as selected proteins are returned to the ER. The retrieval continues from the Golgi apparatus, after the vesicular tubular clusters have delivered their cargo. The retrieval pathway to the er uses sorting signals The retrieval pathway for returning escaped proteins back to the ER depends on ER retrieval signals. Resident ER membrane proteins, for example, contain signals that bind directly to COPI coats and are thus packaged into COPI-coated transport vesicles for retrograde delivery to the ER. The best-characterized retrieval signal of this type consists of two lysines, followed by any two other amino acids, at the extreme C-terminal end of the ER membrane protein. It is called a KKXX sequence, based on the single-letter amino acid code.

1	Soluble ER resident proteins, such as BiP, also contain a short ER retrieval signal at their C-terminal end, but it is different: it consists of a Lys-Asp-Glu-Leu or a similar sequence. If this signal (called the KDEL sequence) is removed from BiP by genetic engineering, the protein is slowly secreted from the cell. If the signal is transferred to a protein that is normally secreted, the protein is now efficiently returned to the ER, where it accumulates. FORWARD PATHWAY RETRIEVAL PATHWAY COPII coat COPI coat vesicular tubular cluster cis cis, medial, and trans

1	Unlike the retrieval signals on ER membrane proteins, which can interact directly with the COPI coat, soluble ER resident proteins must bind to specialized receptor proteins such as the KDEL receptor—a multipass transmembrane protein that binds to the KDEL sequence and packages any protein displaying it into COPI-coated retrograde transport vesicles (Figure 13–25). To accomplish this task, the KDEL receptor itself must cycle between the ER and the Golgi apparatus, and its affinity for the KDEL sequence must differ in these two compartments. The receptor must have a high affinity for the KDEL sequence in vesicular tubular clusters and the Golgi apparatus, so as to capture escaped, soluble ER resident proteins that are present there at low concentration. It must have a low affinity for the KDEL sequence in the ER, however, to unload its cargo in spite of the very high concentration of KDEL-containing soluble resident proteins in the ER.

1	How does the affinity of the KDEL receptor change depending on the compartment in which it resides? The answer is likely related to the lower pH in the Golgi compartments, which is regulated by H+ pumps. As we discuss later, pH-sensitive protein–protein interactions form the basis for many of the protein sorting steps in the cell. Most membrane proteins that function at the interface between the ER and Golgi apparatus, including vand t-SNAREs and some cargo receptors, also enter the retrieval pathway back to the ER. Many proteins are selectively retained in the compartments in which They function

1	The KDEL retrieval pathway only partly explains how ER resident proteins are maintained in the ER. As mentioned, cells that express genetically modified ER resident proteins, from which the KDEL sequence has been experimentally removed, secrete these proteins. But the rate of secretion is much slower than for a normal secretory protein. It seems that a mechanism that is independent of their KDEL signal normally retains ER resident proteins and that only those proteins that escape this retention mechanism are captured and returned via the KDEL receptor. A suggested retention mechanism is that ER resident proteins bind to one another, thus forming complexes that are too big to enter transport vesicles efficiently. Because ER resident proteins are present in the ER at very high concentrations (estimated to be millimolar), relatively low-affinity interactions would suffice to retain most of the proteins in such complexes.

1	Aggregation of proteins that function in the same compartment is a general mechanism that compartments use to organize and retain their resident proteins. Golgi enzymes that function together, for example, also bind to each other and are thereby restrained from entering transport vesicles leaving the Golgi apparatus.

1	Figure 13–25 Retrieval of soluble ER resident proteins. er resident proteins that escape from the er are returned by vesicle transport. (a) The kdel receptor present in both vesicular tubular clusters and the golgi apparatus captures the soluble er resident proteins and carries them in copI-coated transport vesicles back to the er. (recall that the copIcoated vesicles shed their coats as soon as they are formed.) upon binding its ligands in the tubular cluster or golgi, the kdel receptor may change conformation, so as to facilitate its recruitment into budding copI-coated vesicles. (b) The retrieval of er proteins begins in vesicular tubular clusters and continues from later parts of the golgi apparatus. In the environment of the er, the er resident proteins dissociate from the kdel receptor, which is then returned to the golgi apparatus for reuse. we discuss the different compartments of the golgi apparatus shortly. The golgi apparatus consists of an ordered series of compartments

1	Because it could be selectively visualized by silver stains, the Golgi apparatus was one of the first organelles described by early light microscopists. It consists of a collection of flattened, membrane-enclosed compartments called cisternae, that somewhat resemble a stack of pita breads. Each Golgi stack typically consists of four to six cisternae (Figure 13–26), although some unicellular flagellates can have more than 20. In animal cells, tubular connections between corresponding cisternae link many stacks, thus forming a single complex, which is usually located near the cell nucleus and close to the centrosome (Figure 13–27A). This localization depends on microtubules. If microtubules are experimentally depolymerized, the Golgi apparatus reorganizes into individual stacks that are found throughout the cytoplasm, adjacent to ER exit sites. Some cells, including most plant cells, have hundreds of individual Golgi stacks dispersed throughout the cytoplasm where they are typically

1	found throughout the cytoplasm, adjacent to ER exit sites. Some cells, including most plant cells, have hundreds of individual Golgi stacks dispersed throughout the cytoplasm where they are typically found adjacent to ER exit sites (Figure 13–27B).

1	Figure 13–26 The Golgi apparatus. Three-dimensional reconstruction from electron micrographs of the golgi apparatus in a secretory animal cell. The cis face of the golgi stack is that closest to the er. (b) a thin-section electron micrograph of an animal cell. In plant cells, the golgi apparatus is generally more distinct and more clearly separated from other intracellular membranes than in animal cells. (a, redrawn from a. rambourg and y. clermont, Eur. J. Cell Biol. 51:189–200, 1990. with permission from wissenschaftliche verlagsgesellschaft; b, courtesy of brij J. gupta.) Figure 13–27 Localization of the Golgi apparatus in animal and plant cells. The golgi apparatus in a cultured fibroblast stained with a fluorescent antibody that recognizes a golgi resident protein (bright orange). The golgi apparatus is polarized, facing the direction in which the cell was crawling before fixation.

1	The golgi apparatus in a plant cell that is expressing a fusion protein consisting of a resident golgi enzyme fused to green fluorescent protein. (a, courtesy of John henley and Mark Mcniven; b, courtesy of chris hawes.)

1	During their passage through the Golgi apparatus, transported molecules undergo an ordered series of covalent modifications. Each Golgi stack has two distinct faces: a cis face (or entry face) and a trans face (or exit face). Both cis and trans faces are closely associated with special compartments, each composed of a network of interconnected tubular and cisternal structures: the cis Golgi network (CGN) and the trans Golgi network (TGN), respectively. The CGN is a collection of fused vesicular tubular clusters arriving from the ER. Proteins and lipids enter the cis Golgi network and exit from the trans Golgi network, bound for the cell surface or another compartment. Both networks are important for protein sorting: proteins entering the CGN can either move onward in the Golgi apparatus or be returned to the ER. Similarly, proteins exiting from the TGN move onward and are sorted according to their next destination: endosomes, secretory vesicles, or the cell surface. They also can be

1	or be returned to the ER. Similarly, proteins exiting from the TGN move onward and are sorted according to their next destination: endosomes, secretory vesicles, or the cell surface. They also can be returned to an earlier compartment. Some membrane proteins are retained in the part of the Golgi apparatus where they function.

1	As described in Chapter 12, a single species of N-linked oligosaccharide is attached en bloc to many proteins in the ER and then trimmed while the protein is still in the ER. The oligosaccharide intermediates created by the trimming reactions serve to help proteins fold and to help transport misfolded proteins to the cytosol for degradation in proteasomes. Thus, they play an important role in controlling the quality of proteins exiting from the ER. Once these ER functions have been fulfilled, the cell reutilizes the oligosaccharides for new functions. This begins in the Golgi apparatus, which generates the heterogeneous oligosaccharide structures seen in mature proteins. After arrival in the CGN, proteins enter the first of the Golgi processing compartments (the cis Golgi cisternae). They then move to the next compartment (the medial cisternae) and finally to the trans cisternae, where glycosylation is completed. The lumen of the trans cisternae is thought to be continuous with the

1	then move to the next compartment (the medial cisternae) and finally to the trans cisternae, where glycosylation is completed. The lumen of the trans cisternae is thought to be continuous with the TGN, the place where proteins are segregated into different transport packages and dispatched to their final destinations.

1	The oligosaccharide processing steps occur in an organized sequence in the Golgi stack, with each cisterna containing a characteristic mixture of processing enzymes. Proteins are modified in successive stages as they move from cisterna to cisterna across the stack, so that the stack forms a multistage processing unit. Investigators discovered the functional differences between the cis, medial, and trans subdivisions of the Golgi apparatus by localizing the enzymes involved in processing N-linked oligosaccharides in distinct regions of the organelle, both by physical fractionation of the organelle and by labeling the enzymes in electron microscope sections with antibodies (Figure 13–28). The removal of mannose and the addition of N-acetylglucosamine, for example, occur in the cis and medial cisternae, while the addition of galactose and sialic acid occurs in the trans cisterna and trans Golgi network. Figure 13–29 summarizes the functional compartmentalization of the Golgi apparatus.

1	oligosaccharide chains are processed in the golgi apparatus Whereas the ER lumen is full of soluble lumenal resident proteins and enzymes, the resident proteins in the Golgi apparatus are all membrane bound, as the enzymatic reactions apparently occur entirely on membrane surfaces. All of the Golgi glycosidases and glycosyl transferases, for example, are single-pass transmembrane proteins, many of which are organized in multienzyme complexes. Figure 13–28 Molecular compartmentalization of the Golgi apparatus.

1	Figure 13–28 Molecular compartmentalization of the Golgi apparatus. a series of electron micrographs shows the golgi apparatus (a) unstained, (b) stained with osmium, which preferentially labels the cisternae of the cis compartment, and (c and d) stained to reveal the location of specific enzymes. nucleoside diphosphatase is found in the trans golgi cisternae (c), while acid phosphatase is found in the trans golgi network (d). note that usually more than one cisterna is stained. The enzymes are therefore thought to be highly enriched rather than precisely localized to a specific cisterna. (courtesy of daniel s. friend.) 1 µm

1	Two broad classes of N-linked oligosaccharides, the complex oligosaccharides and the high-mannose oligosaccharides, are attached to mammalian glycoproteins. Sometimes, both types are attached (in different places) to the same polypeptide chain. Complex oligosaccharides are generated when the original N-linked oligosaccharide added in the ER is trimmed and further sugars are added; by contrast, high-mannose oligosaccharides are trimmed but have no new sugars added to them in the Golgi apparatus (Figure 13–30). The sialic acids in the = N-acetylneuraminic acid (sialic acid, or NANA) (C) HIGH-MANNOSE OLIGOSACCHARIDE

1	Figure 13–30 The two main classes of asparagine-linked (N-linked) oligosaccharides found in mature mammalian glycoproteins. (a) both complex oligosaccharides and high-mannose oligosaccharides share a common core region derived from the original n-linked oligosaccharide added in the er (see figure 12–50) and typically containing two n-acetylglucosamines (glcnac) and three mannoses (Man). (b) each complex oligosaccharide consists of a core region, together with a terminal region that contains a variable number of copies of a special trisaccharide unit (n-acetylglucosamine–galactose–sialic acid) linked to the core mannoses. frequently, the terminal region is truncated and contains only glcnac and galactose (gal) or just glcnac. In addition, a fucose may be added, usually to the core glcnac attached to the asparagine (asn). Thus, although the steps of processing and subsequent sugar addition are rigidly ordered, complex oligosaccharides can be heterogeneous. Moreover, although the complex

1	to the asparagine (asn). Thus, although the steps of processing and subsequent sugar addition are rigidly ordered, complex oligosaccharides can be heterogeneous. Moreover, although the complex oligosaccharide shown has three terminal branches, two and four branches are also common, depending on the glycoprotein and the cell in which it is made. (c) high-mannose oligosaccharides are not trimmed back all the way to the core region and contain additional mannoses. hybrid oligosaccharides with one Man branch and one glcnac and gal branch are also found (not shown).

1	The three amino acids indicated in (a) constitute the sequence recognized by the oligosaccharyl transferase enzyme that adds the initial oligosaccharide to the protein. ser, serine; Thr, threonine; x, any amino acid, except proline. Figure 13–29 Oligosaccharide processing in Golgi compartments. The localization of each processing step shown was determined by a combination of techniques, including biochemical subfractionation of the golgi apparatus membranes and electron microscopy after staining with antibodies specific for some of the processing enzymes. processing enzymes are not restricted to a particular cisterna; instead, their distribution is graded across the stack, such that early-acting enzymes are present mostly in the cis golgi cisternae and later-acting enzymes are mostly in the trans golgi cisternae. Man, mannose; glcnac, n-acetylglucosamine; gal, galactose; nana, n-acetylneuraminic acid (sialic acid). KEY: = N-acetylneuraminic acid (sialic acid, or NANA)

1	Figure 13–31 Oligosaccharide processing in the ER and the Golgi apparatus. The processing pathway is highly ordered, so that each step shown depends on the previous one. step 1: processing begins in the er with the removal of the glucoses from the oligosaccharide initially transferred to the protein. Then a mannosidase in the er membrane removes a specific mannose. The remaining steps occur in the golgi stack. step 2: golgi mannosidase I removes three more mannoses. step 3: n-acetylglucosamine transferase I then adds an n-acetylglucosamine. step 4: Mannosidase II then removes two additional mannoses. This yields the final core of three mannoses that is present in a complex oligosaccharide. at this stage, the bond between the two n-acetylglucosamines in the core becomes resistant to attack by a highly specific endoglycosidase (Endo H). since all later structures in the pathway are also endo h-resistant, treatment with this enzyme is widely used to distinguish complex from high-mannose

1	by a highly specific endoglycosidase (Endo H). since all later structures in the pathway are also endo h-resistant, treatment with this enzyme is widely used to distinguish complex from high-mannose oligosaccharides. step 5: finally, as shown in figure 13–30, additional n-acetylglucosamines, galactoses, and sialic acids are added. These final steps in the synthesis of a complex oligosaccharide occur in the cisternal compartments of the golgi apparatus: three types of glycosyl transferase enzymes act sequentially, using sugar substrates that have been activated by linkage to the indicated nucleotide; the membranes of the golgi cisternae contain specific carrier proteins that allow each sugar nucleotide to enter in exchange for the nucleoside phosphates that are released after the sugar is attached to the protein on the lumenal face.

1	note that, as a biosynthetic organelle, the golgi apparatus differs from the er: all sugars in the golgi are assembled inside the lumen from sugar nucleotide, whereas in the er, the n-linked precursor oligosaccharide is assembled partly in the cytosol and partly in the lumen, and most lumenal reactions use dolichol-linked sugars as their substrates (see figure 12–51). complex oligosaccharides are of special importance because they bear a negative charge. Whether a given oligosaccharide remains high-mannose or is processed depends largely on its position in the protein. If the oligosaccharide is accessible to the processing enzymes in the Golgi apparatus, it is likely to be converted to a complex form; if it is inaccessible because its sugars are tightly held to the protein’s surface, it is likely to remain in a high-mannose form. The processing that generates complex oligosaccharide chains follows the highly ordered pathway shown in Figure 13–31.

1	Beyond these commonalities in oligosaccharide processing that are shared among most cells, the products of the carbohydrate modifications carried out in the Golgi apparatus are highly complex and have given rise to a new field of study called glycobiology. The human genome, for example, encodes hundreds of different Golgi glycosyl transferases and many glycosidases, which are expressed differently from one cell type to another, resulting in a variety of glycosylated forms of a given protein or lipid in different cell types and at varying stages of differentiation, depending on the spectrum of enzymes expressed by the cell. The complexity of modifications is not limited to N-linked oligosaccharides but also occurs on O-linked sugars, as we discuss next. proteoglycans are assembled in the golgi apparatus

1	proteoglycans are assembled in the golgi apparatus In addition to the N-linked oligosaccharide alterations made to proteins as they pass through the Golgi cisternae en route from the ER to their final destinations, many proteins are also modified in the Golgi apparatus in other ways. Some proteins have sugars added to the hydroxyl groups of selected serines or threonines, H H NHCOCH3 remainder of H NHCOCH3 oligosaccharide side chain remainder of oligosaccharide side chain or, in some cases—such as collagens—to hydroxylated proline and lysine side chains. This O-linked glycosylation (Figure 13–32), like the extension of N-linked oligosaccharide chains, is catalyzed by a series of glycosyl transferase enzymes that use the sugar nucleotides in the lumen of the Golgi apparatus to add sugars to a protein one at a time. Usually, N-acetylgalactosamine is added first, followed by a variable number of additional sugars, ranging from just a few to 10 or more.

1	The Golgi apparatus confers the heaviest O-linked glycosylation of all on mucins, the glycoproteins in mucus secretions, and on proteoglycan core proteins, which it modifies to produce proteoglycans. As discussed in Chapter 19, this process involves the polymerization of one or more glycosaminoglycan chains (long, unbranched polymers composed of repeating disaccharide units; see Figure 19–35) onto serines on a core protein. Many proteoglycans are secreted and become components of the extracellular matrix, while others remain anchored to the extracellular face of the plasma membrane. Still others form a major component of slimy materials, such as the mucus that is secreted to form a protective coating on the surface of many epithelia.

1	The sugars incorporated into glycosaminoglycans are heavily sulfated in the Golgi apparatus immediately after these polymers are made, thus adding a significant portion of their characteristically large negative charge. Some tyrosines in proteins also become sulfated shortly before they exit from the Golgi apparatus. In both cases, the sulfation depends on the sulfate donor 3ʹ-phosphoadenosine-5ʹ-phosphosulfate (PAPS) (Figure 13–33), which is transported from the cytosol into the lumen of the trans Golgi network. what Is the purpose of glycosylation?

1	what Is the purpose of glycosylation? There is an important difference between the construction of an oligosaccharide and the synthesis of other macromolecules such as DNA, RNA, and protein. Whereas nucleic acids and proteins are copied from a template in a repeated series of identical steps using the same enzyme or set of enzymes, complex carbohydrates require a different enzyme at each step, each product being recognized as the exclusive substrate for the next enzyme in the series. The vast abundance of glycoproteins and the complicated pathways that have evolved to synthesize them emphasize that the oligosaccharides on glycoproteins and glycosphingolipids have very important functions.

1	N-linked glycosylation, for example, is prevalent in all eukaryotes, including yeasts. N-linked oligosaccharides also occur in a very similar form in archaeal cell wall proteins, suggesting that the whole machinery required for their synthesis is evolutionarily ancient. N-linked glycosylation promotes protein folding in two ways. First, it has a direct role in making folding intermediates more soluble, thereby preventing their aggregation. Second, the sequential modifications of the N-linked oligosaccharide establish a “glyco-code” that marks the progression of Figure 13–32 Nand O-linked glycosylation. In each case, only the single sugar group that is directly attached to the protein chain is shown. Figure 13–33 The structure of PAPS. protein folding and mediates the binding of the protein to chaperones (discussed in Chapter 12) and lectins—for example, in guiding ER-to-Golgi transport. As we discuss later, lectins also participate in protein sorting in the trans Golgi network.

1	Because chains of sugars have limited flexibility, even a small N-linked oligosaccharide protruding from the surface of a glycoprotein (Figure 13–34) can limit the approach of other macromolecules to the protein surface. In this way, for example, the presence of oligosaccharides tends to make a glycoprotein more resistant to digestion by proteolytic enzymes. It may be that the oligosaccharides on cell-surface proteins originally provided an ancestral cell with a protective coat; compared to the rigid bacterial cell wall, such a sugar coat has the advantage that it leaves the cell with the freedom to change shape and move.

1	The sugar chains have since become modified to serve other purposes as well. The mucus coat of lung and intestinal cells, for example, protects against many pathogens. The recognition of sugar chains by lectins in the extracellular space is important in many developmental processes and in cell–cell recognition: selectins, for example, are transmembrane lectins that function in cell–cell adhesion during blood cell migration, as discussed in Chapter 19. The presence of oligosaccharides may modify a protein’s antigenic and functional properties, making glycosylation an important factor in the production of proteins for pharmaceutical purposes.

1	Glycosylation can also have important regulatory roles. Signaling through the cell-surface signaling receptor Notch, for example, is an important factor in determining the cell’s fate in development (discussed in Chapter 21). Notch is a transmembrane protein that is O-glycosylated by addition of a single fucose to some serines, threonines, and hydroxylysines. Some cell types express an additional glycosyl transferase that adds an N-acetylglucosamine to each of these fucoses in the Golgi apparatus. This addition changes the specificity of Notch for the cell-surface signal proteins that activate it. Transport Through the golgi apparatus May occur by cisternal Maturation

1	It is still uncertain how the Golgi apparatus achieves and maintains its polarized structure and how molecules move from one cisterna to another, and it is likely that more than one mechanism is involved in each case. One hypothesis, called the cisternal maturation model, views the Golgi cisternae as dynamic structures that mature from early to late by acquiring and then losing specific Golgi-resident proteins. According to this view, new cis cisternae continually form as vesicular tubular clusters arrive from the ER and progressively mature to become a medial cisterna and then a trans cisterna (Figure 13–35A). A cisterna therefore moves through the Golgi stack with cargo in its lumen. Retrograde transport of the Golgi enzymes by budding COPI-coated vesicles explains their characteristic distribution. As we discuss later, when a cisterna finally moves forward to become part of the trans Golgi network, various types of coated vesicles bud off it until this network disappears, to be

1	distribution. As we discuss later, when a cisterna finally moves forward to become part of the trans Golgi network, various types of coated vesicles bud off it until this network disappears, to be replaced by a maturing cisterna just behind. At the same time, other transport vesicles are continually retrieving membrane from post-Golgi compartments and returning it to the trans Golgi network.

1	The cisternal maturation model is supported by studies using Golgi enzymes from different cisternae that were fluorescently labeled with different colors. Such studies performed in yeast cells where Golgi cisternae are not stacked reveal that individual Golgi cisternae change their color, thereby demonstrating that they change their complement of resident enzymes as they mature, even though they are not stacked. In further support of the model, electron microscopic observations found that large structures such as procollagen rods in fibroblasts and scales in certain algae move progressively through the Golgi stack. An alternative view holds that Golgi cisternae are long-lived structures that retain their characteristic set of Golgi-resident proteins firmly in place, and cargo proteins are transported from one cisterna to the next by transport vesicles (Figure 13–35B). According to this vesicle transport model, retrograde flow of vesicles

1	Figure 13–34 The three-dimensional structure of a small N-linked oligosaccharide. The structure was determined by x-ray crystallographic analysis of a glycoprotein. This oligosaccharide contains only 6 sugars, whereas there are 14 sugars in the n-linked oligosaccharide that is initially transferred to proteins in the er (see figure 12–47). (a) a backbone model showing all atoms except hydrogens; only the asparagine of the protein is shown. (b) a space-filling model, with the asparagine and sugars indicated using the same color scheme as in (a). (b, courtesy of richard feldmann.) Figure 13–35 Two possible models explaining the organization of the Golgi apparatus and how proteins move through it. It is likely that the transport through the golgi apparatus in the forward direction (red arrows) involves elements of both models.

1	(a) according to the cisternal maturation model, each golgi cisterna matures as it migrates outward through the stack. at each stage, the golgi resident proteins that are carried forward in a maturing cisterna are moved backward to an earlier compartment in copI-coated vesicles. when a newly formed cisterna moves to a medial position, for example, “leftover” cis golgi enzymes would be extracted and transported retrogradely to a new cis cisterna behind. likewise, the medial enzymes would be received by retrograde transport from the cisternae just ahead. In this way, a cis cisterna would mature to a medial and then trans cisterna as it moves outward. (b) In the vesicle transport model, golgi cisternae are static compartments, which contain a characteristic complement of resident enzymes. The passing of molecules from cis to trans through the golgi is accomplished by forward-moving transport vesicles, which bud from one cisterna and fuse with the next in a cis-to-trans direction.

1	retrieves escaped ER and Golgi proteins and returns them to upstream compartments. Directional flow could be achieved because forward-moving cargo molecules are selectively packaged into forward-moving vesicles. Although both forwardand backward-moving vesicles would likely be COPI-coated, the coats may contain different adaptor proteins that confer selectivity on the packaging of cargo molecules. Alternatively, transport vesicles shuttling between Golgi cisternae might not be directional at all, transporting cargo randomly back and forth; directional flow would then occur because of the continual input to the cis cisterna and output from the trans cisterna.

1	The vesicle transport model is supported by experiments that show that cargo molecules are present in small COPI-coated vesicles and that these vesicles can deliver them to Golgi cisternae over large distances. In addition, when experimentally aggregated membrane proteins are introduced into Golgi cisternae, they can be observed staying in place, while soluble cargo, even if present as large aggregates, traverses the Golgi at normal rates.

1	It is likely that aspects of both models are true. A stable core of long-lasting cisternae might exist in the center of each Golgi cisterna, while regions at the rim may undergo continuous maturation, perhaps utilizing Rab cascades that change their identity. As matured pieces of the cisternae are formed, they might break off and fuse with downstream cisternae by homotypic fusion mechanisms, taking large cargo molecules with them. In addition, small COPI-coated vesicles might transport small cargo in the forward direction and retrieve escaped Golgi enzymes and return them to their appropriate upstream cisternae. Golgi golgi Matrix proteins help organize the stack

1	The unique architecture of the Golgi apparatus depends on both the microtubule cytoskeleton, as already mentioned, and cytoplasmic Golgi matrix proteins, which form a scaffold between adjacent cisternae and give the Golgi stack its structural integrity. Some of the matrix proteins, called golgins, form long tethers composed of stiff coiled-coil domains with interspersed hinge regions. Golgins form a forest Figure 13–36 A model of golgin function. of tentacles that can extend 100–400 nm from the surface of the Golgi stack. They filamentous golgins anchored to golgi are thought to help retain Golgi transport vesicles close to the organelle through membranes capture transport vesicles interactions with Rab proteins (Figure 13–36). When the cell prepares to divide, by binding to rab proteins on the vesicle mitotic protein kinases phosphorylate the Golgi matrix proteins, causing the Golgi surface.

1	apparatus to fragment and disperse throughout the cytosol. The Golgi fragments are then distributed evenly to the two daughter cells, where the matrix proteins are dephosphorylated, leading to the reassembly of the Golgi stack. Similarly, during apoptosis, proteolytic cleavage of golgins by caspases ensues (discussed in Chapter 18), fragments the Golgi apparatus as the cell self-destructs. Correctly folded and assembled proteins in the ER are packaged into COPII-coated transport vesicles that pinch off from the ER membrane. Shortly thereafter, the vesicles shed their coat and fuse with one another to form vesicular tubular clusters. In animal cells, the clusters then move on microtubule tracks to the Golgi apparatus, where they fuse with one another to form the cis Golgi network. Any resident ER proteins that escape from the ER are returned there from the vesicular tubular clusters and Golgi apparatus by retrograde transport in COPI-coated vesicles.

1	The Golgi apparatus, unlike the ER, contains many sugar nucleotides, which glycosyl transferase enzymes use to glycosylate lipid and protein molecules as they pass through the Golgi apparatus. The mannoses on the N-linked oligosaccharides that are added to proteins in the ER are often initially removed, and further sugars are added. Moreover, the Golgi apparatus is the site where O-linked glycosylation occurs and where glycosaminoglycan chains are added to core proteins to form proteoglycans. Sulfation of the sugars in proteoglycans and of selected tyrosines on proteins also occurs in a late Golgi compartment.

1	The Golgi apparatus modifies the many proteins and lipids that it receives from the ER and then distributes them to the plasma membrane, lysosomes, and secretory vesicles. The Golgi apparatus is a polarized organelle, consisting of one or more stacks of disc-shaped cisternae. Each stack is organized as a series of at least three functionally distinct compartments, termed cis, medial, and trans cisternae. The cis and trans cisternae are each connected to special sorting stations, called the cis Golgi network and the trans Golgi network, respectively. Proteins and lipids move through the Golgi stack in the cis-to-trans direction. This movement may occur by vesicle transport, by progressive maturation of the cis cisternae as they migrate continuously through the stack, or, most likely, by a combination of these two mechanisms. Continual retrograde vesicle transport from upstream to more downstream cisternae is thought to keep the enzymes concentrated in the cisternae where they are

1	by a combination of these two mechanisms. Continual retrograde vesicle transport from upstream to more downstream cisternae is thought to keep the enzymes concentrated in the cisternae where they are needed. The finished new proteins end up in the trans Golgi network, which packages them in transport vesicles and dispatches them to their specific destinations in the cell.

1	TransporT froM The Trans golgI neTwork To lysosoMes The trans Golgi network sorts all of the proteins that pass through the Golgi apparatus (except those that are retained there as permanent residents) according to their final destination. The sorting mechanism is especially well understood for those proteins destined for the lumen of lysosomes, and in this section we consider this selective transport process. We begin with a brief account of lysosome structure and function. lysosomes are the principal sites of Intracellular digestion

1	lysosomes are the principal sites of Intracellular digestion Lysosomes are membrane-enclosed organelles filled with soluble hydrolytic enzymes that digest macromolecules. Lysosomes contain about 40 types of hydrolytic enzymes, including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases, and sulfatases. All are acid hydrolases; that is, hydro-lases that work best at acidic pH. For optimal activity, they need to be activated by proteolytic cleavage, which also requires an acid environment. The lysosome provides this acidity, maintaining an interior pH of about 4.5–5.0. By this arrange- ment, the contents of the cytosol are doubly protected against attack by the cell’s own digestive system: the membrane of the lysosome keeps the digestive enzymes out of the cytosol, but, even if they leak out, they can do little damage at the cytosolic pH of about 7.2.

1	Like all other membrane-enclosed organelles, the lysosome not only contains a unique collection of enzymes, but also has a unique surrounding membrane. Most of the lysosome membrane proteins, for example, are highly glycosylated, which helps to protect them from the lysosome proteases in the lumen. Transport proteins in the lysosome membrane carry the final products of the digestion of macromolecules—such as amino acids, sugars, and nucleotides—to the cytosol, where the cell can either reuse or excrete them.

1	A vacuolar H+ ATPase in the lysosome membrane uses the energy of ATP hydrolysis to pump H+ into the lysosome, thereby maintaining the lumen at its acidic pH (Figure 13–37). The lysosome H+ pump belongs to the family of V-type ATPases and has a similar architecture to the mitochondrial and chloroplast ATP synthases (F-type ATPases), which convert the energy stored in H+ gradients into ATP (see Figure 11–12). By contrast to these enzymes, however, the vacuolar H+ ATPase exclusively works in reverse, pumping H+ into the organelle. Similar or identical V-type ATPases acidify all endocytic and exocytic organelles, including lysosomes, endosomes, some compartments of the Golgi apparatus, and many transport and secretory vesicles. In addition to providing a low-pH environment that is suitable for reactions occurring in the organelle lumen, the H+ gradient provides a source of energy that drives the transport of small metabolites across the organelle membrane. lysosomes are heterogeneous

1	lysosomes are heterogeneous Lysosomes are found in all eukaryotic cells. They were initially discovered by the biochemical fractionation of cell extracts; only later were they seen clearly in the electron microscope. Although extraordinarily diverse in shape and size, staining them with specific antibodies shows they are members of a single family of organelles. They can also be identified by histochemical techniques that reveal which organelles contain acid hydrolase (Figure 13–38).

1	The heterogeneous morphology of lysosomes contrasts with the relatively uniform structures of many other cell organelles. The diversity reflects the wide variety of digestive functions that acid hydrolases mediate, including the breakdown of intraand extracellular debris, the destruction of phagocytosed microorganisms, and the production of nutrients for the cell. Their morphological diversity, however, also reflects the way lysosomes form. Late endosomes containing material received from both the plasma membrane by endocytosis and newly synthesized lysosomal hydrolases fuse with preexisting lysosomes to form structures that are sometimes referred to as endolysosomes, which then fuse with one another (Figure 13–39). When the majority of the endocytosed material within an endolysosome has been digested so that only resistant or slowly digestible residues remain, these organelles become “classical” lysosomes. These are relatively dense, round, and small, but they can enter the cycle

1	been digested so that only resistant or slowly digestible residues remain, these organelles become “classical” lysosomes. These are relatively dense, round, and small, but they can enter the cycle again by fusing with late endosomes or endolysosomes. Thus, there is no real distinction between endolysosomes and lysosomes: they are the same except that they are in different stages of a maturation cycle. For this reason, lysosomes are sometimes viewed as a heterogeneous collection of distinct organelles, the common feature of which is a high content of

1	Figure 13–38 Histochemical visualization of lysosomes. These electron micrographs show two sections of a cell stained to reveal the location of acid phosphatase, a marker enzyme for lysosomes. The larger membrane-enclosed organelles, containing dense precipitates of lead phosphate, are lysosomes. Their diverse morphology reflects variations in the amount and nature of the material they are digesting. The precipitates are produced when tissue fixed with glutaraldehyde (to fix the enzyme in place) is incubated with a phosphatase substrate in the presence of lead ions. red arrows in the top panel indicate two small vesicles thought to be carrying acid hydrolases from the golgi apparatus. (courtesy of daniel s. friend.) 0.2–0.5 µm pH~7.2 pH~5.0 ACID HYDROLASES: nucleases proteases glycosidases lipases phosphatases sulfatases phospholipases CYTOSOL + H+ pump ATPADPPi H+ Figure 13–37 Lysosomes. The acid hydrolases are hydrolytic enzymes that are active under acidic conditions. an h+ aTpase

1	lipases phosphatases sulfatases phospholipases CYTOSOL + H+ pump ATPADPPi H+ Figure 13–37 Lysosomes. The acid hydrolases are hydrolytic enzymes that are active under acidic conditions. an h+ aTpase in the membrane pumps h+ into the lysosome, maintaining its lumen at an acidic ph.

1	digestion of contents hydrolytically active, low-pH compartments hydrolytic enzymes. It is especially hard to apply a narrower definition than this in plant cells, as we discuss next. plant and fungal vacuoles are remarkably versatile lysosomes

1	plant and fungal vacuoles are remarkably versatile lysosomes Most plant and fungal cells (including yeasts) contain one or several very large, fluid-filled vesicles called vacuoles. They typically occupy more than 30% of the cell volume, and as much as 90% in some cell types (Figure 13–40). Vacuoles are related to animal cell lysosomes and contain a variety of hydrolytic enzymes, but their functions are remarkably diverse. The plant vacuole can act as a storage organelle for both nutrients and waste products, as a degradative compartment, as an economical way of increasing cell size, and as a controller of turgor pressure (the osmotic pressure that pushes outward on the cell wall and keeps the plant from wilting) (Figure 13–41). The same cell may have different vacuoles with distinct functions, such as digestion and storage.

1	The vacuole is important as a homeostatic device, enabling plant cells to withstand wide variations in their environment. When the pH in the environment drops, for example, the flux of H+ into the cytosol is balanced, at least in part, by an increased transport of H+ into the vacuole, which tends to keep the pH in the cytosol constant. Similarly, many plant cells maintain an almost constant turgor pressure despite large changes in the tonicity of the fluid in their immediate environment. They do so by changing the osmotic pressure of the cytosol and vacuole—in part by the controlled breakdown and resynthesis of polymers such as polyphosphate in the vacuole, and in part by altering the transport rates of sugars,

1	Figure 13–39 A model for lysosome maturation. late endosomes fuse with preexisting lysosomes (bottom arrow) or preexisting endolysosomes (top arrow). endolysosomes eventually mature into lysosomes as hydrolases complete the digestion of their contents, which can include intralumenal vesicles. lysosomes also fuse with phagosomes, as we discuss later. Figure 13–40 The plant cell vacuole.

1	Figure 13–40 The plant cell vacuole. (a) a confocal image of cells from an arabidopsis embryo that is expressing an aquaporin—yfp (yellow fluorescent protein) fusion protein in its tonoplast, or vacuole membrane (green); the cell walls have been false-colored orange. each cell contains several large vacuoles. (b) This electron micrograph of cells in a young tobacco leaf shows the cytosol as a thin layer, containing chloroplasts, pressed against the cell wall by the enormous vacuole. (a, courtesy of c. carroll and l. frigerio, based on s. gattolin et al., Mol. Plant 4:180–189, 2011. with permission from oxford university press; b, courtesy of J. burgess.) amino acids, and other metabolites across the plasma membrane and the vacuolar membrane. The turgor pressure regulates the activities of distinct transporters in each membrane to control these fluxes.

1	Humans often harvest substances stored in plant vacuoles—from rubber to opium to the flavoring of garlic. Many stored products have a metabolic function. Proteins, for example, can be preserved for years in the vacuoles of the storage cells of many seeds, such as those of peas and beans. When the seeds germinate, these proteins are hydrolyzed, and the resulting amino acids provide a food supply for the developing embryo. Anthocyanin pigments stored in vacuoles color the petals of many flowers so as to attract pollinating insects, while noxious molecules released from vacuoles when a plant is eaten or damaged provide a defense against predators. Multiple pathways deliver Materials to lysosomes Lysosomes are meeting places where several streams of intracellular traffic converge. A route that leads outward from the ER via the Golgi apparatus delivers most of the lysosome’s digestive enzymes, while at least four paths from different sources feed substances into lysosomes for digestion.

1	The best studied of these degradation paths is the one followed by macromolecules taken up from extracellular fluid by endocytosis. A similar pathway found in phagocytic cells, such as macrophages and neutrophils in vertebrates, is dedicated to the engulfment, or phagocytosis, of large particles and microorganisms to form phagosomes. A third pathway called macropinocytosis specializes in the nonspecific uptake of fluids, membrane, and particles attached to the plasma membrane. We will return to discuss these pathways later in the chapter. A fourth pathway called autophagy originates in the cytoplasm of the cell itself and is used to digest cytosol and worn-out organelles, as we discuss next. The four paths to degradation in lysosomes are illustrated in Figure 13–42.

1	Figure 13–41 The role of the vacuole in controlling the size of plant cells. a plant cell can achieve a large increase in volume without increasing the volume of the cytosol. localized weakening of the cell wall orients a turgor-driven cell enlargement that accompanies the uptake of water into an expanding vacuole. The cytosol is eventually confined to a thin peripheral layer, which is connected to the nuclear region by strands of cytosol stabilized by bundles of actin filaments (not shown). Figure 13–42 Four pathways to degradation in lysosomes. Materials in each pathway are derived from a different source. note that the autophagosome has a double membrane. In all cases, the final step is the fusion with lysosomes.

1	All cell types dispose of obsolete parts by a lysosome-dependent process called autophagy, or “self-eating.” The degradation process is important during normal cell growth and in development, where it helps restructure differentiating cells, but also in adaptive responses to stresses such as starvation and infection. Autophagy can remove large objects—macromolecules, large protein aggregates, and even whole organelles—that other disposal mechanisms such as proteasomal degradation cannot handle. Defects in autophagy may prevent cells from clearing away invading microbes, unwanted protein aggregates and abnormal proteins, and thereby contribute to diseases ranging from infectious disorders to neurodegeneration and cancer.

1	In the initial stages of autophagy, cytoplasmic cargo becomes surrounded by a double membrane that assembles by the fusion of small vesicles of unknown origin, forming an autophagosome (Figure 13–43). A few tens of different proteins have been identified in yeast and animal cells that participate in the process, which must be tightly regulated: either too little or too much can be deleterious. The whole process occurs in the following sequence of steps: 1. Induction by activation of signaling molecules: Protein kinases (including the mTOR complex 1, discussed in Chapter 15) that relay information about the metabolic status of the cell, become activated and signal to the autophagic machinery. 2.

1	2. Nucleation and extension of a delimiting membrane into a crescent-shaped cup: Membrane vesicles, characterized by the presence of ATG9, the only transmembrane protein involved in the process, are recruited to an assembly site, where they nucleate autophagosome formation. ATG9 is not incorporated into the autophagosome: a retrieval pathway must remove it from the assembling structure. 3. Closure of the membrane cup around the target to form a sealed double-membrane-enclosed autophagosome. 4. Fusion of the autophagosome with lysosomes, catalyzed by SNAREs. 5. Digestion of the inner membrane and the lumenal contents of the autophagosome.

1	4. Fusion of the autophagosome with lysosomes, catalyzed by SNAREs. 5. Digestion of the inner membrane and the lumenal contents of the autophagosome. Autophagy can be either nonselective or selective. In nonselective autophagy, a bulk portion of cytoplasm is sequestered in autophagosomes. It might occur, for example, in starvation conditions: when external nutrients are limiting, metabolites derived from the digestion of the captured cytosol might help the cell survive. In selective autophagy specific cargo is packaged into autophagosomes that tend to contain little cytosol, and their shape reflects the shape of the cargo. Selective autophagy mediates the degradation of worn out, or otherwise unwanted, mitochondria, peroxisomes, ribosomes, and ER; it can also be used to destroy invading microbes. Figure 13–43 A model of autophagy.

1	Figure 13–43 A model of autophagy. (a) activation of a signaling pathway initiates a nucleation event in the cytoplasm. a crescent of autophagosomal membrane grows by fusion of vesicles of unknown origin and eventually fuses to form a double-membrane-enclosed autophagosome, which sequesters a portion of the cytoplasm. The autophagosome then fuses with lysosomes containing acid hydrolases that digest its content. during the formation of the autophagosome membrane, a ubiquitinlike protein becomes activated by covalent attachment of a phosphatidylethanolamine lipid anchor. These proteins then mediate vesicle tethering and fusion, leading to the formation of a crescent-shaped membrane structure that assembles around its target (not shown). (b) an electron micrograph of an autophagosome containing a mitochondrion and a peroxisome. (b, courtesy of daniel s. friend, from d.w. fawcett, a Textbook of histology, 12th ed. new york: chapman and hall, 1994. with permission from kluwer.)

1	The selective autophagy of worn out or damaged mitochondria is called mitophagy. As discussed in Chapters 12 and 14, when mitochondria function normally, the inner mitochondrial membrane is energized by an electrochemical H+ gradient that drives ATP synthesis and the import of mitochondrial precursor proteins and metabolites. Damaged mitochondria cannot maintain the gradient, so protein import is blocked. As a consequence, a protein kinase called Pink1, which is normally imported into mitochondria, is instead retained on the mitochondrial surface where it recruits the ubiquitin ligase Parkin from the cytosol. Parkin ubiquitylates mitochondrial outer membrane proteins, which mark the organelle for selective destruction in autophagosomes. Mutations in Pink1 or Parkin cause a form of early-onset Parkinson’s disease, a degenerative disorder of the central nervous system. It is not known why the neurons that die prematurely in this disease are particularly reliant on mitophagy.

1	a Mannose 6-phosphate receptor sorts lysosomal hydrolases in the Trans golgi network We now consider the pathway that delivers lysosomal hydrolases from the TGN to lysosomes. The enzymes are first delivered to endosomes in transport vesicles that bud from the TGN, before they move on to endolysosomes and lysosomes (see Figure 13–39). The vesicles that leave the TGN incorporate the lysosomal proteins and exclude the many other proteins being packaged into different transport vesicles for delivery elsewhere.

1	How are lysosomal hydrolases recognized and selected in the TGN with the required accuracy? In animal cells they carry a unique marker in the form of mannose 6-phosphate (M6P) groups, which are added exclusively to the N-linked oligosaccharides of these soluble lysosomal enzymes as they pass through the lumen of the cis Golgi network (Figure 13–44). Transmembrane M6P receptor proteins, which are present in the TGN, recognize the M6P groups and bind to the lysosomal hydrolases on the lumenal side of the membrane and to adaptor proteins in assembling clathrin coats on the cytosolic side. In this way, the receptors help package the hydrolases into clathrin-coated vesicles that bud from the TGN and deliver their contents to early endosomes.

1	The M6P receptor protein binds to M6P at pH 6.5–6.7 in the TGN lumen and releases it at pH 6, which is the pH in the lumen of endosomes. Thus, after the receptor is delivered, the lysosomal hydrolases dissociate from the M6P receptors, which are retrieved into transport vesicles that bud from endosomes. These vesicles are coated with retromer, a coat protein complex specialized for endosome-to-TGN transport, which returns the receptors to the TGN for reuse (Figure 13–45). Transport in either direction requires signals in the cytoplasmic tail of the M6P receptor that direct this protein to the endosome or back to the TGN. These signals are recognized by the retromer complex that recruits M6P receptors into transport vesicles that bud from endosomes. The recycling of the M6P receptor resembles the recycling of the KDEL receptor discussed earlier, although it differs in the type of coated vesicles that mediate the transport.

1	Not all the hydrolase molecules that are tagged with M6P get to lysosomes. Some escape the normal packaging process in the trans Golgi network and are transported “by default” to the cell surface, where they are secreted into the extracellular fluid. Some M6P receptors, however, also take a detour to the plasma membrane, where they recapture the escaped lysosomal hydrolases and return them by receptor-mediated endocytosis (discussed later) to lysosomes via early and late endosomes. As lysosomal hydrolases require an acidic milieu to work, they can do little harm in the extracellular fluid, which usually has a neutral pH of 7.4.

1	For the sorting system that segregates lysosomal hydrolases and dispatches them to endosomes to work, the M6P groups must be added only to the appropriate glycoproteins in the Golgi apparatus. This requires specific recognition of the hydrolases by the Golgi enzymes responsible for adding M6P. Since all glycoproteins leave the ER with identical N-linked oligosaccharide chains, the signal for Figure 13–44 The structure of mannose 6-phosphate on a lysosomal hydrolase. adding the M6P units to oligosaccharides must reside somewhere in the polypeptide chain of each hydrolase. Genetic engineering experiments have revealed that the recognition signal is a cluster of neighboring amino acids on each protein’s surface, known as a signal patch (Figure 13–46). Since most lysosomal hydrolases contain multiple oligosaccharides, they acquire many M6P groups, providing a high-affinity signal for the M6P receptor. defects in the glcnac phosphotransferase cause a lysosomal storage disease in humans

1	Genetic defects that affect one or more of the lysosomal hydrolases cause a number of human lysosomal storage diseases. The defects result in an accumulation of undigested substrates in lysosomes, with severe pathological consequences, most often in the nervous system. In most cases, there is a mutation in a structural gene that codes for an individual lysosomal hydrolase. This occurs in Hurler’s disease, for example, in which the enzyme required for the breakdown of certain types of glycosaminoglycan chains is defective or missing. The most severe form of lysosomal storage disease, however, is a very rare inherited metabolic disorder called inclusion-cell disease (I-cell disease). In this condition, almost all of the hydrolytic enzymes are missing from the lysosomes of many cell types, and their undigested substrates accumulate in these lysosomes, which consequently form large inclusions in the cells. The consequent pathology is complex, affecting all organ systems, skeletal

1	types, and their undigested substrates accumulate in these lysosomes, which consequently form large inclusions in the cells. The consequent pathology is complex, affecting all organ systems, skeletal integrity, and mental development; individuals rarely live beyond six or seven years.

1	I-cell disease is due to a single gene defect and, like most genetic enzyme deficiencies, it is recessive—that is, it occurs only in individuals having two copies of the defective gene. In patients with I-cell disease, all the hydrolases missing from lysosomes are found in the blood: because they fail to sort properly in the Golgi apparatus, they are secreted rather than transported to lysosomes. The mis-sorting has been traced to a defective or missing GlcNAc phosphotransferase. Because lysosomal enzymes are not phosphorylated in the cis Golgi network, the M6P receptors do not segregate them into the appropriate transport vesicles in the TGN. Instead, the lysosomal hydrolases are carried to the cell surface and secreted. In I-cell disease, the lysosomes in some cell types, such as hepatocytes, contain a normal complement of lysosomal enzymes, implying that there is another

1	Figure 13–45 The transport of newly synthesized lysosomal hydrolases to endosomes. The sequential action of two enzymes in the cis and trans golgi network adds mannose 6-phosphate (M6p) groups to the precursors of lysosomal enzymes (see figure 13–46). The M6p-tagged hydrolases then segregate from all other types of proteins in the Tgn because adaptor proteins (not shown) in the clathrin coat bind the M6p receptors, which, in turn, bind the M6p-modified lysosomal hydrolases. The clathrin-coated vesicles bud off from the Tgn, shed their coat, and fuse with early endosomes. at the lower ph of the endosome, the hydrolases dissociate from the M6p receptors, and the empty receptors are retrieved in retromercoated vesicles to the Tgn for further rounds of transport. In the endosomes, the phosphate is removed from the M6p attached to the hydrolases, which may further ensure that the hydrolases do not return to the Tgn with the receptor.

1	with GlcNAc attached to mannose in oligosaccharide pathway for directing hydrolases to lysosomes that is used by some cell types but not others. Alternative sorting receptors function in these M6P-independent pathways. Similarly, an M6P-independent pathway in all cells sorts the membrane proteins of lysosomes from the TGN for transport to late endosomes, and those proteins are therefore normal in I-cell disease.

1	Targeting of material to lysosomes is not necessarily the end of the pathway. Lysosomal secretion of undigested content enables all cells to eliminate indigestible debris. For most cells, this seems to be a minor pathway, used only when the cells are stressed. Some cell types, however, contain specialized lysosomes that have acquired the necessary machinery for fusion with the plasma membrane. Melanocytes in the skin, for example, produce and store pigments in their lysosomes. These pigment-containing melanosomes release their pigment into the extracellular space of the epidermis by exocytosis. The pigment is then taken up by keratinocytes, leading to normal skin pigmentation. In some genetic disorders, defects in melanosome exocytosis block this transfer process, leading to forms of hypopigmentation (albinism). Under certain conditions, multivesicular bodies can also fuse with the plasma membrane. If that occurs, their intralumenal vesicles are released from cells. Circulating small

1	(albinism). Under certain conditions, multivesicular bodies can also fuse with the plasma membrane. If that occurs, their intralumenal vesicles are released from cells. Circulating small vesicles, also called exosomes, have been observed in the blood and may be used to transport components between cells, although the importance of such a mechanism of potential communication between distant cells is unknown. Some exosomes may derive from direct vesicle budding events at the plasma membrane, which is a topologically equivalent process (see Figure 13–57).

1	Lysosomes are specialized for the intracellular digestion of macromolecules. They contain unique membrane proteins and a wide variety of soluble hydrolytic enzymes that operate best at pH 5, which is the internal pH of lysosomes. An ATP-driven H+ pump in the lysosomal membrane maintains this low pH. Newly synthesized lysosomal proteins transported from the lumen of the ER, through the Golgi apparatus; they are then carried from the trans Golgi network to endosomes by means of clathrin-coated transport vesicles, before moving on to lysosomes.

1	The lysosomal hydrolases contain N-linked oligosaccharides that are covalently modified in a unique way in the cis Golgi so that their mannoses are phosphorylated. These mannose 6-phosphate (M6P) groups are recognized by an M6P receptor protein in the trans Golgi network that segregates the hydrolases and helps package them into budding transport vesicles that deliver their contents to endosomes. The M6P receptors shuttle back and forth between the trans Golgi network and the endosomes. The low pH in endosomes and the removal of the phosphate from the

1	Figure 13–46 The recognition of a lysosomal hydrolase. a glcnac phosphotransferase recognizes lysosomal hydrolases in the golgi apparatus. The enzyme has separate catalytic and recognition sites. The catalytic site binds both high-mannose n-linked oligosaccharides and udp-glcnac. The recognition site binds to a signal patch that is present only on the surface of lysosomal hydrolases. a second enzyme cleaves off the glcnac, leaving the mannose 6-phosphate exposed. M6P group cause the lysosomal hydrolases to dissociate from these receptors, making the transport of the hydrolases unidirectional. A separate transport system uses clathrin-coated vesicles to deliver resident lysosomal membrane proteins from the trans Golgi network to endosomes. TransporT InTo The cell froM The plasMa MeMbrane: endocyTosIs

1	TransporT InTo The cell froM The plasMa MeMbrane: endocyTosIs The routes that lead inward from the cell surface start with the process of endocytosis, by which cells take up plasma membrane components, fluid, solutes, macromolecules, and particulate substances. Endocytosed cargo includes receptor– ligand complexes, a spectrum of nutrients and their carriers, extracellular matrix components, cell debris, bacteria, viruses, and, in specialized cases, even other cells. Through endocytosis, the cell regulates the composition of its plasma membrane in response to changing extracellular conditions.

1	In endocytosis, the material to be ingested is progressively enclosed by a small portion of the plasma membrane, which first invaginates and then pinches off to form an endocytic vesicle containing the ingested substance or particle. Most eukaryotic cells constantly form endocytic vesicles, a process called pinocytosis (“cell drinking”); in addition, some specialized cells contain dedicated pathways that take up large particles on demand, a process called phagocytosis (“cell eating”). Endocytic vesicles form at the plasma membrane by multiple mechanisms that differ in both the molecular machinery used and how that machinery is regulated.

1	Once generated at the plasma membrane, most endocytic vesicles fuse with a common receiving compartment, the early endosome, where internalized cargo is sorted: some cargo molecules are returned to the plasma membrane, either directly or via a recycling endosome, and others are designated for degradation by inclusion in a late endosome. Late endosomes form from a bulbous, vacuolar portion of early endosomes by a process called endosome maturation. This conversion process changes the protein composition of the endosome membrane, patches of which invaginate and become incorporated within the organelles as intralumenal vesicles, while the endosome itself moves from the cell periphery to a location close to the nucleus. As an endosome matures, it ceases to recycle material to the plasma membrane and irreversibly commits its remaining contents to degradation: late endosomes fuse with one another and with lysosomes to form endolysosomes, which degrade their contents, as discussed earlier

1	and irreversibly commits its remaining contents to degradation: late endosomes fuse with one another and with lysosomes to form endolysosomes, which degrade their contents, as discussed earlier (Figure 13–47).

1	Each of the stages of endosome maturation—from the early endosome to the endolysosome—is connected through bidirectional vesicle transport pathways to

1	Figure 13–47 Endosome maturation: the endocytic pathway from the plasma membrane to lysosomes. endocytic vesicles fuse near the cell periphery with an early endosome, which is the primary sorting station. Tubular portions of the early endosome bud off vesicles that recycle endocytosed cargo back to the plasma membrane—either directly, or indirectly via recycling endosomes. recycling endosomes can store proteins until they are needed. conversion of early endosomes to late endosomes is accompanied by loss of the tubular projections. Membrane proteins destined for degradation are internalized in intralumenal vesicles. The developing late endosome, or multivesicular body, moves on microtubules to the cell interior. fully matured late endosomes no longer send vesicles to the plasma membrane, and they fuse with one another and with endolysosomes and lysosomes to degrade their contents. each stage of endosome maturation is connected via transport vesicles with the Tgn, providing a continuous

1	they fuse with one another and with endolysosomes and lysosomes to degrade their contents. each stage of endosome maturation is connected via transport vesicles with the Tgn, providing a continuous supply of newly synthesized lysosomal proteins.

1	0.1 µm the TGN. These pathways allow insertion of newly synthesized materials, such as lysosomal enzymes arriving from the ER, and the retrieval of components, such as the M6P receptor, back into the early parts of the secretory pathway. We next discuss how the cell uses and controls the various features of endocytic trafficking. pinocytic vesicles form from coated pits in the plasma Membrane

1	Virtually all eukaryotic cells continually ingest portions of their plasma membrane in the form of small pinocytic (endocytic) vesicles. The rate at which plasma membrane is internalized in this process of pinocytosis varies between cell types, but it is usually surprisingly high. A macrophage, for example, ingests 25% of its own volume of fluid each hour. This means that it must ingest 3% of its plasma membrane each minute, or 100% in about half an hour. Fibroblasts endocytose at a somewhat lower rate (1% of their plasma membrane per minute), whereas some amoebae ingest their plasma membrane even more rapidly. Since a cell’s surface area and volume remain unchanged during this process, it is clear that the same amount of membrane being removed by endocytosis is being added to the cell surface by the converse process of exocytosis. In this sense, endocytosis and exocytosis are linked processes that can be considered to constitute an endocytic– exocytic cycle. The coupling between

1	surface by the converse process of exocytosis. In this sense, endocytosis and exocytosis are linked processes that can be considered to constitute an endocytic– exocytic cycle. The coupling between exocytosis and endocytosis is particularly strict in specialized structures characterized by high membrane turnover, such as a nerve terminal.

1	The endocytic part of the cycle often begins at clathrin-coated pits. These specialized regions typically occupy about 2% of the total plasma membrane area. The lifetime of a clathrin-coated pit is short: within a minute or so of being formed, it invaginates into the cell and pinches off to form a clathrin-coated vesicle (Figure 13–48). About 2500 clathrin-coated vesicles pinch off from the plasma membrane of a cultured fibroblast every minute. The coated vesicles are even more transient than the coated pits: within seconds of being formed, they shed their coat and fuse with early endosomes. not all pinocytic vesicles are clathrin-coated

1	not all pinocytic vesicles are clathrin-coated In addition to clathrin-coated pits and vesicles, cells can form other types of pinocytic vesicles, such as caveolae (from the Latin for “little cavities”), originally recognized by their ability to transport molecules across endothelial cells that form the inner lining of blood vessels. Caveolae, sometimes seen in the electron microscope as deeply invaginated flasks, are present in the plasma membrane of most vertebrate cell types (Figure 13–49). They are thought to form from lipid rafts in the plasma membrane (discussed in Chapter 10), which are especially rich

1	Figure 13–48 The formation of clathrincoated vesicles from the plasma membrane. These electron micrographs illustrate the probable sequence of events in the formation of a clathrin-coated vesicle from a clathrin-coated pit. The clathrincoated pits and vesicles shown are larger than those seen in normal-sized cells; they are from a very large hen oocyte and they take up lipoprotein particles to form yolk. The lipoprotein particles bound to their membrane-bound receptors appear as a dense, fuzzy layer on the extracellular surface of the plasma membrane—which is the inside surface of the coated pit and vesicle. (courtesy of M.M. perry and a.b. gilbert, J. Cell sci. 39:257–272, 1979. with permission from The company of biologists.) 0.2 µm in cholesterol, glycosphingolipids, and glycosylphosphatidylinositol (GPI)-anchored membrane proteins (see Figure 10–13). The major structural proteins in caveolae are caveolins, a family of unusual integral membrane proteins that each insert a

1	(GPI)-anchored membrane proteins (see Figure 10–13). The major structural proteins in caveolae are caveolins, a family of unusual integral membrane proteins that each insert a hydrophobic loop into the membrane from the cytosolic side but do not extend across the membrane. On their cytosolic side, caveolins are bound to large protein complexes of caving proteins, which are thought to stabilize the membrane curvature.

1	In contrast to clathrin-coated and COPIor COPII-coated vesicles, caveolae are usually static structures. Nonetheless, they can be induced to pinch off and serve as endocytic vesicles to transport cargo to early endosomes or to the plasma membrane on the opposite side of a polarized cell (in a process called transcytosis, which we discuss later). Some animal viruses such as SV40 and papillomavirus (which causes warts) enter cells in vesicles derived from caveolae. The viruses are first delivered to early endosomes and move from there in transport vesicles to the lumen of the ER. The viral genome exits across the ER membrane into the cytosol, from where it is imported into the nucleus to start the infection cycle. Cholera toxin (discussed in Chapters 15 and 19) also enters the cell through caveolae and is transported to the ER before entering the cytosol.

1	Macropinocytosis is another clathrin-independent endocytic mechanism that can be activated in practically all animal cells. In most cell types, it does not operate continually but rather is induced for a limited time in response to cell-surface receptor activation by specific cargoes, including growth factors, integrin ligands, apoptotic cell remnants, and some viruses. These ligands activate a complex signaling pathway, resulting in a change in actin dynamics and the formation of cell-surface protrusions, called ruffles (discussed in Chapter 16). When ruffles collapse back onto the cell, large fluid-filled endocytic vesicles form, called macropinosomes (Figure 13–50), which transiently increase the bulk fluid uptake of a cell by up to tenfold. Macropinocytosis is a dedicated degradative pathway: macropinosomes acidify and then fuse with late endosomes or endolysosomes, without recycling their cargo back to the plasma membrane.

1	cells use receptor-Mediated endocytosis to Import selected extracellular Macromolecules In most animal cells, clathrin-coated pits and vesicles provide an efficient pathway for taking up specific macromolecules from the extracellular fluid. In this process, called receptor-mediated endocytosis, the macromolecules bind to

1	Figure 13–49 Caveolae in the plasma membrane of a fibroblast. (a) This electron micrograph shows a plasma membrane with a very high density of caveolae. (b) This rapid-freeze deep-etch image demonstrates the characteristic “cauliflower” texture of the cytosolic face of the caveolae membrane. The characteristic texture is thought to result from aggregates of caveolins and cavins. a clathrin-coated pit is also seen at the upper right. (courtesy of r.g.w. anderson, from k.g. rothberg et al., Cell 68:673–682, 1992. with permission from elsevier.) complementary transmembrane receptor proteins, which accumulate in coated pits, and then enter the cell as receptor–macromolecule complexes in clathrin-coated vesicles (see Figure 13–48). Because ligands are selectively captured by receptors, receptor-mediated endocytosis provides a selective concentrating mechanism that increases the efficiency of internalization of particular ligands more than a hundredfold. In this way, even minor components

1	endocytosis provides a selective concentrating mechanism that increases the efficiency of internalization of particular ligands more than a hundredfold. In this way, even minor components of the extracellular fluid can be efficiently taken up in large amounts. A particularly well-understood and physiologically important example is the process that mammalian cells use to import cholesterol.

1	Many animal cells take up cholesterol through receptor-mediated endocytosis and, in this way, acquire most of the cholesterol they require to make new membrane. If the uptake is blocked, cholesterol accumulates in the blood and can contribute to the formation in blood vessel (artery) walls of atherosclerotic plaques, deposits of lipid and fibrous tissue that can cause strokes and heart attacks by blocking arterial blood flow. In fact, it was a study of humans with a strong genetic predisposition for atherosclerosis that first revealed the mechanism of receptor-mediated endocytosis.

1	Most cholesterol is transported in the blood as cholesteryl esters in the form of lipid–protein particles known as low-density lipoproteins (LDLs) (Figure 13–51). When a cell needs cholesterol for membrane synthesis, it makes transmembrane receptor proteins for LDL and inserts them into its plasma membrane. Once in the plasma membrane, the LDL receptors diffuse until they associate with clathrin-coated pits that are in the process of forming. There, an endocytosis signal in the cytoplasmic tail of the LDL receptors binds the membrane-bound adaptor protein AP2 after its conformation has been locally unlocked by binding to PI(4,5)P2 on the plasma membrane. Coincidence detection, as discussed earlier, thus imparts both efficiency and selectivity to the process (see Figure 13–9). AP2 then recruits clathrin to initiate endocytosis.

1	Since coated pits constantly pinch off to form coated vesicles, any LDL particles bound to LDL receptors in the coated pits are rapidly internalized in coated vesicles. After shedding their clathrin coats, the vesicles deliver their contents to early endosomes. Once the LDL and LDL receptors encounter the low pH in early endosomes, LDL is released from its receptor and is delivered via late endosomes to lysosomes. There, the cholesteryl esters in the LDL particles are hydrolyzed to free cholesterol, which is now available to the cell for new membrane synthesis (Movie 13.3). If too much free cholesterol accumulates in a cell, the cell shuts off both its own cholesterol synthesis and the synthesis of LDL receptors, so that it ceases both to make or to take up cholesterol.

1	This regulated pathway for cholesterol uptake is disrupted in individuals who inherit defective genes encoding LDL receptors. The resulting high levels of blood cholesterol predispose these individuals to develop atherosclerosis prematurely, and many would die at an early age of heart attacks resulting from coronary artery disease if they were not treated with drugs such as statins that lower the level of blood cholesterol. In some cases, the receptor is lacking altogether. In others, the receptors are defective—in either the extracellular binding site for LDL or the Figure 13–50 Schematic representation of macropinocytosis. cell signaling events lead to a reprogramming of actin dynamics, which in turn triggers the formation of cell-surface ruffles. as the ruffles collapse back onto the cell surface, they nonspecifically trap extracellular fluid and macromolecules and particles contained in it, forming large vacuoles, or macropinosomes, as shown.

1	Figure 13–51 A low-density lipoprotein (LDL) particle. each spherical particle has a mass of 3 × 106 daltons. It contains a core of about 1500 cholesterol molecules esterified to long-chain fatty acids. a lipid monolayer composed of about 800 phospholipid and 500 unesterified cholesterol molecules surrounds the core of cholesteryl esters. a single molecule of apolipoprotein b, a 500,000-dalton beltlike protein, organizes the particle and mediates the specific binding of ldl to cell-surface ldl receptors. intracellular binding site that attaches the receptor AP2 adaptor protein in clathrin-coated pits. In the latter case, normal numbers of LDL receptors are present, but they fail to become localized in clathrin-coated pits. Although LDL binds to the surface of these mutant cells, it is not internalized, directly demonstrating the importance of clathrin-coated pits for the receptor-mediated endocytosis of cholesterol.

1	More than 25 distinct receptors are known to participate in receptor-mediated endocytosis of different types of molecules. They all apparently use clathrin-dependent internalization routes and are guided into clathrin-coated pits by signals in their cytoplasmic tails that bind to adaptor proteins in the clathrin coat. Many of these receptors, like the LDL receptor, enter coated pits irrespective of whether they have bound their specific ligands. Others enter preferentially when bound to a specific ligand, suggesting that a ligand-induced conformational change is required for them to activate the signal sequence that guides them into the pits. Since most plasma membrane proteins fail to become concentrated in clathrin-coated pits, the pits serve as molecular filters, preferentially collecting certain plasma membrane proteins (receptors) over others.

1	Electron-microscope studies of cultured cells exposed simultaneously to different labeled ligands demonstrate that many kinds of receptors can cluster in the same coated pit, whereas some other receptors cluster in different clathrin-coated pits. The plasma membrane of one clathrin-coated pit can accommodate more than 100 receptors of assorted varieties. specific proteins are retrieved from early endosomes and returned to the plasma Membrane

1	specific proteins are retrieved from early endosomes and returned to the plasma Membrane Early endosomes are the main sorting station in the endocytic pathway, just as the cis and trans Golgi networks serve this function in the secretory pathway. In the mildly acidic environment of the early endosome, many internalized receptor proteins change their conformation and release their ligand, as already discussed for the M6P receptors. Those endocytosed ligands that dissociate from their receptors in the early endosome are usually doomed to destruction in lysosomes (although cholesterol is an exception, as just discussed), along with the other soluble contents of the endosome. Some other endocytosed ligands, however, remain bound to their receptors, and thereby share the fate of the receptors.

1	In the early endosome, the LDL receptor dissociates from its ligand, LDL, and is recycled back to the plasma membrane for reuse, leaving the discharged LDL to be carried to lysosomes (Figure 13–52). The recycling transport vesicles bud from long, narrow tubules that extend from the early endosomes. It is likely that the geometry of these tubules helps the sorting process: because tubules have a large membrane area enclosing a small volume, membrane proteins become enriched over soluble proteins. The transport vesicles return the LDL receptor directly to the plasma membrane. The transferrin receptor follows a similar recycling pathway as the LDL receptor, but unlike the LDL receptor it also recycles its ligand. Transferrin is a soluble

1	The transferrin receptor follows a similar recycling pathway as the LDL receptor, but unlike the LDL receptor it also recycles its ligand. Transferrin is a soluble Figure 13–52 The receptor-mediated endocytosis of LDL. note that the ldl dissociates from its receptors in the acidic environment of the early endosome. after a number of steps, the ldl ends up in endolysosomes and lysosomes, where it is degraded to release free cholesterol. In contrast, the ldl receptors are returned to the plasma membrane via transport vesicles that bud off from the tubular region of the early endosome, as shown. for simplicity, only one ldl receptor is shown entering the cell and returning to the plasma membrane. whether it is occupied or not, an ldl receptor typically makes one round trip into the cell and back to the plasma membrane every 10 minutes, making a total of several hundred trips in its 20-hour life-span.

1	protein that carries iron in the blood. Cell-surface transferrin receptors deliver transferrin with its bound iron to early endosomes by receptor-mediated endocytosis. The low pH in the endosome induces transferrin to release its bound iron, but the iron-free transferrin itself (called apotransferrin) remains bound to its receptor. The receptor–apotransferrin complex enters the tubular extensions of the early endosome and from there is recycled back to the plasma membrane. When the apotransferrin returns to the neutral pH of the extracellular fluid, it dissociates from the receptor and is thereby freed to pick up more iron and begin the cycle again. Thus, transferrin shuttles back and forth between the extracellular fluid and early endosomes, avoiding lysosomes and delivering iron to the cell interior, as needed for cells to grow and proliferate. plasma Membrane signaling receptors are down-regulated by degradation in lysosomes

1	plasma Membrane signaling receptors are down-regulated by degradation in lysosomes A second pathway that endocytosed receptors can follow from endosomes is taken by many signaling receptors, including opioid receptors and the receptor that binds epidermal growth factor (EGF). EGF is a small, extracellular signal protein that stimulates epidermal and various other cells to divide. Unlike LDL receptors, EGF receptors accumulate in clathrin-coated pits only after binding their ligand, and most do not recycle but are degraded in lysosomes, along with the ingested EGF. EGF binding therefore first activates intracellular signaling pathways and then leads to a decrease in the concentration of EGF receptors on the cell surface, a process called receptor downregulation, that reduces the cell’s subsequent sensitivity to EGF (see Figure 15–20).

1	Receptor downregulation is highly regulated. The activated receptors are first covalently modified on the cytosolic face with the small protein ubiquitin. Unlike polyubiquitylation, which adds a chain of ubiquitins that typically targets a protein for degradation in proteasomes (discussed in Chapter 6), ubiquitin tagging for sorting into the clathrin-dependent endocytic pathway adds just one or a few single ubiquitin molecules to the protein—a process called monoubiquitylation or multiubiquitylation, respectively. Ubiquitin-binding proteins recognize the attached ubiquitin and help direct the modified receptors into clathrin-coated pits. After delivery to the early endosome, other ubiquitin-binding proteins that are part of ESCRT complexes (ESCRT = Endosome Sorting Complex Required for Transport) recognize and sort the ubiquitylated receptors into intralumenal vesicles, which are retained in the maturing late endosome (see Figure 13–47). In this way, addition of ubiquitin blocks

1	Transport) recognize and sort the ubiquitylated receptors into intralumenal vesicles, which are retained in the maturing late endosome (see Figure 13–47). In this way, addition of ubiquitin blocks receptor recycling to the plasma membrane and directs the receptors into the degradation pathway, as we discuss next.

1	The endosomal compartments can be made visible in the electron microscope by adding a readily detectable tracer molecule, such as the enzyme peroxidase, to the extracellular medium and allowing varying lengths of time for the cell to endocytose the tracer. The distribution of the molecule after its uptake reveals the sequence of events. Within a minute or so after adding the tracer, it starts to appear in early endosomes, just beneath the plasma membrane (Figure 13–53). By 5–15 minutes later, it has moved to late endosomes, close to the Golgi apparatus and near the nucleus.

1	How early endosomes arise is not entirely clear, but their membrane and volume are mainly derived from incoming endocytic vesicles that fuse with one another (Movie 13.4). Early endosomes are relatively small and patrol the cytoplasm underlying the plasma membrane in jerky back-and-forth movements along microtubules, capturing incoming vesicles. Typically, an early endosome receives incoming vesicles for about 10 minutes, during which time membrane and fluid is rapidly recycled to the plasma membrane. Some of the incoming cargo, however, accumulates over the lifetime of the early endosome, eventually to be included in the late endosome. 0.5 µm

1	0.5 µm Figure 13–53 Electron micrograph of an early endosome. The endosome is labeled with endocytosed horseradish peroxidase, a widely used enzyme marker, detected in this case by an electron-dense reaction product. Many tubular extensions protrude from the central vacuolar space of the early endosome, which will later mature to give rise to a late endosome. (from J. Tooze and M. hollinshead, J. Cell Biol. 118:813–830, 1992.)

1	J. Tooze and M. hollinshead, J. Cell Biol. 118:813–830, 1992.) Early endosomes have tubular and vacuolar domains (see Figure 13–53). Most of the membrane surface is in the tubules and most of the volume is in the vacuolar domain. During endosome maturation, the two domains have different fates: the vacuolar portions of the early endosome are retained and transformed into late endosomes; the tubular portions shrink. Maturing endosomes, also called multivesicular bodies, migrate along microtubules toward the cell interior, shedding membrane tubules and vesicles that recycle material to the plasma membrane and TGN, and receiving newly synthesized lysosomal proteins. As they concentrate in a perinuclear region of the cell, the multivesicular bodies fuse with each other, and eventually with endolysosomes and lysosomes (see Figure 13–47).

1	Many changes occur during the maturation process. (1) The endosome changes shape and location, as the tubular domains are lost and the vacuolar domains are thoroughly modified. (2) Rab proteins, phosphoinositide lipids, fusion machinery (SNAREs and tethers), and microtubule motor proteins all participate in a molecular makeover of the cytosolic face of the endosome membrane, changing the functional characteristics of the organelle. (3) A V-type ATPase in the endosome membrane pumps H+ from the cytosol into the endosome lumen and acidifies the organelle. Crucially, the increasing acidity that accompanies maturation renders lysosomal hydrolases increasingly more active, influencing many receptor–ligand interactions, thereby controlling receptor loading and unloading. (4) Intralumenal vesicles sequester endocytosed signaling receptors inside the endosome, thus halting the receptor signaling activity. (5) Lysosome proteins are delivered from the TGN to the maturing endosome. Most of these

1	sequester endocytosed signaling receptors inside the endosome, thus halting the receptor signaling activity. (5) Lysosome proteins are delivered from the TGN to the maturing endosome. Most of these events occur gradually but eventually lead to a complete transformation of the endosome into an early endolysosome.

1	In addition to committing selected cargo for degradation, the maturation process is important for lysosome maintenance. The continual delivery of lysosome components from the TGN to maturing endosomes, ensures a steady supply of new lysosome proteins. The endocytosed materials mix in early endosomes with newly arrived acid hydrolases. Although mild digestion may start here, many hydrolases are synthesized and delivered as proenzymes, called zymogens, which contain extra inhibitory domains that keep the hydrolases inactive until these domains are proteolytically removed at later stages of endosome maturation. Moreover, the pH in early endosomes is not low enough to activate lysosomal hydrolases optimally. By these means, cells can retrieve membrane proteins intact from early endosomes and recycle them back to the plasma membrane. EsCrT protein complexes Mediate the formation of Intralumenal vesicles in Multivesicular bodies

1	EsCrT protein complexes Mediate the formation of Intralumenal vesicles in Multivesicular bodies As endosomes mature, patches of their membrane invaginate into the endosome lumen and pinch off to form intralumenal vesicles. Because of their appearance in the electron microscope such maturing endosomes are also called multivesicular bodies (Figure 13–54).

1	The multivesicular bodies carry endocytosed membrane proteins that are to be degraded. As part of the protein-sorting process, receptors destined for degradation, such as the occupied EGF receptors described previously, selectively partition into the invaginating membrane of the multivesicular bodies. In this way, both the receptors and any signaling proteins strongly bound to them are sequestered away from the cytosol where they might otherwise continue signaling. They also are made fully accessible to the digestive enzymes that eventually will degrade them (Figure 13–55). In addition to endocytosed membrane proteins, multivesicular bodies include the soluble content of early endosomes destined for late endosomes and digestion in lysosomes.

1	As discussed earlier, sorting into intralumenal vesicles requires one or multiple ubiquitin tags, which are added to the cytosolic domains of membrane proteins. These tags initially help guide the proteins into clathrin-coated vesicles in the plasma membrane. Once delivered to the endosomal membrane, the ubiquitin tags are recognized again, this time by a series of cytosolic ESCRT protein 0.5 µm

1	Figure 13–54 Electron micrograph of a multivesicular body. The large amount of internal membrane will be delivered to the lysosome, for digestion. (courtesy of andrew staehelin, from a. driouich, a. Jauneau and l.a. staehelin; Plant Physiol. 113:487–492, 1997. with permission from the american society of plant biologists.) complexes, (ESCRT-0, -I, -II, and -III), which bind sequentially and ultimately mediate the sorting process into the intralumenal vesicles. Membrane invagination into multivesicular bodies also depends on a lipid kinase that phosphorylates phosphatidylinositol to produce PI(3)P, which serves as an additional docking site for the ESCRT complexes; these complexes require both PI(3)P and the presence of ubiquitylated cargo proteins to bind to the endosomal membrane. ESCRT-III forms large multimeric assemblies on the membrane that bend the membrane (Figure 13–56).

1	Mutant cells compromised in ESCRT function display signaling defects. In such cells, activated receptors cannot be down-regulated by endocytosis and packaging into multivesicular bodies. The still-active receptors therefore mediate prolonged signaling, which can lead to uncontrolled cell proliferation and cancer. Processes that shape membranes often use similar machinery. Because of strong similarities in their protein sequences, researchers think that ESCRT complexes are evolutionarily related to components that mediate cell-membrane deformation in cytokinesis in archaea. Similarly, the ESCRT machinery that drives the internal budding from the endosome membrane to form intralumenal vesicles is also used in animal cell cytokinesis and virus budding, which are topologically equivalent, as both processes involve budding away from the cytosolic surface of the membrane (Figure 13–57).

1	The fates of endocytosed receptors—and of any ligands remaining bound to them—vary according to the specific type of receptor. As we discussed, most receptors are recycled and returned to the same plasma membrane domain from which they came; some proceed to a different domain of the plasma membrane, thereby mediating transcytosis; and some progress to lysosomes, where they are degraded. Receptors on the surface of polarized epithelial cells can transfer specific macromolecules from one extracellular space to another by transcytosis. A newborn, for example, obtains antibodies from its mother’s milk (which help protect it against infection) by transporting them across the epithelium of its gut. The lumen of the gut is acidic, and, at this low pH, the antibodies in the milk bind to specific receptors on the apical (absorptive) surface of the gut epithelial cells. The receptor–antibody complexes are internalized via clathrin-coated pits and

1	Figure 13–55 The sequestration of endocytosed proteins into intralumenal vesicles of multivesicular bodies. ubiquitylated membrane proteins are sorted into domains on the endosome membrane, which invaginate and pinch off to form intralumenal vesicles. The ubiquitin marker is removed and returned to the cytosol for reuse before the intralumenal vesicle closes. eventually, proteases and lipases in lysosomes digest all of the internal membranes. The invagination processes are essential for complete digestion of endocytosed membrane proteins: because the outer membrane of the multivesicular body becomes continuous with the lysosomal membrane, which is resistant to lysosomal hydrolases; the hydrolases, for example, could not digest the cytosolic domains of endocytosed transmembrane proteins, such as the egf receptor shown here, if the protein were not localized in intralumenal vesicles.

1	Figure 13–56 Sorting of endocytosed membrane proteins into the intralumenal vesicles of a multivesicular body. a series of complex binding events passes the ubiquitylated cargo proteins sequentially from one escrT complex (escrT-0) to the next, eventually concentrating them in membrane areas that bud into the lumen of the endosome to form intralumenal vesicles. escrT-III assembles into expansive multimeric structures and mediates invagination. The mechanisms of how cargo molecules are shepherded into the vesicles and how the vesicles are formed without including the escrT complexes themselves remain unknown. escrT complexes are soluble in the cytosol, are recruited to the membrane sequentially as needed, and are then released back into the cytosol as the vesicle pinches off.

1	vesicles and are delivered to early endosomes. The complexes remain intact and are retrieved in transport vesicles that bud from the early endosome and subsequently fuse with the basolateral domain of the plasma membrane. On exposure to the neutral pH of the extracellular fluid that bathes the basolateral surface of the cells, the antibodies dissociate from their receptors and eventually enter the baby’s bloodstream. The transcytotic pathway from the early endosome back to the plasma membrane is not direct. The receptors first move from the early endosome to the recycling endosome. The variety of pathways that different receptors follow from early endosomes implies that, in addition to binding sites for their ligands and binding sites for coated pits, many receptors also possess sorting signals that guide them into the appropriate transport pathway (Figure 13–58).

1	Cells can regulate the release of membrane proteins from recycling endosomes, thus adjusting the flux of proteins through the transcytotic pathway according to need. This regulation, the mechanism of which is uncertain, allows recycling endosomes to play an important part in adjusting the concentration of specific plasma membrane proteins. Fat cells and muscle cells, for example, contain large intracellular pools of the glucose transporters that are responsible for the uptake of glucose across the plasma membrane. These membrane transport proteins are stored in specialized recycling endosomes until the hormone insulin stimulates the cell to increase its rate of glucose uptake. In response to the insulin signal, transport vesicles rapidly bud from the recycling endosome and deliver large numbers of glucose transporters to the plasma membrane, thereby greatly increasing the rate of glucose uptake into the cell (Figure 13–59). Similarly, kidney cells regulate the insertion of aquaporins

1	of glucose transporters to the plasma membrane, thereby greatly increasing the rate of glucose uptake into the cell (Figure 13–59). Similarly, kidney cells regulate the insertion of aquaporins and V-ATPase into the plasma membrane to increase water and acid excretion, respectively, both in response to hormones.

1	Phagocytosis is a special form of endocytosis in which a cell uses large endocytic vesicles called phagosomes to ingest large particles such as microorganisms and dead cells. Phagocytosis is distinct, both in purpose and mechanism, from macropinocytosis, which we discussed earlier. In protozoa, phagocytosis is a form of feeding: large particles taken up into phagosomes end up in lysosomes, and the products of the subsequent digestive processes pass into the cytosol to be used as food. However, few cells in multicellular organisms are able to ingest such large particles efficiently. In the gut of animals, for example, extracellular processes break down food particles, and cells import the small products of hydrolysis. Figure 13–57 Conserved mechanism in multivesicular body formation and virus budding. In the two topologically equivalent processes indicated by the arrows, escrT complexes (not shown) shape membranes into buds that bulge away from the cytosol.

1	Figure 13–58 Possible fates for transmembrane receptor proteins that have been endocytosed. Three pathways from the early endosomal compartment in an epithelial cell are shown. retrieved receptors are returned (1) to the same plasma membrane domain from which they came (recycling) or (2) via a recycling endosome to a different domain of the plasma membrane (transcytosis). (3) receptors that are not specifically retrieved from early or recycling endosomes follow the pathway from the endosomal compartment to lysosomes, where they are degraded (degradation). If the ligand that is endocytosed with its receptor stays bound to the receptor in the acidic environment of the endosome, it shares the same fate as the receptor; otherwise, it is delivered to lysosomes. recycling endosomes are a way-station on the transcytotic pathway. In the transcytosis example shown here, an antibody fc receptor on a gut epithelial cell binds antibody and is endocytosed, eventually carrying the antibody to the

1	on the transcytotic pathway. In the transcytosis example shown here, an antibody fc receptor on a gut epithelial cell binds antibody and is endocytosed, eventually carrying the antibody to the basolateral plasma membrane. The receptor is called an fc receptor because it binds the fc part of the antibody (discussed in chapter 24).

1	Figure 13–59 Storage of plasma membrane proteins in recycling endosomes. recycling endosomes can serve as an intracellular storage site for specialized plasma membrane proteins that can be mobilized when needed. In the example shown, insulin binding to the insulin receptor triggers an intracellular signaling pathway that causes the rapid insertion of glucose transporters into the plasma membrane of a fat or muscle cell, greatly increasing its glucose intake.

1	Phagocytosis is important in most animals for purposes other than nutrition, and it is carried out mainly by specialized cells—so-called professional phagocytes. In mammals, two important classes of white blood cells that act as professional phagocytes are macrophages and neutrophils (Movie 13.5). These cells develop from hemopoietic stem cells (discussed in Chapter 22), and they ingest invading microorganisms to defend us against infection. Macrophages also have an important role in scavenging senescent cells and cells that have died by apoptosis (discussed in Chapter 18). In quantitative terms, the clearance of senescent and dead cells is by far the most important: our macrophages, for example, phagocytose more than 1011 senescent red blood cells in each of us every day.

1	The diameter of a phagosome is determined by the size of its ingested particles, and those particles can be almost as large as the phagocytic cell itself (Figure 13–60). Phagosomes fuse with lysosomes, and the ingested material is then degraded. Indigestible substances remain in the lysosomes, forming residual bodies that can be excreted from cells by exocytosis, as mentioned earlier. Some of the internalized plasma membrane components never reach the lysosome, because they are retrieved from the phagosome in transport vesicles and returned to the plasma membrane.

1	Some pathogenic bacteria have evolved elaborate mechanisms to prevent phagosome–lysosome fusion. The bacterium Legionella pneumophila, for example, which causes Legionnaires’ disease (discussed in Chapter 23), injects into its unfortunate host a Rab-modifying enzyme that causes certain Rab proteins to misdirect membrane traffic, thereby preventing phagosome–lysosome fusion. The bacterium, thus spared from lysosomal degradation, remains in the modified phagosome, growing and dividing as an intracellular pathogen, protected from the host’s adaptive immune system.

1	Phagocytosis is a cargo-triggered process. That is, it requires the activation of cell-surface receptors that transmit signals to the cell interior. Thus, to be phagocytosed, particles must first bind to the surface of the phagocyte (although not all particles that bind are ingested). Phagocytes have a variety of cell surface receptors that are functionally linked to the phagocytic machinery of the cell. The best-characterized triggers of phagocytosis are antibodies, which protect us by binding to the surface of infectious microorganisms (pathogens) and initiating a series of events that culminate in the invader being phagocytosed. When antibodies initially attack a pathogen, they coat it with antibody molecules that bind to Fc receptors on the surface of macrophages and neutrophils, activating the receptors to induce the phagocytic cell to extend pseudopods, which engulf the particle and fuse at their tips to form a phagosome (Figure 13–61A). Localized actin polymerization, initiated

1	the receptors to induce the phagocytic cell to extend pseudopods, which engulf the particle and fuse at their tips to form a phagosome (Figure 13–61A). Localized actin polymerization, initiated by Rho family GTPases and their activating Rho-GEFs (discussed in Chapters 15 and 16), shapes the pseudopods. The activated Rho GTPases switch on the kinase activity of local PI kinases to produce PI(4,5)P2 in the membrane (see Figure 13–11), which stimulates actin polymerization. To seal off the phagosome and complete the engulfment, actin is depolymerized by a PI 3-kinase that converts the PI(4,5)P2 to PI(3,4,5)P3, which is required for closure

1	Figure 13–60 Phagocytosis by a macrophage. a scanning electron micrograph of a mouse macrophage phagocytosing two chemically altered red blood cells. The red arrows point to edges of thin processes (pseudopods) of the macrophage that are extending as collars to engulf the red cells. (courtesy of Jean paul revel.) PI(4,5)P2 PI(3,4,5)P3 of the phagosome and may also contribute to reshaping the actin network to help drive the invagination of the forming phagosome (Figure 13–61B). In this way, the ordered generation and consumption of specific phosphoinositides guides sequential steps in phagosome formation.

1	Several other classes of receptors that promote phagocytosis have been characterized. Some recognize complement components, which collaborate with antibodies in targeting microbes for destruction (discussed in Chapter 24). Others directly recognize oligosaccharides on the surface of certain pathogens. Still others recognize cells that have died by apoptosis. Apoptotic cells lose the asymmetric distribution of phospholipids in their plasma membrane. As a consequence, negatively charged phosphatidylserine, which is normally confined to the cytosolic leaflet of the lipid bilayer, is now exposed on the outside of the cell, where it helps to trigger the phagocytosis of the dead cell.

1	Remarkably, macrophages will also phagocytose a variety of inanimate particles—such as glass or latex beads and asbestos fibers—yet they do not phagocytose live cells in their own body. The living cells display “don’t-eat-me” signals in the form of cell-surface proteins that bind to inhibiting receptors on the surface of macrophages. The inhibitory receptors recruit tyrosine phosphatases that antagonize the intracellular signaling events required to initiate phagocytosis, thereby locally inhibiting the phagocytic process. Thus phagocytosis, like many other cell processes, depends on a balance between positive signals that activate the process and negative signals that inhibit it. Apoptotic cells are thought both to gain “eat-me” signals (such as extracellularly exposed phosphatidylserine) and to lose their “don’t-eat-me” signals, causing them to be very rapidly phagocytosed by macrophages.

1	Cells ingest fluid, molecules, and particles by endocytosis, in which localized regions of the plasma membrane invaginate and pinch off to form endocytic vesicles. In most cells, endocytosis internalizes a large fraction of the plasma membrane every hour. The cells remain the same size because most of the plasma membrane components (proteins and lipids) that are endocytosed are continually returned to the cell surface by exocytosis. This large-scale endocytic–exocytic cycle is mediated largely by clathrin-coated pits and vesicles but clathrin-independent endocytic pathways also contribute. While many of the endocytosed molecules are quickly recycled to the plasma membrane, others eventually end up in lysosomes, where they are degraded. Most of the ligands that are endocytosed with their receptors dissociate from their receptors

1	Figure 13–61 A neutrophil reshaping its plasma membrane during phagocytosis. (a) an electron micrograph of a neutrophil phagocytosing a bacterium, which is in the process of dividing. (b) pseudopod extension and phagosome formation are driven by actin polymerization and reorganization, which respond to the accumulation of specific phosphoinositides in the membrane of the forming phagosome: pI(4,5)p2 stimulates actin polymerization, which promotes pseudopod formation, and then pI(3,4,5)p3 depolymerizes actin filaments at the base. (a, courtesy of dorothy f. bainton, phagocytic Mechanisms in health and disease. new york: Intercontinental Medical book corporation, 1971.) in the acidic environment of the endosome and eventually end up in lysosomes, while most of the receptors are recycled via transport vesicles back to the cell surface for reuse. Many cell-surface signaling receptors become tagged with ubiquitin when activated by binding their extracellular ligands. Ubiquitylation guides

1	transport vesicles back to the cell surface for reuse. Many cell-surface signaling receptors become tagged with ubiquitin when activated by binding their extracellular ligands. Ubiquitylation guides activated receptors into clathrin-coated pits, they and their ligands are efficiently internalized and delivered to early endosomes.

1	Early endosomes, rapidly mature into late endosomes. During maturation, patches of the endosomal membrane containing ubiquitylated receptors invaginate and pinch off to form intralumenal vesicles. This process is mediated by ESCRT complexes and sequesters the receptors away from the cytosol, which terminates their signaling activity. Late endosomes migrate along microtubules toward the interior of the cell where they fuse with one another and with lysosomes to form endolysosomes, where degradation occurs. In some cases, both receptor and ligand are transferred to a different plasma membrane domain, causing the ligand to be released at a different surface from where it originated, a process called transcytosis. In some cells, endocytosed plasma membrane proteins and lipids can be stored in recycling endosomes, for as long as necessary until they are needed. TransporT froM The Trans golgI neTwork To The cell exTerIor: exocyTosIs

1	TransporT froM The Trans golgI neTwork To The cell exTerIor: exocyTosIs Having considered the cell’s endocytic and internal digestive systems and the various types of incoming membrane traffic that converge on lysosomes, we now return to the Golgi apparatus and examine the secretory pathways that lead outward to the cell exterior. Transport vesicles destined for the plasma membrane normally leave the TGN in a steady stream as irregularly shaped tubules. The membrane proteins and the lipids in these vesicles provide new components for the cell’s plasma membrane, while the soluble proteins inside the vesicles are secreted to the extracellular space. The fusion of the vesicles with the plasma membrane is called exocytosis. This is the route, for example, by which cells secrete most of the proteoglycans and glycoproteins of the extracellular matrix, as discussed in Chapter 19.

1	All cells require this constitutive secretory pathway, which operates continuously (Movie 13.6). Specialized secretory cells, however, have a second secretory pathway in which soluble proteins and other substances are initially stored in secretory vesicles for later release by exocytosis. This is the regulated secretory pathway, found mainly in cells specialized for secreting products rapidly on demand—such as hormones, neurotransmitters, or digestive enzymes (Figure 13–62). In this section, we consider the role of the Golgi apparatus in both of these pathways and compare the two mechanisms of secretion. Many proteins and lipids are carried automatically from the Trans golgi network (Tgn) to the cell surface

1	A cell capable of regulated secretion must separate at least three classes of proteins before they leave the TGN—those destined for lysosomes (via endosomes), those destined for secretory vesicles, and those destined for immediate delivery to the cell surface (Figure 13–63). We have already discussed how proteins destined for lysosomes are tagged with M6P for packaging into specific departing vesicles, and analogous signals are thought to direct secretory proteins into secretory vesicles. The nonselective constitutive secretory pathway transports most other proteins directly to the cell surface. Because entry into this pathway does not require a particular signal, it is also called the default pathway. Thus, in an unpolarized cell such as a white blood cell or a fibroblast, it seems that any protein in the lumen of the Golgi apparatus is automatically carried by the constitutive pathway to the cell surface unless it is specifically returned to the ER, retained as a resident protein in

1	protein in the lumen of the Golgi apparatus is automatically carried by the constitutive pathway to the cell surface unless it is specifically returned to the ER, retained as a resident protein in the Golgi apparatus itself, or selected for the pathways that lead to regulated secretion or to lysosomes. In polarized cells, where different products have to be delivered to different domains of the cell surface, we shall see that the options are more complex.

1	secretory vesicles bud from the Trans golgi network Cells that are specialized for secreting some of their products rapidly on demand concentrate and store these products in secretory vesicles (often called dense-core secretory granules because they have dense cores when viewed in the electron microscope). Secretory vesicles form from the TGN, and they release their contents to the cell exterior by exocytosis in response to specific signals. The secreted product can be either a small molecule (such as histamine or a neuropeptide) or a protein (such as a hormone or digestive enzyme).

1	Proteins destined for secretory vesicles (called secretory proteins) are packaged into appropriate vesicles in the TGN by a mechanism that involves the selective aggregation of the secretory proteins. Clumps of aggregated, electron-dense material can be detected by electron microscopy in the lumen of the TGN. The signals that direct secretory proteins into such aggregates are not well defined and may be quite diverse. When a gene encoding a secretory protein is artificially expressed in a secretory cell that normally does not make the protein, the foreign protein is appropriately packaged into secretory vesicles. This observation

1	Figure 13–62 The constitutive and regulated secretory pathways. The two pathways diverge in the Tgn. The constitutive secretory pathway operates in all cells. Many soluble proteins are continually secreted from the cell by this pathway, which also supplies the plasma membrane with newly synthesized membrane lipids and proteins. specialized secretory cells also have a regulated secretory pathway, by which selected proteins in the Tgn are diverted into secretory vesicles, where the proteins are concentrated and stored until an extracellular signal stimulates their secretion. The regulated secretion of small molecules, such as histamine and neurotransmitters, occurs by a similar pathway; these molecules are actively transported from the cytosol into preformed secretory vesicles. There they are often bound to specific macromolecules (proteoglycans, for histamine) so that they can be stored at high concentration without generating an excessively high osmotic pressure.

1	Figure 13–63 The three best-understood pathways of protein sorting in the trans Golgi network. (1) proteins with the mannose 6-phosphate (M6p) marker (see figure 13–45) are diverted to lysosomes (via endosomes) in clathrin-coated transport vesicles. (2) proteins with signals directing them to secretory vesicles are concentrated in such vesicles as part of a regulated secretory pathway that is present only in specialized secretory cells. (3) In unpolarized cells, a constitutive secretory pathway delivers proteins with no special features to the cell surface. In polarized cells, such as epithelial cells, however, secreted and plasma membrane proteins are selectively directed to either the apical or the basolateral plasma membrane domain, so a specific signal must mediate at least one of these two pathways, as we discuss later.

1	shows that, although the proteins that an individual cell expresses and packages in secretory vesicles differ, they contain common sorting signals, which function properly even when the proteins are expressed in cells that do not normally make them. It is unclear how the aggregates of secretory proteins are segregated into secretory vesicles. Secretory vesicles have unique proteins in their membrane, some of which might serve as receptors for aggregated protein in the TGN. The aggregates are much too big, however, for each molecule of the secreted protein to be bound by its own cargo receptor, as occurs for transport of the lysosomal enzymes. The uptake of the aggregates into secretory vesicles may therefore more closely resemble the uptake of particles by phagocytosis at the cell surface, where the plasma membrane zippers up around large structures.

1	Initially, most of the membrane of the secretory vesicles that leave the TGN is only loosely wrapped around the clusters of aggregated secretory proteins. Morphologically, these immature secretory vesicles resemble dilated trans Golgi cisternae that have pinched off from the Golgi stack. As the vesicles mature, they fuse with one another and their contents become concentrated (Figure 13–64A), probably as the result of both the continuous retrieval of membrane that is recycled to the TGN, and the progressive acidification of the vesicle lumen that results from the increasing concentration of V-type ATPases in the vesicle membrane that acidify all endocytic and exocytic organelles (see Figure 13–37). The degree of concentration of proteins during the formation and maturation of secretory vesicles is only a small part of the total 200–400-fold concentration of these proteins that occurs after they leave the ER. Secretory and membrane proteins become concentrated as they move from the ER

1	is only a small part of the total 200–400-fold concentration of these proteins that occurs after they leave the ER. Secretory and membrane proteins become concentrated as they move from the ER through the Golgi apparatus because of an extensive retrograde retrieval process mediated by COPI-coated transport vesicles that carry soluble ER resident proteins back to the ER, while excluding the secretory and membrane proteins (see Figure 13–25).

1	Membrane recycling is important for returning Golgi components to the Golgi apparatus, as well as for concentrating the contents of secretory vesicles. The vesicles that mediate this retrieval originate as clathrin-coated buds on the surface of immature secretory vesicles, often being seen even on budding secretory vesicles that have not yet pinched off from the Golgi stack (Figure 13–64B). Because the final mature secretory vesicles are so densely filled with contents, the secretory cell can disgorge large amounts of material promptly by exocytosis when triggered to do so (Figure 13–65). precursors of secretory proteins are proteolytically processed during the formation of secretory vesicles Concentration is not the only process to which secretory proteins are subjected as the secretory vesicles mature. Many protein hormones and small neuropeptides,

1	Figure 13–64 The formation of secretory vesicles. (a) secretory proteins become segregated and highly concentrated in secretory vesicles by two mechanisms. first, they aggregate in the ionic environment of the Tgn; often the aggregates become more condensed as secretory vesicles mature and their lumen becomes more acidic. second, clathrincoated vesicles retrieve excess membrane and lumenal content present in immature secretory vesicles as the secretory vesicles mature. (b) This electron micrograph shows secretory vesicles forming from the Tgn in an insulin-secreting β cell of the pancreas. anti-clathrin antibodies conjugated to gold spheres (black dots) have been used to locate clathrin molecules. The immature secretory vesicles, which contain insulin precursor protein (proinsulin), contain clathrin patches, which are no longer seen on the mature secretory vesicle. (b, courtesy of lelio orci.) as well as many secreted hydrolytic enzymes, are synthesized as inactive precursors.

1	contain clathrin patches, which are no longer seen on the mature secretory vesicle. (b, courtesy of lelio orci.) as well as many secreted hydrolytic enzymes, are synthesized as inactive precursors. Proteolysis is necessary to liberate the active molecules from these precursor proteins. The cleavages occur in the secretory vesicles and sometimes in the extracellular fluid after secretion. Additionally, many of the precursor proteins have an N-terminal pro-peptide that is cleaved off to yield the mature protein. These proteins are synthesized as pre-pro-proteins, the pre-peptide consisting of the ER signal peptide that is cleaved off earlier in the rough ER (see Figure 12–36). In other cases, peptide signaling molecules are made as polyproteins that contain multiple copies of the same amino acid sequence. In still more complex cases, a variety of peptide signaling molecules are synthesized as parts of a single polyprotein that acts as a precursor for multiple end products, which are

1	acid sequence. In still more complex cases, a variety of peptide signaling molecules are synthesized as parts of a single polyprotein that acts as a precursor for multiple end products, which are individually cleaved from the initial polypeptide chain. The same polyprotein may be processed in various ways to produce different peptides in different cell types (Figure 13–66).

1	Why is proteolytic processing so common in the secretory pathway? Some of the peptides produced in this way, such as the enkephalins (five-amino-acid neuropeptides with morphine-like activity), are undoubtedly too short in their mature forms to be co-translationally transported into the ER lumen or to include the necessary signal for packaging into secretory vesicles. In addition, for secreted hydrolytic enzymes—or any other protein whose activity could be harmful inside the cell that makes it—delaying activation of the protein until it reaches a secretory vesicle, or until after it has been secreted, has a clear advantage: the delay prevents the protein from acting prematurely inside the cell in which it is synthesized. secretory vesicles wait near the plasma Membrane until signaled to release Their contents

1	secretory vesicles wait near the plasma Membrane until signaled to release Their contents Once loaded, a secretory vesicle has to reach the site of secretion, which in some cells is far away from the TGN. Nerve cells are the most extreme example. Secretory proteins, such as peptide neurotransmitters (neuropeptides), which will be released from nerve terminals at the end of the axon, are made and packaged into secretory vesicles in the cell body. They then travel along the axon to the nerve terminals, which can be a meter or more away. As discussed in Chapter 16, motor proteins propel the vesicles along axonal microtubules, whose uniform orientation guides the vesicles in the proper direction. Microtubules also guide transport vesicles to the cell surface for constitutive exocytosis.

1	Whereas transport vesicles containing materials for constitutive release fuse with the plasma membrane once they arrive there, secretory vesicles in the regulated pathway wait at the membrane until the cell receives a signal to secrete, and they then fuse. The signal can be an electrical nerve impluse (an action potential) or an extracellular signal molecule, such as a hormone: in either case, it leads to a transient increase in the concentration of free Ca2+ in the cytosol. for rapid exocytosis, synaptic vesicles are primed at the presynaptic plasma Membrane Nerve cells (and some endocrine cells) contain two types of secretory vesicles. As for all secretory cells, these cells package proteins and neuropeptides in dense-cored secretory vesicles in the standard way for release by the regulated secretory pathway. In addition, however, they use another specialized class of tiny (≈50 nm 0.2 µm

1	Figure 13–65 Exocytosis of secretory vesicles. The process is illustrated schematically (top) and in an electron micrograph that shows the release of insulin from a secretory vesicle of a pancreatic β cell. (courtesy of lelio orci, from l. orci, J.-d. vassalli and a. perrelet, sci. am. 259:85–94, 1988.)

1	Figure 13–66 Alternative processing pathways for the prohormone polyprotein proopiomelanocortin. The initial cleavages are made by proteases that cut next to pairs of positively charged amino acids (lys-arg, lys-lys, arg-lys, or arg-arg pairs). Trimming reactions then produce the final secreted products. different cell types produce different concentrations of individual processing enzymes, so that the same prohormone precursor is cleaved to produce different peptide hormones. In the anterior lobe of the pituitary gland, for example, only corticotropin (acTh) and β-lipotropin are produced from proopiomelanocortin, whereas in the intermediate lobe of the pituitary gland mainly α-melanocyte stimulating hormone (α-Msh), γ-lipotropin, β-Msh, and β-endorphin are produced— α-Msh from acTh and the other three from β-lipotropin, as shown. presynaptic (B) 1. DOCKING presynaptic 2. PRIMING I partially plasma membrane plasma membrane

1	presynaptic (B) 1. DOCKING presynaptic 2. PRIMING I partially plasma membrane plasma membrane SNARE bundle 3. PRIMING II4. FUSION PORE OPENING5. FUSION COMPLETE complexin released complexin-Ca2+

1	Figure 13–67 Exocytosis of synaptic vesicles. for orientation at a synapse, see figure 11–36. (a) The trans-snare complex responsible for docking synaptic vesicles at the plasma membrane of nerve terminals consists of three proteins. The v-snare synaptobrevin and the t-snare syntaxin are both transmembrane proteins, and each contributes one α helix to the complex. by contrast to other snares discussed earlier, the t-snare snaP25 is a peripheral membrane protein that contributes two α helices to the four-helix bundle; the two helices are connected by a loop (dashed line) that lies parallel to the membrane and has fatty acyl chains (not shown) attached to anchor it there. The four α helices are shown as rods for simplicity. (b) at the synapse, the basic snare machinery is modulated by the ca2+ sensor synaptotagmin and an additional protein called complexin. synaptic vesicles first dock at the membrane (step 1), and the snare bundle partially assembles (step 2), resulting in a “primed

1	ca2+ sensor synaptotagmin and an additional protein called complexin. synaptic vesicles first dock at the membrane (step 1), and the snare bundle partially assembles (step 2), resulting in a “primed vesicle” that is already drawn close to the membrane. The snare bundle assembles further but the additional binding of complexin prevents fusion (step 3). upon arrival of an action potential, ca2+ enters the cell and binds to synaptotagmin, which releases the block and opens a fusion pore (step 4). further rearrangements complete the fusion reaction (step 5) and release the fusion machinery, which now can be reused. This elaborate arrangement allows the fusion machinery to respond on the millisecond time scale essential for rapid and repetitive synaptic signaling. (a, adapted from r.b. sutton et al., nature 395:347–353, 1998. with permission from Macmillan publishers ltd.; b, adapted from Z.p. pang and T.c. südhof, Curr. Opin. Cell Biol. 22:496–505, 2010. with permission from elsevier.)

1	et al., nature 395:347–353, 1998. with permission from Macmillan publishers ltd.; b, adapted from Z.p. pang and T.c. südhof, Curr. Opin. Cell Biol. 22:496–505, 2010. with permission from elsevier.) diameter) secretory vesicles called synaptic vesicles. These vesicles store small neurotransmitter molecules, such as acetylcholine, glutamate, glycine, and γ-aminobutyric acid (GABA), which mediate rapid signaling from nerve cell to its target cell at chemical synapses. When an action potential arrives at a nerve terminal, it causes an influx of Ca2+ through voltage-gated Ca2+ channels, which triggers the synaptic vesicles to fuse with the plasma membrane and release their contents to the extracellular space (see Figure 11–36). Some neurons fire more than 1000 times per second, releasing neurotransmitters each time.

1	The speed of transmitter release (taking only milliseconds) indicates that the proteins mediating the fusion reaction do not undergo complex, multistep rearrangements. Rather, after vesicles have been docked at the presynaptic plasma membrane, they undergo a priming step, which prepares them for rapid fusion. In the primed state, the SNAREs are partly paired, their helices are not fully wound into the final four-helix bundle required for fusion (Figure 13–67). Proteins called complexins freeze the SNARE complexes in this metastable state. The brake imposed by the complexins is released by another synaptic vesicle protein, synaptotagmin, which contains Ca2+-binding domains. A rise in cytosolic Ca2+ triggers binding of synaptotagmin to phospholipids and to the SNAREs, displacing the complexins. As the SNARE bundle zippers up completely, a fusion pore opens and the neurotransmitters are released. At a typical synapse, only a small number of the docked vesicles are primed and ready for

1	As the SNARE bundle zippers up completely, a fusion pore opens and the neurotransmitters are released. At a typical synapse, only a small number of the docked vesicles are primed and ready for exocytosis. The use of only a small number of vesicles at a time allows each synapse to fire over and over again in quick succession. With each firing, new synaptic vesicles dock and become primed to replace those that have fused and released their contents.

1	For the nerve terminal to respond rapidly and repeatedly, synaptic vesicles need to be replenished very quickly after they discharge. Thus, most synaptic vesicles are generated not from the Golgi membrane in the nerve cell body but by local recycling from the presynaptic plasma membrane in the nerve terminals (Figure 13–68). Similarly, newly made membrane components of the synaptic vesicles are initially delivered to the plasma membrane by the constitutive secretory pathway and then retrieved by endocytosis. But instead of fusing with endosomes, most of the endocytic vesicles immediately fill with neurotransmitter to become synaptic vesicles.

1	The membrane components of a synaptic vesicle include transporters specialized for the uptake of neurotransmitter from the cytosol, where the small-molecule neurotransmitters that mediate fast synaptic signaling are synthesized. Once filled with neurotransmitter, the synaptic vesicles can be used again (see Figure 13–68). Because synaptic vesicles are abundant and relatively uniform in size, they can be purified in large numbers and, consequently, are the best-characterized organelle of the cell, in that all of their membrane components have been identified by quantitative proteomic analyses (Figure 13–69). Extending this analysis to a complete presynaptic terminal, allows us to model the crowded environment in which these reactions occur. secretory vesicle Membrane components are Quickly removed from the plasma Membrane

1	secretory vesicle Membrane components are Quickly removed from the plasma Membrane When a secretory vesicle fuses with the plasma membrane, its contents are discharged from the cell by exocytosis, and its membrane becomes part of the plasma membrane. Although this should greatly increase the surface area of the plasma membrane, it does so only transiently, because membrane components are removed from the surface by endocytosis almost as fast as they are added by exocytosis, a process reminiscent of the endocytic–exocytic cycle discussed earlier. After their removal from the plasma membrane, the proteins of the secretory

1	Figure 13–68 The formation of synaptic vesicles in a nerve cell. These tiny uniform vesicles are found only in nerve cells and in some endocrine cells, where they store and secrete small-molecule neurotransmitters. The import of neurotransmitter directly into the small endocytic vesicles that form from the plasma membrane is mediated by membrane transporters that function as antiports and are driven by an h+ gradient maintained by v-aTpase h+ pumps in the vesicle membrane (discussed in chapter 11).

1	Figure 13–69 Scale models of a brain presynaptic terminal and a synaptic vesicle. The illustrations show sections through a pre-synaptic terminal (a; enlarged in b) and a synaptic vesicle (c) in which proteins and lipids are drawn to scale based on their known stoichiometry and either known or approximated structures. The relative localization of protein molecules in different regions of the presynaptic terminal was inferred from super-resolution imaging and electron microscopy. The model in (a) contains 300,000 proteins of 60 different kinds that vary in abundance from 150 copies to 20,000 copies. In the model in (c), only 70% of the membrane proteins present in the membrane are shown; a complete model would therefore show a membrane that is even more crowded than this picture suggests (Movie 13.7). each synaptic vesicle membrane contains 7000 phospholipid molecules and 5700 cholesterol molecules. each also contains close to 50 different integral membrane protein molecules, which

1	(Movie 13.7). each synaptic vesicle membrane contains 7000 phospholipid molecules and 5700 cholesterol molecules. each also contains close to 50 different integral membrane protein molecules, which vary widely in their relative abundance and together contribute about 600 transmembrane α helices. The transmembrane v-snare synaptobrevin is the most abundant protein in the vesicle (~70 copies per vesicle). by contrast, the v-aTpase, which uses aTp hydrolysis to pump h+ into the vesicle lumen, is present in 1–2 copies per vesicle. The h+ gradient provides the energy for neurotransmitter import by an h+/neurotransmitter antiport, which loads each vesicle with 1800 neurotransmitter molecules, such as glutamate, one of which is shown to scale. (a and b, from b.g. wilhelm et al., science 344:1023–1028, 2014. with permission from aaas; c, adapted from s. Takamori et al., Cell 127:831–846, 2006. with permission from elsevier.) vesicle membrane are either recycled or shuttled to lysosomes for

1	2014. with permission from aaas; c, adapted from s. Takamori et al., Cell 127:831–846, 2006. with permission from elsevier.) vesicle membrane are either recycled or shuttled to lysosomes for degradation. The amount of secretory vesicle membrane that is temporarily added to the plasma membrane can be enormous: in a pancreatic acinar cell discharging digestive enzymes for delivery to the gut lumen, about 900 μm2 of vesicle membrane is inserted into the apical plasma membrane (whose area is only 30 μm2) when the cell is stimulated to secrete.

1	Control of membrane traffic thus has a major role in maintaining the composition of the various membranes of the cell. To maintain each membrane-enclosed compartment in the secretory and endocytic pathways at a constant size, the balance between the outward and inward flows of membrane needs to be precisely regulated. For cells to grow, however, the forward flow needs to be greater than the retrograde flow, so that the membrane can increase in area. For cells to maintain a constant size, the forward and retrograde flows must be equal. We still know very little about the mechanisms that coordinate these flows. some regulated exocytosis events serve to enlarge the plasma Membrane

1	some regulated exocytosis events serve to enlarge the plasma Membrane An important task of regulated exocytosis is to deliver more membrane to enlarge the surface area of a cell’s plasma membrane when such a need arises. A spectacular example is the plasma membrane expansion that occurs during the cellularization process in a fly embryo, which initially is a syncytium—a single cell containing about 6000 nuclei surrounded by a single plasma membrane (see Figure 21–15). Within tens of minutes, the embryo is converted into the same number of cells. This process of cellularization requires a vast amount of new plasma membrane, which is added by a carefully orchestrated fusion of cytoplasmic vesicles, eventually forming the plasma membranes that enclose the separate cells. Similar vesicle fusion events are required to enlarge the plasma membrane when other animal cells or plant cells divide during cytokinesis (discussed in Chapter 17).

1	Many animal cells, especially those subjected to mechanical stresses, frequently experience small ruptures in their plasma membrane. In a remarkable process thought to involve both homotypic vesicle–vesicle fusion and exocytosis, a temporary cell-surface patch is quickly fashioned from locally available internal-membrane sources, such as lysosomes. In addition to providing an emergency barrier against leaks, the patch reduces membrane tension over the wounded area, allowing the bilayer to flow back together to restore continuity and seal the puncture. This membrane repair process, the fusion and exocytosis of vesicles is triggered by the sudden increase of Ca2+, which is abundant in the extracellular space and rushes into the cell as soon as the plasma membrane is punctured. Figure 13–70 shows four examples in which regulated exocytosis leads to plasma membrane expansion. polarized cells direct proteins from the Trans golgi network to the appropriate domain of the plasma Membrane

1	polarized cells direct proteins from the Trans golgi network to the appropriate domain of the plasma Membrane Most cells in tissues are polarized, with two or more molecularly and functionally distinct plasma membrane domains. This raises the general problem of how the Figure 13–70 Four examples of regulated exocytosis leading to plasma membrane enlargement. The vesicles fusing with the plasma membrane during cytokinesis (a) and phagocytosis (b) are thought to be derived from endosomes, whereas those involved in wound repair (c) may be derived from plasma membranes. The vast amount of new plasma membrane inserted during cellularization in a fly embryo occurs by the fusion of cytoplasmic vesicles (d).

1	delivery of membrane from the Golgi apparatus is organized so as to maintain the differences between one cell-surface domain and another. A typical epithelial cell, for example, has an apical domain, which faces either an internal cavity or the outside world and often has specialized features such as cilia or a brush border of microvilli. It also has a basolateral domain, which covers the rest of the cell. The two domains are separated by a ring of tight junctions (see Figure 19–21), which prevent proteins and lipids (in the outer leaflet of the lipid bilayer) from diffusing between the two domains, so that the differences between the two domains are maintained.

1	In principle, differences between plasma membrane domains need not depend on the targeted delivery of the appropriate membrane components. Instead, membrane components could be delivered to all regions of the cell surface indiscriminately but then be selectively stabilized in some locations and selectively eliminated in others. Although this strategy of random delivery followed by selective retention or removal seems to be used in certain cases, deliveries are often specifically directed to the appropriate membrane domain. Epithelial cells lining the gut, for example, secrete digestive enzymes and mucus at their apical surface and components of the basal lamina at their basolateral surface. Such cells must have ways of directing vesicles carrying different cargoes to different plasma membrane domains. Proteins from the ER destined for different domains travel together until they reach the TGN, where they are separated and dispatched in secretory or transport vesicles to the

1	membrane domains. Proteins from the ER destined for different domains travel together until they reach the TGN, where they are separated and dispatched in secretory or transport vesicles to the appropriate plasma membrane domain (Figure 13–71).

1	The apical plasma membrane of most epithelial cells is greatly enriched in glycosphingolipids, which help protect this exposed surface from damage—for example, from the digestive enzymes and low pH in sites such as the gut or stomach, respectively. Similarly, plasma membrane proteins that are linked to the lipid bilayer by a GPI anchor (see Figure 12–52) are found predominantly in the apical plasma membrane. If recombinant DNA techniques are used to attach a GPI anchor to a protein that would normally be delivered to the basolateral surface, the protein is usually delivered to the apical surface instead. GPI-anchored proteins are thought to be directed to the apical membrane because they associate with glycosphingolipids in lipid rafts that form in the membrane of the TGN. As discussed in Chapter 10, lipid rafts form in the TGN and plasma membrane when glycosphingolipids and cholesterol molecules self-associate (see Figure 10–13).

1	Figure 13–71 Two ways of sorting plasma membrane proteins in a polarized epithelial cell. (a) In the direct pathway, proteins destined for different plasma membrane domains are sorted and packaged into different transport vesicles. The lipid-raft-dependent delivery system to the apical domain described in the text is an example of the direct pathway. (b) In the indirect pathway, a protein is retrieved from the inappropriate plasma membrane domain by endocytosis and then transported to the correct domain via early endosomes—that is, by transcytosis. The indirect pathway, for example, is used in liver hepatocytes to deliver proteins to the apical domain that lines bile ducts.

1	Having selected a unique set of cargo molecules, the rafts then bud from the TGN whaT we don’T know into transport vesicles destined for the apical plasma membrane. This process is similar to the selective partitioning of some membrane proteins into the special-• how are targeting and fusion ized lipid domains in caveolae at the plasma membrane discussed earlier. proteins such as snares regulated, Membrane proteins destined for delivery to the basolateral membrane con-so that they can be returned to their tain sorting signals in their cytosolic tail. When present in an appropriate struc-respective donor compartments in an inactive state?

1	tural context, these signals are recognized by coat proteins that package them into appropriate transport vesicles in the TGN. The same basolateral signals that are recognized in the TGN also function in early endosomes to redirect the proteins and endocytic events to keep its back to the basolateral plasma membrane after they have been endocytosed. plasma membrane a constant size? summary • can newly formed daughter cells generate a golgi apparatus de novo, or do they have to inherit it? ion. Whereas the regulated pathways operate only in specialized secretory cells, a constitutive secretory pathway operates in all eukaryotic cells, characterized by continual vesicle transport from the TGN to the plasma membrane. In the regulated their own membranes?

1	pathways, the molecules are stored either in secretory vesicles or in synaptic vesicles, which do not fuse with the plasma membrane to release their contents until they • how does a cell maintain the receive an appropriate signal. Secretory vesicles containing proteins for secretion right amount of every component bud from the TGN. The secretory proteins become concentrated during the forma-(organelles, molecules), and how does tion and maturation of the secretory vesicles. Synaptic vesicles, which are confined it change these amounts as needed to nerve cells and some endocrine cells, form from both endocytic vesicles and from (for example, to greatly expand the endoplasmic reticulum when the cell endosomes, and they mediate the regulated secretion of small-molecule neurotrans needs to produce large amounts of mitters at the axon terminals of nerve cells. secreted proteins)?

1	secreted proteins)? Proteins are delivered from the TGN to the plasma membrane by the constitutive pathway unless they are diverted into other pathways or retained in the Golgi apparatus. In polarized cells, the transport pathways from the TGN to the plasma membrane operate selectively to ensure that different sets of membrane proteins, secreted proteins, and lipids are delivered to the different domains of the plasma membrane. Which statements are true? Explain why or why not. 13–1 In all events involving fusion of a vesicle to a target membrane, the cytosolic leaflets of the vesicle and target bilayers always fuse together, as do the leaflets that are not in contact with the cytosol. 13–2 There is one strict requirement for the exit of a protein from the ER: it must be correctly folded.

1	13–2 There is one strict requirement for the exit of a protein from the ER: it must be correctly folded. 13–3 All the glycoproteins and glycolipids in intracellular membranes have oligosaccharide chains facing the lumenal side, and all those in the plasma membrane have oligosaccharide chains facing the outside of the cell. Discuss the following problems. 13–4 In a nondividing cell such as a liver cell, why must the flow of membrane between compartments be balanced, so that the retrieval pathways match the outward flow? Would you expect the same balanced flow in a gut epithelial cell, which is actively dividing? 13–5 Enveloped viruses, which have a membrane coat, gain access to the cytosol by fusing with a cell membrane. Why do you suppose that these viruses encode their own special fusion protein, rather than making use of a cell's SNAREs?

1	13–6 For fusion of a vesicle with its target membrane to occur, the membranes have to be brought to within 1.5 nm so that the two bilayers can join (Figure Q13–1). Assuming that the relevant portions of the two membranes at the fusion site are circular regions 1.5 nm in diameter, calculate the number of water molecules that would remain between the membranes. (Water is 55.5 M and the volume of a cylinder is πr2h.) Given that an average phospholipid 1.5 nm Figure Q13–1 Close approach of a vesicle and its target membrane in preparation for fusion (problem 13–6).

1	occupies a membrane surface area of 0.2 nm2, how many (A) strain A strain B phospholipids would be present in each of the opposing monolayers at the fusion site? Are there sufficient water molecules to bind to the hydrophilic head groups of this number of phospholipids? (It is estimated that 10–12 water molecules are normally associated with each phospholipid head group at the exposed surface of a membrane.) 13–7 SNAREs exist as complementary partners that carry out membrane fusions between appropriate ves icles and their target membranes. In this way, a vesicle with a particular variety of v-SNARE will fuse only with a membrane that carries the complementary t-SNARE. In some instances, however, fusions of identical membranes (homotypic fusions) are known to occur. For example, when a yeast cell forms a bud, vesicles derived from the mother cell’s vacuole move into the bud where they fuse with one another to form a new vacuole. These vesicles carry both v-SNAREs and t-SNAREs. Are both

1	cell forms a bud, vesicles derived from the mother cell’s vacuole move into the bud where they fuse with one another to form a new vacuole. These vesicles carry both v-SNAREs and t-SNAREs. Are both types of

1	SNAREs essential for this homotypic fusion event? To test this point, you have developed an ingenious assay for fusion of vacuolar vesicles. You prepare vesicles from two different mutant strains of yeast: strain B has a defective gene for vacuolar alkaline phosphatase (Pase); strain A is defective for the protease that converts the pre cursor of alkaline phosphatase (pro-Pase) into its active form (Pase) (Figure Q13–2A). Neither strain has active alkaline phosphatase, but when extracts of the strains are mixed, vesicle fusion generates active alkaline phosphatase, which can be easily measured (Figure Q13–2). Now you delete the genes for the vacuolar v-SNARE, t-SNARE, or both in each of the two yeast strains. You prepare vacuolar vesicles from each and test them for their ability to fuse, as measured by the alkaline phosphatase assay (Figure Q13–2B).

1	What do these data say about the requirements for v-SNAREs and t-SNAREs in the fusion of vacuolar vesicles? Does it matter which kind of SNARE is on which vesicle? 13–8 If you were to remove the ER retrieval signal from protein disulfide isomerase (PDI), which is normally a soluble resident of the ER lumen, where would you expect the modified PDI to be located?

1	13–9 The KDEL receptor must shuttle back and forth between the ER and the Golgi apparatus to accomplish its task of ensuring that soluble ER proteins are retained in the ER lumen. In which compartment does the KDEL receptor bind its ligands more tightly? In which compartment does it bind its ligands more weakly? What is thought to be the basis for its different binding affinities in the two compartments? If you were designing the system, in which compartment would you have the highest concentration of KDEL receptor? Would you predict that the KDEL receptor, which is a transmembrane protein, would itself possess an ER retrieval signal? 13–10 How does the low pH of lysosomes protect the rest of the cell from lysosomal enzymes in case the lysosome breaks? Figure Q13–2 SNARE requirements for vesicle fusion (problem 13–7).

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1	13–11 Melanosomes are specialized lysosomes that store pigments for eventual release by exocytosis. Various cells such as skin and hair cells then take up the pigment, which accounts for their characteristic pigmentation. Mouse mutants that have defective melanosomes often have pale or unusual coat colors. One such light-colored mouse, the Mocha mouse (Figure Q13–3), has a defect in the gene for one of the subunits of the adaptor protein complex AP3, which is associated with coated vesicles budding from the trans Golgi network. How might the loss of AP3 cause a defect in melanosomes? Figure Q13–3 A normal mouse and the Mocha mouse (problem 13–11). In addition to its light coat color, the Mocha mouse has a poor sense of balance. (Courtesy of Margit Burmeister.) harrison sc & kirchhausen T (2010) structural biology: conservation in vesicle coats. nature 466, 1048–1049. pfeffer sr (2013) a prize for membrane magic. Cell 155, 1203–1206.

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1	To maintain their high degree of organization in a universe that is constantly drifting toward chaos, cells have a constant need for a plentiful supply of ATP, as we have explained in Chapter 2. In eukaryotic cells, most of the ATP that powers life processes is produced by specialized, membrane-enclosed, energy-converting organelles. These are of two types. Mitochondria, which occur in virtually all cells of animals, plants, and fungi, burn food molecules to produce ATP by oxidative phosphorylation. Chloroplasts, which occur only in plants and green algae, harness solar energy to produce ATP by photosynthesis. In electron micrographs, the most striking features of both mitochondria and chloroplasts are their extensive internal membrane systems. These internal membranes contain sets of membrane protein complexes that work together to produce most of the cell’s ATP. In bacteria, simpler versions of essentially the same protein complexes produce ATP, but they are located in the cell’s

1	protein complexes that work together to produce most of the cell’s ATP. In bacteria, simpler versions of essentially the same protein complexes produce ATP, but they are located in the cell’s plasma membrane (Figure 14–1).

1	Comparisons of DNA sequences indicate that the energy-converting organelles in present-day eukaryotes originated from prokaryotic cells that were endocytosed during the evolution of eukaryotes (discussed in Chapter 1). This explains why mitochondria and chloroplasts contain their own DNA, which still encodes a subset of their proteins. Over time, these organelles have lost most of their own genomes and become heavily dependent on proteins that are encoded by genes in the nucleus, synthesized in the cytosol, and then imported into the organelle. And the eukaryotic cells now rely on these organelles not only for the ATP they need for biosynthesis, solute transport, and movement, but also for many important biosynthetic reactions that occur inside each organelle.

1	The common evolutionary origin of the energy-converting machinery in mitochondria, chloroplasts, and prokaryotes (archaea and bacteria) is reflected in the fundamental mechanism that they share for harnessing energy. This is known as chemiosmotic coupling, signifying a link between the chemical bond-forming reactions that generate ATP (“chemi”) and membrane transport processes

1	Figure 14–1 The membrane systems of bacteria, mitochondria, and chloroplasts are related. Mitochondria and chloroplasts are cell organelles that have originated from bacteria and have retained the bacterial energy-conversion mechanisms. Like their bacterial ancestors, mitochondria and chloroplasts have an outer and an inner membrane. Each of the membranes colored in this diagram contains energy-harvesting electron-transport chains. the deep invaginations of the mitochondrial inner membrane and the internal membrane system of the chloroplast harbor the machinery for cellular respiration and photosynthesis, respectively.

1	(“osmotic”). The chemiosmotic process occurs in two linked stages, both of which are performed by protein complexes in a membrane. Stage 1: High-energy electrons (derived from the oxidation of food molecules, from pigments excited by sunlight, or from other sources described later) are transferred along a series of electron-transport protein complexes that form an electron-transport chain embedded in a membrane. Each electron transfer releases a small amount of energy that is used to pump protons (H+) and thereby generate a large electrochemical gradient across the membrane (Figure 14–2). As discussed in Chapter 11, such an electrochemical gradient provides a way of storing energy, and it can be harnessed to do useful work when ions flow back across the membrane.

1	Stage 2: The protons flow back down their electrochemical gradient through an elaborate membrane protein machine called ATP synthase, which catalyzes the production of ATP from ADP and inorganic phosphate (Pi). This ubiquitous enzyme works like a turbine in the membrane, driven by protons, to synthesize ATP (Figure 14–3). In this way, the energy derived from food or sunlight in stage 1 is converted into the chemical energy of a phosphate bond in ATP.

1	Electrons move through protein complexes in biological systems via tightly bound metal ions or other carriers that take up and release electrons easily, or by special small molecules that pick electrons up at one location and deliver them to another. For mitochondria, the first of these electron carriers is NAD+, a water-soluble small molecule that takes up two electrons and one H+ derived from food molecules (fats and carbohydrates) to become NADH. NADH transfers these electrons from the sites where the food molecules are degraded to the inner mitochondrial membrane. There, the electrons from the energy-rich NADH are passed from one membrane protein complex to the next, passing to a lower-energy compound at each step, until they reach a final complex in which they combine with molecular oxygen (O2) to produce water. The energy released at each step as the electrons flow down this path from the energy-rich NADH to the low-energy water molecule drives H+ pumps in the inner

1	molecular oxygen (O2) to produce water. The energy released at each step as the electrons flow down this path from the energy-rich NADH to the low-energy water molecule drives H+ pumps in the inner mitochondrial membrane, utilizing three different membrane protein complexes. Together, these complexes generate the proton-motive force harnessed by ATP synthase to produce the ATP that serves as the universal energy currency throughout the cell (see Chapter 2).

1	Figure 14–4 compares the electron-transport processes in mitochondria, which harness energy from food molecules, with those in chloroplasts, which harness energy from sunlight. The energy-conversion systems of mitochondria and chloroplasts can be described in similar terms, and we shall see later in the chapter that two of their key components are closely related. One of these is the ATP synthase, and the other is a proton pump (colored green in Figure 14–4). Among the crucial constituents that are unique to photosynthetic organisms are the two photosystems. These use the green pigment chlorophyll to capture Figure 14–2 Stage 1 of chemiosmotic coupling. Energy from sunlight or the oxidation of food compounds is captured to generate an electrochemical proton gradient across a membrane. the electrochemical gradient serves as a versatile energy store that drives energy-requiring reactions in mitochondria, chloroplasts, and bacteria.

1	Figure 14–3 Stage 2 of chemiosmotic coupling. an atp synthase (yellow) embedded in the lipid bilayer of a membrane harnesses the electrochemical proton gradient across the membrane, using it as a local energy store to drive atp synthesis. the red arrows show the direction of proton movement through the atp synthase.

1	Figure 14–4 Electron-transport processes. (a) the mitochondrion converts energy from chemical fuels. (B) the chloroplast converts energy from sunlight. In both cases, electron flow is indicated by blue arrows. Each of the protein complexes (green) is embedded in a membrane. In the mitochondrion, fats and carbohydrates from food molecules are fed into the citric acid cycle and provide electrons to generate the energy-rich compound NaDh from NaD+. these electrons then flow down an energy gradient as they pass from one complex to the next in the electron-transport chain, until they combine with molecular O2 in the last complex to produce water. the energy released at each stage is harnessed to pump h+ across the membrane. In the chloroplast, by contrast, electrons are extracted from water through the action of light in the photosystem II complex and molecular O2 is released. the electrons pass on to the next complex in the chain, which uses some of their energy to pump protons across the

1	the action of light in the photosystem II complex and molecular O2 is released. the electrons pass on to the next complex in the chain, which uses some of their energy to pump protons across the membrane, before passing to photosystem I, where sunlight generates high-energy electrons that combine with NaDp+ to produce NaDph. NaDph then enters the carbon-fixation cycle along with CO2 to generate carbohydrates.

1	light energy and power the transfer of electrons, not unlike a photocell in a solar panel. The chloroplasts drive electron transfer in the direction opposite to that in mitochondria: electrons are taken from water to produce O2, and these electrons are used (via NADPH, a molecule closely related to the NADH used in mitochondria) to synthesize carbohydrates from CO2 and water. These carbohydrates then serve as the source for all other compounds a plant cell needs. Thus, both mitochondria and chloroplasts make use of an electron-transfer chain to produce an H+ gradient that powers reactions that are critical for the cell. However, chloroplasts generate O2 and take up CO2, whereas mitochondria consume O2 and release CO2 (see Figure 14–4).

1	Mitochondria occupy up to 20% of the cytoplasmic volume of a eukaryotic cell. Although they are often depicted as short, bacterium-like bodies with a diameter of 0.5–1 μm, they are in fact remarkably dynamic and plastic, moving about the cell, constantly changing shape, dividing, and fusing (Movie 14.1). Mitochondria are often associated with the microtubular cytoskeleton (Figure 14–5), which determines their orientation and distribution in different cell types. Thus, in highly polarized cells such as neurons, mitochondria can move long distances (up to a meter or more in the extended axons of neurons), being propelled along the tracks of the microtubular cytoskeleton. In other cells, mitochondria remain fixed at points of high energy demand; for example, in skeletal or cardiac muscle cells, they pack between myofibrils, and in sperm cells they wrap tightly around the flagellum (Figure 14–6).

1	Mitochondria also interact with other membrane systems in the cell, most notably the endoplasmic reticulum (ER). Contacts between mitochondria and ER define specialized domains thought to facilitate the exchange of lipids between the two membrane systems. These contacts also appear to induce mitochondrial fission, which, as we discuss later, is involved in the distribution and partitioning of mitochondria within cells (Figure 14–7).

1	The acquisition of mitochondria was a prerequisite for the evolution of complex animals. Without mitochondria, present-day animal cells would have had to generate all of their ATP through anaerobic glycolysis. When glycolysis converts glucose to pyruvate, it releases only a small fraction of the total free energy that is potentially available from glucose oxidation (see Chapter 2). In mitochondria, the metabolism of sugars is complete: pyruvate is imported into the mitochondrion and ultimately oxidized by O2 to CO2 and H2O, which allows 15 times more ATP to be made from a sugar than by glycolysis alone. As explained later, this became possible only when enough molecular oxygen accumulated in the Earth’s atmosphere to allow organisms to take full advantage, via respiration, of the large amounts of energy potentially available from the oxidation of organic compounds.

1	Mitochondria are large enough to be seen in the light microscope, and they were first identified in the nineteenth century. Real progress in understanding their internal structure and function, however, depended on biochemical procedures developed in 1948 for isolating intact mitochondria, and on electron microscopy, which was first used to look at cells at about the same time. Figure 14–5 The relationship between mitochondria and microtubules. (a) a light micrograph of chains of elongated mitochondria in a living mammalian cell in culture. the cell was stained with a fluorescent dye (rhodamine 123) that specifically labels mitochondria in living cells. (B) an immunofluorescence micrograph of the same cell stained (after fixation) with fluorescent antibodies that bind to microtubules. Note that the mitochondria tend to be aligned along microtubules. (Courtesy of Lan Bo Chen.)

1	Figure 14–6 Localization of mitochondria near sites of high ATP demand in cardiac muscle and a sperm tail. (a) Cardiac muscle in the wall of the heart is the most heavily used muscle in the body, and its continual contractions require a reliable energy supply. It has limited built-in energy stores and has to depend on a steady supply of atp from the copious mitochondria aligned close to the contractile myofibrils (see Figure 16–32). (B) During sperm development, microtubules wind helically around the flagellar axoneme, where they are thought to help localize the mitochondria in the tail to produce the structure shown.

1	Figure 14–7 Interaction of mitochondria with the endoplasmic reticulum. 0 sec 20 sec 30 sec (a) Fluorescence light microscopy shows that tubules of the Er (green) wrap around parts of the mitochondrial network (red) in mammalian cells. the mitochondria then divide at the contact sites. after contact is established, fission occurs within less than a minute, as indicated by time-lapse microscopy. (B) Schematic drawing of an Er tubule wrapped around part of the mitochondrial reticulum. It is thought that Er–mitochondrial contacts also mediate the exchange of lipids between the two membrane systems. (a, adapted from J.r. Friedman et al., Science 334:358–362, 2011.) the Mitochondrion has an Outer Membrane and an Inner Membrane

1	Like the bacteria from which they originated, mitochondria have an outer and an inner membrane. The two membranes have distinct functions and properties, and delineate separate compartments within the organelle. The inner membrane, which surrounds the internal mitochondrial matrix compartment (Figure 14–8), is highly folded to form invaginations known as cristae (the singular is crista), which contain in their membranes the proteins of the electron-transport chain. Where the inner membrane runs parallel to the outer membrane, between the cristae, it is known as the inner boundary membrane. The narrow (20–30 nm) gap between the inner boundary membrane and the outer membrane is known as the intermembrane space. The cristae are about 20 nm-wide membrane discs or tubules that protrude deeply into the matrix and enclose the crista space. The crista membrane is continuous with the inner boundary membrane, and where their membranes join, the membrane forms narrow membrane tubes or slits,

1	into the matrix and enclose the crista space. The crista membrane is continuous with the inner boundary membrane, and where their membranes join, the membrane forms narrow membrane tubes or slits, known as crista junctions.

1	Like the bacterial outer membrane, the outer mitochondrial membrane is freely permeable to ions and to small molecules as large as 5000 daltons. This

1	Figure 14–8 Structure of a mitochondrion. (a) tomographic slice through a three-dimensional map of a mouse heart mitochondrion determined by electron-microscope tomography. the outer membrane envelops the inner boundary membrane. the inner membrane is highly folded into tubular or lamellar cristae, which crisscross the matrix. the dense matrix, which contains most of the mitochondrial protein, appears dark in the electron microscope, whereas the intermembrane space and the crista space appear light due to their lower protein content. the inner boundary membrane follows the outer membrane closely at a distance of ≈20 nm. the inner membrane turns sharply at the cristae junctions, where the cristae join the inner boundary membrane. (B) tomographic surface-rendered portion of a yeast mitochondrion, showing how flattened cristae project into the matrix from the inner membrane (Movie 14.2). (C) Schematic drawing of a mitochondrion showing the outer membrane (gray), and the inner membrane

1	showing how flattened cristae project into the matrix from the inner membrane (Movie 14.2). (C) Schematic drawing of a mitochondrion showing the outer membrane (gray), and the inner membrane (yellow). Note that the inner membrane is compartmentalized into the inner boundary membrane and the crista membrane. there are three distinct spaces: the inner membrane space, the crista space, and the matrix. (a, courtesy of tobias Brandt; B, from K. Davies et al., Proc. Natl Acad. Sci. USA 109:13602–13607, 2012. With permission from the National academy of Sciences.)

1	Figure 14–9 Biochemical fractionation of purified mitochondria into separate components. Large numbers of mitochondria are isolated from homogenized tissue by centrifugation and then suspended in a medium of low osmotic strength. In such a medium, water flows into mitochondria and greatly expands the matrix space (yellow). While the cristae of the inner membrane unfold to accommodate the swelling, the outer membrane— which has no folds—breaks, releasing structures composed of the inner membrane surrounding the matrix. these techniques have made it possible to study the protein composition of the inner membrane (comprising a mixture of cristae, boundary membranes, and cristae junctions), the outer membrane, and the matrix.

1	is because it contains many porin molecules, a special class of β-barrel-type membrane protein that creates aqueous pores across the membrane (see Figure 10–23). As a consequence, the intermembrane space between the outer and inner membrane has the same pH and ionic composition as the cytoplasm, and there is no electrochemical gradient across the outer membrane. If purified mitochondria are gently disrupted and then fractionated (Figure 14–9), the biochemical composition of membranes and mitochondrial compartments can be determined. the Inner Membrane Cristae Contain the Machinery for Electron transport and atp Synthesis Unlike the outer mitochondrial membrane, the inner mitochondrial membrane is a diffusion barrier to ions and small molecules, just like the bacterial inner membrane. However, selected ions, most notably protons and phosphate, as well as essential metabolites such as ATP and ADP, can pass through it by means of special transport proteins.

1	The inner mitochondrial membrane is highly differentiated into functionally distinct regions with different protein compositions. As discussed in Chapter 10, the lateral segregation of membrane regions with different protein and lipid compositions is a key feature of cells. In the inner mitochondrial membrane, the boundary membrane region is thought to contain the machinery for protein import, new membrane insertion, and assembly of the respiratory-chain complexes. The membranes of the cristae, which are continuous with the boundary membrane, contain the ATP synthase enzyme that produces most of the cell’s ATP; they also contain the large protein complexes of the respiratory chain—the name given to the mitochondrion’s electron-transport chain.

1	At the cristae junctions, where the membranes of the cristae join the boundary membrane, special protein complexes provide a diffusion barrier that segregates the membrane proteins in the two regions of the inner membrane; these complexes are also thought to anchor the cristae to the outer membrane, thus maintaining the highly folded topology of the inner membrane. Cristae membranes have one of the highest protein densities of all biological membranes, with a lipid content of 25% and a protein content of 75% by weight. The folding of the inner membrane into cristae greatly increases the membrane area available for oxidative phosphorylation. In highly active cardiac muscle cells, for example, the total area of cristae membranes can be up to 20 times larger than the area of the cell’s plasma membrane. In total, the surface area of cristae membranes in each human body adds up to roughly the size of a football field. the Citric acid Cycle in the Matrix produces NaDh

1	the Citric acid Cycle in the Matrix produces NaDh Together with the cristae that project into it, the matrix is the major working part of the mitochondrion. Mitochondria can use both pyruvate and fatty acids as fuel. Pyruvate is derived from glucose and other sugars, whereas fatty acids are derived from fats. Both of these fuel molecules are transported across the inner mitochondrial membrane by specialized transport proteins, and they are then converted to the crucial metabolic intermediate acetyl CoA by enzymes located in the mitochondrial matrix (see Chapter 2). in medium of low osmolarity the infux of water causes the mitochondrion to swell and the outer membrane to rupture, releasing the contents of the intermembrane space; the inner membrane remains intact FOOD MOLECULES FROM CYTOSOL

1	The acetyl groups in acetyl CoA are oxidized in the matrix via the citric acid cycle, also called the Krebs cycle (see Figure 2–57 and Movie 2.6). The oxidation of these carbon atoms in acetyl CoA produces CO2, which diffuses out of the mitochondrion to be released to the environment as a waste product. More importantly, the citric acid cycle saves a great deal of the bond energy released by this oxidation in the form of electrons carried by NADH. This NADH transfers its electrons from the matrix to the electron-transport chain in the inner mitochondrial membrane, where—through the chemiosmotic coupling process described previously (see Figures 14–2 and 14–3)—the energy that was carried by NADH electrons is converted into phosphate-bond energy in ATP. Figure 14–10 outlines this sequence of reactions schematically.

1	The matrix contains the genetic system of the mitochondrion, including the mitochondrial DNA and the ribosomes. The mitochondrial DNA (see section on genetic systems, p. 800) is organized into compact bodies—the nucleoids—by special scaffolding proteins that also function as transcription regulatory proteins. The large number of enzymes required for the maintenance of the mitochondrial genetic system, as well as for many other essential reactions to be outlined next, accounts for the very high protein concentration in the matrix; at more than 500 mg/mL, this concentration is close to that in a protein crystal. Mitochondria not only generate most of the cell’s ATP; they also provide many other essential resources for biosynthesis and cell growth. Before describing in detail the remarkable machinery of the respiratory chain, we diverge briefly to touch on some of these important roles. Figure 14–10 A summary of the energy-converting metabolism in mitochondria.

1	Figure 14–10 A summary of the energy-converting metabolism in mitochondria. pyruvate and fatty acids enter the mitochondrion (top of the figure) and are broken down to acetyl Coa. the acetyl Coa is metabolized by the citric acid cycle, which reduces NaD+ to NaDh, which then passes its high-energy electrons to the first complex in the electron-transport chain. In the process of oxidative phosphorylation, these electrons pass along the electron-transport chain in the inner membrane cristae to oxygen (O2). this electron transport generates a proton gradient, which drives the production of atp by the atp synthase (see Figure 14–3). Electrons from the oxidation of succinate, a reaction intermediate in the citric acid cycle (see panel 2–9, pp. 106–107), take a separate path to enter this electron-transport chain (not shown, see p. 772).

1	the membranes that comprise the mitochondrial inner membrane—the inner boundary membrane and the crista membrane—contain different mixtures of proteins and they are therefore shaded differently in this diagram.

1	Mitochondria are critical for buffering the redox potential in the cytosol. Cells need a constant supply of the electron acceptor NAD+ for the central reaction in glycolysis that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate (see Figure 2–48). This NAD+ is converted to NADH in the process, and the NAD+ needs to be regenerated by transferring the high-energy NADH electrons somewhere. The NADH electrons will eventually be used to help drive oxidative phosphorylation inside the mitochondrion. But the inner mitochondrial membrane is impermeable to NADH. The electrons are therefore passed from the NADH to smaller molecules in the cytosol that can move through the inner mitochondrial membrane. Once in the matrix, these smaller molecules transfer their electrons to NAD+ to form mitochondrial NADH, after which they are returned to the cytosol for recharging—creating a so-called shuttle system for the NADH electrons.

1	In addition to ATP, biosynthesis in the cytosol requires both a constant supply of reducing power in the form of NADPH and small carbon-rich molecules to serve as building blocks (discussed in Chapter 2). Descriptions of biosynthesis often state that the needed carbon skeletons come directly from the breakdown of sugars, whereas the NADPH is produced in the cytosol by a side pathway for the breakdown of sugars (the pentose phosphate pathway, an alternative to glycolysis). But under conditions where nutrients abound and plenty of ATP is available, mitochondria help to generate both the reducing power and the carbon-rich building blocks (the “carbon skeletons” in Panel 2–1, pp. 90–91) needed for cell growth. For this purpose, excess citrate produced in the mitochondrial matrix by the citric acid cycle (see Panel 2–9, pp. 106–107) is transported down its electrochemical gradient to the cytosol, where it is metabolized to produce essential components of the cell. Thus, for example, as

1	acid cycle (see Panel 2–9, pp. 106–107) is transported down its electrochemical gradient to the cytosol, where it is metabolized to produce essential components of the cell. Thus, for example, as part of a cell’s response to growth signals, large amounts of acetyl CoA are produced in the cytosol from citrate exported from mitochondria, accelerating the production of the fatty acids and sterols that build new membranes (described in Chapter 10). Cancer cells are frequently mutated in ways that enhance this pathway, as part of their program of abnormal growth (see Figure 20–26).

1	The urea cycle is a central metabolic pathway in mammals that converts the ammonia (NH4+) produced by the breakdown of nitrogen-containing compounds (such as amino acids) to the urea excreted in urine. Two critical steps of the urea cycle are carried out in the mitochondria of liver cells, while the remaining steps occur in the cytosol. Mitochondria also play an essential part in the metabolic adaptation of cells to different nutritional conditions. For example, under conditions of starvation, proteins in our bodies are broken down to amino acids, and the amino acids are imported into mitochondria and oxidized to produce NADH for ATP production.

1	The biosynthesis of heme groups—which, as we shall see in the next section, play a central part in electron transfer—is another critical process that is shared between the mitochondrion and the cytoplasm. Iron–sulfur clusters, which are essential not only for electron transfer in the respiratory chain (see p. 766), but also for the maintenance and stability of the nuclear genome, are produced in mitochondria (and chloroplasts). Nuclear genome instability, a hallmark of cancer, can sometimes be linked to the decreased function of cellular proteins that contain iron–sulfur clusters.

1	Mitochondria also have a central role in membrane biosynthesis. Cardiolipin is a two-headed phospholipid (Figure 14–11) that is confined to the inner mitochondrial membrane, where it is also produced. But mitochondria are also a major source of phospholipids for the biogenesis of other cell membranes. Phosphatidylethanolamine, phosphatidylglycerol, and phosphatidic acid are synthesized in the mitochondrion, while phosphatidylinositol, phosphatidylcholine, and phosphatidylserine are primarily synthesized in the endoplasmic reticulum (ER). As described in Chapter 12, most of the cell’s membranes are assembled in the ER. The exchange of lipids between the ER and mitochondria is thought to occur at special sites of close contact (see Figure 14–7) by an as-yet unknown mechanism.

1	Figure 14–11 The structure of cardiolipin. Cardiolipin consists of two covalently linked phospholipid units, with a total of four rather than the usual two fatty acid chains (see Figure 10–3). Cardiolipin is only produced in the mitochondrial inner membrane, where it interacts closely with membrane proteins involved in oxidative phosphorylation and atp transport. In cristae, its two juxtaposed phosphate groups may act as a local proton trap on the membrane surface. Finally, mitochondria are important calcium buffers, taking up calcium from the ER and sarcoplasmic reticulum at special membrane junctions. Cellular calcium levels control muscle contraction (see Chapter 16) and alterations are implicated in neurodegeneration and apoptosis. Clearly, cells and organisms depend on mitochondria in many different ways. We now return to the central function of the mitochondrion in respiratory ATP generation. a Chemiosmotic process Couples Oxidation Energy to atp production

1	Although the citric acid cycle that takes place in the mitochondrial matrix is considered to be part of aerobic metabolism, it does not itself use oxygen. Only the final step of oxidative metabolism consumes molecular oxygen (O2) directly (see Figure 14–10). Nearly all the energy available from metabolizing carbohydrates, fats, and other foodstuffs in earlier stages is saved in the form of energy-rich compounds that feed electrons into the respiratory chain in the inner mitochondrial membrane. These electrons, most of which are carried by NADH, finally combine with O2 at the end of the respiratory chain to form water. The energy released during the complex series of electron transfers from NADH to O2 is harnessed in the inner membrane to generate an electrochemical gradient that drives the conversion of ADP + Pi to ATP. For this reason, the term oxidative phosphorylation is used to describe this final series of reactions (Figure 14–12).

1	The total amount of energy released by biological oxidation in the respiratory chain is equivalent to that released by the explosive combustion of hydrogen when it combines with oxygen in a single step to form water. But the combustion of hydrogen in a single-step chemical reaction, which has a strongly negative ∆G, releases this large amount of energy unproductively as heat. In the respiratory chain, the same energetically favorable reaction H2 + ½ O2 →H2O is divided into small steps (Figure 14–13). This stepwise process allows the cell to store nearly half of the total energy that is released in a useful form. At each step, the electrons, which can be thought of as having been removed from a hydrogen molecule to

1	Figure 14–12 The major net energy conversion catalyzed by the mitochondrion. In the process of oxidative phosphorylation, the mitochondrial inner membrane serves as a device that changes one form of chemical-bond energy to another, converting a major part of the energy of NaDh oxidation into phosphate-bond energy in atp. Figure 14–13 A comparison of biological oxidation with combustion. (a) If hydrogen were simply burned, nearly all of the energy would be released in the form of heat. (B) In biological oxidation reactions, about half of the released energy is stored in a form useful to the cell by means of the electron-transport chain (the respiratory chain) in the crista membrane of the mitochondrion. Only the rest of the energy is released as heat. In the cell, the protons and electrons shown here as being derived from h2 are removed from hydrogen atoms that are covalently linked to NaDh molecules.

1	produce two protons, pass through a series of electron carriers in the inner mitochondrial membrane. At each of three distinct steps along the way (marked by the three electron-transport complexes of the respiratory chain, see below), much of the energy is utilized for pumping protons across the membrane. At the end of the electron-transport chain, the electrons and protons recombine with molecular oxygen into water. Water is a very low-energy molecule and is thus very stable; it can serve as an electron donor only when a large amount of energy from an external source is spent on splitting it into protons, electrons, and molecular oxygen. This is exactly what happens in oxygenic photosynthesis, where the external energy source is the sun, as we shall see later in the section on chloroplasts (p. 782). the Energy Derived from Oxidation Is Stored as an Electrochemical Gradient

1	the Energy Derived from Oxidation Is Stored as an Electrochemical Gradient In mitochondria, the process of electron transport begins when two electrons and a proton are removed from NADH (to regenerate NAD+). These electrons are passed to the first of about 20 different electron carriers in the respiratory chain. The electrons start at a large negative redox potential (see Panel 14–1, p. 765)— that is, at a high energy level—which gradually drops as they pass along the chain. The proteins involved are grouped into three large respiratory enzyme complexes, each composed of protein subunits that sit in the inner mitochondrial membrane. Each complex in the chain has a higher affinity for electrons than its predecessor, and electrons pass sequentially from one complex to the next until they are finally transferred to molecular oxygen, which has the highest electron affinity of all.

1	The net result is the pumping of H+ out of the matrix across the inner membrane, driven by the energetically favorable flow of electrons. This transmembrane movement of H+ has two major consequences: 1. It generates a pH gradient across the inner mitochondrial membrane, with a high pH in the matrix (close to 8) and a lower pH in the intermembrane space. Since ions and small molecules equilibrate freely across the outer mitochondrial membrane, the pH in the intermembrane space is the same as in the cytosol (generally around pH 7.4). 2. It generates a voltage gradient across the inner mitochondrial membrane, creating a membrane potential with the matrix side negative and the crista space side positive.

1	2. It generates a voltage gradient across the inner mitochondrial membrane, creating a membrane potential with the matrix side negative and the crista space side positive. The pH gradient (∆pH) reinforces the effect of the membrane potential (∆V ), because the latter acts to attract any positive ion into the matrix and to push any negative ion out. Together, ∆pH and ∆V make up the electrochemical gradient, which is measured in units of millivolts (mV). This gradient exerts a protonmotive force, which tends to drive H+ back into the matrix (Figure 14–14). force due to proton-motive force due to INTERMEMBRANE membrane potential + + + + + + + + + pH 7.2 inner mitochondrial membrane pH 7.9

1	force due to proton-motive force due to INTERMEMBRANE membrane potential + + + + + + + + + pH 7.2 inner mitochondrial membrane pH 7.9 Figure 14–14 The electrochemical proton gradient across the inner mitochondrial membrane. this gradient is composed of a large force due to the membrane potential (∆V) and a smaller force due to the h+ concentration gradient—that is, the ph gradient (∆ph). Both forces combine to generate the proton-motive force, which pulls h+ back into the mitochondrial matrix. the exact relationship between these forces is expressed by the Nernst equation (see panel 11–1, p. 616).

1	The electrochemical gradient across the inner membrane of a respiring mitochondrion is typically about 180 mV (inside negative), and it consists of a membrane potential of about 150 mV and a pH gradient of about 0.5 to 0.6 pH units (each ∆pH of 1 pH unit is equivalent to a membrane potential of about 60 mV). The electrochemical gradient drives not only ATP synthesis but also the transport of selected molecules across the inner mitochondrial membrane, including the import of selected proteins from the cytoplasm (discussed in Chapter 12).

1	The mitochondrion performs most cellular oxidations and produces the bulk of the animal cell’s ATP. A mitochondrion has two separate membranes: the outer membrane and the inner membrane. The inner membrane surrounds the innermost space (the matrix) of the mitochondrion and forms the cristae, which project into the matrix. The matrix and the inner membrane cristae are the major working parts of the mitochondrion. The membranes that form cristae account for a major part of the membrane surface area in most cells, and they contain the mitochondrion’s electron-transport chain (the respiratory chain).

1	The mitochondrial matrix contains a large variety of enzymes, including those that convert pyruvate and fatty acids to acetyl CoA and those that oxidize this acetyl CoA to CO2 through the citric acid cycle. These oxidation reactions produce large amounts of NADH, whose high-energy electrons are passed to the respiratory chain. The respiratory chain then uses the energy derived from transporting electrons from NADH to molecular oxygen to pump H+ out of the matrix. This produces a large electrochemical proton gradient across the inner mitochondrial membrane, composed of contributions from both a membrane potential and a pH difference. This electrochemical gradient exerts a force to drive H+ back into the matrix. This proton-motive force is harnessed both to produce ATP and for the selective transport of metabolites across the inner mitochondrial membrane.

1	Having considered in general terms how a mitochondrion uses electron transport to generate a proton-motive force, we now turn to the molecular mechanisms that underlie this membrane-based energy-conversion process. In describing the respiratory chain of mitochondria, we accomplish the larger purpose of explaining how an electron-transport process can pump protons across a membrane. As stated at the beginning of this chapter, mitochondria, chloroplasts, archaea, and bacteria use very similar chemiosmotic mechanisms. In fact, these mechanisms underlie the function of all living organisms—including anaerobes that derive all their energy from electron transfers between two inorganic molecules, as we shall see later. We start with some of the basic principles on which all of these processes depend. the redox potential Is a Measure of Electron affinities

1	We start with some of the basic principles on which all of these processes depend. the redox potential Is a Measure of Electron affinities In chemical reactions, any electrons removed from one molecule are always passed to another, so that whenever one molecule is oxidized, another is reduced. As with any other chemical reaction, the tendency of such redox reactions to proceed spontaneously depends on the free-energy change (∆G) for the electron transfer, which in turn depends on the relative affinities of the two molecules for electrons.

1	Because electron transfers provide most of the energy for life, it is worth taking the time to understand them. As discussed in Chapter 2, acids donate protons and bases accept them (see Panel 2–2, p. 93). Acids and bases exist in conjugate acid–base pairs, in which the acid is readily converted into the base by the loss of a proton. For example, acetic acid (CH3COOH) is converted into its conjugate base, the acetate ion (CH3COO–), in the reaction: In an exactly analogous way, pairs of compounds such as NADH and NAD+ are called redox pairs, since NADH is converted to NAD+ by the loss of electrons in the reaction:

1	In an exactly analogous way, pairs of compounds such as NADH and NAD+ are called redox pairs, since NADH is converted to NAD+ by the loss of electrons in the reaction: NADH is a strong electron donor: because two of its electrons are engaged in a covalent bond which releases energy when broken, the free-energy change for passing these electrons to many other molecules is favorable. Energy is required to form this bond from NAD+, two electrons, and a proton (the same amount of energy that was released when the bond was broken). Therefore NAD+, the redox partner of NADH, is of necessity a weak electron acceptor.

1	We can measure the tendency to transfer electrons from any redox pair experimentally. All that is required is the formation of an electrical circuit linking a 1:1 (equimolar) mixture of the redox pair to a second redox pair that has been arbitrarily selected as a reference standard, so that we can measure the voltage difference between them (Panel 14–1). This voltage difference is defined as the redox potential; electrons move spontaneously from a redox pair like NADH/NAD+ with a lower redox potential (a lower affinity for electrons) to a redox pair like O2/H2O with a higher redox potential (a higher affinity for electrons). Thus, NADH is a good molecule for donating electrons to the respiratory chain, while O2 is well suited to act as the “sink” for electrons at the end of the chain. As explained in Panel 14–1, the difference in redox potential, ∆Eʹ0, is a direct measure of the standard free-energy change (∆G°) for the transfer of an electron from one molecule to another.

1	Electron transfers release Large amounts of Energy

1	As just discussed, those pairs of compounds that have the most negative redox potentials have the weakest affinity for electrons and therefore are useful as carriers with a strong tendency to donate electrons. Conversely, those pairs that have the most positive redox potentials have the greatest affinity for electrons and therefore are useful as carriers with a strong tendency to accept electrons. A 1:1 mixture of NADH and NAD+ has a redox potential of –320 mV, indicating that NADH has a strong tendency to donate electrons; a 1:1 mixture of H2O and ½O2 has a redox potential of +820 mV, indicating that O2 has a strong tendency to accept electrons. The difference in redox potential is 1140 mV, which means that the transfer of each electron from NADH to O2 under these standard conditions is enormously favorable, since ∆G° = –109 kJ/mole, and twice this amount of energy is gained for the two electrons transferred per NADH molecule (see Panel 14–1). If we compare this free-energy change

1	enormously favorable, since ∆G° = –109 kJ/mole, and twice this amount of energy is gained for the two electrons transferred per NADH molecule (see Panel 14–1). If we compare this free-energy change with that for the formation of the phosphoanhydride bonds in ATP, where ∆G° = 30.6 kJ/mole (see Figure 2–50), we see that, under standard conditions, the oxidation of one NADH molecule releases more than enough energy to synthesize seven molecules of ATP from ADP and Pi. (In the cell, the number of ATP molecules generated will be lower because the standard conditions are far from the physiological ones; in addition, small amounts of energy are inevitably dissipated as heat along the way.)

1	The electron-transport properties of the membrane protein complexes in the respiratory chain depend upon electron-carrying cofactors, most of which are transition metals such as Fe, Cu, Ni, and Mn, bound to proteins in the complexes. These metals have special properties that allow them to promote both enzyme catalysis and electron-transfer reactions. Most relevant here is the fact that their ions exist in several different oxidation states with closely spaced redox potentials, which enables them to accept or give up electrons readily; this property is

1	HOW REDOX POTENTIALS ARE MEASURED voltmeter salt bridge Areduced and Aoxidized in equimolar amounts 1 M H+ and 1 atmosphere H2 gas examples of redox reactions standard redox potential E0 ° c c e – One beaker (left) contains substance A with an equimolar mixture of the reduced (Areduced) and oxidized (Aoxidized) members of its redox pair. The other beaker contains the hydrogen reference standard (2H+ + 2e – H2), whose redox potential is arbitrarily assigned as zero by international agreement. (A salt bridge formed from a concentrated KCl solution allows K+ and Cl– to move between the beakers, as required to neutralize the charges when electrons fow between the beakers.) The metal wire (dark blue) provides a resistance-free path for electrons, and a voltmeter then measures the redox potential of substance A. If electrons fow from Areduced to H+, as indicated here, the redox pair formed by substance A is said to have a negative redox potential. If they instead fow from H2 to Aoxidized,

1	of substance A. If electrons fow from Areduced to H+, as indicated here, the redox pair formed by substance A is said to have a negative redox potential. If they instead fow from H2 to Aoxidized, the redox pair is said to have a positive redox potential. THE STANDARD REDOX POTENTIAL, E˜0 The standard redox potential for a redox pair, defned as E0 , is measured for a standard state where all of the reactants are at a concentration of 1 M, including H+ . Since biological reactions occur at pH 7, biologists instead defne the standard state as Areduced = Aoxidized and H+ = 10–7 M. This standard redox potential is designated by the symbol E0 , in place of E0. ° PANEL 14–1: Redox Potentials 765

1	CALCULATION OF ˜Go FROM REDOX POTENTIALS E0 ° E0 ° To determine the energy change for an electron transfer, the ˜G o of the reaction (kJ/mole) is calculated as follows: ˜G o = –n(0.096) ˜E0 , where n is the number of electrons transferred across a redox potential change of ˜E0 millivolts (mV), and ˜E0 = (acceptor) – (donor) EXAMPLE: ° ° ° EFFECT OF CONCENTRATION CHANGES As explained in Chapter 2 (see p. 60), the actual free-energy change for a reaction, ˜G, depends on the concentration of the reactants and generally will be different from the standard free-energy change, ˜Go. The standard redox potentials are for a 1:1 mixture of the redox pair. For example, the standard redox potential of –320 mV is for a 1:1 mixture of NADH and NAD+. But when there is an excess of NADH over NAD+, electron transfer from NADH to an electron acceptor becomes more favorable. This is refected by a more negative redox potential and a more negative ˜G for electron transfer.

1	˜E0 = +30 – (–320) = +350 mV The same calculation reveals that the transfer of one electron from ubiquinone to oxygen has an even more favorable ˜Go of –76 kJ/mole. The ˜Go value for the transfer of one electron from NADH to oxygen is the sum of these two values, –110 kJ/mole. ˜Go = –n(0.096) = –1(0.096)(350) = –34 kJ/mole ° °˜E0 ° ° For the transfer of one electron from NADH to ubiquinone: Figure 14–15 The structure of the heme group attached covalently to cytochrome c. the porphyrin ring of the heme is shown in red. there are six different cytochromes in the respiratory chain. Because the hemes in different cytochromes have slightly different structures and are kept in different local environments by their respective proteins, each has a different affinity for an electron, and a slightly different spectroscopic signature. exploited by the membrane protein complexes in the respiratory chain to move electrons both within and between complexes.

1	exploited by the membrane protein complexes in the respiratory chain to move electrons both within and between complexes. Unlike the colorless atoms H, C, N, and O that constitute the bulk of biological molecules, transition metal ions are often brightly colored, which makes the proteins that contain them easy to study by spectroscopic methods using visible light. One family of such colored proteins, the cytochromes, contains a bound heme group, in which an iron atom is tightly held by four nitrogen atoms at the corners of a square in a porphyrin ring (Figure 14–15). Similar porphyrin rings are responsible both for the red color of blood and for the green color of leaves, binding an iron in hemoglobin or a magnesium in chlorophyll, respectively.

1	Iron–sulfur proteins contain a second major family of electron-transfer cofactors. In this case, either two or four iron atoms are bound to an equal number of sulfur atoms and to cysteine side chains, forming iron–sulfur clusters in the protein (Figure 14–16). Like the cytochrome hemes, these clusters carry one electron at a time. The simplest of the electron-transfer cofactors in the respiratory chain—and the only one that is not always bound to a protein—is a quinone (called ubiquinone, or coenzyme Q). A quinone (Q) is a small hydrophobic molecule that is freely mobile in the lipid bilayer. This electron carrier can accept or donate either one or two electrons. Upon reduction (note that reduced quinones are called quinols), it picks up a proton from water along with each electron (Figure 14–17).

1	In the mitochondrial electron-transport chain, six different cytochrome hemes, eight iron–sulfur clusters, three copper atoms, a flavin mononucleotide (another electron-transfer cofactor), and ubiquinone work in a defined sequence to carry electrons from NADH to O2. In total, this pathway involves more than 60 different polypeptides arranged in three large membrane protein complexes, each of which binds several of the above electron-carrying cofactors.

1	As we would expect, the electron-transfer cofactors have increasing affinities for electrons (higher redox potentials) as the electrons move along the respiratory chain. The redox potentials have been fine-tuned during evolution by the protein environment of each cofactor, which alters the cofactor’s normal affinity for electrons. Because iron–sulfur clusters have a relatively low affinity for electrons, they predominate in the first half of the respiratory chain; in contrast, the heme cytochromes predominate further down the chain, where a higher electron affinity is required. NaDh transfers Its Electrons to Oxygen through three Large Enzyme Complexes Embedded in the Inner Membrane

1	NaDh transfers Its Electrons to Oxygen through three Large Enzyme Complexes Embedded in the Inner Membrane Membrane proteins are difficult to purify because they are insoluble in aqueous solutions, and they are easily disrupted by the detergents that are required to solubilize them. But by using mild nonionic detergents, such as octylglucoside or dodecyl maltoside (see Figure 10–28), they can be solubilized and purified in their native form, and even crystallized for structure determination. Each of the three different detergent-solubilized respiratory-chain complexes can be re-inserted

1	Figure 14–16 The structure of an iron–sulfur cluster. these dark brown clusters consist either of four iron and four sulfur atoms, as shown here, or of two irons and two sulfurs linked to cysteines in the polypeptide chain via covalent sulfur bridges, or to histidines. although they contain several iron atoms, each iron–sulfur cluster can carry only one electron at a time. Nine different iron– sulfur clusters participate in electron transport in the respiratory chain. into artificial lipid bilayer vesicles and shown to pump protons across the membrane as electrons pass through them. In the mitochondrion, the three complexes are linked in series, serving as electron-transport-driven H+ pumps that pump protons out of the matrix to acidify the crista space (Figure 14–18): 1.

1	In the mitochondrion, the three complexes are linked in series, serving as electron-transport-driven H+ pumps that pump protons out of the matrix to acidify the crista space (Figure 14–18): 1. The NADH dehydrogenase complex (often referred to as Complex I) is the largest of these respiratory enzyme complexes. It accepts electrons from NADH and passes them through a flavin mononucleotide and eight iron–sulfur clusters to the lipid-soluble electron carrier ubiquinone. The reduced ubiquinol then transfers its electrons to cytochrome c reductase. 2. The cytochrome c reductase (also called the cytochrome b-c1 complex) is a large membrane protein assembly that functions as a dimer. Each monomer contains three cytochrome hemes and an iron–sulfur cluster. The complex accepts electrons from ubiquinol and passes them on to the small, soluble protein cytochrome c, which is located in the crista space and carries electrons one at a time to cytochrome c oxidase. 3.

1	3. The cytochrome c oxidase complex contains two cytochrome hemes and three copper atoms. The complex accepts electrons one at a time from cytochrome c and passes them to molecular oxygen. In total, four electrons and four protons are needed to convert one molecule of oxygen to water. We have previously discussed how the redox potential reflects electron affinities. Figure 14–19 presents an outline of the redox potentials measured along the respiratory chain. These potentials change in three large steps, one across each proton-translocating respiratory complex. The change in redox potential between any two electron carriers is directly proportional to the free energy released when an electron transfers between them. Each complex acts as an energy-conversion device by harnessing some of this free-energy change to pump H+ across the inner membrane, thereby creating an electrochemical proton gradient as electrons pass along the chain.

1	Figure 14–17 Quinone electron carriers. Ubiquinone in the lipid bilayer picks up one h+ (red) from the aqueous environment for each electron (blue) it accepts, in two steps, from respiratory-chain complexes. the first step involves the acquisition of a proton and an electron and converts the ubiquinone into an unstable ubisemiquinone radical. In the second step, it becomes a fully reduced ubiquinone (called ubiquinol), which is freely mobile as an electron carrier in the lipid bilayer of the membrane. When the ubiquinol donates its electrons to the next complex in the chain, the two protons are released. the long hydrophobic tail (green) that confines ubiquinone to the membrane consists of 6–10 five-carbon isoprene units, depending on the organism. the corresponding electron carrier in the photosynthetic membranes of chloroplasts is plastoquinone, which has almost the same structure and works in the same way. For simplicity, we refer to both ubiquinone and plastoquinone in this

1	in the photosynthetic membranes of chloroplasts is plastoquinone, which has almost the same structure and works in the same way. For simplicity, we refer to both ubiquinone and plastoquinone in this chapter as quinone (abbreviated as Q).

1	Figure 14–18 The path of electrons through the three respiratory-chain proton pumps. (Movie 14.3) the approximate size and shape of each complex is shown. During the transfer of electrons from NaDh to oxygen (blue arrows), ubiquinone and cytochrome c serve as mobile carriers that ferry electrons from one complex to the next. During the electron-transfer reactions, protons are pumped across the membrane by each of the respiratory enzyme complexes, as indicated (red arrows).

1	For historical reasons, the three proton pumps in the respiratory chain are sometimes denoted as Complex I, Complex III, and Complex IV, according to the order in which electrons pass through them from NaDh. Electrons from the oxidation of succinate by succinate dehydrogenase (designated as Complex II) are fed into the electron-transport chain in the form of reduced ubiquinone. although embedded in the crista membrane, succinate dehydrogenase does not pump protons and thus does not contribute to the proton-motive force; it is therefore not considered to be an integral part of the respiratory chain.

1	along the mitochondrial electron _400 transport chain. the redox potential _300 (designated Eʹ0) increases as electrons flow down the respiratory chain to oxygen. the _200 standard free-energy change in kilojoules, _100 ∆G°, for the transfer of each of the two can be obtained from the left-hand ordinate [∆G° = –n(0.096) ∆Eʹ0, where n is the number of electrons transferred across a redox potential change of ∆Eʹ0 mV]. Electrons flow through a respiratory through the multiple electron carriers in 500 each complex (blue arrows). as indicated, part of the favorable free-energy change 600 is harnessed by each enzyme complex to 700 pump h+ across the inner mitochondrial membrane (red arrows). the NaDh 800 dehydrogenase pumps up to four h+ per electron, the cytochrome c reductase complex pumps two, whereas the direction of electron fow

1	X-ray crystallography has elucidated the structure of each of the three respiratory-chain complexes in great detail, and we next examine each of them in turn to see how they work. the NaDh Dehydrogenase Complex Contains Separate Modules for Electron transport and proton pumping The NADH dehydrogenase complex is a massive assembly of membrane and nonmembrane proteins that receives electrons from NADH and passes them to ubiquinone. In animal mitochondria, it consists of more than 40 different protein subunits, with a molecular mass of nearly a million daltons. The x-ray structures of the NADH dehydrogenase complex from fungi and bacteria show that it is L-shaped, with both a hydrophobic membrane arm and a hydrophilic arm that projects into the mitochondrial matrix (Figure 14–20).

1	Electron transfer and proton pumping are physically separated in the NADH dehydrogenase complex, with electron transfer occurring in the matrix arm and proton pumping in the membrane arm. The NADH docks near the tip of the matrix arm, where it transfers its electrons via a bound flavin mononucleotide to a string of iron–sulfur clusters that runs down the arm, acting like a wire to carry electrons to a protein-bound molecule of ubiquinone. Electron transfer to the quinone is thought to trigger proton translocation in a set of proton pumps in the membrane arm, and for this to happen the two processes must be energetically and mechanically linked. A mechanical link is thought to be provided by a 6-nm long, amphipathic α helix that runs parallel to the membrane surface on the matrix side of the membrane arm. This helix may act like the connecting rod in a steam engine to generate a mechanical, energy-transducing power stroke that links the quinone-binding site to the proton-translocating

1	membrane arm. This helix may act like the connecting rod in a steam engine to generate a mechanical, energy-transducing power stroke that links the quinone-binding site to the proton-translocating modules in the membrane (see Figure 14–20).

1	The reduction of each quinone by the transfer of two electrons can cause four protons to be pumped out of the matrix into the crista space. In this way, NADH dehydrogenase generates roughly half of the total proton-motive force in mitochondria. Cytochrome c reductase takes Up and releases protons on the Opposite Side of the Crista Membrane, thereby pumping protons As described previously, when a quinone molecule (Q) accepts its two electrons, it also takes up two protons to form a quinol (QH2; see Figure 14–17). In cytochrome c oxidase complex pumps one per electron. Note that NaDh is not the only source of electrons for the respiratory chain. the flavin FaDh2, which is generated by fatty acid oxidation (see Figure 2–56) and by succinate dehydrogenase in the citric acid cycle (see Figure 2–57), also contributes. Its two electrons are passed directly to ubiquinone, bypassing NaDh dehydrogenase.

1	Figure 14–20 The structure of NADH dehydrogenase. (a) the model of the mitochondrial complex shown here is based on the x-ray structure of the smaller bacterial complex, which works in the same way. the matrix arm of NaDh dehydrogenase (also known as Complex I) contains eight iron–sulfur (FeS) clusters that appear to participate in electron transport. the membrane contains more than 70 transmembrane helices, forming three distinct proton-pumping modules, while the matrix arm contains the electron-transport cofactors. (B) NaDh donates two electrons, via a bound flavin mononucleotide (FMN; yellow), to a chain of seven iron–sulfur clusters (red and yellow spheres). From the terminal iron–sulfur cluster, the electrons pass to ubiquinone (orange). Electron transfer results in conformational changes (black arrows) that are thought to be transmitted to a long amphipathic α helix (purple) on the matrix side of the membrane arm, which pulls on discontinuous transmembrane helices (red) in three

1	(black arrows) that are thought to be transmitted to a long amphipathic α helix (purple) on the matrix side of the membrane arm, which pulls on discontinuous transmembrane helices (red) in three membrane subunits, each of which resembles an antiporter (see Chapter 11). this movement is thought to change the conformation of charged residues in the three proton channels, resulting in the translocation of three protons out of the matrix. a fourth proton may be translocated at the interface of the two arms (dotted line). (C) this shows the symbol for NaDh dehydrogenase used throughout this chapter. (adapted from r.G. Efremov, r. Baradaran and L.a. Sazanov, Nature 465:441–445, 2010. pDB code: 3M9S.) the respiratory chain, ubiquinol tranfers electrons from NADH dehydrogenase to cytochrome c reductase. Because the protons in this QH2 molecule are taken up from the matrix and released on the opposite side of the crista membrane, two protons are transferred from the matrix into the crista

1	reductase. Because the protons in this QH2 molecule are taken up from the matrix and released on the opposite side of the crista membrane, two protons are transferred from the matrix into the crista space per pair of electrons transferred (Figure 14–21). This vectorial transfer of protons supplements the electrochemical proton gradient that is created by the NADH dehydrogenase proton pumping just discussed.

1	Cytochrome c reductase is a large assembly of membrane protein subunits. Three subunits form a catalytic core that passes electrons from ubiquinol to cytochrome c, with a structure that has been highly conserved from bacterial ancestors (Figure 14–22). It pumps protons by a vectorial transfer of protons that involves a binding site for a second molecule of ubiquinone; the elaborate Figure 14–21 How a directional release and uptake of protons by a quinone pumps protons across a membrane.

1	Figure 14–21 How a directional release and uptake of protons by a quinone pumps protons across a membrane. two protons are picked up on the matrix side of the inner mitochondrial membrane when the reaction Q + 2e – + 2h+ → Qh2 is catalyzed by the NaDh dehydrogenase complex. this molecule of ubiquinol (Qh2) diffuses rapidly in the plane of the membrane, becoming bound to the crista side of cytochrome c reductase. When its oxidation by cytochrome c reductase generates two protons and two electrons (see Figure 14–17), the two protons are released into the crista space. the flow of electrons is not shown in this diagram. 770 Chapter 14: Energy Conversion: Mitochondria and Chloroplasts electrons out to Figure 14–22 The structure of cytochrome c reductase. Cytochrome c reductase (also known as the cytochrome b-c1 complex) is a dimer of two identical 240,000-dalton halves, each composed of 11 different protein molecules in mammals.

1	A structure graphic of the entire dimer, showing in color the three proteins that form the functional core of the enzyme complex: cytochrome b(green) and cytochrome c1(blue) are colored in one half, and the Rieske protein (purple) containing an Fe2S2 iron–sulfur cluster (red and yellow) is colored in the other. These three protein subunits interact across the two halves. Transfer of electrons through cytochrome c reductase to the small, soluble carrier protein cytochrome c. Electrons entering from ubiquinol near the matrix side of the membrane are captured by the iron–sulfur cluster of the Rieske protein, which moves its iron–sulfur group back and forth to transfer these electrons to heme c(red). Heme c then transfers them to the carrier molecule cytochrome c.

1	As detailed in Figure 14–23, only one of the two electrons from each ubiquinol is transferred through this path. To increase proton pumping, the second ubiquinol electron is passed to a molecule of ubiquinone bound to cytochrome c reductase on the opposite side of the membrane—near the matrix. (C) This shows the symbol for cytochrome c reductase used throughout this chapter. (PDB code: 1EZV.) redox loop mechanism used is called the Q cycle because while one of the electrons received from each QH2 molecule istransferred from ubiquinone through the complex to the carrier protein cytochrome c, the other electron is recycled back into the quinone pool. Through the mechanism illustrated in Figure 14–23, the Q cycle increases the total amount of redox energy that can be stored in the electrochemical proton gradient. As a result, two protons are pumped across the crista membrane for every electron that is transferred from NADH dehydrogenase to cytochrome c.

1	The Cytochrome c Oxidase Complex Pumps Protons and Reduces O2 Using a Catalytic Iron–Copper Center The final link in the mitochondrial electron-transport chain is cytochrome c oxidase. The cytochrome c oxidase complex accepts electrons from the soluble electron carrier cytochrome c, and it uses yet a different, third mechanism to pump protons across the inner mitochondrial membrane. The structure of the mammalian complex is illustrated in Figure 14–24. The atomic-resolution structures, combined with studies of the effect of mutations introduced into the enzyme by genetic engineering of the yeast and bacterial proteins, have revealed the detailed mechanisms of this electron-driven proton pump.

1	Because oxygen has a high affinity for electrons, it can release a large amount of free energy when it is reduced to form water. Thus, the evolution of cellular respiration, in which O2 is converted to water, enabled organisms to harness much more energy than can be derived from anaerobic metabolism. As we discuss later, the availability of the large amount of energy released by the reduction of molecular oxygen to form water is thought to have been essential to the emergence of multicellular life: this would explain why all large organisms respire. The ability of biological systems to use O2 in this way, however, requires sophisticated chemistry. Once a molecule of O2 has picked up one electron, it forms a superoxide radical anion (O2•–) that is dangerously reactive and rapidly takes up an additional three electrons wherever it can get them, with destructive effects on its immediate environment. We can tolerate oxygen in the air we breathe only because the uptake of the first

1	up an additional three electrons wherever it can get them, with destructive effects on its immediate environment. We can tolerate oxygen in the air we breathe only because the uptake of the first electron by the O2 molecule is slow, allowing cells to use enzymes to control electron uptake by oxygen. Thus, cytochrome c oxidase holds

1	Figure 14–23 The two-step mechanism of the cytochrome c reductase Q-cycle. In step 1, ubiquinol reduced by NaDh dehydrogenase docks to the cytochrome c reductase complex. Oxidation of the quinol produces two protons and two electrons. the protons are released into the cristae space. One electron passes via an iron-sulfur cluster to heme c1, and then to the soluble electron carrier protein cytochrome c on the membrane surface. the second electron passes via hemes bL and bh to a ubiquinone (red Q) bound at a separate site near the matrix side of the protein. Uptake of a proton from the matrix produces an ubisemiquinone radical (see Figure 14–17), which remains bound to this site (red Qh• in B).

1	In step 2, a second ubiquinol (blue Qh2) docks and releases two protons and two electrons, as described for step 1. One electron is passed to a second cytochrome c, whereas the other electron is accepted by the ubisemiquinone. the ubisemiquinone takes up a proton from the matrix and is released into the lipid bilayer as fully reduced ubiquinol (red Qh2). On balance, the oxidation of one ubiquinol in the Q cycle pumps two protons through the membrane by a directional release and uptake of protons (see Figure 14–21), while releasing another two into the cristae space. In addition, in each of the two steps and (B), one electron is transferred to a cytochrome c carrier (Movie 14.4).

1	Figure 14–24 The structure of cytochrome c oxidase. the final complex in the mitochondrial electron-transfer chain consists of 13 different protein subunits, with a total mass of 204,000 daltons. (a) the entire dimeric complex is shown, positioned in the crista membrane. the highly conserved subunits I (green), II (purple), and III (blue) are encoded by the mitochondrial genome, and they form the functional core of the enzyme.

1	(B) the functional core of the complex. Electrons pass through this structure from cytochrome c via bound copper ions (blue spheres) and hemes (red) to an O2 molecule bound between heme a3 and a copper ion. the four protons needed to reduce O2 to water are taken up from the matrix; see also Figure 14–25. (C) this shows the symbol for cytochrome c oxidase used throughout this chapter. (pDB code: 2OCC.) 772 Chapter 14: Energy Conversion: Mitochondria and Chloroplasts on to oxygen at a special bimetallic center, where it remains clamped between a heme-linked iron atom and a copper ion until it has picked up a total of four electrons. Only then are the two oxygen atoms of the oxygen molecule safely released as two molecules of water (Figure 14–25).

1	The cytochrome c oxidase reaction accounts for about 90% of the total oxygen uptake in most cells. This protein complex is therefore crucial for all aerobic life. Cyanide and azide are extremely toxic because they bind to the heme iron atoms in cytochrome c oxidase much more tightly than does oxygen, thereby greatly reducing ATP production. the respiratory Chain Forms a Supercomplex in the Crista Membrane

1	the respiratory Chain Forms a Supercomplex in the Crista Membrane By using cryoelectron microscopy to examine proteins that have been very gently isolated, it can be shown that the three protein complexes that form the respiratory chain assemble into an even larger supercomplex in the crista membrane. As illustrated in Figure 14–26, this structure is thought to help the mobile electron carriers ubiquinone (in the crista membrane) and cytochrome c (in the crista space) transfer electrons with high efficiency. The formation of the supercomplex depends on the presence of the mitochondrial lipid cardiolipin (see Figure 14–11), which presumably works like a hydrophobic glue that holds the components together.

1	In addition to the three proton pumps in the supercomplex just discussed, one of the enzymes in the citric acid cycle, succinate dehydrogenase, is embedded in the mitochondrial crista membrane. In the course of oxidizing succinate to fumarate in the matrix, this enzyme complex captures electrons in the form of a tightly bound FADH2 molecule (see Panel 2–9, pp. 106–107) and passes them to

1	Figure 14–25The reaction of O2 with electrons in cytochrome c oxidase. Electrons from cytochrome c pass through the complex via bound copper ions (blue spheres)and hemes (red) to an O2 molecule bound between heme a3 and a copper ion. Iron ions are shown as red spheres. the iron atom in heme aserves as an electron queuing point where electrons are held so that they can be released to an O2 molecule (not shown) that is held at the bimetallic center active site, which is formed by the central iron of the other heme (heme a3) and a closely apposed copper atom. the four protons needed to reduce O2 to water are removed from the matrix. For each O2 molecule that undergoes the reaction 4e – + 4h+ + O2→ 2h2O, another four protons are pumped out of the matrix by mechanisms that are driven by allosteric changes in protein conformation (see Figure 14–28).

1	iron–sulfur cytochrome c Figure 14–26 The respiratory-chain mitochondria. the three proton-pumping CRISTA complexes of the mitochondrial respiratory chain of mammalian mitochondria assemble into large supercomplexes in the crista membrane. Supercomplexes can be isolated by mild detergent treatment of mitochondria, and their structure has been deciphered by single-particle cryoelectron microscopy. the bovine heart supercomplex has a total mass of 1.7 megadaltons. Shown is a schematic of such a complex that consists of NaDh dehydrogenase, cytochrome c reductase, and cytochrome c oxidase, as indicated. the facing quinol-binding sites of NaDh dehydrogenase and cytochrome c reductase, plus the short distance between the cytochrome c-binding sites in cytochrome c reductase and cytochrome c oxidase, facilitate fast, efficient electron transfer. Cofactors active in electron transport are marked as a yellow dot (flavin mononucleotide), red and yellow dots (iron–sulfur clusters), Q (quinone),

1	facilitate fast, efficient electron transfer. Cofactors active in electron transport are marked as a yellow dot (flavin mononucleotide), red and yellow dots (iron–sulfur clusters), Q (quinone), red squares (hemes), and a blue dot (copper atom). Only cofactors participating in the linear flow of electrons from NaDh to water are shown. Blue arrows indicate the path of the electrons through the supercomplex. (adapted from t. athoff et al., EMBO J. 30:4652–4664, 2011.) a molecule of ubiquinone. The reduced ubiquinol then passes its two electrons to the respiratory chain via cytochrome c reductase (see Figure 14–18). Succinate dehydrogenase is not a proton pump, and it does not contribute directly to the electrochemical potential utilized for ATP production in mitochondria. Thus, it is not considered to be an integral part of the respiratory chain.

1	The protons in water are highly mobile: by rapidly dissociating from one water molecule and associating with its neighbor, they can rapidly flit through a hydrogen-bonded network of water molecules (see Figure 2–5). But how can a pro ton move through the hydrophobic interior of a protein embedded in the lipid bilayer? Proton-translocating proteins contain so-called proton wires, which are rows of polar or ionic side chains, or water molecules spaced at short distances, so that the protons can jump from one to the next (Figure 14–27). Along such predefined pathways, protons move up to 40 times faster than through bulk water. The three-dimensional structure of cytochrome c oxidase indicates two different proton-uptake pathways. This confirmed earlier mutagenesis studies, which had shown that replacing the side chains of particular aspartate or arginine residues, whose side chains can bind and release protons, made the cytochrome c oxidase less efficient as a proton pump.

1	But how can electron transport cause allosteric changes in protein conformations that pump protons? From the most basic point of view, if electron transport drives sequential allosteric changes in protein conformation that alter the redox state of the components, these conformational changes can be connected to protein wires that allow the protein to pump H+ across the crista membrane. This type of H+ pumping requires at least three distinct conformations for the pump protein, as schematically illustrated in Figure 14–28. Figure 14–27 Proton movement through water and proteins. (a) protons move rapidly through water, hopping from one h2O molecule to the next by the continuous formation and dissociation of hydronium ions, h3O+ (see Chapter 2). In this diagram, proton jumps are indicated by red arrows.

1	wires.” these are predefined proton paths consisting of suitably spaced amino acid side chains that accept and release protons easily (asp, Glu) or carry a waterlike hydroxyl group (Ser, thr), along with water molecules trapped in the protein interior.

1	The respiratory chain embedded in the inner mitochondrial membrane contains three respiratory enzyme complexes, through which electrons pass on their way from NADH to O2. In these complexes, electrons are transferred along a series of protein-bound electron carriers, including hemes and iron–sulfur clusters. The energy released as the electrons move to lower and lower energy levels is used to pump protons by different mechanisms in the three respiratory enzyme complexes, each coupling lateral electron transport to vectorial proton transport across the membrane. Electrons are shuttled between enzyme complexes by the mobile electron carriers ubiquinone and cytochrome c to complete the electron-transport chain. The path of electron flow is NADH → NADH dehydrogenase complex → ubiquinone → cytochrome c reductase → cytochrome c → cytochrome c oxidase complex → molecular oxygen (O2).

1	As we have just discussed, the three proton pumps of the respiratory chain each contribute to the formation of an electrochemical proton gradient across the inner mitochondrial membrane. This gradient drives ATP synthesis by ATP synthase, a large membrane-bound protein complex that performs the extraordinary feat of converting the energy contained in this electrochemical gradient into biologically useful, chemical-bond energy in the form of ATP (see Figure 14–10). Protons flow down their electrochemical gradient through the membrane part of this proton turbine, thereby driving the synthesis of ATP from ADP and Pi in the extramembranous part of the complex. As discussed in Chapter 2, the formation of ATP from ADP and inorganic phosphate is highly unfavorable energetically. As we shall see, ATP synthase can produce ATP only because of allosteric shape changes in this protein complex that directly couple ATP synthesis to the energetically favorable flow of protons across its membrane.

1	the Large Negative Value of ∆G for atp hydrolysis Makes atp Useful to the Cell An average person turns over roughly 50 kg of ATP per day. In athletes running a marathon, this figure can go up to several hundred kilograms. The ATP produced in mitochondria is derived from the energy available in the intermediates NADH, FADH2, and GTP. These three energy-rich compounds are produced both by the oxidation of glucose (Table 14–1A), and by the oxidation of fats (Table 14–1B; see also Figure 2–56). Glycolysis alone can produce only two molecules of ATP for every molecule of glucose that is metabolized, and this is the total energy yield for the fermentation processes that occur in the absence of O2 (discussed in Chapter 2). In oxidative Figure 14–28 A general model for H+ pumping coupled to electron transport.

1	Figure 14–28 A general model for H+ pumping coupled to electron transport. this mechanism for h+ pumping by a transmembrane protein is thought to be used by NaDh dehydrogenase and cytochrome c oxidase, and by many other proton pumps. the protein is driven through a cycle of three conformations. In one of these conformations, the protein has a high affinity for h+, causing it to pick up an h+ on the inside of the membrane. In another conformation, the protein has a low affinity for h+, causing it to release an h+ on the outside of the membrane. as indicated, the transitions from one conformation to another occur only in one direction, because they are being driven by being allosterically coupled to the energetically favorable process of electron transport (discussed in Chapter 11).

1	phosphorylation, each pair of electrons donated by the NADH produced in mitochondria can provide energy for the formation of about 2.5 molecules of ATP. Oxidative phosphorylation also produces 1.5 ATP molecules per electron pair from the FADH2 produced by succinate dehydrogenase in the mitochondrial matrix, and from the NADH molecules produced by glycolysis in the cytosol. From the product yields of glycolysis and the citric acid cycle, we can calculate that the complete oxidation of one molecule of glucose—starting with glycolysis and ending with oxidative phosphorylation—gives a net yield of about 30 molecules of ATP. Nearly all this ATP is produced by the mitochondrial ATP synthase.

1	In Chapter 2, we introduced the concept of free energy (G). The free-energy change for a reaction, ∆G, determines whether that reaction will occur in a cell. We showed on pp. 60–63 that the ∆G for a given reaction can be written as the sum of two parts: the first, called the standard free-energy change, ∆G°, depends only on the intrinsic characters of the reacting molecules; the second depends only on their concentrations. For the simple reaction A →B, [A] where [A] and [B] denote the concentrations of A and B, and ln is the natural logarithm. ∆G° is the standard reference value, which can be seen to be equal to the value of ∆G when the molar concentrations of A and B are equal (since ln 1 = 0). In Chapter 2, we discussed how the large, favorable free-energy change (large negative ∆G) for ATP hydrolysis is used, through coupled reactions, to drive many other chemical reactions in the cell that would otherwise not occur (see pp. 65–66). The ATP hydrolysis reaction produces two

1	∆G) for ATP hydrolysis is used, through coupled reactions, to drive many other chemical reactions in the cell that would otherwise not occur (see pp. 65–66). The ATP hydrolysis reaction produces two products, ADP and Pi; it is therefore of the type A → B + C, where, as demonstrated in Figure 14–29, [B][C]

1	When ATP is hydrolyzed to ADP and Pi under the conditions that normally exist in a cell, the free-energy change is roughly –46 to –54 kJ/mole (–11 to –13 kcal/mole). This extremely favorable ∆G depends on maintaining a high concentration of ATP compared with the concentrations of ADP and Pi. When ATP, ADP, and Pi are all present at the same concentration of 1 mole/liter (so-called standard conditions), the ∆G for ATP hydrolysis drops to the standard free-energy change (∆G°), which is only –30.5 kJ/mole (–7.3 kcal/mole). At much lower concentrations of ATP relative to ADP and Pi, ∆G becomes zero. At this point, the rate at AT EQUILIBRIUM: synthesis conc. of × conc. of hydrolysis conc. of + synthesis rate constant ×conc. of phosphate ×conc. of ADP synthesis synthesis rate = 2 4 ATPADPPi conc. of conc. of hydrolysis ADP phosphate rate constant thus, = = equilibrium constant K concentration synthesis of ATP rate constant or abbreviated,

1	ADP phosphate rate constant thus, = = equilibrium constant K concentration synthesis of ATP rate constant or abbreviated, At equilibrium the reaction has no net effect on the disorder of For the reaction the universe, so °G = 0. Therefore, at equilibrium, [ATP]the following equation applies: But the concentrations of reactants at equilibrium must satisfy the equilibrium equation: Therefore, at equilibrium, where °G and °Go are in Joules per mole, R is the gas constant (8.3 J/mole K), T is the absolute temperature (K), and all the concentrations are in moles per liter. We thus see that whereas °Go indicates the equilibrium point for a

1	When the concentrations of all reactants are at 1 M, °G = °Go reaction, °G reveals how far the reaction is from equilibrium. °G is (since RT ln 1 = 0). °Go is thus a constant defned as the a measure of the “driving force” for any chemical reaction, just as the standard free-energy change for the reaction. proton-motive force is the driving force for the translocation of protons. which ADP and Pi will join to form ATP will be equal to the rate at which ATP hydrolyzes to form ADP and Pi. In other words, when ∆G = 0, the reaction is at equilibrium (see Figure 14–29).

1	which ADP and Pi will join to form ATP will be equal to the rate at which ATP hydrolyzes to form ADP and Pi. In other words, when ∆G = 0, the reaction is at equilibrium (see Figure 14–29). It is ∆G, not ∆G°, that indicates how far a reaction is from equilibrium and determines whether it can drive other reactions. Because the efficient conversion of ADP to ATP in mitochondria maintains such a high concentration of ATP relative to ADP and Pi, the ATP hydrolysis reaction in cells is kept very far from equilibrium and ∆G is correspondingly very negative. Without this large disequilibrium, ATP hydrolysis could not be used to drive the reactions of the cell. At low ATP concentrations, many biosynthetic reactions would run backward and the cell would die. the atp Synthase Is a Nanomachine that produces atp by rotary Catalysis

1	the atp Synthase Is a Nanomachine that produces atp by rotary Catalysis The ATP synthase is a finely tuned nanomachine composed of 23 or more separate protein subunits, with a total mass of about 600,000 daltons. The ATP synthase can work both in the forward direction, producing ATP from ADP and phosphate in response to an electrochemical gradient, or in reverse, generating an electrochemical gradient by ATP hydrolysis. To distinguish it from other enzymes that hydrolyze ATP, it is also called an F1Fo ATP synthase or F-type ATPase.

1	Resembling a turbine, ATP synthase is composed of both a rotor and a stator (Figure 14–30). To prevent the catalytic head from rotating, a stalk at the periphery of the complex (the stator stalk) connects the head to stator subunits embedded in the membrane. A second stalk in the center of the assembly (the rotor stalk) is connected to the rotor ring in the membrane that turns as protons flow through it, driven by the electrochemical gradient across the membrane. As a result, proton

1	Figure 14–29 The basic relationship between free-energy changes and equilibrium in the ATP hydrolysis reaction. the rate constants in boxes 1 and 2 are determined from experiments in which product accumulation is measured as a function of time (conc., concentration). the equilibrium constant shown here, K, is in units of moles per liter. (See panel 2–7, pp. 102–103, for a discussion of free energy and see Figure 3–44 for a discussion of the equilibrium constant.) flow makes the rotor stalk rotate inside the stationary head, where the catalytic sites that assemble ATP from ADP and Pi are located. Three α and three β subunits of similar structure alternate to form the head. Each of the three β subunits has a catalytic nucleotide-binding site at the α/βinterface. These catalytic sites are all in different conformations, depending on their interaction with the rotor stalk. This stalk acts like a camshaft, the device that opens and closes the valves in a combustion engine. As it rotates

1	all in different conformations, depending on their interaction with the rotor stalk. This stalk acts like a camshaft, the device that opens and closes the valves in a combustion engine. As it rotates within the head, the stalk changes the conformations of the β subunits sequentially. One of the possible conformations of the catalytic sites has high affinity for ADP and Pi, and as the rotor stalk pushes the binding site into a different conformation, these two substrates are driven to form ATP. In this way, the mechanical force exerted by the central rotor stalk is directly converted into the chemical energy of the ATP phosphate bond.

1	Serving as a proton-driven turbine, the ATP synthase is driven by H+ flow into the matrix to spin at about 8000 revolutions per minute, generating three molecules of ATP per turn. In this way, each ATP synthase can produce roughly 400 molecules of ATP per second. proton-driven turbines are of ancient Origin

1	The membrane-embedded rotors of ATP synthases consist of a ring of identical c subunits (Figure 14–31). Each c subunit is a hairpin of two membrane-spanning α helices that contain a proton-binding site defined by a glutamate or aspartate in the middle of the lipid bilayer. The a subunit, which is part of the stator (see Figure 14–30), makes two narrow channels at the interface between the rotor and stator, each spanning half of the membrane and converging on the proton-binding site at the middle of the rotor subunit. Protons flow through the two half-channels down their electrochemical gradient from the crista space back into the matrix. A negatively charged side chain in the binding site accepts a proton arriving from the crista space through the first half-channel, as it rotates past the a subunit. The bound proton then rides round in the ring for a full cycle, whereupon it is thought to be displaced by a positively charged arginine in the a subunit, and escapes

1	Figure 14–30 ATP synthase. the three-dimensional structure of the F1Fo atp synthase, determined by x-ray crystallography. also known as an F-type atpase, it consists of an Fo part (from “oligomycin-sensitive factor”) in the membrane and the large, catalytic F1 head in the matrix. Under mild dissociation conditions, this complex separates into its F1 and Fo components, which can be isolated and studied individually. (a) Diagram of the enzyme complex showing how its globular head portion (green) is kept stationary as proton-flow across the membrane drives a rotor (blue) that turns inside it. (B) In bovine heart mitochondria, the Fo rotor ring in the membrane (light blue) has eight c subunits. It is attached to the γ subunit of the central stalk (dark blue) by the ε subunit (purple). the catalytic F1 head consists of a ring of three α and three β subunits (light and dark green), and it directly converts mechanical energy into chemical-bond energy in atp, as described in the text. the

1	catalytic F1 head consists of a ring of three α and three β subunits (light and dark green), and it directly converts mechanical energy into chemical-bond energy in atp, as described in the text. the elongated peripheral stalk of the stator (orange) is connected to the F1 head by the small δ subunit (red) at one end, and to the a subunit in the membrane (pink oval) at the other. together with the c subunits of the ring rotating past it, the a subunit creates a path for protons through the membrane. (C) the symbol for atp synthase used throughout this book.

1	the closely related atp synthases of mitochondria, chloroplasts, and bacteria synthesize atp by harnessing the proton-motive force across a membrane. this powers the rotation of the rotor against the stator in a counterclockwise direction, as seen from the F1 head. the same enzyme complex can also pump protons against their electrochemical gradient by hydrolyzing atp, which then drives the clockwise rotation of the rotor. the direction of operation depends on the net free-energy change (∆G) for the coupled processes of h+ translocation across the membrane and the synthesis of atp from aDp and pi (Movie 14.5 and Movie 14.6).

1	Measurement of the torque that the atp synthase can produce by atp hydrolysis reveals that the atp synthase is 60 times more powerful than a diesel engine of equal dimensions. (B, courtesy of K. Davies. pDB codes: 2WpD, 2CLy, 2WSS, 2BO5.) through the second half-channel into the matrix. Thus proton flow causes the rotor ring to spin against the stator like a proton-driven turbine. The mitochondrial ATP synthase is of ancient origin: essentially the same enzyme occurs in plant chloroplasts and in the plasma membrane of bacteria or archaea. The main difference between them is the number of c subunits in the rotor ring. In mammalian mitochondria, the ring has 8 subunits. In yeast mitochondria, the number is 10; in bacteria and archaea, it ranges from 11 to 13; in plant chloroplasts, there are 14; and the rings of some cyanobacteria contain 15 c subunits.

1	The c subunits in the rotor ring can be thought of as cogs in the gears of a bicycle. A high gear, with a small number of cogs, is advantageous when the supply of protons is limited, as in mitochondria, but a low gear, with a large number of cogs in the wheel, is preferable when the proton gradient is high. This is the case in chloroplasts and cyanobacteria, where protons produced through the action of sunlight are plentiful. Because each rotation produces three molecules of ATP in the head, the synthesis of one ATP requires around three protons in mitochondria but up to five in photosynthetic organisms. It is the number of c subunits in the ring that defines how many protons need to pass through this marvelous device to make each molecule of ATP, and thereby how high a ratio of ATP to ADP can be maintained by the ATP synthase.

1	In principle, ATP synthase can also run in reverse as an ATP-powered proton pump that converts the energy of ATP back into a proton gradient across the membrane. In many bacteria, the rotor of the ATP synthase in the plasma membrane changes direction routinely, from ATP synthesis mode in aerobic respiration, to ATP hydrolysis mode in anaerobic metabolism. In this latter case, ATP hydrolysis serves to maintain the proton gradient across the plasma membrane, which is used to power many other essential cell functions including nutrient transport and the rotation of bacterial flagella. The V-type ATPases that acidify certain cellular organelles are architecturally similar to the F-type ATP synthases, but they normally function in reverse (see Figure 13–37). Mitochondrial Cristae help to Make atp Synthesis Efficient (A)

1	In the electron microscope, the mitochondrial ATP synthase complexes can be seen to project like lollipops on the matrix side of cristae membranes. Recent studies by cryoelectron microscopy and tomography have shown that this large complex is not distributed randomly in the membrane, but forms long rows of dimers along the cristae ridges (Figure 14–32). The dimer rows induce or stabilize these regions of high membrane curvature, which are otherwise energetically unfavorable. Indeed, the formation of ATP synthase dimers and their assembly into rows are required for cristae formation and have far-reaching consequences for cellular fitness. By contrast with bacterial or chloroplast ATP synthases, which do not form dimers, the mitochondrial complex contains additional subunits, located mostly near the membrane end of the stator stalk. Several of these subunits are found to be dimer-specific. If these subunits are mutated in yeast, the ATP synthase in the membrane remains monomeric, the

1	near the membrane end of the stator stalk. Several of these subunits are found to be dimer-specific. If these subunits are mutated in yeast, the ATP synthase in the membrane remains monomeric, the mitochondria have no cristae, cellular respiration drops by half, and the cells grow more slowly.

1	Figure 14–31 Fo ATP synthase rotor rings. (a) atomic force microscopy image of atp synthase rotors from the cyanobacterium Synechococcus elongatus in a lipid bilayer. Whereas 8 c subunits form the rotor in Figure 14–30, there are 13 c subunits in this ring. (B) the x-ray structure of the Fo ring of the atp synthase from Spirulina platensis, another cyanobacterium, shows that this rotor has 15 c subunits. In all atp synthases, the c subunits are hairpins of two membrane-spanning α helices (one subunit is highlighted in gray). the helices are highly hydrophobic, except for two glutamine and glutamate side chains (yellow) that create proton-binding sites in the membrane. (a, courtesy of thomas Meier and Denys pogoryelov; B, pDB code: 2WIE.)

1	Electron tomography suggests that the proton pumps of the respiratory chain are located in the membrane regions at either side of the dimer rows. Protons pumped into the crista space by these respiratory-chain complexes are thought to diffuse very rapidly along the membrane surface, with the ATP synthase rows creating a proton “sink” at the cristae tips (Figure 14–33). In vitro studies suggest that the ATP synthase needs a proton gradient of about 2 pH units to produce ATP at the rate required by the cell, irrespective of the membrane potential. The H+ gradient across the inner mitochondrial membrane is only 0.5 to 0.6 pH units. The cristae thus seem to work as proton traps that enable the ATP synthase to make efficient use of the protons pumped out of the mitochondrial matrix. As we shall see in the next section, this elaborate arrangement of membrane protein complexes is absent in chloroplasts, where the H+ gradient is much higher.

1	Special transport proteins Exchange atp and aDp through the Inner Membrane Like all biological membranes, the inner mitochondrial membrane contains numerous specific transport proteins that allow particular substances to pass through. One of the most abundant of these is the ADP/ATP carrier protein (Figure 14–34). This carrier shuttles the ATP produced in the matrix through the inner membrane to the intermembrane space, from where it diffuses through the outer mitochondrial membrane to the cytosol. In exchange, ADP passes from the cytosol into the matrix for recycling into ATP. ATP4– has one more negative charge than ADP3–, and the exchange of ATP and ADP is driven by the electrochemical gradient across the inner membrane, so that the more negatively charged ATP is pushed out of the matrix, and the less negatively charged ADP is pulled in. The ADP/ATP carrier is but one member of a mitochondrial carrier family: the inner CYTOSOL pH 7.4 inner membraneMATRIX pH 7.9 ATP synthase

1	CYTOSOL pH 7.4 inner membraneMATRIX pH 7.9 ATP synthase Figure 14–32 Dimers of mitochondrial ATP synthase in cristae membranes. a three-dimensional map of a small mitochondrion obtained by electron microscope tomography shows that atp synthases form long paired rows along cristae ridges. the outer membrane is gray, the inner membrane and cristae membranes have been colored light blue. Each head of an atp synthase is indicated by a yellow sphere. (B) a three-dimensional map of a mitochondrial atp synthase dimer in the crista membrane obtained by subtomogram averaging, with fitted x-ray structures (Movie 14.7). (a, from K. Davies et al., Proc. Natl Acad. Sci. USA 108:14121–14126, 2011. With permission from the National academy of Sciences; B, from K. Davies et al., Proc. Natl Acad. Sci. USA 109:13602–13607, 2012. With permission from the National academy of Sciences.)

1	Figure 14–33 ATP synthase dimers at cristae ridges and ATP production. at the crista ridges, the atp synthases (yellow) form a sink for protons (red). the proton pumps of the electron-transport chain (green) are located in the membrane regions on either side of the crista. as illustrated, protons tend to diffuse along the membrane from their source to the proton sink created by the atp synthase. this allows efficient atp production despite the small h+ gradient between the cytosol and matrix. Red arrows show the direction of the proton flow.

1	Figure 14–34 The ADP/ATP carrier protein. (a) the aDp/atp carrier protein is a small membrane protein that carries the atp produced on the matrix side of the inner membrane to the intermembrane space, and the aDp that is needed for atp synthesis into the matrix. (B) In the aDp/atp carrier, six transmembrane α helices define a cavity that binds either aDp or atp. In this x-ray structure, the substrate is replaced by a tightly bound inhibitor instead (colored). When aDp binds from outside the inner membrane, it triggers a conformational change and is released into the matrix. In exchange, a molecule of atp quickly binds to the matrix side of the carrier and is transported to the intermembrane space. From there the atp diffuses through the outer mitochondrial membrane to the cytoplasm, where it powers the energy-requiring processes in the cell. (B, pDB code: 1OKC.) mitochondrial membrane contains about 20 related carrier proteins exchanging various other metabolites, including the

1	where it powers the energy-requiring processes in the cell. (B, pDB code: 1OKC.) mitochondrial membrane contains about 20 related carrier proteins exchanging various other metabolites, including the phosphate that is required along with ADP for ATP synthesis.

1	In some specialized fat cells, mitochondrial respiration is uncoupled from ATP synthesis by the uncoupling protein, another member of the mitochondrial carrier family. In these cells, known as brown fat cells, most of the energy of oxidation is dissipated as heat rather than being converted into ATP. In the inner membranes of the large mitochondria in these cells, the uncoupling protein allows protons to move down their electrochemical gradient without passing through ATP synthase. This process is switched on when heat generation is required, causing the cells to oxidize their fat stores at a rapid rate and produce heat rather than ATP. Tissues containing brown fat serve as “heating pads,” helping to revive hibernating animals and to protect newborn human babies from the cold.

1	Bacteria use enormously diverse energy sources. Some, like animal cells, are aerobic; they synthesize ATP from sugars they oxidize to CO2 and H2O by glycolysis, the citric acid cycle, and a respiratory chain in their plasma membrane that is similar to the one in the inner mitochondrial membrane. Others are strict anaerobes, deriving their energy either from glycolysis alone (by fermentation, see Figure 2–47) or from an electron-transport chain that employs a molecule other than oxygen as the final electron acceptor. The alternative electron acceptor can be a nitrogen compound (nitrate or nitrite), a sulfur compound (sulfate or sulfite), or a carbon compound (fumarate or carbonate), for example. A series of electron carriers in the plasma membrane that are comparable to those in mitochondrial respiratory chains transfers the electrons to these acceptors.

1	Despite this diversity, the plasma membrane of the vast majority of bacteria contains an ATP synthase that is very similar to the one in mitochondria. In bacteria that use an electron-transport chain to harvest energy, the electron-transport chain pumps H+ out of the cell and thereby establishes a proton-motive force across the plasma membrane that drives the ATP synthase to make ATP. In other bacteria, the ATP synthase works in reverse, using the ATP produced by glycolysis to pump H+ and establish a proton gradient across the plasma membrane.

1	Bacteria, including the strict anaerobes, maintain a proton gradient across their plasma membrane that is harnessed to drive many other processes. It can be used to drive a flagellar motor, for example (Figure 14–35). This gradient is harnessed to pump Na+ out of the bacterium via a Na+–H+ antiporter that takes the place of the Na+-K+ pump of eukaryotic cells. The gradient is also used for the active inward transport of nutrients, such as most amino acids and many sugars: each nutrient is dragged into the cell along with one or more protons through a specific symporter (Figure 14–36; see also Chapter 11). In animal cells, by contrast, most inward transport across the plasma membrane is driven by the Na+ gradient (high Na+ outside, low Na+ inside) that is established by the Na+-K+ pump (see Figure 11–15).

1	Some unusual bacteria have adapted to live in a very alkaline environment and yet must maintain their cytoplasm at a physiological pH. For these cells, any attempt to generate an electrochemical H+ gradient would be opposed by a large H+ concentration gradient in the wrong direction (H+ higher inside than outside). Presumably for this reason, some of these bacteria substitute Na+ for H+ in all of their chemiosmotic mechanisms. The respiratory chain pumps Na+ out of the cell, the transport systems and flagellar motor are driven by an inward flux of Na+, and a Na+-driven ATP synthase synthesizes ATP. The existence of such bacteria demonstrates a critical point: the principle of chemiosmosis is more fundamental than the proton-motive force on which it is normally based. As we discuss next, an ATP synthase coupled to chemiosmotic processes is also a central feature of plants, where it plays critical roles in both mitochondria and chloroplasts.

1	As we discuss next, an ATP synthase coupled to chemiosmotic processes is also a central feature of plants, where it plays critical roles in both mitochondria and chloroplasts. Figure 14–36 The importance of H+-driven transport in bacteria. a proton-motive force generated across the plasma membrane pumps nutrients into the cell and expels Na+. (a) In an aerobic bacterium, a respiratory chain fed by the oxidation of substrates produces an electrochemical proton gradient across the plasma membrane. this gradient is then harnessed to make atp, as well as to transport nutrients (proline, succinate, lactose, and lysine) into the cell and to pump Na+ out of the cell.

1	(B) When the same bacterium grows under anaerobic conditions, it derives its atp from glycolysis. as indicated, the atp synthase in the plasma membrane then hydrolyzes some of this atp to establish an electrochemical proton gradient that drives the same transport processes that depend on respiratory chain proton-pumping in (a). Figure 14–35 The rotation of the bacterial flagellum driven by H+ flow. the flagellum is attached to a series of protein rings (pink), which are embedded in the outer and inner membranes and rotate with the flagellum. the rotation is driven by a flow of protons through an outer ring of proteins (the stator) by mechanisms that may resemble those used by the atp synthase. however, the flow of protons in the flagellar motor is always toward the cytosol, both during clockwise and counterclockwise rotation, whereas in atp synthase this flow reverses with the direction of rotation (Movie 14.8).

1	The large amount of free energy released when H+ flows back into the matrix from the cristae provides the basis for ATP production on the matrix side of mitochondrial cristae membranes by a remarkable protein machine—the ATP synthase. The ATP synthase functions like a miniature turbine, and it is a reversible device that can couple proton flow to either ATP synthesis or ATP hydrolysis. The transmembrane electrochemical gradient that drives ATP production in mitochondria also drives the active transport of selected metabolites across the inner mitochondrial membrane, including an efficient ADP/ATP exchange between the mitochondrion and the cytosol that keeps the cell’s ATP pool highly charged. The resulting high cellular concentration of ATP makes the free-energy change for ATP hydrolysis extremely favorable, allowing this hydrolysis reaction to drive a very large number of energy-requiring processes throughout the cell. The universal presence of ATP synthase in bacteria, mitochondria,

1	favorable, allowing this hydrolysis reaction to drive a very large number of energy-requiring processes throughout the cell. The universal presence of ATP synthase in bacteria, mitochondria, and chloroplasts testifies to the central importance of chemiosmotic mechanisms in cells.

1	All animals and most microorganisms rely on the continual uptake of large amounts of organic compounds from their environment. These compounds provide both the carbon-rich building blocks for biosynthesis and the metabolic energy for life. It is likely that the first organisms on the primitive Earth had access to an abundance of organic compounds produced by geochemical processes, but it is clear that these were used up billions of years ago. Since that time, virtually all of the organic materials required by living cells have been produced by photosynthetic organisms, including plants and photosynthetic bacteria. The core machinery that drives all photosynthesis appears to have evolved more than 3 billion years ago in the ancestors of present-day bacteria; today it provides the only major solar energy storage mechanism on Earth.

1	The most advanced photosynthetic bacteria are the cyanobacteria, which have minimal nutrient requirements. They use electrons from water and the energy of sunlight to convert atmospheric CO2 into organic compounds—a process called carbon fixation. In the course of the overall reaction nH2O + nCO2 → (light) (CH2O)n + nO2, they also liberate into the atmosphere the molecular oxygen that then powers oxidative phosphorylation. In this way, it is thought that the evolution of cyanobacteria from more primitive photosynthetic bacteria eventually made possible the development of the many different aerobic life-forms that populate the Earth today.

1	Plants (including algae) developed much later than cyanobacteria, and their photosynthesis occurs in a specialized intracellular organelle—the chloroplast (Figure 14–37). Chloroplasts use chemiosmotic mechanisms to carry out their energy interconversions in much the same way that mitochondria do. Although much larger than mitochondria, they are organized on the same principles. They have a highly permeable outer membrane; a much less permeable inner membrane, in which membrane transport proteins are embedded; and a narrow intermembrane space in between. Together, these two membranes form the chloroplast envelope (Figure 14–37D). The inner chloroplast membrane surrounds a large space called the stroma, which is analogous to the mitochondrial matrix. The stroma contains many metabolic enzymes and, as for the mitochondrial matrix, it is the place where ATP is made by the head of an ATP synthase. Like the mitochondrion, the chloroplast has its own genome and genetic system. The stroma

1	and, as for the mitochondrial matrix, it is the place where ATP is made by the head of an ATP synthase. Like the mitochondrion, the chloroplast has its own genome and genetic system. The stroma therefore also contains a special set of ribosomes, RNAs, and the chloroplast DNA.

1	An important difference between the organization of mitochondria and chloroplasts is highlighted in Figure 14–38. The inner membrane of the chloroplast is not folded into cristae and does not contain electron-transport chains. Instead, the electron-transport chains, photosynthetic light-capturing systems, and ATP synthase are all contained in the thylakoid membrane, a separate, distinct mem-brane that forms a set of flattened, disc-like sacs, the thylakoids. The thylakoid membrane is highly folded into numerous local stacks of flattened vesicles called grana, interconnected by nonstacked thylakoids. The lumen of each thylakoid is connected with the lumen of other thylakoids, thereby defining a third internal compartment called the thylakoid space. This space represents a separate com-partment in each chloroplast that is not connected to either the intermembrane space or the stroma. Chloroplasts Capture Energy from Sunlight and Use It to Fix Carbon We can group the reactions that occur

1	in each chloroplast that is not connected to either the intermembrane space or the stroma. Chloroplasts Capture Energy from Sunlight and Use It to Fix Carbon We can group the reactions that occur during photosynthesis in chloroplasts into two broad categories: 1. The photosynthetic electron-transfer reactions (also called the “light reactions”) occur in two large protein complexes, called reaction centers, embedded in the thylakoid membrane. A photon (a quantum of light) knocks an electron out of the green pigment molecule chlorophyll in the first reaction center, creating a positively charged chlorophyll ion. This electron then moves along an electron-transport chain and through a sec-ond reaction center in much the same way that an electron moves along the respiratory chain in mitochondria. During this electron-transport process, H+ is pumped across the thylakoid membrane, and the resulting LEAF upper epidermis lower epidermis nucleus cytosol vacuole air space cell wall

1	in mitochondria. During this electron-transport process, H+ is pumped across the thylakoid membrane, and the resulting LEAF upper epidermis lower epidermis nucleus cytosol vacuole air space cell wall mitochondrion (B) (C)(A) 10 mm chloroplast chloroplast (D) 5 µm 0.5 µm

1	Figure 14–37 Chloroplasts in the cell. Schematic cross section through the leaf of a green plant. (B) Light microscopy of a plant leaf cell—here, a mesophyll cell from Zinnia elegans—shows chloroplasts as bright green bodies, measuring several micrometers across, in the transparent cell interior. (C) the electron micrograph of a thin, stained section through a wheat leaf cell shows a thin rim of cytoplasm— containing chloroplasts, the nucleus, and mitochondria—surrounding a large, water-filled vacuole. (D) at higher magnification, electron microscopy reveals the chloroplast envelope membrane and the thylakoid membrane within the chloroplast that is highly folded into grana stacks (Movie 14.9). (B, courtesy of John Innes Foundation; C and D, courtesy of K. plaskitt.)

1	K. plaskitt.) Figure 14–38 A mitochondrion and chloroplast compared. Chloroplasts are generally larger than mitochondria. In addition to an outer and inner envelope membrane, they contain the thylakoid membrane with its internal thylakoid space. the chloroplast thylakoid membrane, which is the site of solar energy conversion in plants and algae, corresponds to the mitochondrial cristae, which are the sites of energy conversion by cellular respiration. Unlike the crista membrane, which is continuous with the inner mitochondrial membrane at cristae junctions, the thylakoid membrane is not connected to the inner chloroplast membrane at any point.

1	electrochemical proton gradient drives the synthesis of ATP in the stroma. As the final step in this series of reactions, electrons are loaded (together with H+) onto NADP+, converting it to the energy-rich NADPH molecule. Because the positively charged chlorophyll in the first reaction center quickly regains its electrons from water (H2O), O2 gas is produced as a by-product. All of these reactions are confined to the chloroplast.

1	2. The carbon-fixation reactions do not require sunlight. Here the ATP and NADPH generated by the light reactions serve as the source of energy and reducing power, respectively, to drive the conversion of CO2 to carbohydrate. These carbon-fixation reactions begin in the chloroplast stroma, where they generate the three-carbon sugar glyceraldehyde 3-phosphate. This simple sugar is exported to the cytosol, where it is used to produce sucrose and many other organic metabolites in the leaves of the plant. The sucrose is then exported to meet the metabolic needs of the nonphotosynthetic plant tissues, serving as a source of both carbon skeletons and energy for growth.

1	Thus, the formation of ATP, NADPH, and O2 (which requires light energy directly) and the conversion of CO2 to carbohydrate (which requires light energy only indirectly) are separate processes (Figure 14–39). However, they are linked by elaborate feedback mechanisms that allow a plant to manufacture sugars only when it is appropriate to do so. Several of the chloroplast enzymes required for carbon fixation, for example, are inactive in the dark and reactivated by light-stimulated electron-transport processes. Carbon Fixation Uses atp and NaDph to Convert CO2 into Sugars

1	Carbon Fixation Uses atp and NaDph to Convert CO2 into Sugars We have seen earlier in this chapter how animal cells produce ATP by using the large amount of free energy released when carbohydrates are oxidized to CO2 and H2O. The reverse reaction, in which plants make carbohydrate from CO2 and H2O, takes place in the chloroplast stroma. The large amounts of ATP and NADPH produced by the photosynthetic electron-transfer reactions are required to drive this energetically unfavorable reaction. Figure 14–39 A summary of the energy-converting metabolism in chloroplasts.

1	Chloroplasts require only water and carbon dioxide as inputs for their light-driven photosynthesis reactions, and they produce the nutrients for most other organisms on the planet. Each oxidation of two water molecules by a photochemical reaction center in the thylakoid membrane produces one molecule of oxygen, which is released into the atmosphere. at the same time, protons are concentrated in the thylakoid space. these protons create a large electrochemical gradient across the thylakoid membrane, which is utilized by the chloroplast atp synthase to produce atp from aDp and phosphate. the electrons withdrawn from water are transferred to a second type of photochemical reaction center to produce NaDph from NaDp+. as indicated, the NaDph and atp are fed into the carbon-fixation cycle to reduce carbon dioxide, thereby producing the precursors for sugars, amino acids, and fatty acids. the CO2 that is taken up from the atmosphere here is the source of the carbon atoms for most organic

1	reduce carbon dioxide, thereby producing the precursors for sugars, amino acids, and fatty acids. the CO2 that is taken up from the atmosphere here is the source of the carbon atoms for most organic molecules on Earth.

1	In a plant cell, a variety of metabolites produced in the chloroplast are exported to the cytoplasm for biosyntheses. Some of the sugar produced is stored in the form of starch granules in the chloroplast, but the rest is transported throughout the plant as sucrose or converted to starch in special storage tissues. these storage tissues serve as a major food source for animals. carbon ribulose 1,5-bisphosphate intermediate 2 molecules of dioxide 3-phosphoglycerate

1	Figure 14–40 illustrates the central reaction of carbon fixation, in which an atom of inorganic carbon is converted to organic carbon: CO2 from the atmosphere combines with the five-carbon compound ribulose 1,5-bisphosphate plus water to yield two molecules of the three-carbon compound 3-phosphoglycerate. This carboxylation reaction is catalyzed in the chloroplast stroma by a large enzyme called ribulose bisphosphate carboxylase, or Rubisco for short. Because the reaction is so slow (each Rubisco molecule turns over only about 3 molecules of substrate per second, compared to 1000 molecules per second for a typical enzyme), an unusually large number of enzyme molecules are needed. Rubisco often constitutes more than 50% of the chloroplast protein mass, and it is thought to be the most abundant protein on Earth. In a global context, Rubisco also keeps the amount of the greenhouse gas CO2 in the atmosphere at a low level.

1	Although the production of carbohydrates from CO2 and H2O is energetically unfavorable, the fixation of CO2 catalyzed by Rubisco is an energetically favorable reaction. Carbon fixation is energetically favorable because a continuous supply of the energy-rich ribulose 1,5-bisphosphate is fed into the process. This compound is consumed by the addition of CO2, and it must be replenished. The energy and reducing power needed to regenerate ribulose 1,5-bisphosphate come from the ATP and NADPH produced by the photosynthetic light reactions.

1	The elaborate series of reactions in which CO2 combines with ribulose 1,5-bisphosphate to produce a simple sugar—a portion of which is used to regenerate ribulose 1,5-bisphosphate—forms a cycle, called the carbon-fixation cycle, or the Calvin cycle (Figure 14–41). This cycle was one of the first metabolic pathways to be worked out by applying radioisotopes as tracers in biochemistry. As indicated, each turn of the cycle converts six molecules of 3-phosphoglycerate to three molecules of ribulose 1,5-bisphosphate plus one molecule of glyceraldehyde 3-phosphate. Glyceraldehyde 3-phosphate, the three-carbon sugar produced by the cycle, then provides the starting material for the synthesis of many other sugars and all of the other organic molecules that form the plant. Sugars Generated by Carbon Fixation Can Be Stored as Starch or Consumed to produce atp

1	Sugars Generated by Carbon Fixation Can Be Stored as Starch or Consumed to produce atp The glyceraldehyde 3-phosphate generated by carbon fixation in the chloroplast stroma can be used in a number of ways, depending on the needs of the plant. During periods of excess photosynthetic activity, much of it is retained in the chloroplast stroma and converted to starch. Like glycogen in animal cells, starch is a large polymer of glucose that serves as a carbohydrate reserve, and it is stored as large granules in the chloroplast stroma. Starch forms an important part of the diet of all animals that eat plants. Other glyceraldehyde 3-phosphate molecules are converted to fat in the stroma. This material, which accumulates as fat droplets, likewise serves as an energy reserve. At night, this stored starch and fat can be broken down to sugars and fatty acids, which are exported to the cytosol to help support the metabolic needs of the plant. Some of the exported sugar enters the

1	Figure 14–40 The initial reaction in carbon fixation. this carboxylation reaction allows one molecule each of carbon dioxide and water to be incorporated into organic carbon molecules. It is catalyzed in the chloroplast stroma by the abundant enzyme ribulose bisphosphate carboxylase, or rubisco. as indicated, the product is two molecules of 3-phosphoglycerate.

1	6 6 6 6 6 6 × 3-phosphoglycerate 6 × 1,3-bisphosphoglycerate 6 × glyceraldehyde 3-phosphate 1 MOLECULE OF GLYCERALDEHYDE 3-PHOSPHATE LEAVES THE CYCLE 5 × glyceraldehyde 3-phosphate 3 ×ribulose 1,5-bisphosphate 3C 5C 3C 3C3C Rubisco ATPADPATPADPNADPHNADP+Pi Pi CARBON-FIXATION (CALVIN) CYCLE NET RESULT: For every 3 molecules of CO2 that enter the cycle, 1 molecule of glyceraldehyde 3-phosphate is produced and 9 molecules of ATP + 6 molecules of NADPH are consumed REGENERATIONCARBOXYLATIONREDUCTION sugars, fats, amino acids glycolytic pathway (see Figure 2–46), where it is converted to pyruvate. Both that pyruvate and the fatty acids can enter the plant cell mitochondria and be fed into the citric acid cycle, ultimately leading to the production of large amounts of ATP by oxidative phosphorylation (Figure 14–42). Plants use this ATP in the same way that animal cells and other nonphotosynthetic organisms do to power a variety of metabolic reactions.

1	The glyceraldehyde 3-phosphate exported from chloroplasts into the cytosol can also be converted into many other metabolites, including the disaccharide sucrose. Sucrose is the major form in which sugar is transported between the cells of a plant: just as glucose is transported in the blood of animals, so sucrose is exported from the leaves to provide carbohydrate to the rest of the plant. the thylakoid Membranes of Chloroplasts Contain the protein Complexes required for photosynthesis and atp Generation

1	We next need to explain how the large amounts of ATP and NADPH required for carbon fixation are generated in the chloroplast. Chloroplasts are much larger and less dynamic than mitochondria, but they make use of chemiosmotic energy conversion in much the same way. As we saw in Figure 14–38, chloroplasts and mitochondria are organized on the same principles, although the chloroplast contains a separate thylakoid membrane system in which its chemiosmotic mechanisms occur. The thylakoid membranes contain two large membrane protein complexes, called photosystems, which endow plants and other photosynthetic organisms with the ability to capture and convert solar energy for their own use. Two other protein complexes in the thylakoid membrane that work together with the photosystems in photophosphorylation—the generation of ATP with sunlight— have mitochondrial equivalents. These are the heme-containing cytochrome

1	Figure 14–41 The carbon-fixation cycle. this central metabolic pathway allows organic molecules to be produced from CO2 and h2O. In the first stage of the cycle (carboxylation), CO2 is added to ribulose 1,5-bisphosphate, as shown in Figure 14–40. In the second stage (reduction), atp and NaDph are consumed to produce glyceraldehyde 3-phosphate molecules. In the final stage (regeneration), some of the glyceraldehyde 3-phosphate produced is used to regenerate ribulose 1,5-bisphosphate. Other glyceraldehyde 3-phosphate molecules are either converted to starch and fat in the chloroplast stroma, or transported out of the chloroplast into the cytosol. the number of carbon atoms in each type of molecule is indicated in yellow. there are many intermediates between glyceraldehyde 3-phosphate and ribulose 5-phosphate, but they have been omitted here for clarity. the entry of water into the cycle is also not shown (but see Figure 14–40).

1	Figure 14–42 How chloroplasts and mitochondria collaborate to supply cells with both metabolites and ATP. (a)the inner chloroplast membrane is impermeable to the atp and NaDph that are produced in the stroma during the light reactions of photosynthesis. these molecules are therefore funneled into the carbon-fixation cycle, where they are used to make sugars. the resulting sugars and their metabolites are either stored within the chloroplast—in the form of starch or fat—or exported to the rest of the plant cell. there, they can enter the energy-generating pathway that ends in atp synthesis linked to oxidative phosphorylation inside the mitochondrion. Unlike the chloroplast, mitochondrial membranes contain a specific transporter that makes them permeable to atp (see Figure 14–34). Note that the O2 released to the atmosphere by photosynthesis in chloroplasts is used for oxidative phosphorylation in mitochondria; similarly, the CO2 released by the citric acid cycle in mitochondria is used

1	the O2 released to the atmosphere by photosynthesis in chloroplasts is used for oxidative phosphorylation in mitochondria; similarly, the CO2 released by the citric acid cycle in mitochondria is used for carbon fixation in chloroplasts. (B) In a leaf, mitochondria (red) tend to cluster close to the chloroplasts (green), as seen in this light micrograph. (B, courtesy of Olivier Grandjean.) b6-f complex, which both functionally and structurally resembles cytochrome CH2 c reductase in the respiratory chain; and the chloroplast ATP synthase, which closely resembles the mitochondrial ATP synthase and works in the same way.

1	The photosystems in the thylakoid membrane are multiprotein assemblies of a complexity comparable to that of the protein complexes in the mitochondrial electron-transport chain. They contain large numbers of specifically bound chlorophyll molecules, in addition to cofactors that will be familiar from our discus-CH2 sion of mitochondria (heme, iron–sulfur clusters, and quinones). Chlorophyll, the green pigment of photosynthetic organisms, has a long hydrophobic tail that makes it behave like a lipid, plus a porphyrin ring that has a central Mg atom and an extensive system of delocalized electrons in conjugated double bonds (Figure 14–43). When a chlorophyll molecule absorbs a quantum of sunlight (a photon), the energy of the photon causes one of these electrons to move from a low-energy molecular orbital to another orbital of higher energy.

1	The excited electron in a chlorophyll molecule tends to return quickly to its ground state, which can occur in one of three ways: 1. By converting the extra energy into heat (molecular motion) or to some combination of heat and light of a longer wavelength (fluorescence); this is what usually happens when light is absorbed by an isolated chlorophyll molecule in solution. 2. By transferring the energy—but not the electron—directly to a neighboring chlorophyll molecule by a process called resonance energy transfer. 3. By transferring the excited electron with its negative charge to another nearby molecule, an electron acceptor, after which the positively charged chlorophyll returns to its original state by taking up an electron from some other molecule, an electron donor.

1	Figure 14–43 The structure of chlorophyll. a magnesium atom is held in a porphyrin ring, which is related to the porphyrin ring that binds iron in heme (see Figure 14–15). Electrons are delocalized over the bonds shaded in blue. The latter two mechanisms occur when chlorophylls are attached to proteins in a chlorophyll–protein complex. The protein coordinates the central Mg atom in the chlorophyll porphyrin, most often through a histidine side chain located in the hydrophobic interior of a membrane, causing each of the chlorophylls in a protein complex to be held at exactly defined distances and orientations. The flow of excitation energy or electrons then depends on both the precise spatial arrangement and the local protein environment of the protein-bound chlorophylls.

1	When excited by a photon, most protein-bound chlorophylls simply transmit the absorbed energy to another nearby chlorophyll by the process of resonance energy transfer. However, in a few specially positioned chlorophylls, the energy difference between the ground state and the excited state is just right for the photon to trigger a light-induced chemical reaction. The special state of such chlorophyll molecules derives from their close interaction with a second chlorophyll molecule in the same chlorophyll–protein complex. Together, these two chlorophylls form a special pair.

1	The photosynthetic electron transfer process starts when a photon of suitable energy ionizes a chlorophyll molecule in such a special pair, dissociating it into an electron and a positively charged chlorophyll ion. The energized electron is passed rapidly to a quinone in the same protein complex, preventing its unproductive reassociation with the chlorophyll ion. This light-induced transfer of an electron from a chlorophyll to a mobile electron carrier is the central charge-separation step in photosynthesis, in which a chlorophyll becomes positively charged and an electron carrier becomes negatively charged (Figure 14–44). The chlorophyll ion is a very strong oxidant that is able to withdraw an electron from a low-energy substrate; in the first step of oxygenic photosynthesis, this low-energy substrate is water.

1	Upon transfer to a mobile carrier in the electron-transport chain, the electron is stabilized as part of a strong electron donor and made available for subsequent reactions. These subsequent reactions require more time to complete, and they result in light-generated energy-rich compounds. a photosystem Consists of an antenna Complex and a reaction Center There are two distinct types of chlorophyll–protein complexes in the photosynthetic membrane. One type, called a photochemical reaction center, contains the special pair of chlorophylls just described. The other type engages exclusively in light absorption and resonance energy transfer and is called an antenna complex. Together, the two types of complex make up a photosystem (Figure 14–45).

1	The role of the antenna complex in the photosystem is to collect the energy of a sufficient number of photons for photosynthesis. Without it, the process would be slow and inefficient, as each reaction-center chlorophyll would absorb only about one light quantum per second, even in broad daylight, whereas hundreds per second are needed for effective photosynthesis. When light excites a chlorophyll molecule in the antenna complex, the energy passes rapidly from one protein-bound chlorophyll to another by resonance energy transfer until it reaches the special pair in the reaction center. The antenna complex is also known as a

1	Figure 14–44 A general scheme for the charge-separation step in a photosynthetic reaction center. In a reaction center, light energy is harnessed to generate electrons that are held at a high energy level by mobile electron carriers in a membrane. Light energy is thereby converted to chemical energy. the process starts when a photon absorbed by the special pair of chlorophylls in the reaction center knocks an electron out of one of the chlorophylls. the electron is taken up by a mobile electron carrier (orange) bound at the opposite membrane surface. a set of intermediary carriers embedded in the reaction center provide the path from the special pair to this carrier (not shown). the physical distance between the positively charged chlorophyll ion and the negatively charged electron carrier stabilizes the charge-separated state for a short time, during which the chlorophyll ion, a strong oxidant, withdraws an electron from a suitable compound (for example, from water, an event we will

1	stabilizes the charge-separated state for a short time, during which the chlorophyll ion, a strong oxidant, withdraws an electron from a suitable compound (for example, from water, an event we will discuss in detail shortly). the electron carrier then diffuses away from the reaction center as a strong electron donor that will transfer its electron to an electron-transport chain.

1	light-harvesting complex, or LHC. In addition to many chlorophyll molecules, an LHC contains orange carotenoid pigments. The carotenoids collect light of a different wavelength from that absorbed by chlorophylls, helping to make the antenna complex more efficient. They also have an important protective role in preventing the formation of harmful oxygen radicals in the photosynthetic membrane. the thylakoid Membrane Contains two Different photosystems Working in Series The excitation energy collected by the antenna complex is delivered to the special pair in the photochemical reaction center. The reaction center is a transmembrane chlorophyll–protein complex that lies at the heart of photosynthesis. It harbors the special pair of chlorophyll molecules, which acts as an irreversible trap for excitation energy (see Figure 14–45).

1	Chloroplasts contain two functionally different although structurally related photosystems, each of which feeds electrons generated by the action of sunlight into an electron-transfer chain. In the chloroplast thylakoid membrane, photo-system I is confined to the unstacked stroma thylakoids, while the stacked grana thylakoids contain photosystem II. The two photosystems were named in order of their discovery, not of their actions in the photosynthetic pathway, and electrons are first activated in photosystem II before being transferred to photosystem I (Figure 14–46). The path of the electron through the two photosystems can be described as a Z-like trajectory and is known as the Z scheme. In the Z scheme, the reaction center of photosystem II first withdraws an electron from water. The electron passes via an electron-transport chain (composed of the electron carrier plastoquinone, the cytochrome b6-f complex, and the protein plastocyanin) to photosystem I, which propels the electron

1	passes via an electron-transport chain (composed of the electron carrier plastoquinone, the cytochrome b6-f complex, and the protein plastocyanin) to photosystem I, which propels the electron across the membrane in a second light-driven charge-separation reaction that leads to NADPH production.

1	Figure 14–45 A photosystem. Each photosystem consists of a reaction center plus a number of light-harvesting antenna complexes. the solar energy for photosynthesis is collected by the antenna complexes, which account for most of the chlorophyll in a plant cell. the energy hops randomly by resonance energy transfer (red arrows) from one chlorophyll molecule to another, until it reaches the reaction center complex, where it ionizes a chlorophyll in the special pair. the chlorophyll special pair holds its electrons at a lower energy than the chlorophyll in the antenna complexes, causing the energy transferred to it from the antenna complex to become trapped there. Note that it is only energy that moves from one chlorophyll molecule to another in the antenna complex, not electrons (Movie 14.10).

1	Figure 14–46 The Z scheme for photosynthesis. the thylakoids of plants and cyanobacteria contain two different photosystems, known as photosystem I and photosystem II, which work in series. Each of the photosystem I and II reaction centers receives excitation energy from its own set of tightly associated antenna complexes, known as LhC-I and LhC-II, by resonance energy transfer. Note that, for historical reasons, the two photosystems were named opposite to the order in which they act, with photosystem II passing its electrons to photosystem I.

1	The Z scheme is necessary to bridge the very large energy gap between water and NADPH (Figure 14–47). A single quantum of visible light does not contain enough energy both to withdraw electrons from water, which holds on to its electrons very tightly (redox potential +820 mV) and therefore is a very poor electron donor, and to force them on to NADP+, which is a very poor electron acceptor (redox potential –320 mV). The Z scheme first evolved in cyanobacteria to enable them to use water as a universally available electron source. Other, simpler photosynthetic bacteria have only one photosystem. As we shall see, they cannot use water as an electron source and must rely on other, more energy-rich substrates instead, from which electrons are more readily withdrawn. The ability to extract electrons from water (and thereby to produce molecular oxygen) was acquired by plants when their ancestors took up the endosymbiotic cyanobacteria that later evolved into chloroplasts (see Figure 1–31).

1	photosystem II Uses a Manganese Cluster to Withdraw Electrons From Water In biology, only photosystem II is able to withdraw electrons from water and to generate molecular oxygen as a waste product. This remarkable specialization of photosystem II is conferred by the unique properties of one of the two chlorophyll molecules of its special pair and by a manganese cluster linked to the protein. These chlorophyll molecules and the manganese cluster form the catalytic core of the photosystem II reaction center, whose mechanism is outlined in Figure 14–48.

1	Water is an inexhaustible source of electrons, but it is also extremely stable; therefore a large amount of energy is required to make it part with its electrons. The only compound in living organisms that is able to achieve this feat after its ionization by light, is the chlorophyll special pair called P680 (P680/P680+ redox potential = +1270 mV). The reaction 2H2O + 4 photons →4H+ + 4e – + O2 is catalyzed by its adjacent manganese cluster. The intermediates remain firmly attached to the manganese cluster until two water molecules have been fully oxidized to O2, thus

1	Figure 14–47 Changes in redox potential during photosynthesis. the redox potential for each molecule is indicated by its position along the vertical axis. photosystem II passes electrons derived from water to photosystem I, which in turn passes them to NaDp+ through ferredoxin-NaDp+ reductase. the net electron flow through the two photosystems is from water to NaDp+, and it produces NaDph as well as an electrochemical proton gradient. this proton gradient is used by the atp synthase to produce atp. Details in this figure will be explained in the subsequent text.

1	ensuring that no dangerous oxygen radicals are released as the reaction proceeds. The protons released by the two water molecules are discharged to the thylakoid space, contributing to the proton gradient across the thylakoid membrane (pH lower in the thylakoid space than in the stroma). The unique protein environment that endows life with this all-important ability to oxidize water has remained essentially unchanged throughout billions of years of evolution (Figure 14–49). All of the oxygen in the Earth’s atmosphere has been generated in this way. Although the exact details of the water-oxidation reaction in photosystem II are still not fully understood, scientists are trying to construct an artificial system that mimics the process. If successful, this might provide a virtually endless supply of clean energy, helping to solve the world’s energy crisis. the Cytochrome b6-f Complex Connects photosystem II to photosystem I

1	the Cytochrome b6-f Complex Connects photosystem II to photosystem I Following the path shown previously in Figure 14–48, the electrons extracted from water by photosystem II are transferred to plastoquinol, a strong electron donor similar to ubiquinol in mitochondria. This quinol, which can diffuse rapidly in the lipid bilayer of the thylakoid membrane, transfers its electrons to the cytochrome b6-f complex, whose structure is homologous to the cytochrome c reductase in mitochondria. The cytochrome b6-f complex pumps H+ into the thylakoid space using the same Q cycle that is utilized in mitochondria (see Figure 14–21), thereby adding to the proton gradient across the thylakoid membrane. The cytochrome b6-f complex forms the connecting link between photosystems II and I in the chloroplast electron-transport chain. It passes its electrons

1	Figure 14–48 The conversion of light energy to chemical energy in the photosystem II complex. (a) Schematic diagram of the photosystem II reaction center, whose special pair of chlorophyll molecules is designated as p680 based on the wavelength of its absorbance maximum (680 nm). (B) Cofactors and pigments at the core of the reaction center. Shown are the manganese (Mn) cluster, the tyrosine side chain that links it to the p680 special pair, four chlorophylls (green), two pheophytins (light blue), two plastoquinones (pink), and an iron atom (red). the path of electrons is shown by blue arrows. In the manganese cluster, four manganese atoms (light blue), one calcium atom (purple), and five oxygen atoms (red) work together to catalyze the oxidation of water. the water-splitting reaction occurs in four successive steps, each requiring the energy of one photon. Each photon turns a p680 reaction-center chlorophyll into a positively charged chlorophyll ion. through an ionized tyrosine side

1	in four successive steps, each requiring the energy of one photon. Each photon turns a p680 reaction-center chlorophyll into a positively charged chlorophyll ion. through an ionized tyrosine side chain (yellow), this chlorophyll ion pulls an electron away from a water molecule bound at the manganese cluster. In this way, a total of four electrons are withdrawn from two water molecules to generate molecular oxygen, which is released into the atmosphere.

1	Each electron that is energized by light passes from the special pair along an electron-transfer chain inside the complex, along the indicated path to the permanently bound plastoquinone Qa and then to plastoquinone QB as electron acceptors. Once QB has picked up two electrons (plus two protons; see Figure 14–17), it dissociates from its binding site in the complex and enters the lipid bilayer as a mobile electron carrier, being immediately replaced by a new, nonreduced molecule of plastoquinone. Note that the chlorophylls and pheophytins form two symmetrical branches of a potential electron-transport chain. Only one branch is active, thus ensuring that the plastoquinones become fully reduced in minimum time.

1	Figure 14–49 The structure of the complete photosystem II complex. this photosystem contains at least 16 protein subunits, along with 36 chlorophylls, two pheophytins, two hemes, and a number of protective carotenoids (colored). Most of these pigments and cofactors are deeply buried, tightly complexed to protein (gray). the path of electrons is indicated by the blue arrows, and is explained in Figure 14–48B. the photosystem II complex presented here is the cyanobacterial complex, which is simpler and more stable than the plant complex, which works in the same way. (pDB code: 3arC.) one at a time to the mobile electron carrier plastocyanin (a small copper-containing protein that takes the place of the cytochrome c in mitochondria), which will transfer them to photosystem I (Figure 14–50). As we discuss next, photosystem I then harnesses a second photon of light to further energize the electrons that it receives.

1	photosystem I Carries Out the Second Charge-Separation Step in the Z Scheme Photosystem I receives electrons from plastocyanin in the thylakoid space and transfers them, via a second charge-separation reaction, to the small protein ferredoxin on the opposite membrane surface (Figure 14–51). Then, in a final step, ferredoxin feeds its electrons to a membrane-associated enzyme complex, the ferredoxin-NADP+ reductase, which uses the electrons to produce NADPH from NADP+ (see Figure 14–50).

1	The redox potential of the NADP+/NADPH pair (–320 mV) is already very low, and reduction of NADP+ therefore requires a compound with an even lower redox potential. This turns out to be a chlorophyll molecule near the stromal membrane surface of photosystem I that has a redox potential of –1000 mV (chlorophyll A0), making it the strongest known electron donor in biology. The reduced NADPH is released into the chloroplast stroma, where it is used for biosynthesis of glyceraldehyde 3-phosphate, amino acid precursors, and fatty acids, much of it to be exported to the cytoplasm. Figure 14–50 Electron flow through the cytochrome b6-f complex to NADPH.

1	Figure 14–50 Electron flow through the cytochrome b6-f complex to NADPH. the cytochrome b6-f complex is the functional equivalent of cytochrome c reductase (the cytochrome b-c1 complex) in mitochondria (see Figure 14–22). Like its mitochondrial homolog, the b6-f complex receives its electrons from a quinone and engages in a complicated Q cycle that pumps two protons across the membrane (details not shown). It hands its electrons, one at a time, to plastocyanin (pC). plastocyanin diffuses along the membrane surface to photosystem I and transfers the electrons via ferredoxin (Fd) to the ferredoxin-NaDp+ reductase (FNr), where they are utilized to produce NaDph. p700 is a special pair of chlorophylls that absorbs light of wavelength 700 nm.

1	Figure 14–51 Structure and function of photosystem I. at the heart of the photosystem I complex assembly is the electron-transfer chain shown. at one end is a special pair of chlorophylls called p700 (because it absorbs light of 700 nm wavelength), receiving electrons from plastocyanin (pC). at the other end are the a0 chlorophylls, which hand the electrons on to ferredoxin via two plastoquinones (pQ; purple) and three iron–sulfur clusters. Even though the roles of photosystems I and II in photosynthesis are very different, their central electron-transfer chains are structurally similar, indicating a common evolutionary origin (see Figure 14–53). Note that in photosystem I both branches of the electron-transfer chain are active, unlike in photosystem II (see Figure 14–48). (pDB code: 3LW5.)

1	Figure 14–52 Summary of electron and proton movements during photosynthesis in the thylakoid membrane. Electrons are withdrawn, through the action of light energy, from a water molecule that is held by the manganese cluster in photosystem II. the electrons pass on to plastoquinone, which delivers them to the cytochrome b6-f complex that resembles the cytochrome c reductase of mitochondria and the b-c complex of bacteria. they are then carried to photosystem I by the soluble electron carrier plastocyanin, the functional equivalent of cytochrome c in mitochondria. From photosystem I they are transferred to ferredoxin-NaDp+ reductase (FNr) by the soluble carrier ferredoxin (Fd; a small protein containing an iron–sulfur center). protons are pumped into the thylakoid space by the cytochrome b6-f complex, in the same way that protons are pumped into mitochondrial cristae by cytochrome c reductase (see Figure 14–21). In addition, the h+ released into the thylakoid space by water oxidation,

1	complex, in the same way that protons are pumped into mitochondrial cristae by cytochrome c reductase (see Figure 14–21). In addition, the h+ released into the thylakoid space by water oxidation, and the h+ consumed during NaDph formation in the stroma, contribute to the generation of the electrochemical h+ gradient across the thylakoid membrane. as illustrated, this gradient drives atp synthesis by an atp synthase that sits in the same membrane (see Figure 14–47).

1	the Chloroplast atp Synthase Uses the proton Gradient Generated by the photosynthetic Light reactions to produce atp The sequence of events that results in light-driven production of ATP and NADPH in chloroplasts and cyanobacteria is summarized in Figure 14–52. Starting with the withdrawal of electrons from water, the light-driven charge-separation steps in photosystems II and I enable the energetically unfavorable (uphill) flow of electrons from water to NADPH (see Figure 14–47). Three small mobile electron carriers—plastoquinone, plastocyanin, and ferredoxin—participate in this process. Together with the electron-driven proton pump of the cytochrome b6-f complex, the photosystems generate a large proton gradient across the thylakoid membrane. The ATP synthase molecules embedded in the thylakoid membranes then harness this proton gradient to produce large amounts of ATP in the chloroplast stroma, mimicking the synthesis of ATP in the mitochondrial matrix.

1	The linear Z scheme for photosynthesis thus far discussed can switch to a circular mode of electron flow through photosystem I and the b6-f complex. Here, the reduced ferredoxin diffuses back to the b6-f complex to reduce plastoquinone, instead of passing its electrons to the ferredoxin-NADP+ reductase enzyme complex. This, in effect, turns photosystem I into a light-driven proton pump, thereby increasing the proton gradient and thus the amount of ATP made by the ATP synthase. An elaborate set of regulatory mechanisms control this switch, which enables the chloroplast to generate either more NADPH (linear mode) or more ATP (circular mode), depending on the metabolic needs of the cell.

1	Evidence for the prokaryotic origins of mitochondria and chloroplasts abounds in their genetic systems, as we will see in the next section. But strong and direct evidence for the evolutionary origins of chloroplasts can also be found in the molecular structures of photosynthetic reaction centers revealed in recent years by crystallography. The positions of the chlorophylls in the special pair and the two branches of the electron-transfer chain are basically the same in photosystem I, photosystem II, and the photochemical reaction centers of photosynthetic bacteria (Movie 14.11). As a result, one can conclude that they all have evolved from a common ancestor. Evidently, the molecular architecture of the photosynthetic reaction center originated only once and has remained essentially unchanged during evolution. By contrast, the less critical antenna systems have evolved in several different ways and are correspondingly diverse in present-day photosynthetic organisms (Figure 14–53).

1	the proton-Motive Force for atp production in Mitochondria and Chloroplasts Is Essentially the Same The proton gradient across the thylakoid membrane depends both on the proton-pumping activity of the cytochrome b6-f complex and on the photosynthetic activity of the two photosystems, which in turn depends on light intensity. In chloroplasts exposed to light, H+ is pumped out of the stroma (pH around 8, similar to the mitochondrial matrix) into the thylakoid space (pH 5–6), creating a gradient of 2–3 pH units across the thylakoid membrane, representing a proton-motive force of about 180 mV. This is very similar to the proton-motive force in respiring mitochondria. However, a membrane potential across the inner mitochondrial membrane makes the largest contribution to the proton-motive force that drives the mitochondrial ATP synthase to make ATP, whereas a H+ gradient predominates for chloroplasts.

1	In contrast to mitochondrial ATP synthase, which forms long rows of dimers along the cristae ridges, the chloroplast ATP synthase is monomeric and located in flat membrane regions (Figure 14–54). Evidently, the H+ gradient across the thylakoid membrane is high enough for ATP synthesis without the need for the elaborate arrangement of ATP synthase seen in mitochondria. The first living cells on Earth may have consumed geochemically produced organic molecules and generated their ATP by fermentation. Because oxygen was not yet present in the atmosphere, such anaerobic fermentation reactions would have dumped organic acids—such as lactic or formic acids, for example—into the Figure 14–53 Evolution of photosynthetic reaction centers. pigments involved in light-harvesting are colored green; those involved in the central photochemical events are colored red.

1	Figure 14–53 Evolution of photosynthetic reaction centers. pigments involved in light-harvesting are colored green; those involved in the central photochemical events are colored red. the primitive photochemical reaction center of purple bacteria contains two related protein subunits, L and M, that bind the pigments involved in the central process of photosynthesis, including a special pair of chlorophyll molecules. Electrons are fed into the excited chlorophylls by a cytochrome. Lh1 is a bacterial antenna complex.

1	photosystem II contains the D1 and D2 proteins, which are homologous to the L and M subunits in (a). the excited p680 chlorophyll in the special pair withdraws electrons from water held by the manganese cluster. LhC-II is the light-harvesting complex that feeds energy into the core antenna proteins. (C) photosystem I contains the psa a and psa B proteins, each of which is equivalent to a fusion of the D1 or D2 protein to a core antenna protein of photosystem II. the loosely bound plastocyanin (pC) feeds electrons into the excited chlorophyll pair. as indicated, in photosystem I, electrons are passed from a bound quinone (Q) through a series of three iron–sulfur centers (red circles). (Modified from K. rhee, E. Morris, J.

1	J. Barber and W. Kühlbrandt, Nature 396:283–286, 1998; and W. Kühlbrandt, Nature 411:896–899, 2001. With permission from Macmillan publishers Ltd.) matrix pH 7.9 intermembrane space pH 7.4 thylakoid space pH 5.5 intermembrane space pH 7.4 environment (see Figure 2–47). Perhaps such acids lowered the pH of the environment, favoring the survival of cells that evolved transmembrane proteins that could pump H+ out of the cytosol, thereby preventing the cell from becoming too acidic (stage 1 in Figure 14–55). One of these pumps may have used the energy available from ATP hydrolysis to eject H+ from the cell; such a proton pump could have been the ancestor of present-day ATP synthases.

1	As the Earth’s supply of geochemically produced nutrients began to dwindle, organisms that could find a way to pump H+ without consuming ATP would have been at an advantage: they could save the small amounts of ATP they derived from the fermentation of increasingly scarce foodstuffs to fuel other important activities. This need to conserve resources might have led to the evolution of electron-transport proteins that allowed cells to use the movement of electrons between molecules of different redox potentials as a source of energy for pumping H+ across the plasma membrane (stage 2 in Figure 14–55). Some of these cells might have used the nonfermentable organic acids that neighboring cells had excreted as waste to provide the electrons needed to feed this electron-transport system. Some present-day bacteria grow on formic acid, for example, using the small amount of redox energy derived from the transfer of electrons from formic acid to fumarate to pump H+.

1	Figure 14–55 How ATP synthesis by chemiosmosis might have evolved in stages. the first stage could have involved the evolution of an atpase that pumped protons out of the cell using the energy of atp hydrolysis. Stage 2 could have involved the evolution of a different proton pump, driven by an electron-transport chain. Stage 3 would then have linked these two systems together to generate a primitive atp synthase that used the protons pumped by the electron-transport chain to synthesize atp. an early bacterium with this final system would have had a selective advantage over bacteria with neither of the systems or only one.

1	Figure 14–54 A comparison of H+ concentrations and the arrangement of ATP synthase in mitochondria and chloroplasts. In both organelles, the ph in the intermembrane space is 7.4, as in the cytoplasm. the ph of the mitochondrial matrix and the ph of the chloroplast stroma are both about 8 (light gray). the ph in the thylakoid space is around 5.5, depending on photosynthetic activity. this results in a high proton-motive force across the thylakoid membrane, consisting largely of the h+ gradient (a high permeability of this membrane to Mg2+ and Cl– ions allows the flow of these ions to dissipate most of the membrane potential).

1	In contrast to chloroplasts, the h+ gradient across the inner mitochondrial membrane is insufficient for atp production, and mitochondria need a membrane potential to bring the proton-motive force to the same level as in chloroplasts. the arrangement of the mitochondrial atp synthase in rows of dimers along the cristae ridges (see Figure 14–32) next to the respiratory-chain proton pumps may help the flow of protons along the membrane surface toward the atp synthase, as the availability of protons is limiting for atp production. In the chloroplast, the atp synthase is distributed randomly in thylakoid membranes. ATP-driven proton pump working in reverse to make ATP

1	ATP-driven proton pump working in reverse to make ATP Eventually, some bacteria would have developed H+-pumping electron-transport systems that were so efficient that they could harvest more redox energy than they needed to maintain their internal pH. Such cells would probably have generated large electrochemical proton gradients, which they could then use to produce ATP. Protons could leak back into the cell through the ATP-driven H+ pumps, essentially running them in reverse so that they synthesized ATP (stage 3 in Figure 14–55). Because such cells would require much less of the dwindling supply of fermentable nutrients, they would have proliferated at the expense of their neighbors. By providing an Inexhaustible Source of reducing power, photosynthetic Bacteria Overcame a Major Evolutionary Obstacle

1	By providing an Inexhaustible Source of reducing power, photosynthetic Bacteria Overcame a Major Evolutionary Obstacle The gradual depletion of nutrients from the environment on the early Earth meant that organisms had to find some alternative source of carbon to make the sugars that serve as the precursors for so many other cell components. Although the CO2 in the atmosphere provides an abundant potential carbon source, to convert it into an organic molecule such as a carbohydrate requires reducing the fixed CO2 with a strong electron donor, such as NADPH, which can generate (CH2O) units from CO2 (see Figure 14–41). Early in cellular evolution, strong reducing agents (electron donors) are thought to have been plentiful. But once an ancestor of ATP synthase began to generate most of the ATP, it would have become imperative for cells to evolve a new way of generating strong reducing agents.

1	A major evolutionary breakthrough in energy metabolism came with the development of photochemical reaction centers that could use the energy of sunlight to produce molecules such as NADPH. It is thought that this occurred early in the process of cellular evolution in the ancestors of the green sulfur bacteria. Present-day green sulfur bacteria use light energy to transfer hydrogen atoms (as an electron plus a proton) from H2S to NADPH, thereby producing the strong reducing power required for carbon fixation. Because the redox potential of H2S is much lower than that of H2O (–230 mV for H2S compared with +820 mV for H2O), one quantum of light absorbed by the single photosystem in these bacteria is sufficient to generate NADPH via a relatively simple photosynthetic electron-transport chain. the photosynthetic Electron-transport Chains of Cyanobacteria produced atmospheric Oxygen and permitted New Life-Forms

1	The next evolutionary step, which is thought to have occurred with the development of the cyanobacteria perhaps 3 billion years ago, was the evolution of organisms capable of using water as the electron source for CO2 reduction. This entailed the evolution of a water-splitting enzyme and also required the addition of a second photosystem, acting in series with the first, to bridge the large gap in redox potential between H2O and NADPH. The biological consequences of this evolutionary step were far-reaching. For the first time, there would have been organisms that could survive on water, CO2, and sunlight (plus a few trace elements). These cells would have been able to spread and evolve in ways denied to the earlier photosynthetic bacteria, which needed H2S or organic acids as a source of electrons. Consequently, large amounts of biologically synthesized, reduced organic materials accumulated and oxygen entered the atmosphere for the first time.

1	Oxygen is highly toxic because the oxidation of biological molecules alters their structure and properties indiscriminately and irreversibly. Most anaerobic bacteria, for example, are rapidly killed when exposed to air. Thus, organisms on the primitive Earth would have had to evolve protective mechanisms against the rising O2 levels in the environment. Late evolutionary arrivals, such as ourselves, have numerous detoxifying mechanisms that protect our cells from the ill effects of oxygen. Even so, an accumulation of oxidative damage to our macromolecules has been postulated to contribute to human aging, as we discuss in the next section.

1	Figure 14–56 Major events during the evolution of living organisms on Earth. With the evolution of the membrane-based process of photosynthesis, organisms were able to make their own organic molecules from CO2 gas. the delay of more than 109 years between the appearance of bacteria that split water and released O2 during photosynthesis and the accumulation of high levels of O2 in the atmosphere is thought to be due to the initial reaction of the oxygen with the abundant ferrous iron (Fe2+) that was dissolved in the early oceans. Only when the ferrous iron was used up would oxygen have started to accumulate in the atmosphere. In response to the rising oxygen levels, nonphotosynthetic oxygen-consuming organisms evolved, and the concentration of oxygen in the atmosphere equilibrated at its present-day level.

1	The increase in atmospheric O2 was very slow at first and would have allowed a gradual evolution of protective devices. For example, the early seas contained large amounts of iron in its reduced, ferrous state (Fe2+), and nearly all the O2 produced by early photosynthetic bacteria would have been used up in oxidizing Fe2+ to ferric Fe3+. This conversion caused the precipitation of huge amounts of stable oxides, and the extensive banded iron formations in sedimentary rocks, beginning about 2.7 billion years ago, help to date the spread of the cyanobacteria. By about 2 billion years ago, the supply of Fe2+ was exhausted, and the deposition of further iron precipitates ceased. Geological evidence reveals how O2 levels in the atmosphere have changed over billions of years, approximating current levels only about 0.5 billion years ago (Figure 14–56).

1	The availability of O2 enabled the rise of bacteria that developed an aerobic metabolism to make their ATP. These organisms could harness the large amount of energy released by breaking down carbohydrates and other reduced organic molecules all the way to CO2 and H2O, as explained when we discussed mitochondria. Components of preexisting electron-transport complexes were modified to produce a cytochrome oxidase, so that the electrons obtained from organic or inorganic substrates could be transported to O2 as the terminal electron acceptor. Some present-day purple photosynthetic bacteria can switch between photosynthesis and respiration depending on the availability of light and O2, with only relatively minor reorganizations of their electron-transport chains.

1	In Figure 14–57, we relate these postulated evolutionary pathways to different types of bacteria. By necessity, evolution is always conservative, taking parts of the old and building on them to create something new. Thus, parts of the electron-transport chains that were derived to service anaerobic bacteria 3–4 billion years ago survive, in altered form, in the mitochondria and chloroplasts of today’s higher eukaryotes. A good example is the overall similarity in structure and function between the cytochrome c reductase that pumps H+ in the central segment of the mitochondrial respiratory chain and the analogous cytochrome b-f complex in the electron-transport chains of both bacteria and chloroplasts, revealing their common evolutionary origin (Figure 14–58).

1	H2S photosynthesis H2O photosynthesis green sulfur bacteria purple sulfur bacteria green flamentous bacteria ˜-proteobacteria (e.g., Bordetella) MITOCHONDRIACHLOROPLASTS EUKARYOTES PROKARYOTES OXIDIZING ATMOSPHERE REDUCING ATMOSPHERE cyanobacteria (e.g., Synechococcus) ancestral fermenting bacteria O2 respiration purple nonsulfur bacteria °-proteobacteria (e.g., Agrobacterium) ˛-proteobacteria (e.g., E. coli)

1	Figure 14–57 Evolutionary scheme showing the postulated origins of mitochondria and chloroplasts and their bacterial ancestors. the consumption of oxygen by respiration is thought to have first developed about 2 billion years ago. Nucleotide-sequence analyses suggest that an endosymbiotic oxygen-evolving cyanobacterium (cyan) gave rise to chloroplasts (dark green), while mitochondria arose from an α-proteobacterium. the nearest relatives of mitochondria (pink) are members of three closely related groups of α-proteobacteria—the rhizobacteria, agrobacteria, and rickettsias—known to form intimate associations with present-day eukaryotic cells. proteobacteria are pink, purple photosynthetic bacteria are purple, and other photosynthetic bacteria are light green.

1	Chloroplasts and photosynthetic bacteria have the unique ability to harness the energy of sunlight to produce energy-rich compounds. This is achieved by the photosystems, in which chlorophyll molecules attached to proteins are excited when hit by a photon. Photosystems are composed of an antenna complex that collects solar energy and a photochemical reaction center, in which the collected energy is funneled to a chlorophyll molecule held in a special position, enabling it to withdraw electrons from an electron donor. Chloroplasts and cyanobacteria contain two distinct photosystems. The two photosystems are normally linked in series in the Z scheme, and they transfer electrons from water to NADP+ to form NADPH, generating a transmembrane electrochemical potential. One of the two photosystems—photosystem II—can split water by removing electrons from this ubiquitous, low-energy compound. All the molecular oxygen (O2) in our atmosphere is a by-product of the water-splitting reaction in

1	II—can split water by removing electrons from this ubiquitous, low-energy compound. All the molecular oxygen (O2) in our atmosphere is a by-product of the water-splitting reaction in this photosystem. The three-dimensional structures of photosystems I and II are strikingly similar to the photosystems of purple photosynthetic bacteria, demonstrating a remarkable degree of conservation over billions of years of evolution.

1	The two photosystems and the cytochrome b6-f complex reside in the thylakoid membrane, a separate membrane system in the central stroma compartment of the chloroplast that is differentiated into stacked grana and unstacked stroma thylakoids. Electron-transport processes in the thylakoid membrane cause protons to be released into the thylakoid space. The backflow of protons through the chloroplast ATP synthase then generates ATP. This ATP is used in conjunction with the NADPH produced by photosynthesis to drive a large number of biosynthetic reactions in the chloroplast stroma, including the carbon-fixation cycle, which generates large amounts of carbohydrates from CO2.

1	In the early evolution of life, cyanobacteria overcame a major obstacle in devising a way to use solar energy to split water and fix carbon dioxide. Cyanobacteria produced both abundant organic nutrients and molecular oxygen, enabling the rise of a multitude of aerobic life-forms. The chloroplasts in plants have evolved from a cyanobacterium that was endocytosed long ago by an aerobic eukaryotic host organism. Figure 14–58 A comparison of three electron-transport chains discussed in this chapter.

1	Figure 14–58 A comparison of three electron-transport chains discussed in this chapter. Bacteria, chloroplasts, and mitochondria all contain a membrane-bound enzyme complex that resembles the cytochrome c reductase of mitochondria. these complexes all accept electrons from a quinone carrier (Q) and pump h+ across their respective membranes. Moreover, in reconstituted in vitro systems, the different complexes can substitute for one another, and the structures of their protein components reveal that they are evolutionarily related. Note that the purple nonsulfur bacteria use a cyclic flow of electrons to produce a large electrochemical proton gradient that drives a reverse electron flow through NaDh dehydrogenase to produce NaDh from NaD+ + h+ + e –.

1	As we discussed in Chapter 1, mitochondria and chloroplasts are thought to have evolved from endosymbiotic bacteria (see Figures 1–29 and 1–31). Both types of organelles still contain their own genomes (Figure 14–59). As we will discuss shortly, they also retain their own biosynthetic machinery for making RNA and organellar proteins.

1	Like bacteria, mitochondria and chloroplasts proliferate by growth and division of an existing organelle. In actively dividing cells, each type of organelle must double in mass in each cell generation and then be distributed into each daughter cell. In addition, nondividing cells must replenish organelles that are degraded as part of the continual process of organelle turnover, or produce additional organelles as the need arises. Organelle growth and proliferation are therefore carefully controlled. The process is complicated because mitochondrial and chloroplast proteins are encoded in two places: the nuclear genome and the separate genomes harbored in the organelles themselves. The biogenesis of mitochondria and chloroplasts thus requires contributions from two separate genetic systems, which must be closely coordinated.

1	Most organellar proteins are encoded by the nuclear DNA. The organelle imports these proteins from the cytosol, after they have been synthesized on cytosolic ribosomes, through the mitochondrial protein translocases of the outer and inner mitochondrial membrane—TOM and TIM. In Chapter 12, we discussed how this happens. Here, we describe the organelle genomes and genetic systems, and consider the consequences of separate organelle genomes for the cell and the organism as a whole. the Genetic Systems of Mitochondria and Chloroplasts resemble those of prokaryotes

1	the Genetic Systems of Mitochondria and Chloroplasts resemble those of prokaryotes As discussed in Chapter 12, it is thought that eukaryotic cells originated through a symbiotic relationship between an archaeon and an aerobic bacterium (a proteobacterium). The two organisms are postulated to have merged to form the ancestor of all nucleated cells, with the archeaon providing the nucleus and the proteobacterium serving as a respiring, ATP-producing endosymbiont—one that would eventually evolve into the mitochondrion (see Figure 12–3). This most likely occurred roughly 1.6 billion years ago, when oxygen had entered the atmosphere in substantial amounts (see Figure 14–56). The chloroplast was derived later, after the plant and animal lineages diverged, through endocytosis of an oxygen-producing cyanobacterium.

1	This endosymbiont hypothesis of organelle development receives strong support from the observation that the genetic systems of mitochondria and chloroplasts are similar to those of present-day bacteria. For example, chloroplast ribosomes are very similar to bacterial ribosomes, both in their structure and in their sensitivity to various antibiotics (such as chloramphenicol, streptomycin, erythromycin, and tetracyclin). In addition, protein synthesis in chloroplasts starts with N-formylmethionine, as in bacteria, and not with methionine as in the cytosol of eukaryotic cells. Although mitochondrial genetic systems are much less similar to those of present-day bacteria than are the genetic systems of chloroplasts, their ribosomes are also sensitive to antibacterial antibiotics, and protein synthesis in mitochondria also starts with N-formylmethionine.

1	Figure 14–59 Staining of nuclear and mitochondrial DNA. In this confocal micrograph of a human fibroblast, the nuclear DNa is stained with the dye DapI (blue) and mitochondrial DNa is visualized with fluorescent antibodies that bind DNa (green). the mitochondria are stained with fluorescent antibodies that recognize a specialized protein translocase specific to the outer mitochondrial membrane (red). Numerous copies of the mitochondrial genome are distributed in distinct nucleoids throughout the mitochondria that snake through the cytoplasm. (From C. Kukat et al., Proc. Natl Acad. Sci. USA 108:13534–13539, 2011. With permission from the National academy of Sciences.) 5 ˜m

1	The processes of organelle DNA transcription, protein synthesis, and DNA replication take place where the genome is located: in the matrix of mitochondria or the stroma of chloroplasts. Although the enzymes that mediate these genetic processes are unique to the organelle, and resemble those of bacteria (or even of bacterial viruses) rather than their eukaryotic analogs, the nuclear genome encodes the vast majority of these enzymes. Indeed, most present-day mitochondrial and chloroplast proteins are encoded by genes that reside in the cell nucleus. Over time, Mitochondria and Chloroplasts have Exported Most of their Genes to the Nucleus by Gene transfer

1	The nature of the organelle genes located in the nucleus of the cell demonstrates that an extensive transfer of genes from organelle to nuclear DNA has occurred in the course of eukaryotic evolution. Such successful gene transfer is expected to be rare, because any gene moved from the organelle needs to adapt to both nuclear transcription and cytoplasmic translation requirements. In addition, the protein needs to acquire a signal sequence that directs it to the correct organelle after its synthesis in the cytosol. By comparing the genes in the mitochondria from different organisms, we can infer that some of the gene transfers to the nucleus occurred relatively recently. The smallest and presumably most highly evolved mitochondrial genomes, for example, encode only a few hydrophobic inner-membrane proteins of the electron-transport chain, plus ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs). Other mitochondrial genomes that have remained more complex tend to contain this same subset

1	proteins of the electron-transport chain, plus ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs). Other mitochondrial genomes that have remained more complex tend to contain this same subset of genes along with others (Figure 14–60). The most complex mitochondrial genomes include genes that encode components of the mitochondrial genetic system, such as RNA polymerase subunits and ribosomal proteins; these same genes are found in the cell nucleus in yeast and all animal cells.

1	The proteins that are encoded by genes in the organellar DNA are synthesized on ribosomes within the organelle, using organelle-produced messenger RNA (mRNA) to specify their amino acid sequence (Figure 14–61). The protein traffic between the cytosol and these organelles seems to be unidirectional: proteins are normally not exported from mitochondria or chloroplasts to the cytosol. An important exception occurs when a cell is about to undergo apoptosis. As will be

1	Figure 14–60 Comparison of mitochondrial genomes. Less complex mitochondrial genomes encode subsets of the proteins and ribosomal rNas that are encoded by larger mitochondrial genomes. In this comparison, there are only five genes that are shared by the six mitochondrial genomes; these encode ribosomal rNas (rns and rnl), cytochrome b (cob), and two cytochrome oxidase subunits (cox1 and cox3). Blue indicates ribosomal rNas; green, ribosomal proteins; and brown, components of the respiratory chain and other proteins. (adapted from M.W. Gray, G. Burger and B.F. Lang, Science 283:1476–1481, 1999.) discussed in detail in Chapter 18, during apoptosis the mitochondrion releases proteins (most notably cytochrome c) from the crista space through its outer mitochondrial membrane, as part of an elaborate signaling pathway that is triggered to cause cells to undergo programmed cell death. the Fission and Fusion of Mitochondria are topologically Complex processes

1	the Fission and Fusion of Mitochondria are topologically Complex processes In mammalian cells, mitochondrial DNA makes up less than 1% of the total cellular DNA. In other cells, however, such as the leaves of higher plants or the very large egg cells of amphibians, a much larger fraction of the cellular DNA may be present in mitochondria or chloroplasts (Table 14–2), and a large fraction of the total RNA and protein synthesis takes place in the organelles. Mitochondria and chloroplasts are large enough to be visible by light microscopy in living cells. For example, mitochondria can be visualized by expressing in cells a genetically engineered fusion of a mitochondrial protein linked to green

1	Figure 14–61 Biogenesis of the respiratory-chain proteins in human mitochondria. Most of the protein components of the mitochondrial respiratory chain are encoded by nuclear DNa, with only a small number encoded by mitochondrial DNa (mtDNa). transcription of mtDNa produces 13 mrNas, all of which encode subunits of the oxidative phosphorylation system, and the 24 rNas (22 transfer rNas and 2 ribosomal rNas) needed for translation of these mrNas on the mitochondrial ribosomes (brown).

1	the mrNas produced by transcription of nuclear genes are translated on cytoplasmic ribosomes (green), which are distinct from the mitochondrial ribosomes. the nuclear-encoded mitochondrial proteins (dark green) are imported into mitochondria through two protein translocases called tOM and tIM, and constitute the vast majority of the approximately 1000 different protein species present in mammalian mitochondria. the nuclear-encoded mitochondrial proteins in humans include the majority of the oxidative phosphorylation system subunits, all proteins needed for expression and maintenance of mtDNa, and all proteins of the mitochondrial ribosomes. the mtDNa-encoded subunits (orange) assemble together with the nuclear subunits to form a functional oxidative phosphorylation system. (adapted from N.G. Larsson, Annu. Rev. Biochem. 79:683–706, 2010.)

1	Figure 14–62 The mitochondrial reticulum is dynamic. (a) In yeast cells, mitochondria form a continuous reticulum on the cytoplasmic side of the plasma membrane (stereo pair). (B) a balance between fission and fusion determines the arrangement of the mitochondria in different cells. (C) time-lapse fluorescent microscopy shows the dynamic behavior of the mitochondrial network in a yeast cell. In addition to shape changes, fission and fusion constantly remodel the network (red arrows). these pictures were taken at 3-minute intervals. (a and C, from J. Nunnari et al., Mol. Biol. Cell 8:1233–1242, 1997. With permission from the american Society for Cell Biology.) fluorescent protein (GFP), or cells can be incubated with a fluorescent dye that is specifically taken up by mitochondria because of their membrane potential. Such images demonstrate that the mitochondria in living cells are dynamic— frequently dividing by fission, fusing, and changing shape (Figure 14–62 and Movie 14.12). The

1	of their membrane potential. Such images demonstrate that the mitochondria in living cells are dynamic— frequently dividing by fission, fusing, and changing shape (Figure 14–62 and Movie 14.12). The fission of mitochondria may be necessary so that small parts of the network can pinch off and reach remote regions of the cell—for example in the thin, extended axon and dendrites of a neuron.

1	The fission and fusion of mitochondria are topologically complex processes that must ensure the integrity of the separate mitochondrial compartments defined by the inner and outer membranes. These processes control the number and shape of mitochondria, which can vary dramatically in different cell types, ranging from multiple spherical or wormlike organelles to a highly branched, net-shaped single organelle called a reticulum. Each depends on its own special set of proteins. The mitochondrial fission machine works by assembling dynamin-related GTPases (discussed in Chapter 13) into helical oligomers that cause local constrictions in tubular mitochondria. GTP hydrolysis then generates the mechanical force that severs the inner and outer mitochondrial membranes in one step (Figure 14–63). Mitochondrial fusion requires two separate machineries, one each for the outer and the inner membrane (Figure 14–64). In addition to GTP hydrolysis for force generation, both mechanisms also depend on

1	fusion requires two separate machineries, one each for the outer and the inner membrane (Figure 14–64). In addition to GTP hydrolysis for force generation, both mechanisms also depend on the mitochondrial proton-motive force for reasons that are still unknown.

1	animal Mitochondria Contain the Simplest Genetic Systems Known Comparisons of DNA sequences in different organisms reveal that, in vertebrates (including ourselves), the mutation rate during evolution has been roughly 100 times greater in the mitochondrial genome than in the nuclear genome. This difference is likely to be due to lower fidelity of mitochondrial DNA replication,

1	Figure 14–63 A model for mitochondrial division. Dynamin-1 (yellow) exists as dimers in the cytosol, which form larger oligomeric structures in a process that requires Gtp hydrolysis. Dynamin assemblies interact with the outer mitochondrial membrane through special adaptor proteins, forming a spiral of Gtp-dynamin around the mitochondrion that causes a constriction. a concerted Gtp-hydrolysis event in the dynamin subunits is then thought to produce the conformational changes that result in fission. (adapted from S. hoppins, L. Lackner and J. Nunnari, Annu. Rev. Biochem. 76:751–780, 2007.) inefficient DNA repair, or both, given that the mechanisms that perform these processes in the organelle are relatively simple compared with those in the nucleus. As discussed in Chapter 4, the relatively high rate of evolution of animal mitochondrial genes makes a comparison of mitochondrial DNA sequences especially useful for estimating the dates of relatively recent evolutionary events, such as

1	high rate of evolution of animal mitochondrial genes makes a comparison of mitochondrial DNA sequences especially useful for estimating the dates of relatively recent evolutionary events, such as the steps in primate evolution.

1	There are 13 protein-encoding genes in human mitochondrial DNA (Figure 14–65). These code for hydrophobic components of the respiratory-chain complexes and of ATP synthase. In contrast, roughly 1000 mitochondrial proteins are encoded in the nucleus, produced on cytosolic ribosomes, and imported by the protein import machinery in the outer and inner membrane (discussed in Chapter 12). It has been suggested that the cytosolic production of hydrophobic membrane proteins and their import into the organelle may present a problem to the cell, and that this is the reason why their genes have remained in the mitochondrion. However, some of the most hydrophobic mitochondrial proteins, such as the c subunit of the ATP synthase rotor ring, are imported from the cytosol in some species (though they are mitochondrially encoded in others). And the parasites Plasmodium falciparum and Leishmania tarentolae, which spend most of their life cycles inside cells of their host organisms, have retained only

1	mitochondrially encoded in others). And the parasites Plasmodium falciparum and Leishmania tarentolae, which spend most of their life cycles inside cells of their host organisms, have retained only two or three mitochondrially encoded proteins.

1	The size range of mitochondrial DNAs is similar to that of viral DNAs. The mitochondrial DNA in Plasmodium falciparum (the human malaria parasite) has less than 6000 nucleotide pairs, whereas the mitochondrial DNAs of some land plants contain more than 300,000 nucleotide pairs (Figure 14–66). In animals, the mitochondrial genome is a simple DNA circle of about 16,600 nucleotide pairs (less than 0.001% of the nuclear genome), and it is nearly the same size in organisms as different from us as Drosophila and sea urchins. The human mitochondrial genome has several surprising features that distinguish it from nuclear, chloroplast, and bacterial genomes: 1.

1	The human mitochondrial genome has several surprising features that distinguish it from nuclear, chloroplast, and bacterial genomes: 1. Dense gene packing. Unlike other genomes, the human mitochondrial genome seems to contain almost no noncoding DNA: nearly every nucleotide seems to be part of a coding sequence, either for a protein or for one of the rRNAs or tRNAs. Since these coding sequences run directly into each other, there is very little room left for regulatory DNA sequences. 2. Relaxed codon usage. Whereas 30 or more tRNAs specify amino acids in the cytosol and in chloroplasts, only 22 tRNAs are required for mitochondrial protein synthesis. The normal codon–anticodon pairing rules are relaxed in mitochondria, so that many tRNA molecules recognize any one of the four nucleotides in the third (wobble) position. Such “2 out of 3” pairing human mtDNA16,569 bporigin ofreplication cytochrome bdehydrogenase dehydrogenasecytochrome oxidasesubunitsATP synthase subunits

1	Figure 14–64 A model for mitochondrial fusion. the fusions of the outer and inner mitochondrial membranes are coordinated sequential events, each of which requires a separate set of protein factors. Outer membrane fusion is brought about by an outer-membrane Gtpase (purple), which forms an oligomeric complex that includes subunits anchored in the two membranes to be fused. Fusion of outer membranes requires Gtp and an h+ gradient across the inner membrane. For fusion of the inner membrane, a dynamin-related protein forms an oligomeric tethering complex (blue) that includes subunits anchored in the two inner membranes to be fused. Fusion of the inner membranes requires Gtp and the electrical component of the potential across the inner membrane. (adapted from S. hoppins, L. Lackner and J. Nunnari, Annu. Rev. Biochem. 76:751–780, 2007.)

1	Figure 14–65 The organization of the human mitochondrial genome. the human mitochondrial genome of ≈16,600 nucleotide pairs contains 2 rrNa genes, 22 trNa genes, and 13 protein-coding sequences. there are two transcriptional promoters, one for each strand of the mitochondrial DNa (mtDNa). the DNas of many other animal mitochondrial genomes have been completely sequenced. Most of these animal mitochondrial DNas encode precisely the same genes as humans, with the gene order being identical for animals ranging from fish to mammals. allows one tRNA to pair with any one of four codons and permits protein synthesis with fewer tRNA molecules. 3. Variant genetic code. Perhaps most surprising, comparisons of mitochondrial gene sequences and the amino acid sequences of the corresponding proteins indicate that the genetic code is different: 4 of the 64 codons have different “meanings” from those of the same codons in other genomes (Table 14–3).

1	The close similarity of the genetic code in all organisms provides strong evidence that they all have evolved from a common ancestor. How, then, do we explain the differences in the genetic code in many mitochondria? A hint comes from the finding that the mitochondrial genetic code in different organisms is not the same. In the mitochondrion with the largest number of genes in Figure 14–60, that of the protozoan Reclinomonas, the genetic code is unchanged from the standard genetic code of the cell nucleus. Yet UGA, which is a stop codon elsewhere, is read as tryptophan in the mitochondria of mammals, fungi, and invertebrates. Similarly, the codon AGG normally codes for arginine, but it codes for stop in the mitochondria of mammals and codes for serine in the mitochondria of Drosophila (see Table 14–3). Such variation suggests that a random drift can occur in the genetic code in mitochondria. Presumably, the unusually small number of proteins encoded by the mitochondrial genome makes

1	Table 14–3). Such variation suggests that a random drift can occur in the genetic code in mitochondria. Presumably, the unusually small number of proteins encoded by the mitochondrial genome makes an occasional change in the meaning of a rare codon tolerable, whereas such a change in a larger genome would alter the function of many proteins and thereby destroy the cell.

1	Interestingly, in many species, one or two tRNAs for mitochondrial protein synthesis are encoded in the nucleus. Some parasites, for example trypanosomes, have not retained any tRNA genes in their mitochondrial DNA. Instead, the required tRNAs are all produced in the cytosol and are thought to be imported into the mitochondrion by special tRNA translocases that are distinct from the mitochondrial protein import system.

1	Figure 14–66 Comparison of various sizes of mitochondrial genomes with the genome of bacterial ancestors. the complete DNa sequences for thousands of mitochondrial genomes have been determined. the lengths of a few of these mitochondrial DNas are shown to scale— as circles for those genomes thought to be circular and lines for linear genomes. the largest circle represents the genome of Rickettsia prowazekii, a small pathogenic bacterium whose genome most closely resembles that of mitochondria. the size of mitochondrial genomes does not correlate well with the number of proteins encoded in them: while human mitochondrial DNa encodes 13 proteins, the 22-fold larger mitochondrial DNa of Arabidopsis thaliana encodes only 32 proteins—that is, about 2.5-fold as many as human mitochondrial DNa. the extra DNa that is found in Arabidopsis, Marchantia, and other plant mitochondria may be “junk DNa”—that is, noncoding DNa with no apparent function. the mitochondrial DNa of the protozoan

1	DNa. the extra DNa that is found in Arabidopsis, Marchantia, and other plant mitochondria may be “junk DNa”—that is, noncoding DNa with no apparent function. the mitochondrial DNa of the protozoan Reclinomonas americana has 98 genes. (adapted from M.W. Gray, G. Burger and B.F. Lang, Science 283:1476–1481, 1999.)

1	The chloroplast genomes of land plants range in size from 70,000 to 200,000 nucleotide pairs. More than 300 chloroplast genomes have now been sequenced. Many are surprisingly similar, even in distantly related plants (such as tobacco and liverwort), and even those of green algae are closely related (Figure 14–67). Chloroplast genes are involved in three main processes: transcription, translation, and photosynthesis. Plant chloroplast genomes typically encode 80–90 proteins and around 45 RNAs, including 37 or more tRNAs. As in mitochondria, most of the organelle-encoded proteins are part of larger protein complexes that also contain one or more subunits encoded in the nucleus and imported from the cytosol.

1	The genomes of chloroplasts and bacteria have striking similarities. Basic regulatory sequences, such as transcription promoters and terminators, are virtually identical. The amino acid sequences of the proteins encoded in chloroplasts are clearly recognizable as bacterial, and several clusters of genes with related functions (such as those encoding ribosomal proteins) are organized in the same way in the genomes of chloroplasts, the bacterium E. coli, and cyanobacteria.

1	The mechanisms by which chloroplasts and bacteria divide are also similar. Both utilize FtsZ proteins, which are self-assembling GTPases related to tubulins (see Chapter 16). Bacterial FtsZ is a soluble protein that assembles into a dynamic ring of membrane-attached protofilaments beneath the plasma membrane in the middle of the dividing cell. The FtsZ ring acts as a scaffold for recruitment of other cell-division proteins and generates a contractile force that results in membrane constriction and eventually in cell division. Presumably, chloroplasts divide in very much the same way. Although both employ membrane-interacting GTPases, the mechanisms by which mitochondria and chloroplasts divide are fundamentally different. The machinery for chloroplast division acts from the inside, as in bacteria, while the dynamin-like GTPases divide mitochondria from the outside (see Figure 14–63). The chloroplasts have remained closer to their bacterial origins than have mitochondria, since the

1	in bacteria, while the dynamin-like GTPases divide mitochondria from the outside (see Figure 14–63). The chloroplasts have remained closer to their bacterial origins than have mitochondria, since the eukaryotic mechanisms of membrane constriction and vesicle formation have been adapted for mitochondrial fission.

1	The RNA editing and RNA processing that is prevalent in chloroplasts owes everything to their eukaryotic hosts. This RNA processing includes the generation of transcript 5ʹ and 3ʹ termini and the cleavage of polycistronic transcripts. In addition, an RNA editing process converts specific C residues to U and can change the amino acid specified by the edited codon. These and other RNA-based 23S 16S 23S 16S liverwort chloroplast DNA 121,024 bp KEY: Figure 14–67 The organization of the liverwort chloroplast genome. the chloroplast genome organization is similar in all higher plants, although the size varies from species to species—depending on how much of the DNa surrounding the genes encoding the chloroplast’s 16S and 23S ribosomal rNas is present in two copies.

1	processes are catalyzed by protein families that are not found in prokaryotes. One can ask why the expression of so few chloroplast genes needs to be so complex. One explanation is that the expression of chloroplast and nuclear genes must be closely coordinated. More generally, the bacterial concept of the operon as a co-regulated set of genes in a single transcription unit has been largely abandoned in chloroplasts. Polycistronic transcripts are cleaved into smaller fragments, which then require splicing or RNA editing to become functional. Organelle Genes are Maternally Inherited in animals and plants

1	Organelle Genes are Maternally Inherited in animals and plants In Saccharomyces cerevisiae (baker’s yeast), when two haploid cells mate, they are equal in size and contribute equal amounts of mitochondrial DNA to the diploid zygote. Mitochondrial inheritance in yeasts is therefore biparental: both parents contribute equally to the mitochondrial gene pool of the progeny. However, during the course of the subsequent asexual, vegetative growth, the mitochondria become distributed more or less randomly to daughter cells. After a few generations, the mitochondria of any given cell contain only the DNA from one or the other parent cell, because only a small sample of the mitochondrial DNA passes from the mother cell to the bud of the daughter cell. This process is known as mitotic segregation, and it gives rise to a distinct form of inheritance that is called non-Mendelian, or cytoplasmic inheritance, in contrast to the Mendelian inheritance of nuclear genes.

1	The inheritance of mitochondria in animals and plants is quite different. In these organisms, the egg cell contributes much more cytoplasm to the zygote than does the male gamete (sperm in animals, pollen in plants). For example, a typical human oocyte contains about 100,000 copies of maternal mitochondrial DNA, whereas a sperm cell contains only a few. In addition, an active process ensures that the sperm mitochondria do not compete with those in the egg. As sperm mature, the DNA is degraded in their mitochondria. Sperm mitochondria are also specifically recognized then eliminated from the fertilized egg cell by autophagy in very much the same way that damaged mitochondria are removed (by ubiquitylation followed by delivery to lysosomes, as discussed in Chapter 13). Because of these two processes, the mitochondrial inheritance in both animals and plants is uniparental. More precisely, the mitochondrial DNA passes from one generation to the next by maternal inheritance.

1	In about two-thirds of higher plants, the chloroplast precursors from the male parent (contained in pollen grains) fail to enter the zygote, so that chloroplast as well as mitochondrial DNA is maternally inherited. In other plants, the chloroplast precursors from the pollen grains enter the zygote, making chloroplast inheritance biparental. In such plants, defective chloroplasts are a cause of variegation: a mixture of normal and defective chloroplasts in a zygote may sort out by mitotic segregation during plant growth and development, thereby producing alternating green and white patches in leaves. Leaf cells in the green patches contain normal chloroplasts, while those in the white patches contain defective chloroplasts (Figure 14–68).

1	In humans, as we have explained, all the mitochondrial DNA in a fertilized egg cell is inherited from the mother. Some mothers carry a mixed population of both mutant and normal mitochondrial genomes. Their daughters and sons will inherit this mixture of normal and mutant mitochondrial DNAs and be healthy unless the process of mitotic segregation results in a majority of defective mitochondria in a particular tissue. Muscle and the nervous system are most at risk. Because they need particularly large amounts of ATP, muscle and nerve cells are particularly dependent on fully functional mitochondria.

1	Numerous diseases in humans are caused by mutations in mitochondrial DNA. These diseases are recognized by their passage from affected mothers to both their daughters and their sons, with the daughters but not the sons producing children with the disease. As expected from the random nature of mitotic segregation, the symptoms of these diseases vary greatly between different family members—including not only the severity and age of onset, but also which tissue is affected. There are also mitochondrial diseases that are caused by mutations in nuclear-encoded mitochondrial proteins; these diseases are inherited in the regular, Mendelian fashion. Figure 14–68 A variegated leaf. In the white patches, the plant cells have inherited a defective chloroplast. (Courtesy of John Innes Foundation.) the accumulation of Mitochondrial DNa Mutations Is a Contributor to aging

1	Mitochondria are marvels of efficiency in energy conversion, and they supply the cells of our body with a readily available source of energy in the form of ATP. But in highly developed, long-lived animals such as ourselves, the cells in our body age and eventually die. A factor in this inevitable process is the accumulation of deletions and point mutations in mitochondrial DNA. Oxidative damage to the cell by reactive oxygen species (ROS) such as H2O2, superoxide, or hydroxyl radicals also increases with age. The mitochondrial respiratory chain is the main source of ROS in animal cells, and animals in which mitochondrial superoxide dismutase—the main ROS scavenger—has been knocked out, die prematurely.

1	The less complex DNA replication and repair systems in mitochondria mean that accidents are corrected less efficiently. This results in a 100-fold higher occurrence of deletions and point mutations than in nuclear DNA. Mathematical modeling suggests that most of these mutations and lesions are acquired in childhood or early adult life, and then proliferate by clonal expansion in later life. Due to mitotic segregation, some cells will accumulate higher levels of faulty mitochondrial DNA than others. Above some threshold, serious deficiencies in respiratory-chain function will develop, producing cells that are senescent. In many organs of the human body, senescent cells with high levels of mitochondrial DNA damage are intermingled with normal cells, resulting in a mosaic of cells with and without respiratory-chain deficiency.

1	The main role of mitochondrial fusion in cellular physiology is most likely to ensure an even distribution of mitochondrial DNA throughout the mitochondrial reticulum, and to prevent the accumulation of damaged DNA in one part of the network. When the fusion machinery is defective, DNA is lost from a subset of the mitochondria in the cell. Loss of mitochondrial DNA leads to a loss of respiratory-chain function, and it can cause disease. All of the considerations just discussed have suggested to some scientists that changes in our mitochondria are major contributors to human aging. However, there are many other processes that tend to go wrong as cells and tissues age, as one might expect given the incredible complexity of human cell biology. Despite intensive research, the issue remains unresolved. Why Do Mitochondria and Chloroplasts Maintain a Costly Separate System for DNa transcription and translation?

1	Why Do Mitochondria and Chloroplasts Maintain a Costly Separate System for DNa transcription and translation? Why do mitochondria and chloroplasts require their own separate genetic systems, when other organelles that share the same cytoplasm, such as peroxisomes and lysosomes, do not? The question is not trivial, because maintaining a separate genetic system is costly: more than 90 proteins—including many ribosomal proteins, aminoacyl-tRNA synthetases, DNA polymerase, RNA polymerase, and RNA-processing and RNA-modifying enzymes—must be encoded by nuclear genes specifically for this purpose. Moreover, as we have seen, the mitochondrial genetic system entails the risk of aging and disease.

1	A possible reason for maintaining this costly and potentially hazardous arrangement is the highly hydrophobic nature of the nonribosomal proteins encoded by organelle genes. This may make their production in and import from the cytoplasm simply too difficult and energy-consuming. It is also possible that the evolution (and eventual elimination) of the organellar genetic systems is still ongoing, but for now there is no alternative for the cell than to maintain separate genetic systems for its nuclear, mitochondrial, and chloroplast genes.

1	Mitochondria are organelles that allow eukaryotes to carry out oxidative phosphorylation, while chloroplasts are organelles that allow plants to carry out photosynthesis. Presumably as a result of their prokaryotic origins, each organelle maintains and reproduces itself in a highly coordinated process that requires the contribution of two separate genetic systems—one in the organelle and the other in the cell nucleus. The vast majority of the proteins in these organelles are encoded by nuclear DNA, synthesized in the cytosol, and then imported individually into the organelle. Other organelle proteins, as well as organelle ribosomal and transfer RNAs, are encoded by the organelle DNA; these are synthesized in the organelle itself.

1	The ribosomes of chloroplasts closely resemble bacterial ribosomes, while the origin of mitochondrial ribosomes is more difficult to trace. Extensive protein similarities, however, suggest that both organelles originated when a primitive eukaryotic cell entered into a stable endosymbiotic relationship with a bacterium. Although some of the genes of these former bacteria still function to make organelle proteins and RNA, most of them have been transferred into the nuclear genome, where they encode bacteria-like enzymes that are synthesized on cytosolic ribosomes and then imported into the organelle. The mitochondrial DNA replication and DNA repair processes are substantially less effective than the corresponding processes in the cell nucleus. Damage therefore accumulates in the genome of mitochondria over time; this damage may be a substantial contributor to the aging of cells and organisms, and it can cause serious diseases. Which statements are true? Explain why or why not.

1	Which statements are true? Explain why or why not. 14–1 The three respiratory enzyme complexes in the mitochondrial inner membrane tend to associate with each other in ways that facilitate the correct transfer of electrons between appropriate complexes. 14–2 The number of c subunits in the rotor ring of ATP synthase defines how many protons need to pass through the turbine to make each molecule of ATP. 14–3 Mutations that are inherited according to Mendelian rules affect nuclear genes; mutations whose inheritance violates Mendelian rules are likely to affect organelle genes. Discuss the following problems. 14–4 In the 1860s, Louis Pasteur noticed that when he added O2 to a culture of yeast growing anaerobically on glucose, the rate of glucose consumption declined dramatically. Explain the basis for this result, which is known as the Pasteur effect.

1	14–5 Heart muscle gets most of the ATP needed to power its continual contractions through oxidative phosphorylation. When oxidizing glucose to CO2, heart muscle consumes O2 at a rate of 10 μmol/min per g of tissue, in order to replace the ATP used in contraction and give a steady-state ATP concentration of 5 μmol/g of tissue. At this rate, What structures are needed to form the barriers that separate and maintain the differentiated membrane domains in a single continuous membrane—as for the cristae and inner boundary membrane in mitochondria? how does a eukaryotic cell regulate the many functions of mitochondria, including atp production? What are the origins and evolutionary history of photosynthetic complexes? are there undiscovered types of photosynthesis present on Earth to help answer this question? Why is the mutation rate so much higher in mitochondria than in the nucleus (and chloroplasts)? Could this high rate have been useful to the cell?

1	Why is the mutation rate so much higher in mitochondria than in the nucleus (and chloroplasts)? Could this high rate have been useful to the cell? What mechanisms and pathways have been used during evolution to transfer genes from the mitochondrion to the nucleus? how many seconds would it take the heart to consume an amount of ATP equal to its steady-state levels? (Complete oxidation of one molecule of glucose to CO2 yields 30 ATP, 26 of which are derived by oxidative phosphorylation using the 12 pairs of electrons captured in the electron carriers NADH and FADH2.) 14–6 Both H+ and Ca2+ are ions that move through the cytosol. Why is the movement of H+ ions so much faster than that of Ca2+ ions? How do you suppose the speed of these two ions would be affected by freezing the solution? Would you expect them to move faster or slower? Explain your answer.

1	14–7 If isolated mitochondria are incubated with a source of electrons such as succinate, but without oxygen, electrons enter the respiratory chain, reducing each of the electron carriers almost completely. When oxygen is then introduced, the carriers become oxidized at different rates (Figure Q14–1). How does this result allow you to order Figure Q14–1 rapid spectrophotometric analysis of the rates of oxidation of electron carriers in the respiratory chain (problem 14–7). Cytochromes a and a3 cannot be distinguished and thus are listed as cytochrome (a + a3). the electron carriers in the respiratory chain? What is their (A) direction of rotation order?

1	14–8 Normally, the flow of electrons to O2 is tightly linked to the production of ATP via the electrochemical gradient. If ATP synthase is inhibited, for example, electrons do not flow down the electron-transport chain and respiration ceases. Since the 1940s, several substances— such as 2,4-dinitrophenol—have been known to uncouple electron flow from ATP synthesis. Dinitrophenol was once prescribed as a diet drug to aid in weight loss. How would an uncoupler of oxidative phosphorylation pro mote weight loss? Why do you suppose dinitrophenol is (B) no longer prescribed? 5 14–9 In actively respiring liver mitochondria, the pH in 4 the matrix is about half a pH unit higher than it is in the cytosol. Assuming that the cytosol is at pH 7 and the matrix is a sphere with a diameter of 1 μm [V = (4/3)πr3], calcu late the total number of protons in the matrix of a respiring liver mitochondrion. If the matrix began at pH 7 (equal to that in the cytosol), how many protons would have to be pumped

1	calcu late the total number of protons in the matrix of a respiring liver mitochondrion. If the matrix began at pH 7 (equal to that in the cytosol), how many protons would have to be pumped out to establish a matrix pH of 7.5 (a difference of 0.5 pH units)?

1	14–10 ATP synthase is the world’s smallest rotary motor. Passage of H+ ions through the membrane-embedded portion of ATP synthase (the Fo component) causes rotation of the single, central, axle-like γ subunit inside the head group. The tripartite head is composed of the three αβ dimers, the β subunit of which is responsible for synthesis of ATP. The rotation of the γ subunit induces conformational changes in the αβ dimers that allow ADP and Pi to be converted into ATP. A variety of indirect evidence had suggested rotary catalysis by ATP synthase, but seeing is believing.

1	To demonstrate rotary motion, a modified form of the α3β3γ complex was used. The β subunits were modified so they could be firmly anchored to a solid support and the γ subunit was modified (on the end that normally inserts into the Fo component in the inner membrane) so that a fluorescently tagged, readily visible filament of actin could be attached (Figure Q14–2A). This arrangement allows rotations of the γ subunit to be visualized as revolutions of the long actin filament. In these experiments, ATP synthase was studied in the reverse of its normal mechanism by allowing it to hydrolyze ATP. At low ATP concentrations, the actin filament was observed to revolve in steps of 120° and then pause for variable lengths of time, as shown in Figure Q14–2B. A. Why does the actin filament revolve in steps with pauses in between? What does this rotation correspond to in terms of the structure of the α3β3γ complex?

1	A. Why does the actin filament revolve in steps with pauses in between? What does this rotation correspond to in terms of the structure of the α3β3γ complex? B. In its normal mode of operation inside the cell, how many ATP molecules do you suppose would be synthesized for each complete 360° rotation of the γ subunit? Explain your answer. 14–11 How much energy is available in visible light? How much energy does sunlight deliver to Earth? How efficient

1	14–11 How much energy is available in visible light? How much energy does sunlight deliver to Earth? How efficient Figure Q14–2 Experimental set-up for observing rotation of the γ subunit of atp synthase (problem 14–10). (a) the immobilized α3β3γ complex. the β subunits are anchored to a solid support and a fluorescent actin filament is attached to the γ subunit. (B) Stepwise revolution of the actin filament. the indicated trace is a typical example from one experiment. the inset shows the positions in the revolution at which the actin filament pauses. (B, from r. yasuda et al., Cell 93:1117–1124, 1998. With permission from Elsevier.) are plants at converting light energy into chemical energy? The answers to these questions provide an important backdrop to the subject of photosynthesis.

1	Each quantum or photon of light has energy hv, where h is Planck’s constant (6.6 × 10–37 kJ sec/photon) and v is the frequency in sec–1. The frequency of light is equal to c/λ, where c is the speed of light (3.0 × 1017 nm/ sec) and λ is the wavelength in nm. Thus, the energy (E) of a photon is A. Calculate the energy of a mole of photons (6 × 1023 photons/mole) at 400 nm (violet light), at 680 nm (red light), and at 800 nm (near-infrared light). B. Bright sunlight strikes Earth at the rate of about 1.3 kJ/sec per square meter. Assuming for the sake of calculation that sunlight consists of monochromatic light of wavelength 680 nm, how many seconds would it take for a mole of photons to strike a square meter?

1	C. Assuming that it takes eight photons to fix one molecule of CO2 as carbohydrate under optimal conditions (8–10 photons is the currently accepted value), calculate how long it would take a tomato plant with a leaf area of 1 square meter to make a mole of glucose from CO2. Assume that photons strike the leaf at the rate calculated above and, furthermore, that all the photons are absorbed and used to fix CO2. D. If it takes 468 kJ/mole to fix a mole of CO2 into carbohydrate, what is the efficiency of conversion of light energy into chemical energy after photon capture? Assume again that eight photons of red light (680 nm) are required to fix one molecule of CO2.

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1	When things change, cells respond. Every cell, from the humble bacterium to the most sophisticated eukaryotic cell, monitors its intracellular and extracellular environment, processes the information it gathers, and responds accordingly. Unicellular organisms, for example, modify their behavior in response to changes in environmental nutrients or toxins. The cells of multicellular organisms detect and respond to countless internal and extracellular signals that control their growth, division, and differentiation during development, as well as their behavior in adult tissues. At the heart of all these communication systems are regulatory proteins that produce chemical signals, which are sent from one place to another in the body or within a cell, usually being processed along the way and integrated with other signals to provide clear and effective communication.

1	The study of cell signaling has traditionally focused on the mechanisms by which eukaryotic cells communicate with each other using extracellular signal molecules such as hormones and growth factors. In this chapter, we describe the features of some of these cell–cell communication systems, and we use them to illustrate the general principles by which any regulatory system, inside or outside the cell, is able to generate, process, and respond to signals. Our main focus is on animal cells, but we end by considering the special features of cell signaling in plants.

1	Long before multicellular creatures roamed the Earth, unicellular organisms had developed mechanisms for responding to physical and chemical changes in their environment. These almost certainly included mechanisms for responding to the presence of other cells. Evidence comes from studies of present-day unicellular organisms such as bacteria and yeasts. Although these cells lead mostly independent lives, they can communicate and influence one another’s behavior. Many bacteria, for example, respond to chemical signals that are secreted by their neighbors and accumulate at higher population density. This process, called quorum sensing, allows bacteria to coordinate their behavior, including their motility, antibiotic production, spore formation, and sexual conjugation. Similarly, yeast cells communicate with one another in preparation for mating. The budding yeast Saccharomyces cerevisiae provides a well-studied example: when a haploid individual is ready to mate, it secretes a peptide

1	communicate with one another in preparation for mating. The budding yeast Saccharomyces cerevisiae provides a well-studied example: when a haploid individual is ready to mate, it secretes a peptide mating factor that signals cells of the opposite mating type to stop proliferating and prepare to mate. The subsequent fusion of two haploid cells of opposite mating type produces a diploid zygote.

1	Intercellular communication achieved an astonishing level of complexity during the evolution of multicellular organisms. These organisms are tight-knit societies of cells, in which the well-being of the individual cell is often set aside for the benefit of the organism as a whole. Complex systems of intercellular communication have evolved to allow the collaboration and coordination of different tissues and cell types. Bewildering arrays of signaling systems govern every conceivable feature of cell and tissue function during development and in the adult. Communication between cells in multicellular organisms is mediated mainly by extracellular signal molecules. Some of these operate over long distances, plasma membrane of target cellCYTOSOL

1	Communication between cells in multicellular organisms is mediated mainly by extracellular signal molecules. Some of these operate over long distances, plasma membrane of target cellCYTOSOL Figure 15–1 A simple intracellular signaling pathway activated by an extracellular signal molecule. The signal molecule usually binds to a receptor protein that is embedded in the plasma membrane of the target cell. The receptor activates one or more intracellular signaling pathways, involving a series of signaling proteins. Finally, one or more of the intracellular signaling proteins alters the activity of effector proteins and thereby the behavior of the cell.

1	signaling to cells far away; others signal only to immediate neighbors. Most cells in multicellular organisms both emit and receive signals. Reception of the signals depends on receptor proteins, usually (but not always) at the cell surface, which bind the signal molecule. The binding activates the receptor, which in turn activates one or more intracellular signaling pathways or systems. These systems depend on intracellular signaling proteins, which process the signal inside the receiving cell and distribute it to the appropriate intracellular targets. The targets that lie at the end of signaling pathways are generally called effector proteins, which are altered in some way by the incoming signal and implement the appropriate change in cell behavior. Depending on the signal and the type and state of the receiving cell, these effectors can be transcription regulators, ion channels, components of a metabolic pathway, or parts of the cytoskeleton (Figure 15–1).

1	The fundamental features of cell signaling have been conserved throughout the evolution of the eukaryotes. In budding yeast, for example, the response to mating factor depends on cell-surface receptor proteins, intracellular GTP-binding proteins, and protein kinases that are clearly related to functionally similar proteins in animal cells. Through gene duplication and divergence, however, the signaling systems in animals have become much more elaborate than those in yeasts; the human genome, for example, contains more than 1500 genes that encode receptor proteins, and the number of different receptor proteins is further increased by alternative RNA splicing and post-translational modifications.

1	Many extracellular signal molecules remain bound to the surface of the signaling cell and influence only cells that contact it (Figure 15–2A). Such contact-dependent signaling is especially important during development and in immune responses. Contact-dependent signaling during development can sometimes operate over relatively large distances if the communicating cells extend long thin processes to make contact with one another. Figure 15–2 Four forms of intercellular signaling. (A) Contact-dependent signaling requires cells to be in direct membrane– membrane contact. (B) Paracrine signaling depends on local mediators that are released into the extracellular space and act on neighboring cells. (C) Synaptic signaling is performed by neurons that transmit signals electrically along their axons and release neurotransmitters at synapses, which are often located far away from the neuronal cell body.

1	(D) Endocrine signaling depends on endocrine cells, which secrete hormones into the bloodstream for distribution throughout the body. Many of the same types of signaling molecules are used in paracrine, synaptic, and endocrine signaling; the crucial differences lie in the speed and selectivity with which the signals are delivered to their targets. In most cases, however, signaling cells secrete signal molecules into the extracellular fluid. Often, the secreted molecules are local mediators, which act only on cells in the local environment of the signaling cell. This is called paracrine signaling (Figure 15–2B). Usually, the signaling and target cells in paracrine signaling are of different cell types, but cells may also produce signals that they themselves respond to: this is referred to as autocrine signaling. Cancer cells, for example, often produce extracellular signals that stimulate their own survival and proliferation.

1	Large multicellular organisms like us need long-range signaling mechanisms to coordinate the behavior of cells in remote parts of the body. Thus, they have evolved cell types specialized for intercellular communication over large distances. The most sophisticated of these are nerve cells, or neurons, which typically extend long, branching processes (axons) that enable them to contact target cells far away, where the processes terminate at the specialized sites of signal transmission known as chemical synapses. When a neuron is activated by stimuli from other nerve cells, it sends electrical impulses (action potentials) rapidly along its axon; when the impulse reaches the synapse at the end of the axon, it triggers secretion of a chemical signal that acts as a neurotransmitter. The tightly organized structure of the synapse ensures that the neurotransmitter is delivered specifically to receptors on the postsynaptic target cell (Figure 15–2C). The details of this synaptic signaling

1	organized structure of the synapse ensures that the neurotransmitter is delivered specifically to receptors on the postsynaptic target cell (Figure 15–2C). The details of this synaptic signaling process are discussed in Chapter 11.

1	A quite different strategy for signaling over long distances makes use of endocrine cells, which secrete their signal molecules, called hormones, into the bloodstream. The blood carries the molecules far and wide, allowing them to act on target cells that may lie anywhere in the body (Figure 15–2D). Extracellular Signal Molecules Bind to Specific Receptors

1	Extracellular Signal Molecules Bind to Specific Receptors Cells in multicellular animals communicate by means of hundreds of kinds of extracellular signal molecules. These include proteins, small peptides, amino acids, nucleotides, steroids, retinoids, fatty acid derivatives, and even dissolved gases such as nitric oxide and carbon monoxide. Most of these signal molecules are released into the extracellular space by exocytosis from the signaling cell, as discussed in Chapter 13. Some, however, are emitted by diffusion through the signaling cell’s plasma membrane, whereas others are displayed on the external surface of the cell and remain attached to it, providing a signal to other cells only when they make contact. Transmembrane signal proteins may operate in this way, or their extracellular domains may be released from the signaling cell’s surface by proteolytic cleavage and then act at a distance.

1	Regardless of the nature of the signal, the target cell responds by means of a receptor, which binds the signal molecule and then initiates a response in the target cell. The binding site of the receptor has a complex structure that is shaped to recognize the signal molecule with high specificity, helping to ensure that the receptor responds only to the appropriate signal and not to the many other signaling molecules surrounding the cell. Many signal molecules act at very low concentrations (typically ≤ 10–8 M), and their receptors usually bind them with high affinity (dissociation constant Kd ≤ 10–8 M; see Figure 3–44).

1	In most cases, receptors are transmembrane proteins on the target-cell surface. When these proteins bind an extracellular signal molecule (a ligand), they become activated and generate various intracellular signals that alter the behavior of the cell. In other cases, the receptor proteins are inside the target cell, and the signal molecule has to enter the cell to bind to them: this requires that the signal molecule be sufficiently small and hydrophobic to diffuse across the target cell’s plasma membrane (Figure 15–3). This chapter focuses primarily on signaling through cell-surface receptors, but we will briefly describe signaling through intracellular receptors later in the chapter. Each Cell Is Programmed to Respond to Specific Combinations of Extracellular Signals

1	Each Cell Is Programmed to Respond to Specific Combinations of Extracellular Signals A typical cell in a multicellular organism is exposed to hundreds of different signal molecules in its environment. The molecules can be soluble, bound to the extracellular matrix, or bound to the surface of a neighboring cell; they can be stimulatory or inhibitory; they can act in innumerable different combinations; and they can influence almost any aspect of cell behavior. The cell responds to this blizzard of signals selectively, in large part by expressing only those receptors and intracellular signaling systems that respond to the signals that are required for the regulation of that cell.

1	Most cells respond to many different signals in the environment, and some of these signals may influence the response to other signals. One of the key challenges in cell biology is to determine how a cell integrates all of this signaling information in order to make decisions—to divide, to move, to differentiate, and so on. Many cells, for example, require a specific combination of extracellular survival factors to allow the cell to continue living; when deprived of these signals, the cell activates a suicide program and kills itself—usually by apoptosis, a form of programmed cell death, as discussed in Chapter 18. Cell proliferation often depends on a combination of signals that promote both cell division and survival, as well as signals that stimulate cell growth (Figure 15–4). On the other hand, differentiation into a nondividing state (called terminal differentiation) frequently requires a different combination of survival and differentiation signals that must override any signal

1	hand, differentiation into a nondividing state (called terminal differentiation) frequently requires a different combination of survival and differentiation signals that must override any signal to divide.

1	In principle, the hundreds of signal molecules that an animal makes can be used in an almost unlimited number of combinations to control the diverse behaviors of its cells in highly specific ways. Relatively small numbers of types of signal molecules and receptors are sufficient. The complexity lies in the ways in which cells respond to the combinations of signals that they receive. A signal molecule often has different effects on different types of target cells. The neurotransmitter acetylcholine (Figure 15–5A), for example, decreases the small, hydrophobic signal molecule

1	A signal molecule often has different effects on different types of target cells. The neurotransmitter acetylcholine (Figure 15–5A), for example, decreases the small, hydrophobic signal molecule Figure 15–3 The binding of extracellular signal molecules to either cell-surface or intracellular receptors. (A) Most signal molecules are hydrophilic and are therefore unable to cross the target cell’s plasma membrane directly; instead, they bind to cell-surface receptors, which in turn generate signals inside the target cell (see Figure 15–1). (B) Some small signal molecules, by contrast, diffuse across the plasma membrane and bind to receptor proteins inside the target cell—either in the cytosol or in the nucleus (as shown here). Many of these small signal molecules are hydrophobic and poorly soluble in aqueous solutions; they are therefore transported in the bloodstream and other extracellular fluids bound to carrier proteins, from which they dissociate before entering the target cell.

1	rate of action potential firing in heart pacemaker cells (Figure 15–5B) and stimulates the production of saliva by salivary gland cells (Figure 15–5C), even though the receptors are the same on both cell types. In skeletal muscle, acetylcholine causes the cells to contract by binding to a different receptor protein (Figure 15–5D). The different effects of acetylcholine in these cell types result from differences in the intracellular signaling proteins, effector proteins, and genes that are activated. Thus, an extracellular signal itself has little information content; it simply induces the cell to respond according to its predetermined state, which depends on the cell’s developmental history and the specific genes it expresses.

1	Figure 15–4 An animal cell’s dependence on multiple extracellular signal molecules. Each cell type displays a set of receptors that enables it to respond to a corresponding set of signal molecules produced by other cells. These signal molecules work in various combinations to regulate the behavior of the cell. As shown here, an individual cell often requires multiple signals to survive (blue arrows) and additional signals to grow and divide (red arrows) or differentiate (green arrows). If deprived of appropriate survival signals, a cell will undergo a form of cell suicide known as apoptosis. The actual situation is even more complex. Although not shown, some extracellular signal molecules act to inhibit these and other cell behaviors, or even to induce apoptosis.

1	Figure 15–5 Various responses induced by the neurotransmitter acetylcholine. (A) The chemical structure of acetylcholine. (B–D) Different cell types are specialized to respond to acetylcholine in different ways. In some cases (B and C), acetylcholine binds to similar receptor proteins (G-protein-coupled receptors; see Figure 15–6), but the intracellular signals produced are interpreted differently in cells specialized for different functions. In other cases (D), the receptor protein is also different (here, an ion-channel-coupled receptor; see Figure 15–6). There Are Three Major Classes of Cell-Surface Receptor Proteins Most extracellular signal molecules bind to specific receptor proteins on the surface of the target cells they influence and do not enter the cytosol or nucleus. These cell-surface receptors act as signal transducers by converting an extracellular ligand-binding event into intracellular signals that alter the behavior of the target cell.

1	Most cell-surface receptor proteins belong to one of three classes, defined by their transduction mechanism. Ion-channel-coupled receptors, also known as transmitter-gated ion channels or ionotropic receptors, are involved in rapid synaptic signaling between nerve cells and other electrically excitable target cells such as nerve and muscle cells (Figure 15–6A). This type of signaling is mediated by a small number of neurotransmitters that transiently open or close an ion channel formed by the protein to which they bind, briefly changing the ion permeability of the plasma membrane and thereby changing the excitability of the postsynaptic target cell. Most ion-channel-coupled receptors belong to a large family of homologous, multipass transmembrane proteins. Because they are discussed in detail in Chapter 11, we will not consider them further here.

1	G-protein-coupled receptors act by indirectly regulating the activity of a separate plasma-membrane-bound target protein, which is generally either an enzyme or an ion channel. A trimeric GTP-binding protein (G protein) mediates the interaction between the activated receptor and this target protein (Figure 15–6B). The activation of the target protein can change the concentration of one or more small intracellular signaling molecules (if the target protein is an enzyme), or it can change the ion permeability of the plasma membrane (if the target protein is an ion channel). The small intracellular signaling molecules act in turn to alter the behavior of yet other signaling proteins in the cell.

1	Enzyme-coupled receptors either function as enzymes or associate directly with enzymes that they activate (Figure 15–6C). They are usually single-pass transmembrane proteins that have their ligand-binding site outside the cell and their catalytic or enzyme-binding site inside. Enzyme-coupled receptors are heterogeneous in structure compared with the other two classes; the great majority, however, are either protein kinases or associate with protein kinases, which phosphorylate specific sets of proteins in the target cell when activated.

1	There are also some types of cell-surface receptors that do not fit easily into any of these classes but have important functions in controlling the specialization of different cell types during development and in tissue renewal and repair in adults. We discuss these in a later section, after we explain how G-protein-coupled receptors and enzyme-coupled receptors operate. First, we continue our general discussion of the principles of signaling via cell-surface receptors. Numerous intracellular signaling molecules relay signals received by cell-surface receptors into the cell interior. The resulting chain of intracellular signaling events ultimately alters effector proteins that are responsible for modifying the behavior of the cell (see Figure 15–1).

1	Some intracellular signaling molecules are small chemicals, which are often called second messengers (the “first messengers” being the extracellular signals). They are generated in large amounts in response to receptor activation and diffuse away from their source, spreading the signal to other parts of the cell. Some, such as cyclic AMP and Ca2+, are water-soluble and diffuse in the cytosol, while others, such as diacylglycerol, are lipid-soluble and diffuse in the plane of the plasma membrane. In either case, they pass the signal on by binding to and altering the behavior of selected signaling or effector proteins.

1	Most intracellular signaling molecules are proteins, which help relay the signal into the cell by either generating second messengers or activating the next signaling or effector protein in the pathway. Many of these proteins behave like molecular switches. When they receive a signal, they switch from an inactive to an active state, until another process switches them off, returning them to their inactive state. The switching off is just as important as the switching on. If a signaling pathway is to recover after transmitting a signal so that it can be ready to transmit another, every activated molecule in the pathway must return to its original, unactivated state.

1	The largest class of molecular switches consists of proteins that are activated or inactivated by phosphorylation (discussed in Chapter 3). For these proteins, the switch is thrown in one direction by a protein kinase, which covalently adds one or more phosphate groups to specific amino acids on the signaling protein, and in the other direction by a protein phosphatase, which removes the phosphate groups (Figure 15–7A). The activity of any protein regulated by phosphorylation depends on the balance between the activities of the kinases that phosphorylate it and of the phosphatases that dephosphorylate it. About 30–50% of human proteins contain covalently attached phosphate, and the human genome encodes about 520 protein kinases and about 150 protein phosphatases. A typical mammalian cell makes use of hundreds of distinct types of protein kinases at any moment.

1	Protein kinases attach phosphate to the hydroxyl group of specific amino acids on the target protein. There are two main types of protein kinase. The great majority are serine/threonine kinases, which phosphorylate the hydroxyl groups of serines and threonines in their targets. Others are tyrosine kinases, which phosphorylate proteins on tyrosines. The two types of protein kinase are closely related members of a large family, differing primarily in the structure of their protein substrate binding sites. Many intracellular signaling proteins controlled by phosphorylation are themselves protein kinases, and these are often organized into kinase cascades. In such a cascade, one protein kinase, activated by phosphorylation, phosphorylates the next protein kinase in the sequence, and so on, relaying the signal onward and, in some cases, amplifying it or spreading it to other signaling pathways.

1	The other important class of molecular switches consists of GTP-binding proteins (discussed in Chapter 3). These proteins switch between an “on” (actively signaling) state when GTP is bound and an “off” state when GDP is bound. In the “on” state, they usually have intrinsic GTPase activity and shut themselves off by hydrolyzing their bound GTP to GDP (Figure 15–7B). There are two major types of GTP-binding proteins. Large, trimeric GTP-binding proteins (also called G proteins) help relay signals from G-protein-coupled receptors that activate them (see Figure 15–6B). Small monomeric GTPases (also called monomeric GTP-binding proteins) help relay signals from many classes of cell-surface receptors.

1	Specific regulatory proteins control both types of GTP-binding proteins. GTPase-activating proteins (GAPs) drive the proteins into an “off” state by increasing the rate of hydrolysis of bound GTP. Conversely, guanine nucleotide exchange factors (GEFs) activate GTP-binding proteins by promoting the release of bound GDP, which allows a new GTP to bind. In the case of trimeric G proteins, the activated receptor serves as the GEF. Figure 15–8 illustrates the regulation of monomeric GTPases. Not all molecular switches in signaling systems depend on phosphorylation or GTP binding. We see later that some signaling proteins are switched on or off by the binding of another signaling protein or a second messenger such as cyclic AMP or Ca2+, or by covalent modifications other than phosphorylation or dephosphorylation, such as ubiquitylation (discussed in Chapter 3).

1	For simplicity, we often portray a signaling pathway as a series of activation steps (see Figure 15–1). It is important to note, however, that most signaling pathways contain inhibitory steps, and a sequence of two inhibitory steps can have the same effect as one activating step (Figure 15–9). This double-negative activation is very common in signaling systems, as we will see when we describe specific pathways later in this chapter. Ideally, an activated intracellular signaling molecule should interact only with the appropriate downstream targets, and, likewise, the targets should only be activated by the appropriate upstream signal. In reality, however, intracellular

1	Figure 15–7 Two types of intracellular signaling proteins that act as molecular switches. (A) A protein kinase covalently adds a phosphate from ATP to the signaling protein, and a protein phosphatase removes the phosphate. Although not shown, many signaling proteins are activated by dephosphorylation rather than by phosphorylation. (B) A GTP-binding protein is induced to exchange its bound GDP for GTP, which activates the protein; the protein then inactivates itself by hydrolyzing its bound GTP to GDP. signaling molecules share the cytoplasm with a crowd of closely related signaling molecules that control a diverse array of cellular processes. It is inevitable that an occasional signaling molecule will bind or modify the wrong partner, potentially creating unwanted cross-talk and interference between signaling systems. How does a signal remain strong, precise, and specific under these noisy conditions?

1	The first line of defense comes from the high affinity and specificity of the interactions between signaling molecules and their correct partners compared to the relatively low affinity of the interactions between inappropriate partners. The binding of a signaling molecule to the correct target is determined by precise and complex interactions between complementary surfaces on the two molecules. Protein kinases, for example, contain active sites that recognize a specific amino acid sequence around the phosphorylation site on the correct target protein, and they often contain additional docking sites that promote a specific, high-affinity interaction with the target. These and related mechanisms help provide a strong and persistent interaction between the correct partners, reducing the likelihood of inappropriate interactions with other proteins.

1	Another important way that cells avoid responses to unwanted background signals depends on the ability of many downstream target proteins to simply ignore such signals. These proteins respond only when the upstream signal reaches a high concentration or activity level. Consider a signaling pathway in which a protein kinase activates some downstream target protein by phosphorylation. If a response is triggered only when more than half of the target proteins are phosphorylated, then there will be little harm done if a small number of them are occasionally phosphorylated by some inappropriate protein kinase. Furthermore, constitutively active protein phosphatases will further reduce the impact of background phosphorylation by rapidly removing much of it. In these and other ways, intracellular signaling systems filter out noise, generating little or no response to low levels of stimuli.

1	Figure 15–8 The regulation of a monomeric GTPase. GTPase-activating proteins (GAPs) inactivate the protein by stimulating it to hydrolyze its bound GTP to GDP, which remains tightly bound to the inactivated GTPase. Guanine nucleotide exchange factors (GEFs) activate the inactive protein by stimulating it to release its GDP; because the concentration of GTP in the cytosol is 10 times greater than the concentration of GDP, the protein rapidly binds GTP and is thereby activated. Figure 15–9 A sequence of two inhibitory signals produces a positive signal. In this simple signaling system, a transcription regulator is kept in an inactive state by a bound inhibitor protein. In response to some upstream signal, a protein kinase is activated and phosphorylates the inhibitor, causing its dissociation from the transcription regulator and thereby activating gene expression.

1	This signaling pathway can be diagrammed as a sequence of four steps, including two sequential inhibitory steps that are equivalent to a single activating step. Cells in a population often exhibit random variation in the concentration or activity of their intracellular signaling molecules. Similarly, individual molecules in a large population of molecules vary in their activity or interactions with other molecules. This signal variability introduces another form of noise that can interfere with the precision and efficiency of signaling. Most signaling systems, however, are built to generate remarkably robust and precise responses even when upstream signals are variable or even when some components of the system are disabled. In many cases, this robustness depends on the presence of backup mechanisms: for example, a signal might employ two parallel pathways to activate a single common downstream target protein, allowing the response to occur even if one pathway is crippled.

1	One simple and effective strategy for enhancing the specificity of interactions between signaling molecules is to localize them in the same part of the cell or even within large protein complexes, thereby ensuring that they interact only with each other and not with inappropriate partners. Such mechanisms often involve scaffold proteins, which bring together groups of interacting signaling proteins into signaling complexes, often before a signal has been received (Figure 15–10A). Because the scaffold holds the proteins in close proximity, they can interact at high local concentrations and be sequentially activated rapidly, efficiently, and selectively in response to an appropriate extracellular signal, avoiding unwanted cross-talk with other signaling pathways.

1	In other cases, such signaling complexes form only transiently in response to an extracellular signal and rapidly disassemble when the signal is gone. They often assemble around a receptor after an extracellular signal molecule has activated it. In many of these cases, the cytoplasmic tail of the activated receptor is phosphorylated during the activation process, and the phosphorylated amino acids then serve as docking sites for the assembly of other signaling proteins (Figure 15–10B). In yet other cases, receptor activation leads to the production of modified phospholipid molecules (called phosphoinositides) in the adjacent plasma membrane, which then recruit specific intracellular signaling proteins to this region of membrane, where they are activated (Figure 15–10C).

1	Simply bringing intracellular signaling proteins together into close proximity is sometimes sufficient to activate them. Thus, induced proximity, where a signal triggers assembly of a signaling complex, is commonly used to relay signals from protein to protein along a signaling pathway. The assembly of such signaling complexes depends on various highly conserved, small interaction domains, which are found in many intracellular signaling proteins. Each of these compact protein modules binds to a particular structural motif in another protein or lipid. The recognized motif in the interacting protein can be a short peptide sequence, a covalent modification (such as a phosphorylated amino acid), or another protein domain. The use of modular interaction domains presumably facilitated the evolution of new signaling pathways; because it can be inserted at many locations in a protein without disturbing the protein’s folding or function, a new interaction domain added to an existing signaling

1	of new signaling pathways; because it can be inserted at many locations in a protein without disturbing the protein’s folding or function, a new interaction domain added to an existing signaling protein could connect the protein to additional signaling pathways.

1	There are many types of interaction domains in signaling proteins. Src homology 2 (SH2) domains and phosphotyrosine-binding (PTB) domains, for example, bind to phosphorylated tyrosines in a particular peptide sequence on activated receptors or intracellular signaling proteins. Src homology 3 (SH3) domains bind to short, proline-rich amino acid sequences. Some pleckstrin homology (PH) domains bind to the charged head groups of specific phosphoinositides that are produced in the plasma membrane in response to an extracellular signal; they Figure 15–10 Three types of intracellular signaling complexes. (A) A receptor and some of the intracellular signaling proteins it activates in sequence are preassembled into a signaling complex on the inactive receptor by a large scaffold protein.

1	A signaling complex assembles transiently on a receptor only after the binding of an extracellular signal molecule has activated the receptor; here, the activated receptor phosphorylates itself at multiple sites, which then act as docking sites for intracellular signaling proteins. Activation of a receptor leads to the increased phosphorylation of specific phospholipids (phosphoinositides) in the adjacent plasma membrane; these then serve as docking sites for specific intracellular signaling proteins, which can now interact with each other. enable the protein they are part of to dock on the membrane and interact with other similarly recruited signaling proteins (see Figure 15–10C). Some signaling proteins consist solely of two or more interaction domains and function only as adaptors to link two other proteins together in a signaling pathway.

1	Interaction domains enable signaling proteins to bind to one another in multiple specific combinations. Like Lego® bricks, the proteins can form linear or branching chains or three-dimensional networks, which determine the route followed by the signaling pathway. As an example, Figure 15–11 illustrates how some interaction domains mediate the formation of a large signaling complex around the receptor for the hormone insulin. Another way of bringing receptors and intracellular signaling proteins together is to concentrate them in a specific region of the cell. An important example is the primary cilium that projects like an antenna from the surface of most vertebrate cells (discussed in Chapter 16). It is usually short and nonmotile and has micro-tubules in its core, and a number of surface receptors and signaling proteins are concentrated there. We shall see later that light and smell receptors are also highly concentrated in specialized cilia.

1	The Relationship Between Signal and Response Varies in Different Signaling Pathways The function of an intracellular signaling system is to detect and measure a specific stimulus in one location of a cell and then generate an appropriately timed and measured response at another location. The system accomplishes this task by sending information in the form of molecular “signals” from the sensor to the target, often through a series of intermediaries that do not simply pass the signal along but process it in various ways. All signaling systems do not work in precisely the same way: each has evolved specialized behaviors that produce a response that is appropriate for the cell function that system controls. In the following paragraphs, we list some of these behaviors and describe how they vary in different systems, as a foundation for more detailed discussions later. 1.

1	1. Response timing varies dramatically in different signaling systems, according to the speed required for the response. In some cases, such as synaptic signaling (see Figure 15–2C), the response can occur within milliseconds. In other cases, as in the control of cell fate by morphogens during development, a full response can require hours or days. 2.

1	2. Sensitivity to extracellular signals can vary greatly. Hormones tend to act at very low concentrations on their distant target cells, which are therefore highly sensitive to low concentrations of signal. Neurotransmitters, on the other hand, operate at much higher concentrations at a synapse, reducing the need for high sensitivity in postsynaptic receptors. Sensitivity is often controlled by changes in the number or affinity of the receptors on the target cell. A particularly important mechanism for increasing the sensitivity of a signaling system is signal amplification, whereby a small number of activated cell-surface receptors evoke a large intracellular response either by producing large amounts of a second messenger or by activating many copies of a downstream signaling protein. 3. Dynamic range of a signaling system is related to its sensitivity. Some systems, like those involved in simple developmental decisions, are responsive

1	Figure 15–11 A specific signaling complex formed using modular interaction domains. This example is based on the insulin receptor, which is an enzyme-coupled receptor (a receptor tyrosine kinase, discussed later). First, the activated receptor phosphorylates itself on tyrosines, and one of the phosphotyrosines then recruits a docking protein called insulin receptor substrate-1 (IRS1) via a PTB domain of IRS1; the PH domain of IRS1 also binds to specific phosphoinositides on the inner surface of the plasma membrane. Then, the activated receptor phosphorylates IRS1 on tyrosines, and one of these phosphotyrosines binds the SH2 domain of the adaptor protein Grb2. Next, Grb2 uses one of its two SH3 domains to bind to a proline-rich region of a protein called Sos, which relays the signal downstream by acting as a GEF (see Figure 15–8) to activate a monomeric GTPase called Ras (not shown). Sos also binds to phosphoinositides in the plasma membrane via its PH domain. Grb2 uses its other SH3

1	by acting as a GEF (see Figure 15–8) to activate a monomeric GTPase called Ras (not shown). Sos also binds to phosphoinositides in the plasma membrane via its PH domain. Grb2 uses its other SH3 domain to bind to a proline-rich sequence in a scaffold protein. The scaffold protein binds several other signaling proteins, and the other phosphorylated tyrosines on IRS1 recruit additional signaling proteins that have SH2 domains (not shown).

1	over a narrow range of extracellular signal concentrations. Other systems, like those controlling vision or the metabolic response to some hormones, are highly responsive over a much broader range of signal strengths. We will see that broad dynamic range is often achieved by adaptation mechanisms that adjust the responsiveness of the system according to the prevailing amount of signal. 4. Persistence of a response can vary greatly. A transient response of less than a second is appropriate in some synaptic responses, for example, while a prolonged or even permanent response is required in cell fate decisions during development. Numerous mechanisms, including positive feedback, can be used to alter the duration and reversibility of a response. 5.

1	5. Signal processing can convert a simple signal into a complex response. In many systems, for example, a gradual increase in an extracellular signal is converted into an abrupt, switchlike response. In other cases, a simple input signal is converted into an oscillatory response, produced by a repeating series of transient intracellular signals. Feedback usually lies at the heart of biochemical switches and oscillators, as we describe later. 6.

1	6. Integration allows a response to be governed by multiple inputs. As discussed earlier, for example, specific combinations of extracellular signals are generally required to stimulate complex cell behaviors such as cell survival and proliferation (see Figure 15–4). The cell therefore has to integrate information coming from multiple signals, which often depends on intracellular coincidence detectors; these proteins are equivalent to AND gates in the microprocessor of a computer, in that they are only activated if they receive multiple converging signals (Figure 15–12). 7.

1	7. Coordination of multiple responses in one cell can be achieved by a single extracellular signal. Some extracellular signal molecules, for example, stimulate a cell to both grow and divide. This coordination generally depends on mechanisms for distributing a signal to multiple effectors, by creating branches in the signaling pathway. In some cases, the branching of signaling pathways can allow one signal to modulate the strength of a response to other signals. Given the complexity that arises from behaviors like signal integration, distribution, and feedback, it is clear that signaling systems rarely depend on a simple linear sequence of steps but are often more like a network, in which information flows not just forward but in multiple directions—and sometimes even backward. A major research challenge is to understand the nature of these networks and the response behaviors they can achieve. The Speed of a Response Depends on the Turnover of Signaling Molecules

1	The Speed of a Response Depends on the Turnover of Signaling Molecules The speed of any signaling response depends on the nature of the intracellular signaling molecules that carry out the target cell’s response. When the response requires only changes in proteins already present in the cell, it can occur very rapidly: an allosteric change in a neurotransmitter-gated ion channel (discussed in Chapter 11), for example, can alter the plasma membrane electrical potential in milliseconds, and responses that depend solely on protein phosphorylation can occur within seconds. When the response involves changes in gene expression and the synthesis of new proteins, however, it usually requires many minutes or hours, regardless of the mode of signal delivery (Figure 15–13).

1	It is natural to think of intracellular signaling systems in terms of the changes produced when an extracellular signal is delivered. But it is just as important to consider what happens when the signal is withdrawn. During development, transient extracellular signals often produce lasting effects: they can trigger a change in the cell’s development that persists indefinitely through cell memory mechanisms, as we discuss later (and in Chapters 7 and 22). In most cases in adult tissues, however, the response fades when a signal ceases. Often the effect is transient Figure 15–12 Signal integration. Extracellular signals A and B activate different intracellular signaling pathways, each of which leads to the phosphorylation of protein Y but at different sites on the protein. Protein Y is activated only when both of these sites are phosphorylated, and therefore it becomes active only when signals A and B are simultaneously present. Such proteins are often called coincidence detectors.

1	because the signal exerts its effects by altering the concentrations of intracellular molecules that are short-lived (unstable), undergoing continual turnover. Thus, once the extracellular signal is gone, the degradation of the old molecules quickly wipes out all traces of the signal’s action. It follows that the speed with which a cell responds to signal removal depends on the rate of destruction, or turnover, of the intracellular molecules that the signal affects.

1	It is also true, although much less obvious, that this turnover rate can determine the promptness of the response when an extracellular signal arrives. Consider, for example, two intracellular signaling molecules, X and Y, both of which are normally maintained at a steady-state concentration of 1000 molecules per cell. The cell synthesizes and degrades molecule Y at a rate of 100 molecules per second, with each molecule having an average lifetime of 10 seconds. Molecule X has a turnover rate that is 10 times slower than that of Y: it is both synthesized and degraded at a rate of 10 molecules per second, so that each molecule has an average lifetime in the cell of 100 seconds. If a signal acting on the cell causes a tenfold increase in the synthesis rates of both X and Y with no change in the molecular lifetimes, at the end of 1 second the concentration of Y will have increased by nearly 900 molecules per cell (10 × 100 – 100), while the concentration of X will have increased by only

1	the molecular lifetimes, at the end of 1 second the concentration of Y will have increased by nearly 900 molecules per cell (10 × 100 – 100), while the concentration of X will have increased by only 90 molecules per cell. In fact, after a molecule’s synthesis rate has been either increased or decreased abruptly, the time required for the molecule to shift halfway from its old to its new equilibrium concentration is equal to its half-life—that is, equal to the time that would be required for its concentration to fall by half if all synthesis were stopped (Figure 15–14).

1	The same principles apply to proteins and small molecules, whether the molecules are in the extracellular space or inside cells. Many intracellular proteins have short half-lives, some surviving for less than 10 minutes. In most cases, these are key regulatory proteins whose concentrations are rapidly controlled in the cell by changes in their rates of synthesis. As we have seen, many cell responses to extracellular signals depend on the conversion of intracellular signaling proteins from an inactive to an active form, rather than on their synthesis or degradation. Phosphorylation or the binding of GTP, for example, commonly activates signaling proteins. Even in these cases, however, the activation must be rapidly and continuously reversed (by dephosphorylation or GTP hydrolysis to GDP, respectively, in these examples) to make rapid signaling possible. These inactivation processes play a crucial part in determining the magnitude, rapidity, and duration of the response.

1	Figure 15–13 Slow and rapid responses to an extracellular signal. Certain types of signal-induced cellular responses, such as increased cell growth and division, involve changes in gene expression and the synthesis of new proteins; they therefore occur slowly, often starting an hour or more after the signal is received. Other responses—such as changes in cell movement, secretion, or metabolism— need not involve changes in gene transcription and therefore occur much more quickly, often starting in seconds or minutes; they may involve the rapid phosphorylation of effector proteins in the cytoplasm, for example. Synaptic responses mediated by changes in membrane potential are even quicker and can occur in milliseconds (not shown). Some signaling systems generate both rapid and slow responses as shown here, allowing the cell to respond quickly to a signal while simultaneously initiating a more long-term, persistent response.

1	minutes after the synthesis rate has minutes after the synthesis rate has been decreased by a factor of 10 been increased by a factor of 10 relative concentration of intracellular molecule Cells Can Respond Abruptly to a Gradually Increasing Signal

1	Some signaling systems are capable of generating a smoothly graded response over a wide range of extracellular signal concentrations (Figure 15–15, blue line); such systems are useful, for example, in the fine tuning of metabolic processes by some hormones. Other signaling systems generate significant responses only when the signal concentration rises beyond some threshold value. These abrupt responses are of two types. One is a sigmoidal response, in which low concentrations of stimulus do not have much effect, but then the response rises steeply and continuously at intermediate stimulus levels (Figure 15–15, red line). Such systems provide a filter to reduce inappropriate responses to low-level background signals but respond with high sensitivity when the stimulus falls within a small range of physiological signal concentrations. A second type of abrupt response is the discontinuous or all-or-none response, in which the response switches on completely (and often irreversibly) when

1	range of physiological signal concentrations. A second type of abrupt response is the discontinuous or all-or-none response, in which the response switches on completely (and often irreversibly) when the signal reaches some threshold concentration (Figure 15–15, green line). Such responses are particularly useful for controlling the choice between two alternative cell states, and they generally involve positive feedback, as we describe in more detail shortly.

1	Cells use a variety of molecular mechanisms to produce a sigmoidal response to increasing signal concentrations. In one mechanism, more than one intracellular signaling molecule must bind to its downstream target protein to induce a response. As we discuss later, for example, four molecules of the second messenger cyclic AMP must bind simultaneously to each molecule of cyclic-AMP-dependent protein kinase (PKA) to activate the kinase. A similar sharpening of response is seen when the activation of an intracellular signaling protein requires phosphorylation at more than one site. Such responses become sharper as the number of required molecules or phosphate groups increases, and if the number is large enough, responses become almost all-or-none (Figure 15–16).

1	Responses are also sharpened when an intracellular signaling molecule activates one enzyme and also inhibits another enzyme that catalyzes the opposite reaction. A well-studied example of this common type of regulation is the stimulation of glycogen breakdown in skeletal muscle cells induced by the hormone adrenaline (epinephrine). Adrenaline’s binding to a G-protein-coupled

1	Figure 15–14 The importance of rapid turnover. The graphs show the predicted relative rates of change in the intracellular concentrations of molecules with differing turnover times when their synthesis rates are either (A) decreased or (B) increased suddenly by a factor of 10. In both cases, the concentrations of those molecules that are normally degraded rapidly in the cell (red lines) change quickly, whereas the concentrations of those that are normally degraded slowly (green lines) change proportionally more slowly. The numbers (in blue) on the right are the half-lives assumed for each of the different molecules.

1	switchlike responses. Some cell responses increase gradually as the concentration of extracellular signal molecule increases, eventually reaching a plateau as the signaling pathway is saturated, resulting in a hyperbolic response curve (blue line). In other cases, the signaling system reduces the response at low signal concentrations and then produces a steeper response at some intermediate signal concentration—resulting in a sigmoidal response curve (red line). In still other cases, the response is more abrupt and switchlike; the cell switches completely between a low and high response, without any stable intermediate response (green line). cell-surface receptor increases the intracellular concentration of cyclic AMP, which both activates an enzyme that promotes glycogen breakdown and inhibits an enzyme that promotes glycogen synthesis.

1	Like intracellular metabolic pathways (discussed in Chapter 2) and the systems controlling gene activity (Chapter 7), most intracellular signaling systems incorporate feedback loops, in which the output of a process acts back to regulate that same process. We discussed the mathematical analysis of feedback loops in Chapter 8. In positive feedback, the output stimulates its own production; in negative feedback, the output inhibits its own production (Figure 15–17). Feedback loops are of great general importance in biology, and they regulate many chemical and physical processes in cells. Those that regulate cell signaling can either operate exclusively within the target cell or involve the secretion of extracellular signals. Here, we focus on those feedback loops that operate entirely within the target cell; even the simplest of these loops can produce complex and interesting effects.

1	Positive feedback in a signaling pathway can transform the behavior of the responding cell. If the positive feedback is of only moderate strength, its effect will be simply to steepen the response to the signal, generating a sigmoidal response like those described earlier; but if the feedback is strong enough, it can produce an all-or-none response (see Figure 15–15). This response goes hand in hand with a further property: once the responding system has switched to the high level of activation, this condition is often self-sustaining and can persist even after the 0.5 1.0 1.5 2.0 1 2 8 16 % of maximum activation of target protein an allosteric protein as a function of effector molecule concentration.

1	The curves show how the sharpness of the activation response increases with an increase in the number of allosteric effector molecules that must be bound simultaneously to activate the target protein. The curves shown are those expected, under certain conditions, if the 0 activation requires the simultaneous binding relative concentration of effector molecule of 1, 2, 8, or 16 effector molecules. signal strength drops back below its critical value. In such a case, the system is said to be bistable: it can exist in either a “switched-off” or a “switched-on” state, and a transient stimulus can flip it from the one state to the other (Figure 15–18A and B).

1	Through positive feedback, a transient extracellular signal can induce longterm changes in cells and their progeny that can persist for the lifetime of the organism. The signals that trigger muscle-cell specification, for example, turn on the transcription of a series of genes that encode muscle-specific transcription regulatory proteins, which stimulate the transcription of their own genes, as well as genes encoding various other muscle-cell proteins; in this way, the decision to become a muscle cell is made permanent. This type of cell memory, which depends on positive feedback, is one of the basic ways in which a cell can undergo a lasting change of character without any alteration in its DNA sequence.

1	Studies of signaling responses in large populations of cells can give the false impression that a response is smoothly graded, even when strong positive feedback is causing an abrupt, discontinuous switch in the response in individual cells. Only by studying the response in single cells is it possible to see its all-or-none character (Figure 15–19). The misleading smooth response in a cell population is due to the random, intrinsic variability in signaling systems that we described earlier: all cells in a population do not respond identically to the same concentration of extracellular signal, especially at intermediate signal concentrations where the receptor is only partially occupied. Negative Feedback is a Common Motif in Signaling Systems

1	Negative Feedback is a Common Motif in Signaling Systems By contrast with positive feedback, negative feedback counteracts the effect of a stimulus and thereby abbreviates and limits the level of the response, making the system less sensitive to perturbations (see Chapter 8). As with positive feedback, however, qualitatively different responses can be obtained when the feedback Figure 15–17 Positive and negative feedback. In these simple examples, a stimulus activates protein A, which, in turn, activates protein B. Protein B then acts back to either increase or decrease the activity of A.

1	Figure 15–18 Some effects of simple feedback. The graphs show the computed effects of simple positive and negative feedback loops (see Chapter 8). In each case, the input signal is an activated protein kinase (S) that phosphorylates and thereby activates another protein kinase (E); a protein phosphatase (I) dephosphorylates and inactivates the activated E kinase. In the graphs, the red line indicates the activity of the E kinase over time; the underlying blue bar indicates the time for which the input signal (activated S kinase) is present. (A) Diagram of the positive feedback loop, in which the activated E kinase acts back to promote its own phosphorylation and activation; the basal activity of the I phosphatase dephosphorylates activated E at a steady, low rate. (B) The top graph shows that, without feedback, the activity of the E kinase is simply proportional (with a

1	E kinase E kinase I phosphatase short lag) to the level of stimulation by the S kinase. The bottom graph shows that, with the positive feedback loop, the transient stimulation by S kinase switches the system from an “off” state to an “on” state, which then persists after the stimulus has been removed. (C) Diagram of the negative feedback loop, in which the activated E kinase phosphorylates and activates the I phosphatase, thereby increasing the rate at which the phosphatase dephosphorylates and inactivates the phosphorylated E kinase. (D) The top graph shows, again, the response in E kinase activity without feedback. The other graphs show the effects on E kinase activity of negative feedback operating after a short or long delay. With a short delay, the system shows a strong, brief response when the signal is abruptly changed, and the feedback then drives the response back down to a lower level. With a long delay, the feedback SIGNAL produces sustained oscillations for as long (D) as

1	when the signal is abruptly changed, and the feedback then drives the response back down to a lower level. With a long delay, the feedback SIGNAL produces sustained oscillations for as long (D) as the stimulus is present.

1	0 0.001 0.01 0.1 1 10 progesterone (µM) increasing concentration of progesterone (A) operates more powerfully. A delayed negative feedback with a long enough delay can produce responses that oscillate. The oscillations may persist for as long as the stimulus is present (Figure 15–18C and D) or they may even be generated spontaneously, without need of an external signal to drive them. Many such oscillators also contain positive feedback loops that generate sharper oscillations. Later in this chapter, we will encounter specific examples of oscillatory behavior in the intracellular responses to extracellular signals; all of them depend on negative feedback, generally accompanied by positive feedback.

1	If negative feedback operates with a short delay, the system behaves like a change detector. It gives a strong response to a stimulus, but the response rapidly decays even while the stimulus persists; if the stimulus is suddenly increased, however, the system responds strongly again, but, again, the response rapidly decays. This is the phenomenon of adaptation, which we now discuss. Cells Can Adjust Their Sensitivity to a Signal

1	In responding to many types of stimuli, cells and organisms are able to detect the same percentage of change in a signal over a wide range of stimulus strengths. The target cells accomplish this through a reversible process of adaptation, or desensitization, whereby a prolonged exposure to a stimulus decreases the cells’ response to that level of stimulus. In chemical signaling, adaptation enables cells to respond to changes in the concentration of an extracellular signal molecule (rather than to the absolute concentration of the signal) over a very wide range of signal concentrations. The underlying mechanism is negative feedback that operates with a short delay: a strong response modifies the signaling machinery involved, such that the machinery resets itself to become less responsive to the same level of signal (see Figure 15–18D, middle graph). Owing to the delay, however, a sudden increase in the signal is able to stimulate the cell again for a short period before the negative

1	to the same level of signal (see Figure 15–18D, middle graph). Owing to the delay, however, a sudden increase in the signal is able to stimulate the cell again for a short period before the negative feedback has time to kick in.

1	Adaptation to a signal molecule can occur in various ways. It can result from inactivation of the receptors themselves. The binding of signal molecules to cell-surface receptors, for example, may induce the endocytosis and temporary sequestration of the receptors in endosomes. In some cases, such signal-induced receptor endocytosis leads to the destruction of the receptors in lysosomes, a process referred to as receptor down-regulation (in other cases, however, activated receptors continue to signal after they have been endocytosed). Receptors can also become inactivated on the cell surface—for example, by becoming phosphorylated—with a short delay following their activation. Adaptation can also occur at sites downstream of the receptors, either by a change in intracellular signaling proteins involved in transducing the extracellular signal or by the production of an inhibitor protein that blocks the signal transduction process. These various adaptation mechanisms are compared in

1	proteins involved in transducing the extracellular signal or by the production of an inhibitor protein that blocks the signal transduction process. These various adaptation mechanisms are compared in Figure 15–20.

1	Though bewildering in their complexity, the multiple cross-regulatory signaling pathways and feedback loops that we describe in this chapter are not just a haphazard tangle, but a highly evolved system for processing and interpreting

1	Figure 15–19 The importance of examining individual cells to detect all-or-none responses to increasing concentrations of an extracellular signal. In these experiments, immature frog eggs (oocytes) were stimulated with increasing concentrations of the hormone progesterone. The response was assessed by analyzing the activation of MAP kinase (discussed later), which is one of the protein kinases activated by phosphorylation in the response. The amount of phosphorylated (activated) MAP kinase in extracts of the oocytes was assessed biochemically. In (A), extracts of populations of stimulated oocytes were analyzed, and the activation of MAP kinase appeared to increase progressively with increasing progesterone concentration. There are two possible ways of explaining this result: (B) MAP kinase could have increased gradually in each individual cell with increasing progesterone concentration; or (C) individual cells could have responded in an all-or-none way, with the gradual increase in

1	could have increased gradually in each individual cell with increasing progesterone concentration; or (C) individual cells could have responded in an all-or-none way, with the gradual increase in total MAP kinase activation reflecting the increasing number of cells responding with increasing progesterone concentration. When extracts of individual oocytes were analyzed, it was found that cells had either very low amounts or very high amounts, but not intermediate amounts, of the activated kinase, indicating that the response was essentially all-or-none at the level of individual cells, as diagrammed in (C). Subsequent studies revealed that this allor-none response is due in part to strong positive feedback in the progesterone signaling system. (Adapted from J.E. Ferrell and E.M. Machleder, Science 280:895– 898, 1998. With permission from AAAS.)

1	Figure 15–20 Some ways in which target cells can become adapted (desensitized) to an extracellular signal molecule. The mechanisms shown here that operate at the level of the receptor often involve phosphorylation or ubiquitylation of the receptor proteins.

1	the vast number of signals that impinge upon animal cells. The whole molecular control network, leading from the receptors at the cell surface to the genes in the nucleus, can be viewed as a computing device; and, like that other biological computing device, the brain, it presents one of the hardest problems in biology. We can identify the components and discover how they work individually. We can understand how small subsets of components work together as regulatory modules, noise filters, or adaptation mechanisms, as we have seen. However, it is a much more difficult task to understand how the system works as a whole. This is not only because the system is complex; it is also because the way in which it behaves is strongly dependent on the quantitative details of the molecular interactions, and, for most animal cells, we have only rough qualitative information. A major challenge for the future of signaling research is to develop more sophisticated quantitative and computational

1	and, for most animal cells, we have only rough qualitative information. A major challenge for the future of signaling research is to develop more sophisticated quantitative and computational methods for the analysis of signaling systems, as described in Chapter 8.

1	Each cell in a multicellular animal is programmed to respond to a specific set of extracellular signal molecules produced by other cells. The signal molecules act by binding to a complementary set of receptor proteins expressed by the target cells. Most extracellular signal molecules activate cell-surface receptor proteins, which act as signal transducers, converting the extracellular signal into intracellular ones that alter the behavior of the target cell. Activated receptors relay the signal into the cell interior by activating intracellular signaling proteins. Some of these signaling proteins transduce, amplify, or spread the signal as they relay it, while others integrate signals from different signaling pathways. Some function as switches that are transiently activated by phosphorylation or GTP binding. Large signaling complexes form by means of modular interaction domains in the signaling proteins, which allow the proteins to form functional signaling networks.

1	Target cells use various mechanisms, including feedback loops, to adjust the ways in which they respond to extracellular signals. Positive feedback loops can help cells to respond in an all-or-none fashion to a gradually increasing concentration of an extracellular signal and to convert a short-lasting signal into a long-lasting, or even irreversible, response. Negative feedback allows cells to adapt to a signal molecule, which enables them to respond to small changes in the concentration of the signal molecule over a large concentration range.

1	G-protein-coupled receptors (GPCRs) form the largest family of cell-surface receptors, and they mediate most responses to signals from the external world, as well as signals from other cells, including hormones, neurotransmitters, and local mediators. Our senses of sight, smell, and taste depend on them. There are more than 800 GPCRs in humans, and in mice there are about 1000 concerned with the sense of smell alone. The signal molecules that act on GPCRs are as varied in structure as they are in function and include proteins and small peptides, as well as derivatives of amino acids and fatty acids, not to mention photons of light and all the molecules that we can smell or taste. The same signal molecule can activate many different GPCR family members; for example, adrenaline activates at least 9 distinct GPCRs, acetylcholine another 5, and the neurotransmitter serotonin at least 14. The different receptors for the same signal are usually expressed in different cell types and elicit

1	least 9 distinct GPCRs, acetylcholine another 5, and the neurotransmitter serotonin at least 14. The different receptors for the same signal are usually expressed in different cell types and elicit different responses.

1	Despite the chemical and functional diversity of the signal molecules that activate them, all GPCRs have a similar structure. They consist of a single polypeptide chain that threads back and forth across the lipid bilayer seven times, forming a cylindrical structure, often with a deep ligand-binding site at its center (Figure 15–21). In addition to their characteristic orientation in the plasma membrane, they all use G proteins to relay the signal into the cell interior. The GPCR superfamily includes rhodopsin, the light-activated protein in the vertebrate eye, as well as the large number of olfactory receptors in the vertebrate nose. Other family members are found in unicellular organisms: the receptors in yeasts that recognize secreted mating factors are an example. It is likely that the GPCRs that mediate cell–cell signaling in multicellular organisms evolved from the sensory receptors in their unicellular eukaryotic ancestors.

1	It is remarkable that almost half of all known drugs work through GPCRs or the signaling pathways GPCRs activate. Of the many hundreds of genes in the human genome that encode GPCRs, about 150 encode orphan receptors, for which the ligand is unknown. Many of them are likely targets for new drugs that remain to be discovered. When an extracellular signal molecule binds to a GPCR, the receptor undergoes a conformational change that enables it to activate a trimeric GTP-binding protein (G protein), which couples the receptor to enzymes or ion channels in the membrane. In some cases, the G protein is physically associated with the receptor before the receptor is activated, whereas in others it binds only after receptor activation. There are various types of G proteins, each specific for a particular set of GPCRs and for a particular set of target proteins in the plasma membrane. They all have a similar structure, however, and operate similarly.

1	G proteins are composed of three protein subunits—α, β, and γ. In the unstimulated state, the α subunit has GDP bound and the G protein is inactive (Figure 15–22). When a GPCR is activated, it acts like a guanine nucleotide exchange factor (GEF) and induces the α subunit to release its bound GDP, allowing GTP to bind in its place. GTP binding then causes an activating conformational change in the Gα subunit, releasing the G protein from the receptor and triggering dissociation of the GTP-bound Gα subunit from the Gβγ pair—both of which then interact with various targets, such as enzymes and ion channels in the plasma membrane, which relay the signal onward (Figure 15–23).

1	The α subunit is a GTPase and becomes inactive when it hydrolyzes its bound GTP to GDP. The time required for GTP hydrolysis is usually short because the GTPase activity is greatly enhanced by the binding of the α subunit to a second protein, which can be either the target protein or a specific regulator of G protein signaling (RGS). RGS proteins act as α-subunit-specific GTPase-activating proteins (GAPs) (see Figure 15–8), and they help shut off G-protein-mediated responses in all eukaryotes. There are about 25 RGS proteins encoded in the human genome, each of which interacts with a particular set of G proteins.

1	Figure 15–21 A G-protein-coupled receptor (GPCR). (A) GPCRs that bind small ligands such as adrenaline have small extracellular domains, and the ligand usually binds deep within the plane of the membrane to a site that is formed by amino acids from several transmembrane segments. GPCRs that bind protein ligands have a large extracellular domain (not shown here) that contributes to ligand binding. (B) The structure of the β2-adrenergic receptor, a receptor for the neurotransmitter adrenaline, illustrates the typical cylindrical arrangement of the seven transmembrane helices in a GPCR. The ligand (orange) binds in a pocket between the helices, resulting in conformational changes on the cytoplasmic surface of the receptor that promote G-protein activation (not shown). (PDB code: 3P0G.) Some G Proteins Regulate the Production of Cyclic AMP

1	Some G Proteins Regulate the Production of Cyclic AMP Cyclic AMP (cAMP) acts as a second messenger in some signaling pathways. An extracellular signal can increase cAMP concentration more than twentyfold in seconds (Figure 15–24). As explained earlier (see Figure 15–14), such a rapid response requires balancing a rapid synthesis of the molecule with its rapid breakdown or removal. Cyclic AMP is synthesized from ATP by an enzyme called

1	Figure 15–22 The structure of an inactive G protein. (A) Note that both the α and the γ subunits have covalently attached lipid molecules (red tails) that help bind them to the plasma membrane, and the α subunit has GDP bound. (B) The three-dimensional structure of the inactive, GDP-bound form of a G protein called Gs, which interacts with numerous GPCRs, including the β2-adrenergic receptor shown in Figures 15–21 and 15–23. The α subunit contains the GTPase domain and binds to one side of the β subunit. The γ subunit binds to the opposite side of the β subunit, and the β and γ subunits together form a single functional unit. The GTPase domain of the α subunit contains two major subdomains: the “Ras” domain, which is related to other GTPases and provides one face of the nucleotide-binding pocket; and the alpha-helical or “AH” domain, which clamps the nucleotide in place. (B, based on D.G. Lombright et al., Nature 379:311– 319, 1996. With permission from Macmillan Publishers Ltd.)

1	D.G. Lombright et al., Nature 379:311– 319, 1996. With permission from Macmillan Publishers Ltd.) Figure 15–23 Activation of a G protein by an activated GPCR. Binding of an extracellular signal molecule to a GPCR changes the conformation of the receptor, which allows the receptor to bind and alter the conformation of a trimeric G protein. The AH domain of the G protein α subunit moves outward to open the nucleotide-binding site, thereby promoting dissociation of GDP. GTP binding then promotes closure of the nucleotide-binding site, triggering conformational changes that cause dissociation of the α subunit from the receptor and from the βγ complex. The GTP-bound α subunit and the βγ complex each regulate the activities of downstream signaling molecules (not shown). The receptor stays active while the extracellular signal molecule is bound to it, and it can therefore catalyze the activation of many G-protein molecules (Movie 15.1).

1	adenylyl cyclase, and it is rapidly and continuously destroyed by cyclic AMP phosphodiesterases (Figure 15–25). Adenylyl cyclase is a large, multipass trans-membrane protein with its catalytic domain on the cytosolic side of the plasma membrane. There are at least eight isoforms in mammals, most of which are regulated by both G proteins and Ca2+. Many extracellular signals work by increasing cAMP concentrations inside the cell. These signals activate GPCRs that are coupled to a stimulatory G protein (Gs). The activated α subunit of Gs binds and thereby activates adenylyl cyclase. Other extracellular signals, acting through different GPCRs, reduce cAMP levels by activating an inhibitory G protein (Gi), which then inhibits adenylyl cyclase.

1	Both Gs and Gi are targets for medically important bacterial toxins. Cholera toxin, which is produced by the bacterium that causes cholera, is an enzyme that catalyzes the transfer of ADP ribose from intracellular NAD+ to the α subunit of Gs. This ADP ribosylation alters the α subunit so that it can no longer hydrolyze its bound GTP, causing it to remain in an active state that stimulates adenylyl cyclase indefinitely. The resulting prolonged elevation in cAMP concentration within intestinal epithelial cells causes a large efflux of Cl– and water into the gut, thereby causing the severe diarrhea that characterizes cholera. Pertussis toxin, which is made by the bacterium that causes pertussis (whooping cough), catalyzes the ADP ribosylation of the α subunit of Gi, preventing the protein from interacting with receptors; as a result, the G protein remains in the inactive GDP-bound state and is unable to regulate its target proteins. These two toxins are widely used in experiments to

1	from interacting with receptors; as a result, the G protein remains in the inactive GDP-bound state and is unable to regulate its target proteins. These two toxins are widely used in experiments to determine whether a cell’s GPCR-dependent response to a signal is mediated by Gs or by Gi.

1	Some of the responses mediated by a Gs-stimulated increase in cAMP concentration are listed in Table 15–1. As the table shows, different cell types respond differently to an increase in cAMP concentration. Some cell types, such as fat cells, activate adenylyl cyclase in response to multiple hormones, all of which thereby stimulate the breakdown of triglyceride (the storage form of fat) to fatty acids. Individuals with genetic defects in the Gs α subunit show decreased responses to certain hormones, resulting in metabolic abnormalities, abnormal bone development, and mental retardation. Cyclic-AMP-Dependent Protein Kinase (PKA) Mediates Most of the Effects of Cyclic AMP In most animal cells, cAMP exerts its effects mainly by activating cyclic-AMPdependent protein kinase (PKA). This kinase phosphorylates specific serines or

1	In most animal cells, cAMP exerts its effects mainly by activating cyclic-AMPdependent protein kinase (PKA). This kinase phosphorylates specific serines or Figure 15–25 The synthesis and degradation of cyclic AMP. In a reaction catalyzed by the enzyme adenylyl cyclase, cyclic AMP (cAMP) is synthesized from ATP through a cyclization reaction that removes two phosphate groups as pyrophosphate (PPi); a pyrophosphatase drives this synthesis by hydrolyzing the released pyrophosphate to phosphate (not shown). Cyclic AMP is short-lived (unstable) in the cell because it is hydrolyzed by specific phosphodiesterases to form 5ʹ-AMP, as indicated. Figure 15–24 An increase in cyclic AMP in response to an extracellular signal.

1	Figure 15–24 An increase in cyclic AMP in response to an extracellular signal. This nerve cell in culture is responding to the neurotransmitter serotonin, which acts through a GPCR to cause a rapid rise in the intracellular concentration of cyclic AMP. To monitor the cyclic AMP level, the cell has been loaded with a fluorescent protein that changes its fluorescence when it binds cyclic AMP. Blue indicates a low level of cyclic AMP, yellow an intermediate level, and red a high level. (A) In the resting cell, the cyclic AMP level is about 5 × 10–8

1	M. (B) Twenty seconds after the addition of serotonin to the culture medium, the intracellular level of cyclic AMP has increased to more than 10–6 M in the relevant parts of the cell, an increase of more than twentyfold. (From B.J. Bacskai et al., Science 260:222–226, 1993. With permission from AAAS.) threonines on selected target proteins, including intracellular signaling proteins and effector proteins, thereby regulating their activity. The target proteins differ from one cell type to another, which explains why the effects of cAMP vary so markedly depending on the cell type (see Table 15–1).

1	In the inactive state, PKA consists of a complex of two catalytic subunits and two regulatory subunits. The binding of cAMP to the regulatory subunits alters their conformation, causing them to dissociate from the complex. The released catalytic subunits are thereby activated to phosphorylate specific target proteins (Figure 15–26). The regulatory subunits of PKA (also called A-kinase) are important for localizing the kinase inside the cell: special A-kinase anchoring proteins (AKAPs) bind both to the regulatory subunits and to a component of the cytoskeleton or a membrane of an organelle, thereby tethering the enzyme complex to a particular subcellular compartment. Some AKAPs also bind other signaling proteins, forming a signaling complex. An AKAP located around the nucleus of heart muscle cells, for example, binds both PKA and a phosphodiesterase that hydrolyzes cAMP. In unstimulated cells, the phosphodiesterase keeps the local cAMP concentration low, so that the bound PKA is

1	muscle cells, for example, binds both PKA and a phosphodiesterase that hydrolyzes cAMP. In unstimulated cells, the phosphodiesterase keeps the local cAMP concentration low, so that the bound PKA is inactive; in stimulated cells, cAMP concentration rapidly rises, overwhelming the phosphodiesterase and activating the PKA. Among the target proteins that PKA phosphorylates and activates in these cells is the adjacent phosphodiesterase, which rapidly lowers the cAMP concentration again. This negative feedback arrangement converts what might otherwise be a prolonged PKA response into a brief, local pulse of PKA activity.

1	Whereas some responses mediated by cAMP occur within seconds (see Figure 15–24), others depend on changes in the transcription of specific genes and take hours to develop fully. In cells that secrete the peptide hormone somatostatin, regulatory inactive complex of Figure 15–26 The activation of cyclicAMP-dependent protein kinase (PKA).

1	The binding of cAMP to the regulatory subunits of the PKA tetramer induces a conformational change, causing these subunits to dissociate from the catalytic subunits, thereby activating the kinase activity of the catalytic subunits. The release of the catalytic subunits requires the binding of more than two cAMP molecules to the regulatory subunits in the tetramer. This requirement greatly sharpens the response of the kinase to changes in cAMP concentration, as discussed earlier (see Figure 15–16). Mammalian cells have at least two types of PKAs: type I is mainly in the cytosol, whereas type II is bound via its regulatory subunits and special anchoring proteins to the plasma membrane, nuclear membrane, mitochondrial outer membrane, and microtubules. In both types, once the catalytic subunits are freed and active, they can migrate into the nucleus (where they can phosphorylate transcription regulatory proteins), while the regulatory subunits remain in the cytoplasm.

1	signal molecule activated ˜ subunit of stimulatory G plasma protein (G ) membranes activated, phosphorylated CREB Figure 15–27 How a rise in intracellular cyclic AMP concentration can alter gene transcription. The binding of an extracellular signal molecule to its GPCR activates adenylyl cyclase via Gs and thereby increases cAMP concentration in the cytosol. This rise activates PKA, and the released catalytic subunits of PKA can then enter the nucleus, where they phosphorylate the transcription regulatory protein CREB. Once phosphorylated, CREB recruits the coactivator CBP, which stimulates gene transcription. In some cases, at least, the inactive CREB protein is bound to the cyclic AMP response element (CRE) in DNA before it is phosphorylated (not shown). See Movie 15.2.

1	for example, cAMP activates the gene that encodes this hormone. The regulatory region of the somatostatin gene contains a short cis-regulatory sequence, called the cyclic AMP response element (CRE), which is also found in the regulatory region of many other genes activated by cAMP. A specific transcription regulator called CRE-binding (CREB) protein recognizes this sequence. When PKA is activated by cAMP, it phosphorylates CREB on a single serine; phosphorylated CREB then recruits a transcriptional coactivator called CREB-binding protein (CBP), which stimulates the transcription of the target genes (Figure 15–27). Thus, CREB can transform a short cAMP signal into a long-term change in a cell, a process that, in the brain, is thought to play an important part in some forms of learning and memory.

1	Many GPCRs exert their effects through G proteins that activate the plasma-membrane-bound enzyme phospholipase C-β (PLCβ). Table 15–2 lists some examples of responses activated in this way. The phospholipase acts on a phosphorylated inositol phospholipid (a phosphoinositide) called phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], which is present in small amounts in the inner half of the plasma membrane lipid bilayer (Figure 15–28). Receptors that activate this inositol phospholipid signaling pathway mainly do so via a G protein called Gq, which activates phospholipase C-β in much the same way that Gs activates adenylyl cyclase. The activated phospholipase then cleaves the PI(4,5)P2 to generate two products: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. At this step, the signaling pathway splits into two branches.

1	IP3 is a water-soluble molecule that leaves the plasma membrane and diffuses rapidly through the cytosol. When it reaches the endoplasmic reticulum (ER), it binds to and opens IP3-gated Ca2+-release channels (also called IP3 receptors) in the ER membrane. Ca2+ stored in the ER is released through the open channels, quickly raising the concentration of Ca2+ in the cytosol (Figure 15–29). The increase in cytosolic Ca2+ propagates the signal by influencing the activity of Ca2+sensitive intracellular proteins, as we describe shortly.

1	At the same time that the IP3 produced by the hydrolysis of PI(4,5)P2 is increasing the concentration of Ca2+ in the cytosol, the other cleavage product of the PI(4,5)P2, diacylglycerol, is exerting different effects. It also acts as a second messenger, but it remains embedded in the plasma membrane, where it has several potential signaling roles. One of its major functions is to activate a protein kinase called protein kinase C (PKC), so named because it is Ca2+-dependent. The initial rise in cytosolic Ca2+ induced by IP3 alters the PKC so that it translocates from the cytosol to the cytoplasmic face of the plasma membrane. There it is activated by the combination of Ca2+, diacylglycerol, and the negatively charged membrane phospholipid phosphatidylserine (see Figure 15–29). Once activated, PKC phosphorylates target proteins that vary depending on the cell type. The principles are the same as discussed earlier for PKA, although most of the target proteins are different.

1	Diacylglycerol can be further cleaved to release arachidonic acid, which can either act as a signal in its own right or be used in the synthesis of other small lipid signal molecules called eicosanoids. Most vertebrate cell types make eicosanoids, including prostaglandins, which have many biological activities. They participate RETICULUMPI 4,5-bisphosphate [PI(4,5)P2] inositol 1,4,5-trisphosphate (IP3) Figure 15–28 The hydrolysis of PI(4,5) P2 by phospholipase C-β. Two second messengers are produced directly from the hydrolysis of PI(4,5)P2: inositol 1,4,5-trisphosphate (IP3), which diffuses through the cytosol and releases Ca2+ from the endoplasmic reticulum, and diacylglycerol, which remains in the membrane and helps to activate protein kinase C (PKC; see Figure 15–29). There are several classes of phospholipase

1	C: these include the β class, which is activated by GPCRs; as we see later, the γ class is activated by a class of enzyme-coupled receptors called receptor tyrosine kinases (RTKs). signal molecule PI 4,5-bisphosphate activated GPCR activated Gqprotein activated phospholipase C-˜diacylglycerol plasma membrane [PI(4,5)P2] activated protein kinase C Ca inositol 1,4,5-trisphosphate (IP3) lumen of endoplasmic reticulum open IP3-gated Ca2+-release channel 2+ GTPP P P P P P ˜°˛ in pain and inflammatory responses, for example, and many anti-inflammatory drugs (such as aspirin, ibuprofen, and cortisone) act in part by inhibiting their synthesis. Ca2+ Functions as a Ubiquitous Intracellular Mediator

1	Ca2+ Functions as a Ubiquitous Intracellular Mediator Many extracellular signals, and not just those that work via G proteins, trigger an increase in cytosolic Ca2+ concentration. In muscle cells, Ca2+ triggers contraction, and in many secretory cells, including nerve cells, it triggers secretion. Ca2+ has numerous other functions in a variety of cell types. Ca2+ is such an effective signaling mediator because its concentration in the cytosol is normally very low (~10–7 M), whereas its concentration in the extracellular fluid (~10–3 M) and in the lumen of the ER [and sarcoplasmic reticulum (SR) in muscle] is high. Thus, there is a large gradient tending to drive Ca2+ into the cytosol across both the plasma membrane and the ER or SR membrane. When a signal transiently opens Ca2+ channels in these membranes, Ca2+ rushes into the cytosol, and the resulting 10–20-fold increase in the local Ca2+ concentration activates Ca2+-responsive proteins in the cell.

1	Some stimuli, including membrane depolarization, membrane stretch, and certain extracellular signals, activate Ca2+ channels in the plasma membrane, resulting in Ca2+ influx from outside the cell. Other signals, including the GPCR-mediated signals described earlier, act primarily through IP3 receptors to stimulate Ca2+ release from intracellular stores in the ER (see Figure 15–29). The ER membrane also contains a second type of regulated Ca2+ channel called the ryanodine receptor (so called because it is sensitive to the plant alkaloid ryanodine), which opens in response to rising Ca2+ levels and thereby amplifies the Ca2+ signal, as we describe shortly.

1	Several mechanisms rapidly terminate the Ca2+ signal and are also responsible for keeping the concentration of Ca2+ in the cytosol low in resting cells. Most importantly, there are Ca2+-pumps in the plasma membrane and the ER membrane that use the energy of ATP hydrolysis to pump Ca2+ out of the cytosol. Cells such as muscle and nerve cells, which make extensive use of Ca2+ signaling, have an additional Ca2+ transporter (a Na+-driven Ca2+ exchanger) in their plasma membrane that couples the efflux of Ca2+ to the influx of Na+. The IP3 receptors and ryanodine receptors of the ER membrane have an important feature: they are both stimulated by low to moderate cytoplasmic Ca2+ concentrations. This Ca2+-induced calcium release (CICR) results in positive feedback,

1	Figure 15–29 How GPCRs increase cytosolic Ca2+ and activate protein kinase C. The activated GPCR stimulates the plasma-membrane-bound phospholipase C-β (PLCβ) via a G protein called Gq. The α subunit and βγ complex of Gq are both involved in this activation. Two second messengers are produced when PI(4,5)P2 is hydrolyzed by activated PLCβ. Inositol 1,4,5-trisphosphate (IP3) diffuses through the cytosol and releases Ca2+ from the ER by binding to and opening IP3-gated Ca2+-release channels (IP3 receptors) in the ER membrane. The large electrochemical gradient for Ca2+ across this membrane causes Ca2+ to escape into the cytosol when the release channels are opened. Diacylglycerol remains in the plasma membrane and, together with phosphatidylserine (not shown) and Ca2+, helps to activate protein kinase C (PKC), which is recruited from the cytosol to the cytosolic face of the plasma membrane. Of the 10 or more distinct isoforms of PKC in humans, at least 4 are activated by diacylglycerol

1	kinase C (PKC), which is recruited from the cytosol to the cytosolic face of the plasma membrane. Of the 10 or more distinct isoforms of PKC in humans, at least 4 are activated by diacylglycerol (Movie 15.3).

1	which has a major impact on the properties of the Ca2+ signal. The importance of this feedback is seen clearly in studies with Ca2+-sensitive fluorescent indicators, such as aequorin or fura-2 (discussed in Chapter 9), which allow researchers to monitor cytosolic Ca2+ in individual cells under a microscope (Figure 15–30 and Movie 15.4).

1	When cells carrying a Ca2+ indicator are treated with a small amount of an extracellular signal molecule that stimulates IP3 production, tiny bursts of Ca2+ are seen in one or more discrete regions of the cell. These Ca2+ puffs or sparks reflect the local opening of small groups of IP3-gated Ca2+-release channels in the ER. Because various Ca2+-binding proteins act as Ca2+ buffers and restrict the diffusion of Ca2+, the signal often remains localized to the site where the Ca2+ enters the cytosol. If the extracellular signal is sufficiently strong and persistent, however, the local Ca2+ concentration can reach a sufficient level to activate nearby IP3 receptors and ryanodine receptors, resulting in a regenerative wave of Ca2+ release that moves through the cytosol (Figure 15–31), much like an action potential in an axon.

1	Figure 15–30 The fertilization of an egg by a sperm triggers a wave of cytosolic Ca2+. This starfish egg was injected with a Ca2+-sensitive fluorescent dye before it was fertilized. A wave of cytosolic Ca2+ (red), released from the ER, sweeps across the egg from the site of sperm entry (arrow). This Ca2+ wave changes the egg cell surface, preventing the entry of other sperm, and it also initiates embryonic development (Movie 15.5). The initial increase in Ca2+ is thought to be caused by a sperm-specific form of PLC (PLCζ) that the sperm brings into the egg cytoplasm when it fuses with the egg; the PLCζ cleaves PI(4,5)P2 to produce IP3, which releases Ca2+ from the egg ER. The released Ca2+ stimulates further Ca2+ release from the ER, producing the spreading wave, as we explain in Figure 15–31. (Courtesy of Stephen A. Stricker.)

1	Figure 15–31 Positive and negative feedback produce Ca2+ waves and oscillations. This diagram shows IP3 receptors and ryanodine receptors on a portion of the ER membrane: active receptors are in green; inactive receptors are in red. When a small amount of cytosolic IP3 activates a cluster of IP3 receptors at one site on the ER membrane (top), the local release of Ca2+ promotes the opening of nearby IP3 and ryanodine receptors, resulting in more Ca2+ release. This positive feedback (indicated by positive signs) produces a regenerative wave of Ca2+ release that spreads across the cell (see Figure 15–30). These waves of Ca2+ release move more quickly across the cell than would be possible by simple diffusion. Also, unlike a diffusing burst of Ca2+ ions, which will become more dilute as it spreads, the regenerative wave produces a high Ca2+ concentration across the entire cell. Eventually, the local Ca2+ concentration inactivates IP3 receptors and ryanodine receptors (middle; indicated by

1	the regenerative wave produces a high Ca2+ concentration across the entire cell. Eventually, the local Ca2+ concentration inactivates IP3 receptors and ryanodine receptors (middle; indicated by red negative signs), shutting down the Ca2+ release. Ca2+-pumps reduce the local cytosolic Ca2+ concentration to its normal low levels. The result is a Ca2+ spike: positive feedback drives a rapid rise in cytosolic Ca2+, and negative feedback sends it back down again. The Ca2+ channels remain refractory to further stimulation for some period of time, delaying the generation of another Ca2+ spike (bottom). Eventually, however, the negative feedback wears off, allowing IP3 to trigger another Ca2+ wave. The end result is repeated Ca2+ oscillations (see Figure 15–32). Under some conditions, these oscillations can be seen as repeating narrow waves of Ca2+ moving across the cell.

1	Another important property of IP3 receptors and ryanodine receptors is that they are inhibited, after some delay, by high Ca2+ concentrations (a form of negative feedback). Thus, the rise in Ca2+ in a stimulated cell leads to inhibition of Ca2+ release; because Ca2+ pumps remove the cytosolic Ca2+, the Ca2+ concentration falls (see Figure 15–31). The decline in Ca2+ eventually relieves the negative feedback, allowing cytosolic Ca2+ to rise again. As in other cases of delayed negative feedback (see Figure 15–18), the result is an oscillation in the Ca2+ concentration. These oscillations persist for as long as receptors are activated at the cell surface, and their frequency reflects the strength of the extracellular stimulus (Figure 15–32). The frequency, amplitude, and breadth of oscillations can also be modulated by other signaling mechanisms, such as phosphorylation, which influence the Ca2+ sensitivity of Ca2+ channels or affect other components in the signaling system.

1	The frequency of Ca2+ oscillations can be translated into a frequency-dependent cell response. In some cases, the frequency-dependent response itself is also oscillatory: in hormone-secreting pituitary cells, for example, stimulation by an extracellular signal induces repeated Ca2+ spikes, each of which is associated with a burst of hormone secretion. In other cases, the frequency-dependent response is non-oscillatory: in some types of cells, for instance, one frequency of Ca2+ spikes activates the transcription of one set of genes, while a higher frequency activates the transcription of a different set. How do cells sense the frequency of Ca2+ spikes and change their response accordingly? The mechanism presumably depends on Ca2+-sensitive proteins that change their activity as a function of Ca2+-spike frequency. A protein kinase that acts as a molecular memory device seems to have this remarkable property, as we discuss next.

1	Ca2+/Calmodulin-Dependent Protein Kinases Mediate Many Responses to Ca2+ Signals Various Ca2+-binding proteins help to relay the cytosolic Ca2+ signal. The most important is calmodulin, which is found in all eukaryotic cells and can constitute as much as 1% of a cell's total protein mass. Calmodulin functions as a multipurpose intracellular Ca2+ receptor, governing many Ca2+-regulated processes. It consists of a highly conserved, single polypeptide chain with four high-affinity Ca2+-binding sites (Figure 15–33A). When activated by Ca2+ binding, it undergoes a conformational change. Because two or more Ca2+ ions must bind before 0.4 nM 0.6 nM 0.9 nM

1	Figure 15–32 Vasopressin-induced Ca2+ oscillations in a liver cell. The cell was loaded with the Ca2+-sensitive protein aequorin and then exposed to increasing concentrations of the peptide signal molecule vasopressin, which activates a GPCR and thereby PLCβ (see Table 15–2). Note that the frequency of the Ca2+ spikes increases with an increasing concentration of vasopressin but that the amplitude of the spikes is not affected. Each spike lasts about 7 seconds. (Adapted from N.M. Woods, K.S.R. Cuthbertson and P.H. Cobbold, Nature 319:600–602, 1986. With permission from Macmillan Publishers Ltd.) peptide portion of target protein calmodulin adopts its active conformation, the protein displays a sigmoidal response to increasing concentrations of Ca2+ (see Figure 15–16).

1	The allosteric activation of calmodulin by Ca2+ is analogous to the activation of PKA by cyclic AMP, except that Ca2+/calmodulin has no enzymatic activity itself but instead acts by binding to and activating other proteins. In some cases, calmodulin serves as a permanent regulatory subunit of an enzyme complex, but usually the binding of Ca2+ instead enables calmodulin to bind to various target proteins in the cell to alter their activity.

1	When an activated molecule of Ca2+/calmodulin binds to its target protein, the calmodulin further changes its conformation, the nature of which depends on the specific target protein (Figure 15–33B). Among the many targets calmodulin regulates are enzymes and membrane transport proteins. As one example, Ca2+/ calmodulin binds to and activates the plasma membrane Ca2+-pump that uses ATP hydrolysis to pump Ca2+ out of cells. Thus, whenever the concentration of Ca2+ in the cytosol rises, the pump is activated, which helps to return the cytosolic Ca2+ level to resting levels. Many effects of Ca2+, however, are more indirect and are mediated by protein phosphorylations catalyzed by a family of protein kinases called Ca2+/calmodulin-dependent kinases (CaM-kinases). Some CaM-kinases phosphorylate transcription regulators, such as the CREB protein (see Figure 15–27), and in this way activate or inhibit the transcription of specific genes.

1	One of the best-studied CaM-kinases is CaM-kinase II, which is found in most animal cells but is especially enriched in the nervous system. It constitutes up to 2% of the total protein mass in some regions of the brain, and it is highly concentrated in synapses. CaM-kinase II has several remarkable properties. To begin with, it has a spectacular quaternary structure: twelve copies of the enzyme are assembled into a stacked pair of rings, with kinase domains on the outside linked to a central hub (Figure 15–34). This structure helps the enzyme function as a molecular memory device, switching to an active state when exposed to Ca2+/ calmodulin and then remaining active even after the Ca2+ signal has decayed. This is because adjacent kinase subunits can phosphorylate each other (a process called autophosphorylation) when Ca2+/calmodulin activates them (Figure 15–34). Once a kinase subunit is autophosphorylated, it remains active even in the absence of Ca2+, thereby prolonging the

1	called autophosphorylation) when Ca2+/calmodulin activates them (Figure 15–34). Once a kinase subunit is autophosphorylated, it remains active even in the absence of Ca2+, thereby prolonging the duration of the kinase activity beyond that of the initial activating Ca2+ signal. The enzyme maintains this activity until a protein phosphatase removes the autophosphorylation and shuts the kinase off. CaM-kinase II activation can thereby serve as a memory trace of a prior Ca2+ pulse, and it seems to have a role in some types of memory and learning in the vertebrate nervous system. Mutant mice that lack a brain-specific form of the enzyme have specific defects in their ability to remember where things are.

1	Figure 15–33 The structure of Ca2+/calmodulin. (A) The molecule has a dumbbell shape, with two globular ends, which can bind to many target proteins. The globular ends are connected by a long, exposed α helix, which allows the protein to adopt a number of different conformations, depending on the target protein it interacts with. Each globular head has two Ca2+binding sites (Movie 15.6). (B) Shown is the major structural change that occurs in Ca2+/calmodulin when it binds to a target protein (in this example, a peptide that consists of the Ca2+/calmodulin-binding domain of a Ca2+/calmodulin-dependent protein kinase). Note that the Ca2+/calmodulin has “jack-knifed” to surround the peptide. When it binds to other targets, it can adopt different conformations. (A, based on x-ray crystallographic data from Y.S. Babu et al., Nature 315:37–40, 1985. With permission from Macmillan Publishers Ltd; B, based on x-ray crystallographic data from W.E. Meador, A.R. Means, and

1	W.E. Meador, A.R. Means, and F.A. Quiocho, Science 257:1251–1255, 1992, and on nuclear magnetic resonance (NMR) spectroscopy data from M. Ikura et al., Science 256:632–638, 1992.)

1	Figure 15–34 The stepwise activation of CaM-kinase II. (A) Each CaM-kinase II protein has two major domains: an amino-terminal kinase domain (green) and a carboxyl-terminal hub domain (blue), linked by a regulatory segment. Six CaM-kinase II proteins are assembled into a giant ring in which the hub domains interact tightly to produce a central structure that is surrounded by kinase domains. The complete enzyme contains two stacked rings, for a total of 12 kinase proteins, but only one ring is shown here for clarity. When the enzyme is inactive, the ring exists in a dynamic equilibrium between two states. The first (upper left) is a compact state, in which the kinase domains interact with the hub, so that the regulatory segment is buried in the kinase active site and thereby blocks catalytic activity. In the second inactive state (upper middle), a kinase domain has popped out and is linked to the central hub by its regulatory segment, which continues to inhibit the kinase but is now

1	activity. In the second inactive state (upper middle), a kinase domain has popped out and is linked to the central hub by its regulatory segment, which continues to inhibit the kinase but is now accessible to Ca2+/calmodulin. If present, Ca2+/calmodulin will bind the regulatory segment and prevent it from inhibiting the kinase, thereby locking the kinase in an active state (upper right). If the adjacent kinase subunit also pops out from the hub, it will also be activated by Ca2+/calmodulin, and the two kinases will then phosphorylate each other on their regulatory segments (lower right). This autophosphorylation further activates the enzyme. It also prolongs the activity of the enzyme in two ways. First, it traps the bound Ca2+/calmodulin so that it does not dissociate from the enzyme until cytosolic Ca2+ levels return to basal values for at least 10 seconds (not shown). Second, it converts the enzyme to a Ca2+-independent form, so that the kinase remains active even after the

1	until cytosolic Ca2+ levels return to basal values for at least 10 seconds (not shown). Second, it converts the enzyme to a Ca2+-independent form, so that the kinase remains active even after the Ca2+/calmodulin dissociates from it (lower left). This activity continues until the action of a protein phosphatase overrides the autophosphorylation activity of CaM-kinase II. (B) This structural model of the enzyme is based on x-ray crystallographic analysis.

1	The remarkable dodecameric structure of the enzyme allows it to achieve a broad range of intermediate activity states in response to different Ca2+ oscillation frequencies: higher frequencies tend to cause more subunits in the enzyme to reach the phosphorylated active state (see Figure 15–35). The behavior of CaM-kinase II is also controlled by the length of the linker segment between the kinase and hub domains. The linker is longer in some isoforms of the enzyme; in these isoforms, the kinase domains tend to pop out of the ring more frequently, making it more sensitive to Ca2+. These and other mechanisms allow the cell to tailor the responsiveness of the enzyme to the needs of different types of neurons. (Adapted from L.H. Chao et al., Cell 146:732–745, 2011. PDB code: 3SOA.)

1	Another remarkable property of CaM-kinase II is that the enzyme can use its intrinsic memory mechanism to decode the frequency of Ca2+ oscillations. This property is thought to be especially important at a nerve cell synapse, where changes in intracellular Ca2+ levels in a postsynaptic cell as a result of neural activity can lead to long-term changes in the subsequent effectiveness of that synapse (discussed in Chapter 11). When CaM-kinase II is exposed to both a protein phosphatase and repetitive pulses of Ca2+/calmodulin at different frequencies that mimic those observed in stimulated cells, the enzyme’s activity increases steeply as a function of pulse frequency (Figure 15–35).

1	G proteins do not act exclusively by regulating the activity of membrane-bound enzymes that alter the concentration of cyclic AMP or Ca2+ in the cytosol. The α subunit of one type of G protein (called G12), for example, activates a guanine nucleotide exchange factor (GEF) that activates a monomeric GTPase of the Rho family (discussed later and in Chapter 16), which regulates the actin cytoskeleton.

1	In some other cases, G proteins directly activate or inactivate ion channels in the plasma membrane of the target cell, thereby altering the ion permeability— and hence the electrical excitability—of the membrane. As an example, acetylcholine released by the vagus nerve reduces the heart rate (see Figure 15–5B). This effect is mediated by a special class of acetylcholine receptors that activate the Gi protein discussed earlier. Once activated, the αsubunit of Gi inhibits adenylyl cyclase (as described previously), while the βγ subunits bind to K+ channels in the heart muscle cell plasma membrane and open them. The opening of these K+ channels makes it harder to depolarize the cell and thereby contributes to the inhibitory effect of acetylcholine on the heart. (These acetylcholine receptors, which can be activated by the fungal alkaloid muscarine, are called muscarinic acetylcholine receptors to distinguish them from the very different nicotinic acetylcholine receptors, which are

1	which can be activated by the fungal alkaloid muscarine, are called muscarinic acetylcholine receptors to distinguish them from the very different nicotinic acetylcholine receptors, which are ion-channel-coupled receptors on skeletal muscle and nerve cells that can be activated by the binding of nicotine, as well as by acetylcholine.)

1	Other G proteins regulate the activity of ion channels less directly, either by stimulating channel phosphorylation (by PKA, PKC, or CaM-kinase, for example) or by causing the production or destruction of cyclic nucleotides that directly activate or inactivate ion channels. These cyclic-nucleotide-gated ion channels have a crucial role in both smell (olfaction) and vision, as we now discuss. Humans can distinguish more than 10,000 distinct smells, which they detect using specialized olfactory receptor neurons in the lining of the nose. These cells use specific GPCRs called olfactory receptors to recognize odors; the receptors are displayed on the surface of the modified cilia that extend from each cell (Figure 15–36). The receptors act through cAMP. When stimulated by odorant binding, Figure 15–35 CaM-kinase II as a frequency decoder of Ca2+ oscillations.

1	(A) At low frequencies of Ca2+ spikes, the enzyme becomes inactive after each spike, as the autophosphorylation induced by Ca2+/calmodulin binding does not maintain the enzyme’s activity long enough for the enzyme to remain active until the next Ca2+ spike arrives. (B) At higher spike frequencies, however, the enzyme fails to inactivate completely between Ca2+ spikes, so its activity ratchets up with each spike. If the spike frequency is high enough, this progressive increase in enzyme activity will continue until the enzyme is autophosphorylated on all subunits and is therefore maximally activated. Although not shown, once enough of its subunits are autophosphorylated, the enzyme can be maintained in a highly active state even with a relatively low frequency of Ca2+ spikes (a form of cell memory). The binding of Ca2+/ calmodulin to the enzyme is enhanced by the CaM-kinase II autophosphorylation (an additional form of positive feedback), helping to generate a more switchlike response

1	memory). The binding of Ca2+/ calmodulin to the enzyme is enhanced by the CaM-kinase II autophosphorylation (an additional form of positive feedback), helping to generate a more switchlike response to repeated Ca2+ spikes. (From

1	P.I. Hanson, T. Meyer, L. Stryer, and H. Schulman, Neuron 12:943–956, 1994. With permission from Elsevier.) they activate an olfactory-specific G protein (known as Golf), which in turn activates adenylyl cyclase. The resulting increase in cAMP opens cyclic-AMP-gated cation channels, thereby allowing an influx of Na+, which depolarizes the olfactory receptor neuron and initiates a nerve impulse that travels along its axon to the brain.

1	There are about 1000 different olfactory receptors in a mouse and about 350 in a human, each encoded by a different gene and each recognizing a different set of odorants. Each olfactory receptor neuron produces only one of these receptors; the neuron responds to a specific set of odorants by means of the specific receptor it displays, and each odorant activates its own characteristic set of olfactory receptor neurons. The same receptor also helps direct the elongating axon of each developing olfactory neuron to the specific target neurons that it will connect to in the brain. A different set of GPCRs acts in a similar way in some vertebrates to mediate responses to pheromones, chemical signals detected in a different part of the nose that are used in communication between members of the same species. Humans, however, are thought to lack functional pheromone receptors.

1	Vertebrate vision employs a similarly elaborate, highly sensitive, signal-detection process. Cyclic-nucleotide-gated ion channels are also involved, but the crucial cyclic nucleotide is cyclic GMP (Figure 15–37) rather than cAMP. As with cAMP, a continuous rapid synthesis (by guanylyl cyclase) and rapid degradation (by cyclic GMP phosphodiesterase) controls the concentration of cyclic GMP in the cytosol.

1	In visual transduction responses, which are the fastest G-protein-mediated responses known in vertebrates, the receptor activation stimulated by light causes a fall rather than a rise in the level of the cyclic nucleotide. The pathway has been especially well studied in rod photoreceptors (rods) in the vertebrate retina. Rods are responsible for noncolor vision in dim light, whereas cone photoreceptors (cones) are responsible for color vision in bright light. A rod photoreceptor is a highly specialized cell with outer and inner segments, a cell body, and a synaptic region where the rod passes a chemical signal to a retinal nerve cell (Figure 15–38). This nerve cell relays the signal to another nerve cell in the retina, which in turn relays it to the brain.

1	The phototransduction apparatus is in the outer segment of the rod, which contains a stack of discs, each formed by a closed sac of membrane that is densely packed with photosensitive rhodopsin molecules. The plasma membrane surrounding the outer segment contains cyclic-GMP-gated cation channels. Cyclic GMP bound to these channels keeps them open in the dark. Paradoxically, light causes a hyperpolarization (which inhibits synaptic signaling) rather than a depolarization of the plasma membrane (which would stimulate synaptic signaling). Hyperpolarization (that is, the membrane potential moves to a more negative value—discussed in Chapter 11) results because the light-induced activation of rhodopsin molecules in the disc membrane decreases the cyclic GMP concentration and closes the cation channels in the surrounding plasma membrane (Figure 15–39).

1	Figure 15–36 Olfactory receptor neurons. (A) A section of olfactory epithelium in the nose. Olfactory receptor neurons possess modified cilia, which project from the surface of the epithelium and contain the olfactory receptors, as well as the signal transduction machinery. The axon, which extends from the opposite end of the receptor neuron, conveys electrical signals to the brain when an odorant activates the cell to produce an action potential. In rodents, at least, the basal cells act as stem cells, producing new receptor neurons throughout life, to replace the neurons that die. (B) A scanning electron micrograph of the cilia on the surface of an olfactory neuron. (B, from E.E. Morrison and R.M. Costanzo, J. Comp. Neurol. 297:1–13, 1990. With permission from Wiley-Liss.) H2NNNNHNCH2OOOHOO_OPGUANINEPHOSPHATESUGAROFigure 15–37 Cyclic GMP.

1	E.E. Morrison and R.M. Costanzo, J. Comp. Neurol. 297:1–13, 1990. With permission from Wiley-Liss.) H2NNNNHNCH2OOOHOO_OPGUANINEPHOSPHATESUGAROFigure 15–37 Cyclic GMP. Figure 15–38 A rod photoreceptor cell. There are about 1000 discs in the outer segment. The disc membranes are not connected to the plasma membrane. The inner and outer segments are specialized parts of a primary cilium (discussed in Chapter 16). A primary cilium extends from the surface of most vertebrate cells, where it serves as a signaling organelle.

1	Rhodopsin is a member of the GPCR family, but the activating extracellular signal is not a molecule but a photon of light. Each rhodopsin molecule contains a covalently attached chromophore, 11-cis retinal, which isomerizes almost instantaneously to all-trans retinal when it absorbs a single photon. The isomerization alters the shape of the retinal, forcing a conformational change in the protein (opsin). The activated rhodopsin molecule then alters the conformation of the G protein transducin (Gt), causing the transducin α subunit to activate cyclic GMP phosphodiesterase. The phosphodiesterase then hydrolyzes cyclic GMP, so that cyclic GMP levels in the cytosol fall. This drop in cyclic GMP concentration decreases the amount of cyclic GMP bound to the plasma membrane cation channels, allowing more of these cyclic-GMP-sensitive channels to close. In this way, the signal quickly passes from the disc membrane to the plasma membrane, and a light signal is converted into an electrical one,

1	more of these cyclic-GMP-sensitive channels to close. In this way, the signal quickly passes from the disc membrane to the plasma membrane, and a light signal is converted into an electrical one, through a hyperpolarization of the rod cell plasma membrane.

1	Rods use several negative feedback loops to allow the cells to revert quickly to a resting, dark state in the aftermath of a flash of light—a requirement for perceiving the shortness of the flash. A rhodopsin-specific protein kinase called rhodopsin kinase (RK) phosphorylates the cytosolic tail of activated rhodopsin on multiple serines, partially inhibiting the ability of the rhodopsin to activate transducin. An inhibitory protein called arrestin (discussed later) then binds to the phosphorylated rhodopsin, further inhibiting rhodopsin’s activity. Mice or humans with a mutation that inactivates the gene encoding RK have a prolonged light response.

1	At the same time as arrestin shuts off rhodopsin, an RGS protein (discussed earlier) binds to activated transducin, stimulating the transducin to hydrolyze its bound GTP to GDP, which returns transducin to its inactive state. In addition, the cation channels that close in response to light are permeable to Ca2+, as well as high rate of low rate of (signals to discs of photoreceptive outer

1	Figure 15–39 The response of a rod photoreceptor cell to light. Rhodopsin molecules in the outer-segment discs absorb photons. Photon absorption closes cation channels in the plasma membrane, which hyperpolarizes the membrane and reduces the rate of neurotransmitter release from the synaptic region. Because the neurotransmitter inhibits many of the postsynaptic retinal neurons, illumination serves to free the neurons from inhibition and thus, in effect, excites them. The neural connections of the retina lie between the light source and the outer segment, and so the light must pass through the synapses and rod cell nucleus to reach the light sensors.

1	to Na+, so that when they close, the normal influx of Ca2+ is inhibited, causing the Ca2+ concentration in the cytosol to fall. The decrease in Ca2+ concentration stimulates guanylyl cyclase to replenish the cyclic GMP, rapidly returning its level to where it was before the light was switched on. A specific Ca2+-sensitive protein mediates the activation of guanylyl cyclase in response to the fall in Ca2+ levels. In contrast to calmodulin, this protein is inactive when Ca2+ is bound to it and active when it is Ca2+-free. It therefore stimulates the cyclase when Ca2+ levels fall following a light response.

1	Negative feedback mechanisms do more than just return the rod to its resting state after a transient light flash; they also help the rod to adapt, stepping down the response when the rod is exposed to light continuously. Adaptation, as we discussed earlier, allows the receptor cell to function as a sensitive detector of changes in stimulus intensity over an enormously wide range of baseline levels of stimulation. It is why we can see faint stars in a dark sky, or a camera flash in bright sunlight. The various trimeric G proteins we have discussed in this chapter fall into four major families, as summarized in Table 15–3.

1	The various trimeric G proteins we have discussed in this chapter fall into four major families, as summarized in Table 15–3. Signaling molecules like cyclic nucleotides and calcium are hydrophilic small molecules that generally act within the cell where they are produced. Some signaling molecules, however, are hydrophobic enough, small enough, or both, to pass readily across the plasma membrane and carry signals to nearby cells. An important and remarkable example is the gas nitric oxide (NO), which acts as a signal molecule in many tissues of both animals and plants.

1	In mammals, one of NO’s many functions is to relax smooth muscle in the walls of blood vessels. The neurotransmitter acetylcholine stimulates NO synthesis by activating a GPCR on the membranes of the endothelial cells that line the interior of the vessel. The activated receptor triggers IP3 synthesis and Ca2+ release (see Figure 15–29), leading to stimulation of an enzyme that synthesizes NO. Because dissolved NO passes readily across membranes, it diffuses out of the cell where it is produced and into neighboring smooth muscle cells, where it causes muscle relaxation and thereby vessel dilation (Figure 15–40). It acts only locally because it has a short half-life—about 5–10 seconds—in the extracellular space before oxygen and water convert it to nitrates and nitrites.

1	The effect of NO on blood vessels provides an explanation for the mechanism of action of nitroglycerine, which has been used for about 100 years to treat patients with angina (pain resulting from inadequate blood flow to the heart muscle). The nitroglycerine is converted to NO, which relaxes blood vessels. This reduces the workload on the heart and, as a consequence, reduces the oxygen requirement of the heart muscle. NO is made by the deamination of the amino acid arginine, catalyzed by enzymes called NO synthases (NOS) (see Figure 15–40). The NOS in endothelial cells is called eNOS, while that in nerve and muscle cells is called nNOS. Both eNOS and nNOS are stimulated by Ca2+. Macrophages, by contrast, make yet another NOS, called inducible NOS (iNOS), that is constitutively active but synthesized only when the cells are activated, usually in response to an infection.

1	In some target cells, including smooth muscle cells, NO binds reversibly to iron in the active site of guanylyl cyclase, stimulating synthesis of cyclic GMP. NO can increase cyclic GMP in the cytosol within seconds, because the normal rate of turnover of cyclic GMP is high: rapid degradation to GMP by a phosphodiesterase constantly balances the production of cyclic GMP by guanylyl cyclase. The drug Viagra® and its relatives inhibit the cyclic GMP phosphodiesterase in the penis, thereby increasing the amount of time that cyclic GMP levels remain elevated in the smooth muscle cells of penile blood vessels after NO production is induced by local nerve terminals. The cyclic GMP, in turn, keeps the blood vessels relaxed and thereby the penis erect. NO can also signal cells independently of cyclic GMP. It can, for example, alter the activity of an intracellular protein by covalently nitrosylating thiol (–SH) groups on specific cysteines in the protein.

1	Figure 15–40 The role of nitric oxide (NO) in smooth muscle relaxation in a blood vessel wall. (A) Simplified cross section of a blood vessel, showing the endothelial cells lining the lumen and the smooth muscle cells around them. (B) The neurotransmitter acetylcholine stimulates blood vessel dilation by activating a GPCR—the muscarinic acetylcholine receptor—on the surface of endothelial cells. This receptor activates a G protein, Gq, thereby stimulating IP3 synthesis and Ca2+ release by the mechanisms illustrated in Figure 15–29. Ca2+ activates nitric oxide synthase, causing the endothelial cells to produce NO from arginine. The NO diffuses out of the endothelial cells and into the neighboring smooth muscle cells, where it activates guanylyl cyclase to produce cyclic GMP. The cyclic GMP triggers a response that causes the smooth muscle cells to relax, increasing blood flow through the vessel.

1	Despite the differences in molecular details, the different intracellular signaling pathways that GPCRs trigger share certain features and obey similar general principles. They depend on relay chains of intracellular signaling proteins and second messengers. These relay chains provide numerous opportunities for amplifying the responses to extracellular signals. In the visual transduction cascade, for example, a single activated rhodopsin molecule catalyzes the activation of hundreds of molecules of transducin at a rate of about 1000 transducin molecules per second. Each activated transducin molecule activates a molecule of cyclic GMP phosphodiesterase, each of which hydrolyzes about 4000 molecules of cyclic GMP per second. This catalytic cascade lasts for about 1 second and results in the hydrolysis of more than 105 cyclic GMP molecules for a single quantum of light absorbed, and the resulting drop in the concentration of cyclic GMP in turn transiently closes hundreds of cation

1	in the hydrolysis of more than 105 cyclic GMP molecules for a single quantum of light absorbed, and the resulting drop in the concentration of cyclic GMP in turn transiently closes hundreds of cation channels in the plasma membrane (Figure 15–41). As a result, a rod cell can respond to even a single photon of light in a way that is highly reproducible in its timing and magnitude.

1	Likewise, when an extracellular signal molecule binds to a receptor that indirectly activates adenylyl cyclase via Gs, each receptor protein may activate many molecules of Gs protein, each of which can activate a cyclase molecule. Each cyclase molecule, in turn, can catalyze the conversion of a large number of ATP molecules to cAMP molecules. A similar amplification operates in the IP3 signaling pathway. In these ways, a nanomolar (10–9 M) change in the concentration of an extracellular signal can induce micromolar (10–6 M) changes in the concentration of a second messenger such as cAMP or Ca2+. Because these messengers function as allosteric effectors to activate specific enzymes or ion channels, a single extracellular signal molecule can alter many thousands of protein molecules within the target cell.

1	Any such amplifying cascade of stimulatory signals requires counterbalancing mechanisms at every step of the cascade to restore the system to its resting state when stimulation ceases. As emphasized earlier, the response to stimulation can be rapid only if the inactivating mechanisms are also rapid. Cells therefore have efficient mechanisms for rapidly degrading (and resynthesizing) cyclic nucleotides and for buffering and removing cytosolic Ca2+, as well as for inactivating the responding enzymes and ion channels once they have been activated. This is not only essential for turning a response off, but is also important for defining the resting state from which a response begins. Each protein in the signaling relay chain can be a separate target for regulation, including the receptor itself, as we discuss next.

1	Each protein in the signaling relay chain can be a separate target for regulation, including the receptor itself, as we discuss next. As discussed earlier, when target cells are exposed to a high concentration of a stimulating ligand for a prolonged period, they can become desensitized, or adapted, in several different ways. An important class of adaptation mechanisms depends on alteration of the quantity or condition of the receptor molecules themselves. For GPCRs, there are three general modes of adaptation (see Figure 15–20): (1) In receptor sequestration, they are temporarily moved to the interior of the cell (internalized) so that they no longer have access to their ligand. (2) In receptor down-regulation, they are destroyed in lysosomes after internalization. (3) In receptor inactivation, they become altered so that they can no longer interact with G proteins.

1	In each case, the desensitization of the GPCRs depends on their phosphorylation by PKA, PKC, or a member of the family of GPCR kinases (GRKs), which includes the rhodopsin-specific kinase RK involved in rod photoreceptor desensitization discussed earlier. The GRKs phosphorylate multiple serines and threonines on a GPCR, but they do so only after ligand binding has activated the receptor, because it is the activated receptor that allosterically activates the GRK. 500 G-protein (transducin) molecules are activated 500 cyclic GMP phosphodiesterase molecules are activated 105 cyclic GMP molecules are hydrolyzed 106–107 Na+ ions per second are prevented from entering the cell for a period of ~1 second membrane potential is altered by 1 mV Figure 15–41 Amplification in the light-induced catalytic cascade in vertebrate rods. The red arrows indicate the steps where amplification occurs, with the thickness of the arrow roughly indicating the magnitude of the amplification.

1	Figure 15–42 The roles of GPCR kinases (GRKs) and arrestins in GPCR desensitization. A GRK phosphorylates only activated receptors because it is the activated GPCR that activates the GRK. The binding of an arrestin to the phosphorylated receptor prevents the receptor from binding to its G protein and also directs its endocytosis (not shown). Mice that are deficient in one form of arrestin fail to desensitize in response to morphine, for example, attesting to the importance of arrestins for desensitization. As with rhodopsin, once a receptor has been phosphorylated by a GRK, it binds with high affinity to a member of the arrestin family of proteins (Figure 15–42).

1	As with rhodopsin, once a receptor has been phosphorylated by a GRK, it binds with high affinity to a member of the arrestin family of proteins (Figure 15–42). The bound arrestin can contribute to the desensitization process in at least two ways. First, it prevents the activated receptor from interacting with G proteins. Second, it serves as an adaptor protein to help couple the receptor to the clathrin-dependent endocytosis machinery (discussed in Chapter 13), inducing receptor-mediated endocytosis. The fate of the internalized GPCR–arrestin complex depends on other proteins in the complex. In some cases, the receptor is dephosphorylated and recycled back to the plasma membrane for reuse. In others, it is ubiquitylated, endocytosed, and degraded in lysosomes (discussed later).

1	Receptor endocytosis does not necessarily stop the receptor from signaling. In some cases, the bound arrestin recruits other signaling proteins to relay the signal onward from the internalized GPCRs along new pathways.

1	GPCRs can indirectly activate or inactivate either plasma-membrane-bound enzymes or ion channels via G proteins. When an activated receptor stimulates a G protein, the G protein undergoes a conformational change that activates its α subunit, thereby triggering release of a βγ complex. Either component can then directly regulate the activity of target proteins in the plasma membrane. Some GPCRs either activate or inactivate adenylyl cyclase, thereby altering the intracellular concentration of the second messenger cyclic AMP. Others activate a phosphoinositide-specific phospholipase C (PLCβ), which generates two second messengers. One is inositol 1,4,5-trisphosphate (IP3), which releases Ca2+ from the ER and thereby increases the concentration of Ca2+ in the cytosol. The other is diacylglycerol, which remains in the plasma membrane and helps activate protein kinase C (PKC). An increase in cytosolic cyclic AMP or Ca2+ levels affects cells mainly by stimulating cAMP-dependent protein

1	which remains in the plasma membrane and helps activate protein kinase C (PKC). An increase in cytosolic cyclic AMP or Ca2+ levels affects cells mainly by stimulating cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinases (CaMkinases), respectively.

1	PKC, PKA, and CaM-kinases phosphorylate specific target proteins and thereby alter the activity of the proteins. Each type of cell has its own characteristic set of target proteins that is regulated in these ways, enabling the cell to make its own distinctive response to the second messengers. The intracellular signaling cascades activated by GPCRs greatly amplify the responses, so that many thousands of target protein molecules are changed for each molecule of extracellular signaling ligand bound to its receptor. The responses mediated by GPCRs are rapidly turned off when the extracellular signal is removed, and activated GPCRs are inactivated by phosphorylation and association with arrestins.

1	Like GPCRs, enzyme-coupled receptors are transmembrane proteins with their ligand-binding domain on the outer surface of the plasma membrane. Instead of having a cytosolic domain that associates with a trimeric G protein, however, their cytosolic domain either has intrinsic enzyme activity or associates directly with an enzyme. Whereas a GPCR has seven transmembrane segments, each subunit of an enzyme-coupled receptor typically has only one. GPCRs and enzyme-coupled receptors often activate some of the same signaling pathways. In this section, we describe some of the important features of signaling by enzyme-coupled receptors, with an emphasis on the most common class of these proteins, the receptor tyrosine kinases.

1	Many extracellular signal proteins act through receptor tyrosine kinases (RTKs). These include many secreted and cell-surface-bound proteins that control cell behavior in developing and adult animals. Some of these signal proteins and their RTKs are listed in Table 15–4. There are about 60 human RTKs, which can be classified into about 20 structural subfamilies, each dedicated to its complementary family of protein ligands. Figure 15–43 shows the basic structural features of a number of the families that operate in mammals. In all cases, the binding of the signal protein to the ligand-binding domain on the extracellular side of the receptor activates the tyrosine kinase domain on the cytosolic side. This leads to phosphorylation of tyrosine side chains on the cytosolic part of the receptor, creating phosphotyrosine docking sites for various intracellular signaling proteins that relay the signal.

1	How does the binding of an extracellular ligand activate the kinase domain on the other side of the plasma membrane? For a GPCR, ligand binding is thought to change the relative orientation of several of the transmembrane α helices, thereby shifting the position of the cytoplasmic loops relative to one another. It is unlikely, however, that a conformational change could propagate across the lipid bilayer EGF receptor SS SS SS insulin receptor, NGF receptor FGF receptor CYTOSOL cysteine-immunoglobulin-like domain Eph receptor kinase insert region fbronectin-type-III-like domain IGF1 PDGF VEGF receptor receptor, receptor MCSF receptor through a single transmembrane α helix. Instead, for most RTKs, ligand binding causes the receptors to dimerize, bringing the two cytoplasmic kinase domains together and thereby promoting their activation (Figure 15–44).

1	Dimerization stimulates kinase activity by a variety of mechanisms. In many cases, such as the insulin receptor, dimerization simply brings the kinase domains close to each other in an orientation that allows them to phosphorylate each other on specific tyrosines in the kinase active sites, thereby promoting conformational changes that fully activate both kinase domains. In other cases, such as the receptor for epidermal growth factor (EGF), the kinase is not activated by phosphorylation but by conformational changes brought about by interactions between the two kinase domains outside their active sites (Figure 15–45).

1	Figure 15–43 Some subfamilies of RTKs. Only one or two members of each subfamily are indicated. Note that in some cases, the tyrosine kinase domain is interrupted by a “kinase insert region” that is an extra segment emerging from the folded kinase domain. The functions of most of the cysteine-rich, immunoglobulinlike, and fibronectin-type-III-like domains are not known. Some of the ligands for the receptors shown are listed in Table 15–4, along with some representative responses that they mediate.

1	Figure 15–44 Activation of RTKs by dimerization. In the absence of extracellular signals, most RTKs exist as monomers in which the internal kinase domain is inactive. Binding of ligand brings two monomers together to form a dimer. In most cases, the close proximity in the dimer leads the two kinase domains to phosphorylate each other, which has two effects. First, phosphorylation at some tyrosines in the kinase domains promotes the complete activation of the domains. Second, phosphorylation at tyrosines in other parts of the receptors generates docking sites for intracellular signaling proteins, resulting in the formation of large signaling complexes that can then broadcast signals along multiple signaling pathways.

1	Mechanisms of dimerization vary widely among different RTK family members. In some cases, as shown here, the ligand itself is a dimer and brings two receptors together by binding them simultaneously. In other cases, a monomeric ligand can interact with two receptors simultaneously to bring them together, or two ligands can bind independently on two receptors to promote dimerization. In some RTKs—notably those in the insulin receptor family—the receptor is always a dimer (see Figure 15–43), and ligand binding causes a conformational change that brings the two internal kinase domains closer together. Although many RTKs are activated by transautophosphorylation as shown here, there are some important exceptions, including the EGF receptor illustrated in Figure 15–45. Phosphorylated Tyrosines on RTKs Serve as Docking Sites for Intracellular Signaling Proteins

1	Phosphorylated Tyrosines on RTKs Serve as Docking Sites for Intracellular Signaling Proteins Once the kinase domains of an RTK dimer are activated, they phosphorylate multiple additional sites in the cytosolic parts of the receptors, typically in disordered regions outside the kinase domain (see Figure 15–44). This phosphorylation creates high-affinity docking sites for intracellular signaling proteins. Each signaling protein binds to a particular phosphorylated site on the activated receptors because it contains a specific phosphotyrosine-binding domain that recognizes surrounding features of the polypeptide chain in addition to the phosphotyrosine.

1	Once bound to the activated RTK, a signaling protein may become phosphorylated on tyrosines and thereby activated. In many cases, however, the binding alone may be sufficient to activate the docked signaling protein, by either inducing a conformational change in the protein or simply bringing it near the protein that is next in the signaling pathway. Thus, receptor phosphorylation serves as a switch to trigger the assembly of an intracellular signaling complex, which can then relay the signal onward, often along multiple routes, to various destinations in the cell. Because different RTKs bind different combinations of these signaling proteins, they activate different responses.

1	Some RTKs use additional docking proteins to enlarge the signaling complex at activated receptors. Insulin and IGF1 receptor signaling, for example, depend on a specialized docking protein called insulin receptor substrate 1 (IRS1). IRS1 associates with phosphorylated tyrosines on the activated receptor and is then phosphorylated at multiple sites, thereby creating many more docking sites than could be accommodated on the receptor alone (see Figure 15–11). Proteins with SH2 Domains Bind to Phosphorylated Tyrosines

1	A whole menagerie of intracellular signaling proteins can bind to the phosphotyrosines on activated RTKs (or on docking proteins such as IRS1). They help to relay the signal onward, mainly through chains of protein–protein interactions mediated by modular interaction domains, as discussed earlier. Some of the docked proteins are enzymes, such as phospholipase C-γ (PLCγ), which functions in the same way as phospholipase C-β—activating the inositol phospholipid signaling pathway discussed earlier in connection with GPCRs (see Figures 15–28 and 15–29). Through this pathway, RTKs can increase cytosolic Ca2+ levels and activate PKC. Another enzyme that docks on these receptors is the cytoplasmic tyrosine kinase Src, which phosphorylates other signaling proteins on tyrosines. Yet another is phosphoinositide 3-kinase (PI 3-kinase), which phosphorylates lipids rather than proteins; as we discuss later, the phosphorylated lipids then serve as docking sites to attract various signaling proteins

1	3-kinase (PI 3-kinase), which phosphorylates lipids rather than proteins; as we discuss later, the phosphorylated lipids then serve as docking sites to attract various signaling proteins to the plasma membrane.

1	The intracellular signaling proteins that bind to phosphotyrosines have varied structures and functions. However, they usually share highly conserved phosphotyrosine-binding domains. These can be either SH2 domains (for Src homology

1	Figure 15–45 Activation of the eGF receptor kinase. In the absence of ligand, the EGF receptor exists primarily as an inactive monomer. EGF binding results in a conformational change that promotes dimerization of the external domains. The receptor kinase domain, unlike that of many RTKs, is not activated by transautophosphorylation. Instead, dimerization orients the internal kinase domains into an asymmetric dimer, in which one kinase domain (the “activator”) pushes against the other kinase domain (the “receiver”), thereby causing an activating conformational change in the receiver. The active receiver domain then phosphorylates multiple tyrosines in the C-terminal tails of both receptors, generating docking sites for intracellular signaling proteins (see Figure 15–44).

1	region) or, less commonly, PTB domains (for phosphotyrosine-binding). By recognizing specific phosphorylated tyrosines, these small interaction domains enable the proteins that contain them to bind to activated RTKs, as well as to many other intracellular signaling proteins that have been transiently phosphorylated on tyrosines (Figure 15–46). As discussed previously, many signaling proteins also contain other interaction domains that allow them to interact specifically with other proteins as part of the signaling process. These domains include the SH3 domain, which binds to proline-rich motifs in intracellular proteins (see Figure 15–11).

1	Not all proteins that bind to activated RTKs via SH2 domains help to relay the signal onward. Some act to decrease the signaling process, providing negative feedback. One example is the c-Cbl protein, which can dock on some activated receptors and catalyze their ubiquitylation, covalently adding one or more ubiquitin molecules to specific sites on the receptor. This promotes the endocytosis and degradation of the receptors in lysosomes—an example of receptor down-regulation (see Figure 15–20). Endocytic proteins that contain ubiquitininteraction motifs (UIMs) recognize the ubiquitylated RTKs and direct them into clathrin-coated vesicles and, ultimately, into lysosomes (discussed in Chapter 13). Mutations that inactivate c-Cbl-dependent RTK down-regulation cause prolonged RTK signaling and thereby promote the development of cancer.

1	As is the case for GPCRs, ligand-induced endocytosis of RTKs does not always decrease signaling. In some cases, RTKs are endocytosed with their bound signaling proteins and continue to signal from endosomes or other intracellular compartments. This mechanism, for example, allows nerve growth factor (NGF) to bind to its specific RTK (called TrkA) at the end of a long nerve cell axon and signal to the cell body of the same cell a long distance away. Here, signaling endocytic Figure 15–46 The binding of SH2containing intracellular signaling proteins to an activated RTK.

1	(A) This drawing of a receptor for platelet-derived growth factor (PDGF) shows five phosphotyrosine docking sites, three in the kinase insert region and two on the C-terminal tail, to which the three signaling proteins shown bind as indicated. The numbers on the right indicate the positions of the tyrosines in the polypeptide chain. These binding sites have been identified by using recombinant DNA technology to mutate specific tyrosines in the receptor. Mutation of tyrosines 1009 and 1021, for example, prevents the binding and activation of PLCγ, so that receptor activation no longer stimulates the inositol phospholipid signaling pathway. The locations of the SH2 (red) and SH3 (blue) domains in the three signaling proteins are indicated. (Additional phosphotyrosine docking sites on this receptor are not shown, including those that bind the cytoplasmic tyrosine kinase Src and two adaptor proteins.) It is unclear how many signaling proteins can bind simultaneously to a single RTK. (B)

1	receptor are not shown, including those that bind the cytoplasmic tyrosine kinase Src and two adaptor proteins.) It is unclear how many signaling proteins can bind simultaneously to a single RTK. (B) The three-dimensional structure of an SH2 domain, as determined by x-ray crystallography. The binding pocket for phosphotyrosine is shaded in orange on the right, and a pocket for binding a specific amino acid side chain (isoleucine, in this case) is shaded in yellow on the left. The RTK polypeptide segment that binds the SH2 domain is shown in yellow (see also Figure 3–40). (C) The SH2 domain is a compact, “plug-in” module, which can be inserted almost anywhere in a protein without disturbing the protein’s folding or function (discussed in Chapter 3). Because each domain has distinct sites for recognizing phosphotyrosine and for recognizing a particular amino acid side chain, different SH2 domains recognize phosphotyrosine in the context of different flanking amino acid sequences. (B,

1	for recognizing phosphotyrosine and for recognizing a particular amino acid side chain, different SH2 domains recognize phosphotyrosine in the context of different flanking amino acid sequences. (B, based on data from G. Waksman et al., Cell 72:779–790, 1993. With permission from Elsevier. PDB code: 2SRC.) vesicles containing TrkA, with NGF bound on the lumenal side and signaling proteins docked on the cytosolic side, are transported along the axon to the cell body, where they signal the cell to survive.

1	Some signaling proteins are composed almost entirely of SH2 and SH3 domains and function as adaptors to couple tyrosine-phosphorylated proteins to other proteins that do not have their own SH2 domains (see Figure 15–11). Adaptor proteins of this type help to couple activated RTKs to the important signaling protein Ras, a monomeric GTPase that, in turn, can activate various downstream signaling pathways, as we now discuss. The GTPase Ras Mediates Signaling by Most RTKs The Ras superfamily consists of various families of monomeric GTPases, but only the Ras and Rho families relay signals from cell-surface receptors (Table 15–5). By interacting with different intracellular signaling proteins, a single Ras or Rho family member can coordinately spread the signal along several distinct downstream signaling pathways, thereby acting as a signaling hub.

1	There are three major, closely related Ras proteins in humans: H-, K-, and N-Ras (see Table 15–5). Although they have subtly different functions, they are thought to work in the same way, and we will refer to them simply as Ras. Like many monomeric GTPases, Ras contains one or more covalently attached lipid groups that help anchor the protein to the cytoplasmic face of the membrane, from where it relays signals to other parts of the cell. Ras is often required, for example, when RTKs signal to the nucleus to stimulate cell proliferation or differentiation, both of which require changes in gene expression. If Ras function is inhibited by various experimental approaches, the cell proliferation or differentiation responses normally induced by the activated RTKs do not occur. Conversely, 30% of human tumors express hyperactive mutant forms of Ras, which contribute to the uncontrolled proliferation of the cancer cells.

1	Like other GTP-binding proteins, Ras functions as a molecular switch, cycling between two distinct conformational states—active when GTP is bound and inactive when GDP is bound (Movie 15.7). As discussed earlier for monomeric GTPases in general, two classes of signaling proteins regulate Ras activity by influencing its transition between active and inactive states (see Figure 15–8). Ras guanine nucleotide exchange factors (Ras-GEFs) stimulate the dissociation of GDP and the subsequent uptake of GTP from the cytosol, thereby activating Ras. Ras GTPase-activating proteins (Ras-GAPs) increase the rate of hydrolysis of bound GTP by Ras, thereby inactivating Ras. Hyperactive mutant forms of Ras are number of activated resistant to GAP-mediated GTPase stimulation and are locked permanently in the GTP-bound active state, which is why they promote the development of cancer.

1	GTP-bound active state, which is why they promote the development of cancer. But how do RTKs normally activate Ras? In principle, they could either activate a Ras-GEF or inhibit a Ras-GAP. Even though some GAPs bind directly (via their SH2 domains) to activated RTKs (see Figure 15–46A), it is the indirect coupling of the receptor to a Ras-GEF that drives Ras into its active state. The loss of function of a Ras-GEF has a similar effect to the loss of function of Ras itself. Activation of the other Ras superfamily proteins, including those of the Rho family, also occurs through the activation of GEFs. The particular GEF determines in which membrane the GTPase is activated and, by acting as a scaffold, it can also determine which downstream proteins the GTPase activates.

1	The GEF that mediates Ras activation by RTKs was discovered by genetic studies of eye development in Drosophila, where an RTK called Sevenless (Sev) is required for the formation of a photoreceptor cell called R7. Genetic screens for components of this signaling pathway led to the discovery of a Ras-GEF called Son-of-sevenless (Sos). Further genetic screens uncovered another protein, now called Grb2, which is an adaptor protein that links the Sev receptor to the Sos protein; the SH2 domain of the Grb2 adaptor binds to the activated receptor, while its two SH3 domains bind to Sos. Sos then promotes Ras activation. Biochemical and cell biological studies have shown that Grb2 and Sos also link activated RTKs to Ras in mammalian cells, revealing that this is a highly conserved mechanism in RTK signaling (Figure 15–47). Once activated, Ras activates various other signaling proteins to relay the signal downstream, as we discuss next.

1	Both the tyrosine phosphorylations and the activation of Ras triggered by activated RTKs are usually short-lived (Figure 15–48). Tyrosine-specific protein phosphatases quickly reverse the phosphorylations, and Ras-GAPs induce activated 0.1 0.05 Figure 15–47 How an RTK activates Ras. Grb2 recognizes a specific phosphorylated tyrosine on the activated receptor by means of an SH2 domain and recruits Sos by means of two SH3 domains. Sos stimulates the inactive Ras protein to replace its bound GDP by GTP, which activates Ras to relay the signal downstream.

1	Figure 15–48 Transient activation of Ras revealed by single-molecule fluorescence resonance energy transfer (FReT). (A) Schematic drawing of the experimental strategy. Cells of a human cancer cell line are genetically engineered to express a Ras protein that is covalently linked to yellow fluorescent protein (YFP). GTP that is labeled with a red fluorescent dye is microinjected into some of the cells. The cells are then stimulated with the extracellular signal protein EGF, and single fluorescent molecules of Ras-YFP at the inner surface of the plasma membrane are followed by video fluorescence microscopy in individual cells. When a fluorescent Ras-YFP molecule becomes activated, it exchanges unlabeled GDP for fluorescently labeled GTP; the energy emitted by the YFP now activates the fluorescent GTP to emit red light (called fluorescence resonance energy transfer, or FRET; see Figure 9–26). Thus, the activation of single Ras molecules can be followed by the emission of red fluorescence

1	GTP to emit red light (called fluorescence resonance energy transfer, or FRET; see Figure 9–26). Thus, the activation of single Ras molecules can be followed by the emission of red fluorescence from a previously yellow-green fluorescent spot at the plasma membrane. As shown in (B), activated Ras molecules can be detected after about 30 seconds of EGF stimulation. The red signal peaks at about 3 minutes and then decreases to baseline by 6 minutes. As Ras-GAP is found to be recruited to the same spots at the plasma membrane as Ras, it presumably plays a major part in rapidly shutting off the Ras signal. (Modified from H. Murakoshi et al., Proc. Natl Acad. Sci. USA 101:7317–7322, 2004. With permission from National Academy of Sciences.)

1	Ras to inactivate itself by hydrolyzing its bound GTP to GDP. To stimulate cells to proliferate or differentiate, these short-lived signaling events must be converted into longer-lasting ones that can sustain the signal and relay it downstream to the nucleus to alter the pattern of gene expression. One of the key mechanisms used for this purpose is a system of proteins called the mitogen-activated protein kinase module (MAP kinase module) (Figure 15–49). The three components of this system form a functional signaling module that has been remarkably well conserved during evolution and is used, with variations, in many different signaling contexts.

1	The three components are all protein kinases. The final kinase in the series is called simply MAP kinase (MAPK). The next one upstream from this is MAP kinase kinase (MAPKK): it phosphorylates and thereby activates MAP kinase. Next above that, receiving an activating signal directly from Ras, is MAP kinase kinase kinase (MAPKKK): it phosphorylates and thereby activates MAPKK. In the mammalian Ras–MAP-kinase signaling pathway, these three kinases are known by shorter names: Raf (= MAPKKK), Mek (= MAPKK), and Erk (=MAPK).

1	Once activated, the MAP kinase relays the signal downstream by phosphorylating various proteins in the cell, including transcription regulators and other protein kinases (see Figure 15–49). Erk, for example, enters the nucleus and phosphorylates one or more components of a transcription regulatory complex. This activates the transcription of a set of immediate early genes, so named because they turn on within minutes after an RTK receives an extracellular signal, even if protein synthesis is experimentally blocked with drugs. Some of these genes encode other transcription regulators that turn on other genes, a process that requires both protein synthesis and more time. In this way, the Ras–MAP-kinase signaling pathway conveys signals from the cell surface to the nucleus and alters the pattern of gene expression. Among the genes activated by this pathway are some that stimulate cell proliferation, such as the genes encoding G1 cyclins (discussed in Chapter 17).

1	Extracellular signals usually activate MAP kinases only transiently, and the period during which the kinase remains active influences the response. When EGF activates its receptors in a neural precursor cell line, for example, Erk MAP kinase activity peaks at 5 minutes and rapidly declines, and the cells later go on to divide. By contrast, when NGF activates its receptors on the same cells, Erk activity remains high for many hours, and the cells stop proliferating and differentiate into neurons. Many factors influence the duration and other features of the signaling response, including positive and negative feedback loops, which can combine to

1	Many factors influence the duration and other features of the signaling response, including positive and negative feedback loops, which can combine to Figure 15–49 The MAP kinase module activated by Ras. The three-component module begins with a MAP kinase kinase kinase called Raf. Ras recruits Raf to the plasma membrane and helps activate it. Raf then activates the MAP kinase kinase Mek, which then activates the MAP kinase Erk. Erk in turn phosphorylates a variety of downstream proteins, including other protein kinases, as well as transcription regulators in the nucleus. The resulting changes in protein activities and gene expression cause complex changes in cell behavior.

1	give responses that are either graded or switchlike and either brief or long lasting. In an example illustrated earlier, in Figure 15–19, MAP kinase activates a complex positive feedback loop to produce an all-or-none, irreversible response when frog oocytes are stimulated to mature by a brief exposure to the extracellular signal molecule progesterone. In many cells, MAP kinases activate a negative feedback loop by increasing the concentration of a protein phosphatase that removes the phosphate from MAP kinase. The increase in the phosphatase results from both an increase in the transcription of the phosphatase gene and the stabilization of the enzyme against degradation. In the Ras–MAP-kinase pathway shown in Figure 15–49, Erk also phosphorylates and inactivates Raf, providing another negative feedback loop that helps shut off the MAP kinase module.

1	Three-component MAP kinase signaling modules operate in all eukaryotic cells, with different modules mediating different responses in the same cell. In budding yeast, for example, one such module mediates the response to mating pheromone, another the response to starvation, and yet another the response to osmotic shock. Some of these MAP kinase modules use one or more of the same kinases and yet manage to activate different effector proteins and hence different responses. As discussed earlier, one way in which cells avoid cross-talk between the different parallel signaling pathways and ensure that each response is specific is to use scaffold proteins (see Figure 15–10A). In budding yeast cells, such scaffolds bind all or some of the kinases in each MAP kinase module to form a complex and thereby help to ensure response specificity (Figure 15–50).

1	Mammalian cells also use this scaffold strategy to prevent cross-talk between different MAP kinase modules. At least five parallel MAP kinase modules can operate in a mammalian cell. These modules make use of at least 12 MAP kinases, 7 MAPKKs, and 7 MAPKKKs. Two of these modules (terminating in MAP kinases called JNK and p38) are activated by different kinds of cell stresses, such as ultraviolet (UV) irradiation, heat shock, and osmotic stress, as well as by inflammatory cytokines; others mainly mediate responses to signals from other cells. Although the scaffold strategy provides precision and avoids cross-talk, it reduces the opportunities for amplification and spreading of the signal to different parts of the cell, which require at least some of the components to be diffusible. It is unclear to what extent the individual components of MAP kinase modules can dissociate from the scaffold during the activation process to permit amplification.

1	Figure 15–50 The organization of two MAP kinase modules by scaffold proteins in budding yeast. Budding yeast have at least six three-component MAP kinase modules involved in a variety of biological processes, including the two responses illustrated here—a mating response and the response to high osmolarity. (A) The mating response is triggered when a mating factor secreted by a yeast of opposite mating type binds to a GPCR. This activates a G protein, the βγ complex of which indirectly activates the MAPKKK (kinase A), which then relays the response onward. Once activated, the MAP kinase (kinase C) phosphorylates and thereby activates several proteins that mediate the mating response, in which the yeast cell stops dividing and prepares for fusion. The three kinases in this module are bound to scaffold protein 1. (B) In a second response, a yeast cell exposed to a high-osmolarity environment is induced to synthesize glycerol to increase its internal osmolarity. This response is mediated

1	to scaffold protein 1. (B) In a second response, a yeast cell exposed to a high-osmolarity environment is induced to synthesize glycerol to increase its internal osmolarity. This response is mediated by an osmolarity-sensing receptor protein and a different MAP kinase module bound to a second scaffold protein. (Note that the kinase domain of scaffold 2 provides the MAPKK activity of this module.) Although both pathways use the same MAPKKK (kinase A, green), there is no cross-talk between them because the kinases in each module are bound to different scaffold proteins, and the osmosensor is bound to the same scaffold protein as the particular kinase it activates.

1	Rho Family GTPases Functionally Couple Cell-Surface Receptors to the Cytoskeleton Besides the Ras proteins, the other class of Ras superfamily GTPases that relays signals from cell-surface receptors is the large Rho family (see Table 15–5). Rho family monomeric GTPases regulate both the actin and microtubule cytoskeletons, controlling cell shape, polarity, motility, and adhesion (discussed in Chapter 16); they also regulate cell-cycle progression, gene transcription, and membrane transport. They play a key part in the guidance of cell migration and nerve axon outgrowth, mediating cytoskeletal responses to the activation of a special class of guidance receptors. We focus on this aspect of Rho family function here.

1	The three best-characterized family members are Rho itself, Rac, and Cdc42, each of which affects multiple downstream target proteins. In the same way as for Ras, GEFs activate and GAPs inactivate the Rho family GTPases; there are more than 80 Rho-GEFs and more than 70 Rho-GAPs in humans. Some of the GEFs and GAPs are specific for one particular family member, whereas others are less specific. Unlike Ras, which is membrane-associated even when inactive (with GDP bound), inactive Rho family GTPases are often bound to guanine nucleotide dissociation inhibitors (GDIs) in the cytosol, which prevent the GTPases from interacting with their Rho-GEFs at the plasma membrane.

1	Signaling by extracellular signaling proteins of the ephrin family provides an example of how RTKs can activate a Rho GTPase. Ephrins bind and thereby activate members of the Eph family of RTKs (see Figure 15–43). One member of the Eph family is found on the surface of motor neurons and helps guide the migrating tip of the axon (called a growth cone) to its muscle target. The binding of a cell-surface ephrin protein activates the Eph receptor, causing the growth cones to collapse, thereby repelling them from inappropriate regions and keeping them on track. The response depends on a Rho-GEF called ephexin, which is stably associated with the cytosolic tail of the Eph receptor. When ephrin binding activates the Eph receptor, the receptor activates a cytoplasmic tyrosine kinase that phosphorylates ephexin on a tyrosine, enhancing the ability of ephexin to activate the Rho protein RhoA. The activated RhoA (RhoA-GTP) then regulates various downstream target proteins, including some

1	ephexin on a tyrosine, enhancing the ability of ephexin to activate the Rho protein RhoA. The activated RhoA (RhoA-GTP) then regulates various downstream target proteins, including some effector proteins that control the actin cytoskeleton, causing the growth cone to collapse (Figure 15–51).

1	Having considered how RTKs use GEFs and monomeric GTPases to relay signals into the cell, we now consider a second major strategy that RTKs use that depends on a quite different intracellular relay mechanism. Figure 15–51 Growth cone collapse mediated by Rho family GTPases. The binding of ephrin A1 proteins on an adjacent cell activates EphA4 RTKs on the growth cone. Phosphotyrosines on the activated Eph receptors recruit and activate a cytoplasmic tyrosine kinase to phosphorylate the receptor-associated Rho-GEF ephexin on a tyrosine. This enhances the ability of the ephexin to activate RhoA. RhoA then induces the growth cone to collapse by stimulating the myosin-dependent contraction of the actin cytoskeleton. PI 3-Kinase Produces Lipid Docking Sites in the Plasma Membrane

1	PI 3-Kinase Produces Lipid Docking Sites in the Plasma Membrane As mentioned earlier, one of the proteins that binds to the intracellular tail of RTK molecules is the plasma-membrane-bound enzyme phosphoinositide 3-kinase (PI 3-kinase). This kinase principally phosphorylates inositol phospholipids rather than proteins, and both RTKs and GPCRs can activate it. It plays a central part in promoting cell survival and growth.

1	Phosphatidylinositol (PI) is unique among membrane lipids because it can undergo reversible phosphorylation at multiple sites on its inositol head group to generate a variety of phosphorylated PI lipids called phosphoinositides. When activated, PI 3-kinase catalyzes phosphorylation at the 3 position of the inositol ring to generate several phosphoinositides (Figure 15–52). The production of PI(3,4,5)P3 matters most because it can serve as a docking site for various intracellular signaling proteins, which assemble into signaling complexes that relay the signal into the cell from the cytosolic face of the plasma membrane (see Figure 15–10C).

1	Notice the difference between this use of phosphoinositides and their use described earlier, in which PI(4,5)P2 is cleaved by PLCβ (in the case of GPCRs) or PLCγ(in the case of RTKs) to generate soluble IP3 and membrane-bound diacylglycerol (see Figures 15–28 and 15–29). By contrast, PI(3,4,5)P3 is not cleaved by either PLC. It is made from PI(4,5)P2 and then remains in the plasma membrane until specific phosphoinositide phosphatases dephosphorylate it. Prominent among these is the PTEN phosphatase, which dephosphorylates the 3 position of the inositol ring. Mutations in PTEN are found in many cancers: by prolonging signaling by PI 3-kinase, they promote uncontrolled cell growth.

1	There are various types of PI 3-kinases. Those activated by RTKs and GPCRs belong to class I. These are heterodimers composed of a common catalytic subunit and different regulatory subunits. RTKs activate class Ia PI 3-kinases, in which the regulatory subunit is an adaptor protein that binds to two phosphotyrosines on activated RTKs through its two SH2 domains (see Figure 15–46A). GPCRs activate class Ib PI 3-kinases, which have a regulatory subunit that binds to the βγ complex of an activated trimeric G protein (Gq) when GPCRs are activated by their extracellular ligand. The direct binding of activated Ras can also activate the common class I catalytic subunit. 6 1 2 3 4 5 diacylglycerol IP3 PLC CATALYZED BY PI 3-KINASE phosphatidylinositol (PI) PI 4-phosphate [PI(4)P] PI 3,4-bisphosphate [PI(3,4)P2] PI 4,5-bisphosphate [PI(4,5)P2] P P P P PP P P P P P P P P P

1	Figure 15–52 The generation of phosphoinositide docking sites by PI 3-kinase. PI 3-kinase phosphorylates the inositol ring on carbon atom 3 to generate the phosphoinositides shown at the bottom of the figure (diverting them away from the pathway leading to IP3 and diacylglycerol; see Figure 15–28). The most important phosphorylation (indicated in red) is of PI(4,5)P2 to PI(3,4,5)P3, which can serve as a docking site for signaling proteins with PI(3,4,5)P3-binding PH domains. Other inositol phospholipid kinases (not shown) catalyze the phosphorylations indicated by the green arrows. PI 3-phosphate PI 3,4,5-trisphosphate [PI(3)P] [PI(3,4,5)P3]

1	PI 3-phosphate PI 3,4,5-trisphosphate [PI(3)P] [PI(3,4,5)P3] Intracellular signaling proteins bind to PI(3,4,5)P3 produced by activated PI 3-kinase via a specific interaction domain, such as a pleckstrin homology (PH) domain, first identified in the platelet protein pleckstrin. PH domains function mainly as protein–protein interaction domains, and it is only a small subset of them that bind to PI(3,4,5)P3; at least some of these also recognize a specific membrane-bound protein as well as the PI(3,4,5)P3, which greatly increases the specificity of the binding and helps to explain why the signaling proteins with PI(3,4,5) P3-binding PH domains do not all dock at all PI(3,4,5)P3 sites. PH domains occur in about 200 human proteins, including the Ras-GEF Sos discussed earlier (see Figure 15–11).

1	One especially important PH-domain-containing protein is the serine/threonine protein kinase Akt. The PI-3-kinase–Akt signaling pathway is the major pathway activated by the hormone insulin. It also plays a key part in promoting the survival and growth of many cell types in both invertebrates and vertebrates, as we now discuss. The PI-3-Kinase–Akt Signaling Pathway Stimulates Animal Cells to Survive and Grow

1	The PI-3-Kinase–Akt Signaling Pathway Stimulates Animal Cells to Survive and Grow As discussed earlier, extracellular signals are usually required for animal cells to grow and divide, as well as to survive (see Figure 15–4). Members of the insulin-like growth factor (IGF) family of signal proteins, for example, stimulate many types of animal cells to survive and grow. They bind to specific RTKs (see Figure 15–43), which activate PI 3-kinase to produce PI(3,4,5)P3. The PI(3,4,5)P3 recruits two protein kinases to the plasma membrane via their PH domains—Akt (also called protein kinase B, or PKB) and phosphoinositide-dependent protein kinase 1 (PDK1), and this leads to the activation of Akt (Figure 15–53). Once activated,

1	PI(3,4,5)P3 PI(4,5)P2 PH domains CYTOSOL activated receptor tyrosine kinase activated PI 3-kinase PDK1 Akt phosphorylation and activation of Akt by PDK1 and mTOR dissociation active Akt Bad inactive apoptosis-inhibitory protein inactivated Bad INHIBITION OF APOPTOSIS PHOSPHORYLATION OF Bad 14-3-3 protein mTOR (in complex 2)P P P P P P P P P P P P P P P P P P

1	Figure 15–53 One way in which signaling through PI 3-kinase promotes cell survival. An extracellular survival signal activates an RTK, which recruits and activates PI 3-kinase. The PI 3-kinase produces PI(3,4,5)P3, which serves as a docking site for two serine/threonine kinases with PH domains—Akt and the phosphoinositide-dependent kinase PDK1—and brings them into proximity at the plasma membrane. The Akt is phosphorylated on a serine by a third kinase (usually mTOR in complex 2), which alters the conformation of the Akt so that it can be phosphorylated on a threonine by PDK1, which activates the Akt. The activated Akt now dissociates from the plasma membrane and phosphorylates various target proteins, including the Bad protein. When unphosphorylated, Bad holds one or more apoptosis-inhibitory proteins (of the Bcl2 family—discussed in Chapter 18) in an inactive state. Once phosphorylated, Bad releases the inhibitory proteins, which now can block apoptosis and thereby promote cell

1	proteins (of the Bcl2 family—discussed in Chapter 18) in an inactive state. Once phosphorylated, Bad releases the inhibitory proteins, which now can block apoptosis and thereby promote cell survival. As shown, the phosphorylated Bad binds to a ubiquitous cytosolic protein called 14-3-3, which keeps Bad out of action.

1	Akt phosphorylates various target proteins at the plasma membrane, as well as in the cytosol and nucleus. The effect on most of the known targets is to inactivate them; but the targets are such that these actions of Akt all conspire to enhance cell survival and growth, as illustrated for one cell survival pathway in Figure 15–53.

1	The control of cell growth by the PI-3-kinase–Akt pathway depends in part on a large protein kinase called TOR (named as the target of rapamycin, a bacterial toxin that inactivates the kinase and is used clinically as both an immunosuppressant and anticancer drug). TOR was originally identified in yeasts in genetic screens for rapamycin resistance; in mammalian cells, it is called mTOR, which exists in cells in two functionally distinct multiprotein complexes. mTOR complex 1 contains the protein raptor; this complex is sensitive to rapamycin, and it stimulates cell growth—both by promoting ribosome production and protein synthesis and by inhibiting protein degradation. Complex 1 also promotes both cell growth and cell survival by stimulating nutrient uptake and metabolism. mTOR complex 2 contains the protein rictor and is insensitive to rapamycin; it helps to activate Akt (see Figure 15–53), and it regulates the actin cytoskeleton via Rho family GTPases.

1	The mTOR in complex 1 integrates inputs from various sources, including extracellular signal proteins referred to as growth factors and nutrients such as amino acids, both of which help activate mTOR and promote cell growth. The growth factors activate mTOR mainly via the PI-3-kinase–Akt pathway. Akt activates mTOR in complex 1 indirectly by phosphorylating, and thereby inhibiting, a GAP called Tsc2. Tsc2 acts on a monomeric Ras-related GTPase called Rheb (see Table 15–5). Rheb in its active form (Rheb-GTP) activates mTOR in complex 1. The net result is that Akt activates mTOR and thereby promotes cell growth (Figure 15–54). We discuss how mTOR stimulates ribosome production and protein synthesis in Chapter 17 (see Figure 17–64).

1	As mentioned earlier, RTKs and GPCRs activate some of the same intracellular signaling pathways. Both, for example, can activate the inositol phospholipid pathway triggered by phospholipase C. Moreover, even when they activate different pathways, the different pathways can converge on the same target proteins. Figure 15–55 illustrates both of these types of signaling overlaps: it summarizes five parallel intracellular signaling pathways that we have discussed so far—one triggered by GPCRs, two triggered by RTKs, and two triggered by both kinds of receptors. Interactions among these pathways allow different extracellular signal molecules to modulate and coordinate each other’s effects. Figure 15–54 Activation of mTOR by the PI-3-kinase–Akt signaling pathway.

1	Figure 15–54 Activation of mTOR by the PI-3-kinase–Akt signaling pathway. (A) In the absence of extracellular growth factors, Tsc2 (a Rheb-GAP) keeps Rheb inactive; mTOR in complex 1 is inactive, and there is no cell growth. (B) In the presence of growth factors, activated Akt phosphorylates and inhibits Tsc2, thereby promoting the activation of Rheb. Activated Rheb (Rheb-GTP) helps activate mTOR in complex 1, which in turn stimulates cell growth. Figure 15–53 shows how growth factors (or survival signals) activate Akt. The Erk MAP kinase (see Figure 15–49) can also phosphorylate and inhibit Tsc2 and thereby activate mTOR. Thus, both the PI-3-kinase–Akt and Ras–MAP-kinase signaling pathways converge on mTOR in complex 1 to stimulate cell growth.

1	Tsc2 is short for tuberous sclerosis protein 2, and it is one component of a heterodimer composed of Tsc1 and Tsc2 (not shown); these proteins are so called because mutations in either gene encoding them cause the genetic disease tuberous sclerosis, which is associated with benign tumors that contain abnormally large cells. PI(3,4,5)P3 Some Enzyme-Coupled Receptors Associate with Cytoplasmic Tyrosine Kinases

1	Some Enzyme-Coupled Receptors Associate with Cytoplasmic Tyrosine Kinases Many cell-surface receptors depend on tyrosine phosphorylation for their activity and yet lack a tyrosine kinase domain. These receptors act through cytoplasmic tyrosine kinases, which are associated with the receptors and phosphorylate various target proteins, often including the receptors themselves, when the receptors bind their ligand. These tyrosine-kinase-associated receptors thus function in much the same way as RTKs, except that their kinase domain is encoded by a separate gene and is noncovalently associated with the receptor polypeptide chain. A variety of receptor classes belong in this category, including the receptors for antigen and interleukins on lymphocytes (discussed in Chapter 24), integrins (discussed in Chapter 19), and receptors for various cytokines and some hormones. As with RTKs, many of these receptors are either preformed dimers or are cross-linked into dimers by ligand binding.

1	Some of these receptors depend on members of the largest family of mammalian cytoplasmic tyrosine kinases, the Src family (see Figures 3–10 and 3–64), which includes Src, Yes, Fgr, Fyn, Lck, Lyn, Hck, and Blk. These protein kinases all contain SH2 and SH3 domains and are located on the cytoplasmic side of the plasma membrane, held there partly by their interaction with transmembrane receptor proteins and partly by covalently attached lipid chains. Different family members are associated with different receptors and phosphorylate overlapping but distinct sets of target proteins. Lyn, Fyn, and Lck, for example, are each associated with different sets of receptors on lymphocytes. In each case, the kinase is activated when an extracellular ligand binds to the appropriate receptor protein. Src itself, as well as several other family members, can also bind to activated RTKs; in these cases, the receptor and cytoplasmic kinases mutually stimulate each other’s catalytic activity, thereby

1	Src itself, as well as several other family members, can also bind to activated RTKs; in these cases, the receptor and cytoplasmic kinases mutually stimulate each other’s catalytic activity, thereby strengthening and prolonging the signal (see Figure 15–51). There are even some G proteins (Gs and Gi) that can activate Src, which is one way that the activation of GPCRs can lead to tyrosine phosphorylation of intracellular signaling proteins and effector proteins.

1	Figure 15–55 Five parallel intracellular signaling pathways activated by GPCRs, RTKs, or both. In this simplified example, the five kinases (shaded yellow) at the end of each signaling pathway phosphorylate target proteins (shaded red), many of which are phosphorylated by more than one of the kinases. The phospholipase C activated by the two types of receptors is different: GPCRs activate PLCβ, whereas RTKs activate PLCγ (not shown). Although not shown, some GPCRs can also activate Ras, but they do so independently of Grb2, via a Ras-GEF that is activated by Ca2+ and diacylglycerol.

1	Another type of cytoplasmic tyrosine kinase associates with integrins, the main receptors that cells use to bind to the extracellular matrix (discussed in Chapter 19). The binding of matrix components to integrins activates intracellular signaling pathways that influence the behavior of the cell. When integrins cluster at sites of matrix contact, they help trigger the assembly of cell–matrix junctions called focal adhesions. Among the many proteins recruited into these junctions is the cytoplasmic tyrosine kinase called focal adhesion kinase (FAK), which binds to the cytosolic tail of one of the integrin subunits with the assistance of other proteins. The clustered FAK molecules phosphorylate each other, creating phosphotyrosine docking sites where the Src kinase can bind. Src and FAK then phosphorylate each other and other proteins that assemble in the junction, including many of the signaling proteins used by RTKs. In this way, the two tyrosine kinases signal to the cell that it has

1	phosphorylate each other and other proteins that assemble in the junction, including many of the signaling proteins used by RTKs. In this way, the two tyrosine kinases signal to the cell that it has adhered to a suitable substratum, where the cell can now survive, grow, divide, migrate, and so on.

1	The largest and most diverse class of receptors that rely on cytoplasmic tyrosine kinases to relay signals into the cell is the class of cytokine receptors, which we consider next. Cytokine Receptors Activate the JAK–STAT Signaling Pathway The large family of cytokine receptors includes receptors for many kinds of local mediators (collectively called cytokines), as well as receptors for some hormones, such as growth hormone and prolactin (Movie 15.8). These receptors are stably associated with cytoplasmic tyrosine kinases called Janus kinases (JAKs) (after the two-faced Roman god), which phosphorylate and activate transcription regulators called STATs (signal transducers and activators of transcription). STAT proteins are located in the cytosol and are referred to as latent transcription regulators because they migrate into the nucleus and regulate gene transcription only after they are activated.

1	Although many intracellular signaling pathways lead from cell-surface receptors to the nucleus, where they alter gene transcription (see Figure 15–55), the JAK–STAT signaling pathway provides one of the more direct routes. Cytokine receptors are dimers or trimers and are stably associated with one or two of the four known JAKs (JAK1, JAK2, JAK3, and Tyk2). Cytokine binding alters the arrangement so as to bring two JAKs into close proximity so that they phosphorylate each other, thereby increasing the activity of their tyrosine kinase domains. The JAKs then phosphorylate tyrosines on the cytoplasmic tails of cytokine receptors, creating phosphotyrosine docking sites for STATs (Figure 15–56). Some adaptor proteins can also bind to some of these sites and couple cytokine receptors to the Ras–MAP-kinase signaling pathway discussed earlier, but these will not be discussed here.

1	There are at least six STATs in mammals. Each has an SH2 domain that performs two functions. First, it mediates the binding of the STAT protein to a phosphotyrosine docking site on an activated cytokine receptor. Once bound, the JAKs phosphorylate the STAT on tyrosines, causing the STAT to dissociate from the receptor. Second, the SH2 domain on the released STAT now mediates its binding to a phosphotyrosine on another STAT molecule, forming either a STAT homodimer or a heterodimer. The STAT dimer then translocates to the nucleus, where, in combination with other transcription regulatory proteins, it binds to a specific cis-regulatory sequence in various genes and stimulates their transcription (see Figure 15–56). In response to the hormone prolactin, for example, which stimulates breast cells to produce milk, activated STAT5 stimulates the transcription of genes that encode milk proteins. Table 15–6 lists some of the more than 30 cytokines and hormones that activate the JAK–STAT

1	cells to produce milk, activated STAT5 stimulates the transcription of genes that encode milk proteins. Table 15–6 lists some of the more than 30 cytokines and hormones that activate the JAK–STAT pathway by binding to cytokine receptors.

1	Negative feedback regulates the responses mediated by the JAK–STAT pathway. In addition to activating genes that encode proteins mediating the cytokine-induced response, the STAT dimers can also activate genes that encode inhibitory proteins that help shut off the response. Some of these proteins bind to and inactivate phosphorylated JAKs and their associated phosphorylated receptors; others bind to phosphorylated STAT dimers and prevent them from binding to their DNA targets. Such negative feedback mechanisms, however, are not enough on their own to turn off the response. Inactivation of the activated JAKs and STATs requires dephosphorylation of their phosphotyrosines.

1	In all signaling pathways that use tyrosine phosphorylation, the tyrosine phosphorylations are reversed by protein tyrosine phosphatases. These phosphatases are as important in the signaling process as the protein tyrosine kinases that add the phosphates. Whereas only a few types of serine/threonine protein phosphatase Figure 15–56 The JAK–STAT signaling pathway activated by cytokines. The binding of the cytokine either causes two separate receptor polypeptide chains to dimerize (as shown) or re-orients the receptor chains in a preformed dimer. In either case, the associated JAKs are brought together so that they can phosphorylate each other on tyrosines to become fully activated, after which they phosphorylate the receptors to generate binding sites for the SH2 domains of STAT proteins. The JAKs also phosphorylate the STAT proteins, which dissociate from the receptor to form dimers and enter the nucleus to control gene expression.

1	catalytic subunits are responsible for removing phosphate groups from phosphorylated serines and threonines on proteins, there are about 100 protein tyrosine phosphatases encoded in the human genome, including some dual-specificity phosphatases that also dephosphorylate serines and threonines. Like tyrosine kinases, the tyrosine phosphatases occur in both cytoplasmic and transmembrane forms. Unlike serine/threonine protein phosphatases, which generally have broad specificity, most tyrosine phosphatases display exquisite specificity for their substrates, removing phosphate groups from only selected phosphotyrosines on a subset of proteins. Together, these phosphatases ensure that tyrosine phosphorylations are short-lived and that the level of tyrosine phosphorylation in resting cells is very low. They do not, however, simply continuously reverse the effects of protein tyrosine kinases; they are often regulated to act only at the appropriate time and place.

1	Having discussed the crucial role of tyrosine phosphorylation and dephosphorylation in the intracellular signaling pathways activated by many enzyme-coupled receptors, we now turn to a class of enzyme-coupled receptors that rely on serine and threonine phosphorylation. These receptor serine/threonine kinases activate an even more direct signaling pathway to the nucleus than does the JAK– STAT pathway. They directly phosphorylate latent transcription regulators called Smads, which then translocate into the nucleus to control gene transcription. Signal Proteins of the TGFβ Superfamily Act Through Receptor Serine/Threonine Kinases and Smads

1	Signal Proteins of the TGFβ Superfamily Act Through Receptor Serine/Threonine Kinases and Smads The transforming growth factor-β (TGFβ) superfamily consists of a large number (33 in humans) of structurally related, secreted, dimeric proteins. They act either as hormones or, more commonly, as local mediators to regulate a wide range of biological functions in all animals. During development, they regulate pattern formation and influence various cell behaviors, including proliferation, specification and differentiation, extracellular matrix production, and cell death. In adults, they are involved in tissue repair and in immune regulation, as well as in many other processes. The superfamily consists of the TGFβ/activin family and the larger bone morphogenetic protein (BMP) family.

1	All of these proteins act through enzyme-coupled receptors that are single-pass transmembrane proteins with a serine/threonine kinase domain on the cytosolic side of the plasma membrane. There are two classes of these receptor serine/threonine kinases—type I and type II—which are structurally similar homodimers. Each member of the TGFβ superfamily binds to a characteristic combination of type-I and type-II receptor dimers, bringing the kinase domains together so that the type-II receptor can phosphorylate and activate the type-I receptor, forming an active tetrameric receptor complex.

1	Once activated, the receptor complex uses a strategy for rapidly relaying the signal to the nucleus that is very similar to the JAK–STAT strategy used by cytokine receptors. The activated type-I receptor directly binds and phosphorylates a latent transcription regulator of the Smad family (named after the first two proteins identified, Sma in C. elegans and Mad in Drosophila). Activated TGFβ/ activin receptors phosphorylate Smad2 or Smad3, while activated BMP receptors phosphorylate Smad1, Smad5, or Smad8. Once one of these receptor-activated Smads (R-Smads) has been phosphorylated, it dissociates from the receptor and binds to Smad4 (called a co-Smad), which can form a complex with any of the five R-Smads. The Smad complex then translocates into the nucleus, where it associates with other transcription regulators and controls the transcription of specific target genes (Figure 15–57). Because the partner proteins in the nucleus vary depending on the cell type and state of the cell,

1	transcription regulators and controls the transcription of specific target genes (Figure 15–57). Because the partner proteins in the nucleus vary depending on the cell type and state of the cell, the genes affected vary.

1	Activated TGFβ receptors and their bound ligand are endocytosed by two distinct routes, one leading to further activation and the other leading to inactivation. The activation route depends on clathrin-coated vesicles and leads to early endosomes (discussed in Chapter 13), where most of the Smad activation occurs. An anchoring protein called SARA (for Smad anchor for receptor activation) has an important role in this pathway; it is concentrated in early endosomes and binds to both activated TGFβ receptors and Smads, increasing the efficiency of receptor-mediated Smad phosphorylation. The inactivation route depends on caveolae (discussed in Chapter 13) and leads to receptor ubiquitylation and degradation in proteasomes.

1	During the signaling response, the Smads shuttle continuously between the cytoplasm and the nucleus: they are dephosphorylated in the nucleus and exported to the cytoplasm, where they can be rephosphorylated by activated receptors. In this way, the effect exerted on the target genes reflects both the concentration of the extracellular signal and the time the signal continues to act on the cell-surface receptors (often several hours). Cells exposed to a morphogen at high concentration, or for a long time, or both, will switch on one set of genes, whereas cells receiving a lower or more transient exposure will switch on another set.

1	As in other signaling systems, negative feedback regulates the Smad pathway. Among the target genes activated by Smad complexes are those that encode inhibitory Smads, either Smad6 or Smad7. Smad7 (and possibly Smad6) binds to the cytosolic tail of the activated receptor and inhibits its signaling ability in at least three ways: (1) it competes with R-Smads for binding sites on the receptor, decreasing R-Smad phosphorylation; (2) it recruits a ubiquitin ligase called Smurf, which ubiquitylates the receptor, leading to receptor internalization and degradation (it is because Smurfs also ubiquitylate and promote the degradation of Smads that they are called Smad ubiquitylation regulatory factors, or Smurfs); and (3) it recruits a protein phosphatase that dephosphorylates and inactivates the receptor. In addition, the inhibitory Smads bind to the co-Smad, Smad4, and inhibit it, either by preventing its binding to R-Smads or by promoting its ubiquitylation and degradation.

1	Although receptor serine/threonine kinases operate mainly through the Smad pathway just described, they can also stimulate other intracellular signaling proteins such as MAP kinases and PI 3-kinase. Conversely, signaling proteins in other pathways can phosphorylate Smads and thereby influence signaling along the Smad pathway. There are various classes of enzyme-coupled receptors, the most common of which are receptor tyrosine kinases (RTKs), tyrosine-kinase-associated receptors, and receptor serine/threonine kinases. Figure 15–57 The Smad-dependent signaling pathway activated by TGFβ.

1	Figure 15–57 The Smad-dependent signaling pathway activated by TGFβ. The TGFβ dimer promotes the assembly of a tetrameric receptor complex containing two copies each of the type-I and type-II receptors. The type-II receptors phosphorylate specific sites on the type-I receptors, thereby activating their kinase domains and leading to phosphorylation of R-Smads such as Smad2 and Smad3. Smads open up to expose a dimerization surface when they are phosphorylated, leading to the formation of a trimeric Smad complex containing two R-Smads and the co-Smad, Smad4. The phosphorylated Smad complex enters the nucleus and collaborates with other transcription regulators to control the transcription of specific target genes.

1	Ligand binding to RTKs causes their dimerization, which leads to activation of their kinase domains. These activated kinase domains phosphorylate multiple tyrosines on the receptors, producing a set of phosphotyrosines that serve as docking sites for a set of intracellular signaling proteins, which bind via their SH2 (or PTB) domains. One such signaling protein serves as an adaptor to couple some activated receptors to a Ras-GEF (Sos), which activates the monomeric GTPase Ras; Ras, in turn, activates a three-component MAP kinase signaling module, which relays the signal to the nucleus by phosphorylating transcription regulatory proteins. Another important signaling protein that can dock on activated RTKs is PI 3-kinase, which phosphorylates specific phosphoinositides to produce lipid docking sites in the plasma membrane for signaling proteins with phosphoinositide-binding PH domains, including the serine/threonine protein kinase Akt (PKB), which plays a key part in the control of cell

1	sites in the plasma membrane for signaling proteins with phosphoinositide-binding PH domains, including the serine/threonine protein kinase Akt (PKB), which plays a key part in the control of cell survival and cell growth. Many receptor classes, including some RTKs, activate Rho family monomeric GTPases, which functionally couple the receptors to the cytoskeleton.

1	Tyrosine-kinase-associated receptors depend on various cytoplasmic tyrosine kinases for their action. These kinases include members of the Src family, which associate with many kinds of receptors, and the focal adhesion kinase (FAK), which associates with integrins at focal adhesions. The cytoplasmic tyrosine kinases then phosphorylate a variety of signaling proteins to relay the signal onward. The largest family of receptors in this class is the cytokine receptor family. When stimulated by ligand binding, these receptors activate JAK cytoplasmic tyrosine kinases, which phosphorylate STATs. The STATs then dimerize, translocate to the nucleus, and activate the transcription of specific genes. Receptor serine/threonine kinases, which are activated by signal proteins of the TGFβsuperfamily, act similarly: they directly phosphorylate and activate Smads, which then oligomerize with another Smad, translocate to the nucleus, and regulate gene transcription.

1	Major changes in the behavior of a cell tend to depend on changes in the expression of numerous genes. Thus, many extracellular signaling molecules carry out their effects, in whole or in part, by initiating signaling pathways that change the activities of transcription regulators. There are numerous examples of gene regulation in both GPCR and enzyme-coupled receptor pathways (see Figures 15–27 and 15–49). In this section, we describe some of the less common signaling mechanisms by which gene expression can be controlled. We begin with several pathways that depend on regulated proteolysis to control the activity and location of latent transcription regulators. We then turn to a class of extracellular signal molecules that do not employ cell-surface receptors but enter the cell and interact directly with transcription regulators to perform their functions. Finally, we briefly discuss some of the mechanisms by which gene expression is controlled by the circadian rhythm: the daily cycle

1	directly with transcription regulators to perform their functions. Finally, we briefly discuss some of the mechanisms by which gene expression is controlled by the circadian rhythm: the daily cycle of light and dark.

1	The Receptor Notch Is a Latent Transcription Regulatory Protein

1	Signaling through the Notch receptor protein is used widely in animal development. As discussed in Chapter 22, it has a general role in controlling cell fate choices and regulating pattern formation during the development of most tissues, as well as in the continual renewal of tissues such as the lining of the gut. It is best known, however, for its role in the production of Drosophila neural cells, which usually arise as isolated single cells within an epithelial sheet of precursor cells. During this process, when a precursor cell commits to becoming a neural cell, it signals to its immediate neighbors not to do the same; the inhibited cells develop into epidermal cells instead. This process, called lateral inhibition, depends on a contact-dependent signaling mechanism that is activated by a single-pass trans-membrane signal protein called Delta, displayed on the surface of the future neural cell. By binding to the Notch receptor protein on a neighboring cell, Delta signals to the

1	by a single-pass trans-membrane signal protein called Delta, displayed on the surface of the future neural cell. By binding to the Notch receptor protein on a neighboring cell, Delta signals to the neighbor not to become neural (Figure 15–58). When this signaling process is defective, a huge excess of neural cells is produced at the expense of epidermal cells, which is lethal.

1	Notch is a single-pass transmembrane protein that requires proteolytic processing to function. It acts as a latent transcription regulator and provides the simplest and most direct signaling pathway known from a cell-surface receptor to the nucleus. When activated by the binding of Delta on another cell, a plasma-membrane-bound protease cleaves off the cytoplasmic tail of Notch, and the released tail translocates into the nucleus to activate the transcription of a set of Notch-response genes. The Notch tail fragment acts by binding to a DNA-binding protein, converting it from a transcriptional repressor into a transcriptional activator.

1	The Notch receptor undergoes three successive proteolytic cleavage steps, but only the last two depend on Delta binding. As part of its normal biosynthesis, it is cleaved in the Golgi apparatus to form a heterodimer, which is then transported to the cell surface as the mature receptor. The binding of Delta to Notch induces a second cleavage in the extracellular domain, mediated by an extracellular protease. A final cleavage quickly follows, cutting free the cytoplasmic tail of the activated receptor (Figure 15–59). Note that, unlike most receptors, the activation of Notch is irreversible; once activated by ligand binding, the protein cannot be used again.

1	This final cleavage of the Notch tail occurs just within the transmembrane segment, and it is mediated by a protease complex called γ-secretase, which is also responsible for the intramembrane cleavage of various other proteins. One of its essential subunits is Presenilin, so called because mutations in the gene encoding it are a frequent cause of early-onset, familial Alzheimer’s disease, a form of presenile dementia. The protease complex is thought to contribute to this and other forms of Alzheimer’s disease by generating extracellular peptide fragments from a transmembrane neuronal protein; the fragments accumulate in excessive amounts and form aggregates of misfolded protein called amyloid plaques, which may injure nerve cells and contribute to their degeneration and loss.

1	Both Notch and Delta are glycoproteins, and their interaction is regulated by the glycosylation of Notch. The Fringe family of glycosyl transferases, in particular, adds extra sugars to the O-linked oligosaccharide (discussed in Chapter 13) on Notch, which alters the specificity of Notch for its ligands. This has provided the first example of the modulation of ligand–receptor signaling by differential receptor glycosylation. Wnt Proteins Bind to Frizzled Receptors and Inhibit the Degradation of β-Catenin Wnt proteins are secreted signal molecules that act as local mediators and morphogens to control many aspects of development in all animals that have been studied. They were discovered independently in flies and in mice: in Drosophila, the Wingless (Wg) gene originally came to light because of its role as a morphogen

1	Figure 15–58 lateral inhibition mediated by Notch and Delta during neural cell development in Drosophila. When individual cells in the epithelium begin to develop as neural cells, they signal to their neighbors not to do the same. This inhibitory, contact-dependent signaling is mediated by the ligand Delta, which appears on the surface of the future neural cell and binds to Notch receptor proteins on the neighboring cells. In many tissues, all the cells in a cluster initially express both Delta and Notch, and a competition occurs, with one cell emerging as winner, expressing Delta strongly and inhibiting its neighbors from doing likewise. In other cases, additional factors interact with Delta or Notch to make some cells susceptible to the lateral inhibition signal and others unresponsive to it.

1	CYTOSOL 2 3 CYTOSOL Delta signaling cell plasma membrane target cell plasma membrane 9 EGF-like domains TRANSPORT TO PLASMA MEMBRANE BINDING TO DELTA, ENDOCYTOSIS OF DELTA– NOTCH-FRAGMENT COMPLEX, AND CLEAVAGE AT SITE 2 CLEAVAGE AT SITE 3 NOTCH TAIL MIGRATES TO NUCLEUS EXTRACELLULAR SPACE ENDOCYTOSIS

1	Figure 15–59 The processing and activation of Notch by proteolytic cleavage. The numbered red arrowheads indicate the sites of proteolytic cleavage. The first proteolytic processing step occurs within the trans Golgi network to generate the mature heterodimeric Notch receptor that is then displayed on the cell surface. The binding to Delta on a neighboring cell triggers the next two proteolytic steps: the complex of Delta and the Notch fragment to which it is bound is endocytosed by the Delta-expressing cell, exposing the extracellular cleavage site in the transmembrane Notch subunit. Note that Notch and Delta interact through their repeated EGF-like domains. The released Notch tail migrates into the nucleus, where it binds to the Rbpsuh protein, which it converts from a transcriptional repressor to a transcriptional activator.

1	in wing development, while in mice, the Int1 gene was found because it promoted the development of breast tumors when activated by the integration of a virus next to it. Both of these genes encode Wnt proteins. Wnts are unusual as secreted proteins in that they have a fatty acid chain covalently attached to their N-terminus, which increases their binding to cell surfaces. There are 19 Wnts in humans, each having distinct, but often overlapping, functions.

1	Wnts can activate at least two types of intracellular signaling pathways. Our primary focus here is the Wnt/β-catenin pathway (also known as the canonical Wnt pathway), which is centered on the latent transcription regulator β-catenin. A second pathway, called the planar polarity pathway, coordinates the polarization of cells in the plane of a developing epithelium and depends on Rho family GTPases. Both of these pathways begin with the binding of Wnts to Frizzled family cell-surface receptors, which are seven-pass transmembrane proteins that resemble GPCRs in structure but do not generally work through the activation of G proteins. Instead, when activated by Wnt binding, Frizzled proteins recruit the scaffold protein Dishevelled, which helps relay the signal to other signaling molecules.

1	The Wnt/β-catenin pathway acts by regulating the proteolysis of the multi-functional protein β-catenin (or Armadillo in flies). A portion of the cell’s β-catenin is located at cell–cell junctions and thereby contributes to the control of cell– cell adhesion (discussed in Chapter 19), while the remaining β-catenin is rapidly degraded in the cytoplasm. Degradation depends on a large protein degradation complex, which binds β-catenin and keeps it out of the nucleus while promoting its degradation. The complex contains at least four other proteins: a protein kinase called casein kinase 1 (CK1) phosphorylates the β-catenin on a serine, priming it for further phosphorylation by another protein kinase called glycogen synthase kinase 3 (GSK3); this final phosphorylation marks the protein for ubiquitylation and rapid degradation in proteasomes. Two scaffold proteins called axin and Adenomatous polyposis coli (APC) hold the protein complex together (Figure 15–60A). APC gets its name from the

1	and rapid degradation in proteasomes. Two scaffold proteins called axin and Adenomatous polyposis coli (APC) hold the protein complex together (Figure 15–60A). APC gets its name from the finding that the gene encoding it is often mutated in a type of benign tumor (adenoma) of the colon; the tumor projects into the lumen as a polyp and can eventually become malignant. (This APC should not be confused with the anaphase-promoting complex, or APC/C, that plays a central part in selective protein degradation during the cell cycle—see Figure 17–15A.)

1	Wnt proteins regulate β-catenin proteolysis by binding to both a Frizzled protein and a co-receptor that is related to the low-density lipoprotein (LDL) receptor (discussed in Chapter 13) and is therefore called an LDL-receptor-related protein (LRP). In a poorly understood process, the activated receptor complex recruits the Dishevelled scaffold and promotes the phosphorylation of the LRP receptor by the two protein kinases, GSK3 and CK1. Axin is brought to the receptor complex and inactivated, thereby disrupting the β-catenin degradation complex in the cytoplasm. In this way, the phosphorylation and degradation of β-catenin are prevented, enabling unphosphorylated β-catenin to accumulate and translocate to the nucleus, where it alters the pattern of gene transcription (Figure 15–60B).

1	Figure 15–60 The Wnt/β-catenin signaling pathway. (A) In the absence of a Wnt signal, β-catenin that is not bound to cell–cell adherens junctions (not shown) interacts with a degradation complex containing APC, axin, GSK3, and CK1. In this complex, β-catenin is phosphorylated by CK1 and then by GSK3, triggering its ubiquitylation and degradation in proteasomes. Wnt-responsive genes are kept inactive by the Groucho co-repressor protein bound to the transcription regulator LEF1/TCF. (B) Wnt binding to Frizzled and LRP clusters the two co-receptors together, and the cytosolic tail of LRP is phosphorylated by GSK3 and then by CK1. Axin binds to the phosphorylated LRP and is inactivated and/or degraded, resulting in disassembly of the degradation complex. The phosphorylation of β-catenin is thereby prevented, and unphosphorylated β-catenin accumulates and translocates to the nucleus, where it binds to LEF1/ TCF, displaces the co-repressor Groucho, and acts as a coactivator to stimulate the

1	prevented, and unphosphorylated β-catenin accumulates and translocates to the nucleus, where it binds to LEF1/ TCF, displaces the co-repressor Groucho, and acts as a coactivator to stimulate the transcription of Wnt target genes. The scaffold protein Dishevelled is required for the signaling pathway to operate; it binds to Frizzled and becomes phosphorylated (not shown), but its precise role is unknown.

1	In the absence of Wnt signaling, Wnt-responsive genes are kept silent by an inhibitory complex of transcription regulatory proteins. The complex includes proteins of the LEF1/TCF family bound to a co-repressor protein of the Groucho family (see Figure 15–60A). In response to a Wnt signal, β-catenin enters the nucleus and binds to the LEF1/TCF proteins, displacing Groucho. The β-catenin now functions as a coactivator, inducing the transcription of the Wnt target genes (see Figure 15–60B). Thus, as in the case of Notch signaling, Wnt/β-catenin signaling triggers a switch from transcriptional repression to transcriptional activation.

1	Among the genes activated by β-catenin is Myc, which encodes a protein (Myc) that is an important regulator of cell growth and proliferation (discussed in Chapter 17). Mutations of the Apc gene occur in 80% of human colon cancers (discussed in Chapter 20). These mutations inhibit the protein’s ability to bind β-catenin, so that β-catenin accumulates in the nucleus and stimulates the transcription of c-Myc and other Wnt target genes, even in the absence of Wnt signaling. The resulting uncontrolled cell growth and proliferation promote the development of cancer. Various secreted inhibitory proteins regulate Wnt signaling in development. Some bind to the LRP receptors and promote their down-regulation, whereas others compete with Frizzled receptors for secreted Wnts. In Drosophila at least, Wnts activate negative feedback loops, in which Wnt target genes encode proteins that help shut the response off; some of these proteins inhibit Dishevelled, and others are secreted inhibitors.

1	Hedgehog Proteins Bind to Patched, Relieving Its Inhibition of Smoothened Hedgehog proteins and Wnt proteins act in similar ways. Both are secreted signal molecules, which act as local mediators and morphogens in many developing invertebrate and vertebrate tissues. Both proteins are modified by covalently attached lipids, depend on secreted or cell-surface-bound heparan sulfate proteoglycans (discussed in Chapter 19) for their action, and activate latent transcription regulators by inhibiting their degradation. They both trigger a switch from transcriptional repression to transcriptional activation, and excessive signaling along either pathway in adult cells can lead to cancer. They even use some of the same intracellular signaling proteins and sometimes collaborate to mediate a response.

1	The Hedgehog proteins were discovered in Drosophila, where this protein family has only one member. Mutation of the Hedgehog gene produces a larva covered with spiky processes (denticles), like a hedgehog. At least three genes encode Hedgehog proteins in vertebrates—Sonic, Desert, and Indian hedgehog. The active forms of all Hedgehog proteins are covalently coupled to cholesterol, as well as to a fatty acid chain. The cholesterol is added during an unusual processing step, in which a precursor protein cleaves itself to produce a smaller, cholesterol-containing signal protein. Most of what we know about the Hedgehog signaling pathway came initially from genetic studies in flies, and it is the fly pathway that we summarize here.

1	The effects of Hedgehog are mediated by a latent transcription regulator called Cubitus interruptus (Ci), the regulation of which is reminiscent of the regulation of β-catenin by Wnts. In the absence of a Hedgehog signal, Ci is ubiquitylated and proteolytically cleaved in proteasomes. Instead of being completely degraded, however, Ci is processed to form a smaller fragment, which accumulates in the nucleus, where it acts as a transcriptional repressor, helping to keep Hedgehog-responsive genes silent. The proteolytic processing of the Ci protein depends on its phosphorylation by three protein kinases—PKA and two kinases also used in the Wnt pathway, namely GSK3 and CK1. As in the Wnt pathway, the proteolytic processing occurs in a multiprotein complex. The complex includes the protein kinase Fused and a scaffold protein Costal2, which stably associates with Ci, recruits the three other kinases, and binds the complex to microtubules, thereby keeping unprocessed Ci out of the nucleus

1	Fused and a scaffold protein Costal2, which stably associates with Ci, recruits the three other kinases, and binds the complex to microtubules, thereby keeping unprocessed Ci out of the nucleus (Figure 15–61A).

1	Patched inactive Smoothened microtubule Costal2 Fused large Ci protein PHOSPHORYLATION OF Ci BY PKA, GSK3, AND CK1 iHog Ig-like domain fbronectin-type-III-like domain vesicle cleaved Ci protein in complex with co-repressor Hedgehog functions by blocking the proteolytic processing of Ci, thereby changing it into a transcriptional activator. It does this by a convoluted signaling process that depends on three transmembrane proteins: Patched, iHog, and Smoothened. Patched is predicted to cross the plasma membrane 12 times, and, although much of it is in intracellular vesicles, some is on the cell surface where it can bind the Hedgehog protein. iHog is also on the cell surface and is thought to serve as a co-receptor for Hedgehog. Smoothened is a seven-pass transmembrane protein with a structure very similar to a GPCR, but it does not seem to act as a Hedgehog receptor or even as an activator of G proteins; it is controlled by Patched and iHog.

1	In the absence of a Hedgehog signal, Patched employs an unknown mechanism to keep Smoothened sequestered and inactive in intracellular vesicles (see Figure 15–61A). The binding of Hedgehog to iHog and Patched inhibits the activity of Patched and induces its endocytosis and degradation. The result is that Smoothened is liberated from inhibition and translocates to the plasma membrane, where it recruits the protein complex containing Ci, Fused, and Costal2.

1	Figure 15–61 Hedgehog signaling in Drosophila. (A) In the absence of Hedgehog, most Patched is in intracellular vesicles (not shown), where it keeps Smoothened inactive and sequestered. The Ci protein is bound in a cytosolic protein degradation complex, which includes the protein kinase Fused and the scaffold protein Costal2. Costal2 recruits three other protein kinases (PKA, GSK3, and CK1; not shown), which phosphorylate Ci. Phosphorylated Ci is ubiquitylated and then cleaved in proteasomes (not shown) to form a transcriptional repressor, which accumulates in the nucleus to help keep Hedgehog target genes inactive.

1	(B) Hedgehog binding to iHog and Patched removes the inhibition of Smoothened by Patched. Smoothened is phosphorylated by PKA and CK1 and translocates to the plasma membrane, where it recruits the complex containing Fused, Costal2, and Ci. Costal2 releases unprocessed Ci, which accumulates in the nucleus and activates the transcription of Hedgehog target genes. Many details in the pathway are poorly understood, including the role of Fused. Costal2 is no longer able to bind the other three kinases, and so Ci is no longer cleaved and can now enter the nucleus and activate the transcription of Hedgehog target genes (Figure 15–61B). Among the genes activated by Ci is Patched itself; the resulting increase in Patched protein on the cell surface inhibits further Hedgehog signaling—providing another example of negative feedback.

1	Many gaps remain in our understanding of the Hedgehog signaling pathway. It is not known, for example, how Patched keeps Smoothened inactive and intracellular. As the structure of Patched resembles a transmembrane transporter protein, it has been proposed that it may transport a small molecule into the cell that keeps Smoothened sequestered in vesicles.

1	Even less is known about the more complex Hedgehog pathway in vertebrate cells. In addition to there being at least three types of vertebrate Hedgehog proteins, there are three Ci-like transcription regulator proteins (Gli1, Gli2, and Gli3) downstream of Smoothened. Gli2 and Gli3 are most similar to Ci in structure and function, and Gli3 has been shown to undergo proteolytic processing like Ci and to act as either a transcriptional repressor or a transcriptional activator. Moreover, in vertebrates, Smoothened, upon activation, becomes localized to the surface of the primary cilium (discussed in Chapter 16), where the Gli proteins are also concentrated, thereby increasing the speed and efficiency of signaling.

1	Hedgehog signaling can promote cell proliferation, and excessive Hedgehog signaling can lead to cancer. Inactivating mutations in one of the two human Patched genes, for example, which lead to excessive Hedgehog signaling, occur frequently in basal cell carcinoma of the skin, the most common form of cancer in Caucasians. A small molecule called cyclopamine, made by a meadow lily, is being used to treat cancers associated with excessive Hedgehog signaling. It blocks Hedgehog signaling by binding tightly to Smoothened and inhibiting its activity. It was originally identified because it causes severe developmental defects in the progeny of sheep grazing on such lilies; these include the presence of a single central eye (a condition called cyclopia), which is also seen in mice that are deficient in Hedgehog signaling.

1	The NFκB proteins are latent transcription regulators that are present in most animal cells and are central to many stressful, inflammatory, and innate immune responses. These responses occur as a reaction to infection or injury and help protect stressed multicellular organisms and their cells (discussed in Chapter 24). An excessive or inappropriate inflammatory response in animals can also damage tissue and cause severe pain, and chronic inflammation can lead to cancer; as in the case of Wnt and Hedgehog signaling, excessive NFκB signaling is found in a number of human cancers. NFκB proteins also have important roles during normal animal development: the Drosophila NFκB family member Dorsal, for example, has a crucial role in specifying the dorsal–ventral axis of the developing fly embryo (discussed in Chapter 22).

1	Various cell-surface receptors activate the NFκB signaling pathway in animal cells. Toll receptors in Drosophila and Toll-like receptors in vertebrates, for example, recognize pathogens and activate this pathway in triggering innate immune responses (discussed in Chapter 24). The receptors for tumor necrosis factor α (TNFα) and interleukin-1 (IL1), which are vertebrate cytokines especially important in inducing inflammatory responses, also activate this signaling pathway. The Toll, Toll-like, and IL1 receptors belong to the same family of proteins, whereas TNF receptors belong to a different family; all of them, however, act in similar ways to activate NFκB. When activated, they trigger a multiprotein ubiquitylation and phosphorylation cascade that releases NFκB from an inhibitory protein complex, so that it can translocate to the nucleus and turn on the transcription of hundreds of genes that participate in inflammatory and innate immune responses.

1	There are five NFκB proteins in mammals (RelA, RelB, c-Rel, NFκB1, and NFκB2), and they form a variety of homodimers and heterodimers, each of which activates its own characteristic set of genes. Inhibitory proteins called IκB bind tightly to the dimers and hold them in an inactive state within the cytoplasm of unstimulated cells. There are three major IκB proteins in mammals (IκB α, β, and ε), and the signals that release NFκB dimers do so by triggering a signaling pathway that leads to the phosphorylation, ubiquitylation, and consequent degradation of the IκB proteins (Figure 15–62).

1	Among the genes activated by the released NFκB is the gene that encodes IκBα. This activation leads to increased synthesis of IκBα protein, which binds to NFκB and inactivates it, creating a negative feedback loop (Figure 15–63A). Experiments on TNFα-induced responses, as well as computer modeling studies of the responses, indicate that the negative feedback produces two types of NFκB responses, depending on the duration of the TNFα stimulus; importantly, the two types of responses induce different patterns of gene expression (Figure 15–63B, C, and D). The negative feedback through IκBα is required for both types of responses: in cells deficient in IκBα, even a short exposure to TNFαinduces a sustained activation of NFκB, without oscillations, and all of the NFκB-responsive genes are activated.

1	Thus far, we have focused on the mechanisms by which extracellular signal molecules use cell-surface receptors to initiate changes in gene expression. We now turn to a class of extracellular signals that bypasses the plasma membrane entirely and controls, in the most direct way possible, transcription regulatory proteins inside the cell. Various small, hydrophobic signal molecules diffuse directly across the plasma membrane of target cells and bind to intracellular receptors that are transcription regulators. These signal molecules include steroid hormones, thyroid hormones, retinoids, and vitamin D. Although they differ greatly from one another in both

1	Figure 15–62 The activation of the NFκb pathway by TNFα. Both TNFα and its receptors are trimers. The binding of TNFα causes a rearrangement of the clustered cytosolic tails of the receptors, which now recruit various signaling proteins, resulting in the activation of a protein kinase that phosphorylates and activates IκB kinase kinase (IKK). IKK is a heterotrimer composed of two kinase subunits (IKKα and IKKβ) and a regulatory subunit called NEMO. IKKβ then phosphorylates IκB on two serines, which marks the protein for ubiquitylation and degradation in proteasomes. The released NFκB translocates into the nucleus, where, in collaboration with coactivator proteins, it stimulates the transcription of its target genes. gene A on, gene B off gene A on, gene B on (A) (B) (C) KEY amount of nuclear NF˜B gene A expression gene B expression

1	Figure 15–63 Negative feedback in the NFκb signaling pathway induces oscillations in NFκb activation. (A) Drawing showing how activated NFκB stimulates the transcription of the IκBα gene, the protein product of which acts back in the cytoplasm to sequester and inhibit NFκB there; if the stimulus is persistent, the newly made IκBα protein will then be ubiquitylated and degraded, liberating active NFκB again so that it can return to the nucleus and activate transcription (see Figure 15–62). (B) A short exposure to TNFα produces a single, short pulse of NFκB activation, beginning within minutes and ending by 1 hour. This response turns on the transcription of gene A but not gene B. (C) A sustained exposure to TNFα for the entire 6 hours of the experiment produces oscillations in NFκB activation that damp down over time. This response turns on the transcription of both genes; gene B turns on only after several hours, indicating that gene B transcription requires prolonged activation of

1	that damp down over time. This response turns on the transcription of both genes; gene B turns on only after several hours, indicating that gene B transcription requires prolonged activation of NFκB, for reasons that are not understood. (D) These time-lapse confocal fluorescence micrographs from a different study of TNFα stimulation show the oscillations of NFκB in a cultured cell, as indicated by its periodic movement into the nucleus (N) of a fusion protein composed of NFκB fused to a red fluorescent protein. In the cell at the center of the micrographs, NFκB is active and in the nucleus at 6, 60, 210, 380, and 480 minutes, but it is exclusively in the cytoplasm at 0, 120, 300, 410, and 510 minutes. (A–C, based on data from A. Hoffmann et al., Science 298:1241–1245, 2002, and adapted from A.Y. Ting and D. Endy, Science 298:1189–1190, 2002; D, from D.E. Nelson et al., Science 306:704–708, 2004. All with permission from AAAS.) chemical structure (Figure 15–64) and function, they all

1	A.Y. Ting and D. Endy, Science 298:1189–1190, 2002; D, from D.E. Nelson et al., Science 306:704–708, 2004. All with permission from AAAS.) chemical structure (Figure 15–64) and function, they all act by a similar mechanism. They bind to their respective intracellular receptor proteins and alter the ability of these proteins to control the transcription of specific genes. Thus, these proteins serve both as intracellular receptors and as intracellular effectors for the signal.

1	The receptors are all structurally related, being part of the very large nuclear receptor superfamily. Many family members have been identified by DNA sequencing only, and their ligand is not yet known; they are therefore referred to as orphan nuclear receptors, and they make up large fractions of the nuclear receptors encoded in the genomes of humans, Drosophila, and the nematode C. elegans. Some mammalian nuclear receptors are regulated by intracellular metabolites rather than by secreted signal molecules; the peroxisome proliferation-activated receptors (PPARs), for example, bind intracellular lipid metabolites and regulate the transcription of genes involved in lipid metabolism and fat-cell differentiation. It seems likely that the nuclear receptors for hormones evolved from such receptors for intracellular metabolites, which would help explain their intracellular location.

1	Steroid hormones—which include cortisol, the steroid sex hormones, vitamin D (in vertebrates), and the molting hormone ecdysone (in insects)—are all made from cholesterol. Cortisol is produced in the cortex of the adrenal glands and influences the metabolism of many types of cells. The steroid sex hormones are made in the testes and ovaries and are responsible for the secondary sex characteristics that distinguish males from females. Vitamin D is synthesized in the skin in response to sunlight; after it has been converted to its active form in the liver or kidneys, it regulates Ca2+ metabolism, promoting Ca2+ uptake in the gut and reducing its excretion in the kidneys. The thyroid hormones, which are made from the amino acid tyrosine, act to increase the metabolic rate of many cell types, while the retinoids, such as retinoic acid, are made from vitamin A and have important roles as local mediators in vertebrate development. Although all of these signal molecules are relatively

1	types, while the retinoids, such as retinoic acid, are made from vitamin A and have important roles as local mediators in vertebrate development. Although all of these signal molecules are relatively insoluble in water, they are made soluble for transport in the bloodstream and other extracellular fluids by binding to specific carrier proteins, from which they dissociate before entering a target cell (see Figure 15–3B).

1	The nuclear receptors bind to specific DNA sequences adjacent to the genes that the ligand regulates. Some of the receptors, such as those for cortisol, are located primarily in the cytosol and enter the nucleus only after ligand binding; others, such as the thyroid and retinoid receptors, are bound to DNA in the nucleus even in the absence of ligand. In either case, the inactive receptors are usually bound to inhibitory protein complexes. Ligand binding alters the conformation of the receptor protein, causing the inhibitory complex to dissociate, while also causing the receptor to bind coactivator proteins that stimulate gene transcription (Figure 15–65). In other cases, however, ligand binding to a nuclear receptor inhibits transcription: some thyroid hormone receptors, for example, act as transcriptional activators in the absence of their hormone and become transcriptional repressors when hormone binds.

1	Thus far, we have focused on the control of gene expression by extracellular signal molecules produced by other cells. We now turn to gene regulation by a more global environmental signal: the cycle of light and darkness that results from the Earth’s rotation. Life on Earth evolved in the presence of a daily cycle of day and night, and many present-day organisms (ranging from archaea to plants and humans) possess an internal rhythm that dictates different behaviors at different times of day. These behaviors range from the cyclical change in metabolic enzyme activities of a bacterium to the elaborate sleep–wake cycles of humans. The internal oscillators that control such diurnal rhythms are called circadian clocks.

1	Having a circadian clock enables an organism to anticipate the regular daily changes in its environment and take appropriate action in advance. Of course, the internal clock cannot be perfectly accurate, and so it must be capable of being reset by external cues such as the light of day. Thus, circadian clocks keep running Figure 15–64 Some signal molecules that bind to intracellular receptors. Note that all of them are small and hydrophobic. The active, hydroxylated form of vitamin D3 is shown. Estradiol and testosterone are steroid sex hormones.

1	ligand ˜ helix of coactivator even when the environmental cues (changes in light and dark) are removed, but the period of this free-running rhythm is generally a little less or more than 24 hours. External signals indicating the time of day cause small adjustments in the running of the clock, so as to keep the organism in synchrony with its environment. Following more drastic shifts, circadian cycles become gradually reset (entrained) by the new cycle of light and dark, as anyone who has experienced jet lag can attest.

1	We might expect that the circadian clock would be a complex multicellular device, with different groups of cells responsible for different parts of the oscillation mechanism. Remarkably, however, in almost all multicellular organisms, including humans, the timekeepers are individual cells. Thus, a clock that operates in each member of a specialized group of brain cells (the SCN cells in the suprachiasmatic nucleus of the hypothalamus) controls our diurnal cycles of sleeping and waking, body temperature, and hormone release. Even if these cells are removed from the brain and dispersed in a culture dish, they will continue to oscillate individually, showing a cyclic pattern of gene expression with a period of approximately 24 hours. In the intact body, the SCN cells receive neural cues from the retina, entraining the SCN cells to the daily cycle of light and dark; they also send information about the time of day to another brain area, the pineal gland, which relays the time signal to

1	the retina, entraining the SCN cells to the daily cycle of light and dark; they also send information about the time of day to another brain area, the pineal gland, which relays the time signal to the rest of the body by releasing the hormone melatonin in time with the clock.

1	Although the SCN cells have a central role as timekeepers in mammals, almost all the other cells in the mammalian body have an internal circadian rhythm, which has the ability to reset in response to light. Similarly, in Drosophila, many different types of cells have a similar circadian clock, which continues to cycle when they have been dissected away from the rest of the fly and can be reset by externally imposed light and dark cycles. The working of circadian clocks, therefore, is a fundamental problem in cell biology. Although we do not yet understand all the details, studies in a wide variety of organisms have revealed the basic principles and molecular components. The key principle is that circadian clocks generally depend on negative feedback loops. As discussed earlier, oscillations in the activity of an intracellular signaling protein can occur if that protein inhibits its own activity with a long delay (see

1	Figure 15–65 The activation of nuclear receptors. All nuclear receptors bind to DNA as either homodimers or heterodimers, but for simplicity we show them as monomers. (A) The receptors all have a related structure, which includes three major domains, as shown. An inactive receptor is bound to inhibitory proteins. Typically, the binding of ligand to the receptor causes the ligand-binding domain of the receptor to clamp shut around the ligand, the inhibitory proteins to dissociate, and coactivator proteins to bind to the receptor’s transcription-activating domain, thereby increasing gene transcription. In other cases, ligand binding has the opposite effect, causing co-repressor proteins to bind to the receptor, thereby decreasing transcription (not shown).

1	The structure of the ligand-binding domain of the retinoic acid receptor is shown in the absence (left) and presence (middle) of ligand (shown in red). When ligand binds, the blue α helix acts as a lid that snaps shut, trapping the ligand in place. The shift in the conformation of the receptor upon ligand binding also creates a binding site for a small α helix (orange) on the surface of coactivator proteins. (PDB codes: 1LBD, 2ZYO, and 2ZXZ.) Tim degraded in response to light mRNAs dissociation of heterodimer

1	Tim degraded in response to light mRNAs dissociation of heterodimer Figure 15–18C and D). In Drosophila and many other animals, including humans, the heart of the circadian clock is a delayed negative feedback loop based on transcription regulators: accumulation of certain gene products switches off the transcription of their own genes, but with a delay, so that the cell oscillates between a state in which the products are present and transcription is switched off, and one in which the products are absent and transcription is switched on (Figure 15–66). The negative feedback underlying circadian rhythms does not have to be based on transcription regulators. In some cell types, the circadian clock is constructed of proteins that govern their own activities through post-translational mechanisms, as we discuss next.

1	The best understood circadian clock is found in the photosynthetic cyanobacterium, Synechococcus elongatus. The core oscillator in this organism is remarkably simple, being composed of just three proteins—KaiA, KaiB, and KaiC. The central player is KaiC, a multifunctional enzyme that catalyzes its own phosphorylation and dephosphorylation in a 24-hour cycle: it gradually phosphorylates itself sequentially at two sites during the day and dephosphorylates itself during the night. This timing depends on interactions with the two other Kai proteins: KaiA binds to unphosphorylated KaiC and stimulates KaiC autophosphosphorylation, first at one site and then, with a delay, at the other. The second phosphorylation promotes the binding of the third protein, KaiB, which blocks the stimulatory effect of KaiA and thereby allows KaiC to dephosphorylate itself, bringing KaiC back to its dephosphorylated state. This clock depends on a negative feedback loop: KaiC drives its own phosphorylation

1	effect of KaiA and thereby allows KaiC to dephosphorylate itself, bringing KaiC back to its dephosphorylated state. This clock depends on a negative feedback loop: KaiC drives its own phosphorylation until, after a delay, it recruits an inhibitor, KaiB, that stimulates KaiC to dephosphorylate itself. Amazingly, when the three Kai proteins are purified and incubated in a test tube with ATP, KaiC phosphorylation and dephosphorylation occur with roughly 24-hour timing over a period of several days (Figure 15–67).

1	Circadian oscillations in KaiC phosphorylation lead to parallel rhythms in the expression of large numbers of genes involved in controlling metabolic activities and cell division (see Figure 15–67). As a result, many aspects of cell behavior are synchronized with the circadian cycle. Even in continuous darkness, cyanobacterial cells generate free-running oscillations of KaiC phosphorylation with roughly 24-hour periods. As in other circadian clocks, the cyanobacterial clock is entrained by the environmental light/dark

1	Figure 15–66 Simplified outline of the mechanism of the circadian clock in Drosophila cells. A central feature of the clock is the periodic accumulation and decay of two transcription regulatory proteins, Tim (short for timeless, based on the phenotype of a gene mutation) and Per (short for period). The mRNAs encoding these proteins rise gradually during the day and are translated in the cytosol, where the two proteins associate to form a heterodimer. After a time delay, the heterodimer dissociates and Tim and Per are transported into the nucleus, where Per represses the Tim and Per genes, resulting in negative feedback that causes the levels of Tim and Per to fall. In addition to this transcriptional feedback, the clock depends on numerous other proteins. For example, the controlled degradation of Per indicated in the diagram imposes delays in the accumulation of Tim and Per, which are crucial to the functioning of the clock. Steps at which specific delays are imposed are shown in

1	of Per indicated in the diagram imposes delays in the accumulation of Tim and Per, which are crucial to the functioning of the clock. Steps at which specific delays are imposed are shown in red.

1	Entrainment (or resetting) of the clock occurs in response to new light–dark cycles. Although most Drosophila cells do not have true photoreceptors, light is sensed by intracellular flavoproteins, also called cryptochromes. In the presence of light, these proteins associate with the Tim protein and cause its degradation, thereby resetting the clock. (Adapted from J.C. Dunlap, Science 311:184–186, 2006.) 1.2 1.0 0.8 0.6 0.4 0.2 cycle. Light is thought to affect the circadian clock indirectly: the activities of Kai proteins are influenced by changes in intracellular redox potential, which occur as a result of increased photosynthetic activity during the day.

1	Some signaling pathways that are especially important in animal development depend on proteolysis to control the activity and location of latent transcription regulatory proteins. Notch receptors are themselves such proteins, which are activated by cleavage when Delta on another cell binds to them; the cleaved cytosolic tail of Notch migrates into the nucleus, where it stimulates the transcription of Notch-responsive genes. In the Wnt/β-catenin signaling pathway, by contrast, the proteolysis of the latent transcription regulatory protein β-catenin is inhibited when a secreted Wnt protein binds to both a Frizzled and LRP receptor protein; as a result, β-catenin accumulates in the nucleus and activates the transcription of Wnt target genes.

1	Hedgehog signaling in flies works much like Wnt signaling. In the absence of a signal, a bifunctional, cytoplasmic transcription regulator, Ci, is proteolytically cleaved to form a transcriptional repressor that keeps Hedgehog target genes silenced. The binding of Hedgehog to its receptors (Patched and iHog) inhibits the proteolytic processing of Ci; as a result, the intact Ci protein accumulates in the nucleus and activates the transcription of Hedgehog-responsive genes. In Notch, Wnt, and Hedgehog signaling, the extracellular signal triggers a switch from transcriptional repression to transcriptional activation.

1	Signaling through the latent transcription regulator NFκB also depends on proteolysis. NFκB proteins are normally held in an inactive state by inhibitory IκB proteins in the cytoplasm. A variety of extracellular stimuli, including proinflammatory cytokines, trigger the phosphorylation and ubiquitylation of IκB, marking it for degradation; this enables the NFκB to translocate to the nucleus and activate

1	Figure 15–67 The core circadian oscillator of cyanobacteria. (A) KaiC is a combined kinase and phosphatase that phosphorylates and dephosphorylates itself on two adjacent sites. In the absence of other proteins, the phosphatase activity is dominant, and the protein is mostly unphosphorylated. The binding of KaiA to KaiC suppresses the phosphatase activity and promotes the kinase activity, leading to KaiC phosphorylation, first at site 1 and then at site 2, resulting in diphosphorylated KaiC. KaiC then dephosphorylates itself slowly at site 1, even in the presence of KaiA, so that KaiC is phosphorylated only at site 2. This form of KaiC interacts with KaiB, which blocks the stimulatory effects of KaiA, thereby reducing the rate of KaiC phosphorylation and allowing dephosphorylation to occur. Diphosphorylated KaiC increases in abundance during the day and peaks around dusk. It activates other proteins that phosphorylate a transcription regulator (RpaA), which then stimulates expression

1	Diphosphorylated KaiC increases in abundance during the day and peaks around dusk. It activates other proteins that phosphorylate a transcription regulator (RpaA), which then stimulates expression of some genes (the dusk genes that peak in early evening) and inhibits expression of other genes (the dawn genes that peak in the morning). When KaiC dephosphorylation gradually occurs during the night, these effects are reversed: dusk genes are turned off and dawn genes are turned on.

1	(B) In this experiment, the three Kai proteins were purified and mixed in a test tube with ATP (which is required for KaiC kinase activity). Every two hours over the next 3 days, the KaiC protein was analyzed by polyacrylamide gel electrophoresis, in which the phosphorylated form of KaiC migrates more slowly (upper band, P-KaiC) than the nonphosphorylated form (lower band, NP-KaiC). The three different phosphorylated forms of KaiC are not distinguished by this method. The phosphorylation of KaiC oscillates with a roughly 24-hour period. (C) The amount of phosphorylated and unphosphorylated KaiC in the experiment in B is plotted on this graph, along with the amount of total protein. (B and C, from M. Nakajima et al., Science 308:414–415, 2005. With permission from AAAS.) the transcription of its target genes. NFκB also activates the transcription of the gene that encodes IκBα, creating a negative feedback loop, which can produce prolonged oscillations in NFκB activity with sustained

1	of its target genes. NFκB also activates the transcription of the gene that encodes IκBα, creating a negative feedback loop, which can produce prolonged oscillations in NFκB activity with sustained extracellular signaling.

1	Some small, hydrophobic signal molecules, including steroid and thyroid hormones, diffuse across the plasma membrane of the target cell and activate intracellular receptor proteins that directly regulate the transcription of specific genes. In many cell types, gene expression is governed by circadian clocks, in which delayed negative feedback produces 24-hour oscillations in the activities of transcription regulators, anticipating the cell's changing needs during the day and night.

1	In plants, as in animals, cells are in constant communication with one another. Plant cells communicate to coordinate their activities in response to the changing conditions of light, dark, and temperature, which guide the plant’s cycle of growth, flowering, and fruiting. Plant cells also communicate to coordinate activities in their roots, stems, and leaves. In this final section, we consider how plant cells signal to one another and how they respond to light. Less is known about the receptors and intracellular signaling mechanisms involved in cell communication in plants than is known in animals, and we will concentrate mainly on how the receptors and intracellular signaling mechanisms differ from those used by animals.

1	Although plants and animals are both eukaryotes, they have evolved separately for more than a billion years. Their last common ancestor is thought to have been a unicellular eukaryote that had mitochondria but no chloroplasts; the plant lineage acquired chloroplasts after plants and animals diverged. The earliest fossils of multicellular animals and plants date from almost 600 million years ago. Thus, it seems that plants and animals evolved multicellularity independently, each starting from a different unicellular eukaryote, some time between 1.6 and 0.6 billion years ago (Figure 15–68).

1	If multicellularity evolved independently in plants and animals, the molecules and mechanisms used for cell communication will have evolved separately and would be expected to be different. There should be some degree of resemblance, however, because the genes in both plants and animals diverged from those contained by their last common unicellular ancestor. Thus, whereas both plants and animals use nitric oxide, cyclic GMP, Ca2+, and Rho family GTPases for signaling, there are no homologs of the nuclear receptor family, Ras, JAK, STAT, TGFβ, Figure 15–68 The proposed divergence of plant and animal lineages from a common unicellular eukaryotic ancestor. The plant lineage acquired chloroplasts after the two lineages diverged. Both lineages independently gave rise to multicellular organisms—plants and animals. (Paintings courtesy of John Innes Foundation.) unicellular ancestor of animals unicellular ancestor of plants CHLOROPLASTS chloroplast

1	Notch, Wnt, or Hedgehog encoded by the completely sequenced genome of Arabidopsis thaliana, the small flowering plant. Similarly, plants do not seem to use cyclic AMP for intracellular signaling. Nevertheless, the general strategies underlying signaling are frequently very similar in plants and animals. Both, for example, use enzyme-coupled cell-surface receptors, as we now discuss. Receptor Serine/Threonine Kinases Are the Largest Class of Cell-Surface Receptors in Plants

1	Receptor Serine/Threonine Kinases Are the Largest Class of Cell-Surface Receptors in Plants Most cell-surface receptors in plants are enzyme-coupled. However, whereas the largest class of enzyme-coupled receptors in animals is the receptor tyrosine kinase (RTK) class, this type of receptor is extremely rare in plants. Instead, plants rely largely on a great diversity of transmembrane receptor serine/threonine kinases, which have a typical serine/threonine kinase cytoplasmic domain and an extracellular ligand-binding domain. The most abundant types of these receptors have a tandem array of extracellular leucine-rich repeat structures and are therefore called leucine-rich repeat (LRR) receptor kinases.

1	There are about 175 LRR receptor kinases encoded by the Arabidopsis genome. These include a protein called Bri1, which forms part of a cell-surface steroid hormone receptor. Plants synthesize a class of steroids that are called brassinosteroids because they were originally identified in the mustard family Brassicaceae, which includes Arabidopsis. These signal molecules regulate the growth and differentiation of plants throughout their life cycle. Binding of a brassinosteroid to a Bri1 cell-surface receptor kinase initiates an intracellular signaling cascade that uses a GSK3 protein kinase and a protein phosphatase to regulate the phosphorylation and degradation of specific transcription regulatory proteins in the nucleus, and thereby specific gene transcription. Mutant plants that are deficient in the Bri1 receptor kinase are insensitive to brassinosteroids and are therefore dwarfs.

1	The LRR receptor kinases are only one of many classes of transmembrane receptor serine/threonine kinases in plants. There are at least six additional families, each with its own characteristic set of extracellular domains. The lectin receptor kinases, for example, have extracellular domains that bind carbohydrate signal molecules. The Arabidopsis genome encodes over 300 receptor serine/threonine kinases, which makes them the largest family of receptors known in plants. Many are involved in defense responses against pathogens. Ethylene Blocks the Degradation of Specific Transcription Regulatory Proteins in the Nucleus

1	Ethylene Blocks the Degradation of Specific Transcription Regulatory Proteins in the Nucleus Various plant growth regulators (also called plant hormones) help to coordinate plant development. They include ethylene, auxin, cytokinins, gibberellins, and abscisic acid, as well as brassinosteroids. Growth regulators are all small molecules made by most plant cells. They diffuse readily through cell walls and can either act locally or be transported to influence cells further away. Each growth regulator can have multiple effects. The specific effect depends on environmental conditions, the nutritional state of the plant, the responsiveness of the target cells, and which other growth regulators are acting.

1	Ethylene is an important example. This small gas molecule (Figure 15–69A) can influence plant development in various ways; it can, for example, promote fruit ripening, leaf abscission, and plant senescence. It also functions as a stress signal in response to wounding, infection, flooding, and so on. When the shoot of a germinating seedling, for instance, encounters an obstacle, ethylene promotes a complex response that allows the seedling to safely bypass the obstacle (Figure 15–69B and C). Plants have various ethylene receptors, which are located in the endoplasmic reticulum and are all structurally related. They are dimeric, multipass transmembrane proteins, with a copper-containing ethylene-binding domain and a domain that interacts with a cytoplasmic protein called CTR1, which is closely related Figure 15–69 The ethylene-mediated triple response that occurs when the growing shoot of a germinating seedling encounters an obstacle underground.

1	(A) The structure of ethylene. (B) In the absence of obstacles, the shoot grows upward and is long and thin. (C) If the shoot encounters an obstacle, such as a piece of gravel in the soil, the seedling responds to the encounter in three ways. First, it thickens its stem, which can then exert more force on the obstacle. Second, it shields the tip of the shoot (at top) by increasing the curvature of a specialized hook structure. Third, it reduces the shoot’s tendency to grow away from the direction of gravity, so as to avoid the obstacle. (Courtesy of Melanie Webb.) in sequence to the Raf MAP kinase kinase kinase discussed earlier (see Figure 15–49). Surprisingly, it is the empty receptors that are active and keep CTR1 active. By an unknown signaling mechanism, active CTR1 stimulates the ubiquitylation and degradation in proteasomes of a nuclear transcription regulator called EIN3, which is required for the transcription of ethylene-responsive genes. In this way, the empty but active

1	ubiquitylation and degradation in proteasomes of a nuclear transcription regulator called EIN3, which is required for the transcription of ethylene-responsive genes. In this way, the empty but active receptors keep ethylene-response genes off. Ethylene binding inactivates the receptors, altering their conformation so that they no longer activate CTR1. The EIN3 protein is no longer ubiquitylated and degraded and can now activate the transcription of the large number of ethylene-responsive genes (Figure 15–70).

1	Regulated Positioning of Auxin Transporters Patterns Plant Growth The plant hormone auxin, which is generally indole-3-acetic acid (Figure 15–71A), binds to receptor proteins in the nucleus. It helps plants grow toward light, grow upward rather than branch out, and grow their roots downward. It also regulates organ initiation and positioning and helps plants flower and bear fruit. Like ethylene (and like some of the animal signal molecules we have described in this chapter), auxin influences gene expression by controlling the degradation of transcription regulators. It works by stimulating the ubiquitylation and degradation of repressor proteins that block the transcription of auxin target genes in unstimulated cells (Figure 15–71B and C). Auxin is unique in the way that it is transported. Unlike animal hormones, which are usually secreted by a specific endocrine organ and transported to target

1	Auxin is unique in the way that it is transported. Unlike animal hormones, which are usually secreted by a specific endocrine organ and transported to target Figure 15–70 The ethylene signaling pathway. (A) In the absence of ethylene, the receptors and CTR1 are active, causing the ubiquitylation and destruction of EIN3, the transcription regulatory protein in the nucleus that is responsible for the transcription of ethylene-responsive genes. (B) The binding of ethylene inactivates the receptors and disrupts the activation of CTR1. The EIN3 protein is not degraded and can therefore activate the transcription of ethylene-responsive genes.

1	Figure 15–71 The auxin signaling pathway. (A) The structure of the auxin indole-3-acetic acid. (B) In the absence of auxin, a transcriptional repressor protein (called Aux/IAA) binds and suppresses a transcription regulatory protein (called auxin-response factor, ARF), which is required for the transcription of auxin-responsive genes. (C) The auxin receptor proteins are mainly located in the nucleus and form part of ubiquitin ligase complexes (not shown). When activated by auxin binding, the receptor–auxin complexes recruit the ubiquitin ligase ARF complexes, which ubiquitylate the Aux/IAA proteins, marking them for degradation in proteasomes. ARF is now free to activate the transcription of auxin-responsive genes. There are many ARFs, Aux/IAA proteins, and auxin receptors that work as illustrated.

1	cells via the circulatory system, auxin has its own transport system. Specific plasma-membrane-bound influx transporter proteins and efflux transporter proteins move auxin into and out of plant cells, respectively. The efflux transporters can be distributed asymmetrically in the plasma membrane to make the efflux of auxin directional. A row of cells with their auxin efflux transporters confined to the basal plasma membrane, for example, will transport auxin from the top of the plant to the bottom.

1	In some regions of the plant, the localization of the auxin transporters, and therefore the direction of auxin flow, is highly dynamic and regulated. A cell can rapidly redistribute transporters by controlling the traffic of vesicles containing them. The auxin efflux transporters, for example, normally recycle between intracellular vesicles and the plasma membrane. A cell can redistribute these transporters on its surface by inhibiting their endocytosis in one domain of the plasma membrane, causing the transporters to accumulate there. One example occurs in the root, where gravity influences the direction of growth. The auxin efflux transporters are normally distributed symmetrically in the cap cells of the root. Within minutes of a change in the direction of the gravity vector, however, the efflux transporters redistribute to one side of the cells, so that auxin is pumped out toward the side of the root pointing downward. Because auxin inhibits root-cell elongation, this redirection

1	the efflux transporters redistribute to one side of the cells, so that auxin is pumped out toward the side of the root pointing downward. Because auxin inhibits root-cell elongation, this redirection of auxin transport causes the root tip to reorient, so that it grows downward again (Figure 15–72).

1	Phytochromes Detect Red Light, and Cryptochromes Detect Blue Light Plant development is greatly influenced by environmental conditions. Unlike animals, plants cannot move when conditions become unfavorable; they have to adapt or they die. The most important environmental influence on plants is of root downward side of root

1	Figure 15–72 Auxin transport and root gravitropism. (A–C) Roots respond to a 90° change in the gravity vector and adjust their direction of growth so that they grow downward again. The cells that respond to gravity are in the center of the root cap, while it is the epidermal cells further back (on the lower side) that decrease their rate of elongation to restore downward growth. (D) The gravity-responsive cells in the root cap redistribute their auxin efflux transporters in response to the displacement of the root. This redirects the auxin flux mainly to the lower part of the displaced root, where it inhibits the elongation of the epidermal cells. The resulting asymmetrical distribution of auxin in the Arabidopsis root tip shown here is assessed indirectly, using an auxin-responsive reporter gene that encodes a protein fused to green fluorescent protein (GFP); the epidermal cells on the downward side of the root are green, whereas those on the upper side are not, reflecting the

1	reporter gene that encodes a protein fused to green fluorescent protein (GFP); the epidermal cells on the downward side of the root are green, whereas those on the upper side are not, reflecting the asymmetrical distribution of auxin. The distribution of auxin efflux transporters in the plasma membrane of cells in different regions of the root (shown as gray rectangles) is indicated in red, and the direction of auxin efflux is indicated by a green arrow. (The fluorescence photograph in D is from

1	T. Paciorek et al., Nature 435:1251–1256, 2005. With permission from Macmillan Publishers Ltd.) light, which is their energy source and has a major role throughout their entire life cycle—from germination, through seedling development, to flowering and senescence. Plants have thus evolved a large set of light-sensitive proteins to monitor the quantity, quality, direction, and duration of light. These are usually referred to as photoreceptors. However, because the term photoreceptor is also used for light-sensitive cells in the animal retina (see Figure 15–38), we shall use the term photoprotein instead.

1	All photoproteins sense light by means of a covalently attached light-absorbing chromophore, which changes its shape in response to light and then induces a change in the protein’s conformation. The best-known plant photoproteins are the phytochromes, which are present in all plants and in some algae but are absent in animals. These are dimeric, cytoplasmic serine/threonine kinases, which respond differentially and reversibly to red and far-red light: whereas red light usually activates the kinase activity of the phytochrome, far-red light inactivates it. When activated by red light, the phytochrome is thought to phosphorylate itself and then to phosphorylate one or more other proteins in the cell. In some light responses, the activated phytochrome translocates into the nucleus, where it activates transcription regulators to alter gene transcription (Figure 15–73). In other cases, the activated phytochrome activates a latent transcription regulator in the cytoplasm, which then

1	it activates transcription regulators to alter gene transcription (Figure 15–73). In other cases, the activated phytochrome activates a latent transcription regulator in the cytoplasm, which then translocates into the nucleus to regulate gene transcription. In still other cases, the photoprotein triggers signaling pathways in the cytosol that alter the cell’s behavior without involving the nucleus.

1	Plants sense blue light using photoproteins of two other sorts, phototropin and cryptochromes. Phototropin is associated with the plasma membrane and is partly responsible for phototropism, the tendency of plants to grow toward light. Phototropism occurs by directional cell elongation, which is stimulated by auxin, but the links between phototropin and auxin are unknown. Cryptochromes are flavoproteins that are sensitive to blue light. They are structurally related to blue-light-sensitive enzymes called photolyases, which are involved in the repair of ultraviolet-induced DNA damage in all organisms, except most mammals. Unlike phytochromes, cryptochromes are also found in animals, where they have an important role in circadian clocks (see Figure 15–66). Although cryptochromes are thought to have evolved from the photolyases, they do not have a role in DNA repair.

1	Plants and animals are thought to have evolved multicellularity and cell communication mechanisms independently, each starting from a different unicellular eukaryote, which in turn evolved from a common unicellular eukaryotic ancestor. Not surprisingly, therefore, the mechanisms used to signal between cells in animals and in plants have both similarities and differences. Whereas animals rely heavily on GPCRs and RTKs, plants rely mainly on enzyme-coupled receptors of the receptor serine/threonine kinase type, especially ones with extracellular leucinerich repeats. Various plant hormones, or growth regulators, including ethylene and auxin, help coordinate plant development. Ethylene acts through intracellular receptors to stop the degradation of specific nuclear transcription regulators, which can then activate the transcription of ethylene-responsive genes. The receptors for some other plant hormones, including auxin, also regulate the degradation of specific transcription regulators,

1	can then activate the transcription of ethylene-responsive genes. The receptors for some other plant hormones, including auxin, also regulate the degradation of specific transcription regulators, although the details vary. Auxin signaling is unusual in that it has its own highly regulated transport system, in which the dynamic positioning of plasma-membrane-bound auxin transporters controls the direction of auxin flow and thereby the direction of plant growth. Light has an important role in regulating plant development. These light responses are mediated by a variety of light-sensitive photoproteins, including phytochromes, which are responsive to red light, and cryptochromes and phototropin, which are sensitive to blue light.

1	Figure 15–73 One way in which phytochromes mediate a light response in plant cells. When activated by red light, the phytochrome, which is a dimeric protein kinase, phosphorylates itself and then moves into the nucleus, where it activates transcription regulatory proteins to stimulate the transcription of red-lightresponsive genes. How does a cell integrate the information received from its many different cell-surface receptors to make all-or-none decisions? Much of what we know about cell signaling comes from biochemical studies of isolated proteins in test tubes. What is the precise quantitative behavior of intracellular signaling networks in an intact cell, or in an intact animal, where countless other signals and cell components might influence signaling specificity and strength? How do intracellular signaling circuits generate specific and dynamic signaling patterns such as oscillations and waves, and how are these patterns sensed and interpreted by the cell?

1	How do intracellular signaling circuits generate specific and dynamic signaling patterns such as oscillations and waves, and how are these patterns sensed and interpreted by the cell? Scaffold proteins and activated receptor tyrosine kinases nucleate the assembly of large intracellular signaling complexes. What is the dynamic behavior of these complexes, and how does this behavior influence downstream signaling? Which statements are true? explain why or why not. 15–1 All second messengers are water-soluble and diffuse freely through the cytosol. 15–2 In the regulation of molecular switches, protein kinases and guanine nucleotide exchange factors (GEFs) always turn proteins on, whereas protein phosphatases and GTPase-activating proteins (GAPs) always turn proteins off. 15–3 Most intracellular signaling pathways provide numerous opportunities for amplifying the responses to extracellular signals.

1	15–3 Most intracellular signaling pathways provide numerous opportunities for amplifying the responses to extracellular signals. 15–4 Binding of extracellular ligands to receptor tyrosine kinases (RTKs) activates the intracellular catalytic domain by propagating a conformational change across the lipid bilayer through a single transmembrane α helix. 15–5 Protein tyrosine phosphatases display exquisite specificity for their substrates, unlike most serine/threonine protein phosphatases, which have rather broad specificity. 15–6 Even though plants and animals independently evolved multicellularity, they use virtually all the same signaling proteins and second messengers for cell–cell communication. Discuss the following problems. 15–7 Suppose that the circulating concentration of hormone is 10–10 M and the Kd for binding to its receptor is 10–8

1	Discuss the following problems. 15–7 Suppose that the circulating concentration of hormone is 10–10 M and the Kd for binding to its receptor is 10–8 M. What fraction of the receptors will have hormone bound? If a meaningful physiological response occurs when 50% of the receptors have bound a hormone molecule, how much will the concentration of hormone have to rise to elicit a response? The fraction of receptors (R) bound to hormone (H) to form a receptor–hormone complex (R–H) is [R–H]/ ([R] + [R–H]) = [R–H]/[R]TOT = [H]/([H] + Kd). 15–8 Cells communicate in ways that resemble human communication. Decide which of the following forms of human communication are analogous to autocrine, paracrine, endocrine, and synaptic signaling by cells. A. A telephone conversation b. Talking to people at a cocktail party C. D.

1	A. A telephone conversation b. Talking to people at a cocktail party C. D. Talking to yourself 15–9 Why do signaling responses that involve changes in proteins already present in the cell occur in milliseconds to seconds, whereas responses that require changes in gene expression require minutes to hours? 15–10 How is it that different cells can respond in different ways to exactly the same signaling molecule even when they have identical receptors? 15–11 Why do you suppose that phosphorylation/ dephosphorylation, as opposed to allosteric binding of small molecules, for example, has evolved to play such a prominent role in switching proteins on and off in signaling pathways?

1	15–12 Consider a signaling pathway that proceeds through three protein kinases that are sequentially activated by phosphorylation. In one case, the kinases are held in a signaling complex by a scaffolding protein; in the other, the kinases are freely diffusible (Figure Q15–1). Discuss the properties of these two types of organization in terms of signal amplification, speed, and potential for cross-talk between signaling pathways. Figure Q15–1 A kinase cascade organized by a scaffolding protein or composed of freely diffusing components (Problem 15–12). 15–13 Describe three ways in which a gradual increase in an extracellular signal can be sharpened by the target cell to produce an abrupt or nearly all-or-none response.

1	15–14 Activation (“maturation”) of frog oocytes is signaled through a MAP kinase signaling module. An increase in the hormone progesterone triggers the module by stimulating the translation of Mos mRNA, which is the frog’s MAP kinase kinase kinase (Figure Q15–2). Maturation is easy to score visually by the presence of a white spot in the middle of the brown surface of the oocyte (see Figure Q15–2). To determine the dose–response curve for pro-gesterone-induced activation of MAP kinase, you place 16 oocytes in each of six plastic dishes and add various concentrations of progesterone. After an overnight incubation, you crush the oocytes, prepare an extract, and determine the state of MAP kinase phosphorylation (hence, activation) by SDS polyacrylamide-gel electrophoresis (Figure Q15–3A). This analysis shows a graded response of MAP kinase to increasing concentrations of progesterone.

1	progesterone Figure Q15–2 Progesterone-induced MAP kinase activation, leading to oocyte Mos maturation (Problem 15–14). (Courtesy of Helfrid Hochegger.) (A) POOLED OOCYTES –+ of frog oocytes (Problem 15–14). pooled oocytes. (B) Phosphorylation of MAP kinase in individual oocytes. MAP kinase 0.001 0.01 0.1 1 10 immunoblotting using progesterone (µM) a MAP-kinase-specific antibody. The first two lanes in each gel contain nonphosphorylated, –+ 0.03µM progesterone and phosphorylated, active MAP kinase (+). (From J.E. Ferrell, Jr., and 0.1 µM progesterone E.M. Machleder, Science 280:895–898, 1998. 0.3 µM progesterone With permission from AAAS.)

1	E.M. Machleder, Science 280:895–898, 1998. 0.3 µM progesterone With permission from AAAS.) Before you crushed the oocytes, you noticed that not all oocytes in individual dishes had white spots. Had some oocytes undergone partial activation and not yet reached the white-spot stage? To answer this question, you repeat the experiment, but this time you analyze MAP kinase activation in individual oocytes. You are surprised to find that each oocyte has either a fully activated or a completely inactive MAP kinase (Figure Q15–3B). How can an all-ornone response in individual oocytes give rise to a graded response in the population? 15–15 Propose specific types of mutations in the gene for the regulatory subunit of cyclic-AMP-dependent protein kinase (PKA) that could lead to either a permanently active PKA or a permanently inactive PKA.

1	15–16 Phosphorylase kinase integrates signals from the cyclic-AMP-dependent and Ca2+-dependent signaling pathways that control glycogen breakdown in liver and muscle cells (Figure Q15–4). Phosphorylase kinase is composed of four subunits. One is the protein kinase that catalyzes the addition of phosphate to glycogen phosphorylase to activate it for glycogen breakdown. The other three subunits are regulatory proteins that control the activity of the Figure Q15–4 Integration of cyclic-AMP-dependent and Ca2+dependent signaling pathways by phosphorylase kinase in liver and muscle cells (Problem 15–16).

1	Figure Q15–4 Integration of cyclic-AMP-dependent and Ca2+dependent signaling pathways by phosphorylase kinase in liver and muscle cells (Problem 15–16). catalytic subunit. Two contain sites for phosphorylation by PKA, which is activated by cyclic AMP. The remaining subunit is calmodulin, which binds Ca2+ when the cytosolic Ca2+ concentration rises. The regulatory subunits control the equilibrium between the active and inactive conformations of the catalytic subunit, with each phosphate and Ca2+ nudging the equilibrium toward the active conformation. How does this arrangement allow phosphorylase kinase to serve its role as an integrator protein for the multiple pathways that stimulate glycogen breakdown?

1	15–17 The Wnt planar polarity signaling pathway normally ensures that each wing cell in Drosophila has a single hair. Overexpression of the Frizzled gene from a heat-shock promoter (hs-Fz) causes multiple hairs to grow from many cells (Figure Q15–5A). This phenotype is suppressed if hs-Fz is combined with a heterozygous deletion (DshΔ) of the Dishevelled gene (Figure Q15–5B). Do these results allow you to order the action of Frizzled and Dishevelled in the signaling pathway? If so, what is the order? Explain your reasoning. Figure Q15–5 Pattern of hair growth on wing cells in genetically different Drosophila (Problem 15–17). (From C.G. Winter et al., Cell 105:81–91, 2001. With permission from Elsevier.) Marks F, Klingmüller U & Müller-Decker K (2009) Cellular Signal Processing: An Introduction to the Molecular Mechanisms of Signal Transduction. New York: Garland Science. Lim W, Mayer B & Pawson T (2015) Cell Signaling: Principles and Mechanisms. New York: Garland Science.

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1	Stepanova AN & Alonso JM (2009) Ethylene signaling and response: where different regulatory modules meet. Curr. Opin. Plant Biol. 12, 548–555. The Cytoskeleton 16 For cells to function properly, they must organize themselves in space and interact mechanically with each other and with their environment. They have to be correctly shaped, physically robust, and properly structured internally. Many have to change their shape and move from place to place. All cells have to be able to rearrange their internal components as they grow, divide, and adapt to changing circumstances. These spatial and mechanical functions depend on a remarkable system of filaments called the cytoskeleton (Figure 16–1).

1	The cytoskeleton’s varied functions depend on the behavior of three families of protein filaments—actin filaments, microtubules, and intermediate filaments. Each type of filament has distinct mechanical properties, dynamics, and biological roles, but all share certain fundamental features. Just as we require our ligaments, bones, and muscles to work together, so all three cytoskeletal filament systems must normally function collectively to give a cell its strength, its shape, and its ability to move.

1	In this chapter, we describe the function and conservation of the three main filament systems. We explain the basic principles underlying filament assembly and disassembly, and how other proteins interact with the filaments to alter their dynamics, enabling the cell to establish and maintain internal order, to shape and remodel its surface, and to move organelles in a directed manner from one place to another. Finally, we discuss how the integration and regulation of the cytoskeleton allows a cell to move to new locations.

1	The three major cytoskeletal filaments are responsible for different aspects of the cell’s spatial organization and mechanical properties. Actin filaments determine the shape of the cell’s surface and are necessary for whole-cell locomotion; they also drive the pinching of one cell into two. Microtubules determine the positions of membrane-enclosed organelles, direct intracellular transport, and form the mitotic spindle that segregates chromosomes during cell division. Intermediate filaments provide mechanical strength. All of these cytoskeletal filaments interact with hundreds of accessory proteins that regulate and link the filaments to other cell components, as well as to each other. The accessory proteins are essential for the controlled assembly of the cytoskeletal filaments in particular locations, and they include the motor proteins, remarkable molecular machines that convert the energy of ATP hydrolysis into mechanical force that can either move organelles along the filaments

1	locations, and they include the motor proteins, remarkable molecular machines that convert the energy of ATP hydrolysis into mechanical force that can either move organelles along the filaments or move the filaments themselves.

1	In this section, we discuss the general features of the proteins that make up the filaments of the cytoskeleton. We focus on their ability to form intrinsically Figure 16–1 The cytoskeleton. (A) A cell in culture has been fixed and labeled to show its cytoplasmic arrays of microtubules (green) and actin filaments (red). (B) This dividing cell has been labeled to show its spindle microtubules (green) and surrounding cage of intermediate filaments (red). The DNA in both cells is labeled in blue. (A, courtesy of Albert Tousson; B, courtesy of Conly Rieder.) polarized and self-organized structures that are highly dynamic, allowing the cell to rapidly modify cytoskeletal structure and function under different conditions. Cytoskeletal Filaments Adapt to Form Dynamic or Stable Structures

1	Cytoskeletal Filaments Adapt to Form Dynamic or Stable Structures Cytoskeletal systems are dynamic and adaptable, organized more like ant trails than interstate highways. A single trail of ants may persist for many hours, extending from the ant nest to a delectable picnic site, but the individual ants within the trail are anything but static. If the ant scouts find a new and better source of food, or if the picnickers clean up and leave, the dynamic structure adapts with astonishing rapidity. In a similar way, large-scale cytoskeletal structures can change or persist, according to need, lasting for lengths of time ranging from less than a minute up to the cell’s lifetime. But the individual macromolecular components that make up these structures are in a constant state of flux. Thus, like the alteration of an ant trail, a structural rearrangement in a cell requires little extra energy when conditions change.

1	Regulation of the dynamic behavior and assembly of cytoskeletal filaments allows eukaryotic cells to build an enormous range of structures from the three basic filament systems. The micrographs in Panel 16–1 illustrate some of these structures. Actin filaments underlie the plasma membrane of animal cells, providing strength and shape to its thin lipid bilayer. They also form many types of cell-surface projections. Some of these are dynamic structures, such as the lamellipodia and filopodia that cells use to explore territory and move around. More stable arrays allow cells to brace themselves against an underlying substratum and enable muscle to contract. The regular bundles of stereocilia on the surface of hair cells in the inner ear contain stable bundles of actin filaments that tilt as rigid rods in response to sound, and similarly organized microvilli on the surface of intestinal epithelial cells vastly increase the apical cell-surface area to enhance nutrient absorption. In

1	as rigid rods in response to sound, and similarly organized microvilli on the surface of intestinal epithelial cells vastly increase the apical cell-surface area to enhance nutrient absorption. In plants, actin filaments drive the rapid streaming of cytoplasm inside cells.

1	Microtubules, which are frequently found in a cytoplasmic array that extends to the cell periphery, can quickly rearrange themselves to form a bipolar mitotic spindle during cell division. They can also form cilia, which function as motile whips or sensory devices on the surface of the cell, or tightly aligned bundles that serve as tracks for the transport of materials down long neuronal axons. In plant cells, organized arrays of microtubules help to direct the pattern of cell wall synthesis, and in many protozoans they form the framework upon which the entire cell is built. Intermediate filaments line the inner face of the nuclear envelope, forming a protective cage for the cell’s DNA; in the cytosol, they are twisted into strong cables that can hold epithelial cell sheets together or help nerve cells to extend long and robust axons, and they allow us to form tough appendages such as hair and fingernails.

1	An important and dramatic example of rapid reorganization of the cytoskeleton occurs during cell division, as shown in Figure 16–2 for a fibroblast growing in a tissue-culture dish. After the chromosomes have replicated, the interphase microtubule array that spreads throughout the cytoplasm is reconfigured into the bipolar mitotic spindle, which segregates the two copies of each chromosome into daughter nuclei. At the same time, the specialized actin structures that enable the fibroblast to crawl across the surface of the dish rearrange so that the cell stops moving and assumes a more spherical shape. Actin and its associated motor protein myosin then form a belt around the middle of the cell, the contractile ring, which constricts like a tiny muscle to pinch the cell in two. When division is complete, the cytoskeletons of the two daughter fibroblasts reassemble into their interphase structures to convert the two rounded-up daughter cells into smaller versions of the flattened,

1	is complete, the cytoskeletons of the two daughter fibroblasts reassemble into their interphase structures to convert the two rounded-up daughter cells into smaller versions of the flattened, crawling mother cell.

1	Many cells require rapid cytoskeletal rearrangements for their normal functioning during interphase as well. For example, the neutrophil, a type of white Actin flaments(also known as microflaments) are helical polymers of the protein actin. They are fexible structures with a diameter of 8 nm that organize into a variety of linear bundles, two-dimensional networks, and three-dimensional gels. Although actin flaments are dispersed throughout the cell, they are most highly concentrated in the cortex, just beneath the plasma membrane. (i) Single actin flament; (ii) microvilli; (iii) stress fbers (red) terminating in focal adhesions (green); (iv) striated muscle. Micrographs courtesy of R. Craig (i and iv); P.T. Matsudaira and D.R. Burgess (ii); K. Burridge (iii). Microtubulesare long, hollow cylinders made of the protein tubulin.

1	Micrographs courtesy of R. Craig (i and iv); P.T. Matsudaira and D.R. Burgess (ii); K. Burridge (iii). Microtubulesare long, hollow cylinders made of the protein tubulin. With an outer diameter of 25 nm, they are much more rigid than actin flaments. Microtubules are long and straight and frequently have one end attached to a microtubule-organizing center (MTOC) called a centrosome. (i) Single microtubule; (ii) cross section at the base of three (green) and organelles (red); (iv) ciliated protozoan. Micrographs courtesy of R. Wade (i); D.T. Woodrow and R.W. Linck (ii); D. Shima (iii); D. Burnette (iv).

1	Micrographs courtesy of R. Wade (i); D.T. Woodrow and R.W. Linck (ii); D. Shima (iii); D. Burnette (iv). Intermediate flaments are ropelike fbers with a diameter of about 10 nm; they are made of intermediate flament proteins, which constitute a large and heterogeneous family. One type of intermediate flament forms a meshwork called the nuclear lamina just beneath the inner nuclear membrane. Other types extend across the cytoplasm, giving cells mechanical strength. In an epithelial tissue, they span the cytoplasm from one cell–cell junction to another, thereby strengthening the entire epithelium. (i) Individual intermediate flaments; (ii) Intermediate flaments (blue) in neurons and (iii) epithelial cell; (iv) nuclear lamina. Micrographs courtesy of R. Quinlan (i); N. L. Kedersha (ii); M. Osborn (iii); U. Aebi (iv).

1	Micrographs courtesy of R. Quinlan (i); N. L. Kedersha (ii); M. Osborn (iii); U. Aebi (iv). blood cell, chases and engulfs bacterial and fungal cells that accidentally gain access to the normally sterile parts of the body, as through a cut in the skin. Like most crawling cells, neutrophils advance by extending a protrusive structure filled with newly polymerized actin filaments. When the elusive bacterial prey moves in a different direction, the neutrophil is poised to reorganize its polarized protrusive structures within seconds (Figure 16–3). The Cytoskeleton Determines Cellular Organization and Polarity

1	The Cytoskeleton Determines Cellular Organization and Polarity In cells that have achieved a stable, differentiated morphology—such as mature neurons or epithelial cells—the dynamic elements of the cytoskeleton must also provide stable, large-scale structures for cellular organization. On specialized epithelial cells that line organs such as the intestine and the lung, cytoskeletal-based cell-surface protrusions including microvilli and cilia are able to maintain a constant location, length, and diameter over the entire lifetime of the cell. For the actin bundles at the cores of microvilli on intestinal epithelial cells, this is only a few days. But the actin bundles at the cores of stereocilia on the hair cells of the inner ear must maintain their stable organization for the entire lifetime of the animal, since these cells do not turn over. Nonetheless, the individual actin filaments

1	Figure 16–2 Diagram of changes in cytoskeletal organization associated with cell division. The crawling fibroblast drawn here has a polarized, dynamic actin cytoskeleton (shown in red) that assembles lamellipodia and filopodia to push its leading edge toward the right. The polarization of the actin cytoskeleton is assisted by the microtubule cytoskeleton (green), consisting of long microtubules that emanate from a single microtubuleorganizing center located in front of the nucleus. When the cell divides, the polarized microtubule array rearranges to form a bipolar mitotic spindle, which is responsible for aligning and then segregating the duplicated chromosomes (brown). The actin filaments form a contractile ring at the center of the cell that pinches the cell in two after the chromosome segregation. After cell division is complete, the two daughter cells reorganize both the microtubule and actin cytoskeletons into smaller versions of those that were present in the mother cell,

1	segregation. After cell division is complete, the two daughter cells reorganize both the microtubule and actin cytoskeletons into smaller versions of those that were present in the mother cell, enabling them to crawl their separate ways.

1	Figure 16–3 A neutrophil in pursuit of bacteria. In this preparation of human blood, a clump of bacteria (white arrow) is about to be captured by a neutrophil. As the bacteria move, the neutrophil quickly reassembles the dense actin network at its leading edge (highlighted in red) to push toward the location of the bacteria (Movie 16.1). Rapid disassembly and reassembly of the actin cytoskeleton in this cell enables it to change its orientation and direction of movement within a few minutes. (From a video recorded by David Rogers.) remain strikingly dynamic and are continuously remodeled and replaced every 48 hours, even within stable cell-surface structures that persist for decades.

1	Besides forming stable, specialized cell-surface protrusions, the cytoskeleton is also responsible for large-scale cellular polarity, enabling cells to tell the difference between top and bottom, or front and back. The large-scale polarity information conveyed by cytoskeletal organization is often maintained over the lifetime of the cell. Polarized epithelial cells use organized arrays of microtubules, actin filaments, and intermediate filaments to maintain the critical differences between the apical surface and the basolateral surface. They also must maintain strong adhesive contacts with one another to enable this single layer of cells to serve as an effective physical barrier (Figure 16–4).

1	Cytoskeletal filaments can reach from one end of the cell to the other, spanning tens or even hundreds of micrometers. Yet the individual protein molecules that form the filaments are only a few nanometers in size. The cell builds the filaments by assembling large numbers of the small subunits, like building a skyscraper out of bricks. Because these subunits are small, they can diffuse rapidly in the cytosol, whereas the assembled filaments cannot. In this way, cells can undergo rapid structural reorganizations, disassembling filaments at one site and reassembling them at another site far away.

1	Actin filaments and microtubules are built from subunits that are compact and globular—actin subunits for actin filaments and tubulin subunits for microtubules—whereas intermediate filaments are made up of smaller subunits that are themselves elongated and fibrous. All three major types of cytoskeletal filaments form as helical assemblies of subunits (see Figure 3–22) that self-associate, using a combination of end-to-end and side-to-side protein contacts. Differences in the structures of the subunits and the strengths of the attractive forces between microvillus terminal web of actin Figure 16–4 Organization of the cytoskeleton in polarized epithelial cells.

1	All the components of the cytoskeleton cooperate to produce the characteristic shapes of specialized cells, including the epithelial cells that line the small intestine, diagrammed here. At the apical (upper) surface, facing the intestinal lumen, bundled actin filaments (red) form microvilli that increase the cell surface area available for absorbing nutrients from food. Below the microvilli, a circumferential band of actin filaments is connected to cell–cell adherens junctions that anchor the cells to each other. Intermediate filaments (blue) are anchored to other kinds of adhesive structures, including desmosomes and hemidesmosomes, that connect the epithelial cells into a sturdy sheet and attach them to the underlying extracellular matrix; these structures are discussed in Chapter 19. Microtubules (green) run vertically from the top of the cell to the bottom and provide a global coordinate system that enables the cell to direct newly synthesized components to their proper

1	19. Microtubules (green) run vertically from the top of the cell to the bottom and provide a global coordinate system that enables the cell to direct newly synthesized components to their proper locations.

1	them produce important differences in the stability and mechanical properties of each type of filament. Whereas covalent linkages between their subunits hold together the backbones of many biological polymers—including DNA, RNA, and proteins—it is weak noncovalent interactions that hold together the three types of cytoskeletal polymers. Consequently, their assembly and disassembly can occur rapidly, without covalent bonds being formed or broken.

1	The subunits of actin filaments and microtubules are asymmetrical and bind to one another head-to-tail so that they all point in one direction. This subunit polarity gives the filaments structural polarity along their length, and makes the two ends of each polymer behave differently. In addition, actin and tubulin subunits are both enzymes that catalyze the hydrolysis of a nucleoside triphosphate— ATP and GTP, respectively. As we discuss later, the energy derived from nucleotide hydrolysis enables the filaments to remodel rapidly. By controlling when and where actin and microtubules assemble, the cell harnesses the polar and dynamic properties of these filaments to generate force in a specific direction, to move the leading edge of a migrating cell forward, for example, or to pull chromosomes apart during cell division. In contrast, the subunits of intermediate filaments are symmetrical, and thus do not form polarized filaments with two different ends. Intermediate filament subunits

1	apart during cell division. In contrast, the subunits of intermediate filaments are symmetrical, and thus do not form polarized filaments with two different ends. Intermediate filament subunits also do not catalyze the hydrolysis of nucleotides. Nevertheless, intermediate filaments can be disassembled rapidly when required. In mitosis , for example, kinases phosphorylate the subunits, leading to their dissociation.

1	Cytoskeletal filaments in living cells are not built by simply stringing subunits together in single file. A thousand tubulin subunits lined up end-to-end, for example, would span the diameter of a small eukaryotic cell, but a filament formed in this way would lack the strength to avoid breakage by ambient thermal energy, unless each subunit in the filament was bound extremely tightly to its two neighbors. Such tight binding would limit the rate at which the filaments could disassemble, making the cytoskeleton a static and less useful structure. To provide both strength and adaptability, microtubules are built of 13 protofilaments—linear strings of subunits joined end-to-end—that associate with one another laterally to form a hollow cylinder. The addition or loss of a subunit at the end of one protofilament makes or breaks a small number of bonds. In contrast, loss of a subunit from the middle of the filament requires breaking many more bonds, while breaking it in two requires

1	end of one protofilament makes or breaks a small number of bonds. In contrast, loss of a subunit from the middle of the filament requires breaking many more bonds, while breaking it in two requires breaking bonds in multiple protofilaments all at the same time (Figure 16–5). The greater energy required to break multiple noncovalent bonds simultaneously allows microtubules to resist thermal breakage, while allowing rapid subunit addition and loss at the filament ends. Helical actin filaments are much thinner and therefore require much less energy to break. However, multiple actin filaments are often bundled together inside cells, providing mechanical strength, while allowing dynamic behavior of filament ends.

1	As with other specific protein–protein interactions, many hydrophobic interactions and noncovalent bonds hold the subunits in a cytoskeletal filament together (see Figure 3–4). The locations and types of subunit–subunit contacts differ for the different filaments. Intermediate filaments, for example, assemble by forming strong lateral contacts between α-helical coiled-coils, which extend over most of the length of each elongated fibrous subunit. Because the individual subunits are staggered in the filament, intermediate filaments form strong, ropelike structures that tolerate stretching and bending to a greater extent than do either actin filaments or microtubules (Figure 16–6).

1	The cell regulates the length and stability of its cytoskeletal filaments, as well as their number and geometry. It does so largely by regulating their attachments to one another and to other components of the cell, so that the filaments can form a wide variety of higher-order structures. Direct covalent modification of the filament subunits regulates some filament properties, but most of the regulation is performed by hundreds of accessory proteins that determine the spatial SINGLE PROTOFILAMENT: THERMALLY UNSTABLE

1	MULTIPLE PROTOFILAMENTS: THERMALLY STABLE distribution and the dynamic behavior of the filaments, converting information received through signaling pathways into cytoskeletal action. These accessory proteins bind to the filaments or their subunits to determine the sites of assembly of new filaments, to regulate the partitioning of polymer proteins between filament and subunit forms, to change the kinetics of filament assembly and disassembly, to harness energy to generate force, and to link filaments to one another or to other cell structures such as organelles and the plasma membrane. In these processes, the accessory proteins bring cytoskeletal structure under the control of extracellular and intracellular signals, including those that trigger the dramatic transformations of the cytoskeleton that occur during each cell cycle. Acting together, the accessory proteins enable a eukaryotic cell to maintain a highly organized but flexible internal structure and, in many cases, to move.

1	Figure 16–5 The thermal stability of cytoskeletal filaments with dynamic ends. A protofilament consisting of a single strand of subunits is thermally unstable, since breakage of a single bond between subunits is sufficient to break the filament. In contrast, formation of a cytoskeletal filament from more than one protofilament allows the ends to be dynamic, while enabling the filaments themselves to be resistant to thermal breakage. In a microtubule, for example, removing a single subunit dimer from the end of the filament requires breaking noncovalent bonds with a maximum of three other subunits, whereas fracturing the filament in the middle requires breaking noncovalent bonds in all thirteen protofilaments.

1	Figure 16–6 Flexibility and stretch in an intermediate filament. Intermediate filaments are formed from elongated fibrous subunits with strong lateral contacts, resulting in resistance to stretching forces. When a tiny mechanical probe is dragged across an intermediate filament, the filament is stretched over three times its length before it breaks, as illustrated by the fluorescently labeled filaments in the photomicrographs. This technique is termed atomic force microscopy (see Figure 9–33). (Adapted from L. Kreplak et al., J. Mol. Biol. 354:569–577, 2005. With permission from Elsevier.)

1	Among the most fascinating proteins that associate with the cytoskeleton are the motor proteins. These proteins bind to a polarized cytoskeletal filament and use the energy derived from repeated cycles of ATP hydrolysis to move along it. Dozens of different motor proteins coexist in every eukaryotic cell. They differ in the type of filament they bind to (either actin or microtubules), the direction in which they move along the filament, and the “cargo” they carry. Many motor proteins carry membrane-enclosed organelles—such as mitochondria, Golgi stacks, or secretory vesicles—to their appropriate locations in the cell. Other motor proteins cause cytoskeletal filaments to exert tension or to slide against each other, generating the force that drives such phenomena as muscle contraction, ciliary beating, and cell division.

1	Cytoskeletal motor proteins that move unidirectionally along an oriented polymer track are reminiscent of some other proteins and protein complexes discussed elsewhere in this book, such as DNA and RNA polymerases, helicases, and ribosomes. All of these proteins have the ability to use chemical energy to propel themselves along a linear track, with the direction of sliding dependent on the structural polarity of the track. All of them generate motion by coupling nucleoside triphosphate hydrolysis to a large-scale conformational change (see Figure 3–75). Bacterial Cell Organization and Division Depend on Homologs of Eukaryotic Cytoskeletal Proteins

1	Bacterial Cell Organization and Division Depend on Homologs of Eukaryotic Cytoskeletal Proteins While eukaryotic cells are typically large and morphologically complex, bacterial cells are usually only a few micrometers long and assume simple shapes such as spheres or rods. Bacteria also lack elaborate networks of intracellular membrane-enclosed organelles. Historically, biologists assumed that a cytoskeleton was not necessary in such simple cells. We now know, however, that bacteria contain homologs of all three of the eukaryotic cytoskeletal filaments. Furthermore, bacterial actins and tubulins are more diverse than their eukaryotic versions, both in the types of assemblies they form and in the functions they carry out.

1	Nearly all bacteria and many archaea contain a homolog of tubulin called FtsZ, which can polymerize into filaments and assemble into a ring (called the Z-ring) at the site where the septum forms during cell division (Figure 16–7). Although the Z-ring persists for many minutes, the individual filaments within it are highly dynamic, with an average filament half-life of about thirty seconds. As the bacte rium divides, the Z-ring becomes smaller until it has completely disassembled. FtsZ filaments in the Z-ring are thought to generate a bending force that drives the membrane invagination necessary to complete cell division. The Z-ring may also serve as a site for localization of enzymes required for building the septum between the two daughter cells.

1	Many bacteria also contain homologs of actin. Two of these, MreB and Mbl, are found primarily in rod-shaped or spiral-shaped cells where they assemble to form dynamic patches that move circumferentially along the length of the cell (Figure 16–8A). These proteins contribute to cell shape by serving as a scaffold to direct the synthesis of the peptidoglycan cell wall, in much the same way that microtubules help organize the synthesis of the cellulose cell wall in higher

1	Figure 16–7 The bacterial FtsZ protein, a tubulin homolog in prokaryotes. (A) A band of FtsZ protein forms a ring in a dividing bacterial cell. This ring has been labeled by fusing the FtsZ protein to green fluorescent protein (GFP), which allows it to be observed in living E. coli cells with a fluorescence microscope. (B) FtsZ filaments and circles, formed in vitro, as visualized using electron microscopy. (C) Dividing chloroplasts (red) from a red alga also cleave using a protein ring made from FtsZ (yellow). (A, from X. Ma, D.W. Ehrhardt and W. Margolin, Proc. Natl Acad. Sci. USA 93:12998–13003, 1996; B, from H.P. Erickson et al., Proc. Natl Acad. Sci. USA 93:519–523, 1996. Both with permission from National Academy of Sciences; C, from S. Miyagishima et al., Plant Cell 13:2257–2268, 2001, with permission from (C)American Society of Plant Biologists.) 1 µm

1	S. Miyagishima et al., Plant Cell 13:2257–2268, 2001, with permission from (C)American Society of Plant Biologists.) 1 µm Figure 16–8 Actin homologs in bacteria determine cell shape. (A) The MreB protein forms abundant patches made up of many short, interwoven linear or helical filaments that are seen to move circumferentially along the length of the bacterium and are associated with sites of cell wall synthesis. (B) The common soil bacterium Bacillus subtilis normally forms cells with a regular rodlike shape when viewed by scanning electron microscopy (left). In contrast, B. subtilis cells lacking the actin homolog MreB or Mbl grow in distorted or twisted shapes and eventually die (center and right). (A, from P. Vats and L. Rothfield, Proc. Natl Acad. Sci. USA 104:17795–17800, 2007. With permission from National Academy of Sciences; B, from A. Chastanet and

1	R. Carballido-Lopez, Front. Biosci. 4S:1582–1606, 2012. With permission Frontiers in Bioscience.) plant cells (see Figure 19–65). As with FtsZ, MreB and Mbl filaments are highly dynamic, with half-lives of a few minutes, and nucleotide hydrolysis accompanies the polymerization process. Mutations disrupting MreB or Mbl expression cause extreme abnormalities in cell shape and defects in chromosome segregation (Figure 16–8B).

1	Relatives of MreB and Mbl have more specialized roles. A particularly intriguing bacterial actin homolog is ParM, which is encoded by a gene on certain bacterial plasmids that also carry genes responsible for antibiotic resistance and cause the spread of multidrug resistance in epidemics. Bacterial plasmids typically encode all the gene products that are necessary for their own segregation, presumably as a strategy to ensure their inheritance and propagation in bacterial hosts following plasmid replication. ParM assembles into filaments that associate at each end with a copy of the plasmid, and growth of the ParM filament pushes the replicated plasmid copies apart (Figure 16–9). This spindle-like structure apparently arises from the selective stabilization of filaments that bind to specialized proteins recruited to the origins of replication on the plasmids. A distant relative of both tubulin and FtsZ, called TubZ, has a similar function in other bacterial species.

1	Thus, self-association of nucleotide-binding proteins into dynamic filaments is used in all cells, and the actin and tubulin families are very ancient, predating the split between the eukaryotic and bacterial kingdoms. At least one bacterial species, Caulobacter crescentus, appears to harbor a protein with significant structural similarity to the third major class of cytoskeletal filaments found in animal cells, the intermediate filaments. A protein called crescentin forms a filamentous structure that influences the unusual crescent shape of this species; when the gene encoding crescentin is deleted, the Caulobacter cells grow as straight rods (Figure 16–10). ParM plasmid origin of ParM ParR monomers replication flaments proteins Figure 16–9 Role of the actin homolog ParM in plasmid segregation in bacteria.

1	Figure 16–9 Role of the actin homolog ParM in plasmid segregation in bacteria. (A) Some bacterial drug-resistance plasmids (orange) encode an actin homolog, ParM, that will spontaneously nucleate to form small, dynamic filaments (green) throughout the bacterial cytoplasm. A second plasmid-encoded protein called ParR (blue) binds to specific DNA sequences in the plasmid and also stabilizes the dynamic ends of the ParM filaments. When the plasmid duplicates, both ends of the ParM filaments become stabilized, and the growing ParM filaments push the duplicated plasmids to opposite ends of the cell. (B) In these bacterial cells harboring a drug-resistance plasmid, the plasmids are labeled in red and the ParM protein in green. Left, a short ParM filament bundle connects the two daughter plasmids shortly after their duplication. Right, the fully assembled ParM filament has pushed the duplicated plasmids to the cell poles. (A, adapted from E.C. Garner,

1	C.S. Campbell and R.D. Mullins, Science 306:1021–1025, 2004; B, from J. Møller-Jensen et al., Mol. Cell 12:1477–1487, 2003. With permission from Elsevier.)

1	The cytoplasm of eukaryotic cells is spatially organized by a network of protein filaments known as the cytoskeleton. This network contains three principal types of filaments: actin filaments, microtubules, and intermediate filaments. All three types of filaments form as helical assemblies of subunits that self-associate using a combination of end-to-end and side-to-side protein contacts. Differences in the structure of the subunits and the manner of their self-assembly give the filaments different mechanical properties. Subunit assembly and disassembly constantly remodel all three types of cytoskeletal filaments. Actin and tubulin (the subunits of actin filaments and microtubules, respectively) bind and hydrolyze nucleoside triphosphates (ATP and GTP, respectively), and assemble head-to-tail to generate polarized filaments capable of generating force. In living cells, accessory proteins modulate the dynamics and organization of cytoskeletal filaments, resulting in complex events such

1	to generate polarized filaments capable of generating force. In living cells, accessory proteins modulate the dynamics and organization of cytoskeletal filaments, resulting in complex events such as cell division or migration, and generating elaborate cellular architecture to form polarized tissues such as epithelia. Bacterial cells also contain homologs of actin, tubulin, and intermediate filaments that form dynamic structures that help control cell shape and division.

1	The actin cytoskeleton performs a wide range of functions in diverse cell types. Each actin subunit, sometimes called globular or G-actin, is a 375-amino-acid polypeptide carrying a tightly associated molecule of ATP or ADP (Figure 16–11A). Actin is extraordinarily well conserved among eukaryotes. The amino acid sequences of actins from different eukaryotic species are usually about 90% identical. Small variations in actin amino acid sequence can cause significant functional differences: In vertebrates, for example, there are three isoforms of actin, termed α, β, and γ, that differ slightly in their amino acid sequences and have distinct functions. α-Actin is expressed only in muscle cells, while βand γ-actins are found together in almost all non-muscle cells. Actin Subunits Assemble Head-to-Tail to Create Flexible, Polar Filaments

1	Actin Subunits Assemble Head-to-Tail to Create Flexible, Polar Filaments Actin subunits assemble head-to-tail to form a tight, right-handed helix, forming a structure about 8 nm wide called filamentous or F-actin (Figure 16–11B and C). Because the asymmetrical actin subunits of a filament all point in the same direction, filaments are polar and have structurally different ends: a slower-growing minus end and a faster-growing plus end. The minus end is also referred to as the “pointed end” and the plus end as the “barbed end,” because of the “arrowhead” appearance of the complex formed between actin filaments and the motor protein myosin (Figure 16–12). Within the filament, the subunits are positioned with their nucleotide-binding cleft directed toward the minus end.

1	Individual actin filaments are quite flexible. The stiffness of a filament can be characterized by its persistence length, the minimum filament length at which random thermal fluctuations are likely to cause it to bend. The persistence length of an actin filament is only a few tens of micrometers. In a living cell, however, Figure 16–10 Caulobacter and crescentin. The sickle-shaped bacterium Caulobacter crescentus expresses a protein, crescentin, with a series of coiled-coil domains similar in size and organization to the domains of eukaryotic intermediate filaments. (A) The crescentin protein forms a fiber (labeled in red) that runs down the inner side of the curving bacterial cell wall. (B) When the gene is disrupted, the bacteria grow as straight rods (bottom). (From N. Ausmees, J.R. Kuhn and

1	C. Jacobs-Wagner, Cell 115:705–713, 2003. With permission from Elsevier.) accessory proteins cross-link and bundle the filaments together, making large-Figure 16–11 The structures of an actin monomer and actin filament. (A) The scale actin structures that are much more rigid than an individual actin filament. ATP or ADP) bound in a deep cleft in the Nucleation Is the Rate-Limiting Step in the Formation of Actin center of the molecule. (B) Arrangement of two protofilaments, held together by lateral contacts, which wind around each

1	The regulation of actin filament formation is an important mechanism by which other as two parallel strands of a helix, cells control their shape and movement. Small oligomers of actin subunits can with a twist repeating every 37 nm. All the assemble spontaneously, but they are unstable and disassemble readily because subunits within the filament have the same each monomer is bound to only one or two other monomers. For a new actin fila-orientation. (C) Electron micrograph of ment to form, subunits must assemble into an initial aggregate, or nucleus, that is negatively stained actin filament. (C, courtesy of Roger Craig.) stabilized by multiple subunit–subunit contacts and can then elongate rapidly by addition of more subunits. This process is called filament nucleation.

1	(C, courtesy of Roger Craig.) stabilized by multiple subunit–subunit contacts and can then elongate rapidly by addition of more subunits. This process is called filament nucleation. Many features of actin nucleation and polymerization have been studied with purified actin in a test tube (Figure 16–13). The instability of smaller actin aggregates creates a kinetic barrier to nucleation. When polymerization is initiated, this barrier results in a lag phase during which no filaments are observed. During this lag phase, however, a few of the small, unstable aggregates succeed in making the transition to a more stable form that resembles an actin filament. This leads to a 0.5 µm

1	Figure 16–12 Structural polarity of the actin filament. (A) This electron micrograph shows an actin filament polymerized from a short actin filament seed that was decorated with myosin motor domains, resulting in an arrowhead pattern. The filament has grown much faster at the barbed (plus) end than at the pointed (minus) end. (B) Enlarged image and model showing the arrowhead pattern. (A, courtesy of Tom Pollard; B, adapted from M. Whittaker, B.O. Carragher and K.A. Milligan, Ultramicro. 54:245–260, 1995.)

1	Figure 16–13 The time course of actin polymerization in a test tube. (A) Polymerization of pure actin subunits into filaments occurs after a lag phase. (B) Polymerization occurs more rapidly in the presence of preformed fragments of actin filaments, which act as nuclei for filament growth. As indicated, the % free subunits after polymerization reflects the critical concentration (Cc), at which there is no net change in polymer. Actin polymerization is often studied by observing the change in the light emission from a fluorescent probe, called pyrene, that has been covalently attached to the actin. Pyrene-actin fluoresces more brightly when it is incorporated into actin filaments.

1	phase of rapid filament elongation during which subunits are added quickly to the ends of the nucleated filaments (Figure 16–13A). Finally, as the concentration of actin monomers declines, the system approaches a steady state at which the rate of addition of new subunits to the filament ends exactly balances the rate of subunit dissociation. The concentration of free subunits left in solution at this point is called the critical concentration, Cc. As explained in Panel 16–2, the value of the critical concentration is equal to the rate constant for subunit loss divided by the rate constant for subunit addition—that is, Cc = koff/kon, which is equal to the dissociation constant, Kd, and the inverse of the equilibrium constant, K (see Figure 3–44). In a test tube, the Cc for actin polymerization—that is, the free actin monomer concentration at which the fraction of actin in the polymer stops increasing—is about 0.2 μM. Inside the cell, the concentration of unpolymerized actin is much

1	is, the free actin monomer concentration at which the fraction of actin in the polymer stops increasing—is about 0.2 μM. Inside the cell, the concentration of unpolymerized actin is much higher than this, and the cell has evolved mechanisms to prevent most of its monomeric actin from assembling into filaments, as we discuss later.

1	The lag phase in filament growth is eliminated if preexisting seeds (such as fragments of actin filaments that have been chemically cross-linked) are added to the solution at the beginning of the polymerization reaction (Figure 16–13B). The cell takes great advantage of this nucleation requirement: it uses special proteins to catalyze filament nucleation at specific sites, thereby determining the location at which new actin filaments are assembled.

1	Due to the uniform orientation of asymmetric actin subunits in the filament, the structures at its two ends are different. This orientation makes the two ends of each polymer different in ways that have a profound effect on filament growth rates. The kinetic rate constants for actin subunit association and dissociation— kon and koff, respectively—are much greater at the plus end than the minus end. This can be seen when an excess of purified actin monomers is allowed to assemble onto polarity-marked filaments—the plus end of the filament elongates up to ten times faster (see Figure 16–12). If filaments are rapidly diluted so that the free subunit concentration drops below the critical concentration, the plus end also depolymerizes faster.

1	It is important to note, however, that the two ends of an actin filament have the same net affinity for actin subunits, if all of the subunits are in the same nucleotide state. Addition of a subunit to either end of a filament of n subunits results in a filament of n + 1 subunits. Thus, the free-energy difference, and therefore the equilibrium constant (and the critical concentration), must be the same for addition of subunits at either end of the polymer. In this case, the ratio of the rate constants, koff/kon, must be identical at the two ends, even though the absolute values of these rate constants are very different at each end (see Panel 16–2).

1	The cell takes advantage of actin filament dynamics and polarity to do mechanical work. Filament elongation proceeds spontaneously when the free-energy change (∆G) for addition of the soluble subunit is less than zero. This is the case when the concentration of subunits in solution exceeds the critical concentration. A cell can couple an energetically unfavorable process to this spontaneous process; thus, the cell can use free energy released during spontaneous filament polymerization to move an attached load. For example, by orienting the fast-growing plus ends of actin filaments toward its leading edge, a motile cell can push its plasma membrane forward, as we discuss later. ATP Hydrolysis Within Actin Filaments Leads to Treadmilling at Steady State

1	ATP Hydrolysis Within Actin Filaments Leads to Treadmilling at Steady State Thus far in our discussion of actin filament dynamics, we have ignored the critical fact that actin can catalyze the hydrolysis of the nucleoside triphosphate ATP. For free actin subunits, this hydrolysis proceeds very slowly; however, it is accelerated when the subunits are incorporated into filaments. Shortly after ATP hydrolysis occurs, the free phosphate group is released from each subunit, but the ADP remains trapped in the filament structure. Thus, two different types of filament structures can exist, one with the “T form” of the nucleotide bound (ATP), and one with the “D form” bound (ADP).

1	When the nucleotide is hydrolyzed, much of the free energy released by cleavage of the phosphate–phosphate bond is stored in the polymer. This makes the free-energy change for dissociation of a subunit from the D-form polymer more negative than the free-energy change for dissociation of a subunit from the T-form polymer. Consequently, the ratio of koff/kon for the D-form polymer, which is numerically equal to its critical concentration [Cc(D)], is larger than the corresponding ratio for the T-form polymer. Thus, Cc(D) is greater than Cc(T). At certain concentrations of free subunits, D-form polymers will therefore shrink while T-form polymers grow.

1	In living cells, most soluble actin subunits are in the T form, as the free concentration of ATP is about tenfold higher than that of ADP. However, the longer the time that subunits have been in the actin filament, the more likely they are to have hydrolyzed their ATP. Whether the subunit at each end of a filament is in the T or the D form depends on the rate of this hydrolysis compared with the rate of subunit addition. If the concentration of actin monomers is greater than the critical concentration for both the T-form and D-form polymer, then subunits will add to the polymer at both ends before the nucleotides in the previously added subunits are hydrolyzed; as a result, the tips of the actin filament will remain in the T form. On the other hand, if the subunit concentration is less than the critical concentrations for both the T-form and D-form polymer, then hydrolysis may occur before the next subunit is added and both ends of the filament will be in the D form and will shrink.

1	the critical concentrations for both the T-form and D-form polymer, then hydrolysis may occur before the next subunit is added and both ends of the filament will be in the D form and will shrink. At intermediate concentrations of actin subunits, it is possible for the rate of subunit addition to be faster than nucleotide hydrolysis at the plus end, but slower than nucleotide hydrolysis at the minus end. In this case, the plus end of the filament remains in the T conformation, while the minus end adopts the D conformation. The filament then undergoes a net addition of subunits at the plus end, while simultaneously losing subunits from the minus end. This leads to the remarkable property of filament treadmilling (Figure 16–14; see Panel 16–2).

1	At a particular intermediate subunit concentration, the filament growth at the plus end exactly balances the filament shrinkage at the minus end. Under these conditions, the subunits cycle rapidly between the free and filamentous states,

1	ON RATES AND OFF RATES A linear polymer of protein molecules, such as an actin flament or a microtubule, assembles (polymerizes) and disassembles (depolymerizes) by the addition and removal of subunits at the ends of the polymer. The rate of addition of these subunits (called monomers) is given by the rate constant kon, which has units of M–1 sec–1. The rate of loss is given by koff (units of sec–1). subunit + polymer (with n subunits) polymer (with n +1 subunits) kon koff NUCLEATION A helical polymer is stabilized by multiple contacts between adjacent subunits. In the case of actin, two actin molecules bind relatively weakly to each other, but addition of a third actin monomer to form a trimer makes the entire group more stable. Further monomer addition can take place onto this trimer, which therefore acts as a nucleus for polymerization. For tubulin, the nucleus is larger and has a more complicated structure (possibly a ring of 13 or more tubulin molecules)—but the principle is the

1	therefore acts as a nucleus for polymerization. For tubulin, the nucleus is larger and has a more complicated structure (possibly a ring of 13 or more tubulin molecules)—but the principle is the same. The assembly of a nucleus is relatively slow, which explains the lag phase seen during polymerization. The lag phase can be reduced or abolished entirely by adding premade nuclei, such as fragments of already polymerized microtubules or actin flaments. THE CRITICAL CONCENTRATION The number of monomers that add to the polymer (actin flament or microtubule) per second will be proportional to the concentration of the free subunit (konC), but the subunits will leave the polymer end at a constant rate (koff) that does not depend on C. As the polymer grows, subunits are used up, and C is observed to drop until it reaches a constant value, called the critical concentration (Cc). At this concentration, the rate of subunit addition equals the rate of subunit loss. At this equilibrium, kon C =

1	to drop until it reaches a constant value, called the critical concentration (Cc). At this concentration, the rate of subunit addition equals the rate of subunit loss. At this equilibrium, kon C = koff so that Cc = (where Kd is the dissociation constant; see Figure 3–44). koff kon = TIME COURSE OF POLYMERIZATION The assembly of a protein into a long helical polymer such as a cytoskeletal flament or a bacterial fagellum typically shows the following time course: The lag phase corresponds to time taken for nucleation. The growth phase occurs as monomers add to the exposed ends of the growing flament, causing flament elongation. The equilibrium phase, or steady state, is reached when the growth of the polymer due to monomer addition precisely balances the shrinkage of the polymer due to disassembly back to monomers. time amount of polymerLAG PHASE GROWTH PHASE EQUILIBRIUM PHASE PLUS AND MINUS ENDS The two ends of an actin flament or microtubule polymerize at different rates. The

1	to disassembly back to monomers. time amount of polymerLAG PHASE GROWTH PHASE EQUILIBRIUM PHASE PLUS AND MINUS ENDS The two ends of an actin flament or microtubule polymerize at different rates. The fast-growing end is called the plus end, whereas the slow-growing end is called the minus end. The difference in the rates of growth at the two ends is made possible by changes in the conformation of each subunit as it enters the polymer. free subunit subunit in polymer SLOW FAST minus end plus end This conformational change affects the rates at which subunits add to the two ends. Even though kon and koff will have different values for the plus and minus ends of the polymer, their ratio koff/kon—and hence Cc—must be the same at both ends for a simple polymerization reaction (no ATP or GTP hydrolysis). This is because exactly the same subunit interactions are broken when a subunit is lost at either end, and the fnal state of the subunit after dissociation is identical. Therefore, the ˜G for

1	This is because exactly the same subunit interactions are broken when a subunit is lost at either end, and the fnal state of the subunit after dissociation is identical. Therefore, the ˜G for subunit loss, which determines the equilibrium constant for its association with the end, is identical at both ends: if the plus end grows four times faster than the minus end, it must also shrink four times faster. Thus, for C > Cc, both ends grow; for C < Cc, both ends shrink. The nucleoside triphosphate hydrolysis that accompanies actin and tubulin polymerization removes this constraint. Kd PANEL 16–2: The Polymerization of Actin and Tubulin 902

1	Each actin molecule carries a tightly bound ATP molecule that is hydrolyzed to a tightly bound ADP molecule soon after its assembly into the polymer. Similarly, each tubulin molecule carries a tightly bound GTP that is converted to a tightly bound GDP molecule soon after the molecule assembles into the polymer. Hydrolysis of the bound nucleotide reduces the binding affnity of the subunit for neighboring subunits and makes it more likely to dissociate from each end of the flament (see Figure 16–44 for a possible mechanism). It is usually the form that adds to the flament and the form that leaves. Considering events at the plus end only: As before, the polymer will grow until C = Cc. For illustrative purposes, we can ignore kD and kToff since they are usually very small, so that polymer growth ceases when

1	As before, the polymer will grow until C = Cc. For illustrative purposes, we can ignore kD and kToff since they are usually very small, so that polymer growth ceases when This is a steady state and not a true equilibrium, because the ATP or GTP that is hydrolyzed must be replenished by a nucleotide exchange reaction of the free subunit ( ). One consequence of the nucleotide hydrolysis that accompanies polymer formation is to change the critical concentration at the two ends of the polymer. Since kDoff and kT refer to different reactions, their ratio on kDoff/kTon need not be the same at both ends of the polymer, so that:

1	Thus, if both ends of a polymer are exposed, polymerization proceeds until the concentration of free monomer reaches a value that is above Cc for the plus end but below Cc for the minus end. At this steady state, subunits undergo a net assembly at the plus end and a net disassembly at the minus end at an identical rate. The polymer maintains a constant length, even though there is a net fux of subunits through the polymer, known as treadmilling. Microtubules depolymerize about 100 times faster from an end containing GDP-tubulin than from one containing GTP-tubulin. A GTP cap favors growth, but if it is lost, then depolymerization ensues. The rate of addition of subunits to a can be faster than the rate at which their bound nucleotide is hydrolyzed. Under such conditions, the end has a “cap” of subunits containing the nucleoside flament or a GTP cap on a microtubule.

1	DYNAMIC INSTABILITY and TREADMILLING are two behaviors observed in cytoskeletal polymers. Both are associated with nucleoside triphosphate hydrolysis. Dynamic instability is believed to predominate in microtubules, whereas treadmilling may predominate in actin flaments. Individual microtubules can therefore alternate between a period of slow growth and a period of rapid disassembly, a phenomenon called dynamic instability. Figure 16–14 Treadmilling of an actin soluble subunits are in T form ( filament, made possible by the ATP hydrolysis that follows subunit addition.

1	Figure 16–14 Treadmilling of an actin soluble subunits are in T form ( filament, made possible by the ATP hydrolysis that follows subunit addition. polymers are a mixture of T form ( (A) Explanation for the different critical concentrations (Cc) at the plus and minus at D minus a growing filament, and then undergo ends. Subunits with bound ATP (T-form nucleotide hydrolysis within the filament. As minus-end addition is slow— plus-end addition is fast— while the total length of the filament remains unchanged. This “steady-state tread-milling” requires a constant consumption of energy in the form of ATP hydrolysis. The Functions of Actin Filaments Are Inhibited by Both Polymer-stabilizing and Polymer-destabilizing Chemicals

1	The Functions of Actin Filaments Are Inhibited by Both Polymer-stabilizing and Polymer-destabilizing Chemicals Chemical compounds that stabilize or destabilize actin filaments are important tools in studies of the filaments’ dynamic behavior and function in cells. The cytochalasins are fungal products that prevent actin polymerization by binding to the plus end of actin filaments. Latrunculin prevents actin polymerization by binding to actin subunits. The phalloidins are toxins isolated from the Amanita mushroom that bind tightly all along the side of actin filaments and stabilize them against depolymerization. All of these compounds cause dramatic changes in the actin cytoskeleton and are toxic to cells, indicating that the function of actin filaments depends on a dynamic equilibrium between filaments and actin monomers (Table 16–1).

1	In a test tube, polymerization of actin is controlled simply by its concentration, as described above, and by pH and the concentrations of salts and ATP. Within a cell, however, actin behavior is also regulated by numerous accessory proteins that bind actin monomers or filaments (summarized in Panel 16–3). At steady state the filament grows, elongation is faster than hydrolysis at the plus end in this example, and the terminal subunits at this end are therefore always in the T form. However, hydrolysis is faster than elongation at the minus end, and so terminal subunits at this end are in the D form. (B) Treadmilling occurs at intermediate concentrations of free subunits. The critical concentration for polymerization on a filament end in the T form is lower than for a filament end in the D form. If the actual subunit concentration is somewhere between these two values, the plus end grows while the minus end shrinks, resulting in treadmilling.

1	nucleates assembly associated with the growing to form a web and remains plus end associated with the minus end binds subunits, prevents assembly proflin binds subunits, – + binds ADP-actin flaments, binds to plus end flament bundling, cross-linking, and attachment to membranes Some of the major accessory proteins of the actin cytoskeleton. Except for the myosin motor proteins, an example of each major type is shown. Each of these is discussed in the text. However, most cells contain more than a hundred different actin-binding proteins, and it is likely that there are important types of actin-associated proteins that are not yet recognized.

1	in vitro, when the monomer concentration is 0.2 μM, filament half-life, a measure of how long an individual actin monomer spends in a filament as it treadmills, is approximately 30 minutes. In a non-muscle vertebrate cell, actin half-life in filaments is only 30 seconds, demonstrating that cellular factors modify the dynamic behavior of actin filaments. Actin-binding proteins dramatically alter actin filament dynamics and organization through spatial and temporal control of monomer availability, filament nucleation, elongation, and depolymerization. In the following sections, we describe the ways in which these accessory proteins modify actin function in the cell.

1	In most non-muscle vertebrate cells, approximately 50% of the actin is in filaments and 50% is soluble—and yet the soluble monomer concentration is 50–200 μM, well above the critical concentration. Why does so little of the actin polymerize into filaments? The reason is that the cell contains proteins that bind to the actin monomers and make polymerization much less favorable (an action similar to that of the drug latrunculin). A small protein called thymosin is the most abundant of these proteins. Actin monomers bound to thymosin are in a locked state, where they cannot associate with either the plus or minus ends of actin filaments and can neither hydrolyze nor exchange their bound nucleotide.

1	How do cells recruit actin monomers from this buffered storage pool and use them for polymerization? The answer depends on another monomer-binding protein called profilin. Profilin binds to the face of the actin monomer opposite the ATP-binding cleft, blocking the side of the monomer that would normally associate with the filament minus end, while leaving exposed the site on the monomer that binds to the plus end (Figure 16–15). When the profilin–actin complex binds a free plus end, a conformational change in actin reduces its affinity for profilin and the profilin falls off, leaving the actin filament one subunit longer. Profilin competes with thymosin for binding to individual actin monomers. Thus, by regulating the local activity of profilin, cells can control the movement of actin subunits from the sequestered thymosin-bound pool onto filament plus ends.

1	Several mechanisms regulate profilin activity, including profilin phosphorylation and profilin binding to inositol phospholipids. These mechanisms can define the sites where profilin acts. For example, profilin is required for filament assembly at the plasma membrane, where it is recruited by an interaction with acidic membrane phospholipids. At this location, extracellular signals can activate profilin to produce local actin polymerization and the extension of actin-rich motile structures such as filopodia and lamellipodia.

1	In addition to the availability of active actin subunits, a second prerequisite for cellular actin polymerization is filament nucleation. Proteins that contain actin monomer binding motifs linked in tandem mediate the simplest mechanism of filament nucleation. These actin-nucleating proteins bring several actin subunits together to form a seed. In most cases, actin nucleation is catalyzed by one of two different types of factors: the Arp 2/3 complex or the formins. The first of these is a complex of proteins that includes two actin-related proteins, or ARPs, each of which is about 45% identical to actin. The Arp 2/3 complex nucleates actin filament growth from the minus end, allowing rapid elongation at the plus end (Figure 16–16A and B). The complex can attach to the side of another actin filament while remaining bound to the minus end of the filament that it has nucleated, thereby building individual filaments into a treelike web (Figure 16–16C and D).

1	Formins are dimeric proteins that nucleate the growth of straight, unbranched filaments that can be cross-linked by other proteins to form parallel bundles. Each formin subunit has a binding site for monomeric actin, and the formin dimer appears to nucleate actin filament polymerization by capturing two monomers.

1	Figure 16–15 Effects of thymosin and profilin on actin polymerization. An actin monomer bound to thymosin is sterically prevented from binding to and elongating the plus end of an actin filament (left). An actin monomer bound to profilin, on the other hand, is capable of elongating a filament (right). Thymosin and profilin cannot both bind to a single actin monomer at the same time. In a cell in which most of the actin monomer is bound to thymosin, the activation of a small amount of profilin can produce rapid filament assembly. As indicated (bottom), profilin binds to actin monomers that are transiently released from the thymosin-bound monomer pool, shuttles them onto the plus ends of actin filaments, and is then released and recycled for further rounds of filament elongation.

1	As the newly nucleated filament grows, the formin dimer remains associated with the rapidly growing plus end while still allowing the addition of new subunits at that end (Figure 16–17). This mechanism of filament assembly is clearly different from that used by the Arp 2/3 complex, which remains stably bound to the filament minus end, preventing subunit addition or loss at that end. Formin-dependent actin filament growth is strongly enhanced by the association of actin monomers with profilin (Figure 16–18). Like profilin activation, actin filament nucleation by Arp 2/3 complexes and formins occurs primarily at the plasma membrane, and the highest density of actin filaments in most cells is at the cell periphery. The layer just beneath the plasma membrane is called the cell cortex, and the actin filaments in this region determine the shape and movement of the cell surface, allowing the cell to change its shape and stiffness rapidly in response to changes in its external environment.

1	Actin filament behavior is regulated by two major classes of binding proteins: those that bind along the side of a filament and those that bind to the ends (see Panel 16–3). Side-binding proteins include tropomyosin, an elongated protein that binds simultaneously to six or seven adjacent actin subunits along each of the two grooves of the helical actin filament. In addition to stabilizing and stiffening 908 Chapter 16: The Cytoskeleton the filament, the binding of tropomyosin can prevent the actin filament from interacting with other proteins; this aspect of tropomyosin function is important in the control of muscle contraction, as we discuss later. An actin filament that stops growing and is not specifically stabilized in the cell will depolymerize rapidly, particularly at its plus end, once the actin molecules

1	Figure 16–16 Nucleation and actin web formation by the Arp 2/3 complex. (A) The structures of Arp2 and Arp3 compared to the structure of actin. Although the face of the molecule equivalent to the plus end (top) in both Arp2 and Arp3 is very similar to the plus end of actin itself, differences on the sides and minus endprevent these actin-related proteins from forming filaments on their own or coassembling into filaments with actin. (B) A model for actin filament nucleation by the Arp 2/3 complex. In the absence of an activating factor, Arp2 and Arp3 are held by their accessory proteins in an orientation that prevents them from nucleating a new actin filament. When an activating factor (indicated by the blue triangle) binds the complex, Arp2 and Arp3 are brought together into a new configuration that resembles the plus end of an actin filament. Actin subunits can then assemble onto this structure, bypassing the rate-limiting step of filament nucleation. (C) The Arp 2/3 complex

1	configuration that resembles the plus end of an actin filament. Actin subunits can then assemble onto this structure, bypassing the rate-limiting step of filament nucleation. (C) The Arp 2/3 complex nucleates filaments most efficiently when it is bound to the side of a preexisting actin filament. The result is a filament branch that grows at a 70° angle relative to the original filament. Repeated rounds of branching nucleation result in a treelike web of actin filaments. (D) Top, electron micrographs of branched actin filaments formed by mixing purified actin subunits with purified Arp 2/3 complexes. Bottom, reconstructed image of a branch where the crystal structures of actin (pink) and the Arp 2/3 complex have been fitted to the electron density. The mother filament runs from top to bottom, and the daughter filament branches off to the right where the Arp 2/3 complex binds to three actin subunits in the mother filament. (D, top, from R.D. Mullins et al., Proc. Natl Acad. Sci. USA

1	and the daughter filament branches off to the right where the Arp 2/3 complex binds to three actin subunits in the mother filament. (D, top, from R.D. Mullins et al., Proc. Natl Acad. Sci. USA 95:6181–6186, 1998, with permission from National Academy of Sciences; botttom, from N. Volkmann et al., Science 293:2456–2459, 2001, with permission from AAAS.) have hydrolyzed their ATP. The binding of plus-end capping protein (also called CapZ for its location in the muscle Z band) stabilizes an actin filament at its plus end by rendering it inactive, greatly reducing the rates of filament growth and depolymerization (Figure 16–19). At the minus end, an actin filament may be capped by the Arp 2/3 complex that was responsible for its nucleation, although many minus ends in a typical cell are released from the Arp 2/3 complex and are uncapped.

1	Tropomodulin, best known for its function in the capping of exceptionally long-lived actin filaments in muscle, binds tightly to the minus ends of actin filaments that have been coated and thereby stabilized by tropomyosin. It can also transiently cap pure actin filaments and significantly reduce their elongation and depolymerization rates. A large family of tropomodulin proteins regulates actin filament length and stability in many cell types. For maximum effect, proteins that bind the side of actin filaments coat the filament completely, and must therefore be present in high amounts. In contrast, end-binding proteins can affect filament dynamics even when they are present at very low levels. Since subunit addition and loss occur primarily at filament ends, one molecule of an end-binding protein per actin filament (roughly one molecule per 200–500 actin subunits) can be enough to transform the architecture of an actin filament network.

1	Another important mechanism of actin filament regulation depends on proteins that break an actin filament into many smaller filaments, thereby generating a large number of new filament ends. The fate of these new ends depends on the presence of other accessory proteins. Under some conditions, newly formed ends nucleate filament elongation, thereby accelerating the assembly of new filament structures. Under other conditions, severing promotes the depolymerization of old filaments, speeding up the depolymerization rate by tenfold or more. In addition, severing changes the physical and mechanical properties of the cytoplasm: stiff, large bundles and gels become more fluid.

1	One class of actin-severing proteins is the gelsolin superfamily. These proteins are activated by high levels of cytosolic Ca2+. Gelsolin interacts with the side of the actin filament and contains subdomains that bind to two different sites: one that is exposed on the surface of the filament and one that is hidden between adjacent Figure 16–18 Profilin and formins. Some members of the formin protein family have unstructured domains or “whiskers” that contain several binding sites for profilin or the profilin–actin complex. These flexible domains serve as a staging area for addition of actin to the growing plus end of the actin filament when formin is bound. Under some conditions, this can enhance the rate of actin filament elongation so that filament growth is faster than that expected for a diffusion-controlled reaction, and faster in the presence of formin and profilin than the rate for pure actin alone (see also Figure 3–78).

1	Figure 16–17 Actin elongation mediated by formins. Formin proteins (green) form a dimeric complex that can nucleate the formation of a new actin filament (red) and remain associated with the rapidly growing plus end as it elongates. The formin protein maintains its binding to one of the two actin subunits exposed at the plus end as it allows each new subunit to assemble. Only part of the large dimeric formin molecule is shown here. Other regions regulate its activity and link it to particular structures in the cell. Many formins are indirectly connected to the cell plasma membrane and aid the insertional polymerization of the actin filament directly beneath the membrane surface. continued rapid growth of actin flament at plus end uncapped population of flaments: growth at plus and minus ends capped population of

1	continued rapid growth of actin flament at plus end uncapped population of flaments: growth at plus and minus ends capped population of Figure 16–19 Filament capping and its effects on filament dynamics. A population of uncapped filaments adds and loses subunits at both the plus and minus ends, resulting in rapid growth or shrinkage, depending on the concentration of available free monomers (green line). In the presence of a protein that caps the plus end (red line), only the minus end is able to add or lose subunits; consequently, flaments: growth filament growth will be slower at all at minus end only monomer concentrations above the critical concentration, and filament shrinkage will be slower at all monomer concentrations below the critical concentration. In addition, the critical concentration for the population shifts to that of the filament minus end.

1	subunits. According to one model, gelsolin binds the side of an actin filament until a thermal fluctuation creates a small gap between neighboring subunits, at which point gelsolin inserts itself into the gap to break the filament. After the severing event, gelsolin remains attached to the actin filament and caps the new plus end. Another important actin-filament destabilizing protein, found in all eukaryotic cells, is cofilin. Also called actin depolymerizing factor, cofilin binds along the length of the actin filament, forcing the filament to twist a little more tightly (Figure 16–20). This mechanical stress weakens the contacts between actin subunits in the filament, making the filament brittle and more easily severed by thermal motions, generating filament ends that undergo rapid disassembly. As a result, most of the actin filaments inside cells are shorter lived than are filaments formed from pure actin in a test tube.

1	Cofilin binds preferentially to ADP-containing actin filaments rather than to ATP-containing filaments. Since ATP hydrolysis is usually slower than filament assembly, the newest actin filaments in the cell still contain mostly ATP and are resistant to depolymerization by cofilin. Cofilin therefore tends to dismantle the older filaments in the cell. As we will discuss later, the cofilin-mediated disassembly of old but not new actin filaments is critical for the polarized, directed growth of the actin network that is responsible for unidirectional cell crawling and the intracellular motility of pathogens. Actin filaments can be protected from cofilin by tropomyosin binding. Thus, the dynamics of actin in different subcellular locations depends on the balance of stabilizing and destabilizing accessory proteins.

1	Figure 16–20 Twisting of an actin filament induced by cofilin. (A) Three-dimensional reconstruction from cryoelectron micrographs of filaments made of pure actin. The bracket shows the span of two twists of the actin helix. (B) Reconstruction of an actin filament coated with cofilin, which binds in a 1:1 stoichiometry to actin subunits all along the filament. Cofilin is a small protein (14 kD) compared to actin (43 kD), and so the filament appears only slightly thicker. The energy of cofilin binding serves to deform the actin filament, twisting it more tightly and reducing the distance spanned by each twist of the helix. (From A. McGough et al., J. Cell Biol. 138:771–781, 1997. With permission from the authors.)

1	Actin filaments in animal cells are organized into several types of arrays: dendritic networks, bundles, and weblike (gel-like) networks (Figure 16–21). Different structures are initiated by the action of distinct nucleating proteins: the actin filaments of dendritic networks are nucleated by the Arp 2/3 complex, while bundles are made of the long, straight filaments produced by formins. The proteins nucleating the filaments in the gel-like networks are not yet well defined.

1	The structural organization of different actin networks depends on specialized accessory proteins. As explained earlier, Arp 2/3 organizes filaments into dendritic networks by attaching filament minus ends to the side of other filaments. Other actin filament structures are assembled and maintained by two classes of proteins: bundling proteins, which cross-link actin filaments into a parallel array, and gel-forming proteins, which hold two actin filaments together at a large angle to each other, thereby creating a looser meshwork. Both bundling and gel-forming proteins generally have two similar actin-filament-binding sites, which can either be part of a single polypeptide chain or contributed by each of two polypeptide chains held together in a dimer (Figure 16–22). The spacing and arrangement of these two filament-binding domains determine the type of actin structure that a given cross-linking protein forms.

1	Each type of bundling protein also determines which other molecules can interact with the cross-linked actin filaments. Myosin II is the motor protein that enables stress fibers and other contractile arrays to contract. The very close packing of actin filaments caused by the small monomeric bundling protein fimbrin apparently excludes myosin, and thus the parallel actin filaments held together by fimbrin are not contractile. On the other hand, α-actinin cross-links oppositely Figure 16–21 Actin arrays in a cell.

1	Figure 16–21 Actin arrays in a cell. A fibroblast crawling in a tissue-culture dish is shown with four areas enlarged to show the arrangement of actin filaments. The actin filaments are shown in red, with arrowheads pointing toward the minus end. Stress fibers are contractile and exert tension. The actin cortex underlies the plasma membrane and consists of gel-like networks or dendritic actin networks that enable membrane protrusion at lamellopodia. Filopodia are spike-like projections of the plasma membrane that allow a cell to explore its environment.

1	Figure 16–22 The modular structures of four actin-cross-linking proteins. Each of the proteins shown has two actin-binding sites (red) that are related in sequence. Fimbrin has two directly adjacent actinbinding sites, so that it holds its two actin filaments very close together (14 nm apart), aligned with the same polarity (see Figure 16–23A). The two actin-binding sites in α-actinin are separated by a spacer around 30 nm long, so that it forms more loosely packed actin bundles (see Figure 16–23A). Filamin has two actin-binding sites with a V-shaped linkage between them, so that it cross-links actin filaments into a network with the filaments oriented almost at right angles to one another (see Figure 16–24). Spectrin is a tetramer of two α and two β subunits, and the tetramer has two actinbinding sites spaced about 200 nm apart (see Figure 10–38).

1	polarized actin filaments into loose bundles, allowing the binding of myosin and formation of contractile actin bundles (Figure 16–23). Because of the very different spacing and orientation of the actin filaments, bundling by fimbrin automatically discourages bundling by α-actinin, and vice versa, so that the two types of bundling protein are mutually exclusive.

1	The bundling proteins that we have discussed so far have straight, stiff connections between their two actin-filament-binding domains. Other actin cross-linking proteins have either a flexible or a stiff, bent connection between their two binding domains, allowing them to form actin filament webs or gels, rather than actin bundles. Filamin (see Figure 16–22) promotes the formation of a loose and highly viscous gel by clamping together two actin filaments roughly at right angles (Figure 16–24A). Cells require the actin gels formed by filamin to extend the thin, sheetlike membrane projections called lamellipodia that help them to crawl across solid surfaces. In humans, mutations in the filamin A gene cause defects in nerve-cell migration during early embryonic development. Cells in the periventricular region of the brain fail to migrate to the cortex and instead form nodules, causing a syndrome called periventricular heterotopia (Figure 16–24B). Interestingly, in addition to binding

1	region of the brain fail to migrate to the cortex and instead form nodules, causing a syndrome called periventricular heterotopia (Figure 16–24B). Interestingly, in addition to binding actin, filamins have been reported to interact with a large number of cellular proteins of great functional diversity, including membrane receptors for signaling molecules, and filamin mutations can also lead to defects in development of bone, the cardiovascular system, and other organs. Thus, filamins may also function as signaling scaffolds by connecting and coordinating a wide variety of cellular processes with the actin cytoskeleton.

1	A very different, well-studied web-forming protein is spectrin, which was first identified in red blood cells. Spectrin is a long, flexible protein made out of four elongated polypeptide chains (two α subunits and two β subunits), arranged so that the two actin-filament-binding sites are about 200 nm apart (compared with 14 nm for fimbrin and about 30 nm for α-actinin; see Figure 16–23). In the red blood cell, spectrin is concentrated just beneath the plasma membrane, where it forms a two-dimensional weblike network held together by short actin filaments whose precise lengths are tightly regulated by capping proteins at each end; spectrin links this web to the plasma membrane because it has separate binding sites for peripheral membrane proteins, which are themselves positioned near the lipid bilayer by integral membrane proteins (see Figure 10–38). The resulting network creates a strong, yet flexible cell cortex that provides mechanical support for the overlying plasma membrane,

1	lipid bilayer by integral membrane proteins (see Figure 10–38). The resulting network creates a strong, yet flexible cell cortex that provides mechanical support for the overlying plasma membrane, allowing the red blood cell to spring back to its original shape after squeezing through a capillary. Close relatives of spectrin are found in the cortex of most other vertebrate cell types, where they also help to shape and stiffen the surface membrane. A particularly striking example of spectrin’s role

1	Figure 16–23 The formation of two types of actin filament bundles. (A) Fimbrin cross-links actin filaments into tight bundles, which exclude the motor protein myosin II from participating in the assembly. In contrast, α-actinin, which is a homodimer, cross-links actin filaments into loose bundles, which allow myosin (not shown) to incorporate into the bundle. Fimbrin and α-actinin tend to exclude one another because of the very different spacing of the actin filament bundles that they form. (B) Electron micrograph of purified α-actinin molecules. (B, courtesy of John Heuser.) in promoting mechanical stability is the long, thin axon of neurons in the nematode worm Caenorhabditis elegans, where spectrin is required to keep them from breaking during the twisting motions the worms make during crawling.

1	The connections of the cortical actin cytoskeleton to the plasma membrane are only partially understood. Members of the ERM family (named for its first three members, ezrin, radixin, and moesin), help organize membrane domains through their ability to interact with transmembrane proteins and the underlying cytoskeleton. In so doing, they not only provide structural links to strengthen the cell cortex, but also regulate the activities of signal transduction pathways. Moesin also increases cortical stiffness to promote cell rounding during mitosis. Measurements by atomic force microscopy indicate that the cell cortex remains soft during mitosis when moesin is depleted. ERM proteins are thought to bind to and organize the cortical actin cytoskeleton in a variety of contexts, thereby affecting the shape and stiffness of the membrane as well as the localization and activity of signaling molecules. Bacteria Can Hijack the Host Actin Cytoskeleton

1	Bacteria Can Hijack the Host Actin Cytoskeleton The importance of accessory proteins in actin-based motility and force production is illustrated beautifully by studies of certain bacteria and viruses that use components of the host-cell actin cytoskeleton to move through the cytoplasm. The cytoplasm of mammalian cells is extremely viscous, containing organelles and cytoskeletal elements that inhibit diffusion of large particles like bacteria or viruses. To move around in a cell and invade neighboring cells, several pathogens, including Listeria monocytogenes (which causes a rare but serious form of food poisoning), overcome this problem by recruiting and activating the Arp 2/3 complex at their surface. The Arp 2/3 complex nucleates the assembly of actin filaments that generate a substantial force and push the bacterium through the

1	Figure 16–24 Filamin cross-links actin filaments into a three-dimensional network and is required for normal neuronal migration. (A) Each filamin homodimer is about 160 nm long when fully extended and forms a flexible, high-angle link between two adjacent actin filaments. A set of actin filaments cross-linked by filamin forms a mechanically strong web or gel. (B) Magnetic resonance imaging of a normal human brain (left) and of a patient with periventricular heterotopia (right) caused by mutation in the filamin A gene. In contrast to the smooth ventricular surface in the normal brain, a rough zone of cortical neurons (arrowheads) is seen along the lateral walls of the ventricles, representing neurons that have failed to migrate to the cortex during brain development. Remarkably, although many neurons are not in the right place, the intelligence of affected individuals is frequently normal or only mildly compromised, and the major clinical syndrome is epilepsy that often starts in the

1	many neurons are not in the right place, the intelligence of affected individuals is frequently normal or only mildly compromised, and the major clinical syndrome is epilepsy that often starts in the second decade of life. (B, adapted from

1	Y. Feng and C.A. Walsh, Nat. Cell Biol. 6:1034–1038, 2004. With permission from Macmillan Publishers Ltd.)

1	Figure 16–25 The actin-based movement of Listeria monocytogenes. (A) Fluorescence micrograph of an infected cell that has been stained to reveal bacteria in red and actin filaments in green. Note the cometlike tail of actin filaments behind each moving bacterium. Regions of overlap between red and green fluorescence appear yellow. (B) Listeria motility can be reconstituted in a test tube with ATP and just four purified proteins: actin, Arp 2/3 complex, capping protein, and cofilin. This micrograph shows the dense actin tails behind bacteria (black). (C) The ActA protein on the bacterial surface activates the Arp 2/3 complex to nucleate new filament assembly along the sides of existing filaments. Filaments grow at their plus end until capped by capping protein. Actin is recycled through the action of cofilin, which enhances depolymerization at the minus ends of the filaments. By this mechanism, polymerization is focused at the rear surface of the bacterium, propelling it forward (see

1	action of cofilin, which enhances depolymerization at the minus ends of the filaments. By this mechanism, polymerization is focused at the rear surface of the bacterium, propelling it forward (see Movie 23.7). (A, courtesy of Julie Theriot and Tim Mitchison; B, from T.P. Loisel et al., Nature 401:613–616, 1999. With permission from Macmillan Publishers Ltd.) cytoplasm at rates of up to 1 μm/sec, leaving behind a long actin “comet tail” (Figure 16–25; see also Figures 23–28 and 23–29). This motility can be reconstituted in a test tube by adding the bacteria to a mixture of pure actin, Arp 2/3 complex, cofilin, and capping protein, illustrating how actin polymerization dynamics generate movement through spatial regulation of filament assembly and disassembly. As we shall see, actin-based movement of this sort also underlies membrane protrusion at the leading edge of motile cells.

1	Actin is a highly conserved cytoskeletal protein that is present in high concentrations in nearly all eukaryotic cells. Nucleation presents a kinetic barrier to actin polymerization, but once formed, actin filaments undergo dynamic behavior due to hydrolysis of the bound nucleotide ATP. Actin filaments are polarized and can undergo treadmilling when a filament assembles at the plus end while simultaneously depolymerizing at the minus end. In cells, actin filament dynamics are regulated at every step, and the varied forms and functions of actin depend on a versatile repertoire of accessory proteins. Approximately half of the actin is kept in a monomeric form through association with sequestering proteins such as thymosin. Nucleation factors such as the Arp 2/3 complex and formins promote formation of branched and parallel filaments, respectively. Interplay between proteins that bind or cap actin filaments and those that promote filament severing or depolymerization can slow or

1	formation of branched and parallel filaments, respectively. Interplay between proteins that bind or cap actin filaments and those that promote filament severing or depolymerization can slow or accelerate the kinetics of filament assembly and disassembly. Another class of accessory proteins assembles the filaments into larger ordered structures by cross-linking them to one another in geometrically defined ways. Connections between these actin arrays and the plasma membrane of cells give an animal cell mechanical strength and permit the elaboration of cortical cellular structures such as lamellipodia, filopodia, and microvilli. By inducing actin filament polymerization at their surface, intracellular pathogens can hijack the host-cell cytoskeleton and move around inside the cell.

1	(A) light chains coiled-coil of two ˜ helices 100 nm N-terminus (B) Figure 16–26 Myosin II. (A) The two globular heads and long tail of a myosin II molecule shadowed with platinum can be seen in this electron micrograph. (B) A myosin II molecule is composed of two heavy chains (each about 2000 amino acids long; green) and four light chains (blue). The light chains are of two distinct types, and one copy of each type is present on each myosin head. Dimerization occurs when the two α helices of the heavy chains wrap around each other to form a coiled-coil, driven by the association of regularly spaced hydrophobic amino acids (see Figure 3–9). The coiled-coil arrangement makes an extended rod in solution, and this part of the molecule forms the tail. (A, courtesy of David Shotton.)

1	A crucial feature of the actin cytoskeleton is that it can form contractile structures that cross-link and slide actin filaments relative to one another through the action of myosin motor proteins. In addition to driving muscle contraction, actin–myosin assemblies perform important functions in non-muscle cells. Actin-Based Motor Proteins Are Members of the Myosin Superfamily

1	Actin-Based Motor Proteins Are Members of the Myosin Superfamily The first motor protein to be identified was skeletal muscle myosin, which generates the force for muscle contraction. This protein, now called myosin II, is an elongated protein formed from two heavy chains and two copies of each of two light chains. Each heavy chain has a globular head domain at its N-terminus that contains the force-generating machinery, followed by a very long amino acid sequence that forms an extended coiled-coil that mediates heavy-chain dimerization (Figure 16–26). The two light chains bind close to the N-terminal head domain, while the long coiled-coil tail bundles itself with the tails of other myosin molecules. These tail–tail interactions form large, bipolar “thick filaments” that have several hundred myosin heads, oriented in opposite directions at the two ends of the thick filament (Figure 16–27).

1	Figure 16–27 The myosin II bipolar thick filament in muscle. (A) Electron micrograph of a myosin II thick filament isolated from frog muscle. Note the central bare zone, which is free of head domains. (B) Schematic diagram, not drawn to scale. The myosin II molecules aggregate by means of their tail regions, with their heads projecting to the outside of the filament. The bare zone in the center of the filament consists entirely of myosin II tails. (C) A small section of a myosin II filament as reconstructed from electron micrographs. An individual myosin molecule is highlighted in green. The cytoplasmic myosin II filaments in non-muscle cells are much smaller, although similarly organized (see Figure 16–39). (A, courtesy of Murray Stewart; C, based on R.A. Crowther, R. Padrón and R. Craig, J. Mol. Biol. 184:429–439, 1985. With permission from Academic Press.)

1	Each myosin head binds and hydrolyzes ATP, using the energy of ATP hydrolysis to walk toward the plus end of an actin filament (Figure 16–28). The opposing orientation of the heads in the thick filament makes the filament efficient at sliding pairs of oppositely oriented actin filaments toward each other, shortening the muscle. In skeletal muscle, in which carefully arranged actin filaments are aligned in “thin filament” arrays surrounding the myosin thick filaments, the ATP-driven sliding of actin filaments results in a powerful contraction. Cardiac and smooth muscle contain myosin II molecules that are similarly arranged, although different genes encode them. Myosin Generates Force by Coupling ATP Hydrolysis to Conformational Changes

1	Myosin Generates Force by Coupling ATP Hydrolysis to Conformational Changes Motor proteins use structural changes in their ATP-binding sites to produce cyclic interactions with a cytoskeletal filament. Each cycle of ATP binding, hydrolysis, and release propels them forward in a single direction to a new binding site along the filament. For myosin II, each step of the movement along actin is generated by the swinging of an 8.5-nm-long α helix, or lever arm, which is structurally stabilized by the binding of light chains. At the base of this lever arm next to the head, there is a pistonlike helix that connects movements at the ATP-binding cleft in the head to small rotations of the so-called converter domain. A small change at this point can swing the helix like a long lever, causing the far end of the helix to move by about 5.0 nm.

1	These changes in the conformation of the myosin are coupled to changes in its binding affinity for actin, allowing the myosin head to release its grip on the actin filament at one point and snatch hold of it again at another. The full mechanochemical cycle of nucleotide binding, nucleotide hydrolysis, and phosphate release (which causes the “power stroke”) produces a single step of movement (Figure 16–29). At low ATP concentrations, the interval between the force-producing step and the binding of the next ATP is long enough that single steps can be observed (Figure 16–30). Sliding of Myosin II Along Actin Filaments Causes Muscles to Contract Muscle contraction is the most familiar and best-understood form of movement in animals. In vertebrates, running, walking, swimming, and flying all depend on the rapid contraction of skeletal muscle on its scaffolding of bone, while involuntary

1	Figure 16–28 Direct evidence for the motor activity of the myosin head. In this experiment, purified myosin heads were attached to a glass slide, and then actin filaments labeled with fluorescent phalloidin were added and allowed to bind to the myosin heads. (A) When ATP was added, the actin filaments began to glide along the surface, owing to the many individual steps taken by each of the dozens of myosin heads bound to each filament. The video frames shown in this sequence were recorded about 0.6 second apart; the two actin filaments shown (one red and one green) were moving in opposite directions at a rate of about 4 μm/sec. (B) Diagram of the experiment. The large red arrows indicate the direction of actin filament movement (Movie 16.2). (A, courtesy of James Spudich.)

1	ATTACHED At the start of the cycle shown in this fgure, plus a myosin head lacking a bound nucleotide is locked end tightly onto an actin flament in a rigor confguration (so named because it is responsible for rigor mortis, the rigidity of death). In an actively contracting muscle, this state is very short-lived, being rapidly terminated by the binding of a molecule of ATP. RELEASED A molecule of ATP binds to the large cleft on the “back” of the head (that is, on the side furthest from the actin flament) and immediately causes a slight change in the conformation of the actin-binding site, reducing the affnity of the head for actin and allowing it to move along the flament. (The space drawn here between the head and actin emphasizes this change, although in reality the head probably remains very close to the actin.)

1	COCKED The cleft closes like a clam shell around the ATP molecule, triggering a movement in the lever arm that causes the head to be displaced along the flament by a distance of about 5 nm. Hydrolysis of ATP occurs, but the ADP and inorganic phosphate (Pi) remain tightly bound to the protein. FORCE-GENERATING Weak binding of the myosin head to a new site on the actin flament causes release of the inorganic phosphate produced by ATP hydrolysis, concomitantly with the tight binding of the head to actin. This release triggers the power stroke—the force-generating change in shape during which the head regains its original conformation. In the course of the power stroke, the head loses its bound ADP, thereby returning to the start of a new cycle. ATTACHED At the end of the cycle, the myosin head is again locked tightly to the actin flament in a rigor confguration. Note that the head has moved to a new position on the actin flament.

1	ATTACHED At the end of the cycle, the myosin head is again locked tightly to the actin flament in a rigor confguration. Note that the head has moved to a new position on the actin flament. Figure 16–29 The cycle of structural changes used by myosin II to walk along an actin filament. In the myosin II cycle, the head remains bound to the actin filament for only about 5% of the entire cycle time, allowing many myosins to work together to move a single actin filament (Movie 16.3). (Based on I. Rayment et al., Science 261:50–58, 1993.) movements such as heart pumping and gut peristalsis depend on the contraction of cardiac muscle and smooth muscle, respectively. All these forms of muscle contraction depend on the ATP-driven sliding of highly organized arrays of actin filaments against arrays of myosin II filaments.

1	Skeletal muscle was a relatively late evolutionary development, and muscle cells are highly specialized for rapid and efficient contraction. The long, thin muscle fibers of skeletal muscle are actually huge single cells that form during 918 Chapter 16: The Cytoskeleton development by the fusion of many separate cells. The large muscle cell retains the many nuclei of the contributing cells. These nuclei lie just beneath the plasma membrane (Figure 16–31). The bulk of the cytoplasm inside is made up of myofibrils, which is the name given to the basic contractile elements of the muscle cell. A myofibril is a cylindrical structure 1–2 μm in diameter that is often as long as the muscle cell itself. It consists of a long, repeated chain of tiny contractile units— called sarcomeres, each about 2.2 μm long—which give the vertebrate myofibril its striated appearance (Figure 16–32).

1	Each sarcomere is formed from a miniature, precisely ordered array of parallel and partly overlapping thin and thick filaments. The thin filaments are composed of actin and associated proteins, and they are attached at their plus ends to a Z disc at each end of the sarcomere. The capped minus ends of the actin filaments extend in toward the middle of the sarcomere, where they overlap with thick filaments, the bipolar assemblies formed from specific muscle isoforms of myosin II (see Figure 16–27). When this region of overlap is examined in cross section by electron microscopy, the myosin filaments are arranged in a regular hexagonal lattice, with the actin filaments evenly spaced between them (Figure 16–33). Cardiac muscle and smooth muscle also contain sarcomeres, although the organization is not as regular as that in skeletal muscle.

1	Figure 16–31 Skeletal muscle cells (also called muscle fibers). (A) These huge multinucleated cells form by the fusion of many muscle cell precursors, called myoblasts. Here, a single muscle cell is depicted. In an adult human, a muscle cell is typically 50 μm in diameter and can be up to several centimeters long. (B) Fluorescence micrograph of rat muscle, showing the peripherally located nuclei (blue) in these giant cells. Myofibrils are stained red. (B, courtesy of Nancy L. Kedersha.)

1	Figure 16–30 The force of a single myosin molecule moving along an actin filament measured using an optical trap. (A) Schematic of the experiment, showing an actin filament with beads attached at both ends and held in place by focused beams of light called optical tweezers (Movie 16.4). The tweezers trap and move the bead, and can also be used to measure the force exerted on the bead through the filament. In this experiment, the filament was positioned over another bead to which myosin II motors were attached, and the optical tweezers were used to determine the effects of myosin binding on movement of the actin filament.

1	These traces show filament movement in two separate experiments. Initially, when the actin filament is unattached to myosin, thermal motion of the filament produces noisy fluctuations in filament position. When a single myosin binds to the actin filament, thermal motion decreases abruptly and a roughly 10-nm displacement results from movement of the filament by the motor. The motor then releases the filament. Because the ATP concentration is very low in this experiment, the myosin remains attached to the actin filament for much longer than it would in a muscle cell. (Adapted from C. Rüegg et al., Physiology 17:213–218, 2002. With permission from the American Physiological Society.)

1	Figure 16–32 Skeletal muscle myofibrils. (A) Low-magnification electron micrograph of a longitudinal section through a skeletal muscle cell of a rabbit, showing the regular pattern of cross-striations. The cell contains many myofibrils aligned in parallel (see Figure 16–31). (B) Detail of the skeletal muscle shown in (A), showing portions of two adjacent myofibrils and the definition of a sarcomere (black arrow). (C) Schematic diagram of a single sarcomere, showing the origin of the dark and light bands seen in the electron micrographs. The Z discs, at each end of the sarcomere, are attachment sites for the plus ends of actin filaments (thin filaments); the M line, or midline, is the location of proteins that link adjacent myosin II filaments (thick filaments) to one another. (D) When the sarcomere contracts, the actin and myosin filaments slide past one another without shortening. (A and B, courtesy of Roger Craig.)

1	Sarcomere shortening is caused by the myosin filaments sliding past the actin thin filaments, with no change in the length of either type of filament (see Figure 16–32C and D). Bipolar thick filaments walk toward the plus ends of two sets of thin filaments of opposite orientations, driven by dozens of independent myosin heads that are positioned to interact with each thin filament. Because there is no coordination among the movements of the myosin heads, it is critical that they remain tightly bound to the actin filament for only a small fraction of each ATPase cycle so that they do not hold one another back. Each myosin thick filament has about 300 heads (294 in frog muscle), and each head cycles about five times per second in the course of a rapid contraction—sliding the myosin and actin filaments past one another at rates of up to 15 μm/sec and enabling the sarcomere to shorten by 10% of its length in less than one-fiftieth of a second. The rapid synchronized shortening of the

1	actin filaments past one another at rates of up to 15 μm/sec and enabling the sarcomere to shorten by 10% of its length in less than one-fiftieth of a second. The rapid synchronized shortening of the thousands of sarcomeres lying end-to-end in each myofibril enables skeletal muscle to contract rapidly enough for running and flying, or for playing the piano.

1	Accessory proteins produce the remarkable uniformity in filament organization, length, and spacing in the sarcomere (Figure 16–34). The actin filament plus ends are anchored in the Z disc, which is built from CapZ and α-actinin; the Z disc caps the filaments (preventing depolymerization), while holding them together in a regularly spaced bundle. The precise length of each thin filament is influenced by a protein of enormous size, called nebulin, which consists almost entirely of a repeating 35-amino-acid actin-binding motif. Nebulin stretches from the Z disc toward the minus end of each thin filament, which is capped and stabilized by tropomodulin. Although there is some slow exchange of actin subunits at both ends of the muscle thin filament, such that the components of the thin filament turn over with a half-life of several days, the actin filaments in sarcomeres are remarkably stable compared with those found in most other cell types, whose dynamic actin filaments turn over with

1	turn over with a half-life of several days, the actin filaments in sarcomeres are remarkably stable compared with those found in most other cell types, whose dynamic actin filaments turn over with half-lives of a few minutes or less.

1	Figure 16–33 Electron micrographs of an insect flight muscle viewed in cross section. The myosin and actin filaments are packed together with almost crystalline regularity. Unlike their vertebrate counterparts, these myosin filaments have a hollow center, as seen in the enlargement on the right. The geometry of the hexagonal lattice is slightly different in vertebrate muscle. (From J. Auber, J. de Microsc. 8:197–232, 1969. With permission from Societé Française de Microscopie Électronique.) plus end minus end of actin nebulin actin (thin flament)

1	J. de Microsc. 8:197–232, 1969. With permission from Societé Française de Microscopie Électronique.) plus end minus end of actin nebulin actin (thin flament) Opposing pairs of an even longer template protein, called titin, position the thick filaments midway between the Z discs. Titin acts as a molecular spring, with a long series of immunoglobulin-like domains that can unfold one by one as stress is applied to the protein. A springlike unfolding and refolding of these domains keeps the thick filaments poised in the middle of the sarcomere and allows the muscle fiber to recover after being overstretched. In C. elegans, whose sarcomeres are longer than those in vertebrates, titin is longer as well, suggesting that it serves also as a molecular ruler, determining in this case the overall length of each sarcomere.

1	The force-generating molecular interaction between myosin thick filaments and actin thin filaments takes place only when a signal passes to the skeletal muscle from the nerve that stimulates it. Immediately upon arrival of the signal, the muscle cell needs to be able to contract very rapidly, with all the sarcomeres shortening simultaneously. Two major features of the muscle cell make extremely rapid contraction possible. First, as previously discussed, the individual myosin motor heads in each thick filament spend only a small fraction of the ATP cycle time bound to the filament and actively generating force, so many myosin heads can act in rapid succession on the same thin filament without interfering with one another. Second, a specialized membrane system relays the incoming signal rapidly throughout the entire cell. The signal from the nerve triggers an action potential in the muscle cell plasma membrane (discussed in Chapter 11), and this electrical excitation spreads swiftly

1	rapidly throughout the entire cell. The signal from the nerve triggers an action potential in the muscle cell plasma membrane (discussed in Chapter 11), and this electrical excitation spreads swiftly into a series of membranous folds—the transverse tubules, or T tubules—that extend inward from the plasma membrane around each myofibril. The signal is then relayed across a small gap to the sarcoplasmic reticulum, an adjacent weblike sheath of modified endoplasmic reticulum that surrounds each myofibril like a net stocking (Figure 16–35A and B).

1	When the incoming action potential activates a Ca2+ channel in the T-tubule membrane, Ca2+ influx triggers the opening of Ca2+-release channels in the sarcoplasmic reticulum (Figure 16–35C). Ca2+ flooding into the cytosol then initiates the contraction of each myofibril. Because the signal from the muscle cell plasma membrane is passed within milliseconds (via the T tubules and sarcoplasmic reticulum) to every sarcomere in the cell, all of the myofibrils in the cell contract at once. The increase in Ca2+ concentration is transient because the Ca2+ is rapidly pumped back into the sarcoplasmic reticulum by an abundant, ATP-dependent Ca2+-pump (also called a Ca2+-ATPase) in its membrane (see Figure 11–13). Typically, the cytoplasmic Ca2+ concentration is restored to resting levels within 30 msec, allowing the myofibrils to relax. Thus, muscle contraction depends on two processes that consume enormous amounts of ATP: filament sliding, driven by the ATPase of the myosin motor domain, and

1	msec, allowing the myofibrils to relax. Thus, muscle contraction depends on two processes that consume enormous amounts of ATP: filament sliding, driven by the ATPase of the myosin motor domain, and Ca2+ pumping, driven by the Ca2+pump.

1	Figure 16–34 Organization of accessory proteins in a sarcomere. Each giant titin molecule extends from the Z disc to the M line—a distance of over 1 μm. Part of each titin molecule is closely associated with a myosin thick filament (which switches polarity at the M line); the rest of the titin molecule is elastic and changes length as the sarcomere contracts and relaxes. Each nebulin molecule is exactly the length of a thin filament. The actin filaments are also coated with tropomyosin and troponin (not shown; see Figure 16–36) and are capped at both ends. Tropomodulin caps the minus end of the actin filaments, and CapZ anchors the plus end at the Z disc, which also contains α-actinin (not shown).

1	The Ca2+-dependence of vertebrate skeletal muscle contraction, and hence its dependence on commands transmitted via nerves, is due entirely to a set of specialized accessory proteins that are closely associated with the actin thin filaments. One of these accessory proteins is a muscle form of tropomyosin, the elongated protein that binds along the groove of the actin filament helix. The other is troponin, a complex of three polypeptides, troponins T, I, and C (named for their tropomyosin-binding, inhibitory, and Ca2+-binding activities, respectively). Troponin I binds to actin as well as to troponin T. In a resting muscle, the troponin I–T complex pulls the tropomyosin out of its normal binding groove into a position along the actin filament that interferes with the binding of myosin heads, thereby preventing any force-generating interaction. When the level of Ca2+ is raised, troponin C—which binds up to four molecules of Ca2+—causes troponin I to release its hold on actin. This

1	heads, thereby preventing any force-generating interaction. When the level of Ca2+ is raised, troponin C—which binds up to four molecules of Ca2+—causes troponin I to release its hold on actin. This allows the tropomyosin molecules to slip back into their normal position so that the myosin heads can walk along the actin filaments (Figure 16–36). Troponin C is closely related to the ubiquitous Ca2+-binding protein calmodulin (see Figure 15–33); it can be thought of as a specialized form of calmodulin that has acquired binding sites for troponin I and troponin T, thereby ensuring that the myofibril responds extremely rapidly to an increase in Ca2+ concentration.

1	In smooth muscle cells, so called because they lack the regular striations of skeletal muscle, contraction is also triggered by an influx of calcium ions, but the regulatory mechanism is different. Smooth muscle forms the contractile portion of the stomach, intestine, and uterus, as well as the walls of arteries and many other structures requiring slow and sustained contractions. Smooth muscle is 0.5 µm

1	Figure 16–35 T tubules and the sarcoplasmic reticulum. (A) Drawing of the two membrane systems that relay the signal to contract from the muscle cell plasma membrane to all of the myofibrils in the cell. (B) Electron micrograph showing a cross section of a T tubule. Note the position of the large Ca2+-release channels in the sarcoplasmic reticulum membrane that connect to the adjacent T-tubule membrane. (C) Schematic diagram showing how a Ca2+-release channel in the sarcoplasmic reticulum membrane is thought to be opened by the activation of a voltage-gated Ca2+ channel (Movie 16.5). (B, courtesy of Clara Franzini-Armstrong.)

1	Figure 16–36 The control of skeletal muscle contraction by troponin. (A) A skeletal-muscle-cell thin filament, showing the positions of tropomyosin and troponin along the actin filament. Each tropomyosin molecule has seven evenly spaced regions with similar amino acid sequences, each of which is thought to bind to an actin subunit in the filament. (B) Reconstructed cryoelectron microscopy image of an actin filament showing the relative position of a superimposed tropomyosin strand in the presence (dark purple) or absence (light purple) of calcium. (A, adapted from G.N. Phillips, J.P. Fillers and C. Cohen, J. Mol. Biol. 192:111–131, 1986. With permission from Academic Press; B, adapted from C. Xu et al., Biophys. J. 77: 985–992, 1999. With permission from Elsevier.) composed of sheets of highly elongated spindle-shaped cells, each with a single nucleus. Smooth muscle cells do not express the troponins. Instead, elevated intracellular Ca2+ levels regulate contraction by a mechanism that

1	elongated spindle-shaped cells, each with a single nucleus. Smooth muscle cells do not express the troponins. Instead, elevated intracellular Ca2+ levels regulate contraction by a mechanism that depends on calmodulin (Figure 16–37). Ca2+-bound calmodulin activates myosin light-chain kinase (MLCK), thereby inducing the phosphorylation of smooth muscle myosin on one of its two light chains. When the light chain is phosphorylated, the myosin head can interact with actin filaments and cause contraction; when it is dephosphorylated, the myosin head tends to dissociate from actin and becomes inactive.

1	The phosphorylation events that regulate contraction in smooth muscle cells occur relatively slowly, so that maximum contraction often requires nearly a second (compared with the few milliseconds required for contraction of a skeletal muscle cell). But rapid activation of contraction is not important in smooth

1	Figure 16–37 Smooth muscle contraction. (A) Upon muscle stimulation by activation of cell-surface receptors, Ca2+ released into the cytoplasm from the sarcoplasmic reticulum (SR) binds to calmodulin (see Figure 15–29). Ca2+-bound calmodulin then binds myosin light-chain kinase (MLCK), which phosphorylates myosin light chain, stimulating myosin activity. Non-muscle myosin is regulated by the same mechanism (see Figure 16–39). (B) Smooth muscle cells in a cross section of cat intestinal wall. The outer layer of smooth muscle is oriented with the long axis of its cells extending parallel along the length of the intestine, and upon contraction will shorten the intestine. The inner layer is oriented circularly around the intestine and when contracted will cause the intestine to become narrower. Contraction of both layers squeezes material through the intestine, much like squeezing toothpaste out of a tube.

1	(C) A model for the contractile apparatus in a smooth muscle cell, with bundles of contractile filaments containing actin and myosin (red) oriented obliquely to the long axis of the cell. Their contraction greatly shortens the cell. Only a few of the many bundles are shown. (B, courtesy of Gwen V. Childs.) muscle: its myosin II hydrolyzes ATP about 10 times more slowly than skeletal muscle myosin, producing a slow cycle of myosin conformational changes that results in slow contraction. The heart is the most heavily worked muscle in the body, contracting about 3 billion (3 × 109) times during the course of a human lifetime (Movie 16.6). Heart cells express several specific isoforms of cardiac muscle myosin and cardiac muscle actin. Even subtle changes in these cardiac-specific contractile proteins—changes that would not cause any noticeable consequences in other tissues—can cause serious heart disease (Figure 16–38).

1	The normal cardiac contractile apparatus is such a highly tuned machine that a tiny abnormality anywhere in the works can be enough to gradually wear it down over years of repetitive motion. Familial hypertrophic cardiomyopathy is a common cause of sudden death in young athletes. It is a genetically dominant inherited condition that affects about two out of every thousand people, and it is associated with heart enlargement, abnormally small coronary vessels, and disturbances in heart rhythm (cardiac arrhythmias). The cause of this condition is either any one of over 40 subtle point mutations in the genes encoding cardiac β myosin heavy chain (almost all causing changes in or near the motor domain) or one of about a dozen mutations in other genes encoding contractile proteins— including myosin light chains, cardiac troponin, and tropomyosin. Minor mis-sense mutations in the cardiac actin gene cause another type of heart condition, called dilated cardiomyopathy, which can also result in

1	light chains, cardiac troponin, and tropomyosin. Minor mis-sense mutations in the cardiac actin gene cause another type of heart condition, called dilated cardiomyopathy, which can also result in early heart failure.

1	Actin and Myosin Perform a Variety of Functions in Non-Muscle Cells Most non-muscle cells contain small amounts of contractile actin–myosin II bundles that form transiently under specific conditions and are much less well organized than muscle fibers. Non-muscle contractile bundles are regulated by myosin phosphorylation rather than the troponin mechanism (Figure 16–39). These contractile bundles function to provide mechanical support to cells, for example, by assembling into cortical stress fibers that connect the cell to the extracellular bipolar flament of 15–20 molecules

1	Figure 16–38 Effect on the heart of a subtle mutation in cardiac myosin. Left, normal heart from a 6-day-old mouse pup. Right, heart from a pup with a point mutation in both copies of its cardiac myosin gene, changing Arg403 to Gln. The arrows indicate the atria. In the heart from the pup with the cardiac myosin mutation, both atria are greatly enlarged (hypertrophic), and the mice die within a few weeks of birth. (From D. Fatkin et al., J. Clin. Invest. 103:147–153, 1999. With permission from The American Society for Clinical Investigation.)

1	Figure 16–39 Light-chain phosphorylation and the regulation of the assembly of myosin II into thick filaments. (A) The controlled phosphorylation by the enzyme myosin light-chain kinase (MLCK) of one of the two light chains (the so-called regulatory light chain, shown in light blue) on non-muscle myosin II in a test tube has at least two effects: it causes a change in the conformation of the myosin head, exposing its actinbinding site, and it releases the myosin tail from a “sticky patch” on the myosin head, thereby allowing the myosin molecules to assemble into short, bipolar, thick filaments. Smooth muscle is regulated by the same mechanism (see Figure 16–37). (B) Electron micrograph of negatively stained short filaments of myosin II that have been induced to assemble in a test tube by phosphorylation of their light chains. These myosin II filaments are much smaller than those found in skeletal muscle cells (see Figure 16–27). (B, courtesy of John Kendrick-Jones.) matrix through

1	by phosphorylation of their light chains. These myosin II filaments are much smaller than those found in skeletal muscle cells (see Figure 16–27). (B, courtesy of John Kendrick-Jones.) matrix through focal adhesions or by forming a circumferential belt in an epithelial cell, connecting it to adjacent cells through adherens junctions (discussed in Chapter 19). As described in Chapter 17, actin and myosin II in the contractile ring generate the force for cytokinesis, the final stage in cell division. Finally, as discussed later, contractile bundles also contribute to the adhesion and forward motion of migrating cells.

1	Non-muscle cells also express a large family of other myosin proteins, which have diverse structures and functions in the cell. Following the discovery of conventional muscle myosin, a second member of the family was found in the freshwater amoeba Acanthamoeba castellanii. This protein had a different tail structure and seemed to function as a monomer, and so it was named myosin I (for one-headed). Conventional muscle myosin was renamed myosin II (for two-headed). Subsequently, many other myosin types were discovered. The heavy chains generally start with a recognizable myosin motor domain at the N-terminus and then diverge widely with a variety of C-terminal tail domains (Figure 16–40). The myosin family includes a number of one-headed and two-headed varieties that are about equally related to myosin I and myosin II, and the nomenclature now reflects their approximate order of discovery (myosin III through at least myosin XVIII). Sequence comparisons among diverse eukaryotes indicate

1	to myosin I and myosin II, and the nomenclature now reflects their approximate order of discovery (myosin III through at least myosin XVIII). Sequence comparisons among diverse eukaryotes indicate that there are at least 37 distinct myosin families in the superfamily. All of the myosins except one move toward the plus end of an actin filament, although they do so at different speeds. The exception is myosin VI, which moves toward the minus end. The myosin tails (and the tails of motor proteins generally) have apparently diversified during evolution to permit the proteins to bind other subunits and to interact with different cargoes.

1	Some myosins are found only in plants, and some are found only in vertebrates. Most, however, are found in all eukaryotes, suggesting that myosins arose early in eukaryotic evolution. The human genome includes about 40 myosin genes. Nine of the human myosins are expressed primarily or exclusively in the hair cells of the inner ear, and mutations in five of them are known to cause hereditary deafness. These extremely specialized myosins are important for the construction and function of the complex and beautiful bundles of actin found in stereocilia that project from the apical surface of these cells (see Figure 9–51); these cellular protrusions tilt in response to sound and convert sound waves into electrical signals.

1	The functions of most of the myosins remain to be determined, but several are well characterized. The myosin I proteins often contain either a second actin-binding site or a membrane-binding site in their tails, and they are generally involved in intracellular organization—including the protrusion of actin-rich structures at the cell surface, such as microvilli (see Panel 16–1 and Figure 16–4), and endocytosis. Myosin V is a two-headed myosin with a large step size (Figure 16–41A) and is involved in organelle transport along actin filaments. In contrast to myosin II motors, which work in ensembles and are attached only transiently to actin filaments so as not to interfere with one another, myosin V moves continuously,

1	Figure 16–40 Myosin superfamily members. Comparison of the domain structure of the heavy chains of some myosin types. All myosins share similar motor domains (shown in dark green), but their C-terminal tails (light green) and N-terminal extensions (light blue) are very diverse. On the right are depictions of the molecular structure for these family members. Many myosins form dimers, with two motor domains per molecule, but a few (such as I, III, and XIV) seem to function as monomers, with just one motor domain. Myosin VI, despite its overall structural similarity to other family members, is unique in moving toward the minus end (instead of the plus end) of an actin filament. The small insertion within its motor head domain, not found in other myosins, is probably responsible for this change in direction.

1	or processively, along actin filaments without letting go. Myosin V functions are well studied in the yeast Saccharomyces cerevisiae, which undergoes a stereotypical pattern of growth and division called budding. Actin cables in the mother cell point toward the bud, where actin is found in patches that concentrate where cell wall growth is taking place. Myosin V motors carry a wide range of cargoes— including mRNA, endoplasmic reticulum, and secretory vesicles—along the actin cables and into the bud. In addition, myosin V mediates the correct partitioning of organelles such as peroxisomes and mitochondria between mother and daughter cells (see Figure 16–41B).

1	Using their neck domain as a lever arm, myosins convert ATP hydrolysis into mechanical work to move along actin filaments in a stepwise fashion. Skeletal muscle is made up of myofibrils containing thousands of sarcomeres assembled from highly ordered arrays of actin and myosin II filaments, together with many accessory proteins. Muscle contraction is stimulated by calcium, which causes the actin-filament-associated protein tropomyosin to move, uncovering myosin binding sites and allowing the filaments to slide past one another. Smooth muscle and non-muscle cells have less well-ordered contractile bundles of actin and myosin, which are regulated by myosin light-chain phosphorylation. Myosin V transports cargo by walking along actin filaments.

1	Microtubules are structurally more complex than actin filaments, but they are also highly dynamic and play comparably diverse and important roles in the cell. Microtubules are polymers of the protein tubulin. The tubulin subunit is itself a heterodimer formed from two closely related globular proteins called α-tubulin and β-tubulin, each comprising 445–450 amino acids, which are tightly bound together by noncovalent bonds (Figure 16–42A). These two tubulin proteins are found only in this heterodimer, and each α or β monomer has a binding site for one molecule of GTP. The GTP that is bound to α-tubulin is physically trapped at the dimer interface and is never hydrolyzed or exchanged; it can therefore be considered to be an integral part of the tubulin heterodimer structure. The nucleotide on the β-tubulin, in contrast, may be in either the GTP or the GDP form and is exchangeable within the soluble (unpolymerized) tubulin dimer.

1	Tubulin is found in all eukaryotic cells, and it exists in multiple isoforms. Yeast and human tubulins are 75% identical in amino acid sequence. In mammals, there are at least six forms of α-tubulin and a similar number of β-tubulins, each encoded by a different gene. The different forms of tubulin are very similar, and they generally copolymerize into mixed microtubules in the test tube. However, they can have distinct locations in cells and tissues and perform subtly different functions. As a striking example, mutations in a particular human β-tubulin gene give rise to a paralytic eye-movement disorder due to loss of ocular nerve function. Numerous human neurological diseases have been linked to specific mutations in different tubulin genes.

1	Figure 16–41 Myosin V carries cargo along actin filaments. (A) The lever arm of myosin V is long, allowing it to take a bigger step along an actin filament than myosin II (see Figure 16–29). (B) Myosin V transports cargo and organelles along actin cables, in this example moving a mitochondrion into the growing bud of a yeast cell. Microtubules Are Hollow Tubes Made of Protofilaments

1	A microtubule is a hollow cylindrical structure built from 13 parallel protofilaments, each composed of αβ-tubulin heterodimers stacked head to tail and then folded into a tube (Figure 16–42B–D). Microtubule assembly generates two new types of protein–protein contacts. Along the longitudinal axis of the microtubule, the “top” of one β-tubulin molecule forms an interface with the “bottom” of the α-tubulin molecule in the adjacent heterodimer. This interface is very similar to the interface holding the α and β monomers together in the dimer subunit, and the binding energy is high. Perpendicular to these interactions, neighboring protofilaments form lateral contacts. In this dimension, the main lateral contacts are between monomers of the same type (α–α and β–β). As longitudinal and lateral contacts are repeated during assembly, a slight stagger in lateral contacts gives rise to the helical microtubule lattice. Because multiple contacts within the lattice hold most of the subunits in a

1	contacts are repeated during assembly, a slight stagger in lateral contacts gives rise to the helical microtubule lattice. Because multiple contacts within the lattice hold most of the subunits in a microtubule in place, the addition and loss of subunits occurs almost exclusively at the microtubule ends (see Figure 16–5). These multiple contacts among subunits make microtubules stiff and difficult to bend. The persistence length of a microtubule is several millimeters, making microtubules the stiffest and straightest structural elements found in most animal cells.

1	The subunits in each protofilament in a microtubule all point in the same direction, and the protofilaments themselves are aligned in parallel (see Figure Figure 16–42 The structure of a microtubule and its subunit. (A) The subunit of each protofilament is a tubulin heterodimer, formed from a tightly linked pair of αand β-tubulin monomers. The GTP molecule in the α-tubulin monomer is so tightly bound that it can be considered an integral part of the protein. The GTP molecule in the β-tubulin monomer, however, is less tightly bound and has an important role in filament dynamics. Both nucleotides are shown in red. (B) One tubulin subunit (αβ-heterodimer) and one protofilament are shown schematically. Each protofilament consists of many adjacent subunits with the same orientation. (C) The microtubule is a stiff hollow tube formed from 13 protofilaments aligned in parallel.

1	(D) A short segment of a microtubule viewed in an electron microscope. (E) Electron micrograph of a cross section of a microtubule showing a ring of 13 distinct protofilaments. (D, courtesy of Richard Wade; E, courtesy of Richard Linck.) Figure 16–43 The preferential growth of microtubules at the plus end.

1	Figure 16–43 The preferential growth of microtubules at the plus end. Microtubules grow faster at one end than at the other. A stable bundle of microtubules obtained from the core of a cilium (called an axoneme) was incubated for a short time with tubulin subunits under polymerizing conditions. Microtubules grew fastest from the plus end of the microtubule bundle, the end at the top in this micrograph. (Courtesy of Gary Borisy.) 16–42). Therefore, the microtubule lattice itself has a distinct structural polarity, with α-tubulins exposed at the minus end and β-tubulins exposed at the plus end. As for actin filaments, the regular, parallel orientation of their subunits gives microtubules structural and dynamic polarity (Figure 16–43), with plus ends growing and shrinking more rapidly.

1	Microtubule dynamics, like those of actin filaments, are profoundly influenced by the binding and hydrolysis of nucleotide—GTP in this case. GTP hydrolysis occurs only within the β-tubulin subunit of the tubulin dimer. It proceeds very slowly in free tubulin subunits but is accelerated when they are incorporated into microtubules. Following GTP hydrolysis, the free phosphate group is released and the GDP remains bound to β-tubulin within the microtubule lattice. Thus, as in the case of actin filaments, two different types of microtubule structures can exist, one with the “T form” of the nucleotide bound (GTP) and one with the “D form” bound (GDP). The energy of nucleotide hydrolysis is stored as elastic strain in the polymer lattice, making the free-energy change for dissociation of a subunit from the D-form polymer more negative than the free-energy change for dissociation of a subunit from the T-form polymer. In consequence, the ratio of koff/kon for GDP-tubulin (its critical

1	a subunit from the D-form polymer more negative than the free-energy change for dissociation of a subunit from the T-form polymer. In consequence, the ratio of koff/kon for GDP-tubulin (its critical concentration [Cc(D)]) is much higher than that of GTP-tubulin. Thus, under physiological conditions, GTP-tubulin tends to polymerize and GDP-tubulin to depolymerize.

1	Whether the tubulin subunits at the very end of a microtubule are in the T or the D form depends on the relative rates of GTP hydrolysis and tubulin addition. If the rate of subunit addition is high—and thus the filament is growing rapidly— then it is likely that a new subunit will be added to the polymer before the nucleotide in the previously added subunit has been hydrolyzed. In this case, the tip of the polymer remains in the T form, forming a GTP cap. However, if the rate of subunit addition is low, hydrolysis may occur before the next subunit is added, and the tip of the filament will then be in the D form. If GTP-tubulin subunits assemble at the end of the microtubule at a rate similar to the rate of GTP hydrolysis, then hydrolysis will sometimes “catch up” with the rate of subunit addition and transform the end to a D form. This transformation is sudden and random, with a certain probability per unit time that depends on the concentration of free GTP-tubulin subunits.

1	Suppose that the concentration of free tubulin is intermediate between the critical concentration for a T-form end and the critical concentration for a D-form end (that is, above the concentration necessary for T-form assembly, but below that for the D form). Now, any end that happens to be in the T form will grow, whereas any end that happens to be in the D form will shrink. On a single microtubule, an end might grow for a certain length of time in a T form, but then suddenly change to the D form and begin to shrink rapidly, even while the free subunit concentration is held constant. At some later time, it might then regain a T-form end and begin to grow again. This rapid interconversion between a growing and shrinking state, at a uniform free subunit concentration, is called dynamic instability (Figure 16–44A and Figure 16–45; see Panel 16–2). The change from growth to shrinkage is called a catastrophe, while the change to growth is called a rescue.

1	In a population of microtubules, at any instant some of the ends are in the T form and some are in the D form, with the ratio depending on the hydrolysis rate and the free subunit concentration. In vitro, the structural difference between a T-form end and a D-form end is dramatic. Tubulin subunits with GTP bound to rapid growth with GTP-capped end random loss of GTP cap rapid shrinkage regain of GTP cap rapid growth with GTP-capped end etc. 50 nmGTP-tubulin dimer ˜°exchangeable GTP straight protoflament GTP HYDROLYSIS CHANGES SUBUNIT CONFORMATION AND WEAKENS BOND IN THE POLYMER DEPOLYMERIZATION GDP-GTP EXCHANGE (A) GROWING SHRINKING CATASTROPHE RESCUE GTP cap less stable region of microtubule containing GDP-tubulin dimers curved protoflament GDP-tubulin dimer GTPGTPGDPGDPGDPGDPGDPGDPGTPGTPGTP

1	Figure 16–44 Dynamic instability due to the structural differences between a growing and a shrinking microtubule end. (A) If the free tubulin concentration in solution is between the critical concentrations of the GTPand GDP-bound forms, a single microtubule end may undergo transitions between a growing state and a shrinking state. A growing microtubule has GTP-containing subunits at its end, forming a GTP cap. If nucleotide hydrolysis proceeds more rapidly than subunit addition, the cap is lost and the microtubule begins to shrink, an event called a “catastrophe.” But GTP-containing subunits may still add to the shrinking end, and if enough add to form a new cap, then microtubule growth resumes, an event called “rescue.” (B) Model for the structural consequences of GTP hydrolysis in the microtubule lattice. The addition of GTP-containing tubulin subunits to the end of a protofilament causes the end to grow in a linear conformation that can readily pack into the cylindrical wall of

1	microtubule lattice. The addition of GTP-containing tubulin subunits to the end of a protofilament causes the end to grow in a linear conformation that can readily pack into the cylindrical wall of the microtubule. Hydrolysis of GTP after assembly changes the conformation of the subunits and tends to force the protofilament into a curved shape that is less able to pack into the microtubule wall. (C) In an intact microtubule, protofilaments made from GDP-containing subunits are forced into a linear conformation by the many lateral bonds within the microtubule wall, given a stable cap of GTP-containing subunits. Loss of the GTP cap, however, allows the GDP-containing protofilaments to relax into their more curved conformation. This leads to a progressive disruption of the microtubule. Above the drawings of a growing and a shrinking microtubule, electron micrographs show actual microtubules in each of these two states. Note particularly the curling, disintegrating GDP-containing

1	Above the drawings of a growing and a shrinking microtubule, electron micrographs show actual microtubules in each of these two states. Note particularly the curling, disintegrating GDP-containing protofilaments at the end of the shrinking microtubule. (C, from E.M. Mandelkow,

1	E. Mandelkow and R.A. Milligan, J. Cell Biol. 114:977–991, 1991. With permission from The Rockefeller University Press.) the β-monomer produce straight protofilaments that make strong and regular lateral contacts with one another. But the hydrolysis of GTP to GDP is associated with a subtle conformational change in the protein, which makes the protofilaments curved (Figure 16–44B). On a rapidly growing microtubule, the GTP cap is thought to constrain the curvature of the protofilaments, and the ends appear straight. But when the terminal subunits have hydrolyzed their nucleotides, this constraint is removed, and the curved protofilaments spring apart. This cooperative release of the energy of hydrolysis stored in the microtubule lattice causes the curled protofilaments to peel off rapidly, and curved oligomers of GDP-containing tubulin are seen near the ends of depolymerizing microtubules (Figure 16–44C).

1	Chemical compounds that impair polymerization or depolymerization of micro-tubules are powerful tools for investigating the roles of these polymers in cells. Whereas colchicine and nocodazole interact with tubulin subunits and lead to microtubule depolymerization, Taxol binds to and stabilizes microtubules, causing a net increase in tubulin polymerization (see Table 16–1). Drugs like these have a rapid and profound effect on the organization of the microtubules in living cells. Both microtubule-depolymerizing drugs (such as nocodazole) and microtubule-polymerizing drugs (such as Taxol) preferentially kill dividing cells, since microtubule dynamics are crucial for correct function of the mitotic spindle (discussed in Chapter 17). Some of these drugs efficiently kill certain types of tumor cells in a human patient, although not without toxicity to rapidly dividing normal cells, including those in the bone marrow, intestine, and hair follicles. Taxol in particular has been widely used to

1	in a human patient, although not without toxicity to rapidly dividing normal cells, including those in the bone marrow, intestine, and hair follicles. Taxol in particular has been widely used to treat cancers of the breast and lung, and it is frequently successful in treatment of tumors that are resistant to other chemotherapeutic agents.

1	Because formation of a microtubule requires the interaction of many tubulin heterodimers, the concentration of tubulin subunits required for spontaneous nucleation of microtubules is very high. Microtubule nucleation therefore requires help from other factors. While αand β-tubulins are the regular building blocks of microtubules, another type of tubulin, called γ-tubulin, is present in much smaller amounts than αand β-tubulin and is involved in the nucleation of microtubule growth in organisms ranging from yeasts to humans. Microtubules are generally nucleated from a specific intracellular location known as a microtubule-organizing center (MTOC) where γ-tubulin is most enriched. Nucleation in many cases depends on the γ-tubulin ring complex (γ-TuRC). Within this complex, two accessory proteins bind directly to the γ-tubulin, along with several other proteins that help create a spiral ring of γ-tubulin molecules, which serves as a template that creates a microtubule with 13

1	proteins bind directly to the γ-tubulin, along with several other proteins that help create a spiral ring of γ-tubulin molecules, which serves as a template that creates a microtubule with 13 protofilaments (Figure 16–46).

1	Figure 16–45 Direct observation of the dynamic instability of microtubules in a living cell. Microtubules in a newt lung epithelial cell were observed after the cell was injected with a small amount of rhodamine-labeled tubulin. Notice the dynamic instability of microtubules at the edge of the cell. Four individual microtubules are highlighted for clarity; each of these shows alternating shrinkage and growth (Movie 16.7). (Courtesy of Wendy C. Salmon and Clare Waterman-Storer.) schematic of ˛-tubulin seven copies of

1	Figure 16–46 Microtubule nucleation by the γ-tubulin ring complex. (A) Two copies of γ-tubulin associate with a pair of accessory proteins to form the γ-tubulin small complex (γ-TuSC). This image was generated by high-resolution electron microscopy of individual purified complexes. (B) Seven copies of the γ-TuSC associate to form a spiral structure in which the last γ-tubulin lies beneath the first, resulting in 13 exposed γ-tubulin subunits in a circular orientation that matches the orientation of the 13 protofilaments in a microtubule. (C) In many cell types, the γ-TuSC spiral associates with additional accessory proteins to form the γ-tubulin ring complex (γ-TuRC), which is likely to nucleate the minus end of a microtubule as shown here. Note the longitudinal discontinuity between two protofilaments, which results from the spiral orientation of the γ-tubulin subunits. Microtubules often have one such “seam” breaking the otherwise uniform helical packing of the protofilaments. (A

1	protofilaments, which results from the spiral orientation of the γ-tubulin subunits. Microtubules often have one such “seam” breaking the otherwise uniform helical packing of the protofilaments. (A and B, from

1	J.M. Kollman et al., Nature 466:879–883, 2010. With permission from Macmillan Publishers Ltd.) Microtubules Emanate from the Centrosome in Animal Cells Many animal cells have a single, well-defined MTOC called the centrosome, which is located near the nucleus and from which microtubules are nucleated at their minus ends, so the plus ends point outward and continuously grow and shrink, probing the entire three-dimensional volume of the cell. A centrosome typically recruits more than fifty copies of γ-TuRC. In addition, γ-TuRC molecules are found in the cytoplasm, and centrosomes are not absolutely required for microtubule nucleation, since destroying them with a laser pulse does not prevent microtubule nucleation elsewhere in the cell. A variety of proteins have been identified that anchor γ-TuRC to the centrosome, but mechanisms that activate microtubule nucleation at MTOCs and at other sites in the cell are poorly understood.

1	Embedded in the centrosome are the centrioles, a pair of cylindrical structures arranged at right angles to each other in an L-shaped configuration (Figure 16–47). A centriole consists of a cylindrical array of short, modified microtubules arranged into a barrel shape with striking ninefold symmetry (Figure 16–48). Together with a large number of accessory proteins, the centrioles organize the pericentriolar material, where microtubule nucleation takes place. As described in Chapter 17, the centrosome duplicates and splits into two parts before mitosis, each containing a duplicated centriole pair. The two centrosomes move to opposite sides of the nucleus when mitosis begins, and they form the two poles of the mitotic spindle (see Panel 17–1).

1	Microtubule organization varies widely among different species and cell types. In budding yeast, microtubules are nucleated at an MTOC that is embedded in the nuclear envelope as a small, multilayered structure called the spindle pole body, also found in other fungi and diatoms. Higher-plant cells appear to nucleate microtubules at sites distributed all around the nuclear envelope and at the cell cortex. Neither fungi nor most plant cells contain centrioles. Despite these differences, all these cells seem to use γ-tubulin to nucleate their microtubules. In cultured animal cells, the aster-like configuration of microtubules is robust, with dynamic plus ends pointing outward toward the cell periphery and stable minus ends collected near the nucleus. The system of microtubules radiating from pair of ++ + (A) (B) of the centrosome

1	Figure 16–47 The centrosome. (A) The centrosome is the major MTOC of animal cells. Located in the cytoplasm next to the nucleus, it consists of an amorphous matrix of fibrous proteins to which the γ-tubulin ring complexes that nucleate microtubule growth are attached. This matrix is organized by a pair of centrioles, as described in the text. (B) A centrosome with attached microtubules. The minus end of each microtubule is embedded in the centrosome, having grown from a γ-tubulin ring complex, whereas the plus end of each microtubule is free in the cytoplasm. (C) In a reconstructed image of the MTOC from a C. elegans cell, a dense thicket of microtubules can be seen emanating from the centrosome. (C, from E.T. O’Toole et al., J. Cell Biol.

1	163:451–456, 2003. With permission from the authors.) the centrosome acts as a device to survey the outlying regions of the cell and to position the centrosome at its center. Even in an isolated cell fragment lacking the centrosome, dynamic microtubules arrange themselves into a star-shaped array with the microtubule minus ends clustered at the center by minus-end-binding proteins (Figure 16–49). This ability of the microtubule cytoskeleton to find the center of the cell establishes a general coordinate system, which is then used to position many organelles within the cell. Highly differentiated cells with complex morphologies such as neurons, muscles, and epithelial cells must use additional measuring mechanisms to establish their more elaborate internal coordinate systems. Thus, for example, when an epithelial cell forms cell–cell junctions and becomes highly polarized, the microtubule minus ends move to a region near the

1	Figure 16–48 A pair of centrioles in the centrosome. (A) An electron micrograph of a thin section of an isolated centrosome showing the mother centriole with its distal appendages and the adjacent daughter centriole, which formed through a duplication event during S phase (see Figure 17–26). In the centrosome, the centriole pair is surrounded by a dense matrix of pericentriolar material from which microtubules nucleate. Centrioles also function as basal bodies to nucleate the formation of ciliary axonemes (see Figure 16–68). (B) Electron micrograph of a cross section through a centriole in the cortex of a protozoan. Each centriole is composed of nine sets of triplet microtubules arranged to form a cylinder. (C) Each triplet contains one complete microtubule (the A microtubule) fused to two incomplete microtubules (the B and C microtubules).

1	(D) The centriolar protein SAS-6 forms a coiled-coil dimer. Nine SAS-6 dimers can self-associate to form a ring. Located at the hub of the centriole cartwheel structure, the SAS-6 ring is thought to generate the ninefold symmetry of the centriole. (A, from from M. Paintrand, et al. J. Struct. Biol. 108:107, 1992. With permission from Elsevier; B, courtesy of Richard Linck; D, courtesy of Michel Steinmetz.) apical plasma membrane. From this asymmetrical location, a microtubule array extends along the long axis of the cell, with plus ends directed toward the basal surface (see Figure 16–4).

1	Microtubule polymerization dynamics are very different in cells than in solutions of pure tubulin. Microtubules in cells exhibit a much higher polymerization rate (typically 10–15 μm/min, relative to about 1.5 μm/min with purified tubulin at similar concentrations), a greater catastrophe frequency, and extended pauses in microtubule growth, a dynamic behavior rarely observed in pure tubulin solutions. These and other differences arise because microtubule dynamics inside the cell are governed by a variety of proteins that bind tubulin dimers or microtubules, as summarized in Panel 16–4.

1	Proteins that bind to microtubules are collectively called microtubule-associated proteins, or MAPs. Some MAPs can stabilize microtubules against disassembly. A subset of MAPs can also mediate the interaction of microtubules with other cell components. This subset is prominent in neurons, where stabilized micro-tubule bundles form the core of the axons and dendrites that extend from the cell body (Figure 16–50). These MAPs have at least one domain that binds to the microtubule surface and another that projects outward. The length of the projecting domain can determine how closely MAP-coated microtubules pack together, as demonstrated in cells engineered to overproduce different MAPs. Cells over-expressing MAP2, which has a long projecting domain, form bundles of stable microtubules that are kept widely spaced, while cells overexpressing tau, a MAP with a much shorter projecting domain, form bundles of more closely packed microtubules (Figure 16–51). MAPs are the targets of several

1	are kept widely spaced, while cells overexpressing tau, a MAP with a much shorter projecting domain, form bundles of more closely packed microtubules (Figure 16–51). MAPs are the targets of several protein kinases, and phosphorylation of a MAP can control both its activity and localization inside cells.

1	Cells contain numerous proteins that bind the ends of microtubules and thereby influence microtubule stability and dynamics. These proteins can influence the Figure 16–50 Localization of MAPs in the axon and dendrites of a neuron. This immunofluorescence micrograph shows the distribution of the proteins tau (green) and MAP2 (orange) in a hippocampal neuron in culture. Whereas tau staining is confined to the axon (long and branched in this neuron), MAP2 staining is confined to the cell body and its dendrites. The antibody used here to detect tau binds only to unphosphorylated tau; phosphorylated tau is also present in dendrites. (Courtesy of James W. Mandell and Gary A. Banker.) Figure 16–49 A microtubule array can find the center of a cell. After the arm of a fish pigment cell is cut off with a needle, the microtubules in the detached cell fragment reorganize so that their minus ends end up near the center of the fragment, buried in a new microtubule-organizing center.

1	nucleates assembly and remains associated with centrosome the minus end remain associated with growing plus ends prevents assembly and can link them to other structures, such as membranes binds subunits, links to intermediate flaments Some of the major accessory proteins of the microtubule cytoskeleton. Except for two classes of motor proteins, an example of each major type is shown. Each of these is discussed in the text. However, most cells contain more than a hundred different microtubule-binding proteins, and — as for the actin-associated proteins — it is likely that there are important types of microtubule-associated proteins that are not yet recognized.

1	rate at which a microtubule switches from a growing to a shrinking state (the frequency of catastrophes) or from a shrinking to a growing state (the frequency of rescues). For example, members of a family of kinesin-related proteins known as catastrophe factors (or kinesin-13) bind to microtubule ends and appear to pry protofilaments apart, lowering the normal activation-energy barrier that prevents a microtubule from springing apart into the curved protofilaments that are characteristic of the shrinking state (Figure 16–52). Another protein, called Nezha or Patronin, protects microtubule minus ends from the effects of catastrophe factors.

1	While very few microtubule minus-end-binding proteins have been characterized, a large subset of MAPs has been identified that are enriched at microtubule plus ends. A particularly ubiquitous example is XMAP215, which has close homologs in organisms that range from yeast to humans. XMAP215 binds free tubulin subunits and delivers them to the plus end, thereby promoting microtubule polymerization and simultaneously counteracting catastrophe factor activity (see Figure 16–52). The phosphorylation of XMAP215 during mitosis inhibits GTP cap on plus end of microtubule Figure 16–51 Organization of microtubule bundles by MAPs. (A) MAP2 binds along the microtubule lattice at one of its ends and extends a long projecting arm with a second microtubule-binding domain at the other end. (B) Tau possesses a shorter microtubule cross-linking domain.

1	Electron micrograph showing a cross section through a microtubule bundle in a cell overexpressing MAP2. The regular spacing of the microtubules (MTs) in this bundle results from the constant length of the projecting arms of the MAP2. Similar cross section through a microtubule bundle in a cell overexpressing tau. Here the microtubules are spaced more closely together than they are in (C) because of tau’s relatively short projecting arm. (C and D, courtesy of J. Chen et al., Nature 360:674–677, 1992. With permission from Macmillan Publishers Ltd.)

1	Figure 16–52 The effects of proteins that bind to microtubule ends. The transition between microtubule growth and shrinkage is controlled in cells by a variety of proteins. Catastrophe factors such as kinesin-13, a member of the kinesin motor protein superfamily, bind to microtubule ends and pry them apart, thereby promoting depolymerization. On the other hand, a MAP such as XMAP215 stabilizes the end of a growing microtubule (XMAP stands for Xenopus microtubuleassociated protein, and the number refers to its molecular mass in kilodaltons). XMAP215 binds tubulin dimers and delivers them to the microtubule plus end, thereby increasing the microtubule growth rate and suppressing catastrophes. its activity and shifts the balance of its competition with catastrophe factors. This shift results in a tenfold increase in the dynamic instability of microtubules during mitosis, a transition that is critical for the efficient construction of the mitotic spindle (discussed in Chapter 17).

1	In many cells, the minus ends of microtubules are stabilized by association with a capping protein or the centrosome, or else they serve as microtubule depolymerization sites. The plus ends, in contrast, efficiently explore and probe the entire cell space. Microtubule-associated proteins called plus-end tracking proteins (+TIPs) accumulate at these active ends and appear to rocket around the cell as passengers at the ends of rapidly growing microtubules, dissociating from the ends when the microtubules begin to shrink (Figure 16–53).

1	The kinesin-related catastrophe factors and XMAP215 mentioned above behave as +TIPs and act to modulate the growth and shrinkage of the microtubule end to which they are attached. Other +TIPs control microtubule positioning by helping to capture and stabilize the growing microtubule end at specific cellular targets, such as the cell cortex or the kinetochore of a mitotic chromosome. EB1 and its relatives, small dimeric proteins that are highly conserved in animals, plants, and fungi, are key players in this process. EB1 proteins do not actively move toward plus ends, but rather recognize a structural feature of the growing plus end (see Figure 16–53). Several of the +TIPs depend on EB1 proteins for their plus-end accumulation and also interact with each other and with the microtubule lattice. By attaching to the plus end, these factors allow the cell to harness the energy of microtubule polymerization to generate pushing forces that can be used for positioning the spindle,

1	lattice. By attaching to the plus end, these factors allow the cell to harness the energy of microtubule polymerization to generate pushing forces that can be used for positioning the spindle, chromosomes, or organelles.

1	As it does with actin monomers, the cell sequesters unpolymerized tubulin subunits to maintain a pool of active subunits at a level near the critical concentration. One molecule of the small protein stathmin (also called Op18) binds to two tubulin heterodimers and prevents their addition to the ends of microtubules (Figure 16–54). Stathmin thus decreases the effective concentration of tubulin subunits that are available for polymerization (an action analogous to that of the drug colchicine), and enhances the likelihood that a growing microtubule will switch to the shrinking state. Phosphorylation of stathmin inhibits its binding to tubulin, and signals that cause stathmin phosphorylation can increase the rate of microtubule elongation and suppress dynamic instability. Stathmin has been implicated in the regulation of both cell proliferation and cell death. Interestingly, mice lacking stathmin develop normally but are less fearful than wild-type mice, reflecting a role for stathmin in

1	in the regulation of both cell proliferation and cell death. Interestingly, mice lacking stathmin develop normally but are less fearful than wild-type mice, reflecting a role for stathmin in neurons of the amygdala, where it is normally expressed at high levels.

1	Severing is another mechanism employed by the cell to destabilize microtubules. To sever a microtubule, thirteen longitudinal bonds must be broken, one for each protofilament. The protein katanin, named after the Japanese word for Figure 16–53 +TIP proteins found at the growing plus ends of microtubules.

1	Figure 16–53 +TIP proteins found at the growing plus ends of microtubules. (A) Frames from a fluorescence time-lapse movie of the edge of a cell expressing fluorescently labeled tubulin that incorporates into microtubules (red) as well as the +TIP protein EB1 tagged with a different color (green). The same microtubule is marked (asterisk) in successive movie frames. When the microtubule is growing (frames 1, 2), EB1 is associated with the tip. When the microtubule undergoes a catastrophe and begins shrinking, EB1 is lost (frames 3, 4). The labeled EB1 is regained when growth of the microtubule is rescued (frame 5). See Movie 16.8. (B) In the fission yeast Schizosaccharomyces pombe, the plus ends of the microtubules (green) are associated with the homolog of EB1 (red) at the two poles of the rod-shaped cells. (A, courtesy of Anna Akhmanova and Ilya Grigoriev; B, courtesy of Takeshi Toda.) 2.5 nm

1	Figure 16–54 Sequestration of tubulin by stathmin. Structural studies with electron microscopy and crystallography suggest that the elongated stathmin protein binds along the side of two tubulin heterodimers. (Adapted from M.O. Steinmetz et al., EMBO J. 19:572–580, 2000. With permission from John Wiley and Sons.) “sword,” accomplishes this demanding task (Figure 16–55). Katanin is made up of two subunits: a smaller subunit that hydrolyzes ATP and performs the actual severing, and a larger one that directs katanin to the centrosome. Katanin releases microtubules from their attachment to a microtubule-organizing center and is thought to contribute to the rapid microtubule depolymerization observed at the poles of spindles during mitosis. It may also be involved in microtubule release and depolymerization in proliferating cells in interphase and in postmitotic cells such as neurons. Two Types of Motor Proteins Move Along Microtubules

1	Like actin filaments, microtubules also use motor proteins to transport cargo and perform a variety of other functions within the cell. There are two major classes of microtubule-based motors, kinesins and dyneins. Kinesin-1, also called “conventional kinesin,” was first purified from squid neurons, where it carries membrane-enclosed organelles away from the cell body toward the axon terminal by walking toward the plus end of microtubules. Kinesin-1 is similar to myosin II in having two heavy chains per active motor; these form two globular head motor domains that are held together by an elongated coiled-coil tail that is responsible for heavy-chain dimerization. One kinesin-1 light chain associates with each heavy chain through its tail domain and mediates cargo binding. Like myosin, kinesin is a member of a large protein superfamily, for which the motor domain is the common element (Figure 16–56). The yeast Saccharomyces cerevisiae has six distinct kinesins. The nematode C. elegans

1	is a member of a large protein superfamily, for which the motor domain is the common element (Figure 16–56). The yeast Saccharomyces cerevisiae has six distinct kinesins. The nematode C. elegans has 20 kinesins, and humans have 45.

1	There are at least fourteen distinct families in the kinesin superfamily. Most of them have the motor domain at the N-terminus of the heavy chain and walk toward the plus end of the microtubule. One family has the motor domain at the C-terminus and walks in the opposite direction, toward the minus end of the microtubule, while kinesin-13 has a central motor domain and does not walk at all, but uses the energy of ATP hydrolysis to depolymerize microtubule ends, as described above (see Figure 16–52). Some kinesin heavy chains are homodimers, and others are heterodimers. Most kinesins have a binding site in the tail for another microtubule; alternatively, they may link the motor to a membrane-enclosed organelle via a light chain or an adaptor protein. Many of the kinesin superfamily members have specific roles in mitotic spindle formation and in chromosome segregation during cell division.

1	In kinesin-1, instead of the rocking of a lever arm, small movements at the nucleotide-binding site regulate the docking and undocking of the motor head domain to a long linker region. This acts to throw the second head forward along Figure 16–55 Microtubule severing by katanin. Taxol stabilized, rhodaminelabeled microtubules were adsorbed on the surface of a glass slide, and purified katanin was added along with ATP. (A) There are a few breaks in the microtubules 30 seconds after the addition of katanin. (B) The same field 3 minutes after the addition of katanin. The filaments have been severed in many places, leaving a series of small fragments at the previous locations of the long microtubules. (From J.J. Hartman et al., Cell 93:277–287, 1998. With permission from Elsevier.)

1	Figure 16–56 Kinesin and kinesinrelated proteins. Structures of four kinesin superfamily members. As in the myosin superfamily, only the motor domains are conserved. Kinesin-1 has the motor domain at the N-terminus of the heavy chain. The middle domain forms a long coiled-coil, mediating dimerization. The C-terminal domain forms a tail that attaches to cargo, such as a membrane-enclosed organelle. Kinesin-5 forms tetramers where two dimers associate by their tails. The bipolar kinesin-5 tetramer is able to slide two microtubules past each other, analogous to the activity of the bipolar thick filaments formed by myosin II. Kinesin-13 has its motor domain located in the middle of the heavy chain. It is a member of a family of kinesins that have lost typical motor activity and instead bind to microtubule ends to promote depolymerization (see Figure 16–52). Kinesin-14 is a C-terminal kinesin that includes the Drosophila protein Ncd and the yeast protein Kar3. These kinesins generally

1	to microtubule ends to promote depolymerization (see Figure 16–52). Kinesin-14 is a C-terminal kinesin that includes the Drosophila protein Ncd and the yeast protein Kar3. These kinesins generally travel in the opposite direction from the majority of kinesins, toward the minus end instead of the plus end of a microtubule.

1	Figure 16–57 The mechanochemical cycle of kinesin. Kinesin-1 is a dimer of two nucleotide-binding motor domains (heads) that are connected through a long coiled-coil tail (see Figure 16–56). The two kinesin motor domains work in a coordinated manner; during a kinesin “step,” the rear head detaches from its tubulin binding site, passes the partner motor domain, and then rebinds to the next available tubulin binding site. Using this “handover-hand” motion, the kinesin dimer can move for long distances on the microtubule without completely letting go of its track.

1	At the start of each step, one of the two kinesin motor domain heads, the rear or lagging head (dark green), is tightly bound to the microtubule and to ATP, while the front or leading head is loosely bound to the microtubule with ADP in its binding site. The forward displacement of the rear motor domain is driven by the dissociation of ADP and binding of ATP in the leading head (between panels 2 and 3 in this drawing). The binding of ATP to this motor domain causes a small peptide called the “neck linker” to shift from a rearward-pointing to a forward-pointing conformation (the neck linker is drawn here as a purple connecting line between the leading motor domain and the intertwined coiled-coil). This shift pulls the rear head forward, once it has detached from the microtubule with ADP bound [detachment requires ATP hydrolysis and phosphate (Pi) release]. The kinesin molecule is now poised for the next step, which proceeds by an exact repeat of the process shown (Movie 16.9).

1	the protofilament to a binding site 8 nm closer to the microtubule plus end, which is the distance between tubulin dimers of a protofilament. The nucleotide-hydrolysis cycles in the two heads are closely coordinated, so that this cycle of linker docking and undocking allows the two-headed motor to move in a hand-overhand (or head-over-head) stepwise manner (Figure 16–57).

1	The dyneins are a family of minus-end directed microtubule motors unrelated to the kinesins. They are composed of one, two, or three heavy chains (that include the motor domain) and a large and variable number of associated intermediate, light-intermediate, and light chains. The dynein family has two major branches (Figure 16–58). The first branch contains the cytoplasmic dyneins, which are homodimers of two heavy chains. Cytoplasmic dynein 1 is encoded by a single gene in almost all eukaryotic cells, but is missing from flowering plants and some algae. It is used for organelle and mRNA trafficking, for positioning the centrosome and nucleus during cell migration, and for construction of the microtubule spindle in mitosis and meiosis. Cytoplasmic dynein 2 is found only in eukaryotic organisms that have cilia and is used to transport material from the tip to the base of the cilia, a process called intraflagellar transport. Axonemal dyneins (also called ciliary dyneins) comprise the

1	that have cilia and is used to transport material from the tip to the base of the cilia, a process called intraflagellar transport. Axonemal dyneins (also called ciliary dyneins) comprise the second branch and include monomers, heterodimers, and heterotrimers, with one, two, or three motor-containing heavy chains, respectively. They are highly specialized for the rapid and efficient sliding movements of microtubules that drive the beating of cilia and flagella (discussed later).

1	Figure 16–58 Dyneins. (A) Freeze-etch electron micrographs of a molecule of cytoplasmic dynein and a molecule of ciliary (axonemal) dynein. Like myosin II and kinesin-1, cytoplasmic dynein is a two-headed molecule. The ciliary dynein shown is from a protozoan and has three heads; ciliary dynein from animals has two heads. Note that the dynein head is very large compared with the head of either myosin or kinesin. (B) Schematic depiction of cytoplasmic dynein showing the two heavy chains (blue and gray) that contain domains for microtubule (MT) binding and ATP hydrolysis, connected by a long stalk. Bound to the heavy chain are multiple intermediate chains (dark green) and light chains (light green) that help to mediate many of dynein’s functions. (A, courtesy of John Heuser; B, adapted from R. Vale, Cell 112:467–480, 2003. With permission from Cell Press.) (B) power stroke, 8 nm

1	Dyneins are the largest of the known molecular motors, and they are also among the fastest: axonemal dyneins attached to a glass slide can move micro-tubules at the rate of 14 μm/sec. The dynein motor is structurally unrelated to myosins and kinesins, but still follows the general rule of coupling nucleotide hydrolysis to microtubule binding and unbinding as well as to a force-generating conformational change (Figure 16–59).

1	A major function of cytoskeletal motors in interphase cells is the transport and positioning of membrane-enclosed organelles (Movie 16.10). Kinesin was originally identified as the protein responsible for fast anterograde axonal transport, the rapid movement of mitochondria, secretory vesicle precursors, and various synapse components down the microtubule highways of the axon to the distant nerve terminals. Cytoplasmic dynein was identified as the motor responsible for transport in the opposite direction, retrograde axonal transport. Although organelles in most cells need not cover such long distances, their polarized transport is equally necessary. A typical microtubule array in an interphase cell is oriented with the minus ends near the center of the cell at the centrosome and the plus ends extending to the cell periphery. Thus, centripetal movements of organelles or vesicles toward the cell center require the action of minus-end directed cytoplasmic dynein motors, whereas

1	the plus ends extending to the cell periphery. Thus, centripetal movements of organelles or vesicles toward the cell center require the action of minus-end directed cytoplasmic dynein motors, whereas centrifugal movements toward the periphery require plus-end directed kinesin motors. Interestingly, in animal cells, nearly all minus-end directed transport is driven by the single cytoplasmic dynein 1 motor, whereas 15 different kinesins are used for plus-end directed transport.

1	Figure 16–59 The power stroke of dynein. (A) The organization of the domains in each dynein heavy chain. This is a huge polypeptide, containing nearly 4000 amino acids. The number of heavy chains in a dynein is equal to its number of motor heads. (B) Illustration of dynein c, a monomeric axonemal dynein found in the unicellular green alga Chlamydomonas reinhardtii. The large dynein motor head is a planar ring containing a C-terminal domain (gray) and six AAA domains, four of which retain ATP-binding sequences, but only one of which (dark red) has the major ATPase activity. Extending from the head are a long, coiled-coil stalk with the microtubule-binding site at the tip, and a tail that attaches to an adjacent microtubule in the axoneme. In the ATP-bound state, the stalk is detached from the microtubule, but ATP hydrolysis causes stalk–microtubule attachment (left). Subsequent release of ADP and phosphate (Pi) then leads to a large conformational “power stroke” involving rotation of

1	the microtubule, but ATP hydrolysis causes stalk–microtubule attachment (left). Subsequent release of ADP and phosphate (Pi) then leads to a large conformational “power stroke” involving rotation of the head and stalk relative to the tail (right). Each cycle generates a step of about 8 nm, thereby contributing to flagellar beating (see Figure 16–65). In the case of cytoplasmic dynein, the tail is attached to a cargo such as a vesicle, and a single power stroke transports the cargo about 8-nm along the microtubule toward its minus end (see Figure 16–60). (C) Electron micrographs of purified monomeric dyneins in two different conformations representing different steps in the mechanochemical cycle. (C, from

1	S.A. Burgess et al., Nature 421:715–718, 2003. With permission from Macmillan Publishers Ltd.)

1	A clear example of the effect of microtubules and microtubule motors on the behavior of intracellular membranes is their role in organizing the endoplasmic reticulum (ER) and the Golgi apparatus. The network of ER membrane tubules aligns with microtubules and extends almost to the edge of the cell (Movie 16.11), whereas the Golgi apparatus is located near the centrosome. When cells are treated with a drug that depolymerizes microtubules, such as colchicine or nocodazole, the ER collapses to the center of the cell, while the Golgi apparatus fragments and disperses throughout the cytoplasm. In vitro, kinesins can tether ER-derived membranes to preformed microtubule tracks and walk toward the microtubule plus ends, dragging the ER membranes out into tubular protrusions and forming a membranous web that looks very much like the ER in cells. Conversely, dyneins are required for positioning the Golgi apparatus near the cell center of animal cells; they do this by moving Golgi vesicles along

1	web that looks very much like the ER in cells. Conversely, dyneins are required for positioning the Golgi apparatus near the cell center of animal cells; they do this by moving Golgi vesicles along microtubule tracks toward the microtubules’ minus ends at the centrosome.

1	The different tails and their associated light chains on specific motor proteins allow the motors to attach to their appropriate organelle cargo. Membrane-associated motor receptors that are sorted to specific membrane-enclosed compartments interact directly or indirectly with the tails of the appropriate kinesin family members. Many viruses take advantage of microtubule motor-based transport during infection and use kinesin to move from their site of replication and assembly to the plasma membrane, from which they are poised to infect neighboring cells. An outer-membrane protein of Vaccinia virus, for example, contains an amino acid motif that mediates binding to kinesin-1 light chain and transport along microtubules to the plasma membrane. Interestingly, this motif is present in over 450 human proteins, one-third of which are associated with human diseases. Thus, kinesin transports a diverse set of cargoes involved in a wide range of important cellular functions.

1	For dynein, a large macromolecular assembly often mediates attachment to membranes. Cytoplasmic dynein, itself a huge protein complex, requires association with a second large protein complex called dynactin to translocate organelles effectively. The dynactin complex includes a short, actin-like filament that forms from the actin-related protein Arp1 (distinct from Arp2 and Arp3, the components of the Arp 2/3 complex involved in the nucleation of conventional actin filaments) (Figure 16–60). A number of other proteins also contribute to dynein cargo binding and motor regulation, and their function is especially important in neurons, where defects in microtubule-based transport have been linked to neurological diseases. A striking example is smooth brain, or lissencephaly, a human disorder in which cells fail to migrate to the cerebral cortex of the developing brain. One type of lissencephaly is caused by defects in Lis1, a dynein-binding protein required for nuclear migration in

1	in which cells fail to migrate to the cerebral cortex of the developing brain. One type of lissencephaly is caused by defects in Lis1, a dynein-binding protein required for nuclear migration in several species. In the normal brain, migration of the nucleus directs the developing neural cell body toward its correct position in the cortex. In the absence of Lis1, however, the nuclei of migrating neurons fail to attach to dynein, resulting in nuclear-migration defects. Dynein is required continuously for neuronal function, as mutations in a dynactin subunit or in the tail region of cytoplasmic dynein lead to neuronal degeneration in humans and mice. These effects are associated with decreased retrograde axonal transport and provide strong evidence for the importance of robust axonal transport in neuronal viability.

1	The cell can regulate the activity of motor proteins and thereby cause either a change in the positioning of its membrane-enclosed organelles or whole-cell movements. Fish melanocytes provide one of the most dramatic examples. These giant cells, which are responsible for rapid changes in skin coloration in several species of fish, contain large pigment granules that can alter their location in response to neuronal or hormonal stimulation (Figure 16–61). The pigment granules aggregate or disperse by moving along an extensive network of microtubules that are anchored at the centrosome by their minus ends. The tracking of individual pigment granules reveals that the inward movement is rapid and smooth, while the outward movement is jerky, with frequent backward steps. Both dynein and kinesin microtubule motors are associated with the pigment granules. The

1	Figure 16–60 Dynactin mediates the attachment of dynein to a membrane-enclosed organelle. Dynein requires the presence of a large number of accessory proteins to associate with membrane-enclosed organelles. Dynactin is a large complex that includes components that bind weakly to microtubules, components that bind to dynein itself, and components that form a small, actin-like filament made of the actin-related protein Arp1. jerky outward movements may result from a tug-of-war between the two opposing microtubule motor proteins, with the stronger kinesin winning out overall. When intracellular cyclic AMP levels decrease, kinesin is inactivated, leaving dynein free to drag the pigment granules rapidly toward the cell center, changing the fish’s color. In a similar way, the movement of other membrane organelles coated with particular motor proteins is controlled by a complex balance of competing signals that regulate both motor protein attachment and activity.

1	Construction of Complex Microtubule Assemblies Requires Microtubule Dynamics and Motor Proteins The construction of the mitotic spindle and the neuronal cytoskeleton are important and fascinating examples of the power of organization by teams of motor proteins interacting with dynamic cytoskeletal filaments. As described in Chapter 17, mitotic spindle assembly depends on reorganization of the interphase array of microtubules to form a bipolar array of microtubules, with their minus ends focused at the poles and their plus ends overlapping in the center or connecting to chromosomes. Spindle assembly depends on the coordinated actions of several motor proteins and other factors that modulate polymerization dynamics (see Figures 17–23 and 17–25).

1	Neurons also contain complex cytoskeletal structures. As they differentiate, neurons send out specialized processes that will either receive electrical signals (dendrites) or transmit electrical signals (axons) (see Figure 16–50). The beautiful and elaborate branching morphology of axons and dendrites enables neurons to form tremendously complex signaling networks, interacting with many other cells simultaneously and making possible the complicated behavior of the higher animals. Both axons and dendrites (collectively called neurites) are filled with bundles of microtubules that are critical to both their structure and their function. In axons, all the microtubules are oriented in the same direction, with their minus end pointing back toward the cell body and their plus end pointing toward the axon terminals (Figure 16–62). The microtubules do not reach from the cell Figure 16–61 Regulated melanosome movements in fish pigment cells.

1	Figure 16–61 Regulated melanosome movements in fish pigment cells. These giant cells, which are responsible for changes in skin coloration in several species of fish, contain large pigment granules, or melanosomes (brown). The melanosomes can change their location in the cell in response to a hormonal or neuronal stimulus. (A) Schematic view of a pigment cell, showing the dispersal and aggregation of melanosomes in response to an increase or decrease in intracellular cyclic AMP (cAMP), respectively. Both redistributions of melanosomes occur along microtubules. (B) Bright-field images of a single cell in a scale of an African cichlid fish, showing its melanosomes either dispersed throughout the cytoplasm (left) or aggregated in the center of the cell (right). (B, courtesy of Leah Haimo.)

1	Figure 16–62 Microtubule organization in a neuron. In a neuron, microtubule organization is complex. In the axon, all microtubules share the same polarity, with the plus ends pointing outward toward the axon terminus. No single microtubule stretches the entire length of the axon; instead, short overlapping segments of parallel microtubules make the tracks for fast axonal transport. In dendrites, the microtubules are of mixed polarity, with some plus ends pointing outward and some pointing inward. Vesicles can associate with both kinesin and dynein and move in either direction along the microtubules in axons and dendrites, depending on which motor is active.

1	body all the way to the axon terminals; each is typically only a few micrometers in length, but large numbers are staggered in an overlapping array. These aligned microtubule tracks act as a highway to transport specific proteins, protein-containing vesicles, and mRNAs to the axon terminals, where synapses are constructed and maintained. The longest axon in the human body reaches from the base of the spinal cord to the foot and is up to a meter in length. By comparison, dendrites are generally much shorter than axons. The microtubules in dendrites lie parallel to one another but their polarities are mixed, with some pointing their plus ends toward the dendrite tip, while others point back toward the cell body, reminiscent of the antiparallel microtubule array of the mitotic spindle.

1	Just as myofibrils are highly specialized and efficient motility machines built from actin and myosin filaments, cilia and flagella are highly specialized and efficient motility structures built from microtubules and dynein. Both cilia and flagella are hairlike cell appendages that have a bundle of microtubules at their core. Flagella are found on sperm and many protozoa. By their undulating motion, they enable the cells to which they are attached to swim through liquid media. Cilia are organized in a similar fashion, but they beat with a whiplike motion that resembles the breaststroke in swimming. Ciliary beating can either propel single cells through a fluid (as in the swimming of the protozoan Paramecium) or can move fluid over the surface of a group of cells in a tissue. In the human body, huge numbers of cilia (109/cm2 or more) line our respiratory tract, sweeping layers of mucus, trapped particles of dust, and bacteria up to the mouth where they are swallowed and ultimately

1	body, huge numbers of cilia (109/cm2 or more) line our respiratory tract, sweeping layers of mucus, trapped particles of dust, and bacteria up to the mouth where they are swallowed and ultimately eliminated. Likewise, cilia along the oviduct help to sweep eggs toward the uterus.

1	The movement of a cilium or a flagellum is produced by the bending of its core, which is called the axoneme. The axoneme is composed of microtubules and their associated proteins, arranged in a distinctive and regular pattern. Nine special doublet microtubules (comprising one complete and one partial micro-tubule fused together so that they share a common tubule wall) are arranged in a ring around a pair of single microtubules (Figure 16–63). Almost all forms of motile eukaryotic flagella and cilia (from protozoans to humans) have this characteristic arrangement. The microtubules extend continuously for the length of the axoneme, which can be 10–200 μm. At regular positions along the length of the microtubules, accessory proteins cross-link the microtubules together.

1	Figure 16–63 The arrangement of microtubules in a flagellum or cilium. (A) Electron micrograph of the flagellum of a green-alga cell (Chlamydomonas) shown in cross section, illustrating the distinctive “9 + 2” arrangement of microtubules. (B) Diagram of the parts of a flagellum or cilium. The various projections from the microtubules link the microtubules together and occur at regular intervals along the length of the axoneme. (C) High-resolution electron tomography image of an outer doublet microtubule showing structural details and features inside the microtubules called microtubule inner proteins (MIPs). (A, courtesy of Lewis Tilney; C, courtesy of Daniela Nicastro.) 942 Chapter 16: The Cytoskeleton

1	Molecules of axonemal dynein form bridges between the neighboring doublet microtubules around the circumference of the axoneme (Figure 16–64). When the motor domain of this dynein is activated, the dynein molecules attached to one microtubule doublet (see Figure 16–59) attempt to walk along the adjacent microtubule doublet, tending to force the adjacent doublets to slide relative to one another, much as actin thin filaments slide during muscle contraction. However, the presence of other links between the microtubule doublets prevents this sliding, and the dynein force is instead converted into a bending motion (Figure 16–65).

1	In humans, hereditary defects in axonemal dynein cause a condition called primary ciliary dyskinesia or Kartagener’s syndrome. This syndrome is characterized by inversion of the normal asymmetry of internal organs (sinus inversus) due to disruption of fluid flow in the developing embryo, male sterility due to immotile sperm, and a high susceptibility to lung infections due to paralyzed cilia being unable to clear the respiratory tract of debris and bacteria. Bacteria also swim using cell-surface structures called flagella, but these do not contain microtubules or dynein and do not wave or beat. Instead, bacterial flagella are long, rigid helical filaments, made up of repeating subunits of the protein flagellin. The flagella rotate like propellers, driven by a special rotary motor embedded in the bacterial cell wall. The use of the same name to denote these two very different types of swimming apparatus is an unfortunate historical accident.

1	Many cells possess a shorter, nonmotile counterpart of cilia and flagella called the primary cilium. Primary cilia can be viewed as specialized cellular compartments or organelles that perform a wide range of cellular functions, but share

1	Figure 16–64 Ciliary dynein. Ciliary (axonemal) dynein is a large protein assembly (nearly 2 million daltons) composed of 9–12 polypeptide chains, the largest of which is the heavy chain of more than 500,000 daltons. (A) The heavy chains form the major portion of the globular head and stem domains, and many of the smaller chains are clustered around the base of the stem. There are two heads in the outer dynein in metazoans (shown here), but three heads in protozoa, each formed from their own heavy chain. The tail of the molecule binds tightly to an A microtubule, while the large globular heads have an ATP-dependent binding site for a B microtubule (see Figure 16–63). When the heads hydrolyze their bound ATP, they move toward the minus end of the B microtubule, thereby producing a sliding force between the adjacent microtubule doublets in a cilium or flagellum (see Figure 16–59). (B) Freeze-etch electron micrograph of a cilium showing the dynein arms projecting at regular intervals

1	force between the adjacent microtubule doublets in a cilium or flagellum (see Figure 16–59). (B) Freeze-etch electron micrograph of a cilium showing the dynein arms projecting at regular intervals from the doublet microtubules. (B, courtesy of John Heuser.)

1	Figure 16–65 The bending of an axoneme. (A) When axonemes are exposed to the proteolytic enzyme trypsin, the linkages holding neighboring doublet microtubules together are broken. In this case, the addition of ATP allows the motor action of the dynein heads to slide one pair of doublet microtubules against the other pair. (B) In an intact axoneme (such as in a sperm), flexible protein links prevent the sliding of the doublet. The motor action therefore causes a bending motion, creating waves or beating motions.

1	many structural features with motile cilia. Both motile and nonmotile cilia are generated during interphase at plasma-membrane-associated structures called basal bodies that firmly root them at the cell surface. At the core of each basal body is a centriole, the same structure found embedded at the center of animal centrosomes, with nine groups of fused triplet microtubules arranged in a cartwheel (see Figure 16–48). Centrioles are multifunctional, contributing to assembly of the mitotic spindle in dividing cells but migrating to the plasma membrane of inter-phase cells to template the nucleation of the axoneme (Figure 16–66). Because no protein translation occurs in cilia, construction of the axoneme requires intraflagellar transport (IFT), a transport system discovered in the green algae Chlamydomonas. Analogous to the axon, motors move cargoes in both anterograde and retrograde directions, in this case driven by kinesin-2 and cytoplasmic dynein 2, respectively.

1	Primary cilia are found on the surface of almost all cell types, where they sense and respond to the exterior environment, functions best understood in the context of smell and sight. In the nasal epithelium, cilia protruding from dendrites of olfactory neurons are the site of both odorant reception and signal amplification. Similarly, the rod and cone cells of the vertebrate retina possess a primary cilium equipped with an expanded tip called the outer segment, which is specialized for converting light into a neural signal (see Figure 15–38). Maintenance of the outer segment requires continuous IFT-mediated transport of large quantities of lipids and proteins into the cilium, at rates of up to 2000 molecules per minute. The links between cilia function and the senses of sight and smell are underscored by Bardet-Biedl syndrome, a set of disorders associated with defects in IFT, the cilium, or the basal body. Patients with Bardet-Biedl syndrome cannot smell and suffer from retinal

1	are underscored by Bardet-Biedl syndrome, a set of disorders associated with defects in IFT, the cilium, or the basal body. Patients with Bardet-Biedl syndrome cannot smell and suffer from retinal degeneration. Other characteristics of this multifaceted disorder include hearing loss, polycystic kidney disease, diabetes, obesity, and polydactyly, suggesting that primary cilia have functions in many aspects of human physiology.

1	Microtubules are stiff polymers of tubulin molecules. They assemble by addition of GTP-containing tubulin subunits to the free end of a microtubule, with one end (the plus end) growing faster than the other. Hydrolysis of the bound GTP takes place Figure 16–66 Primary cilia. (A) Electron micrograph and diagram of the basal body of a mouse neuron primary cilium. The axoneme of the primary cilium (black arrow) is nucleated by the mother centriole at the basal body, which localizes at the plasma membrane near the cell surface.

1	(B) Centrioles function alternately as basal bodies and as the core of centrosomes. Before a cell enters the cell division cycle, the primary cilium is shed or resorbed. The centrioles recruit pericentriolar material and duplicate during S phase, generating two centrosomes, each of which contains a pair of centrioles. The centrosomes nucleate microtubules and localize to the poles of the mitotic spindle. Upon exit from mitosis, a primary cilium again grows from the mother centriole. (A, courtesy of Josef Spacek.) after assembly and weakens the bonds that hold the microtubule together. Microtubules are dynamically unstable and liable to catastrophic disassembly, but they can be stabilized in cells by association with other structures. Microtubule-organizing centers such as centrosomes protect the minus ends of microtubules and continually nucleate the formation of new microtubules. Microtubule-associated proteins (MAPs) stabilize microtubules, and those that localize to the plus end

1	the minus ends of microtubules and continually nucleate the formation of new microtubules. Microtubule-associated proteins (MAPs) stabilize microtubules, and those that localize to the plus end (+TIPs) can alter the dynamic properties of the microtubule or mediate their interaction with other structures. Counteracting the stabilizing activity of MAPs are catastrophe factors, such as kinesin-13 proteins, that act to peel apart microtubule ends. Other kinesin family members as well as dynein use the energy of ATP hydrolysis to move unidirectionally along a microtubule. The motor dynein moves toward the minus end of microtubules, and its sliding of axonemal microtubules underlies the beating of cilia and flagella. Primary cilia are nonmotile sensory organs found on many cell types.

1	All eukaryotic cells contain actin and tubulin. But the third major type of cytoskeletal protein, the intermediate filament, forms a cytoplasmic filament only in some metazoans—including vertebrates, nematodes, and mollusks. Intermediate filaments are particularly prominent in the cytoplasm of cells that are subject to mechanical stress and are generally not found in animals that have rigid exoskeletons, such as arthropods and echinoderms. It seems that intermediate filaments impart mechanical strength to tissues for the squishier animals.

1	Cytoplasmic intermediate filaments are closely related to their ancestors, the much more prevalent nuclear lamins, which are found in many eukaryotes but missing from unicellular organisms. The nuclear lamins form a meshwork lining the inner membrane of the nuclear envelope, where they provide anchorage sites for chromosomes and nuclear pores. Several times during metazoan evolution, lamin genes have apparently duplicated, and the duplicates have evolved to produce ropelike, cytoplasmic intermediate filaments. In contrast to the highly conserved actins and tubulin isoforms that are encoded by a handful of genes, different families of intermediate filaments are much more diverse and are encoded by 70 different human genes with distinct, cell type-specific functions (Table 16–2). (C) 0.1 µm NH2 COOH COOH NH2 staggered tetramer of two coiled-coil dimers lateral association of 8 tetramers addition of 8 tetramers to growing flament

1	(C) 0.1 µm NH2 COOH COOH NH2 staggered tetramer of two coiled-coil dimers lateral association of 8 tetramers addition of 8 tetramers to growing flament Figure 16–67 A model of intermediate filament construction. The monomer shown in (A) pairs with another monomer to form a dimer (B), in which the conserved central rod domains are aligned in parallel and wound together into a coiled-coil.

1	(C) Two dimers then line up side by side to form an antiparallel tetramer of four polypeptide chains. Dimers and tetramers are the soluble subunits of intermediate filaments. (D) Within each tetramer, the two dimers are offset with respect to one another, thereby allowing it to associate with another tetramer. (E) In the final 10-nm ropelike filament, tetramers are packed together in a helical array, which has 16 dimers (32 coiled-coils) in cross section. Half of these dimers are pointing in each direction. An electron micrograph of intermediate filaments is shown on the upper left (Movie 16.12). (Electron micrograph courtesy of Roy Quinlan.) Intermediate Filament Structure Depends on the Lateral Bundling and Twisting of Coiled-Coils

1	Although their aminoand carboxy-terminal domains differ, all intermediate filament family members are elongated proteins with a conserved central α-helical domain containing 40 or so heptad repeat motifs that form an extended coiled-coil structure with another monomer (see Figure 3–9). A pair of parallel dimers then associates in an antiparallel fashion to form a staggered tetramer (Figure 16–67). Unlike actin or tubulin subunits, intermediate filament subunits do not contain a binding site for a nucleotide. Furthermore, since the tetrameric subunit is made up of two dimers pointing in opposite directions, its two ends are the same. The assembled intermediate filament therefore lacks the overall structural polarity that is critical for actin filaments and microtubules. The tetramers pack together laterally to form the filament, which includes eight parallel protofilaments made up of tetramers. Each individual intermediate filament therefore has a cross section of 32 individual

1	pack together laterally to form the filament, which includes eight parallel protofilaments made up of tetramers. Each individual intermediate filament therefore has a cross section of 32 individual α-helical coils. This large number of polypeptides all lined up together, with the strong lateral hydrophobic interactions typical of coiled-coil proteins, gives intermediate filaments a ropelike character. They can be easily bent, with a persistence length of less than one micrometer (compared to several millimeters for microtubules and about ten micrometers for actin), but they are extremely difficult to break and can be stretched to over three times their length (see Figure 16–6).

1	Less is understood about the mechanism of assembly and disassembly of intermediate filaments than of actin filaments and microtubules. In pure protein solutions, intermediate filaments are extremely stable due to tight association of subunits, but some types of intermediate filaments, including vimentin, form highly dynamic structures in cells such as fibroblasts. Protein phosphorylation probably regulates their disassembly, in much the same way that phosphorylation regulates the disassembly of nuclear lamins in mitosis (see Figure 12–18). As evidence for rapid turnover, labeled subunits microinjected into tissue-culture cells incorporate into intermediate filaments within a few minutes. Remodeling of the intermediate filament network accompanies events requiring dynamic cellular reorganization, such as division, migration, and differentiation. Intermediate Filaments Impart Mechanical Stability to Animal Cells

1	Keratins are the most diverse intermediate filament family: there are about 20 found in different types of human epithelial cells and about 10 more that are specific to hair and nails; analysis of the human genome sequence has revealed that there are 54 distinct keratins. Every keratin filament is made up of an equal mixture of type I (acidic) and type II (neutral/basic) keratin proteins; these form a heterodimer filament subunit (see Figure 16–67). Cross-linked keratin networks held together by disulfide bonds can survive even the death of their cells, forming tough coverings for animals, as in the outer layer of skin and in hair, nails, claws, and scales. The diversity in keratins is clinically useful in the diagnosis of epithelial cancers (carcinomas), as the particular set of keratins expressed gives an indication of the epithelial tissue in which the cancer originated and thus can help to guide the choice of treatment.

1	A single epithelial cell may produce multiple types of keratins, and these copolymerize into a single network (Figure 16–68). Keratin filaments impart mechanical strength to epithelial tissues in part by anchoring the intermediate filaments at sites of cell–cell contact, called desmosomes, or cell–matrix contact, called hemidesmosomes (see Figure 16–4). We discuss these important adhesive structures in Chapter 19. Accessory proteins, such as filaggrin, bundle keratin filaments in differentiating cells of the epidermis to give the outermost layers of the

1	Figure 16–68 Keratin filaments in epithelial cells. Immunofluorescence micrograph of the network of keratin filaments (blue) in a sheet of epithelial cells in culture. The filaments in each cell are indirectly connected to those of its neighbors by desmosomes (discussed in Chapter 19). A second protein (red) has been stained to reveal the location of the cell boundaries. (Courtesy of Kathleen Green and Evangeline Amargo.) skin their special toughness. Individuals with mutations in the gene encoding filaggrin are strongly predisposed to dry skin diseases such as eczema.

1	Mutations in keratin genes cause several human genetic diseases. For example, when defective keratins are expressed in the basal cell layer of the epidermis, they produce a disorder called epidermolysis bullosa simplex, in which the skin blisters in response to even very slight mechanical stress, which ruptures the basal cells (Figure 16–69). Other types of blistering diseases, including disorders of the mouth, esophageal lining, and the cornea of the eye, are caused by mutations in the different keratins whose expression is specific to those tissues. All of these maladies are typified by cell rupture as a consequence of mechanical trauma and a disorganization or clumping of the keratin filament cytoskeleton. Many of the specific mutations that cause these diseases alter the ends of the central rod domain, demonstrating the importance of this particular part of the protein for correct filament assembly.

1	Members of another family of intermediate filaments, called neurofilaments, are found in high concentrations along the axons of vertebrate neurons (Figure 16–70). Three types of neurofilament proteins (NF-L, NF-M, and NF-H) coassemble in vivo, forming heteropolymers. The NF-H and NF-M proteins have lengthy C-terminal tail domains that bind to neighboring filaments, generating aligned arrays with a uniform interfilament spacing. During axonal growth, new neurofilament subunits are incorporated all along the axon in a dynamic process that involves the addition of subunits along the filament length as well as the ends. After an axon has grown and connected with its target cell, the diameter of the axon may increase as much as fivefold. The level of neurofilament gene expression seems to directly control axonal diameter, which in turn influences how fast electrical signals travel down the axon. In addition, neurofilaments provide strength and stability to the long cell processes of

1	directly control axonal diameter, which in turn influences how fast electrical signals travel down the axon. In addition, neurofilaments provide strength and stability to the long cell processes of neurons.

1	The neurodegenerative disease amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease) is associated with an accumulation and abnormal assembly of neurofilaments in motor neuron cell bodies and in the axon, aberrations that may interfere with normal axonal transport. The degeneration of the axons leads to muscle weakness and atrophy, which is usually fatal. The overexpression of human NF-L or NF-H in mice results in mice that have an ALS-like disease. However, a causative link between neurofilament pathology and ALS has not been firmly established. basal cell of epidermis

1	basal cell of epidermis Figure 16–69 blistering of the skin caused by a mutant keratin gene. A mutant gene encoding a truncated keratin protein (lacking both the Nand C-terminal domains) was expressed in a transgenic mouse. The defective protein assembles with the normal keratins and thereby disrupts the keratin filament network in the basal cells of the skin. Light micrographs of cross sections of (A) normal and (B) mutant skin show that the blistering results from the rupturing of cells in the basal layer of the mutant epidermis (short red arrows). (C) A sketch of three cells in the basal layer of the mutant epidermis, as observed by electron microscopy. As indicated by the red arrow, the cells rupture between the nucleus and the hemidesmosomes (discussed in Chapter 19), which connect the keratin filaments to the underlying basal lamina. (From P.A. Coulombe et al., J. Cell Biol. 115:1661–1674, 1991. With permission from The Rockefeller University Press.)

1	The vimentin-like filaments are a third family of intermediate filaments. Desmin, a member of this family, is expressed in skeletal, cardiac, and smooth muscle, where it forms a scaffold around the Z disc of the sarcomere (see Figure 16–34). Mice lacking desmin show normal initial muscle development, but adults have various muscle-cell abnormalities, including misaligned muscle fibers. In humans, mutations in desmin are associated with various forms of muscular dystrophy and cardiac myopathy, illustrating the important role of desmin in stabilizing muscle fibers.

1	Besides their well-established role in maintaining the mechanical stability of the nucleus, it is becoming increasingly evident that one class of lamins, the A-type, together with many proteins of the nuclear envelope, are scaffolds for proteins that control myriad cellular processes including transcription, chromatin organization, and signal transduction. The majority of laminopathies are associated with mutant versions of lamin A and include tissue-specific diseases. Skeletal and cardiac abnormalities might be explained by a weakened nuclear envelope leading to cell damage and death, but laminopathies are also thought to arise from pathogenic and tissue-specific alterations in gene expression. Linker Proteins Connect Cytoskeletal Filaments and Bridge the Nuclear Envelope

1	Linker Proteins Connect Cytoskeletal Filaments and Bridge the Nuclear Envelope The intermediate filament network is linked to the rest of the cytoskeleton by members of a family of proteins called plakins. Plakins are large and modular, containing multiple domains that connect cytoskeletal filaments to each other and to junctional complexes. Plectin is a particularly interesting example. In addition to bundling intermediate filaments, it links the intermediate filaments to microtubules, actin filament bundles, and filaments of the motor protein myosin II; it also helps attach intermediate filament bundles to adhesive structures at the plasma membrane (Figure 16–71). Plectin and other plakins can interact with protein complexes that connect the cytoskeleton to the nuclear interior. These complexes consist of SUN proteins 0.5 µm Figure 16–70 Two types of intermediate filaments in cells of the nervous system.

1	Figure 16–70 Two types of intermediate filaments in cells of the nervous system. Freeze-etch electron microscopic image of neurofilaments in a nerve cell axon, showing the extensive cross-linking through protein cross-bridges—an arrangement believed to give this long cell process great tensile strength. The cross-bridges are formed by the long, nonhelical extensions at the C-terminus of the largest neurofilament protein (NF-H). (B) Freeze-etch image of glial filaments in glial cells, showing that these intermediate filaments are smooth and have few cross-bridges. Conventional transmission electron micrograph of a cross section of an axon showing the regular side-to-side spacing of the neurofilaments, which greatly outnumber the microtubules. (A and B, courtesy of Nobutaka Hirokawa; C, courtesy of John Hopkins.)

1	Figure 16–71 Plectin cross-linking of diverse cytoskeletal elements. Plectin (green) is seen here making cross-links from intermediate filaments (blue) to microtubules (red). In this electron micrograph, the dots (yellow) are gold particles linked to anti-plectin antibodies. The entire actin filament network was removed to reveal these proteins. (From

1	T.M. Svitkina et al., J. Cell Biol. 135:991– 1007, 1996. With permission from The Rockefeller University Press.) of the inner nuclear membrane and KASH proteins (also called nesprins) of the outer nuclear membrane (Figure 16–72). SUN and KASH proteins bind to each other within the lumen of the nuclear envelope, forming a bridge that connects the nuclear and cytoplasmic cytoskeletons. Inside the nucleus, the SUN proteins bind to the nuclear lamina or chromosomes, whereas in the cytoplasm, KASH proteins can bind directly to actin filaments and indirectly to microtubules and intermediate filaments through association with motor proteins and plakins, respectively. This linkage serves to mechanically couple the nucleus to the cytoskeleton and is involved in many cellular functions, including chromosome movements inside the nucleus during meiosis, nuclear and centrosome positioning, nuclear migration, and global cytoskeletal organization.

1	Mutations in the gene for plectin cause a devastating human disease that combines epidermolysis bullosa (caused by disruption of skin keratin filaments), muscular dystrophy (caused by disruption of desmin filaments), and neurodegeneration (caused by disruption of neurofilaments). Mice lacking a functional plectin gene die within a few days of birth, with blistered skin and abnormal skeletal and heart muscles. Thus, although plectin may not be necessary for the initial formation and assembly of intermediate filaments, its cross-linking action is required to provide cells with the strength they need to withstand the mechanical stresses inherent to vertebrate life.

1	GTP-binding proteins called septins serve as an additional filament system in all eukaryotes except terrestrial plants. Septins assemble into nonpolar filaments that form rings and cagelike structures, which act as scaffolds to compartmentalize membranes into distinct domains, or recruit and organize the actin and microtubule cytoskeletons. First identified in budding yeast, septin filaments localize to the neck between a dividing yeast mother cell and its growing bud (Figure 16–73A). At this location, septins block the movement of proteins from one side of the bud neck to the other, thereby concentrating cell growth preferentially within the bud. Septins also recruit the actin–myosin machinery that forms the contractile ring required for cytokinesis. In animal cells, septins function in cell division, migration, and vesicle trafficking. In primary cilia, for example, a ring of septin filaments assembles at the base of the cilium and serves as a diffusion barrier at the plasma

1	in cell division, migration, and vesicle trafficking. In primary cilia, for example, a ring of septin filaments assembles at the base of the cilium and serves as a diffusion barrier at the plasma membrane, restricting the movement of membrane proteins and establishing a specific composition in the ciliary membrane (Figure 16–73B and C). Reduction of septin levels impairs primary cilium formation and signaling.

1	There are 7 septin genes in yeast and 13 in human, and septin proteins fall into four groups on the basis of sequence relationships. In a test tube, purified septins assemble into symmetrical hetero-hexamers or hetero–octamers that Figure 16–72 SUN–KASH protein complexes connect the nucleus and cytoplasm through the nuclear envelope. The cytoplasmic cytoskeleton is linked across the nuclear envelope to the nuclear lamina or chromosomes through SUN and KASH proteins (orange and purple, respectively). The SUN and KASH domains of these proteins bind within the lumen of the nuclear envelope. From the inner nuclear envelope, SUN proteins connect to the nuclear lamina or chromosomes. KASH proteins in the outer nuclear envelope connect to the cytoplasmic cytoskeleton by binding microtubule motor proteins, actin filaments, or plectin.

1	mother cell 0.5 µm 10 µm2 µm form nonpolar paired filaments (Figure 16–74). GTP binding is required for the folding of septin polypeptides, but the role of GTP hydrolysis in septin function is not understood. Septin structures assemble and disassemble inside cells, but they are not as dynamic as actin filaments and microtubules. Whereas tubulin and actin have been highly conserved in evolution, intermediate filament proteins are very diverse. There are many tissue-specific forms of intermediate filaments in the cytoplasm of animal cells, including keratin filaments in epithelial cells, neurofilaments in nerve cells, and desmin filaments in muscle cells. The primary function of these filaments is to provide mechanical strength. Septins comprise an additional system of filaments that organize compartments inside cells. Figure 16–74 Septins polymerize to form paired filaments and sheets.

1	Figure 16–74 Septins polymerize to form paired filaments and sheets. (A) Electron micrograph of a septin rod assembled by combining two copies each of the four yeast septins illustrated at the right. The eight-subunit rod is nonpolar because the central pair of subunits (Cdc10) creates a symmetrical dimer. (B) Electron micrograph of paired septin filaments and sheets, assembled from purified septins in the presence of high salt concentrations. (C) Paired septin filaments may assemble by lateral association between filaments, mediated by coiled-coils formed between the paired C-terminal extensions of Cdc3 and Cdc12 that project from each filament. (Images and schematics adapted from A. Bertin et al., Proc. Natl Acad. Sci. USA 105:8274–8279, 2008. With permission from the National Academy of Sciences.)

1	Figure 16–73 Cell compartmentalization by septins. (A) Septins form filaments in the neck region between a mother yeast cell and bud. (B) In this photomicrograph of human cultured cells, the DNA is stained blue and septins are labeled in green. The microtubules of primary cilia are labeled with an antibody that recognizes a modified (acetylated) form of tubulin (red) that is enriched in the axoneme. (C) A magnified image reveals a collar of septin at the base of the cilium. (A, from B. Byers and L. Goetsch, J. Cell Biol. 69:717–721, 1976. With permission from Rockefeller University Press. B and C, from Q. Hu et al., Science 329:436–439, 2010. With permission from AAAS.)

1	A central challenge in cell biology is to understand how multiple individual molecular components collaborate to produce complex cell behaviors. The process of cell migration, which we describe in this final section, relies on the coordinated deployment of the components and processes that we have explored in this chapter: the dynamic assembly and disassembly of cytoskeletal polymers, the regulation and modification of their structure by polymer-associated proteins, and the actions of motor proteins moving along the polymers or exerting tension against them. How does the cell coordinate all these activities to define its polarity and enable it to crawl?

1	Many cells move by crawling over surfaces rather than by using cilia or flagella to swim. Predatory amoebae crawl continuously in search of food, and they can easily be observed to attack and devour smaller ciliates and flagellates in a drop of pond water (see Movie 1.4). In animals, almost all cell locomotion occurs by crawling, with the notable exception of swimming sperm. During embryogenesis, the structure of an animal is created by the migrations of individual cells to specific target locations and by the coordinated movements of whole epithelial sheets (discussed in Chapter 21). In vertebrates, neural crest cells are remarkable for their long-distance migrations from their site of origin in the neural tube to a variety of sites throughout the embryo (see Movie 21.5). Long-distance crawling is fundamental to the construction of the entire nervous system: it is in this way that the actin-rich growth cones at the advancing tips of developing axons travel to their eventual synaptic

1	is fundamental to the construction of the entire nervous system: it is in this way that the actin-rich growth cones at the advancing tips of developing axons travel to their eventual synaptic targets, guided by combinations of soluble signals and signals bound to cell surfaces and extracellular matrix along the way.

1	The adult animal also seethes with crawling cells. Macrophages and neutrophils crawl to sites of infection and engulf foreign invaders as a critical part of the innate immune response. Osteoclasts tunnel into bone, forming channels that are filled in by the osteoblasts that follow after them, in a continuous process of bone remodeling and renewal. Similarly, fibroblasts migrate through connective tissues, remodeling them where necessary and helping to rebuild damaged structures at sites of injury. In an ordered procession, the cells in the epithelial lining of the intestine travel up the sides of the intestinal villi, replacing absorptive cells lost at the tip of the villus. Unfortunately, cell crawling also has a role in many cancers, when cells in a primary tumor invade neighboring tissues and crawl into blood vessels or lymph vessels and then emerge at other sites in the body to form metastases.

1	Cell migration is a complex process that depends on the actin-rich cortex beneath the plasma membrane. Three distinct activities are involved: protrusion, in which the plasma membrane is pushed out at the front of the cell; attachment, in which the actin cytoskeleton connects across the plasma membrane to the substratum; and traction, in which the bulk of the trailing cytoplasm is drawn forward (Figure 16–75). In some crawling cells, such as keratocytes from the fish epidermis, these activities occur simultaneously, and the cells seem to glide forward smoothly without changing shape. In other cells, such as fibroblasts, these activities are more independent, and the locomotion is jerky and irregular.

1	The first step in locomotion, protrusion of a leading edge, frequently relies on forces generated by actin polymerization pushing the plasma membrane outward. Different cell types generate different types of protrusive structures, including filopodia (also known as microspikes) and lamellipodia. These are filled with dense cores of filamentous actin, which excludes membrane-enclosed organelles. The structures differ primarily in the way in which the actin is organized by actin-cross-linking proteins (see Figure 16–22). movement of unpolymerized actin

1	movement of unpolymerized actin Figure 16–75 A model of how forces generated in the actin-rich cortex move a cell forward. The actin-polymerizationdependent protrusion and firm attachment of a lamellipodium at the leading edge of the cell move the edge forward (green arrows at front) and stretch the actin cortex. Contraction at the rear of the cell propels the body of the cell forward (green arrow at back) to relax some of the tension (traction). New focal contacts are made at the front, and old ones are disassembled at the back as the cell crawls forward. The same cycle can be repeated, moving the cell forward in a stepwise fashion. Alternatively, all steps can be tightly coordinated, moving the cell forward smoothly. The newly polymerized cortical actin is shown in red.

1	Filopodia, formed by migrating growth cones of neurons and some types of fibroblasts, are essentially one-dimensional. They contain a core of long, bundled actin filaments, which are reminiscent of those in microvilli but longer and thinner, as well as more dynamic. Lamellipodia, formed by epithelial cells and fibroblasts, as well as by some neurons, are two-dimensional, sheetlike structures. They contain a cross-linked mesh of actin filaments, most of which lie in a plane parallel to the solid substratum. Invadopodia and related structures known as podosomes represent a third type of actin-rich protrusion. These extend in three dimensions and are important for cells to cross tissue barriers, as when a metastatic cancer cell invades the surrounding tissue. Invadopodia contain many of the same actin-regulatory components as filopodia and lamellipodia, and they also degrade the extracellular matrix, which requires the delivery of vesicles containing matrix-degrading proteases.

1	A distinct form of membrane protrusion called blebbing is often observed in vivo or when cells are cultured on a pliable extracellular matrix substratum. Blebs form when the plasma membrane detaches locally from the underlying actin cortex, thereby allowing cytoplasmic flow to push the membrane outward (Figure 16–76). Bleb formation also depends on hydrostatic pressure within the cell, which is generated by the contraction of actin and myosin assemblies. Once blebs have extended, actin filaments reassemble on the bleb membrane to form a new

1	Figure 16–76 Membrane bleb induced by disruption of the actin cortex. On the left is a light micrograph showing a spherical membrane protrusion or bleb induced by laser ablation of a small region of the actin cortex. The cortex is labeled green in the middle image by expression of GFP-actin. (Courtesy of Ewa Paluch.) actin cortex. Recruitment of myosin II and contraction of actin and myosin can then power retraction of membrane blebs. Alternatively, extension of new blebs from old ones can drive cell migration. Lamellipodia Contain All of the Machinery Required for Cell Motility

1	Lamellipodia Contain All of the Machinery Required for Cell Motility Lamellipodia have been particularly well studied in the epithelial cells of the epidermis of fish and frogs; these epithelial cells are known as keratocytes because of their abundant keratin filaments. These cells normally cover the animal by forming an epithelial sheet, and they are specialized to close wounds very rapidly, moving at rates of up to 30 μm/min. When cultured as individual cells, keratocytes assume a distinctive shape with a very large lamellipodium and a small, trailing cell body that is not attached to the substratum (Figure 16–77). Fragments of this lamellipodium can be sliced off with a micropipette. Although the fragments generally lack microtubules and membrane-enclosed organelles, they continue to crawl normally, looking like tiny keratocytes.

1	The dynamic behavior of actin filaments in keratocyte lamellipodia can be studied by labeling a small patch of actin and examining its fate. This reveals that, while the lamellipodia crawl forward, the actin filaments remain stationary with respect to the substratum. The actin filaments in the meshwork are mostly oriented with their plus ends facing forward. The minus ends are frequently attached to the sides of other actin filaments by Arp 2/3 complexes (see Figure 16–16), helping to form the two-dimensional web (Figure 16–78). The web as a whole is undergoing treadmilling, assembling at the front and disassembling at the back, reminiscent of the treadmilling that occurs in individual actin filaments discussed previously (see Figure 16–14).

1	Figure 16–78 Actin filament nucleation and web formation by the Arp 2/3 complex in lamellipodia. (A) A keratocyte with actin filaments labeled in red by fluorescent phalloidin and the Arp 2/3 complex labeled in green with an antibody against one of its subunits. The Arp 2/3 complex is highly concentrated near the front of the lamellipodium, where actin nucleation is most active. (B) Electron micrograph of a platinum-shadowed replica of the leading edge of a keratocyte, showing the dense actin filament meshwork. The labels denote areas enlarged in (C). (C) Close-up views of the marked regions of the actin web at the leading edge shown in (B). Numerous branched filaments can be seen, with the characteristic 70° angle formed when the Arp 2/3 complex nucleates a new actin filament off the side of a preexisting filament (see Figure 16–16). (From T. Svitkina and G. Borisy, J. Cell Biol. 145:1009–1026, 1999. With permission from the authors.)

1	J. Cell Biol. 145:1009–1026, 1999. With permission from the authors.) Figure 16–77 Migratory keratocytes from a fish epidermis. (A) Light micrographs of a keratocyte in culture, taken about 15 seconds apart. This cell is moving at about 15 μm/min (Movie 16.13 and see Movie 1.1). (B) Keratocyte seen by scanning electron microscopy, showing its broad, flat lamellipodium and small cell body, including the nucleus, carried up above the substratum at the rear. (C) Distribution of cytoskeletal filaments in this cell. Actin filaments (red) fill the large lamellipodium and are responsible for the cell’s rapid movement. Microtubules (green) and intermediate filaments (blue) are restricted to the regions close to the nucleus. (A and B, courtesy of Juliet Lee.) 954 Chapter 16: The Cytoskeleton

1	Maintenance of unidirectional motion by lamellipodia is thought to require the cooperation and mechanical integration of several factors. Filament nucleation is localized at the leading edge, with new actin filament growth occurring primarily in that location to push the plasma membrane forward. Most filament depolymerization occurs at sites located well behind the leading edge. Because cofilin (see Figure 16–20) binds cooperatively and preferentially to actin filaments containing ADP-actin (the D form), the new T-form filaments generated at the leading edge should be resistant to depolymerization by cofilin (Figure 16–79). As the filaments age and ATP hydrolysis proceeds, cofilin can efficiently disassemble the older filaments. Thus, the delayed ATP hydrolysis by filamentous actin is thought to provide the basis for a mechanism that maintains an efficient, unidirectional treadmilling process in the lamellipodium (Figure 16–80); it also explains the intracellular movement of bacterial

1	to provide the basis for a mechanism that maintains an efficient, unidirectional treadmilling process in the lamellipodium (Figure 16–80); it also explains the intracellular movement of bacterial pathogens such as Listeria (see Figure 16–25).

1	Myosin Contraction and Cell Adhesion Allow Cells to Pull Themselves Forward Forces generated by actin filament polymerization at the front of a migrating cell are transmitted to the underlying substratum to drive cell motion. For the leading Figure 16–79 Cofilin in lamellipodia. A keratocyte with actin filaments labeled in red by fluorescent phalloidin, and cofilin labeled in green with a fluorescent antibody. Although the dense actin meshwork reaches all the way through the lamellipodium, cofilin is not found at the very leading edge. (B) Close-up view of the region marked with the white rectangle in (A). The actin filaments closest to the leading edge, which are also the ones that have formed most recently and that are most likely to contain ATP-actin (rather than ADP-actin), are generally not associated with cofilin. (From T. Svitkina and G. Borisy, J. Cell Biol. 145:1009–1026, 1999. With permission from the authors.)

1	Figure 16–80 A model for protrusion of the actin meshwork at the leading edge. Two time points during advance of the lamellipodium are illustrated, with newly assembled structures at the later time point shown in a lighter color. Nucleation is mediated by the Arp 2/3 complex at the front. Newly nucleated actin filaments are attached to the sides of preexisting filaments, primarily at a 70° angle. Filaments elongate, pushing the plasma membrane forward because of some sort of anchorage of the array behind. At a steady rate, actin filament plus ends become capped. After newly polymerized actin subunits hydrolyze their bound ATP in the filament lattice, the filaments become susceptible to depolymerization by cofilin. This cycle causes a spatial separation between net filament assembly at the front and net filament disassembly at the rear, so that the actin filament network as a whole can move forward, even though the individual filaments within it remain stationary with respect to the

1	front and net filament disassembly at the rear, so that the actin filament network as a whole can move forward, even though the individual filaments within it remain stationary with respect to the substratum. Not all of the actin disassembles, however, and actin at the rear of the lamellipodium contributes to subsequent steps of migration together with myosin.

1	Figure 16–81 Contribution of myosin II to polarized cell motility.

1	(A) Myosin II bipolar filaments bind to actin filaments in the lamellipodial meshwork and cause network contraction. The myosin-driven reorientation of the actin filaments forms an actin bundle that recruits more myosin II and helps generate the contractile forces required for retraction of the trailing edge of the moving cell. (B) A fragment of the large lamellipodium of a keratocyte can be separated from the main cell body either by surgery with a micropipette or by treating the cell with certain drugs. Many of these fragments continue to move rapidly, with the same overall cytoskeletal organization as the intact keratocytes. Actin (blue) forms a protrusive meshwork at the front of the fragment. Myosin II (pink) is gathered into a band at the rear. (From A. Verkhovsky et al., Curr. Biol. 9:11–20, 1999. With permission from Elsevier.) edge of a migrating cell to advance, protrusion of the membrane must be followed by adhesion to the substratum at the front. Conversely, in order for

1	9:11–20, 1999. With permission from Elsevier.) edge of a migrating cell to advance, protrusion of the membrane must be followed by adhesion to the substratum at the front. Conversely, in order for the cell body to follow, contraction must be coupled with de-adhesion at the rear of the cell. The processes contributing to migration are therefore tightly regulated in space and time, with actin polymerization, dynamic adhesions, and myosin contraction being employed to coordinate movement. Myosin II operates in at least two ways to assist cell migration. The first is by helping to connect the actin cytoskeleton to the substratum through integrin-mediated adhesions. Forces generated by both actin polymerization and myosin activity create tension at attachment sites, promoting their maturation into focal adhesions, which are dynamic assemblies of structural and signaling proteins that link the migrating cell to the extracellular matrix (see Figure 19–59). A second mechanism involves bipolar

1	focal adhesions, which are dynamic assemblies of structural and signaling proteins that link the migrating cell to the extracellular matrix (see Figure 19–59). A second mechanism involves bipolar myosin II filaments, which associate with the actin filaments at the rear of the lamellipodium and pull them into a new orientation—from nearly perpendicular to the leading edge to almost parallel to the leading edge. This sarcomere-like contraction prevents protrusion, and it pinches in the sides of the locomoting lamellipodium, helping to gather in the sides of the cell as it moves forward (Figure 16–81).

1	Actin-mediated protrusions can only push the leading edge of the cell forward if there are strong interactions between the actin network and the focal adhesions that link the cell to the substrate. When these interactions are disengaged, polymerization pressure at the leading edge and myosin-dependent contraction cause the actin network to slip back, resulting in a phenomenon known as retrograde flow (Figure 16–82).

1	The traction forces generated by locomoting cells exert a significant pull on the substratum. By growing cells on a surface coated with tiny flexible posts, the force exerted on the substratum can be calculated by measuring the deflection of each post from its vertical position (Figure 16–83). In a living animal, most crawling cells move across a semiflexible substratum made of extracellular matrix, which can be deformed and rearranged by these cell forces. Conversely, mechanical tension or stretching applied externally to a cell will cause it to assemble stress fibers and focal adhesions, and become more contractile. Although poorly understood, this two-way mechanical interaction between cells and their physical environment is thought to help vertebrate tissues organize themselves. Cell Polarization Is Controlled by Members of the Rho Protein Family

1	Cell Polarization Is Controlled by Members of the Rho Protein Family Cell migration requires long-distance communication and coordination between one end of a cell and the other. During directed migration, it is important that the front end of the cell remain structurally and functionally distinct from the back end. In addition to driving local mechanical processes such as protrusion at the front and retraction at the rear, the cytoskeleton is responsible for coordinating cell shape, organization, and mechanical properties from one end of the cell to the other, a distance that is typically tens of micrometers for animal cells.

1	In many cases, including but not limited to cell migration, large-scale cytoskeletal coordination takes the form of the establishment of cell polarity, where a cell builds different structures with distinct molecular components at the front versus the back, or at the top versus the bottom. Cell locomotion requires an initial polarization of the cell to set it off in a particular direction. Carefully controlled cell-polarization processes are also required for oriented cell divisions in tissues and for formation of a coherent, organized multicellular structure. Genetic studies in yeast, flies, and worms have provided most of our current understanding of the molecular basis of cell polarity. The mechanisms that generate cell polarity in vertebrates are only beginning to be explored. In all known cases, however, the cytoskeleton has a central role, and many of the molecular components have been evolutionarily conserved.

1	The establishment of many kinds of cell polarity depends on the local regulation of the actin cytoskeleton by external signals. Many of these signals seem to converge inside the cell on a group of closely related monomeric GTPases that are members of the Rho protein family—Cdc42, Rac, and Rho. Like other monomeric GTPases, the Rho proteins act as molecular switches that cycle between an active GTP-bound state and an inactive GDP-bound state (see Figure 3–66). Activation of Cdc42 on the inner surface of the plasma membrane triggers actin polymerization and bundling to form filopodia. Activation of Rac promotes actin polymerization at the cell periphery, leading to the formation of sheetlike lamellipodial extensions. Activation of Rho promotes both the bundling of actin filaments with myosin II filaments into stress fibers and the clustering of integrins

1	Figure 16–82 Control of cell– substratum adhesion at the leading edge of a migrating cell. (A) Actin monomers assemble on the barbed end of actin filaments at the leading edge. Transmembrane integrin proteins (blue) help form focal adhesions that link the cell membrane to the substrate. (B) If there is no interaction between the actin filaments and focal adhesions, the actin filament is driven rearward by newly assembled actin. Myosin motors (green) also contribute to filament movement. (C) Interactions between actin-binding adaptor proteins (brown) and integrins link the actin cytoskeleton to the substratum. Myosin-mediated contractile forces are then transmitted through the focal adhesion to generate traction on the extracellular matrix, and new actin polymerization drives the leading edge forward in a protrusion.

1	Figure 16–83 Traction forces exerted by a motile cell. (A) Tiny flexible pillars attached to the substratum bend in response to traction forces. (B) Scanning electron micrograph of a cell on a substratum coated with pillars that are 6.1 μm in height. Pillar deflections are used to calculate force vectors corresponding to inward pulling forces on the underlying substratum. (Adapted from J. Fu et al., Nat. Methods 7:733–736, 2010. With permission from Macmillan Publishers.) and associated proteins to form focal adhesions (Figure 16–84). These dramatic and complex structural changes occur because each of these three molecular switches has numerous downstream target proteins that affect actin organization and dynamics.

1	Some key targets of activated Cdc42 are members of the WASp protein family. Human patients deficient in WASp suffer from Wiskott-Aldrich Syndrome, a severe form of immunodeficiency in which immune system cells have abnormal actin-based motility and platelets do not form normally. Although WASp itself is expressed only in blood cells and immune system cells, other more ubiquitous versions enable activated Cdc42 to enhance actin polymerization in many cell types. WASp proteins can exist in an inactive folded conformation and an activated open conformation. Association with Cdc42-GTP stabilizes the open form of WASp, enabling it to bind to the Arp 2/3 complex and strongly enhance its actin-nucleating activity (see Figure 16–16). In this way, activation of Cdc42 increases actin nucleation.

1	Rac-GTP also activates WASp family members. Additionally, it activates the cross-linking activity of the gel-forming protein filamin and inhibits the contractile activity of the motor protein myosin II. It thereby stabilizes lamellipodia and inhibits the formation of contractile stress fibers (Figure 16–85A). Rho-GTP has a very different set of targets. Instead of activating the Arp 2/3 complex to build actin networks, Rho-GTP turns on formin proteins to construct parallel actin bundles. At the same time, Rho-GTP activates a protein kinase that indirectly inhibits the activity of cofilin, leading to actin filament stabilization. The same protein kinase inhibits a phosphatase acting on myosin light chains (see Figure 16–39). The consequent increase in the net amount of myosin light chain phosphorylation increases the amount of contractile myosin motor protein activity in the cell, enhancing the formation of tension-dependent structures such as stress fibers (Figure 16–85B).

1	In some cell types, Rac-GTP activates Rho, usually at a rate that is slow compared to Rac’s activation of the Arp 2/3 complex. This enables cells to use the Rac pathway to build a new actin structure while subsequently activating the Rho pathway to generate a contractility that builds up tension in this structure. This occurs, for example, during the formation and maturation of cell–cell contacts.

1	Figure 16–84 The dramatic effects of Cdc42, Rac, and Rho on actin organization in fibroblasts. In each case, the actin filaments have been labeled with fluorescent phalloidin. (A) Serum-starved fibroblasts have actin filaments primarily in the cortex, and relatively few stress fibers. (B) Microinjection of a constitutively activated form of Cdc42 causes the protrusion of many long filopodia at the cell periphery. (C) Microinjection of a constitutively activated form of Rac, a closely related monomeric GTPase, causes the formation of an enormous lamellipodium that extends from the entire circumference of the cell. (D) Microinjection of a constitutively activated form of Rho causes the rapid assembly of many prominent stress fibers. (From A. Hall, Science 279:509–514, 1998. With permission from AAAS.)

1	(D) Microinjection of a constitutively activated form of Rho causes the rapid assembly of many prominent stress fibers. (From A. Hall, Science 279:509–514, 1998. With permission from AAAS.) As we will explore in more detail below, the communication between the Rac and Rho pathways also facilitates maintenance of the large-scale differences between the cell front and the cell rear during migration. Extracellular Signals Can Activate the Three Rho Protein Family Members

1	The activation of the monomeric GTPases Rho, Rac, and Cdc42 occurs through an exchange of GTP for a tightly bound GDP molecule, catalyzed by guanine nucleotide exchange factors (GEFs). Of the many GEFs that have been identified in the human genome, some are specific for an individual Rho family GTPase, whereas others seem to act on multiple family members. Different GEFs are restricted to specific tissues and even specific subcellular locations, and they are sensitive to distinct kinds of regulatory inputs. GEFs can be activated by extracellular cues through cell-surface receptors, or in response to intracellular signals. GEFs may also act as scaffolds that direct GTPases to downstream effectors. Interestingly, several of the Rho family GEFs associate with the growing ends of microtubules by binding to one of the +TIPs. This provides a connection between the dynamics of the microtubule cytoskeleton and the large-scale organization of the actin cytoskeleton; such a connection is

1	by binding to one of the +TIPs. This provides a connection between the dynamics of the microtubule cytoskeleton and the large-scale organization of the actin cytoskeleton; such a connection is important for the overall integration of cell shape and movement.

1	External Signals Can Dictate the Direction of Cell Migration Chemotaxis is the movement of a cell toward or away from a source of some diffusible chemical. These external signals act through Rho family proteins to set up large-scale cell polarity by influencing the organization of the cell motility apparatus. One well-studied example is the chemotactic movement of a class of white blood cells, called neutrophils, toward a source of bacterial infection. Receptor proteins on the surface of neutrophils enable them to detect very low concentrations of N-formylated peptides that are derived from bacterial proteins (only prokaryotes begin protein synthesis with N-formylmethionine). Using these receptors, neutrophils are guided to bacterial targets by their ability to detect a difference of only 1% in the concentration of these diffusible peptides on one side of the cell versus the other (Figure 16–86A).

1	In this case, and in the chemotaxis of Dictyostelium amoebae toward a source of cyclic AMP, binding of the chemoattractant to its G-protein-coupled receptor activates phosphoinositide 3-kinases (PI3Ks) (see Figure 15–52), which generate a signaling molecule [PI(3,4,5)P3] that in turn activates the Rac GTPase. Rac

1	Figure 16–85 The contrasting effects of Rac and Rho activation on actin organization. (A) Activation of the small GTPase Rac leads to alterations in actin accessory proteins that tend to favor the formation of actin networks, as in lamellipodia. Several different pathways contribute independently. Rac-GTP activates members of the WASp protein family, which in turn activate actin nucleation and branched web formation by the Arp 2/3 complex. In a parallel pathway, Rac-GTP activates a protein kinase, PAK, which has several targets including the web-forming cross-linker filamin, which is activated by phosphorylation, and the myosin light chain kinase (MLCK), which is inhibited by phosphorylation. Inhibition of MLCK results in decreased phosphorylation of the myosin regulatory light chain and leads to myosin II filament disassembly and a decrease in contractile activity. In some cells, PAK also directly inhibits myosin II activity by phosphorylation of the myosin heavy chain (MHC). (B)

1	leads to myosin II filament disassembly and a decrease in contractile activity. In some cells, PAK also directly inhibits myosin II activity by phosphorylation of the myosin heavy chain (MHC). (B) Activation of the related GTPase Rho leads to nucleation of actin filaments by formins and increases contraction by myosin II, promoting the formation of contractile actin bundles such as stress fibers. Activation of myosin II by Rho requires a Rho-dependent protein kinase called Rock. This kinase inhibits the phosphatase that removes the activating phosphate groups from myosin II light chains (MLC); it may also directly phosphorylate the myosin light chains in some cell types. Rock also activates other protein kinases, such as LIM kinase, which in turn contributes to the formation of stable contractile actin filament bundles by inhibiting the actin depolymerizing factor cofilin. A similar signaling pathway is important for forming the contractile ring necessary for cytokinesis (see Figure

1	actin filament bundles by inhibiting the actin depolymerizing factor cofilin. A similar signaling pathway is important for forming the contractile ring necessary for cytokinesis (see Figure 17–44).

1	5 µm neutrophil Gi G12/13 Rho PI(3,4,5)P3 Rac Rac dominates, polymerization (protrusion) Rho dominates, actin–myosin contraction chemoattractant receptor bacterium back front (A) (B) then activates the Arp 2/3 complex leading to lamellipodial protrusion. Through an unknown mechanism, accumulation of the polarized actin web at the leading edge causes further local enhancement of PI3K activity in a positive feedback loop, strengthening the induction of protrusion. The PI(3,4,5)P3 that activates Rac cannot diffuse far from its site of synthesis, since it is rapidly converted back into PI(4,5)P2 by a constitutively active lipid phosphatase. At the same time, binding of the chemoattractant ligand to its receptor activates another signaling pathway that turns on Rho and enhances myosin-based contractility. The two processes directly inhibit each other, such that Rac activation dominates in the front of the cell and Rho activation dominates in the rear (Figure 16–86B). This enables the cell

1	The two processes directly inhibit each other, such that Rac activation dominates in the front of the cell and Rho activation dominates in the rear (Figure 16–86B). This enables the cell to maintain its functional polarity with protrusion at the leading edge and contraction at the back.

1	Nondiffusible chemical cues attached to the extracellular matrix or to the surface of cells can also influence the direction of cell migration. When these signals activate receptors, they can cause increased cell adhesion and directed actin polymerization. Most long-distance cell migrations in animals, including neuralcrest-cell migration and the travels of neuronal growth cones, depend on a combination of diffusible and nondiffusible signals to steer the locomoting cells or growth cones to their proper destinations.

1	The interconnected cytoskeleton is crucial for cell migration. Although movement is driven primarily by actin polymerization and myosin contractility, septins and intermediate filaments also participate. For example, vimentin intermediate filament networks associate with integrins at focal adhesions, and vimentin-deficient fibroblasts display impaired mechanical stability, migration, and contractile capacity. Furthermore, disruption of linker proteins that connect different cytoskeletal elements, including several plakins and KASH proteins, leads to defects in cell polarization and migration. Thus, interactions among cytoplasmic filament systems, as well as mechanical linkage to the nucleus, are required for complex, whole-cell behaviors such as migration.

1	Cells also use microtubules to help organize persistent movement in a specific direction. In many locomoting cells, the position of the centrosome is influenced by the location of protrusive actin polymerization. Activation of receptors on the protruding front edge of a cell might locally activate dynein motor proteins that move the centrosome by pulling on its microtubules. Several effector proteins downstream of Rac and Rho modulate microtubule dynamics directly: for example, a protein kinase activated by Rac can phosphorylate (and thereby inhibit) the tubulin-binding protein stathmin (see Panel 16–4), thereby stabilizing microtubules.

1	Figure 16–86 Neutrophil polarization and chemotaxis. (A) The pipette tip at the right is leaking a small amount of the bacterial peptide formyl-Met-Leu-Phe, which is recognized by the human neutrophil as the product of a foreign invader. The neutrophil quickly extends a new lamellipodium toward the source of the chemoattractant peptide (top). It then extends this lamellipodium and polarizes its cytoskeleton so that contractile myosin II is located primarily at the rear, opposite the position of the lamellipodium (middle). Finally, the cell crawls toward the source of the peptide (bottom). If a real bacterium were the source of the peptide, rather than an investigator’s pipette, the neutrophil would engulf the bacterium and destroy it (see also Figure 16–3 and Movie 16.14). (B) Binding of bacterial molecules to G-protein-coupled receptors on the neutrophil stimulates directed motility. These receptors are found all over the surface of the cell, but are more likely to be bound to the

1	bacterial molecules to G-protein-coupled receptors on the neutrophil stimulates directed motility. These receptors are found all over the surface of the cell, but are more likely to be bound to the bacterial ligand at the front. Two distinct signaling pathways contribute to the cell’s polarization. At the front of the cell, stimulation of the Rac pathway leads, via the trimeric G protein Gi, to growth of protrusive actin networks. Second messengers within this pathway are short-lived, so protrusion is limited to the region of the cell closest to the stimulant. The same receptor also stimulates a second signaling pathway, via the trimeric G proteins G12 and G13, that triggers the activation of Rho. The two pathways are mutually antagonistic. Since Rac-based protrusion is active at the front of the cell, Rho is activated only at the rear of the cell, stimulating contraction of the cell rear and assisting directed movement. (A, from

1	O.D. Weiner et al., Nat. Cell Biol. 1:75–81, 1999. With permission from Macmillan Publishers Ltd.) In turn, microtubules influence actin rearrangements and cell adhesion. The centrosome nucleates a large number of dynamic microtubules, and its repositioning means that the plus ends of many of these microtubules extend into the protrusive region of the cell. Direct interactions with microtubules help guide focal adhesion dynamics in migrating cells. Microtubules might also influence actin filament formation by delivering Rac-GEFs that bind to the +TIPs traveling on growing microtubule ends. Microtubules also transport cargoes to and from the focal adhesions, thereby affecting their signaling and disassembly. Thus, microtubules reinforce the polarity information that the actin cytoskeleton receives from the outside world, allowing a sensitive response to weak signals and enabling motility to persist in the same direction for a prolonged period.

1	Whole-cell movements and the large-scale shaping and structuring of cells require the coordinated activities of all three basic filament systems along with a large variety of cytoskeletal accessory proteins, including motor proteins. Cell crawling—a widespread behavior important in embryonic development and also in wound healing, tissue maintenance, and immune system function in the adult animal— is a prime example of such complex, coordinated cytoskeletal action. For a cell to crawl, it must generate and maintain an overall structural polarity, which is influenced by external cues. In addition, the cell must coordinate protrusion at the leading edge (by assembly of new actin filaments), adhesion of the newly protruded part of the cell to the substratum, and forces generated by molecular motors to bring the cell body forward. Which statements are true? Explain why or why not.

1	Which statements are true? Explain why or why not. 16–1 The role of ATP hydrolysis in actin polymerization is similar to the role of GTP hydrolysis in tubulin polymerization: both serve to weaken the bonds in the polymer and thereby promote depolymerization. 16–2 Motor neurons trigger action potentials in muscle cell membranes that open voltage-sensitive Ca2+ channels in T tubules, allowing extracellular Ca2+ to enter the cytosol, bind to troponin C, and initiate rapid muscle contraction. 16–3 In most animal cells, minus-end directed microtubule motors deliver their cargo to the periphery of the cell, whereas plus-end directed microtubule motors deliver their cargo to the interior of the cell. Discuss the following problems.

1	Discuss the following problems. 16–4 The concentration of actin in cells is 50–100 times greater than the critical concentration observed for pure actin in a test tube. How is this possible? What prevents the actin subunits in cells from polymerizing into filaments? Why is it advantageous to the cell to maintain such a large pool of actin subunits? 16–5 Detailed measurements of sarcomere length and tension during isometric contraction in striated muscle provided crucial early support for the sliding-filament 2.0 2.2 1.6 1.3 I 3.6 How is the cell cortex regulated locally and globally to coordinate its activities at different places on the cell surface? What determines, for example, where filopodia form? How are actin-regulatory proteins controlled spatially in the cytoplasm to generate multiple distinct types of actin arrays in the same cell? Are there biologically important processes occurring inside a microtubule?

1	Are there biologically important processes occurring inside a microtubule? How can we account for the fact that there are many different kinesins and myosins in the cytoplasm but only one dynein? Mutations in the nuclear lamin proteins cause a large number of diseases called laminopathies. What do we not understand about the nuclear lamina that could account for this fact? Figure Q16–1 Tension as a function of sarcomere length during isometric contraction (Problem 16–5).

1	Figure Q16–1 Tension as a function of sarcomere length during isometric contraction (Problem 16–5). model of muscle contraction. Based on your understanding of the sliding-filament model and the structure of a sarcomere, propose a molecular explanation for the relationship of tension to sarcomere length in the portions of Figure Q16–1 marked I, II, III, and IV. (In this muscle, the length of the myosin filament is 1.6 μm, and the lengths of the actin thin filaments that project from the Z discs are 1.0 μm.) 16–6 At 1.4 mg/mL pure tubulin, microtubules grow at a rate of about 2 μm/min. At this growth rate, how many αβ-tubulin dimers (8 nm in length) are added to the ends of a microtubule each second? Figure Q16–2 Model for microtubule nucleation by pure αβ-tubulin dimers (Problem 16–7).

1	Figure Q16–2 Model for microtubule nucleation by pure αβ-tubulin dimers (Problem 16–7). 16–7 A solution of pure αβ-tubulin dimers is thought to nucleate microtubules by forming a linear protofilament about seven dimers in length. At that point, the probabilities that the next αβ-dimer will bind laterally or to the end of the protofilament are about equal. The critical event for microtubule formation is thought to be the first lateral association (Figure Q16–2). How does lateral association promote the subsequent rapid formation of a microtubule? 16–8 How does a centrosome “know” when it has found the center of the cell?

1	16–8 How does a centrosome “know” when it has found the center of the cell? 16–9 The movements of single motor-protein molecules can be analyzed directly. Using polarized laser light, it is possible to create interference patterns that exert a centrally directed force, ranging from zero at the center to a few piconewtons at the periphery (about 200 nm from the center). Individual molecules that enter the interference pattern are rapidly pushed to the center, allowing them to be captured and moved at the experimenter’s discretion.

1	Using such “optical tweezers,” single kinesin molecules can be positioned on a microtubule that is fixed to a coverslip. Although a single kinesin molecule cannot be seen optically, it can be tagged with a silica bead and tracked indirectly by following the bead (Figure Q16–3A). In the absence of ATP, the kinesin molecule remains at the center of the interference pattern, but with ATP it moves toward the plus end of the microtubule. As kinesin moves along the microtubule, it encounters the force of the interference pattern, which simulates the load kinesin carries during its actual function in the cell. Moreover, the pressure against the silica bead counters the effects of Brownian (thermal) motion, so that the position of the bead more accurately reflects the position of the kinesin molecule on the microtubule. A trace of the movements of a kinesin molecule along a microtubule is shown in Figure Q16–3B.

1	A trace of the movements of a kinesin molecule along a microtubule is shown in Figure Q16–3B. A. As shown in Figure Q16–3B, all movement of kine-sin is in one direction (toward the plus end of the micro-tubule). What supplies the free energy needed to ensure a unidirectional movement along the microtubule? b. What is the average rate of movement of kinesin along the microtubule? C. What is the length of each step that a kinesin takes as it moves along a microtubule? D. From other studies it is known that kinesin has two globular domains that can each bind to β-tubulin, and that kinesin moves along a single protofilament in a microtubule. In each protofilament, the β-tubulin subunit repeats at 8-nm intervals. Given the step length and the interval between β-tubulin subunits, how do you suppose a kine-sin molecule moves along a microtubule? E. Is there anything in the data in Figure Q16–3B that tells you how many ATP molecules are hydrolyzed per step?

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1	Saarikangas J & Barral Y (2011) The emerging functions of septins in metazoans. EMBO Rep. 12, 1118–1126. Abercrombie M (1980) The crawling movement of metazoan cells. Proc. R. Soc. Lond. B 207, 129–147. Gardel ML, Schneider IC, Aratyn-Schaus Y & Waterman CM (2010) Mechanical integration of actin and adhesion dynamics in cell migration. Annu. Rev. Cell Dev. Biol. 26, 315–333. Lo CM, Wang HB, Dembo M & Wang YL (2000) Cell movement is guided by the rigidity of the substrate. Biophys. J. 79, 144–152. Madden K & Snyder M (1998) Cell polarity and morphogenesis in budding yeast. Annu. Rev. Microbiol. 52, 687–744. Parent CA & Devreotes PN (1999) A cell’s sense of direction. Science 284, 765–770. Pollard TD & Borisy GG (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453–465. Rafelski SM & Theriot JA (2004) Crawling toward a unified model of cell mobility: spatial and temporal regulation of actin dynamics. Annu. Rev. Biochem. 73, 209–239.

1	Rafelski SM & Theriot JA (2004) Crawling toward a unified model of cell mobility: spatial and temporal regulation of actin dynamics. Annu. Rev. Biochem. 73, 209–239. Ridley A (2011) Life at the leading edge. Cell 145, 1012–1022. Vitriol EA & Zheng JQ (2012) Growth cone travel in space and time: the cellular ensemble of cytoskeleton, adhesion, and membrane. Neuron 73, 1068–1081. Weiner OD (2002) Regulation of cell polarity during eukaryotic chemotaxis: the chemotactic compass. Curr. Opin. Cell Biol. 14, 196–202. The Cell Cycle 17 The only way to make a new cell is to duplicate a cell that already exists. This simple fact, first established in the middle of the nineteenth century, carries with it a profound message for the continuity of life. All living organisms, from the unicellular bacterium to the multicellular mammal, are products of repeated rounds of cell growth and division extending back in time to the beginnings of life on Earth over three billion years ago.

1	A cell reproduces by performing an orderly sequence of events in which it duplicates its contents and then divides in two. This cycle of duplication and division, known as the cell cycle, is the essential mechanism by which all living things reproduce. In unicellular species, such as bacteria and yeasts, each cell division produces a complete new organism. In multicellular species, long and complex sequences of cell divisions are required to produce a functioning organism. Even in the adult body, cell division is usually needed to replace cells that die. In fact, each of us must manufacture many millions of cells every second simply to survive: if all cell division were stopped—by exposure to a very large dose of x-rays, for example—we would die within a few days.

1	The details of the cell cycle vary from organism to organism and at different times in an organism’s life. Certain characteristics, however, are universal. At a minimum, the cell must accomplish its most fundamental task: the passing on of its genetic information to the next generation of cells. To produce two genetically identical daughter cells, the DNA in each chromosome must first be faithfully replicated to produce two complete copies. The replicated chromosomes must then be accurately distributed (segregated) to the two daughter cells, so that each receives a copy of the entire genome (Figure 17–1). In addition to duplicating their genome, most cells also duplicate their other organelles and macromolecules; otherwise, daughter cells would get smaller with each division. To maintain their size, dividing cells must coordinate their growth (that is, their increase in cell mass) with their division.

1	This chapter describes the events of the cell cycle and how they are controlled and coordinated. We begin with a brief overview of the cell cycle. We then describe the cell-cycle control system, a complex network of regulatory proteins that triggers the different events of the cycle. We next consider in detail the major stages of the cell cycle, in which the chromosomes are duplicated and then segregated into the two daughter cells. Finally, we consider how extracellular signals govern the rates of cell growth and division and how these two processes are coordinated.

1	The most basic function of the cell cycle is to duplicate the vast amount of DNA in the chromosomes and then segregate the copies into two genetically identical daughter cells. These processes define the two major phases of the cell cycle. Chromosome duplication occurs during S phase (S for DNA synthesis), which requires 10–12 hours and occupies about half of the cell-cycle time in a typical mammalian cell. After S phase, chromosome segregation and cell division occur in M phase (M for mitosis), which requires much less time (less than an hour in a mammalian cell). M phase comprises two major events: nuclear division, or mitosis, during which the copied chromosomes are distributed into a pair of daughter nuclei; and cytoplasmic division, or cytokinesis, when the cell itself divides in two (Figure 17–2).

1	At the end of S phase, the DNA molecules in each pair of duplicated chromosomes are intertwined and held tightly together by specialized protein linkages. Early in mitosis at a stage called prophase, the two DNA molecules are gradually disentangled and condensed into pairs of rigid, compact rods called sister chromatids, which remain linked by sister-chromatid cohesion. When the nuclear envelope disassembles later in mitosis, the sister-chromatid pairs become attached to the mitotic spindle, a giant bipolar array of microtubules (discussed in Chapter 16). Sister chromatids are attached to opposite poles of the spindle and, eventually, align at the spindle equator in a stage called metaphase. The destruction of sister-chromatid cohesion at the start of anaphase separates the sister chromatids, which are pulled to opposite poles of the spindle. The spindle is then disassembled, and the segregated chromosomes are packaged into separate nuclei at telophase. Cytokinesis then cleaves the

1	which are pulled to opposite poles of the spindle. The spindle is then disassembled, and the segregated chromosomes are packaged into separate nuclei at telophase. Cytokinesis then cleaves the cell in two, so that each daughter cell inherits one of the two nuclei (Figure 17–3).

1	The Eukaryotic Cell Cycle Usually Consists of Four phases Most cells require much more time to grow and double their mass of proteins and organelles than they require to duplicate their chromosomes and divide. Partly to allow time for growth, most cell cycles have gap phases—a G1 phase between M phase and S phase and a G2 phase between S phase and mitosis. Thus, the eukaryotic cell cycle is traditionally divided into four sequential phases: G1, S, G2, and M. G1, S, and G2 together are called interphase (Figure 17–4, and see Figure 17–3). In a typical human cell proliferating in culture, interphase might occupy 23 hours of a 24-hour cycle, with 1 hour for M phase. Cell growth occurs throughout the cell cycle, except during mitosis. The two gap phases are more than simple time delays to allow cell growth. They also provide time for the cell to monitor the internal and external environment

1	The two gap phases are more than simple time delays to allow cell growth. They also provide time for the cell to monitor the internal and external environment Figure 17–2 The major events of the cell cycle. The major chromosomal events of the cell cycle occur in s phase, when the chromosomes are duplicated, and m phase, when the duplicated chromosomes are segregated into a pair of daughter nuclei (in mitosis), after which the cell itself divides into two (cytokinesis). Figure 17–1 The cell cycle. The division of a hypothetical eukaryotic cell with two chromosomes (one red, and one black) is shown to illustrate how two genetically identical daughter cells are produced in each cycle. Each of the daughter cells will often continue to divide by going through additional cell cycles.

1	Figure 17–3 The events of eukaryotic cell division as seen under a microscope. The easily visible processes of nuclear division (mitosis) and cell division (cytokinesis), collectively called m phase, typically occupy only a small fraction of the cell cycle. The other, much longer, part of the cycle is known as interphase, which includes s phase and the gap phases (discussed in text). The five stages of mitosis are shown: an abrupt change in the biochemical state of the cell occurs at the transition from metaphase to anaphase. a cell can pause in metaphase before this transition point, but once it passes this point, the cell carries on to the end of mitosis and through cytokinesis into interphase.

1	to ensure that conditions are suitable and preparations are complete before the cell commits itself to the major upheavals of S phase and mitosis. The G1 phase is especially important in this respect. Its length can vary greatly depending on external conditions and extracellular signals from other cells. If extracellular conditions are unfavorable, for example, cells delay progress through G1 and may even enter a specialized resting state known as G0 (G zero), in which they can remain for days, weeks, or even years before resuming proliferation. Indeed, many cells remain permanently in G0 until they or the organism dies. If extracellular conditions are favorable and signals to grow and divide are present, cells in early G1 or G0 progress through a commitment point near the end of G1 known as Start (in yeasts) or the restriction point (in mammalian cells). We will use the term Start for both yeast and animal cells. After passing this point, cells are committed to DNA replication, even

1	as Start (in yeasts) or the restriction point (in mammalian cells). We will use the term Start for both yeast and animal cells. After passing this point, cells are committed to DNA replication, even if the extracellular signals that stimulate cell growth and division are removed.

1	Some features of the cell cycle, including the time required to complete certain events, vary greatly from one cell type to another, even in the same organism. The basic organization of the cycle, however, is essentially the same in all eukaryotic Figure 17–4 The four phases of the cell cycle. In most cells, gap phases separate the major events of s phase and m phase. g1 is the gap between m phase and s phase, while g2 is the gap between s phase and m phase.

1	Figure 17–5 Mammalian cells proliferating in culture. The cells in this scanning electron micrograph are rat fibroblasts. Cells at the lower left have rounded up and are in mitosis. (Courtesy of guenter albrecht-Buehler.) cells, and all eukaryotes appear to use similar machinery and control mechanisms to drive and regulate cell-cycle events. The proteins of the cell-cycle control system, for example, first appeared over a billion years ago. Remarkably, they have been so well conserved over the course of evolution that many of them function perfectly when transferred from a human cell to a yeast cell. We can therefore study the cell cycle and its regulation in a variety of organisms and use the findings from all of them to assemble a unified picture of how eukaryotic cells divide.

1	Several model organisms are used in the analysis of the eukaryotic cell cycle. The budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are simple eukaryotes in which powerful molecular and genetic approaches can be used to identify and characterize the genes and proteins that govern the fundamental features of cell division. The early embryos of certain animals, particularly those of the frog Xenopus laevis, are excellent tools for biochemical dissection of cell-cycle control mechanisms, while the fruit fly Drosophila melanogaster is useful for the genetic analysis of mechanisms underlying the control and coordination of cell growth and division in multicellular organisms. Cultured human cells provide an excellent system for the molecular and microscopic exploration of the complex processes by which our own cells divide.

1	How can we tell what stage a cell has reached in the cell cycle? One way is simply to look at living cells with a microscope. A glance at a population of mammalian cells proliferating in culture reveals that a fraction of the cells have rounded up and are in mitosis (Figure 17–5). Others can be observed in the process of cytokinesis. Similarly, looking at budding yeast cells under a microscope is very useful, because the size of the bud provides an indication of cell-cycle stage (Figure 17–6). We can gain additional clues about cell-cycle position by staining cells with DNA-binding fluorescent dyes (which reveal the condensation of chromosomes in mitosis) or with antibodies that recognize specific cell components such as the microtubules (revealing the mitotic spindle). S-phase cells can be identified in the microscope by supplying them with visualizable molecules that are incorporated into newly synthesized DNA, such as the artificial thymidine analog bromodeoxyuridine (BrdU); cell

1	be identified in the microscope by supplying them with visualizable molecules that are incorporated into newly synthesized DNA, such as the artificial thymidine analog bromodeoxyuridine (BrdU); cell nuclei that have incorporated BrdU are then revealed by staining with anti-BrdU antibodies (Figure 17–7).

1	Typically, in a population of cultured mammalian cells that are all proliferating rapidly but asynchronously, about 30–40% will be in S phase at any instant and become labeled by a brief pulse of BrdU. From the proportion of cells in such a population that are labeled, we can estimate the duration of S phase as a fraction of the whole cell-cycle duration. Similarly, from the proportion of cells in mitosis (the mitotic index), we can estimate the duration of M phase. Another way to assess the stage that a cell has reached in the cell cycle is by measuring its DNA content, which doubles during S phase. This approach is greatly facilitated by the use of fluorescent DNA-binding dyes and a flow cytometer, which allows the rapid and automatic analysis of large numbers of cells (Figure 17–8). We can use flow cytometry to determine the lengths of G1, S, and G2 + M phases, by measuring DNA content in a synchronized cell population as it progresses through the cell cycle.

1	Figure 17–6 The morphology of budding yeast cells. In a normal population of proliferating yeast cells, buds vary in size according to the cell-cycle stage. Unbudded cells are in g1. progression through the start transition triggers formation of a tiny bud, which grows in size during the s and m phases until it is almost the size of the mother cell. (Courtesy of Jeff Ubersax.) 20 µm Figure 17–7 Labeling S-phase cells. an immunofluorescence micrograph of BrdU-labeled epithelial cells of the zebrafish gut. The fish was exposed to BrdU, after which the tissue was fixed and prepared for labeling with fluorescent anti-BrdU antibodies (green). all the cells are stained with a red fluorescent dye. (Courtesy of Cécile Crosnier.)

1	Cell division usually begins with duplication of the cell’s contents, followed by distribution of those contents into two daughter cells. Chromosome duplication occurs during S phase of the cell cycle, whereas most other cell components are duplicated continuously throughout the cycle. During M phase, the replicated chromosomes are segregated into individual nuclei (mitosis), and the cell then splits in two (cytokinesis). S phase and M phase are usually separated by gap phases called G1 and G2, when various intracellular and extracellular signals regulate cell-cycle progression. Cell-cycle organization and control have been highly conserved during evolution, and studies in a wide range of systems have led to a unified view of eukaryotic cell-cycle control.

1	For many years, cell biologists watched the puppet show of DNA synthesis, mitosis, and cytokinesis but had no idea of what lay behind the curtain controlling these events. It was not even clear whether there was a separate control system, or whether the processes of DNA synthesis, mitosis, and cytokinesis somehow controlled themselves. A major breakthrough came in the late 1980s with the identification of the key proteins of the control system, along with the realization that they are distinct from the proteins that perform the processes of DNA replication, chromosome segregation, and so on. In this section, we first consider the basic principles upon which the cell-cycle control system operates. We then discuss the protein components of the system and how they work together to time and coordinate the events of the cell cycle. The Cell-Cycle Control system Triggers the major Events of the Cell Cycle

1	The cell-cycle control system operates much like a timer that triggers the events of the cell cycle in a set sequence (Figure 17–9). In its simplest form—as seen in the stripped-down cell cycles of early animal embryos, for example—the control system is rigidly programmed to provide a fixed amount of time for the completion of each cell-cycle event. The control system in these early embryonic divisions is independent of the events it controls, so that its timing mechanisms continue to operate even if those events fail. In most cells, however, the control system does respond to information received back from the processes it controls. If some malfunction prevents the successful completion of DNA synthesis, for example, signals are sent to the control system to delay progression to M phase. Such delays provide time for the machinery to be repaired and also prevent the disaster that might result if the cycle progressed prematurely to the next stage—and segregated incompletely replicated

1	Such delays provide time for the machinery to be repaired and also prevent the disaster that might result if the cycle progressed prematurely to the next stage—and segregated incompletely replicated chromosomes, for example.

1	The cell-cycle control system is based on a connected series of biochemical switches, each of which initiates a specific cell-cycle event. This system of switches possesses many important features that increase the accuracy and reliability of cell-cycle progression. First, the switches are generally binary (on/off ) and launch events in a complete, irreversible fashion. It would clearly be disastrous, for example, if events like chromosome condensation or nuclear-envelope breakdown were only partially initiated or started but not completed. Second, the cell-cycle control system is remarkably robust and reliable, partly because backup mechanisms and other features allow the system to operate effectively under a variety of conditions and even if some components fail. Finally, the control system is highly adaptable and can be modified to suit specific cell types or to respond to specific intracellular or extracellular signals. number of cells 012 relative amount of DNA per cell

1	Figure 17–8 Analysis of DNA content with a flow cytometer. This graph shows typical results obtained for a proliferating cell population when the dna content of its individual cells is determined in a flow cytometer. (a flow cytometer, also called a fluorescence-activated cell sorter, or FaCs, can also be used to sort cells according to their fluorescence—see Figure 8–2). The cells analyzed here were stained with a dye that becomes fluorescent when it binds to dna, so that the amount of fluorescence is directly proportional to the amount of dna in each cell. The cells fall into three categories: those that have an unreplicated complement of dna and are therefore in g1, those that have a fully replicated complement of dna (twice the g1 dna content) and are in g2 or m phase, and those that have an intermediate amount of dna and are in s phase. The distribution of cells indicates that there are greater numbers of cells in g1 than in g2 + m phase, showing that g1 is longer than g2 + m in

1	have an intermediate amount of dna and are in s phase. The distribution of cells indicates that there are greater numbers of cells in g1 than in g2 + m phase, showing that g1 is longer than g2 + m in this population.

1	Are all chromosomes attached to the spindle? Is all DNA replicated? Is environment favorable? G2 M S G1 CONTROLLER ENTER MITOSIS TRIGGER ANAPHASE AND PROCEED TO CYTOKINESIS ENTER CELL CYCLE AND PROCEED TO S PHASE START TRANSITION Is environment favorable? G2/M TRANSITION

1	In most eukaryotic cells, the cell-cycle control system governs cell-cycle progression at three major regulatory transitions (see Figure 17–9). The first is Start (or the restriction point) in late G1, where the cell commits to cell-cycle entry and chromosome duplication. The second is the G2/M transition, where the control system triggers the early mitotic events that lead to chromosome alignment on the mitotic spindle in metaphase. The third is the metaphase-to-anaphase transition, where the control system stimulates sister-chromatid separation, leading to the completion of mitosis and cytokinesis. The control system blocks progression through each of these transitions if it detects problems inside or outside the cell. If the control system senses problems in the completion of DNA replication, for example, it will hold the cell at the G2/M transition until those problems are solved. Similarly, if extracellular conditions are not appropriate for cell proliferation, the control system

1	for example, it will hold the cell at the G2/M transition until those problems are solved. Similarly, if extracellular conditions are not appropriate for cell proliferation, the control system blocks progression through Start, thereby preventing cell division until conditions become favorable.

1	The Cell-Cycle Control system depends on Cyclically activated Cyclin-dependent protein kinases (Cdks) Central components of the cell-cycle control system are members of a family of protein kinases known as cyclin-dependent kinases (Cdks). The activities of these kinases rise and fall as the cell progresses through the cycle, leading to cyclical changes in the phosphorylation of intracellular proteins that initiate or regulate the major events of the cell cycle. An increase in Cdk activity at the G2/M transition, for example, increases the phosphorylation of proteins that control chromosome condensation, nuclear-envelope breakdown, spindle assembly, and other events that occur in early mitosis.

1	Cyclical changes in Cdk activity are controlled by a complex array of enzymes and other proteins. The most important of these Cdk regulators are proteins known as cyclins. Cdks, as their name implies, are dependent on cyclins for their activity: unless they are bound tightly to a cyclin, they have no protein kinase activity (Figure 17–10). Cyclins were originally named because they undergo a cycle of synthesis and degradation in each cell cycle. The levels of the Cdk proteins, by contrast, are constant. Cyclical changes in cyclin protein levels result in the cyclic assembly and activation of cyclin–Cdk complexes at specific stages of the cell cycle. Figure 17–9 The control of the cell cycle.

1	Figure 17–9 The control of the cell cycle. a cell-cycle control system triggers the essential processes of the cycle—such as dna replication, mitosis, and cytokinesis. The control system is represented here as a central arm—the controller—that rotates clockwise, triggering essential processes when it reaches specific transitions on the outer dial (yellow boxes). Information about the completion of cell-cycle events, as well as signals from the environment, can cause the control system to arrest the cycle at these transitions. Figure 17–10 Two key components of the cell-cycle control system. When cyclin forms a complex with Cdk, the protein kinase is activated to trigger specific cell-cycle events. Without cyclin, Cdk is inactive. There are four classes of cyclins, each defined by the stage of the cell cycle at which they bind Cdks and function. All eukaryotic cells require three of these classes (Figure 17–11): 1.

1	There are four classes of cyclins, each defined by the stage of the cell cycle at which they bind Cdks and function. All eukaryotic cells require three of these classes (Figure 17–11): 1. G1/S-cyclins activate Cdks in late G1 and thereby help trigger progression through Start, resulting in a commitment to cell-cycle entry. Their levels fall in S phase. 2. S-cyclins bind Cdks soon after progression through Start and help stimulate chromosome duplication. S-cyclin levels remain elevated until mitosis, and these cyclins also contribute to the control of some early mitotic events. 3. M-cyclins activate Cdks that stimulate entry into mitosis at the G2/M transition. M-cyclin levels fall in mid-mitosis. In most cells, a fourth class of cyclins, the G1-cyclins, helps govern the activities of the G1/S-cyclins, which control progression through Start in late G1.

1	In yeast cells, a single Cdk protein binds all classes of cyclins and triggers different cell-cycle events by changing cyclin partners at different stages of the cycle. In vertebrate cells, by contrast, there are four Cdks. Two interact with G1-cyclins, one with G1/Sand S-cyclins, and one with Sand M-cyclins. In this chapter, we simply refer to the different cyclin–Cdk complexes as G1-Cdk, G1/S-Cdk, S-Cdk, and M-Cdk. Table 17–1 lists the names of the individual Cdks and cyclins.

1	How do different cyclin–Cdk complexes trigger different cell-cycle events? The answer, at least in part, seems to be that the cyclin protein does not simply activate its Cdk partner but also directs it to specific target proteins. As a result, each cyclin–Cdk complex phosphorylates a different set of substrate proteins. The same cyclin–Cdk complex can also induce different effects at different times in the cycle, probably because the accessibility of some Cdk substrates changes during the cell cycle. Certain proteins that function in mitosis, for example, may become available for phosphorylation only in G2.

1	Figure 17–11 Cyclin–Cdk complexes of the cell-cycle control system. The concentrations of the three major cyclin types oscillate during the cell cycle, while the concentrations of Cdks (not shown) do not change and exceed cyclin amounts. In late g1, rising g1/s-cyclin levels lead to the formation of g1/s-Cdk complexes that trigger progression through the start transition. s-Cdk complexes form at the start of s phase and trigger dna replication, as well as some early mitotic events. m-Cdk complexes form during g2 but are held in an inactive state; they are activated at the end of g2 and trigger entry into mitosis at the g2/m transition. a separate regulatory protein complex, the apC/C, initiates the metaphase-toanaphase transition, as we discuss later.

1	Studies of the three-dimensional structures of Cdk and cyclin proteins have revealed that, in the absence of cyclin, the active site in the Cdk protein is partly obscured by a protein loop, like a stone blocking the entrance to a cave (Figure 17–12A). Cyclin binding causes the loop to move away from the active site, resulting in partial activation of the Cdk enzyme (Figure 17–12B). Full activation of the cyclin–Cdk complex then occurs when a separate kinase, the Cdk-activating kinase (CAK), phosphorylates an amino acid near the entrance of the Cdk active site. This causes a small conformational change that further increases the activity of the Cdk, allowing the kinase to phosphorylate its target proteins effectively and thereby induce specific cell-cycle events (Figure 17–12C). The rise and fall of cyclin levels is the primary determinant of Cdk activity during the cell cycle. Several additional mechanisms, however, help control Cdk activity at specific stages of the cycle.

1	Phosphorylation at a pair of amino acids in the roof of the kinase active site inhibits the activity of a cyclin–Cdk complex. Phosphorylation of these sites by a protein kinase known as Wee1 inhibits Cdk activity, while dephosphorylation of these sites by a phosphatase known as Cdc25 increases Cdk activity (Figure 17–13). We will see later that this regulatory mechanism is particularly important in the control of M-Cdk activity at the onset of mitosis. Binding of Cdk inhibitor proteins (CKIs) inactivates cyclin–Cdk complexes. The three-dimensional structure of a cyclin–Cdk–CKI complex reveals that CKI binding stimulates a large rearrangement in the structure of the Cdk active site, rendering it inactive (Figure 17–14). Cells use CKIs primarily to help govern the activities of G1/Sand S-Cdks early in the cell cycle. Regulated proteolysis Triggers the metaphase-to-anaphase Transition

1	Regulated proteolysis Triggers the metaphase-to-anaphase Transition Whereas activation of specific cyclin–Cdk complexes drives progression through the Start and G2/M transitions (see Figure 17–11), progression through the meta-phase-to-anaphase transition is triggered not by protein phosphorylation but by protein destruction, leading to the final stages of cell division. The key regulator of the metaphase-to-anaphase transition is the anaphasepromoting complex, or cyclosome (APC/C), a member of the ubiquitin ligase family of enzymes. As discussed in Chapter 3, these enzymes are used in numerous cell processes to stimulate the proteolytic destruction of specific regulatory proteins. They polyubiquitylate specific target proteins, resulting in their destruction in proteasomes. Other ubiquitin ligases mark proteins for purposes other than destruction (discussed in Chapter 3).

1	Figure 17–12 The structural basis of Cdk activation. These drawings are based on three-dimensional structures of human Cdk2 and cyclin a, as determined by x-ray crystallography. The location of the bound aTp is indicated. The enzyme is shown in three states. (a) In the inactive state, without cyclin bound, the active site is blocked by a region of the protein called the T-loop (red). (B) The binding of cyclin causes the T-loop to move out of the active site, resulting in partial activation of the Cdk2. (C) phosphorylation of Cdk2 (by Cak) at a threonine residue in the T-loop further activates the enzyme by changing the shape of the T-loop, improving the ability of the enzyme to bind its protein substrates (Movie 17.1).

1	Figure 17–13 The regulation of Cdk activity by phosphorylation. The active cyclin–Cdk complex is turned off when the kinase Wee1 phosphorylates two closely spaced sites above the active site. Removal of these phosphates by the phosphatase Cdc25 activates the cyclin– Cdk complex. For simplicity, only one inhibitory phosphate is shown. Cak adds the activating phosphate, as shown in Figure 17–12. Figure 17–14 The inhibition of a cyclin–Cdk complex by a CKI. This drawing is based on the three-dimensional structure of the human cyclin a–Cdk2 complex bound to the CkI p27, as determined by x-ray crystallography. The p27 binds to both the cyclin and Cdk in the complex, distorting the active site of the Cdk. It also inserts into the aTp-binding site, further inhibiting the enzyme activity.

1	The APC/C catalyzes the ubiquitylation and destruction of two major types of proteins. The first is securin, which protects the protein linkages that hold sister-chromatid pairs together in early mitosis. Destruction of securin in meta-phase activates a protease that separates the sisters and unleashes anaphase, as described later. The Sand M-cyclins are the second major targets of the APC/C. Destroying these cyclins inactivates most Cdks in the cell (see Figure 17–11). As a result, the many proteins phosphorylated by Cdks from S phase to early mitosis are dephosphorylated by various phosphatases in the anaphase cell. This dephosphorylation of Cdk targets is required for the completion of M phase, including the final steps in mitosis and then cytokinesis. Following its activation in mid-mitosis, the APC/C remains active in G1 to provide a stable period of Cdk inactivity. When G1/S-Cdk is activated in late G1, the APC/C is turned off, thereby allowing cyclin accumulation in the next

1	the APC/C remains active in G1 to provide a stable period of Cdk inactivity. When G1/S-Cdk is activated in late G1, the APC/C is turned off, thereby allowing cyclin accumulation in the next cell cycle.

1	The cell-cycle control system also uses another ubiquitin ligase called SCF (see Figure 3–71). It has many functions in the cell, but its major role in the cell cycle is to ubiquitylate certain CKI proteins in late G1, thereby helping to control the activation of S-Cdks and DNA replication. SCF is also responsible for the destruction of G1/S-cyclins in early S phase.

1	The APC/C and SCF are both large, multisubunit complexes with some related components (see Figure 3–71), but they are regulated differently. APC/C activity changes during the cell cycle, primarily as a result of changes in its association with an activating subunit—either Cdc20 in mid-mitosis or Cdh1 from late mitosis through early G1. These subunits help the APC/C recognize its target proteins (Figure 17–15A). SCF activity depends on substrate-binding subunits called F-box proteins. Unlike APC/C activity, however, SCF activity is constant during the cell cycle. Ubiquitylation by SCF is controlled instead by changes in the phosphorylation state of its target proteins, as F-box subunits recognize only specifically phosphorylated proteins (Figure 17–15B).

1	In the simple cell cycles of early animal embryos, gene transcription does not occur. Cell-cycle control depends exclusively on post-transcriptional mechanisms that involve the regulation of Cdks and ubiquitin ligases and their target proteins. In the more complex cell cycles of most cell types, however, transcriptional control provides an important additional level of regulation. Changes in cyclin gene transcription, for example, help control cyclin levels in most cells. A variety of methods discussed in Chapter 8 have been used to analyze changes in the expression of all genes in the genome as the cell progresses through the cell cycle. The results of these studies are surprising. In budding yeast, for example, about 10% of the genes encode mRNAs whose levels oscillate during the cell cycle. Some of these genes encode proteins with known cell-cycle functions, but the functions of many others are unknown. The Cell-Cycle Control system Functions as a network of Biochemical switches

1	The Cell-Cycle Control system Functions as a network of Biochemical switches Table 17–2 summarizes some of the major components of the cell-cycle control system. These proteins are functionally linked to form a robust network, which operates essentially autonomously to activate a series of biochemical switches, each of which triggers a specific cell-cycle event. When conditions for cell proliferation are right, various external and internal signals stimulate the activation of G1-Cdk, which in turn stimulates the expression of genes encoding G1/Sand S-cyclins (Figure 17–16). The resulting activation of G1/S-Cdk then drives progression through the Start transition. By mechanisms we discuss later, G1/S-Cdks unleash a wave of S-Cdk activity, which initiates (A) control of proteolysis by APC/C (B) control of proteolysis by SCF

1	Figure 17–15 The control of proteolysis by APC/C and SCF during the cell cycle. (a) The apC/C is activated in mitosis by association with Cdc20, which recognizes specific amino acid sequences on m-cyclin and other target proteins. With the help of two additional proteins called E1 and E2, the apC/C assembles polyubiquitin chains on the target protein. The polyubiquitylated target is then recognized and degraded in a proteasome. (B) The activity of the ubiquitin ligase sCF depends on substrate-binding subunits called F-box proteins, of which there are many different types. The phosphorylation of a target protein, such as the CkI shown, allows the target to be recognized by a specific F-box subunit.

1	chromosome duplication in S phase and also contributes to some early events of mitosis. M-Cdk activation then triggers progression through the G2/M transition and the events of early mitosis, leading to the alignment of sister-chromatid pairs at the equator of the mitotic spindle. Finally, the APC/C, together with its activator Cdc20, triggers the destruction of securin and cyclins, thereby unleashing sister-chromatid separation and segregation and the completion of mitosis. When mitosis is complete, multiple mechanisms collaborate to suppress Cdk activity, resulting in a stable G1 period. We are now ready to discuss these cell-cycle stages in more detail, starting with S phase.

1	Figure 17–16 An overview of the cell-cycle control system. The core of the cell-cycle control system consists of a series of cyclin–Cdk complexes (yellow). The activity of each complex is also influenced by various inhibitory mechanisms, which provide information about the extracellular environment, cell damage, and incomplete cell-cycle events (top). These inhibitory mechanisms are not present in all cell types; many are missing in early embryonic cell cycles, for example. The cell-cycle control system triggers the events of the cell cycle and ensures that they are properly timed and coordinated with each other. The control system responds to various intracellular and extracellular signals and arrests the cycle when the cell either fails to complete an essential cell-cycle process or encounters unfavorable environmental or intracellular conditions.

1	Central components of the control system are the cyclin-dependent protein kinases (Cdks), which depend on cyclin subunits for their activity. Oscillations in the activities of different cyclin–Cdk complexes control various cell-cycle events. Thus, activation of S-phase cyclin–Cdk complexes (S-Cdk) initiates S phase, whereas activation of M-phase cyclin–Cdk complexes (M-Cdk) triggers mitosis. The mechanisms that control the activities of cyclin–Cdk complexes include phosphorylation of the Cdk subunit, binding of Cdk inhibitor proteins (CKIs), proteolysis of cyclins, and changes in the transcription of genes encoding Cdk regulators. The cell-cycle control system also depends crucially on two additional enzyme complexes, the APC/C and SCF ubiquitin ligases, which catalyze the ubiquitylation and consequent destruction of specific regulatory proteins that control critical events in the cycle.

1	The linear chromosomes of eukaryotic cells are vast and dynamic assemblies of DNA and protein, and their duplication is a complex process that takes up a major fraction of the cell cycle. Not only must the long DNA molecule of each chromosome be duplicated accurately—a remarkable feat in itself—but the protein packaging surrounding each region of that DNA must also be reproduced, ensuring that the daughter cells inherit all features of chromosome structure.

1	The central event of chromosome duplication—DNA replication—poses two problems for the cell. First, replication must occur with extreme accuracy to minimize the risk of mutations in the next cell generation. Second, every nucleotide in the genome must be copied once, and only once, to prevent the damaging effects of gene amplification. In Chapter 5, we discuss the sophisticated protein machinery that performs DNA replication with astonishing speed and accuracy. In this section, we consider the elegant mechanisms by which the cell-cycle control system initiates the replication process and, at the same time, prevents it from happening more than once per cycle.

1	DNA replication begins at origins of replication, which are scattered at numerous locations in every chromosome. During S phase, DNA replication is initiated at these origins when a DNA helicase unwinds the double helix and DNA replication enzymes are loaded onto the two single-stranded templates. This leads to the elongation phase of replication, when the replication machinery moves outward from the origin at two replication forks (discussed in Chapter 5).

1	To ensure that chromosome duplication occurs only once per cell cycle, the initiation phase of DNA replication is divided into two distinct steps that occur at different times in the cell cycle (Figure 17–17). The first step occurs in late mitosis and early G1, when a pair of inactive DNA helicases is loaded onto the replication origin, forming a large complex called the prereplicative complex or preRC. This step is sometimes called licensing of replication origins because initiation of DNA synthesis is permitted only at origins containing a preRC. The second step occurs in S phase, when the DNA helicases are activated, resulting in DNA unwinding and the initiation of DNA synthesis. Once a replication origin has been fired in this way, the two helicases move out from the origin with the replication forks, and that origin cannot be reused until a new preRC is assembled there at the end of mitosis. As a result, origins can be activated only once per cell cycle.

1	Figure 17–18 illustrates some of the molecular details underlying the control of the two steps in the initiation of DNA replication. A key player is a large multiprotein complex called the origin recognition complex (ORC), which binds to Figure 17–17 Control of chromosome duplication. preparations for dna replication begin in late mitosis and g1, when the dna helicases are loaded by multiple proteins at the replication origin, forming the prereplicative complex (preRC). s-Cdk activation leads to activation of the dna helicases, which unwind the dna at origins to initiate dna replication. Two replication forks move out from each origin until the entire chromosome is duplicated. duplicated chromosomes are then segregated in m phase. s-Cdk activation in s phase also prevents assembly of new preRCs at any origin until the following g1—thereby ensuring that each origin is activated only once in each cell cycle.

1	replication origins throughout the cell cycle. In late mitosis and early G1, the proteins Cdc6 and Cdt1 collaborate with the ORC to load the inactive DNA helicases around the DNA next to the origin. The resulting large complex is the preRC, and the origin is now licensed for replication. At the onset of S phase, S-Cdk triggers origin activation by phosphorylating specific initiator proteins, which then nucleate the assembly of a large protein complex that activates the DNA helicase and recruits the DNA synthesis machinery. Another protein kinase called DDK is also activated in S phase and helps drive origin activation by phosphorylating specific subunits of the DNA helicase.

1	At the same time as S-Cdk initiates DNA replication, several mechanisms prevent assembly of new preRCs. S-Cdk phosphorylates and thereby inhibits the ORC and Cdc6 proteins. Inactivation of the APC/C in late G1 also helps turn off preRC assembly. In late mitosis and early G1, the APC/C triggers the destruction of a Cdt1 inhibitor called geminin, thereby allowing Cdt1 to be active. When the APC/C is turned off in late G1, geminin accumulates and inhibits the Cdt1 that is not associated with DNA. Also, the association of Cdt1 with a protein at active replication forks stimulates Cdt1 destruction. In these various ways, preRC formation is prevented from S phase to mitosis, thereby ensuring that each origin is fired only once per cell cycle. How, then, is the cell-cycle control system reset to allow replication in the next cell cycle? At the end of mitosis, APC/C activation leads to the inactivation of Cdks and the destruction of geminin. ORC and Cdc6 are dephosphorylated and Cdt1 is

1	to allow replication in the next cell cycle? At the end of mitosis, APC/C activation leads to the inactivation of Cdks and the destruction of geminin. ORC and Cdc6 are dephosphorylated and Cdt1 is activated, allowing preRC assembly to prepare the cell for the next S phase.

1	Chromosome duplication Requires duplication of Chromatin structure The DNA of the chromosomes is extensively packaged in a variety of protein components, including histones and various regulatory proteins involved in the control of gene expression (discussed in Chapter 4). Thus, duplication of a chromosome is not simply a matter of replicating the DNA at its core but also requires the duplication of these chromatin proteins and their proper assembly on the DNA.

1	The production of chromatin proteins increases during S phase to provide the raw materials needed to package the newly synthesized DNA. Most importantly, S-Cdks stimulate a large increase in the synthesis of the four histone subunits that form the histone octamers at the core of each nucleosome. These subunits are assembled into nucleosomes on the DNA by nucleosome assembly factors, which typically associate with the replication fork and distribute nucleosomes on both strands of the DNA as they emerge from the DNA synthesis machinery.

1	Chromatin packaging helps to control gene expression. In some parts of the chromosome, the chromatin is highly condensed and is called heterochromatin, whereas in other regions it has a more open structure and is called euchromatin (discussed in Chapter 4). These differences in chromatin structure depend on a variety of mechanisms, including modification of histone tails and the presence of non-histone proteins. Because these differences are important in gene regulation, Cdc6 ORC (origin recognition complex) P P P P P P P P originCdt1 Mcm helicase + prereplicative complex (preRC) initiator proteins S-Cdk DDK INACTIVATION OF ORC, Cdc6, Cdt1 DNA HELICASE ACTIVATION COMPLETION OF DNA REPLICATION G1 S G2 DNA

1	Figure 17–18 Control of the initiation of DNA replication. The replication origin is bound by the ORC throughout the cell cycle. In early g1, Cdc6 associates with the ORC, and these proteins bind the dna helicase, which contains six closely related subunits called mcm proteins. The helicase also associates with a protein called Cdt1. Using energy provided by aTp hydrolysis, the ORC and Cdc6 proteins load two copies of the dna helicase, in an inactive form, around the dna next to the origin, thereby forming the prereplicative complex (preRC). at the onset of s phase, s-Cdk stimulates the assembly of several initiator proteins on each dna helicase, while another protein kinase, ddk, phosphorylates subunits of the dna helicase. as a result, the dna helicases are activated and unwind the dna. dna polymerase and other replication proteins are recruited to the origin, and dna replication begins. The ORC is displaced by the replication machinery and then rebinds. s-Cdk and other mechanisms

1	dna polymerase and other replication proteins are recruited to the origin, and dna replication begins. The ORC is displaced by the replication machinery and then rebinds. s-Cdk and other mechanisms also inactivate the preRC components ORC, Cdc6, and Cdt1, thereby preventing formation of new preRCs at the origins until the end of mitosis (see text).

1	it is crucial that chromatin structure, like the DNA within, is reproduced accurately during S phase. How chromatin structure is reproduced is not well understood, however. During DNA synthesis, histone-modifying enzymes and various non-histone proteins are probably deposited onto the two new DNA strands as they emerge from the replication fork, and these proteins are thought to reproduce the local chromatin structure of the parent chromosome (see Figure 4–45).

1	At the end of S phase, each replicated chromosome consists of a pair of identical sister chromatids glued together along their length. This sister-chromatid cohesion sets the stage for a successful mitosis because it greatly facilitates the attachment of the two sister chromatids to opposite poles of the mitotic spindle. Imagine how difficult it would be to achieve this bipolar attachment if sister chromatids were allowed to drift apart after S phase. Indeed, defects in sister-chromatid cohesion—in yeast mutants, for example—lead inevitably to major errors in chromosome segregation.

1	Sister-chromatid cohesion depends on a large protein complex called cohesin, which is deposited at many locations along the length of each sister chromatid as the DNA is replicated in S phase. Two of the subunits of cohesin are members of a large family of proteins called SMC proteins (for Structural Maintenance of Chromosomes). Cohesin forms giant ringlike structures, and it has been proposed that these surround the two sister chromatids (Figure 17–19).

1	Sister-chromatid cohesion also results, at least in part, from DNA catenation, the intertwining of sister DNA molecules that occurs when two replication forks meet during DNA synthesis. The enzyme topoisomerase II gradually disentangles the catenated sister DNAs between S phase and early mitosis by cutting one DNA molecule, passing the other through the break, and then resealing the cut DNA (see Figure 5–22). Once the catenation has been removed, sister-chromatid cohesion depends primarily on cohesin complexes. The sudden and synchronous loss of sister cohesion at the metaphase-to-anaphase transition therefore depends primarily on disruption of these complexes, as we describe later.

1	Duplication of the chromosomes in S phase involves the accurate replication of the entire DNA molecule in each chromosome, as well as the duplication of the chromatin proteins that associate with the DNA and govern various aspects of chromosome function. Chromosome duplication is triggered by the activation of S-Cdk, which activates proteins that unwind the DNA and initiate its replication at replication origins. Once a replication origin is activated, S-Cdk also inhibits proteins that are required to allow that origin to initiate DNA replication again. Thus, each origin is fired once and only once in each S phase and cannot be reused until the next cell cycle.

1	Figure 17–19 Cohesin. Cohesin is a protein complex with four subunits. (a) Two subunits, smc1 and smc3, are coiled-coil proteins with an aTpase domain at one end; (B) two additional subunits, scc1 and scc3, connect the aTpase head domains, forming a ring structure that may encircle the sister chromatids as shown in (C). The aTpase domains are required for cohesin loading on the dna.

1	Following the completion of S phase and transition through G2, the cell undergoes the dramatic upheaval of M phase. This begins with mitosis, during which the sister chromatids are separated and distributed (segregated) to a pair of identical daughter nuclei, each with its own copy of the genome. Mitosis is traditionally divided into five stages—prophase, prometaphase, metaphase, anaphase, and telophase—defined primarily on the basis of chromosome behavior as seen in a microscope. As mitosis is completed, the second major event of M phase—cytokinesis—divides the cell into two halves, each with an identical nucleus. Panel 17–1 summarizes the major events of M phase (Movie 17.2, Movie 17.3, Movie 17.4, and Movie 17.5).

1	From a regulatory point of view, mitosis can be divided into two major parts, each governed by distinct components of the cell-cycle control system. First, an abrupt increase in M-Cdk activity at the G2/M transition triggers the events of early mitosis (prophase, prometaphase, and metaphase). During this period, M-Cdk and several other mitotic protein kinases phosphorylate a variety of proteins, leading to the assembly of the mitotic spindle and its attachment to the sister-chromatid pairs. The second major part of mitosis begins at the metaphaseto-anaphase transition, when the APC/C triggers the destruction of securin, liberating a protease that cleaves cohesin and thereby initiates separation of the sister chromatids. The APC/C also promotes the destruction of cyclins, which leads to Cdk inactivation and the dephosphorylation of Cdk targets, which is required for all events of late M phase, including the completion of anaphase, the disassembly of the mitotic spindle, and the

1	to Cdk inactivation and the dephosphorylation of Cdk targets, which is required for all events of late M phase, including the completion of anaphase, the disassembly of the mitotic spindle, and the division of the cell by cytokinesis.

1	In this section, we describe the key mechanical events of mitosis and how M-Cdk and the APC/C orchestrate them. One of the most remarkable features of cell-cycle control is that a single protein kinase, M-Cdk, brings about all of the diverse and complex cell rearrangements that occur in the early stages of mitosis. At a minimum, M-Cdk must induce the assembly of the mitotic spindle and ensure that each sister chromatid in a pair is attached to the opposite pole of the spindle. It also triggers chromosome condensation, the large-scale reorganization of the intertwined sister chromatids into compact, rodlike structures. In animal cells, M-Cdk also promotes the breakdown of the nuclear envelope and rearrangements of the actin cytoskeleton and the Golgi apparatus. Each of these processes is thought to be initiated when M-Cdk phosphorylates specific proteins involved in the process, although most of these proteins have not yet been identified.

1	M-Cdk does not act alone to phosphorylate key proteins involved in early mitosis. Two additional families of protein kinases, the Polo-like kinases and the Aurora kinases, also make important contributions to the control of early mitotic events. The Polo-like kinase Plk, for example, is required for the normal assembly of a bipolar mitotic spindle, in part because it phosphorylates proteins involved in separation of the spindle poles early in mitosis. The Aurora kinase Aurora-A also helps control proteins that govern the assembly and stability of the spindle, whereas Aurora-B controls attachment of sister chromatids to the spindle, as we discuss later. dephosphorylation activates m-Cdk at the Onset of mitosis

1	M-Cdk activation begins with the accumulation of M-cyclin (cyclin B in vertebrate cells; see Table 17–1). In embryonic cell cycles, the synthesis of M-cyclin is constant throughout the cell cycle, and M-cyclin accumulation results from the high stability of the protein in interphase. In most cell types, however, M-cyclin synthesis increases during G2 and M, owing primarily to an increase in M-cyclin gene transcription. The increase in M-cyclin protein leads to a corresponding accumulation of M-Cdk (the complex of Cdk1 and M-cyclin) as the cell approaches mitosis. Although the Cdk in these complexes is phosphorylated at an activating site by the Cdk-activating kinase (CAK), as discussed earlier, the protein kinase Wee1 holds it in an inactive state by inhibitory phosphorylation at two neighboring sites (see Figure 17–13). Thus, by the time the cell reaches the end of G2, it contains an abundant stockpile of M-Cdk that is primed and ready to act but is suppressed by phosphates that

1	neighboring sites (see Figure 17–13). Thus, by the time the cell reaches the end of G2, it contains an abundant stockpile of M-Cdk that is primed and ready to act but is suppressed by phosphates that block the active site of the kinase.

1	What, then, triggers the activation of the M-Cdk stockpile? The crucial event is the activation of the protein phosphatase Cdc25, which removes the inhibitory phosphates that restrain M-Cdk (Figure 17–20). At the same time, the inhibitory activity of the kinase Wee1 is suppressed, further ensuring that M-Cdk activity increases. The mechanisms that unleash Cdc25 activity in early mitosis are not well understood. One possibility is that the S-Cdks that are active in G2 and early prophase stimulate Cdc25.

1	Interestingly, Cdc25 can also be activated, at least in part, by its target, M-Cdk. M-Cdk may also inhibit the inhibitory kinase Wee1. The ability of M-Cdk to activate its own activator (Cdc25) and inhibit its own inhibitor (Wee1) suggests that M-Cdk activation in mitosis involves positive feedback loops (see Figure 17–20). According to this attractive model, the partial activation of Cdc25 (perhaps by S-Cdk) leads to the partial activation of a subpopulation of M-Cdk complexes, which then phosphorylate Cdc25 and Wee1 molecules. This leads to more M-Cdk activation, and so on. Such a mechanism would quickly promote the activation of all M-Cdk complexes in the cell. As mentioned earlier, similar molecular switches operate at various points in the cell cycle to promote the abrupt and complete transition from one cell-cycle state to the next.

1	At the end of S phase, the immensely long DNA molecules of the sister chromatids are tangled in a mass of partially catenated DNA and proteins. Any attempt to pull the sisters apart in this state would undoubtedly lead to breaks in the chromosomes. To avoid this disaster, the cell devotes a great deal of energy in early mitosis to gradually reorganizing the sister chromatids into relatively short, distinct structures that can be pulled apart more easily in anaphase. These chromosomal changes involve two processes: chromosome condensation, in which the chromatids are dramatically compacted; and sister-chromatid resolution, whereby the two sisters are resolved into distinct, separable units (Figure 17–21). Resolution results from the decatenation of the sister DNAs, accompanied by the partial removal of cohesin molecules along the chromosome arms. As a result, when the cell reaches metaphase, the sister chromatids appear in the microscope as compact, rodlike structures that are joined

1	removal of cohesin molecules along the chromosome arms. As a result, when the cell reaches metaphase, the sister chromatids appear in the microscope as compact, rodlike structures that are joined tightly at their centromeric regions and only loosely along their arms.

1	Figure 17–20 The activation of M-Cdk. Cdk1 associates with m-cyclin as the levels of m-cyclin gradually rise. The resulting m-Cdk complex is phosphorylated on an activating site by the Cdk-activating kinase (Cak) and on a pair of inhibitory sites by the Wee1 kinase. The resulting inactive m-Cdk complex is then activated at the end of g2 by the phosphatase Cdc25. Cdc25 is further stimulated by active m-Cdk, resulting in positive feedback. This feedback is enhanced by the ability of m-Cdk to inhibit Wee1. Figure 17–21 The mitotic chromosome. scanning electron micrograph of a human mitotic chromosome, consisting of two sister chromatids joined along their length. The constricted regions are the centromeres. (Courtesy of Terry d. allen.)

1	PROPHASE1centrosome forming mitotic spindle condensing replicated chromosome, consisting of two sister chromatids held together along their length intact nuclear envelope kinetochore At prophase, the replicated chromosomes, each consisting of two closely associated sister chromatids, condense. Outside the nucleus, the mitotic spindle assembles between the two centrosomes, which have replicated and moved apart. For simplicity, only three chromosomes are shown. In diploid cells, there would be two copies of each chromo-some present. In the fuorescence micrograph, chromosomes are stained orange and microtubules are green.PROMETAPHASE 2 Prometaphase starts abruptly with the breakdown of the nuclear envelope. Chromosomes can now attach to spindle microtubules via their kinetochores and undergo active movement. centrosome at spindle pole kinetochore microtubule chromosome in active motion fragments of nuclear envelope METAPHASE 3 At metaphase, the chromosomes are aligned at the equator of

1	active movement. centrosome at spindle pole kinetochore microtubule chromosome in active motion fragments of nuclear envelope METAPHASE 3 At metaphase, the chromosomes are aligned at the equator of the spindle, midway between the spindle poles. The kinetochore microtubules attach sister chromatids to opposite poles of the spindle. centrosome at spindle pole kinetochore microtubule PANeL 17–1: The Principle Stages of M Phase (Mitosis and Cytokinesis) in an Animal Cell 980

1	ANAPHASE4 At anaphase, the sister chromatids synchronously separate to form two daughter chromosomes, and each is pulled slowly toward the spindle pole it faces. The kinetochore microtubules get shorter, and the spindle poles also move apart; both processes contribute to chromosome segregation. TELOPHASE5 During telophase, the two sets of daughter chromo-somes arrive at the poles of the spindle and decondense. A new nuclear envelope reassembles around each set, completing the formation of two nuclei and marking the end of mitosis. The division of the cytoplasm begins with contraction of the contractile ring. CYTOKINESIS6 During cytokinesis, the cytoplasm is divided in two by a contractile ring of actin and myosin flaments, which pinches the cell in two to create two daughters, each with one nucleus. shortening kinetochore microtubule spindle pole moving outward set of daughter chromosomes at spindle pole contractile ring starting to contract centrosomeinterpolar microtubules nuclear

1	one nucleus. shortening kinetochore microtubule spindle pole moving outward set of daughter chromosomes at spindle pole contractile ring starting to contract centrosomeinterpolar microtubules nuclear envelope reassembling around individual chromosomes daughter chromosomes completed nuclear envelope surrounds decondensing chromosomes contractile ring creating cleavage furrow re-formation of interphase array of microtubules nucleated by the centrosome (Micrographs courtesy of Julie Canman and Ted Salmon.) 981

1	The condensation and resolution of sister chromatids depend, at least in part, on a five-subunit protein complex called condensin. Condensin structure is related to that of the cohesin complex that holds sister chromatids together (see Figure 17–19). It contains two SMC subunits like those of cohesin, plus three non-SMC subunits (Figure 17–22). Condensin may form a ringlike structure that somehow uses the energy provided by ATP hydrolysis to promote the compaction and resolution of sister chromatids. Condensin is able to change the coiling of DNA molecules in a test tube, and this coiling activity is thought to be important for chromosome condensation during mitosis. Interestingly, phosphorylation of condensin subunits by M-Cdk stimulates this coiling activity, providing one mechanism by which M-Cdk may promote chromosome restructuring in early mitosis. The mitotic spindle Is a microtubule-Based machine

1	The mitotic spindle Is a microtubule-Based machine The central event of mitosis—chromosome segregation—depends in all eukaryotes on a complex and beautiful machine called the mitotic spindle (see Panel 17–1). The spindle is a bipolar array of microtubules, which pulls sister chromatids apart in anaphase, thereby segregating the two sets of chromosomes to opposite ends of the cell, where they are packaged into daughter nuclei (Movie 17.6). M-Cdk triggers the assembly of the spindle early in mitosis, in parallel with the chromosome restructuring just described. Before we consider how the spindle assembles and how its microtubules attach to sister chromatids, we briefly review the basic features of spindle structure.

1	The core of the mitotic spindle is a bipolar array of microtubules, the minus ends of which are focused at the two spindle poles, and the plus ends of which radiate outward from the poles (Figure 17–23). The plus ends of some microtubules—called the interpolar microtubules—overlap with the plus ends of micro-tubules from the other pole, resulting in an antiparallel array in the spindle mid-zone. The plus ends of other microtubules—the kinetochore microtubules—are attached to sister-chromatid pairs at large protein structures called kinetochores, which are located at the centromere of each sister chromatid. Finally, many spindles also contain astral microtubules that radiate outward from the poles and contact the cell cortex, helping to position the spindle in the cell.

1	In most somatic animal cells, each spindle pole is focused at a protein organelle called the centrosome (see Figures 16–47 and 16–48). Each centrosome consists of a cloud of amorphous material (called the pericentriolar matrix) that surrounds a pair of centrioles (Figure 17–24). The pericentriolar matrix nucleates a radial array of microtubules, with their fast-growing plus ends projecting outward and their minus ends associated with the centrosome. The matrix contains a variety of proteins, including microtubule-dependent motor proteins, coiled-coil proteins that link the motors to the centrosome, structural proteins, and components of the cell-cycle control system. Most important, it contains γ-tubulin ring complexes, which are the components mainly responsible for nucleating microtubules (see Figure 16–46).

1	Some cells—notably the cells of higher plants and the oocytes of many vertebrates—do not have centrosomes, and microtubule-dependent motor proteins and other proteins associate with microtubule minus ends to organize and focus the spindle poles. Figure 17–23 The metaphase mitotic spindle in an animal cell. The plus ends of the microtubules project away from the spindle pole, while the minus ends are anchored at the spindle poles, which in this example are organized by centrosomes. kinetochore microtubules connect the spindle poles with the kinetochores of sister chromatids, while interpolar microtubules from the two poles interdigitate at the spindle equator. astral microtubules radiate out from the poles into the cytoplasm.

1	The function of the mitotic spindle depends on numerous microtubule-dependent motor proteins. As discussed in Chapter 16, these proteins belong to two families—the kinesin-related proteins, which usually move toward the plus end of microtubules, and dyneins, which move toward the minus end. In the mitotic spindle, these motor proteins generally operate at or near the ends of the micro-tubules. Four major types of motor proteins—kinesin-5, kinesin-14, kinesins-4/10, and dynein—are particularly important in spindle assembly and function (Figure 17–25).

1	Kinesin-5 proteins contain two motor domains that interact with the plus ends of antiparallel microtubules in the spindle midzone. Because the two motor domains move toward the plus ends of the microtubules, they slide the two anti-parallel microtubules past each other toward the spindle poles, pushing the poles apart. Kinesin-14 proteins, by contrast, are minus-end directed motors with a single motor domain and other domains that can interact with a neighboring microtubule. They can cross-link antiparallel interpolar microtubules at the spindle midzone and tend to pull the poles together. Kinesin-4 and kinesin-10

1	Figure 17–24 The centrosome. (a) Electron micrograph of an s-phase mammalian cell in culture, showing a duplicated centrosome. Each centrosome contains a pair of centrioles; although the centrioles have duplicated, they remain together in a single complex, as shown in the drawing of the micrograph in (B). One centriole of each centriole pair has been cut in cross section, while the other is cut in longitudinal section, indicating that the two members of each pair are aligned at right angles to each other. The two halves of the replicated centrosome, each consisting of a centriole pair surrounded by pericentriolar matrix, will split and migrate apart to initiate the formation of the two poles of the mitotic spindle when the cell enters m phase. (a, from m. mcgill, d.p. Highfield, T.m. monahan, and B.R. Brinkley, J. Ultrastruct. Res. 57:43–53, 1976. With permission from academic press.) spindle microtubule dynein kinesin-4,10 kinesin-14 kinesin-5 dynein sister chromatids centrosome+ + +

1	and B.R. Brinkley, J. Ultrastruct. Res. 57:43–53, 1976. With permission from academic press.) spindle microtubule dynein kinesin-4,10 kinesin-14 kinesin-5 dynein sister chromatids centrosome+ + + + + + + – – – – – – – plasma membrane Figure 17–25 Major motor proteins of the spindle. Four major classes of microtubule-dependent motor proteins (yellow boxes) contribute to spindle assembly and function (see text). The colored arrows indicate the direction of motor protein movement along a microtubule—blue toward the minus end and red toward the plus end.

1	proteins, also called chromokinesins, are plus-end directed motors that associate with chromosome arms and push the attached chromosome away from the pole (or the pole away from the chromosome). Finally, dyneins are minus-end directed motors that, together with associated proteins, organize microtubules at various locations in the cell. They link the plus ends of astral microtubules to components of the actin cytoskeleton at the cell cortex, for example; by moving toward the minus end of the microtubules, the dynein motors pull the spindle poles toward the cell cortex and away from each other. multiple mechanisms Collaborate in the assembly of a Bipolar mitotic spindle

1	multiple mechanisms Collaborate in the assembly of a Bipolar mitotic spindle The mitotic spindle must have two poles if it is to pull the two sets of sister chromatids to opposite ends of the cell in anaphase. In most animal cells, several mechanisms ensure the bipolarity of the spindle. One depends on centrosomes. A typical animal cell enters mitosis with a pair of centrosomes, each of which nucleates a radial array of microtubules. The two centrosomes provide prefabricated spindle poles that greatly facilitate bipolar spindle assembly. The other mechanisms depend on the ability of mitotic chromosomes to nucleate and stabilize microtubules and on the ability of motor proteins to organize microtubules into a bipolar array. These “self-organization” mechanisms can produce a bipolar spindle even in cells lacking centrosomes.

1	We now describe the steps of spindle assembly, beginning with centrosomedependent assembly in early mitosis. We then consider the self-organization mechanisms that do not require centrosomes and become particularly important after nuclear-envelope breakdown. Centrosome duplication Occurs Early in the Cell Cycle

1	Centrosome duplication Occurs Early in the Cell Cycle Most animal cells contain a single centrosome that nucleates most of the cell’s cytoplasmic microtubules. The centrosome duplicates when the cell enters the cell cycle, so that by the time the cell reaches mitosis there are two centrosomes. Centrosome duplication begins at about the same time as the cell enters S phase. The G1/S-Cdk (a complex of cyclin E and Cdk2 in animal cells; see Table 17–1) that triggers cell-cycle entry also helps initiate centrosome duplication. The two centrioles in the centrosome separate, and each nucleates the formation of a single new centriole, resulting in two centriole pairs within an enlarged pericentriolar matrix (Figure 17–26). This centrosome pair remains together on one side of the nucleus until the cell enters mitosis.

1	There are interesting parallels between centrosome duplication and chromosome duplication. Both use a semiconservative mechanism of duplication, in which the two halves separate and serve as templates for construction of a new half. Centrosomes, like chromosomes, must replicate once and only once per cell cycle, to ensure that the cell enters mitosis with only two copies: an incorrect number of centrosomes could lead to defects in spindle assembly and thus errors in chromosome segregation.

1	The mechanisms that limit centrosome duplication to once per cell cycle are uncertain. In many cell types, experimental inhibition of DNA synthesis blocks centrosome duplication, providing one mechanism by which centrosome number is kept in check. Other cell types, however, including those in the early embryos of flies, sea urchins, and frogs, do not have such a mechanism and centrosome duplication continues if chromosome duplication is blocked. It is not known how such cells limit centrosome duplication to once per cell cycle.

1	Spindle assembly begins in early mitosis, when the two centrosomes move apart along the nuclear envelope, pulled by dynein motor proteins that link astral micro-tubules to the cell cortex (see Figure 17–25). The plus ends of the microtubules between the centrosomes interdigitate to form the interpolar microtubules, and kinesin-5 motor proteins associate with these microtubules and push the centrosomes apart (see Figure 17–25). Also in early mitosis, the number of γ-tubulin ring complexes in each centrosome increases greatly, increasing the ability of the centrosomes to nucleate new microtubules, a process called centrosome maturation.

1	The balance of opposing forces generated by different types of motor proteins determines the final length of the spindle. Dynein and kinesin-5 motors generally promote centrosome separation and increase spindle length. Kinesin-14 proteins do the opposite: they tend to pull the poles together (see Figure 17–25). It is not clear how the cell regulates the balance of opposing forces to generate the appropriate spindle length. M-Cdk and other mitotic protein kinases are required for centrosome separation and maturation. M-Cdk and Aurora-A phosphorylate kinesin-5 motors and stimulate them to drive centrosome separation. Aurora-A and Plk also phosphorylate components of the centrosome and thereby promote its maturation. The Completion of spindle assembly in animal Cells Requires nuclear-Envelope Breakdown

1	The Completion of spindle assembly in animal Cells Requires nuclear-Envelope Breakdown The centrosomes and microtubules of animal cells are located in the cytoplasm, separated from the chromosomes by the double-membrane barrier of the nuclear envelope (discussed in Chapter 12). Clearly, the attachment of sister-chromatid pairs to the spindle requires the removal of this barrier. In addition, many of the motor proteins and microtubule regulators that promote spindle assembly are associated with the chromosomes inside the nucleus, and they require nuclear-envelope breakdown to carry out their functions. Nuclear-envelope breakdown is a complex, multistep process, which is thought to begin when M-Cdk phosphorylates several subunits of the nuclear pore complexes in the nuclear envelope. This phosphorylation initiates the disassembly of nuclear pore complexes and their dissociation from the envelope. M-Cdk

1	Figure 17–26 Centriole replication. The centrosome consists of a centriole pair and associated pericentriolar matrix (green). at a certain point in g1, the two centrioles of the pair separate by a few micrometers. during s phase, a daughter centriole begins to grow near the base of each mother centriole and at a right angle to it. The elongation of the daughter centriole is usually completed in g2. The two centriole pairs remain close together in a single centrosomal complex until the beginning of m phase, when the complex splits in two and the two daughter centrosomes begin to separate. Each centrosome now nucleates its own radial array of microtubules (called an aster), mainly from the mother centriole.

1	also phosphorylates components of the nuclear lamina, the structural framework beneath the envelope. The phosphorylation of these lamina components and of several inner-nuclear-envelope proteins leads to disassembly of the nuclear lamina and the breakdown of the envelope membranes into small vesicles. Most animal cells in interphase contain a cytoplasmic array of microtubules radiating out from the single centrosome. As discussed in Chapter 16, the microtubules of this interphase array are in a state of dynamic instability, in which individual microtubules are either growing or shrinking and stochastically switch between the two states. The switch from growth to shrinkage is called a catastrophe, and the switch from shrinkage to growth is called a rescue. New microtubules are continually being created to balance the loss of those that disappear completely by depolymerization.

1	Entry into mitosis signals an abrupt change in the cell’s microtubules. The interphase array of few, long microtubules radiating from the single centrosome is converted to a larger number of shorter and more dynamic microtubules emanating from both centrosomes. During prophase, and particularly in prometaphase and metaphase (see Panel 17–1), the half-life of microtubules decreases dramatically. This increase in microtubule instability, coupled with the increased ability of centrosomes to nucleate microtubules as mentioned earlier, results in remarkably dense and dynamic arrays of spindle microtubules that are ideally suited for capturing sister chromatids.

1	Microtubule dynamics are controlled in the cell by a variety of regulatory proteins, including microtubule-associated proteins (MAPs) that promote stability and catastrophe factors that destabilize microtubule plus ends. Changes in the activities of these regulatory proteins are responsible for the changes in micro-tubule dynamics that occur during mitosis. Many of these changes result from phosphorylation of specific proteins by M-Cdk and other mitotic protein kinases.

1	Chromosomes are not just passive passengers in the process of spindle assembly. By creating a local environment that favors both microtubule nucleation and microtubule stabilization, they play an active part in spindle formation. The influence of the chromosomes can be demonstrated by using a fine glass needle to reposition them after the spindle has formed. For some cells in metaphase, if a single chromosome is tugged out of alignment, a mass of new spindle micro-tubules rapidly appears around the newly positioned chromosome, while the spindle microtubules at the chromosome’s former position depolymerize. This property of the chromosomes seems to depend, at least in part, on a guanine nucleotide exchange factor (GEF) that is bound to chromatin; the GEF stimulates a small GTPase in the cytosol called Ran to bind GTP in place of GDP. The activated Ran-GTP, which is also involved in nuclear transport (discussed in Chapter 12), releases microtubule-stabilizing proteins from protein

1	cytosol called Ran to bind GTP in place of GDP. The activated Ran-GTP, which is also involved in nuclear transport (discussed in Chapter 12), releases microtubule-stabilizing proteins from protein complexes in the cytosol, thereby stimulating the local nucleation and stabilization of microtubules around chromosomes (Figure 17–27). Local microtubule stabilization is also promoted by the protein kinase Aurora-B, which associates with mitotic chromosomes.

1	Figure 17–27 Activation of the GTPase Ran around mitotic chromosomes. The Ran protein, like other members of the small gTpase family (discussed in Chapter 15), can exist in two conformations depending on whether it is bound to gdp (inactive state) or gTp (active state). The localization of active Ran in mitosis was determined using a protein that emits fluorescence at a specific wavelength when it is activated by Ran-gTp. In the metaphase human cell shown here, Ran activity (yellow and red) is highest around the chromosomes, between the poles of the mitotic spindle (indicated by asterisks). (From p. kaláb et al., Nature 440:697–701, 2006. With permission from macmillan publishers Ltd.) nucleation antiparallel cross-linking outward push focusing of poles by by kinesin-5 by kinesin-4,10 dynein and kinesin-14 – – –

1	Figure 17–28 Spindle self-organization by motor proteins. mitotic chromosomes stimulate the local activation of proteins that nucleate and promote the formation of microtubules in the vicinity of the chromosomes. kinesin-5 motor proteins (see Figure 17–25) organize these microtubules into antiparallel bundles, while plus-end directed kinesins-4 and 10 link the microtubules to chromosome arms and push minus ends away from the chromosomes. dynein and kinesin-14 motors, together with numerous other proteins, focus these minus ends into a pair of spindle poles. The ability of chromosomes to stabilize and organize microtubules enables cells to form bipolar spindles in the absence of centrosomes. Acentrosomal spindle assembly is thought to begin with the formation of microtubules around the chromosomes. Various motor proteins then organize the microtubules into a bipolar spindle, as illustrated in Figure 17–28.

1	Cells that normally lack centrosomes, such as those of higher plants and many animal oocytes, use this chromosome-based self-organization process to form spindles. It is also the process used to assemble spindles in certain animal embryos that have been induced to develop from eggs without fertilization (that is, parthenogenetically); as the sperm normally provides the centrosome when it fertilizes an egg, the mitotic spindles in these parthenogenetic embryos develop without centrosomes (Figure 17–29). Even in cells that normally contain centrosomes, the chromosomes help organize the spindle microtubules and, with the help of various motor proteins, can promote the assembly of a bipolar mitotic spindle if the centrosomes are removed. Although the resulting acentrosomal spindle can segregate chromosomes normally, it lacks astral microtubules, which are responsible for positioning the spindle in animal cells; as a result, the spindle is often mispositioned in the cell.

1	kinetochores attach sister Chromatids to the spindle

1	Following the assembly of a bipolar microtubule array, the second major step in spindle formation is the attachment of the array to the sister-chromatid pairs. Spindle microtubules become attached to each chromatid at its kinetochore, a giant, multilayered protein structure that is built at the centromeric region of the chromatid (Figure 17–30; also see Chapter 4). In metaphase, the plus ends of kinetochore microtubules are embedded head-on in specialized microtubuleattachment sites within the outer region of the kinetochore, furthest from the DNA. The kinetochore of an animal cell can bind 10–40 microtubules, whereas a budding yeast kinetochore can bind only one. Attachment of each microtubule depends on multiple copies of a rod-shaped protein complex called the Ndc80 complex, which is anchored in the kinetochore at one end and interacts with the sides of the microtubule at the other, thereby linking the microtubule to the kinetochore while still allowing the addition or removal of

1	anchored in the kinetochore at one end and interacts with the sides of the microtubule at the other, thereby linking the microtubule to the kinetochore while still allowing the addition or removal of tubulin subunits at this end (Figure 17–31). Regulation of plus-end polymerization and depolymerization at the kinetochore is critical for the control of chromosome movement on the spindle, as we discuss later.

1	Kinetochore attachment to the spindle occurs by a complex sequence of events. At the end of prophase in animal cells, the centrosomes of the growing spindle generally lie on opposite sides of the nuclear envelope. Thus, when the envelope breaks down, the sister-chromatid pairs are bombarded by microtubule

1	Figure 17–29 bipolar spindle assembly without centrosomes in parthenogenetic embryos of the insect Sciara (or fungus gnat). The microtubules are stained green, the chromosomes red. The top fluorescence micrograph shows a normal spindle formed with centrosomes in a normally fertilized Sciara embryo. The bottom micrograph shows a spindle formed without centrosomes in an embryo that initiated development without fertilization. note that the spindle with centrosomes has an aster at each pole of the spindle, whereas the spindle formed without centrosomes does not. Both types of spindles are able to segregate the replicated chromosomes. (From B. de saint phalle and W. sullivan, J. Cell Biol. 141:1383–1391, 1998. With permission from The Rockefeller University press.) plus ends coming from two directions. However, the kinetochores do not instantly achieve the correct ‘end-on’ microtubule attachment to both spindle poles. Instead, detailed studies with light and electron microscopy show that

1	However, the kinetochores do not instantly achieve the correct ‘end-on’ microtubule attachment to both spindle poles. Instead, detailed studies with light and electron microscopy show that most initial attachments are unstable lateral attachments, in which a kinetochore attaches to the side of a passing microtubule, with assistance from kinesin motor proteins in the outer kinetochore. Soon, however, the dynamic microtubule plus ends capture the kinetochores in the correct end-on orientation (Figure 17–32).

1	Another attachment mechanism also plays a part, particularly in the absence of centrosomes. Careful microscopic analysis suggests that short microtubules in the vicinity of the chromosomes become embedded in the plus-end-binding sites of the kinetochore. Polymerization at these plus ends then results in growth of the microtubules away from the kinetochore. The minus ends of these kinetochore microtubules are eventually cross-linked to other minus ends and focused by motor proteins at the spindle pole (see Figure 17–28). The success of mitosis demands that sister chromatids in a pair attach to opposite poles of the mitotic spindle, so that they move to opposite ends of the cell when direction of chromatid movement Figure 17–30 The kinetochore.

1	Figure 17–30 The kinetochore. a fluorescence micrograph of a metaphase chromosome stained with a dna-binding fluorescent dye and with human autoantibodies that react with specific kinetochore proteins. The two kinetochores, one associated with each sister chromatid, are stained red. a drawing of a metaphase chromosome showing its two sister chromatids attached to the plus ends of kinetochore microtubules. Each kinetochore forms a plaque on the surface of the centromere. Electron micrograph of an anaphase chromatid with microtubules attached to its kinetochore. While most kinetochores have a trilaminar structure, the one shown here (from a green alga) has an unusually complex structure with additional layers. (a, courtesy of B.R. Brinkley; C, from J.d. pickett-Heaps and L.C. Fowke, Aust. J. Biol. Sci. 23:71–92, 1970. With permission from CsIRO.)

1	Figure 17–31 Microtubule attachment sites in the kinetochore. (a) In this electron micrograph of a mammalian kinetochore, the chromosome is on the right, and the plus ends of multiple microtubules are embedded in the outer kinetochore on the left. (B) Electron tomography (discussed in Chapter 9) was used to construct a low-resolution three-dimensional image of the outer kinetochore in (a). several microtubules (in multiple colors) are embedded in fibrous material of the kinetochore, which is thought to be composed of the ndc80 complex and other proteins. (C) Each microtubule is attached to the kinetochore by interactions with multiple copies of the ndc80 complex (blue). This complex binds to the sides of the microtubule near its plus end, allowing polymerization and depolymerization to occur while the microtubule remains attached to the kinetochore. (a and B, from Y. dong et al., Nature Cell Biol. 9:516–522, 2007. With permission from macmillan publishers Ltd.) + early prometaphase:

1	while the microtubule remains attached to the kinetochore. (a and B, from Y. dong et al., Nature Cell Biol. 9:516–522, 2007. With permission from macmillan publishers Ltd.) + early prometaphase: lateral kinetochore mid prometaphase: metaphase:late prophase attachments, chromosome arms pushed outwards end-on attachment bi-orientation

1	Figure 17-32 Chromosome attachment to the mitotic spindle in animal cells. (a) In late prophase of most animal cells, the mitotic spindle poles have moved to opposite sides of the nuclear envelope, with an array of overlapping microtubules between them. (B) Following nuclear envelope breakdown, the sister-chromatid pairs are exposed to the large number of dynamic plus ends of microtubules radiating from the spindle poles. In most cases, the kinetochores are first attached to the sides of these microtubules, while at the same time the arms of the chromosomes are pushed outward from the spindle interior, preventing the arms from blocking microtubule access to the kinetochores. (C) Eventually, the laterally-attached sister chromatids are arranged in a ring around the outside of the spindle. most of the microtubules are concentrated in this ring, so that the spindle is relatively hollow inside. (d) dynamic microtubule plus ends eventually encounter the kinetochores in an end-on

1	spindle. most of the microtubules are concentrated in this ring, so that the spindle is relatively hollow inside. (d) dynamic microtubule plus ends eventually encounter the kinetochores in an end-on orientation and are captured and stabilized. (E) stable end-on attachment to both poles results in bi-orientation. additional microtubules are attached to the kinetochore, resulting in a kinetochore fiber containing 10–40 microtubules.

1	they separate in anaphase. How is this mode of attachment, called bi-orientation, achieved? What prevents the attachment of both kinetochores to the same spindle pole or the attachment of one kinetochore to both spindle poles? Part of the answer is that sister kinetochores are constructed in a back-to-back orientation that reduces the likelihood that both kinetochores can face the same spindle pole. Nevertheless, incorrect attachments do occur, and elegant regulatory mechanisms have evolved to correct them.

1	Incorrect attachments are corrected by a system of trial and error that is based on a simple principle: incorrect attachments are highly unstable and do not last, whereas correct attachments become locked in place. How does the kinetochore sense a correct attachment? The answer appears to be tension (Figure 17–33). When a sister-chromatid pair is properly bi-oriented on the spindle, the two kinetochores are pulled in opposite directions by strong poleward forces. Sister-chromatid cohesion resists these poleward forces, creating high levels of tension within the kinetochores. When chromosomes are incorrectly attached—when both sister chromatids are attached to the same spindle pole, for example—tension is low Figure 17–33 Alternative forms of kinetochore attachment to the spindle poles. (a) Initially, a single microtubule from a spindle pole binds to one kinetochore in a sister-chromatid pair. additional microtubules can then bind to the chromosome in various ways.

1	a microtubule from the same spindle pole can attach to the other sister kinetochore, or (C) microtubules from both spindle poles can attach to the same kinetochore. These incorrect attachments are unstable, however, so that one of the two microtubules tends to dissociate. When a microtubule from the opposite pole binds to the second kinetochore, the sister kinetochores are thought to sense tension across their microtubulebinding sites. This triggers an increase in microtubule binding affinity, thereby locking the correct attachment in place.

1	and the kinetochore sends an inhibitory signal that loosens the grip of its microtubule attachment site, allowing detachment to occur. When bi-orientation occurs, the high tension at the kinetochore shuts off the inhibitory signal, strengthening microtubule attachment. In animal cells, tension not only increases the affinity of the attachment site but also leads to the attachment of additional microtubules to the kinetochore. This results in the formation of a thick kinetochore fiber composed of multiple microtubules.

1	The tension-sensing mechanism depends on the protein kinase Aurora-B, which is associated with the kinetochore and is thought to generate the inhibitory signal that reduces the strength of microtubule attachment in the absence of tension. It phosphorylates several components of the microtubule attachment site, including the Ndc80 complex, decreasing the site’s affinity for a microtubule plus end. When bi-orientation occurs, the resulting tension somehow reduces phosphorylation by Aurora-B, thereby increasing the affinity of the attachment site (Figure 17–34).

1	Following their attachment to the two spindle poles, the chromosomes are tugged back and forth, eventually assuming a position equidistant between the two poles, a position called the metaphase plate. In vertebrate cells, the chromosomes then oscillate gently at the metaphase plate, awaiting the signal for the sister chromatids to separate. The signal is produced, with a predictable lag time, after the bi-oriented attachment of the last of the chromosomes. multiple Forces act on Chromosomes in the spindle Multiple mechanisms generate the forces that move chromosomes back and forth after they are attached to the spindle, and produce the tension that is so important for the stabilization of correct attachments. In anaphase, similar forces pull the separated chromatids to opposite ends of the spindle. Three major spindle forces are particularly critical, although their strength and importance vary at different stages of mitosis.

1	The first major force pulls the kinetochore and its associated chromatid along the kinetochore microtubule toward the spindle pole. It is produced by proteins at the kinetochore itself. By an uncertain mechanism, depolymerization at the plus end of the microtubule generates a force that pulls the kinetochore poleward. This force pulls on chromosomes during prometaphase and metaphase but is particularly important for moving sister chromatids toward the poles after they separate in anaphase. Interestingly, this kinetochore-generated poleward force does not require ATP or motor proteins. This might seem implausible at first, but it has been shown that purified kinetochores in a test tube, with no ATP present, can remain attached to depolymerizing microtubules and thereby move. The energy that drives the movement is stored in the microtubule and is released when the microtubule depolymerizes; it ultimately comes from the hydrolysis of GTP that occurs after a tubulin subunit adds to the

1	drives the movement is stored in the microtubule and is released when the microtubule depolymerizes; it ultimately comes from the hydrolysis of GTP that occurs after a tubulin subunit adds to the end of a microtubule (discussed in Chapter 16).

1	Figure 17–34 How tension might increase microtubule attachment to the kinetochore. These diagrams illustrate one speculative mechanism by which bi-orientation might increase microtubule attachment to the kinetochore. a single kinetochore is shown for clarity; the spindle pole is on the right. (a) When a sisterchromatid pair is unattached to the spindle or attached to just one spindle pole, there is little tension between the outer and inner kinetochores. The protein kinase aurora-B is tethered to the inner kinetochore and phosphorylates the microtubule attachment sites, including the ndc80 complex (blue), in the outer kinetochore as shown, thereby reducing the affinity of microtubule binding. microtubules therefore associate and dissociate rapidly, and attachment is unstable. (B) When bi-orientation is achieved, the forces pulling the kinetochore toward the spindle pole are resisted by forces pulling the other sister kinetochore toward the opposite pole, and the resulting tension

1	bi-orientation is achieved, the forces pulling the kinetochore toward the spindle pole are resisted by forces pulling the other sister kinetochore toward the opposite pole, and the resulting tension pulls the outer kinetochore away from the inner kinetochore. as a result, aurora-B is unable to reach the outer kinetochore, and microtubule attachment sites are not phosphorylated. microtubule binding affinity is therefore increased, resulting in the stable attachment of multiple microtubules to both kinetochores. The dephosphorylation of outer kinetochore proteins depends on a phosphatase that is not shown here.

1	How does plus-end depolymerization drive the kinetochore toward the pole? As we discussed earlier (see Figure 17–31C), Ndc80 complexes in the kinetochore make multiple low-affinity attachments along the side of the microtubule. Because the attachments are constantly breaking and re-forming at new sites, the kinetochore remains attached to a microtubule even as the microtubule depolymerizes. In principle, this could move the kinetochore toward the spindle pole.

1	A second poleward force is provided in some cell types by microtubule flux, whereby the microtubules themselves are pulled toward the spindle poles and dismantled at their minus ends. The mechanism underlying this poleward movement is not clear, although it might depend on forces generated by motor proteins and minus-end depolymerization at the spindle pole. In metaphase, the addition of new tubulin at the plus end of a microtubule compensates for the loss of tubulin at the minus end, so that microtubule length remains constant despite the movement of microtubules toward the spindle pole (Figure 17–35). Any kinetochore that is attached to a microtubule undergoing such flux experiences a poleward force, which contributes to the generation of tension at the kinetochore in meta-phase. Together with the kinetochore-based forces discussed above, flux also contributes to the poleward forces that move sister chromatids after they separate in anaphase.

1	A third force acting on chromosomes is the polar ejection force, or polar wind. Plus-end directed kinesin-4 and 10 motors on chromosome arms interact with interpolar microtubules and transport the chromosomes away from the spindle poles (see Figure 17–25). This force is particularly important in prometaphase and metaphase, when it helps push chromosome arms out from the spindle. This force might also help align the sister-chromatid pairs at the metaphase plate (Figure 17–36). One of the most striking aspects of mitosis in vertebrate cells is the continuous oscillatory movement of the chromosomes in prometaphase and metaphase. When studied by video microscopy in newt lung cells, the movements are seen to switch between two states—a poleward state, when the chromosomes are pulled toward the pole, and an away-from-the-pole, or neutral, state, when the poleward forces are turned off and the polar ejection force pushes the chromosomes away

1	Figure 17–35 Microtubule flux in the metaphase spindle. (a) To observe microtubule flux, a very small amount of fluorescent tubulin is injected into living cells so that individual microtubules form with a very small proportion of fluorescent tubulin. such microtubules have a speckled appearance when viewed by fluorescence microscopy. (B) Fluorescence micrograph of a mitotic spindle in a living newt lung epithelial cell. The chromosomes are colored brown, and the tubulin speckles are red. (C) The movement of individual speckles can be followed by time-lapse video microscopy. Images of the thin vertical boxed region (arrow) in (B), taken at sequential times, show that individual speckles move toward the poles at a rate of about 0.75 μm/min, indicating that the microtubules are moving poleward. (d) microtubule length in the metaphase spindle does not change significantly because new tubulin subunits are added at the microtubule plus end at the same rate as tubulin subunits are removed

1	(d) microtubule length in the metaphase spindle does not change significantly because new tubulin subunits are added at the microtubule plus end at the same rate as tubulin subunits are removed from the minus end. (B and C, from T.J. mitchison and E.d. salmon, Nat. Cell Biol. 3:E17–21, 2001. With permission from macmillan publishers Ltd.) interpolar plus-end-directed or astral kinesin-4,10 microtubule motor proteins from the pole. The switch between the two states may depend on the degree of tension in the kinetochore. It has been proposed, for example, that, as chromosomes move toward a spindle pole, an increasing polar ejection force generates tension in the kinetochore nearest the pole, triggering a switch to the away-fromthe-pole state and gradually resulting in the accumulation of chromosomes at the equator of the spindle.

1	The apC/C Triggers sister-Chromatid separation and the Completion of mitosis After M-Cdk has triggered the complex processes leading up to metaphase, the cell cycle reaches its climax with the separation of the sister chromatids at the metaphase-to-anaphase transition (Figure 17–37). Although M-Cdk activity sets the stage for this event, the anaphase-promoting complex (APC/C) discussed earlier throws the switch that initiates sister-chromatid separation by ubiquitylating several mitotic regulatory proteins and thereby triggering their destruction (see Figure 17–15A). During metaphase, cohesins holding the sister chromatids together resist the poleward forces that pull the sister chromatids apart. Anaphase begins with the sudden loss of sister-chromatid cohesion, which allows the sisters to separate and move to opposite poles of the spindle. The APC/C initiates the process by targeting the inhibitory protein securin for destruction. Before anaphase, securin

1	Figure 17–36 How opposing forces may drive chromosomes to the metaphase plate. (a) Evidence for a polar ejection force that pushes chromosomes away from the spindle poles toward the spindle equator. In this experiment, a laser beam severs a prometaphase chromosome that is attached to a single pole by a kinetochore microtubule. The part of the severed chromosome without a kinetochore is pushed rapidly away from the pole, whereas the part with the kinetochore moves toward the pole, reflecting a decreased repulsion. (B) a model of how two opposing forces may cooperate to move chromosomes to the metaphase plate. plus-end-directed motor proteins (kinesin-4 and kinesin-10) on the chromosome arms are thought to interact with microtubules to generate the polar ejection force, which pushes chromosomes toward the spindle equator (see Figure 17–25). poleward forces generated by depolymerization at the kinetochore, together with microtubule flux, are thought to pull chromosomes toward the pole.

1	Figure 17–37 Sister-chromatid separation at anaphase. In the transition from metaphase (a) to anaphase (B), sister chromatids suddenly and synchronously separate and move toward opposite poles of the mitotic spindle—as shown in these light micrographs of Haemanthus (lily) endosperm cells that were stained with gold-labeled antibodies against tubulin. (Courtesy of andrew Bajer.) binds to and inhibits the activity of a protease called separase. The destruction of securin at the end of metaphase releases separase, which is then free to cleave one of the subunits of cohesin. The cohesins fall away, and the sister chromatids separate (Figure 17–38). In addition to securin, the APC/C also targets the Sand M-cyclins for destruction, leading to the loss of most Cdk activity in anaphase. Cdk inactivation allows phosphatases to dephosphorylate the many Cdk target substrates in the cell, as required for the completion of mitosis and cytokinesis.

1	If the APC/C triggers anaphase, what activates the APC/C? The answer is only partly known. As mentioned earlier, APC/C activation requires binding to the protein Cdc20 (see Figure 17–15A). At least two processes regulate Cdc20 and its association with the APC/C. First, Cdc20 synthesis increases as the cell approaches mitosis, owing to an increase in the transcription of its gene. Second, phosphorylation of the APC/C helps Cdc20 bind to the APC/C, thereby helping to create an active complex. Among the kinases that phosphorylate and thus activate the APC/C is M-Cdk. Thus, M-Cdk not only triggers the early mitotic events leading up to metaphase, but it also sets the stage for progression into anaphase. The ability of M-Cdk to promote Cdc20–APC/C activity creates a negative feedback loop: M-Cdk sets in motion a regulatory process that leads to cyclin destruction and thus its own inactivation. Unattached Chromosomes Block sister-Chromatid separation: The spindle assembly Checkpoint

1	Unattached Chromosomes Block sister-Chromatid separation: The spindle assembly Checkpoint Drugs that destabilize microtubules, such as colchicine or vinblastine (discussed in Chapter 16), arrest cells in mitosis for hours or even days. This observation led to the identification of a spindle assembly checkpoint mechanism that is activated by the drug treatment and blocks progression through the metaphase-toanaphase transition. The checkpoint mechanism ensures that cells do not enter anaphase until all chromosomes are correctly bi-oriented on the mitotic spindle. The spindle assembly checkpoint depends on a sensor mechanism that monitors the strength of microtubule attachment at the kinetochore, possibly by sensing tension as described earlier (see Figure 17–34). Any kinetochore that is not properly attached to the spindle sends out a diffusible negative signal that blocks Cdc20–APC/C activation throughout the cell and thus blocks the

1	Figure 17–38 The initiation of sisterchromatid separation by the APC/C. The activation of apC/C by Cdc20 leads to the ubiquitylation and destruction of securin, which normally holds separase in an inactive state. The destruction of securin allows separase to cleave scc1, a subunit of the cohesin complex holding the sister chromatids together (see Figure 17–19). The pulling forces of the mitotic spindle then pull the sister chromatids apart. In animal cells, phosphorylation by Cdks also inhibits separase (not shown). Thus, Cdk inactivation in anaphase (resulting from cyclin destruction) also promotes separase activation by allowing its dephosphorylation. metaphase-to-anaphase transition. When the last sister-chromatid pair is properly bi-oriented, this block is removed, allowing sister-chromatid separation to occur.

1	metaphase-to-anaphase transition. When the last sister-chromatid pair is properly bi-oriented, this block is removed, allowing sister-chromatid separation to occur. The negative checkpoint signal depends on several proteins, including Mad2, which are recruited to unattached kinetochores (Figure 17–39). Detailed structural analyses of Mad2 suggest that the unattached kinetochore acts like an enzyme that catalyzes a change in the conformation of Mad2, so that Mad2, together with other proteins, can bind and inhibit Cdc20–APC/C.

1	In mammalian somatic cells, the spindle assembly checkpoint determines the normal timing of anaphase. The destruction of securin in these cells begins moments after the last sister-chromatid pair becomes bi-oriented on the spindle, and anaphase begins about 20 minutes later. Experimental inhibition of the checkpoint mechanism causes premature sister-chromatid separation and anaphase. Surprisingly, the normal timing of anaphase does not depend on the spindle assembly checkpoint in some cells, such as yeasts and the cells of early frog and fly embryos. Other mechanisms, as yet unknown, must determine the timing of anaphase in these cells.

1	The sudden loss of sister-chromatid cohesion at the onset of anaphase leads to sister-chromatid separation, which allows the forces of the mitotic spindle to pull the sisters to opposite poles of the cell—called chromosome segregation. The chromosomes move by two independent and overlapping processes. The first, anaphase A, is the initial poleward movement of the chromosomes, which is accompanied by shortening of the kinetochore microtubules. The second, anaphase B, is the separation of the spindle poles themselves, which begins after the sister chromatids have separated and the daughter chromosomes have moved some distance apart (Figure 17–40).

1	Chromosome movement in anaphase A depends on a combination of the two major poleward forces described earlier. The first is the force generated by micro-tubule depolymerization at the kinetochore, which results in the loss of tubulin subunits at the plus end as the kinetochore moves toward the pole. The second is provided by microtubule flux, which is the poleward movement of the micro-tubules toward the spindle pole, where minus-end depolymerization occurs. The relative importance of these two forces during anaphase varies in different cell types: in embryonic cells, chromosome movement depends mainly on microtubule flux, for example, whereas movement in yeast and vertebrate somatic cells results primarily from forces generated at the kinetochore.

1	Spindle-pole separation during anaphase B depends on motor-driven mechanisms similar to those that separate the two centrosomes in early mitosis. Plus-end directed kinesin-5 motor proteins, which cross-link the overlapping plus ends of the interpolar microtubules, push the poles apart. In addition, dynein motors that anchor astral microtubule plus ends to the cell cortex pull the poles apart (see Figure 17–25).

1	Although sister-chromatid separation initiates the chromosome movements of anaphase A, other mechanisms also ensure correct chromosome movements in anaphase A and spindle elongation in anaphase B. Most importantly, the completion of a normal anaphase depends on the dephosphorylation of Cdk substrates, which in most cells results from the APC/C-dependent destruction of cyclins. If M-cyclin destruction is prevented—by the production of a mutant form that is not recognized by the APC/C, for example—sister-chromatid separation generally occurs, but the chromosome movements and microtubule behavior of anaphase are abnormal.

1	The relative contributions of anaphase A and anaphase B to chromosome segregation vary greatly, depending on the cell type. In mammalian cells, anaphase B begins shortly after anaphase A and stops when the spindle is about twice its metaphase length; in contrast, the spindles of yeasts and certain protozoa primarily use anaphase B to separate the chromosomes at anaphase, and their spindles elongate to up to 15 times their metaphase length.

1	Figure 17–39 Mad2 protein on unattached kinetochores. This fluorescence micrograph shows a mammalian cell in prometaphase, with the mitotic spindle in green and the sister chromatids in blue. One sister-chromatid pair is attached to only one pole of the spindle. staining with anti-mad2 antibodies indicates that mad2 is bound to the kinetochore of the unattached sister chromatid (red dot, indicated by red arrow). a small amount of mad2 is associated with the kinetochore of the sister chromatid that is attached to the spindle pole (pale dot, indicated by white arrow). (From J.C. Waters et al., J. Cell Biol. 141:1181–1191, 1998. With permission from the authors.) segregated Chromosomes are packaged in daughter nuclei at Telophase

1	By the end of anaphase, the daughter chromosomes have segregated into two equal groups at opposite ends of the cell. In telophase, the final stage of mitosis, the two sets of chromosomes are packaged into a pair of daughter nuclei. The first major event of telophase is the disassembly of the mitotic spindle, followed by the re-formation of the nuclear envelope. Initially, nuclear membrane fragments associate with the surface of individual chromosomes. These membrane fragments fuse to partly enclose clusters of chromosomes and then coalesce to reform the complete nuclear envelope. Nuclear pore complexes are incorporated into the envelope, the nuclear lamina re-forms, and the envelope once again becomes continuous with the endoplasmic reticulum. Once the nuclear envelope has re-formed, the pore complexes pump in nuclear proteins, the nucleus expands, and the mitotic chromosomes are reorganized into their interphase state, allowing gene transcription to resume. A new nucleus has been

1	pore complexes pump in nuclear proteins, the nucleus expands, and the mitotic chromosomes are reorganized into their interphase state, allowing gene transcription to resume. A new nucleus has been created, and mitosis is complete. All that remains is for the cell to complete its division into two.

1	We saw earlier that phosphorylation of various proteins by M-Cdk promotes spindle assembly, chromosome condensation, and nuclear-envelope breakdown in early mitosis. It is thus not surprising that the dephosphorylation of these same proteins is required for spindle disassembly and the re-formation of daughter nuclei in telophase. In principle, these dephosphorylations and the completion of mitosis could be triggered by the inactivation of Cdks, the activation of phosphatases, or both. Although Cdk inactivation—resulting primarily from cyclin destruction—is mainly responsible in most cells, some cells also rely on activation of phosphatases. In budding yeast, for example, the completion of mitosis depends on the activation of a phosphatase called Cdc14, which dephosphorylates a subset of Cdk substrates involved in anaphase and telophase.

1	M-Cdk triggers the events of early mitosis, including chromosome condensation, assembly of the mitotic spindle, and bipolar attachment of the sister-chromatid pairs to microtubules of the spindle. Spindle formation in animal cells depends largely on the ability of mitotic chromosomes to stimulate local microtubule nucleation and stability, as well as on the ability of motor proteins to organize micro-tubules into a bipolar array. Many cells also use centrosomes to facilitate spindle assembly. Anaphase is triggered by the APC/C, which stimulates the destruction of Figure 17–40 The two processes of anaphase in mammalian cells. separated sister chromatids move toward the poles in anaphase a. In anaphase B, the two spindle poles move apart.

1	Figure 17–40 The two processes of anaphase in mammalian cells. separated sister chromatids move toward the poles in anaphase a. In anaphase B, the two spindle poles move apart. the proteins that hold the sister chromatids together. APC/C also promotes cyclin destruction and thus the inactivation of M-Cdk. The resulting dephosphorylation of Cdk targets is required for the events that complete mitosis, including the disassembly of the spindle and the re-formation of the nuclear envelope. The final step in the cell cycle is cytokinesis, the division of the cytoplasm in two. In most cells, cytokinesis follows every mitosis, although some cells, such as early Drosophila embryos and some mammalian hepatocytes and heart muscle cells, undergo mitosis without cytokinesis and thereby acquire multiple nuclei. In most animal cells, cytokinesis begins in anaphase and ends shortly after the completion of mitosis in telophase.

1	The first visible change of cytokinesis in an animal cell is the sudden appearance of a pucker, or cleavage furrow, on the cell surface. The furrow rapidly deepens and spreads around the cell until it completely divides the cell in two. The structure underlying this process is the contractile ring—a dynamic assembly composed of actin filaments, myosin II filaments, and many structural and regulatory proteins. During anaphase, the ring assembles just beneath the plasma membrane (Figure 17–41; see also Panel 17–1). The ring gradually contracts, and, at the same time, fusion of intracellular vesicles with the plasma membrane inserts new membrane adjacent to the ring. This addition of membrane compensates for the increase in surface area that accompanies cytoplasmic division. When ring contraction is completed, membrane insertion and fusion seal the gap between the daughter cells. actin and myosin II in the Contractile Ring generate the Force for Cytokinesis

1	actin and myosin II in the Contractile Ring generate the Force for Cytokinesis In interphase cells, actin and myosin II filaments form a cortical network underlying the plasma membrane. In some cells, they also form large cytoplasmic bundles called stress fibers (discussed in Chapter 16). As cells enter mitosis, these arrays of actin and myosin disassemble; much of the actin reorganizes, and myosin II filaments are released. As the sister chromatids separate in anaphase, actin and myosin II begin to accumulate in the rapidly assembling contractile ring (Figure 17–42), which also contains numerous other proteins that provide structural support or assist in ring assembly. Assembly of the contractile ring results in part from the local formation of new actin filaments, which depends on formin proteins that actin and myosin flaments of the

1	Figure 17–41 Cytokinesis. (a) The actin–myosin bundles of the contractile ring are oriented as shown, so that their contraction pulls the membrane inward. (B) In this low-magnification scanning electron micrograph of a cleaving frog egg, the cleavage furrow is especially prominent, as the cell is unusually large. The furrowing of the cell membrane is caused by the activity of the contractile ring underneath it. (C) The surface of a furrow at higher magnification. (B and C, from H.W. Beams and R.g. kessel, Am. Sci. 64:279–290, 1976. With permission from sigma Xi.) contractile ring of actin and myosin flaments in cleavage furrow (A) 0.5 µm nucleate the assembly of parallel arrays of linear, unbranched actin filaments (discussed in Chapter 16). After anaphase, the overlapping arrays of actin and myosin II filaments contract to generate the force that divides the cytoplasm in two. Once contraction begins, the ring exerts a force large enough to bend a fine glass needle that is inserted in

1	and myosin II filaments contract to generate the force that divides the cytoplasm in two. Once contraction begins, the ring exerts a force large enough to bend a fine glass needle that is inserted in its path. As the ring constricts, it maintains the same thickness, suggesting that its total volume and the number of filaments it contains decrease steadily. Moreover, unlike actin in muscle, the actin filaments in the ring are highly dynamic, and their arrangement changes continually during cytokinesis.

1	The contractile ring is finally dispensed with altogether when cleavage ends, as the plasma membrane of the cleavage furrow narrows to form the midbody. The midbody persists as a tether between the two daughter cells and contains the remains of the central spindle, a large protein structure derived from the anti-parallel interpolar microtubules of the spindle midzone, packed tightly together within a dense matrix material (Figure 17–43). After the daughter cells separate completely, some of the components of the residual midbody often remain on the inside of the plasma membrane of each cell, where they may serve as a mark on the cortex that helps to orient the spindle in the subsequent cell division. Local activation of Rhoa Triggers assembly and Contraction of the Contractile Ring

1	Local activation of Rhoa Triggers assembly and Contraction of the Contractile Ring RhoA, a small GTPase of the Ras superfamily (see Table 15–5), controls the assembly and function of the contractile ring at the site of cleavage. RhoA is activated at the cell cortex at the future division site, where it promotes actin filament formation, myosin II assembly, and ring contraction. It stimulates actin filament formation by activating formins, and it promotes myosin II assembly and contractions by activating multiple protein kinases, including the Rho-activated kinase Rock (Figure 17–44). These kinases phosphorylate the regulatory myosin light chain, a subunit of myosin II, thereby stimulating bipolar myosin II filament formation and motor activity.

1	RhoA is thought to be activated by a guanine nucleotide exchange factor (Rho-GEF), which is found at the cell cortex at the future division site and stimulates the release of GDP and binding of GTP to RhoA (see Figure 17–44). We know little about how the RhoGEF is localized or activated at the division site, although the microtubules of the anaphase spindle seem to be involved, as we discuss next. The microtubules of the mitotic spindle determine the plane of animal Cell division The central problem in cytokinesis is how to ensure that division occurs at the right time and in the right place. Cytokinesis must occur only after the two sets of chromosomes are fully segregated from each other, and the site of division must Figure 17–42 The contractile ring.

1	Figure 17–42 The contractile ring. (a) a drawing of the cleavage furrow in a dividing cell. (B) an electron micrograph of the ingrowing edge of a cleavage furrow of a dividing animal cell. (C) Fluorescence micrographs of a dividing slime mold amoeba stained for actin (red) and myosin II (green). Whereas all of the visible myosin II has redistributed to the contractile ring, only some of the actin has done so; the rest remains in the cortex of the nascent daughter cells. (B, from H.W. Beams and

1	R.g. kessel, Am. Sci. 64:279–290, 1976. With permission from sigma Xi; C, courtesy of Yoshio Fukui.) be placed between the two sets of daughter chromosomes, thereby ensuring that each daughter cell receives a complete set. The correct timing and positioning of cytokinesis in animal cells are achieved by mechanisms that depend on the mitotic spindle. During anaphase, the spindle generates signals that initiate furrow formation at a position midway between the spindle poles, thereby ensuring that division occurs between the two sets of separated chromosomes. Because these signals originate in the anaphase spindle, this mechanism also contributes to the correct timing of cytokinesis in late mitosis. Cytokinesis also occurs at the correct time because dephosphorylation of Cdk substrates, which depends on cyclin destruction in metaphase and anaphase, initiates cytokinesis. We now describe these regulatory mechanisms in more detail, with an emphasis on cytokinesis in animal cells.

1	Studies of the fertilized eggs of marine invertebrates first revealed the importance of spindle microtubules in determining the placement of the contractile ring. After fertilization, these embryos cleave rapidly without intervening periods of growth. In this way, the original egg is progressively divided into smaller and smaller cells. Because the cytoplasm is clear, the spindle can be observed in real time with a microscope. If the spindle is tugged into a new position with a fine glass needle in early anaphase, the incipient cleavage furrow disappears, and a new one develops in accord with the new spindle site—supporting the idea that signals generated by the spindle induce local furrow formation. How does the mitotic spindle specify the site of division? Three general mechanisms have been proposed, and most cells appear to employ a combination of these (Figure 17–45). The first is termed the astral stimulation model, in which the

1	Figure 17–43 The midbody. (a) a scanning electron micrograph of a cultured animal cell dividing; the midbody still joins the two daughter cells. (B) a conventional electron micrograph of the midbody of a dividing animal cell. Cleavage is almost complete, but the daughter cells remain attached by this thin strand of cytoplasm containing the remains of the central spindle. (a, courtesy of guenter albrecht-Buehler; B, courtesy of J.m. mullins.) assembly and contraction of actin–myosin ring Figure 17–44 Regulation of the contractile ring by the GTPase RhoA.

1	Figure 17–44 Regulation of the contractile ring by the GTPase RhoA. Like other Rho family gTpases, Rhoa is activated by a RhogEF protein and inactivated by a Rho gTpase-activating protein (Rhogap). The active gTpbound form of Rhoa is focused at the future cleavage site. By binding formins, activated Rhoa promotes the assembly of actin filaments in the contractile ring. By activating Rho-activated protein kinases, such as Rock, it stimulates myosin II filament formation and activity, thereby promoting contraction of the ring. astral microtubules carry furrow-inducing signals, which are somehow focused in a ring on the cell cortex, halfway between the spindle poles. Evidence for this model comes from ingenious experiments in large embryonic cells, which demonstrate that a cleavage furrow forms midway between two asters, even when the two centrosomes nucleating the asters are not connected to each other by a mitotic spindle (Figure 17–46).

1	A second possibility, called the central spindle stimulation model, is that the spindle midzone, or central spindle, generates a furrow-inducing signal that specifies the site of furrow formation at the cell cortex (see Figure 17–45). The overlapping interpolar microtubules of the central spindle associate with numerous signaling proteins, including proteins that may stimulate RhoA (Figure 17–47). Defects in the functions of these proteins (in Drosophila mutants, for example) result in failure of cytokinesis.

1	A third model proposes that, in some cell types, the astral microtubules promote the local relaxation of actin–myosin bundles at the cell cortex. According to this astral relaxation model, the cortical relaxation is minimal at the spindle equator, thus promoting cortical contraction at that site (see Figure 17–45). In the early embryos of Caenorhabditis elegans, for example, treatments that result in the loss of astral microtubules lead to increased contractile activity throughout the cell cortex, consistent with this model.

1	In some cell types, the site of ring assembly is chosen before mitosis. In budding yeasts, for example, a ring of proteins called septins assembles in late G1 at the future division site. The septins are thought to form a scaffold onto which other components of the contractile ring, including myosin II, assemble. In plant cells, an organized band of microtubules and actin filaments, called the preprophase band, assembles just before mitosis and marks the site where the cell wall will assemble and divide the cell in two, as we now discuss. both nuclei cleavage occurs both between the centrosomes enter mitosis linked by mitotic spindles and between the two centrosomes that are simply adjacent, and four daughter cells are formed

1	both nuclei cleavage occurs both between the centrosomes enter mitosis linked by mitotic spindles and between the two centrosomes that are simply adjacent, and four daughter cells are formed Figure 17–45 Three current models of how the microtubules of the anaphase spindle generate signals that influence the positioning of the contractile ring. no single model explains all the observations, and furrow positioning is probably determined by a combination of these mechanisms, with the importance of the different mechanisms varying in different organisms. see text for details.

1	Figure 17–46 An experiment demonstrating the influence of the position of microtubule asters on the subsequent plane of cleavage in a large egg cell. If the mitotic spindle is mechanically pushed to one side of the cell with a glass bead, the membrane furrowing is incomplete, failing to occur on the opposite side of the cell. subsequent cleavages occur not only at the midzone of each of the two subsequent mitotic spindles (yellow arrowheads), but also between the two adjacent asters that are not linked by a mitotic spindle—but in this abnormal cell share the same cytoplasm (red arrowhead). apparently, the contractile ring that produces the cleavage furrow in these cells always forms in the region midway between two asters, suggesting that the asters somehow alter the adjacent region of cell cortex to induce furrow formation between them. The phragmoplast guides Cytokinesis in Higher plants

1	The phragmoplast guides Cytokinesis in Higher plants In most animal cells, the inward movement of the cleavage furrow depends on an increase in the surface area of the plasma membrane. New membrane is added at the inner edge of the cleavage furrow and is generally provided by small membrane vesicles that are transported on microtubules from the Golgi apparatus to the furrow.

1	Membrane deposition is particularly important for cytokinesis in higher-plant cells. These cells are enclosed by a semirigid cell wall. Rather than a contractile ring dividing the cytoplasm from the outside in, the cytoplasm of the plant cell is partitioned from the inside out by the construction of a new cell wall, called the cell plate, between the two daughter nuclei (Figure 17–48). The assembly of the cell plate begins in late anaphase and is guided by a structure called the phragmoplast, which contains microtubules derived from the mitotic spindle. Motor proteins transport small vesicles along these microtubules from the Golgi apparatus to the cell center. These vesicles, filled with polysaccharide and glycoproteins required for the synthesis of the new cell wall, fuse to form a disc-like, membrane-enclosed structure called the early cell plate. The plate expands outward by further

1	Figure 17–48 Cytokinesis in a plant cell in telophase. In this light micrograph, the early cell plate (between the two arrowheads) has formed in a plane perpendicular to the plane of the page. The microtubules of the spindle are stained with gold-labeled antibodies against tubulin, and the dna in the two sets of daughter chromosomes is stained with a fluorescent dye. note that there are no astral microtubules, because there are no centrosomes in higher-plant cells. (Courtesy of andrew Bajer.)

1	Figure 17–47 Localization of cytokinesis regulators at the central spindle of the human cell. (a) at center is a cultured human cell at the beginning of cytokinesis, showing the locations of the gTpase Rhoa (red) and a protein called Cyk4 (green), which is one of several regulatory proteins that form complexes at the overlapping plus ends of interpolar microtubules. These proteins are thought to generate signals that help control Rhoa activity at the cell cortex. (B) When the same three-dimensional image is viewed in the plane of the contractile ring, as shown here, Rhoa (red) is seen as a ring beneath the cell surface, while the central spindle protein Cyk4 (green) is associated with microtubule bundles scattered throughout the equatorial plane of the cell. (Courtesy of alisa piekny and michael glotzer.) vesicle fusion until it reaches the plasma membrane and the original cell wall and divides the cell in two. Later, cellulose microfibrils are laid down within the matrix of the cell

1	michael glotzer.) vesicle fusion until it reaches the plasma membrane and the original cell wall and divides the cell in two. Later, cellulose microfibrils are laid down within the matrix of the cell plate to complete the construction of the new cell wall (Figure 17–49).

1	membrane-Enclosed Organelles must Be distributed to daughter Cells during Cytokinesis The process of mitosis ensures that each daughter cell receives a full complement of chromosomes. When a eukaryotic cell divides, however, each daughter cell must also inherit all of the other essential cell components, including the membrane-enclosed organelles. As discussed in Chapter 12, organelles such as mitochondria and chloroplasts cannot be assembled de novo from their individual components; they can arise only by the growth and division of the preexisting organelles. Similarly, cells cannot make a new endoplasmic reticulum (ER) unless some part of it is already present.

1	How, then, do the various membrane-enclosed organelles segregate when a cell divides? Organelles such as mitochondria and chloroplasts are usually present in large enough numbers to be safely inherited if, on average, their numbers roughly double once each cycle. The ER in interphase cells is continuous with the nuclear membrane and is organized by the microtubule cytoskeleton. Upon entry into M phase, the reorganization of the microtubules and breakdown of the nuclear envelope releases the ER. In most cells, the ER remains largely intact and is cut in two during cytokinesis. The Golgi apparatus is reorganized and fragmented during mitosis. Golgi fragments associate with the spindle poles and are thereby distributed to opposite ends of the spindle, ensuring that each daughter cell inherits the materials needed to reconstruct the Golgi in telophase. some Cells Reposition Their spindle to divide asymmetrically

1	some Cells Reposition Their spindle to divide asymmetrically Most animal cells divide symmetrically: the contractile ring forms around the equator of the parent cell, producing two daughter cells of equal size and with the same components. This symmetry results from the placement of the mitotic spindle, which in most cases tends to center itself in the cytoplasm. Astral micro-tubules and motor proteins that either push or pull on these microtubules contribute to the centering process. There are many instances in development, however, when cells divide asymmetrically to produce two cells that differ in size, in the cytoplasmic contents they inherit, or in both. Usually, the two different daughter cells are destined to develop

1	Figure 17–49 The special features of cytokinesis in a higher-plant cell. The division plane is established before m phase by a band of microtubules and actin filaments (the preprophase band) at the cell cortex. at the beginning of telophase, after the chromosomes have segregated, a new cell wall starts to assemble inside the cell at the equator of the old spindle. The interpolar microtubules of the mitotic spindle remaining at telophase form the phragmoplast. The plus ends of these microtubules no longer overlap but end at the cell equator. golgi-derived vesicles, filled with cell-wall material, are transported along these microtubules and fuse to form the new cell wall, which grows outward to reach the plasma membrane and original cell wall. The plasma membrane and the membrane surrounding the new cell wall fuse, separating the two daughter cells.

1	along different pathways. To create daughter cells with different fates in this way, the mother cell must first segregate certain components (called cell fate determinants) to one side of the cell and then position the plane of division so that the appropriate daughter cell inherits these components (Figure 17–50). To position the plane of division asymmetrically, the spindle has to be moved in a controlled manner within the dividing cell. It seems likely that changes in local regions of the cell cortex direct such spindle movements and that motor proteins localized there pull one of the spindle poles, via its astral microtubules, to the appropriate region. Genetic analyses in C. elegans and Drosophila have identified some of the proteins required for such asymmetric divisions, and some of these proteins seem to have a similar role in vertebrates.

1	Although nuclear division is usually followed by cytoplasmic division, there are exceptions. Some cells undergo multiple rounds of nuclear division without intervening cytoplasmic division. In the early Drosophila embryo, for example, the first 13 rounds of nuclear division occur without cytoplasmic division, resulting in the formation of a single large cell containing several thousand nuclei, arranged in a monolayer near the surface. A cell in which multiple nuclei share the same cytoplasm is called a syncytium. This arrangement greatly speeds up early development, as the cells do not have to take the time to go through all the steps of cytokinesis for each division. After these rapid nuclear divisions, membranes are created around each nucleus in one round of coordinated cytokinesis called cellularization. The plasma membrane extends inward and, with the help of an actin– myosin ring, pinches off to enclose each nucleus (Figure 17–51).

1	Nuclear division without cytokinesis also occurs in some types of mammalian cells. Megakaryocytes, which produce blood platelets, and some hepatocytes and heart muscle cells, for example, become multinucleated in this way. After cytokinesis, most cells enter G1, in which Cdks are mostly inactive. We end this section by discussing how this state is achieved at the end of M phase. The g1 phase Is a stable state of Cdk Inactivity A key regulatory event in late M phase is the inactivation of Cdks, which is driven primarily by APC/C-dependent cyclin destruction. As described earlier, the inactivation of Cdks in late M phase has many functions: it triggers the events of late mitosis, promotes cytokinesis, and enables the synthesis of prereplicative complexes at DNA replication origins. It also provides a mechanism for resetting the Figure 17–50 An asymmetric cell division segregating cytoplasmic components to only one daughter cell.

1	Figure 17–50 An asymmetric cell division segregating cytoplasmic components to only one daughter cell. These light micrographs illustrate the controlled asymmetric segregation of specific cytoplasmic components to one daughter cell during the first division of a fertilized egg of the nematode

1	C. elegans. The fertilized egg is shown in the left micrographs and the two daughter cells in the right micrographs. The cells above have been stained with a blue, dna-binding, fluorescent dye to show the nucleus (and polar bodies); they are viewed by both differential-interference-contrast and fluorescence microscopy. The cells below are the same cells stained with an antibody against p-granules and viewed by fluorescence microscopy. These small granules are made of Rna and proteins and determine which cells become germ cells. They are distributed randomly throughout the cytoplasm of the unfertilized egg (not shown) but become segregated to the posterior pole of the fertilized egg. The cleavage plane is oriented to ensure that only the posterior daughter cell receives the p-granules when the egg divides. The same segregation process is repeated in several subsequent cell divisions, so that the p-granules end up only in cells that give rise to eggs and sperm. (Courtesy of susan

1	when the egg divides. The same segregation process is repeated in several subsequent cell divisions, so that the p-granules end up only in cells that give rise to eggs and sperm. (Courtesy of susan strome.)

1	Figure 17–51 Mitosis without cytokinesis in the early Drosophila embryo. (a) The first 13 nuclear divisions occur synchronously and without cytoplasmic division to create a large syncytium. most of the nuclei migrate to the cortex, and the plasma membrane extends inward and pinches off to surround each nucleus to form individual cells in a process called cellularization. (B) Fluorescence micrograph of multiple mitotic spindles in a Drosophila embryo before cellularization. The microtubules are stained green and the centrosomes red. note that all the nuclei go through the cycle synchronously; here, they are all in metaphase, with the unlabeled chromosomes seen as a dark band at the spindle equator. (B, courtesy of kristina Yu and William sullivan.) cell-cycle control system to a state of Cdk inactivity as the cell prepares to enter a new cell cycle. In most cells, this state of Cdk inactivity generates a G1 gap phase, during which the cell grows and monitors its environment before

1	of Cdk inactivity as the cell prepares to enter a new cell cycle. In most cells, this state of Cdk inactivity generates a G1 gap phase, during which the cell grows and monitors its environment before committing to a new cell cycle.

1	In early animal embryos, the inactivation of M-Cdk in late mitosis is due almost entirely to the action of Cdc20–APC/C, discussed earlier. Recall, however, that M-Cdk stimulates Cdc20–APC/C activity. Thus, the destruction of M-cyclin in late mitosis soon leads to the inactivation of all APC/C activity in an embryonic cell. This APC/C inactivation immediately after mitosis is especially useful in rapid embryonic cell cycles, as it allows the cell to quickly begin accumulating new M-cyclin for the next cycle (Figure 17–52A).

1	Rapid cyclin accumulation immediately after mitosis is not useful, however, for cells in which a G1 phase is needed to allow control of entry into the next cell cycle. These cells employ several mechanisms to prevent Cdk reactivation after mitosis. One mechanism uses another APC/C-activating protein called Cdh1, mentioned earlier as a close relative of Cdc20 (see Table 17–2). Although both Cdh1 and Cdc20 bind to and activate the APC/C, they differ in one important respect. Whereas M-Cdk activates the Cdc20–APC/C complex, it inhibits the Cdh1–APC/C complex by directly phosphorylating Cdh1. As a result of this relationship, Cdh1– APC/C activity increases in late mitosis after the Cdc20–APC/C complex has initiated the destruction of M-cyclin. M-cyclin destruction therefore continues after mitosis: although Cdc20–APC/C activity has declined, Cdh1–APC/C activity is high (Figure 17–52B).

1	A second mechanism that suppresses Cdk activity in G1 depends on the increased production of CKIs, the Cdk inhibitor proteins discussed earlier. Budding yeast cells, in which this mechanism is best understood, contain a CKI protein called Sic1, which binds to and inactivates M-Cdk in late mitosis and G1 (see Figure 17–52 The creation of a G1 phase by stable Cdk inhibition after mitosis. (a) In early embryonic cell cycles, Cdc20–apC/C activity rises at the end of metaphase, triggering m-cyclin destruction. Because m-Cdk activity stimulates Cdc20–apC/C activity, the loss of m-cyclin leads to apC/C inactivation after mitosis, which allows m-cyclins to begin accumulating again. (B) In cells that have a g1 phase, the drop in m-Cdk activity in late mitosis leads to the activation of Cdh1–apC/C (as well as to the accumulation of Cdk inhibitor proteins; not shown). This ensures a continued suppression of Cdk activity after mitosis, as required for a g1 phase.

1	(A) embryonic cells with no G1 phase Cdc20–APC/C activity (B) cells with G1 phase Cdc20–APC/C activity Table 17–2). Like Cdh1, Sic1 is inhibited by M-Cdk, which phosphorylates Sic1 during mitosis and thereby promotes its ubiquitylation by SCF. Thus, Sic1 and M-Cdk, like Cdh1 and M-Cdk, inhibit each other. As a result, the decline in M-Cdk activity that occurs in late mitosis causes the Sic1 protein to accumulate, and this CKI helps keep M-Cdk activity low after mitosis. A CKI protein called p27 (see Figure 17–14) may serve similar functions in animal cells.

1	In most cells, decreased transcription of M-cyclin genes also inactivates M-Cdks in late mitosis. In budding yeast, for example, M-Cdk promotes the expression of these genes, resulting in a positive feedback loop. This loop is turned off as cells exit from mitosis: the inactivation of M-Cdk by Cdh1 and Sic1 leads to decreased M-cyclin gene transcription and thus decreased M-cyclin synthesis. Gene regulatory proteins that promote the expression of G1/Sand S-cyclins are also inhibited during G1.

1	Thus, Cdh1–APC/C activation, CKI accumulation, and decreased cyclin gene expression act together to ensure that the early G1 phase is a time when essentially all Cdk activity is suppressed. As in many other aspects of cell-cycle control, the use of multiple regulatory mechanisms allows the system to operate with reasonable efficiency even if one mechanism fails. So how does the cell escape from this stable G1 state to initiate a new cell cycle? The answer is that G1/S-Cdk activity, which rises in late G1, releases all the braking mechanisms that suppress Cdk activity, as we describe later, in the last section of this chapter.

1	After mitosis completes the formation of a pair of daughter nuclei, cytokinesis finishes the cell cycle by dividing the cell itself. Cytokinesis depends on a ring of actin and myosin filaments that contracts in late mitosis at a site midway between the segregated chromosomes. In animal cells, the positioning of the contractile ring is determined by signals emanating from the microtubules of the anaphase spindle. Dephosphorylation of Cdk targets, which results from Cdk inactivation in anaphase, triggers cytokinesis at the correct time after anaphase. After cytokinesis, the cell enters a stable G1 state of low Cdk activity, where it awaits signals to enter a new cell cycle.

1	Most eukaryotic organisms reproduce sexually: the genomes of two parents mix to generate offspring that are genetically distinct from either parent. The cells of these organisms are generally diploid: that is, they contain two slightly different copies, or homologs, of each chromosome, one from each parent. Sexual reproduction depends on a specialized nuclear division process called meiosis, which produces haploid cells carrying only a single copy of each chromosome. In many organisms, the haploid cells differentiate into specialized reproductive cells called gametes—eggs and sperm in most species. In these species, the reproductive cycle ends when a sperm and egg fuse to form a diploid zygote, which has the potential to form a new individual. In this section, we consider the basic mechanisms and regulation of meiosis, with an emphasis on how they compare with those of mitosis. meiosis Includes Two Rounds of Chromosome segregation

1	Meiosis reduces the chromosome number by half using many of the same molecular machines and control systems that operate in mitosis. As in the mitotic cell cycle, the cell begins the meiotic program by duplicating its chromosomes in meiotic S phase, resulting in pairs of sister chromatids that are tightly linked along their entire lengths by cohesin complexes. Unlike mitosis, however, two successive rounds of chromosome segregation then occur (Figure 17–53). The first of these divisions (meiosis I) solves the problem, unique to meiosis, of segregating the homologs. The duplicated paternal and maternal homologs pair up alongside each other and become physically linked by the process of genetic recombination. These pairs of homologs, each containing a pair of sister chromatids, then line up on the first meiotic spindle. In the first meiotic anaphase, duplicated homo-logs rather than sister chromatids are pulled apart and segregated into the two daughter nuclei. Only in the second

1	up on the first meiotic spindle. In the first meiotic anaphase, duplicated homo-logs rather than sister chromatids are pulled apart and segregated into the two daughter nuclei. Only in the second division (meiosis II), which occurs without further DNA replication, are the sister chromatids pulled apart and segregated (as in mitosis) to produce haploid daughter nuclei. In this way, each diploid nucleus that enters meiosis produces four haploid nuclei, each of which contains either the maternal or paternal copy of each chromosome, but not both (Movie 17.7).

1	Figure 17–53 Comparison of meiosis and mitosis. For clarity, only one pair of homologous chromosomes (homologs) is shown.

1	(a) meiosis is a form of nuclear division in which a single round of chromosome duplication (meiotic s phase) is followed by two rounds of chromosome segregation. The duplicated homologs, each consisting of tightly bound sister chromatids, pair up and are segregated into different daughter nuclei in meiosis I; the sister chromatids are segregated in meiosis II. as indicated by the formation of chromosomes that are partly red and partly gray, homolog pairing in meiosis leads to genetic recombination (crossing-over) during meiosis I. Each diploid cell that enters meiosis therefore produces four genetically different haploid nuclei, which are distributed by cytokinesis into haploid cells that differentiate into gametes. (B) In mitosis, by contrast, homologs do not pair up, and the sister chromatids are segregated during the single division. Thus, each diploid cell that divides by mitosis produces two genetically identical diploid daughter nuclei, which are distributed by cytokinesis into

1	are segregated during the single division. Thus, each diploid cell that divides by mitosis produces two genetically identical diploid daughter nuclei, which are distributed by cytokinesis into a pair of daughter cells.

1	During mitosis in most organisms, homologous chromosomes behave independently of each other. During meiosis I, however, it is crucial that homologs recognize each other and associate physically in order for the maternal and paternal homologs to be bi-oriented on the first meiotic spindle. Special mechanisms mediate these interactions.

1	The gradual juxtaposition of homologs occurs during a prolonged period called meiotic prophase (or prophase I), which can take hours in yeasts, days in mice, and weeks in higher plants. Like their mitotic counterparts, duplicated meiotic prophase chromosomes first appear as long threadlike structures, in which the sister chromatids are so tightly glued together that they appear as one. It is during early prophase I that the homologs begin to associate along their length in a process called pairing, which, in some organisms at least, begins with interactions between complementary DNA sequences (called pairing sites) in the two homologs. As prophase progresses, the homologs become more closely juxtaposed, forming a four-chromatid structure called a bivalent (Figure 17–54A). In most species, homolog pairs are then locked together by homologous recombination: DNA double-strand breaks are formed at several locations in each sister chromatid, resulting in large numbers of DNA recombination

1	homolog pairs are then locked together by homologous recombination: DNA double-strand breaks are formed at several locations in each sister chromatid, resulting in large numbers of DNA recombination events between the homologs (as described in Chapter 5). Some of these events lead to reciprocal DNA exchanges called crossovers, where the DNA of a chromatid crosses over to become continuous with the DNA of a homologous chromatid (Figure 17–54B; also see Figure 5–54).

1	Homolog pairing Culminates in the Formation of a synaptonemal Complex

1	The paired homologs are brought into close juxtaposition, with their structural axes (axial cores) about 400 nm apart, by a mechanism that depends in most species on the double-strand DNA breaks that occur in sister chromatids. What pulls the axes together? One possibility is that the large protein machine, called a recombination complex, which assembles on a double-strand break in a chromatid, binds the matching DNA sequence in the nearby homolog and helps reel in this partner. This so-called presynaptic alignment of the homologs is followed by synapsis, in which the axial core of a homolog becomes tightly linked to the axial core of its partner by a closely packed array of transverse filaments to create a synaptonemal complex, which bridges the gap, now only 100 nm, between the homologs (Figure 17–55). Although crossing-over begins before the synaptonemal complex assembles, the final steps occur while the DNA is held in the complex.

1	The morphological changes that occur during homolog pairing are the basis for dividing meiotic prophase into five sequential stages—leptotene, zygotene, pachytene, diplotene, and diakinesis (Figure 17–56). Prophase starts with leptotene, when homologs condense and pair and genetic recombination begins. At zygotene, the synaptonemal complex begins to assemble at sites where the homologs are closely associated and recombination events are occurring. At pachytene, the assembly process is complete, and the homologs are synapsed

1	Figure 17–54 Homolog pairing and crossing-over. (a) The structure formed by two closely aligned duplicated homologs is called a bivalent. as in mitosis, the sister chromatids in each homolog are tightly connected along their entire lengths, as well as at their centromeres. at this stage, the homologs are usually joined by a protein complex called the synaptonemal complex (not shown; see Figure 17–55). (B) a later-stage bivalent in which a single crossover has occurred between nonsister chromatids. It is only when the synaptonemal complex disassembles and the paired homologs separate a little at the end of prophase I, as shown, that the crossover is seen microscopically as a thin connection between the homologs called a chiasma. Figure 17–55 Simplified schematic drawing of a synaptonemal complex.

1	Each homolog is organized around a protein axial core, and the synaptonemal axes are linked by rod-shaped transverse filaments. The axial core of each homolog also interacts with the cohesin complexes axial cores that hold the sister chromatids together of the (see Figure 9–35). (modified from homologs k. nasmyth, Annu. Rev. Genet. 35:673– 745, 2001.) cohesin chromatin loops of sister chromatids of one homolog along their entire lengths (see Figure 9–35). The pachytene stage can persist for days or longer, until desynapsis begins at diplotene with the disassembly of the synaptonemal complexes and the concomitant condensation and shortening of the chromosomes. It is only at this stage, after the complexes have disassembled, that the individual crossover events between nonsister chromatids can be seen as inter-homolog connections called chiasmata (singular chiasma), which now play a crucial part in holding the compact homologs together (Figure 17–57). The homologs are now ready to begin

1	be seen as inter-homolog connections called chiasmata (singular chiasma), which now play a crucial part in holding the compact homologs together (Figure 17–57). The homologs are now ready to begin the process of segregation.

1	Figure 17–56 Homolog synapsis and desynapsis during the different stages of prophase I. (a) a single bivalent is shown schematically. at leptotene, the two sister chromatids coalesce, and their chromatid loops extend out from a common axial core. assembly of the synaptonemal complex begins in early zygotene and is complete in pachytene. The complex disassembles in diplotene. (B) an electron micrograph of a synaptonemal complex from a meiotic cell at pachytene in a lily flower. (C and d) Immunofluorescence micrographs of prophase I cells of the fungus Sordaria. partially synapsed bivalents at zygotene are shown in (C) and fully synapsed bivalents are shown in (d). Red arrowheads in (C) point to regions where synapsis is still incomplete. (B, courtesy of Brian Wells; C and d, from a. storlazzi et al., Genes Dev. 17:2675–2687, 2003. With permission from Cold spring Harbor Laboratory press.)

1	Figure 17–58 Comparison of chromosome behavior in meiosis I, meiosis II, and mitosis. Chromosomes behave similarly in mitosis and meiosis II, but they behave very differently in meiosis I. (a) In meiosis I, the two sister kinetochores are located side-by-side on each homolog and attach to microtubules from the same spindle pole. The proteolytic cleavage of cohesin along the sister-chromatid arms unglues the arms and resolves the crossovers, allowing the duplicated homologs to separate at anaphase I, while the residual cohesin at the centromeres keeps the sisters together. Cleavage of centromeric cohesin allows the sister chromatids to separate at anaphase II. (B) In mitosis, by contrast, the two sister kinetochores attach to microtubules from different spindle poles, and the two sister chromatids come apart at the start of anaphase and segregate into separate daughter nuclei.

1	localized at the kinetochores in meiosis I, but we do not know in any detail how these proteins work. They are removed from kinetochores after meiosis I, so that in meiosis II the sister-chromatid pairs can be bi-oriented on the spindle as they are in mitosis. Second, crossovers generate a strong physical linkage between homologs, allowing their bi-orientation at the equator of the spindle—much like cohesion between sister chromatids is important for their bi-orientation in mitosis (and meiosis II). Crossovers hold homolog pairs together only because the arms of the sister chromatids are connected by sister-chromatid cohesion (see Figure 17–58A).

1	Third, cohesion is removed in anaphase I only from chromosome arms and not from the regions near the centromeres, where the kinetochores are located. The loss of arm cohesion triggers homolog separation at the onset of anaphase I. This process depends on APC/C activation, which leads to securin destruction, separase activation, and cohesin cleavage along the arms (see Figure 17–38).

1	Cohesins near the centromeres are protected from separase in meiosis I by a kinetochore-associated protein called shugoshin (from the Japanese word for “guardian spirit”). Shugoshin acts by recruiting a protein phosphatase that removes phosphates from centromeric cohesins. Cohesin phosphorylation is normally required for separase to cleave cohesin; thus, removal of this phosphorylation near the centromere prevents cohesin cleavage. Sister-chromatid pairs therefore remain linked through meiosis I, allowing their correct bi-orientation on the spindle in meiosis II. Shugoshin is inactivated after meiosis I. At the onset of anaphase II, APC/C activation triggers centromeric cohesin cleavage and sisterchromatid separation—much as it does in mitosis. Following anaphase II, nuclear envelopes form around the chromosomes to produce four haploid nuclei, after which cytokinesis and other differentiation processes lead to the production of haploid gametes.

1	Crossing-over has two distinct functions in meiosis: it helps hold homologs together so that they are properly segregated to the two daughter nuclei produced by meiosis I, and it contributes to the genetic diversification of the gametes that are eventually produced. As might be expected, therefore, crossing-over is highly regulated: the number and location of double-strand breaks along each chromosome is controlled, as is the likelihood that a break will be converted into a crossover. On average, the result of this regulation is that each pair of human homologs is linked by about two or three crossovers (Figure 17–59). Although the double-strand breaks that occur in meiosis I can be located almost anywhere along the chromosome, they are not distributed uniformly: they cluster at “hot spots,” where the DNA is accessible, and occur only rarely in “cold spots,” such as the heterochromatin regions around centromeres and telomeres.

1	Figure 17–59 Crossovers between homologs in the human testis. In these immunofluorescence micrographs, antibodies have been used to stain the synaptonemal complexes (red), the centromeres (blue), and the sites of crossing-over (green). note that all of the bivalents have at least one crossover and none have more than four. (modified from a. Lynn et al., Science 296:2222–2225, 2002. With permission from aaas.) At least two kinds of regulation influence the location and number of crossovers that form, neither of which is well understood. Both operate before the synaptonemal complex assembles. One ensures that at least one crossover forms between the members of each homolog pair, as is necessary for normal homolog segregation in meiosis I. In the other, called crossover interference, the presence of one crossover event inhibits another from forming close by, perhaps by locally depleting proteins required for converting a double-strand DNA break into a stable crossover.

1	The sorting of chromosomes that takes place during meiosis is a remarkable feat of intracellular bookkeeping. In humans, each meiosis requires that the starting cell keep track of 92 chromatids (46 chromosomes, each of which has duplicated), distributing one complete set of each type of autosome to each of the four haploid progeny. Not surprisingly, mistakes can occur in allocating the chromosomes during this elaborate process. Mistakes are especially common in human female meiosis, which arrests for years after diplotene: meiosis I is completed only at ovulation, and meiosis II only after the egg is fertilized. Indeed, such chromosome segregation errors during egg development are the most common cause of both spontaneous abortion (miscarriage) and mental retardation in humans.

1	When homologs fail to separate properly—a phenomenon called nondisjunction—the result is that some of the resulting haploid gametes lack a particular chromosome, while others have more than one copy of it. Upon fertilization, these gametes form abnormal embryos, most of which die. Some survive, however. Down syndrome in humans, for example, which is the leading cause of mental retardation, is caused by an extra copy of chromosome 21, usually resulting from nondisjunction during meiosis I in the female ovary. Segregation errors during meiosis I increase greatly with advancing maternal age.

1	Haploid gametes are produced by meiosis, in which a diploid nucleus undergoes two successive cell divisions after one round of DNA replication. Meiosis is dominated by a prolonged prophase. At the start of prophase, the chromosomes have replicated and consist of two tightly joined sister chromatids. Homologous chromosomes then pair up and become progressively more closely juxtaposed as prophase proceeds. The tightly aligned homologs undergo genetic recombination, forming crossovers that help hold each pair of homologs together during metaphase I. Meiosis-specific, kinetochore-associated proteins help ensure that both sister chromatids in a homolog attach to the same spindle pole; other kinetochore-associated proteins ensure that the homologs remain connected at their centromeres during anaphase I, so that homologs rather than sister chromatids are segregated in meiosis I. After meiosis I, meiosis II follows rapidly, without DNA replication, in a process that resembles mitosis, in that

1	I, so that homologs rather than sister chromatids are segregated in meiosis I. After meiosis I, meiosis II follows rapidly, without DNA replication, in a process that resembles mitosis, in that sister chromatids are pulled apart at anaphase.

1	A fertilized mouse egg and a fertilized human egg are similar in size, yet they produce animals of very different sizes. What factors in the control of cell behavior in humans and mice are responsible for these size differences? The same fundamental question can be asked for each organ and tissue in an animal’s body. What factors determine the length of an elephant’s trunk or the size of its brain or its liver? These questions are largely unanswered, but it is nevertheless possible to say what the ingredients of an answer must be. The size of an organ or organism depends on its total cell mass, which depends on both the total number of cells and their size. Cell number, in turn, depends on the amounts of cell division and cell death. Organ and body size are therefore determined by three fundamental processes: cell growth, cell division, and cell survival. Each is tightly regulated—both by intracellular programs and by extracellular signal molecules that control these programs.

1	The extracellular signal molecules that regulate cell growth, division, and survival are generally soluble secreted proteins, proteins bound to the surface of cells, or components of the extracellular matrix. They can be divided operationally into three major classes: 1. Mitogens, which stimulate cell division, primarily by triggering a wave of G1/S-Cdk activity that relieves intracellular negative controls that otherwise block progress through the cell cycle. 2. Growth factors, which stimulate cell growth (an increase in cell mass) by promoting the synthesis of proteins and other macromolecules and by inhibiting their degradation. 3. Survival factors, which promote cell survival by suppressing the form of programmed cell death known as apoptosis.

1	3. Survival factors, which promote cell survival by suppressing the form of programmed cell death known as apoptosis. Many extracellular signal molecules promote all of these processes, while others promote one or two of them. Indeed, the term growth factor is often used inappropriately to describe a factor that has any of these activities. Even worse, the term cell growth is often used to mean an increase in cell number, or cell proliferation. In addition to these three classes of stimulating signals, there are extracellular signal molecules that suppress cell proliferation, cell growth, or both; in general, less is known about them. There are also extracellular signal molecules that activate apoptosis.

1	In this section, we focus primarily on how mitogens and other factors, such as DNA damage, control the rate of cell division. We then turn to the important but poorly understood problem of how a proliferating cell coordinates its growth with cell division so as to maintain its appropriate size. We discuss the control of cell survival and cell death by apoptosis in Chapter 18. Unicellular organisms tend to grow and divide as fast as they can, and their rate of proliferation depends largely on the availability of nutrients in the environment. The cells of a multicellular organism, however, divide only when the organism needs more cells. Thus, for an animal cell to proliferate, it must receive stimulatory extracellular signals, in the form of mitogens, from other cells, usually its neighbors. Mitogens overcome intracellular braking mechanisms that block progress through the cell cycle.

1	One of the first mitogens to be identified was platelet-derived growth factor (PDGF), and it is typical of many others discovered since. The path to its isolation began with the observation that fibroblasts in a culture dish proliferate when provided with serum but not when provided with plasma. Plasma is prepared by removing the cells from blood without allowing clotting to occur; serum is prepared by allowing blood to clot and taking the cell-free liquid that remains. When blood clots, platelets incorporated in the clot are stimulated to release the contents of their secretory vesicles (Figure 17–60). The superior ability of serum to support cell proliferation suggested that platelets contain one or more mitogens. This hypothesis was confirmed by showing that extracts of platelets could serve instead of serum to stimulate fibroblast proliferation. The crucial factor in the extracts was shown to be a protein, which was subsequently purified and named PDGF. In the body, PDGF liberated

1	serve instead of serum to stimulate fibroblast proliferation. The crucial factor in the extracts was shown to be a protein, which was subsequently purified and named PDGF. In the body, PDGF liberated from blood clots helps stimulate cell division during wound healing.

1	PDGF is only one of over 50 animal proteins that are known to act as mitogens. Most of these proteins have a broad specificity. PDGF, for example, can stimulate many types of cells to divide, including fibroblasts, smooth muscle cells, and neuroglial cells. Similarly, epidermal growth factor (EGF ) acts not only on epidermal cells but also on many other cell types, including both epithelial and nonepithelial cells. Some mitogens, however, have a narrow specificity; erythropoietin, for Figure 17–60 A platelet. platelets are miniature cells without a nucleus. They circulate in the blood and help stimulate blood clotting at sites of tissue damage, thereby preventing excessive bleeding. They also release various factors that stimulate wound healing. The platelet shown here has been cut in half to show its secretory vesicles, some of which contain platelet-derived growth factor (pdgF).

1	example, only induces the proliferation of red blood cell precursors. Many mitogens, including PDGF, also have actions other than the stimulation of cell division: they can stimulate cell growth, survival, differentiation, or migration, depending on the circumstances and the cell type. In some tissues, inhibitory extracellular signal proteins oppose the positive regulators and thereby inhibit organ growth. The best-understood inhibitory signal proteins are transforming growth factor-β (TGFβ) and its relatives. TGFβ inhibits the proliferation of several cell types, mainly by blocking cell-cycle progression in G1. In the absence of a mitogenic signal to proliferate, Cdk inhibition in G1 is maintained by the multiple mechanisms discussed earlier, and progression into a new cell cycle is blocked. In some cases, cells partly disassemble their cell-cycle control system and withdraw from the cycle to a specialized nondividing state called G0.

1	Most cells in our body are in G0, but the molecular basis and reversibility of this state vary in different cell types. Most of our neurons and skeletal muscle cells, for example, are in a terminally differentiated G0 state, in which their cell-cycle control system is completely dismantled: the expression of the genes encoding various Cdks and cyclins is permanently turned off, and cell division rarely occurs. Some cell types withdraw from the cell cycle only transiently and retain the ability to reassemble the cell-cycle control system quickly and re-enter the cycle. Most liver cells, for example, are in G0, but they can be stimulated to divide if the liver is damaged. Still other types of cells, including fibroblasts and some lymphocytes, withdraw from and re-enter the cell cycle repeatedly throughout their lifetime.

1	Almost all the variation in cell-cycle length in the adult body occurs during the time the cell spends in G1 or G0. By contrast, the time a cell takes to progress from the beginning of S phase through mitosis is usually brief (typically 12–24 hours in mammals) and relatively constant, regardless of the interval from one division to the next. For the vast majority of animal cells, mitogens control the rate of cell division by acting in the G1 phase of the cell cycle. As discussed earlier, multiple mechanisms act during G1 to suppress Cdk activity. Mitogens release these brakes on Cdk activity, thereby allowing entry into a new cell cycle.

1	As we discuss in Chapter 15, mitogens interact with cell-surface receptors to trigger multiple intracellular signaling pathways. One major pathway acts through the monomeric GTPase Ras, which leads to the activation of a mitogen-activated protein kinase (MAP kinase) cascade (see Figure 15–49). This leads to an increase in the production of transcription regulatory proteins, including Myc. Myc is thought to promote cell-cycle entry by several mechanisms, one of which is to increase the expression of genes encoding G1 cyclins (D cyclins), thereby increasing G1-Cdk (cyclin D–Cdk4) activity. Myc also has a major role in stimulating the transcription of genes that increase cell growth.

1	The key function of G1-Cdk complexes in animal cells is to activate a group of gene regulatory factors called the E2F proteins, which bind to specific DNA sequences in the promoters of a wide variety of genes that encode proteins required for S-phase entry, including G1/S-cyclins, S-cyclins, and proteins involved in DNA synthesis and chromosome duplication. In the absence of mitogenic stimulation, E2F-dependent gene expression is inhibited by an interaction between E2F and members of the retinoblastoma protein (Rb) family. When cells are stimulated to divide by mitogens, active G1-Cdk accumulates and phosphorylates Rb family members, reducing their binding to E2F. The liberated E2F proteins then activate expression of their target genes (Figure 17–61). This transcriptional control system, like so many other control systems that regulate the cell cycle, includes feedback loops that ensure that entry into the activation of transcription regulatory protein CYTOSOL

1	Figure 17–61 Mitogen stimulation of cell-cycle entry. as discussed in Chapter 15, mitogens bind to cell-surface receptors to initiate intracellular signaling pathways. One of the major pathways involves activation of the small gTpase Ras, which activates a map kinase cascade, leading to increased expression of numerous immediate early genes, including the gene encoding the transcription regulatory protein myc. myc increases the expression of many delayed-response genes, including some that lead to increased g1-Cdk activity (cyclin d– Cdk4), which triggers the phosphorylation of members of the Rb family of proteins. This inactivates the Rb proteins, freeing the gene regulatory protein E2F to activate the transcription of g1/s genes, including the genes for a g1/s-cyclin (cyclin E) and s-cyclin (cyclin a). The resulting g1/s-Cdk and s-Cdk activities further enhance Rb protein phosphorylation, forming a positive feedback loop. E2F proteins also stimulate the transcription of their own

1	(cyclin a). The resulting g1/s-Cdk and s-Cdk activities further enhance Rb protein phosphorylation, forming a positive feedback loop. E2F proteins also stimulate the transcription of their own genes, forming another positive feedback loop.

1	cell cycle is complete and irreversible. The liberated E2F proteins, for example, increase the transcription of their own genes. In addition, E2F-dependent transcription of G1/S-cyclin (cyclin E) and S-cyclin (cyclin A) genes leads to increased G1/S-Cdk and S-Cdk activities, which in turn increase Rb protein phosphorylation and promote further E2F release (see Figure 17–61).

1	The central member of the Rb family, the Rb protein itself, was identified originally through studies of an inherited form of eye cancer in children, known as retinoblastoma (discussed in Chapter 20). The loss of both copies of the Rb gene leads to excessive proliferation of some cells in the developing retina, suggesting that the Rb protein is particularly important for restraining cell division in this tissue. The complete loss of Rb does not immediately cause increased proliferation of retinal or other types of cells, in part because Cdh1 and CKIs also help inhibit progression through G1 and in part because other cell types contain Rb-related proteins that provide backup support in the absence of Rb. It is also likely that other proteins, unrelated to Rb, help to regulate the activity of E2F.

1	Additional layers of control promote an overwhelming increase in S-Cdk activity at the beginning of S phase. We mentioned earlier that the APC/C activator Cdh1 suppresses cyclin levels after mitosis. In animal cells, however, G1-and G1/S-cyclins are resistant to Cdh1–APC/C and can therefore act unopposed by the APC/C to promote Rb protein phosphorylation and E2F-dependent gene expression. S-cyclin, by contrast, is not resistant, and its level is initially restrained by Cdh1–APC/C activity. However, G1/S-Cdk also phosphorylates and inactivates Cdh1–APC/C, thereby allowing the accumulation of S-cyclin, further promoting S-Cdk activation. G1/S-Cdk also inactivates CKI proteins that suppress S-Cdk activity. The overall effect of all these interactions is the rapid and complete activation of the S-Cdk complexes required for S-phase initiation. dna damage Blocks Cell division: The dna damage Response

1	dna damage Blocks Cell division: The dna damage Response Progression through the cell cycle, and thus the rate of cell proliferation, is controlled not only by extracellular mitogens but also by other extracellular and intracellular signals. One of the most important influences is DNA damage, which can occur as a result of spontaneous chemical reactions in DNA, errors in DNA replication, or exposure to radiation or certain chemicals (discussed in Chapter 5). It is essential that damaged chromosomes are repaired before attempting to duplicate or segregate them. The cell-cycle control system can readily detect DNA damage and arrest the cycle at either of two transitions—one at Start, which prevents entry into the cell cycle and into S phase, and one at the G2/M transition, which prevents entry into mitosis (see Figure 17–16).

1	DNA damage initiates a signaling pathway by activating one of a pair of related protein kinases called ATM and ATR, which associate with the site of damage and phosphorylate various target proteins, including two other protein kinases called Chk1 and Chk2. These various kinases phosphorylate other target proteins that lead to cell-cycle arrest. A major target is the gene regulatory protein p53, which stimulates transcription of the gene encoding p21, a CKI protein; p21 binds to G1/S-Cdk and S-Cdk complexes and inhibits their activities, thereby helping to block entry into the cell cycle (Figure 17–62 and Movie 17.8).

1	DNA damage activates p53 by an indirect mechanism. In undamaged cells, p53 is highly unstable and is present at very low concentrations. This is largely because it interacts with another protein, Mdm2, which acts as a ubiquitin ligase that targets p53 for destruction by proteasomes. Phosphorylation of p53 after DNA damage reduces its binding to Mdm2. This decreases p53 degradation, which results in a marked increase in p53 concentration in the cell. In addition, the decreased binding to Mdm2 enhances the ability of p53 to stimulate gene transcription (see Figure 17–62).

1	The protein kinases Chk1 and Chk2 also block cell-cycle progression by phosphorylating members of the Cdc25 family of protein phosphatases, thereby inhibiting their function. As described earlier, these phosphatases are particularly important in the activation of M-Cdk at the beginning of mitosis (see Figure 17–20). Chk1 and Chk2 phosphorylate Cdc25 at inhibitory sites that are distinct from the phosphorylation sites that stimulate Cdc25 activity. The inhibition of Cdc25 activity by DNA damage helps block entry into mitosis (see Figure 17–16).

1	The DNA damage response can also be activated by problems that arise when a replication fork fails during DNA replication. When nucleotides are depleted, for example, replication forks stall during the elongation phase of DNA synthesis. To prevent the cell from attempting to segregate partially replicated chromosomes, the same mechanisms that respond to DNA damage detect the stalled replication forks and block entry into mitosis until the problems are resolved. A low level of DNA damage occurs in the normal life of any cell, and this damage accumulates in the cell’s progeny if the DNA damage response is not functioning. Over the long term, the accumulation of genetic damage in cells lacking the DNA damage response leads to an increased frequency of cancer-promoting mutations. Indeed, mutations in the p53 gene occur in at least half of all human cancers (discussed in Chapter 20). This loss of p53 function allows the cancer cell stable, active p53

1	Figure 17–62 How DNA damage arrests the cell cycle in G1. When dna is damaged, various protein kinases are recruited to the site of damage and initiate a signaling pathway that causes cell-cycle arrest. The first kinase at the damage site is either aTm or aTR, depending on the type of damage. additional protein kinases, called Chk1 and Chk2, are then recruited and activated, resulting in the phosphorylation of the transcription regulatory protein p53. mdm2 normally binds to p53 and promotes its ubiquitylation and destruction in proteasomes. phosphorylation of p53 blocks its binding to mdm2; as a result, p53 accumulates to high levels and stimulates transcription of numerous genes, including the gene that encodes the CkI protein p21. The p21 binds and inactivates g1/s-Cdk and s-Cdk complexes, arresting the cell in g1. In some cases, dna damage also induces either the phosphorylation of mdm2 or a decrease in mdm2 production, which causes a further increase in p53 (not shown).

1	to accumulate mutations more readily. Similarly, a rare genetic disease known as ataxia telangiectasia is caused by a defect in ATM, one of the protein kinases that are activated in response to x-ray-induced DNA damage; patients with this disease are very sensitive to x-rays and suffer from increased rates of cancer.

1	What happens if DNA damage is so severe that repair is not possible? The answer differs in different organisms. Unicellular organisms such as budding yeast arrest their cell cycle to try to repair the damage, but the cycle resumes even if the repair cannot be completed. For a single-celled organism, life with mutations is apparently better than no life at all. In multicellular organisms, however, the health of the organism takes precedence over the life of an individual cell. Cells that divide with severe DNA damage threaten the life of the organism, since genetic damage can often lead to cancer and other diseases. Thus, animal cells with severe DNA damage do not attempt to continue division, but instead commit suicide by undergoing apoptosis. Thus, unless the DNA damage is repaired, the DNA damage response can lead to either cell-cycle arrest or cell death. DNA damage-induced apoptosis often depends on the activation of p53. Indeed, it is this apoptosis-promoting function of p53 that

1	damage response can lead to either cell-cycle arrest or cell death. DNA damage-induced apoptosis often depends on the activation of p53. Indeed, it is this apoptosis-promoting function of p53 that is apparently most important in protecting us against cancer.

1	many Human Cells Have a Built-In Limitation on the number of Times They Can divide Many human cells divide a limited number of times before they stop and undergo a permanent cell-cycle arrest. Fibroblasts taken from normal human tissue, for example, go through only about 25–50 population doublings when cultured in a standard mitogenic medium. Toward the end of this time, proliferation slows down and finally halts, and the cells enter a nondividing state from which they never recover. This phenomenon is called replicative cell senescence.

1	Replicative cell senescence in human fibroblasts seems to be caused by changes in the structure of the telomeres, the repetitive DNA sequences and associated proteins at the ends of chromosomes. As discussed in Chapter 5, when a cell divides, telomeric DNA sequences are not replicated in the same manner as the rest of the genome but instead are synthesized by the enzyme telomerase. Telomerase also promotes the formation of protein cap structures that protect the chromosome ends. Because human fibroblasts, and many other human somatic cells, do not produce telomerase, their telomeres become shorter with every cell division, and their protective protein caps progressively deteriorate. Eventually, the exposed chromosome ends are sensed as DNA damage, which activates a p53-dependent cell-cycle arrest (see Figure 17–62). Rodent cells, by contrast, maintain telomerase activity when they proliferate in culture and therefore do not have such a telomere-dependent mechanism for limiting

1	arrest (see Figure 17–62). Rodent cells, by contrast, maintain telomerase activity when they proliferate in culture and therefore do not have such a telomere-dependent mechanism for limiting proliferation. The forced expression of telomerase in normal human fibroblasts, using genetic engineering techniques, blocks this form of senescence. Unfortunately, most cancer cells have regained the ability to produce telomerase and therefore maintain telomere function as they proliferate; as a result, they do not undergo replicative cell senescence.

1	abnormal proliferation signals Cause Cell-Cycle arrest or apoptosis, Except in Cancer Cells Many of the components of mitogenic signaling pathways are encoded by genes that were originally identified as cancer-promoting genes, because mutations in them contribute to the development of cancer. The mutation of a single amino acid in the small GTPase Ras, for example, causes the protein to become permanently overactive, leading to constant stimulation of Ras-dependent signaling pathways, even in the absence of mitogenic stimulation. Similarly, mutations that cause an overexpression of Myc stimulate excessive cell growth and proliferation and thereby promote the development of cancer (discussed in Chapter 20).

1	Surprisingly, however, when a hyperactivated form of Ras or Myc is experimentally overproduced in most normal cells, the result is not excessive proliferation but the opposite: the cells undergo either permanent cell-cycle arrest or apoptosis. The normal cell seems able to detect abnormal mitogenic stimulation, and it responds by preventing further division. Such responses help prevent the survival and proliferation of cells with various cancer-promoting mutations. Although it is not known how a cell detects excessive mitogenic stimulation, such stimulation often leads to the production of a cell-cycle inhibitor protein called Arf, which binds and inhibits Mdm2. As discussed earlier, Mdm2 normally promotes p53 degradation. Activation of Arf therefore causes p53 levels to increase, inducing either cell-cycle arrest or apoptosis (Figure 17–63).

1	How do cancer cells ever arise if these mechanisms block the division or survival of mutant cells with overactive proliferation signals? The answer is that the protective system is often inactivated in cancer cells by mutations in the genes that encode essential components of the blocking mechanisms, such as Arf or p53 or the proteins that help activate them. Cell proliferation is accompanied by Cell growth If cells proliferated without growing, they would get progressively smaller and there would be no net increase in total cell mass. In most proliferating cell populations, therefore, cell growth accompanies cell division. In single-celled organisms stable, active p53 such as yeasts, both cell growth and cell division require only nutrients. In animals, by contrast, both cell growth and cell proliferation depend on extracellular signal molecules, produced by other cells, which we call growth factors and mitogens, respectively.

1	Like mitogens, the extracellular growth factors that stimulate animal cell growth bind to receptors on the cell surface and activate intracellular signaling pathways. These pathways stimulate the accumulation of proteins and other macromolecules, and they do so by both increasing their rate of synthesis and decreasing their rate of degradation. They also trigger increased uptake of nutrients and production of the ATP required to fuel the increased protein synthesis. One of the most important intracellular signaling pathways activated by growth factor receptors involves the enzyme phosphoinositide 3-kinase (PI 3-kinase), which adds a phosphate from ATP to the 3ʹ position of inositol phospholipids in the plasma membrane (discussed in Chapter 15). The activation of PI 3-kinase leads to the activation of a kinase called TOR, which lies at the heart of cell growth regulatory pathways in all eukaryotes. TOR activates many targets in the cell that stimulate metabolic processes , including

1	activation of a kinase called TOR, which lies at the heart of cell growth regulatory pathways in all eukaryotes. TOR activates many targets in the cell that stimulate metabolic processes , including protein synthesis. One target is a protein kinase called S6 kinase (S6K), which phosphorylates ribosomal protein S6, increasing the ability of ribosomes to translate a subset of mRNAs that mostly encode ribosomal components. TOR also indirectly activates a translation initiation factor called eIF4E and directly activates transcription regulators that promote the increased expression of genes encoding ribosomal subunits (Figure 17–64).

1	PI(3,4,5)P3 PI(4,5)P2 growth factor amino acids activated growth factor receptor activated PI 3-kinase TOR S6K 4E-BP S6 eIF4E transcription regulatory proteins P P P P P PP Figure 17–63 Cell-cycle arrest or apoptosis induced by excessive stimulation of mitogenic pathways. abnormally high levels of myc cause the activation of arf, which binds and inhibits mdm2 and thereby increases p53 levels (see Figure 17–62). depending on the cell type and extracellular conditions, p53 then causes either cell-cycle arrest or apoptosis.

1	Figure 17–64 Stimulation of cell growth by extracellular growth factors and nutrients. The occupation of cell-surface receptors by growth factors leads to the activation of pI 3-kinase, which promotes protein synthesis through a complex signaling pathway that leads to the activation of the protein kinase TOR; extracellular nutrients such as amino acids also help activate TOR. TOR phosphorylates multiple proteins to stimulate protein synthesis, as shown; it also inhibits protein degradation (not shown). growth factors also stimulate increased production of the transcription regulatory protein myc (not shown), which activates the transcription of various genes that promote cell metabolism and growth. 4E-Bp is an inhibitor of the translation initiation factor eIF4E. pI(4,5)p2, phosphatidylinositol 4,5-bisphosphate; pI(3,4,5)p3, phosphatidylinositol 3,4,5-trisphosphate.

1	Figure 17–65 Potential mechanisms for coordinating cell growth and division. In proliferating cells, cell size is maintained by mechanisms that coordinate rates of cell division and cell growth. numerous alternative coupling mechanisms are thought to exist, and different cell types appear to employ different combinations of these mechanisms. (a) In many cell types—particularly yeast—the rate of cell division is governed by the rate of cell growth, so that division occurs only when growth rate achieves some minimal threshold; in yeasts, it is mainly the levels of extracellular nutrients that regulate the rate of cell growth and thereby the rate of cell division. (B) In some animal cell types, growth and division can each be controlled by separate extracellular factors (growth factors and mitogens, respectively), and cell size depends on the relative levels of the two types of factors.

1	(C) some extracellular factors can stimulate both cell growth and cell division by simultaneously activating signaling pathways that promote growth and other pathways that promote cell-cycle progression. For proliferating cells to maintain a constant size, they must coordinate their • progression through the cell cycle growth with cell division to ensure that cell size doubles with each division: if cells depends on the phosphorylation grow too slowly, they will get smaller with each division, and if they grow too fast, of hundreds of different proteins by they will get larger with each division. It is not clear how cells achieve this coordi cyclin–Cdk complexes. What are the nation, but it is likely to involve multiple mechanisms that vary in different organ- isms and even in different cell types of the same organism (Figure 17–65).

1	these proteins are phosphorylated at Animal cell growth and division are not always coordinated, however. In many precisely the right time and place? cases, they are completely uncoupled to allow growth without division or division without growth. Muscle cells and nerve cells, for example, can grow dramatically • during s phase, how are histones after they have permanently withdrawn from the cell cycle. Similarly, the eggs of and their modifying enzymes many animals grow to an extremely large size without dividing; after fertilization, controlled to replicate chromatin however, this relationship is reversed, and many rounds of division occur without structure on the duplicated dna? growth. Compared to cell division, there has been surprisingly little study of how cell • What is the structural basis of size is controlled in animals. As a result, it remains a mystery how cell size is deter-chromosome condensation, and how is the process stimulated during mined and why different cell

1	basis of size is controlled in animals. As a result, it remains a mystery how cell size is deter-chromosome condensation, and how is the process stimulated during mined and why different cell types in the same animal grow to be so different in mitosis?

1	size. One of the best-understood cases in mammals is the adult sympathetic neuron, which has permanently withdrawn from the cell cycle. Its size depends on the • What are the mechanisms by which amount of nerve growth factor (NGF) secreted by the target cells it innervates; the greater the amount of NGF the neuron has access to, the larger it becomes. It seems are sensed at the kinetochore by the likely that the genes a cell expresses set limits on the size it can be, while extracel components of the spindle assembly lular signal molecules and nutrients regulate the size within these limits. The chal checkpoint? lenge is to identify the relevant genes and signal molecules for each cell type. • How is cell growth coordinated with cell division to ensure that cell size remains constant?

1	lenge is to identify the relevant genes and signal molecules for each cell type. • How is cell growth coordinated with cell division to ensure that cell size remains constant? In multicellular animals, cell size, cell division, and cell survival are carefully controlled to ensure that the organism and its organs achieve and maintain an appropriate size. Mitogens stimulate the rate of cell division by removing intracellular molecular brakes that restrain cell-cycle progression in G1. Growth factors promote cell growth (an increase in cell mass) by stimulating the synthesis and inhibiting the degradation of macromolecules. To maintain a constant cell size, proliferating cells employ multiple mechanisms to ensure that cell growth is coordinated with cell division. number of cells Which statements are true? explain why or why not. 17–1 Since there are about 1013 cells in an adult human, and about 1010 cells die and are replaced each day, we become new people every three years.

1	17–1 Since there are about 1013 cells in an adult human, and about 1010 cells die and are replaced each day, we become new people every three years. 17–2 In order for proliferating cells to maintain a relatively constant size, the length of the cell cycle must match the time it takes for the cell to double in size. 17–3 While other proteins come and go during the cell cycle, the proteins of the origin recognition complex remain bound to the DNA throughout. 17–4 Chromosomes are positioned on the metaphase plate by equal and opposite forces that pull them toward the two poles of the spindle. 17–5 Meiosis segregates the paternal homologs into sperm and the maternal homologs into eggs. 17–6 If we could turn on telomerase activity in all our cells, we could prevent aging. Discuss the following problems.

1	17–6 If we could turn on telomerase activity in all our cells, we could prevent aging. Discuss the following problems. 17–7 Many cell-cycle genes from human cells function perfectly well when expressed in yeast cells. Why do you suppose that is considered remarkable? After all, many human genes encoding enzymes for metabolic reactions also function in yeast, and no one thinks that is remarkable. 17–8 Hoechst 33342 is a membrane-permeant dye that fluoresces when it binds to DNA. When a population of cells is incubated briefly with Hoechst dye and then sorted in a flow cytometer, which measures the fluorescence of each cell, the cells display various levels of fluorescence as shown in Figure Q17–1. A. Which cells in Figure Q17–1 are in the G1, S, G2, and M phases of the cell cycle? Explain the basis for your answer.

1	A. Which cells in Figure Q17–1 are in the G1, S, G2, and M phases of the cell cycle? Explain the basis for your answer. b. Sketch the sorting distributions you would expect for cells that were treated with inhibitors that block the cell cycle in the G1, S, or M phase. Explain your reasoning. Figure Q17–1 analysis of Hoechst 33342 fluorescence in a population of cells sorted in a flow cytometer (problem 17–8). 17–9 The yeast cohesin subunit Scc1, which is essential for sister-chromatid cohesion, can be artificially regulated for expression at any point in the cell cycle. If expression is turned on at the beginning of S phase, all the cells divide satisfactorily and survive. By contrast, if Scc1 expression is turned on only after S phase is completed, the cells fail to divide and they die, even though Scc1 accumulates in the nucleus and interacts efficiently with chromosomes. Why do you suppose that cohesin must be present during S phase for cells to divide normally?

1	17–10 High doses of caffeine interfere with the DNA damage response in mammalian cells. Why then do you suppose the Surgeon General has not yet issued an appropriate warning to heavy coffee and cola drinkers? A typical cup of coffee (150 mL) contains 100 mg of caffeine (196 g/ mole). How many cups of coffee would you have to drink to reach the dose (10 mM) required to interfere with the DNA damage response? (A typical adult contains about 40 liters of water.) 17–11 How many kinetochores are there in a human cell at mitosis? 17–12 A living cell from the lung epithelium of a newt is shown at different stages in M phase in Figure Q17–2. Order these light micrographs into the correct sequence and identify the stage in M phase that each represents.

1	Figure Q17–2 Light micrographs of a single cell at different stages of m phase (problem 17–12). (Courtesy of Conly L. Rieder.) 17–13 Down syndrome (trisomy 21) and Edwards syndrome (trisomy 18) are the most common autosomal trisomies seen in human infants. Does this fact mean that these chromosomes are the most difficult to segregate properly during meiosis? 17–14 The human genome consists of 23 pairs of chromosomes (22 pairs of autosomes and one pair of sex chromosomes). During meiosis, the maternal and paternal sets of homologs pair, and then are separated into gametes, so that each contains 23 chromosomes. If you assume that the chromosomes in the paired homologs are randomly assorted to daughter cells, how many potential combinations of paternal and maternal homologs can be generated during meiosis? (For the purposes of this calculation, assume that no recombination occurs.)

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1	The growth, development, and maintenance of multicellular organisms depend not only on the production of cells but also on mechanisms to destroy them. The maintenance of tissue size, for example, requires that cells die at the same rate as they are produced. During development, carefully orchestrated patterns of cell death help determine the size and shape of limbs and other tissues. Cells also die when they become damaged or infected, ensuring that they are removed before they threaten the health of the organism. In these and most other cases, cell death is not a random process but occurs by a programmed sequence of molecular events, in which the cell systematically destroys itself from within and is then eaten by other cells, leaving no trace. In most cases, this programmed cell death occurs by a process called apoptosis—from the Greek word meaning “falling off,” as leaves from a tree.

1	Cells dying by apoptosis undergo characteristic morphological changes. They shrink and condense, the cytoskeleton collapses, the nuclear envelope disassembles, and the nuclear chromatin condenses and breaks up into fragments (Figure 18–1A). The cell surface often bulges outward and, if the cell is large, it breaks up into membrane-enclosed fragments called apoptotic bodies. The surface of the cell or apoptotic bodies becomes chemically altered, so that a neighboring cell or a macrophage (a specialized phagocytic cell, discussed in Chapter 22) rapidly engulfs them, before they can spill their contents (Figure 18–1B). In this way, the cell dies neatly and is rapidly cleared away, without causing a damaging inflammatory response. Because the cells are eaten and digested so quickly, there are usually few dead cells to be seen, even when large numbers of cells have died by apoptosis. This is probably why biologists overlooked apoptosis for many years and still might underestimate its

1	are usually few dead cells to be seen, even when large numbers of cells have died by apoptosis. This is probably why biologists overlooked apoptosis for many years and still might underestimate its extent.

1	In contrast to apoptosis, animal cells that die in response to an acute insult, such as trauma or a lack of blood supply, usually do so by a process called cell necrosis. Necrotic cells swell and burst, spilling their contents over their neighbors and eliciting an inflammatory response (Figure 18–1C). In most cases, necrosis is likely to be caused by energy depletion, which leads to metabolic defects and loss of the ionic gradients that normally exist across the cell membrane. One form of necrosis, called necroptosis, is a form of programmed cell death that is triggered by a specific regulatory signal from other cells, although we are only just beginning to understand the underlying mechanisms. Some form of programmed cell death occurs in many organisms, but apoptosis is found primarily in animals. This chapter focuses on the major functions of apoptosis, its mechanism and regulation, and how excessive or insufficient apoptosis can contribute to human disease.

1	The amount of apoptotic cell death that occurs in developing and adult animal tissues is astonishing. In the developing vertebrate nervous system, for example, more than half of many types of nerve cells normally die soon after they are formed. It seems remarkably wasteful for so many cells to die, especially as the vast majority are perfectly healthy at the time they kill themselves. What purposes does this massive cell death serve?

1	In some cases, the answer is clear. Cell death helps sculpt hands and feet during embryonic development: they start out as spade-like structures, and the individual digits separate only as the cells between them die, as illustrated for a mouse paw in Figure 18–2. In other cases, cells die when the structure they form is no longer needed. When a tadpole changes into a frog at metamorphosis, the cells in the tail die, and the tail, which is not needed in the frog, disappears. Apoptosis also functions as a quality-control process in development, eliminating cells that are abnormal, misplaced, nonfunctional, or potentially dangerous to the animal. Striking examples occur in the vertebrate adaptive immune system, where apoptosis eliminates developing T and B lymphocytes that either fail to produce potentially useful antigen-specific receptors or produce self-reactive receptors that make the cells potentially dangerous (discussed in Chapter 24); it also eliminates most of the lymphocytes

1	potentially useful antigen-specific receptors or produce self-reactive receptors that make the cells potentially dangerous (discussed in Chapter 24); it also eliminates most of the lymphocytes activated by an infection, after they have helped destroy the responsible microbes.

1	In adult tissues that are neither growing nor shrinking, cell death and cell division must be tightly regulated to ensure that they are exactly in balance. If part of the liver is removed in an adult rat, for example, liver cell proliferation increases to make up the loss. Conversely, if a rat is treated with the drug phenobarbital— which stimulates liver cell division (and thereby liver enlargement)—and then the phenobarbital treatment is stopped, apoptosis in the liver greatly increases until the liver has returned to its original size, usually within a week or so. Thus, the liver is kept at a constant size through the regulation of both the cell death rate and the cell birth rate. The control mechanisms responsible for such regulation are largely unknown.

1	Animal cells can recognize damage in their various organelles and, if the damage is great enough, they can kill themselves by undergoing apoptosis. An important example is DNA damage, which can produce cancer-promoting mutations if not repaired. Cells have various ways of detecting DNA damage, and undergo apoptosis if they cannot repair it. Apoptosis is triggered by members of a family of specialized intracellular proteases, which cleave specific sequences in numerous proteins inside the cell, thereby bringing about the dramatic changes that lead to cell death and engulfment. These proteases have a cysteine at their active site and cleave their target proteins at specific aspartic acids; they are therefore called caspases (c for cysteine and asp for aspartic acid). Caspases are synthesized in the cell as inactive precursors and are activated only during apoptosis. There are two major classes of apoptotic caspases: initiator caspases and executioner caspases.

1	Figure 18–1 Two distinct forms of cell death. these electron micrographs show cells that have died by apoptosis (A and B) or by necrosis (C). the cells in (A) and (C) died in a culture dish, whereas the cell in (B) died in a developing tissue and has been engulfed by a phagocytic cell. Note that the cells in (A) and (B) have condensed but seem relatively intact, whereas the cell in (C) seems to have exploded. the large vacuoles visible in the cytoplasm of the cell in (A) are a variable feature of apoptosis. (Courtesy of Julia Burne.) Figure 18–2 Sculpting the digits in the developing mouse paw by apoptosis. the paw in this mouse fetus has been stained with a dye that specifically labels cells that have undergone apoptosis. the apoptotic cells appear as bright green dots between the developing digits.

1	the paw in this mouse fetus has been stained with a dye that specifically labels cells that have undergone apoptosis. the apoptotic cells appear as bright green dots between the developing digits. the interdigital cell death has eliminated the tissue between the developing digits, as seen one day later, when there are very few apoptotic cells. (From W. Wood et al., Development 127:5245–5252, 2000. With permission from the Company of Biologists.) initiator caspase (caspases 8,9) DIMERIZATION, ACTIVATION, AND CLEAVAGE executioner caspase (caspases 3,6,7) CLEAVAGE OF MULTIPLE SUBSTRATES

1	DIMERIZATION, ACTIVATION, AND CLEAVAGE executioner caspase (caspases 3,6,7) CLEAVAGE OF MULTIPLE SUBSTRATES Initiator caspases, as their name implies, begin the apoptotic process. They normally exist as inactive, soluble monomers in the cytosol. An apoptotic signal triggers the assembly of large protein platforms that bring multiple initiator caspases together into large complexes. Within these complexes, pairs of caspases associate to form dimers, resulting in protease activation (Figure 18–3). Each caspase in the dimer then cleaves its partner at a specific site in the protease domain, which stabilizes the active complex and is required for the proper function of the enzyme in the cell.

1	The major function of the initiator caspases is to activate the executioner caspases. These normally exist as inactive dimers. When they are cleaved by an initiator caspase at a site in the protease domain, the active site is rearranged from an inactive to an active conformation. One initiator caspase complex can activate many executioner caspases, resulting in an amplifying proteolytic cascade. Once activated, executioner caspases catalyze the widespread protein cleavage events that kill the cell.

1	Various experimental approaches have led to the identification of over a thousand proteins that are cleaved by caspases during apoptosis. Only a few of these proteins have been studied in any detail. These include the nuclear lamins, the cleavage of which causes the irreversible breakdown of the nuclear lamina (discussed in Chapter 12). Another target is a protein that normally holds a DNA-degrading endonuclease in an inactive form; its cleavage frees the endonuclease to cut up the DNA in the cell nucleus (Figure 18–4). Other target proteins include components of the cytoskeleton and cell–cell adhesion proteins that attach cells to their neighbors; the cleavage of these proteins helps the apoptotic cell to round up and detach from its neighbors, making it easier for a neighboring cell to engulf it, or, in the case of an epithelial cell, for the neighbors to extrude the apoptotic cell from the cell sheet. The caspase cascade is not only destructive and self-amplifying but also

1	cell to engulf it, or, in the case of an epithelial cell, for the neighbors to extrude the apoptotic cell from the cell sheet. The caspase cascade is not only destructive and self-amplifying but also irreversible, so that once a cell starts out along the path to destruction, it cannot turn back.

1	How is the initiator caspase first activated in response to an apoptotic signal? The two best-understood activation mechanisms in mammalian cells are called the extrinsic pathway and the intrinsic, or mitochondrial, pathway. Each uses its own initiator caspase and activation system, as we now discuss.

1	Figure 18–3 Caspase activation during apoptosis. An initiator caspase contains a protease domain in its carboxy-terminal region and a small protein interaction domain near its amino terminus. It is initially made in an inactive, monomeric form, sometimes called procaspase. Apoptotic signals trigger the assembly of adaptor proteins carrying multiple binding sites for the caspase amino-terminal domain. Upon binding to the adaptor proteins, the initiator caspases dimerize and are thereby activated, leading to cleavage of a specific site in their protease domains. Each protease domain is then rearranged into a large and small subunit. In some cases (not shown), the adaptor-binding domain of the initiator caspase is also cleaved (see Figure 18–5). Executioner caspases are initially formed as inactive dimers. Upon cleavage at a site in the protease domain by an initiator caspase, the executioner caspase dimer undergoes an activating conformational change. the executioner caspases then

1	as inactive dimers. Upon cleavage at a site in the protease domain by an initiator caspase, the executioner caspase dimer undergoes an activating conformational change. the executioner caspases then cleave a variety of key proteins, leading to the controlled death of the cell.

1	Figure 18–4 DNA fragmentation during apoptosis. (A) In healthy cells, the endonuclease CAD associates with its inhibitor, iCAD. Activation of executioner caspases in the cell leads to cleavage of iCAD, which unleashes the nuclease. Activated CAD cuts the chromosomal DNA between nucleosomes, resulting in the production of DNA fragments that form a ladder pattern (see B) upon gel electrophoresis. (B) Mouse thymus lymphocytes were treated with an antibody against the cell-surface death receptor Fas (discussed in the text), inducing the cells to undergo apoptosis. DNA was extracted at the times indicated above the figure, and the fragments were separated by size by electrophoresis in an agarose gel and stained with ethidium bromide. Because the cleavages occur in the linker regions between nucleosomes, the fragments separate into a characteristic ladder pattern on these gels. Note that in gel electrophoresis, smaller molecules are more widely separated in the lower part of the gel, so

1	nucleosomes, the fragments separate into a characteristic ladder pattern on these gels. Note that in gel electrophoresis, smaller molecules are more widely separated in the lower part of the gel, so that removal of a single nucleosome has a greater apparent effect on their gel mobility. (C) Apoptotic nuclei can be detected using a technique that adds a fluorescent label to DNA ends. In the image shown here, this technique was used in a tissue section of a developing chick leg bud; this cross section through the skin and underlying tissue is from a region between two developing digits, as indicated in the underlying drawing. the procedure is called the tUNEL (tdt-mediated dUtpnick end labeling) technique because the enzyme terminal deoxynucleotidyl transferase (tdt) adds chains of labeled deoxynucleotide (dUtp) to the 3ʹ-Oh ends of DNA fragments. the presence of large numbers of DNA fragments therefore results in bright fluorescent dots in apoptotic cells. (B, from D. McIlroy et al.,

1	deoxynucleotide (dUtp) to the 3ʹ-Oh ends of DNA fragments. the presence of large numbers of DNA fragments therefore results in bright fluorescent dots in apoptotic cells. (B, from D. McIlroy et al., Genes Dev. 14:549–558, 2000. With permission from Cold Spring harbor Laboratory press; C, from V. Zuzarte-Luís and J.M. hurlé, Int. J. Dev. Biol. 46:871–876, 2002. With permission from UBC press.)

1	Cell-Surface Death receptors Activate the Extrinsic pathway of Apoptosis Extracellular signal proteins binding to cell-surface death receptors trigger the extrinsic pathway of apoptosis. Death receptors are transmembrane proteins containing an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular death domain, which is required for the receptors to activate the apoptotic program. The receptors are homotrimers and belong to the tumor necrosis factor (TNF) receptor family, which includes a receptor for TNF itself and the Fas death receptor. The ligands that activate the death receptors are also homotrimers; they are structurally related to one another and belong to the TNF family of signal proteins.

1	A well-understood example of how death receptors trigger the extrinsic pathway of apoptosis is the activation of Fas on the surface of a target cell by Fas ligand on the surface of a killer (cytotoxic) lymphocyte. When activated by the binding of Fas ligand, the death domains on the cytosolic tails of the Fas death receptors bind intracellular adaptor proteins, which in turn bind initiator caspases (primarily caspase-8), forming a death-inducing signaling complex (DISC). Once dimerized and activated in the DISC, the initiator caspases cleave their partners and then activate downstream executioner caspases to induce apoptosis (Figure 18–5). In some cells, the extrinsic pathway recruits the intrinsic apoptotic pathway to amplify the caspase cascade and kill the cell.

1	Many cells produce inhibitory proteins that act to restrain the extrinsic pathway. For example, some cells produce the protein FLIP, which resembles an initiator caspase but has no protease activity because it lacks the key cysteine in its active site. FLIP dimerizes with caspase-8 in the DISC; although caspase-8 appears to be active in these heterodimers, it is not cleaved at the site required for its stable activation, and the apoptotic signal is blocked. Such inhibitory mechanisms help prevent the inappropriate activation of the extrinsic pathway of apoptosis. the Intrinsic pathway of Apoptosis Depends on Mitochondria

1	the Intrinsic pathway of Apoptosis Depends on Mitochondria Cells can also activate their apoptosis program from inside the cell, often in response to stresses, such as DNA damage, or in response to developmental signals. In vertebrate cells, these responses are governed by the intrinsic, or mitochondrial, pathway of apoptosis, which depends on the release into the cytosol of mitochondrial proteins that normally reside in the intermembrane space of these organelles (see Figure 12–19). Some of the released proteins activate a caspase proteolytic cascade in the cytoplasm, leading to apoptosis.

1	A key protein in the intrinsic pathway is cytochrome c, a water-soluble component of the mitochondrial electron-transport chain. When released into the cytosol (Figure 18–6), it takes on a new function: it binds to an adaptor protein called Apaf1 (apoptotic protease activating factor-1), causing the Apaf1 to oligomerize into a wheel-like heptamer called an apoptosome. The Apaf1 proteins in the apoptosome then recruit initiator caspase-9 proteins, which are thought to be activated by proximity in the apoptosome, just as caspase-8 is activated in the DISC. The activated caspase-9 molecules then activate downstream executioner caspases to induce apoptosis (Figure 18–7). Bcl2 proteins regulate the Intrinsic pathway of Apoptosis

1	Bcl2 proteins regulate the Intrinsic pathway of Apoptosis The intrinsic pathway of apoptosis is tightly regulated to ensure that cells kill themselves only when it is appropriate. A major class of intracellular regulators of the intrinsic pathway is the Bcl2 family of proteins, which, like the caspase family, has been conserved in evolution from worms to humans; a human Bcl2 protein, for example, can suppress apoptosis when expressed in the worm Caenorhabditis elegans.

1	Figure 18–5 The extrinsic pathway of apoptosis activated through Fas death receptors. trimeric Fas ligands on the surface of a killer lymphocyte interact with trimeric Fas receptors on the surface of the target cell, leading to clustering of several ligand-bound receptor trimers (only one trimer is shown here for clarity). receptor clustering activates death domains on the receptor tails, which interact with similar domains on the adaptor protein FADD (FADD stands for Fas-associated death domain). Each FADD protein then recruits an initiator caspase (caspase-8) via a death effector domain on both FADD and the caspase, forming a death-inducing signaling complex (DISC). Within the DISC, two adjacent initiator caspases interact and cleave one another to form an activated protease dimer, which then cleaves itself in the region linking the protease to the death effector domain. this stabilizes and releases the active caspase dimer into the cytosol, where it activates executioner caspases

1	then cleaves itself in the region linking the protease to the death effector domain. this stabilizes and releases the active caspase dimer into the cytosol, where it activates executioner caspases by cleaving them.

1	Mammalian Bcl2 family proteins regulate the intrinsic pathway of apoptosis mainly by controlling the release of cytochrome c and other intermembrane mitochondrial proteins into the cytosol. Some Bcl2 family proteins are pro-apoptotic and promote apoptosis by enhancing the release, whereas others are antiapoptotic and inhibit apoptosis by blocking the release. The pro-apoptotic and anti-apoptotic proteins can bind to each other in various combinations to form heterodimers in which the two proteins inhibit each other’s function. The balance between the activities of these two functional classes of Bcl2 family proteins largely determines whether a mammalian cell lives or dies by the intrinsic pathway of apoptosis. As illustrated in Figure 18–8, the anti-apoptotic Bcl2 family proteins, including Bcl2 itself (the founding member of the Bcl2 family) and BclXL, share four distinctive Bcl2 homology (BH) domains (BH1–4). The pro-apoptotic Bcl2 family proteins

1	Figure 18–6 Release of cytochrome c from mitochondria in the intrinsic pathway of apoptosis. Fluorescence micrographs of human cancer cells in culture. (A) the control cells were transfected with a gene encoding a fusion protein consisting of cytochrome c linked to green fluorescent protein (cytochromec-GFp); they were also treated with a red dye that accumulates in mitochondria. the overlapping distribution of the green and red indicates that the cytochromec-GFp is located in mitochondria.

1	(B) Cells expressing cytochrome-c-GFp were irradiated with ultraviolet (UV) light to induce the intrinsic pathway of apoptosis and were photographed 5 hours later. the six cells in the bottom half of this micrograph have released their cytochrome c from mitochondria into the cytosol, whereas the cells in the upper half of the micrograph have not yet done so (Movie 18.1). (From J.C. Goldstein et al., Nat. Cell Biol. 2:156–162, 2000. With permission from Macmillan publishers Ltd.) activation of caspase-9, which cleaves and thereby activates executioner caspases

1	Figure 18–7 The intrinsic pathway of apoptosis. Intracellular apoptotic stimuli cause mitochondria to release cytochrome c, which interacts with Apaf1. the binding of cytochrome c causes Apaf1 to unfold partly, exposing a domain that interacts with the same domain in other activated Apaf1 molecules. Seven activated Apaf1 proteins form a large ring complex called the apoptosome. Each Apaf1 protein contains a caspase recruitment domain (CArD), and these are clustered above the central hub of the apoptosome. the CArDs bind similar domains in multiple caspase-9 molecules, which are thereby recruited into the apoptosome and activated. the mechanism of caspase-9 activation is not clear: it probably results from dimerization and cleavage of adjacent caspase-9 proteins, but it might also depend on interactions between caspase-9 and Apaf1. Once activated, caspase-9 cleaves and thereby activates downstream executioner caspases. Note that the CArD is related in structure and function to the

1	on interactions between caspase-9 and Apaf1. Once activated, caspase-9 cleaves and thereby activates downstream executioner caspases. Note that the CArD is related in structure and function to the death effector domain of caspase-8 (see Figure 18–5). Some scientists use the term “apoptosome” to refer to the complex containing caspase-9.

1	(e.g., Bcl2, BclXL) (e.g., Bax, Bak) (e.g., Bad, Bim, Bid, Puma, Noxa) consist of two subfamilies—the effector Bcl2 family proteins and the BH3-only proteins. The main effector proteins are Bax and Bak, which are structurally similar to Bcl2 but lack the BH4 domain. The BH3-only proteins share sequence homology with Bcl2 in only the BH3 domain.

1	When an apoptotic stimulus triggers the intrinsic pathway, the pro-apoptotic effector Bcl2 family proteins become activated and aggregate to form oligomers in the mitochondrial outer membrane, inducing the release of cytochrome c and other intermembrane proteins by an unknown mechanism (Figure 18–9). In mammalian cells, Bax and Bak are the main effector Bcl2 family proteins, and at least one of them is required for the intrinsic pathway of apoptosis to operate: mutant mouse cells that lack both proteins are resistant to all pro-apoptotic signals that normally activate this pathway. Whereas Bak is bound to the mitochondrial outer membrane even in the absence of an apoptotic signal, Bax is mainly located in the cytosol and translocates to the mitochondria only after an apoptotic signal activates it. As we discuss below, the activation of Bax and Bak usually depends on activated pro-apoptotic BH3-only proteins.

1	The anti-apoptotic Bcl2 family proteins such as Bcl2 itself and BclXL are also located on the cytosolic surface of the outer mitochondrial membrane, where they help prevent inappropriate release of intermembrane proteins. The antiapoptotic Bcl2 family proteins inhibit apoptosis mainly by binding to and inhibiting pro-apoptotic Bcl2 family proteins—either on the mitochondrial membrane or in the cytosol. On the outer mitochondrial membrane, for example, they bind to Bak and prevent it from oligomerizing, thereby inhibiting the release of cytochrome c and other intermembrane proteins. There are at least five mammalian anti-apoptotic Bcl2 family proteins, and every mammalian cell requires at least one to survive. Moreover, a number of these proteins must be inhibited for the intrinsic pathway to induce apoptosis; the BH3-only proteins mediate the inhibition.

1	The BH3-only proteins are the largest subclass of Bcl2 family proteins. The cell either produces or activates them in response to an apoptotic stimulus, and they are thought to promote apoptosis mainly by inhibiting anti-apoptotic Bcl2 family Figure 18–8 The three classes of Bcl2 family proteins. Note that the Bh3 domain is the only Bh domain shared by all Bcl2 family members; it mediates the direct interactions between pro-apoptotic and anti-apoptotic family members. Figure 18–9 The role of pro-apoptotic effector Bcl2 family proteins (mainly Bax and Bak) in the release of mitochondrial intermembrane proteins in the intrinsic pathway of apoptosis. When activated by an apoptotic stimulus, the effector Bcl2 family proteins aggregate on the outer mitochondrial membrane and release cytochrome c and other proteins from the intermembrane space into the cytosol by an unknown mechanism.

1	proteins. Their BH3 domain binds to a long hydrophobic groove on anti-apoptotic Bcl2 family proteins, neutralizing their activity. This binding and inhibition enables the aggregation of Bax and Bak on the surface of mitochondria, which triggers the release of the intermembrane mitochondrial proteins that induce apoptosis (Figure 18–10). Some BH3-only proteins may bind directly to Bax and Bak to help stimulate their aggregation.

1	BH3-only proteins provide the crucial link between apoptotic stimuli and the intrinsic pathway of apoptosis, with different stimuli activating different BH3only proteins. Some extracellular survival signals, for example, block apoptosis by inhibiting the synthesis or activity of certain BH3-only proteins (see Figure 18–12B). Similarly, in response to DNA damage that cannot be repaired, the tumor suppressor protein p53 accumulates (discussed in Chapters 17 and 20) and activates the transcription of genes that encode the BH3-only proteins Puma and Noxa. These BH3-only proteins then trigger the intrinsic pathway, thereby eliminating a potentially dangerous cell that could otherwise become cancerous.

1	As mentioned earlier, in some cells the extrinsic apoptotic pathway recruits the intrinsic pathway to amplify the caspase cascade to kill the cell. The BH3-only protein Bid is the link between the two pathways. Bid is normally inactive. However, when death receptors activate the extrinsic pathway in some cells, the initiator caspase, caspase-8, cleaves Bid, producing an active form of Bid that trans-locates to the outer mitochondrial membrane and inhibits anti-apoptotic Bcl2 family proteins, thereby amplifying the death signal.

1	Figure 18–10 How pro-apoptotic BH3only and anti-apoptotic Bcl2 family proteins regulate the intrinsic pathway of apoptosis. (A) In the absence of an apoptotic stimulus, anti-apoptotic Bcl2 family proteins bind to and inhibit the effector Bcl2 family proteins on the mitochondrial outer membrane (and in the cytosol—not shown). (B) In the presence of an apoptotic stimulus, Bh3-only proteins are activated and bind to the anti-apoptotic Bcl2 family proteins so that they can no longer inhibit the effector Bcl2 family proteins; the latter then become activated, aggregate in the outer mitochondrial membrane, and promote the release of intermembrane mitochondrial proteins into the cytosol. Some activated Bh3only proteins may stimulate mitochondrial protein release more directly by binding to and activating the effector Bcl2 family proteins. Although not shown, the antiapoptotic Bcl2 family proteins are bound to the mitochondrial surface.

1	Because activation of a caspase cascade leads to certain death, the cell employs multiple robust mechanisms to ensure that these proteases are activated only when appropriate. One line of defense is provided by a family of proteins called inhibitors of apoptosis (IAPs). These proteins were first identified in certain insect viruses (baculoviruses), which encode IAP proteins to prevent a host cell that is infected by the virus from killing itself by apoptosis. It is now known that most animal cells also make IAP proteins. All IAPs have one or more BIR (baculovirus IAP repeat) domains, which enable them to bind to and inhibit activated caspases. Some IAPs also polyubiquitylate caspases, marking the caspases for destruction by proteasomes. In this way, the IAPs set an inhibitory threshold that caspases must overcome to trigger apoptosis.

1	In Drosophila at least, the inhibitory barrier provided by IAPs can be neutralized by anti-IAP proteins, which are produced in response to various apoptotic stimuli. There are numerous anti-IAPs in flies, including Reaper, Grim, and Hid, and their only structural similarity is their short, N-terminal, IAP-binding motif, which binds to the BIR domain of IAPs, preventing the domain from binding to a caspase. Deletion of the three genes encoding Reaper, Grim, and Hid blocks apoptosis in flies. Conversely, inactivation of one of the two genes that encode IAPs in Drosophila causes all of the cells in the developing fly embryo to undergo apoptosis. Clearly, the balance between IAPs and anti-IAPs is tightly regulated and is crucial for controlling apoptosis in the fly.

1	The role of mammalian IAP and anti-IAP proteins in apoptosis is less clear. Anti-IAPs are released from the mitochondrial intermembrane space when the intrinsic pathway of apoptosis is activated, blocking IAPs in the cytosol and thereby promoting apoptosis. However, mice appear to develop normally if they are missing either the major mammalian IAP (called XIAP) or the two known mammalian anti-IAPs (called Smac/Diablo and Omi). Worms do not even contain a caspaseinhibiting IAP protein. Apparently, the tight control of caspase activity is achieved by different mechanisms in different animals.

1	Intercellular signals regulate most activities of animal cells, including apoptosis. These extracellular signals are part of the normal “social” controls that ensure that individual cells behave for the good of the organism as a whole—in this case, by surviving when they are needed and killing themselves when they are not. Some extracellular signal molecules stimulate apoptosis, whereas others inhibit it. We have discussed signal proteins such as Fas ligand that activate death receptors and thereby trigger the extrinsic pathway of apoptosis. Other extracellular signal molecules that stimulate apoptosis are especially important during vertebrate development: a surge of thyroid hormone in the bloodstream, for example, signals cells in the tadpole tail to undergo apoptosis at metamorphosis. In mice, locally produced signal proteins stimulate cells between developing fingers and toes to kill themselves (see Figure 18–2). Here, however, we focus on extracellular signal molecules that

1	In mice, locally produced signal proteins stimulate cells between developing fingers and toes to kill themselves (see Figure 18–2). Here, however, we focus on extracellular signal molecules that inhibit apoptosis, which are collectively called survival factors.

1	Most animal cells require continuous signaling from other cells to avoid apoptosis. This surprising arrangement apparently helps ensure that cells survive only when and where they are needed. Nerve cells, for example, are produced in excess in the developing nervous system and then compete for limited amounts of survival factors that are secreted by the target cells that they normally connect to (see Figure 21–81). Nerve cells that receive enough survival signals live, while the others die. In this way, the number of surviving neurons is automatically adjusted so that it is appropriate for the number of target cells they connect with (Figure 18–11). A similar competition for limited amounts of survival factors produced by neighboring cells is thought to control cell numbers in other tissues, both during development and in adulthood.

1	Survival factors usually bind to cell-surface receptors, which activate intracellular signaling pathways that suppress the apoptotic program, often by regulating members of the Bcl2 family of proteins. Some survival factors, for example, stimulate the synthesis of anti-apoptotic Bcl2 family proteins such as Bcl2 itself or BclXL (Figure 18–12A). Others act by inhibiting the function of pro-apoptotic BH3-only proteins such as Bad (Figure M18–12B). In Drosophila, some survival factors act by phosphorylating and inactivating anti-IAP proteins such as Hid, thereby enabling IAP proteins to suppress apoptosis (Figure 18–12C). Some developing neurons, like those illustrated in Figure 18–11, use an ingenious alternative approach: survival-factor receptors stimulate apoptosis—by an unknown mechanism—when they are not occupied, and then stop promoting death when survival factor binds. The end result in all these cases is the same: cell survival depends on survival factor binding.

1	phagocytes remove the Apoptotic Cell Apoptotic cell death is a remarkably tidy process: the apoptotic cell and its fragments do not break open and release their contents, but instead remain intact as they are efficiently eaten—or phagocytosed—by neighboring cells, leaving no trace and therefore triggering no inflammatory response (see Figure 18–1B and Movie 13.5). This engulfment process depends on chemical changes on the surface of the apoptotic cell, which displays signals that recruit phagocytic cells. An especially important change occurs in the distribution of the negatively charged phospholipid phosphatidylserine on the cell surface. This phospholipid is normally located exclusively in the inner leaflet of the lipid bilayer of the plasma membrane (see Figure 10–15), but it flips to the outer leaflet in apoptotic cells. The

1	Figure 18–11 The role of survival factors and cell death in adjusting the number of developing nerve cells to the amount of target tissue. More nerve cells are produced than can be supported by the limited amount of survival factors released by the target cells. therefore, some nerve cells receive an insufficient amount of survival factors to avoid apoptosis. this strategy of overproduction followed by culling helps ensure that all target cells are contacted by nerve cells and that the extra nerve cells are automatically eliminated.

1	Figure 18–12 Three ways that extracellular survival factors can inhibit apoptosis. (A) Some survival factors suppress apoptosis by stimulating the transcription of genes that encode antiapoptotic Bcl2 family proteins such as Bcl2 itself or BclXL. (B) Many others activate the serine/threonine protein kinase Akt, which, among many other targets, phosphorylates and inactivates the pro-apoptotic Bh3-only protein Bad (see Figure 15–53). When not phosphorylated, Bad promotes apoptosis by binding to and inhibiting Bcl2; once phosphorylated, Bad dissociates, freeing Bcl2 to suppress apoptosis. Akt also suppresses apoptosis by phosphorylating and inactivating transcription regulatory proteins that stimulate the transcription of genes encoding proteins that promote apoptosis (not shown). (C) In Drosophila, some survival factors inhibit apoptosis by stimulating the phosphorylation of the anti-IAp protein hid. When not phosphorylated, hid promotes cell death by inhibiting IAps. Once

1	(C) In Drosophila, some survival factors inhibit apoptosis by stimulating the phosphorylation of the anti-IAp protein hid. When not phosphorylated, hid promotes cell death by inhibiting IAps. Once phosphorylated, hid no longer inhibits IAps, which become active and block apoptosis. MAp kinase, mitogenactivated protein kinase.

1	underlying mechanism is poorly understood, but the external exposure of phosphatidylserine is likely to depend on caspase cleavage of some protein involved in phospholipid distribution in the membrane. A variety of soluble “bridging” proteins interact with the exposed phosphatidylserine on the apoptotic cell. These bridging proteins also interact with specific receptors on the surface of a neighboring cell or macrophage, triggering cytoskeletal and other changes that initiate the engulfment process. Macrophages do not phagocytose healthy cells in the animal—despite the fact that healthy cells normally expose some phosphatidylserine on their surfaces. Healthy cells express signal proteins on their surface that interact with inhibitory receptors on macrophages that block phagocytosis. Thus, in addition to expressing cell-surface signals such as phosphatidylserine that stimulate phagocytosis, apoptotic cells must lose or inactivate these “don’t eat me” signals that block phagocytosis.

1	Either Excessive or Insufficient Apoptosis Can Contribute to Disease There are many human disorders in which excessive numbers of cells undergo apoptosis and thereby contribute to tissue damage. Among the most dramatic examples are heart attacks and strokes. In these acute conditions, many cells die by necrosis as a result of ischemia (inadequate blood supply), but some of the less affected cells die by apoptosis. It is hoped that, in the future, drugs that block apoptosis—such as specific caspase inhibitors—will prove useful in saving such cells. There are other conditions where too few cells die by apoptosis. Mutations in mice and humans, for example, that inactivate the genes that encode the Fas death receptor or the Fas ligand prevent the normal death of some lymphocytes, causing these cells to accumulate in excessive numbers in the spleen and lymph glands. In many cases, this leads to autoimmune disease, in which the lymphocytes react against the individual’s own tissues.

1	Decreased apoptosis also makes an important contribution to many tumors, as cancer cells often regulate their apoptotic program abnormally. The Bcl2 gene, for example, was first identified in a common form of lymphocyte cancer in humans, where a chromosome translocation causes excessive production of the Bcl2 protein; indeed, Bcl2 gets its name from this B cell lymphoma. The high level of Bcl2 protein in the lymphocytes that carry the translocation promotes the development of cancer by inhibiting apoptosis, thereby prolonging lymphocyte survival and increasing their number; it also decreases the cells’ sensitivity to anticancer drugs, which commonly work by causing cancer cells to undergo apoptosis.

1	Similarly, the gene encoding the tumor suppressor protein p53 is mutated in about 50% of human cancers so that it no longer promotes apoptosis or cell-cycle arrest in response to DNA damage. The lack of p53 function therefore enables the cancer cells to survive and proliferate even when their DNA is damaged; in this way, the cells accumulate more mutations, some of which make the cancer more malignant (discussed in Chapter 20). As many anticancer drugs induce apoptosis (and cell-cycle arrest) by a p53-dependent mechanism (discussed in Chapters 17 and 20), the loss of p53 function also makes cancer cells less sensitive to these drugs.

1	If decreased apoptosis contributes to many cancers, then we might be able to treat those cancers with drugs that stimulate apoptosis. This line of thinking has recently led to the development of small chemicals that interfere with the function of anti-apoptotic Bcl2 family proteins such as Bcl2 and BclXL. These chemicals bind with high affinity to the hydrophobic groove on anti-apoptotic Bcl2 family proteins, blocking their function in essentially the same way that BH3-only proteins do (Figure 18–13). The intrinsic pathway of apoptosis is thereby stimulated, which in certain tumors increases the amount of cell death.

1	Most human cancers arise in epithelial tissues such as those in the lung, intestinal tract, breast, and prostate. Such cancer cells display many abnormalities in their behavior, including a decreased ability to adhere to the extracellular matrix and to one another at specialized cell–cell junctions. In the next chapter, we discuss the remarkable structures and functions of the extracellular matrix and cell junctions.

1	Animal cells can activate an intracellular death program and kill themselves in a controlled way when they are irreversibly damaged, no longer needed, or are a threat to the organism. In most cases, these deaths occur by apoptosis: the cells shrink, condense, and frequently fragment, and neighboring cells or macrophages rapidly phagocytose the cells or fragments before there is any leakage of cytoplasmic contents. Apoptosis is mediated by proteolytic enzymes called caspases, which cleave specific intracellular proteins to help kill the cell. Caspases are present in all nucleated animal cells as inactive precursors. Initiator caspases are activated when brought into proximity in activation complexes: once activated, they cleave and thereby activate downstream executioner caspases, which then cleave various target proteins in the cell, producing an amplifying, irreversible proteolytic cascade.

1	Cells use at least two distinct pathways to activate initiator caspases and trigger a caspase cascade leading to apoptosis: the extrinsic pathway is activated by extracellular ligands binding to cell-surface death receptors; the intrinsic pathway is activated by intracellular signals generated when cells are stressed. Each pathway uses its own initiator caspases, which are activated in distinct activation complexes: in the extrinsic pathway, the death receptors recruit caspase-8 via adaptor proteins to form the DISC; in the intrinsic pathway, cytochrome c released from the intermembrane space of mitochondria activates Apaf1, which assembles into an apoptosome and recruits and activates caspase-9.

1	Intracellular Bcl2 family proteins and IAP proteins tightly regulate the apoptotic program to ensure that cells kill themselves only when it benefits the animal. Both anti-apoptotic and pro-apoptotic Bcl2 family proteins regulate the intrinsic pathway by controlling the release of mitochondrial intermembrane proteins, while IAP proteins inhibit activated caspases and promote their degradation.

1	Figure 18–13 How the chemical ABT737 inhibits anti-apoptotic Bcl2 family proteins. As shown in Figure 18–10B, an apoptotic signal results in activation of Bh3-only proteins, which interact with a long hydrophobic groove in anti-apoptotic Bcl2 family proteins, thereby preventing them from blocking apoptosis. Using the crystal structure of the groove, the drug shown in (A), called ABt-737, was designed and synthesized to bind tightly in the groove, as shown for the anti-apoptotic Bcl2 family protein, BclXL, in (B). By inhibiting the activity of these proteins, the drug promotes apoptosis in any cell that depends on them for survival. (pDB code: 2YXJ.) how many forms of programmed cell death exist? What are the underlying mechanisms and benefits of each? thousands of caspase substrates have been identified. Which ones are the critical proteins that must be cleaved to trigger the major cell remodeling events underlying apoptosis?

1	thousands of caspase substrates have been identified. Which ones are the critical proteins that must be cleaved to trigger the major cell remodeling events underlying apoptosis? how did the intrinsic pathway of apoptosis evolve, and what is the advantage of having mitochondria play such a central role in regulating apoptosis? how are “don’t eat me” signals eliminated or inactivated during apoptosis to allow the cells to be phagocytosed? Which statements are true? Explain why or why not. 18–1 In normal adult tissues, cell death usually balances cell division. 18–2 Mammalian cells that do not have cytochrome c should be resistant to apoptosis induced by DNA damage. Discuss the following problems.

1	18–2 Mammalian cells that do not have cytochrome c should be resistant to apoptosis induced by DNA damage. Discuss the following problems. 18–3 One important role of Fas and Fas ligand is to mediate the elimination of tumor cells by killer lymphocytes. In a study of 35 primary lung and colon tumors, half the tumors were found to have amplified and overexpressed a gene for a secreted protein that binds to Fas ligand. How do you suppose that overexpression of this protein might contribute to the survival of these tumor cells? Explain your reasoning.

1	18–4 Development of the nematode Caenorhabditis elegans generates exactly 959 somatic cells; it also produces an additional 131 cells that are later eliminated by apoptosis. Classical genetic experiments in C. elegans isolated mutants that led to the identification of the first genes involved in apoptosis. Of the many mutations affecting apoptosis in the nematode, none have ever been found in the gene for cytochrome c. Why do you suppose that such a central effector molecule in apoptosis was not found in the many genetic screens for “death” genes that have been carried out in C. elegans? 18–5 Imagine that you could microinject cytochrome c into the cytosol of wild-type mammalian cells and of cells that were doubly defective for Bax and Bak. Would you expect one, both, or neither type of cell to undergo apoptosis? Explain your reasoning.

1	18–6 In contrast to their similar brain abnormalities, newborn mice deficient in Apaf1 or caspase-9 have distinctive abnormalities in their paws. Apaf1-deficient mice fail to eliminate the webs between their developing digits, whereas caspase-9-deficient mice have normally formed digits (Figure Q18–1). If Apaf1 and caspase-9 function in the same apoptotic pathway, how is it possible for these deficient mice to differ in web-cell apoptosis?

1	Figure Q18–1 Appearance of paws in Apaf1–/– and Casp9–/– newborn mice relative to normal newborn mice (problem +/+ –/– 18–6). (From h. Yoshida et al., Cell 94:739– Casp9 750, 1998. With permission from Elsevier.) 18–7 When human cancer cells are exposed to ultraviolet (UV) light at 90 mJ/cm2, most of the cells undergo apoptosis within 24 hours. Release of cytochrome c from mitochondria can be detected as early as 6 hours after exposure of a population of cells to UV light, and it continues to increase for more than 10 hours thereafter. Does this mean that individual cells slowly release their cytochrome c over this time period? Or, alternatively, do individual cells release their cytochrome c rapidly but with different cells being triggered over the longer time period?

1	To answer this fundamental question, you have fused the gene for green fluorescent protein (GFP) to the gene for cytochrome c, so that you can observe the behavior of individual cells by confocal fluorescence microscopy. In cells that are expressing the cytochrome c–GFP fusion, fluorescence shows the punctate pattern typical of mitochondrial proteins. You then irradiate these cells with UV light and observe individual cells for changes in the punctate pattern. Two such cells (outlined in white) are shown in Figure Q18–2A and B. Release of cytochrome c–GFP is detected as a change from a punctate to a diffuse pattern of fluorescence. Times after UV exposure are indicated as hours:minutes below the individual panels. Which model for cytochrome c release do these observations support? Explain your reasoning. 10:09 10:15 17:10 17:18

1	10:09 10:15 17:10 17:18 Figure Q18–2 time-lapse video fluorescence microscopic analysis of cytochrome c–GFp release from mitochondria of individual cells (problem 18–7). (A) Cells observed for 6 minutes, 10 hours after UV irradiation. (B) Cells observed for 8 minutes, 17 hours after UV irradiation. One cell in (A) and one in (B), each outlined in white, have released their cytochrome c–GFp during the time frame of the observation, which is shown as hours:minutes below each panel. (From

1	J.C. Goldstein et al., Nat. Cell Biol. 2:156–162, 2000. With permission from Macmillan publishers Ltd.) 18–8 Fas ligand is a trimeric, extracellular protein that binds to its receptor, Fas, which is composed of three identical transmembrane subunits (Figure Q18–3). The binding of Fas ligand alters the conformation of Fas so that it binds an adaptor protein, which then recruits and activates caspase-8, triggering a caspase cascade that leads to cell death. In humans, the autoimmune lymphoproliferative syndrome (ALPS) is associated with dominant mutations in Fas that include point mutations and C-terminal 1034 Chapter 18: Cell Death truncations. In individuals that are heterozygous for such mutations, lymphocytes do not die at their normal rate and accumulate in abnormally large numbers, causing a variety of clinical problems. In contrast to these patients, individuals that are heterozygous for mutations that eliminate Fas expression entirely have no clinical symptoms.

1	A. Assuming that the normal and dominant forms of Fas are expressed to the same level and bind Fas ligand equally, what fraction of Fas–Fas ligand complexes on a lymphocyte from a heterozygous ALPS patient would be expected to be composed entirely of normal Fas subunits? B. In an individual heterozygous for a mutation that eliminates Fas expression, what fraction of Fas–Fas ligand complexes would be expected to be composed entirely of normal Fas subunits? C. Why are the Fas mutations that are associated with ALPS dominant, while those that eliminate expression of Fas are recessive? Crawford ED & Wells JA (2011) Caspase substrates and cellular remodeling. Annu. Rev. Biochem. 80, 1055–1087. Czabotar pE, Lessene G, Strasser A et al. (2014) Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat. Rev. Mol. Cell Biol. 15, 49–63.

1	Czabotar pE, Lessene G, Strasser A et al. (2014) Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat. Rev. Mol. Cell Biol. 15, 49–63. Danial NN & korsmeyer SJ (2004) Cell death: critical control points. Cell 116, 205–219. Elliott Mr & ravichandran kS (2010) Clearance of apoptotic cells: implications in health and disease. J. Cell Biol. 189, 1059–1070. Ellis rE, Yuan JY & horvitz rA (1991) Mechanisms and functions of cell death. Annu. Rev. Cell Biol. 7, 663–698. Fadok VA & henson pM (2003) Apoptosis: giving phosphatidylserine recognition an assist—with a twist. Curr. Biol. 13, r655–r657. Green Dr (2011) Means to an End: Apoptosis and Other Cell Death Mechanisms. Cold Spring harbor, New York: Cold Spring harbor Laboratory press. Jacobson MD, Weil M & raff MC (1997) programmed cell death in animal development. Cell 88, 347–354. Jiang X & Wang X (2004) Cytochrome C-mediated apoptosis. Annu. Rev. Biochem. 73, 87–106.

1	Jacobson MD, Weil M & raff MC (1997) programmed cell death in animal development. Cell 88, 347–354. Jiang X & Wang X (2004) Cytochrome C-mediated apoptosis. Annu. Rev. Biochem. 73, 87–106. kerr JF, Wyllie Ah & Currie Ar (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Brit. J. Cancer 26, 239–257. kumar S (2007) Caspase function in programmed cell death. Cell Death Differ. 14, 32–43. Figure Q18–3 The binding of trimeric Fas ligand to Fas (problem 18–8). Lavrik I, Golks A & krammer ph (2005) Death receptor signaling. J. Cell Sci. 118, 265–267. Lessene G, Czabotar pE & Colman pM (2008) BCL-2 family antagonists for cancer therapy. Nat. Rev. Drug Discov. 7, 989–1000. Mace pD & riedl SJ (2010) Molecular cell death platforms and assemblies. Curr. Opin. Cell Biol. 22, 828–836. Nagata S (2005) DNA degradation in development and programmed cell death. Annu. Rev. Immunol. 23, 853–875.

1	Nagata S (2005) DNA degradation in development and programmed cell death. Annu. Rev. Immunol. 23, 853–875. raff MC (1999) Cell suicide for beginners. Nature 396, 119–122. tait SW & Green Dr (2013) Mitochondrial regulation of cell death. Cold Spring Harb. Perspect. Biol. 5, a008706. Vanden Berghe t, Linkermann A, Jouan-Lanhouet S et al. (2014) regulated necrosis: the expanding network of non-apoptotic cell death pathways. Nat. Rev. Mol. Cell Biol. 15, 135–147. Vousden kh (2005) Apoptosis. p53 and pUMA: a deadly duo. Science 309, 1685–1686. Willis SN & Adams JM (2005) Life in the balance: how Bh3-only proteins induce apoptosis. Curr. Opin. Cell Biol. 17, 617–625. Yuan S & Akey CW (2013) Apoptosome structure, assembly, and procaspase activation. Structure 21, 501–515. Cell Junctions and the Extracellular Matrix

1	Yuan S & Akey CW (2013) Apoptosome structure, assembly, and procaspase activation. Structure 21, 501–515. Cell Junctions and the Extracellular Matrix Of all the social interactions between cells in a multicellular organism, the most fundamental are those that hold the cells together. Cells may be linked by direct interactions, or they may be held together within the extracellular matrix, a complex network of proteins and polysaccharide chains that the cells secrete. By one means or another, cells must cohere if they are to form an organized multicellular structure that can withstand and respond to the various external forces that try to pull it apart.

1	The mechanisms of cohesion govern the architecture of the body—its shape, its strength, and the arrangement of its different cell types. The making and breaking of the attachments between cells and the modeling of the extracellular matrix govern the way cells move within the organism, guiding them as the body grows, develops, and repairs itself. Attachments to other cells and to extracellular matrix control the orientation and behavior of the cell’s cytoskeleton, thereby allowing cells to sense and respond to changes in the mechanical features of their environment. Thus, the apparatus of cell junctions and the extracellular matrix is critical for every aspect of the organization, function, and dynamics of multicellular structures. Defects in this apparatus underlie an enormous variety of diseases.

1	The key features of cell junctions and the extracellular matrix are best illustrated by considering two broad categories of tissues that are found in all animals (Figure 19–1). Connective tissues, such as bone or tendon, are formed from an extracellular matrix produced by cells that are distributed sparsely in the matrix. It is the matrix—rather than the cells—that bears most of the mechanical stress to which the tissue is subjected. Direct attachments between one cell and another are relatively rare, but the cells have important attachments to the matrix. These cell–matrix junctions link the cytoskeleton to the matrix, allowing the cells to move through the matrix and monitor changes in its mechanical properties.

1	In epithelial tissues, such as the lining of the gut or the epidermal covering of the skin, cells are tightly bound together into sheets called epithelia. The extracellular matrix is less pronounced, consisting mainly of a thin mat called the basal lamina (or basement membrane) underlying the sheet. Within the epithelium, cells are attached to each other directly by cell–cell junctions, where cytoskeletal filaments are anchored, transmitting stresses across the interiors of the cells, from mechanical stresses are transmitted from cell to cell by cytoskeletal flaments anchored to cell–matrix and cell–cell adhesion sites extracellular matrix directly bears mechanical stresses of tension and compression adhesion site to adhesion site. The cytoskeleton of epithelial cells is also linked to the basal lamina through cell–matrix junctions.

1	Figure 19–2 provides a closer view of epithelial cells to illustrate the major types of cell–cell and cell–matrix junctions that we will discuss in this chapter. The diagram shows the typical arrangement of junctions in a simple columnar epithelium such as the lining of the small intestine of a vertebrate. Here, a single layer of tall cells stands on a basal lamina, with the cells’ uppermost surface, or apex, free and exposed to the extracellular medium. On their sides, or lateral surfaces, the cells make junctions with one another. Two types of anchoring junctions link the cytoskeletons of adjacent cells: adherens junctions are anchorage sites for actin filaments; desmosomes are anchorage sites for intermediate filaments. Two additional types of anchoring junctions link the cytoskeleton of the epithelial cells to the basal lamina: actin-linked cell–matrix junctions anchor actin filaments to the matrix, while hemidesmosomes anchor intermediate filaments to it.

1	Figure 19–1 Two main ways in which animal cells are bound together. In connective tissue, the main stress-bearing component is the extracellular matrix. In epithelial tissue, it is the cytoskeletons of the cells themselves, linked from cell to cell by adhesive junctions. Cell–matrix attachments bond epithelial tissue to the connective tissue beneath it. adherens junction connects actin flament bundle in one cell with that in the next cell desmosome connects intermediate flaments in one cell to those in the next cell gap junction allows the passage of small water-soluble molecules from cell to cell Figure 19–2 A summary of the various cell junctions found in a vertebrate epithelial cell, classified according to their primary functions.

1	Figure 19–2 A summary of the various cell junctions found in a vertebrate epithelial cell, classified according to their primary functions. In the most apical portion of the cell, the relative positions of the junctions are the same in nearly all vertebrate epithelia. the tight junction occupies the most apical position, followed by the adherens junction (adhesion belt) and then by a special parallel row of desmosomes; together these form a structure called a junctional complex. Gap junctions and additional desmosomes are less regularly organized. two types of cell-matrix anchoring junctions tether the basal surface of the cell to the basal lamina. the drawing is based on epithelial cells of the small intestine.

1	Figure 19–3 Transmembrane adhesion proteins link the cytoskeleton to extracellular structures. the external linkage may be either to other cells (cell–cell junctions, mediated typically by cadherins) or to extracellular matrix (cell–matrix junctions, mediated typically by integrins). the internal linkage to the cytoskeleton is generally indirect, via intracellular adaptor proteins, to be discussed later. Two other types of cell–cell junction are shown in Figure 19–2. Tight junctions hold the cells closely together near the apex, sealing the gap between the cells and thereby preventing molecules from leaking across the epithelium. Near the basal end of the cells are channel-forming junctions, called gap junctions, that create passageways linking the cytoplasms of adjacent cells.

1	Each of the four major anchoring junction types depends on transmembrane adhesion proteins that span the plasma membrane, with one end linking to the cytoskeleton inside the cell and the other end linking to other structures outside it (Figure 19–3). These cytoskeleton-linked transmembrane proteins fall neatly into two superfamilies, corresponding to the two basic kinds of external attachment. Proteins of the cadherin superfamily chiefly mediate attachment of cell to cell (Movie 19.1). Proteins of the integrin superfamily chiefly mediate attachment of cells to matrix. There is specialization within each family: some cadherins link to actin and form adherens junctions, while others link to intermediate filaments and form desmosomes; likewise, some integrins link to actin and form actinlinked cell–matrix junctions, while others link to intermediate filaments and form hemidesmosomes (Table 19–1).

1	There are some exceptions to these rules. Some integrins, for example, mediate cell–cell rather than cell–matrix attachment. Moreover, there are other types of cell adhesion molecules that can provide transient cell–cell attachments more flimsy than anchoring junctions, but sufficient to stick cells together in special circumstances. We begin the chapter with a discussion of the major forms of cell–cell junctions. We then consider in turn the extracellular matrix of animals, the structure and function of integrin-mediated cell–matrix junctions, and, finally, the plant cell wall, a special form of extracellular matrix.

1	Cell–cell junctions come in many forms and can be regulated by a variety of mechanisms. The best understood and most common are the two types of cell– cell anchoring junctions, which employ cadherins to link the cytoskeleton of one cell with that of its neighbor. Their primary function is to resist the external forces that pull cells apart. The epithelial cells of your skin, for example, must remain tightly linked when they are stretched, pinched, or poked. Cell–cell anchoring junctions must also be dynamic and adaptable, so that they can be altered or rearranged when tissues are remodeled or repaired, or when there are changes in the forces acting on them. In this section, we focus primarily on the cadherin-based anchoring junctions. We then briefly describe tight junctions and gap junctions. Finally, we consider the more transient cell–cell adhesion mechanisms employed by some cells in the bloodstream. Cadherins Form a Diverse Family of adhesion Molecules

1	Cadherins Form a Diverse Family of adhesion Molecules Cadherins are present in all multicellular animals whose genomes have been analyzed. They are also present in the choanoflagellates, which can exist either as free-living unicellular organisms or as multicellular colonies and are thought to be representatives of the group of protists from which all animals evolved. Other eukaryotes, including fungi and plants, lack cadherins, and they are also absent from bacteria and archaea. Cadherins therefore seem to be part of the essence of what it is to be an animal.

1	The cadherins take their name from their dependence on Ca2+ ions: removing Ca2+ from the extracellular medium causes adhesions mediated by cadherins to come apart. The first three cadherins to be discovered were named according to the main tissues in which they were found: E-cadherin is present on many types of epithelial cells; N-cadherin on nerve, muscle, and lens cells; and P-cadherin on cells in the placenta and epidermis. All are also found in other tissues. These and other classical cadherins are closely related in sequence throughout their extracellular and intracellular domains.

1	There are also a large number of nonclassical cadherins that are more distantly related in sequence, with more than 50 expressed in the brain alone. The nonclassical cadherins include proteins with known adhesive function, such as the diverse protocadherins found in the brain, and the desmocollins and desmogleins that form desmosomes (see Table 19–1). Other family members are involved primarily in signaling. Together, the classical and nonclassical cadherin proteins constitute the cadherin superfamily (Figure 19–4), with more than 180 members in humans. Anchoring junctions between cells are usually symmetrical: if the linkage is to actin in the cell on one side of the junction, it will be to actin in the cell on the other side. In fact, the binding between cadherins is generally homophilic (liketo-like, Figure 19–5): cadherin molecules of a specific subtype on one cell bind to cadherin molecules of the same or closely related subtype on adjacent cells.

1	The spacing between the cell membranes at an anchoring junction is precisely defined and depends on the structure of the participating cadherin molecules. All the members of the superfamily, by definition, have an extracellular portion consisting of several copies of the extracellular cadherin (EC) domain. Homophilic binding occurs at the N-terminal tips of the cadherin molecules—the cadherin domains that lie furthest from the membrane. These terminal domains each form a knob and a nearby pocket, and the cadherin molecules protruding from opposite cell membranes bind by insertion of the knob of one domain into the pocket of the other (Figure 19–6A).

1	Each cadherin domain forms a more-or-less rigid unit, joined to the next cadherin domain by a hinge. Ca2+ ions bind to sites near each hinge and prevent it from flexing, so that the whole string of cadherin domains behaves as a rigid and slightly curved rod. When Ca2+ is removed, the hinges can flex, and the structure becomes floppy (Figure 19–6B). At the same time, the conformation at the N-terminus is thought to change slightly, weakening the binding affinity for the matching cadherin molecule on the opposite cell.

1	Unlike receptors for soluble signal molecules, which bind their specific ligand with high affinity, cadherins (and most other cell–cell adhesion proteins) typically bind to their partners with relatively low affinity. Strong attachments result from the formation of many such weak bonds in parallel. When binding to oppositely oriented partners on another cell, cadherin molecules are often clustered side-to-side with many other cadherin molecules on the same cell (Figure 19–6C). The strength of this junction is far greater than that of any individual intermolecular bond, and yet regulatory mechanisms can easily disassemble the junction by separating the molecules sequentially, just as two pieces of fabric can be joined strongly by Velcro and yet easily peeled apart from the sides. A similar “Velcro principle” also operates at cell–cell and cell–matrix adhesions formed by other types of transmembrane adhesion proteins. Figure 19–4 The cadherin superfamily.

1	Figure 19–4 The cadherin superfamily. the diagram shows some of the diversity among cadherin superfamily members. these proteins all have extracellular portions containing multiple copies of the extracellular cadherin domain (green ovals). In the classical cadherins of vertebrates there are 5 of these domains, and in desmogleins and desmocollins there are 4 or 5, but some nonclassical cadherins have more than 30. the intracellular portions are more varied, reflecting interactions with a wide variety of intracellular ligands, including signaling molecules and adaptor proteins that connect the cadherin to the cytoskeleton. In some cases, such as t-cadherin, a transmembrane domain is not present and the protein is attached to the plasma membrane by a glycosylphosphatidylinositol (GpI) anchor. the differently colored motifs in Fat, Flamingo, and ret represent conserved domains that are also found in other protein families.

1	Figure 19–5 Homophilic versus heterophilic binding. Cadherins in general bind homophilically; some other cell adhesion molecules, discussed later, bind heterophilically. 38.5 nm hinge regions

1	Ca2+ of cell 1 of cell 2 membrane membrane < 0.05 mM Ca2+ (B) plasma membrane of cell 1 plasma membrane of cell 2 Figure 19–6 Cadherin structure and function. (a) the extracellular region of a classical cadherin contains five copies of the extracellular cadherin domain (see Figure 19–4) separated by flexible hinge regions. Ca2+ ions (red dots) bind in the neighborhood of each hinge, preventing it from flexing. as a result, the extracellular region forms a rigid, curved structure as shown here. to generate cell–cell adhesion, the cadherin domain at the N-terminal tip of one cadherin molecule binds the N-terminal domain from a cadherin molecule on another cell. the structure was determined by x-ray diffraction of the crystallized C-cadherin extracellular region. (B) In the absence of Ca2+, increased flexibility in the hinge regions results in a floppier molecule that is no longer oriented correctly to interact with a cadherin on another cell—and adhesion fails. (C) at a typical

1	Ca2+, increased flexibility in the hinge regions results in a floppier molecule that is no longer oriented correctly to interact with a cadherin on another cell—and adhesion fails. (C) at a typical cell–cell junction, an organized array of cadherin molecules functions like Velcro to hold cells together. Cadherins on the same cell are thought to be coupled by side-to-side interactions between their N-terminal head regions, resulting in a linear array like the alternating green and light green cadherins on the lower cell shown here. these arrays are thought to interact with similar linear arrays on an adjacent cell (blue cadherin molecules, top cell). the linear arrays on one cell are perpendicular to those on the other cell, as indicated by the red arrows. Multiple perpendicular arrays on both cells interact to form a tight-knit mat of cadherin proteins. (a, based on t.J. Boggon et al., Science 296:1308–1313, 2002; C, based on O.J. harrison et al. Structure 19:244–256, 2011.)

1	Cadherin-Dependent Cell–Cell adhesion Guides the Organization of Developing tissues Cadherins form specific homophilic attachments, explaining why there are so many different family members. Cadherins are not like glue, making cell surfaces generally sticky. Rather, they mediate highly selective recognition, enabling cells of a similar type to stick together and to stay segregated from other types of cells.

1	Selectivity in the way that animal cells consort with one another was first demonstrated in the 1950s, long before the discovery of cadherins, in experiments in which amphibian embryos were dissociated into single cells. These cells were then mixed up and allowed to reassociate. Remarkably, the dissociated cells often reassembled into structures resembling those of the original embryo (Figure 19–7). These experiments, together with numerous more recent experiments, reveal that selective cell–cell recognition systems make cells of the same differentiated tissue preferentially adhere to one another.

1	Figure 19–7 Sorting out. Cells from different layers of an early amphibian embryo will sort out according to their origins. In the classical experiment shown here, mesoderm cells (green), neural plate cells (blue), and epidermal cells (red) have been disaggregated and then reaggregated in a random mixture. they sort out into an arrangement reminiscent of a normal embryo, with a “neural tube” internally, epidermis externally, and mesoderm in between. (Modified from p.L. townes and J. holtfreter, J. Exp. Zool. 128:53–120, 1955. With permission from Wiley-Liss.)

1	Cadherins play a crucial part in these cell-sorting processes during development. The appearance and disappearance of specific cadherins correlate with steps in embryonic development where cells regroup and change their contacts to create new tissue structures. In the vertebrate embryo, for example, changes in cadherin expression are seen when the neural tube forms and pinches off from the overlying ectoderm: neural tube cells lose E-cadherin and acquire other cadherins, including N-cadherin, while the cells in the overlying ectoderm continue to express E-cadherin (Figure 19–8A and B). Then, when the neural crest cells migrate away from the neural tube, these cadherins become scarcely detectable, and another cadherin (cadherin 7) appears that helps hold the migrating cells together as loosely associated cell groups (Figure 19–8C). Finally, when the cells aggregate to form a ganglion, they switch on expression of N-cadherin again. If N-cadherin is artificially overexpressed in the

1	loosely associated cell groups (Figure 19–8C). Finally, when the cells aggregate to form a ganglion, they switch on expression of N-cadherin again. If N-cadherin is artificially overexpressed in the emerging neural crest cells, the cells fail to escape from the neural tube.

1	Studies with cultured cells further support the idea that the homophilic binding of cadherins controls these processes of tissue segregation. In a line of cultured fibroblasts called L cells, for example, cadherins are not expressed and the cells do not adhere to one another. When these cells are transfected with DNA encoding E-cadherin, E-cadherins on one cell bind to E-cadherins on another, resulting in cell–cell adhesion. If L cells expressing different cadherins are mixed together, they sort out and aggregate separately, indicating that different cadherins preferentially bind to their own type (Figure 19–9A), mimicking what happens when cells derived from tissues that express different cadherins are mixed together. A similar segregation of cells occurs if L cells expressing different amounts of the same cadherin are mixed together (Figure 19–9B). It therefore seems likely that both qualitative and quantitative differences in the expression of cadherins have a role in organizing

1	amounts of the same cadherin are mixed together (Figure 19–9B). It therefore seems likely that both qualitative and quantitative differences in the expression of cadherins have a role in organizing tissues.

1	Figure 19–8 Changing patterns of cadherin expression during construction of the vertebrate nervous system. the figure shows cross sections of the early chick embryo, as the neural tube detaches from the ectoderm and then as neural crest cells detach from the neural tube. (a, B) Immunofluorescence micrographs showing the developing neural tube labeled with antibodies against (a) E-cadherin (blue) and (B) N-cadherin (yellow). (C) as the patterns of gene expression change, the different groups of cells segregate from one another according to the cadherins they express. (Micrographs courtesy of Miwako Nomura and Masatoshi takeichi.) cell expressing high level of E-cadherin cell expressing low level of E-cadherin (B)

1	Figure 19–9 Cadherin-dependent cell sorting. Cells in culture can sort themselves out according to the type and level of cadherins they express. this can be visualized by labeling different populations of cells with dyes of different colors. (a) Cells expressing N-cadherin sort out from cells expressing E-cadherin. (B) Cells expressing high levels of E-cadherin sort out from cells expressing low levels of E-cadherin. the cells expressing high levels adhere more strongly and end up internally. Epithelial–Mesenchymal transitions Depend on Control of Cadherins

1	Epithelial–Mesenchymal transitions Depend on Control of Cadherins The assembly of cells into an epithelium is a reversible process. By switching on expression of adhesion molecules, dispersed unattached mesenchymal cells, such as fibroblasts, can come together to form an epithelium. Conversely, epithelial cells can change their character, disassemble, and migrate away from their parent epithelium as separate cells. Such epithelial–mesenchymal transitions play an important part in normal embryonic development; the origin of the neural crest is one example. These transitions depend in part on transcription regulatory proteins called Slug, Snail, and Twist. Increased expression of Twist, for example, converts epithelial cells to a mesenchymal character, and switching it off does the opposite. Twist exerts its effects, in part, by inhibiting expression of cadherins, including E-cadherin, that hold epithelial cells together.

1	Epithelial–mesenchymal transitions also occur as pathological events during adult life, in cancer. Most cancers originate in epithelia, but become dangerously prone to spread—that is, malignant—only when the cancer cells escape from the epithelium of origin and invade other tissues. Experiments with malignant breast cancer cells in culture show that blocking expression of Twist can convert the cells back toward a nonmalignant character. Conversely, by forcing Twist expression, one can make normal epithelial cells undergo an epithelial–mesenchymal transition and behave like malignant cells. Mutations that disrupt the production or function of E-cadherin are often found in cancer cells and are thought to help make them malignant. Catenins Link Classical Cadherins to the actin Cytoskeleton

1	Catenins Link Classical Cadherins to the actin Cytoskeleton The extracellular domains of cadherins mediate homophilic binding at adherens junctions. The intracellular domains of typical cadherins, including all classical and some nonclassical ones, interact with filaments of the cytoskeleton: actin at adherens junctions and intermediate filaments at desmosomes (see Table 19–1). These cytoskeletal linkages are essential for efficient cell–cell adhesion, as cadherins that lack their cytoplasmic domains cannot stably hold cells together.

1	The linkage of cadherins to the cytoskeleton is indirect and depends on adaptor proteins that assemble on the cytoplasmic tail of the cadherin. At adherens junctions, the cadherin tail binds two such proteins: β-catenin and a distant relative called p120-catenin; a third protein called α-catenin interacts with β-catenin and recruits a variety of other proteins to provide a dynamic linkage to actin filaments (Figure 19–10). At desmosomes, cadherins are linked to intermediate filaments through other adaptor proteins, including a β-catenin-related protein called plakoglobin, as we discuss later.

1	In their mature form, adherens junctions are enormous protein complexes containing hundreds to thousands of cadherin molecules, packed into dense, regular arrays that are linked on the extracellular side by lateral interactions between cadherin domains, as we discussed earlier (see Figure 19–6C). On the cytoplasmic side, a complex network of catenins, actin regulators, and contractile actin bundles holds the cluster of cadherins together and links it to the actin cytoskeleton. Assembling a structure of this complexity is not a simple task, and it involves a complex sequence of events controlled by the actin-regulatory proteins discussed in Chapter 16. The general features of the assembly process are summarized in Figure 19–11. adherens Junctions respond to Forces Generated by the actin Cytoskeleton

1	adherens Junctions respond to Forces Generated by the actin Cytoskeleton Most adherens junctions are linked to contractile bundles of actin filaments and non-muscle myosin II. These junctions are therefore subjected to pulling forces generated by the attached actin. The pulling forces are important for junction assembly and maintenance: disruption of myosin activity, for example, results in Figure 19–10 The linkage of classical cadherins to actin filaments. the cadherins are coupled indirectly to actin filaments through an adaptor protein complex containing p120-catenin, β-catenin, and α-catenin. Other proteins, including vinculin, associate with α-catenin and help provide the linkage to actin. β-Catenin has a second, and very important, function in intracellular signaling, as we discuss in Chapter 15 (see Figure 15–60). For clarity, this diagram does not show the cadherin of the adjacent cell in the junction.

1	(A) (B) recruitment of more (C) contractile actin and the disassembly of many adherens junctions. Furthermore, the contractile forces acting on a junction in one cell are balanced by contractile forces at the junction of the opposite cell, so that no cell pulls others toward it and thereby disrupts the uniform distribution of cells in the tissue.

1	We do not understand the mechanisms responsible for maintaining this balance. Adherens junctions seem to sense the forces acting on them and modify local actin and myosin behavior to balance the forces on both sides of the junction. Evidence for these mechanisms comes from studies of pairs of cultured mammalian cells connected by adherens junctions. If contractile activity in one cell is increased experimentally, the adherens junctions linking the two cells increase in size, and the contractile activity of the second cell increases to match that of the first—resulting in a balance of forces across the junction. These and other experiments suggest that adherens junctions are not simply passive sites of protein–protein binding but are dynamic tension sensors that regulate their behavior in response to changing mechanical conditions. This ability to transduce a mechanical signal into a change in junctional behavior is an example of mechanotransduction. We will see later that it is also

1	response to changing mechanical conditions. This ability to transduce a mechanical signal into a change in junctional behavior is an example of mechanotransduction. We will see later that it is also important at cell–matrix junctions.

1	The mechanotransduction at cell–cell junctions is thought to depend, at least in part, on proteins in the cadherin complex that alter their shape when stretched by tension. The protein α-catenin, for example, is stretched from a folded to an extended conformation when contractile activity increases at the junction. The unfolding exposes a cryptic binding site for another protein, vinculin, which promotes the recruitment of more actin to the junction (Figure 19–12). By mechanisms such as this, pulling on a junction makes it stronger. Furthermore, as noted above, pulling on a junction in one cell will increase the contractile force generated in the attached cell.

1	In some cell types, actin contractility reduces cell–cell adhesion, particularly if large forces are involved. Large actin-based contractile forces might, in some tissues, pull sufficiently hard on the edges of cell–cell adhesions to peel them apart, particularly if contraction is coupled to additional regulatory mechanisms that weaken the adhesion. This mechanism might be important in certain forms of tissue remodeling during development, as we describe next. tissue remodeling Depends on the Coordination of actin-Mediated Contraction With Cell–Cell adhesion Adherens junctions are an essential part of the machinery for modeling the shapes of multicellular structures in the animal body. By indirectly linking the actin filaments in one cell to those in its neighbors, they enable the cells in the tissue to use their actin cytoskeletons in a coordinated way.

1	Adherens junctions occur in various forms. In many nonepithelial tissues, they appear as small punctate or linear attachments that connect the cortical Figure 19–11 Assembly of an adherens junction. (a) assembly begins when two unattached epithelial cell precursors explore their surroundings with membrane protrusions, generated by local nucleation of actin networks. When the cells make contact, small cadherin and catenin clusters take shape at the contact sites and associate with actin, leading to activation of the small monomeric Gtpase rac (not shown), an important actin regulator (see Figure 16–85). (B) rac promotes additional actin protrusions in the vicinity, expanding the size of the contact zone and thereby promoting further recruitment of cadherins and their associated catenin proteins.

1	(C) Eventually, rac is inactivated and replaced by the related Gtpase rho (not shown), which shifts actin remodeling toward the assembly of linear, contractile filament bundles. rho also promotes the assembly of myosin II filaments that associate with bundles of actin filaments to generate contractile activity. this contractile activity generates tension that stimulates further actin recruitment and expansion of the junction, in part through the mechanisms illustrated in Figure 19–12.

1	actin filaments beneath the plasma membranes of two interacting cells. In heart muscle, they anchor the actin bundles of the contractile apparatus and act in parallel with desmosomes to link the contractile cells end-to-end. But the prototypical examples of adherens junctions occur in epithelia, where they often form a continuous adhesion belt (or zonula adherens) just beneath the apical face of the epithelium, encircling each of the interacting cells in the sheet (Figure 19–13). Within each cell, a contractile bundle of actin filaments and myosin II lies adjacent to the adhesion belt, oriented parallel to the plasma membrane and tethered to bundle of actin flaments lateral plasma membranes of adjacent epithelial cells

1	Figure 19–12 Mechanotransduction in an adherens junction. (a) Cell–cell junctions are able to sense increased tension and respond by strengthening their actin linkages. tension sensing is thought to depend in part on α-catenin (see Figure 19–10). (B) When actin filaments are pulled from within the cell by non-muscle myosin II, the resulting force unfolds a domain in α-catenin, thereby exposing an otherwise hidden binding site for the adaptor protein vinculin. Vinculin then promotes additional actin recruitment, strengthening the linkages between the junction and the cytoskeleton.

1	Figure 19–13 Adherens junctions between epithelial cells in the small intestine. these cells are specialized for absorption of nutrients; at their apex, facing the lumen of the gut, they have many microvilli (protrusions that increase the absorptive surface area). the adherens junction takes the form of an adhesion belt, encircling each of the interacting cells. Its most obvious feature is a contractile bundle of actin filaments running along the cytoplasmic surface of the junctional plasma membrane. the actin filament bundles are tethered by intracellular proteins to cadherins, which bind to cadherins on the adjacent cell. In this way, the actin filament bundles in adjacent cells are tied together. For clarity, this drawing does not show most of the other cell–cell and cell–matrix junctions of epithelial cells (see Figure 19–2).

1	it by the cadherins and their associated intracellular adaptor proteins. The actin– myosin bundles are thus linked, via the cadherins, into an extensive transcellular network. Coordinated contraction of this network provides the motile force for a fundamental process in animal morphogenesis—the folding of epithelial cell sheets into tubes, vesicles, and other related structures (Figure 19–14).

1	The coordination of cell–cell adhesion and actin contractility is beautifully illustrated by cellular rearrangements that occur early in the development of the fruit fly Drosophila melanogaster. Soon after gastrulation, the outer epithelium of the embryo is elongated by a process called germ-band extension, in which the cells converge inward toward the dorsal–ventral axis and extend along the anterior–posterior axis (Figure 19–15). Actin-dependent contraction along specific cell boundaries is coordinated with a loss of specific adherens junctions to allow cells to insert themselves between other cells (a process called intercalation), resulting in a longer and narrower epithelium. The mechanisms underlying the loss of adhesion along specific cell boundaries are not clear, but they depend in part on increased degradation of β-catenin, due to its phosphorylation by a protein kinase that is localized specifically at those boundaries.

1	Desmosomes are structurally similar to adherens junctions but contain specialized cadherins that link to intermediate filaments instead of actin filaments. Their main function is to provide mechanical strength. Desmosomes are important Figure 19–14 The folding of an epithelial sheet to form an epithelial tube. the oriented contraction of the bundles of actin and myosin filaments running along adhesion belts causes the epithelial cells to narrow at their apex and helps the epithelial sheet to roll up into a tube. an example is the formation of the neural tube in early vertebrate development (see Figure 19–8).

1	Figure 19–15 Remodeling of cell–cell adhesions in embryonic Drosophila epithelium. Depicted at left is a group of cells in the outer epithelium of a Drosophila embryo. During germ-band extension, cells converge toward each other (middle) on the dorsal–ventral axis and then extend (right) along the anterior–posterior axis. the result is intercalation: cells that were originally far apart along the dorsal–ventral axis (dark green) are inserted between the cells (light green) that separated them. these rearrangements depend on the spatial regulation of actin–myosin contractile bundles, which are localized primarily at the vertical cell boundaries (red, left). Contraction of these bundles is accompanied by removal of E-cadherin (not shown) at the same cell boundaries, resulting in shrinkage and loss of adhesion along the vertical axis (middle). New cadherin-based adhesions (blue, right) then form and expand along horizontal boundaries, resulting in extension of the cells in the

1	and loss of adhesion along the vertical axis (middle). New cadherin-based adhesions (blue, right) then form and expand along horizontal boundaries, resulting in extension of the cells in the anterior–posterior dimension.

1	0.5 µm 100 nm

1	Figure 19–16 Desmosomes. (a) the structural components of a desmosome. On the cytoplasmic surface of each interacting plasma membrane is a dense plaque composed of a mixture of intracellular adaptor proteins. a bundle of keratin intermediate filaments is attached to the surface of each plaque. transmembrane nonclassical cadherins bind to the plaques and interact through their extracellular domains to hold the adjacent membranes together. (B) Some of the molecular components of a desmosome. Desmoglein and desmocollin are nonclassical cadherins. their cytoplasmic tails bind plakoglobin (γ-catenin) and plakophilin (a distant relative of p120-catenin), which in turn bind to desmoplakin. Desmoplakin binds to the sides of intermediate filaments, thereby tying the desmosome to these filaments. (C) an electron micrograph of desmosome junctions between three epidermal cells in the skin of a baby mouse. (D) part of the same tissue at higher magnification, showing a single desmosome, with

1	(C) an electron micrograph of desmosome junctions between three epidermal cells in the skin of a baby mouse. (D) part of the same tissue at higher magnification, showing a single desmosome, with intermediate filaments attached to it. (C and D, from W. he, p. Cowin and D.L. Stokes, Science 302:109–113, 2003. With permission from aaaS.) in vertebrates but are not found, for example, in Drosophila. They are present in most mature vertebrate epithelia and are particularly plentiful in tissues that are subject to high levels of mechanical stress, such as heart muscle and the epidermis, the epithelium that forms the outer layer of the skin.

1	Figure 19–16A shows the general structure of a desmosome, and Figure 19–16B shows some of the proteins that form it. Desmosomes typically appear as buttonlike spots of adhesion, riveting the cells together (Figure 19–16C). Inside the cell, the bundles of ropelike intermediate filaments that are anchored to the desmosomes form a structural framework of great tensile strength (Figure 19–16D), with linkage to similar bundles in adjacent cells, creating a network that extends throughout the tissue (Figure 19–17). The particular type of intermediate filaments attached to the desmosomes depends on the cell type: they are keratin filaments in most epithelial cells, for example, and desmin filaments in heart muscle cells.

1	The importance of desmosomes is demonstrated by some forms of the potentially fatal skin disease pemphigus. Affected individuals make antibodies against one of their own desmosomal cadherin proteins. These antibodies bind to and disrupt the desmosomes that hold their epidermal cells (keratinocytes) together. This results in a severe blistering of the skin, with leakage of body fluids into the loosened epithelium. Sheets of epithelial cells enclose and partition the animal body, lining all its surfaces and cavities, and creating internal compartments where specialized processes occur. The epithelial sheet seems to be one of the inventions that lie at the origin of animal evolution, diversifying in a huge variety of ways but retaining an organization based on a set of conserved molecular mechanisms.

1	Essentially all epithelia are anchored to other tissue on one side—the basal side—and free of such attachment on their opposite side—the apical side. A basal lamina lies at the interface with the underlying tissue, mediating the attachment, while the apical surface of the epithelium is generally bathed by extracellular fluid. Thus, all epithelia are structurally polarized, and so are their individual cells: the basal end of a cell, adherent to the basal lamina below, differs from the apical end, exposed to the medium above. Correspondingly, all epithelia have at least one function in common: they serve as selective permeability barriers, separating the fluid that permeates the tissue on their basal side from fluid with a different chemical composition on their apical side. This barrier function requires that the adjacent cells be sealed together by tight junctions, so that molecules cannot leak freely across the cell sheet.

1	The epithelium of the small intestine provides a good illustration of tight-junction structure and function (see Figure 19–2). This epithelium has a simple columnar structure; that is, it consists of a single layer of tall (columnar) cells. These are of several differentiated types, but the majority are absorptive cells, specialized for uptake of nutrients from the internal cavity, or lumen, of the gut. The absorptive cells have to transport selected nutrients across the epithelium from the lumen into the extracellular fluid on the other side. From there, these nutrients diffuse into small blood vessels to provide nourishment to the organism. This transcellular transport depends on two sets of transport proteins in the plasma membrane of the absorptive cell. One set is confined to the apical surface of the cell (facing the lumen) and actively transports selected molecules into the cell from the gut. The other set is confined to the basolateral (basal and lateral) surfaces of the cell,

1	surface of the cell (facing the lumen) and actively transports selected molecules into the cell from the gut. The other set is confined to the basolateral (basal and lateral) surfaces of the cell, and it allows the same molecules to leave the cell by passive transport into the extracellular fluid on the other side of the epithelium. For this transport activity to be effective, the spaces between the epithelial cells must be tightly sealed, so that the transported molecules cannot leak back into the gut lumen through these spaces (Figure 19–18). Moreover, the transport proteins must be correctly distributed in the plasma membranes: the apical transporters must be delivered to the cell apex and must not be allowed to drift to the basolateral membrane, and the basolateral transporters must be delivered to and remain in the basolateral membrane. Tight junctions, besides sealing the gaps between the cells, also function as “fences” that help prevent apical or basolateral proteins from

1	be delivered to and remain in the basolateral membrane. Tight junctions, besides sealing the gaps between the cells, also function as “fences” that help prevent apical or basolateral proteins from diffusing into the wrong region.

1	The sealing function of tight junctions is easy to demonstrate experimentally: a low-molecular-weight tracer added to one side of an epithelium will generally not pass beyond the tight junction (Figure 19–19). This seal is not absolute, however. Although all tight junctions are impermeable to macromolecules, their permeability to ions and other small molecules varies. Tight junctions in the epithelium lining the small intestine, for example, are 10,000 times more permeable to inorganic ions, such as Na+, than the tight junctions in the epithelium lining the urinary bladder. The movement of ions and other molecules between epithelial cells is called paracellular transport, and tissue-specific differences in transport rates generally result from differences in the proteins that form tight junctions. tight Junctions Contain Strands of transmembrane adhesion proteins

1	tight Junctions Contain Strands of transmembrane adhesion proteins When tight junctions are visualized by freeze-fracture electron microscopy, they are seen as a branching network of sealing strands that completely encircles the Figure 19–17 Desmosomes, hemidesmosomes, and the intermediate filament network. the keratin intermediate filament networks of adjacent cells—in this example, epithelial cells of the small intestine—are indirectly connected to one another through desmosomes, and to the basal lamina through hemidesmosomes.

1	plasma membranes of adjacent cells apical end of each cell in the epithelial sheet (Figure 19–20A and B). In conventional electron micrographs, the outer leaflets of the two interacting plasma membranes are tightly apposed where sealing strands are present (Figure 19–20C). Each sealing strand is composed of a long row of transmembrane homophilic adhesion proteins embedded in each of the two interacting plasma membranes. The extracellular domains of these proteins adhere directly to one another to occlude the intercellular space (Figure 19–21).

1	The main transmembrane proteins forming these strands are the claudins, which are essential for tight-junction formation and function. Mice that lack the claudin-1 gene, for example, fail to make tight junctions between the cells in the epidermal layer of the skin; as a result, the baby mice lose water rapidly by evaporation through the skin and die within a day after birth. Conversely, if nonepithelial cells such as fibroblasts are artificially caused to express claudin genes, they (A) (B) 0.5 µm 0.5 µm

1	Figure 19–18 The role of tight junctions in transcellular transport. For clarity, only the tight junctions are shown. transport proteins are confined to different regions of the plasma membrane in epithelial cells of the small intestine. this segregation permits a vectorial transfer of nutrients across the epithelium from the gut lumen to the blood. In the example shown, glucose is actively transported into the cell by Na+-driven glucose transporters at its apical surface, and it leaves the cell through passive glucose transporters in its basolateral membrane. tight junctions are thought to confine the transport proteins to their appropriate membrane domains by acting as diffusion barriers, or “fences,” within the lipid bilayer of the plasma membrane; these junctions also block the backflow of glucose from the basal side of the epithelium into the gut lumen (see Movie 11.2).

1	Figure 19–19 The role of tight junctions in allowing epithelia to serve as barriers to solute diffusion. (a) the drawing shows how a small extracellular tracer molecule added on one side of an epithelium is prevented from crossing the epithelium by the tight junctions that seal adjacent cells together. adherens junctions and other cell junctions are not shown for clarity. (B) Electron micrographs of cells in an epithelium in which a small, extracellular, electron-dense tracer molecule has been added to either the apical side (on the left) or the basolateral side (on the right). the tight junction blocks passage of the tracer in both directions. (B, courtesy of Daniel Friend.) (A) lateral plasmaridges of transmembrane focal connection focal connection plasma membrane 50 nm0.5 µm intestinal lumenmicrovilli (C)(B) tightjunctioncell 1 cell 2

1	Figure 19–20 The structure of a tight junction between epithelial cells of the small intestine. the junctions are shown (a) schematically, (B) in a freeze-fracture electron micrograph, and (C) in a conventional electron micrograph. In (B), the plane of the micrograph is parallel to the plane of the membrane, and the tight junction appears as a band of branching sealing strands that encircle each cell in the epithelium (see Figure 19–21a). In (C), the junction is seen in cross section as a series of focal connections between the outer leaflets of the two interacting plasma membranes, each connection corresponding to a sealing strand in cross section. (B and C, from N.B. Gilula, in Cell Communication [r.p. Cox, ed.], pp. 1–29. New York: Wiley, 1974.) will form tight-junctional connections with one another. Normal tight junctions also contain a second major transmembrane protein called occludin, which is not essential for the assembly or structure of the tight junction but is important

1	with one another. Normal tight junctions also contain a second major transmembrane protein called occludin, which is not essential for the assembly or structure of the tight junction but is important for limiting junctional permeability. A third transmembrane protein, tricellulin, is required to seal cell membranes together and prevent transepithelial leakage at the points where three cells meet.

1	The claudin protein family has many members (24 in humans), and these are expressed in different combinations in different epithelia to confer particular permeability properties on the epithelial sheet. They are thought to form paracellular pores—selective channels allowing specific ions to cross the tight-junctional barrier, from one extracellular space to another. A specific claudin found in kidney epithelial cells, for example, is needed to let Mg2+ pass between the cells of the kidney tubules so that this ion can be resorbed from the urine into the blood. A mutation in the gene encoding this claudin results in excessive loss of Mg2+ in the urine.

1	Like the cadherin molecules of an adherens junction, the claudins and occludins of a tight junction interact with each other on their extracellular sides to promote junction assembly. Also as in adherens junctions, the organization of adhesion proteins in a tight junction depends on additional proteins that bind the cytoplasmic side of the adhesion proteins. The key organizational proteins at tight junctions are the zonula occludens (ZO) proteins. The three major members of the ZO family—ZO-1, ZO-2, and ZO-3—are large scaffold proteins that provide a structural support on which the tight junction is built. These intracellular molecules cell cell 0.3 µm 12 consist of strings of protein-binding domains, typically including several PDZ domains—segments about 80 amino acids long that can recognize and bind the C-terminal tails of specific partner proteins (Figure 19–22). One domain of these scaffold proteins can attach to a claudin protein, while others can attach to occludin or the actin

1	and bind the C-terminal tails of specific partner proteins (Figure 19–22). One domain of these scaffold proteins can attach to a claudin protein, while others can attach to occludin or the actin cytoskeleton. Moreover, one molecule of scaffold protein can bind to another. In this way, the cell can assemble a mat of intracellular proteins that organizes and positions the sealing strands of the tight junction.

1	The tight-junctional network of sealing strands usually lies just apical to adherens and desmosome junctions that bond the cells together mechanically; the whole assembly is called a junctional complex (see Figure 19–2). The parts of this junctional complex depend on each other for their formation. For example, anticadherin antibodies that block the formation of adherens junctions also block the formation of tight junctions. Tight junctions block the passageways through the gaps between epithelial cells, preventing extracellular molecules from leaking from one side of an epithelium to the other. Another type of junctional structure has a radically different function: it bridges gaps between adjacent cells so as to create direct channels from the Figure 19–21 A model of a tight junction.

1	Figure 19–21 A model of a tight junction. (a) the sealing strands hold adjacent plasma membranes together. the strands are composed of transmembrane proteins that make contact across the intercellular space and create a seal. (B) the molecular composition of a sealing strand. the major extracellular components of the tight junction are members of a family of proteins with four transmembrane domains. One of these proteins, claudin, is the most important for the assembly and structure of the sealing strands, whereas the related protein occludin has the less critical role of determining junction permeability. the two termini of these proteins are both on the cytoplasmic side of the membrane, where they interact with large scaffolding proteins that organize the sealing strands and link the tight junction to the actin cytoskeleton (not shown here, but see Figure 19–22).

1	Figure 19–22 Scaffold proteins at the tight junction. the scaffold proteins ZO-1, ZO-2, and ZO-3 are concentrated beneath the plasma membrane at tight junctions. Each of the proteins contains multiple protein-binding domains, including three pDZ domains, an Sh3 domain, and a GK domain, linked together like beads on a flexible string. these domains enable the proteins to interact with each other and with numerous other partners, as indicated here, to generate a tightly woven protein network that organizes the sealing strands of the tight junction and links them to the actin cytoskeleton. Scaffold proteins with similar structure help organize other junctional complexes, including those at neural synapses. cytoplasm of one to that of the other. These channels are called gap junctions.

1	cytoplasm of one to that of the other. These channels are called gap junctions. Gap junctions are present in most animal tissues, including connective tissues as well as epithelia and heart muscle. Each gap junction appears in conventional electron micrographs as a patch where the membranes of two adjacent cells are separated by a uniform narrow gap of about 2–4 nm (Figure 19–23). The gap is spanned by channel-forming proteins, of which there are two distinct families, called the connexins and the innexins. Connexins are the predominant gap-junction proteins in vertebrates, with 21 isoforms in humans. Innexins are found in the gap junctions of invertebrates.

1	Gap junctions have a pore size of about 1.4 nm, which allows the exchange of inorganic ions and other small water-soluble molecules, but not of macromolecules such as proteins or nucleic acids (Figure 19–24). An electric current injected into one cell through a microelectrode causes an electrical disturbance in the neighboring cell, due to the flow of ions carrying electric charge through gap junctions. This electrical coupling via gap junctions serves an obvious purpose in tissues containing electrically excitable cells: action potentials can spread rapidly from cell to cell, without the delay that occurs at chemical synapses. In vertebrates, for example, electrical coupling through gap junctions synchronizes the contractions of heart muscle cells as well as those of the smooth muscle cells responsible for the peristaltic movements of the intestine. Gap junctions also occur in many tissues whose cells are not electrically excitable. In principle, the sharing of small metabolites and

1	responsible for the peristaltic movements of the intestine. Gap junctions also occur in many tissues whose cells are not electrically excitable. In principle, the sharing of small metabolites and ions provides a mechanism for coordinating the activities of individual cells in such tissues and for smoothing out random fluctuations in small-molecule concentrations in different cells.

1	a Gap-Junction Connexon Is Made of Six transmembrane Connexin Subunits Connexins are four-pass transmembrane proteins, six of which assemble to form a hemichannel, or connexon. When the connexons in the plasma membranes of two cells in contact are aligned, they form a continuous aqueous channel that connects the two cell interiors (Figure 19–25). A gap junction consists of many such connexon pairs in parallel, forming a sort of molecular sieve. Not only does this sieve provide a communication channel between cells, but it also provides a form of cell–cell adhesion that supplements the cadherinand claudin-mediated adhesions we discussed earlier.

1	Figure 19–23 Gap junctions as seen in the electron microscope. (a) thin-section and (B) freeze-fracture electron micrographs of a large and a small gap-junction plaque between fibroblasts in culture. In (B), each gap junction is seen as a cluster of homogeneous intramembrane particles. Each intramembrane particle corresponds to a connexon (see Figure 19–25). (From N.B. Gilula, in Cell Communication [r.p. Cox, ed.], pp. 1–29. New York: Wiley, 1974.) MW1001000500020,000

1	MW1001000500020,000 Figure 19–24 Determining the size of a gap-junction channel. When fluorescent molecules of various sizes are injected into one of two cells coupled by gap junctions, molecules with a molecular weight (MW) of less than about 1000 daltons can pass into the other cell, but larger molecules cannot. thus, the coupled cells share their small molecules (such as inorganic ions, sugars, amino acids, nucleotides, vitamins, and the intracellular signaling molecules cyclic aMp and inositol trisphosphate) but not their macromolecules (proteins, nucleic acids, and polysaccharides). 1.5 nm in diameter (C) 1.4 nm

1	1.5 nm in diameter (C) 1.4 nm Gap junctions in different tissues can have different properties because they are formed from different combinations of connexins, creating channels that differ in permeability and regulation. Most cell types express more than one type of connexin, and two different connexin proteins can assemble into a heteromeric connexon, with its own distinct properties. Moreover, adjacent cells expressing different connexins can form intercellular channels in which the two aligned half-channels are different (see Figure 19–25B).

1	Like conventional ion channels (discussed in Chapter 11), individual gap-junction channels do not remain open all the time; instead, they flip between open and closed states. These changes are triggered by a variety of stimuli, including the voltage difference between the two connected cells, the membrane potential of each cell, and various chemical properties of the cytoplasm, including the pH and concentration of free Ca2+. Some subtypes of gap junctions can also be regulated by extracellular signals such as neurotransmitters. We are only just beginning to understand the physiological functions and structural basis of these various gating mechanisms.

1	Each gap-junctional plaque is a dynamic structure that can readily assemble, disassemble, or be remodeled, and it can contain a cluster of a few to many thousands of connexons (see Figure 19–23B). Studies with fluorescently labeled connexins in living cells show that new connexons are continually added around the periphery of an existing junctional plaque, while old connexons are removed from the middle of it and destroyed (Figure 19–26). This turnover is rapid: the connexin molecules have a half-life of only a few hours.

1	The mechanism of removal of old connexons from the middle of the plaque is not known, but the route of delivery of new connexons to its periphery seems clear: they are inserted into the plasma membrane by exocytosis, like other integral membrane proteins, and then diffuse in the plane of the membrane until they bump into the periphery of a connexon plaque and become trapped. This has a corollary: the plasma membrane away from the gap junction should contain connexons—hemichannels—that have not yet paired with their counterparts on another cell. It is thought that these unpaired hemichannels are normally held in a closed conformation, preventing the cell from losing its small molecules by

1	Figure 19–25 Gap junctions. (a) a drawing of the interacting plasma membranes of two adjacent cells connected by gap junctions. Each lipid bilayer is shown as a pair of red sheets. protein assemblies called connexons (green), each of which is formed by six connexin subunits, penetrate the apposed lipid bilayers. two connexons join across the intercellular gap to form a continuous aqueous channel connecting the two cells. (B) the organization of connexins into connexons, and connexons into intercellular channels. the connexons can be homomeric or heteromeric, and the intercellular channels can be homotypic or heterotypic. (C) the high-resolution structure of a homomeric gap-junction channel, determined by x-ray crystallography of human connexin 26. In this view, we are looking down on the pore, formed from six connexin subunits. the structure illustrates the general features of the channel and suggests a pore size of about 1.4 nm, as predicted from studies of gap-junction permeability

1	formed from six connexin subunits. the structure illustrates the general features of the channel and suggests a pore size of about 1.4 nm, as predicted from studies of gap-junction permeability with molecules of various sizes (see Figure 19–24). (pDB code: 2ZW3.)

1	Figure 19–26 Connexin turnover at a gap junction. Cells were transfected with a slightly modified connexin gene, coding for a connexin with a short amino acid tag containing four cysteines in the sequence Cys-Cys-X-X-Cys-Cys (where X denotes an arbitrary amino acid). this tetracysteine tag can bind strongly to certain small fluorescent dye molecules, which can be added to the culture medium and will readily enter cells by diffusing across the plasma membrane. In the experiment shown, a green dye was added first to label all the connexin molecules in the cells, and the cells were then washed and incubated for 4 or 8 hours. at the end of this time, a red dye was added to the medium and the cells were washed again and fixed. Connexin molecules already present at the beginning of the experiment are labeled green (and take up no red dye because their tetracysteine tags are already saturated with green dye), while connexins synthesized subsequently, during the 4or 8-hour incubation, are

1	are labeled green (and take up no red dye because their tetracysteine tags are already saturated with green dye), while connexins synthesized subsequently, during the 4or 8-hour incubation, are labeled red. the fluorescence images show gap junctions between pairs of cells treated in this way. the central part of the gap-junction plaque is green, indicating that it consists of old connexin molecules, while the periphery is red, indicating that it consists of connexins synthesized during the previous 4 or 8 hours. the longer the time of incubation, the smaller the green central patch of old molecules, and the larger the peripheral ring of new molecules that have been recruited to replace the old ones. (From G. Gaietta et al., Science 296:503–507, 2002. With permission from aaaS.) leakage through them. But there is also evidence that in some circumstances they can open and serve as channels for the release of small signal molecules.

1	In plants, plasmodesmata perform Many of the Same Functions as Gap Junctions The tissues of a plant are organized on different principles from those of an animal. This is because plant cells are imprisoned within tough cell walls composed of an extracellular matrix rich in cellulose and other polysaccharides, as we discuss later. The cell walls of adjacent cells are firmly cemented to those of their neighbors, which eliminates the need for anchoring junctions to hold the cells in place. But a need for direct cell–cell communication remains. Thus, plant cells have only one class of intercellular junctions, plasmodesmata. Like gap junctions, they directly connect the cytoplasms of adjacent cells.

1	In plants, the cell wall between a typical pair of adjacent cells is at least 0.1 μm thick, and so a structure very different from a gap junction is required to mediate communication across it. Plasmodesmata solve the problem. With a few specialized exceptions, every living cell in a higher plant is connected to its living neighbors by these structures, which form fine cytoplasmic channels through the intervening cell walls. As shown in Figure 19–27A, the plasma membrane of one cell is continuous with that of its neighbor at each plasmodesma, which connects the cytoplasms of the two cells by a roughly cylindrical channel with a diameter of 20–40 nm.

1	Running through the center of the channel in most plasmodesmata is a narrower cylindrical structure, the desmotubule, which is continuous with elements of the smooth endoplasmic reticulum (ER) in each of the connected cells (Figure 19–27B–D). Between the outside of the desmotubule and the inner face of the cylindrical channel formed by plasma membrane is an annulus of cytosol through which small molecules can pass from cell to cell. As each new cell wall is assembled during the cytokinesis phase of cell division, plasmodesmata are created within it. They form around elements of smooth ER that become trapped across the developing cell plate (discussed in Chapter 17). They can also be inserted de novo through preexisting cell walls, where they are commonly found in dense clusters called pit fields. When no longer required, plasmodesmata can be removed.

1	In spite of the radical difference in structure between plasmodesmata and gap junctions, they seem to function in remarkably similar ways. Evidence obtained by injecting tracer molecules of different sizes suggests that plasmodesmata allow the passage of molecules with a molecular weight of less than about 800, which cytoplasm plasmodesmataplasma membrane lining plasmodesma, connecting two adjacent cells smooth endoplasmic reticulum desmotubule 100 nm cell walls of adjacent plant cells Figure 19–27 Plasmodesmata. (a) the cytoplasmic channels of plasmodesmata pierce the plant cell wall and connect cells in a plant together. (B) Each plasmodesma is lined with plasma membrane that is common to the two connected cells. It usually also contains a fine tubular structure, the desmotubule, derived from smooth endoplasmic reticulum.

1	(C) Electron micrograph of a longitudinal section of a plasmodesma from a water fern. the plasma membrane lines the pore and is continuous from one cell to the next. Endoplasmic reticulum and its association with the central desmotubule can also be seen. (D) a similar plasmodesma seen in cross section. (C and D, from r. Overall, J. Wolfe and B.E.S. Gunning, in protoplasma 111, pp. 134–150. heidelberg: Springer-Verlag, 1982.) is similar to the molecular-weight cutoff for gap junctions. As with gap junctions, transport through plasmodesmata is regulated. Dye-injection experiments, for example, show that there can be barriers to the movement of even low-molecularweight molecules between certain cells, or groups of cells, that are connected by apparently normal plasmodesmata; the mechanisms that restrict communication in these cases are not understood. Selectins Mediate transient Cell–Cell adhesions in the Bloodstream

1	Selectins Mediate transient Cell–Cell adhesions in the Bloodstream We now complete our overview of cell–cell junctions and adhesion by briefly describing some of the more specialized adhesion mechanisms used in some tissues. In addition to those we have already discussed, at least three other super-families of cell–cell adhesion proteins are important: the integrins, the selectins, and the adhesive immunoglobulin (Ig) superfamily members. We shall discuss integrins in more detail later: their main function is in cell–matrix adhesion, but a few of them mediate cell–cell adhesion in specialized circumstances. Ca2+ dependence provides one simple way to distinguish among these classes of adhesion proteins experimentally. Selectins, like cadherins and integrins, require Ca2+ for their adhesive function; Ig superfamily members do not.

1	Selectins are cell-surface carbohydrate-binding proteins (lectins) that mediate a variety of transient cell–cell adhesion interactions in the bloodstream. Their main role, in vertebrates at least, is in governing the traffic of white blood cells into normal lymphoid organs and any inflamed tissues. White blood cells lead a nomadic life, roving between the bloodstream and the tissues, and this necessitates special adhesive behavior. The selectins control the binding of white blood cells to the endothelial cells lining blood vessels, thereby enabling the blood cells to migrate out of the bloodstream into a tissue.

1	Each selectin is a transmembrane protein with a conserved lectin domain that binds to a specific oligosaccharide on another cell (Figure 19–28A). There are at least three types: L-selectin on white blood cells, P-selectin on blood platelets and on endothelial cells that have been locally activated by an inflammatory response, and E-selectin on activated endothelial cells. In a lymphoid organ, such as a lymph (C) 0.1 µm node or the spleen, the endothelial cells express oligosaccharides that are recognized by L-selectin on lymphocytes, causing the lymphocytes to loiter and become trapped. At sites of inflammation, the roles are reversed: the endothelial cells switch on expression of selectins that recognize the oligosaccharides on white blood cells and platelets, flagging the cells down to help deal with the local emergency. Selectins do not act alone, however; they collaborate with integrins, which strengthen the binding of the blood cells to the endothelium. The cell–cell adhesions

1	help deal with the local emergency. Selectins do not act alone, however; they collaborate with integrins, which strengthen the binding of the blood cells to the endothelium. The cell–cell adhesions mediated by both selectins and integrins are heterophilic—that is, the binding is to a molecule of a different type: selectins bind to specific oligosaccharides on glycoproteins and glycolipids, while integrins bind to specific Ig-family proteins.

1	Selectins and integrins act in sequence to let white blood cells leave the bloodstream and enter tissues (Figure 19–28B). The selectins mediate a weak adhesion because the binding of the lectin domain of the selectin to its carbohydrate ligand is of low affinity. This allows the white blood cell to adhere weakly and reversibly to the endothelium, rolling along the surface of the blood vessel, propelled by the flow of blood. The rolling continues until the blood cell activates its integrins. As we discuss later, these transmembrane molecules can be switched into an adhesive conformation that enables them to latch onto specific macromolecules external to the cell—in the present case, proteins on the surfaces of the endothelial cells. Once it has attached in this way, the white blood cell escapes from the bloodstream into the tissue by crawling out of the blood vessel between adjacent endothelial cells.

1	Members of the Immunoglobulin Superfamily Mediate Ca2+-Independent Cell–Cell adhesion The chief endothelial cell proteins that are recognized by the white blood cell integrins are called ICAMs (intercellular cell adhesion molecules) or VCAMs (vascular cell adhesion molecules). They are members of another large and ancient family of cell-surface molecules—the immunoglobulin (Ig) superfamily. These contain one or more extracellular Ig-like domains that are characteristic of antibody molecules. They have many functions outside the immune system that are unrelated to immune defenses.

1	While ICAMs and VCAMs on endothelial cells both mediate heterophilic binding to integrins, many other Ig superfamily members appear to mediate homophilic binding. An example is the neural cell adhesion molecule (NCAM), which is expressed by various cell types, including most nerve cells, and can take different forms, generated by alternative splicing of an RNA transcript produced from a single gene (Figure 19–29). Some forms of NCAM carry an unusually large

1	Figure 19–28 The structure and function of selectins. (a) the structure of p-selectin. the selectin attaches to the actin cytoskeleton through adaptor proteins that are still poorly characterized. (B) how selectins and integrins mediate the cell–cell adhesions required for a white blood cell to migrate out of the bloodstream into a tissue. First, selectins on endothelial cells bind to oligosaccharides on the white blood cell, so that it becomes loosely attached and rolls along the vessel wall. then the white blood cell activates a cell-surface integrin called LFa1, which binds to a protein called ICaM1 (belonging to the Ig superfamily) on the membrane of the endothelial cell. the white blood cell adheres to the vessel wall and then crawls out of the vessel by a process that requires another immunoglobulin superfamily member called pECaM1 (or CD31), not shown (Movie 19.2). EGF, epidermal growth factor.

1	quantity of sialic acid (with chains containing hundreds of repeating sialic acid units). By virtue of their negative charge, the long polysialic acid chains can interfere with cell adhesion (because like charges repel one another); thus, these forms of NCAM can serve to inhibit adhesion, rather than cause it.

1	A cell of a given type generally uses an assortment of different adhesion proteins to interact with other cells, just as each cell uses an assortment of different receptors to respond to the many soluble extracellular signal molecules in its environment. Although cadherins and Ig superfamily members are frequently expressed on the same cells, the adhesions mediated by cadherins are much stronger, and they are largely responsible for holding cells together, segregating cell collectives into discrete tissues, and maintaining tissue integrity. Molecules such as NCAM seem to contribute more to the fine-tuning of these adhesive interactions during development and regeneration, playing a part in various specialized adhesive phenomena, such as that discussed for blood cells and endothelial cells. Thus, while mutant mice that lack N-cadherin die early in development, those that lack NCAM develop relatively normally but show some mild abnormalities in the development of certain specific

1	cells. Thus, while mutant mice that lack N-cadherin die early in development, those that lack NCAM develop relatively normally but show some mild abnormalities in the development of certain specific tissues, including parts of the nervous system.

1	In epithelia, as well as in some other types of tissue, cells are directly attached to one another through strong cell–cell adhesions, mediated by transmembrane proteins called cadherins, which are anchored intracellularly to the cytoskeleton. Cadherins generally bind to one another homophilically: the head of one cadherin molecule binds to the head of a similar cadherin on an opposite cell. This selectivity enables mixed populations of cells of different types to sort out from one another according to the specific cadherins they express, and it helps to control cell rearrangements during development. The “classical” cadherins at adherens junctions are linked to the actin cytoskeleton by intracellular adaptor proteins called catenins. These form an anchoring complex on the intracellular tail of the cadherin molecule, and are involved not only in physical anchorage but also in the detection of and response to tension and other regulatory signals at the junction.

1	Tight junctions seal the gaps between cells in epithelia, creating a barrier to the diffusion of molecules across the cell sheet and also helping to separate the populations of proteins in the apical and basolateral plasma membrane domains of the epithelial cell. Claudins are the major transmembrane proteins forming gap junctions. Intracellular scaffold proteins organize the claudins and other junctional proteins into a complex protein network that is linked to the actin cytoskeleton. Figure 19–29 Two members of the Ig superfamily of cell–cell adhesion molecules. NCaM is expressed on neurons and many other cell types, and mediates homophilic binding. ICaM is expressed on endothelial cells and some other cell types and binds heterophilically to an integrin on white blood cells. Both NCaM and ICaM are glycoproteins, but their attached carbohydrate chains are not shown.

1	The cells of many animal tissues are coupled by gap junctions, which take the form of plaques of clustered connexons, which usually allow molecules smaller than about 1000 daltons to pass directly from the inside of one cell to the inside of the next. Cells connected by gap junctions share many of their inorganic ions and other small molecules and are therefore chemically and electrically coupled.

1	Three additional classes of transmembrane adhesion proteins mediate more transient cell–cell adhesion: selectins, immunoglobulin (Ig) superfamily members, and integrins. Selectins are expressed on white blood cells, blood platelets, and endothelial cells; they bind heterophilically to carbohydrate groups on cell surfaces, helping to mediate the adhesive interactions between these cells. Ig superfamily proteins also play a part in these interactions, as well as in many other adhesive processes; some of them bind homophilically, some heterophilically. Integrins, though they mainly serve to attach cells to the extracellular matrix, can also mediate cell– cell adhesion by binding to specific Ig superfamily proteins.

1	Tissues are not made up solely of cells. They also contain a remarkably complex and intricate network of macromolecules constituting the extracellular matrix. This matrix is composed of many different proteins and polysaccharides that are secreted locally and assembled into an organized meshwork in close association with the surfaces of the cells that produce them.

1	The classes of macromolecules constituting the extracellular matrix in different animal tissues are broadly similar, but variations in the relative amounts of these different classes of molecules and in the ways in which they are organized give rise to an amazing diversity of materials. The matrix can become calcified to form the rock-hard structures of bone or teeth, or it can form the transparent substance of the cornea, or it can adopt the ropelike organization that gives tendons their enormous tensile strength. It forms the jelly in a jellyfish. Covering the body of a beetle or a lobster, it forms a rigid carapace. Moreover, the extracellular matrix is more than a passive scaffold to provide physical support. It has an active and complex role in regulating the behavior of the cells that touch it, inhabit it, or crawl through its meshes, influencing their survival, development, migration, proliferation, shape, and function.

1	In this section, we describe the major features of the extracellular matrix in animal tissues, with an emphasis on vertebrates. We begin with an overview of the major classes of macromolecules in the matrix, after which we turn to the structure and function of the basal lamina, the thin layer of specialized extracellular matrix that lies beneath all epithelial cells. In the sections that follow, we then describe the varied types of cell–matrix junctions through which cells are connected to the matrix. the Extracellular Matrix Is Made and Oriented by the Cells Within It

1	the Extracellular Matrix Is Made and Oriented by the Cells Within It The macromolecules that constitute the extracellular matrix are mainly produced locally by cells in the matrix. As we discuss later, these cells also help to organize the matrix: the orientation of the cytoskeleton inside the cell can control the orientation of the matrix produced outside. In most connective tissues, the matrix macromolecules are secreted by cells called fibroblasts (Figure 19–30). In certain specialized types of connective tissues, such as cartilage and bone, however, they are secreted by cells of the fibroblast family that have more specific names: chondroblasts, for example, form cartilage, and osteoblasts form bone.

1	The extracellular matrix is constructed from three major classes of macromolecules: (1) glycosaminoglycans (GAGs), which are large and highly charged polysaccharides that are usually covalently linked to protein in the form of proteoglycans; (2) fibrous proteins, which are primarily members of the collagen family; and (3) a large class of noncollagen glycoproteins, which carry conventional asparagine-linked oligosaccharides (described in Chapter 12). All three classes of macromolecule have many members and come in a great variety of shapes and

1	Figure 19–30 Fibroblasts in connective tissue. this scanning electron micrograph shows tissue from the cornea of a rat. the extracellular matrix surrounding the fibroblasts is here composed largely of collagen fibrils. the glycoproteins, hyaluronan, and proteoglycans, which normally form a hydrated gel filling the interstices of the fibrous network, have been removed by enzyme and acid treatment. (Courtesy of t. Nishida.) sizes (Figure 19–31). Mammals are thought to have almost 300 matrix proteins, including about 36 proteoglycans, about 40 collagens, and over 200 glycoproteins, which usually contain multiple subdomains and self-associate to form multimers. Add to this the large number of matrix-associated proteins and enzymes that can modify matrix behavior by cross-linking, degradation, or other mechanisms, and one begins to see that the matrix is an almost infinitely variable material. Each tissue contains its own unique blend of matrix components, resulting in an extracellular

1	or other mechanisms, and one begins to see that the matrix is an almost infinitely variable material. Each tissue contains its own unique blend of matrix components, resulting in an extracellular matrix that is specialized for the needs of that tissue.

1	The proteoglycan molecules in connective tissue typically form a highly hydrated, gel-like “ground substance” in which collagens and glycoproteins are embedded. The polysaccharide gel resists compressive forces on the matrix while permitting the rapid diffusion of nutrients, metabolites, and hormones between the blood and the tissue cells. The collagen fibers strengthen and help organize the matrix, while other fibrous proteins, such as the rubberlike elastin, give it resilience. Finally, the many matrix glycoproteins help cells migrate, settle, and differentiate in the appropriate locations. Glycosaminoglycan (GaG) Chains Occupy Large amounts of Space and Form hydrated Gels

1	Glycosaminoglycan (GaG) Chains Occupy Large amounts of Space and Form hydrated Gels Glycosaminoglycans (GAGs) are unbranched polysaccharide chains composed of repeating disaccharide units. One of the two sugars in the repeating disaccharide is always an amino sugar (N-acetylglucosamine or N-acetylgalactosamine), which in most cases is sulfated. The second sugar is usually a uronic acid (glucuronic or iduronic). Because there are sulfate or carboxyl groups on most of their sugars, GAGs are highly negatively charged (Figure 19–32). Indeed, they are the most anionic molecules produced by animal cells. Four main groups of GAGs are distinguished by their sugars, the type of linkage between the sugars, and the number and location of sulfate groups: (1) hyaluronan, (2) chondroitin sulfate and dermatan sulfate, (3) heparan sulfate, and (4) keratan sulfate.

1	Polysaccharide chains are too stiff to fold into compact globular structures, and they are strongly hydrophilic. Thus, GAGs tend to adopt highly extended conformations that occupy a huge volume relative to their mass (Figure 19–33), and they form hydrated gels even at very low concentrations. The weight of GAGs in connective tissue is usually less than 10% of the weight of proteins, but GAG chains fill most of the extracellular space. Their high density of negative charges attracts a cloud of cations, especially Na+, that are osmotically active, causing large amounts of water to be sucked into the matrix. This creates a swelling pressure, or turgor, that enables the matrix to withstand compressive forces (in contrast to collagen fibrils, which resist stretching forces). The cartilage matrix that lines the knee joint, for example, can support pressures of hundreds of atmospheres in this way.

1	Defects in the production of GAGs can affect many different body systems. In one rare human genetic disease, for example, there is a severe deficiency in the synthesis of dermatan sulfate disaccharide. The affected individuals have a short stature, a prematurely aged appearance, and generalized defects in their skin, joints, muscles, and bones. Figure 19–31 The comparative shapes and sizes of some of the major extracellular matrix macromolecules. protein is shown in green, and glycosaminoglycan (GaG) in red.

1	Figure 19–31 The comparative shapes and sizes of some of the major extracellular matrix macromolecules. protein is shown in green, and glycosaminoglycan (GaG) in red. Figure 19–32 The repeating disaccharide sequence of a heparan sulfate glycosaminoglycan (GAG) chain. these chains can consist of as many as 200 disaccharide units, but are typically less than half that size. there is a high density of negative charges along the chain due to the presence of both carboxyl and sulfate groups. the molecule is shown here with its maximal number of sulfate groups. In vivo, the proportion of sulfated and nonsulfated groups is variable. heparin typically has >70% sulfation, while heparan sulfate has <50%. hyaluronan acts as a Space Filler During tissue Morphogenesis and repair

1	hyaluronan acts as a Space Filler During tissue Morphogenesis and repair Hyaluronan (also called hyaluronic acid or hyaluronate) is the simplest of the GAGs (Figure 19–34). It consists of a regular repeating sequence of up to 25,000 disaccharide units, is found in variable amounts in all tissues and fluids in adult animals, and is especially abundant in early embryos. Hyaluronan is not a typical GAG because it contains no sulfated sugars, all its disaccharide units are identical, its chain length is enormous, and it is not generally linked covalently to any core protein. Moreover, whereas other GAGs are synthesized inside the cell and released by exocytosis, hyaluronan is spun out directly from the cell surface by an enzyme complex embedded in the plasma membrane.

1	Hyaluronan is thought to have a role in resisting compressive forces in tissues and joints. It is also important as a space filler during embryonic development, where it can be used to force a change in the shape of a structure, as a small quantity expands with water to occupy a large volume. Hyaluronan synthesized locally from the basal side of an epithelium can deform the epithelium by creating a cell-free space beneath it, into which cells subsequently migrate. In the developing heart, for example, hyaluronan synthesis helps in this way to drive formation of the valves and septa that separate the heart’s chambers. Similar processes occur in several other organs. When cell migration ends, the excess hyaluronan is generally degraded by the enzyme hyaluronidase. Hyaluronan is also produced in large quantities during wound healing, and it is an important constituent of joint fluid, in which it serves as a lubricant.

1	proteoglycans are Composed of GaG Chains Covalently Linked to a Core protein

1	Except for hyaluronan, all GAGs are covalently attached to protein as proteoglycans, which are produced by most animal cells. Membrane-bound ribosomes make the polypeptide chain, or core protein, of a proteoglycan, which is then threaded into the lumen of the endoplasmic reticulum. The polysaccharide chains are mainly assembled on this core protein in the Golgi apparatus before delivery to the exterior of the cell by exocytosis. First, a special linkage tetrasaccharide is attached to a serine side chain on the core protein to serve as a primer for polysaccharide growth; then, one sugar at a time is added by specific glycosyl transferases (Figure 19–35). While still in the Golgi apparatus, many of the polymerized sugars are covalently modified by a sequential and coordinated series of reactions. Epimerizations alter the configuration of the substituents around individual carbon atoms in the sugar molecule; sulfations increase the negative charge.

1	Proteoglycans are clearly distinguished from other glycoproteins by the nature, quantity, and arrangement of their sugar side chains. By definition, at least one of the sugar side chains of a proteoglycan must be a GAG. Whereas glycoproteins generally contain relatively short, branched oligosaccharide chains that contribute only a small fraction of their weight, proteoglycans can contain as much as globular protein (MW 50,000) glycogen (MW ~400,000) spectrin (MW 460,000) collagen (MW 290,000) Figure 19–33 The relative dimensions and volumes occupied by various macromolecules. Several proteins, a glycogen granule, and a single hydrated molecule of hyaluronan are shown. Figure 19–34 The repeating disaccharide sequence in hyaluronan, a relatively simple GAG. this ubiquitous molecule in vertebrates consists of a single long chain of up to 25,000 sugar monomers. Note the absence of sulfate groups.

1	95% carbohydrate by weight, mostly in the form of long, unbranched GAG chains, each typically about 80 sugars long. In principle, proteoglycans have the potential for almost limitless heterogeneity. Even a single type of core protein can carry highly variable numbers and types of attached GAG chains. Moreover, the underlying repeating sequence of disaccharides in each GAG can be modified by a complex pattern of sulfate groups. The core proteins, too, are diverse, though many of them share some characteristic domains such as the LINK domain, involved in binding to GAGs.

1	Proteoglycans can be huge. The proteoglycan aggrecan, for example, which is a major component of cartilage, has a mass of about 3 × 106 daltons with over 100 GAG chains. Other proteoglycans are much smaller and have only 1–10 GAG chains; an example is decorin, which is secreted by fibroblasts and has a single GAG chain (Figure 19–36). Decorin binds to collagen fibrils and regulates fibril assembly and fibril diameter; mice that cannot make decorin have fragile skin that has reduced tensile strength. The GAGs and proteoglycans of these various types can associate to form even larger polymeric complexes in the extracellular matrix. Molecules of aggrecan, for example, assemble with hyaluronan in cartilage matrix to form aggregates that are as big as a bacterium (Figure 19–37). Moreover, besides associating with one another, GAGs and proteoglycans associate with fibrous matrix proteins such as collagen and with protein meshworks such as the basal lamina, creating extremely complex

1	besides associating with one another, GAGs and proteoglycans associate with fibrous matrix proteins such as collagen and with protein meshworks such as the basal lamina, creating extremely complex composites (Figure 19–38).

1	Not all proteoglycans are secreted components of the extracellular matrix. Some are integral components of plasma membranes and have their core protein either inserted across the lipid bilayer or attached to the lipid bilayer by a glycosylphosphatidylinositol (GPI) anchor. Among the best-characterized plasma membrane proteoglycans are the syndecans, which have a membrane-spanning core protein whose intracellular domain is thought to interact with the actin cytoskeleton and with signaling molecules in the cell cortex. Syndecans are located on the surface of many types of cells, including fibroblasts and epithelial cells. In DECORIN AGGRECAN (MW ~40,000) (MW ~3 x 106)

1	DECORIN AGGRECAN (MW ~40,000) (MW ~3 x 106) Figure 19–35 The linkage between a GAG chain and its core protein in a proteoglycan molecule. a specific link tetrasaccharide is first assembled on a serine side chain. the rest of the GaG chain, consisting mainly of a repeating disaccharide unit, is then synthesized, with one sugar being added at a time. In chondroitin sulfate, the disaccharide is composed of D-glucuronic acid and N-acetyl-D-galactosamine; in heparan sulfate, it is either D-glucuronic acid or L-iduronic acid and N-acetyl-Dglucosamine; in keratan sulfate, it is D-galactose and N-acetyl-D-glucosamine.

1	Figure 19–36 examples of a small (decorin) and a large (aggrecan) proteoglycan found in the extracellular matrix. the figure compares these two proteoglycans with a typical secreted glycoprotein molecule, pancreatic ribonuclease B. all three are drawn to scale. the core proteins of both aggrecan and decorin contain oligosaccharide chains as well as the GaG chains, but these are not shown. aggrecan typically consists of about 100 chondroitin sulfate chains and about 30 keratan sulfate chains linked to a serine-rich core protein of almost 3000 amino acids. Decorin “decorates” the surface of collagen fibrils, hence its name.

1	RIBONUCLEASE (MW ~15,000) short, branched oligosaccharide side chain fibroblasts, syndecans can be found in cell–matrix adhesions, where they modulate integrin function by interacting with fibronectin on the cell surface and with cytoskeletal and signaling proteins inside the cell. As we discuss later, syndecan and other proteoglycans also interact with soluble peptide growth factors, influencing their effects on cell growth and proliferation. Collagens are the Major proteins of the Extracellular Matrix The collagens are a family of fibrous proteins found in all multicellular animals. They are secreted in large quantities by connective-tissue cells, and in smaller quantities by many other cell types. As a major component of skin and bone, collagens are the most abundant proteins in mammals, where they constitute 25% of the total protein mass.

1	The primary feature of a typical collagen molecule is its long, stiff, triple-stranded helical structure, in which three collagen polypeptide chains, called α chains, are wound around one another in a ropelike superhelix (Figure 19–39). 0.5 µm aggrecan aggregate Figure 19–37 An aggrecan aggregate from fetal bovine cartilage. (a) an electron micrograph of an aggrecan aggregate shadowed with platinum. Many free aggrecan molecules are also visible.

1	Figure 19–37 An aggrecan aggregate from fetal bovine cartilage. (a) an electron micrograph of an aggrecan aggregate shadowed with platinum. Many free aggrecan molecules are also visible. (B) a drawing of the giant aggrecan aggregate shown in (a). It consists of about 100 aggrecan monomers (each like the one shown in Figure 19–36) noncovalently bound through the N-terminal domain of the core protein to a single hyaluronan chain. a link protein binds both to the core protein of the proteoglycan and to the hyaluronan chain, thereby stabilizing the aggregate. the link proteins are members of a family of hyaluronan-binding proteins, some of which are cell-surface proteins. the molecular mass of such a complex can be 108 daltons or more, and it occupies a volume equivalent to that of a bacterium, which is about 2 × 10–12 cm3. (a, courtesy of Lawrence rosenberg.) Figure 19–38 Proteoglycans in the extracellular matrix of rat cartilage.

1	Figure 19–38 Proteoglycans in the extracellular matrix of rat cartilage. the tissue was rapidly frozen at –196°C, and fixed and stained while still frozen (a process called freeze substitution) to prevent the GaG chains from collapsing. In this electron micrograph, the proteoglycan molecules are seen to form a fine filamentous network in which a single striated collagen fibril is embedded. the more darkly stained parts of the proteoglycan molecules are the core proteins; the faintly stained threads are the GaG chains. (reproduced from E.B. hunziker and r.K. Schenk, J. Cell Biol. 98:277–282, 1984. With permission from the rockefeller University press.)

1	Figure 19–39 The structure of a typical collagen molecule. (a) a model of part of a single collagen α chain, in which each amino acid is represented by a sphere. the chain is about 1000 amino acids long. It is arranged as a left-handed helix, with three amino acids per turn and with glycine as every third amino acid. therefore, an α chain is composed of a series of triplet Gly-X-Y sequences, in which X and Y can be any amino acid (although X is commonly proline and Y is commonly hydroxyproline, a form of proline that is chemically modified during collagen synthesis in the cell). (B) a model of part of a collagen molecule, in which three α chains, each shown in a different color, are wrapped around one another to form a triple-stranded helical rod. Glycine is the only amino acid small enough to occupy the crowded interior of the triple helix. Only a short length of the molecule is shown; the entire molecule is 300 nm long. (From a model by B.L. trus.)

1	Collagens are extremely rich in proline and glycine, both of which are important in the formation of the triple-stranded helix.

1	The human genome contains 42 distinct genes coding for different collagen α chains. Different combinations of these genes are expressed in different tissues. Although in principle thousands of types of triple-stranded collagen molecules could be assembled from various combinations of the 42 αchains, only a limited number of triple-helical combinations are possible, and roughly 40 types of collagen molecules have been found. Type I is by far the most common, being the principal collagen of skin and bone. It belongs to the class of fibrillar collagens, or fibril-forming collagens: after being secreted into the extracellular space, they assemble into higher-order polymers called collagen fibrils, which are thin structures (10–300 nm in diameter) many hundreds of micrometers long in mature tissues, where they are clearly visible in electron micrographs (Figure 19–40; see also Figure 19–38). Collagen fibrils often aggregate into larger, cablelike bundles, several micrometers in diameter,

1	where they are clearly visible in electron micrographs (Figure 19–40; see also Figure 19–38). Collagen fibrils often aggregate into larger, cablelike bundles, several micrometers in diameter, that are visible in the light microscope as collagen fibers.

1	Collagen types IX and XII are called fibril-associated collagens because they decorate the surface of collagen fibrils. They are thought to link these fibrils to one another and to other components in the extracellular matrix. Type IV is a network-forming collagen, forming a major part of basal laminae, while type VII molecules form dimers that assemble into specialized structures called anchoring fibrils. Anchoring fibrils help attach the basal lamina of multilayered epithelia to the underlying connective tissue and therefore are especially abundant in the skin. There are also a number of “collagen-like” proteins containing short collagen-like segments. These include collagen type XVII, which has a transmembrane domain and is found in hemidesmosomes, and type XVIII, the core protein of a proteoglycan in basal laminae.

1	Many proteins appear to have evolved by repeated duplications of an original DNA sequence, giving rise to a repetitive pattern of amino acids. The genes that encode the αchains of most of the fibrillar collagens provide a good example: they are very large (up to 44 kilobases in length) and contain about 50 exons. Most of 1.5 nm Figure 19–40 A fibroblast surrounded by collagen fibrils in the connective tissue of embryonic chick skin. In this electron micrograph, the fibrils are organized into bundles that run approximately at right angles to one another. therefore, some bundles are oriented longitudinally, whereas others are seen in cross section. the collagen fibrils are produced by fibroblasts. (From C. ploetz, E.I. Zycband and

1	D.E. Birk, J. Struct. Biol. 106:73–81, 1991. With permission from Elsevier.) the exons are 54, or multiples of 54, nucleotides long, suggesting that these collagens originated through multiple duplications of a primordial gene containing 54 nucleotides and encoding exactly six Gly-X-Y repeats (see Figure 19–39). Table 19–2 provides additional details for some of the collagen types discussed in this chapter. Secreted Fibril-associated Collagens help Organize the Fibrils

1	Table 19–2 provides additional details for some of the collagen types discussed in this chapter. Secreted Fibril-associated Collagens help Organize the Fibrils In contrast to GAGs, which resist compressive forces, collagen fibrils form structures that resist tensile forces. The fibrils have various diameters and are organized in different ways in different tissues. In mammalian skin, for example, they are woven in a wickerwork pattern so that they resist tensile stress in multiple directions; leather consists of this material, suitably preserved. In tendons, collagen fibrils are organized in parallel bundles aligned along the major axis of tension. In mature bone and in the cornea, they are arranged in orderly plywoodlike layers, with the fibrils in each layer lying parallel to one another but nearly at right angles to the fibrils in the layers on either side. The same arrangement occurs in tadpole skin (Figure 19–41).

1	The connective-tissue cells themselves determine the size and arrangement of the collagen fibrils. The cells can express one or more genes for the different types of fibrillar collagen molecules. But even fibrils composed of the same mixture of collagens have different arrangements in different tissues. How is this achieved? Part of the answer is that cells can regulate the disposition of the collagen

1	Figure 19–41 Collagen fibrils in the tadpole skin. this electron micrograph shows the plywoodlike arrangement of the fibrils: successive layers of fibrils are laid down nearly at right angles to each other. this organization is also found in mature bone and in the cornea. (Courtesy of Jerome Gross.) 5 µm (A) type IX collagen molecule fbril of type II collagen (B) (C) 100 nm Figure 19–42 Type IX collagen. (a) type IX collagen molecules binding in a periodic pattern to the surface of a fibril containing type II collagen. (B) Electron micrograph of a rotary-shadowed type-II-collagen-containing fibril in cartilage, decorated by type IX collagen molecules. (C) an individual type IX collagen molecule. (B and C, from L. Vaughan et al., J. Cell Biol. 106:991–997, 1988. With permission from the rockefeller University press.) molecules after secretion by guiding collagen fibril formation near the plasma membrane. In addition, cells can influence this organization by secreting, along with

1	the rockefeller University press.) molecules after secretion by guiding collagen fibril formation near the plasma membrane. In addition, cells can influence this organization by secreting, along with their fibrillar collagens, different kinds and amounts of other matrix macromolecules. In particular, they secrete the fibrous protein fibronectin, as we discuss later, and this precedes the formation of collagen fibrils and helps guide their organization.

1	Fibril-associated collagens, such as types IX and XII collagens, are thought to be especially important in organizing collagen fibrils. They differ from fibrillar collagens in the following ways. First, their triple-stranded helical structure is interrupted by one or two short nonhelical domains, which makes the molecules more flexible than fibrillar collagen molecules. Second, they do not aggregate with one another to form fibrils in the extracellular space. Instead, they bind in a periodic manner to the surface of fibrils formed by the fibrillar collagens. Type IX molecules bind to type-II-collagen-containing fibrils in cartilage, the cornea, and the vitreous of the eye (Figure 19–42), whereas type XII molecules bind to type-I-collagen-containing fibrils in tendons and various other tissues.

1	Fibril-associated collagens are thought to mediate the interactions of collagen fibrils with one another and with other matrix macromolecules to help determine the organization of the fibrils in the matrix. Cells help Organize the Collagen Fibrils they Secrete by Exerting tension on the Matrix Cells interact with the extracellular matrix mechanically as well as chemically, and studies in culture suggest that the mechanical interaction can have dramatic effects on the architecture of connective tissue. Thus, when fibroblasts are mixed with a meshwork of randomly oriented collagen fibrils that form a gel in a culture dish, the fibroblasts tug on the meshwork, drawing in collagen from their surroundings and thereby causing the gel to contract to a small fraction of its initial volume. By similar activities, a cluster of fibroblasts surrounds itself with a capsule of densely packed and circumferentially oriented collagen fibers.

1	If two small pieces of embryonic tissue containing fibroblasts are placed far apart on a collagen gel, the intervening collagen becomes organized into a compact band of aligned fibers that connect the two explants (Figure 19–43). The fibroblasts subsequently migrate out from the explants along the aligned collagen fibers. Thus, the fibroblasts influence the alignment of the collagen fibers, and the collagen fibers in turn affect the distribution of the fibroblasts. Fibroblasts may have a similar role in organizing the extracellular matrix inside the body. First they synthesize the collagen fibrils and deposit them in the correct orientation. Then they work on the matrix they have secreted, crawling over it and tugging on it so as to create tendons and ligaments and the tough, dense layers of connective tissue that surround and bind together most organs.

1	Figure 19–43 The shaping of the extracellular matrix by cells. this micrograph shows a region between two pieces of embryonic chick heart (rich in fibroblasts as well as heart muscle cells) that were cultured on a collagen gel for 4 days. a dense tract of aligned collagen fibers has formed between the explants, presumably as a result of the fibroblasts in the explants tugging on the collagen. (From D. Stopak and a.K. harris, Dev. Biol. 90:383–398, 1982. With permission from academic press.)

1	Many vertebrate tissues, such as skin, blood vessels, and lungs, need to be both strong and elastic in order to function. A network of elastic fibers in the extracellular matrix of these tissues gives them the resilience to recoil after transient stretch (Figure 19–44). Elastic fibers are at least five times more extensible than a rubber band of the same cross-sectional area. Long, inelastic collagen fibrils are interwoven with the elastic fibers to limit the extent of stretching and prevent the tissue from tearing.

1	The main component of elastic fibers is elastin, a highly hydrophobic protein (about 750 amino acids long), which, like collagen, is unusually rich in proline and glycine but, unlike collagen, is not glycosylated. Soluble tropoelastin (the biosynthetic precursor of elastin) is secreted into the extracellular space and assembled into elastic fibers close to the plasma membrane, generally in cell-surface infoldings. After secretion, the tropoelastin molecules become highly cross-linked to one another, generating an extensive network of elastin fibers and sheets.

1	The elastin protein is composed largely of two types of short segments that alternate along the polypeptide chain: hydrophobic segments, which are responsible for the elastic properties of the molecule; and alanineand lysine-rich α-helical segments, which are cross-linked to adjacent molecules by covalent attachment of lysine residues. Each segment is encoded by a separate exon. There is still uncertainty concerning the conformation of elastin molecules in elastic fibers and how the structure of these fibers accounts for their rubberlike properties. However, it seems that parts of the elastin polypeptide chain, like the polymer chains in ordinary rubber, adopt a loose “random coil” conformation, and it is the random coil nature of the component molecules cross-linked into the elastic fiber network that allows the network to stretch and recoil like a rubber band (Figure 19–45).

1	Elastin is the dominant extracellular matrix protein in arteries, comprising 50% of the dry weight of the largest artery—the aorta (see Figure 19–44). Mutations in the elastin gene causing a deficiency of the protein in mice or humans result in narrowing of the aorta and other arteries and excessive proliferation of smooth muscle cells in the arterial wall. Apparently, the normal elasticity of an artery is required to restrain the proliferation of these cells.

1	Elastic fibers do not consist solely of elastin. The elastin core is covered with a sheath of microfibrils, each of which has a diameter of about 10 nm. The microfibrils appear before elastin in developing tissues and seem to provide scaffolding to guide elastin deposition. Arrays of microfibrils are elastic in their own right, and in some places they persist in the absence of elastin: they help to hold the lens in its place in the eye, for example. Microfibrils are composed of a number of distinct glycoproteins, including the large glycoprotein fibrillin, which binds to

1	Figure 19–44 elastic fibers. these scanning electron micrographs show (a) a low-power view of a segment of a dog’s aorta and (B) a high-power view of the dense network of longitudinally oriented elastic fibers in the outer layer of the same blood vessel. all the other components have been digested away with enzymes and formic acid. (From K.S. haas et al., Anat. Rec. 230:86–96, 1991. With permission from Wiley-Liss.) elastin and is essential for the integrity of elastic fibers. Mutations in the fibrillin gene result in Marfan’s syndrome, a relatively common human disorder. In the most severely affected individuals, the aorta is prone to rupture; other common effects include displacement of the lens and abnormalities of the skeleton and joints. Affected individuals are often unusually tall and lanky: Abraham Lincoln is suspected to have had the condition. Fibronectin and Other Multidomain Glycoproteins help Organize the Matrix

1	Fibronectin and Other Multidomain Glycoproteins help Organize the Matrix In addition to proteoglycans, collagens, and elastic fibers, the extracellular matrix contains a large and varied assortment of glycoproteins that typically have multiple domains, each with specific binding sites for other matrix macromolecules and for receptors on the surface of cells (Figure 19–46). These proteins therefore contribute to both organizing the matrix and helping cells attach to it. Like the proteoglycans, they also guide cell movements in developing tissues, by serving Figure 19–45 Stretching a network of elastin molecules. the molecules are joined together by covalent bonds (red) to generate a cross-linked network. In this model, each elastin molecule in the network can extend and contract in a manner resembling a random coil, so that the entire assembly can stretch and recoil like a rubber band.

1	Figure 19–46 Complex glycoproteins of the extracellular matrix. Many matrix glycoproteins are large scaffold proteins containing multiple copies of specific protein-interaction domains. Each domain is folded into a discrete globular structure, and many such domains are arrayed along the protein like beads on a string. this diagram shows four representative proteins among the roughly 200 matrix glycoproteins that are found in mammals. Each protein contains multiple repeat domains, with the names listed in the key at the bottom. Fibronectin, for example, contains numerous copies of three different fibronectin repeats (types I–III, labeled here as FN1, FN2, and FN3). two type III repeats near the C-terminus contain important binding sites for cell-surface integrins, whereas other FN repeats are involved in binding fibrin, collagen, and heparin, as indicated (see Figure 19–47). Other matrix proteins contain repeated sequences resembling those of epidermal growth factor (EGF), a major

1	are involved in binding fibrin, collagen, and heparin, as indicated (see Figure 19–47). Other matrix proteins contain repeated sequences resembling those of epidermal growth factor (EGF), a major regulator of cell growth and proliferation; these repeats might serve a similar signaling function in matrix proteins. Other proteins contain domains, such as the insulin-like growth factor-binding protein (IGFBp) repeat, that bind and regulate the function of soluble growth factors. to add more structural diversity, many of these proteins are encoded by rNa transcripts that can be spliced in different ways, adding or removing exons, such as those in fibronectin. Finally, the scaffolding and regulatory functions of many matrix proteins are further expanded by assembly into multimeric forms, as shown at the right: fibronectin forms dimers linked at the C-termini, whereas tenascin and thrombospondin form N-terminally linked hexamers and trimers, respectively. Other domains include four repeats

1	at the right: fibronectin forms dimers linked at the C-termini, whereas tenascin and thrombospondin form N-terminally linked hexamers and trimers, respectively. Other domains include four repeats from thrombospondin (tSpN, tSp1, tSp3, tSp_C). VWC, von Willebrand type C; FBG, fibrinogen-like. (adapted from r.O. hynes and a. Naba, Cold Spring Harb. Perspect. Biol. 4:a004903, 2012.) as tracks along which cells can migrate or as repellents that keep cells out of forbidden areas. They can also bind and thereby influence the function of peptide growth factors and other small molecules produced by nearby cells.

1	The best-understood member of this class of matrix proteins is fibronectin, a large glycoprotein found in all vertebrates and important for many cell– matrix interactions. Mutant mice that are unable to make fibronectin die early in embryogenesis because their endothelial cells fail to form proper blood vessels. The defect is thought to result from abnormalities in the interactions of these cells with the surrounding extracellular matrix, which normally contains fibronectin.

1	Fibronectin is a dimer composed of two very large subunits joined by disulfide bonds at their C-terminal ends. Each subunit contains a series of small repeated domains, or modules, separated by short stretches of flexible polypeptide chain (Figure 19–47). Each domain is usually encoded by a separate exon, suggesting that the fibronectin gene, like the genes encoding many matrix proteins, evolved by multiple exon duplications. In the human genome, there is only one fibronectin gene, containing about 50 exons of similar size, but the transcripts can be spliced in different ways to produce multiple fibronectin isoforms (see Figure 19–46). The major repeat domain in fibronectin is called the type III fibronectin repeat, which is about 90 amino acids long and occurs at least 15 times in each subunit. This repeat is among the most common of all protein domains in vertebrates. Fibronectin Binds to Integrins

1	Fibronectin Binds to Integrins One way to analyze a complex multifunctional protein molecule such as fibronectin is to synthesize individual regions of the protein and test their ability to bind other proteins. By these and other methods, it was possible to show that one region of fibronectin binds to collagen, another to proteoglycans, and another to specific integrins on the surface of various types of cells (see Figure 19–47B). Synthetic peptides corresponding to different segments of the integrin-binding domain were then used to show that binding depends on a specific tripeptide sequence (Arg-Gly-Asp, or RGD) that is found in one of the type III repeats (see Figure 19–47C). Even very short peptides containing this RGD sequence can compete with fibronectin for the binding site on cells, thereby inhibiting the attachment of the cells to a fibronectin matrix.

1	Several extracellular proteins besides fibronectin also have an RGD sequence that mediates cell-surface binding. Many of these proteins are components of the extracellular matrix, while others are involved in blood clotting. Peptides

1	Figure 19–47 The structure of a fibronectin dimer. (a) Electron micrographs of individual fibronectin dimer molecules shadowed with platinum; red arrows mark the joined C-termini. (B) the two polypeptide chains are similar but generally not identical (being made from the same gene but from differently spliced mrNas). they are joined by two disulfide bonds near the C-termini. Each chain is almost 2500 amino acids long and is folded into multiple domains (see Figure 19–46). as indicated, some domains are specialized for binding to a particular molecule. For simplicity, not all of the known binding sites are shown. (C) the three-dimensional structure of the ninth and tenth type III fibronectin repeats, as determined by x-ray crystallography. Both the arg-Glyasp (rGD) and the “synergy” sequences shown in red are important for binding to integrins on cell surfaces. (a, from J. Engel et al., J. Mol. Biol. 150:97–120, 1981. With permission from academic press; C, from Daniel J. Leahy, Annu.

1	shown in red are important for binding to integrins on cell surfaces. (a, from J. Engel et al., J. Mol. Biol. 150:97–120, 1981. With permission from academic press; C, from Daniel J. Leahy, Annu. Rev. Cell Dev. Biol. 13:363–393, 1997. With permission from annual reviews.) 1068 Chapter 19: Cell Junctions and the Extracellular Matrix containing the RGD sequence have been useful in the development of anti-clotting drugs. Some snakes use a similar strategy to cause their victims to bleed: they secrete RGD-containing anti-clotting proteins called disintegrins into their venom.

1	The cell-surface receptors that bind RGD-containing proteins are members of the integrin family, which we describe in detail later. Each integrin specifically recognizes its own small set of matrix molecules, indicating that tight binding requires more than just the RGD sequence. Moreover, RGD sequences are not the only sequence motifs used for binding to integrins: many integrins recognize and bind to other motifs instead. tension Exerted by Cells regulates the assembly of Fibronectin Fibrils

1	Fibronectin can exist both in a soluble form, circulating in the blood and other body fluids, and as insoluble fibronectin fibrils, in which fibronectin dimers are cross-linked to one another by additional disulfide bonds and form part of the extracellular matrix. Unlike fibrillar collagen molecules, however, which can self-assemble into fibrils in a test tube, fibronectin molecules assemble into fibrils only on the surface of cells, and only where those cells possess appropriate fibronectin-binding proteins—in particular, integrins. The integrins provide a linkage from the fibronectin outside the cell to the actin cytoskeleton inside it. The linkage transmits tension to the fibronectin molecules—provided that they also have an attachment to some other structure—and stretches them, exposing cryptic binding sites in the fibronectin molecules (Figure 19–48). This allows them to bind directly to one another and to recruit additional fibronectin molecules to form a fibril (Figure 19–49).

1	cryptic binding sites in the fibronectin molecules (Figure 19–48). This allows them to bind directly to one another and to recruit additional fibronectin molecules to form a fibril (Figure 19–49). This dependence on tension and interaction with cell surfaces ensures that fibronectin fibrils assemble where there is a mechanical need for them and not in inappropriate locations such as the bloodstream.

1	Many other extracellular matrix proteins contain multiple copies of the type III fibronectin repeat (see Figure 19–46), and it is possible that tension exerted on these proteins also uncovers cryptic binding sites and thereby influences their behavior. the Basal Lamina Is a Specialized Form of Extracellular Matrix

1	the Basal Lamina Is a Specialized Form of Extracellular Matrix Thus far in this section we have reviewed the general principles underlying the structure and function of the major classes of extracellular matrix components. We now describe how some of these components are assembled into a specialized type of extracellular matrix called the basal lamina (also known as the basement membrane). This exceedingly thin, tough, flexible sheet of matrix molecules is an essential underpinning of all epithelia. Although small in volume, it has a critical role in the architecture of the body. Like the cadherins, it seems to be one of the defining features common to all multicellular animals, and it seems to have appeared very early in their evolution. The major molecular components of the basal lamina are among the most ancient extracellular matrix macromolecules.

1	Basal laminae are typically 40–120 nm thick. A sheet of basal lamina not only lies beneath epithelial cells but also surrounds individual muscle cells, fat cells, Figure 19–48 Tension-sensing by fibronectin. Some type III fibronectin repeats are thought to unfold when fibronectin is stretched. the unfolding exposes cryptic binding sites that interact with other fibronectin molecules resulting in the formation of fibronectin filaments like those shown in Figure 19–49. (From V. Vogel and M. Sheetz, Nat. Rev. Mol. Cell Biol. 7:265–275, 2006. With permission from Macmillan publishers Ltd.)

1	Figure 19–49 Organization of fibronectin into fibrils at the cell surface. this fluorescence micrograph shows the front end of a migrating mouse fibroblast. Extracellular fibronectin is stained green and intracellular actin filaments are stained red. the fibronectin is initially present as small dotlike aggregates near the leading edge of the cell. It accumulates at focal adhesions (sites of anchorage of actin filaments, discussed later) and becomes organized into fibrils parallel to the actin filaments. Integrin molecules spanning the cell membrane link the fibronectin outside the cell to the actin filaments inside it (see Figure 19–55). tension exerted on the fibronectin molecules through this linkage is thought to stretch them, exposing binding sites that promote fibril formation. (Courtesy of roumen pankov and Kenneth Yamada.) and Schwann cells (which wrap around peripheral nerve cell axons to form myelin). The basal lamina thus separates these cells and epithelia from the

1	(Courtesy of roumen pankov and Kenneth Yamada.) and Schwann cells (which wrap around peripheral nerve cell axons to form myelin). The basal lamina thus separates these cells and epithelia from the underlying or surrounding connective tissue and forms the mechanical connection between them. In other locations, such as the kidney glomerulus, a basal lamina lies between two cell sheets and functions as a selective filter (Figure 19–50). Basal laminae have more than simple structural and filtering roles, however. They are able to determine cell polarity; influence cell metabolism; organize the proteins in adjacent plasma membranes; promote cell survival, proliferation, or differentiation; and serve as highways for cell migration.

1	The mechanical role is nevertheless essential. In the skin, for example, the epithelial outer layer—the epidermis—depends on the strength of the basal lamina to keep it attached to the underlying connective tissue—the dermis. In people with genetic defects in certain basal lamina proteins or in a special type of collagen that anchors the basal lamina to the underlying connective tissue, the epidermis becomes detached from the dermis. This causes a blistering disease called junctional epidermolysis bullosa, a severe and sometimes lethal condition. Laminin and type IV Collagen are Major Components of the Basal Lamina

1	Laminin and type IV Collagen are Major Components of the Basal Lamina The basal lamina is synthesized by the cells on each side of it: the epithelial cells contribute one set of basal lamina components, while cells of the underlying bed of connective tissue (called the stroma, Greek for “bedding”) contribute another set (Figure 19–51). Although the precise composition of the mature basal lamina varies from tissue to tissue and even from region to region in the same lamina, it

1	Figure 19–50 Three ways in which basal laminae are organized. Basal laminae (yellow) surround certain cells (such as skeletal muscle cells), underlie epithelia, and are interposed between two cell sheets (as in the kidney glomerulus). Note that, in the kidney glomerulus, both cell sheets have gaps in them, and the basal lamina has a filtering as well as a supportive function, helping to determine which molecules will pass into the urine from the blood. the filtration also depends on other protein-based structures, called slit diaphragms, that span the intercellular gaps in the epithelial sheet.

1	Figure 19–51 The basal lamina in the cornea of a chick embryo. In this scanning electron micrograph, some of the epithelial cells have been removed to expose the upper surface of the matlike basal lamina. a network of collagen fibrils in the underlying connective tissue interacts with the lower face of the lamina. (Courtesy of robert trelstad.) typically contains the glycoproteins laminin, type IV collagen, and nidogen, along with the proteoglycan perlecan. Other common basal lamina components are fibronectin and type XVIII collagen (an atypical member of the collagen family, forming the core protein of a proteoglycan).

1	Laminin is the primary organizer of the sheet structure, and, early in development, basal laminae consist mainly of laminin molecules. Laminins comprise a large family of proteins, each composed of three long polypeptide chains (α, β, and γ) held together by disulfide bonds and arranged in the shape of an asymmetric bouquet, like a bunch of three flowers whose stems are twisted together at the foot but whose heads remain separate (Figure 19–52). These heterotrimers can self-assemble in vitro into a network, largely through interactions between their heads, although interaction with cells is needed to organize the network into an orderly sheet. Since there are several isoforms of each type of chain, and these can associate in different combinations, many different laminins can be produced, creating basal laminae with distinctive properties. The laminin γ1 chain is, however, a component of most laminin heterotrimers; mice lacking it die during embryogenesis because they are unable to

1	creating basal laminae with distinctive properties. The laminin γ1 chain is, however, a component of most laminin heterotrimers; mice lacking it die during embryogenesis because they are unable to make basal laminae.

1	Type IV collagen is a second essential component of mature basal laminae, and it, too, exists in several isoforms. Like the fibrillar collagens that constitute the bulk of the protein in connective tissues such as bone or tendon, type IV collagen molecules consist of three separately synthesized long protein chains that twist together to form a ropelike superhelix; however, they differ from the fibrillar collagens in that the triple-stranded helical structure is interrupted in more than 20 regions, allowing multiple bends. Type IV collagen molecules interact via their terminal domains to assemble extracellularly into a flexible, feltlike network that gives the basal lamina tensile strength.

1	Laminin and type IV collagen interact with other basal lamina components, such as the glycoprotein nidogen and the proteoglycan perlecan, resulting in a highly cross-linked network of proteins and proteoglycans (Figure 19–53). The laminin molecules that generate the initial sheet structure first join to each other while bound to receptors on the surface of the cells that produce laminin. The cell-surface receptors are primarily members of the integrin family, but another important type of laminin receptor is dystroglycan, a proteoglycan with a core protein that spans the cell membrane, dangling its GAG chains in the extracellular space. Together, these receptors organize basal lamina assembly: they hold the laminin molecules by their feet, leaving the laminin heads positioned to interact so as to form a two-dimensional network. This laminin network then coordinates the assembly of the other basal lamina components.

1	In the kidney glomerulus, an unusually thick basal lamina acts as one of the layers of a molecular filter, helping to prevent the passage of macromolecules from the blood into the urine as urine is formed (see Figure 19–50). The proteoglycan in Figure 19–52 The structure of laminin.

1	(a) the best-understood family member is laminin-111, shown here with some of its binding sites for other molecules (yellow boxes). Laminins are multidomain glycoproteins composed of three polypeptides (α, β, and γ) that are disulfide-bonded into an asymmetric crosslike structure. Each of the polypeptide chains is more than 1500 amino acids long. Five types of α chains, four types of β chains, and three types of γ chains are known, and various combinations of these subunits can assemble to form a large variety of different laminins, which are named according to numbers assigned to each of their three subunits: laminin-111, for example, contains α1, β1, and γ1 subunits. Each isoform tends to have a specific tissue distribution: laminin-332 is found in skin, laminin-211 in muscle, and laminin-411 in endothelial cells of blood vessels. through their binding sites for other proteins, laminin molecules play a central part in organizing basal laminae and anchoring them to cells. (B)

1	laminin-411 in endothelial cells of blood vessels. through their binding sites for other proteins, laminin molecules play a central part in organizing basal laminae and anchoring them to cells. (B) Electron micrographs of laminin molecules shadowed with platinum. (B, from J. Engel et al., J. Mol. Biol. 150:97–120, 1981. With permission from academic press.) the basal lamina is important for this function: when its GAG chains are removed by specific enzymes, the filtering properties of the lamina are destroyed. Type IV collagen also has a role: in a human hereditary kidney disorder (Alport syndrome), mutations in a type IV collagen gene result in an irregularly thickened and dysfunctional glomerular filter. Laminin mutations, too, can disrupt the function of the kidney filter, but in a different way—by interfering with the differentiation of the cells that contact it and support it.

1	The basal lamina can act as a selective barrier to the movement of cells, as well as a filter for molecules. The lamina beneath an epithelium, for example, usually prevents fibroblasts in the underlying connective tissue from making contact with the epithelial cells. It does not, however, stop macrophages, lymphocytes, or nerve processes from passing through it, using specialized protease enzymes to cut a hole for their transit. The basal lamina is also important in tissue regeneration after injury. When cells in tissues such as muscles, nerves, and epithelia are damaged or killed, the basal lamina often survives and provides a scaffold along which regenerating cells can migrate. In this way, the original tissue architecture is readily reconstructed.

1	A particularly striking example of the role of the basal lamina in regeneration comes from studies of the neuromuscular junction, the site where the nerve terminals of a motor neuron form a chemical synapse with a skeletal muscle cell (discussed in Chapter 11). In vertebrates, the basal lamina that surrounds the muscle cell separates the nerve and muscle cell plasma membranes at the synapse, and the synaptic region of the lamina has a distinctive chemical character,

1	Figure 19–53 A model of the molecular structure of a basal lamina. (a) the basal lamina is formed by specific interactions (B) between the proteins laminin, type IV collagen, and nidogen, and the proteoglycan perlecan. Arrows in (B) connect molecules that can bind directly to each other. there are various isoforms of type IV collagen and laminin, each with a distinctive tissue distribution. transmembrane laminin receptors (integrins and dystroglycan) in the plasma membrane are thought to organize the assembly of the basal lamina; only the integrins are shown. (Based on h. Colognato and p.D. Yurchenco, Dev. Dyn. 218:213–234, 2000. With permission from Wiley-Liss.) shell of REGENERATED residual returns to site of original junction nerve cut new acetylcholine receptors become concentrated at site of originalDEGENERATED MUSCLE AND NERVE REGENERATED junctionMUSCLE FIBER with special isoforms of type IV collagen and laminin and a proteoglycan called agrin. After a nerve or muscle injury,

1	at site of originalDEGENERATED MUSCLE AND NERVE REGENERATED junctionMUSCLE FIBER with special isoforms of type IV collagen and laminin and a proteoglycan called agrin. After a nerve or muscle injury, the basal lamina at the synapse has a central role in reconstructing the synapse at the correct location (Figure 19–54). Defects in components of the basal lamina at the synapse are responsible for some forms of muscular dystrophy, in which muscles develop normally but then degenerate later in life.

1	Cells have to Be able to Degrade Matrix, as Well as Make It The ability of cells to degrade and destroy extracellular matrix is as important as their ability to make it and bind to it. Rapid matrix degradation is required in processes such as tissue repair, and even in the seemingly static extracellular matrix of adult animals there is a slow, continuous turnover, with matrix macromolecules being degraded and resynthesized. This allows bone, for example, to be remodeled so as to adapt to changes in the stresses on it. From the point of view of individual cells, the ability to cut through matrix is crucial in two ways: it enables them to divide while embedded in matrix, and it enables them to travel through it. Cells in connective tissues generally need to be able to stretch out in order to divide. If a cell lacks the enzymes needed to degrade the surrounding matrix, it is strongly inhibited from dividing, as well as being hindered from migrating.

1	Localized degradation of matrix components is also required wherever cells have to escape from confinement by a basal lamina. It is needed during normal branching growth of epithelial structures such as glands, for example, to allow the population of epithelial cells to increase, and needed also when white blood cells migrate across the basal lamina of a blood vessel into tissues in response to infection or injury. Matrix degradation is important both for the spread of cancer cells through the body and for their ability to proliferate in the tissues that they invade (discussed in Chapter 20).

1	In general, matrix components are degraded by extracellular proteolytic enzymes (proteases) that act close to the cells that produce them. Many of these proteases belong to one of two general classes. The largest group, with about 50 members in vertebrates, is the matrix metalloproteases, which depend on bound Ca2+ or Zn2+ for activity. The second group is the serine proteases, which have a highly reactive serine in their active site. Together, metalloproteases and serine

1	Figure 19–54 Regeneration experiments demonstrating the special character of the junctional basal lamina at a neuromuscular junction. If a frog muscle and its motor nerve are destroyed, the basal lamina around each muscle cell remains intact and the sites of the old neuromuscular junctions are still recognizable. When the nerve, but not the muscle, is allowed to regenerate (upper right), the junctional basal lamina directs the regenerating nerve to the original synaptic site. When the muscle, but not the nerve, is allowed to regenerate (lower right), the junctional basal lamina causes newly made acetylcholine receptors (blue) to accumulate at the original synaptic site. these experiments show that the junctional basal lamina controls the localization of synaptic components on both sides of the lamina. Some of the molecules responsible for these effects have been identified. Motor neuron axons, for example, deposit agrin in the junctional basal lamina, where it regulates the assembly

1	of the lamina. Some of the molecules responsible for these effects have been identified. Motor neuron axons, for example, deposit agrin in the junctional basal lamina, where it regulates the assembly of acetylcholine receptors and other proteins in the junctional plasma membrane of the muscle cell. reciprocally, muscle cells deposit a particular isoform of laminin in the junctional basal lamina, and this molecule is likely to interact with specific ion channels on the presynaptic membrane of the neuron.

1	proteases cooperate to degrade matrix proteins such as collagen, laminin, and fibronectin. Some metalloproteases, such as the collagenases, are highly specific, cleaving particular proteins at a small number of sites. In this way, the structural integrity of the matrix is largely retained, while the limited amount of proteolysis that occurs is sufficient for cell migration. Other metalloproteases may be less specific, but, because they are anchored to the plasma membrane, they can act just where they are needed; it is this type of matrix metalloprotease that is crucial for a cell’s ability to divide when embedded in matrix.

1	Clearly, the activities of the proteases that degrade the matrix must be tightly controlled, if the fabric of the body is not to collapse in a heap. Numerous mechanisms are therefore employed to ensure that matrix proteases are activated only at the correct time and place. Protease activity is generally confined to the cell surface by specific anchoring proteins, by membrane-associated activators, and by the production of specific protease inhibitors in regions where protease activity is not needed. Matrix proteoglycans and Glycoproteins regulate the activities of Secreted proteins

1	Matrix proteoglycans and Glycoproteins regulate the activities of Secreted proteins The physical properties of extracellular matrix are important for its fundamental roles as a scaffold for tissue structure and as a substrate for cell anchorage and migration. The matrix also has an important impact on cell signaling. Cells communicate with each other by secreting signal molecules that diffuse through the extracellular fluid to influence other cells (discussed in Chapter 15). En route to their targets, the signal molecules encounter the tightly woven meshwork of the extracellular matrix, which contains a high density of negative charges and protein-interaction domains that can interact with the signal molecules, thereby altering their function in a variety of ways.

1	The highly charged heparan sulfate chains of proteoglycans, for example, interact with numerous secreted signal molecules, including fibroblast growth factors (FGFs) and vascular endothelial growth factor (VEGF), which (among other effects) stimulate a variety of cell types to proliferate. By providing a dense array of growth factor binding sites, proteoglycans are thought to generate large local reservoirs of these factors, limiting their diffusion and focusing their actions on nearby cells. Similarly, proteoglycans might help generate steep growth factor gradients in an embryo, which can be important in the patterning of tissues during development. FGF activity can also be enhanced by proteoglycans, which oligomerize the FGF molecules, enabling them to cross-link and activate their cell-surface receptors more effectively.

1	The importance of proteoglycans as regulators of the distribution and activity of signal molecules is illustrated by the severe developmental defects that can occur when specific proteoglycans are inactivated by mutation. In Drosophila, for example, the function of several signal proteins during development is governed by interactions with the membrane-associated proteoglycans Dally and Dally-like. These members of the glypican family are thought to concentrate signal proteins in specific locations and act as co-receptors that collaborate with the conventional cell-surface receptor proteins; as a result, they promote signaling in the correct location and prevent it in the wrong locations. In the Drosophila ovary, for example, Dally is partly responsible for the restricted localization and function of a signaling protein called Dpp, which blocks differentiation of the germ-line stem cells: when the gene encoding Dally is mutated, Dpp activity is greatly reduced and oocyte development

1	function of a signaling protein called Dpp, which blocks differentiation of the germ-line stem cells: when the gene encoding Dally is mutated, Dpp activity is greatly reduced and oocyte development is abnormal.

1	Several matrix proteins also interact with signal proteins. The type IV collagen of basal laminae interacts with Dpp in Drosophila, for example. Fibronectin contains a type III fibronectin repeat that interacts with VEGF, and another domain that interacts with another growth factor called hepatocyte growth factor (HGF), thereby promoting the activities of these factors. As discussed earlier, many matrix glycoproteins contain extensive arrays of binding domains, and the arrangement of these domains is likely to influence the presentation of signal proteins to their target cells (see Figure 19–46). Finally, many matrix glycoproteins contain domains that bind directly to specific cell-surface receptors, thereby generating signals that influence the behavior of the cells, as we describe in the next section.

1	Cells are embedded in an intricate extracellular matrix, which not only binds the cells together but also influences their survival, development, shape, polarity, and migratory behavior. The matrix contains various protein fibers interwoven in a network of glycosaminoglycan (GAG) chains. GAGs are negatively charged polysaccharide chains that (except for hyaluronan) are covalently linked to protein to form proteoglycan molecules. GAGs attract water and occupy a large volume of extracellular space. Proteoglycans are also found on the surface of cells, where they often function as co-receptors to help cells respond to secreted signal proteins. Fiber-forming proteins give the matrix strength and resilience. The fibrillar collagens (types I, II, III, V, and XI) are ropelike, triple-stranded helical molecules that aggregate into long fibrils in the extracellular space, thereby providing tensile strength. They also form structures to which cells can be anchored, often via large multidomain

1	molecules that aggregate into long fibrils in the extracellular space, thereby providing tensile strength. They also form structures to which cells can be anchored, often via large multidomain glycoproteins, such as laminin and fibronectin, that bind to integrins on the cell surface. Elasticity is provided by elastin molecules, which form an extensive cross-linked network of fibers and sheets that can stretch and recoil.

1	The basal lamina is a specialized form of extracellular matrix that underlies epithelial cells or is wrapped around certain other cell types, such as muscle cells. Basal laminae are organized on a framework of laminin molecules, which are linked together by their side-arms and bind to integrins and other receptors in the basal plasma membrane of overlying epithelial cells. Type IV collagen molecules, together with the protein nidogen and the large heparan sulfate proteoglycan perlecan, assemble into a sheetlike mesh that is an essential component of all mature basal laminae. Basal laminae provide mechanical support for epithelia; they form the interface and attachment between epithelia and connective tissue; they serve as filters in the kidney; they act as barriers to keep cells in their proper compartments; they influence cell polarity and cell differentiation; and they guide cell migration during development and tissue regeneration.

1	Cells make extracellular matrix, organize it, and degrade it. The matrix in its turn exerts powerful influences on the cells. The influences are exerted chiefly through transmembrane cell adhesion proteins that act as matrix receptors. These proteins tie the matrix outside the cell to the cytoskeleton inside it, but their role goes far beyond simple passive mechanical attachment. Through them, components of the matrix can affect almost any aspect of a cell’s behavior. The matrix receptors have a crucial role in epithelial cells, mediating their interactions with the basal lamina beneath them. They are no less important in connective-tissue cells, mediating the cells’ interactions with the matrix that surrounds them.

1	Several types of molecules can function as matrix receptors or co-receptors, including the transmembrane proteoglycans. But the principal receptors on animal cells for binding most extracellular matrix proteins are the integrins. Like the cadherins and the key components of the basal lamina, integrins are part of the fundamental architectural toolkit that is characteristic of multicellular animals. The members of this large family of homologous transmembrane adhesion molecules have a remarkable ability to transmit signals in both directions across the plasma membrane. The binding of a matrix component to an integrin can send a message into the interior of the cell, and conditions in the cell interior can send a signal outward to control binding of the integrin to the matrix. Tension applied to an integrin can cause it to tighten its grip on intracellular and extracellular structures, and loss of tension can loosen its hold, so that molecular signaling complexes fall apart on either

1	to an integrin can cause it to tighten its grip on intracellular and extracellular structures, and loss of tension can loosen its hold, so that molecular signaling complexes fall apart on either side of the membrane. In this way, integrins can serve not only to transmit mechanical and molecular signals, but also to convert one type of signal into the other.

1	Integrins are transmembrane heterodimers that Link the Extracellular Matrix to the Cytoskeleton There are many varieties of integrins, but they all conform to a common plan. An integrin molecule is composed of two noncovalently associated glycoprotein subunits called α and β. Both subunits span the cell membrane, with short intracellular C-terminal tails and large N-terminal extracellular domains (Figure 19–55). The extracellular domains bind to specific amino acid sequence motifs in extracellular matrix proteins or, in some cases, in proteins on the surfaces of other cells. The best-understood binding site for integrins is the RGD sequence mentioned earlier (see Figure 19–47), which is found in fibronectin and other extracellular matrix proteins. Some integrins bind a Leu-Asp-Val (LDV) sequence in fibronectin and other proteins. Additional integrin-binding sequences, as yet poorly defined, exist in laminins and collagens.

1	Humans contain 24 types of integrins, formed from the products of 8 different β-chain genes and 18 different α-chain genes, dimerized in different combinations. Each integrin dimer has distinctive properties and functions. Moreover, because the same integrin molecule in different cell types can have different ligand-binding specificities, it seems that additional cell-type-specific factors can interact with integrins to modulate their binding activity. The binding of integrins to their matrix ligands is also affected by the concentration of Ca2+ and Mg2+ in the extracellular medium, reflecting the presence of divalent cation-binding domains in the α and β subunits. The divalent cations can influence both the affinity and the specificity of the binding of an integrin to its extracellular ligands.

1	The intracellular portion of an integrin dimer binds to a complex of several different proteins, which together form a linkage to the cytoskeleton. For all but one of the 24 varieties of human integrins, this intracellular linkage is to actin filaments. These linkages depend on proteins that assemble at the short cytoplasmic tails of the integrin subunits (see Figure 19–55). A large adaptor protein called talin is a component of the linkage in many cases, but numerous additional proteins are also involved. Like the actin-linked cell–cell junctions formed by cadherins, the actin-linked cell–matrix junctions formed by integrins may be small, inconspicuous, and transient, or large, prominent, and durable. Examples of the latter are the focal adhesions that form when fibroblasts have sufficient time to establish strong attachments to the rigid surface of a culture dish, and the myotendinous junctions that attach muscle cells to their tendons.

1	Figure 19–55 The subunit structure of an active integrin molecule, linking extracellular matrix to the actin cytoskeleton. the N-terminal heads of the integrin chains attach directly to an extracellular protein such as fibronectin; the C-terminal intracellular tail of the integrin β subunit binds to adaptor proteins that interact with filamentous actin. the best-understood adaptor is a giant protein called talin, which contains a string of multiple domains for binding actin and other proteins, such as vinculin, that help reinforce and regulate the linkage to actin filaments. One end of talin binds to a specific site on the integrin β subunit cytoplasmic tail; other regulatory proteins, such as kindlin, bind at another site on the tail.

1	In epithelia, the most prominent cell–matrix attachment sites are the hemidesmosomes, where a specific type of integrin anchors the cells to laminin in the basal lamina. Here, uniquely, the intracellular attachment is to keratin intermediate filaments, via the intracellular adaptor proteins plectin and BP230 (Figure 19–56). Integrin Defects are responsible for Many Genetic Diseases Although there is some overlap in the activities of the different integrins—at least five bind laminin, for example—it is the diversity of integrin functions that is more remarkable. Table 19–3 lists some varieties of integrins and the problems that result when individual integrin αor β chains are defective. The β1 subunit forms dimers with at least 12 distinct α subunits and is found on almost all vertebrate cells: α5β1 is a fibronectin receptor and α6β1 is a laminin Figure 19–56 Hemidesmosomes.

1	Figure 19–56 Hemidesmosomes. (a) hemidesmosomes spot-weld epithelial cells to the basal lamina, linking laminin outside the cell to keratin filaments inside it. (B) Molecular components of a hemidesmosome. a specialized integrin (α6β4 integrin) spans the membrane, attaching to keratin filaments intracellularly via adaptor proteins called plectin and Bp230, and to laminin extracellularly. the adhesive complex also contains, in parallel with the integrin, an unusual collagen family member known as collagen type XVII; this has a membrane-spanning domain attached to its extracellular collagenous portion. Defects in any of these components can give rise to a blistering disease of the skin. One such disease, called bullous pemphigoid, is an autoimmune disease in which the immune system develops antibodies against collagen XVII or Bp230.

1	receptor on many types of cells. Mutant mice that cannot make any β1 integrins die early in embryonic development. Mice that are only unable to make the α7 subunit (the partner for β1 in muscle) survive but develop muscular dystrophy (as do mice that cannot make the laminin ligand for the α7β1 integrin).

1	The β2 subunit forms dimers with at least four types of α subunit and is expressed exclusively on the surface of white blood cells, where it has an essential role in enabling these cells to fight infection. The β2 integrins mainly mediate cell– cell rather than cell–matrix interactions, binding to specific ligands on another cell, such as an endothelial cell. The ligands are members of the Ig superfamily of cell–cell adhesion molecules. We have already described an example earlier in the chapter: an integrin of this class (αLβ2, also known as LFA1) on white blood cells enables them to attach firmly to the Ig family protein ICAM1 on vascular endothelial cells at sites of infection (see Figure 19–28B). People with the genetic disease called leukocyte adhesion deficiency fail to synthesize functional β2 subunits. As a consequence, their white blood cells lack the entire family of β2 receptors, and they suffer repeated bacterial infections.

1	The β3 integrins are found on blood platelets (as well as various other cells), and they bind several matrix proteins, including the blood clotting factor fibrinogen. Platelets have to interact with fibrinogen to mediate normal blood clotting, and humans with Glanzmann’s disease, who are genetically deficient in β3 integrins, suffer from defective clotting and bleed excessively.

1	A cell crawling through a tissue—a fibroblast or a macrophage, for example, or an epithelial cell migrating along a basal lamina—has to be able both to make and to break attachments to the matrix, and to do so rapidly if it is to travel quickly. Similarly, a circulating white blood cell has to be able to switch on or off its tendency to bind to endothelial cells in order to crawl out of a blood vessel at a site of inflammation. Furthermore, if force is to be applied where it is needed, the making and breaking of the extracellular attachments in all these cases has to be coupled to the prompt assembly and disassembly of cytoskeletal attachments inside the cell. The integrin molecules that span the membrane and mediate the attachments cannot simply be passive, rigid objects with sticky patches at their two ends. They must be able to switch between an active state, where they readily form attachments, and an inactive state, where they do not.

1	Structural studies, using a combination of electron microscopy and x-ray crystallography, suggest that integrins exist in multiple structural conformations that reflect different states of activity (Figure 19–57). In the inactive state, the external segments of the integrin dimer are folded together into a compact structure that cannot bind matrix proteins. In this state, the cytoplasmic tails of the dimer are Figure 19–57 Integrins exist in two major activity states. Inactive (folded) and active (extended) structures of an integrin molecule, based on data from x-ray crystallography and other methods.

1	Figure 19–57 Integrins exist in two major activity states. Inactive (folded) and active (extended) structures of an integrin molecule, based on data from x-ray crystallography and other methods. Figure 19–58 Activation of integrins by intracellular signaling. Signals received from outside the cell can act through various intracellular mechanisms to stimulate integrin activation. In platelets, as illustrated here, the extracellular signal protein thrombin activates a G-protein-coupled receptor on the cell surface, thereby initiating a signaling pathway that leads to activation of rap1, a member of the monomeric Gtpase family. activated rap1 interacts with the protein rIaM, which then recruits talin to the plasma membrane. together with another protein called kindlin, talin interacts with the integrin β chain to trigger integrin activation. talin then interacts with adaptor proteins such as vinculin, resulting in the formation of an actin linkage (see Figure 19–55).

1	talin regulation depends in part on an interaction between its flexible C-terminal rod domain and the N-terminal head domain that contains the integrin-binding site. this interaction is thought to maintain talin in an inactive state when it is free in the cytoplasm. When talin is recruited by rIaM to the plasma membrane, the talin head domain interacts with a phosphoinositide called pI(4,5)p2 (not shown here, but see Figure 15–28), resulting in dissociation of the rod domain. talin unfolds to expose its binding sites for integrin and other proteins.

1	hooked together, preventing their interaction with cytoskeletal linker proteins. In the active state, the two integrin subunits are unhooked at the membrane to expose the intracellular binding sites for cytoplasmic adaptor proteins, and the external domains unfold and extend, like a pair of legs, to expose a high-affinity matrix-binding site at the tips of the subunits. Thus, the switch from inactive to active states depends on a major conformational change that simultaneously exposes the external and internal ligand-binding sites at the ends of the integrin molecule. External matrix binding and internal cytoskeleton linkages are thereby coupled.

1	Switching between the inactive and active states is regulated by a variety of mechanisms that vary, depending on the needs of the cell. In some cases, activation occurs by an “outside-in” mechanism: the binding of an external matrix protein, such as the RGD sequence of fibronectin, can drive some integrins to switch from the low-affinity inactive state to the high-affinity active state. As a result, binding sites for talin and other cytoplasmic adaptor proteins are exposed on the tail of the β chain. The binding of these adaptor proteins then leads to attachment of actin filaments to the intracellular end of the integrin molecule (see Figure 19–55). In this way, when the integrin catches hold of its ligand outside the cell, the cell reacts by tying the integrin molecule to the cytoskeleton, so that force can be applied at the point of cell attachment.

1	The chain of cause and effect can also operate in reverse, from inside to outside. This “inside-out” integrin-activation process generally depends on intracellular regulatory signals that stimulate the ability of talin and other proteins to interact with the β chain of the integrin. Talin competes with the integrin α chain for its binding site on the tail of the β chain. Thus, when talin binds to the β chain, it blocks the intracellular α–βlinkage, allowing the two legs of the integrin molecule to spring apart. The regulation of “inside-out” integrin activation is particularly well understood in platelets, where an extracellular signal protein called thrombin binds to a specific G-protein-coupled receptor (GPCR) on the cell surface and thereby activates an intracellular signaling pathway that leads to integrin activation (Figure 19–58). It is likely that similar signaling pathways govern integrin activation in numerous other cell types. Integrins Cluster to Form Strong adhesions

1	Integrins Cluster to Form Strong adhesions Integrins, like other cell adhesion molecules, differ from cell-surface receptors for hormones and for other extracellular soluble signal molecules in that they usually bind their ligand with lower affinity and are present at a 10–100-fold higher concentration on the cell surface. The Velcro principle, mentioned earlier in the context of cadherin adhesion (see Figure 19–6C), operates here too. Following their activation, integrins cluster together to create a dense plaque in which many integrin molecules are anchored to cytoskeletal filaments. The resulting protein structure can be remarkably large and complex, as seen in the focal adhesion made by a fibroblast on a fibronectin-coated surface culture dish.

1	The assembly of mature cell–matrix junctional complexes depends on the recruitment of dozens of different scaffolding and signaling proteins. Talin is a major component of many cell–matrix complexes, but numerous other proteins also make important contributions. These include the integrin-linked kinase (ILK) and its binding partners pinch and parvin, which together form a trimeric complex that serves as an organizing hub at many junctions. Cell–matrix junctions also employ several actin-binding proteins, such as vinculin, zyxin, VASP, and α-actinin, to promote the assembly and organization of actin filaments. Another critical component of many cell–matrix junctions is the focal adhesion kinase (FAK), which interacts with multiple components in the junction and serves an important function in signaling, as we describe next. Extracellular Matrix attachments act through Integrins to Control Cell proliferation and Survival

1	Extracellular Matrix attachments act through Integrins to Control Cell proliferation and Survival Like other transmembrane cell adhesion proteins, integrins do more than just create attachments. They also activate intracellular signaling pathways and thereby allow control of almost any aspect of the cell’s behavior according to the nature of the surrounding matrix and the state of the cell’s attachments to it.

1	Many cells will not grow or proliferate in culture unless they are attached to extracellular matrix; nutrients and soluble growth factors in the culture medium are not enough. For some cell types, including epithelial, endothelial, and muscle cells, even cell survival depends on such attachments. When these cells lose contact with the extracellular matrix, they undergo apoptosis. This dependence of cell growth, proliferation, and survival on attachment to a substratum is known as anchorage dependence, and it is mediated mainly by integrins and the intracellular signals they generate. Mutations that disrupt or override this form of control, allowing cells to escape from anchorage dependence, occur in cancer cells and play a major part in their invasive behavior.

1	Our understanding of anchorage dependence has come mainly from studies of cells living on the surface of matrix-coated culture dishes. For connective-tissue cells that are normally surrounded by matrix on all sides, this is a far cry from the natural environment. Walking over a two-dimensional plain is very different from clambering through a three-dimensional jungle. The types of contacts that cells make with a rigid substratum are not the same as those, much less well studied, that they make with the deformable web of fibers of the extracellular matrix, and there are substantial differences in cell behavior in the two contexts. Nevertheless, it is likely that the same basic principles apply. Both in vitro and in vivo, intracellular signals generated at cell–matrix adhesion sites are crucial for cell proliferation and survival. Integrins recruit Intracellular Signaling proteins at Sites of Cell–Matrix adhesion

1	Integrins recruit Intracellular Signaling proteins at Sites of Cell–Matrix adhesion The mechanisms by which integrins signal into the cell interior are complex, involving several pathways, and integrins and conventional signaling receptors often influence one another and work together to regulate cell behavior, as we have already emphasized. The Ras/MAP kinase pathway (see Figure 15–49), for

1	Figure 19–59 Tyrosine phosphorylation at focal adhesions. a fibroblast cultured on a fibronectin-coated substratum and stained with fluorescent antibodies: actin filaments are stained green and activated proteins that contain phosphotyrosine are red, giving orange where the two components overlap. the actin filaments terminate at focal adhesions, where the cell attaches to the substratum by means of integrins. proteins containing phosphotyrosine are also concentrated at these sites, reflecting the local activation of FaK and other protein kinases. Signals generated at such adhesion sites help regulate cell division, growth, and survival. (Courtesy of Keith Burridge.) example, can be activated both by conventional signaling receptors and by integrins, but cells often need both kinds of stimulation of this pathway at the same time to give sufficient activation to induce cell proliferation. Integrins and conventional signaling receptors also cooperate to promote cell survival (discussed

1	of this pathway at the same time to give sufficient activation to induce cell proliferation. Integrins and conventional signaling receptors also cooperate to promote cell survival (discussed in Chapters 15 and 18).

1	One of the best-studied modes of integrin signaling depends on a cytoplasmic protein tyrosine kinase called focal adhesion kinase (FAK). In studies of cells cultured on plastic dishes, focal adhesions are often prominent sites of tyrosine phosphorylation (Figure 19–59), and FAK is one of the major tyrosine-phosphorylated proteins found at these sites. When integrins cluster at cell–matrix contacts, FAK is recruited to the integrin β subunit by intracellular adaptor proteins such as talin or paxillin (which binds to one type of integrin α subunit). The clustered FAK molecules phosphorylate each other on a specific tyrosine, creating a phosphotyrosine docking site for members of the Src family of cytoplasmic tyrosine kinases. In addition to phosphorylating other proteins at the adhesion sites, these kinases then phosphorylate FAK on additional tyrosines, creating docking sites for a variety of additional intracellular signaling proteins. In this way, outside-in signaling from integrins,

1	these kinases then phosphorylate FAK on additional tyrosines, creating docking sites for a variety of additional intracellular signaling proteins. In this way, outside-in signaling from integrins, via FAK and Src family kinases, is relayed into the cell in much the same way as receptor tyrosine kinases generate signals (as discussed in Chapter 15).

1	Cell–Matrix adhesions respond to Mechanical Forces Like the cell–cell junctions we described earlier, cell–matrix junctions can sense and respond to the mechanical forces that act on them. Most cell–matrix junctions, for example, are connected to a contractile actin network that tends to pull the junctions inward, away from the matrix. When cells are attached to a rigid matrix that strongly resists such pulling forces, the cell–matrix junction is able to sense the resulting high tension and trigger a response in which it recruits additional integrins and other proteins to increase the junction’s ability to withstand that tension. Cell attachment to a relatively soft matrix generates less tension and therefore a less robust response. These mechanisms allow cells to sense and respond to differences in the rigidity of extracellular matrices in different tissues.

1	We saw earlier that mechanotransduction at cadherin-based cell–cell junctions likely depends on junctional proteins that change their structure when the junction is stretched by tension (see Figure 19–12). The same is true for cell– matrix junctions. The long C-terminal tail domain of talin, for example, includes a large number of binding sites for the actin-regulatory protein vinculin. Many of these sites are hidden inside folded protein domains but are exposed when those folded talin helices 1 to 12 vinculin as talin unfolds, helix 12 is exposed and can bind to vinculin domains are unfolded by stretching the protein (Figure 19–60). The N-terminal end of talin binds integrin and the C-terminal end binds actin (see Figure 19–55); thus, when actin filaments are pulled by myosin motors inside the cell, the resulting tension stretches the talin rod, thereby exposing vinculin-binding sites. The vinculin molecules then recruit and organize additional actin filaments. Tension thereby

1	inside the cell, the resulting tension stretches the talin rod, thereby exposing vinculin-binding sites. The vinculin molecules then recruit and organize additional actin filaments. Tension thereby increases the strength of the junction.

1	Integrins are the principal cell-surface receptors used by animal cells to bind to the extracellular matrix: they function as transmembrane linkers between the extracellular matrix and the cytoskeleton. Most integrins connect to actin filaments, while those at hemidesmosomes bind to intermediate filaments. Integrin molecules are heterodimers, and the binding of extracellular matrix ligands or intracellular activator proteins such as talin results in a dramatic conformational switch from an inactive to an active state. This creates an allosteric coupling between binding to matrix outside the cell and binding to the cytoskeleton inside it, allowing the integrin to convey signals in both directions across the plasma membrane. Complex assemblies of proteins become organized around the intracellular tails of activated integrins, producing intracellular signals that can influence almost any aspect of cell behavior, from proliferation and survival, as in the phenomenon of anchorage

1	intracellular tails of activated integrins, producing intracellular signals that can influence almost any aspect of cell behavior, from proliferation and survival, as in the phenomenon of anchorage dependence, to cell polarity and guidance of migration. Integrin-based cell–matrix junctions are also capable of mechanotransduction: they can sense and respond to mechanical forces acting across the junction.

1	Each cell in a plant deposits, and is in turn completely enclosed by, an elaborate extracellular matrix called the plant cell wall. It was the thick cell walls of cork, visible in a primitive microscope, that in 1663 enabled Robert Hooke to distinguish and name cells for the first time. The walls of neighboring plant cells, cemented

1	Figure 19–60 Talin is a tension sensor at cell–matrix junctions. tension across cell–matrix junctions stimulates the local recruitment of vinculin and other actin-regulatory proteins, thereby strengthening the junction’s attachment to the cytoskeleton. the experiments presented here tested the hypothesis that tension is sensed by the talin adaptor protein that links integrins to actin filaments (see Figure 19–55). (a) the long, flexible, C-terminal region of talin is divided into a series of folded domains, some of which contain vinculin-binding sites (dark green lines) that are thought to be hidden and therefore inaccessible. One domain near the N-terminus, for example, comprises a folded bundle of 12 α helices containing five vinculin-binding sites. (B) this experiment tested the hypothesis that tension stretches the 12-helix domain, thereby exposing vinculin-binding sites. a fragment of talin containing this domain was attached to an apparatus in which the domain could be

1	hypothesis that tension stretches the 12-helix domain, thereby exposing vinculin-binding sites. a fragment of talin containing this domain was attached to an apparatus in which the domain could be stretched, as shown here. the fragment was labeled at its N-terminus with a tag that sticks to the surface of a glass slide on a microscope stage. the C-terminal end of the fragment was bound to a tiny magnetic bead, so the talin fragment could be stretched using a small magnetic electrode. the solution around the protein contained fluorescently tagged vinculin proteins. after the talin protein was stretched, excess vinculin solution was washed away, and the microscope was used to determine if any fluorescent vinculin proteins were bound to the talin protein. In the absence of stretching (top), most talin molecules did not bind vinculin. When the protein was stretched (bottom), two or three vinculin molecules were bound (only one is shown here for clarity). (adapted from a. del rio et al.,

1	most talin molecules did not bind vinculin. When the protein was stretched (bottom), two or three vinculin molecules were bound (only one is shown here for clarity). (adapted from a. del rio et al., Science 323:638– 641, 2009.) together to form the intact plant (Figure 19–61), are generally thicker, stronger, and, most important of all, more rigid than the extracellular matrix produced by animal cells. In evolving relatively rigid walls, which can be up to many micrometers thick, early plant cells forfeited the ability to crawl about and adopted a sedentary lifestyle that has persisted in all present-day plants.

1	the Composition of the Cell Wall Depends on the Cell type

1	All cell walls in plants have their origin in dividing cells, as the cell plate forms during cytokinesis to create a new partition wall between the daughter cells (discussed in Chapter 17). The new cells are usually produced in special regions called meristems, and they are generally small in comparison with their final size. To accommodate subsequent cell growth, the walls of the newborn cells, called primary cell walls, are thin and extensible, although tough. Once cell growth stops, the wall no longer needs to be extensible: sometimes the primary wall is retained without major modification, but, more commonly, a rigid secondary cell wall is produced by depositing new layers of matrix inside the old ones. These new layers generally have a composition that is significantly different from that of the primary wall. The most common additional polymer in secondary walls is lignin, a complex network of covalently linked phenolic compounds found in the walls of the xylem vessels and fiber

1	that of the primary wall. The most common additional polymer in secondary walls is lignin, a complex network of covalently linked phenolic compounds found in the walls of the xylem vessels and fiber cells of woody tissues.

1	Although the cell walls of higher plants vary in both composition and organization, they are all constructed, like animal extracellular matrices, using a structural principle common to all fiber-composites, including fiberglass and reinforced concrete. One component provides tensile strength, while another, in which the first is embedded, provides resistance to compression. While the principle is the same in plants and animals, the chemistry is different. Unlike the

1	Figure 19–61 Plant cell walls. (a) Electron micrograph of the root tip of a rush, showing the organized pattern of cells that results from an ordered sequence of cell divisions in cells with relatively rigid cell walls. In this growing tissue, the cell walls are still relatively thin, appearing as fine black lines between the cells in the micrograph. (B) Section of a typical cell wall separating two adjacent plant cells. the two dark transverse bands correspond to plasmodesmata that span the wall (see Figure 19–27). (a, courtesy of C. Busby and B. Gunning, Eur. J. Cell Biol. 21:214– 223, 1980. With permission from Elsevier; B, courtesy of Jeremy Burgess.) animal extracellular matrix, which is rich in protein and other nitrogen-containing polymers, the plant cell wall is made almost entirely of polymers that contain no nitrogen, including cellulose and lignin. For a sedentary organism that depends on CO2, H2O, and sunlight, these two abundant biopolymers represent “cheap,” carbon-based

1	of polymers that contain no nitrogen, including cellulose and lignin. For a sedentary organism that depends on CO2, H2O, and sunlight, these two abundant biopolymers represent “cheap,” carbon-based structural materials, helping to conserve the scarce fixed nitrogen available in the soil that generally limits plant growth. Thus trees, for example, make a huge investment in the cellulose and lignin that comprise the bulk of their biomass.

1	In the cell walls of higher plants, the tensile fibers are made from the polysaccharide cellulose, the most abundant organic macromolecule on Earth, tightly linked into a network by cross-linking glycans. In primary cell walls, the matrix in which the cross-linked cellulose network is embedded is composed of pectin, a highly hydrated network of polysaccharides rich in galacturonic acid. Secondary cell walls contain additional molecules to make them rigid and permanent; lignin, in particular, forms a hard, waterproof filler in the interstices between the other components. All of these molecules are held together by a combination of covalent and noncovalent bonds to form a highly complex structure, whose composition, thickness, and architecture depend on the cell type.

1	The plant cell wall thus has a “skeletal” role in supporting the structure of the plant as a whole, a protective role as an enclosure for each cell individually, and a transport role, helping to form channels for the movement of fluid in the plant. When plant cells become specialized, they generally adopt a specific shape and produce specially adapted types of walls, according to which the different types of cells in a plant can be recognized and classified. We focus here, however, on the primary cell wall and the molecular architecture that underlies its remarkable combination of strength, resilience, and plasticity, as seen in the growing parts of a plant. the tensile Strength of the Cell Wall allows plant Cells to Develop turgor pressure

1	The aqueous extracellular environment of a plant cell consists of the fluid contained in the walls that surround the cell. Although the fluid in the plant cell wall contains more solutes than does the water in the plant’s external milieu (for example, soil), it is still hypotonic in comparison with the cell interior. This osmotic imbalance causes the cell to develop a large internal hydrostatic pressure, or turgor pressure, which pushes outward on the cell wall, just as an inner tube pushes outward on a tire. The turgor pressure increases just to the point where the cell is in osmotic equilibrium, with no net influx of water despite the salt imbalance. The turgor pressure generated in this way may reach 10 or more atmospheres, about five times that in the average car tire. This pressure is vital to plants because it is the main driving force for cell expansion during growth, and it provides much of the mechanical rigidity of living plant tissues. Compare the wilted leaf of a

1	is vital to plants because it is the main driving force for cell expansion during growth, and it provides much of the mechanical rigidity of living plant tissues. Compare the wilted leaf of a dehydrated plant, for example, with the turgid leaf of a well-watered one. It is the mechanical strength of the cell wall that allows plant cells to sustain this internal pressure.

1	the primary Cell Wall Is Built from Cellulose Microfibrils Interwoven with a Network of pectic polysaccharides Cellulose gives the primary cell wall tensile strength. Each cellulose molecule consists of a linear chain of at least 500 glucose residues that are covalently linked to one another to form a ribbonlike structure, which is stabilized by hydrogen bonds within the chain (Figure 19–62). In addition, hydrogen bonds between adjacent cellulose molecules cause them to stick together in overlapping parallel arrays, forming bundles of about 40 cellulose chains, all of which have the same polarity. These highly ordered crystalline aggregates, many micrometers long, are called cellulose microfibrils, and they have a tensile strength comparable to that of steel. Sets of microfibrils are arranged in layers, or lamellae, with each microfibril about 20–40 nm from its neighbors and connected to them by long cross-linking

1	Figure 19–62 Cellulose. Cellulose molecules are long, unbranched chains of β1,4-linked glucose units. Each glucose residue is inverted with respect to its neighbors, and the resulting disaccharide repeat occurs hundreds of times in a single cellulose molecule. about 16 individual cellulose molecules assemble to form a strong, hydrogen-bonded cellulose microfibril. glycan molecules, which are attached by hydrogen bonds to the surface of the microfibrils. The primary cell wall consists of several such lamellae arranged in a plywoodlike network (Figure 19–63).

1	glycan molecules, which are attached by hydrogen bonds to the surface of the microfibrils. The primary cell wall consists of several such lamellae arranged in a plywoodlike network (Figure 19–63). The cross-linking glycans are a heterogeneous group of branched polysaccharides that bind tightly to the surface of each cellulose microfibril and thereby help to cross-link the microfibrils into a complex network. There are many classes of cross-linking glycans, but they all have a long linear backbone composed of one type of sugar (glucose, xylose, or mannose) from which short side chains of other sugars protrude. It is the backbone sugar molecules that form hydrogen bonds with the surface of cellulose microfibrils, cross-linking them in the process. Both the backbone and the side-chain sugars vary according to the plant species and its stage of development.

1	Coextensive with this network of cellulose microfibrils and cross-linking glycans is another cross-linked polysaccharide network based on pectins (see Figure 19–63). Pectins are a heterogeneous group of branched polysaccharides that contain many negatively charged galacturonic acid units. Because of their negative charge, pectins are highly hydrated and associated with a cloud of cations, resembling the glycosaminoglycans of animal cells in the large amount of space they occupy (see Figure 19–33). When Ca2+ is added to a solution of pectin molecules, it cross-links them to produce a semirigid gel (it is pectin that is added to fruit juice to make jam set). Certain pectins are particularly abundant in the middle lamella, the specialized region that cements together the walls of adjacent cells (see Figure 19–63); here, Ca2+ cross-links are thought to help hold cell wall components together. Although covalent bonds also play a part in linking the components, very little is known about

1	cells (see Figure 19–63); here, Ca2+ cross-links are thought to help hold cell wall components together. Although covalent bonds also play a part in linking the components, very little is known about their nature. Regulated separation of cells at the middle lamella underlies such processes as the ripening of tomatoes and the abscission (detachment) of leaves in the fall.

1	In addition to the two polysaccharide-based networks that form the bulk of all plant primary cell walls, proteins are present, contributing up to about 5% of the wall’s dry mass. Many of these proteins are enzymes, responsible for wall turnover and remodeling, particularly during growth. Another class of wall proteins, like collagen, contains high levels of hydroxyproline. These proteins are thought to strengthen the wall, and they are produced in greatly increased amounts as a local response to attack by pathogens. From the genome sequence of Arabidopsis, it has been estimated that more than 700 genes are required to synthesize, assemble, and remodel the plant cell wall.

1	Figure 19–63 Scale model of a portion of a primary plant cell wall showing the two major polysaccharide networks. the orthogonally arranged layers of cellulose microfibrils (green) are tied into a network by the cross-linking glycans (red) that form hydrogen bonds with the microfibrils. this network is coextensive with a network of pectin polysaccharides (blue). the network of cellulose and cross-linking glycans provides tensile strength, while the pectin network resists compression. Cellulose, cross-linking glycans, and pectin are typically present in roughly equal amounts in a primary cell wall. the middle lamella is especially rich in pectin, and it cements adjacent cells together.

1	Once a plant cell has left the meristem where it is generated, it can grow dramatically, commonly by more than a thousand times in volume. The manner of this expansion determines the final shape of each cell, and hence the final form of the plant as a whole. Turgor pressure inside the cell drives the expansion, but it is the behavior of the cell wall that governs its direction and extent. Complex wall-remodeling activities are required, as well as the deposition of new wall materials. Because of their crystalline structure, the individual cellulose microfibrils in the wall are unable to stretch, and this gives them a crucial role in the process. For the cell wall to stretch or deform, the microfibrils must either slide past one another or become more widely separated, or both. The orientation of the microfibrils in the innermost layers of the wall governs the direction in which the cell expands. Cells in plants therefore anticipate their future morphology by controlling the

1	orientation of the microfibrils in the innermost layers of the wall governs the direction in which the cell expands. Cells in plants therefore anticipate their future morphology by controlling the orientation of the cellulose microfibrils that they deposit in the wall (Figure 19–64).

1	Unlike most other matrix macromolecules, which are made in the endoplasmic reticulum and Golgi apparatus and are secreted, cellulose is spun out from the surface of the cell by a plasma-membrane-bound enzyme complex (cellulose synthase), which uses as its substrate the sugar nucleotide UDP-glucose supplied from the cytosol. Each enzyme complex, or rosette, has a sixfold symmetry (see Figure 19–65) and contains the protein products of three separate cellulose synthase (CESA) genes. Each CESA protein is essential for the production of a cellulose microfibril. Three CESA genes are required for primary cell wall synthesis and a different three for secondary cell wall synthesis.

1	As they are being synthesized, the nascent cellulose chains assemble into microfibrils. These are spun out on the extracellular surface of the plasma membrane, forming a layer, or lamella, in which all the microfibrils have more or less the same alignment (see Figure 19–63). Each new lamella is deposited internally to the previous one, so that the wall consists of concentrically arranged lamellae, with the oldest on the outside. The most recently deposited microfibrils in elongating cells commonly lie perpendicular to the axis of cell elongation, although the orientation of the microfibrils in the outer lamellae that were laid down earlier may be different (see Figure 19–64B and C). Figure 19–64 Cellulose microfibrils influence the direction of cell elongation.

1	the orientation of cellulose microfibrils in the primary cell wall of an elongating carrot cell is shown in this electron micrograph of a shadowed replica from a rapidly frozen and deep-etched cell wall. the cellulose microfibrils are aligned parallel to one another and perpendicular to the axis of cell elongation. the microfibrils are cross-linked by, and interwoven with, a complex web of matrix molecules (compare with Figure 19–63). (B, C) the cells in (B) and start off with identical shapes (shown here as cubes) but with different net orientations of cellulose microfibrils in their walls. although turgor pressure is uniform in all directions, cell wall loosening allows each cell to elongate only in a direction perpendicular to the orientation of the innermost layer of microfibrils, which have great tensile strength. Cell expansion occurs in concert with the insertion of new wall material. the final shape of an organ, such as a shoot, is determined in part by the direction in which

1	have great tensile strength. Cell expansion occurs in concert with the insertion of new wall material. the final shape of an organ, such as a shoot, is determined in part by the direction in which its component cells can expand. (a, courtesy of Brian Wells and Keith roberts.)

1	An important clue to the mechanism that dictates microfibril orientation came from observations of the microtubules in plant cells. These are frequently arranged in the cortical cytoplasm with the same orientation as the cellulose microfibrils that are currently being deposited in the cell wall in that region. These cortical microtubules form a cortical array close to the cytosolic face of the plasma membrane, held there by poorly characterized proteins. The congruent orientation of the cortical array of microtubules (lying just inside the plasma membrane) and cellulose microfibrils (lying just outside) is seen in many types and shapes of plant cells and is present during both primary and secondary cell wall deposition, suggesting a causal relationship.

1	This suggestion can be tested by treating a plant tissue with a microtubule-depolymerizing drug so as to disassemble the entire system of cortical microtubules. The consequences for subsequent cellulose deposition, however, are not as straightforward as might be expected. The drug treatment does not disrupt the production of new cellulose microfibrils, and in some cases cells can continue to deposit new microfibrils in the preexisting orientation. Any developmental switch in the orientation of the microfibril pattern that would normally occur between successive lamellae, however, is invariably blocked. It seems that a preexisting orientation of microfibrils can be propagated even in the absence of microtubules, but any change in the deposition of cellulose microfibrils requires that intact microtubules be present to determine the new orientation.

1	These observations are consistent with the following model. The cellulose-synthesizing rosettes embedded in the plasma membrane spin out long cellulose molecules. As the synthesis of cellulose molecules and their self-assembly into microfibrils proceeds, the distal end of each microfibril presumably forms indirect cross-links to the previous layer of wall material, orienting the new microfibril in parallel with the old ones as it becomes integrated into the texture of the wall. Since the microfibril is stiff, the rosette at its growing, proximal end has to move as it deposits the new material. Traveling in the plane of the membrane, the rosette moves in the direction defined by the way in which the far end of the microfibril is anchored in the existing wall. In this way, each layer of microfibrils would tend to be spun out from the membrane in the same orientation as the layer laid down previously, with the rosettes following the direction of the preexisting oriented microfibrils

1	microfibrils would tend to be spun out from the membrane in the same orientation as the layer laid down previously, with the rosettes following the direction of the preexisting oriented microfibrils outside the cell. Oriented microtubules inside the cell, however, can force a change in the direction in which the rosettes move: they can create boundaries in the plasma membrane that act like the banks of a canal to constrain rosette movement (Figure 19–65). In this view, cellulose synthesis can occur independently of microtubules; but it is constrained spatially when cortical microtubules are present to define membrane microdomains within which the enzyme complex can move.

1	Figure 19–65 One model of how the orientation of newly deposited cellulose microfibrils might be determined by the orientation of cortical microtubules.

1	(a) the large cellulose synthase complexes, or rosettes, are integral membrane proteins that continuously synthesize cellulose microfibrils on the outer face of the plasma membrane. the distal ends of the stiff microfibrils become integrated into the texture of the wall, and their elongation at the proximal end pushes the synthase complex along in the plane of the membrane. Because the cortical array of microtubules is attached to the plasma membrane in a way that confines this complex to defined membrane channels, the orientation of these microtubules— when they are present—determines the axis along which the new microfibrils are laid down. (B, C) two electron micrographs show the tight association of the cortical microtubules with the plasma membrane. One shows the microtubules in cross section while the other shows a microtubule in longitudinal section. Both emphasize the constant gap of about 20 nm between membrane and microtubule; the connecting molecules responsible remain

1	cross section while the other shows a microtubule in longitudinal section. Both emphasize the constant gap of about 20 nm between membrane and microtubule; the connecting molecules responsible remain obscure. (B and C, courtesy of andrew Staehelin.) 0.1 µm cellulose microfbril being added to preexisting wall plasma membrane microtubule attached to plasma membrane cellulose synthase complex CYTOSOL

1	In this way, plant cells can change their direction of expansion by a sudden What WE DON’t KNOW change in the orientation of their cortical array of microtubules. Because plant cells cannot move (being constrained by their walls), the entire morphology of • What are the regulatory mechanisms a multicellular plant presumably depends on a coordinated, highly patterned that control the rearrangement of deployment of cortical microtubule orientations during plant development. It is cell–cell junctions in epithelia during early development? What roles do not known how these orientations are controlled, although it has been shown that the microtubules can reorient rapidly in response to extracellular stimuli, includ these rearrangements? ing plant growth regulators such as ethylene and auxins (discussed in Chapter 15).

1	ing plant growth regulators such as ethylene and auxins (discussed in Chapter 15). Microtubules are not, however, the only cytoskeletal elements that influence • how do extracellular matrix proteins wall deposition. Local foci of cortical actin filaments can also direct the deposition and carbohydrates influence the of new wall material at specific sites on the cell surface, contributing to the elabo-localization and actions of extracellular rate final shaping of many differentiated plant cells. receptors?

1	coordinate the activation of integrin Plant cells are surrounded by a tough extracellular matrix, or cell wall, which is proteins and their interactions with responsible for many of the unique features of a plant’s lifestyle. The wall is com-cytoskeletal components and their posed of a network of cellulose microfibrils and cross-linking glycans, embedded in response to changes in mechanical a highly cross-linked matrix of pectin polysaccharides. In secondary cell walls, lig-force acting on cell–matrix junctions? nin may be deposited to make them waterproof, hard, and woody. A cortical array of microtubules can control the orientation of newly deposited cellulose microfi molecules have the ability to present brils, which in turn determine the direction of cell expansion and therefore the final ordered arrays of signals to cells, shape of the cell and, ultimately, of the plant as a whole. might the exact spatial relationships between such signals carry a message beyond that of the

1	the final ordered arrays of signals to cells, shape of the cell and, ultimately, of the plant as a whole. might the exact spatial relationships between such signals carry a message beyond that of the individual signals themselves?

1	Which statements are true? explain why or why not. sites of sites for 19–1 Given the numerous processes inside cells that cleavage are regulated by changes in Ca2+ concentration, it seems likely that Ca2+-dependent cell–cell adhesions are also regulated by changes in Ca2+ concentration. 19–2 Tight junctions perform two distinct functions: they seal the space between cells to restrict paracellular flow and they fence off plasma membrane domains to prevent the mixing of apical and basolateral membrane Figure Q19–1 Production of Fab fragments from IgG antibodies by digestion with papain (problem 19–6). proteins.

1	proteins. 19–3 The elasticity of elastin derives from its high con-Fab fragments were generated by digesting the IgG anti-tent of α helices, which act as molecular springs. bodies with papain, a protease, to separate the two binding sites (Figure Q19–1). Why do you suppose it was nec19–4 Integrins can convert mechanical signals into essary to use Fab fragments to block cell aggregation? intracellular molecular signals. 19–7 The food-poisoning bacterium Clostridium per-Discuss the following problems. fringens makes a toxin that binds to members of the claudin family of proteins, which are the main constituents of 19–5 Comment on the following (1922) quote from tight junctions. When the C-terminus of the toxin is bound Warren Lewis, who was one of the pioneers of cell biology.

1	to a claudin, the N-terminus can insert into the adjacent “Were the various types of cells to lose their stickiness for cell membrane, forming holes that kill the cell. The porone another and for the supporting extracellular matrix, tion of the toxin that binds to the claudins has proven to be our bodies would at once disintegrate and flow off into the a valuable reagent for investigating the properties of tight ground in a mixed stream of cells.” junctions. MDCK cells are a common choice for studies 19–6 Cell adhesion molecules were originally identi-of tight junctions because they can form an intact epithefied using antibodies raised against cell-surface compo-lial sheet with high transepithelial resistance. MDCK cells nents to block cell aggregation. In the adhesion-blocking express two claudins: claudin-1, which is not bound by assays, the researchers found it necessary to use antibody the toxin, and claudin-4, which is. fragments, each with a single binding site (so-called Fab

1	two claudins: claudin-1, which is not bound by assays, the researchers found it necessary to use antibody the toxin, and claudin-4, which is. fragments, each with a single binding site (so-called Fab When an intact MDCK epithelial sheet is incufragments), rather than intact IgG antibodies, which are bated with the C-terminal toxin fragment, claudin-4 Y-shaped molecules with two identical binding sites. The disappears, becoming undetectable within 24 hours. In (A) BASOLATERAL (B) APICAL 10,000

1	Figure Q19–2 Effects of Clostridium toxin on the barrier function of MDCK cells (problem 19–7). (A) Addition of toxin from the basolateral side of the epithelial sheet. (B) Addition of toxin from the apical side of the epithelial sheet. For a given voltage, a higher resistance (ohms cm2) gives less paracellular current. the absence of claudin-4, the cells remain healthy and the epithelial sheet appears intact. The mean number of strands in the tight junctions that link the cells also decreases over 24 hours from about four to about two, and they are less highly branched. A functional assay for the integrity of the tight junctions shows that transepithelial resistance decreases dramatically in the presence of the toxin, but the resistance can be restored by washing out the toxin (Figure Q19–2A). Curiously, the toxin produces these effects only when it is added to the basolateral side of the sheet; it has no effect when added to the apical surface (Figure Q19–2B).

1	A. How can it be that two tight-junction strands remain, even though all of the claudin-4 has disappeared? b. Why do you suppose the toxin works when it is added to the basolateral side of the epithelial sheet, but not when added to the apical side?

1	19–8 It is not an easy matter to assign particular functions to specific components of the basal lamina, since the overall structure is a complicated composite material with both mechanical and signaling properties. Nidogen, for example, cross-links two central components of the basal lamina by binding to the laminin γ-1 chain and to type IV collagen. Given such a key role, it was surprising that mice with a homozygous knockout of the gene for nidogen-1 were entirely healthy, with no abnormal phenotype. Similarly, mice homozygous for a knockout of the gene for nidogen-2 also appeared completely normal. By contrast, mice that were homozygous for a defined mutation in the gene for laminin γ-1, which eliminated just the binding site for nidogen, died at birth with severe defects in lung and kidney formation. The mutant portion of the laminin γ-1 chain is thought to have no other function than to bind nidogen, and does not affect laminin structure or its ability to assemble into the basal

1	formation. The mutant portion of the laminin γ-1 chain is thought to have no other function than to bind nidogen, and does not affect laminin structure or its ability to assemble into the basal lamina. How would you explain these genetic observations, which are summarized in Table Q19–1? What would you predict would be the phenotype of a mouse that was homozygous for knockouts of both nidogen genes?

1	19–9 Discuss the following statement: “The basal lamina of muscle fibers serves as a molecular bulletin board, in which adjoining cells can post messages that direct the differentiation and function of the underlying cells.” 19–10 The affinity of integrins for matrix components can be modulated by changes to their cytoplasmic domains: a process known as inside-out signaling. You have identified a key region in the cytoplasmic domains of αIIbβ3 integrin that seems to be required for inside-out signaling (Figure Q19–3). Substitution of alanine for either D723 in the β chain or R995 in the α chain leads to a high level of spontaneous activation, under conditions where the wild-type chains are inactive. Your advisor suggests that you convert the aspartate in the β chain to an arginine (D723R) and the arginine in the α chain to an aspartate (R995D). You compare all three α chains (R995, R995A, and R995D) against all three β chains (D723, D723A, and D723R). You find that all pairs have a

1	and the arginine in the α chain to an aspartate (R995D). You compare all three α chains (R995, R995A, and R995D) against all three β chains (D723, D723A, and D723R). You find that all pairs have a high level of spontaneous activation, except D723 vs R995 (the wild type) and D723R vs R995D, which have low levels. Based on these results, how do you think the αIIbβ3 integrin is held in its inactive state?

1	Figure Q19–3 Schematic representation of αIIbβ3 integrin (problem 19–10). The D723 and R995 residues are indicated. (From P.E. Hughes et al., J. Biol. Chem. 271:6571–6574, 1996. With permission from American Society for Biochemistry and Molecular Biology.) 19–11 The glycosaminoglycan polysaccharide chains that are linked to specific core proteins to form the proteoglycan components of the extracellular space are highly negatively charged. How do you suppose these negatively charged polysaccharide chains help to establish a hydrated gel-like environment around the cell? How would the properties of these molecules differ if the polysaccharide chains were uncharged?

1	19–12 At body temperature, L-aspartate in proteins racemizes to D-aspartate at an appreciable rate. Most proteins in the body have a very low level of D-aspartate, if it can be detected at all. Elastin, however, has a fairly high level of D-aspartate. Moreover, the amount of D-aspartate increases in direct proportion to the age of the person from whom the sample was taken. Why do you suppose that most proteins have little if any D-aspartate, while elastin has levels of D-aspartate that increase steadily with age? 19–13 Your boss is coming to dinner! All you have for a salad is some wilted, day-old lettuce. You vaguely recall that there is a trick to rejuvenating wilted lettuce, but you cannot remember what it is. Should you soak the lettuce in salt water, soak it in tap water, or soak it in sugar water, or maybe just shine a bright light on it and hope that photosynthesis will perk it up?

1	19–14 A plant must be able to respond to changes in the water status of its surroundings. It does so by the flow of water molecules through water channels called aquaporins. The hydraulic conductivity of a single aquaporin is 4.4 × 10–22 m3 per second per MPa (megapascal) of pressure. What does this correspond to in terms of water molecules per second at atmospheric pressure? [Atmospheric pressure is 0.1 MPa (1 bar) and the concentration of water is 55.5 M.] Beckerle M ed. (2002) Cell adhesion. Oxford: Oxford University press. hynes rO & Yamada KM (eds) (2011) Extracellular Matrix Biology (Cold Spring harbor perspectives in Biology). Cold Spring harbor: Cold Spring harbor Laboratory press. Brasch J, harrison OJ, honig B & Shapiro L (2012) thinking outside the cell: how cadherins drive adhesion. Trends Cell Biol. 22, 299–310. Gomez Ga, McLachlan rW & Yap aS (2011) productive tension: force-sensing and homeostasis of cell-cell junctions. Trends Cell Biol. 21, 499–505.

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1	Lecuit t, Lenne pF & Munro E (2011) Force generation, transmission, and integration during cell and tissue morphogenesis. Annu. Rev. Cell Dev. Biol. 27, 157–184. Litjens Sh, de pereda JM & Sonnenberg a (2006) Current insights into the formation and breakdown of hemidesmosomes. Trends Cell Biol. 16, 376–383. Maule aJ, Benitez-alfonso Y & Faulkner C (2011) plasmodesmata— membrane tunnels with attitude. Curr. Opin. Plant Biol. 14, 683–690. McEver rp & Zhu C (2010) rolling cell adhesion. Annu. Rev. Cell Dev. Biol. 26, 363–396. Nakagawa S, Maeda S & tsukihara t (2010) Structural and functional studies of gap junction channels. Curr. Opin. Struct. Biol. 20, 423–430. Shin K, Fogg VC & Margolis B (2006) tight junctions and cell polarity. Annu. Rev. Cell Dev. Biol. 22, 207–236. takeichi M (2007) the cadherin superfamily in neuronal connections and interactions. Nat. Rev. Neurosci. 8, 11–20.

1	takeichi M (2007) the cadherin superfamily in neuronal connections and interactions. Nat. Rev. Neurosci. 8, 11–20. thomason ha, Scothern a, Mcharg S & Garrod Dr (2010) Desmosomes: adhesive strength and signalling in health and disease. Biochem. J. 429, 419–433. The extracellular Matrix of Animals aszodi a, Legate Kr, Nakchbandi I & Fassler r (2006) What mouse mutants teach us about extracellular matrix function. Annu. Rev. Cell Dev. Biol. 22, 591–621. Bulow hE & hobert O (2006) the molecular diversity of glycosaminoglycans shapes animal development. Annu. Rev. Cell Dev. Biol. 22, 375–407. Couchman Jr (2010) transmembrane signaling proteoglycans. Annu. Rev. Cell Dev. Biol. 26, 89–114. Domogatskaya a, rodin S & tryggvason K (2012) Functional diversity of laminins. Annu. Rev. Cell Dev. Biol. 28, 523–553. hynes rO (2009) the extracellular matrix: not just pretty fibrils. Science 326, 1216–1219.

1	hynes rO (2009) the extracellular matrix: not just pretty fibrils. Science 326, 1216–1219. hynes rO & Naba a (2012) Overview of the matrisome—an inventory of extracellular matrix constituents and functions. Cold Spring Harb. Perspect. Biol. 4, a004903. Kielty CM, Sherratt MJ & Shuttleworth Ca (2002) Elastic fibres. J. Cell Sci. 115, 2817–2828. Larsen M, artym VV, Green Ja & Yamada KM (2006) the matrix reorganized: extracellular matrix remodeling and integrin signaling. Curr. Opin. Cell Biol. 18, 463–471. Lu p, takai K, Weaver VM & Werb Z (2011) Extracellular matrix degradation and remodeling in development and disease. Cold Spring Harb. Perspect. Biol. 3, a005058. ricard-Blum S (2011) the collagen family. Cold Spring Harb. Perspect. Biol. 3, a004978. Sasaki t, Fässler r & hohenester E (2004) Laminin: the crux of basement membrane assembly. J. Cell Biol. 164, 959–963. toole Bp (2001) hyaluronan in morphogenesis. Semin. Cell Dev. Biol. 12, 79–87.

1	Sasaki t, Fässler r & hohenester E (2004) Laminin: the crux of basement membrane assembly. J. Cell Biol. 164, 959–963. toole Bp (2001) hyaluronan in morphogenesis. Semin. Cell Dev. Biol. 12, 79–87. Yurchenco pD (2011) Basement membranes: cell scaffoldings and signaling platforms. Cold Spring Harb. Perspect. Biol. 3, a004911. Calderwood Da, Campbell ID & Critchley Dr (2013) talins and kindlins: partners in integrin-mediated adhesion. Nat. Rev. Mol. Cell Biol. 14, 503–517. Campbell ID & humphries MJ (2011) Integrin structure, activation, and interactions. Cold Spring Harb. Perspect. Biol. 3, a004994. hoffman BD, Grashoff C & Schwartz Ma (2011) Dynamic molecular processes mediate cellular mechanotransduction. Nature 475, 316–323. hogg N, patzak I & Willenbrock F (2011) the insider’s guide to leukocyte integrin signalling and function. Nat. Rev. Immunol. 11, 416–426.

1	hogg N, patzak I & Willenbrock F (2011) the insider’s guide to leukocyte integrin signalling and function. Nat. Rev. Immunol. 11, 416–426. Kanchanawong p, Shtengel G, pasapera aM et al. (2010) Nanoscale architecture of integrin-based cell adhesions. Nature 468, 580–584. Luo Bh & Springer ta (2006) Integrin structures and conformational signaling. Curr. Opin. Cell Biol. 18, 579–586. Moser M, Legate Kr, Zent r & Fässler r (2009) the tail of integrins, talin, and kindlins. Science 324, 895–899. ross tD, Coon BG, Yun S et al. (2013) Integrins in mechanotransduction. Curr. Opin. Cell Biol. 25, 613–618. Shattil SJ, Kim C & Ginsberg Mh (2010) the final steps of integrin activation: the end game. Nat. Rev. Mol. Cell Biol. 11, 288–300. The Plant Cell Wall albersheim p, Darvill a, roberts K et al. (2011) plant Cell Walls: From Chemistry to Biology. New York: Garland Science.

1	The Plant Cell Wall albersheim p, Darvill a, roberts K et al. (2011) plant Cell Walls: From Chemistry to Biology. New York: Garland Science. Braidwood L, Breuer C & Sugimoto K (2013) My body is a cage: mechanisms and modulation of plant cell growth. New Phyto. 210, 388–402. Keegstra K (2010) plant cell walls. Plant Physiol. 154, 483–486. Li S, Lei L, Somerville C et al. (2011) Cellulose synthase interactive protein 1 (CSI1) links microtubules and cellulose synthase complexes. Proc. Natl. Acad. Sci. USA 109, 189–190. Lloyd C (2011) Dynamic microtubules and the texture of plant cell walls. Int. Rev. Cell Mol. Biol. 287, 287–329. McFarlane hE, Döring a & perrson S (2014) the cell biology of cellulose synthesis. Annu. Rev. Plant Biol. 65, 69–94. Somerville C (2006) Cellulose synthesis in higher plants. Annu. Rev. Cell Dev. Biol. 22, 53–78.

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1	Wolf S, hématy K & höfte h (2012) Growth control and cell wall signaling in plants. Annu. Rev. Plant Biol. 63, 381–407. About one in five of us will die of cancer, but that is not why we devote a chapter to this disease. Cancer cells break the most basic rules of cell behavior by which multicellular organisms are built and maintained, and they exploit every kind of opportunity to do so. These transgressions help to reveal what the normal rules are and how they are enforced. As a result, cancer research helps to illuminate the fundamentals of cell biology—especially cell signaling (Chapter 15), the cell cycle and cell growth (Chapter 17), programmed cell death (apoptosis, Chapter 18), and the control of tissue architecture (Chapters 19 and 22). Of course, with a deeper understanding of these normal processes, we also gain a deeper understanding of the disease and better tools to treat it.

1	In this chapter, we first consider what cancer is and describe the natural history of the disease from a cellular standpoint. We then discuss the molecular changes that make a cell cancerous. And we end the chapter by considering how our enhanced understanding of the molecular basis of cancer is leading to improved methods for its prevention and treatment.

1	The body of an animal operates as a society or ecosystem, whose individual members are cells that reproduce by cell division and organize themselves into collaborative assemblies called tissues. This ecosystem is very peculiar, however, because self-sacrifice—as opposed to survival of the fittest—is the rule. Ultimately, all of the somatic cell lineages in animals are committed to die: they leave no progeny and instead dedicate their existence to the support of the germ cells, which alone have a chance of continued survival (discussed in Chapter 21). There is no mystery in this, for the body is a clone derived from a fertilized egg, and the genome of the somatic cells is the same as that of the germ-cell lineage that gives rise to sperm or eggs. By their self-sacrifice for the sake of the germ cells, the somatic cells help to propagate copies of their own genes.

1	Thus, unlike free-living cells such as bacteria, which compete to survive, the cells of a multicellular organism are committed to collaboration. To coordinate their behavior, the cells send, receive, and interpret an elaborate set of extracellular signals that serve as social controls, directing cells how to act (discussed in Chapter 15). As a result, each cell behaves in a socially responsible manner—resting, growing, dividing, differentiating, or dying—as needed for the good of the organism.

1	Molecular disturbances that upset this harmony mean trouble for a multicellular society. In a human body with more than 1014 cells, billions of cells experience mutations every day, potentially disrupting the social controls. Most dangerously, a mutation may give one cell a selective advantage, allowing it to grow and divide slightly more vigorously and survive more readily than its neighbors and in this way to become a founder of a growing mutant clone. A mutation that promotes such selfish behavior by individual members of the cooperative can jeopardize the future of the whole enterprise. Over time, repeated rounds of mutation, competition, and natural selection operating within the population of somatic cells can cause matters to go from bad to worse. These are the basic ingredients of cancer: it is a disease in which an individual mutant clone of cells begins by CANCER-CRITICAL GENES: hOW ThEY ARE FOUND AND WhAT ThEY DO

1	CANCER-CRITICAL GENES: hOW ThEY ARE FOUND AND WhAT ThEY DO CANCER PREVENTION AND TREATMENT: PRESENT AND FUTURE prospering at the expense of its neighbors. In the end—as the clone grows, evolves, and spreads—it can destroy the entire cellular society (Movie 20.1). In this section, we discuss the development of cancer as a microevolutionary process that takes place within the course of a human life-span in a subpopulation of cells in the body. But the process depends on the same principles of mutation and natural selection that have driven the evolution of living organisms on Earth for billions of years.

1	Cancer cells are defined by two heritable properties: (1) they reproduce in defiance of the normal restraints on cell growth and division, and (2) they invade and colonize territories normally reserved for other cells. It is the combination of these properties that makes cancers particularly dangerous. An abnormal cell that grows (increases in mass) and proliferates (divides) out of control will give rise to a tumor, or neoplasm—literally, a new growth. As long as the neoplastic cells have not yet become invasive, however, the tumor is said to be benign. For most types of such neoplasms, removing or destroying the mass locally usually achieves a complete cure. A tumor is considered a true cancer if it is malignant; that is, when its cells have acquired the ability to invade surrounding tissue. Invasiveness is an essential characteristic of cancer cells. It allows them to break loose, enter blood or lymphatic vessels, and form secondary tumors called metastases at other sites in the

1	tissue. Invasiveness is an essential characteristic of cancer cells. It allows them to break loose, enter blood or lymphatic vessels, and form secondary tumors called metastases at other sites in the body (Figure 20–1). In general, the more widely a cancer spreads, the harder it becomes to eradicate. It is generally metastases that kill the cancer patient.

1	Cancers are traditionally classified according to the tissue and cell type from which they arise. Carcinomas are cancers arising from epithelial cells, and they are by far the most common cancers in humans. They account for about 80% of cases, perhaps because most of the cell proliferation in adults occurs in epithelia. In addition, epithelial tissues are the most likely to be exposed to the various forms of physical and chemical damage that favor the development of cancer. Sarcomas arise from connective tissue or muscle cells. Cancers that do not fit in either of these two broad categories include the various leukemias and lymphomas, derived from white blood cells and their precursors (hemopoietic cells), as well as cancers derived from cells of the nervous system. Figure 20–2 shows the types of cancers that are common in the United States, together with their incidence and death rates. Each broad category has many subdivisions according to the specific cell type, the location in the

1	of cancers that are common in the United States, together with their incidence and death rates. Each broad category has many subdivisions according to the specific cell type, the location in the body, and the microscopic appearance of the tumor.

1	In parallel with the set of names for malignant tumors, there is a related set of names for benign tumors: an adenoma, for example, is a benign epithelial tumor with a glandular organization; the corresponding type of malignant tumor is an adenocarcinoma (Figure 20–3). Similarly, a chondroma and a chondrosarcoma are, respectively, benign and malignant tumors of cartilage.

1	Most cancers have characteristics that reflect their origin. Thus, for example, the cells of a basal-cell carcinoma, derived from a keratinocyte stem cell in the skin, generally continue to synthesize cytokeratin intermediate filaments, whereas the cells of a melanoma, derived from a pigment cell in the skin, will often (but not always) continue to make pigment granules. Cancers originating from different cell types are, in general, very different diseases. Basal-cell carcinomas of the skin, for example, are only locally invasive and rarely metastasize, whereas melanomas can become much more malignant and often form metastases. Basal-cell carcinomas are readily cured by surgery or local irradiation, whereas malignant melanomas, once they have metastasized widely, are usually fatal.

1	Later, we shall see that there is also a different way to classify cancers, one that cuts across the traditional classification by site of origin: we can classify them in terms of the mutations that make the tumor cells cancerous. The final section of the chapter will show how this information can be crucial to the design and choice of treatments.

1	Figure 20–1 Metastasis. Malignant tumors typically give rise to metastases, making the cancer hard to eradicate. Shown in this fusion image is a whole-body scan of a patient with metastatic non-hodgkin’s lymphoma (NhL). The background image of the body’s tissues was obtained by CT (computed x-ray tomography) scanning. Overlaid on this image, a PET (positron emission tomography) scan reveals the tumor tissue (yellow), detected by its unusually high uptake of radioactively labeled fluorodeoxyglucose (FDG). high FDG uptake occurs in cells with unusually active glucose uptake and metabolism, which is a characteristic of cancer cells (see Figure 20–12). The yellow spots in the abdominal region reveal multiple metastases. (Courtesy of S. Gambhir.) respiratorycancers of system epithelia: carcinomas breast blood: myelomas, leukemias, and lymphomas bones, connective tissue, new cases muscles, and vasculature KEY: deaths/year endocrine system, thyroid

1	KEY: deaths/year endocrine system, thyroid Even when a cancer has metastasized, we can usually trace its origins to a single primary tumor, arising in a specific organ. The primary tumor is thought to derive by cell division from a single cell that initially experienced some heritable change. Subsequently, additional changes accumulate in some of the descendants of this cell, allowing them to outgrow, out-divide, and often outlive their neighbors. By the time it is first detected, a typical human cancer will have been developing for many years and will already contain a billion cancer cells or more (Figure 20–4). Tumors will usually also contain a variety of other cell types; for example, fibroblasts will be present in the supporting connective tissue associated with a carcinoma, in addition to inflammatory and vascular endothelial cells. How can we be sure that the cancer cells are the clonal descendants of a single abnormal cell?

1	One way of proving clonal origin is through molecular analysis of the chromosomes in tumor cells. In almost all patients with chronic myelogenous leukemia (CML), for example, we can distinguish the leukemic white blood cells from the patient’s normal cells by a specific chromosomal abnormality: the so-called Philadelphia chromosome, created by a translocation between the long arms of chromosomes 9 and 22 (Figure 20–5). When the DNA at the site of translocation is cloned and sequenced, it is found that the site of breakage and rejoining of the translocated fragments is identical in all the leukemic cells in any given patient, but that this site differs slightly (by a few hundred or thousand base pairs) from one patient to another. This is the expected result if, and only if, the cancer in each patient arises from a unique accident occurring in a single cell. We will see later

1	Figure 20–2 Cancer incidence and mortality in the United States. The total number of new cases diagnosed in 2012 in the United States was 1,665,540, and total cancer deaths were 585,720. Note that deaths reflect cases diagnosed at many different times and that somewhat less than half of the people who develop cancer die of it. In the world as a whole, the five most common cancers are those of the lung, stomach, breast, colon/rectum, and uterine cervix (included in the figure under the heading of reproductive tract), and the total number of new cancer cases recorded per year is just over 6 million. Skin cancers other than melanomas are not included in these figures, since almost all are cured easily and many are unrecorded.

1	The data for the United Kingdom are similar. however, incidences are different in some other parts of the world, reflecting widespread exposures to different infectious agents and environmental toxins. (Data from American Cancer Society, Cancer Facts and Figures, 2014.) Figure 20–3 Benign versus malignant tumors. A benign glandular tumor (pink cells; an adenoma) remains inside the basal lamina (yellow) that marks the boundary of the normal structure (a duct, in this example). In contrast, a malignant glandular tumor (red cells; an adenocarcinoma) can develop from a benign tumor cell, and it destroys the integrity of the tissue, as shown. There are many different forms that such tumors may take.

1	Figure 20–4 The growth of a typical human tumor, such as a tumor of the breast. The diameter of the tumor is plotted on a logarithmic scale. Years may elapse before the tumor becomes noticeable. The doubling time of a typical breast tumor, for example, is about 100 days. however, particularly virulent tumors may grow much more rapidly. diameter of tumor (mm) how this particular translocation promotes the development of CML by creating a novel hybrid gene encoding a protein that promotes cell proliferation. Many other lines of evidence, from a variety of cancers, point to the same con clusion: most cancers originate from a single aberrant cell. 0.1

1	0.1 If a single abnormal cell is to give rise to a tumor, it must pass on its abnormality to its progeny: the aberration has to be heritable. Thus, the development of a clone of cancer cells depends on genetic changes. The tumor cells contain somatic mutations: they have one or more shared detectable abnormalities in their DNA sequence that distinguish them from the normal cells surrounding the tumor, as in the example of CML just described. (The mutations are called somatic because they occur in the soma, or body cells, not in the germ line). Cancers are also driven by epigenetic changes—persistent, heritable changes in gene expression that result from modifications of chromatin structure without alteration of the cell’s DNA sequence. But somatic mutations that alter DNA sequence appear to be a fundamental and universal feature, and cancer is in this sense a genetic disease.

1	Factors that cause genetic changes tend to provoke the development of cancer. Thus, carcinogenesis (the generation of cancer) can be linked to mutagenesis (the production of a change in the DNA sequence). This correlation is particularly clear for two classes of external agents: (1) chemical carcinogens (which typically cause simple local changes in the nucleotide sequence), and (2) radiation such as x-rays (which typically cause chromosome breaks and translocations) or ultraviolet (UV) light (which causes specific DNA base alterations). As would be expected, people who have inherited a genetic defect in one of several DNA repair mechanisms, causing their cells to accumulate mutations at an elevated rate, run a heightened risk of cancer. Those with the disease xeroderma pigmentosum, for example, have defects in the system that repairs DNA damage induced by UV light, and they have a greatly increased incidence of skin cancers.

1	A Single Mutation Is Not Enough to Change a Normal Cell into a Cancer Cell

1	An estimated 1016 cell divisions occur in a normal human body in the course of a typical lifetime; in a mouse, with its smaller number of cells and its shorter lifespan, the number is about 1012. Even in an environment that is free of mutagens, mutations would occur spontaneously at an estimated rate of about 10–6 mutations per gene per cell division—a value set by fundamental limitations on the accuracy of DNA replication and repair (see pp. 237–238). Thus, in a typical lifetime, every single gene is likely to have undergone mutation on about 1010 separate occasions in a human, or on about 106 occasions in a mouse. Among the resulting mutant cells, we might expect a large number that have sustained deleterious mutations in genes that regulate cell growth and division, causing the cells to disobey the normal restrictions on cell proliferation. From this point of view, the problem of cancer seems to be not why it occurs, but why it occurs so infrequently.

1	Clearly, if a mutation in a single gene were enough to convert a typical healthy cell into a cancer cell, we would not be viable organisms. Many lines of evidence indicate that the development of a cancer typically requires that a substantial number of independent, rare genetic and epigenetic accidents occur in the lineage that emanates from a single cell. One such indication comes from epidemiological studies of the incidence of cancer as a function of age (Figure 20–6). If a Figure 20–5 The translocation between chromosomes 9 and 22 responsible for chronic myelogenous leukemia. The normal structures of chromosomes 9 and 22 are shown at the left. When a translocation occurs between them at the indicated site, the result is the abnormal pair at the right. The smaller of the two resulting abnormal chromosomes (22q–) is called the Philadelphia chromosome, after the city where the abnormality was first recorded.

1	Figure 20–6 Cancer incidence as a function of age. The number of newly 180 diagnosed cases of colon cancer in women in England and Wales in 1 year is plotted as a function of age at diagnosis, relative to the total number of individuals in each age group. The incidence of cancer rises steeply as a function of age. If only a single mutation were required to trigger the cancer and this mutation had an equal chance of occurring at any time, the incidence of this cancer would be the same at all ages. Analyses of this type suggest that the development of a solid tumor instead requires five to eight independent accidents (“hits”) that occur randomly over time. This calculation incidence rate per 100,000 assumes that the mutation rate remains constant as a cancer evolves, where in fact it often increases (see p. 1097). (Data from C. Muir et al., Cancer

1	Incidence in Five Continents, Vol. V. Lyon: International Agency for Research on Cancer, 1987.) single mutation were responsible for cancer, occurring with a fixed probability per year, the chance of developing cancer in any given year of life should be indepen dent of age. In fact, for most types of cancer, the incidence rises steeply with age— as would be expected if cancer is caused by a progressive, random accumulation 20 of a set of mutations in a single lineage of cells. As discussed later, these indirect arguments have now been confirmed by sys-0 tematically sequencing the genomes of the tumor cells from individual cancer patients and cataloging the mutations that they contain.

1	For those cancers known to have a specific external cause, the disease does not usually become apparent until long after exposure to the causal agent. The incidence of lung cancer, for example, does not begin to rise steeply until after decades of heavy smoking (Figure 20–7). Similarly, the incidence of leukemias in Hiroshima and Nagasaki did not show a marked rise until about 5 years after the explosion of the atomic bombs, and industrial workers exposed for a limited period to chemical carcinogens do not usually develop the cancers characteristic of their occupation until 10, 20, or even more years after the exposure. During this long incubation period, the prospective cancer cells undergo a succession of changes, and the same presumably applies to cancers where the initial genetic lesion has no such obvious external cause.

1	The concept that the development of a cancer requires a gradual accumulation of mutations in a number of different genes helps to explain the well-known phenomenon of tumor progression, whereby an initial mild disorder of cell behavior evolves gradually into a full-blown cancer. Chronic myelogenous leukemia again provides a clear example. It begins as a disorder characterized by a nonlethal overproduction of white blood cells and continues in this form for several years before changing into a much more rapidly progressing illness that usually ends in death within a few months. In the early chronic phase, the leukemic cells are distinguished mainly by the chromosomal translocation (the Philadelphia chromosome) mentioned previously, although there may well be other, less visible

1	Figure 20–7 Smoking and the onset of lung cancer. A major increase in cigarette smoking (red line) has caused a dramatic rise in lung cancer deaths (green line), with a lag time of about 35 years. Because global cigarette smoking peaked in 1990, global lung cancer deaths are expected to decline after a similar lag. (Data from R.N. Proctor, Nat. Rev. Cancer 1:82–86, 2001). genetic or epigenetic changes. In the subsequent acute phase, cells that show not only the translocation but also several other chromosomal abnormalities overrun the hemopoietic (blood-forming) system. It appears that cells from the initial mutant clone have undergone further mutations that make them proliferate even more vigorously, so that they come to outnumber both the normal blood cells and their ancestors with the primary chromosomal translocation.

1	Carcinomas and other solid tumors evolve in a similar way (Figure 20–8). Although many such cancers in humans are not diagnosed until a relatively late stage, in some cases it is possible to observe the earlier steps and, as we shall see later, to relate them to specific genetic changes Tumor Progression Involves Successive Rounds of Random Inherited Change Followed by Natural Selection From all the evidence, therefore, it seems that cancers arise by a process in which an initial population of slightly abnormal cells—descendants of a single abnormal ancestor—evolve from bad to worse through successive cycles of random inherited change followed by natural selection. Correspondingly, tumors grow in fits and starts, as additional advantageous inherited changes arise and the cells bearing them flourish. Tumor progression involves a large element of chance and usually takes many years, which may be why the majority of us will die of causes other than cancer.

1	At each stage of progression, some individual cell acquires an additional mutation or epigenetic change that gives it a selective advantage over its neighbors, making it better able to thrive in its environment—an environment that, inside a tumor, may be harsh, with low levels of oxygen, scarce nutrients, and the natural barriers to growth presented by the surrounding normal tissues. The larger the number of tumor cells, the higher the chance that at least one of them will undergo a change that favors it over its neighbors. Thus, as the tumor grows, progression accelerates. The offspring of the best-adapted cells continue to divide, eventually producing the dominant clones in the developing lesion (Figure 20–9).

1	Just as in the evolution of plants and animals, a kind of speciation often occurs: the original cancer cell lineage can diversify to give many genetically different vigorous subclones of cells. These may coexist in the same mass of tumor tissue; or they may migrate and colonize separate environments suited to their individual quirks, where they settle, thrive, and progress as independently evolving metastases. As new mutations arise within each tumor mass, different subclones may gain an advantage and come to predominate, only to be overtaken by others or outgrown by their own sub-subclones. The increasing genetic diversity as a cancer progresses is one of the chief factors that make cures difficult.

1	Figure 20–8 Stages of progression in the development of cancer of the epithelium of the uterine cervix. Pathologists use standardized terminology to classify the types of disorders they see, so as to guide the choice of treatment. (A) In a stratified squamous epithelium, dividing cells are confined to the basal layer. (B) In this low-grade intraepithelial neoplasia (right half of image), dividing cells can be found throughout the lower third of the epithelium; the superficial cells are still flattened and show signs of differentiation, but this is incomplete. (C) In high-grade intraepithelial neoplasia, cells in all the epithelial layers are proliferating and exhibit defective differentiation. (D) True malignancy begins when the cells move through or destroy the basal lamina that underlies the basal layer of epithelium and invade the underlying connective tissue. (Photographs courtesy of Andrew J. Connolly.)

1	Figure 20–9 Clonal evolution. In this schematic diagram, a tumor develops through repeated rounds of mutation and proliferation, giving rise eventually to a clone of fully malignant cancer cells. At each step, a single cell undergoes a mutation that either enhances cell proliferation or decreases cell death, so that its progeny become the dominant clone in the tumor. Proliferation of each clone hastens the occurrence of the next step of tumor progression by increasing the size of the cell population that is at risk of undergoing an additional mutation. The final step depicted here is invasion through the basement membrane, an initial step in metastasis. In reality, there are more than the three steps shown here, and a combination of genetic and epigenetic changes are involved. Not shown here is the fact that, over time, a variety of competing subclones will often arise in a tumor. As we will discuss later, this heterogeneity complicates cancer therapies (see Figure 20–30).

1	Most human cancer cells accumulate genetic changes at an abnormally rapid rate and are said to be genetically unstable. The extent of this instability and its molecular origins differ from cancer to cancer and from patient to patient, as we shall discuss in a later section. The basic phenomenon was evident even before modern molecular analyses. For example, the cells of many cancers show grossly abnormal sets of chromosomes, with duplications, deletions, and translocations that are visible at mitosis (Figure 20–10). When the cells are maintained in culture, these patterns of chromosomal disruption can often be seen to evolve rapidly and in a seemingly haphazard way. And for many years, pathologists have used an abnormal appearance of the cell nucleus to identify and classify cancer cells in tumor biopsies; in particular, cancer cells can contain an unusually large amount of heterochromatin—a condensed form of interphase chromatin that silences genes (see pp. 194–195). This suggested

1	in tumor biopsies; in particular, cancer cells can contain an unusually large amount of heterochromatin—a condensed form of interphase chromatin that silences genes (see pp. 194–195). This suggested that epigenetic changes of chromatin structure can also contribute to the cancer cell phenotype, as recently confirmed by molecular analysis.

1	The genetic instability observed in cancer cells can arise from defects in the ability to repair DNA damage or to correct replication errors of various kinds. These alterations lead to changes in DNA sequence and produce rearrangements such as DNA translocations and duplications. Also common are defects in chromosome segregation during mitosis, which provide another possible source of chromosome instability and changes in karyotype. From an evolutionary perspective, none of this should be a surprise: anything that increases the probability of random changes in gene function heritable from one cell generation to the next—and that is not too deleterious—is likely to speed the evolution of a clone of cells toward malignancy, thereby causing this property to be selected for during tumor progression. epithelial cells growing on accidental production basal lamina of mutant cell cell with cell with

1	epithelial cells growing on accidental production basal lamina of mutant cell cell with cell with Figure 20–10 Chromosomes from a breast tumor displaying abnormalities in structure and number. Chromosomes were prepared from a breast tumor cell in metaphase, spread on a glass slide, and stained with (A) a general DNA stain or (B) a combination of fluorescently labeled DNA molecules that color each normal human chromosome differently (see Figure 4–10). The staining (displayed in false color) shows multiple translocations, including a doubly translocated chromosome (white arrow) that is made up of two pieces of chromosome 8 (green-brown) and a piece of chromosome 17 (purple). The karyotype also contains 48 chromosomes, instead of the normal 46. (Courtesy of Joanne Davidson and Paul Edwards.) Cancer Cells Display an Altered Control of Growth

1	Mutability and large cell population numbers create the opportunities for mutations to occur, but the driving force for development of a cancer has to come from some sort of selective advantage possessed by the mutant cells. Most obviously, a mutation or epigenetic change can confer such an advantage by increasing the rate at which a clone of cells proliferates or by enabling it to continue proliferating when normal cells would stop. Cancer cells that can be grown in culture, or cultured cells artificially engineered to contain the types of mutations encountered in cancers, typically show a transformed phenotype. They are abnormal in their shape, their motility, their responses to growth factors in the culture medium, and, most characteristically, in the way they react to contact with the substratum and with one another. Normal cells will not divide unless they are attached to the substratum; transformed cells will often divide even if held in suspension. Normal cells become inhibited

1	substratum and with one another. Normal cells will not divide unless they are attached to the substratum; transformed cells will often divide even if held in suspension. Normal cells become inhibited from moving and dividing when the culture reaches confluence (where the cells are touching one another); transformed cells continue moving and dividing even after confluence, and so pile up in layer upon layer in the culture dish (Figure 20–11). In addition, transformed cells no longer require all of the positive signals from their surroundings that normal cells require.

1	Their behavior in culture gives a hint of the ways in which cancer cells may misbehave in their natural environment, embedded in a tissue. But cancer cells in the body show other peculiarities that mark them out from normal cells, beyond those just described.

1	Given sufficient oxygen, normal adult tissue cells will generally fully oxidize almost all the carbon in the glucose they take up to CO2, which is lost from the body as a waste product. A growing tumor needs nutrients in abundance to provide the building blocks to make new macromolecules. Correspondingly, most tumors have a metabolism more similar to that of a growing embryo than to that of normal adult tissue. Tumor cells consume glucose avidly, importing it from the blood at a rate that can be as much as 100 times higher than neighboring normal cells. Moreover, only a small fraction of this imported glucose is used for production of ATP by oxidative phosphorylation. Instead, a great deal of lactate is produced, and many of the remaining carbon atoms derived from glucose are diverted for use as raw materials for synthesis of the proteins, nucleic acids, and lipids required for tumor growth (Figure 20–12).

1	This tendency of tumor cells to de-emphasize oxidative phosphorylation even when oxygen is plentiful, while at the same time taking up large quantities of glucose, can be shown to promote cancer cell growth and is called the Warburg contact-inhibited monolayer transformed cells foci of uninhibited of normal cells in lose contact inhibition transformed cells tissue-culture dish Figure 20–11 Loss of contact inhibition by cancer cells in cell culture. Most normal cells stop proliferating once they have carpeted the dish with a single layer of cells: proliferation seems to depend on contact with the dish, and to be inhibited by contacts with other cells—a phenomenon known as “contact inhibition.” Cancer cells, in contrast, usually disregard these restraints and continue to grow, so that they pile up on top of one another, as shown (Movie 20.2). (A) Schematic drawing. (B and C) Light micrographs of normal (B) and transformed (C) fibroblasts. (B and C, courtesy of Lan Bo Chen.)

1	NET PRODUCTS: NET PRODUCTS: ENERGY, CO2, H2O ENERGY, BUILDING BLOCKS, NADPH effect—so named because Otto Warburg first noticed the phenomenon in the early twentieth century. It is this abnormally high glucose uptake that allows tumors to be selectively imaged in whole-body scans (see Figure 20–1), thereby providing a way to monitor cancer progression and responses to treatment. Cancer Cells have an Abnormal Ability to Survive Stress and DNA Damage In a large multicellular organism, there are powerful safety mechanisms that guard against the trouble that can be caused by damaged and deranged cells. For example, internal disorder gives rise to danger signals in the faulty cell, activating protective devices that can eventually lead to apoptosis (see Chapter 18). To survive, cancer cells require additional mutations to elude or break through these defenses against cellular misbehavior.

1	Cancer cells are found to contain mutations that drive the cell into an abnormal state, where metabolic processes may be unbalanced and essential cell components may be produced in ill-matched proportions. States of this type, where the cell’s homeostatic mechanisms are inadequate to cope with an imposed disturbance, are loosely referred to as states of cell stress. As one example, chromosome breakage and other forms of DNA damage are commonly observed during the development of cancer, reflecting the genetic instability that cancer cells display. Thus, to survive and divide without limit, a prospective cancer cell must accumulate mutations that disable the normal safety mechanisms that would otherwise induce a cell that is stressed, in this or in other ways, to commit suicide. In fact, one of the most important properties of many types of cancer cells is that they fail to undergo apoptosis when a normal cell would do so (Figure 20–13).

1	While cancer cells tend to avoid apoptosis, this does not mean that they rarely die. On the contrary, in the interior of a large solid tumor, cell death often occurs on a massive scale: living conditions are difficult, with severe competition among the cancer cells for oxygen and nutrients. Many die, but typically much more by necrosis than by apoptosis (Figure 20–14). The tumor grows because the cell birth rate outpaces the cell death rate, but often by only a small margin. For this reason, the time that a tumor takes to double in size can be far longer than the cell-cycle time of the tumor cells. human Cancer Cells Escape a Built-in Limit to Cell Proliferation Many normal human cells have a built-in limit to the number of times they can divide when stimulated to proliferate in culture: they permanently stop dividing Figure 20–12 The Warburg effect in tumor cells reflects a dramatic change in glucose uptake and sugar metabolism.

1	(A) Cells that are not proliferating will normally oxidize nearly all of the glucose that they import from the blood to produce ATP through the oxidative phosphorylation that takes place in their mitochondria. Only when deprived of oxygen will these cells generate most of their ATP from glycolysis, converting the pyruvate produced to lactate in order to regenerate the NAD+ that they need to keep glycolysis going (see Figure 2–47). (B) Tumor cells, by contrast, will generally produce abundant lactate even in the presence of oxygen. This results from a greatly increased rate of glycolysis that is fed by a very large increase in the rate of glucose import. In this way, tumor cells resemble the rapidly proliferating cells in embryos (and during tissue repair), which likewise require for biosynthesis a large supply of the small-molecule building blocks that can be produced from imported glucose (see also Figure 20–26).

1	1100 Chapter 20: Cancer after a certain number of population doublings (25–50 for human fibroblasts, for example). This cell-division-counting mechanism is termed replicative cell senescence, and it generally depends on the progressive shortening of the telomeres at the ends of chromosomes, a process that eventually changes their structure (discussed in Chapter 17). As discussed in Chapter 5, the replication of telomere DNA during S phase depends on the enzyme telomerase, which maintains a special telomeric DNA sequence that promotes the formation of protein cap structures to protect chromosome ends. Because many proliferating human cells (stem cells being an exception) are deficient in telomerase, their telomeres shorten with every division, and their protective caps deteriorate, creating a DNA damage signal. Eventually, the altered chromosome ends can trigger a permanent cell-cycle arrest, causing a normal cell to die.

1	Human cancer cells avoid replicative cell senescence in one of two ways. They can maintain the activity of telomerase as they proliferate, so that their telomeres do not shorten or become uncapped, or they can evolve an alternate mechanism based on homologous recombination (called ALT) for elongating their chromosome ends. Regardless of the strategy used, the result is that the cancer cells continue to proliferate under conditions when normal cells would stop. The Tumor Microenvironment Influences Cancer Development While the cancer cells in a tumor are the bearers of dangerous mutations and are often grossly abnormal, the other cells in the tumor—especially those of the supporting connective tissue, or stroma—are far from passive bystanders. The

1	Figure 20–13Both increased cell division and decreased apoptosis can contribute to tumorigenesis. In normal tissues, apoptosis balances cell division to maintain homeostasis (see Movie 18.1). During the development of cancer, either an increase in cell division or an inhibition of apoptosis can lead to the increased cell numbers important for tumorigenesis. The cells fated to undergo apoptosis are gray in this diagram. Both an increase in cell division and a decrease in apoptosis normally contribute to tumor growth.

1	Figure 20–14Cross-section of a colon adenocarcinoma that has metastasized to the lung. This tissue slice shows well-differentiated colorectal cancer cells forming cohesive glands in the lung. The metastasis has central pink areas of necrosis where dying cancer cells have outgrown their blood supply. Such anoxic regions are common in the interior of large tumors. (Courtesy of Andrew J. Connolly.) development of a tumor relies on a two-way communication between the tumor cells and the tumor stroma, just as the normal development of epithelial organs relies on communication between epithelial cells and mesenchymal cells (discussed in Chapter 22).

1	The stroma provides a framework for the tumor. It is composed of normal connective tissue containing fibroblasts and inflammatory white blood cells, as well as the endothelial cells that form blood and lymphatic vessels with their attendant pericytes and smooth muscle cells (Figure 20–15). As a carcinoma progresses, the cancer cells induce changes in the stroma by secreting signal proteins that alter the behavior of the stromal cells, as well as proteolytic enzymes that modify the extracellular matrix. The stromal cells in turn act back on the tumor cells, secreting signal proteins that stimulate cancer cell growth and division as well as proteases that further remodel the extracellular matrix. In these ways, the tumor and its stroma evolve together, like weeds and the ecosystem that they invade, and the tumor becomes dependent on its particular stromal cells. Experiments using mice indicate that the growth of some transplanted carcinomas depends on the tumor-associated fibroblasts

1	invade, and the tumor becomes dependent on its particular stromal cells. Experiments using mice indicate that the growth of some transplanted carcinomas depends on the tumor-associated fibroblasts and normal fibroblasts will not do. Such environmental requirements help to protect us from cancer, as we discuss next in considering the critical phenomenon called metastasis.

1	Cancer cells generally need to spread and multiply at new sites in the body in order to kill us, through a process called metastasis. This is the most deadly—and least understood—aspect of cancer, being responsible for 90% of cancer-associated deaths. By spreading through the body, a cancer becomes almost impossible to eradicate by either surgery or local irradiation. Metastasis is itself a multi-step process: the cancer cells first have to invade local tissues and vessels, move through the circulation, leave the vessels, and then establish new cellular colonies at distant sites (Figure 20–16). Each of these events is complex, and most of the molecular mechanisms involved are not yet clear.

1	For a cancer cell to become dangerous, it must break free of constraints that keep normal cells in their proper places and prevent them from invading neighboring tissues. Invasiveness is thus one of the defining properties of malignant tumors, which show a disorganized pattern of growth and ragged borders, with extensions into the surrounding tissue (see, for example, Figure 20–8). Although the underlying molecular changes are not well understood, invasiveness almost certainly requires a disruption of the adhesive mechanisms that normally keep cells tethered to their proper neighbors and to the extracellular matrix. For carcinomas, this change resembles the epithelial–mesenchymal transition (EMT) that occurs in some epithelial tissues during normal development (see p. 1042). The next step in metastasis—the establishment of colonies in distant organs— begins with entry into the circulation: the invasive cancer cells must penetrate the

1	The next step in metastasis—the establishment of colonies in distant organs— begins with entry into the circulation: the invasive cancer cells must penetrate the Figure 20–15 The tumor microenvironment plays a role in tumorigenesis. Tumors consist of many cell types, including cancer cells, endothelial cells, pericytes (vascular smooth muscle cells), fibroblasts, and inflammatory white blood cells. Communication among these and other cell types plays an important part in tumor development. Note, however, that only the cancer cells are thought to be genetically abnormal in a tumor. 1102 Chapter 20: Cancer normal epithelium cells grow as benign tumor in epithelium cells become invasive and enter capillary (fewer than 1 in 1000 cells will survive to form metastases) adhere to blood vessel escape from blood vessel colonize liver, forming wall in liver to form micrometastasis full-blown metastasis

1	Figure 20–16Steps in the process of metastasis. This example illustrates the spread of a tumor from an organ such as the bladder to the liver. Tumor cells may enter the bloodstream directly by crossing the wall of a blood vessel, as diagrammed here, or, more commonly perhaps, by crossing the wall of a lymphatic vessel that ultimately discharges its contents (lymph) into the bloodstream. Tumor cells that have entered a lymphatic vessel often become trapped in lymph nodes along the way, giving rise to lymph-node metastases. Studies in animals show that typically far fewer than one in every thousand malignant tumor cells that enter the bloodstream will colonize a new tissue so as to produce a detectable tumor at a new site.

1	wall of a blood or lymphatic vessel. Lymphatic vessels, being larger and having more flimsy walls than blood vessels, allow cancer cells to enter in small clumps; such clumps may then become trapped in lymph nodes, giving rise to lymph-node metastases. The cancer cells that enter blood vessels, in contrast, seem to do so singly. With modern techniques for sorting cells according to their surface properties, it has become possible in some cases to detect these circulating tumor cells (CTCs) in samples of blood from cancer patients, even though they are only a minute fraction of the total blood-cell population. These cells, in principle at least, provide a useful sample of the tumor-cell population for genetic analysis.

1	Of the cancer cells that enter the lymphatics or bloodstream, only a tiny proportion succeed in making their exit, settling in new sites, and surviving and proliferating there as founders of metastases. Experiments show that fewer than one in thousands, perhaps one in millions, manage this feat. The final step of colonization seems to be the most difficult: like the Vikings who landed on the inhospitable shores of Greenland, the migrant cells may fail to survive in the alien environment; or they may only thrive there for a short while to found a little colony—a micrometastasis—that then dies out (Movie 20.3). Many cancers are discovered before they have managed to found metastatic colonies and can be cured by destruction of the primary tumor. But on occasion, an undetected micrometastasis will remain dormant for many years, only to reveal its presence by erupting into growth to form a large secondary tumor long after the primary tumor has been removed.

1	Many Properties Typically Contribute to Cancerous Growth Clearly, to produce a cancer, a cell must acquire a range of aberrant properties—a collection of subversive new skills—as it evolves. Different cancers require different combinations of these properties. Nevertheless, cancers all share some common features. By definition, they all ignore or misinterpret normal social controls so as to proliferate and spread where normal cells would not. These defining properties are commonly combined with other features that help the miscreants to arise and thrive. A list of the key attributes of cancer cells in general would include the following, all of which we have just discussed: 1. They grow (biosynthesize) when they should not, aided by a metabolism shifted from oxidative phosphorylation toward aerobic glycolysis. 2. They go through the cell-division cycle when they should not. 3.

1	2. They go through the cell-division cycle when they should not. 3. They escape from their home tissues (that is, they are invasive) and survive and proliferate in foreign sites (that is, they metastasize). 4. They have abnormal stress responses, enabling them to survive and continue dividing in conditions of stress that would arrest or kill normal cells, and they are less prone than normal cells to commit suicide by apoptosis. 5. They are genetically and epigenetically unstable. 6. They escape replicative cell senescence, either by producing telomerase or by acquiring another way of stabilizing their telomeres. In the next section of the chapter, we examine the mutations and molecular mechanisms that underlie these and other properties of cancer cells.

1	In the next section of the chapter, we examine the mutations and molecular mechanisms that underlie these and other properties of cancer cells. Cancer cells, by definition, grow and proliferate in defiance of normal controls (that is, they are neoplastic) and are able to invade surrounding tissues and colonize distant organs (that is, they are malignant). By giving rise to secondary tumors, or metastases, they become difficult to eradicate by surgery or local irradiation. Cancers are thought to originate from a single cell that has experienced an initial mutation, but the progeny of this cell must undergo many further changes, requiring additional mutations and epigenetic events, to become cancerous. Tumor progression usually takes many years and reflects the operation of a Darwinian-like process of evolution, in which somatic cells undergo mutation and epigenetic changes accompanied by natural selection.

1	Cancer cells acquire a variety of special properties as they evolve, multiply, and spread. Their mutant genomes enable them to grow and divide in defiance of the signals that normally keep cell proliferation under tight control. As part of the evolutionary process of tumor progression, cancer cells acquire a collection of additional abnormalities, including defects in the controls that permanently stop cell division or induce apoptosis in response to cell stress or DNA damage, and in the mechanisms that normally keep cells from straying from their proper place. All of these changes increase the ability of cancer cells to survive, grow, and divide in their original tissue and then to metastasize, founding new colonies in foreign environments. The evolution of a tumor also depends on other cells present in the tumor microenvironment, collectively called stromal cells, that the cancer attracts and manipulates.

1	Since many changes are needed to confer this collection of asocial behaviors, it is not surprising that most cancer cells are genetically and/or epigenetically unstable. This instability is thought to be selected for in the clones of aberrant cells that are able to produce tumors, because it greatly accelerates the accumulation of the further genetic and epigenetic changes that are required for tumor progression. CANCER-CRITICAL GENES: hOW ThEY ARE FOUND AND WhAT ThEY DO

1	As we have seen, cancer depends on the accumulation of inherited changes in somatic cells. To understand it at a molecular level we need to identify the mutations and epigenetic changes involved and to discover how they give rise to cancerous cell behavior. Finding the relevant cells is often easy; they are favored by natural selection and call attention to themselves by giving rise to tumors. But how do we identify those genes with the cancer-promoting changes among all the other genes in the cancerous cells? A typical cancer depends on a whole set of mutations and epigenetic changes—usually a somewhat different set in each individual patient. In addition, a given cancer cell will also contain a large number of somatic mutations that are accidental by-products—so-called passengers rather than drivers—of its genetic instability, and it can be difficult to distinguish these meaningless changes from those changes that have a causative role in the disease. Despite these difficulties,

1	than drivers—of its genetic instability, and it can be difficult to distinguish these meaningless changes from those changes that have a causative role in the disease. Despite these difficulties, many of the genes that are repeatedly altered in human cancers have been identified over the past 40 years. We will call such genes, for want of a better term, cancer-critical genes, meaning all genes whose alteration contributes to the causation or evolution of cancer by driving tumorigenesis.

1	In this section, we shall first discuss how cancer-critical genes are identified. We shall then examine their functions and the parts they play in conferring on cancer cells the properties outlined in the first part of the chapter. We shall end the section by discussing colon cancer as an extended example, showing how a succession of changes in cancer-critical genes enables a tumor to evolve from one pattern of bad behavior to another that is worse. The Identification of Gain-of-Function and Loss-of-Function Cancer Mutations has Traditionally Required Different Methods

1	Cancer-critical genes are grouped into two broad classes, according to whether the cancer risk arises from too much activity of the gene product or too little. Genes of the first class, in which a gain-of-function mutation can drive a cell toward cancer, are called proto-oncogenes; their mutant, overactive or overexpressed forms are called oncogenes. Genes of the second class, in which a loss-of-function mutation can contribute to cancer, are called tumor suppressor genes. In either case, the mutation may lead toward cancer directly (by causing cells to proliferate when they should not) or indirectly—for example, by causing genetic or epigenetic instability and so hastening the occurrence of other inherited changes that directly stimulate tumor growth. Those genes whose alteration results in genomic instability represent a subclass of cancer-critical genes that are sometimes called genome maintenance genes.

1	As we shall see, mutations in oncogenes and tumor suppressor genes can have similar effects in promoting the development of cancer; overproduction of a signal for cell proliferation, for example, can result from either kind of mutation. Thus, from the point of view of a cancer cell, oncogenes and tumor suppressor genes—and the mutations that affect them—are flip sides of the same coin. The techniques that led to the discovery of these two categories of genes, however, are quite different.

1	The mutation of a single copy of a proto-oncogene that converts it to an oncogene has a dominant, growth-promoting effect on a cell (Figure 20–17A). Thus, we can identify the oncogene by its effect when it is added—by DNA transfection, for example, or through infection with a viral vector—to the genome of a suitable type of tester cell or experimental animal. In the case of the tumor suppressor gene, on the other hand, the cancer-causing alleles produced by the change are generally recessive: often (but not always) both copies of the normal gene must be removed or inactivated in the diploid somatic cell before an effect is seen (Figure 20–17B). This calls for a different experimental approach, one focusing on discovering what is missing in the cancer cell. functionally eliminate the tumor suppressor gene, promoting cell transformation

1	functionally eliminate the tumor suppressor gene, promoting cell transformation We begin by discussing some examples of each class of cancer-critical genes to illustrate basic principles. These examples are chosen also for their historical importance: the experiments that led to their discovery—at different times and by different methods—marked turning points in the understanding of cancer. Retroviruses Can Act as Vectors for Oncogenes That Alter Cell Behavior The search for the genetic causes of human cancer took a devious route, beginning with clues that came from the study of tumor viruses. Although viruses are involved only in a minority of human cancers, a set of viruses that infect animals provided critical early tools for studying cancer.

1	One of the first animal viruses to be implicated in cancer was discovered over 100 years ago in chickens, when an infectious agent that causes connective-tissue tumors, or sarcomas, was characterized as a virus—the Rous sarcoma virus. Like all the other RNA tumor viruses discovered since, it is a retrovirus. When it infects a cell, its RNA genome is copied into DNA by reverse transcription, and the DNA is inserted into the host genome, where it can persist and be inherited by subsequent generations of cells. Something in the DNA inserted by the Rous sarcoma virus made the host cells cancerous, but what was it? The answer was a surprise. It turned out to be a piece of DNA that was unnecessary for the virus’s own survival or reproduction; instead, it was a passenger, a gene called v-Src, that the virus had picked up on its travels. v-Src was unmistakably similar, but not identical, to a gene—c-Src—that was discovered in the normal vertebrate genome. c-Src had evidently been caught up

1	that the virus had picked up on its travels. v-Src was unmistakably similar, but not identical, to a gene—c-Src—that was discovered in the normal vertebrate genome. c-Src had evidently been caught up accidentally by the retrovirus from the genome of a previously infected host cell, and it had undergone mutation in the process to become an oncogene (v-Src).

1	This Nobel Prize-winning finding was followed by a flood of discoveries of other viral oncogenes carried by retroviruses that cause cancer in nonhuman animals. Each such oncogene turned out to have a counterpart proto-oncogene in the normal vertebrate genome. As was the case for Src, these other oncogenes generally differed from their normal counterparts, either in structure or in level of expression. But how did this relate to typical human cancers, most of which are not infectious and in which retroviruses play no part? Figure 20–17 Cancer-critical mutations fall into two readily distinguishable categories, dominant and recessive.

1	Figure 20–17 Cancer-critical mutations fall into two readily distinguishable categories, dominant and recessive. In this diagram, activating mutations are represented by solid red boxes, inactivating mutations by hollow red boxes. (A) Oncogenes act in a dominant manner: a gain-of-function mutation in a single copy of the cancer-critical gene can drive a cell toward cancer. (B) Mutations in tumor suppressor genes, on the other hand, generally act in a recessive manner: the function of both alleles of the cancer-critical gene must be lost to drive a cell toward cancer. Although in this diagram the second allele of the tumor suppressor gene is inactivated by mutation, it is often inactivated instead by loss of the second chromosome. Not shown is the fact that mutation of some tumor suppressor genes can have an effect even when only one of the two gene copies is damaged. Different Searches for Oncogenes Converged on the Same Gene—Ras

1	Different Searches for Oncogenes Converged on the Same Gene—Ras In an attempt to answer the above question, other researchers searched directly for oncogenes in the genomes of human cancer cells. They did this by searching for DNA fragments from cancer cells that could provoke uncontrolled proliferation when introduced into noncancerous cell lines. As tester cells for the assay, cell lines derived from mouse fibroblasts were used. These cells had been previously selected for their ability to proliferate indefinitely in culture, and they are thought to already contain alterations that take them part of the way toward malignancy. For this reason, the addition of a single oncogene can sometimes be enough to produce a dramatic effect.

1	When DNA was extracted from the human tumor cells, broken into fragments, and introduced into the cultured cells, occasional colonies of abnormally proliferating cells began to appear in the culture dish. These cells showed a transformed phenotype, outgrowing the untransformed cells in the culture and piling up in layer upon layer (see Figure 20–11). Each colony was a clone originating from a single cell that had incorporated a DNA fragment that drove cancerous behavior. This fragment, which carried markers of its human origin, could be isolated from the transformed cultured mouse cells. And once isolated and sequenced, it could be recognized: it contained a human version of a gene already known from study of a retrovirus that caused tumors in rats—an oncogene called v-Ras.

1	The newly discovered oncogene was clearly derived by mutation from a normal human gene, one of a small family of proto-oncogenes called Ras. This discovery in the early 1980s of the same oncogene in human tumor cells and in an animal tumor virus was electrifying. The implication that cancers are caused by mutations in a limited number of cancer-critical genes transformed our understanding of the molecular biology of cancer.

1	As discussed in Chapter 15, normal Ras proteins are monomeric GTPases that help transmit signals from cell-surface receptors to the cell interior (see Movie 15.7). The Ras oncogenes isolated from human tumors contain point mutations that create a hyperactive Ras protein that cannot shut itself off by hydrolyzing its bound GTP to GDP. Because this makes the protein hyperactive, its effect is dominant—that is, only one of the cell’s two gene copies needs to change to have an effect. One or another of the three human Ras family members is mutated in perhaps 30% of all human cancers. Ras genes are thus among the most important of all cancer-critical genes. Figure 20–18 summarizes the types of accidents that can convert a proto-oncogene into an oncogene. (1) A small change in DNA sequence such as a point Figure 20–18 The types of accidents that can convert a proto-oncogene into an oncogene.

1	Figure 20–18 The types of accidents that can convert a proto-oncogene into an oncogene. extracellular domain binding of growth factor of receptor triggers intracellular signaling mutation or deletion may produce a hyperactive protein when it occurs within a protein-coding sequence, or lead to protein overproduction when it occurs within a regulatory region for that gene. (2) Gene amplification events, such as those that can be caused by errors in DNA replication, may produce extra gene copies; this can lead to overproduction of the protein. (3) A chromosomal rearrangement— involving the breakage and rejoining of the DNA helix—may either change the protein-coding region, resulting in a hyperactive fusion protein, or alter the control regions for a gene so that a normal protein is overproduced.

1	As one example, the receptor for the extracellular signal protein epidermal growth factor (EGF) can be activated by a deletion that removes part of its extracellular domain, causing it to be active even in the absence of EGF (Figure 20–19). It thus produces an inappropriate stimulatory signal, like a faulty doorbell that rings even when nobody is pressing the button. Mutations of this type are frequently found in the most common type of human brain tumor, called glioblastoma.

1	As another example, the Myc protein, which acts in the nucleus to stimulate cell growth and division (see Chapter 17), generally contributes to cancer by being overproduced in its normal form. In some cases, the gene is amplified— that is, errors of DNA replication lead to the creation of large numbers of gene copies in a single cell. Or a point mutation can stabilize the protein, which normally turns over very rapidly. More commonly, the overproduction appears to be due to a change in a regulatory element that acts on the gene. For example, a chromosomal translocation can inappropriately bring powerful gene regulatory sequences next to the Myc protein-coding sequence, so as to produce unusually large amounts of Myc mRNA. Thus, in Burkitt’s lymphoma, a translocation brings the Myc gene under the control of sequences that normally drive the expression of antibody genes in B lymphocytes. As a result, the mutant B cells tend to proliferate excessively and form a tumor. Different specific

1	the control of sequences that normally drive the expression of antibody genes in B lymphocytes. As a result, the mutant B cells tend to proliferate excessively and form a tumor. Different specific chromosome translocations are common in other cancers.

1	Studies of Rare hereditary Cancer Syndromes First Identified Tumor Suppressor Genes Identifying a gene that has been inactivated in the genome of a cancer cell requires a different strategy from finding a gene that has become hyperactive: one cannot, for example, use a cell transformation assay to identify something that simply is not there. The key insight that led to the discovery of the first tumor suppressor gene came from studies of a rare type of human cancer, retinoblastoma, which arises from cells in the retina of the eye that are converted to a cancerous state by an unusually small number of mutations. As often happens in biology, the discovery arose from examination of a special case, but it turned out to reveal a gene of widespread importance. Retinoblastoma occurs in childhood, and tumors develop from neural precursor cells in the immature retina. About one child in 20,000 is afflicted. One form of the disease is hereditary, and the other is not. In the hereditary form,

1	Figure 20–19 Mutation of the epidermal growth factor (EGF) receptor can make it active even in the absence of EGF, and consequently oncogenic. Only one of the possible types of activating mutations is illustrated here. NORMAL, HEALTHY INDIVIDUAL HEREDITARY RETINOBLASTOMA NONHEREDITARY RETINOBLASTOMA occasional cell inactivates one of its two good Rb genes excessive cell proliferation leading to retinoblastoma excessive cell proliferation leading to retinoblastoma

1	RESULT: MOST PEOPLE WITH INHERITED RESULT: ONLY ABOUT 1 IN 30,000 RESULT: NO TUMOR multiple tumors usually arise independently, affecting both eyes; in the nonhereditary form, only one eye is affected, and by only one tumor. A few individuals with retinoblastoma have a visibly abnormal karyotype, with a deletion of a specific band on chromosome 13 that, if inherited, predisposes an individual to the disease. Deletions of this same region are also encountered in tumor cells from some patients with the nonhereditary disease, which suggested that the cancer was caused by loss of a critical gene in that location.

1	Using the location of this chromosomal deletion, it was possible to clone and sequence the Rb gene. It was then discovered that those who suffer from the hereditary form of the disease have a deletion or loss-of-function mutation present in one copy of the Rb gene in every somatic cell. These cells are predisposed to becoming cancerous, but do not do so if they retain one good copy of the gene. The retinal cells that are cancerous are defective in both copies of Rb because of a somatic event that has eliminated the function of the previously good copy. In patients with the nonhereditary form of the disease, by contrast, the noncancerous cells show no defect in either copy of Rb, while the cancerous cells have become defective in both copies. These nonhereditary retinoblastomas are very rare because they require two independent events that inactivate the same gene on two chromosomes in a single retinal cell lineage (Figure 20–20).

1	The Rb gene is also missing in several common types of sporadic cancer, including carcinomas of lung, breast, and bladder. These more common cancers arise by a more complex series of genetic changes than does retinoblastoma, and they make their appearance much later in life. But in all of them, it seems, loss of Rb function is frequently a major step in the progression toward malignancy. The Rb gene encodes the Rb protein, which is a universal regulator of the cell cycle present in almost all cells of the body (see Figure 17–61). It acts as one of the main brakes on progress through the cell-division cycle, and its loss can allow cells to enter the cell cycle inappropriately, as we discuss later.

1	For tumor suppressor genes, it is their inactivation that is dangerous. This inactivation can occur in many ways, with different combinations of mishaps serving to eliminate or cripple both gene copies. The first copy may, for example, be lost by a small chromosomal deletion or inactivated by a point mutation. The second copy is commonly eliminated by a less specific and more probable mechanism: Figure 20–20 The genetic mechanisms that cause retinoblastoma. In the hereditary form, all cells in the body lack one of the normal two functional copies of the Rb tumor suppressor gene, and tumors occur where the remaining copy is lost or inactivated by a somatic event (either mutation or epigenetic silencing). In the nonhereditary form, all cells initially contain two functional copies of the gene, and the tumor arises because both copies are lost or inactivated through the coincidence of two somatic events in a single line of cells.

1	the chromosome carrying the remaining normal copy may be lost from the cell through errors in chromosome segregation; or the normal gene, along with neighboring genetic material, may be replaced by a mutant version through either a mitotic recombination event or a gene conversion that accompanies it (see p. 286). Figure 20–21 summarizes the range of ways in which the remaining good copy of a tumor suppressor gene can be lost through a DNA sequence change, using the Rb gene as an example. It is important to note that, except for the point mutation mechanism illustrated at the far right, these pathways all produce cells that carry only a single type of DNA sequence in the chromosomal region containing their Rb genes—a sequence that is identical to the sequence in the original mutant chromosome.

1	Epigenetic changes provide another important way to permanently inactivate a tumor suppressor gene. Most commonly, the gene may become packaged into heterochromatin and/or the C nucleotides in CG sequences in its promoter may become methylated in a heritable manner (see pp. 404–405). These mechanisms can irreversibly silence the gene in a cell and in all of its progeny. Analysis of methylation patterns in cancer genomes shows that epigenetic gene silencing is a frequent event in tumor progression, and epigenetic mechanisms are now thought to help inactivate several different tumor suppressor genes in most human cancers (Figure 20–22). Systematic Sequencing of Cancer Cell Genomes has Transformed Our Understanding of the Disease

1	Systematic Sequencing of Cancer Cell Genomes has Transformed Our Understanding of the Disease Methods such as those we have described above shone a spotlight on a set of can-cer-critical genes that were identified in a piecemeal fashion. Meanwhile, the rest of the cancer cell genome remained in darkness: it was a mystery how many other mutations might lurk there, of what types, in which varieties of cancer, at what frequencies, with what variations from patient to patient, and with what consequences. With the sequencing of the human genome and the dramatic advances in DNA sequencing technology (see Panel 8–1, pp. 478–481), it has become possible to see the whole picture—to view cancer cell genomes in their entirety. This transforms our understanding of the disease.

1	Cancer cell genomes can be scanned systematically in several different ways. At one extreme—the most costly, but no longer prohibitively so—one can determine a tumor’s complete genome sequence. More cheaply, one can focus just on the 21,000 or so genes in the human genome that code for protein (the so-called exome), looking for mutations in the cancer cell DNA that alter the amino acid sequence of the product or prevent its synthesis (Figure 20–23). There are also efficient techniques to survey the genome for regions that have undergone

1	Figure 20–21 Six ways of losing the remaining good copy of a tumor suppressor gene through a change in DNA sequences. A cell that is defective in only one of its two copies of a tumor suppressor gene—for example, the Rb gene—usually behaves as a normal, healthy cell; the diagrams below show how this cell may lose the function of the other gene copy as well and thereby progress toward cancer. A seventh possibility, frequently encountered with some tumor suppressors, is that the gene may be silenced by an epigenetic change, without alteration of the DNA sequence, as illustrated in Figure 20–22. (After

1	W.K. Cavenee et al., Nature 305:779–784, 1983. With permission from Macmillan Publishers Ltd.) deletion or duplication, without the need for complete sequence information. The genome can be scanned for epigenetic changes. And finally, alterations in levels of gene expression can be systematically determined by analysis of mRNAs (see Figure 7–3). These approaches generally involve comparing cancer cells with normal controls—ideally, noncancerous cells originating in the same tissue and from the same patient. Figure 20–22 The pathways leading to loss of tumor suppressor gene function in cancer involve both genetic and epigenetic changes. (A) As indicated, the changes that silence tumor suppressor genes can occur in any order. Both DNA methylation and the packaging of a gene into condensed chromatin can prevent its expression in a way that is inherited when a cell divides (see Figure 4–44).

1	(B) The frequency of gene silencing by hypermethylation observed in four different types of cancer. The five genes listed at the top can all function as tumor suppressor genes; BRCA1 and hMLH1 affect genome stability and are in the subclass known as genome maintenance genes. ND, no data. (Adapted from M. Esteller et al., Cancer Res. 61:3225–3229, 2001.)

1	Figure 20–23 The distinct types of DNA sequence changes found in oncogenes compared to tumor suppressor genes. In this diagram, mutations that change an amino acid are denoted by blue arrowheads, whereas mutations that truncate the polypeptide chain are marked by yellow arrowheads. (A) As in this example, oncogene mutations can be detected by the fact that the same nucleotide change is repeatedly found among the missense mutations in a gene. (B) For tumor suppressor genes, by contrast, missense mutations that abort protein synthesis by creating stop codons predominate. (Adapted from B. Vogelstein et al., Science 339:1546–1558, 2013.) chromosome alterations found in the particular primary tumor. As indicated, purple lines connect sites at which two different chromosomes have become joined to create an interchromosomal rearrangment, while green lines connect the sites of rearrangements found within a single chromosome. The intrachromosomal rearrangements can be seen to predominate, and

1	to create an interchromosomal rearrangment, while green lines connect the sites of rearrangements found within a single chromosome. The intrachromosomal rearrangements can be seen to predominate, and most join neighboring sections of DNA that were originally located within 2 million nucleotide pairs of each other. The increases in copy number,

1	CANCER-CRITICAL GENES: hOW ThEY ARE FOUND AND WhAT ThEY DO

1	Cancer genome analysis reveals, first of all, the scale of gross genetic disruption in cancer cells. This varies greatly from one type of cancer and one cancer patient to another, both in severity and in character. In some cases, the karyotype—the set of chromosomes as they appear at mitosis—is normal or nearly so, but many point mutations are detected in individual genes, suggesting a failure of the repair mechanisms that normally correct local errors in the replication or maintenance of DNA sequences. Often, however, the karyotype is severely disordered, with many chromosome breaks and rearrangements. In some breast cancers, for example, genome sequencing reveals an astonishing scene of genetic chaos (Figure 20–24), with hundreds of chromosome breaks and translocations, resulting in many deletions, duplications, and amplifications of parts of the genome. In such cells, the normal machinery for avoidance or repair of DNA double-strand breaks is evidently somehow defective,

1	in many deletions, duplications, and amplifications of parts of the genome. In such cells, the normal machinery for avoidance or repair of DNA double-strand breaks is evidently somehow defective, destabilizing the genome by giving rise to broken chromosomes whose fragments then rejoin in random combinations. From the pattern of changes, one can infer that this disruptive process has occurred repeatedly during the evolution of the tumor, with a progressive increase of genetic disorder. Breast cancers showing the most extreme chromosome disorder are usually hard to treat and have a gloomy prognosis.

1	One survey of more than 3000 individual cancer specimens showed that on average 24 separate blocks of genetic material were duplicated in each tumor, amounting to 17% of the normal genome, and 18 blocks were deleted, amounting to 16% of the normal genome. Many of these changes were found repeatedly, suggesting that they contain cancer-critical genes whose loss (tumor suppressor genes) or gain (oncogenes) confers a selective advantage.

1	Whole-genome analysis also helps to explain some cancers that seem, at first sight, to be exceptions to the general rules. An example is retinoblastoma, with its early onset during childhood. If cancers in general require an accumulation of many genetic changes and are thus diseases of old age, what makes retinoblastoma different? Whole-genome sequencing confirms that in retinoblastoma, the tumor cells contain loss-of-function mutations in the Rb gene; but, astonishingly, they contain practically no mutations or genome rearrangements that affect any other oncogene or tumor suppressor gene. Instead, they contain many epigenetic modifications, which alter the level of expression of many known cancer-critical genes—as many as 15 in one well-analyzed case. Many Mutations in Tumor Cells are Merely Passengers

1	Many Mutations in Tumor Cells are Merely Passengers Cancer cells generally contain many mutations in addition to gross chromosome abnormalities: point mutations can be scattered over the genome as a whole at a rate of about one per million nucleotide pairs, in addition to the abnormalities Figure 20–24 The chromosomal rearrangements in breast cancer cells. The results of an extensive DNA sequencing analysis performed on two different primary tumors are displayed as “Circos plots.” In each plot, the reference DNA sequences of the 22 autosomes and single sex chromosome (X) of a normal human female (3.2 billion nucleotide pairs) are aligned end-to-end to form a circle. Colored lines within the circle are then used to indicate the shown in blue, reveal the amplified DNA sequences (see the highly amplified regions indicated). (Adapted from P.J. Stephens et

1	BREAST CANCER 1 BREAST CANCER 2 al., Nature 462:1005–1010, 2009.) attributed to chromosome breakage and rejoining. Systematic surveys of the pro-tein-coding genes in common solid tumors—such as those of the breast, colon, brain, or pancreas—have revealed that an average of 33 to 66 genes have undergone somatic mutation affecting the sequence of their protein product. Mutations in noncoding regions of the genome are much more numerous, as one would expect from the much larger fraction of the genome that noncoding DNA represents. But they are considerably more difficult to interpret.

1	The high frequency of mutations testifies to the genetic instability of many cancer cells, but it leaves us with a difficult problem. How can we discover which of the mutations are drivers of cancer—that is, causal factors in the development of the disease—and which are merely passengers—mutations that happen to have occurred in the same cell as the driver mutations, thanks to genetic instability, but are irrelevant to the development of the disease? A simple criterion is based on frequency of occurrence. Driver mutations affecting a gene that plays a part in the disease will be seen repeatedly, in many different patients. In contrast, passenger mutations, occurring at more-or-less random locations in the genome and conferring no selective advantage on the cancer cell, are unlikely to be found in the same genes in different patients.

1	Figure 20–25 shows the results of an analysis of this sort for a large sample of colorectal cancers. The different sites in the genome are laid out on a two-dimensional array, with chromosome serial number along one axis and position within each chromosome along the other. The frequency with which mutations are encountered is shown by height above this plane, creating a mutation “landscape” with mountains (sites where mutations are found in a large proportion of the tumors in the sample), hills (where mutations are found less frequently but still more often than would be expected for a random scattering over the genome), and hillocks (sites of occasional mutations, occurring at a frequency no higher than would be expected for mutations scattered at random in each individual tumor). The mountains and the hills are strong candidates to be the sites of driver mutations—in other words, sites of cancer-critical genes; the hillocks are likely to correspond to passengers. Indeed, many of the

1	and the hills are strong candidates to be the sites of driver mutations—in other words, sites of cancer-critical genes; the hillocks are likely to correspond to passengers. Indeed, many of the mountains and hills turn out to be sites of known oncogenes or tumor suppressor genes, whereas the hillocks mostly correspond to genes that have no known or probable role in causation of cancer. Of course, some hillocks may correspond to genes that are mutated in only a few rare patients but are nevertheless cancer-critical for them.

1	About One Percent of the Genes in the human Genome Are Cancer-Critical From studies such as the one just described, it is estimated that the number of driver mutations for an individual case of cancer (the sum of meaningful epigenetic and genetic changes in both coding sequences and regulatory regions) is typically on the order of 10, explaining why cancer progression generally involves an increase in genetic and/or epigenetic instability that enhances the rate of such changes.

1	Figure 20–25 The mutation landscape in colorectal cancer. In this two-dimensional representation of the human genome, the green surface depicts the 22 human autosomes plus the X sex chromosome as being laid out side-by-side in numerical order from left to right, with the DNA sequence of each chromosome running from back to front. The mountains represent the locations of genes mutated with high frequency in different, independent tumors. As indicated, these are suspected driver mutations in the adenomatous polyposis coli (APC), K-Ras, p53, phosphoinositide 3-kinase (PIK3CA), and ubiquitin ligase (FBXW7) proteins. (Adapted from L.D. Wood et al., Science 318:1108–1113, 2007.)

1	By compiling the data for different types of cancer, each with its own range of identified driver mutations, we can develop a comprehensive catalog of genes that are strongly suspected to be cancer-critical. Current estimates put the total number of such genes at about 300, about 1% of the genes in the human genome. These cancer-critical genes are amazingly diverse. Their products include secreted signal proteins, transmembrane receptors, GTP-binding proteins, protein kinases, transcription regulators, chromatin modifiers, DNA repair enzymes, cell–cell adhesion molecules, cell-cycle controllers, apoptosis regulators, scaffold proteins, metabolic enzymes, components of the RNA splicing machinery, and more besides. All these are susceptible to mutations that can contribute, in one way or another, in one tissue or another, to the evolution of cells with the cancerous properties that we listed earlier on page 1103.

1	Clearly, the molecular changes that cause cancer are complex. As we now explain, however, the complexity is not quite as daunting as it may initially seem. Disruptions in a handful of Key Pathways Are Common to Many Cancers

1	Disruptions in a handful of Key Pathways Are Common to Many Cancers Some genes, like Rb and Ras, are mutated in many cases of cancer and in cancers of many different types. The involvement of genes such as Rb and Ras in cancer is no surprise, now that we understand their normal functions: they control fundamental processes of cell division and growth. But even these common culprits feature in considerably less than half of individual cases. What is happening to the control of these processes in the many cases of cancer where, for example, Rb is intact or Ras is not mutated? What part do mutations in the hundreds of other cancer-critical genes play in the development of the disease? With our increasing knowledge of the normal functions of the genes in the human genome, it is becoming easier to see patterns in the cataloged driver mutations and to give some simplifying answers to these questions.

1	Glioblastoma—the commonest type of human brain tumor—provides a good example. Analysis of the genomes of tumor cells from 91 patients identified a total of at least 79 genes that were mutated in more than one individual. The normal functions of most of these genes were known or could be guessed, allowing them to be assigned to specific biochemical or regulatory pathways. Three functional groupings stood out, accounting for a total of 21 of the recurrently mutated genes. One of these groupings consisted of genes in the Rb pathway (that is, Rb itself, along with genes that directly regulate Rb); this pathway governs initiation of the cell-division cycle. Another consisted of genes in the same regulatory subnetwork as Ras—a more loosely defined system of genes referred to as the RTK/Ras/PI3K pathway, after three of its core components; this pathway serves to transmit signals for cell growth and cell division from the cell exterior into the heart of the cell. The third grouping consisted

1	pathway, after three of its core components; this pathway serves to transmit signals for cell growth and cell division from the cell exterior into the heart of the cell. The third grouping consisted of genes in a pathway regulating responses to stress and DNA damage—the p53 pathway. We shall have more to say about each of these pathways below.

1	Out of all tumors, 74% had identifiable mutations in all three pathways. If one were to trace these three pathways further upstream and include all the components, known and unknown, on which they depend, this percentage would almost certainly be even higher. In other words, in almost every case of glioblastoma, there are mutations that disrupt each of three fundamental controls: the control of cell growth, the control of cell division, and the control of responses to stress and DNA damage. Strikingly, in any given tumor-cell clone, there is a strong tendency for no more than one gene to be mutated in each pathway. Evidently, what matters for tumor evolution is the disruption of the control mechanism, and not the genetic means by which that is achieved. Thus, for example, in a patient whose tumor cells have no mutation in Rb itself, there is generally a mutation in some other component of the Rb pathway, producing a similar biological effect.

1	Similar patterns are seen in other types of cancers. A survey of many specimens of the major variety of ovarian cancer, for example, identified 67% of patients as having mutations in the Rb pathway, 45% in the Ras/PI3K pathway (defined more narrowly than in the glioblastoma study), and more than 96% in the p53 pathway. Allowing for additional pathway components not included in the analysis, it seems that most cases of this type of cancer, too, have mutations disrupting the same three controls, leading to misregulated cell growth, misregulated cell proliferation, and abnormal disregard of stress and DNA damage. It seems that these three fundamental controls are subverted in one way or another in virtually every type of cancer. We have devoted an entire chapter to the cell cycle and growth controls (Chapter 17). Some important details of the other two control pathways are reviewed next. Mutations in the PI3K/Akt/mTOR Pathway Drive Cancer Cells to Grow

1	Mutations in the PI3K/Akt/mTOR Pathway Drive Cancer Cells to Grow Cell proliferation is not simply a matter of progression through the cell cycle; it also requires cell growth, which involves complex anabolic processes through which the cell synthesizes all the necessary macromolecules from small-molecule precursors. If a cell divides inappropriately without growing first, it will get smaller at each division and will ultimately die or become too small to divide. Cells appear to require two separate signals to grow and divide (Figure 20–26). Cancer depends, therefore, not only on a loss of restraints on cell-cycle progression, but also on disrupted control of cell growth. The phosphoinositide 3-kinase (PI 3-kinase)/Akt/mTOR intracellular signaling pathway is critical for cell growth control. As described in Chapter 15, various extracellular signal proteins, including insulin and insulin-like growth factors,

1	Figure 20–26 Cells seem to require two types of signals to proliferate. (A) In order to multiply successfully, most normal cells are suspected to require both extracellular signals that drive cell-cycle progression (shown here as blue mitogen) and extracellular signals that drive cell growth (shown here as red growth factor). how mitogens activate the Rb pathway to drive entry into the cell cycle is described in Figure 17–61. (B) Diagram of the signaling system containing Akt that drives cell growth through greatly stimulating glucose uptake and utilization, including a conversion of the excess citric acid produced from sugar intermediates in mitochondria into the acetyl CoA that is needed in the cytosol for lipid synthesis and new membrane production. As indicated, protein synthesis is also increased. This system becomes abnormally activated early in tumor progression. TCA cycle indicates the tricarboxylic acid cycle (citric acid cycle).

1	normally activate this pathway. In cancer cells, however, the pathway is activated by mutation so that the cell can grow in the absence of such signals. The resulting abnormal activation of the protein kinases Akt and mTOR not only stimulates protein synthesis (see Figure 17–64), but also greatly increases both glucose uptake and the production of the acetyl CoA in the cytosol required for cell lipid synthesis, as outlined in Figure 20–26B.

1	The abnormal activation of the PI 3-kinase/Akt/mTOR pathway, which normally occurs early in the process of tumor progression, helps to explain the excessive rate of glycolysis that is observed in tumor cells, known as the Warburg effect, as discussed earlier (see Figure 20–12). As expected from our previous discussion, cancers can activate this pathway in many different ways. Thus, for example, a growth factor receptor can become abnormally activated, as in Figure 20–19. Also very common in cancers is the loss of the PTEN phosphatase, an enzyme that normally suppresses the PI 3-kinase/Akt/mTOR pathway by dephosphorylating the PI (3,4,5) P3 molecules that the PI 3-kinase forms (see pp. 859–861). PTEN is thus a common tumor-suppressor gene.

1	Of course, mutation is not the only way to overactivate the pathway: high levels of insulin in the circulation can have a similar effect. This may explain why the risk of cancer is significantly increased, by a factor of two or more, in people who are obese or have type 2 diabetes. Their insulin levels are abnormally high, driving cancer cell growth without need of mutation in the PI 3-kinase/Akt/mTOR pathway. Mutations in the p53 Pathway Enable Cancer Cells to Survive and Proliferate Despite Stress and DNA Damage

1	Mutations in the p53 Pathway Enable Cancer Cells to Survive and Proliferate Despite Stress and DNA Damage That cancer cells must break the normal rules governing cell growth and cell division is obvious: that is part of the definition of cancer. It is not so obvious why cancer cells should also be abnormal in their response to stress and DNA damage, and yet this too is an almost universal feature. The gene that lies at the center of this response, the p53 gene, is mutated in about 50% of all cases of cancer— a higher proportion than for any other known cancer-critical gene. When we include with p53 the other genes that are closely involved in its function, we find that most cases of cancer harbor mutations in the p53 pathway. Why should this be? To answer, we must first consider the normal function of this pathway.

1	In contrast to Rb, most cells in the body have very little p53 protein under normal conditions: although the protein is synthesized, it is rapidly degraded. Moreover, p53 is not essential for normal development. Mice in which both copies of the gene have been deleted or inactivated typically appear normal in all respects except one—they universally develop cancer before 10 months of age. These observations suggest that p53 has a function that is required only in special circumstances. In fact, cells raise their concentration of p53 protein in response to a whole range of conditions that have only one obvious thing in common: they are, from the cell’s point of view, pathological, putting the cell in danger of death or serious injury. These conditions include DNA damage, putting the cell at risk from a faulty genome; telomere loss or shortening (see p. 1016), also dangerous to the integrity of the genome; hypoxia, depriving the cell of the oxygen it needs to keep its metabolism going;

1	risk from a faulty genome; telomere loss or shortening (see p. 1016), also dangerous to the integrity of the genome; hypoxia, depriving the cell of the oxygen it needs to keep its metabolism going; osmotic stress, causing the cell to swell or shrivel; and oxidative stress, generating dangerous levels of highly reactive free radicals.

1	Yet another form of stress that can activate the p53 pathway arises, it seems, when regulatory signals are so intense or uncoordinated as to drive the cell beyond its normal limits and into a danger zone where its mechanisms of control and coordination break down, as in an engine driven badly or too fast. The p53 concentration rises, for example, when Myc is overexpressed to oncogenic levels. All these circumstances call for desperate action, which may take either of two forms: the cell can block any further progress through the division cycle in order to take time out to repair or recover from the pathological condition; or it can accept that it must die, and do so in a way that minimizes damage to the organism. A good death, from this point of view, is a death by apoptosis. In apoptosis, stable, active p53

1	Figure 20–27 Modes of action of the p53 tumor suppressor. The p53 protein is a cellular stress sensor. In response to hyperproliferative signals, DNA damage, hypoxia, telomere shortening, and various other stresses, the p53 levels in the cell rise. As indicated, this may either arrest cell cycling in a way that allows the cell to adjust and survive, trigger cell suicide by apoptosis, or cause cell “senescence”—an irreversible cell-cycle arrest that stops damaged cells from dividing. the cell is phagocytosed by its neighbors and its contents are efficiently recycled. A bad death is a death by necrosis. In necrosis, the cell bursts or disintegrates and its contents are spilled into the extracellular space, inducing inflammation.

1	The p53 pathway, therefore, behaves as a sort of antenna, sensing the presence of a wide range of dangerous conditions, and when any are detected, triggering appropriate action—either a temporary or permanent arrest of cell cycling (senescence), or suicide by apoptosis (Figure 20–27). These responses serve to prevent deranged cells from proliferating. Cancer cells are indeed generally deranged, and their survival and proliferation thus depend on inactivation of the p53 pathway. If the p53 pathway were active in them, they would be halted in their tracks or die (Movie 20.4).

1	The p53 protein performs its job mainly by acting as a transcription regulator (see Movie 17.8). Indeed, the most common mutations observed in p53 in human tumors are in its DNA-binding domain, where they cripple the ability of p53 to bind to its DNA target sequences. Because p53 binds to DNA as a tetramer, a single mutant subunit within a tetrameric complex can be enough to block its function. Thus, mutations in p53 can have a dominant negative effect, causing loss of p53 function even when the cell also contains a wild-type version of the gene. For this reason, in contrast with other tumor suppressor genes such as Rb, the development of cancer does not always require that both copies of p53 be knocked out.

1	As discussed in Chapter 17, the p53 protein exerts its inhibitory effects on the cell cycle, in part at least, by inducing the transcription of p21, which encodes a protein that binds to and inhibits the cyclin-dependent kinase (Cdk) complexes required for progression through the cell cycle. By blocking the kinase activity of these Cdk complexes, the p21 protein prevents the cell from progressing through S phase and replicating its DNA. The mechanism by which p53 induces apoptosis includes stimulation of the expression of many pro-apoptotic genes, and it will be described in Chapter 18. If the p53 pathway is functional, a cell with unrepaired DNA damage will stop dividing or die; it cannot proliferate. Mutations in the p53 pathway are, therefore, generally present in cancer cells showing genome instability—which is to say, the majority. But how does this genome instability originate? Here too, cancer genome studies are illuminating.

1	In ovarian cancers, for example, chromosome breaks, translocations, and deletions are very common, and these aberrations correlate with a high frequency of mutations and epigenetic silencing in the genes needed for repair of DNA double-strand breaks by homologous recombination, especially Brca1 and Brca2 (see pp. 281–282). In a subset of colorectal cancers with DNA mismatch repair defects, on the other hand, one instead finds many point mutations scattered throughout the genome (see pp. 250–251). In both kinds of cancer, the genome is commonly destabilized, but different types of mutations can bring this about. Cancers of Specialized Tissues Use Many Different Routes to Target the Common Core Pathways of Cancer

1	Cancers of Specialized Tissues Use Many Different Routes to Target the Common Core Pathways of Cancer Mutations in core components of the machinery that regulates cell growth, division, and survival, such as Rb, Ras, PTEN, or p53, are not the only way to pervert the control of these processes. Specialized tissues depend on a variety of pathways, as discussed in Chapter 15, to relay environmental signals to the core control machinery, and each pathway lays the cells open to subversion in a different set of ways. Thus, in different cancers, we can find examples of driver mutations in practically all the major signaling pathways through which cells communicate during development and tissue maintenance (discussed in Chapters 21 and 22).

1	In glioblastoma, for example, most patients have mutations in one or other of a set of cell-surface receptor tyrosine kinases, especially the EGF receptor mentioned earlier (linking into the Ras/PI3K pathway), suggesting that the cells from which the cancer originates are normally controlled by this route. The cells of the prostate gland, on the other hand, respond to the androgen hormone testosterone, and in prostate cancer, components of the androgen receptor signaling pathway (a variety of nuclear hormone receptor signaling; see Chapter 15) are often mutated. In the normal gut lining, Wnt signaling is critical, and Wnt pathway mutations are present in most colorectal cancers. Pancreatic cancers generally have mutations in the transforming growth factor-β (TGFβ) signaling pathway. Activating mutations in the Notch pathway are present in more than 50% of T cell acute lymphocytic leukemias, and so on.

1	Cells are generally regulated by several different types of external signals that must act in combination, representing a “fail-safe” control mechanism that protects the organism as a whole from cancer. These signals are different in different tissues. As expected, therefore, the corresponding cancers often have mutations in several signaling pathways concurrently. This is true of the examples we have just listed, which commonly have mutations in other signaling pathways in addition to the ones that we have singled out. Studies Using Mice help to Define the Functions of Cancer-Critical Genes

1	The ultimate test of a gene’s role in cancer has to come from investigations in the intact, mature organism. The most favored organism for such studies, apart from humans themselves, is the mouse. To explore the function of a candidate oncogene or tumor suppressor gene, one can make a transgenic mouse that overexpresses it or a knockout mouse that lacks it. Using the techniques described in Chapter 8, one can engineer mice in which the misexpression or deletion of the gene is restricted to a specific set of cells, or in which expression of the gene can be switched on at will at a chosen point in time, or both, to see whether and how tumors develop. Moreover, to follow the growth of tumors from day to day in the living organism, the cells of interest can be genetically marked and made visible by expression of a fluorescent or luminescent reporter (Figure 20–28). In these ways, one can begin to clarify the part that each cancer-critical gene plays in cancer initiation or progression.

1	Figure 20–28 Monitoring tumor growth and metastasis in a mouse with a luminescent reporter. A mouse was genetically engineered in a way that allows both copies of its PTEN tumor suppressor gene to be inactivated in the prostate gland, simultaneously with the prostate-specific activation of a gene engineered to produce the enzyme luciferase (derived from fireflies). After an injection of luciferin (the substrate molecule for luciferase) into the mouse’s bloodstream, the cells in the prostate emit light and can be detected by their bioluminescense in a live mouse, as seen in the 67-day-old animal at the left. Cells lacking the PTEN phosphatase enzyme contain elevated amounts of the Akt activator, PI(3,4,5)P3, and this causes the prostate cells to proliferate abnormally, progressing over time to form a cancer. In this way, the process of metastasis could be followed in the same animal over the course of a year. The light intensity in these experiments is proportional to the number of

1	time to form a cancer. In this way, the process of metastasis could be followed in the same animal over the course of a year. The light intensity in these experiments is proportional to the number of prostate-cell descendants, increasing from light blue to green, to yellow, to red in this representation. (Adapted from C.-P. Liao et al., Cancer Res. 67:7525– 7533, 2007.)

1	Figure 20–29 Oncogene collaboration in transgenic mice. The graphs show the incidence of tumors in three types of transgenic mouse strains, one carrying a Myc oncogene, one carrying a Ras oncogene, and one carrying both oncogenes. For these experiments, two lines of transgenic mice were first generated. One carries an inserted copy of an oncogene created by fusing the proto-oncogene Myc with the mouse mammary tumor virus regulatory DNA (which then drives Myc overexpression in the mammary gland). The other line carries an inserted copy of the Ras oncogene under 50 control of the same regulatory element. Both strains of mice develop tumors much more frequently than normal, most often in the mammary or salivary glands. Mice that carry both oncogenes together are obtained by crossing the two strains. These hybrids develop tumors at a far higher rate still, much greater than the sum of the rates for the two oncogenes separately. 0 Nevertheless, the tumors arise only after a delay and only

1	strains. These hybrids develop tumors at a far higher rate still, much greater than the sum of the rates for the two oncogenes separately. 0 Nevertheless, the tumors arise only after a delay and only from a small percentage of tumor-free mice proportion of the cells in the tissues where the two genes are expressed. Further accidental changes, in addition to the two oncogenes, are apparently required for the development of cancer. (After E. Sinn et al., Cell 49:465–475, 1987. With permission from Elsevier.)

1	Transgenic mouse studies confirm, for example, that a single oncogene is generally not enough to turn a normal cell into a cancer cell. Thus, in mice engineered to express a Myc or Ras oncogenic transgene, some of the tissues that express the oncogene may show enhanced cell proliferation, and, over time, occasional cells will undergo further changes to give rise to cancers. Most cells expressing the oncogene, however, do not give rise to cancers. Nevertheless, from the point of view of the whole animal, the inherited oncogene is a serious menace because it creates a high risk that a cancer will arise somewhere in the body. Mice that express both Myc and Ras oncogenes (bred by mating a transgenic mouse carrying a Myc oncogene with one carrying a Ras oncogene) develop cancers earlier and at a much higher rate than either parental strain (Figure 20–29); but, again, the cancers originate as scattered, isolated tumors among noncancerous cells. Thus, even cells expressing these two

1	and at a much higher rate than either parental strain (Figure 20–29); but, again, the cancers originate as scattered, isolated tumors among noncancerous cells. Thus, even cells expressing these two oncogenes must undergo further, randomly generated changes to become cancerous. This strongly suggests that multiple mutations are required for tumorigenesis, as supported by a great deal of other evidence discussed earlier. Experiments using mice with deletions of tumor suppressor genes lead to similar conclusions.

1	Cancers Become More and More heterogeneous as They Progress From simple histology, looking at stained tissue sections, it is clear that some tumors contain distinct sectors, all clearly cancerous, but differing in appearance because they differ genetically: the cancer cell population is heterogeneous. Evidently, within the initial clone of cancerous cells, additional mutations have arisen and thrived, creating diverse subclones. Today, the ability to analyze cancer genomes lets us look much deeper into the process. One approach involves taking samples from different regions of a primary tumor and from the metastases that it has spawned. With modern methods, it is even possible to take representative single cells and analyze their genomes. Such studies reveal a classic picture of Darwinian evolution, occurring on a time scale of months or years rather than millions of years, but governed by the same rules of natural selection (Figure 20–30).

1	One such investigation compared the genomes of 100 individual cells from different regions of a primary tumor of the breast. A large fraction—just over half—of the chosen cells was genetically normal or nearly so: these were connective-tissue cells and other cell types, such as those of the immune system, that were mixed up with the cancer cells. The cancer cells themselves were distinguished by their severely disrupted genomes. The detailed pattern of gene deletions and amplifications in each such cell revealed how closely it was related to the others, and from this data one could draw up a family tree (Figure 20–30B). In this case, three main branches of the tree were seen; that is, the cancer consisted of three major mammary appearance of most epithelial recent common ancestor development number of cells subclones. From the shared abnormalities, one could deduce that their last common ancestor—the presumed founder of the cancer—was already very different from a normal cell, but

1	number of cells subclones. From the shared abnormalities, one could deduce that their last common ancestor—the presumed founder of the cancer—was already very different from a normal cell, but that the first split between branches occurred early, when the tumor was small. This was followed by a large amount of additional change within each branch. A hint of the future could be seen in the smallest of the three major subclones: its cells were distinguished by a massive amplification of a Ras oncogene. Given more time, perhaps they would have out-competed the other cancer cells and taken over the whole tumor.

1	Similar results have been obtained with other cancers. Clearly, cancer cells are constantly mutating, multiplying, competing, evolving, and diversifying as they exploit new ecological niches and react to the treatments that are used against them (Figure 20–30C). Diversification accelerates as they metastasize and colonize new territories, where they encounter new selection pressures. The longer the evolutionary process continues, the harder it becomes to catch them all in the same net and kill them. The Changes in Tumor Cells That Lead to Metastasis Are Still Largely a Mystery

1	The Changes in Tumor Cells That Lead to Metastasis Are Still Largely a Mystery Perhaps the most significant gap in our understanding of cancer concerns invasiveness and metastasis. For a start, it is not clear exactly what new properties a cancer cell must acquire to become metastatic. In some cases, it is possible that invasion and metastasis require no further genetic changes beyond those needed to violate the normal controls on cell growth, cell division, and cell death. On the other hand, it may be that, for some cancers, metastasis requires a large number of additional mutations and epigenetic changes. Clues are coming from comparisons of the genomes of cells of primary tumors with the cells of metastases that they have spawned. The results appear complex and variable from one cancer to another. Nevertheless, some general principles have emerged.

1	As we discussed earlier, it is helpful to distinguish three phases of tumor progression required for a carcinoma to metastasize (see Figure 20–16). First, the cells Figure 20–30 How cancers progress as a series of subclones. (A) Schematic illustration of the pattern of mutation and natural selection in a clone of tumor cells. A family tree of cancer cells sampled from different regions of a single breast tumor, showing how the cells have evolved and diversified from a common ancestor, the cancer founder cell. The genome of each of the indicated 100 cells from a human breast tumor was sequenced to produce an evolutionary tree. About half of these cells were normal cells from the stroma (blue cells). The red cells have greatly amplified their K-Ras gene. Note that many subclones appear to have died out, including the one that contained the founder cells for the three subclones that survive.

1	A depiction of how driver mutations are thought to cause cancer progression over long periods of time, before producing a large enough clone of proliferating cells to be detected as a tumor. The data indicate that driver mutations occur only rarely in a background of long-lived subclones of cells that continually accumulate passenger mutations without gaining a growth advantage. (A, adapted from M. Greaves, Semin. Cancer Biol. 20:65–70, 2010; B, adapted from N. Navin et al., Nature 472:90–94, 2011; C, adapted from S.

1	S. Nik-Zainal et al., Cell 149:994–1007, 2012.) invasiveness survival in the arrest in exit into survival of initial growth persistence causes circulation capillary or remote tissue cells in of cells in of growth entry into other small or organ foreign foreign vessel vessel tissue tissue must escape the normal confines of their parent epithelium and begin to invade the tissue immediately beneath. Second, they must travel via the blood or lymph to lodge in distant sites. Third, they must survive there and multiply. It is the first and last steps in this sequence that are the most difficult to accomplish for most cancers (Figure 20–31).

1	The first step, local invasiveness, requires a relaxation of the mechanisms that normally hold epithelial cells together. As mentioned earlier, this step resembles the normal developmental process known as the epithelial–mesenchymal transition (EMT ), in which epithelial cells undergo a shift in character, becoming less adhesive and more migratory (discussed in Chapter 19). A key part of the EMT process involves switching off expression of the E-cadherin gene. The primary function of the transmembrane E-cadherin protein is in cell–cell adhesion, binding epithelial cells together through adherens junctions (see Figure 19–13). In some carcinomas of the stomach and of the breast, E-cadherin has been identified as a tumor suppressor gene, and a loss of E-cadherin may promote cancer development by facilitating local invasiveness.

1	The initial entry of tumor cells into the circulation is helped by the presence of a dense supply of blood vessels and sometimes lymphatic vessels, which tumors attract to themselves as they grow larger and become hypoxic in their interior. This process, called angiogenesis, is caused by the secretion of angiogenic factors that promote the growth of blood vessels, such as vascular endothelial growth factor (VEGF; see Figure 22–26). An abnormal fragility and leakiness of the new vessels that form may help the cells that have become invasive to enter and then move through the circulation with relative ease.

1	The remaining steps in metastasis, involving exit from a blood or lymphatic vessel and the effective colonization of remote sites, are much harder to study. To discover which of the later steps in metastasis present cancer cells with the greatest difficulties, one can label the cells with a fluorescent dye or green fluorescent protein (GFP), inject them into the bloodstream of a mouse, and then monitor their fate (Movie 20.5). In such experiments, one observes that many cells survive in the circulation, lodge in small vessels, and exit into the surrounding tissue, regardless of whether they come from a tumor that metastasizes or one that does not. Some cells die immediately after they enter foreign tissue; others survive entry into the foreign tissue but fail to proliferate. Still others divide a few times and then stop, forming micrometastases containing ten to several thousand cells. Very few establish full-blown metastases.

1	What, if anything, distinguishes the survivors from the failures? A clue may come from the fact that in many types of tumors, the cancer cells show a kind of heterogeneity that resembles the heterogeneity seen among the cells of those normal tissues that renew themselves continually by a stem-cell strategy, as we discuss next. A Small Population of Cancer Stem Cells May Maintain Many Tumors Self-renewing tissues, where cell division continues throughout life, are the breeding ground for the great majority of human cancers. They include the epidermis Figure 20–31 The barriers to metastasis.

1	Studies of labeled tumor cells leaving a tumor site, entering the circulation, and establishing metastases show which steps in the metastatic process, outlined in Figure 20–16, are difficult or “inefficient,” in the sense that they are steps in which large numbers of cells fail and are lost. It is in these difficult steps that cells from highly metastatic tumors are observed to have much greater success than cells from a nonmetastatic source. It seems that the ability to escape from the parent tissue, and an ability to survive and grow in the foreign tissue, are key properties that cells must acquire to become metastatic. (Adapted from A.F. Chambers et al., Breast Cancer Res. 2:400–407, 2000. With permission from BioMed Central Ltd.) (the outer epithelial layer of the skin), the lining of the digestive and reproductive tracts, and the bone marrow, where blood cells are generated (see Chapter 22). In almost all these tissues, renewal depends on the presence of stem cells, which divide

1	the digestive and reproductive tracts, and the bone marrow, where blood cells are generated (see Chapter 22). In almost all these tissues, renewal depends on the presence of stem cells, which divide to give rise to terminally differentiated cells, which do not divide. This creates a mixture of cells that are genetically identical and closely related by lineage, but are in different states of differentiation. Many tumors seem likewise to consist of cells in varied states of differentiation, with different capacities for cell division and self-renewal.

1	To see the implications, it is helpful to consider how normal stem-cell systems operate. When a normal stem cell divides, each daughter cell has a choice—it can remain a stem cell, or it can commit to a pathway leading to differentiation. A stem-cell daughter remains in place to generate more cells in the future. A committed daughter typically undergoes some rounds of cell proliferation (as a so-called transit amplifying cell) but then stops dividing, terminally differentiates, and eventually is discarded and replaced (it may die by apoptosis, with recycling of its materials, or be shed from the body). On average, the two fates—stem cell or differentiating cell—normally occur with equal probability, so that half the daughters of stem-cell divisions take the one path and half take the other. In a healthy body, feedback controls regulate the process, adjusting this balance of cell-fate choices to correct for any departure from the proper cell population numbers. Thus, the number of stem

1	In a healthy body, feedback controls regulate the process, adjusting this balance of cell-fate choices to correct for any departure from the proper cell population numbers. Thus, the number of stem cells remains approximately constant, and the terminally differentiated cells are continually replaced at a steady rate. Because of the divisions undergone by the transit amplifying cells, the stem cells may be vastly outnumbered by the cells that are committed to terminal differentiation and have lost the capacity for self-renewal. But the stem cells, though few and far between and often relatively slowly dividing, carry the whole responsibility for maintenance of the tissue in the long term.

1	Some cancers seem to be organized in a similar way: they consist of rare cancer stem cells capable of dividing indefinitely, together with much larger numbers of dividing transit amplifying cells that are derived from the cancer stem cells but have a limited capacity for self-renewal (Figure 20–32). These non-stem cells appear to constitute the great majority of the cell population in some tumors. The Cancer Stem-Cell Phenomenon Adds to the Difficulty of Curing Cancer Evidence for the cancer stem-cell phenomenon comes chiefly from experiments in which individual cells from a cancer are tested for their ability to give rise to fresh tumors: a standard assay is to implant the cells into an immunodeficient mouse (Figure 20–33). It has been known for half a century that there is usually only a small chance—typically much less than 1%—that a tumor cell chosen at random and tested in this way will generate a new tumor. This by itself does not prove that

1	Figure 20–32 Cancer stem cells can be responsible for tumor growth and yet remain only a small part of the tumor-cell population. (A) how stem cells produce transit amplifying cells.

1	(B) how a small proportion of cancer stem cells can maintain a tumor. Suppose, for example, that each daughter of a cancer stem cell has a probability slightly greater than 50% of retaining stem-cell potential and a probability slightly less than 50% of becoming a transit amplifying cell that is committed to a program of cell divisions that stops after 10 division cycles. While the number of cancer stem cells will increase slowly but steadily to give a growing tumor, the non-stem cells that they give rise to will always outnumber the stem cells by a large factor—in this example, by a factor of about 1000. (If the cell-divisioncycle and survival times for the two classes of cells are equal.) the tumor cells are heterogeneous: like seeds scattered on difficult ground, each of them may have only a small chance of finding a spot where it can survive and grow. Modern technologies for sorting cells have shown, however, that in some cancers at least, the rate of success in founding new

1	have only a small chance of finding a spot where it can survive and grow. Modern technologies for sorting cells have shown, however, that in some cancers at least, the rate of success in founding new tumors is even lower than it would otherwise be because the cancer cells are heterogeneous in their state of differentiation, and only a small subset of them—the cancer stem cells—have the special properties needed for tumor propagation. For example, in several types of cancer, including breast cancers and leukemias, one can fractionate the tumor cells using monoclonal antibodies that recognize a particular cell-surface marker that is present on the normal stem cells in the tissue of origin of the cancer. The purified cancer cells expressing this marker are found to have a greatly enhanced ability to found new tumors. And the new tumors consist of mixtures of cells that express the marker and cells that do not, all generated from the same founder cell that expressed the marker.

1	Experiments with breast cancer cells have revealed that, instead of following a rigid program from stem cell to transit amplifying cell to terminally differentiated cell, these cancer cells can randomly switch to and fro—with a certain low transition probability—between different states of differentiation that express different molecular markers. In one state, they behave like stem cells, dividing slowly but capable of founding new tumors; in other states, they behave like transit amplifying cells, dividing rapidly but unable to found new tumors in a standard transplant assay. But a single cell in any of these states—given time in culture, or a congenial environment in the body—will give rise to a mixed population that includes all the other states as well.

1	The cancer stem-cell phenomenon, whatever its basis, implies that even when the tumor cells are genetically similar, they are phenotypically diverse. A treatment that wipes out those in one state is likely to allow survival of others that remain a danger. Radiotherapy or a cytotoxic drug, for example, may selectively kill off the rapidly dividing cells, reducing the tumor volume to almost nothing, and yet spare a few slowly dividing cells that go on to resurrect the disease. This greatly adds to the difficulty of cancer therapy, and it is part of the reason why treatments that seem at first to succeed often end in relapse and disappointment. Colorectal Cancers Evolve Slowly Via a Succession of Visible Changes

1	Colorectal Cancers Evolve Slowly Via a Succession of Visible Changes At the beginning of this chapter, we saw that most cancers develop gradually from a single aberrant cell, progressing from benign to malignant tumors by the accumulation of a number of independent genetic and epigenetic changes. We have discussed what some of these changes are in molecular terms and seen how they contribute to cancerous behavior. We now examine one of the common human cancers more closely, using it to illustrate and enlarge upon some of the general principles and molecular mechanisms we have introduced. We take colorectal cancer as our example.

1	Colorectal cancers arise from the epithelium lining the colon (the large intestine) and rectum (the terminal segment of the gut). The organization of this tissue is broadly similar to that of the small intestine, discussed in detail in Chapter 22 (pp. 1217–1221). For both the small and large intestine, the epithelium is renewed at an extraordinarily rapid rate, taking about a week to completely replace most of the epithelial sheet. In both regions, the renewal depends on stem cells that lie in deep pockets of the epithelium, called intestinal crypts. The signals that maintain the stem cells and control the normal organization and renewal of the epithelium are beginning to be quite well understood, as explained in Chapter 22. Mutations that disrupt these signals begin the process of tumor progression for most colorectal cancers (Movie 20.6).

1	Colorectal cancers are common, currently causing nearly 60,000 deaths a year in the United States, or about 10% of total deaths from cancer. Like most cancers, they are not usually diagnosed until late in life (90% occur after the age of 55). However, routine examination of normal adults with a colonoscope (a fiber

1	Figure 20–33 An immunodeficient mouse, as used in transplantation assays to test human cancer cells for their ability to found new tumors. This nude mouse has a mutation that blocks development of the thymus and, as a side effect, robs it of hair. Because it has practically no T cells, it tolerates grafts of cells even from other species. (Courtesy of harlan Sprague Dawley.) optic device for viewing the interior of the colon and rectum) often reveals a small benign tumor, or adenoma, of the gut epithelium in the form of a protruding mass of tissue called a polyp (see Figure 22–4). These adenomatous polyps are believed to be the precursors of a large proportion of colorectal cancers. Because the progression of the disease is usually very slow, there is typically a period of about 10 years in which the slowly growing tumor is detectable but has not yet turned malignant. Thus, when people are screened by colonoscopy in their fifties and the polyps are removed through the colonoscope—a

1	in which the slowly growing tumor is detectable but has not yet turned malignant. Thus, when people are screened by colonoscopy in their fifties and the polyps are removed through the colonoscope—a quick and easy surgical procedure—the subsequent incidence of colorectal cancer is much lower: according to some studies, less than a quarter of what it would be otherwise.

1	In microscopic sections of polyps smaller than 1 cm in diameter, the cells and their arrangement in the epithelium usually appear almost normal. The larger the polyp, the more likely it is to contain cells that look abnormally undifferentiated and form abnormally organized structures. Sometimes, two or more distinct areas can be distinguished within a single polyp, with the cells in one area appearing relatively normal and those in the other appearing clearly cancerous, as though they have arisen as a mutant subclone within the original clone of adenomatous cells. At later stages in the disease, some tumor cells become invasive in a small fraction of the polyps, first breaking through the epithelial basal lamina, then spreading through the layer of muscle that surrounds the gut, and finally metastasizing to lymph nodes via lymphatic vessels and to liver, lung, and other organs via blood vessels. A Few Key Genetic Lesions Are Common to a Large Fraction of Colorectal Cancers

1	A Few Key Genetic Lesions Are Common to a Large Fraction of Colorectal Cancers What are the mutations that accumulate with time to produce this chain of events? Of those genes so far discovered to be involved in colorectal cancer, three stand out as most frequently mutated: the proto-oncogene K-Ras (a member of the Ras gene family), in about 40% of cases; p53, in about 60% of cases; and the tumor suppressor gene Apc (discussed below), in more than 80% of cases. Others are involved in smaller numbers of colon cancers, and some of these are listed in Table 20–1. The role of Apc first came to light through study of certain families showing a rare type of hereditary predisposition to colorectal cancer, called familial

1	Figure 20–34 Colon of familial adenomatous polyposis coli patient compared with normal colon. (A) The normal colon wall is a gently undulating but smooth surface. (B) The polyposis colon is completely covered by hundreds of projecting polyps, each resembling a tiny cauliflower when viewed with the naked eye. (Courtesy of Andrew Wyllie and Mark Arends.) adenomatous polyposis coli (FAP). In this syndrome, hundreds or thousands of polyps develop along the length of the colon (Figure 20–34). These polyps start to appear in early adult life, and if they are not removed, one or more will almost always progress to become malignant; the average time from the first detection of polyps to the diagnosis of cancer is 12 years. The disease can be traced to a deletion or inactivation of the tumor suppressor gene Apc, named after the syndrome. Individuals with FAP have inactivating mutations or deletions of one copy of the Apc gene in all their cells and show loss of heterozygosity in tumors, even

1	gene Apc, named after the syndrome. Individuals with FAP have inactivating mutations or deletions of one copy of the Apc gene in all their cells and show loss of heterozygosity in tumors, even in the benign polyps. Most patients with colorectal cancer do not have the hereditary condition. Nevertheless, in more than 80% of the cases, their cancer cells (but not their normal cells) have inactivated both copies of the Apc gene through mutations acquired during the patient’s lifetime. Thus, by a route similar to that which we discussed for retinoblastoma, mutation of the Apc gene was identified as one of the central ingredients of colorectal cancer.

1	The Apc protein, as we now know, is an inhibitory component of the Wnt signaling pathway (discussed in Chapter 15). It binds to the β-catenin protein, another component of the Wnt pathway, and helps to induce the protein’s degradation. By inhibiting β-catenin in this way, Apc prevents the β-catenin from migrating to the nucleus, where it would act as a transcriptional regulator to drive cell proliferation and maintain the stem-cell state (see Figure 15–60). Loss of Apc results in an excess of free β-catenin and thus leads to an uncontrolled expansion of the stem-cell population. This causes massive increase in the number and size of the intestinal crypts (see Figure 22–4).

1	When the β-catenin gene was sequenced in a collection of colorectal tumors, it was discovered that, many of the tumors that did not have Apc mutations had activating mutations in β-catenin instead. Thus, it is excessive activity in the Wnt signaling pathway that is critical for the initiation of this cancer, rather than any single oncogene or tumor suppressor gene that the pathway contains. This being so, why is the Apc gene in particular so often the most common culprit in colorectal cancer? The Apc protein is large and it interacts not only with β-catenin but also with various other cell components, including microtubules. Loss of Apc appears to increase the frequency of mitotic spindle defects, leading to chromosome abnormalities when cells divide. This additional, independent cancer-promoting effect could explain why Apc mutations feature so prominently in the causation of colorectal cancer.

1	In addition to the hereditary disease (FAP) associated with Apc mutations, there is a second, more common kind of hereditary predisposition to colon carcinoma in which the course of events differs from the one we have described for FAP. In this more common condition, called hereditary nonpolyposis colorectal cancer (HNPCC), the probability of colon cancer is increased without any increase in the number of colorectal polyps (adenomas). Moreover, the cancer cells are unusual, in that they have a normal (or almost normal) karyotype. The majority of colorectal tumors in non-HNPCC patients, in contrast, have gross chromosomal abnormalities, with multiple translocations, deletions, and other aberrations, as well as having many more chromosomes than normal (Figure 20–35).

1	The mutations that predispose HNPCC individuals to colorectal cancer occur in one of several genes that code for central components of the DNA mismatch repair system. These genes are homologous in structure and function to the MutL and MutS genes in bacteria and yeast (see Figure 5–19). Only one of the two copies of the involved gene is defective, so the repair system is still able to remove the inevitable DNA replication errors that occur in the patient’s cells. However, as discussed previously, these individuals are at risk, because the accidental loss or inactivation of the remaining good gene copy will immediately elevate the spontaneous mutation rate by a hundredfold or more (discussed in Chapter 5). These genetically unstable cells then can presumably speed through the standard processes of mutation and natural selection that allow clones of cells to progress to malignancy.

1	This particular type of genetic instability produces invisible changes in the chromosomes—most notably changes in individual nucleotides and short expansions and contractions of monoand dinucleotide repeats such as AAAA… or CACACA…. Once the defect in HNPCC patients was recognized, the epigenetic silencing or mutation of mismatch repair genes was found in about 15% of the colorectal cancers occurring in people with no inherited predisposing mutation.

1	Thus, the genetic instability found in many colorectal cancers can be acquired in at least two ways. The majority of the cancers display a form of chromosomal instability that leads to visibly altered chromosomes, whereas in the others the instability occurs on a much smaller scale and reflects a defect in DNA mismatch repair. Indeed, many carcinomas show either chromosomal instability or defective mismatch repair—but rarely both. These findings clearly demonstrate that genetic instability is not an accidental by-product of malignant behavior but a contributory cause—and that cancer cells can acquire this instability in multiple ways. The Steps of Tumor Progression Can Often Be Correlated with Specific Mutations

1	The Steps of Tumor Progression Can Often Be Correlated with Specific Mutations In what order do K-Ras, p53, Apc, and the other identified colorectal cancer-critical genes mutate, and what contribution does each of them make to the asocial behavior of the cancer cell? There is no single answer, because colorectal cancer can arise by more than one route: thus, we know that in some cases, the first mutation can be in a DNA mismatch repair gene; in others, it can be in a gene regulating cell proliferation. Moreover, as previously discussed, a general feature such as genetic instability or a tendency to proliferate abnormally can arise in a variety of ways, through mutations in different genes.

1	Nevertheless, certain sets of mutations are particularly common in colorectal cancer, and they occur in a characteristic order. Thus, in most cases, mutations inactivating the Apc gene appear to be the first, or at least a very early step, as they are detected at the same high frequency in small benign polyps as in large malignant tumors. Changes that lead to genetic and epigenetic instability are likely also to arise early in tumor progression, since they are needed to drive the later steps. Activating mutations in the K-Ras gene occur later, as they are rare in small polyps but common in larger ones that show disturbances in cell differentiation and histological pattern.

1	Activating mutations in the K-Ras gene occur later, as they are rare in small polyps but common in larger ones that show disturbances in cell differentiation and histological pattern. Inactivating mutations in p53 are thought to come later still, as they are rare in polyps but common in carcinomas (Figure 20–36). We have seen that loss of p53 function allows cancer cells to endure stress and to avoid apoptosis and cell-cycle arrest. Additionally, loss of p53 is related to the heightened activation Figure 20–35 Chromosome complements (karyotypes) of colon cancers showing different kinds of genetic instability. (A) The karyotype of a typical cancer shows many gross abnormalities in chromosome number and structure. Considerable variation can also exist from cell to cell (not shown).

1	(B) The karyotype of a tumor that has a stable chromosome complement with few chromosomal anomalies; the genetic abnormalities are mostly invisible, having been created by defects in DNA mismatch repair. All of the chromosomes in this figure were stained as in Figure 4–10, the DNA of each human chromosome being marked with a different combination of fluorescent dyes. (Courtesy Wael Abdel-Rahman and Paul Edwards.) acquisition of increased genetic and epigenetic instability of oncogenes such as Ras. Experiments in mice show that an initial low level of oncogene activation can give rise to a slowly growing tumor even while p53 is functional: genes such as Ras are, after all, part of the normal machinery of growth control, and moderate activation is not stressful for a cell and does not call the p53 protein into play. Progression of a tumor from slow to rapid, malignant growth, however, involves activation of oncogenes beyond normal physiological limits to a higher, stressful level. If

1	the p53 protein into play. Progression of a tumor from slow to rapid, malignant growth, however, involves activation of oncogenes beyond normal physiological limits to a higher, stressful level. If the p53 protein is present and functional, this should lead to cell-cycle arrest or death. Only by losing p53 function can the cancer cells with hyperactive oncogenes survive and progress.

1	The steps we have just described are only part of the picture. It is important to emphasize that each case of colorectal cancer is different, with its own detailed combination of mutations, and that even for the mutations that are commonly shared, the sequence of occurrence may vary. The same is true for cancers in general. Advances in molecular biology have recently provided the tools to find out precisely which genes are amplified, deleted, mutated, or misregulated by epigenetic mechanisms in the tumor cells of any given patient. As we discuss in the next section, such information promises to become as important for the diagnosis and treatment of cancer as was the breakthrough of being able to identify microorganisms for the treatment of infectious diseases.

1	The molecular analysis of cancer cells reveals two classes of cancer-critical genes: oncogenes and tumor suppressor genes. A set of these genes becomes altered by a combination of genetic and epigenetic accidents to drive tumor progression. Many cancer-critical genes code for components of the social control pathways that regulate when cells grow, divide, differentiate, or die. In addition, a subclass of tumor suppressors can be categorized as “genome maintenance genes,” because their normal role is to help maintain genome integrity. The inactivation of the p53 pathway, which occurs in nearly all human cancers, allows genetically damaged cells to escape apoptosis and continue to proliferate. Inactivation of the Rb pathway also occurs in most human cancers, illustrating how fundamental each of these pathways is for protecting us against cancer.

1	The sequencing of cancer cell genomes reveals that—except for the cancers of childhood—many cancers acquire 10 or so driver mutations over the long course of tumor progression, along with a considerably larger number of passenger mutations of no consequence. The same methods reveal how subclones of cells arise and die out as a tumor ages. Tumors thus contain a heterogeneous mixture of cells, some—the so-called cancer stem cells—being much more dangerous than others.

1	We can often correlate the steps of tumor progression with mutations that activate specific oncogenes and inactivate specific tumor suppressor genes, with colon cancer providing a good example. But different combinations of mutations and epigenetic changes are found in different types of cancer, and even in different patients with the same type of cancer, reflecting the random way in which these inherited changes arise. Nevertheless, many of the same changes are encountered repeatedly, suggesting that there are a limited number of ways to breach our defenses against cancer. Figure 20–36 Suggested typical sequence of genetic changes underlying the development of a colorectal carcinoma. This oversimplified diagram provides a general idea of the way mutation and tumor development are related. But many other mutations are generally involved, and different colon cancers can progress through different sequences of mutations (and/or epigenetic changes).

1	CANCER PREVENTION AND TREATMENT: PRESENT AND FUTURE We can apply the growing understanding of the molecular biology of cancer to sharpen our attack on the disease at three levels: prevention, diagnosis, and treatment. Prevention is always better than cure, and indeed many cancers can be prevented, especially by avoiding smoking. Highly sensitive molecular assays promise new opportunities for earlier and more precise diagnosis, with the aim of detecting primary tumors while they are still small and have not yet metastasized. Cancers caught at these early stages can often be nipped in the bud by surgery or radiotherapy, as we saw for colorectal polyps. Nevertheless, full-blown malignant disease will continue to be common for many years to come, and cancer treatments will continue to be needed.

1	In this section, we first examine the preventable causes of cancer and then consider how advances in our understanding at a molecular level are beginning to transform the treatment of the disease. Epidemiology Reveals That Many Cases of Cancer Are Preventable A certain irreducible background incidence of cancer is to be expected regardless of circumstances. As discussed in Chapter 5, mutations can never be absolutely avoided because they are an inescapable consequence of fundamental limitations on the accuracy of DNA replication and repair. If a person could live long enough, it is inevitable that at least one of his or her cells would eventually accumulate a set of mutations sufficient for cancer to develop.

1	Nevertheless, environmental factors seem to play a large part in determining the risk for cancer. This is demonstrated most clearly by a comparison of cancer incidence in different countries: for almost every cancer that is common in one country, there is another country where the incidence is much lower. Because migrant populations tend to adopt the pattern of cancer incidence typical of their new host country, the differences are thought to be due mostly to environmental, not genetic, factors. From such findings, it has been suggested that 80–90% of cancers should be avoidable, or at least postponable (Figure 20–37).

1	Unfortunately, different cancers have different environmental risk factors, and a population that escapes one such danger is usually exposed to another. This is not, however, inevitable. There are some human subgroups whose way of life substantially reduces the total cancer death rate among individuals of a given age. Under the current conditions in the United States and Europe, approximately one in five people will die of cancer. But the incidence of cancer among strict Mormons in Utah—who avoid alcohol, coffee, cigarettes, drugs, and casual sex—is only about half the incidence for non-practicing members of the same family or for Americans in general. Cancer incidence is also low in certain relatively affluent populations in Africa.

1	Although such observations on human populations indicate that cancer can often be avoided, it has been difficult in most cases—with tobacco as a striking exception—to pinpoint the specific environmental factors responsible for these large population differences or to establish how they act. Nevertheless, several important classes of environmental cancer risk factors have been identified (Figure 20–37B). One thinks first of mutagens. But there are also many other influences—including the amount of food we eat, the hormones that circulate in our bodies, and the irritations, infections, and damage to which we expose our tis-sues—that are no less important and favor development of the disease in other ways. Many quite disparate chemicals are carcinogenic when they are fed to experimental animals or painted repeatedly on their skin. Examples include a range

1	Many quite disparate chemicals are carcinogenic when they are fed to experimental animals or painted repeatedly on their skin. Examples include a range SOUTH AMERICA NORTH AMERICA HAWAII JAPAN CHINA AFRICA prostate, colon, breast prostate, colon, breast stomach naso-pharyngeal Burkitt’s lymphoma Burkitt’s lymphoma (A) EASTERN EUROPE Hodgkin’s disease Figure 20–37 Cancer incidence is related to environmental influences. This map of the world shows the rates of cancer increasing (red arrows) or decreasing (blue arrows) when specific populations move from one location to another. Such observations suggest the importance of environmental factors, including diet, in dictating cancer risk. (B) Some estimated effects of environment and lifestyle on cancer in the United States (US). The table shows both the yearly deaths in the US attributable to each cancer and the estimated percentage of that cancer that could be eliminated through prevention. (B, data from G.A. Colditz, K.Y. Wolin and

1	S. Gehlert, Sci. Transl. Med. 4:127rv4, 2012.) of aromatic hydrocarbons and derivatives of them such as aromatic amines, nitrosamines, and alkylating agents such as mustard gas. Although these chemical carcinogens are diverse in structure, a large proportion of them have at least one shared property—they cause mutations. In one common test for mutagenicity (the Ames test), the carcinogen is mixed with an activating extract prepared from rat liver cells (to mimic the biochemical processing that occurs in an intact animal). The mixture is then added to a culture of specially designed test bacteria and the bacterial mutation rate measured. Most of the compounds scored as mutagenic by this rapid and convenient assay in bacteria also cause mutations or chromosome aberrations when tested on mammalian cells.

1	A few of these carcinogens act directly on DNA. But generally the more potent ones are relatively inert chemically; these chemicals become damaging only after they have been converted to a more reactive molecule by metabolic processes in the liver, catalyzed by a set of intracellular enzymes known as the cytochrome P-450 oxidases. These enzymes normally help to convert ingested toxins into harmless and easily excreted compounds. Unhappily, their activity on certain chemicals generates products that are highly mutagenic. Examples of carcinogens activated in this way include benzo[a]pyrene, a cancer-causing chemical present in coal tar and tobacco smoke and the fungal toxin aflatoxin B1 (Figure 20–38). Fifty Percent of Cancers Could Be Prevented by Changes in Lifestyle

1	Tobacco smoke is the most important carcinogen in the world today. Even though many other chemical carcinogens have been identified, none of these appear to be responsible for anything like the same numbers of human cancer deaths. It is sometimes thought that the main environmental causes of cancer are the products of a highly industrialized way of life—the rise in pollution, the enhanced use of food additives, and so on—but there is little evidence to support this view. The idea may have come in part from the identification of some highly carcinogenic materials used in industry, such as 2-naphthylamine and asbestos. Except for the increase in cancers caused by smoking, however, age-adjusted death rates for most common human cancers have stayed much the same over the past half-century, or, in some cases, have declined significantly (Figure 20–39). Survival rates, moreover, have improved. Thirty years ago, less than 50% of patients lived more than five years from the time of diagnosis;

1	in some cases, have declined significantly (Figure 20–39). Survival rates, moreover, have improved. Thirty years ago, less than 50% of patients lived more than five years from the time of diagnosis; now, more than two-thirds do so.

1	(A) AFLATOXIN AFLATOXIN-2,3-EPOXIDE CARCINOGEN BOUND TO GUANINE IN DNA Figure 20–38 Some known carcinogens. (A) Carcinogen activation. A metabolic transformation must activate many chemical carcinogens before they will cause mutations by reacting with DNA. The compound illustrated here is aflatoxin B1, a toxin from a mold (Aspergillus flavus oryzae) that grows on grain and peanuts when they are stored under humid tropical conditions. Aflatoxin is an important cause of liver cancer in the tropics. (B) Different carcinogens cause different types of cancer. (B, data from Cancer and the Environment: Gene Environment Interactions, National Academies Press, 2002.)

1	Most of the carcinogenic factors that are known to be significant are by no means specific to the modern world. The most potent known carcinogen, by certain assays at least, is aflatoxin B1 (see Figure 20–38). It is produced by fungi that naturally contaminate foods such as tropical peanuts and is an important cause of liver cancer in Africa and Asia. Except for tobacco, chemical toxins and mutagens are of lesser importance as contributory causes of cancer than other factors that are more a matter of personal choice. One important factor is the quantity of food we eat: as mentioned earlier, the risk of cancer is greatly increased in people who are obese. In fact, it is estimated that as many as 50% of all cancers could be avoided by simple, identifiable changes in lifestyle (see Figure 20–37B). Viruses and Other Infections Contribute to a Significant Proportion of human Cancers

1	Viruses and Other Infections Contribute to a Significant Proportion of human Cancers Cancer in humans is not an infectious disease, and most human cancers do not have any infectious cause. However, a small but significant proportion of human cancers, perhaps 15% in the world as a whole, are thought to arise by mechanisms that involve viruses, bacteria, or parasites. Evidence for their involvement comes partly from the detection of viruses in cancer patients and partly from epidemiology. Thus, cancer of the uterine cervix is associated with infection with a papillomavirus, while liver cancer is very common in parts of the world (Africa and Southeast Asia) where hepatitis-B viral infections are common. Chronic infection  VINYL CHLORIDE:  BENZENE:  ARSENIC: skin carcinomas, bladder cancer  ASBESTOS:  RADIUM: Figure 20–39 Age-adjusted cancer death rates, United States, 1930–2008.

1	Figure 20–39 Age-adjusted cancer death rates, United States, 1930–2008. Selected death rates, adjusted to the age distribution of the US population, are plotted for (A) females and (B) males. Note the dramatic rise in lung cancer for both sexes, following the pattern of tobacco smoking, and the fall in deaths from stomach cancer, thought to be related to a fall in rates of infection with Helicobacter pylori. Recent reductions in deaths per 100,000 populationdeaths per 100,000 population0 20 40 60 80 0 20 40 60 80stomach breast lung and bronchus uterus ovary pancreas stomach prostate lung and bronchus leukemia liver pancreas treatment. Age-adjusted data like these are needed to compensate for the inevitable increase in cancer as people live longer, on average. (Adapted from Cancer Facts and Figures, 2012. Data from U.S. Mortality Volumes 1930 to 1959, U.S. Mortality Data 1960 to 2008, National Center for health Statistics, Centers for Disease Control and

1	Prevention. © 2012, American Cancer (A) (B) Society, Inc., Surveillance Research.) with hepatitis-C virus, which has infected 170 million people worldwide, is also clearly associated with the development of liver cancer.

1	The main culprits, as shown in Table 20–2, are the DNA viruses. The DNA tumor viruses cause cancer by the most direct route—by interfering with controls of the cell cycle and apoptosis. To understand this type of viral carcinogenesis, it is important to review the life history of viruses. Many DNA viruses use the host cell’s DNA replication machinery to replicate their own genomes. However, to produce a large number of infectious virus particles within a single host cell, the DNA virus has to commandeer this machinery and drive it hard, breaking through the normal constraints on DNA replication and usually killing the host cell in the process. Many DNA viruses reproduce only in this way. But some have a second option: they can propagate their genome as a quiet, well-behaved passenger in the host cell, replicating in parallel with the host cell’s DNA (either integrated into the host genome, or as an extrachromosomal plasmid) in the course of ordinary cell-division cycles. These viruses

1	host cell, replicating in parallel with the host cell’s DNA (either integrated into the host genome, or as an extrachromosomal plasmid) in the course of ordinary cell-division cycles. These viruses will switch between two modes of existence according to circumstances, remaining latent and harmless for a long time, but then proliferating in occasional cells in a process that kills the host cell and generates large numbers of infectious particles.

1	Neither of these conditions converts the host cell to a cancerous character, nor is it in the interest of the virus to do so. But for viruses with a latent phase, accidents can occur that prematurely activate some of the viral proteins that the virus would normally use in its replicative phase to allow the viral DNA to replicate independently of the cell cycle. As described in the example below, this type of accident can switch on the persistent proliferation of the host cell itself, leading to cancer. Cancers of the Uterine Cervix Can Be Prevented by Vaccination Against human Papillomavirus

1	The papillomaviruses are a prime example of DNA tumor viruses. They are responsible for human warts and are especially important as a cause of carcinoma of the uterine cervix: this is the second commonest cancer of women in the world as a whole, representing about 6% of all human cancers. Human papillomaviruses (HPV) infect the cervical epithelium and maintain themselves in a latent phase in the basal layer of cells as extrachromosomal plasmids, which replicate in step with the chromosomes. Infectious virus particles are generated through a switch to a replicative phase in the outer epithelial layers, as progeny of these cells begin to differentiate before being sloughed from the surface. Here, cell division should normally stop, but the virus interferes with this cell-cycle arrest so as to allow replication of its own genome. Usually, the effect is restricted to the outer layers of cells and is relatively harmless, as in a wart. Occasionally, however, a genetic accident causes the

1	to allow replication of its own genome. Usually, the effect is restricted to the outer layers of cells and is relatively harmless, as in a wart. Occasionally, however, a genetic accident causes the viral genes that encode the proteins that prevent cell-cycle arrest to integrate into the host chromosome and become active in the basal layer, where the stem cells of the epithelium reside (see Figure 22–10). This can lead to cancer, with the viral genes acting as oncogenes (Figure 20–40).

1	The whole process, from initial infection to invasive cancer, is slow, taking many years. It involves a long intermediate stage when the affected patch of cervical epithelium is visibly disordered but the cells have not yet begun to invade the underlying connective tissue—a phenomenon called intraepithelial neoplasia. Many such lesions regress spontaneously. Moreover, at this stage, it is still easy to cure the condition by destroying or surgically removing the abnormal tissue. Fortunately, the presence of such lesions can be detected by scraping off a sample of cells from the surface of the cervix and viewing it under the microscope (the “Pap smear” technique).

1	Better still, a vaccine has now been developed that protects against infection with the relevant strains of human papillomavirus. This vaccine, given to girls before puberty and thus before they become sexually active, has been shown to greatly reduce their risk of ever developing cervical cancer. Because the virus spreads through sexual activity, it is now recommended that both young males and young females be routinely vaccinated. Mass immunization programs have begun in several countries.

1	Figure 20–40 How certain papillomaviruses are thought to give rise to cancer of the uterine cervix. Papillomaviruses have double-stranded circular DNA chromosomes of about 8000 nucleotide pairs. These chromosomes are normally stably maintained in the basal cells of the epithelium as plasmids (red circles), whose replication is regulated so as to keep step with the chromosomes of the host. (A) Normally, the virus perturbs the host cell cycle only when the virus is programmed to produce infectious progeny, in the outer layers of an epithelium. This is relatively harmless.

1	(B) Rare accidents can cause the integration of a fragment of such a plasmid into a chromosome of the host, altering the environment of the viral genes in the basal cells of an epithelium. This can disrupt the normal control of viral gene expression. The unregulated production of certain viral proteins (E6 and E7) interferes with the control of cell division in the basal cells, thereby helping to generate a cancer (bottom). viral proteins mediate the controlled replication of the virus in outer cell layers unregulated production of viral proteins drives cell proliferation in basal cell layer Infectious Agents Can Cause Cancer in a Variety of Ways

1	Infectious Agents Can Cause Cancer in a Variety of Ways In papillomaviruses, the viral genes that are mainly to blame are called E6 and E7. The protein products of these viral oncogenes interact with many host-cell proteins, but, in particular, they bind to two key tumor suppressor proteins of the host cell, putting them both out of action and so permitting the cell to replicate its DNA and divide in an uncontrolled way. One of these host proteins is Rb; the other is p53. Other DNA tumor viruses use similar mechanisms to inhibit Rb and p53, underlining the central importance of inactivating both of these tumor suppressor pathways if a cell is to escape the normal constraints on proliferation.

1	In other cancers, viruses have indirect tumor-promoting actions. The hepatitis-B and C viruses, for example, favor the development of liver cancer by causing chronic inflammation (hepatitis), which stimulates an extensive cell division in the liver that promotes the eventual evolution of tumor cells. In AIDS, the human immunodeficiency virus (HIV) promotes development of an otherwise rare cancer called Kaposi’s sarcoma by destroying the immune system, thereby permitting a secondary infection with a human herpesvirus (HHV-8) that has a direct carcinogenic action. By causing severe inflammation, chronic infection with parasites and bacteria can also promote the development of some cancers. For example, chronic infection of the stomach with the bacterium Helicobacter pylori, which causes ulcers, appears to be a major cause of stomach cancer; dramatic falls in the incidence of stomach cancer over the last half-century (see Figure 20–39) correlate with a decline in the incidence of

1	ulcers, appears to be a major cause of stomach cancer; dramatic falls in the incidence of stomach cancer over the last half-century (see Figure 20–39) correlate with a decline in the incidence of Helicobacter infections.

1	The Search for Cancer Cures Is Difficult but Not hopeless The difficulty of curing a cancer is similar to the difficulty of getting rid of weeds. Cancer cells can be removed surgically or destroyed with toxic chemicals or radiation, but it is hard to eradicate every single one of them. Surgery can rarely ferret out every metastasis, and treatments that kill cancer cells are generally toxic to normal cells as well. Moreover, unlike normal cells, cancer cells can mutate rapidly and will often evolve resistance to the poisons and irradiation used against them.

1	In spite of these difficulties, effective cures using anticancer drugs (alone or in combination with other treatments) have already been found for some formerly highly lethal cancers, including Hodgkin’s lymphoma, testicular cancer, choriocarcinoma, and some leukemias and other cancers of childhood. Even for types of cancer where a cure at present seems beyond our reach, there are treatments that will prolong life or at least relieve distress. But what prospect is there of doing better and finding cures for the most common forms of cancer, which still cause great suffering and so many deaths? Traditional Therapies Exploit the Genetic Instability and Loss of Cell-Cycle Checkpoint Responses in Cancer Cells

1	Anticancer therapies need to take advantage of some molecular peculiarity of cancer cells that distinguishes them from normal cells. One such property is genetic instability, reflecting deficiencies in chromosome maintenance, cell-cycle checkpoints, and/or DNA repair. Remarkably, the most widely used cancer therapies seem to work by exploiting these abnormalities, although this was not known by the scientists who first developed the treatments. Ionizing radiation and most anticancer drugs damage DNA or interfere with chromosome segregation at mitosis, and they preferentially kill cancer cells because cancer cells have a diminished ability to survive the damage. Normal cells treated with radiation, for example, arrest their cell cycle until they have repaired the damage to their DNA, thanks to the cell-cycle checkpoint responses discussed in Chapter 17. Because cancer cells generally have defects in their checkpoint responses, they may continue to divide after irradiation, only to die

1	to the cell-cycle checkpoint responses discussed in Chapter 17. Because cancer cells generally have defects in their checkpoint responses, they may continue to divide after irradiation, only to die after a few days because the genetic damage remains unrepaired. More generally, most cancer cells are physiologically deranged to a stressful degree: they live dangerously. Even though the cells in a tumor have evolved to be unusually tolerant of minor DNA damage, they are hypersensitive to the much greater amount of damage that can be created by radiation and by DNA-damaging drugs. A small increase of genetic damage can be enough to tip the balance between proliferation and death.

1	Unfortunately, while the molecular defects present in cancer cells often enhance their sensitivity to cytotoxic agents, they can also increase their resistance. For example, where a normal cell might die by apoptosis in response to DNA damage, thanks to the stress response mediated by p53, a cancer cell may escape apoptosis because its p53 is lacking. Cancers vary widely in their sensitivity to cytotoxic treatments, some responding to one drug, some to another, probably reflecting the particular kinds of defects that a particular cancer has in DNA repair, cell-cycle checkpoints, and the control of apoptosis.

1	Radiotherapy and traditional cytotoxic drugs are rather weakly selective: they hurt normal cells as well as the cancer cells, and the safety margin is narrow. The dose often cannot be raised high enough to kill all the cancer cells, because this would kill the patient, and curative treatments, where achievable, generally require a combination of several cytotoxic agents. The side effects can be harsh and hard to endure. How can we do better?

1	An ideal treatment is one that is cell-lethal in combination with some lesion that is present in the cancer cells, but harmless to cells where this lesion is absent. Such a treatment is said to be synthetic-lethal (from the original sense of the word synthesis, meaning “putting together”): it kills only in partnership with the cancer-specific mutation. As we become increasingly able to pinpoint the specific alterations in cancer cells that make them different from their normal neighbors, new opportunities for such precisely targeted treatments are coming into view. We end this chapter with some examples of new treatments of this type that are already being put into practice.

1	As we have emphasized, the genetic instability of cancer cells makes the cells both dangerous and vulnerable—dangerous because of the enhancement in their ability to evolve and proliferate, and vulnerable because treatment that leads to still more extreme genetic disruption can take them over the brink and kill them. In some cancers, genetic instability results from an identified fault in one of the many devices on which normal cells depend for DNA repair and maintenance. In this case, a drug is tailored to block a complementary part of the DNA repair machinery can lead to such severe genetic damage that the cancer cells die.

1	Detailed studies of the mechanisms for DNA maintenance discussed in Chapter 5 reveal a surprising amount of apparent redundancy. Thus, knocking out a particular pathway for DNA repair is generally less disastrous than one might expect, because alternate repair pathways exist. For example, stalled DNA replication forks can arise when the fork encounters a single-strand break in a template strand, but cells can avoid the disaster that would otherwise result either by directly repairing these single-strand breaks, or, if that fails, repairing the broken fork that results by homologous recombination (see Figure 5–50). Suppose that the cells in a particular cancer have become genetically unstable by acquiring a mutation that reduces their ability to repair broken replication forks by homologous recombination. Might it be possible to eradicate that cancer by treating it with a drug that inhibits the repair of single-strand breaks, thereby greatly increasing the number of forks that break?

1	recombination. Might it be possible to eradicate that cancer by treating it with a drug that inhibits the repair of single-strand breaks, thereby greatly increasing the number of forks that break? The consequences of such drug treatment might be expected to be relatively harmless for normal cells, but lethal for the cancer.

1	This strategy appears to work to kill the cells in at least one class of cancers— those that have inactivated both copies of either their Brca1 or their Brca2 tumor continues, due to permanently suppressor genes. As described in Chapter 5, Brca2 is an accessory protein that interacts with the Rad51 protein (the RecA analog in humans) in the repair of DNA double-strand breaks by homologous recombination. Brca1 is another protein that is also required for this repair process. Like Rb, the Brca1 and Brca2 genes were discovered as mutations that predispose humans to cancer—in this case, chiefly cancers of the breast and ovaries (though unlike Rb, they seem to be involved in only a small proportion of such cancers). Individuals who inherit one mutant copy of Brca1 or Brca2 develop tumors that have inactivated the second copy of the same gene, presumably because this change makes the cells genetically unstable and speeds tumor progression.

1	While Brca1 and Brca2 are needed for the repair of DNA double-strand breaks, single-strand breaks are repaired by other machinery, involving an enzyme called PARP (polyADP-ribose polymerase). This understanding of the basic mechanisms of DNA repair led to a striking discovery: drugs that block PARP activity kill Brca-deficient cells with extraordinary selectivity. At the same time, PARP inhibition has very little effect on normal cells; in fact, mice that have been engineered to lack PARP1—the major PARP family member involved in DNA repair—remain healthy under laboratory conditions. This result suggests that, while the repair pathway requiring PARP provides a first line of defense against persistent breaks in a DNA strand, these breaks can be repaired efficiently by a genetic recombination pathway in normal cells. In contrast, tumor cells that have acquired their genetic instability by the loss of Brca1 or Brca2 have lost this second line of defense, and they are therefore uniquely

1	pathway in normal cells. In contrast, tumor cells that have acquired their genetic instability by the loss of Brca1 or Brca2 have lost this second line of defense, and they are therefore uniquely sensitive to PARP inhibitors (Figure 20–41).

1	PARP inhibitors are still under clinical trial, but they have produced some striking results, causing tumors to regress in many Brca-deficient patients and delaying progression of their disease, with relatively few disagreeable side effects. These drugs also appear to be applicable to cancers with other mutations that cause defects in the cell’s homologous recombination machinery—a small, though significant, proportion of cancer cases.

1	Figure 20–41 How a tumor’s genetic instability can be exploited for cancer therapy. As explained in Chapter 5, the maintenance of DNA sequences is so critical for life that cells have evolved multiple pathways for repairing DNA damage and reducing DNA replication errors. As illustrated, a DNA replication fork will stall whenever it encounters a break in a DNA template strand. In this example, normal cells have two different repair pathways that help them to avoid the problem, pathways 1 and 2. They are therefore not harmed by treatment with a drug that blocks repair pathway 1. But, because the inactivation of repair pathway 2 was selected for during the evolution of the tumor cell, the tumor cells are killed by the same drug treatment.

1	In the actual case that underlies this example, the function of repair pathway 1 (requiring the PARP protein discussed in the text) is to remove persistent, accidental breaks in a DNA single strand before they are encountered by a moving replication fork. Pathway 2 is the recombination-dependent process (requiring the Brca2 and Brca1 proteins) for repairing stalled replication forks illustrated in Figure 5–50. PARP inhibitors have promise for treating cancers with defective Brca2 or Brca1 tumor suppressor genes. PARP inhibition provides an example of the type of rational, highly selective approach to cancer therapy that is beginning to be possible. Along with other new treatments to be discussed below, it raises high hopes for treating many other cancers. Small Molecules Can Be Designed to Inhibit Specific Oncogenic Proteins

1	An obvious tactic for treating cancer is to attack a tumor expressing an oncogene with a drug designed to specifically block the function of the protein that the oncogene produces. But how can such a treatment avoid hurting the normal cells that depend on the function of the proto-oncogene from which the oncogene has evolved, and why should the drug kill the cancer cells, rather than simply calm them down? One answer may lie in the phenomenon of oncogene dependence. Once a cancer cell has undergone an oncogenic mutation, it will often undergo further mutations, epigenetic changes, or physiological adaptations that make it reliant on the hyperactivity of the initial oncogene, just as drug addicts become reliant on high doses of their drug. Blocking the activity of the oncogenic protein may then kill the cancer cell without significantly harming its normal neighbors. Some remarkable successes have been achieved in this way.

1	As we saw earlier, chronic myelogenous leukemia (CML) is usually associated with a particular chromosomal translocation, visible as the Philadelphia chromosome (see Figure 20–5). This results from chromosome breakage and rejoining at the sites of two specific genes, Abl and Bcr. The fusion of these genes creates a hybrid gene, called Bcr-Abl, that codes for a chimeric protein consisting of the N-terminal fragment of Bcr fused to the C-terminal portion of Abl (Figure 20–42). Abl is a tyrosine kinase involved in cell signaling. The substitution of the Bcr fragment for the normal N-terminus of Abl makes it hyperactive, so that it stimulates inappropriate proliferation of the hemopoietic precursor cells that contain it and prevents these cells from dying by apoptosis—which many of them would normally do. As a result, excessive numbers of white blood cells accumulate in the bloodstream, producing CML.

1	The chimeric Bcr-Abl protein is an obvious target for therapeutic attack. Searches for synthetic drug molecules that can inhibit the activity of tyrosine kinases discovered one, called imatinib (trade name Gleevec®), that blocks Bcr-Abl (Figure 20–43). When the drug was first given to patients with CML, nearly all of them showed a dramatic response, with an apparent disappearance of the cells carrying the Philadelphia chromosome in over 80% of patients. The response appears relatively durable: after years of continuous treatment, many patients have not progressed to later stages of the disease—although imatinib-resistant cancers emerge with a probability of about 5% per year during the early years.

1	Figure 20–42 The conversion of the Abl proto-oncogene into an oncogene in patients with chronic myelogenous leukemia. The chromosome translocation responsible joins the Bcr gene on chromosome 22 to the Abl gene from chromosome 9, thereby generating a Philadelphia chromosome (see Figure 20–5). The resulting fusion protein has the N-terminus of the Bcr protein joined to the C-terminus of the Abl tyrosine protein kinase; in consequence, the Abl kinase domain becomes inappropriately active, driving excessive proliferation of a clone of hemopoietic cells in the bone marrow. Figure 20–43 How imatinib (Gleevec) blocks the activity of Bcr-Abl protein and halts chronic myelogenous leukemia.

1	Figure 20–43 How imatinib (Gleevec) blocks the activity of Bcr-Abl protein and halts chronic myelogenous leukemia. (A) Imatinib sits in the ATP-binding pocket of the tyrosine kinase domain of Bcr-Abl and thereby prevents Bcr-Abl from transferring a phosphate group from ATP onto a tyrosine residue in a substrate protein. This blocks transmission of a signal for cell proliferation and survival. (B) The structure of the complex of imatinib (solid blue object) with the tyrosine kinase domain of the Abl protein (ribbon diagram), as determined by x-ray crystallography. (C) The chemical structure of the drug. It can be given by mouth; it has side effects, but they are usually quite tolerable. (B, from T. Schindler et al., Science 289:1938–1942, 2000. With permission from AAAS.)

1	Results are not so good for those patients who have already progressed to the more acute phase of myeloid leukemia, known as blast crisis, where genetic instability has set in and the march of the disease is far more rapid. These patients show a response at first and then relapse because the cancer cells develop a resistance to imatinib. This resistance is usually associated with secondary mutations in the part of the Bcr-Abl gene that encodes the kinase domain, disrupting the ability of imatinib to bind to Bcr-Abl kinase. Second-generation inhibitors that function effectively against a whole range of imatinib-resistant mutants have now been developed. By combining one or more of these new inhibitors with imatinib as the initial therapy (see below), it seems that CML—at least in the chronic (early) stage—may be on its way to becoming a curable disease.

1	Despite the complications with resistance, the extraordinary success of imatinib is enough to drive home an important principle: once we understand precisely what genetic lesions have occurred in a cancer, we can begin to design effective rational methods to treat it. This success story has fueled efforts to identify small-molecule inhibitors for other oncogenic protein kinases and to use them to attack the appropriate cancer cells. Increasing numbers are being developed. These include molecules that target the EGF receptor and are currently approved for the treatment of some lung cancers, as well as drugs that specifically target the B-Raf oncoprotein in melanomas.

1	Protein kinases have been relatively easy to inhibit with small molecules like imatinib, and many kinase inhibitors are being produced by pharmaceutical companies in the hope that they can be effective as drugs for some forms of cancer. Many cancers lack an oncogenic mutation in a protein kinase. But most tumors contain inappropriately activated signaling pathways, for which a target somewhere in the pathway can hopefully be found (Movie 20.7). As an example, Figure 20–44 displays some of the anticancer drugs and drug targets that are currently being tested for a pathway frequently activated in cancers. Many Cancers May Be Treatable by Enhancing the Immune Response Against the Specific Tumor

1	Many Cancers May Be Treatable by Enhancing the Immune Response Against the Specific Tumor Cancers have complex interactions with the immune system, and its various components may sometimes help as well as hinder tumor progression. But for more than a century it has been a dream of cancer researchers to somehow harness the immune system in a controlled and efficient way to exterminate cancer cells, just as it exterminates infectious organisms. There are finally signs that this dream may one day be realized, at least for some forms of cancer.

1	The simplest type of immunological therapy, conceptually at least, is to inject the patient with antibodies that target the cancer cells. This approach has had some successes. About 25% of breast cancers, for example, express unusually high levels of the Her2 protein, a receptor tyrosine kinase related to the EGF receptor that plays a part in the normal development of mammary epithelium. A monoclonal antibody called trastuzumab (trade name Herceptin®) that binds to Her2 and inhibits its function slows the growth of breast tumors in humans that overexpress Her2, and it is now an approved therapy for these cancers (see Figure 20–44). A related approach uses antibodies to deliver poisons to the cancer cells. Antibodies against proteins that are abundant on the surface of a particular type of cancer cell but rare on normal cells can be armed with a toxin that kills those cells that bind the antibody molecule.

1	A great deal of current excitement centers around a different type of approach, based on the relatively recent recognition that the microenvironment in a tumor is highly immunosuppressive. As a result, the cancer victim’s immune system is prevented from destroying the tumor cells. Recall that, from the thousands of genome sequences thus far determined, we know that a typical cancer cell will contain on the order of 50 proteins with a mutation that alters an amino acid sequence, most of these being “passenger” mutations, as previously explained (see p. 1104). Many of these mutant proteins will be recognized by the patient’s immune system as foreign, but—to allow the cancer cells to survive throughout the course of tumor progression—the cancer cells have evolved a set of anti-immune defenses. These

1	Figure 20–44 Some anticancer drugs and drug targets in the Ras–MAP-kinase signaling pathway. Each of the signaling proteins in this diagram has been identified as a product of a cancer-critical gene, with the exception of Raf1 and Erk. This Ras– MAP-kinase signaling pathway is triggered by a variety of receptor tyrosine kinases (RTKs), including the EGF receptor (see Figures 15–47 and 15–49). Those drugs that are antibodies end in “mab,” while those that are small molecules end in “nib.” (Adapted from B. Vogelstein et al, Science 339:1546–1558, 2013.) defenses include the expression on the cancer cell surface of one or more proteins that bind to inhibitory receptors on activated T cells.

1	The normal immune system is subject to complex controls that keep its activity within safe bounds and prevent autoimmunity from developing. The inhibitory receptors that are expressed on the surface of activated T cells have an important normal function: they control the immune response by down-regulating the T cell response under appropriate circumstances. But in the context of a tumor, the down-regulation is inappropriate, because it prevents the organism from killing the cancer cells that are threatening its survival.

1	In its attack on infectious organisms, the natural immune system usually eliminates every last trace of infection and maintains this immunity in the long term. The challenge is to find ways of recruiting the immune system to attack cancers with similar efficiency and specificity, hunting the cancer cells down by virtue of the tumor-specific antigens that they express. With this aim, a new type of anticancer therapy focuses on overcoming the immunosuppressive environment in a tumor through the use of specific antibodies that prevent the tumor cells from engaging with the inhibitory receptors on T cells. As illustrated in Figure 20–45A, blocking the action of the immune suppressors with such treatments should unleash an immune attack on the cancer cells. Importantly, multiple antigens are recognized as foreign; thus, the cancer cells cannot escape through the mutational loss of a single antigen, making it difficult for the tumor to escape from the T cell attack.

1	This is a potentially dangerous strategy. If one provokes the immune system to recognize the cancer cells as targets for destruction, there is a risk of autoimmune side effects with dire consequences for normal tissues of the body, since the cancer cells and the normal cells are close cousins and share most of their molecular features. Nevertheless, several recent successes seem to hold great promise for the future.

1	One of the many molecules involved in keeping the activity of the normal immune system within safe bounds is a protein called CTLA4 (cytotoxic T-lymphocyte-associated protein 4), which functions as an inhibitory receptor on the surface of T cells. If the function of CTLA4 is blocked, the T cells become more reactive and may mount an attack on cells that they would otherwise leave in peace. In particular, the T cells may attack tumor cells that are recognizably abnormal but whose presence was previously tolerated. With this in mind, cancer immunologists developed a monoclonal antibody, called ipilimumab, that binds to CTLA4 and blocks its action. Injected repeatedly into patients with metastatic melanoma, this antibody increases their median lifespan by several months and, in one large trial, enabled as many as a quarter of them to survive for five years

1	Figure 20–45 Therapies designed to remove the immunosuppressive microenvironment in tumors. (A) The cells in tumors will produce many mutant proteins. As described in Chapter 24, peptides from these proteins will be displayed on MhC complexes on the tumor-cell surface and would normally activate a T cell response that destroys the tumor (see Figure 24–42). however, as schematically illustrated, during the course of tumor progression, the cancer cells have evolved immunosuppressive mechanisms that protect them from such killing. (B) The cells in tumors often protect themselves from immune attack by expressing proteins on their surface that bind to and thereby activate the inhibitory receptors on T cells. As indicated, this makes the tumor susceptible to specific antibody therapies. In this diagram, two such inhibitory receptors are shown, PD1 and a hypothetical protein X. Different tumors are thought to protect themselves by activating different members of a large set of T cell

1	In this diagram, two such inhibitory receptors are shown, PD1 and a hypothetical protein X. Different tumors are thought to protect themselves by activating different members of a large set of T cell inhibitory receptors, some of which are not yet well characterized.

1	or more—far beyond expectations for comparable patients without this treatment. Even more promising are recent clinical trials using a combination of two antibodies, one against CTLA4 and the other against PD1, a second cell-surface receptor on T cells that normally restrains their activity.

1	In clinical trials using such techniques, a substantial fraction of the patients can respond in a dramatic way, with their cancer being driven into remission for years, while the treatment fails to help others with the same type of cancer. One possible explanation is that, while most tumors express proteins that protect them from T-cell attack, these proteins are different for different tumors. Thus, while some tumors will respond dramatically when treated with an antibody that blocks a particular immunosuppressive agent, many others will not. If true, one can foresee an era of personalized immunotherapy, in which each patient’s tumor is molecularly analyzed to determine its particular mechanisms of immunosuppression. The patient would then be treated with a specific cocktail of antibodies designed to remove these blocks (see Figure 20–45). Cancers Evolve Resistance to Therapies

1	Cancers Evolve Resistance to Therapies High hopes have to be tempered with sobering realities. We have seen that genetic instability can provide an Achilles heel that cancer therapies can exploit, but at the same time it can make eradicating the disease more difficult by allowing the cancer cells to evolve resistance to therapeutic drugs, often at an alarming rate. This applies even to the drugs that target genetic instability itself. Thus, PARP inhibitors give valuable remission of illness, but in the long term the disease generally comes back. For example, Brca-deficient cancers can sometimes develop resistance to PARP inhibitors by undergoing a second mutation in an affected Brca gene that restores its function. By then, the cancer is already out of control and it may be too late to affect the course of the disease with additional treatments.

1	There are many different strategies by which cancers can evolve resistance to anticancer drugs. Often, a cancer will be dramatically reduced in size by an initial drug treatment, with all of the detectable tumor cells seeming to disappear. But months or years later the cancer will reappear in an altered form that is resistant to the drug that was at first so successful. In such cases, the initial drug treatment has evidently failed to destroy some tiny fraction of cells in the original tumor-cell population. These cells have escaped death because they carry a protective mutation or epigenetic change, or perhaps simply because they were lurking in a protected environment. They eventually regenerate the cancer by continuing to proliferate, mutating and evolving still further as they do so.

1	In some cases, cells that are exposed to one anticancer drug evolve a resistance not only to that drug but also to other drugs to which they have never been exposed. This phenomenon of multidrug resistance frequently correlates with amplification of a part of the genome that contains a gene called Mdr1 or Abcb1. This gene encodes a plasma-membrane-bound transport ATPase of the ABC transporter superfamily (discussed in Chapter 11), which pumps lipophilic drugs out of the cell (see Movie 11.5). The overproduction of this protein (or some of its other family members) by a cancer cell can prevent the intracellular accumulation of many cytotoxic drugs, making the cell insensitive to them.

1	In the to-and-fro struggle between advanced metastatic cancer and the therapist, as current practice stands, the cancer usually wins in the end. Does it have to be so? As we discuss below, there is reason to think that by attacking a cancer with many weapons at once—instead of using them one after another, each until it fails—it may be possible to do much better. Combination Therapies May Succeed Where Treatments with One Drug at a Time Fail Nowadays, cancers caught at an early stage can often be cured, by surgery, radiation, or drugs. For most cancers that have progressed and metastasized widely, however, cure is still beyond us. Treatments such as those described above can no cell is resistant CANCER CURED = cell resistant to Ato both drugs = cell resistant to A and B (B) give valuable remissions, but sooner or later these are typically followed by relapse.

1	Nevertheless, for some relatively rare forms of advanced cancer, curative therapies have been developed. These generally involve a cocktail of several different anticancer agents: by trial and error, certain combinations of cytotoxic drugs have been found to wipe out the cancer completely. Discovering such combinations has hitherto involved a long, hard search. But now, armed with our new tools for identifying the specific genetic lesions that cancer cells contain, the prospects are better.

1	The logic of combination therapies is the same as that behind the current treatment of HIV-AIDS with a cocktail of three different protease inhibitors: whereas there may always be some cells in the initial population carrying the rare mutations that confer resistance to any one drug treatment, there should be no cell carrying the whole set of rare mutations that would confer resistance to several different drugs delivered simultaneously. In contrast, sequential drug treatments will allow the few cells resistant to the first drug to multiply to large numbers. Within this large population of cells resistant to the first drug, a small number of cells are likely to have arisen that are resistant to the next drug also; and so on (Figure 20–46). We Now have the Tools to Devise Combination Therapies Tailored to the Individual Patient

1	We Now have the Tools to Devise Combination Therapies Tailored to the Individual Patient Efficient, rational combination drug therapy requires three things. First, we have to identify multiple peculiarities of cancer cells that make them vulnerable in ways that normal cells are not. Second, we have to devise drugs (or other treatments) that target each of these vulnerabilities. Third, we have to match the combination of drugs to the specific set of peculiarities present in the cancer cells of the individual patient.

1	The first requirement is already partially met: we now have large catalogs of cancer-critical genes that are commonly mutated in cancer cells. The second requirement is harder, but attainable: we have described some remarkable recent successes, and for cancer researchers there is excitement in the air. It is becoming increasingly possible to use our growing knowledge of cell and molecular biology to design new drugs against designated targets. At the same time, efficient, high-throughput automated methods are available to screen large libraries of chemicals for any that may be effective against cells with a given cancer-related defect. In such searches, the goal is synthetic lethality: a cell death that occurs when and only when a particular drug is put together with a particular cancer cell abnormality. Through these and other approaches, the repertoire of precisely targeted anticancer drugs is rapidly increasing.

1	Figure 20–46 Why multidrug treatments can be more effective than sequential treatments for cancer therapy. (A) Because tumor cells are hypermutable, two single-drug treatments that are given sequentially often allow for the selection of mutant cell clones that are resistant to both drugs. (B) Simultaneous treatment with both drugs can be more effective.

1	This brings us to the third requirement: the therapy—the choice of drugs to be given in combination—must be tailored to the individual patient. Here, too, the prospects are bright. Cancers evolve by a fundamentally random process, and each patient is different; but modern methods of genome analysis now let us characterize the cells from a tumor biopsy in exhaustive detail so as to discover which cancer-critical genes are affected in a particular case. Admittedly, this is not straightforward: the tumor cells in an individual patient are heterogeneous and do not all contain the same genetic lesions. With increased understandings of the pathways of cancer evolution, however, and with the experience gained from many different cases, it should become possible to make good guesses at the optimal therapies to use.

1	From the perspective of the patient, the pace of advance in cancer research can seem frustratingly slow. Each new drug has to be tested in the clinic, first for safety and then for efficacy, before it can be released for general use. And if the drug is to be used in combination with others, the combination therapy must then go through the same long process. Strict ethical rules constrain the conduct of trials, which means that they take time—typically several years. But slow and cautious steps, taken systematically in the right direction, can lead to great advances. There is still far to go, but the examples that we have discussed provide proof of principle and grounds for optimism. From the cancer research effort, we have learned a great deal of what we know about the molecular biology of the normal cell. Now, more and more, we are discovering how to put that knowledge to use in the battle with cancer itself.

1	Our growing understanding of the cell biology of cancers has already begun to lead to better ways of preventing, diagnosing, and treating these diseases. Anticancer therapies can be designed to destroy cancer cells preferentially by exploiting the properties that distinguish cancer cells from normal cells, including the cancer cells’ dependence on oncogenic proteins and the defects they harbor in their DNA repair mechanisms. We now have good evidence that, by increasing our understanding of normal cell control mechanisms and exactly how they are subverted in specific cancers, we can eventually devise drugs to kill cancers precisely by attacking specific molecules critical for the growth and survival of the cancer cells. In addition, great progress has recently been made through sophisticated immunological approaches to cancer therapy. And, as we become better able to determine which genes are altered in the cells of any given tumor, we can begin to tailor treatments more accurately to

1	immunological approaches to cancer therapy. And, as we become better able to determine which genes are altered in the cells of any given tumor, we can begin to tailor treatments more accurately to each individual patient.

1	Which statements are true? Explain why or why not. What is required to enable a cancer cell to metastasize? how can the molecular analysis of an individual tumor be more effectively used to design effective therapies to kill it? Can we identify general features common to all cancer cells—such as their production of misfolded, mutated proteins—that can be used for the targeted destruction of many different types of cancers? Can sensitive and reliable blood tests be devised to detect cancers very early, before they have grown to a size where treatment with a single drug will generally be defeated by the survival of a preexisting resistant variant? how can the observed environmental effects on cancer rates be exploited to reduce avoidable cancers? Can new technologies be devised to reveal exactly how a quiescent micrometastasis converts to a full-blown metastatic tumor?

1	Can new technologies be devised to reveal exactly how a quiescent micrometastasis converts to a full-blown metastatic tumor? 20–1 The chemical carcinogen dimethylbenz[a]anthra-20–4 The main environmental causes of cancer are the cene (DMBA) must be an extraordinarily specific mutagen products of our highly industrialized way of life such as since 90% of the skin tumors it causes have an A-to-T alter-pollution and food additives. ation at exactly the same site in the mutant Ras gene.

1	Discuss the following problems. 20–2 In the cellular regulatory pathways that control 20–5 In contrast to colon cancer, whose incidence cell growth and proliferation, the products of oncogenes increases dramatically with age, incidence of osteosarare stimulatory components and the products of tumor coma—a tumor that occurs most commonly in the long suppressor genes are inhibitory components. bones—peaks during adolescence. Osteosarcomas are rel atively rare in young children (up to age 9) and in adults 20–3 Cancer therapies directed solely at killing the rap-(over 20). Why do you suppose that the incidence of osteoidly dividing cells that make up the bulk of a tumor are sarcoma does not show the same sort of age-dependence unlikely to eliminate the cancer from many patients. as colon cancer? lung cancer mortality, cumulative risk (%)

1	lung cancer mortality, cumulative risk (%) Figure Q20–1 Cumulative risk of lung cancer mortality for nonsmokers, smokers, and former smokers (Problem 20–6). Cumulative risk is the running total of deaths, as a percentage, for each group. Thus, for continuing smokers, 1% died of lung cancer risk of 5%); and 11% more cumulative risk of 16%). 20–6 Mortality due to lung cancer was followed in groups of males in the United Kingdom for 50 years. Figure Q20–1 shows the cumulative risk of dying from lung cancer as a function of age and smoking habits for four groups of males: those who never smoked, those who stopped at age 30, those who stopped at age 50, and those who continued to smoke. These data show clearly that individuals can substantially reduce their cumulative risk of dying from lung cancer by stopping smoking. What do you suppose is the biological basis for this observation?

1	20–7 A small fraction—2 to 3%—of all cancers, across many subtypes, displays a quite remarkable phenomenon: tens to hundreds of rearrangements that primarily involve a single chromosome, or chromosomal region. The breakpoints can be tightly clustered, with several in a few kilobases; the junctions of the rearrangements often involve segments of DNA that were not originally close together on the chromosome. The copy number of various segments within the rearranged chromosome was found to be either zero, indicating deletion, or one, indicating retention. You can imagine two ways in which such multiple, localized rearrangements might happen: a progressive rearrangements model with ongoing inversions, deletions, and duplications involving a localized area, or a catastrophic model in which the chromosome is shattered into fragments that are stitched back together in random order by nonhomologous end joining (Figure Q20–2).

1	A. Which of the two models in Figure Q20–2 accounts more readily for the features of these highly rearranged chromosomes? Explain your reasoning. B. For whichever model you choose, suggest how such multiple rearrangements might arise. (The true mechanism is not known.) C. Do you suppose such rearrangements are likely to be causative events in the cancers in which they are found, or are they probably just passenger events that are unrelated to the cancer? If you think they could be driver events, suggest how such rearrangements might activate an oncogene or inactivate a tumor suppressor gene. 20–8 Virtually all cancer treatments are designed to kill cancer cells, usually by inducing apoptosis. However, one particular cancer—acute promyelocytic leukemia (APL)— has been successfully treated with all-trans-retinoic acid, which causes the promyelocytes to differentiate into neutrophils. How might a change in the state of differentiation of APL cancer cells help the patient?

1	20–9 One major goal of modern cancer therapy is to identify small molecules—anticancer drugs—that can be used to inhibit the products of specific cancer-critical genes. If you were searching for such molecules, would you design inhibitors for the products of oncogenes or the products of tumor suppressor genes? Explain why you would (or would not) select each type of gene. (lost to cell) Figure Q20–2 Two models to explain the multiple, localized chromosome rearrangements found in some cancers (Problem 20–7). The progressive rearrangements model shows a sequence of rearrangements that disrupts the chromosome, generating increasingly complex chromosomal configurations. The chromosome catastrophe model shows the chromosome being fragmented and then reassembled randomly, with some pieces left out.

1	20–10 PolyADP-ribose polymerase (PARP) plays a key role in the repair of DNA single-strand breaks. In the presence of the PARP inhibitor olaparib, single-strand breaks accumulate. When a replication fork encounters a sin-gle-strand break, it converts it to a double-strand break, which in normal cells is then repaired by homologous recombination. In cells defective for homologous recombination, however, inhibition of PARP triggers cell death. Patients who have only one functional copy of the Brca1 gene, which is required for homologous recombination, are at much higher risk for cancer of the breast and ovary. Cancers that arise in these tissues in these patients can be treated successfully with olaparib. Explain how it is that treatment with olaparib kills the cancer cells in these patients, but does not harm their normal cells.

1	20–11 The Tasmanian devil, a carnivorous Australian marsupial, is threatened with extinction by the spread of a fatal disease in which a malignant oral–facial tumor interferes with the animal’s ability to feed. You have been called in to analyze the source of this unusual cancer. It seems clear to you that the cancer is somehow spread from devil to devil, very likely by their frequent fighting, which is accompanied by biting around the face and mouth. To uncover the source of the cancer, you isolate tumors from 11 devils captured in widely separated regions and examine them. As might be expected, the karyotypes of the tumor cells are highly rearranged relative to that of the wild-type devil (Figure Q20–3). Surprisingly, you find that the karyotypes from all 11 tumor samples are very similar. Moreover, one of the Tasmanian devils has an inversion on chromosome 5 that is not present in its facial tumor. How do you suppose this cancer is transmitted from devil to devil? Is it likely to

1	Moreover, one of the Tasmanian devils has an inversion on chromosome 5 that is not present in its facial tumor. How do you suppose this cancer is transmitted from devil to devil? Is it likely to arise as a consequence of an infection by a virus or microorganism? Explain your reasoning.

1	Figure Q20–3 Karyotypes of cells from Tasmanian devils (Problem 20–11). (A) A Tasmanian devil. (B) Normal karyotype for a male Tasmanian devil. The karyotype has 14 chromosomes, including XY. (C) Karyotype of cancer cells found in each of the 11 facial tumors studied. The karyotype has 13 chromosomes, no sex chromosomes, no chromosome 2 pair, one chromosome 6, two chromosomes 1 with deleted long arms, and four highly rearranged marker chromosomes (M1–M4). (A, reproduced courtesy of Museum Victoria; B and C, from A.M. Pearse and K. Swift, Nature 439:549, 2006. With permission from Macmillan Publishers Ltd.) Bishop JM (2004) how to Win the Nobel Prize: An Unexpected Life in Science. Cambridge, MA: harvard University Press. hanahan D & Weinberg RA (2011) hallmarks of cancer: the next generation. Cell 144, 646–674. Vogelstein B, Papadopoulos N, Velculescu VE et al. (2013) Cancer genome landscapes. Science 339, 1546–1558.

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1	Kalos M & June Ch (2013) Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology. Immunity 39, 49–60. Loeb LA (2011) human cancers express mutator phenotypes: origin, consequences and targeting. Nat. Rev. Cancer 11, 450–457. Lord CJ & Ashworth A (2012) The DNA damage response and cancer therapy. Nature 481, 287–294. Pardoll DM (2012) The blockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer 12, 252–264. Peto J (2001) Cancer epidemiology in the last century and the next decade. Nature 411, 390–395. Sawyers C (2004) Targeted cancer therapy. Nature 432, 294–297. Schreiber RD, Old LJ & Smyth MJ (2011) Cancer immunoediting: integrating immunity’s roles in cancer suppression and promotion. Science 331, 1565–1570. Sliwkowski MX & Mellman I (2013) Antibody therapeutics in cancer. Science 341, 1192–1198. Varmus h, Pao W, Politi K et al. (2005) Oncogenes come of age. Cold Spring Harb. Symp. Quant. Biol. 70, 1–9.

1	Varmus h, Pao W, Politi K et al. (2005) Oncogenes come of age. Cold Spring Harb. Symp. Quant. Biol. 70, 1–9. Ward RJ & Dirks PB (2007) Cancer stem cells: at the headwaters of tumor development. Annu. Rev. Pathol. 2, 175–189. Development of Multicellular Organisms 21 An animal or plant starts its life as a single cell—a fertilized egg, or zygote. During development, this cell divides repeatedly to produce many different kinds of cells, arranged in a final pattern of spectacular complexity and precision. The goal of developmental cell biology is to understand the cellular and molecular mechanisms that direct this amazing transformation (Movie 21.1).

1	Plants and animals have very different ways of life, and they use different developmental strategies; in this chapter, we focus mainly on animals. Four processes are fundamental to animal development: (1) cell proliferation, which produces many cells from one; (2) cell–cell interactions, which coordinate the behavior of each cell with that of its neighbors; (3) cell specialization, or differentiation, which creates cells with different characteristics at different positions; and (4) cell movement, which rearranges the cells to form structured tissues and organs (Figure 21–1). It is on the fourth point that plant development differs radically: plant cells are unable to migrate or move independently through the embryo because each one is contained within a cell wall, through which it is cemented to its neighbors, as discussed in Chapter 19.

1	In a developing animal embryo, the four fundamental processes are happening in a kaleidoscopic variety of ways, as they give rise to different parts of the organism. Like the members of an orchestra, the cells in the embryo have to play their individual parts in a highly coordinated manner. In the embryo, however, there is no conductor—no central authority—to direct the performance. Instead, development is a self-assembly process in which the cells, as they grow and proliferate, organize themselves into increasingly complex structures. Each of the millions of cells has to choose for itself how to behave, selectively utilizing the genetic instructions in its chromosomes.

1	At each stage in its development, the cell is presented with a limited set of options, so that its developmental pathway branches repeatedly, reflecting a large set of sequential choices. Like the decisions we make in our own lives, the choices made by the cell are based on its internal state—which largely reflects its history—and on current influences from other cells, especially its close neighbors. To understand development, we need to know how each choice is controlled and how it depends on previous choices. Beyond that, we need to understand how the choices, once made, influence the cell’s chemistry and behavior, and how cell behaviors act synergistically to determine the structure and function of the body. Figure 21–1 The four essential cell processes that allow a multicellular organism to be made.

1	Figure 21–1 The four essential cell processes that allow a multicellular organism to be made. As cells become specialized they change not only their chemistry but also their shape and their attachments to other cells and to the extracellular matrix. They move and rearrange themselves to create the complex architecture of the body, with all its tissues and organs, each structured precisely and defined in size. To understand this process of form generation, or morphogenesis, we will need to take account of the mechanical, as well as the biochemical, interactions between the cells.

1	At first glance, one would no more expect the worm, the flea, the eagle, and the giant squid all to be generated by the same developmental mechanisms than one would suppose that the same methods were used to make a shoe and an airplane. Remarkably, however, research in the past 30 years has revealed that much of the basic machinery of development is essentially the same in all animals—not just in all vertebrates, but in all the major phyla of invertebrates too. Recognizably similar, evolutionarily related molecules define the specialized animal cell types, mark the differences between body regions, and help create the animal body pattern. Homologous proteins are often functionally interchangeable between very different species. Thus, a human protein produced artificially in a fly, for example, can perform the same function as the fly’s own version of that protein (Figure 21–2). Thanks to an underlying unity of mechanisms, developmental biologists have been making great strides toward

1	can perform the same function as the fly’s own version of that protein (Figure 21–2). Thanks to an underlying unity of mechanisms, developmental biologists have been making great strides toward a coherent understanding of animal development.

1	We begin this chapter with an overview of some of the basic mechanisms that operate in animal development. We then discuss, in sequence, how cells in the embryo diversify to form patterns in space, how the timing of developmental events is controlled, how cell movements contribute to morphogenesis, and how the size of an animal is regulated. We end by considering the most challenging aspect of development—the mechanisms that enable a highly complex nervous system to form. misexpression of Eyeless/Pax6 in misexpression of wild type wing precursors Eyeless/Pax6 in leg precursors

1	misexpression of Eyeless/Pax6 in misexpression of wild type wing precursors Eyeless/Pax6 in leg precursors Figure 21–2 Homologous proteins can function interchangeably. (a–c) the eyeless protein (also called pax6) controls eye development in Drosophila and, when misexpressed during development, can cause an eye to form in an abnormal site, such as a wing (B) or a leg (c). the scanning electron micrographs show a patch of eye tissue on the leg of a fly resulting from misexpression of Drosophila Eyeless (e) and of squid Pax6 (F). the homologous protein from a human or practically any animal possessing eyes, when similarly misexpressed in a transgenic fly, has the same effect. the entire eye of a normal Drosophila is shown for comparison in (a) and (D). (B–c, courtesy of Georg halder; D–F, from S. I. tomarev, et al. Proc. Natl Acad. Sci. USA 94:2421–2426, 1997. With permission from National academy of Sciences.)

1	Animals live by eating other organisms. Thus, despite their remarkable diversity, animals as different as worms, mollusks, insects, and vertebrates share anatomical features that are fundamental to this way of life. Epidermal cells form a protective outer layer; gut cells absorb nutrients from ingested food; muscle cells allow movement; and neurons and sensory cells control behavior. These diverse cell types are organized into tissues and organs, forming a sheet of skin covering the exterior, a mouth for feeding, and an internal gut tube to digest food—with muscles, nerves, and other tissues arranged in the space between the skin and the gut tube. Many animals have clearly defined axes—an anteroposterior axis, with mouth and brain anterior and anus posterior; a dorsoventral axis, with back dorsal and belly ventral; and a left-right axis. In this section, we discuss some fundamental mechanisms underlying animal development, beginning with how the basic animal body plan is established.

1	conserved Mechanisms establish the Basic animal Body plan

1	The shared anatomical features of animals develop through conserved mechanisms. After fertilization, the zygote usually divides rapidly, or cleaves, to form many smaller cells; during this cleavage, the embryo, which cannot yet feed, does not grow. This phase of development is initially driven and controlled entirely by the material deposited in the egg by the mother. The embryonic genome remains inactive until a point is reached when maternal mRNAs and proteins rather abruptly begin to be degraded. The embryo’s genome is activated, and the cells cohere to form a blastula—typically a solid or a hollow fluid-filled ball of cells. Complex cell rearrangements called gastrulation (from the Greek “gaster,” meaning “belly”) then transform the blastula into a multilayered structure containing a rudimentary internal gut (Figure 21–3). Some cells of the blastula remain external, constituting the ectoderm, which will give rise to the epidermis and the nervous system; other cells invaginate,

1	a rudimentary internal gut (Figure 21–3). Some cells of the blastula remain external, constituting the ectoderm, which will give rise to the epidermis and the nervous system; other cells invaginate, forming the endoderm, which will give rise to the gut tube and its appendages, such as lung, pancreas, and liver. Another group of cells moves into the space between ectoderm and endoderm and forms the mesoderm, which will give rise to muscles, connective tissues, blood, kidney, and various other components. Further cell movements and accompanying cell differentiations create and refine the embryo’s architecture.

1	Figure 21–3 The early stages of development, as exemplified by a frog.

1	(a) a fertilized egg divides to produce a blastula—a sheet of epithelial cells often surrounding a cavity. During gastrulation, some of the cells tuck into the interior to form the mesoderm (green) and endoderm (yellow). ectodermal cells (blue) remain on the outside. (B) a cross section through the trunk of an amphibian embryo shows the basic animal body plan, with a sheet of ectoderm on the outside, a tube of endoderm on the inside, and mesoderm sandwiched between them. the endoderm forms the epithelial lining of the gut, from the mouth to the anus. It gives rise not only to the pharynx, esophagus, stomach, and intestines, but also to many associated structures. the salivary glands, liver, pancreas, trachea, and lungs, for example, all develop from the wall of the digestive tract and grow to become systems of branching tubes that open into the gut or pharynx. the endoderm forms only the epithelial components of these structures— the lining of the gut and the secretory cells of the

1	to become systems of branching tubes that open into the gut or pharynx. the endoderm forms only the epithelial components of these structures— the lining of the gut and the secretory cells of the pancreas, for example. the supporting muscular and fibrous elements arise from the mesoderm.

1	the mesoderm gives rise to the connective tissues—at first, to the loose mesh of cells in the embryo known as mesenchyme, and ultimately to cartilage, bone, and fibrous tissue, including the dermis (the inner layer of the skin). the mesoderm also forms the muscles, the entire vascular system—including the heart, blood vessels, and blood cells—and the tubules, ducts, and supporting tissues of the kidneys and gonads. the notochord forms from the mesoderm and serves as the core of the future backbone and the source of signals that coordinate the development of surrounding tissues.

1	the ectoderm will form the epidermis (the outer, epithelial layer of the skin) and epidermal appendages such as hair, sweat glands, and mammary glands. It will also give rise to the whole of the nervous system, central and peripheral, including not only neurons and glia but also the sensory cells of the nose, the ear, the eye, and other sense organs. (B, after t. Mohun et al., Cell 22:9–15, 1980. With permission from elsevier.) The ectoderm, mesoderm, and endoderm formed during gastrulation constitute the three germ layers of the early embryo. Many later developmental transformations will produce the elaborately structured organs. But the basic body plan and axes set up in miniature during gastrulation are preserved into adult life, when the organism may be billions of times larger (Movie 21.2). the Developmental potential of cells Becomes progressively restricted

1	Concomitant with the refinement of the body plan, the individual cells become more and more restricted in their developmental potential. During the blastula stages, cells are often totipotent or pluripotent—they have the potential to give rise to all or almost all of the cell types of the adult body. The pluripotency is lost as gastrulation proceeds: a cell located in the endodermal germ layer, for example, can give rise to the cell types that will line the gut or form gut-derived organs such as the liver or pancreas, but it no longer has the potential to form mesoderm-derived structures such as skeleton, heart, or kidney. Such a cell is said to be determined for an endodermal fate. Thus, cell determination starts early and progressively narrows the options as the cell steps through a programmed series of intermediate states—guided at each step by its genome, its history, and its interactions with neighbors. The process reaches its limit when a cell undergoes terminal differentiation

1	series of intermediate states—guided at each step by its genome, its history, and its interactions with neighbors. The process reaches its limit when a cell undergoes terminal differentiation to form one of the highly specialized cell types of the adult body (Figure 21–4). Although there are cell types in the adult that retain some degree of pluripotency, their range of options is generally narrow (discussed in Chapter 22).

1	Underlying the richness and astonishingly complex outcomes of development is cell memory (see p. 404). Both the genes a cell expresses and the way it behaves depend on the cell’s past, as well as on its present circumstances. The cells of our body—the muscle cells, the neurons, the skin cells, the gut cells, and so on—maintain their specialized characters largely because they retain a record of the extracellular signals their ancestors received during development, rather than because they continually receive such instructions from their surroundings. Despite their radically different phenotypes, they retain the same complete genome that was present in the zygote; their differences arise instead from differential gene expression. We have discussed the molecular mechanisms of gene regulation, cell memory, cell division, cell signaling, and cell movement in previous chapters. In this chapter, we shall see how these basic processes are collectively deployed to create an animal.

1	The anatomical features that animals share have undergone many extreme modifications in the course of evolution. As a result, the differences between species are usually more striking to our human eye than the similarities. But at the level of the underlying molecular mechanisms and the macromolecules that mediate them, the reverse is true: the similarities among all animals are profound and extensive. Through more than half a billion years of evolutionary divergence, all animals have retained unmistakably similar sets of genes and proteins that are responsible for generating their body plans and for forming their specialized cells and organs.

1	This astonishing degree of evolutionary conservation was discovered not by broad surveys of animal diversity, but through intensive study of a small number of representative species—the model organisms discussed in Chapter 1. For animal developmental biology, the most important have been the fly Drosophila melanogaster, the frog Xenopus laevis, the roundworm Caenorhabditis elegans, the mouse Mus musculus, and the zebrafish Danio rerio. In discussing the mechanisms of development, we shall draw our examples mainly from these few species. Figure 21–4 The lineage from blastomere to differentiated cell type. as development proceeds, cells become more and more specialized. Blastomeres have the potential to give rise to most or all cell types. Under the influence of signaling molecules and gene regulatory factors, cells acquire more restricted fates until they differentiate into highly specialized cell types, such as the pancreatic β-islet cells that secrete the hormone insulin.

1	Genes Involved in cell–cell communication and transcriptional control are especially Important for animal Development

1	What are the genes that animals share with one another but not with other kingdoms of life? These would be expected to include genes required specifically for animal development but not needed for unicellular existence. Comparison of animal genomes with the genome of budding yeast—a unicellular eukaryote— suggests that three classes of genes are especially important for multicellular organization. The first class includes genes that encode proteins used for cell–cell adhesion and cell signaling; hundreds of human genes encode signal proteins, cell-surface receptors, cell adhesion proteins, or ion channels that are either not present in yeast or present in much smaller numbers. The second class includes genes encoding proteins that regulate transcription and chromatin structure: more than 1000 human genes encode transcription regulators, but only about 250 yeast genes do so. As we shall see, the development of animals is dominated by cell–cell interactions and by differential gene

1	1000 human genes encode transcription regulators, but only about 250 yeast genes do so. As we shall see, the development of animals is dominated by cell–cell interactions and by differential gene expression. The third class of non-coding RNAs has a more uncertain status: it includes genes that encode microR-NAs (miRNAs); there are at least 500 of these in humans. Along with the regulatory proteins, they play a significant part in controlling gene expression during animal development, but the full extent of their importance is still unclear. The loss of individual miRNA genes in C. elegans, where their functions have been well studied, rarely leads to obvious phenotypes, suggesting that the roles of miRNAs during animal development are often subtle, serving to fine-tune the developmental machinery rather than to form its core structures.

1	regulatory DNa Seems Largely responsible for the Differences Between animal Species As discussed in Chapter 7, each gene in a multicellular organism is associated with many thousands of nucleotides of noncoding DNA that contains regulatory elements. These regulatory elements determine when, where, and how strongly the gene is to be expressed, according to the transcription regulators and chromatin structures that are present in the particular cell (Figure 21–5). Consequently, a change in the regulatory DNA, even without any change in the coding DNA, can alter the logic of the gene-regulatory network and change the outcome of development.

1	As discussed in Chapter 4, when we compare the genomes of different animal species, we find that evolution has altered the coding and regulatory DNA to different extents. The coding DNA, for the most part, has been highly conserved, the noncoding regulatory DNA much less so. It seems that changes in regulatory DNA are largely responsible for the dramatic differences between one class of animals and another (see p. 227). We can view the protein products of the coding sequences as a conserved kit of common molecular parts, and the regulatory DNA as instructions for assembly: with different instructions, the same kit of parts can be used to make a whole variety of different body structures. We will return to this important concept later.

1	Figure 21–5 Regulatory DNA defines the gene expression patterns in development. the genome is the same in a muscle cell as in a skin cell, but different genes are active because these cells express different transcription regulators that bind to gene regulatory elements. For example, transcription regulators in skin cells recognize a regulatory element in gene 1, leading to its activation, whereas a different set of regulators is present in muscle cells, binding to and activating gene 3. transcriptional regulators that activate the expression of gene 2 are present in both cell types. Small Numbers of conserved cell–cell Signaling pathways coordinate Spatial patterning

1	Spatial patterning of a developing animal requires that cells become different according to their positions in the embryo, which means that cells must respond to extracellular signals produced by other cells, especially their neighbors. In what is probably the commonest mode of spatial patterning, a group of cells starts out with the same developmental potential, and a signal from cells outside the group then induces one or more members of the group to change their character. This process is called inductive signaling. Generally, the inductive signal is limited in time and space so that only a subset of the cells capable of responding—the cells close to the source of the signal—take on the induced character (Figure 21–6). Some inductive signals depend on cell–cell contact; others act over a longer range and are mediated by molecules that diffuse through the extracellular medium or are transported in the bloodstream (see Figure 15–2).

1	Most of the known inductive events in animal development are governed by a small number of highly conserved signaling pathways, including transforming growth factor-β (TGFβ), Wnt, Hedgehog, Notch, and receptor tyrosine kinase (RTK) pathways (discussed in Chapter 15). The discovery of the limited vocabulary that developing cells use for intercellular communication has emerged over the past 25 years as one of the great simplifying features of developmental biology. through combinatorial control and cell Memory, Simple Signals can Generate complex patterns

1	But how can this small number of signaling pathways generate the huge diversity of cells and patterns? Three kinds of mechanisms are responsible. First, through gene duplication, the basic components of a pathway often come to be encoded by small families of closely related homologous genes. This allows for diversity in the operation of the pathway, according to which family member is employed in a given situation. Notch signaling, for example, may be mediated by Notch1 in one tissue, but by its homolog Notch4 in another. Second, the response of a cell to a given signal protein depends on the other signals that the cell is receiving concurrently (Figure 21–7A). As a result, different combinations of signals can generate a large variety of different responses. Third, and most fundamental, the effect of activating a signaling pathway depends on the previous experiences of the responding cell: past influences leave a lasting mark, registered in the state of the cell’s chromatin and the

1	the effect of activating a signaling pathway depends on the previous experiences of the responding cell: past influences leave a lasting mark, registered in the state of the cell’s chromatin and the selection of transcription regulatory proteins and RNA molecules that the cell contains. This cell memory enables cells with different cells directed to new developmental pathway

1	Figure 21–6 Inductive signaling. Figure 21–7 Two mechanisms for generating different responses to the same inductive signal. (a) In combinatorial signaling, the effect of a signal depends on the presence of other signals received at the same time. (B) through cell memory, previous signals (or other events) can leave a lasting trace that alters the response to the current signal (see Figure 7–54). the memory trace is represented here in the coloring of the cell nucleus. feld of cells morphogen gradient forms source of morphogen cellular response to gradient 0.1 mm histories to respond to the same signals differently (Figure 21–7B). Thus, the same few signaling pathways can be used repeatedly at different times and places with different outcomes, so as to generate patterns of unlimited complexity. Morphogens are Long-range Inductive Signals that exert Graded effects

1	Morphogens are Long-range Inductive Signals that exert Graded effects Signal molecules often govern simple yes–no choices—one outcome when their concentration is high, another when it is low. In many cases, however, the responses are more finely graded: a high concentration of a signal molecule may, for example, direct cells into one developmental pathway, an intermediate concentration into another, and a low concentration into yet another.

1	One common way to generate such different concentrations of a signal molecule is for the molecule to diffuse out from a localized signaling source, creating a concentration gradient. Cells at different distances from the source are driven to behave in a variety of different ways, according to the signal concentration that they experience (Figure 21–8). A signal molecule that imposes a pattern on a whole field of cells in this way is called a morphogen. In the simplest case, a specialized group of cells produces a morphogen at a steady rate, and the morphogen is then degraded as it diffuses away from this source. The speed of diffusion and the half-life of the morphogen will together determine the range and steepness of its resulting gradient (Figure 21–9).

1	This simple mechanism can be modified in various ways. Receptors on the surface of cells along the way, for example, may trap the diffusing morphogen and cause it to be endocytosed and degraded, shortening its effective half-life. Alternatively, the morphogen may bind to molecules in the extracellular matrix such as heparan sulfate proteoglycan (discussed in Chapter 19), thereby greatly reducing its diffusion rate. Lateral Inhibition can Generate patterns of Different cell types Morphogen gradients, and other kinds of inductive signal, exploit an existing asymmetry in the embryo to create further asymmetries and differences between cells: already, at the outset, some cells are specialized to produce the morphogen and thereby impose a pattern on another class of cells that are sensitive to it. But 0 0.1 0.2 0 0.1 0.2 0 0.1 0.2 distance from source (mm)

1	Figure 21–8 Gradient formation and interpretation. a gradient forms by localized production of an inducer—a morphogen—that diffuses away from its source. Different concentrations of morphogen (or different durations of exposure) induce different gene expression patterns and cell fates in responding cells. Diffusive transport can generate gradients only over short distances, and morphogens generally act over distances of 1 mm or less.

1	Figure 21–9 Setting up a signal gradient by diffusion. (a–c) each graph shows six successive stages in the buildup of the concentration of a signal molecule that is produced at a steady rate at the origin, with production starting at time 0. In all cases, the molecule undergoes degradation as it diffuses away from the source, and the graphs are calculated on the assumption that diffusion is occurring along two axes in space (for example, radially from a source in an epithelial sheet). (a) the pattern of the morphogen assuming that the molecule has a half-life of 170 minutes, and that it diffuses with an effective diffusion constant of D = 1 μm2 sec–1, typical of a small protein molecule in extracellular tissues. Note that the gradient is already close to its steady-state form within an hour and that the concentration at steady state falls off exponentially with distance. (B) a threefold increase in the diffusion constant of the morphogen extends its range but lowers its concentration

1	and that the concentration at steady state falls off exponentially with distance. (B) a threefold increase in the diffusion constant of the morphogen extends its range but lowers its concentration next to the source, whereas (c) a threefold increase in morphogen half-life increases its concentration throughout the tissue. effects of the morphogen will depend not just on its concentration at some critical moment, but also on how each target cell integrates its response over time. (courtesy of patrick Müller.)

1	Figure 21–10 Genesis of asymmetry through lateral inhibition and positive feedback. In this example, two cells interact, each producing a substance X that acts on the other cell to inhibit its production of X, an effect known as lateral inhibition. an increase of X in one of the cells leads to a positive feedback that tends to increase X in that cell still further, while decreasing X in its neighbor. this can create a runaway instability, making the two cells become radically different. Ultimately, the system comes to rest in one or the other of two opposite stable states. the final choice of state represents a form of memory: the small influence that initially directed the choice is no longer required to maintain it. what if there is no clear initial asymmetry? Can a regular pattern arise spontaneously within a set of cells that are initially all alike?

1	what if there is no clear initial asymmetry? Can a regular pattern arise spontaneously within a set of cells that are initially all alike? The answer is yes. The fundamental principle underlying such de novo pattern formation is positive feedback: cells can exchange signals in such a way that any small initial discrepancy between cells at different sites becomes self-amplifying, driving the cells toward different fates. This is most clearly illustrated in the phenomenon of lateral inhibition, a form of cell–cell interaction that forces close neighbors to become different and thereby generates fine-grained patterns of different cell types.

1	Consider a pair of adjacent cells that start off in a similar state. Each of these cells can both produce and respond to a certain signal molecule X, with the added rule that the stronger the signal a cell receives, the weaker the signal it generates (Figure 21–10). If one cell produces more X, the other is forced to produce less. This gives rise to a positive feedback loop that tends to amplify any initial difference between the two adjacent cells. Such a difference may arise from a bias imposed by some present or past external factor, or it may simply originate from spontaneous random fluctuations, or “noise”—an inevitable feature of the genetic control circuitry in cells (discussed in Chapter 7). In either case, lateral inhibition means that if cell 1 makes a little more of X, it will thereby cause cell 2 to make less; and because cell 2 makes less X, it delivers less inhibition to cell 1 and so allows the production of X in cell 1 to rise higher still; and so on, until a steady

1	thereby cause cell 2 to make less; and because cell 2 makes less X, it delivers less inhibition to cell 1 and so allows the production of X in cell 1 to rise higher still; and so on, until a steady state is reached where cell 1 produces a lot of X and cell 2 produces very little. In the standard case, the signal molecule X acts in the receiving cell by regulating gene transcription, and the result is that the two cells are driven along different pathways of differentiation.

1	In almost all tissues, a balanced mixture of different cell types is required. Lateral inhibition provides a common way to generate the mixture. As we shall see, lateral inhibition is very often mediated by exchange of signals at cell– cell contacts via the Notch signaling pathway, driving cell diversification by enabling individual cells that express one set of genes to direct their immediate neighbors to express a different set, in exactly the way we have described (see also Figure 15–58).

1	Lateral inhibition mediated by the Notch pathway is not the only example of pattern generation through positive feedback: there are other ways in which, through the same basic principle, a system that starts off homogeneous and symmetrical can pattern itself spontaneously, even in the absence of an external morphogen. Positive feedback processes mediated by diffusible signal molecules can operate over broad arrays of cells to create many types of spatial patterns. Mechanisms of this sort are called reaction-diffusion systems. For example, a substance A (a short-range activator) may stimulate its own production in the cells that contain it and in their immediate neighbors, while also causing these cells to produce a signal I (a long-range inhibitor) that diffuses widely and inhibits the production of A in cells farther away. If the cells all start the same, but one group gains a slight advantage by making a little more A than the rest, the asymmetry can be self-amplifying

1	POSITIVE FEEDBACK: asymmetry is self-amplifying Figure 21–11 Pattern generation by a reaction-diffusion system. From (a) a uniform field of cells, (B) local positive feedback and (c) long-range inhibition can (D) generate patterns within the initially uniform field. the patterns can be complex, resembling the spots of a leopard (as shown) or the stripes of a zebra; or they can be simple, with creation of a single cluster of specialized cells that can, for example, go on to serve as the source of a morphogen gradient. (Figure 21–11). Such short-range activation combined with long-range inhibition can account for the formation of clusters of cells within an initially homogeneous tissue that become specialized as localized signaling centers.

1	Cell diversification does not always depend on extracellular signals: in some cases, daughter cells are born different as a result of an asymmetric cell division, in which some important molecule or molecules are distributed unequally between the two daughters. This asymmetric inheritance ensures that the two daughter cells develop differently (Figure 21–12). Asymmetric division is a common feature of early development, where the fertilized egg already has an internal pattern and cleavage of this large cell segregates different determinants into separate blastomeres. We shall see that asymmetric division also plays a part in some later developmental processes. Initial patterns are established in Small Fields of cells and refined by Sequential Induction as the embryo Grows

1	Initial patterns are established in Small Fields of cells and refined by Sequential Induction as the embryo Grows The signals that organize the spatial pattern of cells in an embryo generally act over short distances and govern relatively simple choices. A morphogen, for example, typically acts over a distance of less than 1 mm—an effective range for diffusion —and directs choices between several developmental options for the cells on which it acts. Yet the organs that eventually develop are much larger and more complex than this.

1	The cell proliferation that follows the initial specification accounts for the size increase, while the refinement of the initial pattern is explained by a series of local inductions plus other interactions that add successive levels of detail on an initially simple sketch. For example, as soon as two types of cells are present in a developing tissue, one of them can produce a signal that induces a subset of the neighboring cells to specialize in a third way. The third cell type can in turn signal 1. asymmetric division: sister cells born different uniform feld of cells long-range inhibitor (red) blocks production of activator by other cells in the neighborhood specialized clusters of cells 2. symmetric division: sister cells become different as a result of Figure 21–12 Two ways of making sister cells different.

1	Figure 21–12 Two ways of making sister cells different. D and E are induced by signal C is induced on A and B, B acting on A back to the other two cell types nearby, generating a fourth and a fifth cell type, and so on (Figure 21–13). This strategy for generating a progressively more complicated pattern is called sequential induction. It is chiefly through sequential inductions that the body plan of a developing animal, after being first roughed out in miniature, becomes elaborated with finer and finer details as development proceeds.

1	The rapid progress in understanding animal development has been one of the great success stories in biology over the last few decades, and it has important practical implications. Some 2 to 5% of all human babies are born with anatomical abnormalities, such as heart malformations, truncated limbs, cleft palate, or spina bifida. Advances in developmental biology help us understand how these defects arise, even if we cannot yet prevent or cure most of them. Less obvious, but even more important from a practical point of view, is that developmental biology provides insights into the workings of cells and tissues in the adult body. Developmental processes do not halt at birth; they continue throughout life, as tissues are maintained and repaired. The fundamental mechanisms of cell growth and division, cell–cell signaling, cell memory, cell adhesion, and cell movement are involved in adult tissue maintenance and repair—just as they are in embryo development.

1	Embryos are simpler than adults, and they allow us to analyze such basic processes more easily. Studies of the early Drosophila embryo, for example, were crucial to the discovery of several conserved signaling pathways, including the Wnt, Hedgehog, and Notch pathways. They also provided the key to understanding the central role of these pathways in the maintenance of normal adult human tissues and in diseases such as cancer. In Chapter 22, we shall consider how other developmental mechanisms operate in the normal adult body, especially in tissues that are continually renewed by means of stem cells—including the gut, skin, and the hematopoietic system. But now, we must look more closely at the way in which an early embryo generates its spatial pattern of specialized cells, beginning with the transformations that create the adult body plan.

1	Animal development is a self-assembly process, in which the cells of the embryo become different from one another and organize themselves into increasingly complex structures. The process begins with a single large cell—the fertilized egg. This cell cleaves to form many smaller cells, producing a blastula. The blastula undergoes gastrulation to generate the three germ layers of the embryo—ectoderm, mesoderm, and endoderm—consisting of cells determined for different fates. As development continues, the cells become more and more narrowly specialized according to their locations and their interactions with one another. Through cell memory, these cell–cell interactions, even though transient, can have lasting effects on each Figure 21–13 Patterning by sequential induction. a series of inductive interactions can generate many types of cells, starting from only a few.

1	Figure 21–13 Patterning by sequential induction. a series of inductive interactions can generate many types of cells, starting from only a few. cell’s internal state. Thus, a succession of simple cues that a cell receives at different times can direct it along a complex developmental pathway. At each step, the cell becomes further restricted in the range of final states open to it. The process reaches its limit when the cell differentiates to form one of the specialized cell types of the adult body.

1	Differences between developing cells arise in various ways and have to be properly coordinated in space. In one common strategy, initially similar cells within a group become different by exposure to different levels of an inductive signal or morphogen emanating from a source outside the group. Neighboring cells can also become different by lateral inhibition, in which a cell signals to its neighbors not to follow the same fate. These cell–cell interactions are mediated by a small number of highly conserved signaling pathways, which are used repeatedly in different organisms and at different times during development. Not all cell diversification arises by cell–cell interactions, however: daughter cells can be born different as a result of asymmetric cell division.

1	Regulators of transcription and chromatin structure bind to regulatory DNA and determine the fate of each cell. Differences of body plan seem to arise to a large extent from differences in the regulatory DNA associated with each gene. This DNA has a central role in defining the sequential program of development, calling genes into action at specific times and places according to the pattern of gene expression that was present in each cell at the previous developmental stage. Development has been most thoroughly studied in a handful of model organisms. But most of the genes and mechanisms thereby identified are used in all animals and repeatedly at different stages of development. Thus, insights from worms, flies, fish, frogs, and mice deeply inform our understanding of embryology, birth defects, and adult tissue maintenance in humans.

1	A developing multicellular organism has to create a pattern in fields of cells where there was little or none before. Some of the early microscopists imagined the entire shape and structure of the human body to be already present in the sperm as a “homunculus,” a miniature human; after fertilization, the homunculus would simply grow and generate a full-sized human. We now know that this view is incorrect and that development is a progression from simple to complex, through a gradual refinement of an animal’s anatomy. To see how the whole sequence of events of spatial patterning and cell determination is set in train, we must return to the egg and the early embryo. Different animals Use Different Mechanisms to establish their primary axes of polarization

1	Different animals Use Different Mechanisms to establish their primary axes of polarization Surprisingly, the earliest steps of animal development are among the most variable, even within a phylum. A frog, a chicken, and a mammal, for example, even though they develop in similar ways later, make eggs that differ radically in size and structure, and they begin their development with different sequences of cell divisions and cell specializations. Gastrulation occurs in all animal embryos, but the details of its timing, of the associated pattern of cell movements, and of the shape and size of the embryo as gastrulation proceeds are highly variable. Likewise, there is great variation in the time and manner in which the primary axes of the body become marked out. However, this polarization of the embryo usually becomes discernible very early, before gastrulation begins: it is the first step of spatial patterning.

1	Three axes generally have to be established. The animal-vegetal (A-V) axis, in most species, defines which parts are to become internal (through the movements of gastrulation) and which are to remain external. (The bizarre name dates from a century ago and has nothing to do with vegetables.) The anteroposterior (A-P) axis specifies the locations of future head and tail. The dorsoventral (D-V) axis specifies the future back and belly. Figure 21–14 The frog egg and its asymmetries. (a) Side view of a Xenopus egg photographed just before fertilization.

1	Figure 21–14 The frog egg and its asymmetries. (a) Side view of a Xenopus egg photographed just before fertilization. (B) the asymmetric distribution of molecules inside the egg, and how this changes following fertilization so as to define a dorsoventral as well as an animal-vegetal asymmetry. Fertilization, through a reorganization of the microtubule cytoskeleton, triggers a rotation of the egg cortex (a layer a few μm deep) through about 30° relative to the core of the egg; the direction of rotation determined by the site of sperm entry. Some components are carried still further to the future dorsal side by active transport along microtubules. the resulting dorsal concentration of Wnt11 mrNa leads to dorsal production of the Wnt11 signal protein and defines the dorsoventral polarity of the future embryo. Vegetally localized Vegt defines the vegetal source of signals that will induce endoderm and mesoderm. (a, courtesy of tony Mills.)

1	At one extreme, the egg is spherically symmetrical, and the axes only become defined during embryogenesis. The mouse comes close to being an example, with little obvious sign of polarity in the egg. Correspondingly, the blastomeres produced by the first few cell divisions seem to be all alike and are remarkably adaptable. If the early mouse embryo is split in two, a pair of identical twins can be produced—two complete, normal individuals from a single cell. Similarly, if one of the cells in a two-cell mouse embryo is destroyed by pricking it with a needle and the resulting “half-embryo” is placed in the uterus of a foster mother to develop, in many cases a perfectly normal mouse will emerge.

1	At the opposite extreme, the structure of the egg defines the future axes of the body. This is the case for most species, including insects such as Drosophila, as we shall see shortly. Many other organisms lie between the two extremes. The egg of the frog Xenopus, for example, has a clearly defined A-V axis even before fertilization: the nucleus near the top defines the animal pole, while the mass of yolk (the embryo’s food supply, destined to be incorporated in the gut) toward the bottom defines the vegetal pole. Several types of mRNA molecules are already localized in the vegetal cytoplasm of the egg, where they produce their protein products. After fertilization, these mRNAs and proteins act in and on the cells in the lower and middle part of the embryo, giving the cells there specialized characters, both by direct effects and by stimulating the production of secreted signal proteins. For example, mRNA encoding the transcription regulator VegT is deposited at the vegetal pole

1	characters, both by direct effects and by stimulating the production of secreted signal proteins. For example, mRNA encoding the transcription regulator VegT is deposited at the vegetal pole during oogenesis. After fertilization, this mRNA is translated, and the resulting VegT protein activates a set of genes that code for signal proteins that induce mesoderm and endoderm, as discussed later.

1	The D-V axis of the Xenopus embryo, by contrast, is defined through the act of fertilization. Following entry of the sperm, the outer cortex of the egg cytoplasm rotates relative to the central core of the egg, so that the animal pole of the cortex becomes slightly shifted to one side (Figure 21–14). Treatments that block the rotation allow cleavage to occur normally but produce an embryo with a central gut and no dorsal structures or D-V asymmetry. Thus, this cortical rotation is required to define the D-V axis of the future body by creating the D-V axis of the egg.

1	The site of sperm entry that biases the direction of the cortical rotation in Xenopus, perhaps through the centrosome that the sperm brings into the egg— inasmuch as the rotation is associated with a reorganization of the microtubules nucleated from the centrosome in the egg cytoplasm. The reorganization leads to a microtubule-based transport of several cytoplasmic components, including the mRNA coding for Wnt11, a member of the Wnt family of signal proteins, moving cytoplasm of animal pole VENTRAL DORSAL it toward the future dorsal side (see Figure 21–14). This mRNA is soon translated and the Wnt11 protein secreted from cells that form in that region of the embryo activates the Wnt signaling pathway (see Figure 15–60). This activation is crucial for triggering the cascade of subsequent events that will organize the dorsoventral axis of the body. (The A-P axis of the embryo will only become clear later, in the process of gastrulation.)

1	Although different animal species use a variety of different mechanisms to specify their axes, the outcome has been relatively well conserved in evolution: head is distinguished from tail, back from belly, and gut from skin. It seems that it does not much matter what tricks the embryo uses to break the initial symmetry and set up this basic body plan. Studies in Drosophila have revealed the Genetic control Mechanisms Underlying Development

1	It is the fly Drosophila, more than any other organism, that has provided the key to our present understanding of how genes govern development. Decades of genetic study culminated in a large-scale genetic screen, focusing especially on the early embryo and searching for mutations that disrupt its pattern. This revealed that the key developmental genes fall into a relatively small set of functional classes defined by their mutant phenotypes. The discovery of these genes and the subsequent analysis of their functions was a famous tour de force and had a revolutionary impact on all of developmental biology, earning its discoverers a Nobel Prize. Some parts of the developmental machinery revealed in this way are conserved between flies and vertebrates, some parts not. But the logic of the experimental approach and the general strategies of genetic control that it revealed have transformed our understanding of multicellular development in general.

1	To understand how the early developmental machinery operates in Drosophila, it is important to note a peculiarity of fly development. Like the eggs of other insects, but unlike most vertebrates, the Drosophila egg—shaped like a cucumber—begins its development with an extraordinarily rapid series of nuclear divisions without cell division, producing multiple nuclei in a common cytoplasm—a syncytium. The nuclei then migrate to the cell cortex, forming a structure called the syncytial blastoderm. After about 6000 nuclei have been produced, the plasma membrane folds inward between them and partitions them into separate cells, converting the syncytial blastoderm into the cellular blastoderm (Figure 21–15).

1	We shall see that the initial patterning of the Drosophila embryo depends on signals that diffuse through the cytoplasm at the syncytial stage and exert their actions on genes in the rapidly dividing nuclei, before the partitioning of the egg into separate cells. Here, there is no need for the usual forms of cell–cell signaling; neighboring regions of the syncytial blastoderm can communicate by means of transcription regulatory proteins that move through the cytoplasm of the giant multinuclear cell. egg-polarity Genes encode Macromolecules Deposited in the egg to Organize the axes of the early Drosophila embryo As in most insects, the main axes of the future body of Drosophila are defined before fertilization by a complex exchange of signals between the developing egg, Figure 21–15 Development of the Drosophila egg from fertilization to the cellular blastoderm stage.

1	Figure 21–15 Development of the Drosophila egg from fertilization to the cellular blastoderm stage. or oocyte, and the follicle cells that surround it in the ovary. In the stages before fertilization, the anteroposterior and dorsoventral axes of the future embryo become defined by four systems of egg-polarity genes that create landmarks— either mRNA or protein—in the developing oocyte. Following fertilization, each landmark serves as a beacon, providing a signal that organizes the developmental process in its neighborhood.

1	The nature of the genes emerged from studies of mutants in which the patterning of the embryo was altered. One class of mutations gave embryos with disrupted polarity—for example, tail-end structures at both ends of the body, with no head-end structures. This class of mutations identified the set of egg-polarity genes. The egg-polarity gene responsible for the signal that organizes the anterior end of the embryo is called Bicoid. A deposit of Bicoid mRNA molecules is localized, before fertilization, at the anterior end of the egg. Upon fertilization, the mRNA is translated to produce Bicoid protein. This protein is an intracellular morphogen and transcription regulator that diffuses away from its source to form a concentration gradient within the syncytial cytoplasm, with its maximum at the head end of the embryo (Figure 21–16). The different concentrations of Bicoid along the A-P axis help determine different cell fates by regulating the transcrip tion of genes in the nuclei of the

1	the head end of the embryo (Figure 21–16). The different concentrations of Bicoid along the A-P axis help determine different cell fates by regulating the transcrip tion of genes in the nuclei of the syncytial blastoderm (discussed in Chapter 7).

1	Of the three other egg-polarity gene systems, two contribute to patterning the syncytial nuclei along the A-P axis and one to patterning them along the D-V axis. Together with the Bicoid group of genes, and acting in a broadly similar way, their gene products mark out three fundamental partitions of body regions—head versus rear, dorsal versus ventral, and endoderm versus mesoderm and ectoderm— as well as a fourth partition, no less fundamental to the body plan of animals: the distinction between germ cells and somatic cells (Figure 21–17).

1	The egg-polarity genes have a further special feature: they are all maternal-effect genes, in that it is the mother’s genome rather than the zygote’s genome that is critical. For example, a fly whose chromosomes are mutant in both copies of the Bicoid gene but who is born from a mother carrying one normal copy of Bicoid develops perfectly normally, without any defects in the head pattern. However, if that offspring is a female, she cannot deposit any functional Bicoid mRNA into her own eggs, which will therefore develop into headless embryos, regardless of the father’s genotype. The egg-polarity genes act first in a hierarchy of gene systems that define a progressively more detailed pattern of body parts. In the next few pages, we begin with the molecular mechanisms that pattern the developing Drosophila embryo and larva along the A-P axis, before considering the patterning along the D-V axis.

1	Figure 21–16 The Bicoid protein gradient. (a) Bicoid mrNa is deposited at the anterior pole during oogenesis. Local translation followed by diffusion generates the Bicoid protein gradient. absence of the Bicoid protein gradient in embryos from Bicoid homozygous mutant mothers. (a and B, courtesy of Stephen Small.)

1	absence of the Bicoid protein gradient in embryos from Bicoid homozygous mutant mothers. (a and B, courtesy of Stephen Small.) Figure 21–17 The organization of the four egg-polarity gradient systems in Drosophila. Nanos is a translational repressor that governs the formation of the abdomen. Localized Nanos mrNa is also incorporated into the germ cells as they form at the posterior of the embryo, and Nanos protein is necessary for germ-line development. Bicoid protein is a transcriptional activator that determines the head and thoracic regions. toll and torso are receptor proteins that are distributed all over the membrane but are activated only at the sites indicated by the coloring, through localized exposure to the extracellular ligands Spaetzle (the ligand for toll) and trunk (the ligand for torso). toll activity determines the mesoderm and torso activity determines the formation of terminal structures.

1	Figure 21–18 The origins of the Drosophila body segments. (a) at 3 hours, the embryo (shown in side view) is at the blastoderm stage and no segmentation is visible, although a fate map can be drawn showing the future segmented regions (color). (B) at 10 hours, all the segments are clearly defined (t1: first thoracic segment; a1: first abdominal segment). See Movie 21.3. (c) the segments of the Drosophila larva and their correspondence with regions in the embryo. (D) the segments of the Drosophila adult and their correspondence with regions in the embryo. three Groups of Genes control Drosophila Segmentation along the a-p axis

1	The body of an insect is divided along its A-P axis into a series of segments. The segments are repetitions of a theme with variations: each segment forms highly specialized structures, but all built according to a similar fundamental plan (Figure 21–18). The gradients of transcription regulators set up along the A-P axis in the early embryo by the egg-polarity genes are the prelude to creation of the segments. These regulators initiate the orderly transcription of segmentation genes, which refine the pattern of gene expression to define the boundaries and ground plan of the individual segments. Segmentation genes are expressed by subsets of cells in the embryo, and their products are the first components that the embryo’s own genome contributes to embryonic development; they are therefore called zygotic-effect genes, to distinguish them from the earlier-acting maternal-effect genes. Mutations in segmentation genes can alter either the number of segments or their basic internal

1	called zygotic-effect genes, to distinguish them from the earlier-acting maternal-effect genes. Mutations in segmentation genes can alter either the number of segments or their basic internal organization.

1	The segmentation genes fall into three groups according to their mutant phenotypes (Figure 21–19). It is convenient to think of the three groups as acting in sequence, although in reality their functions overlap in time. First to be expressed is a set of at least six gap genes, whose products mark out coarse A-P subdivisions of the embryo. Mutations in a gap gene eliminate one or more groups of adjacent segments: in the mutant Krüppel, for example, the larva lacks eight segments. Next comes a set of eight pair-rule genes. Mutations in these genes cause a series of deletions affecting alternate segments, leaving the embryo with only half as many segments as usual; although all the mutants display this two-segment periodicity, they differ in the precise pattern. Finally, there are at least 10 segment-polarity genes, in which mutations produce a normal number of segments but with a part of each segment deleted and replaced by a mirror-image duplicate of all or part of the rest of the

1	10 segment-polarity genes, in which mutations produce a normal number of segments but with a part of each segment deleted and replaced by a mirror-image duplicate of all or part of the rest of the segment.

1	In parallel with the segmentation process, a further set of genes—the homeotic selector, or Hox, genes—serves to define and preserve the differences between one segment and the next, as we describe shortly. The phenotypes of the various segmentation mutants suggest that the segmentation genes form a coordinated system that subdivides the embryo progressively into smaller and smaller domains along the A-P axis, each distinguished by a different pattern of gene expression. Molecular genetics has helped to reveal how this system works. a hierarchy of Gene regulatory Interactions Subdivides the Drosophila embryo

1	a hierarchy of Gene regulatory Interactions Subdivides the Drosophila embryo Like Bicoid, most of the segmentation genes encode transcription regulator proteins. Their control by the egg-polarity genes and their actions on one another and on still other genes can be deciphered by comparing gene expression in normal and mutant embryos. By using appropriate probes to detect RNA transcripts or their protein products, one can observe genes switch on and off in changing patterns. By comparing these patterns in different mutants, one can begin to discern the logic of the entire gene control system. The products of the egg-polarity genes provide the global positional signals in the early embryo (see Figure 21–17). The Bicoid protein, as we have seen, acts as 0.2 mm

1	Figure 21–19 Examples of the phenotypes of mutations affecting egg-polarity genes and the three types of segmentation genes. In each case, the areas shaded in green on the normal larva (left) are deleted in the mutant or are replaced by mirror-image duplicates of the unaffected regions. (Modified from c. Nüsslein-Volhard and e. Wieschaus, Nature 287:795–801, 1980. With permission from Macmillan publishers Ltd.) a morphogen and activates different sets of genes at different positions along the A-P axis: some gap genes are only activated in regions with high levels of Bicoid, others only where levels of Bicoid are lower. After the gap gene products refine their positions by mutual repression, they provide a second tier of positional signals that act more locally to regulate finer details of patterning. Gap genes act by controlling the expression of yet other genes, including the pair-rule genes. The pair-rule genes, in turn, collaborate with one another and with the gap genes to set up

1	patterning. Gap genes act by controlling the expression of yet other genes, including the pair-rule genes. The pair-rule genes, in turn, collaborate with one another and with the gap genes to set up a regular, periodic pattern of expression of the segment-polarity genes, which collaborate with one another to define the internal pattern of each individual segment (Figure 21–20).

1	The initial steps in creation of the segmental pattern occur before cellularization of the syncytial blastoderm and are governed by the combinatorial effects of transcription regulators, as discussed in detail in Chapter 7 for the regulation of the expression of the pair-rule gene Even-skipped (see pp. 394–396). After cellularization, the segment-polarity genes further subdivide each segment into smaller domains. A large subset of the segment-polarity genes codes for components of two signaling pathways—the Wnt pathway and the Hedgehog pathway, including the secreted signal proteins Wingless (the first-named member of the Wnt family) and Hedgehog. (The Hedgehog pathway was first discovered through study of Drosophila segmentation, and it takes its name from the prickly appearance of the surface of the Hedgehog mutant embryo.) Wingless and Hedgehog are synthesized in different bands of cells that serve as signaling centers within each segment. The two proteins mutually maintain each

1	surface of the Hedgehog mutant embryo.) Wingless and Hedgehog are synthesized in different bands of cells that serve as signaling centers within each segment. The two proteins mutually maintain each other’s expression, while regulating the expression of genes such as Engrailed in neighboring cells (Figure 21–21). In such a manner, a series of sequential inductions creates a fine-grained pattern of gene expression within each segment.

1	egg-polarity, Gap, and pair-rule Genes create a transient pattern that Is remembered by Segment-polarity and Hox Genes

1	The gap genes and pair-rule genes are activated within the first few hours after fertilization. Their mRNA products initially appear in patterns that only approximate the final picture; then, within a short time, this fuzzy initial pattern resolves itself into a regular, crisply defined system of stripes. But this pattern itself is unstable and transient: as the embryo proceeds through gastrulation and beyond, the pattern disintegrates. The genes’ actions, however, have passed on an enduring memory of their patterns of expression by inducing the expression of certain segment polarity genes along with Hox genes (discussed shortly). After a period of pattern refinement mediated by cell–cell interactions, the expression patterns of these new groups of patterning genes is stabilized to provide positional labels that serve to maintain the segmental organization of the larva and adult fly.

1	The segment-polarity gene Engrailed provides a good example. Its RNA transcripts form a series of 14 bands in the cellular blastoderm, each approximately one-cell wide. These stripes lie immediately anterior to similar stripes of expression of another segment polarity gene, Wingless. As the cells in the developing embryo continue to grow, divide, and move, a mutually reinforcing signal between the Wingless expressing cells and the Engrailed expressing cells maintains narrow stripes of their expression (see Figure 21–21). After three cell cycles, newly expressed regulators stabilize an Engrailed expression pattern that will last Figure 21–20 An example of the regulatory hierarchy of egg-polarity, segmentation, and Hox genes. as discussed in the text, there are three groups of segmentation genes. the photographs show mrNa expression patterns of representative examples of genes of each type. (courtesy of Stephen Small.)

1	Figure 21–21 Mutual maintenance of Hedgehog and Wingless expression. engrailed is a transcription regulator (blue) that drives the expression of Hedgehog. Hedgehog encodes a secreted protein (red) that activates its signaling pathway in neighboring cells and thereby drives them to express the Wingless gene. In turn, Wingless encodes a secreted protein (green) that acts back on neighbors of the Wingless-expressing cell to maintain their expression of Engrailed and Hedgehog. as indicated, the same control loop repeats along the a-p axis of the fly. (Based on S. Dinardo et al., Curr. Opin. Genet. Dev. 4:529–534, 1994.) throughout the life of the fly, long after the signals that induced and refined it have disappeared. The segment borders will form at the posterior edge of each such Engrailed stripe (Figure 21–22).

1	In addition to regulating the segment-polarity genes, the products of pair-rule genes collaborate with those of gap genes to induce the precisely localized activation of a further set of genes—originally called homeotic selector genes and now often called Hox genes, for reasons that will become clear shortly. It is the Hox genes that permanently distinguish one segment from another. In the next section, we examine these important genes in detail and consider their role in cell memory; we shall see that this role is critical in a wide range of animals, including ourselves. Hox Genes permanently pattern the a-p axis

1	Hox Genes permanently pattern the a-p axis As animal development proceeds, the body becomes more and more complex. But again and again, in every species and at every level of organization, we find that complex structures are made by repeating a few basic themes, with variations. Thus, a limited number of basic differentiated cell types, such as muscle cells or fibroblasts, recur with subtle individual variations in different sites. These cell types are organized into a limited variety of tissue types, such as muscle or tendon, which again are repeated with subtle variations in different regions of the body. From the various tissues, organs such as teeth or digits are built— molars and incisors, fingers and thumbs and toes—a few basic kinds of structure, repeated with variations.

1	Wherever we find this phenomenon of modulated repetition, we can break down the developmental biologist’s problem into two kinds of questions: what is the basic construction mechanism common to all the objects of the given class, and how is this mechanism modified to give the observed variations in different animals? The segments of the insect body provide a good example. We have thus far sketched the way in which the rudiment of a single body segment is constructed and how cells within each segment become different from one another. We now consider how one segment becomes determined, or specified, to be different from another.

1	The first glimpse of the answer to this problem came over 80 years ago, with the discovery of a set of mutations in Drosophila that cause bizarre disturbances in the organization of the adult fly. In the Antennapedia mutant, for example, legs sprout from the head in place of antennae, whereas in the Bithorax mutant, portions of an extra pair of wings appear where normally there should be the much smaller appendages called halteres (Figure 21–23). These mutations transform parts of the body into structures appropriate to other positions, and they are called homeotic mutations (from the Greek “homoios,” meaning similar) because the transformation is between structures of a recognizably similar general type, changing one kind of limb, or one kind of segment, into another. It was eventually discovered that a whole set of genes, the homeotic selector genes, or Hox genes, serve to permanently specify the A-P characters of the whole set of animal segments. These genes are all related to one

1	that a whole set of genes, the homeotic selector genes, or Hox genes, serve to permanently specify the A-P characters of the whole set of animal segments. These genes are all related to one another as members of a multigene family.

1	There are eight Hox genes in the fly, and they all lie in one or the other of two gene clusters known as the Bithorax complex and the Antennapedia complex. wild type loss of Ubx gain of Ubx Figure 21–22 The pattern of expression of Engrailed, a segment-polarity gene. the Engrailed pattern is shown in a 10hour embryo and an adult (whose wings have been removed in this preparation). the pattern is revealed by constructing a strain of Drosophila containing the control sequences of the Engrailed gene coupled to the coding sequence of the reporter LacZ, whose product is detected histochemically through the brown product generated by immunohistochemistry against LacZ (10-hour embryo) or through the blue product generated by a reaction that LacZ catalyzes (adult). Note that the Engrailed pattern, once established, is preserved throughout the animal’s life. (courtesy of tom Kornberg.) Figure 21–23 Homeotic mutations.

1	Figure 21–23 Homeotic mutations. Ultrabithorax, or Ubx, is one of three genes in the Bithorax gene complex (a Hox gene cluster). Ubx is responsible for all of the differences between the second and third thoracic segments. (a, B) Ubx loss-of-function mutations transform the haltere-bearing segment (a) into a wing-bearing segment, resulting in four-winged flies (B). (c) Ubx gain-of-function in the second thoracic segment transforms this wing-bearing segment into a halterebearing segment, resulting in wingless flies. (courtesy of richard Mann.)

1	The genes in the Bithorax complex control the differences among the abdominal and thoracic segments of the body, while those in the Antennapedia complex control the differences among thoracic and head segments. Comparisons with other species show that the same genes are present in essentially all animals, including humans. These comparisons also reveal that the Antennapedia and Bithorax complexes are the two halves of a single entity, called the Hox complex, that has become split in the course of the fly’s evolution, and whose members operate in a coordinated way to exert their control over the head-to-tail pattern of the body.

1	The products of the Hox genes, the Hox proteins, are transcription regulators, all of which possess a highly conserved, 60-amino-acid-long DNA-binding homeodomain (see p. 376). The corresponding motif in the DNA sequence is called a “homeobox,” from which, by abbreviation, the Hox complex takes its name. There are many homeobox-containing genes, but only those located in a Hox complex are Hox genes. The Hox proteins can be viewed as molecular address labels possessed by the cells of each segment: these labels give the cells in each region a positional value—that is, an intrinsic character that differs according to a cell’s location. If the address labels in a developing Drosophila segment are changed, the segment behaves as though it were located somewhere else; if all the Hox genes in an embryo are deleted, the body segments in the larva will all be alike.

1	To a first approximation, each Hox gene is normally expressed in those regions that develop abnormally when that gene is mutated or absent. How does each Hox protein give a segment its permanent identity? All the Hox proteins are similar in their DNA-binding regions, but they are very different in the regions that interact with the other proteins with which the Hox proteins form transcriptional regulatory complexes. The different protein partners act together with the Hox proteins to dictate which DNA binding sites will be recognized, as well as whether the effect on transcription at those sites will be activation or repression. Acting in this way, the Hox proteins modulate the actions of many other transcription regulators. Hundreds of genes are under this type of Hox-modulated control, including genes for cell–cell signaling, transcriptional regulation, cell polarity, cell adhesion, cytoskeletal function, cell growth, and cell death, all conspiring (in ways that are not yet

1	including genes for cell–cell signaling, transcriptional regulation, cell polarity, cell adhesion, cytoskeletal function, cell growth, and cell death, all conspiring (in ways that are not yet understood) to give each segment its distinctive Hox-dependent character.

1	Hox Genes are expressed according to their Order in the Hox complex How, then, is the expression of the Hox genes themselves regulated? The coding sequences of the eight Hox genes in the Antennapedia and Bithorax complexes in Drosophila are interspersed amid a much larger quantity of regulatory DNA. This DNA includes binding sites for the products of the egg-polarity and segmentation genes, thereby serving as an interpreter of the multiple items of spatial information supplied to it by all these transcription regulators. The net result is that the particular set of Hox genes transcribed is appropriate for each location along the A-P body axis.

1	The pattern of Hox gene expression exhibits a remarkable regularity that suggests an additional form of control. The sequence in which the genes are ordered along the chromosome, in both the Antennapedia and the Bithorax complexes, corresponds almost exactly to the order in which they are expressed along the A-P axis of the body (Figure 21–24). This hints at some process of gene activation, perhaps dependent on chromatin structures that propagate along the Hox complexes, switching on one Hox gene after another according to their order along the chromosome. The most “posterior” of the Hox genes that are expressed in a cell generally dominates, driving down expression and activity of the “anterior” genes and dictating the character of the segment. The gene regulatory mechanisms underlying these phenomena are still not well understood, but their consequences are profound. We shall see that the serial organization of gene expression in the Hox complex is a fundamental feature that has

1	these phenomena are still not well understood, but their consequences are profound. We shall see that the serial organization of gene expression in the Hox complex is a fundamental feature that has been highly conserved in the course of animal evolution.

1	trithorax and polycomb Group proteins enable the Hox complexes to Maintain a permanent record of positional Information The spatial pattern of expression of the genes in the Hox complex is set up by signals acting early in development, but the consequences are long lasting. Although the pattern of expression undergoes complex adjustments as development proceeds, the Hox complexes serve to stamp each cell and its progeny with a permanent record of the A-P position that the cell occupied in the early embryo. In this way, the cells of each segment are equipped with a long-term memory of their location along the A-P axis of the body. This memory trace is somehow imprinted on the Hox complexes, and it governs the segment-specific identity not only of the larval segments, but also of the structures of the adult fly.

1	The molecular mechanism of this memory of positional information relies on two types of regulation. One is from the Hox genes themselves: many of the Hox proteins autoactivate the transcription of their own genes, thereby helping to keep the genes on indefinitely. Another crucial input is from two large, complementary sets of proteins, called the Trithorax group and the Polycomb group, which stamp the chromatin of the Hox complex with a heritable record of its embryonic state of activation or repression. These are key general regulators of chromatin structure that can be shown to be critical for cell memory: if genes of the Trithorax or Polycomb group are defective, the pattern of expression of the Hox genes is set up correctly at first, but it is not correctly maintained as the embryo grows older.

1	The two sets of regulators act in opposite ways. Trithorax group proteins are needed to maintain the transcription of Hox genes in cells where transcription has already been switched on. In contrast, Polycomb group proteins form stable complexes that bind to the chromatin of the Hox complex and maintain the repressed state in cells where Hox genes have not been activated at the critical time (Figure 21–25). How such changes in chromatin can store developmental cell memory is discussed in Chapters 4 and 7. the D-V Signaling Genes create a Gradient of the transcription regulator Dorsal As with the patterning along the Drosophila A-P axis just discussed, the patterning along the dorsoventral (D-V) axis begins with maternal gene products that define

1	As with the patterning along the Drosophila A-P axis just discussed, the patterning along the dorsoventral (D-V) axis begins with maternal gene products that define Figure 21–24 The patterns of expression compared to the chromosomal locations of the genes of the Hox complex. the diagram shows the sequence of genes in each of the two subdivisions of the chromosomal complex. this corresponds, with minor deviations, to the spatial sequence in which the genes are expressed, shown in the photograph of a Drosophila embryo at the so-called germ band retraction stage, about 10 hours after fertilization. the embryo has been stained by in situ hybridization with differently labeled probes to detect the mrNa products of different Hox genes in different colors. (photograph courtesy of William McGinnis, adapted from D. Kosman et al., Science 305:846, 2004. With permission from aaaS.)

1	Figure 21–25 The role of genes of the Polycomb group. (a) photograph of a wild-type Drosophila embryo. (B) photograph of a mutant embryo defective for the gene Extra sex combs (Esc) and derived from a mother also lacking this gene. the gene belongs to the Polycomb group. essentially all segments have been transformed to resemble the most posterior abdominal segment. In the mutant, the pattern of expression of the homeotic selector genes, which is roughly normal initially, is unstable in such a way that all these genes soon become switched on all along the body axis. (From G. Struhl, Nature 293:36–41, 1981. With permission from Macmillan publishers Ltd.) this axis in the egg (see Figure 21–17), and it then progresses through zygotic gene products that further subdivide the D-V axis in the embryo.

1	Initially, a protein that is produced by follicle cells underneath the future ventral region of the embryo leads to the localized activation of a transmembrane receptor, called Toll, on the ventral side of the egg membrane. The various maternal genes required for this process are called D-V egg-polarity genes. (Curiously, Drosophila Toll and vertebrate Toll-like proteins also operate in innate immune responses, as discussed in Chapter 24). The localized activation of Toll controls the distribution of Dorsal, a transcription regulator of the NFκB family discussed in Chapter 15. The Toll-regulated activity of Dorsal, like that of NFκB, depends on the translocation of Dorsal from the cytosol, where it is held in an inactive form, to the nucleus, where it regulates gene expression (see Figure 15–62). In the newly laid egg, both Dorsal mRNA and protein are distributed uniformly in the cytosol. After the nuclei in the syncytial blastoderm have migrated to the surface of the embryo, but

1	15–62). In the newly laid egg, both Dorsal mRNA and protein are distributed uniformly in the cytosol. After the nuclei in the syncytial blastoderm have migrated to the surface of the embryo, but before cellularization (see Figure 21–15), Toll receptor activation on the ventral side induces a remarkable redistribution of the Dorsal protein. On the dorsal side, the protein remains in the cytosol, but ventrally it becomes concentrated in the nuclei, with a smooth gradient of nuclear localization between these two extremes (Figure 21–26).

1	Once inside the nucleus, the Dorsal protein acts as a morphogen and turns on or off the expression of different sets of genes depending on Dorsal’s concentration. The expression of each responding gene depends on its regulatory DNA— specifically, on the number and affinity of the binding sites that this DNA contains for Dorsal and other transcription regulators. In this way, the regulatory DNA interprets the positional signal provided by the nuclear Dorsal protein gradient, so as to define a D-V series of territories—distinctive bands of cells that run the length of the embryo. Most ventrally—where the nuclear concentration of Dorsal protein is highest—it switches on, for example, the expression of a gene called Twist, which is specific for mesoderm. Most dorsally, where the nuclear concentration of Dorsal protein is lowest, the cells switch on a gene called Decapentaplegic (Dpp). And in an intermediate region, where the nuclear concentration of Dorsal protein is high enough to

1	concentration of Dorsal protein is lowest, the cells switch on a gene called Decapentaplegic (Dpp). And in an intermediate region, where the nuclear concentration of Dorsal protein is high enough to repress Dpp but too low to activate Twist, the cells switch on another set of genes, including one called Short gastrulation (Sog) (Figure 21–27A).

1	Products of the genes directly regulated by the Dorsal protein generate in turn more local signals, which define finer subdivisions along the D-V axis. These signals act during cellularization and take the form of conventional extracellular Figure 21–26 The concentration gradient of Dorsal protein in the nuclei of the blastoderm. In wild-type Drosophila embryos, the protein is present in the dorsal cytoplasm and absent from the dorsal nuclei; ventrally, it is depleted in the cytoplasm and concentrated in the nuclei. In a mutant in which the toll pathway is activated everywhere and not just ventrally, Dorsal protein is everywhere concentrated in the nuclei; the result is a ventralized embryo. conversely, in a mutant in which the toll signaling pathway is inactivated, Dorsal protein everywhere remains in the cytoplasm and is absent from the nuclei; the result is a dorsalized embryo. (From

1	S. roth, D. Stein and c. Nüsslein-Volhard, Cell 59:1189–1202, 1989. With permission from elsevier.) 1166 Chapter 21: Development of Multicellular Organisms (B) gradients of Dpp and Sog proteins dorsoventral territories are specifed signal proteins. In particular, Dpp codes for a secreted TGFβ-family protein, which forms a local morphogen gradient in the dorsal part of the embryo. Sog encodes another secreted protein that is produced by the neurogenic ectoderm (which gives rise to the nervous system) and acts as an antagonist of Dpp protein. The opposing diffusion gradients of these two signal proteins create a steep gradient of Dpp activity: the highest Dpp activity levels, in combination with certain other factors, cause development of the most dorsal tissue of all—an extraembryonic membrane. Intermediate levels cause development of dorsal epidermis; and the absence of Dpp activity allows the development of neurogenic ectoderm (Figure 21–27B).

1	a hierarchy of Inductive Interactions Subdivides the Vertebrate embryo The molecular genetic analysis of Drosophila development has uncovered how a cascade of transcription regulators and signaling pathways subdivides the embryo. The same principle of progressive pattern refinement is used during the development of all animal embryos, including vertebrates. Remarkably, conservation is not restricted to the general strategy of pattern formation, but also extends to many of the molecules involved. As mentioned previously, the earliest phases of vertebrate development are surprisingly variable, even between closely related species, and it is even hard to say precisely how the axes of an early fly embryo correspond to those of an early frog or mouse embryo. Nevertheless, we shall see that amid this display of evolutionary plasticity, some features of early development turn out to be highly

1	Figure 21–27 How morphogen gradients guide a patterning process along the dorsoventral axis of the Drosophila embryo. (a) Initially, a gradient of Dorsal protein defines three broad territories of gene expression, marked here by the expression of three representative genes—Dpp, Sog, and Twist. (B) Slightly later, the cells expressing Dpp and Sog secrete, respectively, the signal proteins Dpp (a tGFβ family member) and Sog (an antagonist of Dpp). these two proteins then diffuse and interact with one another (and with certain other factors) to create the dorsoventral (D–V) territories shown. conserved. The same is true of later developmental stages also, often to an astonishing degree. From our own anatomy, it is obvious that we are cousins to birds and fish. But looking at molecular mechanisms, we see that we are cousins to flies and worms too.

1	In the following pages, we discuss how vertebrate embryos are patterned by the interplay of signaling molecules and transcription regulators. We begin by discussing the formation and patterning of the embryonic axes in amphibians, taking the frog Xenopus as our example. We have already broached this topic earlier in the chapter. Here, we pick up the thread and draw comparisons with the fly.

1	As noted earlier, the origins of the embryonic axes and the three germ layers in the frog can be traced back to the blastula (see Figure 21–3A). By labeling individual blastomeres, we can track cells through all their divisions, transformations, and migrations and see what they become and where they come from. The precursors of ectoderm, mesoderm, and endoderm are arranged in order along the animal-vegetal axis of the blastula: the endoderm derives from the most vegetal blastomeres, the ectoderm from the most animal, and the mesoderm from a middle set. Within each of these territories, the cells have diverse fates according to their positions along the D-V axis of the later embryo. For ectoderm, epidermal precursors are located ventrally, and future neurons are found dorsally; for mesoderm, precursors for notochord, muscle, kidney, and blood are arranged from dorsal to ventral. All this can be represented by a fate map that shows which cell types derive from which regions of the

1	precursors for notochord, muscle, kidney, and blood are arranged from dorsal to ventral. All this can be represented by a fate map that shows which cell types derive from which regions of the blastula (Figure 21–28). The fate map confronts us with the central question: how are the cells in different positions driven toward their different fates? We have already explained how maternal factors deposited in the developing frog egg define its animal-vegetal axis, and how cortical rotation triggered by fertilization defines the orientation of the dorsoventral axis (see Figure 21–14). But how does the establishment of axes lead on to the subdivision of the embryo into the future body parts?

1	The maternal gene products lead to the formation of signaling centers on the vegetal and dorsal sides of the embryo. The dorsal signaling center in particular has a special place in the history of developmental biology. Experiments in the early twentieth century identified it as a small cluster of cells, located on the dorsal side of the amphibian embryo, with an extraordinary property: when the cells were transplanted to an opposite site, they could trigger a radical reorganization of the neighboring tissue, causing it to form a second whole-body axis (Figure 21–29). The discovery of this signaling center, called the Organizer, led the way to a pioneering analysis of the chain of inductive interactions that establish the framework of the vertebrate body.

1	In contrast to the Drosophila syncytial embryo, the fertilized frog egg undergoes rapid cleavage divisions that result in an embryo consisting of thousands of cells. Patterning must therefore be mediated by extracellular signal molecules Figure 21–28 Blastula fate map in a frog embryo. the endoderm derives from the most vegetal blastomeres (yellow), the ectoderm from the most animal (blue), and the mesoderm from a middle set (green) that contributes also to endoderm and ectoderm. Different cell types derive from different positions along the dorsoventral axis. Figure 21–29 Induction of a secondary axis by the Organizer. an amphibian embryo receives a graft of a small cluster of cells taken from a specific site, called the Organizer region, on the dorsal side of another embryo at the same stage. Signals from the graft organize the behavior of neighboring cells of the host embryo, causing development of a pair of conjoined (Siamese) twins. See Movie 21.4. [after

1	J. holtfreter and V. hamburger, in analysis of Development (B.h. Willier, p.a. Weiss and V. hamburger, eds), pp. 230–296. philadelphia: Saunders, 1955.] 1168 Chapter 21: Development of Multicellular Organisms that diffuse through the embryo from cell to cell, not by transcription regulators that move through the cytoplasm of a syncytium. Not surprisingly, the Organizer is now known to be a major source of secreted protein signals. a competition Between Secreted Signaling proteins patterns the Vertebrate embryo

1	The signal molecules that pattern the frog embryo along the animal-vegetal (A-V) axis belong to the TGFβ family: they are secreted by a signaling center at the vegetal pole and form concentration gradients along the A-V axis. The Nodal protein acts over a relatively short range: cells near the vegetal pole are exposed to high levels of it and respond by switching on genes that promote the development of endoderm; cells further away are exposed to lower levels and activate genes that promote the formation of mesoderm. The cells at the vegetal pole that produce Nodal also produce a more rapidly diffusing TGFβ-like protein called Lefty, which antagonizes Nodal. The result is a high ratio of Lefty to Nodal at the animal pole, where Lefty predominates and Nodal signaling is blocked; this causes the cells there to develop as ectoderm (Figure 21–30A). Thus, a mid-range activation by Nodal, combined with a long-range inhibition by Lefty, sets up the pattern of progenitors along the A-V axis

1	the cells there to develop as ectoderm (Figure 21–30A). Thus, a mid-range activation by Nodal, combined with a long-range inhibition by Lefty, sets up the pattern of progenitors along the A-V axis for the three germ layers—endoderm, mesoderm, and ectoderm.

1	The frog’s dorsal signaling system uses a different set of secreted signals from that of the vegetal signaling system to subdivide the germ-layer territories according to location along the D-V axis of the embryo. It exerts its influence by secreting two inhibitory signal proteins, called Chordin and Noggin. These antagonize the action of bone morphogenetic proteins (BMPs; members of yet another subclass of the TGFβ family), which themselves are secreted throughout the embryo. In this way, Chordin and Noggin form a dorsal-to-ventral gradient that blocks BMP signaling on the dorsal side but allows it to remain high on the ventral side (Figure 21–30B). Ectodermal cells that experience high levels of BMP signaling are driven to epidermal fates, whereas cells that experience little or no BMP signaling remain neural.

1	Knowing the signals that specify the three germ layers and various tissue types of the vertebrate body, one can reproduce this specification in a culture dish. Frog cells taken from the animal-pole region of the embryo, for example, Figure 21–30 How Nodal and bone morphogenic protein (BMP) signaling pattern the embryonic axes. Nodal and its antagonist Lefty pattern the animal-vegetal axis, while BMp and its antagonists chordin and Noggin pattern the dorsoventral axis. In the animal pole region, where Nodal levels are low relative to Lefty, Lefty blocks Nodal from binding to its receptors. In the vegetal region, there is an excess of Nodal, resulting in Nodal pathway activation. along the dorsoventral axis, BMp is widely present but chordin and Noggin are concentrated at the dorsal side: there, they bind to BMp and block its binding to receptors. the resulting patterns of Nodal and BMp activity are illustrated at the bottom of the figure.

1	will differentiate into blood (a ventral mesodermal tissue) when diverted from their original fate by exposure to intermediate concentrations of Nodal and high concentrations of BMP. Similarly, mouse or human embryonic stem cells can be coaxed to generate specific cell types by exposing them in culture to appropriate combinations of signal molecules. In this way, the insights gained through studies of animal development can be used to generate the cell types needed for regenerative medicine, as we discuss in the next chapter. the Insect Dorsoventral axis corresponds to the Vertebrate Ventral-Dorsal axis

1	the Insect Dorsoventral axis corresponds to the Vertebrate Ventral-Dorsal axis The signaling systems that pattern the D-V axis in Drosophila and in vertebrates are similar. In Drosophila, as we saw, Dpp and its inhibitor Sog are responsible, whereas in vertebrates, BMP and its inhibitors Chordin and Noggin do the job. Dpp is a member of the BMP family, while Sog is a homolog of Chordin. Both in flies and frogs, high levels of the inhibitors define the region that is neurogenic, and high levels of BMP/Dpp activity define the region that is not. These and other molecular parallels strongly suggest that this aspect of body patterning has been conserved in evolution from insects to vertebrates. Curiously, however, the axis is inverted: dorsal in the fly corresponds to ventral in the vertebrate (Figure 21–31). At some point in evolution, it seems that the ancestor of one of these classes of animals took to living life upside-down. Hox Genes control the Vertebrate a-p axis

1	Hox Genes control the Vertebrate a-p axis The conservation of developmental mechanisms between Drosophila and vertebrates extends beyond the D-V signaling system. Hox genes are found in almost every animal species studied, where they are often grouped in complexes similar to the insect Hox complex. In mice and humans, for example, there are four such complexes—called the HoxA, HoxB, HoxC, and HoxD complexes—each on a different chromosome. Individual genes in each complex can be recognized by their sequences as counterparts of specific members of the Drosophila set. Indeed, mammalian Hox genes can function in Drosophila as partial replacements for the corresponding Drosophila Hox genes. It appears that each of the four mammalian Hox complexes is, roughly speaking, the equivalent of one complete insect Hox complex (that is, an Antennapedia complex plus a Bithorax complex) (Figure 21–32).

1	The ordering of the genes within each vertebrate Hox complex is essentially the same as in the insect Hox complex, suggesting that all four vertebrate complexes originated by duplications of a single primordial complex and have preserved its basic organization. Most tellingly, when the expression patterns of the Hox genes are examined in the vertebrate embryo, it turns out that the members of each complex are expressed in a head-to-tail series along the axis of the body, just as they are in Drosophila. As in Drosophila, vertebrate Hox gene expression patterns are often aligned with vertebrate segments. This alignment is especially clear in the hindbrain (see Figure 21–32), where the segments are called rhombomeres.

1	The products of the vertebrate Hox genes, the Hox proteins, specify positional values that control the A-P pattern of parts in the hindbrain, neck, and trunk (as well as some other parts of the body). As in Drosophila, when a posterior Hox gene is artificially expressed in an anterior region, it can convert the anterior tissue to

1	Figure 21–31 The vertebrate body plan as a dorsoventral inversion of the insect body plan. Note the correspondence with regard to the circulatory system as well as the gut and nervous system. In insects, the circulatory system is represented by a tubular heart and a main dorsal blood vessel, which pumps blood out into the tissue spaces through one set of apertures and receives blood back from the tissues through another set. Unlike vertebrates, insects have no system of capillary vessels to contain the blood as it percolates through the tissues. Nevertheless, heart development depends on homologous genes in vertebrates and insects, reinforcing the relationship between the two body plans. (after e.L. Ferguson, Curr. Opin. Genet. Dev. 6:424–431, 1996. With permission from elsevier.)

1	Bcd, Lab Pb a posterior character. Conversely, loss of posterior Hox genes allows the posterior tissue where they are normally expressed to adopt an anterior character (Figure 21–33). Because of a redundancy between genes in the four Hox gene clusters, the transformations observed in mouse Hox mutants are not always so straightforward as those in the fly, and they are often incomplete. Nonetheless, it seems clear that the fly and the mouse use essentially the same molecular machinery to impart individual characteristics to successive regions along at least a part of the A-P axis. Just as there are genes that regulate pattern formation and segmental identity, there are genes whose products act as triggers for the development of a specific cell type or even a specific organ, initiating and coordinating the whole complex program of gene expression that is required. An example is the MyoD/myogenin

1	Figure 21–32 The Hox complexes of an insect and a mammal, compared and related to body regions. the genes of the Antennapedia and Bithorax complexes of Drosophila are shown in their chromosomal order in the top line. the corresponding genes of the four mammalian Hox complexes are shown below, also in chromosomal order. the gene expression domains in fly and mammal are indicated in a simplified form by color in the cartoons of animals above and below. there is a remarkable parallelism. however, the details of the patterns depend on developmental stage and vary somewhat from one mammalian Hox complex to another. also, in many cases, genes shown here as expressed in an anterior domain are also expressed more posteriorly, overlapping the domains of more posterior Hox genes.

1	the complexes are thought to have evolved as follows: first, in some common ancestor of worms, flies, and vertebrates, a single primordial homeotic selector gene underwent repeated duplication to form a series of such genes in tandem—the ancestral Hox complex. In the Drosophila sublineage, this single complex became split into separate Antennapedia and Bithorax complexes. Meanwhile, in the lineage leading to the mammals, the whole complex was repeatedly duplicated to give four Hox complexes. the parallelism is not perfect because apparently some individual genes have been duplicated and others lost. Still others have been co-opted for different purposes (genes in parentheses in the top line) over the time that has elapsed since the complexes diverged. (Based on a diagram courtesy of William McGinnis.)

1	Hox gain of function Hox loss of function family of transcription regulators that we encountered in Chapter 7. These proteins drive cells to differentiate into muscle, expressing muscle-specific actins and myosins and all the other specialized cytoskeletal, metabolic, and membrane proteins that a muscle cell needs. Analogously, members of the Achaete/Scute family of transcription regulators drive cells to become neural progenitors. In both these examples, the proteins belong to the basic helix–loop–helix (bHLH) class of transcription regulators (see p. 377), and the same is true for many of the other proteins that induce the differentiation of particular cell types. These master transcription regulators exert their powerful differentiation-inducing activity by binding to many different regulatory sites in the genome and thereby controlling the expression of large numbers of downstream target genes. In one well-studied case, that of an Achaete/Scute family member called Atonal homolog

1	sites in the genome and thereby controlling the expression of large numbers of downstream target genes. In one well-studied case, that of an Achaete/Scute family member called Atonal homolog 1 (Atoh1), the number of direct target genes in the mouse genome is more than 600. It is important to note, however, that even such powerful drivers of cell differentiation can have radically different effects according to the context and history of the cells in which they act: Atoh1, for example, drives differentiation of certain classes of neurons in the brain, of sensory hair cells in the inner ear, and of secretory cells in the lining of the gut.

1	Other genes encoding transcription regulators can drive the formation and assembly of the multiple cell types that constitute an entire organ. A famous example is the transcription regulator Eyeless. When it is artificially expressed in a patch of cells in the leg precursors of Drosophila, a well-organized eye-like organ develops on the leg, with the various eye cell types correctly arranged (see Figure 7–35B); conversely, loss of the Eyeless gene results in flies that lack eyes. Moreover, loss of the Eyeless homolog Pax6 in vertebrates likewise leads to loss of eye structures. Similar organ-selector proteins are known for foregut, heart, pancreas, and other organs. They are all master transcription regulators that directly regulate hundreds of target genes, the products of which then specify and construct the different elements of the appropriate organ. However, as in the example of Atoh1, they usually exert their specific effect only in combination with the right partners, which are

1	and construct the different elements of the appropriate organ. However, as in the example of Atoh1, they usually exert their specific effect only in combination with the right partners, which are only expressed in cells that were appropriately primed during their earlier development.

1	After the establishment of the basic body plan and the generation of organ precursors, many further steps of pattern refinement are required to achieve the adult pattern of terminally differentiated cells in tissues and organs. As we discussed Figure 21–33 Control of anteroposterior pattern by Hox genes in the mouse. (a,B) a normal mouse (wild type) has about 65 vertebrae, differing in structure according to their position along the body axis: 7 cervical (neck), 13 thoracic (with ribs), 6 lumbar [bracketed by yellow asterisks in (B)], 4 sacral [bracketed by red asterisks in (B)], and about 35 caudal (tail). shows a side view and (B) shows a dorsal view; for clarity, the limbs have been removed in each picture.

1	shows a side view and (B) shows a dorsal view; for clarity, the limbs have been removed in each picture. the HoxA10 gene is normally expressed in the lumbar region (together with its paralogs HoxC10 and HoxD10); here it has been artificially expressed in the developing vertebral tissue all along the body axis. as a result, the cervical and thoracic vertebrae are all converted to a lumbar character. (D) conversely, when HoxA10 is removed along with HoxC10 and HoxD10, vertebrae that should normally have a lumbar or sacral character take on a thoracic character instead. (a and c, from M. carapuço et al., Genes Dev. 19:2116–2121, 2005. With permission from cold Spring harbor Laboratory press; B and D, from D.M. Wellik and M.r. capecchi, Science 301:363–367, 2003.) earlier, lateral inhibition mediated by Notch signaling is crucial for both cell diversification and fine-grained patterning in an enormous variety of tissues in all animals.

1	One example is the development of sensory bristles in Drosophila, most easily seen on the fly’s back, but also present on most of its other exposed surfaces. Each of these is a miniature sense organ, consisting of a sensory neuron and a small set of supporting cells. Some bristles respond to chemical stimuli, others to mechanical stimuli, but they are all constructed in a similar way (Figure 21–34). The pro-neural genes Achaete and Scute mentioned earlier mark the patches of epidermis within which bristles will form. Mutations that eliminate the expression of these genes at some of their usual sites block development of bristles at just those sites, and mutations that cause expression in abnormal sites cause bristles to develop there.

1	The initial cells expressing the proneural genes are called proneural cells, and they are primed to take the neurosensory pathway of differentiation, but which of the cells will actually do so depends on competitive interactions among them. In the first round of these interactions, a single cell within each small group of proneural cells is picked to serve as the progenitor of the bristle. This single cell is called the sensory mother cell. It becomes distinct from the other cells of the cluster through lateral inhibition mediated by the Notch signaling pathway. This operates in the way we discussed earlier. The cells in the proneural cluster initially all express both the transmembrane receptor Notch and its transmembrane ligand Delta, along with proteins that regulate the signaling activity of Delta. Wherever Delta activates Notch, an inhibitory signal is transmitted that diminishes the tendency of the Notch-activated cell to specialize as a sensory mother cell.At first, all the

1	of Delta. Wherever Delta activates Notch, an inhibitory signal is transmitted that diminishes the tendency of the Notch-activated cell to specialize as a sensory mother cell.At first, all the cells in the cluster inhibit one another. However, receipt of the signal in a given cell diminishes that cell’s ability to fight back by delivering the inhibitory Delta signal in return. This creates a competitive situation, from which a single cell in each cluster—the future sensory mother cell—eventually emerges as winner, sending a strong inhibitory signal to its immediate neighbors but receiving no such signal in return (Figure 21–35). If a cell that would normally become a sensory mother cell is genetically disabled from doing so, a neighboring proneural cell, freed from lateral inhibition, will become a sensory mother cell instead.

1	The sensory mother cell goes through a short program of further divisions to generate the set of cells that form the final bristle. Notch signaling acts repeatedly at successive stages in this program to drive the descendants of the sensory mother cell along different pathways and assign them to their various specialized fates. However, it does so in conjunction with additional mechanisms that bias the outcome of the competition mediated by lateral inhibition. Determinants that

1	Figure 21–34 The basic structure of a mechanosensory bristle. the lineage of the four cells of the bristle—all descendants of a single sensory mother cell—is shown on the left. the sensory mother cell, once it is specified, generates this set of cells through a short program of division cycles. In each generation of the progeny, lateral inhibition operates again to drive the newborn cells toward different fates: one of the ultimate progeny will become the neuron; another, the shaft of the bristle; others, supporting cells of various sorts. as the sensory mother cell and its progeny divide, certain proteins are allocated preferentially to one of each pair of newborn sister cells, biasing the outcome of the lateral-inhibition competition mediated by Notch signaling. are asymmetrically localized inside the dividing cells have this role in sensory bristle development. They are also important in other contexts, as we now discuss.

1	are asymmetrically localized inside the dividing cells have this role in sensory bristle development. They are also important in other contexts, as we now discuss. Cell diversification does not always have to depend on extracellular signals: in some cases, sister cells are born different as a result of an asymmetric cell division, during which some significant set of molecules is divided unequally between them. This asymmetrically segregated molecule (or set of molecules) then acts as a determinant for one of the cell fates by directly or indirectly altering the pattern of gene expression within the daughter cell that receives it (see Figure 21–12). We have already encountered the asymmetric segregation of molecules in the context of the early frog embryo: VegT RNA is localized in the vegetal region of the fertilized egg. Following cell division, only vegetal daughter cells will inherit VegT RNA.

1	Asymmetric divisions often occur at the beginning of development, but they are also encountered at some later stages. As mentioned for the sensory bristle, they can set the scene for an exchange of Notch signals between the daughter cells, with the signaling occurring after the cells have become separate and reinforcing the differences between them. In the central nervous system, asymmetric divisions have a key role in generating the very large numbers of neurons and glial cells that are needed. A special class of cells becomes committed as neural precursors, but instead of differentiating directly as neurons or glial cells, these undergo a long series of asymmetric divisions through which a succession of additional neurons and glial cells are added to the population. The process is best understood in Drosophila, although there are many hints that something similar occurs also in vertebrate neurogenesis.

1	In the embryonic central nervous system of Drosophila, the nerve-cell precursors, or neuroblasts, are initially singled out from the neurogenic ectoderm by a typical lateral-inhibition mechanism that depends on Notch. Each neuroblast then divides repeatedly in an asymmetric fashion (Figure 21–36). At each division, one daughter remains as a neuroblast, while the other, which is much

1	Figure 21–35 Lateral inhibition. (a) the basic mechanism of Notch-mediated competitive lateral inhibition, illustrated for just two interacting cells. In this diagram, the absence of color on proteins or effector lines indicates inactivity. (B) the outcome of the same process operating in a larger patch of cells. at first, all cells in the patch are equivalent, expressing both the transmembrane receptor Notch and its transmembrane ligand Delta. each cell has a tendency to specialize (as a sensory mother cell), and each sends an inhibitory signal to its neighbors to discourage them from also specializing in that way. this creates a competitive situation. as soon as an individual cell gains any advantage in the competition, that advantage becomes magnified. the winning cell, as it becomes more strongly committed to differentiating as a sensory mother cell, also inhibits its neighbors more strongly. conversely, as these neighbors lose their capacity to differentiate as sensory mothers,

1	more strongly committed to differentiating as a sensory mother cell, also inhibits its neighbors more strongly. conversely, as these neighbors lose their capacity to differentiate as sensory mothers, they also lose their capacity to inhibit other cells from doing so. Lateral inhibition thus makes adjacent cells follow different fates.

1	although the interaction is thought to be normally dependent on cell–cell contacts, the future sensory mother cell may be able to deliver an inhibitory signal to cells that are more than one cell diameter away—for example, by sending out long protrusions to touch them.

1	smaller, becomes specialized as a ganglion mother cell. Each ganglion mother cell will divide only once, giving a pair of neurons, or a neuron plus a glial cell, or a pair of glial cells, with Notch-mediated interactions helping to drive the daughters along different paths. The neuroblast itself becomes smaller at each division, as it parcels out its substance into one ganglion mother cell after another. Eventually, typically after about 12 cycles, the process halts, presumably because the neuroblast becomes too small to pass the cell-size checkpoint in the cell-division cycle. Later, in the larva, neuroblast divisions resume, but now they are accompanied by cell growth, permitting the process to continue indefinitely and to generate the much larger numbers of neurons and glial cells required in the adult fly.

1	In the preceding sections, we have seen that animals contain the same essential cell types, have a similar collection of genes, and share many of the molecular mechanisms of pattern formation. But how can we square this with the radical differences that we see in the body structures of animals as diverse as a worm, a fly, a frog, and a mouse? We asserted earlier, in a general way, that these differences usually seem to reflect differences in the regulatory DNA that calls into play the components of the conserved basic kit of parts. We must now examine the evidence a little more closely.

1	When we compare animal species with similar basic body plans—different vertebrates, for example, such as fish, birds, and mammals—we find that corresponding genes usually have similar sets of regulatory elements: the regulatory DNA sequences have been well conserved and are recognizably homologous in the different animals. The same is true if we compare different species of nematode worms or insects. But, when we compare vertebrate regulatory regions with those of worms or flies, it is hard to see any such resemblance. The protein-coding sequences are unmistakably similar, but the corresponding regulatory DNA sequences appear mostly very different, suggesting that the differences in body plans mainly reflect differences in regulatory DNA. Although variations in the proteins themselves also contribute, differences in regulatory DNA would be enough to generate radically different tissues and body structures even if the proteins were the same.

1	It is not yet possible to trace the genetic steps that have led to all the spectacular diversity of animals. Their lineages have diverged over hundreds of millions of years, and in most cases too many changes have occurred for us to be able to say that this or that feature results from this or that mutation. The picture is clearer, however, for more recent evolutionary events. Studies of both closely related animal populations and plant populations whose members have different morphologies have revealed that dramatic developmental effects can result from subtle changes in regulatory DNA.

1	A well-studied example is the morphological diversity found in stickleback fish. After the last ice age ended about 10,000 years ago, marine sticklebacks colonized many newly formed freshwater streams and lakes. Marine sticklebacks extend sharp spines from their pelvic skeleton. These spines are thought to help protect the fish from soft-mouthed fish predators. In contrast, several populations of freshwater sticklebacks have lost these spines, usually in lakes that lack such predators. The different morphologies reflect differences in control of the expression of a transcription regulator called Pitx1. Whereas marine sticklebacks express the Pitx1 gene in the pelvic bone precursor cells that will form the spikes,

1	Figure 21–36 Neuroblasts and asymmetric cell division in the central nervous system of a fly embryo. the neuroblast originates as a specialized ectodermal cell. It is singled out by lateral inhibition and emerges from the basal (internal) face of the ectoderm. It then goes through repeated division cycles, dividing asymmetrically to generate a series of ganglion mother cells. each ganglion mother cell divides just once to give a pair of differentiated daughters (typically a neuron plus a glial cell). freshwater sticklebacks have lost this expression as a result of a change at the Pitx1 locus. These changes do not lie in the coding sequence. Instead, each is a small deletion of a block of adjacent regulatory DNA that controls Pitx1 expression specifically in the pelvic cells (Figure 21–37).

1	The Pitx1 protein has important functions elsewhere in the body, so that the DNA sequences that encode this protein must be retained. The regulatory DNA responsible for Pitx1 expression at these other sites is also unchanged in the two populations of sticklebacks. The evolution of pelvis development in sticklebacks shows how the modular nature of regulatory DNA elements that we encountered in Chapter 7 (see Figure 7–29) allows independent modification of the different parts of the body, even when formation of those body parts depends on the same proteins. In the recent evolution of plants, changes of body structure can be traced in a similar way to changes in regulatory DNA. For example, these account for a large part of the dramatic difference between the wild teosinte plant and its modern descendant, maize, through some 10,000 years of mutation and selection by Native Americans.

1	Drosophila has been the foremost model organism for the study of the genetics of animal development. Its embryonic pattern is initiated by the products of maternal-effect genes called egg-polarity genes, which operate by setting up graded distributions of transcription regulators in the egg and early embryo. The gradient of Bicoid protein along the A-P axis, for example, helps initiate the orderly expression of gap genes, pair-rule genes, and segment-polarity genes. These three classes of segmentation genes, through a hierarchy of interactions, become expressed in some regions of the embryo and not others, progressively subdividing the embryo along the A-P axis into a regular series of repeating modular units called segments.

1	Superimposed on the pattern of gene expression that repeats itself in every segment, there is a serial pattern of expression of Hox genes that confer on each segment a different identity. These genes are grouped in complexes and are arranged in a sequence that matches their sequence of expression along the A-P axis of the body. Although Hox gene expression is initiated in the embryo, it is subsequently maintained by the action of chromatin-binding proteins of the Polycomb and Trithorax group, which stamp the chromatin of the Hox complex with a heritable record of its embryonic state of repression or activation, respectively. Hox complexes homologous to that of Drosophila are found in virtually every type of animal, where they help pattern the A-P axis of the body.

1	Signaling gradients are also set up along the dorsoventral (D-V) axis. Initially, Toll signaling generates a nuclear gradient of Dorsal protein, which induces an extracellular signaling gradient of the TGFβ-family protein Dpp and its antagonist, Figure 21–37 Morphological diversity in stickleback fish is caused by changes in regulatory elements. (a–D) pelvic spines are present in marine (a) but not in freshwater (c) populations. correspondingly, Pitx1 is expressed in the pelvic area in marine (B) but not in freshwater (D) fish. the lack of expression in the pelvic area of freshwater populations is caused by mutations in an enhancer element. Other enhancers and sites of expression for Pitx1 are the same in marine and freshwater sticklebacks. (courtesy of Michael D. Shapiro.) Sog. This creates a gradient of Dpp activity that helps refine the assignment of different characters to cells at different positions along the D-V axis.

1	Sog. This creates a gradient of Dpp activity that helps refine the assignment of different characters to cells at different positions along the D-V axis. In Xenopus, the polarity of the egg and the site of sperm entry set up the embryonic axes. A gradient generated by the TGFβ-family protein Nodal induces different fates along the animal-vegetal axis, whereas BMP and Chordin—proteins homologous to Drosophila Dpp and Sog, respectively—control the patterning of the D-V axis. This axis is inverted, so that dorsal in the fly corresponds to ventral in the frog.

1	Transcription regulators control the formation of specific cell types. Members of the MyoD/myogenin family drive the process of muscle cell determination, coordinating the many components required, whereas Achaete/Scute transcription regulators control neural fate. Other genes encoding such master transcriptional regulators can regulate the formation of entire organs. Eyeless, for example, is both necessary and sufficient to generate eye structures in Drosophila.

1	To refine the anatomical pattern within such an organ, the cells interact locally, both by diffusible inductive signals and by short-range mechanisms. Often, the cells compete with one another by lateral inhibition. This process results in activation of the Notch signaling pathway in one cell and inhibition in its neighbors, generating two different cell types. Asymmetric cell divisions, in which daughter cells inherit different molecular determinants from the mother cell, provide an additional way to organize a fine-grained diversity of cell types. Evidence from recent evolutionary events indicates that anatomical changes are mostly driven by changes in regulatory DNA sequences that determine when and where developmental genes are expressed. How the striking diversity in body structures has evolved over longer times remains largely unknown, although it seems likely that similar principles apply.

1	Developmental events unfold over minutes, hours, days, weeks, months, or even years, with each organism following its own strict timetable. The cascades of inductive interactions and transcriptional regulatory events described earlier take time, as signals are transmitted and transcription regulators are synthesized and then bind to DNA to activate or repress their target genes. At the beginning of this chapter, we compared development with an orchestral performance. There are many players, and each must do the right thing at the right time; yet there is no leader or conductor to set the tempo and coordinate the timing of all the different events. Each developmental process must thus occur at an appropriate rate, tuned by evolution to fit with the timing of other processes in the embryo or in the environment. The control of timing is one of the most important problems in developmental biology, but also one of the least understood.

1	Developmental processes are complex, but they are built up from simple steps. A first challenge is to understand the timing of these steps. How long does it take, for example, to switch the expression of a gene on or off? This is not like throwing a light switch: it involves delays. First, it takes time to make an mRNA molecule: the RNA polymerase must travel the length of the gene, the primary RNA transcript must be spliced and otherwise processed, and the resulting mRNA must be exported from the nucleus and delivered to the site where it will be translated. This adds up to what one might call the gestation time of the individual molecule. Second, it takes time for the individual mRNA molecules to accumulate to their fully effective concentration; as explained in Chapter 15, this accumulation time is dictated by the average lifetime of the molecules—the longer they last, the higher their ultimate concentration, and the longer the time taken to attain it. Similar delays occur at the

1	time is dictated by the average lifetime of the molecules—the longer they last, the higher their ultimate concentration, and the longer the time taken to attain it. Similar delays occur at the next step, where the mRNA is translated into protein: synthesis of each individual protein molecule involves a gestation delay, and attainment of an effective concentration of protein molecules involves an accumulation delay that depends on the protein’s lifetime. The time for the whole gene switching process is just the sum of the gestation delays and the accumulation delays (basically, the molecular lifetimes) for both the mRNA and the protein molecules. Somewhat counterintuitively, it is the combined length of these delays, rather than the rate of molecular synthesis (the number of molecules synthesized per second), that chiefly determines the switching time.

1	The same additive principle applies to long cascades of gene switching, where gene A activates gene B, and gene B activates gene C, and so on. It also applies in other circumstances, such as in signaling pathways where one protein directly regulates the activation of the next. In all these cases, molecular lifetimes, along with gestation delays, play a key part in determining the pace of development. The lifetimes of mRNA and protein molecules are enormously variable, from a few minutes or hours to days or more, explaining much of the variation we see in the tempo of developmental events.

1	Gene switching delays, however, are not the be-all and end-all of developmental timing. Development involves many other kinds of delay that contribute to timing. Chromatin structure takes time to remodel. Inductive signals take time to diffuse across a field of cells (see Figure 21–9). Cells take time to move and rearrange themselves in space. Nevertheless, the timing of gene switching plays a fundamental part in developmental timing, as illustrated in an especially clear and striking way by a gene-expression oscillator that controls the segmentation of the vertebrate body axis, as we now explain. a Gene-expression Oscillator acts as a clock to control Vertebrate Segmentation

1	a Gene-expression Oscillator acts as a clock to control Vertebrate Segmentation The main body axis of all vertebrates has a repetitive, periodic structure, seen in the series of vertebrae, ribs, and segmental muscles of the neck, trunk, and tail. These segmental structures originate from the mesoderm that lies as a long slab on either side of the embryonic midline. This slab becomes broken up into a regular repetitive series of separate blocks, or somites—cohesive groups of cells, separated by clefts (Figure 21–38A). The somites form (as bilateral pairs) one after another, in a regular rhythm, starting in the region of the head and ending in the tail. Depending on the species, the final number of somites ranges from less than 40 (in a frog or a zebrafish) to more than 300 (in a snake).

1	The posterior, most immature part of the mesodermal slab, called the presomitic mesoderm, supplies the required cells: as the cells proliferate, this mesoderm of oscillation cycle of oscillation cycle tail moves back as new somites form Figure 21–38 Somite formation in the chick embryo. (a) a chick embryo at 40 hours of incubation. (B) how the temporal oscillation of gene expression in the presomitic mesoderm becomes converted into a spatial alternating pattern of gene expression in the formed somites. In the posterior part of the presomitic mesoderm, each cell oscillates with a cycle time of 90 minutes. as cells mature and emerge from the presomitic region, their oscillation is gradually slowed down and finally brought to a halt, leaving them in a state that depends on the phase of the cycle they happen to be in at the critical moment. In this way, a temporal oscillation of gene expression traces out an alternating spatial pattern. (a, from Y.J. Jiang, L. Smithers and

1	J. Lewis, Curr. Biol. 8:r868–r871, 1998. With permission from elsevier.) retreats tailward, extending the embryo (Figure 21–38B). In the process, it deposits a trail of somites formed from cells that group together into blocks as they emerge from the anterior end of the presomitic region. The special character of the presomitic mesoderm is maintained by a combination of fibroblast growth factor (FGF) and Wnt signals, produced by a signaling center at the tail end of the embryo, and the range of these signals seems to define the length of the presomitic mesoderm. The somites emerge with clocklike timing, but what determines the rhythm of the process?

1	In the posterior part of the presomitic mesoderm, the expression of certain genes oscillates in time. Snapshots of gene expression taken by fixing embryos for analysis at different times in the oscillation cycle reveal what is happening, and the oscillations can now also be observed in time-lapse movies of embryos containing fluorescent reporters of individual oscillating genes. One new somite pair is formed in each oscillation cycle, and, in mutants where the oscillations fail to occur, somite segmentation is disrupted: the cells may still break up, belatedly, into separate clusters, but they do so in a haphazard, irregular way. The gene-expression oscillator controlling regular segmentation is called the segmentation clock. The length of one complete oscillation cycle depends on the species: it is 30 minutes in a zebrafish, 90 minutes in a chick, 120 minutes in a mouse.

1	As cells emerge from the presomitic mesoderm to form somites—in other words, as they escape from the influence of the FGF and Wnt signals—their oscillation stops. Some become arrested in one state, some in another, according to the phase of the oscillation cycle at the time they leave the presomitic region. In this way, the temporal oscillation of gene expression in the presomitic mesoderm leaves its trace in a spatially periodic pattern of gene expression in the maturing mesoderm; this in turn dictates how the tissue will break up into physically separate blocks, through effects on the pattern of cell–cell adhesion (see Figure 21–38B).

1	How does the segmentation clock work? The first somite oscillator genes to be discovered were Hes genes, which are key components of the Notch signaling pathway. They are directly regulated by the activated form of Notch, and they code for inhibitory transcription regulators that inhibit the expression of other genes, including Delta. As well as regulating other genes, the products of Hes genes can directly regulate their own expression, creating a remarkably simple negative feedback loop. Autoregulation of certain specific Hes genes (depending on species) is thought to be the basic generator of the oscillations of the somite clock. Although the machinery has been modified in various ways in different species, the underlying principle seems to be conserved. When the key Hes gene is transcribed, the amount of Hes protein product builds up until it is sufficient to block Hes gene transcription; synthesis of the protein ceases; the protein then decays, permitting transcription to begin

1	the amount of Hes protein product builds up until it is sufficient to block Hes gene transcription; synthesis of the protein ceases; the protein then decays, permitting transcription to begin again; and so on, cyclically (Figure 21–39). The period of oscillation, which determines the size of each somite, depends on the delay in the feedback loop. This equals the sum of the gestation delays and accumulation delays (that is, the molecular lifetimes) of the Hes mRNA and protein molecules, according to the additive principle discussed earlier. Mathematical modeling (see Chapter 8) allows us to relate these basic molecular parameters to the cycle time of the segmentation clock: to a first approximation, the cycle period is simply equal to twice the total delay in the negative feedback loop, and thus twice the sum of the delays occurring at each step of the loop.

1	The feedback loop just described is intracellular, and each cell in the presomitic mesoderm can generate oscillations on its own. But these oscillations at the single-cell level are somewhat erratic and imprecise, reflecting the fundamentally noisy, stochastic nature of the control of gene expression, as discussed in Chapter 7. A mechanism is needed to keep all the cells in the presomitic mesoderm that will form a particular somite oscillating in synchrony. This is achieved through cell–cell communication via the Notch signaling pathway, to which the Hes genes are coupled. The gene regulatory circuitry is such that in this context Notch signaling does not drive neighboring cells to be different, as in lateral inhibition, but does just the opposite: it keeps them in unison. In mutants where Notch signaling Figure 21–39 Delayed negative feedback giving rise to oscillating gene expression.

1	(a) a single gene, coding for a transcription regulator that inhibits its own expression, can behave as an oscillator. For oscillation to occur, there must be a delay (or several delays) in the feedback circuit, and the lifetimes of the mrNa and protein (which contribute to the delay) must be short compared with the total delay. the total delay determines the period of oscillation. It is thought that a feedback circuit like this, based on a pair of redundantly acting genes called Her1 and Her7 in the zebrafish—or their counterpart, Hes7, in the mouse—is the pacemaker of the segmentation clock governing somite formation. (B) the predicted oscillation of Her1 and Her7 mrNa and protein, computed using rough estimates of the feedback circuit parameters appropriate to this gene in the zebrafish. concentrations are measured as numbers of molecules per cell. the predicted period is close to the observed period, which is 30 minutes per somite in the zebrafish (depending on temperature).

1	fails, including mutants defective in Delta or Notch itself, the cells drift out of synchrony and somite segmentation is again disrupted. This leads to gross deformity of the vertebral column—an extraordinary display of the consequences of the noisy temporal control of gene expression at the single-cell level, writ large in the structure of the vertebrate body as a whole. Intracellular Developmental programs can help Determine the time-course of a cell’s Development Although signaling between cells plays an essential part in driving the progress of development, this does not mean that cells always need signals from other cells to prod them into changing their character as development proceeds. Some of these changes are intrinsic to the cell (like the ticking of the segmentation clock) and depend on intracellular developmental programs that can operate even when the cell is removed from its normal environment.

1	The best-understood example is in the development of neural precursor cells, or neuroblasts, in the embryonic Drosophila central nervous system. These cells, as we saw, are initially singled out from the neurogenic ectoderm of the embryo by a typical lateral-inhibition mechanism that depends on Notch, and they then proceed through an entirely predictable series of asymmetric cell divisions to generate ganglion mother cells that divide to form neurons and glial cells (see Figure 21–36). The neuroblast changes its internal state as it goes through its set program of divisions, generating different cell types with a reproducible sequence and timing. These successive changes in neuroblast specification occur through the sequential expression of specific transcription regulators. For example, most embryonic neuroblasts sequentially express the transcription regulators Hunchback, Krüppel, Pdm, and Cas in a fixed order (Figure 21–40). When a neuroblast divides, the set of transcription

1	most embryonic neuroblasts sequentially express the transcription regulators Hunchback, Krüppel, Pdm, and Cas in a fixed order (Figure 21–40). When a neuroblast divides, the set of transcription regulators expressed at that time is inherited by the ganglion mother cell and its neural progeny; thus, the differentiated neural cells are endowed with different characters according to their time of birth.

1	Remarkably, when neuroblasts are taken from an embryo and maintained in culture, isolated from their normal surroundings, they step through much the same stereotyped developmental program as if they had been left in the embryo. Moreover, many of the neuroblast transitions occur even when cell division is blocked. The neuroblasts seem to have a built-in timer that determines when each of the transcription regulators is expressed, and this timer can continue to run in the absence of cell division. The molecular basis of the timing is largely unknown; in part, at least, it must depend on the time taken for gene switching, as described above; but it may well also depend on slow progressive changes in chromatin structure. These too can serve to measure the passage of time in the embryo. Figure 21–40 Temporal patterning of neuroblast fate in Drosophila.

1	Figure 21–40 Temporal patterning of neuroblast fate in Drosophila. hunchback, Krüppel, pdm, and cas are transcription regulators that are expressed consecutively in the cell lineage of neuroblasts during development of the Drosophila nervous system. at successive time steps, correlated with cell division, the neuroblast switches its pattern of gene expression. each neuroblast division produces one daughter that remains a neuroblast and expresses the updated set of genes, and one ganglion mother cell that maintains the expression of this gene set and differentiates into specific cell types accordingly. (after B.J. pearson and c.Q. Doe, Nature 425:624–628, 2003.With permission from Macmillan publishers.) cells rarely count cell Divisions to time their Development

1	Many specialized cells in animals develop from proliferating progenitor cells that stop dividing and terminally differentiate after a limited number of cell divisions. In these cases, differentiation is coordinated with withdrawal from the cell cycle, but it is usually not known how the coordination is achieved. It has often been suggested that the cell-division cycle might serve as an intracellular timer to control the timing of cell differentiation. The cell cycle would be the ticking clock that sets the tempo of other developmental processes, with maturational changes in gene expression being dependent on cell-cycle progression. Most of the evidence, however, indicates that this tempting idea is wrong. Although there are examples where cells change their maturation state with each division and the change depends on cell division, this is not the general rule. As we just saw for neuroblasts in the Drosophila embryo, cells in developing animals often carry on with their normal

1	division and the change depends on cell division, this is not the general rule. As we just saw for neuroblasts in the Drosophila embryo, cells in developing animals often carry on with their normal timetable of maturation and differentiation even when cell division is artificially blocked; necessarily, some abnormalities occur, if only because a single undivided cell cannot differentiate in two ways at once. But it seems that most developing cells can change their state without a requirement for cell division. Developmental control genes can switch the cell-division-cycle machinery on or off, and it is the dynamics of these genes, rather than the cell cycle, that sets the tempo of development.

1	Genetic screens are useful for tracking down the genes involved in almost any biological process, and they have been used to search for mutations that alter developmental timing. Such screens were performed in the nematode Caenorhabditis elegans (Figure 21–41). This worm is small, relatively simple, and precisely structured. The anatomy of its development is highly predictable and has been described in extraordinary detail, so that one can map out the exact lineage of every cell in the body and see exactly how the developmental program is altered in a mutant. Genetic screens in C. elegans revealed mutations that disrupt developmental timing in a particularly striking way: in these so-called heterochronic mutants, certain cells in a larva at one stage of development behave as though they were in a larva at a different stage of development, or cells in the adult carry on dividing as though they belonged to a larva (Figure 21–42).

1	Genetic analyses showed that the products of the heterochronic genes act in series, forming regulatory cascades. Unexpectedly, two genes at the top of their respective cascades, called Lin4 and Let7, were found to code not for protein but instead for microRNAs (miRNAs)—short, untranslated, regulatory RNA molecules, 21 or 22 nucleotides long. These act by binding to complementary sequences in the noncoding regions of mRNA molecules transcribed from other heterochronic genes, thereby repressing their translation and promoting their degradation, as discussed in Chapter 7. Increasing levels of Lin4 miRNA govern the progression from first-stage larva cell behaviors to third-stage larva cell behaviors. 1.2 mm Figure 21–41 Caenorhabditis elegans. a side view of an adult hermaphrodite is shown. (From J.e. Sulston and h.r. horvitz, Dev. Biol. 56:110–156, 1977. With permission from academic press.)

1	Figure 21–42 Heterochronic mutations in the Lin14 gene of C. elegans. Only the effects on one of the many altered lineages are shown. a loss-of-function (recessive) mutation in Lin14 causes premature occurrence of the pattern of cell division and differentiation characteristic of a late larva, so that the animal reaches its final state prematurely and with an abnormally small number of cells. the gain-of-function (dominant) mutation has the opposite effect, causing cells to reiterate patterns of cell divisions characteristic of the first larval stage, continuing through as many as five or six molt cycles. the cross denotes a programmed cell death. Green lines represent cells that contain Lin14 protein (which binds to DNa), red lines those that do not. (adapted from V. ambros and h.r. horvitz, Science 226:409–416, 1984. With permission from the authors; and p. arasu, B. Wightman and G. ruvkun, Genes Dev. 5:1825–1833, 1991. With permission from the authors.)

1	Increasing levels of Let7 miRNA govern the progression from late larva to adult. In fact, Lin4 and Let7 were the first miRNAs to be described in any animal: it was through developmental genetic studies in C. elegans that the importance of this whole class of molecules for gene regulation in animals was discovered. More generally, in many animals, miRNAs help regulate the transitions between different stages of development. For example, in flies, fish, and frogs, the maternal mRNAs that are loaded into the egg in the mother are removed during early development when the genome of the embryo begins to be transcribed; at this stage, the embryo begins to express specific miRNAs that target many maternal mRNAs for translational repression and degradation.

1	Thus, miRNAs can sharpen developmental transitions by blocking and removing mRNAs that define an earlier developmental stage. But how is the timing of miRNA expression itself controlled? In the case of the miRNAs that disable maternal mRNAs in frogs and fish, expression is activated at the end of the series of rapid, synchronous divisions that cleave the fertilized egg into many smaller cells. As the division rate of these blastomeres slows, widespread transcription of the embryo’s genome begins (Figure 21–43). This event, where the embryo’s own genome largely takes over control of development from maternal macromolecules, is called the maternal-zygotic transition (MZT), and it occurs with roughly similar timing in most animal species, with the exception of mammals.

1	One trigger for the MZT appears to be the nuclear-to-cytoplasmic ratio. During cleavage, the total amount of cytoplasm in the embryo remains constant, but the number of cell nuclei increases exponentially. As a critical threshold is reached in the ratio of cytoplasm to DNA, the cell cycles lengthen and transcription is initiated. Thus, haploid embryos undergo the MZT one cell cycle later than diploid embryos, which contain twice as much DNA per cell. According to one Figure 21–43 The maternal-zygotic transition in a zebrafish embryo.

1	Maternal mrNas are deposited by the mother into the egg and drive early development. these mrNas are degraded during different stages of embryogenesis, including blastula and gastrula stages, but a relatively abrupt change occurs at the maternal-zygotic transition (MZt). Before this, the embryonic (zygotic) genome is transcriptionally inactive; afterward, zygotic model, the nuclear-to-cytoplasmic ratio might be measured through the titration of a transcription repressor against the increasing amount of nuclear DNA. The total amount of repressor would stay constant during cleavage divisions, but the amount of repressor per genome would decrease, falling by a half with each round of DNA synthesis, until loss of repression allowed the zygotic genome to become transcriptionally active. The newly synthesized transcripts include the miRNAs that recognize many of the transcripts deposited in the egg by the mother, directing their translational repression and rapid degradation.

1	hormonal Signals coordinate the timing of Developmental transitions

1	We have so far emphasized timing mechanisms that operate locally and separately in the different parts of the embryo, or in specific subsystems of the molecular control machinery. Evolution has tuned each of these largely independent processes to run at an appropriate rate, matched to the needs of the organism as a whole. For some purposes, however, this is not enough: a global coordinating signal is required. This is especially true where changes have to occur throughout the body in response to a cue that depends on the environment. For example, when an insect or amphibian undergoes metamorphosis—the transition from larva to adult—almost every part of the body is transformed. The timing of metamorphosis depends on external factors such as the supply of food, which determines when the animal reaches an appropriate size. All the bodily changes have to be triggered together at the right time, even though they are occurring in widely separated sites. The coordination in such cases is

1	animal reaches an appropriate size. All the bodily changes have to be triggered together at the right time, even though they are occurring in widely separated sites. The coordination in such cases is provided by hormones—signal molecules that spread throughout the body.

1	The metamorphosis of amphibians provides a spectacular example. During this developmental transition, amphibians switch from an aquatic to a terrestrial life. Larva-specific organs such as gills and tail disappear, and adult-specific organs such as legs form. This dramatic transformation is triggered by thyroid hormone, produced in the thyroid gland. If the gland is removed or if thyroid hormone action is blocked, metamorphosis does not occur, although growth continues, producing a giant tadpole. Conversely, a dose of thyroid hormone given to a tadpole by an experimenter can trigger metamorphosis prematurely.

1	The thyroid hormone is distributed through the vascular system and induces changes throughout the animal by binding to intracellular nuclear hormone receptors, which regulate hundreds of genes. This does not mean, however, that target tissues all respond in the same way to the hormone: organs differ not only in their levels of thyroid hormone receptors and levels of extracellular proteins that locally regulate the amount of active hormone, but also in the sets of genes that respond. Thyroid hormone induces muscle in the limbs to grow and muscle in the tail to die. The timing of the responses also differs: for example, the legs form early in response to a very low concentration of circulating hormone, but it requires a high level of the hormone to induce resorption of the tail.

1	A surge of thyroid hormone triggers metamorphosis, but how is the timing of the surge controlled? One mechanism depends on coupling hormone synthesis to the size of the thyroid gland, which reflects the size of the tadpole. Only when the gland attains a certain size does it produce enough thyroid hormone to initiate metamorphosis. However, environmental cues other than nutrition also play a part: conditions such as temperature and light are sensed by the nervous system, which regulates the secretion of another tier of hormones (neurohormones) that stimulate the secretion of thyroid hormone. Thus, tadpole-intrinsic factors such as size combine with environmental factors to determine when metamorphosis begins. environmental cues Determine the time of Flowering

1	environmental cues Determine the time of Flowering Another striking example of environmentally controlled developmental timing is the flowering of plants. Flowering involves a transformation of the behavior of the cells at the growing apex of the plant shoot—the apical meristem. During ordinary vegetative growth, these cells behave as stem cells, generating a steady succession of new leaves and new segments of stalk. In flowering, the meristem cells switch to making the components of a flower, with its sepals and petals, its stamens carrying pollen, and its ovary containing the female gametes.

1	To time the switch correctly, the plant has to take account of both past and present conditions. One important cue, for many plants, is day length. To sense this, the plant uses its circadian clock—an endogenous 24-hour rhythm of gene expression—to generate a signal for flowering only when there is light for the appropriate part of the day. The clock itself is influenced by light, and the plant in effect uses the clock to compare past to present lighting conditions. Important parts of the genetic circuitry underlying these phenomena have been identified, including the phytochromes and cryptochromes that act as light receptors (discussed in Chapter 15). The flowering signal that is carried from the leaves to the stem cells via the vasculature depends on the product of Flowering locus T (Ft).

1	But this signal will trigger flowering only if the plant is in a receptive condition from prior long-term cold exposure. Many plants need winter before they will flower—a process called vernalization. Cold over a period of weeks or months progressively reduces the level of expression of a remarkable gene called Flowering locus C (Flc). Flc encodes a transcriptional repressor that suppresses expression of the Ft flowering promoter. How does vernalization shut down Flc so as to lift the block to flowering? The effect involves a noncoding RNA called Coolair that overlaps with the Flc gene and is produced when the temperature is low (Figure 21–44). Together with cold-induced chromatin modifiers, including Polycomb-group proteins, Coolair coordinates the switching of Flc chromatin to a silent state (discussed in Chapters 4 and 7). The degree of silencing depends on the length of cold exposure enabling the plants to distinguish the odd chilly night from the whole of winter.

1	The effect on the chromatin is long lasting, persisting through many rounds of cell division even as the weather grows warmer. Thus vernalization creates a persistent block in production of Flc, enabling the Ft signal to be generated when day length is sufficiently long. Figure 21–44 Temporal control of flowering in Arabidopsis. the Flc gene is active and blocks flowering when plants have been grown without exposure to winterlike temperatures. exposure to a prolonged period of cold leads to the production of the noncoding rNa Coolair, which overlaps with the Flc gene. coolair induces long-term chromatin changes that turn off Flc. these changes persist after the end of the cold period and allow the plant to flower when other environmental conditions are favorable for flowering.

1	Mutations affecting the regulation of Flc expression alter the time of flowering and thus the ability of a plant to flourish in a given climate. The whole control system governing the switch to flowering is thus of vital importance for agriculture, especially in an era of rapid climate change. The example of vernalization suggests a general point about the role of chromatin modification in developmental timing. The plant uses changes in chromatin to record its experience of prolonged cold. It may be that in other organisms—animals as well as plants—slow, progressive changes in chromatin structure provide long-term timers for those mysterious developmental processes that unfold slowly, over a period of days, weeks, months, or years. Such chromatin timers may be among the most important clocks in the embryo, but as yet we understand very little about them.

1	Developmental timing is controlled at many levels. It takes time to switch a gene on or off, and this time delay depends on the lifetimes of the molecules involved, which can vary widely. Cascades of gene regulation involve cascades of delays. Feedback loops can give rise to temporal oscillations in gene expression, and these may serve to generate spatially periodic structures. During vertebrate segmentation, for example, expression of the Hes genes oscillates, and one new pair of somites is formed during each oscillation cycle. Hes genes encode transcription repressor proteins that can act back on expression of the Hes genes themselves. This negative feedback generates oscillations with a period that reflects the delay in the autoregulatory gene switching loop. The period of oscillation of this “segmentation clock” controls the sizes of the somites. Notch signaling between neighboring cells synchronizes their oscillations: when Notch signaling fails, the cells drift out of synchrony

1	of this “segmentation clock” controls the sizes of the somites. Notch signaling between neighboring cells synchronizes their oscillations: when Notch signaling fails, the cells drift out of synchrony because of genetic noise in their individual clocks, and the segmental organization of the vertebral column is disrupted.

1	Timing does not always depend on cell–cell interactions; many developing animal cells have intrinsic developmental programs that play out even in isolated cells in culture. Neuroblasts in Drosophila embryos, for example, go through set programs of asymmetric divisions, generating different neural cell types at each division with a predictable sequence and timing, through a cascade of gene switching events. Studies in both vertebrates and invertebrates show that such programs are rarely governed by the timing of cell division and can unfold even when cell division is blocked. MicroRNAs produced at critical moments sharpen developmental transitions by blocking the translation and promoting the degradation of specific sets of mRNAs. Global coordination of developmental timing is achieved by hormones: as a tadpole grows, for example, thyroid hormone levels surge and trigger its metamorphosis into a frog. Environmental control of developmental timing is especially striking in plants and

1	hormones: as a tadpole grows, for example, thyroid hormone levels surge and trigger its metamorphosis into a frog. Environmental control of developmental timing is especially striking in plants and reveals the presence of molecular timers that act over the long term. In vernalization, for example, prolonged cold induces changes in chromatin that chart the passage through winter so as to allow flowering only in the spring. Slow, progressive changes in chromatin structure are likely to be important timers in the long-term programming of development in animals too.

1	The specialization of cells into distinct types at specific times is important, but it is only one aspect of animal development. Equally important are the movements and deformations that cells go through to assemble into tissues and organs with specific shapes and sizes. Like developmental timing, this process of morphogenesis (“form generation”) is less well understood than the processes of differential gene expression and inductive signaling that lead to cell-type specialization. The cell movements can be readily described, but the underlying molecular mechanisms that coordinate the movements are much harder to decipher.

1	In Chapter 19, we saw how cells cohere to form epithelial sheets or surround themselves with extracellular matrix to create connective tissues. We also discussed how the basic features of tissues, such as the polarity of epithelia, arise from the properties of individual cells. In this section, we consider how the rearrangements of cells during animal development give shape to the embryo and to all the individual organs and appendages of the body.

1	A small number of cell processes are basic to morphogenesis. Individual cells can migrate through the embryo along defined tracks. They can crawl over one another in a coordinated way to elongate, constrict, or thicken a tissue. They can segregate from their neighbors and form physically separate groups. They can change their shape so as to deform an epithelial sheet into a tube or a vesicle. By stretching out while holding on to their companions, specialized sets of cells can form growing tubular networks such as the system of blood or lymph vessels. Mass migrations, as occur in gastrulation, can transform the entire topology of the embryo. Underlying all these processes are changes in cell shape and changes in cell contacts—either with other cells or with extracellular matrix. We begin by considering the migration of individual cells. cell Migration Is Guided by cues in the cell’s environment

1	cell Migration Is Guided by cues in the cell’s environment The birthplace of cells is often far from their ultimate location in the body. Our skeletal muscles, for example, derive from muscle cell precursors, or myoblasts, in somites, from which they migrate into the limbs and other regions. The routes that the migrant cells follow and the selection of sites that they colonize determine the eventual pattern of muscles in the body. The embryonic connective tissues form the framework through which the myoblasts travel, and these tissues provide the cues that guide myoblast distribution. No matter which somite they come from, the myoblasts that migrate into a forelimb bud will form the pattern of muscles appropriate to a forelimb, and those that migrate into a hindlimb bud will form the pattern appropriate to a hindlimb. It is the connective tissue that provides the patterning information.

1	As a migrant cell travels through the embryonic tissues, it repeatedly extends surface projections that probe its immediate surroundings, testing for cues to which it is particularly sensitive by virtue of its specific assortment of cell-surface receptor proteins. Inside the cell, these receptors are connected to the cortical actin and myosin cytoskeleton, which moves the cell along. Some extracellular matrix molecules, such as the protein fibronectin, provide adhesive sites that help the cell advance; others, such as chondroitin sulfate proteoglycan, inhibit locomotion and repel immigration. The nonmigrant cells along the migration pathway may likewise have inviting or repellent macromolecules on their surface; some may even extend filopodia to make their presence known.

1	Among the many guiding influences, a few stand out as especially important. In particular, many types of migrating cells are guided by chemotaxis that depends on a G-protein-coupled receptor (called CXCR4), which is activated by an extracellular ligand called CXCL12. Cells expressing this receptor can snuffle their way along tracks marked out by CXCL12 (Figure 21–45). Chemotaxis toward sources of CXCL12 plays a major part in guiding the migrations of lymphocytes Figure 21–45 CXCL12 guides migrating germ cells. Zebrafish germ cells migrate to domains that express cXcL12. as the sites of cXcL12 expression change, cells follow the cXcL12 track and are guided to the region where the gonad develops at a later developmental stage. (a) at the 4-somite stage, germ cells move from a position that is close to the midline to more lateral regions where cXcL12 is expressed. (B) as the cXcL12 expression retracts, germ cells are guided to more posterior positions.

1	source of CXCL12 and various other white blood cells; of neurons in the developing brain; of myoblasts entering limb buds; of primordial germ cells as they travel toward the developing gonads; and of cancer cells when they metastasize. Detailed studies of primordial-germ-cell migration have shown that CXCL12 signaling does not induce cell migration per se but rather serves to control its direction. In the absence of CXCL12 signaling, germ cells still display the membrane blebbing associated with cell migration, but the position of the cell front where blebs form is randomly chosen (Figure 21–46); if CXCL12 signaling is intact, blebbing is more frequent on the side of the cell that faces the source of CXCL12, resulting in directional migration. the Distribution of Migrant cells Depends on Survival Factors

1	the Distribution of Migrant cells Depends on Survival Factors The final distribution of migrant cells depends not only on the routes they take, but also on whether they survive the journey and thrive in the environment they find at the journey’s end. Specific sites provide survival factors needed for specific types of migrant cells to survive.

1	Among the most important sets of migrant cells in the vertebrate embryo are those of the neural crest. They arise from the border region between the part of the ectoderm that will form epidermis and the part that will form the central nervous system. As the neural ectoderm rolls up to form the neural tube, the neural crest cells break loose from the epithelial sheet along this border region and set out on their long migrations (see Figure 19–8 and Movie 21.5). They settle ultimately in many sites and give rise to a surprising diversity of cell types. Some lodge in the skin and specialize as pigment cells; still others form skeletal tissue in the face. Still others will differentiate into the neurons and glial cells of the peripheral nervous system—not only in the sensory ganglia that lie close to the spinal cord, but also, following a much longer migration, in the wall of the gut.

1	The neural crest cells that give rise to the pigment cells of the skin and those that develop into the nerve cells of the gut depend on a secreted peptide called endothelin-3, which is produced by tissues along the migration pathways and acts as a survival factor for the migrating crest cells. In mutants with a defect in the gene for endothelin-3 or its receptor, many of these migrating crest cells die. As a result, the mutant individuals have nonpigmented (albino) patches of skin and a deficit of nerve cells in the intestine, especially its lower end, the large bowel, which becomes abnormally distended for lack of proper neural control—a potentially lethal condition called megacolon. Figure 21–46 Directional migration by local blebbing. Germ cells migrate via protrusions that define the leading edge of the cell. the persistence and site of the protrusions are biased toward higher levels of cXcL12. thus, germ cells migrate up the cXcL12 gradient.

1	Figure 21–47 Effect of mutations in the Kit gene. Both the baby and the mouse are heterozygous for a loss-of-function mutation that leaves them with only half the normal quantity of Kit gene product. In both cases, pigmentation is defective because pigment cells depend on the gene product as a receptor for a survival factor. (courtesy of r.a. Fleischman, from r.a. Fleischman et al., Proc. Natl Acad. Sci. USA 88:10885–10889, 1991.)

1	Another important survival signal for many types of migratory cells, including primordial germ cells, blood cell precursors, and neural-crest-derived pigment cells, depends on a receptor tyrosine kinase called Kit. This is expressed on the surface of the migrant cells, and a protein ligand, called Steel factor, is produced by the cells of the tissue through which the cells migrate and/or in which they come to settle. Individuals with mutations in the genes for either of these proteins have deficits in pigmentation, blood cells, and germ cells (Figure 21–47). changing patterns of cell adhesion Molecules Force cells Into New arrangements

1	changing patterns of cell adhesion Molecules Force cells Into New arrangements Patterns of gene expression govern embryonic cell movements in many ways. They regulate cell motility, cell shape, and the production of proteins that guide migration. Importantly, they also determine the sets of adhesion molecules that the cells display on their surface. Through changes in its surface molecules, a cell can break old attachments and make new ones. Cells in one region may develop surface properties that make them cohere with one another and become segregated from a neighboring group of cells with different surface chemistry.

1	Experiments done half a century ago on early amphibian embryos showed that the effects of selective cell–cell adhesion can be so powerful that they can bring about an approximate reconstruction of the normal structure of an early postgastrulation embryo after the cells have been artificially dissociated and mixed up. When these cells are reaggregated into a random mixture, the cells spontaneously sort themselves out according to their original germ-layer origins (Figure 21–48). As discussed in Chapter 19, cadherin proteins have a central role in the sorting process (see Figure 19–9). Cadherins belong to a large and varied family of Ca2+-dependent cell–cell adhesion proteins, and they and other cell–cell adhesion proteins are differentially expressed in the various tissues of the early embryo. Antibodies against these proteins interfere with the normal selective adhesion between cells of a similar type.

1	Changes in the patterns of expression of the various cadherins correlate closely with the changing patterns of association among cells during various developmental processes, including gastrulation, neural tube formation, and somite formation. These cell rearrangements are likely to be regulated and driven in part by

1	Figure 21–48 Sorting out by adhesion. cells from different parts of an early amphibian embryo will sort out according to their origins. In the classical experiment shown here, mesoderm cells (green), neural plate cells (blue), and epidermal cells (red) have been disaggregated and then reaggregated in a random mixture. they sort out into an arrangement reminiscent of a normal embryo, with a “neural tube” internally, epidermis externally, and mesoderm in between. (Modified from p.L. townes and J. holtfreter, J. Exp. Zool. 128:53– 120, 1955. With permission from Wiley-Liss.) the cadherin pattern. In particular, cadherins appear to have a major role in controlling the formation and dissolution of epithelial sheets and clusters of cells (see Movie 19.1). They not only glue one cell to another but also provide anchorage for intracellular actin filaments at the sites of cell–cell adhesion. In this way, the pattern of stresses and movements in the developing tissue is regulated according to

1	but also provide anchorage for intracellular actin filaments at the sites of cell–cell adhesion. In this way, the pattern of stresses and movements in the developing tissue is regulated according to the pattern of cell adhesions.

1	The different types of cadherins enable different types of cells to cohere selectively: cells expressing one type of cadherin will maximize their contact with cells expressing the same cadherin and thereby segregate from other cells, creating specific tissue boundaries. Cell mixing can be inhibited and boundaries created and maintained in another way as well: cells of different types can sometimes actively repel one another. The bidirectional activation of Eph receptors and ephrins discussed in Chapter 15 often mediates such repulsion, acting at interfaces between different groups of cells to keep the groups from mixing, and repelling invasion by inappropriate visitors. Ephrin–Eph signaling operates, for example, at the boundaries of the rhombomeres discussed earlier. Neighboring rhombomeres express complementary combinations of ephrins and Eph receptors, and this keeps the cells in adjacent rhombomeres strictly segregated, with a boundary between them that is sharply defined (Figure

1	express complementary combinations of ephrins and Eph receptors, and this keeps the cells in adjacent rhombomeres strictly segregated, with a boundary between them that is sharply defined (Figure 21–49).

1	Groups of Similar cells can perform Dramatic collective rearrangements Cadherin-mediated cell sorting and ephrin–Eph-mediated repulsion exemplify how differences in cell-surface properties can drive tissue arrangements, causing cells that express different sets of genes to separate from one another. However, groups of cells that are all similar can also undergo dramatic rearrangements. During frog gastrulation, for example, cells in one region of the surface epithelium invaginate and migrate as a sheet into the interior of the embryo and converge toward the embryonic midline. The movement is driven mainly by an active rearrangement of the migrating cells, called convergent extension. Here the cells crawl over one another in a coordinated way, displacing their neighbors as they migrate, causing the cell sheet to narrow along one axis (converge) and elongate along another (extend). Strikingly, small, square fragments of tissue from the

1	Figure 21–49 Sorting out by repulsion. ephrin–eph signaling in hindbrain segmentation in a chick embryo. each pair of rhombomeres (segments in the hindbrain) is associated with a branchial arch (a modified gill rudiment) to which it sends innervation. rhombomeres are distinguished from one another by expression of different Hox genes (see Figure 21–32). Mutual repulsion (red bars) between cells that express ephrinB2 in rhombomere 4 and epha4 in rhombomere 5 creates a sharp boundary. lamellipodia attempt to crawl on surfaces of neighboring cells, pulling them inward in direction of arrows appropriate region of the embryo, isolated in culture, will spontaneously narrow and elongate, just as they would in the embryo (Figure 21–50). The alignment of the cell movements depends on the same signaling pathway that is involved in generating planar cell polarity within developing epithelia, as we discuss next.

1	Cells within an epithelium always have an apical–basal polarity (discussed in Chapter 19), but the cells of many epithelia show an additional polarity at right angles to this axis: the cells are all arranged as if they had an arrow written on them, pointing in a specific direction in the plane of the epithelium. This type of polarity is called planar cell polarity. In the wing of a fly, for example, each epithelial cell has a tiny asymmetrical projection, called a wing hair, on its surface, and the hairs all point toward the tip of the wing. Similarly, in the inner ear of a vertebrate, each mechanosensory hair cell has a precisely oriented asymmetric bundle of actin-filled, rodlike protrusions called stereocilia sticking up from its apical plasma membrane as a detector of sound and of forces such as gravity. Tilting the bundle in one direction causes ion channels in the membrane to open, electrically activating the cell; tilting in the opposite direction has the opposite effect. For

1	such as gravity. Tilting the bundle in one direction causes ion channels in the membrane to open, electrically activating the cell; tilting in the opposite direction has the opposite effect. For the ear to function properly, the hair cells must be oriented correctly. Planar cell polarity is also important in the respiratory tract, where every ciliated cell must orient the beating of its cilia so as to sweep mucus upward, away from the lungs.

1	Screens for mutants with misoriented wing hairs in Drosophila have identified a set of genes that is critical for planar cell polarity. Some of these genes code for Figure 21–50 Convergent extension and its cellular basis. (a) Schematic diagram of cell behaviors that underlie convergent extension. the cells form lamellipodia, with which they attempt to crawl over one another. alignment of the lamellipodial movements along a common axis leads to convergent extension. the process depends on the Wnt–Frizzled/ planar-cell-polarity signaling pathway and is cooperative, presumably because cells that are already aligned exert forces that tend to align their neighbors in the same way.

1	(B–G) the pattern of convergent extension of dorsal mesoderm during zebrafish gastrulation at 8.8 (B, e), 9.3 (c, F), and 11.3 (D, G) hours after fertilization. cells that will give rise to the notochord are labeled in green, and cells that will give rise to somites and muscle are labeled in blue. the notochord and somite domains are spatially separate from the start of the recording (B, e), but their boundaries are at first barely visible and only a little later become obvious. convergence narrows the notochord domain to a width of about two cells at the last time point (D, G). (a, after J. Shih and r. Keller, Development 116:901–914, 1992; B–G, after N.S. Glickman et al., Development 130:873–887, 2003. With permission from the company of Biologists.)

1	Figure 21–51 Planar cell polarity. (a) Wing hairs on the wing of a fly. each cell in the wing epithelium forms a small, spiky protrusion or “hair” at its apex, and all the hairs point the same way, toward the tip of the wing. this reflects a planar polarity in the structure of each cell. (B) Sensory hair cells in the inner ear of a mouse similarly have a well-defined planar polarity, manifest in the oriented pattern of stereocilia (actin-filled protrusions) on their surface. the detection of sound depends on the correct, coordinated orientation of the hair cells. (c) a mutation in the gene Flamingo in the fly, coding for a nonclassical cadherin, disrupts the pattern of planar cell polarity in the wing. (D) a mutation in a homologous Flamingo gene in the mouse randomizes the orientation of the planar cell polarity vector of the hair cells in the ear. the mutant mice are deaf. (a and c, from J. chae et al., Development 126:5421–5429, 1999. With permission from the company of Biologists;

1	the planar cell polarity vector of the hair cells in the ear. the mutant mice are deaf. (a and c, from J. chae et al., Development 126:5421–5429, 1999. With permission from the company of Biologists; B and D, from J.a. curtin et al., Curr. Biol. 13:1129–1133, 2003. With permission from elsevier.) components of the Wnt signaling pathway, others code for specialized members of the cadherin superfamily, while the functions of others are uncertain. These components of planar-cell-polarity signaling are assembled at cell–cell junctions in the epithelium in such a way as to exert a polarizing influence that can propagate from cell to cell. Essentially the same system of proteins controls planar cell polarity in vertebrates; mice deficient in homologs of the Drosophila planar polarity genes have a variety of defects, including incorrectly oriented hair cells in the inner ear, making them deaf (Figure 21–51).

1	Animals require specialized types of epithelial surfaces for many functions, including excretion, absorption of nutrients, and gas exchange. Where large surfaces are required, they are often organized as branching tubular structures. The lung is an example. It originates from epithelial buds that grow out from the floor of the foregut and invade neighboring mesenchyme to form the bronchial tree, a system of tubes that branch repeatedly as they extend. Endothelial cells that form the lining of blood vessels invade the same mesenchyme, thereby creating a system of closely apposed airways and blood vessels, as required for gas exchange in the lung (Figure 21–52). This whole process of branching morphogenesis depends on signals that pass in both directions between the growing epithelial buds and the mesenchyme. Genetic studies in mice indicate that FGF proteins and their receptor tyrosine kinases play a central part in these signaling processes. FGF signaling has various roles in

1	buds and the mesenchyme. Genetic studies in mice indicate that FGF proteins and their receptor tyrosine kinases play a central part in these signaling processes. FGF signaling has various roles in development, but it is especially important in the many interactions that occur between a developing epithelium and mesenchyme. Figure 21–52 The airways of the lung,

1	In the case of lung development, FGF10 is expressed in clusters of mesen-shown in a cast of the adult human bronchial tree. resins of different colors chyme cells that lie near the tips of the growing epithelial tubes, and its receptor is expressed in the invading epithelial cells. In FGF10-deficient mutant mice, a pri of the tree of airways. (From r. Warwick mary bud of lung epithelium is formed but fails to grow out into the mesenchyme and p.L. Williams, Gray’s anatomy, 35th ed. to create a branching bronchial tree. Conversely, a microscopic bead soaked in edinburgh: Longman, 1973.) two new centers of FGF10 made by cluster of mesenchyme cells cells at tip of FGF10 and placed near embryonic lung epithelium in culture will induce a bud to form and grow out from the epithelium toward the bead. Evidently, the epithelium invades the mesenchyme only by invitation, in response to FGF10.

1	But what makes the growing epithelial tubes of the lung branch repeatedly as they invade the mesenchyme? This depends on a Sonic hedgehog signal that is sent in the opposite direction, from the epithelial cells at the tips of the buds back to the mesenchyme, as shown in Figure 21–53. In mice lacking Sonic hedgehog, the lung epithelium grows and differentiates, but it forms a sac instead of a branching tree of tubules.

1	FGF signaling acts in a remarkably similar way in the formation of the air-exchange system of insects, which consists of a pattern of fine, air-filled channels called tracheae and tracheoles. These originate from the epidermis covering the surface of the body and extend inward to invade the underlying tissues, branching and narrowing as they go (Figure 21–54). The FGF acts on cells at the tips of the advancing tracheae, causing them to extend filopodia and migrate toward the source of the FGF signal. Because the tip cells remain connected to the remainder of the tracheal epithelium, the pulling force that they generate elongates the tracheal tube.

1	Initially, the pattern of FGF production in fly embryos is defined by the D-V and A-P patterning systems discussed earlier. In later stages of development, however, FGF expression is induced by transcription regulators called hypoxia-inducible factors (HIFs) that are activated by hypoxia (low oxygen levels). In this way, hypoxia stimulates the formation of finer and finer and more extensively branched trachea, until the oxygen supply is sufficient to stop the process. Hypoxia and HIFs have similar roles in vertebrates, especially in the development of blood vessels, as we shall see in the next chapter.

1	Figure 21–54 Branching morphogenesis of airways in a fly. (a) Drosophila embryonic tracheal system. (B) FGF (produced in Drosophila by the Branchless gene) signals from surrounding cells to the tracheal epithelium and activates its FGF receptors, leading to filopodia formation and tube elongation. [a, from G. Manning and M.a. Krasnow, in the Development of Drosophila (a. Martinez-arias and M. Bate, eds), Vol. 1, pp. 609–685. New York: cold Spring harbor Laboratory press, 1993.] Figure 21–53 Branching morphogenesis of the lung. how FGF10 and Sonic hedgehog are thought to induce the growth and branching of the buds of the bronchial tree. Many other signal molecules, such as BMp4, are also expressed in this system, and the suggested branching mechanism is only one of several possibilities.

1	as indicated, FGF10 protein is expressed in clusters of mesenchyme cells near the tips of the growing epithelial tubes, and its receptor is expressed in the epithelial cells themselves. the Sonic hedgehog signal is sent in the opposite direction, from the epithelial cells at the tips of the buds back to the mesenchyme. the patterns of gene expression and their timing suggest that the Sonic hedgehog signal may serve to shut off FGF10 expression in the mesenchyme cells closest to the growing tip of a bud, splitting the FGF10-secreting cluster into two separate clusters, which in turn cause the bud to branch into two. Figure 21–55 The forms of cell behavior an epithelium can Bend During Development to Form a tube or involved in tube formation. FoldingVesicle generates the neural tube, budding underlies the formation of lungs and

1	The creation of systems of tubes such as blood vessels and airways is a complex trachea, cord hollowing occurs during process, and it can involve various additional forms of cell behavior, as sketched the formation of mammalian salivary in Figure 21–55. glands, cell hollowing is involved in the As explained in Chapter 19, the process that converts an epithelial sheet into a formation of tracheal terminal cell tubes, and cell assembly generates the heart tube tube depends on contraction of specific bundles of actin filaments. With the help that forms at the earliest stage of heart of myosin motor proteins, actin filament bundles can shorten, causing the epithe development.

1	lial cells to narrow at their apex. These actin bundles are connected from cell to cell by adherens junctions, and if their contraction is coordinated along a specific axis, the result will be that the sheet bends and rolls up into a tube (Figure 21–56). The vertebrate neural tube, which we discuss in the last section of this chapter, originates in this way. sheet of epithelial cells INVAGINATION OF EPITHELIAL SHEET CAUSED BY AN ORGANIZED TIGHTENING ALONGadhesion belt with ADHESION BELTS IN SELECTED REGIONS OFassociated actin flaments CELL SHEET

1	sheet of epithelial cells INVAGINATION OF EPITHELIAL SHEET CAUSED BY AN ORGANIZED TIGHTENING ALONGadhesion belt with ADHESION BELTS IN SELECTED REGIONS OFassociated actin flaments CELL SHEET Figure 21–56 Bending of an epithelial sheet to form a tube. contraction of apical bundles of actin filaments linked from cell to cell via adherens junctions causes the epithelial cells to narrow at their apex. Depending on whether the contraction is oriented along one axis of the sheet or is equal in all directions, the epithelium will either roll up into a tube or invaginate to form a vesicle. (a) Diagram showing how an apical contraction along one axis of an epithelial sheet can cause the sheet to form a tube. (B) Scanning electron micrograph of a cross section through the trunk of a two-day chick embryo, showing the formation of the neural tube by the process diagrammed in (a). (B, courtesy of Jean-paul revel.)

1	Animal development involves dramatic cell movements, including the guided migration of individual cells, the adhesion and repulsion of groups of cells, and the complex extension, branching, or rolling up of epithelial tissues. Migrant cells, such as those of the neural crest, break loose from their original neighbors and travel through the embryo to colonize new sites. Many migrant cells, including primordial germ cells, are guided by chemotaxis dependent on the receptor CXCR4 and its ligand CXCL12. In general, cells that have similar adhesion molecules on their surfaces cohere and tend to segregate from other cell groups with different surface properties. Selective cell–cell adhesion is often mediated by cadherins; repulsion is often driven by ephrin–Eph signaling. Within an epithelial sheet, cells can rearrange themselves to drive epithelial convergence and extension, as in gastrulation. Many movements are coordinated through a Wnt-dependent planar-polarity signaling pathway that is

1	cells can rearrange themselves to drive epithelial convergence and extension, as in gastrulation. Many movements are coordinated through a Wnt-dependent planar-polarity signaling pathway that is also responsible for orienting cells correctly in various types of epithelium. Elaborate branched tubular structures, such as the airways of the lung, are generated through bidirectional signaling between an epithelial bud and the mesenchyme that it invades, in a process called branching morphogenesis. Epithelial tubes and vesicles can originate in various ways, most simply by the rolling up and pinching off of a segment of epithelium, as in the formation of the neural tube.

1	One of the most fundamental aspects of animal development is one we know surprisingly little about—how the size of an animal or an organ is determined. Why, for example, do we grow to be so much larger than a mouse? Even within a species, size can vary greatly; a Great Dane, for instance, can weigh over 40 times more than a Chihuahua (Figure 21–57). Three variables define the size of an organ or organism: the number of cells, the size of the cells, and the quantity of extracellular material per cell. Size differences can arise from changes in any of these factors (Figure 21–58). If we compare a mouse with a human, for example, we find that the difference lies chiefly in the number of cells, there being roughly 3000 times more cells in a human, corresponding to a body that is roughly 3000 times more massive. Wild and cultivated species of food plants, on the other hand, often differ in body size chiefly because of differences of cell size.

1	The challenge, therefore, is to understand how cell numbers, cell size, and extracellular matrix production are regulated. First of all, we need to identify the signals that drive or inhibit growth. Then we need to discover how the signals themselves are regulated. In many cases, the size of an organ or of the body as a whole seems to be controlled homeostatically, so that the correct size is reached and maintained even in the face of drastic disturbances. This suggests that the developing structure somehow senses its own size and uses this information to regulate the signals for its own growth or shrinkage. In most cases, the nature of this feedback control remains a profound mystery.

1	In other cases, the duration of growth and the final size seem to be dictated by intracellular programs that take no cognizance of the size the structure has attained. These intracellular programs, too, present many mysteries, as we saw in our discussion of developmental timing. Very often, it seems, the sizes and proportions of body parts must depend on combinations of size-measuring feedback controls and intracellular programs, as well as on environmental influences such as nutrition.

1	The variation in control strategies is nicely illustrated by some classic transplantation experiments. If several fetal thymus glands are transplanted into a developing mouse, each grows to its characteristic adult size. In contrast, if multiple fetal spleens are transplanted, each ends up smaller than normal, but collectively they grow to the size of one adult spleen. Thus, thymus growth is regulated by local mechanisms intrinsic to the individual organ, whereas spleen growth is controlled by a feedback mechanism that senses the quantity of spleen tissue in the body as a whole. In neither case is the mechanism known. Figure 21–57 Members of the same species can have dramatically different sizes. the chihuahua weighs 2–5 kilograms, whereas a Great Dane weighs 45–90 kilograms. (courtesy of Deanne Fitzmaurice.) Figure 21–58 Determinants of organ size. the proliferation, Death, and Size of cells Determine Organism Size

1	The nematode worm C. elegans illustrates the different ways in which size differences can arise. This creature follows an astonishingly precise and predictable developmental program. Each individual of a given sex is generated by almost exactly the same sequences of cell divisions and cell deaths, and consequently has precisely the same number of somatic cells—959 in the adult hermaphrodite (the sex of the majority of these animals)—although the number of germ cells is more variable from worm to worm. The stereotyped development makes it possible to trace somatic cell lineages in exhaustive detail. More than 1000 cell divisions generate 1090 somatic cells during hermaphrodite development, but 131 of these cells undergo apoptotic cell death. Thus, precise regulation of both cell division and cell death determines the final numbers of somatic cells in the worm. In fact, genetic screens in C. elegans identified the first genes responsible for apoptosis and its regulation—thereby

1	and cell death determines the final numbers of somatic cells in the worm. In fact, genetic screens in C. elegans identified the first genes responsible for apoptosis and its regulation—thereby revolutionizing our molecular understanding of this form of programmed cell death (discussed in Chapter 18).

1	The final number of somatic cells in the adult worm is already present at sexual maturity (around three days after fertilization), after which no more somatic cells are generated. Yet the worm continues to grow, doubling in size between sexual maturity and death 2–3 weeks later. This doubling results from somatic cell growth: although the cells no longer divide, they continue to go through rounds of DNA synthesis; this endoreplication of the genome makes the cells polyploid. As in all organisms, the size of a cell is proportional to its ploidy—that is, the number of genome copies that it contains: a doubling of ploidy roughly doubles cell volume. By artificial manipulation of somatic cell ploidy, and thereby somatic cell size, the size of the worm as a whole can be increased or decreased. Thus the worm’s final size is set by a combination of programmed cell divisions and cell deaths, along with regulation of the sizes of individual cells through changes in ploidy.

1	In plants, as in animals, cell size increases as ploidy increases (Figure 21–59). This effect has been exploited in the agricultural breeding of plants for large size: most of the major fruits and vegetables that we consume are polyploid.

1	The size of an animal or organ depends on both cell number and cell size—that is, on total cell mass. Remarkably, many animals and organs can somehow assess their total cell mass and regulate it, providing evidence for feedback controls of the sort highlighted earlier in our introductory account of general principles of growth control. In contrast with C. elegans, if cell size is artificially increased or decreased in these cases, cell numbers adjust to maintain a normal total cell mass. This has been beautifully illustrated by experiments done long ago in salamanders, where cell size can be manipulated by altering the animal’s ploidy. As shown in Figure 21–59E, salamanders of different ploidies end up being the same size with very different numbers of cells. The individual cells in a pentaploid salamander,

1	Figure 21–59 Effects of ploidy on cell size and organ size. In all organisms, from bacteria to humans, cell size is proportional to ploidy—the number of copies of the genome per cell. this is illustrated for (a–D) Arabidopsis flowers and (e) for salamanders. In each case, the upper panels show cells in a specific tissue [a petal for Arabidopsis, a pronephric (kidney) tubule for the salamander]; the lower panels show the gross anatomy—flowers for Arabidopsis, the whole body for the salamander. In the case of Arabidopsis flowers, increase in cell size increases organ size. By contrast, the salamander and its individual organs attain their normal standard size regardless of ploidy, because large cell size is compensated for by fewer cells. this indicates that the size of an organism or organ in this species is not controlled simply by counting cell divisions or cell numbers; size must somehow be regulated at the level of total cell mass. [a–D, from c. Breuer et al., Plant Cell

1	or organ in this species is not controlled simply by counting cell divisions or cell numbers; size must somehow be regulated at the level of total cell mass. [a–D, from c. Breuer et al., Plant Cell 19:3655–3668, 2007. With permission from the american Society of plant Biologists; e, adapted from G. Fankhauser, in analysis of Development (B.h. Willier, p.a. Weiss and V. hamburger, eds), pp. 126–150. philadelphia: Saunders, 1955.] for example, are about five times the size of those in a haploid salamander, but there are only one-fifth as many cells. This scaling operates not only in the body as a whole, but in its individual organs.

1	The imaginal discs of Drosophila provide another striking example of homeostatic size control. These are epithelial pouches that grow by cell proliferation during the larval period and, during the pupal stage, form the organs and extremities of the adult fly (Figure 21–60). Experiments have been chiefly done on the wing imaginal disc. Mutations in components of the cell-cycle control machinery can be used to speed up or slow down the rate of cell division in the disc. Remarkably, such mutations can result in an excessive number of abnormally small cells Figure 21–60 The imaginal discs in the Drosophila larva (below) and the structures in the adult (above) that they give rise to. [after J.W. Fristrom et al., in problems in Biology: rNa in Development (e.W. hanley, ed.), p. 382. Salt Lake city: University of Utah press, 1969.]

1	Figure 21–61 Pituitary dwarf and pituitary giant. the “giant” on the right is robert Ladlow (1914–1940), the tallest recorded man at 8 feet 11 inches (2.72 m), together with his father, who was almost 6 feet tall (1.82 m). the dwarf on the left is General tom thumb, which was the stage name of charles Sherwood Stratton (1838–1883). On his 18th birthday, he was measured at 2 feet 8.5 inches (82.6 cm) tall, and at his death, he was 3 feet 4 inches (102 cm).(Images from http://en.wikipedia.org/wiki/ File:robert_Wadlow.jpg. © Bettmann/cOrBIS.) or a reduced number of abnormally large cells, respectively, leaving the size (area) and patterning of the adult wing practically unchanged. Thus, the size of the disc is not regulated so as to contain a set number of cells. Instead, there must be a regulatory mechanism that halts growth when the disc’s total cell mass reaches the appropriate value, so that the size and pattern of the adult wing that develops from the disc are normal. Remarkably,

1	mechanism that halts growth when the disc’s total cell mass reaches the appropriate value, so that the size and pattern of the adult wing that develops from the disc are normal. Remarkably, developing discs—or even disc fragments, taken out of their normal context and transplanted into the abdomen of an adult female—will grow until they reach their normal size. Clearly, the mechanisms that regulate disc size are intrinsic to the disc.

1	We still have very little idea how organisms or organs assess their total cell mass or monitor their own growth. Nevertheless, we are beginning to understand 1 meter some of the signal molecules that drive or halt growth in response to the mysterious cues that convey information about the size attained.

1	We have already seen how some signals act systemically as hormones to regulate the development of the animal as a whole. Some of these serve to regulate growth. In mammals, for instance, growth hormone (GH) is secreted by the pituitary gland into the bloodstream and stimulates growth throughout the body: excessive production of growth hormone leads to gigantism, and too little leads to dwarfism (Figure 21–61). Pituitary dwarfs have bodies and organs that are proportionately small, unlike achondroplastic dwarfs, for example, whose limbs are disproportionately short, usually because of a mutation in a gene encoding an FGF receptor that disrupts normal cartilage development (Figure 21–62).

1	Growth hormone stimulates growth largely by inducing the liver and other organs to produce insulin-like growth factor 1 (IGF1), which acts mainly as a local signal within many tissues to increase cell survival, cell growth, cell proliferation, or some combination of these, depending on the cell type. Large breeds of dogs such as Great Danes owe their great size to high levels of IGF1, while miniature breeds such as Chihuahuas have low levels (see Figure 21–57).

1	Not all growth-regulating extracellular signals stimulate growth; some inhibit it, by promoting cell death or inhibiting cell growth, cell division, or both. Myostatin is a TGFβ family member that specifically inhibits the growth and proliferation of myoblasts—the precursor cells that fuse to form the huge, multinucleated cells of skeletal muscle. When the Myostatin gene is deleted in mice, muscles grow to be several times larger than normal. Remarkably, two breeds of cattle that were bred for large muscles have both turned out to have mutations in the Myostatin gene; whippet dogs mutant for Myostatin develop similarly (Figure 21–63).

1	Figure 21–62 Achondroplasia. this type of dwarfism occurs in one of 10,000–100,000 births; in more than 99% of cases it results from a mutation at an identical site in the genome, corresponding to amino acid 380 in the FGF receptor FGFr3 (a glycine in the transmembrane domain). the mutation is dominant, and almost all cases are due to new, independently occurring mutations, implying an extraordinarily high mutation rate at this particular site in the genome. the defect in FGF signaling causes dwarfism by interfering with the growth of cartilage in developing long bones. (From Velasquez’s painting of Sebastian de Morra. © Museo del prado, Madrid.) Figure 21–63 Myostatin limits muscle growth. a wild-type whippet dog and a bully whippet that lacks myostatin. (a, from http://www.merlinanimalrescue.co.uk/dogs/?m=201211; B, from http://animalslook.com/schwarzenegger-dog/.)

1	Like TGFβ itself, myostatin acts through the Smad intracellular signaling pathway (see Figure 15–57) to inhibit muscle growth specifically. Another intracellular signaling pathway, called the Hippo pathway, inhibits organ and organism growth more generally. It was discovered in Drosophila, but it operates in vertebrates as well. It inhibits growth both by promoting cell death (by blocking an apoptosis inhibitor) and by inhibiting cell-cycle progression (by inhibiting the expression of the cell-cycle gene Cyclin E). Some components of the pathway in Drosophila are shown in Figure 21–64. The organs of animals that are abnormally resistant to Hippo repression can grow to a monstrous size (Figure 21–65).

1	It is important to note that in all species nutritional conditions also play a fundamental part in regulating the pace and extent of growth, and in animals they do so through hormonal signal networks that are highly conserved between vertebrates and invertebrates. Although we do not have space for details here, genetic experiments, especially in Drosophila, have begun to unravel the logic of these controls, and to indicate how they may operate alongside other machinery, such as the Hippo pathway, to determine final size. The sizes of animals and their organs vary widely and largely depend on total cell mass. This in turn depends on the size and number of cells, which are increased through cell growth and cell division, respectively. Cell numbers are reduced by programmed cell death. Each of these processes depends on both intracellular and

1	Figure 21–65 Overcoming Hippo repression increases organ size. (a) Livers from control and Yap-overexpressing mice. In these mice, hippo signaling is insufficient to block Yap. (B) adult heads from control and Yap-overexpressing flies. In the mutant flies, hippo signaling is unable to block Yap. (From J. Dong et al., Cell 130:1120–1133, 2007.With permission from elsevier.) Figure 21–64 Hippo pathway. hippo, a protein kinase, limits growth by phosphorylation and activation of the kinase Warts, which in turn phosphorylates and inactivates the transcriptional coactivator Yorkie (called Yap in vertebrates). When unphosphorylated, Yorkie/Yap drives tissue growth: it activates the transcription of the growth-promoting gene Myc, the cell-cycle progression gene Cyclin E, the antiapoptotic gene Diap, and the microrNa Bantam. hippo-induced phosphorylation of Yorkie/Yap blocks this effect.

1	extracellular signals. The mystery is how these processes are regulated and coordinated to produce and maintain the characteristic final size of the adult organ or animal. Some signals such as survival factors, growth factors, and mitogens stimulate growth by promoting cell survival, cell growth, and cell division, respectively, while other signal molecules do the opposite. Although most of these signals operate locally to help sculpt the size and shape of the animal, its organs, and appendages, others act as hormones to regulate the growth of the animal as a whole. Nutrients can regulate growth through hormonal signals in the entire body. Many animals and organs can, by unknown mechanisms, assess their total cell mass and regulate it. If, for example, cell size is artificially increased or decreased in these cases, cell numbers adjust to maintain a normal total cell mass. Conversely, if cell numbers are artificially increased or decreased, cell size adjusts to compensate.

1	The development of the nervous system poses problems that have little parallel in other tissues. A typical nerve cell, or neuron, has a structure unlike that of any other class of cells, with a long axon and branching dendrites, both of which make many synaptic connections to other cells (Figure 21–66). The central challenge of neural development is to explain how the axons and dendrites grow out, find their right partners, and synapse with them selectively to create a neural network—an electrical signaling system—that functions correctly to guide behavior (Figure 21–67). The problem is formidable: the human brain contains more than 1011 neurons, each of which, on average, has to make connections with a thousand others, according to a regular and predictable wiring plan. The precision required is not so great as in a man-made computer, because the brain performs its computations in a different way and is more tolerant of vagaries in individual components. But the human brain

1	required is not so great as in a man-made computer, because the brain performs its computations in a different way and is more tolerant of vagaries in individual components. But the human brain nevertheless outstrips all other biological structures in its organized complexity.

1	The components of a typical nervous system—the various classes of neurons, glial cells, sensory cells, and muscles—originate in a number of widely separate locations in the embryo. Thus, in the first phase of neural development, the different parts of the nervous system develop according to their own local programs: neurons are born and assigned specific characters according to the place and time of their birth, under the control of inductive signals and transcription regulators, by mechanisms of the types we have already discussed. In the next phase, newborn neurons extend axons and dendrites along specific routes toward their target cells, guided by extracellular signals that attract or repel them. In the third phase, neurons form synapses with other neurons or muscle cells, setting up a provisional but orderly network of connections. In the final phase, which continues into adult life, the synaptic connections are adjusted and refined through mechanisms that usually depend on

1	up a provisional but orderly network of connections. In the final phase, which continues into adult life, the synaptic connections are adjusted and refined through mechanisms that usually depend on synaptic signaling between the cells involved terminal branches of axon make 25 µm synapses on target cells

1	Figure 21–66 A typical neuron of a vertebrate. the arrows indicate the direction in which signals are conveyed. the neuron shown is a basket cell, a type of neuron in the cerebellum. (adapted from S. ramón y cajal, histologie du Système Nerveux de l’homme et des Vertébrés, 1909–1911. paris: Maloine; reprinted, Madrid: c.S.I.c., 1972.) Figure 21–67 The complex organization of nerve cell connections. this drawing depicts a section through a small part of a mammalian brain—the olfactory bulb of a dog—stained by the Golgi technique. the black objects are neurons; the thin lines are axons and dendrites, through which the various sets of neurons are interconnected according to precise rules. (From c. Golgi, Riv. sper. freniat. Reggio-Emilia 1:405–425, 1875.) (Figure 21–68). At all stages, neurons are in intimate contact with various types of non-neuronal supporting cells—the glial cells. Neurons are assigned Different characters according to the time and place of their Birth

1	Neurons are assigned Different characters according to the time and place of their Birth We start our account here with the first phase of neural development: the generation of neural progenitors and their differentiation into hundreds of different neuronal subtypes, along with a much smaller number of glial types. Although the nervous system is exceptional in the extent of cell diversity, the process depends on the same principles that generate different cell types in other organs. We have already discussed some of the underlying machinery in the developing Drosophila nervous system. We turn now to vertebrates. The vertebrate spinal cord, the brain, and the retina of the eye together constitute the central nervous system (CNS). They all originate as parts of the neural tube, whose formation was described earlier (see Figure 21–56). The brain and eyes develop from the anterior neural tube and the spinal cord from the posterior.

1	The developmental anatomy is seen at its simplest in the spinal cord. As it develops, the epithelium forming the walls of the posterior neural tube becomes enormously thickened as the cells proliferate and differentiate, creating a highly organized structure of neurons and glial cells, surrounding a small central channel. Bands of neurons with different future functions—and expressing different genes—are laid out along the dorsoventral axis of the tube. Motor neurons (those that control the muscles) are located ventrally, whereas neurons that process sensory information are found dorsally. This pattern is established by opposing gradients of morphogens. These are secreted by specialized groups of cells that run the length of the ventral and dorsal midlines of the neural tube (Figure 21–69). The two morphogen gradients—consisting of Sonic hedgehog protein from the ventral source and BMP and Wnt from the dorsal source—help induce different groups of proliferating neural progenitor cells

1	The two morphogen gradients—consisting of Sonic hedgehog protein from the ventral source and BMP and Wnt from the dorsal source—help induce different groups of proliferating neural progenitor cells and differentiating neurons to express different combinations of transcription regulators. These regulators in turn drive the production of different combinations of neurotransmitters, receptors, cell–cell adhesion proteins, and other molecules, creating terminally differentiated neurons that will form synaptic connections selectively with the right partners and exchange appropriate signals with them.

1	genesis of neurons outgrowth of axons and dendrites synapse formation Figure 21–68 The four phases of neural development. refnement of synaptic connections 1200 Chapter 21: Development of Multicellular Organisms lumen of groups ofneural tube differentiatingfoor plate and notochord (ventricle) neurons Extracellular morphogen gradients, however, are not the only way to generate cell diversity. As we saw earlier in our discussion of Drosophila neuroblasts (see Figure 21–36), different cell types can also be generated by temporal patterning, in which an intracellular program changes the character of a progenitor cell over time, giving rise to different cell types as development progresses. This mechanism also seems to operate in vertebrate neurogenesis. The most striking illustration comes from study of another part of the CNS—the mammalian cerebral cortex.

1	Although the cerebral cortex is the most complex structure in the human body, it has a simple beginning—from the anterior neural tube. As in the spinal cord, the cells that form the walls of the tube proliferate, and the neuroepithelium thickens and expands as they divide. On a predictable schedule, the divisions of the neuroepithelial cells begin to produce a succession of cells committed to terminal differentiation as neurons. These future neurons are born close to the lumen (the central cavity) of the tube. From here, they migrate outward, losing attachment to the lumenal surface and crawling outward along neighboring cells that continue to span the full thickness of the neuroepithelium. These latter neuroepithelial cells do double duty, functioning as progenitors of neurons and glia, and as supporters of the epithelial architecture. They become stretched out as radial glial cells, forming a scaffold that continues to span the neuroepithelium even as this grows to an enormous

1	and as supporters of the epithelial architecture. They become stretched out as radial glial cells, forming a scaffold that continues to span the neuroepithelium even as this grows to an enormous thickness (Figure 21–70). At the same time, the radial glial cells continue to divide as neural precursors, giving rise to both neurons and glial cells—new radial glial cells as well as glial cells of other types. The newborn neurons, migrating along the radial glial cells, find their appropriate resting places in the developing cortex, where they mature, and from these sites they send out their axons and dendrites. The first-born neurons settle closest to their birthplace near the lumen, while neurons born later crawl past them to settle farther out (Figure 21–71). The successive generations of neurons thus build up as a series of cortical layers, ordered by birthdate and endowed with different intrinsic characters.

1	Strikingly, single cortical progenitor cells isolated in culture generate distinct types of cortical neurons and glial cells, with the timing and characteristics appropriate to specific cortical layers. These observations suggest that the neural progenitors in the developing mammalian cortex, much like the Drosophila neuroblasts, step through an intracellular developmental program that generates the ordered succession of different nerve cell types. Figure 21–69 A schematic cross section of the spinal cord of a chick embryo, showing how cells at different levels along the dorsoventral axis acquire different characters. (a) Signals that direct the dorsoventral pattern. Sonic hedgehog protein from the notochord and the floor plate (the ventral midline of the neural tube) and BMp and Wnt proteins from the roof plate (the dorsal midline) act as morphogens to control gene expression.

1	(B) the resulting patterns of cell fates in the developing spinal cord. Different groups of proliferating neural progenitor cells (in the ventricular zone, close to the lumen of the neural tube) and of differentiating neurons (in the mantle zone, further out) express different combinations of transcription regulators. Neurons expressing different transcription regulators will form connections with different partners and may make different combinations of neurotransmitters and receptors. colors represent different cell types and combinations of regulatory proteins. outer surface of cell body of radial glial cell inner surface of developing neural tube 10 µm the Growth cone pilots axons along Specific routes toward their targets

1	According to the character assigned to it during its early development, a neuron will proceed to make connections with specific partners. This phase of neural development involves a type of morphogenesis unique to the nervous system, in which axons and dendrites extend along specific routes toward their target cells. A typical neuron sends out one long axon and many dendrites, which are usually shorter. The axon projects to distant target cells to which the neuron will eventually send signals. The dendrites will receive incoming signals from axon terminals of other neurons. Axons and dendrites extend by growth at their tip, where one sees an irregular, spiky enlargement called a growth cone (Figure 21–72 and Movie 21.6). The growth cone is both the engine that produces the crawling movement and the steering apparatus that directs the tip along the proper path. Cytoskeletal machinery in the growth cone creates active protrusions, in the form of filopodia and lamellipodia (see Chapter

1	and the steering apparatus that directs the tip along the proper path. Cytoskeletal machinery in the growth cone creates active protrusions, in the form of filopodia and lamellipodia (see Chapter 16 for details): when such a protrusion layers of cortical neurons

1	Figure 21–70 Migration of immature neurons. Before sending out axons and dendrites, newborn neurons often migrate from their birthplace and settle in another location. the diagrams are based on reconstructions from sections of the cerebral cortex (part of the neural tube) of a monkey and rely on a staining technique that picks out at random a small subset of the whole dense mass of neuroepithelial cells. the neurons go through their final cell division close to the inner, lumenal face of the neural tube (in the ventricular proliferative zone) and then migrate outward by crawling along radial glial cells that form a scaffold. each of these latter cells extends from the inner to the outer surface of the tube, a distance that may be as long as 2 cm in the cerebral cortex of the developing brain of a primate. the radial glial cells can be considered as persisting cells of the original columnar epithelium of the neural tube that become extraordinarily stretched as the wall of the tube

1	brain of a primate. the radial glial cells can be considered as persisting cells of the original columnar epithelium of the neural tube that become extraordinarily stretched as the wall of the tube thickens. they also serve as neural stem cells: depending on stage and region, the newborn neurons can be generated from radial glial cells that undergo mitosis while their nuclei are close to the inner surface of the tube, or they can be generated from a nearby class of specialized progenitors in the ventricular proliferative zone. (after p. rakic, J. Comp. Neurol. 145:61–84, 1972. With permission from John Wiley & Sons, Inc.)

1	Figure 21–71 Programmed production of different types of neurons at different times from dividing progenitors in the cerebral cortex of the brain of a mammal. close to one face of the cortical neuroepithelium, progenitor cells divide, in stem-cell fashion, to produce successive generations of neurons (colored here blue, green, red, orange, and black). the neurons migrate out toward the opposite face of the epithelium by crawling along the surfaces of radial glial cells, as shown in Figure 21–70. the first-born neurons settle closest to their birthplace, while neurons born later crawl past them to settle farther out. Successive generations of neurons thus occupy different layers in the cortex and have different intrinsic characters according to their birth dates.

1	contacts an unfavorable surface, it withdraws; when it contacts a more favorable surface, it persists longer, steering the growth cone in that direction. In this way, the growth cone is guided by subtle variations in the properties of the surfaces over which it moves. At the same time, it is sensitive to specific signaling molecules, which—as we discuss next—can either encourage or hinder its advance. a Variety of extracellular cues Guide axons to their targets

1	Growth cones generally travel toward their targets along predictable routes, according to programs stored in the memory of the particular neuron to which they belong (Movie 21.7). In the simplest case, a growth cone can take a route that has been pioneered by other neurites, which they follow by contact guidance. As a result, nerve fibers in a mature animal are usually found grouped together in tight parallel bundles (called fascicles or fiber tracts). Such crawling of growth cones along axons is partly mediated by homophilic cell–cell adhesion molecules— membrane glycoproteins that help a cell displaying them to stick to any other cell that displays the same molecules. As discussed in Chapter 19, many homophilic adhesion molecules fall into one of two main classes: they are members of either the immunoglobulin superfamily, such as N-CAM, or the Ca2+-dependent cadherin family, such as N-cadherin. Members of both families are generally present on the surfaces of growth cones, of axons,

1	the immunoglobulin superfamily, such as N-CAM, or the Ca2+-dependent cadherin family, such as N-cadherin. Members of both families are generally present on the surfaces of growth cones, of axons, and of various other cell types that growth cones crawl over, including glial cells in the central nervous system and muscle cells in the periphery of the body. Growth cones also migrate over components of the extracellular matrix. When tested with neurons growing in a culture dish, some of the matrix molecules, such as laminin, favor axon outgrowth, while others, such as chondroitin sulfate proteoglycans, discourage it. But exactly how the matrix functions to guide axons in intact animals remains to be discovered.

1	Growth cones are generally guided by a succession of different cues at different stages of their journey, as summarized in Figure 21–73. Many of these cues involve specific signaling molecules. Some of these are encountered in the extracellular matrix, while others are attached to the plasma membrane of cells that the growth cones touch. Another important part is played by chemotactic factors; these are proteins secreted from cells that act as beacons at strategic points along the path—some attracting, others repelling. The trajectory of commissural axons— axons that cross from one side of the body to the other—provides a well-studied example.

1	Commissural axons are a general feature of bilaterally symmetrical animals, such as us, because they are required to coordinate behavior of the two sides of the body. In the developing spinal cord of a vertebrate, for example, a large number of neurons send their axonal growth cones ventrally toward the floor plate (the same structure that we encountered earlier as a source of the morphogen Sonic hedgehog—see Figure 21–69). The growth cones cross the floor plate and then turn abruptly through a right angle to follow a longitudinal path up toward the brain, parallel to the floor plate but never again crossing it (Figure 21–74). The first stage of the journey depends on a concentration gradient of the signal protein Netrin, secreted by the cells of the floor plate: the commissural growth cones sniff their way toward its source.

1	Figure 21–72 Internal architecture of a neuronal growth cone, as seen in culture on a flat substratum. the growth cone forms as an expansion of the tip of the growing axon. (a) Image by interference-contrast microscopy. (B) Immunostaining to show microtubules (green). (c) Immunostaining to show actin filaments (red). (D) Diagram of the cytoskeletal machinery. Filopodia form and push forward by assembly of actin filaments at the leading edge of the growth cone. Microtubules stabilize the directional decisions made by the actin-rich protrusions. Filopodia adhering to the flat substratum contract and pull the growth cone forward. (Images by chi-hung Lin, paul Forscher Laboratory, Yale University, New haven, ct.)

1	If commissural growth cones are attracted to the floor plate, why do they cross it and emerge on the other side, instead of staying in the attractive territory? And having crossed it, why do they never cross back again? The answers lie in a change in the responsiveness of the growth cones during their journey. As the growth cones cross the midline, they lose sensitivity to Netrin and become sensitive instead to a signal protein called Slit (see Figure 21–74). Slit is also produced by the floor plate, but it has the opposite effect to that of Netrin: it repels the growth Figure 21–74 The guidance of commissural axons. (a) the pathway taken by commissural axons in the embryonic spinal cord of a vertebrate.

1	Figure 21–74 The guidance of commissural axons. (a) the pathway taken by commissural axons in the embryonic spinal cord of a vertebrate. (B) attraction to the midline. the growth cone is first attracted to the floor plate by Netrin, which is secreted by the floor-plate cells and acts on the receptor Dcc in the axonal membrane. (c) repulsion from the midline after crossing it. as the growth cone crosses the floor plate, Slit comes into play: it binds to its receptors robo1 and robo2 and acts as a repellent to keep the growth cone from re-entering the floor plate. In addition, it blocks responsiveness to the attractant Netrin. Before crossing the midline, the commissural neurons express robo3.1, an alternative splice form of robo3 that is related to robo proteins but blocks Slit signaling. as neurites cross the midline, robo3.1 is lost and growth cones become responsive to Slit and are repelled from the midline.

1	cones, preventing them from re-entering the midline territory. The responses of the growth cone depend on the receptors that it expresses: as commissural neurons approach the floor plate, the Slit receptors are kept inactive by an inhibitory protein (Robo3.1) in the same membrane, allowing the commissural axons to grow to the midline without being repelled. Robo3.1 is lost as the growth cones cross the midline; now the growth cones become sensitive to repulsion by Slit and are thereby prevented from crossing back to the other side. At the same time, signals from the Slit receptors interfere with those from the Netrin receptors, making the growth cones deaf to the signal that attracted them to the floor plate initially. A similar mechanism, using similar proteins, seems to govern midline crossing of commissural axons in other animals, including flies and worms.

1	The guidance of commissural axons illustrates how axons rarely navigate directly to their targets. Instead, they use intermediate targets, or guideposts, and switch their sensitivities as they move from one local guidepost to the next, steering their way through a complex environment to a far-away destination. the Formation of Orderly Neural Maps Depends on Neuronal Specificity In many cases, neurons of a similar type are laid out in a broad array of different positions, but send out axons that come together for their journey and arrive at the target region in a tight bundle. There the axons disperse again, to terminate at different sites in the target territory. This they do in an orderly way, creating a regular mapping from one territory to another—a neural map.

1	The axon projection from the eye to the brain provides an important example. The neurons in the retina that convey visual information back to the brain are called retinal ganglion cells (RGCs). There are more than a million of them in humans, each one reporting on a different part of the visual field. Their axons converge on the optic nerve head at the back of the eye and travel together along the developing optic nerve toward the brain. Their main site of termination, in most vertebrates other than mammals, is the optic tectum—a broad expanse of cells in the midbrain. In connecting with tectal neurons, the RGC axons distribute themselves in a predictable pattern according to the arrangement of their cell bodies in the retina: RGCs that are neighbors in the retina connect with target cells that are neighbors in the tectum. The orderly projection creates a retinotopic map of visual space on the tectum (Figure 21–75).

1	Orderly maps of this sort are found in many brain regions. In the auditory system, for example, the neurons that project from the ear to the brain form a tonotopic map in which brain cells receiving information about sounds of different pitch are ordered along a line, like the keys of a piano. And in the somatosensory Figure 21–75 The neural map from eye to brain in a young zebrafish.

1	Figure 21–75 The neural map from eye to brain in a young zebrafish. (a) Diagrammatic view, looking down on the top of the head. (B) Fluorescence micrograph. Fluorescent tracer dyes have been injected into each eye—red into the anterior part, green into the posterior part. the tracer molecules have been taken up by the neurons in the retina and carried along their axons, revealing the paths they take to the optic tectum in the brain and the map that they form there. (courtesy of chi-Bin chien, from D.h. Sanes, t.a. reh and W.a. harris, Development of the Nervous System. San Diego, ca: academic press, 2000.) genitalia leg toesfoot intra-abdominalpharynx tongue teeth, gums, and jawlower lip lips upper lip facenoseeyethumbindexmiddleringlittlehandwristforearmelbowarmshoulderheadnecktrunkhip

1	Figure 21–76 A map of the body surface in the human brain. the surface of the body is mapped onto the somatosensory region of the cerebral cortex by using an orderly system of nerve cell connections to pair body sites with the brain sites that receive their sensory information. this means that the map in the brain is largely faithful to the topology of the body surface, even though different body regions are represented at different magnifications according to their density of innervation. the homunculus (the “little man” in the brain) has big lips, for example, because the lips are a particularly large and important source of sensory information. the map was determined by stimulating different points in the cortex of conscious patients during brain surgery and recording what they said they felt. (after W. penfield and t. rasmussen, the cerebral cortex of Man. New York: Macmillan, 1950.) system, neurons conveying information about touch map onto the cerebral cortex so as to mark out a

1	felt. (after W. penfield and t. rasmussen, the cerebral cortex of Man. New York: Macmillan, 1950.) system, neurons conveying information about touch map onto the cerebral cortex so as to mark out a “homunculus”—a small, distorted, two-dimensional image of the body surface (Figure 21–76).

1	The retinotopic map of visual space in the optic tectum is the best characterized of all these maps. How does it arise? A famous experiment in the 1940s on frogs provided an important clue. If the optic nerve of a frog is cut, it will regenerate. The retinal axons grow back to the optic tectum, restoring normal vision. If, however, the eye is in addition rotated in its socket at the time of cutting of the nerve, so as to put originally ventral retinal cells in the position of dorsal retinal cells, vision is still restored, but with an awkward flaw: the animal behaves as though it sees the world upside down and left–right inverted (Figure 21–77). If food is dangled in front of it, for example, it will lunge perversely backward. This is because the misplaced retinal cells make the connections appropriate to their original, not their actual, positions. It seems that the retinal ganglion cells (RGCs) have positional values—position-specific biochemical properties representing records of

1	appropriate to their original, not their actual, positions. It seems that the retinal ganglion cells (RGCs) have positional values—position-specific biochemical properties representing records of their original location in the retina, assigned perhaps by earlier morphogen gradients, and making RGCs on opposite sides of the retina intrinsically different.

1	Such nonequivalence among neurons is referred to as neuronal specificity. It is this intrinsic characteristic that guides the retinal axons to their appropriate target sites in the tectum. Those target sites themselves are distinguishable by the Figure 21–77 Neurons in different regions of the retina project axons to different regions in the tectum. Neurons (rGcs) in the anterior retina project axons to the posterior tectum (as shown in Figure 21–75 for zebrafish). regeneration experiments show that retinal neurons have an intrinsic preference for the part of the tectum they normally connect to. If the eye is surgically rotated when the optic nerve is cut, the regenerating retinal axons connect to their original targets, creating an inverted map. (after e. Kandel et al., principles of Neural Science, 5th ed., New York: McGraw hill Medical, 2012.)

1	Figure 21–78 Selectivity of retinal axons growing over tectal membranes. (a) Diagram of an experiment performed with cells from a chick embryo. the culture substratum is coated with alternating stripes of membrane prepared either from posterior tectum or from anterior tectum. axons from posterior retina grow on anterior tectal membrane but are repelled by posterior tectal membrane. axons from anterior retina show different (less selective) behavior. (B) photograph of results. the retinal axons, growing out from the left, are made visible by staining them with a fluorescent marker. the selective pattern of outgrowth shows that anterior tectum differs from posterior tectum, and anterior retina correspondingly differs from posterior retina. In the intact organism, this serves to orient a retinotopic map; the map is refined by subsequent competitive interactions among the anterior and posterior retinal axons, which push the anterior retinal cells off anterior tectal territory. (From J.

1	map; the map is refined by subsequent competitive interactions among the anterior and posterior retinal axons, which push the anterior retinal cells off anterior tectal territory. (From J. Walter et al., Development 101:685–696, 1987.With permission from the company of Biologists.) retinal axons because the tectal cells also carry positional labels. Thus, the neural map depends on a correspondence between two systems of positional markers, one in the retina and the other in the tectum.

1	How are these markers used to make the map? When posterior axons are allowed to grow out over a carpet of anterior or posterior tectal membranes in a culture dish, they show selectivity. Posterior axons strongly prefer the anterior tectal membranes, as in vivo, whereas anterior axons show no preference or prefer posterior tectal membranes (Figure 21–78). The key difference between anterior and posterior tectum is not an attractive factor on the anterior tectum but a repulsive factor on the posterior tectum, to which posterior retinal axons are sensitive but anterior retinal axons are not. If a posterior retinal growth cone touches posterior tectal membrane, it collapses its filopodia and withdraws.

1	In this system, as in others that we have mentioned, the repulsive interactions are mediated by ephrin–Eph signaling—specifically, EphrinA–EphA signaling for the anteroposterior axis (Figure 21–79). An analogous mechanism based on EphB–EphrinB signaling orients the dorsoventral axis of the retinotopic map. These mechanisms serve to orient the map along both axes, but they are not enough by themselves to ensure accurate point-to-point detail. This is brought about through a long process of adjustment that fills in and refines the map through interactions among the RGC axon terminals as they compete for territory on the tectum. This refinement of the pattern of connections involves electrical signaling in the system of developing synapses—a topic that we return to shortly. Both Dendrites and axonal Branches From the Same Neuron avoid One another

1	Both Dendrites and axonal Branches From the Same Neuron avoid One another Axons and dendrites from different neurons can repel one another, or they can cohere; they can collaborate to form synapses, or they can compete. Remarkably, Figure 21–79 Ephrin signaling orients the retinotopic map. (a) Neurons in the posterior retina express epha. as their axons reach the tectum, they are repelled by high levels of ephrina protein in the posterior tectum and project preferentially to the anterior tectum. (B) In epha-mutant mice, posterior retinal axons feel no such repulsion and project more widely within the tectum. (after e. Kandel et al., principles of Neural Science, 5th ed., New York: McGraw hill Medical, 2012.) axons or dendrites can also repel each other when they arise from a single neuron. Such self-avoidance prevents the neuron from making purposeless synapses with itself; it also helps the cell spread out its processes widely so as to innervate a broad territory.

1	Self-avoidance poses a problem. If the same self-recognition molecule were used in every neuron, all neurons in the brain would repel each other. Some classes of neurons do show this sort of mutual repulsion, creating solitary territories—a phenomenon called tiling; but in most cases, axons and dendrites from different neurons can overlap with one another. How then can the processes put out by a single neuron distinguish between self and non-self? This conundrum has been partially resolved by the discovery of a remarkable set of proteins that endow each neuron with a label unlike that of its neighbors. These are the DSCAM proteins in Drosophila and the protocadherins in vertebrates. As described in Chapter 7, DSCAM proteins are extraordinary for the number of isoforms that can be generated by alternative RNA splicing—more than 30,000 variants for DSCAM1 (see Figure 7–57). Diversity arises from alternative exons that code for three highly variable extracellular immunoglobulin domains.

1	by alternative RNA splicing—more than 30,000 variants for DSCAM1 (see Figure 7–57). Diversity arises from alternative exons that code for three highly variable extracellular immunoglobulin domains. Each DSCAM1 isoform engages in homophilic binding (see Figure 19–5), but remarkably, all the variable domains need to be identical for this to occur. Thus, one cell surface will bind to another via DSCAM only when the two cell surfaces express identical isoforms. The result of binding is repulsion, although the detailed mechanisms are poorly understood.

1	If alternative splicing occurs in a random fashion in each cell, neighboring processes from different neurons are unlikely to express the same DSCAM1 variant, so only the processes of the same cell will repel one another. Neurons that lack all DSCAM1 variants have severe defects in neuronal self-avoidance. Engineering Drosophila so that all of its neurons produce a single isoform restores self-avoidance; but now the processes of neighboring neurons express the same isoform and repel each other, resulting in the phenomenon of tiling (Figure 21–80).

1	Vertebrate neurons use a similar self-avoidance strategy to pattern their axons and dendrites, but instead of DSCAMs, they use protocadherins for self/non-self discrimination. The Protocadherin locus encodes 58 related cadherin-like trans-membrane proteins that are expressed in different combinations in single neurons. Homophilic recognition results in self-avoidance of dendrites emanating from the same neuron; neighboring dendrites of different neurons express different protocadherins and thus evade repulsion. Thus, although insect DSCAM and vertebrate protocadherin proteins share no sequence homology, they mediate similar self-avoidance strategies.

1	Eventually, axonal growth cones reach the target region where they must halt and make synapses. These synapses, as a rule, are destined to transmit neural signals in one direction, from axon to target cell. The development of synapses, however, depends on signaling in both directions: signals from the target tissue not only help control which growth cones synapse where (as we discuss shortly), but can also regulate how many of the innervating neurons survive. Many types of vertebrate neurons are produced in excess; up to 50% or more of some of them die soon after they reach their target, even though they appear perfectly normal and healthy up to the time of their death. About half of all the motor neurons that send axons to skeletal muscle, for example, die within a few days after making contact with their target muscle cells. A similar proportion of the sensory neurons that innervate the skin die after their growth cones have arrived there.

1	This large-scale normal neuronal death often seems to reflect the outcome of a competition, in which the target tissue releases a limited amount of a specific neurotrophic factor that the neurons innervating the tissue require to survive; those that do not get enough die by programmed cell death. If the amount of target tissue is increased—for example, by grafting an extra limb bud onto the side of the embryo—more limb-innervating neurons survive; conversely, if the limb bud is cut off, the same neurons all die (Figure 21–81). In this way, although individuals may vary in their bodily proportions, they always retain the right number of motor neurons to innervate all their muscles and the right number of sensory neurons to innervate their body surface. The strategy of overproduction followed by death of surplus cells may seem wasteful, but it provides a simple and effective means to adjust the number of innervating neurons according to the amount of tissue requiring innervation.

1	The first neurotrophic factor to be identified, and still the best characterized, is called nerve growth factor (NGF )—the founding member of the neurotrophin family of signal proteins. It promotes the survival and growth of specific classes of sensory neurons and of sympathetic neurons (a subclass of peripheral neurons that control contractions of smooth muscle and secretion from exocrine glands). Figure 21–80 DSCAM mediates self-avoidance of dendrites. (a) Sensory neurons in the Drosophila peripheral nervous system extend dendrites along the larval body wall. the image shows the dendrites of a regular array of photosensing neurons (red), which allow the larva to detect and avoid harmful light. the posterior epidermal cells of each segment are labeled in blue. there are many neurons, and those shown here spread out their dendrites into overlapping fields.

1	(B) Mutations at the Dscam locus upset the way the various dendrites interact, changing the rules of self-avoidance and the distribution of innervation. (a, courtesy of chun han; B, after D. hattori et al., Annu. Rev. Cell Dev. Biol. 24:597–620, 2008. With permission from annual reviews.) 2.5 day chick section of spinal cord 9 day chick (A) section of spinal cord NGF is produced by the tissues that these neurons innervate. When extra NGF is provided, extra sensory and sympathetic neurons survive, just as if extra target tissue were present. Conversely, in a mouse with a mutation that inactivates the gene for NGF or for its receptor (a receptor tyrosine kinase called TrkA), almost all sympathetic neurons and the NGF-dependent sensory neurons are lost. There are many neurotrophic factors, only a few of which belong to the neurotrophin family, and they act in different combinations to promote the survival and growth of different classes of neurons.

1	Formation of Synapses Depends on two-Way communication Between Neurons and their target cells

1	At journey’s end, the task of a growth cone is to halt its travels and make synapses with specific target cells. Synapses were introduced in Chapter 11, where we discussed channels and the electrical properties of membranes. Two main classes of synapses are found in vertebrates; those made with muscle cells and those made with other neurons. Synapse formation is best understood in the case of the highly specialized connections between motor neurons and skeletal muscle cells—so-called neuromuscular junctions (see Figure 11–38). During synapse formation, the axonal growth cone differentiates into a nerve terminal that contains synaptic vesicles filled with the neurotransmitter acetylcholine, while acetylcholine receptors become clustered in the muscle cell plasma membrane at the site of synapse formation. A synaptic cleft separates the preand postsynaptic plasma membranes, and a thin sheet of basal lamina lies in this space between them (Figure 21–82).

1	Figure 21–81 The survival of motor neurons depends on signals provided by the target muscles. (a) removal of the limb bud shortly after arrival of motor axons results in the death of motor neurons in the spinal cord on the amputated side. (B) transplantation of an extra limb bud increases the survival of motor neurons. (after e. Kandel et al., principles of Neural Science, 5th ed., New York: McGraw hill Medical, 2012.)

1	Formation of the synapse involves two-way communication between the muscle cell and axonal growth cone: each of them, under the influence of the other, must reorganize the molecules on its side of the junction. The growth cone releases the signal protein Agrin, while the muscle expresses the Agrin receptor LRP4. Agrin binding to LRP4 stimulates association of LRP4 with MuSK, a receptor tyrosine kinase. LRP4 also serves as a signal in the reverse direction, from the muscle to the axon (Figure 21–83). During synapse formation, MuSK and LRP4 cluster in the muscle cell plasma membrane in the general neighborhood of the future synapse. As the growth cone approaches, it recognizes LRP4, which stimulates the differentiation of presynaptic structures in the nerve cell. At the same time, Agrin released from the growth cone binds to LRP4 in the muscle cell; this activates MuSK, and promotes a more focused clustering of acetylcholine receptors in the muscle cell membrane. Through these

1	Agrin released from the growth cone binds to LRP4 in the muscle cell; this activates MuSK, and promotes a more focused clustering of acetylcholine receptors in the muscle cell membrane. Through these mechanisms, the reciprocal signaling of LRP4 from muscle to growth cone—and of Agrin from growth cone to muscle—induces the coordinated, localized differentiation of preand postsynaptic structures.

1	Synapse formation between neurons in the CNS is far more challenging, both for the neurons and for the scientists trying to understand the molecular basis of its specificity, and it remains poorly understood.

1	LRP4 SIGNALS BACK TO AXON CLUSTERING OF LRP4 AND MuSK BY AGRIN CLUSTERING OF ACETYLCHOLINE RECEPTORS Agrin Agrin axon terminal axon terminal basal lamina MuSK (Agrin co-receptor) MuSK LRP4 (Agrin receptor) LRP4acetylcholine receptor axon of motor neuron muscle fber (A) (C) (B) new synapse muscle fber Figure 21–83 Reciprocal signaling during neuromuscular synapse differentiation. (a) the agrin receptor Lrp4 and its co-receptor MuSK cluster in the muscle cell membrane in the general neighborhood of the future synapse. (B) as the growth cone approaches, it recognizes Lrp4, which stimulates differentiation of presynaptic structures. reciprocally, agrin is released from the nerve terminal, binds to a complex of Lrp4 and MuSK in the muscle, and (c) promotes the further and more focused clustering of the Lrp4 and acetylcholine receptors in the muscle cell. although the agrin/MuSK/Lrp4 machinery organizes the synapse, the process also depends on electrical signaling via the acetylcholine

1	of the Lrp4 and acetylcholine receptors in the muscle cell. although the agrin/MuSK/Lrp4 machinery organizes the synapse, the process also depends on electrical signaling via the acetylcholine receptors. It is not yet known how Lrp4 signals to the motor axon.

1	Figure 21–82 Formation of the neuromuscular junction. (a) the growth cone of a motor axon approaches the muscle fiber. (B) Initial synapse formation is characterized by the accumulation of synaptic vesicles at the axon terminal and the formation of a specialized basal lamina in the synaptic cleft. (c) as the neuromuscular junction matures, the synaptic cleft accumulates basal lamina and extracellular matrix proteins, synaptic vesicles cluster at presynaptic release sites, and neurotransmitter receptors cluster at postsynaptic sites. Schwann (glial) cells accompany the motor axon and wrap around its terminus outside the region of synaptic contact. (D) transmission electron micrograph of the region of synaptic contact. [D, courtesy of John heuser, from J. Electron Microsc. 60 (Suppl 1), 2011. With permission from Oxford University press.]

1	The two-way exchange of signals between axon growth cones and muscle cells controls the initial formation of neuromuscular junctions, but it is only the first step in the establishment of the final pattern of the synaptic connections. Each muscle cell at first receives synapses from several motor neurons, but in the end it is left innervated by only one. This process of synapse elimination depends on active synaptic communication and electrical activity. If synaptic transmission is blocked by a toxin that binds to the acetylcholine receptors in the muscle cell membrane, or if axonal electrical activity is blocked by a toxin that binds to sodium channels in the axon plasma membrane, the muscle cell retains its multiple innervation beyond the normal time of elimination.

1	The phenomenon of activity-dependent synapse elimination is encountered in almost every part of the developing vertebrate nervous system (Figure 21–84). It has a key role, for example, in the refinement of the retinotopic map discussed earlier. Synapses are first formed in abundance and distributed over a broad target field; then the system of connections is pruned back and remodeled by competitive processes that depend on electrical activity and synaptic signaling. The elimination of synapses in this way is distinct from the elimination of surplus neurons by cell death, and it occurs after the period of normal neuronal death is over. Synapse remodeling during neural development, however, involves more than just synapse elimination; it also involves synapse reinforcement, as we discuss next.

1	Throughout the nervous system, and throughout life, activity-dependent elimination and reinforcement of synapses plays a fundamental part in adjusting the detailed anatomy of the neural network according to functional requirements. The importance of these processes, and their underlying rules, emerged half a century ago from a groundbreaking series of experiments on the developing visual system of young mammals.

1	In the brain of most mammals, axons relaying visual inputs from the two eyes are brought together in a specific neuronal layer in the visual region of the cerebral cortex. Here, they form two overlapping maps of the external visual field, one as perceived through the right eye, the other as perceived through the left. Although there may be a tendency for rightand left-eye inputs to be segregated even before synaptic communication begins, a large proportion of the axons carrying information from the two eyes at early stages form synapses together on shared target neurons in the visual cortex. A period of early electrical signaling activity, is quiet: cell C gets excited simultaneously: cell C gets excited synapse made by B on C synapses made by both A and B is weakened or eliminated on C are strengthened BB

1	Figure 21–84 Synapse modification and its dependence on electrical activity. experiments in several systems indicate that synapses are strengthened or weakened by electrical activity according to the rule shown in the diagram. the underlying principle appears to be that each excitation of a target cell tends to weaken any synapse where the presynaptic axon terminal has been quiet, but to strengthen any synapse where the presynaptic axon terminal has just been active. as a result, any synapse that is repeatedly weakened and rarely strengthened is eventually eliminated altogether.

1	Figure 21–85 Ocular dominance columns in the visual cortex of a monkey’s brain, and their sensitivity to visual experience. (a) Normally, stripes of cortical cells driven by the right eye alternate with stripes, of equal width, driven by the left eye. the stripes, set up before birth, are revealed here by injecting a radioactive tracer molecule into one eye, allowing time for this tracer to be transported to the visual cortex, and detecting radioactivity there by autoradiography, in sections cut parallel to the cortical surface. (B) If one eye is kept covered after birth, during the sensitive period of development, and thus deprived of visual experience, its stripes shrink and those of the active eye expand. In this way, the deprived eye may lose the power of vision almost entirely. (From

1	D.h. hubel, t.N. Wiesel and S. LeVay, Philos. Trans. R. Soc. Lond. B Biol. Sci. 278:377–409, 1977. With permission from the royal Society.) however, occurring spontaneously and independently in each retina before birth, leads to a remarkable pattern of ocular dominance columns in the visual cortex: stripes of cells driven by inputs from the right eye alternating with stripes driven by inputs from the left eye (Figure 21–85).

1	The basis for these phenomena became clear from ingenious experiments interfering artificially with visual experience and altering the coordination of electrical signaling in the two eyes. These studies, and many others subsequently, have highlighted a simple but profoundly important principle that seems to govern synapse reinforcement and elimination throughout the nervous system. When two (or more) neurons synapsing on the same target cell fire at the same time, they reinforce their connections to that cell; when they fire at different times, they compete, so that all but one of them tend to be eliminated. This firing rule is expressed in the catchphrase “neurons that fire together wire together.”

1	The firing rule provides a simple interpretation of the developmental phenomenon we have just described in the mammalian visual system. A pair of axons bringing information from neighboring sites in the left eye will frequently fire together, and therefore wire together, as will a pair of axons from neighboring sites in the right eye; but a right-eye axon and a left-eye axon will rarely fire together, and will instead compete. Indeed, if activity from both eyes is silenced using toxins that block axonal electrical activity or synaptic signaling, as described above, the inputs fail to segregate correctly.

1	The segregation of inputs from the two eyes is only the first of a series of activity-dependent adjustments of visual connections, whose maintenance is extraordinarily sensitive to experience early in life. If, during a certain sensitive period (ending at about 5 years of age in humans), one eye is kept covered for a time so as to deprive it of visual stimulation, while the other eye is allowed normal stimulation, the deprived eye loses its synaptic connections to the cortex and becomes almost entirely, and irreversibly, blind. In accordance with what the firing rule would predict, a competition has occurred in which synapses in the visual cortex made by inactive axons are eliminated while synapses made by active axons are consolidated. In this way, cortical territory is allocated to axons that carry information and is not wasted on those that are silent.

1	Activity-dependent synaptic changes are not confined to early life. They also occur in the adult brain, where many synapses show both functional and morphological alterations with use. This synaptic plasticity is thought to have a fundamental role in learning and memory. Clearly, for the nervous system as for other parts of the body, developmental processes do not end at birth, as we discuss in the next chapter. The development of the nervous system proceeds in four phases. First, neurons and glial cells are generated from dividing neural progenitor cells. Then, the newborn neurons send out axons and dendrites toward their targets. Next, they make synaptic connections with appropriate target cells so that communication can begin. Finally, excessive neurons are eliminated by normal neuronal cell death, after which the system of synaptic connections is refined and remodeled according to the pattern of electrical and synaptic activity in the neural network.

1	Neurons born at different times and places are specialized to express different sets of genes, and they have a cell memory that plays a major role in determining the connections they will form. Their specialization depends not only on spatial patterning by morphogens but also on intrinsic developmental programs that unfold as the neural progenitors proliferate. Axons and dendrites grow out from the neurons by means of growth cones, which follow specific pathways delineated by attractive and repellant signals along the way, including cell-surface and extracellular matrix molecules and soluble signal proteins to which growth cones from different classes of neurons respond differently. In many parts of the nervous system, neural maps are set up—orderly projections of one array of neurons onto another. In the retinotopic system, the map is based on the matching of complementary systems of position-specific cell-surface markers—ephrins and Eph receptors—possessed by the two sets of cells.

1	another. In the retinotopic system, the map is based on the matching of complementary systems of position-specific cell-surface markers—ephrins and Eph receptors—possessed by the two sets of cells. Other cell-surface molecules such as DSCAM proteins in Drosophila and protocadherins in vertebrates mediate self-avoidance between the branches arising from a single neuron, helping the cell spread out its processes.

1	The formation of synapses involves back-and-forth signaling between target cells and the growth cone. After the growth cones have reached their targets and initial connections have formed, individual synapses are eliminated in some places and reinforced in others by mechanisms that depend on synaptic and electrical activity. These mechanisms adjust the architecture of the neural network according to the way in which it is used. Which statements are true? Explain why or why not. What regulates the pace of development? Why does a mouse embryo develop faster than a human embryo, for example? What are the mechanisms that allow cell memory to be stored during development, explaining how each cell’s history determines its future behavior? how do signals move through tissues? What are the roles of the extracellular matrix and of elongated cell projections?

1	how do signals move through tissues? What are the roles of the extracellular matrix and of elongated cell projections? how does a cell know exactly where it is in a multicellular organism? how does it know that its neighbors are the correct ones and that, if not, it should move or kill itself? how do cells respond to tiny gradients of molecules in their environment, as required for knowing their positions? how are morphogen gradients reliably interpreted? What are the genetic changes that allow the repurposing of existing body parts during evolution? For example, how did bat wings evolve from arms? how do cells use genetic instructions to form the shape of something as complex as the human nose?

1	how do cells use genetic instructions to form the shape of something as complex as the human nose? 21–1 In the early cleavage stages, when the embryo 21–5 Changes in the coding regions of genes involved in cannot yet feed, the developmental program is driven and development are primarily responsible for the differences controlled entirely by the material deposited in the egg by between species. the mother. 21–6 The cell cycle is the ticking clock that sets the tempo of developmental processes, with maturational 21–2 Because of the many later developmental trans-changes in gene expression being dependent on cell-cycle formations that produce the elaborately structured organs, progression. the body plan set up during gastrulation bears little resemblance to the body plan in the adult.

1	the body plan set up during gastrulation bears little resemblance to the body plan in the adult. Discuss the following problems. 21–3 As development progresses, individual cells 21–7 Name the four processes that are fundamental to become more and more restricted in the range of cell types animal development, and describe each of them in a single they can give rise to. sentence. 21–4 At different stages of embryonic development, 21–8 What are the three germ layers formed during gas-the same signals are used over and over again by different trulation, and what are the principal structures each gives cells, but with different biological outcomes. rise to in the adult? 21–9 In the early Drosophila embryo, there seems to be no requirement for the usual forms of cell–cell signaling; instead, transcriptional regulators and mRNA molecules move freely between nuclei. How can that be?

1	21–10 Morphogens play a key role in development, creating concentration gradients that inform cells of where they are and how to behave. Examine the simple patterns represented by the flags in Figure Q21–1. Which do you suppose could be created by a gradient of a single morphogen? Which would require gradients of two morphogens? Assuming that such patterns were present in a sheet of cells, explain how they could be created by morphogens. Figure Q21–1 National flags from three countries (problem 21–10).

1	21–11 Two adjacent cells in the nematode worm normally differentiate into an anchor cell (AC) and a ventral uterine precursor (VU) cell, but which of the two becomes the AC and which becomes the VU cell is completely random: the cells have an equal chance of adopting either fate, but they always adopt different fates. Mutations of Lin12 alter these fates. In hyperactive Lin12 mutants, both cells become VU cells, while in inactive Lin12 mutants, both cells become ACs. Thus, Lin12 is central to the decision-making process. In genetic mosaics in which one precursor cell has the hyperactive Lin12 and the other precursor has the inactive Lin12, the cell with the hyperactive Lin12 always becomes the VU cell and the cell with inactive Lin12 always becomes the AC. Assuming that one cell sends a signal and the other cell receives it, explain how these results suggest that Lin12 encodes a protein required to receive the signal. Offer a suggestion for how the fates of these two precursor cells

1	signal and the other cell receives it, explain how these results suggest that Lin12 encodes a protein required to receive the signal. Offer a suggestion for how the fates of these two precursor cells are normally decided in wild-type worms.

1	21–12 It was clear from the early days of studying development that certain “morphogenetic” substances were present in the egg and segregated asymmetrically into cells of the developing embryo. One such investigation in ascidian (sea squirt) embryos examined endodermal alkaline phosphatase, which could be visualized by a histochemical stain. Treatment of embryos with cytochalasin B stopped cell division, but did not block expression of alkaline phosphatase at the appropriate time. Treatment with actinomycin D, which blocks transcription, did not interfere with expression of alkaline phosphatase. Treatment with puromycin, which blocks translation, eliminated expression of alkaline phosphatase. What is the likely nature of the morphogenetic substance that gives rise to alkaline phosphatase?

1	21–13 The mouse HoxA3 and HoxD3 genes are paralogs that occupy equivalent positions in their respective Hox gene clusters and share roughly 50% identity in their pro-tein-coding sequences. Mice with defects in HoxA3 have deficiencies in pharyngeal tissues, whereas mice with defects in HoxD3 have deficiencies in the axial skeleton, suggesting quite different functions for the paralogs. Thus, it came as a surprise when it was found that replacing a defective HoxD3 gene with the normal HoxA3 gene corrected the deficiency, as did the reciprocal experiment of replacing a mutant HoxA3 gene with a normal HoxD3 gene. Neither transplaced gene, however, could supply its normal function; that is, a normal HoxA3 gene at the HoxD3 locus could not correct the deficiency caused by a mutant HoxA3 gene at the HoxA3 locus. The same was true for the HoxD3 gene. If the HoxA3 and HoxD3 genes are equivalent, how do you suppose they can play such distinct roles in development? Why do you suppose they cannot

1	the HoxA3 locus. The same was true for the HoxD3 gene. If the HoxA3 and HoxD3 genes are equivalent, how do you suppose they can play such distinct roles in development? Why do you suppose they cannot perform their normal function in a new location?

1	21–14 The segmentation of somites in vertebrate embryos is thought to depend on oscillations in the expression of the Hes7 gene. Mathematical modeling explains these oscillations in terms of the delays in production of the unstable Hes7 protein, which acts as a transcription regulator to shut off its own expression. Once Hes7 decays, with a half-life of about 20 minutes, its transcription resumes. To test this model, you decide to reduce the total delay by removing one, two, or all three of the introns from the Hes7 gene in mice. Why do you expect that intron removal would reduce the delay? What would you predict would happen to the oscillation time, and somite formation, if the model were correct?

1	21–15 The oscillatory clock that drives somite formation in vertebrates involves three essential components Her7 (an unstable repressor of its own synthesis), Delta (a transmembrane signaling molecule), and Notch (a trans-membrane receptor for Delta). Notch is bound by Delta on neighboring cells, activating the Notch signaling pathway, which then activates Her7 transcription. Normally, this system works flawlessly to create sharply defined somites (Figure Q21–2A). In the absence of Delta, however, only the first five somites form normally, and the rest are poorly defined (Figure Q21–2B). If a pulse of Delta is supplied later, somite formation returns to normal in the regions where Delta was present (Figure Q21–2C). A diagram of the connections between the components of the clock and how they interact in adjacent cells is shown in Figure Q21–2D. In the absence of Delta, why do the cells become unsynchronized? What is it about the presence of Delta that keeps adjacent cells oscillating

1	interact in adjacent cells is shown in Figure Q21–2D. In the absence of Delta, why do the cells become unsynchronized? What is it about the presence of Delta that keeps adjacent cells oscillating in synchrony?

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1	reichardt LF (2006) Neurotrophin-regulated signalling pathways. Philos. Trans. R. Soc. Lond. B Biol. Sci. 361, 1545–1564. Sanes Dh, reh ta & harris Wa (2011) Development of the Nervous System, 3rd ed. San Diego, ca: academic press. Sperry rW (1963) chemoaffinity in the orderly growth of nerve fiber patterns and connections. Proc. Natl Acad. Sci. USA 50, 703–710. Zipursky SL & Sanes Jr (2010) chemoaffinity revisited: dscams, protocadherins, and neural circuit assembly. Cell 143, 343–353.

1	Zipursky SL & Sanes Jr (2010) chemoaffinity revisited: dscams, protocadherins, and neural circuit assembly. Cell 143, 343–353. Cells evolved originally as free-living individuals, and such cells still dominate the Earth and its oceans. But the cells that matter most to us, as human beings, are specialized members of a multicellular community. These cells have lost features needed for independent survival and acquired peculiarities that serve the needs of the body as a whole. Although they share the same genome, they are spectacularly diverse in structure, chemistry, and behavior. There are more than 200 different named cell types in the human body that collaborate with one another to form many different tissues, arranged into organs performing widely varied functions. To understand them, it is not enough to analyze cells in a culture dish: we need also to know how they live, work, and die in their natural habitat, the intact body.

1	In Chapters 7 and 21, we saw how the various cell types become different in the embryo and how cell memory and signals from their neighbors enable them to remain different thereafter. In Chapter 19, we discussed the technology used to build multicellular tissues—the devices that bind cells together and the extracellular materials that give them support. But the adult body is not static: it is a structure in dynamic equilibrium, where new cells are continually being born, differentiating, and dying. Homeostatic mechanisms maintain a proper balance, so that the tissue architecture is preserved despite the constant replacement of old cells by new. In this chapter, we focus on these developmental processes that continue throughout life. In doing so, we shall illustrate some of the diversity of specialized cell types and see how they work together to perform their tasks.

1	We shall examine in particular the role played in many tissues by stem cells— cells that are specialized to provide a fresh supply of differentiated cells where these need to be continually replaced, or when they are required in great number for purposes of repair and regeneration. We shall see that while many tissues renew and repair themselves, some others do not; there, lost cells are lost forever, causing deafness, blindness, dementia, and other ills. In the final section of the chapter, we discuss how stem cells can be generated and manipulated artificially, and we confront the practical question that underlies the current storm of interest in stem-cell technology: How can we use our understanding of the processes of cell differentiation and tissue renewal to improve upon nature, and make good those injuries and failings of the human body that have hitherto seemed to be beyond repair?

1	Among all the self-renewing tissues in a mammal, the champion—for speed at least—is the lining of the small intestine: the long, convoluted portion of the gut tube that is chiefly responsible for absorption of nutrients from the gut lumen. To introduce stem cells, we take the small intestine as our starting point—not only because it renews itself at a greater rate than any other tissue in the body, but also because the molecular mechanisms that control its organization are particularly well understood. It thereby provides a beautiful illustration of the principles of stem-cell systems that have broad applicability. FibRoblaStS and theiR tRanSFoRmationS: the ConneCtive-tiSSue Cell Family blood veSSelS, lymphatiCS, and endothelial CellS a hieRaRChiCal Stem-Cell SyStem: blood Cell FoRmation epithelial cell migration from “birth” at the bottom the lining of the Small intestine is Continually Renewed through Cell proliferation in the Crypts

1	The lining of the small intestine (and of most other regions of the gut) is a sin-gle-layered epithelium, only one cell thick. This epithelium covers the surfaces of the villi that project into the lumen, and it lines the crypts that descend into the underlying connective tissue (Figure 22–1). Dividing cells are restricted to the crypts, and differentiated cells, no longer dividing, pour out of the crypts in a steady stream onto the villi. There are four main types of nondividing differentiated cells—one absorptive and three secretory (Figure 22–2): 1. Absorptive cells (also called brush-border cells or enterocytes) have densely packed microvilli on their exposed surfaces. Their job is to take up nutrients from the gut lumen. To this end, they also produce hydrolytic enzymes that perform some of the final steps of extracellular digestion. They are the majority cell type in the epithelium. 2.

1	2. Goblet cells secrete mucus into the gut lumen that covers the epithelium with a protective coat. 3. Paneth cells form part of the innate immune defense system (discussed in Chapter 24) and secrete proteins that kill bacteria. 4. Enteroendocrine cells, of more than 15 different subtypes, secrete serotonin and peptide hormones that act on neurons and other cell types in the gut wall and regulate the growth, proliferation, and digestive activities of cells of the gut and other tissues. As if on a conveyor belt, the absorptive, goblet, and enteroendocrine cells travel mainly upward from their site of birth in the crypt, by a sliding movement in the plane of the epithelial sheet, to cover the surfaces of the villi. Within 3–5 days (in the mouse) after emerging from the crypts, the cells reach the tips of the villi, where they undergo apoptosis and are finally discarded into the gut lumen (see Figure 22–1 Renewal of the gut lining.

1	(a) the pattern of cell turnover and proliferation in the epithelium that forms the lining of the small intestine. Stem cells (red) lie at the crypt base, interspersed among nondividing differentiated cells (paneth cells). progeny of the stem cells move mainly upward from the crypts onto the villi; after a few quick divisions, they cease dividing and differentiate—some of them while still in the crypt, most of them as they emerge from the crypt. the paneth cells, like the other nondividing differentiated cells, are continually replaced by progeny of the stem cells, but they migrate downward to the crypt base and survive there for many weeks. (b) photograph of a section of part of the lining of the small intestine, showing the crypts and villi. note the mixture of differentiated cell types, all generated from the stem cells; these are primarily absorptive cells, with mucus-secreting goblet cells (stained red) interspersed among them. enteroendocrine cells (not labeled) are less

1	types, all generated from the stem cells; these are primarily absorptive cells, with mucus-secreting goblet cells (stained red) interspersed among them. enteroendocrine cells (not labeled) are less numerous and less easy to identify without special stains.

1	Movie 20.6). The Paneth cells in the crypts are produced in much smaller numbers and have a different migration pattern. They live at the bottom of the crypts, where they too are continually replaced, although not so rapidly, persisting for several weeks (in the mouse) before undergoing apoptosis and being phagocytosed by their neighbors. The central problem is to understand the processes in the crypt that generate a continual supply of all these nondividing, terminally differentiated cell types. Stem Cells of the Small intestine lie at or near the base of each Crypt

1	The general pattern of cell proliferation and migration in the gut lining is revealed by a simple labeling method that uses injected pulses of tritiated (radioactive) thymidine or of a thymidine analog that can be detected in tissue sections. Cells that are in S phase of the division cycle incorporate the marker molecule into their DNA, and their fate can then be followed over subsequent hours and days. If a cell divides after incorporation of the label, the label becomes diluted, halving with each cell cycle. This can be quantified. Experiments based on this labeling method confirm, first of all, that dividing cells are confined to the crypts and that the differentiated cell types listed above do not divide. Second, the most rapidly dividing cells, with a cycle time of about 12 hours in the mouse, are shown to lie in the middle and upper parts of the crypt, and these cells are all fated to differentiate and stop dividing (see Figure 22–1A). Just above the base of the crypt,

1	hours in the mouse, are shown to lie in the middle and upper parts of the crypt, and these cells are all fated to differentiate and stop dividing (see Figure 22–1A). Just above the base of the crypt, interspersed among the Paneth cells, lie cells that divide more slowly. These are the stem cells, which feed some of their progeny into the higher levels of the crypt destined for differentiation, while other progeny remain at the crypt base to continue the whole process. The rapidly dividing cells above these stem cells are derived from them, but already committed to differentiation. These cells are called committed precursors or transit amplifying cells, since their divisions serve to amplify the number of differentiated cells that ultimately result from each stem-cell division.

1	the two daughters of a Stem Cell Face a Choice Stem cells have a critical role in a variety of tissues, and it is useful to list their defining properties: 1. A stem cell is not itself terminally differentiated: that is, it is not at the end of a pathway of differentiation. 2. It can divide without limit (or at least for the lifetime of the animal). 3. When it divides, each daughter has a choice: it can either remain a stem cell, or it can embark on a course that commits it to terminal differentiation (Figure 22–3).

1	Figure 22–2 The four main differentiated cell types found in the epithelial lining of the small intestine. all cells are oriented with the gut lumen at top. broad orange arrows indicate direction of secretion or uptake of materials for each type of cell. all of these cells are generated from undifferentiated multipotent stem cells living near the bottoms of the crypts (see Figure 22–1). Absorptive (brush-border) cells outnumber the other cell types in the epithelium by about 10:1 or more. the microvilli on their apical surface provide a 30-fold increase of surface area, not only for the import of nutrients but also for the anchorage of enzymes that perform the final stages of extracellular digestion, breaking down small peptides and disaccharides into monomers that can be transported across the cell membrane. Goblet cells secrete mucus; these are the commonest of the secretory cell types. Paneth cells secrete (along with some growth factors) cryptdins—proteins of the defensin family

1	the cell membrane. Goblet cells secrete mucus; these are the commonest of the secretory cell types. Paneth cells secrete (along with some growth factors) cryptdins—proteins of the defensin family that kill bacteria. different subtypes of enteroendocrine cells secrete serotonin and peptide hormones into the gut wall (and thence the blood). Cholecystokinin is a hormone released from enteroendocrine cells in response to the presence of nutrients in the gut. it binds to receptors on nearby sensory nerve endings, which relay a signal to the brain to stop the feeling of hunger once one has eaten enough. (after t.l. lentz, Cell Fine Structure. philadelphia: Saunders, 1971;

1	R. krsti´c, illustrated encyclopedia of human histology. berlin: Springer-verlag, 1984.) Stem cells are required wherever there is a recurring need to replace differentiated cells that cannot themselves divide. Although a stem cell must be able to divide, it does not necessarily have to divide rapidly; in fact, many stem cells divide at a relatively slow rate.

1	Stem cells are of many types, specialized for the genesis of different classes of terminally differentiated cells—intestinal stem cells for intestinal epithelium, epidermal stem cells for epidermis, hematopoietic stem cells for blood, and so on. Each stem-cell system nevertheless raises similar fundamental questions. What are the distinguishing features of the stem cell in molecular terms? What conditions serve to keep the stem cell in its proper place and to maintain its stem-cell character? What decides whether a given daughter cell commits to differentiation or remains a stem cell? In a tissue where several distinct types of differentiated cells must be produced, are they all derived from a single type of stem cell, or is there a distinct type of stem cell for each one? wnt Signaling maintains the Gut Stem-Cell Compartment

1	For the gut, the beginnings of an answer to these questions came from studies of cancer of the colon and rectum (the lower end of the gut, also known as the large intestine). Some people have a hereditary predisposition to colorectal cancer and, in advance of the invasive disease, develop large numbers of small precancerous tumors (adenomas) in the lining of this part of the gut (Figure 22–4). The appearance of these tumors suggests that they have arisen from intestinal crypt cells that have failed to halt their proliferation in the normal way. As discussed in Chapter 20, the cause has been traced to mutations in the Apc (adenomatous polyposis coli) gene: the tumors arise from cells that have lost both gene copies. Because Apc codes for a protein that prevents inappropriate activation of the Wnt signaling pathway (see Figure 15–60), this loss of Apc is presumed to mimic the effect of continual exposure to a Wnt signal. The suggestion, therefore, is that Wnt signaling normally keeps

1	the Wnt signaling pathway (see Figure 15–60), this loss of Apc is presumed to mimic the effect of continual exposure to a Wnt signal. The suggestion, therefore, is that Wnt signaling normally keeps crypt cells in a proliferative state, and that a cessation of exposure to Wnt signaling normally makes them stop dividing as they leave the crypt.

1	Stem Cells at the Crypt base are multipotent, Giving Rise to the Full Range of differentiated intestinal Cell types It has long been suspected that all the differentiated cell types in the lining of the intestine derive from a single type of stem cell. But firm proof was lacking, and the precise nature and location of the stem cells were disputed. To solve the problem, and indeed to understand the organization of any stem-cell system, we need to discover how its cells are related to one another—who Figure 22–3 The definition of a stem cell. each daughter produced when a stem cell divides can either remain a stem cell or go on to become terminally differentiated. in many cases, the daughter that opts for terminal differentiation undergoes additional cell divisions before terminal differentiation is completed; such cells are called transit amplifying cells.

1	Figure 22–4 An adenoma in the human colon, compared with normal tissue from an adjacent region of the same person’s colon. the specimen is from a patient with an inherited mutation in one of his two copies of the Apc gene. a mutation in the other Apc gene copy, occurring in a colon epithelial cell during adult life, has given rise to a clone of cells that behave as though the wnt signaling pathway is permanently activated. as a result, the cells of this clone form an adenoma—an enormous, steadily expanding mass of giant cryptlike structures.

1	Figure 22–5 Clonal analysis using a genetic marker. a modern method for tracking cell lineage uses transgenic animals containing two transgenes, which together drive expression of a readily detected and heritable marker protein in a small subset of stem cells. the first transgene (top) carries two adjacent protein-coding sequences, GFP and CreERT2, both expressed under the control of the Lgr promoter that is active only in stem cells and not in their differentiated progeny. GFP encodes green fluorescent protein (see Chapter 9), which is used here simply to confirm expression in the entire stem-cell population. the CreERT2 gene encodes a chimeric form of the Cre recombinase called CreeRt, which consists of Cre recombinase linked to the estrogen receptor protein; this enzyme becomes active as a recombinase only when it binds the artificial estrogen analog tamoxifen.

1	the second transgene (bottom) carries a marker gene, LacZ, under the control of a promoter that is active in all cells. the LacZ gene encodes β-galactosidase, an enzyme that can be detected histochemically in tissues (see Figure 7–28). however, LacZ expression in the transgene shown here is prevented by a blocking sequence (red) that is flanked by LoxP sites (pink; see Figure 5–66). when tamoxifen is provided, CreeRt becomes active––leading to a recombination event that removes the blocking dna sequence (and leaves one LoxP site behind). as a result, the lacZ marker is expressed. because this change is heritable, the marker continues to be expressed in all cells descended from those in which a recombination event has occurred. with a low dose of the inducer molecule tamoxifen, it is possible to activate the marker at random in just a few widely spaced cells, which, in the course of time, give rise to widely separated and easily distinguished clones of progeny (see Figure 22–6).

1	is descended from whom, or, equivalently, what progeny will be produced from any given cell. This can best be done using a heritable marker that can be activated in an individual cell, thus allowing the identification of the clone of progeny descended from that cell. A modern method uses transgenic animals to create a visible genetic mark in just a few widely spaced cells, which, in the course of time, give rise to widely separated and easily distinguished clones of progeny, as explained in Figure 22–5.

1	A search among genes that are strongly upregulated in response to Wnt signaling revealed one, called Lgr5, that is expressed in gut stem cells specifically. The technique described in Figure 22–5 can be used to create a genetic mark in a random subset of Lgr5-expressing cells—a mark that is inherited by the progeny of each cell. These Lgr5 cells divide with a cycle time of about 24 hours, and within a few days marked clones are seen extending from the crypt bases up along the sides of the villi. After as long as 60 days or more, many of these clones still persist, retaining one or more members at the crypt base and extending all the way up to the tips of the villi (Figure 22–6). Moreover, each single clone typically contains all the major differentiated gut cell types—absorptive, goblet, Paneth, and enteroendocrine—in their normal proportions. The Lgr5-expressing cells, therefore, are true stem cells that are multipotent—that is, able to generate a diverse set of differentiated cell

1	Paneth, and enteroendocrine—in their normal proportions. The Lgr5-expressing cells, therefore, are true stem cells that are multipotent—that is, able to generate a diverse set of differentiated cell types.

1	1 day 5 days 60 days Figure 22–6 Lgr5-expressing stem cells and their progeny in the small intestine.

1	the method shown in Figure 22–5 was used here to mark single intestinal stem cells and trace the fates of their progeny. the Lgr5 gene encodes a member of the family of G-protein-linked transmembrane receptors, and it is expressed specifically in stem cells near the crypt base. because the Lgr5 promoter was used to drive expression of CreERT2, treatment with a low dose of tamoxifen resulted in occasional stem cells expressing LacZ. these cells and all of their progeny could subsequently be detected with a blue histochemical stain. all of the blue cells in these images derive from a single Lgr5expressing stem cell. after 60 days, the blue progeny of this cell are seen to extend all the way up a villus. these progeny 100 µm can be shown to include all types of differentiated cells, as well as persistent the two daughters of a Stem Cell do not always have to Lgr5-expressing cells at the crypt base. become different this proves that Lgr5-expressing cells are multipotent stem cells. (From

1	as persistent the two daughters of a Stem Cell do not always have to Lgr5-expressing cells at the crypt base. become different this proves that Lgr5-expressing cells are multipotent stem cells. (From n. barker et If the number of stem cells in a crypt is to remain stable, each stem-cell division al., Nature 449:1003–1007, 2007. with must on average generate one daughter that remains a stem cell and one that permission from macmillan publishers ltd.) becomes committed to differentiation. In principle, this could be achieved in at least two ways (Figure 22–7).

1	One mechanism—the simplest at first sight—would be through asymmetric division: processes internal to the dividing stem cell could distribute regulatory factors asymmetrically to its two daughters, as occurs in Drosophila neuroblast divisions (see Figure 21–36). The factors inherited by one daughter would cause it to remain a stem cell, while those inherited by the other would drive it toward differentiation. This strategy would guarantee that the original stem cell would give rise to precisely one stem cell in every subsequent cell generation.

1	An alternative strategy would be based on a choice that each daughter makes independently of its sister: in normal circumstances, each would have a 50% probability of remaining as a stem cell and a 50% probability of commitment to differentiation. Sometimes the two daughters of a stem cell would thus have opposite fates, sometimes the same. The choice that each cell makes might either be stochastic, like the flip of a coin, or governed by the environment in which the cell finds itself. A strategy of independent choices is more flexible than that of strict asymmetric division. In particular, environmental factors can control the balance of probabilities, adjusting them in favor of the stem-cell option where more stem cells are needed, as they often are, either for growth or for damage repair.

1	Clonal analysis gives a way to distinguish between the two strategies, since they give quite different predictions as to the expected number of clones of different sizes produced from individual stem cells (see Figure 22–7). For the gut, the findings seem clear: the independent-choice theory fits the observations, and the asymmetric-division theory does not. paneth Cells Create the Stem-Cell niche There are about 15 Lgr5-expressing stem cells in each crypt. They are slim and columnar, and they sit at the crypt base interspersed among the Paneth cells (see Figure 22–6). This is the intestinal stem-cell niche: the Paneth cells generate signals, including a strong Wnt signal, that act over a short range to maintain the stem-cell state. Signal proteins from the connective tissue surrounding the crypt base help to reinforce the localizing signal from the Paneth cells; Lgr5 itself is a receptor for one of these proteins, called R-spondin.

1	In the intestine, it seems that the niche created by the Paneth cells has space for only a limited number of stem cells, and when these divide, it is a random POSSIBLE OUTCOMES after frst division cycle of stem cell after frst division cycle of stem cell terminally differentiated cell after second division cycle of stem cell after second division cycle of stem cell POSSIBLE OUTCOMES etc. etc. terminally differentiated cell environmental factors help determine cell fate or or or or or or or or choice determined by asymmetry in dividing stem cell choice determined stochastically and/or by environment localizeddeterminant

1	Figure 22–7 Two ways for a stem cell to produce daughters with different fates: asymmetric division and independent choice. (a) the asymmetric-division strategy gives a clone consisting of precisely one stem cell plus a steadily increasing number of differentiating cells, in proportion to the number of cell divisions. (b) the independent-choice strategy is more variable in its outcome. with a choice made at random by each daughter and with a 50% probability for each one to remain a stem cell or differentiate, there is, for example, a 25% chance at the first division that both daughters will differentiate, so that the clone eventually goes extinct. or, at this division or later, a preponderance of daughters may chance to retain stem-cell character, creating a clone that persists and increases in size. with the help of some mathematics, the probability distribution of clone sizes generated from a single stem cell at any given time can be predicted on this stochastic assumption. the

1	in size. with the help of some mathematics, the probability distribution of clone sizes generated from a single stem cell at any given time can be predicted on this stochastic assumption. the observations in the gut and elsewhere fit the stochastic independent-choice strategy, but not the asymmetric-division strategy.

1	matter which of them are pushed out of the nest and condemned to differentiation and which stay in place as stem cells for the future. In most other stem-cell systems where the question has been examined, it appears that the fates of the daughters of a stem cell are assigned in a similar way, independently and subject to influence from the cells’ environment.

1	The Paneth cells themselves are progeny of the stem cells, suggesting that the intestinal stem-cell system is in some way self-maintaining and self-organizing. This is demonstrated in a striking way by taking single dissociated Lgr5-expressing cells and allowing them to proliferate in culture, embedded in a cell-free matrix rich in the basal-lamina component laminin (mimicking basal lamina). The cells proliferate, forming at first small, round epithelial vesicles. Within a few days, however, one or another of the cells in the vesicle, at random, begins to differentiate as a Paneth cell. This induces its neighbors to behave as stem cells and initiates transformation of the simple vesicle into an organized structure, or organoid (Figure 22–8A,B). Protrusions resembling crypts grow out into the surrounding matrix and contain Paneth cells, Lgr5-expressing stem cells, and the transit amplifying cells derived from them; these cell types are confined to the cryptlike structures. Terminally

1	the surrounding matrix and contain Paneth cells, Lgr5-expressing stem cells, and the transit amplifying cells derived from them; these cell types are confined to the cryptlike structures. Terminally differentiated, nondividing absorptive cells line the other parts of the organoid epithelium, with their microvilli facing the lumen. Goblet and enteroendocrine cells are also present, scattered through the epithelium, and the whole “minigut” structure, with all its cell types, grows and renews itself in much the same way as the lining of the normal intestine.

1	Figure 22–8 Genesis of a minigut from a single Lgr5-expressing cell cultured in a cell-free matrix. (a,b) the founder cell first divides to form a small vesicle. at random, one or more of the cells in this vesicle differentiates as a paneth cell (blue). this cell maintains Lgr5 expression (yellow) in its immediate neighbors, which persist as stem cells that generate the full range of intestinal cell types. (C ) Schematic diagram of the key organizing signals. the paneth cells organize crypts by producing a wnt signal that acts on neighboring cells and keeps them proliferating in the stem-cell state. a repulsive interaction based on ephrin–eph binding causes the crypt cell types (which express ephb, induced by wnt) to segregate from the nondividing differentiated villus cell types (which express ephrinb). both ephrin and eph are cell-surface proteins attached to the plasma membrane; in many tissues, two cells that contain a different member of this pair repel each other when they touch

1	ephrinb). both ephrin and eph are cell-surface proteins attached to the plasma membrane; in many tissues, two cells that contain a different member of this pair repel each other when they touch (see Figure 21–49). (adapted from t. Sato and h. Clevers, Science 340:1190– 1194, 2013. with permission from aaaS.) ephrin–eph Signaling drives Segregation of the different Gut Cell types

1	The remarkable self-organizing behavior of the cultured organoids suggests that some interaction among the different epithelial cells drives them to segregate from one another. The ephrin–Eph signaling pathway (discussed in Chapter 15) appears to be responsible. The cells that live in the crypts express EphB receptor proteins, while absorptive, goblet, and enteroendocrine cells, as they begin to differentiate, switch off expression of this receptor and instead switch on expression of its ligands, cell-surface proteins of the EphrinB family (Figure 22–8C). In various other tissues, cells expressing Eph proteins are repelled by contacts with cells expressing ephrins on their surface (see Figures 21–49 and 21–79). It seems that the same is true in the gut lining, and that this mechanism serves to keep the cells segregated and in their proper places. In EphB knockout mutants, the populations become mixed, so that, for example, Paneth cells wander out onto the villi.

1	notch Signaling Controls Gut Cell diversification and helps maintain the Stem-Cell State If a single type of stem cell generates all the differentiated cell types in the gut lining, what causes the progeny of this stem cell to diversify? Notch signaling has this role in many other systems, where it mediates lateral inhibition—a competitive interaction that drives neighboring cells toward different fates (see Figure 15–58 and Figure 21–35). All the essential components of the Notch pathway are expressed in the crypts; it seems that Wnt signaling maintains them there. If Notch signaling is abruptly blocked, within a few days all the cells in the crypts differentiate as goblet cells, and absorptive cells cease to be produced; conversely, if Notch signaling is artificially activated in all the cells, absorptive cells continue

1	IN CRYPT, to be generated but no goblet cells are produced. This reflects the lateral inhibition mechanism operating in normal animals: the nascent goblet (and other secretory) cells express the Notch ligand Delta and thereby activate Notch in their neighbors, inhibiting them from differentiating as secretory (Figure 22–9). Delta-Notch signaling is crucial not only in the transit amplifying population, but also at the crypt base: the Paneth cells express Delta and this activates Notch in the stem cells, inhibiting differentiation. Without this influence, the stem cells lose their special character and differentiate as secretory cells. Thus maintenance of the intestinal stem-cell state requires a combination of signals, with both Wnt and Notch acting as central players. the epidermal Stem-Cell System maintains a Self-Renewing waterproof barrier

1	Stem-cell systems are organized in many different ways, but they share some underlying principles. Consider the epidermis, for example—the outer, epithelial covering of the body. The epidermis undergoes continual renewal, but, unlike the lining of the gut, it is multilayered or stratified. Stem cells are located in the basal layer, and their progeny move outward toward the exposed surface, differentiating as they go. They end up as lifeless scales or squames, which are eventually shed from the surface of the skin (Figure 22–10). Even though the architecture of this tissue is very different from that of the intestine, many of the same basic principles apply. The stem cells depend for their existence on signals from a specific niche, in this case the basal lamina and underlying connective tissue. The daughters of stem cells that are committed to differentiation undergo several divisions as transit amplifying cells (while still in the basal layer) before differentiating. Finally, a

1	tissue. The daughters of stem cells that are committed to differentiation undergo several divisions as transit amplifying cells (while still in the basal layer) before differentiating. Finally, a stochastic independent-choice mechanism dictates the fates of the daughters of a stem-cell division, allowing for increase in the number of stem cells when needed for growth or wound healing. Most of the same signaling pathways that organize the intestinal stem-cell system are also involved in regulating the epidermal stem-cell system, although with different individual roles.

1	Figure 22–9 How Notch signaling, in combination with Wnt, maintains stem cells and drives cell diversification in the intestine. wnt signaling leads to expression of notch and delta in the cells of the crypt, and delta-notch signaling in the crypt mediates lateral inhibition between adjacent cells. Cells expressing higher levels of delta eventually activate notch in their neighbors, adopt a secretory fate, and stop dividing; their neighbors, with activated notch, are prevented from differentiating and keep on dividing. essentially the same process operates at the crypt base, where the paneth cells express higher levels of delta to prevent stem cells from differentiating, and in the transit amplifying population, where nascent secretory cells express higher levels of delta. division continues in the notch-activated cells as they move up the crypt, until they escape from the influence of wnt and emerge onto the villi to become absorptive cells.

1	squame about to fake off from surface keratin flaments connective tissue of basal cell basal cell dividing

1	Figure 22–10 The multilayered structure of the epidermis, as seen in thin skin of a mouse. (a) the epidermis forms the outer covering of the skin, creating a waterproof barrier that is self-repairing and continually renewed. beneath this lies a relatively thick layer of connective tissue, which includes the tough, collagen-rich dermis (from which leather is made) and the underlying fatty subcutaneous layer or hypodermis. the cells of the epidermis are called keratinocytes, because their characteristic differentiated activity is the synthesis of keratin intermediate filament proteins, which give the epidermis its toughness. these cells change their appearance and properties from one layer to the next, progressing through a regular program of differentiation. those in the innermost layer, attached to an underlying basal lamina, are termed basal cells, and it is usually only these that divide: the basal cell population includes relatively small numbers of stem cells along with larger

1	attached to an underlying basal lamina, are termed basal cells, and it is usually only these that divide: the basal cell population includes relatively small numbers of stem cells along with larger numbers of transit amplifying cells derived from them. above the basal cells are several layers of larger prickle cells, shown in top view in (b), whose numerous desmosomes—each a site of anchorage for thick tufts of keratin filaments—are just visible in the light microscope as tiny prickles around the cell surface. beyond the prickle cells lies the thin, darkly staining granular cell layer, where the cells are sealed together to form a waterproof barrier; this marks the boundary between the inner, metabolically active strata and the outermost layer of the epidermis, consisting of dead cells whose intracellular organelles have disappeared. these outermost cells are reduced to flattened scales, or squames, filled with densely packed keratin, which are eventually shed from the surface of the

1	intracellular organelles have disappeared. these outermost cells are reduced to flattened scales, or squames, filled with densely packed keratin, which are eventually shed from the surface of the skin. the time from exit of a cell from the basal layer to its loss by shedding at the surface is a week or two, depending on body region and species.

1	in addition to the cells destined for keratinization, the deep layers of the epidermis include small numbers of cells (not shown) that invade this tissue and have quite different origins and functions. these immigrants include dendritic cells, called langerhans cells, derived from bone marrow and belonging to the immune system; melanocytes (pigment cells) derived from the neural crest; and merkel cells, which are associated with nerve endings in the epidermis. (b, from R.v. krsti´c, ultrastructure of the mammalian Cell: an atlas. berlin: Springer-verlag, 1979.) tissue Renewal that does not depend on Stem Cells: insulin-Secreting Cells in the pancreas and hepatocytes in the liver

1	Some types of cells can divide even though fully differentiated, allowing for renewal and regeneration without the use of stem cells. The insulin-secreting cells (β cells) of the pancreas are one example. Their mode of renewal has a special importance, because it is the loss of these cells (through autoimmune attack) that is responsible for type 1 (juvenile-onset) diabetes; they are also a significant factor in the type 2 (adult-onset) form of the disease. The β cells are normally sequestered in cell clusters called islets of Langerhans. These islets contain no obvious subset of cells specialized to act as stem cells, yet fresh β cells are continually generated within them. Lineage tracing studies, similar to those described above for the gut, show that the renewal of this population normally occurs by simple duplication of the existing insulin-expressing cells, and not by means of stem cells.

1	Another tissue that can renew by simple duplication of fully differentiated cells is the liver. The main cell type in the liver is the hepatocyte, a large cell that performs the liver’s metabolic functions. Hepatocytes normally live for a year or more and renew themselves through cell division at a very slow rate. Powerful homeostatic mechanisms operate to adjust the rate of cell proliferation or the rate of cell death, or both, so as to keep the organ at its normal size or restore it to that size in case of damage. A dramatic effect is seen if large numbers of hepatocytes are removed surgically or are killed by poisoning with carbon tetrachloride. Within a day or so after either sort of damage, a surge of cell division occurs among the surviving hepatocytes, quickly replacing the lost tissue. If two-thirds of a rat’s liver is removed, for example, a liver of nearly normal size can regenerate from the remainder by hepatocyte proliferation within about two weeks.

1	Both the pancreas and the liver contain small populations of stem cells that can be called into play as a backup mechanism for production of the differentiated cell types in more extreme circumstances. This imparts resilience to the mechanisms of renewal and repair. Some tissues lack Stem Cells and are not Renewable

1	Some tissues lack Stem Cells and are not Renewable The variety among tissues in the capacity for self-renewal is illustrated in a striking way by comparing the olfactory epithelium in the nose, the auditory epithelium of the inner ear, and the photoreceptive epithelium of the retina. These three sensory structures, which like the epidermis develop from the ectodermal layer of the early embryo, differ radically in their self-renewal capabilities. The olfactory epithelium contains a population of stem cells that give rise to differentiated cells that have a limited life-span and are continually replaced. But unlike the epidermis, these differentiated cells (the olfactory receptor cells) are neurons, with cell bodies lying in the olfactory epithelium and axons that extend back to the olfactory lobes in the brain. The continual renewal of this epithelium therefore involves continual production of fresh axons, which have to navigate back to the appropriate sites in the brain.

1	In contrast, in mammals at least, the auditory epithelium and the retinal epithelium lack stem cells, and their sensory receptor cells—the sensory hair cells in the ear, the photoreceptors in the retina—are irreplaceable. If they are destroyed— whether by too much exposure to loud noise, by looking into the beam of a laser, or through degenerative processes in old age—the loss is permanent.

1	Many tissues in the adult mammalian body are continually renewed by stem cells. Stem cells, by definition, are not terminally differentiated and have the ability to divide throughout the organism’s lifetime, yielding some progeny that differentiate and others that remain stem cells. The lining of the gut renews itself more rapidly than any other tissue in the mammalian body and provides a paradigm for the workings of stem-cell systems. In the small intestine, there is a continual upward flow from crypts, where new cells are generated by cell division, onto villi that are composed of nondividing differentiated cells. Wnt signaling maintains cell proliferation in the crypts, and overactivation of the Wnt pathway gives rise to tumors. Stem cells lie at each crypt base and are distinguished by expression of Lgr5 and certain other genes. The Lgr5+ stem cells are multipotent, each capable of generating several different types of differentiated cells as well as new stem cells. The balance of

1	expression of Lgr5 and certain other genes. The Lgr5+ stem cells are multipotent, each capable of generating several different types of differentiated cells as well as new stem cells. The balance of fate choices is adjusted according to need, allowing increase in the number of stem cells where more are needed for growth or repair. In a suitable cell-free culture medium, a single Lgr5+ stem cell can generate a self-organizing “minigut,” containing all the standard intestinal epithelial cell types.

1	Other self-renewing epithelia, such as the epidermis with its multilayered (stratified) architecture, have stem cells and their differentiating progeny arranged in different ways but are governed by similar basic principles. However, tissue renewal and repair does not always have to depend on stem cells. Thus, the population of insulin-producing cells in the pancreas is enlarged and renewed by simple duplication of existing insulin-producing cells. Similarly, in the liver, differentiated hepatocytes remain able to divide throughout life and can dramatically increase their division rate when the need arises. At an opposite extreme, some tissues, such as the sensory epithelia of the ear and the eye, do not undergo any turnover and are not renewable: their cells, once lost, are lost forever. FibRoblaStS and theiR tRanSFoRmationS: the ConneCtive-tiSSue Cell Family

1	FibRoblaStS and theiR tRanSFoRmationS: the ConneCtive-tiSSue Cell Family From epithelia, with their varied patterns of renewal and their enormous variety of protective, absorptive, secretory, sensory, and biosynthetic functions, we turn now to connective tissues. Connective tissues typically consist of cells dispersed in extracellular matrix that they themselves secrete, as discussed in Chapter 19. They originate from the mesodermal (middle) layer of the early embryo, sandwiched between ectoderm and endoderm (see Chapter 21, Figure 21–3).

1	In the adult body, virtually all epithelia are supported by a connective-tissue bed, or stroma; and specialized types of connective tissue, such as bone, cartilage, and tendon, form the supporting framework of the body as a whole. No less important than its mechanical role, connective tissue also contains the blood vessels that bring the oxygen and nourishment on which all cells depend. Cells of the immune system roam through connective tissue, passing in and out of blood vessels and lymphatics, and providing defence against infection; and through the meshes of connective tissue run peripheral nerves. Also embedded in connective tissue are the muscles that enable us to move. In these many ways, the cells that form connective tissue and synthesize its various types of extracellular matrix contribute to the support and repair of almost every tissue and organ.

1	Connective-tissue cells belong to a family of cell types that are related by origin, and they are often remarkably interconvertible. The family includes fibroblasts, cartilage cells, and bone cells, all of which are specialized for the secretion of collagenous extracellular matrix and are jointly responsible for the architectural framework of the body. The connective-tissue family also includes fat cells (adipocytes) and smooth muscle cells. Figure 22–11 illustrates these cell types and the interconversions that are thought to occur between them. The adaptability of the differentiated character of connective-tissue cells is an important feature of responses to many types of damage. Fibroblasts Change their Character in Response to Chemical and physical Signals

1	Fibroblasts Change their Character in Response to Chemical and physical Signals Fibroblasts seem to be the least specialized cells in the connective-tissue family. They are dispersed in connective tissue throughout the body, where they secrete a nonrigid extracellular matrix that is rich in type I or type III collagen, or both, as discussed in Chapter 19. When a tissue is injured, the fibroblasts nearby proliferate, migrate into the wound (Movie 22.1), and produce large amounts of collagenous matrix that helps to isolate and repair the damaged tissue. Their ability to thrive in the face of injury, together with their solitary lifestyle, may explain why fibroblasts are the easiest of cells to grow in culture—a feature that has made them a favorite subject for cell biological studies.

1	Figure 22–11 The family of connective-tissue cells. arrows show the interconversions that are thought to occur within the family. For simplicity, the fibroblast is shown as a single cell type, but it is uncertain how many types of fibroblasts exist and whether the differentiation potential of different types is restricted in different ways.

1	A class of connective-tissue cells in the bone marrow, called bone marrow stromal cells, provides an example of radical connective-tissue versatility. These cells, which can be regarded as a kind of fibroblast, can be isolated from the bone marrow and propagated in culture. Large clones of progeny can be generated in this way from single ancestral stromal cells. Depending on the culture conditions, the members of such a clone either can continue proliferating to produce more cells of the same type, or can differentiate as fat cells, cartilage cells, or bone cells. The fate of the cells depends on physical as well as chemical signals: embedded in a stiff, unyielding matrix, they tend to turn into bone cells, whereas in a softer, more elastic matrix, they tend to turn into fat cells. This effect is mediated by an intracellular pathway that responds to tension in actin–myosin bundles and relays a signal to specific transcription regulators in the nucleus (Figure 22–12). Because of their

1	is mediated by an intracellular pathway that responds to tension in actin–myosin bundles and relays a signal to specific transcription regulators in the nucleus (Figure 22–12). Because of their self-renewing, multipotent character, the bone marrow stromal cells, and other cells with similar properties, are referred to as mesenchymal stem cells.

1	Cartilage and bone are tissues of very different character; but they are closely related in origin, and the formation of the skeleton depends on an intimate partnership between them.

1	Cartilage tissue is structurally simple, consisting of cells of a single type— chondrocytes—embedded in a more or less uniform, highly hydrated matrix consisting of proteoglycans and type II collagen (discussed in Chapter 19). The cartilage matrix is deformable, and the tissue grows by expanding as the chondrocytes divide and secrete more matrix (Figure 22–13). Bone, by contrast, is dense and rigid; it grows by apposition—that is, by deposition of additional matrix on free surfaces. Like reinforced concrete, the bone matrix is predominantly a mixture of tough fibers (type I collagen fibrils), which resist pulling forces, and solid particles (calcium phosphate as hydroxylapatite crystals), which resist compression. The bone matrix is secreted by osteoblasts that lie at the surface of the existing matrix and deposit fresh layers of bone onto it. Some of the osteoblasts remain free at the surface, while others gradually become embedded in their own secretion. This freshly formed material

1	matrix and deposit fresh layers of bone onto it. Some of the osteoblasts remain free at the surface, while others gradually become embedded in their own secretion. This freshly formed material (consisting chiefly of type I collagen) is rapidly converted into hard bone matrix by the deposition of calcium phosphate crystals in it.

1	Once imprisoned in hard matrix, the original bone-forming cell, now called an osteocyte, has no opportunity to divide, although it continues to secrete additional matrix in small quantities around itself. The osteocyte, like the chondrocyte, occupies a small cavity, or lacuna, in the matrix, but unlike the chondrocyte Figure 22–12 Control of fibroblast differentiation by the physical properties of the extracellular matrix. on a stiff matrix, the cells form strong adhesions, spread out, and tend to turn into bone cells. on a soft matrix, where the cells are unable to form strong anchorages, they fail to spread and tend to differentiate as fat cells. these effects depend on transcription regulators (yap and taZ proteins) that move into the cell nucleus in response to tension developed in actin–myosin bundles in the cytoplasm. (based on S. dupont et al., Nature 474:179–183, 2011.) Figure 22–13 The growth of cartilage.

1	Figure 22–13 The growth of cartilage. the tissue expands as the chondrocytes divide and make more matrix. the freshly synthesized matrix with which each cell surrounds itself is shaded dark green. Cartilage may also grow by recruiting fibroblasts from the surrounding tissue and converting them into chondrocytes. osteoblast osteoid (new, uncalcifed bone matrix) old, calcifed bone matrix it is not isolated from its fellows. Tiny channels, or canaliculi, radiate from each lacuna and contain cell processes from the resident osteocyte, enabling it to form gap junctions with adjacent osteocytes (Figure 22–14). Blood vessels and nerves run through the tissue, keeping the bone cells alive and reacting when the bone is damaged.

1	A mature bone has a complex and beautiful architecture, in which dense plates of compact bone tissue enclose spaces spanned by light frameworks of trabecular bone—a filigree of delicate shafts and flying buttresses of bone tissue, with soft marrow in the interstices (Figure 22–15). The creation, maintenance, and repair of this structure depend not only on the cells of the connective-tissue family that synthesize matrix, but also on a separate class of cells called osteoclasts that degrade it, as we explain below. bone is Continually Remodeled by the Cells within it

1	bone is Continually Remodeled by the Cells within it For all its rigidity, bone is by no means a permanent and immutable tissue. Running through the hard extracellular matrix are channels and cavities occupied by living cells, which account for about 15% of the weight of compact bone. These cells are engaged in an unceasing process of remodeling: while osteoblasts deposit new bone matrix, osteoclasts demolish old bone matrix. This mechanism provides for continuous turnover and replacement of the matrix in the interior of the bone. Osteoclasts (Figure 22–16) are large, multinucleated cells that originate, like macrophages, from hematopoietic stem cells in the bone marrow (discussed later

1	Osteoclasts (Figure 22–16) are large, multinucleated cells that originate, like macrophages, from hematopoietic stem cells in the bone marrow (discussed later Figure 22–14 Deposition of bone matrix by osteoblasts. osteoblasts lining the surface of bone secrete the organic matrix of bone (osteoid) and are converted into osteocytes as they become embedded in this matrix. the matrix calcifies soon after it has been deposited. the osteoblasts themselves are thought to derive from osteogenic stem cells that are closely related to fibroblasts.

1	Figure 22–15 Trabecular and compact bone. (a) low-magnification scanning electron micrograph of trabecular bone in a vertebra of an adult man. the soft marrow tissue has been dissolved away. (b) a slice through the head of the femur, with bone marrow and other soft tissue likewise dissolved away, reveals the compact bone of the shaft and the trabecular bone in the interior. because of the way in which bone tissue remodels itself in response to mechanical load, the trabeculae become oriented along the principle axes of stress within the bone. (a, courtesy of alan boyde; b, from J.b. kerr, atlas of Functional histology. mosby, 1999.) to matrix matrix of osteoclast

1	Figure 22–16 Osteoclasts. (a) drawing of an osteoclast in cross section. this giant, multinucleated cell erodes bone matrix. the “ruffled border” is a site of secretion of acids (to dissolve the bone minerals) and hydrolases (to digest the organic components of the matrix). osteoclasts vary in shape, are motile, and often send out processes to resorb bone at multiple sites. they develop from monocytes and can be viewed as specialized macrophages. (b) an osteoclast on bone matrix, seen by scanning electron microscopy. the osteoclast has been crawling over the matrix, eating it away, and leaving a trail of pits where it has done so. (a, from R.v. krstic,´ ultrastructure of the mammalian Cell: an atlas. berlin: Springer-verlag, 1979; b, courtesy of alan boyde.) in this chapter). The precursor cells are released into the bloodstream and collect at sites of bone resorption, where they fuse to form the multinucleated osteoclasts, which cling to surfaces of the bone matrix and eat it away.

1	cells are released into the bloodstream and collect at sites of bone resorption, where they fuse to form the multinucleated osteoclasts, which cling to surfaces of the bone matrix and eat it away. Osteoclasts are capable of tunneling deep into the substance of compact bone, forming cavities that are then invaded by other cells. A blood capillary grows down the center of such a tunnel, and the walls of the tunnel become lined with a layer of osteoblasts (Figure 22–17). These osteoblasts lay down concentric layers of new matrix, which gradually fill the cavity, leaving only a narrow canal surrounding the new blood vessel. At the same time as some tunnels are filling up with bone, others are being bored by osteoclasts, cutting through older concentric systems.

1	Figure 22–17 The remodeling of compact bone. osteoclasts acting together in a small group excavate a tunnel through the old bone, advancing at a rate of about 50 μm per day. osteoblasts enter the tunnel behind them, line its walls, and begin to form new bone, depositing layers of matrix at a rate of 1–2 μm per day. at the same time, a capillary sprouts down the center of the tunnel. the tunnel eventually becomes filled with concentric layers of new bone, with only a narrow central canal remaining. each such canal, besides providing a route of access for osteoclasts and osteoblasts, contains one or more blood vessels that transport the nutrients the bone cells require for survival. typically, about 5–10% of the bone in a healthy adult mammal is replaced in this way each year. (after Z.F.G. Jaworski, b. duck and G. Sekaly, J. Anat. 133:397–405, 1981. with permission from blackwell publishing.) osteoclasts are Controlled by Signals From osteoblasts

1	G. Sekaly, J. Anat. 133:397–405, 1981. with permission from blackwell publishing.) osteoclasts are Controlled by Signals From osteoblasts The osteoblasts that make the matrix also produce the signals that recruit and activate the osteoclasts to degrade it. Disturbance of the balance can lead to osteoporosis, where there is excessive erosion of the bone matrix and weakening of the bone, or to the opposite condition, osteopetrosis, where the bone becomes excessively thick and dense. Hormonal signals have powerful effects on this balance. Chronic use of corticosteroid drugs, for example, can cause osteoporosis as a side effect; but this can be treated by other drugs that redress the balance, including agents that block the factors that osteoblasts secrete to recruit osteoclasts.

1	Local controls allow bone to be deposited in one place while it is resorbed in another. Through such controls over the process of remodeling, bones are endowed with a remarkable ability to adjust their structure in response to longterm variations in the load imposed on them. It is this that makes orthodontics possible, for example: a steady force applied to a tooth with a brace will cause it to move gradually, over many months, through the bone of the jaw, by remodeling of the bone tissue ahead of it and behind it.

1	Bone can also undergo much more rapid and dramatic reconstruction when the need arises. Some cells capable of forming new cartilage persist in the connective tissue that surrounds a bone. If the bone is broken, the cells in the neighborhood of the fracture repair it by a process that resembles the way bones develop in the embryo: cartilage is first laid down to bridge the gap and is then replaced by bone. The capacity for self-repair, so strikingly illustrated by the tissues of the skeleton, is a property of living structures that has no parallel among present-day man-made objects.

1	The family of connective-tissue cells includes fibroblasts, cartilage cells, bone cells, fat cells, and smooth muscle cells. Some classes of fibroblasts, such as the mesenchymal stem cells of bone marrow, seem to be able to transform into any of the other members of the family. These transformations of connective-tissue cell type are regulated by the composition of the surrounding extracellular matrix, by cell shape, and by hormones and growth factors. Cartilage and bone both consist of cells and solid matrix that the cells secrete around themselves—chondrocytes in cartilage, osteoblasts in bone (osteocytes being osteoblasts that have become trapped within the bone matrix). The matrix of cartilage is deformable so that the tissue can grow by swelling, whereas bone is rigid and can grow only by apposition. While osteoblasts secrete bone matrix, they also produce signals that recruit monocytes from the circulation to become osteoclasts, which degrade bone matrix. Through the activities

1	by apposition. While osteoblasts secrete bone matrix, they also produce signals that recruit monocytes from the circulation to become osteoclasts, which degrade bone matrix. Through the activities of these antagonistic classes of cells, bone undergoes a perpetual remodeling through which it can adapt to the load it bears and alter its density in response to hormonal signals. Moreover, adult bone retains an ability to repair itself if fractured, by reactivation of the mechanisms that governed its embryonic development: cells in the neighborhood of the break convert into cartilage, which is later replaced by bone.

1	The term “muscle” includes many cell types, all specialized for contraction but in other respects dissimilar. As noted in Chapter 16, all eukaryotic cells possess a contractile system involving actin and myosin, but muscle cells have developed this apparatus to a high degree. Mammals possess four main categories of cells specialized for contraction: skeletal muscle cells, heart (cardiac) muscle cells, smooth muscle cells, and myoepithelial cells (Figure 22–18). These differ in function, structure, and development. Although all of them generate contractile forces by using organized filament systems based on actin and myosin II, the actin and myosin molecules employed have somewhat different amino acid sequences, are bundle of smooth muscle cells differently arranged in the cell, and are associated with different sets of proteins that control contraction.

1	We focus in this section on skeletal muscle cells, which are responsible for practically all movements that are under voluntary control. These cells can be very large (2–3 cm long and 100 μm in diameter in an adult human) and are often called muscle fibers because of their highly elongated shape. Each one is a syncytium, containing many nuclei within a common cytoplasm. In an intact muscle, they are bundled tightly together, with fibroblasts (and some fat cells) in the interstices between them and blood vessels and nerve fibers running through the tissue. The mechanisms of muscle contraction were discussed in Chapter 16. Here we consider the unusual strategy by which the multinucleate skeletal muscle cells are generated and maintained. myoblasts Fuse to Form new Skeletal muscle Fibers

1	myoblasts Fuse to Form new Skeletal muscle Fibers During development, certain cells, originating from the somites of a vertebrate embryo at a very early stage, become determined as myoblasts, the precursors of skeletal muscle fibers. After a period of proliferation, the myoblasts undergo a dramatic change of state: they stop dividing, switch on the expression of a whole battery of muscle-specific genes required for terminal differentiation, and fuse with one another to form multinucleate skeletal muscle fibers (Figure 22–19). Once differentiation and cell fusion have occurred, the cells do not divide and the nuclei never again replicate their DNA.

1	Figure 22–18 The four classes of muscle cells of a mammal. (a) Schematic drawings (to scale). (b–e) Scanning electron micrographs. Skeletal muscle fibers (b, from a hamster) are giant cells with many nuclei and are formed by cell fusion. the other types of muscle cells are more conventional, generally having only a single nucleus. heart muscle cells (C, from a rat) resemble skeletal muscle fibers in that their actin and myosin filaments are aligned in very orderly arrays to form a series of contractile units called sarcomeres, so that the cells have a striated (striped) appearance. the arrows in (C) point to intercalated discs—end-toend junctions between the heart muscle cells; skeletal muscle cells in long muscles are joined end-to-end in a similar way. Smooth muscle cells (d, from the urinary bladder of a guinea-pig) are so named because they do not appear striated; they belong to the connective-tissue family and are closely related to fibroblasts. note that the smooth muscle is

1	urinary bladder of a guinea-pig) are so named because they do not appear striated; they belong to the connective-tissue family and are closely related to fibroblasts. note that the smooth muscle is shown here at a lower magnification than the other muscle types. the functions of smooth muscle vary greatly, from propelling food along the digestive tract to erecting hairs in response to cold or fear. myoepithelial cells (e, from a secretory alveolus of a lactating rat mammary gland) also have no striations, but unlike all other muscle cells they lie in epithelia and are derived from the ectoderm. they form the dilator muscle of the eye’s iris and serve to expel saliva, sweat, and milk from the corresponding glands. (b, courtesy of Junzo desaki; C, from t. Fujiwara, in Cardiac muscle in handbook of microscopic anatomy [e.d. Canal, ed.]. berlin: Springer-verlag, 1986; d, courtesy of Satoshi nakasiro; e, from t. nagato et al., Cell Tissue Res. 209:1–10, 1980. with permission from

1	in handbook of microscopic anatomy [e.d. Canal, ed.]. berlin: Springer-verlag, 1986; d, courtesy of Satoshi nakasiro; e, from t. nagato et al., Cell Tissue Res. 209:1–10, 1980. with permission from Springer-verlag.)

1	Some myoblasts persist as Quiescent Stem Cells in the adult Even though humans do not normally generate new skeletal muscle fibers in adult life, they still have the capacity to do so, and existing muscle fibers can resume growth when the need arises. Cells capable of serving as myoblasts are retained as small, flattened, and inactive cells lying in close contact with the mature muscle cell and contained within its sheath of basal lamina (Figure 22–20). If the muscle is damaged or stimulated to grow, these satellite cells are activated to proliferate, and their progeny can fuse to repair the damaged muscle or to allow muscle growth. Satellite cells, or some subset of the satellite cells, are thus the stem cells of adult skeletal muscle, normally held in reserve in a quiescent state but available when needed as a self-renewing source of terminally differentiated cells.

1	The process of muscle repair by means of satellite cells is, however, limited in what it can achieve. In one form of muscular dystrophy, for example, a genetic defect in the cytoskeletal protein dystrophin damages differentiated skeletal muscle cells. As a result, satellite cells proliferate to repair the damaged muscle fibers. This regenerative response is, however, unable to keep pace with the damage, and connective tissue eventually replaces the muscle cells, blocking any further possibility of regeneration. A decline of capacity for repair likewise contributes to the weakening of muscle in the elderly. satellite cell activated to divide Figure 22–19 Myoblast fusion in culture.

1	satellite cell activated to divide Figure 22–19 Myoblast fusion in culture. the culture is stained with a fluorescent antibody (green) against skeletal muscle myosin, which marks differentiated muscle cells, and with a dna-specific dye (blue) to show cell nuclei. (a) a short time after a change to a culture medium that favors differentiation, just two of the many myoblasts in the field of view have switched on myosin production and have fused to form a muscle cell with two nuclei (upper right). (b) Somewhat later, almost all the cells have differentiated and fused. (C) high-magnification view, showing characteristic striations (fine transverse stripes) in two of the multinucleate muscle cells. (Courtesy of Jacqueline Gross and terence partridge.) damage to muscle fber

1	Figure 22–20 Satellite cells repair skeletal muscle fibers. (a) the specimen is stained with an antibody (red) against a muscle cadherin, m-cadherin, which is present on both the satellite cell and the muscle fiber and is concentrated at the site where their membranes are in contact. the nuclei of the muscle fiber are stained green, and the nucleus of the satellite cell is stained blue. (b) Schematic of the repair of a damaged muscle fiber by proliferation and fusion of satellite cells. (a, courtesy of terence partridge.)

1	Skeletal muscle fibers are one of four main categories of vertebrate cells specialized for contraction, and they are responsible for all voluntary movement. Each skeletal muscle fiber is a syncytium and develops by the fusion of many myoblasts. Myoblasts proliferate extensively, but once they have fused, they can no longer divide. Fusion generally follows the onset of myoblast differentiation, in which many genes encoding muscle-specific proteins are switched on coordinately. Some myoblasts persist in a quiescent state as satellite cells in adult muscle; when a muscle is damaged, these cells are reactivated to proliferate and to fuse in order to replace the muscle cells that have been lost. They are the stem cells of skeletal muscle, and exhaustion of their regenerative capacity is responsible for some forms of muscular dystrophy as well as for the decline of muscle mass in old age. blood veSSelS, lymphatiCS, and endothelial CellS

1	blood veSSelS, lymphatiCS, and endothelial CellS Almost all tissues depend on a blood supply, and the blood supply depends on endothelial cells, which form the linings of the blood vessels. Endothelial cells have a remarkable capacity to adjust their number and arrangement to suit local requirements. They create an adaptable life-support system, extending by cell migration into almost every region of the body. If it were not for endothelial cells extending and remodeling the network of blood vessels, tissue growth and repair would be impossible. Cancerous tissue is as dependent on a blood supply as is normal tissue, and this has led to a surge of interest in endothelial cell biology, in the hope that it may be possible to block the growth of tumors by attacking the endothelial cells that bring them nourishment.

1	The largest blood vessels are arteries and veins, which have a thick, tough wall of connective tissue and many layers of smooth muscle cells (Figure 22–21). The inner wall is lined by an exceedingly thin single sheet of endothelial cells, the endothelium, separated from the surrounding outer layers by a basal lamina. The amounts of connective tissue and smooth muscle in the vessel wall vary according to the vessel’s diameter and function, but the endothelial lining is always present. In the finest branches of the vascular tree—the capillaries and sinusoids—the walls consist of nothing but endothelial cells and a basal lamina (Figure 22–22), together with a few scattered pericytes. Related to vascular smooth muscle cells, pericytes wrap themselves around the small vessels and strengthen them (Figure 22–23). basal lamina nucleus of endothelial cell basal lamina lumen of capillary tight junction 2 µm loose connective elastic lamina

1	basal lamina nucleus of endothelial cell basal lamina lumen of capillary tight junction 2 µm loose connective elastic lamina Figure 22–21 Diagram of a small artery in cross section. the endothelial cells form the endothelial lining, which although inconspicuous, is the fundamental component. Compare with the capillary in Figure 22–22. Figure 22–22 Capillaries. electron micrograph (left) of a cross section of a small capillary in the pancreas. the wall is formed by a single endothelial cell surrounded by a basal lamina, as seen most clearly in the drawing to the right. (From R.p. bolender, J. Cell Biol. 61:269– 287, 1974. with permission from the Rockefeller university press.)

1	Less obvious than the blood vessels are the lymphatic vessels. These carry no blood and have much thinner and more permeable walls than the blood vessels. They provide a drainage system for the fluid (lymph) that seeps out of the blood vessels, as well as an exit route for white blood cells that have migrated from blood vessels into the tissues. Less happily, they can also provide the path by which cancer cells escape from a primary tumor to invade other tissues. The lymphatics form a branching system of tributaries, all ultimately discharging into a single large lymphatic vessel, the thoracic duct, which opens into a large vein close to the heart. Like blood vessels, lymphatics are lined with endothelial cells.

1	Thus, endothelial cells line the entire blood and lymphatic vascular system, from the heart to the smallest capillary, and they control the passage of materials—and the transit of white blood cells—into and out of the bloodstream. Arteries, veins, capillaries, and lymphatics all develop from small vessels constructed primarily of endothelial cells and a basal lamina: connective tissue and smooth muscle are added later where required, under the influence of signals from the endothelial cells. To understand how the vascular system comes into being and how it adapts to the changing needs of tissues, we have to understand endothelial cells. How do they become so widely distributed, and how do they form channels that connect in just the right way for blood to circulate through the tissues and for lymph to drain back to the bloodstream?

1	Endothelial cells originate at specific sites in the early embryo from precursors that also give rise to blood cells. From these sites, the early embryonic endothelial cells migrate, proliferate, and differentiate to form the first rudiments of blood vessels—a process called vasculogenesis. Subsequent growth and branching of the vessels throughout the body occurs mainly by proliferation and movement of the endothelial cells of these first vessels, in a process called angiogenesis.

1	Angiogenesis occurs in a broadly similar way in the young organism as it grows and in the adult during tissue repair and remodeling. We can watch the behavior of the cells in naturally transparent structures, such as the cornea of the eye or the fin of a tadpole, or in tissue culture, or in the embryo. The embryonic retina, which blood vessels invade according to a predictable timetable, provides a convenient example for experimental study. Each new vessel originates as a capillary sprout from the side of an existing capillary or small venule (Figure 22–24). At the tip of the sprout, leading the way, is an endothelial cell with a distinctive character. This tip cell has a pattern of gene expression somewhat different from that of the endothelial stalk cells following behind it, and while they divide, it does not. The tip cell’s most striking feature is that it puts out many long filopodia, resembling

1	Figure 22–24 Angiogenesis. (a) a new blood capillary forms by the sprouting of an endothelial cell from the wall of an existing small vessel. an endothelial tip cell, with many filopodia, leads the advance of each capillary sprout. the endothelial stalk cells trailing behind the tip cell become hollowed out to form a lumen. (b) blood capillaries sprouting in the retina of an embryonic mouse that had a red dye injected into the bloodstream, revealing the capillary lumen opening up behind the tip cell (Movie 22.2). (b, from h. Gerhardt et al., J. Cell Biol. 161:1163–1177, 2003. with permission from the author.) pericytes clinging 10 µmto outer face of small blood vessel

1	Figure 22–23 Pericytes. the scanning electron micrograph shows pericytes wrapping their processes around a small blood vessel (a post-capillary venule) in the mammary gland of a cat. pericytes are also present around capillaries, but are much more sparsely distributed there. (From t. Fujiwara and y. uehara, Am. J. Anat. 170:39–54, 1984. with permission from wiley-liss.) those of a neuronal growth cone. The column of stalk cells behind it, meanwhile, becomes hollowed out to form a lumen.

1	The endothelial tip cells that pioneer the growth of normal capillaries not only look like neuronal growth cones, but also respond similarly to signals in the environment. In fact, many of the same guidance molecules are involved, including the netrins, slits, and ephrins mentioned in our account of neural development in the previous chapter. The corresponding receptors are expressed in the tip cells and guide the vascular sprouts along specific pathways in the embryo, often in parallel with nerves. Perhaps the most important of the guidance molecules for endothelial cells, however, is one that is chiefly dedicated to the control of vascular development: vascular endothelial growth factor, or VEGF.

1	Almost every cell, in almost every tissue of a vertebrate, is located within 50–100 μm of a blood capillary. What mechanism ensures that the system of blood vessels branches into every nook and cranny? How is it adjusted so perfectly to the local needs of the tissues, not only during normal development but also in pathological circumstances? Wounding, for example, induces a burst of capillary growth in the neighborhood of the damage, to satisfy the high metabolic requirements of the repair process (Figure 22–25). Local irritants and infections also cause a proliferation of new capillaries, most of which regress and disappear when the inflammation subsides. Less benignly, a small sample of tumor tissue implanted in the cornea, which normally lacks blood vessels, causes blood vessels to grow quickly toward the implant from the vascular margin of the cornea; the growth rate of the tumor increases abruptly as soon as the vessels reach it.

1	In all these cases, the invading endothelial cells respond to signals produced by the tissue that they invade. The signals are complex, but a key part is played by vascular endothelial growth factor (VEGF). The regulation of blood vessel growth to match the needs of the tissue depends on the control of VEGF production, through changes in the stability of its mRNA and in its rate of transcription. The latter control is relatively well understood. A shortage of oxygen, in practically any type of cell, causes an increase in the intracellular level of a transcription factor called hypoxia-inducible factor 1α (HIF1α). HIF1α stimulates transcription of Vegf (and of other genes whose products are needed when oxygen is in short supply). The VEGF protein is secreted, diffuses through the tissue, and acts on nearby endothelial cells, stimulating them to proliferate, to produce proteases to help them digest their way through the basal lamina of the parent capillary or venule, and to form

1	and acts on nearby endothelial cells, stimulating them to proliferate, to produce proteases to help them digest their way through the basal lamina of the parent capillary or venule, and to form sprouts. The tip cells of the sprouts detect the VEGF gradient and move toward its source. As the new vessels form, bringing blood to the tissue, the oxygen concentration rises. The HIF1α activity then declines, VEGF production is shut off, and angiogenesis comes to a halt (Figure 22–26).

1	Figure 22–25 New capillary formation in response to wounding. Scanning electron micrographs of casts of the system of blood vessels surrounding the margin of the cornea show the reaction to wounding. the casts are made by injecting a resin into the vessels and letting the resin set; this reveals the shape of the lumen, as opposed to the shape of the cells. Sixty hours after wounding, many new capillaries have begun to sprout toward the site of injury, which is just above the top of the picture. their oriented outgrowth reflects a chemotactic response of the endothelial cells to an angiogenic factor released at the wound. (Courtesy of peter C. burger.) Figure 22–26 The regulatory mechanism controlling blood vessel growth according to a tissue’s need for oxygen. lack of oxygen triggers the secretion of veGF, which stimulates angiogenesis. Signals from endothelial Cells Control Recruitment of pericytes and Smooth muscle Cells to Form the vessel wall

1	The vascular network is continually remodeled as it grows and adapts. A newly formed vessel may enlarge; or it may sprout side branches; or it may regress. Smooth muscle and other connective-tissue cells that pack themselves around the endothelium (see Figure 22–23) help to stabilize vessels as they enlarge. This process of vessel wall formation begins with recruitment of pericytes. Small numbers of these cells travel outward in company with the stalk cells of each endothelial sprout. The recruitment and proliferation of pericytes and smooth muscle cells to form a vessel wall depend on platelet-derived growth factor-B (PDGF-B) secreted by the endothelial cells and on PDGF receptors in the pericytes and smooth muscle cells. In mutants lacking this signal protein or its receptor, these vessel wall cells are missing in many regions. As a result, the embryonic blood vessels develop microaneurysms—microscopic pathological dilatations—that eventually rupture, as well as other abnormalities,

1	wall cells are missing in many regions. As a result, the embryonic blood vessels develop microaneurysms—microscopic pathological dilatations—that eventually rupture, as well as other abnormalities, reflecting the importance of signals exchanged in both directions between the exterior cells of the wall and the endothelial cells.

1	Endothelial cells are the fundamental elements of the vascular system. They form a single cell layer that lines all blood vessels and lymphatics and regulates exchanges between the bloodstream and the surrounding tissues. New vessels originate as endothelial sprouts from the walls of existing small vessels. A specialized motile endothelial tip cell at the leading edge of each sprout puts out filopodia that respond to gradients of guidance molecules in the environment, leading the growth of the sprout in much the same way as the growth cone of a neuron is led. The endothelial stalk cells following behind become hollowed out to form a capillary tube. Signals from endothelial cells organize the growth and development of the connective-tissue cells that form the surrounding layers of the vessel wall.

1	A homeostatic mechanism ensures that blood vessels permeate every region of the body. Cells that are short of oxygen increase their concentration of hypoxia-inducible factor 1α (HIF1α), which stimulates the production of vascular endothelial growth factor (VEGF). VEGF acts on endothelial cells, causing them to proliferate and invade the hypoxic tissue to supply it with new blood vessels. As new vessels enlarge, they recruit increasing numbers of pericytes—cells that cling to the outside of the endothelial tube and mature into the smooth muscle coat that is needed to give the vessel strength. a hieRaRChiCal Stem-Cell SyStem: blood Cell FoRmation

1	The function of blood vessels is to carry blood, and it is to blood itself that we now turn. Blood contains many types of cells, with functions that range from the transport of oxygen to the production of antibodies. Some of these cells stay within the vascular system, while others use the vascular system only as a means of transport and perform their function elsewhere. All blood cells, however, have certain similarities in their life history. They all have limited life-spans and are produced throughout the life of the animal. Most remarkably, they are all generated ultimately from a common stem cell, located (in adult humans) in the bone marrow. This hematopoietic (blood-making) stem cell is thus multipotent, giving rise to all the types of terminally differentiated blood cells as well as some other types of cells, such as the osteoclasts in bone, as mentioned earlier. The hematopoietic system is the most complex of the stem-cell systems in the mammalian body, and it is

1	as well as some other types of cells, such as the osteoclasts in bone, as mentioned earlier. The hematopoietic system is the most complex of the stem-cell systems in the mammalian body, and it is exceptionally important in medical practice.

1	Red blood Cells are all alike; white blood Cells Can be Grouped in three main Classes Blood cells can be classified as red or white. The red blood cells, or erythrocytes, remain within the blood vessels and transport O2 and CO2 bound to hemoglobin. The white blood cells, or leukocytes, combat infection and in some cases phagocytose and digest debris. Leukocytes, unlike erythrocytes, must make their way across the walls of small blood vessels and migrate into tissues to perform their tasks. In addition, the blood contains large numbers of platelets, which are not entire cells but small, detached cell fragments or “minicells” derived from the cortical cytoplasm of large cells called megakaryocytes. Platelets adhere specifically to the endothelial cell lining of damaged blood vessels, where they help to repair breaches and aid in blood clotting.

1	All red blood cells belong in a single class, following the same developmental trajectory as they mature, and the same is true of platelets; but there are many distinct types of white blood cells. White blood cells are traditionally grouped into three major categories—granulocytes, monocytes, and lymphocytes—based on their appearance in the light microscope.

1	Granulocytes contain numerous lysosomes and secretory vesicles (or granules) and are subdivided into three classes according to the morphology and staining properties of these organelles (Figure 22–27). The differences in staining reflect major differences of chemistry and function. Neutrophils (also called polymorphonuclear leukocytes because of their multilobed nucleus) are the most common type of granulocyte; they phagocytose and destroy microorganisms, especially bacteria, and thus have a key role in innate immunity to bacterial infection, as discussed in Chapter 24 (see Movie 16.1). Basophils secrete histamine (and, in some species, serotonin) to help mediate inflammatory reactions; they are closely related to mast cells, which reside in connective tissues but are also generated from the hematopoietic stem cells. Eosinophils help to destroy parasites and modulate allergic inflammatory responses.

1	Once they leave the bloodstream, monocytes (see Figure 22–27D) mature into macrophages, which, together with neutrophils, are the main “professional phagocytes” in the body. As discussed in Chapter 13, both types of phagocytic cells contain specialized lysosomes that fuse with newly formed phagocytic vesicles (phagosomes), exposing phagocytosed microorganisms to a barrage of enzymatically produced, highly reactive molecules of superoxide (O2–) and hypochlorite (ClO–, the active ingredient in bleach), as well as to attack by a concentrated mixture of lysosomal hydrolase enzymes that become activated in the phagosome. Macrophages, however, are much larger and longer-lived than neutrophils. They recognize and remove senescent, dead, and damaged cells in many tissues, and they are unique in being able to ingest large microorganisms such as protozoa.

1	Figure 22–27 White blood cells. (a–d) these electron micrographs show (a) a neutrophil, (b) a basophil, (C) an eosinophil, and (d) a monocyte. electron micrographs of lymphocytes are shown in Figure 24–14. each of the cell types shown here has a different function, which is reflected in the distinctive types of secretory granules and lysosomes it contains. there is only one nucleus per cell, but it has an irregular lobed shape, and in (a), (b), and (C) the connections between the lobes are out of the plane of section. (e) a light micrograph of a blood smear stained with the Romanowsky stain, which colors the white blood cells strongly. (Courtesy 2 µm of dorothy bainton.)

1	Romanowsky stain, which colors the white blood cells strongly. (Courtesy 2 µm of dorothy bainton.) Monocytes also give rise to dendritic cells. Like macrophages, dendritic cells are migratory cells that can ingest foreign substances and organisms, but they do not have as active an appetite for phagocytosis and instead have a crucial role as presenters of foreign antigens to lymphocytes to trigger an immune response. Dendritic cells in the epidermis (called Langerhans cells), for example, ingest foreign antigens and carry these trophies back from the skin to present to lymphocytes in lymph nodes.

1	There are two main classes of lymphocytes, both involved in immune responses: B lymphocytes make antibodies, while T lymphocytes kill virus-infected cells and regulate the activities of other white blood cells. In addition, there are lymphocyte-like cells called natural killer (NK) cells, which kill some types of tumor cells and virus-infected cells. The production of lymphocytes is a specialized topic discussed in detail in Chapter 24. Here we concentrate mainly on the development of the other blood cells, often referred to collectively as myeloid cells. Table 22–1 summarizes the various types of blood cells and their functions. the production of each type of blood Cell in the bone marrow is individually Controlled

1	Table 22–1 summarizes the various types of blood cells and their functions. the production of each type of blood Cell in the bone marrow is individually Controlled Most white blood cells function in tissues other than the blood; blood simply transports them to where they are needed. A local infection or injury in any tissue rapidly attracts white blood cells into the affected region as part of the inflammatory response, which helps fight the infection or heal the wound (Movie 22.3).

1	The inflammatory response is complex and is governed by many different signal molecules produced locally by mast cells, nerve endings, platelets, and white blood cells, as well as by the activation of complement (discussed in Chapter 24). Some of these signal molecules act on the endothelial lining of nearby capillaries, helping white blood cells to first stick and then make an exit from the bloodstream into the tissue where they are needed, as described in Chapter 19 (see Figure 19–28 and Movie 19.2). Damaged or inflamed tissues and local endothelial cells secrete other molecules called chemokines, which act as chemoattractants for specific types of white blood cells, causing them to become polarized and crawl toward the source of the attractant. As a result, large numbers of white blood cells enter the affected tissue (Figure 22–28).

1	Other signal molecules produced during an inflammatory response escape into the blood and stimulate the bone marrow to produce more leukocytes and release them into the bloodstream. The regulation tends to be cell-type specific: some bacterial infections, for example, cause a selective increase in neutrophils, while infections with some protozoa and other parasites cause a selective increase in eosinophils. (For this reason, physicians routinely use differential white blood cell counts to aid in the diagnosis of infectious and other inflammatory diseases.) In other circumstances, erythrocyte production is selectively increased—for example, in response to anemia (lack of hemoglobin) due to blood loss, and in the process of acclimatization when one goes to live at high altitude, where oxygen is scarce. Thus, blood cell formation, or hematopoiesis, necessarily involves complex controls, which regulate the production of each type of blood cell individually to meet changing needs.

1	Figure 22–28 Chemotaxis of white blood cells to damaged tissue. a chemoattractive signal released from a site of damage, which is toward the bottom of the page, causes white blood cells to exit from the capillary by crawling between adjacent endothelial cells, as shown. megakaryocyte process budding off platelets endothelial cell of sinus wall bone marrow Contains multipotent hematopoietic Stem Cells, able to Give Rise to all Classes of blood Cells

1	In the bone marrow, the developing blood cells and their precursors, including the stem cells, are intermingled with one another, as well as with fat cells and other stromal cells (connective-tissue cells), which produce a delicate supporting meshwork of collagen fibers and other extracellular matrix components. In addition, the whole tissue is richly supplied with thin-walled blood vessels, called blood sinuses, into which the new blood cells are discharged. Megakaryocytes are also present; these, unlike other blood cells, remain in the bone marrow when mature and are one of its most striking features, being extraordinarily large (diameter up to 60 μm) with a highly polyploid nucleus. They normally lie close beside blood sinuses, and they extend processes through holes in the endothelial lining of these vessels; platelets pinch off from the processes and are swept away into the blood (Figure 22–29 and Movie 22.4).

1	Because of the complex arrangement of the cells in bone marrow, it is difficult to identify in ordinary tissue sections any but the immediate precursors of the mature blood cells. There is no obvious visible characteristic by which we can recognize the ultimate stem cells. In the case of hematopoiesis, the stem cells were first identified by a functional assay that exploited the wandering lifestyle of blood cells and their precursors.

1	When an animal is exposed to a large dose of x-rays, most of the hematopoietic cells are destroyed and the animal dies within a few days as a result of its inability to manufacture new blood cells. The animal can be saved, however, by a transfusion of cells taken from the bone marrow of a healthy, immunologically compatible donor. Among these cells there are some that can colonize the irradiated host and permanently reequip it with hematopoietic tissue (Figure 22–30). Such experiments prove that the marrow contains hematopoietic stem cells. They also show how we can assay for the presence of hematopoietic stem cells and hence discover the molecular features that distinguish them from other cells.

1	For this purpose, cells taken from bone marrow are sorted (using a fluorescence-activated cell sorter) according to the surface antigens that they display, and the different fractions are transfused back into irradiated mice. If a fraction rescues an irradiated host mouse, it must contain hematopoietic stem cells. In this way, it has been possible to show that the hematopoietic stem cells are characterized by a specific combination of cell-surface proteins, and by appropriate sorting we can obtain virtually pure stem-cell preparations. The stem cells turn out to be a tiny fraction of the bone marrow population—about 1 cell in 50,000–100,000; but this is enough. A single such cell injected into a host mouse with defective hematopoiesis is sufficient to reconstitute its entire hematopoietic system, generating a complete set of blood cell types, as well as fresh stem cells. This and other experiments (using artificial lineage markers) show that the individual hematopoietic stem cell is

1	system, generating a complete set of blood cell types, as well as fresh stem cells. This and other experiments (using artificial lineage markers) show that the individual hematopoietic stem cell is multipotent and can give rise to the complete range of blood cell types, both myeloid and lymphoid, as well as to new stem cells like itself (Figure 22–31).

1	Figure 22–29 A megakaryocyte among other developing blood cells in the bone marrow. the megakaryocyte’s enormous size results from its having a highly polyploid nucleus. one megakaryocyte produces about 10,000 platelets, which split off from long processes that extend through holes in the walls of an adjacent blood sinus. mouse survives; the injected stem cells colonize its hematopoietic tissues and generate a steady supply of new blood cells Figure 22–30 Rescue of an irradiated mouse by a transfusion of bone marrow cells. an essentially similar procedure is used in the treatment of leukemia in human patients by bone marrow transplantation. Commitment is a Stepwise process

1	Commitment is a Stepwise process Hematopoietic stem cells do not jump directly from a multipotent state into a commitment to just one pathway of differentiation; instead, they go through a series of progressive restrictions. The first step, usually, is commitment to either a myeloid or a lymphoid fate. This is thought to give rise to two kinds of progenitor cells, one capable of generating large numbers of all the different types of myeloid cells, and the other giving rise to large numbers of all the different types of lymphoid cells. Further steps give rise to progenitors committed to the production of just one cell type. The steps of commitment correlate with changes in the expression of specific transcription regulators, needed for the production of different subsets of blood cells. divisions of Committed progenitor Cells amplify the number of Specialized blood Cells

1	Hematopoietic progenitor cells generally become committed to a particular pathway of differentiation long before they cease proliferating and terminally differentiate. The committed progenitors go through many rounds of cell division to amplify the ultimate number of cells of the given specialized type. In this way, a single stem-cell division can lead to the production of thousands of differentiated progeny, which explains why the number of stem cells is such a small fraction of the total population of hematopoietic cells. For the same reason, a high rate of blood cell production can be maintained even though the stem-cell division rate is low. The smaller the number of division cycles that the stem cells themselves have to undergo in the course of a lifetime, the lower the risk of generating stem-cell mutations, which would give rise to persistent mutant clones of cells in the body—a particular danger in the hematopoietic system where, as discussed in Chapter 20, a relatively small

1	stem-cell mutations, which would give rise to persistent mutant clones of cells in the body—a particular danger in the hematopoietic system where, as discussed in Chapter 20, a relatively small accumulation of mutations can be sufficient to cause cancer. A low rate of stem-cell division also slows the process of replicative cell senescence (discussed in Chapter 17).

1	Figure 22–31 A tentative scheme of hematopoiesis. the multipotent stem cell normally divides infrequently to generate either more multipotent stem cells, which are self-renewing, or committed progenitor cells, which are limited in the number of times that they can divide before differentiating to form mature blood cells. as they go through their divisions, the progenitors become progressively more specialized in the range of cell types that they can give rise to, as indicated by the branching of this cell-lineage diagram. in adult mammals, all of the cells shown develop mainly in the bone marrow—except for t lymphocytes, which as indicated develop in the thymus, and macrophages and osteoclasts, which develop from blood monocytes. Some dendritic cells may also derive from monocytes.

1	The stepwise nature of commitment means that the hematopoietic system can be viewed as a hierarchical family tree of cells. Multipotent stem cells give rise to committed progenitor cells, which are specified to give rise to only one or a few blood cell types. The committed progenitors divide rapidly, but only a limited number of times, before they terminally differentiate into cells that divide no further and die after several days or weeks. Figure 22–31 depicts the hematopoietic family tree. It should be noted, however, that variations are thought to occur: not all stem cells generate the identical patterns of progeny via precisely the same sequence of steps.

1	Like the stem cells of other tissues, hematopoietic stem cells depend on signals from their niche, in this case created by the specialized connective tissue of the bone marrow. (This is the site in adult humans; during development, and in nonhuman mammals such as the mouse, hematopoietic stem cells can also make their home in other tissues—notably liver and spleen.) When they lose contact with their niche, the hematopoietic stem cells tend to lose their stem-cell potential (Figure 22–32). Evidently the loss of potency is not absolute or instantaneous, however, since the stem cells can still survive journeys via the bloodstream to colonize other sites in the body.

1	While the stem cells depend on contact with bone marrow stromal cells for longterm maintenance, their committed progeny do not, or at least not to the same degree. These cells can thus be dispersed and cultured in a semisolid matrix of dilute agar or methylcellulose, and factors derived from other cells can be added artificially to the medium. The semisolid matrix inhibits migration, so that the progeny of each isolated precursor cell remain together as an easily distinguishable colony. A single committed neutrophil progenitor, for example, may give rise to a clone of thousands of neutrophils. Such culture systems have provided a way to assay for the factors that support hematopoiesis and hence to purify them and explore their actions. These substances are glycoproteins and are usually called colony-stimulating factors (CSFs). Some of these factors circulate in the blood and act as hormones, while others act in the bone marrow as secreted local mediators; still others take the form of

1	colony-stimulating factors (CSFs). Some of these factors circulate in the blood and act as hormones, while others act in the bone marrow as secreted local mediators; still others take the form of membrane-bound signals that act through cell– cell contact.

1	An important example of the latter is a protein called Steel or Stem Cell Factor (SCF ). This is expressed both in the bone marrow stroma (where it helps to define the stem-cell niche) and along pathways of migration, and it occurs both in a membrane-bound and a soluble form. It binds to a receptor tyrosine kinase called Kit, and it is required during development for guidance and survival not only of hematopoietic cells but also of other migratory cell types—specifically, germ cells and pigment cells. erythropoiesis depends on the hormone erythropoietin The best understood of the CSFs that act as hormones is the glycoprotein erythropoietin, which is produced in the kidneys and regulates erythropoiesis, the formation of red blood cells, to which we now turn.

1	The erythrocyte is by far the most common type of cell in the blood (see Table 22–1). When mature, it is packed full of hemoglobin and contains hardly any of the usual cell organelles. In an erythrocyte of an adult mammal, even the nucleus, endoplasmic reticulum, mitochondria, and ribosomes are absent, having been extruded from the cell in the course of its development (Figure 22–33). The erythrocyte therefore cannot grow or divide, and it has a limited life-span—about 120 days in humans or 55 days in mice. Worn-out erythrocytes are phagocytosed and digested by macrophages in the liver and spleen, which remove more than 1011

1	Figure 22–32 Dependence of hematopoietic stem cells on contact with stromal cells. the contact-dependent interaction between the kit receptor and its ligand is one of several signaling mechanisms thought to be involved in hematopoietic stem-cell maintenance. the real system is certainly more complex. moreover, the dependence of hematopoietic cells on contact with stromal cells cannot be absolute, since small numbers of the functional stem cells can be found free in the circulation. SCF, stem-cell factor. Figure 22–33 A developing red blood cell (erythroblast). the cell is shown extruding its nucleus to become an immature erythrocyte (a reticulocyte), which then leaves the bone marrow and passes into the bloodstream. the reticulocyte will lose its mitochondria and ribosomes within a day or two to become a mature erythrocyte. erythrocyte clones develop in the bone marrow on the surface of a macrophage, which phagocytoses and digests the nuclei discarded by the erythroblasts.

1	senescent erythrocytes in each of us each day. Young erythrocytes actively protect themselves from this fate: they have a protein on their surface that binds to an inhibitory receptor on macrophages and thereby prevents their phagocytosis. A lack of oxygen or a shortage of erythrocytes stimulates specialized cells in the kidney to synthesize and secrete increased amounts of erythropoietin into the bloodstream. The erythropoietin, in turn, boosts the production of erythrocytes. The effect is rapid: the rate of release of new erythrocytes into the bloodstream rises steeply 1–2 days after an increase in erythropoietin levels in the bloodstream. Clearly, the hormone must act on cells that are close precursors of the mature erythrocytes.

1	The cells that respond to erythropoietin can be identified by culturing bone marrow cells in a semisolid matrix in the presence of erythropoietin. In a few days, colonies of about 60 erythrocytes appear, each founded by a single committed erythroid progenitor cell. This progenitor depends on erythropoietin for its survival as well as its proliferation. It does not yet contain hemoglobin, and it is derived from an earlier type of committed erythroid progenitor whose survival and proliferation are governed by other factors.

1	The two classes of cells dedicated to phagocytosis, neutrophils and macrophages, develop from a common progenitor cell called a granulocyte/macrophage (GM) progenitor cell. Like the other granulocytes (eosinophils and basophils), neutrophils circulate in the blood for only a few hours before migrating out of capillaries into the connective tissues or other specific sites, where they survive for only a few days. They then die by apoptosis and are phagocytosed by macrophages. Macrophages, in contrast, can persist for months or perhaps even years outside the bloodstream, where they can be activated by local signals to resume proliferation.

1	At least seven distinct CSFs that stimulate neutrophil and macrophage colony formation in culture have been defined, and some or all of these are thought to act in different combinations to regulate the selective production of these cells in vivo. These CSFs are synthesized by various cell types—including endothelial cells, fibroblasts, macrophages, and lymphocytes—and their concentration in the blood typically increases rapidly in response to bacterial infection in a tissue, thereby increasing the number of phagocytic cells released from the bone marrow into the bloodstream. The CSFs not only operate on the precursor cells to promote the production of differentiated progeny, they also activate the specialized functions (such as phagocytosis and target-cell killing) of the terminally differentiated cells. CSFs can be synthesized artificially and are now widely used in human patients to stimulate the regeneration of hematopoietic tissue and to boost resistance to infection.

1	the behavior of a hematopoietic Cell depends partly on Chance CSFs are defined as factors that promote the production of colonies of differentiated blood cells. But precisely what effect does a CSF have on an individual hematopoietic cell? The factor might control the rate of cell division or the number of division cycles that the progenitor cell undergoes before differentiating; it might act late in the hematopoietic lineage to facilitate differentiation; it might act early to influence commitment; or it might simply increase the probability of cell survival (Figure 22–34). By monitoring the fate of isolated individual hematopoietic cells in culture, it has been possible to show that a single CSF, such as granulocyte/ 1. Frequency of stem-cell division 2. Probability of stem-cell death 3. Probability that stem-cell daughter will become a committed progenitor cell of the given type 4. Division cycle time of committed progenitor cell 5. Probability of progenitor-cell death 6.

1	Probability that stem-cell daughter will become a committed progenitor cell of the given type 4. Division cycle time of committed progenitor cell 5. Probability of progenitor-cell death 6. Number of committed progenitorcell divisions before terminal differentiation 7. Lifetime of differentiated cells macrophage CSF, can exert all these effects, although it is still not clear which are most important in vivo.

1	Studies in vitro indicate, moreover, that there is a large element of chance in the way a hematopoietic cell behaves—a reflection, presumably, of “noise” in the genetic control system, as discussed in Chapters 7 and 8. If two sister cells are taken immediately after a cell division and cultured apart under identical conditions, they frequently give rise to colonies that contain different types of blood cells or the same types of blood cells in different numbers. Thus, both the programming of cell division and the process of commitment to a particular path of differentiation seem to involve random events at the level of the individual cell, even though the behavior of the multicellular system as a whole is regulated in a reliable way. The sequence of cell fate restrictions shown earlier, in Figure 22–31, conveys the impression of a program executed with computer-like logic and precision. Individual cells may be more varied, quirky, and erratic, and may sometimes progress by other

1	in Figure 22–31, conveys the impression of a program executed with computer-like logic and precision. Individual cells may be more varied, quirky, and erratic, and may sometimes progress by other decision pathways from the stem-cell state toward terminal differentiation.

1	Regulation of Cell Survival is as important as Regulation of Cell proliferation The default behavior of hematopoietic cells in the absence of CSFs is death by apoptosis (discussed in Chapter 18), and the control of cell survival plays a central part in regulating the numbers of blood cells. The amount of apoptosis in the vertebrate hematopoietic system is enormous: billions of neutrophils die in this way each day in an adult human, for example. In fact, most neutrophils produced in the bone marrow die there without ever functioning. This futile cycle of production and destruction presumably serves to maintain a reserve supply of cells that can be promptly mobilized to fight infection whenever it flares up, or phagocytosed and digested for recycling when all is quiet. Compared with the life of the organism, the lives of cells are cheap.

1	Too little cell death can be as dangerous to the health of a multicellular organism as too much proliferation. As noted in Chapter 18, mutations that inhibit cell death by causing excessive production of the intracellular apoptosis inhibitor Figure 22–34 Some of the parameters through which the production of blood cells of a specific type might be regulated. Studies in culture suggest that various colony-stimulating factors (CSFs) can affect all of these aspects of hematopoiesis. Bcl2 promote the development of cancer in B lymphocytes. Indeed, the capacity for unlimited self-renewal is a dangerous property for any cell to possess. Many cases of leukemia arise through mutations that confer this capacity on committed hematopoietic precursor cells that would normally be fated to differentiate and die after a limited number of division cycles.

1	The many types of blood cells, including erythrocytes, lymphocytes, granulocytes, and macrophages, all derive from a common multipotent stem cell. In the adult, hematopoietic stem cells are found mainly in bone marrow, and they depend on signals from the marrow stromal (connective-tissue) cells to maintain their stem-cell character. The stem cells are few and far between, and they normally divide infrequently to produce more stem cells (self-renewal) and various committed progenitor cells (transit amplifying cells), each able to give rise to only one or a few types of blood cells. The committed progenitor cells divide extensively under the influence of various protein signal molecules (colony-stimulating factors, or CSFs) and then terminally differentiate into mature blood cells, which usually die after several days or weeks.

1	Studies of hematopoiesis have been greatly aided by in vitro assays in which stem cells or committed progenitor cells form clonal colonies when cultured in a semisolid matrix. The progeny of stem cells seem to make their choices between alternative developmental pathways in a partly random manner. Cell death by apoptosis, controlled by the availability of CSFs, also plays a central part in regulating the numbers of mature differentiated blood cells.

1	As we have seen, many of the tissues of the body are not only self-renewing but also self-repairing, and this is largely thanks to stem cells and the feedback controls that regulate their behavior and maintain homeostasis. There are, however, limits to what these natural repair mechanisms can achieve. In most parts of the human brain, for example, nerve cells that die, as in Alzheimer’s disease, are not replaced. Likewise, when heart muscle dies for lack of oxygen, as in a heart attack, it is replaced by scar tissue rather than new heart muscle.

1	Some animals do far better than humans and can regenerate entire organs, such as whole limbs, after amputation. Among the invertebrates, there are some species that can even regenerate all the tissues of the body from a single somatic cell. These phenomena encourage the hope that human cells might be coaxed by artificial measures into similar feats of repair and regeneration, so as to replace the skeletal muscle fibers that degenerate in victims of muscular dystrophy, the nerve cells that die in patients with Parkinson’s disease, the insulin-secreting cells that are lacking in type 1 diabetics, the heart muscle cells that die in a heart attack, and so on. As we learn more about the basic cell biology, these goals, once only a dream, are beginning to seem attainable.

1	In this section, we start with some examples of the remarkable regenerative abilities of some animal species, as an indication of what is possible in principle. We shall then discuss how we can improve upon the natural repair processes of the human body and treat disease by exploiting the properties of the various types of stem cells found in human tissues. In the final section of the chapter, we shall see how a deeper understanding of the molecular biology of cell differentiation and of stem cells has revealed ways to convert one type of cell into another, opening up radically new possibilities.

1	Schmidtea mediterranea is a small freshwater flatworm, or planarian, just under a centimeter long when grown to full size (Figure 22–35). It has an epidermis, a gut, 0.2 mm pharynx a brain, a pair of primitive eyes, a peripheral nervous system, musculature, and excretory and reproductive organs—most of the basic body parts familiar in other animals, although all relatively simple by vertebrate standards and built from about 20–25 distinct differentiated cell types. For more than a century, planarians such as Schmidtea have intrigued biologists because of their extraordinary capacity for regeneration: a small tissue fragment taken from almost any part of the body will reorganize itself and grow to form a complete new animal. This property goes with another: when the animal is starved, it gets smaller and smaller, by reducing its cell numbers while maintaining essentially normal body proportions. This behavior is called degrowth, and it can continue until the animal is as little as

1	it gets smaller and smaller, by reducing its cell numbers while maintaining essentially normal body proportions. This behavior is called degrowth, and it can continue until the animal is as little as one-twentieth or even a smaller fraction of its full size. Supplied with food, it will grow back to full size again. Cycles of degrowth and growth can be repeated indefinitely, without impairing survival or fertility.

1	Underlying this behavior is a process of continual cell turnover. Along with the differentiated cells, which do not divide, there is a population of small, apparently undifferentiated dividing cells called neoblasts. The neoblasts constitute about 20% of the cells in the body and are widely distributed within it; by cell division, they serve as stem cells for the production of new differentiated cells. Differentiated cells, meanwhile, are continually dying by apoptosis, allowing their corpses to be phagocytosed and digested by neighboring cells. Through this cell cannibalism, the constituents of the dying cells can be efficiently recycled. Cell birth continues in a dynamic balance with cell death and cell cannibalism, no matter whether the animal is fed or starved. In conditions of starvation, the balance is evidently tilted toward cell cannibalism, and in conditions of plenty, toward cell birth.

1	A high dose of x-rays halts all cell division, puts a stop to cell turnover, and destroys the capacity for regeneration. The result is death after a delay of several weeks. The animal can be rescued, however, by injecting into it a single neoblast isolated from an unirradiated donor (Figure 22–36). In a certain proportion of cases, the injected cell divides to form a clone of progeny that eventually repopulate the entire body, creating a healthy regenerative individual with an apparently complete set of differentiated cell types as well as dividing neoblasts. Genetic markers prove that these are all derived from the single neoblast that was injected. It follows that at least some neoblasts are totipotent (or at least highly pluripotent) stem cells; that is, cells able to give rise to all (or at least almost all) of the cell types that make up the body of a flatworm, including more neoblasts like themselves.

1	One might think that such powers of regeneration would be a prerogative of small, simple, primitive animals. But some vertebrates, too, especially fish and amphibians, show remarkable regenerative abilities. A newt, for example, can regenerate

1	Figure 22–35 The planarian worm, Schmidtea mediterranea. (a) external view. (b) immunostaining with three different antibodies, revealing the internal anatomy. (a, courtesy of a. Sánchez alvarado; b, from a. Sánchez alvarado, BMC Biol. 10:88, 2012.) injection of a single regeneration of healthy neoblast complete animal a whole amputated limb. In this process, differentiated cells seem to revert to an embryonic character by first forming on the amputation stump a blastema—a small bud resembling an embryonic limb bud. The blastema then grows and its cells differentiate to form a correctly patterned replacement for the limb that has been lost, in what looks like a recapitulation of embryonic limb development (Figure 22–37). A large contribution to the blastema comes from the skeletal muscle cells in the limb stump. These multinucleate cells re-enter the cell cycle, dedifferentiate, and break up into mononucleated cells, which then proliferate within the blastema, before eventually

1	cells in the limb stump. These multinucleate cells re-enter the cell cycle, dedifferentiate, and break up into mononucleated cells, which then proliferate within the blastema, before eventually redifferentiating. But do they redifferentiate only into muscle, or do they behave like neoblasts in the planarian and give rise to the full range of cell types needed to reconstruct the missing part of the limb? Careful lineage tracing, using genetic markers, shows (contrary to previous belief ) that the cells are restricted according to their origins: muscle-derived cells give rise only to muscle, connective-tissue cells only to connective tissues, epidermal cells only to epidermal cells. The cells in the adult vertebrate body are, after all, less adaptable than the cells of the flatworm: by working in concert, they can replace the lost structure, but each cell type is far from totipotent.

1	Why a newt can regenerate a whole limb—as well as many other body parts— but a mammal cannot remains a profound mystery. Stem Cells Can be used artificially to Replace Cells that are diseased or lost: therapy for blood and epidermis Earlier in this chapter, we saw how mice can be irradiated to kill off their hematopoietic cells, and then rescued by a transfusion of new stem cells, which repopulate the bone marrow and restore blood cell production (see Figure 22–30). In the same way, patients with some forms of leukemia or lymphoma can be irradiated or chemically treated to destroy their cancerous cells along with the rest of their hematopoietic tissue, and then can be rescued by a transfusion of healthy, noncancerous hematopoietic stem cells. In favorable cases, these can be sorted out from samples of the patient’s own hematopoietic tissue before it is ablated. They are then transfused back afterward, avoiding problems of immune rejection.

1	Figure 22–36 Regeneration of a planarian from a single somatic cell. (a) the distribution of dividing cells (neoblasts, blue) in the adult body. irradiation blocks all cell division and prevents regeneration, but (b) a single unirradiated neoblast cell injected into the irradiated animal is able to reconstitute all tissues. this eventually produces a complete animal that consists entirely of the progeny of this one cell and can regenerate. (adapted from e.m. tanaka and p.w. Reddien, Dev. Cell 21:172–185, 2011.) Figure 22–37 Newt limb regeneration. the time-lapse sequence shows the stages of regeneration after amputation at the level of the humerus. the sequence spans the events of wound healing, dedifferentiation of stump tissues, blastema formation, and redifferentiation. (Courtesy of Susan bryant and david Gardiner.)

1	Figure 22–38 The continuing production of neurons in an adult mouse brain. the brain is viewed from above, in a cut-away section, to show the region lining the ventricles of the forebrain where neural stem cells are found. these cells continually produce progeny that migrate to the olfactory bulb, where they differentiate as neurons. the constant turnover of neurons in the olfactory bulb is presumably linked in some way to the turnover of the olfactory receptor neurons that project to it from the olfactory epithelium, as mentioned earlier. in adult humans, there is a continuing turnover of neurons in the hippocampus, a region specially concerned with learning and memory. (adapted from b. barres, Cell 97:667–670, 1999. with permission from elsevier.)

1	Another example of the use of stem cells is in the repair of the skin after extensive burns. By culturing cells from undamaged regions of the burned patient’s skin, it is possible to obtain epidermal stem cells quite rapidly in large numbers. These can then be used (through rather long and complicated procedures) to repopulate the damaged body surface. neural Stem Cells Can be manipulated in Culture and used to Repopulate the Central nervous System The central nervous system (the CNS) is the most complex tissue in the body, at an opposite extreme from the epidermis. And yet fish and amphibians can regenerate large parts of the brain, spinal cord, and eyes after they have been cut away. In adult mammals, however, these tissues have very little capacity for self-repair, and stem cells capable of generating new neurons are hard to find—so hard to find, indeed, that for many years they were thought to be absent.

1	We now know, however, that neural stem cells that generate both neurons and glial cells do persist in certain parts of the adult mammalian brain (Figure 22–38). Neuronal turnover occurs on a dramatic scale in certain songbirds’ brains, where large numbers of neurons die each year and are replaced by newborn neurons as part of a process by which the birds refine their song for each new breeding season. In the adult human brain, there is a continuing turnover of neurons in the hippocampus, a region specially concerned with learning and memory. Here, plasticity of adult function is associated with turnover of a specific subset of neurons. About 1400 fresh neurons in this class are generated every day, giving a turnover of 1.75% of the population per year.

1	Fragments taken from self-renewing regions of the adult brain, or from the brain of a fetus, can be dissociated and used to establish cell cultures, where they give rise to floating “neurospheres”—clusters consisting of a mixture of neural stem cells with neurons and glial cells derived from the stem cells. These neurospheres can be propagated through many cell generations, or their cells can be taken at any time and implanted back into the brain of an intact animal. Here they will produce differentiated progeny, in the form of neurons and glial cells.

1	Using slightly different culture conditions, with the right combination of growth factors in the medium, the neural stem cells can be grown as a monolayer and induced to proliferate as an almost pure stem-cell population without attendant differentiated progeny. By a further change in the culture conditions, these cells can be induced at any time to differentiate to give either a mixture of neurons and glial cells (Figure 22–39), or just one of these two cell types, according to the composition of the culture medium.

1	Neural stem cells, whether derived as above or from pluripotent stem cells as described in the next section, can be grafted into an adult brain. Once there, they show a remarkable ability to adjust their behavior to match their new location. Stem cells from the mouse hippocampus, for example, when implanted in the mouse olfactory-bulb-precursor pathway (see Figure 22–38), give rise to neurons that become correctly incorporated into the olfactory bulb. This capacity of neural stem cells and their progeny to adapt to a new environment in animals suggests applications in the treatment for diseases where neurons degenerate, and pure culture of neural stem cells (B) mixture (C) of differentiated neurons (red) and glial cells (green); cell nuclei dissociate cells and dissociate and switch to are blueculture in suspension culture as monolayer medium 3in medium 1 in medium 2 for injuries of the central nervous system. For example, might it be possible to use injected neural stem cells to

1	are blueculture in suspension culture as monolayer medium 3in medium 1 in medium 2 for injuries of the central nervous system. For example, might it be possible to use injected neural stem cells to replace the neurons that die in Parkinson’s disease or to repair accidents that sever the spinal cord?

1	Animals vary in their capacity for regeneration. At one extreme, planarian worms contain stem cells (neoblasts) that support continual turnover of all cell types, and an entire worm can be regenerated from practically any small body fragment or even from a single neoblast cell. Newts can regenerate limbs and other large body parts after amputation, but the cells remain restricted according to their origins: muscle cells in the regenerate derive from muscle, epidermis from epidermis, and so on. In mammals, regeneration is more limited. Nevertheless, it is becoming possible to go beyond the natural limits of wound healing by exploiting stem-cell biology. Thus, certain regions of the nervous system contain stem cells that support production of neurons in these sites throughout life. Neural stem cells can be obtained from these sites or from fetal brains, grown in culture, and then grafted back into other sites in the brain, where they are able to generate neurons appropriate to the new

1	stem cells can be obtained from these sites or from fetal brains, grown in culture, and then grafted back into other sites in the brain, where they are able to generate neurons appropriate to the new location.

1	When cells are transplanted from one site in the mammalian body to another or are removed from the body and maintained in culture, they remain largely faithful to their origins. Each type of specialized cell has a memory of its developmental history and seems fixed in its specialized fate. Some limited transformations can certainly occur, as we saw in our account of the connective-tissue cell family, and some stem cells can generate a variety of differentiated cell types, but the possibilities are restricted. Each type of stem cell serves for the renewal of one particular type of tissue, and the whole pattern of self-renewing and differentiated cells in the adult body is amazingly stable. What, at a fundamental molecular level, is the nature of these stable differences between cell types? Is there any way to override the cell memory mechanisms and force a switch from one state to another that is radically different?

1	We have already discussed these fundamental questions from a general standpoint in Chapter 7. Here we consider them more closely in the context of stem-cell biology, where there has been a recent revolution in our understanding and in our ability to manipulate states of cell differentiation. With further research, these advances would seem to have important practical consequences. Figure 22–39 Neural stem cells. Shown are the steps leading from fetal brain tissue, via neurospheres (a), to a pure culture of neural stem cells (b). these stem cells can be kept proliferating as such indefinitely, or, through a change of medium, can be caused to differentiate (C) into neurons (red) and glial cells (green). neural stem cells with the same properties can also be derived, via a similar series of steps, from embryonic stem (eS) or induced pluripotent stem (ipS) cells (discussed later in this chapter). (micrographs from l. Conti et al., PLoS Biol. 3:1594–1606, 2005.)

1	If we cannot switch the basic character of a specialized cell by changing its environment, can we do so by interfering with its inner workings in a more direct and drastic way? An extreme treatment of this sort is to take the nucleus of the cell and transplant it into the cytoplasm of a large cell of a different type. If the specialized character is defined and maintained by cytoplasmic factors, the transplanted nucleus should switch its pattern of gene expression to conform with that of the host cell. In Chapter 7, we described a famous experiment of this sort, using the frog Xenopus. In this experiment, the nucleus of a differentiated cell (a cell from the lining of a tadpole’s gut) was used to replace the nucleus of an oocyte (an egg-cell precursor arrested in prophase of the first meiotic division, in readiness for fertilization). The resulting hybrid cell went on, in a certain fraction of cases, to develop into a complete normal frog (see Figure 7–2A). This was crucial evidence

1	division, in readiness for fertilization). The resulting hybrid cell went on, in a certain fraction of cases, to develop into a complete normal frog (see Figure 7–2A). This was crucial evidence for what is now a central principle of developmental biology: the cell nucleus, even that of a differentiated cell, contains a complete genome, capable of supporting development of all normal cell types. At the same time, the experiment showed that cytoplasmic factors can indeed reprogram a nucleus: the oocyte cytoplasm can drive the gut cell nucleus back to an early embryonic state, from which it can then step through the changing patterns of gene expression that lead all the way to a complete adult organism.

1	The full story, however, is not quite so simple. First, the reprogramming in such experiments is not perfect. When the transplanted nucleus is taken from a gut cell, for example, a gene that is normally specific to the gut is found to be expressed persistently, even in the muscle cells of the final animal. Second, the experiment succeeds in only a limited proportion of cases, and this success rate becomes lower and lower, the more mature the animal from which the transplanted nucleus is taken: very large numbers of transplantations must be done to score a single success if the nucleus comes from a differentiated cell of an adult frog.

1	Nuclear transplantation can be done in mammals too, with basically similar results. Thus, a nucleus taken from a differentiated cell in the mammary gland of an adult sheep and transplanted into an enucleated sheep’s egg was able to support development of an apparently normal sheep—the famous Dolly. Again, the success rate is low: many transplantations have to be done to obtain one such individual. Reprogramming of a transplanted nucleus involves drastic epigenetic Changes

1	In a typical fully differentiated cell, there seem to be mechanisms maintaining the pattern of gene expression that cytoplasmic factors cannot easily override. An obvious possibility is that the stability of the pattern of gene expression in an adult cell may depend, in part at least, on self-perpetuating modifications of chromatin, as discussed in Chapter 4. As explained in Chapter 7, the phenomenon of X-inactivation in mammals provides a clear example of such epigenetic control. Two X chromosomes exist side by side in each female cell, exposed to the same chemical environment, but while one remains active, the other persists from one cell generation to the next in a condensed inactive state; cytoplasmic factors cannot be responsible for the difference, which must instead reflect mechanisms intrinsic to the individual chromosome. Elsewhere in the genome also, controls at the level of chromatin act in combination with other forms of regulation to govern the expression of each gene.

1	intrinsic to the individual chromosome. Elsewhere in the genome also, controls at the level of chromatin act in combination with other forms of regulation to govern the expression of each gene. Genes can be shut down completely, or switched on constitutively, or maintained in a labile state where they can be readily switched on or off according to changing circumstances.

1	The reprogramming of a nucleus transplanted into an oocyte involves dramatic changes in chromatin. The nucleus swells, increasing its volume 50-fold as the chromosomes decondense; there is a wholesale alteration in patterns of methylation of DNA and histones; the standard histone H1 (the histone that links adjacent nucleosomes) is replaced by a variant form that is peculiar to the oocyte and early embryo; and the preexisting type of histone H3 is also replaced at many sites by a distinct isoform. Evidently, the egg contains factors that reset the state of the chromatin in the nucleus, wiping out old histone modifications on chromatin and imposing new ones. Reprogrammed in this way, the genome becomes competent once again to initiate embryonic development and to give rise to the full range of differentiated cell types. embryonic Stem (eS) Cells Can Generate any part of the body

1	embryonic Stem (eS) Cells Can Generate any part of the body A fertilized egg, or an equivalent cell produced by nuclear transplantation, is a remarkable thing: it can generate a whole new multicellular individual, and that means that it can give rise to every normal type of specialized cell, including even egg or sperm cells for production of the next generation. A cell in such a state is said to be totipotent, and it is said to be pluripotent if it can give rise to most cell types but not absolutely all. Nevertheless, such a progenitor is not a stem cell: it is not self-renewing, but is instead dedicated to a program of progressive differentiation. If it were the only available starting point for study and exploitation of pluripotent cells, the enterprise would require a continual supply of fresh fertilized eggs or fresh nuclear transplantation procedures—an awkward requirement for studies in experimental animals, and unacceptable for practical applications in humans.

1	Here, however, nature has been unexpectedly kind to scientists. It is possible to take an early mouse embryo, at the blastocyst stage, and through cell culture to derive from it a class of stem cells called embryonic stem cells, or ES cells. These originate from the inner cell mass of the early embryo (the cluster of cells that give rise to the body of the embryo proper, as opposed to extraembryonic structures), and they have an extraordinary property: given suitable culture conditions, they will continue proliferating indefinitely and yet retain an unrestricted developmental potential. Their only limitation is that they do not give rise to extraembryonic tissues such as those of the placenta. Thus they are classified as pluripotent, rather than totipotent. But this is a minor restriction. If ES cells are put back into a blastocyst, they become incorporated into the embryo and can give rise to all the tissues and cell types in the body, integrating perfectly into whatever site they

1	If ES cells are put back into a blastocyst, they become incorporated into the embryo and can give rise to all the tissues and cell types in the body, integrating perfectly into whatever site they may come to occupy, and adopting the character and behavior that normal cells would show at that site. They can even give rise to germ cells, from which a new generation of animals can be derived (Figure 22–40).

1	ES cells let us move between cell culture, where we can use powerful techniques for genetic transformation and selection, and the intact organism, where we can discover how such genetic manipulations affect development and physiology. Thus, ES cells have opened the way to efficient genetic engineering in mammals, leading to a revolution in our understanding of mammalian molecular biology. Cells with properties similar to those of mouse ES cells can now be derived from early human embryos and from human fetal germ cells, and even, as we shall explain below, from differentiated cells taken from adult mammalian tissues. In this way, one can obtain a potentially inexhaustible supply of pluripotent

1	Figure 22–40 Production and pluripotency of eS cells. eS cells are derived from the inner cell mass (iCm) of the early embryo. the iCm cells are transferred to a culture dish containing an appropriate medium, where they become converted to eS cells and can be kept proliferating indefinitely without differentiating. the eS cells can be taken at any time—after genetic manipulation, if desired—and injected back into the inner cell mass of another early embryo. there they take part in formation of a well-formed chimeric animal that is a mixture of ordinary and eS-derived cells. the eS-derived cells can differentiate into any of the cell types in the body, including germ cells from which a new generation of mice can be produced. these next-generation progeny are no longer chimeric, but consist of cells that all inherit half their genes from the cultured eS cell line.

1	blastocyst develops in (blastocyst) clump of ES cells injected cells become foster mother into a healthy injected into incorporated in inner chimeric mouse; the ES cells may recipient blastocyst cell mass of host blastocyst contribute to any tissue cells. Grown in culture, these can be manipulated, by suitable choice of culture conditions, to give rise to large quantities of almost any type of differentiated cell, opening the way to important practical applications. Before discussing them, however, we consider the underlying biology. a Core Set of transcription Regulators defines and maintains the eS Cell State What is it that gives ES cells and related types of pluripotent stem cells their extraordinary capabilities? What can they tell us about the fundamental mechanisms underlying stemness, cell differentiation, and the stability of the differentiated state?

1	For some attributes, the answer is simple. For example, an essential feature of ES cells is that they must avoid senescence. As discussed in Chapter 17, this is the fate of fibroblasts and many other types of somatic cells: they are limited in the number of times they will divide, in part at least because they lack telomerase activity, with the result that their telomeres become progressively eroded in each division cycle, leading eventually to cell-cycle arrest. ES cells, by contrast, express high levels of active telomerase, allowing them to escape senescence and continue dividing indefinitely. This is a property shared with other, more specialized types of stem cells, such as those of the adult intestine, which similarly can carry on dividing for hundreds or thousands of cycles.

1	The deeper problem is to explain how the whole complex pattern of gene expression in an ES cell is organized and maintained. As a first step, one can look for genes expressed specifically in ES cells or in the corresponding pluripotent cells of the early embryo. This approach identifies a relatively small number of candidate ES-critical genes; that is, genes that seem to be essential in one way or another for the peculiar character of ES cells. A gene called Oct4, for example, is exclusively expressed in ES cells and in related classes of cells in the intact organism—specifically, in the germ-cell lineage and in the inner cell mass and its precursors. Oct4 codes for a transcription regulator. When it is lost from ES cells, they lose their ES cell character; and when it is missing in an embryo, the cells that should specialize as inner cell mass are diverted into an extraembryonic pathway of differentiation and their development is aborted.

1	Fibroblasts Can be Reprogrammed to Create induced pluripotent Stem Cells (ipS Cells)

1	In Chapter 7, we saw that fibroblasts and some other cell types can be driven to switch their character and differentiate as muscle cells if the master muscle-specific transcription regulator MyoD is artificially expressed in them. Could the same technique be used to convert adult cell types into ES cells, through forced expression of factors such as Oct4? This question was tackled by transfecting fibroblasts with retroviral vectors carrying genes that one might hope to have such an effect. A total of 24 candidate ES-critical genes were tested in this way. None of them was able by itself to cause the conversion; but in certain combinations they could do so. In 2006, the first breakthrough experiments whittled down the requirement to a core set of four factors, all of them transcription regulators: Oct4, Sox2, Klf4, and Myc, known as the OSKM factors for short. When coexpressed, these could reprogram mouse fibroblasts, permanently converting them into cells closely similar to ES cells

1	Oct4, Sox2, Klf4, and Myc, known as the OSKM factors for short. When coexpressed, these could reprogram mouse fibroblasts, permanently converting them into cells closely similar to ES cells (Figure 22–41). ES-like cells created in this way are called induced pluripotent stem cells, or iPS cells. Like ES cells, iPS cells can continue dividing indefinitely in culture, and when incorporated into a mouse blastocyst they can participate in creation of a perfectly formed chimeric animal. In this animal, they can contribute to the development of any tissue and can turn into any differentiated cell type, including functional germ cells from which a new generation of mice can be raised (see Figure 22–40).

1	iPS cells can now be derived from adult human cells and from various other differentiated cell types besides fibroblasts. Numerous methods can be used to drive up regulation of up regulation ofembryonic stem cell proliferationcell genes down regulation of loosening of differentiation genes chromatin structure

1	Figure 22–41 Reprogramming fibroblasts to IPS cells with the OSKM factors. as indicated, the master gene regulator proteins oct4, Sox2, and klf4 (oSk) induce both their own and each other's synthesis (gray shading). this generates a self-sustaining feedback loop that helps to maintain cells in an embryonic stem cell-like state, even after all of the experimentally added oSkm initiators have been removed. myc overexpression speeds up early stages of the reprogramming process through the mechanisms shown (see Figure 17–61). Stable reprogramming also involves the permanently induced expression of the Nanog gene, which produces an additional master transcription regulator. (adapted from

1	J. kim et al., Cell 132:1049–1061, 2008.) expression of the transforming OSKM factors, including methods that leave no trace of foreign DNA in the reprogrammed cell. Variations of the original cocktail of transcription regulators can drive the conversion, with different specialized cell types having somewhat different requirements. Myc overexpression, for example, turns out not to be absolutely necessary, although it enhances the efficiency of the process. And differentiated cell types may express some of the required factors as part of their normal phenotype. For example, cells of the dermal papilla of hair follicles already express Sox2, Klf4, and Myc; to convert them into iPS cells, it is enough to force them artificially to express Oct4. Oct4, indeed, seems to have a central role and to be generally indispensable for the creation of iPS cells. Reprogramming involves a massive upheaval of the Gene Control System

1	Reprogramming involves a massive upheaval of the Gene Control System Converting a differentiated cell into an iPS cell is not like flicking a switch on some predictable, precisely engineered piece of machinery. Only a few of the cells that receive the OSKM factors will actually become iPS cells—one in several thousand in the original experiments, and still only a small minority with more recent, improved techniques. In fact, the success of the original experiments depended on clever selection to pick out those few cells where the conversion had occurred (Figure 22–42).

1	Conversion to an iPS character by the OSKM factors is not only inefficient but also slow: fibroblasts take ten days or more from introduction of the conversion factors before they begin to express markers of the iPS state. This suggests that the transformation involves a long cascade of changes. These changes are being extensively studied, and they affect both the expression of individual genes and the state of the chromatin. The results of one such study are outlined in Figure 22–43. The process begins with a Myc-induced cell proliferation and loosening of chromatin structure that promotes the binding of the other three master regulators to many hundreds of different sites in the genome. At a large proportion of these sites, Oct4, Sox2, and Klf4 all bind in concert. The binding sites include the endogenous Oct4, Sox2, and Klf4 genes themselves, which eventually creates the types of positive feedback loops just described that makes expression of these genes self-sustaining (see Figure

1	the endogenous Oct4, Sox2, and Klf4 genes themselves, which eventually creates the types of positive feedback loops just described that makes expression of these genes self-sustaining (see Figure 22–41). But self-induction of Oct4, Sox2, and Klf4 is only a small part of the transformation that occurs. The three core factors activate some target genes and repress others, producing a cascade of effects that reorganize the gene control system globally and at every level, changing the patterns of histone modification, DNA methylation, and chromatin compaction, as well as the expression of innumerable proteins and noncoding RNAs. By the end of this complex process, the resulting iPS cell is no longer dependent on the artificially generated factors that triggered the change: it has settled into a stable, self-sustaining state of coordinated gene expression, making its own Oct4, Sox2, Klf4, and Myc (and all the other essential ingredients of a pluripotent stem cell) from its own endogenous

1	a stable, self-sustaining state of coordinated gene expression, making its own Oct4, Sox2, Klf4, and Myc (and all the other essential ingredients of a pluripotent stem cell) from its own endogenous copies of the genes.

1	factors O, S, K and M cell state unchanged, resistance gene not expressed Fbx15 G418-resistance gene iPS cell survives promoter rare change of state to iPS character, resistance gene expressed

1	Figure 22–42 A strategy used to select cells that have converted to an iPS character. the experiment makes use of a gene (Fbx15) that is present in all cells but is normally expressed only in eS and early embryonic cells (although not required for their survival). a fibroblast cell line is genetically engineered to contain a gene that produces an enzyme that degrades G418 under the control of the Fbx15 regulatory sequence. G418 is an aminoglycoside antibiotic that blocks protein synthesis in both bacteria and eukaryotic cells. when the oSkm factors are artificially expressed in this cell line, a small proportion of the cells undergo a change of state and activate the Fbx15 regulatory sequence, driving expression of the G418-resistance gene. when G418 is added to the culture medium, these are the only cells that survive and proliferate. when tested, they turn out to have an ipS character.

1	~99% of cells transcription wave 2 ~<1% of cells become IPS cells an experimental manipulation of Factors that modify Chromatin Can increase Reprogramming efficiencies The low efficiency and slow rate of conversion suggest that there is some barrier blocking the switch from the differentiated state to the iPS state in these experiments, and that overcoming this barrier is a difficult process that involves a large element of chance. Likewise, the outcome is variable, with significant differences between the individual lines of transformed cells that are generated, even when the initial differentiated cells are genetically and phenotypically identical. Only some of the candidate iPS lines pass all the tests of pluripotency. At a molecular level, there are differences even among the fully validated iPS cells: although they share many features, they vary in details of their gene expression patterns and, for example, in their patterns of DNA methylation.

1	Overcoming these difficulties will be critical for improving our understanding of how cell specialization is controlled and organized in multicellular organisms; it should also facilitate many medical advances. Thus, intensive research is being carried out on the reprogramming process. One approach aims at obtaining a much clearer picture of the role that chromatin structures play in gene regulation in eukaryotes.

1	From our discussion of nuclear transplantation, one might expect that any reprogramming of a differentiated cell would require a radical and widespread change in the chromatin structure of selected genes. Not only are such changes observed, but a large number of different experiments reveal that the efficiency of the reprogramming process can be substantially increased by altering the activity of proteins that affect chromatin structure. Figure 22–44 categorizes some of the factors that have been shown to enhance the transformation of fibroblasts to iPS cells; those in the top three rows—chromatin remodelers, histone modifying enzymes, and histone variants—are especially well known to have profound effects on the organization of nucleosomes in chromatin (discussed in Chapter 4).

1	We can only touch briefly here on the massive amounts of data that have been accumulating in this exciting research area. The major challenge that remains is to obtain a systems-level model for the complex set of biochemical changes that are involved in reprogramming. For example, which chromatin changes come first, and which then follow? How can these be triggered by the master transcription regulators through their binding to specific DNA sequences, and why do many cells in a population appear resistant to these effects? eS and ipS Cells Can be Guided to Generate Specific adult Cell types and even whole organs We can think of embryonic development in terms of a series of choices presented to cells as they follow a road that leads from the fertilized egg to terminal

1	We can think of embryonic development in terms of a series of choices presented to cells as they follow a road that leads from the fertilized egg to terminal Figure 22–43 A summary of some of the major events that accompany the reprogramming of mouse fibroblasts to iPS cells. by sorting cells at various times after the oSkm induction shown, one can carry out detailed biochemical analyses on the different cell populations shown. this led to the discovery that two major waves of new gene transcription are induced, but that the second wave occurs only in the subset of cells expressing an embryonic marker protein. Some 1500 genes are found to be differentially expressed in these cells, compared to the large majority of cells that fail to progress toward ipS cells. as indicated, major dna methylation changes are observed only after the alteration of chromatin structures.

1	in the first transcription wave, among the genes prominently induced are those for cell proliferation, metabolism, and cytoskeletal organization; in contrast, genes associated with fibroblast development are repressed. in the second transcription wave, genes required for embryonic development and for stem cell maintenance are induced. (adapted from J.m. polo et al., Cell 151:1617–1632, 2012.) specifc miRNAs specifc IncRNAs histone deacetylases specifc histone methyl transferases specifc histone demethylases histone acetyl transferases histone variant H2AZ histone variant macroH2A histone variant H3.3 DNA demethylases differentiation. After their long sojourn in culture, the ES cells or iPS cells and their progeny can still read the signs at each branch in the highway and respond as normal embryonic cells would. If ES or iPS cells are implanted directly into an embryo at a later stage or into an adult tissue, however, they fail to receive the appropriate sequence of cues; their

1	normal embryonic cells would. If ES or iPS cells are implanted directly into an embryo at a later stage or into an adult tissue, however, they fail to receive the appropriate sequence of cues; their differentiation then is not properly controlled, and they will often give rise to a tumor of the type known as a teratoma, containing a mixture of cell types inappropriate to the site in the body.

1	In culture, by exposing the ES or iPS cell to an appropriate sequence of signal proteins and growth factors, delivered with the right timing, it is possible to guide the cell along a pathway that approximates a normal developmental pathway, so as to convert it into one of the standard specialized adult cell types (Figure 22–45 and Movie 22.5). Success requires trial and error, but has now been achieved for many different final specialized states, including neuronal, muscular, and intestinal cell types. In a few cases, it has even been possible, by careful manipulation of the culture conditions, to get ES or iPS cells to interact with one another so as to construct an entire organ, albeit on a small scale (Figure 22–46). retinoic acid insulin, thyroid hormone

1	retinoic acid insulin, thyroid hormone Figure 22–44 Factors that have been observed to enhance reprogramming efficiency. emphasized here are those factors that can alter chromatin states, with those in the top three rows having the most direct effects. an up arrow indicates that reprogramming is increased when the activity of the indicated factor is increased; a down arrow indicates that reprogramming is increased when the activity of the indicated factor is decreased. thus, for example, increased activity of histone acetyl transferases and increased activity of histone deacetylases have opposite effects, as expected from their biochemical activities (see p. 196). Figure 22–45 Production of differentiated cells from eS or iPS cells in culture.

1	these cells can be cultured indefinitely as pluripotent cells when attached as a monolayer to a dish. alternatively they can be detached and allowed to form aggregates called embryoid bodies, which causes the cells to begin to specialize. Cells from embryoid bodies, cultured in media with different factors added, can then be driven to differentiate in various ways. (based on e. Fuchs and J.a. Segre, Cell 100:143–155, 2000. with permission from elsevier.) macrophage colony-interleukin-3, stimulating factor, interleukin-1 macrophage iPS cells (about 1,000 cells) dibutyryl cAMP, retinoic acid smooth muscle cell fbroblast fbroblast fbroblast growth factor 2, growth factor 2, growth epidermal platelet-derived factor growth factor growth factor 1258 Chapter 22: Stem Cells and tissue Renewal aggregate of hollow ball of budding of optic vesicle invaginates cultured ES cells neural cells optic vesicle to form optic cup (B) 100 µm

1	Figure 22–46 Cultured eS cells can give rise to a three-dimensional organ. (a) Remarkably, under appropriate conditions, mouse eS cells in culture can proliferate, differentiate, and interact to form a three-dimensional, eye-like structure, which includes a multilayered retina similar in organization to the one that forms in vivo. (b) Fluorescent micrograph of an optic cup formed by eS cells in culture. the structure includes a developing retina, containing multiple layers of neural cells, which produce a protein (pink) that serves as a marker for retinal tissue. (b, from m. eiraku, n. takata, h. ishibashi et al., Nature 472:51–56, 2011. with permission from macmillan publishers ltd.) Cells of one Specialized type Can be Forced to transdifferentiate directly into another

1	Cells of one Specialized type Can be Forced to transdifferentiate directly into another The route we have just described, from one mode of differentiation to another via conversion to an iPS cell, seems needlessly roundabout. Could we not convert cell type A into cell type B directly, without backtracking to the embryonic-like iPS state? For many years, it has been known that such transdifferentiation can be achieved in a few special cases, such as the conversion of fibroblasts into skeletal muscle cells by forced expression of MyoD (see p. 396). But now, with the insights that have come from the study of ES and iPS cells, ways are being found to bring about such interconversions in a much wider range of cases.

1	An elegant example comes from studies of the heart. By forcing expression of an appropriate combination of factors—not Oct4, Sox2, Klf4, and Myc, but Gata4, Mef2c, and Tbx5—it is possible to convert heart fibroblasts directly into heart muscle cells. This has been done in the living mouse, using retroviral vectors, and the transformation occurs with high efficiency when the vectors carrying the transgenes are injected directly into the heart muscle tissue itself. Although they occupy only a small fraction of the tissue volume, the fibroblasts in the heart outnumber the heart muscle cells, and they survive in large numbers even where the heart muscle cells have died. Thus, in a typical nonfatal heart attack, where heart muscle cells have died for lack of oxygen, the fibroblasts proliferate and make collagenous matrix so as to replace the lost muscle with a fibrous scar. This is a poor sort of repair. By forcing expression of the appropriate factors in the heart, as described above, it

1	and make collagenous matrix so as to replace the lost muscle with a fibrous scar. This is a poor sort of repair. By forcing expression of the appropriate factors in the heart, as described above, it has proved possible, in the mouse at least, to do better than nature and regenerate lost heart muscle by transdifferentiation of heart fibroblasts.

1	We are still a long way from putting this technique into practice as a treatment for heart attacks in humans, but it shows what the future may hold—not only for this medical problem, but for many others. eS and ipS Cells are useful for drug discovery and analysis of disease A large part of the excitement surrounding ES and iPS cells and the technology of transdifferentiation comes from the prospect of using the artificially generated cells for tissue repair. It begins to seem that virtually any type of tissue might be replaceable, allowing treatment of degenerative diseases that have previously had no cure. Research in this area is moving rapidly, but there are many difficulties to be overcome. treatment transplantation of genetically with drugs matched healthy cells

1	treatment transplantation of genetically with drugs matched healthy cells With the advent of iPS cells and direct transdifferentiation, at least one major hurdle has been surmounted, in principle at least: the problem of immune rejection. ES cells, because they are created from early embryos that generally come from unrelated donors, will never be genetically identical to the cells of the patient receiving the transplant. The transplanted cells and their progeny are therefore liable to rejection by the immune system. Both iPS and transdifferentiated cells, in contrast, can be generated from a small sample of the patient’s own tissue and so should escape immune attack when transplanted back into the same individual.

1	Tissue repair by transplantation, however, is not the only application for which ES, iPS, and transdifferentiated cells can be used: there are other ways in which they promise to be more immediately valuable. In particular, they can be used to generate large, homogeneous populations of specialized cells of any chosen type in culture; and these can serve for investigation of disease mechanisms and in the search for new drugs acting on a specific cell type (Figure 22–47).

1	Where a disease has a genetic cause, we can derive iPS cells from sufferers and use these cells to produce the specific cell types that malfunction, to investigate how the malfunction occurs, and to screen for drugs that might help to put it right. Timothy syndrome provides an example. In this rare genetic condition, there is a severe, life-threatening disorder in the rhythm of the heart beat (as well as several other abnormalities), as a result of a mutation in a specific type of Ca2+ channel. To study the underlying pathology, researchers took skin fibroblasts from patients with the disorder, generated iPS cells from the fibroblasts, and drove the iPS cells to differentiate into heart muscle cells. These cells, when compared with heart muscle cells prepared similarly from normal control individuals, showed irregular contractions and abnormal patterns of Ca2+ influx and electrical activity that could be characterized in detail. From this finding, it is a small step to development of

1	individuals, showed irregular contractions and abnormal patterns of Ca2+ influx and electrical activity that could be characterized in detail. From this finding, it is a small step to development of an in vitro assay for drugs that might correct the misbehavior of the heart muscle cells.

1	This approach to drug discovery—where iPS cells are prepared from the individual patient, differentiated into the relevant cell type, and used to test candidate drugs in vitro—would seem to represent a huge advance on the slow, costly traditional methods that involve administration of test compounds to large numbers of people. Figure 22–47 Use of iPS cells for drug discovery and for analysis and treatment of genetic disease. the left side of the diagram shows how differentiated cells that are generated from ipS cells derived from a patient with a genetic disease can be used for analysis of the disease mechanism and for discovery of therapeutic drugs. the right side of the diagram shows how the genetic defect might be repaired in the ipS cells, which could then be induced to differentiate in an appropriate way and grafted back into the patient without danger of immune rejection. (based on d.a. Robinton and G.Q. daley, Nature 481:295–305, 2012).

1	In the adult mammalian body, the various types of stem cells are highly specialized, each giving rise to a limited range of differentiated cell types. Cells become restricted size? how do the cells in each tissue to specific pathways of differentiation during embryonic development. One way know when to terminate their growth to force a return to a pluripotent or totipotent state is by nuclear transplantation: and division, so as to limit the size of the nucleus of a differentiated cell can be injected into an enucleated oocyte, whose an organ or tissue appropriately? cytoplasm reprograms the genome back to an approximation of an early embryonic state. This allows production of an entire new individual. The reversion of the • what is the fundamental molecular genome to this state involves radical, genome-wide changes in chromatin structure difference that distinguishes a stem and DNA methylation. cell?

1	Remarkably, cells taken from the inner cell mass of an early mammalian embryo • how is the correct balance between can be propagated in culture indefinitely in a pluripotent state. When transplanted stem cells, progenitor cells, and back into a host early embryo, these embryonic stem (ES) cells can contribute cells to any tissue, including the germ line. ES cells have been invaluable for genetic engi tissue or organ? neering in mice. Cells with similar properties, called induced pluripotent stem cells (iPS cells), can be generated from adult differentiated cells such as fibroblasts by • what role does chromatin structure forced expression of a cocktail of key transcription regulators. A similar method play in cell memory and in cell reprogramming?

1	can be used to reprogram adult cells directly from one specialized state to another. In principle, iPS cells generated from cells biopsied from an adult human patient • how are molecules inherited could be used for tissue repair in that same individual, avoiding the problem of asymmetrically during cell division? immune rejection. More immediately, they provide a source of specialized cells that can be used to analyze in vitro the effects of mutations affecting human cells and for • how do germ cells avoid aging? screening for drugs for treatment of genetic diseases. Which statements are true? explain why or why not. 22–1 In the small intestine, stem cells in the crypts divide asymmetrically to maintain the population of cells that make up the villi; after each division, one daughter remains a stem cell and the other begins to divide rapidly to produce differentiated progeny. 22–2 Stem cells, being stem cells, are by definition the same in all tissues.

1	22–2 Stem cells, being stem cells, are by definition the same in all tissues. 22–3 Every tissue that can be renewed is renewed from a tissue-specific population of stem cells. 22–4 Disturbance of the balance in the activities of osteoblasts and osteoclasts in favor of osteoclasts can give rise to the condition known as osteoporosis, the brittle-bone syndrome of the elderly. Discuss the following problems.

1	Discuss the following problems. 22–5 In the 1950s, scientists fed 3H-thymidine to rats to label cells that were synthesizing DNA, and then followed the fates of labeled cells for periods of up to a year. They found three patterns of cell labeling in different tissues. Cells in some tissues such as neurons in the central nervous system and the retina did not get labeled. Muscle, kidney, and liver, by contrast, each showed a small number of labeled cells that retained their label, apparently without further division or loss. Finally, cells such as those in the squamous epithelia of the tongue and esophagus were labeled in fairly large numbers, with radioactive pairs of nuclei visible in 12 hours; however, the labeled cells disappeared over time. Which of these three patterns of labeling would you expect to see if the labeled cells were generated by stem cells? Explain your answer.

1	22–6 At any given time, intestinal crypts of mice comprise about 15 stem cells and 10 Paneth cells. After cell division, which occurs about once a day, the daughter cells remain stem cells only if they maintain contact with a Paneth cell. This constant competition for Paneth-cell contact raises the possibility that crypts might become monoclonal over time; that is, the crypt cells at one point in time might derive from only 1 of the 15 stem cells that existed at some earlier time. To test this possibility, you use the so-called confetti marker that upon activation expresses any one of three fluorescent proteins in the stem cells of the crypt. You then examine crypts at various times to determine whether they contain cells with multiple colors or only one color (Figure Q22–1). Do the crypts become monoclonal over time or not? How can you tell?

1	22–7 The origin of new β cells of the pancreas—from stem cells or from preexisting β cells—was not resolved until a decade ago, when the technique of lineage tracing was used to decide the issue. Using transgenic mice that expressed a tamoxifen-activated form of Cre recombinase under the control of the insulin promoter, which is active only in β cells, investigators could remove an inhibitory segment of DNA and thereby allow expression of human placental alkaline phosphatase (HPAP), which can be detected by histochemical staining. After a pulse of tamoxifen that converted about 30% of β cells in young mice to microvilli 22–8 One of the earliest assays for hematopoietic stem cells made use of their ability to form colonies in the spleens of heavily irradiated mice. By varying the amounts

1	Figure Q22–1 Fluorescent cells in crypts in mouse intestines at various times after activation of expression of fluorescent proteins (problem 22–6). the images are taken in the xz plane, which cuts through multiple crypts, as indicated in the schematic drawing. Roughly 50 crypts are visible in each section. Dotted white circles identify some individual crypts. Scale bars are 100 μm. (adapted from h.J. Snippert et al., Cell 143:134–144, 2010. with permission from elsevier.) cells that express HPAP, the investigators followed the percentage of labeled β cells for a year, during which time the total number of β cells in the pancreas increased by 6.5fold. How do you suppose the percentage of β cells would change over time if new β cells were derived from stem cells? What if new β cells were derived from preexisting β cells? Which hypothesis do the results in Figure Q22–2 support?

1	of transplanted bone marrow cells, investigators showed that the number of spleen colonies varied linearly with dose and that the curve passed through the origin, suggesting that single cells were capable of forming individual colonies. However, because colony formation was rare relative to the numbers of transplanted cells, it was possible that undispersed clumps of two or more cells were the actual initiators. A classic paper resolved this issue by exploiting rare, cytologically visible genome rearrangements generated by irradiation. Recipient mice were first irradiated to deplete bone marrow cells, and then they were irradiated a second time after transplantation to generate rare genome rearrangements in the transplanted cell population. Spleen colonies were then screened to find ones that carried genome rearrangements. How do you suppose this experiment distinguishes between colonization by single cells versus cellular aggregates?

1	22–9 It is possible to purify hematopoietic stem cells using a combination of antibodies directed against cellsurface targets. By removing cells that expressed surface markers characteristic of specific lineages such as B cells, granulocytes, myelomonocytic cells, and T cells, investigators generated a population of cells enriched for stem cells. They further enriched this population for putative stem cells by positively selecting for cells that expressed suspected stem-cell surface markers. Spleen colony formation in irradiated mice by these putative stem cells and the unfractionated bone marrow cells is shown in Figure Q22–3. Given that only about 1 in 10 cells lodges in the spleen, do these results support the idea that the enriched population consists mostly of hematopoietic stem cells? What additional information would you need to have to feel confident that the enriched cells are true stem cells? What proportion of bone marrow cells are hematopoietic stem cells?

1	22–10 Generation of induced pluripotent stem (iPS) cells was first accomplished using retroviral vectors to carry the OSKM (Oct4, Sox2, Klf4, and Myc) set of transcription regulators into cells. The efficiency of fibroblast reprogramming was typically low (0.01%), in part because large numbers of retroviruses must integrate to bring about reprogramming and each integration event carries with it the risk of inappropriately disrupting or activating a critical gene. In what other ways, or other forms, do you suppose you might deliver the OSKM transcription regulators so as to avoid these problems? percent of ˜ cellsexpressing HPAP number of colonies cells (problem 22–9).

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1	yamanaka S (2013) the winding road to pluripotency (nobel lecture). Angew. Chem. Int. Ed. Engl. 52, 13900–13909. Infectious diseases currently cause about one-quarter of all human deaths worldwide, more than all forms of cancer combined and second only to cardiovascular diseases. In addition to the continuing heavy burden of ancient diseases such as tuberculosis and malaria, newer infectious diseases continually emerge. The current pandemic (worldwide epidemic) of AIDS (acquired immune deficiency syndrome), was first clinically observed in 1981 and has since caused more than 35 million deaths worldwide. Moreover, some diseases long thought to result from other causes are now recognized to be associated with infections. Most gastric ulcers, for example, are caused not by stress or spicy food, but by infection of the stomach lining by the bacterium Helicobacter pylori.

1	The burden of infectious diseases is not spread equally across the planet. Poorer countries and communities suffer disproportionately, often due to poor public sanitation and health systems. Some infectious diseases, however, occur primarily or exclusively in industrialized communities: Legionnaire’s disease, for example, a bacterial infection of the lungs, commonly spreads through air-conditioning systems. Since the mid-1800s, physicians and scientists have struggled to identify the agents—collectively called pathogens—that are capable of causing infectious diseases. More recently, the advent of microbial genetics and molecular cell biology has greatly enhanced our understanding of the causes and mechanisms of infectious diseases. We now know that pathogens frequently exploit the attributes of their host’s cells in order to infect them. This understanding can give us new insights into normal cell biology, as well as strategies for treating and preventing infectious diseases.

1	Although pathogens are understandably a focus of attention, only a relatively small fraction of the microbial species we encounter are pathogens. Much of the biomass of the Earth is made up of microbes, and they produce everything from the oxygen we breathe to the soil nutrients we use to grow food. Even those species of microbes that colonize the human body do not generally cause disease. The collective of microorganisms that reside in or on an organism is called the microbiota. Many of these microbes have a beneficial effect on the health of the organism, assisting its normal development and physiology. In this chapter, we give an overview of the different kinds of pathogens, as well as those microorganisms that colonize our body without causing trouble. We then discuss the cell biology of infection—the molecular interactions between pathogens and their host. In Chapter 24, we consider how our innate and adaptive immune systems collaborate to defend us against pathogens.

1	We normally think of pathogens as hostile invaders, but a pathogen, like any other organism, is simply exploiting an available niche in which to live and procreate. Living on or in a host organism is a very effective strategy, and it is possible that every organism on Earth is subject to some type of infection (Figure 23–1). A human host is a nutrient-rich, warm, and moist environment, which remains at a uniform temperature and constantly renews itself. It is not surprising that many microorganisms have evolved the ability to survive and reproduce in this desirable niche. In this section, we discuss some of the common features that microorganisms must have in order to colonize the human body or cause disease, and we explore the wide variety of organisms that are known to cause disease. The Human Microbiota Is a Complex Ecological System That Is Important for Our Development and Health

1	The Human Microbiota Is a Complex Ecological System That Is Important for Our Development and Health The human body contains about 1013 human cells, as well as a microbiota consisting of approximately 1014 bacterial, fungal, and protozoan cells, which represent thousands of microbial species—the so-called normal flora. The combined genomes of the various species of the human microbiota, called the microbiome, contain more than 5 × 106 genes—more than 100 times greater than the number of genes in the human genome itself. A consequence of this genomic diversity is that the microbiota expands the range of biochemical and metabolic activities available to the humans.

1	The microbiota is usually confined to the skin, mouth, digestive tract, and vagina. With the exception of microbes colonizing the skin, it consists primarily of anaerobic bacteria, with distinct communities of species inhabiting each body part. These communities vary considerably between individual humans, even between close relatives or identical twins. Although the microbiota of an individual is generally consistent over time, it is influenced by a variety of factors, including age, diet, health status, and antibiotic use.

1	There are various ecological relationships that these microbes have with their host. In mutualism, both the microbe and host benefit. The anaerobic bacteria that inhabit our intestines, for example, gain shelter and a nutrient supply but also contribute to the digestion of our food, produce important nutrients for us, and are essential for the normal development of our gastrointestinal tract and innate and adaptive immune systems. In commensalism, the microbe benefits but offers no benefit and causes no harm: for example, we are infected with many viruses that have no noticeable effect on our health. In parasitism, the microbe benefits to the detriment of the host, as is often the case for pathogens.

1	Many infectious diseases are caused by a single pathogen. There is increasing evidence, however, that an imbalance in the community of microbes that constitute the microbiota can contribute to some diseases, including autoimmune and allergic diseases, obesity, inflammatory bowel disease, and diabetes. Remarkably, in such cases of microbiota imbalance (referred to as dysbiosis), the transfer of the microbiota from a healthy individual to someone suffering from the disease can be beneficial and sometimes curative, as in the case of Clostridium difficile colitis caused by overgrowth of the bacterium. Pathogens Interact with Their Hosts in Different Ways If it is normal for us to live with a community of microbes, why are some of them capable of causing us illness or death? Although the ability of a particular Figure 23–1 Parasitism at many levels.

1	If it is normal for us to live with a community of microbes, why are some of them capable of causing us illness or death? Although the ability of a particular Figure 23–1 Parasitism at many levels. Most animals harbor parasites, an example being the blacklegged tick or deer tick (Ixodes scapularis), shown here on a human finger. Although ticks of this species thrive on white-tailed deer and other wild mammals, they can also live on humans.

1	Ticks themselves harbor their own parasites including the bacterium Borrelia burgdorferi, stained here with a vital dye that labels living bacteria green and dead bacteria red. These spiral-shaped bacteria live in deer ticks and can be transmitted to humans during a tick’s blood meal. Borrelia burgdorferi causes Lyme disease, which is characterized by a bull’s-eye-shaped skin rash and fever; if the infection is left untreated, various complications can result, including arthritis and neurological abnormalities. The idea that parasites have their own parasites was noted by Jonathan Swift in 1733: “So, naturalists observe, a flea Has smaller fleas that on him prey; And these have smaller still to bite ‘em; And so proceed ad infinitum.” (A, from Acorn, White-Footed Mice and Tick Cycle Augment Risks of Lyme Disease in 2012. March 14, 2012. Reprinted with permission of Anita Sil; B, courtesy of M.

1	M. Embers.) microorganism to cause disease depends on many factors, it requires that the pathogen possess specialized pathogenic characteristics that allow it to live in humans.

1	Primary pathogens can cause overt disease in most healthy people. Some primary pathogens cause acute, life-threatening epidemic infections and spread rapidly from one sick or dying host to another; historically important examples include the bacterium Vibrio cholerae, which causes cholera, and the variola and influenza viruses, which cause smallpox and flu, respectively. Others may persistently infect a single individual for years without causing overt disease; examples include the bacterium Mycobacterium tuberculosis (which can cause the life-threatening lung infection tuberculosis) and the intestinal worm Ascaris. Although these potential primary pathogens can make some people critically ill, billions of people carry these foreign organisms in an asymptomatic way, often unaware that they are infected. It is sometimes difficult to draw a line between the asymptomatic presence of such pathogens and the normal microbiota. Some microbes of the normal flora can act as opportunistic

1	they are infected. It is sometimes difficult to draw a line between the asymptomatic presence of such pathogens and the normal microbiota. Some microbes of the normal flora can act as opportunistic pathogens, in that they cause disease only if our immune systems are weakened or if they gain access to a normally sterile part of the body.

1	In order to survive and multiply, a successful pathogen must be able to: (1) enter the host (usually by breaking an epithelial barrier); (2) find a nutritionally compatible niche in the host’s body; (3) avoid, subvert, or circumvent the host’s innate and adaptive immune responses; (4) replicate, using host resources; and (5) exit one host and spread to another. Pathogens have evolved various mechanisms that maximally exploit the biology of their host organisms to help accomplish these tasks. For some pathogens, these mechanisms are adapted to a unique host species, whereas for others the mechanisms are sufficiently general to permit invasion, survival, and replication in a wide variety of hosts. Because pathogens have evolved the ability to interface directly with the molecular machinery of host cells, we have learned a great deal about cell biological principles by studying them.

1	Our constant exposure to pathogens has strongly influenced human evolution. In modern times, humans have learned how to limit the ability of pathogens to infect us through improvements in public health measures and childhood nutrition, vaccines, antimicrobial drugs, and routine testing of blood used for transfusions. As we learn more about the mechanisms by which pathogens cause disease (called pathogenesis), our creativity and resourcefullness will continue to serve as an important addition to our immune systems in fighting infectious diseases. Pathogens Can Contribute to Cancer, Cardiovascular Disease, and Other Chronic Illnesses

1	Some viral and bacterial pathogens can cause or contribute to chronic, life-threatening illnesses that are not normally classified as infectious diseases. An important example is cancer. As discussed in Chapter 20, the oncogene concept—that certain altered genes can trigger cell transformation and tumor development— came initially from studies of the Rous sarcoma virus, which causes a form of cancer (sarcomas) in chickens. One of the viral genes encodes an overactive homolog of the host tyrosine kinase Src (see Figure 3–63), which has been implicated in many kinds of cancer. Several human cancers are also known to have a viral origin. Human papillomavirus, for example, which causes genital warts, is responsible for more than 90% of cervical cancers (see Figure 20–40). The recent development of a vaccine against the most abundant cancer-associated strains of human papillomavirus promises to prevent many of these cancers in the future. In other cases, chronic tissue damage caused by

1	of a vaccine against the most abundant cancer-associated strains of human papillomavirus promises to prevent many of these cancers in the future. In other cases, chronic tissue damage caused by infection can increase the likelihood of cancer. Inflammation caused by the stomach-dwelling bacterium H. pylori can be a major contributor to stomach cancer, as well as to gastric ulcers.

1	The major causes of death in wealthy industrialized nations are cardiovascular diseases. They frequently result from atherosclerosis, the accumulation in blood vessel walls of fatty deposits that can block blood flow and cause heart attacks and strokes. A hallmark of early atherosclerosis is the appearance in blood vessel walls of clumps of macrophages called foam cells, which recruit other white blood cells into the forming atherosclerotic plaque. Foam cells in atherosclerotic plaques often contain the bacterial pathogen Chlamydia pneumoniae, which commonly causes pneumonia in humans and is a significant risk factor for atherosclerosis in humans and animal models. Other bacterial species are also implicated in atherosclerosis, including bacteria usually associated with teeth and gums, such as Porphyromonas gingivalis. As we learn more about the interactions between pathogens and the human body, it seems likely that more chronic conditions will be found to have a link to an infectious

1	as Porphyromonas gingivalis. As we learn more about the interactions between pathogens and the human body, it seems likely that more chronic conditions will be found to have a link to an infectious agent.

1	Pathogens Can Be Viruses, Bacteria, or Eukaryotes Many types of pathogens cause disease in humans. The most familiar are viruses and bacteria. Viruses cause diseases ranging from AIDS and smallpox to the common cold. Viruses are essentially fragments of nucleic acid (DNA or RNA) that generally encode a relatively small number of gene products, wrapped in a protective shell of proteins (Figure 23–2A) and (in some cases) an outer membrane envelope (see Figure 5–62). Much larger and more complex than viruses, bacteria are prokaryotic cells, which perform most of their basic metabolic functions themselves, relying on the host primarily for nutrition (Figure 23–2B).

1	Some other infectious agents are eukaryotic organisms. These range from single-celled fungi and protozoa (Figure 23–2C) to large, complex metazoa such as parasitic worms. One of the most common human parasites, shared by about a billion people at present, is the nematode worm Ascaris lumbricoides, which infects the gut (Figure 23–2D). It closely resembles its harmless nematode cousin Caenorhabditis elegans, which is used as a model organism for genetic and developmental biological research (see Figure 1–39). C. elegans, however, is only about 1 mm in length, whereas Ascaris can reach 30 cm. We now introduce the basic features of each of the major types of pathogens, before we examine the mechanisms that pathogens use to infect their hosts. Figure 23–2 Pathogens in many forms. The structure of the protein coat, or capsid, of poliovirus. This virus was once a common cause of paralysis, but the disease (poliomyelitis) has been greatly reduced by widespread vaccination.

1	The structure of the protein coat, or capsid, of poliovirus. This virus was once a common cause of paralysis, but the disease (poliomyelitis) has been greatly reduced by widespread vaccination. The bacterium Vibrio cholerae, the causative agent of the epidemic, diarrheal disease cholera. (C) The protozoan parasite Trypanosoma brucei (purple) in a field of erythrocytes (red blood cells; pink). This parasite causes African sleeping sickness, a potentially fatal disease of the central nervous system. (D) This clump of Ascaris nematodes was removed from the obstructed intestine of a two-year-old boy. (A, courtesy of Robert Grant, Stephan Crainic, and James M. Hogle; B, photograph courtesy of John Mekalanos; C, CDC, Department of Health and Human Services; D, from J.K. Baird et al., Am. J. Trop. Med. Hyg. 35:314–318, 1986. Photograph by Daniel H. Connor.) lipopolysaccharide (LPS) outer leafet of outer membrane pore protein

1	Figure 23–3 Bacterial shapes and cell-surface structures. (A) Bacteria are traditionally classified by shape. (B and C) They are also classified as Gram positive or Gram negative. (B) Gram-positive bacteria such as Streptococcus and Staphylococcus have a single membrane and a thick cell wall made of cross-linked peptidoglycan. They are called Gram positive because they retain the violet dye used in the Gram-staining procedure. (C) Gram-negative bacteria such as Escherichia coli (E. coli) and Salmonella have two membranes, separated by the periplasm (see Figure 11–17). The peptidoglycan cell wall of these organisms is located in the periplasm and is thinner than in Gram-positive bacteria; they therefore fail to retain the dye in the Gram-staining procedure. The inner membrane of both Gram-positive and Gram-negative bacteria is a phospholipid bilayer. The inner leaflet of the outer membrane of Gram-negativebacteria is also made primarily of phospholipids, whereas the outer leaflet of

1	and Gram-negative bacteria is a phospholipid bilayer. The inner leaflet of the outer membrane of Gram-negativebacteria is also made primarily of phospholipids, whereas the outer leaflet of the outer membrane is composed of a unique glycosylated lipid called lipopolysaccharide (LPS). (D) Cell-surface appendages are important for bacterial behavior. Many bacteria swim using the rotation of helical flagella. The bacterium illustrated has only a single flagellum at one pole; however, many have multiple flagella. Straight pili (also called fimbriae) are used to adhere to various surfaces in the host, as well as to facilitate genetic exchange between bacteria. Some kinds of pili can retract to generate force and thereby help bacteria move along surfaces.

1	Bacteria Are Diverse and Occupy a Remarkable Variety of Ecological Niches

1	Although bacteria generally lack internal membranes, they are highly sophisticated cells whose organization and behaviors have attracted the attention of many scientists. Bacteria are classified broadly by their shape—as rods, spheres (cocci), or spirals (Figure 23–3A)— as well as by their so-called Gram-staining properties, which reflect differences in the structure of the bacterial cell wall. Gram-positive bacteria have a thick layer of peptidoglycan cell wall outside their inner (plasma) membrane (Figure 23–3B), whereas Gram-negative bacteria have a thinner peptidoglycan cell wall. In both cases, the cell wall protects against lysis by osmotic swelling, and it is a target of host antibacterial proteins such as lysozyme and antibiotics such as penicillin. Gram-negative bacteria are also covered outside the cell wall by an outer membrane containing lipopolysaccharide (LPS) (Figure 23–3C). Both peptidoglycan and LPS are unique to bacteria and are recognized as pathogen-associated

1	also covered outside the cell wall by an outer membrane containing lipopolysaccharide (LPS) (Figure 23–3C). Both peptidoglycan and LPS are unique to bacteria and are recognized as pathogen-associated molecular patterns (PAMPs) by the host innate immune system, as discussed in Chapter 24. The surface of bacterial cells can also display an array of appendages, including flagella and pili, which enable bacteria to swim or adhere to desirable surfaces, respectively (Figure 23–3D). Apart from cell shape and structure, differences in ribosomal RNA and genomic DNA sequence are also used for phylogenetic classification. Because bacterial genomes are small—typically between 1,000,000 and 5,000,000 nucleotide pairs (compared to more than 3,000,000,000 for humans)—they are now simple to sequence, making this an important new classification tool.

1	Bacteria also exhibit extraordinary molecular, metabolic, and ecological diversity. At the molecular level, bacteria are far more diverse than eukaryotes, and they can occupy ecological niches having extremes of temperature, salt concentrations, and nutrient limitation. Some bacteria replicate in an environmental reservoir such as water or soil and only cause disease if they happen to encounter a susceptible host; these are called facultative pathogens. Others can only replicate inside the body of their host and are therefore called obligate pathogens. Bacteria also differ in the range of hosts they will infect. Shigella flexneri, for example, which causes epidemic dysentery (bloody diarrhea), will infect only humans and other primates. By contrast, the closely related bacterium Salmonella enterica, which is a common cause of food poisoning in humans, can also infect other vertebrates, including chickens and turtles. A champion generalist is the opportunistic pathogen Pseudomonas

1	enterica, which is a common cause of food poisoning in humans, can also infect other vertebrates, including chickens and turtles. A champion generalist is the opportunistic pathogen Pseudomonas aeruginosa, which can cause disease in a wide variety of plants and animals.

1	Pathogenic bacteria and their closest nonpathogenic relatives often differ in a relatively small number of genes. Genes that contribute to the ability of an organism to cause disease are called virulence genes, and the proteins they encode are called virulence factors. Such virulence genes are often clustered together on the bacterial chromosome; large clusters are called pathogenicity islands. Virulence genes can also be carried on bacteriophages (bacterial viruses) or transposons (see Table 5–4), both of which integrate into the bacterial chromosome, or on extrachromosomal virulence plasmids (Figure 23–4A).

1	Pathogenic bacteria are thought to emerge when groups of virulence genes are transferred together into a previously avirulent bacterium by a process called horizontal gene transfer (to distinguish it from vertical gene transfer from parent to offspring). Horizontal transfer can occur by one of three mechanisms: natural transformation by released naked DNA, transduction by bacteriophages, or sexual exchange by conjugation (Figure 23–4B and Movie 23.1). Sequencing the genomes of large numbers of pathogenic and nonpathogenic bacteria has indicated that horizontal gene transfer has made important contributions to bacterial evolution, enabling species to inhabit new ecological and nutritional niches, as well as to cause disease. Even within a single bacterial species, the amount of chromosomal E. coli Shigella fexneri Salmonella enterica

1	E. coli Shigella fexneri Salmonella enterica Figure 23–4 Genetic differences between pathogenic and nonpathogenic bacteria. (A) Genetic differences between nonpathogenic E. coli and two closely related food-borne pathogens—Shigella flexneri, which causes dysentery, and Salmonella enterica, a common cause of food poisoning. Nonpathogenic E. coli has a single circular chromosome. The chromosome of S. flexneri differs from that of E. coli in a limited number of locations; most of the genes required for pathogenesis (virulence genes) are carried on an extrachromosomal virulence plasmid. The chromosome of S. enterica carries two large inserts (pathogenicity islands) not found in the E. coli chromosome; these inserts each contain many virulence genes.

1	(B) Bacterial pathogens evolve by horizontal gene transfer. This can occur by three mechanisms: natural transformation, in which naked DNA is taken in by competent bacteria; transduction, in which bacterial viruses (bacteriophages) transfer DNA from one bacterium into another; and conjugation, during which plasmid DNA, and even chromosomal DNA, is transferred from a donor to a recipient bacterium. V.cholerae V. cholerae O1 serogroup V. cholerae O1 serogroup Classical V. cholerae O139 serogroup VSP1 VSP2 CTX˜Classical CTX˜El Tor RS1˜O139 antigen SXT 1st through 6th pandemics 7th pandemic V.cholerae variation is astonishing; the genomes of different strains of Escherichia coli can differ by as much as 25%. Such variation has led to the concept that a bacterial species has both a core genome common to all isolates within the species and a larger pan-genome consisting of all genes present in the full spectrum of isolates.

1	Acquisition of genes and gene clusters can drive the rapid evolution of pathogens and turn nonpathogens into pathogens. Consider, for example, Vibrio cholerae—the Gram-negative bacterium that causes the epidemic diarrheal disease cholera. Of the hundreds of strains of Vibrio cholerae, the only ones that cause pandemic human disease are those infected with a mobile bacteriophage (CTXϕ) containing genes encoding the two subunits of the toxin that causes the diarrhea. As summarized in Figure 23–5, seven pandemics of V. cholerae have arisen since 1817. The first six were caused by the periodic reemergence of so-called Classical strains. In addition to the toxin-encoding bacteriophage, these Classical strains shared a similar O1 surface antigen, part of the LPS in the outer membrane (see Figure 23–3C). In 1961, the seventh pandemic began, caused by a new strain named “El Tor,” which arose when an O1-expressing strain acquired two bacteriophages and at least two new pathogenicity islands.

1	23–3C). In 1961, the seventh pandemic began, caused by a new strain named “El Tor,” which arose when an O1-expressing strain acquired two bacteriophages and at least two new pathogenicity islands. El Tor eventually displaced the Classical strains. In 1992, a new strain emerged in which O1 was replaced with another O-antigen variant called O139, which was not recognized by antibodies present in the blood of survivors of previous cholera epidemics. The O139 strain also contains a transposon-like element that encodes antibiotic resistance. As this example makes clear, the rapid evolution of bacterial pathogens can be likened to an arms race which pits the survival of a bacterium against our immune systems and the tools of modern medicine. Similar struggles for survival take place between all pathogens and humans, and understanding these conflicts provides key insights into the evolution of pathogens and greatly informs us how we treat new outbreaks of infectious diseases.

1	Bacterial Virulence Genes Encode Effector Proteins and Secretion Systems to Deliver Effector Proteins to Host Cells What are the gene products that enable a bacterium to cause disease in a healthy host? For pathogenic bacteria that live outside of host cells, called extracellular Figure 23–5 Comparative-genomicsbased model for the evolution of pathogenic Vibrio cholerae strains.

1	Progenitor strains in the wild first acquired the biosynthetic pathway necessary to make the O1 antigen type of carbohydrate chain on the outer-membrane lipopolysaccharide (see Figure 23–3C). Incorporation of the CTXϕ bacteriophage created the Classical pathogenic strains responsible for the first six worldwide epidemics of cholera between 1817 and 1923. Sometime in the twentieth century, an O1 strain in the environment picked up the CTXϕ bacteriophage again, along with an associated bacteriophage RS1ϕ and two pathogenicity islands (VSP1 and VSP2), creating the El Tor strain that emerged as the seventh worldwide pandemic in 1961. In 1992, an El Tor strain was isolated that had picked up a new DNA cassette, enabling it to produce the O139 antigen type of carbohydrate chain rather than the O1 type. This altered the bacterium’s interaction with the human immune system, without diminishing its virulence; this bacterium also picked up a new pathogenicity island (SXT). An electron

1	than the O1 type. This altered the bacterium’s interaction with the human immune system, without diminishing its virulence; this bacterium also picked up a new pathogenicity island (SXT). An electron micrograph of Vibrio cholerae (V. cholerae) is shown in Figure 23–2B.

1	bacterial pathogens, virulence genes often encode secreted toxic proteins (toxins) that interact with host cell structural or signaling proteins to elicit a response that is beneficial to the pathogen. Several of these bacterial toxins are among the most potent of known human poisons. Bacterial toxins are often composed of two protein components—an A subunit with enzymatic activity, and a B subunit that binds to specific receptors on the host cell surface and directs the trafficking of the A subunit to the cytosol by various routes (Figure 23–6). The Vibrio cholerae phage, for example, encodes the two subunits of cholera toxin (Movie 23.2). The A subunit catalyzes the transfer of an ADP-ribose moiety from NAD+ to the trimeric G protein Gs (see Figure 15–23), which activates adenylyl cyclase to make cyclic AMP (see Figure 15–25). ADP-ribosylation prevents inactivation of the G protein and results in the overaccumulation of intracellular cyclic AMP and the release of ions and water into

1	to make cyclic AMP (see Figure 15–25). ADP-ribosylation prevents inactivation of the G protein and results in the overaccumulation of intracellular cyclic AMP and the release of ions and water into the intestinal lumen, leading to the watery diarrhea associated with cholera. The infection then spreads to new hosts via released bacteria, which can contaminate food and water.

1	Some pathogenic bacteria secrete multiple toxins, each of which targets a different signaling pathway in host cells. Anthrax, for example, is an acute infectious disease of sheep, cattle, and occasionally humans. It is caused by contact with spores of the Gram-positive bacterium Bacillus anthracis. Dormant spores can survive in soil for long periods. If inhaled, ingested, or rubbed into breaks in the skin, spores can germinate and the bacteria replicate. The bacteria secrete two toxins with identical B subunits but different A subunits. The B subunits bind to a host cell-surface receptor protein to transfer the two different A subunits into

1	Figure 23–6 Bacterial toxin entry into host cells. Bacterial toxins are often composed of A and B protein subunits. The B (binding) subunit of the toxin interacts with host-cell toxin receptors, enabling endocytosis and intracellular trafficking of B subunit as well as its associated and enzymatically active A subunit(s). In the case of Bacillus anthracis, the B subunit changes conformation in the low pH environment of the endosome to form a pore through which two different A subunits, lethal factor and edema factor, are transported across the membrane of the endosome in an unfolded conformation. In the cases of Vibrio cholerae toxin and Bordetella pertussis toxin, the B and A subunits are transported to the Golgi apparatus and then to the endoplasmic reticulum (ER), where the A subunits are then translocated into the cytosol in an unfolded conformation through a proteintranslocation channel.

1	host cells (see Figure 23–6). The A subunits are called lethal factor and edema factor. The A subunit of edema toxin is an adenylyl cyclase that catalyzes the production of cyclic AMP (see Figure 15–25), leading to an ion imbalance that can cause an accumulation of extracellular fluid (edema) in the skin or lung. The A subunit of lethal toxin is a protease that cleaves several activated members of the mitogen-activated protein kinase kinase (MAP kinase kinase) family (see Figure 15–49), disrupting intracellular signaling and leading to immune cell dysfunction and cell death. Injection of lethal toxin into the bloodstream of an animal causes shock (a large fall in blood pressure) and death.

1	Apart from toxins, bacteria use specialized secretion systems to secrete many other effector proteins that interact with host cells. Gram-negative bacteria have a general secretion system and several classes of accessory secretion systems (types I–VI). A subset of these accessory secretion systems, called contact-dependent secretion systems, is present in many bacteria that contact or live inside host cells. The type III secretion system (Figure 23–7), for example, injects into the host-cell cytoplasm effector proteins that can elicit a variety of host cell responses that enable the bacterium to invade or survive. There is a remarkable degree of structural similarity between the type III syringe and the base of a bacterial flagellum. Because flagella are found in a wider range of bacteria than are type III secretion systems, and the secretion systems appear to be adaptations specific for pathogenesis, it seems likely that the type III secretion systems evolved from flagella. Other

1	than are type III secretion systems, and the secretion systems appear to be adaptations specific for pathogenesis, it seems likely that the type III secretion systems evolved from flagella. Other types of delivery systems used by bacterial pathogens appear to have evolved independently. For example, type IV secretion systems are closely related to the conjugation apparatus that many bacteria use to exchange genetic material.

1	Pathogenic fungi and protozoan parasites are eukaryotes, as are their hosts. Consequently, antifungal and antiparasitic drugs are often less effective and more toxic to the host than are antibiotics that target bacteria. A second characteristic of fungal and parasitic infections that makes them difficult to treat is the tendency of the pathogens to switch among several different forms during their life cycles. A drug that is effective at killing one form can be ineffective at killing another form; therefore the population can survive the treatment.

1	Fungi include both unicellular yeasts (such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, which are used to bake bread and brew beer, and as model organisms for cell biology research) and filamentous, multicellular molds (like those found on moldy fruit or bread). Most of the important pathogenic fungi exhibit dimorphism—the ability to grow in either yeast or mold form. The yeast-to-mold or mold-to-yeast transition is frequently associated with infection.

1	Figure 23–7 Type III secretion systems that can deliver effector proteins into the cytosol of a host cell. (A) Electron micrograph of purified type III secretion systems, each of which consists of over two dozen proteins. (B) The large lower ring is embedded in the bacterial inner membrane, and the smaller upper ring is embedded in the bacterial outer membrane. During infection, docking of the tip of the hollow needle at a host-cell plasma membrane results in the secretion of bacterial translocator proteins (green), which form a pore in the host membrane, through which bacterial effector proteins are then secreted into the host cell. (A, from O. Schraidt et al., PLoS Pathog. 6(4):e1000824, 2010.) in the environment in the host Histoplasma capsulatum, for example, grows as a mold at low temperature in the soil, but it switches to a yeast form when inhaled into the lung, where it can cause the disease histoplasmosis (Figure 23–8).

1	Protozoan parasites are single-celled eukaryotes with more elaborate life cycles than fungi, and they frequently require more than one host. Malaria is the most devastating protozoal disease, infecting more than 200 million people every year and killing upward of 500,000. It is caused by four species of Plasmodium, which are transmitted to humans by the bite of the female Anopheles mosquito. release of infection of red blood cells invasion of gut and growth Figure 23–8 Dimorphism in the pathogenic fungus Histoplasma capsulatum. (A) At low temperature in the soil, H. capsulatum grows as a multicellular filamentous mold consisting of many individual cells connected together. (B) After it is inhaled into the lung of a mammal, the increase in temperature causes a switch to a yeast form consisting of small clumps of round cells. (C) A stained histologic section of a mouse lung infected with

1	H. capsulatum, showing a macrophage containing yeast forms of the pathogen. (A and B, courtesy of Sinem Beyhan and Anita Sil; C, courtesy of Davina Hocking Murray and Anita Sil.) Figure 23–9 The complex life cycle of malaria parasites. (A) The sexual cycle of Plasmodium falciparum requires passage between a human host and an insect host (Movie 23.3). (B)–(D) Blood smears from people with malaria, showing three different forms of the parasite that appear in red blood cells: (B) ring stage; (C) schizont; and (D) gametocyte. (B–D, courtesy of the Centers for Disease Control, Division of Parasitic Diseases, DPDx.)

1	Plasmodium falciparum causes the most serious form of malaria and is the most intensively studied of the malaria-causing parasites. It exists in many distinct forms, and it requires both the human and mosquito hosts to complete its sexual cycle (Figure 23–9). Several of these forms are highly specialized to invade and replicate in specific tissues—the lining of the insect gut, the human liver, and the human red blood cell. Even within a single host cell type, the red blood cell, the Plasmodium parasite undergoes a complex sequence of developmental events, reflected in striking morphological changes (Figure 23–9B–D). All Aspects of Viral Propagation Depend on Host Cell Machinery

1	All Aspects of Viral Propagation Depend on Host Cell Machinery Bacteria, fungal, and protozoan pathogens are living cells themselves. They use their own machinery for DNA replication, transcription, and translation, and, for the most part, they provide their own sources of metabolic energy. Viruses, by contrast, are the ultimate hitchhikers, carrying little more than information in the form of nucleic acid. Most clinically important human viruses have small genomes consisting of double-stranded DNA or single-stranded RNA (Table 23–1), and we now have complete genome sequences of almost all of them.

1	Viral genomes typically encode three types of protein: proteins for replicating the genome, proteins for packaging the genome and delivering it to more host cells, and proteins for modifying the structure or function of the host cell to enhance the replication of the virus (see Figure 7–62). In general, viral replication involves (1) entry into the host cell, (2) disassembly of the infectious virus particle, (3) replication of the viral genome, (4) transcription of viral genes and synthesis of viral proteins, (5) assembly of these viral components into progeny virus particles,

1	Figure 23–10 a simple viral life cycle. The hypothetical simple virus shown here consists of a small double-stranded DNA molecule that codes for only a single viral capsid protein. To reproduce, the viral genome must first enter a host cell, where it is replicated to produce multiple copies, which are transcribed and translated to produce the viral coat protein. The viral genomes can then assemble spontaneously with the coat protein to form a new virus particle, which escapes from the host cell. No known virus is this simple. and (6) release of progeny virions (Figure 23–10). A single virus particle (a virion) that infects a single host cell can produce thousands of progeny.

1	and (6) release of progeny virions (Figure 23–10). A single virus particle (a virion) that infects a single host cell can produce thousands of progeny. Virions come in a wide variety of shapes and sizes (Figure 23–11), and although most have relatively small genomes, genome size can vary considerably. The recently discovered giant viruses of amoebae, called pandoraviruses, are the largest known viruses, with 700 nm particles and double-stranded DNA genomes of over 2,000,000 nucleotide pairs. The virions of poxvirus are also large: they are 250–350 nm long and enclose a genome of double-stranded DNA of about 270,000 nucleotide pairs. At the other end of the size scale are the virions of parvovirus, which are less than 30 nm in diameter and have a single-stranded DNA genome of fewer than 5000 nucleotides.

1	Viral genomes are packaged in a protein coat, called a capsid, which in some viruses is further enclosed by a lipid bilayer membrane, or envelope. The capsid is made of one or several proteins, arranged in regular arrays that generally produce structures with either helical symmetry, which results in a cylindrical structure (for example, influenza, measles, and bunyavirus), or icosahedral symmetry (for example, poliovirus and herpesvirus; see Figure 23–11). Some viruses instead produce capsids with more complicated structures (for example, poxviruses). When the capsid is packaged with the viral genome, the structure is called a nucleocapsid. The nucleocapsids of nonenveloped viruses usually leave an infected cell by lysing it. For enveloped viruses, by contrast, the nucleocapsid is enclosed within a lipid bilayer membrane that the virus acquires in the process of budding from the host-cell plasma membrane, which it does without disrupting the membrane or killing the cell (Figure

1	within a lipid bilayer membrane that the virus acquires in the process of budding from the host-cell plasma membrane, which it does without disrupting the membrane or killing the cell (Figure 23–12). Enveloped viruses can cause persistent infections that may last for years, often without noticeable deleterious effects on the host.

1	Because the host cell performs most of the critical steps in viral replication, the identification of effective antiviral drugs that do not harm the host can be difficult. Probably the most effective strategy for containing viral diseases is through vaccinating of potential hosts. Highly successful vaccination programs have effectively poliovirus HIV Figure 23–11 examples of viral infuenza coronavirus rabies virus mumps virus(AIDS virus) virus (common cold) morphology. As shown, both DNA and shape. eliminated smallpox infection from the planet, and the eradication of poliomyelitis is approaching completion (Figure 23–13).

1	Infectious diseases are caused by pathogens, which include viruses, bacteria, and fungi, as well as protozoan and metazoan parasites. All pathogens must have mechanisms for entering their host and for evading immediate destruction by the host. The great majority of bacteria are not pathogenic to humans. Those that are pathogenic produce specific virulence factors that mediate the bacteria’s interactions with the host; these proteins change the behavior of host cells in ways that promote the replication and spread of the bacteria. Eukaryotic pathogens such as fungi and protozoan parasites typically pass through several different forms during the course of infection; the ability to switch among these forms is usually required for these pathogens to survive in a host and cause disease. In some cases, such as malaria, parasites must pass sequentially through several host species to complete their life cycles. Unlike bacteria and eukaryotic parasites, viruses have no metabolism of their

1	some cases, such as malaria, parasites must pass sequentially through several host species to complete their life cycles. Unlike bacteria and eukaryotic parasites, viruses have no metabolism of their own and no intrinsic ability to produce the proteins encoded by their DNA or RNA genomes; they rely on subverting the machinery of the host cell.

1	reported cases of polio per100,000 population Figure 23–12 acquisition of a viral envelope. (A) Electron micrograph of an animal cell from which six copies of an enveloped virus (Semliki forest virus) are budding. (B) Schematic drawing of the envelope assembly and budding processes. The lipid bilayer that surrounds the viral capsid is derived directly from the plasma membrane of the host cell. In contrast, the proteins in this lipid bilayer (shown in green) are encoded by the viral genome. (A, courtesy of M. Olsen and G. Griffith.) Figure 23–13 effective control of a viral disease through vaccination. The graph shows the number of cases of poliomyelitis reported per year in the United States. The arrows indicate the timing of the introduction of the Salk vaccine (inactivated virus given by injection) and the Sabin vaccine (live attenuated virus given orally).

1	The mechanisms through which pathogens cause disease are as diverse as the pathogens themselves. Nonetheless, all pathogens must carry out certain common tasks: they must gain access to the host, reach an appropriate niche, avoid host defenses, replicate, and exit from the infected host to spread to an uninfected one. In this section, we examine the common strategies that many pathogens use to accomplish these tasks. Pathogens Overcome Epithelial Barriers to Infect the Host The first step in infection is for the pathogen to gain access to the host. A thick covering of skin protects most parts of the human body from the environment. The protective boundaries of some other human tissues (eyes, nasal passages, respiratory tract, mouth, digestive tract, urinary tract, and female genital tract) are less robust. In the lungs and small intestine, for example, the barrier is just a single monolayer of epithelial cells. Nonetheless, all these epithelia serve as barriers to infection.

1	Wounds in barrier epithelia allow pathogens direct access to unoccupied niches within otherwise sterile host tissues. This avenue of entry requires little in the way of pathogen specialization, and many members of the normal flora can cause serious illness if they enter through such wounds. Staphylococci from the skin and nose, or Streptococci from the throat and mouth, are two examples of opportunistic bacterial pathogens that are responsible for many serious infections resulting from breaches in epithelial barriers. The recent emergence of bacterial strains of Staphylococcus that are resistant to the antibiotics commonly used for treatment (for example, methicillin-resistant Staphylococcus aureus, or MRSA, which infects up to 50,000,000 people worldwide) is of particular concern. Papillomaviruses, which cause warts and cervical cancer, also take advantage of breaches in epithelial barriers.

1	Primary pathogens, however, need not wait for a wound to gain access to their host. One efficient way for such a pathogen to cross the skin is to catch a ride in the saliva of a biting arthropod. A diverse group of bacteria, viruses, and protozoa has developed the ability to survive in insects and then use them as vectors to spread from one mammalian host to another. As discussed earlier, the Plasmodium protozoan that causes malaria develops through several forms in its life cycle, including some that are specialized for survival in a human and others that are specialized for survival in a mosquito (see Figure 23–9). Viruses that are spread by insect bites cause yellow fever and Dengue fever, as well as many kinds of viral encephalitis (inflammation of the brain). These viruses replicate in both insect cells and mammalian cells, as required for their transmission by an insect vector.

1	The efficient spread of a pathogen via an insect vector requires that an individual insect consumes a blood meal from an infected host and transfers the pathogen to a naive host. In a few striking cases, the pathogen alters the behavior of the insect so that its transmission to a new host is more likely. An example is the bacterium Yersinia pestis, which causes bubonic plague. It multiplies in the flea’s foregut to form aggregated masses that physically block the digestive tract; during each repeated, but futile, attempt at feeding, some of the bacteria in the foregut are flushed into the bite site, thus transmitting plague to a new host (Figure 23–14).

1	Whereas many epithelial barriers such as the skin and the lining of the mouth and large intestine are densely populated by normal flora, others, including the lining of the lower lung and the bladder, are normally kept nearly sterile. How do these epithelia avoid bacterial colonization? A layer of protective mucus covers the respiratory epithelium, and the coordinated beating of cilia sweeps the mucus and trapped bacteria up and out of the lung. The epithelial lining of the bladder and the upper gastrointestinal tract also has a thick layer of mucus, and

1	Figure 23–14 Plague bacteria within a flea. This light micrograph shows the digestive tract dissected from a flea that had dined about two weeks previously on the blood of an animal infected with the plague bacterium, Yersinia pestis. The bacteria multiplied in the flea gut to produce large cohesive aggregates (red arrows); the bacterial mass on the left is occluding the passage between the esophagus and the midgut. This type of blockage prevents a flea from digesting its blood meals, so that hunger causes it to bite repeatedly, disseminating the infection. (From B.J. Hinnebusch, E.R. Fischer and T.G. Schwan, J. Infect. Dis. 178:1406–1415, 1998.) these organs are periodically flushed by urination and by peristalsis, respectively, which washes away most microbes.

1	Pathogenic bacteria and eukaryotic parasites that infect these epithelial surfaces have evolved specific mechanisms for overcoming these protective mechanisms. Those that infect the urinary tract, for example, adhere tightly to the epithelial lining via specific adhesins, which are proteins or protein complexes that recognize and bind to cell-surface molecules on the epithelium. An important group of adhesins in E. coli strains that infect the kidney are components of the pili—surface projections that can be several micrometers long and thus able to span the thickness of the protective mucus layer; at the tip of each pilus is an adhesin protein that binds tightly to the D-galactose–D-galactose disaccharide on glycolipids on the surface of kidney cells (Figure 23–15). Strains of E. coli that infect the bladder rather than the kidney express a second kind of pilus with a different adhesin protein that binds to bladder epithelial cells. It is the specificity of the adhesin proteins on

1	that infect the bladder rather than the kidney express a second kind of pilus with a different adhesin protein that binds to bladder epithelial cells. It is the specificity of the adhesin proteins on the tips of the two types of pili that is responsible for the bacteria’s colonizing of the different parts of the urinary tract.

1	The stomach is an especially hostile environment for pathogens. Besides the thick layer of mucus and peristaltic washing, it is filled with acid (average pH ≈2), which is lethal to almost all bacteria ingested in food. Yet, it is home to a microbiota of hundreds of resident species, including the bacterium H. pylori, which, as we discussed earlier, is the major cause of stomach ulcers and some stomach cancers. The hypothesis that a persistent bacterial infection could cause stomach ulcers was initially met with skepticism. The young Australian doctor who made the initial discovery finally proved the point: he drank a pure culture of H. pylori and developed inflammation of the stomach, which often precedes the development of ulcers. A short course of antibiotics can now effectively cure a patient of recurrent stomach ulcers. Remarkably, H. pylori is able to persist for life as a commensal in most humans. One way in which it survives in the stomach is by producing the enzyme urease,

1	a patient of recurrent stomach ulcers. Remarkably, H. pylori is able to persist for life as a commensal in most humans. One way in which it survives in the stomach is by producing the enzyme urease, which converts urea to ammonia that neutralizes the acid in its immediate vicinity. The bacterium also uses its flagellum for chemotactic motility, allowing it to seek out the more neutral pH near the surface of gastric epithelial cells. H. pylori virulence proteins that target both epithelial and immune cells help H. pylori persist in the stomach, but they can also induce chronic inflammation, alteration in host gene expression, changes in cell proliferation and apoptosis, and disruption of cell–cell junctions, all of which are predisposing factors for stomach cancer.

1	Extracellular pathogens can cause serious disease without entering host cells. Bordetella pertussis, the bacterium that causes whooping cough, for example, colonizes the respiratory epithelium and circumvents the normal mechanism that Figure 23–15 Pathogenic E. coli in the infected bladder of a mouse.

1	(A) Scanning electron micrograph of uropathogenic E. coli, a common cause of bladder and kidney infections. The bacteria are attached to the surface of epithelial cells lining the infected bladder. (B) A closeup view of one of the bacteria showing the pili on its surface. (C) An E. coli pilus has adaptor proteins on its tip that bind to glycolipids on the surface of kidney cells. (A, from G.E. Soto and S.J. Hultgren, J. Bacteriol. 181:1059–1071, 1999. With permission from the American Society for Microbiology; B, courtesy of D.G. Thanassi and S.J. Hultgren, Methods 20:111–126, 2000. With permission from Academic Press.) (A) P P P Tir injected Tir inserts and folds in plasma membrane HOST CELL type III secretion system enteropathogenic E. coli outer membrane inner membrane intimin intimin actin flaments pedestal phosphorylated Tir host proteins that promote actin polymerization clears the respiratory tract by expressing adhesins that bind ciliated epithelial cells. The adherent

1	actin flaments pedestal phosphorylated Tir host proteins that promote actin polymerization clears the respiratory tract by expressing adhesins that bind ciliated epithelial cells. The adherent bacteria produce toxins that eventually kill the ciliated cells, compromising the host’s ability to clear the infection. The most familiar of these is pertussis toxin, which, like the cholera toxin discused above, has an A subunit that ADP-ribosylates the α subunit of the G protein Gi, inhibiting the G protein from suppressing the activity of the host cell’s adenylyl cyclase, thereby increasing the production of cyclic AMP (see Figure 23–6). This toxin also interferes with the chemotactic pathway that neutrophils use to seek out and destroy invading bacteria (see Figures 16–3 and 16–86). B. pertussis colonization of the respiratory tract causes severe coughing, which helps spread the infection.

1	Not all extracellular pathogens that colonize an epithelium exert their effect through toxins. Enteropathogenic E. coli (EPEC), which causes diarrhea in young children, uses a type III secretion system (see Figure 23–7) to deliver its own special receptor protein (called Tir) into the plasma membrane of a host intestinal epithelial cell (Figure 23–16). The extracellular domain of Tir binds to the bacterial surface protein intimin, triggering actin polymerization in the host cell that results in the formation of a unique cell-surface protrusion called a pedestal; this pushes the tightly adherent bacteria up about 10 μm from the host-cell membrane, thereby promoting bacterial movement along the cell surface. A similar strategy is used by vaccinia virus (the virus that was used as a vaccine to eradicate smallpox) to form mobile pedestals, which promote spread of the virus from cell to cell. The study of how EPEC and vaccinia virus promote actin polymerization has been of major importance

1	eradicate smallpox) to form mobile pedestals, which promote spread of the virus from cell to cell. The study of how EPEC and vaccinia virus promote actin polymerization has been of major importance in understanding how intracellular signaling pathways regulate the cytoskeleton in normal, uninfected cells (discussed in Chapter 16). Although pedestal formation promotes the spread of these pathogens, the sympoms of EPEC infection (severe diarrhea) are caused by the loss of absorptive microvilli and disruption of signaling pathways in epithelial cells, which are triggered by Tir and other effector proteins.

1	Many pathogens have to enter host cells to cause disease. These intracellular pathogens include all viruses and many bacteria and protozoa. Each of these has a preferred niche for replication and survival within host cells. Bacteria and protozoa replicate either in the cytosol or within a membrane-enclosed compartment. While most RNA viruses replicate within the cytosol, most DNA viruses replicate in the nucleus. Life inside a host cell has several advantages. The pathogens are not accessible to antibodies, nor are they easy targets for phagocytic cells (discussed in Chapter 24); furthermore, intracellular bacteria and protozoa are bathed in a rich source of nutrients, and viruses have access to the host cell’s biosynthetic

1	Figure 23–16 Interaction of enteropathogenic E. coli (ePeC) with host intestinal epithelial cells. (A) When EPEC contacts an epithelial cell in the lining of the human gut, it delivers a bacterial protein called Tir into the host cell through a type III secretion system. Tir then inserts into the plasma membrane of the host cell, where it functions as a receptor for the bacterial adhesin protein intimin. Next, a host-cell protein tyrosine kinase phosphorylates the intracellular domain of Tir on tyrosines. Phosphorylated Tir recruits host-cell proteins (including an adaptor protein, a WASp protein, and the Arp 2/3 complex) that trigger actin polymerization (see Figure 16–16). Consequently, a branched network of actin filaments assembles underneath the bacterium, forming an actin pedestal (Movie 23.4). (B) EPEC on a pedestal. In this fluorescence micrograph, the DNA of the EPEC and host cell is labeled in blue, Tir protein is labeled in green, and host-cell actin filaments are labeled

1	(Movie 23.4). (B) EPEC on a pedestal. In this fluorescence micrograph, the DNA of the EPEC and host cell is labeled in blue, Tir protein is labeled in green, and host-cell actin filaments are labeled in red. The inset shows a close-up view of the two upper bacteria on pedestals. (B, from

1	D. Goosney et al., Annu. Rev. Cell Dev. Biol. 16:173–189, 2000. With permission from Annual Reviews.) machinery for their reproduction. This lifestyle, however, requires that the pathogen have mechanisms for entering host cells, for finding a suitable subcellular niche where it can replicate, and for exiting from the infected cell to spread the infection. Below we consider some of the myriad ways that individual intracellular pathogens exploit and modify host cell biology to satisfy these requirements. Viruses Bind to Virus Receptors at the Host Cell Surface The first step for any intracellular pathogen is to bind to the surface of the host target cell. Viruses accomplish this by the binding of viral surface proteins to virus receptors displayed on the host cell. The first virus receptor identified was an

1	E. coli surface protein that is recognized by the bacteriophage lambda; the protein normally functions to transport the sugar maltose from outside the bacterium to the inside where it is used as an energy source. Receptors need not be proteins, however: an envelope protein of herpes simplex virus, for example, binds to heparan sulfate proteoglycans (discussed in Chapter 19) on the surface of certain vertebrate host cells, and simian virus 40 (SV40) binds to a glycolipid. The specificity of virus-receptor interactions often serves as a barrier preventing the spread of a virus from one species to another. Acquiring the ability to bind to a new receptor often requires multiple changes in a virus, but it can be crucial in allowing the cross-species transmission that can result in new disease outbreaks.

1	Viruses that infect animal cells generally exploit cell-surface receptor molecules that are either ubiquitous (such as the sialic-acid-containing oligosaccharides used by the influenza virus) or found uniquely on those cell types in which the virus replicates (such as the neuron-specific proteins used by rabies virus). Although a virus usually uses a single type of host-cell receptor, some viruses use more than one type. An important example is HIV-1, which requires two types of receptors to enter a host cell. Its primary receptor is CD4, a cell-surface protein on helper T cells and macrophages that is involved in immune recognition (discussed in Chapter 24). It also requires a co-receptor, which is either CCR5 (a receptor for β-chemokines) or CXCR4 (a receptor for α-chemokines), depending on the particular variant of the virus; macrophages are susceptible only to HIV variants that use CCR5 for entry, whereas helper T cells are most efficiently infected by variants that use CXCR4

1	on the particular variant of the virus; macrophages are susceptible only to HIV variants that use CCR5 for entry, whereas helper T cells are most efficiently infected by variants that use CXCR4 (Figure 23–17). The viruses that are found within the first few months after HIV infection almost invariably use CCR5, which explains why individuals that carry a defective CCR5 gene are less susceptible to HIV infection. In the later stages of infection, viruses often either switch to use CXCR4 or adapt to use both co-receptors through the accumulation of mutations; in this way, the virus can change the cell types it infects as the disease progresses. It may seem paradoxical that viruses would infect immune cells, as we might expect that virus binding would trigger an immune response; but invasion of an immune cell can be a useful way for a virus to weaken the immune response and travel around the body to infect other immune cells.

1	°-chemokine ˜-chemokine (Sdf1) (Rantes, Mip1°, or Mip1˜) Figure 23–17 Receptor and co-receptors for HIV. All strains of HIV require the CD4 protein as a primary receptor. Early in an infection, most of the viruses use CCR5 as a co-receptor, allowing them to infect macrophages and their precursors, monocytes. As the infection progresses, mutant variants arise that now use CXCR4 as a co-receptor, enabling them to infect helper T cells efficiently. The natural ligand for the chemokine receptors (Sdf1 for CXCR4; Rantes, Mip1α, or Mip1β for CCR5) blocks co-receptor function and prevents viral invasion. Viruses Enter Host Cells by Membrane Fusion, Pore Formation, or Membrane Disruption

1	After recognition and attachment to the host cell surface, the virus must enter the cell to replicate. Some enveloped viruses enter the host cell by fusing their envelope membrane with the plasma membrane. Most viruses, whether enveloped or nonenveloped, activate signaling pathways in the cell that induce endocytosis, commonly via clathrin-coated pits (see Figure 13–7), leading to internalization into endosomes. Large viruses that do not fit into clathrin-coated vesicles, such as poxviruses, often enter cells by macropinocytosis, a process by which membrane ruffles fold over and entrap fluid into macropinosomes (see Figure 13–50). Once inside endosomes, fusion of the viral envelope occurs from the lumenal side of the endosome membrane. The mechanism of membrane fusion mediated by viral spike glycoproteins has similarities with SNARE-mediated membrane fusion during normal vesicular trafficking (discussed in Chapter 13).

1	Enveloped viruses regulate fusion both to ensure that they fuse only with the appropriate host cell membrane and to prevent fusion with one another. For viruses such as HIV-1 that fuse at neutral pH with the plasma membrane (Figure 23–18A), binding to receptors or co-receptors usually triggers a conformational change in a viral envelope protein that exposes a normally buried fusion peptide (see Figure 13–21). Other enveloped viruses, such as influenza A virus, only fuse with a host cell membrane after endocytosis (Figure 23–18B); in this case, it is frequently the acid environment in the late endosome that triggers the conformational change in a viral surface protein that exposes the fusion peptide. The H+ (B) acidifcation, fusion, uncoating fusion with membrane after endocytosis endosomal membrane disruption pore formation(C) (D) endocytosis endocytosis early endosome lysis uncoating, pore formation DNA enters nucleus Figure 23–18 Four virus entry strategies.

1	(A) Some enveloped viruses, such as HIV, fuse directly with the host-cell plasma membrane to release their RNA genome (blue) and capsid proteins (brown) into the cytosol. (B) Other enveloped viruses, such as influenza virus, first bind to cell-surface receptors, triggering receptor-mediated endocytosis; when the endosome acidifies, the virus envelope fuses with the endosomal membrane, releasing the viral RNA genome (blue) and capsid proteins (brown) into the cytosol. (C) Poliovirus, a nonenveloped virus, induces receptor-mediated endocytosis, and then forms a pore in the endosomal membrane to extrude its RNA genome (blue) into the cytosol. (D) Adenovirus, another nonenveloped virus, uses a more complicated strategy: it induces receptor-mediated endocytosis and then disrupts the endosomal membrane, releasing the capsid and its DNA genome into the cytosol; the trimmed-down virus eventually docks onto a nuclear pore and releases its DNA (red) directly into the nucleus (Movie 23.5).

1	pumped into the early endosome also has another effect; it enters the influenza virion through an ion channel in the viral envelope and triggers changes in the viral capsid. These priming steps allow the capsids to disassemble once released into the cytosol after virus fusion with the late endosomal membrane.

1	Nonenveloped viruses use different strategies to enter host cells—strategies that do not rely on membrane fusion. Poliovirus, which causes poliomyelitis, binds to a cell-surface receptor, triggering both receptor-mediated endocytosis (see Figure 13–52) and a conformational change in the viral particle. The conformational change exposes a hydrophobic projection on one of the capsid proteins, which inserts into the endosomal membrane to form a pore. The viral RNA genome then enters the cytosol through the pore, leaving the capsid in the endosome (Figure 23–18C). Other nonenveloped viruses such as adenovirus disrupt the endosomal membrane after they are taken up by receptor-mediated endocytosis. One of the proteins released from the capsid lyses the endosomal membrane, releasing the remainder of the virus into the cytosol. During endosomal trafficking and subsequent transport within the cytosol, adenoviruses undergo multiple uncoating steps, which sequentially remove structural proteins

1	of the virus into the cytosol. During endosomal trafficking and subsequent transport within the cytosol, adenoviruses undergo multiple uncoating steps, which sequentially remove structural proteins and ready the virus particles to release their DNA into the nucleus through nuclear pore complexes (Figure 23–18D).

1	Bacteria are much larger than viruses—too large to be taken up either through pores or by receptor-mediated endocytosis. Instead, they enter host cells by phagocytosis, which is a normal function of phagocytes such as neutrophils, macrophages, and dendritic cells (discussed in Chapter 24). These phagocytes patrol the tissues of the body and ingest and destroy microbes; however, some intracellular bacterial pathogens such as M. tuberculosis use this to their advantage and have evolved to survive and multiply inside macrophages. Some bacterial pathogens can invade host cells that are normally nonphagocytic. One way they do so is by expressing an invasion protein that binds with high affinity to a host-cell receptor, which is often a cell–cell or cell–matrix adhesion protein (discussed in Chapter 19). For example, Yersinia pseudotuberculosis (a bacterium that causes diarrhea and is a close relative of the plague bacterium

1	Y. pestis) expresses a protein called invasin that has an RGD motif that is similar to fibronectin’s and likewise is recognized by host-cell β1 integrins (see Figure 19–55). Listeria monocytogenes, which causes a rare but serious form of food poisoning, invades host cells by expressing a protein that binds to the cell–cell adhesion protein E-cadherin (see Figure 19–6). For both these bacterial species, binding of the bacterial invasion proteins to the host cell adhesion proteins stimulates signaling through members of the Rho family of small GTPases (discussed in Chapter 16). This in turn activates proteins in the WASp family and the Arp 2/3 complex, leading to actin polymerization at the site of bacterial attachment. Actin polymerization, together with the assembly of a clathrin coat (see Figure 13–6), drives the advancement of the host cell’s plasma membrane over the adhesive surface of the microbe, resulting in the phagocytosis of the bacterium—a process known as the zipper

1	(see Figure 13–6), drives the advancement of the host cell’s plasma membrane over the adhesive surface of the microbe, resulting in the phagocytosis of the bacterium—a process known as the zipper mechanism of invasion (Figure 23–19A).

1	A second pathway by which bacteria can invade nonphagocytic cells is known as the trigger mechanism (Figure 23–19B). It is used by various pathogens that cause food poisoning, including Salmonella enterica, and it is initiated when the bacterium injects a set of effector molecules into the host-cell cytosol through a type III secretion system (see Figure 23–7). Some of these effector molecules activate Rho family proteins, which in turn stimulate actin polymerization, as just discussed. Other bacterial effector proteins interact with host-cell cytoskeletal elements more directly, nucleating and stabilizing actin filaments and causing the rearrangement of actin cross-linking proteins. The overall effect is to cause the formation of localized ruffles on the surface of the host cell (Figure 23–19C and D), which fold over and engulf the bacteria by a process that resembles macropinocytosis. The appearance of cells being invaded by use of the trigger mechanism invasin receptors type III

1	23–19C and D), which fold over and engulf the bacteria by a process that resembles macropinocytosis. The appearance of cells being invaded by use of the trigger mechanism invasin receptors type III secretioninvasin (integrins, cadherins) apparatus is similar to the ruffling induced by some extracellular growth factors, suggesting that the bacteria exploit normal intracellular signaling pathways.

1	The uptake of viruses and bacteria into host cells is carried out largely by the host, with the pathogen being a relatively passive participant. In contrast, intracellular eukaryotic parasites, which are typically much larger than other types of intracellular pathogens, invade host cells through a variety of complex pathways that usually require energy expenditure by the parasite.

1	Toxoplasma gondii,a cat parasite that also causes occasional serious human infections, is an example. When this protozoan contacts a host cell, it protrudes an unusual microtubule-based structure called a conoid, which facilitates entry into the host cell (Figure 23–20). The energy for invasion seems to come from actin polymerization in the parasite rather than host cytoskeleton, and invasion also requires at least one unusual parasite myosin motor protein (Class XIV; see Figure 16–40). At the point of contact, the parasite discharges effector proteins from secretory organelles into the host cell, and these proteins target various host pathways to enable invasion, to block an innate immune response, and promote survival. As the parasite moves into the host cell, a membrane derived from the host-cell plasma membrane surrounds it. Remarkably, the parasite removes host transmembrane proteins from the surrounding membrane as it forms, so that the parasite is protected in a

1	derived from the host-cell plasma membrane surrounds it. Remarkably, the parasite removes host transmembrane proteins from the surrounding membrane as it forms, so that the parasite is protected in a membrane-enclosed compartment that does not fuse with lysosomes and does not participate in host-cell membrane trafficking processes (see Figure 23–20). The specialized membrane is selectively porous: it allows the parasite to take up small metabolic intermediates and nutrients from the host cell’s cytosol but excludes macromolecules. Malaria parasites invade human red blood cells using a similar mechanism.

1	Figure 23–19 Mechanisms used by bacteria to induce phagocytosis by host cells that are normally nonphagocytic. In the zipper mechanism, bacteria express an invasion protein that binds with high affinity to a host-cell receptor, which is often a cell–cell or cell–matrix adhesion protein. (B) In the trigger mechanism, bacteria inject a set of effector molecules into the host-cell cytosol through a type III secretion system called SPI1 (Salmonella pathogenicity island 1), inducing membrane ruffling. Both the zipper and trigger mechanisms cause the polymerization of actin at the site of bacterial attachment by activating Rho family small GTPases and the Arp 2/3 complex. (C) A scanning electron micrograph showing a very early stage of Salmonella enterica invasion by the trigger mechanism. Bacteria (pseudocolored yellow) are shown surrounded by a small membrane ruffle.

1	Fluorescence micrograph showing that the large ruffles that engulf the Salmonella bacteria are actin-rich. The bacteria are labeled in green and actin filaments in red; because of the color overlap, the bacteria appear yellow. (C, from Rocky Mountain Laboratories, NIAID, NIH; D, from J.E. Galán, Annu. Rev. Cell Dev. Biol. 17:53–86, 2001. With permission from Annual Reviews.)

1	J.E. Galán, Annu. Rev. Cell Dev. Biol. 17:53–86, 2001. With permission from Annual Reviews.) The protozoan Trypanosoma cruzi, which causes Chagas disease, in Mexico and Central and South America, uses two alternative invasion strategies. In a lysosome-dependent pathway, the parasite attaches to host cell-surface receptors, inducing a local increase in Ca2+ in the host cell’s cytosol. The Ca2+ signal recruits lysosomes to the site of parasite attachment, and the lysosomes fuse with the host cell’s plasma membrane, allowing the parasites rapid access to the lysosomal compartment (Figure 23–21). In a lysosome-independent pathway, the parasite penetrates the host-cell plasma membrane by inducing the membrane to invaginate, without the recruitment of lysosomes. 1. ATTACHMENT TO HOST 2. Ca2+ SIGNAL 3. FUSION OF LYSOSOMES CELL-SURFACE RECEPTORS RECRUITS LYSOSOMES WITH PLASMA MEMBRANE Figure 23–20 The life cycle of the intracellular parasite Toxoplasma gondii.

1	Figure 23–20 The life cycle of the intracellular parasite Toxoplasma gondii. After attachment to a host cell, T. gondii uses its conoid to inject effector proteins that facilitate invasion. As the host cell’s plasma membrane invaginates to surround the parasite, it somehow removes the normal host-cell membrane proteins, so that the compartment (shown in red) does not fuse with lysosomes. After several rounds of replication, the parasite causes the compartment to break down and the host cell to lyse, releasing the progeny parasites to infect other host cells (Movie 23.6). Light micrograph of T. gondii replicating within a membrane-enclosed compartment (a vacuole) in a cultured cell. (B, courtesy of Manuel Camps and John Boothroyd.) HOST CYTOSOL TrypanosomacruziCa2+ Ca2+ compartment derived from lysosomal membranelysosomes lysosomes early endosome 5. SECRETION OF PORE-FORMING PROTEIN REPLICATION 4. INVASION 6. LYSIS OF SURROUNDING MEMBRANE, RELEASE OF PATHOGEN INTO CYTOSOL

1	Figure 23–21 The two alternative strategies that Trypanosoma cruzi uses to invade host cells. In the lysosome-dependent pathway (left), T. cruzi recruits host-cell lysosomes to its site of attachment to the host cell. The lysosomes fuse with the invaginating plasma membrane to create an intracellular compartment constructed almost entirely of lysosomal membrane. After a brief stay in the compartment, the parasite secretes a pore-forming protein that disrupts the surrounding membrane, thereby allowing the parasite to escape into the host-cell cytosol and proliferate. In the lysosome-independent pathway (right), the parasite induces the host plasma membrane to invaginate and pinch off without recruiting lysosomes; then lysosomes fuse with the endosome prior to the parasite’s escape into the cytosol.

1	Figure 23–22 Choices that an intracellular pathogen faces. After entry into a host cell, generally through phagocytosis into a membrane-enclosed compartment, intracellular pathogens can use one of three strategies to survive and replicate. Pathogens that follow strategy (1) include all viruses, Trypanosoma cruzi, Listeria monocytogenes, and Shigella flexneri. Those that follow strategy (2) include Mycobacterium tuberculosis and Legionella pneumophila. Those that follow strategy (3) include Salmonella enterica, Coxiella burnetii, and Leishmania. Some Intracellular Pathogens Escape from the Phagosome into the Cytosol

1	Some Intracellular Pathogens Escape from the Phagosome into the Cytosol The intracellular parasites just discussed raise a general problem that faces all intracellular pathogens, including viruses, bacteria, and eukaryotic parasites: they must find a cell compartment in which they can replicate. After their endocytosis by a host cell, they usually find themselves in an endosomal compartment, which normally would fuse with lysosomes to form a phagolysosome—a dangerous place for pathogens. To survive, pathogens use a variety of strategies. Some escape from the endosomal compartment before such fusion. Others remain in the endosomal compartments but modify it so that it no longer fuses with lysosomes. Still others have evolved to weather the harsh conditions in the phagolysosome (Figure 23–22).

1	Trypanosoma cruzi uses the escape route by secreting a pore-forming toxin that lyses the lysosome membrane, releasing the parasite into the host cell’s cytosol (see Figure 23–21). The bacterium Listeria monocytogenes uses a similar strategy. Following phagocytosis by the zipper mechanism, it secretes a protein called listeriolysin O, which disrupts the phagosomal membrane, releasing the bacteria into the cytosol (Figure 23–23). Many Pathogens Alter Membrane Traffic in the Host Cell to Survive and Replicate The survival and reproduction of many intracellular pathogens requires that they modify membrane (vesicular) traffic in the host cell. They may, for example, prevent the normal fusing of endosomes with lysosomes, or adapt themselves to 1 2 uptake by zipper mechanism 3 listeriolysin secretion 4 listeriolysin-mediated membrane disruption 5 bacterial release and replication 6 secreted listeriolysin now destroyed in host proteasomes phagosome proteasome pH <6.0 pH >6.0

1	Figure 23–23 escape of Listeria monocytogenes by selective destruction of the phagosomal membrane. The bacterium attaches to E-cadherin on the surface of host epithelial cells and induces its own uptake by the zipper mechanism (see Figure 23–19A). Within the phagosome, the bacterium secretes the protein listeriolysin O, which is activated at pH <6 and forms oligomers in the phagosome membrane, thereby creating large pores and eventually disrupting the membrane. Once in the host-cell cytosol, the bacteria begin to replicate and continue to secrete listeriolysin O; because the pH in the cytosol is >6, however, the listeriolysin O there is inactive and is also rapidly degraded by proteasomes. Thus, the host cell’s plasma membrane remains intact. resist the lysosome’s antimicrobial armaments. Intracellular pathogens must also provide a pathway for importing nutrients from the host cytosol into their compartment of choice.

1	Different pathogens have distinct strategies for altering membrane traffic in the host cell (Figure 23–24). M. tuberculosis prevents the early endosome that contains the bacteria from maturing, so the endosome never acidifies or acquires the other characteristics of a late endosome or lysosome. This strategy requires the activity of its type VII secretion system, as well as mycobacterial lipid products that mimic host lipids and influence vesicular traffic. Phagosomes containing Salmonella enterica, in contrast, acidify and acquire markers of late endosomes and lysosomes, but the bacteria slow the process of phagosomal maturation. They do so by injecting effector proteins through a second type III secretion system. These effectors activate host kinesin motor proteins to pull membrane tubules outward from the phagosome along cytoplasmic microtubules, forming a specialized compartment called the Salmonella-containing vacuole (Figure 23–25).

1	Other bacteria seem to find shelter in intracellular compartments that are distinct from those of the usual endocytic system. One example is Legionella pneumophila, which was first recognized as a human pathogen in 1976, when it was found to be the cause of a type of pneumonia known as Legionnaire’s disease. L. pneumophila is normally a parasite of freshwater amoebae, but it is commonly

1	L. pneumophila is normally a parasite of freshwater amoebae, but it is commonly Figure 23–24 Modifications of membrane traffic in host cells by bacterial pathogens. Intracellular bacterial pathogens, including Mycobacterium tuberculosis, Salmonella enterica, and Legionella pneumophila, all replicate in membrane-enclosed compartments, but the compartments differ. M. tuberculosis remains in a compartment that has early endosomal markers and continues to communicate with the plasma membrane via transport vesicles. S. enterica replicates in a compartment that has late endosomal markers and does not communicate with the plasma membrane. L. pneumophila replicates in an unusual compartment that is wrapped in rough endoplasmic reticulum (ER) membrane and communicates with the ER via transport vesicles. TGN, trans Golgi network.

1	Figure 23–25 Salmonella enterica residing in a modified phagosomal compartment called the Salmonella-containing vacuole. These bacteria invade the host cell using an SPI1 type III secretion system to inject effector proteins that induce the trigger mechanism of microbe entry illustrated in Figure 23–19B. Following its engulfment into a phagosome, the bacterium inactivates its SPI1 type III secretion system and activates its SPI2 type III secretion system to inject different effector proteins, which remodel the phagosome into the specialized Salmonella-containing vacuole. One of the injected effector proteins activates host kinesin motor proteins to pull membrane tubules outward toward the plus ends of the microtubules (see Figure 16–42). (B) Fluorescence micrograph showing S. enterica in a Salmonella-containing vacuole. The bacteria are stained green, the microtubules red, and the nucleus blue. (B, courtesy of Stephane Meresse.) blocking of type IV secretion 0.5 µm

1	Figure 23–26 Legionella pneumophila residing in a compartment with characteristics similar to those of the rough endoplasmic reticulum (eR). (A) Electron micrograph showing the unusual coiled structure that the Legionella pneumophila bacterium induces on the surface of a phagocyte during the invasion process. Some other pathogens, including the bacterium Borrelia burgdorferi, which causes Lyme disease, the eukaryotic pathogen Leishmania, and the yeast Candida albicans, can also invade cells using this type of coiling phagocytosis. (B) Following invasion, L. pneumophila uses its type IV secretion system to secrete effector proteins that block phagosome–endosome fusion and phagosome maturation. It also secretes effector proteins that promote the fusion of the phagosome with ER-derived vesicles, thereby creating a Legionella-containing vacuole with characteristics similar to the rough ER. (A, from M.A. Horwitz, Cell 36:27–33, 1984. With permission from Elsevier.) spread to humans by

1	thereby creating a Legionella-containing vacuole with characteristics similar to the rough ER. (A, from M.A. Horwitz, Cell 36:27–33, 1984. With permission from Elsevier.) spread to humans by central air-conditioning systems, which harbor infected amoebae and produce microdroplets of water that are easily inhaled. Once in the lung, the bacteria are engulfed by macrophages by an unusual process called coiling phagocytosis (Figure 23–26A). L. pneumophila uses a type IV secretion system to inject effector proteins into the phagocyte that modulate the activity of proteins that regulate vesicular traffic, including SNARE proteins and Rab and Arf family small GTPases (discussed in Chapter 13). The effector proteins thereby prevent the phagosome from fusing with endosomes and promote its fusion with vesicles derived from the endoplasmic reticulum, converting the phagosome into a compartment that resembles the rough endoplasmic reticulum (Figure 23–26B).

1	Viruses can also alter membrane traffic in the host cell. Enveloped viruses make use of host cell membranes to acquire their own envelope membrane. In the simplest cases, virally encoded glycoproteins are inserted into the endoplasmic reticulum membrane and follow the secretory pathway through the Golgi apparatus to the plasma membrane; the viral capsid proteins and genome assemble into nucleocapsids, which acquire their envelope as they bud off from the plasma membrane (see Figure 23–12). This mechanism is used by many enveloped viruses including HIV-1. Other enveloped viruses such as herpesviruses and vaccinia virus acquire their lipid envelopes in more complex ways (Figure 23–27). Viruses and Bacteria Use the Host-Cell Cytoskeleton for Intracellular Movement

1	Viruses and Bacteria Use the Host-Cell Cytoskeleton for Intracellular Movement As mentioned earlier, many pathogens escape into the cytosol rather than remaining in a membrane-enclosed compartment. The cytosol of mammalian cells is extremely viscous, as it is crowded with protein complexes, organelles, and cytoskeletal filaments, all of which inhibit the diffusion of particles the size of a bacterium or a viral nucleocapsid. Thus, to reach a particular region of the host cell a pathogen must be actively moved there. As with transport of intracellular organelles, pathogens generally use the host cell’s cytoskeleton for their active movement. nucleus Golgi WRAPPING (?) (B)

1	Several bacteria that replicate in the host cell’s cytosol have adopted a remarkable mechanism that depends on actin polymerization for movement. These bacteria include the human pathogens Listeria monocytogenes, Shigella flexneri, Rickettsia rickettsii (which causes Rocky Mountain spotted fever), and Burkholderia pseudomallei (which causes melioidosis, a disease characterized by severe respiratory symptoms). Baculovirus, an insect virus, also uses this mechanism for intracellular movement. All of these pathogens induce the nucleation and assembly of host-cell actin filaments at one pole of the bacterium or virus. The growing filaments generate force and push the pathogens through the cytosol at rates of up to 1 μm/sec (Figure 23–28). New filaments form at the rear of each pathogen and are left behind like a rocket trail as the microbe advances; the filaments depolymerize within a minute or so as they encounter depolymerizing factors in the cytosol. For L. monocytogenes and S.

1	and are left behind like a rocket trail as the microbe advances; the filaments depolymerize within a minute or so as they encounter depolymerizing factors in the cytosol. For L. monocytogenes and S. flexneri, the moving bacteria collide with the plasma membrane and move outward, inducing the formation of long, thin, host-cell protrusions with the bacteria at their tip. As shown in Figure 23–28, a neighboring cell often engulfs these projections, allowing the bacteria to enter the neighbor’s cytoplasm without exposure to the extracellular environment, thereby avoiding antibodies produced by the host’s adaptive immune system. For B. pseudomallei, movement and collision of the bacteria with the plasma membrane promotes cell–cell fusion, which serves a similar purpose of immune avoidance while allowing continued bacterial replication.

1	Figure 23–27 Complex strategies for viral envelope acquisition. (A) Herpesvirus nucleocapsids assemble in the nucleus and then bud through the inner nuclear membrane into the space between the inner and outer nuclear membranes, acquiring a lipid bilayer membrane coat. The virus particles then apparently lose this coat when they fuse with the endoplasmic reticulum membrane to escape into the cytosol. Subsequently, the nucleocapsids bud into the Golgi apparatus and bud out again on the other side, thereby acquiring two new membrane coats in the process. The virus then buds from the cell surface with a single membrane when its outer membrane fuses with the plasma membrane. (B) Vaccinia virus (which is closely related to the virus that causes smallpox and is used to vaccinate against smallpox) assembles in “replication factories” in the cytosol, far away from the plasma membrane. The immature virion, with one membrane, is then surrounded by two additional membranes, both acquired from the

1	assembles in “replication factories” in the cytosol, far away from the plasma membrane. The immature virion, with one membrane, is then surrounded by two additional membranes, both acquired from the Golgi apparatus by a poorly understood wrapping mechanism, to form the intracellular enveloped virion. After fusion of the outermost membrane with the host-cell plasma membrane, the extracellular enveloped virion is released from the host cell.

1	Figure 23–28 The actin-based movement of bacterial pathogens within and between host cells. (A) Following invasion, bacterial pathogens such as Listeria monocytogenes, Shigella flexneri, Rickettsia rickettsii, and Burkholderia pseudomallei induce the assembly of actin-rich tails in the host-cell cytoplasm, which drives rapid bacterial movement. For most of these pathogens, the moving bacteria collide with the host-cell plasma membrane to form membrane-covered protrusions, which are engulfed by neighboring cells—spreading the infection from cell to cell. In contrast, for B. pseudomallei, collision with the plasma membrane promotes cell–cell fusion, creating a conduit through which bacteria can invade neighboring cells (Movie 23.7). The molecular mechanisms of pathogen-induced actin assembly differ for the different pathogens, suggesting that they evolved independently (Figure 23–29).

1	The molecular mechanisms of pathogen-induced actin assembly differ for the different pathogens, suggesting that they evolved independently (Figure 23–29). L. monocytogenes and baculovirus produce proteins that directly bind to and activate the Arp 2/3 complex to initiate the formation of an actin tail and movement (see Figure 16–16). S. flexneri produces an unrelated surface protein that binds to and activates N-WASp, which then activates the Arp 2/3 complex. Rickettsia species produce a protein that directly polymerizes actin by mimicking the function of host formin proteins (see Figure 16–17).

1	Many viral pathogens rely primarily on microtubule-dependent motor proteins rather than actin polymerization to move within the host-cell cytosol. Viruses that infect neurons, such as the neurotropic alpha herpesviruses, which include the virus that causes chickenpox, provide important examples. The virus enters sensory neurons at the tips of their axons, and microtubule-based retrograde “backward” axonal transport carries the nucleocapsids down the axon to the nucleus. The transport is mediated by attachment of viral capsid proteins to the motor protein dynein (see Figure 16–58). After replication and assembly in the nucleus, the enveloped virions are then carried by antegrade “forward” axonal transport along microtubules to the axon tips, with the transport being mediated by the attachment of a different viral capsid protein to a kinesin motor protein (see Figure 16–56). A large number of viruses associate with either dynein or kinesin motor proteins to move along microtubules at

1	of a different viral capsid protein to a kinesin motor protein (see Figure 16–56). A large number of viruses associate with either dynein or kinesin motor proteins to move along microtubules at some stage in their replication. As microtubules serve as oriented tracks for vesicular transport in eukaryotic cells, it is not surprising that many viruses have independently evolved the ability to exploit them for their own transport.

1	Viruses Can Take Over the Metabolism of the Host Cell Viruses use basic host cell machinery for most aspects of their reproduction: they depend on host-cell ribosomes to produce their proteins, and most use host-cell DNA and RNA polymerases for their own replication and transcription. Many viruses encode proteins that modify the host transcription or translation apparatus to favor the synthesis of viral RNAs and proteins over those of the host cell, shifting the synthetic capacity of the cell toward the production of new virus particles. Poliovirus, for example, encodes a protease that specifically cleaves the TATA-binding component of TFIID (see Figure 6–17), shutting off transcription of most of the host cell’s protein-coding genes. Influenza virus produces a protein that blocks both the splicing and the polyadenylation of host-cell RNA transcripts, preventing their export into the cytosol (see Figure 6–38).

1	Viruses also alter translation by the host. Translation initiation for most host-cell mRNAs depends on recognition of their 5ʹ cap by translation initiation factors (see Figure 6–70). This initiation process is often inhibited during viral infection, so that the host-cell ribosomes can be used more efficiently for the synthesis of viral proteins. Some viral genomes encode endonucleases that cleave off the 5ʹ cap from host-cell mRNAs; some go even further by using the liberated 5ʹ caps as primers to synthesize viral mRNAs, a process called cap snatching. Several other viral RNA genomes encode proteases that cleave certain translation initiation factors; these viruses rely on 5ʹ cap-independent translation of their own RNA, using internal ribosome entry sites (IRESs) (see Figure 7–68).

1	A few DNA viruses use host-cell DNA polymerase to replicate their genome. Unfortunately for these viruses, DNA polymerase is expressed at high levels only during S phase of the cell cycle, and most cells that these viruses infect spend most of their time in G1 phase. Adenovirus has evolved a mechanism to drive the host cell into S phase, so that the cell produces large amounts of active DNA polymerase, which then replicates the viral genome; to accomplish this, the adenovirus genome also encodes proteins that inactivate both Rb (see Figure 17–61) and p53 (see Figure 17–62), two key suppressors of cell-cycle progression. As might be expected for any mechanism that encourages unregulated DNA replication, these viruses can promote, under some circumstances, the development of cancer. Other DNA viruses, including poxviruses and mimivirus, encode their own DNA and RNA polymerases, as well as some transcription regulators, allowing them to bypass usual host pathways and replicate outside

1	DNA viruses, including poxviruses and mimivirus, encode their own DNA and RNA polymerases, as well as some transcription regulators, allowing them to bypass usual host pathways and replicate outside the nucleus.

1	RNA viruses must always encode their own replication proteins because host cells lack polymerase enzymes that use RNA as a template. For RNA viruses with a single-stranded genome, the replication strategy depends on whether the RNA is a positive [+] strand, which contains translatable information like mRNA, or a complementary negative [–] strand. When the RNA is a positive [+] strand, the incoming viral genome is used to produce the viral RNA polymerase and viral proteins; the viral polymerase is then used to replicate the viral RNA and to generate mRNAs for the production of more viral proteins. For viruses with a negative [–] strand RNA genome (such as influenza and measles virus), an RNA polymerase enzyme is packaged as a structural protein of the incoming viral capsids.

1	Retroviruses such as HIV-1, which have a positive [+] strand RNA genome, are a special class of RNA virus because they carry with them a viral reverse transcriptase enzyme. After entry to the host cell, the reverse transcriptase uses the viral RNA genome as a template to synthesize a double-stranded DNA copy of the viral genome, which enters into the nucleus and integrates into the host cell’s chromosomes (see Figure 5–62). It is later transcribed by the cell’s DNA-dependent RNA polymerase to produce viral genomes and proteins.

1	The complexity and specificity of the interplay between pathogens and their host cells might suggest that virulence would be difficult to acquire by random mutation. Yet, new pathogens are constantly emerging, and old pathogens are constantly changing in ways that make familiar infections more difficult to prevent or treat. Pathogens have two advantages that enable them to evolve rapidly. First, they replicate very quickly, providing a great deal of material for natural selection to work with. Whereas humans and chimpanzees have acquired a 2% difference in genome sequences over about 8 million years of divergent evolution, poliovirus Figure 23–29 Molecular mechanisms for actin nucleation by various bacterial pathogens. Listeria monocytogenes and Shigella flexneri induce actin nucleation by recruiting and activating the host Arp 2/3 complex (see Figure 16–16), although each uses a different recruitment strategy:

1	L. monocytogenes expresses a surface protein, ActA, that directly binds to and activates the Arp 2/3 complex; S. flexneri expresses a surface protein, IcsA (unrelated to ActA), that recruits the host protein N-WASp, which in turn recruits the Arp 2/3 complex, along with other host proteins, including WIP (WASp-interacting protein). Rickettsia rickettsii uses an entirely different strategy; it expresses a surface protein, Sca2, that directly nucleates actin polymerization by mimicking the activity of host formin proteins. gene b copied to number of parasites levels of antibodies x1000 VSGb gene c copied to expression site infection manages a 2% change in its genome in 5 days—about the time it takes the virus to pass from the human mouth to the gut. Second, selective pressures act rapidly on this genetic variation. The host’s adaptive immune system and modern microbicidal drugs, both of which destroy pathogens that fail to change, are the main sources of these selective pressures.

1	An example of an adaptation to the selective pressure imposed by the adaptive immune system is the phenomenon of antigenic variation. An important adaptive immune response against many pathogens is the host’s production of antibodies that recognize specific molecules (antigens) on the pathogen’s surface (discussed in Chapter 24). Many pathogens have evolved mecanisms that deliberately change these antigens during the course of an infection, enabling them to evade antibodies. Some eukaryotic parasites, for example, undergo programmed rearrangements of the genes encoding their surface antigens. A striking example occurs in Trypanosoma brucei, a protozoan parasite that causes African sleeping sickness and is spread by tsetse flies. (T. brucei is a relative of T. cruzi—see Figure 23–21—but it replicates extracellularly rather than intracellularly.) T. brucei is covered with a single type of glycoprotein, called variant-specific glycoprotein (VSG), which elicits in the host a protective

1	it replicates extracellularly rather than intracellularly.) T. brucei is covered with a single type of glycoprotein, called variant-specific glycoprotein (VSG), which elicits in the host a protective antibody response that rapidly clears most of the parasites. The trypanosome genome, however, contains about 1000 different Vsg genes or pseudogenes, each encoding a VSG with a distinct amino acid sequence. Only one of these genes is expressed at any one time, from one of approximately 20 possible expression sites in the genome. Gene rearrangements that copy different Vsg genes into expression sites repeatedly change the VSG protein displayed on the surface of the pathogen. In this way, a few trypanosomes with an altered VSG escape the initial antibody-mediated clearance, replicate, and cause the disease to recur, leading to a chronic cyclic infection (Figure 23–30).

1	Bacterial pathogens can also rapidly change their surface antigens. As discussed in Chapter 5, Salmonella enterica bacteria switch between expressing either of two versions of the protein flagellin, the structural component of the bacterial flagellum (see Figure 23–3D), in a process called phase variation (see Figure 5–65). Species of the genus Neisseria are also champions at this. These Gram-negative cocci can cause meningitis and sexually transmitted diseases. They undergo genetic recombination very similar to that just described for eukaryotic pathogens, which enables them to vary the pilin protein they use to attach to host cells. By inserting one of the multiple silent copies of variant pilin genes into a single expression locus, they can express many slightly different versions of the protein and repeatedly change the amino acid sequence over time. Neisseria bacteria are

1	Figure 23–30 antigenic variation in trypanosomes. (A) There are about 1000 distinct Vsg genes in Trypanosoma brucei, and they are expressed one at a time from approximately 20 expression sites in the genome. To be expressed, an inactive gene is copied and the copy is moved into an expression site through DNA recombination. Each Vsg gene encodes a different surface protein (antigen). These switching events allow the trypanosome to repeatedly change the surface antigen it expresses. (B) A person infected with trypanosomes expressing VSGa mounts a protective antibody response, which clears most of the parasites expressing this antigen. However, a few of the trypanosomes will have switched to expression of VSGb, which can now proliferate until anti-VSGb antibodies clear them. By that time, however, some parasites will have switched to VSGc, and so the cycle continues.

1	also extremely adept at taking up DNA from their environment by natural transformation and incorporating it into their genomes, further contributing to their extraordinary variability. The end result of this considerable variation is a plethora of different surface compositions with which to bewilder the host adaptive immune system. It is therefore not surprising that it has been difficult to develop an effective vaccine against Neisseria infections, although there are now several that protect against Neisseria meningitidis, a common cause of fatal meningitis.

1	In contrast to the DNA rearrangements in bacteria and parasites, viruses rely on an error-prone replication mechanism for antigenic variation. Retroviral genomes, for example, acquire on average one point mutation every replication cycle, because the viral reverse transcriptase (see Figure 5–62) needed to produce DNA from the viral RNA genome lacks the proofreading activity of DNA polymerases. A typical, untreated HIV infection may eventually produce HIV genomes with every possible point mutation. By a process of mutation and selection within each host, most viruses change over time—from a form that is most efficient at infecting macrophages to one more efficient at infecting T cells, as described earlier (see Figure 23–17). Similarly, once a patient is treated with an antiviral drug, the viral genome can quickly mutate and be selected for its resistance to the drug. Remarkably, only about one-third of the nucleotide positions in the coding sequence of the viral genome are invariant,

1	viral genome can quickly mutate and be selected for its resistance to the drug. Remarkably, only about one-third of the nucleotide positions in the coding sequence of the viral genome are invariant, and nucleotide sequences in some parts of the genome, such as the Env gene (see Figure 7–62), can differ by as much as 30% from one HIV isolate to another. This extraordinary genomic plasticity greatly complicates attempts to develop vaccines against HIV. It has also led to the rapid emergence of new HIV strains. Nucleotide sequence comparisons between various strains of HIV and the very similar simian immunodeficiency virus (SIV) isolated from a variety of monkey species suggest that the most virulent type of HIV, HIV-1, may have jumped from primates to humans multiple independent times, starting as long ago as 1908 (Figure 23–31).

1	Influenza viruses are an important exception to the rule that error-prone replication dominates viral evolution. They are unusual in that their genome consists of several (usually eight) strands of RNA. When two strains of influenza infect the same host, the RNA strands of the two strains can reassort to form a new type of influenza virus. In normal years, influenza is a mild disease in healthy adults, although it can be life-threatening in the very young and very old. Different influenza strains infect fowl such as ducks and chickens, but only a subset of these strains can infect humans, and transmission from fowl to humans is rare. In 1918, however, a particularly virulent variant of avian influenza crossed the species barrier to infect humans, triggering the catastrophic pandemic of 1918 called the Spanish flu, which killed 20–50 million people worldwide. Subsequent influenza pandemics have been triggered by genome reassortment, in which a new RNA segment from an avian form of the

1	called the Spanish flu, which killed 20–50 million people worldwide. Subsequent influenza pandemics have been triggered by genome reassortment, in which a new RNA segment from an avian form of the virus replaced one or more of the viral RNA segments from the human form (Figure 23–32). In 2009, a new H1N1 swine virus emerged that derived genes from pig, avian, and human influenza viruses. Such recombination events allowed the new virus to replicate rapidly and spread through an immunologically naive human population. Generally, within two or three years, the human population develops immunity to a new recombinant strain of virus, and the infection rate drops to a steady-state level. Because the recombination events are unpredictable, it is not possible to know when the next influenza pandemic will occur or how severe it might be.

1	The development of drugs that cure rather than prevent infections has had a major impact on human health. Antibiotics, which are either bactericidal (they kill bacteria) or bacteriostatic (they inhibit bacterial growth without killing), are the most successful class of such drugs. Penicillin was one of the first antibiotics = jumps from monkey and ape to human

1	Figure 23–31 Diversification of HIV-1, HIV-2, and related strains of SIV. HIV comprises different viral families, all descended from SIV (simian immunodeficiency virus). On three separate occasions, SIV was passed from a chimpanzee to a human, resulting in three HIV-1 groups: major (M), outlier (O), and non-M non-O (N). The HIV-1 M group is the most common and is primarily responsible for the global AIDS epidemic. On two separate occasions, SIV was passed from a sooty mangabey monkey to a human, resulting in the two HIV-2 groups. In 2009, a new strain of HIV was discovered that appears to have resulted from SIV passage from a gorilla to a human. 1292 Chapter 23: Pathogens and Infection H2N2 H3N?

1	1292 Chapter 23: Pathogens and Infection H2N2 H3N? used to treat infections in humans, just in time to prevent tens of thousands of deaths from infected battlefield wounds in World War II. Because bacteria (see Figure 1–17) are not closely related evolutionarily to the eukaryotes they infect, much of their basic machinery for DNA replication and transcription, RNA translation, and metabolism differs from that of their host. These differences enable us to develop antibacterial drugs that exhibit selective toxicity, in that they specifically inhibit these processes in bacteria without disrupting them in the host. Most of the antibiotics that we use to treat bacterial infections are small molecules that inhibit macromolecular synthesis in bacteria by targeting bacterial enzymes that either are distinct from their eukaryotic counterparts or are involved in pathways such as cell wall biosynthesis that are absent in animals (Figure 23–33 and see Table 6–4).

1	However, bacteria continuously evolve and strains resistant to antibiotics rapidly develop, often within a few years of the introduction of a new drug. Similar drug resistance also arises rapidly when treating viral infections with antiviral drugs. The virus population in an HIV-infected person treated with the reverse transcriptase inhibitor AZT, for example, will acquire complete resistance to the drug within a few months. The current protocol for treatment of HIV infections involves the simultaneous use of three drugs, which helps to minimize the acquisition of resistance for any one of them. There are three general strategies by which a pathogen can develop drug resistance: (1) it can alter the molecular target of the drug so that it is no longer sensitive to the drug; (2) it can produce an enzyme that modifies or destroys the drug; or (3) it can prevent the drug’s access to the drug target by, for example, actively pumping the drug out of the pathogen (Figure 23–34).

1	Once a pathogen has chanced upon an effective drug-resistance strategy, the newly acquired or mutated genes that confer the resistance are frequently spread throughout the pathogen population by horizontal gene transfer. They may even spread between pathogens of different species. The highly effective but expensive antibiotic vancomycin, for example, is used as a treatment of last resort for many severe, hospital-acquired, Gram-positive bacterial infections that are resistant to most other known antibiotics. Vancomycin prevents one step in bacterial cell wall synthesis—the cross-linking of peptidoglycan chains in the bacterial cell wall (see Figure 23–3B). Resistance can arise if the bacterium synthesizes a cell wall using different subunits that do not bind vancomycin. The most effective form of vancomycin resistance depends on the acquisition of a transposon (see Figure 5–60) containing seven genes, the products of which work together to sense the

1	Figure 23–32 Model for the evolution of pandemic strains of influenza virus by recombination. Influenza A virus is a natural pathogen of birds, particularly waterfowl, and it is always present in wild bird populations. In 1918, a particularly virulent form of the virus crossed the species barrier from birds to humans and caused a devastating worldwide epidemic. This strain was designated H1N1, referring to the specific forms of its main antigens, hemagglutinin (H) and neuraminidase (N). Changes in the virus, rendering it less virulent, and the rise of adaptive immunity in the human population, prevented the pandemic from continuing in subsequent seasons, although H1N1 influenza strains continued to cause serious disease every year in very young and very old people. In 1957, a new pandemic arose when three genes were replaced by equivalent genes from an avian virus (green bars); the new strain (designated H2N2) was not effectively cleared by antibodies in people who had previously

1	arose when three genes were replaced by equivalent genes from an avian virus (green bars); the new strain (designated H2N2) was not effectively cleared by antibodies in people who had previously contracted only H1N1 forms of influenza. In 1968, another pandemic was triggered when two genes were replaced from another avian virus; the new virus was designated H3N2. In 1977, there was a resurgence of H1N1 influenza, which had previously been almost completely replaced by the N2 strains. Molecular sequence information suggests that this minor pandemic may have been caused by an accidental release of an influenza strain that had been held in a laboratory since about 1950. In 2009, a new H1N1 swine virus emerged that had derived five genes from pig influenza viruses, two from avian influenza viruses, and one from a human influenza virus. As indicated, most human influenza today is caused by H1N1 and H3N2 strains.

1	protein synthesis,penicillins 30S ribosome inhibitors protein synthesis,sulfonamides presence of vancomycin, shut down the normal pathway for bacterial cell wall synthesis, and produce a different type of cell wall.

1	Drug-resistance genes acquired by horizontal transfer frequently come from environmental microbial reservoirs. Nearly all antibiotics used to treat bacterial infections today are based on natural products produced by fungi or bacteria. Penicillin, for example, is made by the mold Penicillium, and more than 50% of the antibiotics currently used in the clinic are made by Gram-positive bacteria of the genus Streptomyces, which reside in the soil. It is believed that microorganisms produce antimicrobial compounds, many of which have probably existed on Earth for hundreds of millions of years, as weapons in their competition with other microorganisms in the environment. Surveys of bacteria taken from soil samples that have never been exposed to antibiotic drugs used in modern medicine reveal that the bacteria are typically already resistant to about seven or eight of the antibiotics widely used in clinical practice. When pathogenic microorganisms are faced with the selective pressure

1	that the bacteria are typically already resistant to about seven or eight of the antibiotics widely used in clinical practice. When pathogenic microorganisms are faced with the selective pressure provided by antibiotic treatments, they can apparently draw upon this immense source of genetic material to acquire resistance.

1	Like most other aspects of infectious disease, human behavior has exacerbated the problem of drug resistance. Many patients take antibiotics for symptoms that are typically caused by viruses (flu-like illnesses, colds, sore throats, and earaches) and these drugs have no effects. Persistent and chronic misuse of antibiotics can eventually result in antibiotic-resistant normal flora, which can then transfer the resistance to pathogens. Antibiotics are also misused in agriculture, where they are commonly employed as food additives to promote the growth and health of farm animals. An antibiotic closely related to vancomycin was commonly added to cattle feed in Europe; the resulting resistance in the normal flora of these animals is widely believed to be one of the original sources for vancomycin-resistant bacteria that now threaten the lives of hospitalized patients.

1	Figure 23–33 antibiotic targets. Although there are many antibiotics in clinical use, they have a narrow range of targets, which are highlighted in yellow. A few representative antibiotics in each class are listed. Nearly all antibiotics used to treat human infections fall into one of these categories. The vast majority inhibit either bacterial protein synthesis or bacterial cell wall synthesis.

1	Figure 23–34 Three general mechanisms of antibiotic resistance. (A) A nonresistant wild-type bacterial cell bathed in a drug (red triangles) that binds to and inhibits an essential enzyme (light green) will be killed due to enzyme inhibition. (B) A bacterium that has altered the drug’s target enzyme so that the drug no longer binds to the enzyme will survive and proliferate. In many cases, a single point mutation in the gene encoding the target protein can generate resistance. (C) A bacterium that expresses an enzyme (dark green) that either degrades or covalently modifies the drug will survive and proliferate. Some resistant bacteria, for example, make β-lactamase enzymes, which cleave penicillin and similar molecules. (D) A bacterium that expresses or up-regulates an efflux pump that ejects the drug from the bacterial cytoplasm (using energy derived from either ATP hydrolysis or the electrochemical gradient across the bacterial plasma membrane) will survive and proliferate. Some

1	ejects the drug from the bacterial cytoplasm (using energy derived from either ATP hydrolysis or the electrochemical gradient across the bacterial plasma membrane) will survive and proliferate. Some efflux pumps, such as the TetR efflux pump, are specific for a single drug (in this case, tetracycline), whereas others, called multidrug resistance (MDR) efflux pumps, are capable of exporting a wide variety of structurally dissimilar drugs. Upregulation of an MDR pump can render a bacterium resistant to a very large number of different antibiotics in a single step.

1	Because the acquisition of drug resistance is almost inevitable, it is crucial that we continue to develop innovative treatments for infectious diseases. We must also take additional measures to delay the onset of drug resistance.

1	All pathogens share the ability to interact with host cells in diverse ways that promote the replication and spread of the pathogen. Pathogens often colonize the host by adhering to or invading the epithelial surfaces that line the respiratory, gastrointestinal, and urinary tracts, as well as the other body surfaces in direct contact with the environment. Intracellular pathogens, including all viruses and many bacteria and protozoa, invade host cells by one of several mechanisms. Viruses rely largely on receptor-mediated endocytosis, whereas bacteria exploit cell adhesion and phagocytic pathways; in both cases, the host cell provides the machinery and energy for the invasion. Protozoa, by contrast, employ unique invasion strategies that usually require significant metabolic expense on the part of the invader. Once inside, intracellular pathogens seek out a cell compartment that is favorable for their survival and replication, frequently altering host membrane traffic and exploiting

1	part of the invader. Once inside, intracellular pathogens seek out a cell compartment that is favorable for their survival and replication, frequently altering host membrane traffic and exploiting the host-cell cytoskeleton for intracellular movement. Pathogens evolve rapidly, so that new infectious diseases frequently emerge, and old pathogens acquire new ways to evade

1	What are the genetic and molecular features that differ between pathogens and members of the normal human microbiota? How can our immune system distinguish between the two? To what extent are common host-cell biological pathways and molecules hijacked by diverse microbes? Can host-cell defense molecules be mobilized by drugs to fight infection? our attempts at treatment, prevention, and eradication. Which statements are true? explain why or why not. 23–1 Our adult bodies harbor about 10 times more microbial cells than human cells. 23–2 The microbiomes from healthy humans are all very similar. 23–3 Pathogens must enter host cells to cause disease. 23–4 Viruses replicate their genomes in the nucleus of the host cell. 23–5 You should not take antibiotics for diseases caused by viruses. Discuss the following problems. 23–6 In order to survive and multiply, a successful pathogen must accomplish five tasks. Name them.

1	Discuss the following problems. 23–6 In order to survive and multiply, a successful pathogen must accomplish five tasks. Name them. 23–7 Clostridium difficile infection is the leading cause of hospital-associated gastrointestinal illness. It is typically treated with a course of antibiotics, but the infection recurs in about 20% of cases. C. difficile infections are difficult to eradicate because the bacteria exist in two forms: a replicating, toxin-producing form and a spore form that is resistant to antibiotics. Fecal microbiota transplantation—the transfer of normal gut microbiota from a healthy individual—can resolve >90% of recurrent infections, a much better cure rate than further antibiotic treatment alone. Why do you suppose microbiota transplantation is so effective? 23–8 What are the three general mechanisms for horizontal gene transfer?

1	23–9 The Gram-negative bacterium Yersinia pestis, the causative agent of the plague, is extremely virulent. Upon infection, Y. pestis injects a set of effector proteins into macrophages that suppresses their phagocytic behavior and also interferes with their innate immune responses. One of the effector proteins, YopJ, acetylates serines and threonines on various MAP kinases, including the MAP kinase kinase kinase TAK1, which controls a key signaling step in the innate immune response pathway. To determine how YopJ interferes with TAK1, you transfect human cells with active YopJ (YopJWT) or inactive YopJ (YopJCA) and with FLAG-tagged active TAK1 (TAK1WT) or inactive TAK1 (TAK1K63W), and assay for total TAK1 and for phosphorylated TAK1, using antibodies against the FLAG tag or against phosphorylated TAK1 (Figure Q23–1). How does YopJ block the TAK1 signaling pathway? How do you suppose the serine/threonine acetylase activity of YopJ might interfere with TAK1 activation?

1	IP: ˜-FLAG-TAK1 IB: ˜-pTAK1 IP: ˜-FLAG-TAK1 –76 IB: ˜-FLAG-TAK1

1	Figure Q23–1 effects of YopJ on TaK1 phosphorylation (Problem 23–9). TaK1 was immunoprecipitated (Ip) using antibodies against the FLaG tag (α-FLaG-TaK1). Total TaK1 in the immunoprecipitation was assayed by immunoblot (IB) using the same antibody. phosphorylated TaK1 was assayed by IB using antibodies specific for phospho-TaK1 (α-pTaK1). a scale of protein molecular mass is shown at right in kilodaltons. (From n. paquette et al., Proc. Natl Acad. Sci. USA 109:12710–12715, 2012. With permission from national academy of sciences.) 23–10 The intracellular bacterial pathogen Salmonella typhimurium, which causes gastroenteritis, injects effector proteins to promote its invasion into nonphagocytic host cells by the trigger mechanism. S. typhimurium first stimulates membrane ruffling to promote invasion, and then suppresses membrane ruffling once invasion is complete. This behavior is mediated in part by injection of two effector proteins: SopE, which promotes membrane ruffling and

1	promote invasion, and then suppresses membrane ruffling once invasion is complete. This behavior is mediated in part by injection of two effector proteins: SopE, which promotes membrane ruffling and invasion, and SptP, which blocks the effects of SopE. Both effector proteins target the monomeric GTPase, Rac, which in its active form promotes membrane ruffling. How do you suppose SopE and SptP affect Rac activity? How do you suppose the effects of SopE and SptP are staggered in time if they are injected simultaneously?

1	23–11 John Snow is widely regarded as the father of modern epidemiology. Most famously, he investigated an outbreak of cholera in London in 1854 that killed more than 600 victims before it was finished. Snow recorded where the victims lived, and plotted the data on a map, along with the locations of the water pumps that served as the source of water for the public (Figure Q23–2). He concluded that the disease was most likely spread in the water, although he could find nothing suspicious-looking in it. His conclusion ran counter to the then-current belief that cholera was from “miasmas” in bad air. Very few believed his theory during the next 50 years, with the “bad air” theory persisting until at least 1901. What do you suppose Snow saw in the data that led him to his conclusion? Why do you think most scientists remained skeptical for so long?

1	Figure Q23–2 a map of where the victims of the 1854 cholera outbreak lived, superimposed on a modern Google map of the area (Problem 23–11). The locations of the victims’ houses are indicated in a gradient of colors from blue (indicating few cases) to orange (indicating many cases). public water pumps are shown as red squares. 23–12 Influenza epidemics account for 250,000 to 500,000 deaths globally each year. These epidemics are markedly seasonal, occurring in temperate climates in the northern and southern hemispheres during their respec Figure Q23–3 seasonal patterns of influenza epidemics (Problem 23–12). Cases of influenza at different times of the year are shown for the northern hemisphere (blue), the southern hemisphere (orange), and the tropics (red).

1	tive winters. By contrast, in the tropics, there is significant influenza activity year round, with a peak in the rainy season (Figure Q23–3). Can you suggest some possible explanations for the patterns of influenza epidemics in temperate zones and the tropics? 23–13 Several negative-strand viruses carry their genome as a set of discrete RNA segments. Examples include influenza virus (eight segments), Rift Valley fever virus (three segments), Hantavirus (three segments), and Lassa virus (two segments), to name a few. Why does segmentation of the genome provide a strong evolutionary advantage for these viruses?

1	23–14 Avian influenza viruses readily infect birds, but are transmitted to humans very rarely. Similarly, human influenza viruses spread readily to other humans, but have never been detected in birds. The key to this specificity lies in the viral capsid protein, hemagglutinin, which binds to sialic acid residues on cell-surface glycoproteins, triggering virus entry into the cell (Movie 23.8). Hemagglutinin on human viruses recognizes sialic acid in a 2-6 linkage with galactose, whereas avian hemagglutinin recognizes sialic acid in a 2-3 linkage with galactose. Humans make carbohydrate chains that have only the 2-6 linkage between sialic acid and galactose; birds make only the 2-3 linkage; but pigs make carbohydrate chains with both linkages. How does this situation make pigs ideal hosts for generating new strains of human influenza viruses?

1	23–15 The majority of antibiotics used in the clinic are made as natural products by bacteria. Why do you suppose bacteria make the very agents we use to kill them? 23–16 In the early days of penicillin research, it was discovered that bacteria in the air could destroy the penicillin, a big problem for large-scale production of the drug. How do you suppose this occurs? 23–17 When the Oxford team of Ernst Chain and Norman Heatley had laboriously collected their first two grams of penicillin (probably no more than 2% pure!), Chain injected two normal mice with 1 g each of this preparation, and waited to see what would happen. The mice survived with no apparent ill effects. Their boss, Howard Florey, was furious at what he saw as a waste of good antibiotic. Why was this experiment important? Cossart P, Boquet P, Normark S & Rappuoli R (eds) (2005) Cellular Microbiology, 2nd ed. Washington, DC: ASM Press.

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1	Welch MD & Way M (2013) Arp2/3-mediated actin-based motility: a tail of pathogen abuse. Cell Host Microbe 14, 242–255. The Innate and Adaptive Immune Systems 24

1	As we discussed in Chapter 23, all living organisms serve as hosts for other species, usually in relationships that are benign or even mutually helpful. But all organisms, and all cells in a multicellular organism, need to defend themselves against infection by harmful invaders, collectively called pathogens, which can be microbes (bacteria, viruses, or fungi), or larger parasites. Even bacteria defend themselves against viruses, using intracellular proteins called restriction factors, which block viral propagation. Invertebrates use a variety of defense strategies, including protective barriers, toxic molecules, restriction factors, and phagocytic cells that ingest and destroy invading pathogens. Vertebrates, too, depend on such innate immune responses, but they can also harness more sophisticated and specific mechanisms, called adaptive immune responses. The innate responses occur first, calling the adaptive immune responses into play if required, in which case, both types of

1	sophisticated and specific mechanisms, called adaptive immune responses. The innate responses occur first, calling the adaptive immune responses into play if required, in which case, both types of responses work together to eliminate the pathogen (Figure 24–1).

1	Whereas innate immune responses are general defense reactions that can involve almost any cell type in an organism, the adaptive immune responses are highly specific to the particular pathogen that induced them and depend on a class of white blood cells (leukocytes) called lymphocytes. There are two major classes of lymphocytes that mount adaptive immune responses—B lymphocytes (B cells), which secrete antibodies that bind specifically to the pathogen, and T lymphocytes (T cells), which can either directly kill cells infected with the pathogen or produce secreted or cell-surface signal proteins that stimulate other host cells to help eliminate the pathogen (Figure 24–2). Unlike innate immune responses, which are generally short-lasting, the adaptive responses provide long-lasting protection: a person who recovers from measles or is vaccinated against it, for example, is protected for life against measles by the adaptive immune system, although not against other common viruses, such as

1	a person who recovers from measles or is vaccinated against it, for example, is protected for life against measles by the adaptive immune system, although not against other common viruses, such as those that cause mumps or chickenpox.

1	Both the innate and adaptive immune systems have evolved sensing mechanisms that enable them to recognize harmful invaders (pathogens) and distinguish them from both the host’s own cells and molecules and harmless or beneficial foreign organisms and their molecules. The innate system relies on sensor proteins that recognize particular types or patterns of molecules that are common to pathogens but are absent or sequestered in the host. The adaptive system, by contrast, uses unique genetic mechanisms to produce a virtually limitless diversity of related proteins—receptors on T and B cells and secreted antibodies—that, between them, can bind almost any foreign molecule. This remarkable strategy enables the adaptive immune system to react specifically against any pathogen, even if the animal never encountered it before. But, it also requires that the system learn not to react against self molecules or harmless foreign ones; if these learning mechanisms fail, harmful autoimmune or

1	animal never encountered it before. But, it also requires that the system learn not to react against self molecules or harmless foreign ones; if these learning mechanisms fail, harmful autoimmune or allergic responses result.

1	In this chapter, we focus on vertebrate immune responses and the features that distinguish them from other kinds of cell responses. We begin with innate immune defenses and then discuss the highly specialized properties of the adaptive immune system. The INNATe IMMUNe SYSTeM OVerVIeW OF The ADApTIVe IMMUNe SYSTeM B ceLLS AND Figure 24–1 Innate and adaptive immune responses. Innate immune responses are activated directly by pathogens and defend all multicellular organisms against infection. In vertebrates, pathogens, together with the innate immune responses they activate, also stimulate adaptive immune responses, which then work together with innate immune responses to help fight the infection. The INNATe IMMUNe SYSTeM

1	The INNATe IMMUNe SYSTeM Adaptive immune responses are slow to develop when a vertebrate first encounters a new pathogen. This is because the specific B cells and T cells that can respond to a particular pathogen are initially few in number and must be stimulated to proliferate and differentiate before they can mount effective adaptive immune responses, which can take days. By contrast, a single bacterium that divides every hour can generate almost twenty million progeny in a single day, producing a full-blown infection. Vertebrates, therefore, rely on their innate immune system to defend them against infection during the first critical hours and days of exposure to a new pathogen. Plants and invertebrates lack adaptive immune systems and therefore rely entirely on innate immunity for protection against pathogens. In this section, we consider some of the strategies the innate immune system uses to recognize pathogens and to provide a first line of defense against them.

1	In this section, we consider some of the strategies the innate immune system uses to recognize pathogens and to provide a first line of defense against them. epithelial Surfaces Serve as Barriers to Infection

1	In vertebrates, the first encounters with infectious organisms are typically at the epithelial surfaces that form the skin and line the respiratory, digestive, urinary, and reproductive tracts. These epithelia provide both physical and chemical barriers to invasion by pathogens: tight junctions between epithelial cells bar entry between the cells, and a variety of substances secreted by the cells discourage the attachment and entry of pathogens. The keratinized epithelial cells of the skin, for example, form a thick physical barrier, and the sebaceous glands in the skin secrete fatty acids and lactic acid, which inhibit bacterial growth. In addition, epithelial cells in all tissues, including those in plants and invertebrates, secrete antimicrobial molecules called defensins. Defensins are positively charged, amphipathic peptides that bind to and disrupt the membranes of many pathogens, including enveloped viruses, bacteria, fungi, and parasites.

1	The epithelial cells that line internal organs such as the respiratory and digestive tracts also secrete slimy mucus, which sticks to the epithelial surface and makes it difficult for pathogens to adhere. The beating of cilia on the surface of the epithelial cells lining the respiratory tract and the peristaltic action of the intestine also discourage the adherence of pathogens. Moreover, as we discuss in Chapter 23, healthy skin and gut are populated by enormous numbers of harmless (and often helpful) commensal microbes, collectively called the normal flora, which compete for nutrients with pathogens; some also produce antimicrobial peptides that actively inhibit pathogen proliferation. pattern recognition receptors (prrs) recognize conserved Features of pathogens

1	pattern recognition receptors (prrs) recognize conserved Features of pathogens Pathogens do occasionally breach the epithelial barricades, in which case underlying, nonepithelial cells of the innate immune system provide the next line of defense. These cells sense the presence of pathogens largely through the use of receptor proteins that recognize microbe-associated molecules that either are not present or are sequestered in the host organism. Because these microbial molecules often occur in repeating patterns, they are called pathogen-associated molecular patterns (PAMPs), even though they are not unique to microbes that can cause disease. PAMPs are present in various microbial molecules, including nucleic acids, lipids, polysaccharides, and proteins.

1	The special receptor proteins that recognize PAMPs are called pattern recognition receptors (PRRs). Some PRRs are transmembrane proteins on the surface of many types of host cells, where they recognize extracellular pathogens; on professional phagocytic cells (phagocytes) such as macrophages and neutrophils (discussed in Chapter 22), they can mediate the uptake of the pathogens into phagosomes, which then fuse with lysosomes, where the pathogens are destroyed. Other PRRs are located intracellularly, where they can detect intracellular pathogens such as viruses; these PRRs are either free in the cytosol or associated with Figure 24–2 The two main classes of adaptive immune responses.

1	Figure 24–2 The two main classes of adaptive immune responses. Lymphocytes carry out both classes of adaptive responses—shown here as responses to a viral infection. In one class, B cells secrete antibodies that specifically bind to and neutralize extracellular viruses, by preventing the viruses from infecting host cells. In the other, T cells mediate the response; in this example, they kill the virus-infected host cells. In both cases, innate immune responses help activate the adaptive immune responses through pathways that are not shown.

1	Figure 24–3 A scanning electron micrograph of a mutant fruit fly that died from a fungal infection. The fly is covered with fungal hyphae, as it lacked a Toll receptor, which helps protect Drosophila from fungal infections. (From B. Lemaitre et al., Cell 86:973–983, 1996.) the membranes of the endolysosomal system (discussed in Chapter 13). Still other PRRs are secreted and bind to the surface of extracellular pathogens, marking them for destruction by either phagocytes or blood proteins that are part of the complement system (discussed later). There Are Multiple classes of prrs

1	There Are Multiple classes of prrs The first PRR identified was the Toll receptor in Drosophila, which was well-known for its role in fly development (see Figure 21–17). It was later discovered to be also required for the production of antimicrobial peptides that protect the fly against fungal infections (Figure 24–3). Toll is a transmembrane glycoprotein with a large extracellular domain that contains a series of leucine-rich repeats. Soon it was discovered that both plants and animals have a variety of Toll-like receptors (TLRs) that function as PRRs in innate immune responses against various pathogens. Mammals make at least 10 different TLRs, each recognizing distinct ligands: TLR3, for example, recognizes double-stranded viral RNA in the endosomal lumen (Figure 24–4); TLR4 recognizes lipopolysaccharide (LPS) on the outer

1	Figure 24–4 A Toll-like receptor. The structure of human TLr3 is shown (green), with a double-stranded rNA molecule (dsrNA, blue) bound to it. The receptor is a homodimer in the membrane of endosomes. The binding of dsrNA to the two horseshoe-shaped domains on the lumenal endosome membrane side of the endosome brings the two cytosolic domains together, allowing adaptor proteins in the cytosol to assemble into a large signaling complex (not shown). (B) The crystal structure of a lumenal domain of the receptor, which contains 23 conventional leucine-rich repeats, each of which contributes a β strand to the continuous β sheet (red) that lines the concave surface of the structure. (A, adapted from L. Liu et al., Science 320:379–381, 2008. With permission from AAAS; B, adapted from J. choe, M.S. Kelker and I.A. Wilson, Science 309:581–585, 2006; pDB: 1ZIW.) 0.2 mm membrane of Gram-negative bacteria; TLR5 recognizes the protein that forms the bacterial flagellum; and TLR9 recognizes short,

1	Kelker and I.A. Wilson, Science 309:581–585, 2006; pDB: 1ZIW.) 0.2 mm membrane of Gram-negative bacteria; TLR5 recognizes the protein that forms the bacterial flagellum; and TLR9 recognizes short, unmethylated sequences of bacterial, viral, or protozoan DNA, called CpG motifs, which are uncommon in vertebrate DNA.

1	In addition to TLRs, vertebrates use several other families of PRRs to detect pathogens. One is the large family of NOD-like receptors (NLRs). Like TLRs, NLRs have leucine-rich repeat motifs, but they are exclusively cytoplasmic and recognize a distinct set of bacterial molecules. Individuals who are homozygous for a particular mutant allele of the NLR gene NOD2 have a greatly increased risk of developing Crohn’s disease, a chronic inflammatory disease of the small intestine, possibly triggered by a bacterial infection. Another class of PRRs consists of RIG‑like receptors (RLRs), which are members of the RNA helicase family of proteins. They are also exclusively cytoplasmic and detect viral pathogens. A fourth class of PRRs consists of C‑type lectin receptors (CLRs), which are trans-membrane cell-surface proteins that recognize carbohydrates (which is why they are called lectins) on various microbes. Table 24–1 summarizes some PRRs and their ligands and locations in cells.

1	trans-membrane cell-surface proteins that recognize carbohydrates (which is why they are called lectins) on various microbes. Table 24–1 summarizes some PRRs and their ligands and locations in cells. Collectively, these and other PRRs act as an alarm system to alert the innate and adaptive immune systems that an infection is brewing (Movie 24.1).

1	When a cell-surface or intracellular PRR binds a PAMP, it stimulates the cell to secrete a variety of cytokines and other extracellular signal molecules. Some of these inhibit viral replication, but most induce a local inflammatory response that helps eliminate the pathogen, as we now discuss. Activated prrs Trigger an Inflammatory response at Sites of Infection

1	Activated prrs Trigger an Inflammatory response at Sites of Infection When a pathogen invades a tissue, it activates PRRs on or in various cells of the innate immune system, resulting in an inflammatory response at the site of infection. The inflammatory response depends on changes in local blood vessels and is characterized clinically by local pain, redness, heat, and swelling. The blood vessels dilate and become permeable to fluid and proteins, leading to local swelling and an accumulation of blood proteins that aid in defense. At the same time, the endothelial cells lining the local blood vessels are stimulated to express cell adhesion proteins, which promote the attachment and escape of white blood cells or leukocytes (see Figure 19–29B), adding to the local swelling; initially neutrophils escape, followed later by lymphocytes and monocytes (the blood-borne precursors of macrophages).

1	The activation of PRRs results in the production of a large variety of extracellular signal molecules that mediate the inflammatory response at the site of an infection. These include both lipid signal molecules, such as prostaglandins, and protein (or peptide) signal molecules called cytokines. Some of the most important pro-inflammatory cytokines are tumor necrosis factor‑α (TNFα), interferon‑γ (IFNγ), a variety of chemokines (which recruit leukocytes), and various interleukins (ILs) that we discuss later, including IL1, IL6, IL12, and IL17. In addition, a secreted PRR (mannose-binding lectin) activates the complement system when the PRR binds to a pathogen; fragments of complement proteins released during complement activation stimulate an inflammatory response (discussed shortly; see Figure 24–7).

1	When activated by PAMPs, most cell-surface and intracellular PRRs stimulate the production of multiple pro-inflammatory cytokines by activating intracellular signaling pathways that switch on transcription regulators, including NFκB, to induce the transcription of the relevant cytokine genes (see Figure 15–62). Some PRRs, however, can also stimulate pro-inflammatory cytokine production by a different mechanism: when activated, several cytoplasmic NLRs assemble with adaptor proteins and specific protease precursors of the caspase family (discussed in Chapter 18) to form inflammasomes, in which pro-inflammatory cytokines such as IL1 are cleaved from their inactive precursor proteins by activated caspases. These cytokines are then released from the cell by a poorly understood, unconventional secretion pathway. Inflammasomes closely resemble apoptosomes in their assembly and structure, but, in apoptosomes, procaspases are activated to initiate a proteolytic caspase cascade that leads to

1	secretion pathway. Inflammasomes closely resemble apoptosomes in their assembly and structure, but, in apoptosomes, procaspases are activated to initiate a proteolytic caspase cascade that leads to apoptosis (see Figure 18–7).

1	NLR-dependent inflammasome assembly can also be triggered in the absence of infection if cells are damaged or stressed. Such cells produce “danger signals,” such as altered or misplaced self molecules, which can activate the relevant NLRs: the arthritis caused by uric acid crystals formed in the joints of individuals with gout, who have abnormally high uric acid levels in their blood, is a painful example. phagocytic cells Seek, engulf, and Destroy pathogens

1	phagocytic cells Seek, engulf, and Destroy pathogens In all animals, the recognition of a microbial invader is usually quickly followed by its engulfment by a phagocytic cell. Macrophages are long-lived phagocytes that reside in most vertebrate tissues; they are among the first cells to encounter invading microbes, whose PAMPs activate the macrophages to secrete pro-inflammatory signal molecules. Neutrophils are short-lived phagocytes that are abundant in blood but are not present in healthy tissues; they are rapidly recruited to sites of infection by various attractive molecules, including formylmethionine-containing peptides (which are released by microbes but are not made by mammalian cells), chemokines secreted by activated macrophages, and peptide fragments produced from cleaved, activated complement proteins. The recruited neutrophils contribute their own pro-inflammatory cytokines.

1	In addition to their PRRs, macrophages and neutrophils display a variety of cell-surface receptors that recognize fragments of complement proteins or antibodies bound to the surface of a pathogen. The binding of such a pathogen to these receptors leads to its phagocytosis (Figure 24–5) and an attack on the ingested pathogen once inside a phagolysosome. The phagocytes possess an impressive armory of weapons to kill the invader, including enzymes such as lysozyme and acid hydrolases that can degrade the pathogen’s cell wall. The cells assemble NADPH oxidase complexes on the phagolysosomal membrane, where the complexes catalyze the production of highly toxic oxygen-derived compounds, including superoxide (O2–), hydrogen peroxide, and hydroxyl radicals. A transient increase in oxygen consumption by the phagocytic cells, called the respiratory burst, accompanies the production of these toxic compounds. Whereas macrophages generally survive this killing frenzy and live to kill again,

1	by the phagocytic cells, called the respiratory burst, accompanies the production of these toxic compounds. Whereas macrophages generally survive this killing frenzy and live to kill again, neutrophils do not. Dead and dying neutrophils are a major component of the pus that forms in acute bacterially infected wounds; their half-life in the human bloodstream is only a few hours.

1	Figure 24–5 Antibody-activated phagocytosis. electron micrograph of a neutrophil phagocytosing an antibody-coated bacterium, which is in the process of dividing. The process in which antibody (or complement) coating of a pathogen increases the efficiency with which the pathogen is phagocytosed is called opsonization. (courtesy of Dorothy F. Bainton, from r.c. Williams, Jr. and h.h. Fudenberg, phagocytic Mechanisms in health and Disease. New York: Intercontinental Medical Book corporation, 1971.) If a pathogen is too large to be successfully phagocytosed (if it is a large parasite such as a worm, for example), a group of macrophages, neutrophils, or eosinophils (another type of leukocyte) will gather around the invader. They secrete defensins and other damaging agents and release the toxic products of the respiratory burst. This barrage is often sufficient to destroy the pathogen (Figure 24–6).

1	The blood and other extracellular fluids contain numerous proteins with antimicrobial activity, some of which are produced in response to an infection, while others are produced constitutively. The most important of these are components of the complement system, which consists of about thirty interacting soluble proteins that are mainly made continuously by the liver and are inactive until an infection or another trigger activates them. They were originally identified by their ability to amplify and “complement” the action of antibodies made by B cells, but some are also secreted PRRs, which directly recognize PAMPs on microbes.

1	The early complement components consist of three sets of proteins, belonging to three distinct pathways of complement activation—the classical pathway, the lectin pathway, and the alternative pathway. The early components of all three pathways act locally to cleave and activate C3, which is the pivotal complement component (Figure 24–7); individuals with a C3 deficiency are subject to repeated bacterial infections. The early components are proenzymes, which are activated sequentially by proteolytic cleavage. The cleavage of each proenzyme in the series activates the next component to generate a serine protease, which cleaves the next proenzyme in the series, and so on. Since each activated enzyme cleaves many molecules of the next proenzyme in the chain, the activation of the early components consists of an amplifying proteolytic cascade.

1	Many of these protein cleavages liberate a biologically active small fragment that can attract neutrophils, plus a membrane-binding larger fragment. The binding of the large fragment to a cell membrane, usually the surface of a pathogen, helps stimulate the next reaction in the sequence. In this way, complement activation is largely kept confined to the cell surface where it began. In particular, the large fragment of C3, called C3b, binds covalently to the surface of the pathogen. Here, it recruits protein fragments produced by cleavage of other early complement components to form proteolytic complexes that catalyze the subsequent steps in the complement cascade. The early events in complement activation have diverse functions: C3b-binding receptors on phagocytic cells enhance the ability of these cells to phagocytose the pathogen, and similar receptors on B cells enhance the

1	Figure 24–6 eosinophils attacking a parasite. phagocytes cannot ingest large parasites such as the schistosome larva shown here. When the larva is coated with antibody or complement components, however, eosinophils (and other leukocytes) can recognize it and collectively kill it by secreting a large variety of toxic molecules. (courtesy of Anthony Butterworth.)

1	Figure 24–7 The principal stages in complement activation by the classical, lectin, and alternative pathways. In all three pathways, the reactions of complement activation usually take place on the surface of an invading microbe, such as a bacterium, and lead to the cleavage of c3 and the various consequences shown. As indicated, the complement proteins c1 to c9, mannose-binding lectin (MBL), MBL-associated serine protease (MASp), and factors B and D are the central components of the complement system. The early components are shown within gray arrows, while the late components are shown within a brown arrow. The functions of the protein fragments produced during complement activation are indicated by the black arrows. The various complement proteins that regulate the system are omitted.

1	ability of these cells to make antibodies against various microbial molecules on C3b-coated pathogens; the smaller fragment of C3 (called C3a), as well as small fragments of C4 and C5, act independently as diffusible signals to promote an inflammatory response by recruiting leukocytes to the site of infection. As indicated in Figure 24–7, antibodies bound to the surface of a pathogen activate the classical pathway. Mannose‑binding lectin, mentioned earlier, is a secreted PRR that initiates the lectin pathway of complement activation when it recognizes bacterial or fungal glycolipids and glycoproteins bearing terminal mannose and fucose sugars in a particular spatial conformation. These initial binding events in the classical and lectin pathways cause the recruitment and activation of the early complement components. Finally, molecules on the surface of pathogens will often directly activate the alternative pathway.

1	Host cells produce various plasma membrane molecules that prevent complement reactions from proceeding on their cell surface. The most important of these is the carbohydrate moiety sialic acid, a common constituent of cell-surface glycoproteins and glycolipids (see Figure 10–16). Because pathogens generally lack sialic acid, they are singled out for complement-mediated destruction, while host cells are spared. Some pathogens, including the bacterium Neisseria gonorrhoeae that causes the sexually transmitted disease gonorrhea, coat themselves with a layer of sialic acid to effectively hide from the complement system.

1	Membrane-immobilized C3b, produced by any of the three pathways, triggers a further cascade of reactions that leads to the assembly of the late complement components to form membrane attack complexes. These protein complexes assemble in the pathogen membrane near the site of C3 activation, forming aqueous pores through the membrane (Figure 24–8). For this reason, and because they perturb the structure of the lipid bilayer in their vicinity, they make the membrane leaky and can, in some cases, cause the microbe to lyse.

1	The self-amplifying, inflammatory, and destructive properties of the complement cascade make it essential that key activated components be rapidly inactivated after they are generated, ensuring that the attack does not spread to nearby host cells. Inactivation is achieved in at least two ways. First, specific inhibitor proteins in the blood or on the surface of host cells terminate the cascade, by either binding or cleaving certain complement components once the components have been activated by proteolytic cleavage. Second, many of the activated components in the cascade are unstable; unless they bind immediately to either the next component in the complement cascade or to a nearby membrane, they rapidly inactivate. Virus-Infected cells Take Drastic Measures to prevent Viral replication

1	Virus-Infected cells Take Drastic Measures to prevent Viral replication Because host-cell ribosomes make a virus’s proteins and host-cell lipids form the membranes of enveloped viruses, PAMPs are generally not present on the surface of viruses. Therefore, the only general way that a host cell PRR can recognize the presence of a virus is to detect unusual elements of the viral genome, such as the

1	Figure 24–8 Assembly of the late complement components to form a membrane attack complex. The cleavage of the early complement components (shown within gray arrows in Figure 24–7) results in the formation of c3bcontaining proteolytic complexes called c5 convertases (not shown). These then cleave the first of the late components, c5, to produce c5a and c5b. As illustrated, c5b rapidly assembles with c6 and c7 to form c567, which then binds firmly via c7 to the membrane. One molecule of c8 binds to the complex to form c5678. The binding of a molecule of c9 to c5678 induces a conformational change in c9 that exposes a hydrophobic region and causes c9 to insert into the lipid bilayer of the target membrane. This starts a chain reaction in which the altered c9 binds a second molecule of c9, which can then bind another molecule of c9, and so on. In this way, a ring of c9 molecules forms a large transmembrane channel in the membrane.

1	double-stranded RNA (dsRNA) that is an intermediate in the life cycle of many viruses and is recognized by several PRRs including the Toll-like receptor TLR3; in addition, DNA virus genomes frequently contain significant amounts of the CpG motifs discussed earlier, which can be recognized by TLR9 (see Table 24–1, p. 1300).

1	Mammalian cells are particularly adept at recognizing the presence of dsRNA, which activates intracellular PRRs that induce the host cell to produce and secrete two antiviral cytokines—interferon-α (IFNα) and interferon-β (IFNβ). These interferons are referred to as type I interferons to distinguish them from IFNγ, which is a type II interferon and has different functions, as we discuss later. Type I interferons act in both an autocrine fashion on the infected cells that produced it and a paracrine fashion on uninfected neighbors. They bind to a common cell-surface receptor, which activates the JAK–STAT intracellular signaling pathway (see Figure 15–56) to stimulate specific gene transcription and thereby the production of more than 300 proteins, including many cytokines, reflecting the complexity of the cell’s acute response to a viral infection.

1	The production of type I interferons appears to be a general response of mammalian cells to a viral infection, and viral components other than dsRNA can trigger it. The type I interferons help block viral replication in multiple ways. They activate a latent ribonuclease that nonspecifically degrades single-stranded RNA. They also indirectly activate a protein kinase that phosphorylates and inactivates the protein synthesis initiation factor eIF2 (discussed in Chapter 6), thereby shutting down most protein synthesis in the infected host cell. Apparently, by destroying most of its own RNA and transiently halting most of its protein synthesis, the host cell inhibits viral replication without killing itself. If these measures fail, the cell takes an even more extreme step to prevent the virus from replicating: it kills itself by undergoing apoptosis, often with the help of immune killer cells, as we discuss next. Natural Killer cells Induce Virus-Infected cells to Kill Themselves

1	Natural Killer cells Induce Virus-Infected cells to Kill Themselves Type I interferons also have less direct ways of blocking viral replication. One of these is to enhance the activity of natural killer cells (NK cells), which are leukocytes related to T and B cells but are part of the innate immune system and are recruited early to sites of inflammation. Like cytotoxic T cells of the adaptive immune system (discussed later), NK cells destroy virus-infected cells by inducing the infected cells to kill themselves by undergoing apoptosis (discussed in Chapter 18). We consider how killer cells induce apoptosis later, when we discuss how cytotoxic T cells do it (see Figure 24–43). Although they kill in the same way, the means by which cytotoxic T cells and NK cells distinguish the surface of virus-infected cells from that of uninfected cells are different (Movie 24.2).

1	Both cytotoxic T cells and NK cells recognize the same special class of cell-surface proteins on a host cell to help determine if the cell is virus-infected, but they use distinct receptors to do so. The special cell-surface proteins recognized are called class I MHC proteins, because they are encoded by genes in the major histocompatibility complex; almost all nucleated cells in vertebrates express these genes, and we discuss them in detail later. Cytotoxic T cells use both T cell receptors (TCRs) and co‑receptors to recognize peptide fragments of viral proteins bound to class I MHC proteins on the surface of virus-infected host cells and then induce the infected cells to kill themselves. By contrast, NK cells have cell-surface inhibitory receptors that monitor the level of class I MHC proteins on the surface of other host cells: the high levels of these MHC proteins normally present on healthy cells engage these receptors and thereby inhibit the killing activity of the NK cells. The

1	on the surface of other host cells: the high levels of these MHC proteins normally present on healthy cells engage these receptors and thereby inhibit the killing activity of the NK cells. The NK cells thus focus primarily on host cells expressing abnormally low levels of class I MHC proteins and induce them to kill themselves; these are mainly virus-infected cells and some cancer cells (Figure 24–9). NK cell killing activity is stimulated when various activating receptors on the NK cell surface recognize specific proteins that are greatly increased on the surface of virus-infected cells and some cancer cells.

1	Figure 24–9 A natural killer (NK) cell attacking a cancer cell. This scanning electron micrograph was taken shortly after the NK cell attached to the cancer cell, but before it induced the cell to die by apoptosis. (courtesy of J.c. hiserodt, in Mechanisms of cytotoxicity by Natural Killer cells [r.B. herberman and D. callewaert, eds.]. New York: Academic press, 1995.)

1	The reason that class I MHC protein levels are often low on virus-infected cells is that many viruses have developed a variety of mechanisms to inhibit the expression of these proteins on the surface of the host cells they infect, in order to avoid detection by cytotoxic T cells: some viruses encode proteins that block class I MHC gene transcription; others block the intracellular assembly of pep-tide–MHC complexes; still others block the transport of these complexes to the cell surface. By evading recognition by cytotoxic T cells in these ways, however, a virus incurs the wrath of NK cells, which recognize the infected cells as being different—both because the infected cells express little class I MHC protein and because they express large amounts of other surface proteins that are recognized by the activating receptors on the NK cells (Figure 24–10). Dendritic cells provide the Link Between the Innate and Adaptive Immune Systems

1	Dendritic cells provide the Link Between the Innate and Adaptive Immune Systems Dendritic cells are crucially important components of the innate immune system. They are a heterogeneous class of cells that are widely distributed in the tissues and organs of vertebrates. They express a large variety of PRRs, which enable dendritic cells to recognize and phagocytose invading pathogens and their products and to become activated in the process. The activated dendritic cells cleave the proteins of the pathogen into peptide fragments, which bind to newly synthesized MHC proteins, which then carry the fragments to the dendritic cell surface. The activated cells then migrate to a nearby lymphoid organ such as a lymph node (also called a lymph gland), where they present the peptide–MHC complexes to T cells of the adaptive immune system, activating the T cells to join in the battle against the specific pathogen (Figure 24–11).

1	In addition to the complexes of MHC proteins and microbial peptides displayed on their cell surface, activated dendritic cells also display cell-surface co‑stimulatory proteins that help activate T cells (see Figure 24–11). As we discuss later, the activated dendritic cells also secrete a variety of cytokines that influence the type of response that the T cells make, ensuring that it is appropriate to fight the particular pathogen. In these ways, dendritic cells serve as crucial links between the innate immune system, which provides a rapid first line of defense against invading pathogens, and the adaptive immune system, which mounts slower but more powerful and highly specific responses to attack an invader, as we now discuss. All multicellular organisms possess innate immune defenses against invading pathogens; these defenses include physical and chemical barriers and various defensive cell responses. In vertebrates, these innate defense responses can also

1	Figure 24–10 How an NK cell recognizes its target. An NK cell preferentially attacks infected host cells and cancer cells because these cells have on their surface both activating proteins and, in some cases, abnormally low levels of class I Mhc proteins. (A) The high levels of class I Mhc proteins found on normal host cells activate inhibitory receptors on the NK cell that suppress the killing activity of the NK cell. (B) In contrast, the activating proteins on infected cells and cancer cells bind to activating receptors on the NK cell and stimulate the killing activity of the cell.

1	Figure 24–11 Dendritic cells as functional links between the innate and adaptive immune systems. Dendritic cells pick up invading microbes or their products at the site of an infection. The microbial pAMps activate the dendritic cells to express co-stimulatory proteins and increased amounts of Mhc proteins on their surface and to migrate via lymphatic vessels to a nearby lymph node. In the lymph node, the activated dendritic cells activate T cells that express appropriate receptors for the co-stimulatory proteins and the microbial peptides bound to Mhc proteins on the dendritic cell surface. The activated T cells proliferate, and some of their progeny migrate to the original site of infection, where they help eliminate the microbes, either by activating local macrophages or by killing infected host cells (not shown). In addition, some of the activated T cells help stimulate specific B cells in the lymph node to secrete antibodies against the microbe (not shown).

1	A crucial feature of dendritic cell activation is that the pathogen provides an individual dendritic cell with both the peptides for presentation to T cells and the pAMp signals that activate the dendritic cell to express co-stimulatory proteins. In this way, the individual dendritic cell has all it needs to activate specific T cells that recognize the peptide–Mhc complexes on its surface (Movie 24.3).

1	recruit specific and more powerful adaptive immune responses to help fight the infection. Innate immune responses rely on the ability of host cells to recognize characteristic features of microbial molecules called pathogen‑associated molecular patterns, or PAMPs, which can be associated with a pathogen’s proteins, lipids, sugars, or nucleic acids. PAMPs are mainly recognized by pattern recognition receptors (PRRs), including the Toll‑like receptors (TLRs) found on or in both plant and animal cells. In vertebrates, some PRRs are secreted and can activate complement when they bind microbial PAMPs. The complement system, which can also be activated by antimicrobial antibodies bound to pathogens, consists of a group of blood proteins that are activated in sequence to help fight infections, by disrupting the pathogen's membrane, stimulating an inflammatory response, or targeting the microbe for phagocytosis—mainly by macrophages and neutrophils. The phagocytes use a combination of

1	by disrupting the pathogen's membrane, stimulating an inflammatory response, or targeting the microbe for phagocytosis—mainly by macrophages and neutrophils. The phagocytes use a combination of degradative enzymes, antimicrobial peptides, and oxygen‑derived toxic molecules to kill invading pathogens; in addition, they secrete various signal molecules that help trigger an inflammatory response.

1	Cells infected by a virus produce and secrete type I interferons (IFNα and IFNβ), which induce a complex set of host‑cell responses that inhibit viral replication. The interferons also enhance the killing activity of natural killer (NK) cells. An NK cell kills infected host cells because they express large amounts of surface proteins that activate the NK cell; the killing is especially efficient when infected cells express reduced amounts of class I MHC proteins, which, when present in normal amounts on a host cell surface inhibit the killing activity of NK cells.

1	Dendritic cells of the innate immune system functionally link innate immune responses to adaptive immune responses. The cells become activated when their PRRs pick up microbes and their products at sites of infection and phagocytose them. The activated cells cleave the microbial proteins into peptide fragments, which bind to newly made MHC proteins, which transport the fragments to the cell surface. The activated dendritic cells then carry the peptide–MHC complexes to a lymph organ, where they activate appropriate T cells to make specific adaptive immune responses against the microbes. OVerVIeW OF The ADApTIVe IMMUNe SYSTeM

1	A dramatic “big bang” in immune defense mechanisms occurred when jawed vertebrates evolved and acquired an adaptive immune system. This sophisticated defense system depends on B and T lymphocytes (B and T cells), which, during their development, rearrange particular DNA sequences in various combinations so that, together, the cells can produce an almost limitless variety of B and T cell receptors and antibodies. Collectively, these proteins can bind to essentially any molecule, including small chemicals, carbohydrates, lipids, and proteins; individually, they can distinguish between molecules that are very similar—such as between two proteins that differ in only a single amino acid, or between two optical isomers of the same small molecule. By this strategy, the adaptive immune system can recognize and respond specifically to any pathogen, including new mutant forms. However, because the genetic rearrangement process produces both receptors that can bind to self molecules as well as

1	recognize and respond specifically to any pathogen, including new mutant forms. However, because the genetic rearrangement process produces both receptors that can bind to self molecules as well as receptors that can bind to foreign molecules, vertebrates have had to evolve special mechanisms to ensure that B and T cells do not react against the host’s own molecules and cells—a process called immunological self‑tolerance.

1	Moreover, many harmless foreign substances enter the body, for example, as food or inhaled material, and it would be pointless and potentially dangerous to mount adaptive immune responses against them. Such inappropriate responses are normally avoided because innate immune responses are required to call adaptive immune responses into play and do so only when the innate cells’ PRRs recognize microbial PAMPs, as we discussed earlier. One can trick the adaptive immune system into responding to a harmless foreign molecule, such as a foreign protein, by co-injecting a molecule (often of microbial origin) called an adjuvant, which activates PRRs. This trick is called immunization and is the basis of vaccination. Any substance capable of stimulating B or T cells to make a specific adaptive immune response against it is referred to as an antigen (antibody generator).

1	There are two broad classes of adaptive immune responses—antibody responses and T‑cell‑mediated immune responses, and most pathogens induce both classes of responses. In antibody responses, B cells are activated to secrete antibodies, which are proteins that circulate in the bloodstream and permeate the other body fluids, where they can bind specifically to the foreign antigen that stimulated their production (see Figure 24–2). Binding of antibody neutralizes extracellular viruses and microbial toxins (such as tetanus toxin or cholera toxin) by blocking their ability to bind to receptors on host cells. Antibody binding also marks invading pathogens for destruction, both by making it easier for phagocytes of the innate immune system to ingest and destroy them and by activating the complement system.

1	In T-cell-mediated immune responses, T cells recognize foreign antigens that are bound to MHC proteins on the surface of host cells such as dendritic cells, which are specialized for presenting antigen to T cells and are therefore referred to as professional antigen‑presenting cells (APCs). Because MHC proteins carry fragments of pathogen proteins from inside a host cell to the cell surface, T cells can detect pathogens hiding inside a host cell and either kill the infected cell (see Figure 24–2) or stimulate phagocytes or B cells to help eliminate the pathogens. In this section, we discuss the origins and general properties of B and T cells. In later sections, we consider the specific properties and functions of these cells. B cells Develop in the Bone Marrow, T cells in the Thymus

1	B cells Develop in the Bone Marrow, T cells in the Thymus There are about 2 × 1012 lymphocytes in the human body, making the immune system comparable in cell mass to the liver or the brain. They occur in large numbers in the blood and lymph (the colorless fluid in the lymphatic vessels, which connect the lymph nodes in the body to each other and to the bloodstream). They are also concentrated in lymphoid organs, such as the thymus, lymph nodes, and spleen (Figure 24–12), and many are also found in other organs, including skin, lung, and gut.

1	T cells and B cells derive their names from the organs in which they develop: T cells develop in the thymus, and B cells, in adult mammals, develop in the bone marrow. Both types of cells develop from lymphoid progenitor cells that are produced from multipotent hematopoietic stem cells, which are found mainly in the bone marrow (Figure 24–13). The hematopoietic stem cells give rise to more than just lymphocytes: as discussed in Chapter 22, they produce all of the cells of the Figure 24–12 Human lymphoid organs.

1	Figure 24–12 Human lymphoid organs. Lymphocytes develop from lymphoid progenitor cells in the thymus and bone marrow (yellow), which are therefore called central (or primary) lymphoid organs. The newly formed lymphocytes migrate from these primary organs to peripheral (or secondary) lymphoid organs, where they can react with foreign antigen. Only some of the peripheral lymphoid organs (blue) and lymphatic vessels (green) are shown; many lymphocytes, for example, are found in the skin and respiratory tract. As we discuss later, the lymphatic vessels ultimately empty into the bloodstream (not shown).

1	Figure 24–13 The development of b and T cells. The central lymphoid organs, where lymphocytes develop from lymphoid progenitor cells, are labeled in yellow boxes. The lymphoid progenitor cells develop from multipotent hematopoietic stem cells in the bone marrow. Some lymphoid progenitor cells develop locally in the bone marrow into immature B cells, while others migrate to the thymus (via the bloodstream) where they develop into thymocytes (developing T cells). Foreign antigens activate B cells and T cells mainly in peripheral lymphoid organs, such as lymph nodes or the spleen. hematopoietic system, including erythrocytes, leukocytes, and platelets (see Figure 22–32).

1	hematopoietic system, including erythrocytes, leukocytes, and platelets (see Figure 22–32). Because they are sites where lymphocytes develop from lymphoid progenitor cells, the thymus and bone marrow are referred to as central (primary) lymphoid organs (see Figure 24–12). As we discuss later, most B and T cells die in the central lymphoid organs soon after they develop, without ever functioning. Others, however, mature and migrate via the blood to the peripheral (secondary) lymphoid organs—mainly the lymph nodes, spleen, and epithelium-associated lymphoid tissues in the gastrointestinal tract, respiratory tract, and skin. It is in these peripheral lymphoid organs that foreign antigens activate B and T cells (see Figure 24–13).

1	B and T cells become morphologically distinguishable from each other only after antigen has activated them: resting B and T cells look very similar, even in an electron microscope (Figure 24–14A). After activation by an antigen, both proliferate and mature into effector cells. Effector B cells secrete antibodies; in their most mature form, called plasma cells, they are filled with an extensive rough endoplasmic reticulum that is busily making antibodies (Figure 24–14B). In contrast, effector T cells (Figure 24–14C) contain very little endoplasmic reticulum and secrete a variety of cytokines rather than antibodies. Whereas B-cell-derived antibodies are widely distributed by the bloodstream, T-cell-derived cytokines mainly act locally on neighboring cells, although some are carried via the blood and act on distant host cells.

1	The most remarkable feature of the adaptive immune system is that it can respond to millions of different foreign antigens in a highly specific way. Human B cells, for example, collectively, can make more than 1012 different antibody molecules that react specifically with the antigen that induced their production. How can B cells and T cells respond specifically to such an enormous diversity of foreign antigens? The answer for both B and T cells is the same. As each lymphocyte develops in a central lymphoid organ, it becomes committed to react with a particular antigen before ever being exposed to the antigen. It expresses this commitment in the form of cell-surface receptors that specifically bind the antigen. When a lymphocyte encounters its antigen in a peripheral lymphoid organ, the binding of the antigen to the receptors (with help from co-stimulatory signals, discussed later) Figure 24–14 electron micrographs of resting and effector lymphocytes.

1	(A) This resting lymphocyte could be either a B cell or a T cell, as these cells are difficult to distinguish morphologically until antigen activates them to become effector cells. (B) An effector B cell (a plasma cell). It is filled with an extensive rough endoplasmic reticulum (er), which is distended with antibody molecules that are secreted in large amounts. (c) An effector T cell, which has relatively little rough er but is filled with free ribosomes; it secretes cytokines, but in relatively small amounts. The three cells are shown at the same magnification. (A, courtesy of Dorothy Zucker-Franklin; B, courtesy of carlo Grossi; A and B, from D. Zucker-Franklin et al., Atlas of Blood cells: Function and pathology, 2nd ed. Milan, Italy: edi. ermes, 1988; c, courtesy of Stefanello de petris.) activates the lymphocyte; this causes the lymphocyte to proliferate, thereby producing many more cells with the same receptor—a process called clonal expansion. The encounter with antigen also

1	activates the lymphocyte; this causes the lymphocyte to proliferate, thereby producing many more cells with the same receptor—a process called clonal expansion. The encounter with antigen also causes some of the cells to differentiate into effector cells. An antigen therefore selectively stimulates those cells that express complementary antigen-specific receptors and are thus already committed to respond to it (Figure 24–15). This arrangement, called clonal selection, provides an explanation for immunological memory, whereby we develop lifelong immunity to many common infectious diseases after our initial exposure to the pathogen—either through natural infection or vaccination.

1	It is easy to demonstrate such immunological memory in experimental animals. If an animal is immunized once with antigen A, an immune response (antibody, T-cell-mediated, or both) can be detected after several days; the response rises rapidly and exponentially, and then, more gradually, declines. This is the characteristic course of a primary immune response, occurring on an animal’s first exposure to an antigen. If, after some weeks, months, or even years have elapsed, the animal is immunized again with antigen A, it will usually produce a secondary immune response that differs from the primary response: the lag period is shorter, because there are now many more preexisting B or T cells (or both) with specificity for antigen A, and the response is greater and more efficient. These differences indicate that the animal has “remembered” its first exposure to antigen A. If the animal is given a different antigen (for example, antigen B) instead of a second immunization with antigen A,

1	indicate that the animal has “remembered” its first exposure to antigen A. If the animal is given a different antigen (for example, antigen B) instead of a second immunization with antigen A, the response is typical of a primary, and not a secondary, immune response. The secondary response therefore reflects antigen-specific immunological memory for antigen A (Figure 24–16).

1	Immunological memory depends on both lymphocyte proliferation and differentiation. In an adult animal, the peripheral lymphoid organs contain a mixture of lymphocytes in at least three stages of maturation: naïve cells, effector cells, and memory cells. When naïve cells encounter their specific foreign antigen for the first time, the antigen stimulates some of them to proliferate and differentiate into effector cells, which then carry out an immune response (effector B cells secrete antibody, whereas effector T cells either kill infected cells or influence the response of other immune cells—by secreting cytokines, for example). Some of the antigen-stimulated naïve cells multiply and differentiate into memory cells, which are more easily and more quickly induced to become effector cells by a later encounter with the same antigen: like naïve cells, when memory cells encounter their antigen, they give rise to either effector cells or more memory cells (Figure 24–17).

1	Figure 24–15 Clonal selection. An antigen activates only those lymphocytes that are already committed to respond to it. The committed cell expresses cell-surface receptors that specifically recognize the antigen. The human adaptive immune system consists of many millions of different T and B lymphocyte clones, with cells within a clone expressing the same unique antigen receptor. Before its first encounter with antigen, a clone would usually contain only one or a small number of cells. A particular antigen may activate hundreds of different clones, each expressing a different antigen receptor that binds either a different part of the antigen or the same part with a different binding affinity. Although only B cells are shown here, T cells are selected in a similar way. Note that the antigen receptors on the B cells labeled β in this diagram have the same antigen-binding site as the antibodies secreted by the effector Bβ cells. As we discuss later, B cells require co-stimulatory

1	antigen receptors on the B cells labeled β in this diagram have the same antigen-binding site as the antibodies secreted by the effector Bβ cells. As we discuss later, B cells require co-stimulatory signals from T cells to become activated by antigen to proliferate and differentiate into antibody-secreting cells (not shown).

1	antibodies in blood(arbitrary units, Iog scale) frst immunization second immunization with antigen A with antigen A frst immunization with antigen B Thus, during the primary response, clonal expansion and differentiation of antigen-stimulated naïve cells creates many memory cells, which are able to respond to the same antigen more sensitively, rapidly, and effectively. And, unlike most effector cells, which die within days or weeks, memory cells can persist for the lifetime of the animal, even in the absence of their specific antigen, thereby providing lifelong immunological memory. Although most effector B and T cells die after an immune response is over, some survive as effector cells and help provide long-term protection against the pathogen. A small proportion of the plasma cells produced in a primary B cell response, for example, can survive for many months or years in the bone marrow, where they continue to secrete their specific antibodies into the bloodstream.

1	Pathogens generally enter the body through an epithelial surface, usually through the skin, gut, or respiratory tract. To induce an adaptive immune response, microbes or their products must travel from these entry points to a peripheral lymphoid organ, such as a lymph node or the spleen, which are the sites where lymphocytes are activated (see Figure 24–11). The route and destination depend on the site of entry. Lymphatic vessels carry antigens that enter through the skin or respiratory tract to local lymph nodes; antigens that enter through the gut end up in gut-associated peripheral lymphoid organs such as Peyer’s patches; and the spleen filters out antigens that enter the blood (see Figure 24–12). As discussed earlier (see Figure 24–11), in many cases, activated dendritic cells will carry the antigen from the site of infection to the peripheral lymphoid organ, where they play a crucial part in activating T cells, as we discuss later.

1	But only a tiny fraction of naïve B and T cells can recognize a particular microbial antigen in a peripheral lymphoid organ, a reasonable estimate being between 1/10,000 and 1/1,000,000 of each class of lymphocyte, depending on the antigen. How do these rare cells find an antigen-presenting cell displaying their specific antigen? The answer is that the lymphocytes continuously recirculate between one peripheral lymphoid organ and another via the lymph and blood. In a lymph node, for example, lymphocytes continually leave the bloodstream by squeezing out between specialized endothelial cells lining small veins called postcapillary Figure 24–17 A model for the cellular basis of immunological memory.

1	Figure 24–17 A model for the cellular basis of immunological memory. When stimulated by their specific antigen and co-stimulatory signals, naïve lymphocytes proliferate and differentiate. Most become effector cells, which function and then usually die, while others become memory cells. During a subsequent exposure to the same antigen, the memory cells respond more readily, rapidly, and efficiently than did the naïve cells: they proliferate and give rise to effector cells and to more memory cells. Some memory T cells also develop from a minority of effector T cells (not shown). It is not known how the decision to become an effector cell versus a memory cell is made.

1	Figure 24–16 Immunological memory: primary and secondary antibody responses. The secondary response induced by a second exposure to antigen A is faster and greater than the primary response and is specific for A, indicating that the adaptive immune system has specifically remembered its previous encounter with antigen A. The same type of immunological memory is observed in T-cell-mediated responses (not shown). As we discuss later, the types of antibodies produced in the secondary response are different from those produced in the primary response, and these antibodies bind the antigen more tightly.

1	frst exposure to antigen venules. After percolating through the node, they accumulate in small lymphatic vessels that leave the node and connect with other lymphatic vessels that pass through other lymph nodes downstream (see Figure 24–12). Passing into larger and larger vessels, the lymphocytes eventually enter the main lymphatic vessel (the thoracic duct), which carries them back into the blood (Figure 24–18).

1	The continuous recirculation of a lymphocyte between the blood and lymph ends only if its specific antigen activates it in a peripheral lymphoid organ. In that case, the lymphocyte remains in the peripheral lymphoid organ, where it proliferates and differentiates into either effector cells or memory cells. Many of the effector T cells leave the lymphoid organ via the lymph and migrate through the blood to the site of infection (see Figure 24–11), whereas others stay in the lymphoid organ and help activate (or suppress) other immune cells there. Some effector B cells (plasma cells) remain in the peripheral lymphoid organ and secrete antibodies into the blood for days until they die; others migrate to the bone marrow, where they secrete antibodies into the blood for months or years. The memory T and B cells produced join the recirculating pool of lymphocytes.

1	Lymphocyte recirculation depends on specific interactions between the lymphocyte cell surface and the surface of the endothelial cells lining the blood vessels in the peripheral lymphoid organs. Lymphocytes that enter a lymph node via the blood, for example, adhere weakly to specialized endothelial cells lining the postcapillary venules via homing receptors that belong to the selectin family of cell-surface lectins that bind to specific sugar groups on the endothelial cell surface (see Figure 19–28). The lymphocytes roll slowly along the surface of the endothelial cells until another, much stronger adhesion system, dependent on an integrin protein, is called into play by chemokines secreted by the endothelial cells. Now, the lymphocytes stop rolling, and they crawl out of the blood vessel into the lymph node by using yet another cell adhesion protein called CD31 (Figure 24–19). Although B and T cells initially enter the same region of a lymph node, different chemokines guide them to

1	into the lymph node by using yet another cell adhesion protein called CD31 (Figure 24–19). Although B and T cells initially enter the same region of a lymph node, different chemokines guide them to separate regions of the node—B cells to lymphoid follicles and T cells to the paracortex (Figure 24–20).

1	specialized endothelial cell of postcapillary venule Figure 24–19 Migration of a lymphocyte out of the bloodstream into a lymph node.

1	A circulating lymphocyte adheres weakly to the surface of the specialized endothelial cells lining a postcapillary venule in a lymph node. This initial adhesion is mediated by L-selectin (discussed in chapter 19) on the lymphocyte surface. The adhesion is sufficiently weak to enable the lymphocyte, pushed by the flow of blood, to roll along the surface of the endothelial cells. Stimulated by chemokines secreted by specialized endothelial cells in the node (curved red arrow), the lymphocyte rapidly activates a stronger adhesion system, mediated by an integrin. This strong adhesion enables the cell to stop rolling. The lymphocyte then uses an immunoglobulin-like cell adhesion protein (cD31) to bind to the junctions between adjacent endothelial cells and migrate out of the venule. The subsequent migration of the lymphocyte in the lymph node depends on chemokines produced within the node (straight red arrow). The migration of other types of leukocytes out of the bloodstream into sites of

1	migration of the lymphocyte in the lymph node depends on chemokines produced within the node (straight red arrow). The migration of other types of leukocytes out of the bloodstream into sites of infection occurs in a similar way (see Figure 19–28 and Movie 19.2).

1	Figure 24–18 The path followed by lymphocytes as they continuously recirculate between the lymph and blood. The circulation through a lymph node (yellow) is shown here. Microbial antigens are usually carried into the lymph node by activated dendritic cells (not shown), which enter the node via afferent lymphatic vessels draining an infected tissue (green). B and T cells, by contrast, enter via the blood, migrating out of the bloodstream into the lymph node through postcapillary venules. Unless they encounter their antigen, the B and T cells leave the lymph node via efferent lymphatic vessels, which eventually join the thoracic duct. The thoracic duct empties into a large vein carrying blood to the heart, completing the circulation cycle for T and B cells. A typical circulation cycle for these lymphocytes takes about 12–24 hours.

1	Unless they encounter their antigen, both B and T cells soon leave the lymph node via efferent lymphatic vessels. If they encounter their antigen, however, they are stimulated to display adhesion receptors that trap the cells in the node; the cells accumulate at the junction between the B cell and T cell areas, where the rare antigen-specific B and T cells can interact, leading to their proliferation and differentiation into either effector cells or memory cells. Many of the effector cells leave the node, expressing different chemokine receptors that help guide them to their new destinations—effector plasma B cells to the bone marrow and effector T cells to sites of infection.

1	As discussed earlier, cells of the innate immune system use PRRs to distinguish microbial molecules from self molecules made by the host. The adaptive immune system has the far more difficult recognition task of responding specifically to an almost unlimited number of foreign molecules while not responding to the large number of self molecules. How does it accomplish this feat? It helps that self molecules normally do not induce the innate immune reactions required to activate adaptive immune responses. But even when an infection or tissue injury triggers innate reactions, the vast majority of self molecules normally still fail to induce an adaptive immune response. Why?

1	One important reason is that the adaptive immune system “learns” not to respond to self molecules. Normal mice, for example, cannot mount an immune response against one of their own protein components of the complement system called C5 (see Figure 24–7). However, mutant mice that lack the gene encoding C5 but are otherwise genetically identical to normal mice of the same strain can make a strong immune response to this blood protein when immunized with it. The immunological self-tolerance exhibited by normal mice persists only for as long as the self molecule remains in the body: if a self molecule such as C5 is experimentally removed from an adult mouse, the animal gains the ability to respond to it after a few weeks or months, as new B and T cells develop in the absence of C5. Thus, the adaptive immune system is genetically capable of responding to self molecules, but it learns not to do so.

1	Self-tolerance depends on a number of distinct mechanisms, including the following (Figure 24–21): 1. In receptor editing, developing B cells that recognize self molecules change their antigen receptors so that the cells no longer do so. 2. In clonal deletion, potentially self-reactive B and T cells die by apoptosis when they encounter their particular self molecule.

1	2. In clonal deletion, potentially self-reactive B and T cells die by apoptosis when they encounter their particular self molecule. Figure 24–20 A simplified drawing of a human lymph node. B cells are primarily clustered in structures called lymphoid follicles, whereas T cells are found mainly in the paracortex. chemokines attract both types of lymphocytes into the lymph node from the blood via postcapillary venules (see Figure 24–19). B and T cells then migrate to their respective areas, attracted by different chemokines. If they do not encounter their specific antigen, both B cells and T cells then enter the medullary sinuses and leave the node via the efferent lymphatic vessel. This vessel ultimately empties into the bloodstream, allowing the lymphocytes to begin another cycle of circulation through a peripheral lymphoid organ (see Figure 24–18). During an infection, proliferation of pathogen-specific B cells produces a germinal center in some lymphoid follicles. 3.

1	3. In clonal inactivation (also called clonal anergy), self-reactive B and T cells become functionally inactivated when they encounter their self molecule. 4. In clonal suppression, self-reactive regulatory T cells (discussed later) suppress the activity of other types of potentially self-reactive lymphocytes. Some of these mechanisms—especially the first two, receptor editing in B cells and clonal deletion of B and T cells—operate in central lymphoid organs when newly formed self-reactive B and T cells first encounter their self molecules, and they are largely responsible for the process called central tolerance. Clonal inactivation and clonal suppression, by contrast, operate mainly when mature B and T cells encounter their self molecules in peripheral lymphoid organs, and they are largely responsible for the process called peripheral tolerance. Clonal deletion, however, can also operate peripherally, and clonal inactivation can also operate centrally.

1	Why does the binding of a self molecule lead to tolerance rather than activation? The answer is still not completely known. As we discuss later, the activation of a B or T cell by its antigen in a peripheral lymphoid organ requires more than just antigen binding: it requires co-stimulatory signals, which are provided by a helper T cell (discussed later) in the case of a B cell and by an activated dendritic cell in the case of a naïve T cell. The production of such signals is usually triggered by exposure to a pathogen, but a self-reactive lymphocyte normally encounters its self antigen in the absence of such signals. Under these conditions, the lymphocyte will not only fail to be activated, it will often be rendered tolerant—being either killed or inactivated, or actively suppressed by a regulatory T cell (see Figure 24–21). In peripheral lymphoid organs, both T cell tolerance and activation usually occur on the surface of a dendritic cell.

1	For reasons that are usually unknown, self-tolerance mechanisms sometimes fail, causing T or B cells (or both) to react against the animal’s own molecules. Figure 24–21 Mechanisms of immunological self-tolerance. When a self-reactive immature B cell binds its self molecule in the central lymphoid organ where the cell is produced, it may alter its antigen receptor so that it is no longer self-reactive (cell 1); this process is called receptor editing. Alternatively, when either an immature B or T cell binds its self molecule in a central lymphoid organ, it may die by apoptosis, a process called clonal deletion (cell 2). Because these two forms of tolerance (shown on the left) occur in central lymphoid organs, they are called central tolerance.

1	When a self-reactive naïve B or T cell escapes tolerance in the central lymphoid organ and binds its self molecule in a peripheral lymphoid organ (cell 4), or in another peripheral tissue, it will generally not be activated, because the binding usually occurs in the absence of sufficient co-stimulatory signals; instead, the cell may die by apoptosis (often after a period of proliferation), be inactivated, or be suppressed by a regulatory T cell. These forms of tolerance (shown on the right) are called peripheral tolerance. As discussed later, the cells providing the co-stimulatory signals are T lymphocytes for B cells and usually dendritic cells for T cells (not shown). For T cells at least, both activation and tolerance in a peripheral lymphoid organ usually occurs on the surface of a dendritic cell, although the dendritic cells are different in the two cases.

1	Myasthenia gravis is an example of such an autoimmune disease. Most of the affected individuals make antibodies against the acetylcholine receptors on their own skeletal muscle cells; these receptors are required for the muscle to contract normally in response to nerve stimulation, which releases acetylcholine (see Figure 11–39). The antibodies interfere with the normal functioning of the receptors so that the patients become weak and may die because they cannot breathe. Similarly, in juvenile (type 1) diabetes, adaptive immune reactions against insulin-secreting β cells in the pancreas kill these cells, leading to severe insulin deficiency.

1	Innate immune responses triggered by pathogens at sites of infection help activate adaptive immune responses in peripheral lymphoid organs. The adaptive immune system is composed of many millions of B and T cell clones, with the cells in each clone sharing a unique cell‑surface receptor that enables them to bind a particular pathogen antigen. The binding of antigen to these receptors, with the help of co‑stimulatory signals, stimulates the lymphocyte to proliferate and differentiate into an effector cell that can help eliminate the pathogen. Effector B cells secrete antibodies, which can act over long distances to help eliminate extracellular pathogens and their toxins. Effector T cells, by contrast, produce cell‑surface and secreted co‑stimulatory molecules, which mainly act locally to help other immune cells eliminate the pathogen; in addition, some T cells can induce infected host cells to kill themselves.

1	During a primary adaptive immune response to an antigen, lymphocytes that recognize the antigen proliferate so that there are more of them to respond the next time, during a secondary response to the same antigen; moreover, during a primary response, some lymphocytes differentiate into memory cells, which can respond faster and more efficiently the next time the same pathogen invades. These two mechanisms are largely responsible for immunological memory. Both B and T cells circulate continuously between one peripheral lymphoid organ and another via the blood and lymph; only if they encounter their specific foreign antigen in a peripheral lymphoid organ do they stop migrating, proliferate, and differentiate into effector cells or memory cells. Lymphocytes that would react against self molecules either alter their receptors (in the case of B cells) or are eliminated or inactivated; they can also be suppressed by regulatory T cells. These mechanisms collectively are responsible for

1	molecules either alter their receptors (in the case of B cells) or are eliminated or inactivated; they can also be suppressed by regulatory T cells. These mechanisms collectively are responsible for immunological self‑tolerance, which ensures that the adaptive immune system normally avoids attacking the molecules and cells of the host.

1	Vertebrates inevitably die of infection if they are unable to make antibodies. Antibodies are secreted proteins that defend us against extracellular pathogens in several ways. They bind to viruses and microbial toxins, thereby preventing them from binding to and damaging host cells (see Figure 24–2). When bound to an extracellular pathogen or its products, antibodies also recruit some of the components of the innate immune system, including various types of leukocytes and components of the complement system, which work together to inactivate or eliminate the invaders. Synthesized exclusively by B cells, antibodies are produced in billions of forms, each with a different amino acid sequence. They belong to the class of proteins called immunoglobulins (abbreviated as Igs) and are among the most abundant protein components in the blood. In this section, we discuss the structure and function of immunoglobulins and how they are made in so many different forms.

1	B cells Make Immunoglobulins (Igs) as Both cell-Surface Antigen receptors and Secreted Antibodies The first Igs made by a newly formed B cell are not secreted but are instead inserted into the plasma membrane, where they serve as receptors for antigen. They are called B cell receptors (BCRs), and each B cell has approximately 105 of them in its plasma membrane. Each BCR is stably associated with invariant trans-membrane proteins that activate intracellular signaling pathways when antigen binds to the BCR; we discuss these invariant proteins later, when we consider how B cells are activated with the assistance of helper T cells.

1	Each B cell clone produces a single species of BCR, with a unique antigen-binding site. When an antigen and a helper T cell activate a naïve or a memory B cell, the B cell proliferates and differentiates into an effector cell, which then produces and secretes large amounts of soluble (rather than membrane-bound) Ig. The secreted Ig is now called an antibody, and it has the same unique antigen-binding site as the BCR (Figure 24–22).

1	A typical Ig molecule is bivalent, with two identical antigen-binding sites. It consists of four polypeptide chains—two identical light chains and two identical heavy chains. The N-terminal parts of both light and heavy chains usually cooperate to form the antigen-binding surface, while the more C-terminal parts of the heavy chains form the tail of the Y-shaped protein (Figure 24–23). The tail mediates many of the activities of antibodies, and antibodies with the same antigen-binding sites can have any one of a number of different tail regions, each of which gives the antibody different functional properties, such as the ability to activate complement or to bind to receptor proteins on phagocytic cells that bind a specific type of antibody tail. Mammals Make Five classes of Igs

1	In mammals, there are five major classes of Igs, each of which mediates a characteristic biological response following antigen binding to an antibody: IgA, IgD, IgE, IgG, and IgM, each with its own class of heavy chain—α, δ, ε, γ, and μ, respectively. IgA molecules have α chains, IgG molecules have γ chains, and so on. Moreover, there are four human IgG subclasses (IgG1, IgG2, IgG3, and IgG4), with γ1, γ2, γ3, and γ4 heavy chains, respectively. There are also two IgA subclasses in humans. In addition to the various classes and subclasses of heavy chains, higher vertebrates have two types of light chains, κ and λ, which seem to be functionally indistinguishable. Either type of light chain may be associated with any of the heavy chains, but an individual Ig molecule always contains identical light chains and identical heavy chains: an IgG molecule, for instance, may have either κ or λ light chains, but not one of each. As a result, an Ig’s antigen-binding sites are always identical (see

1	light chains and identical heavy chains: an IgG molecule, for instance, may have either κ or λ light chains, but not one of each. As a result, an Ig’s antigen-binding sites are always identical (see Figure 24–22).

1	The various heavy chains give a distinctive conformation to the tail region of antibodies, so that each class (and subclass) has characteristic properties of its own. IgM is always the first class of Ig that a developing B cell in the bone marrow makes. It forms the BCRs on the surface of immature naïve B cells. After these cells Figure 24–22 The b cell receptors (bCRs) and secreted antibodies made by a b cell clone. The binding of an antigen to Bcrs on either a naïve or memory B cell (together with co-stimulatory signals provided by helper T cells—not shown) activates the cell to proliferate and differentiate into effector B cells. The effector cells produce and secrete antibodies with a unique antigen-binding site, which is the same as that of the cell-surface Bcrs. Because antibodies have two identical antigen-binding sites, they can cross-link antigens, as shown for an antigen with multiple identical antigenic determinants.

1	Figure 24–23 A schematic drawing of a bivalent antibody molecule. The two heavy chains each have a hinge region, which, because of its flexibility, improves the efficiency with which the antibody can cross-link antigens (see Figure 24–22). The two heavy chains also form the tail of the antibody, which determines its functional properties. The heavy and light chains are held together by both covalent S–S bonds (red) and noncovalent bonds (not shown).

1	leave the bone marrow, they start to produce IgD BCRs as well, with the same antigen-binding site as the IgM BCRs. These cells are now called mature naïve B cells, as they can now respond to their specific foreign antigen in peripheral lymphoid organs (Figure 24–24). IgM is also the major class of antibody secreted into the blood in the early stages of a primary antibody response on first exposure to an antigen. In its secreted form, IgM is a wheel-like pentamer composed of five four-chain units, giving it a total of 10 antigen-binding sites that allow it to bind strongly to pathogens; in its antigen-bound form, IgM is highly efficient at activating complement, which is important in early antibody responses to pathogens.

1	The major antibody class in the blood is IgG. These antibodies are four-chain monomers (see Figure 24–23), and they are produced in especially large quantities during secondary antibody responses. The tail region of some subclasses of IgG antibodies that are bound to antigen can activate complement and also bind to specific receptors on macrophages and neutrophils. Largely by means of such Fc receptors (so-named because antibody tails are called Fc regions), these phagocytic cells bind, ingest, and destroy infecting microorganisms that have become coated with the IgG antibodies produced in response to the infection; the activated Fc receptors also signal the phagocyte to secrete pro-inflammatory cytokines (Movie 24.4).

1	The tail region of IgE antibodies binds to another class of Fc receptors on the surface of mast cells in tissues and of basophils in the blood. Because antigen-free IgE antibodies bind with high affinity to such Fc receptors, the antibodies act as antigen receptors on these cells. Antigen binding to the bound antibodies activates the Fc receptors and stimulates the cells to secrete a variety of cytokines and biologically active amines, especially histamine, which causes blood vessels to dilate and become leaky; this helps leukocytes, antibodies, and complement components to enter sites where mast cells have been activated. The release of amines from mast cells and basophils is largely responsible for the symptoms of such allergic reactions as hay fever, asthma, and hives. In addition, mast cells secrete factors that attract and activate leukocytes called eosinophils, which also have Fc receptors that bind IgE molecules and can kill extracellular parasitic worms, especially if the

1	mast cells secrete factors that attract and activate leukocytes called eosinophils, which also have Fc receptors that bind IgE molecules and can kill extracellular parasitic worms, especially if the worms are coated with IgE antibodies (see Figure 24–6).

1	IgA is the principal antibody class in secretions, including saliva, tears, milk, and respiratory and intestinal secretions. Yet another class of Fc receptors, located on the relevant epithelial cells, guides the secretion by binding antigen-free IgA dimers and transporting them across the epithelium. The properties of the various classes of antibodies in humans are summarized in Table 24–2.

1	All classes of Ig can be made in a membrane-bound form, as well as in a soluble, secreted form. The two forms differ only in the C-terminus of their heavy chain. The heavy chains of membrane-bound Ig molecules (BCRs) have a trans-membrane hydrophobic C-terminus, which anchors them in the lipid bilayer of the B cell’s plasma membrane. The heavy chains of secreted antibody molecules, by contrast, have instead a hydrophilic C-terminus, which allows them to escape from the cell. The switch in the character of the Ig molecules made occurs because the activation of B cells by antigen and helper T cells induces a change in the way in which the heavy-chain RNA transcripts are made and processed in the nucleus (see Figure 7–59).

1	Figure 24–24 Stages of b cell development. All of the stages shown occur before the cells bind their specific antigen. The first cells in the B cell lineage that make Ig are called pro-B cells; they make μ heavy chains, which remain in the endoplasmic reticulum until a special type of light chain is made called a surrogate light chain. The surrogate light chains substitute for genuine light chains and assemble with μ chains to form a receptor molecule that inserts into the plasma membrane. The cells are now called pre-B cells. Signaling from this pre-B cell receptor allows the cells to make bona fide light chains, which combine with μ chains to form four-chain IgM molecules that serve as cell-surface Bcrs on immature naïve B cells. After these cells leave the bone marrow, they start to express IgD Bcrs as well, which have the same antigen-binding sites as the IgM Bcrs; it is this mature naïve B cell that reacts with its specific foreign antigen in peripheral lymphoid organs.

1	Ig Light and heavy chains consist of constant and Variable regions Both light and heavy chains have a variable amino acid sequence at their N-terminal ends but a constant sequence at their C-terminal ends. Whereas the constant region and variable region of a light chain are the same size, the constant region of a heavy chain is about three or four times longer, depending on the class (Figure 24–25).

1	The variable regions of the light and heavy chains come together to form the antigen-binding sites, and the variability of their amino acid sequences provides the structural basis for the diversity of these binding sites. The greatest diversity occurs in three small hypervariable regions in the variable regions of both light and heavy chains. Only about 5–10 amino acids in each hypervariable region form the actual antigen-binding site (Figure 24–26). As a result, the size of the antigenic determinant that an Ig molecule recognizes is generally comparably small: it can consist of fewer than 10 amino acids on the surface of a globular protein, for example (see Figure 24–22).

1	Both light and heavy chains are made up of repeating segments—each about 110 amino acids long and each containing one intrachain disulfide bond. Each repeating segment folds independently to form a compact functional unit called an immunoglobulin (Ig) domain. As shown in Figure 24–27A, a light chain consists of one variable (VL) and one constant (CL) domain, whereas a heavy chain has one variable and three or four constant domains: the variable domains of the light and heavy chains pair to form the antigen-binding region. Each Ig domain has a very similar three-dimensional structure, consisting of a sandwich of two β Figure 24–25 Constant and variable regions of Ig chains. The variable regions of the light and heavy chains form the antigen-binding sites, while the constant regions of the heavy chains determine the other biological properties of an Ig protein. The different subclasses of IgG antibodies have different γ-chain constant regions.

1	variable region hypervariable regions Figure 24–26 Ig hypervariable regions. of heavy chain of heavy chain highly schematized drawing of how the three hypervariable regions in each light and heavy chain together form each antigen-binding site of an Ig protein. sheets held together by a disulfide bond; the variable domains are unique in that each has its particular set of hypervariable regions, which are arranged in three hypervariable loops that cluster together at the ends of the variable domains to form the antigen-binding site (Figure 24–27B). Even in the absence of antigen stimulation, a human can probably make more than 1012 different Ig molecules—its preimmune, primary Ig repertoire. The primary repertoire consists of IgM and IgD proteins and is apparently large enough to ensure that there will be an antigen-binding site to fit almost any potential

1	Figure 24–27 Ig domains. (A) The light and heavy chains in an Ig protein are each folded into similar repeating domains. The variable domains (shaded in blue) of the light and heavy chains (VL and Vh) make up the antigen-binding sites, while the constant domains (shaded in gray) of the heavy chains (mainly ch2 and ch3) determine the other biological properties of the protein. The heavy chains of IgM and Ige do not have a hinge region and have an extra constant domain (ch4). hydrophobic interactions between domains on adjacent chains help hold the chains together in the Ig molecule: VL binds to Vh, cL binds to ch1, and so on. (B) X-ray crystallography-based structures of the Ig domains of a light chain (Movie 24.5). Both the variable and constant domains have a similar overall structure, consisting of two β sheets joined by a disulfide bond (red). Note that all the hypervariable regions (black) form loops at the far end of the variable domain, where they come together to form part of

1	consisting of two β sheets joined by a disulfide bond (red). Note that all the hypervariable regions (black) form loops at the far end of the variable domain, where they come together to form part of the antigen-binding site. All Igs are glycosylated on their ch2 domains (not shown); the attached oligosaccharide chains vary from Ig to Ig and can greatly influence the biological properties of the protein, largely by affecting its binding to Fc receptors on immune cells.

1	antigenic determinant, albeit with low affinity—Ka ≈ 105–107 liters/mole. After stimulation by antigen and helper T cells, B cells can switch from making IgM and IgD to making other classes of Ig—a process called class switching. In addition, the binding affinity of these Igs for their antigen progressively increases over time—a process called affinity maturation. Thus, antigen stimulation generates a secondary Ig repertoire, with a greatly increased affinity (Ka up to 1011 liters/mole) and diversity of both Ig classes and antigen-binding sites.

1	How can each of us make so many different Igs? The problem is not quite as formidable as it might first appear. Recall that the variable regions of the Ig light and heavy chains usually combine to form the antigen-binding site. Thus, if we had 1000 genes encoding light chains and 1000 genes encoding heavy chains, we could, in principle, combine their products in 1000 × 1000 different ways to make 106 different antigen-binding sites. Nonetheless, we have evolved special genetic mechanisms to enable our B cells to generate an almost unlimited number of different light and heavy chains in a remarkably economical way. We do so in two steps. First, before antigen stimulation, developing B cells join together separate gene segments in DNA to create the genes that encode the primary repertoire of low-affinity IgM and IgD proteins. Second, after antigen stimulation, the assembled Ig genes can undergo two further changes—mutations that can increase the affinity of their antigen-binding site

1	of low-affinity IgM and IgD proteins. Second, after antigen stimulation, the assembled Ig genes can undergo two further changes—mutations that can increase the affinity of their antigen-binding site and DNA rearrangements that switch the class of Ig made. Together, these changes produce the secondary repertoire of high-affinity IgG, IgE, and IgA proteins.

1	We produce our primary Ig repertoire by joining separate Ig gene segments together during B cell development. Each type of Ig chain—κ light chains, λ light chains, and heavy chains—is encoded by a separate locus on a separate chromosome. Each locus contains a large number of gene segments encoding the V region of an Ig chain, and one or more gene segments encoding the C region. During the development of a B cell in the bone marrow, a complete coding sequence for each of the two Ig chains to be synthesized is assembled by site-specific genetic recombination (discussed in Chapter 5). Once a V-region coding sequence is assembled next to a C-region sequence, it can then be co-transcribed and the resulting RNA transcript processed to produce an mRNA molecule that codes for the complete Ig polypeptide chain.

1	Each light-chain V region, for example, is encoded by a DNA sequence assembled from two gene segments—a long V gene segment and a short joining or J gene segment (Figure 24–28). Each heavy-chain V region is similarly constructed by combining gene segments, but here an additional diversity segment, or D gene segment, is also required (Figure 24–29). In addition to bringing together the separate gene segments of the Ig gene, these rearrangements also activate transcription from the gene promoter through changes in the relative positions of the cis-regulatory DNA sequences acting on the gene. Thus, a complete Ig chain can be synthesized only after the DNA has been rearranged.

1	The large number of inherited V, J, and D gene segments available for encoding Ig chains contributes substantially to Ig diversity, and the combinatorial joining of these segments (called combinatorial diversification) greatly increases this contribution. Any of the 35 or so functional V segments in our κ light-chain locus, for example, can be joined to any of the 5 J segments (see Figure 24–28), so that this locus can encode at least 175 (35 × 5) different κ-chain V regions. Similarly, any of the 40 V segments in the human heavy-chain locus can be joined to any of the 23 or so D segments and to any of the 6 J segments to encode at least 5520 (40 × 23 × 6) different heavy-chain V regions. By this mechanism alone, called V(D)J recombination, a human can produce 295 different VL regions (175 κand 120 λ) and 5520 different VH regions. In principle, these could then be combined to make over 1.5 × 106 (295 × 5520) different antigen-binding sites.

1	V(D)J recombination is mediated by an enzyme complex called V(D)J recombinase, which recognizes recombination signal sequences in the DNA that flanks each gene segment to be joined. Although the process ensures that only appropriate gene segments recombine, a variable number of nucleotides are often lost from the ends of the recombining gene segments, and one or more randomly regions of DNA to be joined chosen nucleotides are also inserted. This random loss and gain of nucleotides at joining sites is called junctional diversification, and it enormously increases the diversity of V-region coding sequences created by V(D)J recombination (up to about 108-fold), specifically in the third hypervariable region. This increased diversification comes at a price, however. In many cases, it shifts the reading frame to produce a nonfunctional gene, in which case the developing B cell fails to make a functional Ig molecule and consequently dies in the bone marrow. Once a B cell makes a functional

1	reading frame to produce a nonfunctional gene, in which case the developing B cell fails to make a functional Ig molecule and consequently dies in the bone marrow. Once a B cell makes a functional heavy chain and light chain that form an antigen-binding site, it turns off the V(D)J recombination process, thereby ensuring that the cell makes Ig of only one antigen-binding specificity.

1	B cells making BCRs that bind strongly to self antigens in the bone marrow would be dangerous. Such B cells maintain expression of an active V(D)J recombinase and are activated by such self-binding to undergo a second round of V(D)J recombination in a light-chain locus, thereby changing the specificity of its BCR—the process of receptor editing discussed earlier; self-reactive B cells that fail to change their specificity die by apoptosis, in the process of clonal deletion (see Figure 24–21). As mentioned earlier, with the passage of time following an infection or vaccination, there is usually a progressive increase in the affinity of the antibodies produced against the pathogen. This phenomenon of affinity maturation is due to

1	Figure 24–28 The V–J joining process involved in making a human κ light chain. In the “germ-line” DNA (where the Ig gene segments are not rearranged and are therefore not being expressed), the cluster of five J gene segments is separated from the c-region coding sequence by a short intron and from the 35 or so functional V gene segments by thousands of nucleotide pairs. During the development of a B cell, a randomly chosen V gene segment (V3 in this case) is moved to lie precisely next to one of the J gene segments (J3 in this case). The “extra” J gene segments (J4 and J5) and the intron sequence are transcribed (along with the joined V3 and J3 gene segments and the c-region coding sequence) and then removed by rNA splicing to generate mrNA molecules with contiguous V3, J3, and C sequences, as shown. These mrNAs are then translated into κ light chains. A J gene segment encodes the 15 or so c-terminal amino acids of the V region, and a short sequence containing the V–J segment

1	as shown. These mrNAs are then translated into κ light chains. A J gene segment encodes the 15 or so c-terminal amino acids of the V region, and a short sequence containing the V–J segment junction encodes the third hypervariable region, which is the most variable part of the light-chain V region.

1	Figure 24–29 The human heavy-chain locus. There are 40 V segments, about 23 D segments, 6 J segments, and an ordered cluster of c-region coding sequences, each cluster encoding a different class of heavy chain. The D segment (and part of the J segment) encodes amino acids in the third hypervariable region, which is the most variable part of the heavy-chain V region. The genetic mechanisms involved in producing a heavy chain are the same as those shown in Figure 24–28 for light chains, except that two DNA rearrangement steps are required instead of one: first a D segment joins to a J segment, and then a V segment joins to the rearranged DJ segment. The rearrangements lead to the production of a VDJc mrNA that encodes a complete Ig heavy chain. The figure is not drawn to scale: the total length of the heavy-chain locus is over two megabases. Moreover, a number of details are omitted: for example, the exons encoding each c-region Ig domain and the hinge region (see Figure 24–27) and the

1	of the heavy-chain locus is over two megabases. Moreover, a number of details are omitted: for example, the exons encoding each c-region Ig domain and the hinge region (see Figure 24–27) and the different subclasses of cγ-coding segments are not shown.

1	the accumulation of point mutations in both heavy-chain and light-chain V-region coding sequences. The mutations occur long after the coding regions have been assembled. After B cells have been stimulated by antigen and helper T cells in a peripheral lymphoid organ, some of the activated B cells proliferate rapidly in the lymphoid follicles and form germinal centers (see Figure 24–20). Here, the B cells mutate at the rate of about one mutation per V-region coding sequence per cell generation. Because this is about a million times greater than the spontaneous mutation rate in other genes and occurs in somatic cells rather than germ cells, the process is called somatic hypermutation.

1	Very few of the altered Igs generated by hypermutation will have an increased affinity for the antigen. But, because the same Ig genes produce both BCRs and secreted antibodies, the antigen will stimulate preferentially those few B cells that do make BCRs with increased affinity for the antigen. Clones of these altered B cells will preferentially survive and proliferate, especially as the amount of antigen decreases to very low levels late in the response. Most other B cells in the germinal center will die by apoptosis. Thus, as a result of repeated cycles of somatic hyper-mutation followed by antigen-driven proliferation of selected clones of effector and memory B cells, antibodies of increasingly higher affinity become abundant during an adaptive immune response, providing progressively better protection against the pathogen (Movie 24.6).

1	A breakthrough in understanding the molecular mechanism of somatic hyper-mutation came with the identification of an enzyme that is required for the process. It is called activation-induced deaminase (AID) because it is expressed specifically in activated B cells and deaminates cytosine (C) to uracil (U) during transcription of V-region coding DNA. The deamination produces U:G mismatches in the DNA double helix, and the repair of these mismatches produces various types of mutations, depending on the repair pathway used. Somatic hypermutation affects only actively transcribed DNA, because AID works only on single-stranded DNA (which is transiently exposed during transcription) and because proteins involved in the transcription of V-region coding sequences are required to recruit the AID enzyme. AID is also required for activated B cells to switch from IgM and IgD production to the production of the other classes of Ig, as we now discuss. B cells can Switch the class of Ig They Make

1	After a developing B cell leaves the bone marrow, before it interacts with antigen, it expresses both IgM and IgD BCRs on its surface, both with the same antigen-binding sites (see Figure 24–24). Stimulation by antigen and helper T cells activates many of these mature naïve B cells to become IgM-secreting effector cells, so that IgM antibodies dominate the primary antibody response. Later in the immune response, however, when activated B cells are undergoing somatic hypermutation, the combination of antigen and helper-T-cell-derived cytokines (discussed later) stimulates many of the B cells to switch from making membrane-bound IgM and IgD to making IgG, IgE, or IgA, in the process of class switching. Some of these cells become memory cells that express the corresponding class of Ig as BCRs on their surface, while others become effector cells that secrete the Ig molecules as antibodies. The IgG, IgE, and IgA molecules retain their original antigen-binding site and are collectively

1	BCRs on their surface, while others become effector cells that secrete the Ig molecules as antibodies. The IgG, IgE, and IgA molecules retain their original antigen-binding site and are collectively referred to as secondary classes of Igs, because they are produced only after antigen stimulation, dominate secondary antibody responses, and make up the secondary Ig repertoire.

1	As discussed earlier, the constant region of an Ig heavy chain determines the class of the Ig. Thus, the ability of B cells to switch the class of antibody they make without changing the antigen-binding site implies that the same assembled VH-region coding sequence (which specifies the antigen-binding part of the heavy chain) can sequentially associate with different CH-coding sequences. This has important functional implications. It means that, in an individual animal, a particular antigen-binding site that has been selected by environmental antigens can be distributed among the various classes of antibodies, thereby acquiring the different biological properties of each class. Cµ C° TRANSCRIPTION, RNA SPLICING,5ˆ

1	When a B cell switches from making IgM and IgD to one of the secondary classes of Ig, an irreversible change occurs in the DNA—a process called class switch recombination. It entails the deletion of all the CH-coding sequences between the assembled VDJ-coding sequence and the particular CH-coding sequence that the cell is destined to express. Class switch recombination differs from V(D)J recombination in several ways. (1) It happens after antigen stimulation, mainly in germinal centers, and depends on helper T cells. (2) It uses different recombination signal sequences, called switch sequences, which flank the different CH-coding segments. (3) It involves cutting and joining the switch sequences, which are noncoding sequences, and leaves the assembled VH-region coding sequence unchanged (Figure 24–30). (4) Most importantly, the molecular mechanism is different. It depends on AID, which is also involved in somatic hypermutation, rather than on the V(D)J recombinase. The cytokines that

1	24–30). (4) Most importantly, the molecular mechanism is different. It depends on AID, which is also involved in somatic hypermutation, rather than on the V(D)J recombinase. The cytokines that activate class switching induce the production of transcription regulators that activate transcription from the relevant switch sequences, allowing the recruitment of AID to these sites.

1	Once bound, AID initiates switch recombination by deaminating some cytosines to uracil in the vicinity of these switch sequences. Excision of these uracils is thought to lead to double-strand breaks in the participating switch regions, which are then joined by a form of nonhomologous end joining (discussed in Chapter 5). Thus, whereas the primary Ig repertoire in humans (and mice) is generated by V(D)J joining mediated by V(D)J recombinase, the secondary antibody repertoire is generated by somatic hypermutation and class switch recombination, both of which are mediated by AID. Figure 24–31 lists the main mechanisms that we have discussed in this chapter that diversify Igs.

1	Each B cell clone makes Ig molecules with a unique antigen‑binding site. Initially, the Ig molecules are inserted into the plasma membrane and serve as B cell receptors (BCRs) for antigen. Antigen binding to the BCRs, together with co‑stimulatory signals from helper T cells, activates the B cells to proliferate and differentiate into either memory cells or antibody‑secreting effector cells. The effector cells secrete large amounts of antibodies with the same antigen‑binding site as the BCRs. A typical Ig molecule is composed of four polypeptide chains—two identical heavy chains and two identical light chains. Parts of both the heavy and light chains form the two identical antigen‑binding sites. There are multiple classes of Ig (IgA, IgD, IgE, IgG, and IgM), each with a distinctive heavy chain, which determines the

1	Figure 24–30 An example of the DNA rearrangement that occurs in class switch recombination. A B cell making IgM molecules with a V region encoded by a particular assembled VDJ DNA sequence is stimulated to switch to making IgA molecules with the same V region. In the process, it deletes the DNA between the VDJ sequence and the cα-coding sequence. Specific DNA sequences (switch sequences) located upstream of each ch-coding sequence (except cδ, as B cells don't switch from cμ to cδ) can recombine with one another, with the deletion of the intervening DNA, as shown here. As discussed in the text, the recombination process depends on AID, the same enzyme that is involved in somatic hypermutation. When switching from IgM to IgG or Ige, the c-region coding sequences downstream of cγ or cδ, which remain after the DNA deletion, are removed during rNA splicing. combinatorial joining of gene segments combinatorial joining of L and H chains

1	combinatorial joining of gene segments combinatorial joining of L and H chains Figure 24–31 The main mechanisms of Ig diversification in mice and humans. Those shaded in green occur during B cell development in the bone marrow, whereas the two mechanisms shaded in red occur when B cells are stimulated by foreign antigen and helper T cells in germinal centers in peripheral lymphoid organs, either late in a primary response or in a secondary response. biological properties of the Ig class. Each light and heavy chain is composed of a number of Ig domains. The amino acid sequence variation in the variable domains of both light and heavy chains is concentrated in several small hypervariable regions, which form loops at one end of these domains to produce the antigen‑binding site.

1	Igs are encoded by loci on three different chromosomes, each of which is responsible for producing a different polypeptide chain—a κ light chain, a λ light chain, or a heavy chain. Each locus contains separate gene segments that encode different parts of the variable region of the particular Ig chain. Each light‑chain locus contains one or more constant‑ (C‑) region coding sequences and sets of variable (V) and joining (J) gene segments. The heavy‑chain locus contains sets of C‑region coding sequences and sets of V, diversity (D), and J gene segments.

1	During B cell development in the bone marrow, before antigen stimulation, separate gene segments are brought together by site‑specific recombination that depends on a V(D)J recombinase. A VL gene segment recombines with a JL gene segment to produce a DNA sequence coding for the V region of a light chain, and a VH gene segment recombines with a D and a JH gene segment to produce a DNA sequence coding for the V region of a heavy chain. Each of the newly assembled V‑region coding sequences is then co‑transcribed with the appropriate C‑region sequence to produce an RNA molecule that codes for the complete Ig polypeptide chain.

1	By randomly combining inherited gene segments that code for the variable regions during B cell development, humans can make hundreds of different light chains and thousands of different heavy chains. Because the antigen‑binding site is formed where the hypervariable loops of the VL and VH domains come together in the final Ig molecule, the heavy and light chains can potentially pair to form Igs with millions of different antigen‑binding sites. A loss or gain of nucleotides at the site of gene‑segment joining increases this number enormously. The Igs made by such V(D)J recombination before antigen stimulation are IgMs and IgDs with low affinity for binding antigen, and they constitute the primary Ig repertoire.

1	Igs are further diversified following antigen stimulation in peripheral lymphoid organs by the AID‑ and helper‑T‑cell‑dependent processes of somatic hypermutation and class switch recombination, which together produce the high‑affinity IgG, IgE, and IgA Igs that constitute the secondary Ig repertoire. The process of class switching allows the same antigen‑binding site to be incorporated into antibodies that have different tails and therefore different biological properties.

1	Like antibody responses, T-cell-mediated immune responses are exquisitely antigen-specific, and they are at least as important as antibodies in defending vertebrates against infection. Indeed, most adaptive immune responses, including most antibody responses, require helper T cells for their initiation. Most importantly, unlike B cells, T cells can help eliminate pathogens that have entered the interior of host cells, where they are invisible to B cells and antibodies. Much of the rest of this chapter is concerned with how T cells accomplish this feat.

1	T cell responses differ from B cell responses in at least two crucial ways. First, a T cell is activated by foreign antigen to proliferate and differentiate into effector cells only when the antigen is displayed on the surface of an antigen‑presenting cell (APC), usually a dendritic cell in a peripheral lymphoid organ. One reason T cells require APCs for activation is that the form of antigen they recognize is different from that recognized by the Igs produced by B cells. Whereas Igs can recognize antigenic determinants on the surface of pathogens and soluble folded proteins, for example, T cells can only recognize fragments of protein antigens that have been produced by partial proteolysis inside a host cell. As mentioned earlier, newly formed MHC proteins capture these peptide fragments and carry them to the surface of the host cell, where T cells can recognize them.

1	The second difference is that, once activated, effector T cells act mainly at short range, either within a secondary lymphoid organ or after they have migrated to a site of infection. Effector B cells, by contrast, secrete antibodies that can act far away. Effector T cells interact directly with another host cell in the body, which they either kill (if it is an infected host cell, for example) or signal in some way (if it is a B cell or macrophage, for example). We will refer to such host cells as target cells. As is the case with APCs, target cells must display an antigen bound to an MHC protein on their surface for a T cell to recognize them.

1	There are three main classes of T cells—cytotoxic T cells, helper T cells, and regulatory T cells. When activated, they function as effector cells (see Figure 24–17), each with their own distinct activities. Effector cytotoxic T cells directly kill cells that are infected with a virus or some other intracellular pathogen. Effector helper T cells help stimulate the responses of other immune cells—mainly macrophages, dendritic cells, B cells, and cytotoxic T cells; as we will see, there are a variety of functionally distinct subtypes of helper T cells. Effector regulatory T cells suppress the activity of other immune cells. In this section, we describe these classes and subclasses of T cells and their respective functions. We discuss how they recognize foreign antigens on the surface of APCs or target cells and the crucial part played by MHC proteins in the recognition process. We begin by considering the cell-surface receptors that T cells use to recognize antigen.

1	T cell receptors (TCRs), unlike Igs made by B cells, exist only in membrane-bound form. They are composed of two transmembrane, disulfide-linked polypeptide chains, each of which contains two Ig-like domains—one variable and one constant. On most T cells, the TCRs have one α chain and one β chain (Figure 24–32).

1	The genetic loci that encode the αand β chains are located on different chromosomes. Like an Ig heavy-chain locus (see Figure 24–29), the TCR loci contain separate V, D, and J gene segments (or just V and J gene segments in the case of the α-chain locus), which are brought together by site-specific recombination during T cell development in the thymus. With one exception, T cells use the same mechanisms to generate antigen-binding site diversity of their TCRs as B cells use to generate antigen-binding site diversity of their Igs, and they use the same V(D)J recombinase; thus, humans or mice deficient in this recombinase cannot make functional B or T cells. The mechanism that does not operate in TCR diversification is antigen-driven somatic hypermutation. Thus, the affinities of TCRs tend to be low (Ka ≈ 105–107 liters/mole). Various co-receptors and cell–cell adhesion proteins, however, greatly strengthen the binding of a T cell to an APC or target cell.

1	Figure 24–32 A T cell receptor (TCR) heterodimer. (A) Schematic drawing showing that the receptor is composed of an α and a β polypeptide chain. each chain has a large extracellular part that is folded into two Ig-like domains—one variable (V) and one constant (c). A Vα and a Vβ domain (shaded in blue) form the antigen-binding site. Unlike Igs, which have two binding sites for antigen, Tcrs have only one. The αβ-heterodimer is noncovalently associated with a large set of invariant membrane-bound proteins (not shown), which help activate the T cell when the Tcrs bind their specific antigen (see Figure 24–45B). A typical T cell has about 30,000 Tcrs on its surface. (B) The three-dimensional structure of the extracellular part of a Tcr. The antigen-binding site is formed by the hypervariable loops of both the Vα and Vβ domains (black), and it is similar in its overall dimensions and geometry to the antigen-binding site of an Ig molecule. (B, based on K.c. Garcia et al., Science

1	loops of both the Vα and Vβ domains (black), and it is similar in its overall dimensions and geometry to the antigen-binding site of an Ig molecule. (B, based on K.c. Garcia et al., Science 274:209–219, 1996.)

1	Instead of making α and β chains, a minority of T cells makes a different but related type of TCR heterodimer, composed of γ chains and δ chains. Although these γ/δ T cells normally make up only 5–10% of the T cells in human blood, they can be the dominant T cell population in epithelia (in the skin and gut, for example). They have some properties in common with natural killer (NK) cells and with an enlarging category of T-like cells that have features of both innate and adaptive immune cells, which are sometimes collectively referred to as innate lymphoid cells. The cells in all these categories tend to be enriched in mucosal tissues, respond early to infection, display little immunological memory, and, compared with B and T cells, have surface receptors of restricted diversity. We will not discuss them further.

1	As with BCRs, TCRs are tightly associated in the plasma membrane with a number of invariant membrane-bound proteins that are involved in passing the signal from an antigen-activated receptor to the cell interior. We will discuss these proteins in more detail later, when we consider some of the molecular events involved in T and B cell activation. First, we consider the special ways in which T cells recognize foreign antigen on the surface of an APC or target cell.

1	Generally, naïve T cells, including naïve helper and cytotoxic T cells, proliferate and differentiate into effector cells and memory cells only when they see their specific antigen on the surface of an activated dendritic cell in a peripheral lymphoid organ (Figure 24–33). The activated dendritic cell displays the antigen in a complex with MHC proteins on its surface, along with co-stimulatory proteins (see Figure 24–11). The memory T cells that develop, however, can be activated by the same antigen–MHC complex on the surface of other types of APCs (target cells), including macrophages and B cells—as well as by dendritic cells.

1	Immature dendritic cells are located in most tissues—underlying epithelial layers of the skin and gut, for example—where they are constantly sampling and processing proteins in their environment. They become activated to mature when their pattern recognition receptors (PRRs) encounter pathogen associated molecular patterns (PAMPs) on an invading pathogen or its products. The pathogen or products are ingested, and the microbial proteins are cleaved into peptide fragments, which are loaded onto MHC proteins, as we discuss later. The activated dendritic cells then migrate via the lymph from the site of infection to local lymph nodes or gut-associated lymphoid organs, where they present the foreign antigens, displayed as peptide–MHC complexes on the dendritic cell surface, for recognition by the relevant T cells (see Figure 24–11).

1	Activated dendritic cells display three types of protein molecules on their surface that have a role in activating a T cell to become an effector cell or memory cell (Figure 24–34): (1) MHC proteins, which present foreign peptides to the TCRs; (2) co‑stimulatory proteins, which bind to complementary receptors on the T cell surface; and (3) cell–cell adhesion molecules, which enable a T cell to bind to the dendritic cell for long enough to become activated, typically several hours. In addition, activated dendritic cells secrete a variety of cytokines that influence the type of effector helper T cell that develops, and different types of dendritic cells promote different outcomes (discussed later). T cells recognize Foreign peptides Bound to Mhc proteins

1	T cells recognize Foreign peptides Bound to Mhc proteins MHC proteins capture and display peptide fragments of foreign proteins for presentation to T cells. There are two main classes of MHC proteins, which are structurally and functionally distinct. Class I MHC proteins mainly present foreign peptides to cytotoxic T cells, whereas class II MHC proteins mainly present foreign peptides to helper and regulatory T cells (Figure 24–35). Some class-I-like MHC proteins present microbial lipid and glycolipid antigens to T cells, but they are not encoded within the MHC region of the genome, and we will not consider them further. Figure 24–33 Immunofluorescence micrograph of a dendritic cell in culture. These Apcs derive their name from their long processes, or “dendrites.” The cell has been labeled with a monoclonal antibody that recognizes a surface antigen on these cells. (courtesy of David Katz.)

1	Both class I and class II MHC proteins are heterodimers, in which two extracellular domains form a peptide‑binding groove, which always has a variable small peptide bound in it. In class I MHC proteins, the two domains that form the pep-tide-binding groove are provided by the transmembrane αchain, which is noncovalently associated with a small subunit called β2-microglobulin; in class II MHC proteins, a different α chain and a large noncovalently associated β chain each contribute an extracellular domain to form the peptide-binding groove (Figure 24–36). A TCR binds to both the peptide and the ridges of the binding groove. Humans have three major class I proteins, called HLA‑A, HLA‑B, and HLA‑C, and three class II proteins, called HLA‑DR, HLA‑DP, and HLA‑DQ (HLA stands for human-leukocyte-associated, as these proteins were first demonstrated on human leukocytes). Figure 24–37 shows how the genes that encode these proteins are arranged on human chromosome 6.

1	There are important differences between the class I and class II MHC proteins with regard to the cell types that express them and the origin of the peptides in their peptide-binding grooves. Almost all of our nucleated cells express class I proteins. Their peptide-binding groove displays one of a diverse collection of peptides (typically 8–10 amino acids in length). In a healthy cell, the peptides originate from the cell’s own cytosolic and nuclear proteins that have undergone partial degradation in proteasomes in the processes of normal protein turnover and quality control mechanisms. Some of the peptide fragments produced in this way are actively transported into the lumen of the endoplasmic reticulum (ER), through a specialized transporter in the ER membrane, where they are loaded onto newly synthesized class I MHC α chains; once a peptide binds, the α chain can assemble with its partner chain. The resulting self-peptide–MHC complex is then transported through the Golgi apparatus

1	newly synthesized class I MHC α chains; once a peptide binds, the α chain can assemble with its partner chain. The resulting self-peptide–MHC complex is then transported through the Golgi apparatus to the cell surface. Such complexes are not dangerous, however, because the cytotoxic T cells that could recognize fragment MHC of foreign MHC of foreign protein

1	Figure 24–34 The three general types of proteins on the surface of an activated dendritic cell involved in activating a T cell. Although only membrane-bound co-stimulatory molecules are shown, activated dendritic cells also secrete soluble co-stimulatory molecules. The invariant polypeptide chains that are always stably associated with the Tcr are not shown; they are illustrated in Figure 24–45B and Movie 24.7. Figure 24–35 Recognition by T cells of foreign peptides bound to MHC proteins. cytotoxic T cells recognize foreign peptides in association with class I Mhc proteins, whereas helper T cells and regulatory T cells recognize foreign peptides in association with class II Mhc proteins. In both cases, the T cell recognizes the peptide–Mhc complexes on the surface of an Apc—either a dendritic cell or a target cell. Some regulatory T cells recognize self peptides in association with class II Mhc proteins (not shown).

1	Figure 24–36 Class I and class II MHC proteins. (A) The α chain of the class I molecule has three extracellular domains, α1, α2, and α3, each encoded by a separate exon. The α chain is noncovalently associated with a smaller polypeptide chain, β2-microglobulin, which is not encoded within the Mhc region of the genome. The α3 domain and β2-microglobulin are Ig-like. While β2-microglobulin is invariant, the α chain is extremely polymorphic, mainly in the α1 and α2 domains. (B) In class II Mhc proteins, both the α chain and the β chain are encoded within the Mhc and are polymorphic, mainly in the α1 and β1 domains; the α2 and β2 domains are Ig-like. Thus, there are striking similarities between class I and class II Mhc proteins. In both, the two outermost domains (shaded in blue) are polymorphic and interact to form a groove that binds peptide fragments. (c) The three-dimensional structure of the peptide-binding groove of a human class I Mhc protein is viewed from above, with bound

1	and interact to form a groove that binds peptide fragments. (c) The three-dimensional structure of the peptide-binding groove of a human class I Mhc protein is viewed from above, with bound peptide shown schematically; a peptide must be bound in the groove for the Mhc protein to assemble and be transported to the cell surface. The sides of the groove are formed by two α helices, and the floor is formed by a β pleated sheet. The S–S disulfide bond is shown in red (Movie 24.8 and Movie 24.9). (c, adapted from p.J. Bjorkman et al., Nature 329:506–512, 1987. With permission from Macmillan publishers Ltd.) them have been either eliminated or inactivated, or suppressed by regulatory T cells in the process of self-tolerance. By contrast, in a cell infected by a pathogen such as a virus, the pathogen proteins will be processed in the same way, and peptides derived from them will be displayed on the infected cell surface bound to class I MHC proteins; there, they are recognized by cytotoxic T

1	proteins will be processed in the same way, and peptides derived from them will be displayed on the infected cell surface bound to class I MHC proteins; there, they are recognized by cytotoxic T cells expressing the appropriate TCRs, thereby targeting the infected cell for destruction (Figure 24–38).

1	In general, only antigen-presenting cells (APCs) express class II MHC proteins. Dendritic cells are referred to as professional APCs, as they are specialized for this function and only they can activate naïve T cells. Other immune cells that are targets of effector T cell regulation, including B cells and macrophages, are nonprofessional APCs. All APCs load their newly synthesized class II MHC proteins with peptides derived mainly from extracellular proteins that are endocytosed and delivered to endosomes. The newly synthesized class II MHC proteins initially contain an invariant chain, which occupies the peptide-binding groove

1	Figure 24–37 Human MHC genes. This simplified schematic drawing shows the location of the genes that encode the transmembrane subunits of class I (light green) and class II (dark green) Mhc proteins. The genes shown encode three types of class I Mhc proteins (hLA-A, hLA-B, and hLA-c) and three types of class II Mhc proteins (hLA-Dp, hLA-DQ, and hLA-Dr). An individual can therefore make six types of class I Mhc proteins (three encoded by maternal genes and three by paternal genes) and more than six types of class II Mhc proteins. Because of the extreme polymorphism of the Mhc genes, the chances are very low that the maternal and paternal alleles will be the same. The number of class II Mhc proteins that can be made is greater than six because there are two DR β genes and because maternally encoded and paternally encoded polypeptide chains can sometimes pair. The entire region shown spans about seven million base pairs and contains other genes that are not shown.

1	and prevents it from prematurely binding a peptide until the class II MHC protein reaches specialized vesicles, which fuse with endosomes. Here, the invariant chain is removed and peptide fragments (typically 12–20 amino acids long) produced from endocytosed proteins can bind to the groove of the class II MHC proteins, which are then transported to the plasma membrane for display on the surface of the APC. In a healthy host cell, class II MHC protein grooves are loaded with self-peptides derived from normal proteins and will be ignored by T cells because of self-tolerance mechanisms. During an infection, however, pathogen proteins are also endocytosed and processed in the same way, enabling APCs to present pathogen peptides bound to class II MHC proteins to T cells expressing an appropriate TCR (Figure 24–39).

1	The distinction just discussed between the antigen-processing pathways for loading peptides onto class I and class II MHC proteins is not absolute. Dendritic cells, for example, need to be able to activate cytotoxic T cells to kill virus-infected cells even when the virus does not infect dendritic cells themselves. To do so, specialized subsets of dendritic cells use a process called cross-presentation, which begins when these noninfected dendritic cells phagocytose virus-infected host cells or their fragments. The ingested viral proteins are then released by an unknown mechanism from phagolysosomes into the cytosol, where they are degraded in proteasomes; the resulting protein fragments are then transported into the ER lumen, where they load onto assembling class I MHC proteins. Cross-presentation in dendritic cells is not confined to endocytosed pathogens and their products: it also operates to activate cytotoxic T cells against tumor antigens of cancer cells and the MHC proteins of

1	in dendritic cells is not confined to endocytosed pathogens and their products: it also operates to activate cytotoxic T cells against tumor antigens of cancer cells and the MHC proteins of foreign organ grafts.

1	Figure 24–38 The processing of an extracellular foreign protein for presentation to cytotoxic T cells. An effector cytotoxic T cell kills a virus-infected cell when it recognizes fragments of an internal viral protein bound to class I Mhc proteins on the surface of the infected cell. Not all viruses enter the cell in the way that this enveloped rNA virus does, but fragments of internal viral proteins always follow the pathway shown. Only a small proportion of the viral proteins synthesized in the cytosol are degraded and transported to the cell surface, but this is sufficient to attract an attack by a cytotoxic T cell. Several chaperone proteins in the er lumen aid the folding and assembly of class I Mhc proteins (not shown). The assembly of class I Mhc proteins and their transport to the cell surface require the binding of either a self or foreign peptide (Movie 24.10).

1	Figure 24–39 The processing of an extracellular protein antigen for presentation to a helper T cell. This simplified depiction shows how peptide–class-II-Mhc complexes are formed in endosomes and delivered via vesicles to the cell surface. Viral envelope glycoproteins can also be processed by this pathway for presentation to helper T cells (not shown): these glycoproteins are normally made in the er and transported via the Golgi for insertion into the plasma membrane; although most of these glycoproteins will be incorporated into the envelope of budding viral particles, some will be endocytosed and enter endosomes, from where they can enter the class II Mhc processing pathway. RECOGNITION BY HELPER T CELL DELIVERY OF PEPTIDE– MHC COMPLEX TO PLASMA MEMBRANE FOR RECOGNITION BY HELPER T CELL Golgi apparatus trans Golgi class II MHC protein late endosome early endosome folded protein antigenplasma membrane DENDRITIC OR TARGET CELL LIMITED PROTEOLYSIS OF ANTIGEN AND INVARIANT CHAIN LEAVES

1	Golgi apparatus trans Golgi class II MHC protein late endosome early endosome folded protein antigenplasma membrane DENDRITIC OR TARGET CELL LIMITED PROTEOLYSIS OF ANTIGEN AND INVARIANT CHAIN LEAVES FRAGMENT OF INVARIANT CHAIN IN BINDING GROOVE OF MHC PROTEIN RELEASE OF INVARIANT CHAIN FRAGMENT AND BINDING OF ANTIGEN-DERIVED PEPTIDE INVARIANT CHAIN DIRECTS CLASS II MHC PROTEIN TO LATE ENDOSOME ENDOCYTOSIS AND DELIVERY TO ENDOSOME fragment of invariant chain CYTOSOL antigenic peptide

1	During an infection, only a small fraction of the many thousands of MHC proteins on the surface of an APC or target cell will have pathogen peptides bound to them. This is sufficient, however: fewer than 50 copies of such a peptide–MHC complex on a dendritic cell, for example, can activate a helper T cell that has a TCR that binds the complex with a high-enough affinity. Table 24–3 compares the properties of class I and class II MHC proteins. Mhc proteins Are the Most polymorphic human proteins Known

1	Mhc proteins Are the Most polymorphic human proteins Known Although any individual can make only a small number of different class I and class II MHC proteins, together, these proteins must be able to present peptide fragments from almost any foreign protein to T cells. Thus, unlike the antigen-binding site of an Ig protein, the peptide-binding groove of each MHC protein must be able to bind a very large number of different peptides. The genes encoding class I and class II MHC proteins (see Figure 24–37) are the most polymorphic known in higher vertebrates: in the human population, for example, there are more than 2000 allelic variants of these genes. The corresponding variations in the MHC proteins are concentrated in the floor and walls of the peptide-binding grooves and allow MHC molecules in different individuals to bind different arrays of peptides.

1	It is thought that infectious diseases have been an important driving force for generating this remarkable MHC polymorphism. In the evolutionary war between pathogens and the adaptive immune system, pathogens will tend to change their proteins through mutation so that the peptides derived from them will not fit in the MHC peptide-binding grooves. When a pathogen succeeds, it can sweep through a population as an epidemic. In such circumstances, the few individuals who produce a new allelic form of MHC protein that can bind peptides derived from the altered pathogen will have a large selective advantage. This type of selection will tend to promote and maintain a large diversity of MHC proteins in the population. In West Africa, for example, individuals with a specific MHC allele (HLA-B53) have a reduced susceptibility to a severe form of malaria that is endemic there; although this allele is rare elsewhere, it is found in 25% of the West African population.

1	The extensive diversity of human MHC proteins is the main reason that individuals who receive a foreign organ transplant must be treated with strong immunosuppressive drugs to prevent the immunological rejection of the grafted organ. Of all the foreign proteins that the graft expresses, the MHC proteins are by far the most powerful stimulators of the recipient’s T cells, which would rapidly destroy the graft if they were not prevented from doing so by such drugs. Foreign MHC proteins are powerful T cell stimulants because T cells respond to them in the same way they respond to self MHC proteins that have foreign peptides bound to them; for this reason, the proportion of a person’s T cells that can specifically recognize any foreign MHC protein is relatively high. cD4 and cD8 co-receptors on T cells Bind to Invariant parts of Mhc proteins

1	cD4 and cD8 co-receptors on T cells Bind to Invariant parts of Mhc proteins The affinity of TCRs for peptide–MHC complexes on an APC is usually too low by itself to mediate a functional interaction between the two cells. T cells normally require accessory receptors to help stabilize the interaction by increasing the overall strength of the cell–cell adhesion. Unlike TCRs or MHC proteins, the accessory receptors are invariant and do not bind to foreign peptides. Once bound to the surface of a dendritic cell, for example, a T cell increases the strength of the binding by activating an integrin adhesion protein (discussed in Chapter 19), which then binds more strongly to an Ig-like protein on the surface of the dendritic cell. This increased adhesion enables the T cell to remain bound long enough to become activated.

1	When an accessory receptor has a direct role in activating the T cell by generating its own intracellular signals, it is called a co-receptor. The most important and best understood of the co-receptors on T cells are the CD4 and CD8 proteins, both of which are single-pass transmembrane proteins with extracellular Ig-like domains. Like TCRs, they recognize MHC proteins, but, unlike TCRs, they bind to invariant parts of the MHC protein, far away from the peptide-binding groove. CD4 is expressed on both helper T cells and regulatory T cells and binds to class II MHC proteins, whereas CD8 is expressed on cytotoxic T cells and binds to class I MHC proteins (Figure 24–40).

1	CD4 and CD8 contribute to T cell recognition by helping the T cell to focus on particular MHC proteins, and thereby on particular types of target cells. Thus, the recognition of class I MHC proteins by CD8 allows cytotoxic T cells to focus on any type of infected host cell, while the recognition of class II MHC proteins by CD4 allows helper and regulatory T cells to focus on the target immune cells that they help or suppress, respectively. The cytoplasmic tail of the CD4 and CD8 proteins is associated with a member of the Src family of cytoplasmic tyrosine kinases

1	Figure 24–40 CD4 and CD8 co-receptors on the surface of T cells. cytotoxic T cells (Tc) express cD8, which recognizes class I Mhc proteins, whereas helper T cells (Th) and regulatory T cells (not shown) express cD4, which recognizes class II Mhc proteins. Note that the co-receptors bind to the same Mhc protein that the Tcr has engaged, so that they are brought together with Tcrs during the antigen-recognition process. Whereas the Tcr binds to the variable (polymorphic) parts of the Mhc protein that form the peptide-binding groove, the co-receptor binds to the invariant part, well away from the binding groove. (discussed in Chapter 15) called Lck, which phosphorylates various intracellular proteins on tyrosines and thereby participates in the activation of the T cell (discussed later).

1	(discussed in Chapter 15) called Lck, which phosphorylates various intracellular proteins on tyrosines and thereby participates in the activation of the T cell (discussed later). The AIDS virus (HIV) uses CD4 molecules (as well as chemokine receptors) to enter helper T cells (see Figure 23–17). AIDS patients are susceptible to infection by microbes that are not normally dangerous because HIV depletes helper T cells. As a result, most AIDS patients die of infection within several years of the onset of symptoms, unless they are treated with a combination of anti-HIV drugs. HIV also uses CD4 and chemokine receptors to enter macrophages, which also have both types of receptors on their surface.

1	T cell development begins when bone-marrow-derived lymphoid progenitor cells enter the thymus from the bloodstream. There, the cells receive a variety of signals from thymus stromal cells, epithelial cells, macrophages, and dendritic cells, which promote their stepwise development into mature thymocytes. At one step, the progenitor cells are induced to express V(D)J recombinase and begin to rearrange their TCR gene segments. Soon thereafter, the cells express both CD4 and CD8 co-receptors, and these so-called double‑positive thymocytes migrate inward and interact with thymus dendritic cells or epithelial cells expressing self peptides bound to class I and class II MHC proteins. If the TCR on the thymocyte binds with high affinity to these complexes, a strong signal will be transmitted, causing the cell to undergo apoptosis. This process, called negative selection, is an example of clonal deletion (see Figure 24–21), and it eliminates thymocytes that could potentially attack normal

1	causing the cell to undergo apoptosis. This process, called negative selection, is an example of clonal deletion (see Figure 24–21), and it eliminates thymocytes that could potentially attack normal host cells and tissues and thereby cause an autoimmune disease if the cells were to continue to mature and leave the thymus.

1	If its TCR is unable to bind at all to a self-peptide–MHC complex in the thymus, the thymocyte will fail to receive the signals it needs to survive and will die of “neglect;” without the ability to recognize self-MHC proteins, a T cell would generally be of no use, as T cells can only see pathogen-derived peptides in the context of self-MHC proteins. Thymocytes that express a TCR that binds with an appropriate affinity to a self peptide bound to either a class I MHC protein (using CD8 as a co-receptor) or a class II MHC protein (using CD4 as a co-receptor) will receive an optimal signal to survive and continue to mature, a process called positive selection (Figure 24–41). As part of this maturation process, and depending on the TCR’s preference for class I or class II MHC proteins, the CD4 or CD8 co-receptor that is not needed is silenced by DNA methylation of the respective gene; this results in the development of CD4 or CD8 single‑positive thymocytes, which exit the thymus as naïve

1	or CD8 co-receptor that is not needed is silenced by DNA methylation of the respective gene; this results in the development of CD4 or CD8 single‑positive thymocytes, which exit the thymus as naïve T cells and enter the recirculating pool of T cells—the CD4 cells as either helper or regulatory T cells and the CD8 cells as cytotoxic T cells.

1	Although naïve helper and cytotoxic T cells constantly receive survival signals in the form of self peptides bound to MHC proteins that the T cells bind weakly, a T cell is only activated to proliferate and mount an immune response if its TCR binds with high affinity to a peptide–MHC complex and receives co-stimulatory signals at the same time. Generally, this happens only when the T cell encounters an activated dendritic cell (in a peripheral lymphoid organ) that expresses an MHC protein with a foreign peptide derived from a pathogen in its binding groove. Only then will the naïve T cell proliferate and differentiate into an effector or memory T cell.

1	Negative selection in the thymus is a major mechanism for ensuring that peripheral T cells do not react with host cells expressing MHC proteins with peptides derived from self proteins in their peptide-binding grooves. This mechanism, however, requires that the APCs in the thymus display an array of peptides on their MHC molecules that will reflect the self proteins in peripheral tissues, as well as in the thymus. The thymus, however, would not be expected to produce many of the proteins that are specifically expressed in other organs. As an example, it would not be expected to produce insulin, and yet it is crucial to delete thymocytes with TCRs that could recognize insulin-derived peptides bound to MHC

1	DEATH BY DEFAULT NEGATIVE SELECTION (signaled death) DEATH BY DEFAULT apoptotic cell TH POSITIVE SELECTION (signaled survival) SURVIVAL, MATURATION, and EMIGRATION TH TC TC Treg Treg proteins on the surface of insulin-secreting β cells in the pancreas. Any failure to do so would result in the T-cell-dependent destruction of the β cells and, as a consequence, cause type 1 (or juvenile) diabetes.

1	The mechanism that enables the deletion of all such cells in the thymus depends on a subpopulation of epithelial cells in the thymus that express a transcriptional regulator called AIRE (autoimmune regulator). By a poorly understood mechanism, the AIRE protein promotes the production of small amounts of mRNA from many genes that encode such “organ-specific” proteins, including the insulin gene. When the peptides derived from the proteins encoded by these genes are bound by MHC proteins and displayed on the surface of the epithelial cells in the thymus medulla, this is sufficient to provoke the deletion of the potentially self-reactive thymocytes. Mutations that inactivate the AIRE gene cause a severe multiorgan autoimmune disease in both mice and humans, indicating the importance of AIRE in self-tolerance. cytotoxic T cells Induce Infected Target cells to Kill Themselves

1	Cytotoxic T cells (TC cells), like the NK cells discussed earlier, protect us against intracellular pathogens, including viruses, bacteria, and parasites, that multiply in the cytoplasm of a host cell. TC cells kill infected host cells before the pathogen can escape to infect neighboring host cells. Before it can kill, however, a naïve TC cell has to become an effector cell by activation on an APC, usually an activated dendritic cell that has pathogen-derived peptides bound to class I MHC proteins—a process that depends on helper T cells. The effector TC cell can then recognize any target cell harboring the same pathogen and expressing some of the same peptide–MHC complexes on its surface: its TCRs cluster, along with CD8 co-receptors, adhesion molecules, and intracellular signaling proteins (discussed later), at the interface between the two cells, forming an immunological synapse. In this process, the effector TC cell reorganizes its cytoskeleton to focus its killing apparatus on

1	(discussed later), at the interface between the two cells, forming an immunological synapse. In this process, the effector TC cell reorganizes its cytoskeleton to focus its killing apparatus on the target cell, secreting its toxic proteins into a confined space (Figure 24–42); in this way, it avoids killing neighboring cells. A similar synapse forms when an effector helper T cell interacts with its target cell, except that the co-receptor is CD4 (Movie 24.11).

1	Figure 24–41 Positive and negative selection in the thymus. Developing thymocytes with Tcrs that would potentially enable them to respond to peptides in association with self Mhc proteins after they leave the thymus are positively selected: the binding of their Tcrs to self peptides bound to self Mhc proteins in the thymus signals such cells to survive, mature, and migrate to peripheral lymphoid organs. All of the other thymocytes undergo apoptosis—either because they do not express Tcrs that recognize self Mhc proteins with self peptides bound or because they recognize such complexes too well and undergo negative selection. The regulatory T cells (Treg cells) that are positively selected in the thymus are called natural Treg cells to distinguish them from induced Treg cells, which develop in peripheral lymphoid organs from naive helper T cells (Th cells), as we discuss shortly.

1	Figure 24–42 effector cytotoxic T cells killing target cells in culture. (A) electron micrograph showing an effector cytotoxic T cell (Tc cell) binding to a target cell. The Tc cells were obtained from mice immunized with the target cells, which are foreign tumor cells. (B) electron micrograph showing a Tc cell and a tumor cell that the Tc cell has killed. In an animal, as opposed to a culture dish, the killed target cell would be phagocytosed by neighboring cells (especially macrophages) long before it disintegrated in the way that it has here. (c) Immunofluorescence micrograph of a Tc cell and tumor cell after immunofluorescence staining with anti-tubulin antibodies. Note that the centrosome in the Tc cell is located at the point of cell–cell contact with the target cell—an immunological synapse. The secretory granules (not visible) in the Tc cell are initially transported along microtubules to the centrosome, which then moves to the synapse, delivering the granules to where they

1	synapse. The secretory granules (not visible) in the Tc cell are initially transported along microtubules to the centrosome, which then moves to the synapse, delivering the granules to where they can release their contents. (A and B, from D. Zagury et al., Eur. J. Immunol. 5:818–822, 1975. With permission from John Wiley & Sons, Inc; c, from B. Geiger, D. rosen and G. Berke, J. Cell Biol. 95:137–143, 1982. With permission from the authors.)

1	An effector TC cell (or an NK cell) can employ one of two strategies to kill the target, both of which operate by inducing the target cell to activate caspases and kill itself by undergoing apoptosis. One mechanism uses a protein called Fas ligand on the killer-cell surface, which binds to a transmembrane receptor protein called Fas on the target cell; this mechanism is discussed in Chapter 18 (see Figure 18–5). The other mechanism is the main one used by both NK cells and TC cells to kill an infected target cell. The killer cell stores various toxic proteins within secretory vesicles in its cytoplasm that it releases into the synaptic space by exocytosis. The toxic proteins include perforin and proteases called granzymes. The perforin is homologous to complement component C9 and polymerizes in Figure 24–43 The main way that an the target-cell plasma membrane (see Figure 24–8), forming a transmembrane effector TC cell (or NK cell) kills an pore that disrupts the membrane and allows

1	in Figure 24–43 The main way that an the target-cell plasma membrane (see Figure 24–8), forming a transmembrane effector TC cell (or NK cell) kills an pore that disrupts the membrane and allows the granzymes to enter the target infected target cell. This simplified drawing shows how the killer cell releases cell. Once in the cytosol, the granzymes help activate caspases, thereby inducing perforin and granzymes onto the surface apoptosis (Figure 24–43).

1	of an infected target cell by localized exocytosis at an immunological synapse. The high concentration of ca2+ in the extracellular fluid causes the perforin to assemble into transmembrane channels in the target-cell plasma membrane, allowing the granzymes to enter the target-cell cytosol. The granzymes cleave and activate procaspases to initiate a caspase cascade, leading to apoptosis (see Figure 18–3). A single cytotoxic cell can kill multiple target cells in sequence. It remains a mystery why the released perforins do not form pores in the plasma membrane of the killer cell itself target cell (Movie 24.12 and Movie 24.13). effector helper T cells help Activate Other cells of the Innate and Adaptive Immune Systems

1	In contrast to TC cells, helper T cells (TH cells) are crucial for defense against both extracellular and intracellular pathogens, and they express CD4 rather than CD8 co-receptors and recognize foreign peptides bound to class II rather than class I MHC proteins. Once naïve TH cells are induced on activated dendritic cells to become effector cells, they can help activate other cells: they help activate B cells to become antibody-secreting cells and later to undergo Ig class switching and somatic hypermutation; they help activate macrophages to destroy any intracellular pathogens multiplying within the macrophage’s phagosomes; they help induce naïve TC cells to become effector cells that can kill infected target cells; and they stimulate the activated dendritic cell that activated them to maintain the dendritic cell in an activated state. In each case, the effector TH cell recognizes the same complex of foreign peptide and class II MHC protein on the target-cell surface that it

1	to maintain the dendritic cell in an activated state. In each case, the effector TH cell recognizes the same complex of foreign peptide and class II MHC protein on the target-cell surface that it initially recognized on the activated dendritic cell. As discussed later, the TH cell stimulates the target cell both by secreting a variety of cytokines and by displaying co-stimulatory proteins on its surface.

1	Naïve helper T cells can Differentiate Into Different Types of effector T cells When activated by binding to a foreign peptide bound to a class II MHC protein on an activated dendritic cell, a naïve TH cell can differentiate into several distinct types of effector T cells, depending on the nature of the pathogen and the cytokines they encounter. These cells include four subtypes of helper cells—TH1, TH2, TFH, and TH17 cells—and regulatory (suppressor) T cells. Figure 24–44 summarizes both the cytokines that induce these effector T cells and some of the cytokines the effector cells secrete, as well as the master transcription regulators that control the effector cell’s development. Naïve TH cells activated by dendritic cells secreting the cytokine interleukin-12 (IL12) develop into TH1 cells. These effector cells produce interferon‑γ (IFNγ),

1	Naïve TH cells activated by dendritic cells secreting the cytokine interleukin-12 (IL12) develop into TH1 cells. These effector cells produce interferon‑γ (IFNγ), Figure 24–44 Differentiation of naïve helper T cells into different types of effector helper cells or regulatory T cells in a peripheral lymphoid organ. The cytokines produced by the activating dendritic cell (and by other cells in the environment) mainly determine which type of effector T cell develops, as indicated. Some of the main cytokines produced by each type of effector cell are also shown, and the master transcription regulator for each subset is indicated in the nucleus. There is increasing evidence that some of the effector cells are plastic and can change the cytokines they produce in response to changes in their environment (not shown).

1	which is critical for the activation of macrophages to destroy pathogens that either invaded the macrophage or were ingested by it; the IFNγ can also induce B cells to switch the class of Ig they are making. Naïve TH cells activated in the presence of IL4 develop into TH2 cells. These effector cells are important for the control of extracellular pathogens, including parasites. They stimulate B cells to undergo somatic hypermutation and to switch the class of Ig they produce: for example, the TH2 cells themselves produce IL4, which can induce B cells to switch from making IgM and IgD to making IgE antibodies, which can bind to mast cells, as discussed earlier. Naïve TH cells activated in the presence of IL6 and IL21 develop into follicular helper T cells (TFH), which are located in lymphoid follicles and secrete a variety of cytokines, including IL4 and IL21; these cells are especially important for stimulating B cells to undergo Ig class switching and somatic hyper-mutation. Naïve TH

1	follicles and secrete a variety of cytokines, including IL4 and IL21; these cells are especially important for stimulating B cells to undergo Ig class switching and somatic hyper-mutation. Naïve TH cells activated in the presence of IL6 and TGFβ develop into TH17 cells. These effector cells secrete IL17, which recruits neutrophils and stimulates epithelial cells and fibroblasts in the skin and gut to produce pro-inflammatory cytokines. TH17 cells are important in controlling extracellular bacterial and fungal infections and in wound healing, but they can also have a major role in autoimmune diseases and allergy.

1	In some cases, naïve TH cells that encounter their antigen in a peripheral lymphoid organ in the presence of TGFβ and the absence of IL6 develop into induced regulatory T cells (Treg cells), which suppress rather than help immune cells; as mentioned earlier, natural Treg cells develop in the thymus during thymocyte development (see Figure 24–41). In either case, the Treg cells suppress the development, activation, or function of most other types of immune cells, by means of both secreted suppressive cytokines such as IL10 and TGFβ and inhibitory proteins on the Treg cell surface. Induced Treg cells seem mainly to suppress immune responses to foreign antigens—preventing responses to harmless ingested or inhaled antigens and limiting responses against pathogens to avoid excessive responses that cause unwanted pathology; natural Treg cells are needed to prevent immune responses to self molecules (see Figure 24–21). Treg cells express the transcription regulator FoxP3, which serves as

1	that cause unwanted pathology; natural Treg cells are needed to prevent immune responses to self molecules (see Figure 24–21). Treg cells express the transcription regulator FoxP3, which serves as both a marker of these cells and a master controller of their development: if the gene encoding this protein is inactivated in mice or humans, the individuals fail to produce Treg cells and develop a fatal autoimmune disease involving multiple organs—findings that establish the crucial importance of Treg cells in self-tolerance.

1	Foreign antigen binding to BCRs or TCRs initiates the process whereby the T and B cells are stimulated to proliferate and differentiate into effector or memory cells. As mentioned earlier, these antigen receptors do not act on their own: they are stably associated with invariant transmembrane polypeptide chains that are required to relay the signal into the cell. In B cells, these are called Igα and Igβ (Figure 24–45A), while in T cells they exist in a complex called CD3, composed of four types of polypeptide chains (Figure 24–45B). In both cases, the associated proteins help convert extracellular antigen binding to the TCR or BCR into intracellular signals, and they do so in similar ways.

1	Antigen binding to BCRs or TCRs clusters these receptors and their associated invariant chains (and CD4 or CD8 co-receptors in the case of TCRs). This clustering activates a Src family cytoplasmic tyrosine kinase to phosphorylate tyro-sines on the cytoplasmic tails of some of the invariant chains. The phosphotyrosines then serve as docking sites for a second cytoplasmic tyrosine kinase, which becomes phosphorylated and activated by the first kinase; the second kinase then relays the signal downstream by phosphorylating other intracellular signaling proteins on tyrosines. Some of these early events in the signaling pathway activated by BCRs are shown in Figure 24–46.

1	Signaling through BCRs or TCRs and their associated proteins alone is not sufficient to activate a lymphocyte to proliferate and differentiate. Extracellular co-stimulatory signals produced by another cell are also required, and they are provided by membrane-bound proteins (see Figure 24–34) and secreted cytokines. Indeed, signaling through the BCR or TCR with insufficient co-stimulation can either eliminate the lymphocyte (clonal deletion) or inactivate it, with both of these mechanisms contributing to self-tolerance (see Figure 24–21). For a naïve T cell, an activated dendritic cell provides the co-stimulatory signals; these include the transmembrane B7 proteins, which are recognized by the co-receptor protein CD28 on the surface of the T cell (Figure 24–47A). For a B cell, an effector TH cell provides the co-stimulatory signals; these include the transmembrane CD40 ligand, which binds to CD40 receptors on the B cell (Figure 24–47B). The CD40 ligand on effector TH cells acts in two

1	TH cell provides the co-stimulatory signals; these include the transmembrane CD40 ligand, which binds to CD40 receptors on the B cell (Figure 24–47B). The CD40 ligand on effector TH cells acts in two other situations: (1) it acts back on CD40 receptors on the dendritic cell surface to increase and sustain the activation of the dendritic cell, creating a positive feedback loop; and (2) it acts as a co-stimulatory signal on the surface of an effector TH1 cell, allowing the T cell to help activate an infected macrophage to destroy the pathogens it harbors.

1	In addition to receptors for co-stimulatory proteins, both B and T cells have inhibitory proteins on their surface that help regulate the cell’s activity, preventing excessive or inappropriate responses. Two such proteins expressed by T cells have attracted great attention because of their roles in suppressing the ability of T cells to inhibit cancer progression: CTLA4 and PD1 proteins inhibit T cell activity in different ways, and monoclonal antibodies against either or especially both can relieve the inhibition and allow T cells to dramatically destroy the tumors in some patients with metastatic cancer (see Figure 20–45). Figure 24–45 The invariant chains associated with bCRs and TCRs.

1	Figure 24–45 The invariant chains associated with bCRs and TCRs. (A) each Bcr is associated with two invariant heterodimers, each composed of an Igα and an Igβ polypeptide chain linked by a sulfide bond (red). (B) each Tcr is associated with an invariant cD3 complex composed of two disulfide-bonded ζ chains, two ε chains, and one δ and one γ chain; these chains form homodimers or heterodimers, as shown.

1	Figure 24–46 early signaling events in a b cell activated by the binding of specific foreign antigen to its bCRs. If the antigen is on the surface of a pathogen or is a soluble macromolecule with two or more identical antigenic determinants (as shown), it cross-links adjacent Bcrs, causing them and their associated invariant chains to cluster, as shown. A Src-like cytoplasmic tyrosine kinase (which can be Fyn or Lyn) is associated with the cytosolic tail of Igβ; it joins the cluster and phosphorylates both the Igα and Igβ invariant chains (for simplicity, only the phosphorylation on Igβ is shown). A transmembrane protein tyrosine phosphatase called CD45 is also required to remove inactivating phosphates from these Src-like kinases (not shown). The resulting phosphotyrosines on Igα and Igβ serve as docking sites for another Src-like tyrosine kinase called Syk, which becomes phosphorylated and thereby activated to relay the signal downstream.

1	The pathway from Tcrs is similar (including a requirement for cD45), except that the first Src-like kinase is Lck, which is associated with a cD4 or cD8 co-receptor and phosphorylates tyrosines on all the cD3 polypeptide chains shown in Figure 24–45B; the second Src-like kinase is ZAP70, which is homologous to the Syk kinase in B cells (Movie 24.14). Many cell-Surface proteins Belong to the Ig Superfamily

1	Many cell-Surface proteins Belong to the Ig Superfamily Most of the proteins that mediate antigen recognition and cell–cell recognition in the immune system contain one or more Ig or Ig-like domains, suggesting that the proteins have a common evolutionary history. Included in this very large Ig superfamily are antibodies, TCRs, MHC proteins, the CD4, CD8, and CD28 co-receptors, the B7 co-stimulatory proteins, and most of the invariant polypeptide chains associated with TCRs and BCRs, as well as the various Fc receptors on lymphocytes and other leukocytes. Many of these proteins are dimers or higher oligomers, in which Ig or Ig-like domains of one chain interact with those in another (Figure 24–48).

1	Figure 24–47 Comparison of the co-stimulatory proteins required to activate a helper T cell and a b cell in response to the same foreign protein. (A) A naïve helper T cell is activated by a peptide fragment of a foreign protein bound to a class II Mhc protein on the surface of an activated dendritic cell. The co-stimulatory protein on the dendritic cell (a B7 protein— either cD80 or cD86) binds to the cD28 co-receptor on the T cell, providing a necessary co-stimulatory signal to the T cell; in addition, cytokines secreted by the dendritic cell (or other nearby cells) influence what subtype of effector helper cell the T cell becomes (see Figure 24–44). (B) Once activated to become an effector cell, the helper T cell can help activate B cells that have the same peptide–Mhc protein complexes on their surface as the dendritic cell that activated the T cell. These B cells have Bcrs that bind an antigenic determinant on the surface of a folded foreign protein and endocytose the protein

1	on their surface as the dendritic cell that activated the T cell. These B cells have Bcrs that bind an antigenic determinant on the surface of a folded foreign protein and endocytose the protein (red arrow); the protein is then cleaved into peptides, which are carried to the B cell surface by class II Mhc proteins, where some of them can be recognized by the Tcrs on the helper T cell (see Figure 24–39). Note that the Bcrs and Tcrs recognize different antigenic determinants of the protein. As indicated, the co-stimulatory protein used by the effector helper T cell is cD40 ligand, which binds to the cD40 co-receptor on the B cell; the T cell also secretes cytokines such as IL4 to help stimulate the B cell to undergo somatic hypermutation and class switching (not shown). The cD4 co-receptor on Th cells is omitted in both (A) and (B) for simplicity.

1	Figure 24–48 Some of the cell-surface proteins discussed in this chapter that belong to the Ig superfamily. The Ig and Ig-like domains are shaded in gray, except for the antigen-binding domains (not all of which are Ig domains—the class I and class II Mhc proteins are the exception), which are shaded in blue. The Ig superfamily also includes many cell-surface proteins involved in cell–cell interactions outside the immune system, such as the neural cell adhesion molecule (N-cAM) discussed in chapter 19 and the receptors for various protein growth factors discussed in chapter 15 (not shown). There are more than 750 members of the Ig superfamily in humans.

1	In both vertebrates and invertebrates, many proteins in the Ig superfamily are also found outside immune systems, where they often function in cell–cell recognition and adhesion processes, both during development and in adult tissues. It seems likely that the entire gene superfamily evolved from a primordial gene coding for a single Ig-like domain, similar to that encoding β2-microglobulin (see Figure 24–36). In present-day family members, a separate exon usually encodes the amino acids in each Ig-like domain, consistent with the likelihood that new family members arose during evolution by exon and gene duplications.

1	There are three main functionally distinct classes of T cells. Cytotoxic T cells (TC cells) directly kill infected cells by secreting perforins and granzymes that induce the infected cells to undergo apoptosis. Helper T cells (TH cells) help activate cytotoxic T cells to kill their target cells, B cells to make antibody responses, macrophages to destroy the microorganisms they harbor, and dendritic cells to activate T cells. Regulatory T cells (Treg cells) produce suppressive proteins (such as the cytokines IL10 and TGFβ) to inhibit other immune cells.

1	All T cells express cell‑surface antigen receptors (TCRs), which are encoded by genes that are assembled from multiple gene segments during T cell development in the thymus. TCRs recognize peptide fragments of foreign proteins that are displayed in association with MHC proteins on the surface of antigen‑presenting cells (APCs) and target cells. Naïve T cells are activated in peripheral lymphoid organs by activated dendritic cells, which secrete cytokines and express peptide–MHC complexes, co‑stimulatory proteins, and various cell–cell adhesion molecules on their cell surface.

1	Class I MHC proteins present foreign peptides to TC cells, whereas class II MHC proteins present foreign peptides to TH cells and Treg cells. Whereas class I MHC proteins are expressed on almost all nucleated vertebrate cells, class II MHC proteins are normally restricted to APCs, including dendritic cells, macrophages, and B lymphocytes. Both classes of MHC proteins have a single peptide‑binding groove, which binds a large set of small peptide fragments produced intracellularly by normal protein‑degradation processes: class I MHC proteins mainly bind fragments produced in the cytosol, whereas class II MHC proteins mainly bind fragments produced in endocytic compartments. The peptide–MHC complexes are transported to the cell surface, where complexes that contain a peptide derived from a foreign protein are recognized by TCRs, which interact with both the peptide and the walls of the peptide‑binding groove. T cells also express CD4 or CD8 co‑receptors, which recognize invariant regions

1	protein are recognized by TCRs, which interact with both the peptide and the walls of the peptide‑binding groove. T cells also express CD4 or CD8 co‑receptors, which recognize invariant regions of MHC proteins: TH cells and Treg cells express CD4, which recognizes class II MHC proteins; TC cells express CD8, which recognizes class I MHC proteins.

1	A combination of positive and negative selection operates during T cell development in the thymus to help ensure that only T cells with potentially useful TCRs survive, mature, and emigrate, while all of the others die by apoptosis. The naïve TH and TC cells that leave the thymus constantly receive survival signals when their TCRs recognize self‑peptide–MHC complexes, but they can only be activated when their TCRs encounter foreign peptides in the grooves of MHC proteins on an activated dendritic cell. The natural Treg cells that leave the thymus suppress self‑reactive lymphocytes to help maintain self‑tolerance.

1	The production of an effector T cell from a naïve T cell requires multiple signals from an activated dendritic cell. MHC–peptide complexes on the dendritic cell surface provide one signal, by binding to both TCRs and a CD4 co‑receptor on a TH or Treg cell. Co‑stimulatory proteins on the dendritic cell surface and secreted cytokines are the other signals. When naïve TH cells are initially activated on a dendritic cell, they differentiate into TH1, TH2, TFH, or TH17 effector helper cells or into induced Treg cells, depending mainly on the cytokines in their environment. TH1 cells secrete interferon‑γ (IFNγ) to activate macrophages and to induce B cells to switch the class of Ig they make; TH2 and TFH cells secrete other cytokines that also induce B cells to switch Ig class; and TH17 cells secrete IL17 to promote inflammatory responses What initiates an autoimmune disease such as type 1 diabetes or multiple sclerosis?

1	What initiates an autoimmune disease such as type 1 diabetes or multiple sclerosis? When a naïve or memory T or B cell is activated by antigen and co-stimulatory signals, how does it decide whether to become an effector cell or memory cell? Are there cells that are pre-committed to becoming either effector or memory cells, for example, or is the decision determined solely by extracellular signals? Why do some of us make Ige antibodies against harmless antigens and thereby develop hay fever and allergic asthma, while most of us do not, and why is the proportion of such allergic individuals increasing? how does a cytotoxic T cell (or NK cell) avoid being killed by the perforin and granzymes that it secretes to kill a target cell?

1	how does a cytotoxic T cell (or NK cell) avoid being killed by the perforin and granzymes that it secretes to kill a target cell? and wound healing. The effector helper TH cells recognize the same complex of foreign peptide and class II MHC protein on the target‑cell surface as they initially recognized on the dendritic cell that activated them. They activate their target cells by producing a combination of membrane‑bound and secreted co‑stimulatory proteins. Treg cells suppress immune cells using cell‑surface and secreted inhibitory proteins. Both T cells and B cells require multiple signals for activation. Antigen binding to the TCRs or BCRs provides one signal, while co‑stimulatory proteins binding to co‑receptors and cytokines binding to their complementary receptors provide the others. Effector TH cells provide the co‑stimulatory signals for B cells, whereas APCs provide them for T cells. Which statements are true? explain why or why not.

1	Which statements are true? explain why or why not. 24–1 T cells whose receptors strongly bind a self-peptide–MHC complex are killed off in peripheral lymphoid organs when they encounter the self peptide on an antigen-presenting dendritic cell. 24–2 To guarantee that the antigen-presenting cells in the thymus will display a complete repertoire of self peptides to allow elimination of self-reactive T cells, the thymus recruits dendritic cells from all over the body. 24–3 The antibody diversity created by the combinatorial joining of V, D, and J segments by V(D)J recombination pales in comparison to the enormous diversity created by the random gain and loss of nucleotides at V, D, and J joining sites. Discuss the following problems. 24–4 Why do living trees not rot? Redwood trees, for example, can live for centuries, but once they die they decay fairly quickly. What might this suggest?

1	Discuss the following problems. 24–4 Why do living trees not rot? Redwood trees, for example, can live for centuries, but once they die they decay fairly quickly. What might this suggest? 24–5 It would be disastrous if a complement attack were not confined to the surface of the pathogen that is the target of the attack. Yet, the proteolytic cascade involved in the attack liberates biologically active molecules at several steps: one that diffuses away and one that remains bound to the target surface. How does the complement reaction remain localized when active products leave the surface?

1	[Hint: imagine what would happen if the G:U mismatch created by AID was replicated several times; how would the sequences of the final mutations relate to the original G-C base pair?] 24–7 For many years it was a complete mystery how cytotoxic T cells could see a viral protein that seemed to be present only in the nucleus of the virus-infected cell. The answer was revealed in a classic paper that took advantage of a clone of T cells whose T cell receptor was directed against an antigen assoicated with the nuclear protein of the 1968 strain of influenza virus. The authors of the paper found that when they incubated high concentrations of certain peptides derived from the viral nuclear protein, the cells became sensitive to lysis by subsequent incubation with the cytotoxic T cells. Using various peptides from the 1968 strain and the 1934 strain (with which the cytotoxic T cells did not react), the authors defined the particular peptide responsible for the T cell response (Figure Q24–1).

1	A. Which part of the viral protein gives rise to the peptide that is recognized by the clone of cytotoxic T cells? 24–6 Based on its sequence similarity to Apobec1, which deaminates Cs to Us in RNA, activation-induced deaminase (AID) was originally proposed to work on RNA. But definitive experiments in E. coli demonstrated that AID deaminates Cs to Us in DNA. The authors of the paper expressed AID in bacteria and followed mutations in a selectable gene. They found that AID expression increased mutations about fivefold above the background level in the absence of AID expression. More importantly, they found that 80% of the induced mutations were G→A or C→T. Does this fit with your expectation if AID-induced mutations arose by deamination of C to U in the DNA?

1	Figure Q24–1 Viral nuclear protein recognition by cytotoxic T cells (problem 24–7). (A) Sequences of a segment of the nuclear protein from the 1968 and 1934 strains of influenza virus. peptides used in the experiments in (B) are highlighted by pink bars. The amino acid differences between the viral proteins are highlighted in blue. (B) cytotoxic T-cell-mediated lysis of target cells. The target cells were untreated (none), infected with virus (1968 or 1934 strain), or preincubated with high concentrations of the indicated viral peptide. Why do not all viral peptides sensitize the target cells for lysis by the cytotoxic T cells? b. It is thought the MHC molecules come to the cell surface with peptides already bound. If that is so, how do you imagine that these experiments worked?

1	b. It is thought the MHC molecules come to the cell surface with peptides already bound. If that is so, how do you imagine that these experiments worked? 24–8 Working out the rules by which T cells interact with their target cells was complicated. Some of the key observations came from studying the way cytotoxic T cells killed cells infected with choriomeningitis virus (LCMV). (A) Figure Q24–3 Scanning of the T cell repertoire by dendritic cells (problem 24–9). (A) contacts between different T cells and one dendritic cell. T cells are green and dendritic cells are red. The dendritic cell labeled with of images. Times are shown At first glance, it would seem a dangerous strategy ing the same k-type MHC protein, but they did not lyse as hours: minutes. (B) plot of T cell contacts for individual dendritic cells over time. (A, from p Bousso and proteins (Figure Q24–2). Similarly, cytotoxic T cells from e. robey, Nat. Immunol.

1	(A, from p Bousso and proteins (Figure Q24–2). Similarly, cytotoxic T cells from e. robey, Nat. Immunol. 4:579–581, 2003. With permission from Macmillan d-type mice lysed infected d-type cells, but not infected minutes publishers Ltd.) k-type cells. LCMV can kill both k-type and d-type mice. cytotoxic fbroblasts T cells added to Figure Q24–2 pattern of killing of LcMV-infected fibroblasts by cytotoxic T cells from an LcMV-infected k-type mouse (problem 24–8). A. If homozygous d-type mice were bred to homozygous k-type mice to generate d-type/k-type heterozygous progeny, would you expect that cytotoxic T cells from these heterozygotes, when infected with LCMV, to be able to lyse infected d-type cells? How about infected k-type cells? Explain your answers.

1	b. Oddly enough, LCMV infection does not kill mice that lack a thymus—such as “nude” mice, so called because they also lack hair. If a thymus is transplanted back into a nude mouse, it will die when infected with LCMV. Suppose that a d-type/k-type heterozygous nude mouse was given a thymus from an d-type donor. Would you expect its cytotoxic T cells to be able to lyse infected d-type cells? How about infected k-type cells? Explain your answers.

1	24–9 Before exposure to a foreign antigen, T cells with receptors specific for the antigen are a tiny fraction of the T cells—on the order of 1 in 105 or 1 in 106 T cells. After exposure to the antigen, only a small number of dendritic cells typically display the antigen on their surface. How long does it take for such antigen-presenting dendritic cells to interact with the antigen-specific T cells, which is the key first step in T cell activation and clonal expansion? The dynamics of the search process were examined by labeling dendritic cells red and T cells green, so that contacts in an for the thymus to actively promote the survival, matura tion, and emigration of developing T cells that bind weakly to self peptides bound to self MHC molecules. Would it not be safer to get rid of these T cells, along with those that bind strongly to such self-peptide–MHC complexes, as this would seem a more secure way to avoid autoimmune reactions?

1	24–11 CD4 proteins on helper and regulatory T cells serve as co-receptors that bind to invariant parts of class II MHC proteins. CD4 is thought to increase the adhesion between T cells and antigen-presenting cells (APCs) that are initially connected only weakly by the T cell receptor bound to its specific peptide–MHC complex. To test this possibility, you label cell-surface MHC molecules with a fluorescently labeled peptide so that you can detect individual peptide–MHC complexes at the interface between the APCs and the T cells in a culture dish. To detect T cell responses—the sign of a productive contact—you load them with a Ca2+ indicator dye, as cytosolic Ca2+ increases when lymphocytes are active. You now count the peptide– MHC complexes at a large number of interfaces (immunological synapses) and measure the resulting uptake of Ca2+ in the adherent T cells (Figure Q24–4, red circles). When you repeat the experiment in the presence of blocking antibodies against CD4, you get a

1	synapses) and measure the resulting uptake of Ca2+ in the adherent T cells (Figure Q24–4, red circles). When you repeat the experiment in the presence of blocking antibodies against CD4, you get a different result (blue circles). Do these results support or refute the notion that CD4 augments T cell receptor binding? Explain your answer.

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1	Zhu J, Yamane H & Paul We (2010) Peripheral cD4+ T-cell differentiation regulated by networks of cytokines and transcription factors. Annu. Rev. Immunol. 28, 445–489. ABC transporters A large family of membrane transport proteins that use the energy of ATP hydrolysis to transfer peptides or small molecules across membranes. (Figure 11–16) acetyl CoA Small water-soluble activated carrier molecule. Consists of an acetyl group linked to coenzyme A (CoA) by an easily hydrolyzable thioester bond. (Figure 2–38) acetylcholine receptor (AChR) Membrane protein that responds to binding of acetylcholine (ACh). The nicotinic AChR is a transmitter-gated ion channel that opens in response to ACh. The muscarinic AChR is not an ion channel, but a G-protein-coupled cell-surface receptor.

1	acid A proton donor. Substance that releases protons (H+) when dissolved in water, forming hydronium ions (H3O+) and lowering the pH. (Panel 2–2, pp. 92–93) acid hydrolases Hydrolytic enzymes—including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases, and sulfatases—that work best at acidic pH; these enzymes are found within the lysosome.

1	action potential Rapid, transient, self-propagating electrical excitation in the plasma membrane of a cell such as a neuron or muscle cell. Action potentials, or nerve impulses, make possible long-distance signaling in the nervous system. (Figure 11–31) activated carrier Small diffusible molecule that stores easily exchangeable energy in the form of one or more energy-rich covalent bonds. Examples are ATP, acetyl CoA, FADH2, NADH, and NADPH. (Figure 2–31) activation energy The extra energy that must be acquired by atoms or molecules in addition to their ground-state energy in order to reach the transition state required for them to undergo a particular chemical reaction. (Figure 2–21) activation-induced deaminase (AID) The enzyme catalyzing the processes of somatic hypermutation and immunoglobulin class switching in activated B cells.

1	active site Region of an enzyme surface to which a substrate molecule binds in order to undergo a catalyzed reaction. (Figure 1–7) active transport Movement of a molecule across a membrane or other barrier driven by energy other than that stored in the electrochemical or concentration gradient of the transported molecule. adaptation (1) Adaptation (desensitization): adjustment of sensitivity following repeated stimulation. The mechanism that allows a cell to react to small changes in stimuli even against a high background level of stimulation. (2) Evolutionary adaptation: an evolved trait.

1	adaptive immune system System of lymphocytes providing highly specific and long-lasting defense against pathogens in vertebrates. It consists of two major classes of lymphocytes: B lymphocytes (B cells), which secrete antibodies that bind specifically to the pathogen or its products, and T lymphocytes (T cells), which can either directly kill cells infected with the pathogen or produce secreted or cell-surface signal proteins that stimulate other host cells to help eliminate the pathogen. (Figure 24–2) adaptor protein, adaptor General term for a protein that functions solely to link two or more different proteins together in an intracellular signaling pathway or protein complex. (Figure 15–11) adenylyl cyclase (adenylate cyclase) Membrane-bound enzyme that catalyzes the formation of cyclic AMP from ATP. An important component of some intracellular signaling pathways.

1	adherens junction Cell junction in which the cytoplasmic face of the plasma membrane is attached to actin filaments. Examples include adhesion belts linking adjacent epithelial cells and focal contacts on the lower surface of cultured fibroblasts. adhesins Specific proteins or protein complexes of pathogenic bacteria that recognize and bind cell-surface molecules on the host cells to enable tight adhesion and colonization of tissues. adhesion belt Adherens junctions in epithelia that form a continuous belt (zonula adherens) just beneath the apical face of the epithelium, encircling each of the interacting cells in the sheet.

1	adhesion belt Adherens junctions in epithelia that form a continuous belt (zonula adherens) just beneath the apical face of the epithelium, encircling each of the interacting cells in the sheet. ADP (adenosine 5′-diphosphate) Nucleotide produced by hydrolysis of the terminal phosphate of ATP. Regenerates ATP when phosphorylated by an energy-generating process such as oxidative phosphorylation. (Figure 2–33) aerobic respiration Process by which a cell obtains energy from sugars or other organic molecules by allowing their carbon and hydrogen atoms to combine with the oxygen in air to produce CO2 and H2O, respectively. affinity maturation Progressive increase in the affinity of antibodies for the immunizing antigen with the passage of time after immunization. Agrin Signal protein released by an axonal growth cone during formation of the synapse between it and a muscle cell.

1	Agrin Signal protein released by an axonal growth cone during formation of the synapse between it and a muscle cell. AIRE (autoimmune regulator) A protein expressed by a subpopulation of epithelial cells in the thymus that stimulates the production of small amounts of self proteins characteristic of other organs, exposing developing thymocytes to these proteins for the purpose of self-tolerance. Akt Serine/threonine protein kinase that acts in the PI-3kinase/Akt intracellular signaling pathway involved especially in signaling cells to grow and survive. Also called protein kinase B (PKB). allele One of several alternative forms of a gene. In a diploid cell, each gene will typically have two alleles, occupying the corresponding position (locus) on homologous chromosomes.

1	allele One of several alternative forms of a gene. In a diploid cell, each gene will typically have two alleles, occupying the corresponding position (locus) on homologous chromosomes. allosteric protein A protein that can adopt at least two distinct conformations, and for which the binding of a ligand at one site causes a conformational change that alters the activity of the protein at a second site; this allows one type of molecule in a cell to alter the fate of a molecule of another type, a feature widely exploited in enzyme regulation.

1	allostery (adjective allosteric) Change in a protein’s conformation brought about by the binding of a regulatory ligand (at a site other than the protein’s catalytic site), or by covalent modification. The change in conformation alters the activity of the protein and can form the basis of directed movement. (Figures 3–57 and 16–29) alpha helix (α helix) Common folding pattern in proteins, in which a linear sequence of amino acids folds into a right-handed helix stabilized by internal hydrogen-bonding between backbone atoms. (Figure 3–7) alternative RNA splicing Production of different RNAs from the same gene by splicing the transcript in different ways. (Figure 7–57) amino acid Organic molecule containing both an amino group and a carboxyl group. Those that serve as building blocks of proteins are alpha amino acids, having both the amino and carboxyl groups linked to the same carbon atom. (NH2CHRCOOH, Panel 3–1, pp. 112–113) aminoacyl-tRNA synthetase Enzyme that attaches the correct

1	are alpha amino acids, having both the amino and carboxyl groups linked to the same carbon atom. (NH2CHRCOOH, Panel 3–1, pp. 112–113) aminoacyl-tRNA synthetase Enzyme that attaches the correct amino acid to a tRNA molecule to form an aminoacyltRNA. (Figure 6–54)

1	AMPA receptor Glutamate-gated ion channel in the mammalian central nervous system that carries most of the depolarizing current responsible for excitatory postsynaptic potentials. amphiphilic Having both hydrophobic and hydrophilic regions, as in a phospholipid or a detergent molecule. amyloid fibrils Self-propagating, stable β-sheet aggregates built from hundreds of identical polypeptide chains that become layered one over the other to create a continuous stack of β sheets. The unbranched fibrous structure can contribute to human diseases when not controlled. anaphase (1) Stage of mitosis during which sister chromatids separate and move away from each other. (2) Anaphase I and II: stages of meiosis during which chromosome homolog pairs separate (I), and then sister chromatids separate (II). (Panel 17–1, pp. 980–981) anaphase A Stage of mitosis during which chromosome segregation occurs as chromosomes move toward the two spindle poles.

1	anaphase B Stage of mitosis during which chromosome segregation occurs as spindle poles separate and move apart. anaphase-promoting complex (APC/C; cyclosome) Ubiquitin ligase that catalyzes the ubiquitylation and destruction of securin and Mand S-cyclins, initiating the separation of sister chromatids in the metaphase-to-anaphase transition during mitosis. anchorage dependence Dependence of cell growth, proliferation, and survival on attachment to a substratum. anchoring junction Cell junction that attaches cells to neighboring cells or to the extracellular matrix. (Table 19–1, p. 1037) angiogenesis Growth of new blood vessels by sprouting from existing ones. antenna complex Part of a photosystem that captures light energy and channels it into the photochemical reaction center. It consists of protein complexes that bind large numbers of chlorophyll molecules and other pigments.

1	Antennapedia complex One of two gene clusters in Drosophila that contain Hox genes; genes in the Antennapedia complex control the differences among the thoracic and head segments of the body. anti-apoptotic Bcl2 family proteins Proteins (e.g., Bcl2, BclXL) on the cytosolic surface of the outer mitochondrial membrane that bind and inhibit pro-apoptotic Bcl2 family proteins and thereby help prevent inappropriate activation of the intrinsic pathway of apoptosis. anti-IAP Produced in response to various apoptotic stimuli and, by binding to IAPs and preventing their binding to a caspase, neutralize the inhibition of apoptosis provided by IAPs. antibiotic Substance such as penicillin or streptomycin that is toxic to microorganisms. Often a natural product of a particular microorganism or plant.

1	antibiotic Substance such as penicillin or streptomycin that is toxic to microorganisms. Often a natural product of a particular microorganism or plant. antibody Protein secreted by activated B cells in response to a pathogen or foreign molecule. Binds tightly to the pathogen or foreign molecule, inactivating it or marking it for destruction by phagocytosis or complement-induced lysis. (Figure 24–23) antibody response Adaptive immune response in which B cells are activated to secrete antibodies that circulate in the bloodstream or enter other body fluids, where they can bind specifically to the foreign antigen that stimulated their production. anticodon Sequence of three nucleotides in a transfer RNA (tRNA) molecule that is complementary to a three-nucleotide codon in a messenger RNA (mRNA) molecule. antigen A molecule that can induce an adaptive immune response or that can bind to an antibody or T cell receptor.

1	antigen A molecule that can induce an adaptive immune response or that can bind to an antibody or T cell receptor. antigen-presenting cell Cell that displays foreign antigen complexed with an MHC protein on its surface for presentation to T lymphocytes. antigenic determinant Specific region of an antigen that binds to an antibody or a complementary receptor on the surface of a B cell (BCR) or T cell (TCR). antigenic variation Ability to change the antigens displayed on the cell surface; a property of some pathogenic microorganisms that enables them to evade attack by the adaptive immune system. antiparallel Describes the relative orientation of the two strands in a DNA double helix or two paired regions of a polypeptide chain; the polarity of one strand is opposite to that of the other. antiporter Carrier protein that transports two different ions or small molecules across a membrane in opposite directions, either simultaneously or in sequence. (Figure 11–8)

1	antiporter Carrier protein that transports two different ions or small molecules across a membrane in opposite directions, either simultaneously or in sequence. (Figure 11–8) Apaf1 Adaptor protein of the intrinsic apoptotic pathway; on binding cytochrome c, oligomerizes to form an apoptosome. apical Referring to the tip of a cell, a structure, or an organ. The apical surface of an epithelial cell is the exposed free surface, opposite to the basal surface. The basal surface rests on the basal lamina that separates the epithelium from other tissue. apoptosis Form of programmed cell death, in which a “suicide” program is activated within an animal cell, leading to rapid cell death mediated by intracellular proteolytic enzymes called caspases. apoptosome Heptamer of Apaf1 proteins that forms on activation of the intrinsic apoptotic pathway; it recruits and activates initiator caspases that subsequently activate downstream executioner caspases to induce apoptosis.

1	aquaporin (water channel) Channel protein embedded in the plasma membrane that greatly increases the cell’s permeability to water, allowing transport of water, but not ions, at a high rate across the membrane. archaeon (plural arch[a]ea) (archaebacterium) Single-celled organism without a nucleus, superficially similar to bacteria. At a molecular level, more closely related to eukaryotes in genetic machinery than are bacteria. Archaea and bacteria together make up the prokaryotes. (Figure 1–17) ARF proteins Monomeric GTPase in the Ras superfamily responsible for regulating both COPI coat assembly and clathrin coat assembly. (Table 15–5, p. 854) ARP (actin-related protein) complex (Arp 2/3 complex) Complex of proteins that nucleates actin filament growth from the minus end.

1	ARP (actin-related protein) complex (Arp 2/3 complex) Complex of proteins that nucleates actin filament growth from the minus end. arrestin Member of a family of proteins that contributes to GPCR desensitization by preventing the activated receptor from interacting with G proteins and serving as an adaptor to couple the receptor to clathrin-dependent endocytosis. (Figure 15–42) astral microtubule In the mitotic spindle, any of the microtubules radiating from the aster which are not attached to a kinetochore of a chromosome. asymmetric cell division Cell division in which some important molecule or molecules are distributed unequally between the two daughter cells, causing these cells to become different from each other. ATM (ataxia telangiectasia mutated protein) Protein kinase activated by double-strand DNA breaks. If breaks are not repaired, ATM initiates a signal cascade that culminates in cell cycle arrest. Related to ATR.

1	ATP (adenosine 5′-triphosphate) Nucleoside triphosphate composed of adenine, ribose, and three phosphate groups. The principal carrier of chemical energy in cells. The terminal phosphate groups are highly reactive in the sense that their hydrolysis, or transfer to another molecule, takes place with the release of a large amount of free energy. (Figure 2–33) ATP synthase (F1Fo ATPase) Transmembrane enzyme complex in the inner membrane of mitochondria and the thylakoid membrane of chloroplasts. Catalyzes the formation of ATP from ADP and inorganic phosphate during oxidative phosphorylation and photosynthesis, respectively. Also present in the plasma membrane of bacteria. ATR (ataxia telangiectasia and Rad3 related protein) Protein kinase activated by DNA damage. If damage remains unrepaired, ATR helps initiate a signal cascade that culminates in cell cycle arrest. Related to ATM.

1	autoimmune disease Pathological state in which the body mounts a disabling adaptive immune response against one or more of its own molecules. autophagosome Organelle surrounded by a double membrane contains engulfed cytoplasmic cargo in the initial stages of autophagy. autophagy Digestion of cytoplasm and worn-out organelles by the cell’s own lysosomes. auxin Plant hormone, commonly indole-3-acetic acid, with numerous roles in plant growth and development. axon Long nerve cell projection that can rapidly conduct nerve impulses over long distances so as to deliver signals to other cells. axoneme Bundle of microtubules and associated proteins that forms the core of a cilium or a flagellum in eukaryotic cells and is responsible for their movements. bacterial artificial chromosome (BAC) Cloning vector that can accommodate large pieces of DNA, typically up to 1 million base pairs.

1	bacterial artificial chromosome (BAC) Cloning vector that can accommodate large pieces of DNA, typically up to 1 million base pairs. bacteriorhodopsin Pigmented protein found in the plasma membrane of a salt-loving archaeon, Halobacterium salinarium (Halobacterium halobium). Pumps protons out of the cell in response to light. bacterium (plural bacteria) (eubacterium) Member of the domain bacteria, one of the three main branches of the tree of life (archaea, bacteria, and eukaryotes). Bacteria and archaea both lack a distinct nuclear compartment, and together comprise the prokaryotes. (Figure 1–17) Bak A main effector Bcl2 family protein of the intrinsic pathway of apoptosis in mammalian cells that is bound to the mitochondrial outer membrane even in the absence of an apoptotic signal; activation is usually by activated pro-apoptotic BH3-only proteins. basal Situated near the base. Opposite the apical surface.

1	basal Situated near the base. Opposite the apical surface. basal lamina (plural basal laminae) Thin mat of extracellular matrix that separates epithelial sheets, and many other types of cells such as muscle or fat cells, from connective tissue. Sometimes called basement membrane. (Figure 19–51) base (1) A substance that can reduce the number of protons in solution, either by accepting H+ ions directly, or by releasing OH– ions, which then combine with H+ to form H2O. (2) The purines and pyrimidines in DNA and RNA are organic nitrogenous bases and are often referred to simply as bases. (Panel 2–2, pp. 92–93) base excision repair DNA repair pathway in which single faulty bases are removed from the DNA helix and replaced. Compare nucleotide excision repair. (Figure 5–41) base pair Two nucleotides in an RNA or DNA molecule that are held together by hydrogen bonds—for example, G paired with C, and A paired with T or U.

1	basement membrane Thin mat of extracellular matrix that separates epithelial sheets, and many other types of cells such as muscle or fat cells, from connective tissue. Also called basal lamina. (Figure 19–51) Bax A main effector Bcl2 family protein of the intrinsic pathway of apoptosis in mammalian cells; located mainly in the cytosol and translocates to the mitochondria only after activation, usually by activated pro-apoptotic BH3-only proteins. B cell receptor (BCR) The transmembrane immunoglobulin protein on the surface of a B cell that serves as its receptor for antigen. Bcl2 Anti-apoptotic Bcl2 family protein of the outer mitochondrial membrane that binds and inhibits pro-apoptotic Bcl2 family proteins and prevents inappropriate activation of the intrinsic pathway of apoptosis.

1	Bcl2 family Family of intracellular proteins that either promote or inhibit apoptosis by regulating the release of cytochrome c and other mitochondrial proteins from the intermembrane space into the cytosol. BclXL Anti-apoptotic Bcl2 family protein of the outer mitochondrial membrane that binds and inhibits pro-apoptotic Bcl2 family proteins and prevents inappropriate activation of the intrinsic pathway of apoptosis. benign Of tumors: self-limiting in growth, and noninvasive.

1	benign Of tumors: self-limiting in growth, and noninvasive. beta sheet (β sheet) Common structural motif in proteins in which different sections of the polypeptide chain run alongside each other, joined together by hydrogen-bonding between atoms of the polypeptide backbone. Also known as a β pleated sheet. (Figure 3–7) beta-catenin (β-catenin) Multifunctional cytoplasmic protein involved in cadherin-mediated cell–cell adhesion, linking cadherins to the actin cytoskeleton. Can also act independently as a transcription regulatory protein. Has an important role in animal development as part of a Wnt signaling pathway. BH3-only proteins The largest subclass of Bcl2 family proteins. Produced or activated in response to an apoptotic stimulus and promote apoptosis mainly by inhibiting antiapoptotic Bcl2 family proteins.

1	bi-orientation The attachment of sister chromatids to opposite poles of the mitotic spindle, so that they move to opposite ends of the cell when they separate in anaphase. binding site Region on the surface of one molecule (usually a protein or nucleic acid) that can interact with another molecule through noncovalent bonding. BiP Endoplasmic reticulum (ER)-resident chaperone protein member of the family of hsp70-type chaperone proteins. Bithorax complex One of two gene clusters in Drosophila that contain Hox genes; genes in the Bithorax complex control the differences among the abdominal and thoracic segments of the body. bivalent A four-chromatid structure formed during meiosis, consisting of a duplicated chromosome tightly paired with its homologous duplicated chromosome. blastomere One of the many cells formed by the cleavage of a fertilized egg.

1	blastomere One of the many cells formed by the cleavage of a fertilized egg. blastula Early stage of an animal embryo, usually consisting of a hollow ball of epithelial cells surrounding a fluid-filled cavity, before gastrulation begins. blebbing Membrane protrusion formed when the plasma membrane detaches locally from the underlying actin cortex, allowing cytoplasmic flow and hydrostatic pressure within the cell to push the membrane outward. bone Dense and rigid connective tissue comprising a mixture of tough fibers (type I collagen fibrils), which resist pulling forces, and solid particles (calcium phosphate as hydroxylapatite crystals), which resist compression. brassinosteroids Class of steroid signal molecules in plants that regulate the growth and differentiation of plants throughout their life cycle via binding to a cell-surface receptor kinase to initiate a signaling cascade.

1	bright-field microscope Normal light microscope in which the image is obtained by simple transmission of light through the object being viewed. buffer Solution of weak acid or weak base that resists the pH change that would otherwise occur when small quantities of acid or base are added. C3 The pivotal complement protein that is activated by the early components of all three complement pathways (the classical pathway, the lectin pathway, and the alternative pathway). (Figure 24–7) Ca2+ pump (calcium pump, Ca2+ ATPase) Transport protein in the membrane of sarcoplasmic reticulum of muscle cells (and elsewhere). Pumps Ca2+ out of the cytoplasm into the sarcoplasmic reticulum using the energy of ATP hydrolysis.

1	Ca2+-activated K+ channel Opens in response to the raised concentration of Ca2+ in nerve cells that occurs in response to an action potential. Increased K+ permeability makes the membrane harder to depolarize, increasing the delay between action potentials and decreasing the response of the cell to constant, prolonged stimulation (adaptation). Ca2+/calmodulin-dependent kinase (CaM-kinase) Serine/ threonine protein kinase that is activated by Ca2+/calmodulin. Indirectly mediates the effects of an increase in cytosolic Ca2+ by phosphorylating specific target proteins. (Figure 15–33) cadherin Member of the large cadherin superfamily of transmembrane adhesion proteins. Mediates homophilic Ca2+-dependent cell–cell adhesion in animal tissues. (Figure 19–3 and Table 19–1, p. 1037) cadherin superfamily Family of classical and nonclassical cadherin proteins with more than 180 members in humans.

1	calmodulin Ubiquitous intracellular Ca2+-binding protein that undergoes a large conformation change when it binds Ca2+, allowing it to regulate the activity of many target proteins. In its activated (Ca2+-bound) form, it is called Ca2+/calmodulin. (Figure 15–33) calnexin Carbohydrate-binding chaperone protein in the endoplasmic reticulum (ER) membrane that binds to oligosaccharides on incompletely folded proteins and retains them in the ER. calreticulin Carbohydrate-binding chaperone protein in the endoplasmic reticulum (ER) lumen that binds to oligosaccharides on incompletely folded proteins and retains them in the ER.

1	calreticulin Carbohydrate-binding chaperone protein in the endoplasmic reticulum (ER) lumen that binds to oligosaccharides on incompletely folded proteins and retains them in the ER. CaM-kinase II Multifunctional Ca2+/calmodulin-dependent protein kinase that phosphorylates itself and various target proteins when activated. Found in most animal cells but is especially abundant at synapses in the brain, and is involved in some forms of synaptic plasticity in vertebrates. (Figure 15–34) cancer stem cells Rare cancer cells capable of dividing indefinitely. cancer-critical genes Genes whose alteration contributes to the causation or evolution of cancer by driving tumorigenesis.

1	cancer-critical genes Genes whose alteration contributes to the causation or evolution of cancer by driving tumorigenesis. capsid Protein coat of a virus, formed by the self-assembly of one or more types of protein subunit into a geometrically regular structure. (Figure 3–27) carbohydrate layer The carbohydrate-rich zone on the eukaryotic cell surface attributable to glycoproteins, glycolipids, and proteoglycans of the plasma membrane. carbon-fixation reaction Process by which inorganic carbon (as atmospheric CO2) is incorporated into organic molecules. The second stage of photosynthesis. (Figure 14–40) carcinogenesis The generation of cancer. carcinoma Cancer of epithelial cells. The most common form of human cancer. cargo The membrane components and soluble molecules carried by transport vesicles. cartilage Form of connective tissue composed of cells (chondrocytes) embedded in a matrix rich in type II collagen and chondroitin sulfate proteoglycan.

1	cartilage Form of connective tissue composed of cells (chondrocytes) embedded in a matrix rich in type II collagen and chondroitin sulfate proteoglycan. caspase Intracellular protease that is involved in mediating the intracellular events of apoptosis. catalyst Substance that can lower the activation energy of a reaction (thus increasing its rate), without itself being consumed by the reaction. caveola (plural caveolae) Invaginations at the cell surface that bud off internally to form pinocytic vesicles. Thought to form from lipid rafts, regions of membrane rich in certain lipids. caveolins Family of unusual integral membrane proteins that are the major structural proteins in caveolae, CD4 Co-receptor protein on helper T cells and regulatory T cells that binds to a nonvariable part of class II MHC proteins (on antigen-presenting cells) outside the peptide-binding groove. (Figure 24–40)

1	CD8 Co-receptor protein on cytotoxic T cells that binds to a nonvariable part of class I MHC proteins (on antigen-presenting cells and infected target cells) outside the peptide-binding groove. (Figure 24–40) Cdc20 Activating subunit of the anaphase-promoting complex (APC/C). Cdc25 Protein phosphatase that dephosphorylates Cdks and increases their activity. Cdc42 Member of the Rho family of monomeric GTPases that regulate the actin and microtubule cytoskeletons, cell-cycle progression, gene transcription, and membrane transport. Cdc6 Protein essential in the preparation of DNA for replication. With Cdt1 it binds to an origin recognition complex on chromosomal DNA and helps load the Mcm proteins onto the complex to form the prereplicative complex. Cdh1 Activating subunit of the anaphase-promoting complex (APC/C). Cdk inhibitor protein (CKI) Protein that binds to and inhibits cyclin–Cdk complexes, primarily involved in the control of G1 and S phases.

1	Cdk inhibitor protein (CKI) Protein that binds to and inhibits cyclin–Cdk complexes, primarily involved in the control of G1 and S phases. Cdk-activating kinase (CAK) Protein kinase that phosphorylates Cdks in cyclin–Cdk complexes, activating the Cdk. cDNA clone Clone containing double-stranded cDNA molecules derived from the protein-coding mRNA molecules present in a cell. cDNA library Collection of cloned DNA molecules representing complementary DNA copies of the mRNA produced by a cell. Cdt1 Protein essential in the preparation of DNA for replication. With Cdc6 it binds to origin recognition complexes on chromosomes and helps load the Mcm proteins on to the complex, forming the prereplicative complex. cell cortex Specialized layer of cytoplasm on the inner face of the plasma membrane. In animal cells it is an actin-rich layer responsible for movements of the cell surface.

1	cell cortex Specialized layer of cytoplasm on the inner face of the plasma membrane. In animal cells it is an actin-rich layer responsible for movements of the cell surface. cell cycle (cell-division cycle) Reproductive cycle of a cell: the orderly sequence of events by which a cell duplicates its chromosomes and, usually, the other cell contents, and divides into two. (Figure 17–4) cell determination Process whereby a cell progressively loses the potential to form other cell types, as development proceeds. cell doctrine The proposal that all living organisms are composed of one or more cells and that all cells arise from the division of other living cells. cell memory Retention by cells and their descendants of persistently altered patterns of gene expression, without any change in DNA sequence. See also epigenetic inheritance.

1	cell memory Retention by cells and their descendants of persistently altered patterns of gene expression, without any change in DNA sequence. See also epigenetic inheritance. cell plate Flattened membrane-bounded structure that forms by fusing vesicles in the cytoplasm of a dividing plant cell and is the precursor of the new cell wall. cell-cycle control system Network of regulatory proteins that governs progression of a eukaryotic cell through the cell cycle. cellulose Long, unbranched chains of glucose; major constituent of plant cell walls. cellulose microfibril Highly ordered crystalline aggregate formed from bundles of about 40 cellulose chains, arranged with the same polarity and stuck together in overlapping parallel arrays by hydrogen bonds between adjacent cellulose molecules.

1	central (primary) lymphoid organ Organ in which T or B lymphocytes are produced from precursor cells. In adult mammals, these are the thymus and bone marrow, respectively. (Figure 24–12) centriole Short cylindrical array of microtubules, closely similar in structure to a basal body. A pair of centrioles is usually found at the center of a centrosome in animal cells. (Figure 16–48) centromere Constricted region of a mitotic chromosome that holds sister chromatids together. This is also the site on the DNA where the kinetochore forms so as to capture microtubules from the mitotic spindle. (Figure 4–43) centrosome Centrally located organelle of animal cells that is the primary microtubule-organizing center (MTOC) and acts as the spindle pole during mitosis. In most animal cells it contains a pair of centrioles. (Figures 16–47 and 17–24) cerebral cortex Outermost layer of the hemispheres of the brain; the most complex structure in the human body.

1	CG island Region of DNA in vertebrate genomes with a greater than average density of CG sequences; these regions generally remain unmethylated. channel (membrane channel) Transmembrane protein complex that allows inorganic ions or other small molecules to diffuse passively across the lipid bilayer. (Figure 11–3) channelrhodopsin Photosensitive protein forming a cation channel across the membrane that opens in response to light. charge separation In photosynthesis, the light-induced transfer of a high-energy electron from chlorophyll to an acceptor molecule resulting in the formation of a positive charge on the chlorophyll and a negative charge on a mobile electron carrier. chemical biology Name given to a strategy that uses large-scale screening of hundreds of thousands of small molecules in biological assays to identify chemicals that affect a particular biological process and that can then be used to study it.

1	chemical carcinogens Disparate chemicals that are carcinogenic—due to the ability to cause mutations—when fed to experimental animals or painted repeatedly on their skin. chemical group Certain combinations of atoms—such as methyl (–CH3), hydroxyl (–OH), carboxyl (–COOH), carbonyl (–C=O), phosphate (–PO32–), sulfhydryl (–SH), and amino (–NH2) groups—that have distinct chemical and physical properties and influence the behavior of the molecule in which the group occurs. chemiosmotic coupling (chemiosmosis) Mechanism in which an electrochemical proton gradient across a membrane (composed of a pH gradient plus a membrane potential) is used to drive an energy-requiring process, such as ATP production or the rotation of bacterial flagella. chemotaxis Movement of a cell toward or away from some diffusible chemical.

1	chemotaxis Movement of a cell toward or away from some diffusible chemical. chiasma (plural chiasmata) X-shaped connection visible between paired homologous chromosomes during meiosis. Represents a site of chromosomal crossing-over, a form of genetic recombination. chlorophyll Light-absorbing green pigment that plays a central part in photosynthesis in bacteria, plants, and algae. chloroplast Organelle in green algae and plants that contains chlorophyll and carries out photosynthesis. cholera toxin Secreted toxic protein of Vibrio cholerae responsible for causing the watery diarrhea associated with cholera. Comprises an A subunit with enzymatic activity and a B subunit that binds to host-cell receptors to direct subunit A to the host-cell cytosol.

1	cholesterol An abundant lipid molecule with a characteristic four-ring steroid structure. An important component of the plasma membranes of animal cells. (Figure 10–4) chromatin Complex of DNA, histones, and non-histone proteins found in the nucleus of a eukaryotic cell. The material of which chromosomes are made. chromatin immunoprecipitation Technique by which chromosomal DNA bound by a particular protein can be isolated and identified by precipitating it by means of an antibody against the protein. (Figures 8–66 and 8–67) chromosome Structure composed of a very long DNA molecule and associated proteins that carries part (or all) of the hereditary information of an organism. Especially evident in plant and animal cells undergoing mitosis or meiosis, during which each chromosome becomes condensed into a compact rodlike structure visible in the light microscope.

1	cilium (plural cilia) Hairlike extension of a eukaryotic cell containing a core bundle of microtubules. Many cells contain a single nonmotile cilium, while others contain large numbers that perform repeated beating movements. Compare flagellum. circadian clock Internal cyclical process that produces a particular change in a cell or organism with a period of around 24 hours, for example the sleep-wakefulness cycle in humans. cis face Face on the same or near side. cis Golgi network (CGN) Network of fused vesicular tubular clusters that is closely associated with the cis face of the Golgi apparatus and is the compartment at which proteins and lipids enter the Golgi.

1	cis-regulatory sequences DNA sequences to which transcription regulators bind to control the rate of gene transcription. In nearly all cases, these sequences must be on the same chromosome (that is, in cis) to the genes they control. (Figure 7–18) cisternal maturation model One hypothesis for how the Golgi apparatus achieves and maintains its polarized structure and how molecules move from one cisterna to another. This model views the cisternae as dynamic structures that mature from early to late by acquiring and then losing specific Golgiresident proteins as they move through the Golgi stack with cargo.

1	citric acid cycle [tricarboxylic acid (TCA) cycle, Krebs cycle] Central metabolic pathway found in aerobic organisms. Oxidizes acetyl groups derived from food molecules, generating the activated carriers NADH and FADH2, some GTP, and waste CO2. In eukaryotic cells, it occurs in the mitochondria. (Panel 2–9, pp. 106–107) clamp loader Protein complex that utilizes ATP hydrolysis to load the sliding clamp on to a primer–template junction in the process of DNA replication.

1	class I MHC protein One of two classes of major histocompatibility complex (MHC) protein. Found on the surface of almost all vertebrate cell types, where it can present foreign peptides derived from a pathogen such as a virus to cytotoxic T cells. (Figures 24–35 and 24–36A) class II MHC protein One of two classes of major histocompatibility complex (MHC) protein. Found on the surface of various antigen-presenting cells, where it presents peptides to helper and regulatory T cells. (Figures 24–35 and 24–36B) class switching Change from making one class of immunoglobulin (for example, IgM) to making another class (for example, IgG) that many B cells undergo during the course of an adaptive immune response. Involves DNA rearrangements called class-switch recombination. (Figure 24–30) class-switch recombination An irreversible change at the DNA level when a B cell switches from making IgM and IgD to making one of the secondary classes of immunoglobulin.

1	classical cadherins Family of cadherin proteins, including E-cadherin, N-cadherin, and P-cadherin, that are closely related in sequence throughout their extracellular and intracellular domains. clathrin Protein that assembles into a polyhedral cage on the cytosolic side of a membrane so as to form a clathrin-coated pit, which buds off by endocytosis to form an intracellular clathrin-coated vesicle. (Figure 13–6) clathrin-coated pits Specialized regions typically occupying about 2% of the total plasma membrane area at which the endocytic pathway often begins. clathrin-coated vesicles Coated vesicles that transport material from the plasma membrane and between endosomal and Golgi compartments. cleavage (1) Physical splitting of a cell into two. (2) Specialized type of cell division seen in many early embryos whereby a large cell becomes subdivided into many smaller cells without growth.

1	cleavage (1) Physical splitting of a cell into two. (2) Specialized type of cell division seen in many early embryos whereby a large cell becomes subdivided into many smaller cells without growth. clonal selection From a population of T and B lymphocytes with a vast repertoire of randomly generated antigen-specific receptors, a given foreign antigen activates (selects) only those lymphocyte clones that display a receptor that fits the antigen. Explains how the adaptive immune system can respond to millions of different antigens in a highly specific way. (Figure 24–15) co-receptor In immunology: an accessory receptor on B cells or T cells that does not bind antigen but binds to a co-stimulatory signal and helps activate the lymphocyte, by helping to activate an intracellular signaling pathway. co-stimulatory signal In immunology: a secreted or membrane-bound signal protein that helps activate an antigen-responding B cell or T cell.

1	co-stimulatory signal In immunology: a secreted or membrane-bound signal protein that helps activate an antigen-responding B cell or T cell. co-translational Occurring as translation proceeds. Examples include the import of a protein into the endoplasmic reticulum before the polypeptide chain is completely synthesized (co-translational translocation, Figure 12–32), and the folding of a nascent protein into its secondary and tertiary structure as it emerges from a ribosome. (Figure 6–79) coat-recruitment GTPases Members of a family of monomeric GTPases that have important roles in vesicle transport, being responsible for coat assembly at the membrane. coated vesicle Small membrane-enclosed organelle with a cage of proteins (the coat) on its cytosolic surface. Formed by the pinching off of a coated region of membrane (coated pit). Some coats are made of clathrin, others are made from other proteins.

1	codon Sequence of three nucleotides in a DNA or mRNA molecule that represents the instruction for incorporation of a specific amino acid into a growing polypeptide chain. coenzyme Small molecule tightly associated with an enzyme that participates in the reaction that the enzyme catalyzes, often by forming a covalent bond to the substrate. Examples include biotin, NAD+, and coenzyme A.

1	cohesin, cohesin complex Complex of proteins that holds sister chromatids together along their length before their separation. (Figure 17–19) coiled-coil Especially stable rodlike protein structure formed by two or more α helices coiled around each other. (Figure 3–9) collagen Fibrous protein rich in glycine and proline that is a major component of the extracellular matrix in animals, conferring tensile strength. Exists in many forms: type I, the most common, is found in skin, tendon, and bone; type II is found in cartilage; type IV is present in basal laminae. (Figures 3–23 and 19–40) collagen fibril A higher-order collagen polymer of fibrillar collagens that assemble into thin structures (10–300 nm in diameter) many hundreds of micrometers long in mature tissues. colony-stimulating factor (CSF) General name for numerous signal molecules that control differentiation of blood cells.

1	colony-stimulating factor (CSF) General name for numerous signal molecules that control differentiation of blood cells. colorectal cancer Cancer arising from the epithelium lining the colon (the large intestine) and rectum (the terminal segment of the gut). column chromatography Technique for separation of a mixture of substances in solution by passage through a column containing a porous solid matrix. Substances are retarded to different extents by their interaction with the matrix and can be collected separately from the column. Depending on the matrix, separation can be according to charge, hydrophobicity, size, or the ability to bind to other molecules. commensalism Ecological relationship between microbes and their host in which the microbe benefits but offers no benefit and causes no harm. committed precursor Cell derived from a stem cell that divides for a limited number of times before terminally differentiating; also known as a transit amplifying cell.

1	complement system System of blood proteins that can be activated by antibody–antigen complexes or pathogens to help eliminate the pathogens, by directly causing their lysis, by promoting their phagocytosis, or activating an inflammatory response. (Figure 24–7) complementary (1) Of nucleic acid sequences: capable of forming a perfect base-paired duplex with each other. (FIgure 4–4) (2) Of other interacting molecules, such as an enzyme and its substrate: having biochemical or structural features that marry up, so that noncovalent bonding is facilitated. (Figure 2–3) complementation test Test to determine whether two mutations that produce similar phenotypes are in the same or different genes. (Panel 8–2, pp. 487) complex oligosaccharides Broad class of N-linked oligosaccharides, attached to mammalian glycoproteins in the endoplasmic reticulum and modified in the Golgi apparatus, containing N-acetylglucosamine, galactose, sialic acid, and fucose residues.

1	condensin, condensin complex Complex of proteins involved in chromosome condensation prior to mitosis. Target for M-Cdk. (Figure 17–22) conditional mutation Mutation that changes a protein or RNA molecule so that its function is altered only under some conditions, such as at an unusually high or unusually low temperature. cone photoreceptor (cone) Photoreceptor cell in the vertebrate retina that is responsible for color vision in bright light. confocal microscope Type of light microscope that produces a clear image of a given plane within a solid object. It uses a laser beam as a pinpoint source of illumination and scans across the plane to produce a two-dimensional “optical section.” (Figure 9–19) conformation The folded, three-dimensional structure of a polypeptide chain.

1	connective tissue Any supporting tissue that lies between other tissues and consists of cells embedded in a relatively large amount of extracellular matrix. Includes bone, cartilage, and loose connective tissue.

1	connexin Protein component of gap junctions, a four-pass transmembrane protein. Six connexins assemble in the plasma membrane to form a connexon, or “hemichannel.” (Figure 19–25) connexon Water-filled pore in the plasma membrane formed by a ring of six connexin protein subunits. Half of a gap junction: connexons from two adjoining cells join to form a continuous channel through which ions and small molecules can pass. (Figure 19–25) consensus nucleotide sequence A summary or “average” of a large number of individual nucleotide sequences derived by comparing many sequences with the same basic function and tallying up the most common nucleotides found at each position. (Figure 6–12) consensus sequence Average or most typical form of a sequence that is reproduced with minor variations in a group of related DNA, RNA, or protein sequences. Indicates the nucleotide or amino acid most often found at each position. Preservation of a sequence implies that it is functionally important.

1	conservative site-specifc recombination A type of DNA recombination that takes place between short, specific sequences of DNA and occurs without the gain or loss of nucleotides. It does not require extensive homology between the recombining DNA molecules. constant region In immunology: region of an immunoglobulin or T cell receptor chain that has a constant amino acid sequence. constitutive secretory pathway Pathway present in all cells by which molecules such as plasma membrane proteins are continually delivered to the plasma membrane from the Golgi apparatus in vesicles that fuse with the plasma membrane. The default route to the plasma membrane if no other sorting signals are present. (Figure 13–63) contact-dependent signaling Form of intercellular signaling in which signal molecules remain bound to the surface of the signaling cell and influence only cells that contact it.

1	contractile ring Ring containing actin and myosin that forms under the surface of animal cells undergoing cell division. Contracts to pinch the two daughter cells apart. (Figure 17–42) convergent extension Rearrangement of cells within a tissue that causes it to extend in one dimension and shrink in another. (Figure 21–50) COPI-coated vesicles Coated vesicles that transport material early in the secretory pathway, budding from Golgi compartments. COPII-coated vesicles Coated vesicles that transport material early in the secretory pathway, budding from the endoplasmic reticulum. copy number variations (CNVs) A difference between two individuals in the same population in the number of copies of a particular block of DNA sequence. This variation arises from occasional duplications and deletions of these sequences. cortex The cytoskeletal network in the cortical region of the cytosol just beneath the plasma membrane.

1	cortex The cytoskeletal network in the cortical region of the cytosol just beneath the plasma membrane. coupled reaction Linked pair of chemical reactions in which the free energy released by one serves to drive the other. (Figure 2–29) covalent bond Stable chemical link between two atoms produced by sharing one or more pairs of electrons. (Panel 2–1, pp. 90–91) CRE-binding (CREB) protein Transcription regulator that recognizes the cyclic AMP response element (CRE) in the regulatory region of genes activated by cAMP. On activation by PKA, phosphorylated CREB recruits a transcriptional coactivator (CREB-binding protein; CBP) to stimulate transcription of target genes. CRISPR A defense mechanism in bacteria using small noncoding RNA molecules (crRNAs) to seek out and destroy invading viral genomes through complementary base-pairing and targeted nuclease digestion. crista (plural cristae) A specialized invagination of the inner mitochondrial membrane.

1	crista (plural cristae) A specialized invagination of the inner mitochondrial membrane. cross-linking glycan One of a heterogeneous group of branched polysaccharides that help to cross-link cellulose microfibrils into a complex network. Has a long linear backbone of one sugar type (glucose, xylose, or mannose) with short side chains of other sugars. cross-presentation A process in which extracellular proteins taken up by specialized dendritic cells can give rise to peptides that can be presented by class I MHC proteins to cytotoxic T cells. crRNAs Small noncoding RNAs (≈30 nucleotides) that are the effectors of CRISPR-mediated immunity in bacteria.

1	crRNAs Small noncoding RNAs (≈30 nucleotides) that are the effectors of CRISPR-mediated immunity in bacteria. cryoelectron microscopy Technique for examining a thin film of an aqueous suspension of biological material that has been frozen rapidly enough to create vitreous ice. The specimen is then kept frozen and transferred to the electron microscope. Image contrast is low, but is generated solely by the macromolecular structures present. cryptochrome Plant flavoprotein sensitive to blue light. Structurally related to blue-light-sensitive enzymes called photolyases (involved in the repair of ultraviolet-induced DNA damage) but do not have a role in DNA repair. Also found in animals, where they have an important role in circadian clocks. Cubitus interruptus (Ci) Latent transcription regulator that mediates the effects of Hedgehog.

1	Cubitus interruptus (Ci) Latent transcription regulator that mediates the effects of Hedgehog. cyclic AMP (cAMP) Nucleotide that is generated from ATP by adenylyl cyclase in response to various extracellular signals. It acts as a small intracellular signaling molecule, mainly by activating cAMP-dependent protein kinase (PKA). It is hydrolyzed to AMP by a phosphodiesterase. (Figure 15–25) cyclic AMP phosphodiesterase Specific enzyme that rapidly and continuously destroys cyclic AMP, forming 5′-AMP. (Figure 15–25). cyclic AMP-dependent protein kinase (protein kinase A, PKA) Enzyme that phosphorylates target proteins in response to a rise in intracellular cyclic AMP. (Figure 15–26) cyclic GMP (cGMP) Nucleotide that is generated from GTP by guanylyl cyclase in response to various extracellular signals. cyclic GMP phosphodiesterase Specific enzyme that rapidly hydrolyzes and degrades cyclic GMP.

1	cyclic GMP phosphodiesterase Specific enzyme that rapidly hydrolyzes and degrades cyclic GMP. cyclin Protein that periodically rises and falls in concentration in step with the eukaryotic cell cycle. Cyclins activate crucial protein kinases (called cyclin-dependent protein kinases, or Cdks) and thereby help control progression from one stage of the cell cycle to the next. cyclin-dependent kinase (Cdk) Protein kinase that has to be complexed with a cyclin protein in order to act. Different cyclin–Cdk complexes trigger different steps in the cell-division cycle by phosphorylating specific target proteins. (Figure 17–10) cyclin–Cdk complex Protein complex formed periodically during the eukaryotic cell cycle as the level of a particular cyclin increases. A cyclin-dependent kinase (Cdk) then becomes partially activated. (Figures 17–10 and 17–11, and Table 17–1, p. 969) cytochrome Colored heme-containing protein that transfers electrons during respiration and photosynthesis.

1	cytochrome c Soluble component of the mitochondrial electron-transport chain. Its release into the cytosol from the mitochondrial intermembrane space also initiates apoptosis. (Figure 14–26) cytochrome c oxidase complex Third of the three electron-driven proton pumps in the respiratory chain. It accepts electrons from cytochrome c and generates water using molecular oxygen as an electron acceptor. (Figure 14–18) cytochrome c reductase Second of the three electron-driven proton pumps in the respiratory chain. Accepts electrons from ubiquinone and passes them to cytochrome c. (Figure 14–18) cytokine Extracellular signal protein or peptide that acts as a local mediator in cell–cell communication.

1	cytokine receptor Cell-surface receptor that binds a specific cytokine or hormone and acts through the JAK–STAT signaling pathway. (Figure 15–56) cytokinesis Division of the cytoplasm of a plant or animal cell into two, as distinct from the associated division of its nucleus (which is mitosis). Part of M phase. (Panel 17–1, pp. 980–981) cytoplasm Contents of a cell that are contained within its plasma membrane but, in the case of eukaryotic cells, outside the nucleus. cytoplasmic tyrosine kinase Enzyme activated by certain cell-surface receptors (tyrosine-kinase-associated receptors) that transmits the receptor signal onward by phosphorylating target cytoplasmic proteins on tyrosine side chains. cytoskeleton System of protein filaments in the cytoplasm of a eukaryotic cell that gives the cell shape and the capacity for directed movement. Its most abundant components are actin filaments, microtubules, and intermediate filaments.

1	cytosol Contents of the main compartment of the cytoplasm, excluding membrane-bounded organelles such as endoplasmic reticulum and mitochondria. cytotoxic T cell (TC cell) Type of T cell responsible for killing host cells infected with a virus or another type of intracellular pathogen. (Figure 24–42) dark-field microscopy Type of light microscopy in which oblique rays of light focused on the specimen do not enter the objective lens, but light that is scattered by components in the living cell can be collected to produce a bright image on a dark background. (Figure 9–7) death receptor Transmembrane receptor protein that can signal the cell to undergo apoptosis when it binds its extracellular ligand. (Figure 18–5) death-inducing signaling complex (DISC) Activation complex in which initiator caspases interact and are activated following binding of extracellular ligands to cell-surface death receptors in the extrinsic pathway of apoptosis.

1	default pathway The transport pathway of proteins directly to the cell surface via the nonselective constitutive secretory pathway, entry into which does not require a particular signal. defensin Positively charged, amphipathic, antimicrobial peptide—secreted by epithelial cells—that binds to and disrupts the membranes of many pathogens. delayed K+ channel Neuronal voltage-gated K+ channel that opens following membrane depolarization but during the falling phase of an action potential due to slower activation kinetics than Na+ channels; opening permits K+ efflux, driving the membrane potential back toward its original negative value, ready to transmit a second impulse. Delta Single-pass transmembrane signal protein displayed on the surface of cells that binds to the Notch receptor protein on a neighboring cell, activating a contact-dependent signaling mechanism. dendrite Extension of a nerve cell, often elaborately branched, that receives stimuli from other nerve cells.

1	dendrite Extension of a nerve cell, often elaborately branched, that receives stimuli from other nerve cells. dendritic cell The most potent type of antigen-presenting cell, which takes up antigen and processes it for presentation to T cells. It is required for activating naïve T cells. (Figure 24–11) deoxyribonucleic acid (DNA) Polynucleotide formed from covalently linked deoxyribonucleotide units. The store of hereditary information within a cell and the carrier of this information from generation to generation. (Figure 4–3 and Panel 2–6, pp. 100–101) depolarization Deviation in the electric potential across the plasma membrane towards a positive value. A depolarized cell has a potential that is positive outside and negative inside.

1	desmosome Anchoring cell–cell junction, usually formed between two epithelial cells. Characterized by dense plaques of protein into which intermediate filaments in the two adjoining cells insert. (Figure 19–2) detergent Small amphiphilic molecule, more soluble in water than lipids, that disrupts hydrophobic associations and destroys the lipid bilayer thereby solubilizing membrane proteins. D gene segment A short DNA sequence that encodes a part of the variable region of an immunoglobulin heavy chain or the β chain of a T cell receptor (TCR).

1	D gene segment A short DNA sequence that encodes a part of the variable region of an immunoglobulin heavy chain or the β chain of a T cell receptor (TCR). diacylglycerol (DAG) Lipid produced by the cleavage of inositol phospholipids in response to extracellular signals. Composed of two fatty acid chains linked to glycerol, it serves as a small signaling molecule to help activate protein kinase C (PKC). (Figure 15–28) dideoxy sequencing The standard enzymatic method of DNA sequencing. (Panel 8–1, p. 478) differential-interference-contrast microscope Type of light microscope that exploits the interference effects that occur when light passes through parts of a cell of different refractive indices. Used to view unstained living cells. differentiation Process by which a cell undergoes a change to an overtly specialized cell type. diffusion The net drift of molecules through space due to random thermal movements.

1	differentiation Process by which a cell undergoes a change to an overtly specialized cell type. diffusion The net drift of molecules through space due to random thermal movements. Dishevelled Scaffold protein recruited to the Frizzled family of cell-surface receptors upon their activation by Wnt binding that helps relay the signal to other signaling molecules. DNA cloning (1) The act of making many identical copies (typically billions) of a DNA molecule—the amplification of a particular DNA sequence. (2) Also, the isolation of a particular stretch of DNA (often a particular gene) from the rest of the cell’s genome. DNA helicase Enzyme that is involved in opening the DNA helix into its single strands for DNA replication. DNA library Collection of cloned DNA molecules, representing either an entire genome (genomic library) or complementary DNA copies of the mRNA produced by a cell (cDNA library).

1	DNA library Collection of cloned DNA molecules, representing either an entire genome (genomic library) or complementary DNA copies of the mRNA produced by a cell (cDNA library). DNA ligase Enzyme that joins the ends of two strands of DNA together with a covalent bond to make a continuous DNA strand. DNA methylation Addition of methyl groups to DNA. Extensive methylation of the cytosine base in CG sequences is used in plants and animals to help keep genes in an inactive state. DNA microarray A large array of short DNA molecules (each of known sequence) bound to a glass microscope slide or other suitable support. Used to monitor expression of thousands of genes simultaneously: mRNA isolated from test cells is converted to cDNA, which in turn is hybridized to the microarray. (Figure 8–64) DNA polymerase Enzyme that synthesizes DNA by joining nucleotides together using a DNA template as a guide.

1	DNA polymerase Enzyme that synthesizes DNA by joining nucleotides together using a DNA template as a guide. DNA primase Enzyme that synthesizes a short strand of RNA on a DNA template, producing a primer for DNA synthesis. (Figure 5–10) DNA repair A set of processes for repairing the many accidental lesions that occur continually in DNA. DNA replication Process by which a copy of a DNA molecule is made. DNA supercoiling A conformation with loops or coils that DNA adopts in response to superhelical tension; conversely, creating various loops or coils in the helix can create such tension.

1	DNA supercoiling A conformation with loops or coils that DNA adopts in response to superhelical tension; conversely, creating various loops or coils in the helix can create such tension. DNA topoisomerase (topoisomerase) Enzyme that binds to DNA and reversibly breaks a phosphodiester bond in one or both strands. Topoisomerase I creates transient single-strand breaks, allowing the double helix to swivel and relieving superhelical tension. Topoisomerase II creates transient double-strand breaks, allowing one double helix to pass through another and thus resolving tangles. (Figures 5–21 and 5–22) DNA tumor virus General term for a variety of different DNA viruses that can cause tumors. DNA-only transposon Transposable element that exists as DNA throughout its life cycle. Many move by cut-and-paste transposition. See also transposon. dolichol Isoprenoid lipid molecule that anchors the precursor oligosaccharide in the endoplasmic reticulum membrane during protein glycosylation.

1	dolichol Isoprenoid lipid molecule that anchors the precursor oligosaccharide in the endoplasmic reticulum membrane during protein glycosylation. domain (protein domain) Portion of a protein that has a tertiary structure of its own. Larger proteins are generally composed of several domains, each connected to the next by short flexible regions of polypeptide chain. Homologous domains are recognized in many different proteins. Dorsal protein Transcription regulator of the NFκB family regulating gene expression and involved in establishing the dorsoventral axis in the embryo. double helix The three-dimensional structure of DNA, in which two antiparallel DNA chains, held together by hydrogen-bonding between the bases, are wound into a helix. (Figure 4–5) drivers Mutations that are causal factors in the development of cancer.

1	dynamic instability Sudden conversion from growth to shrinkage, and vice versa, in a protein filament such as a microtubule or actin filament. (Panel 16–2, pp. 902–903) dynamin Cytosolic GTPase that binds to the neck of a clathrin-coated vesicle in the process of budding from the membrane, and which is involved in completing vesicle formation. dynein Large motor protein that undergoes ATP-dependent movement along microtubules. E2F protein Transcription regulatory protein that switches on many genes that encode proteins required for entry into the S phase of the cell cycle. early endosome Common receiving compartment with which most endocytic vesicles fuse and where internalized cargo is sorted either for return to the plasma membrane or for degradation by inclusion in a late endosome. ectoderm Embryonic epithelial tissue that is the precursor of the epidermis and nervous system.

1	ectoderm Embryonic epithelial tissue that is the precursor of the epidermis and nervous system. edema factor One of the two A subunits of anthrax toxin; an adenylyl cyclase that catalyzes production of cAMP, leading to ion imbalance and consequent edema in the skin or lung. effector Bcl2 family proteins Pro-apoptotic proteins of the intrinsic pathway of apoptosis that in response to an apoptotic stimulus become activated and aggregate to form oligomers in the mitochondrial outer membrane, inducing the release of cytochrome c and other intermembrane proteins. Bax and Bak are the main effector Bcl2 family proteins in mammalian cells. effector cell Cell that carries out the final response or function in a particular process. The main effector cells of the immune system, for example, are activated lymphocytes and phagocytes that help eliminate pathogens.

1	egg-polarity genes Genes in the Drosophila egg that define the anteroposterior and dorsoventral axes of the future embryo through the creation of landmarks (mRNA or protein) in the egg that provide signals organizing the developmental process. elastic fiber Extensible fiber formed by the protein elastin in many animal connective tissues, such as in skin, blood vessels, and lungs, which gives them their stretchability and resilience. elastin Extracellular protein that forms extensible fibers (elastic fibers) in connective tissues. electrochemical gradient Combined influence of a difference in the concentration of an ion on two sides of a membrane and the electrical charge difference across the membrane (membrane potential). Ions or charged molecules can move passively only down their electrochemical gradient. electron microscope Microscope that uses a beam of electrons to create the image.

1	electron microscope Microscope that uses a beam of electrons to create the image. electron microscope (EM) tomography Technique for viewing three-dimensional specimens in the electron microscope in which multiple views are taken from different directions by tilting the specimen holder. The views are combined computationally to give a three-dimensional image. electron-transport chain Series of reactions in which electron carrier molecules pass electrons “down the chain” from higher to successively lower energy levels. The energy released during such electron movement can be used to power various processes. Electron-transport chains present in the inner mitochondrial membrane (called the respiratory chain) and in the thylakoid membrane of chloroplasts generate a proton gradient across the membrane that is used to drive ATP synthesis. See especially Figures 14–18 and 14–52.

1	electrostatic attraction A noncovalent, ionic bond between two molecules carrying groups of opposite charge. (Panel 2–3, pp. 94–95) embryonic stem cells (ES cells) Cells derived from the inner cell mass of the early mammalian embryo. Capable of giving rise to all the cells in the body. Can be grown in culture, genetically modified, and inserted into a blastocyst to develop a transgenic animal. endocrine cell Specialized animal cell that secretes a hormone into the blood. Usually part of a gland, such as the thyroid or pituitary gland. endocytic vesicle Vesicle formed as material ingested by the cell during endocytosis is progressively enclosed by a small portion of the plasma membrane, which first invaginates and then pinches off to form the vesicle. endocytosis Uptake of material into a cell by an invagination of the plasma membrane and its internalization in a membrane-enclosed vesicle. See also pinocytosis and phagocytosis.

1	endocytosis Uptake of material into a cell by an invagination of the plasma membrane and its internalization in a membrane-enclosed vesicle. See also pinocytosis and phagocytosis. endoderm Embryonic tissue that is the precursor of the gut and associated organs. endoplasmic reticulum (ER) Labyrinthine membrane-bounded compartment in the cytoplasm of eukaryotic cells, where lipids are synthesized and membrane-bound proteins and secretory proteins are made. (Figure 12–33) endosome maturation Process by which early endosomes mature to late endosomes and endolysosomes; in the conversion process, the endosome membrane protein composition changes, the endosome moves from the cell periphery to close to the nucleus, and the endosome ceases to recycle material to the plasma membrane and irreversibly commits its remaining contents to degradation. endothelial cell Flattened cell type that forms a sheet (the endothelium) lining all blood and lymphatic vessels.

1	endothelial cell Flattened cell type that forms a sheet (the endothelium) lining all blood and lymphatic vessels. entropy (S) Thermodynamic quantity that measures the degree of disorder or randomness in a system; the higher the entropy, the greater the disorder. (Panel 2–7, pp. 102–103) enveloped virus Virus with a capsid surrounded by a lipid bilayer membrane (the envelope), which is often derived from the host-cell plasma membrane when the virus buds from the cell. (Figure 23–12) enzyme Protein that catalyzes a specific chemical reaction.

1	enzyme-coupled receptor A major type of cell-surface receptor that has a cytoplasmic domain that either has enzymatic activity or is associated with an intracellular enzyme. In either case, the enzymatic activity is stimulated by an extracellular ligand binding to the receptor. (Figure 15–6) ephrin One of a family of membrane-bound protein ligands for the Eph receptor tyrosine kinases (RTKs) that, among many other functions, stimulate repulsion or attraction responses that guide the migration of cells and nerve cell axons during animal development. epidermis Epithelial layer covering the outer surface of the body. Has different structures in different animal groups. The outer layer of plant tissue is also called the epidermis.

1	epidermis Epithelial layer covering the outer surface of the body. Has different structures in different animal groups. The outer layer of plant tissue is also called the epidermis. epigenetic inheritance Inheritance of phenotypic changes in a cell or organism that do not result from changes in the nucleotide sequence of DNA. Can be due to positive feedback loops of transcription regulators or to heritable modifications in chromatin such as DNA methylation or histone modifications. (Figure 7–53) epistasis analysis Analysis to discover the order in which the genes act, by investigating if a mutation in one gene can mask the effect of a mutation in another gene when both mutations are present in the same organism or cell. epithelial tissues Tissues, such as the lining of the gut or the epidermal covering of the skin, in which cells are closely bound together into sheets called epithelia.

1	epithelial tissues Tissues, such as the lining of the gut or the epidermal covering of the skin, in which cells are closely bound together into sheets called epithelia. epithelium (plural epithelia) Sheet of cells covering the outer surface of a structure or lining a cavity. equilibrium State in a chemical reaction where there is no net change in free energy to drive the reaction in either direction. The ratio of product to substrate reaches a constant value at chemical equilibrium. (Figure 2–30) equilibrium constant (K) The ratio of forward and reverse rate constants for a reaction. Equal to the association or affinity constant (Ka) for a simple binding reaction (A + B AB). See also affinity constant, dissociation constant. (See page 62) ER lumen Space enclosed by the membrane of the endoplasmic reticulum (ER).

1	AB). See also affinity constant, dissociation constant. (See page 62) ER lumen Space enclosed by the membrane of the endoplasmic reticulum (ER). ER resident protein Protein that remains in the endoplasmic reticulum (ER) or its membranes and carries out its function there, as opposed to proteins that are present in the ER only in transit. ER retention signal Short amino acid sequence on a protein that prevents it from moving out of the endoplasmic reticulum (ER). Found on proteins that are resident in the ER and function there. ER signal sequence N-terminal signal sequence that directs proteins to enter the endoplasmic reticulum (ER). Cleaved off by signal peptidase after entry. ER tail-anchored proteins Membrane proteins anchored in the endoplasmic reticulum (ER) membrane by a single transmembrane α helix contained at their C-terminus.

1	ER tail-anchored proteins Membrane proteins anchored in the endoplasmic reticulum (ER) membrane by a single transmembrane α helix contained at their C-terminus. erythrocyte Small hemoglobin-containing blood cell of vertebrates that transports oxygen to, and carbon dioxide from, tissues. Also called a red blood cell. erythropoietin A hormone produced by the kidney that stimulates the production of red blood cells in bone marrow. ESCRT protein complexes Four protein complexes (ESCRT-0, ESCRT-1, ESCRT-2, and ESCRT-3) that act sequentially to shepherd mono-ubiquitylated membrane proteins on endosomal membranes into intralumenal vesicles. ESCRT-3 complex catalyzes the pinching-off reaction. ethylene Small gas molecule that is a plant growth regulator influencing plant development in various ways including promoting fruit ripening, leaf abscission, and plant senescence and functioning as a stress signal in response to wounding, infection, and flooding.

1	euchromatin Region of an interphase chromosome that stains diffusely; “normal” chromatin, as opposed to the more condensed heterochromatin. eukaryote Organism composed of one or more cells that have a distinct nucleus. Member of one of the three main divisions of the living world, the other two being bacteria and archaea. (Figure 1–17) eukaryotic initiation factor (eIF) Protein that helps load initiator tRNA on to the ribosome, thus initiating translation. excitatory neurotransmitter Neurotransmitter that opens cation channels in the postsynaptic membrane, causing an influx of Na+, and in many cases Ca2+, that depolarizes the postsynaptic membrane toward the threshold potential for firing an action potential. executioner caspases Apoptotic caspases that catalyze the widespread cleavage events during apoptosis that kill the cell. exocytosis Excretion of material from the cell by vesicle fusion with the plasma membrane; can occur constitutively or be regulated.

1	exocytosis Excretion of material from the cell by vesicle fusion with the plasma membrane; can occur constitutively or be regulated. exon Segment of a eukaryotic gene that consists of a sequence of nucleotides that will be represented in mRNA or in a final transfer, ribosomal, or other mature RNA molecule. In protein-coding genes, exons encode the amino acids in the protein. An exon is usually adjacent to a noncoding DNA segment called an intron. (Figure 4–15) exosome Large protein complex with an interior rich in 3′-to-5′ RNA exonucleases; degrades RNA molecules to produce ribonucleotides. extracellular pathogens Pathogens that disturb host cells and can cause serious disease without replicating in host cells. extracellular signal molecule Any secreted or cell-surface chemical signal that binds to receptors and regulates the activity of the cell expressing the receptor.

1	extracellular signal molecule Any secreted or cell-surface chemical signal that binds to receptors and regulates the activity of the cell expressing the receptor. extrinsic pathway Pathway of apoptosis triggered by extracellular signal proteins binding to cell-surface death receptors. facultative pathogens Bacteria that replicate in an environmental reservoir such as water or soil and only cause disease if they happen to encounter a susceptible host. FAD/FADH2 (flavin adenine dinucleotide/reduced flavin adenine dinucleotide) Electron carrier system that functions in the citric acid cycle and fatty acid oxidation. One molecule of FAD gains two electrons plus two protons in becoming the activated carrier FADH2. (Figure 2–39) Fas (Fas protein, Fas death receptor) Transmembrane death receptor that initiates apoptosis when it binds its extracellular ligand (Fas ligand). (Figure 18–5)

1	Fas (Fas protein, Fas death receptor) Transmembrane death receptor that initiates apoptosis when it binds its extracellular ligand (Fas ligand). (Figure 18–5) Fas ligand Ligand that activates the cell-surface death receptor, Fas, triggering the extrinsic pathway of apoptosis. fat Energy-storage lipid in cells. Composed of triglycerides— fatty acids esterified with glycerol. fate map Representation showing which cell types will later derive from which regions of a tissue; e.g. from the blastula. (Figure 21–28) Fc receptor One of a family of cell-surface receptors that bind the tail region (Fc region) of an antibody molecule. Different Fc receptors are specific for different classes of antibodies, such as IgG, IgA, or IgE.

1	feedback inhibition The process in which a product of a reaction feeds back to inhibit a previous reaction in the same pathway. (Figures 3–55 and 3–56) fermentation Anaerobic energy-yielding metabolic pathway involving the oxidation of organic molecules. Anaerobic glycolysis refers to the process whereby pyruvate is converted into lactate or ethanol, with the conversion of NADH to NAD+. (Figure 2–47) fibril-associated collagen Mediates the interactions of collagen fibrils with one another and with other matrix macromolecules to help determine the organization of the fibrils in the matrix. This collagen (including types IX and XII) has a flexible triple-stranded helical structure and binds to the surface of the fibrils rather than forming aggregates.

1	fibrillar collagen Class of fibril-forming collagens (including type I collagen, the most common type and the principal collagen of skin and bone) that have long ropelike structures with few or no interruptions and which assemble into collagen fibrils. fibroblast Common cell type found in connective tissue. Secretes an extracellular matrix rich in collagen and other extracellular matrix macromolecules. Migrates and proliferates readily in wounded tissue and in tissue culture. fibronectin Extracellular matrix protein involved in adhesion of cells to the matrix and guidance of migrating cells during embryogenesis. Integrins on the cell surface are receptors for fibronectin.

1	filopodium (plural filopodia) (microspike) Thin, spike-like protrusion with an actin filament core, generated on the leading edge of a crawling animal cell. (Figure 16–21) firing rule Important principle governing synapse reinforcement and elimination during development of the nervous system: when two (or more) neurons synapsing on the same target cell fire at the same time, they reinforce their connections to that cell; when they fire at different times, they compete, so that all but one of them tend to be eliminated. flagellum (plural flagella) Long, whiplike protrusion whose undulations drive a cell through a fluid medium. Eukaryotic flagella are longer versions of cilia. Bacterial flagella are smaller and completely different in construction and mechanism of action. Compare cilium.

1	fluorescence microscope Microscope designed to view material stained with fluorescent dyes or proteins. Similar to a light microscope but the illuminating light is passed through one set of filters before the specimen, to select those wavelengths that excite the dye, and through another set of filters before it reaches the eye, to select only those wavelengths emitted when the dye fluoresces. (Figure 9–12) fluorescence recovery after photobleaching (FRAP) Technique for monitoring the kinetic parameters of a protein by analyzing how fluorescent protein molecules move into an area of the cell bleached by a beam of laser light. (Figure 9–29) fluorescence resonance energy transfer (FRET) Technique for monitoring the closeness of two fluorescently labeled molecules (and thus their interaction) in cells. Also known as Förster resonance energy transfer. (Figure 9–26) focal adhesion kinase (FAK) Cytoplasmic tyrosine kinase present at cell–matrix junctions (focal adhesions) in association

1	in cells. Also known as Förster resonance energy transfer. (Figure 9–26) focal adhesion kinase (FAK) Cytoplasmic tyrosine kinase present at cell–matrix junctions (focal adhesions) in association with the cytoplasmic tails of integrins.

1	focal adhesion kinase (FAK) Cytoplasmic tyrosine kinase present at cell–matrix junctions (focal adhesions) in association with the cytoplasmic tails of integrins. follicular helper T cell (TFH) Type of T cell located in lymphoid follicles that secretes various cytokines to stimulate B cells to undergo antibody class switching and somatic hypermutation. formin Dimeric protein that nucleates the growth of straight, unbranched actin filaments that can be cross-linked by other proteins to form parallel bundles. free energy (G) (Gibbs free energy) The energy that can be extracted from a system to drive reactions. Takes into account changes in both energy and entropy. (Panel 2–7, pp. 102–103) free ribosome Ribosome that is free in the cytosol, unattached to any membrane. free-energy change (∆G) see ΔG.

1	free-energy change (∆G) see ΔG. Frizzled Family of cell-surface receptors that are seven-pass transmembrane proteins that resemble GPCRs in structure but do not generally work through the activation of G proteins. Activated by Wnt binding to recruit the scaffold protein Dishevelled, which helps relay the signal to other signaling molecules. fungus (plural fungi) Kingdom of eukaryotic organisms that includes the yeasts, molds, and mushrooms. Many plant diseases and a relatively small number of animal diseases are caused by fungi. fusion protein Engineered protein that combines two or more normally separate polypeptides. Produced from a recombinant gene. ∆G Change in the free energy during a reaction: the free energy of the product molecules minus the free energy of the starting molecules. A large negative value of ∆G indicates that the reaction has a strong tendency to occur. (Panel 2–7, pp. 102–103)

1	G0 State of withdrawal from the eukaroytic cell-division cycle by entry into a quiescent digression from the G1 phase. A common, sometimes permanent, state for differentiated cells. G1 phase Gap 1 phase of the eukaryotic cell-division cycle, between the end of mitosis and the start of DNA synthesis. (Figure 17–4) G1-Cdk Cyclin–Cdk complex formed in vertebrate cells by a G1-cyclin and the corresponding cyclin-dependent kinase (Cdk). (Table 17–1, p. 969) G1-cyclin Cyclin present in the G1 phase of the eukaryotic cell cycle. Forms complexes with Cdks that help govern the activity of the G1/S-cyclins, which control progression to S phase. G1/S-Cdk Cyclin–Cdk complex formed in vertebrate cells by a G1/S-cyclin and the corresponding cyclin-dependent kinase (Cdk). (Figure 17–11 and Table 17–1, p. 969)

1	G1/S-Cdk Cyclin–Cdk complex formed in vertebrate cells by a G1/S-cyclin and the corresponding cyclin-dependent kinase (Cdk). (Figure 17–11 and Table 17–1, p. 969) G1/S-cyclin Cyclin that activates Cdks in late G1 of the eukaryotic cell cycle and thereby helps trigger progression through Start, resulting in a commitment to cell-cycle entry. Its level falls at the start of S phase. (Figure 17–11) G2 phase Gap 2 phase of the eukaryotic cell-division cycle, between the end of DNA synthesis and the beginning of mitosis. (Figure 17–4)

1	G2 phase Gap 2 phase of the eukaryotic cell-division cycle, between the end of DNA synthesis and the beginning of mitosis. (Figure 17–4) G2/M transition Point in the eukaryotic cell cycle at which the cell checks for completion of DNA replication before triggering the early mitotic events that lead to chromosome alignment on the spindle. (Figure 17–9) ganglioside Any glycolipid having one or more sialic acid residues in its structure. Found in the plasma membrane of eukaryotic cells and especially abundant in nerve cells. (Figure 10–16) gap gene In Drosophila development, a gene that is expressed in specific broad regions along the anteroposterior axis of the early embryo, and which helps designate the main divisions of the insect body. (Figure 21–20) gap junction Communicating channel-forming cell–cell junction present in most animal tissues that allows ions and small molecules to pass from the cytoplasm of one cell to the cytoplasm of the next.

1	gastrulation Important stage in animal embryogenesis during which the embryo is transformed from a ball of cells to a structure with a gut (a gastrula). gated transport Movement of proteins between the cytosol and the nucleus through nuclear pore complexes in the nuclear envelope that function as selective gates. geminin Protein that prevents the formation of new prereplicative complexes during S phase and mitosis, thus ensuring that the chromosomes are replicated only once in each cell cycle. gene Region of DNA that is transcribed as a single unit and carries information for a discrete hereditary characteristic, usually corresponding to (1) a single protein (or set of related proteins generated by variant post-transcriptional processing), or (2) a single RNA (or set of closely related RNAs).

1	gene control region The set of linked DNA sequences regulating expression of a particular gene. Includes promoter and cis-regulatory sequences required to initiate transcription of the gene and control the rate of transcription. (Figure 7–17) gene conversion Process by which DNA sequence information can be transferred from one DNA helix (which remains unchanged) to another DNA helix whose sequence is altered. It often accompanies general recombination events. (Figure 5–59) gene family The set of genes in an organism related in DNA sequence due to their derivation from the same ancestor.

1	gene segments In immunology: short DNA sequences that are joined together during B cell and T cell development to produce the coding sequences for immunoglobulins and T cell receptors, respectively. (Figure 24–28) general transcription factor Any of the proteins whose assembly at all promoters of a given type is required for the binding and activation of RNA polymerase and the initiation of transcription. (Table 6–3, p. 311) genetic code The set of rules specifying the correspondence between nucleotide triplets (codons) in DNA or RNA and amino acids in proteins. (Figure 6–48) genetic instability Abnormally increased spontaneous mutation rate, such as occurs in cancer cells. genetic screen Procedure for discovery of genes affecting a specific phenotype by surveying large numbers of mutagenized individuals. genetics The study of the genes of an organism on the basis of heredity and variation.

1	genetics The study of the genes of an organism on the basis of heredity and variation. genome The totality of genetic information belonging to a cell or an organism; in particular, the DNA that carries this information. genome annotation Process attempting to mark out all the genes (protein-coding and noncoding) in a genome and ascribing functions to each. genomic imprinting Phenomenon in which a gene is either expressed or not expressed in the offspring depending on which parent it is inherited from. (Figure 7–49) genomic library Collection of cloned DNA molecules representing an entire genome.

1	genotype Genetic constitution of an individual cell or organism. The particular combination of alleles found in a specific individual. (Panel 8–2, p. 486) germ cell A cell in the germ line of an organism, which includes the haploid gametes and their specified diploid precursor cells. Germ cells contribute to the formation of a new generation of organisms and are distinct from somatic cells, which form the body and leave no descendants. germ layer One of the three primary tissue layers (endoderm, mesoderm, and ectoderm) of an animal embryo. (Figure 21–3) glial cell Supporting non-neural cell of the nervous system. Includes oligodendrocytes and astrocytes in the vertebrate central nervous system and Schwann cells in the peripheral nervous system.

1	glycogen Polysaccharide composed exclusively of glucose units. Used to store energy in animal cells. Large granules of glycogen are especially abundant in liver and muscle cells. (Figure 2–51 and Panel 2–4, pp. 96–97) glycolipid Lipid molecule with a sugar residue or oligosaccharide attached. (Panel 2–5, pp. 98–99) glycolysis Ubiquitous metabolic pathway in the cytosol in which sugars are incompletely degraded with production of ATP. Literally, “sugar splitting.” (Figure 2–46 and Panel 2–8, pp. 104–105) glycoprotein Any protein with one or more saccharide or oligosaccharide chains covalently linked to amino acid side chains. Most secreted proteins and most proteins exposed on the outer surface of the plasma membrane are glycoproteins.

1	glycosaminoglycan (GAG) Long, linear, highly charged polysaccharide composed of a repeating pair of sugars, one of which is always an amino sugar. Mainly found covalently linked to a protein core in extracellular matrix proteoglycans. Examples include chondroitin sulfate, hyaluronan, and heparin. (Figure 19–32) glycosylphosphatidylinositol anchor (GPI anchor) Lipid linkage by which some membrane proteins are bound to the membrane. The protein is joined, via an oligosaccharide linker, to a phosphatidylinositol anchor during its travel through the endoplasmic reticulum. (Figure 12–52) Golgi apparatus (Golgi complex) Complex organelle in eukaryotic cells, centered on a stack of flattened, membrane-enclosed spaces, in which proteins and lipids transferred from the endoplasmic reticulum are modified and sorted. It is the site of synthesis of many cell wall polysaccharides in plants and extracellular matrix glycosaminoglycans in animal cells. (Figure 13–26)

1	GPCR kinase (GRK) Member of a family of enzymes that phosphorylates multiple serines and threonines on a GPCR to produce receptor desensitization. (Figure 15–42) G protein (trimeric GTP-binding protein) A trimeric GTP-binding protein with intrinsic GTPase activity that couples GPCRs to enzymes or ion channels in the plasma membrane. (Table 15–3, p. 846) G-protein-coupled receptor (GPCR) A seven-pass cell-surface receptor that, when activated by its extracellular ligand, activates a G protein, which in turn activates either an enzyme or ion channel in the plasma membrane. (Figures 15–6 and 15–21) Gq Class of G protein that couples GPCRs to phospholipase C-β to activate the inositol phospholipid signaling pathway. Gram negative Description for bacteria that do not stain with Gram stain as a result of having a thinner peptidoglycan cell wall outside their inner (plasma) membrane, and on an additional outer membrane.

1	Gram positive Description for bacteria that stain positive with Gram stain due to a thick layer of peptidoglycan cell wall outside their inner (plasma) membrane. Gram staining A technique for classifying bacteria based on differences in the structure of the bacterial cell wall and outer surface.

1	Gram staining A technique for classifying bacteria based on differences in the structure of the bacterial cell wall and outer surface. granulocyte Category of white blood cell distinguished by conspicuous cytoplasmic granules. Includes neutrophils, basophils, and eosinophils. Arises from a granulocyte/ macrophage (GM) progenitor cell. (Figure 22–27) granulocyte/macrophage (GM) progenitor cell Committed progenitor cell in the bone marrow that gives rise to neutrophils and macrophages. (Figure 22–31) green fluorescent protein (GFP) Fluorescent protein isolated from a jellyfish. Widely used as a marker in cell biology. (Figure 9–24) growth cone Migrating motile tip of a growing nerve cell axon or dendrite. (Figure 21–72) growth factor Extracellular signal protein that can stimulate a cell to grow. They often have other functions as well, including stimulating cells to survive or proliferate. Examples include epidermal growth factor (EGF) and platelet-derived growth factor (PDGF).

1	growth hormone (GH) Mammalian hormone secreted by the pituitary gland into the bloodstream that stimulates growth throughout the body. GTP (guanosine 5′-triphosphate) Nucleoside triphosphate produced by the phosphorylation of GDP (guanosine diphosphate). Like ATP, it releases a large amount of free energy on hydrolysis of its terminal phosphate group. Has a special role in microtubule assembly, protein synthesis, and cell signaling. (Figure 2–58) GTP-binding protein Also called GTPase; an enzyme that converts GTP to GDP.

1	GTP-binding protein Also called GTPase; an enzyme that converts GTP to GDP. GTPase An enzyme that converts GTP to GDP. GTPases fall into two large families. Large trimeric G proteins are composed of three different subunits and mainly couple GPCRs to enzymes or ion channels in the plasma membrane. Small monomeric GTP-binding proteins (also called monomeric GTPases) consist of a single subunit and help relay signals from many types of cell-surface receptors and have roles in intracellular signaling pathways, regulating intracellular vesicle trafficking, and signaling to the cytoskeleton. Both trimeric G proteins and monomeric GTPases cycle between an active GTP-bound form and an inactive GDP-bound form and frequently act as molecular switches in intracellular signaling pathways. See page 820.

1	GTPase-activating protein (GAP) Protein that binds to a GTPase and inhibits it by stimulating its GTPase activity, causing the enzyme to hydrolyze its bound GTP to GDP. (Figure 15–8) guanine nucleotide exchange factor (GEF) Protein that binds to a GTPase and activates it by stimulating it to release its tightly bound GDP, thereby allowing it to bind GTP in its place. (Figure 15–8) haplotype block Combination of alleles and DNA markers that has been inherited in a large, linked block on one chromosome of a homologous pair—undisturbed by genetic recombination—across many generations. Hedgehog protein Secreted extracellular signal molecule that has many different roles controlling cell differentiation and gene expression in animal embryos and adult tissues. Excessive Hedgehog signaling can lead to cancer.

1	helper T cell (TH cell) Type of T cell that helps activate B cells to make antibodies, cytotoxic T cells to become effector cells, and macrophages to kill ingested pathogens. They can also help activate dendritic cells. heterochromatin Chromatin that is highly condensed even in interphase; generally transcriptionally inactive. (Compare with euchromatin.) heterochronic Describes genes involved in developmental timing; mutation results in cells of a specific fate behaving as cells at a different stage of development. high-mannose oligosaccharides Broad class of N-linked oligosaccharides, attached to mammalian glycoproteins in the endoplasmic reticulum, containing two N-acetylglucosamine residues and many mannose residues. high-performance liquid chromatography (HPLC) Type of chromatography that uses columns packed with tiny beads of matrix; the solution to be separated is pushed through under high pressure.

1	high-performance liquid chromatography (HPLC) Type of chromatography that uses columns packed with tiny beads of matrix; the solution to be separated is pushed through under high pressure. histone One of a group of small abundant proteins, rich in arginine and lysine, that combine to form the nucleosome cores around which DNA is wrapped in eukaryotic chromosomes. (Figure 4–24) histone chaperone (chromatin assembly factor) Protein that binds free histones, releasing them once they have been incorporated into newly replicated chromatin. (Figure 4–27) histone H1 “Linker” (as opposed to “core”) histone protein that binds to DNA where it exits from a nucleosome and helps package nucleosomes into the 30-nm chromatin fiber. (Figure 4–30)

1	Holliday junction (cross-strand exchange) X-shaped structure observed in DNA undergoing recombination, in which the two DNA molecules are held together at the site of crossing-over, also called a cross-strand exchange. (Figure 5–55) homeotic selector gene In Drosophila development, a gene that defines and preserves the differences between body segments. homolog One of two or more genes that are similar in sequence as a result of derivation from the same ancestral gene. The term covers both orthologs and paralogs. (Figure 1–21) See homologous chromosomes. homologous Genes, proteins, or body structures that are similar as a result of a shared evolutionary origin. homologous chromosomes (homologs) The maternal and paternal copies of a particular chromosome in a diploid cell.

1	homologous chromosomes (homologs) The maternal and paternal copies of a particular chromosome in a diploid cell. homologous recombination (general recombination) Genetic exchange between a pair of identical or very similar DNA sequences, typically those located on two copies of the same chromosome. Also a DNA repair mechanism for double-strand breaks. (Figures 5–48, 5–50, and 5–54) homophilic Binding between molecules of the same kind, especially those involved in cell–cell adhesion. (Figure 19–5) horizontal gene transfer Gene transfer between bacteria via natural transformation by released naked DNA, transduction by bacteriophages, or sexual exchange by conjugation. hormone Signal molecule secreted by an endocrine cell into the bloodstream, which can then carry the signal to distant target cells. Hox complex A gene complex consisting of a series of Hox genes

1	hormone Signal molecule secreted by an endocrine cell into the bloodstream, which can then carry the signal to distant target cells. Hox complex A gene complex consisting of a series of Hox genes Hox genes Genes coding for transcription regulators, each gene containing a homeodomain, and specifying body-region differences. Hox mutations typically cause homeotic transformations. Hox proteins Transcription regulator proteins encoded by Hox genes; possess a highly conserved, 60-amino-acid-long DNA-binding homeodomain. HPV Human papillomavirus; infects the cervical epithelium and is important as a cause of carcinoma of the uterine cervix.

1	hyaluronan (hyaluronic acid) Type of nonsulfated glycosaminoglycan with a regular repeating sequence of up to 25,000 identical disaccharide units, not linked to a core protein. Found in the fluid lubricating joints and in many other tissues. (Figures 19–33 and 19–34) hybridization In molecular biology, the process whereby two complementary nucleic acid strands form a base-paired duplex DNA-DNA, DNA-RNA, or RNA-RNA molecule. Forms the basis of a powerful technique for detecting specific nucleotide sequences. (Figures 5–47 and 8–33) hybridoma Hybrid cell line generated by fusion of a tumor cell and another cell type. Monoclonal antibodies are produced by hybridoma lines obtained by fusing antibody-secreting B cells with cells of a B lymphocyte tumor. (Figure 8–4) hydrogen bond Noncovalent bond in which an electropositive hydrogen atom is partially shared by two electronegative atoms. (Panel 2–3, pp. 94–95) hydronium ion (H3O+) Water molecule associated with an additional proton. The

1	bond in which an electropositive hydrogen atom is partially shared by two electronegative atoms. (Panel 2–3, pp. 94–95) hydronium ion (H3O+) Water molecule associated with an additional proton. The form generally taken by protons in aqueous solution.

1	hydrophilic Dissolving readily in water. Literally, “water loving.” hydrophobic (lipophilic) Not dissolving readily in water. Literally, “water-fearing.” hydrophobic force Force exerted by the hydrogen-bonded network of water molecules that brings two nonpolar surfaces together by excluding water between them. (Panel 2–3, pp. 94–95) hypervariable region In immunology: any of the three small parts of the variable region of an immunoglobulin or T cell receptor chain that show the highest variability from molecule to molecule and contribute to the antigen-binding site. (Figure 24–26) hypoxia-inducible factor 1α (HIF1α). Transcription regulator, the intracellular levels of which increase in response to a shortage of oxygen, that stimulates transcription of the VEGF gene to promote angiogenesis. IκB Inhibitory proteins that bind tightly to NFκB dimers and hold them in an inactive state within the cytoplasm of unstimulated cells.

1	IκB Inhibitory proteins that bind tightly to NFκB dimers and hold them in an inactive state within the cytoplasm of unstimulated cells. Ig superfamily Large and diverse family of proteins that contain immunoglobulin or immunoglobulin-like domains. Most are involved in cell–cell interactions or antigen recognition. (Figure 24–48) IgA Immunoglobulin A; the principal class of antibody in secretions, including saliva, tears, milk, and respiratory and intestinal secretions. IgD Immunoglobulin D; produced by immature naïve B cells after leaving the bone marrow. Transmembrane IgD and IgM proteins, with the same antigen-binding site, form the B cell receptors (BCRs) on these cells.

1	IgE Immunoglobulin E; binds with high affinity via its tail region to a class of Fc receptors on the surface of mast cells (tissues) or basophils (blood), where it acts as an antigen receptor; antigen binding stimulates the secretion of cytokines and biologically active amines, which help attract white blood cells, antibodies, and complement proteins to the site of activation. IgG Immunoglobulin G; the major antibody class in the blood, produced in especially large quantities during secondary antibody responses. The tail region of some IgG subclasses can bind to specific Fc receptors on macrophages and neutrophils. Antigen–IgG complexes can activate complement.

1	IgM Immunoglobulin M; the first class of immunoglobulin that a developing B cell in the bone marrow makes, forming B-cell receptors on its surface. IgM antibodies are the major class of antibody secreted into the blood in the early stages of a primary antibody response on first exposure to an antigen, where their pentameric structure (with 10 antigen-binding sites) allows strong binding to pathogens. When bound to antigen, it is highly efficient activation of complement. iHog Protein with four or five immunoglobulin-like domains and two or three fibronectin-type-III-like domains; located on the cell surface and thought to serve as co-receptors for Hedgehog proteins.

1	image processing Computer based techniques in microscopy that process digital images in order to extract latent information. Enables compensation for some optical faults in microscopes, enhanced contrast to improve detection of small differences in light intensity, and subtraction of background irregularities in the optical system. imaginal disc Group of cells that are set aside, apparently undifferentiated, in the Drosophila embryo and which will develop into an adult structure, e.g., eye, leg, wing. Overt differentiation occurs at metamorphosis. (Figure 21–60) immunization Method of inducing adaptive immune responses to pathogens or foreign molecules, usually involving the co-injection of an adjuvant, a molecule (often of microbial origin) that helps activate innate immune responses required for the adaptive responses.

1	immunoglobulin (Ig) superfamily Large and diverse family of proteins that contain immunoglobulin domains or immunoglobulin-like domains. Most are involved in cell–cell interactions or antigen recognition. (Figure 24–48) immunoglobulin domain (Ig domain) Characteristic protein domain of about 100 amino acids that is found in immunoglobulin light and heavy chains. Similar domains, known as immunoglobulin-like (Ig-like) domains, are present in many other proteins, which, together with Igs, constitute the Ig superfamily. (Figure 24–27) immunogold electron microscopy Method to localize specific macromolecules using a primary antibody that binds to the molecule of interest and is then detected with a secondary antibody to which a colloidal gold particle has been attached. The gold particle is electron-dense and can be seen as a black dot in the electron microscope. (Figure 9–45) immunological memory Long-lived property of the adaptive immune system that follows a primary immune response

1	electron-dense and can be seen as a black dot in the electron microscope. (Figure 9–45) immunological memory Long-lived property of the adaptive immune system that follows a primary immune response to many antigens, such that a subsequent encounter with the same antigen will provoke a more rapid and stronger secondary immune response. (Figure 24–16) immunological self-tolerance The lack of response of the adaptive immune system to an antigen. Tolerance to self molecules is crucial to avoid autoimmune diseases. (Figure 24–21) immunological synapse The highly organized interface that develops between a T cell and an antigen-presenting cell (APC) or target cell it is in contact with, formed by T-cell receptors binding to antigen–MHC complexes on the APC and cell-adhesion proteins binding to their counterparts on the APCs.

1	induced fit A principle for increasing the specificity of substrate recognition by proteins and RNAs. In protein synthesis, a ribosome, or enzyme folds around a codon– anticodon interaction and only when the match is correct is the subsequent reaction allowed to proceed. induced pluripotent stem cells (iPS cells) Cells that are induced by artificial expression of specific transcription regulators to look and behave like the pluripotent embryonic stem cells that are derived from embryos. induced regulatory T cell A regulatory T cell (Treg cell) that develops from naive helper T cells when they are activated in the presence of TGFβ in the absence of IL6. inflammasome Intracellular protein complex formed after activation of cytoplasmic NOD-like receptors with adaptor proteins. It contains a caspase enzyme that cleaves pro-inflammatory cytokines from their precursor proteins.

1	inflammatory response Local response of a tissue to injury or infection—characterized clinically by redness, swelling, heat, and pain. Caused by invasion of white blood cells, which are attracted by and secrete various cytokines. inhibitors of apoptosis (IAPs) Intracellular protein inhibitors of apoptosis. inhibitory G protein (Gi) Trimeric G protein that can regulate ion channels and inhibit the enzyme adenylyl cyclase in the plasma membrane. See also G protein. (Table 15–3, p. 846) inhibitory neurotransmitter Neurotransmitter that opens transmitter-gated Cl– or K+ channels in the postsynaptic membrane of a nerve or muscle cell and thus tends to inhibit the generation of an action potential. initial segment Specialized membrane region at the base of a nerve axon (adjacent to the cell body) that is rich in voltage-gated Na+ channels plus other classes of ion channels that all contribute to the encoding of membrane depolarization into action potential frequency.

1	initiator caspases Apoptotic caspases that begin the apoptotic process, activating the executioner caspases. initiator tRNA Special tRNA that intiates translation. It always carries the amino acid methionine, forming the complex MettRNAi. (Figure 6–70) innate immune response An early immune response in all organisms to a pathogen, which includes the production of antimicrobial molecules and the activation of phagocytic cells. Such a response is not specific for the pathogen, in contrast to an adaptive immune response. inner membrane Mitochondrial membrane that encloses the matrix space and forms extensive invaginations called cristae. inner mitochondrial membrane Mitochondrial membrane that encloses the matrix space and forms extensive invaginations called cristae. inner nuclear membrane One of two concentric membranes comprising the nuclear envelope; contnuous with the outer nuclear membrane; contains specific proteins as anchoring sites for chromatin and the nuclear lamina.

1	inositol 1,4,5-trisphosphate (IP3) Small intracellular signaling molecule produced during activation of the inositol phospholipid signaling pathway. Acts to release Ca2+ from the endoplasmic reticulum. (Figures 15–28 and 15–29) inositol phospholipid signaling pathway Intracellular signaling pathway that starts with the activation of phospholipase C and the generation of IP3 and diacylglycerol (DAG) from inositol phospholipids in the plasma membrane. The DAG helps to activate protein kinase C. (Figures 15–28 and 15–29) integrin Transmembrane adhesion protein that is involved in the attachment of cells to the extracellular matrix and to each other. (Figure 19–3 and Table 19–1, p. 1037) interaction domain Compact protein module, found in many intracellular signaling proteins, that binds to a particular structural motif (e.g., a short peptide sequence, a covalent modification, or another protein domain) in another protein or lipid.

1	interferon-α (IFNα) and interferon-β (IFNβ) Cytokines (type I interferons) produced by mammalian cells as a general response to a viral infection. intermembrane space Compartment of mitochondrion between by the outer and inner mitochondrial membranes. internal ribosome entry site (IRES) Specific site in a eukaryotic mRNA, other than at the 5′ end, at which translation can be initiated. (Figure 7–68) interphase Long period of the cell cycle between one mitosis and the next. Includes G1 phase, S phase, and G2 phase. (Figure 17–4) interpolar microtubule In the mitotic or meiotic spindle, a microtubule interdigitating at the equator with the microtubules emanating from the other pole. (Figure 17–23) intracellular pathogens Pathogens, including all viruses and many bacteria and protozoa, that enter and replicate inside host cells to cause disease.

1	intrinsic pathway (mitochondrial pathway) Pathway of apoptosis activated from inside the cell in response to stress or developmental signals; depends on the release into the cytosol of mitochondrial proteins normally resident in the mitochondrial intermembrane space. intron Noncoding region of a eukaryotic gene that is transcribed into an RNA molecule but is then excised by RNA splicing during production of the mRNA or other functional RNA. (Figure 4–15) invadopodia Actin-rich protrusions extending in three-dimensions that are important for cells to cross tissue barriers by degrading the extracellular matrix.

1	ion channel Transmembrane protein complex that forms a water-filled channel across the lipid bilayer through which specific inorganic ions can diffuse down their electrochemical gradients. (Figure 11–22) ion-channel-coupled receptor (transmitter-gated ion channel, ionotropic receptor) Ion channel found at chemical synapses in the postsynaptic plasma membranes of nerve and muscle cells. Opens only in response to the binding of a specific extracellular neurotransmitter. The resulting inflow of ions leads to the generation of a local electrical signal in the postsynaptic cell. (Figures 15–6 and 11–35) ion-sensitive indicators Molecules whose light emission reflects the local concentration of a particular ion; some are luminescent (emitting light spontaneously) while others are fluorescent (emitting light on exposure to light).

1	IP3-gated Ca2+-release channel (IP3 receptor) Gated Ca2+ channel in the ER membrane that opens on binding cytosolic IP3, releasing stored Ca2+ into the cytosol. (Figure 15–29) iron–sulfur cluster Electron-transporting group consisting of either two or four iron atoms bound to an equal number of sulfur atoms, found in a class of electron-transport proteins. (Figure 14–16) J gene segment Short DNA sequences that encodes part of the variable region of light and heavy immunoglobulin chains and of α and β chains of T cell receptors. (Figures 24–28 and 24–29) JAK–STAT signaling pathway Signaling pathway activated by cytokines and some hormones, providing a rapid route from the plasma membrane to the nucleus to alter gene transcription. Involves cytoplasmic Janus kinases (JAKs), and signal transducers and activators of transcription (STATs).

1	Janus kinases (JAKs) Cytoplasmic tyrosine kinases associated with cytokine receptors, which phosphorylate and activate transcription regulators called STATs. junctional diversification The random loss and gain of nucleotides at joining sites during V(D)J recombination that occurs during B and T cell development when the cells are assembling the gene segments that encode their antigen receptors. It enormously increases the diversity of V-region coding sequences. K+ leak channel K+-transporting ion channel in the plasma membrane of animal cells that remains open even in a “resting” cell. karyotype Display of the full set of chromosomes of a cell, arranged with respect to size, shape, and number. keratin Type of intermediate filament, commonly produced by epithelial cells. kinase cascade Intracellular signaling pathway in which one protein kinase, activated by phosphorylation, phosphorylates the next protein kinase in the sequence, and so on, relaying the signal onward.

1	kinesin Member of one of the two main classes of motor proteins that use the energy of ATP hydrolysis to move along microtubules. (Figure 16–56) kinesin-1 Motor protein associated with microtubules that transports cargo within the cell; also called “conventional kinesin.” kinetic proofreading A principle for increasing the specificity of catalysis. In the synthesis of DNA, RNA, and proteins, it refers to a time delay that begins with an irreversible step (such as ATP or GTP hydrolysis) and during which incorrect base pairs are more likely to dissociate than correct pairs. kinetochore Large protein complex that connects the centromere of a chromosome to microtubules of the mitotic spindle. (FIgure 17–30) kinetochore microtubule In the mitotic or meiotic spindle, a microtubule that connects the spindle pole to the kinetochore of a chromosome.

1	lagging strand One of the two newly synthesized strands of DNA found at a replication fork. The lagging strand is made in discontinuous lengths that are later joined covalently. (Figure 5–7) lamellipodium (plural lamellipodia) Flattened, sheetlike protrusion supported by a meshwork of actin filaments, which is extended at the leading edge of a crawling animal cell. (Figures 16–77 and 16–79) laminin Extracellular matrix fibrous protein found in basal laminae, where it forms a sheetlike network. (Figures 19–52 and 19–53) lampbrush chromosome Huge chromosome paired in preparation for meiosis, found in immature amphibian eggs; consisting of large loops of chromatin extending out from a linear central axis. (Figure 4–47) late endosome Compartment formed from a bulbous, vacuolar portion of early endosomes by a process called endosome maturation; late endosomes fuse with one another and with lysosomes to form endolysosomes that degrade their contents.

1	LDL-receptor-related protein (LRP) Co-receptor bound by Wnt proteins in the regulation of β-catenin proteolysis. leading strand One of the two newly synthesized strands of DNA found at a replication fork. The leading strand is made by continuous synthesis in the 5′-to-3′ direction. (Figure 5–7) lectin Protein that binds tightly to a specific sugar. Abundant lectins from plant seeds are used as affinity reagents to purify glycoproteins or to detect them on the surface of cells. Legionnaire’s disease Type of pneumonia resulting from infection with Legionella pneumophila, a parasite of freshwater amoebae that is spread to humans by air-conditioning systems that harbor infected amoebae and produce microdroplets of water that are easily inhaled. lethal factor One of the two A subunits of anthrax toxin; a protease that cleaves several activated members of the MAP kinase kinase family and causes a large fall in blood pressure and death on entry into the bloodstream of an animal.

1	leucine-rich repeat (LRR) receptor kinases Common type of receptor serine/threonine kinase in plants that contains a tandem array of leucine-rich repeat sequences in its extracellular portion. leukemia Cancer of white blood cells. leukocyte General name for all the nucleated blood cells lacking hemoglobin. Also called white blood cells. Includes lymphocytes, granulocytes, and monocytes. (Figure 22–27) ligand Any molecule that binds to a specific site on a protein or other molecule. From Latin ligare, to bind. light microscope One of a class of microscopes that uses visible light to create the image. lignin Network of cross-linked phenolic compounds that forms a supporting network throughout the cell walls of xylem and woody tissue in plants. limit of resolution In microscopy, the smallest distance apart at which two point objects can be resolved as separate. Just under 0.2 μm for conventional light microscopy, a limit determined by the wavelength of light.

1	linkage In ligand binding, the conformational coupling between two separate ligand-binding sites on a protein, such that a conformational change in the protein induced by binding of one ligand affects the binding of a second ligand. lipid bilayer (phospholipid bilayer) Thin double sheet of phospholipid molecules that forms the core structure of all cell membranes. The two layers of lipid molecules are packed with their hydrophobic tails pointing inward and their hydrophilic heads outward, exposed to water. (Figure 10–1 and Panel 2–5, pp. 98–99) lipid droplets Storage form in cells for excess lipids; comprised of a single monolayer of phospholipids and proteins that surrounds neutral lipids that can be retrieved from droplets as required by the cell.

1	lipid raft Small region of a membrane enriched in sphingolipids and cholesterol. (Figure 10–13) liposome Artificial phospholipid bilayer vesicle formed from an aqueous suspension of phospholipid molecules. (Figure 10–9) local mediator Extracellular signal molecule that acts on neighboring cells. long noncoding RNA (lncRNA) One of a large group (≈8000 in humans) of RNAs longer than 200 nucleotides and not coding for protein. The functions, if any, of most lncRNAs is unknown but individual lncRNA are known to play important roles in the cell, for example, in telomerase function and genomic imprinting. In a general sense, lncRNAs are believed to act as scaffolds, holding together proteins and nucleic acids to speed up a wide variety of reactions in the cell.

1	long-term depression (LTD) A long-lasting (hours or more) decrease in the sensitivity of certain synapses in the brain triggered by NMDA receptor activation. As the opposing process to long-term potentiation, it is thought to be involved in learning and memory. long-term potentiation (LTP) Long-lasting increase (days to weeks) in the sensitivity of certain synapses in the brain, induced by a short burst of repetitive firing in the presynaptic neurons. (Figure 11–44) loss of heterozygosity The result of errant homologous recombination that uses the homolog from the other parent instead of the sister chromatid as the template, converting the sequence of the repaired DNA to that of the other homolog.

1	low-density lipoprotein (LDL) Large complex composed of a single protein molecule and many esterified cholesterol molecules, together with other lipids. The form in which cholesterol is transported in the blood and taken up into cells. (Figure 13–51) lumen The space inside a hollow structure. In cells: the cavity enclosed by an organelle membrane. In tissues: the cavity enclosed by a sheet of cells. lymphocyte White blood cell responsible for the specificity of adaptive immune responses. Two main types: B cells, which produce antibody, and T cells, which interact directly with other effector cells of the immune system and with infected cells. T cells develop in the thymus and are responsible for cell-mediated immunity. B cells develop in the bone marrow in mammals and are responsible for the production of circulating antibodies.

1	lymphoid organ An organ containing large numbers of lymphocytes. Lymphocytes are produced in primary lymphoid organs and respond to antigen in peripheral lymphoid organs. (Figure 24–12) lymphoma Cancer of lymphocytes, in which the cancer cells are mainly found in lymphoid organs (rather than in the blood, as in leukemias). lysosomal storage diseases Genetic diseases resulting from defects in or a lack of one or more functional hydrolases in lysosomes of some cells, leading to accumulation of undigested substrates in lysosomes and consequent cell pathology. lysosome Membrane-enclosed organelle in eukaryotic cells containing digestive enzymes, which are typically most active at the acid pH found in the lumen of lysosomes. (Figure 13–37) lysozyme Enzyme that catalyzes the cutting of polysaccharide chains in the cell walls of bacteria.

1	M-Cdk (M-phase Cdk) Cyclin-Cdk complex formed in vertebrate cells by an M-cyclin and the corresponding cyclindependent kinase (Cdk). (Figure 17–11 and Table 17–1, p. 969) M-cyclin A cyclin found in all eukaryotic cells that promotes the events of mitosis. (Figure 17–11) M6P receptor proteins Transmembrane receptor proteins present in the trans Golgi network that recognize the mannose 6-phosphate (M6P) groups added exclusively to lysosomal enzymes, marking the enzymes for packaging and delivery to early endosomes. macromolecule Polymers constructed of long chains of covalently linked, small organic (carbon-containing) molecules. The principal building blocks from which a cell is constructed and the components that confer the most distinctive properties of living things. macrophage Phagocytic cell derived from blood monocytes, resident in most tissues but able to roam. It has both scavenger and antigen-presenting functions in immune responses.

1	macrophage Phagocytic cell derived from blood monocytes, resident in most tissues but able to roam. It has both scavenger and antigen-presenting functions in immune responses. macropinocytosis Clathrin-independent, dedicated degradative endocytic pathway induced in most cell types by cell-surface receptor activation by specific cargoes. malaria Protozoal disease caused by four species of Plasmodium, which are transmitted to humans by the bite of the female Anopheles mosquito. malignant Of tumors and tumor cells: invasive and/or able to undergo metastasis. A malignant tumor is a cancer. (Figure 20–3)

1	malignant Of tumors and tumor cells: invasive and/or able to undergo metastasis. A malignant tumor is a cancer. (Figure 20–3) MAP kinase module (mitogen-activated protein kinase module) An intracellular signaling module composed of three protein kinases, acting in sequence, with MAP kinase as the third. Typically activated by a Ras protein in response to extracellular signals. (Figure 15–49) master transcription regulator A transcription regulator specifically required for formation of a particular cell type. Artificial expression of master transcription regulators (alone or in combination with others) will often convert one cell type into another. maternal inheritance A form of inheritance observed when following mitochondria in animals and plants, where mitochondrial DNA is inherited only through the female germ line.

1	maternal inheritance A form of inheritance observed when following mitochondria in animals and plants, where mitochondrial DNA is inherited only through the female germ line. maternal-effect gene Gene that acts in the mother to specify maternal mRNAs and proteins in the egg. Maternal-effect mutations affect the development of the embryo even if the embryo itself has not inherited the mutated gene. maternal-zygotic transition (MZT) Event in animal development where the embryo’s own genome largely takes over control of development from maternally deposited macromolecules. proteolytic enzyme present in the extracellular matrix that degrades matrix proteins. Includes the collagenases. matrix space Large internal compartment of the mitochondrion. mechanosensitive channels Transmembrane ion channels that open in response to a mechanical stress on the lipid bilayer in which they are embedded.

1	mechanosensitive channels Transmembrane ion channels that open in response to a mechanical stress on the lipid bilayer in which they are embedded. megakaryocyte Large myeloid cell with a multilobed nucleus that remains in the bone marrow when mature. Buds off platelets from long cytoplasmic processes. (Figures 22–29) meiosis I The first of two rounds of chromosome segregation following meiotic chromosome duplication; segregates the homologs, each composed of a tightly linked pair of sister chromatids. meiosis II The second of two rounds of chromosome segregation following meiotic chromosome duplication; segregates the sister chromatids of each homolog.

1	membrane potential Voltage difference across a membrane due to a slight excess of positive ions on one side and of negative ions on the other. A typical membrane potential for an animal cell plasma membrane is –60 mV (inside negative relative to the surrounding fluid). (Figure 11–23) membrane protein Amphiphilic protein of diverse structure and function that associates with the lipid bilayer of cell membranes. (Figure 10–17) membrane transport protein Membrane protein that mediates the passage of ions or molecules across a membrane. The two main classes are transporters (also called carriers or permeases) and channels. (Figure 11–4) membrane-associated protein Membrane protein not extending into the hydrophobic interior of the lipid bilayer but bound to either face of the membrane by noncovalent interactions with other membrane proteins. (Figure 10–17) membrane-bending proteins Attach to specific membrane regions as needed and act to control local membrane curvature and thus

1	by noncovalent interactions with other membrane proteins. (Figure 10–17) membrane-bending proteins Attach to specific membrane regions as needed and act to control local membrane curvature and thus confer on membranes their characteristic three-dimensional shapes.

1	membrane-bound ribosome Ribosome attached to the cytosolic face of the endoplasmic reticulum. The site of synthesis of proteins that enter the endoplasmic reticulum. (Figure 12–38) memory cell In immunology: a T or B lymphocyte generated following antigen stimulation that is more easily and more quickly induced to become an effector cell or another memory cell by a later encounter with the same antigen. (Figure 24–17) mesoderm Embryonic tissue that is the precursor to muscle, connective tissue, skeleton, and many of the internal organs. (Figure 21–3) messenger RNA (mRNA) RNA molecule that specifies the amino acid sequence of a protein. Produced in eukaryotes by processing of an RNA molecule made by RNA polymerase as a complementary copy of DNA. It is translated into protein in a process catalyzed by ribosomes. (Figure 6–20) metabolism The sum total of the chemical processes that take place in living cells. All of catabolism plus anabolism. (Figure 2–14) metabotropic receptors

1	catalyzed by ribosomes. (Figure 6–20) metabolism The sum total of the chemical processes that take place in living cells. All of catabolism plus anabolism. (Figure 2–14) metabotropic receptors Neurotransmitter receptors that regulate ion channels indirectly through the activation of second-messenger molecules.

1	metaphase plate Imaginary plane at right angles to the mitotic spindle and midway between the spindle poles; the plane in which chromosomes are positioned at metaphase. (Panel 17–1, pp. 980–981) metaphase-to-anaphase transition Transition in the eukaryotic cell cycle preceding sister-chromatid separation at anaphase. If the cell is not ready to proceed to anaphase, the cell cycle is halted at this point. (Figure 17–9, and Panel 17–1, pp. 980–981) metastases Secondary tumors, at sites in the body additional to that of the primary tumor, resulting from cancer cells breaking loose, entering blood or lymphatic vessels, and colonizing separate environments. metastasis The spread of cancer cells from their site of origin to other sites in the body. (Figures 20–1 and 20–16)

1	metastasis The spread of cancer cells from their site of origin to other sites in the body. (Figures 20–1 and 20–16) MHC complex (major histocompatibility complex) Cluster of genes in one vertebrate chromosome (chromosome 6 in humans) that code for a set of highly polymorphic cell-surface glycoproteins (MHC proteins). (Figure 24–37) microbiome The combined genomes of the various species of a defined microbiota. microbiota The collective of microorganisms that reside in or on an organism. microelectrode A piece of fine glass tubing, pulled to an even finer tip, that is used to inject electric current into cells or to study the intracellular concentrations of common inorganic ions (such as H+, Na+, K+, Cl–, and Ca2+) in a single living cell by insertion of its tip directly into the cell interior through the plasma membrane.

1	microRNAs (miRNAs) Short (~21 nucleotide) eukaryotic RNAs, produced by the processing of specialized RNA transcripts coded in the genome, that regulate gene expression through base-pairing with mRNA. (Figure 7–75) microsome Small vesicle derived from endoplasmic reticulum that is produced by fragmentation when cells are homogenized. (Figure 12–34) microtubule flux Movement of individual tubulin molecules in the microtubules of the spindle toward the poles by loss of tubulin at their minus ends. Helps to generate the poleward movement of sister chromatids after they separate in anaphase. (Figure 17–35) microtubule-associated protein (MAP) Any protein that binds to microtubules and modifies their properties. Many different kinds have been found, including structural proteins, such as MAP2, and motor proteins, such as dynein. [Not to be confused with the “MAP” (mitogen-activated protein kinase) of “MAP kinase.”] microtubule-organizing center (MTOC) Region in a cell, such as a

1	as MAP2, and motor proteins, such as dynein. [Not to be confused with the “MAP” (mitogen-activated protein kinase) of “MAP kinase.”] microtubule-organizing center (MTOC) Region in a cell, such as a centrosome or a basal body, from which microtubules grow.

1	midbody Structure formed at the end of cleavage that can persist for some time as a tether between the two daughter cells in animals. (Figure 17–43) mitochondrial hsp70 Part of a multisubunit protein assembly bound to the matrix side of the TIM23 complex that acts as a motor to pull mitochondrial precursor proteins into the matrix space. mitochondrial matrix Large internal compartment of the mitochondrion. The corresponding compartment in a chloroplast is known as the stroma. mitochondrial precursor proteins Proteins first fully synthesized in the cytosol and then translocated into mitochondrial subcompartments as directed by one or more signal sequences. mitochondrion (plural mitochondria) Membrane-bounded organelle, about the size of a bacterium, that carries out oxidative phosphorylation and produces most of the ATP in eukaryotic cells. (Figure 1–28) mitogen Extracellular signal molecule that stimulates cells to proliferate.

1	mitotic chromosome Highly condensed duplicated chromosome as seen at mitosis, consisting of two sister chromatids held together at the centromere. mitotic spindle Bipolar array of microtubules and associated molecules that forms in a eukaryotic cell during mitosis and serves to move the duplicated chromosomes apart. (Figure 17–23 and Panel 17–1, pp. 980–981) model organism A species that has been studied intensively over a long period and thus serves as a “model” for deriving fundamental biological principles. molecular chaperone (chaperone) Protein that helps guide the proper folding of other proteins, or helps them avoid misfolding. Includes heat-shock proteins (hsp). monoallelic gene expression Expression of only one of the two copies of a gene in a diploid genome, occurring, for example, as a result of imprinting or X-chromosome inactivation.

1	monoallelic gene expression Expression of only one of the two copies of a gene in a diploid genome, occurring, for example, as a result of imprinting or X-chromosome inactivation. monoclonal antibody Antibody secreted by a hybridoma cell line. Because the hybridoma is generated by the fusion of a single B cell with a single tumor cell, each hybridoma produces antibodies that are all identical. (Page 444) monocyte Type of white blood cell that leaves the bloodstream and matures into a macrophage in tissues. (Figure 22–27) monomeric GTPase A single-subunit enzyme that converts GTP to GDP (also called small monomeric GTP-binding proteins). Cycles between an active GTP-bound form and an inactive GDP-bound form and frequently acts as a molecular switch in intracellular signaling pathways.

1	morphogen Diffusible signal molecule that can impose a pattern on a field of cells by causing cells in different places to adopt different fates. (Figure 21–8) morphogenesis Developmental process in which cells undergo movements and deformations in order to assemble into tissues and organs with specific shapes and sizes. motor protein Protein that uses energy derived from nucleoside triphosphate hydrolysis to propel itself along a linear track (protein filament or other polymeric molecule). mRNA degradation control Regulation by a cell of gene expression by selectively preserving or destroying certain mRNA molecules in the cytoplasm. mTOR The mammalian version of the large protein kinase called TOR, involved in cell signaling; mTOR exists in two functionally distinct multiprotein complexes.

1	mTOR The mammalian version of the large protein kinase called TOR, involved in cell signaling; mTOR exists in two functionally distinct multiprotein complexes. multidrug resistance An observed phenomenon in which cells exposed to one anticancer drug evolve a resistance not only to that drug, but also to other drugs to which they have never been exposed. multidrug resistance (MDR) protein Type of ABC transporter protein that can pump hydrophobic drugs (such as some anticancer drugs) out of the cytoplasm of eukaryotic cells. multipass transmembrane protein Membrane protein in which the polypeptide chain crosses the lipid bilayer more than once. (Figure 10–17) multivesicular bodies Intermediates in the endosome maturation process; early endosomes that are on their way to becoming late endosomes. mutation Heritable change in the nucleotide sequence of a chromosome. (Panel 8–2, pp. 486–487) mutation rate The rate at which changes (mutations) occur in DNA sequences.

1	mutation Heritable change in the nucleotide sequence of a chromosome. (Panel 8–2, pp. 486–487) mutation rate The rate at which changes (mutations) occur in DNA sequences. mutualism Ecological relationship between microbes and their host in which both the microbe and host benefit.

1	mutualism Ecological relationship between microbes and their host in which both the microbe and host benefit. Myc Transcription regulatory protein that is activated when a cell is stimulated to grow and divide by extracellular signals. It activates the transcription of many genes, including those that stimulate cell growth. (Figure 17–61) myelin sheath Insulating layer of specialized cell membrane wrapped around vertebrate axons. Produced by oligodendrocytes in the central nervous system and by Schwann cells in the peripheral nervous system. (Figure 11–33) myeloid cell Any white blood cell other than a lymphocyte. (Figure 22–31) myoblast Mononucleated, undifferentiated muscle precursor cell. A skeletal muscle cell is formed by the fusion of multiple myoblasts. (Figure 22–19) myofibril Long, highly organized bundle of actin, myosin, and other proteins in the cytoplasm of muscle cells that contracts by a sliding filament mechanism.

1	myosin Type of motor protein that uses the energy of ATP hydrolysis to move along actin filaments. Na+-K+ pump (Na+-K+ ATPase) Transmembrane carrier protein found in the plasma membrane of most animal cells that pumps Na+ out of and K+ into the cell, using energy derived from ATP hydrolysis. (Figure 11–15) NAD+/NADH (nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide) Electron carrier system that participates in oxidation–reduction reactions, such as the oxidation of food molecules. NAD+ accepts the equivalent of a hydride ion (H–, a proton plus two electrons) to become the activated carrier NADH. The NADH formed donates its high-energy electrons to the ATP-generating process of oxidative phosphorylation. (Figure 2–36) NADH dehydrogenase complex First of the three electron-driven proton pumps in the mitochondrial respiratory chain, also known as Complex I. It accepts electrons from NADH and passes them to a quinone. (Figure 14–18)

1	NADP+/NADPH (nicotinamide adenine dinucleotide phosphate/reduced nicotinamide adenine dinucleotide phosphate) Electron carrier system closely related to NAD+/ NADH, but used almost exclusively in reductive biosynthetic, rather than catabolic, pathways. (Figure 2–36) naïve cell In immunology: a T or B lymphocyte that proliferates and differentiates into an effector cell or memory cell when it encounters its specific foreign antigen for the first time. (Figure 24–17) natural killer cell (NK cell) Cytotoxic cell of the innate immune system that can kill virus-infected cells and some cancer cells. natural regulatory T cell A regulatory T cell (Treg cell) that develops in the thymus and helps maintain self-tolerance. negative selection Process by which thymocytes expressing a T cell receptor with high affinity for a self peptide bound to a self-MHC protein are eliminated by undergoing apoptosis.

1	negative selection Process by which thymocytes expressing a T cell receptor with high affinity for a self peptide bound to a self-MHC protein are eliminated by undergoing apoptosis. negative staining A technique in electron microscopy enabling fine detail of isolated macromolecules to be seen. Samples are prepared such that a very thin film of heavy-metal salt covers everywhere except where excluded by the presence of macromolecules, which allow electrons to pass through, creating a reverse or negative image of the molecule. Nernst equation Equation that computes relates the electrical potential (voltage) generated by differences in ion concentrations across a membrane. Netrin Signal protein, secreted by cells of the neural tube floor plate, responsible for attracting growth cones of commissural axons toward and across the midline.

1	Netrin Signal protein, secreted by cells of the neural tube floor plate, responsible for attracting growth cones of commissural axons toward and across the midline. neural crest Collection of cells located along the line where the neural tube pinches off from the surrounding epidermis in the vertebrate embryo. Neural crest cells migrate to give rise to a variety of tissues, including neurons and glia of the peripheral nervous system, pigment cells of the skin, and the bones of the face and jaws. (Figure 19–8) neural map Regular mapping of neurons of a similar type from one territory to another, such that there are orderly projections of one array of neurons onto another.

1	neural tube Tube of ectoderm that will form the brain and spinal cord in a vertebrate embryo. (Figure 21–56) neurofilament Type of intermediate filament found in nerve cells. (Figure 16–72) neuromuscular junction Specialized chemical synapse between an axon terminal of a motor neuron and a skeletal muscle cell. (Figure 11–37) neuron (nerve cell) Impulse-conducting cell of the nervous system, with extensive processes specialized to receive, conduct, and transmit signals. (Figures 11–28 and 21–66) neuronal specificity Nonequivalence among neurons; an intrinsic characteristic that guides axons to their appropriate target sites. neurotransmitter Small signal molecule secreted by the presynaptic nerve cell at a chemical synapse to relay the signal to the postsynaptic cell. Examples include acetylcholine, glutamate, GABA, glycine, and many neuropeptides.

1	neurotrophic factor Factor released in limited amounts by a target tissue that the neurons innervating that tissue require to survive. neurotrophin Family of signal proteins that promote the survival and growth of specific classes of neurons. neutrophil White blood cell that is specialized for the uptake of particulate material by phagocytosis. Enters tissues that become infected or inflamed. (Figure 24–5) NFκB protein Latent transcription regulator that is activated by various intracellular signaling pathways when cells are stimulated during immune, inflammatory, or stress responses. Also has important roles in animal development. (Figure 15–62) nitric oxide (NO) Gaseous signal molecule that is widely used in cell–cell communication in both animals and plants. (Figure 15–40) nitrogen fixation Biochemical process carried out by certain bacteria that reduces atmospheric nitrogen (N2) to ammonia, leading eventually to various nitrogen-containing metabolites.

1	NMDA receptor Subclass of glutamate-gated ion channel in the mammalian central nervous system critical for longterm potentiation and long-term depression. NMDA-receptor channels are doubly gated, opening only when glutamate is bound to the receptor and, simultaneously, the membrane is strongly depolarized. NO synthase (NOS) Enzyme that synthesizes nitric oxide (NO) by the deamination of arginine. (Figure 15–40B) NOD-like receptors (NLRs) Large family of pattern recognition receptors (PRRs) with leucine-rich repeat motifs; they are exclusively cytoplasmic and recognize a distinct set of microbial molecules. nonclassical cadherins Large family of cadherins that are more distantly related in sequence than classical cadherins and include proteins involved in adhesion (including protocadherins, desmocollins, and desmogleins) and signaling.

1	noncoding RNA An RNA molecule that is the final product of a gene and does not code for protein. These RNAs serve as enzymatic, structural, and regulatory components for a wide variety of processes in the cell. nondisjunction Event occurring occasionally during meiosis in which a pair of homologous chromosomes fails to separate so that the resulting germ cell has either too many or too few chromosomes. nonenveloped virus Virus consisting of a nucleic acid core and a protein capsid only. (Figure 23–18C,D) nonhomologous end joining A DNA repair mechanism for double-strand breaks in which the broken ends of DNA are brought together and rejoined by DNA ligation, generally with the loss of one or more nucleotides at the site of joining.

1	nonretroviral retrotransposons Type of transposable element that moves by being first transcribed into an RNA copy that is converted to DNA by reverse transcriptase then inserted elsewhere in the genome. The mechanism of insertion differs from that of the retroviral-like transposons. (Table 5–4, p. 288) nonsense-mediated mRNA decay Mechanism for degrading aberrant mRNAs containing in-frame internal stop codons before they can be translated into protein. (Figure 6–76) normal flora The human microbiota consisting of approximately 1014 bacterial, fungal, and protozoan cells, representing thousands of microbial species. Notch Transmembrane receptor protein (and latent transcription regulator) involved in many cell-fate choices in animal development, for example in the specification of nerve cells from ectodermal epithelium. Its ligands are cell-surface proteins such as Delta and Serrate. (Figure 15–59)

1	NSF Hexameric ATPase that disassembles a complex of a v-SNARE and a t-SNARE. (Figure 13–20) nuclear envelope Double membrane (two bilayers) surrounding the nucleus. Consists of an outer and inner membrane and is perforated by nuclear pores. The outer membrane is continuous with the endoplasmic reticulum. (Figures 4–9 and 12–7) nuclear export receptors Bind to both the export signal and nuclear pore complex proteins to guide their cargo through the nuclear pore complex to the cytosol. nuclear export signal Sorting signal contained in the structure of molecules and complexes, such as nuclear RNPs and new ribosomal subunits, that are transported from the nucleus to the cytosol through nuclear pore complexes. (Figure 12–13) nuclear import receptors Recognize nuclear localization signals to initiate nuclear import of proteins containing the appropriate nuclear localization signal. nuclear lamin Protein subunit of the intermediate filaments that form the nuclear lamina.

1	nuclear lamin Protein subunit of the intermediate filaments that form the nuclear lamina. nuclear lamina Fibrous meshwork of proteins on the inner surface of the inner nuclear membrane. It is made up of a network of intermediate filaments formed from nuclear lamins.

1	nuclear localization signal (NLS) Signal sequence or signal patch found in proteins destined for the nucleus that enables their selective transport into the nucleus from the cytosol through the nuclear pore complexes. (Figures 12–9 and 12–13) nuclear magnetic resonance (NMR) spectroscopy NMR is the resonant absorption of electromagnetic radiation at a specific frequency by atomic nuclei in a magnetic field, due to flipping of the orientation of their magnetic dipole moments. The NMR spectrum provides information about the chemical environment of the nuclei. NMR is used widely to determine the three-dimensional structure of small proteins and other small molecules. The principles of NMR are also used for medical diagnostic purposes in magnetic resonance imaging (MRI). (Figure 8–22) nuclear pore complex (NPC) Large multiprotein structure forming an aqueous channel (the nuclear pore) through the nuclear envelope that allows selected molecules to move between nucleus and cytoplasm.

1	pore complex (NPC) Large multiprotein structure forming an aqueous channel (the nuclear pore) through the nuclear envelope that allows selected molecules to move between nucleus and cytoplasm. (Figure 12–8) nuclear receptor superfamily Intracellular receptors for hydrophobic signal molecules such as steroid and thyroid hormones and retinoic acid. The receptor-ligand complex acts as a transcription factor in the nucleus. (Figure 15–65) nuclear transport receptor (karyopherin) Protein that escorts macromolecules either into or out of the nucleus: nuclear import receptor or nuclear export receptor. (Figure 12–13) nucleolus A prominent structure in the nucleus where rRNA is transcribed and ribosomal subunits are assembled. (Figure 4–9) nucleoporin Any of a number of different proteins that make up nuclear pore complexes.

1	nucleosome Beadlike structure in eukaryotic chromatin, composed of a short length of DNA wrapped around an octameric core of histone proteins. The fundamental structural unit of chromatin. (Figures 4–22 and 4–23) nucleotide Nucleoside with one or more phosphate groups joined in ester linkages to the sugar moiety. DNA and RNA are polymers of nucleotides. (Panel 2–6, pp. 100–101) nucleotide excision repair Type of DNA repair that corrects damage of the DNA double helix, such as that caused by chemicals or UV light, by cutting out the damaged region on one strand and resynthesizing it using the undamaged strand as template. Compare base excision repair. (Figure 5–41) O-linked glycosylation Addition of one or more sugars to a hydroxyl group on a protein. obligate pathogens Bacteria that can only replicate inside their host.

1	O-linked glycosylation Addition of one or more sugars to a hydroxyl group on a protein. obligate pathogens Bacteria that can only replicate inside their host. olfactory receptors G-protein-coupled receptors on the modified cilia of olfactory receptor neurons that recognize odors. The receptors activate adenylyl cyclase via an olfactoryspecific G protein (Golf) and resultant increases in cAMP open cyclic-AMP-gated cation channels, allowing Na+ influx and depolarization and initiation of a nerve impulse. oligodendrocyte Glial cell in the vertebrate central nervous system that forms a myelin sheath around axons. Compare Schwann cell.

1	oligodendrocyte Glial cell in the vertebrate central nervous system that forms a myelin sheath around axons. Compare Schwann cell. oncogene An altered gene whose product can act in a dominant fashion to help make a cell cancerous. Typically, an oncogene is a mutant form of a normal gene (proto-oncogene) involved in the control of cell growth or division. (Figure 20–17) open reading frame (ORF) A continuous nucleotide sequence free from stop codons in at least one of the three reading frames (and thus with the potential to code for protein). opportunistic pathogens Microbes of the normal flora that can cause disease only if the immune systems are weakened or if they gain access to a normally sterile part of the body.

1	opportunistic pathogens Microbes of the normal flora that can cause disease only if the immune systems are weakened or if they gain access to a normally sterile part of the body. optogenetics Use of genetically engineered channelrhodopsin and other light-responsive ion channels and transporters to modulate neuron function and hence analyze the neurons and circuits underlying complex functions, including behaviors in whole animals. (Figure 11–32) organelle Subcellular compartment or large macromolecular complex, often membrane-enclosed, that has a distinct structure, composition, and function. Examples are nucleus, nucleolus, mitochondrion, Golgi apparatus, and centrosomes. (Figure 1–25) Organizer Specialized tissue at the dorsal lip of the blastopore in an amphibian embryo; a source of signals that help to orchestrate formation of the embryonic body axis.

1	Organizer Specialized tissue at the dorsal lip of the blastopore in an amphibian embryo; a source of signals that help to orchestrate formation of the embryonic body axis. origin recognition complex (ORC) Large protein complex that is bound to the DNA at origins of replication in eukaryotic chromosomes throughout the cell cycle. (Figure 5–31) orthologs Genes or proteins from different species that are similar in sequence because they are descendants of the same gene in the last common ancestor of those species. Compare paralogs. (Figure 1–21) osteoblast Cell that secretes matrix of bone. (Figure 22–14) osteoclast Macrophage-like cell that erodes bone, enabling it to be remodeled during growth and in response to stresses throughout life. (Figure 22–16) osteocyte Nondividing cell in bone that develops from an osteoblast and is embedded in bone matrix. (Figure 22–14) outer membrane Mitochondrial membrane that is in contact with the cytosol.

1	outer mitochondrial membrane Membrane that separates the organelle from the cytosol. outer nuclear membrane One of two concentric membranes comprising the nuclear envelope; surrounds the inner nuclear membrane and is continuous with the inner nuclear membrane and the membrane of the endoplasmic reticulum. OXA complex Protein translocator in the inner mitochondrial membrane that mediates insertion of inner membrane proteins.

1	oxidation (verb oxidize) Loss of electrons from an atom, as occurs during the addition of oxygen to a molecule or when a hydrogen is removed. Opposite of reduction. (Figure 2–20) oxidative phosphorylation Process in bacteria and mitochondria in which ATP formation is driven by the transfer of electrons through the electron transport chain to molecular oxygen. Involves the intermediate generation of a proton gradient (pH gradient) across a membrane and a chemiosmotic coupling of that gradient to the ATP synthase. (Figures 14–12) oxidative phosphorylation Process in bacteria and mitochondria in which ATP formation is driven by the transfer of electrons through the electron-transport chain to molecular oxygen. Involves the intermediate generation of an electrochemical proton gradient across a membrane and a chemiosmotic coupling of that gradient to the ATP synthase. (Figure 14–10)

1	P-type pumps A class of ATP-driven pumps comprising structurally and functionally related multipass transmembrane proteins that phosphorylate themselves during the pumping cycle. The class includes many of the ion pumps responsible for setting up and maintaining gradients of Na+, K+, H+, and Ca2+ across cell membranes. (Figure 11–12) p53 A transcription regulatory protein that is activated by damage to DNA and is involved in blocking further progression through the cell cycle. (Figures 20–37 and 20–40) p53 Tumor suppressor gene that is mutated in about half of human cancers. Encodes a transcription regulator that is activated by damage to DNA and is involved in blocking further progression through the cell cycle. (Figure 20–27) pair-rule gene In Drosophila development, a gene expressed in a series of regular transverse stripes along the body of the embryo and which helps to determine its segments. (Figure 21–19) pairing In meiosis, the lining up of the two homologous chromosomes

1	in a series of regular transverse stripes along the body of the embryo and which helps to determine its segments. (Figure 21–19) pairing In meiosis, the lining up of the two homologous chromosomes along their length. (Figure 17–54) papillomaviruses Class of viruses responsible for human warts and a prime example of DNA tumor viruses, being a cause of cancer of the uterine cervix.

1	paracrine signaling Short-range cell–cell communication via secreted signal molecules that act on neighboring cells. (Figure 15–2) paralogs Genes or proteins that are similar in sequence because they are the result of a gene duplication event occurring in an ancestral organism. Those in two different organisms are less likely to have the same function than are orthologs. Compare orthologs. (Figure 1–21) parasitism Ecological relationship between microbes and their host in which the microbe benefits to the detriment of the host, as is often the case for pathogens. passengers Mutations that have occurred in the same cell as driver mutations, but which are irrelevant to the development of the cancer.

1	passengers Mutations that have occurred in the same cell as driver mutations, but which are irrelevant to the development of the cancer. passive transport (facilitated diffusion) Transport of a solute across a membrane down its concentration gradient or its electrochemical gradient, using only the energy stored in the gradient. (Figure 11–4) patch-clamp recording Electrophysiological technique in which a tiny electrode tip is sealed onto a patch of cell membrane, thereby making it possible to record the flow of current through individual ion channels in the patch. (Figure 11–34) Patched Transmembrane protein predicted to cross the plasma membrane 12 times; much is in intracellular vesicles and some is on the cell surface where it binds the Hedgehog protein. pathogen (adjective pathogenic) An organism, cell, virus, or prion that causes disease.

1	pathogen (adjective pathogenic) An organism, cell, virus, or prion that causes disease. pathogen-associated molecular patterns (PAMPs) Microbe-associated molecules, either not present or sequestered in the host organism, that often occur in repeating patterns that are recognized by pattern recognition receptors (PRRs) in or on cells of the innate immune system. PAMPs are present in various microbial molecules, including nucleic acids, lipids, polysaccharides, and proteins. pattern recognition receptor (PRR) Receptor present on or in cells of the innate immune system that recognizes and is activated by microbial pathogen-associated molecular patterns (PAMPs).

1	pattern recognition receptor (PRR) Receptor present on or in cells of the innate immune system that recognizes and is activated by microbial pathogen-associated molecular patterns (PAMPs). PDZ domain Protein-binding domain present in many scaffold proteins, and often used as a docking site for intracellular tails of transmembrane proteins. (Figure 19–22) pectin Mixture of polysaccharides rich in galacturonic acid which forms a highly hydrated matrix in which cellulose is embedded in plant cell walls. (Figure 19–63) peripheral (secondary) lymphoid organ Lymphoid organ in which T cells and B cells interact and respond to foreign antigens. Examples are spleen, lymph nodes, and mucosalassociated lymphoid organs. (Figure 24–12) peroxins Form a protein translocator that participates in the import of proteins into peroxisomes.

1	peroxisome Small membrane-bounded organelle that uses molecular oxygen to oxidize organic molecules. Contains some enzymes that produce and others that degrade hydrogen peroxide (H2O2). (Figure 12–27) pH scale Common measure of the acidity of a solution: “p” refers to power of 10, “H” to hydrogen. Defined as the negative logarithm of the hydrogen ion concentration in moles per liter (M). pH = –log [H+]. Thus a solution of pH 3 will contain 10–3 M hydrogen ions. pH less than 7 is acidic and pH greater than 7 is alkaline. phagocytosis Process by which unwanted cells, debris, and other bulky particulate material is endocytosed (“eaten”) by a cell. Prominent in carnivorous cells, such as Amoeba proteus, and in vertebrate macrophages and neutrophils. From Greek phagein, to eat.

1	phagosome Large intracellular membrane-enclosed vesicle that is formed as a result of phagocytosis. Contains ingested extracellular material. (Figure 13–61) phase variation The random switching of phenotype and expression of proteins involved in infection at frequencies much higher than mutation rates.

1	phase-contrast microscope Type of light microscope that exploits the interference effects that occur when light passes through material of different refractive indices. Used to view living cells. (Figure 9–7) phenotype The observable character (including both physical appearance and behavior) of a cell or organism. (Panel 8–2, p. 486) phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2, PIP2] Membrane inositol phospholipid (a phosphoinositide) that is cleaved by phospholipase C into IP3 and diacylglycerol at the beginning of the inositol phospholipid signaling pathway. It can also be phosphorylated by PI 3-kinase to produce PIP3 docking sites for signaling proteins in the PI-3-kinase–Akt signaling pathway. (Figures 15–28 and 15–53) phosphoglyceride Phospholipid derived from glycerol, abundant in biomembranes. (Figures 10–2 and 10–3) phosphoinositide A lipid containing a phosphorylated inositol derivative. Minor component of the plasma membrane, but important in demarking different

1	in biomembranes. (Figures 10–2 and 10–3) phosphoinositide A lipid containing a phosphorylated inositol derivative. Minor component of the plasma membrane, but important in demarking different membranes and for intracellular signal transduction in eukaryotic cells. (Figure 15–52) phosphoinositide 3-kinase (PI 3-kinase) Membrane-bound enzyme that is a component of the PI-3-kinase–Akt intracellular signaling pathway. It phosphorylates phosphatidylinositol 4,5-bisphosphate at the 3 position on the inositol ring to produce PIP3 docking sites in the membrane for other intracellular signaling proteins. (Figure 15–53) phosphoinositides (PIPs; phosphatidylinositol phosphates) A lipid containing a phosphorylated inositol derivative. Minor component of the plasma membrane, but important in demarking different membranes and for intracellular signal transduction in eukaryotic cells. (Figure 13–10) phospholipase C (PLC) Membrane-bound enzyme that cleaves inositol phospholipids to produce IP3 and

1	different membranes and for intracellular signal transduction in eukaryotic cells. (Figure 13–10) phospholipase C (PLC) Membrane-bound enzyme that cleaves inositol phospholipids to produce IP3 and diacylglycerol in the inositol phospholipid signaling pathway. PLCβ is activated by GPCRs via specific G proteins, while PLCγ is activated by RTKs. (Figure 15–55) phospholipid The main category of lipids used to construct biomembranes. Generally composed of two fatty acids linked through glycerol (or sphingosine) phosphate to one of a variety of polar groups. (FIgure 10–3, and Panel 2–5, pp. 98–99) phosphorylation Reaction in which a phosphate group is covalently coupled to another molecule.

1	photoactivation Technique for studying intracellular processes in which an inactive form of a molecule of interest is introduced into the cell, and is then activated by a focused beam of light at a precise spot in the cell. (Figure 9–28) photochemical reaction center The part of a photosystem that converts light energy into chemical energy in photosynthesis. (Figure 14–44) photosynthetic electron-transfer reactions Light-driven reactions in photosynthesis in which electrons move along an electron-transport chain in a membrane, generating ATP and NADPH. photosystem Multiprotein complex involved in photosynthesis that captures the energy of sunlight and converts it to useful forms of energy: a reaction center plus an antenna (Figure 14–45) phototropin Photoprotein associated with the plant plasma membrane that senses blue light and is partly responsible for phototropism.

1	phragmoplast Structure made of microtubules and actin filaments that forms in the prospective plane of division of a plant cell and guides formation of the cell plate. (Figure 17–49) phytochrome Plant photoprotein that senses light via a covalently attached light-absorbing chromophore, which changes its shape in response to light and then induces a change in the protein’s conformation. Plant phytochromes are dimeric, cytoplasmic serine/threonine kinases, which respond differentially and reversibly to red and far-red light to alter cell behavior.

1	PI-3-kinase–Akt pathway Intracellular signaling pathway that stimulates animal cells to survive and grow. (Figure 15–53) pinocytosis Literally, “cell drinking.” Type of endocytosis in which soluble materials are continually taken up from the environment in small vesicles and moved into endosomes along with the membrane-bound molecules. Compare phagocytosis. (Figure 13–48) piRNAs (piwi-interacting RNAs) A class of small noncoding RNAs made in the germ line that, in complex with Piwi proteins, keep in check the movement of transposable elements by transcriptionally silencing transposon genes and destroying RNAs produced by them.

1	planar cell polarity Type of cellular asymmetry seen in some epithelia, such that each cell has a polarity vector oriented in the plane of the epithelium. (Figure 21–51) plant growth regulator (plant hormone) Signal molecule that helps coordinate growth and development. Examples are ethylene, auxins, gibberellins, cytokinins, abscisic acid, and the brassinosteroids. plasma membrane The membrane that surrounds a living cell. (Figure 10–1) plasmid vector Small, circular molecules of double-stranded DNA derived from plasmids that occur naturally in bacterial cells; widely used for gene cloning. plasmodesma (plural plasmodesmata) Plant equivalent of a gap junction. Communicating cell–cell junction in plants in which a channel of cytoplasm lined by plasma membrane connects two adjacent cells through a small pore in their cell walls.

1	platelet Cell fragment, lacking a nucleus, that breaks off from a megakaryocyte in the bone marrow and is found in large numbers in the bloodstream. Helps initiate blood clotting when blood vessels are injured. (Figure 22–29) pleckstrin homology domain (PH domain) Protein domain found in some intracellular signaling proteins. Some PH domains in intracellular signaling proteins bind to phosphatidylinositol 3,4,5-trisphosphate produced by PI 3-kinase, bringing the signaling protein to the plasma membrane when PI 3-kinase is activated. pluripotent Describes a cell that has the potential to give rise to all or almost all of the cell types of the adult body. polarized In epithelia, that the basal end of a cell, adherent to the basal lamina below, differs from the apical end, exposed to the medium above; thus, all epithelia and their individual cells are structurally polarized.

1	Polycomb group Set of proteins critical for cell memory for some genes. They form complexes as part of the chromatin of the Hox complex, where they maintain a repressed state in cells where Hox genes have not been activated. polymerase chain reaction (PCR) Technique for amplifying specific regions of DNA by the use of sequence-specific primers and multiple cycles of DNA synthesis, each cycle being followed by a brief heat treatment to separate complementary strands. (Figure 8–36) polymorphisms Describes genome sequences that coexist as two or more sequence variants at high frequency in a population. polypeptide Linear polymer of amino acids. Proteins are large polypeptides, and the two terms can be used interchangeably. (Panel 3–1, pp. 112–113) polypeptide backbone Repeating sequence of atoms along the core of the polypeptide chain. polyribosome mRNA engaged with multiple ribosomes in the act of translation.

1	polyribosome mRNA engaged with multiple ribosomes in the act of translation. polytene chromosome Giant chromosome in which the DNA has undergone repeated replication and the many copies have stayed together in precise alignment. (Figures 4–50 and 4–51) porin Channel-forming proteins of the outer membranes of bacteria, mitochondria, and chloroplasts. position effect variegation Alteration in gene expression resulting from change in the position of the gene in relation to other chromosomal domains, especially heterochromatic domains. When an active gene is placed next to heterochromatin, the inactivating influence of the heterochromatin can spread to affect the gene to a variable degree, giving rise to position effect variegation. (Figure 4–31) positional value A cell’s internal record of its positional information in a multicellular organism; an intrinsic character that differs according to a cell’s location.

1	positive feedback Control mechanism whereby the end product of a reaction or pathway stimulates its own production or activation. positive selection In immunology: process of thymocyte maturation in which thymocytes expressing a T cell receptor with appropriate affinity for a self peptide bound to a self MHC protein is signaled to survive and continue development. post-transcriptional controls Any control on gene expression that is exerted at a stage after transcription has begun. (Figure 7–54) post-translational Occurring after completion of translation.

1	preprophase band Circumferential band of microtubules and actin filaments that forms around a plant cell under the plasma membrane prior to mitosis and cell division. (Figure 17–49) prereplicative complex (preRC) Multiprotein complex that is assembled at origins of replication during late mitosis and early G1 phases of the cell cycle; a prerequisite to license the assembly of a preinitiation complex, and the subsequent initiation of DNA replication. (Figures 17–17 and 17–18) primary cell wall The first cell wall produced by a developing plant cell; it is thin and flexible, allowing room for cell growth. (Figure 19–63) primary cilium Short, single, nonmotile cilium lacking dynein that arises from a centriole and projects from the surface of many animal cell types. Some signaling proteins are concentrated in the primary cilium. (Figure 15–38) primary Ig repertoire The billions of IgM and IgD immunoglobulin molecules made by the B cells of an adaptive immune system in the absence of

1	are concentrated in the primary cilium. (Figure 15–38) primary Ig repertoire The billions of IgM and IgD immunoglobulin molecules made by the B cells of an adaptive immune system in the absence of antigen stimulation.

1	primary immune response Adaptive immune response to an antigen that is made on first encounter with that antigen. (Figure 24–16) primary pathogens Pathogens that can cause overt disease in most healthy people. Some cause acute, life-threatening epidemic infections and spread rapidly between hosts; other potential primary pathogens may persistently infect a single individual for years without causing overt disease, the host often being unaware that they are infected. primary structure Linear sequence of monomer units in a polymer, such as the amino acid sequence of a protein. primary tumor Tumor at the original site at which a cancer first arose. Secondary tumors develop elsewhere by metastasis.

1	primary tumor Tumor at the original site at which a cancer first arose. Secondary tumors develop elsewhere by metastasis. prion disease Transmissible spongiform encephalopathy— such as Kuru and Creutzfeldt–Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE, or “mad cow disease”) in cows—that is caused and transmitted by an infectious, abnormally folded protein (prion). (Figure 3–33) pro-inflammatory cytokine Any cytokine that stimulates an inflammatory response. programmed cell death A form of cell death in which a cell kills itself by activating an intracellular death program.

1	prokaryote Single-celled microorganism whose cells lack a well-defined, membrane-enclosed nucleus. Either a bacterium or an archaeon. (Figure 1–17) promoter Nucleotide sequence in DNA to which RNA polymerase binds to begin transcription. (Figure 7–17) proteasome Large protein complex in the cytosol with proteolytic activity that is responsible for degrading proteins that have been marked for destruction by ubiquitylation or by some other means. (Figures 6–83 and 6–84) protein The major macromolecular constituent of cells. A linear polymer of amino acids linked together by peptide bonds in a specific sequence. (Figure 3–1) protein activity control The selective activation, inactivation, degradation, or compartmentalization of specific proteins after they have been made. One of the means by which a cell controls which proteins are active at a given time or location in the cell.

1	protein glycosylation Process of transferring a single saccharide or preformed precursor oligosaccharide to proteins. protein kinase Enzyme that transfers the terminal phosphate group of ATP to one or more specific amino acids (serine, threonine, or tyrosine) of a target protein. protein kinase C (PKC) Ca2+-dependent protein kinase that, when activated by diacylglycerol and an increase in the concentration of cytosolic Ca2+, phosphorylates target proteins on specific serine and threonine residues. (Figure 15–29) protein phosphatase Enzyme that catalyzes phosphate removal from amino acids of a target protein. protein subunit An individual protein chain in a protein composed of more than one chain. protein translocation Process of moving a protein across a membrane.

1	protein subunit An individual protein chain in a protein composed of more than one chain. protein translocation Process of moving a protein across a membrane. protein translocator Membrane-bound protein that mediates the transport of another protein across a membrane. (Figure 12–21) protein tyrosine phosphatase Enzyme that removes phosphate groups from phosphorylated tyrosine residues on proteins. proteoglycan Molecule consisting of one or more glycosaminoglycan chains attached to a core protein. (Figure 19–38) proteomics Study of all the proteins, including all the covalently modified forms of each, produced by a cell, tissue, or organism. Proteomics often investigates changes in this larger set of proteins—in “the proteome”—caused by changes in the environment or by extracellular signals. proto-oncogene Normal gene, usually concerned with the regulation of cell proliferation, that can be converted into a cancer-promoting oncogene by mutation.

1	proto-oncogene Normal gene, usually concerned with the regulation of cell proliferation, that can be converted into a cancer-promoting oncogene by mutation. protofilament Linear string of microtubule subunits joined end to end; multiple protofilaments associate with one another laterally to construct and provide strength and adaptability to microtubules. proton (H+) Positively charged subatomic particle that forms part of an atomic nucleus. Hydrogen has a nucleus composed of a single proton (H+). proton-motive force The force exerted by the electrochemical proton gradient that moves protons across a membrane. protozoan parasite Parasitic, nonphotosynthetic, single-celled, motile eukaryotic organism, for example Plasmodium. pseudogene Nucleotide sequence of DNA that has accumulated multiple mutations that have rendered an ancestral gene inactive and nonfunctional.

1	pseudogene Nucleotide sequence of DNA that has accumulated multiple mutations that have rendered an ancestral gene inactive and nonfunctional. purified cell-free system Fractionated cell homogenate that retains a particular biological function of the intact cell, and in which biochemical reactions and cell processes can be more easily studied. purifying selection Natural selection operating in a population to slow genome changes and reduce divergence by eliminating individuals carrying deleterious mutations. quantitative RT-PCR (reverse transcription–polymerase chain reaction) Technique in which a population of mRNAs is converted into cDNAs via reverse transcription, and the cDNAs are then amplified by PCR. The quantitative part relies on a direct relationship between the rate at which the PCR product is generated and the original concentration of the mRNA species of interest.

1	quaternary structure Three-dimensional relationship of the different polypeptide chains in a multisubunit protein or protein complex. quinone (Q) Small, lipid-soluble, mobile electron carrier molecule found in the respiratory and photosynthetic electron-transport chains. (Figure 14–17) Rab cascade An ordered recruitment of sequentially acting Rab proteins into Rab domains on membranes, which changes the identity of an organelle and reassigns membrane dynamics. Rab effectors Molecules that bind activated, membrane-bound Rab proteins and act as downstream mediators of vesicle transport, membrane tethering, and fusion. Rab proteins Monomeric GTPase in the Ras superfamily present in plasma and organelle membranes in its GTP-bound state, and as a soluble cytosolic protein in its GDP-bound state. Involved in conferring specificity on vesicle docking. (Table 15–5, p. 854)

1	Rac Member of the Rho family of monomeric GTPases that regulate the actin and microtubule cytoskeletons, cell-cycle progression, gene transcription, and membrane transport. Rad51 Eukaryotic protein that catalyzes synapsis of DNA strands during genetic recombination. Called RecA in E. coli. Ran (Ran protein) Monomeric GTPase of the Ras superfamily present in both cytosol and nucleus. Required for the active transport of macromolecules into and out of the nucleus through nuclear pore complexes. (Table 15–5, p. 854) rapidly inactivating K+ channel Neuronal voltage-gated K+ channel, open when the membrane is depolarized, with a specific voltage sensitivity and kinetics of inactivation that induce a reduced rate of action potential firing at levels of stimulation only just above the threshold required, thereby resulting in a firing rate proportional to the strength of the depolarizing stimulus.

1	Ras A small family of proto-oncogenes that are frequently mutated in cancers, each of which produces a Ras monomeric GTPase. Ras (Ras protein) Monomeric GTPase of the Ras superfamily that helps to relay signals from cell-surface receptor tyrosine kinase receptors to the nucleus, frequently in response to signals that stimulate cell division. Named for the ras gene, first identified in viruses that cause rat sarcomas. (Figure 3–67) Ras superfamily Large superfamily of monomeric GTPases (also called small GTP-binding proteins) of which Ras is the prototypical member. (Table 15–5, p. 854) Ras-GAPs Ras GTPase-activating proteins; increase the rate of hydrolysis of bound GTP by Ras, thereby inactivating Ras. Ras-GEFs Ras guanine nucleotide exchange factors; stimulate the dissociation of GDP and the subsequent uptake of GTP from the cytosol, thereby activating Ras.

1	Ras-GEFs Ras guanine nucleotide exchange factors; stimulate the dissociation of GDP and the subsequent uptake of GTP from the cytosol, thereby activating Ras. Ras–MAP-kinase signaling pathway Intracellular signaling pathway that relays signals from activated receptor tyrosine kinases to effector proteins in the cell including transcription regulators in the nucleus. Rb gene The gene that is defective in both copies in individuals with retinoblastoma; its protein product plays a central role in cell-cycle control. reading frame The phase in which nucleotides are read in sets of three to encode a protein. An mRNA molecule can be read in any one of three reading frames, only one of which will give the required protein. (Figure 6–49)

1	RecA (RecA protein) Prototype for a class of DNA-binding proteins that catalyze synapsis of DNA strands during genetic recombination. (Figure 5–49) receptor Any protein that binds a specific signal molecule (ligand) and initiates a response in the cell. Some are on the cell surface, while others are inside the cell. (Figure 15–3) receptor editing Process by which a developing B cell that recognizes a self molecule changes its antigen receptors so that the cell no longer does so.

1	receptor serine/threonine kinase Cell-surface receptor with an extracellular ligand-binding domain and an intracellular kinase domain that phosphorylates signaling proteins on serine or threonine residues in response to ligand binding. The TGFβ receptor is an example. (Figure 15–57) receptor tyrosine kinase (RTK) Cell-surface receptor with an extracellular ligand-binding domain and an intracellular kinase domain that phosphorylates signaling proteins on tyrosine residues in response to ligand binding. (Figure 15–43 and Table 15–4, p. 850) receptor-mediated endocytosis Internalization of receptor– ligand complexes from the plasma membrane by endocytosis. (Figure 13–52) recombinant DNA technology Collection of techniques by which DNA segments from different sources are combined to make a new DNA, often called a recombinant DNA. Recombinant DNAs are widely used in the cloning of genes, in the genetic modification of organisms, and in the production of large amounts of rare proteins.

1	recycling endosome Organelle that provides an intermediate stage on the passage of recycled receptors back to the cell membrane. Regulates plasma membrane insertion of some proteins. (Figure 13–58) red blood cell Small hemoglobin-containing blood cell of vertebrates that transports oxygen to, and carbon dioxide from, tissues. Also called an erythrocyte.

1	redox pair Pair of molecules in which one acts as an electron donor and one as an electron acceptor in an oxidation– reduction reaction: for example, NADH (electron donor) and NAD+ (electron acceptor). (Panel 14–1, p. 765) redox potential The affinity of a redox pair for electrons, generally measured as the voltage difference between an equimolar mixture of the pair and a standard reference. NADH/ NAD+ has a low redox potential and O2/H2 has a high redox potential (high affinity for electrons). (Panel 14–1, p. 765) redox reaction Reaction in which one component becomes oxidized and the other reduced; an oxidation–reduction reaction. (Panel 14–1, p. 765) reduction (verb reduce) Addition of electrons to an atom, as occurs during the addition of hydrogen to a biological molecule or the removal of oxygen from it. Opposite of oxidation. (Figure 2–20) regulated nuclear transport Mechanisms controlling export of mRNAs from the nucleus to the cytosol that can be used to regulate gene

1	removal of oxygen from it. Opposite of oxidation. (Figure 2–20) regulated nuclear transport Mechanisms controlling export of mRNAs from the nucleus to the cytosol that can be used to regulate gene expression. Also includes the selective import of proteins and RNA molecules into the nucleus.

1	regulated secretory pathway A second secretory pathway found mainly in cells specialized for secreting products rapidly on demand—such as hormones, neurotransmitters, or digestive enzymes—in which soluble proteins and other substances are initially stored in secretory vesicles for later release. (Figure 13–62) regulator of G protein signaling (RGS) A GAP protein that binds to a trimeric G protein and enhances its GTPase activity, thus helping to limit G-protein-mediated signaling. (Figure 15–8) regulatory site Region of an enzyme surface to which a regulatory molecule binds and thereby influences the catalytic events at the separate active site. regulatory T cell (Treg) A type of T cell that suppresses the development, activation, or function of other immune cells via secreted cytokines or cell-surface inhibitory proteins.

1	regulatory T cell (Treg) A type of T cell that suppresses the development, activation, or function of other immune cells via secreted cytokines or cell-surface inhibitory proteins. replication fork Y-shaped region of a replicating DNA molecule at which the two strands of the DNA are being separated and the daughter strands are being formed. (Figures 5–7 and 5–18) replication origin Location on a DNA molecule at which duplication of the DNA begins. (Figures 4–19 and 5–23) replicative cell senescence Phenomenon observed in primary cell cultures in which cell proliferation slows down and finally irreversibly halts.

1	respiratory chain (electron-transport chain) Electron-transport chain present in the inner mitochondrial membrane that generates an electrochemical gradient across the membrane that is used to drive ATP synthesis. (Figures 14–4 and 14–10) resting membrane potential Electrical potential across the plasma membrane of a cell at rest, i.e. a cell that has not been stimulated to open additional ion channels than those that are normally open.

1	restriction nuclease One of a large number of nucleases that can cleave a DNA molecule at any site where a specific short sequence of nucleotides occurs. Extensively used in recombinant DNA technology. (Figure 8–24) restriction point Important transition at the end of G1 in the eukaryotic cell cycle; commits the cell to enter S phase. The term was originally used for this transition in the mammalian cell cycle; in this book we use the term Start. (Figure 17–9) retinoblastoma A rare type of human cancer arising from cells in the retina of the eye that are converted to a cancerous state by an unusually small number of mutations. Studies of retinoblastoma led to the discovery of the first tumor suppressor gene.

1	retinoblastoma protein (Rb protein) Tumor suppressor protein involved in the regulation of cell division. Mutated in the cancer retinoblastoma, as well as in many other tumors. Its normal activity is to regulate the eukaryotic cell cycle by binding to and inhibiting the E2F proteins, thus blocking progression to DNA replication and cell division. (Figure 17–61) retroviral-like retrotransposons A large family of transposons that move themselves in and out of chromosomes by a mechanism similar to that used by retroviruses, being first transcribed into an RNA copy that is converted to DNA by reverse transcriptase then inserted elsewhere in the genome. (Table 5–4, p. 288) retrovirus RNA-containing virus that replicates in a cell by first making an RNA–DNA intermediate and then a double-strand DNA molecule that becomes integrated into the cell’s DNA. (Figure 5–62) reverse genetics Approach to discovering gene function that starts from the DNA (gene) and its protein product and then

1	DNA molecule that becomes integrated into the cell’s DNA. (Figure 5–62) reverse genetics Approach to discovering gene function that starts from the DNA (gene) and its protein product and then creates mutants to analyze the gene’s function.

1	reverse transcriptase Enzyme first discovered in retroviruses that makes a double-strand DNA copy from a single-strand RNA template molecule. RGD sequence Tripeptide sequence of arginine-glycineaspartic acid that forms a binding site for integrins; present in fibronectin and some other extracellular proteins. (Figure 19–47C) Rheb A monomeric Ras-related GTPase that in its active form (Rheb-GTP) activates mTOR, which promotes cell growth. Rho Member of the Rho family of monomeric GTPases that regulate the actin and microtubule cytoskeletons, cell-cycle progression, gene transcription, and membrane transport.

1	Rho Member of the Rho family of monomeric GTPases that regulate the actin and microtubule cytoskeletons, cell-cycle progression, gene transcription, and membrane transport. Rho family Family of monomeric GTPases within the Ras superfamily involved in signaling the rearrangement of the cytoskeleton. Includes Rho, Rac, and Cdc42. (Table 15–5, p. 854) rhodopsin Seven-span membrane protein of the GPCR family that acts as a light sensor in rod photoreceptor cells in the vertebrate retina. Contains the light-sensitive prosthetic group retinol. (Figure 15–39) ribosomal RNA (rRNA) Any one of a number of specific RNA molecules that form part of the structure of a ribosome and participate in the synthesis of proteins. Often distinguished by their sedimentation coefficient (e.g., 28S rRNA, 5S rRNA).

1	ribosome Particle composed of rRNAs and ribosomal proteins that catalyzes the synthesis of protein using information provided by mRNA. (Figure 6–64) ribozyme An RNA molecule with catalytic activity. RNA (ribonucleic acid) Polymer formed from covalently linked ribonucleotide monomers. See also messenger RNA, ribosomal RNA, transfer RNA. (Figure 6–4) RNA editing Type of RNA processing that alters the nucleotide sequence of an RNA transcript after it is synthesized by inserting, deleting, or altering individual nucleotides. RNA interference (RNAi) As originally described, mechanism by which an experimentally introduced double-stranded RNA induces sequence-specific destruction of complementary mRNAs. The term RNAi is often used to include the inhibition of gene expression by microRNAs (miRNAs) and piwi RNAs (piRNAs), which are encoded in the cell’s own genome.

1	RNA polymerase Enzyme that catalyzes the synthesis of an RNA molecule on a DNA template from ribonucleoside triphosphate precursors. (Figure 6–9) RNA primer Short stretch of RNA synthesized on a DNA template. It is required by DNA polymerases to start their DNA synthesis. RNA processing control Regulation by a cell of gene expression by controlling the processing of RNA transcripts, which includes their splicing. RNA splicing Process in which intron sequences are excised from RNA transcripts. A major process in the nucleus of eukaryotic cells leading to formation of messenger RNAs (mRNAs). RNA transport and localization control Regulation by a cell of gene expression by selecting which completed mRNAs are exported from the nucleus to the cytosol and determining where in the cytosol they are localized. RNA world Hypothesis that early life on Earth was based primarily on RNA molecules that both stored genetic information and catalyzed biochemical reactions.

1	RNA world Hypothesis that early life on Earth was based primarily on RNA molecules that both stored genetic information and catalyzed biochemical reactions. RNA-seq Sequencing the entire repertoire of RNA from a cell or tissue; also known as deep RNA sequencing. robustness The ability of biological regulatory systems to function normally in the face of perturbations such as exposure to frequent and/or extreme variations in external conditions or the concentrations or activities of key components. rod photoreceptor (rod) Photoreceptor cell in the vertebrate retina that is responsible for noncolor vision in dim light. rough endoplasmic reticulum (rough ER) Endoplasmic reticulum with ribosomes on its cytosolic surface. Involved in the synthesis of secreted and membrane-bound proteins. rRNA gene Gene that specifies a ribosomal RNA (rRNA).

1	rRNA gene Gene that specifies a ribosomal RNA (rRNA). ryanodine receptor A regulated Ca2+ channel in the ER membrane that opens in response to rising Ca2+ levels and thus amplifies the Ca2+ signal. SAM complex Protein translocator that helps β-barrel proteins to fold properly in the outer mitochondrial membrane. Sar1 protein Monomeric GTPase responsible for regulating COPII coat assembly at the endoplasmic reticulum membrane. sarcoma Cancer of connective tissue. scaffold protein Protein that binds groups of intracellular signaling proteins into a signaling complex, often anchoring the complex at a specific location in the cell. (Figure 15–10) scanning electron microscope Type of electron microscope that produces an image of the surface of an object. (Figure 9–50) S-Cdk Cyclin–Cdk complex formed in vertebrate cells by an S-cyclin and the corresponding cyclin-dependent kinase (Cdk). (Figure 17–11 and Table 17–1, p. 969)

1	S-Cdk Cyclin–Cdk complex formed in vertebrate cells by an S-cyclin and the corresponding cyclin-dependent kinase (Cdk). (Figure 17–11 and Table 17–1, p. 969) SCF Family of ubiquitin ligases formed as a complex of several different proteins. One is involved in regulating the eukaryotic cell cycle, directing the destruction of inhibitors of S-Cdks in late G1 and thus promoting the activation of S-Cdks and DNA replication. (Figures 3–71 and 17–15) Schwann cell Glial cell responsible for forming myelin sheaths in the peripheral nervous system. Compare oligodendrocyte. (Figure 11–33) S-cyclin Member of a class of cyclins that accumulate during late G1 phase and bind Cdks soon after progression through Start; they help stimulate DNA replication and chromosome duplication. Levels remain high until late mitosis, after which these cyclins are destroyed. (Figure 17–11)

1	Sec61 complex Three-subunit core of the protein translocator that transfers polypeptide chains across the endoplasmic reticulum membrane. second messenger (small intracellular mediator) Small intracellular signaling molecule that is formed or released for action in response to an extracellular signal and helps to relay the signal within the cell. Examples include cyclic AMP, cyclic GMP, IP3, Ca2+, and diacylglycerol. secondary cell wall Permanent rigid cell wall that is laid down underneath the thin primary cell wall in certain plant cells that have completed their growth. secondary Ig repertoire Immunoglobulins produced by B cells after antigenand helper-T-cell-induced somatic hypermutation and class switching. Compared to the primary Ig repertoire, these Igs have a greatly increased diversity of both Ig classes and antigen-binding sites and have increased affinity for antigen.

1	secondary immune response The adaptive immune response that occurs in response to a second or subsequent exposure to an antigen. The response is more rapid in onset and stronger than the primary immune response. (Figure 24–16) secondary structure Regular local folding pattern of a polymeric molecule; in proteins, α helices and β sheets. secretion system Specialized bacterial systems that secrete effector proteins that interact with host cells.

1	secretion system Specialized bacterial systems that secrete effector proteins that interact with host cells. secretory vesicle Membrane-enclosed organelle in which molecules destined for secretion are stored prior to release. Sometimes called secretory granule because darkly staining contents make the organelle visible as a small solid object. (Figures 13–65) securin Protein that binds to the protease separase and thereby prevents its cleavage of the protein linkages that hold sister chromatids together in early mitosis. Securin is destroyed at the metaphase-to-anaphase transition. (Figure 17–38) segment Divisions of an insect body along its anteroposterior axis, each forming highly specialized structures, but all built according to a similar fundamental plan.

1	segment-polarity gene In Drosophila development, a gene involved in specifying the anteroposterior organization of each body segment. (Figure 21–19) segmentation clock The gene-expression oscillator controlling regular segmentation during vertebrate embryonic development. segmentation genes Genes expressed by subsets of cells in the embryo that refine the pattern of gene expression so as to define the boundaries and ground plan of the individual body segments.

1	segmentation genes Genes expressed by subsets of cells in the embryo that refine the pattern of gene expression so as to define the boundaries and ground plan of the individual body segments. selectin Member of a family of cell-surface carbohydrate-binding proteins that mediate transient, Ca2+-dependent cell–cell adhesion in the bloodstream—for example between white blood cells and the endothelium of the blood vessel wall. (Figure 19–28) selectivity filter The part of an ion channel structure that determines which ions it can transport. (Figures 11–24 and 11–25) sensory bristles Miniature sense organs present on most exposed surfaces of Drosophila, consisting of a sensory neuron and supporting cells and responding to chemical or mechanical stimuli.

1	separase Protease that cleaves the cohesin protein linkages that hold sister chromatids together. Acts at anaphase, enabling chromatid separation and segregation. (Figure 17–38) septum Structure formed during bacterial cell division by the inward growth of the cell wall and plasma membrane and that divides the cell into two. sequential induction Development process that generates a progressively more complicated pattern. A series of local inductions whereby one of two cell types present in a developing tissue can produce a signal to induce neighboring cells to specialize in a third way; the third cell type can then signal back to the other two cell types nearby to generate a fourth and a fifth cell type, and so on. serine protease Type of protease that has a reactive serine in the active site. (Figures 3–12 and 3–39) serine/threonine kinase Enzyme that phosphorylates specific proteins on serine or threonines.

1	SH2 domain Src homology region 2, a protein domain present in many signaling proteins. Binds a short amino acid sequence containing a phosphotyrosine. (Panel 3–2, pp. 142–143) side chain The part of an amino acid that differs between amino acid types. The side chains give each type of amino acid its unique physical and chemical properties. (Panel 3–1, pp. 112–113) signal patch Protein-sorting signal that consists of a specific three-dimensional arrangement of atoms on the folded protein’s surface. (Figure 13–46) signal peptidase Enzyme that removes a terminal signal sequence from a protein once the sorting process is complete. (Figure 12–35) signal sequence Short continuous sequence of amino acids that determines the eventual location of a protein in the cell. An example is the N-terminal sequence of 20 or so amino acids that directs nascent secretory and transmembrane proteins to the endoplasmic reticulum. (Table 12–3, p. 648) signal-recognition particle (SRP)

1	is the N-terminal sequence of 20 or so amino acids that directs nascent secretory and transmembrane proteins to the endoplasmic reticulum. (Table 12–3, p. 648) signal-recognition particle (SRP) Ribonucleoprotein particle that binds an ER signal sequence on a partially synthesized polypeptide chain and directs the polypeptide and its attached ribosome to the endoplasmic reticulum. (Figure 12–36) signaling center Cluster of specialized cells in developing tissues that serves as a source of developmental signals—for example, the generation of a morphogen gradient.

1	single-nucleotide polymorphism (SNP) A variation between individuals in a population due to a relatively common difference in a specific nucleotide at a defined point in the DNA sequence.

1	single-particle reconstruction Computational procedure in electron microscopy in which images of many identical molecules are obtained and digitally combined to produce an averaged three-dimensional image, thereby revealing structural details that are hidden by noise in the original images. (Figures 9–54 and 9–55) single-pass transmembrane protein Membrane protein in which the polypeptide chain crosses the lipid bilayer only once. (Figure 10–24) single-strand DNA-binding (SSB) protein Protein that binds to the single strands of the opened-up DNA double helix, preventing helical structures from reforming while the DNA is being replicated. (Figure 5–15) sister chromatids Tightly linked pair of chromosomes that arise from chromosome duplication during S phase. They separate during M phase and segregate into different daughter cells. (Figure 17–21) sliding clamp Protein complex that holds the DNA polymerase on DNA during DNA replication. (Figure 5–17)

1	Slit Signal protein, secreted by cells of the neural tube floor plate, responsible for repelling the growth cones of commissural axons after they have crossed the midline, thereby ensuring these neurons do not re-cross the midline.

1	Smad family Latent transcription regulators that are phosphorylated and activated by receptor serine/threonine kinases and carry the signal from the cell surface to the nucleus. (Figure 15–57) small interfering RNAs (siRNAs) Short (21–26 nucleotide) double-stranded RNAs that inhibit gene expression by directing destruction of complementary mRNAs. Production of siRNAs is usually triggered by exogenously introduced double-stranded RNA. (Figure 7–77) small nuclear RNA (snRNA) Small RNA molecules that are complexed with proteins to form the ribonucleoprotein particles (snRNPs) involved in RNA splicing. (Figures 6–28 and 6–29) small nucleolar RNA (snoRNA) Small RNAs found in the nucleolus, with various functions, including guiding the modifications of precursor rRNA. (Table 6–1, p. 305, and Figure 6–41) smooth endoplasmic reticulum (smooth ER) Region of the endoplasmic reticulum not associated with ribosomes. Involved in detoxification reactions, Ca2+ storage, and lipid synthesis.

1	and Figure 6–41) smooth endoplasmic reticulum (smooth ER) Region of the endoplasmic reticulum not associated with ribosomes. Involved in detoxification reactions, Ca2+ storage, and lipid synthesis. (Figure 12–33)

1	Smoothened Seven-pass transmembrane protein with a structure very similar to a GPCR but does not seem to act as a Hedgehog receptor or as an activator of G proteins; it is controlled by the Patched and iHog proteins. SNARE proteins (SNAREs) Members of a large family of transmembrane proteins present in organelle membranes and the vesicles derived from them. SNAREs catalyze the many membrane fusion events in cells. They exist in pairs—a v-SNARE in the vesicle membrane that binds specifically to a complementary t-SNARE in the target membrane.

1	sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE) Type of electrophoresis used to separate proteins by size. The protein mixture to be separated is first treated with a powerful negatively charged detergent (SDS) and with a reducing agent (β-mercaptoethanol), before being run through a polyacrylamide gel. The detergent and reducing agent unfold the proteins, free them from association with other molecules, and separate the polypeptide subunits. somatic cell Any cell of a plant or animal other than cells of the germ line. From Greek soma, body. somatic hypermutation In immunology: accumulation of point mutations in the assembled variable-region-coding sequences of immunoglobulin genes that occurs when B cells are activated to form memory cells. Results in the production of antibodies with altered antigen-binding sites, some of which bind antigen with increased affinity; it is responsible for affinity maturation in antibody responses.

1	somatic mutations In cancer, one or more detectable abnormalities in the DNA sequence of tumor cells that distinguish them from the normal somatic cells surrounding the tumor. somite One of a series of paired blocks of mesoderm that form during early development and lie on either side of the notochord in a vertebrate embryo. They give rise to the segments of the body axis, including the vertebrae, muscles, and associated connective tissue. (Figure 21–38) sorting signal Signal sequence or signal patch that directs the delivery of a protein to a specific location, such as a particular intracellular compartment. spectrin Abundant protein associated with the cytosolic side of the plasma membrane in red blood cells, forming a network that supports the membrane. Also present in other cells. (Figure 10–38) S phase Period of a eukaryotic cell cycle in which DNA is synthesized. (Figure 17–4) spinal cord Bundle of neurons and support cells that extends from the brain.

1	S phase Period of a eukaryotic cell cycle in which DNA is synthesized. (Figure 17–4) spinal cord Bundle of neurons and support cells that extends from the brain. spindle assembly checkpoint Regulatory system that operates during mitosis to ensure that all chromosomes are properly attached to the spindle before sister-chromatid separation starts. (Figure 17–9 and Panel 17–1, pp. 980–981) spliceosome Large assembly of RNA and protein molecules that performs pre-mRNA splicing in eukaryotic cells. Src (Src protein family) Family of cytoplasmic tyrosine kinases (pronounced “sark”) that associate with the cytoplasmic domains of some enzyme-linked cell-surface receptors (for example, the T cell antigen receptor) that lack intrinsic tyrosine kinase activity. They transmit a signal onward by phosphorylating the receptor itself and specific intracellular signaling proteins on tyrosines. (Figure 3–10)

1	SRP (signal-recognition particle) receptor Component in the endoplasmic reticulum (ER) membrane that guides the signal recognition particle to the ER membrane. starch Polysaccharide composed exclusively of glucose units, used as an energy-storage material in plant cells. (Figure 2–51) Start (restriction point) Important transition at the end of G1 in the eukaryotic cell cycle. Passage through Start commits the cell to enter S phase. The term was originally used for this point in the yeast cell cycle only; the equivalent point in the mammalian cell cycle was called the restriction point. In this book we use Start for both. (Figure 17–9) start-transfer signal Short amino acid sequence that enables a polypeptide chain to start being translocated across the endoplasmic reticulum membrane through a protein translocator. Multipass membrane proteins sometimes have both N-terminal (signal sequence) and internal start-transfer signals. (Figure 12–42)

1	STAT (signal transducer and activator of transcription) Latent transcription regulator that is activated by phosphorylation by Janus kinases (JAKs) and enters the nucleus in response to signaling from receptors of the cytokine receptor family. (Figure 15–56) stem cell Undifferentiated cell that can continue dividing indefinitely, throwing off daughter cells that can either commit to differentiation or remain a stem cell (in the process of self-renewal). (Figure 22–3) stem-cell niche The specialized microenvironment in a tissue in which self-renewing stem cells can be maintained. steroid hormones Hormones, including cortisol, estrogen, and testosterone, that are hydrophobic lipid molecules derived from cholesterol that activate intracellular nuclear receptors.

1	steroid hormones Hormones, including cortisol, estrogen, and testosterone, that are hydrophobic lipid molecules derived from cholesterol that activate intracellular nuclear receptors. stimulatory G protein (Gs) G protein that, when activated, activates the enzyme adenylyl cyclase and thus stimulates the production of cyclic AMP. See also G protein. (Table 15–3, p. 846) stochastic Random. Involving chance, probability, or random variables. stop-transfer signal Hydrophobic amino acid sequence that halts translocation of a polypeptide chain through the endoplasmic reticulum membrane, thus anchoring the protein chain in the membrane. (Figure 12–42) strand exchange Reaction in which one of the single-strand 3′ ends from one duplex DNA molecule penetrates another duplex and searches it for homologous sequences through base-pairing. Also called strand invasion.

1	strand-directed mismatch repair A proofreading system that removes DNA replication errors missed by the DNA polymerase proofreading exonuclease. Detects the potential for DNA helix distortion from noncomplementary base pairs then recognizes and excises the mismatch in the newly synthesized strand and resynthesizes the excised segment using the old strand as a template. stress fibers Cortical fibers of contractile actin-myosin II bundles that connect the cell to the extracellular matrix or adjacent cells through focal adhesions or a circumferential belt and adherens junctions. stroma (1) “Bedding”: the connective tissue in which a glandular or other epithelium is embedded. Stromal cells provide the environment necessary for the development of other cells within the tissue. (2) The large interior space of a chloroplast, containing enzymes that incorporate CO2 into sugars. (Figure 14–38) substrate Molecule on which an enzyme acts.

1	superresolution Describes several approaches in light microscopy that bypass the limit imposed by the diffraction of light and successfully allow objects as small as 20 nm to be imaged and clearly resolved. survival factor Extracellular signal that promotes cell survival by inhibiting apoptosis. (Figure 18–12) symporter Carrier protein that transports two types of solute across the membrane in the same direction. (Figure 11–8) synapse Communicating cell–cell junction that allows signals to pass from a nerve cell to another cell. In a chemical synapse, the signal is carried by a diffusible neurotransmitter. (Figure 19–22) In an electrical synapse, a direct connection is made between the cytoplasms of the two cells via gap junctions. (Figure 11–34 and 19–23) synapse elimination Process by which each muscle cell at first receives synapses from several motor neurons, but is ultimately left innervated by only one.

1	synaptic plasticity Changes in the strength with which a chemical synapse transmits a signal. It is thought to be important in memory formation, where concentrations of postsynaptic AMPA receptor are modulated in response to a synapse’s activity. synaptic signaling Intercellular signaling performed by neurons that transmit signals electrically along their axons and release neurotransmitters at synapses, which are often located far away from the neuronal cell body. synaptic vesicle Small neurotransmitter-filled secretory vesicle found at the axon terminals of nerve cells. Its contents are released into the synaptic cleft by exocytosis when an action potential reaches the axon terminal.

1	synaptonemal complex Structure that holds paired homologous chromosomes tightly together in pachytene of prophase I in meiosis and promotes the final steps of crossing-over. (Figures 17–55 and 17–56) syncytium Mass of cytoplasm containing many nuclei enclosed by a single plasma membrane. Typically the result either of cell fusion or of a series of incomplete division cycles in which the nuclei divide but the cell does not. TATA box Sequence in the promoter region of many eukaryotic genes that binds a general transcription factor (TFIID) and hence specifies the position at which transcription is initiated. (Figure 6–14) T cell receptor (TCR) Transmembrane receptor for antigen on the surface of T lymphocytes, consisting of an immunoglobulin-like heterodimer. (Figure 24–32) T-cell-mediated immune response Any adaptive immune response mediated by antigen-specific T cells.

1	T-cell-mediated immune response Any adaptive immune response mediated by antigen-specific T cells. telomerase Enzyme that elongates telomere sequences in DNA, which occur at the ends of eukaryotic chromosomes. telomere End of a chromosome, associated with a characteristic DNA sequence that is replicated in a special way. Counteracts the tendency of the chromosome otherwise to shorten with each round of replication. From Greek telos, end, and meros, portion. telomere End of a chromosome, associated with a characteristic DNA sequence that is replicated in a special way. Counteracts the tendency of the chromosome otherwise to shorten with each round of replication. From Greek telos, end.

1	telophase Final stage of mitosis in which the two sets of separated chromosomes decondense and become enclosed by nuclear envelopes. (Panel 17–1, pp. 980–981) template Single strand of DNA or RNA whose nucleotide sequence acts as a guide for the synthesis of a complementary strand. (Figure 1–3) terminal differentiation The limit of cell determination when a cell forms one of the highly specialized cell types of the adult body. terminally differentiated A cell at the limit of cell determination, being one of the highly specialized cell types of the adult body. terminator Signal in bacterial DNA that halts transcription; in eukaryotes, transcription terminates after cleavage and polyadenylation of the newly synthesized RNA. tertiary structure Complex three-dimensional form of a folded polymer chain, especially a protein or RNA molecule.

1	tertiary structure Complex three-dimensional form of a folded polymer chain, especially a protein or RNA molecule. TH1 cell A type of effector helper T cell that secretes interferon-γ to help activate macrophages and induces B cells to switch the class of antibody they make. (Figure 24–44) TH17 cell A type of effector helper T cell that secretes IL17, which recruits neutrophils and stimulates an inflammatory response. (Figure 24–44)

1	TH17 cell A type of effector helper T cell that secretes IL17, which recruits neutrophils and stimulates an inflammatory response. (Figure 24–44) TH2 cell A type of effector helper T cell that helps activate B cells to produce antibodies, to undergo somatic hypermutation, and switch the class of immunoglobulin produced. (Figure 24–44) thylakoid Flattened sac of membrane in a chloroplast that contains chlorophyll and other pigments and carries out the light-trapping reactions of photosynthesis. Stacks of thylakoids form the grana of chloroplasts. (Figures 14–35 and 14–36) thylakoid membrane Chloroplast membrane system that contains the large membrane protein complexes for photosynthesis and photophosphorylation. thymocytes Developing T cells in the thymus. tight junction Cell–cell junction that seals adjacent epithelial cells together, preventing the passage of most dissolved molecules from one side of the epithelial sheet to the other. (Figures 19–2 and 19–21)

1	TIM complexes Protein translocators in the mitochondrial inner membrane. The TIM23 complex mediates the transport of proteins into the matrix and the insertion of some proteins into the inner membrane; the TIM22 complex mediates the insertion of a subgroup of proteins into the inner membrane. (Figure 12–21) Toll A transmembrane receptor protein. On the ventral side of the Drosophila egg membrane, its activation controls the distribution of Dorsal, a transcription regulator of the NFκB family. Toll-like receptors (TLRs) Family of pattern recognition receptors (PRRs) on or in cells of the innate immune system. They recognize pathogen-associated immunostimulants (PAMPs) associated with microbes. (Figure 24–4) TOM complex Multisubunit protein complex that transports proteins across the mitochondrial outer membrane. (Figure 12–21) TOR Large, serine/threonine protein kinase that is activated by the PI-3-kinase–Akt signaling pathway and promotes cell growth.

1	TOR Large, serine/threonine protein kinase that is activated by the PI-3-kinase–Akt signaling pathway and promotes cell growth. totipotent Describes a cell that is able to give rise to all the different cell types in an organism. trans face Face on the other (far) side. trans Golgi network (TGN) Network of interconnected tubular and cisternal structures closely associated with the trans face of the Golgi apparatus and the compartment from which proteins and lipids exit the Golgi, bound for the cell surface or another compartment.

1	transcellular transport Transport of solutes, such as nutrients, across an epithelium, by means of membrane transport proteins in the apical and basal faces of the epithelial cells. (Figure 11–11) transcription (DNA transcription) Copying of one strand of DNA into a complementary RNA sequence by the enzyme RNA polymerase. (Figures 6–1 and 6–8) transcription regulators General name for any protein that binds to a specific DNA sequence (known as a cis-regulatory sequence) to influence the transcription of a gene. transcriptional control Regulation by a cell of gene expression by controlling when and how often a given gene is transcribed.

1	transcriptional control Regulation by a cell of gene expression by controlling when and how often a given gene is transcribed. transcytosis Uptake of material at one face of a cell by endocytosis, its transfer across a cell in vesicles, and discharge from another face by exocytosis. (Figure 13–58) transfer RNA (tRNA) Set of small RNA molecules used in protein synthesis as an interface (adaptor) between mRNA and amino acids. Each type of tRNA molecule is covalently linked to a particular amino acid. (Figure 6–50) transferrin receptor Cell-surface receptor for transferrin (a soluble protein that carries iron) that delivers iron to the cell interior via receptor-mediated endocytosis and recycling of the receptor–transferrin complex. transformed A cell with an altered phenotype that behaves in many ways like a cancer cell (i.e., unregulated proliferation, anchorage-independent growth in culture).

1	transformed A cell with an altered phenotype that behaves in many ways like a cancer cell (i.e., unregulated proliferation, anchorage-independent growth in culture). transforming growth factor-β superfamily (TGFβ superfamily) Large family of structurally related secreted proteins that act as hormones and local mediators to control a wide range of functions in animals, including during development. It includes the TGFβ/activin and bone morphogenetic protein (BMP) subfamilies. (Figure 15–57) transgene The foreign or modified gene that has been added to create a transgenic organism. transgenic organism Plant or animal that has stably incorporated one or more genes from another cell or organism (through insertion, deletion, and/or replacement) and can pass them on to successive generations. (Figures 8–53 and 8–70) transit amplifying cell Cell derived from a stem cell that divides a limited number of times before terminally differentiating.

1	transition state Structure that forms transiently in the course of a chemical reaction and has the highest free energy of any reaction intermediate. Its formation is a rate-limiting step in the reaction. (Figure 3–47) translation (RNA translation) Process by which the sequence of nucleotides in an mRNA molecule directs the incorporation of amino acids into protein. Occurs on a ribosome. (Figures 6–1 and 6–64) translational control Regulation by a cell of gene expression by selecting which mRNAs in the cytoplasm are translated by ribosomes. translocon The assembly of a translocator associated with other membrane complexes, such as enzymes that modify the growing polypeptide chain. transmembrane adhesion proteins Cytoskeletonlinked transmembrane molecules with one end linking to the cytoskeleton inside the cell and the other end linking to other structures outside it.

1	transmembrane protein Membrane protein that extends through the lipid bilayer, with part of its mass on either side of the membrane. (Figure 10–17) transmitter-gated ion channel (ion-channel-coupled receptor, ionotropic receptor) Ion channel found at chemical synapses in the postsynaptic plasma membranes of nerve and muscle cells. Opens only in response to the binding of a specific extracellular neurotransmitter. The resulting inflow of ions leads to the generation of a local electrical signal in the postsynaptic cell. (Figures 11–36 and 15–6) transport vesicle Membrane-enclosed transport containers that bud from specialized coated regions of donor membrane and pass from one cell compartment to another as part of the cell’s membrane transport processes; vesicles can be spherical, tubular, or irregularly shaped.

1	transporter (carrier protein, permease) Membrane transport protein that binds to a solute and transports it across the membrane by undergoing a series of conformational changes. Transporters can transport ions or molecules passively down an electrochemical gradient or can link the conformational changes to a source of metabolic energy such as ATP hydrolysis to drive active transport. Compare channel protein. See also membrane transport protein. (Figure 11–3) transposable element (transposon) Segment of DNA that can move from one genome position to another by transposition. (Table 5–4, p. 288) transposition (transpositional recombination) Movement of a DNA sequence from one genome site to another. (Table 5–4, p. 288) treadmilling Process by which a polymeric protein filament is maintained at constant length by addition of protein subunits at one end and loss of subunits at the other. (Panel 16–2, pp. 902–903)

1	Trithorax group Set of proteins critical for cell memory that maintains the transcription of Hox genes in cells where transcription has already been switched on. t-SNAREs Transmembrane SNARE protein, usually composed of three proteins and found on target membranes where it interacts with v-SNAREs on vesicle membranes. tubulin The protein subunit of microtubules. (Panel 16–1, p. 891, and Figure 16–42) γ-tubulin ring complex (γ-TuRC) Protein complex containing γ-tubulin and other proteins that is an efficient nucleator of microtubules and caps their minus ends. tumor progression Process by which an initial mildly disordered cell behavior gradually evolves into a full-blown cancer. (Figures 20–8 and 20–9) tumor suppressor gene Gene that appears to help prevent formation of a cancer. Loss-of-function mutations in such genes favor the development of cancer. (Figure 20–17) tumor virus Virus that can help make the cell it infects cancerous.

1	turgor pressure Large hydrostatic pressure developed inside a plant cell as the result of the intake of water by osmosis; it is the force driving cell expansion in plant growth and it maintains the rigidity of plant stems and leaves. two-dimensional gel electrophoresis Technique combining two different separation procedures—separation by charge (isoelectric focusing) in the first dimension, then separation by size in a direction at a right angle to that of the first step—to resolve up to 2000 proteins in the form of a two-dimensional protein map. type III fibronectin repeat The major repeat domain in fibronectin, it is about 90 amino acids long and occurs at least 15 times in each subunit. The repeat is among the most common of all protein domains in vertebrates.

1	type III secretion system One of several secretion systems in Gram negative bacteria; delivers effector proteins into host cells in a contact-dependent manner. (Figure 23–7) type IV collagen An essential component of mature basal laminae consisting of three long protein chains twisted into a ropelike superhelix with multiple bends. Separate molecules assemble into a flexible, felt-like network that gives the basal lamina tensile strength. tyrosine kinase Enzyme that phosphorylates specific proteins on tyrosines. See also cytoplasmic tyrosine kinase. tyrosine-kinase-associated receptor Cell-surface receptor that functions similarly to RTKs, except that the kinase domain is encoded by a separate gene and is noncovalently associated with the receptor polypeptide chain.

1	ubiquitin Small, highly conserved protein present in all eukaryotic cells that becomes covalently attached to lysines of other proteins. Attachment of a short chain of ubiquitins to such a lysine can tag a protein for intracellular proteolytic destruction by a proteasome. (Figure 3–69) ubiquitin ligase Any one of a large number of enzymes that attach ubiquitin to a protein, often marking it for destruction in a proteasome. The process catalyzed by a ubiquitin ligase is called ubiquitylation. (Figure 3–71) unfolded protein response Cellular response triggered by an accumulation of misfolded proteins in the endoplasmic reticulum. Involves expansion of the ER and increased transcription of genes that code for endoplasmic reticulum chaperones and degradative enzymes. (Figure 12–51) uniporter Carrier protein that transports a single solute from one side of the membrane to the other. (Figure 11–8)

1	V(D)J recombination Somatic recombination process by which gene segments are brought together to form a functional gene for a polypeptide chain of an immunoglobulin or T cell receptor. (Figure 24–28) vacuole Large fluid-filled compartment found in most plant and fungal cells, typically occupying more than a third of the cell volume. (Figure 13–41) van der Waals attraction Type of (individually weak) noncovalent bond that is formed at close range between nonpolar atoms. (Table 2–1, p. 45 and Panel 2–3, pp. 94–95) variable region Region of an immunoglobulin or T cell receptor polypeptide chain that is the most variable and contributes to the antigen-binding site. (Figures 24–25 and 24–32) vascular endothelial growth factor (VEGF) Secreted protein that stimulates the growth of blood vessels. (Table 15–4, p. 850, and Figure 22–26) vesicle transport model One hypothesis for how the Golgi apparatus achieves and maintains its polarized structure and how molecules move from one cisterna

1	(Table 15–4, p. 850, and Figure 22–26) vesicle transport model One hypothesis for how the Golgi apparatus achieves and maintains its polarized structure and how molecules move from one cisterna to another. This model holds that Golgi cisternae are long-lived structures that retain their characteristic set of Golgi-resident proteins firmly in place, and cargo proteins are transported from one cisterna to the next by transport vesicles.

1	vesicular transport Transport of proteins from one cell compartment to another by means of membrane-bounded intermediaries such as vesicles or organelle fragments. V gene segment A DNA sequence encoding most of the variable region of an immunoglobulin or T cell receptor polypeptide chain. There are many different V gene segments, one of which becomes joined to a D or J gene segment by somatic recombination when an individual lymphoid progenitor cell begins to differentiate into a B or T lymphocyte. (Figure 24–28) virulence factor Protein, encoded by a virulence gene, that contributes to an organism’s ability to cause disease. virulence gene Gene that contributes to an organism’s ability to cause disease.

1	virulence gene Gene that contributes to an organism’s ability to cause disease. virus Particle consisting of nucleic acid (RNA or DNA) enclosed in a protein coat and capable of replicating within a host cell and spreading from cell to cell. (Figure 23–11) virus receptor Molecule on the host cell surface to which virus surface proteins bind to enable binding of virus to the cell surface. voltage-gated cation channel Type of ion channel found in the membranes of electrically excitable cells (such as nerve, endocrine, egg, and muscle cells). Opens in response to a shift in membrane potential past a threshold value. voltage-gated K+ channel Ion channel in the membrane of nerve cells that opens in response to membrane depolarization, enabling K+ efflux and rapid restoration of the negative membrane potential.

1	voltage-gated K+ channel Ion channel in the membrane of nerve cells that opens in response to membrane depolarization, enabling K+ efflux and rapid restoration of the negative membrane potential. voltage-gated Na+ channel Ion channel in the membrane of nerve and skeletal muscle cells that opens in response to a stimulus causing sufficient depolarization, allowing Na+ to enter the cell down its electrochemical gradient v-SNAREs Transmembrane SNARE protein, comprising a single polypeptide chain, usually found in vesicle membranes where it interacts with t-SNAREs in target membranes. V-type pumps Turbine-like protein machines constructed from multiple different subunits that use the energy of ATP hydrolysis to drive transport across a membrane. The V-type proton pump transfers H+ into organelles such as lysosomes to acidify their interior. (Figure 11–12)

1	WASp protein Key target of activated Cdc42. Exists in an inactive folded conformation and an activated open conformation; association with Cdc42 stabilizes the open form, enabling binding to the Arp 2/3 complex and enhancing actinnucleating activity. Wee1 Protein kinase that inhibits Cdk activity by phosphorylating amino acids in the Cdk active site. Important in regulating entry into M phase of the cell cycle. Western blotting Technique by which proteins are separated by electrophoresis and immobilized on a paper sheet and then analyzed, usually by means of a labeled antibody. Also called immunoblotting. white blood cell General name for all the nucleated blood cells lacking hemoglobin. Also called leukocytes. Includes lymphocytes, granulocytes, and monocytes. (Figure 22–27) Wnt protein Member of a family of secreted signal proteins that have many different roles in controlling cell differentiation, proliferation, and gene expression in animal embryos and adult tissues.

1	Wnt/β-catenin pathway Signaling pathway activated by binding of a Wnt protein to its cell-surface receptors. The pathway has several branches. In the major (canonical) branch, activation causes increased amounts of β-catenin to enter the nucleus, where it regulates the transcription of genes controlling cell differentiation and proliferation. Overactivation of the Wnt/β-catenin pathway can lead to cancer. (Figure 15–60) X-inactivation Inactivation of one copy of the X chromosome in the somatic cells of female mammals. X-inactivation center (XIC) Site in an X chromosome at which inactivation is initiated and spreads outward. x-ray crystallography Technique for determining the three-dimensional arrangement of atoms in a molecule based on the diffraction pattern of x-rays passing through a crystal of the molecule. (Figure 8–21) zygote Diploid cell produced by fusion of a male and female gamete. A fertilized egg.

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1	Leadership in SurgeryStephen Markowiak, Hollis Merrick, Shiela Beroukhim, Jeremy J. Laukka, Amy Lightner, Munier Nazzal, Lee Hammerling, James R. Macho, and F. Charles Brunicardi 1chapterINTRODUCTIONThe field of surgery has evolved greatly from its roots, and sur-gical practice now requires the mastery of modern leadership principles and skills as much as the acquisition of medical knowledge and surgical technique. Historically, surgeons took sole responsibility for their patients and directed proceedings in the operating room with absolute authority, using a command-and-control style of leadership. Modern surgical practice has now evolved from single provider–based care toward a team-based approach, which requires collaborative leadership skills. Surgical care benefits from the collaboration of surgeons, anes-thesiologists, internists, radiologists, pathologists, radiation oncologists, nurses, pharmacists, social workers, therapists, hospital staff, and administrators. Occupying a

1	of surgeons, anes-thesiologists, internists, radiologists, pathologists, radiation oncologists, nurses, pharmacists, social workers, therapists, hospital staff, and administrators. Occupying a central role on the healthcare team, surgeons1 have the potential to improve patient outcomes, reduce medical errors, and improve patient satisfaction through their leadership of the multidisciplinary team. Thus, in the landscape of modern healthcare systems, it is imperative that surgical training programs include formal instruction on leadership principles and skills to cultivate their trainees’ leadership capabilities.Many medical and surgical communities, including residency training programs, acknowledge the need for improved physician leadership. Specifically, surveyed surgical residents felt a lack of confidence in multiple areas of leadership, particularly in conflict resolution.2 Surgical trainees identify leadership skills as important, but they report themselves as “not competent” or

1	a lack of confidence in multiple areas of leadership, particularly in conflict resolution.2 Surgical trainees identify leadership skills as important, but they report themselves as “not competent” or “minimally competent” in this regard.2,3 While a small number of surgical training programs have implemented 1formal curricula focused on teaching leadership principles, it is now imperative that all surgical training programs teach these important skills to their trainees.4,5 Interviews of academic chairpersons identified several critical leadership success factors,6 including mastery of visioning, communication, change management, emotional intelligence, team building, business skills, personnel management, and systems thinking. These chairpersons stated that the ability of emotional intelligence was “fundamental to their success and its absence the cause of their failures,” regardless of medical knowledge.6 Thus, residency programs need to include leadership training to prepare future

1	was “fundamental to their success and its absence the cause of their failures,” regardless of medical knowledge.6 Thus, residency programs need to include leadership training to prepare future surgeons for success in modern healthcare delivery.In the United States, the Accreditation Council for Graduate Medical Education (ACGME) has established six core competencies—patient care, medical knowledge, prac-tice-based learning and improvement, interpersonal and com-munication skills, professionalism, and systems-based practice (Table 1-1)4—that each contain principles of leadership. The ACGME has mandated the teaching of these core competencies but has not established a formal guide on how to teach the lead-ership skills described within the core competencies. Therefore, this chapter offers a review of fundamental principles of leader-ship and an introduction of the concept of a leadership training program for future surgeons.DEFINITIONS OF LEADERSHIPMany different definitions of

1	offers a review of fundamental principles of leader-ship and an introduction of the concept of a leadership training program for future surgeons.DEFINITIONS OF LEADERSHIPMany different definitions of leadership have been described. Former First Lady Rosalynn Carter once observed that “A leader takes people where they want to go. A great leader takes people Introduction 3Definitions of Leadership 3Levels of Leadership / 4Fundamental Principles of Leadership 4Vision / 5Generating Belief in Your Vision / 6Willingness / 7Time Management / 10Self-Care and Wellness / 11Recruitment / 11Creating a Culture of Empathy, Patient-Family-Centered Care, and Personalized Surgery / 11Why We Lead 11Choosing to Become a Leader / 11Leadership’s Effect on Healthcare Cost and Clinical Outcomes / 11The Importance of Diversity and Leadership 12Leadership Styles 12Formal Leadership Training Programs in Surgery 13History of Leadership Training and the Multifactor Leadership Questionnaire / 13Designing

1	of Diversity and Leadership 12Leadership Styles 12Formal Leadership Training Programs in Surgery 13History of Leadership Training and the Multifactor Leadership Questionnaire / 13Designing the Program / 14Practicing Leadership Skills and Assessing Leadership Formally With Objective Structured Clinical Examination (OSCE) and Simulation / 14Evaluation of Surgeon Performance / 16Mentoring and Development 18Mentoring / 18Modeling Leadership for Medical Students and the “Hidden Curriculum” / 18Tools to Measure Leadership Outcomes in Healthcare / 19Leadership Training for the Prospective Surgeon / 20Early Career Development and Establishing Oneself 20Senior Faculty Development: Transitioning to Departmental Leadership and Legacy Building 21Conclusion 21Brunicardi_Ch01_p0001-p0026.indd 329/01/19 10:58 AM 4Table 1-1Accreditation Council for Graduate Medical Education core competenciesCORE COMPETENCYDESCRIPTIONPatient careTo be able to provide compassionate and effective

1	329/01/19 10:58 AM 4Table 1-1Accreditation Council for Graduate Medical Education core competenciesCORE COMPETENCYDESCRIPTIONPatient careTo be able to provide compassionate and effective healthcare in the modern-day healthcare environmentMedical knowledgeTo effectively apply current medical knowledge in patient care and to be able to use medical tools (i.e., PubMed) to stay current in medical educationPractice-based learning and improvementTo critically assimilate and evaluate information in a systematic manner to improve patient care practicesInterpersonal and communication skillsTo demonstrate sufficient communication skills that allow for efficient information exchange in physician-patient interactions and as a member of a healthcare teamProfessionalismTo demonstrate the principles of ethical behavior (i.e., informed consent, patient confidentiality) and integrity that promote the highest level of medical careSystems-based practiceTo acknowledge and understand that each

1	of ethical behavior (i.e., informed consent, patient confidentiality) and integrity that promote the highest level of medical careSystems-based practiceTo acknowledge and understand that each individual practice is part of a larger healthcare delivery system and to be able to use the system to support patient careKey Points1 Effective surgical leadership improves patient care, safety, and clinical outcomes.2 A fundamental principle of leadership is to provide a vision that people can live up to, thereby providing direction and purpose to the constituency.3 Surgical leaders have the willingness to lead through an active and passionate commitment to the vision.4 Surgical leaders have the willingness to commit to lifelong learning.5 Surgical leaders have the willingness to communicate effec-tively and resolve conflict.6 Surgical leaders must practice effective time management.7 Different leadership styles are tools to use based on the team dynamic.8 Surgical trainees can be taught

1	effec-tively and resolve conflict.6 Surgical leaders must practice effective time management.7 Different leadership styles are tools to use based on the team dynamic.8 Surgical trainees can be taught leadership principles in formal leadership training programs to enhance their ability to lead.9 Mentorship provides wisdom, guidance, and insight essen-tial for the successful development of a surgical leader.of business. In business, the processes of customer satisfaction, product development, and organization efficiency are the equiva-lent of patient satisfaction, medical advancement, and efficient delivery of care. Jim Collins, author of Good to Great, studied the success and leadership styles of Fortune 500 companies over a 30-year period. He found that leadership is strongly correlated with corporate success, and most importantly for our study, that leadership strength can be broken down by level and characteristic (See figure 1-1).8Of 11 particularly outstanding organizations

1	with corporate success, and most importantly for our study, that leadership strength can be broken down by level and characteristic (See figure 1-1).8Of 11 particularly outstanding organizations identified, great leadership was the single major defining characteristic that distinguished them from their peers. These organizations were led by what Collins called the “Level 5 Leader,” one whose per-sonal humility and professional will drove team success. Under this system of leadership study, surgeon-leaders begin at the bottom level and, through study, hard work, and professional development, advance to the ultimate level of leadership.8FUNDAMENTAL PRINCIPLES OF LEADERSHIPLeadership is a complex concept. Surgeons should strive to adopt leadership qualities that provide the best outcomes for their patients, based on the following fundamental principles: vision, willingness, time management, conflict resolution, where they don’t necessarily want to go, but where they ought to be.”

1	for their patients, based on the following fundamental principles: vision, willingness, time management, conflict resolution, where they don’t necessarily want to go, but where they ought to be.” Leadership does not always have to come from a position of authority. Former American president John Quincy Adams stated, “If your actions inspire others to dream more, learn more, do more, and become more, you are a leader.” Another defini-tion is that leadership is the process of using social influence to enlist the aid and support of others in a common task.7Levels of LeadershipWhen working toward organizational success, strong leader-ship is a critical component. The best study of the relationship between leadership skill and organizational success is in the field LEVEL 5 EXECUTIVEBuilds enduring greatnessthrough a paradoxical combinationof personal humility plus professional will.LEVEL 4 EFFECTIVE LEADERCatalyzes commitment to and vigorous pursuitof a clear and compelling vision;

1	enduring greatnessthrough a paradoxical combinationof personal humility plus professional will.LEVEL 4 EFFECTIVE LEADERCatalyzes commitment to and vigorous pursuitof a clear and compelling vision; stimulatesthe group to high performance standards.LEVEL 3 COMPETENT MANAGEROrganizes people and resources toward the effectiveand efficient pursuit of predetermined objectives.LEVEL 2 CONTRIBUTING TEAM MEMBERContributes to the achievement of groupobjectives; works effectively with others in a group setting.LEVEL 1 HIGHLY CAPABLE INDIVIDUALMakes productive contributions through talent, knowledge,skills, and good work habits.Figure 1-1. Levels of leadership as defined by Jim Collins in Good to Great. (Reproduced with permission from Collins J: Good to Great. Boston, MA: Harper Collins; 2011.)Brunicardi_Ch01_p0001-p0026.indd 429/01/19 10:58 AM 5LEADERSHIP IN SURGERYCHAPTER 1recruitment, and culture (See Table 1-2). Surgeon-leaders will develop a team of faculty, residents, and other

1	429/01/19 10:58 AM 5LEADERSHIP IN SURGERYCHAPTER 1recruitment, and culture (See Table 1-2). Surgeon-leaders will develop a team of faculty, residents, and other healthcare per-sonnel who are aligned on mission, vision, and values. The team and leader must be willing to address complex problems with honest communication and well-developed conflict resolution skills. A culture must be established where faculty and staff will work towards the advancement of the medical arts and the greater good of society.9VisionThe first and most fundamental principle of leadership is to establish a vision that people can live up to, thus providing direction and purpose to the constituency. Creating a vision is a declaration of the near future that inspires and conjures motivation.10 A classic example of a powerful vision that held effective impact is President Kennedy’s declaration in 1961 that “. . . this nation should commit itself to achieving the goal, before this decade is out, of landing a

1	of a powerful vision that held effective impact is President Kennedy’s declaration in 1961 that “. . . this nation should commit itself to achieving the goal, before this decade is out, of landing a man on the moon and returning him safely to the earth.” Following his declaration of this vision with a timeline to achieve it, the United Sates mounted a remarkable unified effort, and by the end of the decade, Neil Armstrong took his famous walk and the vision had been accomplished (Fig. 1-2).On a daily basis, surgeons are driven by a powerful vision: the vision that our surgical care will improve patients’ lives. The great surgical pioneers, such as Hunter, Lister (Fig. 1-3), Halsted, von Langenbeck, Billroth, Kocher (Fig. 1-4), Carrel, Gibbon, Blalock, Wangensteen, Moore, Rhoads, Huggins, Murray, Kountz, Longmire, Starzl, and DeBakey (Fig. 1-5), each possessed a vision that revolutionized the field of surgery. In the 19th century, Joseph Lister changed the practice of surgery with his

1	Murray, Kountz, Longmire, Starzl, and DeBakey (Fig. 1-5), each possessed a vision that revolutionized the field of surgery. In the 19th century, Joseph Lister changed the practice of surgery with his application of Pasteur’s germ theory. He set a young boy’s open compound leg fracture, a condition with a 90% mortality 2Figure 1-2. Apollo 11 Lunar Module moon walk. Astronaut Edwin “Buzz” Aldrin walks by the footpad of the Apollo 11 Lunar Module, July 1969. (Reproduced with permission from AP Photo/NASA. © 2018 The Associated Press.)Figure 1-3. Joseph Lister directing use of carbolic acid spray in one of his earliest antiseptic surgical operations, circa 1865. (Used with permission from Getty Images.)rate at that time, using carbolic acid dressings and aseptic surgical technique. The boy recovered, and Lister gathered nine more patients. His famous publication on the use of aseptic technique introduced the modern era of sterile technique. Emil Theodor Kocher was the first to master the

1	and Lister gathered nine more patients. His famous publication on the use of aseptic technique introduced the modern era of sterile technique. Emil Theodor Kocher was the first to master the thyroidectomy, thought to be an impossible operation at the time, and went on to perform thousands of thyroidectomies with a mortality of less than 1%. He was awarded the Nobel Prize in Physiology or Medicine in 1909 for describing the thyroid’s physiologic role in metabolism. Michael E. DeBakey’s powerful vision led to the development of numerous groundbreaking procedures that helped pioneer the field of cardiovascular surgery. For example, envisioning an artificial artery for arterial bypass operations, Dr. DeBakey invented the Dacron graft, which has helped millions of patients suffering from vascular disease and enabled the development of endovascular surgery. Dr. Frederick Banting, the youngest recipient of the Nobel Prize in Physiology or Medicine, had a vision to discover the biochemical

1	disease and enabled the development of endovascular surgery. Dr. Frederick Banting, the youngest recipient of the Nobel Prize in Physiology or Medicine, had a vision to discover the biochemical link between diabetes and glucose homeostasis. His vision and perseverance led to the discovery of insulin.11 In retrospect, the power and clarity of their visions were remarkable, and their willingness and dedication were inspiring. By studying their careers and accomplishments, surgical trainees can be inspired by the potential impact of a well-developed vision.Table 1-2The fundamental principles of leadershipLEADERSHIP SKILLDESCRIPTION AND APPLICATION IN THE FIELD OF MEDICINEVisionThe act of establishing tangible goals of care for patients on both a daily basis as well as for long-term purposes.Effective communicationEstablishing an open, respectful, and nonjudgmental forum for communication among different members of the healthcare team and with the patient.Willingness to leadTaking on full

1	communicationEstablishing an open, respectful, and nonjudgmental forum for communication among different members of the healthcare team and with the patient.Willingness to leadTaking on full responsibility for the care of patients and remaining ethical, professional, and committed despite the especially challenging rigors of joining the field of surgery.Willingness to learnA commitment to lifelong learning of the latest scientific, medical, and surgical updates to deliver optimized patient care.Conflict resolutionThe art of resolving conflicts in a peaceful and ethical manner in team settings.Brunicardi_Ch01_p0001-p0026.indd 529/01/19 10:58 AM 6BASIC CONSIDERATIONSPART IFigure 1-5. Michael E. DeBakey. (Reproduced with permission from AP Photo/David J. Phillip. © 2018 The Associated Press.)Generating Belief in Your VisionSurgical leaders with great visions will inevitably require help from colleagues, other healthcare professionals, scientists, administrators, patients, and

1	Press.)Generating Belief in Your VisionSurgical leaders with great visions will inevitably require help from colleagues, other healthcare professionals, scientists, administrators, patients, and nonmedical personnel. To get this help, surgical leaders must inspire their team and understand motivation. For the surgeon-leader, it is critical to know that people do not follow leaders because of what they do; people follow leaders because of why they do what they do. The people who help the leader execute the vision are motivated by the leader’s beliefs and attitudes more than the leader’s policy or agenda. This concept, based on Simon Sinek’s Start With Why, is rooted in understanding of the anatomy and function of the human brain.13 See figure 1-6.For example, take a surgeon-leader who wants to imple-ment a new perioperative checklist to reduce surgical errors. The “what” is very simple: a checklist to reduce errors. The operating room team may make a rational decision to adapt the

1	wants to imple-ment a new perioperative checklist to reduce surgical errors. The “what” is very simple: a checklist to reduce errors. The operating room team may make a rational decision to adapt the checklist; however, it is also possible that the checklist may be perceived as “another piece of paperwork” and rejected, or that the checklist may have its implementation fought, undermined, delayed, or ignored. A surgeon-leader who does not understand how people are motivated might argue rationally, telling the team that the checklist was created with great care, that all of the best evidence was incorporated in its creation, and that the checklist is short and efficient. This is the “how,” and once again it appeals to the rational and analytical side of the team. With these arguments, the surgeon-leader’s vision remains susceptible to rejection for many of the same reasons. A leader who understands how to motivate a team towards a vision will start with “why.” Before ever discussing

1	surgeon-leader’s vision remains susceptible to rejection for many of the same reasons. A leader who understands how to motivate a team towards a vision will start with “why.” Before ever discussing the checklist in detail with the team, the leader will speak of their shared mission to offer the best patient care possible, ask the team to imagine how they might want a family member treated, and emphasize that a careless error could lead to patient harm and embarrassment for the team. With these argu-ments, which constitute an emotional appeal to the team’s belief system, the leader can expect this vision for better patient care via a new surgical checklist to be adapted by the team. The team will be receptive to implementing a new checklist, not because they believe in the checklist as a tool, but because they believe in the surgeon-leader’s vision for optimizing patient care.There is a biological reason why this concept works. “Why,” “how,” and “what” are correlated to the functions

1	tool, but because they believe in the surgeon-leader’s vision for optimizing patient care.There is a biological reason why this concept works. “Why,” “how,” and “what” are correlated to the functions Figure 1-4. Emil Theodor Kocher. (Reproduced with permission from the National Library of Medicine.)Leaders must learn to develop a vision to provide direction for their team. The vision can be as straightforward as provid-ing quality of care or as lofty as defining a new field of sur-gery, such as atomic surgery and personalized medicine. One can start developing such vision by brainstorming the answers to two simple questions: “Which disease needs to be cured?” and “How can it be cured?”12 The answers represent a vision and should be recorded succinctly in a laboratory notebook or journal. Committing pen to paper enables the surgical trainee to define his or her vision in a manner that can be shared with others.WhatHowWhyNeocortexLimbic BrainWhen a leader's vision appeals to “why,” it

1	Committing pen to paper enables the surgical trainee to define his or her vision in a manner that can be shared with others.WhatHowWhyNeocortexLimbic BrainWhen a leader's vision appeals to “why,” it triggers an emotional response in the limbic brain and increases the likelihood that a vision will be embraced. Persuasion using “what” and “how” appeals to the neocortex and is more easily rejected.Figure 1-6. When leaders seek to generate belief in their vision, it is best to appeal to the team with “why” statements. (Data from Sinek S. Start with why: how great leaders inspire everyone to take action. London: Portfolio/Penguin; 2013.)Brunicardi_Ch01_p0001-p0026.indd 629/01/19 10:58 AM 7LEADERSHIP IN SURGERYCHAPTER 1of separate anatomical levels in the human brain. The neo-cortex is, evolutionarily, the newest area of our brains, and it is responsible for the analytical and rational thoughts and decisions that we make. It corresponds to the “what” and the “how.” When the

1	is, evolutionarily, the newest area of our brains, and it is responsible for the analytical and rational thoughts and decisions that we make. It corresponds to the “what” and the “how.” When the surgeon-leader in the previous example started with the checklist and its rational arguments, the leader was appealing to their team’s neocortex, and the vision was rejected. However, when the surgeon started with the “why,” the vision for better patient care was emotionally accepted by the team, who became receptive to the checklist as a tool for achieving the vision.13Surgery is a field that requires extraordinary dedication and great personal sacrifice. The very nature of vision—steps forward into a better future—implies that change and difficult work will be required of the leader. See figure 1-7. For this reason, surgeon-leaders should establish visions about which they are deeply pas-sionate and committed so that when obstacles are encountered the leader has the strength of will to

1	1-7. For this reason, surgeon-leaders should establish visions about which they are deeply pas-sionate and committed so that when obstacles are encountered the leader has the strength of will to progress forward. Leaders should be selective about which options they pursue. Each oppor-tunity and idea requires great effort to execute; ultimately only a few can be brought to completion. Therefore, leaders should understand what drives their organization’s economic engine: the ideas and opportunities that will bring patients better care, bring the organization more patients, and create new treatments, etc. Thousands of hospitals, companies, innovators, and physicians are addressing many of the same problems in healthcare, such as growing burdens of chronic disease, an aging population, and ris-ing health costs. The best opportunities lie where talent and ability align, so leaders and organizations should be cognizant of choos-ing projects for which they have the potential to be the “best

1	health costs. The best opportunities lie where talent and ability align, so leaders and organizations should be cognizant of choos-ing projects for which they have the potential to be the “best in the world” at doing. Once the vision is set and the project is chosen, it is up to the leader to generate momentum.Momentum is either a cumulative effect of continuous steps towards improvement or, alternatively, in the negative sense, movements towards failure or stagnation. The “flywheel effect,” depicted in Fig. 1-8, demonstrates the building of momentum with (a) initial steps forward, (b) an accumulation of visible results, (c) realignment of the team in the new direc-tion (accounting for new information and data), and then (d) an accumulation of momentum followed by more steps forward. Careful attention to the aforementioned principles is essential in building a successful surgical career, department, or division.8The Flywheel Effect of Building and

1	followed by more steps forward. Careful attention to the aforementioned principles is essential in building a successful surgical career, department, or division.8The Flywheel Effect of Building and SustainingMomentumStepsforwardVisibleresultsMomentumbuildsThe Teamaligns andadjustsFigure 1-8. The “flywheel effect.” (Data from Collins J: Good to Great. Boston, MA: Harper Collins; 2011.)WHAT YOU ARE DEEPLYPASSIONATE ABOUTWHAT DRIVESYOURECONOMICENGINEWHAT YOU CANBE THE BEST INTHE WORLD ATFigure 1-7. Leaders should be selective about where they expend their efforts, as demonstrated by Jim Collins in “Good to Great.” (Reproduced with permission from Collins J: Good to Great. Boston, MA: Harper Collins; 2011.)WillingnessThe Willingness Principle represents the active commitment of the leader toward his or her vision. To do so, a surgical leader must be willing to lead, commit to lifelong learning, communi-cate effectively, and resolve conflict.To Lead. A key characteristic of all great

1	toward his or her vision. To do so, a surgical leader must be willing to lead, commit to lifelong learning, communi-cate effectively, and resolve conflict.To Lead. A key characteristic of all great leaders is the will-ingness to serve as the leader. Dr. Martin Luther King Jr, who championed the civil rights movement with a powerful vision of equality for all based on a commitment to nonviolent methods,14 did so at a time when his vocalization of this vision ensured harassment, imprisonment, and threats of violence against him-self, his colleagues, and his family and friends (Fig. 1-9). King, a young, highly educated pastor, had the security of employ-ment and family, yet was willing to accept enormous respon-sibility and personal risk and did so in order to lead a nation toward his vision of civil rights, for which he was awarded the Nobel Peace Prize in 1964.Willingness to lead is a necessity in any individual who desires to become a surgeon. By entering into the surgical the-ater, a

1	of civil rights, for which he was awarded the Nobel Peace Prize in 1964.Willingness to lead is a necessity in any individual who desires to become a surgeon. By entering into the surgical the-ater, a surgeon accepts the responsibility to care for and operate on patients, despite the risks and burdens involved. They do so, believing fully in the improved quality of life that can be achieved. Surgeons must embrace the responsibility of leading surgical teams that care for their patients, as well as leading sur-gical trainees to become future surgeons. A tremendous sacrifice is required for the opportunity to learn patient care. Surgical trainees accept the hardships of residency with its accompanying steep learning curve, anxiety, long work hours, and time spent away from family and friends. The active, passionate commit-ment to excellent patient care reflects a natural willingness to lead based on altruism and a sense of duty toward those receiving care. Thus, to ensure delivery of the

1	The active, passionate commit-ment to excellent patient care reflects a natural willingness to lead based on altruism and a sense of duty toward those receiving care. Thus, to ensure delivery of the utmost level of care, surgical trainees should commit to developing and refining leadership skills. These skills include a commitment to lifelong learn-ing, effective communication, and conflict resolution.To Learn. Surgeons and surgical trainees, as leaders, must possess willingness to commit to continuous learning. Modern surgery is an ever-changing field with dynamic and evolving healthcare systems and constant scientific discovery and inno-vation. Basic and translational science relating to surgical care is growing at an exponential rate. The sequencing of the human 3Brunicardi_Ch01_p0001-p0026.indd 729/01/19 10:58 AM 8BASIC CONSIDERATIONSPART Igenome and the enormous advances in molecular biology and signaling pathways are leading to the transformation of pre-cision medicine and

1	729/01/19 10:58 AM 8BASIC CONSIDERATIONSPART Igenome and the enormous advances in molecular biology and signaling pathways are leading to the transformation of pre-cision medicine and personalized surgery in the 21st century (see Chapter 15).15 Performing prophylactic mastectomies with immediate reconstruction for BRCA1 mutations and thyroidecto-mies with thyroid hormone replacement for RET proto-oncogene mutations are two of many examples of genomic information guiding surgical care. Technologic advances in minimally inva-sive surgery and robotic surgery as well as electronic records and other information technologies are revolutionizing the craft of surgery. The expansion of minimally invasive and endovas-cular surgery over the past three decades required surgeons to retrain in new techniques using new skills and equipment. In this short time span, laparoscopy and endovascular operations are now recognized as the standard of care for many surgical diseases, resulting in shorter

1	techniques using new skills and equipment. In this short time span, laparoscopy and endovascular operations are now recognized as the standard of care for many surgical diseases, resulting in shorter hospital stay, quicker recovery, and a kinder and gentler manner of practicing surgery. Remarkably, during the last century, the field of surgery has progressed at an exponential pace and will continue to do so with the advent of using genomic analyses to engineer cancer cells with molecular imaging agents that will guide personalized surgery, which will transform the field of surgery during this century. Therefore, surgical leadership training should emphasize and facilitate the continual pursuit of knowledge.Willingness to learn encompasses the surgeon’s commit-ment to lifelong learning. This has been exemplified by the surgeons of the past several decades who have dedicated their peak practicing years to perfecting minimally invasive surgical Figure 1-9. Dr. Martin Luther King Jr

1	This has been exemplified by the surgeons of the past several decades who have dedicated their peak practicing years to perfecting minimally invasive surgical Figure 1-9. Dr. Martin Luther King Jr acknowledges the crowd at the Lincoln Memorial for his “I Have a Dream” speech during the March on Washington, D.C., August 28, 1963. (Reproduced with permission from AP Photo. © 2018 The Associated Press.)techniques, including the use of robotic surgery. The field con-tinues to advance, offering many advantages to patients includ-ing faster recovery, sometimes decreased pain depending on procedure type, and shorter hospital stays.16-18Fortunately, surgical organizations and societies provide surgeons and surgical trainees a means to acquire new knowl-edge on a continuous basis. There are numerous local, regional, national, and international meetings of surgical organizations that provide ongoing continuing medical education credits, also required for the renewal of most medical licenses.

1	local, regional, national, and international meetings of surgical organizations that provide ongoing continuing medical education credits, also required for the renewal of most medical licenses. The American Board of Surgery requires all surgeons to complete meaningful continuing medical education to maintain certification.19 These societies and regulatory bodies enable surgeons and surgical trainees to commit to continual learning and ensure their competence in a dynamic and rapidly growing field.Surgeons and trainees now benefit from the rapid expan-sion of web-based education as well as mobile handheld tech-nology. These are powerful tools to minimize nonproductive time in the hospital and make learning and reinforcement of medical knowledge accessible. Currently web-based resources provide quick access to a vast collection of surgical texts, lit-erature, and surgical videos. Surgeons and trainees dedicated to continual learning should be well versed in the utilization of these

1	provide quick access to a vast collection of surgical texts, lit-erature, and surgical videos. Surgeons and trainees dedicated to continual learning should be well versed in the utilization of these information technologies to maximize their education. The next evolution of electronic surgical educational materials will likely include simulation training similar to laparoscopic and Da Vinci device training modules. The ACGME, acknowl-edging the importance of lifelong learning skills and moderniza-tion of information delivery and access methods, has included them as program requirements for residency accreditation.To Communicate Effectively. The complexity of modern healthcare delivery systems requires a higher level and collab-orative style of communication. Effective communication directly impacts patient care. In 2000, the U.S. Institute of Medi-cine published To Err Is Human: Building a Safer Health System, which raised awareness concerning the magnitude of medical errors. This

1	impacts patient care. In 2000, the U.S. Institute of Medi-cine published To Err Is Human: Building a Safer Health System, which raised awareness concerning the magnitude of medical errors. This work showcased medical errors as the eighth leading cause of death in the United States with an estimated 100,000 deaths annually.20 Subsequent studies examining medical errors have identified communication errors as one of the most com-mon causes of medical error.21-23 In fact, the Joint Commission identifies miscommunication as the leading cause of sentinel events. Information transfer and communication errors cause delays in patient care, waste surgeon and staff time, and cause serious adverse patient events.23 Effective communication among surgeons, nurses, ancillary staff, and patients is not only a crucial element to improved patient outcomes, but it also leads to less medical litigation.24-26 A strong correlation exists between communication and patient outcomes.Establishing a

1	is not only a crucial element to improved patient outcomes, but it also leads to less medical litigation.24-26 A strong correlation exists between communication and patient outcomes.Establishing a collaborative atmosphere is important since communication errors leading to medical mishaps are not simply failures to transmit information. Communication errors “are far more complex and relate to hierarchical differences, concerns with upward influence, conflicting roles and role ambiguity, and interpersonal power and conflict.”22,27-29 Errors frequently originate from perceived limited channels of com-munication and hostile, critical environments. To overcome these barriers, surgeons and surgical trainees should learn to communicate in an open, universally understood manner and remain receptive to any team member’s concerns. A survey of physicians, nurses, and ancillary staff identified effective communication as a key element of a successful leader.30 As

1	manner and remain receptive to any team member’s concerns. A survey of physicians, nurses, and ancillary staff identified effective communication as a key element of a successful leader.30 As 45Brunicardi_Ch01_p0001-p0026.indd 829/01/19 10:58 AM 9LEADERSHIP IN SURGERYCHAPTER 1leaders, surgeons, and surgical trainees who facilitate an open, effective, and collaborative style of communication can reduce errors and enhance patient care. A prime example is that suc-cessful communication of daily goals of patient care from the team leader improves patient outcomes. In one recent study, the modest act of explicitly stating daily goals in a standard-ized fashion significantly reduced patient length of intensive care unit stay and increased resident and nurse understanding of goals of care.31 Implementing standardized daily team briefings in the wards and preoperative units led to improvements in staff turnover rates, employee satisfaction, and prevention of wrong-site surgery.27 In

1	Implementing standardized daily team briefings in the wards and preoperative units led to improvements in staff turnover rates, employee satisfaction, and prevention of wrong-site surgery.27 In cardiac surgery, improving communication in the operating room and transition to the postanesthesia care unit was an area identified to decrease risk for adverse outcomes.32 Behaviors associated with ineffective communication, including absence from the operating room when needed, playing loud music, making inappropriate comments, and talking to others in a raised voice or a condescending tone, were identified as patient hazards; conversely, behaviors associated with effec-tive collaborative communication, such as leading the time-out process and closed-loop communication technique, resulted in improved patient outcomes.One model to ensure open communication is through standardization of established protocols. A commonly accepted protocol is the “time out” that is now required in the modern

1	improved patient outcomes.One model to ensure open communication is through standardization of established protocols. A commonly accepted protocol is the “time out” that is now required in the modern operating room. During the time-out protocol, all team mem-bers introduce themselves and state a body of critical informa-tion needed to safely complete the intended operation. This same standardization can be taught outside the operating room. Within the Kaiser system, certain phrases have been given a uni-versal meaning: “I need you now” by members of the team is an understood level of urgency and generates a prompt physician response 100% of the time.27 As mentioned earlier, standardized forms can be useful tools in ensuring universally understood communication during sign-out. The beneficial effect of stan-dardized team communication further demonstrates how effec-tive communication can improve patient care and is considered a vital leadership skill.Effective communication with

1	effect of stan-dardized team communication further demonstrates how effec-tive communication can improve patient care and is considered a vital leadership skill.Effective communication with patients in the mod-ern era, necessitates understanding that many patients access health information via the internet and that patients are often ill equipped to evaluate the individual source.33,34 Discrepancies exist between surgeon’s self-perceived ability to communicate and patient’s actual satisfaction. A patient’s perceived interac-tion with their physician has an enormous impact on patient health outcomes, malpractice, and financial reimbursement;35-40 specifically, the association between poor doctor–patient com-munication and a patient’s perception that their doctor does not care about them. Good bedside manner has been shown to decrease litigation even in situations of error or undesirable out-come.39-40 Physicians who demonstrate concern, actively know their patients, and share

1	them. Good bedside manner has been shown to decrease litigation even in situations of error or undesirable out-come.39-40 Physicians who demonstrate concern, actively know their patients, and share responsibility for decision-making are more likely to be trusted by their patients.26,41,42 Strong doctor–patient relationships and effective communication skills have been incentivized by the Agency for Healthcare Research and Quality and the Centers for Medicare & Medicaid Services through their Hospital Consumer Assessment of Healthcare Providers and Systems (HCAHPS) and Clinical and Group Consumer Assessment of Healthcare Providers and Systems (CGCAHPS) programs, which measure patient satisfaction.43To Resolve Conflict. Great leaders are able to achieve their vision through their ability to resolve conflict. Delivery of modern surgical care is complex; numerous conflicts arise on a daily basis when surgeons and surgical trainees provide high-quality care. Therefore, the techniques for

1	resolve conflict. Delivery of modern surgical care is complex; numerous conflicts arise on a daily basis when surgeons and surgical trainees provide high-quality care. Therefore, the techniques for conflict resolution are essential for surgical leaders.To properly use conflict resolution techniques, it is impor-tant for the surgeon and surgical trainee to always remain objec-tive and seek personal flexibility and self-awareness. The gulf between self-perception and the perception of others can be profound; in a study of cooperation and collaboration among operating room staff, the quality of their own collaboration was rated at 80% by surgeons, yet was rated at only 48% by oper-ating room nurses.44 Systematic inclusion of modern conflict resolution methods that incorporate the views of all members of a multidisciplinary team help maintain objectivity. Reflection is often overlooked in surgical residency training, but it is a critical component of learning conflict resolution skills.

1	members of a multidisciplinary team help maintain objectivity. Reflection is often overlooked in surgical residency training, but it is a critical component of learning conflict resolution skills. Introspection allows the surgeon to understand the impact of his or her actions and biases. Objectivity is the basis of effective conflict resolu-tion, which can improve satisfaction among team members and help deliver optimal patient care.Modern conflict resolution techniques are based on objec-tivity, willingness to listen, and pursuit of principle-based solu-tions.45 For example, an effective style of conflict resolution is the utilization of the “abundance mentality” model, which attempts to achieve a solution that benefits all involved and is based on core values of the organization, as opposed to the uti-lization of the traditional fault-finding model, which identifies sides as right or wrong.46 Application of the abundance mental-ity in surgery elevates the conflict above the affected

1	to the uti-lization of the traditional fault-finding model, which identifies sides as right or wrong.46 Application of the abundance mental-ity in surgery elevates the conflict above the affected parties and focuses on the higher unifying goal of improved patient care. “Quality Improvement” (previously or alternatively “Morbidity and Mortality”) conferences are managed in this style and have the purpose of practice improvement and improving overall quality of care within the system, as opposed to placing guilt or blame on the surgeon or surgical trainees for the complication being reviewed. The traditional style of command-and-control technique based on fear and intimidation is no longer welcome in any healthcare system and can lead to sanctions, lawsuits, and removal of hospital privileges or position of leadership.Another intuitive method that can help surgical trainees learn to resolve conflict is the “history and physical” model of conflict resolution. This model is based on the

1	or position of leadership.Another intuitive method that can help surgical trainees learn to resolve conflict is the “history and physical” model of conflict resolution. This model is based on the seven steps of caring for a surgical patient that are well known to the surgical trainee47: (a) the “history” is the equivalent of gathering subjec-tive information from involved parties with appropriate empa-thy and listening; (b) the “laboratory/studies” are the equivalent of collecting objective data to validate the subjective informa-tion; (c) a “differential diagnosis” is formed out of possible root causes of the conflict; (d) the “assessment/plan” is developed in the best interest of all involved parties; the plan, including risks and benefits, is openly discussed in a compassionate style of com-munication; (e) “preoperative preparation” includes the acquisi-tion of appropriate consultations for clearances, consideration of equipment and supplies needed for implementation, and the

1	of com-munication; (e) “preoperative preparation” includes the acquisi-tion of appropriate consultations for clearances, consideration of equipment and supplies needed for implementation, and the “informed consent” from the involved parties; (f) the “operation” is the actual implementation of the agreed-upon plan, including a time-out; (g) and “postoperative care” involves communicat-ing the operative outcome, regular postoperative follow-up, and the correction of any complications that arise. This seven-step method is an example of an objective, respectful method of con-flict resolution.47 Practicing different styles of conflict resolu-tion and effective communication in front of the entire group of Brunicardi_Ch01_p0001-p0026.indd 929/01/19 10:58 AM 10BASIC CONSIDERATIONSPART Isurgical trainees attending the leadership training program is an effective means of teaching conflict resolution techniques.Time ManagementIt is important for leaders to practice effective time

1	trainees attending the leadership training program is an effective means of teaching conflict resolution techniques.Time ManagementIt is important for leaders to practice effective time management. Time is the most precious resource, as it cannot be bought, saved, or stored. Thus, management of time is essential for a productive and balanced life for those in the organization. The effective use of one’s time is best done through a formal time management program to improve one’s ability to lead by setting priorities and making choices to achieve goals. The efficient use of one’s time helps to improve both productivity and quality of life.48-50It is important for surgeons and surgical trainees to learn and use a formal time-management program. There are ever-increasing demands placed on surgeons and surgical trainees to deliver the highest quality care in highly regu-lated environments. Furthermore, strict regulations on limita-tion of work hours demand surgical trainees learn patient

1	and surgical trainees to deliver the highest quality care in highly regu-lated environments. Furthermore, strict regulations on limita-tion of work hours demand surgical trainees learn patient care in a limited amount of time.48-50 All told, these demands are enormously stressful and can lead to burnout, drug and/or alcohol abuse, and poor performance.48-50 A time-motion study of general surgery trainees analyzed residents’ self-reported time logs to determine resident time expenditure on educa-tional/service-related activities (Fig. 1-10).50 Surprisingly, senior residents were noted to spend 13.5% of their time on low-service, low-educational value activities. This time, prop-erly managed, could be used to either reduce work hours or improve educational efficiency in the context of new work hour restrictions. It is therefore critical that time be used wisely on effectively achieving one’s goals.Parkinson’s law, proposed in 1955 by the U.K. politi-cal analyst and historian Cyril

1	new work hour restrictions. It is therefore critical that time be used wisely on effectively achieving one’s goals.Parkinson’s law, proposed in 1955 by the U.K. politi-cal analyst and historian Cyril Northcote Parkinson, states that work expands to fill the time available for its completion, thus leading individuals to spend the majority of their time on insignificant tasks.51 Pareto’s 80/20 principle states that 80% of goals are achieved by 20% of effort and that achieving the final 20% requires 80% of their effort. Therefore, proper planning for undertaking any goal needs to include an analysis of how much effort will be needed to complete the task.49 Formal time management programs help surgeons and surgical trainees bet-ter understand how their time is spent, enabling them to increase productivity and achieve a better-balanced lifestyle.Various time allocation techniques have been described.49 A frequently used basic technique is the “prioritized list,” also known as the ABC

1	productivity and achieve a better-balanced lifestyle.Various time allocation techniques have been described.49 A frequently used basic technique is the “prioritized list,” also known as the ABC technique. Individuals list and assign relative 6Low educationLow serviceHigh serviceHigh educationTime Motion StudyLow education, lowservice value (Ex: Waiting duringmandatory in-house call)High education, lowservice value (Ex: Teaching conferences)High education, highservice value (Ex: Operating with a mentor)Low education,high service value (Ex: Repeatedly performing History & Physicals)values to their tasks. The use of the lists and categories serves solely as a reminder, thus falling short of aiding the user in allo-cating time wisely. Another technique is the “time management matrix technique.”49 This technique plots activities on two axes: importance and urgency, yielding four quadrants (Fig. 1-11). Congruous with the Pareto’s 80/20 principle and Parkinson’s law, the time management

1	This technique plots activities on two axes: importance and urgency, yielding four quadrants (Fig. 1-11). Congruous with the Pareto’s 80/20 principle and Parkinson’s law, the time management matrix technique channels efforts into quadrant II (important but nonurgent) activities. The activi-ties in this quadrant are high yield and include planning, creative activity, building relationships, and maintaining productivity. Too often, surgeons spend a majority of their time attending to quadrant I (important and urgent) tasks. Quadrant I tasks include emergencies and unplanned or disorganized situations that require intensive and often inefficient effort. While most surgeons and surgical trainees have to deal with emergencies, they often develop the habit of inappropriately assigning activi-ties into quadrant I; excess time spent on quadrant I tasks leads to stress or burnout for the surgeon and distracts from long-term goals. Efficient time management allows surgeons and surgical trainees

1	into quadrant I; excess time spent on quadrant I tasks leads to stress or burnout for the surgeon and distracts from long-term goals. Efficient time management allows surgeons and surgical trainees to be proactive about shifting energy from quadrant I tasks to quadrant II, emphasizing preplanning and creativity over always attending to the most salient issue at hand, depend-ing on the importance and not the urgency.Finally, “the six areas of interest” is an alternative effec-tive time management model that can help surgeons and surgi-cal trainees achieve their goals, live a better-balanced lifestyle, and improve the quality of their lives.49 The process begins by performing a time-motion study in which the activities of 6-hour increments of time over a routine week are chronicled. At the end of the week, the list of activities is analyzed to determine how the 168 hours in 1 week have been spent. The surgical trainee then selects six broad categories of areas of interest

1	At the end of the week, the list of activities is analyzed to determine how the 168 hours in 1 week have been spent. The surgical trainee then selects six broad categories of areas of interest ImportantUrgentQuadrant IQuadrant IIIQuadrant IIQuadrant IVNonurgentTime Management MatrixNonimportantFigure 1-11. Time management. (Data from Covey S. The Seven Habits of Highly Effective People. New York, NY: Simon & Schuster; 1989.)Figure 1-10. Surgery resident time-motion study.Brunicardi_Ch01_p0001-p0026.indd 1029/01/19 10:58 AM 11LEADERSHIP IN SURGERYCHAPTER 1(i.e., family, clinical care, education, health, community service, hobbies) and sets a single activity goal in each category every day and monitors whether those goals are achieved. This tech-nique is straightforward and improves one’s quality of life by setting and achieving a balanced set of goals of personal inter-est, while eliminating time-wasting activities.A formal time management program is essential for modern

1	one’s quality of life by setting and achieving a balanced set of goals of personal inter-est, while eliminating time-wasting activities.A formal time management program is essential for modern leadership. The practice and use of time management strategies can help surgeons and surgical trainees achieve and maintain their goals of excellent clinical care for their patients, while maintaining a more balanced lifestyle.Self-Care and WellnessThe challenges of practicing medicine place unique stresses on surgeons. A departmental program for improving wellness and teaching self-care can help alleviate these stresses. Acknowl-edging these stresses is an important step for any leader to help peers at risk. Quality of life surveys have identified individual protective factors that can be implemented prophylactically. These factors for improving self-care and wellness include regu-lar exercise programs, maintenance of routine medical care, and health screening. The following may not apply to

1	prophylactically. These factors for improving self-care and wellness include regu-lar exercise programs, maintenance of routine medical care, and health screening. The following may not apply to all physicians; however, religious practices, reflective writing, and maximizing work-life balance have also been demonstrated to be protective.52Surgeons and physicians overall experience increased rates of suicide, depression, substance abuse, marital and family prob-lems, and other stress-related health effects as compared to the general population. Suicide rates in physicians are higher among those who are divorced, widowed, or never married. Depression is a common challenge, with rates as high as 30% among trainees, and higher when lifetime risk is considered. Drug and alcohol abuse among physicians mirrors the general population; however, physicians have higher rates of prescription drug abuse. The abil-ity to self-medicate likely contributes to prescription drug abuse by physicians.

1	physicians mirrors the general population; however, physicians have higher rates of prescription drug abuse. The abil-ity to self-medicate likely contributes to prescription drug abuse by physicians. Divorce and marriage unhappiness among physi-cians has been attributed to the “psychology of postponement,” compulsive personality traits that are reinforced and selected for during medical training, and lack of work-life balance. Residents, due to their inexperience, may be at higher risk than practicing physicians. For physicians who do not seek professional help, fear of losing their medical license is the most commonly provided reason. Departmental wellness programs may provide an alterna-tive source of support for these surgeons.52-54The past 10 years have seen a significant increase in atten-tion to the issue of physician wellness. Physician wellness has become an issue transcending specialties and resulting in signif-icant research. The creation of wellness and self-care programs

1	atten-tion to the issue of physician wellness. Physician wellness has become an issue transcending specialties and resulting in signif-icant research. The creation of wellness and self-care programs within departments represents an opportunity for surgeons to demonstrate leadership qualities.52-54RecruitmentThe challenges of modern medicine and ever-larger medical centers have created a reality where no single surgeon-leader can exercise complete control—it takes a team of leaders with shared vision, mission, and goals. To this end, the previously discussed “level 5 leader” who embodies personal humility and professional will is essential.8 Previous generations whose lead-ers and departments were composed of self-proclaimed giants dominated and suppressed alternative points of view, com-munication, and innovation. In recent years, there has been a change to building teams with authentic leaders who have high ethical standards and well-developed nontechnical skills, who lead by

1	com-munication, and innovation. In recent years, there has been a change to building teams with authentic leaders who have high ethical standards and well-developed nontechnical skills, who lead by example, and who never compromise excellence. The surgeon-leader must build a team where talented individuals are placed in the right job for their skills. The essence of a leader is one who enables others to succeed. Team work is imperative to change, and trust is the make-or-break component. Simply put, teams that trust each other work well, and teams that do not trust each other do not work well.9Creating a Culture of Empathy, Patient-Family-Centered Care, and Personalized SurgeryCreating the right culture is the most challenging of all the sur-geon-leader’s tasks. Modern surgical departments should focus on creating a culture of empathy, patient–family-centered care, and personalized surgery. Instilling a positive culture requires both discipline and consistency because it may take

1	should focus on creating a culture of empathy, patient–family-centered care, and personalized surgery. Instilling a positive culture requires both discipline and consistency because it may take consider-able time to change how people think, feel, and behave.9,55,56 Organizational culture is built around the leader’s vision and values. Coming up with strong values requires genuine com-mitment. A leader should realize that staying true to his or her values can be challenging when conflicts arise.57WHY WE LEADChoosing to Become a LeaderThere are many benefits to becoming a leader. Humankind has pondered the question of whether leaders are born or made for millennia. The best evidence to date indicates that leaders are both born and made. Leadership potential is a skill that all per-sons are born with, to some degree, and that can be formally trained, learned through observation, and honed with practice.13 The positive effects of a leader on others are innumerable, including a leader’s

1	born with, to some degree, and that can be formally trained, learned through observation, and honed with practice.13 The positive effects of a leader on others are innumerable, including a leader’s positive influence on innovation, diversity, culture, and quality. For modern surgeons, leadership skills are essential for the delivery of quality patient care; therefore, it is the duty of the surgeon to study leadership.For the surgeon studying to be a better leader, effective leadership also has many individual benefits, including rec-ognition from one’s peers, promotion, and autonomy. Mod-ern leaders are increasingly required to be humble about their accomplishments in order to be successful and effective.8 Beyond recognition, promotion, and autonomy there are more selfless reasons for surgeons to desire leadership. Leadership is a tool to help make a difference. Leadership is a good path towards a career as an educator, which offers the leader a sense of accomplishment and

1	for surgeons to desire leadership. Leadership is a tool to help make a difference. Leadership is a good path towards a career as an educator, which offers the leader a sense of accomplishment and satisfaction in seeing others succeed. Some choose to become leaders out of a sense of selfless ser-vice, taking on leadership for the benefit of others, or out of a desire to solve problems. Leadership may come with material rewards, including wealth and power, which motivate some.Whatever the motivation, surgeons, in their role as lead-ers of patient care teams, have a duty to develop some skill in leadership. It would be best for individuals, departments, and patients if all surgeons sought to develop leadership skills and experience in some area of administration, patient care, educa-tion, or research. The benefits to the individual are numerous.Leadership’s Effect on Healthcare Cost and Clinical OutcomesMuch attention has rightly been paid to historical leaders’ impact on humanity.

1	or research. The benefits to the individual are numerous.Leadership’s Effect on Healthcare Cost and Clinical OutcomesMuch attention has rightly been paid to historical leaders’ impact on humanity. Surgical leaders of the past have made great contributions on which we may build. All surgeons have a responsibility to be leaders, whether at the team level or in Brunicardi_Ch01_p0001-p0026.indd 1129/01/19 10:58 AM 12BASIC CONSIDERATIONSPART Ian administrative or organizational capacity. To that end, it is worth noting the benefits of formal leadership education.Large observational studies using trained observers assessed the effects of different surgical leadership styles on operative cases. Team cohesion and collective efficiency were reduced when leaders utilized abusive supervision or over-controlling methods. Abusive supervision alone was associated with decreased “psychological safety.”58 Surgeons perceived as having positive leadership characteristics by their staff have lower

1	over-controlling methods. Abusive supervision alone was associated with decreased “psychological safety.”58 Surgeons perceived as having positive leadership characteristics by their staff have lower 30-day all-cause mortality.59 This is likely due to creating a culture of safety where the staff can speak up if they notice an error and feel they have the latitude to do what is best for the patient quickly and autonomously.59,60With increased recognition and attention on human error, nontechnical skills, including leadership, play a role in patient safety. The landmark study, “To Err Is Human,” estimated that almost 100,000 people die each year due to medical errors.20 In the surgical setting, 40% to 50% of errors may be attributed to communication breakdown. The Multifactor Leadership Ques-tionnaire scores subjects on their demonstration of transforma-tional leadership behaviors. Transformational leaders exhibit the qualities of charisma, inspired motivation, intellectual stimulation,

1	scores subjects on their demonstration of transforma-tional leadership behaviors. Transformational leaders exhibit the qualities of charisma, inspired motivation, intellectual stimulation, and individualized consideration. In video analysis of complex surgical operations, surgeons scoring even a single point higher on the transformational leadership score exhibited 3 times more information sharing behaviors, 5 times more posi-tive voice behaviors, and 10 times more supportive behaviors, all while displaying poor behaviors 12.5 times less frequently than their peers.60 Exhibiting the characteristics of transforma-tional leadership clearly has much to offer the surgeon-leader in preventing serious errors.58-60The field of trauma contains the largest body of formal study demonstrating the positive effects of leadership on clini-cal results. Strong leadership skills improve both the speed of resuscitation and completion of the initial trauma evaluation.61-63 There is no one optimal style

1	effects of leadership on clini-cal results. Strong leadership skills improve both the speed of resuscitation and completion of the initial trauma evaluation.61-63 There is no one optimal style of leadership covering all situa-tions; some call for a more empowering leadership style while others call for a more directive style. The optimal style of lead-ership varies based on team composition, with less experienced teams better responding to the directive style, while more expe-rienced teams work faster with trust and an empowering style. The formally educated surgical leader should be able to switch easily between styles based on the situation at hand.56,58,60-64Leadership styles affect responses to patient safety con-cerns and protect the organization as a whole. The surgical leader adopts a supervisory capacity while creating a culture of safety. In detail, frontline staff must be encouraged to partici-pate in safety improvement. Staff ownership of safety must be established and

1	a supervisory capacity while creating a culture of safety. In detail, frontline staff must be encouraged to partici-pate in safety improvement. Staff ownership of safety must be established and upheld. In order to assure this outcome, whistle-blowers must be protected. A culture of psychological safety, organizational fairness, and continuous learning is required. Subordinates require appropriate authority, autonomy, and lati-tude to do their jobs and care for patients.60Formal leadership training has been well studied within the Veteran’s Health Administration system using the Surgical Care Improvement Program. The Medical Team Training Pro-gram, for instance, has been shown to result in a 18% decrease in 30-day mortality65 and a 17% decrease in 30-day morbidity.66Also at the organizational level, leaders using an empowering style may improve process of care protocols and increase efficiency. Operating room turnover times specifically have been shown to be reducible.67 Value-based

1	level, leaders using an empowering style may improve process of care protocols and increase efficiency. Operating room turnover times specifically have been shown to be reducible.67 Value-based purchasing benchmarks, such as hospital-acquired infections, which affect reimbursement, can be reduced or eliminated depending on the measure.68,69 Medical errors may be reduced, and significant medical errors may have their effects mitigated. Patient satis-faction may be improved. The overall financial performance of the institution can be affected in a positive manner.69,70There are positive correlations between mutual respect, clinical leadership, and surgical safety. Traditional command and control style leadership negatively impacts psychological safety resulting in the development of more modern leadership styles. The best clinical processes have the potential to break down when there is a toxic work environment and lack of psy-chological safety within the team.The Importance of

1	more modern leadership styles. The best clinical processes have the potential to break down when there is a toxic work environment and lack of psy-chological safety within the team.The Importance of Diversity and LeadershipThe past quarter century has seen a steady increase in diversity within the field of surgery. Women, as of 2015, represent 38% of surgical trainees and 10% of academic professors currently, but have doubled their representation in the past 20 years.71 Some fields, such as head and neck surgery and plastic surgery72 have studied their own subspecialty groups with similar find-ings. African Americans comprise both 6% of medical school graduates, 6% surgical trainees, and 2% to 4% of professors of surgery nationwide.73 Hispanics represent 5% of graduat-ing medical students, 9% of general surgery trainees, and 4% to 5% of persons at all levels of academic surgery.73 Physician diversity is crucial and may help to address disparities in social determinants of

1	students, 9% of general surgery trainees, and 4% to 5% of persons at all levels of academic surgery.73 Physician diversity is crucial and may help to address disparities in social determinants of health.74Studies indicate that the bottleneck in diversity occurs at the level of the medical school application pool, which in turn is caused by educational deficiencies at the primary, secondary, and collegiate level.73,75-78 As an attempted solution, the University of Michigan developed a “pipeline” program that pairs grade-school and high-school students with physicians for experiential learn-ing and the development of mentoring, presentation skills, and networking.75 It is important for departments of surgery to develop a diversity program for recruitment of residents and faculty. Multi-institutional blinded studies indicate that the implementa-tion of formal leadership and diversity training improves diversity leadership and strategic human resource management.74,78LEADERSHIP STYLESThe

1	blinded studies indicate that the implementa-tion of formal leadership and diversity training improves diversity leadership and strategic human resource management.74,78LEADERSHIP STYLESThe principles of leadership can be practiced in a variety of styles. Just as there are many definitions of leadership, many classifications of styles exist as well. A landmark study by Daniel Goleman in Harvard Business Review identified six distinct leadership styles, based on different components of emotional intelligence.79 Emotional intelligence is the ability to recognize, understand, and control the emotions in others and ourselves. By learning different styles, surgeons and trainees can recognize their own leadership style and the effect on the team dynamic. Furthermore, it teaches when the situation may demand change in style for the best outcome. The six leadership styles identified are coercive, authoritative, affiliative, demo-cratic, pacesetting, and coaching.The coercive leader demands

1	may demand change in style for the best outcome. The six leadership styles identified are coercive, authoritative, affiliative, demo-cratic, pacesetting, and coaching.The coercive leader demands immediate compliance. This style reflects the command and control style that has histori-cally dominated surgery. Excessive coercive leadership erodes team members’ sense of responsibility, motivation, sense of participation in a shared vision, and ultimately, performance. The phrase, “Do what I tell you!” brings to mind the coercive leader. However, it is effective in times of crisis to deliver clear, Brunicardi_Ch01_p0001-p0026.indd 1229/01/19 10:58 AM 13LEADERSHIP IN SURGERYCHAPTER 1concise instruction. This style should be used sparingly and is best suited for emergencies.The authoritative leader embodies the phrase “Come with me,” focusing on mobilizing the team toward a common, grand vision. This type of leader allows the team freedom to innovate, experiment, and devise its own

1	leader embodies the phrase “Come with me,” focusing on mobilizing the team toward a common, grand vision. This type of leader allows the team freedom to innovate, experiment, and devise its own means. Goleman’s research indicates this style is often the most effective. These leaders display self-confidence, empathy, and proficiency in initiating new ideas and leading people in a new direction. This is best used when a shift in paradigm is needed.The affiliative leader creates harmony and builds emo-tional bonds. This requires employment of empathy, building relationships, and emphasis on communication. An affiliative leader frequently gives positive feedback. This style can allow poor performance to go uncorrected if too little constructive/critical advice is given. Affiliative leadership is most useful when motivating people during stressful circumstances or heal-ing rifts in a team.The coaching style of leadership focuses on developing people for the future. Coaching is leadership

1	is most useful when motivating people during stressful circumstances or heal-ing rifts in a team.The coaching style of leadership focuses on developing people for the future. Coaching is leadership through mentor-ship. The coach gives team members challenging tasks, coun-sels, encourages, and delegates. Unlike the affiliative leader who focuses on positive feedback, the coach helps people iden-tify their weaknesses and improve their performance, and ties their work into their long-term career aspirations. This leader-ship style builds team capabilities by helping motivated learners improve. However, this style does not work well when team members are defiant and unwilling to change or learn, or if the leader lacks proficiency.The democratic leader forges consensus through participa-tion. This leadership style listens to and values each member’s input. It is not the best choice in an emergency situation, when time is limited, or when teammates cannot contribute informed guidance to the

1	leadership style listens to and values each member’s input. It is not the best choice in an emergency situation, when time is limited, or when teammates cannot contribute informed guidance to the leader. It can also be exasperating if a clear vision does not arise from the collaborative process. This style is most appropriate when it is important to obtain team consensus, quell conflict, or create harmony.The pacesetter leader sets high standards for performance and exemplifies them. These leaders identify poor performers and demand more from them. However, unlike the coach, the pacesetter does not build the skills of those who are not keep-ing up. Rather, a pacesetter will either take over the task him-self or delegate the task to another team member. This style can be summed up best by the phrase, “Do as I do, now.” This leadership style works well when it is important to obtain high-quality results and there is a motivated, capable team. However, pacesetters can easily become

1	by the phrase, “Do as I do, now.” This leadership style works well when it is important to obtain high-quality results and there is a motivated, capable team. However, pacesetters can easily become micromanagers who have diffi-culty delegating tasks to team members, which leads to burn out on the part of the leader. Additionally, team members can feel overwhelmed and demoralized by the demands for excellence without an empathic counter balance.Each of the above styles of leadership has strengths and weakness. Importantly, leaders who are the most successful do not rely only on one leadership style alone. They use sev-eral of them seamlessly depending on the situation and the team members at hand. Therefore, the more styles a leader has mastered, the better, with particular emphasis on the authorita-tive, affiliative, democratic, and coaching styles. Each leader-ship style is a tool that is ultimately employed to guide a team to realizing a vision or goal. Thus, leadership training

1	the authorita-tive, affiliative, democratic, and coaching styles. Each leader-ship style is a tool that is ultimately employed to guide a team to realizing a vision or goal. Thus, leadership training programs should teach the proper use of all leadership styles while adher-ing to the principles of leadership.7FORMAL LEADERSHIP TRAINING PROGRAMS IN SURGERYHistory of Leadership Training and the Multifactor Leadership QuestionnaireSince it has been shown that effective leadership can improve patient outcomes, leadership principles and skills should be taught to surgical trainees using formal leadership training programs. The importance of teaching leadership skills is reflected by the ACGME mandated core competencies (see Table 1-1). However, surgical trainees, most notably chief residents, find themselves in various leadership roles without ever having experienced formal-ized leadership training, which has been shown to result in a self-perceived lack of leadership ability.2 When

1	find themselves in various leadership roles without ever having experienced formal-ized leadership training, which has been shown to result in a self-perceived lack of leadership ability.2 When surveyed on 18 core leadership skills (Table 1-3), 92% of residents rated all 18 skills as important, but over half rated themselves as “minimally” or “not competent” in 10 out of 18 skills.2 Increasingly, residents and junior faculty are requesting leadership training and wish to close the gap between perceived need for training and the implementa-tion of formal leadership training programs.80-86A number of leadership workshops have been created. Extracurricular leadership programs have been designed mostly Table 1-3Eighteen leadership training modulesSKILLSIMPORTANCE MEAN SCORECOMPETENCE MEAN SCORE*Academic program development3.22.4*Leadership training3.82.3*Leadership theory3.22.1*Effective communication3.72.7*Conflict resolution3.83*Management principles3.72.7*Negotiation3.72.8*Time

1	SCORE*Academic program development3.22.4*Leadership training3.82.3*Leadership theory3.22.1*Effective communication3.72.7*Conflict resolution3.83*Management principles3.72.7*Negotiation3.72.8*Time management42.8*Private or academic practice, managed care3.62*Investment principles3.52.2*Ethics3.63.2Billing, coding, and compliance3.51.7*Program improvement32*Writing proposals3.32.2*Writing reports3.42.4*Public speaking3.72.7*Effective presentations3.72.7*Risk management3.52.1*Total3.62.5**P <0.001 by Student t-test between mean importance and mean competence scores.Reproduced with permission from Itani KMF, Liscum K, Brunicardi FC: Physician leadership is a new mandate in surgical training, Am J Surg. 2004 Mar;187(3):328-331.Brunicardi_Ch01_p0001-p0026.indd 1329/01/19 10:58 AM 14BASIC CONSIDERATIONSPART Ifor physicians with an MBA or management background but have not been incorporated into the core residency training program.80 Also, there are many institutions that have published

1	physicians with an MBA or management background but have not been incorporated into the core residency training program.80 Also, there are many institutions that have published experiences with leadership retreats or seminars for residents or young physicians.81-84 The ACGME hosts multiple leader-ship skills workshops for chief residents, mostly targeted toward pediatricians, family practitioners, and psychiatrists.85 Similarly, the American College of Surgeons leads an annual 3-day lead-ership conference focusing on leadership attributes, consensus development, team building, conflict resolution, and translation of leadership principles into clinical practice.86-87 These pro-grams were all received well by participants and represent a call for a formal leadership program for all surgical trainees.An innovative leadership curriculum first implemented in 2000, prior to work-hour restrictions, taught general surgery trainees’ collaborative leadership skills at a time when the

1	surgical trainees.An innovative leadership curriculum first implemented in 2000, prior to work-hour restrictions, taught general surgery trainees’ collaborative leadership skills at a time when the tradi-tional command-and-control leadership style predominated.2,89,90 Surgical residents participated in 18-hour-long modules based on the leadership principles and skills listed in Table 1-2, taught by the surgical faculty. A number of leadership techniques, including time management techniques and applied conflict resolution techniques described earlier, were designed and implemented as part of this leadership training program. Within 6 months of implementation, residents’ self-perceived total commitment to the highest personal and professional standards, communication skills, visualization of clear missions of patient care, and leadership of others toward that mission increased sig-nificantly.2,89,90 Remarkably, the positive impact of this leader-ship curriculum was significant when

1	of clear missions of patient care, and leadership of others toward that mission increased sig-nificantly.2,89,90 Remarkably, the positive impact of this leader-ship curriculum was significant when measured using tools, such as the Multifactor Leadership Questionnaire (MLQ), social skills inventory, personality inventory, and internal strength scorecard.2,89,90 The MLQ is a well-validated instrument that objectively quantifies leadership beliefs and self-perceived out-comes across medical and nonmedical disciplines. Based on the MLQ, surgical residents more often use a passive-avoidance style of leadership that emphasizes taking corrective action only after a problem is “significant and obvious.” This tool can also be used to track progress toward more effective, collaborative styles of leadership. These studies demonstrated the ability to measure leadership behavior of surgical trainees in a standard-ized, quantifiable format.2,89,90 Taken together, these studies sup-port the concept

1	These studies demonstrated the ability to measure leadership behavior of surgical trainees in a standard-ized, quantifiable format.2,89,90 Taken together, these studies sup-port the concept that leadership skills can and should be taught to surgical trainees, and there are validated tools to measure outcomes.Designing the ProgramSuccess in designing a formal leadership development program can be achieved through the following method. First, select the right participants at the right time in their career. Junior sur-geons new to practice are ideal; however, they should be given a chance to get their clinical and research interests off the ground before they are asked to lead others. Candidates, should be open to taking on leadership roles and have the right combination of introspection and humility that lends to professional develop-ment. High-quality speakers from the business, legal, creative, and medical worlds should be brought as guest speakers. Topics could include leadership

1	and humility that lends to professional develop-ment. High-quality speakers from the business, legal, creative, and medical worlds should be brought as guest speakers. Topics could include leadership overall, strategy, finance, management skills, feedback, and coaching. Constructive criticism is essential because prospective leaders will need guidance and mentoring. Surgeons who have been through a formal leadership training program will become proficient at team-building skills and man-agement and will become self-empowered individuals.918Formal leadership training is not restricted to faculty alone. Leadership training should begin early and continue throughout residency. Surgical residents’ leadership styles have been studied in environments where they are given an assistant to supervise, as if they were an attending. Most residents were able to adapt to difficult operative challenges, in this setting, by providing a more directed style of leadership to their assistants. When faced

1	as if they were an attending. Most residents were able to adapt to difficult operative challenges, in this setting, by providing a more directed style of leadership to their assistants. When faced with a less challenging task, or when the surgery resident’s confidence was particularly high, their leadership score was also high. For the surgical resident preparing to move on to the attending level, such skills are necessary to develop.92Nontechnical surgical skills, such as leadership, demon-strate a number of desired effects for the operative team. Patient safety, including all cause 30-day mortality, is improved by stronger nontechnical skills.59 Development of clear and effec-tive communication, situational awareness, team skills, and decision-making all are correlated with reduced surgical errors. Interruptions, such as needing to answer a page during an opera-tion, are the only nontechnical factors in surgical error that are not directly attributable to leadership style.93Surgical

1	errors. Interruptions, such as needing to answer a page during an opera-tion, are the only nontechnical factors in surgical error that are not directly attributable to leadership style.93Surgical leaders have a responsibility to make ethical deci-sions. At this time, there is no standard curriculum to formally train surgical residents in ethics, despite interest from a majority of residency program directors.94-97 Several solutions have been proposed. A case-based approach to ethics training appears to have some merit, where monthly hour long ethical dilemmas are discussed in an informal, nonhierarchical setting.98 In another study, an ICU-based simulation model demonstrated promise for teaching compassion and end-of-life ethics to surgical resi-dents. In this model, surgery residents have their first end-of-life conversations with standardized patients simulating the surgical ICU environment.99,100Practicing Leadership Skills and Assessing Leadership Formally With Objective

1	have their first end-of-life conversations with standardized patients simulating the surgical ICU environment.99,100Practicing Leadership Skills and Assessing Leadership Formally With Objective Structured Clinical Examination (OSCE) and SimulationThe past decade has seen a demonstrable increase in our knowl-edge of how to develop leadership skills, particularly through simulation, as well as leadership evaluation through OSCE and other tools. Multiple groups have assessed multidisciplinary teams, typically composed of nurses, anesthesia groups, and surgeons for the leadership associated nontechnical skills of communication, teamwork, and situational awareness. Through increasingly validated instruments and assessment tools, these nontechnical skills have been found to be trainable.101 The OSCE has been established as the gold standard102 for the train-ing and assessment of a wide range of clinical and nontechnical skills with high reliability and validity.103-106The OSCE was developed

1	OSCE has been established as the gold standard102 for the train-ing and assessment of a wide range of clinical and nontechnical skills with high reliability and validity.103-106The OSCE was developed by Harden, at the Ninewells Hospital in Dundee, Scotland, and first published in 1975.107 He subsequently coined the term “OSCE” in his 1979 publica-tion “Assessment of Clinical Competence Using an Objective Structured Clinical Examination (OSCE).”108 The purpose of the OSCE was to address the lack of a reliable method to evalu-ate the clinical abilities of physicians and featured a compre-hensive assessment of history-taking and physical examination skills. Early versions also assessed nontechnical skills, patient interaction, and professionalism. Since its inception, the OSCE has matured, been subjected to rigorous tests of reliability and validity, and has seen widespread adoption.109-111OSCEs remain a critical portion of resident evaluation. They have been well validated for teaching

1	subjected to rigorous tests of reliability and validity, and has seen widespread adoption.109-111OSCEs remain a critical portion of resident evaluation. They have been well validated for teaching leadership skills in Brunicardi_Ch01_p0001-p0026.indd 1429/01/19 10:58 AM 15LEADERSHIP IN SURGERYCHAPTER 1trauma and interacting with simulated patients in difficult sce-narios. OSCEs can be tailored to a variety of circumstances, including practicing breaking bad news or discussing end of life care, dealing with angry or aggressive patients, and simulating disagreements with other providers or family members.109-112 The potential for OSCEs to train, test, and perfect nontechnical skills, such as leadership, is extraordinary.A pilot project for the Medical Council of Canada was conducted by the University of Toronto and published in 1988 describing the use of an OSCE for evaluating the clinical skills of international medical graduates applying to Canadian resi-dency.113 Effective

1	by the University of Toronto and published in 1988 describing the use of an OSCE for evaluating the clinical skills of international medical graduates applying to Canadian resi-dency.113 Effective communication and language proficiency have been key components since the beginning. A compre-hensive review of this program 2 years later confirmed the reliability and validity of using an OSCE for this purpose.114 The Medical Council of Canada has subsequently mandated a requirement for an OSCE evaluation of all international gradu-ates applying for positions in Canada. In place for the past two decades, the program has ensured a baseline proficiency of skill, attitude, knowledge, and other nontechnical skills.115OSCEs quickly gained acceptance as an established tool to assess learners in a comprehensive manner and became the inspiration for the creation of the USMLE Step 2 Clinical Skills (CS) examination, required for all U.S. medical students prior to licensure.116 Indeed, medical

1	a comprehensive manner and became the inspiration for the creation of the USMLE Step 2 Clinical Skills (CS) examination, required for all U.S. medical students prior to licensure.116 Indeed, medical students whose schools use OSCE as practice do better on USMLE Step 2.117 The USMLE Step 2 CS examination meets the criteria, discussed in the following section, for a thorough and well-designed OSCE examination, due to its 12-station design which takes 8 hours to complete. It has been found to be a valid and comprehensive evaluation of a student’s clinical abilities, admittedly at massive expense to medical students.118 In the United States, osteopathic medical students take the OSCE-style Level 2 Performance Evaluation.119Although station number and total duration are not com-pletely agreed upon, data indicate that the OSCE examination should be between 3 and 6 hours and 8 to 10 stations in length in order to obtain reliable (r = ≥0.7) communication, history, and physical examination

1	upon, data indicate that the OSCE examination should be between 3 and 6 hours and 8 to 10 stations in length in order to obtain reliable (r = ≥0.7) communication, history, and physical examination skills. A guideline was that at least seven cases are needed in any domain to achieve reliability. The testing period may be spread over several sessions making up an aggregate score in order to maintain validity. Many medical schools prepare their students for clinical practice with OSCE-style examinations throughout the year, which, taken together, are summative of a high-quality, multistation, valid OSCE. Checklists are typically the standard scoring tools; however, checklists alone may not be as reliable as a more comprehen-sive review by more experienced clinicians—particularly when assessing more advanced students and residents.120 All of the licensure examinations, discussed previously, meet the criteria for a well-designed OSCE based on number of stations and time duration.Beginning

1	more advanced students and residents.120 All of the licensure examinations, discussed previously, meet the criteria for a well-designed OSCE based on number of stations and time duration.Beginning in 2003, the ACGME mandated the use of OSCEs within residency programs. At the time, residents were wary of its adoption, particularly fearing its use as a tool for determining promotion. Residents’ perceptions of the examina-tion, over time, did change to reflect an acceptance of its use for grading both technical and nontechnical skills.121-123In the United States, the OSCE assesses technical and nontechnical skills in an accurate and valid fashion. The OSCE demonstrates a rapid progression of technical skills highly cor-related to a postgraduate year, whereas clinical skills improve at a more moderate rate121 (Fig. 1-12).10.5123PGY Level45z score0–0.5–1Technical stationsClinical stationsFigure 1-12. Resident assessment by year of training by OSCE. Technical skills assessment demonstrates

1	moderate rate121 (Fig. 1-12).10.5123PGY Level45z score0–0.5–1Technical stationsClinical stationsFigure 1-12. Resident assessment by year of training by OSCE. Technical skills assessment demonstrates a rapid and continual pro-gression through 5 years of training, whereas clinical evaluations show only modest improvement over the same time period. (Data from Turner JL, Dankoski ME. Objective structured clinical exams: a critical review, Fam Med. 2008 Sep;40(8):574-578.)The past 2 to 3 years has seen an explosion in simula-tion technology and research. In one particularly strong study, multiple teams were assessed for hemorrhage and airway emer-gencies. The Non-Technical Skills for Surgeons (NOTSS) tool was used to assess teams prior to and during simulation. For surgeons, higher NOTSS scores were associated with a quicker resolution of the simulation crisis.125 Advances are being made in using simulation to solve difficult to teach physical examina-tion skills such as breast lump

1	scores were associated with a quicker resolution of the simulation crisis.125 Advances are being made in using simulation to solve difficult to teach physical examina-tion skills such as breast lump detection and prostate or rectal cancers.126,127 Studies assessing these new simulation tools have also indicated that many attending level surgeons would benefit from continued simulation practice both for keeping operative skills fresh and preventing the decline of physical exam skills, for instance during dedicated research time.126-128The Objective Structured Assessment of Technical Skills (OSATS) was initially developed as a bench station examination. It was later applied to intraoperative skill assessment, and appears to be an additional option for programs seeking a validated and reliable method for mixing technical skills assessment into simu-lations of nontechnical exercises to create a more “real world” simulation.129-133 With recent focus on milestones and proficiency-based

1	method for mixing technical skills assessment into simu-lations of nontechnical exercises to create a more “real world” simulation.129-133 With recent focus on milestones and proficiency-based promotion, as compared to time-based promotion of resi-dents, there is a need to reliably assess intraoperative skill.134 Digitization and modern computing have created new opportu-nities for simulation and education. One proposed method is a real-time, mobile web system featuring consistent and accurate assessment of the residents’ performance. The platform enabled timely recording of data, was efficient in terms of how much fac-ulty time it took to complete an assessment (average 2 minutes), and from a validity standpoint did trend well overall with resident postgraduate year. The system itself fulfilled the ACGME and American Board of Surgery mandate for program assessment of resident performance in the operating room.134Nontechnical skills often erode during stressful events, particularly in

1	the ACGME and American Board of Surgery mandate for program assessment of resident performance in the operating room.134Nontechnical skills often erode during stressful events, particularly in surgery where bleeding, complexity of the opera-tion, time-constraints, and equipment problems can have a nega-tive effect. Additionally, roadblocks with insurance and other third parties, critical illness, and delivering bad news add differ-ent kinds of stress.135 Indeed, video analysis of real operations indicates that attending surgeons typically take over, change their style of leadership, and decrease their teaching in the oper-ating room once unintended events occur.136Brunicardi_Ch01_p0001-p0026.indd 1529/01/19 10:58 AM 16BASIC CONSIDERATIONSPART IBy using simulated patients, patient-centered models, and intensive and immersive training, nontechnical skills including communication can improve interview techniques.137,138 Post com-munication skills training at the 12-month follow-up

1	models, and intensive and immersive training, nontechnical skills including communication can improve interview techniques.137,138 Post com-munication skills training at the 12-month follow-up demonstrated that the training was effective, and with real clinical practice after the training communication skills had improved even more.139Lastly, there appears to be a positive feedback loop tying nontechnical leadership skills with self-perceived operating room prowess. Those surgeons who rate their own technical skills highly are also more likely to engage in positive leader-ship skills, including teaching in the operating room, handle difficult situations, and provide more clear instructions.140 Simulation may be particularly critical for preventing techni-cal skill decline in residents on dedicated research time or for attending surgeons whose research, clinic, or administrative duties decrease the amount of time they can spend in the operating room. Simulation represents the future of

1	research time or for attending surgeons whose research, clinic, or administrative duties decrease the amount of time they can spend in the operating room. Simulation represents the future of medicine and an excellent opportunity for research and development. Medicine, including surgery, has much ground to make up in regards to simulation training compared to other high-risk fields, such as the military, space, and aeronautics. Modern surgical leaders should recognize surgical simulation as criti-cal to their organization’s success.Evaluation of Surgeon PerformanceMultiple organizations are evaluating the technical and nontechnical skills of surgeons in a real-time basis. We have included this com-prehensive list of organizations (Table 1-4) with a brief description of their purpose and mechanisms of evaluation. Several of these involve technical skills evaluations and most involve nontechnical Table 1-4Multiple organizations have been created to evaluate both the technical and

1	and mechanisms of evaluation. Several of these involve technical skills evaluations and most involve nontechnical Table 1-4Multiple organizations have been created to evaluate both the technical and nontechnical skills of surgeons141ORGANIZATION NAMEDESCRIPTIONMAIN SKILLS, CONDITIONS, OR QUALITIES EVALUATEDEVALUATION OF TECHNICAL SKILLS?EVALUATION OF NONTECHNICAL SKILLS?Hospital Consumer Assessment of Healthcare Providers and Systems (HCAHPS)A public reporting initiative that measures patient perspectives on and satisfaction with hospital care based on qualities of healthcare that patients view as important.Communication with nurses, communication with doctors, responsiveness of hospital staff, pain management, communication about medicines, discharge information, care transitionNoYesClinical and Group Consumer Assessment of Healthcare Providers and Systems (CGCAHPS)A public reporting initiative that measures patient perspectives on and satisfaction with care provided in an office

1	and Group Consumer Assessment of Healthcare Providers and Systems (CGCAHPS)A public reporting initiative that measures patient perspectives on and satisfaction with care provided in an office setting based on qualities of healthcare that patients view as important.Access to care, provider communication, test results, office staff, overall provider ratingNoYesDatix, Incident ReportingA database of incidents that improves reliability of physicians by improving rates of reporting, promoting ownership of mistakes, and improving patient safety.System issues, patient safety and quality issues, provider behavior, leadership styleYesYesPatient Advocacy Reporting System (PARS)A system that compiles patient complaints into a complaint index for each physician for comparison with other medical group members and to help identify high-malpractice-risk physicians who may benefit from peer intervention.Unprofessional behavior deemed as disrespectful and rudeNoYesCo-worker Observation Reporting

1	group members and to help identify high-malpractice-risk physicians who may benefit from peer intervention.Unprofessional behavior deemed as disrespectful and rudeNoYesCo-worker Observation Reporting SystemA system in which physicians document coworker unprofessional conduct in order to provide nonjudgmental and timely feedback and to encourage self-reflection and change.Unprofessional behavior deemed as disrespectful and unsafeYesYes(Continued)Brunicardi_Ch01_p0001-p0026.indd 1629/01/19 10:58 AM 17LEADERSHIP IN SURGERYCHAPTER 1Table 1-4Multiple organizations have been created to evaluate both the technical and nontechnical skills of surgeons141ORGANIZATION NAMEDESCRIPTIONMAIN SKILLS, CONDITIONS, OR QUALITIES EVALUATEDEVALUATION OF TECHNICAL SKILLS?EVALUATION OF NONTECHNICAL SKILLS?American Board of Surgery (ABS) Maintenance of Certification (MOC) ProgramA program that documents a surgeon’s ongoing commitment to professionalism, lifelong learning, and practice improvement through

1	Board of Surgery (ABS) Maintenance of Certification (MOC) ProgramA program that documents a surgeon’s ongoing commitment to professionalism, lifelong learning, and practice improvement through self-report.Restrictions on medical license, restrictions on hospital privileges, continuing medical education, self-assessment of continuing medical education, cognitive expertise, ongoing participation in quality assessment program relevant to the surgeon’s practiceYesYesHospital CompareA database that is part of the Centers for Medicare & Medicaid Services (CMS) Hospital Quality Initiative and provides information on hospital performance and quality of care based on consumer perspectives so that patients can assess and compare hospitals.Hospital Compare is based on data from HCAHPS and evaluates hospitals by the same guidelines as HCAHPSNoYesFederation of State Medical Boards (FSMB)An organization representing all state medial and osteopathic boards in the United States that license

1	hospitals by the same guidelines as HCAHPSNoYesFederation of State Medical Boards (FSMB)An organization representing all state medial and osteopathic boards in the United States that license physicians and sponsors the United States Medical Licensing Examination.Medical knowledge, patient complaints, violations of the lawYesYesInternet clinical scoresA database of direct patient opinions of physicians, provided through various sources, including Healthgrades.com, RateMDs.com, and Yelp.Professionalism, communication, timelinessNoYesHospital-Acquired Condition Reduction ProgramA government program that provides incentives for hospitals to reduce the number of undesirable patient conditions resulting from their stay in the hospital and that could have been avoided by adjusting hospital reimbursement rates accordingly.Foreign objects retained after surgery, air embolism, blood incompatibility, pressure ulcers, falls, poor glycemic control, catheter-associate infections, surgical site

1	rates accordingly.Foreign objects retained after surgery, air embolism, blood incompatibility, pressure ulcers, falls, poor glycemic control, catheter-associate infections, surgical site infections, deep vein thrombosis, pulmonary embolism, pneumothoraxYesNoAmerican College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP)A program that collects information on and provides a risk-adjusted ranking of preventable surgical complication rates to encourage providers to improve care.Surgical complications rates, surgical site infections, urinary tract infections, readmission rates, surgical outcomesYesNoCenters for Medicare & Medicaid Services Surgical Care Improvement Project (CMS SCIP)A collaborative healthcare organization that collects data on surgical complication rates based on established guidelines.Rates of infection, cardiac, venous thromboembolism, vascular, and respiratory, complications of surgeryYesNo(Continued)Brunicardi_Ch01_p0001-p0026.indd 1729/01/19

1	based on established guidelines.Rates of infection, cardiac, venous thromboembolism, vascular, and respiratory, complications of surgeryYesNo(Continued)Brunicardi_Ch01_p0001-p0026.indd 1729/01/19 10:58 AM 18BASIC CONSIDERATIONSPART Iskills. Additionally, most have been tied to performance evalua-tions and even salary and discipline up to and including loss of licensure. To our knowledge, this is the first comprehensive listing of the various agencies that evaluate surgeon performance.141MENTORING AND DEVELOPMENTMentoringA formal leadership training program for surgical trainees should include mentoring. Mentoring is the active process by which an experienced, empathetic person guides another indi-vidual in the development and self-recognition of their own vision, learning, core competencies, and professional develop-ment. Halstead established the concept of a surgical mentor who directly provided the trainees with professional and technical guidance. Halstead’s concept went beyond

1	and professional develop-ment. Halstead established the concept of a surgical mentor who directly provided the trainees with professional and technical guidance. Halstead’s concept went beyond a simple preceptor-ship by emphasizing clinical decision making based on scien-tific evidence. His goal was to develop surgeons who would go on to become outstanding leaders and innovators in the field. Although surgery has changed dramatically since Halstead’s era, mentorship remains crucial in surgical training. In addition to teaching technical skills, clinical judgment, and scientific inquiry, modern-day mentors must also model effective com-munication, empathy, humanism, and the prioritization of com-peting professional and personal activities.The mentor must also be an experienced and trusted advi-sor committed to the success of the mentee. A greater level of trust and commitment distinguishes the mentor from the teacher. More than a teacher, a mentor is a coach. The goal of a teacher is

1	advi-sor committed to the success of the mentee. A greater level of trust and commitment distinguishes the mentor from the teacher. More than a teacher, a mentor is a coach. The goal of a teacher is to pass on a defined level of knowledge for each stage of a student’s education. The underlying premise is a limited level of advancement for the student. The coach, on the other hand, has the sole purpose to make his or her student the best at their game, with an unlimited level of advancement. Modern men-torship implies a partnership between the mentor and the men-tee. Surgical residency program chairs and program directors must recruit and develop faculty “coaches” to mentor residents to optimize their potential. Emeritus Chair of the University of California, Los Angeles Head and Neck Surgery, Dr. Paul Ward, said it best: “We strive to produce graduates of our resi-dency program who are among those who change the way we think and practice.”142 Having more than 25 former residents

1	Surgery, Dr. Paul Ward, said it best: “We strive to produce graduates of our resi-dency program who are among those who change the way we think and practice.”142 Having more than 25 former residents become chairs of academic head and neck surgical programs, Dr. Ward embodied the role as a surgeon’s coach. The respon-sibilities of an effective mentor are summarized by Barondess: “Mentoring, to be effective, requires of the mentor empathy, maturity, self-confidence, resourcefulness, and willingness to commit time and energy to another. The mentor must be able to offer guidance for a new and evolving professional life, to stimulate and challenge, to encourage self-realization, to fos-ter growth, and to make more comprehensible the landscape in which the protégé stands.”143One of the major goals of mentors is to assess the aptitudes and abilities of mentees with regard to the appropriateness of their vision for their surgical career. Proper selection of the appropriate mentor can bring to

1	of mentors is to assess the aptitudes and abilities of mentees with regard to the appropriateness of their vision for their surgical career. Proper selection of the appropriate mentor can bring to the mentee much needed wisdom, guidance, and resources and can expand the scope of his or her vision. In addition, the mentor can refine the leadership skills taught to mentees in formal training programs. Highly successful surgeons most often have had excellent surgical mentors. It is impressive to note that more than 50% of United States’ Nobel laureates have served under other Nobel laureates in the capacity of student, postdoctoral fellow, or junior collaborator.144 In academic medicine, evidence-based studies have shown benefits to the mentees that include enhanced research productivity, higher likelihood of obtaining research grants, and greater success in obtaining desired positions in practice or at academic institutions.145 Mentoring provides benefits to the mentors themselves,

1	higher likelihood of obtaining research grants, and greater success in obtaining desired positions in practice or at academic institutions.145 Mentoring provides benefits to the mentors themselves, including refinement of their own personal leadership skills and a strong sense of satisfaction and accomplishment.Mentorship is essential to accomplish the successful development of surgical trainees and to help cultivate their vision. Therefore, formal leadership training programs that have a goal of training the future leaders in surgery should include mentoring.Modeling Leadership for Medical Students and the “Hidden Curriculum”Medical students enter school with great empathy, excitement, optimism, and an idealistic vision. They have self-selected to enter a profession of healing and achieved entry into a highly coveted graduate training program with centuries of tradition. Yet, these medical students are naive to the actual practice of medicine and its professional norms. Along the

1	entry into a highly coveted graduate training program with centuries of tradition. Yet, these medical students are naive to the actual practice of medicine and its professional norms. Along the way to becoming a doctor, many medical students lose some of the optimism, empathy, and excite-ment, particularly during their first and third years of school. Some students come to see the patient-physician relationship as an after-thought to providing care.145,146 Through the “hidden curriculum,” formal leadership training, and modeling of professional behavior, surgical residents, and attendings can help medical students to real-ize their vision of becoming empathic physicians.Traditionally, medical schools and professors have unknowingly relied on a hidden curriculum to mold these ide-alistic students into capable professionals. The hidden curricu-lum is the informal social norms learned by students implicitly, based on their observations of resident and attending behavior. The hidden

1	students into capable professionals. The hidden curricu-lum is the informal social norms learned by students implicitly, based on their observations of resident and attending behavior. The hidden curriculum has always been present in education, for better or worse, and may be unmasked and studied, but cannot be eliminated. Medical students actively engage in seeking out mentors, and naturally and subconsciously look to their men-tors for cues on how to conduct themselves as physicians, the same way in which a child learns how to behave from a parent or older sibling. Whether or not the witnessed behavior is a positive example of professionalism, the student will begin to perceive that behavior as normal and acceptable. For better or worse, the professional norms of medicine (the Hippocratic oath, respect to patients and colleagues, ethical conduct, personal accountability, empathy, and altruism) are modeled in every personal encounter. It is imperative that all resident and attending

1	oath, respect to patients and colleagues, ethical conduct, personal accountability, empathy, and altruism) are modeled in every personal encounter. It is imperative that all resident and attending surgeons under-stand that the medical students are observing them closely. When resident and attending surgeons model professional behavior, the hidden curriculum becomes a useful tool for professional devel-opment.147-150 This consistent modeling of professional behavior is one necessary component of leadership.During their clinical years, medical students experience both an exponential growth in knowledge and a measurable decline in empathy towards their patients. Initially, medical stu-dents are filled with excitement and wonder during their first patient encounters. The rapid pace of clinical work, acquisition of knowledge, and intense experiences create stress for the stu-dent, both positively and negatively. Scrubbing into the operat-ing room, witnessing the passing of a patient,

1	work, acquisition of knowledge, and intense experiences create stress for the stu-dent, both positively and negatively. Scrubbing into the operat-ing room, witnessing the passing of a patient, helping deliver a baby, and studying for boards are impactful milestones that each student experiences in a matter of months. Due to the 9Brunicardi_Ch01_p0001-p0026.indd 1829/01/19 10:58 AM 19LEADERSHIP IN SURGERYCHAPTER 1challenges of their work, students naturally have doubts about their own career choices and abilities, even as they experience growth and success. However, as students gain knowledge and abilities, they also come to see commonly encountered clinical problems as routine work. As familiarity and comfort with clini-cal problems increases, the excitement and wonder experienced by the student decreases. It is during this time that a decline in student empathy occurs, typically in their third year of medical school.151 In medicine, even routine clinical work still requires

1	by the student decreases. It is during this time that a decline in student empathy occurs, typically in their third year of medical school.151 In medicine, even routine clinical work still requires extraordinary attention to detail, and compassionate care must be delivered to every patient, every time. This attention to detail and compassionate delivery of care are the hallmark of the true professional. It is important that surgical residents and attend-ings always model positive behavior.Previously, medical schools instructed students in anat-omy, physiology, pathology, and clinical medicine, but left the acquisition of professionalism to the informal hidden curricu-lum. The Carnegie Report, published in 2010 at the 100-year anniversary of the Flexner Report, called for medical education to promote “the progressive formation of the physician’s profes-sional identity.”152 To this end, many medical schools nation-wide emphasize early professional education and an integrated curriculum.

1	to promote “the progressive formation of the physician’s profes-sional identity.”152 To this end, many medical schools nation-wide emphasize early professional education and an integrated curriculum. The Liaison Committee on Medical Education (LCME) sets standards for administrative and faculty leader-ship that manage the curricular model and educational affairs of students; however, formal leadership education is not explicitly required at this time. However, career exploration, mentoring, and advising are instrumental responsibilities of each medical school and a requirement of the LCME. Establishing a leader-ship program that is perpetual and coexists within an integrated curriculum will support this endeavor.153 A longitudinal lead-ership program beginning at the onset of medical school can establish a pattern of ethical behavior, professionalism, balance, Table 1-5Leadership assessment toolboxMETHOD OF LEADERSHIP MEASUREMENTDESCRIPTIONMultifactor Leadership Questionnaire (MLQ)The

1	can establish a pattern of ethical behavior, professionalism, balance, Table 1-5Leadership assessment toolboxMETHOD OF LEADERSHIP MEASUREMENTDESCRIPTIONMultifactor Leadership Questionnaire (MLQ)The MLQ is a questionnaire based on the differences between transformation and transactional approaches of leadership. It identifies leadership qualities through the rater’s beliefs about effective leadership.NEO Five-Factor Personality Inventory (NEO)NEO explores different facets of five different personality traits—neuroticism, extraversion, openness to experience, agreeableness, and conscientiousness—through a questionnaire.Surgeon’s Leadership Inventory (SLI)The SLI is a questionnaire based on literature on leadership in surgery and surgeon’s leadership behaviors observed in the operating room. It includes eight elements of surgeon’s leadership in the operating room, which are maintaining standards, managing resources, making decisions, directing, training, supporting others, communicating,

1	It includes eight elements of surgeon’s leadership in the operating room, which are maintaining standards, managing resources, making decisions, directing, training, supporting others, communicating, and coping with pressure.Patient feedbackPatient complaints are inversely related to leadership effectiveness and can thus be used as opportunities to improve and as a measure of leadership.Objective Structured Clinical Examination (OSCE)The OSCE can be administered in a controlled environment with attending feedback on various aspects of leadership tackled in the practice cases. Videotaped sessions provide further opportunities for improvement as residents will be able to later observe their own behaviors and reflect on ways to improve their approach to the case presented.Consumer Assessment of Healthcare Providers and Systems (CAHPS)CAHPS surveys are based on aspects of healthcare that matter most to patients, such as physician communication. The results are made public and can be used

1	of Healthcare Providers and Systems (CAHPS)CAHPS surveys are based on aspects of healthcare that matter most to patients, such as physician communication. The results are made public and can be used to shed light on areas of leadership physicians can improve on to work towards a patient-centered approach to care.Reproduced with permission from Jacobs LA: Practical Ethics for the Surgeon. Philadelphia, PA: Wolters Kluwer; 2018.wellness, and strong character. Indeed, many medical schools are shifting to a new, integrated style curriculum with early pro-fessional development as the standard.154This chapter has emphasized, in an intentional way, the importance of establishing a vision and goals. Throughout the process of becoming a physician, medical students will trade some of their idealism and optimism for a refined vision and a set of goals that become a part of their professional identity. This newly matured vision will guide the future these students create. The future leaders of

1	and optimism for a refined vision and a set of goals that become a part of their professional identity. This newly matured vision will guide the future these students create. The future leaders of medicine and surgery are current medical students. To foster a climate of professionalism and empathy, medical students should be taught in an environment of formal leadership training, from the first day of medical school through graduation. In addition to leadership training, medical students will inevitably acquire some of the traits and habits of their resi-dent and attending mentors through the hidden curriculum. In this way, the modeling of professional behaviors by surgical residents and attendings can serve to reinforce professionalism and may help to ward off the empathy decline experienced by medical students in their clinical years.Tools to Measure Leadership Outcomes in HealthcareThere is evidence that leadership training improves healthcare quality. The ACGME, via its core

1	by medical students in their clinical years.Tools to Measure Leadership Outcomes in HealthcareThere is evidence that leadership training improves healthcare quality. The ACGME, via its core competencies, has recognized technical skills, surgical judgement, and nontechnical skills as qualities essential to develop in residents.155,156 The objec-tive measurement of nontechnical skills is difficult. Table 1-5 includes a list of methods for assessing nontechnical skills cur-rently in use by some residency programs. The Consumer Assess-ment of Healthcare Providers and Systems (CAHPS) survey is in the early phases of being applied to individual physicians, but it has been applied to hospitals as a whole for several years.Brunicardi_Ch01_p0001-p0026.indd 1929/01/19 10:58 AM 20BASIC CONSIDERATIONSPART ILeadership can be evaluated through instruments such as the Multifactor Leadership Questionnaire, the NEO Five–Factor Inventory, and the Surgeon’s Leadership Inventory. The Mul-tifactor

1	can be evaluated through instruments such as the Multifactor Leadership Questionnaire, the NEO Five–Factor Inventory, and the Surgeon’s Leadership Inventory. The Mul-tifactor Leadership Questionnaire (MLQ) analyzes leadership aptitude as either a transactional or a transformational style.157 Leadership based on transaction focuses on completing and rewarding the tasks, whereas leadership based on transforma-tion focuses more on the motivation for completing the tasks and emphasizes a positive and encouraging working environ-ment for the team.158,159 In a study applying the questionnaire to five surgeons in a single hospital, surgeons who scored higher on the transformational section were more focused on promot-ing an open environment for all the attendings, residents, nurses and other staff in the operating room. This transformational style correlated with greater communication. These findings are important in showing that lack of communication is often a leading factor in surgical

1	in the operating room. This transformational style correlated with greater communication. These findings are important in showing that lack of communication is often a leading factor in surgical errors.The use of an MLQ in 2008 studying surgical residents showed a significant association between transformational lead-ership and overall perceived team effectiveness and resident sat-isfaction.158-159 The questionnaire also found that the residents, as leaders, placed less value on the individual needs of their colleagues, possibly reflecting a high sense of independence and frequent changes in teams due to rotations among services. This finding helped identify an area of leadership training on which the program can focus to help further develop a more supportive team atmosphere amongst the residents. In 2011, a study administered the NEO Five-Factor Personality Inventory (NEO) to a group of surgical residents. NEO, which assesses personality on five broad strokes, including neuroticism,

1	In 2011, a study administered the NEO Five-Factor Personality Inventory (NEO) to a group of surgical residents. NEO, which assesses personality on five broad strokes, including neuroticism, open-ness, agreeableness, extroversion, and conscientiousness, found that the surgeons scored above the national average on most of the factors tested but below average on agreeableness. This is a measure of altruism and tolerance, among other related factors. This result corresponded with the MLQ administered to the same group of residents and therefore highlighted areas of lead-ership that required modification.158,159The Surgeon’s Leadership Inventory (SLI) is a helpful guide for residency programs.160 The SLI grades surgeons on eight different elements of leadership, as listed in Table 1-6. As with the MLQ and NEO questionnaires, the SLI can be used to assess the growth of leadership ability in surgery residents. Table 6 provides a list and description of the different elements assessed by the

1	the MLQ and NEO questionnaires, the SLI can be used to assess the growth of leadership ability in surgery residents. Table 6 provides a list and description of the different elements assessed by the SLI.141LEADERSHIP TRAINING FOR THE PROSPECTIVE SURGEONProspective surgeons such as medical students and premedical students may have no better source for developing the personal attributes necessary for a successful career than current surgical attending surgeons and current residents. When surveyed, these doctors emphasized accountability, resilience, and high personal standards for oneself as critical tools. Prospective surgeons are advised to pursue perfectionism and be self-critical, cautioning against taking these traits to far towards neurotic behavior. Criti-cal leadership skills of teamwork and learning to take initiative are mandatory in modern medicine and must be learned early. Innovation is highly desirable.162Residents, on the other hand, are closer to becoming inde-pendent.

1	and learning to take initiative are mandatory in modern medicine and must be learned early. Innovation is highly desirable.162Residents, on the other hand, are closer to becoming inde-pendent. To some extent, they have already been selected for their leadership, innovation, and resiliency through the process Table 1-6Surgeons Leadership Inventory (SLI)ELEMENTDESCRIPTIONMaintaining standardsPracticing safe and quality patient care by following established protocols and asking for help when neededMaking decisionsMaking informed judgments and communicating decisions with relevant personnelManaging resourcesAppropriately assigning resources and tasks to team membersDirectingClearly communicating expectations and instructions and demonstrating confidence in leadership abilityTrainingEducating and training team members when the opportunity arisesSupporting othersOffering assistance where appropriate and encouraging open communicationCommunicatingSharing information in a timely manner and

1	and training team members when the opportunity arisesSupporting othersOffering assistance where appropriate and encouraging open communicationCommunicatingSharing information in a timely manner and encouraging input from othersCoping with pressureShowing flexibility when required to meet goalsData correlating patient complaints in a large number of hospitals show that improved leadership is associated with better hospital climate, improved performance, and a lower number of complaints.161Reproduced with permission from Jacobs LA: Practical Ethics for the Surgeon. Philadelphia, PA: Wolters Kluwer; 2018.of the match. During training their progression from novice to expert is necessarily rapid. A graded tool for all procedure based specialties including surgery – OpTrust – has been recently vali-dated to facilitate the resident’s transition to leadership across five domains including questioning, planning, instruction, prob-lem solving, and leadership.163As emphasized throughout this

1	vali-dated to facilitate the resident’s transition to leadership across five domains including questioning, planning, instruction, prob-lem solving, and leadership.163As emphasized throughout this chapter, the concept of training leadership skills early applies particularly to junior fac-ulty and residents. The resident-surgeon-manager conference is one model for integrating department members of various experience levels into a results-based leadership conference. In this conference, various stakeholders including attorneys, per-sons with business experience, and risk management experts are brought in as guest participants. Exercises were immersive and included case-based discussions, role-playing, simulation, and interactive lecture. Topics included teamwork, learning negoti-ating techniques, time management, risk management, balance, giving feedback, and creating immediate, goal-oriented action plans.86EARLY CAREER DEVELOPMENT AND ESTABLISHING ONESELFA variety of methods have been

1	time management, risk management, balance, giving feedback, and creating immediate, goal-oriented action plans.86EARLY CAREER DEVELOPMENT AND ESTABLISHING ONESELFA variety of methods have been proposed for the professional development of new attending surgeons. “Speed Mentoring”— 10-minute pairings of senior and junior surgeons answering pre-set questions—have been studied at national conferences with promising results. These sessions could be spread out over several days and integrated into a busy surgeon’s schedule.164Brunicardi_Ch01_p0001-p0026.indd 2029/01/19 10:58 AM 21LEADERSHIP IN SURGERYCHAPTER 1A study of department chairs and award-winning surgeon-scientists identified perseverance and team leadership skills as critical factors for development in the young attending surgeon. Chairs advocated protected time for research, financial support, and mentorship as departmental level support that the surgeon scientist should actively seek out in their first position. The

1	surgeon. Chairs advocated protected time for research, financial support, and mentorship as departmental level support that the surgeon scientist should actively seek out in their first position. The sur-geon-scientist compared to the pure clinician faces a different set of challenges, particularly the financial challenge of funding research and clinical duties competing for time and attention with research interests.165One study addressed surgeon behavior in the operating room to assess the leadership style most associated with strong leadership. Based off of this study, surgeons who are trained to collaborate, consult others appropriately, be polite (simple “please” and “thank you”), and create a safe space for their operating room staff to voice concerns will demonstrate good leadership. However, surgeons who demonstrate nonconstruc-tive criticism, destructive humor, steer conversation away from the current case, and express frustration will be perceived as demonstrating poor

1	However, surgeons who demonstrate nonconstruc-tive criticism, destructive humor, steer conversation away from the current case, and express frustration will be perceived as demonstrating poor leadership. Under this system, surgeon behavior can be categorized—conductor, elucidator, delegator, engagement facilitator, tone setter, being human, and safe space maker—in order to provide individual feedback for professional development.166SENIOR FACULTY DEVELOPMENT: TRANSITIONING TO DEPARTMENTAL LEADERSHIP AND LEGACY BUILDINGThe presence of experienced, senior academic surgeons within a department represents an opportunity. The formal develop-ment of a plan for late career transitioning through departmental leadership roles all the way to emeritus status naturally initi-ates a constructive process when thought out years in advance. The plan should be agreeable to the senior faculty member in question as well as departmental leadership and hospital stakeholders. Once in place, the senior

1	when thought out years in advance. The plan should be agreeable to the senior faculty member in question as well as departmental leadership and hospital stakeholders. Once in place, the senior academic surgeon and department will both thrive thanks to a shared vision, mutual understanding, and clear goals and transition points. Depart-mental leadership can use the transition plan to look ahead at the future of their department years down the line.167Recognition of senior academic surgeons with departmen-tal leadership, promotions, and emeritus status is a privilege earned by the academician over a lifetime of work; however, for the department it represents an opportunity to shape the values and culture of the faculty body as a whole. The continued vis-ibility, model, and influence of such leaders will have a trickle-down effect on the rest of the department. Surgical leaders are part of a large and extraordinary network facilitated by men-torship and decades of professional

1	of such leaders will have a trickle-down effect on the rest of the department. Surgical leaders are part of a large and extraordinary network facilitated by men-torship and decades of professional collaboration. Exceptional senior academic surgeons may often experience the “multiplier-effect” whereby one excellent leader trains several, who go on to train several more until the culture of surgery nationwide is influenced.168Although there are no mandatory ages for which surgeons must retire as in other professions, such as airline pilots, the issue of aging and when to cease practice has been controver-sial. There are some, however few, reports of physicians prac-ticing after the decline of their skill and becoming dangerous. As a whole, the profession has been unable to prevent this. Nationwide, from 1975 to 2015, the number of physicians prac-ticing after age 65 has increased by 374%. Some hospitals and healthcare organizations have implemented mandatory cognitive and physical

1	Nationwide, from 1975 to 2015, the number of physicians prac-ticing after age 65 has increased by 374%. Some hospitals and healthcare organizations have implemented mandatory cognitive and physical evaluations as a condition of continued practice. In the absence of more robust professional initiatives our field may see legislative oversight in the future.169 The authors believe that a formally planned transition emphasizing the values of leader-ship and legacy-building offers a more palatable alternative.CONCLUSIONAlthough there are several definitions of leadership and a vari-ety of leadership styles, all share the common goal of improving patient care in the modern era. All forms of leadership require a vision and willingness—the willingness to assume the respon-sibility to lead, continue learning, practice effective commu-nication styles, and resolve conflict. Effective leadership can change surgical departments and improve patient care through innovation. A growing body of

1	continue learning, practice effective commu-nication styles, and resolve conflict. Effective leadership can change surgical departments and improve patient care through innovation. A growing body of evidence suggests the mastery of leadership requires practice through intentional curricula and reinforcement through mentorship.Surgical leadership is bred through its training programs. Thus, innovation in surgical training programs is needed to enhance the development of leadership skills of surgical train-ees, to prepare them for practice in modern healthcare systems, and to optimize patient care, as well as compliance with require-ments set forth by regulatory institutions governing surgery and surgical education. A growing body of literature supports the value of effective leadership in improving patient care, produc-tivity, and the work environment while it validates the ability to measure the impact of leadership training. Therefore, it is of paramount importance to teach modern

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1	Systemic Response to Injury and Metabolic SupportSiobhan A. Corbett This chapter is dedicated to Dr. Stephen Lowry, my mentor and friend.2chapterOVERVIEW: INJURY-ASSOCIATED SYSTEMIC INFLAMMATORY RESPONSEThe inflammatory response to injury occurs as a consequence of the local or systemic release of “damage-associated” molecules to mobilize the necessary resources required for the restoration of homeostasis. Minor host insults result in a localized inflam-matory response that is transient and, in most cases, benefi-cial. Major host insults follow a different trajectory. A subset of these patients will die within 24 hours of hospital admis-sion, succumbing to overwhelming tissue injury and immediate organ damage. With advances in prehospital care and improved trauma management, these numbers have diminished. A second subgroup of patients who suffer a major host insult succumb to secondary organ damage remote from the injury site and die later (weeks) in their hospital course. They form

1	have diminished. A second subgroup of patients who suffer a major host insult succumb to secondary organ damage remote from the injury site and die later (weeks) in their hospital course. They form an increasing percentage of the in hospital trauma-related deaths. A dysreg-ulated, overwhelming systemic inflammatory response to the injury/hemorrhage and associated ischemia/reperfusion events has been implicated as the cause of multiple organ failure in these patients. Moreover, it has been linked to immune suppres-sion that increases the risk of infectious complications and poor outcome. Finally, a third subgroup, characterized by extended length of stay in the ICU, complicated postdischarge courses, and failure to regain/recover to their preinjury status, has been described and also linked to persistent inflammation and sup-pressed host protective immunity. The term persistent inflamma-tion, immunosuppression, and catabolism syndrome (PICS) has Overview: Injury-Associated Systemic

1	to persistent inflammation and sup-pressed host protective immunity. The term persistent inflamma-tion, immunosuppression, and catabolism syndrome (PICS) has Overview: Injury-Associated Systemic Inflammatory Response 27The Detection of Cellular Injury 28The Detection of Injury is Mediated by Members of the Damage-Associated Molecular Pattern Family / 28DAMPs Are Ligands for Pattern Recognition Receptors / 32Central Nervous System Regulation of Inflammation in Response to Injury 34Neuroendocrine Response to Injury / 35The Cellular Stress Responses 39Reactive Oxygen Species and the Oxidative Stress Response / 39The Unfolded Protein Response / 39Autophagy / 40Apoptosis / 40Necroptosis / 40Pyroptosis / 41Mediators of Inflammation 42Cytokines / 42Eicosanoids / 47Plasma Contact System / 48Serotonin / 50Histamine / 50Cellular Response to Injury 50Cytokine Receptor Families and Their Signaling Pathways / 50JAK-STAT Signaling / 50Suppressors of Cytokine Signaling / 50Tumor Necrosis

1	/ 48Serotonin / 50Histamine / 50Cellular Response to Injury 50Cytokine Receptor Families and Their Signaling Pathways / 50JAK-STAT Signaling / 50Suppressors of Cytokine Signaling / 50Tumor Necrosis Factor Superfamily / 51Transforming Growth Factor-β Family of Receptors / 51Transcriptional and Translational Regulation of The Injury Response 52Transcriptional Events Following Blunt Trauma / 52Transcriptional Regulation of Gene Expression / 52Epigenetic Regulation of Transcription / 53Translation Regulation of Inflammatory Gene Expression / 53Cell-Mediated Inflammatory Response 54Neutrophils / 54Monocyte/Macrophages / 54Lymphocytes and T-Cell Immunity / 55Dendritic Cells / 55Platelets / 56Mast Cells / 56Endothelium-Mediated Injury 56Vascular Endothelium / 56Neutrophil-Endothelium Interaction / 56Chemokines / 57Nitric Oxide / 58Prostacyclin / 58Endothelins / 59Platelet Activating Factor / 59Natriuretic Peptides / 60Surgical Metabolism 60Metabolism During Fasting / 60Metabolism

1	Interaction / 56Chemokines / 57Nitric Oxide / 58Prostacyclin / 58Endothelins / 59Platelet Activating Factor / 59Natriuretic Peptides / 60Surgical Metabolism 60Metabolism During Fasting / 60Metabolism After Injury / 62Lipid Metabolism After Injury / 63Ketogenesis / 64Carbohydrate Metabolism / 65Protein and Amino Acid Metabolism / 66Nutrition in The Surgical Patient 66Estimation of Energy Requirements / 66Vitamins and Minerals / 68Overfeeding / 68Enteral Nutrition 68Rationale for Enteral Nutrition / 68Early vs. Late Feeding / 68Intermittent vs. Continuous Enteral Feeding / 69Enteral Formulas / 69Access for Enteral Nutritional Support / 71Parenteral Nutrition 72Rationale for Parenteral Nutrition / 73Total Parenteral Nutrition / 73Peripheral Parenteral Nutrition / 73Initiation of Parenteral Nutrition / 73Complications of Parenteral Nutrition / 74Brunicardi_Ch02_p0027-p0082.indd 2701/03/19 6:49 PM 28been applied to this group.1 Recent data suggest that severely injured patients who

1	Nutrition / 73Complications of Parenteral Nutrition / 74Brunicardi_Ch02_p0027-p0082.indd 2701/03/19 6:49 PM 28been applied to this group.1 Recent data suggest that severely injured patients who are destined to die from their injuries, whether late in their hospital course or after discharge, differ from survivors only in the degree and duration of their dysregu-lated acute inflammatory response.1-3As trauma is the leading cause of mortality and morbidity for individuals under age 45, understanding the complex path-ways that regulate the local and systemic inflammatory response following severe traumatic injury is necessary to develop appro-priate and targeted therapeutic strategies that will improve out-comes for these patients.In this chapter, we will review what is known about the soluble and cellular effectors of the injury-induced inflamma-tory response, how the signals are sensed, transduced, and mod-ulated, and how their dysregulation is associated with alterations in the

1	soluble and cellular effectors of the injury-induced inflamma-tory response, how the signals are sensed, transduced, and mod-ulated, and how their dysregulation is associated with alterations in the immune system. We will also discuss how these events are monitored regulated by the central nervous system. Finally, we will review how injury reprograms cellular metabolism, in an attempt to mobilize energy and structural stores to meet the challenge of restoring homeostasis.THE DETECTION OF CELLULAR INJURYThe Detection of Injury is Mediated by Members of the Damage-Associated Molecular Pattern FamilyTraumatic injury activates the innate immune system to pro-duce a systemic inflammatory response (SIR) in an attempt to limit damage and to restore homeostasis. It includes two general responses: (a) an acute proinflammatory response resulting from innate immune system recognition of ligands, and (b) an anti-inflammatory response that may serve to modulate the proin-flammatory phase and

1	(a) an acute proinflammatory response resulting from innate immune system recognition of ligands, and (b) an anti-inflammatory response that may serve to modulate the proin-flammatory phase and direct a return to homeostasis (Fig. 2-1). This is accompanied by a suppression of adaptive immunity.4 Rather than occurring sequentially, recent data indicate that all three responses are simultaneously and rapidly induced follow-ing severe traumatic injury.3The degree of the systemic inflammatory response follow-ing trauma is proportional to injury severity and is an inde-pendent predictor of subsequent organ dysfunction and resultant 1mortality. Recent work has provided insight into the mecha-nisms by which immune activation in this setting is triggered. The clinical features of the injury-mediated systemic inflamma-tory response, characterized by increased body temperature, heart rate, respirations, and white blood cell count, are similar to those observed with infection (Table 2-1).

1	systemic inflamma-tory response, characterized by increased body temperature, heart rate, respirations, and white blood cell count, are similar to those observed with infection (Table 2-1). However, it is widely accepted that systemic inflammation following trauma is sterile, resulting from endogenous molecules that are produced as a consequence of tissue damage or cellular stress.5 Termed damage-associated molecular patterns (DAMPs) or alarmins, DAMPs interact with specific cell receptors that are located both on the cell surface and intracellularly.6Trauma DAMPs are structurally diverse endogenous mol-ecules that are immunologically active. Table 2-2 includes a par-tial list of DAMPs that are released either passively from necrotic/damaged cells or actively from physiologically “stressed” cells by upregulation or overexpression. Once they are outside the cell, DAMPs promote the activation of innate immune cells, as well as the recruitment and activation of antigen-presenting cells,

1	cells by upregulation or overexpression. Once they are outside the cell, DAMPs promote the activation of innate immune cells, as well as the recruitment and activation of antigen-presenting cells, which are engaged in host defense.7 The best-characterized DAMP with significant preclinical evidence for posttrauma release, as well as a direct link to the systemic inflammatory response, is high-mobility group protein B1 (HMGB1). Additional evidence for other important DAMP molecules that participate in postin-jury inflammation is also presented.High-Mobility Group Protein B1. The best-characterized DAMP in the context of the injury-associated inflamma-tory response is high-mobility group B1 (HMGB1) protein. HMGB1 is highly conserved across species. It is a constitutively expressed, nonhistone chromosomal protein that participates in a variety of nuclear events, including DNA repair and transcrip-tion. Inflammatory signaling can redirect HMGB1 to the cytosol in both monocytes and

1	chromosomal protein that participates in a variety of nuclear events, including DNA repair and transcrip-tion. Inflammatory signaling can redirect HMGB1 to the cytosol in both monocytes and macrophages, as a result of posttransla-tional modification. HMGB1 is released passively from dam-aged or necrotic cells and is detected rapidly in the circulation within 30 minutes post injury. It can also be actively secreted from immune-competent cells stimulated by bacterial-derived lipoproteins (e.g., endotoxin) or by inflammatory cytokines (e.g., tumor necrosis factor). For example, macrophages release HMGB1 following the activation of the inflammasomes.8,9 Key Points1 Endogenous damage-associated molecular patterns (DAMPs) are produced following tissue and cellular injury. These molecules interact with immune and nonimmune cell receptors to initiate a “sterile” systemic inflammatory response following severe traumatic injury.2 In many cases, DAMP molecules are sensed by pattern rec-ognition

1	with immune and nonimmune cell receptors to initiate a “sterile” systemic inflammatory response following severe traumatic injury.2 In many cases, DAMP molecules are sensed by pattern rec-ognition receptors (PRRs), which are the same receptors that cells use to sense invading pathogens. This explains in part, the similar clinical picture of systemic inflammation observed in injured and/or septic patients.3 The central nervous system receives information with regard to injury-induced inflammation via soluble mediators as well as direct neural projections that transmit informa-tion to regulatory areas in the brain. The resulting neuro-endocrine reflex plays an important modulatory role in the immune response.4 Inflammatory signals activate key cellular stress responses (the oxidative stress response; the heat shock protein response; the unfolded protein response; autophagy; pyroptosis), which serve to mobilize cellular defenses and resources in an attempt to restore homeostasis.5 The

1	response; the heat shock protein response; the unfolded protein response; autophagy; pyroptosis), which serve to mobilize cellular defenses and resources in an attempt to restore homeostasis.5 The cells, mediators, signaling mechanisms, and pathways that comprise and regulate the systemic inflammatory response are closely networked and tightly regulated by tran-scriptional events as well as by epigenetic mechanisms, post-translational modification, and microRNA synthesis.6 Management of critically ill and injured patients is optimized with the use of evidence-based and algorithm-driven therapy.7 Nutritional assessments, whether clinical or laboratory guided, and intervention should be considered at an early juncture in all surgical and critically ill patients.Brunicardi_Ch02_p0027-p0082.indd 2801/03/19 6:49 PM 29SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2MOFPICSInjuryImmunehomeostasisEarly deathDischarge to LTACPoor quality of lifeIndolent deathCatabolic

1	2801/03/19 6:49 PM 29SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2MOFPICSInjuryImmunehomeostasisEarly deathDischarge to LTACPoor quality of lifeIndolent deathCatabolic statePersistence inflammationProgressive immunosuppressionAnti-inflammatoryPro-inflammatoryRapid recoveryChronic critical illnessTimeUncomplicatedclinical courseFigure 2-1. Postinjury systemic response can follow multiple trajectories. MOF = multiple organ failure; PICS = persistent inflammation, immunosuppression, and catabolism syndrome; LTAC = long-term acute care facility. (Reproduced with permission from Loftus TJ, Mira JC, Ozrazgat-Baslanti T, et al: Sepsis and Critical Illness Research Center investigators: protocols and standard operating procedures for a prospec-tive cohort study of sepsis in critically ill surgical patients, BMJ Open. 2017 Aug 1;7(7):e015136.)Table 2-1Clinical spectrum of infection and systemic inflammatory response syndrome (SIRS)TERMDEFINITIONInfectionIdentifiable source of

1	ill surgical patients, BMJ Open. 2017 Aug 1;7(7):e015136.)Table 2-1Clinical spectrum of infection and systemic inflammatory response syndrome (SIRS)TERMDEFINITIONInfectionIdentifiable source of microbial insultSIRSTwo or more of following criteria are met: Temperature ≥38°C (100.4°F) or ≤36°C (96.8°F) Heart rate ≥90 beats per minute Respiratory rate ≥20 breaths per minute or Paco2 ≤ 32 mmHg or mechanical ventilation Abnormal white blood cell count (≥12,000/μL or ≤4000/μL or ≥10% immature band forms)SepsisIdentifiable source of infection + SIRSSevere sepsisSepsis + organ dysfunctionSeptic shockSepsis + cardiovascular collapse (requiring vasopressor support)Paco2 = partial pressure of arterial carbon dioxide.Table 2-2Damage-associated molecular patterns (DAMPs) and their receptorsDAMP MOLECULEPUTATIVE RECEPTOR(S)HMGB-1TLRs (2,4,9), RAGEHeat shock proteinsTLR2, TLR4, CD40, CD14,SiglecsS100 proteinTLR4, RAGEMitochondrial DNATLR9HyaluranTLR2, TLR4, CD44BiglycanTLR2 and TLR4Formyl

1	MOLECULEPUTATIVE RECEPTOR(S)HMGB-1TLRs (2,4,9), RAGEHeat shock proteinsTLR2, TLR4, CD40, CD14,SiglecsS100 proteinTLR4, RAGEMitochondrial DNATLR9HyaluranTLR2, TLR4, CD44BiglycanTLR2 and TLR4Formyl peptides (mitochondrial)Formyl peptide receptor 1IL-1αIL-1 receptorOnce outside the cell, HMGB1 has been shown to signal via the Toll-like receptors (TLR2, TLR4, TLR9), the receptor for advanced glycosylation end products (RAGE), CD24, and others. The activation of TLRs by HMGB1 occurs mainly in myeloid cells, whereas RAGE is thought to be the receptor tar-get for HMGB1 in endothelial cells.The diverse proinflammatory biological responses that result from HMGB1 signaling include: (a) the release of cytokines and chemokines from macrophage/monocytes and dendritic cells; (b) neutrophil activation and chemotaxis; (c) alterations in epithelial barrier function, including increased permeability; and (d) increased procoagulant activity on plate-let surfaces; among others.10 In addition, HMGB1

1	and chemotaxis; (c) alterations in epithelial barrier function, including increased permeability; and (d) increased procoagulant activity on plate-let surfaces; among others.10 In addition, HMGB1 binding to TLR4 triggers the proinflammatory cytokine release that medi-ates “sickness behavior.”11The biologic function of HMGB1 is regulated by its redox state. For example, a thiol at C106 is required for HMGB1 to promote macrophage TNF release, while a disulfide bond between C23 and C45 confers proinflammatory properties. With all three cysteines in the thiol (reduced) state, HMGB1 loses its DAMP function, but gains the capacity to serve as a chemotactic mediator. Importantly, shifts between the redox states have been demonstrated and indicate that redox state dynamics are impor-tant regulators of HMGB1.12Brunicardi_Ch02_p0027-p0082.indd 2901/03/19 6:49 PM 30BASIC CONSIDERATIONSPART IHMGB1 levels in human subjects following injury corre-late with the Injury Severity Score and

1	regulators of HMGB1.12Brunicardi_Ch02_p0027-p0082.indd 2901/03/19 6:49 PM 30BASIC CONSIDERATIONSPART IHMGB1 levels in human subjects following injury corre-late with the Injury Severity Score and complement activation, as well as with increases in circulating inflammatory mediators such as tumor necrosis factor.13 Exogenous administration of HMGB1 to normal animals produces fever, weight loss, epithe-lial barrier dysfunction, and, possibly, death. Further supporting the HMGB1 role in sterile inflammation, traumatic brain injury (TBI) induced by a cortical injury model has been shown to result in acute lung injury with increased alveolar hemorrhage, neutrophil infiltration, and poor oxygenation. This acute lung injury (ALI) was accompanied by a doubling in serum HMGB1 concentrations along with evidence that neocortical brain cells were a source of HMGB1 following TBI.14 More recently, in an animal model of hemorrhagic shock, HMGB1 release from intestinal epithelium was linked to

1	along with evidence that neocortical brain cells were a source of HMGB1 following TBI.14 More recently, in an animal model of hemorrhagic shock, HMGB1 release from intestinal epithelium was linked to acute lung injury.15 Finally, increased plasma levels of HMGB1 have been shown to cor-relate with immune suppression and increased infection risk in patients undergoing major surgical procedures.16 The identifica-tion of the receptor for advanced glycation end products as the receptor for HMGB1 in this setting has identified new therapeu-tic strategy to ameliorate ALI following TBI.17A Role for Mitochondrial DAMPs in the Injury-Mediated Inflammatory Response. Mitochondrial proteins and/or DNA can act as DAMPs by triggering an inflammatory response to cellular necrosis and stress. Specifically, mitochondrial DNA (mtDNA) released from damaged or dysfunctional mitochon-dria leads both to inflammasome activation and activation of the stimulator of interferon gene pathway (STING).18Cell-free

1	mitochondrial DNA (mtDNA) released from damaged or dysfunctional mitochon-dria leads both to inflammasome activation and activation of the stimulator of interferon gene pathway (STING).18Cell-free mtDNA (cf-mtDNA) has been shown to be thousands of times higher in trauma patients when compared to normal volunteers. In addition, direct injection of mitochon-dria lysates in an animal model causes remote organ damage, including liver, and lung inflammation.19 These data suggest that with cellular stress or tissue injury, cf-mtDNA released from damaged/stressed mitochondria contribute to the sterile inflammatory response in injured patients. From an evolution-ary perspective, given that eukaryotic mitochondria derive from bacterial origin, it would make sense that they retain bacterial features capable of eliciting a strong response that is typically associated with a pathogen trigger. In addition, the mitochon-drial transcription factor A (TFAM), a highly abundant mito-chondrial protein,

1	capable of eliciting a strong response that is typically associated with a pathogen trigger. In addition, the mitochon-drial transcription factor A (TFAM), a highly abundant mito-chondrial protein, is functionally and structurally homologous to HMGB1. It has also been shown be released in high amounts from damaged cells where it acts in conjunction with mtDNA to activate TLR9 signaling.20Following trauma, cf-mtDNA levels appear to be higher in nonsurvivors when compared to survivors and correlate with the development of both SIRS and sepsis post injury.21,22 Cf-mtDNA has also been linked both ex vivo and in vivo to the formation of neutrophil extracellular traps, which are also associated with sterile inflammation and are a possible cause of secondary tissue injury.23,24 Reducing cf-mtDNA, perhaps by targeting enzymes capable of digesting circulating mtDNA is an attractive therapeutic option to prevent development of inflam-matory complications of trauma.25Heat-Shock Proteins as

1	perhaps by targeting enzymes capable of digesting circulating mtDNA is an attractive therapeutic option to prevent development of inflam-matory complications of trauma.25Heat-Shock Proteins as DAMPs. Heat shock proteins (HSPs) are a large and diverse family of intracellular proteins that are expressed during times of inflammation and oxidative stress or following tissue injury.26 Very highly conserved across species, HSPs function as molecular chaperones to monitor and maintain appropriate protein folding.27 They accomplish this task through the promotion of protein refolding, the targeting of misfolded proteins for degradation, or the sequestering of partially folded proteins for movement to appropriate membrane compartments. HSPs are also capable of binding foreign proteins and thereby function as intracellular chaperones for ligands such as bacterial DNA and endotoxin.HSPs are presumed to protect cells from the effects of traumatic stress and, when released by damaged cells, alert

1	function as intracellular chaperones for ligands such as bacterial DNA and endotoxin.HSPs are presumed to protect cells from the effects of traumatic stress and, when released by damaged cells, alert the immune system of the tissue damage by activating both innate and acquired immunity.28 HSPs are also released from intact cells via a nonclassical secretory pathway, both via “secretory lysosomes” as well as the exosomal pathway. For example, HSP70-containing exosomes have been implicated in postshock inflammation.29 Once outside the cell, free HSPs can bind to pattern-recognition receptors (PRR) as well as other cell surface receptors to modu-late the inflammatory response. Recently, the role of free HSP-mediated proinflammatory properties via TLR2 and TLR4 has been questioned, as it has been suggested that the presence of con-taminating endotoxin in bacterially-produced HSP preparations may explain at least some of these inflammatory effect results.30 However, the additional evidence

1	suggested that the presence of con-taminating endotoxin in bacterially-produced HSP preparations may explain at least some of these inflammatory effect results.30 However, the additional evidence suggests that the immunos-timulatory properties may be dependent on how HSPs arrive outside the cell. In the context of massive cell damage or large exosome release, HSPs may serve as proinflammatory DAMPs. In contrast, HSPs released by active secretion may exert anti-inflammatory immune dampening signals (Table 2-3).31,32 New receptors for HSP have been identified that are members of the sialic acid-binding immunoglobulin-like lectins (siglecs), which may explain these effects. Two members of the family, Siglec-5 and Siglec-14, with similar binding sites for HSP70, exhibit oppo-site intracellular events in response to HSP binding, being either pro-(Siglec-14) or anti-(Siglec-5) inflammatory.33,34From a clinical perspective, extracellular HSPs have been demonstrated to be elevated almost

1	events in response to HSP binding, being either pro-(Siglec-14) or anti-(Siglec-5) inflammatory.33,34From a clinical perspective, extracellular HSPs have been demonstrated to be elevated almost immediately post injury in polytraumatized patients (up to 10 times normal) with the degree of elevation being correlated with the severity of illness.35 Moreover, in the setting of polytrauma, plasma HSP70 levels have been shown to correlate inversely with HLA-DRA expression, a marker of immunosuppression.36Extracellular Matrix Molecules Act as DAMPs. Recent work has explored the role of extracellular matrix (ECM) proteins in the TLR-mediated inflammatory response that follows tissue injury. These molecules, which are sequestered under normal conditions, can be released in a soluble form with proteolytic digestion of the ECM. Proteoglycans, glycosaminoglycans, and glycoproteins such as fibronectin have all been implicated as key players in the DAMP/TLR interaction. Proteoglycans, in

1	with proteolytic digestion of the ECM. Proteoglycans, glycosaminoglycans, and glycoproteins such as fibronectin have all been implicated as key players in the DAMP/TLR interaction. Proteoglycans, in particu-lar, have also been shown to activate the intracellular inflamma-somes that trigger sterile inflammation. These molecules, which consist of a protein core with one or more covalently attached glycosaminoglycan chains, can be membrane-bound, secreted, or proteolytically cleaved and shed from the cell surface.Biglycan is one of the first proteoglycans to be described as a TLR ligand.37 It consists of a protein core containing leucine-rich repeat regions, with two glycosaminoglycan (GAG) side-chains (chondroitin sulfate or dermatan sulfate). While biglycan typically exists in a matrix bound form, with tissue injury it is released from the ECM in a soluble form where it interacts with TLR2 or TLR4 to generate an immediate inflammatory response.Various proinflammatory cytokines and

1	bound form, with tissue injury it is released from the ECM in a soluble form where it interacts with TLR2 or TLR4 to generate an immediate inflammatory response.Various proinflammatory cytokines and chemokines includ-ing tumor necrosis factor (TNF)-α and interleukin (IL)-1β are Brunicardi_Ch02_p0027-p0082.indd 3001/03/19 6:49 PM 31SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2downstream effector molecules of biglycan/TLR2/4 signaling. Among these, the mechanism of biglycan-mediated autonomous synthesis and secretion of mature IL-1β is unique. Usually, release of mature IL-1β from the cell requires two signals: one that is needed to initiate synthesis (TLR2/4-mediated), and the other to process pro-IL-1β to its mature form (inflammasome-mediated). How is it possible for biglycan to provide both signals? Current evidence indicates that when soluble biglycan binds to the TLR, it simultaneously serves as a ligand for a purinergic receptor, which facilitates the

1	for biglycan to provide both signals? Current evidence indicates that when soluble biglycan binds to the TLR, it simultaneously serves as a ligand for a purinergic receptor, which facilitates the inflammasome activation required for IL-1β processing.38 These data support the idea that DAMP-mediated signals can initiate a robust inflammatory response.S100 Proteins as DAMPs. S100 proteins are a group of cal-cium-binding proteins that participate in the regulation of intra-cellular calcium. There are at least 25 members identified to date, with diverse functions that are cell-type dependent. While regulation and management of calcium storage is a primary function of S100 proteins, additional specialized roles include cytoskeletal organization, protein trafficking and transcriptional regulation. They are loosely grouped according to their func-tional capability: those that work exclusively inside the cell, outside the cell, or in both locations.8,39Similar to both HMGB1 and HSP, S100

1	They are loosely grouped according to their func-tional capability: those that work exclusively inside the cell, outside the cell, or in both locations.8,39Similar to both HMGB1 and HSP, S100 proteins are released passively from damaged cells as well as actively via nonclassical protein secretion mechanisms. For example, S100A8/A9 (also called calprotectin) is released by activated neutrophils and macrophages, although the exact mechanisms that regulate the active release of S100 proteins remain unclear.40 In addition, S100A8/A9 secretion is linked to the release of neu-trophil extracellular traps (NETs), a fibrillar matrix of DNA and granule proteins that are extruded from activated neutrophils and that serve an antimicrobial function.41 Similar to HMGB1, S100 protein functions can be modulated by their redox state.Extracellular S100A8/A9 functions as an endogenous agonist to bind TLR4 and RAGE, serving as a strong proin-flammatory mediator. It can induce both inflammatory cytokine

1	by their redox state.Extracellular S100A8/A9 functions as an endogenous agonist to bind TLR4 and RAGE, serving as a strong proin-flammatory mediator. It can induce both inflammatory cytokine production and activation of leukocyte migration, as well as pro-mote apoptosis and autophagy in distinct cell types. S100A8/A9 is increased following polytrauma, and in some studies, higher levels of S100A8/A9 have been correlated with patient survival.9 More recent work in a second severely injured patient cohort (median ISS of 39) showed that the most seriously injured patients demonstrated a significantly smaller overall increase in S100A8/A9 when compared to the other patients, and that lower S100A8/A9 levels were associated with infectious risk.42A second S100 family protein, S100B, is highly expressed in astrocytes and is an important biomarker for traumatic brain injury. When assessed within 3 hours of injury, it is a highly sensitive marker that is helpful in identifying those patients

1	expressed in astrocytes and is an important biomarker for traumatic brain injury. When assessed within 3 hours of injury, it is a highly sensitive marker that is helpful in identifying those patients with mild TBI who do not require imaging.43 In a recent cohort of 100 patients with severe traumatic brain injury, serum S100B levels were significantly higher in those patients who had an unfavorable outcome 3 months post injury compared with those who had made a good recovery. The patients who died also had significantly higher S100B levels than the survivors.8,44Heme as DAMP. Heme is the oxygen-binding moiety found in hemoglobin and other hemoproteins in the muscle and mito-chondria. It is a very highly conserved molecule composed of a tetrapyrrole ring surrounding a single iron. When red blood cells are damaged, hemoglobin is released, where it is bound by plasma proteins such as haptoglobin. In turn, the hemoglobin-haptoglobin complexes are scavenged by the reticuloendothelial system

1	cells are damaged, hemoglobin is released, where it is bound by plasma proteins such as haptoglobin. In turn, the hemoglobin-haptoglobin complexes are scavenged by the reticuloendothelial system in the liver and spleen to form bilirubin as the ultimate end product. When the amount of free hemoglobin exceeds the binding capacity of haptoglobin and other specialized binding proteins, it is loosely bound to other plasma proteins where it can be readily oxidized. Ultimately, this can result in the release of the prosthetic heme group from hemoglobin, generating labile heme, which is a pro-oxidant.45In vitro experiments demonstrate that labile heme induces cell activation, via both TLR4-dependent processes and the inflammasome, resulting in cytokine release.46,47 Moreover, Table 2-3The immunomodulatory functions of heat shock proteins (HSPs)CELL LOCATIONRECOGNIZED AS DAMPIMMUNOMODULATORY FUNCTIONHSP90Cytoplasm, endoplasmic reticulumCan function both inside and outside the cellMay act as

1	functions of heat shock proteins (HSPs)CELL LOCATIONRECOGNIZED AS DAMPIMMUNOMODULATORY FUNCTIONHSP90Cytoplasm, endoplasmic reticulumCan function both inside and outside the cellMay act as DAMP chaperone to activate innate immune responseBinds and optimizes RNA polymerase II action to regulate gene transcriptionStabilizes glucocorticoid receptor in the cytoplasmImportant for processing and membrane expression of TLRChaperones include IKKFacilitates antigen presentation to dendritic cellsHSP70Can function both inside and outside the cellEndoplasmic reticulum homolog is BiPExogenous HSP70 elicits cellular calcium flux, NF-κB activation, cytokine productionCan have anti-inflammatory actions when expression is increasedInhibits TLR-mediated cytokine production via NF-κBReduces dendritic cell capacity for T-cell stimulationBiP sequesters proteins important to the unfolded protein responseHSP60MitochondriaExogenous HSP60 inhibits NF-κB activationPlays a role in intracellular protein

1	cell capacity for T-cell stimulationBiP sequesters proteins important to the unfolded protein responseHSP60MitochondriaExogenous HSP60 inhibits NF-κB activationPlays a role in intracellular protein traffickingModulates cytokine synthesisBiP = binding immunoglobulin protein; DAMP = damage-associated molecular pattern; IKK = IκB kinase; NF-κB, nuclear factor-κB; TLR = Toll-like receptorBrunicardi_Ch02_p0027-p0082.indd 3101/03/19 6:49 PM 32BASIC CONSIDERATIONSPART Iheme-induced neutrophils activation leads to extracellular traps (NETs) release through a mechanism dependent on reactive oxygen species.48 However, unlike the other DAMPs discussed, labile heme can also have direct cytotoxic effects on cells by a direct interaction with membrane phospholipids and the catal-ysis of membrane lipid peroxidation, leading to programmed cell death. In macrophages, labile heme can induce necroptosis, rather than apoptosis.45DAMPs Are Ligands for Pattern Recognition ReceptorsThe inflammatory

1	lipid peroxidation, leading to programmed cell death. In macrophages, labile heme can induce necroptosis, rather than apoptosis.45DAMPs Are Ligands for Pattern Recognition ReceptorsThe inflammatory response that occurs following traumatic injury is similar to that observed with pathogen exposure. Not surprisingly, surface and cytoplasmic receptors that medi-ate the innate immune response to microbial infection have also been implicated in the activation of sterile inflamma-tion. In support of this idea, genes have been identified that are dysregulated acutely both in response to a microbial ligand administered to human volunteers and in response to traumatic injury in a large patient population.49 The classes of receptors that are important for sensing damaged cells and cell debris are part of the larger group of germ-line encoded pattern recogni-tion receptors (PRRs). The best described ligands for these receptors are microbial components, the pathogen-associated molecular patterns

1	of the larger group of germ-line encoded pattern recogni-tion receptors (PRRs). The best described ligands for these receptors are microbial components, the pathogen-associated molecular patterns (PAMPs). The PRRs of the innate immune system are varied and include Toll-like receptors (TLRs), calcium-dependent (C-type) lectin receptors (CLRs), the nucleotide-binding domain, leucine-rich repeat–containing (NBD-LRR) proteins (NLRs; also nucleotide-binding and oligomerization domain [NOD]-like receptors), receptors for advanced glycation end-products (RAGE), and retinoic acid–inducible gene (RIG)-I-like receptors (RLRs). Following receptor ligation, intracel-lular signaling modulates transcriptional and posttranslational events necessary for host defense by coordinating the synthesis and release of cytokines and chemokines to either initiate or suppress the inflammatory response. The best described of these receptors, the TLRs, NLRs, CLRs and RAGE, are dis-cussed in the following

1	and release of cytokines and chemokines to either initiate or suppress the inflammatory response. The best described of these receptors, the TLRs, NLRs, CLRs and RAGE, are dis-cussed in the following section.Toll-Like Receptors. The Toll-like receptors are evolutionarily conserved type 1 transmembrane proteins that are the best-char-acterized PRRs in mammalian cells. They were first identified in Drosophila, where a mutation in the Toll gene led to its iden-tification as a key component in their immune defense against fungal infection. The first human TLR, TLR4, was identified shortly thereafter. Now, more than 10 human TLR family mem-bers have been identified, with distinct ligands that include lipid, carbohydrate, peptide, and nucleic-acid components of various pathogens. TLRs are expressed by both immune and nonimmune cells. At first, the expression of TLR was thought to be isolated to professional antigen-presenting cells such as dendritic cells and macrophages. However, mRNA for

1	by both immune and nonimmune cells. At first, the expression of TLR was thought to be isolated to professional antigen-presenting cells such as dendritic cells and macrophages. However, mRNA for TLR family members have been detected in most cells of myeloid lineage, as well as NK cells.50 In addition, activation of T cells increases their TLR expression and induces their survival and clonal expan-sion. Direct engagement of TLR in Treg cells promotes their expansion and reprograms them to differentiate into T helper cells, which in turn provides help to effector cells. In addition, B cells express a distinct subset of the TLR family that deter-mines their ability to respond to DAMPs; however, the signifi-cance of restricted TLR expression in these cells is not yet clear.All TLRs consist of a ligand-binding domain, char-acterized by multiple leucine-rich repeats (LRRs), and a carboxy-terminal, intracellular Toll/interleukin (IL) 1 recep-tor (TIR) domain. The LRR domains recognize

1	of a ligand-binding domain, char-acterized by multiple leucine-rich repeats (LRRs), and a carboxy-terminal, intracellular Toll/interleukin (IL) 1 recep-tor (TIR) domain. The LRR domains recognize bacterial and viral PAMPs in the extracellular environment (TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11) or in the endolysosomes (TLR3, TLR7, TLR8, TLR9, and TLR10). While the role of TLRs in sepsis has been well described, more recent data indi-cate that a subset of the TLRs—TLR4 in particular—also rec-ognize DAMPs released from injured cells and tissues.51 Among the DAMP ligands for surface TLR are HMGB1, HSPs, S100 proteins, and several others. Endosomal TLR ligands include mtDNA and other mitochondrial proteins.What we know about TLR signaling events has largely been derived from the TLR-mediated response to bacterial pathogens. However, it is largely accepted that the intracellu-lar adaptors required for signal transmission by TLRs are con-served and utilized for “damage” sensing of

1	response to bacterial pathogens. However, it is largely accepted that the intracellu-lar adaptors required for signal transmission by TLRs are con-served and utilized for “damage” sensing of endogenous (“self”) ligands as well.52,53 The intracellular domain structure of TLRs is highly conserved and is characterized by a cytoplasmic Toll/IL-1R homology (TIR) domain. Binding of ligand to the recep-tor results in a receptor dimer, either a homodimer (e.g., TLR4/TLR4) or heterodimer (e.g., TLR2/TLR1), which recruits a number of adaptor proteins to the TIR domains through TIR-TIR interaction.54 With one exception (TLR3), the universal adaptor protein central to the TLR signaling complex is myeloid dif-ferentiation factor 88 (MyD88), a member of the interleukin-1 receptor subfamily. MyD88 works through the recruitment of a second TIR-containing adaptor, MyD88 adaptor-like protein (Mal, also termed Toll/interleukin-1 receptor-containing adaptor protein, or TIRAP) in the context of TLR4 and

1	through the recruitment of a second TIR-containing adaptor, MyD88 adaptor-like protein (Mal, also termed Toll/interleukin-1 receptor-containing adaptor protein, or TIRAP) in the context of TLR4 and TLR2 signaling, which serves as a bridge between MyD88 and activated TLRs to initiate signal transduction. It is interesting that Mal’s adaptor function requires cleavage of the carboxy-terminal portion of the protein by caspase-1, a key effector of the inflammasome.55 This finding suggests an important synergy between TLRs and the inflammasome that may potentiate TLR-mediated signaling.Signaling through the MyD88-dependent pathway occurs once the receptor is ligated at the cell surface.54 Receptor liga-tion, dimerization, and recruitment of the MyD88/Mal com-plex results in the activation of numerous cytoplasmic protein kinases, including IL-1 receptor–associated kinases, resulting in an interaction with tumor necrosis factor receptor–associated factor 6 (TRAF6). TRAF6, an E3 ubiquitin

1	numerous cytoplasmic protein kinases, including IL-1 receptor–associated kinases, resulting in an interaction with tumor necrosis factor receptor–associated factor 6 (TRAF6). TRAF6, an E3 ubiquitin ligase, forms a com-plex with two other proteins, which together activate the com-plex that subsequently phosphorylates IκB kinase (IKK)-β and the MAP kinases (MAPKs). Ultimately, the phosphorylation of IκB leads to its degradation, which frees NF-κB and allows its translocation to the nucleus and the transcription of NF-κB tar-get genes. Simultaneously, MAP kinase activation is critical for activation of the activator protein-1 (AP-1) transcription factor, and thus production of inflammatory cytokines.Two other TIR domain-containing adaptor proteins, TIR-domain-containing adapter-inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM), are important to TLR-signaling events that are involved in the MyD88-independent sig-naling pathways, activated by TLR3 and TLR4. One

1	interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM), are important to TLR-signaling events that are involved in the MyD88-independent sig-naling pathways, activated by TLR3 and TLR4. One distinction of MyD88-dependent and -independent TLR signaling is that TLR4/TRIF transduction begins after the signaling complex is internalized into endosomes. The MyD88-independent pathway acts through TRIF to activate NF-κB, similar to the MyD88-dependent pathway. However, TRIF can also recruit other sig-naling molecules to phosphorylate interferon-regulatory factor 3 (IRF3), which induces expression of type I IFN genes.5422Brunicardi_Ch02_p0027-p0082.indd 3201/03/19 6:49 PM 33SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2The initiation of transcription by TLR activation leads to the upregulation of a large cohort of target genes that include interferons α and β (IFNα/β), nitric oxide synthase 2 (NOS2A), and tumor necrosis factor (TNF), which play critical roles in

1	leads to the upregulation of a large cohort of target genes that include interferons α and β (IFNα/β), nitric oxide synthase 2 (NOS2A), and tumor necrosis factor (TNF), which play critical roles in initiating innate immune responses to cellular injury and stress. Given the importance of TLR triggering of the innate immune response to immune homeostasis, it is no surprise that the pro-cess is tightly regulated. TLR signaling is controlled at multiple levels, both posttranscriptionally via ubiquitination, phosphory-lation, and micro RNA actions that affect mRNA stability, and by the localization of the TLRs and their signaling complexes within the cell.TLR expression is significantly increased following blunt traumatic injury.50,51 A recent study of patients undergoing “high-risk” surgical procedures examined immune parameters, includ-ing TLR expression, that were associated with the development of SIRS. The investigators demonstrated that patients who developed postoperative SIRS

1	procedures examined immune parameters, includ-ing TLR expression, that were associated with the development of SIRS. The investigators demonstrated that patients who developed postoperative SIRS exhibited increased TLR4 and TLR5 expres-sion on a subgroup of CD14+ monocytes when compared to those patients with an uneventful recovery.56 Moreover, the upregulation of TLR in these patients was associated with increased expression of IL-6. Interestingly, the authors hypothesize that preoperatively, a subset of monocytes may already be primed to act in this way and thus may identify a vulnerable patient group.Nucleotide-Binding Oligomerization Domain (NOD)-like Receptor (NLR) Family. The nucleotide-binding oligo-merization domain-like receptors (NLR) are a large family of proteins composed of intracellular PRRs that sense both endog-enous (DAMPs) and exogenous (PAMPs) molecules to trigger innate immune activation. The best characterized of the NLRs is the NLR family pyrin domain-containing

1	PRRs that sense both endog-enous (DAMPs) and exogenous (PAMPs) molecules to trigger innate immune activation. The best characterized of the NLRs is the NLR family pyrin domain-containing 3 (NLRP3), which is highly expressed in peripheral blood leukocytes. It forms the key “sensing” component of the larger, multiprotein inflamma-some complex, which is composed of NLRP3; the adapter pro-tein apoptosis-associated speck-like protein containing a CARD (ASC); and the effector protein, caspase 1.57 Activation of the NLRP3 inflammasome is tightly regulated, both transcription-ally and at the posttranslational level. An initial priming event (typically via TLR/nuclear factor [NF]-κB signals) upregulates NLRP3 expression. The receptor then resides in the cytoplasm in an inactive form due to an internal interaction between two adjacent domains. When phagocytosed DAMPs are sensed by NLRP3, this second event releases the self-repression. The pro-tein can then oligomerize and recruit other complex

1	interaction between two adjacent domains. When phagocytosed DAMPs are sensed by NLRP3, this second event releases the self-repression. The pro-tein can then oligomerize and recruit other complex members. The net result is the auto-activation of pro-caspase 1 to caspase 1. This event is pivotal to all known inflammasome signaling pathways.57,58 The caspase-1 products assemble to form the IL-1 converting enzyme (ICE), which cleaves the proforms of IL-1β, IL-18, and IL-33 to form their active, mature forms required for secretion from the cell.59The inflammasome-activated cytokines, IL-1β and IL-18, are potent proinflammatory molecules that promote key immune responses essential to host defense. Both IL-1β and IL-18 lack a signal sequence, which is usually necessary for the secretion of cellular proteins. More than 20 proteins in addition to IL-1β and IL-18 undergo unconventional protein secretion independent of the ER and Golgi, including a number of the DAMP molecules.60 Currently, the

1	proteins. More than 20 proteins in addition to IL-1β and IL-18 undergo unconventional protein secretion independent of the ER and Golgi, including a number of the DAMP molecules.60 Currently, the mechanisms responsible for unconventional protein secretion are not understood; however, the process is also evident in yeast under conditions of cellular stress. It makes evolutionary sense that a mechanism for rapid secretion of stored proteins essential to the stress response is highly conserved.Evidence suggests that genetic variations in the NLRP3 gene might affect the magnitude of immune inflammatory responses following trauma. Single nucleotide polymor-phisms within the NLRP3 gene were found to be associated with increased risk of sepsis and MODS in patients with major trauma.61 In an animal model of burn injury, early inflamma-some activation has been detected in a variety of immune cells (NK cells, CD4/ CD8 T cells, and B cells), as determined by the assessment of caspase 1 cleavage

1	model of burn injury, early inflamma-some activation has been detected in a variety of immune cells (NK cells, CD4/ CD8 T cells, and B cells), as determined by the assessment of caspase 1 cleavage by flow cytometry.62 Further, inhibition of caspase 1 activity in vivo results in increased burn mortality, suggesting that inflammasome activation may play an unanticipated protective role in the host response to injury that may be linked to increased production of specific cytokines.CNS trauma induces inflammasome activation in the ner-vous system. Moreover, exosomes containing inflammasome protein cargo are secreted into cerebral spinal fluid and can be detected in patients with TBI.63 In an animal model of TBI, controlled cortical impact, exosomes containing inflammasome proteins are detected in the serum and appear to be linked to TBI-related acute lung injury.64C-Type Lectin and Lectin-Like Receptors. Macrophages and dendritic cells possess receptors that detect molecules released from

1	in the serum and appear to be linked to TBI-related acute lung injury.64C-Type Lectin and Lectin-Like Receptors. Macrophages and dendritic cells possess receptors that detect molecules released from damaged or dying cells in order to retrieve and process antigens for T cell presentation. A key family of receptors that directs this process is the C-type lectin (CLR) and C-type lec-tin-like (CTLR) receptor family that includes the selectin and the mannose receptor families. CLR and CTLR bind carbohy-drates in both a calcium-dependent (CLR) and -independent (CTLR) fashion. Best described for their sensing of PAMPs, the CLRs can also act to promote the endocytosis and clear-ance of cell debris, which can be processed and presented to T cells.65,66 CTLR receptor recognition of DAMPs of intracellular origin, such as F-actin and the ribonucleoprotein SAP-130, can trigger multiple signaling pathways leading to NF-κB, type I interferon (IFN), and/or inflammasome activation. Expression of the

1	origin, such as F-actin and the ribonucleoprotein SAP-130, can trigger multiple signaling pathways leading to NF-κB, type I interferon (IFN), and/or inflammasome activation. Expression of the CTLR, MINCLE (macrophage-inducible C-type lectin), is increased after exposure to proinflammatory stimuli or cell stress. When MINCLE senses self-damage in association with ischemia-reperfusion injury, it promotes proinflammatory cyto-kine, chemokine, and nitric oxide production.67Receptor for Advanced Glycation End Products (RAGE). Another key player in the sterile inflammatory response to injury is the transmembrane receptor, the receptor for advanced glycation endproducts, or RAGE. Highly conserved across spe-cies, RAGE is a member of the immunoglobulin superfamily that is constitutively expressed at high levels in the lung, with low/absent expression in other adult cell types. However, pro-inflammatory stimuli and the presence of RAGE ligands can increase RAGE expression on immune cells such

1	at high levels in the lung, with low/absent expression in other adult cell types. However, pro-inflammatory stimuli and the presence of RAGE ligands can increase RAGE expression on immune cells such as neutrophils, macrophages, and lymphocytes.68 RAGE also exists as a solu-ble form (sRAGE) composed only of the extracellular domain, which can bind to and sequester RAGE ligands, without conse-quent signaling events.RAGE binds diverse ligands, including HMGB1 and S100, as well as components of the extracellular matrix such as collagen. As a receptor, RAGE recognizes the three-dimensional structure of its ligands that allow it to bind a diverse reper-toire of molecules, independent of their amino acid sequence. Brunicardi_Ch02_p0027-p0082.indd 3301/03/19 6:49 PM 34BASIC CONSIDERATIONSPART ISignaling via RAGE is mediated via multiple pathways lead-ing to transcriptional activation and release of proinflammatory mediators.69 Animal models have linked RAGE to acute lung injury in

1	via RAGE is mediated via multiple pathways lead-ing to transcriptional activation and release of proinflammatory mediators.69 Animal models have linked RAGE to acute lung injury in ischemia-reperfusion models.70 In clinical studies, high sRAGE levels have be linked to prolonged mechanical ventila-tion post lung transplant as well as worse outcomes following TBI-associated acute lung injury.42 These events likely represent a role for an HMGB1-RAGE axis in these pathologic processes.Soluble Pattern Recognition Molecules: The Pentraxins.68 Soluble pattern recognition molecules (PRMs) are a molecu-larly diverse group of molecules that share a conserved mode of action defined by complement activation, agglutination and neutralization, and opsonization. The best described of the PRMs are the pentraxins. PRMs can be synthesized at sites of injury and inflammation by macrophages and dendritic cells, while neutrophils can store PRMs and release them rapidly fol-lowing activation. In addition,

1	PRMs can be synthesized at sites of injury and inflammation by macrophages and dendritic cells, while neutrophils can store PRMs and release them rapidly fol-lowing activation. In addition, epithelial tissues (the liver in par-ticular) serve as a reservoir source for systemic mass release. The short pentraxin, C-reactive protein (CRP), was the first PRM to be identified. Serum amyloid protein (SAP), which has 51% sequence similarity to human CRP, also contains the pentraxin molecular signature. CRP and SAP plasma levels are low (≤3 mg/L) under normal circumstances. However, CRP is synthesized by the liver in response to interleukin-6, increasing serum levels more than a 1000-fold. Thus, CRP is considered part of the acute-phase protein response in humans. For this reason, C-reactive protein has been studied as a marker of the proinflammatory response in many clinical settings, includ-ing appendicitis, vasculitis, and ulcerative colitis. CRP and SAP are ancient immune molecules that

1	has been studied as a marker of the proinflammatory response in many clinical settings, includ-ing appendicitis, vasculitis, and ulcerative colitis. CRP and SAP are ancient immune molecules that share many functional properties with antibodies: they bind bacterial polysaccharides, ECM components, apoptotic cells, and nuclear materials, as well as all three classes of Fcγ receptors (FcγR). Both molecules also participate in the activation and regulation of complement pathways. In this way, short pentraxins can link immune cells to the complement system.71Finally, there is significant data to support a role for pen-traxin 3 (PTX3), a long pentraxin family member, in the “sterile” inflammatory response associated with cellular stress. While CRP is produced solely in the liver, PTX3 is produced by vari-ous cells in peripheral tissues, including immune cells. PTX3 plasma concentrations increase rapidly in various inflammatory conditions, including sepsis. Further, in a recent prospective

1	by vari-ous cells in peripheral tissues, including immune cells. PTX3 plasma concentrations increase rapidly in various inflammatory conditions, including sepsis. Further, in a recent prospective study of polytraumatized patients, serum PTX3 concentrations were highly elevated, peaking at 24 hours. Further, PTX3 con-centrations at admission were associated with injury severity, while higher PTX3 serum concentrations 24 hours after admis-sion correlated with lower probability for survival.72CENTRAL NERVOUS SYSTEM REGULATION OF INFLAMMATION IN RESPONSE TO INJURYThe central nervous system (CNS) communicates with the body through ordered systems of sensory and motor neurons, which receive and integrate information to generate a coordinated response. Rather than being an immune-privileged organ, recent work indicates that the CNS receives information with regard to injury-induced inflammation both via soluble media-tors as well as direct neural projections that transmit informa-tion to

1	recent work indicates that the CNS receives information with regard to injury-induced inflammation both via soluble media-tors as well as direct neural projections that transmit informa-tion to regulatory areas in the brain (Fig. 2-2). How does the TNFIL-1Central nervous systemACTHglucocorticoidsSensory vagusSympatheticParasympathetic(Motor vagus)AcetylcholineInjuryinflammationInflammatorycascadeInjury siteEPI, NOREPIFigure 2-2. Neural circuit relaying messages of localized injury to the brain (nucleus tractus solitarius). The brain follows with a hor-mone release (adrenocorticotropic hormone [ACTH], glucocorticoids) into the systemic circulation and by sympathetic response. The vagal response rapidly induces acetylcholine release directed at the site of injury to curtail the inflammatory response elicited by the activated immunocytes. This vagal response occurs in real time and is site specific. EPI = epinephrine; IL-1 = interleukin-1; NOREPI = norepinephrine; TNF = tumor necrosis

1	response elicited by the activated immunocytes. This vagal response occurs in real time and is site specific. EPI = epinephrine; IL-1 = interleukin-1; NOREPI = norepinephrine; TNF = tumor necrosis factor. (Adapted with permission from Tracey KJ: The inflammatory reflex, Nature. 2002 Dec 19-26;420(6917):853-859.)Brunicardi_Ch02_p0027-p0082.indd 3401/03/19 6:49 PM 35SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2CNS sense inflammation? DAMPs and inflammatory molecules convey stimulatory signals to the CNS via multiples routes. For example, soluble inflammatory signaling mol-ecules from the periphery can reach neurons and glial cells directly through the fenestrated endothelium of the circumven-tricular organs (CVO) or via a leaky blood-brain barrier in path-ological settings following a traumatic brain injury.73 In addition, inflammatory stimuli can interact with receptors located on the brain endothelial cells to generate a variety of proinflammatory mediators

1	settings following a traumatic brain injury.73 In addition, inflammatory stimuli can interact with receptors located on the brain endothelial cells to generate a variety of proinflammatory mediators (cytokines, chemokines, adhesion molecules, proteins of the complement system, and immune receptors) that directly impact the brain parenchyma. Not surprising, this response is countered by potent anti-inflammatory signaling, a portion of which is provided by the HPA axis and the release of systemic glucocorticoids. Inflammatory stimuli in the CNS result in behavioral changes, such as increased sleep, lethargy, reduced appetite, and the most common feature of infection, fever.Information regarding peripheral inflammation and tis-sue damage can also be signaled to the brain via afferent neu-ral fibers, particularly those of the vagus nerve.74 These afferent fibers can interconnect with neurons that project to the hypo-thalamus to modulate the HPA axis. In addition, afferent vagal nerve

1	fibers, particularly those of the vagus nerve.74 These afferent fibers can interconnect with neurons that project to the hypo-thalamus to modulate the HPA axis. In addition, afferent vagal nerve impulses modulate cells in the brain stem, at the dorsal motor nucleus of the vagus, from which efferent pregangli-onic parasympathetic originate. Axons from these cells, which comprise the visceromotor component of the vagus nerve, form an “inflammatory reflex” that feeds back to the periphery to regulate inflammatory signaling events.75 Mechanistic insight into the “inflammatory reflex” was provided by the observa-tion in several experimental model systems, that vagal stimu-lation reduced proinflammatory cytokine production from the spleen.75,76 This effect was dependent on both vagal efferent signals and on splenic catecholaminergic nerve fibers that origi-nated in the celiac plexus and terminated in the T cell–rich area of the spleen. The vagal efferent fibers that terminated within the

1	signals and on splenic catecholaminergic nerve fibers that origi-nated in the celiac plexus and terminated in the T cell–rich area of the spleen. The vagal efferent fibers that terminated within the celiac ganglion were found to synapse on the cell bodies of the catecholaminergic splenic nerves. Vagal stimulation resulted in the firing of these adrenergic nerves, resulting in the activation of β2-adrenergic receptors on a subset of acetylcholine (ACh)-producing T cells. The ACh released from this T cell popula-tion targets α-7 nicotinic ACh receptors (a7nAChR) expressed by splenic macrophages.77 Macrophage ACh receptor ligation blocks cell activation, inhibiting cytokine production and shifting the macrophages towards an M2 anti-inflammatory phenotype. Moreover, ACh-receptor binding inhibits intracellular signaling including the nuclear translocation of NF-κB and the activation of the inflammasome. In a rat model of hemorrhagic shock with reperfusion, vagal nerve stimulation post

1	intracellular signaling including the nuclear translocation of NF-κB and the activation of the inflammasome. In a rat model of hemorrhagic shock with reperfusion, vagal nerve stimulation post injury resulted in a decrease in the inflammatory response to hemorrhage.78Neuroendocrine Response to InjuryTraumatic injury results in complex neuroendocrine signaling from the brain that serves to enhance immune defense and rap-idly mobilize substrates necessary to meet essential energy and structural needs. The two principle neuroendocrine pathways that orchestrate the host response are the hypothalamic-pitu-itary-adrenal (HPA) axis, which results in the release of gluco-corticoid hormones, and the sympathetic nervous system, which results in release of the catecholamines, epinephrine (EPI), and norepinephrine (EPI). Virtually every hormone of the HPA axis influences the physiologic response to injury and stress (Table 2-4), but some with direct influence on the inflammatory 3Table 2-4Hormones

1	(EPI). Virtually every hormone of the HPA axis influences the physiologic response to injury and stress (Table 2-4), but some with direct influence on the inflammatory 3Table 2-4Hormones regulated by the hypothalamus, pituitary, and autonomic systemHypothalamic RegulationCorticotropin-releasing hormoneThyrotropin-releasing hormoneGrowth hormone–releasing hormoneLuteinizing hormone–releasing hormoneAnterior Pituitary RegulationAdrenocorticotropic hormoneCortisolThyroid-stimulating hormoneThyroxineTriiodothyronineGrowth hormoneGonadotrophinsSex hormonesInsulin-like growth factorSomatostatinProlactinEndorphinsPosterior Pituitary RegulationVasopressinOxytocinAutonomic SystemNorepinephrineEpinephrineAldosteroneRenin-Angiotensin SystemInsulinGlucagonEnkephalinsresponse or immediate clinical impact are highlighted here, including growth hormone (GH), macrophage inhibitory factor (MIF), aldosterone, and insulin.The Hypothalamic-Pituitary-Adrenal Axis. One of the main mechanisms by which the

1	are highlighted here, including growth hormone (GH), macrophage inhibitory factor (MIF), aldosterone, and insulin.The Hypothalamic-Pituitary-Adrenal Axis. One of the main mechanisms by which the brain responds to injury-associated stress is through activation of the hypothalamic-pituitary-adrenal (HPA) axis. Following injury, corticotrophin-releasing hormone (CRH) is secreted from the paraventricular nucleus (PVN) of the hypothalamus. This action is mediated in part by circulating cytokines produced as a result of the innate immune response to injury. These include tumor necrosis factor-α (TNF-α) IL-1β, IL-6, and the type I interferons (IFN-α/β). Cytokines that are produced as a result of the adaptive immune response (IL-2 and IFN-γ) are also capable of increasing cortisol release. Direct neu-ral input via afferent vagal fibers that interconnect with neurons projecting to the hypothalamus can also trigger CRH release. CRH acts on the anterior pituitary to stimulate the secretion of

1	neu-ral input via afferent vagal fibers that interconnect with neurons projecting to the hypothalamus can also trigger CRH release. CRH acts on the anterior pituitary to stimulate the secretion of adrenocorticotropin hormone (ACTH) into the systemic circula-tion. Interestingly, the cytokines that act on the hypothalamus are also capable of stimulating ACTH release from the anterior pituitary so that marked elevations in ACTH and in cortisol can occur that are proportional in magnitude to the injury severity. Additionally, pain, anxiety, vasopressin, angiotensin II, chole-cystokinin, vasoactive intestinal peptide, and catecholamines all contribute to ACTH release in the injured patient.Brunicardi_Ch02_p0027-p0082.indd 3501/03/19 6:49 PM 36BASIC CONSIDERATIONSPART IACTH acts on the zona fasciculate of the adrenal glands to synthesize and secrete glucocorticoids (Fig. 2-3). Cortisol is the major glucocorticoid in humans and is essential for survival during significant physiologic

1	fasciculate of the adrenal glands to synthesize and secrete glucocorticoids (Fig. 2-3). Cortisol is the major glucocorticoid in humans and is essential for survival during significant physiologic stress. The resulting increase in cortisol levels following trauma have several important anti-inflammatory actions.Cortisol elicits its many actions through a cytosolic recep-tor, the glucocorticoid receptor (GR). Because it is lipid soluble, cortisol can diffuse through the plasma membrane to interact with its receptor, which is sequestered in the cytoplasm in a com-plex with heat shock proteins (Fig. 2-4). Upon ligand binding, the GR is activated and can employ a number of mechanisms to modulate proinflammatory gene transcription and signaling events, with a “net” anti-inflammatory effect.79 For example, the activated GR complex can interact with transcription factors to sequester them in the cytoplasm, promote their degradation, or inhibit them through other mechanisms. Affected target

1	For example, the activated GR complex can interact with transcription factors to sequester them in the cytoplasm, promote their degradation, or inhibit them through other mechanisms. Affected target genes include proinflammatory cytokines, growth factors, adhesion molecules, and nitric oxide. In addition, glucocorticoids can negatively affect the access of the transcription factor, nuclear factor-κB (NF-κB), to the promoter regions of its target genes via a mechanism that involves histone deacetylase 2. In this way, glucocorticoids can inhibit a major mechanism by which TLR ligation induces proinflammatory gene expression.80 The GR complex can also bind to specific nucleotide sequences (termed glucocorticoid response elements) to promote the 17-˜-OH-progesterone11-DeoxycortisolCortisolCholesterolACTHPregnenolone17-˜-OH-PregnenoloneDehydroepiandrosteroneAndrostenedioneTestosteroneEstradiolSex

1	response elements) to promote the 17-˜-OH-progesterone11-DeoxycortisolCortisolCholesterolACTHPregnenolone17-˜-OH-PregnenoloneDehydroepiandrosteroneAndrostenedioneTestosteroneEstradiolSex steroidsGlucocorticoid11-DeoxycorticosteroneCorticosteroneAldosteroneMineralocorticoidProgesteroneFigure 2-3. Steroid synthesis from cholesterol. Adrenocorticotropic hormone (ACTH) is a principal regulator of steroid synthesis. The end products are mineralocorticoids, glucocorticoids, and sex steroids.HSPRProtein synthesisCytoplasmic membraneDNAmRNASRSSSSSSHSPRSNucleusFigure 2-4. Simplified schematic of steroid transport into the nucleus. Steroid molecules (S) diffuse readily across cytoplasmic membranes. Intracellularly, the receptors (R) are rendered inactive by being coupled to heat shock protein (HSP). When S and R bind, HSP dissoci-ates, and the S-R complex enters the nucleus, where the S-R complex induces DNA transcription, resulting in protein synthesis. mRNA = messenger

1	shock protein (HSP). When S and R bind, HSP dissoci-ates, and the S-R complex enters the nucleus, where the S-R complex induces DNA transcription, resulting in protein synthesis. mRNA = messenger RNA.Brunicardi_Ch02_p0027-p0082.indd 3601/03/19 6:49 PM 37SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2transcription of genes, which have anti-inflammatory functions. These include interleukin-10 and interleukin-1-receptor antago-nists. Further, GR complex activation can indirectly influence TLR activity via an interaction with signaling pathways such as the mitogen-activated protein kinase and transforming growth factor–activated kinase-1 (TAK1) pathways. Finally, a recent report demonstrated that GCs target suppressor of cytokine signaling 1 (SOCS1) and type 1 interferons to regulate TLR-induced signaling events.81Adrenal insufficiency represents a clinical syndrome high-lighted largely by inadequate amounts of circulating cortisol and aldosterone. Classically, adrenal

1	regulate TLR-induced signaling events.81Adrenal insufficiency represents a clinical syndrome high-lighted largely by inadequate amounts of circulating cortisol and aldosterone. Classically, adrenal insufficiency is described in patients with atrophic adrenal glands caused by exogenous steroid administration who undergo a stressor such as surgery. These patients subsequently manifest signs and symptoms such as tachycardia, hypotension, weakness, nausea, vomiting, and fever. However, it is now apparent that severe traumatic injury associated with an extended proinflammatory response can increase the risk of critical illness–related corticosteroid insuf-ficiency, or CIRCI.In the postinjury setting, CIRCI describes a phenomenon in which an exaggerated proinflammatory response is associated with a blunted adrenocortical response.82 Factors that have been linked to CIRCI include dysregulation of the HPA axis with altered adrenal synthesis of cortisol, altered cortisol metabo-lism, and

1	with a blunted adrenocortical response.82 Factors that have been linked to CIRCI include dysregulation of the HPA axis with altered adrenal synthesis of cortisol, altered cortisol metabo-lism, and tissue resistance to corticosteroids with inadequate glucocorticoid receptor activity. As a consequence, cortisol levels prove insufficient for the severity of stress. Investigators have determined that CIRCI in trauma patients occurs more fre-quently than previously thought.83 In one recent study, CIRCI occurred in 38 of 70 patients with multiple injuries. In most cases, the diagnosis was made within the first 48 hours follow-ing injury.84 Laboratory findings in adrenal insufficiency include hypoglycemia from decreased gluconeogenesis, hyponatremia from impaired renal tubular sodium resorption, and hyperka-lemia from diminished kaliuresis. Recommended guidelines to diagnose CIRCI include measuring delta cortisol (change in baseline cortisol at 60 min of <9 µg/dL) after cosyntropin (250 µg)

1	and hyperka-lemia from diminished kaliuresis. Recommended guidelines to diagnose CIRCI include measuring delta cortisol (change in baseline cortisol at 60 min of <9 µg/dL) after cosyntropin (250 µg) administration and a random plasma cortisol of <10 µg/dL. Treatment strategies remain controversial in the set-ting of trauma.85,86Macrophage Migration Inhibitory Factor Modulates Cortisol Function. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine expressed by a variety of cells and tissues, including the anterior pituitary, macrophages and T lymphocytes. MIF is also classified as an atypical chemo-kine that binds to the CXC4 receptor.87 Several important func-tions of MIF in innate and adaptive immune responses and in inflammation have been described supporting the idea that MIF may function to counteract the anti-inflammatory activity of glucocorticoids.88 For example, MIF has been reported to play a central role in the exacerbation of inflammation associated

1	idea that MIF may function to counteract the anti-inflammatory activity of glucocorticoids.88 For example, MIF has been reported to play a central role in the exacerbation of inflammation associated with acute lung injury, where it has been detected in the affected lungs and in alveolar macrophages. MIF has also been reported to upregulate the expression of Toll-like receptor 4 (TLR4) in macrophages,89 and an early increase in plasma MIF has been detected in severely injured patients and was found to corre-late with NF-κB translocation and respiratory burst in PMNs derived from severely injured patients. Further, nonsurvivors were shown to have higher serum MIF concentrations early after injury than survivors.90 These data suggest that targeting MIF with available small molecule inhibitors may be a novel therapeutic strategy for preventing early PMN activation and subsequent organ failure in severely injured patients.Growth Hormone, Insulin-Like Growth Factor, and Ghrelin. Growth

1	may be a novel therapeutic strategy for preventing early PMN activation and subsequent organ failure in severely injured patients.Growth Hormone, Insulin-Like Growth Factor, and Ghrelin. Growth hormone (GH) is a neurohormone expressed primarily by the pituitary gland that has both metabolic and immunomodulatory effects. GH promotes both protein synthe-sis and insulin resistance while enhancing the mobilization of fat stores. GH secretion is upregulated by hypothalamic GH–releasing hormone and downregulated by somatostatin. GH pri-marily exerts its downstream effects through direct interaction with GH receptors and through the enhanced hepatic synthesis of insulin-like growth factor-1 (IGF-1), an anabolic growth fac-tor that is known to improve the metabolic rate, gut mucosal function, and protein loss after traumatic injury. Less than 5% of IGF-1 circulates free in the plasma, with the remainder bound principally to one of six IGF-binding proteins (IGFBPs), the majority to IGFBP-3.

1	protein loss after traumatic injury. Less than 5% of IGF-1 circulates free in the plasma, with the remainder bound principally to one of six IGF-binding proteins (IGFBPs), the majority to IGFBP-3. In the liver, IGF-1 stimulates protein syn-thesis and glycogenesis; in adipose tissue, it increases glucose uptake and lipid utilization; and in skeletal muscles, it mediates glucose uptake and protein synthesis. In addition to its effects on cellular metabolism, GH enhances phagocytic activity of immu-nocytes through increased lysosomal superoxide production. It also increases the proliferation of T-cell populations.91The catabolic state that follows severe injury has been linked to the suppression of the growth hormone-IGF-IGFBP axis, as critical illness is associated with decreased circulat-ing IGF-1 levels. Not surprising, the administration of exog-enous recombinant human GH (rhGH) has been studied in a prospective, randomized trial of critically ill patients where it was associated

1	IGF-1 levels. Not surprising, the administration of exog-enous recombinant human GH (rhGH) has been studied in a prospective, randomized trial of critically ill patients where it was associated with increased mortality, prolonged ventilator dependence, and increased susceptibility to infection.92 More recently, circulating GH levels were examined on admission in 103 consecutive critically ill adult patients. In this study, cir-culating GH levels were increased by about sevenfold in the 24 nonsurvivors when compared with survivors, and they were an independent predictor of mortality, along with APACHE II/SAPS II scores. In distinct contrast, the effect of rhGH adminis-tration in severely burned children, both acutely and following prolonged treatment, has been proven to be beneficial. Pediatric burn patients receiving rhGH demonstrated markedly improved growth and lean body mass, while hypermetabolism was signifi-cantly attenuated.93 This finding was associated with significant

1	Pediatric burn patients receiving rhGH demonstrated markedly improved growth and lean body mass, while hypermetabolism was signifi-cantly attenuated.93 This finding was associated with significant increases in serum GH, IGF-I, and IGFBP-3.Ghrelin, a natural ligand for the GH-secretagogue receptor 1a (GHS-R1a), is an appetite stimulant that is secreted by the stomach. GHS-R1a is expressed in a variety of tissues in differ-ent concentrations including the immune cells, Band T-cells, and neutrophils. Ghrelin seems to play a role in promoting GH secretion, and in glucose homeostasis, lipid metabolism, and immune function. In a rodent gut ischemia/reperfusion model, ghrelin administration inhibits proinflammatory cytokine release, reduces neutrophil infiltration, ameliorates intestinal barrier dysfunction, attenuates organ injury, and improves sur-vival. It is interesting that this effect was dependent on an intact vagus nerve, and that intracerebroventricular injection of ghre-lin was

1	dysfunction, attenuates organ injury, and improves sur-vival. It is interesting that this effect was dependent on an intact vagus nerve, and that intracerebroventricular injection of ghre-lin was also protective.94 These data suggest that the effect of ghrelin is mediated via the central nervous system, most likely through the “cholinergic anti-inflammatory pathway.” High ghrelin levels were demonstrated in critically ill patients as compared to healthy controls, independent of the presence of Brunicardi_Ch02_p0027-p0082.indd 3701/03/19 6:49 PM 38BASIC CONSIDERATIONSPART Iinflammatory markers. Moreover, the high ghrelin levels were a positive predictor of ICU-survival in septic patients, match-ing previous results from animal models. Based on these data, ghrelin seems to exert anti-inflammatory effects that are medi-ated by diverse pathways. Recent work has linked ghrelin to a novel pathway mediated by upregulation of uncoupling protein 2 (UCP2) particularly in the setting of

1	effects that are medi-ated by diverse pathways. Recent work has linked ghrelin to a novel pathway mediated by upregulation of uncoupling protein 2 (UCP2) particularly in the setting of traumatic brain injury.95The Role of Catecholamines in Postinjury Inflammation. Injury-induced activation of the sympathetic nervous system results in secretion of acetylcholine from the preganglionic sympathetic fibers innervating the adrenal medulla. The adre-nal medulla is a special case of autonomic innervation and is considered a modified postganglionic neuron. Thus, acetyl-choline signaling to the resident chromaffin cells ensures that a surge of epinephrine (EPI) and norepinephrine (NE) release into the circulation takes place in a ratio that is tightly regulated by both central and peripheral mechanisms. Circulating levels of EPI and NE are threeto fourfold elevated, an effect that per-sists for an extended time. The release of EPI can be modulated by transcriptional regulation of

1	mechanisms. Circulating levels of EPI and NE are threeto fourfold elevated, an effect that per-sists for an extended time. The release of EPI can be modulated by transcriptional regulation of phenylethanolamine N-methyl-transferase (PNMT), which catalyzes the last step of the cat-echolamine biosynthesis pathway methylating NE to form EPI. PNMT transcription, a key step in the regulation of epinephrine production, is activated in response to stress and tissue hypoxia by hypoxia-inducible factor 1α (HIF1A).Catecholamine release almost immediately prepares the body for the “fight or flight” response with well-described effects on the cardiovascular and pulmonary systems, and on metabolism. These include increased heart rate, myocardial contractility, conduction velocity, and blood pressure; the redi-rection of blood flow to skeletal muscle; increased cellular metabolism throughout the body; and mobilization of glucose from the liver via glycogenolysis, gluconeogenesis, lipolysis, and

1	the redi-rection of blood flow to skeletal muscle; increased cellular metabolism throughout the body; and mobilization of glucose from the liver via glycogenolysis, gluconeogenesis, lipolysis, and ketogenesis. To compound the resulting hyperglycemia, insulin release is decreased mainly through the stimulation of α-adrenergic pancreatic receptors. Hyperglycemia, as will be discussed, contributes to the proinflammatory response and to further mitochondrial dysfunction.The goal of this well-orchestrated catecholamine response is to reestablish and maintain the systems’ homeostasis, includ-ing the innate immune system. Circulating catecholamines can directly influence inflammatory cytokine production.96 Data indicate that basal EPI levels condition the activity and respon-siveness of cytokine-secreting cells, which may explain large inter-individual variability in innate cytokine profiles observed following injury. Epinephrine infusion at higher doses has been found to inhibit production

1	cells, which may explain large inter-individual variability in innate cytokine profiles observed following injury. Epinephrine infusion at higher doses has been found to inhibit production of tumor necrosis factor (TNF)-alpha in vivo and to enhance the production of the anti-inflammatory cytokine interleukin IL-10.97 Additionally, in vitro studies indi-cate that stress levels of glucocorticoids and epinephrine, acting in concert, can inhibit production of IL-12, a potent stimulator of Th1 responses. Further, they have been shown in vitro to decrease Th1 cytokine production and increase Th2 cytokine production to a significantly greater degree compared to either adrenal hormone alone. Thus, catecholamines secreted from the adrenal, specifically epinephrine, play a role in both innate proinflammatory cytokine regulation, as well as adaptive Th responses, and may act in concert with cortisol during the injury response to modulate cytokine activity.98How are these effects explained? It is

1	cytokine regulation, as well as adaptive Th responses, and may act in concert with cortisol during the injury response to modulate cytokine activity.98How are these effects explained? It is well established that a variety of human immune cells (e.g., mononuclear cells, macrophages, and granulocytes) express adrenergic receptors that are members of the family of G-protein coupled recep-tors that act through the activation of intracellular second mes-sengers such as cAMP and calcium ions influx (discussed in more detail in the following section). These second messengers can regulate a variety of immune cell functions, including the release of inflammatory cytokines and chemokines.The sympathetic nervous system also has direct immune-modulatory properties via its innervation of lymphoid tissues that contain resting and activated immune cells. The close prox-imity of sympathetic nerve terminals to immune cells responding to antigens (e.g., in the spleen) allows for a high concentration of

1	that contain resting and activated immune cells. The close prox-imity of sympathetic nerve terminals to immune cells responding to antigens (e.g., in the spleen) allows for a high concentration of norepinephrine to be localized within the microenvironment of antigen-activated immune cells. Norepinephrine can then interact with b2-adrenergic receptors expressed by CD4+ T and B lymphocytes, many of which also express α2-adrenergic receptors. Additionally, endogenous catecholamine expression has been detected in these cells (both CD4+ CD25+ T cells and phagocytes) as has the machinery for catecholamine synthesis. For example, monocytes contain inducible mRNA for the catecholamine-generating enzymes, tyrosine-hydroxylase, and dopamine-b-hydroxylase, and there is data to suggest that cells can regulate their own catecholamine synthesis in response to extracellular cues. Immune cell release of NE provides a way in which cells may exert additional regulation of inflammatory cell activation.

1	regulate their own catecholamine synthesis in response to extracellular cues. Immune cell release of NE provides a way in which cells may exert additional regulation of inflammatory cell activation. For example, mature dendritic cells express both functional αand b-adrenergic receptor (AR) types, as do monocytes and monocyte-derived macrophages, whereas B cells and Th1 cells express b2-AR exclusively.99 Exposure of PBMCs to NE triggers a distinct genetic profile that indicates a modulation of Th cell function. Thus, stimulation of AR results in varied signaling events to regulate both immune cell pheno-type as well as mature cell function.100Aldosterone. Aldosterone is a mineralocorticoid released by the zona glomerulosa of the adrenal cortex. It binds to the mineralocorticoid receptor (MR) of principal cells in the col-lecting duct of the kidney where it can stimulate expression of genes involved in sodium reabsorption and potassium excretion to regulate extracellular volume and

1	(MR) of principal cells in the col-lecting duct of the kidney where it can stimulate expression of genes involved in sodium reabsorption and potassium excretion to regulate extracellular volume and blood pressure. Mineralo-corticoid receptors (MR) have also been shown to have effects on cell metabolism and immunity. For example, recent studies show aldosterone interferes with insulin signaling pathways and reduces expression of the insulin-sensitizing factors, adiponec-tin and peroxisome proliferator activated receptor−γ (PPAR-γ), which contribute to insulin resistance. In the immune system, monocytes, lymphocytes, dendritic cells, and neutrophils have all been shown to possess a MR that binds aldosterone with high specificity, regulating sodium and potassium flux, as well as plasminogen activator inhibitor-1 and p22 phox expression in these cells.101 In dendritic cells, MR activation by aldoste-rone induces the secretion of proinflammatory cytokines. Fur-ther, aldosterone inhibits

1	activator inhibitor-1 and p22 phox expression in these cells.101 In dendritic cells, MR activation by aldoste-rone induces the secretion of proinflammatory cytokines. Fur-ther, aldosterone inhibits cytokine-mediated NF-κB activation in neutrophils, which also possess a functional MR.Insulin. Hyperglycemia and insulin resistance are hallmarks of injury and critical illness due to the catabolic effects of circu-lating mediators, including catecholamines, cortisol, glucagon, and growth hormone. The increase in these circulating progly-cemic factors, particularly epinephrine, induces glycogenoly-sis, lipolysis, and increased lactate production independent of available oxygen in a process that is termed “aerobic glycoly-sis.” Although there is an increase in insulin production at the Brunicardi_Ch02_p0027-p0082.indd 3801/03/19 6:49 PM 39SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2same time, severe stress is frequently associated with insulin resistance, leading to

1	3801/03/19 6:49 PM 39SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2same time, severe stress is frequently associated with insulin resistance, leading to decreased glucose uptake in the liver and the periphery contributing to acute hyperglycemia. Insulin is a hormone secreted by the pancreas, which mediates an overall host anabolic state through hepatic glycogenesis and glycolysis, peripheral glucose uptake, lipogenesis, and protein synthesis.102The insulin receptor (IR) is widely expressed and con-sists of two isoforms, which can form homoor hetero-dimers with insulin binding. Dimerization leads to receptor autophos-phorylation and activation of intrinsic tyrosine kinase activity. Downstream signaling events are dependent on the recruitment of the adaptor proteins, insulin receptor substrate (IRS-1), and Shc to the IR. Systemic insulin resistance likely results from proinflammatory signals, which modulate the phosphorylation of IRS-1 to affect its

1	proteins, insulin receptor substrate (IRS-1), and Shc to the IR. Systemic insulin resistance likely results from proinflammatory signals, which modulate the phosphorylation of IRS-1 to affect its function.Hyperglycemia during critical illness is predictive of increased mortality in critically ill trauma patients.103 It can modulate the inflammatory response by altering leukocyte func-tions and the resulting decreases in phagocytosis, chemotaxis, adhesion, and respiratory burst activities are associated with an increased risk for infection. In addition, glucose administration results in a rapid increase in NF-κB activation and proinflamma-tory cytokine production. Insulin therapy to manage hypergly-cemia has grown in favor and has been shown to be associated with both decreased mortality and a reduction in infectious complications in select patient populations. However, the trend towards tight glycemic control in the intensive care unit failed to show benefit when examined in several

1	and a reduction in infectious complications in select patient populations. However, the trend towards tight glycemic control in the intensive care unit failed to show benefit when examined in several reviews.104 Thus, the ideal blood glucose range within which to maintain critically ill patients and to avoid hypoglycemia has yet to be determined.THE CELLULAR STRESS RESPONSESReactive Oxygen Species and the Oxidative Stress ResponseReactive oxygen and nitrogen species (ROS, RNS, respectively) are small molecules that are highly reactive due to the presence of unpaired outer orbit electrons. They can cause cellular injury to host cells and invading pathogens through the oxidation of cell membrane substrates, cellular proteins, and DNA. ROS has also been shown to have important roles as signaling messengers, particularly in the immune system.105,106Oxygen radicals (superoxide anion, hydroxyl radical, hydrogen peroxide) are produced as a by-product of oxygen metabolism. The main areas of

1	messengers, particularly in the immune system.105,106Oxygen radicals (superoxide anion, hydroxyl radical, hydrogen peroxide) are produced as a by-product of oxygen metabolism. The main areas of ROS production are oxida-tive processes involving the mitochondrial electron transport chain as well as those mediated by NADPH oxidases (NOX), a large class of ROS producing enzymes. Additional metabolic enzymes such as lipoxygenases, cytochrome P-450 and b5, and cyclooxygenases also produce ROS as by-products of their reactions.107 The synthesis of ROS is regulated at several check-points and via complex signaling mechanisms, including Ca2+ signaling, phosphorylation, and small G protein activation, which influence both the recruitment of the molecules required for NOX function and the synthesis of ROS in the mitochondria. Not surprisingly, NOX activation is triggered by a number of inflammatory mediators (e.g., TNF, chemokines, lysophospho-lipids, complement, and leukotrienes).Host cells are

1	ROS in the mitochondria. Not surprisingly, NOX activation is triggered by a number of inflammatory mediators (e.g., TNF, chemokines, lysophospho-lipids, complement, and leukotrienes).Host cells are protected from the damaging effects of ROS through a number of mechanisms. The best described of these is via the upregulation and/or activation of endogenous antioxidant enzymes such as superoxide dismutases, catalases, and glutaredoxins. Pyruvate kinase also provides negative feed-back for ROS synthesis as do molecules that react nonenzy-matically with ROS. Under normal physiologic conditions, ROS production is balanced effectively by these antioxidative strate-gies. As a consequence, ROS can act as signaling molecules through their ability to modulate cysteine residues by oxidation, and thus influence the functionality of target proteins.108 ROS can also contribute to transcriptional activity both indirectly through its effects on transcription factor lifespan, and directly through the

1	influence the functionality of target proteins.108 ROS can also contribute to transcriptional activity both indirectly through its effects on transcription factor lifespan, and directly through the oxidation of DNA.The role for ROS has been well described in phagocytes, which utilize these small molecules for pathogen killing. A sec-ond important role for ROS is in the regulation of the inflamma-some. As discussed previously, the inflammasome mediates the activation of inflammatory capsases leading to the production and secretion of mature cytokines in macrophages.109 Impor-tantly, the best described inflammasome, NLRP3, is redox sen-sitive. Increased intracellular ROS enables the assembly of the protein complex.110 ROS also appears to be involved in adap-tive immunity by influencing immune cell response.106 ROS can alter thiol group oxidative states on the cell surface and, in turn, affect cell signaling. Moreover, intracellular ROS can inhibit DNA transcription. ROS has been

1	immune cell response.106 ROS can alter thiol group oxidative states on the cell surface and, in turn, affect cell signaling. Moreover, intracellular ROS can inhibit DNA transcription. ROS has been described as a prime source of phosphatase activation in both B and T lymphocytes, which can regulate the function of key receptors and intracellu-lar signaling molecules in these cells by affecting phosphoryla-tion events. Finally, large amounts of ROS cannot only suppress cell function, but also can result in cell death.111The Unfolded Protein ResponseSecreted, membrane-bound, and organelle-specific proteins fold in the lumen of the endoplasmic reticulum (ER) where they also receive their posttranslational modifications. Cellular stress dis-rupts the quality control required for this process leading to the accumulation of misfolded or unfolded proteins. These occur-rences are sensed by a highly conserved array of signaling pro-teins in the ER that try to reestablish appropriate folding,

1	leading to the accumulation of misfolded or unfolded proteins. These occur-rences are sensed by a highly conserved array of signaling pro-teins in the ER that try to reestablish appropriate folding, while at the same time decreasing protein synthesis.112 The important proteins involved in this process include inositol requiring enzyme 1 (IRE1), protein kinase RNA (PKR)–like ER kinase (PERK), and activating transcription factor 6 (ATF6). Together, these proteins form a complex that generates the unfolded pro-tein response (UPR). The UPR is a mechanism by which ER distress signals are sent to the nucleus to modulate transcription in an attempt to restore homeostasis. While obviously important to secretory epithelial cells, the UPR is also important to cells of the immune system.113Significant protein misfolding results in an alarm signal that, if not addressed, can result in cell death. Genes activated in the UPR result not only in the inhibition of translation, but also other

1	protein misfolding results in an alarm signal that, if not addressed, can result in cell death. Genes activated in the UPR result not only in the inhibition of translation, but also other potentially immunomodulatory events including induction of the acute phase response, activation of NF-κB, and the generation of antibody-producing B cells.114 Activation of the UPR is also an alternative mechanism for activation of the inflammasome115 and can increase proinflammatory cytokine production.116Markers of ER stress during critical illness have been demonstrated conclusively in burn patients,114,117 and in animal models they have been detected following hemorrhagic shock, correlating with the degree of organ dysfunction. Burn injury in particular leads to the marked reduction in ER calcium levels 4Brunicardi_Ch02_p0027-p0082.indd 3901/03/19 6:49 PM 40BASIC CONSIDERATIONSPART Iand activation of UPR sensing proteins. Moreover, recent data in a series of burn patients strongly links the

1	4Brunicardi_Ch02_p0027-p0082.indd 3901/03/19 6:49 PM 40BASIC CONSIDERATIONSPART Iand activation of UPR sensing proteins. Moreover, recent data in a series of burn patients strongly links the UPR to insulin resistance and hyperglycemia in these patients.117 Thus, a better understanding of the UPR, which is triggered by severe inflam-mation, may allow the identification of novel therapeutic targets for injury-associated insulin resistance.118Fibroblast growth factor-21 (FGF21), a recently identi-fied hormone that regulates systemic metabolic homeostasis, is upregulated following mitochondrial damage and may be part of an integrated stress response that includes ER stress and the UPR.119 In animal models, induction of ER stress with chemical ER stressors results in increased FGF21 expression. A recent study examining FGF21 in critically ill patients demonstrated that serum FGF21 concentrations were eight-fold higher in the critically ill patients as compared with the matched

1	expression. A recent study examining FGF21 in critically ill patients demonstrated that serum FGF21 concentrations were eight-fold higher in the critically ill patients as compared with the matched controls, regardless of the presence of sepsis. While FGF21 concentrations gradually decreased over time, they remained highly elevated at all studied time points and cor-related with patient mortality.120 These data support the idea that the UPR may play an important role in the response to severe injury.AutophagyUnder normal circumstances, cells need to have a way of dis-posing of damaged organelles and debris aggregates that are too large to be managed by proteosomal degradation. In order to accomplish this housekeeping task, cells utilize a process referred to as “macroautophagy” (autophagy), which is thought to have originated as a stress response.121 The steps of autoph-agy include the engulfment of cytoplasm/organelle by an “isola-tion membrane,” which is also called a phagophore.

1	which is thought to have originated as a stress response.121 The steps of autoph-agy include the engulfment of cytoplasm/organelle by an “isola-tion membrane,” which is also called a phagophore. The edges of the phagophore then fuse to form the autophagosome, a dou-ble-membraned vesicle that sequesters the cytoplasmic material and is a characteristic feature of autophagy. The autophagosome then fuses with a lysosome to form an autolysosome, where the contents, together with the inner membrane, are degraded. This process is controlled by numerous autophagy-specific genes and by the specific kinase, mammalian target of rapamycin (mTOR).As noted previously, autophagy is a normal cellular pro-cess that occurs in quiescent cells for cellular maintenance. However, under conditions of hypoxia and low cellular energy, autophagy is induced in an attempt to provide additional nutri-ents for energy production. The induction of autophagy pro-motes a shift from aerobic respiration to glycolysis

1	and low cellular energy, autophagy is induced in an attempt to provide additional nutri-ents for energy production. The induction of autophagy pro-motes a shift from aerobic respiration to glycolysis and allows cellular components of the autophagosome to be hydrolyzed to energy substrates. Increased levels of autophagy are typical in activated immune cells and are a mechanism for the disposal of ROS and phagocytosed debris.Recent data support the idea that autophagy plays an important role in the immune response.122 Autophagy is stimu-lated by Th1 cytokines and with activation of TLR in macro-phages but is inhibited by Th2 cytokines. It is also recognized as an important regulator of cytokine secretion, particularly those cytokines of the IL-1 family that are dependent on inflamma-some processing for activation. For example, autophagosomes can sequester and degrade pro-IL-1β and inflammasome com-ponents. In animal models of sepsis, inhibition of autophagy results in increased

1	processing for activation. For example, autophagosomes can sequester and degrade pro-IL-1β and inflammasome com-ponents. In animal models of sepsis, inhibition of autophagy results in increased proinflammatory cytokine levels that corre-late with increased mortality.123 These data suggest that autoph-agy is a protective mechanism whereby the cell can regulate the levels of cytokine production.ApoptosisApoptosis (regulated cell death) is an energy-dependent, orga-nized mechanism for clearing senescent or dysfunctional cells, including macrophages, neutrophils, and lymphocytes, without promoting an inflammatory response. This contrasts with cel-lular necrosis, which results in a disorganized sequence of intra-cellular molecular releases with subsequent immune activation and inflammatory response. Systemic inflammation modulates apoptotic signaling in active immunocytes, which subsequently influences the inflammatory response through the loss of effec-tor cells.Apoptosis proceeds

1	response. Systemic inflammation modulates apoptotic signaling in active immunocytes, which subsequently influences the inflammatory response through the loss of effec-tor cells.Apoptosis proceeds primarily through two pathways: the extrinsic pathway and the intrinsic pathway. The extrinsic path-way is activated through the binding of death receptors (e.g., Fas, TNFR), which leads to the recruitment of Fas-associated death domain protein and subsequent activation of caspase 3 (Fig. 2-5). On activation, caspases are the effectors of apoptotic signaling because they mediate the organized breakdown of nuclear DNA. The intrinsic pathway proceeds through protein mediators (e.g., Bcl-2, Bcl-2-associated death promoter, Bcl-2–associated X protein, Bim) that influence mitochondrial mem-brane permeability. Increased membrane permeability leads to the release of mitochondrial cytochrome C, which ultimately activates caspase 3 and thus induces apoptosis. These pathways do not function in a

1	permeability. Increased membrane permeability leads to the release of mitochondrial cytochrome C, which ultimately activates caspase 3 and thus induces apoptosis. These pathways do not function in a completely autonomous manner because there is significant interaction and crosstalk between mediators of both extrinsic and intrinsic pathways. Apoptosis is modulated by several regulatory factors, including inhibitor of apoptosis proteins and regulatory caspases (e.g., caspases 1, 8, 10).Apoptosis during sepsis may influence the ultimate com-petency of the acquired immune response. In a murine model of peritoneal sepsis, increased lymphocyte apoptosis was associ-ated with mortality, which may be due to a resultant decrease in IFN-γ release. In postmortem analysis of patients who expired from overwhelming sepsis, there was an increase in lymphocyte apoptosis, whereas macrophage apoptosis did not appear to be affected. Clinical trials have observed an association between the degree of

1	from overwhelming sepsis, there was an increase in lymphocyte apoptosis, whereas macrophage apoptosis did not appear to be affected. Clinical trials have observed an association between the degree of lymphopenia and disease severity in sepsis. In addition, after the phagocytosis of apoptotic cells by macro-phages, anti-inflammatory mediators such as IL-10 are released that may exacerbate immune suppression during sepsis. Neutro-phil apoptosis is inhibited by inflammatory products, including TNF, IL-1, IL-3, IL-6, GM-CSF, and IFN-γ. This retardation in regulated cell death may prolong and exacerbate secondary injury through neutrophil free radical release as the clearance of senescent cells is delayed.124NecroptosisCellular necrosis refers to the premature uncontrolled death of cells in living tissue typically caused by accidental exposure to external factors, such as ischemia, inflammation or trauma, which result in extreme cellular stress. Necrosis is character-ized by the loss of

1	living tissue typically caused by accidental exposure to external factors, such as ischemia, inflammation or trauma, which result in extreme cellular stress. Necrosis is character-ized by the loss of plasma membrane integrity and cellular col-lapse with extrusion of cytoplasmic contents, but the cell nuclei typically remain intact. Recent data have defined a process by which necrosis occurs through a series of well-described steps that are dependent on a signaling pathway that involves the receptor-interacting protein kinase (RIPK) complex. Termed “necroptosis,” it occurs in response to specific stimuli, such as TNFand TLR-mediated signals.125 For example, ligation of the tumor necrosis factor receptor 1 (TNFR1) under conditions in which caspase-8 is inactivated (e.g., by pharmacological Brunicardi_Ch02_p0027-p0082.indd 4001/03/19 6:49 PM 41SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2agents) results in the over-generation of ROS and a metabolic collapse. The net

1	Brunicardi_Ch02_p0027-p0082.indd 4001/03/19 6:49 PM 41SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2agents) results in the over-generation of ROS and a metabolic collapse. The net result is programmed necrosis (necroptosis). The effect of cell death by necroptosis on the immune response is not yet known. However, it is likely that the “DAMP” sig-nature that occurs in response to necroptotic cell death is an important contributor to the systemic inflammatory response. Evidence to support this concept was provided by investiga-tors who examined the role of necroptosis in murine model of sepsis. They demonstrated that Ripk3−/− mice were capable of recovering body temperature better, exhibited lower circu-lating DAMP levels, and survived at higher rates than their WT littermates.126 These data suggest that the cellular damage that occurs with programmed necrosis exacerbates the sepsis-associated systemic inflammatory response.PyroptosisPyroptosis is a form of regulated

1	These data suggest that the cellular damage that occurs with programmed necrosis exacerbates the sepsis-associated systemic inflammatory response.PyroptosisPyroptosis is a form of regulated cell death that is dependent on the activity of the proinflammatory caspase enzymes associ-ated with the inflammasome and is thus an inflammatory form of cell death.127 Pyropotosis shares some features with apopo-tosis, including DNA fragmentation and positive annexin V staining, among others. However, it is associated with the acti-vation of caspase-1 and the formation of caspase-1–dependent pores that allow early permeabilization of the cell membrane, electrolyte movement into the cells, and, finally, osmotic lysis of the cell.128 As a form of cell death, pyroptosis seems to be largely observed in macrophages, dendritic cells, and neutro-phils, although it has been documented in other cells as well, especially if they express high levels of caspase-1. As noted, pyroptosis is linked to activation

1	dendritic cells, and neutro-phils, although it has been documented in other cells as well, especially if they express high levels of caspase-1. As noted, pyroptosis is linked to activation of the inflammasome, which can occur in response to diverse cell alarm signals, including DAMPs. Not surprising, the mechanism of cell death leads to the release of additional intracellular DAMPs, including HMGB1 and S100 proteins.A recent study examined pyroptosis in peripheral blood mononuclear cells in a cohort of 60 trauma patients.129 The investigators found that the percentages of pyroptotic PBMCs were significantly higher in trauma patients than those in healthy CD95TNFR-1(p55)TNFR-2(p75)FADDDDDDDDDEDDDDEDDDDEDDEDFADDCaspase 8Caspase 2CaspaseCascadeApoptosisNIKI-˜B/NF-˜BNF-˜BMEKK1JNKc-JunRIPRAIDDTRAF2IAPTRADDDDDDDDTRADDDEDDDDDFADDRecruitedTRAF1TRAF2IAPFigure 2-5. Signaling pathway for tumor necrosis factor receptor 1 (TNFR-1) (55 kDa) and TNFR-2 (75 kDa) occurs by the recruitment of several

1	2-5. Signaling pathway for tumor necrosis factor receptor 1 (TNFR-1) (55 kDa) and TNFR-2 (75 kDa) occurs by the recruitment of several adapter proteins to the intracellular receptor complex. Optimal signaling activity requires receptor trimerization. TNFR-1 initially recruits TNFR-associated death domain (TRADD) and induces apoptosis through the actions of proteolytic enzymes known as caspases, a pathway shared by another receptor known as CD95 (Fas). CD95 and TNFR-1 possess similar intracellular sequences known as death domains (DDs), and both recruit the same adapter proteins known as Fas-associated death domains (FADDs) before activating caspase 8. TNFR-1 also induces apoptosis by activating caspase 2 through the recruitment of receptor-interacting protein (RIP). RIP also has a func-tional component that can initiate nuclear factor-κB (NF-κB) and c-Jun activation, both favoring cell survival and proinflammatory functions. TNFR-2 lacks a DD component but recruits adapter proteins

1	component that can initiate nuclear factor-κB (NF-κB) and c-Jun activation, both favoring cell survival and proinflammatory functions. TNFR-2 lacks a DD component but recruits adapter proteins known as TNFR-associated factors 1 and 2 (TRAF1, TRAF2) that interact with RIP to mediate NF-κB and c-Jun activation. TRAF2 also recruits additional proteins that are antiapoptotic, known as inhibitor of apoptosis proteins (IAPs). DED = death effector domain; I-κB = inhibitor of κB; I-κB/NF-κB = inactive complex of NF-κB that becomes activated when the I-κB portion is cleaved; JNK = c-Jun N-terminal kinase; MEKK1 = mitogen-activated protein/extracellular regulatory protein kinase kinase kinase-1; NIK = NF-κB–inducing kinase; RAIDD = RIP-associated interleukin-1b-converting enzyme and ced-homologue-1–like protein with death domain, which activates proapoptotic caspases. (Adapted with permission from Vincent JL: Marshall JC, Cohen J: Update in Intensive Care and Emergency Medicine: Vol. 31: Immune

1	protein with death domain, which activates proapoptotic caspases. (Adapted with permission from Vincent JL: Marshall JC, Cohen J: Update in Intensive Care and Emergency Medicine: Vol. 31: Immune Response in Critical Illness. Berlin: Springer-Verlag; 2002.)Brunicardi_Ch02_p0027-p0082.indd 4101/03/19 6:49 PM 42BASIC CONSIDERATIONSPART Icontrols and correlated with injury severity. Moreover, increase in pyroptotic PBMCs significantly correlated with elevated cytokine levels (IL-10, IL-18, and MCP-1) and was a strong predictor for the development of sepsis.MEDIATORS OF INFLAMMATIONCytokinesCytokines are a class of protein signaling compounds that are essential for both innate and adaptive immune responses. Cytokines mediate a broad sequence of cellular responses, including cell migration, DNA replication, cell turnover, and immunocyte proliferation (Table 2-5). When function-ing locally at the site of injury and infection, cytokines mediate the eradication of invading microorganisms

1	DNA replication, cell turnover, and immunocyte proliferation (Table 2-5). When function-ing locally at the site of injury and infection, cytokines mediate the eradication of invading microorganisms and also promote wound healing. However, an exaggerated proinflammatory cyto-kine response to inflammatory stimuli may result in hemody-namic instability (i.e., septic shock) and metabolic derangements (i.e., muscle wasting). Anti-inflammatory cytokines also are released, at least in part, as an opposing influence on the proin-flammatory cascade. These anti-inflammatory mediators may also result in immunocyte dysfunction and host immunosuppres-sion. Cytokine signaling after an inflammatory stimulus can best be represented as a finely tuned balance of opposing influences and should not be oversimplified as a “black and white” proin-flammatory/anti-inflammatory response. A brief discussion of the important cytokine molecules is included below.Tumor Necrosis Factor-α. Tumor necrosis factor-α

1	as a “black and white” proin-flammatory/anti-inflammatory response. A brief discussion of the important cytokine molecules is included below.Tumor Necrosis Factor-α. Tumor necrosis factor-α (TNF-α) is a potent inflammatory mediator that is rapidly mobilized in response to stressors such as injury and infection. It is primar-ily synthesized by immune cells, such as macrophages, den-dritic cells, and T lymphocytes, and is generated in a precursor form that is expressed as a trimer on the surface of activated cells. After being processed by the metalloproteinase, TNF-α-converting enzyme (TACE, also known as ADAMS 17), a smaller, soluble form of TNF is released, which mediates its bio-logical activities through types 1 and 2 TNF receptors (TNFR-1; TNFR-2).130 Transmembrane TNF-α also binds to TNFR-1 and -2, but its biological activities are likely mediated through TNFR-2. While the two receptors share homology in their ligand-binding regions, there are distinct differences that regu-late

1	TNFR-1 and -2, but its biological activities are likely mediated through TNFR-2. While the two receptors share homology in their ligand-binding regions, there are distinct differences that regu-late their biologic function. For example, TNFR-1 is expressed by a wide variety of cells, but it is typically sequestered in the Golgi. Following appropriate cell signaling, TNFR-1 is mobi-lized to the cell surface, where it sensitizes cells to TNF or it can be cleaved from the surface in the form of a soluble recep-tor that can neutralize TNF.131 In contrast, TNFR-2 expression is confined principally to immune cells where it resides in the plasma membrane. Both TNF receptors are capable of binding intracellular adaptor proteins that lead to activation of complex signaling processes and mediate the effects of TNF.Although the circulating half-life of soluble TNF is brief, it acts upon almost every differentiated cell type, eliciting a wide range of important cellular responses. Moreover, it is

1	effects of TNF.Although the circulating half-life of soluble TNF is brief, it acts upon almost every differentiated cell type, eliciting a wide range of important cellular responses. Moreover, it is one of the first cytokines to be released following trauma. In particular, TNF elicits many metabolic and immunomodulatory activities. It stimulates muscle breakdown and cachexia through increased catabolism, insulin resistance, and redistribution of amino acids to hepatic circulation as fuel substrates. TNF also mediates coagulation activation, cell migration, and macrophage phago-cytosis and enhances the expression of adhesion molecules, prostaglandin E2, platelet-activating factor, glucocorticoids, and eicosanoids. TNF-α increases endothelial cell permeability and activates macrophages, NK cells and lymphocytes to induce the secretion of various cytokines. While TNF is clearly play-ing a role in injury-induced inflammation, reports are conflict-ing whether postinjury TNF concentrations

1	cells and lymphocytes to induce the secretion of various cytokines. While TNF is clearly play-ing a role in injury-induced inflammation, reports are conflict-ing whether postinjury TNF concentrations correlated with the development of multiple organ dysfunction syndrome.132Interleukin-1 Family. The IL-1 family of proteins contains 11 members. The best-studied of these are IL-1α and IL-1β and IL-1 receptor antagonist (IL-1Ra), but member cytokines also include IL-18, IL-33, IL-36, IL-3,7 and IL-38. IL-1α and IL-1β, which are encoded by two distinct IL-1 genes, share similar bio-logic functions despite limited sequence homology. They uti-lize the same cell surface receptor, termed IL-1 receptor type 1 (IL-1RI), which is present on nearly all cells. Once bound to its receptor, IL-1 initiates signaling events that result in the synthe-sis and release of a variety of inflammatory mediators. Members of the IL-1 family are expressed as proforms (pIL-1) that are matured through enzymatic

1	signaling events that result in the synthe-sis and release of a variety of inflammatory mediators. Members of the IL-1 family are expressed as proforms (pIL-1) that are matured through enzymatic cleavage. The IL-1α precursor is constitutively expressed and stored in a variety of healthy cells, including epithelium and endothelium, and its expression can be increased in response to proinflammatory or stress-associated stimuli.133Both the precursor and mature forms of IL-α have nearly identical biologic activities as measured by their ability to trig-ger IL-6 and TNF release. With appropriate signals, IL-1α can move both to the cell membrane, where it can act on adjacent cells bearing the IL-1R and to the nucleus where it can stim-ulate gene transcription. Pro-IL-1α can also be released pas-sively from damaged injured cells in its active form. In this way, IL-1α is believed to function as a DAMP, which promotes the synthesis of inflammatory mediators, such as chemokines and eicosanoids.

1	from damaged injured cells in its active form. In this way, IL-1α is believed to function as a DAMP, which promotes the synthesis of inflammatory mediators, such as chemokines and eicosanoids. These mediators attract neutrophils to the injured site, facilitate their exit from the vasculature, and promote their activation. Once they have reached their target, neutrophil lifes-pan is extended by the presence of IL-1α.8,134IL-1β is a multifunctional proinflammatory cytokine whose expression and synthesis is tightly regulated and con-fined to activated cells, such as monocytes, tissue macrophages, and dendritic cells.134 In contrast to IL-1α, IL-1β is synthesized as an inactive precursor, pro-IL-1β, which is processed by the inflammasome in response to various stimuli, including cyto-kines and foreign pathogens, via pattern recognition receptors such as TLR4 as well as ROS. Mature IL-1β is then released from the cell via an unconventional secretory pathway.135 IL-1β has a spectrum of

1	and foreign pathogens, via pattern recognition receptors such as TLR4 as well as ROS. Mature IL-1β is then released from the cell via an unconventional secretory pathway.135 IL-1β has a spectrum of proinflammatory effects that are largely simi-lar to those induced by TNF, and injection of IL-1β alone is sufficient to induce an acute inflammatory response. High doses of either IL-1β or TNF are associated with profound hemody-namic compromise. Interestingly, low doses of both IL-1β and TNF administered together elicit hemodynamic events similar to those elicited by high doses of either mediator, which sug-gests a synergistic effect.There are two primary receptor types for IL-1: IL-1R1 and IL-1R2. IL-1R1 is widely expressed and mediates inflammatory signaling on ligand binding. IL-1R2 is proteolytically cleaved from the membrane surface to soluble form on activation and thus serves as another mechanism for competition and regula-tion of IL-1 activity. IL-1α or IL-1β bind first to the

1	proteolytically cleaved from the membrane surface to soluble form on activation and thus serves as another mechanism for competition and regula-tion of IL-1 activity. IL-1α or IL-1β bind first to the IL-1R1, which is considered the ligand-binding chain. This is followed by recruitment of a transmembrane co-receptor, termed the 5Brunicardi_Ch02_p0027-p0082.indd 4201/03/19 6:49 PM 43SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2Table 2-5Cytokines and their sourcesCYTOKINESOURCECOMMENTTNFMacrophages/monocytesKupffer cellsNeutrophilsNK cellsAstrocytesEndothelial cellsT lymphocytesAdrenal cortical cellsAdipocytesKeratinocytesOsteoblastsMast cellsDendritic cellsAmong earliest responders after injury; half-life <20 min; activates TNF receptors 1 and 2; induces significant shock and catabolismIL-1Macrophages/monocytesB and T lymphocytesNK cellsEndothelial cellsEpithelial cellsKeratinocytesFibroblastsOsteoblastsDendritic cellsAstrocytesAdrenal cortical

1	significant shock and catabolismIL-1Macrophages/monocytesB and T lymphocytesNK cellsEndothelial cellsEpithelial cellsKeratinocytesFibroblastsOsteoblastsDendritic cellsAstrocytesAdrenal cortical cellsMegakaryocytesPlateletsNeutrophilsNeuronal cellsTwo forms (IL-1 α and IL-1 β); similar physiologic effects as TNF; induces fevers through prostaglandin activity in anterior hypothalamus; promotes β-endorphin release from pituitary; half-life <6 minIL-2T lymphocytesPromotes lymphocyte proliferation, immunoglobulin production, gut barrier integrity; half-life <10 min; attenuated production after major blood loss leads to immunocompromise; regulates lymphocyte apoptosisIL-3T lymphocytesMacrophagesEosinophilsMast cellsIL-4T lymphocytesMast cellsBasophilsMacrophagesB lymphocytesEosinophilsStromal cellsInduces B-lymphocyte production of IgG4 and IgE, mediators of allergic and anthelmintic response; downregulates TNF, IL-1, IL-6, IL-8IL-5T lymphocytesEosinophilsMast cellsBasophilsPromotes

1	cellsInduces B-lymphocyte production of IgG4 and IgE, mediators of allergic and anthelmintic response; downregulates TNF, IL-1, IL-6, IL-8IL-5T lymphocytesEosinophilsMast cellsBasophilsPromotes eosinophil proliferation and airway inflammationIL-6MacrophagesB lymphocytesNeutrophilsBasophilsMast cellsFibroblastsEndothelial cellsElicited by virtually all immunogenic cells; long half-life; circulating levels proportional to injury severity; prolongs activated neutrophil survival(Continued)Brunicardi_Ch02_p0027-p0082.indd 4301/03/19 6:49 PM 44BASIC CONSIDERATIONSPART ITable 2-5Cytokines and their sourcesCYTOKINESOURCECOMMENTAstrocytesSynovial cellsAdipocytesOsteoblastsMegakaryocytesChromaffin cellsKeratinocytesIL-8Macrophages/monocytesT lymphocytesBasophilsMast cellsEpithelial cellsPlateletsChemoattractant for neutrophils, basophils, eosinophils, lymphocytesIL-10T lymphocytesB lymphocytesMacrophagesBasophilsMast cellsKeratinocytesProminent anti-inflammatory cytokine; reduces mortality

1	for neutrophils, basophils, eosinophils, lymphocytesIL-10T lymphocytesB lymphocytesMacrophagesBasophilsMast cellsKeratinocytesProminent anti-inflammatory cytokine; reduces mortality in animal sepsis and ARDS modelsIL-12Macrophages/monocytesNeutrophilsKeratinocytesDendritic cellsB lymphocytesPromotes Th1 differentiation; synergistic activity with IL-2IL-13T lymphocytesPromotes B-lymphocyte function; structurally similar to IL-4; inhibits nitric oxide and endothelial activationIL-15Macrophages/monocytesEpithelial cellsAnti-inflammatory effect; promotes lymphocyte activation; promotes neutrophil phagocytosis in fungal infectionsIL-18MacrophagesKupffer cellsKeratinocytesAdrenal cortical cellsOsteoblastsSimilar to IL-12 in function; levels elevated in sepsis, particularly gram-positive infections; high levels found in cardiac deathsIFN-γT lymphocytesNK cellsMacrophagesMediates IL-12 and IL-18 function; half-life of days; found in wounds 5–7 d after injury; promotes ARDSGM-CSFT

1	infections; high levels found in cardiac deathsIFN-γT lymphocytesNK cellsMacrophagesMediates IL-12 and IL-18 function; half-life of days; found in wounds 5–7 d after injury; promotes ARDSGM-CSFT lymphocytesFibroblastsEndothelial cellsStromal cellsPromotes wound healing and inflammation through activation of leukocytesIL-21T lymphocytesPreferentially secreted by Th2 cells; structurally similar to IL-2 and IL-15; activates NK cells, B and T lymphocytes; influences adaptive immunityHMGB1Monocytes/lymphocytesHigh mobility group box chromosomal protein; DNA transcription factor; late (downstream) mediator of inflammation (ARDS, gut barrier disruption); induces “sickness behavior”ARDS = acute respiratory distress syndrome; GM-CSF = granulocyte-macrophage colony-stimulating factor; IFN = interferon; Ig = immunoglobulin; IL = interleukin; NK = natural killer; Th1 = helper T cell subtype 1; Th2 = helper T cell subtype 2; TNF = tumor necrosis factor.(Continued)accessory protein (IL-1RAcP). A

1	Ig = immunoglobulin; IL = interleukin; NK = natural killer; Th1 = helper T cell subtype 1; Th2 = helper T cell subtype 2; TNF = tumor necrosis factor.(Continued)accessory protein (IL-1RAcP). A complex is formed of IL-1RI plus IL-1 plus the coreceptor. The signal is initiated with recruit-ment of the adaptor protein MyD88 to the Toll-IL-1 receptor (TIR) domains of the receptor complex and signal transduction via intermediates, which are homologous to the signal cascade initiated by TLRs. These events culminate in the activation of NF-kB and its nuclear translocation.136Recent animal studies have implicated postinjury IL-1β in the exacerbation of traumatic brain injury.137 In a mouse model of polytrauma, which included both cortical brain injury and tibial fracture, mice that received both injuries demonstrated increased neuroinflammation, brain damage, and behavioral deficits compared to mice given an isolated-TBI. These changes correlated with increased IL-1β levels in the brain.

1	injuries demonstrated increased neuroinflammation, brain damage, and behavioral deficits compared to mice given an isolated-TBI. These changes correlated with increased IL-1β levels in the brain. Treatment Brunicardi_Ch02_p0027-p0082.indd 4401/03/19 6:49 PM 45SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2with IL-1R antagonist post injury reduced volume loss in the injured cortex as well as markers of axonal injury, resulting in improved outcome in these animals.IL-18 is also a member of the IL-1 superfamily of cytokines.138 First noted as an IFN-γ-inducing factor produced by LPS-stim-ulated macrophages, IL-18 expression is found both in immune cells and nonimmune cells at low to intermediate levels. How-ever, activated macrophages and Kupffer cells produce large amounts of mature IL-18. Similar to IL-1β, IL-18 is synthe-sized and stored as an inactive precursor form (pro-IL-18), and activation requires activation of the inflammasome resulting the processing of

1	of mature IL-18. Similar to IL-1β, IL-18 is synthe-sized and stored as an inactive precursor form (pro-IL-18), and activation requires activation of the inflammasome resulting the processing of pro-IL-18 by caspase-1.139 It then exits the cell through a nontraditional secretory pathway. The IL-18 receptor (IL-18R) is composed of two subunits, IL-18Rα and IL-18Rβ, and is a member of the IL-1R superfamily that is structurally similar in their cytoplasmic domains to the TLR. One unique biological property of IL-18 is the potential, in conjunction with IL-12, to promote the Th1 response.IL-18 induces IFNγ production by CD4+ T cells. IFNγ, in turn, activates macrophages to produce inflammatory cytokines. Independent of its ability to induce interferon, IL-18 can act similarly to other proinflammatory cytokines by acting directly to increase in cell adhesion molecule expression, nitric oxide synthesis, and chemokine production by macrophages.140In a cohort of critically ill patients with

1	cytokines by acting directly to increase in cell adhesion molecule expression, nitric oxide synthesis, and chemokine production by macrophages.140In a cohort of critically ill patients with acute lung injury and ARDS, inflammasome-related mRNA transcripts (CASP1, IL1B, and IL18) were increased in peripheral blood. Moreover, plasma IL-18 were also elevated and served as a marker of mor-tality risk.141 Recent studies suggest that IL-18 therapy may hold promise as effective therapy in promoting immune recovery after severe surgical stress.142IL-33, a second important IL-1 family member, is mainly expressed in surface epithelium and endothelium, where it is normally bound via an N-terminal chromatin-binding motif.143 Nuclear localization is important for its function and perhaps its regulation. Expression in mice of an IL-33 that lacks the nuclear localization sequence, results lethal inflammation, suggesting that nuclear localization acts to prevent unregulated extracel-lular release.144

1	Expression in mice of an IL-33 that lacks the nuclear localization sequence, results lethal inflammation, suggesting that nuclear localization acts to prevent unregulated extracel-lular release.144 Similar to HMGB1 and other IL-1 family mem-bers, IL-33 lacks a signal sequence for active secretion so that its release is injury-dependent. Once released from damaged cells, full length IL-33 is biologically active, but it can be further pro-cessed by inflammatory proteases to a mature form that exhibits tento thirtyfold higher activity. Il-33 can binds to a member of the IL-1R family, ST2, leading to activation of NF-kB-mediated transcriptional events. ST2+ cells include macrophages, mast cells, Th2 cells, and tissue regulatory T cells (Tregs) which are important controllers of immune homeostasis.Interleukin-2 Family. Interleukin-2 (IL-2) is a multifunc-tional cytokine produced primarily by CD4+ T cells after antigen activation, which plays pivotal roles in the immune response. Other

1	Family. Interleukin-2 (IL-2) is a multifunc-tional cytokine produced primarily by CD4+ T cells after antigen activation, which plays pivotal roles in the immune response. Other cellular sources for IL-2 include CD8+ and NK T cells, mast cells, and activated dendritic cells. Discovered as a T cell growth factor, IL-2 also promotes CD8+ T cell and natural killer cell cytolytic activity and modulates T cell differentiation pro-grams in response to antigen. Thus, IL-2 promotes naive CD4+ T cell differentiation into T helper 1 (Th1) and T helper 2 (Th2) cells while inhibiting T helper 17 (Th17) and T follicular helper (Tfh) cell differentiation. Moreover, IL-2 is essential for the development and maintenance of T regulatory (Treg) cells and for activation-induced cell death, thereby mediating tolerance and limiting inappropriate immune reactions. The upregulation of IL-2 requires calcium as well as protein kinase C signaling, which leads to the activation of transcription factors such as

1	tolerance and limiting inappropriate immune reactions. The upregulation of IL-2 requires calcium as well as protein kinase C signaling, which leads to the activation of transcription factors such as nuclear factor of activated T cells (NFAT) and NF-κB. MicroRNAs also play a role in the regulation of IL-2 expression.145IL-2 binds to IL-2 receptors (IL-2R), which are expressed on leukocytes. IL-2Rs are formed from various combinations of three receptor subunits: IL-2Rα, IL-2Rβ, and IL-2Rγ. These subunits form in low, medium, and high affinity forms of the receptor depending on the subunit combination. IL-2Rγ has been renamed the common cytokine receptor γ chain (γc), which is now known to be shared by IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Constitutive IL-2 receptor expression is low and is inducible by T cell receptor ligation and cytokine stimulation. Importantly, the transcription of each receptor subunit is indi-vidually regulated via a complex process to effect tight control of

1	is inducible by T cell receptor ligation and cytokine stimulation. Importantly, the transcription of each receptor subunit is indi-vidually regulated via a complex process to effect tight control of surface expression. Once the receptor is ligated, the major IL-2 signaling pathways that are engaged include Janus Kinase (JAK)-signal transducer and activator of transcription (STAT), Shc-Ras-MAPK, and phosphoinositol-3-kinase (PI3-K)-AKT. Partly due to its short half-life of <10 minutes, IL-2 is not read-ily detectable after acute injury. IL-2 receptor blockade induces immunosuppressive effects and can be pharmacologically used for organ transplantation. Attenuated IL-2 expression observed during major injury or blood transfusion may contribute to the relatively immunosuppressed state of the surgical patient.146Interleukin-6 Family. Following burn or traumatic injury, damage-associated molecular patterns (DAMPs) from dam-aged or dying cells stimulate TLRs to produce IL-6, a

1	state of the surgical patient.146Interleukin-6 Family. Following burn or traumatic injury, damage-associated molecular patterns (DAMPs) from dam-aged or dying cells stimulate TLRs to produce IL-6, a proin-flammatory cytokine that plays a central role in host defense. IL-6 levels in the circulation are detectable by 60 minutes post injury, peak between 4 and 6 hours, and can persist for as long as 10 days. Further, plasma levels of IL-6 are proportional to the degree of injury. In the liver, IL-6 strongly induces a broad spec-trum of acute-phase proteins such as C-reactive protein (CRP) and fibrinogen, among others, while it reduces expression of albumin, cytochrome P 450, and transferrin. In lymphocytes, IL-6 induces B cell maturation into immunoglobulin-producing cells and regulates Th17/Treg balance. IL-6 modulates T cell behavior by inducing the development of Th17 cells and inhib-iting Treg cell differentiation in conjunction with transforming growth factor-β. IL-6 also promotes

1	balance. IL-6 modulates T cell behavior by inducing the development of Th17 cells and inhib-iting Treg cell differentiation in conjunction with transforming growth factor-β. IL-6 also promotes angiogenesis and increased vascular permeability, which are associated with local inflam-matory responses. To date, ten IL-6 family cytokines have been identified, including IL-6, oncostatin M, neuropoietin, IL-11, IL-27, and IL-31.147The interleukin-6 receptor (IL-6R, gp80) is expressed on hepatocytes, monocytes, B cells, and neutrophils in humans. However, many other cells respond to IL-6 through a process known as trans-signaling.148 In this case, soluble IL-6Rs (sIL-6R) exist in the serum and bind to IL-6, forming an IL-6/sIL-6R complex. The soluble receptor is produced by proteo-lytic cleavage from the surface of neutrophils in a process that is stimulated by C-reactive protein, complement factors, and leu-kotrienes. The IL6/sIL6R complex can then bind to the gp130 receptor, which is

1	from the surface of neutrophils in a process that is stimulated by C-reactive protein, complement factors, and leu-kotrienes. The IL6/sIL6R complex can then bind to the gp130 receptor, which is expressed ubiquitously on cells. Upon IL-6 stimulation, gp130 transduces two major signaling pathways: the JAK-STAT3 pathway and the SHP2-Gab-Ras-Erk-MAPK pathway, which is regulated by cytoplasmic suppressor of cytokine signaling (SOCS3). These signaling events can lead to increased expression of adhesion molecules as well as proinflammatory chemokines and cytokines. High plasma IL-6 levels have been associated with mortality during Brunicardi_Ch02_p0027-p0082.indd 4501/03/19 6:49 PM 46BASIC CONSIDERATIONSPART Iintra-abdominal sepsis.149 Moreover, prolonged (more than 3 days) elevation of IL-6 concentrations has been reported to correlate with the occurrence of complications and mortality following severe traumatic injury. More recently, a meta-analy-sis analyzed the predictive value of

1	concentrations has been reported to correlate with the occurrence of complications and mortality following severe traumatic injury. More recently, a meta-analy-sis analyzed the predictive value of IL-6 for the development of complications and mortality after trauma and found that the con-centration of IL-6 in the first 24 hours after trauma was predic-tive for the development of multiple organ failure and death.150Interleukin-10 Family. We have talked almost exclusively about the factors that initiate the proinflammatory response fol-lowing cellular stress or injury. The reestablishment of immune homeostasis following these events requires the resolution of inflammation and the initiation of tissue repair processes. Interleukin-10 (IL-10) plays a central role in this anti-inflammatory response by regulating the duration and magnitude of inflamma-tion in the host.151The IL-10 family currently has six members, includ-ing IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26. IL-10 is produced

1	by regulating the duration and magnitude of inflamma-tion in the host.151The IL-10 family currently has six members, includ-ing IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26. IL-10 is produced by a variety of immune cells of both myeloid and lymphoid origin. Its synthesis is up-regulated during times of stress and systemic inflammation; however, each cell type that produces IL-10 does so in response to different stimuli, allow-ing for tight control of its expression. IL-10 exerts effects by binding to the IL-10 receptor (IL-10R), which is a tetramer formed from two distinct subunits, IL-10R1 and IL-10R2. Specifically, IL-10 binds first to the IL10R1 subunit, which then recruits IL-10R2, allowing the receptor complex to form. While IL-10R2 is widely expressed, IL-10R1 expression is confined to leukocytes so that the effects of IL-10 are confined to the immune system. Once receptor ligation occurs, signaling proceeds by the activation of JAK1 and STAT3. In particular, STAT3 in

1	confined to leukocytes so that the effects of IL-10 are confined to the immune system. Once receptor ligation occurs, signaling proceeds by the activation of JAK1 and STAT3. In particular, STAT3 in conjunction with IL-10 is absolutely required for the transcription of genes responsible for the anti-inflammatory response (AIR). IL-10 inhibits the secretion of proinflamma-tory cytokines, including TNF and IL-1, partly through the downregulation of NF-κB and thereby functions as a nega-tive feedback regulator of the inflammatory cascade.151 In macrophages, IL-10 suppresses the transcription of 20% of all LPS-induced genes. Further, experimental models of inflam-mation have shown that neutralization of IL-10 increases TNF production and mortality, whereas restitution of circulating IL-10 reduces TNF levels and subsequent deleterious effects. Increased plasma levels of IL-10 also have been associated with mortality and disease severity after traumatic injury.Interleukin-12

1	IL-10 reduces TNF levels and subsequent deleterious effects. Increased plasma levels of IL-10 also have been associated with mortality and disease severity after traumatic injury.Interleukin-12 Family. Interleukin-12 (IL-12) is unique among the cytokines in being the only heterodimeric cytokine. This family, which includes IL-12, IL-23, IL-27, and IL-35, consists of an α-chain that is structurally similar to the IL-6 cytokine and a β-chain that is similar to the class I receptor for cytokines. The individual IL-12 family members are formed from various combinations of the α and β subunits. Despite the sharing of individual subunits, and the similarities of their receptors, the IL-12 cytokines have different biological func-tions. IL-12 and IL-23 are considered proinflammatory, stimu-latory cytokines with key roles in the development of Th1 and Th17 subsets of helper T cells. In contrast, both IL-27 and IL-35 appear to have immunoregulatory functions that are associated with cytokine

1	cytokines with key roles in the development of Th1 and Th17 subsets of helper T cells. In contrast, both IL-27 and IL-35 appear to have immunoregulatory functions that are associated with cytokine inhibition in specific Treg cell populations, par-ticularly the Th17 cells.152 The effects of these cytokines require specific receptor chains that are also shared among the cyto-kines. The complexity of signaling is evidenced by the fact that these receptor chains can function both as dimers and as mono-mers. Ligation of the IL-12 receptors initiate signaling events via the JAK-STAT pathway.IL-12 synthesis and release is increased during endotox-emia and sepsis.153 Together with IL-18, it stimulates lympho-cytes to increase secretion of IFN-γ. IL-12 also stimulates NK cell cytotoxicity and helper T cell differentiation in this setting. IL-12 release is inhibited by IL-10, and its deficiency inhibits phagocytosis in neutrophils. In experimental models of inflam-matory stress, IL-12

1	and helper T cell differentiation in this setting. IL-12 release is inhibited by IL-10, and its deficiency inhibits phagocytosis in neutrophils. In experimental models of inflam-matory stress, IL-12 neutralization conferred a mortality benefit in mice during endotoxemia.IL-23, an important IL-12 family member, is a heterodi-meric cytokine comprised of a unique p19 subunit linked to a p40 subunit that is common with IL-12. IL-23 appears to be an important survival signal for a specific subset of T helper (Th) cells, Th-17 cells, where it provides a secondary stimu-lus for Th-17 differentiation.152 The Th-17 population of cells has recently been demonstrated to expand following traumatic injury and may mark an early phenotypic shift in cell population that has prognostic significance.154,155Interleukin-17 Family. IL-17A (also called IL-17) is the major effector cytokine predominantly produced by a subset of helper T cells, the T helper (Th)-17 cells.156 It is the founding member of the

1	Family. IL-17A (also called IL-17) is the major effector cytokine predominantly produced by a subset of helper T cells, the T helper (Th)-17 cells.156 It is the founding member of the IL-17 family of cytokines, which includes IL-17A through F. The original described activity for IL-17A was to promote the differentiation of bone marrow progenitor cells along the granulopoietic lineage. Subsequent studies have confirmed that IL-17A is required for increasing circulating neutrophil numbers following stress. In the setting of infection, it is now known that IL-17 acts in conjunction with IL-23 to upregulate granulocyte-colony stimulating factor to promote granulopoi-esis. IL-17A has also been shown to regulate the production of specific chemokines in both gut and lung epithelial cells and thus can modulate both the emigration of neutrophils into these tissues and their activation at the site. IL-17 also induces the expression of matrix metalloproteinases, which can make the extracellular

1	can modulate both the emigration of neutrophils into these tissues and their activation at the site. IL-17 also induces the expression of matrix metalloproteinases, which can make the extracellular matrix more accessible for immune cell recruitment.156IL-17 has the ability to induce the expression of impor-tant proinflammatory cytokines, including IL-1β, IL-6, and TNF from macrophages and other cells, and in this way, cre-ates a self-sustaining loop that enhances its own production and strengthens its overall effects.157 Recent data supports a pivotal role for IL-17 in the posttrauma immune response and has iden-tified associations between increased IL-17 expression associ-ated with Th17immune response outcomes following blunt trauma.154Interferons. Interferons were first recognized as soluble mediators that inhibited viral replication through the activa-tion of specific antiviral genes in infected cells. Interferons are categorized into three types based on receptor specificity and

1	mediators that inhibited viral replication through the activa-tion of specific antiviral genes in infected cells. Interferons are categorized into three types based on receptor specificity and sequence homology. The two major types, type I and type II are discussed in the following section.Type I interferon family is composed of twenty distinct proteins. These include IFN-α, IFN-β, and IFN-ω, which are structurally related and bind to a common receptor.158 They are likely produced by most cell types and tissues after the detec-tion of PAMPs/ DAMPs by cytosolic or membrane receptors, including TLR in macrophages and dendritic cells. Type 1 IFNs bind to a heterodimeric transmembrane receptor interferon (α and β) receptor 1, resulting in STAT activation and nuclear trans-location. In the nucleus, dimeric STATs recruit an additional Brunicardi_Ch02_p0027-p0082.indd 4601/03/19 6:49 PM 47SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2transcriptional factor to form a complex

1	dimeric STATs recruit an additional Brunicardi_Ch02_p0027-p0082.indd 4601/03/19 6:49 PM 47SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2transcriptional factor to form a complex capable of binding to interferon-stimulated response elements, inducing hundreds of IFN-stimulated genes.Type I interferons influence adaptive immune responses by inducing the maturation of dendritic cells and by stimulat-ing class I MHC expression. IFN-α and IFN-β also enhance immune responses by increasing the cytotoxicity of natural killer cells both in culture and in vivo. Further, they have been implicated in the enhancement of chemokine synthesis, particu-larly those that recruit myeloid cells and lymphoid cells. Thus, IFN/STAT signaling has important effects on the mobilization, tissue recruitment, and activation of immune cells that compose the inflammatory infiltrate. In contrast, type I IFNs appear to inhibit inflammasome activity, possibly via IL-10.159The single type II interferon,

1	and activation of immune cells that compose the inflammatory infiltrate. In contrast, type I IFNs appear to inhibit inflammasome activity, possibly via IL-10.159The single type II interferon, IFN-γ is secreted by various T cells, NK cells, and antigen-presenting cells in response to bacterial antigens and cytokines. It functions as a key regula-tor of macrophage activation toward the “M1” proinflammatory phenotype.160 In response to IFN-γ, macrophages produce high levels of proinflammatory cytokines such as Il-1β, IL-12, IL-23, and TNF-α as well as reactive nitrogen and oxygen species. As a consequence, macrophages demonstrate enhanced phagocyto-sis and killing. In addition, IFN-γ signaling generates additional cytokines and inflammatory factors to sustain inflammation and help to maintain Th1 responses.IFN-γ regulation of macrophage activity may contribute to acute lung injury after major surgery or trauma. A dimin-ished IFN-γ level, as seen in knockout mice, is associated with

1	Th1 responses.IFN-γ regulation of macrophage activity may contribute to acute lung injury after major surgery or trauma. A dimin-ished IFN-γ level, as seen in knockout mice, is associated with increased susceptibility to both viral and bacterial pathogens. In addition, IFN-γ promotes differentiation of T cells to the helper T cell subtype 1 and also enhances B-cell isotype switching to immunoglobulin G.158Receptors of all IFN subtypes belong to the class II of cytokine receptors and utilize JAK-STAT signaling pathway for nuclear signaling, although different STAT activation (e.g., STAT1 and STAT2) is favored by individual receptors.Granulocyte-Macrophage Colony-Stimulating Fac-tor/Interleukin-3/Interleukin-5 Family. Granulocytemacrophage colony-stimulating factor (GM-CSF), IL-3, and IL-5 compose a small family of cytokines that regulates the growth and activation of immune cells. They are largely the products of activated T cells, which when released, stimulate the behavior of myeloid

1	a small family of cytokines that regulates the growth and activation of immune cells. They are largely the products of activated T cells, which when released, stimulate the behavior of myeloid cells by inducing cytokine expression and antigen presentation. In this way, GM-CSF, IL-3, and IL-5 are able to link the innate and acquired immune responses. With the exception of eosinophils, GM-CSF/IL-3/IL-5 are not essential for constitutive hematopoietic cell function. Rather, they play an important role when the host is stressed by serving to increase the numbers of activated and sensitized cells required to bol-ster host defense.161 Currently, GM-CSF is in clinical trials for administration to children with an injury severity score >10 fol-lowing blunt or penetrating trauma. The goal of the study is to provide evidence of the effectiveness of GM-CSF as an agent that can ameliorate posttraumatic immune suppression.Receptors for the GM–CSF/IL-3/IL-5 family of cytokines are expressed at very

1	is to provide evidence of the effectiveness of GM-CSF as an agent that can ameliorate posttraumatic immune suppression.Receptors for the GM–CSF/IL-3/IL-5 family of cytokines are expressed at very low level on hematopoietic cells. Similar to the other cytokine receptors discussed, they are heterodimers composed of a cytokine-specific α subunit and a common β sub-unit (βc), which is shared by all three receptors and is required for high affinity signal transduction. The binding of cytokine to its receptor activates JAK2-STAT, MAPK, and PI3-K—mediated signaling events to regulate a variety of important cell behaviors, including effector function in mature cells.EicosanoidsOmega-6 Polyunsaturated Fat Metabolites: Arachidonic Acid. Eicosanoids are derived primarily by oxidation of the membrane phospholipid, arachidonic acid (AA), which is relatively abundant in the membrane lipids of inflammatory cells. The major precursor of arachidonic acid is the omega-6 (n-6) polyunsaturated fatty acid

1	phospholipid, arachidonic acid (AA), which is relatively abundant in the membrane lipids of inflammatory cells. The major precursor of arachidonic acid is the omega-6 (n-6) polyunsaturated fatty acid (PUFA) linolenic acid, a major source of which is soybean oil. Not surprising, an excess of linolenic acid is thought to promote inflammation via increased availability of AA, and in turn, eicosanoids.Eicosanoids generated from AA include prostaglandins, thromboxanes, and leukotrienes. When a cell senses the proper stimulus, AA is released from phospholipids or diacylglycerols by the enzymatic activation of phospholipase A2 (Fig. 2-6A). Prostanoids, which include all of the prostaglandins (PG) and the thromboxanes, result from the sequential action of the cyclooxy-genase (COX) enzyme and terminal synthetases on arachidonic acid. In contrast, arachidonic acid may be oxidized along the lipoxygenase pathway via the central enzyme 5-lipoxygenase, to produce several classes of leukotrienes and

1	synthetases on arachidonic acid. In contrast, arachidonic acid may be oxidized along the lipoxygenase pathway via the central enzyme 5-lipoxygenase, to produce several classes of leukotrienes and lipoxins, which have anti-inflammatory functions. In general, the effects of eico-sanoids are mediated via specific receptors, which are members of a superfamily of G protein-coupled receptors.Eicosanoids are not stored within cells but are instead generated rapidly in response to many proinflammatory stimuli, including hypoxic injury, direct tissue injury, endotoxin (lipo-polysaccharide), norepinephrine, vasopressin, angiotensin II, bradykinin, serotonin, acetylcholine, cytokines, and histamine. They have a broad range of physiologic roles, including neuro-transmission, and vasomotor regulation. Eicosanoids are also involved in immune cell regulation (Table 2-6), by modulating the intensity and duration of inflammatory responses.Glucocorticoids, NSAIDs, and leukotriene inhibitors can

1	Eicosanoids are also involved in immune cell regulation (Table 2-6), by modulating the intensity and duration of inflammatory responses.Glucocorticoids, NSAIDs, and leukotriene inhibitors can successfully block the end products of eicosanoid pathways to modulated inflammation.The production of eicosanoids is celland stimulus-specific. Therefore, the signaling events that are initiated will depend on the concentrations and types of eicosanoids gener-ated, as well as the unique complement of receptors expressed by their target cells. For example, prostaglandin E2 (PGE2) suppresses the effector function of macrophages (i.e., phago-cytosis and intracellular pathogen killing) via a mechanism that is dependent on increased cAMP levels. PGE2 also modulates chemokine production and enhances local accumulation of reg-ulatory T cells and myeloid-derived suppressor cells. Prostacy-clin (PGI2) has an inhibitory effect on Th1 and Th2-mediated immune responses, while enhancing Th17 differentiation

1	accumulation of reg-ulatory T cells and myeloid-derived suppressor cells. Prostacy-clin (PGI2) has an inhibitory effect on Th1 and Th2-mediated immune responses, while enhancing Th17 differentiation and cytokine production. Leukotrienes are potent mediators of capil-lary leakage as well as leukocyte adherence, neutrophil activa-tion, bronchoconstriction, and vasoconstriction. Leukotriene B4 is synthesized from arachidonic acid in response to acute Ca2+ signaling induced by inflammatory mediators.162 High affinity leukotriene receptors (BLT1) are expressed primarily in leu-kocytes, including granulocytes, eosinophils, macrophages, and differentiated T cells, whereas the low affinity receptor is expressed in many cell types. Leukotrienes, most notably leu-kotriene B4 (LTB4), has been implicated in the development of both acute lung injury and acute kidney injury following hemor-rhagic shock in animal models.163,164Brunicardi_Ch02_p0027-p0082.indd 4701/03/19 6:49 PM 48BASIC

1	implicated in the development of both acute lung injury and acute kidney injury following hemor-rhagic shock in animal models.163,164Brunicardi_Ch02_p0027-p0082.indd 4701/03/19 6:49 PM 48BASIC CONSIDERATIONSPART IPhospholipidPhospholipase A2CorticosteroidsCyclooxygenaseLipoxygenaseProstaglandinsPGD2PGE2PGF2˜PGI2ThromboxaneTXA2Hydroxyeicosatetraenoic acid(HETE)LeukotrienesLTA4LTB4LTC4LTD4LTE4Hydroperoxyeicosatetraenoic acid(HPETE)Cyclic endoperoxides(PGG2,PGH2)Arachidonic acidAFree eicosapentaenoic acidCyclooxygenaseLipoxygenase3-seriesprostaglandins5-seriesleukotrienesPGG3LTA5LTC5LTB5PGH35-HPEPEAnti-inflammatory andinflammation resolvingE-seriesresolvinsBFigure 2-6. Schematic diagram of (A) arachidonic acid and (B) eicosapentaenoic acid metabolism. LT = leukotriene; PG = prostaglandin; TXA2 = thromboxane A2; HPEPE = hydroperoxyeicosapentaenoic acid.Omega-3 Polyunsaturated Fat Metabolites: All-cis-5, 8, 11, 14, 17-Eicosapentaenoic Acid. The second major family of PUFAs is the

1	TXA2 = thromboxane A2; HPEPE = hydroperoxyeicosapentaenoic acid.Omega-3 Polyunsaturated Fat Metabolites: All-cis-5, 8, 11, 14, 17-Eicosapentaenoic Acid. The second major family of PUFAs is the omega-3 fatty acid, α-linolenic acid, which is found primarily in cold water fish. α-Linolenic acid is the metabolic precursor of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Omega-3 PUFAs are also sub-strates for the cyclooxygenase and lipoxygenase enzymes that produce eicosanoids, but the mediators produced have a differ-ent structure from the AA-derived mediators, and this influences their actions (Fig. 2-6B). For example, omega-3 fatty acids are reported to have specific anti-inflammatory effects, including inhibition of NF-κB activity, TNF release from hepatic Kupffer cells, and leukocyte adhesion and migration. Key derivatives of omega-3 PUFAs have also been identified and synthesized. These include resolvins, protectins, and maresins. In a vari-ety of model systems,

1	and leukocyte adhesion and migration. Key derivatives of omega-3 PUFAs have also been identified and synthesized. These include resolvins, protectins, and maresins. In a vari-ety of model systems, resolvins have been shown to attenuate the inflammatory phenotypes of a number of immune cells by decreasing neutrophil recruitment, reducing synthesis of pro-inflammatory cytokines and regulating transcription factor activation.165,166The ratio of dietary omega-6 to omega-3 PUFA is reflected in the membrane composition of various cells, including cells of the immune system, which has potential implications for the inflammatory response. For example, a diet that is rich in omega-6 PUFA will result in cells whose membranes are “omega-6 PUFA rich.” When omega-6 PUFAs are the main plasma membrane lipid available for phospho-lipase activity, more proinflammatory PUFAs (i.e., 2-series prostaglandins) are generated. Many lipid preparations are soy-based and thus primarily composed of omega-6 fatty

1	available for phospho-lipase activity, more proinflammatory PUFAs (i.e., 2-series prostaglandins) are generated. Many lipid preparations are soy-based and thus primarily composed of omega-6 fatty acids. These are thought to be “inflammation-enhancing.” Nutritional supplementation with omega-3 fatty acid has the potential to dampen inflammation by shifting the cell mem-brane composition in factor of omega 3-PUFAs. In a study of surgical patients, preoperative supplementation with omega-3 fatty acid was associated with reduced need for mechanical ventilation, decreased hospital length of stay, and decreased mortality with a good safety profile.167Plasma Contact SystemComplement. Following traumatic injury, there is almost immediate activation of the complement system, which is a major effector mechanism of the innate immune system. The complement system was thought to act initially as the required “first line of defense” for the host against pathogens, by bind-ing and clearing them from

1	mechanism of the innate immune system. The complement system was thought to act initially as the required “first line of defense” for the host against pathogens, by bind-ing and clearing them from the circulation. Recent data indicate Brunicardi_Ch02_p0027-p0082.indd 4801/03/19 6:49 PM 49SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2depicted as a linear process in which parallel pathways are activated, it actually functions more like a central node that is tightly networked with other systems. Then, depending on the activating signal, several initiation and regulatory events act in concert to heighten immune surveillance.Complement activation proceeds via distinct pathways. Pathway initiation occurs by the binding and activation of a specific recognition unit to its designated ligand. The classical pathway, which is often referred to as “antibody-dependent” is initiated by direct binding of C1q to its common ligands, which include IgM/IgG aggregates. Alternately, C1q

1	ligand. The classical pathway, which is often referred to as “antibody-dependent” is initiated by direct binding of C1q to its common ligands, which include IgM/IgG aggregates. Alternately, C1q can activate complement by binding to soluble pattern recognition molecules such as pentraxins (e.g., C-reactive protein [CRP]). In a series of subsequent activation and amplification steps, the pathway ulti-mately leads to the generation of C3a and C5a, which are potent anaphylotoxins, in addition to C3b, which acts as an opsonin. An additional product, C5b, initiates the formation of the mem-brane attack complex, which becomes inserted into cell mem-brane to form a lytic pore. The subsequent effect of complement signaling is neutrophil activation leading to ROS production, as well as protease and vasoactive mediator secretion.168 The complement cascade also results in the release of proinflamma-tory cytokines synergistically with TLR-signaling, which con-tributes to systemic inflammation and

1	vasoactive mediator secretion.168 The complement cascade also results in the release of proinflamma-tory cytokines synergistically with TLR-signaling, which con-tributes to systemic inflammation and generalized capillary leak following severe injury.An additional means of complement activation via the lectin pathway is initiated by mannose-binding lectins (MBL) or ficolins, which bind specific carbohydrate structures. This pathway has been speculated to be a major mechanism for complement activation post injury by interactions with matrix fragments and mitochondrial DAMPs. In addition, both low pH and serine proteases of the coagulation cascade may contribute to complement activation. As a consequence, high levels of acti-vated complement components may help to continue to drive systemic inflammation post injury.169Kallikrein-Kinin System. The kallikrein-kinin system, also referred to as the “contact” system, is a group of proteins that contribute to both coagulation and inflammation.

1	post injury.169Kallikrein-Kinin System. The kallikrein-kinin system, also referred to as the “contact” system, is a group of proteins that contribute to both coagulation and inflammation. Prekalli-krein circulates in the plasma bound to high molecular weight kininogen (HK). A variety of stimuli lead to the binding of prekallikrein-HK complex to Hageman factor (factor XII) to initiate the intrinsic clotting cascade. This results in formation of the serine protease kallikrein, which is both proinflamma-tory and procoagulant. HK is cleaved by kallikrein to form bradykinin (BK).The kinins (e.g., BK) mediate several physiologic pro-cesses, including vasodilation, increased capillary perme-ability, tissue edema, and neutrophil chemotaxis.170 They also increase renal vasodilation and consequently reduce renal per-fusion pressure. Kinin receptors are members of the rhodopsin family of G-protein-coupled receptors and are located on vas-cular endothelium and smooth muscle cells. Kinin receptors

1	reduce renal per-fusion pressure. Kinin receptors are members of the rhodopsin family of G-protein-coupled receptors and are located on vas-cular endothelium and smooth muscle cells. Kinin receptors are rapidly upregulated following TLR4 and cytokine signal-ing and appear to have important effects on both immune cell behavior and on immune mediators.171 For example, activation of the kinin receptor, B1, results in increased neutrophil che-motaxis, while increased B2 receptor expression causes activa-tion of arachidonic-prostaglandin pathways. Bradykinin and kallikrein levels are increased following hemorrhagic shock and tissue injury. The degree of elevation in the levels of these mediators has been associated with the magnitude of injury and mortality.Table 2-6Systemic stimulatory and inhibitory actions of eicosanoidsORGAN/FUNCTIONSTIMULATORINHIBITORPancreas Glucose-stimulated insulin secretion Glucagon secretion12-HPETEPGD2, PGE2PGE2Liver Glucagon-stimulated glucose

1	and inhibitory actions of eicosanoidsORGAN/FUNCTIONSTIMULATORINHIBITORPancreas Glucose-stimulated insulin secretion Glucagon secretion12-HPETEPGD2, PGE2PGE2Liver Glucagon-stimulated glucose production PGE2Fat Hormone-stimulated lipolysis PGE2Bone ResorptionPGE2, PGE-m, 6-K-PGE1, PGF1α, PGI2 Pituitary Prolactin Luteinizing hormone Thyroid-stimulating hormone Growth hormonePGE1PGE1, PGE2, 5-HETEPGA1, PGB1, PGE1, PGE1PGE1 Parathyroid Parathyroid hormonePGE2PGF2Lung BronchoconstrictionPGF2α TXA2, LTC4, LTD4, LTE4PGE2Kidney Stimulation of renin secretionPGE2, PGI2 Gastrointestinal system Cytoprotective effectPGE2 Immune response Suppression of lymphocyte activityPGE2 Hematologic system Platelet aggregationTXA2PGI25-HETE = 5-hydroxyeicosatetraenoic acid; 12-HPETE = 12-hydroxyperoxyeicosatetraenoic acid; 6-K-PGE1 = 6-keto-prostaglandin E1; LT = leukotriene; PG = prostaglandin; PGE-m = 13,14-dihydro-15-keto-PGE2 (major urine metabolite of PGE2); TXA2 = thromboxane A2.that

1	acid; 6-K-PGE1 = 6-keto-prostaglandin E1; LT = leukotriene; PG = prostaglandin; PGE-m = 13,14-dihydro-15-keto-PGE2 (major urine metabolite of PGE2); TXA2 = thromboxane A2.that complement is also participates in the elimination of immune complexes as well as damaged and dead cells. In addi-tion, complement is recognized as contributing to mobiliza-tion of hematopoietic stem/progenitor cells (HSPC) and lipid metabolism.168 Although complement activation is typically Brunicardi_Ch02_p0027-p0082.indd 4901/03/19 6:49 PM 50BASIC CONSIDERATIONSPART ISerotoninSerotonin is a monoamine neurotransmitter (5-hydroxytrypta-mine; 5-HT) derived from tryptophan. Serotonin is synthesized by neurons in the CNS as well as by intestinal enterochromaf-fin cells, which are the major source of plasma 5-HT. Once in the plasma, 5-HT is taken up rapidly into platelets via the serotonin transporter (SERT), where it is either stored in the dense granules in millimolar concentrations or targeted for

1	5-HT. Once in the plasma, 5-HT is taken up rapidly into platelets via the serotonin transporter (SERT), where it is either stored in the dense granules in millimolar concentrations or targeted for deg-radation. It is interesting that the surface expression of SERT on platelets is sensitive to plasma 5-HT levels, which in turn modulates platelet 5-HT content. Receptors for serotonin are widely distributed in the periphery and are found in the GI tract, cardiovascular system, and some immune cells.172 Serotonin is a potent vasoconstrictor and also modulates cardiac inotropy and chronotropy through nonadrenergic cyclic adenosine mono-phosphate (cAMP) pathways. Serotonin is released at sites of injury, primarily by platelets. Recent work has demonstrated an important role for platelet 5-HT in the local inflammatory response to injury. Using mice that lack the nonneuronal iso-form of tryptophan hydroxylase (Tph1), the rate-limiting step for 5-HT synthesis in the periphery, investigators

1	in the local inflammatory response to injury. Using mice that lack the nonneuronal iso-form of tryptophan hydroxylase (Tph1), the rate-limiting step for 5-HT synthesis in the periphery, investigators demonstrated fewer neutrophils rolling on mesenteric venules.173 Tph1-/mice, in response to an inflammatory stimulus, also showed decreased neutrophil extravasation. Together, these data indicate an impor-tant role for nonneuronal 5-HT in neutrophil recruitment to sites of inflammation and injury.HistamineHistamine is a short-acting endogenous amine that is widely distributed throughout the body. It is synthesized by histidine decarboxylase (HDC), which decarboxylates the amino acid his-tidine. Histamine is either rapidly released or stored in neurons, skin, gastric mucosa, mast cells, basophils, and platelets and plasma levels are increased with hemorrhagic shock, trauma, thermal injury, and sepsis.174 Not surprisingly, circulating cyto-kines can increase immune cell expression of HDC to

1	and platelets and plasma levels are increased with hemorrhagic shock, trauma, thermal injury, and sepsis.174 Not surprisingly, circulating cyto-kines can increase immune cell expression of HDC to further contribute to histamine synthesis. There are four histamine receptor (HR) subtypes with varying physiologic roles, but they are all members of the rhodopsin family of G-protein coupled receptors. H1R binding mediates vasodilation, bronchocon-striction, intestinal motility, and myocardial contractility. H1R knockout mice demonstrate significant immunologic defects, including impaired B and T cell responses.H2R binding is best described for its stimulation of gastric parietal cell acid secretion. However, H2R can also modulate a range of immune system activities, such as mast cell degranula-tion, antibody synthesis, Th1 cytokine production, and T-cell proliferation. H3R was initially classified as a presynaptic auto-receptor in the peripheral and central nervous system (CNS). However,

1	antibody synthesis, Th1 cytokine production, and T-cell proliferation. H3R was initially classified as a presynaptic auto-receptor in the peripheral and central nervous system (CNS). However, data using H3R knockout mice demonstrates that it also participates in inflammation in the CNS. H3R knockout mice display increased severity of neuroinflammatory diseases, which correlates with dysregulation of blood-brain barrier per-meability and increased expression of macrophage inflammatory protein 2, IFN-inducible protein 10, and CXCR3 by peripheral T cells. H4R is expressed primarily in bone marrow, but it has also been detected in leukocytes, including neutrophils, eosino-phils, mast cells, dendritic cells, T cells, and basophils. H4R is emerging as an important modulator of chemoattraction and cytokine production in these cells. Thus, it is clear that cells of both the innate and adaptive immune response can be regulated by histamine, which is up-regulated following injury.175CELLULAR

1	and cytokine production in these cells. Thus, it is clear that cells of both the innate and adaptive immune response can be regulated by histamine, which is up-regulated following injury.175CELLULAR RESPONSE TO INJURYCytokine Receptor Families and Their Signaling PathwaysCytokines act on their target cells by binding to specific membrane receptors. These receptor families have been organized by struc-tural motifs and include type 1 cytokine receptors, type II cytokine receptors, chemokine receptors, tumor necrosis factor receptors (TNFR), and transforming growth factor receptors (TGFR). In addition, there are cytokine receptors that belong to the immuno-globulin receptor superfamilies. Several of these receptors have characteristic signaling pathways that are associated with them. These will be briefly reviewed in the following section.JAK-STAT SignalingA major subgroup of cytokines, comprising roughly 60 factors, bind to receptors termed type I/II cytokine receptors. Cytokines that

1	will be briefly reviewed in the following section.JAK-STAT SignalingA major subgroup of cytokines, comprising roughly 60 factors, bind to receptors termed type I/II cytokine receptors. Cytokines that bind these receptors include type I IFNs, IFN-γ, many inter-leukins (e.g., IL-6, IL-10, IL-12, and IL-13), and hematopoietic growth factors. These cytokines play essential rolls in the initia-tion, maintenance, and modulation of innate and adaptive immu-nity for host defense. All type I/II cytokine receptors selectively associate with the Janus kinases (JAK1, JAK2, JAK3, TYK2), which represent a family of tyrosine kinases that mediate the signal transduction for these receptors. As such, the JAK-STAT signaling pathway is considered a central communication hub for the immune system.176JAKs are constitutively bound to the cytokine receptors, and on ligand binding and receptor dimerization, activated JAKs phosphorylate the receptor to recruit signal transducer and acti-vator of transcription

1	constitutively bound to the cytokine receptors, and on ligand binding and receptor dimerization, activated JAKs phosphorylate the receptor to recruit signal transducer and acti-vator of transcription (STAT) molecules (Fig. 2-7). Activated STAT proteins further dimerize and translocate into the nucleus where they modulate the transcription of target genes. Rather than being a strictly linear pathway, it is likely that individual cytokines activate more than one JAK-STAT combination. The molecular implications for this in terms of cytokine signaling are still being unraveled, but the development of JAK-specific inhibitors (jakinibs) is moving the field forward quickly.177 Inter-estingly, STAT-DNA binding can be observed within minutes of cytokine binding. STATs have also been shown to modulate gene transcription via epigenetic mechanisms. Thus, JAKs and STATs are central players in the regulation of key immune cell function, by providing a signaling platform for proinflamma-tory

1	modulate gene transcription via epigenetic mechanisms. Thus, JAKs and STATs are central players in the regulation of key immune cell function, by providing a signaling platform for proinflamma-tory cytokines (IL-6 via JAK1 and STAT3); anti-inflammatory cytokines (IL-10 via STAT3) and integrating signals required for helper and regulatory T cell development and differentiation. The JAK/STAT pathway is inhibited by the action of phospha-tase, the export of STATs from the nucleus, as well the interac-tion of antagonistic proteins.178 JAK/STAT signaling has also been implicated in the secondary muscle wasting that occurs with chronic, persistent inflammation.179Suppressors of Cytokine SignalingSuppressor of cytokine signaling (SOCS) molecules are a fam-ily of proteins that function as a negative feedback loop for types I and II cytokine receptors by terminating JAK/STAT signaling. There are currently eight family members (SOCS1-7 and CIS [cytokine-inducible SH2-containing protein]) that

1	feedback loop for types I and II cytokine receptors by terminating JAK/STAT signaling. There are currently eight family members (SOCS1-7 and CIS [cytokine-inducible SH2-containing protein]) that are associated with cytokine receptor signaling. Pattern recognition receptors, including both TLR and C-type lectin receptors, also activate SOCS.180 Interestingly, induction of SOCS proteins is also achieved through activators of JAK/STAT signaling, creating an inhibitory feedback loop through which cytokines Brunicardi_Ch02_p0027-p0082.indd 5001/03/19 6:49 PM 51SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2JAKJAKPPJAKJAKPSTATSTATPPPSTATPSTATSTATSTATSOCSPSTATNuclear translocationNucleusReceptordimerizationFigure 2-7. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway also requires dimer-ization of monomeric units. STAT molecules possess “docking” sites that allow for STAT dimerization. The STAT complexes translocate into the nucleus

1	(JAK/STAT) signaling pathway also requires dimer-ization of monomeric units. STAT molecules possess “docking” sites that allow for STAT dimerization. The STAT complexes translocate into the nucleus and serve as gene transcription fac-tors. JAK/STAT activation occurs in response to cytokines (e.g., interleukin-6) and cell stressors, and has been found to induce cell proliferation and inflammatory function. Intracellular molecules that inhibit STAT function, known as suppressors of cytokine sig-naling (SOCSs), have been identified. P = phosphate.can effectively self-regulate by extinguishing their own signal. SOCS molecules can positively and negatively influence the activation of macrophages and dendritic cells and are crucial for T-cell development and differentiation.181 All SOCS proteins are able to regulate receptor signaling through the recruitment of proteasomal degradation components to their target proteins, whether the target is a specific receptor or an associated adaptor

1	are able to regulate receptor signaling through the recruitment of proteasomal degradation components to their target proteins, whether the target is a specific receptor or an associated adaptor molecule. Once associated with the SOCS complex, target pro-teins are readily ubiquinated and targeted for proteasomal deg-radation. SOCS1 and SOCS3 can also exert an inhibitory effect on JAK-STAT signaling via their N-terminal kinase inhibitory region (KIR) domain, which acts as a pseudo-substrate for JAK.SOCS3 has been shown to be a positive regulator of TLR4 responses in macrophages via inhibition of IL-6 receptor-medi-ated STAT3 activation.181,182 A deficiency of SOCS activity may render a cell hypersensitive to certain stimuli, such as inflamma-tory cytokines and growth hormones. Interestingly, in a murine model, SOCS knockout resulted in a lethal phenotype in part because of unregulated interferon signaling.Chemokine Receptors Are Members of the G-ProteinCoupled Receptor Family All

1	in a murine model, SOCS knockout resulted in a lethal phenotype in part because of unregulated interferon signaling.Chemokine Receptors Are Members of the G-ProteinCoupled Receptor Family All chemokine receptors are mem-bers of the G-protein-coupled seven transmembrane family of receptors (GPCR), which is one of the largest and most diverse of the membrane protein families. GPCRs function by detect-ing a wide spectrum of extracellular signals, including photons, ions, small organic molecules, and entire proteins. After ligand binding, GPCRs undergo conformational changes, causing the recruitment of heterotrimeric G proteins to the cytoplasmic sur-face (Fig. 2-8). Heterotrimeric G proteins are composed of three subunits, Gα, Gβ, and Gγ, each of which have numerous mem-bers, adding to the complexity of the signaling. When signaling, however, G proteins perform functionally as dimers because the signal is communicated either by the Gα subunit or the Gβγ complex. The GPCR family includes

1	of the signaling. When signaling, however, G proteins perform functionally as dimers because the signal is communicated either by the Gα subunit or the Gβγ complex. The GPCR family includes the receptors for catechol-amines, bradykinins, and leukotrienes, in addition to a variety of other ligands important to the inflammatory response.183 In gen-eral, GPCRs can be classified according to their pharmacological properties into four main families: class A rhodopsin-like, class B secretin-like, class C metabotropic glutamate/pheromone, and frizzled receptors. As noted above, GPCR activation by ligand binding results in an extracellular domain shift, which is then transmitted to cytoplasmic portion of the receptor to facilitate coupling to its principle effector molecules, the heterotrimeric G proteins. Although there are more than 20 known Gα subunits, they have been divided into four families based on sequence similarity, which has served to define both receptor and effec-tor coupling.

1	G proteins. Although there are more than 20 known Gα subunits, they have been divided into four families based on sequence similarity, which has served to define both receptor and effec-tor coupling. These include Gαs and Gαi, which signal through the activation (Gαs) or inhibition (Gαi) of adenylate cyclase to increase or decrease cyclic adenosine monophosphate (cAMP) levels, respectively. Increased intracellular cAMP can activate gene transcription through the activity of intracellular signal transducers such as protein kinase A. The Ga subunits also include the Gq pathway, which stimulates phospholipase C-β to produce the intracellular messengers inositol triphosphate and diacylglycerol. Inositol triphosphate triggers the release of calcium from intracellular stores, while diacylglycerol recruits protein kinase C to the plasma membrane for activation. Finally, Gα12/13 appears to act through Rho and Ras-mediated signaling.Tumor Necrosis Factor SuperfamilySignaling pathway for tumor

1	recruits protein kinase C to the plasma membrane for activation. Finally, Gα12/13 appears to act through Rho and Ras-mediated signaling.Tumor Necrosis Factor SuperfamilySignaling pathway for tumor necrosis factor receptor 1 (TNFR-1) (55 kDa) and TNFR-2 (75 kDa) occurs by the recruitment of sev-eral adapter proteins to the intracellular receptor complex. Opti-mal signaling activity requires receptor trimerization. TNFR-1 initially recruits TNFR-associated death domain (TRADD) and induces apoptosis through the actions of proteolytic enzymes known as caspases, a pathway shared by another receptor, CD95 (Fas). CD95 and TNFR-1 possess similar intracellular sequences known as death domains (DDs), and both recruit the same adapter proteins (Fas-associated death domains [FADDs]) before activating caspase 8. TNFR-1 also induces apoptosis by activating caspase 2 through the recruitment of receptor-inter-acting protein (RIP). RIP also has a functional component that can initiate nuclear factor

1	caspase 8. TNFR-1 also induces apoptosis by activating caspase 2 through the recruitment of receptor-inter-acting protein (RIP). RIP also has a functional component that can initiate nuclear factor kB (NF-kB) and c-Jun activation, both favoring cell survival and proinflammatory functions. TNFR-2 lacks a DD component but recruits adapter proteins known as TNFR-associated factors 1 and 2 (TRAF1, TRAF2) that inter-act with RIP to mediate NF-kB and c-Jun activation. TRAF2 also recruits additional proteins that are antiapoptotic, known as inhibitors of apoptosis proteins (IAPs).Transforming Growth Factor-a Family of ReceptorsTransforming growth factor-β1 (TGF-β1) is a pleiotropic cyto-kine expressed by immune cells that has potent immunoregula-tory activities. Specifically, recent data indicate that TGF-β is Brunicardi_Ch02_p0027-p0082.indd 5101/03/19 6:49 PM 52BASIC CONSIDERATIONSPART Iessential for T cell homeostasis, as mice deficient in TGF-β1 develop a multiorgan autoimmune

1	that TGF-β is Brunicardi_Ch02_p0027-p0082.indd 5101/03/19 6:49 PM 52BASIC CONSIDERATIONSPART Iessential for T cell homeostasis, as mice deficient in TGF-β1 develop a multiorgan autoimmune inflammatory disease and die a few weeks after birth, an effect that is dependent upon the presence of mature T cells. The receptors for TGF-β ligands are the TGF-β superfamily of receptors, which are type I transmem-brane proteins that contain intrinsic serine/threonine kinase activ-ity. These receptors comprise two subfamilies, the type I and the type II receptors that are distinguished by the presence of a glycine/serine-rich membrane domain found in the type I receptors. Each TGF-β ligand binds a characteristic combination of type I and type II receptors, both of which are required for signaling. Whether the type I or the type II receptor binds first is ligand-dependent, and the second type I or type II receptor is then recruited to form a het-eromeric signaling complex. When TGF-β binds to

1	Whether the type I or the type II receptor binds first is ligand-dependent, and the second type I or type II receptor is then recruited to form a het-eromeric signaling complex. When TGF-β binds to the TGF-βR, heterodimerization activates the receptor which then directly recruits and activates a receptor-associated Smad (Smad 2 or 3) through phosphorylation. An additional “common” Smad is then recruited. The activated Smad-complex translocates into the nucleus and, with other nuclear cofactors, regulates the transcrip-tion of target genes. TGF-β can also induce the rapid activation of the Ras-extracellular signal-regulated kinase (ERK) signaling pathway in addition to other MAPK pathways (JNK, p38MAPK). How does TGF-β inhibit immune responses? One of the most important effects is the suppression of interleukin-2 production by T cells. It also inhibits T cell proliferation.184 More recently, it was noted that TGF-β can regulate the maturation of differenti-ated dendritic cells and

1	suppression of interleukin-2 production by T cells. It also inhibits T cell proliferation.184 More recently, it was noted that TGF-β can regulate the maturation of differenti-ated dendritic cells and dendritic cell-mediated T-cell responses. Importantly, TGF-β can induce “alternative activation” macro-phages, designated M2 macrophages, which express a wide array of anti-inflammatory molecules, including IL-10 and arginase1.TRANSCRIPTIONAL AND TRANSLATIONAL REGULATION OF THE INJURY RESPONSETranscriptional Events Following Blunt TraumaInvestigators have examined the transcriptional response in cir-culating leukocytes in a large series of patients who suffered severe blunt trauma. This work identified an overwhelming 5GRELigandProtein kinase CactivationCA2+ releaseRGESecond messengers(cAMP, IP3)LigandERCell membraneCytoplasmG-protein receptors(vasoactive polypeptides, mitogens, phospholipids, neurotransmitters, prostaglandins)Figure 2-8. G-protein–coupled receptors are transmembrane

1	IP3)LigandERCell membraneCytoplasmG-protein receptors(vasoactive polypeptides, mitogens, phospholipids, neurotransmitters, prostaglandins)Figure 2-8. G-protein–coupled receptors are transmembrane pro-teins. The G-protein receptors respond to ligands such as adrenaline and serotonin. On ligand binding to the receptor (R), the G protein (G) undergoes a conformational change through guanosine triphosphate–guanosine diphosphate conversion and in turn activates the effector (E) component. The E component subse-quently activates second messengers. The role of inositol triphosphate (IP3) is to induce release of calcium from the endoplasmic reticulum (ER). cAMP = cyclic adenosine triphosphate.shift in the leukocyte transcriptome, with more than 80% of the cellular functions and pathways demonstrating some altera-tion in gene expression. In particular, changes in gene expres-sion for pathways involved in the systemic inflammatory, innate immune, compensatory anti-inflammatory, and adaptive

1	some altera-tion in gene expression. In particular, changes in gene expres-sion for pathways involved in the systemic inflammatory, innate immune, compensatory anti-inflammatory, and adaptive immune responses were simultaneous and marked. Moreover, they occurred rapidly (within 4–12 hours), and were prolonged for days and weeks. When different injuries (i.e., blunt trauma, burn injury, human model of endotoxemia) were compared, the patterns of gene expression were surprisingly similar, sug-gesting that the stress response to both injury and inflammation is highly conserved and may follow a universal pathway that includes common denominators. Finally, delayed clinical recov-ery and organ injury were not associated with a distinct pattern of transcriptional response elements.3 These data describe a new paradigm based on the observation of a rapid and coordinated transcriptional response to severe traumatic injury that involves both the innate and adaptive immune systems. Further, the

1	describe a new paradigm based on the observation of a rapid and coordinated transcriptional response to severe traumatic injury that involves both the innate and adaptive immune systems. Further, the data support the idea that individuals who are destined to die from their injuries are characterized primarily by the degree and dura-tion of their dysregulated inflammatory response rather than a “unique signature” indicative of a “second hit.”Transcriptional Regulation of Gene ExpressionMany genes are regulated at the point of DNA transcription and thus influence whether messenger RNA (mRNA) and its subse-quent product are expressed (Fig. 2-9). Gene expression relies on the coordinated action of transcription factors and coactiva-tors (i.e., regulatory proteins), which are complexes that bind to highly specific DNA sequences upstream of the target gene known as the promoter region. Enhancer sequences of DNA mediate gene expression, whereas repressor sequences are non-coding regions that

1	to highly specific DNA sequences upstream of the target gene known as the promoter region. Enhancer sequences of DNA mediate gene expression, whereas repressor sequences are non-coding regions that bind proteins to inhibit gene expression. For example, nuclear factor κB (NF-κB), one of the best-described transcription factors, has a central role in regulating the gene products expressed after inflammatory stimuli (Fig. 2-10). The NF-κB family of transcription factors is composed of five mem-bers that share a common domain. They form numerous homo or heterodimers that are normally retained in the cytosol through Brunicardi_Ch02_p0027-p0082.indd 5201/03/19 6:49 PM 53SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2TranscriptionDNAmRNAmRNAProteinInactivemRNAInactiveproteinCytoplasmCell membraneNucleusFigure 2-9. Gene expression and protein synthesis can occur within a 24-hour period. The process can be regulated at various stages: transcription, messenger RNA (mRNA)

1	membraneNucleusFigure 2-9. Gene expression and protein synthesis can occur within a 24-hour period. The process can be regulated at various stages: transcription, messenger RNA (mRNA) processing, or pro-tein packaging. At each stage, it is possible to inactivate the mRNA or protein, rendering these molecules nonfunctional.Ligand(e.g.: TNF, IL-1)PI-˜B kinaseDegradation of I-˜BUbiquitinizationI-˜Bp65p50I-˜Bp65p50I-˜Bp65p50Pp65p50Nuclear translocationNucleusNF-˜B activationFigure 2-10. Inhibitor of κB (I-κB) binding to the p50-p65 subunits of nuclear factor κB (NF-κB) inactivates the molecule. Ligand binding to the receptor activates a series of downstream signaling molecules, of which I-κB kinase is one. The phosphorylated NF-κB complex further undergoes ubiquitinization and proteosome degradation of I-κB, activating NF-κB, which translocates into the nucleus. Rapid resynthesis of I-κB is one method of inactivating the p50-p65 complex. IL-1 = interleukin-1; P = phosphate; TNF =

1	degradation of I-κB, activating NF-κB, which translocates into the nucleus. Rapid resynthesis of I-κB is one method of inactivating the p50-p65 complex. IL-1 = interleukin-1; P = phosphate; TNF = tumor necrosis factor.the inhibitory binding of inhibitor of κB (I-κB). In response to an inflammatory stimulus (e.g., TNF, IL-1, or DAMP) a sequence of intracellular mediator phosphorylation reactions leads to the degradation of I-κB and subsequent release of NF-κB to allow nuclear translocation and the initiation of transcription.Epigenetic Regulation of TranscriptionThe DNA access of protein machineries involved in transcription processes is tightly regulated by histones, which are a family of basic proteins that associate with DNA in the nucleus. Histone proteins help to condense the DNA into tightly packed nucleo-somes that limit transcription. Emerging evidence indicates that transcriptional activation of many proinflammatory genes requires nucleosome remodeling, a process that is

1	into tightly packed nucleo-somes that limit transcription. Emerging evidence indicates that transcriptional activation of many proinflammatory genes requires nucleosome remodeling, a process that is regulated by the histone modifying enzymes.185 There are at least seven identified chromatin modifications, including acetylation, meth-ylation, phosphorylation, ubiquitinylation, sumoylation, ADP ribosylation, deimination, and proline isomerization. Alteration of chromatin packing in this way makes the DNA more or less accessible for transcription. Recently, the development of chro-matin immunoprecipitation (ChIP) coupled to massively parallel DNA sequencing technology (ChIP-Seq) has enabled the map-ping of histone modifications in living cells. In this way, it has allowed the identification of the large number of posttranslational histone modifications that are “written” and “erased” by histone-modifying enzymes. The role of histone modifications in the reg-ulation of gene expression is

1	of the large number of posttranslational histone modifications that are “written” and “erased” by histone-modifying enzymes. The role of histone modifications in the reg-ulation of gene expression is referred to as “epigenetic” control.The addition of an acetyl group to lysine residues on his-tones is an epigenetic mark associated with gene activation. These acetyl groups are reversibly maintained by histone acet-yltransferases (HATs) and histone deacetylases (HDACs).186 Hemorrhagic shock has been shown to alter the acetylation of histone proteins via an imbalance of HDAC/HAT activity in the heart, lung, and liver in a tissue-specific pattern. In animal mod-els, postshock administration of the HDAC inhibitor valproic acid improved overall survival.187 Valproic acid is currently in Phase 2 clinical trials for the treatment of hemorrhaghic shock.188Translation Regulation of Inflammatory Gene ExpressionOnce mRNA transcripts are generated, they can also be regu-lated by a variety of

1	2 clinical trials for the treatment of hemorrhaghic shock.188Translation Regulation of Inflammatory Gene ExpressionOnce mRNA transcripts are generated, they can also be regu-lated by a variety of mechanisms, including (a) splicing, which can cleave mRNA and remove noncoding regions; (b) capping, which modifies the 5’ ends of the mRNA sequence to inhibit breakdown by exonucleases; (c) and the addition of a polyad-enylated tail, which adds a noncoding sequence to the mRNA, to regulated the half-life of the transcript. Recent data has identified microRNAs (miRNAs) as important translational regulators of gene expression via their binding to partially complementary sequences in the 3’-untranslated region (3’-UTR) of target mRNA transcripts.189 Binding of miRNA to the Brunicardi_Ch02_p0027-p0082.indd 5301/03/19 6:49 PM 54BASIC CONSIDERATIONSPART ImRNA usually results in gene silencing. MicroRNAs are endog-enous, single-stranded RNAs of approximately 22 nucleotides in length that are

1	5301/03/19 6:49 PM 54BASIC CONSIDERATIONSPART ImRNA usually results in gene silencing. MicroRNAs are endog-enous, single-stranded RNAs of approximately 22 nucleotides in length that are highly conserved in eukaryotes. MicroRNAs are encoded either singly or can be transcribed in a “polycis-tronic” clusters and produced by an elaborate expression and processing mechanism. After a primary miRNA transcript is generated by RNA polymerase II or III, it is processed in the nucleus to produce a short hairpin precursor miRNA transcript. The precursor is then transported into the cytoplasm where the final mature miRNA is generated by a protein termed Dicer. The mature double-stranded miRNA is then incorporated into the RNA-induced silencing complex (RISC) in the cytoplasm. Once programmed with a small RNA, RISC can silence targeted genes by one of several distinct mechanisms, working at (a) the level of protein synthesis through translation inhibition, (b) the transcript level through mRNA

1	a small RNA, RISC can silence targeted genes by one of several distinct mechanisms, working at (a) the level of protein synthesis through translation inhibition, (b) the transcript level through mRNA degradation, or (c) the level of the genome itself through the formation of heterochromatin or by DNA elimination. MiRNAs are involved in TLR signaling in the innate immune system by targeting multiple molecules in the TLR signaling pathways.190 Traumatic brain injury alters serum miRNA profiles that may be useful both as biomarkers for severe TBI and as therapeutic targets.189CELL-MEDIATED INFLAMMATORY RESPONSENeutrophilsNeutrophils (PMNs) are among the first responders to sites of infection and injury and as such are potent mediators of acute inflammation.191 Mobilization of PMNs from the bone mar-row is facilitated by reduction in bone-marrow expression of stromal cell-derived factor-1 (SDF1, also CXCL12) and subse-quent expression of both SDF1 and its receptor CXCR4 in target

1	the bone mar-row is facilitated by reduction in bone-marrow expression of stromal cell-derived factor-1 (SDF1, also CXCL12) and subse-quent expression of both SDF1 and its receptor CXCR4 in target tissues.192 This and other chemotactic mediators induce PMN adherence to the vascular endothelium and promote eventual cell migration into the injured tissue. Early signals for PMN recruitment include endogenous “self” molecules released from damaged tissues, like the DAMPs described previously and also include histone proteins as well as adenosine triphos-phate. DAMP molecules can also induce secretion of powerful chemokines such as IL-8 (CXCL8), which can bind to tissue glycosaminoglycans, creating a gradient for PMN migration. PMNs generally have short half-lives (4 to 10 hours). However, inflammatory signals may promote their longevity in target tis-sues, which can contribute to their potential detrimental effects and subsequent bystander injury. In addition, following sterile trauma,

1	signals may promote their longevity in target tis-sues, which can contribute to their potential detrimental effects and subsequent bystander injury. In addition, following sterile trauma, large numbers of immature PMNs are recruited from the bone marrow into the circulation.193Once primed and activated by inflammatory stimuli, includ-ing TNF, IL-1, and microbial pathogens, PMNs are capable of amplifying the inflammatory response as well as releasing toxic effectors such as ROS and proteolytic enzymes into the extracel-lular space.193 Neutrophils can also dump their granule contents into the extracellular space, and many of these proteins also have important effects on the innate and adaptive immune responses. When highly activated, neutrophils can extrude a meshwork of chromatin fibers, composed of DNA and histones that are deco-rated with granule contents. Termed neutrophils extracellular traps or NETs, they were first described as effective mechanism whereby neutrophils can

1	composed of DNA and histones that are deco-rated with granule contents. Termed neutrophils extracellular traps or NETs, they were first described as effective mechanism whereby neutrophils can immobilize bacteria to facilitate their killing. In the setting of tissue injury, NETS may allow continued presentation of auto-antigens to the host immune system, which can contribute to further tissue injury.194 NETS may also serve to prime T cells, making their threshold for activation lower.Neutrophils do facilitate the recruitment of monocytes into inflamed tissues. These recruited cells are capable of phagocy-tosing apoptotic neutrophils to contribute to resolution of the inflammatory response and to promote tissue repair.195 How-ever, at least some portion of the neutrophil population from the injury site is capable of reentering the blood stream and return-ing to the bone marrow in a process regulated by chemokine CXC receptor 4.196 Whether reverse migration of neutrophils is beneficial

1	injury site is capable of reentering the blood stream and return-ing to the bone marrow in a process regulated by chemokine CXC receptor 4.196 Whether reverse migration of neutrophils is beneficial to the host or likely to cause distant organ injury needs further investigation.Monocyte/MacrophagesMonocytes and macrophages are mononuclear phagocytes that play a critical role in inflammation and the injury response.197 Monocytes are leukocytes derived from bone marrow pro-genitors that circulate in the bloodstream and given the right stimuli, exit the vasculature, and differentiate into monocyte-derived macrophages (e.g., alveolar macrophages or Kupffer cells) upon migrating into appropriate tissues. Macrophages represent the large number of phagocytes that are resident in tissues under resting conditions. Distinct from monocytes, they are derived from embryonic precursors and can repopulate their numbers either by self-renewal or from monocytes derived from the bone marrow.198

1	resting conditions. Distinct from monocytes, they are derived from embryonic precursors and can repopulate their numbers either by self-renewal or from monocytes derived from the bone marrow.198 Together, monocytes/macrophages are the main effector cells that sense and respond to “danger signals,” primarily through mechanisms that include phagocytosis of cel-lular debris, release of inflammatory mediators, and recruitment of additional immune cells to injury sites. Moreover, these cells fulfill homeostatic roles beyond host defense by performing important functions in the remodeling of tissues, both during development and in the adult animal. SDF1 has also been impli-cated in the recruitment of monocytes to sites of tissue injury.199 Importantly, SDF1 forms a complex with HMGB1, a DAMP molecule, which potently increases its chemotactic function. In conjunction with CXCR4, the SDF1-HMGB1 complex induces early monocyte migration into injured tissues, where they play an important role

1	molecule, which potently increases its chemotactic function. In conjunction with CXCR4, the SDF1-HMGB1 complex induces early monocyte migration into injured tissues, where they play an important role coordinating between innate and adaptive immunity.In tissues, mononuclear phagocytes are quiescent. How-ever, they respond to external cues (e.g., PAMPs, DAMPs, acti-vated lymphocytes) by changing their phenotype.200 In response to various signals, macrophages may undergo classical M1 acti-vation (stimulated by TLR ligands and IFN-γ) or alternative M2 activation (stimulated by type II cytokines IL-4/IL-13); these states mirror the Th1–Th2 polarization of T cells described in the following section. The M1 phenotype is characterized by the expression of high levels of proinflammatory cytokines, like TNF-α, IL-1 and IL-6, in addition to the synthesis of ROS and RNS. Activated macrophages can also secrete HMGB1 and in this way, can recruit additional macrophages to form a self-activating

1	like TNF-α, IL-1 and IL-6, in addition to the synthesis of ROS and RNS. Activated macrophages can also secrete HMGB1 and in this way, can recruit additional macrophages to form a self-activating loop.In contrast, M2 macrophages are considered to be involved in the promotion of wound repair and the restoration of immune homeostasis through their expression of arginase-1 and IL-10, in addition to a variety of PRR (e.g., scavenging molecules).201 In truth, this classification system is overly simplistic. In fact, macrophages are highly heterogeneous and possess specialized properties that are precisely adapted to individual tissues. Thus, they are likely to also possess individualized response to local tissue damage.197Brunicardi_Ch02_p0027-p0082.indd 5401/03/19 6:49 PM 55SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2TH1Injury Severity:IL-12, IL-18, IFN-, TNF, IL-1, IL-21, TGF-˜IL-4, IL-5, IL-6, IL-10,

1	5401/03/19 6:49 PM 55SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2TH1Injury Severity:IL-12, IL-18, IFN-, TNF, IL-1, IL-21, TGF-˜IL-4, IL-5, IL-6, IL-10, (Glucocorticoids)IL-2IL-3IL-6IL-12IFN-TNF-°GM-CSFTNF-˜IL-3IL-4IL-5IL-6IL-9IL-10IL-13TNF-°GM-CSFCell-mediatedImmunityAntibody-mediatedImmunityless severemore severeTH2Figure 2-11. Specific immunity mediated by helper T lympho-cytes subtype 1 (TH1) and subtype 2 (TH2) after injury. A TH1 response is favored in lesser injuries, with intact cell-mediated and opsonizing antibody immunity against microbial infections. This cell-mediated immunity includes activation of monocytes, B lym-phocytes, and cytotoxic T lymphocytes. A shift toward the TH2 response from naive helper T cells is associated with injuries of greater magnitude and is not as effective against microbial infec-tions. A TH2 response includes the activation of eosinophils, mast cells, and B-lymphocyte immunoglobulin 4 and immunoglobulin E production. (Primary

1	and is not as effective against microbial infec-tions. A TH2 response includes the activation of eosinophils, mast cells, and B-lymphocyte immunoglobulin 4 and immunoglobulin E production. (Primary stimulants and principal cytokine prod-ucts of such responses are in bold characters.) Interleukin-4 (IL-4) and IL-10 are known inhibitors of the TH1 response. Interferon-γ (IFN-γ) is a known inhibitor of the TH2 response. Although not cytokines, glucocorticoids are potent stimulants of a TH2 response, which may partly contribute to the immunosuppressive effects of cortisol. GM-CSF = granulocyte-macrophage colony-stimulating factor; IL = interleukin; TGF = transforming growth factor; TNF = tumor necrosis factor. (Adapted with permission from Lin E, Calvano SE, Lowry SF. Inflammatory cytokines and cell response in surgery, Surgery 2000 Feb;127(2):117-126.)In a mouse model of hemorrhagic shock/reperfusion, mac-rophages play a key role in the recruitment of hematopoietic stem cells from the

1	and cell response in surgery, Surgery 2000 Feb;127(2):117-126.)In a mouse model of hemorrhagic shock/reperfusion, mac-rophages play a key role in the recruitment of hematopoietic stem cells from the bone marrow by secreting granulocyte-monocyte stimulating factor (GM-CSF) in response to circu-lating HMGB1.202 In the lung, alveolar macrophages sense DAMPs and extracellular matrix fragments via pattern recog-nition receptors. In response, they upregulate their expression of TLR4, which primes the cell for response against potential infection.203 At the same time, they release proinflammatory cytokines and ROS, which contribute injury to the alveolar epithelial cells. More recently, data indicate that an imbalance of M1/M2 macrophage populations in the lung contribute to acute lung injury following hemorrhagic shock (HS).204 In this study, investigators demonstrated that HS/resuscitation resulted in a significant decrease in M2 phenotype macrophages, with a delayed increase in M1.

1	following hemorrhagic shock (HS).204 In this study, investigators demonstrated that HS/resuscitation resulted in a significant decrease in M2 phenotype macrophages, with a delayed increase in M1. Augmenting the M2 population prior to injury lessened the degree of lung injury as assessed histologically.Lymphocytes and T-Cell ImmunityThe expression of genes associated with the adaptive immune response is rapidly altered following severe blunt trauma.3 In fact, significant injury is associated with adaptive immune sup-pression that is characterized by altered cell–mediated immu-nity. This correlates with both a decrease in the overall number of lymphocytes as well as the balance between the NK and T cell populations.205CD4+ T cells (helper) play central roles in the function of the immune system through their effects on B cell anti-body production, their enhancement of specific TReg cell func-tions, and their assistance with macrophage activation. CD4+ Th cells are functionally divided

1	through their effects on B cell anti-body production, their enhancement of specific TReg cell func-tions, and their assistance with macrophage activation. CD4+ Th cells are functionally divided into subsets, which include Th1, Th2, and Th17 cells. Each of these groups produces spe-cific effector cytokines that are under unique transcriptional control. The specific functions of these cells include the rec-ognition and killing of intracellular pathogens (cellular immu-nity, Th1 cells); regulation of antibody production (humoral immunity, TH2 cells); and maintenance of mucosal immu-nity and barrier integrity (Th17 cells). Historically, activi-ties have been characterized as proinflammatory (Th1) and anti-inflammatory (Th2) respectively, as determined by their distinct cytokine signatures (Fig. 2-11). Given the proinflam-matory action of IL-17A produced by Th17 cells, they could also be placed in this category. However, it is clear that the Th17 differentiation is more complex and may

1	(Fig. 2-11). Given the proinflam-matory action of IL-17A produced by Th17 cells, they could also be placed in this category. However, it is clear that the Th17 differentiation is more complex and may involve the two distinct phenotypes, a pathogenic phenotype characterized by increased IL-17 production and a more regulatory phenotype in which IL-10 expression is increased.206Recent evidence suggests that the population of Th17 cells is altered following severe traumatic injury. Mass cytometry by time-of-flight (CyTOF) was used to collect single cell phenotyp-ing data on circulating peripheral blood mononuclear cells from a cohort of severely injured trauma patients.155 The investigators identified an expansion of Th17 cells at all time points follow-ing injury and was associated with an increase in the cytokine profile associated with a Th17 phenotype. This supports prior work also demonstrating a robust type 17 immune response early (within the first 24 hours) among nonsurvivors,

1	increase in the cytokine profile associated with a Th17 phenotype. This supports prior work also demonstrating a robust type 17 immune response early (within the first 24 hours) among nonsurvivors, which also identified a Th17 profile more consistent with “pathogenic” Th17 cells.154Successful recovery from injury also depends upon a bal-anced Th1/Th2 response. Following injury, however, there is a reduction in Th1 cell differentiation and cytokine production in favor of an increased population of Th2 lymphocytes and their signaling products. As a consequence, both macrophage activation and proinflammatory cytokine synthesis are inhib-ited. This imbalance, which may be associated with decreased IL-12 production by activated monocytes/macrophages, has been associated with increased risk of infectious complications following surgery and trauma. What are the systemic mecha-nisms responsible for this shift? Several events have been impli-cated, including the direct effect of

1	risk of infectious complications following surgery and trauma. What are the systemic mecha-nisms responsible for this shift? Several events have been impli-cated, including the direct effect of glucocorticoids on monocyte IL-12 production and T cell IL-12 receptor expression. In addi-tion, sympathoadrenal catecholamine production has also been demonstrated to reduce IL-12 production and proinflammatory cytokine synthesis. Finally, more recent work has implicated circulating immature myeloid cells, termed myeloid-derived suppressor cells, that have immune suppressive activity par-ticularly through their increased expression of arginase.208 These cells have the potential to deplete the microenvironment of argi-nine, leading to further T cell dysfunction.Dendritic CellsRecent studies have focused on the cellular components of the immune system in the context of polytrauma. While the Brunicardi_Ch02_p0027-p0082.indd 5501/03/19 6:49 PM 56BASIC CONSIDERATIONSPART Iactivation of

1	have focused on the cellular components of the immune system in the context of polytrauma. While the Brunicardi_Ch02_p0027-p0082.indd 5501/03/19 6:49 PM 56BASIC CONSIDERATIONSPART Iactivation of granulocytes and monocyte/macrophages follow-ing trauma has been well described, more recent work has dem-onstrated that dendritic cells (DC) are also activated in response to damage signals, to stimulate both the innate and the adaptive immune responses.Dendritic cells are the most important antigen-presenting cells (APCs) for initiating T-cell responses against protein antigens. Primary “danger signals” that are recognized and activated by DC include debris from damaged or dying cells (e.g., HMGB1, nucleic acids including single nucleotides, and degradation products of the extracellular matrix). DC are fre-quently referred to as “professional APCs” since their principal function is to capture, process, and present both endogenous and exogenous antigens, which, along with their

1	matrix). DC are fre-quently referred to as “professional APCs” since their principal function is to capture, process, and present both endogenous and exogenous antigens, which, along with their co-stimulatory molecules, are capable of inducing a primary immune response in resting naive T lymphocytes. In addition, they have the capac-ity to further regulate the immune response, both positively and negatively, through the upregulation and release of immuno-modulatory molecules such as the chemokine CCL5 (RANTES) and the CXC chemokine CXCL5. Finally, they have been impli-cated both in the induction and maintenance of immune toler-ance as well as in the acquisition of immune memory.209 There are distinct classes and subsets of DC, which are functionally heterogeneous. Different levels of damage-sensing receptors (e.g., TLR) that dictate a preferential response to DAMPs at that site. While relatively small in number relative to the total leukocyte population, the diverse distribution of DC

1	receptors (e.g., TLR) that dictate a preferential response to DAMPs at that site. While relatively small in number relative to the total leukocyte population, the diverse distribution of DC in virtually all body tissues underlines their potential for a collaborative role in the initiation of the trauma-induced sterile systemic inflam-matory response. Data support a phenotypic alteration in these cells following traumatic injury.210PlateletsPlatelets are small (2 µm), circulating fragments of a larger cell precursor, the megakaryocyte that is located chiefly within the bone marrow. Although platelets lack a nucleus, they contain both mRNA and a large number of cytoplasmic and surface pro-teins that equip them for diverse functionality. While their role in hemostasis is well described, more recent work suggest that platelets play a role in both local and systemic inflammatory responses, particularly following ischemia reperfusion. Plate-lets express functional scavenger and Toll-like

1	recent work suggest that platelets play a role in both local and systemic inflammatory responses, particularly following ischemia reperfusion. Plate-lets express functional scavenger and Toll-like receptors (TLR) that are important detectors of both pathogens and “damage”-associated molecules.211 At the site of tissue injury, complex interactions between platelets, endothelial cells, and circulating leukocytes facilitate cellular activation by the numerous local alarmins and immune mediators. For example, platelet-specific TLR4 activation can cause thrombocytes to bind to and activate neutrophils to extrude their DNA to form neutrophil extracel-lular traps or NETs, an action that facilitates the capacity of the innate immune system to trap bacteria but also leads to local endothelial cell damage.212Once activated, platelets adopt an initial proinflamma-tory phenotype by expressing and releasing a variety of adhe-sion molecules, cytokines, and other immune modulators, including high

1	damage.212Once activated, platelets adopt an initial proinflamma-tory phenotype by expressing and releasing a variety of adhe-sion molecules, cytokines, and other immune modulators, including high mobility group 1 protein (HMGB1), interleukin (IL)-1β, and CD40 ligand (CD40L, CD154). However, acti-vated platelets also express large amounts of the immunosup-pressive factor, transforming growth factor-β (TGFβ), that has been implicated in Treg cell homeostasis. Recently, in a large animal model of hemorrhage, TGF-b levels were shown to be significantly increased 2 hours post injury, suggesting a pos-sible mechanism for injury-related immune dysfunction.213 And, while soluble CD154 was not increased following hemorrhage and traumatic brain injury in that study, in a murine model of mesenteric ischemia-reperfusion injury, platelet expression of CD40 and CD154 was linked to remote organ damage.Mast CellsMast cells are important in the primary response to injury because they are located in

1	ischemia-reperfusion injury, platelet expression of CD40 and CD154 was linked to remote organ damage.Mast CellsMast cells are important in the primary response to injury because they are located in tissues. TNF release from mast cells has been found to be crucial for neutrophil recruitment and pathogen clearance. Mast cells are also known to play an impor-tant role in the anaphylactic response to allergens. On activation from stimuli including allergen binding, infection, and trauma, mast cells produce histamine, cytokines, eicosanoids, proteases, and chemokines, which leads to vasodilatation, capillary leak-age, and immunocyte recruitment. Mast cells are thought to be important cosignaling effector cells of the immune system via the release of IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, and IL-14, as well as macrophage migration–inhibiting factor.214ENDOTHELIUM-MEDIATED INJURYVascular EndotheliumUnder physiologic conditions, the vascular endothelium has important anticoagulant properties

1	as well as macrophage migration–inhibiting factor.214ENDOTHELIUM-MEDIATED INJURYVascular EndotheliumUnder physiologic conditions, the vascular endothelium has important anticoagulant properties and forms a critical barrier to regulate the tissue migration of circulating cells. Following injury, endothelial cells are differentially modulated, resulting in a procoagulant shift that may lead to microthrombosis and organ injury. Recent work has associated postinjury vascular dysfunc-tion (traumatic endotheliopathy) with circulating levels of syn-decan-1, a surrogate marker for disruption of the endothelial cell glycocalyx.215 In a cohort of over 400 severely injured patients, higher syndecan-1 measurements correlated with ISS and plasma catecholamine levels and, ultimately, with mortality in this group. The authors’ hypothesize that the increased disruption of the endothelial glycocalyx results in endothelial cell injury and an altered phenotype resulting in a prothrombotic state that

1	in this group. The authors’ hypothesize that the increased disruption of the endothelial glycocalyx results in endothelial cell injury and an altered phenotype resulting in a prothrombotic state that leads to microvascular thrombosis and ensuing organ dysfunction.Neutrophil-Endothelium InteractionThe regulated inflammatory response to infection facilitates neutrophil and other immunocyte migration to compromised regions through the actions of increased vascular permeabil-ity, chemoattractants, and increased endothelial adhesion fac-tors referred to as selectins that are elaborated on cell surfaces (Table 2-7). In response to inflammatory stimuli released from sentinel leukocytes in the tissues, including chemokines, throm-bin, leukotrienes, histamine, and TNF, vascular endothelium are activated and their surface protein expression is altered. Within 10 to 20 minutes, prestored reservoirs of the adhesion molecule P-selectin are mobilized to the cell surface where it can mediate

1	are activated and their surface protein expression is altered. Within 10 to 20 minutes, prestored reservoirs of the adhesion molecule P-selectin are mobilized to the cell surface where it can mediate neutrophil recruitment (Fig. 2-12). After 2 hours, endothelial cell transcriptional processes provide additional surface expression of E-selectin. E-selectin and P-selectin bind P-selectin glycoprotein ligand-1 (PSGL-1) on the neutrophils to orchestrate the capture and rolling of these leukocytes and allow targeted immunocyte extravasation. Immobilized chemo-kines on the endothelial surface create a chemotactic gradient to further enhance immune cell recruitment.216 Also important are secondary leukocyte-leukocyte interactions in which PGSL-1 and L-selectin binding facilitates further leukocyte tethering. Brunicardi_Ch02_p0027-p0082.indd 5601/03/19 6:49 PM 57SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2Table 2-7Molecules that mediate leukocyte-endothelial adhesion,

1	tethering. Brunicardi_Ch02_p0027-p0082.indd 5601/03/19 6:49 PM 57SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2Table 2-7Molecules that mediate leukocyte-endothelial adhesion, categorized by familyADHESION MOLECULEACTIONORIGININDUCERS OF EXPRESSIONTARGET CELLSSelectins L-selectin P-selectin E-selectinFast rollingSlow rollingVery slow rollingLeukocytesPlatelets and endotheliumEndotheliumNativeThrombin, histamineCytokinesEndothelium, platelets, eosinophilsNeutrophils, monocytesNeutrophils, monocytes, lymphocytesImmunoglobulins ICAM-1 ICAM-2 VCAM-1 PECAM-1Firm adhesion/transmigrationFirm adhesionFirm adhesion/transmigrationAdhesion/ transmigrationEndothelium, leukocytes, fibroblasts, epitheliumEndothelium, plateletsEndotheliumEndothelium, platelets, leukocytesCytokinesNativeCytokinesNativeLeukocytesLeukocytesMonocytes, lymphocytesEndothelium, platelets, leukocytesβ2-(CD18) Integrins CD18/11a CD18/11b (Mac-1) CD18/11cFirm adhesion/transmigrationFirm

1	lymphocytesEndothelium, platelets, leukocytesβ2-(CD18) Integrins CD18/11a CD18/11b (Mac-1) CD18/11cFirm adhesion/transmigrationFirm adhesion/transmigrationAdhesionLeukocytesNeutrophils, monocytes, natural killer cellsNeutrophils, monocytes, natural killer cellsLeukocyte activationLeukocyte activationLeukocyte activationEndotheliumEndotheliumEndotheliumβ1-(CD29) Integrins VLA-4Firm adhesion/transmigrationLymphocytes, monocytesLeukocyte activationMonocytes, endothelium, epitheliumICAM-1 = intercellular adhesion molecule-1; ICAM-2 = intercellular adhesion molecule-2; Mac-1 = macrophage antigen 1; PECAM-1 = platelet-endothelial cell adhesion molecule-1; VCAM-1 = vascular cell adhesion molecule-1; VLA-4 = very late antigen-4.Although there are distinguishable properties among individ-ual selectins in leukocyte rolling, effective rolling most likely involves a significant degree of functional overlap.217ChemokinesChemokines are a family of small proteins (8–13 kDa) that were first

1	selectins in leukocyte rolling, effective rolling most likely involves a significant degree of functional overlap.217ChemokinesChemokines are a family of small proteins (8–13 kDa) that were first identified through their chemotactic and activating effects on inflammatory cells. They are produced at high levels follow-ing nearly all forms of injury in all tissues, where they are key attractants for immune cell extravasation. There are more than 50 different chemokines and 20 chemokine receptors that have been identified. Chemokines are released from endothelial cells, mast cells, platelets, macrophages, and lymphocytes. They are soluble proteins, which when secreted, bind to glycosamino-glycans on the cell surface or in the extracellular matrix. In this way, the chemokines can form a fixed chemical gradient that promotes immune cell exit to target areas. Supporting the idea of their importance in leukocyte recruitment post injury, a sub-set of chemokines are elevated early following

1	chemical gradient that promotes immune cell exit to target areas. Supporting the idea of their importance in leukocyte recruitment post injury, a sub-set of chemokines are elevated early following traumatic injury in both survivors and nonsurvivors.218Chemokines are distinguished (in general) from cyto-kines by virtue of their receptors, which are members of the G-protein–coupled receptor superfamily. Most chemokine receptors recognize more than one chemokine ligand leading to redundancy in chemokine signaling.The chemokines are subdivided into families based on their amino acid sequences at their N-terminus. For example, CC chemokines contain two N-terminus cysteine residues that are immediately adjacent (hence the “C-C” designation) while the N-terminal cysteines in CXC chemokines are separated by a single amino acid. The CXC chemokines are particularly impor-tant for neutrophil (PMN) proinflammatory function. Members of the CXC chemokine family, which include IL-8 (CXCL8), induce

1	separated by a single amino acid. The CXC chemokines are particularly impor-tant for neutrophil (PMN) proinflammatory function. Members of the CXC chemokine family, which include IL-8 (CXCL8), induce neutrophil migration and secretion of cytotoxic granu-lar contents and metabolites. Additional chemokine families include the Cand CX3C-chemokines.216Recent studies support the idea that a subset of chemo-kines, monokine induced by γ-interferon (MIG), monocyte chemotactic protein 1 (MCP-1), and interferon γ-induced pro-teins 10 (IP-10) may work in concert to regulate the inflam-matory response post injury and may serve as biomarkers for clinical outcome in trauma patients.218,219 These investigators propose that MIG, MCP-1, and IP-10 function as a “chemo-kine switch” in which the relative levels of each chemokine may promote its own expression, which suppresses the expres-sion of the other two according to the severity and type of injury.220 In this way, the authors propose that the

1	levels of each chemokine may promote its own expression, which suppresses the expres-sion of the other two according to the severity and type of injury.220 In this way, the authors propose that the balance between these three chemokines, by regulating inflammatory mediator production (e.g., IL-6) may help to correlate with long-term outcomes.Brunicardi_Ch02_p0027-p0082.indd 5701/03/19 6:49 PM 58BASIC CONSIDERATIONSPART ICaptureFastrollingSlowrollingArrest50–150 µm/sec20–50 µm/sec10–20 µm/sec0–10 µm/secVelocity:LeukocyteSeconds0Velocity (µm/second)1501000501234EndotheliumFigure 2-12. Simplified sequence of selectin-mediated neutrophil-endothelium interaction after an inflammatory stimulus. CAPTURE (tethering), predominantly mediated by cell L-selectin with contri-bution from endothelial P-selectin, describes the initial recognition between leukocyte and endothelium, in which circulating leuko-cytes marginate toward the endothelial surface. FAST ROLLING (20 to 50 μm/s) is a

1	P-selectin, describes the initial recognition between leukocyte and endothelium, in which circulating leuko-cytes marginate toward the endothelial surface. FAST ROLLING (20 to 50 μm/s) is a consequence of rapid L-selectin shedding from cell surfaces and formation of new downstream L-selectin to endo-thelium bonds, which occur in tandem. SLOW ROLLING (10 to 20 μm/s) is predominantly mediated by P-selectins. The slowest rolling (3 to 10 μm/s) before arrest is predominantly mediated by E-selec-tins, with contribution from P-selectins. ARREST (firm adhesion) leading to transmigration is mediated by β-integrins and the immu-noglobulin family of adhesion molecules. In addition to interact-ing with the endothelium, activated leukocytes also recruit other leukocytes to the inflammatory site by direct interactions, which are mediated in part by selectins. (Adapted with permission from Lin E, Calvano SE, Lowry SF. Selectin neutralization: does it make biological sense? Crit Care Med. 1999

1	by direct interactions, which are mediated in part by selectins. (Adapted with permission from Lin E, Calvano SE, Lowry SF. Selectin neutralization: does it make biological sense? Crit Care Med. 1999 Sep;27(9):2050-2053.)Nitric OxideNitric oxide (NO) was initially known as endothelium-derived relaxing factor due to its effect on vascular smooth muscle. Normal vascular smooth muscle cell relaxation is maintained by a constant output of NO that is regulated in the endothe-lium by both flowand receptor-mediated events. NO can also reduce microthrombosis by reducing platelet adhesion and aggregation (Fig. 2-13) and interfering with leukocyte adhe-sion to the endothelium. NO easily traverses cell membranes and has a short half-life of a few seconds. Endogenous NO formation is derived largely from the action of NO synthase (NOS), which is constitutively expressed in endothelial cells (NOS3, eNOS). Nitric oxide synthase generates NO by cata-lyzing the degradation of L-arginine to

1	largely from the action of NO synthase (NOS), which is constitutively expressed in endothelial cells (NOS3, eNOS). Nitric oxide synthase generates NO by cata-lyzing the degradation of L-arginine to L-citrulline and NO, in the presence of oxygen and NADPH. There are two addi-tional isoforms of NOS: neuronal NOS (NOS1, nNOS) and inducible NOS (iNOS/NOS2), which is expressed in response to cytokines and bacterial products. The vasodilatory effects of NO are mediated by guanylyl cyclase, an enzyme that is found in vascular smooth muscle cells and most other cells of the body. When NO is formed by endothelium, it rapidly diffuses into adjacent cells where it binds to and activates gua-nylyl cyclase. This enzyme catalyzes the dephosphorylation of GTP to cGMP, which serves as a second messenger for many important cellular functions, particularly for signaling smooth muscle relaxation.NO synthesis is increased due to the upregulation of iNOS expression in response to proinflammatory

1	for many important cellular functions, particularly for signaling smooth muscle relaxation.NO synthesis is increased due to the upregulation of iNOS expression in response to proinflammatory media-tors such as TNF-α, and IL-1β, as well as microbial products.221,222 In fact, studies in both animal models and humans have shown that severe systemic injury and asso-ciated hemorrhage produce an early upregulation of iNOS in the liver, lung, spleen, and vascular system. In these cir-cumstances, NO is reported to function as an immunoregu-lator, which is capable of modulating cytokine production and immune cell development.223 In particular, recent data supports a role for iNOS/eNOS in the regulation of T-cell dysfunction in the setting of trauma as evidenced by sup-pressed proliferative and TH1 cytokine release. In particular, the formation of S-nitrosothiols, which can serve as a molec-ular switch to regulate protein functions, may explain many signaling effects of both iNOSand

1	and TH1 cytokine release. In particular, the formation of S-nitrosothiols, which can serve as a molec-ular switch to regulate protein functions, may explain many signaling effects of both iNOSand eNOS-derived NO in the immune system with regard to T-cell activation and signaling through the T cell receptor. In T cells, NO effects have been implicated in the regulation of the immune synapse as well as the regulation of mitochondrial bioenergetics indicating that NO may play an important role as a link between innate and adaptive immunity.224Inhibition of NO production seemed initially to be a promising strategy in patients with severe sepsis. However, a randomized clinical trial in patients with septic shock determined that treatment with a nonselective NOS inhibitor was associated with an increase in mortality compared with placebo.225 More recent data utilizing an ovine model of perito-nitis demonstrated that selective iNOS inhibition reduced pul-monary artery hypertension and gas

1	increase in mortality compared with placebo.225 More recent data utilizing an ovine model of perito-nitis demonstrated that selective iNOS inhibition reduced pul-monary artery hypertension and gas exchange impairment and promoted higher visceral organ blood flow, coinciding with lower plasma cytokine concentrations.226 These data suggest that specific targeting of iNOS in the setting of sepsis may remain a viable therapeutic option.Recent work using an animal model of traumatic brain injury (TBI) showed that acute TBI results in endothelial dys-function in a remote vascular bed.227 The investigators linked the effect of TBI with impaired nitric oxide (NO) production and also with an increase in arterial arginase activity, implicat-ing the depletion of L-arginine by arginase with the decreased NO production.ProstacyclinThe immune effects of prostacyclin (PGI2) have been discussed previously. The best-described effects of PGI2 are in the car-diovascular system, however, where it is

1	NO production.ProstacyclinThe immune effects of prostacyclin (PGI2) have been discussed previously. The best-described effects of PGI2 are in the car-diovascular system, however, where it is produced by vascular endothelial cells. Prostacyclin is a potent vasodilator that also inhibits platelet aggregation. In the pulmonary system, PGI2 reduces pulmonary blood pressure as well as bronchial hyper-responsiveness. In the kidneys, PGI2 modulates renal blood flow and glomerular filtration rate. Prostacyclin acts through its receptor (a G-protein–coupled receptor of the rhodopsin family) to stimulate the enzyme, adenylate cyclase, allowing the syn-thesis of cyclic adenosine monophosphate (cAMP) from ATP. Brunicardi_Ch02_p0027-p0082.indd 5801/03/19 6:49 PM 59SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2cAMPcAMPcGMPcGMPNONOPGI2AAPlateletETETPGI2Big ETL-arginineEndotheliumSmooth muscleRelaxationFigure 2-13. Endothelial interac-tion with smooth muscle cells and with

1	AND METABOLIC SUPPORTCHAPTER 2cAMPcAMPcGMPcGMPNONOPGI2AAPlateletETETPGI2Big ETL-arginineEndotheliumSmooth muscleRelaxationFigure 2-13. Endothelial interac-tion with smooth muscle cells and with intraluminal platelets. Prosta-cyclin (prostaglandin I2, or PGI2) is derived from arachidonic acid (AA), and nitric oxide (NO) is derived from L-arginine. The increase in cyclic adenosine monophosphate (cAMP) and cyclic guanosine mono-phosphate (cGMP) results in smooth muscle relaxation and inhibition of platelet thrombus formation. Endo-thelins (ETs) are derived from “big ET,” and they counter the effects of prostacyclin and NO.This leads to a cAMP-mediated decrease in intracellular cal-cium and subsequent smooth muscle relaxation.During systemic inflammation, endothelial prostacyclin expression is impaired, and thus the endothelium favors a more procoagulant profile. Exogenous prostacyclin analogues, both intravenous and inhaled, have been utilized to improve oxygen-ation in patients with

1	is impaired, and thus the endothelium favors a more procoagulant profile. Exogenous prostacyclin analogues, both intravenous and inhaled, have been utilized to improve oxygen-ation in patients with acute lung injury. Early clinical studies with prostacyclin have delivered some encouraging results.228 However, a recent study examining the administration of epo-prostenol in the setting of severe injury and TBI demonstrated and attenuation of the inflammatory response as measured by serologic markers had no effect on long-term outcome.229EndothelinsEndothelins (ETs) are potent mediators of vasoconstriction ET-1, synthesized primarily by endothelial cells, is the most potent endogenous vasoconstrictor, and is estimated to be 10 times more potent than angiotensin II. ET release is upregu-lated in response to hypotension, LPS, injury, thrombin, TGF-b, IL-1, angiotensin II, vasopressin, catecholamines, and anoxia. ETs release is transcriptionally regulated and occurs at the ablu-minal side

1	in response to hypotension, LPS, injury, thrombin, TGF-b, IL-1, angiotensin II, vasopressin, catecholamines, and anoxia. ETs release is transcriptionally regulated and occurs at the ablu-minal side of endothelial cells. Very little is stored in cells; thus, a plasma increase in ET is associated with a marked increase in production. Three endothelin receptors have been identified and function via the G-protein–coupled receptor mechanism. ETB receptors are associated with increased NO and prostacyclin production, which may serve as a feedback mechanism. Atrial ETA receptor activation has been associated with increased inotropy and chronotropy. ET-1 infusion is associated with increased pulmonary vascular resistance and pulmonary edema and may contribute to pulmonary abnormalities during sepsis. At low levels, in conjunction with NO, ETs regulate vascular tone. However, at increased concentrations, ETs can disrupt the normal blood flow and distribution and may compromise oxy-gen delivery

1	At low levels, in conjunction with NO, ETs regulate vascular tone. However, at increased concentrations, ETs can disrupt the normal blood flow and distribution and may compromise oxy-gen delivery to the tissue. Recent data links endothelin expres-sion in pulmonary vasculature with persistent inflammation associated with the development of pulmonary hypertension.230 Endothelin expression is linked to posttranslational and tran-scriptional initiation of the unfolded protein response in the affected cells, which results in the production of inflammatory cytokines.231 Persistent endothelin-1 stimulation may play a role in decreased vascular reactivity that is evident following hemor-rhagic shock.232Platelet Activating FactorPhosphotidylcholine is a major lipid constituent of the plasma membrane. Its enzymatic processing by cytosolic phospho-lipase A2 (cPLA2) or calcium-independent phospholipase A2 (iPLA2) generates powerful small lipid molecules, which func-tion as intracellular second

1	Its enzymatic processing by cytosolic phospho-lipase A2 (cPLA2) or calcium-independent phospholipase A2 (iPLA2) generates powerful small lipid molecules, which func-tion as intracellular second messengers. One of these is arachi-donic acid, the precursor molecule for eicosanoids. Another is platelet-activating factor (PAF). During acute inflammation, PAF is released by immune cells following the activation of PLA2. The receptor for PAF (PAFR), which is constitutively expressed by platelets, leukocytes, and endothelial cells, is a G-protein–coupled receptor of the rhodopsin family. Ligand binding to the PAFR promotes the activation and aggregation of platelets and leukocytes, leukocyte adherence, motility, chemotaxis, and invasion, as well as ROS generation.233 Addi-tionally, PAF activation of human PMNs induces extrusion of neutrophil extracellular traps (NETs), while platelet activa-tion induces IL-1 via a novel posttranscriptional mechanism. Finally, PAFR ligation results not only

1	of human PMNs induces extrusion of neutrophil extracellular traps (NETs), while platelet activa-tion induces IL-1 via a novel posttranscriptional mechanism. Finally, PAFR ligation results not only in the upregulation of numerous proinflammatory genes, including COX-2, iNOS, and IL-6, but also in the generation of lipid intermediates such as arachidonic acid and lysophospholipids through the Brunicardi_Ch02_p0027-p0082.indd 5901/03/19 6:49 PM 60BASIC CONSIDERATIONSPART IBrainRBCWBCNerveKidneyMuscleHeartKidneyMuscleAminoacidsGlycerol16gFattyacid160g40gFatty acid120gKetone60gGlucose180g36g144g36gLactate + PyruvateLIVERGlycogen75gGluconeogenesisOxidationFuel utilization in short-term fasting man (70 kg)Muscleprotein75gFat storestriglycerides160gFigure 2-14. Fuel utilization in a 70-kg man during short-term fasting with an approximate basal energy expenditure of 1800 kcal. During starvation, muscle proteins and fat stores provide fuel for the host, with the latter being most abundant.

1	during short-term fasting with an approximate basal energy expenditure of 1800 kcal. During starvation, muscle proteins and fat stores provide fuel for the host, with the latter being most abundant. RBC = red blood cell; WBC = white blood cell. activation of Phospholipase A2. Antagonists to PAF recep-tors have been experimentally shown to mitigate the effects of ischemia and reperfusion injury. Of note, human sepsis is associated with a reduction in the levels of PAF-acetylhydro-lase, which inactivates PAF by removing an acetyl group. Indeed, PAF-acetylhydrolase administration in patients with severe sepsis has yielded some reduction in multiple organ dysfunction and mortality234; however, larger phase III clinical trials failed to show benefit.Natriuretic PeptidesThe natriuretic peptides, atrial natriuretic factor (ANP) and brain natriuretic peptide (BNP), are a family of peptides that are released primarily by atrial and ventricular tissue respectively, but are also synthesized by

1	atrial natriuretic factor (ANP) and brain natriuretic peptide (BNP), are a family of peptides that are released primarily by atrial and ventricular tissue respectively, but are also synthesized by the gut, kidney, brain, adrenal glands, and endothelium. The functionally active forms of the peptides are C-terminal fragments of a larger pro-hormone, and both Nand C-terminal fragments are detectable in the blood (referred to a N-terminal pro-BNP and pro-ANF, respectively). ANF and BNP share most biological properties, including diuretic, natri-uretic, vasorelaxant, and cardiac remodeling properties that are affected by signaling through a common receptor: the guanylyl cyclase(GC-) A receptor. They are both increased in the setting of cardiac disorders; however, evidence indicates some distinctions in the setting of inflammation. For example, elevated proBNP has been detected in septic patients in the absence of myocardial dysfunction and appears to have prognostic significance.235 More

1	in the setting of inflammation. For example, elevated proBNP has been detected in septic patients in the absence of myocardial dysfunction and appears to have prognostic significance.235 More recently, investigators examined changes in N-terminal pro-BNP (NT-proBNP) in a cohort of severely injured patients and deter-mined that persistently high level of NT-proBNP in major trauma patients is indicative of poor outcome.236SURGICAL METABOLISMThe initial hours after surgical or traumatic injury are metaboli-cally associated with a reduced total body energy expenditure and urinary nitrogen wasting. With adequate resuscitation and stabilization of the injured patient, a reprioritization of substrate use ensues to preserve vital organ function and to support repair of injured tissue. This phase of recovery also is characterized by functions that participate in the restoration of homeostasis, such as augmented metabolic rates and oxygen consumption, enzymatic preference for readily oxidizable

1	recovery also is characterized by functions that participate in the restoration of homeostasis, such as augmented metabolic rates and oxygen consumption, enzymatic preference for readily oxidizable substrates such as glucose, and stimulation of the immune system. Understanding of the collective alterations in amino acid (protein), carbohy-drate, and lipid metabolism characteristic of the surgical patient lays the foundation upon which metabolic and nutritional sup-port can be implemented.Metabolism During FastingFuel metabolism during unstressed fasting states has historically served as the standard to which metabolic alterations after acute injury and critical illness are compared (Fig. 2-14). To maintain basal metabolic needs (i.e., at rest and fasting), a normal healthy adult requires approximately 22 to 25 kcal/kg per day drawn from carbohydrate, lipid, and protein sources. This requirement can substantially increase during severe stress states, such as those seen in patients with

1	22 to 25 kcal/kg per day drawn from carbohydrate, lipid, and protein sources. This requirement can substantially increase during severe stress states, such as those seen in patients with burn injuries.In the healthy adult, principal sources of fuel during short-term fasting (<5 days) are derived from muscle protein and body fat, with fat being the most abundant source of energy (Table 2-8). The normal adult body contains 300 to 400 g of carbohydrates in the form of glycogen, of which 75 to 100 g are stored in the liver. Approximately 200 to 250 g of glycogen are stored within skeletal, cardiac, and smooth muscle cells. The greater glycogen stores within the muscle are not read-ily available for systemic use due to a deficiency in glucose-6-phosphatase but are available for the energy needs of muscle cells. Therefore, in the fasting state, hepatic glycogen stores are rapidly and preferentially depleted, which results in a fall of serum glucose concentration within hours (<16

1	needs of muscle cells. Therefore, in the fasting state, hepatic glycogen stores are rapidly and preferentially depleted, which results in a fall of serum glucose concentration within hours (<16 hours).During fasting, a healthy 70-kg adult will utilize 180 g of glucose per day to support the metabolism of obligate glycolytic cells such as neurons, leukocytes, erythrocytes, and the renal medullae. Other tissues that use glucose for fuel are skeletal muscle, intestinal mucosa, fetal tissues, and solid tumors.Brunicardi_Ch02_p0027-p0082.indd 6001/03/19 6:49 PM 61SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2RBCWBCNerveKidneyMuscleMuscleProteinpyruvateKetoneLactate + PyruvateFattyacidGlucoseKetoneGlucoseAlanineGlucose-alanine cycleCori cycleLIVERGluconeogenesisFigure 2-15. The recycling of peripheral lactate and pyruvate for hepatic gluconeogenesis is accomplished by the Cori cycle. Alanine within skeletal muscles can also be used as a precursor for hepatic gluconeogenesis.

1	of peripheral lactate and pyruvate for hepatic gluconeogenesis is accomplished by the Cori cycle. Alanine within skeletal muscles can also be used as a precursor for hepatic gluconeogenesis. During starvation, such fatty acid provides fuel sources for basal hepatic enzymatic function. RBC = red blood cell; WBC = white blood cell.Glucagon, norepinephrine, vasopressin, and angiotensin II can promote the utilization of glycogen stores (glycogenolysis) during fasting. Although glucagon, epinephrine, and cortisol directly promote gluconeogenesis, epinephrine and cortisol also promote pyruvate shuttling to the liver for gluconeogen-esis. Precursors for hepatic gluconeogenesis include lactate, glycerol, and amino acids such as alanine and glutamine. Lac-tate is released by glycolysis within skeletal muscles, as well as by erythrocytes and leukocytes. The recycling of lactate and pyruvate for gluconeogenesis is commonly referred to as the Cori cycle, which can provide up to 40% of plasma

1	skeletal muscles, as well as by erythrocytes and leukocytes. The recycling of lactate and pyruvate for gluconeogenesis is commonly referred to as the Cori cycle, which can provide up to 40% of plasma glucose during starvation (Fig. 2-15).Lactate production from skeletal muscle is insufficient to maintain systemic glucose needs during short-term fasting (simple starvation). Therefore, significant amounts of protein must be degraded daily (75 g/d for a 70-kg adult) to provide the amino acid substrate for hepatic gluconeogenesis. Proteolysis during starvation, which results primarily from decreased insu-lin and increased cortisol release, is associated with elevated urinary nitrogen excretion from the normal 7 to 10 g/d up to 30 g or more per day.237 Although proteolysis during starvation occurs mainly within skeletal muscles, protein degradation in solid organs also occurs.In prolonged starvation, systemic proteolysis is reduced to approximately 20 g/d and urinary nitrogen excretion

1	occurs mainly within skeletal muscles, protein degradation in solid organs also occurs.In prolonged starvation, systemic proteolysis is reduced to approximately 20 g/d and urinary nitrogen excretion stabilizes at 2 to 5 g/d (Fig. 2-16). This reduction in proteolysis reflects the adaptation by vital organs (e.g., myocardium, brain, renal cortex, and skeletal muscle) to using ketone bodies as their prin-cipal fuel source. In extended fasting, ketone bodies become an important fuel source for the brain after 2 days and gradually become the principal fuel source by 24 days.Enhanced deamination of amino acids for gluconeogen-esis during starvation consequently increases renal excretion of ammonium ions. The kidneys also participate in gluconeo-genesis by the use of glutamine and glutamate and can become the primary source of gluconeogenesis during prolonged star-vation, accounting for up to one-half of systemic glucose production.Table 2-8A. Body fuel reserves in a 70-kg man and B. Energy

1	can become the primary source of gluconeogenesis during prolonged star-vation, accounting for up to one-half of systemic glucose production.Table 2-8A. Body fuel reserves in a 70-kg man and B. Energy equivalent of substrate oxidationA. COMPONENTMASS (kg)ENERGY (kcal)DAYS AVAILABLEWater and minerals4900Protein6.024,00013.0Glycogen0.28000.4Fat15.0140,00078.0Total70.2164,80091.4B. SUBSTRATEO2 CONSUMED (L/g)CO2 PRODUCED (L/g)RESPIRATORY QUOTIENTkcal/gRECOMMENDED DAILY REQUIREMENTGlucose0.750.751.04.07.2 g/kg per dayDextrose———3.4—Lipid2.01.40.79.01.0 g/kg per dayProtein1.00.80.84.00.8 g/kg per dayBrunicardi_Ch02_p0027-p0082.indd 6101/03/19 6:50 PM 62BASIC CONSIDERATIONSPART IWOUNDRBCWBCNerveKidneyMuscleHeartKidneyMuscleAminoacidsGlycerol17gFattyacid170g40gFatty acid130gKetone60gGlucose360g180g180gLactate+PyruvateKIDNEYGluconeogenesisGluconeogenesisLIVEROxidationFuel utilization following traumaMuscleProtein250gFat storesTriglycerides170gFigure 2-17. Acute injury is associated with

1	utilization following traumaMuscleProtein250gFat storesTriglycerides170gFigure 2-17. Acute injury is associated with significant alterations in substrate utilization. There is enhanced nitrogen loss, indicative of catabolism. Fat remains the primary fuel source under these circumstances. RBC = red blood cell; WBC = white blood cell.Fuel utilization in long-term fasting man (70 kg)HeartKidneyMuscleAminoacidsGlycerol18gFattyacid180g45gFatty acid135gKetone68gGlucose80g44g36gLactate + PyruvateKIDNEYGluconeogenesis15g5g40g40g10g (100 mEq) in urine44g36g58gMuscleProtein20gFat storesTriglycerides180gBrainRBCWBCNerveKidneyMuscleLIVERGlycogenGluconeogenesisOxidationFigure 2-16. Fuel utilization in extended starvation. Liver glycogen stores are depleted, and there is adaptive reduction in proteolysis as a source of fuel. The brain uses ketones for fuel. The kidneys become important participants in gluconeogenesis. RBC = red blood cell; WBC = white blood cell. Lipid stores within adipose tissue

1	as a source of fuel. The brain uses ketones for fuel. The kidneys become important participants in gluconeogenesis. RBC = red blood cell; WBC = white blood cell. Lipid stores within adipose tissue provide 40% or more of caloric expenditure during starvation. Energy requirements for basal enzymatic and muscular functions (e.g., gluconeogenesis, neural transmission, and cardiac contraction) are met by the mobilization of triglycerides from adipose tissue. In a resting, fasting, 70-kg person, approximately 160 g of free fatty acids and glycerol can be mobilized from adipose tissue per day. Free fatty acid release is stimulated in part by a reduction in serum insulin levels and in part by the increase in circulating glucagon and catecholamine. Such free fatty acids, like ketone bodies, are used as fuel by tissues such as the heart, kidney (renal cortex), muscle, and liver. The mobilization of lipid stores for energy importantly decreases the rate of glycolysis, gluconeogenesis, and

1	are used as fuel by tissues such as the heart, kidney (renal cortex), muscle, and liver. The mobilization of lipid stores for energy importantly decreases the rate of glycolysis, gluconeogenesis, and proteolysis, as well as the overall glucose requirement to sustain the host. Furthermore, ketone bodies spare glucose utili-zation by inhibiting the enzyme pyruvate dehydrogenase.Metabolism After InjuryInjuries or infections induce unique neuroendocrine and immu-nologic responses that differentiate injury metabolism from that of unstressed fasting (Fig. 2-17). The magnitude of metabolic expenditure over time appears to be directly proportional to the severity of insult, with thermal injuries and severe infections having the highest energy demands (Fig. 2-18). Of note, the first few days following both sepsis and trauma are not hyper-metabolic states, with the more severe insults associated with increased “metabolic hibernation.” However, by week 2, the total energy expenditure increases

1	both sepsis and trauma are not hyper-metabolic states, with the more severe insults associated with increased “metabolic hibernation.” However, by week 2, the total energy expenditure increases dramatically.238 The increase Brunicardi_Ch02_p0027-p0082.indd 6201/03/19 6:50 PM 63SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 201020304050% REE100752550125150175200225Days after injuryMajor burnsSepsis/peritonitisSkeletal traumaNormalrangeElective surgeryStarvationFigure 2-18. Influence of injury severity on resting metabolism (resting energy expenditure, or REE). The shaded area indicates normal REE. (Reproduced with permission from Long CL, Schaffel N, Geiger JW, et al: Metabolic response to injury and illness: estimation of energy and protein needs from indirect calorim-etry and nitrogen balance, JPEN J Parenter Enteral Nutr. 1979 Nov-Dec;3(6):452-456.)DietarytriglyceridesIntestinal lumenLymphatic ductPancreaticlipaseGutenterocyteMonoglyceridesTriglyceridesMonoglyceride +

1	nitrogen balance, JPEN J Parenter Enteral Nutr. 1979 Nov-Dec;3(6):452-456.)DietarytriglyceridesIntestinal lumenLymphatic ductPancreaticlipaseGutenterocyteMonoglyceridesTriglyceridesMonoglyceride + 2 Fatty acyI-CoAFatty acidsChylomicron+ ProteinFigure 2-19. Pancreatic lipase within the small intestinal brush borders hydrolyzes triglycerides into monoglycerides and fatty acids. These components readily diffuse into the gut enterocytes, where they are re-esterified into triglycerides. The resynthesized triglycerides bind car-rier proteins to form chylomicrons, which are transported by the lymphatic system. Shorter triglycerides (those with <10 carbon atoms) can bypass this process and directly enter the portal circulation for transport to the liver. CoA = coenzyme A.in energy expenditure is mediated in part by sympathetic acti-vation and catecholamine release, which has been replicated by the administration of catecholamines to healthy human sub-jects. Lipid metabolism after injury is

1	mediated in part by sympathetic acti-vation and catecholamine release, which has been replicated by the administration of catecholamines to healthy human sub-jects. Lipid metabolism after injury is intentionally discussed first because this macronutrient becomes the primary source of energy during stressed states.239Lipid Metabolism After InjuryLipids are not merely nonprotein, noncarbohydrate fuel sources that minimize protein catabolism in the injured patient. Lipid metabolism potentially influences the structural integrity of cell membranes as well as the immune response during systemic inflammation. Adipose stores within the body (triglycerides) are the predominant energy source (50% to 80%) during critical ill-ness and after injury. Fat mobilization (lipolysis) occurs mainly in response to catecholamine stimulus of the hormone-sensitive triglyceride lipase. Other hormonal influences that potentiate lipolysis include adrenocorticotropic hormone (ACTH), cat-echolamines, thyroid

1	to catecholamine stimulus of the hormone-sensitive triglyceride lipase. Other hormonal influences that potentiate lipolysis include adrenocorticotropic hormone (ACTH), cat-echolamines, thyroid hormone, cortisol, glucagon, growth hor-mone release, and reduction in insulin levels.240Lipid Absorption. Although the process is poorly understood, adipose tissue provides fuel for the host in the form of free fatty acids and glycerol during critical illness and injury. Oxidation of 1 g of fat yields approximately 9 kcal of energy. Although the liver is capable of synthesizing triglycerides from carbohy-drates and amino acids, dietary and exogenous sources provide the major source of triglycerides. Dietary lipids are not readily absorbable in the gut but require pancreatic lipase and phospho-lipase within the duodenum to hydrolyze the triglycerides into free fatty acids and monoglycerides. The free fatty acids and monoglycerides are then readily absorbed by gut enterocytes, which resynthesize

1	within the duodenum to hydrolyze the triglycerides into free fatty acids and monoglycerides. The free fatty acids and monoglycerides are then readily absorbed by gut enterocytes, which resynthesize triglycerides by esterification of the mono-glycerides with fatty acyl coenzyme A (acyl-CoA) (Fig. 2-19). Long-chain triglycerides (LCTs), defined as those with 12 car-bons or more, generally undergo this process of esterification Brunicardi_Ch02_p0027-p0082.indd 6301/03/19 6:50 PM 64BASIC CONSIDERATIONSPART Iand enter the circulation through the lymphatic system as chy-lomicrons. Shorter fatty acid chains directly enter the portal circulation and are transported to the liver by albumin carriers. Hepatocytes use free fatty acids as a fuel source during stress states but also can synthesize phospholipids or triglycerides (i.e., very-low-density lipoproteins) during fed states. Systemic tissue (e.g., muscle and the heart) can use chylomicrons and triglycerides as fuel by hydrolysis with

1	phospholipids or triglycerides (i.e., very-low-density lipoproteins) during fed states. Systemic tissue (e.g., muscle and the heart) can use chylomicrons and triglycerides as fuel by hydrolysis with lipoprotein lipase at the luminal surface of capillary endothelium.241 Trauma or sepsis suppresses lipoprotein lipase activity in both adipose tissue and muscle, presumably mediated by TNF.Lipolysis and Fatty Acid Oxidation. Periods of energy demand are accompanied by free fatty acid mobilization from adipose stores. This is mediated by hormonal influences (e.g., catecholamines, ACTH, thyroid hormones, growth hormone, and glucagon) on triglyceride lipase through a cAMP pathway (Fig. 2-20). In adipose tissues, triglyceride lipase hydrolyzes triglycerides into free fatty acids and glycerol. Free fatty acids enter the capillary circulation and are transported by albumin to tissues requiring this fuel source (e.g., heart and skeletal mus-cle). Insulin inhibits lipolysis and favors triglyceride

1	fatty acids enter the capillary circulation and are transported by albumin to tissues requiring this fuel source (e.g., heart and skeletal mus-cle). Insulin inhibits lipolysis and favors triglyceride synthesis by augmenting lipoprotein lipase activity as well as intracellu-lar levels of glycerol-3-phosphate. The use of glycerol for fuel depends on the availability of tissue glycerokinase, which is abundant in the liver and kidneys.Free fatty acids absorbed by cells conjugate with acyl-CoA within the cytoplasm. The transport of fatty acyl-CoA from the outer mitochondrial membrane across the inner mito-chondrial membrane occurs via the carnitine shuttle (Fig. 2-21). Medium-chain triglycerides (MCTs), defined as those 6 to 12 carbons in length, bypass the carnitine shuttle and readily cross the mitochondrial membranes. This accounts in part for the fact that MCTs are more efficiently oxidized than LCTs. Ideally, the rapid oxidation of MCTs makes them less prone to fat deposition,

1	cross the mitochondrial membranes. This accounts in part for the fact that MCTs are more efficiently oxidized than LCTs. Ideally, the rapid oxidation of MCTs makes them less prone to fat deposition, particularly within immune cells and the reticu-loendothelial system—a common finding with lipid infusion in parenteral nutrition.242 However, exclusive use of MCTs as fuel in animal studies has been associated with higher metabolic demands and toxicity, as well as essential fatty acid deficiency.Within the mitochondria, fatty acyl-CoA undergoes b-oxidation, which produces acetyl-CoA with each pass through the cycle. Each acetyl-CoA molecule subsequently enters the tricarboxylic acid (TCA) cycle for further oxidation to yield 12 adenosine triphosphate (ATP) molecules, carbon dioxide, and water. Excess acetyl-CoA molecules serve as precursors for ketogenesis. Unlike glucose metabolism, oxidation of fatty acids requires proportionally less oxygen and produces less car-bon dioxide. This is

1	Excess acetyl-CoA molecules serve as precursors for ketogenesis. Unlike glucose metabolism, oxidation of fatty acids requires proportionally less oxygen and produces less car-bon dioxide. This is frequently quantified as the ratio of carbon dioxide produced to oxygen consumed for the reaction and is known as the respiratory quotient (RQ). An RQ of 0.7 would imply greater fatty acid oxidation for fuel, whereas an RQ of 1 indicates greater carbohydrate oxidation (overfeeding). An RQ of 0.85 suggests the oxidation of equal amounts of fatty acids and glucose.KetogenesisCarbohydrate depletion slows the entry of acetyl-CoA into the TCA cycle secondary to depleted TCA intermediates and enzyme activity. Increased lipolysis and reduced systemic carbo-hydrate availability during starvation diverts excess acetyl-CoA Hormone-receptoractivationCapillaryAdiposecellFFAcAMPProtein kinaseTriglyceride lipaseTriglycerideDiglycerideMonoglycerideGlycerolFFAFFAFFAFigure 2-20. Fat mobilization in adipose

1	excess acetyl-CoA Hormone-receptoractivationCapillaryAdiposecellFFAcAMPProtein kinaseTriglyceride lipaseTriglycerideDiglycerideMonoglycerideGlycerolFFAFFAFFAFigure 2-20. Fat mobilization in adipose tissue. Triglyceride lipase activation by hormonal stimulation of adipose cells occurs through the cyclic adenosine monophosphate (cAMP) pathway. Triglycerides are serially hydrolyzed with resultant free fatty acid (FFA) release at every step. The FFAs diffuse readily into the capillary bed for transport. Tissues with glycerokinase can use glycerol for fuel by forming glycerol-3-phosphate. Glycerol-3-phosphate can esterify with FFAs to form triglycerides or can be used as a precursor for renal and hepatic gluconeogenesis. Skeletal muscle and adipose cells have little glycerokinase and thus do not use glycerol for fuel.Brunicardi_Ch02_p0027-p0082.indd 6401/03/19 6:50 PM 65SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2Carnitineacyltransferase

1	little glycerokinase and thus do not use glycerol for fuel.Brunicardi_Ch02_p0027-p0082.indd 6401/03/19 6:50 PM 65SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2Carnitineacyltransferase ITransportproteinCytosolMitochondriaFFAAcetyl-CoABeta OxidationCarnitineCarnitineacyltransferase IICoACarnitineMitochondrialmembraneORCCarnitineORCCoAROCCoAORCCarnitineCoAFigure 2-21. Free fatty acids (FFAs) in the cells form fatty acyl-coenzyme A (CoA) with CoA. Fatty acyl-CoA cannot enter the inner mitochondrial membrane and requires carnitine as a carrier protein (carnitine shuttle). Once inside the mitochondria, carnitine dissociates, and fatty acyl-CoA is reformed. The carnitine molecule is transported back into the cytosol for reuse. The fatty acyl-CoA undergoes β-oxidation to form acetyl-CoA for entry into the tri-carboxylic acid cycle. “R” represents a part of the acyl group of acyl-CoA.toward hepatic ketogenesis. A number of extrahepatic tissues, but not the liver itself, are

1	for entry into the tri-carboxylic acid cycle. “R” represents a part of the acyl group of acyl-CoA.toward hepatic ketogenesis. A number of extrahepatic tissues, but not the liver itself, are capable of using ketones for fuel. Ketosis represents a state in which hepatic ketone production exceeds extrahepatic ketone utilization.The rate of ketogenesis appears to be inversely related to the severity of injury. Major trauma, severe shock, and sepsis attenuate ketogenesis by increasing insulin levels and by caus-ing rapid tissue oxidation of free fatty acids. Minor injuries and infections are associated with modest elevations in plasma free fatty acid concentrations and ketogenesis. However, in minor stress states ketogenesis does not exceed that in nonstressed starvation.Carbohydrate MetabolismIngested and enteral carbohydrates are primarily digested in the small intestine, where pancreatic and intestinal enzymes reduce the complex carbohydrates to dimeric units. Disaccharidases (e.g.,

1	and enteral carbohydrates are primarily digested in the small intestine, where pancreatic and intestinal enzymes reduce the complex carbohydrates to dimeric units. Disaccharidases (e.g., sucrase, lactase, and maltase) within intestinal brush bor-ders dismantle the complex carbohydrates into simple hexose units, which are transported into the intestinal mucosa. Glu-cose and galactose are primarily absorbed by energy-dependent active transport coupled to the sodium pump. Fructose absorp-tion, however, occurs by concentration-dependent facilitated diffusion. Neither fructose and galactose within the circulation nor exogenous mannitol (for neurologic injury) evokes an insu-lin response. Intravenous administration of low-dose fructose in fasting humans has been associated with nitrogen conservation, but the clinical utility of fructose administration in human injury remains to be demonstrated.Discussion of carbohydrate metabolism primarily refers to the utilization of glucose. The

1	conservation, but the clinical utility of fructose administration in human injury remains to be demonstrated.Discussion of carbohydrate metabolism primarily refers to the utilization of glucose. The oxidation of 1 g of carbohy-drate yields 4 kcal, but sugar solutions such as those found in intravenous fluids or parenteral nutrition provide only 3.4 kcal/g of dextrose. In starvation, glucose production occurs at the expense of protein stores (i.e., skeletal muscle). Hence, the primary goal for maintenance glucose administration in surgical patients is to minimize muscle wasting. The exog-enous administration of small amounts of glucose (approxi-mately 50 g/d) facilitates fat entry into the TCA cycle and reduces ketosis. Unlike in starvation in healthy subjects, in septic and trauma patients provision of exogenous glucose never has been shown to fully suppress amino acid degrada-tion for gluconeogenesis. This suggests that during periods of stress, other hormonal and proinflammatory

1	provision of exogenous glucose never has been shown to fully suppress amino acid degrada-tion for gluconeogenesis. This suggests that during periods of stress, other hormonal and proinflammatory mediators have a profound influence on the rate of protein degradation and that some degree of muscle wasting is inevitable. The administra-tion of insulin, however, has been shown to reverse protein catabolism during severe stress by stimulating protein synthe-sis in skeletal muscles and by inhibiting hepatocyte protein degradation. Insulin also stimulates the incorporation of ele-mental precursors into nucleic acids in association with RNA synthesis in muscle cells.In cells, glucose is phosphorylated to form glucose-6-phosphate. Glucose-6-phosphate can be polymerized during glycogenesis or catabolized in glycogenolysis. Glucose catabolism occurs by cleavage to pyruvate or lactate (pyruvic acid pathway) or by decarboxylation to pentoses (pentose shunt) (Fig. 2-22).Excess glucose from

1	or catabolized in glycogenolysis. Glucose catabolism occurs by cleavage to pyruvate or lactate (pyruvic acid pathway) or by decarboxylation to pentoses (pentose shunt) (Fig. 2-22).Excess glucose from overfeeding, as reflected by RQs >1.0, can result in conditions such as glucosuria, thermogenesis, and conversion to fat (lipogenesis). Excessive glucose adminis-tration results in elevated carbon dioxide production, which may be deleterious in patients with suboptimal pulmonary function, as well as hyperglycemia, which may contribute to infectious risk and immune suppression.Injury and severe infections acutely induce a state of peripheral glucose intolerance, despite ample insulin produc-tion at levels several-fold above baseline. This may occur in part due to reduced skeletal muscle pyruvate dehydrogenase activity after injury, which diminishes the conversion of pyru-vate to acetyl-CoA and subsequent entry into the TCA cycle. The three-carbon structures (e.g., pyruvate and lactate)

1	dehydrogenase activity after injury, which diminishes the conversion of pyru-vate to acetyl-CoA and subsequent entry into the TCA cycle. The three-carbon structures (e.g., pyruvate and lactate) that consequently accumulate are shunted to the liver as substrate for gluconeogenesis. Furthermore, regional tissue catheteriza-tion and isotope dilution studies have shown an increase in net splanchnic glucose production by 50% to 60% in septic patients and a 50% to 100% increase in burn patients.242 The increase in plasma glucose levels is proportional to the severity of injury, and this net hepatic gluconeogenic response is believed to be under the influence of glucagon. Unlike in the nonstressed sub-ject, in the hypermetabolic, critically ill patient the hepatic glu-coneogenic response to injury or sepsis cannot be suppressed by exogenous or excess glucose administration but rather persists. Hepatic gluconeogenesis, arising primarily from alanine and glutamine catabolism, provides a ready

1	or sepsis cannot be suppressed by exogenous or excess glucose administration but rather persists. Hepatic gluconeogenesis, arising primarily from alanine and glutamine catabolism, provides a ready fuel source for tissues such as those of the nervous system, wounds, and erythrocytes, which do not require insulin for glucose transport. The elevated glucose concentrations also provide a necessary energy source for leukocytes in inflamed tissues and in sites of microbial invasions.Brunicardi_Ch02_p0027-p0082.indd 6501/03/19 6:50 PM 66BASIC CONSIDERATIONSPART IThe shunting of glucose away from nonessential organs such as skeletal muscle and adipose tissues is mediated by cat-echolamines. Experiments with infusing catecholamines and glucagon in animals have demonstrated elevated plasma glu-cose levels as a result of increased hepatic gluconeogenesis and peripheral insulin resistance. Interestingly, although glucocorti-coid infusion alone does not increase glucose levels, it does

1	glu-cose levels as a result of increased hepatic gluconeogenesis and peripheral insulin resistance. Interestingly, although glucocorti-coid infusion alone does not increase glucose levels, it does pro-long and augment the hyperglycemic effects of catecholamines and glucagon when glucocorticoid is administered concurrently with the latter.Glycogen stores within skeletal muscles can be mobilized by epinephrine activation of b-adrenergic receptors, GTP-binding proteins (G-proteins), which subsequently activates the second messenger, cAMP. The cAMP activates phosphorylase kinase, which in turn leads to conversion of glycogen to glucose-1-phosphate. Phosphorylase kinase also can be activated by the second messenger, calcium, through the breakdown of phos-phatidylinositol phosphate, which is the case in vasopressin-mediated hepatic glycogenolysis.243Protein and Amino Acid MetabolismThe average protein intake in healthy young adults ranges from 80 to 120 g/d, and every 6 g of protein yields

1	case in vasopressin-mediated hepatic glycogenolysis.243Protein and Amino Acid MetabolismThe average protein intake in healthy young adults ranges from 80 to 120 g/d, and every 6 g of protein yields approximately 1 g of nitrogen. The degradation of 1 g of protein yields approxi-mately 4 kcal of energy, similar to the yield in carbohydrate metabolism. After injury, the initial systemic proteolysis, medi-ated primarily by glucocorticoids, increases urinary nitrogen excretion to levels in excess of 30 g/d, which roughly corre-sponds to a loss in lean body mass of 1.5% per day. An injured individual who does not receive nutrition for 10 days can theo-retically lose 15% lean body mass. Therefore, amino acids can-not be considered a long-term fuel reserve, and indeed excessive protein depletion (i.e., 25% to 30% of lean body weight) is not compatible with sustaining life.244Protein catabolism after injury provides substrates for gluconeogenesis and for the synthesis of acute phase proteins.

1	(i.e., 25% to 30% of lean body weight) is not compatible with sustaining life.244Protein catabolism after injury provides substrates for gluconeogenesis and for the synthesis of acute phase proteins. Radiolabeled amino acid incorporation studies and protein anal-yses confirm that skeletal muscles are preferentially depleted acutely after injury, whereas visceral tissues (e.g., the liver and kidney) remain relatively preserved. The accelerated urea excretion after injury also is associated with the excretion of intracellular elements such as sulfur, phosphorus, potassium, magnesium, and creatinine. Conversely, the rapid utilization of elements such as potassium and magnesium during recovery from major injury may indicate a period of tissue healing.The net changes in protein catabolism and synthesis corre-spond to the severity and duration of injury (Fig. 2-23). Elective operations and minor injuries result in lower protein synthesis and moderate protein breakdown. Severe trauma, burns,

1	corre-spond to the severity and duration of injury (Fig. 2-23). Elective operations and minor injuries result in lower protein synthesis and moderate protein breakdown. Severe trauma, burns, and sepsis are associated with increased protein catabolism. The rise in urinary nitrogen and negative nitrogen balance can be detected early after injury and peak by 7 days. This state of pro-tein catabolism may persist for as long as 3 to 7 weeks. The patient’s prior physical status and age appear to influence the degree of proteolysis after injury or sepsis. Activation of the ubiquitin-proteosome system in muscle cells is one of the major path-ways for protein degradation during acute injury. This response is accentuated by tissue hypoxia, acidosis, insulin resistance, and elevated glucocorticoid levels.NUTRITION IN THE SURGICAL PATIENTThe goal of nutritional support in the surgical patient is to prevent or reverse the catabolic effects of disease or injury. Although several important biologic

1	IN THE SURGICAL PATIENTThe goal of nutritional support in the surgical patient is to prevent or reverse the catabolic effects of disease or injury. Although several important biologic parameters have been used to measure the efficacy of nutritional regimens, the ultimate validation for nutritional support in surgical patients should be improvement in clinical outcome and restoration of function.Estimation of Energy RequirementsAll patients admitted to the hospital should have their nutritional status assessed. Overall nutritional assessment is undertaken to 7GlycogenGlucoseGlucose-6-Phosphate6-PhosphogluconatePyruvicacidLacticacidPentosemonophosphateshuntTricarboxylic acidFigure 2-22. Simplified schema of glucose catabolism through the pen-tose monophosphate pathway or by breakdown into pyruvate. Glucose-6-phosphate becomes an important “crossroad” for glucose metabolism.Brunicardi_Ch02_p0027-p0082.indd 6601/03/19 6:50 PM 67SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER

1	Glucose-6-phosphate becomes an important “crossroad” for glucose metabolism.Brunicardi_Ch02_p0027-p0082.indd 6601/03/19 6:50 PM 67SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2Nitrogen excretion (g/d)128041620242832DaysMajor burnsSevere sepsisSkeletal traumaInfection010203040Elective surgeryPartial starvationTotal starvationFigure 2-23. The effect of injury sever-ity on nitrogen wasting. (Reproduced with permission from Long CL, Schaffel N, Geiger JW, et al: Metabolic response to injury and illness: estimation of energy and protein needs from indirect calorimetry and nitrogen balance, JPEN J Parenter Enteral Nutr. 1979 Nov-Dec;3(6):452-456.)determine the severity of nutrient deficiencies or excess and to aid in predicting nutritional requirements. Pertinent information is obtained by determining the presence of weight loss, chronic illnesses, or dietary habits that influence the quantity and quality of food intake. Social habits predisposing to malnutrition and the

1	is obtained by determining the presence of weight loss, chronic illnesses, or dietary habits that influence the quantity and quality of food intake. Social habits predisposing to malnutrition and the use of medications that may influence food intake or urina-tion should also be investigated. Physical examination seeks to assess loss of muscle and adipose tissues, organ dysfunction, and subtle changes in skin, hair, or neuromuscular function reflecting frank or impending nutritional deficiency. Anthropo-metric data (i.e., weight change, skinfold thickness, and arm cir-cumference muscle area) and biochemical determinations (i.e., creatinine excretion, albumin level, prealbumin level, total lym-phocyte count, and transferrin level) may be used to substantiate the patient’s history and physical findings. This information, in conjunction with nutritional risk assessment scoring, can iden-tify patients who may benefit from early nutritional support.For critically ill and injured patients,

1	findings. This information, in conjunction with nutritional risk assessment scoring, can iden-tify patients who may benefit from early nutritional support.For critically ill and injured patients, validated scoring systems such as the Nutritional Risk Screening (NRS)245 or the Nutrition Risk in the Critically Ill (NUTRIC)246 score should be employed to make this determination and should be performed in conjunction with assessment of GI tract function and risk of aspiration. Appreciation for the stresses and natural history of the disease process, in combination with nutritional assess-ment, remains the basis for identifying patients in acute or anticipated need of nutritional support. Currently, specialized enteral nutrition can be avoided in patients who are deemed to be a low nutritional risk with low disease severity for up to one week. However, their nutritional status should be reassessed regularly.247A fundamental goal of nutritional support is to meet the energy requirements for

1	risk with low disease severity for up to one week. However, their nutritional status should be reassessed regularly.247A fundamental goal of nutritional support is to meet the energy requirements for essential metabolic processes and tissue repair. Failure to provide adequate nonprotein energy sources will lead to consumption of lean tissue stores. The requirement for energy may be measured by indirect calorimetry, which is the gold standard in hospitalized patients and is recommended for the critically ill.243 However, the use of indirect calorimetry, particularly in the critically ill patient, may not be available or feasible in this setting. Moreover, it may lead to an overestima-tion of caloric requirements, which has been associated with increased risk of infectious complications.248In the absence of indirect calorimetry, resting energy expenditure may also be estimated using a published predictive equation. Adjusted for the type of surgical stress, such equations are suitable

1	the absence of indirect calorimetry, resting energy expenditure may also be estimated using a published predictive equation. Adjusted for the type of surgical stress, such equations are suitable for estimating energy requirements in the major-ity of hospitalized patients. Alternately, a simple weight-based equation of 25 to 30 kcal/kg (using dry or usual body weight) per day is appropriate with a low risk of overfeeding and is con-sistent with current recommendations from ASPEN (American Society of Parenteral and Enteral Nutrition) in the ICU setting.247 After trauma or sepsis, energy substrate demands are increased during the recovery phase and may necessitate greater nonpro-tein calories beyond calculated energy expenditure (Table 2-9). These additional nonprotein calories provided after injury are usually 1.2 to 2.0 times greater than calculated resting energy expenditure, depending on the type of injury. It is seldom appro-priate to exceed this level of nonprotein energy intake

1	injury are usually 1.2 to 2.0 times greater than calculated resting energy expenditure, depending on the type of injury. It is seldom appro-priate to exceed this level of nonprotein energy intake during the height of the catabolic phase. Currently, standard enteral nutri-tion delivers 49% to 53% of calories as carbohydrate and 29% to 30% of calories as fat, which is consistent with current recom-mendations. For parenteral nutrition, dextrose-containing stock solutions are prepared and available in different concentrations. The percentage of calories that is contributed by dextrose should be determined on a per-patient basis according to the severity of injury/illness and the estimated caloric needs. Lipid emul-sions can be included in the total mixture or be administered separately in 10% or 20% solutions with 1.1 kcal/ml and 2 kcal/ml, respectively.The second objective of nutritional support is to meet the substrate requirements for protein synthesis. Protein nutritional support is

1	or 20% solutions with 1.1 kcal/ml and 2 kcal/ml, respectively.The second objective of nutritional support is to meet the substrate requirements for protein synthesis. Protein nutritional support is especially important for maintaining immune func-tion and lean body mass and is more closely linked to positive outcomes than total caloric intake. Although the mean protein requirement of healthy individuals is defined as 0.8 g/kg per day by the Food and Nutrition Board of the U.S. National Research Council, current recommendations for protein dosing exceed this Brunicardi_Ch02_p0027-p0082.indd 6701/03/19 6:50 PM 68BASIC CONSIDERATIONSPART Iamount (1.2–2 gm protein/kg per day), especially for the critically ill and injured. Higher protein intake seems to support improved nitrogen balance and high-protein nutritional support is currently recommended for patients with body mass index >30.247Vitamins and MineralsThe requirements for vitamins and essential trace minerals usu-ally can be

1	and high-protein nutritional support is currently recommended for patients with body mass index >30.247Vitamins and MineralsThe requirements for vitamins and essential trace minerals usu-ally can be met easily in the average patient with an uncom-plicated postoperative course. Therefore, vitamins usually are not given in the absence of preoperative deficiencies. Patients maintained on elemental diets or parenteral hyperalimenta-tion require complete vitamin and mineral supplementation. Commercial enteral diets contain varying amounts of essential minerals and vitamins. It is necessary to ensure that adequate replacement is available in the diet or by supplementation. Numerous commercial vitamin preparations are available for intravenous or intramuscular use, although most do not contain vitamin K and some do not contain vitamin B12 or folic acid. Supplemental trace minerals may be given intravenously via commercial preparations. Essential fatty acid supplementation also may be

1	vitamin K and some do not contain vitamin B12 or folic acid. Supplemental trace minerals may be given intravenously via commercial preparations. Essential fatty acid supplementation also may be necessary, especially in patients with depletion of adipose stores.OverfeedingOverfeeding usually results from overestimation of caloric needs, as occurs when actual body weight is used to calculate the BEE in patient populations such as the critically ill with sig-nificant fluid overload and the obese. Indirect calorimetry can be used to quantify energy requirements but frequently overes-timates BEE by 10% to 15% in stressed patients, particularly if they are receiving ventilatory support. In these instances, esti-mated dry weight should be obtained from preinjury records or family members. Adjusted lean body weight also can be cal-culated. Overfeeding may contribute to clinical deterioration via increased oxygen consumption, increased carbon dioxide production and prolonged need for

1	Adjusted lean body weight also can be cal-culated. Overfeeding may contribute to clinical deterioration via increased oxygen consumption, increased carbon dioxide production and prolonged need for ventilatory support, fatty liver, suppression of leukocyte function, hyperglycemia, and increased risk of infection.ENTERAL NUTRITIONRationale for Enteral NutritionEnteral nutrition (EN) is preferred over parenteral nutrition (PN) based on the lower cost of enteral feeding and the associ-ated risks of the intravenous route, including vascular access complications.249 Of further consideration are the consequences of gastrointestinal tract disuse, which include diminished solu-ble IgA production and cytokine production as well as bacterial overgrowth and altered mucosal barrier function and immune defenses. In support of this idea, recent meta-analysis demon-strated a significant reduction in infectious complications in criti-cally ill or injured patients receiving EN when compared to PN as

1	defenses. In support of this idea, recent meta-analysis demon-strated a significant reduction in infectious complications in criti-cally ill or injured patients receiving EN when compared to PN as well as ICU length of stay.250 However, no increase in overall survival was noted. While EN is recommended as the first choice for nutritional support in patients who can tolerate it, a recent large trial from Europe comparing early isocaloric EN vs. PN in adult critically ill patients with shock did not reduce mortality or the risk of secondary infections but was associated with a greater risk of digestive complications including intestinal ischemia.251The benefits of enteral feeding in patients undergoing elective surgery appear to be linked to their preoperative nutri-tional status. Historical studies comparing postoperative enteral and parenteral nutrition in patients undergoing gastrointestinal surgery have demonstrated reduced infectious complications and acute phase protein production

1	studies comparing postoperative enteral and parenteral nutrition in patients undergoing gastrointestinal surgery have demonstrated reduced infectious complications and acute phase protein production in those fed by the enteral route. Yet prospectively randomized studies of patients with adequate nutritional status (albumin ≥4 g/dL) undergoing gas-trointestinal surgery demonstrate no differences in outcome and complications between those administered enteral nutrition and those given maintenance intravenous fluids alone in the initial days after surgery.252Early vs. Late FeedingCurrent recommendations support early enteral nutrition (within 48 hours) in critically ill patients, but with a caveat.253 Early “full nutrition” is likely to be harmful and is associated with a higher infection rate. The aim therefore is a caloric target below the actual energy expenditure, with the goal of providing >80% of estimated total energy goals gradually by 3 to 4 days. Early EN may be protective of

1	The aim therefore is a caloric target below the actual energy expenditure, with the goal of providing >80% of estimated total energy goals gradually by 3 to 4 days. Early EN may be protective of the enteral epithelial barrier func-tion and help to maintain the diversity of the microbiome. While early caloric limitation seems to benefit the critically ill patient when compared to overfeeding, the restriction likely creates a significant shortfall in protein provision considering the low protein-to-calorie ratio of most enteral products.254In this regard, it is important to distinguish “permissive underfeeding” in which the total calories provided average 1500 kcal/d with 40 gm/d of protein from hypocaloric nutri-tion which has the same total calories with 140 gm/d protein. Hypocaloric nutrition is currently recommended for critically ill obese patients, but some investigators argue that this nutritional strategy may also benefit nonobese patients especially during Table 2-9Caloric

1	nutrition is currently recommended for critically ill obese patients, but some investigators argue that this nutritional strategy may also benefit nonobese patients especially during Table 2-9Caloric adjustments above basal energy expenditure (BEE) in hypermetabolic conditionsCONDITIONkcal/kg PER DAYADJUSTMENT ABOVE BEEGRAMS OF PROTEIN/ kg PER DAYNONPROTEIN CALORIES: NITROGENNormal/moderate malnutrition25–301.11.0150:1Mild stress25–301.21.2150:1Moderate stress301.41.5120:1Severe stress30–351.62.090–120:1Burns35–402.02.590–100:1Brunicardi_Ch02_p0027-p0082.indd 6801/03/19 6:50 PM 69SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2the early acute period of critical illness.255 This recommendation excludes those patients whose pre-ICU weight loss indicate that they are malnourished.As the patient enters the recovery period of their illness, total protein and caloric requirements are likely to significantly increase. Based on our understanding of starvation, increased

1	the patient enters the recovery period of their illness, total protein and caloric requirements are likely to significantly increase. Based on our understanding of starvation, increased extrinsic delivery of both calories and protein are likely to be required during this period.For patients undergoing elective surgery, healthy patients without malnutrition who are undergoing uncomplicated sur-gery can tolerate 10 days of partial starvation (i.e., maintenance intravenous fluids only) before any clinically significant protein catabolism occurs. Earlier intervention is likely indicated for patients in whom preoperative protein-calorie malnutrition has been identified. Other clinical scenarios for which the benefits of enteral nutritional support have been substantiated include permanent neurologic impairment, oropharyngeal dysfunction, short-bowel syndrome, and bone marrow transplantation.Initiation of enteral nutrition should occur as soon as fea-sible after adequate resuscitation, most

1	impairment, oropharyngeal dysfunction, short-bowel syndrome, and bone marrow transplantation.Initiation of enteral nutrition should occur as soon as fea-sible after adequate resuscitation, most readily determined by adequate urine output. The presence of bowel sounds and the passage of flatus or stool are not absolute prerequisites for ini-tiation of enteral nutrition, but in the setting of gastroparesis feedings should be administered distal to the pylorus. Gastric residuals of 200 mL or more in a 4to 6-hour period or abdomi-nal distention requires cessation of feeding and adjustment of the infusion rate. Concomitant gastric decompression with dis-tal small-bowel feedings may be appropriate in certain patients such as closed-head injury patients with gastroparesis. There is no evidence to support withholding enteric feedings for patients after bowel resection or for those with low-output enterocutane-ous fistulas of <500 mL/d. In fact, a recent systematic review of studies of early

1	to support withholding enteric feedings for patients after bowel resection or for those with low-output enterocutane-ous fistulas of <500 mL/d. In fact, a recent systematic review of studies of early enteral feeding (within 24 hours of gastro-intestinal surgery) showed no effect on anastomotic leak and a reduction in mortality. Early enteral feeding is also associated in reduced incidence of fistula formation in patients with open abdomen. Enteral feeding should also be offered to patients with short-bowel syndrome or clinical malabsorption, but necessary calories, essential minerals, and vitamins should be supple-mented using parenteral modalities.Intermittent vs. Continuous Enteral FeedingEnteral nutrition can be administered either continuously or intermittently; however, the standard choice for critically injured adults is continuous enteral feeding (CEF) due to the lower complication rates.247 Data also suggest that CEF may promote protein anabolism by inhibiting protein

1	choice for critically injured adults is continuous enteral feeding (CEF) due to the lower complication rates.247 Data also suggest that CEF may promote protein anabolism by inhibiting protein breakdown.256Enteral FormulasFor most critically ill patients, the choice of enteral formula will be determined by a number of factors and will include a clinical judgment as to the “best fit” for the patients’ needs. In general, feeding formulas to consider are GI tolerance-promot-ing, anti-inflammatory, immune-modulating, organ support-ive, and standard enteral nutrition. In addition, guidelines from professional nutrition societies identify certain populations of patients who can benefit from formulations with specific pharmaconutrients.257 For many others, each physician must use his or her own clinical judgment about what formula will best meet the patient’s needs.The functional status of the gastrointestinal tract deter-mines the type of enteral solutions to be used. Patients with an intact

1	judgment about what formula will best meet the patient’s needs.The functional status of the gastrointestinal tract deter-mines the type of enteral solutions to be used. Patients with an intact gastrointestinal tract will tolerate complex solutions, but patients who have not been fed via the gastrointestinal tract for prolonged periods are less likely to tolerate complex carbohy-drates. In those patients who are having difficulty tolerating standard enteral formulas, peptideand medium-chain triglyc-eride-based formulas with prebiotics can lessen GI tolerance problems. Additionally, in patients with demonstrated malab-sorption issues, such as with inflammatory bowel diseases or short bowel syndrome, current guidelines endorse the provision of hydrolyzed protein formulas to improve absorption. Guide-lines have not yet been made with regard to the fiber content of enteral formulas. However, recent evidence indicates that supplementation of enteral formulas with soluble dietary fiber may

1	Guide-lines have not yet been made with regard to the fiber content of enteral formulas. However, recent evidence indicates that supplementation of enteral formulas with soluble dietary fiber may be beneficial for improving stool consistency in patients suffering from diarrhea.Factors that influence the choice of enteral formula also include the extent of organ dysfunction (e.g., renal, pulmonary, hepatic, or gastrointestinal), the nutrients needed to restore optimal function and healing, and the cost of specific products. There are still no conclusive data to recommend one category of product over another, and nutritional support committees typically develop the most cost-efficient enteral formulary for the most commonly encountered disease categories within the institution.As discussed extensively in the first sections of this chap-ter, surgery and trauma result in a significant “sterile” inflamma-tory response that impacts for the innate and adaptive immune systems. The provision

1	extensively in the first sections of this chap-ter, surgery and trauma result in a significant “sterile” inflamma-tory response that impacts for the innate and adaptive immune systems. The provision of immune-modulating nutrients, termed “immunonutrition,” is one mechanism by which the immune response can be supported and an attempt made to lower infec-tious risk. At present, the best studied of immune-nutrients are glutamine, arginine, and omega-3 PUFAs.“Immunonutrients.” As discussed extensively in the first sections of this chapter, surgery and trauma result in a sig-nificant “sterile” inflammatory response that impacts both the innate and adaptive immune systems. The provision of immune-modulating nutrients, termed “immunonutrition,” is one mecha-nism by which the immune response can be supported and an attempt made to lower infectious risk. Studies have shown that a variety of nutrients, including amino acids (glutamine and arginine); lipids (omega-3 PUFAs); and micronutrients

1	be supported and an attempt made to lower infectious risk. Studies have shown that a variety of nutrients, including amino acids (glutamine and arginine); lipids (omega-3 PUFAs); and micronutrients (e.g., vitamin C and selenium) can provide support to the immune system. While current evidence does not support their use uni-versally, benefit may exist for individual patients.258 At present, the best studied of immune-nutrients are glutamine, arginine, and omega-3 PUFAs.Glutamine is the most abundant amino acid in the human body, comprising nearly two thirds of the free intracel-lular amino acid pool. Considered a nonessential amino acid, glutamine is a necessary substrate for nucleotide synthesis in most dividing cells and hence provides a major fuel source for enterocytes. It also serves as an important fuel source for immu-nocytes. During stress states, peripheral glutamine stores are rapidly depleted, and the amino acid is preferentially shunted as a fuel source toward the visceral

1	as an important fuel source for immu-nocytes. During stress states, peripheral glutamine stores are rapidly depleted, and the amino acid is preferentially shunted as a fuel source toward the visceral organs and tumors, respectively.259 These situations create, at least experimentally, a glutamine-depleted environment with potential immune consequences, thus generating interest in both enteral and parenteral glutamine supplementation. However, recently reported data from two large randomized controlled clinical trials in which critically ill patients received glutamine supplementation demonstrated Brunicardi_Ch02_p0027-p0082.indd 6901/03/19 6:50 PM 70BASIC CONSIDERATIONSPART Iincreased 6-month mortality in the patients who received glutamine.260 Therefore, glutamine supplementation in the criti-cally ill patient is not currently recommended.247Arginine, also a nonessential amino acid in healthy sub-jects, first attracted attention for its immunoenhancing proper-ties, wound-healing

1	the criti-cally ill patient is not currently recommended.247Arginine, also a nonessential amino acid in healthy sub-jects, first attracted attention for its immunoenhancing proper-ties, wound-healing benefits, and association with improved survival in animal models of sepsis and injury.261 However, arginine can be metabolized to nitric oxide, via nitric oxide synthase (NOS). If NOS is upregulated, with arginine as avail-able substrate, NO production can also increase, which can have a negative impact on the critically ill patient. As with glutamine, the benefits of experimental arginine supplementa-tion during stress states are diverse. In clinical studies involv-ing critically ill and injured patients and patients who have undergone surgery for certain malignancies, enteral adminis-tration of arginine has led to net nitrogen retention and pro-tein synthesis, whereas isonitrogenous diets have not. Some of these studies also provide in vitro evidence of enhanced immunocyte function.

1	of arginine has led to net nitrogen retention and pro-tein synthesis, whereas isonitrogenous diets have not. Some of these studies also provide in vitro evidence of enhanced immunocyte function. The clinical utility of arginine supple-mentation in improving overall patient outcome remains an area of investigation.262As previously discussed, omega-3 polyunsaturated fatty acids (PUFAs, canola oil, or fish oil) displaces omega-6 fatty acids in cell membranes, which theoretically reduces the pro-inflammatory response from prostaglandin production. Hence, there has been significant interest in reducing the ratio of omega-6 to omega-3. The data regarding supplementation of enteral feedings with fish oil as a source for omega-3 PUFAs has been mixed, however, with no demonstrated improvement in respiratory complications in severe trauma patients and pos-sible benefits in patients with mild sepsis.166Standard Polymeric Formulas. Most polymeric formulas provide a caloric density from 1 to 2

1	respiratory complications in severe trauma patients and pos-sible benefits in patients with mild sepsis.166Standard Polymeric Formulas. Most polymeric formulas provide a caloric density from 1 to 2 kcal/mL, and approxi-mately 1500 to 1800 mL are required to meet daily require-ments. These compositions provide baseline carbohydrates, protein, electrolytes, water, fat, and fat-soluble vitamins (some do not have vitamin K). These contain no fiber bulk and there-fore leave minimum residue. These solutions usually are consid-ered to be the standard or first-line formulas for stable patients with an intact gastrointestinal tract. Normal digestive function is required for this formula.Fiber-Containing Formulas. Isotonic formulas with fiber contain soluble and insoluble fiber, which is most often soy based. Physiologically, fiber-based solutions delay intestinal transit time and may reduce the incidence of diarrhea compared with nonfiber solutions. It is most beneficial in this regard in

1	soy based. Physiologically, fiber-based solutions delay intestinal transit time and may reduce the incidence of diarrhea compared with nonfiber solutions. It is most beneficial in this regard in patients who have a high number of loose stools.263 Fiber stimu-lates pancreatic lipase activity and is degraded by gut bacteria into short-chain fatty acids (SCFA), an important fuel for colo-nocytes. Recent data have also demonstrated the expression of SCFA receptors on leukocytes, suggesting that fiber fermenta-tion by the colonic microbiome may indirectly regulate immune cell function. Another potential plus of fiber-containing formu-las is the inclusion of prebiotic fibers with the goal of positively impacting bacterial targets in the gut as well as gut barrier func-tion. While there has been limited research in this area to deter-mine the possible impact on clinical outcomes, addition of these fermentable soluble fiber additives is something that should be considered in the ICU patient

1	limited research in this area to deter-mine the possible impact on clinical outcomes, addition of these fermentable soluble fiber additives is something that should be considered in the ICU patient as a measure that can aid in the maintenance or restoration of a healthy balance of commensal gut bacteria.Immune-Enhancing Formulas. Immune-enhancing formulas are fortified with special nutrients that are purported to enhance various aspects of immune or solid organ function as previously discussed. Such additives include glutamine, arginine, omega-3 fatty acids, and nucleotides.264 Although several trials have pro-posed that one or more of these additives reduce surgical com-plications and improve outcome, these results have not been uniformly corroborated by other trials. The Canadian Clinical Practice Guidelines currently do not recommend the addition of arginine supplements for critically ill patients due to the poten-tial for harm when used in septic patients.265 Omega-3 PUFAs results

1	Practice Guidelines currently do not recommend the addition of arginine supplements for critically ill patients due to the poten-tial for harm when used in septic patients.265 Omega-3 PUFAs results from the EDEN-Omega study demonstrated that twice-daily enteral supplementation of n-3 fatty acids, γ-linolenic acid, and antioxidants did not improve the primary end point of ventilator-free days or other clinical outcomes in patients with acute lung injury and may be harmful.266 Glutamine supplemen-tation should be strictly guided by the individual patient condi-tion for the reasons discussed previously.Calorie-Dense Formulas. The primary distinction of calorie-dense formulas is a greater caloric value for the same volume. Most commercial products of this variety provide 1.5 to 2 kcal/mL and therefore are suitable for patients requiring fluid restriction or those unable to tolerate large-volume infusions. As expected, these solutions have higher osmolality than standard formulas and are

1	therefore are suitable for patients requiring fluid restriction or those unable to tolerate large-volume infusions. As expected, these solutions have higher osmolality than standard formulas and are suitable for intragastric feedings.High-Protein Formulas/Bariatric Formulas. High-protein formulas are available in isotonic and nonisotonic mixtures and are proposed for critically ill or trauma patients with high protein requirements. These formulas have nonprotein-calorie to nitrogen ratios between 80:1 and 120:1. While some obser-vational studies show improved outcomes with higher protein intakes in critically ill patients, there is limited data from ran-domized trials that prevents making strong conclusions about the dose of protein in critically ill patients.As discussed previously, there has been support for high-protein, hypocaloric feeding in obese patients. As such, enteral formulas termed “bariatric formulas” have been developed. As an example, one product has 1 kcal/mL of

1	there has been support for high-protein, hypocaloric feeding in obese patients. As such, enteral formulas termed “bariatric formulas” have been developed. As an example, one product has 1 kcal/mL of formula, with 37% of the calories coming from protein. As the evidence for high-protein, hypocaloric feeding is low grade, it is unclear whether clinical outcomes with respect to survival and infectious com-plications is improved, and more data is required for definitive recommendation.267Elemental Formulas. Elemental formulas contain predigested nutrients and provide proteins in the form of small peptides. Complex carbohydrates are limited, and fat content, in the form of MCTs and LCTs, is minimal. The primary advantage of such a formula is ease of absorption, but the inherent scarcity of fat, associated vitamins, and trace elements limits its long-term use as a primary source of nutrients. Due to its high osmolarity, dilution, or slow infusion rates usually are necessary, particu-larly

1	associated vitamins, and trace elements limits its long-term use as a primary source of nutrients. Due to its high osmolarity, dilution, or slow infusion rates usually are necessary, particu-larly in critically ill patients. These formulas have been used frequently in patients with malabsorption, gut impairment, and pancreatitis, but their cost is significantly higher than that of standard formulas. To date, there has been no evidence of their benefit in routine use.Renal-Failure Formulas. The primary benefits of renal for-mulas are the lower fluid volume and concentrations of potas-sium, phosphorus, and magnesium needed to meet daily calorie requirements. This type of formulation almost exclusively Brunicardi_Ch02_p0027-p0082.indd 7001/03/19 6:50 PM 71SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2contains essential amino acids and has a high nonprotein-calorie to nitrogen ratio; however, it does not contain trace elements or vitamins. Current guidelines suggest that

1	METABOLIC SUPPORTCHAPTER 2contains essential amino acids and has a high nonprotein-calorie to nitrogen ratio; however, it does not contain trace elements or vitamins. Current guidelines suggest that patients with chronic kidney disease (CKD) who require enteral feeding should be placed on “standard enteral formulations.” Moreover, standard recommendations for both protein and calories support are also recommended.263Hepatic-Failure Formulas. Close to 50% of the proteins in hepatic-failure formulas are branched-chain amino acids (e.g., leucine, isoleucine, and valine). The goal of such a formula is to reduce aromatic amino acid levels and increase the levels of branched-chain amino acids, which can potentially reverse encephalopathy in patients with hepatic failure.268 The use of these formulas is controversial, however, because no clear bene-fits have been proven by clinical trials. Protein restriction should be avoided in patients with end-stage liver disease because such patients

1	is controversial, however, because no clear bene-fits have been proven by clinical trials. Protein restriction should be avoided in patients with end-stage liver disease because such patients have significant protein energy malnutrition that pre-disposes them to additional morbidity and mortality.269 Similar to patients with CKD, standard formulations are recommended initially unless the patient develops hepatic encephalopathy that is refractory to standard treatment. With regard to protein sup-plementation, data indicate that providing 1.5 gm protein/kg per day improves clinical outcomes in these patients.270Access for Enteral Nutritional SupportThe available techniques and repertoire for enteral access have provided multiple options for feeding the gut. Presently used methods and preferred indications are summarized in Table 2-10.271Nasoenteric Tubes. Nasogastric feeding should be reserved for those with intact mentation and protective laryngeal reflexes to minimize risks of

1	preferred indications are summarized in Table 2-10.271Nasoenteric Tubes. Nasogastric feeding should be reserved for those with intact mentation and protective laryngeal reflexes to minimize risks of aspiration. Even in intubated patients, naso-gastric feedings often can be recovered from tracheal suction. Nasojejunal feedings are associated with fewer pulmonary complications including risk of pneumonia, but access past the pylorus requires greater effort to accomplish. Therefore, routine use of small bowel feedings is preferred in units where small bowel access is readily feasible. Where there may be difficul-ties obtaining access, small bowel feedings may be considered a priority for those patients at high risk for intolerance to enteral nutrition (e.g., high gastric residuals).Blind insertion of nasogastric feeding tubes is fraught with misplacement, and air instillation with auscultation is inaccurate for ascertaining proper positioning. Radiographic confirmation is usually

1	insertion of nasogastric feeding tubes is fraught with misplacement, and air instillation with auscultation is inaccurate for ascertaining proper positioning. Radiographic confirmation is usually required to verify the position of the nasogastric feed-ing tube.Several methods have been recommended for the passage of nasoenteric feeding tubes into the small bowel, including use of prokinetic agents, right lateral decubitus positioning, gastric insufflation, tube angulation, and application of clock-wise torque. However, the successful placement of feeding tubes by these methods is highly variable and operator depen-dent. Furthermore, it is time consuming, and success rates for intubation past the duodenum into the jejunum by these methods are <20%. Fluoroscopy-guided intubation past the pylorus has a >90% success rate, and more than half of these intubations result in jejunal placement. Similarly, endoscopy-guided placement past the pylorus has high success rates, but attempts to

1	pylorus has a >90% success rate, and more than half of these intubations result in jejunal placement. Similarly, endoscopy-guided placement past the pylorus has high success rates, but attempts to advance the tube beyond the second portion of the duodenum using a standard gastroduodenoscope is unlikely to be successful.Table 2-10Options for enteral feeding accessACCESS OPTIONCOMMENTSNasogastric tubeShort-term use only; aspiration risks; nasopharyngeal trauma; frequent dislodgmentNasoduodenal/nasojejunal tubeShort-term use; lower aspiration risks in jejunum; placement challenges (radiographic assistance often necessary)Percutaneous endoscopic gastrostomy (PEG)Endoscopy skills required; may be used for gastric decompression or bolus feeds; aspiration risks; can last 12–24 mo; slightly higher complication rates with placement and site leaksSurgical gastrostomyRequires general anesthesia and small laparotomy; procedure may allow placement of extended duodenal/jejunal feeding ports;

1	higher complication rates with placement and site leaksSurgical gastrostomyRequires general anesthesia and small laparotomy; procedure may allow placement of extended duodenal/jejunal feeding ports; laparoscopic placement possibleFluoroscopic gastrostomyBlind placement using needle and T-prongs to anchor to stomach; can thread smaller catheter through gastrostomy into duodenum/jejunum under fluoroscopyPEG-jejunal tubeJejunal placement with regular endoscope is operator dependent; jejunal tube often dislodges retrograde; two-stage procedure with PEG placement, followed by fluoroscopic conversion with jejunal feeding tube through PEGDirect percutaneous endoscopic jejunostomy (DPEJ)Direct endoscopic tube placement with enteroscope; placement challenges; greater injury risksSurgical jejunostomyCommonly carried out during laparotomy; general anesthesia; laparoscopic placement usually requires assistant to thread catheter; laparoscopy offers direct visualization of catheter

1	jejunostomyCommonly carried out during laparotomy; general anesthesia; laparoscopic placement usually requires assistant to thread catheter; laparoscopy offers direct visualization of catheter placementFluoroscopic jejunostomyDifficult approach with injury risks; not commonly doneSmall-bowel feeding is more reliable for delivering nutri-tion than nasogastric feeding. Furthermore, the risks of aspi-ration pneumonia can be reduced by 25% with small-bowel feeding compared with nasogastric feeding. The disadvantages of the use of nasoenteric feeding tubes are clogging, kinking, and inadvertent displacement or removal of the tube as well as nasopharyngeal complications. If nasoenteric feeding will be required for longer than 30 days, access should be converted to a percutaneous one.272Brunicardi_Ch02_p0027-p0082.indd 7101/03/19 6:50 PM 72BASIC CONSIDERATIONSPART IPercutaneous Endoscopic Gastrostomy. The most common indications for percutaneous endoscopic gastrostomy (PEG) include

1	7101/03/19 6:50 PM 72BASIC CONSIDERATIONSPART IPercutaneous Endoscopic Gastrostomy. The most common indications for percutaneous endoscopic gastrostomy (PEG) include impaired swallowing mechanisms, oropharyngeal or esophageal obstruction, and major facial trauma. It is frequently used for debilitated patients requiring caloric supplementation, hydration, or frequent medication dosing. It is also appropriate for patients requiring passive gastric decompression. Relative contraindications for PEG placement include ascites, coagu-lopathy, gastric varices, gastric neoplasm, and lack of a suitable abdominal site. Most tubes are 18F to 28F in size and may be used for 12 to 24 months.Identification of the PEG site requires endoscopic transil-lumination of the anterior stomach against the abdominal wall. A 14-gauge angiocatheter is passed through the abdominal wall into the fully insufflated stomach. A guidewire is threaded through the angiocatheter, grasped by snares or forceps, and

1	abdominal wall. A 14-gauge angiocatheter is passed through the abdominal wall into the fully insufflated stomach. A guidewire is threaded through the angiocatheter, grasped by snares or forceps, and pulled out through the mouth. The tapered end of the PEG tube is secured to the guidewire and is pulled into position out of the abdominal wall. The PEG tube is secured without tension against the abdominal wall, and many have reported using the tube within hours of placement. It has been the practice of some to connect the PEG tube to a drainage bag for passive decom-pression for 24 hours before use, allowing more time for the stomach to seal against the peritoneum.If endoscopy is not available or technical obstacles pre-clude PEG placement, the interventional radiologist can attempt the procedure percutaneously under fluoroscopic guidance by first insufflating the stomach against the abdominal wall with a nasogastric tube. If this also is unsuccessful, surgical gas-trostomy tube

1	the procedure percutaneously under fluoroscopic guidance by first insufflating the stomach against the abdominal wall with a nasogastric tube. If this also is unsuccessful, surgical gas-trostomy tube placement can be considered, particularly with minimally invasive methods. When surgery is contemplated, it may be wise to consider directly accessing the small bowel for nutrition delivery.Although PEG tubes enhance nutritional delivery, facili-tate nursing care, and are superior to nasogastric tubes, serious complications occur in approximately 3% of patients. These complications include wound infection, necrotizing fasciitis, peritonitis, aspiration, leaks, dislodgment, bowel perforation, enteric fistulas, bleeding, and aspiration pneumonia.273 For patients with significant gastroparesis or gastric outlet obstruc-tion, feedings through PEG tubes are hazardous. In such cases, the PEG tube can be used for decompression and allow access for converting the PEG tube to a transpyloric

1	or gastric outlet obstruc-tion, feedings through PEG tubes are hazardous. In such cases, the PEG tube can be used for decompression and allow access for converting the PEG tube to a transpyloric feeding tube.Percutaneous Endoscopic Gastrostomy-Jejunostomy and Direct Percutaneous Endoscopic Jejunostomy. Although gastric bolus feedings are more physiologic, patients who can-not tolerate gastric feedings or who have significant aspiration risks should be fed directly past the pylorus. In the percutane-ous endoscopic gastrostomy-jejunostomy (PEG-J) method, a 9F to 12F tube is passed through an existing PEG tube, past the pylorus, and into the duodenum. This can be achieved by endoscopic or fluoroscopic guidance. With weighted catheter tips and guidewires, the tube can be further advanced past the ligament of Treitz. However, the incidence of long-term PEG-J tube malfunction has been reported to be >50% as a result of ret-rograde tube migration into the stomach, kinking, or clogging.Direct

1	the ligament of Treitz. However, the incidence of long-term PEG-J tube malfunction has been reported to be >50% as a result of ret-rograde tube migration into the stomach, kinking, or clogging.Direct percutaneous endoscopic jejunostomy (DPEJ) tube placement uses the same techniques as PEG tube placement but requires an enteroscope or colonoscope to reach the jejunum. DPEJ tube malfunctions are probably less frequent than PEG-J tube malfunctions, and kinking or clogging is usually averted by placement of larger-caliber catheters. The success rate of DPEJ tube placement is variable because of the complexity of endo-scopic skills required to locate a suitable jejunal site. In such cases where endoscopic means are not feasible, surgical jeju-nostomy tube placement is more appropriate, especially when minimally invasive techniques are available.Surgical Gastrostomy and Jejunostomy. For a patient undergoing complex abdominal or trauma surgery, thought should be given during surgery to the

1	when minimally invasive techniques are available.Surgical Gastrostomy and Jejunostomy. For a patient undergoing complex abdominal or trauma surgery, thought should be given during surgery to the possible routes for sub-sequent nutritional support because laparotomy affords direct access to the stomach or small bowel. The only absolute contra-indication to feeding jejunostomy is distal intestinal obstruction. Relative contraindications include severe edema of the intestinal wall, radiation enteritis, inflammatory bowel disease, ascites, severe immunodeficiency, and bowel ischemia. Needle-catheter jejunostomies also can be done with a minimal learning curve. The biggest drawback usually is possible clogging and knotting of the 6F catheter.274Abdominal distention and cramps are common adverse effects of early enteral nutrition. Some have also reported impaired respiratory mechanics as a result of intolerance to enteral feedings. These are mostly correctable by temporarily discontinuing

1	effects of early enteral nutrition. Some have also reported impaired respiratory mechanics as a result of intolerance to enteral feedings. These are mostly correctable by temporarily discontinuing feedings and resuming at a lower infusion rate.Pneumatosis intestinalis and small-bowel necrosis are infrequent but significant problems in patients receiving jejunal tube feedings. Several contributing factors have been proposed, including the hyperosmolarity of enteral solutions, bacte-rial overgrowth, fermentation, and accumulation of metabolic breakdown products. The common pathophysiology is believed to be bowel distention and consequent reduction in bowel wall perfusion. Risk factors for these complications include cardio-genic and circulatory shock, vasopressor use, diabetes mellitus, and chronic obstructive pulmonary disease. Therefore, enteral feedings in the critically ill patient should be delayed until adequate resuscitation has been achieved. As alternatives, dilut-ing standard

1	chronic obstructive pulmonary disease. Therefore, enteral feedings in the critically ill patient should be delayed until adequate resuscitation has been achieved. As alternatives, dilut-ing standard enteral formula, delaying the progression to goal infusion rates, or using monomeric solutions with low osmolal-ity requiring less digestion by the gastrointestinal tract all have been successfully used.PARENTERAL NUTRITIONParenteral nutrition is the continuous infusion of a hyperosmo-lar solution containing carbohydrates, proteins, fat, and other necessary nutrients through an indwelling catheter inserted into the superior vena cava. To obtain the maximum benefit, the calorie to protein ratio must be adequate (at least 100 to 150 kcal/g nitrogen), and both carbohydrates and proteins must be infused simultaneously. When the sources of calories and nitrogen are given at different times, there is a significant decrease in nitrogen utilization. These nutrients can be given in quantities

1	be infused simultaneously. When the sources of calories and nitrogen are given at different times, there is a significant decrease in nitrogen utilization. These nutrients can be given in quantities considerably greater than the basic caloric and nitro-gen requirements, and this method has proved to be highly suc-cessful in achieving growth and development, positive nitrogen balance, and weight gain in a variety of clinical situations. Clini-cal trials and meta-analysis of studies of parenteral feeding in the perioperative period have suggested that preoperative nutri-tional support may benefit some surgical patients, particularly those with extensive malnutrition.Historically, short-term use of parenteral nutrition (PN) in critically ill patients (i.e., duration of <7 days) when enteral Brunicardi_Ch02_p0027-p0082.indd 7201/03/19 6:50 PM 73SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2nutrition (EN) may have been instituted was associated with higher rates of

1	enteral Brunicardi_Ch02_p0027-p0082.indd 7201/03/19 6:50 PM 73SYSTEMIC RESPONSE TO INJURY AND METABOLIC SUPPORTCHAPTER 2nutrition (EN) may have been instituted was associated with higher rates of infectious complications. It appears, however, that the increased mortality associated with PN may have been associated with excessive caloric delivery. More recent data have shown no mortality difference between EN and PN when caloric delivery was reduced and matched. A recent meta-analysis confirmed this result and noted no increase in infectious complications.250 That being said, the risk/benefit for PN in the ICU is much smaller and in a patient with low nutritional risk provides little benefit over the first week of hospitalization in the ICU.Rationale for Parenteral NutritionThe principal indications for parenteral nutrition are malnu-trition, sepsis, or surgical or traumatic injury in seriously ill patients for whom use of the gastrointestinal tract for feedings is not possible.247

1	indications for parenteral nutrition are malnu-trition, sepsis, or surgical or traumatic injury in seriously ill patients for whom use of the gastrointestinal tract for feedings is not possible.247 Parenteral nutrition should not be used based solely on the medical diagnosis or disease state. Rather, PN use is recommended for those critically ill or injured patients who are at high nutritional risk, when EN is not possible. Alter-nately, PN can also be used to supplement EN after 1 week of use if use of EN is unable to meet >60% of energy and protein requirements.247The safe and successful use of parenteral nutrition requires proper selection of patients with specific nutritional needs, expe-rience with the technique, and an awareness of the associated complications. In patients with significant malnutrition, paren-teral nutrition can rapidly improve nitrogen balance, which may enhance immune function. Routine postoperative use of paren-teral nutrition is not shown to have clinical

1	malnutrition, paren-teral nutrition can rapidly improve nitrogen balance, which may enhance immune function. Routine postoperative use of paren-teral nutrition is not shown to have clinical benefit and may be associated with a significant increase in complication rate. As with enteral nutrition, the fundamental goals are to provide suf-ficient calories and nitrogen substrate to promote tissue repair and to maintain the integrity or growth of lean tissue mass.Total Parenteral NutritionTotal parenteral nutrition (TPN), also referred to as central parenteral nutrition, requires access to a large-diameter vein to deliver the entire nutritional requirements of the individual. Dextrose content of the solution is high (15%–25%), and all other macronutrients and micronutrients are deliverable by this route.Peripheral Parenteral NutritionThe lower osmolarity of the solution used for peripheral par-enteral nutrition (PPN), secondary to reduced levels of dex-trose (5% to 10%) and protein (3%),

1	this route.Peripheral Parenteral NutritionThe lower osmolarity of the solution used for peripheral par-enteral nutrition (PPN), secondary to reduced levels of dex-trose (5% to 10%) and protein (3%), allows its administration via peripheral veins. Some nutrients cannot be supplemented because they cannot be concentrated into small volumes. There-fore, PPN is not appropriate for repleting patients with severe malnutrition. It can be considered if central routes are not avail-able or if supplemental nutritional support is required. Typi-cally, PPN is used for short periods (<2 weeks). Beyond this time, TPN should be instituted.Initiation of Parenteral NutritionThe basic solution for parenteral nutrition contains a final con-centration of 15% to 25% dextrose and 3% to 5% crystalline amino acids. The solutions usually are prepared in sterile condi-tions in the pharmacy from commercially available kits contain-ing the component solutions and transfer apparatus. Preparation in the pharmacy

1	The solutions usually are prepared in sterile condi-tions in the pharmacy from commercially available kits contain-ing the component solutions and transfer apparatus. Preparation in the pharmacy under laminar flow hoods reduces the incidence of bacterial contamination of the solution. Proper preparation with suitable quality control is absolutely essential to avoid sep-tic complications.The proper provision of electrolytes and amino acids must take into account routes of fluid and electrolyte loss, renal function, metabolic rate, cardiac function, and the under-lying disease state.Intravenous vitamin preparations also should be added to parenteral formulas. Vitamin deficiencies are rare occurrences if such preparations are used. In addition, because vitamin K is not part of any commercially prepared vitamin solution, it should be supplemented on a weekly basis. During prolonged parenteral nutrition with fat-free solutions, essential fatty acid deficiency may become clinically apparent

1	prepared vitamin solution, it should be supplemented on a weekly basis. During prolonged parenteral nutrition with fat-free solutions, essential fatty acid deficiency may become clinically apparent and manifests as dry, scaly der-matitis and loss of hair. The syndrome may be prevented by periodic infusion of a fat emulsion at a rate equivalent to 10% to 15% of total calories. Essential trace minerals may be required after prolonged TPN and may be supplied by direct addition of commercial preparations. The most frequent presentation of trace mineral deficiencies is the eczematoid rash develop-ing both diffusely and at intertriginous areas in zinc-deficient patients. Other rare trace mineral deficiencies include a micro-cytic anemia associated with copper deficiency, and glucose intolerance presumably related to chromium deficiency. The latter complications are seldom seen except in patients receiving parenteral nutrition for extended periods. The daily administra-tion of commercially

1	presumably related to chromium deficiency. The latter complications are seldom seen except in patients receiving parenteral nutrition for extended periods. The daily administra-tion of commercially available trace mineral supplements will obviate most such problems.Depending on fluid and nitrogen tolerance, parenteral nutrition solutions generally can be increased over 2 to 3 days toward the desired infusion rate. Current recommendations sug-gest that hypocaloric nutrition (high protein with lower caloric dosing) be considered in the critically ill or injured over the first week in the ICU. The suggested target dose is <20 kcal/kg per day or <80% of estimated caloric needs with adequate pro-tein (>1.2 g/kg per day). This strategy is suggested to minimize risk of both hyperglycemia and insulin resistance, which may reduce infectious complications. Insulin may be supplemented as necessary to ensure glucose tolerance, with a targeted blood glucose range of 140 or 150 to 180 mg/dL for the

1	resistance, which may reduce infectious complications. Insulin may be supplemented as necessary to ensure glucose tolerance, with a targeted blood glucose range of 140 or 150 to 180 mg/dL for the general ICU population.247 Administration of additional intravenous fluids and electrolytes may occasionally be necessary in patients with persistently high fluid losses.The patient should be carefully monitored for development of electrolyte, volume, acid-base, and septic complications. Vital signs and urinary output should be measured regularly, and the patient should be weighed regularly. Frequent adjust-ments of the volume and composition of the solutions are neces-sary during the course of therapy. Samples for measurement of electrolytes are drawn daily until levels are stable and every 2 or 3 days thereafter. Blood counts, blood urea nitrogen level, lev-els of liver function indicators, and phosphate and magnesium levels are determined at least weekly.The urine or capillary blood

1	2 or 3 days thereafter. Blood counts, blood urea nitrogen level, lev-els of liver function indicators, and phosphate and magnesium levels are determined at least weekly.The urine or capillary blood glucose level is checked every 6 hours, and serum glucose concentration is checked at least once daily during the first few days of the infusion and at fre-quent intervals thereafter. Relative glucose intolerance, which often manifests as glycosuria, may occur after initiation of par-enteral nutrition. If blood glucose levels remain elevated or gly-cosuria persists, the dextrose concentration may be decreased, the infusion rate slowed, or regular insulin added to each bottle. The rise in blood glucose concentration observed after initiating parenteral nutrition may be temporary, as the normal pancreas Brunicardi_Ch02_p0027-p0082.indd 7301/03/19 6:50 PM 74BASIC CONSIDERATIONSPART Iincreases its output of insulin in response to the continuous car-bohydrate infusion. In patients with

1	pancreas Brunicardi_Ch02_p0027-p0082.indd 7301/03/19 6:50 PM 74BASIC CONSIDERATIONSPART Iincreases its output of insulin in response to the continuous car-bohydrate infusion. In patients with diabetes mellitus, additional insulin may be required.Potassium is essential to achieve positive nitrogen bal-ance and replace depleted intracellular stores. In addition, a significant shift of potassium ion from the extracellular to the intracellular space may take place because of the large glucose infusion, with resultant hypokalemia, metabolic alkalosis, and poor glucose utilization. In some cases, as much as 240 mEq of potassium ion daily may be required. Hypokalemia may cause glycosuria, which would be treated with potassium, not insulin. Thus, before giving insulin, the serum potassium level must be checked to avoid exacerbating the hypokalemia.Patients with insulin-dependent diabetes mellitus may exhibit wide fluctuations in blood glucose levels while receiving parenteral nutrition.

1	must be checked to avoid exacerbating the hypokalemia.Patients with insulin-dependent diabetes mellitus may exhibit wide fluctuations in blood glucose levels while receiving parenteral nutrition. This may require protocol-driven intrave-nous insulin therapy. In addition, partial replacement of dex-trose calories with lipid emulsions may alleviate these problems in selected patients.Lipid emulsions derived from soybean or safflower oils are widely used as an adjunctive nutrient to prevent the development of essential fatty acid deficiency, although recent data support reducing the overall omega-6 PUFA load in favor of omega-3 PUFA or MCT. Current recommendations are to limit intrave-nous fat emulsion infusion over the first week of hospitalization to a maximum of 100 g per week delivered in two divided doses. This is based on standard emulsions that are soy-based. As data is acquired for omega-3 PUFAs, including fish-oil or olive-oil based emulsions, these recommendations may alter.The

1	two divided doses. This is based on standard emulsions that are soy-based. As data is acquired for omega-3 PUFAs, including fish-oil or olive-oil based emulsions, these recommendations may alter.The delivery of parenteral nutrition requires central intra-venous access. Temporary or short-term access can be achieved with a 16-gauge percutaneous catheter inserted into a subclavian or internal jugular vein and threaded into the superior vena cava. More permanent access with the intention of providing long-term or home parenteral nutrition can be achieved by placement of a catheter with a subcutaneous port for access by tunneling a catheter with a substantial subcutaneous length or threading a long catheter through the basilic or cephalic vein into the supe-rior vena cava.Complications of Parenteral NutritionTechnical Complications. One of the more common and seri-ous complications associated with long-term parenteral feed-ing is sepsis secondary to contamination of the central venous

1	Parenteral NutritionTechnical Complications. One of the more common and seri-ous complications associated with long-term parenteral feed-ing is sepsis secondary to contamination of the central venous catheter. Contamination of solutions should also be considered, but it is rare when proper pharmacy protocols have been fol-lowed. Central line-associated blood stream infections (CLA-BSI) occur as a consequence of hematogenous seeding of the cath-eter with bacteria. One of the earliest signs of systemic sepsis from CVA-BSI may be the sudden development of glucose intolerance (with or without temperature increase) in a patient who previously has been maintained on parenteral alimen-tation without difficulty. When this occurs, or if high fever (> 38.5°C [101.3°F]) develops without obvious cause, a dili-gent search for a potential septic focus is indicated. Other causes of fever should also be investigated. If fever persists, the infu-sion catheter should be removed and submitted for

1	cause, a dili-gent search for a potential septic focus is indicated. Other causes of fever should also be investigated. If fever persists, the infu-sion catheter should be removed and submitted for culture. If the catheter is the cause of the fever, removal of the infectious source is usually followed by rapid defervescence. Some centers are now replacing catheters considered at low risk for infec-tion over a guidewire. However, if blood cultures are positive and the catheter tip is also positive, then the catheter should be removed and placed in a new site. Should evidence of infec-tion persist over 24 to 48 hours without a definable source, the catheter should be replaced into the opposite subclavian vein or into one of the internal jugular veins, and the infusion should be restarted.275The use of multilumen catheters may be associated with a slightly increased risk of infection. This is most likely asso-ciated with greater catheter manipulation and intensive use. The rate of

1	use of multilumen catheters may be associated with a slightly increased risk of infection. This is most likely asso-ciated with greater catheter manipulation and intensive use. The rate of catheter infection is highest for those placed in the femoral vein, lower for those in the jugular vein, and lowest for those in the subclavian vein. When catheters are indwelling for <3 days, infection risks are negligible. If indwelling time is 3 to 7 days, the infection risk is 3% to 5%. Indwelling times of >7 days are associated with a catheter infection risk of 5% to 10%. Strict adherence to barrier precautions also reduces the rate of infection as can the implementation of procedure checklists to ensure compliance with evidence-based guidelines shown to reduce infectious risk.276Other complications related to catheter placement include the development of pneumothorax, hemothorax, hydrothorax, subclavian artery injury, thoracic duct injury, cardiac arrhyth-mia, air embolism, catheter

1	related to catheter placement include the development of pneumothorax, hemothorax, hydrothorax, subclavian artery injury, thoracic duct injury, cardiac arrhyth-mia, air embolism, catheter embolism, and cardiac perforation with tamponade. All of these complications may be avoided by strict adherence to proper techniques. Further, the use of ultra-sonographic guidance during CV line placement has been dem-onstrated to significantly decrease the failure rate, complication rate, and number of attempts required for successful access.277Metabolic Complications. Hyperglycemia may develop with normal rates of infusion in patients with impaired glucose toler-ance or in any patient if the hypertonic solutions are adminis-tered too rapidly. This is a particularly common complication in patients with latent diabetes and in patients subjected to severe surgical stress or trauma. Treatment of the condition consists of volume replacement with correction of electrolyte abnormali-ties and the

1	with latent diabetes and in patients subjected to severe surgical stress or trauma. Treatment of the condition consists of volume replacement with correction of electrolyte abnormali-ties and the administration of insulin. This complication can be avoided with careful attention to daily fluid balance and frequent monitoring of blood glucose levels and serum electrolytes.Increasing experience has emphasized the importance of not overfeeding the parenterally nourished patient. This is particularly true for the depleted patient in whom excess calo-rie infusion may result in carbon dioxide retention and respi-ratory insufficiency. In addition, excess feeding also has been related to the development of hepatic steatosis or marked gly-cogen deposition in selected patients. Cholestasis and forma-tion of gallstones are common in patients receiving long-term parenteral nutrition. Mild but transient abnormalities of serum transaminase, alkaline phosphatase, and bilirubin levels occur in many

1	of gallstones are common in patients receiving long-term parenteral nutrition. Mild but transient abnormalities of serum transaminase, alkaline phosphatase, and bilirubin levels occur in many parenterally nourished patients. Failure of the liver enzymes to plateau or return to normal over 7 to 14 days should suggest another etiology.Intestinal Atrophy. Lack of intestinal stimulation is associ-ated with intestinal mucosal atrophy, diminished villous height, bacterial overgrowth, reduced lymphoid tissue size, reduced immunoglobulin A production, and impaired gut immunity. The full clinical implications of these changes are not well real-ized, although bacterial translocation has been demonstrated in animal models. The most efficacious method to prevent these changes is to provide at least some nutrients enterally. In patients requiring TPN, it may be feasible to infuse small amounts of feedings via the gastrointestinal tract.Brunicardi_Ch02_p0027-p0082.indd 7401/03/19 6:50 PM

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1	2007;35(5): 1277-1283. doi: 10.1097/01.CCM.0000261893.72811.0F 236. Qian A, Zhang M, Zhao G. Dynamic detection of N-terminal pro-B-type natriuretic peptide helps to predict the outcome of patients with major trauma. Eur J Trauma Emerg Surg. 2015;41(1):57-64. doi: 10.1007/s00068-014-0406-7 237. Mitch WE, Price SR. Mechanisms activating proteolysis to cause muscle atrophy in catabolic conditions. J Ren Nutr. 2003;13(2):149-152. doi: 10.1053/jren.2003.50019 238. Uehara M, Plank LD, Hill GL. Components of energy expen-diture in patients with severe sepsis and major trauma: a basis for clinical care. Crit Care Med. 1999;27(7):1295-1302. 239. Guirao X. Impact of the inflammatory reaction on intermediary metab-olism and nutrition status. Nutrition. 2002;18(11-12):949-952. 240. Souba WW. Nutritional support. N Engl J Med. 1997;336(1): 41-48. doi: 10.1056/NEJM199701023360107 241. Bistrian BR. Clinical aspects of essential fatty acid metabo-lism: Jonathan Rhoads Lecture. JPEN J Parenter Enteral

1	support. N Engl J Med. 1997;336(1): 41-48. doi: 10.1056/NEJM199701023360107 241. Bistrian BR. Clinical aspects of essential fatty acid metabo-lism: Jonathan Rhoads Lecture. JPEN J Parenter Enteral Nutr. 2003;27(3):168-175. 242. Dahn MS, Mitchell RA, Lange MP, Smith S, Jacobs LA. Hepatic metabolic response to injury and sepsis. Surgery. 1995;117(5):520-530. 243. Vidal-Puig A, O’Rahilly S. Metabolism. Controlling the glucose factory. Nature. 2001;413(6852):125-126. doi: 10.1038/35093198 244. Volpi E, Sheffield-Moore M, Rasmussen BB, Wolfe RR. Basal muscle amino acid kinetics and protein synthesis in healthy young and older men. JAMA. 2001;286(10):1206-1212. 245. Kondrup J, Rasmussen HH, Hamberg O, Stanga Z, Ad Hoc EWG. Nutritional risk screening (NRS 2002): a new method based on an analysis of controlled clinical trials. Clin Nutr. 2003;22(3):321-336. 246. Heyland DK, Dhaliwal R, Jiang X, Day AG. Identifying critically ill patients who benefit the most from nutrition therapy: the

1	of controlled clinical trials. Clin Nutr. 2003;22(3):321-336. 246. Heyland DK, Dhaliwal R, Jiang X, Day AG. Identifying critically ill patients who benefit the most from nutrition therapy: the development and initial validation of a novel risk assessment tool. Crit Care. 2011;15(6):R268. doi: 10.1186/cc10546 247. Taylor BE, McClave SA, Martindale RG, et al. Guide-lines for the provision and assessment of nutrition support therapy in the adult critically ill patient: Society of Critical Care Medicine (SCCM) and American Society for Paren-teral and Enteral Nutrition (A.S.P.E.N.). Crit Care Med. 2016;44(2):390-438. doi: 10.1097/CCM.0000000000001525 248. Singer P, Anbar R, Cohen J, et al. The tight calorie control study (TICACOS): a prospective, randomized, controlled pilot study of nutritional support in critically ill patients. Intensive Care Med. 2011;37(4):601-609. doi: 10.1007/s00134-011-2146-z 249. Heslin MJ, Brennan MF. Advances in perioperative nutrition: cancer. World J Surg.

1	support in critically ill patients. Intensive Care Med. 2011;37(4):601-609. doi: 10.1007/s00134-011-2146-z 249. Heslin MJ, Brennan MF. Advances in perioperative nutrition: cancer. World J Surg. 2000;24(12):1477-1485. 250. Elke G, van Zanten AR, Lemieux M, et al. Enteral versus parenteral nutrition in critically ill patients: an updated systematic review and meta-analysis of randomized con-trolled trials. Crit Care. 2016;20(1):117. doi: 10.1186/s13054-016-1298-1 251. Reignier J, Boisrame-Helms J, Brisard L, et al. Enteral ver-sus parenteral early nutrition in ventilated adults with shock: a randomised, controlled, multicentre, open-label, parallel-group study (NUTRIREA-2). Lancet. 2018;391(10116):133-143. doi: 10.1016/S0140-6736(17)32146-3 252. Heslin MJ, Latkany L, Leung D, et al. A prospective, ran-domized trial of early enteral feeding after resection of upper gastrointestinal malignancy. Ann Surg. 1997;226(4):567-577; discussion 77-80. 253. Reintam Blaser A, Berger MM. Early or

1	ran-domized trial of early enteral feeding after resection of upper gastrointestinal malignancy. Ann Surg. 1997;226(4):567-577; discussion 77-80. 253. Reintam Blaser A, Berger MM. Early or late feeding after ICU admission? Nutrients. 2017;9(12):E1278. doi: 10.3390/nu9121278 254. Oshima T, Deutz NE, Doig G, Wischmeyer PE, Pichard C. Protein-energy nutrition in the ICU is the power couple: a hypothesis forming analysis. Clin Nutr. 2016;35(4):968-974. doi: 10.1016/j.clnu.2015.10.016 255. Hoffer LJ. High-protein hypocaloric nutrition for non-obese critically ill patients. Nutr Clin Pract. 2018;33(3):325-332. doi: 10.1002/ncp.10091 256. Di Girolamo FG, Situlin R, Fiotti N, Biolo G. Intermittent vs. continuous enteral feeding to prevent catabolism in acutely ill adult and pediatric patients. Curr Opin Clin Nutr Metab Care. 2017;20(5):390-395. doi: 10.1097/MCO.0000000000000397Brunicardi_Ch02_p0027-p0082.indd 8101/03/19 6:50 PM 82BASIC CONSIDERATIONSPART I 257. Bankhead R, Boullata J,

1	Curr Opin Clin Nutr Metab Care. 2017;20(5):390-395. doi: 10.1097/MCO.0000000000000397Brunicardi_Ch02_p0027-p0082.indd 8101/03/19 6:50 PM 82BASIC CONSIDERATIONSPART I 257. Bankhead R, Boullata J, Brantley S, et al. Enteral nutrition practice recommendations. JPEN J Parenter Enteral Nutr. 2009;33(2):122-167. doi: 10.1177/0148607108330314 258. Heyland DK, Murch L, Cahill N, et al. Enhanced protein-energy provision via the enteral route feeding protocol in critically ill patients: results of a cluster randomized trial. Crit Care Med. 2013;41(12):2743-2753. doi: 10.1097/CCM.0b013e31829efef5 259. Exner R, Tamandl D, Goetzinger P, et al. Perioperative GLY-GLN infusion diminishes the surgery-induced period of immunosuppression: accelerated restoration of the lipo-polysaccharide-stimulated tumor necrosis factor-alpha response. Ann Surg. 2003;237(1):110-115. doi: 10.1097/01.SLA.0000041040.98684.CB 260. van Zanten AR, Hofman Z, Heyland DK. Consequences of the REDOXS and METAPLUS trials: the

1	necrosis factor-alpha response. Ann Surg. 2003;237(1):110-115. doi: 10.1097/01.SLA.0000041040.98684.CB 260. van Zanten AR, Hofman Z, Heyland DK. Consequences of the REDOXS and METAPLUS trials: the end of an era of glutamine and antioxidant supplementation for critically ill patients? JPEN J Parenter Enteral Nutr. 2015;39(8):890-892. doi: 10.1177/0148607114567201 261. Luiking YC, Ten Have GA, Wolfe RR, Deutz NE. Arginine de novo and nitric oxide production in disease states. Am J Physiol Endocrinol Metab. 2012;303(10):E1177-E1189. doi: 10.1152/ajpendo.00284.2012 262. Drover JW, Dhaliwal R, Weitzel L, Wischmeyer PE, Ochoa JB, Heyland DK. Perioperative use of arginine-supplemented diets: a systematic review of the evidence. J Am Coll Surg. 2011;212(3):385-399, 99 e1. doi: 10.1016/j.jamcollsurg.2010.10.016 263. Brown B, Roehl K, Betz M. Enteral nutrition for-mula selection: current evidence and implications for practice. Nutr Clin Pract. 2015;30(1):72-85. doi:

1	e1. doi: 10.1016/j.jamcollsurg.2010.10.016 263. Brown B, Roehl K, Betz M. Enteral nutrition for-mula selection: current evidence and implications for practice. Nutr Clin Pract. 2015;30(1):72-85. doi: 10.1177/0884533614561791 264. Marik PE, Flemmer M. Immunonutrition in the surgical patient. Minerva Anestesiol. 2012;78(3):336-342. 265. Guidelines CCP. Enteral Feeding Guidelines. 2013. 266. Pontes-Arruda A, Martins LF, de Lima SM, et al. Enteral nutri-tion with eicosapentaenoic acid, gamma-linolenic acid and antioxidants in the early treatment of sepsis: results from a mul-ticenter, prospective, randomized, double-blinded, controlled study: the INTERSEPT study. Crit Care. 2011;15(3):R144. doi: 10.1186/cc10267 267. Choban P, Dickerson R, Malone A, et al. A.S.P.E.N. Clinical guidelines: nutrition support of hospitalized adult patients with obesity. JPEN J Parenter Enteral Nutr. 2013;37(6):714-744. doi: 10.1177/0148607113499374 268. Btaiche IF. Branched-chain amino acids in patients with

1	support of hospitalized adult patients with obesity. JPEN J Parenter Enteral Nutr. 2013;37(6):714-744. doi: 10.1177/0148607113499374 268. Btaiche IF. Branched-chain amino acids in patients with hepatic encephalopathy. 1982. Nutr Clin Pract. 2003;18(1):97-100. 269. Patton KM, Aranda-Michel J. Nutritional aspects in liver dis-ease and liver transplantation. Nutr Clin Pract. 2002;17(6): 332-340. 270. Koretz RL, Avenell A, Lipman TO. Nutritional support for liver disease. Cochrane Database Syst Rev. 2012(5):CD008344. doi: 10.1002/14651858.CD008344.pub2 271. DiSario JA, Baskin WN, Brown RD, et al. Endoscopic approaches to enteral nutritional support. Gastrointest Endosc. 2002;55(7):901-908. 272. Heyland DK, Drover JW, Dhaliwal R, Greenwood J. Optimiz-ing the benefits and minimizing the risks of enteral nutrition in the critically ill: role of small bowel feeding. JPEN J Parenter Enteral Nutr. 2002;26(6 suppl):S51-S55; discussion S56S57. 273. Scolapio JS. Methods for decreasing risk of

1	of enteral nutrition in the critically ill: role of small bowel feeding. JPEN J Parenter Enteral Nutr. 2002;26(6 suppl):S51-S55; discussion S56S57. 273. Scolapio JS. Methods for decreasing risk of aspiration pneu-monia in critically ill patients. JPEN J Parenter Enteral Nutr. 2002;26(6 suppl):S58-S61; discussion S61. 274. Vanek VW. Ins and outs of enteral access: part 2—long term access—esophagostomy and gastrostomy. Nutr Clin Pract. 2003;18(1):50-74. 275. Mermel LA, Allon M, Bouza E, et al. Clinical practice guide-lines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America. Clin Infect Dis. 2009;49(1):1-45. doi: 10.1086/599376 276. Quality AfHRa. Tools for reducing central line-associated blood stream infections. Available at: http://www.ahrq.gov/legacy/qual/clabsitools/clabsitools.htm#purpose 277. Maecken T, Grau T. Ultrasound imaging in vascular access. Crit Care Med. 2007;35(5 suppl):S178-S185. doi:

1	Available at: http://www.ahrq.gov/legacy/qual/clabsitools/clabsitools.htm#purpose 277. Maecken T, Grau T. Ultrasound imaging in vascular access. Crit Care Med. 2007;35(5 suppl):S178-S185. doi: 10.1097/01.CCM.0000260629.86351.A5Brunicardi_Ch02_p0027-p0082.indd 8201/03/19 6:50 PM

1	Introduction 83Body Fluids 83Total Body Water / 83Fluid Compartments / 83Composition of Fluid Compartments / 83Osmotic Pressure / 84Body Fluid Changes 85Normal Exchange of Fluid and Electrolytes / 85Classification of Body Fluid Changes / 85Disturbances in Fluid Balance / 85Volume Control / 86Concentration Changes / 86Composition Changes: Etiology and Diagnosis / 88Acid-Base Balance / 91Metabolic Derangements / 91Fluid and Electrolyte Therapy 93Parenteral Solutions / 93Alternative Resuscitative Fluids / 94Correction of Life-Threatening Electrolyte Abnormalities / 94Preoperative Fluid Therapy / 96Intraoperative Fluid Therapy / 97Postoperative Fluid Therapy / 97Fluid Management in Enhanced Recovery After Surgery (ERAS) Pathways / 97Special Considerations for the Postoperative Patient / 98Electrolyte Abnormalities in Specific Surgical Patients 98Neurologic Patients / 98Malnourished Patients: Refeeding Syndrome / 98Acute Renal Failure Patients / 99Cancer Patients / 99Fluid and

1	/ 98Electrolyte Abnormalities in Specific Surgical Patients 98Neurologic Patients / 98Malnourished Patients: Refeeding Syndrome / 98Acute Renal Failure Patients / 99Cancer Patients / 99Fluid and Electrolyte Management of the Surgical PatientMatthew D. Neal 3chapterINTRODUCTIONFluid and electrolyte management is paramount to the care of the surgical patient. Changes in both fluid volume and electro-lyte composition occur preoperatively, intraoperatively, and postoperatively, as well as in response to trauma and sepsis. The sections that follow review the normal anatomy of body fluids, electrolyte composition and concentration abnormalities and treatments, common metabolic derangements, and alternative resuscitative fluids. These concepts are then discussed in relationship to management of specific surgical patients and their commonly encountered fluid and electrolyte abnormalities.BODY FLUIDSTotal Body WaterWater constitutes approximately 50% to 60% of total body weight. The

1	of specific surgical patients and their commonly encountered fluid and electrolyte abnormalities.BODY FLUIDSTotal Body WaterWater constitutes approximately 50% to 60% of total body weight. The relationship between total body weight and total body water (TBW) is relatively constant for an individual and is primarily a reflection of body fat. Lean tissues such as muscle and solid organs have higher water content than fat and bone. As a result, young, lean males have a higher proportion of body weight as water than elderly or obese individuals. In an average young adult male, TBW accounts for 60% of total body weight, whereas in an average young adult female, it is 50%.1 The lower percent-age of TBW in females correlates with a higher percentage of adipose tissue and lower percentage of muscle mass in most. Estimates of percentage of TBW should be adjusted downward approximately 10% to 20% for obese individuals and upward by 10% for malnourished individuals. The highest percentage of TBW

1	mass in most. Estimates of percentage of TBW should be adjusted downward approximately 10% to 20% for obese individuals and upward by 10% for malnourished individuals. The highest percentage of TBW is found in newborns, with approximately 80% of their total body weight comprised of water. This decreases to approximately 65% by 1 year of age and thereafter remains fairly constant.1Fluid CompartmentsTBW is divided into three functional fluid compartments: plasma, extravascular interstitial fluid, and intracellular fluid (Fig. 3-1). The extracellular fluids (ECF), plasma, and intersti-tial fluid together compose about one-third of the TBW, and the intracellular compartment composes the remaining two thirds. The extracellular water composes 20% of the total body weight and is divided between plasma (5% of body weight) and inter-stitial fluid (15% of body weight). Intracellular water makes up approximately 40% of an individual’s total body weight, with the largest proportion in the

1	plasma (5% of body weight) and inter-stitial fluid (15% of body weight). Intracellular water makes up approximately 40% of an individual’s total body weight, with the largest proportion in the skeletal muscle mass.Composition of Fluid CompartmentsThe normal chemical composition of the body fluid compart-ments is shown in Fig. 3-2. The ECF compartment is bal-anced between sodium, the principal cation, and chloride and bicarbonate, the principal anions. The intracellular fluid compartment is composed primarily of the cations potassium and magnesium, the anions phosphate and sulfate, and proteins. The concentration gradient between compartments is maintained by adenosine triphosphate–driven sodium-potassium pumps located within the cell membranes. The composition of the plasma and interstitial fluid differs only slightly in ionic compo-sition. The slightly higher protein content (organic anions) in plasma results in a higher plasma cation composition relative to the interstitial fluid,

1	fluid differs only slightly in ionic compo-sition. The slightly higher protein content (organic anions) in plasma results in a higher plasma cation composition relative to the interstitial fluid, as explained by the Gibbs-Donnan equilib-rium equation. Proteins add to the osmolality of the plasma and contribute to the balance of forces that determine fluid balance across the capillary endothelium. Although the movement of ions and proteins between the various fluid compartments is restricted, water is freely diffusible. Water is distributed evenly throughout all fluid compartments of the body so that a given volume of water increases the volume of any one compartment 2Brunicardi_Ch03_p0083-p0102.indd 8308/12/18 10:07 AM 84Figure 3-1. Functional body fluid compartments. TBW = total body water.Key Points1 Proper management of fluid and electrolytes facilitates cru-cial homeostasis that allows cardiovascular perfusion, organ system function, and cellular mechanisms to respond to

1	body water.Key Points1 Proper management of fluid and electrolytes facilitates cru-cial homeostasis that allows cardiovascular perfusion, organ system function, and cellular mechanisms to respond to sur-gical illness.2 Knowledge of the compartmentalization of body fluids forms the basis for understanding pathologic shifts in these fluid spaces in disease states. Although difficult to quantify, a deficiency in the functional extracellular fluid compart-ment often requires resuscitation with isotonic fluids in sur-gical and trauma patients.3 Alterations in the concentration of serum sodium have pro-found effects on cellular function due to water shifts between the intracellular and extracellular spaces.4 Different rates of compensation between respiratory and metabolic components of acid-base homeostasis require fre-quent laboratory reassessment during therapy.5 Although active investigation continues, alternative resusci-tation fluids have limited clinical utility, other than the

1	homeostasis require fre-quent laboratory reassessment during therapy.5 Although active investigation continues, alternative resusci-tation fluids have limited clinical utility, other than the cor-rection of specific electrolyte abnormalities.6 Enhanced recovery after surgery (ERAS) protocols have markedly changed perioperative fluid management and are being used more frequently. ERAS minimizes perioperative fluid administration and focuses on early enteral intake to reduce morbidity associated with IV fluid administration.7 Most acute surgical illnesses are accompanied by some degree of volume loss or redistribution. Consequently, iso-tonic fluid administration is the most common initial intra-venous fluid strategy, while attention is being given to alterations in concentration and composition.8 Some surgical patients with neurologic illness, malnutrition, acute renal failure, or cancer require special attention to well-defined, disease-specific abnormalities in fluid and electrolyte

1	surgical patients with neurologic illness, malnutrition, acute renal failure, or cancer require special attention to well-defined, disease-specific abnormalities in fluid and electrolyte status.% of Total body weightPlasma 5%Interstitialfluid 15%Intracellularvolume 40%Volume of TBWExtracellular volumePlasmaInterstitialIntracellular volume14,000 mL3500 mL10,500 mL28,000 mL42,000 mLMale (70 kg)10,000 mL2500 mL7500 mL20,000 mL30,000 mLFemale (60 kg)relatively little. Sodium, however, is confined to the ECF com-partment, and because of its osmotic and electrical properties, it remains associated with water. Therefore, sodium-containing fluids are distributed throughout the ECF and add to the volume of both the intravascular and interstitial spaces. Although the administration of sodium-containing fluids expands the intra-vascular volume, it also expands the interstitial space by approx-imately three times as much as the plasma.Osmotic PressureThe physiologic activity of electrolytes in

1	fluids expands the intra-vascular volume, it also expands the interstitial space by approx-imately three times as much as the plasma.Osmotic PressureThe physiologic activity of electrolytes in solution depends on the number of particles per unit volume (millimoles per liter, or mmol/L), the number of electric charges per unit volume (milli-equivalents per liter, or mEq/L), and the number of osmotically active ions per unit volume (milliosmoles per liter, or mOsm/L). The concentration of electrolytes usually is expressed in terms of the chemical combining activity, or equivalents. An equiva-lent of an ion is its atomic weight expressed in grams divided by the valence:Equivalent = atomic weight (g)/valenceFor univalent ions such as sodium, 1 mEq is same as 1 mmol. For divalent ions such as magnesium, 1 mmol equals 2 mEq. The number of milliequivalents of cations must be bal-anced by the same number of milliequivalents of anions. How-ever, the expression of molar equivalents alone does

1	magnesium, 1 mmol equals 2 mEq. The number of milliequivalents of cations must be bal-anced by the same number of milliequivalents of anions. How-ever, the expression of molar equivalents alone does not allow a physiologic comparison of solutes in a solution.The movement of water across a cell membrane depends primarily on osmosis. To achieve osmotic equilibrium, water moves across a semipermeable membrane to equalize the con-centration on both sides. This movement is determined by the concentration of the solutes on each side of the membrane. Osmotic pressure is measured in units of osmoles (osm) or milliosmoles (mOsm) that refer to the actual number of osmotically active particles. For example, 1 mmol of sodium chloride contributes to 2 mOsm (one from sodium and one from chloride). The principal determinants of osmolality are the concentrations of sodium, glucose, and urea (blood urea nitrogen, or BUN):Calculated serum osmolality = 2 sodium + (glucose/18) + (BUN/2.8)The osmolality

1	principal determinants of osmolality are the concentrations of sodium, glucose, and urea (blood urea nitrogen, or BUN):Calculated serum osmolality = 2 sodium + (glucose/18) + (BUN/2.8)The osmolality of the intracellular and extracellular fluids is maintained between 290 and 310 mOsm in each compart-ment. Because cell membranes are permeable to water, any Brunicardi_Ch03_p0083-p0102.indd 8408/12/18 10:07 AM 85FLUID AND ELECTROLYTE MANAGEMENT OF THE SURGICAL PATIENTCHAPTER 3change in osmotic pressure in one compartment is accompanied by a redistribution of water until the effective osmotic pressure between compartments is equal. For practical clinical purposes, most significant gains and losses of body fluid are directly from the extracellular compartment.BODY FLUID CHANGESNormal Exchange of Fluid and ElectrolytesThe healthy person consumes an average of 2000 mL of water per day, approximately 75% from oral intake and the rest extracted from solid foods. Daily water losses include

1	of Fluid and ElectrolytesThe healthy person consumes an average of 2000 mL of water per day, approximately 75% from oral intake and the rest extracted from solid foods. Daily water losses include 800 to 1200 mL in urine, 250 mL in stool, and 600 mL in insensible losses. Insensible losses of water occur through both the skin (75%) and lungs (25%) and can be increased by such factors as fever, hypermetabolism, and hyperventilation. Sensible water losses such as sweating or pathologic loss of gastrointestinal (GI) fluids vary widely, but these include the loss of electrolytes as well as water (Table 3-1). To clear the products of metabo-lism, the kidneys must excrete a minimum of 500 to 800 mL of urine per day, regardless of the amount of oral intake.The typical individual consumes 3 to 5 g of dietary salt per day, with the balance maintained by the kidneys. With hypo-natremia or hypovolemia, sodium excretion can be reduced to as little as 1 mEq/d or maximized to as much as 5000 mEq/d to

1	dietary salt per day, with the balance maintained by the kidneys. With hypo-natremia or hypovolemia, sodium excretion can be reduced to as little as 1 mEq/d or maximized to as much as 5000 mEq/d to achieve balance except in people with salt-wasting kidneys. Sweat is hypotonic, and sweating usually results in only a small sodium loss. GI losses are isotonic to slightly hypotonic and contribute little to net gain or loss of free water when measured and appropriately replaced by isotonic salt solutions.Classification of Body Fluid ChangesDisorders in fluid balance may be classified into three general categories: disturbances in (a) volume, (b) concentration, and (c) composition. Although each of these may occur simultaneously, each is a separate entity with unique mechanisms demanding individual correction. Isotonic gain or loss of salt solution results in extracellular volume changes, with little impact on intracellular fluid volume. If free water is added or lost from the ECF, water

1	correction. Isotonic gain or loss of salt solution results in extracellular volume changes, with little impact on intracellular fluid volume. If free water is added or lost from the ECF, water will pass between the ECF and intracellular fluid until solute concentration or osmolarity is equalized between the compartments. Unlike with sodium, the concentration of most other ions in the ECF can be altered without significant change in the total number of osmotically active particles, producing only a compositional change.Disturbances in Fluid BalanceExtracellular volume deficit is the most common fluid disorder in surgical patients and can be either acute or chronic. Acute volume deficit is associated with cardiovascular and central ner-vous system signs, whereas chronic deficits display tissue signs, such as a decrease in skin turgor and sunken eyes, in addition to cardiovascular and central nervous system signs (Table 3-2). Laboratory examination may reveal an elevated BUN level if the

1	signs, such as a decrease in skin turgor and sunken eyes, in addition to cardiovascular and central nervous system signs (Table 3-2). Laboratory examination may reveal an elevated BUN level if the deficit is severe enough to reduce glomerular filtration and hemoconcentration. Urine osmolality usually will be higher than serum osmolality, and urine sodium will be low, typically <20 mEq/L. Serum sodium concentration does not necessar-ily reflect volume status and therefore may be high, normal, or low when a volume deficit is present. The most common cause of volume deficit in surgical patients is a loss of GI flu-ids (Table 3-3) from nasogastric suction, vomiting, diarrhea, or Figure 3-2. Chemical composition of body fluid compartments.PlasmaCATIONS154 mEq/L154 mEq/LCI−SO42–PO43–ProteinOrganicacids103HCO3−273516Na+142K+4Ca2+5Mg2+3ANIONSInterstitialfluidCATIONS153 mEq/L153 mEq/LCI−PO43–SO42–ProteinOrganicacids114HCO3−30531Na+144K+4Ca2+3Mg2+2ANIONSIntracellularfluidCATIONS200 mEq/L200

1	mEq/L153 mEq/LCI−PO43–SO42–ProteinOrganicacids114HCO3−30531Na+144K+4Ca2+3Mg2+2ANIONSIntracellularfluidCATIONS200 mEq/L200 mEq/LK+150ProteinHCO3−SO42–HPO43–1015040Mg2+40Na+10ANIONSBrunicardi_Ch03_p0083-p0102.indd 8508/12/18 10:07 AM 86BASIC CONSIDERATIONSPART Ienterocutaneous fistula. In addition, sequestration secondary to soft tissue injuries, burns, and intra-abdominal processes such as peritonitis, obstruction, or prolonged surgery can also lead to massive volume deficits.Extracellular volume excess may be iatrogenic or second-ary to renal dysfunction, congestive heart failure, or cirrhosis. Both plasma and interstitial volumes usually are increased. Symptoms are primarily pulmonary and cardiovascular (see Table 3-2). In fit patients, edema and hyperdynamic circula-tion are common and well tolerated. However, the elderly and patients with cardiac disease may quickly develop congestive heart failure and pulmonary edema in response to only a moder-ate volume excess.Volume

1	common and well tolerated. However, the elderly and patients with cardiac disease may quickly develop congestive heart failure and pulmonary edema in response to only a moder-ate volume excess.Volume ControlVolume changes are sensed by both osmoreceptors and baro-receptors. Osmoreceptors are specialized sensors that detect even small changes in fluid osmolality and drive changes in thirst and diuresis through the kidneys.2 For example, when plasma osmolality is increased, thirst is stimulated and water consumption increases, although the exact cell mechanism is not known.3 Additionally, the hypothalamus is stimulated to secrete vasopressin, which increases water reabsorption in the kidneys. Together, these two mechanisms return the plasma osmolality to normal. Baroreceptors also modulate volume in response to changes in pressure and circulating volume through specialized pressure sensors located in the aortic arch and carotid sinuses.4 Baroreceptor responses are both neural, through

1	volume in response to changes in pressure and circulating volume through specialized pressure sensors located in the aortic arch and carotid sinuses.4 Baroreceptor responses are both neural, through sympathetic and parasympathetic pathways, and hormonal, through substances including renin-angiotensin, aldosterone, atrial natriuretic pep-tide, and renal prostaglandins. The net result of alterations in renal sodium excretion and free water reabsorption is restoration of volume to the normal state.Concentration ChangesChanges in serum sodium concentration are inversely propor-tional to TBW. Therefore, abnormalities in TBW are reflected by abnormalities in serum sodium levels.Hyponatremia. A low serum sodium level occurs when there is an excess of extracellular water relative to sodium. Extracel-lular volume can be high, normal, or low (Fig. 3-3). In most cases of hyponatremia, sodium concentration is decreased as a consequence of either sodium depletion or dilution.5 Dilutional

1	Extracel-lular volume can be high, normal, or low (Fig. 3-3). In most cases of hyponatremia, sodium concentration is decreased as a consequence of either sodium depletion or dilution.5 Dilutional hyponatremia frequently results from excess extracellular water and therefore is associated with a high extracellular volume status. Excessive oral water intake or iatrogenic intravenous (IV) excess free water administration can cause hyponatremia. Post-operative patients are particularly prone to increased secretion of antidiuretic hormone (ADH), which increases reabsorption 3Table 3-2Signs and symptoms of volume disturbancesSYSTEMVOLUME DEFICITVOLUME EXCESSGeneralizedWeight lossWeight gain Decreased skin turgorPeripheral edemaCardiacTachycardiaIncreased cardiac output Orthostasis/hypotensionIncreased central venous pressure Collapsed neck veinsDistended neck veins MurmurRenalOliguria— Azotemia GIIleusBowel edemaPulmonary—Pulmonary edemaTable 3-1Water exchange (60to 80-kg man)ROUTESAVERAGE

1	central venous pressure Collapsed neck veinsDistended neck veins MurmurRenalOliguria— Azotemia GIIleusBowel edemaPulmonary—Pulmonary edemaTable 3-1Water exchange (60to 80-kg man)ROUTESAVERAGE DAILY VOLUME (mL)MINIMAL (mL)MAXIMAL (mL)H2O gain: Sensible: Oral fluids800–150001500/h Solid foods500–70001500 Insensible: Water of oxidation250125800 Water of solution00500H2O loss: Sensible: Urine800–15003001400/h Intestinal0–25002500/h Sweat004000/h Insensible: Lungs and skin6006001500Brunicardi_Ch03_p0083-p0102.indd 8608/12/18 10:07 AM 87FLUID AND ELECTROLYTE MANAGEMENT OF THE SURGICAL PATIENTCHAPTER 3of free water from the kidneys with subsequent volume expan-sion and hyponatremia. This is usually self-limiting in that both hyponatremia and volume expansion decrease ADH secretion. Additionally, a number of drugs can cause water retention and subsequent hyponatremia, such as antipsychotics and tricyclic antidepressants as well as angiotensin-converting enzyme

1	ADH secretion. Additionally, a number of drugs can cause water retention and subsequent hyponatremia, such as antipsychotics and tricyclic antidepressants as well as angiotensin-converting enzyme inhib-itors. The elderly are particularly susceptible to drug-induced hyponatremia. Physical signs of volume overload usually are absent, and laboratory evaluation reveals hemodilution. Deple-tional causes of hyponatremia are associated with either a decreased intake or increased loss of sodium-containing fluids. A concomitant ECF volume deficit is common. Causes include decreased sodium intake, such as consumption of a low-sodium diet or use of enteral feeds, which are typically low in sodium; Table 3-3Composition of GI secretionsTYPE OF SECRETIONVOLUME (mL/24 h)NA (mEq/L)K (mEq/L)CL (mEq/L)HCO3− (mEq/L)Stomach1000–200060–9010–30100–1300Small intestine2000–3000120–1405–1090–12030–40Colon—6030400Pancreas600–800135–1455–1070–9095–115Bile300–800135–1455–1090–11030–40Figure 3-3. Evaluation of

1	(mEq/L)Stomach1000–200060–9010–30100–1300Small intestine2000–3000120–1405–1090–12030–40Colon—6030400Pancreas600–800135–1455–1070–9095–115Bile300–800135–1455–1090–11030–40Figure 3-3. Evaluation of sodium abnormalities. ADH = antidiuretic hormone; SIADH = syndrome of inappropriate secretion of antidi-uretic hormone.HighIncreased intakePostoperative ADH secretionDrugsHighIatrogenic sodium administrationMineralocorticoid excessAldosteronismCushing’s diseaseCongenital adrenal hyperplasiaHyponatremiaVolume statusNormalHyperglycemia˜Plasma lipids/proteinsSIADHWater intoxicationDiureticsHypernatremiaVolume statusNormalNonrenal water lossSkinGIRenal water lossRenal diseaseDiureticsDiabetes insipidusLowDecreased sodium intakeGI lossesRenal lossesDiureticsPrimary renal diseaseLowNonrenal water lossSkinGI Renal water lossRenal (tubular) diseaseOsmotic diureticsDiabetes insipidusAdrenal failureBrunicardi_Ch03_p0083-p0102.indd 8708/12/18 10:07 AM 88BASIC CONSIDERATIONSPART IGI losses from

1	lossSkinGI Renal water lossRenal (tubular) diseaseOsmotic diureticsDiabetes insipidusAdrenal failureBrunicardi_Ch03_p0083-p0102.indd 8708/12/18 10:07 AM 88BASIC CONSIDERATIONSPART IGI losses from vomiting, prolonged nasogastric suctioning, or diarrhea; and renal losses due to diuretic use or primary renal disease.Hyponatremia also can be seen with an excess of solute relative to free water, such as with untreated hyperglycemia or mannitol administration. When hyponatremia in the presence of hyperglycemia is being evaluated, the corrected sodium concen-tration should be calculated as follows:For every 100-mg/dL increment in plasma glucose above normal, the plasma sodium should decrease by 1.6 mEq/LLastly, extreme elevations in plasma lipids and proteins can cause pseudohyponatremia because there is no true decrease in extracellular sodium relative to water.Signs and symptoms of hyponatremia (Table 3-4) are dependent on the degree of hyponatremia and the rapidity with which it

1	because there is no true decrease in extracellular sodium relative to water.Signs and symptoms of hyponatremia (Table 3-4) are dependent on the degree of hyponatremia and the rapidity with which it occurred. Clinical manifestations primarily have a central nervous system origin and are related to cellular water intoxication and associated increases in intracranial pressure. Oliguric renal failure also can be a rapid complication in the setting of severe hyponatremia.A systematic review of the etiology of hyponatremia should reveal its cause in a given instance. Hyperosmolar causes, including hyperglycemia or mannitol infusion and pseu-dohyponatremia, should be easily excluded. Next, depletional versus dilutional causes of hyponatremia are evaluated. In the absence of renal disease, depletion is associated with low urine sodium levels (<20 mEq/L), whereas renal sodium wasting shows high urine sodium levels (>20 mEq/L). Dilutional causes of hyponatremia usually are associated with

1	is associated with low urine sodium levels (<20 mEq/L), whereas renal sodium wasting shows high urine sodium levels (>20 mEq/L). Dilutional causes of hyponatremia usually are associated with hypervolemic cir-culation. A normal volume status in the setting of hyponatremia should prompt an evaluation for a syndrome of inappropriate secretion of ADH.Hypernatremia. Hypernatremia results from either a loss of free water or a gain of sodium in excess of water. Like hypo-natremia, it can be associated with an increased, normal, or decreased extracellular volume (see Fig. 3-3). Hypervolemic hypernatremia usually is caused either by iatrogenic adminis-tration of sodium-containing fluids, including excess sodium bicarbonate, or mineralocorticoid as seen in hyperaldosteronism, Cushing’s syndrome, and congenital adrenal hyperplasia. Urine sodium concentration is typically >20 mEq/L, and urine osmo-larity is >300 mOsm/L. Normovolemic hypernatremia can result from renal causes, including diabetes

1	congenital adrenal hyperplasia. Urine sodium concentration is typically >20 mEq/L, and urine osmo-larity is >300 mOsm/L. Normovolemic hypernatremia can result from renal causes, including diabetes insipidus, diuretic use, and renal disease, or from nonrenal water loss from the GI tract or skin, although the same conditions can result in hypovolemic hypernatremia. When hypovolemia is present, the urine sodium concentration is <20 mEq/L and urine osmolarity is <300 to 400 mOsm/L. Nonrenal water loss can occur secondary to rela-tively isotonic GI fluid losses such as that caused by diarrhea, to hypotonic skin fluid losses such as loss due to fever, or to losses via tracheotomies during hyperventilation. Additionally, thyrotoxicosis can cause water loss, as can the use of hyper-tonic glucose solutions for peritoneal dialysis. With nonrenal water loss, the urine sodium concentration is <15 mEq/L, and the urine osmolarity is >400 mOsm/L.Symptomatic hypernatremia usually occurs only in

1	solutions for peritoneal dialysis. With nonrenal water loss, the urine sodium concentration is <15 mEq/L, and the urine osmolarity is >400 mOsm/L.Symptomatic hypernatremia usually occurs only in patients with impaired thirst or restricted access to fluid because thirst will result in increased water intake. Symptoms are rare until the serum sodium concentration exceeds 160 mEq/L but, once present, are associated with significant morbidity and mortality. Because symptoms are related to hyperosmolarity, central nervous system effects predominate (see Table 3-4). Water shifts from the intracellular to the extracellular space in response to a hyperosmolar extracellular space, which results in cellular dehydration. This can put traction on the cerebral vessels and lead to subarachnoid hemorrhage. Central nervous system symptoms can range from restlessness and irritability to seizures, coma, and death. The classic signs of hypovolemic hypernatremia (tachycardia, orthostasis, and

1	hemorrhage. Central nervous system symptoms can range from restlessness and irritability to seizures, coma, and death. The classic signs of hypovolemic hypernatremia (tachycardia, orthostasis, and hypotension) may be present, as well as the unique findings of dry, sticky mucous membranes.Composition Changes: Etiology and DiagnosisPotassium Abnormalities. The average dietary intake of potassium is approximately 50 to 100 mEq/d, which in the absence of hypokalemia is excreted primarily in the urine. Extra-cellular potassium is maintained within a narrow range, princi-pally by renal excretion of potassium, which can range from 10 to 700 mEq/d. Although only 2% of the total body potassium (4.5 mEq/L × 14 L = 63 mEq) is located within the extracellular compartment, this small amount is critical to cardiac and neu-romuscular function; thus, even minor changes can have major effects on cardiac activity. The intracellular and extracellular distribution of potassium is influenced by a number

1	to cardiac and neu-romuscular function; thus, even minor changes can have major effects on cardiac activity. The intracellular and extracellular distribution of potassium is influenced by a number of factors, including surgical stress, injury, acidosis, and tissue catabolism.Table 3-4Clinical manifestations of abnormalities in serum sodium levelBODY SYSTEMHYPONATREMIACentral nervous systemHeadache, confusion, hyperactive or hypoactive deep tendon reflexes, seizures, coma, increased intracranial pressureMusculoskeletalWeakness, fatigue, muscle cramps/twitchingGIAnorexia, nausea, vomiting, watery diarrheaCardiovascularHypertension and bradycardia if intracranial pressure increases significantlyTissueLacrimation, salivationRenalOliguriaBODY SYSTEMHYPERNATREMIACentral nervous systemRestlessness, lethargy, ataxia, irritability, tonic spasms, delirium, seizures, comaMusculoskeletalWeaknessCardiovascularTachycardia, hypotension, syncopeTissueDry sticky mucous membranes, red swollen tongue,

1	lethargy, ataxia, irritability, tonic spasms, delirium, seizures, comaMusculoskeletalWeaknessCardiovascularTachycardia, hypotension, syncopeTissueDry sticky mucous membranes, red swollen tongue, decreased saliva and tearsRenalOliguriaMetabolicFeverBrunicardi_Ch03_p0083-p0102.indd 8808/12/18 10:07 AM 89FLUID AND ELECTROLYTE MANAGEMENT OF THE SURGICAL PATIENTCHAPTER 3Hyperkalemia Hyperkalemia is defined as a serum potassium concentration above the normal range of 3.5 to 5.0 mEq/L. It is caused by excessive potassium intake, increased release of potassium from cells, or impaired potassium excretion by the kidneys (Table 3-5).6 Increased intake can be either from oral or IV supplementation, or from red cell lysis after transfu-sion. Hemolysis, rhabdomyolysis, and crush injuries can dis-rupt cell membranes and release intracellular potassium into the ECF. Acidosis and a rapid rise in extracellular osmolality from hyperglycemia or IV mannitol can raise serum potassium levels by causing

1	cell membranes and release intracellular potassium into the ECF. Acidosis and a rapid rise in extracellular osmolality from hyperglycemia or IV mannitol can raise serum potassium levels by causing a shift of potassium ions to the extracellular compartment.7 Because 98% of total body potassium is in the intracellular fluid compartment, even small shifts of intracel-lular potassium out of the intracellular fluid compartment can lead to a significant rise in extracellular potassium. A number of medications can contribute to hyperkalemia, particularly in the presence of renal insufficiency, including potassium-sparing diuretics, angiotensin-converting enzyme inhibitors, and non-steroidal anti-inflammatory drugs (NSAIDs). Spironolactone and angiotensin-converting enzyme inhibitors interfere with aldosterone activity, inhibiting the normal renal mechanism of potassium excretion. Acute and chronic renal insufficiency also impairs potassium excretion.Symptoms of hyperkalemia are primarily GI,

1	aldosterone activity, inhibiting the normal renal mechanism of potassium excretion. Acute and chronic renal insufficiency also impairs potassium excretion.Symptoms of hyperkalemia are primarily GI, neuromus-cular, and cardiovascular (Table 3-6). GI symptoms include nausea, vomiting, intestinal colic, and diarrhea. Neuromuscu-lar symptoms range from weakness to ascending paralysis to respiratory failure. Early cardiovascular signs may be appar-ent from electrocardiogram (ECG) changes and eventually lead to hemodynamic symptoms of arrhythmia and cardiac arrest. ECG changes that may be seen with hyperkalemia include high peaked T waves (early), widened QRS complex, flattened P wave, prolonged PR interval (first-degree block), sine wave formation, and ventricular fibrillation.Hypokalemia Hypokalemia is much more common than hyper-kalemia in the surgical patient. It may be caused by inadequate potassium intake; excessive renal potassium excretion; potas-sium loss in pathologic GI

1	is much more common than hyper-kalemia in the surgical patient. It may be caused by inadequate potassium intake; excessive renal potassium excretion; potas-sium loss in pathologic GI secretions, such as with diarrhea, fis-tulas, vomiting, or high nasogastric output; or intracellular shifts from metabolic alkalosis or insulin therapy (see Table 3-5). Table 3-5 Etiology of potassium abnormalitiesHyperkalemiaIncreased intake Potassium supplementation Blood transfusions Endogenous load/destruction: hemolysis, rhabdomyolysis, crush injury, gastrointestinal hemorrhageIncreased release Acidosis Rapid rise of extracellular osmolality (hyperglycemia or mannitol)Impaired excretion Potassium-sparing diuretics Renal insufficiency/failureHypokalemiaInadequate intake Dietary, potassium-free intravenous fluids, potassium deficient TPNExcessive potassium excretion Hyperaldosteronism MedicationsGI losses Direct loss of potassium from GI fluid (diarrhea) Renal loss of potassium (to conserve sodium

1	fluids, potassium deficient TPNExcessive potassium excretion Hyperaldosteronism MedicationsGI losses Direct loss of potassium from GI fluid (diarrhea) Renal loss of potassium (to conserve sodium in response to gastric losses)Table 3-6Clinical manifestations of abnormalities in potassium, magnesium, and calcium levelsINCREASED SERUM LEVELSSYSTEMPOTASSIUMMAGNESIUMCALCIUMGINausea/vomiting, colic, diarrheaNausea/vomitingAnorexia, nausea/vomiting, abdominal painNeuromuscularWeakness, paralysis, respiratory failureWeakness, lethargy, decreased reflexesWeakness, confusion, coma, bone painCardiovascularArrhythmia, arrestHypotension, arrestHypertension, arrhythmia, polyuriaRenal——PolydipsiaDECREASED SERUM LEVELSSYSTEMPOTASSIUMMAGNESIUMCALCIUMGIIleus, constipation——NeuromuscularDecreased reflexes, fatigue, weakness, paralysisHyperactive reflexes, muscle tremors, tetany, seizuresHyperactive reflexes, paresthesias, carpopedal spasm, seizuresCardiovascularArrestArrhythmiaHeart

1	reflexes, fatigue, weakness, paralysisHyperactive reflexes, muscle tremors, tetany, seizuresHyperactive reflexes, paresthesias, carpopedal spasm, seizuresCardiovascularArrestArrhythmiaHeart failureBrunicardi_Ch03_p0083-p0102.indd 8908/12/18 10:07 AM 90BASIC CONSIDERATIONSPART IThe change in potassium associated with alkalosis can be calcu-lated by the following formula:Potassium decreases by 0.3 mEq/L for every 0.1 increase in pH above normal.Additionally, drugs such as amphotericin, aminoglyco-sides, cisplatin, and ifosfamide that induce magnesium depletion cause renal potassium wastage.8,9 In cases in which potassium deficiency is due to magnesium depletion,10 potassium repletion is difficult unless hypomagnesemia is first corrected.The symptoms of hypokalemia (see Table 3-6), like those of hyperkalemia, are primarily related to failure of normal con-tractility of GI smooth muscle, skeletal muscle, and cardiac mus-cle. Findings may include ileus, constipation, weakness,

1	like those of hyperkalemia, are primarily related to failure of normal con-tractility of GI smooth muscle, skeletal muscle, and cardiac mus-cle. Findings may include ileus, constipation, weakness, fatigue, diminished tendon reflexes, paralysis, and cardiac arrest. In the setting of ECF depletion, symptoms may be masked initially and then worsened by further dilution during volume repletion. ECG changes suggestive of hypokalemia include U waves, T-wave flattening, ST-segment changes, and arrhythmias (with digitalis therapy).Calcium Abnormalities. The vast majority of the body’s calcium is contained within the bone matrix, with <1% found in the ECF. Serum calcium is distributed among three forms: protein found (40%), complexed to phosphate and other anions (10%), and ionized (50%). It is the ionized fraction that is responsible for neuromuscular stability and can be measured directly. When total serum calcium levels are measured, the albumin concentration must be taken into

1	It is the ionized fraction that is responsible for neuromuscular stability and can be measured directly. When total serum calcium levels are measured, the albumin concentration must be taken into consideration:Adjust total serum calcium down by 0.8 mg/dL for every 1 g/dL decrease in albumin.Unlike changes in albumin, changes in pH will affect the ionized calcium concentration. Acidosis decreases protein bind-ing, thereby increasing the ionized fraction of calcium.Daily calcium intake is 1 to 3 g/d. Most of this is excreted via the bowel, with urinary excretion relatively low. Total body calcium balance is under complex hormonal control, but distur-bances in metabolism are relatively long term and less important in the acute surgical setting. However, attention to the critical role of ionized calcium in neuromuscular function often is required.Hypercalcemia Hypercalcemia is defined as a serum calcium level above the normal range of 8.5 to 10.5 mEq/L or an increase in the ionized

1	ionized calcium in neuromuscular function often is required.Hypercalcemia Hypercalcemia is defined as a serum calcium level above the normal range of 8.5 to 10.5 mEq/L or an increase in the ionized calcium level above 4.2 to 4.8 mg/dL. Primary hyperparathyroidism in the outpatient setting and malignancy in hospitalized patients, from either bony metastasis or secre-tion of parathyroid hormone–related protein, account for most cases of symptomatic hypercalcemia.11 Symptoms of hypercal-cemia (see Table 3-6), which vary with the degree of sever-ity, include neurologic impairment, musculoskeletal weakness and pain, renal dysfunction, GI symptoms of nausea, vomiting, and abdominal pain. Cardiac symptoms can be manifested as hypertension, cardiac arrhythmias, and a worsening of digitalis toxicity. ECG changes in hypercalcemia include shortened QT interval, prolonged PR and QRS intervals, increased QRS volt-age, T-wave flattening and widening, and atrioventricular block (which can progress

1	ECG changes in hypercalcemia include shortened QT interval, prolonged PR and QRS intervals, increased QRS volt-age, T-wave flattening and widening, and atrioventricular block (which can progress to complete heart block and cardiac arrest).Hypocalcemia Hypocalcemia is defined as a serum calcium level below 8.5 mEq/L or a decrease in the ionized calcium level below 4.2 mg/dL. The causes of hypocalcemia include pancreatitis, massive soft tissue infections such as necrotizing fasciitis, renal failure, pancreatic and small bowel fistulas, hypo-parathyroidism, toxic shock syndrome, abnormalities in mag-nesium levels, and tumor lysis syndrome. In addition, transient hypocalcemia commonly occurs after removal of a parathyroid adenoma due to atrophy of the remaining glands and avid bone remineralization, and sometimes requires high-dose calcium supplementation.12 Additionally, malignancies associated with increased osteoblastic activity, such as breast and prostate can-cer, can lead to

1	and sometimes requires high-dose calcium supplementation.12 Additionally, malignancies associated with increased osteoblastic activity, such as breast and prostate can-cer, can lead to hypocalcemia from increased bone formation.13 Calcium precipitation with organic anions is also a cause of hypocalcemia and may occur during hyperphosphatemia from tumor lysis syndrome or rhabdomyolysis. Pancreatitis may sequester calcium via chelation with free fatty acids. Massive blood transfusion with citrate binding is another mechanism.14,15 Hypocalcemia rarely results solely from decreased intake because bone reabsorption can maintain normal levels for pro-longed periods.Asymptomatic hypocalcemia may occur when hypopro-teinemia results in a normal ionized calcium level. Conversely, symptoms can develop with a normal serum calcium level during alkalosis, which decreases ionized calcium. In general, neuromuscular and cardiac symptoms do not occur until the ion-ized fraction falls below 2.5 mg/dL

1	with a normal serum calcium level during alkalosis, which decreases ionized calcium. In general, neuromuscular and cardiac symptoms do not occur until the ion-ized fraction falls below 2.5 mg/dL (see Table 3-6). Clinical findings may include paresthesias of the face and extremities, muscle cramps, carpopedal spasm, stridor, tetany, and seizures. Patients will demonstrate hyperreflexia and may exhibit positive Chvostek’s sign (spasm resulting from tapping over the facial nerve) and Trousseau’s sign (spasm resulting from pressure applied to the nerves and vessels of the upper extremity with a blood pressure cuff). Hypocalcemia may lead to decreased cardiac contractility and heart failure. ECG changes of hypocal-cemia include prolonged QT interval, T-wave inversion, heart block, and ventricular fibrillation.Phosphorus Abnormalities. Phosphorus is the primary intra-cellular divalent anion and is abundant in metabolically active cells. Phosphorus is involved in energy production during

1	fibrillation.Phosphorus Abnormalities. Phosphorus is the primary intra-cellular divalent anion and is abundant in metabolically active cells. Phosphorus is involved in energy production during gly-colysis and is found in high-energy phosphate products such as adenosine triphosphate. Serum phosphate levels are tightly controlled by renal excretion.Hyperphosphatemia Hyperphosphatemia can be due to decreased urinary excretion, increased intake, or endogenous mobilization of phosphorus. Most cases of hyperphosphatemia are seen in patients with impaired renal function. Hypoparathy-roidism or hyperthyroidism also can decrease urinary excretion of phosphorus and thus lead to hyperphosphatemia. Increased release of endogenous phosphorus can be seen in association with any clinical condition that results in cell destruction, includ-ing rhabdomyolysis, tumor lysis syndrome, hemolysis, sepsis, severe hypothermia, and malignant hyperthermia. Excessive phosphate administration from IV

1	that results in cell destruction, includ-ing rhabdomyolysis, tumor lysis syndrome, hemolysis, sepsis, severe hypothermia, and malignant hyperthermia. Excessive phosphate administration from IV hyperalimentation solutions or phosphorus-containing laxatives may also lead to elevated phosphate levels. Most cases of hyperphosphatemia are asymp-tomatic, but significant prolonged hyperphosphatemia can lead to metastatic deposition of soft tissue calcium-phosphorus complexes.Hypophosphatemia Hypophosphatemia can be due to a decrease in phosphorus intake, an intracellular shift of phospho-rus, or an increase in phosphorus excretion. Decreased GI uptake due to malabsorption or administration of phosphate binders and decreased dietary intake from malnutrition are causes of chronic hypophosphatemia. Most acute cases are due to an intracellular Brunicardi_Ch03_p0083-p0102.indd 9008/12/18 10:07 AM 91FLUID AND ELECTROLYTE MANAGEMENT OF THE SURGICAL PATIENTCHAPTER 3shift of phosphorus in

1	Most acute cases are due to an intracellular Brunicardi_Ch03_p0083-p0102.indd 9008/12/18 10:07 AM 91FLUID AND ELECTROLYTE MANAGEMENT OF THE SURGICAL PATIENTCHAPTER 3shift of phosphorus in association with respiratory alkalosis, insulin therapy, refeeding syndrome, and hungry bone syn-drome. Clinical manifestations of hypophosphatemia usually are absent until levels fall significantly. In general, symptoms are related to adverse effects on the oxygen availability of tissue and to a decrease in high-energy phosphates, and can be mani-fested as cardiac dysfunction or muscle weakness.Magnesium Abnormalities. Magnesium is the fourth most common mineral in the body and, like potassium, is found pri-marily in the intracellular compartments. Approximately one-half of the total body content of 2000 mEq is incorporated in bone and is slowly exchangeable. Of the fraction found in the extracellular space, one-third is bound to serum albumin. There-fore, the plasma level of magnesium may be a

1	2000 mEq is incorporated in bone and is slowly exchangeable. Of the fraction found in the extracellular space, one-third is bound to serum albumin. There-fore, the plasma level of magnesium may be a poor indicator of total body stores in the presence of hypoalbuminemia. Mag-nesium should be replaced until levels are in the upper limit of normal. The normal dietary intake is approximately 20 mEq/d and is excreted in both the feces and urine. The kidneys have a remarkable ability to conserve magnesium, with renal excretion <1 mEq/d during magnesium deficiency.Hypermagnesemia Hypermagnesemia is rare but can be seen with severe renal insufficiency and parallel changes in potas-sium excretion. Magnesium-containing antacids and laxatives can produce toxic levels in patients with renal failure. Excess intake in conjunction with total parenteral nutrition (TPN), or, rarely, massive trauma, thermal injury, and severe acidosis, may be associated with symptomatic hypermagnesemia. Clini-cal

1	Excess intake in conjunction with total parenteral nutrition (TPN), or, rarely, massive trauma, thermal injury, and severe acidosis, may be associated with symptomatic hypermagnesemia. Clini-cal examination (see Table 3-6) may find nausea and vomit-ing; neuromuscular dysfunction with weakness, lethargy, and hyporeflexia; and impaired cardiac conduction leading to hypo-tension and arrest. ECG changes are similar to those seen with hyperkalemia and include increased PR interval, widened QRS complex, and elevated T waves.Hypomagnesemia Magnesium depletion is a common prob-lem in hospitalized patients, particularly in the critically ill.16 The kidney is primarily responsible for magnesium homeostasis through regulation by calcium/magnesium receptors on the renal tubular cells that respond to serum magnesium concentrations.17 Hypomagnesemia may result from alterations of intake, renal excretion, and pathologic losses. Poor intake may occur in cases of starvation, alcoholism, prolonged IV

1	serum magnesium concentrations.17 Hypomagnesemia may result from alterations of intake, renal excretion, and pathologic losses. Poor intake may occur in cases of starvation, alcoholism, prolonged IV fluid therapy, and TPN with inadequate supplementation of magnesium. Losses are seen in cases of increased renal excretion from alcohol abuse, diuretic use, administration of amphotericin B, and primary aldosteronism, as well as GI losses from diarrhea, malabsorp-tion, and acute pancreatitis. The magnesium ion is essential for proper function of many enzyme systems. Depletion is characterized by neuromuscular and central nervous system hyperactivity. Symptoms are similar to those of calcium defi-ciency, including hyperactive reflexes, muscle tremors, tetany, and positive Chvostek’s and Trousseau’s signs (see Table 3-6). Severe deficiencies can lead to delirium and seizures. A number of ECG changes also can occur and include prolonged QT and PR intervals, ST-segment depression, flattening

1	signs (see Table 3-6). Severe deficiencies can lead to delirium and seizures. A number of ECG changes also can occur and include prolonged QT and PR intervals, ST-segment depression, flattening or inversion of P waves, torsades de pointes, and arrhythmias. Hypomagnese-mia is important not only because of its direct effects on the nervous system but also because it can produce hypocalcemia and lead to persistent hypokalemia. When hypokalemia or hypo-calcemia coexists with hypomagnesemia, magnesium should be aggressively replaced to assist in restoring potassium or calcium homeostasis.Acid-Base BalanceAcid-Base Homeostasis. The pH of body fluids is maintained within a narrow range despite the ability of the kidneys to gen-erate large amounts of HCO3− and the normal large acid load produced as a by-product of metabolism. This endogenous acid load is efficiently neutralized by buffer systems and ultimately excreted by the lungs and kidneys.Important buffers include intracellular proteins

1	as a by-product of metabolism. This endogenous acid load is efficiently neutralized by buffer systems and ultimately excreted by the lungs and kidneys.Important buffers include intracellular proteins and phos-phates and the extracellular bicarbonate–carbonic acid system. Compensation for acid-base derangements can be by respiratory mechanisms (for metabolic derangements) or metabolic mecha-nisms (for respiratory derangements). Changes in ventilation in response to metabolic abnormalities are mediated by hydrogen-sensitive chemoreceptors found in the carotid body and brain stem. Acidosis stimulates the chemoreceptors to increase venti-lation, whereas alkalosis decreases the activity of the chemore-ceptors and thus decreases ventilation. The kidneys provide compensation for respiratory abnormalities by either increasing or decreasing bicarbonate reabsorption in response to respira-tory acidosis or alkalosis, respectively. Unlike the prompt change in ventilation that occurs with

1	abnormalities by either increasing or decreasing bicarbonate reabsorption in response to respira-tory acidosis or alkalosis, respectively. Unlike the prompt change in ventilation that occurs with metabolic abnormalities, the compensatory response in the kidneys to respiratory abnor-malities is delayed. Significant compensation may not begin for 6 hours and then may continue for several days. Because of this delayed compensatory response, respiratory acid-base derange-ments before renal compensation are classified as acute, whereas those persisting after renal compensation are categorized as chronic. The predicted compensatory changes in response to metabolic or respiratory derangements are listed in Table 3-7.18 If the predicted change in pH is exceeded, then a mixed acid-base abnormality may be present (Table 3-8).Metabolic DerangementsMetabolic Acidosis. Metabolic acidosis results from an increased intake of acids, an increased generation of acids, or an increased loss of

1	may be present (Table 3-8).Metabolic DerangementsMetabolic Acidosis. Metabolic acidosis results from an increased intake of acids, an increased generation of acids, or an increased loss of bicarbonate (Table 3-9). The body responds by several mechanisms, including producing buffers (extracellular bicarbonate and intracellular buffers from bone and muscle), increasing ventilation (Kussmaul’s respirations), and increas-ing renal reabsorption and generation of bicarbonate. The kid-ney also will increase secretion of hydrogen and thus increase urinary excretion of NH4+ (H+ + NH3+ = NH4+). Evaluation of a patient with a low serum bicarbonate level and metabolic 4Table 3-7 Predicted changes in acid-base disordersDISORDERPREDICTED CHANGEMetabolic Metabolic acidosis Metabolic alkalosisRespiratory Acute respiratory acidosis Chronic respiratory acidosis Acute respiratory alkalosis Chronic respiratory alkalosisPco2 = 1.5 × HCO3− + 8Pco2 = 0.7 × HCO3− + 21Δ pH = (Pco2 – 40) × 0.008Δ pH =

1	respiratory acidosis Chronic respiratory acidosis Acute respiratory alkalosis Chronic respiratory alkalosisPco2 = 1.5 × HCO3− + 8Pco2 = 0.7 × HCO3− + 21Δ pH = (Pco2 – 40) × 0.008Δ pH = (Pco2 – 40) × 0.003 Δ pH = (40 – Pco2) × 0.008Δ pH = (40 – Pco2) × 0.017Pco2 = partial pressure of carbon dioxide.Brunicardi_Ch03_p0083-p0102.indd 9108/12/18 10:07 AM 92BASIC CONSIDERATIONSPART Iacidosis includes determination of the anion gap (AG), an index of unmeasured anions.AG = (Na) – (Cl + HCO3)The normal AG is <12 mmol/L and is due primarily to the albumin effect, so that the estimated AG must be adjusted for albumin (hypoalbuminemia reduces the AG).19Corrected AG = actual AG – (2.5[4.5 – albumin])Metabolic acidosis with an increased AG occurs either from ingestion of exogenous acid such as from ethylene gly-col, salicylates, or methanol, or from increased endogenous acid production of the following:• β-Hydroxybutyrate and acetoacetate in ketoacidosis• Lactate in lactic acidosis• Organic

1	ethylene gly-col, salicylates, or methanol, or from increased endogenous acid production of the following:• β-Hydroxybutyrate and acetoacetate in ketoacidosis• Lactate in lactic acidosis• Organic acids in renal insufficiencyA common cause of severe metabolic acidosis in surgi-cal patients is lactic acidosis. In circulatory shock, lactate is produced in the presence of hypoxia from inadequate tissue perfusion. The treatment is to restore perfusion with volume resuscitation rather than to attempt to correct the abnormality with exogenous bicarbonate. With adequate perfusion, the lactic acid is rapidly metabolized by the liver, and the pH level returns to normal. In clinical studies of lactic acidosis and ketoacidosis, the administration of bicarbonate has not reduced morbidity or mortality or improved cellular function.20 Administered bicar-bonate can combine with the excess hydrogen ions to form car-bonic acid; this is then converted to CO2 and water, which thus raises the partial

1	or improved cellular function.20 Administered bicar-bonate can combine with the excess hydrogen ions to form car-bonic acid; this is then converted to CO2 and water, which thus raises the partial pressure of CO2 (Pco2). This hypercarbia could compound ventilation abnormalities in patients with underlying acute respiratory distress syndrome. This CO2 can diffuse into cells, but bicarbonate remains extracellular, which thus worsens intracellular acidosis. Clinically, lactate levels may not be useful in directing resuscitation, although lactate levels may be higher in nonsurvivors of serious injury.21Metabolic acidosis with a normal AG results from exog-enous acid administration (HCl or NH4+), from loss of bicar-bonate due to GI disorders such as diarrhea and fistulas or ureterosigmoidostomy, or from renal losses. In these settings, the bicarbonate loss is accompanied by a gain of chloride; thus, the AG remains unchanged. To determine whether the loss of bicarbonate has a renal cause,

1	or from renal losses. In these settings, the bicarbonate loss is accompanied by a gain of chloride; thus, the AG remains unchanged. To determine whether the loss of bicarbonate has a renal cause, the urinary (NH4+) can be mea-sured. A low urinary (NH4+) in the face of hyperchloremic acidosis would indicate that the kidney is the site of loss, and evaluation for renal tubular acidosis should be undertaken. Prox-imal renal tubular acidosis results from decreased tubular reab-sorption of HCO3−, whereas distal renal tubular acidosis results from decreased acid excretion. The carbonic anhydrase inhibitor acetazolamide also causes bicarbonate loss from the kidneys.Metabolic Alkalosis Normal acid-base homeostasis prevents metabolic alkalosis from developing unless both an increase in bicarbonate generation and impaired renal excretion of bicar-bonate occur (Table 3-10). Metabolic alkalosis results from the loss of fixed acids or the gain of bicarbonate and is worsened by potassium depletion.

1	generation and impaired renal excretion of bicar-bonate occur (Table 3-10). Metabolic alkalosis results from the loss of fixed acids or the gain of bicarbonate and is worsened by potassium depletion. The majority of patients also will have hypokalemia because extracellular potassium ions exchange with intracellular hydrogen ions and allow the hydrogen ions to buffer excess HCO3–. Hypochloremic and hypokalemic meta-bolic alkalosis can occur from isolated loss of gastric contents in infants with pyloric stenosis or adults with duodenal ulcer disease. Unlike vomiting associated with an open pylorus, which involves a loss of gastric as well as pancreatic, biliary, Table 3-9 Etiology of metabolic acidosisIncreased Anion Gap Metabolic AcidosisExogenous acid ingestion Ethylene glycol Salicylate MethanolEndogenous acid production Ketoacidosis Lactic acidosis Renal insufficiencyNormal Anion GapAcid administration (HCl)Loss of bicarbonateGI losses (diarrhea, fistulas)UreterosigmoidostomyRenal

1	acid production Ketoacidosis Lactic acidosis Renal insufficiencyNormal Anion GapAcid administration (HCl)Loss of bicarbonateGI losses (diarrhea, fistulas)UreterosigmoidostomyRenal tubular acidosisCarbonic anhydrase inhibitorTable 3-8 Respiratory and metabolic components of acid-base disorders ACUTE UNCOMPENSATEDCHRONIC (PARTIALLY COMPENSATED)TYPE OF ACID-BASE DISORDERpHPco2 (RESPIRATORY COMPONENT)PLASMA HCO3−a (METABOLIC COMPONENT)pHPco2 (RESPIRATORY COMPONENT)PLASMA HCO3−a (METABOLIC COMPONENT)Respiratory acidosis↓↓↑↑N↓↑↑↑Respiratory alkalosis↑↑↓↓N↑↓↓↓Metabolic acidosis↓↓N↓↓↓↓↓Metabolic alkalosis↑↑N↑↑↑↑↑aMeasured as standard bicarbonate, whole blood buffer base, CO2 content, or CO2 combining power. The base excess value is positive when the standard bicarbonate is above normal and negative when the standard bicarbonate is below normal.N = normal; Pco2 = partial pressure of carbon dioxide.Brunicardi_Ch03_p0083-p0102.indd 9208/12/18 10:07 AM 93FLUID AND ELECTROLYTE MANAGEMENT OF

1	when the standard bicarbonate is below normal.N = normal; Pco2 = partial pressure of carbon dioxide.Brunicardi_Ch03_p0083-p0102.indd 9208/12/18 10:07 AM 93FLUID AND ELECTROLYTE MANAGEMENT OF THE SURGICAL PATIENTCHAPTER 3and intestinal secretions, vomiting with an obstructed pylorus results only in the loss of gastric fluid, which is high in chloride and hydrogen, and therefore results in a hypochloremic alkalo-sis. Initially, the urinary bicarbonate level is high in compensa-tion for the alkalosis. Hydrogen ion reabsorption also ensues, with an accompanied potassium ion excretion. In response to the associated volume deficit, aldosterone-mediated sodium reabsorption increases potassium excretion. The resulting hypo-kalemia leads to the excretion of hydrogen ions in the face of alkalosis, a paradoxic aciduria. Treatment includes replacement of the volume deficit with isotonic saline and then potassium replacement once adequate urine output is achieved.Respiratory

1	face of alkalosis, a paradoxic aciduria. Treatment includes replacement of the volume deficit with isotonic saline and then potassium replacement once adequate urine output is achieved.Respiratory Derangements. Under normal circumstances, blood Pco2 is tightly maintained by alveolar ventilation, con-trolled by the respiratory centers in the pons and medulla oblongata.Respiratory Acidosis Respiratory acidosis is associated with the retention of CO2 secondary to decreased alveolar ventila-tion. The principal causes are listed in Table 3-11. Because compensation is primarily a renal mechanism, it is a delayed response. Treatment of acute respiratory acidosis is directed at the underlying cause. Measures to ensure adequate ventila-tion are also initiated. This may entail patient-initiated volume expansion using noninvasive bilevel positive airway pressure or may require endotracheal intubation to increase minute ven-tilation. In the chronic form of respiratory acidosis, the partial

1	volume expansion using noninvasive bilevel positive airway pressure or may require endotracheal intubation to increase minute ven-tilation. In the chronic form of respiratory acidosis, the partial pressure of arterial CO2 remains elevated, and the bicarbonate concentration rises slowly as renal compensation occurs.Respiratory Alkalosis In the surgical patient, most cases of respiratory alkalosis are acute and secondary to alveolar hyper-ventilation. Causes include pain, anxiety, and neurologic dis-orders, including central nervous system injury and assisted ventilation. Drugs such as salicylates, fever, gram-negative bacteremia, thyrotoxicosis, and hypoxemia are other possibili-ties. Acute hypocapnia can cause an uptake of potassium and phosphate into cells and increased binding of calcium to albu-min, leading to symptomatic hypokalemia, hypophosphatemia, and hypocalcemia with subsequent arrhythmias, paresthesias, muscle cramps, and seizures. Treatment should be directed at the

1	calcium to albu-min, leading to symptomatic hypokalemia, hypophosphatemia, and hypocalcemia with subsequent arrhythmias, paresthesias, muscle cramps, and seizures. Treatment should be directed at the underlying cause, but direct treatment of the hyperventila-tion using controlled ventilation may also be required.FLUID AND ELECTROLYTE THERAPYParenteral SolutionsA number of commercially available electrolyte solutions are available for parenteral administration. The most commonly used solutions are listed in Table 3-12. The type of fluid admin-istered depends on the patient’s volume status and the type of concentration or compositional abnormality present. Plasma-Lyte, lactated Ringer’s solution, and normal saline are consid-ered isotonic and are useful in replacing GI losses and correcting extracellular volume deficits. Lactated Ringer’s is slightly hypotonic in that it contains 130 mEq of lactate. Lactate is used rather than bicarbonate because it is more stable in IV fluids during

1	extracellular volume deficits. Lactated Ringer’s is slightly hypotonic in that it contains 130 mEq of lactate. Lactate is used rather than bicarbonate because it is more stable in IV fluids during storage. It is converted into bicarbonate by the liver after infusion, even in the face of hemorrhagic shock.Sodium chloride is mildly hypertonic, containing 154 mEq of sodium that is balanced by 154 mEq of chloride. The high chloride concentration imposes a significant chloride load on the kidneys and may lead to a hyperchloremic metabolic acido-sis. Sodium chloride is an ideal solution, however, for correcting volume deficits associated with hyponatremia, hypochloremia, and metabolic alkalosis.Plasma-Lyte is the crystalloid preparation that most closely resembles the electrolyte composition of human plasma. In addi-tion to the favorable, isotonic electrolyte composition, Plasma-Lyte contains a number of additional buffers that create a favorable profile for addressing acidosis.22 These

1	of human plasma. In addi-tion to the favorable, isotonic electrolyte composition, Plasma-Lyte contains a number of additional buffers that create a favorable profile for addressing acidosis.22 These characteristics have resulted in Plasma-Lyte emerging as one of the most popular isotonic fluids for use in surgery. It should be noted that Plasma-Lyte contains small quantities of potassium; although the likeli-hood of inducing hyperkalemia is very low, care should be taken when using Plasma-Lyte in patients at risk for renal impairment. The less concentrated sodium solutions, such as 0.45% sodium chloride, are useful for replacement of ongoing GI losses as well as for maintenance fluid therapy in the post-operative period. This solution provides sufficient free water for insensible losses and enough sodium to aid the kidneys in adjustment of serum sodium levels. The addition of 5% dextrose Table 3-10 Etiology of metabolic alkalosisIncreased bicarbonate generation1. Chloride losing

1	and enough sodium to aid the kidneys in adjustment of serum sodium levels. The addition of 5% dextrose Table 3-10 Etiology of metabolic alkalosisIncreased bicarbonate generation1. Chloride losing (urinary chloride >20 mEq/L)Mineralocorticoid excessProfound potassium depletion2. Chloride sparing (urinary chloride <20 mEq/L)Loss from gastric secretions (emesis or nasogastric suction)Diuretics3. Excess administration of alkaliAcetate in parenteral nutritionCitrate in blood transfusionsAntacidsBicarbonateMilk-alkali syndromeImpaired bicarbonate excretion1. Decreased glomerular filtration2. Increased bicarbonate reabsorption (hypercarbia or potassium depletion)Table 3-11Etiology of respiratory acidosis: hypoventilationNarcoticsCentral nervous system injuryPulmonary: significant Secretions Atelectasis Mucus plug Pneumonia Pleural effusionPain from abdominal or thoracic injuries or incisionsLimited diaphragmatic excursion from intra-abdominal pathology Abdominal distention Abdominal

1	plug Pneumonia Pleural effusionPain from abdominal or thoracic injuries or incisionsLimited diaphragmatic excursion from intra-abdominal pathology Abdominal distention Abdominal compartment syndrome AscitesBrunicardi_Ch03_p0083-p0102.indd 9308/12/18 10:07 AM 94BASIC CONSIDERATIONSPART I(50 g of dextrose per liter) supplies 200 kcal/L, and dextrose is always added to solutions containing <0.45% sodium chloride to maintain osmolality and thus prevent the lysis of red blood cells that may occur with rapid infusion of hypotonic fluids. The addition of potassium is useful once adequate renal function and urine output are established.Alternative Resuscitative FluidsA number of alternative solutions for volume expansion and resuscitation are available (Table 3-13).23 Hypertonic saline solutions (3.5% and 5%) are used for correction of severe sodium deficits and are discussed elsewhere in this chap-ter. Hypertonic saline (7.5%) has been used as a treatment modality in patients with closed

1	(3.5% and 5%) are used for correction of severe sodium deficits and are discussed elsewhere in this chap-ter. Hypertonic saline (7.5%) has been used as a treatment modality in patients with closed head injuries. It has been shown to increase cerebral perfusion and decrease intracranial pressure, thus decreasing brain edema.24 However, there have also been concerns about increased bleeding because hypertonic saline is an arteriolar vasodilator. A recent meta-analysis of hypertonic saline in severe traumatic brain injury revealed that, in a total of 11 eligible studies, there was no mortality benefit associated with hypertonic saline compared to other solutions.25 Colloids also are used in surgical patients, and their effectiveness as vol-ume expanders compared with isotonic crystalloids has long been debated. Due to their molecular weight, they are confined to the intravascular space, and their infusion results in more effi-cient transient plasma volume expansion. However, under

1	has long been debated. Due to their molecular weight, they are confined to the intravascular space, and their infusion results in more effi-cient transient plasma volume expansion. However, under con-ditions of severe hemorrhagic shock, capillary membrane permeability increases; this permeability permits colloids to enter the interstitial space, which can worsen edema and impair tissue oxygenation. Four major types of colloids are available—albumin, dextrans, hetastarch, and gelatins—and are described by their molecular weight and size in Table 3-13. In a large randomized trial of patients admitted to an intensive care unit with hypovolemic shock, administration of colloid showed no improvement in mortality at 30 days as compared to crystalloid resuscitation.26 Interestingly, there was a suggestion of improve-ment of 90-day mortality and more days alive without mechani-cal ventilation in patients receiving colloid; however, these were not trial primary endpoints and were considered

1	a suggestion of improve-ment of 90-day mortality and more days alive without mechani-cal ventilation in patients receiving colloid; however, these were not trial primary endpoints and were considered exploratory. Furthermore, a Cochrane Database systematic review on the topic concluded that there is no available evidence from ran-domized trials to support colloid use over crystalloid to reduce the risk of death following trauma, burns, or surgery.27 Colloids are markedly more expensive than crystalloids, and certain col-loids, such as hydroxyethyl starch, have been associated with increased morbidity including the need for renal replacement therapy.28 Taken together, the use of colloid for resuscitation of critically ill and surgical patients has limited application.Correction of Life-Threatening Electrolyte AbnormalitiesSodium Hypernatremia Treatment of hypernatremia usually consists of treatment of the associated water deficit. In hypovolemic patients, volume should be restored with

1	Electrolyte AbnormalitiesSodium Hypernatremia Treatment of hypernatremia usually consists of treatment of the associated water deficit. In hypovolemic patients, volume should be restored with normal saline before the concentration abnormality is addressed. Once adequate volume has been achieved, the water deficit is replaced using a hypotonic fluid such as 5% dextrose, 5% dextrose in onequarter normal saline, or enterally administered water. The for-mula used to estimate the amount of water required to correct hypernatremia is as follows:Waterdeficit(L)serumsodium140=−×140TBWEstimate TBW as 50% of lean body mass in men and 40% in women5Table 3-12 Electrolyte solutions for parenteral administration ELECTROLYTE COMPOSITION (mEq/L)SOLUTIONNaClKHCO3−CaMgmOsmExtracellular fluid14210342753280–310Lactated Ringer’s1301094283 2730.9% Sodium chloride154154 308D5 0.45% Sodium chloride7777 407D5W 2533% Sodium chloride513513 1026Plasma-Lyte14098527 3295D5 = 5% dextrose; D5W = 5%

1	Ringer’s1301094283 2730.9% Sodium chloride154154 308D5 0.45% Sodium chloride7777 407D5W 2533% Sodium chloride513513 1026Plasma-Lyte14098527 3295D5 = 5% dextrose; D5W = 5% dextrose in water.Table 3-13 Alternative resuscitative fluidsSOLUTIONMOLECULAR WEIGHTOSMOLALITY (mOsm/L)SODIUM (mEq/L)Hypertonic saline (7.5%)—25651283Albumin 5%70,000300130–160Albumin 25%70,0001500130–160Dextran 4040,000308154Dextran 7070,000308154Hetastarch450,000310154Hextend670,000307143Gelofusine30,000NA154NA = not available.Brunicardi_Ch03_p0083-p0102.indd 9408/12/18 10:07 AM 95FLUID AND ELECTROLYTE MANAGEMENT OF THE SURGICAL PATIENTCHAPTER 3The rate of fluid administration should be titrated to achieve a decrease in serum sodium concentration of no more than 1 mEq/h and 12 mEq/d for the treatment of acute symp-tomatic hypernatremia. Even slower correction should be under-taken for chronic hypernatremia (0.7 mEq/h) because overly rapid correction can lead to cerebral edema and herniation. The

1	of acute symp-tomatic hypernatremia. Even slower correction should be under-taken for chronic hypernatremia (0.7 mEq/h) because overly rapid correction can lead to cerebral edema and herniation. The type of fluid used depends on the severity and ease of correc-tion. Oral or enteral replacement is acceptable in most cases, or IV replacement with halfor quarter-normal saline can be used. Caution also should be exercised when using 5% dextrose in water to avoid overly rapid correction. Frequent neurologic evaluation as well as frequent evaluation of serum sodium levels also should be performed. Hypernatremia is less common than hyponatremia, but has a worse prognosis, and is an independent predictor of mortality in critical illness.29Hyponatremia Most cases of hyponatremia can be treated by free water restriction and, if severe, the administration of sodium. In patients with normal renal function, symptomatic hyponatremia usually does not occur until the serum sodium level is ≤120 mEq/L.

1	water restriction and, if severe, the administration of sodium. In patients with normal renal function, symptomatic hyponatremia usually does not occur until the serum sodium level is ≤120 mEq/L. If neurologic symptoms are present, 3% normal saline should be used to increase the sodium by no more than 1 mEq/L per hour until the serum sodium level reaches 130 mEq/L or neurologic symptoms are improved. Correction of asymptomatic hyponatremia should increase the sodium level by no more than 0.5 mEq/L per hour to a maxi-mum increase of 12 mEq/L per day, and even more slowly in chronic hyponatremia. The rapid correction of hyponatremia can lead to pontine myelinolysis,30 with seizures, weakness, paresis, akinetic movements, and unresponsiveness, and may result in permanent brain damage and death. Serial magnetic resonance imaging may be necessary to confirm the diagnosis.31Potassium Hyperkalemia Treatment options for symptomatic hyperka-lemia are listed in Table 3-14. The goals of therapy

1	Serial magnetic resonance imaging may be necessary to confirm the diagnosis.31Potassium Hyperkalemia Treatment options for symptomatic hyperka-lemia are listed in Table 3-14. The goals of therapy include reducing the total body potassium, shifting potassium from the extracellular to the intracellular space, and protecting the cells from the effects of increased potassium. For all patients, exogenous sources of potassium should be removed, including potassium supplementation in IV fluids and enteral and paren-teral solutions. Potassium can be removed from the body using a cation-exchange resin such as Kayexalate that binds potas-sium in exchange for sodium. It can be administered either orally, in alert patients, or rectally. Immediate measures also should include attempts to shift potassium intracellularly with glucose and bicarbonate infusion. Nebulized albuterol (10 to 20 mg) may also be used. Use of glucose alone will cause a rise in insulin secretion, but in the acutely ill, this

1	intracellularly with glucose and bicarbonate infusion. Nebulized albuterol (10 to 20 mg) may also be used. Use of glucose alone will cause a rise in insulin secretion, but in the acutely ill, this response may be blunted, and therefore both glucose and insulin may be necessary. Circulatory overload and hypernatremia may result from the administration of Kayexalate and bicarbonate, so care should be exercised when administering these agents to patients with fragile cardiac function. When ECG changes are present, calcium chloride or calcium gluconate (5 to 10 mL of 10% solution) should be administered immediately to counteract the myocardial effects of hyperkalemia. Calcium infusion should be used cautiously in patients receiving digitalis because digitalis toxicity may be precipitated. All of the aforementioned mea-sures are temporary, lasting from 1 to approximately 4 hours. Dialysis should be considered in severe hyperkalemia when conservative measures fail.Hypokalemia Treatment for

1	of the aforementioned mea-sures are temporary, lasting from 1 to approximately 4 hours. Dialysis should be considered in severe hyperkalemia when conservative measures fail.Hypokalemia Treatment for hypokalemia consists of potas-sium repletion, the rate of which is determined by the symptoms (Table 3-15). Oral repletion is adequate for mild, asymptomatic hypokalemia. If IV repletion is required, usually no more than 10 mEq/h is advisable in an unmonitored setting. This amount can be increased to 40 mEq/h when accompanied by continu-ous ECG monitoring, and even more in the case of imminent cardiac arrest from a malignant arrhythmia-associated hypoka-lemia. Caution should be exercised when oliguria or impaired renal function is coexistent.Calcium Hypercalcemia Treatment is required when hypercalcemia is symptomatic, which usually occurs when the serum level exceeds 12 mg/dL. The critical level for serum calcium is 15 mg/dL, when symptoms noted earlier may rapidly progress to death. The

1	is symptomatic, which usually occurs when the serum level exceeds 12 mg/dL. The critical level for serum calcium is 15 mg/dL, when symptoms noted earlier may rapidly progress to death. The initial treatment is aimed at repleting the associated volume deficit and then inducing a brisk diuresis with normal saline. Treatment of hypercalcemia associated with malignancies is discussed later in this chapter.Hypocalcemia Asymptomatic hypocalcemia can be treated with oral or IV calcium (see Table 3-15). Acute symptomatic hypocalcemia should be treated with IV 10% calcium gluconate to achieve a serum concentration of 7 to 9 mg/dL. Associated deficits in magnesium, potassium, and pH must also be cor-rected. Hypocalcemia will be refractory to treatment if coex-isting hypomagnesemia is not corrected first. Routine calcium supplementation is no longer recommended in association with massive blood transfusions.32Phosphorus Hyperphosphatemia Phosphate binders such as sucralfate or

1	is not corrected first. Routine calcium supplementation is no longer recommended in association with massive blood transfusions.32Phosphorus Hyperphosphatemia Phosphate binders such as sucralfate or aluminum-containing antacids can be used to lower serum phosphorus levels. Calcium acetate tablets also are useful when hypocalcemia is simultaneously present. Dialysis usually is reserved for patients with renal failure.Hypophosphatemia Depending on the level of depletion and tolerance to oral supplementation, a number of enteral and par-enteral repletion strategies are effective for the treatment of hypophosphatemia (see Table 3-15).Magnesium Hypermagnesemia Treatment for hypermagnesemia consists of measures to eliminate exogenous sources of magnesium, Table 3-14Treatment of symptomatic hyperkalemiaPotassium removal Kayexalate Oral administration is 15–30 g in 50–100 mL of 20% sorbitol Rectal administration is 50 g in 200 mL of 20% sorbitol DialysisShift potassium Glucose 1 ampule of

1	removal Kayexalate Oral administration is 15–30 g in 50–100 mL of 20% sorbitol Rectal administration is 50 g in 200 mL of 20% sorbitol DialysisShift potassium Glucose 1 ampule of D50 and regular insulin 5–10 units IV Bicarbonate 1 ampule IVCounteract cardiac effects Calcium gluconate 5–10 mL of 10% solutionD50 = 50% dextrose.Brunicardi_Ch03_p0083-p0102.indd 9508/12/18 10:07 AM 96BASIC CONSIDERATIONSPART ITable 3-15Electrolyte replacement therapy protocolPotassiumSerum potassium level <4.0 mEq/L: Asymptomatic, tolerating enteral nutrition: KCl 40 mEq per enteral access × 1 dose Asymptomatic, not tolerating enteral nutrition: KCl 20 mEq IV q2h × 2 doses Symptomatic: KCl 20 mEq IV q1h × 4 doses Recheck potassium level 2 h after end of infusion; if <3.5 mEq/L and asymptomatic, replace as per above protocolMagnesiumMagnesium level 1.0–1.8 mEq/L: Magnesium sulfate 0.5 mEq/kg in normal saline 250 mL infused IV over 24 h × 3 d Recheck magnesium level in 3 dMagnesium level <1.0

1	replace as per above protocolMagnesiumMagnesium level 1.0–1.8 mEq/L: Magnesium sulfate 0.5 mEq/kg in normal saline 250 mL infused IV over 24 h × 3 d Recheck magnesium level in 3 dMagnesium level <1.0 mEq/L: Magnesium sulfate 1 mEq/kg in normal saline 250 mL infused IV over 24 h × 1 d, then 0.5 mEq/kg in normal saline 250 mL infused IV over 24 h × 2 d Recheck magnesium level in 3 dIf patient has gastric access and needs a bowel regimen: Milk of magnesia 15 mL (approximately 49 mEq magnesium) q24h per gastric tube; hold for diarrheaCalciumIonized calcium level <4.0 mg/dL: With gastric access and tolerating enteral nutrition: Calcium carbonate suspension 1250 mg/5 mL q6h per gastric access; recheck ionized calcium level in 3 d Without gastric access or not tolerating enteral nutrition: Calcium gluconate 2 g IV over 1 h × 1 dose; recheck ionized calcium level in 3 dPhosphatePhosphate level 1.0–2.5 mg/dL: Tolerating enteral nutrition: Neutra-Phos 2 packets q6h per gastric tube or

1	Calcium gluconate 2 g IV over 1 h × 1 dose; recheck ionized calcium level in 3 dPhosphatePhosphate level 1.0–2.5 mg/dL: Tolerating enteral nutrition: Neutra-Phos 2 packets q6h per gastric tube or feeding tube No enteral nutrition: KPHO4 or NaPO4 0.15 mmol/kg IV over 6 h × 1 dose Recheck phosphate level in 3 dPhosphate level <1.0 mg/dL: Tolerating enteral nutrition: KPHO4 or NaPO4 0.25 mmol/kg over 6 h × 1 dose Recheck phosphate level 4 h after end of infusion; if <2.5 mg/dL, begin Neutra-Phos 2 packets q6h Not tolerating enteral nutrition: KPHO4 or NaPO4 0.25 mmol/kg (LBW) over 6 h × 1 dose; recheck phosphate level 4 h after end of infusion; if <2.5 mg/dL, then KPHO4 or NaPO4 0.15 mmol/kg (LBW) IV over 6 h × 1 dose3 mmol KPHO4 = 3 mmol Phos and 4.4 mEq K+ = 1 mL3 mmol NaPO4 = 3 mmol Phos and 4 mEq Na+ = 1 mLNeutra-Phos 1 packet = 8 mmol Phos, 7 mEq K+, 7 mEq Na+Use patient’s lean body weight (LBW) in kilograms for all calculations.Disregard protocol if patient has renal failure,

1	Phos and 4 mEq Na+ = 1 mLNeutra-Phos 1 packet = 8 mmol Phos, 7 mEq K+, 7 mEq Na+Use patient’s lean body weight (LBW) in kilograms for all calculations.Disregard protocol if patient has renal failure, is on dialysis, or has a creatinine clearance <30 mL/min.correct concurrent volume deficits, and correct acidosis if present. To manage acute symptoms, calcium chloride (5 to 10 mL) should be administered to immediately antagonize the cardiovascular effects. If elevated levels or symptoms persist, hemodialysis may be necessary.Hypomagnesemia Correction of magnesium depletion can be oral if asymptomatic and mild. Otherwise, IV repletion is indicated and depends on severity (see Table 3-15) and clini-cal symptoms. For those with severe deficits (<1.0 mEq/L) or those who are symptomatic, 1 to 2 g of magnesium sulfate may be administered IV over 15 minutes. Under ECG monitoring, it may be given over 2 minutes if necessary to correct torsades de pointes (irregular ventricular rhythm). Caution

1	g of magnesium sulfate may be administered IV over 15 minutes. Under ECG monitoring, it may be given over 2 minutes if necessary to correct torsades de pointes (irregular ventricular rhythm). Caution should be taken when giving large amounts of magnesium because magnesium toxicity may develop. The simultaneous administration of cal-cium gluconate will counteract the adverse side effects of a rap-idly rising magnesium level and correct hypocalcemia, which is frequently associated with hypomagnesemia.Preoperative Fluid TherapyThe administration of maintenance fluids should be all that is required in an otherwise healthy individual who may be under orders to receive nothing by mouth for some period before the time of surgery. This does not, however, include replenishment of a preexisting deficit or ongoing fluid losses. The following is a frequently used formula for calculating the volume of mainte-nance fluids in the absence of preexisting abnormalities:For the first 0–10 kgFor the next

1	or ongoing fluid losses. The following is a frequently used formula for calculating the volume of mainte-nance fluids in the absence of preexisting abnormalities:For the first 0–10 kgFor the next 10–20 kgFor weight >20 kgGive 100 mL/kg per dayGive an additional 50 mL/kg per dayGive an additional 20 mL/kg per dayFor example, a 60-kg female would receive a total of 2300 mL of fluid daily: 1000 mL for the first 10 kg of body Brunicardi_Ch03_p0083-p0102.indd 9608/12/18 10:07 AM 97FLUID AND ELECTROLYTE MANAGEMENT OF THE SURGICAL PATIENTCHAPTER 3weight (10 kg × 100 mL/kg per day), 500 mL for the next 20 kg (10 kg × 50 mL/kg per day), and 800 mL for the last 40 kg (40 kg × 20 mL/kg per day).An alternative approach is to replace the calculated daily water losses in urine, stool, and insensible loss with a hypotonic saline solution rather than water alone, which allows the kid-ney some sodium excess to adjust for concentration. Although there should be no “routine” maintenance fluid

1	loss with a hypotonic saline solution rather than water alone, which allows the kid-ney some sodium excess to adjust for concentration. Although there should be no “routine” maintenance fluid orders, both of these methods would yield an appropriate choice of 5% dex-trose in 0.45% sodium chloride at 100 mL/h as initial therapy, with potassium added for patients with normal renal function. However, many surgical patients have volume and/or electrolyte abnormalities associated with their surgical disease. Preopera-tive evaluation of a patient’s volume status and preexisting elec-trolyte abnormalities is an important part of overall preoperative assessment and care. Volume deficits should be considered in patients who have obvious GI losses, such as through emesis or diarrhea, as well as in patients with poor oral intake second-ary to their disease. Less obvious are those fluid losses known as third-space or nonfunctional ECF losses that occur with GI obstruction, peritoneal or bowel

1	in patients with poor oral intake second-ary to their disease. Less obvious are those fluid losses known as third-space or nonfunctional ECF losses that occur with GI obstruction, peritoneal or bowel inflammation, ascites, crush injuries, burns, and severe soft tissue infections such as nec-rotizing fasciitis. The diagnosis of an acute volume deficit is primarily clinical (see Table 3-2), although the physical signs may vary with the duration of the deficit. Cardiovascular signs of tachycardia and orthostasis predominate with acute volume loss, usually accompanied by oliguria and hemoconcentration. Acute volume deficits should be corrected as much as possible before the time of operation.Once a volume deficit is diagnosed, prompt fluid replace-ment should be instituted, usually with an isotonic crystalloid, depending on the measured serum electrolyte values. Patients with cardiovascular signs of volume deficit should receive a bolus of 1 to 2 L of isotonic fluid followed by a

1	an isotonic crystalloid, depending on the measured serum electrolyte values. Patients with cardiovascular signs of volume deficit should receive a bolus of 1 to 2 L of isotonic fluid followed by a continuous infu-sion. Close monitoring during this period is imperative. Resus-citation should be guided by the reversal of the signs of volume deficit, such as restoration of acceptable values for vital signs, maintenance of adequate urine output (0.5 to 1 mL/kg per hour in an adult), and correction of base deficit. Patients whose vol-ume deficit is not corrected after this initial volume challenge and those with impaired renal function and the elderly should be considered for more intensive monitoring in an intensive care unit setting. In these patients, early invasive monitoring of cen-tral venous pressure or cardiac output may be necessary.If symptomatic electrolyte abnormalities accompany vol-ume deficit, the abnormality should be corrected to the point that the acute symptom is

1	venous pressure or cardiac output may be necessary.If symptomatic electrolyte abnormalities accompany vol-ume deficit, the abnormality should be corrected to the point that the acute symptom is relieved before surgical interven-tion. For correction of severe hypernatremia associated with a volume deficit, an unsafe rapid fall in extracellular osmolarity from 5% dextrose infusion is avoided by slowly correcting the hypernatremia with 0.45% saline or even lactated Ringer’s solu-tion rather than 5% dextrose alone. This will safely and slowly correct the hypernatremia while also correcting the associated volume deficit.Intraoperative Fluid TherapyWith the induction of anesthesia, compensatory mechanisms are lost, and hypotension will develop if volume deficits are not appropriately corrected before the time of surgery. Hemody-namic instability during anesthesia is best avoided by correct-ing known fluid losses, replacing ongoing losses, and providing adequate maintenance fluid therapy

1	before the time of surgery. Hemody-namic instability during anesthesia is best avoided by correct-ing known fluid losses, replacing ongoing losses, and providing adequate maintenance fluid therapy preoperatively. In addition to measured blood loss, major open abdominal surgeries are associated with continued extracellular losses in the form of bowel wall edema, peritoneal fluid, and the wound edema dur-ing surgery. Large soft tissue wounds, complex fractures with associated soft tissue injury, and burns are all associated with additional third-space losses that must be considered in the operating room. These represent distributional shifts, in that the functional volume of ECF is reduced but fluid is not externally lost from the body. These functional losses have been referred to as parasitic losses, sequestration, or third-space edema because the lost volume no longer participates in the normal functions of the ECF.Until the 1960s saline solutions were withheld during sur-gery.

1	parasitic losses, sequestration, or third-space edema because the lost volume no longer participates in the normal functions of the ECF.Until the 1960s saline solutions were withheld during sur-gery. Administered saline was retained and was felt to be an inappropriate challenge to a physiologic response of intraopera-tive salt intolerance. Basic and clinical research began to change this concept,33,34 eventually leading to the current concept that saline administration is necessary to restore the obligate ECF losses noted earlier. Although no accurate formula can predict intraoperative fluid needs, replacement of ECF during surgery often requires 500 to 1000 mL/h of a balanced salt solution to support homeostasis. The addition of albumin or other colloidcontaining solutions to intraoperative fluid therapy is not neces-sary. Manipulation of colloid oncotic forces by albumin infusion during major vascular surgery showed no advantage in support-ing cardiac function or avoiding the

1	fluid therapy is not neces-sary. Manipulation of colloid oncotic forces by albumin infusion during major vascular surgery showed no advantage in support-ing cardiac function or avoiding the accumulation of extravas-cular lung water.35Postoperative Fluid TherapyPostoperative fluid therapy should be based on the patient’s current estimated volume status and projected ongoing fluid losses. Any deficits from either preoperative or intraoperative losses should be corrected, and ongoing requirements should be included along with maintenance fluids. Third-space losses, although difficult to measure, should be included in fluid replacement strategies. In the initial postoperative period, an isotonic solution should be administered. The adequacy of resuscitation should be guided by the restoration of acceptable values for vital signs and urine output and, in more complicated cases, by the correction of base deficit or lactate. Adjuncts to assessing volume status in the postoperative patient

1	of acceptable values for vital signs and urine output and, in more complicated cases, by the correction of base deficit or lactate. Adjuncts to assessing volume status in the postoperative patient include such tools as a straight leg raise, point-of-care ultrasound, and assessment of respiratory variation via use of an arterial cath-eter in a mechanically ventilated patient. After the initial 24 to 48 hours, fluids can be changed to 5% dextrose in 0.45% saline in patients unable to tolerate enteral nutrition. If normal renal function and adequate urine output are present, potassium may be added to the IV fluids. Daily fluid orders should begin with assessment of the patient’s volume status and assessment of electrolyte abnormalities. There is rarely a need to check elec-trolyte levels in the first few days of an uncomplicated post-operative course. However, postoperative diuresis may require attention to replacement of urinary potassium loss. All measured losses, including losses

1	in the first few days of an uncomplicated post-operative course. However, postoperative diuresis may require attention to replacement of urinary potassium loss. All measured losses, including losses through vomiting, nasogastric suction-ing, drains, and urine output, as well as insensible losses, are replaced with the appropriate parenteral solutions as previously reviewed.Fluid Management in Enhanced Recovery After Surgery (ERAS) PathwaysAs pioneered by the Danish surgeon Henrik Kehlet, ERAS path-ways have been designed to guide the perioperative management Brunicardi_Ch03_p0083-p0102.indd 9708/12/18 10:07 AM 98BASIC CONSIDERATIONSPART Iof various types of surgical procedures. ERAS consists of a mul-timodal strategy to maximize and maintain preoperative organ function, and implementation of ERAS protocols has resulted in a decrease in length of stay, improved patient satisfaction, cost savings, and a reduction in complications. A full discussion of ERAS is outside the scope of

1	of ERAS protocols has resulted in a decrease in length of stay, improved patient satisfaction, cost savings, and a reduction in complications. A full discussion of ERAS is outside the scope of this chapter; however, it is impor-tant to note that perioperative fluid management is a major tenet of ERAS protocols. The 2011 European Society of Anaesthesi-ology guidelines were among the first formal recommendations to alter standard recommendations for preoperative enteral intake. These recommendations include allowance of clear liq-uids up to 2 hours prior to surgery.36 Many ERAS protocols include the use of carbohydrate and electrolyte-rich fluids to enhance hydration and metabolic response to surgery. In addi-tion to preoperative enteral hydration, a major focus of ERAS protocols is the restriction of intraand postoperative sodium and intravenous fluids. Fluid overload has been associated with prolonged ileus and coagulation abnormalities.37 Goal-directed fluid therapy has been shown to

1	of intraand postoperative sodium and intravenous fluids. Fluid overload has been associated with prolonged ileus and coagulation abnormalities.37 Goal-directed fluid therapy has been shown to reduce postoperative morbidity and length of stay independent of the other multimodal compo-nents of ERAS, making minimizing fluids a major target of intervention.38 Postoperatively, early enteral intake is advised, with prompt discontinuation of intravenous fluids. These strate-gies targeting euvolemia have been shown to be safe and improve outcomes, making ERAS a rapidly evolving strat-egy that will continue to influence the perioperative fluid and electrolyte management of surgical patients.39,40Special Considerations for the Postoperative PatientVolume excess is a common disorder in the postoperative period. The administration of isotonic fluids in excess of actual needs may result in excess volume expansion. This may be due to the overestimation of third-space losses or to ongoing GI losses

1	period. The administration of isotonic fluids in excess of actual needs may result in excess volume expansion. This may be due to the overestimation of third-space losses or to ongoing GI losses that are difficult to measure accurately. The earliest sign of volume overload is weight gain. The average postopera-tive patient who is not receiving nutritional support should lose approximately 0.25 to 0.5 lb/d (0.11 to 0.23 kg/d) from catabo-lism. Additional signs of volume excess may also be present as listed in Table 3-2. Peripheral edema may not necessarily be associated with intravascular volume overload because overex-pansion of total ECF may exist in association with a deficit in the circulating plasma volume.Volume deficits also can be encountered in surgical patients if preoperative losses were not completely corrected, intraoperative losses were underestimated, or postoperative losses were greater than appreciated. The clinical manifestations are described in Table 3-2 and include

1	were not completely corrected, intraoperative losses were underestimated, or postoperative losses were greater than appreciated. The clinical manifestations are described in Table 3-2 and include tachycardia, orthostasis, and oliguria. Hemoconcentration also may be present. Treat-ment will depend on the amount and composition of fluid lost. In most cases of volume depletion, replacement with an isotonic fluid will be sufficient while alterations in concentration and composition are being evaluated.ELECTROLYTE ABNORMALITIES IN SPECIFIC SURGICAL PATIENTSNeurologic PatientsSyndrome of Inappropriate Secretion of Antidiuretic Hormone. The syndrome of inappropriate secretion of antidi-uretic hormone (SIADH) can occur after head injury or surgery to the central nervous system, but it also is seen in association with administration of drugs such as morphine, nonsteroidals, and oxytocin, and in a number of pulmonary and endocrine dis-eases, including hypothyroidism and glucocorticoid

1	is seen in association with administration of drugs such as morphine, nonsteroidals, and oxytocin, and in a number of pulmonary and endocrine dis-eases, including hypothyroidism and glucocorticoid deficiency. Additionally, it can be seen in association with a number of malignancies, most often small cell cancer of the lung but also pancreatic carcinoma, thymoma, and Hodgkin’s disease.41 SIADH should be considered in patients who are euvolemic and hyponatremic with elevated urine sodium levels and urine osmolality. ADH secretion is considered inap-propriate when it is not in response to osmotic or volume-related conditions. Correction of the underlying problem should be attempted when possible. In most cases, restriction of free water will improve the hyponatremia. The goal is to achieve net water balance while avoiding volume depletion that may compromise renal function. Furosemide also can be used to induce free water loss. If hyponatremia persists after fluid restriction, the

1	net water balance while avoiding volume depletion that may compromise renal function. Furosemide also can be used to induce free water loss. If hyponatremia persists after fluid restriction, the addition of isotonic or hypertonic fluids may be effective. The adminis-tration of isotonic saline may sometimes worsen the problem if the urinary sodium concentration is higher than the infused sodium concentration. The use of loop diuretics may be helpful in this situation by preventing further urine concentration. In chronic SIADH, when long-term fluid restriction is difficult to maintain or is ineffective, demeclocycline and lithium can be used to induce free water loss.Diabetes Insipidus. Diabetes insipidus (DI) is a disorder of ADH stimulation and is manifested by dilute urine in the case of hypernatremia. Central DI results from a defect in ADH secre-tion, and nephrogenic DI results from a defect in end-organ responsiveness to ADH. Central DI is frequently seen in asso-ciation with

1	of hypernatremia. Central DI results from a defect in ADH secre-tion, and nephrogenic DI results from a defect in end-organ responsiveness to ADH. Central DI is frequently seen in asso-ciation with pituitary surgery, closed head injury, and anoxic encephalopathy.42 Nephrogenic DI occurs in association with hypokalemia, administration of radiocontrast dye, and use of certain drugs such as aminoglycosides and amphotericin B. In patients tolerating oral intake, volume status usually is normal because thirst stimulates increased intake. However, volume depletion can occur rapidly in patients who are incapable of oral intake. The diagnosis can be confirmed by documenting a paradoxical increase in urine osmolality in response to a period of water deprivation. In mild cases, free water replacement may be adequate therapy. In more severe cases, vasopressin can be added. The usual dosage of vasopressin is 5 U subcutaneously every 6 to 8 hours. However, serum electrolytes and osmolality should

1	may be adequate therapy. In more severe cases, vasopressin can be added. The usual dosage of vasopressin is 5 U subcutaneously every 6 to 8 hours. However, serum electrolytes and osmolality should be monitored to avoid excess vasopressin administration with resulting iatrogenic SIADH.Cerebral Salt Wasting. Cerebral salt wasting is a diagnosis of exclusion that occurs in patients with a cerebral lesion and renal wasting of sodium and chloride with no other identifiable cause.43 Natriuresis in a patient with a contracted extracellular volume should prompt the possible diagnosis of cerebral salt wasting. Hyponatremia is frequently observed but is nonspecific and occurs as a secondary event, which differentiates it from SIADH.Malnourished Patients: Refeeding SyndromeRefeeding syndrome is a potentially lethal condition that can occur with rapid and excessive feeding of patients with severe underlying malnutrition due to starvation, alcoholism, delayed nutritional support, anorexia nervosa,

1	lethal condition that can occur with rapid and excessive feeding of patients with severe underlying malnutrition due to starvation, alcoholism, delayed nutritional support, anorexia nervosa, or massive weight loss in obese patients.44 With refeeding, a shift in metabolism from fat to carbohydrate substrate stimulates insulin release, which 678Brunicardi_Ch03_p0083-p0102.indd 9808/12/18 10:07 AM 99FLUID AND ELECTROLYTE MANAGEMENT OF THE SURGICAL PATIENTCHAPTER 3results in the cellular uptake of electrolytes, particularly phos-phate, magnesium, potassium, and calcium. However, severe hyperglycemia may result from blunted basal insulin secretion. The refeeding syndrome can be associated with enteral or par-enteral refeeding, and symptoms from electrolyte abnormalities include cardiac arrhythmias, confusion, respiratory failure, and even death. To prevent the development of refeeding syndrome, underlying electrolyte and volume deficits should be corrected. Additionally, thiamine

1	arrhythmias, confusion, respiratory failure, and even death. To prevent the development of refeeding syndrome, underlying electrolyte and volume deficits should be corrected. Additionally, thiamine should be administered before the initia-tion of feeding. Caloric repletion should be instituted slowly and should gradually increase over the first week.45 Vital signs, fluid balance, and electrolytes should be closely monitored and any deficits corrected as they evolve.Acute Renal Failure PatientsA number of fluid and electrolyte abnormalities are specific to patients with acute renal failure. With the onset of renal failure, an accurate assessment of volume status must be made. If prerenal azotemia is present, prompt correction of the underlying volume deficit is mandatory. Once acute tubular necrosis is established, measures should be taken to restrict daily fluid intake to match urine output and insensible and GI losses. Oliguric renal failure requires close monitoring of serum

1	tubular necrosis is established, measures should be taken to restrict daily fluid intake to match urine output and insensible and GI losses. Oliguric renal failure requires close monitoring of serum potassium levels. Measures to correct hyperkalemia as reviewed in Table 3-14 should be instituted early, including consideration of early hemodialysis. Hyponatremia is common in established renal failure as a result of the breakdown of proteins, carbohydrates, and fats, as well as the administration of free water. Dialysis may be required for severe hyponatremia. Hypocalcemia, hypermagnesemia, and hyperphosphatemia also are associated with acute renal failure. Hypocalcemia should be verified by measuring ionized calcium, because many patients also are hypoalbuminemic. Phosphate binders can be used to control hyperphosphatemia, but dialy-sis may be required in more severe cases. Metabolic acidosis is commonly seen with renal failure, as the kidneys lose their ability to clear acid

1	can be used to control hyperphosphatemia, but dialy-sis may be required in more severe cases. Metabolic acidosis is commonly seen with renal failure, as the kidneys lose their ability to clear acid by-products. Bicarbonate can be useful, but dialysis often is needed. Although dialysis may be either intermittent or continuous, renal recovery may be improved by continuous renal replacement.46Cancer PatientsFluid and electrolyte abnormalities are common in patients with cancer. The causes may be common to all patient populations or may be specific to cancer patients and their treatment.47 Hypo-natremia is frequently hypovolemic due to renal loss of sodium caused by diuretics or salt-wasting nephropathy as seen with some chemotherapeutic agents such as cisplatin. Cerebral salt wasting also can occur in patients with intracerebral lesions. Normovolemic hyponatremia may occur in association with SIADH from cervical cancer, lymphoma, and leukemia, or from certain chemotherapeutic agents.

1	can occur in patients with intracerebral lesions. Normovolemic hyponatremia may occur in association with SIADH from cervical cancer, lymphoma, and leukemia, or from certain chemotherapeutic agents. Hypernatremia in can-cer patients most often is due to poor oral intake or GI volume losses, which are common side effects of chemotherapy. Central DI also can lead to hypernatremia in patients with central ner-vous system lesions.Hypokalemia can develop from GI losses associated with diarrhea caused by radiation enteritis or chemotherapy, or from tumors such as villous adenomas of the colon. Tumor lysis syn-drome can precipitate severe hyperkalemia from massive tumor cell destruction.Hypocalcemia can be seen after removal of a thyroid or parathyroid tumor or after a central neck dissection, which can damage the parathyroid glands. Hungry bone syndrome produces acute and profound hypocalcemia after parathyroid surgery for secondary or tertiary hyperparathyroidism because calcium is rapidly

1	can damage the parathyroid glands. Hungry bone syndrome produces acute and profound hypocalcemia after parathyroid surgery for secondary or tertiary hyperparathyroidism because calcium is rapidly taken up by bones. Prostate and breast cancer can result in increased osteoblastic activity, which decreases serum calcium by increasing bone formation. Acute hypocalce-mia also can occur with hyperphosphatemia because phospho-rus complexes with calcium. Hypomagnesemia is a side effect of ifosfamide and cisplatin therapy. Hypophosphatemia can be seen in hyperparathyroidism, due to decreased phosphorus reabsorption, and in oncogenic osteomalacia, which increases the urinary excretion of phosphorus. Other causes of hypophos-phatemia in cancer patients include renal tubular dysfunction from multiple myeloma, Bence Jones proteins, and certain che-motherapeutic agents. Acute hypophosphatemia can occur as rapidly proliferating malignant cells take up phosphorus in acute leukemia. Tumor lysis

1	myeloma, Bence Jones proteins, and certain che-motherapeutic agents. Acute hypophosphatemia can occur as rapidly proliferating malignant cells take up phosphorus in acute leukemia. Tumor lysis syndrome or the use of bisphosphonates to treat hypercalcemia also can result in hyperphosphatemia.Malignancy is the most common cause of hypercalcemia in hospitalized patients and is due to increased bone resorption or decreased renal excretion. Bone destruction occurs from bony metastasis as seen in breast or renal cell cancer but also can occur in multiple myeloma. With Hodgkin’s and non-Hodgkin’s lymphoma, hypercalcemia results from increased calcitriol for-mation, which increases both absorption of calcium from the GI tract and mobilization from bone. Humoral hypercalcemia of malignancy is a common cause of hypercalcemia in cancer patients. As in primary hyperparathyroidism, a parathyroid-related protein is secreted that binds to parathyroid receptors, stimulating calcium resorption from

1	common cause of hypercalcemia in cancer patients. As in primary hyperparathyroidism, a parathyroid-related protein is secreted that binds to parathyroid receptors, stimulating calcium resorption from bone and decreasing renal excretion of calcium. The treatment of hypercalcemia of malig-nancy should begin with saline volume expansion, which will decrease renal reabsorption of calcium as the associated volume deficit is corrected. Once an adequate volume status has been achieved, a loop diuretic may be added. Unfortunately, these measures are only temporary, and additional treatment is often necessary. A variety of drugs are available with varying times of onset, durations of action, and side effects.48 The bisphos-phonates etidronate and pamidronate inhibit bone resorption and osteoclastic activity. They have a slow onset of action, but effects can last for 2 weeks. Calcitonin also is effective, inhibit-ing bone resorption and increasing renal excretion of calcium. It acts quickly,

1	activity. They have a slow onset of action, but effects can last for 2 weeks. Calcitonin also is effective, inhibit-ing bone resorption and increasing renal excretion of calcium. It acts quickly, within 2 to 4 hours, but its use is limited by the development of tachyphylaxis. Corticosteroids may decrease tachyphylaxis in response to calcitonin and can be used alone to treat hypercalcemia. Gallium nitrates are potent inhibitors of bone resorption. They display a long duration of action but can cause nephrotoxicity. Mithramycin is an antibiotic that blocks osteoclastic activity, but it can be associated with liver, renal, and hematologic abnormalities, which limits its use to the treatment of Paget’s disease of bone. For patients with severe, refractory hypercalcemia who are unable to tolerate volume expansion due to pulmonary edema or congestive heart failure, dialysis is an option.Tumor lysis syndrome results when the release of intracel-lular metabolites overwhelms the kidneys’

1	tolerate volume expansion due to pulmonary edema or congestive heart failure, dialysis is an option.Tumor lysis syndrome results when the release of intracel-lular metabolites overwhelms the kidneys’ excretory capacity. This rapid release of uric acid, potassium, and phosphorus can result in marked hyperuricemia, hyperkalemia, hyperphospha-temia, hypocalcemia, and acute renal failure. It is typically seen with poorly differentiated lymphomas and leukemias but also can occur with a number of solid tumor malignancies. Tumor lysis syndrome most commonly develops during treatment Brunicardi_Ch03_p0083-p0102.indd 9908/12/18 10:07 AM 100BASIC CONSIDERATIONSPART Iwith chemotherapy or radiotherapy. Once it develops, volume expansion should be undertaken and any associated electrolyte abnormalities corrected. In this setting, hypocalcemia should not be treated unless it is symptomatic to avoid metastatic calci-fications. Dialysis may be required for management of impaired renal function

1	corrected. In this setting, hypocalcemia should not be treated unless it is symptomatic to avoid metastatic calci-fications. Dialysis may be required for management of impaired renal function or correction of electrolyte abnormalities.REFERENCESEntries highlighted in bright blue are key references. 1. Aloia JF, Vaswani A, Flaster E, et al. Relationship of body water compartment to age, race and fat-free mass. J Lab Clin Med. 1998;132:483. 2. Bourque CW, Oliet SHR. Osmoreceptors in the central nervous system. Annu Rev Physiol. 1997;59:601. 3. Verbalis JG. How does the brain sense osmolality? J Am Soc Nephrol. 2007;18(12):3056. 4. Stauss HM. Baroreceptor reflex function. Am J Physiol Regul Integr Comp Physiol. 2002;283:R284. 5. Miller M. Syndromes of excess antidiuretic hormone release. Crit Care Clin. 2001;17:11. 6. Kapoor M, Chan G. Fluid and electrolyte abnormalities. Crit Care Clin. 2001;17:571. 7. Adrogue HJ, Lederer ED, Suki WN, et al. Determinants of plasma potassium in diabetic

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1	significant increase in the rate of renal replacement therapy after HES use. 29. Overgaard-Steensen C, Ring T. Clinical review: practical approach to hyponatremia and hypernatremia in critically ill patients. Crit Care. 2013;17(1):206. 30. Norenberg MD. Central pontine myelinolysis: historical and mechanistic considerations. Metab Brain Dis. 2010;25(1):97. 31. Graff-Radford J, Fugate JE, Kauffmann TJ. Clinical and radio-logic correlations of central pontine myelinolysis syndrome. Mayo Clin Proc. 2011;86(11):1063. 32. American College of Surgeons. Shock. In: American College of Surgeons Advanced Trauma Life Support Manual. 9th ed. Chicago: American College of Surgeons; 2012. 33. Shires GT, Williams J, Brown F. Acute changes in extracel-lular fluids associated with major surgical procedures. Ann Surg. 1961;154:803. This early paper by Shires and colleagues produced important early observations of fluid shifts and resuscitation strategies fol-lowing surgery and shaped some of the early

1	Ann Surg. 1961;154:803. This early paper by Shires and colleagues produced important early observations of fluid shifts and resuscitation strategies fol-lowing surgery and shaped some of the early questions in the field of resuscitation science after surgery. 34. Shires GT, Jackson DE. Postoperative salt tolerance. Arch Surg. 1962;84:703. 35. Shires GT III, Peitzman AB, Albert SA, et al. Response of extravascular lung water to intraoperative fluids. Ann Surg. 1983;197:515. 36. Smith I, Kranke P, Murat I, et al. Perioperative fasting in adults and children: guidelines from the European Society of Anaesthesiology. Eur J Anaesthesiol. 2011;28:556-569. Index guidelines published by the European Society of Anaes-thesiology were among the first to support the use of ERAS principles and provided a template for the design of the rapidly evolving practice of ERAS worldwide. 37. Horosz B, Nawrocka K, Malec-Milewska M. Anaesthetic peri-operative management according to the ERAS protocol.

1	a template for the design of the rapidly evolving practice of ERAS worldwide. 37. Horosz B, Nawrocka K, Malec-Milewska M. Anaesthetic peri-operative management according to the ERAS protocol. Anaes-thesiol Intensive Ther. 2016;48(1):49-54. 38. Rollins KE, Lobo DN. Intraoperative goal-directed fluid therapy in elective major abdominal surgery: a meta-analysis of randomized controlled trials. Ann Surg. 2016 Mar;263(3):465-476. Meta-analysis of randomized controlled trials demonstrated a marked reduction in morbidity, hospital length of stay (LOS), Brunicardi_Ch03_p0083-p0102.indd 10008/12/18 10:07 AM 101FLUID AND ELECTROLYTE MANAGEMENT OF THE SURGICAL PATIENTCHAPTER 3ICU LOS, and time to passage of feces utilizing goal-directed fluid therapy (GDFT) for intraoperative fluid management. Of note, in those studies where a multimodal ERAS pathway was utilized, the benefits of GDFT were reduced, although reduc-tion in major morbidities remained. These data suggest that the multimodal

1	Of note, in those studies where a multimodal ERAS pathway was utilized, the benefits of GDFT were reduced, although reduc-tion in major morbidities remained. These data suggest that the multimodal benefits of ERAS may extend beyond just fluid management. 39. Miller TE, Roche AM, Mythen M. Fluid management and goal-directed therapy as an adjunct to enhanced recovery after sur-gery (ERAS). Can J Anaesth. 2015 Feb;62(2):158-168. 40. Liu VX, Rosas E, Hwang J, et al. Enhanced recovery after surgery program implementation in 2 surgical populations in an integrated health care delivery system. JAMA Surg. 2017 Jul 19;152(7):e171032. 41. Ellison DH, Burl T. Clinical practice. The syndrome of inap-propriate antidiuresis. N Engl J Med. 2007;356(20):2064. 42. Tisdall M, Crocker M, Watkiss J, et al. Disturbances of sodium in critically ill adult neurologic patients: a clinical review. J Neurosurg Anesthesiol. 2006;18(1):57. 43. Yee AH, Burns JD, Wijdicks EF. Cerebral salt wasting:

1	J, et al. Disturbances of sodium in critically ill adult neurologic patients: a clinical review. J Neurosurg Anesthesiol. 2006;18(1):57. 43. Yee AH, Burns JD, Wijdicks EF. Cerebral salt wasting: patho-physiology, diagnosis, and treatment. Neurosurg Clin N Am. 2010;21(2):339. 44. Kozar RA, McQuiggan MM, Moore FA. Nutritional support in trauma patients. In: Shikora SA, Martindale RG, Schwaitzberg SD, eds. Nutritional Considerations in the Intensive Care Unit. 1st ed. Dubuque, IA: Kendall/Hunt Publishing; 2002:229. 45. Boateng AA, Sriram K, Mequid MM, et al. Refeeding syn-drome: treatment considerations based on collective analysis of literature case reports. Nutrition. 2010;26(2):156. 46. Glassford NJ, Bellomo R. Acute kidney injury: how can we facilitate recovery? Curr Opin Crit Care. 2011;17(6):562. 47. Kapoor M, Chan GZ. Fluid and electrolyte abnormalities. Crit Care Clin. 2002;17:503. 48. Clines GA. Mechanisms and treatment of hypercalce-mia of malignancy. Curr Opin Endocrinol

1	2011;17(6):562. 47. Kapoor M, Chan GZ. Fluid and electrolyte abnormalities. Crit Care Clin. 2002;17:503. 48. Clines GA. Mechanisms and treatment of hypercalce-mia of malignancy. Curr Opin Endocrinol Diabetes Obes. 2011;18(6):339.Brunicardi_Ch03_p0083-p0102.indd 10108/12/18 10:07 AM

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1	Hemostasis, Surgical Bleeding, and TransfusionRonald Chang, John B. Holcomb, Evan Leibner, Matthew Pommerening, and Rosemary A. Kozar 4chapterBIOLOGY OF HEMOSTASISHemostasis is a complex process whose function is to limit blood loss from an injured vessel. Four major physiologic events participate in the hemostatic process: vascular constric-tion, platelet plug formation, fibrin formation, and fibrinolysis. Although each tends to be activated in order, the four processes are interrelated so that there is a continuum and multiple rein-forcements. The process is shown schematically in Fig. 4-1.Vascular ConstrictionVascular constriction is the initial response to vessel injury. It is more pronounced in vessels with medial smooth muscles and is dependent on local contraction of smooth muscle. Vasoconstric-tion is subsequently linked to platelet plug formation. Throm-boxane A2 (TXA2) is produced locally at the site if injury via the release of arachidonic acid from platelet membranes and

1	Vasoconstric-tion is subsequently linked to platelet plug formation. Throm-boxane A2 (TXA2) is produced locally at the site if injury via the release of arachidonic acid from platelet membranes and is a potent constrictor of smooth muscle. Similarly, endothelin synthesized by injured endothelium and serotonin (5-hydroxy-tryptamine [5-HT]) released during platelet aggregation are potent vasoconstrictors. Lastly, bradykinin and fibrinopeptides, which are involved in the coagulation schema, are also capable of contracting vascular smooth muscle.The extent of vasoconstriction varies with the degree of vessel injury. A small artery with a lateral incision may remain open due to physical forces, whereas a similarly sized vessel that is completely transected may contract to the extent that bleeding ceases spontaneously.Platelet FunctionPlatelets are anucleate fragments of megakaryocytes. The nor-mal circulating number of platelets ranges between 150,000 and 400,000/μL. Up to 30% of

1	bleeding ceases spontaneously.Platelet FunctionPlatelets are anucleate fragments of megakaryocytes. The nor-mal circulating number of platelets ranges between 150,000 and 400,000/μL. Up to 30% of circulating platelets may be sequestered in the spleen. If not consumed in a clotting reaction, platelets are normally removed by the spleen and have an aver-age life span of 7 to 10 days.Platelets play an integral role in hemostasis by forming a hemostatic plug and by contributing to thrombin formation (Fig. 4-2). Platelets do not normally adhere to each other or to the vessel wall but can form a plug that aids in cessation of bleeding when vascular disruption occurs. Injury to the intimal layer in the vascular wall exposes subendothelial collagen to which platelets adhere. This process requires von Willebrand factor (vWF), a protein in the subendothelium that is lacking in patients with von Willebrand’s disease. vWF binds to glycopro-tein (GP) I/IX/V on the platelet membrane. Following

1	von Willebrand factor (vWF), a protein in the subendothelium that is lacking in patients with von Willebrand’s disease. vWF binds to glycopro-tein (GP) I/IX/V on the platelet membrane. Following adhesion, platelets initiate a release reaction that recruits other platelets from the circulating blood to seal the disrupted vessel. Up to this point, this process is known as primary hemostasis. Platelet aggregation is reversible and is not associated with secretion. Additionally, heparin does not interfere with this reaction, and thus, hemostasis can occur in the heparinized patient. Adenosine diphosphate (ADP) and serotonin are the principal mediators in platelet aggregation.Arachidonic acid released from the platelet membranes is converted by cyclooxygenase to prostaglandin G2 (PGG2) and then to prostaglandin H2 (PGH2), which, in turn, is converted to TXA2. TXA2 has potent vasoconstriction and platelet aggrega-tion effects. Arachidonic acid may also be shuttled to adjacent endothelial

1	to prostaglandin H2 (PGH2), which, in turn, is converted to TXA2. TXA2 has potent vasoconstriction and platelet aggrega-tion effects. Arachidonic acid may also be shuttled to adjacent endothelial cells and converted to prostacyclin (PGI2), which is a vasodilator and acts to inhibit platelet aggregation. Platelet cyclooxygenase is irreversibly inhibited by aspirin and revers-ibly blocked by nonsteroidal anti-inflammatory agents but is not affected by cyclooxygenase-2 (COX-2) inhibitors.In the second wave of platelet aggregation, a release reaction occurs in which several substances including ADP, Ca2+, serotonin, TXA2, and α-granule proteins are discharged. Biology of Hemostasis 103Vascular Constriction / 103Platelet Function / 103Coagulation / 104Fibrinolysis / 106Congenital Factor Deficiencies 106Coagulation Factor Deficiencies / 106Platelet Functional Defects / 107Acquired Hemostatic Defects 108Platelet Abnormalities / 108Acquired Hypofibrinogenemia / 110Myeloproliferative Diseases

1	Factor Deficiencies / 106Platelet Functional Defects / 107Acquired Hemostatic Defects 108Platelet Abnormalities / 108Acquired Hypofibrinogenemia / 110Myeloproliferative Diseases / 110Coagulopathy of Liver Disease / 110Coagulopathy of Trauma / 111Acquired Coagulation Inhibitors / 112Anticoagulation and Bleeding / 112Topical Hemostatic Agents / 115Transfusion 115Background / 115Replacement Therapy/ 115Indications for Replacement of Blood and Its Elements / 117Volume Replacement / 117New Concepts in Resuscitation / 117Prehospital Transfusion / 119Whole Blood Resuscitation / 121Fibrinogen Replacement / 121Complications of Transfusion (Table 4-9) / 121Tests of Hemostasis and Blood Coagulation 123Evaluation of Excessive Intraoperative or Postoperative Bleeding 124Brunicardi_Ch04_p0103-p0130.indd 10329/01/19 11:05 AM 104Figure 4-1. Biology of hemostasis. The four phys-iologic processes that interrelate to limit blood loss from an injured vessel are illustrated and include vascular

1	10329/01/19 11:05 AM 104Figure 4-1. Biology of hemostasis. The four phys-iologic processes that interrelate to limit blood loss from an injured vessel are illustrated and include vascular constriction, platelet plug formation, fibrin clot formation, and fibrinolysis.Key Points1 The life span of platelets ranges from 7 to 10 days. Drugs that interfere with platelet function include aspirin, clopido-grel, prasugrel, dipyridamole, and the glycoprotein IIb/IIIa (GP IIb/IIIa) inhibitors. Approximately 5 to 7 days should pass from the time the drug is stopped until an elective pro-cedure is performed.2 Laboratory evidence of trauma-induced coagulopathy is found in up to one-third of severely injured patients at admission. It is distinct from disseminated intravascular coagulopathy and iatrogenic causes of coagulopathy such as hemodilution. Several non–mutually exclusive mechanisms have been proposed. However, the relationship between laboratory coagulation abnormalities and clinically

1	causes of coagulopathy such as hemodilution. Several non–mutually exclusive mechanisms have been proposed. However, the relationship between laboratory coagulation abnormalities and clinically evident coagulopathic bleeding is unclear.3 Direct oral anticoagulants have no readily available method for monitoring anticoagulation. A new monoclonal antibody has been approved to reverse coagulopathy due to dabiga-tran, and agents are currently in clinical trials for the reversal of direct factor Xa oral anticoagulants.4 When determining the need for bridging of therapeutic anti-coagulation in the preoperative and postoperative setting, the patient’s risk of bleeding should be carefully considered against the risk of thromboembolism and used to guide the need for reversal of anticoagulation therapy preoperatively and the timing of its reinstatement postoperatively.5 Damage control resuscitation has three basic components: permissive hypotension, minimizing crystalloid-based resus-citation,

1	preoperatively and the timing of its reinstatement postoperatively.5 Damage control resuscitation has three basic components: permissive hypotension, minimizing crystalloid-based resus-citation, and the administration of balanced ratios of blood products.6 The need for massive transfusion should be anticipated, and guidelines should be in place to provide early and balanced amounts of red blood cells, plasma, and platelets.Fibrinogen is a required cofactor for this process, acting as a bridge for the GP IIb/IIIa receptor on the activated platelets. The release reaction results in compaction of the platelets into a plug, a process that is no longer reversible. Thrombospondin, another protein secreted by the α-granule, stabilizes fibrino-gen binding to the activated platelet surface and strengthens the platelet-platelet interactions. Platelet factor 4 (PF4) and α-thromboglobulin are also secreted during the release reac-tion. PF4 is a potent heparin antagonist. The second wave of

1	strengthens the platelet-platelet interactions. Platelet factor 4 (PF4) and α-thromboglobulin are also secreted during the release reac-tion. PF4 is a potent heparin antagonist. The second wave of platelet aggregation is inhibited by aspirin and nonsteroidal anti-inflammatory drugs, by cyclic adenosine monophosphate (cAMP), and by nitric oxide. As a consequence of the release reaction, alterations occur in the phospholipids of the platelet membrane that allow calcium and clotting factors to bind to the platelet surface, forming enzymatically active complexes. The altered lipoprotein surface (sometimes referred to as platelet factor 3) catalyzes reactions that are involved in the conversion of prothrombin (factor II) to thrombin (factor IIa) by activated factor X (Xa) in the presence of factor V and calcium, and it is involved in the reaction by which activated factor IX (IXa), fac-tor VIII, and calcium activated factor X. Platelets may also play a role in the initial activation of

1	factor V and calcium, and it is involved in the reaction by which activated factor IX (IXa), fac-tor VIII, and calcium activated factor X. Platelets may also play a role in the initial activation of factors XI and XII.CoagulationHemostasis involves a complex interplay and combination of interactions between platelets, the endothelium, and multiple circulating or membrane-bound coagulation factors. While overly simplistic and not reflective of the depth or complexity of these interactions, the coagulation cascade has traditionally been depicted as two possible pathways converging into a single Common pathwayIntrinsic pathwayClotting factorsVIII, IX, X, XI, XIIFibrin1. Vascular phase(Vasoconstriction)2. Platelet phase(Platelets aggregate)3. Coagulation phase (Clot formation)(Clot retraction)4. Fibrinolysis(Clot destruction)Extrinsic pathwayClotting factorsVIIProthrombinThrombinCA2+vCA2+Brunicardi_Ch04_p0103-p0130.indd 10429/01/19 11:05 AM 105HEMOSTASIS, SURGICAL BLEEDING, AND

1	Fibrinolysis(Clot destruction)Extrinsic pathwayClotting factorsVIIProthrombinThrombinCA2+vCA2+Brunicardi_Ch04_p0103-p0130.indd 10429/01/19 11:05 AM 105HEMOSTASIS, SURGICAL BLEEDING, AND TRANSFUSIONCHAPTER 4common pathway (Fig. 4-3). While this pathway reflects the basic process and sequences that lead to the formation of a clot, the numerous feedback loops, endothelial interplay, and platelet functions are not included. The intrinsic pathway begins with the activation of factor XII that subsequently activates factors XI, IX, and VIII. In this pathway, each of the primary factors is “intrinsic” to the circulating plasma, whereby no surface is required to initi-ate the process. In the extrinsic pathway, tissue factor (TF) is released or exposed on the surface of the endothelium, binding to circulating factor VII, facilitating its activation to VIIa. Each of these pathways continues on to a common sequence that begins with the activation of factor X to Xa (in the presence of VIIIa).

1	to circulating factor VII, facilitating its activation to VIIa. Each of these pathways continues on to a common sequence that begins with the activation of factor X to Xa (in the presence of VIIIa). Subsequently, Xa (with the help of factor Va) converts factor II (prothrombin) to thrombin and then factor I (fibrinogen) to fibrin. Clot formation occurs after fibrin monomers are cross-linked to polymers with the assistance of factor XIII.One convenient feature of depicting the coagulation cas-cade with two merging arms is that commonly used laboratory tests segregate abnormalities of clotting to one of the two arms. An elevated activated partial thromboplastin time (aPTT) is associated with abnormal function of the intrinsic arm of the cascade (II, IX, X, XI, XII), while the prothrombin time (PT) is associated with the extrinsic arm (II, VII, X). Vitamin K defi-ciency or warfarin use affects factors II, VII, IX, and X.Expanding from the basic concept of Fig. 4-3, the cell-based model of

1	(PT) is associated with the extrinsic arm (II, VII, X). Vitamin K defi-ciency or warfarin use affects factors II, VII, IX, and X.Expanding from the basic concept of Fig. 4-3, the cell-based model of hemostasis, divided into the initiation, ampli-fication, and propagation phases, provides a more complete picture of clot formation. During initiation, the primary pathway for coagulation is initiated by TF exposure following suben-dothelial injury. TF binds to VIIa, and this complex catalyzes the activation of factor X to Xa and IX to IXa, which in turn activates factor V to Va. This “prothrombinase” complex gener-ates small amounts of thrombin from prothrombin in a calcium-dependent process. During amplification, platelets adhere to extracellular matrix components at the site of injury and become activated upon exposure to thrombin and other stimuli. Finally, during the propagation phase, “tenase” (factor VIIIa/IXa) and prothrombinase (factor Va/Xa) complexes are assembled on the

1	and become activated upon exposure to thrombin and other stimuli. Finally, during the propagation phase, “tenase” (factor VIIIa/IXa) and prothrombinase (factor Va/Xa) complexes are assembled on the surfaces of activated platelets. This results in large-scale genera-tion of thrombin (“thrombin burst”) and fibrin.In building on the redundancy inherent in the coagulation system, factor VIIIa combines with IXa to form the intrinsic factor complex. Factor IXa is responsible for the bulk of the conversion of factor X to Xa. This complex (VIIIa-IXa) is 50 times more effective at catalyzing factor X activation than is the extrinsic (TF-VIIa) complex and five to six orders of mag-nitude more effective than factor IXa alone.Once formed, thrombin leaves the membrane surface and converts fibrinogen by two cleavage steps into fibrin and two small peptides termed fibrinopeptides A and B. Removal of fibrinopeptide A permits end-to-end polymerization of the fibrin molecules, whereas cleavage of

1	by two cleavage steps into fibrin and two small peptides termed fibrinopeptides A and B. Removal of fibrinopeptide A permits end-to-end polymerization of the fibrin molecules, whereas cleavage of fibrinopeptide B allows side-to-side polymerization of the fibrin clot. This latter step is Platelet hemostaticfunctionVasoconstrictionADP, serotonin,Ca2+, fibrinogenADP, serotonin,Ca2+, fibrinogenSubendothelial collagenPlatelet adhesion secretionPlatelet aggregation secretionPlatelet aggregationPlatelet-fibrinthrombus(Reversible)(Irreversible)Coagulation activationvia tissue factor-factor VIIaIXa, XaComplexes onactivated plateletsThrombin+FibrinogenVascular endothelialinjuryFigure 4-2. Schematic of platelet activation and thrombus function.Figure 4-3. Schematic of the coagulation system. HMW = high molecular weight.Tissue factor-Factor VIIaInflammationComplement activationFibrinolysisPhysiologicFactor VFactor VaCa2+PhospholipidCa2+Ca2+Prothrombin(factor II) Thrombin(factor

1	system. HMW = high molecular weight.Tissue factor-Factor VIIaInflammationComplement activationFibrinolysisPhysiologicFactor VFactor VaCa2+PhospholipidCa2+Ca2+Prothrombin(factor II) Thrombin(factor IIa)IntrinsicSurfaceFactor XIIFactor XIIa KallikreinPrekallikreinHMW kininogenSurfaceFactor XIaFactor IXaFactor XIFactor IXExtrinsicVascular injuryTissue factor +factor VIIFactor XaFactor XCa2+FibrinFactor XIIIFibrinFactor XIIIaX-Linked fibrinFibrinogenFactor VIIIaCa2+PhospholipidFactor VIIIBrunicardi_Ch04_p0103-p0130.indd 10529/01/19 11:05 AM 106BASIC CONSIDERATIONSPART Ifacilitated by thrombin-activatable fibrinolysis inhibitor (TAFI), which acts to stabilize the resultant clot.In seeking to balance profound bleeding with overwhelm-ing clot burden, several related processes exist to prevent prop-agation of the clot beyond the site of injury.1 First, feedback inhibition on the coagulation cascade deactivates the enzyme complexes leading to thrombin formation. Thrombomodulin (TM)

1	prevent prop-agation of the clot beyond the site of injury.1 First, feedback inhibition on the coagulation cascade deactivates the enzyme complexes leading to thrombin formation. Thrombomodulin (TM) presented by the endothelium serves as a “thrombin sink” by forming a complex with thrombin, rendering it no longer available to cleave fibrinogen. This then activates protein C (APC) and reduces further thrombin generation by inhibiting factors V and VIII. Second, tissue plasminogen activator (tPA) is released from the endothelium following injury, cleaving plasminogen to initiate fibrinolysis. APC then consumes plas-minogen activator inhibitor-1 (PAI-1), leading to increased tPA activity and fibrinolysis. Building on the anticoagulant response to inhibit thrombin formation, tissue factor pathway inhibitor (TFPI) is released, blocking the TF-VIIa complex and reducing the production of factors Xa and IXa. Antithrombin III (AT-III) then neutralizes all of the procoagulant serine proteases

1	inhibitor (TFPI) is released, blocking the TF-VIIa complex and reducing the production of factors Xa and IXa. Antithrombin III (AT-III) then neutralizes all of the procoagulant serine proteases and also inhibits the TF-VIIa complex. The most potent mechanism of thrombin inhibition involves the APC system. APC forms a complex with its cofactor, protein S, on a phospholipid surface. This complex then cleaves factors Va and VIIIa so that they are no longer able to participate in the formation of TF-VIIa or pro-thrombinase complexes. This is of interest clinically in the form of a genetic mutation, called factor V Leiden. In this setting, factor V is resistant to cleavage by APC, thereby remaining active as a procoagulant. Patients with factor V Leiden are pre-disposed to venous thromboembolic events.Degradation of fibrin clot is accomplished by plasmin, a serine protease derived from the proenzyme plasminogen. Plas-min formation occurs as a result of one of several plasminogen

1	events.Degradation of fibrin clot is accomplished by plasmin, a serine protease derived from the proenzyme plasminogen. Plas-min formation occurs as a result of one of several plasminogen activators. tPA is made by the endothelium and other cells of the vascular wall and is the main circulating form of this family of enzymes. tPA is selective for fibrin-bound plasminogen so that endogenous fibrinolytic activity occurs predominately at the site of clot formation. The other major plasminogen activa-tor, urokinase plasminogen activator (uPA), also produced by endothelial cells as well as by urothelium, is not selective for fibrin-bound plasminogen. Of note, the thrombin-TM complex activates TAFI, leading to a mixed effect on clot stability. In addition to inhibiting fibrinolysis directly, removal of the termi-nal lysine on the fibrin molecule by TAFI renders the clot more susceptible to lysis by plasmin.FibrinolysisFibrin clot breakdown (lysis) allows restoration of blood flow during the

1	of the termi-nal lysine on the fibrin molecule by TAFI renders the clot more susceptible to lysis by plasmin.FibrinolysisFibrin clot breakdown (lysis) allows restoration of blood flow during the healing process following injury and begins at the same time clot formation is initiated. Fibrin polymers are degraded by plasmin, a serine protease derived from the pro-enzyme plasminogen. Plasminogen is converted to plasmin by one of several plasminogen activators, including tPA. Plasmin then degrades the fibrin mesh at various places, leading to the production of circulating fragments, termed fibrin degradation products (FDPs), cleared by other proteases or by the kidney and liver (Fig. 4-4). Fibrinolysis is directed by circulating kinases, tissue activators, and kallikrein present in vascular endothelium. tPA is synthesized by endothelial cells and released by the cells on thrombin stimulation. Bradykinin, a potent endothelial-dependent vasodilator, is cleaved from high molecular weight

1	endothelium. tPA is synthesized by endothelial cells and released by the cells on thrombin stimulation. Bradykinin, a potent endothelial-dependent vasodilator, is cleaved from high molecular weight kininogen by kallikrein and enhances the release of tPA. Both tPA and plasminogen bind to fibrin as it forms, and this trimo-lecular complex cleaves fibrin very efficiently. After plasmin is generated, however, it cleaves fibrin somewhat less efficiently.As with clot formation, fibrinolysis is also kept in check through several robust mechanisms. tPA activates plasmino-gen more efficiently when it is bound to fibrin, so that plasmin is formed selectively on the clot. Plasmin is inhibited by α2-antiplasmin, a protein that is cross-linked to fibrin by factor XIII, which helps to ensure that clot lysis does not occur too quickly. Any circulating plasmin is also inhibited by α2-antiplasmin and circulating tPA or urokinase. Clot lysis yields FDPs including E-nodules and D-dimers. These smaller

1	lysis does not occur too quickly. Any circulating plasmin is also inhibited by α2-antiplasmin and circulating tPA or urokinase. Clot lysis yields FDPs including E-nodules and D-dimers. These smaller fragments interfere with normal platelet aggregation, and the larger fragments may be incorporated into the clot in lieu of normal fibrin monomers. This may result in an unstable clot as seen in cases of severe coagu-lopathy such as hyperfibrinolysis associated with trauma-induced coagulopathy or disseminated intravascular coagulopathy. The presence of D-dimers in the circulation may serve as a marker of thrombosis or other conditions in which a significant activa-tion of the fibrinolytic system is present. Another inhibitor of the fibrinolytic system is TAFI, which removes lysine residues from fibrin that are essential for binding plasminogen.CONGENITAL FACTOR DEFICIENCIESCoagulation Factor DeficienciesInherited deficiencies of all of the coagulation factors are seen. However, the three

1	fibrin that are essential for binding plasminogen.CONGENITAL FACTOR DEFICIENCIESCoagulation Factor DeficienciesInherited deficiencies of all of the coagulation factors are seen. However, the three most frequent are factor VIII deficiency (hemophilia A or von Willebrand’s disease), factor IX defi-ciency (hemophilia B or Christmas disease), and factor XI deficiency. Hemophilia A and hemophilia B are inherited as sex-linked recessive disorders with males being affected almost exclusively. The clinical severity of hemophilia A and hemo-philia B depends on the measurable level of factor VIII or factor IX in the patient’s plasma. Plasma factor levels less than 1% of normal are considered severe disease, factor levels between 1% and 5% moderately severe disease, and levels between 5% and 30% mild disease. Patients with severe hemophilia have spontaneous bleeds, frequently into joints, leading to crippling arthropathies. Intracranial bleeding, intramuscular hematomas, retroperitoneal

1	30% mild disease. Patients with severe hemophilia have spontaneous bleeds, frequently into joints, leading to crippling arthropathies. Intracranial bleeding, intramuscular hematomas, retroperitoneal hematomas, and gastrointestinal, genitourinary, and retropharyngeal bleeding are added clinical sequelae seen with severe disease. Patients with moderately severe hemophilia have less spontaneous bleeding but are likely to bleed severely EndotheliumPlateletThrombinPlasminogentPAPlasminFibrinFDPFigure 4-4. Formation of fibrin degradation products (FDPs). tPA = tissue plasminogen activator.Brunicardi_Ch04_p0103-p0130.indd 10629/01/19 11:05 AM 107HEMOSTASIS, SURGICAL BLEEDING, AND TRANSFUSIONCHAPTER 4after trauma or surgery. Mild hemophiliacs do not bleed sponta-neously and have only minor bleeding after major trauma or sur-gery. Since platelet function is normal in hemophiliacs, patients may not bleed immediately after an injury or minor surgery as they have a normal response with

1	bleeding after major trauma or sur-gery. Since platelet function is normal in hemophiliacs, patients may not bleed immediately after an injury or minor surgery as they have a normal response with platelet activation and forma-tion of a platelet plug. At times, the diagnosis of hemophilia is not made in these patients until after their first minor procedure (e.g., tooth extraction or tonsillectomy).Patients with hemophilia A or B are treated with factor VIII or factor IX concentrate, respectively. Recombinant factor VIII is strongly recommended for patients not treated previously and is generally recommended for patients who are both human immunodeficiency virus (HIV) and hepatitis C virus (HCV) seronegative. For factor IX replacement, the preferred products are recombinant or high-purity factor IX. In general, activity levels should be restored to 30% to 40% for mild hemorrhage, 50% for severe bleeding, and 80% to 100% for life-threatening bleeding. Up to 20% of hemophiliacs with

1	factor IX. In general, activity levels should be restored to 30% to 40% for mild hemorrhage, 50% for severe bleeding, and 80% to 100% for life-threatening bleeding. Up to 20% of hemophiliacs with factor VIII defi-ciency develop inhibitors that can neutralize FVIII. For patients with low titers, inhibitors can be overcome with higher doses of factor VIII. For patients with high titer inhibitors, alternate treat-ments should be used and may include porcine factor VIII, pro-thrombin complex concentrates, activated prothrombin complex concentrates, or recombinant factor VIIa. For patients undergo-ing elective surgical procedures, a multidisciplinary approach with preoperative planning and replacement is recommended.2von Willebrand’s Disease. von Willebrand’s disease (vWD), the most common congenital bleeding disorder, is characterized by a quantitative or qualitative defect in vWF, a large glycopro-tein responsible for carrying factor VIII and platelet adhesion. The latter is important

1	bleeding disorder, is characterized by a quantitative or qualitative defect in vWF, a large glycopro-tein responsible for carrying factor VIII and platelet adhesion. The latter is important for normal platelet adhesion to exposed subendothelium and for aggregation under high shear condi-tions. Patients with vWD have bleeding that is characteristic of platelet disorders such as easy bruising and mucosal bleed-ing. Menorrhagia is common in women. vWD is classified into three types. Type I is a partial quantitative deficiency; type II is a qualitative defect; type III is total deficiency. For bleeding, type I patients usually respond well to desmopressin (DDAVP). Type II patients may respond, depending on the particular defect. Type III patients are usually unresponsive. These patients may require vWF concentrates.3Factor XI Deficiency. Factor XI deficiency, an autosomal recessive inherited condition sometimes referred to as hemo-philia C, is more prevalent in the Ashkenazi Jewish

1	may require vWF concentrates.3Factor XI Deficiency. Factor XI deficiency, an autosomal recessive inherited condition sometimes referred to as hemo-philia C, is more prevalent in the Ashkenazi Jewish population but found in all races. Spontaneous bleeding is rare, but bleeding may occur after surgery, trauma, or invasive procedures. Treat-ment of patients with factor XI deficiency who present with bleeding or in whom surgery is planned and who are known to have bled previously is with fresh frozen plasma (FFP). Each milliliter of plasma contains 1 unit of factor XI activity, so the volume needed depends on the patient’s baseline level, the desired level, and the plasma volume. Antifibrinolytics may be useful in patients with menorrhagia. Factor VIIa is recom-mended for patients with anti-factor XI antibodies, although thrombosis has been reported.4 There has been renewed interest in factor XI inhibitors as antithrombotic agents because patients with factor XI deficiency generally have

1	XI antibodies, although thrombosis has been reported.4 There has been renewed interest in factor XI inhibitors as antithrombotic agents because patients with factor XI deficiency generally have only minimal bleeding risk unless a severe deficiency is present and seem to be pro-tected from thrombosis.5Deficiency of Factors II (Prothrombin), V, and X. Inher-ited deficiencies of factors II, V, and X are rare. These deficiencies are inherited as autosomal recessive. Significant bleeding in homozygotes with less than 1% of normal activ-ity is encountered. Bleeding with any of these deficiencies is treated with FFP. Similar to factor XI, FFP contains one unit of activity of each per milliliter. However, factor V activity is decreased because of its inherent instability. The half-life of prothrombin (factor II) is long (approximately 72 hours), and only about 25% of a normal level is needed for hemostasis. Prothrombin complex concentrates can be used to treat defi-ciencies of prothrombin or

1	(factor II) is long (approximately 72 hours), and only about 25% of a normal level is needed for hemostasis. Prothrombin complex concentrates can be used to treat defi-ciencies of prothrombin or factor X. Daily infusions of FFP are used to treat bleeding in factor V deficiency, with a goal of 20% to 25% activity. Factor V deficiency may be coinherited with factor VIII deficiency. Treatment of bleeding in individuals with the combined deficiency requires factor VIII concentrate and FFP. Some patients with factor V deficiency are also lacking the factor V normally present in platelets and may need platelet transfusions as well as FFP.Factor VII Deficiency. Inherited factor VII deficiency is a rare autosomal recessive disorder. Clinical bleeding can vary widely and does not always correlate with the level of FVII coagulant activity in plasma. Bleeding is uncommon unless the level is less than 3%. The most common bleeding manifesta-tions involve easy bruising and mucosal bleeding,

1	with the level of FVII coagulant activity in plasma. Bleeding is uncommon unless the level is less than 3%. The most common bleeding manifesta-tions involve easy bruising and mucosal bleeding, particularly epistaxis or oral mucosal bleeding. Postoperative bleeding is also common, reported in 30% of surgical procedures.6 Treat-ment is with FFP or recombinant factor VIIa. The half-life of recombinant factor VIIa is only approximately 2 hours, but excellent hemostasis can be achieved with frequent infusions. The half-life of factor VII in FFP is up to 4 hours.Factor XIII Deficiency. Congenital factor XIII (FXIII) defi-ciency, originally recognized by Duckert in 1960, is a rare autosomal recessive disease usually associated with a severe bleeding diathesis.7 The male-to-female ratio is 1:1. Although acquired FXIII deficiency has been described in association with hepatic failure, inflammatory bowel disease, and myeloid leukemia, the only significant association with bleeding in chil-dren

1	acquired FXIII deficiency has been described in association with hepatic failure, inflammatory bowel disease, and myeloid leukemia, the only significant association with bleeding in chil-dren is the inherited deficiency.8 Bleeding is typically delayed because clots form normally but are susceptible to fibrinolysis. Umbilical stump bleeding is characteristic, and there is a high risk of intracranial bleeding. Spontaneous abortion is usual in women with factor XIII deficiency unless they receive replace-ment therapy. Replacement can be accomplished with FFP, cryoprecipitate, or a factor XIII concentrate. Levels of 1% to 2% are usually adequate for hemostasis.Platelet Functional DefectsInherited platelet functional defects include abnormalities of platelet surface proteins, abnormalities of platelet granules, and enzyme defects. The major surface protein abnormalities are thrombasthenia and Bernard-Soulier syndrome. Thrombasthe-nia, or Glanzmann thrombasthenia, is a rare genetic platelet

1	granules, and enzyme defects. The major surface protein abnormalities are thrombasthenia and Bernard-Soulier syndrome. Thrombasthe-nia, or Glanzmann thrombasthenia, is a rare genetic platelet disorder, inherited in an autosomal recessive pattern, in which the platelet glycoprotein IIb/IIIa (GP IIb/IIIa) complex is either lacking or present but dysfunctional. This defect leads to faulty platelet aggregation and subsequent bleeding. The disorder was first described by Dr. Eduard Glanzmann in 1918.9 Bleeding in thrombasthenic patients must be treated with platelet transfu-sions. Bernard-Soulier syndrome is caused by a defect in the GP Ib/IX/V receptor for vWF, which is necessary for platelet adhesion to the subendothelium. Transfusion of normal platelets is required for bleeding in these patients.Brunicardi_Ch04_p0103-p0130.indd 10729/01/19 11:05 AM 108BASIC CONSIDERATIONSPART IThe most common intrinsic platelet defect is storage pool disease. It involves loss of dense granules

1	patients.Brunicardi_Ch04_p0103-p0130.indd 10729/01/19 11:05 AM 108BASIC CONSIDERATIONSPART IThe most common intrinsic platelet defect is storage pool disease. It involves loss of dense granules (storage sites for ADP, adenosine triphosphate [ATP], Ca2+, and inorganic phosphate) and α-granules. Dense granule deficiency is the most prevalent of these. It may be an isolated defect or occur with partial albinism in Hermansky-Pudlak syndrome. Bleed-ing is variable, depending on the severity of the granule defect. Bleeding is caused by the decreased release of ADP from these platelets. A few patients have been reported who have decreased numbers of both dense and α-granules. They have a more severe bleeding disorder. Patients with mild bleeding as a consequence of a form of storage pool disease can be treated with DDAVP. It is likely that the high levels of vWF in the plasma after DDAVP somehow compensate for the intrinsic platelet defect. With more severe bleeding, platelet transfusion

1	can be treated with DDAVP. It is likely that the high levels of vWF in the plasma after DDAVP somehow compensate for the intrinsic platelet defect. With more severe bleeding, platelet transfusion is required.ACQUIRED HEMOSTATIC DEFECTSPlatelet AbnormalitiesAcquired congenital abnormalities of platelets are much more common than acquired defects and may be quantitative or quali-tative, although some patients have both types of defects. Quan-titative defects may be a result of failure of production, shortened survival, or sequestration. Failure of production is generally a result of bone marrow disorders such as leukemia, myelodys-plastic syndrome, severe vitamin B12 or folate deficiency, che-motherapeutic drugs, radiation, acute ethanol intoxication, or viral infection. If a quantitative abnormality exists and treatment is indicated either due to symptoms or the need for an invasive procedure, platelet transfusion is utilized. The etiologies of both qualitative and quantitative defects

1	exists and treatment is indicated either due to symptoms or the need for an invasive procedure, platelet transfusion is utilized. The etiologies of both qualitative and quantitative defects are reviewed in Table 4-1.Quantitative Defects. Shortened platelet survival is seen in immune thrombocytopenia, disseminated intravascular coagu-lation, or disorders characterized by platelet thrombi such as thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Immune thrombocytopenia may be idiopathic or associated with other autoimmune disorders or low-grade B-cell malignancies, and it may also be secondary to viral infections (including HIV) or drugs. Secondary immune thrombocytopenia often presents with a very low platelet count, petechiae and pur-pura, and epistaxis. Large platelets are seen on peripheral smear. Initial treatment consists of corticosteroids, intravenous gamma globulin, or anti-D immunoglobulin in patients who are Rh posi-tive. Both gamma globulin and anti-D

1	are seen on peripheral smear. Initial treatment consists of corticosteroids, intravenous gamma globulin, or anti-D immunoglobulin in patients who are Rh posi-tive. Both gamma globulin and anti-D immunoglobulin are rapid in onset. Platelet transfusions are not usually needed unless cen-tral nervous system bleeding or active bleeding from other sites occurs. Survival of the transfused platelets is usually short.Primary immune thrombocytopenia is also known as idio-pathic thrombocytopenic purpura (ITP). In children, it is usually acute in onset, short lived, and typically follows a viral illness. In contrast, ITP in adults is gradual in onset, chronic in nature, and has no identifiable cause. Because the circulating platelets in ITP are young and functional, bleeding is less for a given platelet count than when there is failure of platelet production. The patho-physiology of ITP is believed to involve both impaired platelet production and T cell–mediated platelet destruction.10

1	platelet count than when there is failure of platelet production. The patho-physiology of ITP is believed to involve both impaired platelet production and T cell–mediated platelet destruction.10 Manage-ment options are summarized in Table 4-2.11 Treatment of drug-induced immune thrombocytopenia may simply entail withdrawal of the offending drug, but corticosteroids, gamma globulin, and anti-D immunoglobulin may hasten recovery of the count.12,13Table 4-1Etiology of acquired platelet disordersA. Quantitative Disorders1. Failure of production: related to impairment in bone marrow functiona. Leukemiab. Myeloproliferative disordersc. B12 or folate deficienciesd. Chemotherapy or radiation therapye. Acute alcohol intoxicationf. Viral infections2. Decreased survivala. Immune-mediated1) Idiopathic thrombocytopenia (ITP)2) Heparin-induced thrombocytopenia3) Autoimmune disorders or B-cell malignancies4) Secondary thrombocytopeniab. Disseminated intravascular coagulation (DIC)c. Related to

1	thrombocytopenia (ITP)2) Heparin-induced thrombocytopenia3) Autoimmune disorders or B-cell malignancies4) Secondary thrombocytopeniab. Disseminated intravascular coagulation (DIC)c. Related to platelet thrombi1) Thrombocytopenic purpura (TTP)2) Hemolytic uremic syndrome (HUS)3. Sequestrationa. Portal hypertensionb. Sarcoidc. Lymphomad. Gaucher’s DiseaseB. Qualitative Disorders1. Massive transfusion2. Therapeutic platelet inhibitors3. Disease statesa. Myeloproliferative disordersb. Monoclonal gammopathiesc. Liver diseaseTable 4-2Management of idiopathic thrombocytopenic purpura (ITP) in adultsFirst line: a. Corticosteroids: Longer courses of corticosteroids are preferred over shorter courses of corticosteroids b. Intravenous immunoglobulin (IVIG) or anti-D immunoglobulin: the dose should initially be 1 g/kg as a one-time dose. This dosage may be repeated if necessarySecond line: a. Splenectomy b. Rituximab, an anti-CD 20 monoclonal antibody c. Thrombopoietin (TPO) receptor

1	should initially be 1 g/kg as a one-time dose. This dosage may be repeated if necessarySecond line: a. Splenectomy b. Rituximab, an anti-CD 20 monoclonal antibody c. Thrombopoietin (TPO) receptor agonists d. Immunosuppressive agentsThird line: (failing first and second line therapy) a. Thrombopoietin (TPO) receptor agonists b. Combination of first and second line therapies c. Combination chemotherapyBrunicardi_Ch04_p0103-p0130.indd 10829/01/19 11:05 AM 109HEMOSTASIS, SURGICAL BLEEDING, AND TRANSFUSIONCHAPTER 4Heparin-induced thrombocytopenia (HIT) is a form of drug-induced immune thrombocytopenia. It is an immunologic event during which antibodies against platelet factor 4 (PF4) formed during exposure to heparin affect platelet activation and endothelial function with resultant thrombocytopenia and intravascular thrombosis.14 The platelet count typically begins to fall 5 to 7 days after heparin has been started, but if it is a reexposure, the decrease in count may occur within 1

1	and intravascular thrombosis.14 The platelet count typically begins to fall 5 to 7 days after heparin has been started, but if it is a reexposure, the decrease in count may occur within 1 to 2 days. HIT should be suspected if the platelet count falls to less than 100,000 or if it drops by 50% from baseline in a patient receiv-ing heparin. While HIT is more common with full-dose unfrac-tionated heparin (1% to 3%), it can also occur with prophylactic doses or with low molecular weight heparins. Interestingly, approximately 17% of patients receiving unfractionated hepa-rin and 8% receiving low molecular weight heparin develop antibodies against PF4, yet a much smaller percentage develop thrombocytopenia, and even fewer develop clinical HIT.15 In addition to mild to moderate thrombocytopenia, this disorder is characterized by a high incidence of thrombosis that may be arterial or venous. Importantly, the absence of thrombo-cytopenia in these patients does not preclude the diagnosis of

1	this disorder is characterized by a high incidence of thrombosis that may be arterial or venous. Importantly, the absence of thrombo-cytopenia in these patients does not preclude the diagnosis of HIT. The 4Ts scoring system by Lo et al can be used to assess the pretest probability of HIT and incorporates the timing and magnitude of the platelet count fall, new thrombosis, and the likelihood of other reasons for thrombocytopenia.16 A low probability 4Ts score is quite accurate in excluding HIT, but patients with intermediate or high probability scores require further evaluation.Laboratory testing should include an anti–platelet fac-tor 4–heparin enzyme-linked immunosorbent assay (ELISA). Unfortunately, this test, like the 4Ts, has a high negative predic-tive value but a low positive predictive value. While a negative ELISA essentially rules out HIT, a positive ELISA does not confirm HIT. To increase the specificity of this assay, it can be restricted to IgG antibodies or obtained in

1	value. While a negative ELISA essentially rules out HIT, a positive ELISA does not confirm HIT. To increase the specificity of this assay, it can be restricted to IgG antibodies or obtained in conjunction with a functional assay such as the serotonin release assay and the heparin-induced platelet activation test. Both of these are avail-able only at specialized laboratories and should only be used as second-line diagnostic assays.l7The initial treatment of suspected HIT is to stop heparin and begin an alternative anticoagulant. Stopping heparin with-out addition of another anticoagulant is not adequate to prevent thrombosis in this setting. Alternative anticoagulants are pri-marily thrombin inhibitors. The most recent guideline by the American College of Chest Physicians recommends lepiru-din, argatroban, or danaparoid for patients with normal renal function and argatroban for patients with renal insufficiency.18 Because of warfarin’s early induction of a hypercoagulable state,

1	lepiru-din, argatroban, or danaparoid for patients with normal renal function and argatroban for patients with renal insufficiency.18 Because of warfarin’s early induction of a hypercoagulable state, warfarin should be instituted only once full anticoagula-tion with an alternative agent has been accomplished and the platelet count has begun to recover.These are also disorders in which thrombocytopenia is a result of platelet activation and formation of platelet thrombi. In thrombotic thrombocytopenic purpura (TTP), large vWF mol-ecules interact with platelets, leading to activation. These large molecules result from inhibition of a metalloproteinase enzyme, ADAM-S13, which cleaves the large vWF molecules.19 TTP is classically characterized by thrombocytopenia, microangio-pathic hemolytic anemia, fever, and renal and neurologic signs or symptoms. The finding of schistocytes on a peripheral blood smear aids in the diagnosis. Plasma exchange with replacement of FFP is the treatment for

1	anemia, fever, and renal and neurologic signs or symptoms. The finding of schistocytes on a peripheral blood smear aids in the diagnosis. Plasma exchange with replacement of FFP is the treatment for acute TTP.20 Additionally, rituximab, a monoclonal antibody against the CD20 protein on B lympho-cytes, is indicated in relapsing and/or refractory TTP.21Hemolytic uremic syndrome (HUS) often occurs second-ary to infection by Escherichia coli 0157:H7 or other Shiga toxin-producing bacteria. The metalloproteinase is normal in these cases. HUS is usually associated with some degree of renal failure, with many patients requiring renal replacement therapy. Neurologic symptoms are less frequent. A number of patients develop features of both TTP and HUS. This may occur with autoimmune diseases, especially systemic lupus erythematosus and HIV infection, or in association with certain drugs (such as ticlopidine, mitomycin C, gemcitabine) or immunosuppressive agents (such as cyclosporine and

1	especially systemic lupus erythematosus and HIV infection, or in association with certain drugs (such as ticlopidine, mitomycin C, gemcitabine) or immunosuppressive agents (such as cyclosporine and tacrolimus). Discontinuation of the involved drug is the mainstay of therapy. Plasmapheresis is frequently used, but it is not clear what etiologic factor is being removed by the pheresis.Sequestration is another important cause of thrombocyto-penia and usually involves trapping of platelets in an enlarged spleen typically related to portal hypertension, sarcoid, lym-phoma, or Gaucher’s disease. The total body platelet mass is essentially normal in patients with hypersplenism, but a much larger fraction of the platelets are in the enlarged spleen. Platelet survival is mildly decreased. Bleeding is less than anticipated from the count because sequestered platelets can be mobilized to some extent and enter the circulation. Platelet transfusion does not increase the platelet count as much as

1	is less than anticipated from the count because sequestered platelets can be mobilized to some extent and enter the circulation. Platelet transfusion does not increase the platelet count as much as it would in a normal person because the transfused platelets are similarly sequestered in the spleen. Splenectomy is not indicated to correct the throm-bocytopenia of hypersplenism caused by portal hypertension.Thrombocytopenia and platelet dysfunction are the most common abnormalities of hemostasis that result in bleeding in the surgical patient. The patient may have a reduced platelet count as a result of a variety of disease processes, as discussed earlier. In these circumstances, the marrow usually demon-strates a normal or increased number of megakaryocytes. By contrast, when thrombocytopenia occurs in patients with leu-kemia or uremia and in patients on cytotoxic therapy, there are generally a reduced number of megakaryocytes in the marrow. Thrombocytopenia also occurs in surgical

1	occurs in patients with leu-kemia or uremia and in patients on cytotoxic therapy, there are generally a reduced number of megakaryocytes in the marrow. Thrombocytopenia also occurs in surgical patients as a result of massive blood loss with product replacement deficient in platelets. Thrombocytopenia may also be induced by heparin administration during cardiac and vascular cases, as in the case of HIT, or may be associated with thrombotic and hemorrhagic complications. When thrombocytopenia is present in a patient for whom an elective operation is being considered, manage-ment is contingent upon the extent and cause of platelet reduc-tion and extent of platelet dysfunction.Early platelet administration has now become part of mas-sive transfusion protocols.22,23 Platelets are also administered preoperatively to rapidly increase the platelet count in surgical patients with underlying thrombocytopenia or platelet dysfunc-tion. One unit of platelet concentrate contains approximately 5.5 ×

1	preoperatively to rapidly increase the platelet count in surgical patients with underlying thrombocytopenia or platelet dysfunc-tion. One unit of platelet concentrate contains approximately 5.5 × 1010 platelets and would be expected to increase the cir-culating platelet count by about 10,000/μL in the average 70-kg person. Fever, infection, hepatosplenomegaly, and the pres-ence of antiplatelet alloantibodies decrease the effectiveness of platelet transfusions. In patients who are refractory to standard platelet transfusion, the use of human leukocyte antigen (HLA)-compatible platelets coupled with special processors has proved effective.Brunicardi_Ch04_p0103-p0130.indd 10929/01/19 11:05 AM 110BASIC CONSIDERATIONSPART IQualitative Platelet Defects. Impaired platelet function often accompanies thrombocytopenia but may also occur in the presence of a normal platelet count. The importance of this is obvious when one considers that 80% of overall clot strength is related to platelet

1	thrombocytopenia but may also occur in the presence of a normal platelet count. The importance of this is obvious when one considers that 80% of overall clot strength is related to platelet function. The life span of platelets ranges from 7 to 10 days, placing them at increased risk for impairment by medical disorders and prescription and over-the-counter medications. Impairment of ADP-stimulated aggregation occurs with massive transfusion of blood products. Ure-mia may be associated with increased bleeding time and impaired aggregation. Defective aggregation and platelet dys-function are also seen in patients with severe trauma, thrombo-cythemia, polycythemia vera, and myelofibrosis.Drugs that interfere with platelet function include aspirin, clopidogrel, prasugrel, dipyridamole, and GP IIb/IIIa inhibitors. Aspirin, clopidogrel, and prasugrel all irreversibly inhibit plate-let function. Clopidogrel and prasugrel do so through selective irreversible inhibition of ADP-induced platelet

1	IIb/IIIa inhibitors. Aspirin, clopidogrel, and prasugrel all irreversibly inhibit plate-let function. Clopidogrel and prasugrel do so through selective irreversible inhibition of ADP-induced platelet aggregation.24 Aspirin works through irreversible acetylation of platelet pros-taglandin synthase.There are no prospective randomized trials in general sur-gical patients to guide the timing of surgery in patients on aspi-rin, clopidogrel, or prasugrel.25 The general recommendation is that approximately 5 to 7 days should pass from the time the drug is stopped until an elective procedure is performed.26 Tim-ing of urgent and emergent surgeries is even more unclear. Pre-operative platelet transfusions may be beneficial, but there are no good data to guide their administration. However, functional tests such as thromboelastography (TEG) with platelet mapping are becoming available that may better demonstrate defects in platelet function and may serve to guide the timing of operation or when

1	tests such as thromboelastography (TEG) with platelet mapping are becoming available that may better demonstrate defects in platelet function and may serve to guide the timing of operation or when platelet transfusions might be indicated.Other disorders associated with abnormal platelet func-tion include uremia, myeloproliferative disorders, monoclonal gammopathies, and liver disease. In the surgical patient, plate-let dysfunction of uremia can often be corrected by dialysis or the administration of DDAVP. Platelet transfusion may not be helpful if the patient is uremic when the platelets are given and only serve to increase antibodies. Platelet dysfunction in myelo-proliferative disorders is intrinsic to the platelets and usually improves if the platelet count can be reduced to normal with chemotherapy. If possible, surgery should be delayed until the count has been decreased. These patients are at risk for both bleeding and thrombosis. Platelet dysfunction in patients with

1	normal with chemotherapy. If possible, surgery should be delayed until the count has been decreased. These patients are at risk for both bleeding and thrombosis. Platelet dysfunction in patients with monoclonal gammopathies is a result of interaction of the mono-clonal protein with platelets. Treatment with chemotherapy or, occasionally, plasmapheresis to lower the amount of monoclo-nal protein improves hemostasis.Acquired HypofibrinogenemiaDisseminated Intravascular Coagulation (DIC). DIC is an acquired syndrome characterized by systemic activation of coagulation pathways that result in excessive thrombin genera-tion and the diffuse formation of microthrombi. This distur-bance ultimately leads to consumption and depletion of platelets and coagulation factors with the resultant classic picture of dif-fuse bleeding. Fibrin thrombi developing in the microcirculation may cause microvascular ischemia and subsequent end-organ failure if severe. There are many different conditions that

1	picture of dif-fuse bleeding. Fibrin thrombi developing in the microcirculation may cause microvascular ischemia and subsequent end-organ failure if severe. There are many different conditions that pre-dispose a patient to DIC, and the presence of an underlying condition is required for the diagnosis. For example, injuries resulting in embolization of materials such as brain matter, bone marrow, or amniotic fluid can act as potent thromboplastins that activate the DIC cascade.27 Additional etiologies include malig-nancy, organ injury (such as severe pancreatitis), liver failure, certain vascular abnormalities (such as large aneurysms), snake bites, illicit drugs, transfusion reactions, transplant rejection, and sepsis.28 In fact, DIC frequently accompanies sepsis and may be associated with multiple organ failure. The important interplay between sepsis and coagulation abnormalities was demonstrated by Dhainaut et al who showed that activated protein C was effective in septic patients

1	multiple organ failure. The important interplay between sepsis and coagulation abnormalities was demonstrated by Dhainaut et al who showed that activated protein C was effective in septic patients with DIC, though this has subsequently been disproven.29 The diagnosis of DIC is made based on an inciting etiology with associated thrombo-cytopenia, prolongation of the prothrombin time, a low fibrino-gen level, and elevated fibrin markers (FDPs, D-dimer, soluble fibrin monomers). A scoring system developed by the Interna-tional Society for Thrombosis and Hemostasis has been shown to have high sensitivity and specificity for diagnosing DIC as well as a strong correlation between an increasing DIC score and mortality, especially in patients with infections.30The most important facets of treatment are relieving the patient’s causative primary medical or surgical problem and maintaining adequate perfusion. If there is active bleeding, hemostatic factors should be replaced with FFP, which is

1	are relieving the patient’s causative primary medical or surgical problem and maintaining adequate perfusion. If there is active bleeding, hemostatic factors should be replaced with FFP, which is usually sufficient to correct the hypofibrinogenemia, although cryopre-cipitate, fibrinogen concentrates, or platelet concentrates may also be needed. Given the formation of microthrombi in DIC, heparin therapy has also been proposed. Heparin may be indi-cated in cases where thrombosis predominates, such as arterial or venous thromboembolism and severe purpura fulminans.31Primary Fibrinolysis. Other than due to trauma, an acquired hypofibrinogenic state in the surgical patient can be a result of pathologic fibrinolysis. This may occur in patients following prostate resection when urokinase is released during surgical manipulation of the prostate or in patients undergoing extracor-poreal bypass. The severity of fibrinolytic bleeding is dependent on the concentration of breakdown products in

1	during surgical manipulation of the prostate or in patients undergoing extracor-poreal bypass. The severity of fibrinolytic bleeding is dependent on the concentration of breakdown products in the circula-tion. Antifibrinolytic agents, such as ε-aminocaproic acid and tranexamic acid, interfere with fibrinolysis by inhibiting plas-minogen activation.Myeloproliferative DiseasesPolycythemia, or an excess of red blood cells, places surgical patients at risk. Spontaneous thrombosis is a complication of polycythemia vera, a myeloproliferative neoplasm, and can be explained in part by increased blood viscosity, increased plate-let count, and an increased tendency toward stasis. Paradoxi-cally, a significant tendency toward spontaneous hemorrhage also is noted in these patients. Thrombocytosis can be reduced by the administration of low-dose aspirin, phlebotomy, and hydroxyurea.32Coagulopathy of Liver DiseaseThe liver plays a key role in hemostasis because it is responsible for the synthesis

1	be reduced by the administration of low-dose aspirin, phlebotomy, and hydroxyurea.32Coagulopathy of Liver DiseaseThe liver plays a key role in hemostasis because it is responsible for the synthesis of many of the coagulation factors (Table 4-3). Patients with liver disease, therefore, have decreased production of several key nonendothelial cell-derived coagulation factors as well as natural anticoagulant proteins, causing a disturbance in the balance between procoagulant and anticoagulant path-ways. This disturbance in coagulation mechanisms causes a complex paradigm of both increased bleeding risk and increased 1Brunicardi_Ch04_p0103-p0130.indd 11029/01/19 11:05 AM 111HEMOSTASIS, SURGICAL BLEEDING, AND TRANSFUSIONCHAPTER 4thrombotic risk. The most common coagulation abnormalities associated with liver dysfunction are thrombocytopenia and impaired humoral coagulation function manifested as prolonga-tion of the prothrombin time and international normalized ratio (INR). The etiology

1	with liver dysfunction are thrombocytopenia and impaired humoral coagulation function manifested as prolonga-tion of the prothrombin time and international normalized ratio (INR). The etiology of thrombocytopenia in patients with liver disease is typically related to hypersplenism, reduced produc-tion of thrombopoietin, and immune-mediated destruction of platelets. The total body platelet mass is often normal in patients with hypersplenism, but a much larger fraction of the platelets is sequestered in the enlarged spleen. Bleeding may be less than anticipated because sequestered platelets can be mobilized to some extent and enter the circulation. Thrombopoietin, the pri-mary stimulus for thrombopoiesis, may be responsible for some cases of thrombocytopenia in cirrhotic patients, although its role is not well delineated. Finally, immune-mediated thrombocyto-penia may also occur in cirrhotics, especially those with hepatitis C and primary biliary cirrhosis.33 In addition to

1	although its role is not well delineated. Finally, immune-mediated thrombocyto-penia may also occur in cirrhotics, especially those with hepatitis C and primary biliary cirrhosis.33 In addition to thrombocytope-nia, these patients also exhibit platelet dysfunction via defective interactions between platelets and the endothelium, and possibly due to uremia and changes in endothelial function in the setting of concomitant renal insufficiency. Hypocoagulopathy is fur-ther exacerbated with low platelet counts because platelets help facilitate thrombin generation by assembling coagulation factors on their surfaces. In conditions mimicking intravascular flow, low hematocrit and low platelet counts contributed to decreased adhesion of platelets to endothelial cells, although increased vWF, a common finding in cirrhotic patients, may offset this change in patients with cirrhosis.34 Hypercoagulability of liver disease has recently gained increased attention, with more evi-dence demonstrating

1	finding in cirrhotic patients, may offset this change in patients with cirrhosis.34 Hypercoagulability of liver disease has recently gained increased attention, with more evi-dence demonstrating the increased incidence of thromboem-bolism despite thrombocytopenia and a hypocoagulable state on conventional blood tests.35,36 This is attributed to decreased production of liver-synthesized proteins C and S, antithrombin, and plasminogen levels, as well as elevated levels of endothe-lial-derived vWF and factor VIII, a potent driver of thrombin generation.37,38 Given the concomitant hypoand hypercoagu-lable features seen in patients with liver disease, conventional coagulation tests may be difficult to interpret, and whole blood functional tests such as thromboelastography (TEG) or ROTEM may be more informative of the status of clot formation and stability in cirrhotic patients. Small studies have indicated that TEG provides a better assessment of bleeding risk than standard tests of

1	may be more informative of the status of clot formation and stability in cirrhotic patients. Small studies have indicated that TEG provides a better assessment of bleeding risk than standard tests of hemostasis in patients with liver disease; however, no large studies have directly tested this, and future larger trials are needed.39Before instituting any therapy to ameliorate thrombocy-topenia, the actual need for correction should be strongly con-sidered. In general, correction based solely on a low platelet count should be discouraged. Most often, treatment should be withheld for invasive procedures and surgery. When required, platelet transfusions are the mainstay of therapy; however, the effect typically lasts only several hours. Risks associated with transfusions in general and the development of antiplate-let antibodies in a patient population likely to need recurrent correction should be considered. A less well-accepted option is splenectomy or splenic embolization to reduce

1	development of antiplate-let antibodies in a patient population likely to need recurrent correction should be considered. A less well-accepted option is splenectomy or splenic embolization to reduce hypersplenism. In addition to the risks associated with these techniques, reduced splenic blood flow can reduce portal vein flow with subsequent portal vein thrombosis. Results are mixed following insertion of a transjugular intrahepatic portosystemic shunt (TIPS). There-fore, treatment of thrombocytopenia should not be the primary indication for a TIPS procedure.Decreased production or increased destruction of coagula-tion factors as well as vitamin K deficiency can all contribute to a prolonged PT and INR in patients with liver disease. As liver dysfunction worsens, so does the liver’s synthetic func-tion, which results in decreased production of coagulation fac-tors. Additionally, laboratory abnormalities may mimic those of DIC. Elevated D-dimers have been reported to increase the risk

1	func-tion, which results in decreased production of coagulation fac-tors. Additionally, laboratory abnormalities may mimic those of DIC. Elevated D-dimers have been reported to increase the risk of variceal bleeding. The absorption of vitamin K is dependent on bile production. Therefore, liver patients with impaired bile production and cholestatic disease may be at risk for vitamin K deficiency.Similar to thrombocytopenia, correction of coagulopathy should be reserved for treatment of active bleeding and prophy-laxis for invasive procedures and surgery. Treatment of coagu-lopathy caused by liver disease is usually done with FFP, but because the coagulopathy is usually not a result of decreased levels of factor V, complete correction is not usually possible. If the fibrinogen is less than 200 mg/dL, administration of cryo-precipitate may be helpful. Cryoprecipitate is also a source of factor VIII for the rare patient with a low factor VIII level.Coagulopathy of TraumaTraditional

1	than 200 mg/dL, administration of cryo-precipitate may be helpful. Cryoprecipitate is also a source of factor VIII for the rare patient with a low factor VIII level.Coagulopathy of TraumaTraditional teaching regarding trauma-related coagulopathy attributed its development to acidosis, hypothermia, and dilution of coagulation factors. Recent data, however, have shown that over one-third of severely injured patients have laboratory-based evidence of coagulopathy at the time of admission,40 a phenotype called trauma-induced coagulopathy (TIC). TIC is independent of traditional (iatrogenic) causes of posttraumatic coagulopathy such as hemodilution, is precipitated by tissue injury and/or hemorrhagic shock, and is associated with signifi-cantly higher risk of mortality, especially in the first 24 hours after injury. Furthermore, TIC is a separate and distinct process from disseminated intravascular coagulopathy with its own specific components of hemostatic failure.As shown in Fig. 4-5,

1	24 hours after injury. Furthermore, TIC is a separate and distinct process from disseminated intravascular coagulopathy with its own specific components of hemostatic failure.As shown in Fig. 4-5, several non–mutually exclusive mechanisms have been proposed as the etiology of TIC,41 includ-ing activated protein C-mediated clotting factor deactivation,42 endothelial injury and “auto-heparinization” due to shedding of endothelial heparin sulfate and chondroitin sulfate into the circulation,43 platelet dysfunction,44 and hyperfibrinolysis.45 Hemorrhagic shock was previously thought to be an essential component of TIC, but isolated traumatic brain injury46 and pulmonary contusions47 have been shown to induce laboratory-defined TIC in the absence of shock, possibly due to a high pro-portion of endothelium in these organs. Traumatic brain injury may also induce TIC via a consumptive mechanism by the release of large amounts of tissue factor into the circulation.48 2Table 4-3Coagulation

1	of endothelium in these organs. Traumatic brain injury may also induce TIC via a consumptive mechanism by the release of large amounts of tissue factor into the circulation.48 2Table 4-3Coagulation factors synthesized by the liverVitamin K–dependent factors: II (prothrombin factor), VII, IX, XFibrinogenFactor VFactor VIIIFactors XI, XII, XIIIAntithrombin IIIPlasminogenProtein C and protein SBrunicardi_Ch04_p0103-p0130.indd 11129/01/19 11:05 AM 112BASIC CONSIDERATIONSPART IHowever, the relationship between laboratory-based coagula-tion abnormalities and true clinically evident coagulopathic bleeding is unclear. With the widespread application of damage control resuscitation, the frequency of clinical coagulopathy has decreased.Interestingly, the converse of hyperfibrinolysis, known as fibrinolytic shutdown, has also been associated with increased mortality after trauma.49 In a multicenter study of 2540 trauma patients, those with intermediate fibrinolytic activity (“physiologic,”

1	fibrinolytic shutdown, has also been associated with increased mortality after trauma.49 In a multicenter study of 2540 trauma patients, those with intermediate fibrinolytic activity (“physiologic,” 0.8% to 2.9% lysis) on admission had the lowest mortality (14%). Shutdown (<0.8% lysis) patients had increased mortality (22%), often due to late causes such as mul-tiple organ failure, while patients with hyperfibrinolysis (≥3% lysis) had the greatest mortality (34%) and most often died due to hemorrhage.50Acquired Coagulation InhibitorsAmong the most common acquired coagulation inhibitors is the antiphospholipid syndrome (APLS), which includes the lupus anticoagulant and anticardiolipin antibodies. These antibodies may be associated with either venous or arterial thrombosis, or both. In fact, patients presenting with recurrent thrombosis should be evaluated for APLS. Antiphospholipid antibodies are very common in patients with systemic lupus but may also be seen in association with

1	fact, patients presenting with recurrent thrombosis should be evaluated for APLS. Antiphospholipid antibodies are very common in patients with systemic lupus but may also be seen in association with rheumatoid arthritis and Sjögren’s syndrome. There are also individuals who will have no autoimmune disor-ders but develop transient antibodies in response to infections or those who develop drug-induced APLS. The hallmark of APLS is a prolonged aPTT in vitro but an increased risk of thrombosis in vivo.Anticoagulation and BleedingSpontaneous bleeding can be a complication of any antico-agulant therapy whether it is heparin, low molecular weight heparins, warfarin, factor Xa inhibitors, or new direct thrombin inhibitors. The risk of spontaneous bleeding related to heparin is reduced with a continuous infusion technique. Therapeutic anticoagulation is more reliably achieved with a low molecu-lar weight heparin. However, laboratory testing is more chal-lenging with these medications, as they

1	infusion technique. Therapeutic anticoagulation is more reliably achieved with a low molecu-lar weight heparin. However, laboratory testing is more chal-lenging with these medications, as they are not detected with conventional coagulation testing. However, their more reli-able therapeutic levels (compared to heparin) make them an attractive option for outpatient anticoagulation and more costeffective for the inpatient setting. If monitoring is required (e.g., in the presence of renal insufficiency or severe obesity), the drug effect should be determined with an assay for anti-Xa activity.Warfarin is used for long-term anticoagulation in various clinical conditions, including deep vein thrombosis, pulmonary embolism, valvular heart disease, atrial fibrillation, recurrent systemic emboli, recurrent myocardial infarction, prosthetic heart valves, and prosthetic implants. Due to the interaction of the P450 system, the anticoagulant effect of the warfarin is reduced (e.g., increased dose

1	recurrent myocardial infarction, prosthetic heart valves, and prosthetic implants. Due to the interaction of the P450 system, the anticoagulant effect of the warfarin is reduced (e.g., increased dose required) in patients receiving barbiturates as well as in patients with diets low in vitamin K. Increased warfarin requirements may also be needed in patients taking contraceptives or estrogen-containing compounds, corti-costeroids, and adrenocorticotropic hormone (ACTH). Medica-tions that can alter warfarin requirements are shown in Table 4-4.Although warfarin use is often associated with a signifi-cant increase in morbidity and mortality in acutely injured and emergency surgery patients, with rapid reversal, these com-plications can be reduced. There are several reversal options that include vitamin K administration, plasma, cryoprecipi-tate, recombinant factor VIIa, and factor concentrates. The 2012 CHEST guidelines for the Management of Anticoagulant Therapy Antithrombotic Therapy

1	vitamin K administration, plasma, cryoprecipi-tate, recombinant factor VIIa, and factor concentrates. The 2012 CHEST guidelines for the Management of Anticoagulant Therapy Antithrombotic Therapy and Prevention of Thrombo-sis recommends patients with major life-threatening bleeding TRAUMATICCLINICALCOAGULOPATHICBLEEDING?PlateletactivationEGLsheddingEndothelial activationAcidosisHypothermiaHemorrhageShock˜˜˜tPA &°PAI-1Hypo-perfusionClotting factorconsumptionPre-injuryanticoagulantmedicationIatrogenicresuscitationinjuryHemodilution˜Acidosis˜HypothermiaAuto-heparinization°Clotting factoractivityPlatelet dysfunctionHyperfibrinolysis˜Catecholamines˜APCFigure 4-5. Illustration of the pathophysiologic mechanism responsible for the acute coagulopathy of trauma. APC = activated protein C; EGL = endothelial glycocalyx; PAI-1 = plasminogen activator inhibitor 1; tPA = tissue plasminogen activator; TIC = trauma-induced coagulopathy. (Reproduced with permission from Chang R, Cardenas JC, Wade CE,

1	glycocalyx; PAI-1 = plasminogen activator inhibitor 1; tPA = tissue plasminogen activator; TIC = trauma-induced coagulopathy. (Reproduced with permission from Chang R, Cardenas JC, Wade CE, et al: Advances in the understanding of trauma-induced coagulopathy. Blood. 2016 Aug 25;128(8):1043-1049.)Table 4-4Medications that can alter warfarin dosing↓ warfarin effect↑ warfarin requirementsBarbiturates, oral contraceptives, estrogen-containing compounds, corticosteroids, adrenocorticotropic hormone↑ warfarin effect↓ warfarin requirementsPhenylbutazone, clofibrate, anabolic steroids, L-thyroxine, glucagons, amiodarone, quinidine, cephalosporinsBrunicardi_Ch04_p0103-p0130.indd 11229/01/19 11:05 AM 113HEMOSTASIS, SURGICAL BLEEDING, AND TRANSFUSIONCHAPTER 4due to warfarin receive reversal with vitamin K and a rapid reversal agent such as plasma or prothrombin complex concen-trate (PCC).51 Vitamin K is given to sustain the effects of the plasma or PCC due to their short half-lives. In major

1	vitamin K and a rapid reversal agent such as plasma or prothrombin complex concen-trate (PCC).51 Vitamin K is given to sustain the effects of the plasma or PCC due to their short half-lives. In major bleeds, Vitamin K 10 mg given as a slow IV infusion is utilized for more rapid onset compared to the oral form. Studies have shown that PCC is superior to plasma for speed of reversal and has decreased risk of fluid overload, but it is equivalent in adverse and thromboembolic events and costlier.52,53 Prothrombin com-plex concentrate is available in two forms: three-factor PCC (factors II, IX, and X) and four-factor PCC (factors II, VII, IX, and X). Four-factor PCCs have been shown to have a more reli-able correction of INR compared to three-factor PCCs.54,55Direct oral anticoagulants (DOACs) include direct throm-bin inhibitors and factor Xa inhibitors and have no readily available method of detection of the degree of anticoagula-tion. More concerning is the difficulty in the reversal of

1	include direct throm-bin inhibitors and factor Xa inhibitors and have no readily available method of detection of the degree of anticoagula-tion. More concerning is the difficulty in the reversal of these new anticoagulants. Recently, idarucizumab, a humanized monoclonal antibody fragment that binds dabigatran, has been approved for use for reversal of the thrombin inhibitor, dabiga-tran, and dabigatran-related coagulopathy. Clinical studies have demonstrated normalization of laboratory tests.56,57Factor Xa inhibitors such as rivaroxaban, apixaban, and edoxaban currently lack a specific antidote. Two novel anti-dotes, andexanet alfa and ciraparantag (PER977), are currently undergoing clinical trials. Andexanet alfa is a recombinant human FXa variant,58,59 and ciraparantag is a cationic small molecule.60 These are both being evaluated for reversal of the factor Xa inhibitors. Until these agents are approved, attempts to reverse Factor Xa inhibitors should include four factor PCCs.61 In

1	molecule.60 These are both being evaluated for reversal of the factor Xa inhibitors. Until these agents are approved, attempts to reverse Factor Xa inhibitors should include four factor PCCs.61 In less urgent states, these drugs can be held for 36 to 48 hours prior to surgery without increased risk of bleeding in those with normal renal function. Alternatively, activated clotting time (stand alone or with rapid TEG) or ecarin clotting time can be obtained in those on dabigatran, and anti-factor Xa assays can be obtained in those taking rivaroxaban.Bleeding complications in patients on anticoagulants include hematuria, soft tissue bleeding, intracerebral bleeding, skin necrosis, and abdominal bleeding. Bleeding secondary to anticoagulation therapy is also a common cause of rectus sheath hematomas.Surgical intervention may prove necessary in patients receiving anticoagulation therapy. Increasing experience suggests that surgical treatment can be undertaken without full reversal of the

1	intervention may prove necessary in patients receiving anticoagulation therapy. Increasing experience suggests that surgical treatment can be undertaken without full reversal of the anticoagulant, depending on the procedure being performed.62 When the aPTT is less than 1.3 times control in a heparinized patient, or when the INR is less than 1.5 in a patient on warfarin, reversal of anticoagulation therapy may not be necessary. How-ever, meticulous surgical technique is mandatory, and the patient must be observed closely throughout the postoperative period.Certain surgical procedures should not be performed in concert with anticoagulation. In particular, cases where even minor bleeding can cause great morbidity, such as the central nervous system and the eye, surgery should be avoided. Emer-gency operations are occasionally necessary in patients who have been heparinized. The first step in these patients is to dis-continue heparin. For more rapid reversal, protamine sulfate is

1	Emer-gency operations are occasionally necessary in patients who have been heparinized. The first step in these patients is to dis-continue heparin. For more rapid reversal, protamine sulfate is effective. However, significant adverse reactions, especially in patients with severe fish allergies, may be encountered when administering protamine.63 Symptoms include hypotension, flushing, bradycardia, nausea, and vomiting. Prolongation of the aPTT after heparin neutralization with protamine may also be a result of the anticoagulant effect of protamine. In the elective surgical patient who is receiving warfarin-derivative therapy sufficient to effect anticoagulation, the drug can be discontinued several days before operation and the prothrombin concentration then checked.64 Rapid reversal of anticoagulation can be accom-plished with plasma or prothrombin complex concentrates in the emergent situation. An example of a warfarin reversal guideline using four-factor prothrombin complex

1	of anticoagulation can be accom-plished with plasma or prothrombin complex concentrates in the emergent situation. An example of a warfarin reversal guideline using four-factor prothrombin complex concentrate for patients with major or life-threatening bleeding or intracranial bleeding is shown in Fig. 4-6. Parenteral administration of vitamin K also is indicated in elective surgical treatment of patients with biliary obstruction or malabsorption who may be vitamin K deficient. However, if low levels of factors II, VII, IX, and X (vitamin K–dependent factors) exist as a result of hepatocellular dysfunc-tion, vitamin K administration is ineffective.The perioperative management of patients receiving long-term oral anticoagulation therapy is an increasingly common problem. The American College of Chest Physicians Evidence-Based Clinical Practice Guidelines from 2012 suggests periopera-tive “bridging” of anticoagulation.65 However, recent studies have found an increased risk of major

1	of Chest Physicians Evidence-Based Clinical Practice Guidelines from 2012 suggests periopera-tive “bridging” of anticoagulation.65 However, recent studies have found an increased risk of major bleeding without a change in thromboembolism rate when comparing bridging to no-bridg-ing for elective operations or procedures.66 Additional clinical trials are currently underway, but at the current time, physicians should carefully balance risks of bleeding vs. venous thromboembolism risks for individual patients when deciding on bridging of anticoagulation for procedures.67 For patients in whom the risk of venous thromboembolism out-weighs the risk of bleeding, a heparin infusion should be held for 4 to 6 hours before the procedure and restarted within 12 to 24 hours of the end of its completion. High-risk indications include mechanical heart valves, recent (within 30 days) myo-cardial infarction, stroke, or pulmonary embolism. Lower risk indications, such as thromboembolic events greater

1	High-risk indications include mechanical heart valves, recent (within 30 days) myo-cardial infarction, stroke, or pulmonary embolism. Lower risk indications, such as thromboembolic events greater than 30 days prior, hypercoagulable history, and atrial fibrillation, do not require such stringent restarting strategies.Cardiopulmonary Bypass. Under normal conditions, homeo-stasis of the coagulation system is maintained by complex inter-actions between the endothelium, platelets, and coagulation factors. In patients undergoing cardiopulmonary bypass (CPB), contact with circuit tubing and membranes results in abnormal platelet and clotting factor activation, as well as activation of inflammatory cascades, that ultimately results in excessive fibri-nolysis and a combination of both quantitative and qualitative platelet defects. Platelets undergo reversible alterations in mor-phology and their ability to aggregate, which causes sequestra-tion in the filter, partially degranulated platelets,

1	and qualitative platelet defects. Platelets undergo reversible alterations in mor-phology and their ability to aggregate, which causes sequestra-tion in the filter, partially degranulated platelets, and platelet fragments. This multifactorial coagulopathy is compounded by the effects of shear stress in the system, induced hypothermia, hemodilution, and anticoagulation.68While on pump, activated clotting time measurements are obtained along with blood gas measurements; however, con-ventional coagulation assays and platelet counts are not nor-mally performed until rewarming and after a standard dose of protamine has been given. TEG may give a better estimate of the extent of coagulopathy and may also be used to anticipate transfusion requirements if bleeding is present.68Empiric treatment with FFP and cryoprecipitate is often used for bleeding patients; however, there are no universally accepted transfusion thresholds. Platelet concentrates are given for bleeding patients in the

1	with FFP and cryoprecipitate is often used for bleeding patients; however, there are no universally accepted transfusion thresholds. Platelet concentrates are given for bleeding patients in the immediate postoperative period; however, studies have shown that indiscriminate plate-let therapy conferred no therapeutic advantage.69 It is in these 34Brunicardi_Ch04_p0103-p0130.indd 11329/01/19 11:05 AM 114BASIC CONSIDERATIONSPART Ipatients where rapid coagulation testing is required to assist with directed transfusion therapy.70 Many institutions now use antifibrinolytics, such as ε-aminocaproic acid and tranexamic acid, at the time of anesthesia induction after several studies have shown that such treatment reduced postoperative bleed-ing and reoperation. Aprotinin, a protease inhibitor that acts as an antifibrinolytic agent, has been shown to reduce transfusion requirements associated with cardiac surgery.71 Desmopressin acetate stimulates release of factor VIII from endothelial

1	that acts as an antifibrinolytic agent, has been shown to reduce transfusion requirements associated with cardiac surgery.71 Desmopressin acetate stimulates release of factor VIII from endothelial cells and may also be effective in reducing blood loss during cardiac surgery.Local Hemostasis. Significant surgical bleeding is usually caused by ineffective local hemostasis. The goal is therefore to prevent further blood loss from a disrupted vessel that has been incised or transected. Hemostasis may be accomplished by interrupting the flow of blood to the involved area or by direct closure of the blood vessel wall defect.Mechanical Procedures. The oldest mechanical method of bleeding cessation is application of direct digital pressure, either at the site of bleeding or proximally to permit more definitive action. An extremity tourniquet that occludes a major vessel proximal to the bleeding site or the Pringle maneuver for liver bleeding are good examples. Direct digital pressure is very

1	definitive action. An extremity tourniquet that occludes a major vessel proximal to the bleeding site or the Pringle maneuver for liver bleeding are good examples. Direct digital pressure is very effective and has the advantage of being less traumatic than hemostatic or even “atraumatic” clamps.When a small vessel is transected, a simple ligature is usu-ally sufficient. However, for larger pulsating arteries, a transfix-ion suture to prevent slipping is indicated. All sutures represent foreign material, and selection should be based on their intrinsic characteristics and the state of the wound. Direct pressure applied by “packing” a wound with gauze or laparotomy pads affords the best method of controlling diffuse bleeding from large areas, such as in the trauma situation. Packing bone wax on the raw surface to effect pressure can control bleeding from cut bone.Thermal Agents. Heat achieves hemostasis by denaturation of protein that results in coagulation of large areas of tissue.

1	wax on the raw surface to effect pressure can control bleeding from cut bone.Thermal Agents. Heat achieves hemostasis by denaturation of protein that results in coagulation of large areas of tissue. Elec-trocautery generates heat by induction from an alternating cur-rent source, which is then transmitted via conduction from the instrument directly to the tissue. The amplitude setting should be high enough to produce prompt coagulation, but not so high as to set up an arc between the tissue and the cautery tip. This avoids thermal injury outside of the operative field and also prevents exit of current through electrocardiographic leads, other moni-toring devices, or permanent pacemakers or defibrillators. A negative grounding plate should be placed beneath the patient to avoid severe skin burns, and caution should be used with certain Major bleeding/life-threatening bleeding orintracranial hemorrhage (lCH)Administer phytonadione 10 mg IV immediatelyCheck PT/INR, PTT, fibrinogen,

1	skin burns, and caution should be used with certain Major bleeding/life-threatening bleeding orintracranial hemorrhage (lCH)Administer phytonadione 10 mg IV immediatelyCheck PT/INR, PTT, fibrinogen, platelets, Hgb/HctEvaluate for relative contraindications to PCC4 (Kcentra®)• Thrombotic event in the past 3 months – myocardial infarction,stroke, pulmonary embolism, deep vein thrombosis• Very high risk of thrombosis, such as patients with clinical orlaboratory evidence of overt disseminated intravascularcoagulopathy, heparin-induced thrombocytopenia (HIT), high-riskthrombophilia, or antiphospholipid syndromeCheck PT/INR 1 hr, 6 hrs, and 24 hrs after completion of Kcentra®• If INR >1.5 at 1 hr, consider switching to FFP therapy• If INR >1.5 at 6 hrs, repeat phytonadione 10 mg IV over 30 min• If INR >1.5 at 24 hrs, repeat phytonadione 10 mg IV over 30 minINR 1.5–3.9Kcentra®25 units/kg(maximum 2500 units)INR 4–6Kcentra®35 units/kg(maximum 3500 units)INR >6Kcentra®50 units/kg(maximum 5000

1	If INR >1.5 at 24 hrs, repeat phytonadione 10 mg IV over 30 minINR 1.5–3.9Kcentra®25 units/kg(maximum 2500 units)INR 4–6Kcentra®35 units/kg(maximum 3500 units)INR >6Kcentra®50 units/kg(maximum 5000 units)Administer FFPYesNoContraindication toKcentra®Figure 4-6. Example of a warfarin reversal guideline using four-factor prothrombin complex concentrate for patients with major or life-threatening bleeding or intracranial bleeding.Brunicardi_Ch04_p0103-p0130.indd 11429/01/19 11:05 AM 115HEMOSTASIS, SURGICAL BLEEDING, AND TRANSFUSIONCHAPTER 4anesthetic agents (diethyl ether, divinyl ether, ethyl chloride, ethylene, and cyclopropane) because of the hazard of explosion.A direct current also can result in hemostasis. Because the protein moieties and cellular elements of blood have a nega-tive surface charge, they are attracted to a positive pole where a thrombus is formed. Direct currents in the 20to 100-mA range have successfully controlled diffuse bleeding from raw surfaces, as has

1	surface charge, they are attracted to a positive pole where a thrombus is formed. Direct currents in the 20to 100-mA range have successfully controlled diffuse bleeding from raw surfaces, as has argon gas.Topical Hemostatic AgentsTopical hemostatic agents can play an important role in help-ing to facilitate surgical hemostasis. These agents are classified based on their mechanism of action, and many act at specific stages in the coagulation cascade and take advantage of natural physiologic responses to bleeding.72 The ideal topical hemo-static agent has significant hemostatic action, minimal tissue reactivity, nonantigenicity, in vivo biodegradability, ease of sterilization, low cost, and can be tailored to specific needs.73Achneck et al have published a comprehensive overview of absorbable, biologic, and synthetic agents.74 Absorbable agents include gelatin foams (Gelfoam), oxidized cellulose (Surgicel), and microfibrillar collagens (Avitene). Both gelatin foam and oxidized cellulose

1	biologic, and synthetic agents.74 Absorbable agents include gelatin foams (Gelfoam), oxidized cellulose (Surgicel), and microfibrillar collagens (Avitene). Both gelatin foam and oxidized cellulose provide a physical matrix for clotting initia-tion, while microfibrillar collagens facilitate platelet adherence and activation. Biologic agents include topical thrombin, fibrin sealants (FloSeal), and platelet sealants (Vitagel). Human or recombinant thrombin derivatives, which facilitate the forma-tion of fibrin clots and subsequent activation of several clotting factors, take advantage of natural physiologic processes, thereby avoiding foreign body or inflammatory reactions.73 Caution must be taken in judging vessel caliber in the wound because thrombin entry into larger caliber vessels can result in systemic exposure to thrombin with a risk of disseminated intravascular clotting or death. They are particularly effective in controlling capillary bed bleeding when pressure or ligation is

1	result in systemic exposure to thrombin with a risk of disseminated intravascular clotting or death. They are particularly effective in controlling capillary bed bleeding when pressure or ligation is insufficient; however, the bovine derivatives should be used with caution due to the potential immunologic response and worsened coagulopathy. Fibrin sealants are prepared from cryoprecipitate (homologous or synthetic) and have the advantage of not promoting inflam-mation or tissue necrosis.75 A recent study by Koea et al dem-onstrated in a prospective multicenter randomized trial that a fibrin sealant patch was safe and highly effective in controlling parenchymal bleeding following hepatectomy regardless of the type of resection.76 Platelet sealants are a mixture of collagen and thrombin combined with plasma-derived fibrinogen and platelets from the patient, which requires the additional need for centrifugation and processing.Topical agents are not a substitute for meticulous surgical

1	with plasma-derived fibrinogen and platelets from the patient, which requires the additional need for centrifugation and processing.Topical agents are not a substitute for meticulous surgical technique and only function as adjuncts to help facilitate surgi-cal hemostasis. The advantages and disadvantages of each agent must be considered, and use should be limited to the minimum amount necessary to minimize toxicity, adverse reactions, inter-ference with wound healing, and procedural costs.TRANSFUSIONBackgroundHuman blood replacement therapy was accepted in the late nine-teenth century. This was followed by the introduction of blood grouping by Landsteiner who identified the major A, B, and O groups in 1900, resulting in widespread use of blood products in World War I. Levine and Stetson in 1939 followed with the concept of Rh grouping. These and other breakthroughs in blood product preservation, storage, and infectious disease screening established the foundation from which the field

1	1939 followed with the concept of Rh grouping. These and other breakthroughs in blood product preservation, storage, and infectious disease screening established the foundation from which the field of transfusion medicine has grown. Whole blood was considered the standard in transfusion until the late 1970s when component therapy began to take prominence. This change in practice was driven in part by blood bank economics, increasing the availability of specific blood products for patients with isolated deficien-cies (anemia, thrombocytopenia, clotting factor deficiencies), especially those associated with chemotherapy.77 However, this change occurred for all patients simultaneously without studies to identify the clinical ramifications in different patient popu-lations requiring blood product therapy. Interestingly, whole blood transfusion (the ultimate balanced transfusion product) is making a resurgence in both military and civilian practice for resuscitation of acute

1	blood product therapy. Interestingly, whole blood transfusion (the ultimate balanced transfusion product) is making a resurgence in both military and civilian practice for resuscitation of acute hemorrhage.Replacement TherapyTyping and Crossmatching. Serologic compatibility for A, B, O, and Rh groups is established routinely. Crossmatching between the donors’ red blood cells and the recipients’ sera (the major crossmatch) is performed. Rh-negative recipients should be transfused only with Rh-negative red blood cells. However, this group represents only 15% of the population. Therefore, the administration of Rh-positive red blood cells is acceptable if Rh-negative red blood cells blood is not available. However, Rh-positive red blood cells should not be transfused to Rhnegative females who are of childbearing age.In emergency situations, universal donor type O-negative red blood cells and type AB plasma may be transfused to all recip-ients. Platelets do not require crossmatching. Due

1	are of childbearing age.In emergency situations, universal donor type O-negative red blood cells and type AB plasma may be transfused to all recip-ients. Platelets do not require crossmatching. Due to a shortage of type AB plasma, low anti-B titer type A plasma has become widely adopted for emergency (uncrossmatched) transfusion.78 In the United States, 85% of individuals are type A or type O, mak-ing type A plasma compatible with the vast majority of poten-tial recipients. Uncrossmatched plasma is routinely transfused as part of platelet transfusions, with major transfusion reactions reported rarely,79 and type AB plasma currently carries a higher risk of TRALI compared to other plasma types.80 Many cen-ters have transitioned to low titer type A plasma for emergency transfusions, with no increase in adverse events.81 O negative and type-specific red blood cells are equally safe for emergency transfusion. In patients known to have clinically significant cold agglutinins, blood should

1	increase in adverse events.81 O negative and type-specific red blood cells are equally safe for emergency transfusion. In patients known to have clinically significant cold agglutinins, blood should be administered through a blood warmer. If these antibodies are present in high titer, hypother-mia is contraindicated.In patients who have been multiply transfused and who have developed alloantibodies or who have autoimmune hemo-lytic anemia with pan-red blood cell antibodies, typing and crossmatching is often difficult, and sufficient time should be allotted preoperatively to accumulate blood that might be required during the operation. Crossmatching should always be performed before the administration of dextran because it inter-feres with the typing procedure.81aBanked Whole Blood. Interest in whole blood as an ideal therapy for acute traumatic hemorrhagic shock has increased in the last several years with multiple reports of successful use in military and civilian trauma patients.

1	in whole blood as an ideal therapy for acute traumatic hemorrhagic shock has increased in the last several years with multiple reports of successful use in military and civilian trauma patients. However, there is still limited access in most civilian centers.Red Blood Cells and Frozen Red Blood Cells. Red blood cells are the traditional product of choice for most clinical Brunicardi_Ch04_p0103-p0130.indd 11529/01/19 11:05 AM 116BASIC CONSIDERATIONSPART Isituations requiring resuscitation, although deficits in oxygen delivery are rarely related to inadequate red cells. Concentrated suspensions of red blood cells can be prepared by removing most of the supernatant plasma after centrifugation. The prepa-ration reduces but does not eliminate reactions caused by plasma components. With sequential changes in storage solutions, the shelf life of red blood cells is now 42 days. However, recent evidence has demonstrated that the age of red cells may play a significant role in the

1	With sequential changes in storage solutions, the shelf life of red blood cells is now 42 days. However, recent evidence has demonstrated that the age of red cells may play a significant role in the inflammatory response and incidence of multiple organ failure.82 The changes in the red blood cells that occur during storage include reduction of intracellular ADP and 2,3-diphosphoglycerate (2,3-DPG), which alters the oxygen dissociation curve of hemoglobin, resulting in a decrease in oxy-gen transport. Stored RBCs progressively become acidotic with elevated levels of lactate, potassium, and ammonia. Addition-ally, the in vitro hemostatic potential of plasma83 and platelet84 products also decrease with storage.The morphologic and biochemical changes that occur over time in red cells may contribute to worsened outcomes. This limits the ability to bank large amounts of blood, particu-larly rarer blood types, for use in times of high demand and blood supply shortage, such as on the

1	may contribute to worsened outcomes. This limits the ability to bank large amounts of blood, particu-larly rarer blood types, for use in times of high demand and blood supply shortage, such as on the battlefield and after mass casualty events. Storage solutions, however, do not fully sup-press the metabolic and physical changes associated with aging RBCs. Newer evidence suggests that cryopreservation of red blood cells may provide a safe alternative means of storage. Cryopreservation uses the beneficial effects of ultra-low tem-peratures to suppress molecular motion and arrest metabolic and biochemical reactions. Frozen (cryopreserved) red blood cells have a shelf life of ten years at -80°C with improved cel-lular viability and maintenance of ATP and 2,3 DPG concen-trations.85 A trial of stable trauma patients randomized to old (>14 storage days) red blood cells, young (≤14 storage days) red blood cells, and cryopreserved red blood cells found that cryopreserved red blood cells were

1	stable trauma patients randomized to old (>14 storage days) red blood cells, young (≤14 storage days) red blood cells, and cryopreserved red blood cells found that cryopreserved red blood cells were as safe and effective as stan-dard red blood cells.85 Cryopreserved red blood cells required a thawing and preparation period of about 90 minutes, limiting immediate availability for emergency use. A recent study sug-gests that the post-thaw characteristics of cryopreserved units may not, however, be comparable to fresh red cells.86 Additional research needs to be done to optimize the process, but frozen cells likely represent a viable option for storage in the future.Leukocyte-Reduced and Leukocyte-Reduced/Washed Red Blood Cells. These products are prepared by filtration that removes about 99.9% of the white blood cells and most of the platelets (leukocyte-reduced red blood cells) and, if necessary, by additional saline washing (leukocyte-reduced/washed red blood cells). Leukocyte

1	99.9% of the white blood cells and most of the platelets (leukocyte-reduced red blood cells) and, if necessary, by additional saline washing (leukocyte-reduced/washed red blood cells). Leukocyte reduction prevents almost all febrile, nonhemolytic transfusion reactions (fever and/or rigors), allo-immunization to HLA class I antigens, and platelet transfu-sion refractoriness and cytomegalovirus transmission. In most Western nations, it is the standard red blood cell transfusion product. Supporters of universal leukocyte reduction argue that allogenic transfusion of white cells predisposes to postoperative bacterial infection and multiorgan failure. Reviews of random-ized trials and meta-analyses have not provided convincing evi-dence either way,87,88 although a large Canadian retrospective study suggests a decrease in mortality and infections.89Platelet Concentrates. The indications for platelet transfu-sion include thrombocytopenia caused by massive blood loss and replacement with

1	study suggests a decrease in mortality and infections.89Platelet Concentrates. The indications for platelet transfu-sion include thrombocytopenia caused by massive blood loss and replacement with platelet-poor products, thrombocytopenia caused by inadequate production, and qualitative platelet dis-orders. Platelets are stored at room temperature under constant agitation to prevent clumping and have a shelf life of 5 days from time of donation due to risk of bacterial overgrowth. One unit of platelet concentrate has a volume of approximately 50 mL. Platelet preparations are capable of transmitting infec-tious diseases and can account for allergic reactions similar to those caused by red blood cell transfusion. A therapeutic level of platelets is in the range of 50,000 to 100,000/μL, but is very dependent on the clinical situation. Recent evidence suggests that earlier use of platelets may improve outcomes in bleeding patients.90In rare cases, in patients who become alloimmunized

1	but is very dependent on the clinical situation. Recent evidence suggests that earlier use of platelets may improve outcomes in bleeding patients.90In rare cases, in patients who become alloimmunized through previous transfusion or patients who are refractory from sensitization through prior pregnancies, HLA-matched platelets can be used.Plasma. Plasma is the usual source of the vitamin K–dependent factors, the only source of factor V, and carries similar infectious risks as other component therapies. Several plasma products are available. Fresh frozen plasma (FFP) is frozen within hours of donation and can be stored for up to two years at -18°C, but requires 20 to 30 minutes to thaw prior to use, limiting immedi-ate availability. Thawed FFP can be relabeled as thawed plasma, which is immediately transfusable and can be stored for up to 5 days at 2° to 4°C. Liquid plasma is never frozen and can be stored for up to 26 days at 2° to 4°C. In vitro studies demonstrate that liquid plasma

1	transfusable and can be stored for up to 5 days at 2° to 4°C. Liquid plasma is never frozen and can be stored for up to 26 days at 2° to 4°C. In vitro studies demonstrate that liquid plasma has a better hemostatic profile than thawed plasma.91 Freeze-dried (lyophilized) plasma (FDP) has been recently “rediscovered” as an ideal resuscitation product for patients in remote and austere environments. FDP is distributed as a powder that is shelf-stable for up to 2 years at room tem-perature and relatively stable at temperature extremes.92 It was used extensively as a primary resuscitation fluid during World War II, but production was stopped due to risk of disease trans-mission. FDP is currently manufactured by updated processes in France, Germany, and South Africa. Several noncomparative studies in the literature have documented its ease of use, rapid reconstitution within minutes, clinical efficacy similar to other plasma products, and lack of apparent adverse events.93,94 The Israeli

1	in the literature have documented its ease of use, rapid reconstitution within minutes, clinical efficacy similar to other plasma products, and lack of apparent adverse events.93,94 The Israeli Defense Force has reported successful use of FDP at the point of injury,95 just as it was used in World War II. Beside limited use by U.S. Special Forces under the U.S. Federal Drug Administration’s (FDA) Investigational New Drug (IND) pro-gram, no FDP product is currently approved for general use in the United States. These products have the advantage of being pathogen reduced, have expanded storage capabilities, and can be quickly reconstituted.96Tranexamic Acid. Tranexamic acid (TXA; trade name: Cyk-lokapron) is an antifibrinolytic that inhibits both plasminogen activation and plasmin activity, thus preventing clot breakdown rather than promoting new clot formation. It occupies the lysine-binding sites on plasminogen, thus preventing its binding to lysine residues on fibrin. This reduces

1	thus preventing clot breakdown rather than promoting new clot formation. It occupies the lysine-binding sites on plasminogen, thus preventing its binding to lysine residues on fibrin. This reduces plasminogen activation to plasmin. Similarly, blockade of lysine-binding sites on circu-lating plasmin prevents binding to fibrin and thus prevents clot breakdown. TXA is 10 times more potent in vitro than aminoca-proic acid. At therapeutically relevant concentrations, TXA does not affect platelet count or aggregation or coagulation param-eters. It is excreted largely unchanged in urine and has a half-life of about 2 hours in circulation. It has been used to decrease bleeding and the need for blood transfusions in coronary artery Brunicardi_Ch04_p0103-p0130.indd 11629/01/19 11:05 AM 117HEMOSTASIS, SURGICAL BLEEDING, AND TRANSFUSIONCHAPTER 4bypass grafting (CABG), orthotopic liver transplantation, hip and knee arthroplasty, and other surgical settings. TXA has been used to treat injured

1	SURGICAL BLEEDING, AND TRANSFUSIONCHAPTER 4bypass grafting (CABG), orthotopic liver transplantation, hip and knee arthroplasty, and other surgical settings. TXA has been used to treat injured patients in both civilian and military settings.97,98 A recent practice guideline by the Eastern Associa-tion for the Surgery of Trauma (EAST) conditionally recom-mended the use of tranexamic acid as a hemostatic adjunct in severely injured patients when used early after injury.99 There is some controversy if its use should be empiric in patients with hemorrhage or based on documented hyperfibrinolysis. Results of prehospital studies with TXA are not yet available, but there are five ongoing trials. The true risk of venous thrombotic events is also not well established. Therefore, tranexamic acid should not be used with active intravascular clotting and should not be given with activated prothrombin complex concentrate or factor IX complex concentrates.Indications for Replacement of Blood and

1	not be used with active intravascular clotting and should not be given with activated prothrombin complex concentrate or factor IX complex concentrates.Indications for Replacement of Blood and Its ElementsImprovement in Oxygen-Carrying Capacity. Oxygencarrying capacity is primarily a function of the red blood cells. Thus, transfusion of red blood cells should augment oxygen-carrying capacity. Additionally, hemoglobin is fundamental to arterial oxygen content and thus oxygen delivery. Despite this obvious association, there is little evidence that actually sup-ports the premise that transfusion of red blood cells equates with enhanced cellular delivery and utilization. The reasons for this apparent discrepancy are related to changes that occur with stor-age of blood. The decrease in 2,3-DPG and P50 impair oxygen offloading, and deformation of the red cells impairs microcir-culatory perfusion.100Treatment of Anemia: Transfusion Triggers. The concept of transfusion triggers refers

1	2,3-DPG and P50 impair oxygen offloading, and deformation of the red cells impairs microcir-culatory perfusion.100Treatment of Anemia: Transfusion Triggers. The concept of transfusion triggers refers primarily to the nonactively bleed-ing ICU patient. A 1988 National Institutes of Health Consensus Report challenged the dictum that a hemoglobin value of less than 10 g/dL or a hematocrit level less than 30% indicates a need for preoperative red blood cell transfusion. This was veri-fied in a prospective randomized controlled trial in critically ill patients that compared a restrictive transfusion threshold to a more liberal strategy and demonstrated that maintaining hemo-globin levels between 7 and 9 g/dL had no adverse effect on mortality. In fact, patients with APACHE II scores of ≤20 or patients age <55 years actually had a lower mortality.101One unresolved issue related to transfusion triggers is the safety of maintaining a hemoglobin of 7 g/dL in a patient with ischemic heart

1	or patients age <55 years actually had a lower mortality.101One unresolved issue related to transfusion triggers is the safety of maintaining a hemoglobin of 7 g/dL in a patient with ischemic heart disease. Data on this subject are mixed, and many studies have significant design flaws, including their ret-rospective nature. However, the majority of the published data favors a restrictive transfusion trigger for patients with non–ST-elevation acute coronary syndrome, with many reporting worse outcomes in those patients receiving transfusions.102,103 Recent guidelines from the American Association of Blood Banks (AABB) recommend a minimum threshold of 7 g/dL for hemodynamically stable patients and 8 g/dL for patients under-going cardiac surgery, orthopedic surgery, and those with pre-existing cardiovascular disease.104 However, both the SCCM/EAST and AABB guidelines recommend taking into account patient-specific characteristics and the overall clinical context when considering RBC

1	cardiovascular disease.104 However, both the SCCM/EAST and AABB guidelines recommend taking into account patient-specific characteristics and the overall clinical context when considering RBC transfusions in non-acutely hemorrhag-ing patients. Patients with symptomatic anemia should be trans-fused one RBC unit at a time, and isolated asymptomatic anemia in and of itself is rarely an indication for RBC transfusion.Volume ReplacementThe most common indication for blood transfusion in surgical patients is the replenishment of the blood volume; however, the quantification of actual intravascular volume deficit is often difficult to accurately and quickly determine. Measure-ments of hemoglobin or hematocrit levels are frequently used to assess blood loss, but can be occasionally misleading in the face of acute loss.105 Both the amount and the rate of bleeding are factors in the development of signs and symptoms of blood loss.Loss of blood in the operating room can be roughly evalu-ated by

1	the face of acute loss.105 Both the amount and the rate of bleeding are factors in the development of signs and symptoms of blood loss.Loss of blood in the operating room can be roughly evalu-ated by estimating the amount of blood in the wound and on the drapes, weighing the sponges, and quantifying blood suctioned from the operative field. Significant blood loss will require a balanced resuscitation including red blood cells, FFP, and plate-lets (detailed later in this chapter) (Table 4-5).New Concepts in ResuscitationTraditional resuscitation algorithms were sequentially based on crystalloid followed by red blood cells and then plasma and platelet transfusions, and they have been in widespread use since the 1970s. No quality clinical data supported this concept. Recently the damage control resuscitation (DCR)105a strategy, with simultaneous measures to acquire mechanical hemorrhage control, has become the standard for treatment of substantial traumatic hemorrhage. DCR emphasizes

1	resuscitation (DCR)105a strategy, with simultaneous measures to acquire mechanical hemorrhage control, has become the standard for treatment of substantial traumatic hemorrhage. DCR emphasizes rapid maneuvers that promote hemostasis (balanced resuscitation with early delivery of plasma and platelets) while limiting iatrogenic insults that exacerbate bleeding (i.e., minimization of crystalloid and artifi-cial colloid, permissive hypotension), combined with multiple adjuncts for hemorrhage control.Rationale. In urban civilian trauma systems, nearly half of all deaths happen before a patient reaches the hospital.106 Patients who survive to an emergency center have a high incidence of truncal hemorrhage, and deaths in this group of patients may be potentially preventable. Truncal hemorrhage patients in shock often present with the early coagulopathy of trauma in the emer-gency department and are at significant risk of dying.107-109Many of these patients have suffered substantial bleeding,

1	in shock often present with the early coagulopathy of trauma in the emer-gency department and are at significant risk of dying.107-109Many of these patients have suffered substantial bleeding, generally defined as requiring the administration of ≥3 units of red blood cells within any hour of admission, and may have received a massive transfusion (MT), traditionally defined as ≥10 units of red blood cells in 24 hours.110 The traditional defi-nition is admittedly arbitrary and fails to identify many patients who truly receive large volume transfusions in a short period of time, further promoting survival bas. Newer definitions evaluating massive transfusion do so by taking into account both volume transfused as well as the rate at which transfu-sions are given. The critical administration threshold (CAT) has been prospectively validated and shown to be a superior predictor of mortality when compared to the conventional defi-nition of MT.110 By this measure, CAT-positive status is

1	threshold (CAT) has been prospectively validated and shown to be a superior predictor of mortality when compared to the conventional defi-nition of MT.110 By this measure, CAT-positive status is defined by transfusion of 3 units of red blood cells within a 60-minute period, and this is additive for each additional time this measure is reached. CAT-positive status is associated with a two-fold increase in risk of mortality. CAT is more sensitive than tra-ditional definitions of bleeding and allows for both earlier and more accurate identification of injured patients at greatest risk of death.Although 25% of all severely injured trauma admissions receive a unit of blood early after admission, only a small Brunicardi_Ch04_p0103-p0130.indd 11729/01/19 11:05 AM 118BASIC CONSIDERATIONSPART ITable 4-5Replacement of clotting factorsFACTORNORMAL LEVELLIFE SPAN IN VIVO (HALF-LIFE)FATE DURING COAGULATIONLEVEL REQUIRED FOR SAFE HEMOSTASISIDEAL AGENT ACD BANK BLOOD (4°C [39.2°F])IDEAL AGENT

1	4-5Replacement of clotting factorsFACTORNORMAL LEVELLIFE SPAN IN VIVO (HALF-LIFE)FATE DURING COAGULATIONLEVEL REQUIRED FOR SAFE HEMOSTASISIDEAL AGENT ACD BANK BLOOD (4°C [39.2°F])IDEAL AGENT FOR REPLACING DEFICITI (fibrinogen)200–400 mg/100 mL72 hConsumed60–100 mg/100 mLVery stableBank blood; concentrated fibrinogenII (prothrombin)20 mg/100 mL (100% of normal level)72 hConsumed15%–20%StableBank blood; concentrated preparationV (proaccelerin, accelerator globulin, labile factor)100% of normal level36 hConsumed5%–20%Labile (40% of normal level at 1 wk)Fresh frozen plasma; blood under 7 dVII (proconvertin, serum prothrombin conversion accelerator, stable factor)100% of normal level5 hSurvives5%–30%StableBank blood; concentrated preparationVIII (antihemophilic factor, antihemophilic globulin)100% of normal level (50%–150% of normal level)6–12 hConsumed30%Labile (20%–40% of normal level at 1 wk)Fresh frozen plasma; concentrated antihemophilic factor; cryoprecipitateIX (Christmas factor,

1	of normal level (50%–150% of normal level)6–12 hConsumed30%Labile (20%–40% of normal level at 1 wk)Fresh frozen plasma; concentrated antihemophilic factor; cryoprecipitateIX (Christmas factor, plasma thromboplastin component)100% of normal level24 hSurvives20%–30%StableFresh-frozen plasma; bank blood; concentrated preparationX (Stuart-Prower factor)100% of normal level40 hSurvives15%–20%StableBank blood; concentrated preparationXI (plasma thromboplastin antecedent)100% of normal levelProbably 40–80 hSurvives10%Probably stableBank bloodXII (Hageman factor)100% of normal levelUnknownSurvivesDeficit produces no bleeding tendencyStableReplacement not requiredXIII (fibrinase, fibrin-stabilizing factor)100% of normal level4–7 dSurvivesProbably <1%StableBank bloodPlatelets150,000–400,000/μL8–11 dConsumed60,000–100,000/μLVery labile (40% of normal level at 20 h; 0 at 48 h)Fresh blood or plasma; fresh platelet concentrate (not frozen plasma)ACD = acid-citrate-dextrose.Reproduced with

1	dConsumed60,000–100,000/μLVery labile (40% of normal level at 20 h; 0 at 48 h)Fresh blood or plasma; fresh platelet concentrate (not frozen plasma)ACD = acid-citrate-dextrose.Reproduced with permission from Kinney JM, Egdahl RH, Zuidema GD: Manual of Preoperative and Postoperative Care, 2nd ed. Philadelphia, PA: WB Saunders/Elsevier; 1971.Brunicardi_Ch04_p0103-p0130.indd 11829/01/19 11:05 AM 119HEMOSTASIS, SURGICAL BLEEDING, AND TRANSFUSIONCHAPTER 4percentage of patients receive a massive transfusion. In the military setting, the percentage of massive transfusion patients almost doubles.111Damage Control Resuscitation. Prior to DCR, resuscitation guidelines advocated volume replacement with crystalloid, fol-lowed by packed red blood cell and only later plasma or platelets.112 This conventional massive transfusion practice was based on a several small uncontrolled retrospective studies that used blood products containing increased amounts of plasma, which are no longer

1	This conventional massive transfusion practice was based on a several small uncontrolled retrospective studies that used blood products containing increased amounts of plasma, which are no longer available.113 Because of the known early coagulopathy of trauma, the current approach to managing the exsanguinating patient involves early implementation of DCR. Although most of the attention to hemorrhagic shock resuscitation has centered on higher ratios of plasma and platelets, DCR is actually composed of four basic components: permissive hypotension, minimizing crystalloid-based resusci-tation, the immediate release and administration of predefined balanced blood products (red blood cells, plasma, and platelets) in ratios similar to those of whole blood, and the use of hemo-static adjuncts.The shift to DCR began in earnest in 2007 when a retro-spective study of 246 military casualties reported that patients with high plasma:RBC ratio (median 1:1.4) had substantially reduced mortality

1	shift to DCR began in earnest in 2007 when a retro-spective study of 246 military casualties reported that patients with high plasma:RBC ratio (median 1:1.4) had substantially reduced mortality (19% vs. 65%) compared to patients with low plasma:RBC ratio (median 1:8).114 Subsequent observational studies among civilian and military trauma patients corrobo-rated these findings.115-118 In particular, the prospective, obser-vational, multicenter, major trauma transfusion (PROMMTT) study119 found that hemorrhagic death occurred rapidly (median of 2 to 3 hours after hospital arrival) and that plasma:RBC and platelet:RBC ratios significantly varied during massive trans-fusion. Increased plasma:RBC (adjusted hazard ratio [HR] 0.31, 95% confidence interval [CI] 0.16-0.58) and increased platelet:RBC (adjusted HR 0.55, 95% CI 0.31-0.98) were associated with reduced 6-hour mortality, when risk of hemor-rhagic death was highest. After 6 hours, however, increasing plasma:RBC and platelet:RBC were

1	(adjusted HR 0.55, 95% CI 0.31-0.98) were associated with reduced 6-hour mortality, when risk of hemor-rhagic death was highest. After 6 hours, however, increasing plasma:RBC and platelet:RBC were no longer associated with reduced mortality due to increasing competing risk for non-hemorrhagic death (e.g., traumatic brain injury). The Pragmatic Randomized Optimal Platelet and Plasma Ratios (PROPPR) trial120 randomized 680 bleeding trauma patients across 12 highest-level trauma centers to resuscitation with 1:1:1 vs. 1:1:2 plasma to platelets to RBCs. Although there was no significant difference in mortality at 24 hours (13% vs. 17%) or 30 days (22% vs. 26%), the 1:1:1 group had significantly decreased mortality due to hemorrhage at 24 hours (9% vs. 15%) and more patients achieving hemostasis (86% vs. 78%). Despite fears that resuscitation with increased plasma volumes would lead to more inflammatory complications, there were no between-group differences in 23 prespecified secondary

1	(86% vs. 78%). Despite fears that resuscitation with increased plasma volumes would lead to more inflammatory complications, there were no between-group differences in 23 prespecified secondary outcomes, including acute respiratory distress syndrome, sepsis, multiple organ failure, and venous thromboembolism. A recent system-atic review/meta-analysis and practice management guideline from EAST reported reduced mortality (31% vs. 38%) in 5292 patients receiving high (≥1:1) versus low (<1:2) plasma to RBC, and reduced mortality (28% vs. 43%) in 1607 patients receiving high versus low platelet to RBC.99 The authors therefore recom-mend high and balanced ratio (≥1:1) of plasma and platelet to RBC for resuscitation of severely injured trauma patients.The mechanism for these benefits are unclear. While cor-rection of hypovolemia as well as augmention of the patient’s hemostatic potential with clotting factors and platelets are impor-tant, other plasma proteins likely play key roles as well.

1	While cor-rection of hypovolemia as well as augmention of the patient’s hemostatic potential with clotting factors and platelets are impor-tant, other plasma proteins likely play key roles as well. Recently, plasma resuscitation has been shown to reverse endothelial injury in animal models of hemorrhagic shock, particularly by repair of the endothelial glycocalyx layer (EGL).121,122 The EGL is the primary determinant of vascular permeability.123 Hemorrhage results in shedding of EGL components and vascular perme-ability. Crystalloid and artificial colloid-based resuscitation increases the hydrostatic pressure without repairing the EGL, which likely contributes to the myriad of edema-related com-plications seen in the pre-DCR era. Plasma, on the other hand, repairs the EGL, limiting extravascular leakage and edema. However, the exact protein moieties that mediate these benefits have yet to be identified and remain an area of investigation. Nevertheless, several studies have reported

1	leakage and edema. However, the exact protein moieties that mediate these benefits have yet to be identified and remain an area of investigation. Nevertheless, several studies have reported decreased inflam-matory and edema-related complications with increased plasma and decreased crystalloid utilization. In trauma patients, there are strong correlations between increasing circulating levels of glycocalyx components such as syndecan-1 and trauma severity, coagulopathy, and mortality,124-126 although it remains unclear if these relationships are causative or merely associative. Finally, the use of DCR principles to guide transfusion of substantial nontraumatic hemorrhage is intuitive, although there is little evidence in the literature to support this practice.It is essential that the trauma center has an established mechanism to deliver these products quickly and in the correct amounts to these critically injured patients.99 An example of an adult massive transfusion clinical

1	the trauma center has an established mechanism to deliver these products quickly and in the correct amounts to these critically injured patients.99 An example of an adult massive transfusion clinical guideline specifying the early use of component therapy is shown in Table 4-6. Specific rec-ommendations for the administration of component ther-apy during a massive transfusion are shown in Table 4-7.Because only a small percentage of trauma patients require a massive transfusion and because blood products in general are in short supply, there is a need for early prediction models.127 A comparison of results from existing models in both civilian and military studies is shown in Table 4-8.128-132 While compel-ling, many of these models require laboratory data, complicated injury severity scores, or calculated values that are not readily available or feasible to obtain in the urgent setting of bleeding. The Assessment of Blood Consumption (ABC) score is a sim-plified score to predict

1	scores, or calculated values that are not readily available or feasible to obtain in the urgent setting of bleeding. The Assessment of Blood Consumption (ABC) score is a sim-plified score to predict massive transfusion after trauma using immediately available data (heart rate, blood pressure, FAST exam, mechanism of injury).132 The ABC score has been vali-dated across multiple trauma centers; however, it may be limited in some centers by the variable use of and operator-dependent FAST examination. In using the ABC score as it was intended, less than 5% of patients who will require massive transfusion will be missed; and 85% of all major trauma patients will be correctly identified.Prehospital TransfusionIn bleeding patients, earlier initiation of appropriate therapy improves outcomes. For example, decreased overall blood product use and increased 30-day survival was observed after moving four units of universal donor, ready-to-transfuse plasma from the blood bank to the emergency

1	For example, decreased overall blood product use and increased 30-day survival was observed after moving four units of universal donor, ready-to-transfuse plasma from the blood bank to the emergency department and using the plasma as a primary resuscitation fluid.133 A prehospital retrospective study that analyzed 1677 severely injured trauma patients who were transported by helicopter found that in-flight plasma transfusion was associated with less deranged physiol-ogy on admission and reduced early mortality in the most criti-cally ill patients.134 Prehospital RBC transfusion has also been 56Brunicardi_Ch04_p0103-p0130.indd 11929/01/19 11:05 AM 120BASIC CONSIDERATIONSPART ITable 4-6Adult transfusion clinical practice guidelineA. Initial Transfusion of Red Blood Cells (RBCs):1. Notify blood bank immediately of urgent need for RBCs.O negative uncrossmatched (available immediately).As soon as possible, switch to O negative for females and O positive for males.Type-specific

1	blood bank immediately of urgent need for RBCs.O negative uncrossmatched (available immediately).As soon as possible, switch to O negative for females and O positive for males.Type-specific uncrossmatched (available in approximately 5–10 min).Completely crossmatched (available in approximately 40 min).2. A blood sample must be sent to blood bank for a type and cross.3. The Emergency Release of Blood form must be completed. If the blood type is not known and blood is needed immediately, O-negative RBCs should be issued.4. RBCs will be transfused in the standard fashion. All patients must be identified (name and number) prior to transfusion.5. Patients who are unstable or receive 1–2 RBCs and do not rapidly respond should be considered candidates for the massive transfusion (MT) guideline.B. Adult Massive Transfusion Guideline:1. The Massive Transfusion Guideline (MTG) should be initiated as soon as it is anticipated that a patient will require massive transfusion. The blood bank should

1	Massive Transfusion Guideline:1. The Massive Transfusion Guideline (MTG) should be initiated as soon as it is anticipated that a patient will require massive transfusion. The blood bank should strive to deliver plasma, platelets, and RBCs in a 1:1:1 ratio. To be effective and minimize further dilutional coagulopathy, the 1:1:1 ratio must be initiated early, ideally with the first 2 units of transfused RBCs. Crystalloid infusion should be minimized.2. Once the MTG is activated, the blood bank will have 6 RBCs, 6 FFP, and a 6-pack of platelets packed in a cooler available for rapid transport. If 6 units of thawed FFP are not immediately available, the blood bank will issue units that are ready and notify appropriate personnel when the remainder is thawed. Every attempt should be made to obtain a 1:1:1 ratio of plasma:platelets:RBCs.3. Once initiated, the MT will continue until stopped by the attending physician. MT should be terminated once the patient is no longer actively

1	made to obtain a 1:1:1 ratio of plasma:platelets:RBCs.3. Once initiated, the MT will continue until stopped by the attending physician. MT should be terminated once the patient is no longer actively bleeding.4. No blood components will be issued without a pickup slip with the recipient’s medical record number and name.5. Basic laboratory tests should be drawn immediately on ED arrival and optimally performed on point-of-care devices, facilitating timely delivery of relevant information to the attending clinicians. These tests should be repeated as clinically indicated (e.g., after each cooler of products has been transfused). Suggested laboratory values are:• CBC• INR, fibrinogen• pH and/or base deficit• TEG, where availableCBC = complete blood count; ED = emergency department; FFP = fresh frozen plasma; INR = international normalized ratio; TEG = thromboelastography.Table 4-7Component therapy administration during massive transfusionFresh frozen plasma (FFP)As soon as the need for

1	frozen plasma; INR = international normalized ratio; TEG = thromboelastography.Table 4-7Component therapy administration during massive transfusionFresh frozen plasma (FFP)As soon as the need for massive transfusion is recognized.For every 6 red blood cells (RBCs), give 6 FFP (1:1 ratio).PlateletsFor every 6 RBCs and plasma, give one 6-pack of platelets. 6 random-donor platelet packs = 1 apheresis platelet unit.Platelets are in every cooler.Keep platelet counts >100,000.CryoprecipitateAfter first 6 RBCs, check fibrinogen level. If ≤200 mg/dL, give 20 units cryoprecipitate (2 g fibrinogen). Repeat as needed, depending on fibrinogen level, and request appropriate amount of cryoprecipitate.Table 4-8Comparison of massive transfusion prediction studiesAUTHORVARIABLESROC AUC VALUEMcLaughlin et al128SBP, HR, pH, Hct0.839Yücel et al129SBP, HR, BD, Hgb, male, + FAST, long bone/pelvic fracture0.892Moore et al130SBP, pH, ISS >250.804Schreiber et al131Hgb ≤11, INR >1.5, penetrating

1	et al128SBP, HR, pH, Hct0.839Yücel et al129SBP, HR, BD, Hgb, male, + FAST, long bone/pelvic fracture0.892Moore et al130SBP, pH, ISS >250.804Schreiber et al131Hgb ≤11, INR >1.5, penetrating injury0.80Cotton et al132HR, SBP, FAST, penetrating injury0.83–0.90AUC = area under the curve; BD = base deficit; FAST = focused assessment with sonography for trauma; Hct = hematocrit; Hgb = hemoglobin; HR = heart rate; INR = international normalized ratio; ISS = injury severity score; ROC = receiver operating characteristic; SBP = systolic blood pressure.Brunicardi_Ch04_p0103-p0130.indd 12029/01/19 11:05 AM 121HEMOSTASIS, SURGICAL BLEEDING, AND TRANSFUSIONCHAPTER 4Nonhemolytic Reactions. Febrile, nonhemolytic reactions are defined as an increase in temperature (>1°C) associated with a transfusion and are fairly common (approximately 1% of all transfusions). Preformed cytokines in donated blood and recipi-ent antibodies reacting with donated antibodies are postulated eti-ologies. The incidence

1	and are fairly common (approximately 1% of all transfusions). Preformed cytokines in donated blood and recipi-ent antibodies reacting with donated antibodies are postulated eti-ologies. The incidence of febrile reactions can be greatly reduced by the use of leukocyte-reduced blood products. Pretreatment with acetaminophen reduces the severity of the reaction.Bacterial contamination of infused blood is rare. Gram-negative organisms, which are capable of growth at 4°C, are the most common cause. Most cases, however, are associated with the administration of platelets that are stored at 20°C or, even more commonly, with apheresis platelets stored at room tem-perature. Cases from FFP thawed in contaminated water baths have also been reported.150 Bacterial contamination can result in sepsis and death in up 25% of patients.151 Clinical manifesta-tions includes systemic signs such as fever and chills, tachycar-dia and hypotension, and gastrointestinal symptoms (abdominal cramps, vomiting,

1	and death in up 25% of patients.151 Clinical manifesta-tions includes systemic signs such as fever and chills, tachycar-dia and hypotension, and gastrointestinal symptoms (abdominal cramps, vomiting, and diarrhea). If the diagnosis is suspected, the transfusion should be discontinued and the blood cultured. Emergency treatment includes oxygen, adrenergic blocking agents, and antibiotics.Allergic Reactions. Allergic reactions are relatively frequent, occurring in about 1% of all transfusions. Reactions are usually mild and consist of rash, urticaria, and flushing. In rare instances, anaphylactic shock develops. Allergic reactions are caused by the transfusion of antibodies from hypersensitive donors or the trans-fusion of antigens to which the recipient is hypersensitive. Allergic reactions can occur after the administration of any blood product but are commonly associated with FFP and platelets. Treatment and prophylaxis consist of the administration of antihistamines. In more serious

1	can occur after the administration of any blood product but are commonly associated with FFP and platelets. Treatment and prophylaxis consist of the administration of antihistamines. In more serious cases, epinephrine or steroids may be indicated.Respiratory Complications. Respiratory compromise may be associated with transfusion-associated circulatory overload (TACO), which is an avoidable complication. It can occur with rapid infusion of blood, plasma expanders, and crystalloids, par-ticularly in older patients with underlying heart disease. Central venous pressure monitoring should be considered whenever large amounts of fluid are administered. Overload is manifested by a rise in venous pressure, dyspnea, and cough. Rales can gen-erally be heard at the lung bases. Treatment consists of diuresis, slowing the rate of blood administration, and minimizing fluids while blood products are being transfused.The syndrome of TRALI is defined as noncardiogenic pulmonary edema related to

1	of diuresis, slowing the rate of blood administration, and minimizing fluids while blood products are being transfused.The syndrome of TRALI is defined as noncardiogenic pulmonary edema related to transfusion.152 It can occur with the administration of any plasma-containing blood product. Symptoms are similar to circulatory overload with dyspnea and associated hypoxemia. However, TRALI is characterized as noncardiogenic and is often accompanied by fever, rigors, and bilateral pulmonary infiltrates on chest X-ray. It most com-monly occurs within 1 to 2 hours after the onset of transfusion but virtually always before 6 hours. Toy et al reported a decrease in the incidence of TRALI with the reduction transfusion of plasma from female donors, due to a combination of reduced transfusion of strong cognate HLA class II antibodies and HNA antibodies in patients with risk factors for acute lung injury.153 TRALI now occurs less than 1 in 10,000 units transfused and is usually self-limited with

1	cognate HLA class II antibodies and HNA antibodies in patients with risk factors for acute lung injury.153 TRALI now occurs less than 1 in 10,000 units transfused and is usually self-limited with supportive therapy. Treatment of TRALI entails discontinuation of any transfusion, notification of the transfusion service, and pulmonary support, which may vary from supplemental oxygen to mechanical ventilation.associated with similar findings.135 In the military setting, imple-mentation of prehospital transfusion protocols in conjunction with other measures, including more rapid transport times, was also associated with reduced mortality.136Whole Blood ResuscitationMilitary experience with whole blood for the resuscitation of traumatic hemorrhage is extensive, going back to the American Civil War. In the modern era, more than 10,000 whole blood units were transfused during Operations Enduring Freedom and Iraqi Freedom. One key advantage of whole blood ver-sus component therapy is that

1	War. In the modern era, more than 10,000 whole blood units were transfused during Operations Enduring Freedom and Iraqi Freedom. One key advantage of whole blood ver-sus component therapy is that platelets are often unavailable in the remote and austere settings. Two retrospective studies of military casualties treated at a combat support hospital and forward surgical teams found that whole blood was associated with improved survival compared to component (plasma and RBC) therapy.137,138 Whole blood has higher hematocrit, clotting factor activity, and platelet count compared to 1:1:1 component therapy due to relatively less diluent volume in whole blood. During the Vietnam War, low anti-A and anti-B titer whole blood was transfused universally with a low incidence of hemo-lytic reactions (1 per 9600 units).139 An in vitro study found that the hemostatic potential of whole blood was preserved for up to 14 days with cold storage.140 Pilot trials have reported success-ful use of

1	(1 per 9600 units).139 An in vitro study found that the hemostatic potential of whole blood was preserved for up to 14 days with cold storage.140 Pilot trials have reported success-ful use of crossmatched modified whole blood (leukoreduced and platelet-poor)141 and uncrossmatched low-titer whole blood (leukoreduced, containing platelets)142 in the initial resuscita-tion of civilian trauma patients. In the future, whole blood may return as the therapy of choice for the initial resuscitation of substantial hemorrhage.143Fibrinogen ReplacementFibrinogen is the first coagulation factor to fall to critically low levels during major hemorrhage, and low systemic concentra-tions of fibrinogen are associated with increased severity of injury and coagulopathy and are independently predictive of mortality.144,145 Additionally, fibrinogen levels drop in the pre-hospital phase of injury, suggesting early administration by fibrinogen concentrate (not FDA-approved) or cryoprecipitate is needed.146

1	Additionally, fibrinogen levels drop in the pre-hospital phase of injury, suggesting early administration by fibrinogen concentrate (not FDA-approved) or cryoprecipitate is needed.146 Fibrinogen concentrate is stored as a lyophilized powder at room temperature and can be reconstituted quickly allowing for rapid administration without delays for thawing or crossmatching.147 In contrast to plasma, viral inactivation steps are routinely included in the manufacturing process for fibrino-gen concentrate, thus minimizing the risk of viral transmission. A pilot trial of massively transfused trauma patients randomized to a massive transfusion protocol or a massive transfusion pro-tocol with early cryoprecipitate found that early cryoprecipitate delivery was feasible and that these patients had higher fibrino-gen levels at all time points during resuscitation, although there was no mortality difference.148 A randomized control trial in Austria of prehospital fibrinogen concentrate versus

1	higher fibrino-gen levels at all time points during resuscitation, although there was no mortality difference.148 A randomized control trial in Austria of prehospital fibrinogen concentrate versus placebo has been completed with publication of results pending.149Complications of Transfusion (Table 4-9)Transfusion-related complications are primarily related to blood-induced proinflammatory responses. Transfusion-related149a events are estimated to occur in approximately 10% of all trans-fusions, but less than 0.5% are serious in nature. Transfusionrelated deaths, although exceedingly rare, do occur and are related primarily to transfusion-related acute lung injury (TRALI), ABO hemolytic transfusion reactions, and bacterial contamination of platelets.Brunicardi_Ch04_p0103-p0130.indd 12129/01/19 11:05 AM 122BASIC CONSIDERATIONSPART IHemolytic Reactions. Hemolytic reactions can be classified as either acute or delayed. Acute hemolytic reactions occur with the administration of

1	12129/01/19 11:05 AM 122BASIC CONSIDERATIONSPART IHemolytic Reactions. Hemolytic reactions can be classified as either acute or delayed. Acute hemolytic reactions occur with the administration of ABO-incompatible blood and can be fatal in up to 6% of cases. Contributing factors include errors in the laboratory of a technical or clerical nature or the administra-tion of the wrong blood type. Immediate hemolytic reactions are characterized by intravascular destruction of red blood cells and consequent hemoglobinemia and hemoglobinuria. DIC can be initiated by antibody-antigen complexes activating factor XII and complement, leading to activation of the coagulation cas-cade. Finally, acute renal insufficiency results from the toxicity associated with free hemoglobin in the plasma, resulting in tubu-lar necrosis and precipitation of hemoglobin within the tubules.Delayed hemolytic transfusion reactions occur 2 to 10 days after transfusion and are characterized by extravascular

1	resulting in tubu-lar necrosis and precipitation of hemoglobin within the tubules.Delayed hemolytic transfusion reactions occur 2 to 10 days after transfusion and are characterized by extravascular hemolysis, mild anemia, and indirect (unconjugated) hyperbili-rubinemia. They occur when an individual has a low antibody titer at the time of transfusion, but the titer increases after trans-fusion as a result of an anamnestic response. Reactions to non-ABO antigens involve immunoglobulin G-mediated clearance by the reticuloendothelial system.If the patient is awake, the most common symptoms of acute transfusion reactions are pain at the site of transfusion, facial flushing, and back and chest pain. Associated symptoms include fever, respiratory distress, hypotension, and tachycardia. In anesthetized patients, diffuse bleeding and hypotension are the hallmarks. A high index of suspicion is needed to make the diag-nosis. The laboratory criteria for a transfusion reaction are

1	In anesthetized patients, diffuse bleeding and hypotension are the hallmarks. A high index of suspicion is needed to make the diag-nosis. The laboratory criteria for a transfusion reaction are hemo-globinuria and serologic criteria that show incompatibility of the donor and recipient blood. A positive Coombs’ test indicates Table 4-9Transfusion-related complicationsABBREVIATIONCOMPLICATIONSIGNS AND SYMPTOMSFREQUENCYMECHANISMPREVENTIONNHTRFebrile, nonhemolytic transfusion reactionFever0.5%–1.5% of transfusionsPreformed cytokinesHost Ab to donor lymphocytesUse leukocyte-reduced bloodStore platelets <5 d Bacterial contaminationHigh fever, chillsHemodynamic changesDICEmesis, diarrheaHemoglobinuria<0.01% of blood<0.05% of plateletsInfusion of contaminated blood Allergic reactionsRash, hivesItching0.1%–0.3% of unitsSoluble transfusion constituentsProvide antihistamine prophylaxisTACOTransfusion-associated circulatory overloadPulmonary edema1:200–1:10,00 of transfused patientsLarge volume

1	of unitsSoluble transfusion constituentsProvide antihistamine prophylaxisTACOTransfusion-associated circulatory overloadPulmonary edema1:200–1:10,00 of transfused patientsLarge volume of blood transfused into an older patient with CHFIncrease transfusion timeAdminister diureticsMinimize associated fluidsTRALITransfusion-related acute lung injuryAcute (<6 h) hypoxemiaBilateral infiltrates ± Tachycardia, hypotension Anti-HLA or anti-HNA Ab in transfused blood attacks circulatory and pulmonary leukocytesLimit female donors Hemolytic reaction, acuteFeverHypotensionDICHemoglobinuriaHemoglobinemiaRenal insufficiency1:33,000–1:1,500,000 unitsTransfusion of ABO-incompatible bloodPreformed IgM Ab to ABO AgTransfuse appropriately matched blood Hemolytic reaction, delayed (2–10 d)AnemiaIndirect hyperbilirubinemiaDecreased haptoglobin levelPositive result on direct Coombs’ test IgG mediatedIdentify patient’s Ag to prevent recurrenceAb = antibody; Ag = antigen; CHF = congestive heart failure; DIC

1	haptoglobin levelPositive result on direct Coombs’ test IgG mediatedIdentify patient’s Ag to prevent recurrenceAb = antibody; Ag = antigen; CHF = congestive heart failure; DIC = disseminated intravascular coagulation; HLA = human leukocyte antigen; HNA = anti-human neutrophil antigen; IgG = immunoglobulin G; IgM = immunoglobulin M.Brunicardi_Ch04_p0103-p0130.indd 12229/01/19 11:05 AM 123HEMOSTASIS, SURGICAL BLEEDING, AND TRANSFUSIONCHAPTER 4transfused cells coated with patient antibody and is diagnostic. Delayed hemolytic transfusions may also be manifested by fever and recurrent anemia. Jaundice and decreased haptoglobin usu-ally occur, and low-grade hemoglobinemia and hemoglobinuria may be seen. The Coombs’ test is usually positive, and the blood bank must identify the antigen to prevent subsequent reactions.If an immediate hemolytic transfusion reaction is sus-pected, the transfusion should be stopped immediately, and a sample of the recipient’s blood drawn and sent along with

1	subsequent reactions.If an immediate hemolytic transfusion reaction is sus-pected, the transfusion should be stopped immediately, and a sample of the recipient’s blood drawn and sent along with the suspected unit to the blood bank for comparison with the pretransfusion samples. Urine output should be monitored and adequate hydration maintained to prevent precipitation of hemo-globin within the tubules. Delayed hemolytic transfusion reac-tions do not usually require specific intervention.Transmission of Disease. Malaria, Chagas’ disease, brucel-losis, and, very rarely, syphilis are among the diseases that have been transmitted by transfusion. Malaria can be transmitted by all blood components. The species most commonly implicated is Plasmodium malariae. The incubation period ranges from 8 to 100 days; the initial manifestations are shaking chills and spiking fever. Cytomegalovirus (CMV) infection resembling infectious mononucleosis also has occurred.Transmission of hepatitis C and

1	8 to 100 days; the initial manifestations are shaking chills and spiking fever. Cytomegalovirus (CMV) infection resembling infectious mononucleosis also has occurred.Transmission of hepatitis C and HIV-1 has been dra-matically minimized by the introduction of better antibody and nucleic acid screening for these pathogens. The residual risk among allogeneic donations is now estimated to be less than 1 per 1,000,000 donations. The residual risk of hepatitis B is approximately 1 per 300,000 donations.154 Hepatitis A is very rarely transmitted because there is no asymptomatic carrier state. Improved donor selection and testing are responsible for the decreased rates of transmission. Recent concerns about the rare transmission of these and other pathogens, such as West Nile virus, are being addressed by current trials of “pathogen inactivation systems” that reduce infectious levels of all viruses and bacteria known to be transmittable by transfusion. Prion dis-orders (e.g.,

1	are being addressed by current trials of “pathogen inactivation systems” that reduce infectious levels of all viruses and bacteria known to be transmittable by transfusion. Prion dis-orders (e.g., Creutzfeldt-Jakob disease) also are transmissible by transfusion, but there is currently no information on inactivation of prions in blood products for transfusion.Recently, there is heightened concern of transmission of Zika virus by blood product transfusion. Studies in endemic areas have shown rates of Zika infection detected in donor blood as high as 2.8%.155 Although no such cases have been reported in the United States, transmission of Zika virus via platelet products have been reported in Brazil.156 Zika virus may result in serious birth defects including microcephaly when infection occurs in pregnant women. Because the majority of cases in adults produce nonspecific or no symptoms, Zika screening cannot be accomplished by questionnaires.157 The Centers for Disease Control and

1	occurs in pregnant women. Because the majority of cases in adults produce nonspecific or no symptoms, Zika screening cannot be accomplished by questionnaires.157 The Centers for Disease Control and Prevention has issued guidelines for screen-ing of Zika virus in donated blood. Although no tests have been FDA-approved, laboratory testing is currently being performed under the FDA’s IND program.TESTS OF HEMOSTASIS AND BLOOD COAGULATIONThe initial approach to assessing hemostatic function is a careful review of the patient’s clinical history (including previous abnor-mal bleeding or bruising), drug use, and basic laboratory testing.Conventional Coagulation Tests. Common screening labo-ratory testing includes platelet count, PT or INR, and aPTT. Platelet dysfunction can occur at either extreme of platelet count. The normal platelet count ranges from 150,000 to 400,000/μL. Whereas a platelet count greater than 1,000,000/μL may be associated with bleeding or thrombotic complications,

1	extreme of platelet count. The normal platelet count ranges from 150,000 to 400,000/μL. Whereas a platelet count greater than 1,000,000/μL may be associated with bleeding or thrombotic complications, increased bleeding complications may be observed with major surgical procedures when the platelets are below 50,000/μL and with minor surgical procedures when counts are below 30,000/μL, and spontaneous hemorrhage can occur when the counts fall below 20,000/μL. Despite a lack of evidence supporting their use, platelet transfusions are still recommended in ophthalmo-logic and neurosurgical procedures when the platelet count is less than 100,000/μL.The PT and aPTT are variations of plasma recalcifica-tion times initiated by the addition of a thromboplastic agent. The PT reagent contains thromboplastin and calcium that, when added to plasma, leads to the formation of a fibrin clot. The PT test measures the function of factors I, II, V, VII, and X. Factor VII is part of the extrinsic pathway,

1	and calcium that, when added to plasma, leads to the formation of a fibrin clot. The PT test measures the function of factors I, II, V, VII, and X. Factor VII is part of the extrinsic pathway, and the remaining factors are part of the common pathway. Factor VII has the shortest half-life of the coagulation factors, and its synthesis is vitamin K dependent. The PT test is best suited to detect abnor-mal coagulation caused by vitamin K deficiencies and warfarin therapy.Due to variations in thromboplastin activity, it can be dif-ficult to accurately assess the degree of anticoagulation on the basis of PT alone. To account for these variations, the INR is now the method of choice for reporting PT values. The Interna-tional Sensitivity Index (ISI) is unique to each batch of thrombo-plastin and is furnished by the manufacturer to the hematology laboratory. Human brain thromboplastin has an ISI of 1, and the optimal reagent has an ISI between 1.3 and 1.5.The INR is a calculated number

1	and is furnished by the manufacturer to the hematology laboratory. Human brain thromboplastin has an ISI of 1, and the optimal reagent has an ISI between 1.3 and 1.5.The INR is a calculated number derived from the follow-ing equation:INR = (measured PT/normal PT)ISIThe aPTT reagent contains a phospholipid substitute, acti-vator, and calcium, which in the presence of plasma leads to fibrin clot formation. The aPTT measures function of factors I, II, and V of the common pathway and factors VIII, IX, X, and XII of the intrinsic pathway. Heparin therapy is often monitored by following aPTT values with a therapeutic target range of 1.5 to 2.5 times the control value (approximately 50 to 80 seconds). Low molecular weight heparins are selective Xa inhibitors that may mildly elevate the aPTT, but therapeutic monitoring is not routinely recommended.Additional medications may significantly impair hemo-static function, such as antiplatelet agents (clopidogrel and GP IIb/IIIa inhibitors),

1	but therapeutic monitoring is not routinely recommended.Additional medications may significantly impair hemo-static function, such as antiplatelet agents (clopidogrel and GP IIb/IIIa inhibitors), anticoagulant agents (hirudin, chondroitin sul-fate, dermatan sulfate), and thrombolytic agents (streptokinase, tPA). If abnormalities in any of the coagulation studies cannot be explained by known medications, congenital abnormalities of coagulation or comorbid disease should be considered.Unfortunately, while conventional coagulation tests (PT, aPTT) capture the classic intrinsic and extrinsic coagulation cas-cade, they do not reflect the complexity of in vivo coagulation.158 Although they are useful to follow warfarin and heparin thera-pies, they poorly reflect the status of actively bleeding patients. This is not surprising given that these tests use only plasma and not whole blood to provide their assessment of the patient’s clot-ting status. To better assess the complex and rapidly

1	patients. This is not surprising given that these tests use only plasma and not whole blood to provide their assessment of the patient’s clot-ting status. To better assess the complex and rapidly changing hemostatic function of an actively bleeding patient, many cen-ters have moved to whole blood viscoelastic testing.Brunicardi_Ch04_p0103-p0130.indd 12329/01/19 11:05 AM 124BASIC CONSIDERATIONSPART IViscoelastic Assays. Viscoelastic assays, such as TEG or rotational thromboelastometry (ROTEM), monitor hemostasis as a dynamic process rather than revealing information from isolated conventional coagulation screens.159 Both tests measure the viscoelastic properties of blood as clotting is induced under a low-shear environment. The patterns of change in shear elas-ticity enable determination of the kinetics of clot formation and growth as well as the strength and stability of the formed clot. The strength and stability provide information about the ability of the clot to perform the

1	of the kinetics of clot formation and growth as well as the strength and stability of the formed clot. The strength and stability provide information about the ability of the clot to perform the work of hemostasis, while the kinet-ics determines the adequacy of quantitative factors available for clot formation.Continuous improvements in this technique have made this test a valuable tool for medical personnel interested in coagulation. A sample of celite-activated whole blood is placed into a prewarmed cuvette, and the clotting process is activated with reagents, such as kaolin for standard TEG, and kaolin plus tissue factor for rapid TEG. Both TEG and ROTEM employ a vertical pin which is lowered into the activated blood sample. In TEG, the cuvette oscillates in an arc around the stationary pin. As the blood clots, fibrin strands and platelet aggregates form between the pin and inner walls of the cuvette. The resulting torque on the pin is measured and converted to an electrical

1	pin. As the blood clots, fibrin strands and platelet aggregates form between the pin and inner walls of the cuvette. The resulting torque on the pin is measured and converted to an electrical signal. In ROTEM, the cuvette is stationary while the pin oscil-lates within the sample. The extent to which the pin can oscillate is reduced as the blood clots, and this is measured by the angle of deflection of a beam of light directed at the pin.160 In TEGs, the strength of a clot is graphically represented over time as a characteristic cigar-shaped figure (Fig. 4-7).Several parameters are generated from the TEG tracing. The r-value (reaction time) represents the time between the start of the assay and initial clot formation. This reflects clotting fac-tor activity and initial fibrin formation and is increased with fac-tor deficiency or severe hemodilution. The k-time (clot kinetics) is the time needed to reach specified clot strength and repre-sents the interactions of clotting factors and

1	is increased with fac-tor deficiency or severe hemodilution. The k-time (clot kinetics) is the time needed to reach specified clot strength and repre-sents the interactions of clotting factors and platelets. As such, the k-time is prolonged with hypofibrinogenemia and signifi-cant factor deficiency. Prolonged r-value and k-time are com-monly addressed with plasma transfusions. The alpha or angle (∝) is the slope of the tracing and reflects clot acceleration. The angle reflects the interactions of clotting factors and platelets. The slope is decreased with hypofibrinogenemia and platelet dysfunction. Decreased angles are treated with cryoprecipitate transfusion or fibrinogen administration. The maximal ampli-tude (mA) is the greatest height of the tracing and represents clot strength. Its height is reduced with dysfunction or deficiencies in platelets or fibrinogen. Decreased mA is addressed with platelet transfusion and, in cases where the angle is also decreased, with cryoprecipitate

1	is reduced with dysfunction or deficiencies in platelets or fibrinogen. Decreased mA is addressed with platelet transfusion and, in cases where the angle is also decreased, with cryoprecipitate (or fibrinogen) as well. The G-value is a para-metric measure derived from the mA value and reflects overall clot strength or firmness. An increased G-value is associated with hypercoagulability, whereas a decrease is seen with hypo-coagulable states. Finally, the LY30 is the amount of lysis occur-ring in the clot, and the value is the percentage of amplitude reduction at 30 minutes after mA is achieved. The LY30 rep-resents clot stability and when increased fibrinolysis is present.TEG and ROTEM are the only tests measuring all dynamic steps of clot formation until eventual clot lysis or retraction. TEG has also been shown to identify on admission those patients likely to develop thromboembolic complications after injury and postoperatively.161Recent trauma data have shown TEG to be useful in

1	TEG has also been shown to identify on admission those patients likely to develop thromboembolic complications after injury and postoperatively.161Recent trauma data have shown TEG to be useful in pre-dicting early transfusion of red blood cells, plasma, platelets, and cryoprecipitate.162 TEG can also predict the need for life-saving interventions shortly after arrival, 24-hour and 30-day mortality, and can be used to guide administration of TXA to injured patients with hyperfibrinolysis.163,164 Lastly, some cen-ters have demonstrated that the graphic display options allow for more rapid return of results and may be less expensive than standard coagulation panels. Given the strong association of viscoelastic tests with clinical outcomes, some centers now use TEG rather than conventional coagulation tests to evaluate injured patients in the emergency department.165EVALUATION OF EXCESSIVE INTRAOPERATIVE OR POSTOPERATIVE BLEEDINGExcessive bleeding during or after a surgical procedure may

1	tests to evaluate injured patients in the emergency department.165EVALUATION OF EXCESSIVE INTRAOPERATIVE OR POSTOPERATIVE BLEEDINGExcessive bleeding during or after a surgical procedure may be the result of ineffective hemostasis, blood transfusion, unde-tected hemostatic defect, consumptive coagulopathy, and/or fibrinolysis. Excessive bleeding from the operative field unas-sociated with bleeding from other sites usually suggests inad-equate mechanical hemostasis.Massive blood transfusion is a well-known cause of throm-bocytopenia. Bleeding following massive transfusion can occur because of hypothermia, dilutional coagulopathy, platelet dys-function, fibrinolysis, or hypofibrinogenemia. Another cause of hemostatic failure related to the administration of blood is a hemolytic transfusion reaction. The first sign of a transfusion reaction may be diffuse bleeding. The pathogenesis of this bleed-ing is thought to be related to the release of ADP from hemolyzed red blood cells, resulting

1	reaction. The first sign of a transfusion reaction may be diffuse bleeding. The pathogenesis of this bleed-ing is thought to be related to the release of ADP from hemolyzed red blood cells, resulting in diffuse platelet aggregation, after which the platelet clumps are removed out of the circulation.Transfusion purpura occurs when the donor platelets are of the uncommon HPA-1 group. This is an uncommon cause of thrombocytopenia and associated bleeding after transfusion. The platelets sensitize the recipient, who makes antibody to the foreign platelet antigen. The foreign platelet antigen does not completely disappear from the recipient circulation but attaches to the recipient’s own platelets. The antibody then destroys the recipient’s own platelets. The resultant thrombocytopenia and bleeding may continue for several weeks. This uncommon cause of thrombocytopenia should be considered if bleeding follows transfusion by 5 or 6 days. Platelet transfusions are of little help in the

1	bleeding may continue for several weeks. This uncommon cause of thrombocytopenia should be considered if bleeding follows transfusion by 5 or 6 days. Platelet transfusions are of little help in the management of this syndrome because the new donor platelets usually are subject to the binding of antigen and dam-age from the antibody. Corticosteroids may be of some help in reducing the bleeding tendency. Posttransfusion purpura is self-limited, and the passage of several weeks inevitably leads to subsidence of the problem.DIC is characterized by systemic activation of the coagu-lation system, which results in the deposition of fibrin clots and microvascular ischemia and may contribute to the development CoagulationLYFibrinolysisRMAKAngleFigure 4-7. Illustration of a thromboelastogram (TEG) tracing. K = clot kinetics; LY = lysis.Brunicardi_Ch04_p0103-p0130.indd 12429/01/19 11:05 AM 125HEMOSTASIS, SURGICAL BLEEDING, AND TRANSFUSIONCHAPTER 4of multiorgan failure. Consumption and

1	tracing. K = clot kinetics; LY = lysis.Brunicardi_Ch04_p0103-p0130.indd 12429/01/19 11:05 AM 125HEMOSTASIS, SURGICAL BLEEDING, AND TRANSFUSIONCHAPTER 4of multiorgan failure. Consumption and subsequent exhaustion of coagulation proteins and platelets due to the ongoing acti-vation of the coagulation system may induce severe bleeding complications.Lastly, severe hemorrhagic disorders due to thrombo-cytopenia have occurred as a result of gram-negative sepsis. Defibrination and hemostatic failure also may occur with meningococcemia, Clostridium perfringens sepsis, and staph-ylococcal sepsis. Hemolysis appears to be one mechanism in sepsis leading to defibrination.REFERENCESEntries highlighted in bright blue are key references. 1. Brohi K, Cohen MJ, Davenport RA. Acute coagulopathy of trauma: mechanism, identification and effect. Curr Opin Crit Care. 2007;13:680-685. 2. Kulkarani R. Comprehensive care of the patient with haemo-philia and inhibitors undergoing surgery: practical

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1	rationale for the use of balanced salt solutions. Crit Care Med. 1976;4:46. 113. Harrigan C, Lucas CE, Ledgerwood AM, et al. Serial changes in primary hemostasis after massive transfusion. Surgery. 1985;98:836. 114. Borgman MA, Spinella PC, Perkins JG, et al. The ratio of blood products transfused affects mortality in patients receiving massive transfusions at a combat support hospital. J Trauma. 2007;63(4):805-813. 115. Holcomb JB, Wade CE, Michalek JE, et al. Increased plasma and platelet to red blood cell ratios improves outcome in 466 massively transfused civilian trauma patients. Ann Surg. 2008;248(3):447-458. 116. Mitra B, Mori A, Cameron PA, et al. Fresh frozen plasma (FFP) use during massive blood transfusion in trauma resus-citation. Injury. 2010;41(1):35-39. 117. Peiniger S, Nienaber U, Lefering R, et al. Balanced massive transfusion ratios in multiple injury patients with traumatic brain injury. Crit Care. 2011;15(1):R68. 118. Cotton BA, Reddy N, Hatch QM, et al. Damage

1	Nienaber U, Lefering R, et al. Balanced massive transfusion ratios in multiple injury patients with traumatic brain injury. Crit Care. 2011;15(1):R68. 118. Cotton BA, Reddy N, Hatch QM, et al. Damage control resuscitation is associated with a reduction in resuscitation volumes and improvement in survival in 390 damage control laparotomy patients. Ann Surg. 2011;254(4):598-605. 119. Holcomb JB, del Junco DJ, Fox EE, et al. The prospective, obser-vational, multicenter, major trauma transfusion (PROMMTT) study: comparative effectiveness of a time-varying treatment with competing risks. JAMA Surg. 2013;148:127-136. 120. Holcomb JB, Tilley BC, Baraniuk S, et al. Transfusion of plasma, platelets, and red blood cells in a 1:1:1 vs a 1:1:2 ratio and mortality in patients with severe trauma: the PROPPR randomized clinical trial. JAMA. 2015;313:471-482. This is a prospective randomized study at 12 level 1 trauma centers of massively bleeding trauma patients, com-paring two balanced transfusion

1	randomized clinical trial. JAMA. 2015;313:471-482. This is a prospective randomized study at 12 level 1 trauma centers of massively bleeding trauma patients, com-paring two balanced transfusion groups. Fewer patients bled to death in the 1:1:1 group. 121. Kozar RA, Peng Z, Zhang R, et al. Plasma restoration of endothelial glycocalyx in a rodent model of hemorrhagic shock. Anesth Analg. 2011;112(6):1289-1295. 122. Potter DR, Baimukanova G, Keating SM, et al. Fresh frozen plasma and spray-dried plasma mitigate pulmonary vascu-lar permeability and inflammation in hemorrhagic shock. J Trauma Acute Care Surg. 2015;78(6 Suppl 1):S7-S17. 123. Woodcock TE, Woodcock TM. Revised Starling equation and the glycocalyx model of transvascular fluid exchange: an improved paradigm for prescribing intravenous fluid therapy. Br J Anaesth. 2012;108(3):384-394. 124. Johansson PI, Stensballe J, Rasmussen LS, Ostrowski SR. A high admission syndecan-1 level, a marker of endothelial gly-cocalyx degradation,

1	fluid therapy. Br J Anaesth. 2012;108(3):384-394. 124. Johansson PI, Stensballe J, Rasmussen LS, Ostrowski SR. A high admission syndecan-1 level, a marker of endothelial gly-cocalyx degradation, is associated with inflammation, protein C depletion, fibrinolysis, and increased mortality in trauma patients. Ann Surg. 2011;254(2):194-200. 125. Ostrowski SR, Johansson PI. Endothelial glycocalyx degra-dation induces endogenous heparinization in patients with severe injury and early traumatic coagulopathy. J Trauma Acute Care Surg. 2012;73(1):60-66. 126. Rahbar E, Cardenas JC, Baimukanova G, et al. Endothelial glycocalyx shedding and vascular permeability in severely injured trauma patients. J Transl Med. 2015;13:117. 127. Johansson PI, Stensballe J, Oliveri R, Wade CE, Ostrowski SR, Holcomb JB. How I treat patients with massive hemor-rhage. Blood. 2014;124(20):3052-3058. 128. McLaughlin DF, Niles SE, Salinas J, et al. A predictive model for massive transfusion in combat casualty patients.

1	How I treat patients with massive hemor-rhage. Blood. 2014;124(20):3052-3058. 128. McLaughlin DF, Niles SE, Salinas J, et al. A predictive model for massive transfusion in combat casualty patients. J Trauma. 2008;64(2 Suppl):S57. 129. Yücel N, Lefering R, Maegele M, et al. Trauma-Associated Severe Hemorrhage (TASH) score: probability of mass trans-fusion as surrogate for life threatening hemorrhage after mul-tiple trauma. J Trauma. 2006;60:1228. 130. Moore FA, Nelson T, McKinley BA, et al. Massive transfu-sion in trauma patients: tissue hemoglobin oxygen saturation predicts poor outcome. J Trauma. 2008;64:1010. 131. Schreiber MA, Perkins J, Kiraly L, et al. Early predictors of massive transfusion in combat casualties. J Am Coll Surg. 2007;205:541. 132. Cotton BA, Dossett LA, Haut ER, et al. Multicenter valida-tion of a simplified score to predict massive transfusion in trauma. J Trauma. 2010;69(Suppl 1):S33-S39. 133. Radwan ZA, Bai Y, Matijevic N, et al. An emergency depart-ment

1	et al. Multicenter valida-tion of a simplified score to predict massive transfusion in trauma. J Trauma. 2010;69(Suppl 1):S33-S39. 133. Radwan ZA, Bai Y, Matijevic N, et al. An emergency depart-ment thawed plasma protocol for severely injured patients. JAMA Surg. 2013;148(2):170-175. 134. Holcomb JB, Donathan DP, Cotton BA, et al. Prehospital transfusion of plasma and red blood cells in trauma patients. Prehosp Emerg Care. 2015;19(1):1-9. 135. Brown JB, Sperry JL, Fombona A, Billiar TR, Peitzman AB, Guyette FX. Pre-trauma center red blood cell transfusion is associated with improved early outcomes in air medical trauma patients. J Am Coll Surg. 2015;220(5):797-808. 136. O’Reilly DJ, Morrison JJ, Jansen JO, Apodaca AN, Rasmus-sen TE, Midwinter MJ. Prehospital blood transfusion in the en route management of severe combat trauma: a matched cohort study. J Trauma Acute Care Surg. 2014;77(3 Suppl 2):S114-S120. 137. Spinella PC, Perkins JG, Grathwohl KW, Beekley AC, Holcomb JB. Warm fresh

1	management of severe combat trauma: a matched cohort study. J Trauma Acute Care Surg. 2014;77(3 Suppl 2):S114-S120. 137. Spinella PC, Perkins JG, Grathwohl KW, Beekley AC, Holcomb JB. Warm fresh whole blood is independently asso-ciated with improved survival for patients with combat-related traumatic injuries. J Trauma. 2009;66(4 Suppl):S69-S76. 138. Nessen SC, Eastridge BJ, Cronk D, et al. Fresh whole blood use by forward surgical teams in Afghanistan is associated with improved survival compared to component therapy without platelets. Transfusion 2013;53 Suppl 1:107S-113S. 139. Neel S. Chapter 9: The military blood program. In: Medical Support of the U.S. Army in Vietnam. Washington D.C.: Office Brunicardi_Ch04_p0103-p0130.indd 12829/01/19 11:05 AM 129HEMOSTASIS, SURGICAL BLEEDING, AND TRANSFUSIONCHAPTER 4of the Surgeon General, Department of the Army; 1991; 114-126. Available at: http://history.amedd.army.mil/books-docs/vietnam/medicalsupport/chapter9.html. Accessed June 28,

1	AND TRANSFUSIONCHAPTER 4of the Surgeon General, Department of the Army; 1991; 114-126. Available at: http://history.amedd.army.mil/books-docs/vietnam/medicalsupport/chapter9.html. Accessed June 28, 2016. 140. Strandenes G, Austlid I, Apelseth TO, et al. Coagulation function of stored whole blood is preserved for 14 days in austere conditions: A ROTEM feasibility study during a Norwegian antipiracy mission and comparison to equal ratio reconstituted blood. J Trauma Acute Care Surg. 2015;78(6 Suppl 1):S31-38. 141. Cotton BA, Podbielski J, Camp E, et al. A randomized con-trolled pilot trial of modified whole blood versus component therapy in severely injured patients requiring large volume transfusions. Ann Surg. 2013;258(4):527-532. This is the first randomized study of whole blood in trauma patients. This small pilot study showed feasibility and suggested benefit. 142. Yazer MH, Jackson B, Sperry JL, et al. Initial safety and fea-sibility of cold-stored uncrossmatched whole blood

1	trauma patients. This small pilot study showed feasibility and suggested benefit. 142. Yazer MH, Jackson B, Sperry JL, et al. Initial safety and fea-sibility of cold-stored uncrossmatched whole blood transfu-sion in civilian trauma patients. J Trauma Acute Care Surg. 2016;81(1):21-26. 143. Stubbs JR, Zielinski MD, Jenkins D. The state of the science of whole blood: lessons learned at Mayo Clinic. Transfusion. 2016;56(Suppl 2):S173-181. 144. Rourke C, Curry N, Khan S, et al. Fibrinogen levels dur-ing trauma hemorrhage response to replacement therapy, and association with patient outcomes. J Thromb Haemost. 2012;10(7):1342-1351. 145. Inaba K, Karamanos E, Lustenberger T, et al. Impact of fibrin-ogen levels on outcomes after injury in patients requiring a massive transfusion. J Am Coll Surg. 2013;216(2):290-297. 146. Floccard B, Rugeri L, Faure A, et al. Early coagulopathy in trauma patients: an on-scene and hospital admission study. Injury. 2012;43(1):26-32. 147. Levy JH, Welsby I,

1	2013;216(2):290-297. 146. Floccard B, Rugeri L, Faure A, et al. Early coagulopathy in trauma patients: an on-scene and hospital admission study. Injury. 2012;43(1):26-32. 147. Levy JH, Welsby I, Goodnough LT. Fibrinogen as a therapeu-tic target for bleeding: a review of critical levels and replace-ment therapy. Transfusion. 2014 May;54(5):1389-1405. 148. Curry N, Rourke C, Davenport R, et al. Early cryoprecipitate for major haemorrhage in trauma: a randomised controlled feasibility trial. Br J Anaesth. 2015;115(1):76-83. 149. www.clinical trials.gov. Fibrinogen concentrate in trauma patients presumed to bleed; NCT01475344. Accessed December 28, 2016.149a. Meyer DE, Reynolds JW, Hobbs R, et al. The Incidence of Transfusion-Related Acute Lung Injury at a Large, Urban Tertiary Medical Center: A Decade’s Experience. Anesth Analg. 2018;127(2):444-449. 150. Pandey S, Vyas GN. Adverse-effects of plasma transfusion. Transfusion. 2012;52:65S-79S. 151. Goodnough LT, Brecher ME, Kanter MH.

1	A Decade’s Experience. Anesth Analg. 2018;127(2):444-449. 150. Pandey S, Vyas GN. Adverse-effects of plasma transfusion. Transfusion. 2012;52:65S-79S. 151. Goodnough LT, Brecher ME, Kanter MH. Transfusion medi-cine: blood transfusion. N Engl J Med. 1999;340:438. 152. Looney MR, Gropper MA, Matthay MA. Transfusion-related acute lung injury. Chest. 2004;126:249. 153. Toy P, Gajic O, Bacchetti P, et al. Transfusion-related acute lung injury: incidence and risk factors. Blood. 2012;119(7):1757-1767. 154. Zou S, Stramer SL, Dodd RY. Donor testing and risk: cur-rent prevalence, incidence, and residual risk of transfusion-transmissible agents in US allogeneic donations. Transfusion Med Rev. 2012;26(2):119-128. 155. Musso D, Nhan T, Robin E, et al. Potential for Zika virus transmission through blood transfusion demonstrated during an outbreak in French Polynesia, November 2013 to February 2014. Euro Surveill. 2014;19(14). 156. Jimenez A, Shaz BH, Bloch EM. Zika Virus and the blood sup-ply:

1	blood transfusion demonstrated during an outbreak in French Polynesia, November 2013 to February 2014. Euro Surveill. 2014;19(14). 156. Jimenez A, Shaz BH, Bloch EM. Zika Virus and the blood sup-ply: what do we know? Transfus Med Rev. 2017;31(1):1-10. 157. Bierlaire D, Mauguin S, Broult J, Musso D. Zika virus and blood transfusion: the experience of French Polynesia. Trans-fusion. 2017;57(3pt2):729-733. 158. Hoffman M, Monroe DM. Coagulation 2006: a modern view of hemostasis. Hematol Oncol Clin North Am. 2007;21:1-11. 159. Mallet SV, Cox DJA. Thromboelastography: a review article. Br J Anaesth. 1992;69:307. 160. Jackson GN, Ashpole KJ, Yentis SM. The TEG vs the ROTEM thromboelastography/ thromboelastometry systems. Anaesthe-sia. 2009;64(2):212-215. 161. Cotton BA, Radwan ZA, Matijevic N, et al. Admission rapid thromboelastography (rTEG) predicts development of pulmonary embolism in trauma patients. J Trauma. 2012;72(6):1470-1477. 162. Cotton BA, Faz G, Hatch Q, et al. Rapid

1	N, et al. Admission rapid thromboelastography (rTEG) predicts development of pulmonary embolism in trauma patients. J Trauma. 2012;72(6):1470-1477. 162. Cotton BA, Faz G, Hatch Q, et al. Rapid thromboelastogra-phy (r-TEG) delivers real-time results that predict transfusion within one hour of admission. J Trauma. 2011;71(2):407-417. 163. Schöchl H, Cotton BA, Inaba K, et al. FIBTEM provides early prediction of massive transfusion in trauma. Crit Care. 2011;15:R265-R271. 164. Cotton BA, Harvin JA, Kostousouv V, et al. Hyperfibrinoly-sis on admission is an uncommon but highly lethal event associated with shock and pre-hospital fluid administration. J Trauma. 2012;72(2):365-370. 165. Holcomb JB, Minei KM, Scerbo ML, et al. Admission rapid thromboelastography (r-TEG) can replace conventional coag-ulation tests in the emergency department: experience with 1974 consecutive trauma patients. Ann Surg. 2012;256(3): 476-486.Brunicardi_Ch04_p0103-p0130.indd 12929/01/19 11:05 AM

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1	ShockBrian S. Zuckerbraun, Andrew B. Peitzman, and Timothy R. Billiar 5chapter“Shock is the manifestation of the rude unhinging of the machinery of life.”1—Samuel V. Gross, 1872EVOLUTION IN UNDERSTANDING SHOCKOverviewShock, at its most rudimentary definition and regardless of its etiology, is the failure to meet the metabolic needs of the cell and the consequences that ensue. The initial cellular injury that occurs is reversible; however, the injury will become irre-versible if tissue perfusion is prolonged or severe enough such that, at the cellular level, compensation is no longer pos-sible. Our understanding of shock and the disease processes that result in shock made its most significant advances throughout the 20th century as our appreciation for the physiology and pathophysiology of shock matured. Most notably, our compre-hension of the sympathetic and neuroendocrine stress responses on the cardiovascular system has flourished. The clinical mani-festations of these physiologic

1	shock matured. Most notably, our compre-hension of the sympathetic and neuroendocrine stress responses on the cardiovascular system has flourished. The clinical mani-festations of these physiologic responses are most often what lead practitioners to the diagnosis of shock as well as guide the management of patients in shock. However, hemodynamic parameters such as blood pressure and heart rate are relatively insensitive measures of shock, and additional considerations must be used to help aid in early diagnosis and treatment of patients in shock. The general approach to the management of patients in shock has been empiric: assuring a secure airway with adequate ventilation, control of hemorrhage in the bleeding patient, and restoration of vascular volume and tissue perfusion.Historical BackgroundIntegral to our understanding of shock is the appreciation that our bodies attempt to maintain a state of homeostasis. Claude Bernard suggested in the mid-19th century that the organism

1	BackgroundIntegral to our understanding of shock is the appreciation that our bodies attempt to maintain a state of homeostasis. Claude Bernard suggested in the mid-19th century that the organism attempts to maintain constancy in the internal environment against external 1forces that attempt to disrupt the milieu interieur.2 Walter B. Cannon carried Bernard’s observations further and introduced the term homeostasis, emphasizing that an organism’s ability to survive was related to maintenance of homeostasis.3 The failure of physiologic systems to buffer the organism against external forces results in organ and cellular dysfunction, what is clini-cally recognized as shock. He first described the “fight or flight response,” generated by elevated levels of catecholamines in the bloodstream. Cannon’s observations on the battlefields of World War I led him to propose that the initiation of shock was due to a disturbance of the nervous system that resulted in vasodilation and hypotension. He

1	observations on the battlefields of World War I led him to propose that the initiation of shock was due to a disturbance of the nervous system that resulted in vasodilation and hypotension. He proposed that secondary shock, with its attendant capillary permeability leak, was caused by a “toxic factor” released from the tissues.In a series of critical experiments, Alfred Blalock docu-mented that the shock state in hemorrhage was associated with reduced cardiac output due to volume loss, not a “toxic factor.”4 In 1934, Blalock proposed four categories of shock: hypovole-mic, vasogenic, cardiogenic, and neurogenic. Hypovolemic shock, the most common type, results from loss of circulating blood volume. This may result from loss of whole blood (hemorrhagic shock), plasma, interstitial fluid (bowel obstruction), or a combi-nation. Vasogenic shock results from decreased resistance within capacitance vessels, usually seen in sepsis. Neurogenic shock is a form of vasogenic shock in which

1	(bowel obstruction), or a combi-nation. Vasogenic shock results from decreased resistance within capacitance vessels, usually seen in sepsis. Neurogenic shock is a form of vasogenic shock in which spinal cord injury or spinal anesthesia causes vasodilation due to acute loss of sympathetic vascular tone. Cardiogenic shock results from failure of the heart as a pump, as in arrhythmias or acute myocardial infarction (MI).This categorization of shock based on etiology persists today (Table 5-1). In recent clinical practice, further classifi-cation has described six types of shock: hypovolemic, septic (vasodilatory), neurogenic, cardiogenic, obstructive, and trau-matic shock. Obstructive shock is a form of cardiogenic shock that results from mechanical impediment to circulation leading to depressed cardiac output rather than primary cardiac failure. Evolution in Understanding Shock 131Overview / 131Historical Background / 131Current Definitions and Challenges/ 133Pathophysiology of

1	depressed cardiac output rather than primary cardiac failure. Evolution in Understanding Shock 131Overview / 131Historical Background / 131Current Definitions and Challenges/ 133Pathophysiology of Shock 133Neuroendocrine and Organ-Specific Responses to Hemorrhage/ 134Afferent Signals / 134Efferent Signals / 135Circulatory Homeostasis / 136Metabolic Effects 136Cellular Hypoperfusion / 137Immune and Inflammatory Responses 137Cytokines/Chemokines / 138Complement / 140Neutrophils / 140Cell Signaling / 140Forms of Shock 141Hypovolemic/Hemorrhagic / 141Traumatic Shock / 145Septic Shock (Vasodilatory Shock) / 145Cardiogenic Shock / 148Obstructive Shock / 150Neurogenic Shock / 151Endpoints in Resuscitation 152Assessment of Endpoints in Resuscitation / 152Brunicardi_Ch05_p0131-p0156.indd 13129/01/19 11:06 AM 132Table 5-1Classification of shockHypovolemicCardiogenicSeptic (vasogenic)NeurogenicTraumaticObstructiveKey Points1 Shock is defined as a failure to meet the metabolic demands of

1	11:06 AM 132Table 5-1Classification of shockHypovolemicCardiogenicSeptic (vasogenic)NeurogenicTraumaticObstructiveKey Points1 Shock is defined as a failure to meet the metabolic demands of cells and tissues and the consequences that ensue.2 A central component of shock is decreased tissue perfusion. This may be a direct consequence of the etiology of shock, such as in hypovolemic/hemorrhagic, cardiogenic, or neuro-genic etiologies, or may be secondary to elaborated or released molecules or cellular products that result in endothelial/ cellular activation, such as in septic shock or traumatic shock.3 Physiologic responses to shock are based upon a series of affer-ent (sensing) signals and efferent responses that include neu-roendocrine, metabolic, and immune/inflammatory signaling.4 The mainstay of treatment of hemorrhagic/hypovolemic shock includes volume resuscitation with blood products. In the case of hemorrhagic shock, timely control of bleeding is essential and influences

1	mainstay of treatment of hemorrhagic/hypovolemic shock includes volume resuscitation with blood products. In the case of hemorrhagic shock, timely control of bleeding is essential and influences outcome.5 Prevention of hypothermia, acidemia, and coagulopathy are essential in the management of patients in hemorrhagic shock.6 The mainstay of treatment of septic shock is fluid resuscita-tion, initiation of appropriate antibiotic therapy, and control of the source of infection. This includes drainage of infected fluid collections, removal of infected foreign bodies, and debridement of devitalized tissues.7 A combination of physiologic parameters and markers of organ perfusion/tissue oxygenation are used to determine if patients are in shock and to follow the efficacy of resuscitation.This includes etiologies such as pulmonary embolism or tension pneumothorax. In traumatic shock, soft tissue and bony injury lead to the activation of inflammatory cells and the release of circulating

1	includes etiologies such as pulmonary embolism or tension pneumothorax. In traumatic shock, soft tissue and bony injury lead to the activation of inflammatory cells and the release of circulating factors, such as cytokines and intracellular molecules that modulate the immune response. Recent investigations have revealed that the inflammatory mediators released in response to tissue injury (damage-associated molecular patterns [DAMPs]) are recognized by many of the same cellular receptors (pattern recognition receptors [PRRs]) and activate similar signaling pathways as do bacterial products elaborated in sepsis (pathogen-associated molecular patterns), such as lipopolysaccharide.5 These effects of tissue injury are combined with the effects of hemorrhage, creating a more complex and amplified deviation from homeostasis.In the midto later 20th century, further development of experimental models contributed significantly to the under-standing of the pathophysiology of shock. In 1947,

1	deviation from homeostasis.In the midto later 20th century, further development of experimental models contributed significantly to the under-standing of the pathophysiology of shock. In 1947, Wiggers developed a sustainable, irreversible model of hemorrhagic shock based on uptake of shed blood into a reservoir to maintain a set level of hypotension.6 G. Tom Shires added further under-standing of hemorrhagic shock with a series of clinical stud-ies demonstrating that a large extracellular fluid deficit, greater than could be attributed to vascular refilling alone, occurred in severe hemorrhagic shock.7,8 The phenomenon of fluid redistri-bution after major trauma involving blood loss was termed third spacing and described the translocation of intravascular volume into the peritoneum, bowel, burned tissues, or crush injury sites. These seminal studies form the scientific basis for the current treatment of hemorrhagic shock with red blood cells and lac-tated Ringer’s solution or isotonic

1	burned tissues, or crush injury sites. These seminal studies form the scientific basis for the current treatment of hemorrhagic shock with red blood cells and lac-tated Ringer’s solution or isotonic saline.As resuscitation strategies evolved and patients survived the initial consequences of hemorrhage, new challenges of sustained shock became apparent. During the Vietnam War, aggressive fluid resuscitation with red blood cells and crystal-loid solution or plasma resulted in survival of patients who pre-viously would have succumbed to hemorrhagic shock. Renal failure became a less frequent clinical problem; however, a new disease process, acute fulminant pulmonary failure, appeared as an early cause of death after seemingly successful surgery to control hemorrhage. Initially called DaNang lung or shock lung, the clinical problem became recognized as acute respiratory dis-tress syndrome (ARDS). This led to new methods of prolonged mechanical ventilation. Our current concept of ARDS is a

1	or shock lung, the clinical problem became recognized as acute respiratory dis-tress syndrome (ARDS). This led to new methods of prolonged mechanical ventilation. Our current concept of ARDS is a com-ponent in the spectrum of multiple organ system failure.Studies and clinical observations over the past two decades have extended the early observations of Canon, that “restoration of blood pressure prior to control of active bleeding may result in loss of blood that is sorely needed,” and challenged the appro-priate endpoints in resuscitation of uncontrolled hemorrhage.9 Core principles in the management of patients in hemorrhagic shock include: (a) control of active hemorrhage must occur promptly (delay in control of bleeding increases mortality and recent battlefield data would suggest that in the young and otherwise healthy population commonly injured in combat, that control of bleeding is the paramount priority); (b) volume resuscitation with blood products (red blood cells, plasma,

1	in the young and otherwise healthy population commonly injured in combat, that control of bleeding is the paramount priority); (b) volume resuscitation with blood products (red blood cells, plasma, and platelets) with limited volume of crystalloid must occur while operative control of bleeding is achieved; (c) unrecognized or inadequately corrected hypoperfusion increases morbidity and mortality (i.e., inadequate resuscitation results in avoidable early deaths from shock); and (d) excessive fluid resuscitation may exacerbate bleeding (i.e., uncontrolled resuscitation is harmful). Thus, both inadequate or unrestrained volume resus-citation are harmful.Likewise, observations in the management of septic shock have led to consensus statements and evolving guideline based management.10 Core principles in the management of patients in septic shock include: (a) septic shock is an emergency, and treatment/resuscitation should begin as early as possible; (b) specific anatomic diagnosis of

1	principles in the management of patients in septic shock include: (a) septic shock is an emergency, and treatment/resuscitation should begin as early as possible; (b) specific anatomic diagnosis of infection requiring emergent source control be identified or excluded as rapidly as possible and that any required source control intervention be imple-mented as soon as medically and logistically practical; (c) ini-tiation of broad spectrum antibiotics within 1 hour of diagnosis; (d) in the resuscitation from sepsis-induced hypoperfusion, at Brunicardi_Ch05_p0131-p0156.indd 13229/01/19 11:06 AM 133SHOCKCHAPTER 5least 30 mL/kg of intravenous crystalloid fluid be given within the first 3 hours, and additional fluids be guided by frequent reassessment of hemodynamic status; (e) vasopressors (norepi-nephrine) should be added to achieve a mean arterial pressure of 65 mmHg if fluid resuscitation is inadequate.Current Definitions and ChallengesA modern definition and approach to shock

1	(norepi-nephrine) should be added to achieve a mean arterial pressure of 65 mmHg if fluid resuscitation is inadequate.Current Definitions and ChallengesA modern definition and approach to shock acknowledges that shock consists of inadequate tissue perfusion marked by decreased delivery of required metabolic substrates and inade-quate removal of cellular waste products. This involves failure of oxidative metabolism that can involve defects of oxygen (O2) delivery, transport, and/or utilization. Current challenges include moving beyond fluid resuscitation based upon endpoints of tissue oxygenation and using therapeutic strategies at the cellular and molecular level. This approach will help to identify compensated patients or patients early in the course of their disease, initiate appropriate treatment, and allow for con-tinued evaluation for the efficacy of resuscitation and adjuncts.Current investigations focus on determining the cellular events that often occur in parallel to result

1	treatment, and allow for con-tinued evaluation for the efficacy of resuscitation and adjuncts.Current investigations focus on determining the cellular events that often occur in parallel to result in organ dysfunc-tion, shock irreversibility, and death. This chapter will review our current understanding of the pathophysiology and cellular responses of shock states. Current and experimental diagnostic and therapeutic modalities for the different categories of shock are reviewed, with a focus on hemorrhagic/hypovolemic shock and septic shock.PATHOPHYSIOLOGY OF SHOCKRegardless of etiology, the initial physiologic responses in shock are driven by tissue hypoperfusion and the developing cellular energy deficit. This imbalance between cellular supply and demand leads to neuroendocrine and inflammatory responses, the magnitude of which is usually proportional to the degree and duration of shock. The specific responses will differ based on the etiology of shock, as certain physiologic

1	inflammatory responses, the magnitude of which is usually proportional to the degree and duration of shock. The specific responses will differ based on the etiology of shock, as certain physiologic responses may be limited by the inciting pathology. For exam-ple, the cardiovascular response driven by the sympathetic ner-vous system is markedly blunted in neurogenic or septic shock. Additionally, decreased perfusion may occur as a consequence of cellular activation and dysfunction, such as in septic shock and to a lesser extent traumatic shock (Fig. 5-1). Many of the organ-specific responses are aimed at maintaining perfusion in the cerebral and coronary circulation. These are regulated at multiple levels including (a) stretch receptors and baroreceptors in the heart and vasculature (carotid sinus and aortic arch), (b) chemoreceptors, (c) cerebral ischemia responses, (d) release of endogenous vasoconstrictors, (e) shifting of fluid into the intra-vascular space, and (f) renal

1	(carotid sinus and aortic arch), (b) chemoreceptors, (c) cerebral ischemia responses, (d) release of endogenous vasoconstrictors, (e) shifting of fluid into the intra-vascular space, and (f) renal reabsorption and conservation of salt and water.Furthermore, the pathophysiologic responses vary with time and in response to resuscitation. In hemorrhagic shock, the body can compensate for the initial loss of blood volume primar-ily through the neuroendocrine response to maintain hemody-namics. This represents the compensated phase of shock. With continued hypoperfusion, which may be unrecognized, cellular death and injury are ongoing, and the decompensation phase of shock ensues. Microcirculatory dysfunction, parenchymal tissue damage, and inflammatory cell activation can perpetuate hypo-perfusion. Ischemia/reperfusion injury will often exacerbate the initial insult. These effects at the cellular level, if untreated, will lead to compromise of function at the organ system level, thus

1	Ischemia/reperfusion injury will often exacerbate the initial insult. These effects at the cellular level, if untreated, will lead to compromise of function at the organ system level, thus leading to the “vicious cycle” of shock (Fig. 5-2). Persis-tent hypoperfusion results in further hemodynamic derange-ments and cardiovascular collapse. This has been termed the irreversible phase of shock and can develop quite insidiously and may only be obvious in retrospect. At this point, there has occurred extensive enough parenchymal and microvascular injury such that volume resuscitation fails to reverse the pro-cess, leading to death of the patient. In experimental animal models of hemorrhagic shock (modified Wiggers model), this is 23Figure 5-1. Pathways leading to decreased tissue perfusion and shock. Decreased tissue perfusion can result directly from hemorrhage/hypovolemia, cardiac failure, or neurologic injury. Decreased tissue perfusion and cellu-lar injury can then result in immune

1	and shock. Decreased tissue perfusion can result directly from hemorrhage/hypovolemia, cardiac failure, or neurologic injury. Decreased tissue perfusion and cellu-lar injury can then result in immune and inflammatory responses. Alternatively, elaboration of microbial products during infection or release of endogenous cellular products from tissue injury can result in cellular activation to subsequently influ-ence tissue perfusion and the develop-ment of shock. HMGB1 = high mobility group box 1; LPS = lipopolysaccharide; RAGE = receptor for advanced glycation end products.Bacterial products(i.e., LPS)Direct effectPattern recognition receptor activation(Toll-like receptors, RAGE)TissueinjuryDamage associatedmolecular patterns(i.e., HMGB1, heparan sulfate)Released/elaboratedmediators ofinflammationCellular activationDecreased tissue perfusionCellular hypoxia/ischemiaShockDisruptionhost-microbeequilibriumAcute heart failureNeurogenicHemorrhageTraumaCellular

1	ofinflammationCellular activationDecreased tissue perfusionCellular hypoxia/ischemiaShockDisruptionhost-microbeequilibriumAcute heart failureNeurogenicHemorrhageTraumaCellular effectBrunicardi_Ch05_p0131-p0156.indd 13329/01/19 11:06 AM 134BASIC CONSIDERATIONSPART Irepresented by the “uptake phase” or “compensation endpoint” when shed blood must be returned to the animal to sustain the hypotension at the set level to prevent further hypotension and death.11 If shed blood volume is slowly returned to maintain the set level of hypotension, eventually the injury progresses to irreversible shock, where further volume will not reverse the process and the animal dies (Fig. 5-3).Neuroendocrine and Organ-Specific Responses to HemorrhageThe goal of the neuroendocrine response to hemorrhage is to maintain perfusion to the heart and the brain, even at the expense of other organ systems. Peripheral vasoconstriction occurs, and fluid excretion is inhibited. The mechanisms include autonomic

1	is to maintain perfusion to the heart and the brain, even at the expense of other organ systems. Peripheral vasoconstriction occurs, and fluid excretion is inhibited. The mechanisms include autonomic control of peripheral vascular tone and cardiac contractility, hormonal response to stress and volume depletion, and local microcirculatory mechanisms that are organ specific and regu-late regional blood flow. The initial stimulus is loss of circulat-ing blood volume in hemorrhagic shock. The magnitude of the neuroendocrine response is based on both the volume of blood lost and the rate at which it is lost.Afferent SignalsAfferent impulses transmitted from the periphery are processed within the central nervous system (CNS) and activate the reflexive effector responses or efferent impulses. These effec-tor responses are designed to expand plasma volume, maintain peripheral perfusion and tissue O2 delivery, and restore homeo-stasis. The afferent impulses that initiate the body’s intrinsic

1	effec-tor responses are designed to expand plasma volume, maintain peripheral perfusion and tissue O2 delivery, and restore homeo-stasis. The afferent impulses that initiate the body’s intrinsic adaptive responses and converge in the CNS originate from a variety of sources. The initial inciting event usually is loss of circulating blood volume. Other stimuli that can produce the neuroendocrine response include pain, hypoxemia, hypercarbia, acidosis, infection, change in temperature, emotional arousal, or hypoglycemia. The sensation of pain from injured tissue is trans-mitted via the spinothalamic tracts, resulting in activation of the hypothalamic-pituitary-adrenal axis, as well as activation of the autonomic nervous system (ANS) to induce direct sympathetic stimulation of the adrenal medulla to release catecholamines.Baroreceptors also are an important afferent pathway in initiation of adaptive responses to shock. Volume receptors, sensitive to changes in both chamber pressure and

1	to release catecholamines.Baroreceptors also are an important afferent pathway in initiation of adaptive responses to shock. Volume receptors, sensitive to changes in both chamber pressure and wall stretch, are present within the atria of the heart. They become activated with low volume hemorrhage or mild reductions in right atrial pressure. Receptors in the aortic arch and carotid bodies respond to alterations in pressure or stretch of the arterial wall, respond-ing to larger reductions in intravascular volume or pressure. These receptors normally inhibit induction of the ANS. When activated, these baroreceptors diminish their output, thus dis-inhibiting the effect of the ANS. The ANS then increases its output, principally via sympathetic activation at the vasomotor centers of the brain stem, producing centrally mediated constric-tion of peripheral vessels.Chemoreceptors in the aorta and carotid bodies are sensi-tive to changes in O2 tension, H+ ion concentration, and car-bon dioxide

1	producing centrally mediated constric-tion of peripheral vessels.Chemoreceptors in the aorta and carotid bodies are sensi-tive to changes in O2 tension, H+ ion concentration, and car-bon dioxide (CO2) levels. Stimulation of the chemoreceptors results in vasodilation of the coronary arteries, slowing of the heart rate, and vasoconstriction of the splanchnic and skeletal circulation. In addition, a variety of protein and nonprotein mediators are produced at the site of injury as part of the inflam-matory response, and they act as afferent impulses to induce a host response. These mediators include histamine, cytokines, Decreased cardiac outputDecreased tissue perfusion˜ Venousreturn˜ CoronaryperfusionMetabolicacidosisCellularhypoxiaParenchymal cell injuryEndothelial activation/microcirculatory damageCellularaggregationIntracellularfluid lossFigure 5-2. The “vicious cycle of shock.” Regardless of the etiol-ogy, decreased tissue perfusion and shock results in a feed-forward loop that can

1	damageCellularaggregationIntracellularfluid lossFigure 5-2. The “vicious cycle of shock.” Regardless of the etiol-ogy, decreased tissue perfusion and shock results in a feed-forward loop that can exacerbate cellular injury and tissue dysfunction.Figure 5-3. Rat model of hemorrhagic shock through the phases of compensa-tion, decompensation, and irreversibility. The percentages shown above the curve represent survival rates. (Reproduced with permission from Shah NS, Kelly E, Billiar TR, et al. Utility of clinical param-eters of tissue oxygenation in a quantita-tive model of irreversible hemorrhagic shock, Shock. 1998 Nov;10(5):343-346.)Rat hemorrhagic shock model24-hour survival following resuscitationMean arterial pressureCompensatedDecompensatedIrreversible% Shedblood returnTransition to acute irreversible shockTransition to subacute lethal shockABDeath100%90%50%30%10%0%10%20%30%40%50%0%Compensation endpoint8040ABBrunicardi_Ch05_p0131-p0156.indd 13429/01/19 11:06 AM

1	to acute irreversible shockTransition to subacute lethal shockABDeath100%90%50%30%10%0%10%20%30%40%50%0%Compensation endpoint8040ABBrunicardi_Ch05_p0131-p0156.indd 13429/01/19 11:06 AM 135SHOCKCHAPTER 5eicosanoids, and endothelins, among others that are discussed in greater detail later in this chapter in the “Immune and Inflam-matory Responses” section.Efferent SignalsCardiovascular Response. Changes in cardiovascular func-tion are a result of the neuroendocrine response and ANS response to shock, and they constitute a prominent feature of both the body’s adaptive response mechanism and the clinical signs and symptoms of the patient in shock. Hemorrhage results in diminished venous return to the heart and decreased cardiac output. This is compensated by increased cardiac heart rate and contractility, as well as venous and arterial vasoconstriction. Stimulation of sympathetic fibers innervating the heart leads to activation of β1-adrenergic receptors that increase heart rate and

1	contractility, as well as venous and arterial vasoconstriction. Stimulation of sympathetic fibers innervating the heart leads to activation of β1-adrenergic receptors that increase heart rate and contractility in this attempt to increase cardiac output. Increased myocardial O2 consumption occurs as a result of the increased workload; thus, myocardial O2 supply must be maintained, or myocardial dysfunction will develop. The car-diovascular response in hemorrhage/hypovolemia differs from the responses elicited with the other etiologies of shock. These are compared in Table 5-2.Direct sympathetic stimulation of the peripheral circula-tion via the activation of α1-adrenergic receptors on arterioles induces vasoconstriction and causes a compensatory increase in systemic vascular resistance and blood pressure. The arte-rial vasoconstriction is not uniform; marked redistribution of blood flow results. Selective perfusion to tissues occurs due to regional variations in arteriolar resistance,

1	blood pressure. The arte-rial vasoconstriction is not uniform; marked redistribution of blood flow results. Selective perfusion to tissues occurs due to regional variations in arteriolar resistance, with blood shunted away from less essential organ beds such as the intestine, kidney, and skin. In contrast, the brain and heart have autoreg-ulatory mechanisms that attempt to preserve their blood flow despite a global decrease in cardiac output. Direct sympathetic stimulation also induces constriction of venous vessels, decreas-ing the capacitance of the circulatory system and accelerating blood return to the central circulation.Increased sympathetic output induces catecholamine release from the adrenal medulla. Catecholamine levels peak within 24 to 48 hours of injury and then return to baseline. Persis-tent elevation of catecholamine levels beyond this time suggests ongoing noxious afferent stimuli. The majority of the circulating epinephrine is produced by the adrenal medulla, while

1	Persis-tent elevation of catecholamine levels beyond this time suggests ongoing noxious afferent stimuli. The majority of the circulating epinephrine is produced by the adrenal medulla, while norepi-nephrine is derived from synapses of the sympathetic nervous system. Catecholamine effects on peripheral tissues include stimulation of hepatic glycogenolysis and gluconeogenesis to increase circulating glucose availability to peripheral tissues, an increase in skeletal muscle glycogenolysis, suppression of insulin release, and increased glucagon release.Hormonal Response. The stress response includes activation of the ANS as discussed previously in the “Afferent Signals” section, as well as activation of the hypothalamic-pituitary-adrenal axis. Shock stimulates the hypothalamus to release corticotropin-releasing hormone, which results in the release of adrenocorticotropic hormone (ACTH) by the pituitary. ACTH subsequently stimulates the adrenal cortex to release cortisol. Cortisol acts

1	hormone, which results in the release of adrenocorticotropic hormone (ACTH) by the pituitary. ACTH subsequently stimulates the adrenal cortex to release cortisol. Cortisol acts synergistically with epinephrine and glucagon to induce a catabolic state. Cortisol stimulates gluconeogenesis and insulin resistance, resulting in hyperglycemia as well as muscle cell protein breakdown and lipolysis to provide sub-strates for hepatic gluconeogenesis. Cortisol causes retention of sodium and water by the nephrons of the kidney. In the setting of severe hypovolemia, ACTH secretion occurs independently of cortisol negative feedback inhibition.The renin-angiotensin system is activated in shock. Decreased renal artery perfusion, β-adrenergic stimulation, and increased renal tubular sodium concentration cause the release of renin from the juxtaglomerular cells. Renin catalyzes the conversion of angiotensinogen (produced by the liver) to angiotensin I, which is then converted to angiotensin II by

1	cause the release of renin from the juxtaglomerular cells. Renin catalyzes the conversion of angiotensinogen (produced by the liver) to angiotensin I, which is then converted to angiotensin II by angiotensin-converting enzyme (ACE) produced in the lung. While angiotensin I has no significant functional activity, angiotensin II is a potent vasoconstrictor of both splanchnic and peripheral vascular beds, and also stimulates the secretion of aldosterone, ACTH, and antidiuretic hormone (ADH). Aldo-sterone, a mineralocorticoid, acts on the nephron to promote reabsorption of sodium, and as a consequence, water. Potassium and hydrogen ions are lost in the urine in exchange for sodium.The pituitary also releases vasopressin or ADH in response to hypovolemia, changes in circulating blood volume sensed by baroreceptors and left atrial stretch receptors, and increased plasma osmolality detected by hypothalamic osmo-receptors. Epinephrine, angiotensin II, pain, and hyperglycemia increase

1	sensed by baroreceptors and left atrial stretch receptors, and increased plasma osmolality detected by hypothalamic osmo-receptors. Epinephrine, angiotensin II, pain, and hyperglycemia increase production of ADH. ADH levels remain elevated for about 1 week after the initial insult, depending on the sever-ity and persistence of the hemodynamic abnormalities. ADH acts on the distal tubule and collecting duct of the nephron to increase water permeability, decrease water and sodium losses, and preserve intravascular volume. Also known as arginine vasopressin, ADH acts as a potent mesenteric vasoconstrictor, shunting circulating blood away from the splanchnic organs during hypovolemia.12 This may contribute to intestinal isch-emia and predispose to intestinal mucosal barrier dysfunction in Table 5-2Hemodynamic responses to different types of shockTYPE OF SHOCKCARDIAC INDEXSVRVENOUS CAPACITANCECVP/PCWPSvo2CELLULAR/METABOLIC

1	and predispose to intestinal mucosal barrier dysfunction in Table 5-2Hemodynamic responses to different types of shockTYPE OF SHOCKCARDIAC INDEXSVRVENOUS CAPACITANCECVP/PCWPSvo2CELLULAR/METABOLIC EFFECTSHypovolemic↓↑↓↓↓EffectSeptic↑↑↓↑↑↓↑↓CauseCardiogenic↓↓↑↑→↑↓EffectNeurogenic↑↓→↓↓EffectThe hemodynamic responses are indicated by arrows to show an increase (↑), severe increase (↑↑), decrease (↓), severe decrease (↓↓), varied response (↑↓), or little effect →). CVP = central venous pressure; PCWP = pulmonary capillary wedge pressure; Svo2 = mixed venous oxygen saturation; SVR = systemic vascular resistance.Brunicardi_Ch05_p0131-p0156.indd 13529/01/19 11:06 AM 136BASIC CONSIDERATIONSPART Ishock states. Vasopressin also increases hepatic gluconeogen-esis and increases hepatic glycolysis.In septic states, endotoxin directly stimulates arginine vasopressin secretion independently of blood pressure, osmotic, or intravascular volume changes. Proinflammatory cytokines also contribute to

1	septic states, endotoxin directly stimulates arginine vasopressin secretion independently of blood pressure, osmotic, or intravascular volume changes. Proinflammatory cytokines also contribute to arginine vasopressin release. Interestingly, patients on chronic therapy with ACE inhibitors are more at risk of developing hypotension and vasodilatory shock with open heart surgery. Low plasma levels of arginine vasopressin were confirmed in these patients.13Circulatory HomeostasisPreload. At rest, the majority of the blood volume is within the venous system. Venous return to the heart generates ventricular end-diastolic wall tension, a major determinant of cardiac out-put. Gravitational shifts in blood volume distribution are quickly corrected by alterations in venous capacity. With decreased arte-riolar inflow, there is active contraction of the venous smooth muscle and passive elastic recoil in the thin-walled systemic veins. This increases venous return to the heart, thus maintain-ing

1	arte-riolar inflow, there is active contraction of the venous smooth muscle and passive elastic recoil in the thin-walled systemic veins. This increases venous return to the heart, thus maintain-ing ventricular filling.Most alterations in cardiac output in the normal heart are related to changes in preload. Increases in sympathetic tone have a minor effect on skeletal muscle beds but produce a dra-matic reduction in splanchnic blood volume, which normally holds 20% of the blood volume.The normal circulating blood volume is maintained within narrow limits by the kidney’s ability to manage salt and water balance with external losses via systemic and local hemody-namic changes and hormonal effects of renin, angiotensin, and ADH. These relatively slow responses maintain preload by altering circulating blood volume. Acute responses to intravas-cular volume include changes in venous tone, systemic vascular resistance, and intrathoracic pressure, with the slower hormonal changes less

1	circulating blood volume. Acute responses to intravas-cular volume include changes in venous tone, systemic vascular resistance, and intrathoracic pressure, with the slower hormonal changes less important in the early response to volume loss. Furthermore, the net effect of preload on cardiac output is influ-enced by cardiac determinants of ventricular function, which include coordinated atrial activity and tachycardia.Ventricular Contraction. The Frank-Starling curve describes the force of ventricular contraction as a function of its preload. This relationship is based on force of contraction being deter-mined by initial muscle length. Intrinsic cardiac disease will shift the Frank-Starling curve and alter mechanical performance of the heart. In addition, cardiac dysfunction has been demon-strated experimentally in burns and in hemorrhagic, traumatic, and septic shock.Afterload. Afterload is the force that resists myocardial work during contraction. Arterial pressure is the major

1	demon-strated experimentally in burns and in hemorrhagic, traumatic, and septic shock.Afterload. Afterload is the force that resists myocardial work during contraction. Arterial pressure is the major component of afterload influencing the ejection fraction. This vascular resistance is determined by precapillary smooth muscle sphinc-ters. Blood viscosity also will increase vascular resistance. As afterload increases in the normal heart, stroke volume can be maintained by increases in preload. In shock, with decreased circulating volume and therefore diminished preload, this com-pensatory mechanism to sustain cardiac output is impeded. The stress response with acute release of catecholamines and sym-pathetic nerve activity in the heart increases contractility and heart rate.Microcirculation. The microvascular circulation plays an integral role in regulating cellular perfusion and is significantly influenced in response to shock. The microvascular bed is innervated by the sympathetic

1	microvascular circulation plays an integral role in regulating cellular perfusion and is significantly influenced in response to shock. The microvascular bed is innervated by the sympathetic nervous system and has a pro-found effect on the larger arterioles. Following hemorrhage, larger arterioles vasoconstrict; however, in the setting of sep-sis or neurogenic shock, these vessels vasodilate. Addition-ally, a host of other vasoactive proteins, including vasopressin, angiotensin II, and endothelin-1, also lead to vasoconstriction to limit organ perfusion to organs such as skin, skeletal muscle, kidneys, and the GI tract to preserve perfusion of the myocar-dium and CNS.Flow in the capillary bed is heterogeneous in shock states, which likely is secondary to multiple local mechanisms, including endothelial cell swelling, dysfunction, and activa-tion marked by the recruitment of leukocytes and platelets.14 Together, these mechanisms lead to diminished capillary perfu-sion that may persist

1	endothelial cell swelling, dysfunction, and activa-tion marked by the recruitment of leukocytes and platelets.14 Together, these mechanisms lead to diminished capillary perfu-sion that may persist after resuscitation. In hemorrhagic shock, correction of hemodynamic parameters and restoration of O2 delivery generally leads to restoration of tissue O2 consumption and tissue O2 levels. In contrast, regional tissue dysoxia often persists in sepsis, despite similar restoration of hemodynamics and O2 delivery. Whether this defect in O2 extraction in sepsis is the result of heterogeneous impairment of the microcirculation (intraparenchymal shunting) or impaired tissue parenchymal cell oxidative phosphorylation and O2 consumption by the mito-chondria is not resolved.15 Interesting data suggest that in sepsis the response to limit O2 consumption by the tissue parenchymal cells is an adaptive response to the inflammatory signaling and decreased perfusion.16An additional pathophysiologic

1	that in sepsis the response to limit O2 consumption by the tissue parenchymal cells is an adaptive response to the inflammatory signaling and decreased perfusion.16An additional pathophysiologic response of the microcir-culation to shock is failure of the integrity of the endothelium of the microcirculation and development of capillary leak, intracellular swelling, and the development of an extracellu-lar fluid deficit. Seminal work by Shires helped to define this phenomenon.8,17 There is decreased capillary hydrostatic pres-sure secondary to changes in blood flow and increased cellular uptake of fluid. The result is a loss of extracellular fluid vol-ume. The cause of intracellular swelling is multifactorial, but dysfunction of energy-dependent mechanisms, such as active transport by the sodium-potassium pump contributes to loss of membrane integrity.Capillary dysfunction also occurs secondary to activation of endothelial cells by circulating inflammatory mediators gen-erated in

1	the sodium-potassium pump contributes to loss of membrane integrity.Capillary dysfunction also occurs secondary to activation of endothelial cells by circulating inflammatory mediators gen-erated in septic or traumatic shock. This exacerbates endothelial cell swelling and capillary leak, as well as increases leukocyte adherence. This results in capillary occlusion, which may persist after resuscitation, and is termed no-reflow. Further ischemic injury ensues as well as release of inflammatory cytokines to compound tissue injury. Experimental models have shown that neutrophil depletion in animals subjected to hemorrhagic shock produces fewer capillaries with no-reflow and lower mortality.14METABOLIC EFFECTSCellular metabolism is based primarily on the hydrolysis of adenosine triphosphate (ATP). The splitting of the phosphoan-hydride bond of the terminal or g-phosphate from ATP is the source of energy for most processes within the cell under nor-mal conditions. The majority of ATP is

1	(ATP). The splitting of the phosphoan-hydride bond of the terminal or g-phosphate from ATP is the source of energy for most processes within the cell under nor-mal conditions. The majority of ATP is generated in our bodies through aerobic metabolism in the process of oxidative phos-phorylation in the mitochondria. This process is dependent on the availability of O2 as a final electron acceptor in the electron transport chain. As O2 tension within a cell decreases, there is a decrease in oxidative phosphorylation, and the generation Brunicardi_Ch05_p0131-p0156.indd 13629/01/19 11:06 AM 137SHOCKCHAPTER 5of ATP slows. When O2 delivery is so severely impaired such that oxidative phosphorylation cannot be sustained, the state is termed dysoxia.18 When oxidative phosphorylation is insuffi-cient, the cells shift to anaerobic metabolism and glycolysis to generate ATP. This occurs via the breakdown of cellular glyco-gen stores to pyruvate. Although glycolysis is a rapid process, it is not

1	the cells shift to anaerobic metabolism and glycolysis to generate ATP. This occurs via the breakdown of cellular glyco-gen stores to pyruvate. Although glycolysis is a rapid process, it is not efficient, allowing for the production of only 2 mol of ATP from 1 mol of glucose. This is compared to complete oxidation of 1 mol of glucose that produces 38 mol of ATP. Additionally, under hypoxic conditions in anaerobic metabo-lism, pyruvate is converted into lactate, leading to an intracel-lular metabolic acidosis.There are numerous consequences secondary to these met-abolic changes. The depletion of ATP potentially influences all ATP-dependent cellular processes. This includes maintenance of cellular membrane potential, synthesis of enzymes and pro-teins, cell signaling, and DNA repair mechanisms. Decreased intracellular pH also influences vital cellular functions such as normal enzyme activity, cell membrane ion exchange, and cellular metabolic signaling.19 These changes also will lead to

1	Decreased intracellular pH also influences vital cellular functions such as normal enzyme activity, cell membrane ion exchange, and cellular metabolic signaling.19 These changes also will lead to changes in gene expression within the cell. Furthermore, acido-sis leads to changes in calcium metabolism and calcium signal-ing. Compounded, these changes may lead to irreversible cell injury and death.Epinephrine and norepinephrine have a profound impact on cellular metabolism. Hepatic glycogenolysis, gluconeogen-esis, ketogenesis, skeletal muscle protein breakdown, and adi-pose tissue lipolysis are increased by catecholamines. Cortisol, glucagon, and ADH also contribute to the catabolism during shock. Epinephrine induces further release of glucagon, while inhibiting the pancreatic β-cell release of insulin. The result is a catabolic state with glucose mobilization, hyperglycemia, pro-tein breakdown, negative nitrogen balance, lipolysis, and insulin resistance during shock and injury. The

1	of insulin. The result is a catabolic state with glucose mobilization, hyperglycemia, pro-tein breakdown, negative nitrogen balance, lipolysis, and insulin resistance during shock and injury. The relative underuse of glu-cose by peripheral tissues preserves it for the glucose-dependent organs such as the heart and brain.Cellular HypoperfusionHypoperfused cells and tissues experience what has been termed oxygen debt, a concept first proposed by Crowell in 1961.20 The O2 debt is the deficit in tissue oxygenation over time that occurs during shock. When O2 delivery is limited, O2 consumption can be inadequate to match the metabolic needs of cellular respira-tion, creating a deficit in O2 requirements at the cellular level. The measurement of O2 deficit uses calculation of the difference between the estimated O2 demand and the actual value obtained for O2 consumption. Under normal circumstances, cells can “repay” the O2 debt during reperfusion. The magnitude of the O2 debt correlates with

1	the estimated O2 demand and the actual value obtained for O2 consumption. Under normal circumstances, cells can “repay” the O2 debt during reperfusion. The magnitude of the O2 debt correlates with the severity and duration of hypoperfusion. Surrogate values for measuring O2 debt include base deficit and lactate levels and are discussed later in the “Hypovolemic/ Hemorrhagic” section.In addition to induction of changes in cellular metabolic pathways, shock also induces changes in cellular gene expres-sion. The DNA binding activity of a number of nuclear tran-scription factors is altered by hypoxia and the production of O2 radicals or nitrogen radicals that are produced at the cellular level by shock. Expression of other gene products such as heat shock proteins, vascular endothelial growth factor, inducible nitric oxide synthase (iNOS), heme oxygenase-1, and cytokines also are clearly increased by shock.21 Many of these shock-induced gene products, such as cytokines, have the ability

1	factor, inducible nitric oxide synthase (iNOS), heme oxygenase-1, and cytokines also are clearly increased by shock.21 Many of these shock-induced gene products, such as cytokines, have the ability to subsequently alter gene expression in specific target cells and tissues. The involvement of multiple pathways emphasizes the complex, integrated, and overlapping nature of the response to shock.IMMUNE AND INFLAMMATORY RESPONSESThe inflammatory and immune responses are a complex set of interactions between circulating soluble factors and cells that can arise in response to trauma, infection, ischemia, toxic, or autoimmune stimuli.21 The processes are well regulated and can be conceptualized as an ongoing surveillance and response sys-tem that undergoes a coordinated escalation following injury to heal disrupted tissue or restore host-microbe equilibrium, as well as active suppression back to baseline levels. Failure to adequately control the activation, escalation, or suppression of the

1	to heal disrupted tissue or restore host-microbe equilibrium, as well as active suppression back to baseline levels. Failure to adequately control the activation, escalation, or suppression of the inflammatory response can lead to systemic inflammatory response syndrome and potentiate multiple organ failure.Both the innate and adaptive branches of the immune system work in concert to rapidly respond in a specific and effective manner to challenges that threaten an organism’s well-being. Each arm of the immune system has its own set of func-tions, defined primarily by distinct classes of effector cells and their unique cell membrane receptor families. Alterations in the activity of the innate host immune system can be responsible for both the development of shock (i.e., septic shock follow-ing severe infection and traumatic shock following tissue injury with hemorrhage) and the pathophysiologic sequelae of shock such as the proinflammatory changes seen following hypoperfu-sion (see

1	severe infection and traumatic shock following tissue injury with hemorrhage) and the pathophysiologic sequelae of shock such as the proinflammatory changes seen following hypoperfu-sion (see Fig. 5-1). When the predominantly paracrine mediators gain access to the systemic circulation, they can induce a variety of metabolic changes that are collectively referred to as the host inflammatory response. Understanding of the intricate, redun-dant, and interrelated pathways that comprise the inflammatory response to shock continues to expand. Despite limited under-standing of how our current therapeutic interventions impact the host response to illness, inappropriate or excessive inflam-mation appears to be an essential event in the development of ARDS, multiple organ dysfunction syndrome (MODS), and posttraumatic immunosuppression that can prolong recovery.22Following direct tissue injury or infection, there are several mechanisms that lead to the activation of the active inflamma-tory and

1	and posttraumatic immunosuppression that can prolong recovery.22Following direct tissue injury or infection, there are several mechanisms that lead to the activation of the active inflamma-tory and immune responses. These include release of bioactive peptides by neurons in response to pain and the release of intra-cellular molecules by broken cells, such as heat shock proteins, mitochondrial products, heparan sulfate, high mobility group box 1, and RNA. Only recently has it been realized that the release of intracellular products from damaged and injured cells can have paracrine and endocrine-like effects on distant tissues to activate the inflammatory and immune responses.23 This hypothesis, which was first proposed by Matzinger, is known as danger signaling. Under this novel paradigm of immune function, endogenous molecules are capable of signaling the presence of danger to surrounding cells and tissues. These molecules that are released from cells are known as damage associated

1	of immune function, endogenous molecules are capable of signaling the presence of danger to surrounding cells and tissues. These molecules that are released from cells are known as damage associated molecular patterns (DAMPs, Table 5-3). DAMPs are recognized by cell surface receptors to effect intracellular signaling that primes and amplifies the immune response. These receptors are known as pattern recognition receptors (PRRs) and include the Toll-like receptors (TLRs) and the receptor for advanced glycation end products. Interestingly, TLRs and PRRs were first recognized for their role in signaling as part of the immune response to the Brunicardi_Ch05_p0131-p0156.indd 13729/01/19 11:06 AM 138BASIC CONSIDERATIONSPART Ientry of microbes and their secreted products into a normally sterile environment. These bacterial products, including lipo-polysaccharide, are known as pathogen-associated molecular patterns. The salutary consequences of PRR activation most likely relate to the

1	environment. These bacterial products, including lipo-polysaccharide, are known as pathogen-associated molecular patterns. The salutary consequences of PRR activation most likely relate to the initiation of the repair process and the mobi-lization of antimicrobial defenses at the site of tissue disruption. However, in the setting of excessive tissue damage, the inflam-mation itself may lead to further tissue damage, amplifying the response both at the local and systemic level.21 PRR activation leads to intracellular signaling and release of cellular products, including cytokines (Fig. 5-4).Before the recruitment of leukocytes into sites of injury, tissue-based macrophages or mast cells act as sentinel respond-ers, releasing histamines, eicosanoids, tryptases, and cytokines (Fig. 5-5). Together these signals amplify the immune response by further activation of neurons and mast cells, as well as increasing the expression of adhesion molecules on the endothe-lium. Furthermore, these

1	Together these signals amplify the immune response by further activation of neurons and mast cells, as well as increasing the expression of adhesion molecules on the endothe-lium. Furthermore, these mediators cause leukocytes to release platelet-activating factor, further increasing the stickiness of the endothelium. Additionally, the coagulation and kinin cascades impact the interaction of endothelium and leukocytes.Cytokines/ChemokinesThe immune response to shock encompasses the elaboration of mediators with both proinflammatory and anti-inflammatory properties (Table 5-4). Furthermore, new mediators, new rela-tionships between mediators, and new functions of known mediators are continually being identified. As new pathways are uncovered, understanding of the immune response to injury and the potential for therapeutic intervention by manipulating the immune response following shock will expand. What seems clear at present, however, is that the innate immune response can help restore

1	the potential for therapeutic intervention by manipulating the immune response following shock will expand. What seems clear at present, however, is that the innate immune response can help restore homeostasis, or if it is excessive, promote cel-lular and organ dysfunction.Multiple mediators have been implicated in the host immune response to shock. It is likely that some of the most important mediators have yet to be discovered and the roles of many known mediators have not been defined. A compre-hensive description of all of the mediators and their complex interactions is beyond the scope of this chapter. For a general overview, a brief description of the more extensively studied mediators, as well as some of the known effects of these sub-stances, see the following discussion. A more comprehensive review can be found in Chapter 2, “Systemic Response to Injury and Metabolic Support.”Tumor necrosis factor alpha (TNF-a) was one of the first cytokines to be described, and it is one of

1	review can be found in Chapter 2, “Systemic Response to Injury and Metabolic Support.”Tumor necrosis factor alpha (TNF-a) was one of the first cytokines to be described, and it is one of the earliest cytokines released in response to injurious stimuli. Monocytes, macro-phages, and T cells release this potent proinflammatory cyto-kine. TNF-a levels peak within 90 minutes of stimulation and return frequently to baseline levels within 4 hours. Release of TNF-a may be induced by bacteria or endotoxin and leads to the development of shock and hypoperfusion, most commonly observed in septic shock. Production of TNF-a also may be induced following other insults, such as hemorrhage and isch-emia. TNF-a levels correlate with mortality in animal models of hemorrhage.24 In contrast, the increase in serum TNF-a levels reported in trauma patients is far less than that seen in septic patients.25 Once released, TNF-a can produce peripheral vaso-dilation, activate the release of other cytokines,

1	in serum TNF-a levels reported in trauma patients is far less than that seen in septic patients.25 Once released, TNF-a can produce peripheral vaso-dilation, activate the release of other cytokines, induce proco-agulant activity, and stimulate a wide array of cellular metabolic changes. During the stress response, TNF-a contributes to the muscle protein breakdown and cachexia.Interleukin-1 (IL-1) has actions similar to those of TNF-a. IL-1 has a very short half-life (6 minutes) and primarily acts in a paracrine fashion to modulate local cellular responses. Sys-temically, IL-1 produces a febrile response to injury by activat-ing prostaglandins in the posterior hypothalamus, and causes anorexia by activating the satiety center. This cytokine also aug-ments the secretion of ACTH, glucorticoids, and β-endorphins. In conjunction with TNF-a, IL-1 can stimulate the release of other cytokines such as IL-2, IL-4, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, and

1	glucorticoids, and β-endorphins. In conjunction with TNF-a, IL-1 can stimulate the release of other cytokines such as IL-2, IL-4, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, and interferon-g.IL-2 is produced by activated T cells in response to a vari-ety of stimuli and activates other lymphocyte subpopulations and natural killer cells. The lack of clarity regarding the role of IL-2 in the response to shock is intimately associated with that of understanding immune function after injury. Some investigators have postulated that increased IL-2 secretion promotes shock-induced tissue injury and the development of shock. Others have demonstrated that depressed IL-2 production is associated with, Table 5-3Endogenous damage-associated molecular pattern moleculesMitochondrial DNAHyaluronan oligomersHeparan sulfateExtra domain A of fibronectinHeat shock proteins 60, 70, Gp96Surfactant Protein Aβ-Defensin 2FibrinogenBiglycanHigh mobility group box 1Uric

1	DNAHyaluronan oligomersHeparan sulfateExtra domain A of fibronectinHeat shock proteins 60, 70, Gp96Surfactant Protein Aβ-Defensin 2FibrinogenBiglycanHigh mobility group box 1Uric acidInterleukin-1αS-100sNucleolinNeuropeptidesTissue-based macrophages/mast cells Histamines,leukotrienes,chemokines,TNF TraumaDAMPs (HMGB1,heparan sulfate,uric acid)Macrophages Bacteria andbacterialproductsComplementNeutrophilsDegranulationChemokines,TNFDefensinsTNF,Interferon-LymphocytesAntigenStimulation/activationProductionFigure 5-4. A schema of information flow between immune cells in early inflammation following tissue injury and infection. Cells require multiple inputs and stimuli before activation of a full response. DAMPs = damage associated molecular patterns; HMGB1 = high mobility group box 1; TNF = tumor necrosis factor.Brunicardi_Ch05_p0131-p0156.indd 13829/01/19 11:06 AM 139SHOCKCHAPTER 5and perhaps contributes to, the depression in immune func-tion after hemorrhage that may increase

1	= tumor necrosis factor.Brunicardi_Ch05_p0131-p0156.indd 13829/01/19 11:06 AM 139SHOCKCHAPTER 5and perhaps contributes to, the depression in immune func-tion after hemorrhage that may increase the susceptibility of patients who develop shock to suffer infections.26,27 It has been postulated that overly exuberant proinflammatory activation promotes tissue injury, organ dysfunction, and the subsequent immune dysfunction/suppression that may be evident later.22 Emphasizing the importance of temporal changes in the produc-tion of mediators, both the initial excessive production of IL-2 and later depressed IL-2 production are probably important in the progression of shock.MKK3p 38MKK 7JNKDAMPLPS signalingHemorrhagic shockIschemia/reperfusionTissue traumaToxic exposureDanger signalingInjuryNecrosisCD14HMGB-1HMGB-1LPSActivatedTLR4TRAMTRIFIRF 3MyD88-dependentpathwayMyD88-independentpathwayNuclear membraneOtherco-receptorsSecretion fromstressed cellsBreakdownof matrixMyD88MALIRAK 4IRAK

1	3MyD88-dependentpathwayMyD88-independentpathwayNuclear membraneOtherco-receptorsSecretion fromstressed cellsBreakdownof matrixMyD88MALIRAK 4IRAK 1TRAF 6TAK 1IKK 1IKK 2IRF 3MD-2NEMOI˜Bp 50p 65NF-˜Bp 50p 65TBK 1Cell membraneCell membraneTLR4TLR4TLR4MD-2LBPFigure 5-5. Signaling via the pattern recognition receptor TLR4. LPS signaling via TLR4 requires the cofactors LPS binding protein (LBP), MD-2, and CD14. Endogenous danger signals released from a variety of sources also signal in a TLR4-dependent fashion, although it is as yet unknown what cofactors may be required for this activity. Once TLR4 is activated, an intracellular signaling cascade is initiated that involves both a MyD88-dependent and independent pathway. DAMP = damage associated molecular pattern; LPS = lipopolysaccharide; MD-2 = myeloid differentiation factor-2; MyD88 = myeloid differentiation primary response gene 88; NF-kB = nuclear factor kB; TLR4 = Toll-like receptor-4. (Reproduced with permission from Mollen KP, Levy

1	= myeloid differentiation factor-2; MyD88 = myeloid differentiation primary response gene 88; NF-kB = nuclear factor kB; TLR4 = Toll-like receptor-4. (Reproduced with permission from Mollen KP, Levy RM, Prince JM, et al. Systemic inflammation and end organ damage following trauma involves functional TLR4 signaling in both bone marrow-derived cells and parenchymal cells, J Leukoc Biol. 2008 Jan;83(1):80-88.)Brunicardi_Ch05_p0131-p0156.indd 13929/01/19 11:06 AM 140BASIC CONSIDERATIONSPART IIL-6 is elevated in response to hemorrhagic shock, major operative procedures, or trauma. Elevated IL-6 levels correlate with mortality in shock states. IL-6 contributes to lung, liver, and gut injury after hemorrhagic shock.28 Thus, IL-6 may play a role in the development of diffuse alveolar damage and ARDS. IL-6 and IL-1 are mediators of the hepatic acute phase response to injury, and enhance the expression and activity of comple-ment, C-reactive protein, fibrinogen, haptoglobin, amyloid A, and

1	ARDS. IL-6 and IL-1 are mediators of the hepatic acute phase response to injury, and enhance the expression and activity of comple-ment, C-reactive protein, fibrinogen, haptoglobin, amyloid A, and alpha1-antitrypsin, and promote neutrophil activation.29IL-10 is considered an anti-inflammatory cytokine that may have immunosuppressive properties. Its production is increased after shock and trauma, and it has been associated with depressed immune function clinically, as well as an increased susceptibility to infection.30 IL-10 is secreted by T cells, mono-cytes, and macrophages, and inhibits proinflammatory cytokine secretion, O2 radical production by phagocytes, adhesion mol-ecule expression, and lymphocyte activation.30,31 Administra-tion of IL-10 depresses cytokine production and improves some aspects of immune function in experimental models of shock and sepsis.32,33Recent studies point to the importance of chemokines, a specific set of cytokines, that have the ability to induce

1	some aspects of immune function in experimental models of shock and sepsis.32,33Recent studies point to the importance of chemokines, a specific set of cytokines, that have the ability to induce che-motaxis of leukocytes. Chemokines bind to specific chemokine receptors and transduce chemotactic signals to leukocytes. The significance of this large family of chemoattractant cytokines in immunology is difficult to understate, as almost every facet of the immune system is influenced by chemokines, including immune system development, immune surveillance, immune priming, effector responses, and immune regulation.34ComplementThe complement cascade can be activated by injury, shock, and severe infection, and contributes to host defense and pro-inflammatory activation. Significant complement consumption occurs after hemorrhagic shock.35 In trauma patients, the degree of complement activation is proportional to the magnitude of injury and may serve as a marker for severity of injury. Patients

1	occurs after hemorrhagic shock.35 In trauma patients, the degree of complement activation is proportional to the magnitude of injury and may serve as a marker for severity of injury. Patients in septic shock also demonstrate activation of the complement pathway, with elevations of the activated complement proteins C3a and C5a. Activation of the complement cascade can con-tribute to the development of organ dysfunction. Activated complement factors C3a, C4a, and C5a are potent mediators of increased vascular permeability, smooth muscle cell contraction, histamine and arachidonic acid by-product release, and adher-ence of neutrophils to vascular endothelium. Activated comple-ment acts synergistically with endotoxin to induce the release of TNF-a and IL-1. The development of ARDS and MODS in trauma patients correlates with the intensity of complement activation.36 Complement and neutrophil activation may corre-late with mortality in multiply injured patients.NeutrophilsNeutrophil

1	in trauma patients correlates with the intensity of complement activation.36 Complement and neutrophil activation may corre-late with mortality in multiply injured patients.NeutrophilsNeutrophil activation is an early event in the upregulation of the inflammatory response; neutrophils are the first cells to be recruited to the site of injury. Polymorphonuclear leuko-cytes (PMNs) remove infectious agents, foreign substances that have penetrated host barrier defenses, and nonviable tis-sue through phagocytosis. However, activated PMNs and their products may also produce cell injury and organ dysfunction. Activated PMNs generate and release a number of substances that may induce cell or tissue injury, such as reactive O2 spe-cies, lipid-peroxidation products, proteolytic enzymes (elastase, cathepsin G), and vasoactive mediators (leukotrienes, eico-sanoids, and platelet-activating factor). Oxygen-free radicals, such as superoxide anion, hydrogen peroxide, and hydroxyl radical, are

1	cathepsin G), and vasoactive mediators (leukotrienes, eico-sanoids, and platelet-activating factor). Oxygen-free radicals, such as superoxide anion, hydrogen peroxide, and hydroxyl radical, are released and induce lipid peroxidation, inactivate enzymes, and consume antioxidants (such as glutathione and tocopherol). Ischemia-reperfusion activates PMNs and causes PMN-induced organ injury. In animal models of hemorrhagic shock, activation of PMNs correlates with irreversibility of shock and mortality, and neutrophil depletion prevents the pathophysiologic sequelae of hemorrhagic and septic shock. Human data corroborate the activation of neutrophils in trauma and shock and suggest a role in the development of MODS.37 Plasma markers of PMN activation, such as elastase, correlate with severity of injury in humans.Interactions between endothelial cells and leukocytes are important in the inflammatory process. The vascular endothe-lium contributes to regulation of blood flow, leukocyte

1	of injury in humans.Interactions between endothelial cells and leukocytes are important in the inflammatory process. The vascular endothe-lium contributes to regulation of blood flow, leukocyte adher-ence, and the coagulation cascade. Extracellular ligands such as intercellular adhesion molecules, vascular cell adhesion mol-ecules, and the selectins (E-selectin, P-selectin) are expressed on the surface of endothelial cells, and are responsible for leuko-cyte adhesion to the endothelium. This interaction allows acti-vated neutrophils to migrate into the tissues to combat infection, but also can lead to PMN-mediated cytotoxicity and microvas-cular and tissue injury.Cell SignalingA host of cellular changes occur following shock. Although many of the intracellular and intercellular pathways that are important in shock are being elucidated, undoubtedly there are many more that have yet to be identified. Many of the media-tors produced during shock interact with cell surface receptors on

1	that are important in shock are being elucidated, undoubtedly there are many more that have yet to be identified. Many of the media-tors produced during shock interact with cell surface receptors on target cells to alter target cell metabolism. These signaling pathways may be altered by changes in cellular oxygenation, redox state, high-energy phosphate concentration, gene expres-sion, or intracellular electrolyte concentration induced by shock. Cells communicate with their external environment through the use of cell surface membrane receptors, which, once bound by a ligand, transmit their information to the interior of the cell through a variety of signaling cascades. These signaling path-ways may subsequently alter the activity of specific enzymes, the expression or breakdown of important proteins, or affect intracellular energy metabolism. Intracellular calcium (Ca2+) homeostasis and regulation represents one such pathway. Intra-cellular Ca2+ concentrations regulate many aspects

1	proteins, or affect intracellular energy metabolism. Intracellular calcium (Ca2+) homeostasis and regulation represents one such pathway. Intra-cellular Ca2+ concentrations regulate many aspects of cellular metabolism; many important enzyme systems require Ca2+ for Table 5-4Inflammatory mediators of shockPROINFLAMMATORYANTI-INFLAMMATORYInterleukin-1α/βInterleukin-4Interleukin-2Interleukin-10Interleukin-6Interleukin-13Interleukin-8Prostaglandin E2InterferonTGFβTNF PAF PAF = platelet activating factor; TGFβ = transforming growth factor β; TNF = tumor necrosis factor.Brunicardi_Ch05_p0131-p0156.indd 14029/01/19 11:06 AM 141SHOCKCHAPTER 5full activity. Profound changes in intracellular Ca2+ levels and Ca2+ transport are seen in models of shock.38 Alterations in Ca2+ regulation may lead to direct cell injury, changes in transcription factor activation, alterations in the expression of genes impor-tant in homeostasis, and the modulation of the activation of cells by other shock-induced

1	direct cell injury, changes in transcription factor activation, alterations in the expression of genes impor-tant in homeostasis, and the modulation of the activation of cells by other shock-induced hormones or mediators.39,40A proximal portion of the intracellular signaling cascade consists of a series of kinases that transmit and amplify the sig-nal through the phosphorylation of target proteins. The O2 radi-cals produced during shock and the intracellular redox state are known to influence the activity of components of this cascade, such as protein tyrosine kinases, mitogen activated kinases, and protein kinase C.41-43 Either through changes in these sig-naling pathways, changes in the activation of enzyme systems through Ca2+-mediated events, or direct conformational changes to oxygen-sensitive proteins, O2 radicals also regulate the activ-ity of a number of transcription factors that are important in gene expression, such as nuclear factor kB, APETALA1, and hypoxia-inducible

1	proteins, O2 radicals also regulate the activ-ity of a number of transcription factors that are important in gene expression, such as nuclear factor kB, APETALA1, and hypoxia-inducible factor 1.44,45 It is therefore becoming increas-ingly clear that oxidant-mediated direct cell injury is merely one consequence of the production of O2 radicals during shock.The study of the effects of shock on the regulation of gene expression as an important biologic effect was stimulated by the work of Buchman and colleagues.46 The effects of shock on the expression and regulation of numerous genes and gene products has been studied in both experimental animal models and human patients. These studies include investigations into single genes of interest as well as large-scale genomic and proteomic analysis.47-49 Changes in gene expression are critical for adaptive and survival cell signaling. Polymorphisms in gene promoters that lead to a differential level of expression of gene products are also

1	Changes in gene expression are critical for adaptive and survival cell signaling. Polymorphisms in gene promoters that lead to a differential level of expression of gene products are also likely to contribute significantly to varied responses to similar insults.50,51 In a recent study, the genetic responses to traumatic injury in humans or endotoxin delivery to healthy human volunteers demonstrated that severe stresses produce a global reprioritiza-tion affecting >80% of the cellular functions and pathways.52 The similarities in genomic responses among different injuries revealed a fundamental human response to stressors involving dysregulated immune responses (Fig. 5-6). Furthermore, in trau-matic injury patients, complications like nosocomial infections and organ failure were not associated with any genomic evi-dence of a second hit and differed only in the magnitude and duration of this genomic reprioritization.FORMS OF SHOCKHypovolemic/HemorrhagicThe most common cause of shock in

1	with any genomic evi-dence of a second hit and differed only in the magnitude and duration of this genomic reprioritization.FORMS OF SHOCKHypovolemic/HemorrhagicThe most common cause of shock in the surgical or trauma patient is loss of circulating volume from hemorrhage. Acute blood loss results in reflexive decreased baroreceptor stimu-lation from stretch receptors in the large arteries, resulting in decreased inhibition of vasoconstrictor centers in the brain stem, increased chemoreceptor stimulation of vasomotor centers, and diminished output from atrial stretch receptors. These changes increase vasoconstriction and peripheral arterial resistance. Hypovolemia also induces sympathetic stimulation, leading to epinephrine and norepinephrine release, activation of the renin-angiotensin cascade, and increased vasopressin release. Periph-eral vasoconstriction is prominent, while lack of sympathetic effects on cerebral and coronary vessels and local autoregula-tion promote maintenance of

1	and increased vasopressin release. Periph-eral vasoconstriction is prominent, while lack of sympathetic effects on cerebral and coronary vessels and local autoregula-tion promote maintenance of cardiac and CNS blood flow.Diagnosis. Treatment of shock is initially empiric. A secure airway must be confirmed or established in obtunded patients. The priority is the initiation of volume infusion while the search for the cause of the hypotension is pursued. Shock in a trauma patient or postoperative patient should be presumed to be due to hemorrhage until proven otherwise. The clinical signs of shock may be evidenced by agitation, cool clammy extremities, tachy-cardia, weak or absent peripheral pulses, and hypotension. Such apparent clinical shock results from at least 25% to 30% loss of the blood volume. However, substantial volumes of blood may be lost before the classic clinical manifestations of shock are evident. Thus, when a patient is significantly tachycardic or hypotensive, this

1	blood volume. However, substantial volumes of blood may be lost before the classic clinical manifestations of shock are evident. Thus, when a patient is significantly tachycardic or hypotensive, this represents both significant blood loss and physiologic decompensation. The clinical and physiologic response to hemorrhage has been classified according to the magnitude of volume loss. Loss of up to 15% of the circulat-ing volume (700 to 750 mL for a 70-kg patient) may produce little in terms of obvious symptoms, while loss of up to 30% of the circulating volume (1.5 L) may result in mild tachycar-dia, tachypnea, and anxiety. Hypotension, marked tachycardia (i.e., pulse greater than 110 to 120 bpm), and confusion may not be evident until more than 30% of the blood volume has been lost; loss of 40% of circulating volume (2 L) is immedi-ately life threatening and generally requires operative control of bleeding (Table 5-5). Young healthy patients with vigor-ous compensatory mechanisms may

1	of 40% of circulating volume (2 L) is immedi-ately life threatening and generally requires operative control of bleeding (Table 5-5). Young healthy patients with vigor-ous compensatory mechanisms may tolerate larger volumes of blood loss while manifesting fewer clinical signs despite the presence of significant peripheral hypoperfusion. These patients may maintain a near-normal blood pressure until a precipitous cardiovascular collapse occurs. Elderly patients may be tak-ing medications that either promote bleeding (e.g., warfarin or aspirin), or mask the compensatory responses to bleeding (e.g., β-blockers). In addition, atherosclerotic vascular disease, diminishing cardiac compliance with age, inability to elevate heart rate or cardiac contractility in response to hemorrhage, and overall decline in physiologic reserve decrease the elderly Dysregulated adaptive immune responseMagnitude of responseMagnitude of responseUncomplicated outcomeComplicated outcomeDysregulated innate immune

1	decline in physiologic reserve decrease the elderly Dysregulated adaptive immune responseMagnitude of responseMagnitude of responseUncomplicated outcomeComplicated outcomeDysregulated innate immune responseFigure 5-6. The concurrent dysregulated innate immune responses that promote inflammation and dysregulated adaptive immune responses that result in immunosuppression occur in patients follow-ing traumatic injury. However, these genetic responses can result in complicated outcomes in trauma patients if the magnitude or dura-tion of these responses are pronounced. (Reproduced with permission from Xiao W, Mindrinos MN, Seok J, et al. A genomic storm in criti-cally injured humans, J Exp Med. 2011 Dec 19;208(13):2581-2590.)Brunicardi_Ch05_p0131-p0156.indd 14129/01/19 11:06 AM 142BASIC CONSIDERATIONSPART Ipatient’s ability to tolerate hemorrhage. Recent data in trauma patients suggest that a systolic blood pressure (SBP) of less than 110 mmHg is a clinically relevant definition of

1	CONSIDERATIONSPART Ipatient’s ability to tolerate hemorrhage. Recent data in trauma patients suggest that a systolic blood pressure (SBP) of less than 110 mmHg is a clinically relevant definition of hypotension and hypoperfusion based upon an increasing rate of mortality below this pressure (Fig. 5-7).53In addressing the sensitivity of vital signs and identifying major thoracoabdominal hemorrhage, a study retrospectively identified patients with injury to the trunk and an abbreviated injury score of 3 or greater who required immediate surgical intervention and transfusion of at least 5 units of blood within the first 24 hours. Ninety-five percent of patients had a heart rate greater than 80 bpm at some point during their postinjury course. However, only 59% of patients achieved a heart rate greater than 120 bpm. Ninety-nine percent of all patients had a recorded blood pressure of less than 120 mmHg at some point. Ninety-three percent of all patients had a recorded SBP of less than 100

1	greater than 120 bpm. Ninety-nine percent of all patients had a recorded blood pressure of less than 120 mmHg at some point. Ninety-three percent of all patients had a recorded SBP of less than 100 mmHg.54 A more recent study corroborated that tachy-cardia was not a reliable sign of hemorrhage following trauma and was present in only 65% of hypotensive patients.55Serum lactate and base deficit are measurements that are helpful to both estimate and monitor the extent of bleeding and shock. The amount of lactate that is produced by anaerobic res-piration is an indirect marker of tissue hypoperfusion, cellular O2 debt, and the severity of hemorrhagic shock. Several stud-ies have demonstrated that the initial serum lactate and serial lactate levels are reliable predictors of morbidity and mortality with hemorrhage following trauma (Fig. 5-8).56 Similarly, base deficit values derived from arterial blood gas analysis provide clinicians with an indirect estimation of tissue acidosis from

1	mortality with hemorrhage following trauma (Fig. 5-8).56 Similarly, base deficit values derived from arterial blood gas analysis provide clinicians with an indirect estimation of tissue acidosis from hypoperfusion. Davis and colleagues stratified the extent of base deficit into mild (–3 to –5 mmol/L), moderate (–6 to –9 mmol/L), and severe (less than –10 mmol/L), and from this established a correlation between base deficit upon admission with transfu-sion requirements, the development of multiple organ failure, and death (Fig. 5-9).57 Both base deficit and lactate correlate with the extent of shock and patient outcome, but interestingly do not firmly correlate with each other.58-60 Evaluation of both values may be useful in trauma patients with hemorrhage.Although hematocrit changes may not rapidly reflect the total volume of blood loss, admission hematocrit has been shown to be associated with 24 hour fluid and transfusion requirements and more strongly associated with PRBC

1	may not rapidly reflect the total volume of blood loss, admission hematocrit has been shown to be associated with 24 hour fluid and transfusion requirements and more strongly associated with PRBC transfu-sion than either tachycardia, hypotension, or acidosis.61 It must be noted that lack of a depression in the initial hematocrit does not rule out substantial blood loss or ongoing bleeding.In management of trauma patients, understanding the pat-terns of injury of the patient in shock will help direct the evalu-ation and management. Identifying the sources of blood loss in patients with penetrating wounds is relatively simple because potential bleeding sources will be located along the known or suspected path of the wounding object. Patients with penetrating injuries who are in shock usually require operative intervention. Patients who suffer multisystem injuries from blunt trauma have multiple sources of potential hemorrhage. Blood loss sufficient to cause shock is generally of a large

1	require operative intervention. Patients who suffer multisystem injuries from blunt trauma have multiple sources of potential hemorrhage. Blood loss sufficient to cause shock is generally of a large volume, and there are a limited number of sites that can harbor sufficient extravascular blood volume to induce hypotension (e.g., external, intratho-racic, intra-abdominal, retroperitoneal, and long bone fractures). In the nontrauma patient, the GI tract must always be considered as a site for blood loss. Substantial blood loss externally may be suspected from prehospital medical reports documenting a sub-stantial blood loss at the scene of an accident, history of massive Table 5-5Classification of hemorrhage CLASSPARAMETERIIIIIIIVBlood loss (mL)<750750–15001500–2000>2000Blood loss (%)<1515–3030–40>40Heart rate (bpm)<100>100>120>140Blood pressureNormalOrthostaticHypotensionSevere hypotensionCNS symptomsNormalAnxiousConfusedObtundedbpm = beats per minute; CNS = central nervous

1	(%)<1515–3030–40>40Heart rate (bpm)<100>100>120>140Blood pressureNormalOrthostaticHypotensionSevere hypotensionCNS symptomsNormalAnxiousConfusedObtundedbpm = beats per minute; CNS = central nervous system.3025201510506065707580859095100105Systolic BP in the ED110115120125130135140145150155160121086420% MortalityBase deficit% MortalityBDFigure 5-7. The relationship between systolic blood pressure and mortality in trauma patients with hemorrhage. These data suggest that a systolic blood pressure of less than 110 mmHg is a clinically relevant definition of hypotension and hypoperfusion based upon an increasing rate of mortal-ity below this pressure. Base deficit (BD) is also shown on this graph. ED = emergency department. (Reproduced with permission from Eastridge BJ, Salinas J, McManus JG, et al. Hypotension begins at 110 mm Hg: redefining “hypotension” with data, J Trauma. 2007 Aug;63(2):291-297.)Brunicardi_Ch05_p0131-p0156.indd 14229/01/19 11:06 AM 143SHOCKCHAPTER 5blood loss from

1	al. Hypotension begins at 110 mm Hg: redefining “hypotension” with data, J Trauma. 2007 Aug;63(2):291-297.)Brunicardi_Ch05_p0131-p0156.indd 14229/01/19 11:06 AM 143SHOCKCHAPTER 5blood loss from wounds, visible brisk bleeding, or presence of a large hematoma adjacent to an open wound. Injuries to major arteries or veins with associated open wounds may cause massive blood loss rapidly. Direct pressure must be applied and sustained to minimize ongoing blood loss. Tourniquets should be used for extremity bleeding stopped by direct pressure and applied in the prehospital setting as needed.62,63 Persistent bleeding from uncontrolled smaller vessels can, over time, precipitate shock if inadequately treated.When major blood loss is not immediately visible in the setting of trauma, internal (intracavitary) blood loss should be suspected. Each pleural cavity can hold 2 to 3 L of blood and can therefore be a site of significant blood loss. Diagnostic and therapeutic tube thoracostomy may be

1	blood loss should be suspected. Each pleural cavity can hold 2 to 3 L of blood and can therefore be a site of significant blood loss. Diagnostic and therapeutic tube thoracostomy may be indicated in unstable patients based on clinical findings and clinical suspicion. In a more stable patient, a chest radiograph may be obtained to look for evidence of hemothorax. Major retroperitoneal hem-orrhage typically occurs in association with pelvic fractures, which is confirmed by pelvic radiography in the resuscitation bay. Intraperitoneal hemorrhage is probably the most common source of blood loss that induces shock. The physical exam for detection of substantial blood loss or injury is insensitive and unreliable; large volumes of intraperitoneal blood may be present before physical examination findings are apparent. Findings with intra-abdominal hemorrhage include abdominal distension, abdominal tenderness, or visible abdominal wounds. Hemodynamic abnormalities generally stimulate a search

1	findings are apparent. Findings with intra-abdominal hemorrhage include abdominal distension, abdominal tenderness, or visible abdominal wounds. Hemodynamic abnormalities generally stimulate a search for blood loss before the appearance of obvious abdominal findings. Adjunctive tests are essential in the diagnosis of intraperitoneal bleeding; intraperitoneal blood may be rapidly identified by diagnostic ultrasound or diagnostic peritoneal lavage. Further-more, patients that have sustained high-energy blunt trauma that are hemodynamically stable or that have normalized their vital signs in response to initial volume resuscitation should undergo computed tomography scans to assess for head, chest, and/or abdominal bleeding.Treatment. Control of ongoing hemorrhage is an essential component of the resuscitation of the patient in shock. As mentioned in the previous “Diagnosis” section, treatment of hemorrhagic shock is instituted concurrently with diagnostic evaluation to identify a

1	of the resuscitation of the patient in shock. As mentioned in the previous “Diagnosis” section, treatment of hemorrhagic shock is instituted concurrently with diagnostic evaluation to identify a source. Patients who fail to respond to initial resuscitative efforts should be assumed to have ongo-ing active hemorrhage from large vessels and require prompt operative intervention. Based on trauma literature, patients with ongoing hemorrhage demonstrate increased survival if the elapsed time between the injury and control of bleeding is decreased. Although there are no randomized controlled trials, retrospective studies provide compelling evidence in this regard. To this end, Clarke and colleagues64 demonstrated that trauma Serum lactateMuscle lactateLiver lactateTime (hours)Serum lactate (mg/100 ml)Tissue lactate (M/gm tissue)012345510152025306090120150Figure 5-8. Progressive increases in serum lactate, muscle lactate, and liver lactate in a baboon model of hemorrhagic shock. (Repro-duced

1	ml)Tissue lactate (M/gm tissue)012345510152025306090120150Figure 5-8. Progressive increases in serum lactate, muscle lactate, and liver lactate in a baboon model of hemorrhagic shock. (Repro-duced with permission from Peitzman AB, Corbett WA, Shires GT, et al: Cellular function in liver and muscle during hemorrhagic shock in primates, Surg Gynecol Obstet. 1985 Nov;161(5):419-424.)Extracellular BEA, mmol/L10059590858075706560555045403530252015100102–6–14–22–38% MortalityBase excess = –11.8LD50% Mortality = e˜1 + e˜x 10010090807060504030201001020% Predicted death on the basis of linear logistic model from BEAECF% Observed death0–0.19–0.17–4.5–7.4–9.7–11.8–14–16.4–19.2–23.530405060708090100Figure 5-9. The relationship between base deficit (negative base excess) and mortality in trauma patients. BEA = base excess arterial; ECF = extracellular fluid. (Reproduced with permission from Siegel JH, Rivkind AI, Dalal S, et al: Early physiologic predictors of injury severity and death in blunt

1	BEA = base excess arterial; ECF = extracellular fluid. (Reproduced with permission from Siegel JH, Rivkind AI, Dalal S, et al: Early physiologic predictors of injury severity and death in blunt multiple trauma, Arch Surg. 1990 Apr;125(4):498-508.)Brunicardi_Ch05_p0131-p0156.indd 14329/01/19 11:06 AM 144BASIC CONSIDERATIONSPART Ipatients with major injuries isolated to the abdomen requiring emergency laparotomy had an increased probability of death with increasing length of time in the emergency department for patients who were in the emergency department for 90 minutes or less. This probability increased approximately 1% for each 3 minutes in the emergency department.The appropriate priorities in these patients are as follows: (a) control the source of blood loss, (b) perform IV volume resuscitation with blood products in the hypotensive patient, and (c) secure the airway. In trauma, identifying the body cavity har-boring active hemorrhage will help focus operative efforts;

1	volume resuscitation with blood products in the hypotensive patient, and (c) secure the airway. In trauma, identifying the body cavity har-boring active hemorrhage will help focus operative efforts; how-ever, because time is of the essence, rapid treatment is essential, and diagnostic laparotomy or thoracotomy may be indicated. The actively bleeding patient cannot be resuscitated until control of ongoing hemorrhage is achieved. Our current understanding has led to the management strategy known as damage control resuscitation.65 This strategy begins in the emergency depart-ment, continues into the operating room, and into the intensive care unit (ICU). Initial resuscitation is limited to keep SBP around 80 to 90 mmHg. This prevents renewed bleeding from recently clotted vessels. Resuscitation and intravascular volume resuscitation is accomplished with blood products and lim-ited crystalloids, which is addressed further later in this section. Too little volume allowing persistent

1	and intravascular volume resuscitation is accomplished with blood products and lim-ited crystalloids, which is addressed further later in this section. Too little volume allowing persistent severe hypoten-sion and hypoperfusion is dangerous, yet too vigorous of a vol-ume resuscitation may be just as deleterious. Control of hemorrhage is achieved in the operating room (or angiography suite once surgical causes of hemorrhage have been ruled out), and efforts to warm patients and to prevent coagulopathy using multiple blood products and pharmacologic agents are used in both the operating room and ICU.Cannon and colleagues first made the observation that attempts to increase blood pressure in soldiers with uncontrolled sources of hemorrhage is counterproductive, with increased bleeding and higher mortality.3 This work was the foundation for the “hypotensive resuscitation” strategies. Several laboratory studies confirmed the observation that attempts to restore normal blood pressure with

1	mortality.3 This work was the foundation for the “hypotensive resuscitation” strategies. Several laboratory studies confirmed the observation that attempts to restore normal blood pressure with fluid infusion or vasopressors was rarely achievable and resulted in more bleeding and higher mortality.66 A prospective, randomized clinical study compared delayed fluid resuscitation (upon arrival in the operating room) with standard fluid resuscitation (with arrival by the paramedics) in hypotensive patients with penetrating torso injury.67 The authors reported that delayed fluid resuscitation resulted in lower patient mortality. Further laboratory studies demonstrated that fluid restriction in the setting of profound hypotension resulted in early deaths from severe hypoperfusion. These studies also showed that aggressive crystalloid resuscitation attempting to normalize blood pressure resulted in marked hemodilution, with hematocrits of 5%.66 Reasonable conclusions in the setting of

1	also showed that aggressive crystalloid resuscitation attempting to normalize blood pressure resulted in marked hemodilution, with hematocrits of 5%.66 Reasonable conclusions in the setting of uncontrolled hemorrhage include (a) any delay in surgery for control of hemorrhage increases mortality; (b) with uncontrolled hemorrhage, attempting to achieve normal blood pressure may increase mortality, particularly with penetrating injuries and short transport times; (c) a goal of SBP of 80 to 90 mmHg may be adequate in the patient with penetrating injury; and (d) profound hemodilution should be avoided by early transfusion of red blood cells. For the patient with blunt injury, where the major cause of death is a closed head injury, the increase in mortality with hypotension in the setting of brain injury must be avoided. In this setting, a SBP of 110 mmHg would seem to be more appropriate.Patients who respond to initial resuscitative effort but then deteriorate hemodynamically frequently

1	brain injury must be avoided. In this setting, a SBP of 110 mmHg would seem to be more appropriate.Patients who respond to initial resuscitative effort but then deteriorate hemodynamically frequently have injuries that require operative intervention. The magnitude and duration of their response will dictate whether diagnostic maneuvers can be performed to identify the site of bleeding. However, hemo-dynamic deterioration generally denotes ongoing bleeding for which some form of intervention (i.e., operation or interven-tional radiology) is required. Patients who have lost significant intravascular volume, but whose hemorrhage is controlled or has abated, often will respond to resuscitative efforts if the depth and duration of shock have been limited.A subset of patients exists who fail to respond to resuscita-tive efforts despite adequate control of ongoing hemorrhage. These patients have ongoing fluid requirements despite ade-quate control of hemorrhage, have persistent hypotension

1	respond to resuscita-tive efforts despite adequate control of ongoing hemorrhage. These patients have ongoing fluid requirements despite ade-quate control of hemorrhage, have persistent hypotension despite restoration of intravascular volume necessitating vasopressor support, and may exhibit a futile cycle of uncorrectable hypothermia, hypoperfusion, acidosis, and coagulopathy that cannot be interrupted despite maximum ther-apy. These patients have deteriorated to decompensated or irre-versible shock with peripheral vasodilation and resistance to vasopressor infusion. Mortality is almost inevitable once the patient manifests shock in its terminal stages. Unfortunately, this is often diagnosed in retrospect.Fluid resuscitation is a major adjunct to physically con-trolling hemorrhage in patients with shock. The ideal type of fluid to be used continues to be debated; however, crystalloids continue to be the mainstay fluid of choice. Several studies have demonstrated increased risk of

1	patients with shock. The ideal type of fluid to be used continues to be debated; however, crystalloids continue to be the mainstay fluid of choice. Several studies have demonstrated increased risk of death in bleeding trauma patients treated with colloid compared to patients treated with crystalloid.68 In patients with severe hemorrhage, restoration of intravascular volume should be achieved with blood products.69Ongoing studies continue to evaluate the use of hypertonic saline as a resuscitative adjunct in bleeding patients.70 The ben-efit of hypertonic saline solutions may be immunomodulatory. Specifically, these effects have been attributed to pharmacologic effects resulting in decreased reperfusion-mediated injury with decreased O2 radical formation, less impairment of immune function compared to standard crystalloid solution, and less brain swelling in the multi-injured patient. The reduction of total volume used for resuscitation makes this approach appealing as a resuscitation

1	compared to standard crystalloid solution, and less brain swelling in the multi-injured patient. The reduction of total volume used for resuscitation makes this approach appealing as a resuscitation agent for combat injuries and may contribute to a decrease in the incidence of ARDS and multiple organ failure.Transfusion of packed red blood cells and other blood products is essential in the treatment of patients in hemorrhagic shock. Current recommendations in stable ICU patients aim for a target hemoglobin of 7 to 9 g/dL71,72; however, no prospective randomized trials have compared restrictive and liberal transfu-sion regimens in trauma patients with hemorrhagic shock. The current standard in severely injured patients is termed dam-age control resuscitation and consists of transfusion with red blood cells, fresh frozen plasma (FFP), and platelet units given in equal number.73 Civilian and military trauma data show that the development of coagulopathy of trauma is predictive of

1	with red blood cells, fresh frozen plasma (FFP), and platelet units given in equal number.73 Civilian and military trauma data show that the development of coagulopathy of trauma is predictive of mortality.74 Data collected from a U.S. Army combat support hospital helped to propagate this practice, showing in patients that received massive transfusion of packed red blood cells (>10 units in 24 hours) that a high plasma to RBC ratio (1:1.4 units) was independently associated with improved survival.75 A number of civilian studies have demonstrated similar results.76 Similarly, platelet transfusion is important. The Pragmatic Randomized Optimal Platelet and Plasma Ratios (PROPPR) 45Brunicardi_Ch05_p0131-p0156.indd 14429/01/19 11:06 AM 145SHOCKCHAPTER 5trial has demonstrated that damage-control resuscitation, a massive transfusion strategy targeting a balanced delivery of plasma-platelet-red blood cell in a ratio of 1:1:1, results in improved survival at 3 hours and a reduction in

1	resuscitation, a massive transfusion strategy targeting a balanced delivery of plasma-platelet-red blood cell in a ratio of 1:1:1, results in improved survival at 3 hours and a reduction in deaths caused by exsanguination in the first 24 hours.77 Studies have demon-strated that low platelet counts in trauma patients was associ-ated with increased mortality78 and that increased platelet use appears to improve outcome.79,80 The benefit of platelet trans-fusion may be most pronounced in trauma patients with brain injury.81 It has been suggested that platelets should be transfused in the bleeding patient to maintain counts above 50 × 109/L. In addition, transfusion of whole blood is gaining in popularity, and ongoing studies are evaluating the benefit of this approach.There is a potential role for other coagulation factor based products, such as fibrinogen concentrates and prothrombin complex concentrates. Use of these agents may be guided by a drop in fibrinogen levels to less than 1

1	role for other coagulation factor based products, such as fibrinogen concentrates and prothrombin complex concentrates. Use of these agents may be guided by a drop in fibrinogen levels to less than 1 g/L, or less specifically by thromboelastogram findings to suggest hyperfibrinolysis. Data also support the use of antifibrinolytic agents in bleeding trauma patients, specifically tranexamic acid (a synthetic lysine analogues that acts as a competitive inhibitor of plasmin and plasminogen). The multinational Clinical Randomization of an Antifibrinolytic in Significant Haemorrhage 2 (CRASH-2) trial suggested that early use of tranexamic acid limits rebleeding and reduces mortality82 (Fig 5-10). In the past, coagulopathy associated with the bleeding patient was presumed to be due solely to dilution and depletion of clotting factors and plate-lets. We now understand that an acute coagulopathy of trauma occurs as an immediate consequence of injury, with abnormal admission coagulation as a

1	and depletion of clotting factors and plate-lets. We now understand that an acute coagulopathy of trauma occurs as an immediate consequence of injury, with abnormal admission coagulation as a predictor of high mortality.83 Tradi-tional measurement of platelets, INR, and PTT may not reflect the coagulopathy of trauma or response to therapy effectively. Recently, viscoelastic functional testing such as thromboelas-tography (TEG) has been utilized as a quicker, more compre-hensive determination of coagulopathy and fibrinolysis in the injured patient. Holcomb et al recently reported that TEG pre-dicted patients with substantial bleeding and red cell transfusion better than conventional coagulopathy tests, and it also predicted the need for platelet transfusion better than platelet count and the need for plasma transfusion better than fibrinogen levels.84OR (95% CI) of tranexamic acidTime to treatment (h)0.58765432101.01.52.02.53.0Figure 5-10. Early treatment (within 3 hours) of trauma

1	the need for plasma transfusion better than fibrinogen levels.84OR (95% CI) of tranexamic acidTime to treatment (h)0.58765432101.01.52.02.53.0Figure 5-10. Early treatment (within 3 hours) of trauma patients with tranexamic acid reduces mortality. However, later treatment exacerbated outcome. (Reproduced with permission from Roberts I, Shakur H, Afolabi A, et al. The importance of early treatment with tranexamic acid in bleeding trauma patients: an exploratory analysis of the CRASH-2 randomised controlled trial, Lancet. 2011 Mar 26;377(9771):1096-1101.)Additional resuscitative adjuncts in patients with hemor-rhagic shock include minimization of heat loss and maintaining normothermia. The development of hypothermia in the bleeding patient is associated with acidosis, hypotension, and coagulopa-thy. Hypothermia in bleeding trauma patients is an independent risk factor for bleeding and death. This likely is secondary to impaired platelet function and impairments in the coagulation

1	coagulopa-thy. Hypothermia in bleeding trauma patients is an independent risk factor for bleeding and death. This likely is secondary to impaired platelet function and impairments in the coagulation cascade. Several studies have investigated the induction of con-trolled hypothermia in patients with severe shock based on the hypothesis of limiting metabolic activity and energy require-ments, creating a state of “suspended animation.” These studies are promising and continue to be evaluated in large trials.Traumatic ShockThe systemic response after trauma, combining the effects of soft tissue injury, long bone fractures, and blood loss, is clearly a different physiologic insult than simple hemorrhagic shock. Multiple organ failure, including acute respiratory distress syn-drome (ARDS), develops relatively often in the blunt trauma patient, but rarely after pure hemorrhagic shock (such as a GI bleed). The hypoperfusion deficit in traumatic shock is magnified by the proinflammatory

1	develops relatively often in the blunt trauma patient, but rarely after pure hemorrhagic shock (such as a GI bleed). The hypoperfusion deficit in traumatic shock is magnified by the proinflammatory activation that occurs following the induc-tion of shock. In addition to ischemia or ischemia-reperfusion, accumulating evidence demonstrates that even simple hemor-rhage induces proinflammatory activation that results in many of the cellular changes typically ascribed only to septic shock.85,86 At the cellular level, this may be attributable to the release of cellular products termed damage associated molecu-lar patterns (DAMPs, i.e., riboxynucleic acid, uric acid, and high mobility group box 1) that activate the same set of cell surface receptors as bacterial products, initiating similar cell signaling.5,87 These receptors are termed pattern recognition receptors (PRRs) and include the TLR family of proteins. Examples of traumatic shock include small volume hemorrhage accompanied by soft

1	signaling.5,87 These receptors are termed pattern recognition receptors (PRRs) and include the TLR family of proteins. Examples of traumatic shock include small volume hemorrhage accompanied by soft tissue injury (femur fracture, crush injury), or any combination of hypovolemic, neurogenic, cardiogenic, and obstructive shock that precipitate rapidly progressive proin-flammatory activation. In laboratory models of traumatic shock, the addition of a soft tissue or long bone injury to hemorrhage produces lethality with significantly less blood loss when the animals are stressed by hemorrhage. Treatment of traumatic shock is focused on correction of the individual elements to diminish the cascade of proinflammatory activation, and includes prompt control of hemorrhage, adequate volume resus-citation to correct O2 debt, debridement of nonviable tissue, stabilization of bony injuries, and appropriate treatment of soft tissue injuries.Septic Shock (Vasodilatory Shock)In the peripheral

1	to correct O2 debt, debridement of nonviable tissue, stabilization of bony injuries, and appropriate treatment of soft tissue injuries.Septic Shock (Vasodilatory Shock)In the peripheral circulation, profound vasoconstriction is the typical physiologic response to the decreased arterial pressure and tissue perfusion with hemorrhage, hypovolemia, or acute heart failure. This is not the characteristic response in vasodila-tory shock. Vasodilatory shock is the result of dysfunction of the endothelium and vasculature secondary to circulating inflam-matory mediators and cells or as a response to prolonged and severe hypoperfusion. Thus, in vasodilatory shock, hypotension results from failure of the vascular smooth muscle to constrict appropriately. Vasodilatory shock is characterized by peripheral vasodilation with resultant hypotension and resistance to treat-ment with vasopressors. Despite the hypotension, plasma cate-cholamine levels are elevated, and the renin-angiotensin system

1	peripheral vasodilation with resultant hypotension and resistance to treat-ment with vasopressors. Despite the hypotension, plasma cate-cholamine levels are elevated, and the renin-angiotensin system 6Brunicardi_Ch05_p0131-p0156.indd 14529/01/19 11:06 AM 146BASIC CONSIDERATIONSPART Iis activated in vasodilatory shock. The most frequently encoun-tered form of vasodilatory shock is septic shock. Other causes of vasodilatory shock include hypoxic lactic acidosis, carbon monoxide poisoning, decompensated and irreversible hemor-rhagic shock, terminal cardiogenic shock, and postcardiotomy shock (Table 5-6). Thus, vasodilatory shock seems to represent the final common pathway for profound and prolonged shock of any etiology.88Despite advances in intensive care, the mortality rate for severe sepsis remains at 30% to 50%. In the United States, 750,000 cases of sepsis occur annually, one-third of which are fatal.89 Sepsis accounts for 9.3% of deaths in the United States, as many yearly as

1	sepsis remains at 30% to 50%. In the United States, 750,000 cases of sepsis occur annually, one-third of which are fatal.89 Sepsis accounts for 9.3% of deaths in the United States, as many yearly as MI. Septic shock is a by-product of the body’s response to disruption of the host-microbe equilibrium, result-ing in invasive or severe localized infection.In the attempt to eradicate the pathogens, the immune and other cell types (e.g., endothelial cells) elaborate soluble medi-ators that enhance macrophage and neutrophil killing effec-tor mechanisms, increase procoagulant activity and fibroblast activity to localize the invaders, and increase microvascular blood flow to enhance delivery of killing forces to the area of invasion. When this response is overly exuberant or becomes systemic rather than localized, manifestations of sepsis may be evident. These findings include enhanced cardiac output, peripheral vasodilation, fever, leukocytosis, hyperglycemia, and tachycardia. In septic

1	rather than localized, manifestations of sepsis may be evident. These findings include enhanced cardiac output, peripheral vasodilation, fever, leukocytosis, hyperglycemia, and tachycardia. In septic shock, the vasodilatory effects are due, in part, to the upregulation of the inducible isoform of nitric oxide synthase (iNOS or NOS 2) in the vessel wall. iNOS produces large quantities of nitric oxide for sustained periods of time. This potent vasodilator suppresses vascular tone and renders the vasculature resistant to the effects of vasoconstricting agents. Additionally, endothelial activation or injury likely contributes to some degree of vascular dysfunction.Diagnosis. Attempts to standardize terminology have led to the establishment of criteria for the diagnosis of sepsis in the hospitalized adult. These criteria include manifestations of the host response to infection in addition to identification of an offending organism. The terms sepsis and septic shock are used to quantify the

1	adult. These criteria include manifestations of the host response to infection in addition to identification of an offending organism. The terms sepsis and septic shock are used to quantify the magnitude of the systemic inflammatory reac-tion. Patients with sepsis have evidence of an infection, as well as systemic signs of inflammation (e.g., fever, leukocytosis, and tachycardia). Hypoperfusion with signs of organ dysfunction is termed severe sepsis. Septic shock requires the presence of the aforementioned signs, associated with more significant evidence of tissue hypoperfusion and systemic hypotension. Beyond the hypotension, maldistribution of blood flow and shunting in the microcirculation further compromise delivery of nutrients to the tissue beds.90,91 The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) have refined the defi-nitions and utilize a Sequential (Sepsis-related) Organ Failure Assessment (SOFA) score to help determine signs of organ

1	Definitions for Sepsis and Septic Shock (Sepsis-3) have refined the defi-nitions and utilize a Sequential (Sepsis-related) Organ Failure Assessment (SOFA) score to help determine signs of organ dys-function and to guide management.92Recognizing septic shock begins with defining the patient at risk. The clinical manifestations of septic shock will usually become evident and prompt the initiation of treatment before bacteriologic confirmation of an organism or the source of an organism is identified. In addition to fever, tachycardia, and tachypnea, signs of hypoperfusion such as confusion, malaise, oliguria, or hypotension may be present. These should prompt an aggressive search for infection, including a thorough physical examination, inspection of all wounds, evaluation of intravas-cular catheters or other foreign bodies, obtaining appropriate cultures, and adjunctive imaging studies, as needed.Treatment. Evaluation of the patient in septic shock begins with an assessment of the

1	catheters or other foreign bodies, obtaining appropriate cultures, and adjunctive imaging studies, as needed.Treatment. Evaluation of the patient in septic shock begins with an assessment of the adequacy of their airway and ven-tilation. Severely obtunded patients and patients whose work of breathing is excessive require intubation and ventilation to prevent respiratory collapse. Because vasodilation and decrease in total peripheral resistance may produce hypotension, fluid resuscitation and restoration of circulatory volume with bal-anced salt solutions is essential. The Surviving Sepsis Campaign has updated treatment recommendations and care bundles with a most recent goal for care within the first hour93,94 (Table 5-7). Serum lactate should be measured as a marker of shock. Fluid resuscitation should begin within the first hour and should be at least 30 mL/kg for hypotensive patients. Incremental fluid boluses should be continued based upon endpoint of resuscita-tion, including

1	should begin within the first hour and should be at least 30 mL/kg for hypotensive patients. Incremental fluid boluses should be continued based upon endpoint of resuscita-tion, including clearance of lactate. Starch-based colloid solu-tions should be avoided as recent evidence suggests that these fluids may be deleterious in the setting of sepsis.90,95,96 Blood cultures should be obtained. Empiric antibiotics must be chosen carefully based on the most likely pathogens (gram-negative rods, gram-positive cocci, and anaerobes) because the portal of entry of the offending organism and its identity may not be evi-dent until culture data return or imaging studies are completed. Knowledge of the bacteriologic profile of infections in an indi-vidual unit can be obtained from most hospital infection control departments and will suggest potential responsible organisms. Antibiotics should be tailored to cover the responsible organ-isms once culture data are available, and if appropriate, the

1	control departments and will suggest potential responsible organisms. Antibiotics should be tailored to cover the responsible organ-isms once culture data are available, and if appropriate, the Table 5-6Causes of septic and vasodilatory shockSystemic response to infectionNoninfectious systemic inflammation Pancreatitis BurnsAnaphylaxisAcute adrenal insufficiencyProlonged, severe hypotension Hemorrhagic shock Cardiogenic shock Cardiopulmonary bypassMetabolic Hypoxic lactic acidosis Carbon monoxide poisoningTable 5-7Surviving Sepsis Campaign bundles of care to be initiated within the first hour after presentation in the patient with sepsis93,94Measure serum lactate level. Remeasure if the initial lactate is >2 mmol/LObtain blood culture prior to administration of antibioticsAdminister broad spectrum antibioticsRapid administration of 30 mL/kg crystalloid for hypotension or lactate ≥4 mmol/LUse vasopressors if the patient is hypotensive during or after fluid resuscitation to maintain a

1	spectrum antibioticsRapid administration of 30 mL/kg crystalloid for hypotension or lactate ≥4 mmol/LUse vasopressors if the patient is hypotensive during or after fluid resuscitation to maintain a mean arterial pressure ≥65 mmHgBrunicardi_Ch05_p0131-p0156.indd 14629/01/19 11:06 AM 147SHOCKCHAPTER 5spectrum of coverage narrowed. Long-term, empiric, broad-spectrum antibiotic use should be minimized to reduce the development of resistant organisms and to avoid the potential complications of fungal overgrowth and antibiotic-associated colitis from overgrowth of Clostridium difficile. IV antibiotics will be insufficient to adequately treat the infectious episode in the settings of infected fluid collections, infected foreign bodies, and devitalized tissue. These situations require source control and involve percutaneous drainage and operative management to target a focus of infection. These situations may require mul-tiple operations to ensure proper wound hygiene and healing.After

1	control and involve percutaneous drainage and operative management to target a focus of infection. These situations may require mul-tiple operations to ensure proper wound hygiene and healing.After first-line therapy of the septic patient with antibiot-ics, IV fluids, and intubation if necessary, vasopressors may be necessary to treat patients with septic shock. Catecholamines are the vasopressors used most often, with norepinephrine being the first-line agent followed by epinephrine. Occasionally, patients with septic shock will develop arterial resistance to catechol-amines. Arginine vasopressin, a potent vasoconstrictor, is often efficacious in this setting and is often added to norepinephrine.The majority of septic patients have hyperdynamic physi-ology with supranormal cardiac output and low systemic vas-cular resistance. On occasion, septic patients may have low cardiac output despite volume resuscitation and even vasopres-sor support. Dobutamine therapy is recommended for

1	and low systemic vas-cular resistance. On occasion, septic patients may have low cardiac output despite volume resuscitation and even vasopres-sor support. Dobutamine therapy is recommended for patients with cardiac dysfunction as evidenced by high filling pres-sures and low cardiac output or clinical signs of hypoperfusion after achievement of restoration of blood pressure following fluid resuscitation. Mortality in this group is high. Despite the increasing incidence of septic shock over the past several decades, the overall mortality rates have changed little. Stud-ies of interventions, including immunotherapy, resuscitation to pulmonary artery endpoints with hemodynamic optimization (cardiac output and O2 delivery, even to supranormal values), and optimization of mixed venous O2 measurements up to 72 hours after admission to the ICU, have not changed mortality.The advances made in the treatment of patients with sep-sis and septic shock and collaborative groups such as the

1	measurements up to 72 hours after admission to the ICU, have not changed mortality.The advances made in the treatment of patients with sep-sis and septic shock and collaborative groups such as the Sur-viving Sepsis Campaign continue to evaluate, modify, and put forth recommendations based upon data. Negative results from previous studies have led to the suggestion that earlier inter-ventions directed at improving global tissue oxygenation may be of benefit. To this end, Rivers and colleagues reported that goal-directed therapy of septic shock and severe sepsis initiated in the emergency department and continued for 6 hours signifi-cantly improved outcome.97 This approach involved adjustment of cardiac preload, afterload, and contractility to balance O2 delivery with O2 demand. They found that goal-directed therapy during the first 6 hours of hospital stay (initiated in the emer-gency department) had significant effects, such as higher mean venous O2 saturation, lower lactate levels,

1	that goal-directed therapy during the first 6 hours of hospital stay (initiated in the emer-gency department) had significant effects, such as higher mean venous O2 saturation, lower lactate levels, lower base deficit, higher pH, and decreased 28-day mortality (49.2% vs. 33.3%) compared to the standard therapy group. The frequency of sud-den cardiovascular collapse was also significantly less in the group managed with goal-directed therapy (21.0% vs. 10.3%). Interestingly, the goal-directed therapy group received more IV fluids during the initial 6 hours, but the standard therapy group required more IV fluids by 72 hours. The authors emphasize that continued cellular and tissue decompensation is subclinical and often irreversible when obvious clinically. Goal-directed therapy allowed identification and treatment of these patients with insidious illness (global tissue hypoxia in the setting of normal vital signs).Several multicenter trials have been performed to further refine these

1	and treatment of these patients with insidious illness (global tissue hypoxia in the setting of normal vital signs).Several multicenter trials have been performed to further refine these finding. In the Protocolized Care for Early Septic Shock (ProCESS) trial, a multicenter, randomized trial in which patients were identified early in the emergency department as having septic shock and received antibiotics and other nonre-suscitation aspects of care promptly, the investigators found no significant advantage, with respect to mortality or morbid-ity, of protocol-based resuscitation over bedside care that was provided according to the treating physician’s judgment. They also found no significant benefit of the mandated use of central venous catheterization and central hemodynamic monitoring in all patients. This last finding was recapitulated in the Pro-tocolised Management in Sepsis (ProMISe) trial.98,99 Failure of these more recent trials to show a benefit of early goal-directed

1	in all patients. This last finding was recapitulated in the Pro-tocolised Management in Sepsis (ProMISe) trial.98,99 Failure of these more recent trials to show a benefit of early goal-directed protocols vs. standard of care may be secondary to the gener-alized improvement in early recognition of sepsis and institu-tion of protocolized care by efforts such as the Surviving Sepsis Campaign.Hyperglycemia and insulin resistance are typical in criti-cally ill and septic patients, including patients without underly-ing diabetes mellitus. A recent study reported significant positive impact of tight glucose management on outcome in critically ill patients.100 The two treatment groups in this randomized, pro-spective study were assigned to receive intensive insulin therapy (maintenance of blood glucose between 80 and 110 mg/dL) or conventional treatment (infusion of insulin only if the blood glu-cose level exceeded 215 mg/dL, with a goal between 180 and 200 mg/dL). The mean morning glucose

1	glucose between 80 and 110 mg/dL) or conventional treatment (infusion of insulin only if the blood glu-cose level exceeded 215 mg/dL, with a goal between 180 and 200 mg/dL). The mean morning glucose level was significantly higher in the conventional treatment as compared to the inten-sive insulin therapy group (153 vs. 103 mg/dL). Mortality in the intensive insulin treatment group (4.6%) was significantly lower than in the conventional treatment group (8.0%), representing a 42% reduction in mortality. This reduction in mortality was most notable in the patients requiring longer than 5 days in the ICU. Furthermore, intensive insulin therapy reduced episodes of septicemia by 46%, reduced duration of antibiotic therapy, and decreased the need for prolonged ventilatory support and renal replacement therapy.Another treatment protocol that has been demonstrated to increase survival in patients with ARDS investigated the use of lower ventilatory tidal volumes compared to traditional tidal

1	therapy.Another treatment protocol that has been demonstrated to increase survival in patients with ARDS investigated the use of lower ventilatory tidal volumes compared to traditional tidal volumes.101 The majority of the patients enrolled in this multicenter, randomized trial developed ARDS secondary to pneumonia or sepsis. The trial compared traditional ventilation treatment, which involved an initial tidal volume of 12 mL/kg of predicted body weight, with ventilation with a lower tidal volume, which involved an initial tidal volume of 6 mL/kg of predicted body weight. The trial was stopped after the enroll-ment of 861 patients because mortality was lower in the group treated with lower tidal volumes than in the group treated with traditional tidal volumes (31.0% vs. 39.8%, P = .007), and the number of days without ventilator use during the first 28 days after randomization was greater in this group (mean ± SD, 12 ± 11 vs. 10 ± 11; P = .007). The investigators concluded that in

1	and the number of days without ventilator use during the first 28 days after randomization was greater in this group (mean ± SD, 12 ± 11 vs. 10 ± 11; P = .007). The investigators concluded that in patients with acute lung injury and ARDS, mechanical ventilation with a lower tidal volume than is traditionally used results in decreased mortality and increases the number of days without ventilator use. Additional strategies in ARDS manage-ment include higher levels of positive end expiratory pressure (PEEP), alveolar recruitment maneuvers, and prone position-ing. Prone positioning has become part of many standardized protocols. This is supported by several studies, including a Brunicardi_Ch05_p0131-p0156.indd 14729/01/19 11:06 AM 148BASIC CONSIDERATIONSPART Imeta-analysis demonstrating that prone positioning is associ-ated with significantly reduced mortality from ARDS in the low tidal volume era.102The use of corticosteroids in the treatment of sepsis and septic shock has been

1	that prone positioning is associ-ated with significantly reduced mortality from ARDS in the low tidal volume era.102The use of corticosteroids in the treatment of sepsis and septic shock has been controversial for decades. The observa-tion that severe sepsis often is associated with adrenal insuf-ficiency or glucocorticoid receptor resistance has generated renewed interest in therapy for septic shock with corticosteroids. A single IV dose of 50 mg of hydrocortisone improved mean arterial blood pressure response relationships to norepinephrine and phenylephrine in patients with septic shock and was most notable in patients with relative adrenal insufficiency. A study evaluated therapy with hydrocortisone (50 mg IV every 6 hours) and fludrocortisone (50 µg orally once daily) vs. placebo for 1 week in patients with septic shock.103 As in earlier studies, the authors performed corticotropin tests on these patients to document and stratify patients by relative adrenal insufficiency. In

1	1 week in patients with septic shock.103 As in earlier studies, the authors performed corticotropin tests on these patients to document and stratify patients by relative adrenal insufficiency. In this study, 7-day treatment with low doses of hydrocortisone and fludrocortisone significantly and safely lowered the risk of death in patients with septic shock and relative adrenal insuf-ficiency. In an international, multicenter, randomized trial of corticosteroids in sepsis (CORTICUS study; 499 analyzable patients), steroids showed no benefit in intent to treat mortality or shock reversal.104 This study suggested that hydrocortisone therapy cannot be recommended as routine adjuvant therapy for septic shock. However, if SBP remains less than 90 mmHg despite appropriate fluid and vasopressor therapy, hydrocorti-sone at 200 mg/day for 7 days in four divided doses or by con-tinuous infusion should be considered.Additional adjunctive immune modulation strategies have been developed for the

1	therapy, hydrocorti-sone at 200 mg/day for 7 days in four divided doses or by con-tinuous infusion should be considered.Additional adjunctive immune modulation strategies have been developed for the treatment of septic shock. These include the use of antiendotoxin antibodies, anticytokine anti-bodies, cytokine receptor antagonists, immune enhancers, a non– isoform-specific nitric oxide synthase inhibitor, and O2 radical scavengers. These compounds are each designed to alter some aspect of the host immune response to shock that is hypothesized to play a key role in its pathophysiology. However, most of these strategies have failed to demonstrate efficacy in human patients despite utility in well-controlled animal experiments. It is unclear whether the failure of these compounds is due to poorly designed clinical trials, inadequate understanding of the interactions of the complex host immune response to injury and infection, or animal models of shock that poorly represent the human

1	to poorly designed clinical trials, inadequate understanding of the interactions of the complex host immune response to injury and infection, or animal models of shock that poorly represent the human disease.Cardiogenic ShockCardiogenic shock is defined clinically as circulatory pump failure leading to diminished forward flow and subsequent tis-sue hypoxia, in the setting of adequate intravascular volume. Hemodynamic criteria include sustained hypotension (i.e., SBP <90 mmHg for at least 30 minutes), reduced cardiac index (<2.2 L/min per square meter), and elevated pulmonary artery wedge pressure (>15 mmHg).105 Mortality rates for cardiogenic shock are 50% to 80%. Acute, extensive MI is the most common cause of cardiogenic shock; a smaller infarction in a patient with existing left ventricular dysfunction also may precipitate shock. Cardiogenic shock complicates 5% to 10% of acute MIs. Con-versely, cardiogenic shock is the most common cause of death in patients hospitalized with

1	dysfunction also may precipitate shock. Cardiogenic shock complicates 5% to 10% of acute MIs. Con-versely, cardiogenic shock is the most common cause of death in patients hospitalized with acute MI. Although shock may develop early after MI, it typically is not found on admission. Seventy-five percent of patients who have cardiogenic shock complicating acute MIs develop signs of cardiogenic shock within 24 hours after onset of infarction (average 7 hours).Recognition of the patient with occult hypoperfusion is critical to prevent progression to obvious cardiogenic shock with its high mortality rate; early initiation of therapy to maintain blood pressure and cardiac output is vital. Rapid assessment, adequate resuscitation, and reversal of the myocardial ischemia are essential in optimizing outcome in patients with acute MI. Prevention of infarct extension is a critical component. Large segments of nonfunctional but viable myocardium contribute to the development of cardiogenic shock

1	outcome in patients with acute MI. Prevention of infarct extension is a critical component. Large segments of nonfunctional but viable myocardium contribute to the development of cardiogenic shock after MI. In the setting of acute MI, expeditious restoration of cardiac output is mandatory to minimize mortality; the extent of myocardial salvage possible decreases exponentially with increased time to restoration of coronary blood flow. The degree of coronary flow after percuta-neous transluminal coronary angioplasty correlates with inhos-pital mortality (i.e., 33% mortality with complete reperfusion, 50% mortality with incomplete reperfusion, and 85% mortality with absent reperfusion).106 Inadequate cardiac function can be a direct result of cardiac injury, including profound myocardial contusion, blunt cardiac valvular injury, or direct myocardial damage (Table 5-8).105-108 The pathophysiology of cardiogenic shock involves a vicious cycle of myocardial ischemia that causes myocardial

1	blunt cardiac valvular injury, or direct myocardial damage (Table 5-8).105-108 The pathophysiology of cardiogenic shock involves a vicious cycle of myocardial ischemia that causes myocardial dysfunction, which results in more myocar-dial ischemia. When sufficient mass of the left ventricular wall is necrotic or ischemic and fails to pump, the stroke volume decreases. An autopsy series of patients dying from cardiogenic shock have found damage to 40% of the left ventricle.109 Isch-emia distant from the infarct zone may contribute to the systolic dysfunction in patients with cardiogenic shock. The majority of these patients have multivessel disease, with limited vasodila-tor reserve and pressure-dependent coronary flow in multiple areas of the heart. Myocardial diastolic function is impaired in cardiogenic shock as well. Decreased compliance results from myocardial ischemia, and compensatory increases in left ven-tricular filling pressures progressively occur.Diminished cardiac output

1	in cardiogenic shock as well. Decreased compliance results from myocardial ischemia, and compensatory increases in left ven-tricular filling pressures progressively occur.Diminished cardiac output or contractility in the face of adequate intravascular volume (preload) may lead to under-perfused vascular beds and reflexive sympathetic discharge. Table 5-8Causes of cardiogenic shockAcute myocardial infarctionPump failureMechanical complications Acute mitral regurgitation Acute ventricular septal defect Free wall rupture Pericardial tamponadeArrhythmiaEnd-stage cardiomyopathyMyocarditisSevere myocardial contusionLeft ventricular outflow obstruction Aortic stenosis Hypertrophic obstructive cardiomyopathyObstruction to left ventricular filling Mitral stenosis Left atrial myxomaAcute mitral regurgitationAcute aortic insufficiencyMetabolicDrug reactionsBrunicardi_Ch05_p0131-p0156.indd 14829/01/19 11:06 AM 149SHOCKCHAPTER 5Increased sympathetic stimulation of the heart, either through

1	regurgitationAcute aortic insufficiencyMetabolicDrug reactionsBrunicardi_Ch05_p0131-p0156.indd 14829/01/19 11:06 AM 149SHOCKCHAPTER 5Increased sympathetic stimulation of the heart, either through direct neural input or from circulating catecholamines, increases heart rate, myocardial contraction, and myocardial O2 consump-tion, which may not be relieved by increases in coronary artery blood flow in patients with fixed stenoses of the coronary arter-ies. Diminished cardiac output may also decrease coronary artery blood flow, resulting in a scenario of increased myocar-dial O2 demand at a time when myocardial O2 supply may be limited. Acute heart failure may also result in fluid accumula-tion in the pulmonary microcirculatory bed, decreasing myocar-dial O2 delivery even further.Diagnosis. Rapid identification of the patient with pump failure and institution of corrective action are essential in pre-venting the ongoing spiral of decreased cardiac output from injury causing increased

1	identification of the patient with pump failure and institution of corrective action are essential in pre-venting the ongoing spiral of decreased cardiac output from injury causing increased myocardial O2 needs that cannot be met, leading to progressive and unremitting cardiac dysfunc-tion. In evaluation of possible cardiogenic shock, other causes of hypotension must be excluded, including hemorrhage, sepsis, pulmonary embolism, and aortic dissection. Signs of circulatory shock include hypotension, cool and mottled skin, depressed mental status, tachycardia, and diminished pulses. Cardiac exam may include dysrhythmia, precordial heave, or distal heart tones. Confirmation of a cardiac source for the shock requires electrocardiogram and urgent echocardiography. Other useful diagnostic tests include chest radiograph, arterial blood gases, electrolytes, complete blood count, and cardiac enzymes. Inva-sive cardiac monitoring, which generally is not necessary, can be useful to exclude right

1	include chest radiograph, arterial blood gases, electrolytes, complete blood count, and cardiac enzymes. Inva-sive cardiac monitoring, which generally is not necessary, can be useful to exclude right ventricular infarction, hypovolemia, and possible mechanical complications.Making the diagnosis of cardiogenic shock involves the identification of cardiac dysfunction or acute heart failure in a susceptible patient. In the setting of blunt traumatic injury, hemorrhagic shock from intra-abdominal bleeding, intrathoracic bleeding, and bleeding from fractures must be excluded, before implicating cardiogenic shock from blunt cardiac injury. Rela-tively few patients with blunt cardiac injury will develop cardiac pump dysfunction. Those who do generally exhibit cardiogenic shock early in their evaluation. Therefore, establishing the diag-nosis of blunt cardiac injury is secondary to excluding other etiologies for shock and establishing that cardiac dysfunction is present. Invasive hemodynamic

1	Therefore, establishing the diag-nosis of blunt cardiac injury is secondary to excluding other etiologies for shock and establishing that cardiac dysfunction is present. Invasive hemodynamic monitoring with a pulmonary artery catheter may uncover evidence of diminished cardiac out-put and elevated pulmonary artery pressure.Treatment. After ensuring that an adequate airway is present and ventilation is sufficient, attention should be focused on sup-port of the circulation. Intubation and mechanical ventilation often are required, if only to decrease work of breathing and facilitate sedation of the patient. Rapidly excluding hypovolemia and establishing the presence of cardiac dysfunction are essen-tial. Treatment of cardiac dysfunction includes maintenance of adequate oxygenation to ensure adequate myocardial O2 deliv-ery and judicious fluid administration to avoid fluid overload and development of cardiogenic pulmonary edema. Electrolyte abnormalities, commonly hypokalemia and

1	ensure adequate myocardial O2 deliv-ery and judicious fluid administration to avoid fluid overload and development of cardiogenic pulmonary edema. Electrolyte abnormalities, commonly hypokalemia and hypomagnesemia, should be corrected. Pain is treated with IV morphine sulfate or fentanyl. Significant dysrhythmias and heart block must be treated with antiarrhythmic drugs, pacing, or cardioversion, if necessary. Early consultation with cardiology is essential in current management of cardiogenic shock, particularly in the setting of acute MI.105When profound cardiac dysfunction exists, inotropic support may be indicated to improve cardiac contractility and cardiac output. Dobutamine primarily stimulates cardiac β1-receptors to increase cardiac output but may also vasodilate peripheral vascular beds, lower total peripheral resistance, and lower systemic blood pressure through effects on β2-receptors. Ensuring adequate preload and intravascular volume is therefore essential prior to

1	vascular beds, lower total peripheral resistance, and lower systemic blood pressure through effects on β2-receptors. Ensuring adequate preload and intravascular volume is therefore essential prior to instituting therapy with dobutamine. Dopamine stimulates receptors (vasoconstriction), β1-receptors (cardiac stimulation), and β2-receptors (vasodilation), with its effects on β-receptors predominating at lower doses. Dopamine may be preferable to dobutamine in treatment of cardiac dysfunction in hypotensive patients. Tachycardia and increased peripheral resistance from dopamine infusion may worsen myocardial ischemia. Titration of both dopamine and dobutamine infusions may be required in some patients.Epinephrine stimulates αand β-receptors and may increase cardiac contractility and heart rate; however, it also may have intense peripheral vasoconstrictor effects that impair further cardiac performance. Catecholamine infusions must be carefully controlled to maximize coronary perfusion,

1	however, it also may have intense peripheral vasoconstrictor effects that impair further cardiac performance. Catecholamine infusions must be carefully controlled to maximize coronary perfusion, while minimizing myocardial O2 demand. Balancing the beneficial effects of impaired cardiac performance with the potential side effects of excessive reflex tachycardia and peripheral vasocon-striction requires serial assessment of tissue perfusion using indices such as capillary refill, character of peripheral pulses, adequacy of urine output, or improvement in laboratory param-eters of resuscitation such as pH, base deficit, and lactate. Inva-sive monitoring generally is necessary in these unstable patients. The phosphodiesterase inhibitors amrinone and milrinone may be required on occasion in patients with resistant cardiogenic shock. These agents have long half-lives and induce thrombocy-topenia and hypotension, and use is reserved for patients unre-sponsive to other treatment.Patients

1	patients with resistant cardiogenic shock. These agents have long half-lives and induce thrombocy-topenia and hypotension, and use is reserved for patients unre-sponsive to other treatment.Patients whose cardiac dysfunction is refractory to car-diotonics may require mechanical circulatory support with an intra-aortic balloon pump.110 Intra-aortic balloon pumping increases cardiac output and improves coronary blood flow by reduction of systolic afterload and augmentation of diastolic perfusion pressure. Unlike vasopressor agents, these beneficial effects occur without an increase in myocardial O2 demand. An intra-aortic balloon pump can be inserted at the bedside in the ICU via the femoral artery through either a cutdown or using the percutaneous approach. Aggressive circulatory support of patients with cardiac dysfunction from intrinsic cardiac disease has led to more widespread application of these devices and more familiarity with their operation by both physicians and critical care

1	patients with cardiac dysfunction from intrinsic cardiac disease has led to more widespread application of these devices and more familiarity with their operation by both physicians and critical care nurses.Preservation of existing myocardium and preservation of cardiac function are priorities of therapy for patients who have suffered an acute MI. Ensuring adequate oxygenation and O2 delivery, maintaining adequate preload with judicious volume restoration, minimizing sympathetic discharge through adequate relief of pain, and correcting electrolyte imbalances are all straightforward nonspecific maneuvers that may improve exist-ing cardiac function or prevent future cardiac complications. Anticoagulation and aspirin are given for acute MI. Although thrombolytic therapy reduces mortality in patients with acute MI, its role in cardiogenic shock is less clear. Patients in car-diac failure from an acute MI may benefit from pharmaco-logic or mechanical circulatory support in a manner similar

1	with acute MI, its role in cardiogenic shock is less clear. Patients in car-diac failure from an acute MI may benefit from pharmaco-logic or mechanical circulatory support in a manner similar to that of patients with cardiac failure related to blunt cardiac Brunicardi_Ch05_p0131-p0156.indd 14929/01/19 11:06 AM 150BASIC CONSIDERATIONSPART Iinjury. Additional pharmacologic tools may include the use of β-blockers to control heart rate and myocardial O2 consumption, nitrates to promote coronary blood flow through vasodilation, and ACE inhibitors to reduce ACE-mediated vasoconstrictive effects that increase myocardial workload and myocardial O2 consumption.Current guidelines of the American Heart Association recommend percutaneous transluminal coronary angiography for patients with cardiogenic shock, ST elevation, left bundle-branch block, and age less than 75 years.111,112 Early definition of coronary anatomy and revascularization is the pivotal step in treatment of patients with

1	shock, ST elevation, left bundle-branch block, and age less than 75 years.111,112 Early definition of coronary anatomy and revascularization is the pivotal step in treatment of patients with cardiogenic shock from acute MI.113 When feasible, percutaneous transluminal coronary angioplasty (generally with stent placement) is the treatment of choice. Coronary artery bypass grafting seems to be more appropriate for patients with multiple vessel disease or left main coronary artery disease.Obstructive ShockAlthough obstructive shock can be caused by a number of dif-ferent etiologies that result in mechanical obstruction of venous return (Table 5-9), in trauma patients this is most commonly due to the presence of tension pneumothorax. Cardiac tamponade occurs when sufficient fluid has accumulated in the pericardial sac to obstruct blood flow to the ventricles. The hemodynamic abnormalities in pericardial tamponade are due to elevation of intracardiac pressures with limitation of ventricular

1	in the pericardial sac to obstruct blood flow to the ventricles. The hemodynamic abnormalities in pericardial tamponade are due to elevation of intracardiac pressures with limitation of ventricular filling in diastole with resultant decrease in cardiac output. Acutely, the pericardium does not distend; thus small volumes of blood may produce cardiac tamponade. If the effusion accumulates slowly (e.g., in the setting of uremia, heart failure, or malignant effusion), the quantity of fluid producing cardiac tamponade may reach 2000 mL. The major determinant of the degree of hypotension is the pericardial pressure. With either cardiac tamponade or tension pneumothorax, reduced filling of the right side of the heart from either increased intrapleural pressure secondary to air accumulation (tension pneumothorax) or increased intra-pericardial pressure precluding atrial filling secondary to blood accumulation (cardiac tamponade) results in decreased cardiac output associated with increased

1	pneumothorax) or increased intra-pericardial pressure precluding atrial filling secondary to blood accumulation (cardiac tamponade) results in decreased cardiac output associated with increased central venous pressure.Diagnosis and Treatment. The diagnosis of tension pneu-mothorax should be made on clinical examination. The classic findings include respiratory distress (in an awake patient), hypo-tension, diminished breath sounds over one hemithorax, hyper-resonance to percussion, jugular venous distention, and shift of mediastinal structures to the unaffected side with tracheal deviation. In most instances, empiric treatment with pleural decompression is indicated rather than delaying to wait for radiographic confirmation. When a chest tube cannot be imme-diately inserted, such as in the prehospital setting, the pleural space can be decompressed with a large caliber needle. Immedi-ate return of air should be encountered with rapid resolution of hypotension. Unfortunately, not all of

1	prehospital setting, the pleural space can be decompressed with a large caliber needle. Immedi-ate return of air should be encountered with rapid resolution of hypotension. Unfortunately, not all of the clinical manifestations of tension pneumothorax may be evident on physical examina-tion. Hyperresonance may be difficult to appreciate in a noisy resuscitation area. Jugular venous distention may be absent in a hypovolemic patient. Tracheal deviation is a late finding and often is not apparent on clinical examination. Practically, three findings are sufficient to make the diagnosis of tension pneu-mothorax: respiratory distress or hypotension, decreased lung sounds, and hypertympany to percussion. Chest X-ray findings that may be visualized include deviation of mediastinal struc-tures, depression of the hemidiaphragm, and hypo-opacification with absent lung markings. As discussed previously, definitive treatment of a tension pneumothorax is immediate tube thora-costomy. The chest tube

1	of the hemidiaphragm, and hypo-opacification with absent lung markings. As discussed previously, definitive treatment of a tension pneumothorax is immediate tube thora-costomy. The chest tube should be inserted rapidly, but care-fully, and should be large enough to evacuate any blood that may be present in the pleural space. Most recommend place-ment is in the fourth intercostal space (nipple level) at the ante-rior axillary line.Cardiac tamponade results from the accumulation of blood within the pericardial sac, usually from penetrating trauma or chronic medical conditions such as heart failure or uremia. Although precordial wounds are most likely to injure the heart and produce tamponade, any projectile or wounding agent that passes in proximity to the mediastinum can potentially produce tamponade. Blunt cardiac rupture, a rare event in trauma vic-tims who survive long enough to reach the hospital, can produce refractory shock and tamponade in the multiply-injured patient. The

1	tamponade. Blunt cardiac rupture, a rare event in trauma vic-tims who survive long enough to reach the hospital, can produce refractory shock and tamponade in the multiply-injured patient. The manifestations of cardiac tamponade, such as total circula-tory collapse and cardiac arrest, may be catastrophic, or they may be more subtle. A high index of suspicion is warranted to make a rapid diagnosis. Patients who present with circulatory arrest from cardiac tamponade require emergency pericardial decompression, usually through a left thoracotomy. The indi-cations for this maneuver are discussed in Chapter 7. Cardiac tamponade also may be associated with dyspnea, orthopnea, cough, peripheral edema, chest pain, tachycardia, muffled heart tones, jugular venous distention, and elevated central venous pressure. Beck’s triad consists of hypotension, muffled heart tones, and neck vein distention. Unfortunately, absence of these clinical findings may not be sufficient to exclude cardiac injury

1	venous pressure. Beck’s triad consists of hypotension, muffled heart tones, and neck vein distention. Unfortunately, absence of these clinical findings may not be sufficient to exclude cardiac injury and cardiac tamponade. Muffled heart tones may be difficult to appreciate in a busy trauma center, and jugular venous disten-tion and central venous pressure may be diminished by coexis-tent bleeding. Therefore, patients at risk for cardiac tamponade whose hemodynamic status permits additional diagnostic tests frequently require additional diagnostic maneuvers to confirm cardiac injury or tamponade.Invasive hemodynamic monitoring may support the diag-nosis of cardiac tamponade if elevated central venous pressure, pulsus paradoxus (i.e., decreased systemic arterial pressure with inspiration), or elevated right atrial and right ventricular pres-sure by pulmonary artery catheter are present. These hemody-namic profiles suffer from lack of specificity, the duration of time required to obtain

1	or elevated right atrial and right ventricular pres-sure by pulmonary artery catheter are present. These hemody-namic profiles suffer from lack of specificity, the duration of time required to obtain them in critically injured patients, and their inability to exclude cardiac injury in the absence of tam-ponade. Chest radiographs may provide information on the pos-sible trajectory of a projectile, but rarely are diagnostic because Table 5-9Causes of obstructive shockPericardial tamponadePulmonary embolusTension pneumothoraxIVC obstruction Deep venous thrombosis Gravid uterus on IVC NeoplasmIncreased intrathoracic pressure Excess positive end-expiratory pressure NeoplasmIVC = inferior vena cava.Brunicardi_Ch05_p0131-p0156.indd 15029/01/19 11:06 AM 151SHOCKCHAPTER 5the acutely filled pericardium distends poorly. Echocardiogra-phy has become the preferred test for the diagnosis of cardiac tamponade. Good results in detecting pericardial fluid have been reported, but the yield in

1	pericardium distends poorly. Echocardiogra-phy has become the preferred test for the diagnosis of cardiac tamponade. Good results in detecting pericardial fluid have been reported, but the yield in detecting pericardial fluid depends on the skill and experience of the ultrasonographer, body habitus of the patient, and absence of wounds that preclude visualization of the pericardium. Standard two-dimensional or transesopha-geal echocardiography are sensitive techniques to evaluate the pericardium for fluid, and are typically performed by examiners skilled at evaluating ventricular function, valvular abnormali-ties, and integrity of the proximal thoracic aorta. Unfortunately, these skilled examiners are rarely immediately available at all hours of the night, when many trauma patients present; there-fore, waiting for this test may result in inordinate delays. In addition, although both ultrasound techniques may demonstrate the presence of fluid or characteristic findings of tamponade

1	there-fore, waiting for this test may result in inordinate delays. In addition, although both ultrasound techniques may demonstrate the presence of fluid or characteristic findings of tamponade (large volume of fluid, right atrial collapse, poor distensibility of the right ventricle), they do not exclude cardiac injury per se. Pericardiocentesis to diagnose pericardial blood and potentially relieve tamponade may be used. Performing pericardiocentesis under ultrasound guidance has made the procedure safer and more reliable. An indwelling catheter may be placed for sev-eral days in patients with chronic pericardial effusions. Needle pericardiocentesis may not evacuate clotted blood and has the potential to produce cardiac injury, making it a poor alternative in busy trauma centers.Diagnostic pericardial window represents the most direct method to determine the presence of blood within the pericar-dium. The procedure is best performed in the operating room under general anesthesia. It

1	pericardial window represents the most direct method to determine the presence of blood within the pericar-dium. The procedure is best performed in the operating room under general anesthesia. It can be performed through either the subxiphoid or transdiaphragmatic approach. Adequate equip-ment and personnel to rapidly decompress the pericardium, explore the injury, and repair the heart should be present. Once the pericardium is opened and tamponade relieved, hemody-namics usually improve dramatically and formal pericardial exploration can ensue. Exposure of the heart can be achieved by extending the incision to a median sternotomy, performing a left anterior thoracotomy, or performing bilateral anterior tho-racotomies (“clamshell”).Neurogenic ShockNeurogenic shock refers to diminished tissue perfusion as a result of loss of vasomotor tone to peripheral arterial beds. Loss of vasoconstrictor impulses results in increased vascular capacitance, decreased venous return, and decreased

1	tissue perfusion as a result of loss of vasomotor tone to peripheral arterial beds. Loss of vasoconstrictor impulses results in increased vascular capacitance, decreased venous return, and decreased cardiac output. Neurogenic shock is usually secondary to spinal cord injuries from vertebral body fractures of the cervical or high thoracic region that disrupt sympathetic regulation of peripheral vascular tone (Table 5-10). Rarely, a spinal cord injury without bony fracture, such as an epidural hematoma impinging on the spinal cord, can produce neurogenic shock. Sympathetic input to the heart, which normally increases heart rate and cardiac contractility, and input to the adrenal medulla, which increases catecholamine release, may also be disrupted, preventing the typical reflex tachycardia that occurs with hypovolemia. Acute spinal cord injury results in activation of multiple secondary injury mechanisms: (a) vascular compromise to the spinal cord with loss of autoregulation, vasospasm,

1	occurs with hypovolemia. Acute spinal cord injury results in activation of multiple secondary injury mechanisms: (a) vascular compromise to the spinal cord with loss of autoregulation, vasospasm, and thrombosis; (b) loss of cellular membrane integrity and impaired energy metabo-lism; and (c) neurotransmitter accumulation and release of free radicals. Importantly, hypotension contributes to the worsening of acute spinal cord injury as the result of further reduction in blood flow to the spinal cord. Management of acute spinal cord injury with attention to blood pressure control, oxygenation, and hemodynamics, essentially optimizing perfusion of an already ischemic spinal cord, seems to result in improved neurologic outcome. Patients with hypotension from spinal cord injury are best monitored in an ICU and carefully followed for evidence of cardiac or respiratory dysfunction.Diagnosis. Acute spinal cord injury may result in bradycardia, hypotension, cardiac dysrhythmias, reduced cardiac

1	in an ICU and carefully followed for evidence of cardiac or respiratory dysfunction.Diagnosis. Acute spinal cord injury may result in bradycardia, hypotension, cardiac dysrhythmias, reduced cardiac output, and decreased peripheral vascular resistance. The severity of the spi-nal cord injury seems to correlate with the magnitude of cardio-vascular dysfunction. Patients with complete motor injuries are over five times more likely to require vasopressors for neuro-genic shock compared to those with incomplete lesions.114 The classic description of neurogenic shock consists of decreased blood pressure associated with bradycardia (absence of reflex-ive tachycardia due to disrupted sympathetic discharge), warm extremities (loss of peripheral vasoconstriction), motor and sen-sory deficits indicative of a spinal cord injury, and radiographic evidence of a vertebral column fracture. Patients with multisys-tem trauma that includes spinal cord injuries often have head injuries that may make

1	of a spinal cord injury, and radiographic evidence of a vertebral column fracture. Patients with multisys-tem trauma that includes spinal cord injuries often have head injuries that may make identification of motor and sensory defi-cits difficult in the initial evaluation. Furthermore, associated injuries may occur that result in hypovolemia, further compli-cating the clinical presentation. In a subset of patients with spi-nal cord injuries from penetrating wounds, most of the patients with hypotension had blood loss as the etiology (74%) rather than neurogenic causes, and few (7%) had the classic findings of neurogenic shock.115 In the multiply injured patient, other causes of hypotension including hemorrhage, tension pneumothorax, and cardiogenic shock, must be sought and excluded.Treatment. After the airway is secured and ventilation is ade-quate, fluid resuscitation and restoration of intravascular volume often will improve perfusion in neurogenic shock. Most patients with

1	the airway is secured and ventilation is ade-quate, fluid resuscitation and restoration of intravascular volume often will improve perfusion in neurogenic shock. Most patients with neurogenic shock will respond to restoration of intravas-cular volume alone, with satisfactory improvement in perfusion and resolution of hypotension. Administration of vasoconstric-tors will improve peripheral vascular tone, decrease vascular capacitance, and increase venous return, but should only be con-sidered once hypovolemia is excluded as the cause of the hypo-tension and the diagnosis of neurogenic shock is established. If the patient’s blood pressure has not responded to what is felt to be adequate volume resuscitation, dopamine may be used first. A pure α-agonist, such as phenylephrine, may be used primar-ily or in patients unresponsive to dopamine. Specific treatment for the hypotension is often of brief duration, as the need to administer vasoconstrictors typically lasts 24 to 48 hours. On the

1	or in patients unresponsive to dopamine. Specific treatment for the hypotension is often of brief duration, as the need to administer vasoconstrictors typically lasts 24 to 48 hours. On the other hand, life-threatening cardiac dysrhythmias and hypo-tension may occur up to 14 days after spinal cord injury.The duration of the need for vasopressor support for neurogenic shock may correlate with the overall prognosis or chances of improvement in neurologic function. Appropriate rapid restoration of blood pressure and circulatory perfusion may improve perfusion to the spinal cord, prevent progressive Table 5-10Causes of neurogenic shockSpinal cord traumaSpinal cord neoplasmSpinal/epidural anestheticBrunicardi_Ch05_p0131-p0156.indd 15129/01/19 11:06 AM 152BASIC CONSIDERATIONSPART Ispinal cord ischemia, and minimize secondary cord injury. Res-toration of normal blood pressure and adequate tissue perfusion should precede any operative attempts to stabilize the vertebral fracture.ENDPOINTS

1	ischemia, and minimize secondary cord injury. Res-toration of normal blood pressure and adequate tissue perfusion should precede any operative attempts to stabilize the vertebral fracture.ENDPOINTS IN RESUSCITATIONShock is defined as inadequate perfusion to maintain normal organ function. With prolonged anaerobic metabolism, tissue acidosis and O2 debt accumulate. Thus, the goal in the treatment of shock is restoration of adequate organ perfusion and tissue oxygenation. Resuscitation is complete when O2 debt is repaid, tissue acidosis is corrected, and aerobic metabo-lism restored. Clinical confirmation of this endpoint remains a challenge.Resuscitation of the patient in shock requires simultaneous evaluation and treatment; the etiology of the shock often is not initially apparent. Hemorrhagic shock, septic shock, and trau-matic shock are the most common types of shock encountered on surgical services. To optimize outcome in bleeding patients, early control of the hemorrhage and

1	shock, septic shock, and trau-matic shock are the most common types of shock encountered on surgical services. To optimize outcome in bleeding patients, early control of the hemorrhage and adequate volume resuscita-tion, including both red blood cells and crystalloid solutions, are necessary. Expedient operative resuscitation is mandatory to limit the magnitude of activation of multiple mediator sys-tems and to abort the microcirculatory changes, which may evolve insidiously into the cascade that ends in irreversible hemorrhagic shock. Attempts to stabilize an actively bleeding patient anywhere but in the operating room are inappropriate. Any intervention that delays the patient’s arrival in the operat-ing room for control of hemorrhage increases mortality, thus the important concept of operating room resuscitation of the critically injured patient.Recognition by care providers of the patient who is in the compensated phase of shock is equally important, but more dif-ficult based on

1	operating room resuscitation of the critically injured patient.Recognition by care providers of the patient who is in the compensated phase of shock is equally important, but more dif-ficult based on clinical criteria. Compensated shock exists when inadequate tissue perfusion persists despite normalization of blood pressure and heart rate. Even with normalization of blood pressure, heart rate, and urine output, 80% to 85% of trauma patients have inadequate tissue perfusion, as evidenced by increased lactate or decreased mixed venous O2 saturation.56,116 Persistent, occult hypoperfusion is frequent in the ICU, with a resultant significant increase in infection rate and mortality in major trauma patients. Patients failing to reverse their lactic acidosis within 12 hours of admission (acidosis that was persis-tent despite normal heart rate, blood pressure, and urine output) developed an infection three times as often as those who normal-ized their lactate levels within 12 hours of

1	that was persis-tent despite normal heart rate, blood pressure, and urine output) developed an infection three times as often as those who normal-ized their lactate levels within 12 hours of admission. In addi-tion, mortality was fourfold higher in patients who developed infections. Both injury severity score and occult hypotension (lactic acidosis) longer than 12 hours were independent predic-tors of infection.117 Thus, recognition of subclinical hypoperfu-sion requires information beyond vital signs and urinary output.Endpoints in resuscitation can be divided into systemic or global parameters, tissue-specific parameters, and cellu-lar parameters. Global endpoints include vital signs, cardiac output, pulmonary artery wedge pressure, O2 delivery and con-sumption, lactate, and base deficit (Table 5-11).Assessment of Endpoints in ResuscitationInability to repay O2 debt is a predictor of mortality and organ failure; the probability of death has been directly cor-related to the

1	deficit (Table 5-11).Assessment of Endpoints in ResuscitationInability to repay O2 debt is a predictor of mortality and organ failure; the probability of death has been directly cor-related to the calculated O2 debt in hemorrhagic shock. Direct measurement of the O2 debt in the resuscitation of patients is difficult. The easily obtainable parameters of arterial blood pressure, heart rate, urine output, central venous pressure, and pulmonary artery occlusion pressure are poor indicators of the adequacy of tissue perfusion. Therefore, surrogate parameters have been sought to estimate the O2 debt; serum lactate and base deficit have been shown to correlate with O2 debt.Lactate. Lactate is generated by conversion of pyruvate to lactate by lactate dehydrogenase in the setting of insufficient O2. Lactate is released into the circulation and is predominantly taken up and metabolized by the liver and kidneys. The liver accounts for approximately 50% and the kidney for about 30% of whole body

1	Lactate is released into the circulation and is predominantly taken up and metabolized by the liver and kidneys. The liver accounts for approximately 50% and the kidney for about 30% of whole body lactate uptake. Elevated serum lactate is an indi-rect measure of the O2 debt, and therefore an approximation of the magnitude and duration of the severity of shock. The admission lactate level, highest lactate level, and time interval to normalize the serum lactate are important prognostic indi-cators for survival. For example, in a study of 76 consecutive patients, 100% survival was observed among the patients with normalization of lactate within 24 hours, 78% survival when lactate normalized between 24 and 48 hours, and only 14% sur-vivorship if it took longer than 48 hours to normalize the serum lactate.56 In contrast, individual variability of lactate may be too great to permit accurate prediction of outcome in any individual case. Base deficit and volume of blood transfusion required

1	serum lactate.56 In contrast, individual variability of lactate may be too great to permit accurate prediction of outcome in any individual case. Base deficit and volume of blood transfusion required in the first 24 hours of resuscitation may be better predictors of mortality than the plasma lactate alone.Base Deficit. Base deficit is the amount of base in millimoles that is required to titrate 1 L of whole blood to a pH of 7.40 with the sample fully saturated with O2 at 37°C (98.6°F) and a partial pressure of CO2 of 40 mmHg. It usually is measured by arterial blood gas analysis in clinical practice as it is readily and quickly available. The mortality of trauma patients can be stratified according to the magnitude of base deficit measured in the first 24 hours after admission.60 In a retrospective study of over 3000 trauma admissions, patients with a base deficit worse than 15 mmol/L had a mortality of 70%. Base deficit can be stratified into mild (3 to 5 mmol/L), moderate (6 to 14

1	study of over 3000 trauma admissions, patients with a base deficit worse than 15 mmol/L had a mortality of 70%. Base deficit can be stratified into mild (3 to 5 mmol/L), moderate (6 to 14 mmol/L), and severe (15 mmol/L) categories, with a trend toward higher mortality with worsening base deficit in patients with trauma. Both the magnitude of the perfusion deficit as indicated by the base deficit and the time required to correct it are major factors determining outcome in shock.Indeed, when elevated base deficit persists (or lactic acidosis) in the trauma patient, ongoing bleeding is often the 7Table 5-11Endpoints in resuscitationSystemic/global Lactate Base deficit Cardiac output Oxygen delivery and consumptionTissue specific Gastric tonometry Tissue pH, oxygen, carbon dioxide levels Near infrared spectroscopyCellular Membrane potential Adenosine triphosphateBrunicardi_Ch05_p0131-p0156.indd 15229/01/19 11:06 AM 153SHOCKCHAPTER 5etiology. Trauma patients admitted with a base

1	infrared spectroscopyCellular Membrane potential Adenosine triphosphateBrunicardi_Ch05_p0131-p0156.indd 15229/01/19 11:06 AM 153SHOCKCHAPTER 5etiology. Trauma patients admitted with a base deficit greater than 15 mmol/L required twice the volume of fluid infusion and six times more blood transfusion in the first 24 hours com-pared to patients with mild acidosis. Transfusion requirements increased as base deficit worsened and ICU and hospital lengths of stay increased. Mortality increased as base deficit worsened; the frequency of organ failure increased with greater base deficit.57 The probability of trauma patients developing ARDS has been reported to correlate with severity of admission base deficit and lowest base deficit within the first 24 hours postinjury.59 Persistently high base deficit is associated with abnormal O2 utilization and higher mortality. Monitoring base deficit in the resuscitation of trauma patients assists in assess-ment of O2 transport and efficacy of

1	base deficit is associated with abnormal O2 utilization and higher mortality. Monitoring base deficit in the resuscitation of trauma patients assists in assess-ment of O2 transport and efficacy of resuscitation.58Factors that may compromise the utility of the base defi-cit in estimating O2 debt are the administration of bicarbonate, hypothermia, hypocapnia (overventilation), heparin, ethanol, and ketoacidosis. However, the base deficit remains one of the most widely used estimates of O2 debt for its clinical relevance, accuracy, and availability.Near Infrared Spectroscopy. Near infrared (NIR) spectros-copy can measure tissue oxygenation and redox state of cyto-chrome a,a3 on a continuous, noninvasive basis. The NIR probe emits multiple wavelengths of light in the NIR spectrum (650 to 1100 nm). Photons are then either absorbed by the tissue or reflected back to the probe. Maximal exercise in laboratory stud-ies resulted in reduction of cytochrome a,a3; this correlated with tissue

1	1100 nm). Photons are then either absorbed by the tissue or reflected back to the probe. Maximal exercise in laboratory stud-ies resulted in reduction of cytochrome a,a3; this correlated with tissue lactate elevation. NIR spectroscopy can be used to com-pare tissue oxyhemoglobin levels (indicating tissue O2 supply to cytochrome a,a3 with mitochondrial O2 consumption), thus demonstrating flow-independent mitochondrial oxidative dys-function and the need for further resuscitation. Trauma patients with decoupled oxyhemoglobin and cytochrome a,a3 have redox dysfunction and have been shown to have a higher incidence of organ failure (89 vs. 13%).121,122Tissue PH, Oxygen, and Carbon Dioxide Concentration. Tissue probes with optical sensors have been used to measure tissue pH and partial pressure of O2 and CO2 in subcutaneous sites, muscle, and the bladder. These probes may use transcuta-neous methodology with Clark electrodes or direct percutaneous probes.123,124 The percutaneous probes

1	of O2 and CO2 in subcutaneous sites, muscle, and the bladder. These probes may use transcuta-neous methodology with Clark electrodes or direct percutaneous probes.123,124 The percutaneous probes can be inserted through an 18-gauge catheter and hold promise as continuous monitors of tissue perfusion.Right Ventricular End-Diastolic Volume Index. Right ven-tricular end-diastolic volume index (RVEDVI) seems to more accurately predict preload for cardiac index than does pulmonary artery wedge pressure.125 Chang and colleagues reported that 50% of trauma patients had persistent splanchnic ischemia that was reversed by increasing RVEDVI. RVEDVI is a parameter that seems to correlate with preload-related increases in cardiac output. More recently, these authors have described left ven-tricular power output as an endpoint (LVP >320 mmHg·L/min per square meter), which is associated with improved clearance of base deficit and a lower rate of organ dysfunction following

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1	lung injury at a combat support hospital: a prospective observational study. J Trauma. 2010;69 Suppl 1:S81-86. 108. Aji J, Hollenberg S. Cardiogenic shock: giving the heart a break. Crit Care Med. 2006;34(4):1248-1249. 109. Alonso DR, Scheidt S, Post M, Killip T. Pathophysiology of cardiogenic shock. Quantification of myocardial necrosis, clinical, pathologic and electrocardiographic correlations. Circulation. 1973;48(3):588-596. 110. Goldstein DJ, Oz MC. Mechanical support for postcardi-otomy cardiogenic shock. Semin Thorac Cardiovasc Surg. 2000;12(3):220-228. 111. Thygesen K, Alpert JS, Jaffe AS, et al. Third universal definition of myocardial infarction. J Am Coll Cardiol. 2012;60(16):1581-1598. 112. Gibbons RJ, Smith SC, Jr., Antman E. American College of Cardiology/American Heart Association clinical practice guidelines: part II: evolutionary changes in a continuous quality improvement project. Circulation. 2003;107(24):3101-3107. 113. Menon V, Hochman JS. Management of

1	Heart Association clinical practice guidelines: part II: evolutionary changes in a continuous quality improvement project. Circulation. 2003;107(24):3101-3107. 113. Menon V, Hochman JS. Management of cardiogenic shock complicating acute myocardial infarction. Heart. 2002;88(5): 531-537. 114. Levi L, Wolf A, Belzberg H. Hemodynamic parameters in patients with acute cervical cord trauma: description, intervention, and prediction of outcome. Neurosurgery. 1993;33(6):1007-1016; discussion 1016-1007. 115. Zipnick RI, Scalea TM, Trooskin SZ, et al. Hemodynamic responses to penetrating spinal cord injuries. J Trauma. 1993;35(4):578-582; discussion 582-573. 116. Abou-Khalil B, Scalea TM, Trooskin SZ, Henry SM, Hitchcock R. Hemodynamic responses to shock in young trauma patients: need for invasive monitoring. Crit Care Med. 1994;22(4):633-639. 117. Claridge JA, Crabtree TD, Pelletier SJ, Butler K, Sawyer RG, Young JS. Persistent occult hypoperfusion is associated with a significant increase

1	monitoring. Crit Care Med. 1994;22(4):633-639. 117. Claridge JA, Crabtree TD, Pelletier SJ, Butler K, Sawyer RG, Young JS. Persistent occult hypoperfusion is associated with a significant increase in infection rate and mortality in major trauma patients. J Trauma. 2000;48(1):8-14; discussion 14-15. 118. Ivatury RR, Simon RJ, Havriliak D, Garcia C, Greenbarg J, Stahl WM. Gastric mucosal pH and oxygen delivery and oxygen consumption indices in the assessment of adequacy of resuscitation after trauma: a prospective, randomized study. J Trauma. 1995;39(1):128-134; discussion 134-136. 119. Maynard N, Beale R, Smithies M, Bihari D. Gastric intramu-cosal pH in critically ill patients. Lancet. 1992;339(8792): 550-551. 120. Gomersall CD, Joynt GM, Freebairn RC, Hung V, Buckley TA, Oh TE. Resuscitation of critically ill patients based on the results of gastric tonometry: a prospective, randomized, controlled trial. Crit Care Med. 2000;28(3):607-614. 121. Cairns CB, Moore FA, Haenel JB, et

1	of critically ill patients based on the results of gastric tonometry: a prospective, randomized, controlled trial. Crit Care Med. 2000;28(3):607-614. 121. Cairns CB, Moore FA, Haenel JB, et al. Evidence for early supply independent mitochondrial dysfunction in patients developing multiple organ failure after trauma. J Trauma. 1997;42(3):532-536. 122. Cohn SM, Crookes BA, Proctor KG. Near-infrared spectros-copy in resuscitation. J Trauma. 2003;54(5 Suppl):S199-202. 123. Knudson MM, Bermudez KM, Doyle CA, Mackersie RC, Hopf HW, Morabito D. Use of tissue oxygen tension measure-ments during resuscitation from hemorrhagic shock. J Trauma. 1997;42(4):608-614; discussion 614-616. 124. McKinley BA, Marvin RG, Cocanour CS, Moore FA. Tissue hemoglobin O2 saturation during resuscitation of traumatic shock monitored using near infrared spectrometry. J Trauma. 2000;48(4):637-642. 125. Cheatham ML, Nelson LD, Chang MC, Safcsak K. Right ven-tricular end-diastolic volume index as a predictor of

1	shock monitored using near infrared spectrometry. J Trauma. 2000;48(4):637-642. 125. Cheatham ML, Nelson LD, Chang MC, Safcsak K. Right ven-tricular end-diastolic volume index as a predictor of preload status in patients on positive end-expiratory pressure. Crit Care Med. 1998;26(11):1801-1806. 126. Chang MC, Meredith JW, Kincaid EH, Miller PR. Maintain-ing survivors’ values of left ventricular power output dur-ing shock resuscitation: a prospective pilot study. J Trauma. 2000;49(1):26-33; discussion 34-37.Brunicardi_Ch05_p0131-p0156.indd 15629/01/19 11:06 AM

1	Surgical InfectionsRobert E. Bulander, David L. Dunn, and Greg J. Beilman 6chapterHISTORICAL BACKGROUNDAlthough treatment of infection has long been an integral part of the surgeon’s practice, the body of knowledge that led to the present field of surgical infectious disease was derived from the evolution of germ theory and antisepsis. Application of the latter to clinical practice, concurrent with the development of anesthe-sia, was pivotal in allowing surgeons to expand their repertoire to encompass complex procedures that previously were associ-ated with extremely high rates of morbidity and mortality due to postoperative infections. However, until recently the occurrence of infection related to the surgical wound was the rule rather than the exception. In fact, the development of modalities to effectively prevent and treat infection has occurred only within the last several decades.A number of observations by 19th century physicians and investigators were critical to our current

1	to effectively prevent and treat infection has occurred only within the last several decades.A number of observations by 19th century physicians and investigators were critical to our current understanding of the pathogenesis, prevention, and treatment of surgical infections. In 1846, Ignaz Semmelweis, a Magyar physician, took a post at the Allgemein Krankenhaus in Vienna. He noticed that the mortality rate from puerperal (“childbed”) fever was nearly three times higher in the teaching ward than in the ward where patients were delivered by midwives. He also made the observa-tion that women who delivered prior to arrival on the teaching ward had a negligible mortality rate. When a colleague died from overwhelming infection resulting from a knife scratch received during an autopsy of a woman who had died of puer-peral fever, Semmelweis observed that pathologic changes in his friend were identical to those of women dying from this postpartum disease. He hypothesized that puerperal fever

1	who had died of puer-peral fever, Semmelweis observed that pathologic changes in his friend were identical to those of women dying from this postpartum disease. He hypothesized that puerperal fever was caused by putrid material carried on the examining fingers of medical students and physicians who cared for women dying of the disease, and who often went from the autopsy room to the wards. The low mortality rate in the midwives’ ward, Sem-melweis realized, was because midwives did not participate in autopsies. Fired with the zeal of his revelation, he posted a notice on the door to the ward requiring all caregivers to rinse their hands thoroughly in chlorine water prior to entering the area. This simple intervention reduced the mortality rate from puerperal fever on the teaching ward to 1.5%, surpassing the record of the midwives. In 1861, he published his classic work on childbed fever based on records from his practice. Unfor-tunately, Semmelweis’ ideas were not well accepted by the

1	surpassing the record of the midwives. In 1861, he published his classic work on childbed fever based on records from his practice. Unfor-tunately, Semmelweis’ ideas were not well accepted by the authorities of the time.1 Increasingly frustrated by the indiffer-ence of the medical profession, he began writing open letters to well-known obstetricians in Europe and was committed to an asylum due to concerns that he was losing his mind. He died shortly thereafter. His achievements were only recognized after Pasteur’s description of the germ theory of disease.Louis Pasteur performed a body of work during the lat-ter part of the 19th century that provided the underpinnings of modern microbiology, at the time known as germ theory. His work in humans followed experiments identifying infectious agents in silkworms. He was able to elucidate the principle that contagious diseases are caused by specific microbes and that these microbes are foreign to the infected organism. Using this principle,

1	in silkworms. He was able to elucidate the principle that contagious diseases are caused by specific microbes and that these microbes are foreign to the infected organism. Using this principle, he developed techniques of sterilization criti-cal to oenology and identified several bacteria responsible for human illnesses, including Staphylococcus and Streptococcus pneumoniae (pneumococcus).Joseph Lister, the son of a wine merchant, was appointed professor of surgery at the Glasgow Royal Infirmary in 1859. In his early practice, he noted that more than half of his patients undergoing amputation died because of postoperative infection. After hearing of Pasteur’s work, Lister experimented with the use of a solution of carbolic acid, which he knew was being used to treat sewage. He first reported his findings to the British Medical Association in 1867 using dressings saturated with car-bolic acid on 12 patients with compound fractures; 10 recovered Historical Background 157Pathogenesis of

1	his findings to the British Medical Association in 1867 using dressings saturated with car-bolic acid on 12 patients with compound fractures; 10 recovered Historical Background 157Pathogenesis of Infection 159Host Defenses / 159Definitions / 160Microbiology of Infectious Agents 161Bacteria / 161Fungi / 162Viruses / 162Prevention and Treatment of Surgical Infections 163General Principles / 163Source Control / 163Appropriate Use of Antimicrobial Agents / 164Infections of Significance in Surgical Patients 169Surgical Site Infections / 169Intra-Abdominal Infections / 171Organ-Specific Infections / 172Infections of the Skin and Soft Tissue / 173Postoperative Nosocomial Infections / 174Sepsis / 175Resistant Organisms / 177Blood-Borne Pathogens / 177Biologic Warfare Agents 178Bacillus anthracis (Anthrax) / 178Yersinia pestis (Plague) / 178Smallpox / 178Francisella tularensis (Tularemia) / 179Brunicardi_Ch06_p0157-p0182.indd 15701/03/19 4:46 PM 158without amputation, one survived

1	(Anthrax) / 178Yersinia pestis (Plague) / 178Smallpox / 178Francisella tularensis (Tularemia) / 179Brunicardi_Ch06_p0157-p0182.indd 15701/03/19 4:46 PM 158without amputation, one survived with amputation, and one died of causes unrelated to the wound. In spite of initial resistance, his methods were quickly adopted throughout much of Europe.From 1878 until 1880, Robert Koch was the district medi-cal officer for Wollstein, an area in Prussia where anthrax was endemic. Performing experiments in his home, without the ben-efit of scientific equipment and academic contact, Koch devel-oped techniques for culture of Bacillus anthracis and proved the ability of this organism to cause anthrax in healthy animals. He developed the following four postulates to identify the asso-ciation of organisms with specific diseases: (a) the suspected pathogenic organism should be present in all cases of the disease and absent from healthy animals, (b) the suspected pathogen should be isolated from a

1	with specific diseases: (a) the suspected pathogenic organism should be present in all cases of the disease and absent from healthy animals, (b) the suspected pathogen should be isolated from a diseased host and grown in a pure culture in vitro, (c) cells from a pure culture of the suspected organism should cause disease in a healthy animal, and (d) the organism should be reisolated from the newly diseased animal and shown to be the same as the original. He used these same techniques to identify the organisms responsible for cholera and tuberculosis. During the next century, Koch’s postulates, as they came to be called, became critical to the understanding of surgi-cal infections.2The first intra-abdominal operation to treat infection via “source control” (i.e., surgical intervention to eliminate the source of infection) was appendectomy. This operation was pioneered by Charles McBurney at the New York College of Physicians and Surgeons, among others.3 McBurney’s classic report on

1	eliminate the source of infection) was appendectomy. This operation was pioneered by Charles McBurney at the New York College of Physicians and Surgeons, among others.3 McBurney’s classic report on early operative intervention for appendicitis was pre-sented before the New York Surgical Society in 1889. Appen-dectomy for the treatment of appendicitis, previously an often fatal disease, was popularized after the 1902 coronation of King Edward VII of England was delayed due to his falling ill with appendicitis. Edward insisted on carrying out his sched-ule, despite worsening abdominal pain. Sir Frederick Treves, a prominent London surgeon, was among the consultants in atten-dance upon Edward. As the prince’s condition deteriorated, and as he continued to insist that he would go to Westminster Abbey to be crowned, Treves told him, “Then Sire, you will go as a corpse.” Edward relented, Treves drained a large periappendi-ceal abscess, and the king lived.4During the 20th century the

1	Abbey to be crowned, Treves told him, “Then Sire, you will go as a corpse.” Edward relented, Treves drained a large periappendi-ceal abscess, and the king lived.4During the 20th century the development of effective anti-microbials added a new dimension to modern surgical practice. Sir Alexander Fleming, after serving in the British Army Medical Corps during World War I, continued his work on the natural antibacterial action of the blood and antiseptics. In 1928, while studying influenza virus, he noted a zone of inhibition around a mold colony (Penicillium notatum) that serendipitously grew on a plate of Staphylococcus, and he named the active substance penicillin. Penicillin, along with the sulfonamide antibiotics, were among the first of hundreds of potent antimicrobials that became a critical component of the armamentarium to prevent and treat aggressive, lethal surgical infections.5Concurrent with the development of antimicrobial agents were advances in the field of clinical

1	a critical component of the armamentarium to prevent and treat aggressive, lethal surgical infections.5Concurrent with the development of antimicrobial agents were advances in the field of clinical microbiology. Many new microbes were identified, including numerous anaerobes. The autochthonous microflora of the skin, gastrointestinal tract, and other parts of the body that the surgeon encountered in the pro-cess of an operation were characterized in great detail. However, it remained unclear whether these organisms were commensals or pathogens. Subsequently, the initial clinical observations of surgeons such as Frank Meleney, William Altemeier, and others provided the key when they observed that aerobic and anaerobic host flora could synergize to cause serious soft tissue and severe intra-abdominal infection.6,7 Thus, the concepts that resident Key Points1 Sepsis is a life-threatening syndrome reflecting both an infection and the systemic host response to it. It has a broad variety of

1	infection.6,7 Thus, the concepts that resident Key Points1 Sepsis is a life-threatening syndrome reflecting both an infection and the systemic host response to it. It has a broad variety of presentations and manifestations that hold in com-mon some form of organ dysfunction. Outcomes in patients with sepsis are improved with an organized approach to therapy that addresses rapid resuscitation, antibiotics, and source control.2 Source control is a key concept in the treatment of most surgically relevant infections. Infected or necrotic material must be drained or removed as part of the treatment plan in this setting. Delays in adequate source control are associated with worsened outcomes.3 Principles relevant to appropriate antibiotic prophylaxis for surgery: (a) select an agent with activity against organisms commonly found at the site of surgery, (b) administer the ini-tial dose of the antibiotic within 30 minutes prior to incision, (c) redose the antibiotic during long operations

1	against organisms commonly found at the site of surgery, (b) administer the ini-tial dose of the antibiotic within 30 minutes prior to incision, (c) redose the antibiotic during long operations based upon the half-life of the agent to ensure adequate tissue levels, and (d) limit the antibiotic regimen to no more than 24 hours after surgery for routine prophylaxis.4 When using antimicrobial agents for therapy of serious infection, several principles should be followed: (a) identify likely sources of infection, (b) select an agent (or agents) that will have efficacy against likely organisms for these sources, (c) begin therapy rapidly with broad coverage, as inadequate or delayed antibiotic therapy results in increased mortality, (d) when possible, obtain cultures early and use results to refine therapy, (e) if no infection is identified after 3 days, strongly consider discontinuation of antibiotics, based upon the patient’s clinical course, and (f) discontinue antibiotics after an

1	to refine therapy, (e) if no infection is identified after 3 days, strongly consider discontinuation of antibiotics, based upon the patient’s clinical course, and (f) discontinue antibiotics after an appropriate course of therapy.5 The incidence of surgical site infections can be reduced by appropriate patient preparation, timely perioperative antibi-otic administration, maintenance of perioperative normo-thermia and normoglycemia, and appropriate wound management.6 The keys to good outcomes in patients with necrotizing soft tissue infection are early recognition and appropriate debridement of infected tissue with repeated debridement until no further signs of infection are present.7 Transmission of HIV and other infections spread by blood and body fluids from patient to healthcare worker can be minimized by practicing universal precautions, which include routine use of barriers when anticipating contact with blood or body fluids, washing of hands and other skin surfaces immediately

1	can be minimized by practicing universal precautions, which include routine use of barriers when anticipating contact with blood or body fluids, washing of hands and other skin surfaces immediately after contact with blood or body fluids, and careful handling and disposal of sharp instruments dur-ing and after use.Brunicardi_Ch06_p0157-p0182.indd 15801/03/19 4:46 PM 159SURGICAL INFECTIONSCHAPTER 6microbes were nonpathogenic until they entered a sterile body cavity at the time of surgery, and that many, if not most, surgical infections were polymicrobial in nature, became critical ideas.8,9 These tenets became firmly established after microbiology lab-oratories demonstrated the invariable presence of aerobes and anaerobes in peritoneal cultures obtained at the time of surgery for intra-abdominal infection due to perforated viscus or gangre-nous appendicitis. Clinical trials provided ample evidence that optimal therapy for these infections required effective source control and the

1	infection due to perforated viscus or gangre-nous appendicitis. Clinical trials provided ample evidence that optimal therapy for these infections required effective source control and the administration of antimicrobial agents directed against both types of pathogens.William Osler made an observation in 1904 in his treatise The Evolution of Modern Medicine that was to have profound implications for the future of treatment of infection: “Except on few occasions, the patient appears to die from the body’s response to infection rather than from it.”10 The discovery of cytokines began to allow insight into the human organism’s response to infection, and led to an explosion in our understand-ing of the host inflammatory response. Expanding knowledge of the multiple pathways activated during the response to invasion by infectious organisms has permitted the design of new thera-pies targeted at modifying the inflammatory response to infec-tion, which seems to cause much of the organ

1	the response to invasion by infectious organisms has permitted the design of new thera-pies targeted at modifying the inflammatory response to infec-tion, which seems to cause much of the organ dysfunction and failure. Preventing and treating this process of multiple organ failure during infection is one of the major challenges of modern critical care and surgical infectious disease.PATHOGENESIS OF INFECTIONHost DefensesThe mammalian host possesses several layers of endogenous defense mechanisms that serve to prevent microbial invasion, limit proliferation of microbes within the host, and contain or eradicate invading microbes. These defenses are integrated and redundant so that the various components function as a com-plex, highly regulated system that is extremely effective in cop-ing with microbial invaders. They include site-specific defenses that function at the tissue level, as well as components that freely circulate throughout the body in both blood and lymph. Systemic host

1	with microbial invaders. They include site-specific defenses that function at the tissue level, as well as components that freely circulate throughout the body in both blood and lymph. Systemic host defenses invariably are recruited to a site of infec-tion, a process that begins immediately upon introduction of microbes into a sterile area of the body. Perturbation of one or more components of these defenses (e.g., via immunosuppres-sants, foreign body, chronic illness, or burns) may have substan-tial negative impact on resistance to infection.Entry of microbes into the mammalian host is precluded by a number of barriers that possess either an epithelial (integu-ment) or mucosal (respiratory, gut, and urogenital) surface. Barrier function, however, is not solely limited to physical characteristics. Host barrier cells may secrete substances that limit microbial proliferation or prevent invasion. Also, resident or commensal microbes adherent to the physical surface and to each other may

1	Host barrier cells may secrete substances that limit microbial proliferation or prevent invasion. Also, resident or commensal microbes adherent to the physical surface and to each other may preclude invasion, particularly of virulent organ-isms; this is termed colonization resistance.11The most extensive physical barrier is the integument or skin. In addition to the physical barrier posed by the epithelial surface, the skin harbors its own resident microflora that may block the attachment and invasion of noncommensal microbes. Microbes also are held in check by chemicals secreted by seba-ceous glands and by the constant shedding of epithelial cells. The endogenous microflora of the integument primarily com-prises gram-positive aerobic microbes belonging to the genera Staphylococcus and Streptococcus, as well as Corynebacterium and Propionibacterium species. These organisms plus Entero-coccus faecalis and faecium, Escherichia coli and other Entero-bacteriaceae, and yeast such as

1	and Streptococcus, as well as Corynebacterium and Propionibacterium species. These organisms plus Entero-coccus faecalis and faecium, Escherichia coli and other Entero-bacteriaceae, and yeast such as Candida albicans can be isolated from the infraumbilical regions of the body. Diseases of the skin (e.g., eczema and dermatitis) are associated with overgrowth of skin commensal organisms, and barrier breaches invariably lead to the introduction of these microbes.The respiratory tract possesses several host defense mech-anisms that facilitate the maintenance of sterility in the distal bronchi and alveoli. In the upper respiratory tract, respiratory mucus traps larger particles, including microbes. This mucus is then passed into the upper airways and oropharynx by cili-ated epithelial cells, where the mucus is cleared via coughing. Smaller particles arriving in the lower respiratory tract are cleared via phagocytosis by pulmonary alveolar macrophages. Any process that diminishes these host

1	the mucus is cleared via coughing. Smaller particles arriving in the lower respiratory tract are cleared via phagocytosis by pulmonary alveolar macrophages. Any process that diminishes these host defenses can lead to development of bronchitis or pneumonia.The urogenital, biliary, pancreatic ductal, and distal respi-ratory tracts do not possess resident microflora in healthy indi-viduals, although microbes may be present if these barriers are affected by disease (e.g., malignancy, inflammation, calculi, or foreign body), or if microorganisms are introduced from an external source (e.g., urinary catheter or pulmonary aspiration). In contrast, significant numbers of microbes are encountered in many portions of the gastrointestinal tract, with vast numbers being found within the oropharynx and distal colon or rectum, although the specific organisms differ.One would suppose that the entire gastrointestinal tract would be populated via those microbes found in the oropharynx, but this is not

1	distal colon or rectum, although the specific organisms differ.One would suppose that the entire gastrointestinal tract would be populated via those microbes found in the oropharynx, but this is not the case.11 This is because after ingestion these organisms routinely are killed in the highly acidic, low-motility environment of the stomach during the initial phases of diges-tion. Thus, only small numbers of microbes populate the gas-tric mucosa (∼102 to 103 colony-forming units [CFU]/mL). This population expands in the presence of drugs or disease states that diminish gastric acidity. Microbes that are not destroyed within the stomach enter the small intestine, in which a certain amount of microbial proliferation takes place, such that approxi-mately 105 to 108 CFU/mL are present in the terminal ileum.The relatively low-oxygen, static environment of the colon is accompanied by the exponential growth of microbes that com-prise the most extensive host endogenous microflora. Anaerobic

1	terminal ileum.The relatively low-oxygen, static environment of the colon is accompanied by the exponential growth of microbes that com-prise the most extensive host endogenous microflora. Anaerobic microbes outnumber aerobic species approximately 100:1 in the distal colon, and approximately 1011 to 1012 CFU/g are pres-ent in feces. Large numbers of facultative and strict anaerobes (Bacteroides fragilis, distasonis, and thetaiotaomicron, Bifido-bacterium, Clostridium, Eubacterium, Fusobacterium, Lactoba-cillus, and Peptostreptococcus species) as well as several orders of magnitude fewer aerobic microbes (E coli and other Entero-bacteriaceae, E faecalis and faecium, C albicans and other Candida spp.) are present. Intriguingly, although colonization resistance on the part of this extensive, well-characterized host microflora effectively prevents invasion of enteric pathogens such as Salmonella, Shigella, Vibrio, and other enteropathogenic bacterial species, these same organisms provide

1	well-characterized host microflora effectively prevents invasion of enteric pathogens such as Salmonella, Shigella, Vibrio, and other enteropathogenic bacterial species, these same organisms provide the initial inoc-ulum for infection should perforation of the gastrointestinal tract occur. It is of great interest that only some of these microbial species predominate in established intra-abdominal infections.Once microbes enter a sterile body compartment (e.g., the pleural or peritoneal cavity) or tissue, additional host defenses act to limit and/or eliminate these pathogens. Initially, several Brunicardi_Ch06_p0157-p0182.indd 15901/03/19 4:46 PM 160BASIC CONSIDERATIONSPART Iprimitive and relatively nonspecific host defenses act to con-tain the nidus of infection, which may include microbes as well as debris, devitalized tissue, and foreign bodies, depending on the nature of the injury. These defenses include the physi-cal barrier of the tissue itself, as well as the capacity of

1	as well as debris, devitalized tissue, and foreign bodies, depending on the nature of the injury. These defenses include the physi-cal barrier of the tissue itself, as well as the capacity of pro-teins such as lactoferrin and transferrin to sequester the critical microbial growth factor iron, thereby limiting microbial growth. In addition, fibrinogen within the inflammatory fluid has the ability to trap large numbers of microbes during the process in which it polymerizes into fibrin. Within the peritoneal cavity, unique host defenses exist, including a diaphragmatic pump-ing mechanism whereby particles—including microbes—within peritoneal fluid are expunged from the abdominal cavity via specialized structures (stomata) on the undersurface of the dia-phragm that lead to thoracic lymphatic channels. Concurrently, containment by the omentum and intestinal ileus serve to wall off infections. However, the latter processes and fibrin trapping have a high likelihood of contributing to the

1	channels. Concurrently, containment by the omentum and intestinal ileus serve to wall off infections. However, the latter processes and fibrin trapping have a high likelihood of contributing to the formation of an intra-abdominal abscess.Microbes also immediately encounter a series of host defense mechanisms that reside within the vast majority of tissues of the body. These include resident macrophages and low levels of complement (C) proteins and immunoglobulins (e.g., antibodies).12 The response in macrophages is initiated by genome-encoded pattern recognition receptors that respond to invading microbes. With exposure to a foreign organism, these receptors recognize microbial pathogen-associated molecular patterns (PAMPs) and endogenous danger-associated molecular patterns (DAMPs). Toll-like receptors (TLRs) are a well-defined example of a PAMP that plays an important role in pathogen signaling.13 Resident macrophages secrete a wide array of sub-stances in response to the

1	Toll-like receptors (TLRs) are a well-defined example of a PAMP that plays an important role in pathogen signaling.13 Resident macrophages secrete a wide array of sub-stances in response to the aforementioned processes, some of which appear to regulate the cellular components of the host defense response. This results in recruitment and proliferation of inflammatory cells. Macrophage cytokine synthesis is upreg-ulated. Secretion of tumor necrosis factor-alpha (TNF-α), of interleukins (IL)-1β, 6, and 8; and of gamma interferon (IFN-γ) occurs within the tissue milieu, and depending on the magnitude of the host defense response, the systemic circulation.14 Concur-rently, a counterregulatory response is initiated consisting of binding protein (TNF-BP), cytokine receptor antagonists (e.g., IL-1ra), and anti-inflammatory cytokines (IL-4 and IL-10).The interaction of microbes with these first-line host defenses leads to microbial opsonization (C1q, C3bi, and IgFc), phagocytosis, and both

1	IL-1ra), and anti-inflammatory cytokines (IL-4 and IL-10).The interaction of microbes with these first-line host defenses leads to microbial opsonization (C1q, C3bi, and IgFc), phagocytosis, and both extracellular (C5b6-9 membrane attack complex) and intracellular microbial destruction (via cellular ingestion into phagocytic vacuoles). Concurrently, the classical and alternate complement pathways are activated both via direct contact with and via IgM and IgG binding to microbes, leading to the release of a number of different biologically active com-plement protein fragments (C3a, C4a, C5a), acting to markedly enhance vascular permeability. Bacterial cell wall components and a variety of enzymes expelled from leukocyte phagocytic vacuoles during microbial phagocytosis and killing act in this capacity as well.Simultaneously, the release of substances to which poly-morphonuclear leukocytes (PMNs) in the bloodstream are attracted takes place. These consist of C5a, microbial cell wall

1	this capacity as well.Simultaneously, the release of substances to which poly-morphonuclear leukocytes (PMNs) in the bloodstream are attracted takes place. These consist of C5a, microbial cell wall peptides containing N-formyl-methionine, and macrophage secretion of cytokines such as IL-8. This process of host defense recruitment leads to further influx of inflammatory fluid into the area of incipient infection and is accompanied by diapedesis of large numbers of PMNs, a process that begins within several minutes and may peak within hours or days. The magnitude of the response and eventual outcome is generally related to several factors: (a) the initial number of microbes, (b) the rate of microbial proliferation in relation to containment and killing by host defenses, (c) microbial virulence, and (d) the potency of host defenses. In regard to the latter, drugs or disease states that diminish any or multiple components of host defenses are asso-ciated with higher rates and potentially

1	and (d) the potency of host defenses. In regard to the latter, drugs or disease states that diminish any or multiple components of host defenses are asso-ciated with higher rates and potentially more grave infections.DefinitionsSeveral possible outcomes can occur subsequent to microbial invasion and the interaction of microbes with resident and recruited host defenses: (a) eradication; (b) containment, often leading to the presence of purulence, the hallmark of chronic infections (e.g., a furuncle in the skin and soft tissue or abscess within the parenchyma of an organ or potential space); (c) locoregional infection (cellulitis, lymphangitis, and aggressive soft tissue infection) with or without distant spread of infec-tion (metastatic abscess); or (d) systemic infection (bactere-mia or fungemia). Obviously, the latter represents the failure of resident and recruited host defenses at the local level, and is associated with significant morbidity and mortality. Disease progression

1	or fungemia). Obviously, the latter represents the failure of resident and recruited host defenses at the local level, and is associated with significant morbidity and mortality. Disease progression commonly occurs such that serious locoregional infection is associated with concurrent systemic infection. A chronic abscess also may intermittently drain and/or be associ-ated with bacteremia.Infection is defined by the presence of microorganisms in host tissue or the bloodstream. The classic findings of rubor, calor, and dolor in areas such as the skin or subcutaneous tis-sue are common at the site of infection. Most infections in nor-mal individuals with intact host defenses are associated with these local manifestations, plus systemic manifestations such as elevated temperature, elevated white blood cell (WBC) count, tachycardia, or tachypnea. The systemic manifestations noted previously comprise what has been termed the systemic inflammatory response syndrome (SIRS). SIRS reflects a

1	white blood cell (WBC) count, tachycardia, or tachypnea. The systemic manifestations noted previously comprise what has been termed the systemic inflammatory response syndrome (SIRS). SIRS reflects a pro-inflammatory state in response to a variety of disease processes, including infection, pancreatitis, polytrauma, malignancy, and burns. There are a variety of systemic manifestations of infec-tion, with the classic factors of fever, tachycardia, and tachypnea broadened to include a variety of other variables (Table 6-1).15The definition of sepsis is evolving. Earlier models described sepsis as SIRS caused by infection. This was based upon the idea that sepsis is mediated by the production of a cascade of proinflammatory mediators produced in response to exposure to microbial products. These products include lipo-polysaccharide (endotoxin, LPS) derived from gram-negative organisms; peptidoglycans and teichoic acids from grampositive organisms; many different microbial cell wall

1	These products include lipo-polysaccharide (endotoxin, LPS) derived from gram-negative organisms; peptidoglycans and teichoic acids from grampositive organisms; many different microbial cell wall compo-nents, such as mannan from yeast and fungi; and many others.There are several issues, however, with basing a sepsis diagnosis on the presence of SIRS. One problem is that it is insufficiently specific. Patients can exhibit SIRS criteria without the presence of the more whole-body dysregulation consistent with sepsis, and conversely can suffer from sepsis without meet-ing SIRS criteria. Patients with SIRS do not necessarily prog-ress to sepsis and do not necessarily have worsened outcomes because of the SIRS diagnosis; in other words, SIRS is not inher-ently life-threatening. Another issue is that the SIRS criteria can vary and are inconsistently applied. Numerous definitions exist, specifying differing physiologic and laboratory criteria for the Brunicardi_Ch06_p0157-p0182.indd

1	is that the SIRS criteria can vary and are inconsistently applied. Numerous definitions exist, specifying differing physiologic and laboratory criteria for the Brunicardi_Ch06_p0157-p0182.indd 16001/03/19 4:46 PM 161SURGICAL INFECTIONSCHAPTER 6diagnosis. This creates difficulty in clinical, epidemiological, and research settings. Further, sepsis is not a purely inflamma-tory phenomenon, as both proand anti-inflammatory cascades have been shown to be activated in septic patients. Basing a diagnosis upon inflammatory markers alone disregards nonin-flammatory organ dysfunction, which may not manifest as SIRS but can contribute to mortality. A final concern is that defining sepsis using SIRS criteria implies that SIRS, sepsis, severe sep-sis, and septic shock exist upon a continuum, and while SIRS and sepsis have common features, the former does not necessar-ily lead to the latter. This being said, SIRS criteria have utility in that they point toward an organism experiencing

1	and while SIRS and sepsis have common features, the former does not necessar-ily lead to the latter. This being said, SIRS criteria have utility in that they point toward an organism experiencing physiological stress. The presence of SIRS warrants further investigation by the clinician.16An international consensus panel proposed new defini-tions of sepsis and septic shock in 2016. What is known as the Sepsis-3 model defines sepsis as life-threatening organ dysfunc-tion caused by a dysregulated host response to infection. Organ dysfunction is quantified by an increase of ≥2 points on the Sequential Organ Failure Assessment (SOFA). The SOFA score looks at PaO2/FiO2 ratio, bilirubin, platelet count, mean arterial pressure (MAP), Glasgow Coma Scale (GCS) score, creatinine level, and urine output (Table 6-2). An increase in SOFA score of 2 or more is correlated with a 10% in-hospital mortality risk, which is suggestive of the life-threatening nature of sepsis. An abbreviated version of the

1	(Table 6-2). An increase in SOFA score of 2 or more is correlated with a 10% in-hospital mortality risk, which is suggestive of the life-threatening nature of sepsis. An abbreviated version of the scoring system, the quick SOFA (qSOFA) is recommended as a screening and mon-itoring tool for patients with suspected sepsis. The qSOFA sug-gests potentially life-threatening sepsis when at least two of the following parameters are met: altered mental status, systolic blood pressure of 100 mmHg or less, and respiratory rate greater than 22 breaths/minute. The qSOFA can readily identify patients at risk of poor outcome from sepsis without reliance upon labo-ratory or imaging data.16Under the older nomenclature, severe sepsis was char-acterized as sepsis combined with the presence of new-onset organ failure. The Sepsis-3 definitions consider the term “severe sepsis” to be redundant, as by this definition all sepsis involves organ dysfunction. Under the Sepsis-3 guidelines, septic shock is a

1	failure. The Sepsis-3 definitions consider the term “severe sepsis” to be redundant, as by this definition all sepsis involves organ dysfunction. Under the Sepsis-3 guidelines, septic shock is a subset of sepsis in which circulatory and cellular metabolic derangements are profound enough to significantly increase the risk of death. Sepsis is the most common cause of death in non-coronary critical care units and the 11th most common cause of death overall in the United States, with a mortality rate of 10.3 cases per 100,000 population in 2010.17 Septic shock is the most severe manifestation of infection, with an attendant mortality rate in excess of 40%. It can be identified by persistent arterial hypo-tension requiring vasopressors to maintain mean arterial pressure (MAP) ≥65, and by serum lactate >2 mmol/L (18 mg/dL) despite adequate volume resuscitation.16,18,19MICROBIOLOGY OF INFECTIOUS AGENTSA partial list of common pathogens that cause infections in sur-gical patients is provided

1	lactate >2 mmol/L (18 mg/dL) despite adequate volume resuscitation.16,18,19MICROBIOLOGY OF INFECTIOUS AGENTSA partial list of common pathogens that cause infections in sur-gical patients is provided in Table 6-3.BacteriaBacteria are responsible for the majority of surgical infections. Specific species are identified using Gram stain and growth characteristics on specific media. The Gram stain is an important evaluation that allows rapid classification of bacteria by color. This color is related to the staining characteristics of the bacterial cell wall: gram-positive bacteria stain blue and gram-negative bacteria stain red. Bacteria are classified based upon a num-ber of additional characteristics, including morphology (cocci and bacilli), the pattern of division (single organisms, groups of organisms in pairs [diplococci], clusters [staphylococci], and chains [streptococci]), and the presence and location of spores.Gram-positive bacteria that frequently cause infections in surgical

1	of organisms in pairs [diplococci], clusters [staphylococci], and chains [streptococci]), and the presence and location of spores.Gram-positive bacteria that frequently cause infections in surgical patients include aerobic skin commensals (Staphylo-coccus aureus and epidermidis and Streptococcus pyogenes) and enteric organisms such as E faecalis and faecium. Aerobic skin commensals cause a large percentage of surgical site infec-tions (SSIs), either alone or in conjunction with other patho-gens; enterococci can cause nosocomial infections (urinary tract infections [UTIs] and bacteremia) in immunocompromised or chronically ill patients, but are of relatively low virulence in healthy individuals.There are many pathogenic gram-negative bacterial spe-cies that are capable of causing infection in surgical patients. Most gram-negative organisms of interest to the surgeon are bacilli belonging to the family Enterobacteriaceae, including Escherichia coli, Klebsiella pneumoniae, Serratia

1	in surgical patients. Most gram-negative organisms of interest to the surgeon are bacilli belonging to the family Enterobacteriaceae, including Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, and Enterobacter, Citrobacter, and Acinetobacter species. Other gram-negative bacilli of note include Pseudomonas, including P aeruginosa and fluorescens, and Stenotrophomonas species.1Table 6-1Criteria for systemic inflammatory response syndrome (SIRS)General variables Fever (core temp >38.3°C) Hypothermia (core temp <36°C) Heart rate >90 bpm Tachypnea Altered mental status Significant edema or positive fluid balance (>20 mL/kg over 24 hours) Hyperglycemia in the absence of diabetesInflammatory variables Leukocytosis (WBC >12,000) Leukopenia (WBC <4,000) Bandemia (>10% band forms) Plasma C-reactive protein >2 s.d. above normal value Plasma procalcitonin >2 s.d. above normal valueHemodynamic variables Arterial hypotension (SBP <90 mmHg, MAP <70, or SBP decrease >40 mmHg)Organ

1	C-reactive protein >2 s.d. above normal value Plasma procalcitonin >2 s.d. above normal valueHemodynamic variables Arterial hypotension (SBP <90 mmHg, MAP <70, or SBP decrease >40 mmHg)Organ dysfunction variables Arterial hypoxemia Acute oliguria Creatinine increase Coagulation abnormalities Ileus Thrombocytopenia HyperbilirubinemiaTissue perfusion variables Hyperlactatemia Decreased capillary fillingbpm = beats per minute; MAP = mean arterial pressure; SBP = systolic blood pressure; s.d. = standard deviations; SvO2 = venous oxygen saturation; WBC = white blood cell count.Brunicardi_Ch06_p0157-p0182.indd 16101/03/19 4:46 PM 162BASIC CONSIDERATIONSPART IAnaerobic organisms divide poorly or are unable to grow in air, as most do not possess the enzyme catalase, which allows for metabolism of reactive oxygen species. Anaerobes are the predominant indigenous flora in many areas of the human body, with the particular species being dependent on the site. For example, Propionibacterium

1	of reactive oxygen species. Anaerobes are the predominant indigenous flora in many areas of the human body, with the particular species being dependent on the site. For example, Propionibacterium acnes and other species are a major component of the skin microflora and cause the infectious mani-festation of acne. As noted previously, large numbers of anaer-obes contribute to the microflora of the oropharynx and colon.Infection due to Mycobacterium tuberculosis was once one of the most common causes of death in Europe, causing one in four deaths in the 17th and 18th centuries. In the 19th and 20th centuries, thoracic surgical intervention was often required for severe pulmonary disease, now an increasingly uncommon occur-rence in developed countries. This organism and other related organisms (M avium-intracellulare and M leprae) are known as acid-fast bacilli. Other acid-fast bacilli include Nocardia. These organisms typically are slow growing, sometimes necessitating observation in

1	(M avium-intracellulare and M leprae) are known as acid-fast bacilli. Other acid-fast bacilli include Nocardia. These organisms typically are slow growing, sometimes necessitating observation in culture for weeks to months prior to final identi-fication, although deoxyribonucleic acid (DNA)-based analysis is increasingly available to provide a means for preliminary, rapid detection.FungiFungi are typically identified by use of special stains (e.g., potas-sium hydroxide, India ink, methenamine silver, or Giemsa). Initial identification is assisted by observation of the form of branching and septation in stained specimens or in culture. Final identification is based on growth characteristics in special media, similar to bacteria, as well as on the capacity for growth at a different temperature (25°C vs. 37°C). Fungi of relevance to surgeons include those that cause nosocomial infections in surgical patients as part of polymicrobial infections or fungemia (e.g., C albicans and related

1	(25°C vs. 37°C). Fungi of relevance to surgeons include those that cause nosocomial infections in surgical patients as part of polymicrobial infections or fungemia (e.g., C albicans and related species), rare causes of aggressive soft tissue infections (e.g., Mucor, Rhizopus, and Absidia spp.), and opportunistic pathogens that cause infection in the immuno-compromised host (e.g., Aspergillus fumigatus, niger, terreus, and other spp., Blastomyces dermatitidis, Coccidioides immitis, and Cryptococcus neoformans). Agents currently available for antifungal therapy are described in Table 6-4.VirusesDue to their small size and necessity for growth within cells, viruses are difficult to culture, requiring a longer time than is typically optimal for clinical decision making. Previously, viral infection was identified by indirect means (i.e., the host anti-body response); more modern techniques identify the presence of viral DNA or ribonucleic acid (RNA) using methods such as polymerase chain

1	was identified by indirect means (i.e., the host anti-body response); more modern techniques identify the presence of viral DNA or ribonucleic acid (RNA) using methods such as polymerase chain reaction. Similar to many fungal infections, most clinically relevant viral infections in surgical patients occur in the immunocompromised host, particularly those receiv-ing immunosuppression to prevent rejection of a solid organ allograft. Relevant viruses include adenoviruses, cytomegalo-virus, Epstein-Barr virus, herpes simplex virus, and varicella-zoster virus. Surgeons must be aware of the manifestations of hepatitis B and C viruses, as well as human immunodeficiency Table 6-2Sequential Organ Failure Assessment scoreSYSTEMSCORE01234RespiratoryPaO2/FiO2, mmHg (kPa)≥400 (53.3)<400 (53.3)<300 (40)<200 (26.7) with respiratory support<100 (13.3) with respiratory supportCoagulationPlatelets, × 103/μL≥150<150<100<50<20HepaticBilirubin, mg/dL (μmol/L)<1.2 (20)1.2–1.9 (20–32)2–5.9 (33–101)6–11.9

1	(26.7) with respiratory support<100 (13.3) with respiratory supportCoagulationPlatelets, × 103/μL≥150<150<100<50<20HepaticBilirubin, mg/dL (μmol/L)<1.2 (20)1.2–1.9 (20–32)2–5.9 (33–101)6–11.9 (102–204)>12 (204)CardiovascularMAP ≥70 mmHgMAP <70 mmHgDopamine <5 or dobutamineDopamine 5.1–15 or epinephrine ≤0.1 or norepinephrine ≤0.1Dopamine >15 or epinephrine >0.1 or norepinephrine >0.1CNSGCS score1513–1410–126–9<6RenalCreatinine, mg/dL (μmol/L)<1.2 (110)1.2–1.9 (110–170)2–3.4 (171–299)3.5–4.9 (300–440)>5 (440)Urine output, mL/24 hours<500<200MAP = mean arterial pressure; PaO2 = partial pressure of oxygen; FiO2 = fraction of inspired oxygen; CNS = central nervous system; GCS = Glasgow Coma ScaleCatecholamine doses in μg/kg/minuteReproduced with permission from Vincent JL, Moreno R, Takala J, et al: The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive

1	et al: The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine, Intensive Care Med. 1996 Jul;22(7):707-710.Brunicardi_Ch06_p0157-p0182.indd 16201/03/19 4:46 PM 163SURGICAL INFECTIONSCHAPTER 6virus infections, including their capacity to be transmitted to healthcare workers (see “General Principles”). Prophylactic and therapeutic use of antiviral agents is discussed elsewhere in this textbook.PREVENTION AND TREATMENT OF SURGICAL INFECTIONSGeneral PrinciplesManeuvers to diminish the presence of exogenous (surgeon and operating room environment) and endogenous (patient) microbes are termed prophylaxis and consist of a variety of mechanical and chemical modalities. The Centers for Disease Control and Prevention (CDC) publishes updated, evidence-based guidelines on best practices for prevention of surgical site infections. Important

1	and chemical modalities. The Centers for Disease Control and Prevention (CDC) publishes updated, evidence-based guidelines on best practices for prevention of surgical site infections. Important principles in prophylaxis can be grouped into factors pertaining to skin preparation, antimicrobial therapy, and patient physiological management.Patient skin preparation should begin the night before a planned surgical procedure with a full body bath or shower using soap or an antiseptic agent. Hair removal from an opera-tive site should be performed in the operating room with clippers rather than with a razor, to avoid creating nicks in the skin that could foster bacterial growth. Prior to incision, the skin should be cleansed with an alcohol-based antiseptic agent. There is no clear evidence that use of antimicrobial-containing fluids for either irrigation or soaking prosthetic materials is beneficial in preventing infections. Preoperative antimicrobial therapy should be administered when

1	use of antimicrobial-containing fluids for either irrigation or soaking prosthetic materials is beneficial in preventing infections. Preoperative antimicrobial therapy should be administered when appropriate, based on clinical guidelines, and occur within a time frame that allows bactericidal con-centration of the agent in tissues before the incision is made. Physiological management of the intraoperative patient includes maintenance of euglycemia (serum glucose <200 mg/dL) and normothermia, and optimization of tissue oxygenation.20Source ControlThe primary precept of surgical infectious disease therapy con-sists of drainage of all purulent material, debridement of all infected, devitalized tissue and debris, and/or removal of foreign bodies at the site of infection, plus remediation of the underlying cause of infection.21 This is termed source control. A dis-crete, walled-off purulent fluid collection (i.e., an abscess) 2Table 6-3Common pathogens in surgical patientsGram-positive

1	the underlying cause of infection.21 This is termed source control. A dis-crete, walled-off purulent fluid collection (i.e., an abscess) 2Table 6-3Common pathogens in surgical patientsGram-positive aerobic cocci Staphylococcus aureus Staphylococcus epidermidis Streptococcus pyogenes Streptococcus pneumoniae Enterococcus faecium, E faecalisGram-negative aerobic bacilli Escherichia coli Haemophilus influenzae Klebsiella pneumoniae Proteus mirabilis Enterobacter cloacae, E aerogenes Serratia marcescens Acinetobacter calcoaceticus Citrobacter freundii Pseudomonas aeruginosa Stenotrophomonas maltophiliaAnaerobes Gram-positive Clostridium difficile Clostridium perfringens, C tetani, C septicum Peptostreptococcus spp. Gram-negative Bacteroides fragilis Fusobacterium spp.Other bacteria Mycobacterium avium-intracellulare Mycobacterium tuberculosis Nocardia asteroids Legionella pneumophila Listeria monocytogenesFungi Aspergillus fumigatus, A niger, A terreus, A flavus Blastomyces

1	avium-intracellulare Mycobacterium tuberculosis Nocardia asteroids Legionella pneumophila Listeria monocytogenesFungi Aspergillus fumigatus, A niger, A terreus, A flavus Blastomyces dermatitidis Candida albicans Candida glabrata, C paropsilosis, C krusei Coccidiodes immitis Cryptococcus neoformans Histoplasma capsulatum Mucor/RhizopusViruses Cytomegalovirus Epstein-Barr virus Hepatitis A, B, C viruses Herpes simplex virus Human immunodeficiency virus Varicella zoster virusTable 6-4Antifungal agents and their characteristicsANTIFUNGALADVANTAGESDISADVANTAGESAmphotericin BBroad-spectrum, inexpensiveRenal toxicity, premeds, IV onlyLiposomal Amphotericin BBroad-spectrumExpensive, IV only, renal toxicityAzolesFluconazoleIV and PO availabilityNarrow-spectrum, drug interactionsItraconazoleIV and PO availabilityNarrow spectrum, no CSF penetrationDrug interactions, decreased cardiac contractilityPosaconazoleBroad-spectrum, zygomycete activityPO onlyVoriconazoleIV and PO availability,

1	and PO availabilityNarrow spectrum, no CSF penetrationDrug interactions, decreased cardiac contractilityPosaconazoleBroad-spectrum, zygomycete activityPO onlyVoriconazoleIV and PO availability, broad-spectrumIV diluent accumulates in renal failure, Visual disturbancesEchinocandinsAnidulofungin, Caspofungin, micafunginBroad-spectrumIV only, poor CNS penetrationBrunicardi_Ch06_p0157-p0182.indd 16301/03/19 4:46 PM 164BASIC CONSIDERATIONSPART Irequires drainage, either surgically or via percutaneous drain insertion. An ongoing source of contamination (e.g., bowel per-foration) or the presence of an aggressive, rapidly spreading infection (e.g., necrotizing soft tissue infection) invariably requires expedient, aggressive operative intervention, both to remove contaminated material and infected tissue (e.g., radical debridement or amputation) and to remove the initial cause of infection (e.g., bowel resection). Delay in operative interven-tion, whether due to misdiagnosis or the need

1	tissue (e.g., radical debridement or amputation) and to remove the initial cause of infection (e.g., bowel resection). Delay in operative interven-tion, whether due to misdiagnosis or the need for additional diagnostic studies, is associated with increased morbidity and occasional mortality. Other treatment modalities such as antimi-crobial agents, albeit critical, are of secondary importance to effective surgery with regard to treatment of surgical infections. Rarely, if ever, can an aggressive surgical infection be cured only by the administration of antibiotics, and never in the face of an ongoing source of contamination.22Appropriate Use of Antimicrobial AgentsA classification of antimicrobial agents, mechanisms of action, and spectrums of activity is shown in Table 6-5. As discussed previously, prophylaxis consists of the administration of an anti-microbial agent or agents prior to initiation of certain specific types of surgical procedures in order to reduce the number of

1	previously, prophylaxis consists of the administration of an anti-microbial agent or agents prior to initiation of certain specific types of surgical procedures in order to reduce the number of microbes that enter the tissue or body cavity. Agents are selected according to their activity against microbes likely to be present at the surgical site, based on knowledge of host microflora. For example, patients undergoing elective colorectal surgery should receive antimicrobial prophylaxis directed against skin flora, gram-negative aerobes, and anaerobic bacteria. There are a wide variety of agents that meet these criteria with recently published guidelines.23By definition, prophylaxis is limited to the time prior to and during the operative procedure; in the vast majority of cases only a single dose of antibiotic is required, and only for certain types of procedures (see “Surgical Site Infections”). However, patients who undergo complex, prolonged procedures in which the duration of the

1	dose of antibiotic is required, and only for certain types of procedures (see “Surgical Site Infections”). However, patients who undergo complex, prolonged procedures in which the duration of the operation exceeds the serum drug half-life should receive an additional dose or doses of the antimicrobial agent.23 There is no evidence that administration of postopera-tive doses of an antimicrobial agent provides additional benefit, and this practice should be discouraged, as it is costly and is associated with increased rates of microbial drug resistance. Guidelines for prophylaxis are provided in Table 6-6.Empiric therapy is the use of antimicrobial agents when the risk of a surgical infection is high, based on the underlying disease process (e.g., ruptured appendicitis), or when signifi-cant contamination during surgery has occurred (e.g., inad-equate bowel preparation or considerable spillage of colon contents). Obviously, prophylaxis merges into empiric therapy in situations in which

1	contamination during surgery has occurred (e.g., inad-equate bowel preparation or considerable spillage of colon contents). Obviously, prophylaxis merges into empiric therapy in situations in which the risk of infection increases markedly because of intraoperative findings. Empiric therapy also is often employed in critically ill patients in whom a potential site of infection has been identified and severe sepsis or septic shock occurs. Empiric therapy should be limited to a short course of treatment (3 to 5 days) and should be curtailed as soon as pos-sible based on microbiologic data (i.e., absence of positive cul-tures) coupled with improvements in the clinical course of the patient.Empiric therapy can merge into therapy of established infection in some patients. However, among surgical patients, the manner in which therapy is employed, particularly in rela-tion to the use of microbiologic data (culture and antibiotic sensitivity patterns), differs depending on whether the

1	surgical patients, the manner in which therapy is employed, particularly in rela-tion to the use of microbiologic data (culture and antibiotic sensitivity patterns), differs depending on whether the infection is monomicrobial or polymicrobial. Monomicrobial infections frequently are nosocomial infections occurring in postoperative patients, such as UTIs, pneumonia, or bacteremia. Evidence of systemic inflammatory response syndrome (fever, tachycardia, tachypnea, or elevated leukocyte count) in such individuals, coupled with evidence of local infection (e.g., an infiltrate on chest roentgenogram plus a positive Gram stain in bronchoal-veolar lavage samples) should lead the surgeon to initiate empiric antibiotic therapy. An appropriate approach to antimi-crobial treatment involves de-escalation therapy, where initial antimicrobial selection is broad, with a narrowing of agents based on patient response and culture results. Initial drug selec-tion must be based on initial evidence

1	therapy, where initial antimicrobial selection is broad, with a narrowing of agents based on patient response and culture results. Initial drug selec-tion must be based on initial evidence (gram-positive vs. gram-negative microbes, yeast), coupled with institutional and unit-specific drug sensitivity patterns. It is important to ensure that antimicrobial coverage chosen is adequate, since delay in appropriate antibiotic treatment has been shown to be associated with significant increases in mortality. A critical component of this approach is appropriate collection of culture specimens to allow for thorough analysis, since within 48 to 72 hours culture and sensitivity reports will allow refinement of the antibiotic regimen to select the most efficacious agent.Although the primary therapeutic modality to treat polymicrobial surgical infections is source control, antimicro-bial agents play an important role. Culture results are of lesser importance in managing these types of infections,

1	modality to treat polymicrobial surgical infections is source control, antimicro-bial agents play an important role. Culture results are of lesser importance in managing these types of infections, as it has been repeatedly demonstrated that only a limited cadre of microbes predominate in the established infection, selected from a large number present at the time of initial contamination. Invariably it is difficult to identify all microbes that comprise the initial polymicrobial inoculum. For this reason, the antibiotic regimen should not be modified solely on the basis of culture informa-tion, as it is less important than the clinical course of the patient. As long as appropriately broad-spectrum coverage for aerobic and anaerobic microbes is provided, a worsening of the patient’s clinical course should direct the surgeon to investigate whether effective source control has been achieved.24 Duration of anti-biotic administration should be decided at the time the drug regimen is

1	clinical course should direct the surgeon to investigate whether effective source control has been achieved.24 Duration of anti-biotic administration should be decided at the time the drug regimen is prescribed. As mentioned previously, prophylaxis is limited to a single dose administered immediately prior to creating the incision. Empiric therapy should be limited to 3 to 5 days or less and should be curtailed if the presence of a local site or systemic infection is not revealed.25 In fact, prolonged use of empirical antibiotic therapy in culture-negative critically ill patients is associated with increased mortality, highlighting the need to discontinue therapy when there is no proven evidence of infection.26Therapy for monomicrobial infections follows standard guidelines: 3 to 5 days for UTIs, 7 to 8 days for pneumonia, and 7 to 14 days for bacteremia. Longer courses of therapy in this setting do not result in improved care and are associated with increased risk of superinfection

1	for UTIs, 7 to 8 days for pneumonia, and 7 to 14 days for bacteremia. Longer courses of therapy in this setting do not result in improved care and are associated with increased risk of superinfection by resistant organisms.27-29 There is some evidence that measuring and monitoring serum procalcitonin trends in the setting of infection allows earlier cessation of antibiotics without decrement in the rate of clini-cal cure.30 Antibiotic therapy for osteomyelitis, endocarditis, or prosthetic infections in which it is hazardous to remove the device consists of prolonged courses of treatment for 6 to 12 weeks. The specific agents are selected based on analysis of the degree to which the organism is killed in vitro using the minimum inhibitory concentration (MIC) of a standard pure 34Brunicardi_Ch06_p0157-p0182.indd 16401/03/19 4:46 PM 165SURGICAL INFECTIONSCHAPTER 6Table 6-5Antimicrobial agentsANTIBIOTIC CLASS, GENERIC NAMETRADE NAMEMECHANISM OF ACTIONORGANISMS PyogenesMSSAMRSAS

1	16401/03/19 4:46 PM 165SURGICAL INFECTIONSCHAPTER 6Table 6-5Antimicrobial agentsANTIBIOTIC CLASS, GENERIC NAMETRADE NAMEMECHANISM OF ACTIONORGANISMS PyogenesMSSAMRSAS epidermidisEnterococcusVREE coliP aeruginosaANAEROBESPenicillinsCell wall synthesis inhibitors (bind penicillin-binding protein)Penicillin G1000+/–0001NafcillinNallpen, Unipen110+/–00000PiperacillinPipracil1000+/–011+/–Penicillin/a-lactamase inhibitor combinationsCell wall synthesis inhibitors/β-lactamase inhibitorsAmpicillin/sulbactamUnasyn110+/–1+/–101Ticarcillin/clavulanateTimentin110+/–+/–0111Piperacillin/tazobactamZosyn1101+/–0111First-generation cephalosporinsCell wall synthesis inhibitorsCefazolin, cephalexinAncef, Keflex110+/–00100Second-generation cephalosporinsCell wall synthesis inhibitorsCefoxitinMefoxin110+/–00101CefotetanCefotan110+/–00101CefuroximeCeftin110+/–00100Thirdand fourth-generation cephalosporinsCell wall synthesis

1	cephalosporinsCell wall synthesis inhibitorsCefoxitinMefoxin110+/–00101CefotetanCefotan110+/–00101CefuroximeCeftin110+/–00100Thirdand fourth-generation cephalosporinsCell wall synthesis inhibitorsCeftriaxoneRocephin110+/–00100CeftazidimeFortaz1+/–0+/–00110CefepimeMaxipime110+/–00110CefotaximeCefotaxime110+/–001+/–0CeftarolineTeflaro111100100(Continued)Brunicardi_Ch06_p0157-p0182.indd 16501/03/19 4:46 PM 166BASIC CONSIDERATIONSPART ICarbapenemsCell wall synthesis inhibitorsImipenem-cilastatinPrimaxin1101+/–0111MeropenemMerrem110100111ErtapenemInvanz1101001+/–1AztreonamAzactam000000110AminoglycosidesAlteration of cell membrane, binding and inhibition of 30S ribosomal subunitGentamicin010+/–10110Tobramycin, amikacin010+/–00110FluoroquinolonesInhibit topo-isomerase II and IV (DNA synthesis inhibition)CiprofloxacinCipro+/–10100110LevofloxacinLevaquin1101001+/–0GlycopeptidesCell wall synthesis inhibition (peptidoglycan synthesis

1	topo-isomerase II and IV (DNA synthesis inhibition)CiprofloxacinCipro+/–10100110LevofloxacinLevaquin1101001+/–0GlycopeptidesCell wall synthesis inhibition (peptidoglycan synthesis inhibition)VancomycinVancocin111110000Quinupristin-dalfopristinSynercidInhibits 2 sites on 50S ribosome (protein synthesis inhibition)11111100+/–Table 6-5Antimicrobial agentsANTIBIOTIC CLASS, GENERIC NAMETRADE NAMEMECHANISM OF ACTIONORGANISMS PyogenesMSSAMRSAS epidermidisEnterococcusVREE coliP aeruginosaANAEROBES(Continued)Brunicardi_Ch06_p0157-p0182.indd 16601/03/19 4:46 PM 167SURGICAL INFECTIONSCHAPTER 6LinezolidZyvoxInhibits 50S ribosomal activity11111100+/–DaptomycinCubicinBinds bacterial membrane, results in depolarization, lysis111111000RifampinInhibits DNA-dependent RNA polymerase1111+/–0000ClindamycinCleocinInhibits 50S ribosomal activity110000001MetronidazoleFlagylProduction of toxic intermediates (free radicals)000000001MacrolidesInhibit 50S ribosomal activity (protein synthesis

1	50S ribosomal activity110000001MetronidazoleFlagylProduction of toxic intermediates (free radicals)000000001MacrolidesInhibit 50S ribosomal activity (protein synthesis inhibition)Erythromycin1+/–0+/–00000AzithromycinZithromax110000000ClarithromycinBiaxin110000000Trimethoprim-sulfamethoxazoleBactrim, SeptraInhibits sequential steps of folate metabolism+/–10/–00100TetracyclinesBind 30S ribosomal unit (protein synthesis inhibition)MinocyclineMinocin11000000+/–DoxycyclineVibromycin1+/–000010+/–=TigacyclineTygacil111111101E coli = Escherichia coli; MRSA = methicillin-resistant Staphylococcus aureus; MSSA = methicillin-sensitive S aureus; P aeruginosa = Pseudomonas aeruginosa; S epidermidis = Staphylococcus epidermidis; S pyogenes = Streptococcus pyogenes; VRE = vancomycin-resistant Enterococcus1 = reliable activity; +/– = variable activity; 0 = no activity.The sensitivities printed here are generalizations. The clinician should confirm sensitivity patterns at the locale where the patient

1	= reliable activity; +/– = variable activity; 0 = no activity.The sensitivities printed here are generalizations. The clinician should confirm sensitivity patterns at the locale where the patient is being treated since these patterns may vary widely depending on location.Brunicardi_Ch06_p0157-p0182.indd 16701/03/19 4:46 PM 168BASIC CONSIDERATIONSPART ITable 6-6Prophylactic use of antibioticsSITEANTIBIOTICALTERNATIVE (E.G., PENICILLIN ALLERGIC)Cardiovascular surgeryCefazolin, cefuroximeVancomycin, clindamycinGastroduodenal areaSmall intestine, nonobstructedCefazolinClindamycin or vancomycin + aminoglycoside or aztreonem or fluoroquinoloneBiliary tract: open procedure, laparoscopic high riskCefazolin, cefoxitin, cefotetan, ceftriaxone, ampicillin-sulbactamClindamycin or vancomycin + aminoglycoside or aztreonem or fluoroquinoloneMetronidazole + aminoglycoside or fluoroquinoloneBiliary tract: laparoscopic low riskNoneNoneAppendectomy, uncomplicatedCefoxitin, cefotetan, cefazolin +

1	aminoglycoside or aztreonem or fluoroquinoloneMetronidazole + aminoglycoside or fluoroquinoloneBiliary tract: laparoscopic low riskNoneNoneAppendectomy, uncomplicatedCefoxitin, cefotetan, cefazolin + metronidazoleClindamycin + aminoglycoside or aztreonem or fluoroquinoloneMetronidazole + aminoglycoside or fluoroquinoloneColorectal surgery, obstructed small intestineCefazolin or ceftriaxone plus metronidazole, ertapenem, cefoxitin, cefotetan, ampicillin-sulbactamClindamycin + aminoglycoside or aztreonem or fluoroquinolone, metronidazole + aminoglycoside or fluoroquinoloneHead and neck; clean contaminatedCefazolin or cefuroxime + metronidazole, ampicillin-sulbactamClindamycinNeurosurgical proceduresCefazolinClindamycin, vancomycinOrthopedic surgeryCefazolin, ceftriaxoneClindamycin, vancomycinBreast, herniaCefazolinClindamycin, vancomycinData from Pieracci FM, Barie PS. Management of severe sepsis of abdominal origin, Scand J Surg. 2007;96(3):184-196.inoculum of 105 CFU/mL of the

1	herniaCefazolinClindamycin, vancomycinData from Pieracci FM, Barie PS. Management of severe sepsis of abdominal origin, Scand J Surg. 2007;96(3):184-196.inoculum of 105 CFU/mL of the organism isolated from the site of infection or bloodstream. Sensitivities are reported in rela-tion to the achievable blood level of each antibiotic in a panel of agents. The least toxic, least expensive agent to which the organism is most sensitive should be selected. Serious or recru-descent infection may require therapy with two or more agents, particularly if a multidrug-resistant pathogen is causative, limit-ing therapeutic options to drugs to which the organism is only moderately sensitive. Commonly, an agent may be administered intravenously for 1 to 2 weeks, followed by treatment with an oral drug. However, this should only be undertaken in patients who demonstrate progressive clinical improvement, and the oral agent should be capable of achieving high serum levels as well (e.g.,

1	an oral drug. However, this should only be undertaken in patients who demonstrate progressive clinical improvement, and the oral agent should be capable of achieving high serum levels as well (e.g., fluoroquinolones).The 2016 Surgical Infection Society guidelines on man-agement of intra-abdominal infection recommend antibiotic duration of no more than 24 hours in patients with traumatic bowel perforation who receive surgical treatment within 12 hours, gastroduodenal perforations operated upon within 24 hours, ischemic nonperforated bowel, and gangrenous acute appen-dicitis or cholecystitis without perforation. More extensive intraperitoneal infection (perforated appendicitis, for example) should have treatment limited to 4 days. Patients with a greater degree of contamination may require longer courses of therapy; as in all facets of clinical practice, the therapeutic plan must be individualized to the patient. In the later phases of postopera-tive antibiotic treatment of serious

1	longer courses of therapy; as in all facets of clinical practice, the therapeutic plan must be individualized to the patient. In the later phases of postopera-tive antibiotic treatment of serious intra-abdominal infection, the absence of an elevated white blood cell (WBC) count, lack of band forms of PMNs on peripheral smear, and lack of fever (<38°C [100.5°F]) provide close to complete assurance that infection has been eradicated.31 There is also emerging data that suggest following a patient’s procalcitonin level may provide the clinician with useful information regarding whether an infection has resolved and allow more expedient cessation of therapy.32,33 Patients who do not improve with 5 to 7 days of antibiotic therapy should be reevaluated for inadequate source control or a new extra-abdominal source of infection.Allergy to antimicrobial agents must be considered prior to prescribing them. First, it is important to ascertain whether a patient has had any type of allergic

1	extra-abdominal source of infection.Allergy to antimicrobial agents must be considered prior to prescribing them. First, it is important to ascertain whether a patient has had any type of allergic reaction in association with administration of a particular antibiotic. However, one should take care to ensure that the purported reaction consists of true allergic symptoms and signs, such as urticaria, bron-chospasm, or other similar manifestations, rather than indiges-tion or nausea. Penicillin allergy is quite common, the reported incidence ranging from 0.7% to 10%. Although avoiding the use of any β-lactam drug is appropriate in patients who mani-fest significant allergic reactions to penicillins, the incidence of cross-reactivity appears low for all related agents, with 1% cross-reactivity for carbapenems, 5% to 7% cross-reactivity for cephalosporins, and extremely small or nonexistent cross-reactivity for monobactams.34Severe allergic manifestations, such as anaphylaxis, to a

1	for carbapenems, 5% to 7% cross-reactivity for cephalosporins, and extremely small or nonexistent cross-reactivity for monobactams.34Severe allergic manifestations, such as anaphylaxis, to a specific class of agents generally preclude the use of any agents in that class, except under circumstances in which use of a certain drug represents a lifesaving measure. In some centers, patients undergo intradermal testing using a dilute solution of a particular antibiotic to determine whether a severe allergic reac-tion would be elicited by parenteral administration. A pathway, including such intradermal testing, has been effective in reduc-tion of vancomycin use to 16% in surgical patients with reported allergy to penicillin.35 This type of testing rarely is employed because it is simpler to select an alternative class of agent. Should administration of a specific agent to which the patient is Brunicardi_Ch06_p0157-p0182.indd 16801/03/19 4:46 PM 169SURGICAL INFECTIONSCHAPTER 6allergic

1	an alternative class of agent. Should administration of a specific agent to which the patient is Brunicardi_Ch06_p0157-p0182.indd 16801/03/19 4:46 PM 169SURGICAL INFECTIONSCHAPTER 6allergic become necessary, desensitization using progressively higher doses of antibiotic can be undertaken, providing the ini-tial testing does not cause severe allergic manifestations.Misuse of antimicrobial agents is rampant in both the inpa-tient and outpatient settings, and is associated with an enormous financial impact on healthcare costs, adverse reactions due to drug toxicity and allergy, the occurrence of new infections such as Clostridium difficile colitis, and the development of multiagent drug resistance among nosocomial pathogens. Each of these factors has been directly correlated with overall drug administration. It has been estimated that in the United States in excess of $20 billion is spent on antibiotics each year.36 The responsible practitioner limits prophylaxis to the period

1	drug administration. It has been estimated that in the United States in excess of $20 billion is spent on antibiotics each year.36 The responsible practitioner limits prophylaxis to the period dur-ing the operative procedure, does not convert prophylaxis into empiric therapy except under well-defined conditions, sets the duration of antibiotic therapy from the outset, curtails antibi-otic administration when clinical and microbiologic evidence does not support the presence of an infection, and limits therapy to a short course in every possible instance. Prolonged treat-ment associated with drains and tubes has not been shown to be beneficial.INFECTIONS OF SIGNIFICANCE IN SURGICAL PATIENTSSurgical Site InfectionsSurgical site infections (SSIs) are infections of the tissues, organs, or spaces exposed by surgeons during performance of an invasive procedure. SSIs are classified into incisional and organ/space infections, and the former are further subclas-sified into superficial (limited

1	exposed by surgeons during performance of an invasive procedure. SSIs are classified into incisional and organ/space infections, and the former are further subclas-sified into superficial (limited to skin and subcutaneous tissue) and deep incisional categories.37,38 The development of SSIs is related to three factors: (a) the degree of microbial contamina-tion of the wound during surgery; (b) the duration of the proce-dure; and (c) host factors such as diabetes, malnutrition, obesity, immune suppression; and a number of other underlying disease states. Table 6-7 lists risk factors for development of SSIs. By definition, an incisional SSI has occurred if a surgical wound drains purulent material or if the surgeon judges it to be infected and opens it.Surgical wounds are classified based on the presumed mag-nitude of the bacterial load at the time of surgery (Table 6-8).39 Clean wounds (class I) include those in which no infection is present; only skin microflora potentially

1	based on the presumed mag-nitude of the bacterial load at the time of surgery (Table 6-8).39 Clean wounds (class I) include those in which no infection is present; only skin microflora potentially contaminate the wound, and no hollow viscus that contains microbes is entered. Class I D wounds are similar except that a prosthetic device (e.g., mesh or valve) is inserted. Clean/contaminated wounds (class II) include those in which a hollow viscus such as the respiratory, alimentary, or genitourinary tracts with indigenous bacterial flora is opened under controlled circumstances without significant spillage of contents.While elective colorectal cases have classically been included as class II cases, a number of studies in the last decade have documented higher SSI rates (9–25%). One study iden-tified two-thirds of infections presenting after discharge from hospital, highlighting the need for careful follow-up of these patients.40 Infection is also more common in cases involving entry into

1	two-thirds of infections presenting after discharge from hospital, highlighting the need for careful follow-up of these patients.40 Infection is also more common in cases involving entry into the rectal space.41 In a recent single-center quality improvement study using a multidisciplinary approach, one group of clinicians has demonstrated the ability to decrease SSI from 9.8% to 4.0%.425Table 6-7Risk factors for development of surgical site infectionsPatient factors Older age Immunosuppression Obesity Diabetes mellitus Chronic inflammatory process Malnutrition Smoking Renal failure Peripheral vascular disease Anemia Radiation Chronic skin disease Carrier state (e.g., chronic Staphylococcus carriage) Recent operationLocal factors Open compared to laparoscopic surgery Poor skin preparation Contamination of instruments Inadequate antibiotic prophylaxis Prolonged procedure Local tissue necrosis Blood transfusion Hypoxia, hypothermiaMicrobial factors Prolonged hospitalization (leading to

1	of instruments Inadequate antibiotic prophylaxis Prolonged procedure Local tissue necrosis Blood transfusion Hypoxia, hypothermiaMicrobial factors Prolonged hospitalization (leading to nosocomial organisms) Toxin secretion Resistance to clearance (e.g., capsule formation)Table 6-8Wound class, representative procedures, and expected infection ratesWOUND CLASSEXAMPLES OF CASESEXPECTED INFECTION RATESClean (class I)Hernia repair, breast biopsy1–2%Clean/contaminated (class II)Cholecystectomy, elective GI surgery (not colon)2.1–9.5%Clean/contaminated (class II)Colorectal surgery4–14%Contaminated (class III)Penetrating abdominal trauma, large tissue injury, enterotomy during bowel obstruction3.4–13.2%Dirty (class IV)Perforated diverticulitis, necrotizing soft tissue infections3.1–12.8%Brunicardi_Ch06_p0157-p0182.indd 16901/03/19 4:46 PM 170BASIC CONSIDERATIONSPART IContaminated wounds (class III) include open acciden-tal wounds encountered early after injury, those with extensive

1	16901/03/19 4:46 PM 170BASIC CONSIDERATIONSPART IContaminated wounds (class III) include open acciden-tal wounds encountered early after injury, those with extensive introduction of bacteria into a normally sterile area of the body due to major breaks in sterile technique (e.g., open cardiac massage), gross spillage of viscus contents such as from the intestine, or incision through inflamed, albeit nonpurulent tis-sue. Dirty wounds (class IV) include traumatic wounds in which a significant delay in treatment has occurred and in which necrotic tissue is present, those created in the presence of overt infection as evidenced by the presence of purulent material, and those created to access a perforated viscus accompanied by a high degree of contamination. The microbiology of SSIs is reflective of the initial host microflora such that SSIs fol-lowing creation of a class I wound are invariably caused by skin microbes found on that portion of the body, while SSIs subsequent to a class

1	of the initial host microflora such that SSIs fol-lowing creation of a class I wound are invariably caused by skin microbes found on that portion of the body, while SSIs subsequent to a class II wound created for the purpose of elec-tive colon resection may be caused by either skin microbes or colonic microflora, or both.Surgical management of the wound is a critical determi-nant of the propensity to develop an SSI. In healthy individuals, classes I and II wounds may be closed primarily, while skin closure of class III and IV wounds is associated with high rates of incisional SSIs (∼25–50%). The superficial aspects of these latter types of wounds should be packed open and allowed to heal by secondary intention, although selective use of delayed primary closure has been associated with a reduction in inci-sional SSI rates.43 One clear example based on data from clini-cal trials is that class III wounds in healthy patients undergoing appendectomy for perforated or gangrenous

1	with a reduction in inci-sional SSI rates.43 One clear example based on data from clini-cal trials is that class III wounds in healthy patients undergoing appendectomy for perforated or gangrenous appendicitis can be primarily closed as long as antibiotic therapy directed against aerobes and anaerobes is administered. This practice leads to SSI rates of approximately 3% to 4%.44Recent investigations have studied the effect of additional maneuvers in an attempt to further reduce the rate of SSIs. The adverse effects of hyperglycemia on WBC function have been well described.45 A number of studies in patients undergoing several different types of surgery describe increased risk of SSI in patients with hyperglycemia, and the 2017 CDC guidelines for prevention of surgical site infection recommend maintaining blood glucose <200 mg/dL (11.1 mmol/L) in all patients during the perioperative period.46-48The respective effects of body temperature and the level of inhaled oxygen during surgery on

1	maintaining blood glucose <200 mg/dL (11.1 mmol/L) in all patients during the perioperative period.46-48The respective effects of body temperature and the level of inhaled oxygen during surgery on SSI rates also have been studied, and both hypothermia and hypoxia during surgery are associated with a higher rate of SSI. There is conflicting evi-dence regarding whether supplying higher levels of inhaled oxy-gen to perioperative patients reduces the rate of SSI. Although an initial study provided evidence that patients who received high levels of inhaled oxygen during colorectal surgery devel-oped fewer SSIs,49 a later meta-analysis suggested that the over-all benefit is small and may not warrant use.50 The 2017 CDC guidelines, however, support administration of increased FiO2 during surgery and after extubation in patients with normal pul-monary function receiving general anesthesia as there has been some evidence of benefit.20,51 Further evaluation via multicenter studies is needed

1	and after extubation in patients with normal pul-monary function receiving general anesthesia as there has been some evidence of benefit.20,51 Further evaluation via multicenter studies is needed prior to implementation of hyperoxia as stan-dard therapy, but it is clear that intraoperative hypothermia and hypoxia should be prevented.Effective therapy for incisional SSIs consists solely of incision and drainage without the additional use of antibiotics. Antibiotic therapy is reserved for patients in whom evidence of significant cellulitis is present, or who concurrently manifest a systemic inflammatory response syndrome. The open wound often is allowed to heal by secondary intention, with dressings being changed as the clinical team deems appropriate. The use of topical antibiotics and antiseptics to further wound healing remains unproven, although anecdotal studies indicate their potential utility in complex wounds that do not heal with routine measures.52 Despite a paucity of

1	and antiseptics to further wound healing remains unproven, although anecdotal studies indicate their potential utility in complex wounds that do not heal with routine measures.52 Despite a paucity of prospective studies, vacuum-assisted closure is increasingly used in management of large, complex open wounds and can be applied to wounds in loca-tions that are difficult to manage with dressings (Fig. 6-1).53,54 One also should consider obtaining wound cultures in patients who develop SSIs and who have been hospitalized or reside in long-term care facilities due to the increasing incidence of infec-tion caused by multidrug-resistant organisms.In the United States, hospitals are required to conduct sur-veillance for the development of SSIs for a period of 30 days ABFigure 6-1. Negative pressure wound therapy in a patient after amputation for wet gangrene (A) and in a patient with enterocutaneous fistula (B). It is possible to adapt these dressings to fit difficult anatomy and provide

1	wound therapy in a patient after amputation for wet gangrene (A) and in a patient with enterocutaneous fistula (B). It is possible to adapt these dressings to fit difficult anatomy and provide appropriate wound care while reducing frequency of dressing change. It is important to evaluate the wound under these dressings if the patient demonstrates signs of sepsis with an unidentified source, since typical clues of wound sepsis such as odor and drainage are hidden by the suction apparatus.Brunicardi_Ch06_p0157-p0182.indd 17001/03/19 4:46 PM 171SURGICAL INFECTIONSCHAPTER 6after the operative procedure.55 Such surveillance has been associated with greater awareness and a reduction in SSI rates, probably in large part based upon the impact of observation and promotion of adherence to appropriate care standards. Begin-ning in 2012, all hospitals receiving reimbursement from the Centers for Medicare & Medicaid Services (CMS) are required to report SSIs.A recent refinement of risk indexes

1	care standards. Begin-ning in 2012, all hospitals receiving reimbursement from the Centers for Medicare & Medicaid Services (CMS) are required to report SSIs.A recent refinement of risk indexes has been implemented through the National Healthcare Safety Network, a secure, web-based system of surveillance used by the CDC for surveillance of healthcare-associated infections. This refinement utilized data reported from 847 hospitals in nearly one million patients over a 2-year period to develop procedure-specific risk indices for SSIs.56SSIs are associated with considerable morbidity and occasional lethality, as well as substantial healthcare costs and patient inconvenience and dissatisfaction.57 A number of healthcare organizations within the United States are interested in evaluating performance of hospitals and physicians with respect to implementing processes that support delivery of stan-dard of care. One major process of interest is reduction in SSIs, since the morbidity (and

1	performance of hospitals and physicians with respect to implementing processes that support delivery of stan-dard of care. One major process of interest is reduction in SSIs, since the morbidity (and subsequent cost) of this complication is high. Several of these organizations are noted in Table 6-9. Appropriate guidelines in this area incorporating the principles discussed previously have been developed and disseminated.58 However, observers have noted that adherence to these guide-lines has been poor.59 Most experts believe that better adherence to evidence-based practice recommendations and implementing systems of care with redundant safeguards will result in reduc-tion of surgical complications and better patient outcomes. More important, the CMS, the largest third-party insurance payer in the United States, has required reporting by hospitals of many processes related to reduction of surgical infections, including appropriate use of perioperative antibiotics. This information,

1	in the United States, has required reporting by hospitals of many processes related to reduction of surgical infections, including appropriate use of perioperative antibiotics. This information, which is reported publicly by hospitals, has led to significant improvement in reported rates of these process measures. How-ever, the effect of this approach on the incidence of SSIs is not known at this time.Intra-Abdominal InfectionsMicrobial contamination of the peritoneal cavity is termed peri-tonitis or intra-abdominal infection and is classified according to etiology. Primary microbial peritonitis occurs when microbes invade the normally sterile confines of the peritoneal cavity via hematogenous dissemination from a distant source of infec-tion or direct inoculation. This process is more common among patients who retain large amounts of peritoneal fluid due to ascites, and among those individuals who are being treated for renal failure via peritoneal dialysis. These infections

1	more common among patients who retain large amounts of peritoneal fluid due to ascites, and among those individuals who are being treated for renal failure via peritoneal dialysis. These infections invariably are monomicrobial and rarely require surgical intervention. The diagnosis is established based on identification of risk factors as noted previously, physical examination that reveals diffuse tenderness and guarding without localized findings, absence of a surgically treatable source of infection on an imaging study, and the presence of more than 250 neutrophils/mL in fluid obtained via paracentesis.60 Cultures typically will demonstrate the presence of gram-positive organisms in patients undergoing peritoneal dialysis. In patients without this risk factor, the most common etiologic organisms are E coli, K pneumoniae, and S pneumoniae. Treatment consists of administration of an anti-biotic to which the organism is sensitive; often 14 to 21 days of therapy are required. Removal

1	organisms are E coli, K pneumoniae, and S pneumoniae. Treatment consists of administration of an anti-biotic to which the organism is sensitive; often 14 to 21 days of therapy are required. Removal of indwelling devices, if present, may be required for effective therapy of recurrent infections.Secondary microbial peritonitis occurs subsequent to con-tamination of the peritoneal cavity due to perforation or severe inflammation and infection of an intra-abdominal organ. Exam-ples include appendicitis, perforation of any portion of the gas-trointestinal tract, or diverticulitis. As noted previously, effective therapy requires source control to resect or repair the diseased organ; debridement of necrotic, infected tissue and debris; and administration of antimicrobial agents directed against aerobes and anaerobes.61 This type of antibiotic regimen should be cho-sen because in most patients the precise diagnosis cannot be established until exploratory laparotomy is performed, and the most

1	aerobes and anaerobes.61 This type of antibiotic regimen should be cho-sen because in most patients the precise diagnosis cannot be established until exploratory laparotomy is performed, and the most morbid form of this disease process is colonic perforation, due to the large number of microbes present. A combination of agents or single agents with a broad spectrum of activity can be used for this purpose; conversion of a parenteral to an oral regi-men when the patient’s ileus resolves provides results similar to those achieved with intravenous antibiotics. Effective source control and antibiotic therapy is associated with low failure rates and a mortality rate of approximately 5% to 6%; inability to control the source of infection is associated with mortality greater than 40%.62The response rate to effective source control and use of appropriate antibiotics has remained approximately 70% to 90% over the past several decades.63 Patients in whom stan-dard therapy fails typically

1	response rate to effective source control and use of appropriate antibiotics has remained approximately 70% to 90% over the past several decades.63 Patients in whom stan-dard therapy fails typically develop one or more of the follow-ing: an intra-abdominal abscess, leakage from a gastrointestinal anastomosis leading to postoperative peritonitis, or tertiary (persistent) peritonitis. The latter is a poorly understood entity that is more common in immunosuppressed patients in whom peritoneal host defenses do not effectively clear or sequester Table 6-9Quality improvement organizations of interest to surgeons in the United StatesABBREVIATIONORGANIZATIONWEBSITENSQIPNational Surgical Quality Improvement Programacsnsqip.orgIHIInstitute for Healthcare Improvementwww.ihi.orgCMSCenters for Medicare & Medicaid Serviceswww.medicare.govwww.cms.gov/NCQANational Committee for Quality Assurancewww.ncqa.orgSISSurgical Infection Societywww.sisna.orgCDCCenters for Disease Control and

1	for Medicare & Medicaid Serviceswww.medicare.govwww.cms.gov/NCQANational Committee for Quality Assurancewww.ncqa.orgSISSurgical Infection Societywww.sisna.orgCDCCenters for Disease Control and Preventionwww.cdc.gov/HAI/ssi/ssi.htmlBrunicardi_Ch06_p0157-p0182.indd 17101/03/19 4:46 PM 172BASIC CONSIDERATIONSPART Ithe initial secondary microbial peritoneal infection. Microbes such as E faecalis and faecium, S epidermidis, C albicans, and P aeruginosa commonly are identified, typically in combina-tion, and their presence may be due to their lack of responsive-ness to the initial antibiotic regimen, coupled with diminished activity of host defenses. Unfortunately, even with effective antimicrobial agent therapy, this disease process is associated with mortality rates in excess of 50%.64Formerly, the presence of an intra-abdominal abscess mandated surgical reexploration and drainage. Today, the vast majority of such abscesses can be effectively diagnosed via abdominal computed

1	50%.64Formerly, the presence of an intra-abdominal abscess mandated surgical reexploration and drainage. Today, the vast majority of such abscesses can be effectively diagnosed via abdominal computed tomographic (CT) imaging techniques and drained percutaneously. Surgical intervention is reserved for those individuals who harbor multiple abscesses, those with abscesses in proximity to vital structures such that percutaneous drainage would be hazardous, and those in whom an ongoing source of contamination (e.g., enteric leak) is identified. The necessity of antimicrobial agent therapy and precise guidelines that dictate duration of catheter drainage have not been estab-lished. A short course (3 to 5 days) of antibiotics that possess aerobic and anaerobic activity seems reasonable so long as the patient has good clinical response to therapy, and most practi-tioners leave the drainage catheter in situ until it is clear that cavity collapse has occurred, output is less than 10 to 20 mL/d,

1	the patient has good clinical response to therapy, and most practi-tioners leave the drainage catheter in situ until it is clear that cavity collapse has occurred, output is less than 10 to 20 mL/d, no evidence of an ongoing source of contamination is present, and the patient’s clinical condition has improved.33Organ-Specific InfectionsHepatic abscesses are rare, currently accounting for approximately 15 per 100,000 hospital admissions in the United States. Pyogenic abscesses account for approximately 80% of cases, the remaining 20% being equally divided among parasitic and fungal forms.65 Formerly, pyogenic liver abscesses mainly were caused by pyle-phlebitis due to neglected appendicitis or diverticulitis. Today, manipulation of the biliary tract to treat a variety of diseases has become a more common cause, although in nearly 50% of patients no cause is identified. The most common aerobic bacteria iden-tified in recent series include E coli, K pneumoniae, and other enteric bacilli,

1	a more common cause, although in nearly 50% of patients no cause is identified. The most common aerobic bacteria iden-tified in recent series include E coli, K pneumoniae, and other enteric bacilli, enterococci, and Pseudomonas spp., while the most common anaerobic bacteria are Bacteroides spp., anaero-bic streptococci, and Fusobacterium spp. C albicans and other related yeast cause the majority of fungal hepatic abscesses. Small (<1 cm), multiple abscesses should be sampled and treated with a 4to 6-week course of antibiotics. Larger abscesses are generally amenable to percutaneous drainage, with parameters for antibiotic therapy and drain removal similar to those men-tioned previously. Splenic abscesses are extremely rare and are treated in a similar fashion. Recurrent hepatic or splenic abscesses may require operative intervention—unroofing and marsupialization or splenectomy, respectively.Secondary pancreatic infections (e.g., infected pancreatic necrosis or pancreatic abscess)

1	abscesses may require operative intervention—unroofing and marsupialization or splenectomy, respectively.Secondary pancreatic infections (e.g., infected pancreatic necrosis or pancreatic abscess) occur in approximately 10% to 15% of patients who develop severe pancreatitis with necro-sis. The surgical treatment of this disorder was pioneered by Bradley and Allen, who noted significant improvements in out-come for patients undergoing repeated pancreatic debridement of infected pancreatic necrosis.66 Care of patients with severe acute pancreatitis includes staging with dynamic, contrast-enhanced helical CT scan to evaluate the extent of pancreatitis (unless significant renal dysfunction exists, in which case one should forego the use of contrast material) coupled with the use of one of several prognostic scoring systems. Patients who exhibit clinical signs of instability (e.g., oliguria, hypoxemia, large-volume fluid resuscitation) should be carefully monitored in the ICU and undergo

1	prognostic scoring systems. Patients who exhibit clinical signs of instability (e.g., oliguria, hypoxemia, large-volume fluid resuscitation) should be carefully monitored in the ICU and undergo follow-up contrast CT examination when renal function has stabilized to evaluate for development of local pancreatic complications (Fig. 6-2). Routine use of pro-phylactic antibiotics to prevent infected pancreatic necrosis is not indicated. Early enteral feeding using nasojejunal feeding tubes placed past the ligament of Treitz has been associated with decreased development of infected pancreatic necrosis, possibly due to a decrease in gut translocation of bacteria.67,68The presence of secondary pancreatic infection should be suspected in patients whose systemic inflammatory response (fever, elevated WBC count, or organ dysfunction) fails to resolve, or in those individuals who initially recuperate, only to develop sepsis syndrome 2 to 3 weeks later. CT-guided aspira-tion of fluid from the

1	WBC count, or organ dysfunction) fails to resolve, or in those individuals who initially recuperate, only to develop sepsis syndrome 2 to 3 weeks later. CT-guided aspira-tion of fluid from the pancreatic bed for performance of Gram stain and culture analysis can be useful. A positive Gram stain or culture from CT-guided aspiration, or identification of gas within the pancreas on CT scan, mandate surgical intervention.The approach of open necrosectomy with repeated debridements, although life-saving, is associated with sig-nificant morbidity and prolonged hospitalization. Efforts to reduce the amount of surgical injury, while still preserving the improved outcomes associated with debridement of the infected sequestrum, have led to a variety of less invasive approaches, including endoscopic and laparoscopic techniques.69 There are a limited number of randomized trials reporting the use of these new techniques. An important concept common to all of these approaches, however, is the

1	and laparoscopic techniques.69 There are a limited number of randomized trials reporting the use of these new techniques. An important concept common to all of these approaches, however, is the attempt to delay surgical interven-tion, since a number of trials have identified increased mortality when intervention occurs during the first 2 weeks of illness.Data supporting the use of endoscopic approaches to infected pancreatic necrosis include nearly a dozen case series and a randomized trial.70,71 The reported mortality rate was 5%, with a 30% complication rate. Most authors noted the common requirement for multiple endoscopic debridements (similar to the open approach), with a median of four sessions required. Fewer series report experience with the laparoscopic approach, either transgastric or transperitoneal, entering the necrosis through the transverse mesocolon or gastrocolic ligament. Lap-aroscopic intervention is limited by the difficulty in achieving Figure

1	either transgastric or transperitoneal, entering the necrosis through the transverse mesocolon or gastrocolic ligament. Lap-aroscopic intervention is limited by the difficulty in achieving Figure 6-2. Contrast-enhanced CT scan of pancreas 1.5 weeks after presentation showing large central peripancreatic fluid col-lection (arrow).Brunicardi_Ch06_p0157-p0182.indd 17201/03/19 4:46 PM 173SURGICAL INFECTIONSCHAPTER 6Figure 6-3. Infected pancreatic necrosis. (A) Open necrosectomy specimen with pancreatic stent in situ. It is important to gently debride only necrotic pancreatic tissue, relying on repeated opera-tion to ensure complete removal. (B) For video-assisted retroperito-neal debridement (VARD), retroperitoneal access is gained through radiologic placement of a drain, followed by dilation 2 to 3 days later. (C) Retroperitoneal cavity seen through endoscope during VARD.BCmultiple debridements and the technical expertise required to achieve an adequate debridement. In 9 case series,

1	2 to 3 days later. (C) Retroperitoneal cavity seen through endoscope during VARD.BCmultiple debridements and the technical expertise required to achieve an adequate debridement. In 9 case series, mortality in a total of 65 patients was 6%.72Debridement of necrosis through a lumbar approach has been advocated by a number of authors. This approach, devel-oped with experience in a large number of patients,73 has been subjected to a single-center, randomized, prospective trial.74 This approach includes delay of intervention when possible until 4 weeks after the onset of disease. Patients receive transgastric or preferably retroperitoneal drainage of the sequestrum. If patients do not improve over 72 hours, they are treated with video-assisted retroperitoneal drainage (VARD), consisting of dilation of the retroperitoneal drain tract and debridement of the pancreatic bed (Fig. 6-3). Repeat debridements are performed as clinically indi-cated, with most patients requiring multiple

1	of dilation of the retroperitoneal drain tract and debridement of the pancreatic bed (Fig. 6-3). Repeat debridements are performed as clinically indi-cated, with most patients requiring multiple debridements. In the trial reported, patients randomized to VARD (n = 43) compared to those randomized to the standard open necrosectomy (n = 45) had a decreased incidence of the composite endpoint of compli-cations and death (40% vs. 69%), with comparable mortality rate, hospital, and ICU lengths of stay. Patients randomized to VARD had fewer incisional hernias and occurrences of new-onset diabe-tes, as well as less need for pancreatic enzyme supplementation.It is apparent that patients with infected pancreatic necro-sis can safely undergo procedures that are more minimal than the gold-standard open necrosectomy with good outcomes. However, to obtain good outcomes these approaches require an experienced multidisciplinary team consisting of interventional radiologists, gastroenterologists,

1	open necrosectomy with good outcomes. However, to obtain good outcomes these approaches require an experienced multidisciplinary team consisting of interventional radiologists, gastroenterologists, surgeons, and others. Impor-tant concepts for successful management include careful pre-operative planning, delay (if possible) to allow maturation of the fluid collection, and the willingness to repeat procedures as necessary until nonviable tissue has been removed.Infections of the Skin and Soft TissueThese infections can be classified according to whether sur-gical intervention is required. For example, superficial skin and skin structure infections such as cellulitis, erysipelas, and lymphangitis invariably are effectively treated with antibiotics alone, although a search for a local underlying source of infec-tion should be undertaken. Generally, drugs that possess activity against the causative gram-positive skin microflora are selected. Furuncles or boils may drain spontaneously or

1	source of infec-tion should be undertaken. Generally, drugs that possess activity against the causative gram-positive skin microflora are selected. Furuncles or boils may drain spontaneously or require surgical incision and drainage. Antibiotics are prescribed if significant cellulitis is present or if cellulitis does not rapidly resolve after surgical drainage. Community-acquired methicillin-resistant S aureus (MRSA) infection should be suspected if infection persists after treatment with adequate drainage and administra-tion of first-line antibiotics. These infections may require more aggressive drainage and altered antimicrobial therapy.75Aggressive soft tissue infections are rare, difficult to diag-nose, and require immediate surgical intervention plus adminis-tration of antimicrobial agents. Failure to rapidly recognize and treat these infections results in an extremely high mortality rate (∼80–100%), and even with expedient therapy mortality rates are high (16–24%).76 Eponyms

1	agents. Failure to rapidly recognize and treat these infections results in an extremely high mortality rate (∼80–100%), and even with expedient therapy mortality rates are high (16–24%).76 Eponyms and differing classifications in the past has led to a hodgepodge of terminology—such as Meleney’s synergistic gangrene, Fournier’s gangrene, rapidly spreading cellulitis, gas gangrene, and necrotizing fasciitis—regarding these serious infections. Today it seems best to delin-eate them based on the soft tissue layer(s) of involvement 6Brunicardi_Ch06_p0157-p0182.indd 17301/03/19 4:46 PM 174BASIC CONSIDERATIONSPART I(e.g., skin and superficial soft tissue, deep soft tissue, and mus-cle) and the pathogen(s) that cause them.Patients at risk for these types of infections include those who are elderly, immunosuppressed, or diabetic, and/or who suf-fer from peripheral vascular disease, though extremely aggressive necrotizing soft tissue infections (often caused by streptococci) have been

1	elderly, immunosuppressed, or diabetic, and/or who suf-fer from peripheral vascular disease, though extremely aggressive necrotizing soft tissue infections (often caused by streptococci) have been described among healthy individuals as well. The com-mon thread among these host factors appears to be compromise of the fascial blood supply, and if this is coupled with the introduc-tion of exogenous microbes, the result can be devastating.Initially, the diagnosis is established solely upon a constel-lation of clinical findings, not all of which are present in every patient. Not surprisingly, patients often develop sepsis syndrome or septic shock without an obvious cause. The extremities, perineum, trunk, and torso are most commonly affected, in that order. Careful examination should be undertaken for an entry site such as a small break or sinus in the skin from which grayish, turbid semipurulent material (“dishwater pus”) can be expressed, as well as for the presence of skin changes

1	for an entry site such as a small break or sinus in the skin from which grayish, turbid semipurulent material (“dishwater pus”) can be expressed, as well as for the presence of skin changes (bronze hue or brawny induration), blebs, or crepitus. The patient often develops pain at the site of infection that appears to be out of proportion to any of the physical manifestations. Any of these findings man-dates immediate surgical intervention, which should consist of incision and direct visualization of potentially infected tissue (including deep soft tissue, fascia, and underlying muscle) and radical resection of affected areas. Radiologic studies should not be undertaken in patients in whom the diagnosis seriously is con-sidered, as they delay surgical intervention and frequently pro-vide confusing information. Unfortunately, surgical extirpation of infected tissue frequently entails amputation and/or disfigur-ing procedures; the surgeon must bear in mind that incomplete procedures are

1	information. Unfortunately, surgical extirpation of infected tissue frequently entails amputation and/or disfigur-ing procedures; the surgeon must bear in mind that incomplete procedures are associated with higher rates of morbidity and mortality and debride all nonviable tissue (Fig. 6-4).During the procedure, a Gram stain should be performed on tissue fluid. Antimicrobial agents directed against gram-positive and gram-negative aerobes and anaerobes (e.g., van-comycin plus a carbapenem), as well as high-dose aqueous penicillin G (16,000,000 to 20,000,000 U/d), the latter to treat clostridial pathogens, should be administered. Approximately 50% of such infections are polymicrobial, the remainder being caused by a single organism such as S pyogenes, P aeruginosa, or C perfringens. The microbiology of these polymicrobial infections is similar to that of secondary microbial peritonitis, with the exception that gram-positive cocci are more commonly encountered. Most patients should be

1	of these polymicrobial infections is similar to that of secondary microbial peritonitis, with the exception that gram-positive cocci are more commonly encountered. Most patients should be returned to the operat-ing room on a scheduled basis to determine if disease progres-sion has occurred. If so, additional resection of infected tissue and debridement should take place. Antibiotic therapy can be refined based on culture and sensitivity results, particularly in the case of monomicrobial soft tissue infections. Hyperbaric oxygen therapy may be of use in patients with infection caused by gas-forming organisms (e.g., C perfringens), although the evidence to support efficacy is limited to underpowered studies and case reports. In the absence of such infection, hyperbaric oxygen therapy has not been shown to be effective.77Postoperative Nosocomial InfectionsSurgical patients are prone to develop a wide variety of nosoco-mial infections during the postoperative period, which include SSIs,

1	been shown to be effective.77Postoperative Nosocomial InfectionsSurgical patients are prone to develop a wide variety of nosoco-mial infections during the postoperative period, which include SSIs, UTIs, pneumonia, and bacteremia. SSIs are discussed ear-lier, and the latter types of nosocomial infections are related to prolonged use of indwelling tubes and catheters for the purpose of urinary drainage, ventilation, and venous and arterial access, respectively.The presence of a postoperative UTI should be considered based on urinalysis demonstrating WBCs or bacteria, a positive test for leukocyte esterase, or a combination of these elements. The diagnosis is established after >104 CFU/mL of microbes are identified by culture techniques in symptomatic patients, or >105 CFU/mL in asymptomatic individuals. Treatment for 3 to 5 days with a single antibiotic directed against the most common organ-isms (e.g., E Coli, K pneumoniae) that achieves high levels in the urine is appropriate. Initial

1	individuals. Treatment for 3 to 5 days with a single antibiotic directed against the most common organ-isms (e.g., E Coli, K pneumoniae) that achieves high levels in the urine is appropriate. Initial therapy is directed by Gram stain results and is refined as culture results become available. Postop-erative surgical patients should have indwelling urinary catheters removed as quickly as possible to avoid the development of a UTI.Prolonged mechanical ventilation is associated with nos-ocomial pneumonia. These patients present with more severe disease, are more likely to be infected with drug-resistant pathogens, and suffer increased mortality compared to patients who develop community-acquired pneumonia. The diagnosis of pneumonia is established by presence of purulent sputum, elevated leukocyte count, fever, and new chest X-ray abnor-malities, such as consolidation. The presence of two of the clini-cal findings, plus chest X-ray findings, significantly increases the likelihood of

1	leukocyte count, fever, and new chest X-ray abnor-malities, such as consolidation. The presence of two of the clini-cal findings, plus chest X-ray findings, significantly increases the likelihood of pneumonia.78 Consideration should be given to performing bronchoalveolar lavage to obtain samples for Gram stain and culture. Some authors advocate quantitative cultures as a means to identify a threshold for diagnosis.79 Surgical patients should be weaned from mechanical ventilation as soon as feasi-ble, based on oxygenation and inspiratory effort, as risk of pneu-monia increases with increased time on mechanical ventilation.Infection associated with indwelling intravascular cathe-ters is a common problem among hospitalized patients. Because of the complexity of many surgical procedures, these devices are increasingly used for physiologic monitoring, vascular access, drug delivery, and hyperalimentation. Among the sev-eral million catheters inserted each year in the United States,

1	these devices are increasingly used for physiologic monitoring, vascular access, drug delivery, and hyperalimentation. Among the sev-eral million catheters inserted each year in the United States, approximately 25% will become colonized, and approximately 5% will be associated with bacteremia. Duration of catheteriza-tion, insertion or manipulation under emergency or nonsterile conditions, use for hyperalimentation, and the use of multilu-men catheters increase the risk of infection. Use of a central line insertion protocol that includes full barrier precautions and chlorhexidine skin prep has been shown to decrease the inci-dence of infection.80 Although no randomized trials have been performed, peripherally inserted central venous catheters have a catheter-related infection rate similar to those inserted in the subclavian or jugular veins.81Many patients who develop intravascular catheter infec-tions are asymptomatic, often exhibiting solely an elevation in the blood WBC count.

1	to those inserted in the subclavian or jugular veins.81Many patients who develop intravascular catheter infec-tions are asymptomatic, often exhibiting solely an elevation in the blood WBC count. Blood cultures obtained from a peripheral site and drawn through the catheter that reveals the presence of the same organism increase the index of suspicion for the pres-ence of a catheter infection. Obvious purulence at the exit site of the skin tunnel, severe sepsis syndrome due to any type of organism when other potential causes have been excluded, or bacteremia due to gram-negative aerobes or fungi should lead to catheter removal. Selected catheter infections due to low-virulence microbes such as S epidermidis can be effectively treated in approximately 50% to 60% of patients with a 14to 21-day course of an antibiotic, which should be considered when no other vascular access site exists.82 The use of antibi-otic-bonded catheters and chlorhexidine sponges at the insertion

1	with a 14to 21-day course of an antibiotic, which should be considered when no other vascular access site exists.82 The use of antibi-otic-bonded catheters and chlorhexidine sponges at the insertion Brunicardi_Ch06_p0157-p0182.indd 17401/03/19 4:46 PM 175SURGICAL INFECTIONSCHAPTER 6FIGURE 6-4. Necrotizing soft tissue infection. (A) This patient presented with hypotension due to severe late necrotizing fasci-itis and myositis due to β-hemolytic streptococcal infection. The patient succumbed to his disease after 16 hours despite aggressive debridement. (B) This patient presented with spreading cellulites and pain on motion of his right hip 2 weeks after total colectomy. Cellulitis on right anterior thigh is outlined. (C) Classic dishwater edema of tissues with necrotic fascia. (D) Right lower extremity after debridement of fascia to viable muscle.site has been associated with lower rates of colonization.83 Use of ethanol or antimicrobial catheter “locks” have shown prom-ise in

1	Right lower extremity after debridement of fascia to viable muscle.site has been associated with lower rates of colonization.83 Use of ethanol or antimicrobial catheter “locks” have shown prom-ise in reducing incidence of infection in dialysis catheters.84 The surgeon should carefully consider the need for any type of vascular access devices, rigorously attend to their maintenance to prevent infection, and remove them as quickly as possible. Use of systemic antibacterial or antifungal agents to prevent catheter infection is of no utility and is contraindicated.SepsisAs previously discussed, sepsis is increasing in incidence, with more than 1.1 million cases estimated per year in the United States with an annual cost of $24 billion. This rate is expected to increase as the population of aged in the United States increases. One third of sepsis cases occur in surgical pop-ulations and sepsis is a major cause of morbidity and mortality.85 The treatment of sepsis has improved over the

1	aged in the United States increases. One third of sepsis cases occur in surgical pop-ulations and sepsis is a major cause of morbidity and mortality.85 The treatment of sepsis has improved over the last decade, with mortality rates dropping to under 30%. Factors contributing to this improvement relate both to recent randomized prospective trials demonstrating improved outcomes with new therapies, and to improvements in the process of care delivery to the sepsis patient. The “Surviving Sepsis Campaign,” a multidisciplinary group that develops treatment recommendations, published guidelines incorporating evidence-based sepsis treatment strate-gies most recently in 2016.15,86 These guidelines are summarized in Table 6-10.ABCDBrunicardi_Ch06_p0157-p0182.indd 17501/03/19 4:46 PM 176BASIC CONSIDERATIONSPART IPatients presenting with sepsis should receive resuscitation fluids early in the course of therapy. While former guidelines advocated fluids until the patient’s central venous

1	CONSIDERATIONSPART IPatients presenting with sepsis should receive resuscitation fluids early in the course of therapy. While former guidelines advocated fluids until the patient’s central venous pressure was 8 to 12 mmHg, newer guidelines recommend using dynamic monitoring systems (such as ultrasound) as well as assessment of physiological response to fluids by evaluating variables such as heart rate, blood pressure, and urine output to determine ade-quate resuscitation volumes. Resuscitation endpoints include achieving a goal mean arterial pressure of ≥65 mmHg, urine output of ≥0.5 mL/kg per hour, and normalization of serum lac-tate. Delaying this resuscitative step for as little as 3 hours has been shown to result in worse outcomes.87 Resuscitation may necessitate placement of a central venous catheter.A number of studies have demonstrated the importance of early empiric antibiotic therapy in patients who develop sep-sis or nosocomial infection; the Surviving Sepsis guidelines

1	venous catheter.A number of studies have demonstrated the importance of early empiric antibiotic therapy in patients who develop sep-sis or nosocomial infection; the Surviving Sepsis guidelines advocate for initiation of treatment within the first hour of the patient’s care. This therapy should be initiated as soon as pos-sible with broad-spectrum antibiotics directed against the most likely organisms. Use of institutionand unit-specific sensitivity patterns are critical in selecting an appropriate agent for patients with nosocomial infection. Obtain appropriate cultures before Table 6-10Summary of Surviving Sepsis Campaign guidelinesInitial Evaluation and Infection IssuesInitial resuscitation: Begin resuscitation immediately in patients with hypotension or elevated serum lactate with resuscitation goal of at least 30 mL/kg IV crystalloid given in the first 3 hours.Ongoing fluid administration should be guided by physiologic response as measured by clinical variables (e.g., heart

1	resuscitation goal of at least 30 mL/kg IV crystalloid given in the first 3 hours.Ongoing fluid administration should be guided by physiologic response as measured by clinical variables (e.g., heart rate, blood pressure, urine output) and/or other invasive or noninvasive monitoring.Resuscitation goals include mean arterial pressure >65 mmHg, urine output >0.5 mL/kg per h, and mixed venous oxygen saturation >65%.Target resuscitation to normalize lactate in patients with elevated lactate levels.Diagnosis: Obtain appropriate cultures prior to antibiotics, but do not delay antibiotic therapy. Imaging studies should be performed promptly to confirm a source of infection.Antibiotic therapy: Begin IV antibiotic therapy as early as possible and within the first hour after recognition of severe sepsis/septic shock. Use broad spectrum antibiotic regimen with penetration into presumed source, reassess regimen daily with de-escalation as appropriate, discontinue antibiotics in 7 to 10 days for

1	sepsis/septic shock. Use broad spectrum antibiotic regimen with penetration into presumed source, reassess regimen daily with de-escalation as appropriate, discontinue antibiotics in 7 to 10 days for most infections, stop antibiotics for noninfectious issues. Consider the use of serial procalcitonin levels, which may allow earlier cessation of antibiotic therapy.Source control: Establish anatomic site of infection as rapidly as possible; implement source control measures as soon as possible after initial resuscitation. Remove intravascular access devices if potentially infected.Hemodynamic Support and Adjunctive TherapyFluid therapy: Fluid resuscitate using crystalloid, with continued fluid challenges so long as hemodynamic parameters continue to improve (i.e., for so long as the patient remains fluid-responsive). Albumin may be used as an adjunct if large volumes of crystalloid are required, but hydroxyethyl starch and gelatin-based fluids should not be used.Vasopressors/Inotropic

1	remains fluid-responsive). Albumin may be used as an adjunct if large volumes of crystalloid are required, but hydroxyethyl starch and gelatin-based fluids should not be used.Vasopressors/Inotropic Therapy: Maintain MAP of >65 mmHg. Centrally-administered norepinephrine is the first-line choice. Add vasopressin if needed to raise MAP or to reduce norepinephrine requirement. Epinephrine is an alternative to vasopressin but has greater risk of reduced splanchnic blood flow. Dopamine is an appropriate alternative only in select patients (bradycardia, low risk of arrhythmia), and there is no role for low-dose “renal protection” dopamine. Phenylephrine is not recommended. Insert arterial catheters for patients requiring vasopressors. Consider dobutamine infusion for persistent hypotension after appropriate resuscitation and use of vasopressor agents.Steroids: Consider intravenous hydrocortisone (dose <300 mg/day) for adult septic shock when hypotension responds poorly to fluids and

1	after appropriate resuscitation and use of vasopressor agents.Steroids: Consider intravenous hydrocortisone (dose <300 mg/day) for adult septic shock when hypotension responds poorly to fluids and vasopressors.Other Supportive TherapyBlood product administration: Transfuse red blood cells when hemoglobin decreases to <7.0 g/dL in the absence of extenuating circumstances (e.g., myocardial ischemia, hemorrhage). It is not necessary to use fresh frozen plasma to correct INR abnormalities in the absence of bleeding. Consider prophylactic transfusion of platelets when counts are less than 10,000/mL in the absence of bleeding, <20,000/mL if there is a risk of bleeding, and <50,000 in the setting of active bleeding or need for procedure.Mechanical ventilation: Target an initial tidal volume of 6 mL/kg body weight and plateau pressure of <30 cm H2O in patients with acute lung injury. Use PEEP to avoid lung collapse. Adopt a conservative fluid strategy. In the setting of sepsis-induced ARDS

1	6 mL/kg body weight and plateau pressure of <30 cm H2O in patients with acute lung injury. Use PEEP to avoid lung collapse. Adopt a conservative fluid strategy. In the setting of sepsis-induced ARDS with PaO2/FiO2 ratio <150, use prone ventilation over continued supine position or high-frequency oscillatory ventilation. Use a weaning protocol to evaluate the potential for discontinuing mechanical ventilation. Pulmonary artery catheter placement is not indicated for routine monitoring.Sedation: Minimize sedation using specific titration endpoints.Glucose control: Use protocolized approach to blood glucose management targeting upper blood glucose target of 180 mg/dL.Prophylaxis: Use stress ulcer (proton pump inhibitor or H2 blocker) and deep venous thrombosis (low-dose unfractionated or fractionated heparin) prophylaxis.Limitation of support: Discuss advance care planning with patients and families and set realistic expectations.Data from Rhodes A, Evans LE, Alhazzani W, et al:

1	or fractionated heparin) prophylaxis.Limitation of support: Discuss advance care planning with patients and families and set realistic expectations.Data from Rhodes A, Evans LE, Alhazzani W, et al: Surviving Sepsis Campaign: International Guidelines for Management of Sepsis and Septic Shock: 2016, Intensive Care Med. 2017 Mar;43(3):304-377.Brunicardi_Ch06_p0157-p0182.indd 17601/03/19 4:46 PM 177SURGICAL INFECTIONSCHAPTER 6starting antibiotics so that appropriate de-escalation of therapy can take place when results return, but only if doing so does not delay the initiation of treatment.In patients who require vasopressor therapy, the first-line agent should be norepinephrine. This can be augmented with vasopressin, if needed, to achieve MAP ≥65 mmHg. It is impor-tant to titrate therapy based on other parameters such as mixed venous oxygen saturation and plasma lactate levels to reduce the risk of vasopressor-induced perfusion deficits. Patients who have persistently poor perfusion

1	based on other parameters such as mixed venous oxygen saturation and plasma lactate levels to reduce the risk of vasopressor-induced perfusion deficits. Patients who have persistently poor perfusion despite adequate fluid resus-citation may require addition of inotropic agents (epinephrine, dobutamine) or adjunctive therapy with low-dose corticoste-roids (hydrocortisone 200 mg/day).86Patients with acute lung injury associated with sepsis should receive mechanical ventilation with tidal volumes of 6 mL/kg and pulmonary airway plateau pressures of ≤30 cm H2O. Finally, red blood cell transfusion should be reserved for patients with hemoglobin of <7 g/dL, with a more liberal trans-fusion strategy reserved for those patients with severe coronary artery disease, ongoing blood loss, or severe hypoxemia.86Resistant OrganismsPenicillin was first available for widespread clinical use in the 1940s, and within a year resistant strains of S aureus had emerged. There are two major factors

1	severe hypoxemia.86Resistant OrganismsPenicillin was first available for widespread clinical use in the 1940s, and within a year resistant strains of S aureus had emerged. There are two major factors responsible for antibiotic resistance. First, there may be a genetic component innate to an organism that prevents the effect of a particular antibiotic. For instance, if an organism does not have a target receptor specific to the mechanism of action of a particular antibiotic, the antibi-otic will not be effective against this organism. A good example is penicillin and gram-negative organisms, as these microbes lack penicillin-binding proteins. The second component driv-ing resistance is inducible and related to natural selection. Over generations of exposure to a particular antibiotic, selection pres-sure will drive proliferation of more organisms resistant to that antibiotic. This acquired antibiotic resistance can be mutational, leading to changes in the chromosomal makeup of the

1	selection pres-sure will drive proliferation of more organisms resistant to that antibiotic. This acquired antibiotic resistance can be mutational, leading to changes in the chromosomal makeup of the microbe, or it can be extrachromosomal, induced by transfer of exog-enous genetic material in the form of a plasmid or transposon. In either case, cellular mechanisms of resistance that develop include target site modification, changes in bacterial permeabil-ity or antibiotic uptake, activation of drug efflux systems, and drug deactivation. Given that millions of kilograms of antibiot-ics are used annually in people, in agriculture, and for animal use, environmental selection pressures are high, and antibiotic resistance has now been described in all classes of antibiotics in common use. Antibiotic resistance comes at a high cost, with a significant increase in mortality associated with infection from resistant organisms, and an economic cost of billions of dollars per year.There are

1	Antibiotic resistance comes at a high cost, with a significant increase in mortality associated with infection from resistant organisms, and an economic cost of billions of dollars per year.There are several drug-resistant organisms of interest to the surgeon. MRSA most commonly occurs as a hospitalassociated infection in chronically ill patients who have received multiple courses of antibiotics. However, strains of MRSA have emerged in the community among patients without preexisting risk factors for disease.75 These strains, which produce a toxin known as Panton-Valentine leukocidin, make up an increasingly high percentage of surgical site infections since they are resis-tant to commonly employed prophylactic antimicrobial agents.88 Extended spectrum β-lactamase (ESBL)-producing strains of enterobacteriaceae, originally geographically localized and infrequent, have become much more widespread and common in the last decade.89 These strains, typically Klebsiella species or E coli,

1	of enterobacteriaceae, originally geographically localized and infrequent, have become much more widespread and common in the last decade.89 These strains, typically Klebsiella species or E coli, produce a plasmid-mediated inducible β-lactamase. Commonly encountered plasmids also confer resistance to many other antibiotic classes. A common laboratory finding with ESBL is sensitivity to first-, second-, or third-generation cephalosporins, with resistance to other agents. Unfortunately, use of this seemingly active agent leads to rapid induction of resistance and failure of antibiotic therapy. The appropriate anti-biotic choice in this setting is a carbapenem.While Enterococcus was considered a low-virulence organ-ism in the past, infections caused by E faecium and faecalis have been found to be increasingly severe, especially in the immu-nocompromised host. The last decade has seen increased iso-lation of a vancomycin-resistant strain of Enterococcus. This resistance is

1	been found to be increasingly severe, especially in the immu-nocompromised host. The last decade has seen increased iso-lation of a vancomycin-resistant strain of Enterococcus. This resistance is transposon-mediated via the vanA gene and is typically seen in E faecium strains. A real infection control con-cern is potential for transfer of genetic material to S aureus in a host coinfected with both organisms. This is thought to be the mechanism behind emerging cases of vancomycin resistance in S aureus.90Blood-Borne PathogensThe risk of human immunodeficiency virus (HIV) transmission from patient to surgeon is low. As of May 2011, there had been six cases of surgeons with HIV seroconversion from a possible occupational exposure, with no new cases reported since 1999. Of the numbers of healthcare workers with likely occupationally acquired HIV infection (n = 200), surgeons were one of the lower risk groups (compared to nurses at 60 cases and nonsur-geon physicians at 19 cases).91 The

1	workers with likely occupationally acquired HIV infection (n = 200), surgeons were one of the lower risk groups (compared to nurses at 60 cases and nonsur-geon physicians at 19 cases).91 The estimated risk of transmis-sion from a needlestick from a source with HIV-infected blood is estimated at 0.3%. Transmission of HIV (and other infections spread by blood and body fluid) from patient to healthcare worker can be minimized by observation of universal precau-tions, including: (a) routine use of barriers (gloves, gown, mask, eye protection) when anticipating contact with blood or body fluids, (b) washing hands and other skin surfaces immediately after contact with blood or body fluids, and (c) careful handling and disposal of sharp instruments during and after use.Postexposure prophylaxis for HIV has significantly decreased the risk of seroconversion for healthcare workers with occupational exposure to HIV. Steps to initiate postexposure prophylaxis should be initiated within hours for

1	HIV has significantly decreased the risk of seroconversion for healthcare workers with occupational exposure to HIV. Steps to initiate postexposure prophylaxis should be initiated within hours for the most effec-tive preventive therapy. Postexposure prophylaxis with a three-drug regimen should be initiated for healthcare workers with significant exposure to patients with an HIV-positive status. If a patient’s HIV status is unknown, it may be advisable to begin postexposure prophylaxis while testing is carried out, particu-larly if the patient is at high risk for infection due to HIV (e.g., has had a history of intravenous drug use). Generally, postexpo-sure prophylaxis is not warranted for exposure to sources with unknown status, such as deceased persons or needles from a sharps container.92The risks of acquiring HIV infection for surgeons are related to the prevalence of HIV infection in the patient popula-tion, the probability of transmission from a percutaneous injury suffered

1	risks of acquiring HIV infection for surgeons are related to the prevalence of HIV infection in the patient popula-tion, the probability of transmission from a percutaneous injury suffered while caring for an infected patient, the number of such injuries sustained, and the use of postexposure prophylaxis. Average risk of HIV seroconversion is 0.3% from a percutane-ous exposure, and 0.09% from a mucous membrane exposure. The overall risk is influenced by the degree of viral inoculum 7Brunicardi_Ch06_p0157-p0182.indd 17701/03/19 4:46 PM 178BASIC CONSIDERATIONSPART Itransmitted from patient to surgeon, with greater risk of sero-conversion associated with hollow-bore needle injury, with larger-volume blood transmission, with direct introduction of infected blood into an artery or vein, and in exposure to blood with higher viral load. One study in Glasgow, Scotland, cal-culated annual risks and found a range in seroconversion rates from 1 in 200,000 for general surgeons not utilizing

1	in exposure to blood with higher viral load. One study in Glasgow, Scotland, cal-culated annual risks and found a range in seroconversion rates from 1 in 200,000 for general surgeons not utilizing postexpo-sure prophylaxis to as low as 1 in 10,000,000 with use of routine postexposure prophylaxis after significant exposures.92,93Hepatitis B virus (HBV) is a DNA virus that affects only humans. Primary infection with HBV generally is self-limited, but it can cause fulminant hepatitis or progress to a chronic car-rier state. Death from chronic liver disease or hepatocellular cancer occurs in roughly 30% of chronically infected persons. Surgeons and other healthcare workers are at high risk for this blood-borne infection and should receive the HBV vaccine; children are routinely vaccinated in the United States.94 This vaccine has contributed to a significant decline in the number of new cases of HBV per year in the United States, from approxi-mately 250,000 annually in the 1980s to 3350 in

1	United States.94 This vaccine has contributed to a significant decline in the number of new cases of HBV per year in the United States, from approxi-mately 250,000 annually in the 1980s to 3350 in 2010.95,96Hepatitis C virus (HCV), previously known as non-A, non-B hepatitis, is a RNA flavivirus first identified in the late 1980s. This virus is confined to humans and chimpanzees. A chronic carrier state develops in 75% to 80% of patients with the infection, with chronic liver disease occurring in three-fourths of this subgroup. The number of new infections per year has declined since the 1980s due to routine testing of blood donors for the virus. Fortunately, HCV is not transmitted efficiently through occupational exposures to blood, with the seroconver-sion rate after accidental needlestick approximately 1.8%.97 To date, a vaccine to prevent HCV infection has not been devel-oped. Experimental studies in chimpanzees with HCV immu-noglobulin using a model of needlestick injury have

1	approximately 1.8%.97 To date, a vaccine to prevent HCV infection has not been devel-oped. Experimental studies in chimpanzees with HCV immu-noglobulin using a model of needlestick injury have failed to demonstrate a protective effect, and no effective antiviral agents for postexposure prophylaxis are available. Treatment of patients with HCV infection historically included ribavirin and pegylated gamma interferon; the development of novel direct-acting antiviral agents such as sofosbuvir, boceprevir, and tela-previr has led to changes in this strategy.98,99BIOLOGIC WARFARE AGENTSSeveral infectious organisms have been studied by the United States and the former Soviet Union and presumably other entities for potential use as biologic weapons. Programs involving biologic agents in the United States were halted by presidential decree in 1971. However, concern remains that these agents could be used by rogue states or terrorist organi-zations as weapons of mass destruction, as they are

1	States were halted by presidential decree in 1971. However, concern remains that these agents could be used by rogue states or terrorist organi-zations as weapons of mass destruction, as they are relatively inexpensive to make in terms of infrastructure development. Given these concerns, physicians, including surgeons, should familiarize themselves with the manifestations of infection due to these pathogens. The typical agent is selected for the ability to be spread via the inhalational route, as this is the most efficient mode of mass exposure. Several potential agents are discussed in the following sections.Bacillus anthracis (Anthrax)Anthrax is a zoonotic disease occurring in domesticated and wild herbivores. The first identification of inhalational anthrax as a disease occurred among woolsorters in England in the late 1800s. The largest recent epidemic of inhalational anthrax occurred in 1979 in Sverdlovsk, Russia, after accidental release of anthrax spores from a military

1	among woolsorters in England in the late 1800s. The largest recent epidemic of inhalational anthrax occurred in 1979 in Sverdlovsk, Russia, after accidental release of anthrax spores from a military facility. Inhalational anthrax develops after a 1to 6-day incubation period, with nonspe-cific symptoms, including malaise, myalgia, and fever. Over a short period of time these symptoms worsen, with development of respiratory distress, chest pain, and diaphoresis. Character-istic chest roentgenographic findings include a widened medi-astinum and pleural effusions. Rapid antigen tests are under development for identification of this gram-positive rod, so a key element of establishing the diagnosis is eliciting an expo-sure history. Postexposure prophylaxis consists of administra-tion of either ciprofloxacin or doxycycline.100 If an isolate is demonstrated to be penicillin-sensitive, the patient should be switched to amoxicillin. Inhalational exposure followed by the development of symptoms

1	ciprofloxacin or doxycycline.100 If an isolate is demonstrated to be penicillin-sensitive, the patient should be switched to amoxicillin. Inhalational exposure followed by the development of symptoms is associated with a high mortality rate. Treatment options include combination therapy with cip-rofloxacin, clindamycin, and rifampin. Clindamycin is added to block toxin production, while rifampin penetrates into the central nervous system and intracellular locations.Yersinia pestis (Plague)Plague is caused by the gram-negative organism Y pestis. The naturally occurring disease in humans is transmitted via flea bites from rodents. It was the first biologic warfare agent, and was used in the Crimean city of Caffa by the Tartar army, whose soldiers catapulted bodies of plague victims at the Genoese. When plague is used as a biologic warfare agent, clinical manifestations include epidemic pneumonia with blood-tinged sputum if aerosolized bacteria are used, or bubonic plague if fleas are

1	Genoese. When plague is used as a biologic warfare agent, clinical manifestations include epidemic pneumonia with blood-tinged sputum if aerosolized bacteria are used, or bubonic plague if fleas are used as carriers. Individuals who develop a painful enlarged lymph node lesion, termed a “bubo,” associ-ated with fever, severe malaise, and exposure to fleas should be suspected to have plague. Diagnosis is confirmed via aspirate of the bubo and a direct antibody stain to detect plague bacil-lus, whose morphology is a bipolar, safety-pin-shaped gram-negative rod. Postexposure prophylaxis for patients exposed to plague consists of doxycycline. Treatment of the pneumonic or bubonic/septicemic form includes administration of either strep-tomycin, an aminoglycoside, doxycycline, a fluoroquinolone, or chloramphenicol.101SmallpoxVariola, the causative agent of smallpox, was a major cause of infectious morbidity and mortality until its eradication in the late 1970s. Even in the absence of

1	or chloramphenicol.101SmallpoxVariola, the causative agent of smallpox, was a major cause of infectious morbidity and mortality until its eradication in the late 1970s. Even in the absence of laboratory-preserved virus, the prolonged viability of variola virus has been dem-onstrated in scabs up to 13 years after collection. The potential for reverse genetic engineering using the known sequence of smallpox also makes it a potential biologic weapon. This has resulted in the United States undertaking a vaccination program for key healthcare workers.102 Variola virus is highly infectious in the aerosolized form; after an incubation period of 10 to 12 days, clinical manifestations of malaise, fever, vomiting, and headache appear, followed by development of a characteristic centripetal rash (which is found to predominate on the face and extremities). The fatality rate may reach 30%. Postexposure prophylaxis with smallpox vaccine has been noted to be effec-tive for up to 4 days

1	rash (which is found to predominate on the face and extremities). The fatality rate may reach 30%. Postexposure prophylaxis with smallpox vaccine has been noted to be effec-tive for up to 4 days postexposure. Cidofovir, an acyclic nucleo-side phosphonate analogue, has demonstrated activity in animal models of poxvirus infections and may offer promise for the treatment of smallpox.103Brunicardi_Ch06_p0157-p0182.indd 17801/03/19 4:46 PM 179SURGICAL INFECTIONSCHAPTER 6Francisella tularensis (Tularemia)The principal reservoir of this gram-negative aerobic organism is the tick. After inoculation, this organism proliferates within macrophages. Tularemia is considered a potential bioterrorist threat due to a very high infectivity rate after aerosolization. Patients with tularemia pneumonia develop a cough and dem-onstrate pneumonia on chest roentgenogram. Enlarged lymph nodes occur in approximately 85% of patients. The organism can be cultured from tissue samples, but this is difficult,

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1	development of tertiary peritoni-tis on a surgical intensive care unit. J Gastrointest Surg. 2009;13(7):1358-1367. 65. Pang TC, Fung T, Samra J, et al. Pyogenic liver abscess: an audit of 10 years’ experience. World J Gastroenterol. 2011;17(12):1622-1630. 66. Bradley EL III, Allen K. A prospective longitudinal study of observation versus surgical intervention in the management of necrotizing pancreatitis. Am J Surg. 1991;161:19. 67. Charbonney E, Nathens AB. Severe acute pancreatitis: a review. Surg Infect (Larchmt). 2008;9(6):573-578. 68. Freeman ML, Werner J, van Santvoort HC, et al. Interven-tions for necrotizing pancreatitis: summary of a multidis-ciplinary consensus conference. Pancreas. 2012;41(8): 1176-1194. 69. Wysocki AP, McKay CJ, Carter CR. Infected pancreatic necro-sis: minimizing the cut. ANZ J Surg. 2010;80(1-2):58-70. 70. Haghshenasskashani A, Laurence JM, Kwan V, et al. Endo-scopic necrosectomy of pancreatic necrosis: a systematic review. Surg Endosc.

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1	improves outcome. Gastroenterology. 2011;141(4):1254-1263. 74. van Santvoort HC, Besselink MG, Bakker OJ, et al. A step-up approach or open necrosectomy for necrotizing pancreatitis. N Engl J Med. 2010;362(16):1491-1502. A study assessing a minimally invasive approach to pancreatic debridement. 75. Beilman GJ, Sandifer G, Skarda D, et al. Emerging infections with community-associated methicillin-resistant Staphylococ-cus aureus in outpatients at an army community hospital. Surg Infect (Larchmt). 2005;6(1):87-92. 76. Kao LS, Lew DF, Arab SN, et al. Local variations in the epidemiology, microbiology, and outcome of necrotizing soft-tissue infections: a multicenter study. Am J Surg. 2011; 202(2):139-145. 77. George ME, Rueth NM, Skarda DE, et al. Hyperbaric oxygen does not improve outcome in patients with necrotizing soft tissue infection. Surg Infect (Larchmt). 2009;10(1):21-28. 78. Klompas M. Does this patient have ventilator-associated pneu-monia? JAMA. 2007

1	not improve outcome in patients with necrotizing soft tissue infection. Surg Infect (Larchmt). 2009;10(1):21-28. 78. Klompas M. Does this patient have ventilator-associated pneu-monia? JAMA. 2007 11;297(14):1583-1593. 79. Riaz OJ, Malhotra AK, Aboutanos MB, et al. Bronchoal-veolar lavage in the diagnosis of ventilator-associated pneu-monia: to quantitate or not, that is the question. Am Surg. 2011;77(3):297-303. 80. O’Grady NP, Alexander M, Burns LA, et al. Guidelines for the prevention of intravascular catheter-related infections. Clin Infect Dis. 2011;52(9):e162-e193. 81. Safdar N, Maki DG. Risk of catheter-related bloodstream infection with peripherally inserted central venous catheters used in hospitalized patients. Chest. 2005;128(2):489-495. 82. Marr KA, Sexton DJ, Conlon PJ, et al. Catheter-related bac-teremia and outcome of attempted catheter salvage in patients undergoing hemodialysis. Ann Intern Med. 1997;127:275. 83. O’Grady NP, Alexander M, Burns LA, et al. Guidelines for

1	Catheter-related bac-teremia and outcome of attempted catheter salvage in patients undergoing hemodialysis. Ann Intern Med. 1997;127:275. 83. O’Grady NP, Alexander M, Burns LA, et al. Guidelines for the prevention of intravascular catheter-related infections. Clin Infect Dis. 2011;52(9):e162-e193. 84. Broom JK, Krishnasamy R, Hawley CM, et al. A randomised controlled trial of Heparin versus EthAnol Lock THerapY for the prevention of Catheter Associated infecTion in Haemo-dialysis patients—the HEALTHY-CATH trial. BMC Nephrol. 2012;13:146. 85. Moore LJ, Moore FA. Epidemiology of sepsis in surgical patients. Surg Clin North Am. 2012;92(6):1425-1443. 86. Rhodes A, Evans L, Alhazzani W, et al. Surviving Sepsis campaign: international guidelines for management of sepsis and septic shock: 2016. Intensive Care Med. 2017;43:304-377. Updated recommendations and best practice guidelines. 87. Otero RM, Nguyen HB, Huang DT, et al. Early goal-directed therapy in severe sepsis and septic shock

1	Intensive Care Med. 2017;43:304-377. Updated recommendations and best practice guidelines. 87. Otero RM, Nguyen HB, Huang DT, et al. Early goal-directed therapy in severe sepsis and septic shock revisited: con-cepts, controversies, and contemporary findings. Chest. 2006;130(5):1579-1595. 88. Miller LG, McKinnell JA, Vollmer ME, Spellberg B. Impact of methicillin-resistant Staphylococcus aureus prevalence among S aureus isolates on surgical site infection risk after coronary artery bypass surgery. Infect Control Hosp Epide-miol. 2011;32(4):342-350. 89. Han JH, Nachamkin I, Zaoutis TE, et al. Risk factors for gastrointestinal tract colonization with extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Kleb-siella species in hospitalized patients. Infect Control Hosp Epidemiol. 2012;33(12):1242-1245. 90. Calfee DP. Methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, and other Gram-positives in healthcare. Curr Opin Infect Dis.

1	Hosp Epidemiol. 2012;33(12):1242-1245. 90. Calfee DP. Methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, and other Gram-positives in healthcare. Curr Opin Infect Dis. 2012;25(4):385-394. 91. Centers for Disease Control and Prevention. Surveillance of occupationally acquired HIV/AIDS in healthcare personnel, as of December 2010. Available at https://www.cdc.gov/HAI/organisms/hiv/Surveillance-Occupationally-Acquired-HIV-AIDS.html. Accessed July 31, 2017. 92. Updated U.S. Public Health Service guidelines for the manage-ment of occupational exposures to HIV and recommendations for postexposure prophylaxis. Downloaded from Centers for Disease Control and Prevention, Human Immunodeficiency Virus in Healthcare Settings, https://www.cdc.gov/hai/organ-isms/hiv/hiv.html. Accessed July 31, 2017. 93. Goldberg D, Johnston J, Cameron S, et al. Risk of HIV trans-mission from patients to surgeons in the era of post-exposure prophylaxis. J Hosp Infect.

1	Accessed July 31, 2017. 93. Goldberg D, Johnston J, Cameron S, et al. Risk of HIV trans-mission from patients to surgeons in the era of post-exposure prophylaxis. J Hosp Infect. 2000;44:99-105. 94. Recommended Adult Immunization Schedule-United States. Available at: http://www.cdc.gov/vaccines/schedules/hcp/adult.html. Accessed July 31, 2017. 95. Centers for Disease Control and Prevention. Hepatitis B vaccination–United States, 1982–2002. MMWR. 2002;51:549. 96. Centers for Disease Control, Viral hepatitis statistics and surveillance. Available at http://www.cdc.gov/hepatitis/Statistics/2010Surveillance/Table3.1.htm. Accessed July 31, 2017. 97. MacCannell T, Laramie AK, Gomaa A, Perz JF. Occupational exposure of health care personnel to hepatitis B and hepatitis C: prevention and surveillance strategies. Clin Liver Dis. 2010; 14(1):23-36. 98. Katz LH, Goldvaser H, Gafter-Gvili A, Tur-Kaspa R. Extended peginterferon plus ribavirin treatment for 72 weeks versus standard peginterferon

1	strategies. Clin Liver Dis. 2010; 14(1):23-36. 98. Katz LH, Goldvaser H, Gafter-Gvili A, Tur-Kaspa R. Extended peginterferon plus ribavirin treatment for 72 weeks versus standard peginterferon plus ribavirin treatment for 48 weeks in chronic hepatitis C genotype 1 infected slow-responder adult patients. Cochrane Database Syst Rev. 2012;9:CD008516. 99. Cholongitas E, Papatheodoridis GV. Sofosbuvir: a novel oral agent for chronic hepatitis C. Ann Gastroenterol. 2014;27(4):331-337. 100. Inglesby TV, O’Toole T, Henderson DA, et al. Anthrax as a biological weapon, 2002: updated recommendations for man-agement. JAMA. 2002;287:2236-2252. 101. Inglesby TV, Dennis DT, Henderson DA, et al. Plague as a bio-logical weapon; medical and public health management. Work-ing group on civilian biodefense. JAMA. 2000;283:2281-2290. 102. Russell PK, Gronvall GK. U.S. medical countermeasure devel-opment since 2001: a long way yet to go. Biosecur Bioterror. 2012;10(1):66-76. 103. DeClercq E. Cidofovir in

1	JAMA. 2000;283:2281-2290. 102. Russell PK, Gronvall GK. U.S. medical countermeasure devel-opment since 2001: a long way yet to go. Biosecur Bioterror. 2012;10(1):66-76. 103. DeClercq E. Cidofovir in the treatment of poxvirus infections. Antiviral Res. 2002;55:1-13.Brunicardi_Ch06_p0157-p0182.indd 18101/03/19 4:46 PM

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1	TraumaClay Cothren Burlew and Ernest E. Moore 7chapterINTRODUCTIONTrauma, or injury, is defined as cellular disruption caused by environmental energy that is beyond the body’s resilience, which is compounded by cell death due to ischemia/reperfu-sion. Trauma is the most common cause of death for all indi-viduals between the ages of 1 and 44 years, and is the third most common cause of death regardless of age.1 It is also the leading cause of years of productive life lost. Unin-tentional injuries account for over 135,000 deaths per year, with homicides, suicides, and other causes are responsible for another 60,000 deaths each year. However, death rate under-estimates the magnitude of the societal toll. For example, in 2014 there were almost 200,000 injury-related deaths, but 37.2 million injured patients treated in emergency depart-ments (EDs). Injury-related medical expenditures are esti-mated to be $117 billion each year in the United States.2 The aggregate lifetime cost for all

1	injured patients treated in emergency depart-ments (EDs). Injury-related medical expenditures are esti-mated to be $117 billion each year in the United States.2 The aggregate lifetime cost for all injured patients is estimated to be in excess of $260 trillion. For these reasons, trauma must be considered a major public health issue. The American Col-lege of Surgeons Committee on Trauma addresses this issue by assisting in the development of trauma centers and sys-tems. The organization of trauma systems has had a signifi-cant favorable impact on patient outcomes, although system integration and maldistribution of trauma centers remain chal-lenges.3-5INITIAL EVALUATION AND RESUSCITATION OF THE INJURED PATIENTPrimary SurveyThe Advanced Trauma Life Support (ATLS) course of the American College of Surgeons Committee on Trauma was developed in the late 1970s, based on the premise that appro-priate and timely care can improve the outcome for the injured 1patient.6 ATLS provides a structured

1	of Surgeons Committee on Trauma was developed in the late 1970s, based on the premise that appro-priate and timely care can improve the outcome for the injured 1patient.6 ATLS provides a structured approach to the trauma patient with standard algorithms of care; it emphasizes the “golden hour” concept that timely, prioritized interventions are necessary to prevent death and disability. The ATLS for-mat and basic tenets are followed throughout this chapter, with some modifications. The initial management of seriously injured patients consists of phases that include the primary survey/concurrent resuscitation, the secondary survey/diag-nostic evaluation, definitive care, and the tertiary survey. The first step in patient management is performing the primary survey, the goal of which is to identify and treat conditions that constitute an immediate threat to life. The ATLS course refers to the primary survey as assessment of the “ABCs” (Airway with cervical spine protection, Breathing,

1	identify and treat conditions that constitute an immediate threat to life. The ATLS course refers to the primary survey as assessment of the “ABCs” (Airway with cervical spine protection, Breathing, and Circulation). The timing of emergent intubation in the hypovolemic patient remains controversial because of the risk of further compro-mising cardiac function. Although the concepts within the primary survey are presented in a sequential fashion, in reality they are pursued simultaneously in coordinated team resuscitation. Life-threatening injuries must be identified (Table 7-1) and treated before progressing to the secondary survey.Airway Management With Cervical Spine Protection. Ensuring a patent airway is the first priority in the primary survey. This is essential because efforts to restore cardio-vascular integrity will be futile unless the oxygen content of the blood is adequate. Simultaneously, all patients with blunt trauma require cervical spine immobilization until injury

1	restore cardio-vascular integrity will be futile unless the oxygen content of the blood is adequate. Simultaneously, all patients with blunt trauma require cervical spine immobilization until injury is excluded. This is typically accomplished by applying a hard cervical collar or placing sandbags on both sides of the head with the patient’s forehead taped across the bags. Soft collars do not effectively immobilize the cervical spine. For penetrating neck wounds, however, cervical collars are not recommended because they provide no benefit and may interfere with assess-ment and treatment.7,82Introduction 183Initial Evaluation and Resuscitation of the Injured Patient 183Primary Survey / 183Secondary Survey / 195Mechanisms and Patterns of Injury / 195Regional Assessment and Special Diagnostic Tests / 196General Principles of Management 205Transfusion Practices / 206Prophylactic Measures / 208Operative Approaches and Exposure / 209Damage Control Surgery / 215Treatment of Specific

1	Tests / 196General Principles of Management 205Transfusion Practices / 206Prophylactic Measures / 208Operative Approaches and Exposure / 209Damage Control Surgery / 215Treatment of Specific Injuries 217Head Injuries / 217Cervical Injuries / 219Chest Injuries / 222Abdominal Injuries / 225Pelvic Fracture Hemorrhage Control / 234Extremity Vascular Injuries, Fractures, and Compartment Syndromes / 235Surgical Intensive Care Management 237Postinjury Resuscitation / 237Abdominal Compartment Syndrome / 238Special Populations 240Pregnant Patients / 240Geriatric Patients / 243Pediatric Patients / 244Brunicardi_Ch07_p0183-p0250.indd 18310/12/18 6:16 PM 184Table 7-1Immediately life-threatening injuries to be identified during the primary surveyAirwayAirway obstructionAirway injuryBreathingTension pneumothoraxOpen pneumothoraxMassive air leak from tracheobronchial injuryFlail chest with underlying pulmonary contusionCirculationHemorrhagic shockMassive hemothoraxMassive

1	injuryBreathingTension pneumothoraxOpen pneumothoraxMassive air leak from tracheobronchial injuryFlail chest with underlying pulmonary contusionCirculationHemorrhagic shockMassive hemothoraxMassive hemoperitoneumMechanically unstable pelvis fracture with bleedingExtremity blood lossCardiogenic shockCardiac tamponadeNeurogenic shockDisabilityIntracranial hemorrhageCervical spine injuryPatients who are conscious, without tachypnea, and have a normal voice are unlikely to require early airway intervention. Exceptions are penetrating injuries to the neck with an expanding hematoma; evidence of chemical or thermal injury to the mouth, nares, or hypopharynx; extensive subcutaneous air in the neck; complex maxillofacial trauma; or airway bleeding. Although these patients may initially have an adequate airway, it may become compromised if soft tissue swelling, hematoma forma-tion, or edema progresses. In these cases, preemptive intubation should be performed before airway access becomes

1	an adequate airway, it may become compromised if soft tissue swelling, hematoma forma-tion, or edema progresses. In these cases, preemptive intubation should be performed before airway access becomes challenging.Patients who have an abnormal voice, abnormal breathing sounds, tachypnea, or altered mental status require further air-way evaluation. Blood, vomit, the tongue, teeth, foreign objects, and soft tissue swelling can cause airway obstruction; suction-ing affords immediate relief in many patients. In the comatose patient, the tongue may fall backward and obstruct the hypo-pharynx; this can be relieved by either a chin lift or jaw thrust. An oral airway or a nasal trumpet is also helpful in maintaining airway patency, although the former is usually not tolerated by an awake patient. Establishing a definitive airway (i.e., endotra-cheal intubation) is indicated in patients with apnea; inability to protect the airway due to altered mental status; impending airway compromise due to

1	Establishing a definitive airway (i.e., endotra-cheal intubation) is indicated in patients with apnea; inability to protect the airway due to altered mental status; impending airway compromise due to inhalation injury, hematoma, facial bleeding, soft tissue swelling, or aspiration; and inability to maintain oxygenation. Altered mental status is the most com-mon indication for intubation. Agitation or obtundation, often attributed to intoxication or drug use, may actually be due to hypoxia. But the timing of endotracheal intubation may be criti-cal in the hypovolemic patient because positive airway pres-sure may further compromise cardiac function and precipitate cardiac arrest; thus, Circulation may take priority over Airway.Options for endotracheal intubation include nasotracheal, orotracheal, or operative routes. Nasotracheal intubation can be accomplished only in patients who are breathing spontaneously. Although nasotracheal intubation is frequently used by prehospital providers,

1	or operative routes. Nasotracheal intubation can be accomplished only in patients who are breathing spontaneously. Although nasotracheal intubation is frequently used by prehospital providers, the technique is limited in the ED to those patients requiring emergent airway support in whom chemical paralysis Key Points1 Trauma is the most common cause of death for all indi-viduals between the ages of 1 and 44 years and is the third most common cause of death regardless of age.2 The initial management of seriously injured patients usu-ally follows the primary survey (the “ABCs”—Airway with cervical spine protection, Breathing, and Circulation), although at times restoring Circulatory volume may pro-ceed active Airway intervention; the goals of the primary survey are to identify and treat conditions that constitute an immediate threat to life.3 All patients with blunt injury should be assumed to have unstable cervical spine injuries until proven otherwise; one must maintain cervical spine

1	that constitute an immediate threat to life.3 All patients with blunt injury should be assumed to have unstable cervical spine injuries until proven otherwise; one must maintain cervical spine precautions and in-line stabilization.4 Patients with ongoing hemodynamic instability, whether “nonresponders” or “transient responders,” require prompt intervention; one must consider the dominant causes of acute shock, i.e., hemorrhagic, cardiogenic, and neuro-genic shock.5 Patients with trauma-induced coagulopathy (TIC) are at risk for massive transfusion and need to be identified early.6 Indications for immediate operative intervention for pene-trating cervical injury include hemodynamic instability and significant external arterial hemorrhage; the management algorithm for hemodynamically stable patients is based on the presenting symptoms and anatomic location of injury, with the neck being divided into three distinct zones.7 The gold standard for determining if there is a blunt descending

1	patients is based on the presenting symptoms and anatomic location of injury, with the neck being divided into three distinct zones.7 The gold standard for determining if there is a blunt descending aortic injury is computed tomography angiog-raphy (CTA) scanning; indications are primarily based on injury mechanism.8 The abdomen is a diagnostic black box. Physical examina-tion and FAST ultrasound can identify patients requiring emergent laparotomy. Computed tomography (CT) scan-ning is the mainstay of evaluation in the remaining patients to more precisely identify the site and magnitude of injury.9 Manifestation of the “bloody vicious cycle” (the lethal combination of coagulopathy, hypothermia, and metabolic acidosis) is the most common indication for damage con-trol surgery. The primary objectives of damage control laparotomy are to control bleeding and limit gastrointesti-nal spillage.10 Blunt injuries to the carotid and vertebral arteries are usu-ally managed with systemic

1	primary objectives of damage control laparotomy are to control bleeding and limit gastrointesti-nal spillage.10 Blunt injuries to the carotid and vertebral arteries are usu-ally managed with systemic antithrombotic therapy.11 The abdominal compartment syndrome may be primary (i.e., due to the injury of abdominal organs, bleeding, and packing) or secondary (i.e., due to reperfusion visceral edema, retroperitoneal edema, and ascites).Brunicardi_Ch07_p0183-p0250.indd 18410/12/18 6:16 PM 185TRAUMACHAPTER 7AFigure 7-1. Cricothyroidotomy is recommended for emergent surgical establishment of a patent airway. A vertical skin incision avoids injury to the anterior jugular veins, which are located just lateral to the midline. Hemorrhage from these vessels obscures vision and prolongs the procedure. When a transverse incision is made in the cricothyroid membrane, the blade of the knife should be angled inferiorly to avoid injury to the vocal cords. A. Use of a tracheostomy hook stabilizes

1	When a transverse incision is made in the cricothyroid membrane, the blade of the knife should be angled inferiorly to avoid injury to the vocal cords. A. Use of a tracheostomy hook stabilizes the thyroid cartilage and facilitates tube insertion. B. A 6.0 endotracheal tube is inserted after digital confirmation of airway access.Figure 7-2. A “clothesline” injury can partially or completely tran-sect the anterior neck structures, including the trachea. With com-plete tracheal transection, the endotracheal tube is placed directly into the distal aperture, with care taken not to push the trachea into the mediastinum.incision, with sharp division of the subcutaneous tissues. Visu-alization may be improved by having an assistant retract lat-erally on the neck incision using retractors. The cricothyroid membrane is verified by digital palpation and opened in a hori-zontal direction. The airway may be stabilized before incision of the membrane using a tracheostomy hook; the hook should be

1	membrane is verified by digital palpation and opened in a hori-zontal direction. The airway may be stabilized before incision of the membrane using a tracheostomy hook; the hook should be placed under the thyroid cartilage to elevate the airway. A 6.0 endotracheal tube (maximum diameter in adults) is then advanced through the cricothyroid opening and sutured into place. In patients under the age of 11, cricothyroidotomy is rela-tively contraindicated due to the risk of subglottic stenosis, and tracheostomy should be performed.Emergent tracheostomy is indicated in patients with laryn-gotracheal separation or laryngeal fractures, in whom cricothy-roidotomy may cause further damage or result in complete loss of the airway. This procedure is best performed in the operating room (OR) where there is optimal lighting and availability of advanced equipment (e.g., sternal saw). In these cases, often after a “clothesline” injury, direct visualization and instrumenta-tion of the trachea usually

1	is optimal lighting and availability of advanced equipment (e.g., sternal saw). In these cases, often after a “clothesline” injury, direct visualization and instrumenta-tion of the trachea usually is done through the traumatic anterior neck defect or after a generous collar skin incision (Fig. 7-2). If the trachea is completely transected, a nonpenetrating clamp should be placed on the distal aspect to prevent tracheal retrac-tion into the mediastinum; this is particularly important before placement of the endotracheal tube.Breathing and Ventilation. Once a secure airway is obtained, adequate oxygenation and ventilation must be ensured. All injured patients should receive supplemental oxygen and be monitored by pulse oximetry. The following conditions con-stitute an immediate threat to life due to inadequate ventilation and should be recognized during the primary survey: tension pneumothorax, open pneumothorax, flail chest with underlying pulmonary contusion, massive hemothorax, and

1	due to inadequate ventilation and should be recognized during the primary survey: tension pneumothorax, open pneumothorax, flail chest with underlying pulmonary contusion, massive hemothorax, and major air leak due to a tracheobronchial injury.Tension pneumothorax is presumed in any patient mani-festing respiratory distress and hypotension in combination with any of the following physical signs: tracheal deviation away from the affected side, lack of or decreased breath sounds on cannot be used. Orotracheal intubation is the preferred technique to establish a definitive airway. Because all patients are presumed to have cervical spine injuries, manual in-line cervical immobilization is essential.6 Correct endotracheal placement is verified with direct laryngoscopy, capnography, audible bilateral breath sounds, and finally a chest film. The GlideScope®, a video laryngoscope that uses fiberoptics to visualize the vocal cords, is being employed more frequently.9 Advantages of orotracheal

1	breath sounds, and finally a chest film. The GlideScope®, a video laryngoscope that uses fiberoptics to visualize the vocal cords, is being employed more frequently.9 Advantages of orotracheal intubation include the direct visualization of the vocal cords, ability to use larger-diameter endotracheal tubes, and applicability to apneic patients. The disadvantage of orotracheal intubation is that conscious patients usually require neuromuscular blockade, which may result in the inability to intubate, aspiration, or medication complications. Those who attempt rapid-sequence induction must be thoroughly familiar with the procedure (see Chapter 13).Patients in whom attempts at intubation have failed or who are precluded from intubation due to extensive facial inju-ries require operative establishment of an airway. Cricothy-roidotomy (Fig. 7-1) is performed through a generous vertical 3Brunicardi_Ch07_p0183-p0250.indd 18510/12/18 6:16 PM 186BASIC CONSIDERATIONSPART Ithe affected side,

1	of an airway. Cricothy-roidotomy (Fig. 7-1) is performed through a generous vertical 3Brunicardi_Ch07_p0183-p0250.indd 18510/12/18 6:16 PM 186BASIC CONSIDERATIONSPART Ithe affected side, and subcutaneous emphysema on the affected side. Patients may have distended neck veins due to impedance of venous return, but the neck veins may be flat due to concur-rent systemic hypovolemia. Tension pneumothorax and simple pneumothorax have similar signs, symptoms, and examination findings, but hypotension qualifies the pneumothorax as a ten-sion pneumothorax. Although immediate needle thoracostomy decompression with a 14-gauge angiocatheter may be indicated in the field, tube thoracostomy in the midaxillary line should be performed immediately in the ED before a chest radiograph is obtained (Fig. 7-3). Recent studies suggest that preferred loca-tion for needle decompression may be the fifth intercostal space in the anterior axillary line due to body habitus.10 In cases of tension

1	(Fig. 7-3). Recent studies suggest that preferred loca-tion for needle decompression may be the fifth intercostal space in the anterior axillary line due to body habitus.10 In cases of tension pneumothorax, the parenchymal tear in the lung acts as a one-way valve, with each inhalation allowing additional air to accumulate in the pleural space. The normally negative intra-pleural pressure becomes positive, which depresses the ipsilat-eral hemidiaphragm and shifts the mediastinal structures into the contralateral chest. Subsequently, the contralateral lung is compressed and the heart rotates about the superior and inferior vena cava; this decreases venous return and ultimately cardiac output, which culminates in cardiovascular collapse.An open pneumothorax or “sucking chest wound” occurs with full-thickness loss of the chest wall, permitting free com-munication between the pleural space and the atmosphere (Fig. 7-4). This compromises ventilation due to equilibration of atmospheric and

1	full-thickness loss of the chest wall, permitting free com-munication between the pleural space and the atmosphere (Fig. 7-4). This compromises ventilation due to equilibration of atmospheric and pleural pressures, which prevents lung inflation Figure 7-3. A. Tube thoracostomy is performed in the midaxillary line at the fourth or fifth intercostal space (inframammary crease) to avoid iatrogenic injury to the liver or spleen. B. Heavy scissors are used to cut through the intercostal muscle into the pleural space. This is done on top of the rib to avoid injury to the intercostal bun-dle located just beneath the rib. C. The incision is digitally explored to confirm intrathoracic location and identify pleural adhesions. D. A 28Fr chest tube is directed superiorly and posteriorly with the aid of a large clamp.Figure 7-4. A. Full-thickness loss of the chest wall results in an open pneumothorax. B. The defect is temporarily managed with an occlusive dressing that is taped on three sides,

1	of a large clamp.Figure 7-4. A. Full-thickness loss of the chest wall results in an open pneumothorax. B. The defect is temporarily managed with an occlusive dressing that is taped on three sides, which allows accumulated air to escape from the pleural space and thus prevents a tension pneumothorax. Repair of the chest wall defect and tube thoracostomy remote from the wound is definitive treatment.and alveolar ventilation, and results in hypoxia and hypercar-bia. Complete occlusion of the chest wall defect without a tube thoracostomy may convert an open pneumothorax to a tension pneumothorax. Temporary management of this injury includes covering the wound with an occlusive dressing that is taped on three sides. This acts as a flutter valve, permitting effective ven-tilation on inspiration while allowing accumulated air to escape from the pleural space on the untapped side, so that a tension pneumothorax is prevented. Definitive treatment is closure of the chest wall defect and tube

1	while allowing accumulated air to escape from the pleural space on the untapped side, so that a tension pneumothorax is prevented. Definitive treatment is closure of the chest wall defect and tube thoracostomy remote from the wound.Flail chest occurs when three or more contiguous ribs are fractured in at least two locations. Paradoxical movement of this free-floating segment of chest wall is usually evident in patients with spontaneous ventilation, due to the negative intra-pleural pressure of inspiration. The additional work of breathing and chest wall pain caused by the flail segment is rarely suf-ficient to compromise ventilation. Instead, it is the decreased compliance and increased shunt fraction caused by the associ-ated pulmonary contusion that is the source of acute respiratory failure. Pulmonary contusions often progress during the first 12 hours. Resultant hypoventilation and hypoxemia may require intubation and mechanical ventilation. The patient’s initial chest

1	failure. Pulmonary contusions often progress during the first 12 hours. Resultant hypoventilation and hypoxemia may require intubation and mechanical ventilation. The patient’s initial chest Brunicardi_Ch07_p0183-p0250.indd 18610/12/18 6:17 PM 187TRAUMACHAPTER 7radiograph often underestimates the extent of the pulmonary parenchymal damage (Fig. 7-5); close monitoring and frequent clinical reevaluation are warranted.Major air leak occurs from tracheobronchial injuries. Type I injuries are those occurring within 2 cm of the carina.11,12 These may not be associated with a pneumothorax due to the envelopment in the mediastinal pleura. Type II injuries are more distal injuries within the tracheobronchial tree and hence mani-fest with a pneumothorax. Bronchoscopy confirms the extent of the injury and its location, and directs management.Circulation With Hemorrhage Control. With a secure airway and adequate ventilation established, circulatory status is the next focus. An initial

1	of the injury and its location, and directs management.Circulation With Hemorrhage Control. With a secure airway and adequate ventilation established, circulatory status is the next focus. An initial approximation of the patient’s cardiovascular status can be obtained by palpating peripheral pulses. In gen-eral, systolic blood pressure (SBP) must be 60 mmHg for the carotid pulse to be palpable, 70 mmHg for the femoral pulse, and 80 mmHg for the radial pulse. Any episode of hypotension (defined as a SBP <90 mmHg) is assumed to be caused by hem-orrhage until proven otherwise. Patients with rapid massive blood loss may have paradoxical bradycardia.13 Blood pressure and pulse should be measured at least every 5 minutes in patients with significant blood loss until normal vital sign values are restored.Intravenous (IV) access for fluid resuscitation and medi-cation administration is obtained with two peripheral catheters, 16-gauge or larger in adults. For patients in whom peripheral

1	are restored.Intravenous (IV) access for fluid resuscitation and medi-cation administration is obtained with two peripheral catheters, 16-gauge or larger in adults. For patients in whom peripheral angiocatheter access is difficult, intraosseous (IO) needles Figure 7-5. A. Admission chest film may not show the full extent of the patient’s pulmonary parenchymal injury. B. This patient’s left pulmonary contusion blossomed 12 hours later, and its associ-ated opacity is noted on repeat chest radiograph.should be rapidly placed in the proximal humerus or tibia (Fig. 7-6).14,15 All medications administered IV may be admin-istered in a similar dosage intraosseously. Although safe for emergent use, the needle should be removed once alternative access is established to prevent potential osteomyelitis. Blood should be drawn simultaneously for a bedside hemoglobin level and routine trauma laboratory tests. In the seriously injured patient arriving in shock, an arterial blood gas for base deficit

1	Blood should be drawn simultaneously for a bedside hemoglobin level and routine trauma laboratory tests. In the seriously injured patient arriving in shock, an arterial blood gas for base deficit (BD), cross-matching for possible blood component (RBC and plasma) transfusion, and a coagulation panel/viscoelastic hemo-statis assay (e.g., TEG, ROTEM) should be obtained. In these patients, secondary large bore (7 to 9 Fr) cannulae should be obtained via the femoral or subclavian veins; Cordis introducer catheters are preferred over triple-lumen catheters. In general, initial access in trauma patients is best secured in the groin so that placement of the catheter will not interfere with the perfor-mance of other diagnostic and therapeutic thoracic procedures. A rule of thumb to consider for secondary access is placement of femoral access for thoracic trauma and jugular or subclavian access for abdominal trauma. Internal jugular or subclavian catheters provide a more reliable measurement of

1	access is placement of femoral access for thoracic trauma and jugular or subclavian access for abdominal trauma. Internal jugular or subclavian catheters provide a more reliable measurement of central venous pressure (CVP), which may be helpful in determining the vol-ume status of the patient and in excluding cardiac tamponade. Saphenous vein cutdowns at the ankle can also provide excel-lent access (Fig. 7-7). The saphenous vein is reliably found 1 cm anterior and 1 cm superior to the medial malleolus. Stan-dard 14-gauge catheters can be quickly placed, even in an exsanguinating patient with collapsed veins. In severely injured children younger than 6 years of age, the preferred venous access is peripheral intravenous catheters followed by an IO needle. Central venous catheter placement or saphenous vein cutdown may be considered as the third choice of access based upon provider experience. Inadvertent femoral artery cannula-tion, however, may result in limb-threatening arterial

1	or saphenous vein cutdown may be considered as the third choice of access based upon provider experience. Inadvertent femoral artery cannula-tion, however, may result in limb-threatening arterial spasm.External control of any visible hemorrhage should be achieved promptly while circulating volume is restored. For Figure 7-6. Intraosseous infusions are indicated for patients in whom one or two attempts at IV access have failed. A. The proxi-mal tibia or humerus is the preferred location. Alternatively, the distal femur can be used if the tibia is fractured. B. The position is satisfactory if bone marrow can be aspirated and saline can be eas-ily infused without evidence of extravasation.Brunicardi_Ch07_p0183-p0250.indd 18710/12/18 6:17 PM 188BASIC CONSIDERATIONSPART Iopen wounds with ongoing bleeding, manual compression should be done with a single 4 × 4 gauze and a gloved hand. Covering the wound with excessive dressings may permit ongoing unrecognized blood loss that is hidden

1	ongoing bleeding, manual compression should be done with a single 4 × 4 gauze and a gloved hand. Covering the wound with excessive dressings may permit ongoing unrecognized blood loss that is hidden underneath the dressing. Blind clamping of bleeding vessels should be avoided because of the risk to adjacent structures, including nerves. This is particularly true for penetrating injuries of the neck, thoracic outlet, and groin, where bleeding may be torrential and aris-ing deep within the wound. In these situations, a gloved finger placed through the wound directly onto the bleeding vessel can apply enough pressure to control active bleeding. The surgeon performing this maneuver must then walk with the patient to the OR for definitive treatment. For bleeding of the extremi-ties, it is tempting to apply tourniquets for hemorrhage control, Figure 7-7. Saphenous vein cutdowns are excellent sites for fluid resuscitation access. A. The vein is consistently found 1 cm ante-rior and 1 cm

1	tempting to apply tourniquets for hemorrhage control, Figure 7-7. Saphenous vein cutdowns are excellent sites for fluid resuscitation access. A. The vein is consistently found 1 cm ante-rior and 1 cm superior to the medial malleolus. B. Proximal and distal traction sutures are placed with the distal suture ligated. C. A 14-gauge IV catheter is introduced and secured with sutures and tape to prevent dislodgment.Figure 7-8. More than 1500 mL of blood in the pleural space is considered a massive hemothorax. Chest film findings reflect the positioning of the patient. A. In the supine position, blood tracks along the entire posterior section of the chest and is most notable pushing the lung away from the chest wall. B. In the upright position, blood is visible dependently in the right pleural space.but digital occlusion will usually control the bleeding; complete vascular occlusion with a tourniquet risks permanent neuromus-cular impairment. Patients in shock have a lower tolerance to

1	space.but digital occlusion will usually control the bleeding; complete vascular occlusion with a tourniquet risks permanent neuromus-cular impairment. Patients in shock have a lower tolerance to warm ischemia, and an occluded extremity is prone to small vessel thrombosis. For patients with open fractures, fracture reduction with stabilization via splints will limit bleeding both externally and into the subcutaneous tissues. Scalp lacerations through the galea aponeurotica tend to bleed profusely; these can be temporarily controlled with skin staples, Raney clips, or a full-thickness continuous running nylon stitch.During the circulation section of the primary survey, four life-threatening injuries must be identified promptly: (a) mas-sive hemothorax, (b) cardiac tamponade, (c) massive hemo-peritoneum, and (d) mechanically unstable pelvic fractures with bleeding. Massive hemoperitoneum and mechanically unstable pelvic fractures are discussed in “Emergent Abdomi-nal Exploration” and

1	hemo-peritoneum, and (d) mechanically unstable pelvic fractures with bleeding. Massive hemoperitoneum and mechanically unstable pelvic fractures are discussed in “Emergent Abdomi-nal Exploration” and “Pelvic Fractures and Emergent Hemor-rhage Control,” respectively. Critical tools used to differentiate these in the multisystem trauma patient are the chest and pelvis radiographs, and extended focused abdominal sonography for trauma (eFAST) (see “Regional Assessment and Special Diag-nostic Tests”). A massive hemothorax (life-threatening injury number one) is defined as >1500 mL of blood or, in the pediatric population, >25% of the patient’s blood volume in the pleural space (Fig. 7-8). Although it may be estimated on chest radio-graph, tube thoracostomy is the only reliable means to quantify the amount of hemothorax. After blunt trauma, a hemothorax is usually due to multiple rib fractures with severed intercostal vessels, but occasionally bleeding is from lacerated lung paren-chyma,

1	the amount of hemothorax. After blunt trauma, a hemothorax is usually due to multiple rib fractures with severed intercostal vessels, but occasionally bleeding is from lacerated lung paren-chyma, which is usually associated with an air leak. After pen-etrating trauma, a great vessel or pulmonary hilar vessel injury should be presumed. In either scenario, a massive hemothorax is an indication for operative intervention, but tube thoracostomy is critical to facilitate lung reexpansion, which may improve oxygenation and cardiac performance as well as tamponade venous bleeding. In patients arriving in shock with a high risk of pelvic fracture (e.g., autopedestrian accident), the pelvis should be presumptively stabilized with a sheet or binder.Brunicardi_Ch07_p0183-p0250.indd 18810/12/18 6:17 PM 189TRAUMACHAPTER 7Cardiac tamponade occurs most commonly after penetrat-ing thoracic wounds, although occasionally blunt rupture of the heart, particularly the atrial appendage, is seen.

1	6:17 PM 189TRAUMACHAPTER 7Cardiac tamponade occurs most commonly after penetrat-ing thoracic wounds, although occasionally blunt rupture of the heart, particularly the atrial appendage, is seen. Acutely, <100 mL of pericardial blood may cause pericardial tamponade.16 The classic Beck’s triad—dilated neck veins, muffled heart tones, and a decline in arterial pressure—is usually not appreciated in the trauma bay because of the noisy environment and associ-ated hypovolemia. Because the pericardium is not acutely dis-tensible, the pressure in the pericardial sac will rise to match that of the injured chamber. When this pressure exceeds that of the right atrium, right atrial filling is impaired and right ventricular preload is reduced. This ultimately leads to decreased right ven-tricular output. Additionally, increased intrapericardial pressure impedes myocardial blood flow, which leads to subendocardial ischemia and a further reduction in cardiac output.Diagnosis of hemopericardium is

1	Additionally, increased intrapericardial pressure impedes myocardial blood flow, which leads to subendocardial ischemia and a further reduction in cardiac output.Diagnosis of hemopericardium is best achieved by ultra-sound of the pericardium (Fig. 7-9). Early in the course of tamponade, blood pressure and cardiac output will transiently improve with fluid administration due to increased central venous pressure. In patients with any hemodynamic distur-bance, a pericardial drain can be placed using ultrasound guid-ance (Fig. 7-10). Removing as little as 15 to 20 mL of blood will Figure 7-9. Subxiphoid pericardial ultrasound reveals a large peri-cardial fluid collection. LV = left ventricle; RV = right ventricle.Figure 7-10. Pericardiocentesis is indicated for patients with evidence of pericardial tamponade. A. Access to the pericardium is obtained through a subxiphoid approach, with the needle angled 45° up from the chest wall and toward the left shoulder. B. Seldinger technique is used

1	tamponade. A. Access to the pericardium is obtained through a subxiphoid approach, with the needle angled 45° up from the chest wall and toward the left shoulder. B. Seldinger technique is used to place a pigtail catheter. Blood can be repeatedly aspirated with a syringe, or the tubing may be attached to a gravity drain. Evacuation of unclotted pericardial blood prevents subendocardial ischemia and stabilizes the patient for transport to the operating room for sternotomy.often temporarily stabilize the patient’s hemodynamic status and alleviate the subendocardial ischemia that can be associ-ated with lethal arrhythmias; this allows safe transport to the OR for sternotomy. Pericardiocentesis is successful in decompress-ing tamponade in approximately 80% of cases17; the majority of failures are due to the presence of clotted blood within the pericardium. Patients with a persistent SBP <60 mmHg warrant resuscitative thoracotomy (RT) with opening of the pericardium for rapid decompression

1	are due to the presence of clotted blood within the pericardium. Patients with a persistent SBP <60 mmHg warrant resuscitative thoracotomy (RT) with opening of the pericardium for rapid decompression and control of bleeding.The utility of RT has been debated for decades. Current indi-cations are based on 40 years of prospective data (Table 7-2).18-20 RT is associated with the highest survival rate after isolated cardiac injury; 35% of patients presenting in shock and 20% without vital signs (i.e., no pulse or obtainable blood pressure) are salvaged after Table 7-2Current indications and contraindications for emergency department thoracotomyIndicationsSalvageable postinjury cardiac arrest:Patients sustaining witnessed penetrating trauma to the torso with <15 min of prehospital CPRPatients sustaining witnessed blunt trauma with <10 min of prehospital CPRPatients sustaining witnessed penetrating trauma to the neck or extremities with <5 min of prehospital CPRPersistent severe postinjury

1	witnessed blunt trauma with <10 min of prehospital CPRPatients sustaining witnessed penetrating trauma to the neck or extremities with <5 min of prehospital CPRPersistent severe postinjury hypotension (SBP ≤60 mmHg) due to:Cardiac tamponadeHemorrhage—intrathoracic, intra-abdominal, extremity, cervicalAir embolismContraindicationsPenetrating trauma: CPR >15 min and no signs of life (pupillary response, respiratory effort, motor activity)Blunt trauma: CPR >10 min and no signs of life or asystole without associated tamponadeCPR = cardiopulmonary resuscitation; SBP = systolic blood pressure.Brunicardi_Ch07_p0183-p0250.indd 18910/12/18 6:17 PM 190BASIC CONSIDERATIONSPART Iisolated penetrating injury to the heart. For all penetrating wounds, survival rate is 15%. Conversely, patient outcome is limited when RT is done for blunt trauma, with 2% survival among patients in shock and <1% survival among those with no vital signs. Thus, patients undergoing cardiopulmonary resuscitation (CPR)

1	is limited when RT is done for blunt trauma, with 2% survival among patients in shock and <1% survival among those with no vital signs. Thus, patients undergoing cardiopulmonary resuscitation (CPR) upon arrival to the ED should undergo RT selectively based on injury and duration of CPR (Fig. 7-11). RT is best accomplished using a generous left anterolateral thoracotomy, with the skin incision started to the right of the sternum (Fig. 7-12). A longitudinal peri-cardiotomy anterior to the phrenic nerve is used to release cardiac tamponade and permits access to the heart for cardiac repair and open cardiac massage. The cardiac wound should be repaired prior to vigorous efforts of myocardial resuscitation (e.g., epinephrine, calcium). Cross-clamping of the aorta improves central circula-tion, augments cerebral and coronary blood flow, and limits further abdominal blood loss (Fig. 7-13). The patient must sustain a SBP of 70 mmHg after RT and associated interventions to be considered

1	augments cerebral and coronary blood flow, and limits further abdominal blood loss (Fig. 7-13). The patient must sustain a SBP of 70 mmHg after RT and associated interventions to be considered resuscitable, and hence transported to the OR.18,19Disability and Exposure. The Glasgow Coma Scale (GCS) score should be determined for all injured patients (Table 7-3). It is calculated by adding the scores of the best motor response, best verbal response, and the best eye response. Scores range from 3 (the lowest) to 15 (normal). Scores of 13 to 15 indicate mild head injury, 9 to 12 moderate injury, and ≤8 severe injury. The GCS is a quantifiable determination of neurologic function that is useful for triage, treatment, and prognosis.Neurologic evaluation, including spinal cord integrity, is critical before administration of neuromuscular blockade for intubation. Subtle changes in mental status can be caused by hypoxia, hypercarbia, or hypovolemia, or may be an early sign of increasing

1	critical before administration of neuromuscular blockade for intubation. Subtle changes in mental status can be caused by hypoxia, hypercarbia, or hypovolemia, or may be an early sign of increasing intracranial pressure. An abnormal mental status should prompt an immediate reevaluation of the patient’s ABCs and consideration of central nervous system injury. Deterioration in mental status may be subtle and may not progress in a predict-able fashion. For example, previously calm, cooperative patients may become anxious and combative as they become hypoxic. However, a patient who is agitated and combative from drugs or alcohol may become somnolent if hypovolemic shock develops. Patients with neurogenic shock are typified by hypotension with relative bradycardia, and are often first recognized due to paraly-sis, decreased rectal tone, or priapism. Patients with high spinal cord disruption are at greatest risk for neurogenic shock due to physiologic disruption of sympathetic fibers;

1	due to paraly-sis, decreased rectal tone, or priapism. Patients with high spinal cord disruption are at greatest risk for neurogenic shock due to physiologic disruption of sympathetic fibers; treatment consists of volume loading and a dopamine infusion, which is both inotropic and chronotropic, as well as a vasoconstrictor.Seriously injured patients must have all of their clothing removed to avoid overlooking limbor life-threatening injuries, but warmed blankets should be placed immediately to avoid hypothermia.Shock Classification and Initial Fluid Resuscitation. Classic signs and symptoms of shock are tachycardia, hypoten-sion, tachypnea, altered mental status, diaphoresis, and pallor PatientUndergoingCPR–No Signs ofLife*Blunt TraumaCPR <10 min----------Penetrating Torso TraumaCPR <15 min----------Penetrating Non-Torso TraumaCPR <5 minResuscitative ThoracotomyYesDeadCardiacActivity?NoTamponade?NoRepair HeartAssessViabilityYesORThoracicHemorrhageAir

1	Torso TraumaCPR <15 min----------Penetrating Non-Torso TraumaCPR <5 minResuscitative ThoracotomyYesDeadCardiacActivity?NoTamponade?NoRepair HeartAssessViabilityYesORThoracicHemorrhageAir EmboliTamponadeExtrathoracicHemorrhageSBP <70,apply aorticX-clampHilar X-clampControlNoYes*no respiratory ormotor effort, electricalactivity, or pupillaryactivityProfoundRefractoryShockFigure 7-11. Algorithm directing the use of resuscitative thoracotomy (RT) in the injured patient undergoing cardiopulmonary resuscitation (CPR). OR = operating room; SBP = systolic blood pressure.Brunicardi_Ch07_p0183-p0250.indd 19010/12/18 6:17 PM 191TRAUMACHAPTER 7Figure 7-12. A. Resuscitative tho-racotomy (RT) is performed through the fifth intercostal space using the anterolateral approach. B and C. The pericardium is opened anterior to the phrenic nerve, and the heart is rotated out for evaluation. D. Open cardiac massage should be performed with a hinged, clapping motion of the hands, with sequential closing

1	is opened anterior to the phrenic nerve, and the heart is rotated out for evaluation. D. Open cardiac massage should be performed with a hinged, clapping motion of the hands, with sequential closing from palms to fingers. The two-handed technique is strongly recommended because the one-handed massage technique poses the risk of myocar-dial perforation with the thumb.Figure 7-13. A. Aortic cross-clamp is applied with the left lung retracted superiorly, below the inferior pulmonary ligament, just above the diaphragm. B. The flaccid aorta is identified as the first structure encountered on top of the spine when approached from the left chest.(Table 7-4). In general, the quantity of acute blood loss cor-relates with physiologic abnormalities. For example, patients in class II shock are tachycardic, but they do not exhibit a reduction in blood pressure until over 1500 mL of blood loss, or class III shock. Physical findings should be used as an aid in the evaluation of the patient’s

1	tachycardic, but they do not exhibit a reduction in blood pressure until over 1500 mL of blood loss, or class III shock. Physical findings should be used as an aid in the evaluation of the patient’s response to treatment. The goal of fluid resuscitation is to re-establish tissue perfusion. Fluid resuscitation usually begins with isotonic crystalloid, typically Ringer’s lactate. However, for patients arriving in shock (persistent SBP <90 mmHg in an adult), instead of crystalloid the current practice is to activate a massive trans-fusion protocol (MTP) in which red blood cells (RBC) and fresh frozen plasma (FFP) are administered. The details of a MTP are discussed later. Patients who have a good response to Brunicardi_Ch07_p0183-p0250.indd 19110/12/18 6:17 PM 192BASIC CONSIDERATIONSPART Ifluid infusion (i.e., normalization of vital signs, clearing of the sensorium) and evidence of good peripheral perfusion (warm extremities with normal capillary refill) are presumed to have adequate

1	infusion (i.e., normalization of vital signs, clearing of the sensorium) and evidence of good peripheral perfusion (warm extremities with normal capillary refill) are presumed to have adequate overall perfusion. Urine output is a reliable indicator of organ perfusion but requires time to quantitate. Adequate urine output is 0.5 mL/kg per hour in an adult, 1 mL/kg per hour in a child, and 2 mL/kg per hour in an infant <1 year of age. Because measurement of this resuscitation-related vari-able is time dependent, it is generally more useful in the OR and intensive care unit (ICU) setting, than in initial evaluation in the trauma bay.There are several caveats to be considered when evaluat-ing the injured patient for shock. Tachycardia (HR >110 bpm) is often the earliest sign of ongoing blood loss, but the critical issue is change in HR over time. Individuals in good physical condition with a resting pulse rate in the 50s may manifest a relative tachycardia in the 90s; although clinically

1	loss, but the critical issue is change in HR over time. Individuals in good physical condition with a resting pulse rate in the 50s may manifest a relative tachycardia in the 90s; although clinically significant, this does not meet the standard definition of tachycardia. Con-versely, patients on cardiac medications such as β-blockers may not be capable of increasing their heart rate to compensate for hypovolemia. Bradycardia can occur with rapid severe blood loss13; this is an ominous sign, often heralding impending car-diovascular collapse. Other physiologic stresses, aside from hypovolemia, may produce tachycardia, such as hypoxia, pain, anxiety, and stimulant drugs (cocaine, amphetamines). As noted previously, decreased SBP is not a reliable early sign of hypo-volemia because blood loss must exceed 30% before hypoten-sion is evident. Additionally, younger patients may maintain their SBP due to sympathetic tone despite severe intravascular deficits until they are on the verge of

1	loss must exceed 30% before hypoten-sion is evident. Additionally, younger patients may maintain their SBP due to sympathetic tone despite severe intravascular deficits until they are on the verge of cardiac arrest. Pregnant Table 7-3Glasgow Coma ScaleaADULTSINFANTS/CHILDRENEye opening4SpontaneousSpontaneous3To voiceTo voice2To painTo pain1NoneNoneVerbal5OrientedAlert, normal vocalization4ConfusedCries, but consolable3Inappropriate wordsPersistently irritable2Incomprehensible wordsRestless, agitated, moaning1NoneNoneMotor response6Obeys commandsSpontaneous, purposeful5Localizes painLocalizes pain4WithdrawsWithdraws3Abnormal flexionAbnormal flexion2Abnormal extensionAbnormal extension1NoneNoneaScore is calculated by adding the scores of the best motor response, best verbal response, and eye opening. Scores range from 3 (the lowest) to 15 (normal).Table 7-4Signs and symptoms of advancing stages of hemorrhagic shockCLASS ICLASS IICLASS IIICLASS IVBlood loss (mL)Up to

1	response, and eye opening. Scores range from 3 (the lowest) to 15 (normal).Table 7-4Signs and symptoms of advancing stages of hemorrhagic shockCLASS ICLASS IICLASS IIICLASS IVBlood loss (mL)Up to 750750–15001500–2000>2000Blood loss (% BV)Up to 15%15%–30%30%–40%>40%Pulse rate<100>100>120>140Blood pressureNormalNormalDecreasedDecreasedPulse pressure (mmHg)Normal or increasedDecreasedDecreasedDecreasedRespiratory rate14–20>20–3030–40>35Urine output (mL/h)>30>20–305–15NegligibleCNS/mental statusSlightly anxiousMildly anxiousAnxious and confusedConfused and lethargicBV = blood volume; CNS = central nervous system.Brunicardi_Ch07_p0183-p0250.indd 19210/12/18 6:17 PM 193TRAUMACHAPTER 7patients have a progressive increase in circulating blood volume over gestation; therefore, they must lose a relatively larger vol-ume of blood before manifesting signs and symptoms of hypo-volemia (see “Special Populations”).Based on the initial response to fluid resuscitation, hypo-volemic injured

1	lose a relatively larger vol-ume of blood before manifesting signs and symptoms of hypo-volemia (see “Special Populations”).Based on the initial response to fluid resuscitation, hypo-volemic injured patients can be separated into three broad cat-egories: responders, transient responders, and nonresponders. Individuals who are stable or have a good response to initial fluid therapy as evidenced by normalization of vital signs, men-tal status, and urine output are unlikely to have significant ongo-ing hemorrhage, and further diagnostic evaluation for occult injuries can proceed in an orderly fashion (see “Secondary Sur-vey”). At the other end of the spectrum are patients classified as “nonresponders” who have persistent hypotension despite aggressive resuscitation. These patients mandate immediate identification of the source of hypotension with appropriate intervention to prevent a fatal outcome. Transient responders are those who respond initially to volume loading with improve-ment

1	identification of the source of hypotension with appropriate intervention to prevent a fatal outcome. Transient responders are those who respond initially to volume loading with improve-ment in vital signs, but subsequently deteriorate hemodynami-cally. This group of patients can be challenging to triage for definitive management.Persistent Hypotension. Patients with ongoing hemodynamic instability, whether “nonresponders” or “transient responders,” require systematic evaluation and prompt intervention. The spectrum of disease in patients with persistent hypotension ranges from easily reversible problems such as a tension pneu-mothorax to multisystem injury with a number of sites of ongo-ing active hemorrhage. One must first consider the dominant categories of postinjury shock that may be the underlying cause: hemorrhagic, cardiogenic, and neurogenic. In patients with per-sistent hypotension and tachycardia, cardiogenic or hemorrhagic shock are the likely causes. Ultrasound

1	may be the underlying cause: hemorrhagic, cardiogenic, and neurogenic. In patients with per-sistent hypotension and tachycardia, cardiogenic or hemorrhagic shock are the likely causes. Ultrasound evaluation of the pericar-dium, pleural cavities, and abdomen in combination with plain radiographs of the chest and pelvis will usually identify the source of shock. In patients with persistent hypotension follow-ing blunt trauma, the pelvis should be wrapped with a sheet for stabilization until radiography can be done; external blood loss should be controlled, and extremity fractures should be splinted to minimize further blood loss. Evaluation of the CVP or ultra-sound of the IVC may further assist in distinguishing between cardiogenic and hypovolemic shock. Base deficit measurement is critical; a base deficit of >8 mmol/L implies ongoing cellular shock.21,22 Serum lactate also is used to monitor the patient’s physiologic response to resuscitation.23 Evolving technology, such as

1	is critical; a base deficit of >8 mmol/L implies ongoing cellular shock.21,22 Serum lactate also is used to monitor the patient’s physiologic response to resuscitation.23 Evolving technology, such as measurement of the critical reserve index, may provide noninvasive monitoring.24The differential diagnosis of cardiogenic shock in the trauma patient is: (a) tension pneumothorax, (b) pericardial tam-ponade, (c) blunt cardiac injury, (d) myocardial infarction, and (e) bronchovenous air embolism. Tension pneumothorax, the most frequent cause of cardiac failure, and pericardial tampon-ade have been discussed earlier. Although as many as one-third of patients sustaining significant blunt chest trauma experience some degree of blunt cardiac injury (BCI), few such injuries result in hemodynamic embarrassment. Patients with electro-cardiographic (ECG) abnormalities or dysrhythmias require continuous ECG monitoring and antidysrhythmic treatment as indicated. Unless myocardial infarction is

1	embarrassment. Patients with electro-cardiographic (ECG) abnormalities or dysrhythmias require continuous ECG monitoring and antidysrhythmic treatment as indicated. Unless myocardial infarction is suspected, there is no role for routine serial measurement of cardiac enzyme levels—they lack specificity and do not predict significant dysrhythmias.25 In patients with no identified injuries who are being considered for discharge from the ED, the combination of a normal EKG and troponin level rules out significant BCI.26 The patient with hemodynamic instability from BCI requires appropriate resuscitation and may benefit from hemodynamic monitoring to optimize preload and guide inotropic support. Echocardiography (ECHO) is performed to exclude valvular or septal injuries; the most common ECHO finding in BCI is right ventricular dyskinesia due to the anterior orientation of the right ventricle. Transthoracic and transesophageal ECHO are now becoming routine in the trauma bay and surgical

1	finding in BCI is right ventricular dyskinesia due to the anterior orientation of the right ventricle. Transthoracic and transesophageal ECHO are now becoming routine in the trauma bay and surgical intensive care unit (SICU).27,28 Rarely, patients with refractory cardio-genic shock may require placement of an intra-aortic balloon pump to enhance coronary perfusion and decrease myocardial work. Acute myocardial infarction may be the cause of a motor vehicle collision or other trauma in older patients. Although optimal initial management includes treatment for the evolving infarction, such as thrombolytic therapy, anticoagulation, and emergent angioplasty, these decisions must be individualized in accordance with the patient’s other injuries.Bronchovenous air embolism is a frequently overlooked lethal complication of pulmonary injury. Air emboli can occur after blunt or penetrating trauma, when air from an injured bron-chus enters an adjacent injured pulmonary vein and returns air to

1	lethal complication of pulmonary injury. Air emboli can occur after blunt or penetrating trauma, when air from an injured bron-chus enters an adjacent injured pulmonary vein and returns air to the left heart. Air accumulation in the left ventricle impedes diastolic filling, and during systole air is pumped into the coro-nary arteries, disrupting coronary perfusion. The typical case is a patient with a penetrating thoracic injury who is hemody-namically stable but experiences sudden cardiac arrest after being intubated and placed on positive pressure ventilation. The patient should be placed immediately in Trendelenburg’s posi-tion to trap the air in the apex of the left ventricle. Emergency thoracotomy is followed by cross-clamping of the pulmonary hilum on the side of the injury to prevent further introduction of air (Fig. 7-14). Air is aspirated from the apex of the left ventricle and then the aortic root with an 18-gauge needle and 50-mL syringe. Vigorous massage is used to force

1	further introduction of air (Fig. 7-14). Air is aspirated from the apex of the left ventricle and then the aortic root with an 18-gauge needle and 50-mL syringe. Vigorous massage is used to force the air bubbles through the coronary arteries; if this is unsuccessful, a tuber-culin syringe is used to aspirate air bubbles from the coronary artery (most common is the right). Once circulation is restored, the patient should be kept in Trendelenburg’s position with the pulmonary hilum clamped until the pulmonary venous injury is controlled operatively.Persistent hypotension due to uncontrolled hemorrhage is associated with high mortality. A rapid search for the source or sources of hemorrhage includes visual inspection with knowledge of the injury mechanism, eFAST, and chest and pelvic radiographs. During diagnostic evaluation, type O RBCs (O-negative for women of childbearing age) and thawed AB plasma should be administered at a ratio of 2:1. Type-specific RBCs should be administered as

1	During diagnostic evaluation, type O RBCs (O-negative for women of childbearing age) and thawed AB plasma should be administered at a ratio of 2:1. Type-specific RBCs should be administered as soon as available. The acute coagulopathy of trauma is now well recognized and underscores the importance of preemptive blood component administration.29 The resurgent interest in viscoelastic hemostatic assays (throm-belastography [TEG] and thrombelastometry [ROTEM]) has facilitated the appropriate and timely use of clotting adjuncts, including the prompt recognition of fibrinolysis.30 In patients with clear indications for operation, essential radiographs should be taken, and the patient should be transported to the OR imme-diately. Such patients include those with blunt trauma and mas-sive hemothorax, those with penetrating trauma and an initial chest tube output of >1 L, and those with abdominal trauma and ultrasound evidence of extensive hemoperitoneum. In patients with gunshot wounds to

1	those with penetrating trauma and an initial chest tube output of >1 L, and those with abdominal trauma and ultrasound evidence of extensive hemoperitoneum. In patients with gunshot wounds to the chest or abdomen, a chest and 4Brunicardi_Ch07_p0183-p0250.indd 19310/12/18 6:17 PM 194BASIC CONSIDERATIONSPART Iabdominal film, with radiopaque markers at the wound sites, should be obtained to determine the trajectory of the bullet or location of a retained fragment prior to transport to the OR. For example, a patient with a gunshot wound to the upper abdo-men should have a chest radiograph to ensure that the bullet did not traverse the diaphragm causing intrathoracic injury. Simi-larly, a chest radiograph is important in a patient with a gunshot wound to the right chest to evaluate the left hemithorax. If a patient arrives with a penetrating weapon remaining in place, the weapon should not be removed in the ED because it could be tamponading a lacerated blood vessel (Fig. 7-15). The

1	hemithorax. If a patient arrives with a penetrating weapon remaining in place, the weapon should not be removed in the ED because it could be tamponading a lacerated blood vessel (Fig. 7-15). The sur-geon should extract the offending instrument in the controlled environment of the OR, ideally once an incision has been made with adequate exposure for vascular control. In situations where knives are embedded in the head or neck, preoperative imaging may be useful to anticipate arterial injuries.In patients with persistent hypotension and no clear operative indications, one should systematically evaluate the five potential sources of blood loss: scalp, chest, abdomen, pelvis, and extremities. Significant bleeding at the scene may be reported by paramedics, but its quantification is unreliable. Examination should exclude active bleeding from a scalp lac-eration that may be readily controlled with clips or staples. Thoracoabdominal trauma should be evaluated with a combi-nation of eFAST,

1	Examination should exclude active bleeding from a scalp lac-eration that may be readily controlled with clips or staples. Thoracoabdominal trauma should be evaluated with a combi-nation of eFAST, chest radiograph, and pelvic radiograph. If the FAST results are negative and no other source of hypo-tension is obvious, diagnostic peritoneal aspiration should be entertained.31 Extremity examination and radiographs should be used to identify associated fractures. Fracture-related blood loss, when additive, may be a potential source of the patient’s hemodynamic instability. Each rib fracture can produce 100 to 200 mL of blood loss; for tibial fractures, 300 to 500 mL; for Figure 7-14. A. A Satinsky clamp is used to clamp the pulmonary hilum to prevent further bronchovenous air embolism. B. Sequential sites of aspiration include the left ventricle, the aortic root, and the right coronary artery.Figure 7-15. If a weapon is still in place, it should be removed in the operating room because it

1	sites of aspiration include the left ventricle, the aortic root, and the right coronary artery.Figure 7-15. If a weapon is still in place, it should be removed in the operating room because it could be tamponading a lacerated blood vessel.Brunicardi_Ch07_p0183-p0250.indd 19410/12/18 6:17 PM 195TRAUMACHAPTER 7femur fractures, 800 to 1000 mL; and for pelvic fractures >2000 mL. Although no single injury can account for the patient’s hemo-dynamic instability, the sum of the injuries may result in life-threatening blood loss. The diagnostic measures advocated earlier are those that can be easily performed in the trauma bay. Transport of a hypotensive patient out of the ED for CT scan-ning is hazardous; monitoring is compromised, and the envi-ronment is suboptimal for dealing with acute problems. Fast track CT scanning should be used in all patients manifesting evidence of shock. The surgeon must accompany the patient and be prepared to abort the CT scan with diversion to the OR. This

1	Fast track CT scanning should be used in all patients manifesting evidence of shock. The surgeon must accompany the patient and be prepared to abort the CT scan with diversion to the OR. This dilemma is becoming less common in many trauma centers where CT scanning is available in the ED.The concept of hypotensive resuscitation remains contro-versial, and it is primarily relevant for patients with penetrating vascular injuries. Experimental work suggests that an endog-enous sealing clot of an injured artery may be disrupted at a SBP of >90 mmHg32; thus, many believe that this should be the preoperative blood pressure target for patients with potential noncompressible arterial injuries. On the other hand, optimal management of traumatic brain injury (TBI) includes maintain-ing the SBP >100 mmHg,33 and thus, hypotensive resuscitation is not appropriate for most blunt trauma patients.Secondary SurveyOnce the immediate threats to life have been addressed, a thor-ough history is obtained,

1	mmHg,33 and thus, hypotensive resuscitation is not appropriate for most blunt trauma patients.Secondary SurveyOnce the immediate threats to life have been addressed, a thor-ough history is obtained, and the patient is examined in a sys-tematic fashion. The patient and surrogates should be queried to obtain an AMPLE history (Allergies, Medications, Past illnesses or Pregnancy, Last meal, and Events related to the injury). The physical examination should be literally head to toe, with special attention to the patient’s back, axillae, and perineum, because injuries here are easily overlooked. All seriously injured patients should undergo digital rectal examination to evaluate for sphincter tone, presence of blood, rectal perforation, or a high-riding prostate; this is particularly critical in patients with suspected spinal cord injury, pelvic fracture, or transpelvic gun-shot wounds. Vaginal examination with a speculum should be performed in women with pelvic fractures to exclude an open

1	patients with suspected spinal cord injury, pelvic fracture, or transpelvic gun-shot wounds. Vaginal examination with a speculum should be performed in women with pelvic fractures to exclude an open fracture. Specific injuries, their associated signs and symptoms, diagnostic options, and treatments are discussed in detail later in this chapter.Adjuncts to the physical examination include vital sign and ECG monitoring, nasogastric tube placement, Foley cath-eter placement, radiographs, hemoglobin, base deficit measure-ments, urinalysis, and repeat FAST exam. A nasogastric tube should be inserted in all intubated patients to decrease the risk of gastric aspiration, but it may not be necessary in the awake patient. Nasogastric tube placement in patients with complex mid-facial fractures is contraindicated; rather, a tube should be placed orally if required. Nasogastric tube evaluation of stomach contents for blood may suggest occult gastroduodenal injury, or the errant path of the

1	is contraindicated; rather, a tube should be placed orally if required. Nasogastric tube evaluation of stomach contents for blood may suggest occult gastroduodenal injury, or the errant path of the nasogastric tube on a chest film may indicate a left diaphragm injury. A Foley catheter should be inserted in patients unable to void to decompress the blad-der, obtain a urine specimen, and monitor urine output. Gross hematuria demands evaluation of the genitourinary system for injury. Foley catheter placement may be deferred until urologic evaluation in patients with signs of urethral injury: blood at the meatus, perineal or scrotal hematomas, or a high-riding prostate. Although policies vary at individual institutions, most agree that patients in extremis should undergo one attempt at Foley catheter placement; if the catheter does not pass easily, a supra-pubic cystostomy should be considered.Selective radiography and laboratory tests are done early in the evaluation after the primary

1	catheter placement; if the catheter does not pass easily, a supra-pubic cystostomy should be considered.Selective radiography and laboratory tests are done early in the evaluation after the primary survey. For patients with severe blunt trauma, chest and pelvic radiographs should be obtained. Historically, a lateral cervical spine radiograph was also obtained, hence the reference to the big three films, but currently patients preferentially undergo CT scanning of the spine rather than plain film radiography. For patients with truncal gunshot wounds, anteroposterior and at times lateral radiographs of the chest and abdomen are warranted. It is important to mark the entrance and exit sites of penetrating wounds with ECG pads, metallic clips, or staples so that the trajectory of the missile can be estimated. Limited one-shot extremity radiographs may also be appropriate to assist in application of a splint. In critically injured patients, blood samples for a routine trauma panel (type

1	can be estimated. Limited one-shot extremity radiographs may also be appropriate to assist in application of a splint. In critically injured patients, blood samples for a routine trauma panel (type and crossmatch, complete blood count, blood chemistries, coagulation studies, and arterial blood gas analysis) should be sent to the laboratory. For less severely injured patients, only a complete blood count and urinalysis may be required. Because older patients may pres-ent in subclinical shock, even with minor injuries, routine analy-sis of an arterial blood gas in patients over the age of 55 should be considered. Repeat FAST is mandatory if there are any signs of abdominal injury or unexplained blood loss.Many trauma patients cannot provide specific information about the mechanism of their injury. Emergency medical ser-vice personnel and police are trained to evaluate an injury scene and should be questioned while they are present in the ED. For automobile collisions, the speed of the

1	injury. Emergency medical ser-vice personnel and police are trained to evaluate an injury scene and should be questioned while they are present in the ED. For automobile collisions, the speed of the vehicles involved, angle of impact, location of the patient within the vehicle, use of restraints, airbag deployment, condition of the steer-ing wheel and windshield, amount of intrusion, ejection of the patient from the vehicle, and fate of other passengers should be ascertained. For other injury mechanisms, critical information includes such things as height of a fall, surface impact, helmet use, and weight of an object by which the patient was crushed. In patients sustaining gunshot wounds, bullet characteris-tics, distance, and presumed path of the bullet are important, if known. For patients with stab wounds, the length and type of object is helpful. Finally, some patients experience a com-bination of blunt and penetrating trauma. Do not assume that someone who was stabbed was not

1	with stab wounds, the length and type of object is helpful. Finally, some patients experience a com-bination of blunt and penetrating trauma. Do not assume that someone who was stabbed was not also assaulted; the patient may have a multitude of injuries and cannot be presumed to have only injuries associated with the more obvious penetrat-ing wound. In short, these details of information are critical to the clinician to determine overall mechanism of injury and anticipate associated injury patterns.Mechanisms and Patterns of InjuryIn general, more energy is transferred over a wider area during blunt trauma than from a penetrating wound. As a result, blunt trauma is associated with multiple widely distributed injuries, whereas in penetrating wounds the damage is localized to the path of the bullet or knife. In blunt trauma, organs that can-not yield to impact by elastic deformation are most likely to be injured, namely, the solid organs (liver, spleen, and kidneys). For penetrating

1	the bullet or knife. In blunt trauma, organs that can-not yield to impact by elastic deformation are most likely to be injured, namely, the solid organs (liver, spleen, and kidneys). For penetrating trauma, organs with the largest surface area are most prone to injury (small bowel, liver, and colon). Addition-ally, because bullets and knives usually follow straight lines, adjacent structures are commonly injured.Patients who have sustained blunt trauma separate them into categories according to their risk for multiple injuries: those Brunicardi_Ch07_p0183-p0250.indd 19510/12/18 6:17 PM 196BASIC CONSIDERATIONSPART Isustaining high energy transfer injuries and those sustaining low energy transfer injuries. Injuries involving high energy transfer include auto-pedestrian accidents, motor vehicle collisions in which the car’s change of velocity (ΔV) exceeds 20 mph or in which the patient has been ejected, motorcycle collisions, and falls from heights >20 ft.34 In fact, for motor

1	motor vehicle collisions in which the car’s change of velocity (ΔV) exceeds 20 mph or in which the patient has been ejected, motorcycle collisions, and falls from heights >20 ft.34 In fact, for motor vehicle collisions the vari-ables strongly associated with life-threatening injuries, and hence reflective of the magnitude of the mechanism, are death of another occupant in the vehicle, extrication time of >20 minutes, ΔV >20 mph, lack of restraint use, and lateral impact.34 Low-energy trauma, such as being struck with a club or falling from a bicycle, usually does not result in widely distributed injuries. However, potentially lethal injuries of internal organs can occur because the net energy transfer to any given location may be substantial.In blunt trauma, particular constellations of injury or injury patterns are associated with specific injury mechanisms. For example, when an unrestrained driver sustains a frontal impact, the head strikes the windshield, the chest and upper

1	of injury or injury patterns are associated with specific injury mechanisms. For example, when an unrestrained driver sustains a frontal impact, the head strikes the windshield, the chest and upper abdomen hit the steering column, and the legs or knees contact the dashboard. The resultant injuries can include facial fractures, cervical spine fractures, injury of the descending thoracic aorta, myocardial contusion, injury to the spleen and liver, and fractures of the pel-vis and lower extremities. When such patients are evaluated, the discovery of one of these injuries should prompt a search for the others. Collisions with side impact also carry the risk of cervical spine and thoracic trauma, diaphragm rupture, and crush injuries of the pelvic ring, but solid organ injury usually is limited to either the liver or spleen based on the direction of impact. However, patients ejected from a vehicle or thrown a significant distance from a motorcycle have the risk of any injury

1	is limited to either the liver or spleen based on the direction of impact. However, patients ejected from a vehicle or thrown a significant distance from a motorcycle have the risk of any injury pattern.Penetrating injuries are classified according to the wound-ing agent (i.e., stab wound, gunshot wound, or shotgun wound). Gunshot wounds are subdivided further into highand low-velocity injuries because the speed of the bullet is much more important than its weight in determining potential kinetic energy transfer. High-velocity gunshot wounds (bullet speed >2000 ft/s) are infrequent in the civilian setting. Shotgun injuries are divided into close-range (<20 feet) and long-range wounds. Close-range shotgun wounds are tantamount to high-velocity wounds because the entire energy of the load is delivered to a small area, often with devastating results. In contrast, long-range shotgun blasts result in a diffuse pellet pattern in which many pellets miss the victim, and those that do strike

1	is delivered to a small area, often with devastating results. In contrast, long-range shotgun blasts result in a diffuse pellet pattern in which many pellets miss the victim, and those that do strike are dis-persed and are of comparatively low energy.Regional Assessment and Special Diagnostic TestsBased on mechanism, location of injuries identified on physi-cal examination, screening radiographs, and the patient’s over-all condition, additional diagnostic studies often are indicated. However, the seriously injured patient is in constant jeopardy when undergoing special diagnostic testing; therefore, the sur-geon must be in attendance and must be prepared to alter plans as circumstances demand. Hemodynamic, respiratory, and men-tal status will determine the most appropriate course of action. With these issues in mind, additional diagnostic tests are dis-cussed on an anatomic basis.Head. Evaluation of the head includes examination for injuries to the scalp, eyes, ears, nose, mouth,

1	With these issues in mind, additional diagnostic tests are dis-cussed on an anatomic basis.Head. Evaluation of the head includes examination for injuries to the scalp, eyes, ears, nose, mouth, facial bones, and intracra-nial structures. Palpation of the head is done to identify scalp lacerations, which should be evaluated for depth, and pres-ence of associated depressed or open skull fractures. The eye examination includes not only pupillary size and reactivity, but also examination for visual acuity and for hemorrhage within the globe. Ocular entrapment, caused by orbital fractures with impingement of the ocular muscles, is evident when the patient cannot move his or her eyes through an entire range of motion. It is important to perform the eye examination early because significant orbital swelling may prevent later evaluation. A lat-eral canthotomy may be needed to relieve periorbital pressure. The tympanic membrane is examined to identify hemotym-panum, otorrhea, or rupture, which

1	swelling may prevent later evaluation. A lat-eral canthotomy may be needed to relieve periorbital pressure. The tympanic membrane is examined to identify hemotym-panum, otorrhea, or rupture, which may signal an underlying head injury. Otorrhea, rhinorrhea, raccoon eyes, and Battle’s sign (ecchymosis behind the ear) suggest a basilar skull fracture. Although such fractures may not require treatment, there is an association with blunt cerebrovascular injuries, cranial nerve injuries, and risk of meningitis.Anterior facial structures should be examined to rule out fractures. This entails palpating for bony step-off of the facial bones and instability of the midface (by grasping the upper pal-ate and seeing if this moves separately from the patient’s head). A good question to ask awake patients is whether their bite feels normal to them; abnormal dental closure suggests malalignment of facial bones and the possibility for a mandible or maxillary fracture. Nasal fractures, which may be

1	is whether their bite feels normal to them; abnormal dental closure suggests malalignment of facial bones and the possibility for a mandible or maxillary fracture. Nasal fractures, which may be evident on direct inspec-tion or palpation, typically bleed vigorously. This may result in the patient having airway compromise due to blood running down the posterior pharynx, or there may be vomiting provoked by swallowed blood. Nasal packing or balloon tamponade may be necessary to control bleeding. Examination of the oral cavity includes inspection for open fractures, loose or fractured teeth, and sublingual hematomas.All patients with a significant closed head injury (GCS score <14) should undergo CT scanning of the head. Addition-ally, elderly patients or those patients on antiplatelet agents or anticoagulation should be imaged despite a GCS of 15.35,36 For penetrating injuries, plain skull films may be helpful in the trauma bay to determine the trajectory of the bullet. The presence of

1	or anticoagulation should be imaged despite a GCS of 15.35,36 For penetrating injuries, plain skull films may be helpful in the trauma bay to determine the trajectory of the bullet. The presence of later-alizing findings (e.g., a unilateral dilated pupil unreactive to light, asymmetric movement of the extremities either spontaneously or in response to noxious stimuli, or unilateral Babinski’s reflex) suggests an intracranial mass lesion or major structural damage.Such intracranial lesions following trauma include hemato-mas, contusions, hemorrhage into ventricular and subarachnoid spaces, and diffuse axonal injury (DAI). Epidural hematomas occur when blood accumulates between the skull and dura, and are caused by disruption of the middle meningeal artery or other small arteries in that potential space, typically after a skull frac-ture (Fig. 7-16). Subdural hematomas occur between the dura and cortex and are caused by venous disruption or laceration of the parenchyma of the brain. Due

1	space, typically after a skull frac-ture (Fig. 7-16). Subdural hematomas occur between the dura and cortex and are caused by venous disruption or laceration of the parenchyma of the brain. Due to associated parenchy-mal injury, subdural hematomas have a much worse prognosis than epidural collections. Hemorrhage into the subarachnoid space may cause vasospasm and further reduce cerebral blood flow. Intraparenchymal hematomas and contusions can occur anywhere within the brain. DAI results from high-speed decel-eration injury and represents direct axonal damage from shear effects. CT scan may demonstrate blurring of the gray and white matter interface and multiple small punctate hemorrhages, but magnetic resonance imaging is a more accurate test. Although prognosis for these injuries is extremely variable, early evidence of DAI is associated with a poor outcome. Stroke syndromes should prompt a search for carotid or vertebral artery injury using multislice CTA (Fig.

1	injuries is extremely variable, early evidence of DAI is associated with a poor outcome. Stroke syndromes should prompt a search for carotid or vertebral artery injury using multislice CTA (Fig. 7-17).Brunicardi_Ch07_p0183-p0250.indd 19610/12/18 6:17 PM 197TRAUMACHAPTER 7Significant intracranial penetrating injuries usually are produced by bullets from handguns, but an array of other weap-ons or instruments can injure the cerebrum via the orbit or through the thinner temporal region of the skull. Although the diagnosis usually is obvious, in some instances wounds in the auditory canal, mouth, and nose can be elusive. Prognosis is variable, but virtually all supratentorial wounds that injure both hemispheres are fatal.Neck. All blunt trauma patients should be assumed to have cer-vical spine injuries until proven otherwise. During the physi-cal examination, one must maintain cervical spine precautions and in-line stabilization. Due to the devastating consequences of quadriplegia, a

1	spine injuries until proven otherwise. During the physi-cal examination, one must maintain cervical spine precautions and in-line stabilization. Due to the devastating consequences of quadriplegia, a diligent evaluation for occult cervical spine injuries is mandatory. In the awake patient, the presence of posterior midline pain or tenderness should provoke a thor-ough radiologic evaluation. Additionally, intubated patients, patients with distracting injuries, significant mechanism, or another identified spine fracture should undergo CT imaging.37 A ligamentous injury may not be visible with standard imaging techniques. Flexion and extension views or magnetic resonance imaging (MRI) are obtained to further evaluate patients at risk or those with persistent symptoms.Spinal cord injuries can vary in severity. Complete injuries cause either quadriplegia or paraplegia, depending on the level of injury. These patients have a complete loss of motor func-tion and sensation two or more levels

1	vary in severity. Complete injuries cause either quadriplegia or paraplegia, depending on the level of injury. These patients have a complete loss of motor func-tion and sensation two or more levels below the bony injury. Patients with high spinal cord disruption are at risk for shock due to physiologic disruption of sympathetic fibers. Significant neurologic recovery is rare. However, there are several partial or incomplete spinal cord injury syndromes where the prognosis is better. Central cord syndrome typically occurs in older persons who experience hyperextension injuries. Motor function, pain, and temperature sensation are preserved in the lower extremi-ties but diminished in the upper extremities. Some functional ABFigure 7-16. Epidural hematomas (A) have a distinctive convex shape on computed tomo-graphic scan, whereas subdural hematomas (B) are concave along the surface of the brain.Figure 7-17. A. A right middle cerebral infarct noted on a computed tomographic scan of the

1	on computed tomo-graphic scan, whereas subdural hematomas (B) are concave along the surface of the brain.Figure 7-17. A. A right middle cerebral infarct noted on a computed tomographic scan of the head. Such a finding should prompt imaging to rule out an associated extracranial cerebrovascular injury. B. An inter-nal carotid artery pseudoaneurysm documented by angiography.Brunicardi_Ch07_p0183-p0250.indd 19710/12/18 6:17 PM 198BASIC CONSIDERATIONSPART Irecovery usually occurs, but is often not a return to normal. Anterior cord syndrome is characterized by diminished motor function, pain, and temperature sensation below the level of the injury, but position sensing, vibratory sensation, and crude touch are maintained. Prognosis for recovery is poor. Brown-Séquard syndrome is usually the result of a penetrating injury in which one-half of the spinal cord is transected. This lesion is characterized by the ipsilateral loss of motor function, proprio-ception, and vibratory sensation,

1	result of a penetrating injury in which one-half of the spinal cord is transected. This lesion is characterized by the ipsilateral loss of motor function, proprio-ception, and vibratory sensation, whereas pain and temperature sensation are lost on the contralateral side.During the primary survey, identification of injuries to the neck with exsanguination, expanding hematomas, airway obstruc-tion, or aerodigestive injuries is a priority. A more subtle injury that may not be identified is a fracture of the larynx due to blunt trauma. Signs and symptoms include hoarseness, subcutaneous emphysema (Fig. 7-18), or a palpable fracture. Penetrating inju-ries of the anterior neck that violate the platysma are potentially life-threatening because of the density of critical structures in this region. Although operative exploration is appropriate for overt injuries, selective nonoperative management has been proven safe (Fig. 7-19).38 Indications for immediate operative intervention for

1	region. Although operative exploration is appropriate for overt injuries, selective nonoperative management has been proven safe (Fig. 7-19).38 Indications for immediate operative intervention for penetrating cervical injury include hemodynamic instability, sig-nificant external hemorrhage, or evidence of aerodigestive injury. The management algorithm for hemodynamically stable patients is based on the presenting symptoms and anatomic location of injury, with the neck being divided into three distinct zones (Fig. 7-20). Zone I is inferior to the clavicles encompassing the thoracic outlet structures, zone II is between the thoracic outlet and the angle of the mandible, and zone III is above the angle of the mandible. Due to technical difficulties of injury expo-sure and varying operative approaches, a precise preoperative diagnosis is desirable for symptomatic zone I and III injuries. Therefore, these patients should ideally undergo diagnostic imag-ing before operation if they remain

1	approaches, a precise preoperative diagnosis is desirable for symptomatic zone I and III injuries. Therefore, these patients should ideally undergo diagnostic imag-ing before operation if they remain hemodynamically stable. Management of patients is further divided into those who are symptomatic and those who are not (see Fig 7-19). Specific symptoms or signs that should be identified include dysphagia, hoarseness, hematoma, venous bleeding, minor hemoptysis, and 5PenetratingNeck InjuryHemodynamically UnstableUncontrolled HemorrhageHard signs: massive hemoptysis, rapidly expanding hematomaOperativeExplorationObserveHemodynamically StableSoft signs: dysphagia, venous bleeding,subcutaneous emphysema, hematoma,hoarseness, stridor, odynophagiaAsymptomaticZone IZone IIZone IIIangioembolizationfor Zone III+Zone IZone IIZone IIITranscervical GSWAll OthersCTAneck/chest+angiographyesophagrambronchoscopyCTAneck/chest+ angiographyesophagrambronchoscopy+Figure 7-19. Algorithm for the management

1	Zone III+Zone IZone IIZone IIITranscervical GSWAll OthersCTAneck/chest+angiographyesophagrambronchoscopyCTAneck/chest+ angiographyesophagrambronchoscopy+Figure 7-19. Algorithm for the management of penetrating neck injuries. CTA = computed tomographic angiography; GSW = gunshot wound.Figure 7-18. A laryngeal fracture results in air tracking around the trachea along the prevertebral space (arrows).Brunicardi_Ch07_p0183-p0250.indd 19810/12/18 6:17 PM 199TRAUMACHAPTER 7subcutaneous emphysema. Symptomatic patients, without overt injuries, should undergo CTA with further evaluation or operation based upon the imaging findings. Overall, less than 15% of pen-etrating cervical trauma requires neck exploration.39 Asymptom-atic patients are typically observed for 6 to 12 hours. The one caveat is asymptomatic patients with a transcervical gunshot wound; these patients should undergo CTA to determine the tra-jectory of the bullet; further studies are performed based on prox-imity to major

1	is asymptomatic patients with a transcervical gunshot wound; these patients should undergo CTA to determine the tra-jectory of the bullet; further studies are performed based on prox-imity to major structures.39 Such additional imaging includes angiography, soluble contrast esophagram followed by barium esophagram, esophagoscopy, or bronchoscopy. Angiographic diagnosis, particularly of zone III injuries, can then be managed by selective angioembolization.Chest. Blunt trauma to the chest may involve the chest wall, tho-racic spine, heart, lungs, thoracic aorta and great vessels, and rarely the esophagus. Most of these injuries can be evaluated by physi-cal examination and chest radiography, with supplemental CT scanning to exclude vascular injury. Any patient who undergoes an intervention in the ED—endotracheal intubation, central line placement, tube thoracostomy—needs a repeat chest radiograph to document the adequacy of the procedure. This is particularly true in patients undergoing

1	the ED—endotracheal intubation, central line placement, tube thoracostomy—needs a repeat chest radiograph to document the adequacy of the procedure. This is particularly true in patients undergoing tube thoracostomy for a pneumothorax or hemothorax. Patients with persistent pneumothorax, large air leaks after tube thoracostomy, or difficulty ventilating should undergo fiber-optic bronchoscopy to exclude a tracheobronchial injury or presence of a foreign body. Patients with hemothorax must have a chest radiograph documenting complete evacuation of the chest; a persistent hemothorax that is not drained by two chest tubes is termed a caked hemothorax and mandates immediate thoracotomy IIIIIIIIFigure 7-20. For the purpose of evaluating penetrating injuries, the neck is divided into three zones. Zone I is to the level of the clavicu-lar heads and is also known as the thoracic outlet. Zone II is located between the clavicles and the angle of the mandible. Zone III is above the angle of the

1	Zone I is to the level of the clavicu-lar heads and is also known as the thoracic outlet. Zone II is located between the clavicles and the angle of the mandible. Zone III is above the angle of the mandible.(Fig. 7-21). Pneumomediastinum following blunt trauma that is identified on CT imaging is a poor predictor of aerodigestive injury; selective workup is appropriate.40Occult thoracic vascular injury must be diligently sought due to the high mortality of a missed lesion. Widening of the mediastinum on initial anteroposterior chest radiograph, caused by a hematoma around an injured vessel that is contained by the mediastinal pleura, suggests an injury of the great vessels. The mediastinal abnormality may suggest the location of the arterial injury (i.e., left-sided hematomas are associated with descend-ing blunt aortic injuries [BAI], whereas right-sided hematomas are seen with innominate injuries) (Fig. 7-22). Posterior rib frac-tures, sternal fractures with laceration of small

1	with descend-ing blunt aortic injuries [BAI], whereas right-sided hematomas are seen with innominate injuries) (Fig. 7-22). Posterior rib frac-tures, sternal fractures with laceration of small vessels, and mediastinal venous bleeding also can produce similar hemato-mas. Other chest radiographic findings suggestive of a BAI are summarized in Table 7-5 (Fig. 7-23). However, at least 7% of patients with a descending BAI have a normal chest radio-graph.41 Therefore, screening CTA is performed based on the mechanism of injury: high-energy deceleration motor vehicle collision with frontal or lateral impact (>30 mph frontal impact and >23 mph lateral impact), motor vehicle collision with ejection, falls of >25 ft, or direct impact (horse kick to chest, snowmobile, or ski collision with tree).42,43 In >95% of patients who survive to reach the ED, the BAI occurs just distal to the left subclavian artery, where it is tethered by the ligamen-tum arteriosum (Fig. 7-24). In 2% to 5% of patients

1	In >95% of patients who survive to reach the ED, the BAI occurs just distal to the left subclavian artery, where it is tethered by the ligamen-tum arteriosum (Fig. 7-24). In 2% to 5% of patients the injury occurs in the ascending aorta, in the transverse arch, or at the diaphragm. Reconstructions with multislice CTA obviate the need for invasive arteriography.42For penetrating thoracic trauma, physical examination, plain posteroanterior and lateral chest radiographs with metallic markings of wounds, and pericardial ultrasound will identify the majority of injuries.44 Injuries of the esophagus and tra-chea are the exceptions. Bronchoscopy should be performed to evaluate the trachea in patients with a persistent air leak from the chest tube or mediastinal air. Patients at risk for an esopha-geal injury should undergo bedside esophagoscopy or soluble contrast esophagography followed by barium examination to look for extravasation of contrast.45 As with neck injuries, 6Figure

1	an esopha-geal injury should undergo bedside esophagoscopy or soluble contrast esophagography followed by barium examination to look for extravasation of contrast.45 As with neck injuries, 6Figure 7-21. Persistence of a hemothorax despite two tube tho-racostomies is termed a caked hemothorax and is an indication for prompt thoracotomy.Brunicardi_Ch07_p0183-p0250.indd 19910/12/18 6:18 PM 200BASIC CONSIDERATIONSPART Ihemodynamically stable patients with transmediastinal gunshot wounds should undergo CT scanning to determine the path of the bullet; trajectory in proximity to vascular or visceral struc-tures dictates the need for angiography, endoscopy, or opera-tive plan. If there is a suspicion of a subclavian artery injury, brachial-brachial indices should be measured, but >60% of patients with an injury may not have a pulse deficit.46 Therefore, CTA should be performed based on injury proximity to intra-thoracic vasculature. Finally, with GSWs identified on the chest, penetrating

1	with an injury may not have a pulse deficit.46 Therefore, CTA should be performed based on injury proximity to intra-thoracic vasculature. Finally, with GSWs identified on the chest, penetrating trauma should not be presumed to be isolated to the thorax. Injury to contiguous body cavities (i.e., the abdomen and neck) must be excluded; plain radiographs are a rapid, effective screening modality to identify retained bullet fragments.Abdomen. The abdomen is a diagnostic black box. Fortunately, with few exceptions, it is not necessary to determine in the ED which intra-abdominal organs are injured, only whether an explor-atory laparotomy is necessary. However, physical examina-tion of the abdomen can be unreliable in making this determination, and drugs, alcohol, and head and spinal cord injuries can complicate the clinical evaluation. The presence of abdominal rigidity and hemodynamic compromise is an undisputed indication 7Figure 7-22. Location of the hematoma within the mediastinal

1	can complicate the clinical evaluation. The presence of abdominal rigidity and hemodynamic compromise is an undisputed indication 7Figure 7-22. Location of the hematoma within the mediastinal silhouette suggests the type of great vessel injury. A predominant hematoma on the left suggests the far more common descending torn aorta (A; arrows), whereas a hematoma on the right indicates a relatively unusual but life-threatening innominate artery injury (B; arrows).Table 7-5Findings on chest radiograph suggestive of a descending thoracic aortic tear1. Widened mediastinum2. Abnormal aortic contour3. Tracheal shift4. Nasogastric tube shift5. Left apical cap6. Left or right paraspinal stripe thickening7. Depression of the left main bronchus8. Obliteration of the aorticopulmonary window9. Left pulmonary hilar hematomaFigure 7-23. Chest film findings associated with descending torn aorta include apical capping (A; arrows) and tracheal shift (B; arrows).Brunicardi_Ch07_p0183-p0250.indd

1	pulmonary hilar hematomaFigure 7-23. Chest film findings associated with descending torn aorta include apical capping (A; arrows) and tracheal shift (B; arrows).Brunicardi_Ch07_p0183-p0250.indd 20010/12/18 6:18 PM 201TRAUMACHAPTER 7Figure 7-24. Imaging to diagnose descending torn aorta includes computed tomographic angiography (A), with three-dimensional reconstructions (B, anterior; C, posterior) demonstrating the proxi-mal and distal extent of the injury (arrows).for prompt surgical exploration. For the remainder of patients, a variety of diagnostic adjuncts are used to identify abdominal injury.The diagnostic approach differs for penetrating trauma and blunt abdominal trauma. As a rule, laparotomy is warranted for gunshot or shotgun wounds that penetrate the peritoneal cavity because most have significant internal injuries. The standard has been that anterior truncal gunshot wounds between the fourth intercostal space and the pubic symphysis whose trajectory as determined by

1	most have significant internal injuries. The standard has been that anterior truncal gunshot wounds between the fourth intercostal space and the pubic symphysis whose trajectory as determined by radiograph or wound location indicates perito-neal penetration should undergo laparotomy (Fig. 7-25). How-ever, there has been increased use of CT scanning to facilitate nonoperative management of abdominal GSWs.47 The exception is penetrating trauma isolated to the right upper quadrant; in hemodynamically stable patients with trajectory confined to the liver by CT scan, nonoperative observation may be reasonable.48 In obese patients, if the gunshot wound is thought to be tangen-tial through the subcutaneous tissues, CT scan can delineate the track and exclude peritoneal violation. Laparoscopy is another option to assess peritoneal penetration for tangential wounds it should not be done in unstable patients. In the scenario of tan-gential high energy GSWs, however, it is possible to sustain a

1	another option to assess peritoneal penetration for tangential wounds it should not be done in unstable patients. In the scenario of tan-gential high energy GSWs, however, it is possible to sustain a transmitted intraperitoneal hollow visceral injury due to a blast insult. Gunshot wounds to the back or flank are more difficult to evaluate because of the retroperitoneal location of the injured abdominal organs. Triple-contrast CT scan can delineate the trajectory of the bullet and identify peritoneal violation or retro-peritoneal entry, and associated injuries.In contrast to gunshot wounds, stab wounds that penetrate the peritoneal cavity are less likely to injure intra-abdominal organs. Anterior abdominal stab wounds (from costal margin to inguinal ligament and bilateral midaxillary lines) should be explored under local anesthesia in the ED to determine if the fascia has been violated. Injuries that do not penetrate the peri-toneal cavity do not require further evaluation, and the

1	should be explored under local anesthesia in the ED to determine if the fascia has been violated. Injuries that do not penetrate the peri-toneal cavity do not require further evaluation, and the patient Brunicardi_Ch07_p0183-p0250.indd 20110/12/18 6:18 PM 202BASIC CONSIDERATIONSPART Imay be discharged from the ED. Patients with fascial pen-etration must be further evaluated for intra-abdominal injury because there is up to a 50% chance of requiring laparotomy. Debate remains over whether the optimal diagnostic approach is serial examination, diagnostic peritoneal lavage (DPL), or CT scanning. The most recent evidence supports serial examination and laboratory evaluation.49,50 Patients with stab wounds to the right upper quadrant can undergo CT scanning to determine tra-jectory and confinement to the liver for potential nonoperative care.48 Those with stab wounds to the flank and back should undergo contrasted CT to assess for the potential risk of retro-peritoneal injuries of the

1	to the liver for potential nonoperative care.48 Those with stab wounds to the flank and back should undergo contrasted CT to assess for the potential risk of retro-peritoneal injuries of the colon, duodenum, and urinary tract.Penetrating thoracoabdominal wounds may cause occult injury to the diaphragm. Patients with gunshot or stab wounds to the left lower chest should be evaluated with diagnostic lapa-roscopy or DPL to exclude diaphragmatic injury. In general, penetrating right diaphragm injury is ignored unless there is a major underlying liver injury with a risk of biliopleural fistula. Diagnostic laparoscopy may be preferred in patients with a posi-tive chest radiograph (hemothorax or pneumothorax) or in those who would not tolerate a DPL. For patients undergoing DPL evaluation, laboratory value cutoffs to rule out diaphragm injury are different from traditional values formerly used for abdomi-nal stab wounds (Table 7-6). An RBC count of >10,000/μL is considered a positive finding

1	value cutoffs to rule out diaphragm injury are different from traditional values formerly used for abdomi-nal stab wounds (Table 7-6). An RBC count of >10,000/μL is considered a positive finding and an indication for abdominal evaluation; patients with a DPL RBC count between 1000/μL and 10,000/μL should undergo laparoscopy or thoracoscopy.PenetratingAbdominalTraumaHemodynamicallyUnstableHemodynamicallyStableOperatingRoomGSWSWAnteriorAbdomenRUQTangential*,Back/FlankCTScan AASWwith+ LWE** Back/FlankSerialExams/Labs+*Tangential GSWs may also be evaluated with diagnostic laparoscopy.** A positive local wound exploration is defined as violation of the posterior fascia. CTScanLeft-sided thoracoabdominalDPL vs.laparoscopy++Evisceration/PeritonitisFigure 7-25. Algorithm for the evaluation of penetrating abdominal injuries. AASW = anterior abdominal stab wound; CT = computed tomography; DPL = diagnostic peritoneal lavage; GSW = gunshot wound; LWE = local wound exploration; RUQ = right upper

1	abdominal injuries. AASW = anterior abdominal stab wound; CT = computed tomography; DPL = diagnostic peritoneal lavage; GSW = gunshot wound; LWE = local wound exploration; RUQ = right upper quadrant; SW = stab wound.Table 7-6Criteria for “positive” finding on diagnostic peritoneal lavageABDOMINAL TRAUMATHORACOABDOMINAL STAB WOUNDSRed blood cell count>100,000/mL>10,000/mLWhite blood cell count>500/mL>500/mLAmylase level>19 IU/L>19 IU/LAlkaline phosphatase level>2 IU/L>2 IU/LBilirubin level>0.01 mg/dL>0.01 mg/dLBlunt abdominal trauma is now evaluated initially by FAST examination, and this has supplanted DPL (Fig. 7-26). FAST is not 100% sensitive, however, so diagnostic peritoneal aspiration is warranted in hemodynamically unstable patients without a defined source of blood loss to rule out abdominal hemorrhage.31 FAST is used to identify free intraperitoneal fluid (Fig. 7-27) in Morrison’s pouch, the left upper quadrant, and the pelvis. Although this method is sensitive for

1	to rule out abdominal hemorrhage.31 FAST is used to identify free intraperitoneal fluid (Fig. 7-27) in Morrison’s pouch, the left upper quadrant, and the pelvis. Although this method is sensitive for detecting Brunicardi_Ch07_p0183-p0250.indd 20210/12/18 6:18 PM 203TRAUMACHAPTER 7HemodynamicallystableFAST +DPALaparotomyEquivocalPeritonitis?FAST +Candidate fornonoperativemanagementorpatient withcirrhosisIndications for CT:-Altered mental status-Confounding injury-Gross hematuria-Pelvic fracture-Abdominal tenderness-Unexplained Hct <35%Abdominal CTRepeat FASTin 30 minutesNoNoNoNoYesYesYesYesYesNoNoFigure 7-26. Algorithm for the initial evaluation of a patient with suspected blunt abdominal trauma. CT = computed tomography; DPA = diagnostic peritoneal aspiration; FAST = focused abdominal sonography for trauma; Hct = hematocrit.Figure 7-27. Focused abdominal sonography for trauma imag-ing detects intra-abdominal hemorrhage. Hemorrhage is presumed when a fluid stripe is visible

1	sonography for trauma; Hct = hematocrit.Figure 7-27. Focused abdominal sonography for trauma imag-ing detects intra-abdominal hemorrhage. Hemorrhage is presumed when a fluid stripe is visible between the right kidney and liver (A), between the left kidney and spleen (B), or in the pelvis (C).Brunicardi_Ch07_p0183-p0250.indd 20310/12/18 6:18 PM 204BASIC CONSIDERATIONSPART IFigure 7-28. Computed tomography images reveal critical infor-mation about solid organ injuries, such as associated contrast extravasation from a grade IV laceration of the spleen (A; arrow) and the amount of subcapsular hematoma in a grade III liver lacera-tion (B; arrows).intraperitoneal fluid of >250 mL, it does not reliably deter-mine the source of hemorrhage nor grade solid organ inju-ries.51 Patients with fluid on FAST examination, considered a “positive FAST,” who do not have immediate indications for laparotomy (hemodynamically stable, no evidence of perito-nitis) undergo CT scanning to quantify their

1	on FAST examination, considered a “positive FAST,” who do not have immediate indications for laparotomy (hemodynamically stable, no evidence of perito-nitis) undergo CT scanning to quantify their injuries. Injury grading using the American Association for the Surgery of Trauma (AAST) grading scale (Table 7-7) is an important component of nonoperative management of solid organ inju-ries. Additional findings that should be noted on CT scan in patients with solid organ injury include contrast extravasa-tion (i.e., a “blush”), the amount of intra-abdominal hemor-rhage, and presence of pseudoaneurysms (Fig. 7-28). CT also is indicated for hemodynamically stable patients for whom the physical examination is unreliable. Despite the increasing diagnostic accuracy of multidetector CT scanners, identifica-tion of intestinal injuries remains a limitation. Bowel injury is suggested by findings of thickened bowel wall, “streak-ing” in the mesentery, free fluid without associated solid organ

1	identifica-tion of intestinal injuries remains a limitation. Bowel injury is suggested by findings of thickened bowel wall, “streak-ing” in the mesentery, free fluid without associated solid organ injury, or free intraperitoneal air.52,53 Patients with free intra-abdominal fluid without solid organ injury are closely monitored for evolving signs of peritonitis; if patients have a significant closed head injury or cannot be serially examined, DPL should be performed to exclude bowel injury. If DPL is pursued, an infraumbilical approach is used (Fig. 7-29). After placement of the catheter, a 10-mL syringe is connected and the abdominal contents aspirated (termed a diagnostic perito-neal aspiration). The aspirate is considered positive if >10 mL of blood is aspirated. If <10 mL is withdrawn, a liter of normal saline is instilled. The effluent is withdrawn via siphoning and sent to the laboratory for RBC count, white blood cell (WBC) count, and determination of amylase, bilirubin, and

1	a liter of normal saline is instilled. The effluent is withdrawn via siphoning and sent to the laboratory for RBC count, white blood cell (WBC) count, and determination of amylase, bilirubin, and alkaline phosphatase levels. Values representing positive findings are summarized in Table 7-6.Table 7-7American Association for the Surgery of Trauma grading scales for solid organ injuriesSUBCAPSULAR HEMATOMALACERATIONLiver Injury GradeGrade I<10% of surface area<1 cm in depthGrade II10%–50% of surface area1–3 cmGrade III>50% of surface area or >10 cm in depth>3 cmGrade IV25%–75% of a hepatic lobeGrade V>75% of a hepatic lobeGrade VIHepatic avulsionSplenic Injury GradeGrade I<10% of surface area<1 cm in depthGrade II10%–50% of surface area1–3 cmGrade III>50% of surface area or >10 cm in depth>3 cmGrade IV>25% devascularizationHilumGrade VShattered spleenComplete devascularizationPelvis. Blunt injury to the pelvis may produce mechanically unstable fractures with major hemorrhage (Fig.

1	cmGrade IV>25% devascularizationHilumGrade VShattered spleenComplete devascularizationPelvis. Blunt injury to the pelvis may produce mechanically unstable fractures with major hemorrhage (Fig. 7-30). Plain radiographs will reveal gross abnormalities, but CT scanning is necessary to determine the precise geometry. Sharp spicules of bone can lacerate the bladder, rectum, or vagina. Alterna-tively, bladder rupture may result from a direct blow to the torso if the bladder is full. CT cystography is performed if the urinalysis demonstrates RBCs. Urethral injuries are suspected if examination reveals blood at the meatus, scrotal or perineal hematomas, or a high-riding prostate on rectal examination. Urethrograms should be obtained for stable patients before placing a Foley catheter to avoid false passage and subsequent stricture. Major vascular injuries of the external iliacs caus-ing bleeding are uncommon in blunt pelvic trauma; however, thrombosis of either the arteries or veins in the

1	passage and subsequent stricture. Major vascular injuries of the external iliacs caus-ing bleeding are uncommon in blunt pelvic trauma; however, thrombosis of either the arteries or veins in the iliofemoral system may occur, and CTA should be performed for evalu-ation if there is a pulse differential. Life-threatening hemor-rhage can be associated with pelvic fractures and may initially Brunicardi_Ch07_p0183-p0250.indd 20410/12/18 6:18 PM 205TRAUMACHAPTER 7Figure 7-29. Diagnostic peritoneal lavage is performed through an infraumbilical incision unless the patient has a pelvic fracture or is preg-nant. A. The linea alba is sharply incised, and the catheter is directed into the pelvis. B. The abdominal contents should initially be aspirated using a 10-mL syringe.preclude definitive imaging. Treatment algorithms for patients with complex pelvic fractures and hemodynamic instability are presented later in the chapter.Extremities. Blunt or penetrating trauma to the extremities requires

1	Treatment algorithms for patients with complex pelvic fractures and hemodynamic instability are presented later in the chapter.Extremities. Blunt or penetrating trauma to the extremities requires an evaluation for fractures, ligamentous disruption, and neurovascular injury. Plain radiographs are used to evaluate fractures, whereas ligamentous injuries, particularly those of the knee and shoulder, can be imaged with magnetic resonance imaging. Physical examination identifies the majority of arte-rial injuries, and findings are classified as either hard signs or soft signs of vascular injury (Table 7-8). In general, hard signs constitute indications for operative exploration, whereas soft signs are indications for further testing or observation. Bony fractures or knee dislocations should be realigned before defini-tive vascular examination. In management of vascular trauma, controversy exists regarding the treatment of patients with soft signs of injury, particularly those with injuries

1	realigned before defini-tive vascular examination. In management of vascular trauma, controversy exists regarding the treatment of patients with soft signs of injury, particularly those with injuries in proximity to major vessels. It is known that some of these patients will have arterial injuries that require repair. The most common approach has been to measure SBP using Doppler ultrasonography and compare the value for the injured side with that for the unin-jured side, termed the A-A index.54 If the pressures are within 10% of each other, a significant injury is unlikely, and no fur-ther evaluation is performed. If the difference is >10%, CTA or arteriography is indicated. Others argue that there are occult injuries, such as pseudoaneurysms or injuries of the profunda femoris or peroneal arteries, which may not be detected with this technique. If hemorrhage occurs from these injuries, com-partment syndrome and limb loss may occur. Although busy trauma centers continue to debate

1	arteries, which may not be detected with this technique. If hemorrhage occurs from these injuries, com-partment syndrome and limb loss may occur. Although busy trauma centers continue to debate this issue, the surgeon who is obliged to treat the occasional injured patient may be better served by performing CTA in selected patients with soft signs. In patients with hard signs of vascular injury, on-table angi-ography may be useful to localize the arterial injury and thus, limit tissue dissection. For example, a patient with an absent popliteal pulse and femoral shaft fracture due to a bullet that entered the lateral hip and exited below the medial knee could have injured either the femoral or popliteal artery anywhere along its course (Fig. 7-31).GENERAL PRINCIPLES OF MANAGEMENTOver the past 25 years there has been a remarkable change in management practices and operative approach for the injured patient. With the advent of CT scanning, nonoperative man-agement of solid organ injuries

1	25 years there has been a remarkable change in management practices and operative approach for the injured patient. With the advent of CT scanning, nonoperative man-agement of solid organ injuries has replaced routine operative exploration. Those patients who do require operation may be treated with less radical resection techniques, such as splen-orrhaphy or partial nephrectomy. Colonic injuries, previously mandating colostomy, are now repaired primarily in virtually all cases. Additionally, the type of anastomosis has shifted from a double-layer closure to a continuous running single-layer clo-sure; this method is technically equivalent to and faster than the interrupted multilayer techniques.55 Adoption of damage con-trol surgical techniques in physiologically deranged patients has resulted in limited initial operative time, with definitive injury repair delayed until after resuscitation in the surgical intensive care unit (SICU) with physiologic restoration.56 Abdominal drains,

1	in limited initial operative time, with definitive injury repair delayed until after resuscitation in the surgical intensive care unit (SICU) with physiologic restoration.56 Abdominal drains, once considered mandatory for parenchymal injuries and some anastomoses, have disappeared; fluid collections are managed by percutaneous techniques. Newer endovascular Brunicardi_Ch07_p0183-p0250.indd 20510/12/18 6:18 PM 206BASIC CONSIDERATIONSPART ITable 7-8Signs and symptoms of peripheral arterial injuryHARD SIGNS (OPERATION MANDATORY)SOFT SIGNS (FURTHER EVALUATION INDICATED)Pulsatile hemorrhageProximity to vasculatureAbsent pulsesSignificant hematomaAcute ischemiaAssociated nerve injuryA-A index of <0.9Thrill or bruitA-A index = systolic blood pressure on the injured side compared with that on the uninjured side.techniques such as stenting of arterial injuries and angioem-bolization are routine adjuncts. Blunt cerebrovascular injuries have been recognized as a significant, preventable

1	on the uninjured side.techniques such as stenting of arterial injuries and angioem-bolization are routine adjuncts. Blunt cerebrovascular injuries have been recognized as a significant, preventable source of neurologic morbidity and mortality. The use of preperitoneal pelvic packing for unstable pelvic fractures as well as early frac-ture immobilization with external fixators are paradigm shifts in management. Recently resuscitative endovascular balloon occlusion (REBOA) has been added to the armamentarium for life-threatening pelvic fracture bleeding57,58 (Fig. 7-32). Finally, the institution of massive transfusion protocols balances the benefit of blood component therapy against immunologic risk. Viscoelastic hemostatic assays (TEG and ROTEM) have been shown to be superior to traditional laboratory tests and have been central to the evolving concept of goal-directed hemo-stasis.59 These many conceptual changes have significantly improved survival of critically injured patients; they

1	laboratory tests and have been central to the evolving concept of goal-directed hemo-stasis.59 These many conceptual changes have significantly improved survival of critically injured patients; they have been promoted and critically reviewed by academic trauma centers via forums such as the American College of Surgeons Com-mittee on Trauma, the American Association for the Surgery of Trauma, the International Association of Trauma Surgery and Intensive Care, the Pan-American Trauma Congress, and other surgical organizations.Transfusion PracticesInjured patients with life-threatening hemorrhage develop acute coagulopathy of trauma (ACOT).60,61 The mechanism for inad-equate clot formation remains uncertain, but it is believed to involve activation of protein C, which impairs Va and VIIa, glycocalyx breakdown, which releases heparin sulfate, immune Figure 7-30. The three types of mechanically unstable pelvis fractures are lateral compression (A), anteroposterior compression (B), and

1	glycocalyx breakdown, which releases heparin sulfate, immune Figure 7-30. The three types of mechanically unstable pelvis fractures are lateral compression (A), anteroposterior compression (B), and vertical shear (C).Brunicardi_Ch07_p0183-p0250.indd 20610/12/18 6:18 PM 207TRAUMACHAPTER 7activation with the releases of DAMPs, DNA, histone, poly-phosphate, and PMN elastase, and complement activation. Fibrinolysis is an important component of the ACOT; hyper-fibrinolysis and fibrinolysis shutdown are both associated with increased mortality.62 Fresh whole blood, arguably the optimal replacement, has not been available in the United States since the early 1980s. Rather, its component parts, packed red blood cells (PRBCs), fresh frozen plasma, platelets, and cryoprecipi-tate, are administered. Specific transfusion triggers for indi-vidual blood components remain debated.63 Although current critical care guidelines indicate that PRBC transfusion should occur once the patient’s

1	administered. Specific transfusion triggers for indi-vidual blood components remain debated.63 Although current critical care guidelines indicate that PRBC transfusion should occur once the patient’s hemoglobin level is <7 g/dL,64 in the acute phase of resuscitation a hemoglobin of 10 g/dL is suggested to facilitate hemostasis via platelet margination.65 The traditional Figure 7-31. On-table angiography in the operating room isolates the area of vascular injury to the superficial femoral artery in a patient with a femoral fracture after a gunshot wound to the lower extremity.ABthresholds for blood component replacement in the patient mani-festing a coagulopathy have been INR >1.5, PTT >1.5 normal, platelet count >50,000/μL, and fibrinogen >100 mg/dl. However, these guidelines have been replaced by TEG and ROTEM cri-teria in many trauma centers. Such guidelines are designed to limit the transfusion of immunologically active blood compo-nents and decrease the risk of

1	have been replaced by TEG and ROTEM cri-teria in many trauma centers. Such guidelines are designed to limit the transfusion of immunologically active blood compo-nents and decrease the risk of transfusion-associated lung injury and secondary multiple organ failure.66,67In the critically injured patient requiring large amounts of blood component therapy, a massive transfusion protocol should be followed (Fig. 7-33). This approach calls for administration of various components in a specific ratio during transfusion to achieve restoration of blood volume to reverse shock and cor-rect coagulopathy. Although the optimal ratio is unknown, cur-rent evidence suggests a presumptive 1:2 red cell:plasma ratio in patients at risk for massive transfusion (10 units of PRBCs in 6 hours).67-72 Because complete typing and cross-matching takes up to 45 minutes, patients requiring emergent transfusions are given type O-negative RBCs. Similarly, without time for blood typing, AB plasma is the universal

1	typing and cross-matching takes up to 45 minutes, patients requiring emergent transfusions are given type O-negative RBCs. Similarly, without time for blood typing, AB plasma is the universal donor, although A plasma appears to be a safe option. Blood typing, and to a lesser extent cross-matching, is essential to avoid life-threatening intravas-cular hemolytic transfusion reactions. Trauma centers and their associated blood banks must have the capability of transfusing tremendous quantities of blood components because it is not unusual to have >50 component units transfused during one procedure and have the patient survive. Massive transfusion protocols, established preemptively, permit coordination of the activities of surgeons, anesthesiologists, and blood bankers to facilitate transfusion of the appropriate blood products.Postinjury coagulopathy due to shock is aggravated by core hypothermia and metabolic acidosis, termed the bloody vicious cycle,56 and now commonly referred to as

1	of the appropriate blood products.Postinjury coagulopathy due to shock is aggravated by core hypothermia and metabolic acidosis, termed the bloody vicious cycle,56 and now commonly referred to as the lethal triad. The pathophysiology is multifactorial and includes inhibition of temperature-dependent enzyme-acti-vated coagulation cascades, platelet dysfunction, endothelial abnormalities, and fibrinolytic activity. Such coagulopathy may be insidious, so the surgeon must be cognizant of subtle signs such as excessive bleeding from the cut edges of skin. Point-of-care viscoelastic assays (TEG and ROTEM), which provide a comprehensive assessment of clot capacity and fibri-nolysis, can provide useful information within 15 minutes. In contrast, traditional laboratory tests of coagulation capability (i.e., INR, PTT, fibrinogen levels, and platelet count) requires at least 45 minutes. Using damage control techniques to limit operative time and provide physiologic restoration in the SICU can be

1	(i.e., INR, PTT, fibrinogen levels, and platelet count) requires at least 45 minutes. Using damage control techniques to limit operative time and provide physiologic restoration in the SICU can be lifesaving (see “Damage Control Surgery”).8Figure 7-32. A. Resuscitative endovascular balloon of the aorta (REBOA) is introduced through a 7Fr arterial sheath in the common femoral artery using ultrasound guidance. B. For patients with pelvic fractures and hemodynamic instabil-ity, the balloon is positioned in zone III just above the aortic bifurcation; a mixture of saline and contrast is used to inflate the balloon, which can be visualized on bedside plain radiography.Brunicardi_Ch07_p0183-p0250.indd 20710/12/18 6:18 PM 208BASIC CONSIDERATIONSPART IFigure 7-33. Denver Health Medical Center’s Massive Transfusion Protocol (MTP). ACT = activated clotting time; Cryo = cryoprecipitate; FFP = fresh frozen plasma; MA = maximum amplitude; PRBC = packed red blood cells; PTT = partial

1	Center’s Massive Transfusion Protocol (MTP). ACT = activated clotting time; Cryo = cryoprecipitate; FFP = fresh frozen plasma; MA = maximum amplitude; PRBC = packed red blood cells; PTT = partial thromboplastin time; SBP = systolic blood pressure; TEG = thromboelastography.Prophylactic MeasuresAll injured patients undergoing an operation should receive preoperative antibiotics. The type of antibiotic is determined by the anticipated source of contamination in the abdomen or other operative region; additional doses should be adminis-tered during the procedure based on blood loss and the half-life of the antibiotic. Extended postoperative antibiotic therapy is administered only for contaminated open fractures. Tetanus prophylaxis is administered to all patients according to pub-lished guidelines.Trauma patients are at risk for venous thromboembolism and its associated morbidity and mortality. In fact, pulmo-nary embolus can occur much earlier in the patient’s hospital course than

1	guidelines.Trauma patients are at risk for venous thromboembolism and its associated morbidity and mortality. In fact, pulmo-nary embolus can occur much earlier in the patient’s hospital course than previously believed.73 Patients at higher risk for venous thromboembolism are those with multiple fractures of the pelvis and lower extremities, those with TBI or spinal cord injury, and those requiring ligation of large veins in the abdo-men and lower extremities. Morbidly obese patients and those over 55 years of age are at additional risk. Administration of low molecular weight heparin (LMWH) is initiated as soon MASSIVE TRANSFUSION PROTOCOL FOR TRAUMAAny of these in the ED:• Penetrating Torso Injury• Major Pelvic Fracture• FAST ˜ >1 Body Region• SBP 70 OR• SBP 71–90 AND HR š108 if ... your patient has these VS in the FIELD or EDACTIVATE MTPCaCl2 1 gr. i.v.Transfuse RBC 4 Unitsand FFP 2 UnitsOrder Citrated Rapid TEGContinue to component transfusion based on TEG Resultsif

1	if ... your patient has these VS in the FIELD or EDACTIVATE MTPCaCl2 1 gr. i.v.Transfuse RBC 4 Unitsand FFP 2 UnitsOrder Citrated Rapid TEGContinue to component transfusion based on TEG Resultsif patient is bleedingANDACT>128 sec Angle<65°MA<55 mmLY30 š10%FFP 2 UnitsCryo10 UnitsPlatelets1 UnitTXA1 gmReassess via Citrated Rapid TEGMassive Transfusion ProtocolTriggers: SBP <70 with penetrating torso injury, major pelvic injury, FAST + SBP <71-90 mmHg and HR >108 with penetrating torso injury, major pelvic injury, FAST + ** order citrated rapid TEGContinued Treatment of ShockHemorrhage Control, Correct Hypothermia, Correct AcidosisNormalize Ca++Empiric Transfusion Until Lab Results AvailableShipmentPRBCsFFPPlateletsCryo142242110TEG Based Resuscitation*rapidTEG-ACT >128 sec ˜ 2 units thawed plasmarapidTEG-MA <55mm ˜ 1 unit of apheresis plateletsrapidTEG-angle <66 degrees ˜ 10 units pooled cryoprecipitaterapidTEG EPL >9% ˜ 1g tranexamic acid*Transfusion Triggers if TEGis

1	2 units thawed plasmarapidTEG-MA <55mm ˜ 1 unit of apheresis plateletsrapidTEG-angle <66 degrees ˜ 10 units pooled cryoprecipitaterapidTEG EPL >9% ˜ 1g tranexamic acid*Transfusion Triggers if TEGis UnavailablePT, PTT > 1.5 control ˜2 units thawed plasmaPlatelet count <50,000/mcL ˜1 unit of apheresis plateletsFibrinogen <100 mg/dL ˜10 units pooled cryoprecipitateBrunicardi_Ch07_p0183-p0250.indd 20810/12/18 6:18 PM 209TRAUMACHAPTER 7Figure 7-34. A. Unilateral neck exploration is performed through an incision along the anterior border of the sternocleidomastoid muscle; exposure of the carotid artery requires early division of the facial vein. B. The distal internal carotid artery is exposed by dividing the ansa cervicalis, which permits mobilization of the hypoglossal nerve. C. Further exposure is facilitated by resection of the posterior belly of the digastric muscle.as bleeding has been controlled and there is stable intracranial pathology. In high-risk patients, antiplatelet

1	exposure is facilitated by resection of the posterior belly of the digastric muscle.as bleeding has been controlled and there is stable intracranial pathology. In high-risk patients, antiplatelet therapy should be added.74 Removable inferior vena caval filters should be con-sidered if there are prolonged contraindications to administra-tion of LMWH. Additionally, pulsatile compression stockings (also termed sequential compression devices) are used routinely unless there is a fractured lower extremity or vascular injury.A final prophylactic measure that is usually not consid-ered is thermal protection. Hemorrhagic shock impairs perfu-sion and metabolic activity throughout the body, with resultant decrease in heat production and body temperature. Removing the patient’s clothes causes a second thermal insult, and infu-sion of cold PRBCs or room temperature crystalloid exacerbates the problem. As a result, injured patients can become hypother-mic, with temperatures below 34°C (93.2°F)

1	thermal insult, and infu-sion of cold PRBCs or room temperature crystalloid exacerbates the problem. As a result, injured patients can become hypother-mic, with temperatures below 34°C (93.2°F) upon arrival in the OR. Hypothermia aggravates coagulopathy and provokes myocardial irritability. Therefore, prevention must begin in the ED by maintaining a comfortable ambient temperature, cover-ing patients with warm blankets, and administering warmed IV fluids and blood products. Additionally, in the OR a Bair Hug-ger® warmer (the upper body or lower body blanket) and heated inhalation via the ventilatory circuit is instituted. For cases of severe hypothermia (temperature <30°C [86°F]), arteriovenous rewarming should be considered.Operative Approaches and ExposureCervical Exposure. Operative exposure for midline structures of the neck (e.g., trachea, thyroid, bilateral carotid sheaths) is obtained through a collar incision; this is typically performed two finger breadths above the sternal

1	for midline structures of the neck (e.g., trachea, thyroid, bilateral carotid sheaths) is obtained through a collar incision; this is typically performed two finger breadths above the sternal notch, but can be varied based on the level of anticipated injury. After subplatysmal flap elevation, the strap muscles are divided in the midline to gain access to the central neck compartment. More superior and lateral structures are accessed by extending the collar incision upward along the sternocleidomastoid muscle; this may be done bilaterally if neces-sary. For unilateral injuries, neck exploration is done through an incision extending from the mastoid down to the clavicle, along the anterior border of the sternocleidomastoid muscle (Fig. 7-34). Brunicardi_Ch07_p0183-p0250.indd 20910/12/18 6:19 PM 210BASIC CONSIDERATIONSPART IBFigure 7-36. A. A “clamshell” thoracotomy provides exposure to bilateral thoracic cavities. B. Sternal transection requires individual ligation of both the

1	6:19 PM 210BASIC CONSIDERATIONSPART IBFigure 7-36. A. A “clamshell” thoracotomy provides exposure to bilateral thoracic cavities. B. Sternal transection requires individual ligation of both the proximal and distal internal mammary arteries on the undersurface of the sternum.123Figure 7-35. Options for thoracic exposure include the most versa-tile incision, the anterolateral thoracotomy (1), as well as a median sternotomy (2) and a “trap door” thoracotomy (3). Any thoracic incision may be extended into a supraclavicular or anterior neck incision for wider exposure.The carotid sheath, containing the carotid artery, jugular vein, and vagus nerve, is opened widely to examine these structures. The facial vein, which marks the carotid bifurcation, is usually ligated for exposure of the internal carotid artery and the hypoglossal nerve is the next structure encountered.Exposure of the distal carotid artery in zone III is difficult (see Fig. 7-34). The first step is division of the ansa

1	carotid artery and the hypoglossal nerve is the next structure encountered.Exposure of the distal carotid artery in zone III is difficult (see Fig. 7-34). The first step is division of the ansa cervicalis to facilitate mobilization of the hypoglossal nerve. Next, the poste-rior portion of the digastric muscle, which overlies the internal carotid, is transected. The glossopharyngeal and vagus nerves are also mobilized and retracted as necessary. If accessible, the styloid process and attached muscles are removed. In desperate situations, anterior displacement of the mandible (subluxation) may be helpful or the vertical ramus of the mandible may be divided. However, the latter maneuver often entails resection of the parotid gland, and the facial nerve is at risk of injury.Thoracic Incisions. An anterolateral thoracotomy, with the patient placed supine, is the most versatile incision for emer-gent thoracic exploration. The location of the incision is in the fifth interspace, in the

1	anterolateral thoracotomy, with the patient placed supine, is the most versatile incision for emer-gent thoracic exploration. The location of the incision is in the fifth interspace, in the inframammary line (Fig. 7-35). If access is needed to both pleural cavities, the original incision can be extended across the sternum with a Lebsche knife, into a “clam-shell” thoracotomy (Fig. 7-36). If the sternum is divided, the internal mammary arteries should be ligated to prevent blood loss. The heart, lungs, descending aorta, pulmonary hilum, and esophagus are accessible with this approach. For control of the great vessels, the superior portion of the sternum may be divided vertically with extension of the incision into the neck considered. A method advocated for access to the proximal left subclavian artery is through a fourth interspace anterolateral thoracotomy, superior sternal extension, and left supraclavicular incision (“trap door” thoracotomy). Although the trap door procedure is

1	artery is through a fourth interspace anterolateral thoracotomy, superior sternal extension, and left supraclavicular incision (“trap door” thoracotomy). Although the trap door procedure is appro-priate after resuscitative thoracotomy, the proximal left subcla-vian artery can be accessed more easily via a sternotomy with a supraclavicular extension. If the left subclavian artery is injured outside the thoracic outlet, vascular control can be obtained via the sternotomy and definitive repair done through the supraclavicular incision. Emergent median sternotomy is optimal for anterior stab wounds to the heart. Typically, these patients have pericardial tamponade and may undergo placement of a pericardial drain before a semiurgent median sternotomy is performed. Patients in extremis, however, should undergo anterolateral thoracotomy.Median sternotomy with cervical extension is used for rapid exposure in patients with presumed proximal subclavian, innominate, or proximal carotid artery

1	should undergo anterolateral thoracotomy.Median sternotomy with cervical extension is used for rapid exposure in patients with presumed proximal subclavian, innominate, or proximal carotid artery injuries. Care must be taken to avoid injury to the phrenic and vagus nerves that pass over the subclavian artery and to the recurrent laryngeal nerve passing posteriorly. Posterolateral thoracotomies are used for exposure of injuries to the trachea or main stem bronchi near the carina or the upper esophagus (right posterolateral thoracotomy) and tears of the descending thoracic aorta or lower esophagus (left posterolateral thoracotomy).Emergent Abdominal Exploration. Abdominal exploration in adults is performed using a midline incision because of its versatility. For children under the age of 6, a transverse inci-sion may be advantageous. Making the incision is faster with a scalpel than with an electrosurgical unit; incisional abdominal wall bleeding should be ignored until intra-abdominal

1	a transverse inci-sion may be advantageous. Making the incision is faster with a scalpel than with an electrosurgical unit; incisional abdominal wall bleeding should be ignored until intra-abdominal sources of hemorrhage are controlled. Liquid and clotted blood are Brunicardi_Ch07_p0183-p0250.indd 21010/12/18 6:19 PM 211TRAUMACHAPTER 7Figure 7-37. A sagittal view of packs placed to control hepatic hemorrhage. LAP = laparotomy.Figure 7-38. The Pringle maneuver, performed with a vascular clamp, occludes the hepatic pedicle containing the portal vein, hepatic artery, and common bile duct.evacuated with multiple laparotomy pads to identify the major source(s) of active bleeding. After blunt trauma, the spleen and liver should be palpated first and packed if fractured, and the infracolic mesentery should be inspected for zone I vascular injury. In contrast, after a penetrating wound the search for bleeding should pursue the trajectory of the penetrating device. If the patient has an

1	mesentery should be inspected for zone I vascular injury. In contrast, after a penetrating wound the search for bleeding should pursue the trajectory of the penetrating device. If the patient has an SBP of <70 mmHg when the abdomen is opened, digital pressure or a clamp should be placed on the aorta at the diaphragmatic hiatus. After the source of hemorrhage is localized, direct digital occlusion (vascular injury) or laparot-omy pad packing (solid organ injury) is used to control bleed-ing (Fig. 7-37). If the liver is the source in a hemodynamically Figure 7-39. To mobilize the spleen, an incision is made into the endoabdominal fascia 1 cm lateral to the reflection of the peritoneum onto the spleen (A). While the spleen is gently rotated medially, a plane is developed between the pancreas and left kidney (B). With complete mobilization, the spleen can reach the level of the abdominal incision.unstable patient, additional control of bleeding is obtained by clamping the hepatic pedicle

1	and left kidney (B). With complete mobilization, the spleen can reach the level of the abdominal incision.unstable patient, additional control of bleeding is obtained by clamping the hepatic pedicle with a vascular clamp or Rummel tourniquet (termed the Pringle maneuver) (Fig. 7-38). Similarly, clamping the splenic hilum may be required for hilar bleeding. When the spleen is mobilized, it should be gently rotated medi-ally to expose the lateral peritoneum; this peritoneum and endo-abdominal fascia are incised, which allows blunt dissection of the spleen and pancreas as a composite from the retroperito-neum anterior to Gerota’s fascia (Fig. 7-39).Rapid exposure of the intra-abdominal vasculature can prove challenging in the face of exsanguinating hemorrhage. Proximal control of the aorta is obtained at the diaphragmatic hiatus; if an aortic injury is supraceliac, transecting the left crus of diaphragm or extending the laparotomy via a left thoracotomy may be necessary.

1	of the aorta is obtained at the diaphragmatic hiatus; if an aortic injury is supraceliac, transecting the left crus of diaphragm or extending the laparotomy via a left thoracotomy may be necessary. Brunicardi_Ch07_p0183-p0250.indd 21110/12/18 6:19 PM 212BASIC CONSIDERATIONSPART IAn alternative for a contained hematoma is placement of a trans-femoral REBOA into zone I. The first decision is whether the patient has a supracolic or an infracolic vascular injury. Supracolic injuries (aorta, celiac axis, proximal superior mesenteric artery [SMA], and left renal arteries) are best approached via a left medial visceral rotation (Fig. 7-40). This is done by incising the lateral peritoneal reflection (white line of Toldt) beginning at the distal descending colon and extending the incision along the colonic splenic flexure, around the posterior aspect of the spleen, and behind the gastric fundus, ending at the esophagus. The left colon, spleen, pancreas, and stomach are then rotated toward

1	the colonic splenic flexure, around the posterior aspect of the spleen, and behind the gastric fundus, ending at the esophagus. The left colon, spleen, pancreas, and stomach are then rotated toward the midline. The authors prefer to leave the kidney in situ when mobilizing the viscera because this exaggerates the separation of the renal vessels from the SMA. The operative approach for SMA injuries is based on the level of injury. Fullen zone I SMA injuries, located posterior to the pancreas, are best exposed by a left medial visceral rotation. Fullen zone II SMA injuries, extending from the pancreatic edge to the middle colic branch, on the other hand, are approached via the lesser sac along the inferior edge of the pancreas at the base of the transverse mesocolon; the pancreatic body may be divided to gain proximal vascular access. More distal SMA injuries, Fullen zones III and IV, are approached directly within the mesentery. A venous injury behind the pancreas, from the junction of

1	be divided to gain proximal vascular access. More distal SMA injuries, Fullen zones III and IV, are approached directly within the mesentery. A venous injury behind the pancreas, from the junction of the superior mesenteric, splenic, and portal veins, is accessed by dividing the neck of the pancreas. Inferior vena cava injuries are approached by a right medial visceral rotation (Fig. 7-41). Proximal control is Figure 7-41. A right medial visceral rotation is used to expose the infrahepatic vena cava.Figure 7-40. A left medial visceral rotation is used to expose the abdominal aorta.obtained just above the iliac bifurcation with direct pressure via a sponge stick; the injury is identified by cephalad dissection along the anterior surface of the inferior vena cava. A Satinsky clamp can be used to control anterior caval wounds.Injuries of the iliac vessels pose a unique problem for emergent vascular control due to the number of vessels, their close proximity, and cross circulation.

1	be used to control anterior caval wounds.Injuries of the iliac vessels pose a unique problem for emergent vascular control due to the number of vessels, their close proximity, and cross circulation. Proximal control at the infrarenal aorta arrests the arterial bleeding and avoids splanch-nic and renal ischemia; however, venous injuries are not con-trolled with aortic clamping. Tamponade with digital pressure or with a folded laparotomy pad held directly over the bleeding site usually will establish hemostasis sufficient to prevent exsan-guination. If hemostasis is not adequate to expose the vessel proximal and distal to the injury, sponge sticks can be strategi-cally placed on either side of the injury and carefully adjusted to improve hemostasis. Alternatively, complete pelvic vascular isolation (Fig. 7-42) may be required to control hemorrhage for adequate visualization of the injuries. The right common iliac artery obscures the bifurcation of the vena cava and the right iliac vein;

1	(Fig. 7-42) may be required to control hemorrhage for adequate visualization of the injuries. The right common iliac artery obscures the bifurcation of the vena cava and the right iliac vein; the iliac artery may require division to expose venous injuries in this area (Fig. 7-43). The artery must be repaired after the venous injury is treated, however, because of limb-threatening ischemia.Once overt hemorrhage is controlled, sources of enteric contamination are identified by serially running along the small and large bowel, looking at all surfaces. Associated hematomas should be unroofed to rule out adjacent bowel injury. The ante-rior and posterior aspects of the stomach should be inspected, which requires opening the lesser sac for complete visualiza-tion. Duodenal injuries should be evaluated with a wide Kocher maneuver. During exploration of the lesser sac, visualization and palpation of the pancreas is done to exclude injury. Palpat-ing the anterior surface is not sufficient

1	evaluated with a wide Kocher maneuver. During exploration of the lesser sac, visualization and palpation of the pancreas is done to exclude injury. Palpat-ing the anterior surface is not sufficient because the investing Brunicardi_Ch07_p0183-p0250.indd 21210/12/18 6:19 PM 213TRAUMACHAPTER 7fascia may mask a pancreatic injury; mobilization, including evaluation of the posterior aspect, is critical. After injuries are identified, whether to use damage control techniques or per-form primary repair of injuries is based on the patient’s intraop-erative physiologic status (see “Damage Control Surgery” and “Treatment of Specific Injuries”). In a patient with multisys-tem trauma, enteral access via gastrostomy or jejunostomy tube should be considered. If abdominal closure is indicated after the patient’s injuries are addressed, the abdomen is irrigated with warm saline and the midline fascia is closed with a running heavy absorbable suture. The skin is closed selectively based on the

1	the patient’s injuries are addressed, the abdomen is irrigated with warm saline and the midline fascia is closed with a running heavy absorbable suture. The skin is closed selectively based on the amount of intra-abdominal contamination.Figure 7-42. Pelvic vascular isolation. A. Initially, clamps are placed on the aorta, inferior vena cava, and bilateral external iliac vessels. B. With continued dissection, the clamps can be moved progressively closer to the vascular injury to limit unwarranted ischemia.Figure 7-43. The right common iliac artery can be divided to expose the bifurcation of the inferior vena cava and the right common iliac vein.Table 7-9Options for the treatment of vascular injuriesObservationLigationLateral suture repairEnd-to-end primary anastomosisInterposition graftsAutogenous veinPolytetrafluoroethylene graftDacron graftTranspositionsExtra-anatomic bypassEndovascularStentsEmbolizationVascular Repair Techniques. Initial control of vascular inju-ries is accomplished

1	veinPolytetrafluoroethylene graftDacron graftTranspositionsExtra-anatomic bypassEndovascularStentsEmbolizationVascular Repair Techniques. Initial control of vascular inju-ries is accomplished digitally by applying enough direct pres-sure to stop the hemorrhage. Sharp dissection is used to define the injury and mobilize sufficient length for proximal and distal control. Fogarty thromboembolectomy should be done proxi-mally and distally to optimize collateral blood flow. Heparin-ized saline (50 units/mL) is then injected into the proximal and distal ends of the injured vessel to prevent small clot formation on the exposed intima and media. Ragged edges of the injury site should be debrided using sharp dissection. Intravascular shunts are used when there are multiple life-threatening injuries or the arterial injury is anticipated to require saphenous vein interposition reconstruction.Options for the treatment of vascular injuries are listed in Table 7-9. Arterial repair should always be

1	or the arterial injury is anticipated to require saphenous vein interposition reconstruction.Options for the treatment of vascular injuries are listed in Table 7-9. Arterial repair should always be done for the aorta, carotid, innominate, brachial, superior mesenteric, proper hepatic, renal, iliac, femoral, and popliteal arteries. Named arter-ies that usually tolerate ligation include the right or left hepatic artery and the celiac artery. In the lower extremities, at least one artery with distal runoff should be salvaged. Arterial injuries that may be treated nonoperatively include small pseudoaneurysms, intimal dissections, small intimal flaps, and small arteriovenous Brunicardi_Ch07_p0183-p0250.indd 21310/12/18 6:19 PM 214BASIC CONSIDERATIONSPART IFigure 7-45. The parachute technique is helpful for accurate placement of the posterior sutures of an anastomosis when the arte-rial end is fixed and an interposition graft is necessary. Traction must be maintained on both ends of the

1	is helpful for accurate placement of the posterior sutures of an anastomosis when the arte-rial end is fixed and an interposition graft is necessary. Traction must be maintained on both ends of the suture to prevent loosening and leakage of blood. Six stitches should be placed before the graft is pulled down to the artery.Figure 7-44. Small arteries repaired with an end-to-end anastomo-sis are prone to stricture. Enlarging the anastomosis by beveling the cut ends of the injured vessel can minimize this problem. A curved hemostat is a useful adjunct to create the curve.fistulas in the extremities. Follow-up imaging is performed 1 to 2 weeks after injury to confirm healing. Venous repair should be performed for injuries of the superior vena cava, the inferior vena cava proximal to the renal veins, and the portal vein, although the portal vein may be ligated in extreme cases. The SMV should be repaired optimally, but >80% of patients will survive follow-ing ligation. Similarly, the left

1	veins, and the portal vein, although the portal vein may be ligated in extreme cases. The SMV should be repaired optimally, but >80% of patients will survive follow-ing ligation. Similarly, the left renal vein can usually be ligated adjacent to the IVC due to collateral decompression.The type of operative repair for a vascular injury is based on the extent and location of injury. Lateral suture repair is preferred for arterial injuries with minimal loss of tissue. End-to-end primary anastomosis is performed if the vessel can be repaired without tension. Arterial defects of 1 to 2 cm often can be bridged by mobilizing the severed ends of the vessel after ligating small branches. The aorta, subclavian artery, brachial artery, and popliteal artery however, are difficult to mobilize for additional length. To avoid postoperative stenosis, particularly in smaller arteries, beveling or spatulation should be used so that the completed anastomosis is slightly larger in diameter than the native

1	length. To avoid postoperative stenosis, particularly in smaller arteries, beveling or spatulation should be used so that the completed anastomosis is slightly larger in diameter than the native artery (Fig. 7-44). The authors emphasize the parachute technique to ensure precision placement of the posterior suture line (Fig. 7-45). If this technique is used, traction must be main-tained on both ends of the suture, or leakage from the posterior aspect of the suture line may occur.Interposition grafts are used when end-to-end anastomosis cannot be accomplished without tension despite mobilization. For vessels <6 mm in diameter (e.g., internal carotid, brachial, superficial femoral, and popliteal arteries), autogenous greater saphenous vein (GSV) from the contralateral groin should be used because polytetrafluoroethylene (PTFE) grafts of <6 mm have a prohibitive rate of thrombosis. When GSV is not avail-able, autologous options include the cephalic and basilic veins. Larger arteries

1	because polytetrafluoroethylene (PTFE) grafts of <6 mm have a prohibitive rate of thrombosis. When GSV is not avail-able, autologous options include the cephalic and basilic veins. Larger arteries (e.g., subclavian, innominate, aorta, common iliac) are bridged by PTFE grafts. PTFE is preferred over Dacron because of the reported decreased risk of infection.75 Aortic or iliac arterial injuries may be complicated by enteric contami-nation from colon or small bowel injuries. There is a natural reluctance to place artificial grafts in such circumstances, but graft infections are rare, and the time required to perform an axillofemoral bypass is excessive. Therefore, after the control of hemorrhage, bowel contamination is contained and the abdomen irrigated before placing PTFE grafts.76 After placement of the graft, it is covered with peritoneum or omentum before defini-tive treatment of the enteric injuries.Transposition procedures can be used when an artery has a bifurcation and one

1	placement of the graft, it is covered with peritoneum or omentum before defini-tive treatment of the enteric injuries.Transposition procedures can be used when an artery has a bifurcation and one vessel can be ligated safely. Injuries of the proximal internal carotid can be treated by mobilizing the adja-cent external carotid, dividing it distal to the internal injury, and performing an end-to-end anastomosis between it and the distal internal carotid (Fig. 7-46). The proximal stump of the internal carotid is oversewn, with care taken to avoid a blind pocket where a clot may form. Injuries of the common and external iliac arteries can be handled in a similar fashion (Fig. 7-47), while maintaining flow in at least one internal iliac artery.Venous injuries should be repaired when technically fea-sible. Small injuries without loss of tissue can be treated with lateral suture repair. More complex repairs with interposition grafts may thrombose, but this typically occurs gradually over 1

1	fea-sible. Small injuries without loss of tissue can be treated with lateral suture repair. More complex repairs with interposition grafts may thrombose, but this typically occurs gradually over 1 to 2 weeks. During this time adequate collateral circulation develops, which is sufficient to avoid acute venous hyperten-sion. Therefore, it is reasonable to use ringed PTFE for venous interposition grafting and accept a gradual, but eventual, throm-bosis while allowing time for collateral circulation to develop. Such an approach is reasonable for venous injuries of the supe-rior vena cava, suprarenal vena cava, SMV, and popliteal vein because ligation of these is associated with significant morbid-ity. In the remainder of venous injuries, the vein may be ligated. In such patients, chronic venous hypertensive complications in the lower extremities often can be avoided by (a) temporary use of elastic bandages (Ace wraps) applied from the toes to the hips at the end of the procedure, and (b)

1	hypertensive complications in the lower extremities often can be avoided by (a) temporary use of elastic bandages (Ace wraps) applied from the toes to the hips at the end of the procedure, and (b) judicious elevation of the lower extremities. These measures should be maintained for Brunicardi_Ch07_p0183-p0250.indd 21410/12/18 6:19 PM 215TRAUMACHAPTER 7Figure 7-46. Carotid transposition is an effective approach for treating injuries of the proximal internal carotid artery.ABCFigure 7-47. Transposition procedures can be used for iliac artery injuries to eliminate the dilemma of placing an interposition polytetra-fluoroethylene graft in the presence of enteric contamination. A. Right common iliac artery transposed to left common iliac artery. B. Left internal iliac artery transposed to the distal right common iliac artery. C. Right internal iliac artery transposed to the right external iliac artery.1 week; if the patient has no peripheral edema with ambulation, these maneuvers are no

1	distal right common iliac artery. C. Right internal iliac artery transposed to the right external iliac artery.1 week; if the patient has no peripheral edema with ambulation, these maneuvers are no longer required.Damage Control SurgeryThe recognition of the bloody vicious cycle and the introduction of damage control surgery (DCS) have improved the survival of critically injured patients. Conceptually, the bloody vicious cycle, first described in 1981, is the lethal combination of coag-ulopathy, hypothermia, and metabolic acidosis (Fig. 7-48).56 Hypothermia from evaporative and conductive heat loss and diminished heat production occurs despite the use of warming blankets and blood warmers. The metabolic acidosis of shock is exacerbated by aortic clamping, administration of vasopres-sors, massive RBC transfusions, and impaired myocardial per-formance. The ACOT, described previously, is compounded by hemodilution, hypothermia, and acidosis. Once the cycle starts, each component

1	massive RBC transfusions, and impaired myocardial per-formance. The ACOT, described previously, is compounded by hemodilution, hypothermia, and acidosis. Once the cycle starts, each component magnifies the other, which leads to a down-ward spiral and ultimately a fatal arrhythmia. The purpose of DCS is to limit operative time so that the patient can be returned to the SICU for physiologic restoration and the cycle thereby broken. Indications to limit the initial operation and institute DCS techniques include a combination of refractory hypother-mia (temperature <35°C), profound acidosis (arterial pH <7.2, base deficit >15 mmol/L), and refractory coagulopathy.56,77 The decision to abbreviate a trauma laparotomy is made intraopera-tively as the patient’s clinical course becomes clearer and labo-ratory values become available.The goal of DCS is to control surgical bleeding and limit GI spillage. The operative techniques used are temporary mea-sures, with definitive repair of injuries

1	labo-ratory values become available.The goal of DCS is to control surgical bleeding and limit GI spillage. The operative techniques used are temporary mea-sures, with definitive repair of injuries delayed until the patient is physiologically replete. Controlling surgical bleeding while preventing ischemia is of utmost importance during DCS. Aortic injuries must be repaired using an interposition PTFE Brunicardi_Ch07_p0183-p0250.indd 21510/12/18 6:19 PM 216BASIC CONSIDERATIONSPART Igraft. Although celiac artery injuries may be ligated, the SMA must maintain flow, and the early insertion of an intravascu-lar shunt is advocated. Similarly, perfusion of the iliac system and infrainguinal vessels can be restored with a vascular shunt, with interposition graft placement delayed. Arterial reconstruc-tion following shunt placement should be done optimally within 6 hours.78 Venous injuries are preferentially treated with ligation in damage control situations, except for the suprarenal

1	reconstruc-tion following shunt placement should be done optimally within 6 hours.78 Venous injuries are preferentially treated with ligation in damage control situations, except for the suprarenal inferior vena cava and popliteal vein. For extensive solid organ injuries to the spleen or one kidney, excision is indicated rather than an attempt at operative repair. For hepatic injuries, perihe-patic packing of the liver will usually tamponade bleeding (see Fig. 7-37). Translobar gunshot wounds of the liver are best con-trolled with balloon catheter tamponade, whereas deep lacera-tions can be controlled with Foley catheter inflation deep within the injury track (Fig. 7-49). For thoracic injuries requiring DCS several options exist. For bleeding peripheral pulmonary inju-ries, wedge resection using a stapler is performed. In penetrating injuries, pulmonary tractotomy is used to divide the parenchyma (Fig. 7-50); individual vessels and bronchi are then ligated using a 3-0 polydioxanone

1	using a stapler is performed. In penetrating injuries, pulmonary tractotomy is used to divide the parenchyma (Fig. 7-50); individual vessels and bronchi are then ligated using a 3-0 polydioxanone suture (PDS), and the track is left open. Patients who sustain more proximal injuries may require formal pulmonary resection, but pneumonectomy is poorly tolerated. Recent experimental work suggests inhaled nitric oxide (NO) will reduce right heart failure following pneumonectomy.79 Car-diac injuries may be temporarily controlled using a running 3-0 nonabsorbable polypropylene suture or skin staples. Pledgeted repair should be performed for the relatively thin right ventricle.The second key component of DCS is limiting enteric content spillage. Small GI injuries (stomach, duodenum, small intestine, and colon) may be controlled using a rapid whipstitch of 3-0 PDS. Complete transection of the bowel or segmental damage is controlled using a GIA stapler, often with resection of the injured

1	and colon) may be controlled using a rapid whipstitch of 3-0 PDS. Complete transection of the bowel or segmental damage is controlled using a GIA stapler, often with resection of the injured segment. Alternatively, open ends of the bowel may be ligated using umbilical tapes to limit spillage. Pancreatic injuries, regardless of location, are packed and the evaluation of ductal integrity postponed. Urologic injuries may require cath-eter diversion. Before the patient is returned to the SICU, the abdomen must be closed temporarily. Temporary closure of the abdomen is accomplished using an antimicrobial surgical incise drape (Ioban, 3M Health Care, St Paul, MN) (Fig. 7-51). In this technique, the bowel is covered with a fenestrated subfascial sterile drape (45 × 60 cm Steri-Drape 3M Health Care), and two Jackson-Pratt drains are placed along the fascial edges; this is Severe TraumaBlood LossTissue InjuryMassive RBCTransfusionIatrogenicFactorsCellularShockCoreHypothermiaMetabolic

1	Health Care), and two Jackson-Pratt drains are placed along the fascial edges; this is Severe TraumaBlood LossTissue InjuryMassive RBCTransfusionIatrogenicFactorsCellularShockCoreHypothermiaMetabolic AcidosisHypocalcemiaImmuno-ActivationActivation/Consumptionof Complement SystemProgressiveSystemicCoagulopathyAcuteEndogenousCoagulopathyClotting FactorDeficienciesPreexistingDiseasesFFP resistantFFP sensitiveFigure 7-48. The bloody vicious cycle. FFP = fresh frozen plasma; RBC = red blood cell.Brunicardi_Ch07_p0183-p0250.indd 21610/12/18 6:19 PM 217TRAUMACHAPTER 7Figure 7-49. A. An intrahepatic balloon used to tamponade hemorrhage from transhepatic penetrating injuries is made by placing a red rubber catheter inside a 1-inch Penrose drain, with both ends of the Penrose drain ligated. B. Once placed inside the injury tract, the balloon is inflated with saline until hemorrhage stops. C. A Foley catheter with a 30-mL balloon can be used to halt hemorrhage from deep lacerations to the

1	Once placed inside the injury tract, the balloon is inflated with saline until hemorrhage stops. C. A Foley catheter with a 30-mL balloon can be used to halt hemorrhage from deep lacerations to the liver.Figure 7-50. Pulmonary tractotomy divides the pulmonary paren-chyma using a gastrointestinal anastomosis (GIA) stapler. The opened track permits direct access to injured vessels or bronchi for individual ligation.then covered using an Ioban drape, which allows closed suction to control reperfusion-related ascitic fluid egress while provid-ing adequate space for bowel expansion to prevent abdomi-nal compartment syndrome. During the initial DCS stage, the subfascial sterile drape is not covered by a blue towel so that the status of the bowel and hemorrhage control can be assessed. The use of direct peritoneal resuscitation following DCS should be considered; 18 Fr round Blake drains may be placed intraop-eratively for the instillation of the dialysate solution.80 Return to the OR within

1	direct peritoneal resuscitation following DCS should be considered; 18 Fr round Blake drains may be placed intraop-eratively for the instillation of the dialysate solution.80 Return to the OR within 24 hours is planned once the patient clini-cally improves, as evidenced by normothermia, normalization of coagulation test results, and correction of acidosis.TREATMENT OF SPECIFIC INJURIESHead InjuriesIntracranial Injuries. CT scanning, performed on all patients with a significant closed head injury (GCS score <14), identi-fies and quantitates intracranial lesions as well as intracranial hypertension. Patients with intracranial hemorrhage, including epidural hematoma, subdural hematoma, subarachnoid hemor-rhage, intracerebral hematoma or contusion, and diffuse axo-nal injury, are admitted to the SICU. In patients with abnormal findings on CT scans and GCS scores of ≤8, intracranial pres-sure (ICP) should be monitored using fiber-optic intraparenchy-mal devices or intraventricular

1	to the SICU. In patients with abnormal findings on CT scans and GCS scores of ≤8, intracranial pres-sure (ICP) should be monitored using fiber-optic intraparenchy-mal devices or intraventricular catheters.33,81 Although an ICP of 10 mmHg is the upper limit of normal, therapy is targeted to maintain an ICP of <20 mmHg.33 The newest neurosurgery guidelines additionally recommend maintaining the cerebral perfusion pressure (CPP) at >60 mmHg. Indications for opera-tive intervention to remove space-occupying hematomas are based on the clot volume, amount of midline shift, location of the clot, GCS score, and ICP.29 A shift of >5 mm typically is considered an indication for evacuation, but this is not an abso-lute rule. Smaller hematomas that are in treacherous locations, such as the posterior fossa, may require drainage due to brain stem compression or impending herniation. Removal of small hematomas may also improve ICP and cerebral perfusion in patients with elevated ICP that is

1	fossa, may require drainage due to brain stem compression or impending herniation. Removal of small hematomas may also improve ICP and cerebral perfusion in patients with elevated ICP that is refractory to medical therapy. Patients with diffuse cerebral edema resulting in excessive ICP may require a decompressive craniectomy, although a recent AAST multicenter trial questioned the benefits.82,83 Patients with open or depressed skull fractures, with or without sinus involvement, may require operative intervention. Penetrating injuries to the head may require operative intervention for hem-orrhage control, evacuation of blood, skull fracture fixation, or debridement.General surgeons in communities without emergency neu-rosurgical coverage should have a working knowledge of burr hole placement in the event that emergent evacuation is required for a life-threatening epidural hematoma (Fig. 7-52).84 The typi-cal clinical course of an epidural hematoma is an initial loss of consciousness, a

1	in the event that emergent evacuation is required for a life-threatening epidural hematoma (Fig. 7-52).84 The typi-cal clinical course of an epidural hematoma is an initial loss of consciousness, a lucid interval, and recurrent loss of conscious-ness with an ipsilateral fixed and dilated pupil. While decom-pression of subdural hematomas may be delayed, epidural hematomas require evacuation within 70 minutes.83 The final stages of this sequence are caused by blood accumulation that forces the temporal lobe medially, with resultant compression of the third cranial nerve and eventually the brain stem. The burr hole is made on the side of the dilated pupil to decompress the intracranial space. After stabilization, the patient is transferred to a facility with neurosurgical capability for formal craniotomy.In addition to operative intervention, postinjury care directed at limiting secondary injury to the brain is critical. The goal of resuscitation and management in patients with head

1	formal craniotomy.In addition to operative intervention, postinjury care directed at limiting secondary injury to the brain is critical. The goal of resuscitation and management in patients with head injuries is to avoid hypotension (SBP of <100 mmHg) and hypoxia (partial pressure of arterial oxygen of <60 or arterial oxygen saturation of <90%).33 Attention, therefore, is focused on maintaining cerebral perfusion rather than merely lowering Brunicardi_Ch07_p0183-p0250.indd 21710/12/18 6:20 PM 218BASIC CONSIDERATIONSPART IABCDFigure 7-51. Temporary closure of the abdomen entails covering the bowel with a fenestrated subfascial 45 × 60 cm sterile drape (A), placing Jackson-Pratt drains along the fascial edge (B), and then occluding with an Ioban drape (C, D).ICP. Resuscitation efforts aim for a euvolemic state and a SBP of >100 mmHg. Cerebral perfusion pressure (CPP) is equal to the mean arterial pressure minus the ICP, with a target range of >60 mmHg.33 CPP can be increased by

1	aim for a euvolemic state and a SBP of >100 mmHg. Cerebral perfusion pressure (CPP) is equal to the mean arterial pressure minus the ICP, with a target range of >60 mmHg.33 CPP can be increased by either lowering ICP or raising mean arterial pressure. Sedation, osmotic diuresis, paralysis, ventricular drainage, and barbiturate coma are used in sequence, with coma induction being the last resort. The role of decompressive craniectomy for refractory ICH remains contro-versial.82 The partial pressure of carbon dioxide (PCO2) should be maintained in a normal range (35–40 mmHg), but for tem-porary management of acute intracranial hypertension, induc-ing cerebral vasoconstriction by hyperventilation to a PCO2 of Brunicardi_Ch07_p0183-p0250.indd 21810/12/18 6:20 PM 219TRAUMACHAPTER 7<30 mmHg is occasionally warranted. Moderate hypothermia (32°–33°C [89.6°–91.4°F]) has been shown experimentally to improve neurologic outcomes, but clinical studies have not vali-dated this concept.33,85,86

1	is occasionally warranted. Moderate hypothermia (32°–33°C [89.6°–91.4°F]) has been shown experimentally to improve neurologic outcomes, but clinical studies have not vali-dated this concept.33,85,86 Patients with intracranial hemorrhage should be monitored for postinjury seizures, and prophylactic anticonvulsant therapy is indicated for 7 days after injury.33Maxillofacial Injuries. Maxillofacial injuries are common with multisystem trauma and require coordinated management by the trauma surgeon and the specialists in otolaryngology, plastic surgery, ophthalmology, and oral and maxillofacial surgery. Delay in addressing these systems that control vision, hearing, smelling, breathing, eating, and phonation may pro-duce dysfunction and disfigurement with serious psychological impact. The maxillofacial complex is divided into three regions; the upper face containing the frontal sinus and brain; the mid-face containing the orbits, nose, and zygomaticomaxillary com-plex; and the lower face

1	complex is divided into three regions; the upper face containing the frontal sinus and brain; the mid-face containing the orbits, nose, and zygomaticomaxillary com-plex; and the lower face containing the mandible. High-impact kinetic energy is required to fracture the frontal sinus, orbital rims, and mandible, whereas low-impact forces will injure the nasal bones and zygoma.The most common scenario, which at times may be life-threatening, is bleeding from facial fractures.87 Temporizing measures include nasal packing, Foley catheter tamponade of posterior nasal bleeding, and oropharyngeal packing. Prompt angioembolization will halt exsanguinating hemorrhage. Frac-tures of tooth-bearing bone are considered open fractures and require antibiotic therapy and semiurgent repair to preserve the Figure 7-52. A burr hole is made for decompression of an epidural hematoma as a life-saving maneuver. One or more branches of the external carotid artery usually must be ligated to gain access to the

1	Figure 7-52. A burr hole is made for decompression of an epidural hematoma as a life-saving maneuver. One or more branches of the external carotid artery usually must be ligated to gain access to the skull. No attempt should be made to control intracranial hemor-rhage through the burr hole. Rather, the patient’s head should be wrapped with a bulky absorbent dressing and the patient transferred to a neurosurgeon for definitive care.Figure 7-53. Three-dimensional computed tomography scan illustrating Le Fort II maxillary (L) and alveolar (A) fractures, and fracture of the mandible (M) at the midline and at the weaker con-dyle (C). (Used with permission from Vincent D. Eusterman, MD, DDS.)airway as well as the functional integrity of the occlusion (bite) and the aesthetics of the face. Orbital fractures may compro-mise vision, produce muscle injury causing diplopia, or change orbital volume to produce a sunken appearance to the orbit. Nose and nasoethmoidal fractures should be assessed

1	fractures may compro-mise vision, produce muscle injury causing diplopia, or change orbital volume to produce a sunken appearance to the orbit. Nose and nasoethmoidal fractures should be assessed carefully to identify damage to the lacrimal drainage system or to the cribriform plate producing cerebrospinal fluid rhinorrhea. After initial stabilization, a systematic physical examination of the head and neck should be performed that also includes cranial nerve examination and three-dimensional CT scanning of the maxillofacial complex (Fig. 7-53).Cervical InjuriesSpine. Treatment of injuries to the cervical spine is based on the level of injury, the stability of the spine, the presence of subluxation, the extent of angulation, the level of neurologic deficit, and the overall condition of the patient. In general, phy-sician-supervised axial traction, via cervical tongs or the more commonly used halo vest, is used to reduce subluxations and stabilize the injury. Immobilization of injuries

1	patient. In general, phy-sician-supervised axial traction, via cervical tongs or the more commonly used halo vest, is used to reduce subluxations and stabilize the injury. Immobilization of injuries also is achieved with spinal orthoses (braces), particularly in those with asso-ciated thoracolumbar injuries. Surgical fusion typically is performed in patients with neurologic deficit, those with angu-lation of >11° or translation of >3.5 mm, and those who remain unstable after halo placement. Indications for immediate opera-tive intervention are deterioration in neurologic function and fractures or dislocations with incomplete deficit. Historically, methylprednisolone was administered to patients with acute spi-nal cord injury after blunt injury, with clinical data suggesting a small benefit to initiating a 24-hour infusion if started within 3 hours and a 48-hour infusion if started within 3 to 8 hours.88 Current guidelines, however, no longer recommend steroids for acute injuries.89

1	to initiating a 24-hour infusion if started within 3 hours and a 48-hour infusion if started within 3 to 8 hours.88 Current guidelines, however, no longer recommend steroids for acute injuries.89 The role and timing of operative surgical decompression after acute spinal cord injury is debated, and Brunicardi_Ch07_p0183-p0250.indd 21910/12/18 6:20 PM 220BASIC CONSIDERATIONSPART Ithe concept of damage control has been suggested. However, evidence supports urgent decompression of bilateral locked fac-ets in patients with incomplete tetraplegia or with neurologic deterioration. Performing surgery within 24 hours may decrease length of stay and complications.90 Complete injuries of the spi-nal cord remain essentially untreatable. Yet, approximately 3% of patients who present with flaccid quadriplegia have concus-sive injuries, and these patients represent the very few who seem to have miraculous recoveries.Vascular. Cervical vascular injuries due to either blunt or pen-etrating trauma

1	have concus-sive injuries, and these patients represent the very few who seem to have miraculous recoveries.Vascular. Cervical vascular injuries due to either blunt or pen-etrating trauma can result in devastating neurologic sequelae or exsanguination. Penetrating injuries to the carotid artery and internal jugular vein usually are obvious on operative neck exploration. The principles of vascular repair techniques (dis-cussed previously) apply to carotid injuries, and options for repair include end-to-end primary repair (often possible with mobilization of the common carotid), graft interposition, and transposition procedures. All carotid injuries should be repaired except in patients who present in coma with a delay in transport. Prompt revascularization of the internal carotid artery, using a temporary Pruitt-Inahara shunt, should be considered in patients arriving in profound shock. Otherwise, carotid shunting should be done selectively as in elective carotid endarterectomy, but

1	using a temporary Pruitt-Inahara shunt, should be considered in patients arriving in profound shock. Otherwise, carotid shunting should be done selectively as in elective carotid endarterectomy, but the patient should be systemically anticoagulated. Currently, we administer heparin with an ACT target of 250 sec. Tangential wounds of the internal jugular vein should be repaired by lateral venorrhaphy, but extensive wounds are efficiently addressed by ligation. However, it is not advisable to ligate both jugular veins due to potential intracranial hypertension. Vertebral artery inju-ries due to penetrating trauma are difficult to control operatively because of the artery’s protected location within the foramen transversarium. Although exposure from an anterior approach can be accomplished by removing the anterior elements of the bony canal and the tough fascia covering the artery between the elements, typically the most efficacious control of such inju-ries is angioembolization. Fogarty

1	removing the anterior elements of the bony canal and the tough fascia covering the artery between the elements, typically the most efficacious control of such inju-ries is angioembolization. Fogarty catheter balloon occlusion, however, is useful for controlling acute bleeding if encountered during neck exploration.Blunt injury to the carotid or vertebral arteries may cause dissection, thrombosis, or pseudoaneurysm, typically in the surgi-cally inaccessible distal internal carotid (Fig. 7-54).91 Early recog-nition and management of these injuries is paramount because patients treated with antithrombotics have a stroke rate of <1% compared with stroke rates of 20% in untreated patients.92 Because treatment must be instituted during the latent period between injury and onset of neurologic sequelae, diagnostic imaging is performed based on identified risk factors (Fig. 7-55).92 After identification of an injury, antithrombotics are administered if the patient does not have

1	neurologic sequelae, diagnostic imaging is performed based on identified risk factors (Fig. 7-55).92 After identification of an injury, antithrombotics are administered if the patient does not have contraindications (intra-cranial hemorrhage, falling hemoglobin level with solid organ injury or complex pelvic fractures). Heparin, started without a loading dose at 15 units/kg per hour, is titrated to achieve a PTT between 40 and 50 seconds or antiplatelet agents are initiated (aspirin 325 mg/d or clopidogrel 75 mg/d). The types of 9Figure 7-54. The Denver grading scale for blunt cerebrovascular injuries. Grade I: irregularity of the vessel wall, dissection/intramural hematoma with <25% luminal stenosis. Grade II: visualized intraluminal thrombus or raised intimal flap, or dissection/intramural hematoma with 25% or more luminal narrowing. Grade III: pseudoaneurysm. Grade IV: vessel occlusion. CAI = carotid artery injury; VAI = vertebral artery injury.Brunicardi_Ch07_p0183-p0250.indd

1	hematoma with 25% or more luminal narrowing. Grade III: pseudoaneurysm. Grade IV: vessel occlusion. CAI = carotid artery injury; VAI = vertebral artery injury.Brunicardi_Ch07_p0183-p0250.indd 22010/12/18 6:20 PM 221TRAUMACHAPTER 7Yes Yes NoNoMulti-Slice CTA*Surgically Accessible?Grade I Injury Antithrombotic Therapy: Heparin (PTT 40–50 sec)or Antiplatelet Therapy**NoDiscontinue AntithromboticsRepeat CTA in 7–10 days***Injury Healed? Yes NoPositive Equivocal Finding or High Clinical Suspicion ArteriogramNoYes Negative Grade II–IV Injury Grade V Injury Operative Repair Endovascular Treatment Yes StopStop Surgically Accessible?Potential arterial hemorrhage from neck/nose/mouthCervical bruit in pt <50 yrs oldExpanding cervical hematomaFocal neurologic defect: TIA, hemiparesis, vertebrobasilar symptoms, Horner’s SyndromeNeurologic deÿcit inconsistent with head CTStroke on CT or MRISigns/Symptoms of BCVIThe Denver Health Medical Center BCVI screening guideline.High energy transfer

1	symptoms, Horner’s SyndromeNeurologic deÿcit inconsistent with head CTStroke on CT or MRISigns/Symptoms of BCVIThe Denver Health Medical Center BCVI screening guideline.High energy transfer mechanismDisplaced mid-face fracture (LeFort II or III)Mandible fractureComplex skull fracture/basilar skull fracture/occipital condyle fractureSevere Traumatic Brain Injury (TBI) with GCS <6Cervical spine fracture, subluxation or ligamentous injury at any levelNear hanging with anoxic injury/ strangulations with LOCClothesline type injury or seat belt abrasion with signiÿcant swelling, pain, or altered MSTBI with thoracic injuriesScalp deglovingThoracic vascular injuriesBlunt cardiac ruptureUpper rib fractures (1–3)Risk Factors for BCVIAntithrombotics for 3–6 months and reimageConsider endovascular stenting for severe luminal narrowing, symptomatology, or markedly expanding pseudoaneurysm* CTA with multidetector-row CT, 64-channel optimal. If fewer than 16 channels, interpret CTA with caution;

1	stenting for severe luminal narrowing, symptomatology, or markedly expanding pseudoaneurysm* CTA with multidetector-row CT, 64-channel optimal. If fewer than 16 channels, interpret CTA with caution; digital subtraction arteriogra-phy is the gold standard.** Antiplatelet therapy is typically ASA 325 mg.*** If CC fistula and symptomatic, consider angiography and endovascular therapy. If asymptomatic CC fistula, reimage with CTA at 3–4 weeks.Figure 7-55. Screening and treatment algorithm for blunt cerebrovascular injuries (BCVIs). ASA = acetylsalicylic acid; BRB = bright red blood; CHI = closed head injury; C-spine = cervical spine; CT = computed tomography; DAI = diffuse axonal injury; GCS = Glasgow Coma Scale score; MRI = magnetic resonance imaging; MS = mental status; Neg = negative; pt = patient; PTT = partial thromboplastin time; TIA = transient ischemic attack.antithrombotic treatment appear equivalent in published studies to date, and the duration of treatment is empirically

1	pt = patient; PTT = partial thromboplastin time; TIA = transient ischemic attack.antithrombotic treatment appear equivalent in published studies to date, and the duration of treatment is empirically recommended to be 6 months.93,94 The role of carotid stenting for grade II or III internal carotid artery injuries remain controversial; current litera-ture suggests stenting be reserved for symptomatic patients or markedly enlarging pseudoaneurysms.95 Thrombosis of the internal jugular veins caused by blunt trauma can occur unilaterally or bilaterally and is often discovered incidentally because most patients are asymptomatic. Bilateral thrombosis can aggravate cerebral edema in patients with serious head injuries; stent placement should be considered in such patients if ICP remains elevated.Brunicardi_Ch07_p0183-p0250.indd 22110/12/18 6:20 PM 222BASIC CONSIDERATIONSPART IAerodigestive. Fractures of the larynx and trachea may mani-fest as cervical emphysema. Fractures documented by

1	22110/12/18 6:20 PM 222BASIC CONSIDERATIONSPART IAerodigestive. Fractures of the larynx and trachea may mani-fest as cervical emphysema. Fractures documented by CT scan are usually repaired. Common injuries include thyroid carti-lage fractures, rupture of the thyroepiglottic ligament, disrup-tion of the arytenoids or vocal cord tears, and cricoid fractures. After debridement of devitalized tissue, tracheal injuries are repaired end-to-end using a single layer of interrupted absorb-able sutures. Associated injuries of the esophagus are common in penetrating injuries due to its close proximity. After debride-ment and repair, vascularized tissue is interposed between the repaired esophagus and trachea, and a closed suction drain is placed. The sternocleidomastoid muscle or strap muscles are useful for interposition and help prevent postoperative fistulas.Chest InjuriesThe most common injuries from both blunt and penetrating thoracic trauma are hemothorax and pneumothorax. More than

1	are useful for interposition and help prevent postoperative fistulas.Chest InjuriesThe most common injuries from both blunt and penetrating thoracic trauma are hemothorax and pneumothorax. More than 85% of patients can be definitively treated with a chest tube. The indications for thoracotomy include significant initial or ongoing hemorrhage from the tube thoracostomy and specific imaging-identified diagnoses (Table 7-10). One caveat concerns the patient who presents after a delay. Even when the initial chest tube output is 1.5 L, if the output ceases and the lung is reexpanded, the patient may be managed nonoperatively if hemodynamically stable.Great Vessels. Over 90% of thoracic great vessel injuries are due to penetrating trauma, although blunt injury to the innomi-nate, subclavian, or descending aorta may cause a pseudoaneu-rysm or frank rupture.46,96,97 Simple lacerations of the ascending or transverse aortic arch can be repaired with lateral aortor-rhaphy. Repair of posterior

1	or descending aorta may cause a pseudoaneu-rysm or frank rupture.46,96,97 Simple lacerations of the ascending or transverse aortic arch can be repaired with lateral aortor-rhaphy. Repair of posterior aortic injuries, complex ascending or transverse injuries, or those requiring interposition grafting of the arch, require full cardiopulmonary bypass. Innominate artery injuries are repaired using the bypass exclusion tech-nique,97 which avoids the need for cardiopulmonary bypass. Bypass grafting from the proximal aorta to the distal innominate with a prosthetic tube graft is performed before the postinjury hematoma is entered. The PTFE graft is anastomosed end-to-side from the proximal undamaged aorta and anastomosed end-to-end to the innominate artery (Fig. 7-56). The origin of the ABCFigure 7-56. A. Angiography reveals a 1-cm pseudoaneurysm of the innominate artery origin. B. In the first stage of the bypass exclusion technique, a 12-mm polytetrafluoroethylene graft is anastomosed end

1	7-56. A. Angiography reveals a 1-cm pseudoaneurysm of the innominate artery origin. B. In the first stage of the bypass exclusion technique, a 12-mm polytetrafluoroethylene graft is anastomosed end to side from the proximal undamaged aorta, tunneled under the vein, and anastomosed end to end to the innominate artery. C. The origin of the innominate is then oversewn at its base to exclude the pseudoaneurysm.Table 7-10Indications for operative treatment of thoracic injuries• Initial tube thoracostomy drainage of >1000 mL (penetrating injury) or >1500 mL (blunt injury)• Ongoing tube thoracostomy drainage of >200 mL/h for 3 consecutive hours in noncoagulopathic patients• Caked hemothorax despite placement of two chest tubes• Great vessel injury (endovascular techniques may be used in selected patients)• Pericardial tamponade• Cardiac herniation• Massive air leak from the chest tube with inadequate ventilation• Tracheal or main stem bronchial injury diagnosed by endoscopy or imaging• Open

1	patients)• Pericardial tamponade• Cardiac herniation• Massive air leak from the chest tube with inadequate ventilation• Tracheal or main stem bronchial injury diagnosed by endoscopy or imaging• Open pneumothorax• Esophageal perforation• Air embolisminnominate is then oversewn at its base to exclude the pseu-doaneurysm or other injury. Subclavian artery injuries can be repaired using lateral arteriorrhaphy or PTFE graft interposition; due to its multiple branches and tethering of the artery, end-to-end primary anastomosis is not advocated if there is a significant segmental loss.Descending BAI may require urgent intervention. How-ever, operative intervention for intracranial or intra-abdominal hemorrhage or unstable pelvic fractures takes precedence. To prevent aortic rupture, pharmacologic therapy with a selective β1-antagonist, esmolol, should be instituted in the trauma bay, with a target SBP of <100 mmHg and heart rate of <100/min.41,98 Endovascular stenting is now the mainstay of

1	therapy with a selective β1-antagonist, esmolol, should be instituted in the trauma bay, with a target SBP of <100 mmHg and heart rate of <100/min.41,98 Endovascular stenting is now the mainstay of treatment.99,100 While endograft sizing has improved, the major question is long-term outcome in younger patients. Open repair of the descending aorta is accomplished using partial left heart bypass to prevent spinal cord and splanchnic ischemia and reduce left ventricular afterload (Fig. 7-57).101 Nonoperative management for grade I Brunicardi_Ch07_p0183-p0250.indd 22210/12/18 6:20 PM 223TRAUMACHAPTER 7LAFigure 7-57. When repairing a tear of the descending thoracic aorta, perfusion of the spinal cord while the aorta is clamped is achieved by using partial left heart bypass. The venous cannula is inserted into the left superior pulmonary vein because it is less prone to tearing than the left atrium (LA).Figure 7-58. A variety of techniques may be necessary to repair cardiac wounds.

1	cannula is inserted into the left superior pulmonary vein because it is less prone to tearing than the left atrium (LA).Figure 7-58. A variety of techniques may be necessary to repair cardiac wounds. Generally, pledget support is used for the relatively thin-walled right ventricle.intimal aortic injuries is accomplished with antiplatelet agents and blood pressure control.102Heart. Blunt and penetrating cardiac injuries have widely dif-fering presentations and therefore disparate treatments. Survivable penetrating cardiac injuries consist of wounds that can be repaired operatively; most are stab wounds. Before repair of the injury is attempted, hemorrhage should be controlled; injuries to the atria can be clamped with a Satinsky vascular clamp, whereas digital pressure is used to occlude the majority of ventricular wounds. Foley catheter occlusion of larger stellate lesions is described, but even minimal traction may enlarge the original injury. Temporary control of hemorrhage, and at

1	majority of ventricular wounds. Foley catheter occlusion of larger stellate lesions is described, but even minimal traction may enlarge the original injury. Temporary control of hemorrhage, and at times definitive repair, may be accomplished with skin staples for left ventricular lacerations; the myocardial edges of the laceration must coapt in diastole for stapling to be technically feasible. Definitive repair of cardiac injuries is performed with either running 3-0 polypropylene suture or interrupted, pledgeted 2-0 polypropylene suture (Fig. 7-58).103 Use of pledgets may be particularly important in the right ventricle to prevent sutures from pulling through the thinner myocardium. Injuries adjacent to coronary arteries should be repaired using horizontal mattress sutures because use of running sutures results in coronary occlu-sion and distal infarction. Gunshot wounds may result in stellate lesions or contused, extremely friable myocardium adjacent to the wound. When the edges of

1	running sutures results in coronary occlu-sion and distal infarction. Gunshot wounds may result in stellate lesions or contused, extremely friable myocardium adjacent to the wound. When the edges of such complex wounds cannot be fully approximated and hence the repair is not hemostatic, the authors have used surgical adhesive (BioGlue) to achieve hemo-stasis.104 Occasionally, interior structures of the heart may be damaged. Intraoperative auscultation or postoperative hemody-namic assessment usually identifies such injuries.105 ECHO can diagnose the injury and quantitate its effect on cardiac output. Immediate repair of valvular damage or septal defects rarely is necessary and would require cardiopulmonary bypass, but struc-tural intracardiac lesions may progress, and thus patients must have a follow-up ECHO.Patients with blunt cardiac injury typically present with persistent tachycardia or conduction disturbances, but occa-sionally present with tamponade due to atrial or right

1	must have a follow-up ECHO.Patients with blunt cardiac injury typically present with persistent tachycardia or conduction disturbances, but occa-sionally present with tamponade due to atrial or right ventricu-lar rupture. There are no pathognomonic ECG findings, and cardiac enzyme levels do not correlate with the risk of cardiac complications.25 Therefore, patients for whom there is high clinical suspicion of cardiac contusion and who are hemody-namically stable should be monitored for dysrhythmias for 24 hours by telemetry. Patients with hemodynamic instability should undergo ECHO to evaluate for wall motion abnormali-ties (particularly hypokinesis of the right ventricle), pericardial fluid, valvular dysfunction, chordae rupture, or diminished ejection fraction. If such findings are noted or if vasoactive agents are required, cardiac function can be continuously moni-tored using a pulmonary artery catheter and serial SICU trans-thoracic or transesophageal ECHO.Trachea, Bronchi, and

1	or if vasoactive agents are required, cardiac function can be continuously moni-tored using a pulmonary artery catheter and serial SICU trans-thoracic or transesophageal ECHO.Trachea, Bronchi, and Lung Parenchyma. Less than 1% of all injured patients sustain intrathoracic tracheobronchial Brunicardi_Ch07_p0183-p0250.indd 22310/12/18 6:20 PM 224BASIC CONSIDERATIONSPART Iinjuries, and only a small number require operative intervention. Although penetrating injuries may occur throughout the tracheo-bronchial system, blunt injuries most commonly occur within 2.5 cm of the carina. For patients with a massive air leak requir-ing emergent exploration, initial control of the injury to provide effective ventilation is obtained by passing an endotracheal tube either beyond the injury or into the contralateral mainstem bron-chus. Principles of repair are similar to those for repair of cervi-cal tracheal injuries. Devitalized tissue is debrided, and primary end-to-end anastomosis with 3-0 PDS

1	contralateral mainstem bron-chus. Principles of repair are similar to those for repair of cervi-cal tracheal injuries. Devitalized tissue is debrided, and primary end-to-end anastomosis with 3-0 PDS suture is performed. Dis-section should be limited to the area of injury to prevent disrup-tion of surrounding bronchial vasculature and ensuing ischemia and stricture. Suture lines should be encircled with vascularized tissue, either pericardium, intercostal muscle, or pleura. Expect-ant management is employed for bronchial injuries that are less than one-third the circumference of the airway and have no evi-dence of a persistent major air leak.11,12 In patients with periph-eral bronchial injuries, indicated by persistent air leaks from the chest tube and documented by endoscopy, bronchoscopically directed fibrin glue sealing may be useful.The majority of pulmonary parenchymal injuries are sus-pected based upon identification of a pneumothorax; the vast majority can be managed with a tube

1	directed fibrin glue sealing may be useful.The majority of pulmonary parenchymal injuries are sus-pected based upon identification of a pneumothorax; the vast majority can be managed with a tube thoracostomy. Identified parenchymal injuries encountered during thoracic exploration for a massive hemothorax are managed without resection as much as possible. Bronchovenous fistula is a constant threat and should be minimized by prompt control of a major air leak. Peripheral lacerations with persistent bleeding can be managed with stapled wedge resection. For central injuries, the current treatment is pulmonary tractotomy, which permits selective ligation of individual bronchioles and bleeders, pre-vents the development of an intraparenchymal hematoma or air embolism, and reduces the need for formal lobar resection (see Fig. 7-50).106,107 A stapling device, preferably the longest stapler available (e.g., GIA-100), is inserted directly into the injury track and positioned along the thinnest

1	lobar resection (see Fig. 7-50).106,107 A stapling device, preferably the longest stapler available (e.g., GIA-100), is inserted directly into the injury track and positioned along the thinnest section of overly-ing parenchyma. The injury track is thus filleted open, which allows direct access to the bleeding vessels and leaking bronchi. The majority of injuries are definitively managed with selective ligation, and the defect is left open. Occasionally, tractotomy reveals a more proximal vascular on bronchial injury that must be treated with formal lobectomy. Injuries severe enough to mandate pneumonectomy usually are fatal because of right heart decompensation.108One parenchymal injury that may be discovered during thoracic imaging is a posttraumatic pulmonary pseudocyst, col-loquially termed a pneumatocele.109 Traumatic pneumatoceles typically follow a benign clinical course and are treated with aggressive pain management, pulmonary toilet, and serial chest radiography to monitor

1	termed a pneumatocele.109 Traumatic pneumatoceles typically follow a benign clinical course and are treated with aggressive pain management, pulmonary toilet, and serial chest radiography to monitor for resolution of the lesion. If the patient has persistent fever or leukocytosis, however, chest CT is done to evaluate for an evolving abscess because pneumatoceles may become infected. CT-guided catheter drainage may be required in such cases because 25% of patients do not respond to anti-biotic therapy alone. Surgery, ranging from partial resection to anatomic lobectomy, is indicated for unresolving complex pneumatoceles or infected lesions refractory to antibiotic ther-apy and drainage.The most common complication after thoracic injury is development of an empyema. Management is based on CT diagnostic criteria.110 Percutaneous drainage is indicated for a single loculation without appreciable rind. While fibrinolytics are often used for empyema, there is a paucity of data to support

1	CT diagnostic criteria.110 Percutaneous drainage is indicated for a single loculation without appreciable rind. While fibrinolytics are often used for empyema, there is a paucity of data to support their use. Early decortication via video-assisted thoracic surgery should be done promptly in patients with multiple loculations or a pleural rind of >1 cm.111 Antibiotic treatment is based on definitive culture results, but presumptive antibiotics should cover MRSA in the SICU.Esophagus. Due to the proximity of the structures, esophageal injuries often occur with tracheobronchial injuries, particularly in cases of penetrating trauma. Operative options are based on the extent and location of esophageal injury. With sufficient mobilization, a primary single-layer end-to-end anastomosis may be performed after appropriate debridement. As with cer-vical repairs, if there are two suture lines in close approximation (trachea or bronchi and esophagus) interposition of a vascular-ized pedicle is

1	performed after appropriate debridement. As with cer-vical repairs, if there are two suture lines in close approximation (trachea or bronchi and esophagus) interposition of a vascular-ized pedicle is warranted to prevent fistula formation. Perfora-tions at the gastroesophageal junction may be treated with repair and Nissan fundoplication or, for destructive injuries, segmen-tal resection and gastric pull-up. Small esophageal injuries can be managed with stenting. With large destructive injuries or delayed presentation of injuries, esophageal exclusion with wide drainage, diverting loop esophagostomy, and placement of a gastrostomy tube should be considered.Chest Wall and Diaphragm. Virtually all chest wall injuries, consisting of rib fractures and laceration of intercostal vessels, are treated nonoperatively with pain control, pulmonary toilet or venti-latory management, and drainage of the pleural space as indicated. Early institution of effective pain control is essential. The

1	treated nonoperatively with pain control, pulmonary toilet or venti-latory management, and drainage of the pleural space as indicated. Early institution of effective pain control is essential. The authors advocate preemptive rib blocks with 0.25% bupivacaine hydrochlo-ride (Marcaine) in the trauma bay, followed by thoracic wall pain catheters.112 Epidural anesthesia is reserved for multiple segmen-tal fractures. Persistent hemorrhage from a chest tube after blunt trauma most often is due to injured intercostal arteries; for unusual persistent bleeding (see Table 7-10), thoracotomy with direct liga-tion or angioembolization may be required to arrest hemorrhage. In cases of extensive flail chest segments, markedly displaced bicortical rib fractures, or loss of 20% of the thoracic volume, open reduction and internal fixation of the fracture with plates may be warranted. Chest wall defects, particularly those seen with open pneumothorax, are repaired using local approximation of tissues

1	reduction and internal fixation of the fracture with plates may be warranted. Chest wall defects, particularly those seen with open pneumothorax, are repaired using local approximation of tissues or tissue transfer for coverage. Scapular and sternal fractures rarely require operative intervention but are markers for significant tho-racoabdominal force during injury; significant displacement may benefit from sternal plating (Fig. 7-59). Careful examination and imaging should exclude associated injuries, including blunt cardiac injury and descending BAI. On the other hand, clavicle fractures are often isolated injuries and should be managed with pain control and immobilization. The exception is posterior dislocation of the clavicular head, which may injure the subclavian vessels.Blunt diaphragmatic injuries usually result in a linear tear, and most injuries are large, whereas penetrating injuries are variable in size and location depending on the agent of injury. Regardless of the

1	diaphragmatic injuries usually result in a linear tear, and most injuries are large, whereas penetrating injuries are variable in size and location depending on the agent of injury. Regardless of the etiology, acute injuries are usually repaired through an abdominal approach to manage potential associated visceral injury. After delineation of the injury, the chest should be evacuated of all blood and particulate matter, and a thora-costomy tube placed if not previously done. Allis clamps are used to approximate the diaphragmatic edges, and the defect is closed with a running No. 1 polypropylene suture. Occasionally, large avulsions or shotgun wounds with extensive tissue loss will require polypropylene or biologic mesh to bridge the defect. Brunicardi_Ch07_p0183-p0250.indd 22410/12/18 6:20 PM 225TRAUMACHAPTER 7Alternatively, transposition of the diaphragm cephalad one to two intercostal spaces may allow repair without undue tension.73Abdominal InjuriesLiver and Extrahepatic

1	6:20 PM 225TRAUMACHAPTER 7Alternatively, transposition of the diaphragm cephalad one to two intercostal spaces may allow repair without undue tension.73Abdominal InjuriesLiver and Extrahepatic Biliary Tract. The liver’s large size makes it the organ most susceptible to blunt trauma, and it is frequently involved in upper torso penetrating wounds. Nonop-erative management of solid organ injuries is pursued in hemo-dynamically stable patients who do not have overt peritonitis or other indications for laparotomy. Patients with >grade II injuries should be admitted to the SICU with frequent hemodynamic monitoring, determination of hemoglobin, and abdominal exam-ination. The only absolute contraindication to nonoperative management is hemodynamic instability from intraperitoneal hemorrhage. Factors such as high injury grade, large hemo-peritoneum, contrast extravasation, or pseudoaneurysms may predict complications or failure of nonoperative management. Angioembolization and endoscopic

1	Factors such as high injury grade, large hemo-peritoneum, contrast extravasation, or pseudoaneurysms may predict complications or failure of nonoperative management. Angioembolization and endoscopic retrograde cholangiopan-creatography (ERCP) are useful adjuncts that can improve the success rate of nonoperative management.113,114 The indication for angiography to control hepatic hemorrhage is transfusion of 4 units of RBCs in 6 hours or 6 units of RBCs in 24 hours attributable to the liver.In the 15% of patients for whom emergent laparotomy is mandated, the primary goal is to arrest hemorrhage. Initial control of hemorrhage is best accomplished using perihepatic packing and manual compression. The edges of the liver laceration should be opposed for local pressure control of bleeding. Hemorrhage from most major hepatic injuries can be controlled with effec-tive perihepatic packing. The right costal margin is elevated, and the pads are strategically placed over and around the bleeding

1	from most major hepatic injuries can be controlled with effec-tive perihepatic packing. The right costal margin is elevated, and the pads are strategically placed over and around the bleeding site (see Fig. 7-37). Additional pads should be placed between the liver, diaphragm, and anterior chest wall until the bleeding has been controlled. Sometimes 10 to 15 pads may be required to control the hemorrhage from an extensive right lobar injury. Packing of injuries of the left lobe is not as effective because there is insufficient abdominal and thoracic wall anterior to the left lobe to provide adequate compression with the abdomen open. Fortunately, hemorrhage from the left lobe usually can be controlled by mobilizing the lobe and compressing it between the surgeon’s hands. With extensive injuries and major hemorrhage, a Pringle maneuver should be done immediately. Intermittent release of the Pringle is helpful to attenuate hepatic cellular loss. ABFigure 7-59. Significant sternal

1	injuries and major hemorrhage, a Pringle maneuver should be done immediately. Intermittent release of the Pringle is helpful to attenuate hepatic cellular loss. ABFigure 7-59. Significant sternal displacement (A; arrows) can be reduced and stabilized with sternal plating (B).If the patient has persistent bleeding despite packing, injuries to the hepatic artery, portal vein, and retrohepatic vasculature should be considered. A Pringle maneuver can help delineate the source of hemorrhage. Hemorrhage from hepatic artery and portal vein injuries will halt with the application of a vascular clamp across the portal triad, whereas bleeding from the hepatic veins and the retrohepatic vena cava will continue despite a Pringle maneuver.Injuries of the portal triad vasculature should be addressed immediately. In general, ligation from the celiac axis to the level of the common hepatic artery at the gastroduodenal arterial branch is tolerated due to extensive collaterals, but the proper hepatic

1	In general, ligation from the celiac axis to the level of the common hepatic artery at the gastroduodenal arterial branch is tolerated due to extensive collaterals, but the proper hepatic artery should be repaired. The right or left hepatic artery, or in urgent situations the portal vein, may be selectively ligated; occasionally, lobar necrosis will necessitate delayed anatomic resection. If the right hepatic artery is ligated, cholecystectomy also should be performed. If the vascular injury is a stab wound with clean transection of the vessels, primary end-to-end repair is done. If the injury is destructive, temporary shunting should be performed followed by interposition reversed saphenous vein graft (RSVG). Blunt avulsions of the portal structures are par-ticularly problematic if located at the hepatic plate, flush with the liver; hemorrhage control at the liver can be attempted with directed packing or Fogarty catheters. If injury to the portal triad vasculature is more proximal,

1	at the hepatic plate, flush with the liver; hemorrhage control at the liver can be attempted with directed packing or Fogarty catheters. If injury to the portal triad vasculature is more proximal, at the superior border of the pancreatic body or even retropancreatic, the pancreas must be transected to gain access for hemorrhage control and repair.If massive venous hemorrhage is seen from behind the liver despite use of the Pringle maneuver, the patient likely has a hepatic vein or retrohepatic vena cava injury. If bleeding can be controlled with perihepatic packing, the packing should be left undisturbed and the patient observed in the SICU. Placement of a hepatic vein stent by interventional radiology may be considered. If bleeding continues despite repeated attempts at packing, then direct repair, with or without hepatic vascular isolation, should be attempted. Three techniques have been used to accomplish hepatic vascular isolation: (a) direct repair with suprahepatic and

1	then direct repair, with or without hepatic vascular isolation, should be attempted. Three techniques have been used to accomplish hepatic vascular isolation: (a) direct repair with suprahepatic and infrahepatic clamping of the vena cava and stapled assisted parenchymal resection115; (b) temporary shunting of the retrohe-patic vena cava; and (c) venovenous bypass (Fig. 7-60).116A number of methods for the definitive control of hepatic parenchymal hemorrhage have been developed. Minor lac-erations may be controlled with manual compression applied directly to the injury site. Topical hemostatic techniques include Brunicardi_Ch07_p0183-p0250.indd 22510/12/18 6:20 PM 226BASIC CONSIDERATIONSPART Ithe use of an electrocautery (with the device set at 100 watts), argon beam coagulator, microcrystalline collagen, thrombin-soaked gelatin foam sponge, fibrin glue, and BioGlue. Suturing of the hepatic parenchyma with a blunt tipped 0 chromic suture (e.g., a “liver suture”) can be an effective

1	collagen, thrombin-soaked gelatin foam sponge, fibrin glue, and BioGlue. Suturing of the hepatic parenchyma with a blunt tipped 0 chromic suture (e.g., a “liver suture”) can be an effective hemostatic technique. A running suture is used to approximate the edges of shal-low lacerations, whereas deeper lacerations are approximated using interrupted horizontal mattress sutures placed parallel to the edge of the laceration. When the suture is tied, tension is adequate when visible hemorrhage ceases or the liver blanches around the suture. Caution must be used to prevent hepatic necrosis. This technique of placing large liver sutures controls bleeding through reapproximation of the liver laceration rather than direct ligation of bleeding vessels. Aggressive finger frac-ture to identify bleeding vessels followed by individual clip or suture ligation was advocated previously but currently has a limited role in hemostasis. Hepatic lobar arterial ligation may be appropriate for patients with

1	vessels followed by individual clip or suture ligation was advocated previously but currently has a limited role in hemostasis. Hepatic lobar arterial ligation may be appropriate for patients with recalcitrant arterial hemorrhage from deep within the liver and is a reasonable alternative to a deep hepatotomy, particularly in unstable patients. Omen-tum can be used to fill large defects in the liver. The tongue of omentum not only obliterates potential dead space with viable tissue but also provides an excellent source of macrophages. Additionally, the omentum can provide buttressing support for parenchymal sutures.Translobar penetrating injuries are particularly challeng-ing because the extent of the injury cannot be fully visualized. As discussed in “Damage Control Surgery,” options include intraparenchymal tamponade with a Foley catheter or balloon occlusion (see Fig. 7-49).117 If tamponade is successful with either modality, the balloon is left inflated for 24 to 48 hours Figure

1	intraparenchymal tamponade with a Foley catheter or balloon occlusion (see Fig. 7-49).117 If tamponade is successful with either modality, the balloon is left inflated for 24 to 48 hours Figure 7-60. Venovenous bypass permits hepatic vascular isolation with continued venous return to the heart. IMV = inferior mesenteric vein; IVC = inferior vena cava; SMV = superior mesenteric vein.followed by sequential deflation and removal at a second lapa-rotomy. Hepatotomy with ligation of individual bleeders occa-sionally may be required; however, division of the overlying viable hepatic tissue may cause considerable blood loss in the coagulopathic patient. Finally, angioembolization is an effective adjunct in any of these scenarios and should be considered early in the course of treatment.Several centers have reported patients with devastat-ing hepatic injuries or necrosis of the entire liver who have undergone successful hepatic transplantation.118 Clearly this is dramatic therapy, and the

1	centers have reported patients with devastat-ing hepatic injuries or necrosis of the entire liver who have undergone successful hepatic transplantation.118 Clearly this is dramatic therapy, and the patient must have all other injuries delineated, particularly those of the central nervous system, and have an excellent chance of survival excluding the hepatic injury. Because donor availability will limit such procedures, hepatic transplantation for trauma will continue to be performed only in extraordinary circumstances.Cholecystectomy is performed for injuries of the gallblad-der and after operative ligation of the right hepatic artery. Inju-ries of the extrahepatic bile ducts are a challenge due to their small size and thin walls. Because of the proximity of other portal structures and the vena cava, associated vascular inju-ries are common. These factors may preclude primary repair. Small lacerations with no accompanying loss or devitalization of adjacent tissue can be treated by the

1	vena cava, associated vascular inju-ries are common. These factors may preclude primary repair. Small lacerations with no accompanying loss or devitalization of adjacent tissue can be treated by the insertion of a T-tube through the wound or by lateral suturing using 6-0 monofila-ment absorbable suture. Virtually all transections and any injury associated with significant tissue loss will require a Roux-en-Y choledochojejunostomy. The anastomosis is performed using a single-layer interrupted technique with 5-0 monofilament absorbable suture. To reduce anastomotic tension, the jejunum should be sutured to the areolar tissue of the hepatic pedicle or porta hepatis. Injuries of the hepatic ducts are almost impos-sible to satisfactorily repair under emergent circumstances. One approach is to intubate the duct for external drainage and attempt a repair when the patient recovers or attempt stenting via ERC. Alternatively, the duct can be ligated if the opposite lobe is normal and

1	is to intubate the duct for external drainage and attempt a repair when the patient recovers or attempt stenting via ERC. Alternatively, the duct can be ligated if the opposite lobe is normal and uninjured.Patients undergoing perihepatic packing for extensive liver injuries typically are returned to the OR for pack removal 24 hours after initial injury. Earlier exploration may be indi-cated in patients with evidence of ongoing hemorrhage. Signs of rebleeding are usually conspicuous, and include a falling hemoglobin, accumulation of blood clots under the temporary abdominal closure device, and bloody output from drains; the magnitude of hemorrhage is reflected in ongoing hemodynamic instability and metabolic monitoring. Postoperative hemorrhage should be reevaluated in the OR once the patient’s coagulopa-thy is corrected. Alternatively, angioembolization is appropri-ate for complex injuries. Patients with hepatic ischemia due to prolonged intraoperative use of the Pringle maneuver have

1	coagulopa-thy is corrected. Alternatively, angioembolization is appropri-ate for complex injuries. Patients with hepatic ischemia due to prolonged intraoperative use of the Pringle maneuver have an expected elevation but subsequent resolution of transaminase levels, whereas patients requiring hepatic artery ligation may have frank hepatic necrosis. Although febrile patients should be evaluated for infectious complications, patients with complex hepatic injuries typically have intermittent “liver fever” for the first 5 days after injury.Aside from hemorrhage and hepatic necrosis, additional complications after significant hepatic trauma include bilomas, arterial pseudoaneurysms, and biliary fistulas (Fig. 7-61). Bilomas are loculated collections of bile, which may or may not be infected. If infected, they should be treated like an abscess via percutaneous drainage. Although small, sterile bilomas Brunicardi_Ch07_p0183-p0250.indd 22610/12/18 6:20 PM 227TRAUMACHAPTER 7Figure

1	If infected, they should be treated like an abscess via percutaneous drainage. Although small, sterile bilomas Brunicardi_Ch07_p0183-p0250.indd 22610/12/18 6:20 PM 227TRAUMACHAPTER 7Figure 7-61. Complications after hepatic trauma include bilomas (A; arrow), hepatic duct injuries (B), and hepatic necrosis after hepatic artery ligation or embolization (C).eventually will be reabsorbed, larger fluid collections should be drained. Biliary ascites, due to the disruption of a major bile duct, often requires reoperation and wide drainage. Primary repair of the injured intrahepatic duct is unlikely to be success-ful. Resectional debridement is indicated for the removal of peripheral portions of nonviable hepatic parenchyma.Pseudoaneurysms and biliary fistulas are rare complica-tions in patients with hepatic injuries. Because hemorrhage from hepatic injuries often is treated without isolating individ-ual bleeding vessels, arterial pseudoaneurysms may develop, with the potential for

1	patients with hepatic injuries. Because hemorrhage from hepatic injuries often is treated without isolating individ-ual bleeding vessels, arterial pseudoaneurysms may develop, with the potential for rupture. Rupture into a bile duct results in hemobilia, which is characterized by intermittent episodes of right upper quadrant pain, upper GI hemorrhage, and jaun-dice. If the aneurysm ruptures into a portal vein, portal venous hypertension with bleeding esophageal varices may occur. Either scenario is best managed with hepatic arteriography and embolization. Biliovenous fistulas, causing jaundice due to rapid increases in serum bilirubin levels, should be treated with ERCP and sphincterotomy. Rarely, a biliary fistulous commu-nication will form with intrathoracic structures in patients with associated diaphragm injuries, resulting in a bronchobiliary or pleurobiliary fistula. Due to the pressure differential between the biliary tract (positive) and the pleural cavity (negative), the

1	associated diaphragm injuries, resulting in a bronchobiliary or pleurobiliary fistula. Due to the pressure differential between the biliary tract (positive) and the pleural cavity (negative), the majority require operative closure. Occasionally, endoscopic sphincterotomy with stent placement will be required to address the pressure differential, and the pleurobiliary fistula will close spontaneously.Spleen. Until the 1970s, splenectomy was considered manda-tory for all splenic injuries. Recognition of the immune function of the spleen refocused efforts on operative splenic salvage in the 1980s.119,120 After demonstrated success in pediatric patients, nonoperative management has become the preferred means of splenic salvage for all patients. The identification of contrast extravasation as a risk factor for failure of nonoperative man-agement led to liberal use of angioembolization. The role of selective angioembolization (SAE) continues to be defined, but appears warranted in high

1	as a risk factor for failure of nonoperative man-agement led to liberal use of angioembolization. The role of selective angioembolization (SAE) continues to be defined, but appears warranted in high grade injuries, particularly those with contrast blush.121 It is clear, however, that up to 15% to 20% of patients with splenic trauma warrant early splenectomy and that failure of nonoperative management often represents inappro-priate patient selection.122,123 Indications for early intervention in adults include initiation of blood transfusion within the first 12 hours and hemodynamic instability. Unlike hepatic injuries, which usually rebleed within 48 hours, delayed hemorrhage or rupture of the spleen can occur up to weeks after injury.Splenic injuries are managed operatively by splenectomy, partial splenectomy, or splenic repair (splenorrhaphy), based on the extent of the injury and the physiologic condition of the patient. Splenectomy is indicated for significant hilar injuries,

1	partial splenectomy, or splenic repair (splenorrhaphy), based on the extent of the injury and the physiologic condition of the patient. Splenectomy is indicated for significant hilar injuries, pulverized splenic parenchyma, or any >grade II injury in a patient with coagulopathy or multiple life-threatening inju-ries. The authors use autotransplantation of splenic implants (Fig. 7-62) to achieve partial immunocompetence in younger patients who do not have an associated enteric injury. Drains are not used. Partial splenectomy can be employed in patients in whom only the superior or inferior pole has been injured. Hem-orrhage from the raw splenic edge is controlled with horizontal mattress sutures, with gentle compression of the parenchyma (Fig. 7-63). During splenorrhaphy hemostasis is achieved by topical methods (electrocautery; argon beam coagulation; appli-cation of thrombin-soaked gelatin foam sponges, fibrin glue, or BioGlue), envelopment of the injured spleen in absorbable mesh,

1	by topical methods (electrocautery; argon beam coagulation; appli-cation of thrombin-soaked gelatin foam sponges, fibrin glue, or BioGlue), envelopment of the injured spleen in absorbable mesh, and pledgeted suture repair.After splenectomy or splenorrhaphy, postoperative hemor-rhage may be due to an improperly ligated or unrecognized short gastric artery, or recurrent bleeding from the splenic parenchyma if splenic repair was used. An immediate postsplenectomy Brunicardi_Ch07_p0183-p0250.indd 22710/12/18 6:20 PM 228BASIC CONSIDERATIONSPART Iincrease in platelets and WBCs is normal; however, beyond postoperative day 5, a WBC count above 15,000/mm3 and a platelet/WBC ratio of <20 are associated with sepsis and should prompt a thorough search for underlying infection.124 A com-mon infectious complication after splenectomy is a subphrenic abscess, which should be managed with percutaneous drain-age. Additional sources of morbidity include a concurrent or unrecognized iatrogenic injury

1	complication after splenectomy is a subphrenic abscess, which should be managed with percutaneous drain-age. Additional sources of morbidity include a concurrent or unrecognized iatrogenic injury to the pancreatic tail during rapid splenectomy resulting in pancreatic ascites or fistula, or gastric perforation during short gastric vessel ligation. Enthusiasm for splenic salvage was driven by the rare, but often fatal, compli-cation of overwhelming postsplenectomy sepsis. Overwhelm-ing postsplenectomy sepsis is caused by encapsulated bacteria, Streptococcus pneumoniae, Haemophilus influenzae, and Neis-seria meningitidis, which are resistant to antimicrobial treat-ment. In patients undergoing splenectomy, prophylaxis against these bacteria is provided via vaccines administered optimally at >14 days postinjury.125Stomach and Small Intestine. Little controversy exists regarding the repair of injuries to the stomach or small bowel because of a rich blood supply. Gastric wounds can be

1	at >14 days postinjury.125Stomach and Small Intestine. Little controversy exists regarding the repair of injuries to the stomach or small bowel because of a rich blood supply. Gastric wounds can be oversewn with a running single-layer suture line or closed with a stapler. If a single-layer closure is chosen, full-thickness bites should be taken to ensure hemostasis from the well-vascularized gastric Figure 7-63. Interrupted pledgeted sutures may effectively control hemorrhage from the cut edge of the spleen.Figure 7-62. Autologous splenic transplantation is performed by placing sections of splenic parenchyma, 40 × 40 × 3 mm in size, into pouches in the greater omentum.wall. The most commonly missed gastric injury is the posterior wound of a through and through penetrating injury. Injuries also can be overlooked if the wound is located within the mesen-tery of the lesser curvature or high in the fundus. To delineate a questionable injury, the stomach can be digitally occluded at the

1	also can be overlooked if the wound is located within the mesen-tery of the lesser curvature or high in the fundus. To delineate a questionable injury, the stomach can be digitally occluded at the pylorus while methylene blue-colored saline is instilled via a nasogastric (NG) tube. Alternatively, air can be introduced via the NG tube with the abdomen filled with saline. Partial gastrectomy may be required for destructive injuries, with resec-tions of the distal antrum or pylorus reconstructed using a Bill-roth procedure. Patients with injuries that damage both Latarjet nerves or vagi should undergo a drainage procedure (see Chap-ter 26). Small intestine injuries can be repaired using a trans-verse running 3-0 PDS suture if the injury is less than one-third the circumference of the bowel. Destructive injuries or multiple penetrating injuries occurring close together are treated with segmental resection followed by end-to-end anastomosis using a continuous, single-layer 3-0

1	the bowel. Destructive injuries or multiple penetrating injuries occurring close together are treated with segmental resection followed by end-to-end anastomosis using a continuous, single-layer 3-0 polypropylene suture.55 Mesenteric injuries may result in an ischemic segment of intestine, which mandates resection.Following repair of GI tract injuries, patients may develop a postoperative ileus. Return of bowel function is indicated by a decrease in gastrostomy or nasogastric tube output. The topic of nutrition is well covered in other chapters (see Chapter 2), but a few issues warrant mention. Multiple studies have con-firmed the importance of early total enteral nutrition (TEN) in the trauma population, particularly its impact in reducing septic complications.126 The route of enteral feedings (stomach vs. small bowel) tends to be less important because gut toler-ance appears equivalent unless there is upper GI tract pathol-ogy. Although early enteral nutrition is the goal, evidence

1	(stomach vs. small bowel) tends to be less important because gut toler-ance appears equivalent unless there is upper GI tract pathol-ogy. Although early enteral nutrition is the goal, evidence of bowel function should be apparent before advancing to goal tube feedings. Overzealous jejunal feeding can lead to small bowel necrosis in the patient recovering from profound shock. Patients undergoing monitoring for nonoperative manage-ment of grade III or higher solid organ injuries should receive nothing by mouth for at least 48 hours in case they require an operation. Although there is general reluctance to initiate TEN in patients with an open abdomen, a recent multicenter trial demonstrates that TEN in the postinjury open abdomen is feasible.127 For those patients without a bowel injury, TEN was associated with higher fascial closure rates, decreased Brunicardi_Ch07_p0183-p0250.indd 22810/12/18 6:21 PM 229TRAUMACHAPTER 7complications, and decreased mortality. TEN in patients with

1	TEN was associated with higher fascial closure rates, decreased Brunicardi_Ch07_p0183-p0250.indd 22810/12/18 6:21 PM 229TRAUMACHAPTER 7complications, and decreased mortality. TEN in patients with bowel injuries does not appear to alter fascial closure rates, complications, or mortality; hence, EN appears to be neither advantageous nor detrimental in these patients. Once resuscita-tion is complete, initiation of TEN, even at trophic levels (20 mL/h), should be considered in all injured patients with an open abdomen.Duodenum and Pancreas. The spectrum of injuries to the duodenum includes hematomas, perforation (blunt blow-outs, lacerations from stab wounds, or blast injury from gunshot wounds), and combined pancreaticoduodenal injuries. The majority of duodenal hematomas are managed nonoperatively with nasogastric suction and parenteral nutrition. Patients with suspected associated perforation, suggested by clinical deterio-ration or imaging with retroperitoneal free air or contrast

1	with nasogastric suction and parenteral nutrition. Patients with suspected associated perforation, suggested by clinical deterio-ration or imaging with retroperitoneal free air or contrast extrav-asation, should undergo operative exploration. A marked drop in nasogastric tube output heralds resolution of the hematoma, which typically occurs within 2 weeks; repeat imaging to con-firm these clinical findings is optional. If the patient shows no clinical or radiographic improvement within 3 weeks, operative evaluation is warranted.Small duodenal perforations or lacerations should be treated by primary repair using a running single-layer suture of 3-0 monofilament.128 The wound should be closed in a direc-tion that results in the largest residual lumen. Challenges arise when there is a substantial loss of duodenal tissue. Extensive injuries of the first portion of the duodenum (proximal to the duct of Santorini) can be repaired by debridement and end-to-end anastomosis because of the

1	loss of duodenal tissue. Extensive injuries of the first portion of the duodenum (proximal to the duct of Santorini) can be repaired by debridement and end-to-end anastomosis because of the mobility and rich blood supply of the distal gastric atrium and pylorus. In contrast, the second portion is tethered to the head of the pancreas by its blood supply and the ducts of Wirsung and Santorini; therefore, no more than 1 cm of duodenum can be mobilized away from the pancreas. Moreover, suture repair using an end-to-end anastomosis in the second portion often results in an unacceptably narrow lumen. Therefore, defects in the second portion of the duodenum should be “patched” with a Roux-en-Y duodenojejunostomy. Duodenal injuries with tissue loss distal to the papilla of Vater and proximal to the superior mesenteric vessels are best treated by Roux-en-Y duodenojejunostomy with the distal portion of the duodenum oversewn (Fig. 7-64). In particular, injuries in the distal third and fourth

1	the superior mesenteric vessels are best treated by Roux-en-Y duodenojejunostomy with the distal portion of the duodenum oversewn (Fig. 7-64). In particular, injuries in the distal third and fourth portions of the duodenum (behind the mesenteric vessels) should be resected, and a duodenojejunostomy should be per-formed on the D3 side of the superior mesenteric vessels.Optimal management of pancreatic trauma is determined by where the parenchymal damage is located and whether the intra-pancreatic common bile duct and main pancreatic duct remain intact. Patients with pancreatic contusions (defined as injuries that leave the ductal system intact) can be treated nonoperatively or with closed suction drainage if undergoing laparotomy for other indications. Patients with proximal pancreatic injuries, defined as those that lie to the right of the superior mesenteric vessels, are also managed with closed suction drainage.129 In contrast, distal pancreatic injuries are managed based upon

1	injuries, defined as those that lie to the right of the superior mesenteric vessels, are also managed with closed suction drainage.129 In contrast, distal pancreatic injuries are managed based upon ductal integrity. Pan-creatic duct disruption can be identified through direct exploration of the parenchymal laceration, operative pancreatography, ERCP, or magnetic resonance cholangiopancreatography. Patients with distal ductal disruption undergo distal pancreatectomy, preferably with splenic preservation.Injuries to the pancreatic head add substantial complex-ity because the intrapancreatic portion of the common bile duct Figure 7-64. Roux-en-Y duodenojejunostomy is used to treat duo-denal injuries between the papilla of Vater and superior mesenteric vessels when tissue loss precludes primary repair.traverses this area and often converges with the pancreatic duct. In contrast to diagnosis of pancreatic duct injuries, identifica-tion of intrapancreatic common bile duct disruption is

1	repair.traverses this area and often converges with the pancreatic duct. In contrast to diagnosis of pancreatic duct injuries, identifica-tion of intrapancreatic common bile duct disruption is relatively simple. The first method is to squeeze the gallbladder and look for bile leaking from the pancreatic wound. Otherwise, cholan-giography, optimally via the cystic duct, is diagnostic. Definitive treatment of this injury entails division of the common bile duct superior to the first portion of the duodenum, with ligation of the distal duct and reconstruction with a Roux-en-Y choledochojeju-nostomy. For injuries to the head of the pancreas that involve the main pancreatic duct but not the intrapancreatic bile duct, there are few options. Distal pancreatectomy alone is rarely indicated due to the extended resection of normal gland and the resultant risk of pancreatic insufficiency. Central pancreatectomy pre-serves the common bile duct, and mobilization of the pancreatic body permits

1	to the extended resection of normal gland and the resultant risk of pancreatic insufficiency. Central pancreatectomy pre-serves the common bile duct, and mobilization of the pancreatic body permits drainage into a posterior wall pancreaticogas-trostomy or a Roux-en-Y pancreaticojejunostomy (Fig. 7-65). Although this approach avoids a pancreaticoduodenectomy (Whipple procedure), the complexity may make the pancre-aticoduodenectomy more appropriate in patients with multiple injuries and is usually done in a damage control scenario. Some injuries of the pancreatic head do not involve either the pancre-atic or common bile duct; if no clear ductal injury is present, drains are placed. Rarely, patients sustain destructive injuries to the head of the pancreas or combined pancreaticoduodenal inju-ries that require pancreaticoduodenectomy. Examples of such injuries include transection of both the intrapancreatic bile duct and the main pancreatic duct in the head of the pancreas, avul-sion of

1	that require pancreaticoduodenectomy. Examples of such injuries include transection of both the intrapancreatic bile duct and the main pancreatic duct in the head of the pancreas, avul-sion of the papilla of Vater from the duodenum, and destruction Brunicardi_Ch07_p0183-p0250.indd 22910/12/18 6:21 PM 230BASIC CONSIDERATIONSPART Iof the entire second portion of the duodenum. In these cases of extensive injuries, damage control principles are often employed.In contrast to proximal injuries, pancreatic resection con-tinues to be advocated for major ductal disruption in the more distal pancreas. Several options exist for treating injuries of the pancreatic body and tail. In stable patients, spleen-preserving distal pancreatectomy should be performed. An alternative, which preserves both the spleen and distal transected end of the pancreas, is either a Roux-en-Y pancreaticojejunostomy or pan-creaticogastrostomy. If the patient is physiologically compro-mised, distal pancreatectomy with

1	spleen and distal transected end of the pancreas, is either a Roux-en-Y pancreaticojejunostomy or pan-creaticogastrostomy. If the patient is physiologically compro-mised, distal pancreatectomy with splenectomy is the preferred approach. Regardless of the choice of definitive procedure, the pancreatic duct in the proximal edge of transected pancreas should be individually ligated or occluded with a TA stapler. Application of fibrin glue over the stump may be advantageous.Pyloric exclusion may be used to divert the GI stream after high-risk, complex duodenal repairs, particularly with adjacent pancreatic injuries (Fig. 7-66).130 If the duodenal repair breaks down, the resultant fistula is an end fistula, which is easier to manage and more likely to close than a lateral fistula. To per-form a pyloric exclusion, first a gastrostomy is made on the greater curvature near the pylorus. The pylorus is then grasped with a Babcock clamp, via the gastrostomy, and oversewn with an O polypropylene

1	a pyloric exclusion, first a gastrostomy is made on the greater curvature near the pylorus. The pylorus is then grasped with a Babcock clamp, via the gastrostomy, and oversewn with an O polypropylene suture. A gastrojejunostomy restores GI tract continuity. Vagotomy is not necessary because a risk of marginal ulceration has not been documented. Perhaps surpris-ingly, the sutures maintain diversion for only 3 to 4 weeks. Alternatively, the most durable pyloric closure is a double exter-nal staple line across the pylorus using a TA stapler.Complications should be expected after major pancre-aticoduodenal injuries.131 Delayed hemorrhage is rare but may Figure 7-65. For injuries of the pancreatic head that involve the pancreatic duct but spare the common bile duct, central pancreatic resection with Roux-en-Y pancreaticojejunostomy prevents pancreatic insufficiency.occur with pancreatic necrosis or abdominal infection; this usu-ally can be managed by angioembolization. If closed suction

1	with Roux-en-Y pancreaticojejunostomy prevents pancreatic insufficiency.occur with pancreatic necrosis or abdominal infection; this usu-ally can be managed by angioembolization. If closed suction drains have been inserted for major pancreatic trauma, these should remain in place until the patient is tolerating an oral diet or enteral nutrition. Pancreatic fistula is diagnosed after post-operative day 5 in patients with drain output of >30 mL/d and a drain amylase level three times the serum value. Pancreatic fistula develops in over 20% of patients with combined injuries and should be managed similar to fistulas after elective surgery (see Chapter 33). Similarly, a duodenal fistula, presumptively an end fistula if a pyloric exclusion has been done, will typi-cally heal in 6 to 8 weeks with adequate drainage and control of intra-abdominal sepsis. Pancreatic pseudocysts in patients man-aged nonoperatively suggest a missed injury, and ERCP should be done to evaluate the integrity of the

1	adequate drainage and control of intra-abdominal sepsis. Pancreatic pseudocysts in patients man-aged nonoperatively suggest a missed injury, and ERCP should be done to evaluate the integrity of the pancreatic duct. Late pseudocysts may be a complication of operative management and are treated much like those in patients with pancreatitis (see Chapter 33). Intra-abdominal abscesses are common and rou-tinely managed with percutaneous drainage.Colon and Rectum. Currently, three methods for treating colonic injuries are used: primary repair, end colostomy, and primary repair with diverting loop ileostomy. Primary repairs include lateral suture repair or resection of the damaged seg-ment with reconstruction by ileocolostomy or colocolostomy. All suturing and anastomoses are performed using a running single-layer technique (Fig. 7-67).55 The advantage of defini-tive treatment must be balanced against the possibility of anas-tomotic leakage if suture lines are created under suboptimal

1	a running single-layer technique (Fig. 7-67).55 The advantage of defini-tive treatment must be balanced against the possibility of anas-tomotic leakage if suture lines are created under suboptimal conditions. Alternatively, although use of an end colostomy requires a second operation, an unprotected suture line with Brunicardi_Ch07_p0183-p0250.indd 23010/12/18 6:21 PM 231TRAUMACHAPTER 7Figure 7-66. A. Pyloric exclusion is used to treat combined injuries of the duodenum and the head of the pancreas as well as isolated duodenal injuries when the duodenal repair is less than optimal. B and C. The pylorus is oversewn through a gastrotomy, which is subsequently used to create a gastrojejunostomy. The authors frequently use operatively placed feeding jejunostomy tube feedings for these patients.Figure 7-67. Technique for bowel repair and anastomosis. A. The running, single-layer suture is started at the mesenteric border. B. Stitches are spaced 3 to 4 mm from the edge of the bowel and

1	7-67. Technique for bowel repair and anastomosis. A. The running, single-layer suture is started at the mesenteric border. B. Stitches are spaced 3 to 4 mm from the edge of the bowel and advanced 3 to 4 mm, including all layers except the mucosa. C. The continuous suture is tied near the antimesenteric border.Brunicardi_Ch07_p0183-p0250.indd 23110/12/18 6:21 PM 232BASIC CONSIDERATIONSPART IFigure 7-68. Loop colostomy will completely divert the fecal flow, allowing the low rectal injury to heal. For extensive wounds, presacral drains are inserted through a perianal incision (box) and advanced along Waldeyer’s fascia (dashed line).the potential for breakdown is avoided. Numerous large ret-rospective and several prospective studies have now clearly demonstrated that primary repair is safe and effective in virtu-ally all patients with penetrating wounds.132 Colostomy is still appropriate in a few patients, but the current dilemma is how to select which patients should undergo the

1	safe and effective in virtu-ally all patients with penetrating wounds.132 Colostomy is still appropriate in a few patients, but the current dilemma is how to select which patients should undergo the procedure. Cur-rently, the overall physiologic status of the patient, rather than local factors, directs decision making. Patients with devastating left colon injuries requiring damage control have a leak rate of over 40%, and may be candidates for temporary colostomy.133 Diverting ileostomy with colocolostomy is another option for these high-risk patients.Rectal injuries are similar to colonic injuries with respect to the ecology of the luminal contents, overall structure, and blood supply of the wall, but access to extraperitoneal injuries is limited due to the surrounding bony pelvis. Therefore, indirect treatment with intestinal diversion usually is required. The current options are loop ileostomy and sigmoid loop colostomy. The latter is preferred because it is quick and easy to

1	Therefore, indirect treatment with intestinal diversion usually is required. The current options are loop ileostomy and sigmoid loop colostomy. The latter is preferred because it is quick and easy to perform, and provides essentially total fecal diversion. For sigmoid colostomy, technical elements include: (a) adequate mobilization of the sigmoid colon so that the loop will rest on the abdominal wall without tension, (b) maintenance of the spur of the colostomy (the common wall of the proximal and distal limbs after maturation) above the level of the skin with a one-half-inch Penrose drain or similar device, (c) longitudinal incision in the tenia coli, and (d) immediate matu-ration in the OR (Fig. 7-68). If the injury is accessible (e.g., in the posterior intraperitoneal portion of the rectum), repair of the injury should also be attempted. However, it is not necessary to explore the extraperitoneal rectum to repair a distal perforation. If the rectal injury is extensive, another

1	rectum), repair of the injury should also be attempted. However, it is not necessary to explore the extraperitoneal rectum to repair a distal perforation. If the rectal injury is extensive, another option is to divide the rectum at the level of the injury, oversew or staple the distal rec-tal pouch if possible, and create an end colostomy (Hartmann’s procedure). Extensive injuries may warrant presacral drainage with Penrose drains placed along Waldeyer’s fascia via a peri-anal incision (see Fig. 7-68), but routine presacral drainage and rectal washout is no longer practiced. In rare instances in which destructive injuries are present, an abdominoperineal resection may be necessary to avert lethal pelvic sepsis.Complications related to colorectal injuries include intra-abdominal abscess, fecal fistula, wound infection, and stomal complications. Intra-abdominal abscesses occur in approxi-mately 10% of patients, and most are managed with percu-taneous drainage. Fistulas occur in 1% to 3%

1	fistula, wound infection, and stomal complications. Intra-abdominal abscesses occur in approxi-mately 10% of patients, and most are managed with percu-taneous drainage. Fistulas occur in 1% to 3% of patients and usually present as an abscess or wound infection with subse-quent continuous drainage of fecal output; the majority will heal spontaneously with routine care (see Chapter 29). Stomal com-plications (necrosis, stenosis, obstruction, and prolapse) occur in 5% of patients and may require either immediate or delayed reoperation. Stomal necrosis should be carefully monitored because spread beyond the mucosa may result in septic compli-cations, including necrotizing fasciitis of the abdominal wall. Penetrating injuries that involve both the rectum and adjacent bony structures are prone to development of osteomyelitis. Bone biopsy is performed for diagnosis and bacteriologic analysis, and treatment entails long-term IV antibiotic therapy and occa-sionally debridement.Abdominal and

1	to development of osteomyelitis. Bone biopsy is performed for diagnosis and bacteriologic analysis, and treatment entails long-term IV antibiotic therapy and occa-sionally debridement.Abdominal and Pelvic Vasculature. Injury to the major arter-ies and veins in the abdomen can be a technical challenge.132-137 Although penetrating trauma indiscriminately affects all blood vessels, blunt trauma most commonly involves renal vasculature and occasionally the abdominal aorta. Patients with a penetrat-ing aortic wound who survive to reach the OR frequently have a contained hematoma within the retroperitoneum. Due to lack of mobility of the abdominal aorta, few injuries are amenable to primary repair. Supraceliac aortic wounds are particularly challenging due to the need for proximal control. Small lateral perforations may be controlled with 4-0 polypropylene suture or a PTFE patch, but end-to-end interposition grafting with a PTFE tube graft is the most common repair. Blunt injuries are

1	Small lateral perforations may be controlled with 4-0 polypropylene suture or a PTFE patch, but end-to-end interposition grafting with a PTFE tube graft is the most common repair. Blunt injuries are typi-cally extensive intimal tears of the infrarenal aorta with resultant thrombosis, and are exposed via a direct approach; most require an interposition graft. To avoid future vascular-enteric fistulas, the vascular suture lines should be covered with omentum.Brunicardi_Ch07_p0183-p0250.indd 23210/12/18 6:21 PM 233TRAUMACHAPTER 7Penetrating wounds to the superior mesenteric artery (SMA) are typically encountered after exploration for a gun-shot wound, with “black bowel” and associated supramesocolic hematoma being pathognomonic. Blunt avulsions of the SMA are rare but should be considered in patients with a seat belt sign who have midepigastric pain or tenderness and associated hypotension. For injuries of the SMA, temporary damage con-trol with a Pruitt-Inahara shunt can prevent

1	in patients with a seat belt sign who have midepigastric pain or tenderness and associated hypotension. For injuries of the SMA, temporary damage con-trol with a Pruitt-Inahara shunt can prevent extensive bowel necrosis. For definitive repair, end-to-end interposition RSVG from the proximal SMA to the SMA past the point of injury can be performed if there is no associated pancreatic injury. Alterna-tively, if the patient has an associated pancreatic injury, the graft should be tunneled from the distal aorta beneath the duodenum to the distal SMA. For proximal SMV injuries, digital compres-sion for hemorrhage control is followed by attempted venorrha-phy; ligation is an option in a life-threatening situation, but the resultant bowel edema requires aggressive fluid resuscitation. Temporary abdominal closure and a second-look operation to evaluate bowel viability should be done.Transpelvic gunshot wounds or blunt injuries with associ-ated pelvic fractures are the most common scenarios in

1	closure and a second-look operation to evaluate bowel viability should be done.Transpelvic gunshot wounds or blunt injuries with associ-ated pelvic fractures are the most common scenarios in patients with iliac artery injuries. As with abdominal vascular injuries, a Pruitt-Inahara shunt can be used for temporary shunting of the vessel for damage control. Definitive interposition graft-ing with excision of the injured segment is appropriate (see “Vascular Repair Techniques”). Careful monitoring for distal embolic events and reperfusion injury necessitating fasciotomy is imperative.In general, outcome after pelvic vascular injuries is related to (a) the technical success of the vascular reconstruction and (b) associated soft tissue and nerve injuries. Vascular repairs rarely fail after the first 12 hours, whereas soft tissue infection is a limb threat for several weeks. Following aortic interposition grafting, the patient’s SBP should not exceed 120 mmHg for at least the first 72 hours

1	first 12 hours, whereas soft tissue infection is a limb threat for several weeks. Following aortic interposition grafting, the patient’s SBP should not exceed 120 mmHg for at least the first 72 hours postoperatively. Patients requiring ligation of an inferior vena cava injury often develop marked bilateral lower extremity edema. To limit the associated morbidity, the patient’s legs should be wrapped with elastic bandages from the toes to the hips and elevated. For superior mesenteric vein injuries, either ligation or thrombosis after venorrhaphy results in marked bowel edema; fluid resuscitation should be aggressive and abdominal pressure monitoring routine in these patients. Prosthetic graft infections are rare complications, but prevention of bacteremia is imperative82; administration of antibiotics perioperatively and treatment of secondary infections is indicated. Long-term arte-rial graft complications such as stenosis or pseudoaneurysms are uncommon, and routine graft

1	of antibiotics perioperatively and treatment of secondary infections is indicated. Long-term arte-rial graft complications such as stenosis or pseudoaneurysms are uncommon, and routine graft surveillance rarely is performed. Consequently, long-term administration of antiplatelet agents or antithrombotics is not routine.Genitourinary Tract. Historically, when undergoing lapa-rotomy for trauma, all penetrating wounds to the kidneys were explored; recently, routine exploration of Gerota’s fascia has been questioned.138,139 Parenchymal renal injuries are treated with hemostatic and reconstructive techniques similar to those used for injuries of the liver and spleen: topical methods (electrocautery; argon beam coagulation; application of thrombin-soaked gela-tin foam sponge, fibrin glue, or BioGlue) and pledgeted suture repair. However, two caveats are recognized: the collecting sys-tem should be closed separately, and the renal capsule should be preserved to close over the repair of the

1	and pledgeted suture repair. However, two caveats are recognized: the collecting sys-tem should be closed separately, and the renal capsule should be preserved to close over the repair of the collecting system (Fig. 7-69). Renal vascular injuries are common after penetrat-ing trauma and may be deceptive due to tamponade by Gerota’s Figure 7-69. When renorrhaphy is undertaken, effective repair is assisted by attention to several key points: A. Vascular occlusion controls bleeding and permits adequate visualization. B. The renal capsule is carefully preserved. C. The collecting system is closed separately with absorbable suture. D. The preserved capsule is closed over the collecting system repair.Brunicardi_Ch07_p0183-p0250.indd 23310/12/18 6:21 PM 234BASIC CONSIDERATIONSPART Ifascia, which results in delayed hemorrhage. Arterial reconstruc-tion using graft interposition should be attempted within 5 hours of injury for renal preservation. For destructive parenchymal or irreparable

1	which results in delayed hemorrhage. Arterial reconstruc-tion using graft interposition should be attempted within 5 hours of injury for renal preservation. For destructive parenchymal or irreparable renovascular injuries, nephrectomy may be the only option; a normal contralateral kidney must be palpated because unilateral renal agenesis occurs in 0.1% of patients.Over 90% of blunt renal injuries are treated nonoperatively. Hematuria typically resolves within a few days with bed rest, although rarely bleeding is so persistent that bladder irrigation to dispel blood clots is warranted. Persistent gross hematuria may require embolization, whereas urinomas can be drained percutane-ously. Operative intervention after blunt trauma is limited to reno-vascular injuries and destructive parenchymal injuries that result in hypotension. The renal arteries and veins are uniquely susceptible to traction injury caused by blunt trauma. As the artery is stretched, the inelastic intima and media may

1	injuries that result in hypotension. The renal arteries and veins are uniquely susceptible to traction injury caused by blunt trauma. As the artery is stretched, the inelastic intima and media may rupture, which causes throm-bus formation and resultant stenosis or occlusion. The success rate for renal artery repair is limited, but an attempt is reasonable if the injury is <5 hours old or if the patient has a solitary kidney or bilat-eral injuries.140 Image-guided endoluminal stent placement is now employed for many of these injuries recognized by CT scanning. Reconstruction after blunt renal injuries may be difficult, however, because the injury is typically at the level of the aorta. If repair is not possible within this time frame, leaving the kidney in situ does not necessarily lead to the late sequelae of hypertension or abscess formation. The renal vein may be torn or completely avulsed from the vena cava due to blunt trauma. Typically, the large hematoma causes hypotension,

1	to the late sequelae of hypertension or abscess formation. The renal vein may be torn or completely avulsed from the vena cava due to blunt trauma. Typically, the large hematoma causes hypotension, which leads to operative intervention. During laparotomy for blunt trauma, expanding or pulsatile perinephric hematomas should be explored. If necessary, emergent vascular control can be obtained by placing a curved vascular clamp across the hilum from an inferior approach. Techniques of repair and hemostasis are similar to those described earlier.Injuries to the ureters are uncommon but may occur in patients with pelvic fractures and penetrating trauma. An injury may not be identified until a complication (i.e., a urinoma) becomes apparent. If an injury is suspected during operative exploration but is not clearly identified, methylene blue or indigo carmine is administered IV with observation for extrava-sation. Injuries are repaired using 5-0 absorbable monofilament, and mobilization of

1	but is not clearly identified, methylene blue or indigo carmine is administered IV with observation for extrava-sation. Injuries are repaired using 5-0 absorbable monofilament, and mobilization of the kidney may reduce tension on the anas-tomosis. Distal ureteral injuries can be treated by reimplanta-tion facilitated with a psoas hitch and/or Boari flap. In damage control circumstances, the ureter can be ligated on both sides of the injury and a nephrostomy tube placed.Bladder injuries are subdivided into those with intraperito-neal extravasation and those with extraperitoneal extravasation. Ruptures or lacerations of the intraperitoneal bladder are opera-tively closed with a running, single-layer, 3-0 absorbable mono-filament suture. Laparoscopic repair is becoming common in patients not requiring laparotomy for other injuries. Extraperito-neal ruptures are treated nonoperatively with bladder decompres-sion for 2 weeks. Urethral injuries are managed by bridging the defect with a

1	not requiring laparotomy for other injuries. Extraperito-neal ruptures are treated nonoperatively with bladder decompres-sion for 2 weeks. Urethral injuries are managed by bridging the defect with a Foley catheter, with or without direct suture repair. Strictures are not uncommon but can be managed electively.Female Reproductive Tract. Gynecologic injuries are rare. Occasionally the vaginal wall will be lacerated by a bone frag-ment from a pelvic fracture. Although repair is not mandated, it should be performed if physiologically feasible. More impor-tant, however, is recognition of the open fracture, need for possible drainage, and potential for pelvic sepsis. Penetrating injuries to the vagina, uterus, fallopian tubes, and ovaries are also uncommon, and routine hemostatic techniques are used. Repair of a transected fallopian tube can be attempted but prob-ably is unjustified because a suboptimal repair will increase the risk of tubal pregnancy. Transection at the injury site with

1	used. Repair of a transected fallopian tube can be attempted but prob-ably is unjustified because a suboptimal repair will increase the risk of tubal pregnancy. Transection at the injury site with proximal ligation and distal salpingectomy is a more prudent approach.Pelvic Fracture Hemorrhage ControlPatients with pelvic fractures who are hemodynamically unsta-ble are a diagnostic and therapeutic challenge for the trauma team. These injuries often occur in conjunction with other life-threatening injuries, and there is no universal agreement among clinicians on management. Current management algorithms in the United States incorporate variable time frames for bony stabilization and fixation, as well as hemorrhage control by preperitoneal pelvic packing and/or angioembolization. Early institution of a multidisciplinary approach with the involvement of trauma surgeons, orthopedic surgeons, interventional radiolo-gists, the director of the blood bank, and anesthesiologists is imperative

1	of a multidisciplinary approach with the involvement of trauma surgeons, orthopedic surgeons, interventional radiolo-gists, the director of the blood bank, and anesthesiologists is imperative due to high associated mortality rates (Fig. 7-70).Evaluation in the ED focuses on identification of injuries mandating operative intervention (e.g., massive hemothorax or hemoperitoneum) and injuries related to the pelvic fracture that alter management (e.g., injuries to the iliac artery, rectum, ure-thra, or bladder). Immediate temporary stabilization with sheeting of the pelvis or application of commercially available compres-sion devices should be performed in hemodynamically unstable patients. In patients with profound shock who are at high risk based upon mechanism (e.g., autopedestrian accident), pelvic sta-bilization should be done before radiographic confirmation. If the patient’s primary source of bleeding is the fracture-related pelvic hematoma, several options exist for hemorrhage

1	pelvic sta-bilization should be done before radiographic confirmation. If the patient’s primary source of bleeding is the fracture-related pelvic hematoma, several options exist for hemorrhage control. Because 85% of bleeding due to pelvic fractures is venous or bony in ori-gin, the authors advocate immediate external fixation and pre-peritoneal pelvic packing.141 Anterior external fixation decreases pelvic volume, which promotes tamponade of venous bleeding and prevents secondary hemorrhage from the shifting of bony ele-ments. Pelvic packing, in which six laparotomy pads (four in chil-dren) are placed directly into the paravesical space through a small anterior suprapubic incision, provides tamponade for the bleeding (Fig. 7-71). Pelvic packing also eliminates the often difficult deci-sion by the trauma surgeon: OR vs. interventional radiology? All patients can be rapidly transported to the OR, and packing can be accomplished in under 30 minutes. In the authors’ experience, this

1	by the trauma surgeon: OR vs. interventional radiology? All patients can be rapidly transported to the OR, and packing can be accomplished in under 30 minutes. In the authors’ experience, this results in hemodynamic stability and abrupt cessation of the need for ongoing blood transfusion in the majority of cases.141 Patients also can undergo additional procedures such as laparotomy, tho-racotomy, external fixation of extremity fractures, fasciotomy, revascularization, or craniotomy. Following pelvic packing, angi-ography is reserved for patients with evidence of ongoing pelvic bleeding after admission to the SICU (>4 units of RBCs in the first 12 postoperative hours after the coagulopathy is corrected). Patients undergo standard posttrauma resuscitative SICU care, and the pelvic packs are removed within 48 hours; prior to unpacking, however, the patient’s coagulopathy should be corrected to pre-vent the need for repacking of the pelvic space. Repacking of the pelvic space is

1	packs are removed within 48 hours; prior to unpacking, however, the patient’s coagulopathy should be corrected to pre-vent the need for repacking of the pelvic space. Repacking of the pelvic space is associated with an infection rate of 47% and should be avoided; directed hemostasis with topical agents, suture repair, or electrocautery at pelvic pack removal should be performed.Another clinical challenge is the open pelvic fracture. In many instances, the wounds are located in the perineum, and the risk of pelvic sepsis and osteomyelitis is high. To reduce Brunicardi_Ch07_p0183-p0250.indd 23410/12/18 6:21 PM 235TRAUMACHAPTER 7the risk of infection, performance of a diverting sigmoid colos-tomy is recommended. The pelvic wound is manually debrided and then irrigated daily with a high-pressure pulsatile irrigation system until granulation tissue covers the wound. The wound is then left to heal by secondary intention with a wound vacuum-assisted wound closure (VAC) device.Extremity

1	pulsatile irrigation system until granulation tissue covers the wound. The wound is then left to heal by secondary intention with a wound vacuum-assisted wound closure (VAC) device.Extremity Vascular Injuries, Fractures, and Compartment SyndromesPatients with injured extremities often require a multidisci-plinary approach with involvement of trauma, orthopedic, and plastic surgeons to address vascular injuries, fractures, soft tis-sue injuries, and compartment syndromes. Immediate stabili-zation of fractures or unstable joints is done in the ED using Hare traction, knee immobilizers, or plaster splints. In patients with open fractures, the wound should be covered with povido-neiodine (Betadine)-soaked gauze and antibiotics administered. Options for fracture fixation include external fixation or open reduction and internal fixation with plates or intramedullary nails. Vascular injuries, either isolated or in combination with fractures, require emergent repair. Common combined injuries

1	or open reduction and internal fixation with plates or intramedullary nails. Vascular injuries, either isolated or in combination with fractures, require emergent repair. Common combined injuries include clavicle/first rib fractures and subclavian artery injuries, dislocated shoulder/proximal humeral fractures and axillary artery injuries, supracondylar fractures/elbow dislocations and brachial artery injuries, femur fracture and superficial femoral artery injuries, and knee dislocation and popliteal vessel inju-ries. On-table angiography in the OR facilitates rapid interven-tion and is warranted in patients with evidence of limb threat on arrival. Arterial access for on-table lower extremity angiography can be obtained percutaneously at the femoral vessels with a standard arterial catheter, via femoral vessel exposure and direct cannulation, or with superficial femoral artery (SFA) exposure just above the medial knee. Controversy exists regarding which should be done first, fracture

1	via femoral vessel exposure and direct cannulation, or with superficial femoral artery (SFA) exposure just above the medial knee. Controversy exists regarding which should be done first, fracture fixation or arterial repair. The authors prefer placement of temporary intravascular shunts first to reestablish arterial flow and minimize ischemia during frac-ture treatment, with definitive vascular repair following. Rarely, immediate amputation may be considered due to the severity of orthopedic and neurovascular injuries. This is particularly true if primary nerve transection is present in addition to fracture and arterial injury.142 Collaborative decision making by the trauma, orthopedic, and plastic/reconstructive team is essential.FAST ExamPositiveNegative2 units RBCs/ED trauma bayHD StableHD StableHD StableHD UnstableHD UnstableResuscitate in the SICU + CT scansOperating Room:Exploratory LaparotomyIf Pelvic Hematoma Evident ˜Pelvic Fixation and Pelvic PackingHD UnstableOngoing

1	StableHD StableHD StableHD UnstableHD UnstableResuscitate in the SICU + CT scansOperating Room:Exploratory LaparotomyIf Pelvic Hematoma Evident ˜Pelvic Fixation and Pelvic PackingHD UnstableOngoing Transfusion Requirements after Pelvic Packing?(>4 units RBCs from pelvic source with normal coags in 12 hours)NoAngiographySICUYesDenver Health Unstable Pelvic Fracture ManagementResuscitate with 2 L crystalloid – measure base deÿcit – rule out thoracic source – sheet the pelvis.Transfuse fresh frozen plasma (FFP) and RBC 1:2; 1 apheresis unit of platelets for each 5 units RBCs; perform thromboelastography.Immediate notiÿcation: Attending Trauma Surgeon, Attending Orthopedic Surgeon, Operating Room, Blood BankPlace 7Fr Terumo arterial sheath if SBP <90 mmHg; consider REBOA if SBP <80 mmHg Operating Room:Pelvic Fixation and Pelvic PackingReultrasound AbdomenFigure 7-70. Management algorithm for patients with pelvic fractures with hemodynamic instability. CT = computed tomography; ED =

1	Operating Room:Pelvic Fixation and Pelvic PackingReultrasound AbdomenFigure 7-70. Management algorithm for patients with pelvic fractures with hemodynamic instability. CT = computed tomography; ED = emergency department; FAST = focused abdominal sonography for trauma; HD = hemodynamic; PLT = platelets; PRBCs = packed red blood cells; SICU = surgical intensive care unit.Brunicardi_Ch07_p0183-p0250.indd 23510/12/18 6:21 PM 236BASIC CONSIDERATIONSPART IOperative intervention for vascular injuries should follow standard principles of repair (see “Vascular Repair Techniques”). For subclavian or axillary artery repairs, 6-mm PTFE graft or RSVG are used depending on the location. Because associated injuries of the brachial plexus are common, a thorough neuro-logic examination of the extremity is mandated before operative intervention. Operative approach for a brachial artery injury is via a medial upper extremity longitudinal incision; proximal control may be obtained at the axillary

1	is mandated before operative intervention. Operative approach for a brachial artery injury is via a medial upper extremity longitudinal incision; proximal control may be obtained at the axillary artery, and an S-shaped exten-sion through the antecubital fossa provides access to the distal brachial artery. The injured vessel segment is excised, and an end-to-end interposition RSVG graft is performed. Upper extremity fasciotomy is rarely required because of the rich collateral per-fusion via the profunda. For SFA injuries, external fixation of the femur typically is performed, followed by end-to-end RSVG of the injured SFA segment. Close monitoring for calf compart-ment syndrome is mandatory. Preferred access to the popliteal space for an acute injury is the medial, one-incision approach with detachment of the semitendinosus, semimembranosus, and gracilis muscles (Fig. 7-72). Another option is a medial approach Figure 7-71. A. Pelvic packing is performed through a 6to 8-cm midline

1	with detachment of the semitendinosus, semimembranosus, and gracilis muscles (Fig. 7-72). Another option is a medial approach Figure 7-71. A. Pelvic packing is performed through a 6to 8-cm midline incision made from the pubic symphysis cephalad, with division of the midline fascia. B. The pelvic hematoma often dissects the preperitoneal and paravesical space down to the presacral region, which facilitates packing; alternatively, blunt digital dissection opens the preperitoneal space for packing. C. Three standard surgical laparotomy pads are placed on each side of the bladder, deep within the preperitoneal space; the fascia is closed with an O polydioxanone monofilament suture and the skin with staples.Figure 7-72. A. The popliteal space is commonly accessed using a single medial incision (the detached semitendinosus, semimembranosus, and gracilis muscles are identified by different suture types). B. Alternatively, a medial approach with two incisions may be used. Insertion of a

1	(the detached semitendinosus, semimembranosus, and gracilis muscles are identified by different suture types). B. Alternatively, a medial approach with two incisions may be used. Insertion of a Pruitt-Inahara shunt (arrow) provides temporary restoration of blood flow, which prevents ischemia during fracture treatment.Brunicardi_Ch07_p0183-p0250.indd 23610/12/18 6:21 PM 237TRAUMACHAPTER 7Table 7-11Arterial vasospasm treatment guidelineStep 1: Intra-arterial alteplase (tissue plasminogen activator) 5 mg/20 mL bolusIf spasm continues, proceed to step 2.Step 2: Intra-arterial nitroglycerin 200 μg/20 mL bolusRepeat same dose once as needed.If spasm continues, proceed to step 3.Step 3: Interarterial verapamil 10 mg/10 mL bolusIf spasm continues, proceed to step 4.Step 4: Interarterial papaverine drip 60 mg/50 mL given over 15 minwith two incisions using a longer RSVG, but this requires interval ligation of the popliteal artery and geniculate branches. Rarely, with open wounds a straight

1	drip 60 mg/50 mL given over 15 minwith two incisions using a longer RSVG, but this requires interval ligation of the popliteal artery and geniculate branches. Rarely, with open wounds a straight posterior approach with an S-shaped incision can be used. If the patient has an associated popliteal vein injury, this should be repaired first with a PTFE interposition graft while the artery is shunted. For an isolated popliteal artery injury, RSVG is performed with an end-to-end anastomosis. Compart-ment syndrome is common, and presumptive four-compartment fasciotomies are warranted in patients with combined arterial and venous injury. Once the vessel is repaired and restoration of arte-rial flow documented, completion angiography should be done in the OR if there is no palpable distal pulse. Vasoparalysis with verapamil, nitroglycerin, and papaverine may be used to treat vasoconstriction (Table 7-11).Compartment syndromes, which can occur anywhere in the extremities, involve an acute

1	Vasoparalysis with verapamil, nitroglycerin, and papaverine may be used to treat vasoconstriction (Table 7-11).Compartment syndromes, which can occur anywhere in the extremities, involve an acute increase in pressure inside a closed space, which impairs blood flow to the structures within. Causes of compartment syndrome include arterial hemorrhage into a compartment, venous ligation or thrombosis, crush inju-ries, and reperfusion injury. In conscious patients, pain is the prominent symptom, and active or passive motion of muscles in the involved compartment increases the pain. Paresthesias may also be described. In the lower extremity, numbness between the first and second toes is the hallmark of early compartment syndrome in the exquisitely sensitive anterior compartment and its enveloped deep peroneal nerve. Progression to paralysis can occur, and loss of pulses is a late sign. In comatose or obtunded patients, the diagnosis is more difficult to secure. In patients with a compatible

1	deep peroneal nerve. Progression to paralysis can occur, and loss of pulses is a late sign. In comatose or obtunded patients, the diagnosis is more difficult to secure. In patients with a compatible history and a tense extremity, compartment pressures should be measured with a hand-held Stryker device. Fasciotomy is indicated in patients with a gradient of <30 mmHg (gradient = diastolic pressure − compartment pressure), an abso-lute compartment pressure >30 mmHg, ischemic periods of >6 hours, or combined arterial and venous injuries. The lower extremity is most frequently involved, and compartment release is performed using a two-incision, four-compartment fasciotomy (Fig. 7-73). Of note, the soleus muscle must be detached from the tibia to decompress the deep flexor compartment.SURGICAL INTENSIVE CARE MANAGEMENTPostinjury ResuscitationICU management of the trauma patient, either with direct admis-sion from the ED or after emergent operative intervention, is considered in distinct

1	INTENSIVE CARE MANAGEMENTPostinjury ResuscitationICU management of the trauma patient, either with direct admis-sion from the ED or after emergent operative intervention, is considered in distinct phases because there are differing goals and priorities. The period of acute resuscitation, typically last-ing for the first 12 to 24 hours after injury, combines several key principles: optimizing tissue perfusion, ensuring normother-mia, and restoring coagulation status.143 There are a multitude of management algorithms aimed at accomplishing these goals, the majority of which involve goal-directed resuscitation with initial volume loading to attain adequate preload, followed by judicious use of inotropic agents or vasopressors.144 Although the optimal hemoglobin level remains debated, during shock resuscitation a hemoglobin level of >10 g/dL is generally accepted to optimize hemostasis and ensure adequate oxygen delivery. After the first 24 hours of resuscitation, a more judicious

1	during shock resuscitation a hemoglobin level of >10 g/dL is generally accepted to optimize hemostasis and ensure adequate oxygen delivery. After the first 24 hours of resuscitation, a more judicious transfusion trigger of a hemoglobin level of <7 g/dL in the euvolemic patient limits the adverse inflammatory effects of stored RBCs. Recent trends have focused on limiting crystalloid loading. In fact, optimiz-ing crystalloid administration is a challenging aspect of early care (i.e., balancing cardiac performance against generation of an abdominal compartment syndrome and generalized tissue edema). Although early colloid administration is appealing, evi-dence to date does not support this concept.Invasive monitoring with pulmonary artery catheters has been supplanted with specialized catheters that measure arte-rial pulse contour analysis; in mechanically ventilated patients, stroke volume (SV) and continuous cardiac output can be mea-sured. A patient’s volume status may be ascertained

1	that measure arte-rial pulse contour analysis; in mechanically ventilated patients, stroke volume (SV) and continuous cardiac output can be mea-sured. A patient’s volume status may be ascertained by measur-ing the SV following either 10 cc/kg volume bolus or following a passive leg raise, which augments preload by 250 to 500 mL. A change in SV of ≥10% suggests preload responsiveness, and additional resuscitation fluid should be given. Although norepi-nephrine is the agent of choice for patients with low systemic vascular resistance who are unable to maintain a mean arterial pressure of >60 mmHg, patients may have an element of myo-cardial dysfunction requiring inotropic support. The role of rela-tive adrenal insufficiency is another controversial area.Optimal early resuscitation is mandatory and determines when the patient can (a) undergo additional necessary imaging, and (b) be returned to the OR after initial damage control surgery for definitive repair of injuries. Specific goals

1	and determines when the patient can (a) undergo additional necessary imaging, and (b) be returned to the OR after initial damage control surgery for definitive repair of injuries. Specific goals of resuscitation before repeated “semi-elective” transport include a core tempera-ture of >35°C (95°F), base deficit of <6 mmol/L, and normal coagulation indices. Although correction of metabolic acidosis is desirable, how quickly this should be accomplished requires careful consideration. Adverse sequelae of excessive crystalloid resuscitation include increased intracranial pressure, worsening pulmonary edema, and intra-abdominal visceral and retroperi-toneal edema resulting in secondary abdominal compartment syndrome. Therefore, it should be the overall trend of the resus-citation rather than a rapid reduction of the base deficit that is the goal. The goal is to normalize lactate within 24 hours.145In general, wounds sustained from trauma should be examined daily for progression of healing

1	rapid reduction of the base deficit that is the goal. The goal is to normalize lactate within 24 hours.145In general, wounds sustained from trauma should be examined daily for progression of healing and signs of infection. Complex soft tissue wounds of the abdomen, such as degloving injuries after blunt trauma (termed Morel-Lavallee lesions146), shotgun wounds, and other destructive blast injuries, are par-ticularly difficult to manage. Following initial debridement of devitalized tissue, wound care includes wet-to-dry dressing changes twice daily or application of a VAC device. Repeated operative debridement may be necessary, and early involvement of the reconstructive surgery service for possible flap cover-age is advised. Midline laparotomy wounds are inspected 48 hours postoperatively by removing the sterile surgical dressing. If an ileostomy or colostomy is required, one should inspect it Brunicardi_Ch07_p0183-p0250.indd 23710/12/18 6:21 PM 238BASIC CONSIDERATIONSPART IFigure

1	by removing the sterile surgical dressing. If an ileostomy or colostomy is required, one should inspect it Brunicardi_Ch07_p0183-p0250.indd 23710/12/18 6:21 PM 238BASIC CONSIDERATIONSPART IFigure 7-73. A. The anterior and lateral compartments are approached from a lateral incision, with identification of the fascial raphe between the two compartments. Care must be taken to avoid the superficial peroneal nerve running along the raphe. B. To decompress the deep flexor compartment, which contains the tibial nerve and two of the three arteries to the foot, the soleus muscle must be detached from the tibia.daily to ensure that it is viable. If the patient develops a high-grade fever, the wound should be inspected sooner to exclude an early necrotizing infection. If a wound infection is identified—as evidenced by erythema, pain along the wound, or purulent drainage—the wound should be widely opened by removing skin staples. After ensuring that the midline fascia is intact with digital

1	evidenced by erythema, pain along the wound, or purulent drainage—the wound should be widely opened by removing skin staples. After ensuring that the midline fascia is intact with digital palpation, the wound is initially managed with wet-to-dry dressing changes. The most common intra-abdominal complica-tions are anastomotic failure and abscess. The choice between percutaneous and operative therapy is based on the location, timing, and extent of the collection.Abdominal Compartment SyndromeThe abdominal compartment syndrome is classified as patho-logic intra-abdominal hypertension due to intra-abdominal injury (primary) or splanchnic reperfusion after massive resus-citation (secondary). Secondary abdominal compartment syn-drome may result from any condition requiring extensive crystalloid resuscitation, including extremity trauma, chest trauma, or even postinjury sepsis. The sources of increased intra-abdominal pressure include bowel edema, ascites, bleed-ing, and packs. A diagnosis

1	resuscitation, including extremity trauma, chest trauma, or even postinjury sepsis. The sources of increased intra-abdominal pressure include bowel edema, ascites, bleed-ing, and packs. A diagnosis of intra-abdominal hypertension cannot reliably be made by physical examination; therefore, it is obtained by measuring the intraperitoneal pressure. The most common technique is to measure the patient’s bladder pressure. Fifty milliliters of saline is instilled into the bladder via the aspi-ration port of the Foley catheter with the drainage tube clamped, and a three-way stopcock and water manometer is placed at the level of the pubic symphysis. Bladder pressure is then measured on the manometer in centimeters of water (Table 7-12) and 10Brunicardi_Ch07_p0183-p0250.indd 23810/12/18 6:21 PM 239TRAUMACHAPTER 7Table 7-12Abdominal compartment syndrome grading systemBLADDER PRESSUREGRADEmmHgcmH2OI10–1513–20II16–2521–35III26–3536–47IV >35 >48˜ Renal blood flow˜ UOPHypoxemia ˛ Airway

1	PM 239TRAUMACHAPTER 7Table 7-12Abdominal compartment syndrome grading systemBLADDER PRESSUREGRADEmmHgcmH2OI10–1513–20II16–2521–35III26–3536–47IV >35 >48˜ Renal blood flow˜ UOPHypoxemia ˛ Airway pressures ˜ Compliance ˛ PA pressures ˛ CVP readings˜ CO˜ VEDV˜ SV˛ SVR˛ ICPSplanchnicischemiaExtremityischemiaIncreased abdominal pressureIntrathoracic pressure˛Venous return˜Compression of kidneysFigure 7-74. Abdominal compartment syndrome is defined by the end organ sequelae of intra-abdominal hypertension. CO = cardiac output; CVP = central venous pressure; ICP = intracranial pressure; PA = pulmonary artery; SV = stroke volume; SVR = systemic vascular resistance; UOP = urine output; VEDV = ventricular end diastolic volume.correlated with the physiologic sequelae. Conditions in which the bladder pressure is unreliable include bladder rupture, exter-nal compression from pelvic packing, neurogenic bladder, and adhesive disease.Increased abdominal pressure affects multiple organ systems

1	bladder pressure is unreliable include bladder rupture, exter-nal compression from pelvic packing, neurogenic bladder, and adhesive disease.Increased abdominal pressure affects multiple organ systems (Fig. 7-74). Abdominal compartment syndrome, as noted earlier, is defined as intra-abdominal hypertension suf-ficient to produce physiologic deterioration and manifests via such end-organ sequelae as decreased urine output, increased pulmonary inspiratory pressures, decreased cardiac preload, and decreased cardiac output. Because any of these clinical symptoms of abdominal compartment syndrome may be attributed to the primary injury, a heightened awareness of this syndrome must be maintained. Organ fail-ure can occur over a wide range of recorded bladder pres-sures. Generally, no specific bladder pressure prompts therapeutic intervention, except when the pressure is >35 mmHg. Rather, emergent decompression is carried out when intra-abdominal hypertension reaches a level at which end-organ

1	pressure prompts therapeutic intervention, except when the pressure is >35 mmHg. Rather, emergent decompression is carried out when intra-abdominal hypertension reaches a level at which end-organ dysfunction occurs. Mortality is directly affected by the timing of decompression, with 70% mortality in patients with a delay in decompression, and nearly uniform mortality in those not undergoing decompression. Decompression is usually performed operatively, either in the ICU if the patient 11is hemodynamically unstable or in the OR. ICU bedside lapa-rotomy is easily accomplished, avoids transport of hemody-namically compromised patients, and requires minimal equipment (e.g., scalpel, suction device, cautery, and dress-ings for temporary abdominal closure). In patients with sig-nificant intra-abdominal fluid as the primary component of abdominal compartment syndrome, rather than bowel or ret-roperitoneal edema, decompression can be accomplished effectively via a percutaneous drain. These

1	fluid as the primary component of abdominal compartment syndrome, rather than bowel or ret-roperitoneal edema, decompression can be accomplished effectively via a percutaneous drain. These patients are iden-tified by bedside ultrasound, and the morbidity of a laparot-omy is avoided. When operative decompression is required with egress of the abdominal contents, temporary coverage is obtained using a fenestrated subfascial 45 × 60 cm sterile drape and Ioban application (see Fig. 7-49).The performance of damage control surgery and recogni-tion of abdominal compartment syndrome have dramatically improved patient survival, but at the cost of an open abdomen. Several management points deserve attention. Despite having a widely open abdomen, patients can develop recurrent abdomi-nal compartment syndrome, which increases their morbidity and mortality; therefore, bladder pressure should be monitored every 4 hours, with significant increases in pressures alerting the clini-cian to the possible

1	syndrome, which increases their morbidity and mortality; therefore, bladder pressure should be monitored every 4 hours, with significant increases in pressures alerting the clini-cian to the possible need for repeat operative decompression. Patients with an open abdomen lose between 500 and 2500 mL per day of abdominal effluent. Appropriate volume compensa-tion for this albumin-rich fluid remains controversial, with regard to both the amount administered (replacement based on clinical indices vs. routine 0.5 mL replacement for every milliliter lost) as well as the type of replacement (crystalloid vs. colloid).Following resuscitation and management of specific inju-ries, the goal of the operative team is to close the abdomen as quickly as possible. Multiple techniques have been introduced to obtain fascial closure of the open abdomen to minimize mor-bidity and cost of care. Historically, for patients who could not be closed at repeat operation, approximation of the fascia with mesh

1	to obtain fascial closure of the open abdomen to minimize mor-bidity and cost of care. Historically, for patients who could not be closed at repeat operation, approximation of the fascia with mesh (prosthetic or biologic) was used, with planned reopera-tion. Another option was split-thickness skin grafts applied directly to the exposed bowel for coverage; removal of the skin Brunicardi_Ch07_p0183-p0250.indd 23910/12/18 6:21 PM 240BASIC CONSIDERATIONSPART Igrafts was planned 9 to 12 months after the initial surgery, with definitive repair of the hernia by component separation. How-ever, delayed abdominal wall reconstruction was resource inva-sive, with considerable patient morbidity. The advent of VAC technology has revolutionized fascial closure. The authors cur-rently use a sequential closure technique with the wound VAC device that is based on constant fascial tension and return to the OR every 48 hours until closure is complete (Fig. 7-75).147 The authors’ success rate with

1	closure technique with the wound VAC device that is based on constant fascial tension and return to the OR every 48 hours until closure is complete (Fig. 7-75).147 The authors’ success rate with this approach exceeds 95%. This is important because among patients not attaining fascial closure, 20% suffer GI tract complications that prolong their hospital course. These include intra-abdominal abscess, enteric fistula, and bowel perforations (Fig. 7-76). Management requires fre-quent operative or percutaneous drainage of abscesses, control of fistulas, and prolonged nutritional support.SPECIAL POPULATIONSPregnant PatientsDuring pregnancy, 7% of women are injured. Motor vehicle col-lisions and falls are the leading causes of injury, accounting for 70% of cases. Fetal death after trauma most frequently occurs after motor vehicle collisions, but only 11% of fetal deaths are due to the death of the mother; therefore, early trauma resusci-tation and management is directed not only at the

1	frequently occurs after motor vehicle collisions, but only 11% of fetal deaths are due to the death of the mother; therefore, early trauma resusci-tation and management is directed not only at the mother but also at the fetus. Domestic violence is also common, affecting between 10% and 30% of pregnant women and resulting in fetal mortality of 5%.Pregnancy results in physiologic changes that may impact postinjury evaluation (Table 7-13). Heart rate increases by 10 to 15 beats per minute during the first trimester and remains elevated until delivery. Blood pressure diminishes during the first two trimesters due to a decrease in systemic vascular resis-tance and rises again slightly during the third trimester (mean values: first = 105/60, second = 102/55, third = 108/67). Intra-vascular volume is increased by up to 8 L, which results in a relative anemia but also a relative hypervolemia. Consequently, a pregnant woman may lose 35% of her blood volume before exhibiting signs of shock.

1	is increased by up to 8 L, which results in a relative anemia but also a relative hypervolemia. Consequently, a pregnant woman may lose 35% of her blood volume before exhibiting signs of shock. Pregnant patients have an increase in tidal volume and minute ventilation but a decreased functional residual capacity; this results in a diminished PCO2 and respi-ratory alkalosis. Also, pregnant patients may desaturate more rapidly, particularly in the supine position and during intuba-tion. Supplemental oxygen is always warranted in the trauma patient but is particularly critical in the injured pregnant patient because the oxygen dissociation curve is shifted to the left for the fetus compared to the mother (i.e., small changes in maternal oxygenation result in larger changes for the fetus because the fetus is operating in the steep portion of the dissociation curve). Anatomic changes contribute to these pulmonary functional alterations and are relevant in terms of procedures. With the

1	because the fetus is operating in the steep portion of the dissociation curve). Anatomic changes contribute to these pulmonary functional alterations and are relevant in terms of procedures. With the gravid uterus enlarged, DPL should be performed in a supra-umbilical site with the catheter directed cephalad. In addition, the upward pressure on the diaphragm calls for caution when placing a thoracostomy tube; standard positioning may result in an intra-abdominal location or perforation of the diaphragm.Other physiologic changes during pregnancy affect the GI, renal, and hematologic systems. The lower esophageal sphincter has decreased competency, which increases the risk for aspira-tion. Liver function test values increase, with the alkaline phos-phatase level nearly doubling. The high levels of progesterone impair gallbladder contractions, which results in bile stasis and an increased incidence of gallstone formation; this may not affect the trauma bay evaluation but becomes

1	high levels of progesterone impair gallbladder contractions, which results in bile stasis and an increased incidence of gallstone formation; this may not affect the trauma bay evaluation but becomes important in a pro-longed ICU stay. Plasma albumin level decreases from a normal of around 4.3 g/dL to an average of 3.0 g/dL. Renal blood flow increases by 30% during pregnancy, which causes a decrease in serum level of blood urea nitrogen and creatinine. The uterus may also compress the ureters and bladder, causing hydronephro-sis and hydroureter. Finally, as noted earlier there is a relative anemia during pregnancy, but a hemoglobin level of <11 g/dL is considered abnormal. Additional hematologic changes include a moderate leukocytosis (up to 20,000 mm3) and a rela-tive hypercoagulable state due to increased levels of factors VII, VIII, IX, X, and XII and decreased fibrinolytic activity.During evaluation in the ED, the primary and secondary surveys commence, with mindfulness that the

1	due to increased levels of factors VII, VIII, IX, X, and XII and decreased fibrinolytic activity.During evaluation in the ED, the primary and secondary surveys commence, with mindfulness that the mother always receives priority while conditions are still optimized for the fetus.148 This management includes provision of supplemental oxygen (to prevent maternal and fetal hypoxia), fluid resuscita-tion (the hypervolemia of pregnancy may mask signs of shock), and placement of the patient in the left lateral decubitus posi-tion (or tilting of the backboard to the left) to avoid caval com-pression. Assessment of the fetal heart rate is the most valuable information regarding fetal viability. Fetal monitoring should initially be assessed with bedside FAST ultrasound to document the heart rate of the fetus; subsequent monitoring should be per-formed with a cardiotocographic device that measures both con-tractions and fetal heart tones (FHTs). Because change in heart rate is the primary

1	of the fetus; subsequent monitoring should be per-formed with a cardiotocographic device that measures both con-tractions and fetal heart tones (FHTs). Because change in heart rate is the primary response of the fetus to hypoxia or hypo-tension, anything above an FHT of 160 is a concern, whereas bradycardia (FHT of <120) is considered fetal distress. Indica-tions for emergent cesarean section include: (a) severe mater-nal shock or impending death (if the fetus is delivered within 5 minutes, survival is estimated at 70%), (b) uterine injury or significant fetal distress (anticipated survival rates of >70% if FHTs are present and fetal gestational age is >28 weeks).149If possible, a member of the obstetrics team should be present during initial evaluation. Vaginal bleeding can signal early cervical dilation and labor, abruptio placentae, or placenta previa. Amniotic sac rupture can result in prolapse of the umbil-ical cord with fetal compromise. Strong contractions are associ-ated with

1	cervical dilation and labor, abruptio placentae, or placenta previa. Amniotic sac rupture can result in prolapse of the umbil-ical cord with fetal compromise. Strong contractions are associ-ated with true labor and should prompt consideration of delivery and resuscitation of the neonate. Focused prenatal history-taking should elicit a history of pregnancy-induced hypertension, ges-tational diabetes, congenital heart disease, preterm labor, or placental abnormalities. Asking the patient when the baby first moved and if she is currently experiencing movement of the fetus is important. Determining fetal age is key for consider-ations of viability. Gestational age may be estimated by noting fundal height, with the fundus approximating the umbilicus at 20 weeks and the costal margin at 40 weeks. Discrepancy in dates and size may be due to uterine rupture or hemorrhage.Initial evaluation for abdominopelvic trauma in pregnant patients should proceed in the standard manner. Ultrasound (FAST)

1	Discrepancy in dates and size may be due to uterine rupture or hemorrhage.Initial evaluation for abdominopelvic trauma in pregnant patients should proceed in the standard manner. Ultrasound (FAST) of the abdomen should evaluate the four windows (pericardial, right and left upper quadrant, and bladder) and additionally assess FHTs, fetal movement, and sufficiency of amniotic fluid. DPL can be performed in pregnant women via a supraumbilical, open technique. Trauma radiography of pregnant patients presents a conundrum. Radiation damage has three distinct phases of damage and effect: preimplantation, dur-ing the period of organogenesis from 3 to 16 weeks, and after 16 weeks. Generally, it is accepted that “safe” doses of radiation Brunicardi_Ch07_p0183-p0250.indd 24010/12/18 6:21 PM 241TRAUMACHAPTER 7Figure 7-75. The authors’ sequential closure technique for the open abdomen. A. Multiple white sponges (solid arrow), stapled together, are placed on top of the bowel underneath the

1	241TRAUMACHAPTER 7Figure 7-75. The authors’ sequential closure technique for the open abdomen. A. Multiple white sponges (solid arrow), stapled together, are placed on top of the bowel underneath the fascia. Interrupted No. 1 polydioxanone sutures are placed approximately 5 cm apart (dashed arrow), which puts the fascia under moderate tension over the white sponge. B. After the sticky clear plastic vacuum-assisted closure (VAC) dressing is placed over the white sponges and adjacent 5 cm of skin, the central portion is removed by cutting along the wound edges. C and D. Black VAC sponges are placed on top of the white sponges and plastic-protected skin with standard occlusive dressing and suction. E. On return to the operating room (OR) 48 hours later, fascial sutures are placed from both the superior and inferior directions until tension precludes further closure; skin is closed over the fascial closure with skin staples. F. White sponges (fewer in number) are again applied and

1	both the superior and inferior directions until tension precludes further closure; skin is closed over the fascial closure with skin staples. F. White sponges (fewer in number) are again applied and fascial retention sutures are placed with planned return to the OR in 48 hours.Brunicardi_Ch07_p0183-p0250.indd 24110/12/18 6:22 PM 242BASIC CONSIDERATIONSPART IFigure 7-76. Complications after split-thickness skin graft closure of the abdomen include enterocutaneous fistulas (intubated here with a red rubber catheter) (A; arrow), rupture of the graft with exposure of the bowel mucosa (B), enteroatmospheric fistulas with a large ventral hernias and loss of abdominal domain (C).from radiography are <5 rad.150 A chest radiograph results in a dose of 0.07 mrad; CT scan of the chest, <1 rad; and CT scan of the abdomen, 3.5 rad. It is important, therefore, to limit radio-graphs to those that are essential and to shield the pelvis with a lead apron when possible. If clinically warranted,

1	and CT scan of the abdomen, 3.5 rad. It is important, therefore, to limit radio-graphs to those that are essential and to shield the pelvis with a lead apron when possible. If clinically warranted, however, a radiograph should be obtained.The vast majority of injuries are treated similarly whether the patient is pregnant or not. Following standard protocols for nonoperative management of blunt trauma avoids the risks associated with general anesthesia. A particular challenge in the pregnant trauma patient is a major pelvic fracture. Because uter-ine and retroperitoneal veins may dilate to 60 times their origi-nal size, hemorrhage from these vessels may be torrential. Fetal loss may be related to both maternal shock and direct injury to the uterus or fetal head. Penetrating injuries in this patient population also carry a high risk. The gravid uterus is a large target, and any penetrating injury to the abdomen may result in fetal injury depending on trajectory and uterine size. Gunshot

1	patient population also carry a high risk. The gravid uterus is a large target, and any penetrating injury to the abdomen may result in fetal injury depending on trajectory and uterine size. Gunshot wounds to the abdomen are associated with a 70% injury rate to the uterus and 35% mortality rate of the fetus. If the bul-let traverses the uterus and the fetus is viable, cesarean section should be performed. On the other hand, stab wounds do not often penetrate the thick wall of the uterus.Any patient with a viable pregnancy should be monitored after trauma, with the length of monitoring determined by the injury mechanism and patient physiology. Patients who are Brunicardi_Ch07_p0183-p0250.indd 24210/12/18 6:22 PM 243TRAUMACHAPTER 7symptomatic, defined by the presence of uterine irritability or contractions, abdominal tenderness, vaginal bleeding, or blood pressure instability, should be monitored in the hospital for at least 24 hours. In addition, patients at high risk for fetal

1	or contractions, abdominal tenderness, vaginal bleeding, or blood pressure instability, should be monitored in the hospital for at least 24 hours. In addition, patients at high risk for fetal loss (those experiencing vehicle ejection or involved in motorcycle or pedestrian collisions and those with maternal tachycardia, Injury Severity Score of >9, gestational period of >35 weeks, or history of prior assault) also warrant careful monitoring.151 Patients without these risk factors who are asymptomatic can be monitored for 6 hours in the ED and sent home if no problems develop. They should be counseled regarding warning signs that mandate prompt return to the ED.Geriatric PatientsElderly trauma patients (>65 years of age) are hospitalized twice as often as those in any other age group, and this population accounts for one quarter of all trauma admissions. Although the physiology of aging separates older trauma patients from the younger generation (Table 7-14), treatment must remain

1	and this population accounts for one quarter of all trauma admissions. Although the physiology of aging separates older trauma patients from the younger generation (Table 7-14), treatment must remain indi-vidualized (some octogenarians look and physiologically act 50 years old, whereas others appear closer to 100 years). No chronologic age is associated with a higher morbidity or mor-tality, but a patient’s comorbidities do impact the individual’s postinjury course and outcome. For example, recognition that a patient is taking β-blockers affects the physician’s evaluation of vital signs in the ED and impacts treatment course in the ICU. Early monitoring of arterial blood gas values will identify occult shock. A base deficit of >6 mmol/L is associated with a twofold higher risk of mortality in patients over the age of 55 than in younger patients (67% vs. 30%).152Although the published literature on geriatric traumatic brain injury is relatively sparse and uncontrolled with regard to

1	in patients over the age of 55 than in younger patients (67% vs. 30%).152Although the published literature on geriatric traumatic brain injury is relatively sparse and uncontrolled with regard to management, some interesting points are noted. First, as anticipated, outcomes are worse in this age group than in their younger counterparts. Based on data from the Traumatic Coma Databank, mortality in patients with severe head injury more than doubles after the age of 55. Moreover, 25% of patients with a normal GCS score of 15 had intracranial bleeding, with an associated mortality of 50%.140 Just as there is no absolute age that predicts outcome, admission GCS score is a poor predictor of individual outcome. Therefore, the majority of trauma centers advocate an initial aggressive approach with reevaluation at the 72-hour mark to determine subsequent care.One of the most common sequelae of blunt thoracic trauma is rib fractures. In the aging population, perhaps due to osteo-porosis, less

1	reevaluation at the 72-hour mark to determine subsequent care.One of the most common sequelae of blunt thoracic trauma is rib fractures. In the aging population, perhaps due to osteo-porosis, less force is required to cause a fracture. In fact, in one study, 50% of patients >65 years old sustained rib fractures from a fall of <6 ft, compared with only 1% of patients <65 years of age. Concurrent pulmonary contusion is noted in up to 35% of patients, and pneumonia complicates the injuries in 10% to 30% of patients with rib fractures, not surprisingly leading to longer ICU stays.153,154 Additionally, mortality increases linearly with the number of rib fractures. Patients who sustain more than six rib fractures have pulmonary morbidity rates of >50% and over-all mortality rates of >20%.Chronologic age is not the best predictor of outcome, but the presence of preexisting conditions, which affect a patient’s physiologic age, is associated with increased mortality rates. Injury Severity

1	age is not the best predictor of outcome, but the presence of preexisting conditions, which affect a patient’s physiologic age, is associated with increased mortality rates. Injury Severity Score is probably the best overall predictor of patient outcome in the elderly; however, for any given individ-ual its sensitivity may not be precise, and there is a time delay in obtaining sufficient information to calculate the final score. In addition to preexisting conditions and severity of injury, the occurrence of complications compounds the risk for mortality.Table 7-13Physiologic effects of pregnancyCardiovascularIncrease in heart rate by 10–15 bpm(a) Decreased systemic vascular resistance resulting in:(b) Increased intravascular volumeDecreased blood pressure during the first two trimestersPulmonaryElevated diaphragmIncreased tidal volumeIncreased minute ventilationDecreased functional residual capacityHematopoieticRelative anemiaLeukocytosisHypercoagulability(a) Increased levels of

1	diaphragmIncreased tidal volumeIncreased minute ventilationDecreased functional residual capacityHematopoieticRelative anemiaLeukocytosisHypercoagulability(a) Increased levels of factors VII, VIII, IX, X, XII(b) Decreased fibrinolytic activityOtherDecreased competency of lower esophageal sphincterIncreased enzyme levels on liver function testsImpaired gallbladder contractionsDecreased plasma albumin levelDecreased blood urea nitrogen and creatinine levelsHydronephrosis and hydroureterTable 7-14Physiologic effects of agingCardiovascularAtherosclerotic disease that limits cardiac response to stressProgressive stiffening and loss of elasticity of the myocardiumDiminished stroke volume, systolic contraction, and diastolic relaxationDecrease in cardiac output of 0.5% per yearThickening and calcification of the cardiac valves, which results in valvular incompetencePulmonaryLoss of complianceProgressive loss of alveolar size and surface areaAir trapping and atelectasisIntracranialLoss of

1	calcification of the cardiac valves, which results in valvular incompetencePulmonaryLoss of complianceProgressive loss of alveolar size and surface areaAir trapping and atelectasisIntracranialLoss of cerebral volume, resulting in:(a) Increased risk of tearing of bridging veins with smaller injuries(b) Accumulation of a significant amount of blood before symptoms occur(c) muscle lossSenescence of the sensesOtherDecline in creatinine clearance by 80%–90%Osteoporosis, which causes a greater susceptibility to fracturesBrunicardi_Ch07_p0183-p0250.indd 24310/12/18 6:22 PM 244BASIC CONSIDERATIONSPART IPediatric PatientsTwenty million children, or almost one in four children, are injured each year, with an associated cost of treating the injured child of $16 billion per year. Injury is the leading cause of death among children over the age of 1 year, with 15,000 to 25,000 pediatric deaths per year. Disability after traumatic injury is more devastating, with rates 3 to 10 times that of the

1	cause of death among children over the age of 1 year, with 15,000 to 25,000 pediatric deaths per year. Disability after traumatic injury is more devastating, with rates 3 to 10 times that of the death rate. Pediatric trauma involves different mechanisms, different constellations of injury, and the potential for long-term problems related to growth and development. As with adult trauma, over 85% of pediatric trauma has a blunt mecha-nism, with boys injured twice as often as girls.155 Falls are the most common cause of injury in infants and toddlers. In chil-dren, bicycle mishaps are the most common cause of severe injury, whereas motor vehicle-related injury predominates in adolescence. Although unintentional injuries are by far the most common type of injuries in childhood, the number of intentional injuries, such as firearm-related injury and child abuse, is increasing.ED preparation for the pediatric trauma patient includes assembling age-appropriate equipment (e.g., intubation

1	of intentional injuries, such as firearm-related injury and child abuse, is increasing.ED preparation for the pediatric trauma patient includes assembling age-appropriate equipment (e.g., intubation equip-ment; IV catheters, including intraosseous needles and 4F single-lumen lines), laying out the Broselow Pediatric Emergency Tape (which allows effective approximation of the patient’s weight, medication doses, size of endotracheal tube, and chest tube size), and turning on heat lamps. Upon the pediatric patient’s arrival, the basic tenets of the ABCs apply, with some caveats. In children, the airway is smaller and more cephalad in position compared with that of adults, and in children younger than 10 years, the larynx is funnel shaped rather than cylindrical as in adults. Addi-tionally, the child’s tongue is much larger in relation to the oro-pharynx. Therefore, a small amount of edema or obstruction can significantly reduce the diameter of the airway (thus increasing the work of

1	the child’s tongue is much larger in relation to the oro-pharynx. Therefore, a small amount of edema or obstruction can significantly reduce the diameter of the airway (thus increasing the work of breathing), and the tongue may posteriorly obstruct the airway, causing intubation to be difficult. During intubation, a Miller (straight) blade rather than a Macintosh (curved) blade may be more effective due to the acute angle of the cephalad, funnel-shaped larynx. Administration of atropine before rapid-sequence intubation will prevent bradycardia. Adequate ventila-tion is critical because oxygen consumption in infants and young children is twice that in adults; onset of hypoxemia, followed by cardiac arrest, may be precipitous. Because gastric distension can inhibit adequate ventilation, placement of a nasogastric tube may facilitate effective gas exchange. Approximately one-third of pre-ventable deaths in children are related to airway management; therefore, if airway control cannot be

1	of a nasogastric tube may facilitate effective gas exchange. Approximately one-third of pre-ventable deaths in children are related to airway management; therefore, if airway control cannot be obtained using a standard endotracheal method, surgical establishment of an airway should be considered. In children older than 11 years, standard crico-thyroidotomy is performed. Due to the increased incidence of subglottic stenosis in younger patients, needle cricothyroidotomy with either a 14or 16-gauge catheter is advocated, although it is rarely used. Alternatively, tracheostomy may be performed. In children, the standard physiologic response to hypovolemia is peripheral vasoconstriction and reflex tachycardia; this may mask significant hemorrhagic injury because children can compensate for up to a 25% loss of circulating blood volume with minimal external signs. “Normal” values for vital signs should not neces-sarily make one feel more secure about the child’s volume status. Volume

1	for up to a 25% loss of circulating blood volume with minimal external signs. “Normal” values for vital signs should not neces-sarily make one feel more secure about the child’s volume status. Volume restoration is based on the child’s weight; two to three boluses of 20 mL/kg of crystalloid is appropriate. Hypotension in children may be due to TBI rather than hypovolemia and should be considered in the appropriate clinic scenario.156After initial evaluation based on the trauma ABCs, iden-tification and management of specific injuries proceeds. Acute traumatic brain injury is the most common cause of death and disability in any pediatric age group. Although falls are the most common mechanism overall, severe brain injury most often is due to child abuse (in children <2 years) or motor vehicle col-lisions (in those >2 years). Head CT should be performed to determine intracranial pathology, followed by skull radiogra-phy to diagnose skull fractures. As in adults, CPP is monitored, and

1	col-lisions (in those >2 years). Head CT should be performed to determine intracranial pathology, followed by skull radiogra-phy to diagnose skull fractures. As in adults, CPP is monitored, and appropriate resuscitation is critical to prevent the second-ary insults of hypoxemia and hypovolemia. Although some data indicate that the pediatric brain recovers from traumatic injury better than the adult brain, this advantage may be eliminated if hypotension is allowed to occur.As is true in adults, the vast majority of thoracic trauma is also blunt. However, because a child’s skeleton is not completely calcified, it is more pliable. Significant internal organ damage may occur without overlying bony fractures. For example, adult patients with significant chest trauma have a 70% incidence of rib fractures, whereas only 40% of children with significant chest trauma do. Pneumothorax is treated similarly in the pediatric population; patients who are asymptomatic with a pneumothorax of <15% are

1	fractures, whereas only 40% of children with significant chest trauma do. Pneumothorax is treated similarly in the pediatric population; patients who are asymptomatic with a pneumothorax of <15% are admitted for observation, whereas those who have a pneumothorax of >15% or who require positive pressure ventilation undergo tube decompression. Presence of a hemothorax in this age group may be particularly problematic because the child’s chest may contain his or her entire blood volume. If the chest tube output is initially 20% of the patient’s blood volume (80 mL/kg) or is persistently >1 to 2 mL/kg per hour, thoracotomy should be considered. Aortic injuries are rare in children, and tracheobronchial injuries are more amenable to nonoperative management. Penetrating thoracic trauma, although uncom-mon, has 35% operative intervention rate, which is consid-erably higher than that of the adult population.157 Thoracic injuries are second only to brain injuries as the main cause of death

1	uncom-mon, has 35% operative intervention rate, which is consid-erably higher than that of the adult population.157 Thoracic injuries are second only to brain injuries as the main cause of death according to the National Pediatric Trauma Registry; however, the overall mortality rate of 15% correlates with the levels in many adult studies.The evaluation for abdominal trauma in the pediatric patient is similar to that in the adult. FAST is valid in the pedi-atric age group to detect intra-abdominal fluid. The mecha-nism of injury often correlates with specific injury patterns. A child sustaining a blow to the epigastrium (e.g., hitting the handlebars during a bike accident) should be evaluated for a duodenal hematoma and/or a pancreatic transection. After a motor vehicle collision in which the patient was wearing a passenger restraint, injuries comprising the “lap belt com-plex” or “seat belt syndrome” (i.e., abdominal wall contusion, small bowel perforation, flexion-distraction injury

1	the patient was wearing a passenger restraint, injuries comprising the “lap belt com-plex” or “seat belt syndrome” (i.e., abdominal wall contusion, small bowel perforation, flexion-distraction injury of the lumbar spine, diaphragm rupture, and occasionally abdomi-nal aortic dissection) may exist. Nonoperative management of solid organ injuries, first used in children, is the current standard of care in the hemodynamically stable patient. If the patient shows clinical deterioration or hemodynamic lability, has a hollow viscus injury, or requires >40 mL/kg of packed RBCs, continued nonoperative management is not an option. Success rates of nonoperative management approach 95%,158 with an associated 10% to 23% transfusion rate. Findings of a hepatic or splenic blush on CT imaging does not uniformly require intervention; patient physiology should dictate embo-lization or operative intervention.159Brunicardi_Ch07_p0183-p0250.indd 24410/12/18 6:22 PM 245TRAUMACHAPTER 7REFERENCESEntries

1	require intervention; patient physiology should dictate embo-lization or operative intervention.159Brunicardi_Ch07_p0183-p0250.indd 24410/12/18 6:22 PM 245TRAUMACHAPTER 7REFERENCESEntries highlighted in bright blue are key references. 1. Minino AM, Heron MP, Murphy SL, et al. Deaths: final data for 2004. Natl Vital Stat Rep. 2007 Aug 21;55(19):1-120. Available at: http://www.cdc.gov/nchs/data/nvsr/nvsr65/nvsr65_04.pdf. Accessed June 8, 2017. 2. National Center for Injury Prevention and Control. CDC Injury Fact Book. Atlanta: Centers for Disease Control and Preven-tion, November 2006. Available at: http://www.cdc.gov/ncipc/fact_book/InjuryBook2006.pdf. Accessed October 29, 2012. 3. Brasel KJ. Epidemiology. In: Moore EE, Mattox KL, Feliciano DV, eds. Trauma. 8th ed. New York: McGraw-Hill; 2017. 4. Eastman AB. Wherever the dart lands: toward the ideal trauma system. J Am Coll Surg. 2010;211(2):153-168. 5. MacKenzie EJ, Rivara FP, Jurkovich GJ, et al. A national eval-uation of the

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1	al. WTA Study Group. Defining the limits of resuscitative emergency depart-ment thoracotomy: a contemporary Western Trauma Associa-tion perspective. J Trauma. 2011;70(2):334-339. 20. Moore HB, Moore EE, Burlew CC, et al. Establishing bench-marks for resuscitation of traumatic circulatory arrest: suc-cess-to-rescue and survival among 1,708 patients. J Am Coll Surg. 2016;223(1):42-50. 21. Ouellet JF, Roberts DJ, Tiruta C, et al. Admission base deficit and lactate levels in Canadian patients with blunt trauma: are they useful markers of mortality? J Trauma Acute Care Surg. 2012;72(6):1532-1535. 22. Callaway DW, Shapiro NI, Donnino MW, Baker C, Rosen CL. Serum lactate and base deficit as predictors of mortal-ity in normotensive elderly blunt trauma patients. J Trauma. 2009;66(4):1040-1044. 23. Johnson M, Alarhayem A, Convertino V, et al. Comparison of compensatory reserve and arterial lactate as markers of shock and resuscitation. J Trauma Acute Care Surg. 2017

1	2009;66(4):1040-1044. 23. Johnson M, Alarhayem A, Convertino V, et al. Comparison of compensatory reserve and arterial lactate as markers of shock and resuscitation. J Trauma Acute Care Surg. 2017 Oct;83(4):603-608. 24. Stewart CL, Mulligan J, Grudic GZ, Talley ME, Jurkovich GJ, Moulton SL. The compensatory reserve index follow-ing injury: results of a prospective clinical trial. Shock. 2016 Sep;46(3 Suppl 1):61-67. 25. Clancy K, Velopulos C, Bilaniuk JW, et al. Screening for blunt cardiac injury: an Eastern Association for the Surgery of Trauma practice management guideline. J Trauma Acute Care Surg. 2012;73:S301-S306. 26. Velmahos GC, Karaiskakis M, Salim A, et al. Normal electro-cardiography and serum troponin I levels preclude the pres-ence of clinically significant blunt cardiac injury. J Trauma. 2003;54(1):45-50. 27. Ferrada P, Murthi S, Anand RJ, Bochicchio GV, Scalea T. Transthoracic focused rapid echocardiographic examination: real-time evaluation of fluid status in

1	injury. J Trauma. 2003;54(1):45-50. 27. Ferrada P, Murthi S, Anand RJ, Bochicchio GV, Scalea T. Transthoracic focused rapid echocardiographic examination: real-time evaluation of fluid status in critically ill trauma patients. J Trauma. 2011;70:56-62. 28. Ferrada P, Evans D, Wolfe L, et al. Findings of a randomized con-trolled trial using limited transthoracic echocardiogram (LTTE) as a hemodynamic monitoring tool in the trauma bay. J Trauma Acute Care Surg. 2014;76(1):31-37; discussion 37-88. 29. Kutcher ME, Howard BM, Sperry JL, et al. Evolving beyond the vicious triad: differential mediation of traumatic coagu-lopathy by injury, shock, and resuscitation. J Trauma Acute Care Surg. 2015;78(3):516-523. 30. Gonzalez E, Moore EE, Moore HB. Management of trauma-induced coagulopathy with thrombelastography. Crit Care Clin. 2017;33(1):119-134. Review. 31. Dolich MO, McKenney MG, Varela JE, et al. 2,576 ultrasounds for blunt abdominal trauma. J Trauma. 2001;50:108-112. 32. Sondeen JL,

1	thrombelastography. Crit Care Clin. 2017;33(1):119-134. Review. 31. Dolich MO, McKenney MG, Varela JE, et al. 2,576 ultrasounds for blunt abdominal trauma. J Trauma. 2001;50:108-112. 32. Sondeen JL, Coppes VG, Holcomb JB. Blood pressure at which rebleeding occurs after resuscitation in swine with aor-tic injury. J Trauma. 2003;54(Suppl):S110-S117. 33. Carney N, Totten AM, O’Reilly C, et al. Guidelines for the management of severe traumatic brain injury. 4th ed. 2016. Available at: https://braintrauma.org/uploads/03/12/Guidelines_for_Management_of_Severe_TBI_4th_Edi-tion.pdf. Accessed July 9, 2017. 34. Ryb GE, Dischinger PC, Kufera JA, et al. Delta V, principal direction of force, and restraint use contributions to motor vehicle crash mortality. J Trauma. 2007;63:1000-1005. 35. Ivascu FA, Howells GA, Junn FS, Bair HA, Bendick PJ, Janczyk RJ. Predictors of mortality in trauma patients with intracranial hemorrhage on preinjury aspirin or clopidogrel. J Trauma.

1	FA, Howells GA, Junn FS, Bair HA, Bendick PJ, Janczyk RJ. Predictors of mortality in trauma patients with intracranial hemorrhage on preinjury aspirin or clopidogrel. J Trauma. 2008;65(4):785-788. 36. Moore MM, Pasquale MD, Badellino M. Impact of age and anticoagulation: need for neurosurgical intervention in trauma patients with mild traumatic brain injury. J Trauma Acute Care Surg. 2012;73(1):126-130. 37. Inaba K, Byerly S, Bush LD, et al; WTA C-Spine Study Group. Cervical spinal clearance: a prospective Western Trauma Asso-ciation Multi-institutional Trial. J Trauma Acute Care Surg. 2016;81(6):1122-1130. 38. Sekharan J, Dennis JW, Veldenz HC, et al. Continued experience with physical examination alone for evaluation and manage-ment of penetrating zone 2 neck injuries: results of 145 cases. J Vasc Surg. 2000;32:483-489. 39. Inaba K, Branco BC, Menaker J, et al. Evaluation of multi-detector computed tomography for penetrating neck injury: a prospective multicenter study. J Trauma

1	cases. J Vasc Surg. 2000;32:483-489. 39. Inaba K, Branco BC, Menaker J, et al. Evaluation of multi-detector computed tomography for penetrating neck injury: a prospective multicenter study. J Trauma Acute Care Surg. 2012;72:576-583.Brunicardi_Ch07_p0183-p0250.indd 24510/12/18 6:22 PM 246BASIC CONSIDERATIONSPART I 40. Chouliaras K, Bench E, Talving P, et al. Pneumomediasti-num following blunt trauma: worth an exhaustive workup? J Trauma Acute Care Surg. 2015;79(2):188-192; discussion 192-193. 41. Fabian TC, Richardson JD, Croce MA, et al. Prospective study of blunt aortic injury: multicenter trial of the American Associ-ation for the Surgery of Trauma. J Trauma. 1997;42:374-380. 42. Dyer DS, Moore EE, Ilke DN, et al. Thoracic aortic injury: how predictive is mechanism and is chest computed tomog-raphy a reliable screening tool? A prospective study of 1,561 patients. J Trauma. 2000;48:673-682. 43. Siegel JH, Smith JA, Siddiqi SQ. Change in velocity and energy dissipation on impact

1	tomog-raphy a reliable screening tool? A prospective study of 1,561 patients. J Trauma. 2000;48:673-682. 43. Siegel JH, Smith JA, Siddiqi SQ. Change in velocity and energy dissipation on impact in motor vehicle crashes as a function of the direction of crash: key factors in the production of thoracic aortic injuries, their pattern of associated injuries and patient survival. A Crash Injury Research Engineering Network (CIREN) study. J Trauma. 2004;57(4):760-777. 44. Karmy-Jones R, Namias N, Coimbra R, et al. Western Trauma Association critical decisions in trauma: penetrating chest trauma. J Trauma Acute Care Surg. 2014;77(6):994-1002. 45. Arantes V, Campolina C, Valerio SH, et al. Flexible esopha-goscopy as a diagnostic tool for traumatic esophageal injuries. J Trauma. 2009;66(6):1677-1682. 46. Cox CS Jr, Allen GS, Fischer RP, et al. Blunt vs. penetrating subclavian artery injury: presentation, injury pattern, and out-come. J Trauma. 1999;46:445-449. 47. Peponis T, Kasotakis G, Yu J,

1	CS Jr, Allen GS, Fischer RP, et al. Blunt vs. penetrating subclavian artery injury: presentation, injury pattern, and out-come. J Trauma. 1999;46:445-449. 47. Peponis T, Kasotakis G, Yu J, et al. Selective nonoperative management of abdominal gunshot wounds from heresy to adoption: a multicenter study of the Research Consortium of New England Centers for Trauma (ReCoNECT). J Am Coll Surg. 2017;224(6):1036-1045. 48. Demetriades D, Hadjizacharia P, Constantinou C, et al. Selec-tive nonoperative management of penetrating abdominal solid organ injuries. Ann Surg. 2006;244:620-628. 49. Biffl WL, Cothren CC, Brasel KJ, et al. A prospective observa-tional multicenter study of the optimal management of patients with anterior abdominal stab wounds. J Trauma. 2008;64:250. 50. Biffl WL, Kaups KL, Pham TN, et al. Validating the West-ern Trauma Association algorithm for managing patients with anterior abdominal stab wounds: a Western Trauma Associa-tion multicenter trial. J Trauma.

1	Kaups KL, Pham TN, et al. Validating the West-ern Trauma Association algorithm for managing patients with anterior abdominal stab wounds: a Western Trauma Associa-tion multicenter trial. J Trauma. 2011;71(6):1494-1502. 51. Ochsner MG, Knudson MM, Pachter HL, et al. Significance of minimal or no intraperitoneal fluid visible on CT scan associ-ated with blunt liver and splenic injuries: a multicenter analy-sis. J Trauma. 2000;49:505-510. 52. Yu J, Fulcher AS, Turner MA, Cockrell C, Halvorsen RA. Blunt bowel and mesenteric injury: MDCT diagnosis. Abdom Imaging. 2011;36(1):50-61. 53. LeBedis CA, Anderson SW, Soto JA. CT imaging of blunt traumatic bowel and mesenteric injuries. Radiol Clin North Am. 2012;50(1):123-136. 54. Fox N, Rajani RR, Bokhari F, et al. Evaluation and manage-ment of penetrating lower extremity arterial trauma: an Eastern Association for the Surgery of Trauma practice management guideline. J Trauma Acute Care Surg. 2012;73:S315-S320. 55. Burch JM, Franciose RJ, Moore

1	lower extremity arterial trauma: an Eastern Association for the Surgery of Trauma practice management guideline. J Trauma Acute Care Surg. 2012;73:S315-S320. 55. Burch JM, Franciose RJ, Moore EE, et al. Single-layer con-tinuous vs. two-layer interrupted intestinal anastomosis—a prospective randomized study. Ann Surg. 2000;231:832-837. 56. Moore EE. Thomas G. Orr Memorial Lecture. Staged lapa-rotomy for the hypothermia, acidosis, and coagulopathy syn-drome. Am J Surg. 1996;172:405-410. 57. Morrison JJ, Galgon RE, Jansen JO, Cannon JW, Rasmussen TE, Eliason JL. A systematic review of the use of resuscita-tive endovascular balloon occlusion of the aorta in the man-agement of hemorrhagic shock. J Trauma Acute Care Surg. 2016;80(2):324-334. 58. Moore LJ, Brenner M, Kozar RA, et al. Implementation of resuscitative endovascular balloon occlusion of the aorta as an alternative to resuscitative thoracotomy for noncom-pressible truncal hemorrhage. J Trauma Acute Care Surg. 2015;79(4):523-530;

1	of resuscitative endovascular balloon occlusion of the aorta as an alternative to resuscitative thoracotomy for noncom-pressible truncal hemorrhage. J Trauma Acute Care Surg. 2015;79(4):523-530; discussion 530-532. 59. Gonzalez E, Moore EE, Moore HB. Management of trauma-induced coagulopathy with thrombelastography. Crit Care Clin. 2017;33(1):119-134. Review. 60. Cohen MJ, Call M, Nelson M, et al. Critical role of acti-vated protein C in early coagulopathy and later organ fail-ure, infection and death in trauma patients. Ann Surg. 2012;255(2):379-385. 61. Gonzalez E, Moore EE, Moore HB, Chapman MP, Silliman CC, Banerjee A. Trauma-induced coagulopathy: an institu-tion’s 35-year perspective on practice and research. Scand J Surg. 2014;103(2):89-103. 62. Moore HB, Moore EE, Liras IN, et al. Acute fibrinolysis shut-down after injury occurs frequently and increases mortality: a multicenter evaluation of 2,540 severely injured patients. J Am Coll Surg. 2016;222(4):347-355. 64. Hebert PC,

1	Acute fibrinolysis shut-down after injury occurs frequently and increases mortality: a multicenter evaluation of 2,540 severely injured patients. J Am Coll Surg. 2016;222(4):347-355. 64. Hebert PC, Wells G, Blajchman MA, et al. A multicenter, ran-domized, controlled clinical trial of transfusion requirements in critical care. New Engl J Med. 1999;340:409-417. 65. West MA, Shapiro MB, Nathens AB, et al. Inflammation and the host response to injury, a large-scale collaborative project: patient-oriented research core-standard operating procedures for clinical care. IV. Guidelines for transfusion in the trauma patient. J Trauma. 2006;61:436-439. 66. Tariket S, Sut C, Hamzeh-Cognasse H, et al. Transfusion-related acute lung injury: transfusion, platelets and biological response modifiers. Expert Rev Hematol. 2016;9(5):497-508. 67. Moore FA, Moore EE, Sauaia A. Blood transfusion: an inde-pendent risk factor for postinjury multiple organ failure. Arch Surg. 1997;132:620-624. 68. Kashuk JL,

1	Rev Hematol. 2016;9(5):497-508. 67. Moore FA, Moore EE, Sauaia A. Blood transfusion: an inde-pendent risk factor for postinjury multiple organ failure. Arch Surg. 1997;132:620-624. 68. Kashuk JL, Moore EE, Sauaia A, et al. Postinjury life-threat-ening coagulopathy: is 1:1 fresh frozen plasma: packed red blood cells the answer? J Trauma. 2008;65:261-270. 69. Davenport R, Curry N, Manson J, et al. Hemostatic effects of fresh frozen plasma may be maximal at red cell ratios of 1:2. J Trauma. 2011;70(1):90-95. 70. Stanworth SJ, Morris TP, Gaarder C, et al. Reappraising the concept of massive transfusion in trauma. Crit Care. 2010;14(6):R239. 71. Dzik WH, Blajchman MA, Fergusson D, et al. Clinical review: Canadian National Advisory Committee on Blood and Blood Products–Massive transfusion consensus conference 2011: report of the panel. Crit Care. 2011;15(6):242. 72. Holcomb JB, Tilley BC, Baraniuk S, et al; PROPPR Study Group. Transfusion of plasma, platelets, and red blood cells in a 1:1:1

1	conference 2011: report of the panel. Crit Care. 2011;15(6):242. 72. Holcomb JB, Tilley BC, Baraniuk S, et al; PROPPR Study Group. Transfusion of plasma, platelets, and red blood cells in a 1:1:1 vs a 1:1:2 ratio and mortality in patients with severe trauma: the PROPPR randomized clinical trial. JAMA. 2015;313(5):471-482. 73. Menaker J, Stein DM, Scalea TM. Incidence of early pulmo-nary embolism after injury. J Trauma. 2007;63:620-624. 74. Harr JN, Moore EE, Chin TL, et al. Platelets are dominant contributors to hypercoagulability after injury. J Trauma Acute Care Surg. 2013;74(3):756-762; discussion 762-765. 75. Prager M, Polterauer P, Böhmig HJ, et al. Collagen vs. gelatin-coated Dacron vs. stretch polytetrafluoroethylene in abdominal aortic bifurcation graft surgery: results of a seven-year prospective, randomized multicenter trial. Surgery. 2001;130(3):408-414. 76. Asensio JA, Feliciano DV. Abdominal vascular injury. In: Moore EE, Mattox KL, Feliciano DV, eds. Trauma. 8th ed. New

1	prospective, randomized multicenter trial. Surgery. 2001;130(3):408-414. 76. Asensio JA, Feliciano DV. Abdominal vascular injury. In: Moore EE, Mattox KL, Feliciano DV, eds. Trauma. 8th ed. New York: McGraw-Hill; 2017. 77. Coleman JJ, Brewer BL, Feliciano DV. Trauma damage con-trol. In: Moore EE, Mattox KL, Feliciano DV, eds. Trauma. 8th ed. New York: McGraw-Hill; 2017. 78. Mathew S, Smith BP, Cannon JW, Reilly PM, Schwab CW, Seamon MJ. Temporary arterial shunts in damage Brunicardi_Ch07_p0183-p0250.indd 24610/12/18 6:22 PM 247TRAUMACHAPTER 7control: experience and outcomes. J Trauma Acute Care Surg. 2017;82(3):512-517. 79. Lubitz AL, Sjoholm LO, Goldberg A, et al. Acute right heart failure after hemorrhagic shock and trauma pneumonectomy—a management approach: a blinded randomized controlled ani-mal trial using inhaled nitric oxide. J Trauma Acute Care Surg. 2017;82(2):243-251. 80. Smith JW, Matheson PJ, Franklin GA, Harbrecht BG, Richardson JD, Garrison RN. Randomized controlled

1	ani-mal trial using inhaled nitric oxide. J Trauma Acute Care Surg. 2017;82(2):243-251. 80. Smith JW, Matheson PJ, Franklin GA, Harbrecht BG, Richardson JD, Garrison RN. Randomized controlled trial evaluating the efficacy of peritoneal resuscitation in the man-agement of trauma patients undergoing damage control sur-gery. J Am Coll Surg. 2017;224(4):396-404. 81. Dawes AJ, Sacks GD, Cryer HG, et al; Los Angeles County Trauma Consortium. Intracranial pressure monitoring and inpatient mortality in severe traumatic brain injury: a pro-pensity score-matched analysis. J Trauma Acute Care Surg. 2015;78(3):492-501; discussion 501-502. 82. Nirula R, Millar D, Greene T, et al. Decompressive craniectomy for medical management for refractory intracranial hyperten-sion: an AAST-MITC propensity score analysis. J Trauma Acute Care Surg. 2014;76(4):944-952; discussion 952-955. 83. Cooper DJ, Rosenfeld JV, Murray L, et al; DECRA Trial Investigators; Australian and New Zealand Intensive Care Society

1	analysis. J Trauma Acute Care Surg. 2014;76(4):944-952; discussion 952-955. 83. Cooper DJ, Rosenfeld JV, Murray L, et al; DECRA Trial Investigators; Australian and New Zealand Intensive Care Society Clinical Trials Group. Decompressive craniec-tomy in diffuse traumatic brain injury. N Engl J Med. 2011;364(16):1493-1502. 84. Rinker C, McMurry F, Groeneweg V, et al. Emergency craniotomy in a rural level III trauma center. J Trauma. 1998;44:984-989. 85. Hutchison JS, Ward RE, Lacroix J, et al; Hypothermia Pedi-atric Head Injury Trial Investigators and the Canadian Critical Care Trials Group. Hypothermia therapy after traumatic brain injury in children. N Engl J Med. 2008;358(23):2447-2456. 86. Kramer C, Freeman WD, Hoffman-Snyder C, et al. Therapeu-tic hypothermia for severe traumatic brain injury: a critically appraised topic. Neurologist. 2012;18(3):173-177. 87. Cogbill T, Cothren CC, Ahearn MK, et al. Management of severe hemorrhage associated with maxillofacial injuries: a

1	brain injury: a critically appraised topic. Neurologist. 2012;18(3):173-177. 87. Cogbill T, Cothren CC, Ahearn MK, et al. Management of severe hemorrhage associated with maxillofacial injuries: a multicenter perspective. J Trauma. 2008;64:250. 88. Bracken MB, Shepard MJ, Holford TR, et al. Administration of methylprednisolone for 24 or 48 hours or tirilazad mesyl-ate for 48 hours in the treatment of acute spinal cord injury. Results of the Third National Acute Spinal Cord Injury Ran-domized Controlled Trial. National Acute Spinal Cord Injury Study. JAMA. 1997;277:1597-1604. 89. Stahel PF, Vanderheiden T, Finn MA. Management strategies for acute spinal cord injury: current options and future per-spectives. Curr Opin Crit Care. 2012;18(6):651-660. 90. Fehlings MG, Perrin RG: The timing of surgical intervention in the treatment of spinal cord injury: a systematic review of recent clinical evidence. Spine. 2006;31:S28-S35. 91. Biffl WL, Moore EE, Offner PJ, et al. Blunt carotid arte-rial

1	intervention in the treatment of spinal cord injury: a systematic review of recent clinical evidence. Spine. 2006;31:S28-S35. 91. Biffl WL, Moore EE, Offner PJ, et al. Blunt carotid arte-rial injuries: implications of a new grading scale. J Trauma. 1999;47:845-853. 92. Burlew CC, Biffl WL, Moore EE, Barnett CC, Johnson JL, Bensard DD. Blunt cerebrovascular injuries: redefining screen-ing criteria in the era of noninvasive diagnosis. J Trauma Acute Care Surg. 2012;72(2):330-335. 93. Cothren CC, Moore EE, Biffl WL, et al. Anticoagulation is the gold standard therapy for blunt carotid injuries to reduce stroke rate. Arch Surg. 2004;139:540-545. 94. Shahan CP, Croce MA, Fabian TC, Magnotti LJ. Impact of continuous evaluation of technology and therapy: 30 years of research reduces stroke and mortality from blunt cerebrovas-cular injury. J Am Coll Surg. 2017;224(4):595-599. 95. Burlew CC, Biffl WL, Moore EE, et al. Endovascular stenting is rarely necessary for the management of blunt

1	and mortality from blunt cerebrovas-cular injury. J Am Coll Surg. 2017;224(4):595-599. 95. Burlew CC, Biffl WL, Moore EE, et al. Endovascular stenting is rarely necessary for the management of blunt cerebrovascu-lar injuries. J Am Coll Surg. 2014;218(5):1012-1017. 96. Weinberg JA, Moore AH, Magnotti LJ, et al. Contemporary management of civilian penetrating cervicothoracic arterial injuries. J Trauma Acute Care Surg. 2016;81(2):302-306. 97. Johnston RH, Wall MJ, Mattox KL. Innominate artery trauma: a thirty-year experience. J Vasc Surg. 1993;17:134-139. 98. Shalhub S, Starnes BW, Brenner ML, et al. Blunt abdominal aortic injury: a Western Trauma Association multicenter study. J Trauma Acute Care Surg. 2014;77(6):879-885; discussion 885. 99. Harris DG, Rabin J, Starnes BW, et al. Evolution of lesion-specific management of blunt thoracic aortic injury. J Vasc Surg. 2016;64(2):500-505. 100. Fox N, Schwartz D, Salazar JH, et al. Evaluation and man-agement of blunt traumatic aortic injury:

1	of lesion-specific management of blunt thoracic aortic injury. J Vasc Surg. 2016;64(2):500-505. 100. Fox N, Schwartz D, Salazar JH, et al. Evaluation and man-agement of blunt traumatic aortic injury: a practice manage-ment guideline from the Eastern Association for the Surgery of Trauma. J Trauma Acute Care Surg. 2015;78(1):136-146. 101. Moore EE, Burch JM, Moore JB. Repair of the torn descend-ing thoracic aorta using the centrifugal pump with partial left heart bypass. Ann Surg. 2004;240:38-43. 102. Rabin J, DuBose J, Sliker CW, O’Connor JV, Scalea TM, Griffith BP. Parameters for successful nonoperative manage-ment of traumatic aortic injury. J Thorac Cardiovasc Surg. 2014;147(1):143-149. 103. Wall MJ, Tsai PI, Mattox KL. Heart and thoracic vascular injury. In: Moore EE, Mattox KL, Feliciano DV, eds. Trauma. 8th ed. New York: McGraw-Hill; 2017. 104. Jones EL, Burlew CC, Moore EE. BioGlue hemostasis of penetrating cardiac wounds in proximity to the left ante-rior descending coronary

1	DV, eds. Trauma. 8th ed. New York: McGraw-Hill; 2017. 104. Jones EL, Burlew CC, Moore EE. BioGlue hemostasis of penetrating cardiac wounds in proximity to the left ante-rior descending coronary artery. J Trauma Acute Care Surg. 2012;72(3):796-798. 105. Cothren CC, Moore EE. Traumatic ventricular septal defect. Surgery. 2007;142:776-777. 106. Wall MJ Jr, Hirshberg A, Mattox KL. Pulmonary tractotomy with selective vascular ligation for penetrating injuries to the lung. Am J Surg. 1994;168:665-669. 107. Cothren C, Moore EE, Biffl WL, et al. Lung-sparing tech-niques are associated with improved outcome compared with anatomic resection for severe lung injuries. J Trauma. 2002;53:483-487. 108. Cryer HG, Mavroudis C, Yu J, et al. Shock, transfusion, and pneumonectomy. Death is due to right heart failure and increased pulmonary vascular resistance. Ann Surg. 1990;212:197-201. 109. Luo L, Yin L, Liu Z, Xiang Z. Posttraumatic pulmonary pseudocyst: computed tomography findings and man-agement in

1	failure and increased pulmonary vascular resistance. Ann Surg. 1990;212:197-201. 109. Luo L, Yin L, Liu Z, Xiang Z. Posttraumatic pulmonary pseudocyst: computed tomography findings and man-agement in 33 patients. J Trauma and Acute Care Surg. 2012;73(5):1225-1228. 110. Moore HB, Moore EE, Burlew CC, et al. Western Trauma Association critical decisions in trauma: management of parapneumonic effusion. J Trauma Acute Care Surg. 2012;73:1372-1379. 111. de Souza A, Offner PJ, Moore EE, et al. Optimal management of complicated empyema. Am J Surg. 2000;180:507-511. 112. Truitt MS, Murry J, Amos J, et al. Continuous intercostal nerve blockade for rib fractures: ready for primetime? J Trauma. 2011;71(6):1548-1552. 113. Kozar RA, Moore FA, Cothren CC, et al. Risk factors for hepatic morbidity following nonoperative management: mul-ticenter study. Arch Surg. 2006;141:451-458. 114. Malhotra AK, Fabian TC, Croce MA, et al. Blunt hepatic injury: a paradigm shift from operative to nonoperative

1	following nonoperative management: mul-ticenter study. Arch Surg. 2006;141:451-458. 114. Malhotra AK, Fabian TC, Croce MA, et al. Blunt hepatic injury: a paradigm shift from operative to nonoperative man-agement in the 1990s. Ann Surg. 2000;231:804-813. 115. Peitzman AB, Marsh JW. Advanced operative techniques in the management of complex liver injury. J Trauma Acute Care Surg. 2012;73(3):765-770. 116. Biffl WL, Moore EE, Franciose RJ. Venovenous bypass and hepatic vascular isolation as adjuncts in the repair of destruc-tive wounds to the retrohepatic inferior vena cava. J Trauma. 1998;45:400-403.Brunicardi_Ch07_p0183-p0250.indd 24710/12/18 6:22 PM 248BASIC CONSIDERATIONSPART I 117. Poggetti RS, Moore EE, Moore FA, et al. Balloon tampon-ade for bilobar transfixing hepatic gunshot wounds. J Trauma. 1992;33:694-697. 118. Delis SG, Bakoyiannis A, Selvaggi G, et al. Liver transplanta-tion for severe hepatic trauma: experience from a single center. World J Gastroenterol.

1	gunshot wounds. J Trauma. 1992;33:694-697. 118. Delis SG, Bakoyiannis A, Selvaggi G, et al. Liver transplanta-tion for severe hepatic trauma: experience from a single center. World J Gastroenterol. 2009;15(13):1641-1644. 119. Pickhardt B, Moore EE, Moore FA, et al. Operative splenic salvage in adults: a decade perspective. J Trauma. 1989;29:1386-1391. 120. Feliciano DV, Spjut-Patrinely V, Burch JM, et al. Splenorrha-phy: the alternative. Ann Surg. 1990;211:569-580. 121. Bhullar IS, Tepas JJ 3rd, Siragusa D, Loper T, Kerwin A, Frykberg ER. To nearly come full circle: nonoperative man-agement of high-grade IV-V blunt splenic trauma is safe using a protocol with routine angioembolization. J Trauma Acute Care Surg. 2017;82(4):657-664. 122. McIntyre LK, Schiff M, Jurkovich GJ. Failure of nonopera-tive management of splenic injuries: causes and consequences. Arch Surg. 2005;140:563-568. 123. Lopez JM Jr, McGonagill PW, Gross JL, et al. Subcapsular hematoma in blunt splenic injury: a

1	nonopera-tive management of splenic injuries: causes and consequences. Arch Surg. 2005;140:563-568. 123. Lopez JM Jr, McGonagill PW, Gross JL, et al. Subcapsular hematoma in blunt splenic injury: a significant predictor of failure of nonoperative management. J Trauma Acute Care Surg. 2015;79(6):957-959; discussion 959-960. 124. Toutouzas KG, Velmahos GC, Kaminski A, et al. Leukocytosis after posttraumatic splenectomy: a physiologic event or sign of sepsis? Arch Surg. 2002;137:924-928. 125. Howdieshell TR, Heffernan D, Dipiro JT; Therapeutic Agents Committee of the Surgical Infection Society. Surgical infec-tion society guidelines for vaccination after traumatic injury. Surg Infect (Larchmt). 2006;7(3):275-303. 126. Todd SR, Kozar RA, Moore FA. Nutrition support in adult trauma patients. Nutr Clin Pract. 2006;21:421-429. 127. Burlew CC, Moore EE, Cuschieri J, et al; the WTA Study Group. Who should we feed? Western Trauma Asso-ciation multi-institutional study of enteral nutrition in

1	Nutr Clin Pract. 2006;21:421-429. 127. Burlew CC, Moore EE, Cuschieri J, et al; the WTA Study Group. Who should we feed? Western Trauma Asso-ciation multi-institutional study of enteral nutrition in the open abdomen after injury. J Trauma Acute Care Surg. 2012;73:1380-1387. 128. Schroeppel TJ, Saleem K, Sharpe JP, et al. Penetrating duode-nal trauma: a 19-year experience. J Trauma Acute Care Surg. 2016;80(3):461-465. 129. Biffl WL, Moore EE, Croce M, et al. Western Trauma Asso-ciation critical decisions in trauma: management of pancreatic injuries. J Trauma Acute Care Surg. 2013;75(6):941-946. 130. Vaughn GD, Frazier OH, Graham D, et al. The use of pyloric exclusion in the management of severe duodenal injuries. Am J Surg. 1977;134:785. 131. Krige JE, Kotze UK, Setshedi M, Nicol AJ, Navsaria PH. Surgical management and outcomes of combined pancreati-coduodenal injuries: analysis of 75 consecutive cases. J Am Coll Surg. 2016;222(5):737-749. 132. Nelson R, Singer M. Primary repair for

1	PH. Surgical management and outcomes of combined pancreati-coduodenal injuries: analysis of 75 consecutive cases. J Am Coll Surg. 2016;222(5):737-749. 132. Nelson R, Singer M. Primary repair for penetrating colon inju-ries. Cochrane Database Syst Rev. 2003;(3):CD002247. 133. Burlew CC, Moore EE, Cuschieri J, et al; WTA Study Group. Sew it up! A Western Trauma Association multi-institutional study of enteric injury management in the postinjury open abdomen. J Trauma. 2011;70(2):273-277. 134. Asensio JA, Britt LD, Borzotta A, et al. Multi-institutional experience with the management of superior mesenteric artery injuries. J Am Coll Surg. 2001;193:354-356. 135. Burch JM, Richardson RJ, Martin RR, et al. Penetrating iliac vascular injuries: experience with 233 consecutive patients. J Trauma. 1990;30:1450-1459. 136. Mullins RJ, Lucas CE, Ledgerwood AM. The natural his-tory following venous ligation for civilian injuries. J Trauma. 1980;20:737-743. 137. Jurkovich GJ, Hoyt DB, Moore FA, et

1	1990;30:1450-1459. 136. Mullins RJ, Lucas CE, Ledgerwood AM. The natural his-tory following venous ligation for civilian injuries. J Trauma. 1980;20:737-743. 137. Jurkovich GJ, Hoyt DB, Moore FA, et al. Portal triad injuries. J Trauma. 1995;39:426-434. 138. Voelzke BB, McAninch JW. Renal gunshot wounds: clinical management and outcome. J Trauma. 2009;66(3):593-600. 139. Rostas J, Simmons JD, Frotan MA, Brevard SB, Gonzalez RP. Intraoperative management of renal gunshot injuries: is man-datory exploration of Gerota’s fascia necessary? Am J Surg. 2016;211(4):783-786. 140. Knudson MM, Harrison PB, Hoyt DB, et al. Outcome after major renovascular injuries: a Western Trauma Association multicenter report. J Trauma. 2000;49:1116-1122. 141. Burlew CC, Moore EE, Stahel PF, et al. Preperitoneal pelvic packing reduces mortality in patients with life-threatening hemorrhage due to unstable pelvic fractures. J Trauma Acute Care Surg. 2017;82(2):233-242. 142. Bosse MJ, MacKenzie EJ, Kellam JF, et

1	packing reduces mortality in patients with life-threatening hemorrhage due to unstable pelvic fractures. J Trauma Acute Care Surg. 2017;82(2):233-242. 142. Bosse MJ, MacKenzie EJ, Kellam JF, et al. An analysis of outcomes of reconstruction or amputation of leg-threatening injuries. N Engl J Med. 2002;347:1924-1931. 143. Einersen PM, Moore EE, Chapman MP, et al. Rapid throm-belastography thresholds for goal-directed resuscitation of patients at risk for massive transfusion. J Trauma Acute Care Surg. 2017;82(1):114-119. 144. Moore FA, McKinley BA, Moore EE, et al. Inflammation and the host response to injury, a large-scale collaborative proj-ect: patient-oriented research core—standard operating proce-dures for clinical care. III. Guidelines for shock resuscitation. J Trauma, 2006;61:82-89. 145. Dezman ZD, Comer AC, Smith GS, Narayan M, Scalea TM, Hirshon JM. Failure to clear elevated lactate predicts 24-hour mortality in trauma patients. J Trauma Acute Care Surg.

1	Trauma, 2006;61:82-89. 145. Dezman ZD, Comer AC, Smith GS, Narayan M, Scalea TM, Hirshon JM. Failure to clear elevated lactate predicts 24-hour mortality in trauma patients. J Trauma Acute Care Surg. 2015;79(4):580-585. 146. Nickerson TP, Zielinski MD, Jenkins DH, Schiller HJ. The Mayo Clinic experience with Morel-Lavallée lesions: estab-lishment of a practice management guideline. J Trauma Acute Care Surg. 2014;76(2):493-497. 147. Burlew CC, Moore EE, Biffl WL, Bensard DD, Johnson JL, Barnett CC. One hundred percent fascial approximation can be achieved in the postinjury open abdomen with a sequential clo-sure protocol. J Trauma Acute Care Surg. 2012;72(1):235-241. 148. Sela HY, Weiniger CF, Hersch M, Smueloff A, Laufer N, Einav S. The pregnant motor vehicle accident casualty: adher-ence to basic workup and admission guidelines. Ann Surg. 2011;254(2):346-352. 149. Drukker L, Hants Y, Sharon E, Sela HY, Grisaru-Granovsky S. Perimortem cesarean section for maternal and fetal salvage:

1	to basic workup and admission guidelines. Ann Surg. 2011;254(2):346-352. 149. Drukker L, Hants Y, Sharon E, Sela HY, Grisaru-Granovsky S. Perimortem cesarean section for maternal and fetal salvage: concise review and protocol. Acta Obstet Gynecol Scand. 2014;93(10):965-972. 150. ACOG Committee on Obstetric Practice. ACOG Commit-tee Opinion. Number 299, September 2004. Guidelines for diagnostic imaging during pregnancy. Obstet Gynecol. 2004;104:647-651. 151. Curet MJ, Schermer CR, Demarest GB, et al. Predictors of outcome in trauma during pregnancy: identification of patients who can be monitored for less than 6 hours. J Trauma. 2000;49:18-24. 152. Bruijns SR, Guly HR, Bouamra O, Lecky F, Lee WA. The value of traditional vital signs, shock index, and age-based markers in predicting trauma mortality. J Trauma Acute Care Surg. 2013;74(6):1432-1437. 153. Bulger EM, Arneson MA, Mock CN, et al. Rib fractures in the elderly. J Trauma. 2000;48:1040-1046. 154. Pieracci FM, Lin Y, Rodil M, et

1	mortality. J Trauma Acute Care Surg. 2013;74(6):1432-1437. 153. Bulger EM, Arneson MA, Mock CN, et al. Rib fractures in the elderly. J Trauma. 2000;48:1040-1046. 154. Pieracci FM, Lin Y, Rodil M, et al. A prospective, controlled clinical evaluation of surgical stabilization of severe rib frac-tures. J Trauma Acute Care Surg. 2016;80(2):187-194. 155. Tepas JJ. The national pediatric trauma registry: a legacy of commitment to control childhood injury. Semin Pediatr Surg. 2004;13:126-132. 156. Gardner AR, Diz DI, Tooze JA, Miller CD, Petty J. Injury pat-terns associated with hypotension in pediatric trauma patients: Brunicardi_Ch07_p0183-p0250.indd 24810/12/18 6:22 PM 249TRAUMACHAPTER 7a national trauma database review. J Trauma Acute Care Surg. 2015;78(6):1143-1148. 157. Mollberg NM, Tabachnick D, Lin FJ, et al. Age-associated impact on presentation and outcome for penetrating thoracic trauma in the adult and pediatric patient populations. J Trauma Acute Care Surg.

1	NM, Tabachnick D, Lin FJ, et al. Age-associated impact on presentation and outcome for penetrating thoracic trauma in the adult and pediatric patient populations. J Trauma Acute Care Surg. 2014;76(2):273-277; discussion 277-278. 158. Linnaus ME, Langlais CS, Garcia NM, et al. Failure of non-operative management of pediatric blunt liver and spleen injuries: a prospective Arizona-Texas-Oklahoma-Memphis-Arkansas Consortium study. J Trauma Acute Care Surg. 2017;82(4):672-679. 159. Ingram MC, Siddharthan RV, Morris AD, et al. Hepatic and splenic blush on computed tomography in children fol-lowing blunt abdominal trauma: is intervention necessary? J Trauma Acute Care Surg. 2016;81(2):266-270.Brunicardi_Ch07_p0183-p0250.indd 24910/12/18 6:22 PM

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1	BurnsJeffrey H. Anderson, Samuel P. Mandell, and Nicole S. Gibran 8chapterSurgical care of the burned patient has evolved into a special-ized field incorporating the interdisciplinary skills of burn sur-geons, nurses, burn therapists, and other healthcare specialists. However, recent mass casualty events have been a reminder that healthcare systems may be rapidly pressed to care for large numbers of burn patients. Naturally, general surgeons may be at the forefront in these events, so it is crucial that they are com-fortable with the care of burned patients and well equipped to provide the standard of care.BACKGROUNDBurn injury historically carried a poor prognosis. With advances in fluid resuscitation1 and the advent of early excision of the burn wound,2 survival has become an expectation even for patients with severe burns.3 Continued improvements in critical care and progress in skin bioengineering herald a future in which func-tional and psychologic outcomes are equally important

1	even for patients with severe burns.3 Continued improvements in critical care and progress in skin bioengineering herald a future in which func-tional and psychologic outcomes are equally important as sur-vival alone. With this shift in priority, the American Burn Association (ABA) has emphasized referral to specialized burn centers after early stabilization. Specific criteria should guide transfer of patients with more complex injuries or other medical needs to a burn center (Table 8-1). The ABA has published stan-dards of care4 and created a verification process to ensure that burn centers meet those standards.5 Because of increased prehospital safety measures, burn patients are trans-ferred longer distances for definitive care at regional burn centers6; data from one burn center with a particularly wide catch-ment area confirmed that even transport times averaging several hours did not affect the long-term outcomes of burn patients.7INITIAL EVALUATIONInitial evaluation of the

1	a particularly wide catch-ment area confirmed that even transport times averaging several hours did not affect the long-term outcomes of burn patients.7INITIAL EVALUATIONInitial evaluation of the burned patient should follow the same initial priorities of all trauma patients and involves four crucial assessments: airway management, evaluation of other injuries, 1estimation of burn size, and diagnosis of CO and cyanide poi-soning. With direct thermal injury to the upper airway or smoke inhalation, rapid and severe airway edema is potentially lethal. Anticipating the need for intubation and establishing an early airway are critical. Signs of impending respiratory compromise include a hoarse voice, wheezing, or stridor; subjective dyspnea is a particularly concerning symptom and should trigger prompt elective endotracheal intubation. Perioral burns and singed nasal hairs alone do not indicate an upper airway injury, but are signs that the oral cavity and pharynx should be further

1	trigger prompt elective endotracheal intubation. Perioral burns and singed nasal hairs alone do not indicate an upper airway injury, but are signs that the oral cavity and pharynx should be further evaluated for mucosal injury. Orotracheal intubation is the preferred method for securing the airway. Nasotracheal intubation may be use-ful for patients with associated facial trauma when experienced providers are present, but it should be avoided if oral intubation is safe and easy.Burned patients are trauma patients and evaluated with a primary survey in accordance with Advanced Trauma Life Sup-port guidelines. Concurrently with the primary survey, large-bore peripheral intravenous (IV) catheters should be placed and fluid resuscitation should be initiated; for a burn larger than 40% total body surface area (TBSA), two large-bore IVs are ideal. IV place-ment through burned skin is safe and effective but requires atten-tion to securing the catheters. Central venous access and intraosseous

1	surface area (TBSA), two large-bore IVs are ideal. IV place-ment through burned skin is safe and effective but requires atten-tion to securing the catheters. Central venous access and intraosseous (IO) access should be considered when peripheral access cannot be easily obtained. Rarely, IV resuscitation is indi-cated in patients with burns smaller than 15% who can usually hydrate orally. Pediatric patients with burns larger than 15% may require IO access in emergent situations if venous access cannot be attained. An early and comprehensive secondary survey must be performed on all burn patients, but especially those with a his-tory of associated mechanical trauma such as a motor vehicle collision. Also, patients from structural fires in which the manner of egress is not known should be carefully evaluated for injuries from a possible jump or fall. Urgent radiology studies, such as a chest X-ray, should be performed in the emergency department, but nonurgent skeletal evaluation (i.e.,

1	evaluated for injuries from a possible jump or fall. Urgent radiology studies, such as a chest X-ray, should be performed in the emergency department, but nonurgent skeletal evaluation (i.e., extremity X-rays) can be Background 251Initial Evaluation 251Burn Classification 252Burn Depth 253Prognosis 254Resuscitation 254Inhalation Injury and Ventilator Management 255Treatment of The Burn Wound 257Nutrition 257Complications in Burn Care 258Surgery 259Wound Coverage 259Rehabilitation 260Late Complications: Hypertrophic Scar, Contractures, and Heterotopic Ossification 260Psychological Recovery 261Prevention 261Burn Disasters 261Future Areas of Study 262Brunicardi_Ch08_p0251-p0270.indd 25128/12/18 11:51 AM 252Table 8-1Guidelines for referral to a burn centerPartial-thickness burns greater than 10% TBSABurns involving the face, hands, feet, genitalia, perineum, or major jointsThird-degree burns in any age groupElectrical burns, including lightning injuryChemical burnsInhalation

1	greater than 10% TBSABurns involving the face, hands, feet, genitalia, perineum, or major jointsThird-degree burns in any age groupElectrical burns, including lightning injuryChemical burnsInhalation injuryBurn injury in patients with complicated preexisting medical disordersPatients with burns and concomitant trauma in which the burn is the greatest risk. If the trauma is the greater immediate risk, the patient may be stabilized in a trauma center before transfer to a burn center.Burned children in hospitals without qualified personnel for the care of childrenBurn injury in patients who will require special social, emotional, or rehabilitative interventionTBSA = total body surface area.done in the intensive care unit (ICU) to avoid hypothermia and delayed resuscitation. Hypothermia is a common prehospital complication that contributes to resuscitation failure. Patients should be wrapped with clean blankets in transport. Cooling should be avoided in patients with moderate or large

1	a common prehospital complication that contributes to resuscitation failure. Patients should be wrapped with clean blankets in transport. Cooling should be avoided in patients with moderate or large (>20% TBSA) burns. Patients with acute burn injuries should never receive prophylactic antibiotics. This intervention has been clearly demonstrated to promote development of fungal infections and resistant organisms and was abandoned in the mid-1980s.8 A tetanus booster should be administered in the emergency department depending on patient immunization status.The importance of pain management for these patients has been widely recognized over the past 25 years. While pain man-agement is a priority for burn patients, it is important to acknowl-edge the opioid crisis and the recent push toward decreasing opiate use in general.9 In order to limit opiate-related morbidity, we recommend responsible opiate use in conjunction with mul-timodal pain control and development of a weaning plan

1	decreasing opiate use in general.9 In order to limit opiate-related morbidity, we recommend responsible opiate use in conjunction with mul-timodal pain control and development of a weaning plan start-ing at opioid commencement. Clear expectations around pain medication use should be set with patients. Anxiety is another component of the psychological response of burn patients, seen with both wound care and general postinjury hospital course. Benzodiazepines are a staple in the treatment of acute anxiety; however, they can contribute significantly to hospital delirium.10 We recommend conservative benzodiazepine use to mitigate the effects of anxiety while minimizing deliriogenic effects of benzodiazepines.2Most burn resuscitation formulas estimate fluid require-ments based on burn size measured as a percentage of TBSA (%TBSA). The “rule of nines” is a crude but quick and effec-tive method of estimating burn size (Fig. 8-1). In adults, the anterior and posterior trunk each account for

1	as a percentage of TBSA (%TBSA). The “rule of nines” is a crude but quick and effec-tive method of estimating burn size (Fig. 8-1). In adults, the anterior and posterior trunk each account for 18%, each lower extremity is 18%, each upper extremity is 9%, and the head is 9%. In children under 3 years old, the head accounts for a larger relative surface area and should be taken into account when esti-mating burn size. For smaller or odd-shaped burns, the “rule of the palm” where the palmar surface of the hand, including the digits, is 1% TBSA is useful. Diagrams such as the Lund and Browder chart give a more accurate accounting of the true burn size in children and adults. The importance of an accurate burn size assessment cannot be overemphasized. Superficial or first-degree burns should not be included when calculating burn size, and thorough cleaning of soot and debris is mandatory to avoid confusing soiled skin with burns. Examination of referral data suggests that physicians

1	not be included when calculating burn size, and thorough cleaning of soot and debris is mandatory to avoid confusing soiled skin with burns. Examination of referral data suggests that physicians inexperienced with burns tend to overestimate the size of small burns and underestimate the size of large burns, with potentially detrimental effects on pretrans-fer resuscitation.11BURN CLASSIFICATIONBurns are commonly classified as thermal, electrical, or chemi-cal burns, with thermal burns consisting of flame, contact, or scald burns. Flame burns are the most common cause for hos-pital admission of burns, and have the highest mortality. This is primarily related to their association with structural fires and the accompanying inhalation injury and/or CO poisoning.12Electrical burns make up 3% of U.S. hospital admissions but have special concerns,12 including cardiac arrhythmia and compartment syndrome with concurrent rhabdomyolysis. A baseline ECG is recommended in all patients with an

1	3% of U.S. hospital admissions but have special concerns,12 including cardiac arrhythmia and compartment syndrome with concurrent rhabdomyolysis. A baseline ECG is recommended in all patients with an electrical injury, and a normal ECG in a low-voltage injury (<1000 V) may preclude hospital admission. Because compartment syn-drome and rhabdomyolysis are common in high-voltage elec-trical injuries, vigilance must be maintained for neurologic or vascular compromise, and fasciotomies should be performed even in cases of moderate clinical suspicion. Long-term neuro-logic symptoms13 and cataract development14 are not uncommon with high-voltage electrical injuries, and neurologic and oph-thalmologic consultation should be obtained to define baseline patient function.15Chemical burns also comprise 3% of admitted burn patients12 and result in potentially severe burns. Typically, acid chemical burns result in coagulation necrosis and alkali chemical burns cause liquefactive necrosis (with an

1	3% of admitted burn patients12 and result in potentially severe burns. Typically, acid chemical burns result in coagulation necrosis and alkali chemical burns cause liquefactive necrosis (with an exception of hydrofluoric acid, which also causes liquefactive necrosis).16,17 Key Points1 Follow American Burn Association criteria for referral of a patient to a regional burn center.2 Never administer prophylactic antibiotics other than tetanus vaccination.3 Early excision and grafting of full-thickness and deep partial-thickness burns improve outcomes.4 Intravenous fluid resuscitation for patients with burns >20% of total body surface area (children with burns >15% of total body surface area) should be titrated to mean arterial pres-sure (MAP) >60 mmHg and appropriate urine output.Brunicardi_Ch08_p0251-p0270.indd 25228/12/18 11:51 AM 253BURNSCHAPTER 8The most important components of initial therapy are careful removal of the toxic substance from the patient and irrigation of the

1	25228/12/18 11:51 AM 253BURNSCHAPTER 8The most important components of initial therapy are careful removal of the toxic substance from the patient and irrigation of the affected area with water for a minimum of 30 minutes. In cases of exposure to dry chemicals, such as concrete or pow-dered forms of lye, the substance should be swept from the patient to avoid a thermal reaction with water. The offending agents in chemical burns can be systemically absorbed and may cause specific metabolic derangements. Formic acid has been known to cause hemolysis and hemoglobinuria, and hydroflu-oric acid causes hypocalcemia. Hydrofluoric acid is a particu-larly common offender due to its widespread industrial uses. Calcium-based therapies are the mainstay of treating hydroflu-oric acid burns, with topical application of calcium gluconate onto wounds18 and IV administration of calcium gluconate for systemic hypocalcemia symptoms. Intra-arterial calcium gluco-nate infusion provides effective

1	topical application of calcium gluconate onto wounds18 and IV administration of calcium gluconate for systemic hypocalcemia symptoms. Intra-arterial calcium gluco-nate infusion provides effective treatment of progressive tissue injury and intense pain.19,20 Patients undergoing intra-arterial therapy need continuous cardiac monitoring. Persistent refrac-tory hypocalcemia with electrocardiac abnormalities may signal the need for emergent excision of the burned areas.21BURN DEPTHBased on the original burn depth classification by Dupuytren in 1832,22 burn wounds are commonly classified as superficial (first-degree), partial-thickness (second-degree), full-thickness (third-degree), and fourth-degree burns, which affect underlying soft tissue. Fifth-degree burns (through muscle to bone) and sixth degree burns (charring bone) were also described although are less common. Partial-thickness burns are classified as either superficial or deep partial-thickness burns by depth of involved dermis.

1	degree burns (charring bone) were also described although are less common. Partial-thickness burns are classified as either superficial or deep partial-thickness burns by depth of involved dermis. Clinically, first-degree burns are painful but do not blis-ter, second-degree burns have dermal involvement and are extremely painful with weeping and blisters, and third-degree burns are leathery, painless, and nonblanching. Jackson described three zones of tissue injury following burn injury.23 The zone of coagulation is the most severely burned portion and is typically in the center of the wound. As the name implies, the affected tissue is coagulated and sometimes frankly necrotic, much like a full thickness burn, and will need excision and graft-ing. Peripheral to that is a zone of stasis, with variable degrees of vasoconstriction and resultant ischemia, much like a second-degree burn. Appropriate resuscitation and wound care may pre-vent conversion to a deeper wound, but infection or

1	with variable degrees of vasoconstriction and resultant ischemia, much like a second-degree burn. Appropriate resuscitation and wound care may pre-vent conversion to a deeper wound, but infection or suboptimal perfusion may result in an increase in burn depth. This is clini-cally relevant because many superficial partial-thickness burns will heal with nonoperative management, and the majority of deep partial-thickness burns benefit from excision and skin grafting. The outermost area of a burn is called the zone of hyperemia, which will heal with minimal or no scarring and is most like a superficial partial thickness burn or first-degree burn.Unfortunately, even experienced burn surgeons have lim-ited ability to accurately predict the healing potential of partial-thickness burns soon after injury; one reason is that burn wounds evolve over the 48 to 72 hours after injury. Numerous burn depth assessment tools have been developed with the idea that

1	of partial-thickness burns soon after injury; one reason is that burn wounds evolve over the 48 to 72 hours after injury. Numerous burn depth assessment tools have been developed with the idea that 39%4.5%4.5%9%9%18%1%1%18%18%18%FrontBackFigure 8-1. The “rule of nines” can be used as a quick reference for estimating a patient’s burn size by dividing the body into regions to which total body surface area is allocated in multiples of nine.Brunicardi_Ch08_p0251-p0270.indd 25328/12/18 11:51 AM 254BASIC CONSIDERATIONSPART Iearlier burn depth definition will expedite appropriate surgical decision-making. One of the most effective ways to determine burn depth is full-thickness biopsy, but this has several limita-tions; not only is the procedure painful and potentially scar-ring, but accurate interpretation of the histopathology requires a specialized pathologist and may have slow turnaround times.24 Laser Doppler can measure skin perfusion to predict burn depth with sensitivities and

1	interpretation of the histopathology requires a specialized pathologist and may have slow turnaround times.24 Laser Doppler can measure skin perfusion to predict burn depth with sensitivities and specificity of up to 83% and 97%, respectively.25 Noncontact ultrasound has been postulated as a painless modality to predict nonhealing wounds and has the advantage of easily performed serial measurements.26 Unfortu-nately, none of these newer therapies have proven adequately superior to justify their cost and as yet have not substituted serial examination by experienced burn surgeons.PROGNOSISThe Baux score (mortality risk equals age plus %TBSA) was used for many years to predict mortality in burns. Analysis of multiple risk factors for burn mortality has validated age and burn size as the strongest predictors of mortality.27 Advance-ments in burn care have lowered overall mortality to the point that the original Baux score may no longer be accurate. How-ever, the Revised Baux Score, which

1	predictors of mortality.27 Advance-ments in burn care have lowered overall mortality to the point that the original Baux score may no longer be accurate. How-ever, the Revised Baux Score, which accounts for age, burn size, and inhalation injury, has been found to be independently associated with mortality.28 As such, age, burn size, and inhala-tion injury continue to be the most robust indicators for burn mortality.29 Age even as a single variable strongly predicts mortality in burns,30 and in-hospital mortality in elderly burn patients is a function of age regardless of other comorbidities.31 In nonelderly patients, comorbidities such as preinjury human immunodeficiency virus (HIV), metastatic cancer, and kidney or liver disease may influence mortality and length of stay.32 A large database study of 68,661 burn patients found that the vari-ables with the highest predictive value for mortality were age, %TBSA, inhalation injury, coexistent trauma, and pneumonia.33 A more recent study

1	study of 68,661 burn patients found that the vari-ables with the highest predictive value for mortality were age, %TBSA, inhalation injury, coexistent trauma, and pneumonia.33 A more recent study analyzing 506,628 burn inpatients between 1998 and 2008 demonstrated an association between burn size, age, inhalation injury, and mortality. Other factors associated with mortality included African American race, female gender, and treatment in urban private hospitals (as opposed to urban academic hospitals).34Mortality is not the only outcome of interest in the burn population. Burn injury can significantly impact the subsequent quality of life for survivors, including but not limited to appear-ance, mobility, functional status, and ability to work. One study found that burn injury reduces short term quality of life by 30% and long-term quality of life by approximately 11%.35 Predic-tors of poorer physical and mental health 12 months removed from burn injury include older age, female

1	short term quality of life by 30% and long-term quality of life by approximately 11%.35 Predic-tors of poorer physical and mental health 12 months removed from burn injury include older age, female gender, and greater %TBSA burn size.36 One factor impacting quality of life is itching—a late and bothersome consequence of burn injury that affects both adult and pediatric population.37,38 Other factors dis-cussed later in this chapter include hypertrophic scarring, con-tracture, and heterotopic ossification. Finally, return to work or school has been a useful tool to evaluate recovery and prognosis. A recent meta-analysis found that approximately 28% of burn survivors never return to work.39 A recent study using an inter-ventional bundle involving the patient, the employee, worker’s compensation, and burn clinic staff demonstrated a return to work rate of 93%.40 The return to school for pediatric patients is actually very prompt, averaging about 10 days after discharge. However, further

1	and burn clinic staff demonstrated a return to work rate of 93%.40 The return to school for pediatric patients is actually very prompt, averaging about 10 days after discharge. However, further study is needed to determine whether atten-dance and performance suffer despite early reentry to school.41 It is important to recognize these potential quality-of-life issues in burn patients and take necessary steps to diminish the impact that burn injury has on quality of life both in the hospital and following discharge.RESUSCITATIONA myriad of formulas exist for calculating fluid needs during burn resuscitation, suggesting that no one formula benefits all patients. The most commonly used formula, the Parkland or Baxter formula, consists of 3 to 4 mL/kg per % burn of Lac-tated Ringer’s, of which half is given during the first 8 hours after burn and the remaining half is given over the subsequent 16 hours.1 The most recent American Burn Association con-sensus formula recommends 2 mL/kg per %

1	half is given during the first 8 hours after burn and the remaining half is given over the subsequent 16 hours.1 The most recent American Burn Association con-sensus formula recommends 2 mL/kg per % burn of Lactated Ringers given the tendency toward excessive fluid administra-tion with the traditional formulas.42 The concept behind continu-ous fluid requirements is simple. The burn (and/or inhalation injury) drives an inflammatory response that leads to capillary leak; as plasma leaks into the extravascular space, crystalloid administration maintains the intravascular volume. Therefore, if a patient receives a large fluid bolus in a prehospital setting or emergency department, the fluid has likely leaked into the interstitium, and the patient still requires ongoing burn resusci-tation according to the estimates. Continuation of fluid volumes should depend on the time since injury, urine output, and mean arterial pressure (MAP). As the capillary leak closes, the patient will require

1	to the estimates. Continuation of fluid volumes should depend on the time since injury, urine output, and mean arterial pressure (MAP). As the capillary leak closes, the patient will require less volume to maintain these two resuscitation endpoints. Children under 20 kg have the additional require-ment that they do not have sufficient glycogen stores to main-tain an adequate glucose level in response to the inflammatory response. Specific pediatric formulas have been described, but the simplest approach is to deliver a weight-based maintenance IV fluid with glucose supplementation in addition to the calcu-lated resuscitation with lactated Ringer’s.It is important to remember that any formula for burn resuscitation is merely a guideline, and fluid must be titrated based on appropriate response to therapy. A number of parame-ters are widely used to gauge burn resuscitation, but the most common remain the simple outcomes of blood pressure and urine output. As in any critically ill

1	response to therapy. A number of parame-ters are widely used to gauge burn resuscitation, but the most common remain the simple outcomes of blood pressure and urine output. As in any critically ill patient, a target MAP of 60 mmHg ensures optimal end-organ perfusion. Goals for urine out-put should be 30 mL/h in adults and 1 to 1.5 mL/kg per h in pediatric patients. Because blood pressure and urine output may not correlate perfectly with true tissue perfusion, the search continues for other adjunctive parameters that may more accu-rately reflect adequate resuscitation. Some centers have found serum lactate to be a better predictor of mortality in severe burns,43,44 and others have found that base deficit predicts even-tual organ dysfunction and mortality.45,46 Because burned patients with normal blood pressure and serum lactate levels may have compromised gastric mucosal perfusion, continuous measurement of mucosal pH with its logistical difficulties has garnered limited

1	patients with normal blood pressure and serum lactate levels may have compromised gastric mucosal perfusion, continuous measurement of mucosal pH with its logistical difficulties has garnered limited popularity.47,48 Invasive monitoring with pul-monary artery catheters typically results in significant excessive fluid administration without improved cardiac output or preload measurements; use of invasive monitoring seems to have vari-able effects on long-term outcomes.49Actual administrated fluid volumes typically exceed vol-umes predicted by standard formulas.50 One survey of burn 4Brunicardi_Ch08_p0251-p0270.indd 25428/12/18 11:51 AM 255BURNSCHAPTER 8centers showed that 58% of patients end up getting more fluids than would be predicted by Baxter’s formula.51 Comparison of modern-day patients with historical controls shows that over-resuscitation may be a relatively recent trend.52 One theory is that increased opioid analgesic use results in peripheral vaso-dilation and

1	patients with historical controls shows that over-resuscitation may be a relatively recent trend.52 One theory is that increased opioid analgesic use results in peripheral vaso-dilation and hypotension and the need for greater volumes of bolused resuscitative fluids.53 A classic study by Navar et al showed that burned patients with inhalation injury required an average of 5.76 mL/kg per % burn, vs. 3.98 mL/kg per % burn for patients without inhalation injury, and this has been corroborated by subsequent studies.54,55 Prolonged mechanical ventilation may also play a role in increased fluid needs.56 A multicenter study found that age, weight, %TBSA, and intu-bation on admission were significant predictors of more fluid delivery during the resuscitation period. Those patients receiv-ing higher fluid volumes were at increased risk of complications and death.57 Common complications include abdominal com-partment syndrome, extremity compartment syndrome, intraoc-ular compartment syndrome,

1	fluid volumes were at increased risk of complications and death.57 Common complications include abdominal com-partment syndrome, extremity compartment syndrome, intraoc-ular compartment syndrome, and pleural effusions. Monitoring bladder pressures can provide valuable information about devel-opment of intra-abdominal hypertension.The use of colloid as part of the burn resuscitation has gen-erated much interest over the years. In late resuscitation when the capillary leak has closed, colloid administration may decrease overall fluid volumes and potentially may decrease associated complications such as intra-abdominal hypertension.58 A recent meta-analysis accounting for statistical heterogeneity among studies included demonstrated a trend toward mortality ben-efit for patients receiving albumin.59 However, albumin use has never been shown to definitively improve mortality in burn patients and has controversial effects on mortality in critically ill patients.60-63 Still, many burn

1	albumin.59 However, albumin use has never been shown to definitively improve mortality in burn patients and has controversial effects on mortality in critically ill patients.60-63 Still, many burn centers including ours continue to use albumin as an adjunct during burn resuscitation.64 Attempts to minimize fluid volumes in burn resuscitation have included study of hypertonic solutions. A recent meta-analysis evaluating hyperosmotic vs. isoosmotic fluid resuscitation demonstrates decreased total fluid load (vol/%TBSA per weight) over the first 24 hours with use of hyperosmotic fluid with no difference in total fluid, urine output, creatinine, or mortality.65 A described downside of hypertonic fluid administration is hyperchloremic acidosis.66Other adjuncts are being increasingly used during initial burn resuscitation. High-dose ascorbic acid (vitamin C) may decrease fluid volume requirements and ameliorate respira-tory embarrassment during resuscitation, although no mortality benefit

1	initial burn resuscitation. High-dose ascorbic acid (vitamin C) may decrease fluid volume requirements and ameliorate respira-tory embarrassment during resuscitation, although no mortality benefit has been noted thus far in two trials.67,68 Plasmapheresis has also been associated with decreased fluid requirements and increased urine output in patients who require higher resuscita-tive volumes than predicted to maintain adequate urine output and MAP.69 It is postulated that plasmapheresis may filter out inflammatory mediators, thus decreasing ongoing vasodilation and capillary leak.70One adjunct that has found increasing utility in surgi-cal ICUs has been the application of bedside ultrasound.71 Ultrasound offers the potential to make rapid, noninvasive assessments during acute changes in clinical condition. For burn patients, bedside ultrasonography may be indicated for evalu-ation of volume status, gross assessment of cardiac function, and diagnosis of pneumothorax. Determining

1	in clinical condition. For burn patients, bedside ultrasonography may be indicated for evalu-ation of volume status, gross assessment of cardiac function, and diagnosis of pneumothorax. Determining patient cardiac function and volume status may guide fluid resuscitation. Car-diac function can be evaluated with three common heart views: the parasternal long axis, parasternal short axis, and apical four-chamber views.72 Whereas no study has used ultrasound to guide fluid resuscitation in burn patients, volume status can be estimated by examination of cardiac function and evaluation of the inferior vena cava (IVC) diameter with changes in respira-tion, as has been done in patients with hemorrhage and shock.73 Ultrasound also allows timely diagnosis of pneumothorax.74 A high-frequency probe with an adequate window between ribs permits identification of lung parenchyma against the chest well. A pneumothorax appears as a transition on ultrasound between lung parenchyma, which has a

1	probe with an adequate window between ribs permits identification of lung parenchyma against the chest well. A pneumothorax appears as a transition on ultrasound between lung parenchyma, which has a heterogeneous appear-ance, and air, which has a hypoechoic appearance. Further stud-ies are warranted to identify indications for the use of ultrasound in burned patients.Machine learning and bedside computer decision support are other adjuncts gaining traction in caring for burn patients. These modalities can enhance patient care and aid in diagnosis, treatment, and research.75 The use of bedside computer decision support has been particularly appealing for resuscitation of burn patients in the first 48 hours and has been shown to improve fluid management during initial resuscitation.76The role of blood transfusion in critically injured patients has undergone a reevaluation in recent years.77,78 Blood transfu-sions are considered to be immunomodulatory and potentially immunosuppressive,

1	of blood transfusion in critically injured patients has undergone a reevaluation in recent years.77,78 Blood transfu-sions are considered to be immunomodulatory and potentially immunosuppressive, which is one explanation to the links between blood transfusions and increased infection and shorter time to recurrence after oncologic surgery.79,80 A large multi-center study of blood transfusions in burn patients found that increased numbers of transfusions were associated with increased infections and higher mortality in burn patients, even when cor-recting for burn severity.81 A follow-up study implementing a restrictive transfusion policy in burned children showed that a hemoglobin threshold of 7 g/dL had no more adverse outcomes vs. a traditional transfusion trigger of 10 g/dL. In addition, costs incurred to the institution were significantly less.82 A recent ran-domized control trial in patients with >20% TBSA compared outcomes of a restrictive to a liberal red blood cell transfusion

1	costs incurred to the institution were significantly less.82 A recent ran-domized control trial in patients with >20% TBSA compared outcomes of a restrictive to a liberal red blood cell transfusion strategy (hemoglobin 7–8 vs. 10–11, respectively). There were no differences in blood stream infection, organ dysfunction, ven-tilator days, time to wound healing, or 30-day mortality between both groups.83 These data, in concert with other reported compli-cations such as transfusion-related lung injury,84 have led to rec-ommendations that blood transfusions be used only when there is an apparent physiologic need. Attempts to minimize blood transfusion in nonburned critically ill patients have led to use of erythropoietin by some centers. However, burn patients often have elevated erythropoietin levels, and a randomized study in burn patients showed that recombinant human erythropoietin did not effectively prevent anemia or decrease the number of transfusions given.85 Promising animal

1	levels, and a randomized study in burn patients showed that recombinant human erythropoietin did not effectively prevent anemia or decrease the number of transfusions given.85 Promising animal studies demonstrating erythropoietin-mediated prevention of secondary burn progres-sion have yet to be validated in humans.86INHALATION INJURY AND VENTILATOR MANAGEMENTInhalation injuries are commonly seen in tandem with burn inju-ries and are known to increase mortality in burned patients.87,88 Smoke inhalation is present in as many as 35% of hospital-ized burn patients and may triple the hospital stay compared to isolated burn injuries.89 Mortality for inhalation injury has been reported to be as high as 25%, with this increasing to 50% in patients with ≥20% TBSA burns.90 The pneumonia rate in Brunicardi_Ch08_p0251-p0270.indd 25528/12/18 11:51 AM 256BASIC CONSIDERATIONSPART Ipatients with inhalation injury has been reported to be three times higher than those without inhalation injury, and

1	25528/12/18 11:51 AM 256BASIC CONSIDERATIONSPART Ipatients with inhalation injury has been reported to be three times higher than those without inhalation injury, and it has been associated with increased length of stay, increased ven-tilator days, and need for tracheostomy.91,92 The combination of burns, inhalation injury, and pneumonia increases mortal-ity by up to 60% over burns alone.93 Subsequent development of the adult respiratory distress syndrome (ARDS) is common in these patients and may be caused in part by recruitment of alveolar leukocytes with an enhanced endotoxin-activated cyto-kine response.94 When ARDS complicates burns and inhalation injury, mortality approaches 66%; in one study, patients with burns ≥60% TBSA in combination with inhalation injury and ARDS had 100% mortality.95Smoke inhalation causes injury in two ways: by direct heat injury to the upper airways and inhalation of combustion prod-ucts into the lower airways. Direct injury to the upper airway

1	mortality.95Smoke inhalation causes injury in two ways: by direct heat injury to the upper airways and inhalation of combustion prod-ucts into the lower airways. Direct injury to the upper airway causes airway swelling that typically leads to maximal edema in the first 24 to 48 hours after injury and often requires a short course of endotracheal intubation for airway protection. Com-bustion products found in smoke, most commonly from syn-thetic substances in structural fires, cause lower airway injury. These irritants cause direct mucosal injury, which in turn leads to mucosal sloughing, edema, reactive bronchoconstriction, and finally obstruction of the lower airways. Injury to both the epi-thelium and pulmonary alveolar macrophages causes release of prostaglandins, chemokines, and other inflammatory mediators; neutrophil migration; increased tracheobronchial blood flow; and, finally, increased capillary permeability. All of these com-ponents of acute lung injury increase the risk of

1	inflammatory mediators; neutrophil migration; increased tracheobronchial blood flow; and, finally, increased capillary permeability. All of these com-ponents of acute lung injury increase the risk of pneumonia and ARDS following an inhalation injury.The physiologic effects of smoke inhalation are numerous. Inhalation injury decreases lung compliance96 and increases air-way resistance work of breathing.97 Inhalation injury in the pres-ence of burns also increases overall metabolic demands.98 The most common physiologic derangement seen with inhalation injury is increased fluid requirement during resuscitation. Since severe inhalation injury may result in mucosal sloughing with obstruction of smaller airways, bronchoscopy findings including carbon deposits, erythema, edema, bronchorrhea, and a hemor-rhagic appearance may be useful for staging inhalation injury. The Abbreviated Injury Score—a scale from 0 to 4, with 0 representing no injury and 4 representing massive injury—is commonly

1	a hemor-rhagic appearance may be useful for staging inhalation injury. The Abbreviated Injury Score—a scale from 0 to 4, with 0 representing no injury and 4 representing massive injury—is commonly used for grading inhalation injury. Higher grades of bronchoscopic inhalation injury have been associated with increased incidence of ARDS, increased ventilator days, higher rate of multiple organ dysfunction syndrome, and higher mortality.99 Bronchoscopic evaluation can also help isolate organisms early in the course of a potential pneumonia. Bronchoalveolar lavage (BAL) within 24 hours after an inhalation injury demonstrates a high rate of positive quantitative cultures,100 suggesting that pneumonia develops soon after the acute lung injury. Bacterial contamina-tion from urgent intubation may contribute to early development of pneumonia in patients with inhalation injury.100 Early evalu-ation with bronchoscopy can identify causative organisms and guide appropriate antibiotic

1	may contribute to early development of pneumonia in patients with inhalation injury.100 Early evalu-ation with bronchoscopy can identify causative organisms and guide appropriate antibiotic therapy.Because bronchoscopy is an invasive test, attempts have been made to utilize other diagnostic modalities, such as tho-racic computed tomography (CT) scans101 and xenon ventilationperfusion scanning.102,103 However, these are generally not uti-lized unless otherwise indicated, and the best tools available for diagnosing inhalation injury remain clinical presentation and bronchoscopic evaluation. Decreased PaO2:FiO2 ratio (<350) on admission may not only predict inhalation injury but also indi-cate increased fluid needs more accurately than bronchoscopic grading of the severity of inhalation.104Treatment of inhalation injury consists primarily of support-ive care. Aggressive pulmonary toilet and routine use of nebulized bronchodilators such as albuterol are recommended. Nebulized

1	of inhalation injury consists primarily of support-ive care. Aggressive pulmonary toilet and routine use of nebulized bronchodilators such as albuterol are recommended. Nebulized N-acetylcysteine is an antioxidant free radical scavenger designed to decrease the toxicity of high oxygen concentrations. Aero-solized heparin aims to prevent formation of fibrin plugs and decrease the formation of airway casts and has been associated with increased number of ventilator-free days.105 A recent meta-analysis demonstrated improved mortality with the use of inhaled anticoagulation regimens.106 Aerosolized tissue plasminogen activator107 and recombinant human antithrombin108 have shown promise in sheep models but have not yet seen widespread clinical use. Administration of intrabronchial surfactant has been used as a salvage therapy in patients with severe burns and inhalation injury.109 Inhaled nitric oxide may also be useful as a last effort in burn patients with severe lung injury who are

1	has been used as a salvage therapy in patients with severe burns and inhalation injury.109 Inhaled nitric oxide may also be useful as a last effort in burn patients with severe lung injury who are failing other means of ventilator support.110 The use of steroids has traditionally been avoided due to the worse outcomes in burn patients111; however, some data demonstrate selectively improved outcomes with septic shock requiring vasopressor circulatory.112An important contributor to early mortality in burn patients and often seen in patients with inhalation injury is carbon mon-oxide (CO) poisoning. This clear, odorless gas has an affinity for hemoglobin is approximately 200 to 250 times more than that of oxygen. Carboxyhemoglobin decreases the levels of nor-mal oxygenated hemoglobin and can quickly lead to anoxia and death.113 CO also causes uncoupling of oxidative phosphory-lation in mitochondria, free radical generation, and increased systemic inflammatory response via platelet

1	can quickly lead to anoxia and death.113 CO also causes uncoupling of oxidative phosphory-lation in mitochondria, free radical generation, and increased systemic inflammatory response via platelet activation—all of which may increase cardiac and neurologic morbidity and mortality in CO toxicity.114 Unexpected neurologic or cardiac symptoms should raise the level of suspicion, and an arterial carboxyhemoglobin level must be obtained because pulse oxim-etry can be falsely elevated. Administration of 100% normo-baric oxygen is the gold standard for treating CO poisoning and reduces the half-life of CO from 250 minutes in room air to 40 to 60 minutes.115 Some authors have proposed hyperbaric oxygen as an adjunctive therapy for CO poisoning.116 However, a recent meta-analysis offers mixed results regarding the suc-cess and long-term outcomes of hyperbaric oxygen, and its asso-ciated logistical difficulties and complications have limited its usefulness for patients with moderate or large

1	regarding the suc-cess and long-term outcomes of hyperbaric oxygen, and its asso-ciated logistical difficulties and complications have limited its usefulness for patients with moderate or large burns.117 Patients who sustain a cardiac arrest as a result of their CO poisoning have an extremely poor prognosis regardless of the success of initial resuscitation attempts.118Hydrogen cyanide toxicity may also be a component of an overwhelming smoke inhalation injury. Cyanide inhibits cyto-chrome oxidase, which is required for oxidative phosphorylation.119 Afflicted patients may have a persistent, severe lactic acidosis, neurologic symptoms, pulmonary edema, or cardiac sequelae (ST elevation on electrocardiogram).120,121 Classic signs of cya-nide poisoning—including bitter almond breath and cherry-red skin changes—are rare and should not be used as the sole diagnostic criteria. Treatment consists of sodium thiosulfate, hydroxocobalamin, and 100% oxygen. Sodium thiosulfate works as a

1	and cherry-red skin changes—are rare and should not be used as the sole diagnostic criteria. Treatment consists of sodium thiosulfate, hydroxocobalamin, and 100% oxygen. Sodium thiosulfate works as a substrate for the metabolism cyanide into a nontoxic deriva-tive, but it works slowly and is not effective for acute therapy.121 Hydroxocobalamin—a vitamin B12 precursor—quickly Brunicardi_Ch08_p0251-p0270.indd 25628/12/18 11:51 AM 257BURNSCHAPTER 8complexes with cyanide, is excreted by the kidney, and is rec-ommended for immediate therapy.122 In the majority of patients, lactic acidosis will resolve with ventilation, and sodium thio-sulfate treatment becomes unnecessary.123 Given the unknown side-effects of hydroxocobalamin administration, it should be reserved only for patients with a strong suspicion of cyanide poisoning.New ventilator strategies have contributed to the improved mortality with ARDS. Although ARDS still contributes to mor-tality in burn patients, treatments have

1	a strong suspicion of cyanide poisoning.New ventilator strategies have contributed to the improved mortality with ARDS. Although ARDS still contributes to mor-tality in burn patients, treatments have improved so that mor-tality is primarily from multisystem organ failure rather than isolated respiratory causes.124 The ARDS Network Study find-ing that low tidal volume (6 cc/kg) or “lung-protective ventila-tion” had a 22% lower mortality than patients with traditional tidal volumes (12 cc/kg)124 has dramatically changed the man-agement of patients with acute lung injury. A similar approach had previously been shown to improve outcomes in pediatric burn patients.125 In patients with refractory hypoxemia despite lung-protective ventilation, prone positioning may improve oxygenation and mortality.126,127 No specific studies have exam-ined prone positioning in burned patients, and in fact exclusion criteria from a large prone positioning trial included patients with ≥20% TBSA.127 Select

1	No specific studies have exam-ined prone positioning in burned patients, and in fact exclusion criteria from a large prone positioning trial included patients with ≥20% TBSA.127 Select reports demonstrate the feasibility of prone positioning in burn patients,128 although they pres-ent logistical challenges and caution must be used in patients with frontal and facial burns who are already at risk for loss of the grafts, invasive catheters, and the endotracheal tube. Highfrequency percussive ventilation (HFPV) has shown early promise in patients with inhalation injury.129 One study showed notable decreases in both morbidity and mortality with HFPV, especially in patients with burns <40% TBSA and inhalation injury.130 A randomized controlled trial between low-tidal vol-ume ventilation and HFPV in burn patients requiring mechani-cal ventilation demonstrated no significant difference in primary clinical outcomes.131 A related technique is high-frequency oscillatory ventilation (HFOV),

1	HFPV in burn patients requiring mechani-cal ventilation demonstrated no significant difference in primary clinical outcomes.131 A related technique is high-frequency oscillatory ventilation (HFOV), which has been used primarily as a salvage modality in patients refractory to more conventional measures.132 However, two recent studies and a recent metaanalysis have concluded that HFOV yields no mortality benefit and in fact may actually increase patient mortality in patients with ARDS.133-135 Extracorporeal membrane oxygenation (ECMO) is typically reserved for salvage situations, although utilization of ECMO for burn patients is increasing and out-comes have been shown to be similar to other ECMO patients.136TREATMENT OF THE BURN WOUNDMultitudes of topical therapies exist for the treatment of burn wounds, many of which contain antimicrobial properties. A recent Cochrane Database Review nicely summarizes the data surrounding antisepsis for burns; however, much of the data is

1	treatment of burn wounds, many of which contain antimicrobial properties. A recent Cochrane Database Review nicely summarizes the data surrounding antisepsis for burns; however, much of the data is inconclusive.137 Silver sulfadiazine is one of the most widely used in clinical practice. Silver sulfadiazine has a wide range of antimicrobial activity, primarily as prophylaxis against burn wound infections rather than treatment of existing infec-tions. It has the added benefits of being inexpensive, being easily applied, and having soothing qualities. It is not signifi-cantly absorbed systemically and thus has minimal metabolic derangements. Silver sulfadiazine has a reputation for causing neutropenia, but this association is more likely due to neutro-phil margination from the inflammatory response following burn injury. True allergic reactions to the sulfa component of silver sulfadiazine are rare, and at-risk patients can have a small test patch applied to identify a burning sensation

1	following burn injury. True allergic reactions to the sulfa component of silver sulfadiazine are rare, and at-risk patients can have a small test patch applied to identify a burning sensation or rash. Silver sulfadiazine destroys skin grafts and is contraindicated on burns or donor sites in proximity to newly grafted areas. Also, silver sulfadiazine may retard epithelial migration in healing partial-thickness wounds.Mafenide acetate, either in cream or solution form, is an effective topical antimicrobial. It is effective even in the pres-ence of eschar and can be used in both treating and prevent-ing wound infections; the solution formulation is an excellent antimicrobial for fresh skin grafts. Use of mafenide acetate may be limited by pain with application to partial-thickness burns. As mafenide is a carbonic anhydrase inhibitor, a his-torically described side effect is metabolic acidosis. However, multiple studies have been performed using mafenide to treat burn wounds without any

1	mafenide is a carbonic anhydrase inhibitor, a his-torically described side effect is metabolic acidosis. However, multiple studies have been performed using mafenide to treat burn wounds without any significant incidence of metabolic acidosis.138,139Silver nitrate has broad-spectrum antimicrobial activity as a topical solution. The solution used must be dilute (0.5%), and prolonged topical application leads to electrolyte extravasation with resulting hyponatremia. A rare complication is methemo-globinemia.140 Although inexpensive, silver nitrate solution causes black stains, and laundry costs may offset any fiscal benefit to the hospital. Although there is no definitive evidence regarding use in the burn population, Dakin’s solution (0.5% sodium hypochlorite solution) is an acceptable alternative as an inexpensive topical antimicrobial.For smaller burns or larger burns that are nearly healed, topical ointments such as bacitracin, neomycin, and polymyxin B can be used. These are also

1	as an inexpensive topical antimicrobial.For smaller burns or larger burns that are nearly healed, topical ointments such as bacitracin, neomycin, and polymyxin B can be used. These are also useful for superficial partial-thickness facial burns as they can be applied and left open to air without dressing coverage. Meshed skin grafts in which the interstices are nearly closed are another indication for use of these agents, preferably with greasy gauze to help retain the ointment in the affected area. All three have been reported to cause nephrotoxicity and should be used sparingly in large burns. Recent media coverage of methicillin-resistant Staphy-lococcus aureus (MRSA) has led to widespread use by commu-nity practitioners of mupirocin for new burns. Unless the patient has known risk factors for MRSA, mupirocin should only be used in culture-positive burn wound infections to prevent emer-gence of further resistance.Silver-impregnated dressings are increasingly being used for donor

1	for MRSA, mupirocin should only be used in culture-positive burn wound infections to prevent emer-gence of further resistance.Silver-impregnated dressings are increasingly being used for donor sites, skin grafts, and partial-thickness burns because of their potential to avoid daily dressing changes. These may be more comfortable for the patient, reduce the number of dressing changes, and shorten hospital length of stay, but they limit serial wound examinations. Biologic membranes such as Biobrane (Smith & Nephew Global Products) provide a prolonged barrier under which wounds may heal. Because of the occlusive nature of these dressings, these are typically used only on fresh, super-ficial, partial-thickness burns that are clearly not contaminated.NUTRITIONNutritional support may be more important in patients with large burns than in any other patient population. Not only does adequate nutrition play a role in acute issues such as immune responsiveness, but the hypermetabolic response

1	in patients with large burns than in any other patient population. Not only does adequate nutrition play a role in acute issues such as immune responsiveness, but the hypermetabolic response in burn injury may raise baseline metabolic rates by as much as 200%.141 This can lead to catabolism of muscle proteins and decreased lean body mass that may delay functional recovery.142 Early enteral Brunicardi_Ch08_p0251-p0270.indd 25728/12/18 11:51 AM 258BASIC CONSIDERATIONSPART Ifeeding for patients with burns >20% TBSA is safe and may reduce loss of lean body mass,143 slow the hypermetabolic response,144 and result in more efficient protein metabolism.145,146 Early enteral feeds have also been associated with shorter dura-tion of ICU stay and decreased rates of wound infection.147 If the enteral feeds are started within the first few hours after admis-sion, gastric ileus may be avoided. Adjuncts such as metoclo-pramide promote gastrointestinal motility; if other measures for gastric

1	enteral feeds are started within the first few hours after admis-sion, gastric ileus may be avoided. Adjuncts such as metoclo-pramide promote gastrointestinal motility; if other measures for gastric feeding are unsuccessful, advancing the tube into the small bowel with nasojejunal feeding can be attempted.148 In endotracheally intubated patients, trips to the operating room do not necessitate holding enteral feedings.149 Immune-modulating supplements such as glutamine may decrease infectious com-plications in burn patients,150,151 although the effect on mortality and wound closure remains unknown. One proposed mechanism for glutamine’s immune modulating properties is via preven-tion of T-cell suppression in mesenteric lymph nodes.152 There is currently a multicenter randomized control trial recruiting to determine the effect of glutamine on mortality, blood stream infections, and health-related quality of life (https://clinicaltri-als.gov/ct2/show/NCT00985205). Micronutrient

1	trial recruiting to determine the effect of glutamine on mortality, blood stream infections, and health-related quality of life (https://clinicaltri-als.gov/ct2/show/NCT00985205). Micronutrient supplementa-tion with antioxidant vitamins (vitamin E and ascorbic acid) and trace minerals (selenium, zinc, and copper) optimizes wound healing, enhances immune function, and fights oxida-tive stress.153Calculating the appropriate caloric needs of the burn patient can be challenging. A commonly used formula in non-burned patients is the Harris-Benedict equation, which calcu-lates caloric needs using factors such as gender, age, height, and weight. This formula uses an activity factor for specific inju-ries, and for burns, the basal energy expenditure is multiplied by two. The Harris-Benedict equation may be inaccurate in burns of <40% TBSA, and in these patients, the Curreri formula may be more appropriate. This formula estimates caloric needs to be 25 kcal/kg per d plus 40 kcal/%TBSA per d.

1	may be inaccurate in burns of <40% TBSA, and in these patients, the Curreri formula may be more appropriate. This formula estimates caloric needs to be 25 kcal/kg per d plus 40 kcal/%TBSA per d. Indirect calorim-etry can also be used to calculate resting energy expenditure, but in burn patients, a “metabolic cart” has not been documented to be more beneficial than the predictive equations.154 Titrating caloric needs closely is important because overfeeding patients will lead to storage of fat instead of muscle anabolism.155Modifying the hypermetabolic response is an area of intense study. β-Blocker use in pediatric patients decreases heart rate and resting energy expenditure and abrogates protein catabolism, even in long-term use.156 There may be benefits to β-blockade in adult patients,157 and many centers use β-blockers routinely in the adult population with limited safety and efficacy data.158 Some data suggests that β-blocker use in the adult burn population has a greater

1	and many centers use β-blockers routinely in the adult population with limited safety and efficacy data.158 Some data suggests that β-blocker use in the adult burn population has a greater incidence of iatrogenic hypotension and bradycardia. As such, it is important to monitor hemodynamic status when starting β-blockers in these populations.159The anabolic steroid oxandrolone has been extensively studied in burn patients as well and has demonstrated improve-ments in lean body mass and bone density in severely burned children.160 The weight gain and functional improvements seen with oxandrolone may persist even after stopping administration of the drug.161,162 A double-blind, randomized study of oxandro-lone showed decreased length of stay, improved hepatic pro-tein synthesis, and no adverse effects on endocrine function, although the authors noted a rise in transaminases with unclear clinical significance.163 Oxandrolone therapy has also been asso-ciated with overall decreased

1	adverse effects on endocrine function, although the authors noted a rise in transaminases with unclear clinical significance.163 Oxandrolone therapy has also been asso-ciated with overall decreased mortality in patients with large burns.164Hyperglycemia has been associated with increased mortal-ity after burn injury,165 and intensive insulin therapy in critically ill patients has shown benefit, presumably from avoidance of hyperglycemia.166 However, in burn patients, the insulin itself may have a metabolic benefit, with improvements in lean body mass and amelioration of the inflammatory response to burn injury.167,168 Oral hypoglycemic agents such as metformin also help to avoid hyperglycemia and may contribute to prevention of muscle catabolism.169COMPLICATIONS IN BURN CAREThere are several complications commonly associated with treatment of burn patients. Though not always avoidable, main-taining vigilance for typical complications and using appro-priate techniques for prevention

1	complications commonly associated with treatment of burn patients. Though not always avoidable, main-taining vigilance for typical complications and using appro-priate techniques for prevention may limit the frequency and severity of complications. Ventilator-associated pneumonia, as in all critically ill patients, is a significant problem in burned patients. However, it is so common in patients with inhalation injury that a better nomenclature may be postinjury pneumonia. Unfortunately, commonly used scores in critical illness such as the Clinical Pulmonary Infection Score (CPIS) have not been shown to be reliable in burn patients. Quantitative broncho-scopic cultures in the setting of clinical suspicion of pneumo-nia should guide treatment of pneumonia.170 Simple measures such as elevating the head of the bed and maintaining excel-lent oral hygiene and pulmonary toilet are recommended to help decrease the risk of postinjury pneumonia. There is some question as to whether early

1	the head of the bed and maintaining excel-lent oral hygiene and pulmonary toilet are recommended to help decrease the risk of postinjury pneumonia. There is some question as to whether early tracheostomy decreases infectious morbidity in burn patients and whether it improves long-term outcomes. There do not seem to be any major differences in the rates of pneumonia with early tracheostomy, though there may be reduced development of subglottic stenosis compared with prolonged endotracheal intubation.171,172 Practical consid-erations such as protection of facial skin grafts may influence the decision for tracheostomy placement. One major consider-ation in deciding whether to perform a tracheostomy has been the presence of eschar at the insertion site, which complicates tracheostomy site care and increases the risk of airway infec-tion. Bedside percutaneous dilatational tracheostomy is a facile method for performing tracheostomy and is reported to be as safe as open tracheostomy in the

1	and increases the risk of airway infec-tion. Bedside percutaneous dilatational tracheostomy is a facile method for performing tracheostomy and is reported to be as safe as open tracheostomy in the burn population.173Massive resuscitation of burned patients may lead to an abdominal compartment syndrome characterized by increased airway pressures with hypoventilation and decreased urine output and hemodynamic compromise. Decompressive lapa-rotomy is the standard of care for refractory abdominal com-partment syndrome but carries an especially poor prognosis in burn patients.174 Adjunctive measures such as minimizing fluid, performing torso escharotomies, decreasing tidal volumes, and chemical paralysis should be initiated before resorting to decompressive laparotomy. Patients undergoing massive resus-citation also develop elevated intraocular pressures and may require lateral canthotomy.175Deep vein thrombosis (DVT) and prophylaxis in the burn population has received increasing attention

1	resus-citation also develop elevated intraocular pressures and may require lateral canthotomy.175Deep vein thrombosis (DVT) and prophylaxis in the burn population has received increasing attention in the literature recently. Up to 25% of burn patients develop DVT, and fatal pulmonary emboli have been reported in burn patients.176,177 A recent prospective trial demonstrated an 8% incidence of DVT in patients with 30% to 60% TBSA burns not receiving low molecular weight heparin prophylaxis with no evidence of DVT in patients receiving prophylaxis. There were no complications Brunicardi_Ch08_p0251-p0270.indd 25828/12/18 11:51 AM 259BURNSCHAPTER 8from low molecular weight heparin prophylaxis.178 Thus, it appears that heparin prophylaxis is safe in burn patients and may help prevent thrombotic complications.Unfortunately, the use of both prophylactic and therapeu-tic heparin may be associated with heparin-associated throm-bocytopenia (HIT). One study of HIT in burn patients showed an

1	complications.Unfortunately, the use of both prophylactic and therapeu-tic heparin may be associated with heparin-associated throm-bocytopenia (HIT). One study of HIT in burn patients showed an incidence of 1.6% in heparinized burn patients. Thrombotic complications included DVT, pulmonary embolus, and even arterial thrombosis requiring limb amputation. Nonheparin anticoagulation for HIT commonly caused bleeding complica-tions requiring transfusion.179 Although rare, a high index of suspicion for HIT should be maintained in thrombocytopenic burn patients, particularly if the platelet counts drop at hospital days 7 to 10.Burn patients often require central venous access for fluid resuscitation and hemodynamic monitoring. Because of the ana-tomic relation of their burns to commonly used access sites, burn patients may be at higher risk for catheter-related blood-stream infections. The 2012 Centers for Disease Control and Prevention National Healthcare Safety Network report indicates

1	access sites, burn patients may be at higher risk for catheter-related blood-stream infections. The 2012 Centers for Disease Control and Prevention National Healthcare Safety Network report indicates that American burn centers have higher infectious complica-tion rates than any other ICUs.180 Because burn patients may commonly exhibit leukocytosis with a documented bloodstream infection, practice has been to rewire lines over a guide wire and to culture the catheter tip. However, this may increase the risk of catheter-related infections in burned patients, and a new site should be used if at all possible.181SURGERYFull-thickness burns with a rigid eschar can form a tourniquet effect as the edema progresses, leading to compromised venous outflow and eventually arterial inflow. The resulting compart-ment syndrome is most common in circumferential extremity burns, but abdominal and thoracic compartment syndromes also occur. Warning signs of impending compartment syndrome may include

1	compart-ment syndrome is most common in circumferential extremity burns, but abdominal and thoracic compartment syndromes also occur. Warning signs of impending compartment syndrome may include paresthesias, pain, decreased capillary refill, and progression to loss of distal pulses; in an intubated patient, the surgeon should anticipate the compartment syndrome and perform frequent neurovascular evaluations. Abdominal com-partment syndrome should be suspected with decreased urine output, increased ventilator airway pressures, and hypotension. Hypoventilation, increased airway pressures, and hypotension may also characterize thoracic compartment syndrome. Escha-rotomies are rarely needed within the first 8 hours following injury and should not be performed unless indicated because of the terrible aesthetic sequelae. When indicated, they are usu-ally performed at the bedside, preferably with electrocautery to minimize blood loss. Extremity incisions are made on the lateral and medial

1	terrible aesthetic sequelae. When indicated, they are usu-ally performed at the bedside, preferably with electrocautery to minimize blood loss. Extremity incisions are made on the lateral and medial aspects of the limbs in an anatomic position and may extend onto thenar and hypothenar eminences of the hand. Digital escharotomies do not usually result in any meaningful salvage of functional tissue and are not recommended. Inad-equate perfusion despite proper escharotomies may indicate the need for fasciotomy, but this procedure should not be routinely performed as part of the eschar release. Thoracic escharotomies should be placed along the anterior axillary lines with bilateral subcostal and subclavicular extensions. Extension of the ante-rior axillary incisions down the lateral abdomen typically will allow adequate release of abdominal eschar.The strategy of early excision and grafting in burned patients revolutionized survival outcomes in burn care. Not only did it improve

1	abdomen typically will allow adequate release of abdominal eschar.The strategy of early excision and grafting in burned patients revolutionized survival outcomes in burn care. Not only did it improve mortality, but early excision also decreased reconstruction surgery, hospital length of stay, and costs of care.182-184 Once the initial resuscitation is complete and the patient is hemodynamically stable, attention should be turned to excising the burn wound. Burn excision and wound coverage should ideally start within the first several days, and in larger burns, serial excisions can be performed as patient condition allows. Excision is performed with repeated tangential slices using a Watson or Goulian blade until viable, diffusely bleeding tissue remains. It is appropriate to leave healthy dermis, which will appear white with punctate areas of bleeding. Excision to fat or fascia may be necessary in deeper burns. The downside of tangential excision is a high blood loss, though this may

1	dermis, which will appear white with punctate areas of bleeding. Excision to fat or fascia may be necessary in deeper burns. The downside of tangential excision is a high blood loss, though this may be ame-liorated using techniques such as instillation of an epinephrine tumescence solution underneath the burn. Pneumatic tourni-quets are helpful in extremity burns, and compresses soaked in a dilute epinephrine solution are necessary adjuncts after excision. A fibrinogen and thrombin spray sealant (Tisseel Fibrin Sealant; Baxter, Deerfield, IL) also has beneficial effects on both hemo-stasis and graft adherence to the wound bed. The use of these techniques has markedly decreased the number of blood trans-fusions given during burn surgery.185 For patients with clearly deep burns and concern for excessive blood loss, fascial exci-sion may be employed. In this technique, electrocautery is used to excise the burned tissue and the underlying subcutaneous tis-sue down to muscle fascia. This

1	for excessive blood loss, fascial exci-sion may be employed. In this technique, electrocautery is used to excise the burned tissue and the underlying subcutaneous tis-sue down to muscle fascia. This technique markedly decreases blood loss but results in a cosmetically inferior appearance due to the loss of subcutaneous tissue. For excision of burns in dif-ficult anatomic areas, such as the face, eyelids, or hands, a pres-surized water dissector may offer more precision but is time consuming, has a steep learning curve, and is expensive.186WOUND COVERAGESince full-thickness burns are impractical for most burn wounds, split-thickness sheet autografts harvested with a power dermatome make the most durable wound coverings and have a decent cosmetic appearance. In larger burns, meshed auto-grafted skin provides a larger area of wound coverage. This also allows drainage of blood and serous fluid to prevent accumula-tion under the skin graft with subsequent graft loss. Areas of cosmetic

1	skin provides a larger area of wound coverage. This also allows drainage of blood and serous fluid to prevent accumula-tion under the skin graft with subsequent graft loss. Areas of cosmetic importance such as the face, neck, and hands should be grafted with nonmeshed sheet grafts to ensure optimal appear-ance and function. Unfortunately, even extensive meshing of skin grafts in patients with limited donor sites may not provide adequate amounts of skin. One emerging technique for large burns with limited donor sites is the Meek micrografting tech-nique, or “postage-stamp” technique, where expansion ratios of up to 9:1 are able to be achieved. This technique has a con-siderable learning curve and requires further investigation to determine whether it is the optimal technique for large surface area burns with limited donor sites.187 Options for temporary wound coverage include human cadaveric allograft, which is incorporated into the wound but is rejected by the immune sys-tem and must

1	area burns with limited donor sites.187 Options for temporary wound coverage include human cadaveric allograft, which is incorporated into the wound but is rejected by the immune sys-tem and must be eventually replaced. This allows temporary biologic wound coverage until donor sites heal enough so that they may be reharvested. Xenograft appears to function as well as allograft for temporary wound coverage and is considerably less expensive.The search for a perfect permanent synthetic skin sub-stitute remains elusive. Integra (Integra LifeSciences Cor-poration, Plainsboro, NJ) is a bilayer product with a porous Brunicardi_Ch08_p0251-p0270.indd 25928/12/18 11:51 AM 260BASIC CONSIDERATIONSPART Icollagen-chondroitin 6-sulphate inner layer that is attached to an outer silastic sheet, which helps prevent fluid loss and infection as the inner layer becomes vascularized, creating an artificial neodermis. At approximately 2 weeks after placement, the silas-tic layer can be removed and a

1	helps prevent fluid loss and infection as the inner layer becomes vascularized, creating an artificial neodermis. At approximately 2 weeks after placement, the silas-tic layer can be removed and a thin autograft can be placed over the neodermis. This results in faster healing of the more superfi-cial donor sites and has been increasingly utilized for treatment of complex wounds and injuries.188 Alloderm (LifeCell Corpo-ration, The Woodlands, TX) is a dermal substitute consisting of cryopreserved acellular human dermis. NovoSorb™ Biode-gradable Temporizing Matrix (PolyNovo Limited, Melbourne, Australia) is a biodegradable polyurethane dermal substitute newly available and recently approved by the United States Food and Drug Administration (FDA). These dermal substitutes should also be used in combination with thin split-thickness skin grafts for final wound coverage.189Epidermal skin substitutes such as cultured epithelial auto-grafts are an option in patients with massive burns and

1	in combination with thin split-thickness skin grafts for final wound coverage.189Epidermal skin substitutes such as cultured epithelial auto-grafts are an option in patients with massive burns and very limited donor sites.190,191 Their clinical use has been limited by a long turnaround time for culturing, as well as the fragility of the cultured skin, which creates great difficulty with intraopera-tive handling and graft take. There are promising developments in skin culturing techniques and engineered skin development, but no other products are FDA approved and commercially available.188Thighs make convenient anatomic donor sites; they are eas-ily harvested and relatively hidden from an aesthetic standpoint. The thicker skin of the back is useful in older patients, who have thinner skin elsewhere and may have difficulty with healing of donor sites. The buttocks are an excellent donor site in infants and toddlers; silver sulfadiazine can be applied to the donor site with a diaper as

1	elsewhere and may have difficulty with healing of donor sites. The buttocks are an excellent donor site in infants and toddlers; silver sulfadiazine can be applied to the donor site with a diaper as coverage. The scalp is also an excellent donor site; the skin is thick and the many hair follicles allow rapid heal-ing, with the added advantage of being completely hidden once hair regrows. Epinephrine tumescence is necessary for harvest-ing the scalp, for both hemostasis of this hypervascular area and also to create a smooth contoured surface for harvesting.The list of commonly used donor site dressings is long and includes simple transparent films to hydrocolloids, petrolatum gauzes, and silver-impregnated dressings. Donor sites close to fresh grafts may be dressed with a porous nonadherent gauze, and both the donors and grafts are soaked with an antimicrobial solution. Principles behind choosing a dressing should balance ease of care, comfort, infection control, and cost. The choice

1	gauze, and both the donors and grafts are soaked with an antimicrobial solution. Principles behind choosing a dressing should balance ease of care, comfort, infection control, and cost. The choice of donor site dressing is largely institution dependent, and few data support the clear superiority of any single treatment plan.REHABILITATIONRehabilitation is an integral part of the clinical care plan for the burn patient and should be initiated on admission. Immediate and ongoing physical and occupational therapy is mandatory to prevent functional loss. Patients who are unable to actively participate should have passive range-of-motion exercises done at least twice a day. This includes patients with burns over joints, such as with hand burns. Patients should be taught exer-cises they can do themselves to maintain full range of motion. Patients with foot and extremity burns should be instructed to walk independently without crutches or other assistive devices to prevent extremity

1	can do themselves to maintain full range of motion. Patients with foot and extremity burns should be instructed to walk independently without crutches or other assistive devices to prevent extremity swelling, desensitize the burned areas, and prevent disuse atrophy; when patients are not ambulating, they must elevate the affected extremity to minimize swelling. If postoperative immobilization is used for graft protection, the graft should be evaluated early and at frequent intervals so that active exercise can be resumed at the earliest possible occasion. The transition to outpatient care should also include physical and occupational therapy, with introduction of exercises designed to accelerate return to activities of daily living as well as specific job-related tasks. Tight-fitting pressure garments provide vascu-lar support in burns that are further along in the healing process. Whether they prevent hypertrophic scar formation has been long debated. However, they do provide

1	pressure garments provide vascu-lar support in burns that are further along in the healing process. Whether they prevent hypertrophic scar formation has been long debated. However, they do provide vascular support that many patients find more comfortable.LATE COMPLICATIONS: HYPERTROPHIC SCAR, CONTRACTURES, AND HETEROTOPIC OSSIFICATIONOnce patients have recovered from their acute burns, many face management of the hypertrophic burn scars. In patients with healed burns or donor sites, hypertrophic scar-related morbid-ity includes pruritus, erythema, pain, thickened tight skin, and even contractures. Within these scars, there is believed to be an increased inflammatory response, irregular neovascularization, aberrant cytokine and Toll-like receptor expression, abundant collagen production, and abnormal extracellular matrix struc-ture. Treatment for these scars has included nonsurgical therapies such as compression garments, silicone gel sheeting, massage, physical therapy, and

1	and abnormal extracellular matrix struc-ture. Treatment for these scars has included nonsurgical therapies such as compression garments, silicone gel sheeting, massage, physical therapy, and corticosteroid. Surgical excision and scar revision represent more invasive scar management approaches that are often necessary for functional and aesthetic recovery.192Laser-based therapies provide additional treatment options for symptomatic hypertrophic scars. Two of the most common ones are the pulsed dye laser (PDL) and the ablative carbon dioxide (CO2) laser. The PDL causes photothermolysis of hemoglobin, resulting in coagulative necrosis.193 It obliterates small capillaries close to the skin and has had success treating congenital, cutaneous vascular malformations. The CO2 laser has been used for treatment of acne and recently has been gain-ing acceptance for its use to treat hypertrophic burn scars.194 It works by ablating microscopic columns of tissue to flatten scars and is also believed

1	treatment of acne and recently has been gain-ing acceptance for its use to treat hypertrophic burn scars.194 It works by ablating microscopic columns of tissue to flatten scars and is also believed to stimulate matrix metalloproteinases and other signaling pathways to induce collagen reorganization. Lasers are ultimately believed to help with scar remodeling and collagen reorganization. CO2 laser therapy has been shown to decrease symptoms associated with hypertrophic scarring, including scar appearance, pliability, contracture, neuropathic pain, and pruritus. A recent prospective study utilizing PDL and CO2 laser therapy demonstrated improved signs and symptoms of hypertrophic scars based on the Vancouver Scar Scale and the University of North Carolina 4P Scar Scale.195 Outpatient and office-based treatment sessions are tolerated well by most patients. There is wide practice variation on when to start ther-apy and the number of treatments, but the literature has general support for

1	treatment sessions are tolerated well by most patients. There is wide practice variation on when to start ther-apy and the number of treatments, but the literature has general support for starting treatment at 6 to 12 months and offering three treatments. More research is needed to determine the full potential of laser therapy to provide burn survivors a less inva-sive treatment of hypertrophic scars with improved symptoms and quality of life.Contractures are another long-term complication of burn injury that can result in significant morbidity. Contractures result from both wound contracture and scar contracture and prevents range of motion of a particular joint. Factors influenc-ing contracture development include burn depth and activation Brunicardi_Ch08_p0251-p0270.indd 26028/12/18 11:51 AM 261BURNSCHAPTER 8of dermal fibroblasts, myofibroblasts, fibrocytes, and helper T cells.196 Despite aggressive physiotherapy, contractures have been reported to develop in as many as

1	11:51 AM 261BURNSCHAPTER 8of dermal fibroblasts, myofibroblasts, fibrocytes, and helper T cells.196 Despite aggressive physiotherapy, contractures have been reported to develop in as many as one-third of burn patients. A recent study of 1865 patients demonstrated that the shoulder is the most affected joint, followed by the elbow, wrist, ankle, and knee.197 A similar study in the pediatric population yielded similar results. Gender, race, and %TBSA were associated with contracture development in the adult population. Age and length of stay in the ICU were associated with contracture develop-ment, severity of contracture, and total number of contractures in the pediatric population. Treatment of contractures includes both nonsurgical and surgical options, ranging from pressure garments and splints to laser therapy and contracture excision.Heterotopic ossification (HO) is another long-term mor-bidity associated with burn injury. HO is the pathologic devel-opment of lamellar bone in

1	and splints to laser therapy and contracture excision.Heterotopic ossification (HO) is another long-term mor-bidity associated with burn injury. HO is the pathologic devel-opment of lamellar bone in peripheral tissue. Its incidence has been reported to be between 1% and 3% of burn patients.198 Symptoms include decreased range of motion, pain, and swell-ing overlying the affected joints. Often times, the pathologic bone formation can be visualized radiographically with plain X-rays. Risk factors include >30% TBSA, arm burns, arm grafts, ventilator days, and number of trips to the operat-ing room.199 Treatment includes aggressive physiotherapy, NSAIDs, bisphosphonates, radiation therapy, and rarely surgi-cal excision. A risk scoring system has been developed to pre-dict which burn patients are at risk of developing HO based on admission criteria; however, further validation is warranted.200PSYCHOLOGICAL RECOVERYPsychological rehabilitation is equally important in the burn patient.

1	are at risk of developing HO based on admission criteria; however, further validation is warranted.200PSYCHOLOGICAL RECOVERYPsychological rehabilitation is equally important in the burn patient. Depression, posttraumatic stress disorder (PTSD), concerns about image, and anxiety about returning to society constitute predictable barriers to progress in both the inpa-tient and outpatient setting. Psychological distress occurs in as many as 38% of burn patients and persists in severity long after discharge.201 Rates of depression vary between 4% and 54% following injury, although these numbers vary dramatically based on the methodology used to diagnose depression.202 Still, depressive symptoms have been documented in up to 43% of patients 2 years following injury and have been associated with the female gender. Factors such as gender, extraversion, capac-ity for forgiveness, the event as a disaster or nondisaster, alcohol use, and peritraumatic emotional response have been identified as

1	the female gender. Factors such as gender, extraversion, capac-ity for forgiveness, the event as a disaster or nondisaster, alcohol use, and peritraumatic emotional response have been identified as contributing factors to PTSD.203 Despite the psychological impact of burn injury, many patients will be able to quickly return to work or school, and goals should be set accordingly. The involvement of clinical psychologists and psychiatrists is invaluable in providing guidance and coping techniques to lessen the significant psychological burden of burn injury.PREVENTIONDespite many areas of progress in prevention over the past cen-tury, burns continue to be a common source of injury. The cor-nerstone for burn prevention programs has been “The Five Step Process,” a systematic method of assessing, implementing, and evaluating burn hazards and subsequent intervention impact, and The Five E’s—engineering/environment, enforcement, education, emergency response, and economic initiative.204 It

1	implementing, and evaluating burn hazards and subsequent intervention impact, and The Five E’s—engineering/environment, enforcement, education, emergency response, and economic initiative.204 It has been shown that patients who live in environments opti-mal for sustaining burn injury have decreased knowledge of burn prevention strategies.205 Some successful initiatives have included school-based education and community-based inter-ventions targeting simple home safety measures. A 6-year study of second-graders demonstrated both shortand long-term retainment of information related to burn, fire, and life safety following multiple educational sessions. Smoke alarms are known to decrease mortality from structural fires, but not all homes are equipped with proper smoke alarms, particularly in low-income households. Mandatory smoke alarm installa-tion via community initiatives can be successful but seems to be contingent on close, long-term follow-up to ensure proper maintenance and

1	in low-income households. Mandatory smoke alarm installa-tion via community initiatives can be successful but seems to be contingent on close, long-term follow-up to ensure proper maintenance and function.206,207 Regulation of hot water heater temperatures has had some success and may be even more effec-tive in conjunction with community-based programs emphasiz-ing education and in-home inspections.208,209 A recent systematic review of prevention in lowand middle-income countries iden-tified multiple successful prevention programs.210 Burn profes-sionals have also demonstrated incomplete knowledge on best practices for fire safety and burn prevention.211 As such, appro-priate education of burn professionals participating in preven-tion programs is necessary.BURN DISASTERSAlthough rare, burn disasters can be devastating to those involved due to the sudden nature of the event, the difficulty of managing personnel and resources,212 a deficit of staff expe-rience in burn management, and

1	disasters can be devastating to those involved due to the sudden nature of the event, the difficulty of managing personnel and resources,212 a deficit of staff expe-rience in burn management, and relatively small resource availability for a potentially large number of patients.213 The American Burn Association has estimated that up to 30% of patients in mass casualty incidents suffer from burn injury.214 A recent review of the literature between 1990 and 2016 identi-fied 752 burn disasters world-wide, defined as an incident with ≥50 burn injuries and/or ≥30 burn-related deaths. The major-ity occurred in Asia and the Middle East and are thought to be secondary to rapid industrialization, inadequate fire-prevention strategies, and poor building codes. There was a significant increase in terrorist-related incidences from 2000 to 2015. Finally, the authors demonstrated that international adoption of the U.S. Health and Human Services guidelines on bed avail-ability for burns and trauma

1	incidences from 2000 to 2015. Finally, the authors demonstrated that international adoption of the U.S. Health and Human Services guidelines on bed avail-ability for burns and trauma dramatically underestimated the number of beds needed for burn disasters.215Preparedness is paramount for reacting quickly, efficiently, and effectively to a burn disaster. General surgeons not trained in burn care may feel uncomfortable longitudinally caring for severely burned patients following a burn disaster. However, due to resource limitations, they should be prepared to care for burn patients for the first 72 hours of resuscitation or until the patients can be transferred to a center that specializes in burn care. This will involve initial evaluation, resuscitation, and potential inter-ventions including central line placement, intubation, and escha-rotomies. Coordination for burn disasters should take place at three levels: institutional/intrafacility, interfacility/intrastate, and

1	including central line placement, intubation, and escha-rotomies. Coordination for burn disasters should take place at three levels: institutional/intrafacility, interfacility/intrastate, and interstate/regional. It is important to have multiple stakeholders involved in the development of a disaster plan, from the burn surgeons to the emergency department personnel to the emer-gency medical personnel who are first responders.216 Resource utilization can be guided by The American Burn Association Age/TBSA survival grid, which stratifies patients into benefit-to-resource categories (outpatient, high, low, and expectant) based on age and %TBSA.3 This allows providers to allocate resources during burn disasters based on the severity of injury Brunicardi_Ch08_p0251-p0270.indd 26128/12/18 11:51 AM 262BASIC CONSIDERATIONSPART Iand expected survival.214 Another important consideration is the involvement of nonburn hospitals in the planning for burn disas-ters as burn centers do not

1	11:51 AM 262BASIC CONSIDERATIONSPART Iand expected survival.214 Another important consideration is the involvement of nonburn hospitals in the planning for burn disas-ters as burn centers do not possess enough resources to be sole providers in these events. Multiple strategies have been adopted by local burn centers, including the development of a consortium of hospitals surrounding one burn center in New Jersey to allow transfer of patients when resource capacity is in jeopardy.217Interest in mass burn casualty disaster planning invariably includes a discussion of radiation burns. Radioactive material results in both acute injury from immediate exposure and more prolonged injury from delayed exposure to radioactive fallout or contamination. When a 10-kiloton nuclear bomb is deto-nated, people at a distance 0.7 miles from ground zero absorb 4.5 Gy. At 60 days, the median lethal radiation dose (LD50) is 3.5 Sv; with aggressive medical care, this dose might be doubled to nearly 7 Sv.

1	people at a distance 0.7 miles from ground zero absorb 4.5 Gy. At 60 days, the median lethal radiation dose (LD50) is 3.5 Sv; with aggressive medical care, this dose might be doubled to nearly 7 Sv. To put this in context, radiation expo-sure from a diagnostic CT of the chest or abdomen is 5 mSv, and the average annual background absorbed radiation dose is 3.6 mSv. Radiation is known to impact several organ systems and result in several syndromes based on increasing exposure doses. These syndromes include hematologic (1–8 Sv expo-sure), gastrointestinal (8–30 Sv exposure), and cardiovascular/neurologic syndromes (>30 Sv exposure), with the latter two being nonsurvivable.218-220After initial evaluation and decontamination by removing clothing, a useful way to estimate exposure is by determining the time to emesis. Patients who do not experience emesis within 4 hours of exposure are unlikely to have severe clinical effects. Emesis within 2 hours suggests a dose of at least 3 Sv, and

1	the time to emesis. Patients who do not experience emesis within 4 hours of exposure are unlikely to have severe clinical effects. Emesis within 2 hours suggests a dose of at least 3 Sv, and emesis within 1 hour suggests at least 4 Sv. The hematologic system follows a similar dose-dependent temporal pattern for predicting radiation exposure, mortality, and treatment. These have been determined based on the Armed Forces Radiobiology Research Institute’s Biodosimetry Assessment Tool, which can be downloaded from www.afrri.usuhs.mil.The combination of radiation exposure and burn wounds has the potential to increase mortality compared with traditional burns. Early closure of wounds before radiation depletes circu-lating lymphocytes may be needed for wound healing (which occurs within 48 hours). Also, in radiation injuries combined with burn or trauma, laboratory lymphocyte counts may be unreliable.218-221 A significant difference between burn/traumatic injuries and radiation injures is

1	Also, in radiation injuries combined with burn or trauma, laboratory lymphocyte counts may be unreliable.218-221 A significant difference between burn/traumatic injuries and radiation injures is that burn/traumatic injuries can result in higher mortality when not treated within hours.Decontamination and triage are vital to maximize the num-ber of survivors. Initial decontamination requires removal of clothing and washing wounds with water. Irrigation fluid should be collected to prevent radiation spread into the water supply. Work by many professional organizations, including the ABA, has focused on nationwide triage for disasters and will be vital to save as many lives as possible. Yet, it is likely that expectant or comfort care could be offered to more patients than typically seen in civilian hospitals, due to resource availability after the disaster.Finally, agents used in warfare—including white phos-phorus and sulfur mustard—can cause significant morbidity and mortality. White

1	civilian hospitals, due to resource availability after the disaster.Finally, agents used in warfare—including white phos-phorus and sulfur mustard—can cause significant morbidity and mortality. White phosphorus oxidizes when exposed to the atmosphere, creating the highly corrosive phosphorus pentox-ide. Absorption of even small amounts of white phosphorus can result in hypocalcemia and hyperphosphatemia and their subse-quent cardiac side effects. Treatment consists of removal of all clothing, irrigation with cool liquid (as phosphorus pentoxide liquidizes above 44°C), application of saline soaked gauze to prevent drying out and reignition, and potential surgical excision.222,223 Sulfur mustard, more commonly known as mus-tard gas, is another chemical warfare agent that can cause lesions similar to burn lesions. The gas infiltrates the skin surface, caus-ing degranulation of mast cells, leukocyte invasion, and sub-sequent blistering of the skin. Treatment includes scrubbing to

1	similar to burn lesions. The gas infiltrates the skin surface, caus-ing degranulation of mast cells, leukocyte invasion, and sub-sequent blistering of the skin. Treatment includes scrubbing to relieve the remaining skin of sulfur mustard, irrigation, and traditional burn therapy depending on the depth of the lesion.FUTURE AREAS OF STUDYIt has long been anecdotally noted that two patients of simi-lar ages and burn size may have very divergent responses to their burn injuries. Attention is being increasingly turned to identifying genetic differences among burn patients and how they affect response to injury. Specific allele variants have been linked with increased mortality in burned patients.224 It may be that genetic differences may predispose burn patients to severe sepsis,225 perhaps by downregulating the immune response.226 The Inflammation and the Host Response to Injury trial was a prospective, multicenter, federally funded study that aimed to define specific genetic pathways

1	by downregulating the immune response.226 The Inflammation and the Host Response to Injury trial was a prospective, multicenter, federally funded study that aimed to define specific genetic pathways that differ in the response to both burns and traumatic injury.227 Blood and tissue samples from a strictly defined patient population were analyzed using gene arrays to determine whether differential expression in cer-tain genetic pathways affects clinical outcomes.228 Although data from this study are still being analyzed, some interesting findings suggest that sepsis, trauma, and burn patients share common gene expression patterns, starting early after injury.229 These genes can upregulate proinflammatory pathways as well as disrupt antigen presentation pathways. A better understand-ing of these common genomic responses may allow for the tar-geted treatment of immunologic and signal pathways to help improve patient survival from burn injuries.Another area of increasing interest includes

1	common genomic responses may allow for the tar-geted treatment of immunologic and signal pathways to help improve patient survival from burn injuries.Another area of increasing interest includes integration of technology to burn size estimation and resuscitation. These include the use of smart device applications to assist with esti-mation of burn size and resuscitation recommendations.230-232 Further investigation is needed to determine the applicability of these models to burn estimation and resuscitation. However, as these models can include hourly updates and recommendations, they nudge clinicians to frequently reconsider fluid parameters during the critical stages of resuscitation.With the dramatic progress in improving survival follow-ing a major burn injury during the twentieth century, under-standing and addressing functional and psychological outcomes is critical to the well-being of burn survivors. Since 1993, the National Institute of Disability and Rehabilitation Research

1	under-standing and addressing functional and psychological outcomes is critical to the well-being of burn survivors. Since 1993, the National Institute of Disability and Rehabilitation Research has funded four burn model systems to identify long-term sequelae of burn injuries and to develop ways to improve outcomes for survivors. Ongoing outcome studies are crucial for dismantling barriers that our patients face in returning to their communities and to the workplace or to school.REFERENCESEntries highlighted in bright blue are key references. 1. Baxter CR, Shires T. Physiological response to crystal-loid resuscitation of severe burns. Ann N Y Acad Sci. 1968;150(3):874-894. doi:10.1111/j.1749-6632.1968.tb14738.xBrunicardi_Ch08_p0251-p0270.indd 26228/12/18 11:51 AM 263BURNSCHAPTER 8 2. Janzekovic Z. A new concept in the early excision and imme-diate grafting of burns. J Trauma. 1970;10(12):1103-1108. Available at: http://www.ncbi.nlm.nih.gov/pubmed/4921723. Accessed May 7,

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1	ABA Plan. J Burn Care Rehabil. 2005;26(2):102-106. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15756109. Accessed May 7, 2018. 215. Dai A, Carrougher GJ, Mandell SP, Fudem G, Gibran NS, Pham TN. Review of recent large-scale burn disasters world-wide in comparison to preparedness guidelines. J Burn Care Res. 2017;38(1):36-44. doi:10.1097/BCR.0000000000000441 216. Kearns RD, Conlon KM, Valenta AL, et al. Disaster planning: the basics of creating a burn mass casualty disaster plan for a burn center. J Burn Care Res. 2014;35(1):e1-e13. doi:10.1097/BCR.0b013e31829afe25 217. Conlon KM, Ruhren C, Johansen S, et al. Developing and implementing a plan for large-scale burn disaster response in New Jersey. J Burn Care Res. 35(1):e14-20. doi:10.1097/BCR.0b013e3182779b59 218. Wolbarst AB, Wiley AL, Nemhauser JB, Christensen DM, Hen-dee WR. Medical response to a major radiologic emergency: a primer for medical and public health practitioners. Radiology. 2010;254(3):660-677.

1	AB, Wiley AL, Nemhauser JB, Christensen DM, Hen-dee WR. Medical response to a major radiologic emergency: a primer for medical and public health practitioners. Radiology. 2010;254(3):660-677. doi:10.1148/radiol.09090330 219. Flynn DF, Goans RE. Nuclear terrorism: triage and medical management of radiation and combined-injury casualties. Surg Clin North Am. 2006;86(3):601-636. doi:10.1016/j.suc.2006.03.005 220. DiCarlo AL, Maher C, Hick JL, et al. Radiation injury after a nuclear detonation: medical consequences and the need for scarce resources allocation. Disaster Med Public Health Prep. 2011;5 Suppl 1:S32-S44. doi:10.1001/dmp.2011.17 221. Palmer JL, Deburghgraeve CR, Bird MD, Hauer-Jensen M, Kovacs EJ. Development of a combined radiation and burn injury model. J Burn Care Res. 2011;32(2):317-323. doi:10.1097/BCR.0b013e31820aafa9 222. Barillo DJ, Cancio LC, Goodwin CW. Treatment of white phosphorus and other chemical burn injuries at one burn center over a 51-year period. Burns.

1	doi:10.1097/BCR.0b013e31820aafa9 222. Barillo DJ, Cancio LC, Goodwin CW. Treatment of white phosphorus and other chemical burn injuries at one burn center over a 51-year period. Burns. 2004;30(5):448-452. doi:10.1016/j.burns.2004.01.032 223. Aviv U, Kornhaber R, Harats M, Haik J. The burning issue of white phosphorus: a case report and review of the literature. Disaster Mil Med. 2017;3:6. doi:10.1186/s40696-017-0034-y 224. Barber RC, Aragaki CC, Chang L-YE, et al. CD14-159 C allele is associated with increased risk of mortality after burn injury. Shock. 2007;27(3):232-237. doi:10.1097/01.shk.0000239770.10528.9a 225. Barber RC, Chang L-YE, Arnoldo BD, et al. Innate immu-nity SNPs are associated with risk for severe sepsis after burn injury. Clin Med Res. 2006;4(4):250-255. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17210974. Accessed May 7, 2018. 226. Moore CB, Medina MA, van Deventer HW, et al. Down-regulation of immune signaling genes in patients with large surface burn injury.

1	Accessed May 7, 2018. 226. Moore CB, Medina MA, van Deventer HW, et al. Down-regulation of immune signaling genes in patients with large surface burn injury. J Burn Care Res. 2007;28(6):879-887. doi:10.1097/BCR.0b013e318159a41e 227. Xiao W, Mindrinos MN, Seok J, et al. A genomic storm in critically injured humans. J Exp Med. 2011;208(13): 2581-2590. doi:10.1084/jem.20111354 228. Klein MB, Silver G, Gamelli RL, et al. Inflammation and the host response to injury: an overview of the multicenter study of the genomic and proteomic response to burn injury. J Burn Care Res. 2006;27(4):448-451. doi:10.1097/01.BCR.0000227477.33877.E6 229. Sood RF, Gibran NS, Arnoldo BD, et al. Early leukocyte gene expression associated with age, burn size, and inhalation injury in severely burned adults. J Trauma Acute Care Surg. 2016;80(2):250-257. doi:10.1097/TA.0000000000000905 230. Godwin Z, Tan J, Bockhold J, Ma J, Tran NK. Development and evaluation of a novel smart device-based application for burn

1	Acute Care Surg. 2016;80(2):250-257. doi:10.1097/TA.0000000000000905 230. Godwin Z, Tan J, Bockhold J, Ma J, Tran NK. Development and evaluation of a novel smart device-based application for burn assessment and management. Burns. 2015;41(4): 754-760. doi:10.1016/j.burns.2014.10.006 231. Barnes J, Duffy A, Hamnett N, et al. The Mersey Burns App: evolving a model of validation. Emerg Med J. 2015;32(8): 637-641. doi:10.1136/emermed-2013-203416 232. Wurzer P, Parvizi D, Lumenta DB, et al. Smartphone appli-cations in burns. Burns. 2015;41(5):977-989. doi:10.1016/j.burns.2014.11.010Brunicardi_Ch08_p0251-p0270.indd 26928/12/18 11:51 AM

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1	Wound HealingMunier Nazzal, Mohamed F. Osman, Heitham Albeshri, Darren B. Abbas, and Carol A. Angel 9chapterHISTORY OF WOUND HEALINGThe earliest accounts of wound healing date back to about 2000 b.c., when the Sumerians employed two modes of treatment: a spiritual method consisting of incantations, and a physical method of applying poultice-like materials to the wound. The Egyptians were the first to differentiate between infected and diseased wounds compared to noninfected wounds. The 1650 b.c. Edwin Smith Surgical Papyrus, a copy of a much older document, describes at least 48 different types of wounds. A later document (Ebers Papyrus, 1550 b.c.) relates the use of con-coctions containing honey (antibacterial properties), lint (absor-bent properties), and grease (barrier) for treating wounds. These same properties are still considered essential in contemporary daily wound management.The Greeks, equipped with the knowledge bequeathed by the Egyptians, went even further and classified

1	These same properties are still considered essential in contemporary daily wound management.The Greeks, equipped with the knowledge bequeathed by the Egyptians, went even further and classified wounds as acute or chronic in nature. Galen of Pergamum (120–201 a.d.), appointed as the doctor to the Roman gladiators, had an enormous number of wounds to deal with following gladiato-rial combats. He emphasized the importance of maintaining a moist environment to ensure adequate healing. It took almost 19 centuries for this important concept to be proven scientifi-cally, when it was shown that the epithelialization rate increases by 50% in a moist wound environment when compared to a dry wound environment.1The next major stride in the history of wound heal-ing was the discovery of antiseptics and their importance in reducing wound infections. Ignaz Philipp Semmelweis, a Hungarian obstetrician (1818–1865), noted that the inci-dence of puerperal fever was much lower if medical students,

1	and their importance in reducing wound infections. Ignaz Philipp Semmelweis, a Hungarian obstetrician (1818–1865), noted that the inci-dence of puerperal fever was much lower if medical students, following cadaver-dissection class and prior to attending child-birth, washed their hands with soap and hypochlorite. Louis Pasteur (1822–1895) was instrumental in dispelling the theory of spontaneous generation of germs and proving that germs existed in and were always introduced from the environment. Joseph Lister probably made one of the most significant con-tributions to wound healing. On a visit to Glasgow, Scotland, Lister noted that some areas of the city’s sewer system were less murky than the rest. He discovered that the water from pipes that were dumping waste containing carbolic acid (phenol) was clear. In 1865, Lister began soaking his surgical instruments in phenol and spraying the operating rooms, reducing the post-operative mortality rates from 50% to 15%. After attending an

1	(phenol) was clear. In 1865, Lister began soaking his surgical instruments in phenol and spraying the operating rooms, reducing the post-operative mortality rates from 50% to 15%. After attending an impressive lecture by Lister in 1876, Robert Wood Johnson left the meeting and began 10 years of research that would ulti-mately result in the production of an antiseptic dressing in the form of cotton gauze impregnated with iodoform. Since then, several other materials have been used to impregnate cotton gauze to achieve antisepsis.The 1960s and 1970s led to the development of polymeric dressings. These polymeric dressings can be custom made to specific parameters, such as permeability to gases (occlusive vs. semi-occlusive), varying degrees of absorbency, and different physical forms. Due to the ability to customize, the available range of materials that aid in wound care has grown exponen-tially to include an ever-expanding variety. Currently, the prac-tice of wound healing encompasses

1	the ability to customize, the available range of materials that aid in wound care has grown exponen-tially to include an ever-expanding variety. Currently, the prac-tice of wound healing encompasses manipulation and/or use of, among others, inflammatory cytokines, growth factors, and bio-engineered tissue. It is the combination of all these modali-ties that enables optimal wound healing. The role of organism in the perpetuation of nonhealing of chronic wounds 1History of Wound Healing 271Phases of Wound Healing 272Hemostasis and Inflammation / 272Proliferation / 273Matrix Synthesis / 274Maturation and Remodeling / 275Epithelialization / 275Role of Growth Factors in Normal Healing / 276Wound Contraction / 276Heritable Diseases of Connective Tissue 276Ehlers-Danlos Syndrome / 276Osteogenesis Imperfecta / 278Epidermolysis Bullosa / 279Acrodermatitis Enteropathica / 279Healing in Specific Tissues 279Gastrointestinal Tract / 279Bone / 280Cartilage / 281Tendon / 281Nerve / 281Fetal Wound

1	Imperfecta / 278Epidermolysis Bullosa / 279Acrodermatitis Enteropathica / 279Healing in Specific Tissues 279Gastrointestinal Tract / 279Bone / 280Cartilage / 281Tendon / 281Nerve / 281Fetal Wound Healing / 281Classification of Wounds 282Factors Affecting Wound Healing / 283Chronic Wounds / 289Excess Healing 291Treatment of Wounds 294Local Care / 294Antibiotics / 295Dressings / 295Skin Replacements / 296Cellular and Tissue-Based Products in Chronic Wound and Ulcer Management / 297Oxygen Therapy in Wound Healing / 299Biofilm and Chronic Wound Healing / 299Brunicardi_Ch09_p0271-p0304.indd 27101/03/19 4:49 PM 272has been better understood. Although wounds are classified under one entity, it is believed that they behave differently based on the host and organism involved. The future of wound healing is in “precision medicine” in which treatment strategies will be based on the host, the underlying mechanism, and the organisms in the wound bed and tissue.PHASES OF WOUND HEALINGWound

1	wound healing is in “precision medicine” in which treatment strategies will be based on the host, the underlying mechanism, and the organisms in the wound bed and tissue.PHASES OF WOUND HEALINGWound healing is a complex process of overlapping phases that is initiated by an injury or wound. Normal wound healing is divided into phases defined by characteristic cellular popula-tions and biochemical activities: (a) hemostasis and inflammation, (b) proliferation, and (c) maturation and remodeling. An approximate timeline of these events is depicted in Fig. 9-1. This sequence of events in most circumstances spans the time from injury to resolution of acute wounds. All wounds need to progress through this series of cellular and biochemical events that characterize the phases of healing in order to suc-cessfully reestablish tissue integrity. However, multiple factors can interfere with this sequence and can lead to lengthy healing (chronic wounds) or nonhealing.Hemostasis and

1	in order to suc-cessfully reestablish tissue integrity. However, multiple factors can interfere with this sequence and can lead to lengthy healing (chronic wounds) or nonhealing.Hemostasis and InflammationHemostasis precedes and initiates inflammation with the ensuing release of chemotactic factors from the wound site (Fig. 9-2A). Wounding by definition disrupts tissue integrity, leading to divi-sion of blood vessels and direct exposure of extracellular matrix to platelets. Exposure of subendothelial collagen to platelets results in platelet aggregation, degranulation, and activation of the coagulation cascade. Platelet α granules release a number of wound-active substances, such as platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), platelet-activating factor (PAF), fibronectin, and serotonin. In addition to achieving hemostasis, the fibrin clot serves as scaffolding for the migration into the wound of inflammatory cells such as poly-morphonuclear leukocytes

1	fibronectin, and serotonin. In addition to achieving hemostasis, the fibrin clot serves as scaffolding for the migration into the wound of inflammatory cells such as poly-morphonuclear leukocytes (PMNs, neutrophils) and monocytes.Cellular infiltration after injury follows a character-istic, predetermined sequence (see Fig. 9-1). PMNs are the 2first infiltrating cells to enter the wound site, peaking at 24 to 48 hours. Increased vascular permeability, local prostaglan-din release, and the presence of chemotactic substances such as complement factors, interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), TGF-β, platelet factor 4, or bacterial prod-ucts all stimulate neutrophil migration.The postulated primary role of neutrophils is phagocytosis of bacteria and tissue debris. PMNs are also a major source of cytokines early during inflammation, especially TNF-α3 which may have a significant influence on subsequent angiogenesis and collagen synthesis (see Fig. 9-2B). PMNs also release

1	a major source of cytokines early during inflammation, especially TNF-α3 which may have a significant influence on subsequent angiogenesis and collagen synthesis (see Fig. 9-2B). PMNs also release pro-teases such as collagenases, which participate in matrix and ground substance degradation in the early phase of wound heal-ing. Other than their role in limiting infections, these cells do not appear to play a role in collagen deposition or acquisition of mechanical wound strength. On the contrary, neutrophil fac-tors have been implicated in delaying the epithelial closure of wounds.4The second population of inflammatory cells that invades the wound consists of macrophages, which are recognized as being essential to successful healing.5 Derived from circulat-ing monocytes, macrophages achieve significant numbers in the wound by 48 to 96 hours post injury and remain present until wound healing is complete.Macrophages, like neutrophils, participate in wound debridement via phagocytosis and

1	significant numbers in the wound by 48 to 96 hours post injury and remain present until wound healing is complete.Macrophages, like neutrophils, participate in wound debridement via phagocytosis and contribute to microbial stasis via oxygen radical and nitric oxide synthesis (see Fig. 9-2B,C). The macrophage’s central function is activation and recruitment of other cells via mediators such as cytokines and growth fac-tors, as well as directly by cell-cell interaction and intercellular adhesion molecules (ICAM). By releasing such mediators as TGF-β, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), epithelial growth factor (EGF), and lac-tate, macrophages regulate cell proliferation, matrix synthesis, and angiogenesis.6,7 Macrophages also play a significant role in regulating angiogenesis and matrix deposition and remodeling (Table 9-1).T lymphocytes comprise another population of inflam-matory/immune cells that routinely invades the wound. Less Key

1	role in regulating angiogenesis and matrix deposition and remodeling (Table 9-1).T lymphocytes comprise another population of inflam-matory/immune cells that routinely invades the wound. Less Key Points1 Wound healing is a complex cellular and biochemical cascade that leads to restitution of integrity and function.2 All tissues heal by similar mechanisms, and the process undergoes phases of inflammation, cellular migration, pro-liferation, matrix deposition, and remodeling.3 Factors that impede normal healing include local, systemic, and technical conditions that the surgeon must take into account.4 Clinically, excess healing can be as significant a problem as impaired healing; genetic, technical, and local factors play a major role.5 Optimal outcome of acute wounds relies on complete eval-uation of the patient and of the wound and application of best practices and techniques.6 Antibiotics should be used only in the presence of infec-tion; colonization and contamination does not mean

1	of the patient and of the wound and application of best practices and techniques.6 Antibiotics should be used only in the presence of infec-tion; colonization and contamination does not mean there is infection.7 Dressing should facilitate the major changes taking place during healing to produce an optimally healed wound and take into consideration the comorbid conditions associated with chronic wounds.8 Cellular and tissue-based products are additional mea-sures, and these products might accelerate the rate of heal-ing but will not replace basic wound care.9 Chronic wounds have a decrease in oxygen supply to the wound, which contributes to delayed healing; oxygen therapy might aid in the healing of certain types of wounds.10 Biofilm is the term used for the bacterial growth on a chronic wound that is encapsulated by a protective layer made up of the host and bacterial proteins; this layer makes it difficult to heal chronic wounds and control infection.Brunicardi_Ch09_p0271-p0304.indd

1	that is encapsulated by a protective layer made up of the host and bacterial proteins; this layer makes it difficult to heal chronic wounds and control infection.Brunicardi_Ch09_p0271-p0304.indd 27201/03/19 4:49 PM 273WOUND HEALINGCHAPTER 9numerous than macrophages, T-lymphocyte numbers peak at about 1 week post injury and truly bridge the transition from the inflammatory to the proliferative phase of healing. Though known to be essential to wound healing, the role of lymphocytes in wound healing is not fully defined.8 A significant body of data supports the hypothesis that T lymphocytes play an active role in the modulation of the wound environment. Depletion of most wound T lymphocytes decreases wound strength and col-lagen content,9 while selective depletion of the CD8+ suppressor subset of T lymphocytes enhances wound healing. However, depletion of the CD4+ helper subset has no effect.10 Lympho-cytes also exert a downregulating effect on fibroblast collagen synthesis by

1	subset of T lymphocytes enhances wound healing. However, depletion of the CD4+ helper subset has no effect.10 Lympho-cytes also exert a downregulating effect on fibroblast collagen synthesis by cell-associated interferon IFN-γ, TNF-α, and IL-1. This effect is lost if the cells are physically separated, suggest-ing that extracellular matrix synthesis is regulated not only via soluble factors but also by direct cell-cell contact between lym-phocytes and fibroblasts.11ProliferationThe proliferative phase is the second phase of wound healing and roughly spans days 4 through 12 (see Fig. 9-2C). It is dur-ing this phase that tissue continuity is reestablished. Fibroblasts and endothelial cells are the last cell populations to infiltrate the healing wound, and the strongest chemotactic factor for fibroblasts is PDGF.12,13 Upon entering the wound environment, recruited fibroblasts first need to proliferate, and then become activated, to carry out their primary function of matrix synthesis

1	for fibroblasts is PDGF.12,13 Upon entering the wound environment, recruited fibroblasts first need to proliferate, and then become activated, to carry out their primary function of matrix synthesis remodeling. This activation is mediated mainly by the cytokines and growth factors released from wound macrophages.Fibroblasts isolated from wounds synthesize more colla-gen than nonwound fibroblasts, they proliferate less, and they actively carry out matrix contraction. Although it is clear that the cytokine-rich wound environment plays a significant role in this phenotypic alteration and activation, the exact mediators are only partially characterized.14,15 Additionally, lactate, which accumulates in significant amounts in the wound environment over time (∼10 mmol), is a potent regulator of collagen syn-thesis through a mechanism involving adenosine diphosphate (ADP)-ribosylation.16,17Endothelial cells also proliferate extensively during this phase of healing. These cells participate in

1	collagen syn-thesis through a mechanism involving adenosine diphosphate (ADP)-ribosylation.16,17Endothelial cells also proliferate extensively during this phase of healing. These cells participate in the formation of new capillaries (angiogenesis), a process essential to successful wound healing. Endothelial cells migrate from intact venules close to the wound. Their migration, replication, and new capil-lary tubule formation is under the influence of such cytokines and growth factors as TNF-α, TGF-β, and VEGF. Although Figure 9-1. The cellular, biochemical, and mechanical phases of wound healing.Phases of healing0246810121416MaturationProliferationInflammationmonths0246810121416Relative number of cellsNeutrophilsMacrophagesFibroblastsLymphocytes0246810121416Relative amount ofmatrix synthesisDays postwoundingCollagen ICollagen IIIWound-breakingstrengthFibronectinBrunicardi_Ch09_p0271-p0304.indd 27301/03/19 4:50 PM 274BASIC CONSIDERATIONSPART Imany cells produce VEGF, macrophages

1	postwoundingCollagen ICollagen IIIWound-breakingstrengthFibronectinBrunicardi_Ch09_p0271-p0304.indd 27301/03/19 4:50 PM 274BASIC CONSIDERATIONSPART Imany cells produce VEGF, macrophages represent a major source in the healing wound, and VEGF receptors are located specifically on endothelial cells.18,19Matrix SynthesisBiochemistry of Collagen. Collagen, the most abundant pro-tein in the body, plays a critical role in the successful comple-tion of adult wound healing. Its deposition, maturation, and subsequent remodeling are essential to the functional integrity of the wound.Although there are at least 18 types of collagen described, the main ones of interest to wound repair are types I and III. Type I collagen is the major component of extracellular matrix in skin. Type III, which is also normally present in skin, becomes more prominent and important during the repair process.Biochemically, each chain of collagen is composed of a glycine residue in every third position. The second

1	normally present in skin, becomes more prominent and important during the repair process.Biochemically, each chain of collagen is composed of a glycine residue in every third position. The second position in the triplet is made up of proline or lysine during the translation process. The polypeptide chain that is translated from mRNA contains approximately 1000 amino acid residues and is called protocollagen. Release of protocollagen into the endoplasmic reticulum results in the hydroxylation of proline and lysine by specific hydroxylases (Fig. 9-3). Prolyl hydroxylase requires oxygen and iron as cofactors, α-ketoglutarate as co-substrate, and ascorbic acid (vitamin C) as an electron donor. In the endo-plasmic reticulum, the protocollagen chain is also glycosylated by the linking of galactose and glucose at specific hydroxyly-sine residues. These steps of hydroxylation and glycosylation alter the hydrogen bonding forces within the chain, imposing steric changes that force the

1	of galactose and glucose at specific hydroxyly-sine residues. These steps of hydroxylation and glycosylation alter the hydrogen bonding forces within the chain, imposing steric changes that force the protocollagen chain to assume an α-helical configuration. Three α-helical chains entwine to form a right-handed superhelical structure called procollagen. At both ends, this structure contains nonhelical peptide domains called registration peptides. Although initially joined by weak, ionic bonds, the procollagen molecule becomes much stronger by the covalent cross-linking of lysine residues.Extracellularly, the nonhelical registration peptides are cleaved by a procollagen peptidase, and the procollagen strands undergo further polymerization and cross-linking. The resulting collagen monomer is further polymerized and cross-linked by the formation of intraand intermolecular covalent bonds.Collagen synthesis, as well as posttranslational modifica-tions, are highly dependent on systemic

1	is further polymerized and cross-linked by the formation of intraand intermolecular covalent bonds.Collagen synthesis, as well as posttranslational modifica-tions, are highly dependent on systemic factors such as an adequate oxygen supply; the presence of sufficient nutrients (amino acids and carbohydrates) and cofactors (vitamins and trace metals); and the local wound environment (vascular supply and lack of infection). Addressing these factors and reversing nutritional defi-ciencies can optimize collagen synthesis and deposition.Proteoglycan Synthesis. Glycosaminoglycans comprise a large portion of the “ground substance” that makes up granula-tion tissue. Rarely found free, they couple with proteins to form proteoglycans. The polysaccharide chain is made up of repeat-ing disaccharide units composed of glucuronic or iduronic acid and a hexosamine, which is usually sulfated. The disaccharide

1	to form proteoglycans. The polysaccharide chain is made up of repeat-ing disaccharide units composed of glucuronic or iduronic acid and a hexosamine, which is usually sulfated. The disaccharide EpidermisFibrinPlateletsClotNeutrophilsLymphocytesScabCollagenMacrophageEndothelialbudsDisruptedblood vesselRed bloodcellsDermisABCEpidermisDermisEpidermisDermisFibroblastFigure 9-2. The phases of wound healing viewed histologically. A. The hemostatic/inflammatory phase. B. Latter inflammatory phases reflecting infiltration by mononuclear cells and lympho-cytes. C. The proliferative phase, with associated angiogenesis and collagen synthesis.Table 9-1Macrophage activities during wound healingACTIVITYMEDIATORSPhagocytosisReactive oxygen speciesNitric oxideDebridementCollagenase, elastaseCell recruitment and activationGrowth factors: PDGF, TGF-β, EGF, IGFCytokines: TNF-α, IL-1, IL-6FibronectinMatrix synthesisGrowth factors: TGF-β, EGF, PDGFCytokines: TNF-α, IL-1, IFN-γEnzymes: arginase,

1	recruitment and activationGrowth factors: PDGF, TGF-β, EGF, IGFCytokines: TNF-α, IL-1, IL-6FibronectinMatrix synthesisGrowth factors: TGF-β, EGF, PDGFCytokines: TNF-α, IL-1, IFN-γEnzymes: arginase, collagenaseProstaglandinsNitric oxideAngiogenesisGrowth factors: FGF, VEGFCytokines: TNF-αNitric oxideEGF = epithelial growth factor; FGF = fibroblast growth factor; IGF = insulin-like growth factor; IFN-γ = interferon-γ; IL = interleukin; PDGF = platelet-derived growth factor; TGF-β = transforming growth factor-β; TNF-α = tumor necrosis factor-α; VEGF = vascular endothelial growth factor.Brunicardi_Ch09_p0271-p0304.indd 27401/03/19 4:50 PM 275WOUND HEALINGCHAPTER 9composition of proteoglycans varies from about 10 units in the case of heparin sulfate to as much as 2000 units in the case of hyaluronic acid.The major glycosaminoglycans present in wounds are dermatan and chondroitin sulfate. Fibroblasts synthesize these compounds, increasing their concentration greatly during the first 3

1	hyaluronic acid.The major glycosaminoglycans present in wounds are dermatan and chondroitin sulfate. Fibroblasts synthesize these compounds, increasing their concentration greatly during the first 3 weeks of healing. The interaction between collagen and proteoglycans is being actively studied. It is thought that the assembly of collagen subunits into fibrils and fibers is depen-dent upon the lattice provided by the sulfated proteoglycans. Furthermore, it appears that the extent of sulfation is critical in determining the configuration of the collagen fibrils. As scar collagen is deposited, the proteoglycans are incorporated into the collagen scaffolding. However, with scar maturation and collagen remodeling, the content of proteoglycans gradually diminishes.Maturation and RemodelingThe maturation and remodeling of the scar begins during the fibroplastic phase and is characterized by a reorganization of previously synthesized collagen. Collagen is broken down by matrix

1	RemodelingThe maturation and remodeling of the scar begins during the fibroplastic phase and is characterized by a reorganization of previously synthesized collagen. Collagen is broken down by matrix metalloproteinases (MMPs), and the net wound collagen content is the result of a balance between collagenolysis and collagen synthesis. There is a net shift toward collagen synthesis and eventually the reestablishment of extracellular matrix com-posed of a relatively acellular collagen-rich scar.Wound strength and mechanical integrity in the fresh wound are determined by both the quantity and quality of the newly deposited collagen. The deposition of matrix at the wound site follows a characteristic pattern: fibronectin and collagen type III constitute the early matrix scaffolding; glycosamino-glycans and proteoglycans represent the next significant matrix components; and collagen type I is the final matrix. By several weeks post injury, the amount of collagen in the wound reaches a

1	and proteoglycans represent the next significant matrix components; and collagen type I is the final matrix. By several weeks post injury, the amount of collagen in the wound reaches a plateau, but the tensile strength continues to increase for sev-eral more months.20 Fibril formation and fibril cross-linking result in decreased collagen solubility, increased strength, and increased resistance to enzymatic degradation of the collagen matrix. Fibrillin, a glycoprotein secreted by fibroblasts, is essen-tial for the formation of elastic fibers found in connective tis-sue. Scar remodeling continues for many (6 to 12) months post injury, gradually resulting in a mature, avascular, and acellular scar. The mechanical strength of the scar never achieves that of the uninjured tissue.There is a constant turnover of collagen in the extracellular matrix, both in the healing wound as well as during normal tissue homeostasis. Collagenolysis is the result of collagenase activity, a class of MMPs

1	constant turnover of collagen in the extracellular matrix, both in the healing wound as well as during normal tissue homeostasis. Collagenolysis is the result of collagenase activity, a class of MMPs that require activation. Both collagen synthesis and lysis are strictly controlled by cytokines and growth factors. Some factors affect both aspects of collagen remodeling. For example, TGF-β increases new collagen transcription and also decreases collagen breakdown by stimulating synthesis of tis-sue inhibitors of metalloproteinase.21 This balance of collagen deposition and degradation is the ultimate determinant of wound strength and integrity.EpithelializationEpithelialization is the final step in establishing tissue integrity. This process is characterized primarily by proliferation and migration of epithelial cells adjacent to the wound (Fig. 9-4). The process begins within 1 day of injury and is seen as thicken-ing of the epidermis at the wound edge. Marginal basal cells at the edge

1	of epithelial cells adjacent to the wound (Fig. 9-4). The process begins within 1 day of injury and is seen as thicken-ing of the epidermis at the wound edge. Marginal basal cells at the edge of the wound lose their firm attachment to the underly-ing dermis, enlarge, and begin to migrate across the surface of the provisional matrix. Fixed basal cells in a zone near the cut edge undergo a series of rapid mitotic divisions, and these cells appear to migrate by moving over one another in a leapfrog fashion until the defect is covered.22 Once the defect is bridged, the migrating epithelial cells lose their flattened appearance, become more columnar in shape, and increase their mitotic activity. Layering of the epithelium is reestablished, and the surface layer eventually keratinizes.23LysyloxidaseALDOL condensationCollagen genesPre-mRNAmRNA transcriptionmRNA processingmRNA translationOHOHTriple helix formationRibosomeon roughendoplasmicreticulumGolgiSecretory vesicleCell membraneProline

1	condensationCollagen genesPre-mRNAmRNA transcriptionmRNA processingmRNA translationOHOHTriple helix formationRibosomeon roughendoplasmicreticulumGolgiSecretory vesicleCell membraneProline and Lysine hydroxylationCollagen mRNASyndesineAldimineCH OCH OH OH OHOHOProcollagen peptidaseExtracellular space˜-1˜-1˜-2NonenzymaticNH2CCCCFigure 9-3. The steps of collagen synthesis. mRNA = messenger RNA.Brunicardi_Ch09_p0271-p0304.indd 27501/03/19 4:50 PM 276BASIC CONSIDERATIONSPART IReepithelialization is complete in less than 48 hours in the case of approximated incised wounds but may take sub-stantially longer in the case of larger wounds, where there is a significant epidermal/dermal defect. If only the epithelium and superficial dermis are damaged, such as occurs in split-thickness skin graft donor sites or in superficial second-degree burns, then repair consists primarily of reepithelialization with minimal or no fibroplasia and granulation tissue formation. The

1	skin graft donor sites or in superficial second-degree burns, then repair consists primarily of reepithelialization with minimal or no fibroplasia and granulation tissue formation. The stimuli for reepithelialization remain incompletely defined; however, it appears that the process is mediated by a combination of a loss of contact inhibition; exposure to constituents of the extracel-lular matrix, particularly fibronectin; and cytokines produced by immune mononuclear cells.24,25 In particular EGF, TGF-β, basic fibroblast growth factor (bFGF), PDGF, and IGF-1 have been shown to promote epithelialization.Role of Growth Factors in Normal HealingGrowth factors and cytokines are polypeptides produced in nor-mal and wounded tissue that stimulate cellular migration, pro-liferation, and function. They often are named for the cells from which they were first derived (e.g., platelet-derived growth fac-tor, PDGF) or for their initially identified function (e.g., fibro-blast growth factor, FGF).

1	They often are named for the cells from which they were first derived (e.g., platelet-derived growth fac-tor, PDGF) or for their initially identified function (e.g., fibro-blast growth factor, FGF). These names are often misleading because growth factors have been demonstrated to have mul-tiple functions. Most growth factors are extremely potent and produce significant effects in nanomolar concentrations.They may act in an autocrine manner (where the growth factor acts on the cell producing it), a paracrine manner (by release into the extracellular environment, where it acts on the immediately neighboring cells), or in an endocrine manner (where the effect of the substance is distant to the site of release, and the substance is carried to the effector site through the blood stream). In addition to the concentration of the growth factor, the timing of release is as important to determine their effective-ness. As these growth factors exert their effects by cell-surface receptor binding,

1	to the concentration of the growth factor, the timing of release is as important to determine their effective-ness. As these growth factors exert their effects by cell-surface receptor binding, the appropriate receptor on the responding cells must be present at the time of release in order for the bio-logic effect to occur. Table 9-2 summarizes the principal growth factors found in healing wounds and their known effects on cells participating in the healing process. Growth factors have diver-gent actions on different cells; they can be chemoattractive to one cell type while stimulating replication of a different cell type. Little is known about the ratio of growth factor concentra-tions, which may be as important as the absolute concentration of individual growth factors.Growth factors act on cells via surface receptor binding. Various receptor types have been described, such as ion chan-nels, G-protein linked, or enzyme linked. The response elicited in the cell is usually one of

1	act on cells via surface receptor binding. Various receptor types have been described, such as ion chan-nels, G-protein linked, or enzyme linked. The response elicited in the cell is usually one of phosphorylation or dephosphoryla-tion of second-messenger molecules through the action of phos-phatases or kinases, resulting in activation or deactivation of proteins in the cytosol or nucleus of the target cell. Phosphoryla-tion of nuclear proteins is followed by the initiation of transcrip-tion of target genes.26 The signal is stopped by internalization of the receptor-ligand complex.Wound ContractionAll wounds undergo some degree of contraction. For wounds that do not have surgically approximated edges, the area of the wound will be decreased by this action (healing by secondary intention). The myofibroblast has been postulated as the major cell responsible for contraction, and it differs from the normal fibroblast in that it possesses a cytoskeletal structure. Typically this cell

1	The myofibroblast has been postulated as the major cell responsible for contraction, and it differs from the normal fibroblast in that it possesses a cytoskeletal structure. Typically this cell contains α-smooth muscle actin in thick bundles called stress fibers, giving myofibroblasts contractile capability.27 The α-smooth muscle actin is undetectable until day 6, and then it is increasingly expressed for the next 15 days of wound healing.28 After 4 weeks, this expression fades, and the cells are believed to undergo apoptosis.29 A puzzling point is that the identification of myofibroblasts in the wound does not correspond directly to the initiation of wound contraction, which starts almost immedi-ately after injury. It is believed that fibroblasts might play a role in contraction. In vitro, fibroblasts placed in a collagen lattice actively move in the lattice and contract it without expressing stress fibers. It is postulated that the movement of cells with concomitant reorganization

1	fibroblasts placed in a collagen lattice actively move in the lattice and contract it without expressing stress fibers. It is postulated that the movement of cells with concomitant reorganization of the cytoskeleton is responsible for contraction.30HERITABLE DISEASES OF CONNECTIVE TISSUEHeritable diseases of connective tissue consist of a group of generalized, genetically determined, primary disorders of one of the elements of connective tissue: collagen, elastin, or muco-polysaccharide. Five major types, Ehlers-Danlos syndrome, Marfan’s syndrome, osteogenesis imperfecta, epidermolysis bullosa, and acrodermatitis enteropathica, will be discussed, as each provides unique challenges to the surgeon.Ehlers-Danlos SyndromeEhlers-Danlos syndrome (EDS) is a group of 10 disorders that present as a defect in collagen formation. Over half of the Hair follicleSweat glandBlood vesselsWoundEpidermisDermisRegeneratingepitheliumEpithelialislandEpidermisDermisHair follicleSweat glandBlood

1	present as a defect in collagen formation. Over half of the Hair follicleSweat glandBlood vesselsWoundEpidermisDermisRegeneratingepitheliumEpithelialislandEpidermisDermisHair follicleSweat glandBlood vesselsEpidermisDermisFigure 9-4. The healing by epithelialization of superficial cutane-ous wounds.Brunicardi_Ch09_p0271-p0304.indd 27601/03/19 4:50 PM 277WOUND HEALINGCHAPTER 9Table 9-2Growth factors participating in wound healingGROWTH FACTORWOUND CELL ORIGINCELLULAR AND BIOLOGIC EFFECTSPDGFPlatelets, macrophages, monocytes, smooth muscle cells, endothelial cellsChemotaxis: fibroblasts, smooth muscle, monocytes, neutrophilsMitogenesis: fibroblasts, smooth muscle cellsStimulation of angiogenesisStimulation of collagen synthesisEnhance reepithelizationModulate tissue remodelingFGFFibroblasts, endothelial cells, keratinocytes, smooth muscle cells, chondrocytesStimulation of angiogenesis (by stimulation of endothelial cell proliferation and migration)Mitogenesis: mesoderm and

1	endothelial cells, keratinocytes, smooth muscle cells, chondrocytesStimulation of angiogenesis (by stimulation of endothelial cell proliferation and migration)Mitogenesis: mesoderm and neuroectodermHGFFibroblastsStimulates fibroblasts, keratinocytes, chondrocytes, myoblastsSuppresses inflammation, granulation tissue formation, angiogenesis, reepithelializationKeratinocyte growth factorKeratinocytes, fibroblastsSignificant homology with FGF; stimulates keratinocytesEGFPlatelets, macrophages, monocytes (also identified in salivary glands, duodenal glands, kidney, and lacrimal glands)Stimulates proliferation and migration of all epithelial cell typesTGF-αKeratinocytes, platelets, macrophagesHomology with EGF; binds to EGF receptorMitogenic and chemotactic for epidermal and endothelial cellsTGF-β (three isoforms: β1, β2, β3)Platelets, T lymphocytes, macrophages, monocytes, neutrophils, fibroblasts, keratinocytesStimulates angiogenesisStimulates leukocyte chemotaxisTGF-β1 stimulates wound

1	(three isoforms: β1, β2, β3)Platelets, T lymphocytes, macrophages, monocytes, neutrophils, fibroblasts, keratinocytesStimulates angiogenesisStimulates leukocyte chemotaxisTGF-β1 stimulates wound matrix production (fibronectin, collagen glycosaminoglycans); regulation of inflammationTGF-β3 inhibits scar formationInsulin-like growth factors (IGF-1, IGF-2)Platelets (IGF-1 in high concentrations in liver; IGF-2 in high concentrations in fetal growth); likely the effector of growth hormone actionPromote protein/extracellular matrix synthesisIncrease membrane glucose transportVascular endothelial growth factorMacrophages, fibroblasts, endothelial cells, keratinocytesMitogen for endothelial cells (not fibroblasts)Stimulates angiogenesisProinflammatoryIL-1IL-4IL-6ActivinAngiopoitein-1/-2CX3CL1Macrophages, leukocytes, keratinocytes, fibroblastsLeukocytesFibroblasts, endothelial cells, macrophages, keratinocytesKeratinocytes, fibroblastsEndothelial cellsMacrophages, endothelial

1	leukocytes, keratinocytes, fibroblastsLeukocytesFibroblasts, endothelial cells, macrophages, keratinocytesKeratinocytes, fibroblastsEndothelial cellsMacrophages, endothelial cellsProinflammatoryStimulates angiogenesis, reepithelialization, tissue remodelingEnhances collagen synthesisStimulates inflammation, angiogenesis, reepithelialization, collagen deposition, tissue remodelingStimulates granulation tissue formation, keratinocyte differentiation, reepithelializationStimulates angiogenesisStimulates inflammation, angiogenesis, collagen depositionGranulocyte-macrophage colony-stimulating factorMacrophage/monocytes, endothelial cells, fibroblastsStimulates macrophage differentiation/proliferationCX3CL1 = chemokine (C-X3-C motif) ligand; EGF = epidermal growth factor; FGF = fibroblast growth factor; HGF = hepatocyte growth factor; IL = interleukin; PDGF = platelet-derived growth factor; TGF = transforming growth factor.Brunicardi_Ch09_p0271-p0304.indd 27701/03/19 4:50 PM 278BASIC

1	factor; HGF = hepatocyte growth factor; IL = interleukin; PDGF = platelet-derived growth factor; TGF = transforming growth factor.Brunicardi_Ch09_p0271-p0304.indd 27701/03/19 4:50 PM 278BASIC CONSIDERATIONSPART Iaffected patients manifest genetic defects encoding α-chains of collagen type V, causing it to be either quantitatively or struc-turally defective. These changes lead to “classic” EDS with phenotypic findings that include thin, friable skin with promi-nent veins, easy bruising, poor wound healing, atrophic scar formation, recurrent hernias, and hyperextensible joints. Gas-trointestinal problems include bleeding, hiatal hernia, intesti-nal diverticulae, and rectal prolapse. Small blood vessels are fragile, making suturing difficult during surgery. Large vessels may develop aneurysms, varicosities, or arteriovenous fistulas or may spontaneously rupture.31-33 Table 9-3 presents a descrip-tion of EDS subtypes, including a recently recognized autoso-mal recessive form

1	aneurysms, varicosities, or arteriovenous fistulas or may spontaneously rupture.31-33 Table 9-3 presents a descrip-tion of EDS subtypes, including a recently recognized autoso-mal recessive form characterized by tenascin-X deficiency. The defect is a quantitative loss of protein, resulting in phenotypic changes similar to those observed in other types of EDS.EDS must be considered in every child with recurrent her-nias and coagulopathy, especially when accompanied by platelet abnormalities and low coagulation factor levels. Inguinal her-nias in these children resemble those seen in adults. Great care should be taken to avoid tearing the skin and fascia. The trans-versalis fascia is thin, and the internal ring is greatly dilated. Like adults, hernia repair in these patients with the use of mesh or felt may result in a lower incidence of recurrence.34Closing wounds in patients with EDS might represent a major challenge to the surgeon. Dermal wounds should be closed in two layers,

1	use of mesh or felt may result in a lower incidence of recurrence.34Closing wounds in patients with EDS might represent a major challenge to the surgeon. Dermal wounds should be closed in two layers, approximated with the sutures under ten-sion, and the stitches should be left in place twice as long as usual. In addition, external fixation with adhesive tape can help reinforce the scar and prevent stretching.35Marfan’s Syndrome. Patients with Marfan’s syndrome have tall stature, arachnodactyly, lax ligaments, myopia, sco-liosis, pectus excavatum, and aneurysm of the ascending aorta. Patients who suffer from this syndrome also are prone to her-nias. Skin may be hyperextensible but shows no delay in wound healing.36,37The genetic defect associated with Marfan’s syndrome is a mutation in the FBN1 gene, which encodes for fibrillin. Pre-viously, it was thought that structural alteration of the micro-fibrillar system was responsible for the phenotypic changes seen with the disease. However,

1	FBN1 gene, which encodes for fibrillin. Pre-viously, it was thought that structural alteration of the micro-fibrillar system was responsible for the phenotypic changes seen with the disease. However, recent research indicates an intricate role that FBN1 gene products play in TGF-β signal-ing. These extracellular matrix molecules normally bind and regulate TGF-β signaling; abnormal FBN1 gene function may cause an increase in TGF-β signaling, particularly in the aortic wall.38Osteogenesis ImperfectaPatients with osteogenesis imperfecta (OI) have brittle bones, osteopenia, low muscle mass, hernias, and ligament and joint laxity. OI is a result of a mutation in type I collagen. Mutations in prolidase, an enzyme responsible for cleaving c-terminal pro-line and hydroxyproline, may have a role in the disease. There are four major OI subtypes with mild to lethal manifestations. Patients experience dermal thinning and increased bruisability. Scarring is normal, and the skin is not

1	a role in the disease. There are four major OI subtypes with mild to lethal manifestations. Patients experience dermal thinning and increased bruisability. Scarring is normal, and the skin is not hyperextensible. Surgery can be successful but difficult in these patients, as the bones fracture easily under minimal stress.31,34 Table 9-4 lists the vari-ous features associated with the clinical subtypes of OI.Table 9-3Clinical, genetic, and biochemical aspects of Ehlers-Danlos subtypesTYPECLINICAL FEATURESINHERITANCEBIOCHEMICAL DEFECTISkin: soft, hyperextensible, easy bruising, fragile, atrophic scars; hypermobile joints; varicose veins; premature birthsADNot knownIISimilar to type I, except less severeADNot knownIIISkin: soft, not hyperextensible, normal scars; small and large joint hypermobilityADNot knownIVSkin: thin, translucent, visible veins, normal scarring, no hyperextensibility; no joint hypermobility; arterial, bowel, and uterine ruptureADType III collagen defectVSimilar to

1	knownIVSkin: thin, translucent, visible veins, normal scarring, no hyperextensibility; no joint hypermobility; arterial, bowel, and uterine ruptureADType III collagen defectVSimilar to type IIXLRNot knownVISkin: hyperextensible, fragile, easy bruising; hypermobile joints; hypotonia; kyphoscoliosisARLysyl hydroxylase deficiencyVIISkin: soft, mild hyperextensibility, no increased fragility; extremely lax joints with dislocationsADType I collagen gene defectVIIISkin: soft, hyperextensible, easy bruising, abnormal scars with purple discoloration; hypermobile joints; generalized periodontitisADNot knownIXSkin: soft, lax; bladder diverticula and rupture; limited pronation and supination; broad clavicle; occipital hornsXLRLysyl oxidase defect with abnormal copper useXSimilar to type II with abnormal clotting studiesARFibronectin defectTNxHypermobile joints, skin fragilityARAbsence of tenascin X proteinAD = autosomal dominant; AR = autosomal recessive; XLR = X-linked recessive.Reproduced with

1	clotting studiesARFibronectin defectTNxHypermobile joints, skin fragilityARAbsence of tenascin X proteinAD = autosomal dominant; AR = autosomal recessive; XLR = X-linked recessive.Reproduced with permission from Cohen IK, Diegelmann RF, Lindblad WJ: Wound Healing: Biochemical and Clinical Aspects. Philadelphia, PA: WB Saunders/Elsevier; 1992.Brunicardi_Ch09_p0271-p0304.indd 27801/03/19 4:50 PM 279WOUND HEALINGCHAPTER 9Epidermolysis BullosaEpidermolysis bullosa (EB) is classified into four major sub-types: EB simplex, junctional EB, dystrophic EB, and Kindler’s syndrome. The first three are determined by location in various skin layers; the last can present as multiple blisters throughout different layers of skin. There are identified genetic defects for each subtype, but the overall phenotype is remarkably similar. The disease manifestations include impairment in tissue adhe-sion within the epidermis, basement membrane, or dermis, resulting in tissue separation and blistering with

1	is remarkably similar. The disease manifestations include impairment in tissue adhe-sion within the epidermis, basement membrane, or dermis, resulting in tissue separation and blistering with minimal trauma. Characteristic features of EB are blistering and ulceration. The recessively inherited dystrophic type is characterized by defects in the COL7A1 gene, encoding type 7 collagen, important for connecting the epidermis to the dermis, and therefore pheno-typically resulting in blistering.39 Management of nonhealing wounds in patients with EB is a challenge, as their nutritional status is compromised because of oral erosions and esophageal obstruction. Surgical interventions include esophageal dilata-tion and gastrostomy tube placement. Dermal incisions must be meticulously placed to avoid further trauma to skin.34,40 The skin requires nonadhesive pads covered by a “bulky” dressing to avoid blistering.Acrodermatitis EnteropathicaAcrodermatitis enteropathica (AE) is an autosomal

1	avoid further trauma to skin.34,40 The skin requires nonadhesive pads covered by a “bulky” dressing to avoid blistering.Acrodermatitis EnteropathicaAcrodermatitis enteropathica (AE) is an autosomal recessive disease of children that causes an inability to absorb sufficient zinc from breast milk or food. The AE mutation affects zinc uptake in the intestine by preventing zinc from binding to the cell surface and its translocation into the cell. Recently, the genetic defect has been localized on chromosome 8q24.3 identi-fied as the SLC39A4 gene, expressed in the intestinal lumen and upregulated based on zinc stores.41 Zinc deficiency is associated with impaired granulation tissue formation, as zinc is a neces-sary cofactor for DNA polymerase and reverse transcriptase, and its deficiency may impair healing due to inhibition of cell proliferation.AE is characterized by impaired wound healing as well as erythematous pustular dermatitis involving the extremities and the areas around the

1	may impair healing due to inhibition of cell proliferation.AE is characterized by impaired wound healing as well as erythematous pustular dermatitis involving the extremities and the areas around the bodily orifices. Diagnosis is confirmed by the presence of an abnormally low blood zinc level (<100 mg/dL). Oral supplementation with 100 to 400 mg zinc sulfate orally per day is curative for impaired healing.42,43HEALING IN SPECIFIC TISSUESGastrointestinal TractRepair and healing of the gastrointestinal tract is essential for the normal functions of the GI tract such as absorptive, bar-rier, and motor functions. Full-thickness GI injury healing remains an unresolved clinical issue. Healing of full-thickness GI wounds begins with a surgical or mechanical reapposition of the bowel ends, which is most often the initial step in the repair process. Sutures or staples are principally used, although various other means such as buttons, plastic tubes, and various wrappings have been attempted

1	is most often the initial step in the repair process. Sutures or staples are principally used, although various other means such as buttons, plastic tubes, and various wrappings have been attempted with variable success. Failure of healing results in dehiscence, leaks, and fistulas, which carry significant morbidity and mortality. Conversely, excessive heal-ing can be just as troublesome, resulting in stricture formation and stenosis of the lumen.The gross anatomic features of the GI tract are remarkably constant throughout most of its length. Within the lumen, the epithelium is supported by the lamina propria and underlying muscularis mucosa. The submucosa lies radially and circumfer-entially outside of these layers, is comprised of abundant col-lagenous and elastic fibers, and supports neural and vascular structures. Further toward the peritoneal surface of the bowel are the inner and outer muscle layers and ultimately a peritoneal extension, the serosa. The submucosa is the layer

1	neural and vascular structures. Further toward the peritoneal surface of the bowel are the inner and outer muscle layers and ultimately a peritoneal extension, the serosa. The submucosa is the layer that imparts the greatest tensile strength and greatest suture-holding capac-ity, a characteristic that should be kept in mind during surgical repair of the GI tract. Additionally, serosal healing is essential for quickly achieving a watertight seal from the luminal side of the bowel. The importance of the serosa is underscored by the significantly higher rates of anastomotic failure observed clinically in segments of bowel that are extraperitoneal and lack serosa (i.e., the esophagus and rectum).Injuries to all parts of the GI tract undergo the same sequence of healing as cutaneous wounds. However, there are some significant differences (Table 9-5). Mesothelial (serosal) and mucosal healing can occur without scarring. The early integ-rity of the anastomosis is dependent on formation of a

1	there are some significant differences (Table 9-5). Mesothelial (serosal) and mucosal healing can occur without scarring. The early integ-rity of the anastomosis is dependent on formation of a fibrin seal on the serosal side, which achieves water tightness, and on the suture-holding capacity of the intestinal wall, particularly the sub-mucosal layer. There is a significant decrease in marginal strength during the first week due to an early and marked collagenolysis. The lysis of collagen is carried out by collagenase derived from neutrophils, macrophages, and intraluminal bacteria. Recently, it has been shown that strains of Pseudomonas aeruginosa undergo phenotypic shifts characterized by higher collagenase secretion in an injured/anastomosed bowel environment.44 Collagenase activ-ity occurs early in the healing process, and during the first 3 to 5 days, collagen breakdown far exceeds collagen synthesis. The integrity of the anastomosis represents equilibrium between col-lagen lysis,

1	early in the healing process, and during the first 3 to 5 days, collagen breakdown far exceeds collagen synthesis. The integrity of the anastomosis represents equilibrium between col-lagen lysis, which occurs early, and collagen synthesis, which takes a few days to initiate (Fig. 9-5). Collagenase is expressed following injury in all segments of the GI tract, but it is much more marked in the colon compared to the small bowel. Colla-gen synthesis in the GI tract is carried out by both fibroblasts and smooth muscle cells. Colon fibroblasts produce greater amounts of collagen than skin fibroblasts, reflecting different phenotypic features, as well as different responses to cytokines and growth factors among these different fibroblast populations. Ultimate anastomotic strength is not always related to the absolute amount of collagen, and the structure and arrangement of the collagen matrix may be more important.45Table 9-4Osteogenesis imperfecta: clinical and genetic featuresTYPECLINICAL

1	to the absolute amount of collagen, and the structure and arrangement of the collagen matrix may be more important.45Table 9-4Osteogenesis imperfecta: clinical and genetic featuresTYPECLINICAL FEATURESINHERITANCEIMild bone fragility, blue scleraDominantII“Prenatal lethal”; crumpled long bones, thin ribs, dark blue scleraDominantIIIProgressively deforming; multiple fractures; early loss of ambulationDominant/recessiveIVMild to moderate bone fragility; normal or gray sclera; mild short statureDominantReproduced with permission from Cohen IK, Diegelmann RF, Lindblad WJ: Wound Healing: Biochemical and Clinical Aspects. Philadelphia, PA: WB Saunders/Elsevier; 1992.Brunicardi_Ch09_p0271-p0304.indd 27901/03/19 4:50 PM 280BASIC CONSIDERATIONSPART ITechnical Considerations. Traditional teaching holds that in order for an anastomosis to heal without complications it must be tension free, have an adequate blood supply, receive adequate nutrition, and be free of sepsis. Although sound

1	teaching holds that in order for an anastomosis to heal without complications it must be tension free, have an adequate blood supply, receive adequate nutrition, and be free of sepsis. Although sound prin-ciples for all wound healing, there are several considerations unique to GI anastomotic healing. From a technical viewpoint, the ideal method of suturing two ends of bowel together has not yet been identified. Although debate exists concerning meth-ods of creating an anastomosis, clinically there has been no convincing evidence that any given technique has any advan-tage over another (i.e., hand-sutured vs. stapled, continuous vs. interrupted sutures, absorbable vs. nonabsorbable sutures, or singlevs. two-layer closure). A recent meta-analysis revealed that stapled ileocolic anastomoses have fewer leak rates than hand-constructed ones, but this might not apply to colo-colic or small bowel anastomoses.46Fluid third spacing, tissue edema, and increased intraab-dominal pressure

1	have fewer leak rates than hand-constructed ones, but this might not apply to colo-colic or small bowel anastomoses.46Fluid third spacing, tissue edema, and increased intraab-dominal pressure secondary to overzealous fluid administration can result in blood flow compromise in small vessels at the edge of anastomosis and thus interfere with GI healing.47,48BoneFollowing any type of injury to bone, several changes take place at the site of injury to restore structural and functional integrity. Most of the phases of healing resemble those observed in der-mal healing, but some notable individual characteristics apply to Table 9-5Comparison of wound healing in the gastrointestinal tract and skin GI TRACTSKINWound environmentpHVaries throughout GI tract in accordance with local exocrine secretionsUsually constant except during sepsis or local infection MicroorganismsAerobic and anaerobic, especially in the colon and rectum; problematic if they contaminate the peritoneal cavitySkin

1	secretionsUsually constant except during sepsis or local infection MicroorganismsAerobic and anaerobic, especially in the colon and rectum; problematic if they contaminate the peritoneal cavitySkin commensals rarely cause problems; infection usually results from exogenous contamination or hematogenous spread Shear stressIntraluminal bulk transit and peristalsis exert distracting forces on the anastomosisSkeletal movements may stress the suture line but pain usually acts as a protective mechanism preventing excess movement Tissue oxygenationDependent on intact vascular supply and neocapillary formationCirculatory transport of oxygen as well as diffusionCollagen synthesisCell typeFibroblasts and smooth muscle cellsFibroblasts Lathyrogensd-Penicillamine has no effect on collagen cross-linkingSignificant inhibition of cross-linking with decreased wound strength SteroidsContradictory evidence exists concerning their negative effect on GI healing; increased abscess in the anastomotic line

1	inhibition of cross-linking with decreased wound strength SteroidsContradictory evidence exists concerning their negative effect on GI healing; increased abscess in the anastomotic line may play a significant roleSignificant decrease in collagen accumulationCollagenase activity—Increased presence throughout GI tract after transection and reanastomosis; during sepsis, excess enzyme may promote dehiscence by decreasing suture-holding capacity of tissueNot as significant a role in cutaneous woundsWound strength—Rapid recovery to preoperative level.Less rapid than GI tissueScar formationAgeDefinite scarring seen in fetal wound sitesUsually heals without scar formation in the fetusDaysTensile strengthResultant curveStrength of newcollagen increaseswith synthesisStrength of collagendecreases due tolysisFigure 9-5. Diagrammatic representation of the concept of GI wound healing as a fine balance between collagen synthesis and collagenolysis. The “weak” period when collagenolysis exceeds

1	due tolysisFigure 9-5. Diagrammatic representation of the concept of GI wound healing as a fine balance between collagen synthesis and collagenolysis. The “weak” period when collagenolysis exceeds col-lagen synthesis can be prolonged or exacerbated by any factors that upset the equilibrium. (Reproduced with permission from Dunphy JE: Fundamentals of Wound Management in Surgery. New York, NY: Chirurgecom, Inc.; 1976.)Brunicardi_Ch09_p0271-p0304.indd 28001/03/19 4:50 PM 281WOUND HEALINGCHAPTER 9bone injuries. The initial stage of hematoma formation consists of an accumulation of blood at the fracture site, which also con-tains devitalized soft tissue, dead bone, and necrotic marrow. The next stage accomplishes the liquefaction and degradation of nonviable products at the fracture site. The normal bone adja-cent to the injury site can then undergo revascularization, with new blood vessels growing into the fracture site. This is similar to the formation of granulation in soft tissue.

1	The normal bone adja-cent to the injury site can then undergo revascularization, with new blood vessels growing into the fracture site. This is similar to the formation of granulation in soft tissue. The symptoms associated with this stage are characteristic of inflammation, with clinical evidence of swelling and erythema.Three to 4 days following injury, soft tissue forms a bridge between the fractured bone segments in the next stage (soft callus stage). The soft tissue is deposited where neovasculariza-tion has taken place and serves as an internal splint, preventing damage to the newly laid blood vessels and achieving a fibrocar-tilaginous union. The soft callus is formed externally along the bone shaft and internally within the marrow cavity. Clinically, this phase of healing is characterized by the cessation of pain and inflammatory signs.The next phase consists of mineralization of the soft callus and conversion to bone (hard callus stage). This may take up to 2 to 3 months and

1	by the cessation of pain and inflammatory signs.The next phase consists of mineralization of the soft callus and conversion to bone (hard callus stage). This may take up to 2 to 3 months and leads to complete bony union. The bone is now considered strong enough to allow weight bearing and will appear healed on radiographs. Then remodeling phase follows, in which the excessive callus is reabsorbed and the marrow cav-ity is recanalized. Remodeling allows for the correct transmis-sion of forces and restores the contours of the bone.As in dermal healing, the process of osseous union is mediated by soluble growth factors and cytokines. The most extensively studied group is the bone morphogenic proteins (BMPs), which belong to the TGF-β superfamily. By stimulat-ing the differentiation of mesenchymal cells into chondroblasts and osteoblasts, BMPs directly affect bone and cartilage repair. Other growth factors such as PDGF, TGF-β, TNF-α, and bFGF also participate in bony repair by mediating

1	cells into chondroblasts and osteoblasts, BMPs directly affect bone and cartilage repair. Other growth factors such as PDGF, TGF-β, TNF-α, and bFGF also participate in bony repair by mediating the inflammatory and proliferative phases of healing.CartilageCartilage consists of cells (chondrocytes) surrounded by an extracellular matrix made up of several proteoglycans, colla-gen fibers, and water. Unlike bone, cartilage is very avascular and depends on diffusion for transmittal of nutrients across the matrix. Additionally, the hypervascular perichondrium contrib-utes substantially to the nutrition of the cartilage. Therefore, injuries to cartilage may be associated with permanent defects due to tenuous blood supply.The healing response of cartilage depends on the depth of injury. In a superficial injury, there is disruption of the pro-teoglycan matrix and injury to the chondrocytes. There is no inflammatory response, but an increase in synthesis of proteo-glycan and collagen dependent

1	injury, there is disruption of the pro-teoglycan matrix and injury to the chondrocytes. There is no inflammatory response, but an increase in synthesis of proteo-glycan and collagen dependent entirely on the chondrocyte. The healing power of cartilage is often inadequate, and over-all regeneration is incomplete. Therefore, superficial cartilage injuries are slow to heal and often result in persistent structural defects.In contrast to superficial injuries, deep injuries involve the underlying bone and soft tissue. This leads to the exposure of vascular channels of the surrounding damaged tissue that may help in the formation of granulation tissue. Hemorrhage allows for the initiation of the inflammatory response and the subse-quent mediator activation of cellular function for repair. As the granulation tissue is laid down, fibroblasts migrate toward the wound and synthesize fibrous tissue that undergoes chondrifica-tion. Gradually, hyaline cartilage is formed, which restores the

1	As the granulation tissue is laid down, fibroblasts migrate toward the wound and synthesize fibrous tissue that undergoes chondrifica-tion. Gradually, hyaline cartilage is formed, which restores the structural and functional integrity of the injured site.TendonTendons and ligaments are specialized structures that link mus-cle and bone, and bone and bone, respectively. They consist of parallel bundles of collagen interspersed with spindle cells. Tendons and ligaments can be subjected to a variety of injuries, such as laceration, rupture, and contusion. Due to the mobility of the underlying bone or muscles, the damaged ends usually separate. Tendon and ligament healing progresses in a similar fashion as in other areas of the body (i.e., through hematoma formation, organization, laying down of reparative tissue, and scar formation). Matrix is characterized by accumulation of types I and III collagen along with increased water, DNA, and glycosaminoglycan content. As the collagen fibers

1	of reparative tissue, and scar formation). Matrix is characterized by accumulation of types I and III collagen along with increased water, DNA, and glycosaminoglycan content. As the collagen fibers are orga-nized, transmission of forces across the damaged portion can occur. Restoration of the mechanical integrity may never be equal to that of the undamaged tendon.Tendon vasculature has a clear effect on healing. Hypovas-cular tendons tend to heal with less motion and more scar for-mation than tendons with better blood supply. The specialized cells, tenocytes, are metabolically very active and retain a large regenerative potential, even in the absence of vascularity. Cells on the tendon surface are identical to those within the sheath and play a role in tendon healing as well.NerveNerve injuries are very common, with an estimated 200,000 repairs performed every year in the United States. Peripheral nerves are a complex arrangement of axons, nonneuronal cells, and extracellular

1	injuries are very common, with an estimated 200,000 repairs performed every year in the United States. Peripheral nerves are a complex arrangement of axons, nonneuronal cells, and extracellular elements. There are three types of nerve inju-ries: neurapraxia (focal demyelination), axonotmesis (interrup-tion of axonal continuity but preservation of Schwann cell basal lamina), and neurotmesis (complete transection). Following all types of injury, the nerve ends progress through a predictable pattern of changes involving three crucial steps: (a) survival of axonal cell bodies; (b) regeneration of axons that grow across the transected nerve to reach the distal stump; and (c) migration and connection of the regenerating nerve ends to the appropriate nerve ends or organ targets.Phagocytes remove the degenerating axons and myelin sheath from the distal stump (Wallerian degeneration). Regen-erating axonal sprouts extend from the proximal stump and probe the distal stump and the surrounding

1	the degenerating axons and myelin sheath from the distal stump (Wallerian degeneration). Regen-erating axonal sprouts extend from the proximal stump and probe the distal stump and the surrounding tissues. Schwann cells envelope and help in remyelinating the regenerating axons. Functional units are formed when the regenerating axons con-nect with the appropriate end targets. Several factors play a role in nerve healing, such as growth factors, cell adhesion mol-ecules, and nonneuronal cells and receptors. Growth factors include nerve growth factor, brain-derived neurotrophic factor, basic and acidic fibroblastic growth factors, and neuroleukin. Cell adhesion molecules involved in nerve healing include nerve adhesion molecule, neuron-glia adhesion molecule, myelin adhesion glycoprotein, and N-cadherin. This complex interplay of growth factors and adhesion molecules helps in nerve regeneration.Fetal Wound HealingThe main characteristic that distinguishes the healing of fetal wounds from

1	N-cadherin. This complex interplay of growth factors and adhesion molecules helps in nerve regeneration.Fetal Wound HealingThe main characteristic that distinguishes the healing of fetal wounds from that of adult wounds is the lack of scar forma-tion. Understanding how fetal wounds achieve integrity without Brunicardi_Ch09_p0271-p0304.indd 28101/03/19 4:50 PM 282BASIC CONSIDERATIONSPART Ievidence of scarring holds promise for the possible manipula-tion of unwanted fibrosis or excessive scar formation in adults.Although early fetal healing is characterized by the absence of scarring and resembles tissue regeneration, there is a phase of transition during gestational life when a more adult-like healing pattern emerges. This so-called “transition wound” occurs at the beginning of the third trimester, and during this period, there is scarless healing; however, there is a loss of the ability to regenerate skin appendages.49 Eventually a classic, adult-patterned healing with scar

1	third trimester, and during this period, there is scarless healing; however, there is a loss of the ability to regenerate skin appendages.49 Eventually a classic, adult-patterned healing with scar formation occurs exclusively, although overall healing continues to be faster than in adults.There are a number of characteristics that may influence the differences between fetal and adult wounds. These include wound environment, inflammatory responses, differential growth factor profiles, and wound matrix.Wound Environment. The fetus is bathed in a sterile, tem-perature-stable fluid environment, although this alone does not explain the observed differences. Experiments have demon-strated that scarless healing may occur outside of the amniotic fluid environment, and conversely, scars can form in utero.50,51Inflammation. The extent and robustness of the inflammatory response correlates directly with the amount of scar formation in all healing wounds. Reduced fetal inflammation due to the

1	in utero.50,51Inflammation. The extent and robustness of the inflammatory response correlates directly with the amount of scar formation in all healing wounds. Reduced fetal inflammation due to the immaturity of the fetal immune system may partially explain the lack of scarring observed. Not only is the fetus neutrope-nic, but fetal wounds also contain lower numbers of PMNs and macrophages.52Growth Factors. Fetal wounds are notable for the absence of TGF-β, which may have a significant role in scarring. Blocking TGF-β1 or TGF-β2 using neutralizing antibodies considerably reduces scar formation in adult wounds. Exogenous application of TGF-β3 downregulates TGF-β1 and TGF-β2 levels at the wound site with a resultant reduction in scarring.53 Thus, the balance between the concentration and/or activity of TGF-β iso-forms may be important for regulating scar production.Wound Matrix. The fetal wound is characterized by excessive and extended hyaluronic acid production, a

1	concentration and/or activity of TGF-β iso-forms may be important for regulating scar production.Wound Matrix. The fetal wound is characterized by excessive and extended hyaluronic acid production, a high-molecular-weight glycosaminoglycan that is produced primarily by fibro-blasts. Although adult wounds also produce hyaluronic acid, its synthesis is sustained only in the fetal wound. Components of amniotic fluid, most specifically fetal urine, have a unique abil-ity to stimulate hyaluronic acid production.54 Fetal fibroblasts produce more collagen than adult fibroblasts, and the increased level of hyaluronic acid may aid in the orderly organization of collagen. As a result of these findings, hyaluronic acid is used topically to enhance healing and to inhibit postoperative adhesion formation.55 The collagen pattern of fetal wounds is reticular in nature and resembles surrounding tissue, while adult patterns express large bundles of parallel collagen fibrils ori-ented perpendicular to

1	The collagen pattern of fetal wounds is reticular in nature and resembles surrounding tissue, while adult patterns express large bundles of parallel collagen fibrils ori-ented perpendicular to the surface.56CLASSIFICATION OF WOUNDSWounds are classified as either acute or chronic. By definition, an acute wound becomes chronic if healing is not achieved after 4 weeks of treatment. Acute wounds heal in a predictable man-ner and time frame as previously mentioned. The process occurs with few, if any, complications, and the end result is a well-healed wound. Surgical wounds can heal in several ways. An incised wound that is clean and closed by sutures is said to heal by primary intention. Often, because of bacterial contamination or tissue loss, a wound will be left open to heal by granula-tion tissue formation and contraction; this constitutes healing by secondary intention. Delayed primary closure, or healing by tertiary intention, represents a combination of the first two, con-sisting

1	tissue formation and contraction; this constitutes healing by secondary intention. Delayed primary closure, or healing by tertiary intention, represents a combination of the first two, con-sisting of the placement of sutures, allowing the wound to stay open for a few days, and the subsequent closure of the sutures (Fig. 9-6).The healing spectrum of acute wounds is broad (Fig. 9-7). In examining the acquisition of mechanical integrity and strength during healing, the normal process is characterized by a con-stant and continual increase that reaches a plateau at some point after injury. In regular wounds, the maximal wound strength is reached after about 6 weeks of healing. A fully healed wound achieves only 75% to 80% of a normal tissue. Wounds with delayed healing are characterized by decreased wound-breaking strength in comparison to wounds that heal at a normal rate; however, they eventually achieve the same integrity and strength as wounds that heal normally. Conditions such as

1	decreased wound-breaking strength in comparison to wounds that heal at a normal rate; however, they eventually achieve the same integrity and strength as wounds that heal normally. Conditions such as nutritional deficiencies, infections, or severe trauma cause delayed heal-ing, which reverts to normal with correction of the underlying pathophysiology. Impaired healing is characterized by a failure to achieve mechanical strength equivalent to normally healed wounds. Patients with compromised immune systems such as those with diabetes, chronic steroid usage, or tissues damaged by radiotherapy are prone to this type of impaired healing. The surgeon must be aware of these situations and exercise great care in the placement of incision and suture selection, postop-erative care, and adjunctive therapy to maximize the chances of healing without supervening complications.In general, wounds heal by a combination of mecha-nisms, including connective tissue deposition, contraction, and Primary

1	therapy to maximize the chances of healing without supervening complications.In general, wounds heal by a combination of mecha-nisms, including connective tissue deposition, contraction, and Primary IntentionSecondary IntentionTertiary IntentionContractionContractionConnective TissueRepairConnective TissueRepairEpithelializationEpithelializationFigure 9-6. Different clinical approaches to the closure and heal-ing of acute wounds.Brunicardi_Ch09_p0271-p0304.indd 28201/03/19 4:50 PM 283WOUND HEALINGCHAPTER 9epithelialization, depending on wound type. Surgically closed wounds need mostly epithelialization for healing, while open wounds require a combination of tissue contraction, connective tissue deposition, and epithelialization to a lesser extent. Chronic ulcers heal by secondary intention similar to open wounds.Normal healing is affected by both systemic and local fac-tors (Table 9-6). The clinician must be familiar with these fac-tors and should attempt to counteract their

1	intention similar to open wounds.Normal healing is affected by both systemic and local fac-tors (Table 9-6). The clinician must be familiar with these fac-tors and should attempt to counteract their deleterious effects. Complications occurring in wounds with higher risk can lead to failure of healing or the development of chronic, nonhealing wounds.Factors Affecting Wound HealingAdvanced Age. Most surgeons believe that aging produces intrinsic physiologic changes that result in delayed or impaired wound healing. Clinical experience with elderly patients tends to support this belief. Studies of hospitalized surgical patients show a direct correlation between older age and poor wound healing outcomes such as dehiscence and incisional hernia.57,58 However, these statistics fail to take into account underlying illnesses or diseases as a possible source of impaired wound healing in the elderly. The increased incidence of cardiovascu-lar disease, metabolic diseases (diabetes mellitus,

1	into account underlying illnesses or diseases as a possible source of impaired wound healing in the elderly. The increased incidence of cardiovascu-lar disease, metabolic diseases (diabetes mellitus, malnutrition, and vitamin deficiencies), and cancer, and the widespread use of drugs that impair wound healing may all contribute to the higher incidence of wound problems in the elderly. However, more recent clinical experience suggests that major operative interventions can be accomplished safely in the elderly.The results of animal studies regarding the effects of aging on wound healing have yielded contradictory results. In healthy human volunteers, there was a significant delay of 1.9 days in the epithelialization of superficial skin defects in those older than 70 years of age when compared to younger volunteers.59 In the same volunteers, using a micro-model of fibroplasia, no dif-ference in DNA or hydroxyproline wound accumulation could be demonstrated between the young and elderly

1	to younger volunteers.59 In the same volunteers, using a micro-model of fibroplasia, no dif-ference in DNA or hydroxyproline wound accumulation could be demonstrated between the young and elderly groups; how-ever, the young volunteers had a significantly higher amount of total α-amino nitrogen in their wounds, a reflection of total protein content of the wound. Thus, although wound collagen synthesis does not seem to be impaired with advanced age, non-collagenous protein accumulation at wounded sites is decreased with aging, which may impair the mechanical properties of scar-ring in elderly patients. Generally, in a relatively healthy person age will cause a delay in healing rather than nonhealing.Hypoxia, Anemia, and Hypoperfusion. Low oxygen tension has a profoundly deleterious effect on all aspects of wound heal-ing. Fibroplasia, although stimulated initially by the hypoxic wound environment, is significantly impaired by local hypoxia. Optimal collagen synthesis requires oxygen as

1	all aspects of wound heal-ing. Fibroplasia, although stimulated initially by the hypoxic wound environment, is significantly impaired by local hypoxia. Optimal collagen synthesis requires oxygen as a cofactor, partic-ularly for the hydroxylation steps. Increasing subcutaneous oxy-gen tension levels by increasing the fraction of inspired oxygen (Fio2) of inspired air for brief periods during and immediately following surgery results in enhanced collagen deposition and in decreased rates of wound infection after elective surgery.60-62Major factors affecting local oxygen delivery include hypoperfusion either for systemic reasons (low volume or car-diac failure) or due to local causes (arterial insufficiency, local vasoconstriction, or excessive tension on tissues). The level of vasoconstriction of the subcutaneous capillary bed is exquisitely responsive to fluid status, temperature, and hyperactive sympa-thetic tone as is often induced by postoperative pain. Correction of these factors

1	of the subcutaneous capillary bed is exquisitely responsive to fluid status, temperature, and hyperactive sympa-thetic tone as is often induced by postoperative pain. Correction of these factors can have a remarkable influence on wound out-come, particularly on decreasing wound infection rates.61-63 Mild to moderate normovolemic anemia does not appear to adversely affect wound oxygen tension and collagen synthesis. However, profound anemia with 15% less hematocrit can interfere with wound healing.63Steroids and Chemotherapeutic Drugs. Large doses or chronic usage of glucocorticoids reduce collagen synthesis and wound strength.64 The major effect of steroids is to inhibit the inflammatory phase of wound healing (angiogenesis, neutro-phil and macrophage migration, and fibroblast proliferation) and the release of lysosomal enzymes. The stronger the anti-inflammatory effect of the steroid compound used, the greater the inhibitory effect on wound healing. Steroids used after the first 3 to

1	and the release of lysosomal enzymes. The stronger the anti-inflammatory effect of the steroid compound used, the greater the inhibitory effect on wound healing. Steroids used after the first 3 to 4 days after injury do not affect wound healing as severely as when they are used in the immediate postoperative 3Normal healingDelayed healingImpaired healing chronicTimeWound mechanical strengthFigure 9-7. The acquisition of wound mechanical strength over time in normal, delayed, and impaired healing.Table 9-6Factors affecting wound healingSystemic Age Nutrition Trauma Metabolic diseases Immunosuppression Connective tissue disorders SmokingLocal Mechanical injury Infection Edema Ischemia/necrotic tissue Topical agents Ionizing radiation Low oxygen tension Foreign bodiesBrunicardi_Ch09_p0271-p0304.indd 28301/03/19 4:50 PM 284BASIC CONSIDERATIONSPART Iperiod. Therefore, if possible, their use should be delayed, or alternatively, forms with lesser anti-inflammatory effects should be

1	28301/03/19 4:50 PM 284BASIC CONSIDERATIONSPART Iperiod. Therefore, if possible, their use should be delayed, or alternatively, forms with lesser anti-inflammatory effects should be administered.In addition to their effect on collagen synthesis, steroids also inhibit epithelialization and contraction and contribute to increased rates of wound infection, regardless of the time of administration.64 Steroid-delayed healing of cutaneous wounds can be stimulated to epithelialize by topical application of vitamin A.64,65 Collagen synthesis of steroid-treated wounds also can be stimulated by vitamin A.All chemotherapeutic antimetabolite drugs adversely affect wound healing by inhibiting early cell proliferation, wound DNA and protein synthesis, attenuation of the inflamma-tory phase, decrease fibrin deposition, and delay wound contrac-tion, all of which are critical to successful healing. The effect is worse if these agents are given preoperatively; so a delay in the use of such drugs

1	fibrin deposition, and delay wound contrac-tion, all of which are critical to successful healing. The effect is worse if these agents are given preoperatively; so a delay in the use of such drugs for about 2 weeks after injury appears to lessen wound healing impairment.66 Extravasation of most che-motherapeutic agents is associated with tissue necrosis, marked ulceration, and protracted healing at the affected site.67Metabolic Disorders. Diabetes mellitus is the best known of the metabolic disorders contributing to increased rates of wound infection and failure.68 Uncontrolled diabetes results in reduced inflammation, angiogenesis, and collagen synthe-sis. Additionally, the largeand small-vessel disease that is the hallmark of advanced diabetes contributes to hypoperfusion and local hypoxemia. Defects in granulocyte function, capillary ingrowth, and fibroblast proliferation all have been described in diabetes. Obesity, insulin resistance, hyperglycemia, and dia-betic renal failure

1	Defects in granulocyte function, capillary ingrowth, and fibroblast proliferation all have been described in diabetes. Obesity, insulin resistance, hyperglycemia, and dia-betic renal failure contribute significantly and independently to the impaired wound healing observed in diabetics.69 In wound studies on experimental diabetic animals, insulin restores colla-gen synthesis and granulation tissue formation to normal levels if given during the early phases of healing.70 In clean, nonin-fected, and well-perfused experimental wounds in human dia-betic volunteers, type 1 diabetes mellitus was noted to decrease wound collagen accumulation in the wound, independent of the degree of glycemic control. Type 2 diabetic patients showed no effect on collagen accumulation when compared to healthy, age-matched controls.71 Furthermore, the diabetic wound appears to be lacking in sufficient growth factor levels, which signal normal healing. It remains unclear whether decreased collagen synthesis or

1	controls.71 Furthermore, the diabetic wound appears to be lacking in sufficient growth factor levels, which signal normal healing. It remains unclear whether decreased collagen synthesis or an increased breakdown due to an abnormally high proteolytic wound environment is responsible.Careful preoperative correction of blood sugar levels improves the outcome of wounds in diabetic patients. Increas-ing the inspired oxygen tension, judicious use of antibiotics, and correction of other coexisting metabolic abnormalities all can result in improved wound healing. Additionally, revasculariza-tion to local areas with chronic ulcers will aid in the healing process.Uremia also has been associated with disordered wound healing and impairs defenses for infection. Experimentally, uremic animals demonstrate decreased wound collagen synthe-sis and breaking strength, causing delayed healing of intestinal anastomosis and abdominal wounds. The contribution of uremia alone to this impairment, rather than

1	decreased wound collagen synthe-sis and breaking strength, causing delayed healing of intestinal anastomosis and abdominal wounds. The contribution of uremia alone to this impairment, rather than that of associated malnutri-tion, is difficult to assess.69 The clinical use of dialysis to correct the metabolic abnormalities and nutritional restoration should impact greatly on the wound outcome of such patients. In some uremic patients, wounds might be associated with abnormal deposition of calcium and phosphate in the tissue leading to uremic gangrene syndrome (calciphylaxis). In such patients, the wounds are extremely painful and difficult to heal.Obesity is the largest growing public health problem in the United States and the world. Over 60% of Americans are overweight or obese. Uncomplicated obesity (i.e., in the absence of comorbid conditions such as cardiovascular disease, diabetes, or respiratory insufficiency) has by itself significant deleterious effects on wound healing.

1	obesity (i.e., in the absence of comorbid conditions such as cardiovascular disease, diabetes, or respiratory insufficiency) has by itself significant deleterious effects on wound healing. Visceral adiposity is active metaboli-cally and immunologically and, through generation of proin-flammatory cytokines and adipokines, leads to the development of the metabolic syndrome. Many of these molecules have effects on cells participating in the healing response. In nondia-betic obese rodents, wounds are mechanically weaker, and there is less dermal and reparative scar collagen. Preadipocytes infil-trate the dermis, and although they can evolve into fibroblasts, their regulatory mechanisms appear different from those of dermal or wound fibroblasts. Many studies indicate that obese patients have high rates of perioperative complications, with estimates as high as 30% for wound dehiscence, 17% for surgi-cal site infections, 30% for incisional hernias, 19% for seromas, 13% for hematomas, and 10%

1	rates of perioperative complications, with estimates as high as 30% for wound dehiscence, 17% for surgi-cal site infections, 30% for incisional hernias, 19% for seromas, 13% for hematomas, and 10% for fat necrosis.72-74 Increased subcutaneous fat was associated with a tenfold increased risk of surgery-related complications including anastomotic leaks, abdominal collection, and wound infections.75 In many studies, obesity is a constant and major risk factor for hernia formation and recurrence after repair. The mechanism by which obesity impairs wound healing awaits complete delineation.Nutrition. The importance of nutrition in the recovery from traumatic or surgical injury has been recognized by clinicians since the time of Hippocrates. Poor nutritional intake or lack of individual nutrients significantly alters many aspects of wound healing. The clinician must pay close attention to the nutritional status of patients with wounds, since wound failure or wound infections may be no more

1	significantly alters many aspects of wound healing. The clinician must pay close attention to the nutritional status of patients with wounds, since wound failure or wound infections may be no more than a reflection of poor nutrition. Although the full interaction of nutrition and wound healing is still not fully understood, efforts are being made to develop wound-specific nutritional interventions and institute the phar-macologic use of individual nutrients as modulators of wound outcomes.Experimental rodents fed either a 0% or 4% protein diet have impaired collagen deposition with a secondary decrease in skin and fascial wound-breaking strength and increased wound infection rates. Induction of energy-deficient states by providing only 50% of the normal caloric requirement leads to decreased granulation tissue formation and matrix protein deposition in rats. Acute fasting in rats markedly impairs collagen synthesis while decreasing procollagen mRNA.76Clinically, it is extremely rare

1	granulation tissue formation and matrix protein deposition in rats. Acute fasting in rats markedly impairs collagen synthesis while decreasing procollagen mRNA.76Clinically, it is extremely rare to encounter pure energy or protein malnutrition, and the vast majority of patients exhibit combined protein-energy malnutrition. Such patients have diminished hydroxyproline accumulation (an index of collagen deposition) into subcutaneously implanted polytetrafluoroeth-ylene tubes when compared to normally nourished patients (Fig. 9-8). Furthermore, malnutrition correlates clinically with enhanced rates of wound complications and increased wound failure following diverse surgical procedures. This reflects impaired healing response as well as reduced cell-mediated immunity, phagocytosis, and intracellular killing of bacte-ria by macrophages and neutrophils during protein-calorie malnutrition.76Two additional nutrition-related factors warrant discus-sion. First, the degree of nutritional

1	intracellular killing of bacte-ria by macrophages and neutrophils during protein-calorie malnutrition.76Two additional nutrition-related factors warrant discus-sion. First, the degree of nutritional impairment need not be Brunicardi_Ch09_p0271-p0304.indd 28401/03/19 4:50 PM 285WOUND HEALINGCHAPTER 9long-standing in humans, as opposed to the experimental situa-tion. Thus, patients with brief preoperative illnesses or reduced nutrient intake in the period immediately preceding the injury or operative intervention will demonstrate impaired fibroplasias.77,78 Second, brief and not necessarily intensive nutritional interven-tion, either via the parenteral or enteral route, can reverse or prevent the decreased collagen deposition noted with malnutri-tion or with postoperative starvation.79The possible role of single amino acids in enhanced wound healing has been studied for the last several decades. Arginine appears most active in terms of enhancing wound fibroplasia. Arginine

1	possible role of single amino acids in enhanced wound healing has been studied for the last several decades. Arginine appears most active in terms of enhancing wound fibroplasia. Arginine deficiency results in decreased wound-breaking strength and wound-collagen accumulation in chow-fed rats. Rats that are given 1% arginine HCl supplementation, and there-fore are not arginine-deficient, have enhanced wound-breaking strength and collagen synthesis when compared to chow-fed controls.80 Studies have been carried out in healthy human vol-unteers to examine the effect of arginine supplementation on collagen accumulation. Young, healthy, human volunteers (age 25–35 years) were found to have significantly increased wound-collagen deposition following oral supplementation with either 30 g of arginine aspartate (17 g of free arginine) or 30 g of argi-nine Hall (24.8 g of free arginine) daily for 14 days.81 In a study of healthy older humans (age 67–82 years), daily supplements of 30 g of

1	arginine aspartate (17 g of free arginine) or 30 g of argi-nine Hall (24.8 g of free arginine) daily for 14 days.81 In a study of healthy older humans (age 67–82 years), daily supplements of 30 g of arginine aspartate for 14 days resulted in significantly enhanced collagen and total protein deposition at the wound site when compared to controls given placebos. There was no enhanced DNA synthesis present in the wounds of the arginine-supplemented subjects, suggesting that the effect of arginine is not mediated by an inflammatory mode of action.82 In this and later studies, arginine supplementation, whether administered orally or parenterally, had no effect on the rate of epithelializa-tion of a superficial skin defect. This further suggests that the main effect of arginine on wound healing is to enhance wound collagen deposition. Recently, a dietary supplemental regimen of arginine, β-hydroxy-β-methyl butyrate, and glutamine was found to significantly and specifically enhance collagen

1	is to enhance wound collagen deposition. Recently, a dietary supplemental regimen of arginine, β-hydroxy-β-methyl butyrate, and glutamine was found to significantly and specifically enhance collagen depo-sition in elderly, healthy human volunteers when compared to an isocaloric, isonitrogenous supplement (Fig. 9-9).83 As increases in breaking strength during the first weeks of healing are directly related to new collagen synthesis, arginine supple-mentation may result in an improvement in wound strength as a consequence of enhanced collagen deposition.The vitamins most closely involved with wound healing are vitamin C and vitamin A. Scurvy or vitamin C deficiency leads to a defect in wound healing, particularly via a failure in collagen synthesis and cross-linking. Biochemically, vitamin C is required for the conversion of proline and lysine to hydroxy-proline and hydroxylysine, respectively. Vitamin C deficiency has also been associated with an increased incidence of wound infection,

1	is required for the conversion of proline and lysine to hydroxy-proline and hydroxylysine, respectively. Vitamin C deficiency has also been associated with an increased incidence of wound infection, and if wound infection does occur, it tends to be more severe. These effects are believed to be due to an associated impairment in neutrophil function, decreased complement activ-ity, and decreased walling-off of bacteria secondary to insuffi-cient collagen deposition. The recommended dietary allowance is 60 mg daily. This provides a considerable safety margin for most healthy nonsmokers. In severely injured or extensively burned patients, this requirement may increase to as high as 2 g daily. There is no evidence that excess vitamin C is toxic; however, there is no evidence that supratherapeutic doses of vitamin C are of any benefit.84Vitamin A deficiency impairs wound healing, while sup-plemental vitamin A benefits wound healing in nondeficient humans and animals. Vitamin A increases

1	doses of vitamin C are of any benefit.84Vitamin A deficiency impairs wound healing, while sup-plemental vitamin A benefits wound healing in nondeficient humans and animals. Vitamin A increases the inflammatory OHP (nmol/mg)1.52.01.00.50.0Adequatefood intakeInadequatefood intake1.51.00.5OHP (µg/cm)0.0WellnourishedMalnourishedFigure 9-8. Effect of malnutrition on collagen deposition in exper-imental human wounds. OHP = hydroxyproline.54321ASPLYSOHPExperimentalControl˜ANFigure 9-9. Ratios of 14-day to 7-day values for aspartate (ASP), lysine (LYS), hydroxyproline (OHP), and α-amino nitrogen (αAN) in volunteers given dietary supplements of arginine, β-hydroxy-β-methylbutyrate, and glutamine. P <.05. (Reproduced with per-mission from Williams JZ, Abumrad NN, Barbul A. Effect of a specialized amino acid mixture on human collagen deposition, Ann Surg. 2002 Sep;236(3):369-374.)Brunicardi_Ch09_p0271-p0304.indd 28501/03/19 4:50 PM 286BASIC CONSIDERATIONSPART Iresponse in wound healing,

1	amino acid mixture on human collagen deposition, Ann Surg. 2002 Sep;236(3):369-374.)Brunicardi_Ch09_p0271-p0304.indd 28501/03/19 4:50 PM 286BASIC CONSIDERATIONSPART Iresponse in wound healing, probably by increasing the labil-ity of lysosomal membranes. There is an increased influx of macrophages, with an increase in their activation and increased collagen synthesis. Vitamin A directly increases collagen pro-duction and epidermal growth factor receptors when it is added in vitro to cultured fibroblasts. As mentioned before, supple-mental vitamin A can reverse the inhibitory effects of cortico-steroids on wound healing. Vitamin A also can restore wound healing that has been impaired by diabetes, tumor formation, cyclophosphamide, and radiation. Serious injury or stress leads to increased vitamin A requirements. In the severely injured patient, supplemental doses of vitamin A have been recom-mended. Doses ranging from 25,000 to 100,000 IU per day have been advocated.The connections

1	vitamin A requirements. In the severely injured patient, supplemental doses of vitamin A have been recom-mended. Doses ranging from 25,000 to 100,000 IU per day have been advocated.The connections between specific minerals and trace ele-ments and deficits in wound healing are complex. Frequently, deficiencies are multiple and include macronutrient deficien-cies. As with some of the vitamins described earlier, the specific trace element may function as a cofactor or part of an enzyme that is essential for homeostasis and wound healing. Clinically, preventing deficiencies is often easier to accomplish than diag-nosing them.Zinc is the most well-known element in wound heal-ing and has been used empirically in dermatologic conditions for centuries. It is essential for wound healing in animals and humans. There are over 150 known enzymes for which zinc is either an integral part or an essential cofactor, and many of these enzymes are critical to wound healing.85 With zinc deficiency, there

1	and humans. There are over 150 known enzymes for which zinc is either an integral part or an essential cofactor, and many of these enzymes are critical to wound healing.85 With zinc deficiency, there is decreased fibroblast proliferation, decreased collagen synthesis, impaired overall wound strength, and delayed epithe-lialization. These defects are reversed by zinc supplementation. To date, no study has shown improved wound healing with zinc supplementation in patients who are not zinc deficient.86Infections. Wound infections continue to represent a major medical problem, both in terms of how they affect the outcome of surgical procedures (surgical site infections), and for their impact on the length of hospital stay and medical costs.87 Many otherwise successful surgical operations fail because of the development of wound infections. The occurrence of infections is of major concern when implants are used, and their occur-rence may lead to the removal of the prosthetic material, thus

1	because of the development of wound infections. The occurrence of infections is of major concern when implants are used, and their occur-rence may lead to the removal of the prosthetic material, thus subjecting the patient to further operations and severe risk of morbidity and mortality. Infections can weaken an abdominal closure or hernia repair and result in wound dehiscence or recur-rence of the hernia. Cosmetically, infections can lead to disfig-uring, unsightly, or delayed closures.Exhaustive studies have been undertaken that examine the appropriate prophylactic treatment of operative wounds. Bacte-rial contaminants normally present on skin are prevented from entry into deep tissues by intact epithelium. Surgery breaches the intact epithelium, allowing bacteria access to these tissues and the bloodstream. Antibiotic prophylaxis is most effective when adequate concentrations of antibiotic are present in the tissues at the time of incision, and assurance of adequate pre-operative

1	and the bloodstream. Antibiotic prophylaxis is most effective when adequate concentrations of antibiotic are present in the tissues at the time of incision, and assurance of adequate pre-operative antibiotic dosing and timing has become a significant hospital performance measure.88 The addition of antibiotics after operative contamination has occurred clearly is ineffective in preventing postoperative wound infections.Studies that compare operations performed with and without antibiotic prophylaxis demonstrate that classes II, III, and IV procedures (see below) treated with appropriate prophylactic antibiotics have only one third the wound infection rate of previously reported untreated series.89 More recently, repeat dosing of antibiotics has been shown to be essential in decreasing postoperative wound infections in operations with durations exceeding the biochemical half-life (t1/2) of the anti-biotic or in which there is large-volume blood loss and fluid replacement.90,91 In

1	postoperative wound infections in operations with durations exceeding the biochemical half-life (t1/2) of the anti-biotic or in which there is large-volume blood loss and fluid replacement.90,91 In lengthy cases, those in which prosthetic implants are used, or when unexpected contamination is encoun-tered, additional doses of antibiotic may be administered for 24 hours postoperatively.Selection of antibiotics for use in prophylaxis should be tailored to the type of surgery to be performed, operative con-taminants that might be encountered during the procedure, and the profile of resistant organisms present at the institution where the surgery is performed. The continuing widespread appear-ance of methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant enterococci (VRE) has significantly restricted the selection of these agents for routine use. Surgery-specific treatment guidelines are provided in Table 9-7.90Patients with prosthetic heart valves or any implanted

1	has significantly restricted the selection of these agents for routine use. Surgery-specific treatment guidelines are provided in Table 9-7.90Patients with prosthetic heart valves or any implanted vascular or orthopedic prostheses should receive antibiotic prophylaxis prior to any procedure in which significant bac-teremia is anticipated. Dental procedures require prophylaxis with broad-spectrum penicillins or amoxicillin, while urologic instrumentation should be pretreated with a second-generation cephalosporin. Patients with prostheses who undergo gastroin-testinal surgery should receive anaerobic coverage combined with a cephalosporin. Nasal screening and decolonization for S aureus carriers are recommended for selected procedures (i.e., cardiac, orthopedic, neurosurgical procedures with implants).The incidence of wound infection is about 5% to 10% nationwide and has not changed during the last few decades. Quantitatively, it has been shown that if the wound is contami-nated with

1	implants).The incidence of wound infection is about 5% to 10% nationwide and has not changed during the last few decades. Quantitatively, it has been shown that if the wound is contami-nated with >105 microorganisms per gram of tissue, the risk of wound infection is markedly increased, but this threshold may be much lower in the presence of foreign materials. The source of pathogens for the infection is usually the endogenous flora of the patient’s skin, mucous membranes, or from hollow organs. The most common organisms responsible for wound infections in order of frequency are Staphylococcus species, coagulase-negative Streptococcus, enterococci, and Escherichia coli. The incidence of wound infection bears a direct relationship to the degree of contamination that occurs during the operation from the disease process itself (clean—class I, clean contaminated—class II, contaminated—class III, and dirty—class IV). Many factors contribute to the development of postoperative wound

1	operation from the disease process itself (clean—class I, clean contaminated—class II, contaminated—class III, and dirty—class IV). Many factors contribute to the development of postoperative wound infections. Most surgical wound infections become apparent within 7 to 10 days postoperatively, although a small number manifest years after the original operative intervention. With the hospital stay becoming shorter and shorter, many infections are detected in the outpatient setting, leading to underreporting of the true incidence of wound infections absent intensive sur-veillance. There has been much debate about the actual defini-tion of wound infection. The narrowest definition would include wounds that drain purulent material with bacteria identified on culture. The broader definition would include all wounds drain-ing pus, whether or not the bacteriologic studies are positive; wounds that are opened by the surgeon; and wounds that the surgeon considers infected.92Anatomically, wound

1	include all wounds drain-ing pus, whether or not the bacteriologic studies are positive; wounds that are opened by the surgeon; and wounds that the surgeon considers infected.92Anatomically, wound infections can be classified as superficial incisional, deep incisional, and organ/space wound infections, involving fascia, muscle, or the abdominal cavity. Brunicardi_Ch09_p0271-p0304.indd 28601/03/19 4:50 PM 287WOUND HEALINGCHAPTER 9Table 9-7Antimicrobial prophylaxis for surgeryNATURE OF OPERATIONCOMMON PATHOGENSRECOMMENDED ANTIMICROBIALSADULT DOSAGE BEFORE SURGERY1CardiacStaphylococcus aureus, S. epidermidisCefazolin orCefuroxime orVancomycin41–2 g IV2,31.5 g IV31 g IVGastrointestinalEsophageal/gastroduodenalEnteric gram-negative bacilli, gram-positive cocciHigh risk5 only: cefazolin61–2 g IV2Biliary tractEnteric gram-negative bacilli, enterococci, clostridiaHigh risk7 only: cefazolin6,81–2 g IV2ColorectalEnteric gram-negative bacilli, anaerobes, enterococciOral: neomycin +

1	g IV2Biliary tractEnteric gram-negative bacilli, enterococci, clostridiaHigh risk7 only: cefazolin6,81–2 g IV2ColorectalEnteric gram-negative bacilli, anaerobes, enterococciOral: neomycin + erythromycin base9 or metronidazole9Parenteral: cefoxitin6 orCefotetan6 orCefazolin +Metronidazole6 orAmpicillin/sulbactamsee note 91–2 g IV1–2 g IV1–2 g IV20.5 g IV3 g IVAppendectomy, nonperforated11Same as for colorectalCefoxitin6 or cefotetan6 orCefazolin6 +Metronidazole1–2 g IV1–2 g IV20.5 g IVGenitourinaryCystoscopy aloneEnteric gram-negative bacilli, enterococciHigh risk only12: ciprofloxacin10 orTrimethoprim-sulfamethoxazole500 mg PO or 400 mg IV1 DS tabletCystoscopy with manipulation or upper tract instrumentation13Enteric gram-negative bacilli, enterococciCiprofloxacin10 orTrimethoprim-sulfamethoxazole500 mg PO or 400 mg IV1 DS tabletOpen or laparoscopic surgery14Enteric gram-negative bacilli, enterococciCefazolin61–2 g IV2Gynecologic and obstetricVaginal, abdominal, or laparoscopic

1	mg PO or 400 mg IV1 DS tabletOpen or laparoscopic surgery14Enteric gram-negative bacilli, enterococciCefazolin61–2 g IV2Gynecologic and obstetricVaginal, abdominal, or laparoscopic hysterectomyEnteric gram-negative bacilli, anaerobes, group B streptococci, enterococciCefazolin6 or cefoxitin6 or cefotetan6 or Ampicillin/sulbactam6,101–2 g IV23 g IVCesarean sectionSame as for hysterectomyCefazolin61–2 g IV2Abortion, surgicalSame as for hysterectomyDoxycycline300 mg PO15Head and neck surgeryIncisions through oral or pharyngeal mucosaAnaerobes, enteric gram-negative bacilli, S. aureusClindamycin orCefazolin +Metronidazole orAmpicillin/sulbactam10600–900 mg IV1–2 g IV20.5 g IV3 g IVNeurosurgeryS. aureus, S. epidermidisCefazolin1–2 g IV2OphthalmicS. epidermidis, S. aureus, streptococci, enteric gram-negative bacilli, Pseudomonas spp.Gentamicin, tobramycin, ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, ofloxacin or neomycin-gramicidin-polymyxin BOR cefazolinMultiple drops

1	enteric gram-negative bacilli, Pseudomonas spp.Gentamicin, tobramycin, ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, ofloxacin or neomycin-gramicidin-polymyxin BOR cefazolinMultiple drops topically over 2 to 24 hours100 mg subconjuctivallyOrthopedicS. aureus, S. epidermidisCefazolin16 or Vancomycin2,161–2 g IV2, 1 g IVThoracic (noncardiac)S. aureus, S. epidermidis, streptococci, enteric gram-negative bacilliCefazolin orAmpicillin/sulbactam10 or Vancomycin41–2 g IV23 g IV1 g IV(Continued)Brunicardi_Ch09_p0271-p0304.indd 28701/03/19 4:50 PM 288BASIC CONSIDERATIONSPART IAbout three-fourths of all wound infections are superficial, involving skin and subcutaneous tissue only. Clinical diagnosis is easy when a postoperative wound looks edematous and ery-thematous and is tender. Often the presentation is more subtle, and the development of postoperative fever, usually low-grade; the development of a mild and unexplained leukocytosis; or the presence of undue incisional pain

1	Often the presentation is more subtle, and the development of postoperative fever, usually low-grade; the development of a mild and unexplained leukocytosis; or the presence of undue incisional pain should direct attention to the wound. Inspection of the wound is most useful in detecting sub-tle edema around the suture or staple line, manifested as a waxy appearance of the skin, which characterizes the early phase of infection. If a wound infection is suspected, several stitches or staples around the most suspicious area should be removed with insertion of a cotton-tipped applicator into the subcutane-ous area to open a small segment of the incision. This causes minimal if any discomfort to the patient. Presence of pus man-dates further opening of the subcutaneous and skin layers to the full extent of the infected pocket. Samples should be taken for aerobic and anaerobic cultures, with very few patients requir-ing antibiotic therapy. Patients who are immunosuppressed (diabetics and

1	full extent of the infected pocket. Samples should be taken for aerobic and anaerobic cultures, with very few patients requir-ing antibiotic therapy. Patients who are immunosuppressed (diabetics and those on steroids or chemotherapeutic agents), who have evidence of tissue penetration or systemic toxicity, or who have had prosthetic devices inserted (vascular grafts, heart valves, artificial joints, or mesh) should be treated with systemic antibiotics.92Deep wound infections arise immediately adjacent to the fascia, either above or below it, and often have an intra-abdominal component. Most intra-abdominal infections do not, however, communicate with the wound. Deep infections present with fever and leukocytosis. The incision may drain pus spon-taneously, or the intra-abdominal extension may be recognized following the drainage of what was thought to be a superficial wound infection, but pus draining between the fascial sutures will be noted. Sometimes wound dehiscence will occur.The

1	may be recognized following the drainage of what was thought to be a superficial wound infection, but pus draining between the fascial sutures will be noted. Sometimes wound dehiscence will occur.The most dangerous of the deep infections is necrotizing fasciitis. It results in high mortality, particularly in the elderly. This is an invasive process that involves the fascia and leads to secondary skin necrosis. Pathophysiologically, it is a septic thrombosis of the vessels between the skin and the deep layers. Table 9-7Antimicrobial prophylaxis for surgeryNATURE OF OPERATIONCOMMON PATHOGENSRECOMMENDED ANTIMICROBIALSADULT DOSAGE BEFORE SURGERY1VascularArterial surgery involving a prosthesis, the abdominal aorta, or a groin incisionS. aureus, S. epidermidis, enteric gram-negative bacilliCefazolin orVancomycin41–2 g IV21 g IVLower extremity amputation for ischemiaS. aureus, S. epidermidis, enteric gram-negative bacilli, clostridiaCefazolin or Vancomycin41–2 g IV21 g IV1Parenteral

1	orVancomycin41–2 g IV21 g IVLower extremity amputation for ischemiaS. aureus, S. epidermidis, enteric gram-negative bacilli, clostridiaCefazolin or Vancomycin41–2 g IV21 g IV1Parenteral prophylactic antimicrobials can be given as a single IV dose begun within 60 min before the operation. For prolonged operations (>3 h) or those with major blood loss, or in patients with extensive burns, additional intraoperative doses should be given at intervals 1–2 times the half-life of the drug (ampicillin/sulbactam q2 h, cefazolin q4 h, cefuroxime q4 h, cefoxitin q2 h, clindamycin q6 h, vancomycin q12 h) for the duration of the procedure in a patient with normal renal function. If vancomycin or a fluoroquinolone is used, the infusion should be started 60–120 min before the initial incision to minimize the possibility of an infusion reaction close to the time of induction of anesthesia and to have adequate tissue levels at the time of incision.2The recommended dose of cefazolin is 1 g for patients

1	the possibility of an infusion reaction close to the time of induction of anesthesia and to have adequate tissue levels at the time of incision.2The recommended dose of cefazolin is 1 g for patients who weigh 80 kg and 2 g for those >80 kg. Morbidly obese patients may need higher doses.3Some experts recommend an additional dose when patients are removed from bypass during open heart surgery.4Vancomycin can be used in hospitals in which methicillin-resistant Staphylococcus aureus (MRSA) and S. epidermidis are a frequent cause of postoperative wound infection, in patients previously colonized with MRSA, or for those who are allergic to penicillin or cephalosporins. Rapid IV administration may cause hypotension, which could be especially dangerous during induction of anesthesia. Even when the drug is given over 60 min, hypotension may occur; treatment with diphenhydramine (Benadryl and others) and further slowing of the infusion rate may be helpful. Some experts would give 15 mg/kg of

1	the drug is given over 60 min, hypotension may occur; treatment with diphenhydramine (Benadryl and others) and further slowing of the infusion rate may be helpful. Some experts would give 15 mg/kg of vancomycin to patients weighing more than 75 kg up to a maximum of 1.5 g with a slower infusion rate (90 min for 1.5 g). For procedures in which gram-negative bacilli are common pathogens, many experts would add another drug such as an aminoglycoside (gentamicin, tobramycin, or amikacin), aztreonam, or a fluoroquinolone.5Morbid obesity, GI obstruction, decreased gastric acidity or gastrointestinal motility, gastric bleeding, malignancy or perforation, or immunosuppression.6For patients allergic to penicillin and cephalosporins, clindamycin or vancomycin with either gentamicin, ciprofloxacin, levofloxacin, or aztreonam is a reasonable alternative. Fluoroquinolones should not be used for prophylaxis in cesarean section.7Age >70 y, acute cholecystitis, nonfunctioning gallbladder, obstructive

1	or aztreonam is a reasonable alternative. Fluoroquinolones should not be used for prophylaxis in cesarean section.7Age >70 y, acute cholecystitis, nonfunctioning gallbladder, obstructive jaundice, or common duct stones.8Cefotetan, cefoxitin, and ampicillin/sulbactam are reasonable alternatives.9In addition to mechanical bowel preparation, 1 g of neomycin plus 1 g of erythromycin at 1 p.m., 2 p.m., and 11 p.m. or 2 g of neomycin plus 2 g of metronidazole at 7 p.m. and 11 p.m. the day before an 8 a.m. operation.10Due to increasing resistance of E. coli to fluoroquinolones and ampicillin/sulbactam, local sensitivity profiles should be reviewed prior to use.11For a ruptured viscous, therapy is often continued for about 5 d.12Urine culture positive or unavailable, preoperative catheter, transrectal prostate biopsy, or placement of prosthetic material.13Shock wave lithotripsy, ureteroscopy.14Including percutaneous renal surgery, procedures with entry into the urinary tract, and those

1	transrectal prostate biopsy, or placement of prosthetic material.13Shock wave lithotripsy, ureteroscopy.14Including percutaneous renal surgery, procedures with entry into the urinary tract, and those involving implantation of a prosthesis. If manipulation of bowel is involved, prophylaxis is given according to colorectal guidelines.15Divided into 100 mg before procedure and 200 mg after.16If a tourniquet is to be used in the procedure, the entire dose of antibiotic must be infused prior to its inflation.Reprinted with special permission from Treatment Guidelines from The Medical Letter, October 2012; Vol. 10(122):73. www.medicalletter.org.(Continued)Brunicardi_Ch09_p0271-p0304.indd 28801/03/19 4:50 PM 289WOUND HEALINGCHAPTER 9The skin demonstrates hemorrhagic bullae and subsequent frank necrosis, with surrounding areas of inflammation and edema. The fascial necrosis is usually wider than the skin involvement or than the surgeon estimates on clinical grounds. The patient is toxic

1	necrosis, with surrounding areas of inflammation and edema. The fascial necrosis is usually wider than the skin involvement or than the surgeon estimates on clinical grounds. The patient is toxic and has high fever, tachycardia, and marked hypovo-lemia, which if uncorrected, progresses to cardiovascular col-lapse. Bacteriologically, this is a mixed infection, and samples should be obtained for Gram stain smears and cultures to aid in diagnosis and treatment. As soon as bacteriologic studies have been obtained, high-dose penicillin treatment needs to be started (20–40 million U/d intravenously) due to concern over the presence of Clostridia perfringens and other related species; broad-spectrum antibiotics should be added and the regimen modified based on culture results. Cardiovascular resuscitation with electrolyte solutions, blood, and/or plasma is carried out as expeditiously as possible prior to induction of anesthesia. The aim of surgical treatment is thorough removal of all

1	resuscitation with electrolyte solutions, blood, and/or plasma is carried out as expeditiously as possible prior to induction of anesthesia. The aim of surgical treatment is thorough removal of all necrosed skin and fascia. If viable skin overlies necrotic fascia, multiple longitudinal skin incisions can be made to allow for excision of the devitalized fascia. Although removal of all necrotic tissue is the goal of the first surgical intervention, the distinction between necrotic and simply edematous tissue often is difficult. Careful inspection every 12 to 24 hours will reveal any new necrotic areas, and these need further debridement and excision. When all necrotic tissue has been removed and the infection has been controlled, the wounds may be covered with homoor xeno-grafts until definitive reconstruction and autografting can take place.The mere presence of bacteria in an open wound, either acute or chronic, does not constitute an infection, because large numbers of bacteria can

1	reconstruction and autografting can take place.The mere presence of bacteria in an open wound, either acute or chronic, does not constitute an infection, because large numbers of bacteria can be present in the normal situation. In addition, the bacteria identified by cultures may not be repre-sentative of the bacteria causing the actual wound infection. There seems to be confusion as to what exactly constitutes wound infection. For purposes of clarity, we have to differ-entiate between contamination, colonization, and infection. Contamination is the presence of bacteria without multiplica-tion, colonization is multiplication without host response, and infection is the presence of host response in reaction to depo-sition and multiplication of bacteria. The presence of a host response helps to differentiate between infection and coloniza-tion as seen in chronic wounds. The host response that helps in diagnosing wound infection comprises cellulitis, abnormal discharge, delayed healing,

1	to differentiate between infection and coloniza-tion as seen in chronic wounds. The host response that helps in diagnosing wound infection comprises cellulitis, abnormal discharge, delayed healing, change in pain, abnormal granula-tion tissue, bridging, and abnormal color and odor.As discussed previously, neutrophils play a major role in preventing wound infections. Chronic granulomatous disease (CGD) comprises a genetically heterogeneous group of dis-eases in which the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxide enzyme is deficient. This defect impairs the intracellular killing of microorganisms, leav-ing the patient liable to infection by bacteria and fungi. Afflicted patients have recurrent infections and form granulomas, which can lead to obstruction of the gastric antrum and genitourinary tracts and poor wound healing. Surgeons become involved when the patient develops infectious or obstructive complications.The nitro blue tetrazolium (NBT)

1	of the gastric antrum and genitourinary tracts and poor wound healing. Surgeons become involved when the patient develops infectious or obstructive complications.The nitro blue tetrazolium (NBT) reduction test is used to diagnose CGD. Normal neutrophils can reduce this com-pound, while neutrophils from affected patients do not, facili-tating the diagnosis via a colorimetric test. Clinically, patients develop recurrent infections such as pneumonia, lymphadenitis, hepatic abscess, and osteomyelitis. Organisms most commonly responsible are S. aureus, Aspergillus, Klebsiella, Serratia, or Candida. When CGD patients require surgery, a preoperative pulmonary function test should be considered since they are predisposed to obstructive and restrictive lung disease. Wound complications, mainly infection, are common. Sutures should be removed as late as possible since the wounds heal slowly. Abscess drains should be left in place for a prolonged period until the infection is completely

1	infection, are common. Sutures should be removed as late as possible since the wounds heal slowly. Abscess drains should be left in place for a prolonged period until the infection is completely resolved.93Hyperglycemia has been shown to be a significant risk factor of postoperative infections.94 Tight blood glucose con-trol, beginning preoperatively and continued into the operating room and beyond, has been associated with significant reduc-tion in infectious complications, in particular following cardiac surgery.95,96 Too tight of a glycemic control (80–100 mg/dL) appears to be associated with more complications and is as effec-tive, if not less than, moderate control (120–180 mg/dL).97,98Another host factor that has been implicated in the devel-opment of superficial surgical site infection relates to the state of the subcutaneous capillary bed. Thomas K. Hunt had shown through several decades of work that this capillary bed is exqui-sitely sensitive to hypovolemia,99

1	site infection relates to the state of the subcutaneous capillary bed. Thomas K. Hunt had shown through several decades of work that this capillary bed is exqui-sitely sensitive to hypovolemia,99 hypothermia,100 and stress, leading to rapid vasoconstriction with secondary impaired oxy-gen delivery and increased rates of infection.61 Maintenance of euvolemia, core temperature above 36°C to 36.5°C, and pain control have all been shown singly and additively to reduce rates of wound infections.63 Another suggestion has been to increase inspired Fio2 to 0.8 for the duration of the operation and in the immediate postoperative period, as a means of increasing sub-cutaneous tissue oxygen delivery. Although successful in most studies,62,101 there have also been negative results from such a single approach102; this suggests that addressing volume, tem-perature, pain control, and oxygen delivery in concert may be the more fruitful approach to reduce surgical wound infections.Chronic

1	a single approach102; this suggests that addressing volume, tem-perature, pain control, and oxygen delivery in concert may be the more fruitful approach to reduce surgical wound infections.Chronic WoundsChronic wounds are defined as wounds that have failed to pro-ceed through the orderly process that produces satisfactory ana-tomic and functional integrity or that have proceeded through the repair process without producing an adequate anatomic and functional result. The majority of wounds that have not healed in 3 months are considered chronic, although a duration as low as 4 weeks has been used to indicate chronicity. Skin ulcers, which usually occur in traumatized or vascular compromised soft tis-sue, are also considered chronic in nature, and proportionately are the major component of chronic wounds. In addition to the factors discussed earlier that can delay wound healing, other causative mechanisms may also play a role in the etiology of chronic wounds. Repeated trauma, poor

1	of chronic wounds. In addition to the factors discussed earlier that can delay wound healing, other causative mechanisms may also play a role in the etiology of chronic wounds. Repeated trauma, poor perfusion or oxygen-ation, and/or excessive inflammation contribute to the causation and the perpetuation of the chronicity of wounds.Unresponsiveness to normal regulatory signals also has been implicated as a predictive factor of chronic wounds. This may come about as a failure of normal growth factor synthesis,103 and thus an increased breakdown of growth factors within a wound environment that is markedly proteolytic because of overexpression of protease activity or a failure of the normal antiprotease inhibitor mechanisms.104 Fibroblasts from chronic wounds also have been found to have decreased proliferative potential, perhaps because of senescence105 or decreased expres-sion of growth factor receptors.106 Chronic wounds occur due to various etiologic factors, and several of the most

1	proliferative potential, perhaps because of senescence105 or decreased expres-sion of growth factor receptors.106 Chronic wounds occur due to various etiologic factors, and several of the most common are discussed later.Brunicardi_Ch09_p0271-p0304.indd 28901/03/19 4:50 PM 290BASIC CONSIDERATIONSPART IMalignant transformation of chronic ulcers can occur in any long-standing wound (Marjolin’s ulcer). Any wound that does not heal for a prolonged period of time is prone to malig-nant transformation. Malignant wounds are differentiated clini-cally from nonmalignant wounds by the presence of overturned wound edges (Fig. 9-10). In patients with suspected malignant transformations, biopsy of the wound edges must be performed to rule out malignancy. Cancers arising de novo in chronic wounds include both squamous and basal cell carcinomas.Ischemic Arterial Ulcers. These wounds occur due to a lack of blood supply and are typically extremely painful in patients with pure ischemic ulcers. They

1	both squamous and basal cell carcinomas.Ischemic Arterial Ulcers. These wounds occur due to a lack of blood supply and are typically extremely painful in patients with pure ischemic ulcers. They usually are associated with other symptoms of peripheral vascular disease, such as history of intermittent claudication, rest pain, and color or trophic changes. These wounds commonly are present at the most distal portions of the extremities such as the interdigital clefts, although more proxi-mal locations are also encountered. On examination, there may be diminished or absent pulses with decreased ankle-brachial index and poor formation of granulation tissue. Other signs of peripheral ischemia, such as dryness of skin, hair loss, scaling, and pallor can be present. The wound itself usually is shallow with smooth mar-gins, and a pale base and surrounding skin may be present. The management of these wounds is two-pronged and includes revas-cularization and wound care.107 Nonhealing of these

1	shallow with smooth mar-gins, and a pale base and surrounding skin may be present. The management of these wounds is two-pronged and includes revas-cularization and wound care.107 Nonhealing of these wounds is the norm unless successful revascularization is performed. In patients with ischemia and bed confinement, prevention of ulcer devel-opment is extremely important. Removal of restrictive stockings (in patients with critical ischemia), frequent repositioning, and surveillance are vital to preventing these ulcers.108Venous Stasis Ulcers. Although there is unanimous agree-ment that venous ulcers are due to venous stasis and increased venous pressure, there is less consensus as to what are the exact pathophysiologic pathways that lead to ulceration and impaired healing. On the microvascular level, there is alteration and distention of the dermal capillaries with leakage of fibrinogen into the tissues; polymerization of fibrinogen into fibrin cuffs leads to perivascular cuffing that

1	level, there is alteration and distention of the dermal capillaries with leakage of fibrinogen into the tissues; polymerization of fibrinogen into fibrin cuffs leads to perivascular cuffing that can impede oxygen exchange, thus contributing to ulceration. These same fibrin cuffs and the leakage of macromolecules such as fibrinogen and α2-macroglobulin trap growth factors and impede wound healing.103 Another hypothesis suggests that neutrophils adhere to the cap-illary endothelium and cause plugging with diminished dermal blood flow. Venous hypertension and capillary damage lead to extravasation of hemoglobin. The products of this breakdown are irritating and cause pruritus and skin damage. The result-ing brownish pigmentation of skin combined with the loss of subcutaneous fat produces characteristic changes called lipoder-matosclerosis. Regardless of the pathophysiologic mechanisms, the clinically characteristic picture is that of an ulcer that fails to reepithelialize despite the

1	characteristic changes called lipoder-matosclerosis. Regardless of the pathophysiologic mechanisms, the clinically characteristic picture is that of an ulcer that fails to reepithelialize despite the presence of adequate granulation tis-sue in a patient with skin color changes in the area of ulceration and signs of venous hypertension.Venous stasis occurs due to increased venous hyperten-sion caused by either venous insufficiency or venous outflow obstruction. Venous insufficiency can be due to any combina-tion of deep, superficial, and perforator vein valvular reflux. The higher the ambulatory venous hypertension, the higher the chance of ulceration. Chronic venous ulcers are commonly painless. Stasis ulcers tend to occur at the sites of incompetent Figure 9-10. Typical appearance of the malignant transformation of a long-standing chronic wound. (Used with permission by Dr. Robert S. Kirsner, University of Miami.)Brunicardi_Ch09_p0271-p0304.indd 29001/03/19 4:50 PM 291WOUND

1	the malignant transformation of a long-standing chronic wound. (Used with permission by Dr. Robert S. Kirsner, University of Miami.)Brunicardi_Ch09_p0271-p0304.indd 29001/03/19 4:50 PM 291WOUND HEALINGCHAPTER 9perforators, the most common being above the medial malleo-lus, over Cockett’s perforator. Upon examination, the typical location combined with a history of venous incompetence and other skin changes is diagnostic. The wound usually is shallow with irregular margins and pigmented surrounding skin.The cornerstone of treatment of venous ulcers is compres-sion therapy, although the best method to achieve it remains controversial. Compression can be accomplished via rigid or flexible means. The most commonly used method is the rigid, zinc oxide–impregnated, nonelastic bandage. Others have proposed a four-layered bandage approach as a more optimal method of obtaining graduated compression.109 Wound care in these patients focuses on maintaining a moist wound environ-ment, which

1	have proposed a four-layered bandage approach as a more optimal method of obtaining graduated compression.109 Wound care in these patients focuses on maintaining a moist wound environ-ment, which can be achieved with hydrocolloids. Other, more modern approaches include use of vasoactive substances and growth factor application, as well as the use of skin substitutes. Recently, sprayed allogeneic keratinocytes and fibroblasts plus four-layer bandages have been shown to hasten healing when compared to compression alone.110 Addressing the causes of venous hypertension aids in the healing of venous ulcers.109 Unfortunately, recurrences are frequent despite preventative measures, largely because of patients’ lack of compliance.111Diabetic Wounds. Ten percent to 25% of diabetic patients run the risk of developing ulcers. There are approximately 50,000 to 60,000 amputations performed in diabetic patients each year in the United States. The major contributors to the formation of diabetic

1	the risk of developing ulcers. There are approximately 50,000 to 60,000 amputations performed in diabetic patients each year in the United States. The major contributors to the formation of diabetic ulcers include neuropathy, foot deformity, and isch-emia. It is estimated that 60% to 70% of diabetic ulcers are due to neuropathy, 15% to 20% are due to ischemia, and another 15% to 20% are due to a combination of both. The neuropathy is both sensory and motor and is secondary to persistently elevated glucose levels. The loss of sensory function allows unrecognized injury to occur from ill-fitting shoes, foreign bodies, or other trauma. The motor neuropathy or Charcot’s foot leads to col-lapse or dislocation of the interphalangeal or metatarsophalan-geal joints, causing pressure on areas with little protection. There is also severe microand macrovascular circulatory impairment.Once ulceration occurs, the chances of healing are poor and the chances of recurrent ulceration are high. The

1	little protection. There is also severe microand macrovascular circulatory impairment.Once ulceration occurs, the chances of healing are poor and the chances of recurrent ulceration are high. The treatment of diabetic wounds involves local and systemic measures.112 Achievement of adequate blood sugar levels is very impor-tant. Most diabetic wounds are infected, and eradication of the infectious source is paramount to the success of healing. Treatment should address the possible presence of osteomyeli-tis and should employ antibiotics that achieve adequate levels both in soft tissue and bone. Wide debridement of all necrotic or infected tissue is another cornerstone of treatment. Offloading of the ulcerated area by using specialized orthotic shoes or casts allows for ambulation while protecting the fragile wound environment. Topical application of PDGF and granulocyte-macrophage colony-stimulating factor has met with limited but significant success in achieving closure.113 The

1	protecting the fragile wound environment. Topical application of PDGF and granulocyte-macrophage colony-stimulating factor has met with limited but significant success in achieving closure.113 The application of engineered skin allograft substitutes, although expensive, also has shown some significant success.114 Preven-tion and specifically foot care play an important role in the man-agement of diabetics.115Decubitus or Pressure Ulcers. The incidence of pressure ulcers ranges from 2.7% to 9% in the acute care setting, in com-parison to 2.4% to 23% in long-term care facilities. A pressure ulcer is a localized area of tissue necrosis that develops when soft tissue is compressed between a bony prominence and an external surface. Excessive pressure causes capillary collapse and impedes the delivery of nutrients to body tissues. Pressure ulcer formation is accelerated in the presence of friction, shear forces, and moisture. Other contributory factors in the patho-genesis of pressure

1	the delivery of nutrients to body tissues. Pressure ulcer formation is accelerated in the presence of friction, shear forces, and moisture. Other contributory factors in the patho-genesis of pressure ulcers include immobility, altered activity levels, altered mental status, chronic conditions, and altered nutritional status. The four stages of pressure ulcer formation are as follows: stage I, no blanching erythema of intact skin; stage II, partial-thickness skin loss involving epidermis or der-mis or both; stage III, full-thickness skin loss, but not through the fascia; and stage IV, full-thickness skin loss with extensive involvement of muscle and bone.The treatment of established pressure ulcers is most suc-cessful when carried out in a multidisciplinary manner by involving wound care teams consisting of physicians, nurses, dietitians, physical therapists, and nutritionists. Care of the ulcer itself comprises debridement of all necrotic tissue, maintenance of a favorable moist wound

1	consisting of physicians, nurses, dietitians, physical therapists, and nutritionists. Care of the ulcer itself comprises debridement of all necrotic tissue, maintenance of a favorable moist wound environment that will facilitate healing, relief of pressure, and addressing host issues such as nutritional, metabolic, and circulatory status. Debridement is most efficiently carried out surgically, but enzymatic proteo-lytic preparations and hydrotherapy also are used. The wound bed should be kept moist by employing dressings that absorb secretions but do not desiccate the wound.116 Operative repair, usually involving flap rotation, has been found to be useful in obtaining closure. Unfortunately, recurrence rates are extremely high, owing to the population at risk and the inability to fully address the causative mechanisms.117EXCESS HEALING Clinically, excess healing can be as significant as delayed or nonhealing. It is likely that more operative interventions are required for correction

1	the causative mechanisms.117EXCESS HEALING Clinically, excess healing can be as significant as delayed or nonhealing. It is likely that more operative interventions are required for correction of the morbidity associated with excessive healing than are required for wound failure. The clinical manifestations of exuberant healing are protean and dif-fer in the skin (mutilating or debilitating scars, burn contrac-tions), tendons (frozen repairs), the GI tract (strictures or stenoses), solid organs (cirrhosis, pulmonary fibrosis), or the peritoneal cavity (adhesive disease).Hypertrophic scars (HTSs) and keloids represent an over-abundance of fibroplasia in the dermal healing process. HTSs rise above the skin level but stay within the confines of the origi-nal wound and often regress over time. Keloids rise above the skin level as well, but they extend beyond the border of the original wound and rarely regress spontaneously (Fig. 9-11). Both HTSs and keloids occur after trauma to the skin

1	Keloids rise above the skin level as well, but they extend beyond the border of the original wound and rarely regress spontaneously (Fig. 9-11). Both HTSs and keloids occur after trauma to the skin and may be tender, pruritic, and cause a burning sensation. Keloids are 15 times more common in darker-pigmented ethnicities, with individuals of African, Spanish, and Asian ethnicities being especially susceptible. Men and women are equally affected. Genetically, the predilection to keloid formation appears to be autosomal dominant with incomplete penetration and variable expression.117,118HTSs usually develop within 4 weeks after trauma. The risk of HTS increases if epithelialization takes longer than 21 days, independent of site, age, and race. Rarely elevated more than 4 mm above the skin level, HTSs stay within the boundaries of the wound. They usually occur across areas of tension and flexor surfaces, which tend to be at right angles to joints or skin creases. The lesions are

1	skin level, HTSs stay within the boundaries of the wound. They usually occur across areas of tension and flexor surfaces, which tend to be at right angles to joints or skin creases. The lesions are initially erythematous and raised and over time may evolve into pale, flatter scars.4Brunicardi_Ch09_p0271-p0304.indd 29101/03/19 4:50 PM 292BASIC CONSIDERATIONSPART IKeloids can result from surgery, burns, skin inflamma-tion, acne, chickenpox, zoster, folliculitis, lacerations, abrasions, tattoos, vaccinations, injections, insect bites, or ear piercing, or may arise spontaneously. Keloids tend to occur 3 months to years after the initial insult, and even minor injuries can result in large lesions. They vary in size from a few millimeters to large, pedun-culated lesions with a soft to rubbery or hard consistency. While they project above surrounding skin, they rarely extend into underlying subcutaneous tissues. Certain body sites have a higher incidence of keloid formation, including

1	or hard consistency. While they project above surrounding skin, they rarely extend into underlying subcutaneous tissues. Certain body sites have a higher incidence of keloid formation, including the skin of the earlobe as well as the deltoid, presternal, and upper back regions. They rarely occur on eyelids, genitalia, palms, soles, or across joints. Keloids rarely involute spontaneously, and surgical intervention can lead to recurrence, often with a worse result (Table 9-8).Histologically, both HTSs and keloids demonstrate increased thickness of the epidermis with an absence of rete ridges. There is an abundance of collagen and glycoprotein deposition. Normal skin has distinct collagen bundles, mostly parallel to the epithelial surface, with random connections between bundles by fine fibrillar strands of collagen. In HTS, the collagen bundles are flatter and more random, and the fibers are in a wavy pattern. In keloids, the collagen bundles are virtu-ally nonexistent, and the fibers

1	strands of collagen. In HTS, the collagen bundles are flatter and more random, and the fibers are in a wavy pattern. In keloids, the collagen bundles are virtu-ally nonexistent, and the fibers are connected haphazardly in loose sheets with a random orientation to the epithelium. The collagen fibers are larger and thicker, and myofibroblasts are generally absent.119Keloidal fibroblasts have normal proliferation param-eters but synthesize collagen at a rate 20 times greater than that observed in normal dermal fibroblasts, and 3 times higher than fibroblasts derived from HTS. Abnormal amounts of extracel-lular matrix such as fibronectin, elastin, and proteoglycans also are produced. The synthesis of fibronectin, which promotes clot generation, granulation tissue formation, and reepithelializa-tion, decreases during the normal healing process; however, production continues at high levels for months to years in HTSs and keloids. This perturbed synthetic activity is mediated by altered

1	decreases during the normal healing process; however, production continues at high levels for months to years in HTSs and keloids. This perturbed synthetic activity is mediated by altered growth factor expression. TGF-β expression is higher in HTS, and both HTSand keloid-derived fibroblasts respond to lower concentrations of TGF-β than do normal dermal fibro-blasts. HTSs also express increased levels of insulin-like growth factor-1, which reduces collagenase mRNA activity and increases mRNA for types I and II procollagen.120 Keloid fibroblasts have enhanced expression of TGF-β1 and TGF-β2, VEGF, and plasminogen activator inhibitor-1 and an increased number of PDGF receptors; they also have upregulated anti-apoptotic gene expression, which can be differentially expressed within different areas of the same scar.The underlying mechanisms that cause HTSs and keloids are not known. The immune system appears to be involved in the formation of both HTSs and keloids, although the exact

1	areas of the same scar.The underlying mechanisms that cause HTSs and keloids are not known. The immune system appears to be involved in the formation of both HTSs and keloids, although the exact relationship is unknown. Much is inferred from the presence of various immune cells in HTSs and keloids. For example, in both HTSs and keloids, keratinocytes express human leuko-cyte antigen (HLA)-2 and ICAM-1 receptors, which are absent Figure 9-11. Recurrent keloid on the neck of a 17-year-old patient that had been revised several times. (Reproduced with permission from Cohen IK, Diegelmann RF, Lindblad WJ: Wound Healing: Biochemical and Clinical Aspects. Philadelphia, PA: WB Saunders/ Elsevier; 1992.)Table 9-8Characteristics of keloids and hypertrophic scars KELOIDHYPERTROPHIC SCARIncidenceRareFrequentEthnic groupsAfrican American, Asian, HispanicNo predilectionPrior injuryYesYesSite predilectionNeck, chest, ear lobes, shoulders, upper backAnywhereGeneticsAutosomal dominant with incomplete

1	groupsAfrican American, Asian, HispanicNo predilectionPrior injuryYesYesSite predilectionNeck, chest, ear lobes, shoulders, upper backAnywhereGeneticsAutosomal dominant with incomplete penetrationNoTimingSymptom-free interval; may appear years after injury4–6 weeks post injurySymptomsPain, pruritus, hyperesthesia, growth beyond wound marginsRaised, some pruritus, respects wound confinesRegressionNoFrequent spontaneousContractureRareFrequentHistologyHypocellular, thick, wavy collagen fibers in random orientationParallel orientation of collagen fibersBrunicardi_Ch09_p0271-p0304.indd 29201/03/19 4:50 PM 293WOUND HEALINGCHAPTER 9in normal scar keratinocytes. Keloids also have increased deposition of immunoglobulins IgG, IgA, and IgM, and their formation correlates with serum levels of IgE. Antinuclear antibodies against fibroblasts, epithelial cells, and endothelial cells are found in keloids, but not HTSs. HTSs have higher T lymphocyte and Langerhans cell contents. There is also a

1	Antinuclear antibodies against fibroblasts, epithelial cells, and endothelial cells are found in keloids, but not HTSs. HTSs have higher T lymphocyte and Langerhans cell contents. There is also a larger number of mast cells present in both HTSs and keloids compared to normal scars. Another recently described cell pop-ulation is the fibrocyte, a leukocyte subpopulation derived from peripheral mononuclear cells. Present in large numbers at the site of excess scarring, fibrocytes can stimulate fibroblast num-bers and collagen synthesis. They also generate large numbers of cytokines, growth factors, and extracellular matrix proteins, which are characteristically upregulated in keloid tissue. Other mechanisms that may cause abnormal scarring include mechani-cal tension (although keloids often occur in areas of minimal tension) and prolonged irritation and/or inflammation that may lead to the generation of abnormal concentrations of profibrotic cytokines.Treatment goals for keloid and HTS

1	occur in areas of minimal tension) and prolonged irritation and/or inflammation that may lead to the generation of abnormal concentrations of profibrotic cytokines.Treatment goals for keloid and HTS include restoration of function to the area, relief of symptoms, and prevention of recurrence. Many patients seek intervention due to cosmetic concerns. Because the underlying mechanisms causing keloids and HTSs remain unknown, many different modalities of treat-ment have been used without consistent success.121Excision alone of keloids is subject to a high recurrence rate, ranging from 45% to 100%. Inclusion of the dermal advancing edge that characterizes keloids, use of incisions in skin tension lines, and tension-free closure all have been pro-posed to decrease recurrence rates. There are fewer recurrences when surgical excision is combined with other modalities such as intralesional corticosteroid injection, topical application of silicone sheets, or the use of radiation or pressure.

1	fewer recurrences when surgical excision is combined with other modalities such as intralesional corticosteroid injection, topical application of silicone sheets, or the use of radiation or pressure. Surgery is recommended for debulking large lesions or as second-line therapy when other modalities have failed. Silicone application is relatively painless and should be maintained for 24 hours a day for about 3 months to prevent rebound hypertrophy. It may be secured with tape or worn beneath a pressure garment. The mechanism of action is not understood, but increased hydration of the skin, which decreases capillary activity, inflammation, hyperemia, and collagen deposition, may be involved. Silicone is more effective than other occlusive dressings and is an espe-cially good treatment for children and others who cannot toler-ate the pain involved in other modalities.102Intralesional corticosteroid injections decrease fibroblast proliferation, collagen and glycosaminoglycan synthesis, the

1	and others who cannot toler-ate the pain involved in other modalities.102Intralesional corticosteroid injections decrease fibroblast proliferation, collagen and glycosaminoglycan synthesis, the inflammatory process, and TGF-β levels. When used alone, however, there is a variable rate of response and recurrence; therefore, steroids are recommended as first-line treatment for keloids and second-line treatment for HTSs if topical thera-pies have failed. Intralesional injections are more effective on younger scars. They may soften, flatten, and give symptomatic relief to keloids, but they cannot make the lesions disappear, and they cannot narrow wide HTSs. Success is enhanced when used in combination with surgical excision. Serial injections every 2 to 3 weeks are required. Complications include skin atrophy, hypopigmentation, telangiectasias, necrosis, and ulceration.Although radiation destroys fibroblasts, it has variable, unreliable results and produces poor results, with 10% to 100%

1	skin atrophy, hypopigmentation, telangiectasias, necrosis, and ulceration.Although radiation destroys fibroblasts, it has variable, unreliable results and produces poor results, with 10% to 100% recurrence when used alone. It is more effective when combined with surgical excision. The timing, duration, and dosage for radiation therapy are still controversial, but doses ranging from 1500 to 2000 rads appear effective. Given the risks of hyperpig-mentation, pruritus, erythema, paresthesias, pain, and possible secondary malignancies, radiation should be reserved for adults with scars resistant to other modalities.Pressure aids collagen maturation, flattens scars, and improves thinning and pliability. It reduces the number of cells in a given area, possibly by creating ischemia, which decreases tissue metabolism and increases collagenase activity. External compression is used to treat HTSs, especially after burns. Ther-apy must begin early, and a pressure between 24 and 30 mmHg must be

1	tissue metabolism and increases collagenase activity. External compression is used to treat HTSs, especially after burns. Ther-apy must begin early, and a pressure between 24 and 30 mmHg must be achieved in order to exceed capillary pressure, yet pre-serve peripheral blood circulation. Garments should be worn for 23 to 24 hours a day for up to 1 or more years to avoid rebound hypertrophy. Scars older than 6 to 12 months respond poorly.Topical retinoids also have been used as treatment for both HTSs and keloids, with reported responses of 50% to 100%. Intralesional injections of IFN-γ, a cytokine released by T lymphocytes, reduce collagen types I, II, and III by decreasing mRNA and possibly by reducing levels of TGF-β. As mono-therapy, IFN-γ has failed because of high recurrence rates due to resistance to repeated injections. More recently, imiquimod, an immunomodulator that induces IFN-γ and other cytokines at the site of application, has been recommended following exci-sion.

1	due to resistance to repeated injections. More recently, imiquimod, an immunomodulator that induces IFN-γ and other cytokines at the site of application, has been recommended following exci-sion. Intralesional injections of chemotherapeutic agents such as 5-fluorouracil have been used both alone and in combina-tion with steroids. The use of bleomycin or mitomycin C has been reported to achieve some success in older scars resistant to steroids.Peritoneal Scarring. Peritoneal adhesions are fibrous bands of tissues formed between organs that are normally separated and/or between organs and the internal body wall. Most intra-abdominal adhesions are a result of peritoneal injury, either by a prior surgical procedure or due to intra-abdominal infection. Postmortem examinations demonstrate adhesions in 67% of patients with prior surgical procedures and in 28% with a history of intra-abdominal infection. Intra-abdominal adhesions are the most common cause (65%–75%) of small bowel obstruction,

1	in 67% of patients with prior surgical procedures and in 28% with a history of intra-abdominal infection. Intra-abdominal adhesions are the most common cause (65%–75%) of small bowel obstruction, especially in the ileum. Operations in the lower abdomen have a higher chance of producing small bowel obstruction. Follow-ing rectal surgery, left colectomy, or total colectomy, there is an 11% chance of developing small bowel obstruction within 1 year, and this rate increases to 30% by 10 years. Adhesions also are a leading cause of secondary infertility in women and can cause substantial abdominal and pelvic pain. Adhesions account for 2% of all surgical admissions and 3% of all lapa-rotomies in general surgery.122Adhesions form when the peritoneal surface is damaged due to surgery, thermal or ischemic injury, inflammation, or foreign body reaction. The injury disrupts the protective meso-thelial cell layer lining the peritoneal cavity and the underlying connective tissue. The injury

1	or ischemic injury, inflammation, or foreign body reaction. The injury disrupts the protective meso-thelial cell layer lining the peritoneal cavity and the underlying connective tissue. The injury elicits an inflammatory response consisting of hyperemia, fluid exudation, release, and activation of white blood cells and platelets in the peritoneal cavity, activa-tion of inflammatory cytokines, and the onset of the coagulation and complement cascades. Fibrin deposition occurs between the damaged but opposed serosal surfaces. These filmy adhesions often are transient and degraded by proteases of the fibrinolytic system, with restoration of the normal peritoneal surface. If insufficient fibrinolytic activity is present, permanent fibrous adhesions will form by collagen deposition within 1 week of the injury (Fig. 9-12).Brunicardi_Ch09_p0271-p0304.indd 29301/03/19 4:50 PM 294BASIC CONSIDERATIONSPART IExtensive research has been done on the effect of surgery and peritonitis on the

1	1 week of the injury (Fig. 9-12).Brunicardi_Ch09_p0271-p0304.indd 29301/03/19 4:50 PM 294BASIC CONSIDERATIONSPART IExtensive research has been done on the effect of surgery and peritonitis on the fibrinolytic and inflammatory cascades within the peritoneal cavity. During normal repair, fibrin is prin-cipally degraded by the fibrinolytic protease plasmin, which is derived from inactive plasminogen through the action of two plasminogen activators (PA): tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Fibrino-lytic activity in peritoneal fluid is reduced after abdominal sur-gery due to initial decreases in tPA levels and later to increases in plasminogen activator inhibitor-1 (PAI-1), which are induced by various cytokines, including TNF-α, IL-1, and interleukin-6 (IL-6).123There are two major strategies for adhesion prevention or reduction. Surgical trauma is minimized within the peritoneum by careful tissue handling, avoiding desiccation and

1	interleukin-6 (IL-6).123There are two major strategies for adhesion prevention or reduction. Surgical trauma is minimized within the peritoneum by careful tissue handling, avoiding desiccation and ischemia, and spare use of cautery, laser, and retractors. Fewer adhesions form with laparoscopic surgical techniques due to reduced tis-sue trauma. The second major advance in adhesion prevention has been the introduction of barrier membranes and gels, which separate and create barriers between damaged mesothelial sur-faces, allowing for adhesion-free healing. Currently, only three products are Food and Drug Administration (FDA) approved for reducing adhesion formation: Interceed (oxidized regen-erated cellulose, indicated only in pelvic surgery), Seprafilm (a film composed of hyaluronic acid and carboxymethylcel-lulose) that is usually applied below the incision, and Adept (4% icodextrin, a corn starch derivative in electrolyte solution, also for use mainly in pelvic surgery). However,

1	and carboxymethylcel-lulose) that is usually applied below the incision, and Adept (4% icodextrin, a corn starch derivative in electrolyte solution, also for use mainly in pelvic surgery). However, use of these substances directly over bowel anastomoses is contraindicated due to an elevated risk of leak.124 There have been innumerable studies investigating different molecules in hopes of preventing adhesion formation, but most of the success is limited to animal models, and clinically significant results in humans have yet to be achieved.TREATMENT OF WOUNDSLocal Care (Fig. 9-13)Management of acute wounds begins with obtaining a careful history of the events surrounding the injury. The history is fol-lowed by a meticulous examination of the wound. Examination should assess the depth and configuration of the wound, the extent of nonviable tissue, and the presence of foreign bodies and other contaminants. The wound/ulcer should be described based on location, dimensions, presence of

1	configuration of the wound, the extent of nonviable tissue, and the presence of foreign bodies and other contaminants. The wound/ulcer should be described based on location, dimensions, presence of infection, drainage and type of drainage, base characteristics, presence or absence of necrosis, presence of pain, and description of edges. Possible etiology should be mentioned, and the presence of sys-temic factors and circulation should be evaluated.After completion of the history, examination, and admin-istration of tetanus prophylaxis, the wound should be meticu-lously anesthetized. Lidocaine (0.5%–1%) or bupivacaine (0.25%–0.5%) combined with a 1:100,000 to 1:200,000 dilution of epinephrine provides satisfactory anesthesia and hemostasis. 5ThrombinCoagulationFibrinogenPeritoneal fluidInflammationFibrin++PAI-1, PAI-2tPA, uPAFibrin residuesFibrinolysisFibrinolysisdegradationRestitutionFibroblasts and capillariesFibrous adhesionPeritoneal

1	fluidInflammationFibrin++PAI-1, PAI-2tPA, uPAFibrin residuesFibrinolysisFibrinolysisdegradationRestitutionFibroblasts and capillariesFibrous adhesionPeritoneal injuryBleedingMacrophagesmesotheliumPlateletsTFFigure 9-12. Fibrin formation and degradation in peritoneal tis-sue repair and adhesion formation. PAI-1, PAI-2 = types 1 and 2 plasminogen activator inhibitor; TF = tissue factor; tPA = tissue plasminogen activator; uPA = urokinase plasminogen activator.Figure 9-13. Algorithm for management of acute wounds.Management of Acute Wounds1. Examinationa) Depth? Underlying structures injuredb) Configuration?c) Nonviable tissue?4. Follow-upa) Cellulitis/drainage?b) Suture removal -4–5 days for face -7–10 days other skin3. Approximationa) Deep layers -Fascial layers only -Absorbable sutureb) Superficial layers -Meticulous alignment -Nonabsorbable sutures in skin -Staples -Monofilament -Dermal glues2. Preparationa) Anesthetic -Lidocaine w or w/o epinephrineb) Exploration -Underlying

1	Superficial layers -Meticulous alignment -Nonabsorbable sutures in skin -Staples -Monofilament -Dermal glues2. Preparationa) Anesthetic -Lidocaine w or w/o epinephrineb) Exploration -Underlying structures injuredc) Cleansing -Pulsed irrigation, saline onlyd) Hemostasise) Debride nonviable tissuef) Betadine on surrounding sking) Antibiotics (rare)h) TetanusBrunicardi_Ch09_p0271-p0304.indd 29401/03/19 4:50 PM 295WOUND HEALINGCHAPTER 9Epinephrine should not be used in wounds of the fingers, toes, ears, nose, or penis, due to the risk of tissue necrosis secondary to terminal arteriole vasospasm in these structures.Irrigation to visualize all areas of the wound and remove foreign material is best accomplished with normal saline (with-out additives). High-pressure wound irrigation is more effective in achieving complete debridement of foreign material and non-viable tissues. Iodine, povidone-iodine, hydrogen peroxide, and organically based antibacterial preparations have all been shown

1	in achieving complete debridement of foreign material and non-viable tissues. Iodine, povidone-iodine, hydrogen peroxide, and organically based antibacterial preparations have all been shown to impair wound healing due to injury to wound neutrophils and macrophages, and thus should not be used. All hematomas present within wounds should be carefully evacuated and any remaining bleeding sources controlled with ligature or cautery. If the injury has resulted in the formation of a marginally viable flap of skin or tissue, this should be resected or revascularized prior to further wound repair and closure.After the wound has been anesthetized, explored, irri-gated, and debrided, the area surrounding the wound should be cleaned, inspected, and the surrounding hair clipped. The area surrounding the wound should be prepared with povi-done iodine, chlorhexidine, or similar bacteriostatic solutions and draped with sterile towels. Having ensured hemostasis and adequate debridement of nonviable

1	the wound should be prepared with povi-done iodine, chlorhexidine, or similar bacteriostatic solutions and draped with sterile towels. Having ensured hemostasis and adequate debridement of nonviable tissues and removal of any remaining foreign bodies, irregular, macerated, or beveled wound edges should be debrided in order to provide a fresh edge for reapproximation. Although plastic surgical techniques such as Wor Z-plasty are seldom recommended for acute wounds, great care must be taken to realign wound edges properly. This is particularly important for wounds that cross the vermilion border, eyebrow, or hairline. Initial sutures that realign the edges of these different tissue types will speed and greatly enhance the aesthetic outcome of the wound repair.In general, the smallest suture required to hold the vari-ous layers of the wound in approximation should be selected in order to minimize suture-related inflammation. Nonabsorbable or slowly absorbing monofilament sutures are most

1	required to hold the vari-ous layers of the wound in approximation should be selected in order to minimize suture-related inflammation. Nonabsorbable or slowly absorbing monofilament sutures are most suitable for approximating deep fascial layers, particularly in the abdomi-nal wall. Subcutaneous tissues should be closed with braided absorbable sutures, with care to avoid placement of sutures in fat. Multiple layer closure of the abdominal wall is preferable. However, additional layers with sutures might increase the risk of wound infection.In areas of significant tissue loss, rotation of adjacent mus-culocutaneous flaps or free flaps may be required to provide sufficient tissue mass for closure. In cases of significant superfi-cial tissue loss, split-thickness skin grafting (placed in a delayed manner to assure an adequate tissue bed) may be required and will speed formation of an intact epithelial barrier to fluid loss and infection. In acute, contaminated wounds with skin loss, use

1	manner to assure an adequate tissue bed) may be required and will speed formation of an intact epithelial barrier to fluid loss and infection. In acute, contaminated wounds with skin loss, use of porcine skin xenografts or skin cadaveric allografts might be needed to avoid infection.After closing deep tissues and replacing significant tissue deficits, skin edges should be reapproximated for cosmesis and to aid in rapid wound healing. Skin edges may be quickly reap-proximated with stainless steel staples or nonabsorbable mono-filament sutures. Care must be taken to remove these from the wound prior to epithelialization of the skin tracts where sutures or staples penetrate the dermal layer. Failure to remove the sutures or staples prior to 7 to 10 days after repair will result in a cosmetically inferior wound. Where wound cosmesis is impor-tant, the aforementioned problems may be avoided by place-ment of buried dermal sutures using absorbable braided sutures. This method of wound

1	inferior wound. Where wound cosmesis is impor-tant, the aforementioned problems may be avoided by place-ment of buried dermal sutures using absorbable braided sutures. This method of wound closure allows for a precise reapproxi-mation of wound edges and may be enhanced by application of wound closure tapes to the surface of the wound. Intradermal absorbable sutures do not require removal.The development of octyl-cyanoacrylate tissue glues has shown good results for the management of simple, linear wounds with viable skin edges. These new glues are less prone to brittleness and have superior burst-strength characteristics. Studies have shown them to be suitable for use in contaminated situations without significant risk of infection. When used in the previously mentioned types of wounds, these glues appear to provide superb cosmetic results and result in significantly less trauma than sutured repair, particularly when used in pediatric patients.AntibioticsAntibiotics should be used

1	these glues appear to provide superb cosmetic results and result in significantly less trauma than sutured repair, particularly when used in pediatric patients.AntibioticsAntibiotics should be used only when there is an obvious wound infection. Most wounds are contaminated or colonized with bacteria. The presence of a host response constitutes an infection and justifies the use of antibiotics. Signs of infec-tion to look for include erythema, cellulitis, swelling, and puru-lent discharge. Indiscriminate use of antibiotics should be avoided to prevent emergence of multidrug-resistant bacteria.Antibiotic treatment of acute wounds must be based on organisms suspected to be found within the infected wound and the patient’s overall immune status. When a single specific organism is suspected, treatment may be commenced using a single antibiotic. Conversely, when multiple organisms are suspected, as with enteric contamination or when a patient’s immune function is impaired by diabetes,

1	treatment may be commenced using a single antibiotic. Conversely, when multiple organisms are suspected, as with enteric contamination or when a patient’s immune function is impaired by diabetes, chronic disease, or medication, treatment should commence with a broad-spectrum antibiotic or several agents in combination. Antibiotics also can be delivered topically as part of irrigations or dressings, although their efficacy is questionable.DressingsThe main purpose of wound dressings is to provide the ideal envi-ronment for wound healing. The dressing should facilitate the major changes taking place during healing to produce an opti-mally healed wound. Although the ideal dressing still is not a clinical reality, technological advances are promising (Table 9-9).Dressings should take into account the type of wound and the comorbid conditions and associated factors such as edema and circulation. In patients with edema, dressing should compress the edematous area to aid in healing. In

1	account the type of wound and the comorbid conditions and associated factors such as edema and circulation. In patients with edema, dressing should compress the edematous area to aid in healing. In patients with significant circulation compromise, a compressing dress-ing should be avoided. The dressing should maintain 67Table 9-9Desired characteristics of wound dressingsPromote wound healing (maintain moist environment)ConformabilityPain controlOdor controlNonallergenic and nonirritatingPermeability to gasSafetyNontraumatic removalCost-effectivenessConvenienceBrunicardi_Ch09_p0271-p0304.indd 29501/03/19 4:50 PM 296BASIC CONSIDERATIONSPART Ian adequate moist environment but should help absorption excessive drainage. Occlusion of a wound with dressing mate-rial helps healing by controlling the level of hydration and oxy-gen tension within the wound. It also allows transfer of gases and water vapor from the wound surface to the atmosphere. Occlusion affects both the dermis and

1	the level of hydration and oxy-gen tension within the wound. It also allows transfer of gases and water vapor from the wound surface to the atmosphere. Occlusion affects both the dermis and epidermis, and it has been shown that exposed wounds are more inflamed and develop more necrosis than covered wounds. Occlusion also helps in dermal collagen synthesis and epithelial cell migration and lim-its tissue desiccation. Since it may enhance bacterial growth, occlusion is contraindicated in infected and/or highly exudative wounds.Dressings can be classified as primary or secondary. A pri-mary dressing is placed directly on the wound and may provide absorption of fluids and prevent desiccation, infection, and adhe-sion of a secondary dressing. A secondary dressing is one that is placed on the primary dressing for further protection, absorption, compression, and occlusion. Although the ideal dressing does not exist, many types of dressings help achieve certain goals, so knowledge of the

1	primary dressing for further protection, absorption, compression, and occlusion. Although the ideal dressing does not exist, many types of dressings help achieve certain goals, so knowledge of the wound and the dressing function is essential to make it possible to choose the appropriate dressing.Absorbent Dressings. This type of dressing helps con-trol exudate without soaking through the dressing, which can increase infection potential.Nonadherent Dressings. Nonadherent dressings are impreg-nated with paraffin, petroleum jelly, or water-soluble jelly for use as nonadherent coverage. A secondary dressing must be placed on top to seal the edges and prevent desiccation and infection.Occlusive and Semiocclusive Dressings. Occlusive and semiocclusive dressings provide a good environment for clean, minimally exudative wounds. These film dressings are water-proof and impervious to microbes but permeable to water vapor and oxygen.Hydrophilic and Hydrophobic Dressings. These dressings are

1	clean, minimally exudative wounds. These film dressings are water-proof and impervious to microbes but permeable to water vapor and oxygen.Hydrophilic and Hydrophobic Dressings. These dressings are components of a composite dressing. Hydrophilic dressing aids in absorption, whereas a hydrophobic dressing is water-proof and prevents absorption.Hydrocolloid and Hydrogel Dressings. Hydrocolloid and hydrogel dressings attempt to combine the benefits of occlusion and absorbency. Hydrocolloids and hydrogels form complex structures with water, and fluid absorption occurs with particle swelling, which aids in atraumatic removal of the dressing. Absorption of exudates by the hydrocolloid dressing leaves a yellowish-brown gelatinous mass after dressing removal that can be washed off. Hydrogel is a cross-linked polymer that has high water content. Hydrogels allow a high rate of evaporation without compromising wound hydration, which makes them useful in burn wound treatment.Alginates. Alginates

1	cross-linked polymer that has high water content. Hydrogels allow a high rate of evaporation without compromising wound hydration, which makes them useful in burn wound treatment.Alginates. Alginates are derived from brown algae and con-tain long chains of polysaccharides containing mannuronic and glucuronic acid. The ratios of these sugars vary with the species of algae used, as well as the season of harvest. Processed as the calcium forms, alginates turn into soluble sodium alginate through ion exchange in the presence of wound exudates. The polymers gel, swell, and absorb a great deal of fluid. Alginates are being used when there is skin loss, in open surgical wounds with medium exudation, and on full-thickness chronic wounds. Alginate widely used as primary dressing and can be reinforced with other forms of dressing such as compression dressing.Absorbable Materials. Absorbable materials are mainly used within wounds as hemostats and include collagen, gelatin, oxi-dized cellulose,

1	with other forms of dressing such as compression dressing.Absorbable Materials. Absorbable materials are mainly used within wounds as hemostats and include collagen, gelatin, oxi-dized cellulose, and oxidized regenerated cellulose.Medicated Dressings. Medicated dressings have long been used as a drug-delivery system. Agents delivered in the dress-ings include benzoyl peroxide, zinc oxide, neomycin, and bacitracin-zinc. These agents have been shown to increase epi-thelialization by 28%.The type of dressing to be used depends on the amount of wound drainage. A nondraining wound can be covered with semiocclusive dressing. Drainage of less than 1 to 2 mL/d may require a semiocclusive or absorbent nonadherent dressing. Moderately draining wounds (3–5 mL/d) can be dressed with a nonadherent primary layer plus an absorbent secondary layer plus an occlusive dressing to protect normal tissue. Heavily draining wounds (>5 mL/d) require a similar dressing as mod-erately draining wounds, but with

1	layer plus an absorbent secondary layer plus an occlusive dressing to protect normal tissue. Heavily draining wounds (>5 mL/d) require a similar dressing as mod-erately draining wounds, but with the addition of a highly absor-bent secondary layer.Mechanical Devices. Mechanical therapy augments and improves on certain functions of dressings, in particular the absorption of exudates and control of odor. The negative pres-sure dressing systems assists in wound closure by applying localized negative pressure to the surface and margins of the wound. The negative-pressure therapy is applied to a special foam dressing cut to the dimensions of the wound and posi-tioned in the wound cavity or over a flap or graft. The continu-ous negative pressure is very effective in removing exudates from the wound. This form of therapy has been found to be effective for chronic open wounds (diabetic ulcers and stages III and IV pressure ulcers), acute and traumatic wounds,125 flaps and grafts, and subacute

1	This form of therapy has been found to be effective for chronic open wounds (diabetic ulcers and stages III and IV pressure ulcers), acute and traumatic wounds,125 flaps and grafts, and subacute wounds (i.e., dehisced incisions), although more randomized trials need to be carried out to con-firm efficacy. Different types of sponges are available to use on wounds with negative pressure systems.Skin ReplacementsAll wounds require coverage in order to prevent evaporative losses and infection and to provide an environment that pro-motes healing. Both acute and chronic wounds may demand use of skin replacement, and several options are available.Conventional Skin Grafts. Skin grafts have long been used to treat both acute and chronic wounds. Split(partial-) thickness grafts consist of the epidermis plus part of the dermis, whereas full-thickness grafts retain the entire epidermis and dermis. Autologous grafts (autografts) are transplants from one site on the body to another; allogeneic

1	plus part of the dermis, whereas full-thickness grafts retain the entire epidermis and dermis. Autologous grafts (autografts) are transplants from one site on the body to another; allogeneic grafts (allografts, homografts) are transplants from a living nonidentical donor or cadaver to the host; and xenogeneic grafts (heterografts) are taken from another species (e.g., porcine). Split-thickness grafts require less blood supply to restore skin function. The dermal component of full-thickness grafts lends mechanical strength and resists wound contraction better, resulting in improved cosmesis. Allo-geneic and xenogeneic grafts require the availability of tissue, are subject to rejection, and may contain pathogens.The use of skin grafts or bioengineered skin substitutes and other innovative treatments (e.g., topically applied growth factors, systemic agents, and gene therapy) cannot be effec-tive unless the wound bed is adequately prepared. This may include debridement to remove necrotic

1	(e.g., topically applied growth factors, systemic agents, and gene therapy) cannot be effec-tive unless the wound bed is adequately prepared. This may include debridement to remove necrotic or fibrinous tissue, control of edema, revascularization of the wound bed, reduc-tion in the bacterial burden, with minimal exudate. Temporary Brunicardi_Ch09_p0271-p0304.indd 29601/03/19 4:50 PM 297WOUND HEALINGCHAPTER 9placement of allografts or xenografts may be used to prepare the wound bed.Cellular and Tissue-Based Products in Chronic Wound and Ulcer ManagementWound management and ulcer healing are among the most chal-lenging problems in medical practices. The lack of large clini-cal trials, the heterogeneity of wound causes, and mechanisms of chronicity add to the complexity of wound management. In most cases, management is based on the experi-ence of the providers and the availability of the treatment modalities within the health facilities. In spite of advances in wound care, the basic

1	In most cases, management is based on the experi-ence of the providers and the availability of the treatment modalities within the health facilities. In spite of advances in wound care, the basic principles of wound management remain to be essential to healing. Additional measures and products might accelerate the rate of healing but will not replace basic wound care. The basic principles include achieving optimal blood flow, control of infection, removal of debris, proper dress-ing, offloading the injured area, and compression therapy in certain cases. Once these basic principles have been achieved, advanced treatment modalities such as cellular and tissue-based products (CTP) can be considered for enhanced healing. CTPs presumably act by altering the biology of wounds and ulcers or by preparing the wound/ulcer bed for healing and other potential procedures. CTPs are divided into two categories: dermoindu-cive and democonductive.126 As the name indicates, dermoindu-cive products help

1	the wound/ulcer bed for healing and other potential procedures. CTPs are divided into two categories: dermoindu-cive and democonductive.126 As the name indicates, dermoindu-cive products help provide cells and factors that will activate healing within the wound by inducing tissue growth or inducing granulation within the wound. Such products include Apligraph (Organogenesis, Canton, MA), Theraskin (Soluble systems, LLC, Newport News, VA), and Dermagraft (Organogenesis Canton, MA). None of these products have achieved healing in all wounds, but generally they have improved the percentage of healing, or achieved healing over a shorter period of time. In a pivotal Apligraf study, for example, it was found that patients who received Apligraf achieved 56% healing over 65 days com-pared to 38% healing over 90 days in those who received saline dressing.127 On the other hand, the dermoconductive products provide scaffolding to a wound ending in a neodermis by allow-ing migration of

1	to 38% healing over 90 days in those who received saline dressing.127 On the other hand, the dermoconductive products provide scaffolding to a wound ending in a neodermis by allow-ing migration of surrounding tissues across the wound, and this helps healing. An example of such products is Integra, which is composed of type 1 bovine collagen, shark chrondroitin 6-sulfate, and a silicone layer and helps to prepare the wound bed for subsequent skin grafting.128 In our experience, we use Integra for wound bed preparation in superficial wounds but also, in some cases, in deeper wounds. In addition, we also use it to cover exposed bone, especially smaller areas and tendons; how-ever, it might require more than one application in some cases to achieve optimal wound bed preparation. In a systematic review of the literature, the authors reviewed 15 randomized trials, one prospective comparative study, and five systematic reviews. The authors concluded that living cell-based skin sub-stitutes

1	review of the literature, the authors reviewed 15 randomized trials, one prospective comparative study, and five systematic reviews. The authors concluded that living cell-based skin sub-stitutes have a convincing supportive body of evidence in wound healing with some promise for acellular skin substitutes.129 In their review, the authors indicated that the evidence they based their review on was of low quality. Stem cell–based therapy is gaining more traction in the management of difficult wounds. Although it was initially used as an attempt to achieve scarless healing, stem cell therapy has gained more popularity in recent years as a means for enhanced healing and skin regeneration, in addition to reduction of scar formation. Similar to other fields, stem cells in wound healing produce growth factors and che-mokines that can differentiate into different cells and tissues. Stem cells in wound healing are mostly derived from human amniotic membrane of placental tissue. Examples of

1	growth factors and che-mokines that can differentiate into different cells and tissues. Stem cells in wound healing are mostly derived from human amniotic membrane of placental tissue. Examples of such prod-ucts include Epiphex (MiMedx Group Inc, Marietta CA) and Grafix (Osirix Therapeutics Inc, Columbia, MD), among others. It is believed that the growth factors in the amniotic membranes are preserved through different processes, thus assisting with wound healing. Multiple growth factors derived from the amni-otic membrane are thought to contribute to wound healing, including vascular endothelia growth factor, platelet-derived growth factor, epidermal growth factor, transforming growth factor, and others.130,131 A significant drawback of such products is that they are extremely expensive. Furthermore, it is recom-mended that these products should be used weekly to achieve optimal healing, which adds to the cost of using such products.Originally, these products were devised to provide

1	Furthermore, it is recom-mended that these products should be used weekly to achieve optimal healing, which adds to the cost of using such products.Originally, these products were devised to provide cover-age of extensive wounds because of the limited availability of autografts, which remains the ideal tissue coverage. However, CTPs have now gained acceptance as natural dressings. Manu-factured by tissue engineering, they combine novel materials with living cells to provide functional skin substitutes, providing a bridge between dressings and skin grafts.Skin substitutes have the theoretical advantages of being readily available and not requiring painful harvests like in skin grafts, and they may be applied freely or with surgical sutur-ing. In addition, they promote healing, either by stimulating host cytokine generation or by providing cells that may also produce growth factors locally. Their disadvantages include limited sur-vival, high cost, and the need for multiple applications

1	host cytokine generation or by providing cells that may also produce growth factors locally. Their disadvantages include limited sur-vival, high cost, and the need for multiple applications (Table 9-10). Allografting, albeit with a very thin graft, may at times be required to accomplish complete coverage.A variety of skin substitutes are available, each with its own set of advantages and disadvantages; however, the ideal skin substitute has yet to be developed (Table 9-11). The devel-opment of the newer composite substitutes, which provide both the dermal and epidermal components essential for permanent skin replacement, may represent an advance toward that goal.Cultured epithelial autografts (CEAs) represent expanded autologous or homologous keratinocytes. CEAs are expanded from a biopsy of the patient’s own skin, will not be rejected, and can stimulate reepithelialization as well as the growth of underlying connective tissue. Keratinocytes harvested from a biopsy roughly the size of

1	of the patient’s own skin, will not be rejected, and can stimulate reepithelialization as well as the growth of underlying connective tissue. Keratinocytes harvested from a biopsy roughly the size of a postage stamp are cultured with fibroblasts and growth factors and grown into sheets that can cover large areas and give the appearance of normal skin. Until the epithelial sheets are sufficiently expanded, the wound must be covered with an occlusive dressing or a temporary allograft or xenograft. The dermis regenerates very slowly, if at all, for full-thickness wounds because the sheets are very fragile, are difficult to work with, are susceptible to infection, and do not resist contracture well, leading to poor cosmetic results.8Table 9-10Desired features of tissue-engineered skinRapid reestablishment of functional skin (epidermis/dermis)Receptive to body’s own cells (e.g., rapid “take” and integration)Graftable by a single, simple procedureGraftable on chronic or acute

1	skinRapid reestablishment of functional skin (epidermis/dermis)Receptive to body’s own cells (e.g., rapid “take” and integration)Graftable by a single, simple procedureGraftable on chronic or acute woundsEngraftment without use of extraordinary clinical intervention (i.e., immunosuppression)Brunicardi_Ch09_p0271-p0304.indd 29701/03/19 4:50 PM 298BASIC CONSIDERATIONSPART ICEAs are available from cadavers, unrelated adult donors, or neonatal foreskins. Fresh or cryopreserved cultured alloge-neic keratinocytes can be left in place long enough to be super-seded by multiplying endogenous skin cells because, unlike allografts containing epidermal Langerhans cells, they do not express major histocompatibility antigens. Cryopreserved CEAs are readily available “off the shelf,” and provide growth factors that may aid healing. However, like autologous keratinocyte sheets, the grafts lack the strength provided by a dermal compo-nent and pose a risk of disease transmission.Viable fibroblasts

1	factors that may aid healing. However, like autologous keratinocyte sheets, the grafts lack the strength provided by a dermal compo-nent and pose a risk of disease transmission.Viable fibroblasts can be grown on bioabsorbable or non-bioabsorbable meshes to yield living dermal tissue that can act as a scaffold for epidermal growth. Fibroblasts stimulated by growth factors can produce type I collagen and glycosamino-glycans (e.g., chondroitin sulfates), which adhere to the wound surface to permit epithelial cell migration, as well as adhesive ligands (e.g., the matrix protein fibronectin), which promote cell adhesion. This approach has the virtue of being less timeconsuming and expensive than culturing keratinocyte sheets. There are a number of commercially available, bioengineered dermal replacements approved for use in burn wound treatment as well as other indications.Bioengineered skin substitutes have evolved from kera-tinocyte monolayers to dermal equivalents to split-thickness

1	replacements approved for use in burn wound treatment as well as other indications.Bioengineered skin substitutes have evolved from kera-tinocyte monolayers to dermal equivalents to split-thickness products with a pseudo-epidermis, and most recently, to prod-ucts containing both epidermal and dermal components that resemble the three-dimensional structure and function of nor-mal skin (see Table 9-11). Indicated for use with standard com-pression therapy in the treatment of venous insufficiency ulcers and for the treatment of neuropathic diabetic foot ulcers, these bilayered skin equivalents also are being used in a variety of wound care settings.Growth Factor Therapy. As discussed previously, it is believed that nonhealing wounds result from insufficient or inadequate growth factors in the wound environment. A simplistic solu-tion would be to flood the wound with single or multiple growth factors in order to “jump-start” healing and reepithelialization. Although there is a large body

1	wound environment. A simplistic solu-tion would be to flood the wound with single or multiple growth factors in order to “jump-start” healing and reepithelialization. Although there is a large body of work demonstrating the effects of growth factors in animals, translation of these data into clinical practice has met with limited success. Growth factors for clinical use may be either recombinant or homologous/autologous. Autol-ogous growth factors are harvested from the patient’s own plate-lets, yielding an unpredictable combination and concentration of factors, which are then applied to the wound. This approach allows treatment with patient-specific factors at an apparently physiologic ratio of growth factor concentrations. Disappoint-ingly, a recent meta-analysis failed to demonstrate any value for autologous platelet-rich plasma in the treatment of chronic wounds.132 Recombinant molecular biologic means permit the purification of high concentrations of individual growth factors.

1	any value for autologous platelet-rich plasma in the treatment of chronic wounds.132 Recombinant molecular biologic means permit the purification of high concentrations of individual growth factors. Current FDA-approved formulations, as well as those used exper-imentally, deliver concentrations approximately 103 times higher than those observed physiologically.At present, only platelet-derived growth factor BB (PDGF-BB) is currently approved by the FDA for treatment of diabetic foot ulcers. Application of recombinant human PDGF-BB in a gel suspension to these wounds increases the incidence of total healing and decreases healing time. Several other growth factors have been tested clinically and show some promise, but currently none are approved for use. A great deal more needs to be discovered about the concentration, temporal release, and receptor cell population before growth factor therapy is to make a consistent impact on wound healing.Gene or Cell Therapy. Given the disappointing

1	about the concentration, temporal release, and receptor cell population before growth factor therapy is to make a consistent impact on wound healing.Gene or Cell Therapy. Given the disappointing results from the application of purified growth factors onto wounds, the pos-sible therapeutic potential of gene therapy has been recognized and studied. Direct access to the open wound bed, which char-acterizes almost all chronic wounds, has facilitated this therapy. Gene delivery to wounds includes traditional approaches such as viral vectors and plasmid delivery or, more recently, electro-poration and microseeding.Although a variety of genes expressing interleukin-8, PDGF, IGF-1, keratinocyte growth factor, and laminin-5 have been successfully delivered to wounds in both animal and human models, the effects have been modest and specific to unique wound situations. Delivering extra genes into the wound bed presents the challenge of expression of the necessary signals to turn the genes on and

1	the effects have been modest and specific to unique wound situations. Delivering extra genes into the wound bed presents the challenge of expression of the necessary signals to turn the genes on and off at appropriate times so that dys-regulated, hypertrophic, and abnormal healing does not occur. Elaborate systems have been created for topical use as on/off switches for genes. The more important question is which genes to express, in what temporal sequence, and in what regions of the wound bed, as it is unlikely that a single gene coding for one protein can significantly affect overall healing. There is growing consensus that delivery of genes is not going to represent the universal solution. Although gene therapy replaces missing or Table 9-11Advantages and disadvantages of various bioengineered skin substitutesSKIN SUBSTITUTEADVANTAGESDISADVANTAGESCultured allogeneic keratinocyte graftNo biopsy needed“Off the shelf” availabilityProvides wound coveragePromotes healingUnstableDoes not

1	skin substitutesSKIN SUBSTITUTEADVANTAGESDISADVANTAGESCultured allogeneic keratinocyte graftNo biopsy needed“Off the shelf” availabilityProvides wound coveragePromotes healingUnstableDoes not prevent wound contractureInadequate cosmesisPossibility of disease transmissionFragileBioengineered dermal replacementPrevents contractureGood prep for graft applicationLimited ability to drive reepithelializationLargely serves as temporary dressingCultured bilayer skin equivalentMore closely mimics normal anatomyDoes not need secondary procedureEasily handledCan be sutured, meshed, etc.CostShort shelf lifeTrue engraftment questionableBrunicardi_Ch09_p0271-p0304.indd 29801/03/19 4:50 PM 299WOUND HEALINGCHAPTER 9defective genes, most acute wounds already have and express the necessary genes for successful healing and the wound envi-ronment produces signals adequate to the activation of these genes. What, if any, are the deficiencies in gene expression or activity in failed wounds is

1	for successful healing and the wound envi-ronment produces signals adequate to the activation of these genes. What, if any, are the deficiencies in gene expression or activity in failed wounds is unknown.Another approach is to deliver multiple genes coding for proteins that can act synergistically and even in a timed sequence, as would occur during normal healing. This would involve the use of activated cells that participate in the heal-ing sequence that could be delivered in an activated state to the wound environment. Use of mesenchymal stem cells as a delivery vector for many genes simultaneously is the latest such approach. The feasibility of applying bone marrow-derived, umbilical cord-derived, adipose-derived, and epidermal stem cells that can differentiate into various cells that participate in the wound healing response also has been documented. These cells, as part of their differentiation and activation in the wound, have been shown to produce a variety of growth factors

1	participate in the wound healing response also has been documented. These cells, as part of their differentiation and activation in the wound, have been shown to produce a variety of growth factors includ-ing VEGF, PDGF, bFGF, and MMP-9. The challenges remain how to maintain the viability and activity of the transplanted cells, how to document that the observed effects are due to the delivered cells, and what are the mechanisms necessary for reg-ulating or ending their activity.Oxygen Therapy in Wound HealingOxygen is required for almost all steps of wound healing and is also an important factor in the body’s defense against bacterial infection.133 In addition to its role in healing, oxygen plays an essential role in the production of reactive oxygen species such as superoxide that are angiogenesis stimulators and are bacterio-static. Chronic wounds have a decreased oxygen supply, and for a long time lack of oxygen was recognized as a potential cause of delayed healing. To counteract

1	stimulators and are bacterio-static. Chronic wounds have a decreased oxygen supply, and for a long time lack of oxygen was recognized as a potential cause of delayed healing. To counteract this delay, supple-mental oxygen therapy was used to improve healing, and both local oxygen therapy and systemic therapy were used for that purpose. Local therapy included oxygen dressings and topical oxygen therapy, while systemic therapy included supplemental inspired oxygen therapy and pressurized oxygen treatment.134 Of the different methods of oxygen therapy, pressurized oxy-gen, also termed hyperbaric oxygen therapy (HBOT) is the most used modality and the most investigated. In HBOT, oxygen is delivered under pressure, more than atmospheric pressure, lead-ing to a higher concentration of oxygen in tissues. The Undersea and Hyperbaric Medical Society (UHMS) defined HBOT as an intervention that involves breathing near 100% oxygen intermit-tently under high pressure achieved by a pressurized

1	in tissues. The Undersea and Hyperbaric Medical Society (UHMS) defined HBOT as an intervention that involves breathing near 100% oxygen intermit-tently under high pressure achieved by a pressurized chamber to more than sea level pressure (sea level pressure = 1 atmosphere absolute [ATA]). The therapeutic pressure should be at least 1.4 ATA. Chambers can be single occupancy or multiple occu-pancy.135 Although there are numerous indications and potential indications for HBOT, there are 14 accepted indications by Undersea and Hyperbaric Medical Society, and the FDA. Indi-cations related to wounds and ulcers include clostridial myone-crosis, crush injury, radiation-induced soft tissue and bone necrosis, necrotizing soft tissue infections, diabetic ulcers Wag-ner III or higher, refractory osteomyelitis, and thermal burns.135 Two systemic reviews were published on the effect of HBOT on wound healing. The first one was published in 2003 on all stud-ies done up to 2001.136 Generally, the

1	osteomyelitis, and thermal burns.135 Two systemic reviews were published on the effect of HBOT on wound healing. The first one was published in 2003 on all stud-ies done up to 2001.136 Generally, the review showed beneficial effects of HBOT on different disease processes. HBOT was found to improve graft survival, complete healing of grafts, and lessen infection in patients with a graft. Patients with osteoradionecrosis showed improved bone changes. The effect on tissue radionecrosis was described as positive, but not all studies showed statistical significance. HBOT was found to have improved amputation rates in patients with gas gangrene with better healing. In addition, HBOT was found to reduce mortality rates from necrotizing fasciitis, and significantly decreased wound size in nonhealing diabetic wounds. In the sec-ond systemic review, a total of 29 studies published between 2001 and 2016 were included. A total of 14 studies were related to chronic wounds: 12 studies in acute

1	diabetic wounds. In the sec-ond systemic review, a total of 29 studies published between 2001 and 2016 were included. A total of 14 studies were related to chronic wounds: 12 studies in acute wounds, 1 study on both acute and chronic wounds, and 2 experimental studies. Out of those 29 studies, 18 (62%) showed at least one positive out-come.134 One of the drawbacks of such studies in general is the lack of heterogeneity and the lack of accurate randomization. Perhaps, HBOT in diabetic foot ulcers attracted more attention than other forms of skin ulcers. In patients with diabetic foot ulcers, studies showed that HBOT led to significantly more healing wounds at 1 year,137 greater reduction in the wound bed,138 greater healing with less proximal amputation,139 and more complete wound healing at 1 month after HBOT.140Biofilm and Chronic Wound HealingTraditionally, nonhealing of chronic wounds has been associ-ated with numerous risk factors, including longer duration of ulcers, advanced

1	at 1 month after HBOT.140Biofilm and Chronic Wound HealingTraditionally, nonhealing of chronic wounds has been associ-ated with numerous risk factors, including longer duration of ulcers, advanced age, increased body mass index, venous reflux, arterial and venous disease, nutritional deficiencies, diabetes mellitus, and smoking. Chronic bacterial infection is another factor that has been associated with nonhealing wounds.Chronic wounds, in general, behave differently in relation to bacterial growth when compared to acute wounds. Chronic wounds develop bacterial growth that is resistant to invasion by antibiotics and is protected from the host immune defenses. Biofilm is the term used for the bacterial growth on a chronic wound that is encapsulated by a protective layer made up of the host and bacterial proteins. Bjarnsholt et al have suggested a simplified definition of the biofilm as “an aggregate of bacteria tolerant to treatment and the host defense.” It has been found that more

1	and bacterial proteins. Bjarnsholt et al have suggested a simplified definition of the biofilm as “an aggregate of bacteria tolerant to treatment and the host defense.” It has been found that more than 60% of chronic wounds have a biofilm.141 Biofilms lead to a chronic inflammatory process that will interfere with healing.142 Biofilms are formed in three stages. The first stage—the reversible bacterial adhesion stage—is formed by adhesion of bacteria to the surface of the wound. The second stage is the permanent adhesion or maturation stage in which the organisms permanently attach, proliferate, and maturate in the wound surface. This stage depends on a micro-bial-to-microbial cell communication system, called quorum sensing, in which small signaling molecules are released and gradually increase in concentration. The signaling molecules regulate gene expression and help form the biofilm. Lastly, in the third stage, organisms secrete a surrounding protective matrix called the

1	increase in concentration. The signaling molecules regulate gene expression and help form the biofilm. Lastly, in the third stage, organisms secrete a surrounding protective matrix called the extracellular polymeric substance (EPS). Once a biofilm colony forms, it will continuously shed bacteria to uncolonized areas, causing more biofilm colonies to form.143,144The presence of biofilms protects bacteria from the host defenses. Bacteria will then begin to exhibit phenotypic and genotypic plurality: the former allows bacteria to adapt to dif-ferent growth conditions such as nutrient availability, pH, and oxidizing potential within the biofilm, while the latter allows for virulence and bacterial resistance to drugs. The genetic plurality is passed horizontally among bacteria in the wound, adding to the resistance to treatment and allowing the bacteria to avoid the defense mechanisms of the host such as bacterial phagocytosis, 910Brunicardi_Ch09_p0271-p0304.indd 29901/03/19 4:50 PM

1	adding to the resistance to treatment and allowing the bacteria to avoid the defense mechanisms of the host such as bacterial phagocytosis, 910Brunicardi_Ch09_p0271-p0304.indd 29901/03/19 4:50 PM 300BASIC CONSIDERATIONSPART Ineutrophil degranulation, and formation of reactive oxygen spe-cies.145-147 In some cases, the biofilm allows bacteria to become quiescent and thus become less sensitive to antimicrobials that typically affect dividing bacterial cells.144,148Biofilms can form and recover from debridement rap-idly. Fully mature biofilm colonies can form within 2 to 4 days depending on the species and growth conditions. Common bacterial species such as Staphylococcus, Streptococcus, and Pseudomonas can attach to the surface of the wound in min-utes, forming adherent microcolonies in 2 to 4 hours. Bacterial species develops the extracellular polymeric substances (EPS) and resistance to disinfectants, antiseptics, and antibiotics in 6 to 12 hours. The biofilm then develops into

1	in 2 to 4 hours. Bacterial species develops the extracellular polymeric substances (EPS) and resistance to disinfectants, antiseptics, and antibiotics in 6 to 12 hours. The biofilm then develops into mature colonies in 2 to 4 days. Furthermore, it rapidly recovers form mechanical disruption, such as debridement, within 24 hours.144The presence of biofilms leads to delayed wound healing by stimulating chronic inflammation in the wound. The host responds to the biofilms by mobilizing macrophages and neutro-phils to the biofilm area, resulting in secretions of high levels of reactive oxygen species and proteases that can cause damage to the normal and healing tissue. The resulting increase in exudate production provides a source of nutrients to the biofilms, lead-ing to more resistance to healing.149The best method of treating wounds with biofilms is not well defined. It is believed that frequent debridement to mechanically remove the biofilm remains to be the best method of treatment.

1	best method of treating wounds with biofilms is not well defined. It is believed that frequent debridement to mechanically remove the biofilm remains to be the best method of treatment. The frequency of debridement is not well defined, although a weekly debridement has been suggested to improve healing. More than one method of debridement might be needed in wounds resistant to healing. A few products have been sug-gested to control and remove biofilms, such as surfactant and some wound cleansing formulas. In addition, topical broad-spectrum antimicrobials such as silver, iodine, honey, and others have been suggested to aid in the treatment of biofilms. Using a various combination of debridement methods, cleansing agents, and antimicrobials may be needed in resistant chronic wounds. An effective therapy to combat biofilm should include effective removal of the biofilm, antimicrobial eradication of bacteria/organisms, and prevention of biofilm recurrence, and these treat-ment options

1	therapy to combat biofilm should include effective removal of the biofilm, antimicrobial eradication of bacteria/organisms, and prevention of biofilm recurrence, and these treat-ment options may need to be repeated multiple times to achieve effective therapy. Many agents and devices thought to be effec-tive in eradicating and treating biofilms are under evaluation or development; however, their long-term effect is yet to be proven.REFERENCESEntries highlighted in bright blue are key references. 1. Winter GD. Formation of the scab and the rate of epithelialisa-tion of superficial wounds in the skin of the young domestic pig. Nature. 1962;193:293-294. 2. Gulliver G, ed. The Works of John Hunter. London: Longman; 1837. 3. Feiken E, Romer J, Eriksen J, et al. Neutrophils express tumor necrosis factor-alpha during mouse skin wound healing. J Invest Dermatol. 1995;105(1):120-123. 4. Dovi JV, He L-K, DiPietro LA. Accelerated wound closure in neutrophil-depleted mice. J Leukoc Biol.

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1	oxygen in lower extremity wound healing. Clin Podiatr Med Surg. 2007;24(3):529-546. 127. Veves A, Falanga V, Armostrong DG, Sabolinski ML; Apligraf Diabetic Foot Ulcer Study. Graftskin, a human skin equivalent, is effective in the management of noninfected neuropathic dia-betic foot ulcers: a prospective randomized multicenter clini-cal trial. Diabetes Care. 2001;24(2):290-295. 128. Kim PJ, Attinger CE, Steinberg JS, Evans KK. Integra bilayer wound matrix application for complex lower extremity soft tissue reconstruction. Surg Technol Inter. 2014;24:65-73. 129. Felder JM, Goyal SS, Attinger CE. A systematic review of skin substitutes for foot ulcers. Plast Recontr Surg. 2012;130(1):145-164. 130. Gu C, Huang S, Gao D, et al. Angiogenic effect of mesenchy-mal stem cells as a therapeutic target for enhancing diabetic wound healing. Int J Low Extrem Wounds. 2014;13(2):88-93. 131. Zelen CM, Snyder RJ, Serena TE, Li WW. The use of human amnion/chroion membrane in the clinical setting for

1	for enhancing diabetic wound healing. Int J Low Extrem Wounds. 2014;13(2):88-93. 131. Zelen CM, Snyder RJ, Serena TE, Li WW. The use of human amnion/chroion membrane in the clinical setting for lower extremity repair: a review. Clin Podiatr Med Surg. 2015;32:135-146. 132. Martinez-Zapata MJ, Marti-Carvajal AJ, Sola I, et al. Autol-ogous platelet-rich plasma for treating chronic wounds. Cochrane Database Syst Rev. 2012;10:CD006899. 133. Rodriguez PG, Felix FN, Woodley DT, Shim EK. The role of oxygen in wound healing: a review of the literature. Dermatol Surg. 2008:34:1159-1169. 134. De Smet GH, Kroese LF, Menon AG, et al. Oxygen thera-pies and their effects on wound healing. Wound Rep Reg. 2017;25(4):591-608. 135. Weaver LK, ed. The Undersea and Hyperbaric Medical Soci-ety Manual on Hyperbaric Oxygen Therapy Indications. 13th ed. North Palm Beach, FL: Best Publishing Company; 2014, ix-xi. 136. Wang C, Schwaitzberg S, Berliner E, Zarin DA, Lau J. Hyper-baric oxygen for treating wounds:

1	Oxygen Therapy Indications. 13th ed. North Palm Beach, FL: Best Publishing Company; 2014, ix-xi. 136. Wang C, Schwaitzberg S, Berliner E, Zarin DA, Lau J. Hyper-baric oxygen for treating wounds: a systemic review of the literature. Arch Surg. 2003;138:272-280. 137. Londahl M, Katzman P, Nilson A, Hammarlund C. Hyperbaric oxygen therapy facilitates healing of chronic wound ulcers in patients with diabetes. Diabetes Care. 2010;33(5):998-1003. 138. Ma L, Li P, Shi Z, Hou T, Chen X, Du J. A prospective, random-ized, controlled study of hyperbaric oxygen therapy: effects on healing and oxidative stress of ulcer tissue in patients with diabetic foot ulcers. Ostomy Wound Manag. 2103;59(3):18-24. 139. Duzgun AP, Satir HZ, Ozozan O, Saylam B, Kulah B, Coskun F. Effect of hyperbaric oxygen therapy on diabetic foot ulcers. J Foot Ankle Surg. 2008;47(6):515-519. 140. Kaur S, Pawar M, Banerjee N, Garg R. Evaluation of the efficacy of hyperbaric oxygen therapy in the management of chronic

1	therapy on diabetic foot ulcers. J Foot Ankle Surg. 2008;47(6):515-519. 140. Kaur S, Pawar M, Banerjee N, Garg R. Evaluation of the efficacy of hyperbaric oxygen therapy in the management of chronic nonhealing ulcer and role of periwound transcu-taneous oximetry as predictor of wound healing response: a randomized prospective controlled trial. J Anaesthesiol Clin Pharmacol. 2012:28(1):70-75. 141. Bjarnsholt T, Cooper R, Fletcher J, et al. Management of biofilm. WoundsInternational.com. Available at: http://www .woundsinternational.com/wuwhs/view/position-document-management-of-biofilm. Accessed June 26, 2018. 142. Wolcott R, Rhoads D, Dowd S. Biofilms and chronic wound inflammation. J Wound Care. 2008;17(8):333-341. 143. Scali C, Kunimoto B. An update on chronic wounds and the role of biofilms. J Cutan Med Surg. 2013;17(6):371-376. 144. Philips PL, Wolcott RD, Fletcher J, Schultz GS. Biofilms made easy. WoundsInternational.com. Available at: http://www

1	wounds and the role of biofilms. J Cutan Med Surg. 2013;17(6):371-376. 144. Philips PL, Wolcott RD, Fletcher J, Schultz GS. Biofilms made easy. WoundsInternational.com. Available at: http://www .woundsinternational.com/made-easys/view/biofilms-made-easy. Accessed June 26, 2018. 145. Thomson CH. Biofilms: do they affect wound healing? Int Wound J. 2011;8(1):63-67. 146. Bjarnshot T, Bjarnsholt T, Kirketerp-Moller K, Jensen P, et al. Why chronic wounds won’t heal: a novel hypothesis. Wound Repair Regen. 2008;16(1):2-10. 147. Leid J, Wilson C, Shirtliff M, Hassett DJ, Parsek MR, Jeffers AK. The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from INF-gamma-mediated mac-rophage killing. J Immunol. 2005;175(1):7512-7518. 148. Hall-Stoodley L, Stoodley P. Evolving concepts in biofilm infections. Cell Microbiol. 2009;11(7):1034-1043. 149. Lawrence JR, Swerhone GD, Kuhlicke U, Neu TR. In situ evidence of microdomains in the polymer matrix of bacterial microcolonies.

1	in biofilm infections. Cell Microbiol. 2009;11(7):1034-1043. 149. Lawrence JR, Swerhone GD, Kuhlicke U, Neu TR. In situ evidence of microdomains in the polymer matrix of bacterial microcolonies. Can J Microbiol. 2007;53(3):450-458.Brunicardi_Ch09_p0271-p0304.indd 30301/03/19 4:50 PM

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1	ONCOLOGY AND SURGICAL PRACTICEAs the population ages, oncology is becoming a larger portion of surgical practice. The surgeon often is responsible for the initial diagnosis and management of solid tumors. Knowledge of cancer epidemiology, etiology, staging, and natural history is required for initial patient assessment, as well as to determina-tion of the optimal surgical therapy.Modern cancer therapy is multidisciplinary, involving the coordinated care of patients by surgeons, medical oncologists, radiation oncologists, reconstructive surgeons, pathologists, radi-ologists, and primary care physicians. Primary (or defini-tive) surgical therapy refers to en bloc resection of tumor with adequate margins of normal tissues and regional lymph nodes as necessary. Adjuvant therapy refers to radiation therapy and systemic therapies, including chemotherapy, immunotherapy, hormonal therapy, and, increasingly, biologic therapy. The pri-mary goal of surgical and radiation therapy is local and

1	therapy and systemic therapies, including chemotherapy, immunotherapy, hormonal therapy, and, increasingly, biologic therapy. The pri-mary goal of surgical and radiation therapy is local and regional control. On the other hand, the primary goal of adjuvant therapy is systemic control by treatment of distant foci of subclinical dis-ease to prevent distant recurrence. Surgeons must be familiar with adjuvant therapies to coordinate multidisciplinary care and to determine the best sequence of therapy. They must also be aware of the potential for patients to receive effective systemic therapies prior to surgery as a means of reducing tumor volume.Recent advances in molecular biology are revolutionizing medicine. New information is being translated rapidly into clinical use, with the development of new prognostic and predictive markers and new biologic therapies. Increasingly cancer therapy is becoming personalized, incorporating information about each patient’s tumor characteristics,

1	of new prognostic and predictive markers and new biologic therapies. Increasingly cancer therapy is becoming personalized, incorporating information about each patient’s tumor characteristics, patient’s own genome, as well as host immune responses and tumor microenvironment, into clinical decisionmaking. It is therefore essential that surgeons understand the prin-ciples of molecular oncology to appropriately interpret these new contributions and incorporate them into practice.12OncologyWilliam E. Carson III, Funda Meric-Bernstam, and Raphael E. Pollock 10chapterOncology and Surgical Practice 305Epidemiology 306Basic Principles of Cancer Epidemiology / 306Cancer Incidence and Mortality in the United States / 306Global Statistics on Cancer Incidence and Mortality / 307Cancer Biology 310Hallmarks of Cancer / 310Cell Proliferation and Transformation / 310Cancer Initiation / 310Cell-Cycle Dysregulation in Cancer / 311Oncogenes / 313Alterations in Apoptosis in Cancer Cells / 314Autophagy

1	of Cancer / 310Cell Proliferation and Transformation / 310Cancer Initiation / 310Cell-Cycle Dysregulation in Cancer / 311Oncogenes / 313Alterations in Apoptosis in Cancer Cells / 314Autophagy in Cancer Cells / 315Cancer Invasion / 315Angiogenesis / 316Metastasis / 316Epithelial-Mesenchymal Transition / 317Cancer Stem Cells / 318Cancer Etiology 318Cancer Genomics / 318Tumor Heterogeneity and Molecular Evolution / 319Genes Associated With Hereditary Cancer Risk / 320APC Gene and Familial Adenomatous Polyposis / 324PTEN and Cowden Disease / 325RET Proto-Oncogene and Multiple Endocrine Neoplasia Type 2 / 325Chemical Carcinogens / 326Physical Carcinogens / 326Viral Carcinogens / 326Cancer Risk Assessment 328Cancer Screening 330Cancer Diagnosis 332Cancer Staging 333Tumor Markers 334Prognostic and Predictive Tissue Markers / 334Serum Markers / 335Circulating Tumor Cells / 336Bone Marrow Micrometastases / 337Surgical Approaches to Cancer Therapy 337Multidisciplinary Approach to Cancer /

1	and Predictive Tissue Markers / 334Serum Markers / 335Circulating Tumor Cells / 336Bone Marrow Micrometastases / 337Surgical Approaches to Cancer Therapy 337Multidisciplinary Approach to Cancer / 337Surgical Management of Primary Tumors / 337Surgical Management of the Regional Lymph Node Basin / 338Surgical Management of Distant Metastases / 339Chemotherapy 340Clinical Use of Chemotherapy / 340Principles of Chemotherapy / 340Anticancer Agents / 340Combination Chemotherapy / 341Drug Toxicity / 341Administration of Chemotherapy / 341Hormonal Therapy 342Targeted Therapy 342Immunotherapy 342Gene Therapy 345Mechanisms of Intrinsic and Acquired Drug Resistance 345Radiation Therapy 346Physical Basis of Radiation Therapy / 346Biologic Basis of Radiation Therapy / 346Radiation Therapy Planning / 347Side Effects / 348Cancer Prevention 348Trends in Oncology 349Cancer Screening and Diagnosis / 349Surgical Therapy / 349Systemic Therapy / 349Brunicardi_Ch10_p0305-p0354.indd 30522/02/19 2:14

1	/ 347Side Effects / 348Cancer Prevention 348Trends in Oncology 349Cancer Screening and Diagnosis / 349Surgical Therapy / 349Systemic Therapy / 349Brunicardi_Ch10_p0305-p0354.indd 30522/02/19 2:14 PM 306Key Points1 Modern cancer therapy is multidisciplinary, involving coor-dinated care by surgeons, medical oncologists, radiation oncologists, reconstructive surgeons, pathologists, radiolo-gists, and primary care physicians.2 Understanding cancer biology is essential to successfully implement personalized cancer therapy.3 The following alterations are critical for malignant cancer growth: self-sufficiency of growth signals, insensitivity to growth-inhibitory signals, evasion of apoptosis, potential for limitless replication, angiogenesis, invasion and metastasis, reprogramming of energy metabolism, and evading immune destruction.EPIDEMIOLOGYBasic Principles of Cancer EpidemiologyThe term incidence refers to the number of new cases occur-ring. Incidence is usually expressed as the

1	metabolism, and evading immune destruction.EPIDEMIOLOGYBasic Principles of Cancer EpidemiologyThe term incidence refers to the number of new cases occur-ring. Incidence is usually expressed as the number of new cases per 100,000 persons per year. Mortality refers to the number of deaths occurring and is expressed as the number of deaths per 100,000 persons per year. Incidence and mortality data are usually available through cancer registries. Mortality data are also available as public records in many countries where deaths are registered as vital statistics, often with the cause of death. In areas where cancer registries do not exist, mortality data are used to extrapolate incidence rates. These numbers are likely to be less accurate than registry data, as the relationship between incidence and cause-specific death is likely to vary significantly among countries owing to the variation in health care delivery.The incidence of cancer varies by geography. This is due in part to genetic

1	and cause-specific death is likely to vary significantly among countries owing to the variation in health care delivery.The incidence of cancer varies by geography. This is due in part to genetic differences and in part to differences in environ-mental and dietary exposures. Epidemiologic studies that moni-tor trends in cancer incidence and mortality have tremendously enhanced our understanding of the etiology of cancer. Further-more, analysis of trends in cancer incidence and mortality allows us to monitor the effects of different preventive and screening measures, as well as the evolution of therapies for specific cancers.The two types of epidemiologic studies that are conducted most often to investigate the etiology of cancer and the effect of prevention modalities are cohort studies and case-control stud-ies. Cohort studies follow a group of people who initially do not have a disease over time and measure the rate of development of a disease. In cohort studies, a group that is

1	and case-control stud-ies. Cohort studies follow a group of people who initially do not have a disease over time and measure the rate of development of a disease. In cohort studies, a group that is exposed to a certain environmental factor or intervention usually is compared to a group that has not been exposed (e.g., smokers vs. nonsmokers). A case-control study compares a group of patients affected with a disease to a group of individuals without the disease and looks back retrospectively to compare how frequently the exposure to a risk factor is present in each group to determine the rela-tionship between the risk factor and the disease. The results are expressed in terms of an odds ratio, or relative risk. A relative risk <1 indicates a protective effect of the exposure, whereas a relative risk >1 indicates an increased risk of developing the disease with exposure.Cancer Incidence and Mortality in the United StatesIn the year 2017, it is estimated that 1.7 million new cancer cases

1	risk >1 indicates an increased risk of developing the disease with exposure.Cancer Incidence and Mortality in the United StatesIn the year 2017, it is estimated that 1.7 million new cancer cases will be diagnosed in the United States, excluding carci-noma in situ of any site except bladder, and excluding basal cell and squamous cell carcinomas of the skin.1 In addition, 63,410 cases of carcinoma in situ of the breast, and 74,680 of melanoma in situ are expected.1It is estimated that in 2017 an estimated 600,920 people will die of cancer in the United States, corresponding to about 1650 deaths per day.1 The estimated new cancer cases and deaths by cancer type are shown in Table 10-1.1 The most com-mon causes of cancer death in men are cancers of the lung and bronchus, colon and rectum, and prostate; in women, the most common cancers are of the lung and bronchus, breast, and colon and rectum.1 These four cancers account for almost half of total cancer deaths among men and women.The

1	and prostate; in women, the most common cancers are of the lung and bronchus, breast, and colon and rectum.1 These four cancers account for almost half of total cancer deaths among men and women.The annual age-adjusted cancer incidence rates among males and females for selected cancer types are shown in Fig. 10-1.1 Incidence rates are declining for most cancer sites (Fig. 10-2).1 Incidence rates for thyroid cancer have begun to stabilize recently, possibly due to changes in clinical practice guidelines that were initiated in 2009 and included more conservative indications for biopsy. The age-adjusted incidence rate of breast cancer started to decrease from 2001 to 2004.2 This decrease in breast cancer inci-dence has at least temporally been associated with the first report of the Women’s Health Initiative, which documented an increased risk of coronary artery disease and breast cancer with the use of hormone replacement therapy; this was followed by a drop in the use of hormone

1	Health Initiative, which documented an increased risk of coronary artery disease and breast cancer with the use of hormone replacement therapy; this was followed by a drop in the use of hormone replacement therapy by postmenopausal women in the United States.2 Unfortunately, there was a slight increase in breast cancer incidence from 2004 to 2013. This was driven wholly by nonwhite women; rates increased by about 2% per year among women other than white or black and by 0.5% per year among black women. Thus, rates have risen slightly in women as a whole from 2013 to 2017, although rates remained stable in white women.Declines in colorectal cancer incidence have been mainly attributed to increased screening that allows for removal of pre-cancerous polyps. Prostate cancer rates rapidly increased and decreased between 1995 and 1998. These trends are thought to be attributable to increased use of prostate-specific antigen (PSA) screening.3 Due to growing concerns about overdiagno-sis and

1	and decreased between 1995 and 1998. These trends are thought to be attributable to increased use of prostate-specific antigen (PSA) screening.3 Due to growing concerns about overdiagno-sis and overtreatment, a U.S. Preventive Services Task Force recommended against routine use of PSA testing to screen for prostate cancer. As a result, there was more than 10% annual reduction in prostate cancer incidence from 2010 to 2013. Dif-ferences in lung cancer incidence patterns between women and men are thought to reflect historical differences in tobacco use. Differences in smoking prevalence is also thought to contribute to regional differences in lung cancer incidence. Lung cancer incidence is fourfold higher in Kentucky, which has the highest smoking prevalence, compared with Utah, which has the lowest smoking prevalence.1The 5-year survival rates for selected cancers are listed in Table 10-2. From 2010 to 2014, cancer death rates decreased by 1.8% per year in men and by 1.4% per year in

1	the lowest smoking prevalence.1The 5-year survival rates for selected cancers are listed in Table 10-2. From 2010 to 2014, cancer death rates decreased by 1.8% per year in men and by 1.4% per year in women.1 These declines in mortality have been consistent in the past decade and larger than what was observed in the previous decade.3 Over the Brunicardi_Ch10_p0305-p0354.indd 30622/02/19 2:14 PM 307ONCOLOGYCHAPTER 10Table 10-1Estimated new cancer cases and deaths, United States, 2017a ESTIMATED NEW CASESESTIMATED DEATHS ESTIMATED NEW CASESESTIMATED DEATHSAll cancers1,688,780600,920Genital system279,80059,100Oral cavity and pharynx49,6709700 Uterine cervix12,8204210Digestive system310,440157,700 Uterine corpus61,38010,920 Esophagus16,94015,690 Ovary22,44014,080 Stomach28,00010,960 Vulva60201150 Small intestine10,1901390 Vagina and other genital, female48101240 Colon and rectum95,520 / 39,91050,260b Prostate161,36026,730 Anus, anal canal, and anorectum82001100 Testis8850410 Liver

1	intestine10,1901390 Vagina and other genital, female48101240 Colon and rectum95,520 / 39,91050,260b Prostate161,36026,730 Anus, anal canal, and anorectum82001100 Testis8850410 Liver and intrahepatic bile duct40,71028,920 Penis and other genital, male2120360 Gallbladder and other biliary11,7403830Urinary system146,65032,190 Pancreas53,67043,090 Urinary bladder79,03016,870 Other digestive organs55602460 Kidney and renal pelvis63,99014,400Respiratory system243,170160,420 Ureter and other urinary organs3630920 Larynx13,3603660Eye and orbit3130330 Lung and bronchus220,500155,870Brain and other nervous system23,80016,700 Other respiratory organs7310890Endocrine system59,2503010Bones and joints32601550 Thyroid56,8702010Soft tissue (including heart)12,3904990 Other endocrine23801000Skin (excluding basal and squamous)95,36013,590Lymphoma80,50021,210 Melanoma87,1109730Multiple myeloma30,28012,590 Other nonepithelial82503860Leukemia62,13024,500Breast255,18041,070Other and unspecified primary

1	basal and squamous)95,36013,590Lymphoma80,50021,210 Melanoma87,1109730Multiple myeloma30,28012,590 Other nonepithelial82503860Leukemia62,13024,500Breast255,18041,070Other and unspecified primary sitesc33,77042,270aRounded to the nearest 10, cases exclude basal cell and squamous cell skin cancers and in situ carcinoma except bladder. About 63,410 cases of carcinoma in situ of the female breast and 74,680 cases of melanoma in situ were diagnosed in 2017.bDeaths for colon and rectum cancers are combined because a large number of deaths from rectal cancer are misclassified as colon.cMore deaths than cases may reflect lack of specificity in recording underlying cause of death on death certificate and/or an undercount in the case estimate.Modified with permission from Siegel RL, Miller KD, Jemal A: Cancer Statistics, 2017, CA Cancer J Clin. 2017 Jan;67(1):7-30.past 3 decades, the 5-year relative survival rate for all cancers combined has increased by 20% among whites and 24% among blacks.

1	A: Cancer Statistics, 2017, CA Cancer J Clin. 2017 Jan;67(1):7-30.past 3 decades, the 5-year relative survival rate for all cancers combined has increased by 20% among whites and 24% among blacks. Improvements in survival for the most common cancers have been similar across both sexes but are more pronounced among patients age 50 to 64 years than among those older than 65 years. This difference may reflect reduced efficacy of new therapies in the elderly or perhaps lower utilization.1 Progress has been rapid for hematopoietic and lymphoid malignancies due to improved treatment protocols and the discovery of tar-geted therapies. The decrease in lung cancer death rates in men is thought to be due to a decrease in tobacco use, whereas the decreases in death rates from breast, colorectal cancer, and prostate cancer likely reflect advances in early detection and treatment. For instance, there is potential for lung cancer to be diagnosed at an earlier stage through the use of screening with

1	and prostate cancer likely reflect advances in early detection and treatment. For instance, there is potential for lung cancer to be diagnosed at an earlier stage through the use of screening with low-dose computed tomography.Global Statistics on Cancer Incidence and MortalityThe five most common cancers for men worldwide are lung, prostate, colorectal cancer, stomach, and liver and for women are breast, colorectal, cervix, lung, and stomach.4 Notably, for several cancer types there is wide geographical variability in cancer incidence (Fig. 10-3). The mortality rates for different cancers also vary significantly among countries. This is attribut-able not only to variations in incidence but also to variations in survival after a cancer diagnosis. The survival rates are influ-enced by treatment patterns as well as by variations in cancer screening practices, which affect the stage of cancer at diagno-sis. For example, the 5-year survival rate for stomach cancer is much higher in Japan,

1	patterns as well as by variations in cancer screening practices, which affect the stage of cancer at diagno-sis. For example, the 5-year survival rate for stomach cancer is much higher in Japan, where the cancer incidence is high enough to warrant mass screening, which is presumed to lead to earlier diagnosis. In the case of prostate cancer, on the other Brunicardi_Ch10_p0305-p0354.indd 30722/02/19 2:14 PM 308BASIC CONSIDERATIONSPART Ihand, the mortality rates diverge much less than the incidence rates among countries. Survival rates for prostate cancer are much higher in North America than in developing countries.5 It is possible that the extensive screening practices in the United States allow discovery of cancers at an earlier, more curable stage; however, it is also possible that this screening leads to discovery of more latent, less biologically aggressive cancers, which may not have caused death even if they had not been identified.In 2008 (the last date for which complete

1	this screening leads to discovery of more latent, less biologically aggressive cancers, which may not have caused death even if they had not been identified.In 2008 (the last date for which complete data are available), about 1 million new cases of stomach cancer were estimated to have occurred (988,000 cases, 7.8% of the total), making it the fourth most common malignancy in the world, behind cancers of the lung, breast, and colorectal cancer. The incidence of stomach cancer varies significantly among different regions of the world. The difference in risk by country is presumed to be primarily due to differences in dietary factors. The risk is increased by high consumption of preserved salted foods, such as meats and pick-les, and decreased by high intake of fruits and vegetables.5 There also is some international variation in the incidence of infection with Helicobacter pylori, which is known to play a major role in gastric cancer development.5 Fortunately, a steady decline is being

1	also is some international variation in the incidence of infection with Helicobacter pylori, which is known to play a major role in gastric cancer development.5 Fortunately, a steady decline is being observed in the incidence and mortality rates of gastric cancer. This may be related to improvements in preservation and storage of foods as well as due to changes in the prevalence of H pylori.5 More than 70% of cases (713,000 cases) occur in developing countries, and half the cases in the world occur in Eastern Asia (mainly in China).4 Age-standardized incidence rates are about twice as high for men as for women, ranging from 3.9 in Northern Africa to 42.4 in Eastern Asia for men, and from 2.2 in Southern Africa to 18.3 in Eastern Asia for women. Stomach cancer is the second leading cause of cancer death in both sexes worldwide.Overall, the incidence of breast cancer is rising in most countries. Incidence varies from 19.3 per 100,000 women in Eastern Africa to 89.7 per 100,000 women in

1	cancer death in both sexes worldwide.Overall, the incidence of breast cancer is rising in most countries. Incidence varies from 19.3 per 100,000 women in Eastern Africa to 89.7 per 100,000 women in Western Europe, and are high in developed regions of the world (except Japan) and low in most of the developing regions.4 Although breast cancer has been linked to cancer susceptibility genes, mutations in these genes account for only 5% to 10% of breast tumors, which suggests that the wide geographic variations in breast cancer incidence are not due to geographic variations in the prevalence of these genes. Most of the differences, therefore, are attributed to differences in reproductive factors, diet, alcohol, obesity, physical activity, and other environmental differences. Indeed, breast cancer risk increases significantly in females MalesFemalesBreastLung & bronchusColon & rectumUterine corpusNon-Hodgkin lymphomaThyroidMelanoma of the skinPancreasLeukemiaKidney & renal pelvisAll

1	cancer risk increases significantly in females MalesFemalesBreastLung & bronchusColon & rectumUterine corpusNon-Hodgkin lymphomaThyroidMelanoma of the skinPancreasLeukemiaKidney & renal pelvisAll Sites252,710105,51064,01061,38042,47034,94032,16025,84025,70023,380852,63030%12%8%7%5%4%4%3%3%3%100%MalesFemalesLung & bronchusProstateColon & rectumPancreasLiver & intrahepatic bile ductLeukemiaEsophagusUrinary bladderNon-Hodgkin lymphomaBrain & other nervous systemAll SitesLung & bronchusBreastColon & rectumPancreasOvaryLeukemiaNon-Hodgkin lymphomaUterine corpusLiver & intrahepatic bile ductBrain & other nervous systemAll Sites71,28040,61023,11020,79014,08010,92010,2009,3108,6907,080282,50025%14%8%7%5%4%4%3%3%3%100%84,59027,15026,73022,30019,61014,30012,72012,24011,4509,620318,42027%9%8%7%6%4%4%4%4%3%100%ProstateLung & bronchusColon & rectumUrinary bladderMelanoma of the skinNon-Hodgkin lymphomaKidney & renal pelvisOral cavity & pharynxLeukemiaLiver & intrahepatic bile ductAll

1	& bronchusColon & rectumUrinary bladderMelanoma of the skinNon-Hodgkin lymphomaKidney & renal pelvisOral cavity & pharynxLeukemiaLiver & intrahepatic bile ductAll Sites161,360116,99071,42060,49052,17040,61040,08036,29035,72029,200836,15019%14%9%7%6%5%5%4%4%3%100%Estimated new casesEstimated deathsFigure 10-1. Ten leading cancer types with the estimated new cancer cases and deaths by sex in the United States, 2013. *Excludes basal and squamous cell skin cancers and in situ carcinomas except those of the urinary bladder. Estimates are rounded to the nearest 10 (Modified with permission from Siegel RL, Miller KD, Jemal A: Cancer Statistics, 2017, CA Cancer J Clin. 2017 Jan;67(1):7-30.)Brunicardi_Ch10_p0305-p0354.indd 30822/02/19 2:14 PM 309ONCOLOGYCHAPTER 10who have migrated from Asia to America.5 The range of breast cancer mortality rates is much less (approximately 6 to 19 per 100,000) because of the more favorable survival of breast cancer in developed regions. As a result,

1	to America.5 The range of breast cancer mortality rates is much less (approximately 6 to 19 per 100,000) because of the more favorable survival of breast cancer in developed regions. As a result, breast cancer ranks as the fifth cause of death from cancer overall (458,000 deaths), but it is still the most frequent cause of cancer death in women in both developing (269,000 deaths, 12.7% of total) and developed regions (estimated 189,000 deaths).4There is a 25-fold variation in colon cancer incidence world-wide.5 The incidence of colon and rectal cancer is higher in devel-oped countries than in developing countries. The incidence rates are highest in North America, Australia and New Zealand, and Western Europe, and especially in Japanese men.5 In contrast, the incidence is relatively low in North Africa, South America, and Eastern, Southeastern, and Western Asia. These geographic differences are thought to reflect environmental exposures and are presumed to be related mainly to dietary

1	North Africa, South America, and Eastern, Southeastern, and Western Asia. These geographic differences are thought to reflect environmental exposures and are presumed to be related mainly to dietary differences in con-sumption of animal fat, meat, and fiber.5Worldwide liver cancer is the fifth most common cancer in men (523,000 cases, 7.9% of the total) and the seventh in women (226,000 cases, 6.5% of the total). Almost 85% of liver cancer cases occur in developing countries, and particularly in men.4 The overall sex ratio of male to female is 2:4. The regions of high incidence are Eastern and Southeastern Asia, Middle and Western Africa, as well as Melanesia and Micronesia/Polynesia (particularly in men). Low rates are estimated in developed regions, with the exception of Southern Europe. There were an estimated 694,000 deaths from liver cancer in 2008 (477,000 in men, 217,000 in women), and because of its high fatality (overall ratio of mortality to incidence of 0.93), liver cancer

1	There were an estimated 694,000 deaths from liver cancer in 2008 (477,000 in men, 217,000 in women), and because of its high fatality (overall ratio of mortality to incidence of 0.93), liver cancer is the third most common cause of death from cancer worldwide. The geographical distribution of the mortality rates is similar to that observed for incidence. Worldwide, the major risk factors for liver cancer are infection with hepatitis B and C viruses and consumption of foods contaminated with aflatoxin. Hepatitis B immunization in children has recently been shown to reduce the incidence of liver cancer.5In summary, the incidence rates of many common cancers vary widely by geography. This is due in part to genetic dif-ferences, including racial and ethnic differences. It is due also in part to differences in environmental and dietary exposures, factors that can potentially be altered. Therefore, establishment of regional and international databases is critical to improving our

1	in part to differences in environmental and dietary exposures, factors that can potentially be altered. Therefore, establishment of regional and international databases is critical to improving our understanding of the etiology of cancer and will ultimately assist in the initiation of targeted strategies for global cancer prevention. Furthermore, the monitoring of cancer mortality rates and 5-year cancer-specific survival rates will identify regions where there are inequities of health care, so that access to health care can be facilitated and guidelines for treatment can be established.Liver*ThyroidUterine corpusMelanoma of the skinColorectumLung & bronchusBreastFemale197519801985199019952000200520102013025Year of diagnosis5075100125150175200225250025Year of diagnosisLiver*ThyroidMelanoma of the skinUrinary bladderColorectumLung & bronchusProstateMaleRate per 100,000 population5075100125150175200225250197519801985199019952000200520102013Figure 10-2. Trends in cancer incidence rates

1	of the skinUrinary bladderColorectumLung & bronchusProstateMaleRate per 100,000 population5075100125150175200225250197519801985199019952000200520102013Figure 10-2. Trends in cancer incidence rates for selected cancer by sex among males and females for selected cancer types, United States, 1975 to 2009. Rates are age adjusted to the 2000 U.S. standard population. +Liver includes intrahepatic bile duct. (Modified with permission from Siegel RL, Miller KD, Jemal A: Cancer Statistics, 2017, CA Cancer J Clin. 2017 Jan;67(1):7-30.)Brunicardi_Ch10_p0305-p0354.indd 30922/02/19 2:14 PM 310BASIC CONSIDERATIONSPART ITable 10-2Five-year relative survival rates adjusted to normal life expectancy by year of diagnosis, United States, 1975–2008 RELATIVE 5-YEAR SURVIVAL RATES (%)CANCER TYPE1975–19771987–19892005-2011All cancers495669Brain222935Breast (female)758491Uterine cervix697069Colon516169Uterine corpus878383Esophagus51020Hodgkin’s

1	5-YEAR SURVIVAL RATES (%)CANCER TYPE1975–19771987–19892005-2011All cancers495669Brain222935Breast (female)758491Uterine cervix697069Colon516169Uterine corpus878383Esophagus51020Hodgkin’s disease727988Kidney505774Larynx666663Leukemia344362Liver3518Lung and bronchus121318Melanoma of the skin828893Multiple myeloma252849Non-Hodgkin’s lymphoma475172Oral cavity535466Ovary363846Pancreas248Prostate688399Rectum485868Stomach152030Testis839597Thyroid929598Urinary bladder737979Data from Siegel RL, Miller KD, Jemal A: Cancer Statistics, 2017, CA Cancer J Clin. 2017 Jan;67(1):7-30.CANCER BIOLOGYHallmarks of CancerAlthough there are >100 types of cancer, it has been proposed that there are six essential alterations in cell physiology that dic-tate malignant growth: self-sufficiency of growth signals, insen-sitivity to growth-inhibitory signals, evasion of apoptosis (programmed cell death), potential for limitless replication, angiogenesis, and invasion and metastasis.6 Recently two additional

1	insen-sitivity to growth-inhibitory signals, evasion of apoptosis (programmed cell death), potential for limitless replication, angiogenesis, and invasion and metastasis.6 Recently two additional hallmarks have emerged—reprogramming of energy metabolism and evading immune destruction.7 These hallmarks of cancer are being pursued as targets for cancer ther-apy (Fig. 10-4).Cell Proliferation and TransformationIn normal cells, cell growth and proliferation are under strict control. In cancer cells, cells become unresponsive to normal growth controls, which leads to uncontrolled cell division. Human cells require several genetic changes for neoplastic transformation. Cell type–specific differences also exist in the process by which a normal cell is transformed into a cancer-ous one. Abnormally proliferating, transformed cells outgrow normal cells in the culture dish (i.e., in vitro) and commonly display several abnormal characteristics.8 These include loss of contact inhibition (i.e.,

1	proliferating, transformed cells outgrow normal cells in the culture dish (i.e., in vitro) and commonly display several abnormal characteristics.8 These include loss of contact inhibition (i.e., cells continue to proliferate after a con-fluent monolayer is formed); an altered appearance and poor adherence to other cells or to the substratum; loss of anchorage dependence for growth; immortalization; and gain of tumorige-nicity (i.e., the ability to give rise to tumors when injected into an appropriate host).Cancer InitiationTumorigenesis is proposed to have three steps: initiation, promo-tion, and progression. Initiating events such as gain of function of genes known as oncogenes or loss of function of genes known as tumor-suppressor genes may lead a single cell to acquire a dis-tinct growth advantage. Although tumors usually arise from a sin-gle cell or clone, it is thought that sometimes not a single cell but 3Brunicardi_Ch10_p0305-p0354.indd 31022/02/19 2:14 PM

1	a dis-tinct growth advantage. Although tumors usually arise from a sin-gle cell or clone, it is thought that sometimes not a single cell but 3Brunicardi_Ch10_p0305-p0354.indd 31022/02/19 2:14 PM 311ONCOLOGYCHAPTER 10rather a large number of cells in a target organ may have under-gone the initiating genetic event. Thus, many normal-appearing cells may have an increased malignant potential. This is referred to as a field effect. The initiating events are usually genetic and occur as deletions of tumor-suppressor genes or amplification or mutation of oncogenes. Subsequent events can lead to accumula-tions of additional deleterious mutations in the clone.Cancer is thought to be a disease of clonal progression as tumors arise from a single cell and accumulate mutations that confer on the tumor an increasingly aggressive behavior. Most tumors go through a progression from benign lesions to in situ tumors to invasive cancers (e.g., atypical ductal hyperplasia to ductal carcinoma in situ

1	an increasingly aggressive behavior. Most tumors go through a progression from benign lesions to in situ tumors to invasive cancers (e.g., atypical ductal hyperplasia to ductal carcinoma in situ to invasive ductal carcinoma of the breast). Fearon and Vogelstein proposed the model for colorec-tal tumorigenesis presented in Fig. 10-5.9 Colorectal tumors arise from the mutational activation of oncogenes coupled with muta-tional inactivation of tumor-suppressor genes, the latter being the predominant change.9 Mutations in at least four or five genes are required for formation of a malignant tumor, while fewer changes suffice for a benign tumor. Although genetic mutations often occur in a preferred sequence, a tumor’s biologic properties are determined by the total accumulation of its genetic changes.Gene expression is a multistep process that starts from transcription of a gene into messenger ribonucleic acid (mRNA) and then translation of this sequence into the functional protein. There

1	changes.Gene expression is a multistep process that starts from transcription of a gene into messenger ribonucleic acid (mRNA) and then translation of this sequence into the functional protein. There are several controls at each level. In addition to alterations at the genome level (e.g., amplifications of a gene), alterations at the transcription level (e.g., methylation of the DNA leading to transcriptional silencing) or at the level of mRNA process-ing, mRNA stability, mRNA translation, or protein stability, all can alter the levels of critical proteins and thus contribute to tumorigenesis. Alternatively, changes in the genomic sequence can lead to a mutated product with altered function.Cell-Cycle Dysregulation in CancerThe proliferative advantage of tumor cells is a result of their ability to bypass a quiescent state. Cancer cells often show alterations in signal transduction pathways that lead to prolif-eration in response to external signals. Mutations or alterations in the

1	ability to bypass a quiescent state. Cancer cells often show alterations in signal transduction pathways that lead to prolif-eration in response to external signals. Mutations or alterations in the expression of cell-cycle proteins, growth factors, growth factor receptors, intracellular signal transduction proteins, and nuclear transcription factors all can lead to disturbance of the basic regulatory mechanisms that control the cell cycle, allow-ing unregulated cell growth and proliferation.The cell cycle is divided into four phases (Fig. 10-6).10 During the synthetic or S phase, the cell generates a single copy of its genetic material, whereas in the mitotic or M phase, the cellular components (including copies of DNA) are partitioned between two daughter cells. The G1 and G2 phases represent gap phases, during which the cells prepare themselves for All cancers< 11.2< 0.3< 0.5< 0.7< 1.0< 11.2< 0.4< 0.7< 1.0< 1.6< 4.9Liver cancerStomach cancer< 13.4< 16.6< 22.5< 31.9< 2.2< 3.1< 4.1<

1	gap phases, during which the cells prepare themselves for All cancers< 11.2< 0.3< 0.5< 0.7< 1.0< 11.2< 0.4< 0.7< 1.0< 1.6< 4.9Liver cancerStomach cancer< 13.4< 16.6< 22.5< 31.9< 2.2< 3.1< 4.1< 6.0< 11.4BreastFigure 10-3. Estimated cancer incidence worldwide in 2008. Age-standardized incidence rates per 100,000 for all cancers (upper left), breast cancer (upper right), liver cancer (lower left), and stomach cancer (lower right). (Modified with permission from Ferlay J, Shin HR, Bray F, et al: GLOBOCAN 2008 v2.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 10 [Internet]. Lyon, France: International Agency for Research on Cancer; 2010. Available from: http://globocan.iarc.fr.)Brunicardi_Ch10_p0305-p0354.indd 31122/02/19 2:14 PM 312BASIC CONSIDERATIONSPART Icompletion of the S and M phases, respectively. When cells cease proliferation, they exit the cell cycle and enter the quies-cent state referred to as G0. The two key classes of regulatory molecules that regulate

1	the S and M phases, respectively. When cells cease proliferation, they exit the cell cycle and enter the quies-cent state referred to as G0. The two key classes of regulatory molecules that regulate cellular progress through the cell cycle are the cyclins and the cyclin-dependent kinases (CDKs), which associate to form an activated heterodimer. CDKs are expressed constitutively and have a catalytic activity (phosphorylation of downstream proteins), whereas the cyclins serve a regula-tory function and are synthesized at specific times during the cell cycle. Two families of genes, the cip/kip (CDK interact-ing protein/Kinase inhibitory protein) family and the INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame) family Aerobic glycolysisinhibitorsSustainingproliferativesignalingEvadinggrowthsuppressorsEnablingreplicativeimmortalityActivatinginvasion &metastasisInducingangiogenesisGenomeinstability

1	Frame) family Aerobic glycolysisinhibitorsSustainingproliferativesignalingEvadinggrowthsuppressorsEnablingreplicativeimmortalityActivatinginvasion &metastasisInducingangiogenesisGenomeinstability &mutationResistingcelldeathDeregulatingcellularenergeticsTumor-promotinginflammationAvoidingimmunedestructionEGFRinhibitorsCyclin-dependentkinase inhibitorsPARPinhibitorsInhibitors ofVEGF signalingInhibitors ofHGF/c-MetTelomeraseInhibitorsSelective anti-inflammatory drugsProapoptoticBH3 mimeticsImmune activatinganti-CTLA4 oranti-PD-1 mAbFigure 10-4. Hallmarks of cancer and their therapeutic implications. Drugs that interfere with each of the acquired capabilities necessary for tumor growth and progression are in clinical trials and in some cases approved for clinical use in treating forms of human cancer. The drugs listed are illustrative examples. (Reproduced with permission from Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation, CCell. 2011 Mar 4;144(5):646-674.)First Hit:

1	cancer. The drugs listed are illustrative examples. (Reproduced with permission from Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation, CCell. 2011 Mar 4;144(5):646-674.)First Hit: APCSomatic Mutation(Sporadic CRC)First Hit: APCGermline Mutation(FAP)K-rasGenomic InstabilitySmad 4p53OtherAlterations˜-CateninNormalEpitheliumDysplasticACFEarlyAdenomaIntermediateAdenomaLateAdenomaCarcinomaMetastasisFigure 10-5. The adenoma-carcinoma model of human colorectal carcinogenesis. The neoplastic process is initiated by mutations in the adenomatous polyposis coli (APC) or b-catenin genes. Tumor progression results from mutations in other genes (e.g., K-ras, Smad 4 and p53) and the development of genomic instability. Patients with familial adenomatous polyposis inherit mutations in the APC gene and develop multiple aberrant crypt foci. Some of these may progress to cancer as they acquire other genetic mutations. (Reproduced with permission from Li C-J, Zhang, X, Fan G-W. Updates in

1	APC gene and develop multiple aberrant crypt foci. Some of these may progress to cancer as they acquire other genetic mutations. (Reproduced with permission from Li C-J, Zhang, X, Fan G-W. Updates in colorectal cancer stem cell research, J Cancer Res Ther. 2014 Dec;10 Suppl:233-239.)Brunicardi_Ch10_p0305-p0354.indd 31222/02/19 2:14 PM 313ONCOLOGYCHAPTER 10prevent the progression of the cell cycle. Mutation or altered expression of these genes can lead to tumor formation.OncogenesNormal cellular genes that contribute to cancer when abnormal are called oncogenes. The normal counterpart of such a gene is referred to as a proto-oncogene. Oncogenes are usually designated by three-letter abbreviations, such as myc or ras. Oncogenes are further designated by the prefix “v-” for virus or “c-” for cell or chromosome, corresponding to the origin of the oncogene when it was first detected. Proto-oncogenes can be activated (show increased activity) or overexpressed (expressed at increased

1	for cell or chromosome, corresponding to the origin of the oncogene when it was first detected. Proto-oncogenes can be activated (show increased activity) or overexpressed (expressed at increased pro-tein levels) by translocation (e.g., abl), promoter insertion (e.g., c-myc), mutation (e.g., ras), or amplification (e.g., HER2/neu). More than 100 oncogenes have been identified.Oncogenes may be growth factors (e.g., platelet-derived growth factor), growth factor receptors (e.g., HER2), intracel-lular signal transduction molecules (e.g., ras), nuclear tran-scription factors (e.g., c-myc), or other molecules involved in the regulation of cell growth and proliferation. Growth factors are ubiquitous proteins that are produced and secreted by cells locally and that stimulate cell proliferation by binding specific cell-surface receptors on the same cells (autocrine stimula-tion) or on neighboring cells (paracrine stimulation). Persis-tent overexpression of growth factors can lead to

1	by binding specific cell-surface receptors on the same cells (autocrine stimula-tion) or on neighboring cells (paracrine stimulation). Persis-tent overexpression of growth factors can lead to uncontrolled autostimulation and neoplastic transformation. Alternatively, growth factor receptors can be aberrantly activated (turned on) through mutations or overexpressed (continually presenting cells with growth-stimulatory signals, even in the absence of growth factors), which leads cells to respond as if growth factor levels are altered. The growth-stimulating effect of growth fac-tors and other mitogens is mediated through postreceptor signal Cell with duplicated chromosomesChromosome separationMitosisCell divisionCell with chromosomes in the nucleusDNA synthesisChromosome duplicationMG1SG2CDKCyclinFigure 10-6. Schematic representation of the phases of the cell cycle. Mitogenic growth factors can drive a quiescent cell from G0 into the cell cycle. Once the cell cycle passes beyond the

1	10-6. Schematic representation of the phases of the cell cycle. Mitogenic growth factors can drive a quiescent cell from G0 into the cell cycle. Once the cell cycle passes beyond the restric-tion point, mitogens are no longer required for progression into and through S phase. The DNA is replicated in S phase, and the chromo-somes are condensed and segregated in mitosis. In early G1 phase, certain signals can drive a cell to exit the cell cycle and enter a quies-cent phase. Cell-cycle checkpoints have been identified in G1, S, G2, and M phases. CDK = cyclin-dependent kinase.transduction molecules. These molecules mediate the passage of growth signals from the outside to the inside of the cell and then to the cell nucleus, initiating the cell cycle and DNA transcrip-tion. Aberrant activation or expression of cell-signaling mol-ecules, cell-cycle molecules, or transcription factors may play an important role in neoplastic transformation. Protein tyrosine kinases account for a large

1	or expression of cell-signaling mol-ecules, cell-cycle molecules, or transcription factors may play an important role in neoplastic transformation. Protein tyrosine kinases account for a large portion of known oncogenes. One of the best-studied oncogenes, HER2 is discussed as an example later.HER2, also known as neu or c-erbB-2, is a member of the epidermal growth factor receptor (EGFR) family and is one of the best-characterized tyrosine kinases. Unlike other recep-tor tyrosine kinases, HER2/neu does not have a direct soluble ligand. It plays a key role in signaling, however, because it is the preferred partner in heterodimer formation with all the other EGFR family members (EGFR/c-erbB-1, HER2/c-erbB-3, and HER3/c-erbB-4), which bind at least 30 ligands, including epidermal growth factor (EGF), transforming growth factor α (TGFα), heparin-binding EGF-like growth factor, amphiregu-lin, and heregulin.11 Heterodimerization with HER2 potenti-ates recycling of receptors rather than

1	factor (EGF), transforming growth factor α (TGFα), heparin-binding EGF-like growth factor, amphiregu-lin, and heregulin.11 Heterodimerization with HER2 potenti-ates recycling of receptors rather than degradation, enhances signal potency and duration, increases affinity for ligands, and increases catalytic activity.11HER2 can interact with different members of the HER fam-ily and activate mitogenic and antiapoptotic pathways (Fig. 10-7). The specificity and potency of the intracellular signals are affected by the identity of the ligand, the composition of the receptors, and the phosphotyrosine-binding proteins associated with the erbB molecules. The Rasand Shc-activated mitogen-activated protein kinase (MAPK) pathway is a target of all erbB ligands, which increase the transcriptional activity of early response genes such as c-myc, c-fos, and c-jun.12 MAPK-independent pathways such as the phosphoinositide-3 kinase (PI3K) pathway also are activated by most erbB dimers, although the

1	of early response genes such as c-myc, c-fos, and c-jun.12 MAPK-independent pathways such as the phosphoinositide-3 kinase (PI3K) pathway also are activated by most erbB dimers, although the potency and kinetics of activation may differ. Stimulation of the PI3K pathway through HER2 signaling also can lead to activation of survival molecule Akt, which suppresses apoptosis through mul-tiple mechanisms.The mutant rat neu gene was first recognized as an oncogene in neuroblastomas from carcinogen-treated rats.13 The HER2 gene is frequently amplified and the protein over-expressed in many cancers, including breast, ovarian, lung, gastric, and oral cancers. Overexpression of HER2 results in ligand-independent activation of HER2 kinase, which leads to mitogenic signaling. HER2 overexpression is associated with increased cell proliferation and anchorage-independent growth as well as resistance to proapoptotic stimuli. Further, overex-pression of HER2 increases cell migration and upregulates

1	associated with increased cell proliferation and anchorage-independent growth as well as resistance to proapoptotic stimuli. Further, overex-pression of HER2 increases cell migration and upregulates the activities of matrix metalloproteinases (MMPs) and in vitro invasiveness. In animal models, HER2 increases tumorigenic-ity, angiogenesis, and metastasis. These results all suggest that HER2 plays a key role in cancer biology. More recently, HER2 mutations have also been reported in human cancer, including 3% of patients with lung cancer.14-17 A phase 2 study of the irre-versible kinase inhibitor neratinib showed it to have efficacy in HER2-mutated breast cancer lacking HER amplification.18The critical role of HER2 in cancer biology has been lever-aged for therapeutics, leading to several HER2-targeted drugs with different mechanism of action approved by the Food and Drug Administration (FDA): monoclonal antibodies trastu-zumab and pertuzumab, small molecule inhibitor lapatinib, and

1	HER2-targeted drugs with different mechanism of action approved by the Food and Drug Administration (FDA): monoclonal antibodies trastu-zumab and pertuzumab, small molecule inhibitor lapatinib, and antibody-drug conjugate ado-trastuzumab emtansine. Anti-HER2 agents first showed efficacy in the metastatic setting but Brunicardi_Ch10_p0305-p0354.indd 31322/02/19 2:14 PM 314BASIC CONSIDERATIONSPART Iare now routinely used as adjuvant therapy of breast cancer and also as neoadjuvant treatments (“up-front chemotherapy”).Alterations in Apoptosis in Cancer CellsApoptosis is a genetically regulated program to dispose of cells. Cancer cells must avoid apoptosis if tumors are to arise. The growth of a tumor mass is dependent not only on an increase in proliferation of tumor cells but also on a decrease in their apoptotic rate. Apoptosis is distinguished from necrosis because it leads to several characteristic changes. Soon after undergoing apoptosis, membrane phosphatidylserine translocates

1	in their apoptotic rate. Apoptosis is distinguished from necrosis because it leads to several characteristic changes. Soon after undergoing apoptosis, membrane phosphatidylserine translocates from the inner face of the plasma membrane to the cell surface where it can be detected via the use of a fluorescent conjugate of Annexin V, a protein that exhibits a high affinity for phosphatidylserine. Late in apoptosis there are characteristic changes in nuclear morphology, such as chromatin condensation, nuclear frag-mentation, and DNA laddering, as well as membrane blebbing. Apoptotic cells are then engulfed and degraded by phagocytic cells. The effectors of apoptosis are a family of proteases called caspases (cysteine-dependent and aspartate-directed proteases). The initiator caspases (e.g., 8, 9, and 10), which are upstream, cleave the downstream executioner caspases (e.g., 3, 6, and 7) that carry out the destructive functions of apoptosis.Two principal molecular pathways signal apoptosis

1	9, and 10), which are upstream, cleave the downstream executioner caspases (e.g., 3, 6, and 7) that carry out the destructive functions of apoptosis.Two principal molecular pathways signal apoptosis by cleaving the initiator caspases with the potential for crosstalk: the mitochondrial pathway and the death receptor pathway. In the mitochondrial (or intrinsic) pathway, death results from the release of cytochrome c from the mitochondria. Cytochrome c, procaspase 9, and apoptotic protease activating factor 1 (Apaf-1) form an enzyme complex, referred to as the apoptosome, that activates the effector caspases. In addition to these proteins, the mitochondria contain other proapoptotic proteins such as Smac/DIABLO (second mitochondria-derived activator of cas-pase/direct inhibitor of apoptosis-binding protein with low pI). The mitochondrial pathway can be stimulated by many factors, including DNA damage, reactive oxygen species, or the with-drawal of survival factors. The permeability of

1	protein with low pI). The mitochondrial pathway can be stimulated by many factors, including DNA damage, reactive oxygen species, or the with-drawal of survival factors. The permeability of the mitochon-drial membrane determines whether the apoptotic pathway will proceed. The Bcl-2 family of regulatory proteins includes both proapoptotic proteins (e.g., Bax, BAD, and Bak) and antiapop-totic proteins (e.g., Bcl-2 and Bcl-xL). The activity of the Bcl-2 proteins is centered on the mitochondria, where they regulate LigandsPI3KILKEZH2GSK3MDM2ForkheadCaspase-9CaspasesFas-LAktIKKBadI˜Bp27p214E-BP1TSC1/2sosGrb2ShcsrcMEKKMEKSEKMAPKSAPKELKMYCCREBJUNS6KS6elF4EAlterations in geneexpressionMigrationGrowthProliferationAngiogenesisSurvivalNF-˜BBcl-xLPLC-°IP31,2 diacyl-glycerolCa++mobilizationProteinkinase CRaf-1AdhesionmTORHER1/3/4HER2FAKRasFigure 10-7. The HER2 signaling pathway. HER2 can interact with different members of the HER family and activate mitogenic and antiapoptotic pathways. 4E-BP1 =

1	CRaf-1AdhesionmTORHER1/3/4HER2FAKRasFigure 10-7. The HER2 signaling pathway. HER2 can interact with different members of the HER family and activate mitogenic and antiapoptotic pathways. 4E-BP1 = eIF4E binding protein 1; CREB = cyclic adenosine monophosphate element binding; eIF4E = eukaryotic initiation factor 4E; EZH = enhancer of zeste homolog; FAK = focal adhesion kinase; Fas-L = Fas ligand; GSK3 = glycogen synthase kinase-3; HER = human epidermal growth receptor; IKK = IκB kinase; ILK= integrin-linked kinase; IP3 = inositol triphosphate; IκB = inhibitor of NF-κB; MAPK = mitogen-activated protein kinase; MDM2 = mouse double minute 2 homologue; MEK = mitogen-activated protein/extracel-lular signal regulated kinase kinase; MEKK = MEK kinase; mTOR = mammalian target of rapamycin; NF-κB = nuclear factor κB; PI3K = phosphoinositide-3 kinase; PLC-γ = phospholipase Cγ; SAPK = stress-activated protein kinase; SEK = SAPK/extracellular signal regulated kinase kinase; TSC = tuberous

1	NF-κB = nuclear factor κB; PI3K = phosphoinositide-3 kinase; PLC-γ = phospholipase Cγ; SAPK = stress-activated protein kinase; SEK = SAPK/extracellular signal regulated kinase kinase; TSC = tuberous sclerosis complex. (Modified with permission from Meric-Bernstam F, Hung MC. Advances in targeting human epidermal growth factor receptor-2 signaling for cancer therapy, Clin Cancer Res. 2006 Nov 1;12(21):6326-6330.)Brunicardi_Ch10_p0305-p0354.indd 31422/02/19 2:14 PM 315ONCOLOGYCHAPTER 10membrane permeability. Growth factors promote survival sig-naling through the PI3K/Akt pathway, which phosphorylates and inactivates proapoptotic BAD. In contrast, growth fac-tor withdrawal may promote apoptosis through signaling by unphosphorylated BAD. The heat shock proteins, including Hsp70 and Hsp27, are also involved in inhibition of down-stream apoptotic pathways by blocking formation of the apop-tosome complex and inhibiting release of cytochrome c from the mitochondria.19The second principal

1	are also involved in inhibition of down-stream apoptotic pathways by blocking formation of the apop-tosome complex and inhibiting release of cytochrome c from the mitochondria.19The second principal apoptotic pathway is the death recep-tor pathway, sometimes referred to as the extrinsic pathway. Cell-surface death receptors include Fas/APO1/CD95, tumor necrosis factor receptor 1, and KILL-ER/DR5, which bind their ligands Fas-L, tumor necrosis factor (TNF), and TNF-related apoptosis-inducing ligand (TRAIL), respectively. When the receptors are bound by their ligands, they form a death-induc-ing signaling complex (DISC). At the DISC, procaspase 8 and procaspase 10 are cleaved, yielding active initiator caspases.20 The death receptor pathway may be regulated at the cell sur-face by the expression of “decoy” receptors for Fas (DcR3) and TRAIL (TRID and TRUNDD). The decoy receptors are closely related to the death receptors but lack a functional death domain; therefore, they bind death

1	of “decoy” receptors for Fas (DcR3) and TRAIL (TRID and TRUNDD). The decoy receptors are closely related to the death receptors but lack a functional death domain; therefore, they bind death ligands but do not transmit a death signal. Another regulatory group is the FADD-like interleukin-1 protease-inhibitory proteins (FLIPs). FLIPs have homology to caspase 8; they bind to the DISC and inhibit the activation of caspase 8. Finally, inhibitors of apoptosis proteins (IAPs) block caspase 3 activation and have the ability to regulate both the death receptor and the mitochondrial pathway.In human cancers, aberrations in the apoptotic program include increased expression of Fas and TRAIL decoy receptors; increased expression of antiapoptotic Bcl-2; increased expres-sion of the IAP-related protein survivin; increased expression of c-FLIP; mutations or downregulation of proapoptotic Bax, cas-pase 8, APAF1, XAF1, and death receptors CD95, TRAIL-R1, and TRAIL-R2; alterations of the p53 pathway;

1	survivin; increased expression of c-FLIP; mutations or downregulation of proapoptotic Bax, cas-pase 8, APAF1, XAF1, and death receptors CD95, TRAIL-R1, and TRAIL-R2; alterations of the p53 pathway; overexpression of growth factors and growth factor receptors; and activation of the PI3K/Akt survival pathway.20Autophagy in Cancer CellsAutophagy (self-eating) is a major cellular pathway for protein and organelle turnover. The autophagic pathway is a mechanism for the delivery of cellular materials to lysosomes for degra-dation. This process leads to the basal turnover of cell com-ponents and provides energy and macromolecular precursors. This process helps maintain a balance between anabolism and catabolism for normal cell growth and development. Inability to activate autophagy in response to nutrient deprivation, or consti-tutive activation of autophagy in response to stress, can lead to cell death; thus, autophagy is sometimes referred to as a second form of programmed cell death.

1	to nutrient deprivation, or consti-tutive activation of autophagy in response to stress, can lead to cell death; thus, autophagy is sometimes referred to as a second form of programmed cell death. Autophagy plays an essential role during starvation, cellular differentiation, cell death, and aging. Autophagy is also involved in the elimination of cancer cells by triggering a nonapoptotic cell death program, which suggests a negative role in tumor development. Mouse models that are heterozygotes for the beclin 1 gene, an important gene for autophagy, have altered autophagic response and show a high incidence of spontaneous tumors, which establishes a role for autophagy in tumor suppression.21 This also suggests that mutations in other genes operating in this pathway may con-tribute to tumor formation through deregulation of autophagy. However, autophagy also acts as a stress response mechanism to protect cancer cells from low nutrient supply or therapeu-tic insults. Thus, in cancer,

1	formation through deregulation of autophagy. However, autophagy also acts as a stress response mechanism to protect cancer cells from low nutrient supply or therapeu-tic insults. Thus, in cancer, autophagy can have opposing and context-dependent roles, and interventions to both stimulate and inhibit autophagy have been proposed as possible antican-cer treatments. Studies on the molecular controls of autophagy are ongoing and are expected to generate novel therapeutic strategies. Chloroquin is an antimalarial drug that acts as an autophagic inhibitor by blocking the autophagosome and has been tested for its anticancer properties.Cancer InvasionA feature of malignant cells is their ability to invade the sur-rounding normal tissue. Tumors in which the malignant cells appear to lie exclusively above the basement membrane are referred to as in situ cancer, whereas tumors in which the malig-nant cells are demonstrated to breach the basement membrane, penetrating into surrounding stroma, are

1	above the basement membrane are referred to as in situ cancer, whereas tumors in which the malig-nant cells are demonstrated to breach the basement membrane, penetrating into surrounding stroma, are termed invasive cancer. The ability to invade involves changes in adhesion, initiation of motility, and proteolysis of the extracellular matrix (ECM).Cell-to-cell adhesion in normal cells involves interactions between cell-surface proteins. Calcium adhesion molecules of the cadherin family (E-cadherin, P-cadherin, and N-cadherin) are thought to enhance the cells’ ability to bind to one another and suppress invasion. Migration occurs when cancer cells pen-etrate and attach to the basal matrix of the tissue being invaded; this allows the cancer cell to pull itself forward within the tissue. Attachment to glycoproteins of the ECM such as fibronectin, laminin, and collagen is mediated by tumor cell integrin recep-tors. Integrins are a family of glycoproteins that form heterodi-meric receptors

1	to glycoproteins of the ECM such as fibronectin, laminin, and collagen is mediated by tumor cell integrin recep-tors. Integrins are a family of glycoproteins that form heterodi-meric receptors for ECM molecules. The integrins can form at least 25 distinct pairings of their α and β subunits, and each pairing is specific for a unique set of ligands. In addition to regulating cell adhesion to the ECM, integrins relay molecular signals regarding the cellular environment that influence shape, survival, proliferation, gene transcription, and migration.Factors that are thought to play a role in cancer cell motil-ity include autocrine motility factor, autotaxin, scatter factor (also known as hepatocyte growth factor), TGFα, EGF, and insulin-like growth factors. Also, serine, cysteine, and aspar-tic proteinases and MMPs have all been implicated in cancer invasion. Urokinase and tissue plasminogen activators (uPA and tPA) are serine proteases that convert plasminogen into plasmin. Plasmin, in

1	proteinases and MMPs have all been implicated in cancer invasion. Urokinase and tissue plasminogen activators (uPA and tPA) are serine proteases that convert plasminogen into plasmin. Plasmin, in return, can degrade several ECM components. Plas-min also may activate MMPs. uPA has been more closely corre-lated with tissue invasion and metastasis than tPA. Plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2) are produced in tissues and counteract the activity of plasminogen activators.MMPs comprise a family of metal-dependent endopepti-dases. Upon activation, MMPs degrade a variety of ECM com-ponents. Although MMPs often are referred to by their common names, which reflect the ECM component for which they have specificity, a sequential numbering system has been adopted for standardization. For example, collagenase-1 is now referred to as MMP-1. The MMPs are further classified as secreted and membrane-type MMPs. Most of the MMPs are synthesized as inactive zymogens (pro-MMP) and

1	For example, collagenase-1 is now referred to as MMP-1. The MMPs are further classified as secreted and membrane-type MMPs. Most of the MMPs are synthesized as inactive zymogens (pro-MMP) and are activated by proteolytic removal of the propeptide domain outside the cell by other active MMPs or serine proteinases.MMPs are upregulated in almost every type of cancer. Some of the MMPs are expressed by cancer cells, whereas others are expressed by the tumor stromal cells. Experimental models have demonstrated that MMPs promote cancer progression by Brunicardi_Ch10_p0305-p0354.indd 31522/02/19 2:14 PM 316BASIC CONSIDERATIONSPART Iincreasing cancer cell growth, migration, invasion, angiogenesis, and metastasis. MMPs exert these effects by cleaving not only structural components of the ECM but also growth factor–binding proteins, growth factor precursors, cell adhesion molecules, and other proteinases to provide a growth advantage. The activity of MMPs is regulated by their endogenous

1	but also growth factor–binding proteins, growth factor precursors, cell adhesion molecules, and other proteinases to provide a growth advantage. The activity of MMPs is regulated by their endogenous inhibitors and tissue inhibitors of MMPs (TIMP-1, TIMP-2, TIMP-3, and TIMP-4).AngiogenesisAngiogenesis is the establishment of new blood vessels from a preexisting vascular bed. This neovascularization is essential for tumor growth and metastasis. Tumors develop an angiogenic phenotype as a result of accumulated genetic alterations and in response to local selection pressures such as hypoxia. Many of the common oncogenes and tumor-suppressor genes have been shown to play a role in the induction of angiogenesis.In response to the angiogenic switch, pericytes retract and the endothelium secretes several growth factors such as basic fibroblast growth factor, platelet-derived growth fac-tor (PDGF), and insulin-like growth factor. The basement membrane and stroma around the capillary are

1	several growth factors such as basic fibroblast growth factor, platelet-derived growth fac-tor (PDGF), and insulin-like growth factor. The basement membrane and stroma around the capillary are proteolytically degraded, a process that is mediated in most part by uPA. The endothelium then migrates through the degraded matrix, ini-tially as a solid cord and later forming lumina. Finally, sprout-ing tips anastomose to form a vascular network surrounded by a basement membrane.Angiogenesis is mediated by factors produced by various cells, including tumor cells, endothelial cells, stromal cells, and inflammatory cells. The first proangiogenic factor was identi-fied by Folkman and colleagues in 1971.22 Since then, several other factors have been shown to be proangiogenic or antian-giogenic. Of the angiogenic stimulators, the best studied are the vascular endothelial growth factors (VEGFs). The VEGF fam-ily consists of six growth factors (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental

1	angiogenic stimulators, the best studied are the vascular endothelial growth factors (VEGFs). The VEGF fam-ily consists of six growth factors (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor) and three receptors (VEGFR1 or Flt-1, VEGFR2 or KDR/FLK-1, and VEGFR3 or Flt-4).23 Neuropilin 1 and 2 also may act as recep-tors for VEGF.24 VEGF is induced by hypoxia and by different growth factors and cytokines, including EGF, PDGF, TNF-α, TGFβ, and interleukin-1β. VEGF has various functions, includ-ing increasing vascular permeability, inducing endothelial cell proliferation and tube formation, and inducing endothelial cell synthesis of proteolytic enzymes such as uPA, PAI-1, uroki-nase plasminogen activator receptor, and MMP-1. Furthermore, VEGF may mediate blood flow by its effects on the vasodila-tor nitric oxide and act as an endothelial survival factor, thus protecting the integrity of the vasculature. The proliferation of new lymphatic vessels, lymphangiogenesis, is

1	effects on the vasodila-tor nitric oxide and act as an endothelial survival factor, thus protecting the integrity of the vasculature. The proliferation of new lymphatic vessels, lymphangiogenesis, is also thought to be controlled by the VEGF family. Signaling in lymphatic cells is thought to be modulated by VEGFR3.25 Experimental studies with VEGF-C and VEGF-D have shown that they can induce tumor lymphangiogenesis and direct metastasis via the lymphatic vessels and lymph nodes.25,26PDGFs A, B, C, and D also play important roles in angio-genesis. PDGFs are produced by tumor cells as well as support-ing cells in the tumor microenvironment. PDGFs can enhance endothelial cell proliferation directly, and they can also upreg-ulate VEGF expression in vascular smooth muscle cells, pro-moting endothelial cell survival via a paracrine effect.23 The angiopoietins, angiopoietin-1 and angiopoietin-2 (Ang-1 and Ang-2), are thought to regulate blood vessel maturation. Ang-1 and Ang-2 both bind to

1	cell survival via a paracrine effect.23 The angiopoietins, angiopoietin-1 and angiopoietin-2 (Ang-1 and Ang-2), are thought to regulate blood vessel maturation. Ang-1 and Ang-2 both bind to the angiopoietin-1 receptor (also known as tyrosine-protein kinase receptor TIE-2), but only the binding of Ang-1 activates signal transduction; thus Ang-2 is an Ang-1 antagonist. Ang-1, via the Tie-2 receptor, induces remodel-ing and stabilization of blood vessels. Therefore, the balance between these factors determines the angiogenetic capacity of a tumor.Tumor angiogenesis is regulated by several factors in a coordinated fashion. In addition to upregulation of proangiogenic molecules, angiogenesis also can be encouraged by suppression of naturally occurring inhibitors. Such inhibitors of angiogen-esis include thrombospondin 1 and angiostatin. Angiogenesis is a prerequisite not only for primary tumor growth but also for metastasis. Angiogenesis in the primary tumor, as determined by microvessel

1	include thrombospondin 1 and angiostatin. Angiogenesis is a prerequisite not only for primary tumor growth but also for metastasis. Angiogenesis in the primary tumor, as determined by microvessel density, has been demonstrated to be an inde-pendent predictor of distant metastatic disease and survival in several cancers. Expression of angiogenic factors such as VEGFs has had prognostic value in many studies. These findings further emphasize the importance of angiogenesis in cancer biology.MetastasisMetastases arise from the spread of cancer cells from the pri-mary site and the formation of new tumors in distant sites. The metastatic process consists of a series of steps that need to be completed successfully (Fig. 10-8).27 First, the primary cancer must develop access to the circulation through either the blood circulatory system or the lymphatic system. After the cancer cells are shed into the circulation, they must survive. Next, the circulating cells lodge in a new organ and

1	through either the blood circulatory system or the lymphatic system. After the cancer cells are shed into the circulation, they must survive. Next, the circulating cells lodge in a new organ and extravasate into the new tissue. Next, the cells need to initiate growth in the new tis-sue and eventually establish vascularization to sustain the new tumor. Overall, metastasis is an inefficient process, although the initial steps of hematogenous metastasis (the arrest of tumor cells in the organ and extravasation) are believed to be per-formed efficiently. Only a small subset of cancer cells is then able to initiate micrometastases, and an even smaller portion goes on to grow into macrometastases.Metastases can sometimes arise several years after the treatment of primary tumors. For example, although most breast cancer recurrences occur within the first 10 years after the initial treatment and recurrences are rare after 20 years, breast cancer recurrences have been reported decades after

1	although most breast cancer recurrences occur within the first 10 years after the initial treatment and recurrences are rare after 20 years, breast cancer recurrences have been reported decades after the original tumor. This phenomenon is referred to as dormancy, and it remains one of the biggest challenges in cancer biology. Persistence of solitary cancer cells in a secondary site such as the liver or bone marrow is one possible contributor to dormancy.28 Another explanation of dormancy is that cells remain viable in a qui-escent state and then become reactivated by a physiologically perturbing event. Interestingly, primary tumor removal has been proposed to be a potentially perturbing factor.29 An alternate explanation is that cells establish preangiogenic metastases in which they continue to proliferate but that the proliferative rate is balanced by the apoptotic rate. Therefore, when these small metastases acquire the ability to become vascularized, substan-tial tumor growth can

1	to proliferate but that the proliferative rate is balanced by the apoptotic rate. Therefore, when these small metastases acquire the ability to become vascularized, substan-tial tumor growth can be achieved at the metastatic site, leading to clinical detection. More recently, it has been proposed that dormancy may be the result of the host losing immunologic con-trol of subclinical metastatic foci of disease either through loss of immune cell populations with antigen-specific capabilities or via the mutation of tumor cells such that their immunogenicity is altered.30Several types of tumors metastasize in an organ-specific pattern. One explanation for this is mechanical and is based on Brunicardi_Ch10_p0305-p0354.indd 31622/02/19 2:14 PM 317ONCOLOGYCHAPTER 10the different circulatory drainage patterns of the tumors. When different tumor types and their preferred metastasis sites were compared, 66% of organ-specific metastases were explained on the basis of blood flow alone. The

1	drainage patterns of the tumors. When different tumor types and their preferred metastasis sites were compared, 66% of organ-specific metastases were explained on the basis of blood flow alone. The other explanation for pref-erential metastasis is what is referred to as the “seed and soil” theory, the dependence of the seed (the cancer cell) on the soil (the secondary organ). According to this theory, once cells have reached a secondary organ, their growth efficiency in that organ is based on the compatibility of the cancer cell’s biology with its new microenvironment. For example, breast cancer cells may grow more efficiently in bone than in some other organs because of favorable molecular interactions that occur in the bone microenvironment. The ability of cancer cells to grow in a specific site likely depends on features inherent to the cancer cell, features inherent to the organ, and the interplay between the cancer cell and its microenvironment.31Many of the oncogenes discovered

1	site likely depends on features inherent to the cancer cell, features inherent to the organ, and the interplay between the cancer cell and its microenvironment.31Many of the oncogenes discovered to date, such as HER2 and ras, are thought to potentiate not only malignant transforma-tion but also one or more of the steps required in the metastatic process. Experimental models have suggested a role for several molecules, including RhoC, osteopontin and interleukin-11, and Twist, in tumor metastasis. Metastasis also may involve the loss of metastasis-suppressor genes. Laboratory work involving cancer cell lines that have been selected to have a higher meta-static potential have led to the realization that these more highly metastatic cells have a different gene expression profile than their less metastatic parental counterparts. This in turn has led to the currently held belief that the ability of a primary tumor to metastasize may be predictable by analysis of its gene expres-sion

1	less metastatic parental counterparts. This in turn has led to the currently held belief that the ability of a primary tumor to metastasize may be predictable by analysis of its gene expres-sion profile. Indeed, several studies have focused on identify-ing a gene expression profile or a molecular signature that is associated with metastasis. It has been shown that such a gene expression profile can be used to predict the probability that the patient will remain free of distant metastasis.32 This suggests that the metastatic potential of a tumor is already predetermined by the genetic alterations that the cancer cells acquire early in tumorigenesis. Notably, this hypothesis differs from the mul-tistep tumorigenesis theory in that the ability to metastasize is considered an inherent quality of the tumor from the beginning. It is assumed that metastasis develops not from a few rare cells in the primary tumor that acquire the ability to metastasize but that all cells in tumors with such

1	of the tumor from the beginning. It is assumed that metastasis develops not from a few rare cells in the primary tumor that acquire the ability to metastasize but that all cells in tumors with such molecular signatures develop the ability to metastasize. The reality probably lies in between since some early genetic changes detectable in the entire tumor can give tumors an advantage in the metastatic process, whereas additional genetic changes can give a clone of cells additional advantages, thus allowing them to succeed in metastasis.Epithelial-Mesenchymal TransitionA regulatory program referred to as epithelial-mesenchymal transition (EMT) is a fundamental event in morphogenesis. During EMT, epithelial cells are converted to migratory and Figure 10-8. A schematic representation of the metastatic process. A. The metastatic process begins with an in situ cancer surrounded by an intact basement membrane. B. Invasion requires reversible changes in cell-cell and cell-extracellular matrix

1	process. A. The metastatic process begins with an in situ cancer surrounded by an intact basement membrane. B. Invasion requires reversible changes in cell-cell and cell-extracellular matrix adherence, destruction of proteins in the matrix and stroma, and motility. C. Metastasizing cells can enter the circulation via the lymphatics. D. They can also directly enter the circulation. E. Intravascular survival of the tumor cells and extravasation of the circulatory system follow. F. Metastatic single cells can colonize sites and remain dormant for years as occult micrometastases. G. Subsequent progression and neovascularization leads to clinically detectable metastases and progressively growing, angiogenic metastases. (Reproduced with permission Steeg PS. Metastasis suppressors alter the signal transduction of cancer cells, Nat Rev Cancer. 2003 Jan;3(1):55-63.)Brunicardi_Ch10_p0305-p0354.indd 31722/02/19 2:14 PM 318BASIC CONSIDERATIONSPART Iinvasive mesenchymal cells.33 EMT has also

1	transduction of cancer cells, Nat Rev Cancer. 2003 Jan;3(1):55-63.)Brunicardi_Ch10_p0305-p0354.indd 31722/02/19 2:14 PM 318BASIC CONSIDERATIONSPART Iinvasive mesenchymal cells.33 EMT has also been implicated as the mechanism through which epithelial cells acquire the abil-ity to migrate, invade, resist apoptosis, and metastasize in the setting of cancer. EMT is a developmental process, and a set of pleiotropically acting transcriptional factors, including Snail, Twist, Slug, and Zeb1/2 orchestrate EMT. Several of these tran-scription factors can directly repress E-cadherin gene expression reducing levels of this key suppressor of motility and invasive-ness in cancer cells. This process also entails the induction of mesenchymal markers such as vimentin. It has been proposed that the process of invasion and metastases requires significant plasticity, suggesting that EMT is required for invasion, intrava-sation, and extravasation. Thus, suppression of EMT regulators (and consequently

1	of invasion and metastases requires significant plasticity, suggesting that EMT is required for invasion, intrava-sation, and extravasation. Thus, suppression of EMT regulators (and consequently EMT reversion, or MET) can be important for metastatic outgrowth.34-36Cancer Stem CellsStem cells are cells that have the ability to perpetuate them-selves through self-renewal and to generate mature cells of a particular tissue through differentiation.37 It has been proposed that stem cells themselves may be the target of transformation. It was first documented for leukemia and multiple myeloma that only a small subset of cancer cells is capable of extensive prolif-eration. It has subsequently also been shown for many solid can-cers that only a small proportion of cells is clonogenic in culture and in vivo. Similarly, in many solid tumor types only a small proportion of cells is clonogenic in culture and in vivo. If indeed tumor growth and metastasis are driven by a small population of

1	and in vivo. Similarly, in many solid tumor types only a small proportion of cells is clonogenic in culture and in vivo. If indeed tumor growth and metastasis are driven by a small population of cancer stem cells, this may alter our current approaches to cancer therapy. Currently available drugs can shrink metastatic tumors but often cannot eradicate them. The failure of these treatments usually is attributed to the acquisition of drug resistance by the cancer cells; however, the cancer stem cell hypothesis raises the possibility that existing therapies may simply fail to kill cancer stem cells effectively. Therapeutic approaches targeting stem cells specifically are under study.CANCER ETIOLOGYCancer GenomicsOne widely held opinion is that cancer is a genetic disease that arises from an accumulation of genomic alterations that leads to the selection of cells with increasingly aggressive behavior. These alterations may lead either to a gain of function by onco-genes or to a loss of

1	accumulation of genomic alterations that leads to the selection of cells with increasingly aggressive behavior. These alterations may lead either to a gain of function by onco-genes or to a loss of function by tumor-suppressor genes. These acquired gene alterations present within the tumor are termed somatic mutations to distinguish them from germline mutations that are inherited from parents and transmitted to offspring. Somatic mutations in a cancer genome may consist of several classes of DNA sequence changes. These include substitutions of one base by another; insertions or deletions of small or large segments of DNA; rearrangements, in which the DNA sequence has been broken and then rejoined to another DNA segment; copy number losses that may result in complete absence of a DNA sequence; and copy number gains over and above the two copies present in the normal diploid genome.Somatic mutations in a cancer cell genome have accumu-lated over the lifetime of the patient (Fig.

1	sequence; and copy number gains over and above the two copies present in the normal diploid genome.Somatic mutations in a cancer cell genome have accumu-lated over the lifetime of the patient (Fig. 10-9).38 DNA in normal cells is continuously damaged by internal and external mutagens. Most of this damage is repaired; however, a small fraction may remain as fixed mutations. Mutation rates increase in the pres-ence of substantial exogenous mutagenic exposures, such as tobacco carcinogens or various forms of radiation, including ultraviolet light. These exposures are associated with increased rates of lung and skin cancer, respectively, and somatic muta-tions within such cancers often exhibit the distinctive mutational signatures known to be associated with the mutagen.39 The rates of somatic mutations are also increased in several rare inher-ited diseases, such as Fanconi anemia, ataxia telangiectasia, and xeroderma pigmentosum, which are associated with increased risks of cancer.40,41

1	mutations are also increased in several rare inher-ited diseases, such as Fanconi anemia, ataxia telangiectasia, and xeroderma pigmentosum, which are associated with increased risks of cancer.40,41 The rest of the somatic mutations in a can-cer cell have been acquired after the cancer cell already shows phenotypic evidence of neoplastic change. Whether the somatic AdulthoodEarly clonalexpansionBenigntumorEarly invasivecancerLate invasivecancerChemotherapy-resistantrecurrenceFertilized eggIntrinsicmutation processesPassenger mutationDriver mutationChemotherapyresistance mutationEnvironmentaland lifestyle exposures10s–1,000s of mitosesdepending on the organ10s–100s of mitosesdepending on the cancer10s–100,000 of morepassenger mutationsMutatorphenotypeChemotherapy1–10 or moredriver mutationsGestationInfancyChildhoodFigure 10-9. Accumulation of somatic mutations acquired by the cancer cell. Mutations may be acquired while the cell lineage is phenotypi-cally normal, reflecting intrinsic

1	10-9. Accumulation of somatic mutations acquired by the cancer cell. Mutations may be acquired while the cell lineage is phenotypi-cally normal, reflecting intrinsic mutations acquired during normal cell division as well as the effects of exogenous mutagens. Other processes such as example DNA repair defects may contribute to the mutational burden. Passenger mutations do not have any effect on the cancer cell, but driver mutations cause clonal expansion. Relapse after chemotherapy can be associated with resistance mutations that may predate the initiation of treatment. (Reproduced with permission from Stratton MR, Campbell PJ, Futreal PA. The cancer genome, Nature. 2009 Apr 9;458(7239):719-724.)Brunicardi_Ch10_p0305-p0354.indd 31822/02/19 2:14 PM 319ONCOLOGYCHAPTER 10mutation rate is always higher during this part of the lineage is controversial. This is clearly the case for some cancers. For instance, colorectal and endometrial cancers with defective DNA mismatch repair due to

1	always higher during this part of the lineage is controversial. This is clearly the case for some cancers. For instance, colorectal and endometrial cancers with defective DNA mismatch repair due to abnormalities in genes such as MLH1 and MSH2, exhibit increased rates of single nucleotide changes and small insertions/deletions at repetitive noncoding polynucleotide tracts known as microsatellites.42 It has been proposed that one early step in tumor progression is the development of a “mutator phenotype” that is the result of mutations in genes that normally function in the maintenance of genetic stability. This hypothesis was formulated in order to account for the disparity between the low frequency of spontaneous mutations in normal cells as com-pared to the large number of mutations seen in human tumors.To date about 300 genes that have been reported to be mutated and causally implicated in cancer development.43 Ninety percent of cancer genes are mutated at the somatic or tumor

1	seen in human tumors.To date about 300 genes that have been reported to be mutated and causally implicated in cancer development.43 Ninety percent of cancer genes are mutated at the somatic or tumor level, 20% show germline mutations, and 10% show both. The most common class of genomic alterations among the known cancer genes is a chromosomal translocation that creates a chi-meric gene. Many more cancer genes have been found in leuke-mias, lymphomas, and sarcomas than in other types of cancer; and these genes are usually altered by chromosomal transloca-tion. The most common cancer genes are protein kinases. Several domains that are involved in DNA binding and transcriptional regulation are also common in proteins encoded by cancer genes. Somatic mutations in a cancer genome may be classified according to its consequences for cancer development. “Driver” mutations confer a growth advantage to the cells carrying them and have been positively selected during the evolution of the cancer.

1	according to its consequences for cancer development. “Driver” mutations confer a growth advantage to the cells carrying them and have been positively selected during the evolution of the cancer. The remainder of mutations are “bystanders” or “pas-sengers” that do not confer growth advantage. It is likely that most somatic mutations are passenger mutations. Each tumor may have dozens to hundreds of genomic alterations, making it critical to determine which alterations are indeed drivers, and potentially better therapeutic targets.There have been many recent advancements in large-scale databases and tools to catalogue and interpret genomic vari-ants in cancer patient populations. Currently, the NCI Genomic Data Commons provides a unified data repository, The Cancer Genome Atlas (TCGA), the NCI Center for Cancer Genomics (CCG), and the childhood cancer initiative entitled Therapeu-tically Applicable Research to Generate Effective Treatments (TARGET), as well as a suite of tools for

1	the NCI Center for Cancer Genomics (CCG), and the childhood cancer initiative entitled Therapeu-tically Applicable Research to Generate Effective Treatments (TARGET), as well as a suite of tools for users to interact with the GDC data and provide their own data. Other cancer genome repositories include the Catalogue of Somatic Mutations in Can-cer (COSMIC) and the International Cancer Genome Consor-tium (ICGC).44 The Precision Medicine Initiative launched in 2016 that includes the All of Us Research Program, which will collect genetic data, biologic samples, and other clinical informa-tion from at least 1 million volunteer participants. To facilitate the clinical and biologic interpretation of genomic variants in cancer genomes, several open-access tools have been developed and expanded, including MuSiC, MutSigCV, and OncodriveFM. Other curated database resources used to annotate clinical pheno-types to variants observed in cancer genomes include ClinVAR, Clinical Interpretation of

1	including MuSiC, MutSigCV, and OncodriveFM. Other curated database resources used to annotate clinical pheno-types to variants observed in cancer genomes include ClinVAR, Clinical Interpretation of Variants in Cancer (CiVic), and the Precision Medicine Knowledgebase (PMKB). These resources and tools are being utilized to conduct pan-cancer analyses to characterize genomic variation and other molecular aberra-tions observed across tumors to define cancer drivers, clinically actionable targets, and prognostic and predictive signatures. This information is being integrated into clinical practice in many UnknownKIF5B-RETROS1 fusionsNRASMAP2K1AKT1PIK3CABRAFHER2ALKfusionsKRASEGFRFigure 10-10. Molecular subsets of lung adenocarcinoma. Pie chart shows the percentage of tumors with each potentially action-able alteration. (Reproduced with permission from Pao W, Hutchinson KE. Chipping away at the lung cancer genome, Nat Med. 2012 Mar 6;18(3):349-351.)Hereditary:Nonhereditary:TumorTumorFigure

1	action-able alteration. (Reproduced with permission from Pao W, Hutchinson KE. Chipping away at the lung cancer genome, Nat Med. 2012 Mar 6;18(3):349-351.)Hereditary:Nonhereditary:TumorTumorFigure 10-11. Tumor heterogeneity. A. Patients with tumors with similar histologies may differ in genetic mutation status and other molecular features B. Cells within the primary tumor can acquire or lose genomic alterations in different metastatic sites. C. Intra-tumoral spatial heterogeneity: common initiating genomic events usually exist in all tumor cells but additional spatially separated heterogeneous somatic mutations or copy number changes may accumulate within an individual tumor. (Reproduced with permis-sion from Knudson AG: Two genetic hits (more or less) to cancer, Nat Rev Cancer. 2001 Nov;1(2):157-162.)tumor types, such as lung cancer, where molecular drivers are being taken into consideration when selecting systemic therapies (Fig. 10-10). TCGA data was recently leveraged in a study

1	types, such as lung cancer, where molecular drivers are being taken into consideration when selecting systemic therapies (Fig. 10-10). TCGA data was recently leveraged in a study to identify TRK fusions in tumors from six different cancer types, which led to the development of novel TRK inhibitor therapies.Tumor Heterogeneity and Molecular EvolutionThere is increasing recognition that tumors are heterogeneous; this represents an important challenge to utilizing genomic alterations to personalize cancer therapy (Fig. 10-11).45 First, Brunicardi_Ch10_p0305-p0354.indd 31922/02/19 2:14 PM 320BASIC CONSIDERATIONSPART Ithere may be significant heterogeneity between cancer patients, such that patients with tumors that seem similar histologically, may differ in genomic alterations and in malignant potential.46-48 Second, during cancer progression, subclones frequently arise, resulting in differences in the proportion and pattern of genomic alterations between the primary tumor and the

1	potential.46-48 Second, during cancer progression, subclones frequently arise, resulting in differences in the proportion and pattern of genomic alterations between the primary tumor and the metastases or local-regional recurrences.45 Third, there may also be signifi-cant heterogeneity within any one tumor deposit, with spatially separated heterogeneous somatic mutations and chromosomal imbalances.49 Such spatial heterogeneity of subclones within the primary tumor or metastases provides an additional challenge, as it has been proposed that sequencing of a biopsy specimen or only a portion of the tumor could miss therapeutically rel-evant genomic alterations. The genomic alterations found in a tumor can also change under the selective pressure of a targeted therapy, adding to the challenge of implementing genomically informed personalized therapy.Genes Associated With Hereditary Cancer RiskMost of our information on human cancer genes has been gained from hereditary cancers. In the

1	of implementing genomically informed personalized therapy.Genes Associated With Hereditary Cancer RiskMost of our information on human cancer genes has been gained from hereditary cancers. In the case of hereditary cancers, the individual carries a particular germline mutation in every cell. To date, over 70 genes have been associated with hereditary cancers (Table 10-3).43 A few of these hereditary cancer genes are oncogenes, but most are tumor-suppressor genes. Although hereditary cancer syndromes are rare, somatic mutations that occur in sporadic cancer have been found to disrupt the cellular pathways altered in hereditary cancer syndromes, which sug-gests that these pathways are critical to normal cell growth, cell cycle, and proliferation. Recently, the results of a genome-wide association study of breast cancer in over 120,000 cases and 100,000 controls identified 65 new loci that are associated with overall breast cancer risk.50The following factors may suggest the presence of

1	study of breast cancer in over 120,000 cases and 100,000 controls identified 65 new loci that are associated with overall breast cancer risk.50The following factors may suggest the presence of a hereditary cancer51:1. Tumor development at a much younger age than usual2. Presence of bilateral disease3. Presence of multiple primary malignancies4. Presentation of a cancer in the less affected sex (e.g., male breast cancer)5. Clustering of the same cancer type in relatives6. Occurrence of cancer in association with other conditions such as mental retardation or pathognomonic skin lesionsIt is crucial that all surgeons caring for cancer patients be aware of hereditary cancer syndromes, because a patient’s genetic background has significant implications for patient and family counseling, planning of surgical therapy, and cancer screening and prevention. Some of the more commonly encoun-tered hereditary cancer syndromes are discussed here.rb1Gene. The retinoblastoma gene rb1 was the first

1	of surgical therapy, and cancer screening and prevention. Some of the more commonly encoun-tered hereditary cancer syndromes are discussed here.rb1Gene. The retinoblastoma gene rb1 was the first tumor sup-pressor to be cloned. The rb1 gene product, the Rb protein, is a regulator of transcription that controls the cell cycle, differen-tiation, and apoptosis in normal development.52 Retinoblastoma has long been known to occur in hereditary and nonhereditary forms. Interestingly, although most children with an affected parent develop bilateral retinoblastoma, some develop unilateral retinoblastoma. Furthermore, some children with an affected parent are not affected themselves but then have an affected child, which indicates that they are rb1 mutation carriers. These findings led to the theory that a single mutation is not sufficient for tumorigenesis. Alfred Knudson hypothesized that hereditary retinoblastoma involves two mutations, of which one is germ-line and one somatic, whereas

1	that a single mutation is not sufficient for tumorigenesis. Alfred Knudson hypothesized that hereditary retinoblastoma involves two mutations, of which one is germ-line and one somatic, whereas nonhereditary retinoblastoma is due to two somatic mutations (Fig. 10-12).53 Thus, both heredi-tary and nonhereditary forms of retinoblastoma involve the same number of mutations, a hypothesis known as Knudson’s “two-hit” hypothesis. A “hit” may be a point mutation, a chro-mosomal deletion referred to as allelic loss, or a loss of hetero-zygosity, or silencing of an existing gene. Approximately 40% of retinoblastomas are hereditary and due to germline mutations in the RB1 gene. Children with hereditary RB are also at risk for developing a midline intracranial tumor, most commonly pineoblastoma.p53 and Li-Fraumeni Syndrome. Li-Fraumeni syndrome (LFS) was first defined on the basis of observed clustering of malignancies, including early-onset breast cancer, soft tissue sarcomas, brain tumors,

1	and Li-Fraumeni Syndrome. Li-Fraumeni syndrome (LFS) was first defined on the basis of observed clustering of malignancies, including early-onset breast cancer, soft tissue sarcomas, brain tumors, adrenocortical tumors, and leukemia.54 Criteria for classic LFS in an individual (the proband) include: (a) a bone or soft tissue sarcoma when younger than 45 years, (b) a first-degree relative with cancer before age 45 years, and (c) another firstor second-degree relative with either a sar-coma diagnosed at any age or any cancer diagnosed before age 45 years.55 Approximately 70% of LFS families have been shown to have germline mutations in the tumor-suppressor gene p53.56 Breast carcinoma, soft tissue sarcoma, osteosarcoma, brain tumors, adrenocortical carcinoma, Wilms’ tumor, and phyllodes tumor of the breast are strongly associated; pancre-atic cancer is moderately associated; and leukemia and neuro-blastoma are weakly associated with germline p53 mutations.57 Mutations of p53 have not

1	of the breast are strongly associated; pancre-atic cancer is moderately associated; and leukemia and neuro-blastoma are weakly associated with germline p53 mutations.57 Mutations of p53 have not been detected in approximately 30% of LFS families, and it is hypothesized that genetic alterations in other proteins interacting with p53 function may play a role in these families.Of the known genes in human cancer, p53 is the most commonly mutated within cancer cells. The p53 protein regu-lates cell-cycle progression as well as apoptotic cell death as part of stress response pathways after exposure to ionizing or ultraviolet (UV) irradiation, chemotherapy, acidosis, growth factor deprivation, or hypoxia. When cells are exposed to stress-ors, p53 acts as a transcription factor for genes that induce cell-cycle arrest or apoptosis. A majority of p53 mutations are found within a central DNA recognition motif and disrupt DNA bind-ing by p53. Families with germline missense mutations in the

1	induce cell-cycle arrest or apoptosis. A majority of p53 mutations are found within a central DNA recognition motif and disrupt DNA bind-ing by p53. Families with germline missense mutations in the DNA-binding domain show a more highly penetrant phenotype than families with other p53 mutations.58 Furthermore, proband cancers are linked with significantly younger age at diagnosis in patients with missense mutations in the DNA-binding domain.58 It has become apparent that children and adults with LFS will benefit from intensive surveillance aimed at early detection of cancers and a modified version of the “Toronto protocol” that includes a combination of physical exams, blood tests, and imaging is recommended.BRCA1, BRCA2, and Hereditary Breast-Ovarian Cancer Syndrome. It is estimated that 5% to 10% of breast cancers are hereditary. Of women with early-onset breast cancer (age 40 years or younger), nearly 10% have a germline mutation in one of the breast cancer genes BRCA1 or BRCA2.59

1	5% to 10% of breast cancers are hereditary. Of women with early-onset breast cancer (age 40 years or younger), nearly 10% have a germline mutation in one of the breast cancer genes BRCA1 or BRCA2.59 Mutation carriers are more prevalent among women who have a firstor second-degree relative with premenopausal breast cancer or ovarian cancer at any age. The likelihood of a BRCA mutation is higher in patients who belong to a population in which founder Brunicardi_Ch10_p0305-p0354.indd 32022/02/19 2:14 PM 321ONCOLOGYCHAPTER 10Table 10-3Selected genes associated with hereditary cancerSYMBOLNAMETUMOR TYPES (GERMLINE MUTATIONS)CANCER SYNDROMEALKanaplastic lymphoma kinase (Ki-1)NeuroblastomaFamilial neuroblastomaAPCadenomatous polyposis of the colon geneColorectal, pancreatic, desmoid, hepatoblastoma, glioma, other CNSAdenomatous polyposis coli; Turcot syndromeATMataxia telangiectasia mutatedLeukemia, lymphoma, medulloblastoma, gliomaAtaxia-telangiectasiaBLMBloom syndromeLeukemia,

1	hepatoblastoma, glioma, other CNSAdenomatous polyposis coli; Turcot syndromeATMataxia telangiectasia mutatedLeukemia, lymphoma, medulloblastoma, gliomaAtaxia-telangiectasiaBLMBloom syndromeLeukemia, lymphoma, skin squamous cell, other cancersBloom syndromeBMPR1Abone morphogenetic protein receptor, type IAGastrointestinal polypsJuvenile polyposisBRCA1familial breast/ovarian cancer gene 1Breast, ovarianHereditary breast/ovarian cancerBRCA2familial breast/ovarian cancer gene 2Breast, ovarian, pancreaticHereditary breast/ovarian cancerBRIP1BRCA1 interacting protein C-terminal helicase 1AML, leukemia, breastFanconi anemia J, breast cancer susceptibilityBUB1BBUB1 budding uninhibited by benzimidazoles 1 homolog beta (yeast)RhabdomyosarcomaMosaic variegated aneuploidyCDH1cadherin 1, type 1, E-cadherin (epithelial) (ECAD)Gastric, lobular cancerFamilial gastric carcinomaCDK4cyclin-dependent kinase 4MelanomaFamilial malignant melanomaCDKN2Acyclin-dependent kinase inhibitor 2A (p16[INK4a])

1	E-cadherin (epithelial) (ECAD)Gastric, lobular cancerFamilial gastric carcinomaCDK4cyclin-dependent kinase 4MelanomaFamilial malignant melanomaCDKN2Acyclin-dependent kinase inhibitor 2A (p16[INK4a]) geneMelanoma, pancreaticFamilial malignant melanomaCDKN2a(p14)cyclin-dependent kinase inhibitor 2A– p14ARF proteinMelanoma, pancreaticFamilial malignant melanomaCHEK2CHK2 checkpoint homolog (S pombe)BreastFamilial breast cancerCYLDfamilial cylindromatosis geneCylindromaFamilial cylindromatosisDDB2damage-specific DNA binding protein 2Skin basal cell, skin squamous cell, melanomaXeroderma pigmentosum (E)DICER1dicer 1, ribonuclease type IIIPleuropulmonary blastomaFamilial pleuropulmonary blastomaEGFRepidermal growth factor receptor (erythroblastic leukemia viral [v-erb-b] oncogene homolog, avian)NSCLCFamilial lung cancerERCC2, 3, 4, 5excision repair cross-complementing rodent repair deficiency, complementation groupSkin basal cell, skin squamous cell, melanomaXeroderma pigmentosum (D, B, F,

1	lung cancerERCC2, 3, 4, 5excision repair cross-complementing rodent repair deficiency, complementation groupSkin basal cell, skin squamous cell, melanomaXeroderma pigmentosum (D, B, F, G)EXT1multiple exostoses type 1 geneexostoses, osteosarcomaexostoses, osteosarcomaFANCA, C, D2, E, F, GFanconi anemia, complementation groupAML, leukemiaFanconi anemia A, C, D2, E, F, GFHfumarate hydrataseleiomyomatas, renalHereditary leiomyomatosis and renal cell cancerGPC3glypican 3Wilms’ tumorSimpson-Golabi-Behmel syndromeHRASv-Ha-ras Harvey rat sarcoma viral oncogene homologv-Ha-ras Harvey rat sarcoma viral oncogene homologCostello syndromeHRPT2Hyperparathyroidism 2 (parafibromin)parathyroid adenoma, multiple ossifying jaw fibromaHyperparathyroidism-jaw tumor syndrome(Continued)Brunicardi_Ch10_p0305-p0354.indd 32122/02/19 2:14 PM 322BASIC CONSIDERATIONSPART ITable 10-3Selected genes associated with hereditary cancerSYMBOLNAMETUMOR TYPES (GERMLINE MUTATIONS)CANCER SYNDROMEKITv-kit

1	32122/02/19 2:14 PM 322BASIC CONSIDERATIONSPART ITable 10-3Selected genes associated with hereditary cancerSYMBOLNAMETUMOR TYPES (GERMLINE MUTATIONS)CANCER SYNDROMEKITv-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homologGIST, epitheliomaFamilial gastrointestinal stromal tumorMADH4Homolog of Drosophila Mothers Against Decapentaplegic 4 geneGastrointestinal polypsJuvenile polyposisMEN1multiple endocrine neoplasia type 1 geneParathyroid adenoma, pituitary adenoma, pancreatic islet cell, carcinoidParathyroid adenoma, pituitary adenoma, pancreatic islet cell, carcinoidMLH1E coli MutL homolog geneColorectal, endometrial, ovarian, CNSHereditary nonpolyposis colorectal cancer, Turcot syndromeMPLmyeloproliferative leukemia virus oncogene, thrombopoietin receptorMPDFamilial essential thrombocythemiaMSH2mutS homolog 2 (E coli)colorectal, endometrial, ovarianHereditary non-polyposis colorectal cancerMSH6mutS homolog 6 (E coli)colorectal, endometrial, ovarianHereditary non-polyposis

1	thrombocythemiaMSH2mutS homolog 2 (E coli)colorectal, endometrial, ovarianHereditary non-polyposis colorectal cancerMSH6mutS homolog 6 (E coli)colorectal, endometrial, ovarianHereditary non-polyposis colorectal cancerMUTYHmutY homolog (E coli)ColorectalAdenomatous polyposis coliNBS1Nijmegen breakage syndrome 1 (nibrin)NHL, glioma, medulloblastoma, rhabdomyosarcomaNijmegen breakage syndromeNF1neurofibromatosis type 1 geneNeurofibroma, gliomaNeurofibromatosis type 1NF2neurofibromatosis type 2 geneMeningioma, acoustic neuromaNeurofibromatosis type 2PALB2partner and localizer of BRCA2Wilms tumor, medulloblastoma, AML, breastFanconi anemia N, breast cancer susceptibilityPHOX2Bpaired-like homeobox 2bNeuroblastomaFamilial neuroblastomaPMS1PMS1 postmeiotic segregation increased 1 (S cerevisiae)Colorectal, endometrial, ovarianHereditary non-polyposis colorectal cancerPMS2PMS2 postmeiotic segregation increased 2 (S cerevisiae)Colorectal, endometrial, ovarian, medulloblastoma, gliomaHereditary

1	endometrial, ovarianHereditary non-polyposis colorectal cancerPMS2PMS2 postmeiotic segregation increased 2 (S cerevisiae)Colorectal, endometrial, ovarian, medulloblastoma, gliomaHereditary nonpolyposis colorectal cancer, Turcot syndromePRKAR1Aprotein kinase, cAMP-dependent, regulatory, type I, alpha (tissue specific extinguisher 1)Myxoma, endocrine, papillary thyroidCarney complexPTCHHomolog of Drosophila Patched geneSkin basal cell, medulloblastomaNevoid basal cell carcinoma syndromePTENphosphatase and tensin homolog geneHamartoma, glioma, prostate, endometrialCowden syndrome, Bannayan-Riley-Ruvalcaba syndromeRB1retinoblastoma geneRetinoblastoma, sarcoma, breast, small cell lungFamilial retinoblastomaRECQL4RecQ protein-like 4Osteosarcoma, skin basal and squamous cellRothmund-Thompson syndromeRETret proto-oncogeneMedullary thyroid, papillary thyroid, pheochromocytomaMultiple endocrine neoplasia 2A/2BSBDSShwachman-Bodian-Diamond syndrome proteinAML, MDSSchwachman-Diamond

1	syndromeRETret proto-oncogeneMedullary thyroid, papillary thyroid, pheochromocytomaMultiple endocrine neoplasia 2A/2BSBDSShwachman-Bodian-Diamond syndrome proteinAML, MDSSchwachman-Diamond syndrome(Continued)(Continued)Brunicardi_Ch10_p0305-p0354.indd 32222/02/19 2:14 PM 323ONCOLOGYCHAPTER 10Table 10-3Selected genes associated with hereditary cancerSYMBOLNAMETUMOR TYPES (GERMLINE MUTATIONS)CANCER SYNDROMESDH5chromosome 11 open reading frame 79ParagangliomaFamilial paragangliomaSHD, B, Dsuccinate dehydrogenase complexParaganglioma, pheochromocytomaFamilial paragangliomaSMARCB1SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily b, member 1Malignant rhabdoidRhabdoid predisposition syndromeSTK11serine/threonine kinase 11 gene (LKB1)Jejunal hamartoma, ovarian, testicular, pancreaticPeutz-Jeghers syndromeSUFUsuppressor of fused homolog (Drosophila)MedulloblastomaMedulloblastoma predispositionTCF1transcription factor 1, hepatic (HNF1)Hepatic adenoma,

1	testicular, pancreaticPeutz-Jeghers syndromeSUFUsuppressor of fused homolog (Drosophila)MedulloblastomaMedulloblastoma predispositionTCF1transcription factor 1, hepatic (HNF1)Hepatic adenoma, hepatocellular carcinomaFamilial Hepatic AdenomaTP53tumor protein p53Breast, sarcoma, adrenocortical carcinoma, glioma, multiple other tumor typesLi-Fraumeni syndromeTSC1tuberous sclerosis 1 geneHamartoma, renal cellTuberous sclerosis 1TSC2tuberous sclerosis 2 geneHamartoma, renal cellTuberous sclerosis 2TSHRthyroid stimulating hormone receptorThyroid adenoma VHLvon Hippel-Lindau syndrome geneRenal, hemangioma, pheochromocytomavon Hippel-Lindau syndromeWRNWerner syndrome (RECQL2)Osteosarcoma, meningioma, othersWerner syndromeWT1Wilms’ tumor 1 geneWilms’Denys-Drash syndrome, Frasier syndrome, Familial Wilms tumorXPA, Cxeroderma pigmentosum, complementation groupSkin basal cell, skin squamous cell, melanomaXeroderma pigmentosum (A C)A = amplification; AEL = acute eosinophilic leukemia; AL = acute

1	Wilms tumorXPA, Cxeroderma pigmentosum, complementation groupSkin basal cell, skin squamous cell, melanomaXeroderma pigmentosum (A C)A = amplification; AEL = acute eosinophilic leukemia; AL = acute leukemia; ALCL = anaplastic large-cell lymphoma; ALL = acute lymphocytic leukemia; AML = acute myelogenous leukemia; AML = acute myelogenous leukemia (primarily treatment associated); APL = acute promyelocytic leukemia; B-ALL = B-cell acute lymphocytic leukemia; B-CLL = B-cell lymphocytic leukemia; B-NHL = B-cell non-Hodgkin’s lymphoma; CLL = chronic lymphatic leukemia; CML = chronic myeloid leukemia; CMML = chronic myelomonocytic leukemia; CNS = central nervous system; D = large deletion; DFSP = dermatofibrosarcoma protuberans; DLBL = diffuse large B-cell lymphoma; DLCL = diffuse large-cell lymphoma; Dom = dominant; E = epithelial; F = frameshift; GIST = gastrointestinal stromal tumor; JMML = juvenile myelomonocytic leukemia; L = leukemia/lymphoma; M = mesenchymal; MALT =

1	large-cell lymphoma; Dom = dominant; E = epithelial; F = frameshift; GIST = gastrointestinal stromal tumor; JMML = juvenile myelomonocytic leukemia; L = leukemia/lymphoma; M = mesenchymal; MALT = mucosa-associated lymphoid tissue lymphoma; MDS = myelodysplastic syndrome; Mis = Missense; MLCLS = mediastinal large cell lymphoma with sclerosis; MM = multiple myeloma; MPD = myeloproliferative disorder; N = nonsense; NHL = non-Hodgkin’s lymphoma; NK/T = natural killer T cell; NSCLC = non-small cell lung cancer; O = other; PMBL = primary mediastinal B-cell lymphoma; pre-B All = pre-B-cell acute lymphoblastic leukemia; Rec = recessive; S = splice site; T = translocation; T-ALL = T-cell acute lymphoblastic leukemia; T-CLL = T-cell chronic lymphocytic leukemia; TGCT = testicular germ cell tumor; T-PLL = T-cell prolymphocytic leukemia.Adapted with permission from Futreal PA, Coin L, Marshall M, et al. A census of human cancer genes. Nat Rev Cancer. 2004 Mar;4(3):177-183.(Continued)mutations

1	T-PLL = T-cell prolymphocytic leukemia.Adapted with permission from Futreal PA, Coin L, Marshall M, et al. A census of human cancer genes. Nat Rev Cancer. 2004 Mar;4(3):177-183.(Continued)mutations may be prevalent, such as in the Ashkenazi Jewish population. For a female BRCA1 mutation carrier, the cumula-tive risks of developing breast cancer and ovarian cancer by age 70 have been estimated to be 87% and 44%, respectively.60 The cumulative risks of breast cancer and ovarian cancer by age 70 in families with BRCA2 mutation have been estimated to be 84% and 27%, respectively.61 Although male breast cancer can occur with either BRCA1 or BRCA2 mutation, the majority of families (76%) with both male and female breast cancer have mutations in BRCA2.61 Besides breast and ovar-ian cancer, BRCA1 and BRCA2 mutations may be associated with increased risks for several other cancers. BRCA1 mutations confer a fourfold increased risk for colon cancer and threefold increased risk for prostate

1	BRCA1 and BRCA2 mutations may be associated with increased risks for several other cancers. BRCA1 mutations confer a fourfold increased risk for colon cancer and threefold increased risk for prostate cancer.60 BRCA2 mutations confer a fivefold increased risk for prostate cancer, sevenfold in men younger than 65 years.62 Furthermore, BRCA2 mutations confer Brunicardi_Ch10_p0305-p0354.indd 32322/02/19 2:14 PM 324BASIC CONSIDERATIONSPART Ia fivefold increased risk for gallbladder and bile duct cancers, fourfold increased risk for pancreatic cancer, and threefold increased risk for gastric cancer and malignant melanoma.62BRCA1 was the first breast cancer susceptibility gene identi-fied and has been mapped to 17q21. BRCA2, mapped to 13q12.3, was reported shortly afterward. BRCA1 and BRCA2 encode large nuclear proteins, 208 kDa and 384 kDa, respectively, that have been implicated in processes fundamental to all cells, including DNA repair and recombination, checkpoint control of the

1	encode large nuclear proteins, 208 kDa and 384 kDa, respectively, that have been implicated in processes fundamental to all cells, including DNA repair and recombination, checkpoint control of the cell cycle, and transcription.63 Although early studies suggested that the two proteins function together as a complex, subsequent data demonstrated that they have distinct functions.64,65 In fact, breast cancers arising from BRCA1 or BRCA2 mutations are different at the molecular level and have been found to have distinct gene expression profiles.66 BRCA1-associated tumors are more likely to be estrogen receptor negative, whereas BRCA2-associated tumors are more likely to be estrogen receptor positive. Cur-rently, studies are ongoing to determine whether BRCA1 and BRCA2 status can be used to guide systemic therapy choices for breast cancer. Some targeted therapies are showing activity in BRCA mutation carriers with breast cancer such as PARP poly(ADP-ribose) polymerase-inhibitors.APC Gene

1	systemic therapy choices for breast cancer. Some targeted therapies are showing activity in BRCA mutation carriers with breast cancer such as PARP poly(ADP-ribose) polymerase-inhibitors.APC Gene and Familial Adenomatous PolyposisPatients affected with familial adenomatous polyposis (FAP) characteristically develop hundreds to thousands of polyps in the colon and rectum. The polyps usually appear in adolescence and, if left untreated, progress to colorectal cancer. FAP is asso-ciated with benign extracolonic manifestations that may be use-ful in identifying new cases, including congenital hypertrophy of the retinal pigment epithelium, epidermoid cysts, and osteo-mas. In addition to colorectal cancer, patients with FAP are at risk for upper intestinal neoplasms (gastric and duodenal pol-yps, duodenal and periampullary cancer), hepatobiliary tumors (hepatoblastoma, pancreatic cancer, and cholangiocarcinoma), thyroid carcinomas, desmoid tumors, and medulloblastomas.The product of the

1	duodenal and periampullary cancer), hepatobiliary tumors (hepatoblastoma, pancreatic cancer, and cholangiocarcinoma), thyroid carcinomas, desmoid tumors, and medulloblastomas.The product of the adenomatous polyposis coli tumor-sup-pressor gene (APC) plays an important role in cell-cell interac-tions, cell adhesion, regulation of β-catenin, and maintenance of cytoskeletal microtubules. Alterations in APC lead to dys-regulation of several physiologic processes that govern colonic epithelial cell homeostasis, including cell-cycle progression, migration, differentiation, and apoptosis. Mutations in the APC have been identified in FAP and in 80% of sporadic colorectal cancers.67 Furthermore, APC mutations are the earliest known genetic alterations in colorectal cancer progression, which emphasizes its importance in cancer initiation. The germline mutations in APC may arise from point mutations, insertions, or deletions that lead to a premature stop codon and a truncated, functionally

1	its importance in cancer initiation. The germline mutations in APC may arise from point mutations, insertions, or deletions that lead to a premature stop codon and a truncated, functionally inactive protein. The risk of developing specific manifestations of FAP is correlated with the position of the FAP mutations, a phenomenon referred to as genotype-phenotype correlation. For example, desmoids usually are associated with mutations between codons 1403 and 1578.68,69 Mutations in the extreme 5′ or 3′ ends of APC, or in the alternatively spliced region of exon 9, are associated with an attenuated version of FAP. Better understanding of the genotype-phenotype correla-tions may assist in patient counseling and therapeutic planning.Mismatch Repair Genes and Hereditary Nonpolyposis Colorectal Cancer. Hereditary nonpolyposis colorectal cancer (HNPCC), also referred to as Lynch syndrome, is an autoso-mal dominant hereditary cancer syndrome that predisposes to a wide spectrum of cancers,

1	Cancer. Hereditary nonpolyposis colorectal cancer (HNPCC), also referred to as Lynch syndrome, is an autoso-mal dominant hereditary cancer syndrome that predisposes to a wide spectrum of cancers, including colorectal cancer without polyposis. Some have proposed that HNPCC consists of at least two syndromes: Lynch syndrome 1, which entails hereditary predisposition for colorectal cancer with early age of onset (approximately age 44 years) and an excess of synchronous and metachronous colonic cancers; and Lynch syndrome 2, featur-ing a similar colonic phenotype accompanied by a high risk for carcinoma of the endometrium, transitional cell carcinoma of the ureter and renal pelvis, and carcinomas of the stomach, small bowel, ovary, and pancreas.70 The diagnostic criteria for HNPCC are referred to as the Amsterdam criteria, or the 3-2-1-0 rule. The classic Amsterdam criteria were revised to include other HNPCC-related cancers (Table 10-4).71 These criteria are met when three or more family

1	to as the Amsterdam criteria, or the 3-2-1-0 rule. The classic Amsterdam criteria were revised to include other HNPCC-related cancers (Table 10-4).71 These criteria are met when three or more family members have histologically ver-ified, HNPCC-associated cancers (one of whom is a first-degree relative of the other two), two or more generations are involved, at least one individual was diagnosed before age 50 years, and no individuals have FAP.71During DNA replication, DNA polymerases may intro-duce single nucleotide mismatches or small insertion or deletion loops. These errors are corrected through a process referred to as mismatch repair. When mismatch repair genes are inactivated, DNA mutations in other genes that are critical to cell growth and proliferation accumulate rapidly. In HNPCC, germline mutations have been identified in several genes that play a key role in DNA nucleotide mismatch repair: hMLH1 (human mutL homologue 1), hMSH2 (human mutS homologue 2), hMSH6, A

1	In HNPCC, germline mutations have been identified in several genes that play a key role in DNA nucleotide mismatch repair: hMLH1 (human mutL homologue 1), hMSH2 (human mutS homologue 2), hMSH6, A Heterogeneity among patientsB Heterogeneity betweenprimary and metastatic sitesC Intratumoral spatialheterogeneityFigure 10-12. “Two-hit” tumor formation in both hereditary and nonhereditary cancers. A “one-hit” clone is a precursor to the tumor in nonhereditary cancer, whereas all cells are one-hit clones in hereditary cancer. (Reproduced with permission from Meric-Bernstam F, Mills GB. Overcoming implementation challenges of personalized cancer therapy, Nat Rev Clin Oncol. 2012 Sep;9(9):542-548.)Brunicardi_Ch10_p0305-p0354.indd 32422/02/19 2:14 PM 325ONCOLOGYCHAPTER 10and hPMS1 and hPMS2 (human postmeiotic segregation 1 and 2), of which hMLH1 and hMSH2 are the most common.72-77 The hallmark of HNPCC is microsatellite instability, which occurs on the basis of unrepaired mismatches and

1	postmeiotic segregation 1 and 2), of which hMLH1 and hMSH2 are the most common.72-77 The hallmark of HNPCC is microsatellite instability, which occurs on the basis of unrepaired mismatches and small inser-tion or deletion loops. Microsatellite instability can be tested by comparing the DNA of a patient’s tumor with DNA from adja-cent normal epithelium, amplifying the DNA with polymerase chain reaction (PCR) using a standard set of markers, compar-ing the amplified genomic DNA sequences, and classifying the degree of microsatellite instability as high, low, or stable. Such microsatellite instability testing may help select patients who are more likely to have germline mutations. An analysis of patients with early-onset colorectal cancer (age less than 50) showed that 8% had an unsuspected germline mutation in a mismatch repair gene and could be considered as having Lynch syndrome. Thus, genetic counseling and testing with a multigene panel should be considered for such patients.78PTEN

1	germline mutation in a mismatch repair gene and could be considered as having Lynch syndrome. Thus, genetic counseling and testing with a multigene panel should be considered for such patients.78PTEN and Cowden DiseaseSomatic deletions or mutations in the tumor-suppressor gene PTEN (phosphatase and tensin homologue deleted on chromosome 10) have been observed in a number of glioma breast, prostate, and renal carcinoma cell lines and several primary tumor specimens.79PTEN encodes a 403-amino-acid protein, tyrosine phos-phatase. PTEN negatively controls the PI3K signaling pathway for the regulation of cell growth and survival by dephosphory-lating phosphoinositol 3,4,5-triphosphate; thus, mutation of PTEN leads to constitutive activation of the PI3K/Akt signaling pathway. The “hot spot” for PTEN mutations has been identi-fied in exon 5. Forty-three percent of CD mutations have been identified in this exon, which contains the tyrosine phosphatase core domain. This suggests that the PTEN

1	PTEN mutations has been identi-fied in exon 5. Forty-three percent of CD mutations have been identified in this exon, which contains the tyrosine phosphatase core domain. This suggests that the PTEN catalytic activity is vital for its biologic function. PTEN was identified as the sus-ceptibility gene for the autosomal dominant syndrome Cowden disease (CD) or multiple hamartoma syndrome.80 Trichilem-momas, benign tumors of the hair follicle infundibulum, and mucocutaneous papillomatosis are pathognomonic of CD. Other common features include thyroid adenomas and multinodular goiters, breast fibroadenomas, and hamartomatous GI polyps. The diagnosis of CD is made when an individual or family has a combination of pathognomonic major and/or minor criteria pro-posed by the International Cowden Consortium.81 CD is associ-ated with an increased risk of breast and thyroid cancers. Breast cancer develops in 25% to 50% of affected women.81p16 and Hereditary Malignant Melanoma. The gene p16, also

1	CD is associ-ated with an increased risk of breast and thyroid cancers. Breast cancer develops in 25% to 50% of affected women.81p16 and Hereditary Malignant Melanoma. The gene p16, also known as INK4A, CDKN1, CDKN2A, and MTS1, is a tumor suppressor that acts by binding CDK4 and CDK6 and inhibit-ing the catalytic activity of the CDK4-CDK6/cyclin D complex that is required for phosphorylation of Rb and subsequent cell-cycle progression. Studies suggest that germline mutations in p16 can be found in 20% of melanoma-prone families.82 Muta-tions in p16 that alter its ability to inhibit the catalytic activity of the CDK4-CDK6/cyclin D complex not only increase the risk of melanoma by 75-fold but also increase the risk of pancre-atic cancer by 22-fold.83 Interestingly, p16 mutations that do not appear to alter its function increase the risk of melanoma by 38-fold and do not increase the risk of pancreatic cancer.83 Genomic characterization of primary tumors has revealed that p16 is

1	do not appear to alter its function increase the risk of melanoma by 38-fold and do not increase the risk of pancreatic cancer.83 Genomic characterization of primary tumors has revealed that p16 is inactivated through point mutation, promoter methyla-tion, or deletion in a significant portion of sporadic tumors, including cancers of the pancreas, esophagus, head and neck, stomach, breast, and colon, as well as melanomas.E-cadherin and Hereditary Diffuse Gastric Cancer. E-cadherin is a cell adhesion molecule that plays an important role in nor-mal architecture and function of epithelial cells. The adhesive function of E-cadherin is dependent on interaction of its cyto-plasmic domain with βand γ-catenins and may be regulated by phosphorylation of β-catenin.Hereditary diffuse gastric carcinoma is an autosomal domi-nant cancer syndrome that results from germline mutations in the E-cadherin gene, CDH1. Carriers of CDH1 mutations have a 70% to 80% chance of developing gastric cancer.84

1	is an autosomal domi-nant cancer syndrome that results from germline mutations in the E-cadherin gene, CDH1. Carriers of CDH1 mutations have a 70% to 80% chance of developing gastric cancer.84 Furthermore, mutations of CDH1 have been described in sporadic cancers of the ovary, endometrium, breast, and thyroid. However, frequent mutations have been identified in only two particular tumors: dif-fuse gastric carcinomas and lobular breast carcinomas. Invasive lobular breast carcinomas often show inactivating mutations in combination with a loss of heterozygosity of the wild-type CDH1 allele.85 Interestingly, in gastric carcinomas the predominant mutations are exon skipping causing in-frame deletions, whereas most mutations identified in lobular breast cancers are premature stop codons; this suggests a genotype-phenotype correlation.RET Proto-Oncogene and Multiple Endocrine Neoplasia Type 2The RET (rearranged during transfection) gene encodes for a transmembrane receptor tyrosine kinase

1	suggests a genotype-phenotype correlation.RET Proto-Oncogene and Multiple Endocrine Neoplasia Type 2The RET (rearranged during transfection) gene encodes for a transmembrane receptor tyrosine kinase that plays a role in pro-liferation, migration, and differentiation of cells derived from the neural crest. Gain-of-function mutations in the RET gene are associated with medullary thyroid carcinoma in isolation or mul-tiple endocrine neoplasia type 2 (MEN2) syndromes. MEN2A is associated with medullary thyroid carcinoma and pheochro-mocytoma (in 50%) or parathyroid adenoma (in 20%), whereas MEN2B is associated with medullary thyroid carcinoma, mar-fanoid habitus, mucosal neuromas, and ganglioneuromatosis of the gastrointestinal tract.86 RET mutations lead to uncontrolled growth of the thyroid C cells, and in familial medullary cancer, C-cell hyperplasia progresses to bilateral, multicentric medul-lary thyroid cancer. Mutations in the RET gene have also been identified in half of sporadic

1	C cells, and in familial medullary cancer, C-cell hyperplasia progresses to bilateral, multicentric medul-lary thyroid cancer. Mutations in the RET gene have also been identified in half of sporadic medullary thyroid cancers.Genetic Modifiers of Risk. Individuals carrying identi-cal germline mutations vary in regard to cancer penetrance (whether cancer will develop or not) and cancer phenotype (the tissues involved). It is thought that this variability may be due to environmental influences or, if genetic, to genetic modifiers Table 10-4Revised criteria for hereditary nonpolyposis colon cancer (HNPCC) (Amsterdam criteria II)Three or more relatives with an HNPCC-associated cancer (colorectal cancer, endometrial cancer, cancer of the small bowel, ureter, or renal pelvis), one of whom is a first-degree relative of the other twoAt least two successive generations affectedAt least one case diagnosed before age 50 yFamilial adenomatous polyposis excludedTumors verified by pathologic

1	is a first-degree relative of the other twoAt least two successive generations affectedAt least one case diagnosed before age 50 yFamilial adenomatous polyposis excludedTumors verified by pathologic examinationReproduced with permission from Vasen HF, Watson P, Mecklin JP, et al: New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology. 1999 Jun;116(6):1453-1456.Brunicardi_Ch10_p0305-p0354.indd 32522/02/19 2:14 PM 326BASIC CONSIDERATIONSPART Iof risk. Similarly, genetic modifiers of risk also can play a role in determining whether an individual will develop cancer after exposure to carcinogens.Chemical CarcinogensThe first report indicating that cancer could be caused by envi-ronmental factors was by John Hill, who in 1761 noted the association between nasal cancer and excessive use of tobacco snuff.87 Currently, approximately 60% to 90% of cancers are thought to be due

1	factors was by John Hill, who in 1761 noted the association between nasal cancer and excessive use of tobacco snuff.87 Currently, approximately 60% to 90% of cancers are thought to be due to environmental factors. Any agent that can contribute to tumor formation is referred to as a carcinogen and can be a chemical, physical, or viral agent. Chemicals are clas-sified into three groups based on how they contribute to tumor formation. The first group of chemical agents, the genotoxins, can initiate carcinogenesis by causing a mutation. The second group, the cocarcinogens, by themselves cannot cause cancer but potentiate carcinogenesis by enhancing the potency of geno-toxins. The third group, tumor promoters, enhances tumor for-mation when given after exposure to genotoxins.The International Agency for Research on Cancer (IARC) maintains a registry of human carcinogens that is available through the World Wide Web (http://www.iarc.fr). The compounds are categorized into five groups based

1	Agency for Research on Cancer (IARC) maintains a registry of human carcinogens that is available through the World Wide Web (http://www.iarc.fr). The compounds are categorized into five groups based on an analysis of epidemio-logic studies, animal models, and short-term mutagenesis tests. Group 1 contains what are considered to be proven human carcinogens, based on formal epidemiologic studies among workers who were exposed for long periods (several years) to the chemicals.88 Group 2A contains what are considered to be probable human carcinogens. Suggestive epidemiologic evidence exists for compounds in this group, but the data are insufficient to establish causality. There is evidence of carcinogenicity, however, from animal studies carried out under conditions relevant to human exposure. Group 2B contains what are considered to be possible carcinogens because these substances are associated with a clear statistically and biologically significant increase in the incidence of

1	exposure. Group 2B contains what are considered to be possible carcinogens because these substances are associated with a clear statistically and biologically significant increase in the incidence of malignant tumors in more than one animal species or strain. Group 3 agents are not classifiable, and Group 4 agents are prob-ably not carcinogenic to humans. Selected substances that have been classified as proven carcinogens (group 1) by the IARC in an expert panel review are listed in Table 10-5.89Physical CarcinogensPhysical carcinogenesis can occur through induction of inflam-mation and cell proliferation over a period of time or through exposure to physical agents that induce DNA damage. Foreign bodies can cause chronic irritation that can expose cells to carci-nogenesis due to other environmental agents. In animal models, for example, subcutaneous implantation of a foreign body can lead to the development of tumors that have been attributed to chronic irritation from the foreign

1	environmental agents. In animal models, for example, subcutaneous implantation of a foreign body can lead to the development of tumors that have been attributed to chronic irritation from the foreign objects. In humans, clinical sce-narios associated with chronic irritation and inflammation such as chronic nonhealing wounds, burns, and inflammatory bowel syn-drome have all been associated with an increased risk of cancer. H pylori infection is associated with gastritis and gastric cancer, and thus, its carcinogenicity may be considered physical carci-nogenesis. Infection with the liver fluke Opisthorchis viverrini similarly leads to local inflammation and cholangiocarcinoma.The induction of lung and mesothelial cancers by asbes-tos fibers and nonfibrous particles such as silica are other examples of foreign body-induced physical carcinogenesis.90 Animal experiments have demonstrated that the dimensions and durability of the asbestos and other fibrous minerals are the key determinants

1	examples of foreign body-induced physical carcinogenesis.90 Animal experiments have demonstrated that the dimensions and durability of the asbestos and other fibrous minerals are the key determinants of their carcinogenicity.91 Short fibers can be inactivated by phagocytosis, whereas long fibers (>10 μm) are cleared less effectively and are encompassed by proliferat-ing epithelial cells. The long fibers support cell proliferation and have been shown to preferentially induce tumors. Asbestosassociated biologic effects also may be mediated through reactive oxygen and nitrogen species. Furthermore, an inter-action occurs between asbestos and silica and components of cigarette smoke. Polycyclic aromatic hydrocarbons (PAHs) in cigarette smoke are metabolized by epithelial cells and form DNA adducts. If PAH is coated on asbestos, PAH uptake is increased.90 Both PAH and asbestos impair lung clearance, potentially increasing uptake further. Therefore, physical car-cinogens may be synergistic

1	If PAH is coated on asbestos, PAH uptake is increased.90 Both PAH and asbestos impair lung clearance, potentially increasing uptake further. Therefore, physical car-cinogens may be synergistic with chemical carcinogens.Radiation is the best-known agent of physical carcinogens and is classified as ionizing radiation (X-rays, gamma rays, and alpha and beta particles) or nonionizing radiation (UV). The car-cinogenic potential of ionizing radiation was recognized soon after Wilhelm Conrad Roentgen’s discovery of X-rays in 1895. Within the next 20 years, a large number of radiation-related skin cancers were reported. Long-term follow-up of survivors of the atomic bombing of Hiroshima and Nagasaki revealed that virtually all tissues exposed to radiation are at risk for cancer.Radiation can induce a spectrum of DNA lesions that includes damage to the nucleotide bases and cross-linking, and DNA singleand double-strand breaks (DSBs). Misrepaired DSBs are the principal lesions of importance in

1	a spectrum of DNA lesions that includes damage to the nucleotide bases and cross-linking, and DNA singleand double-strand breaks (DSBs). Misrepaired DSBs are the principal lesions of importance in the induction of chromosomal abnormalities and gene mutations. DSBs in irradiated cells are repaired primarily by a nonhomologous end-joining process, which is error prone; thus, DSBs facilitate the production of chromosomal rearrangements and other large-scale changes such as chromosomal deletions. It is thought that radiation may initiate cancer by inactivating tumor-suppressor genes. Activation of oncogenes appears to play a lesser role in radiation carcinogenesis.Although it has been assumed that the initial genetic events induced by radiation constitute direct mutagenesis from radiation, other indirect effects may contribute to carcinogenesis. For exam-ple, radiation induces genomic instability in cells that persists for at least 30 generations after irradiation. Therefore, even if

1	other indirect effects may contribute to carcinogenesis. For exam-ple, radiation induces genomic instability in cells that persists for at least 30 generations after irradiation. Therefore, even if cells do not acquire mutations at initial irradiation, they remain at risk for developing new mutations for several generations. Moreover, even cells that have not been directly irradiated appear to be at risk, a phenomenon referred to as the bystander effect.Nonionizing UV radiation is a potent DNA-damaging agent and is known to induce skin cancer in experimental ani-mals. Most nonmelanoma human skin cancers are thought to be induced by repeated exposure to sunlight, which leads to a series of mutations that allow the cells to escape normal growth control. Patients with inherited xeroderma pigmentosum lack one or more DNA repair pathways, which confers susceptibility to UV-induced cancers, especially on sun-exposed body parts. Patients with ataxia telangiectasia mutated syndrome also have

1	lack one or more DNA repair pathways, which confers susceptibility to UV-induced cancers, especially on sun-exposed body parts. Patients with ataxia telangiectasia mutated syndrome also have a radiation-sensitive phenotype.Viral CarcinogensOne of the first observations that cancer may be caused by trans-missible agents was by Peyton Rous in 1910 when he demon-strated that cell-free extracts from sarcomas in chickens could transmit sarcomas to other animals injected with these extracts.92 This was subsequently discovered to represent viral transmission of cancer by the Rous sarcoma virus. At present, several human viruses are known to have oncogenic properties, and several Brunicardi_Ch10_p0305-p0354.indd 32622/02/19 2:14 PM 327ONCOLOGYCHAPTER 10Table 10-5Group 1 chemical carcinogens and evidence for carcinogenicity in humans and for genotoxicity as the main mechanism TUMOR SITES OR TYPES WITH SUFFICIENT EVIDENCE IN HUMANSEVIDENCE OF GENOTOXICITY AS THE MAIN

1	chemical carcinogens and evidence for carcinogenicity in humans and for genotoxicity as the main mechanism TUMOR SITES OR TYPES WITH SUFFICIENT EVIDENCE IN HUMANSEVIDENCE OF GENOTOXICITY AS THE MAIN MECHANISM4-AminobiphenylUrinary bladderStrongBenzidineUrinary bladderStrongDyes metabolized to benzidine⋅⋅Stronga4,4’-Methylenebis(2-chloroaniline)⋅⋅Stronga2-NapthylamineUrinary bladderStrongOrtho-toluidineUrinary bladderModerateAuramine productionUrinary bladderWeak/lack of databMagenta productionUrinary bladderWeak/lack of databBenzo[α]pyrene⋅⋅StrongaSoot (chimney sweeping)Skin, lungModerateCoal gasificationLungStrongCoal-tar distillationSkinStrongCoke productionLungStrongCoal-tar pitches (paving, roofing)LungStrongAluminum productionLung, urinary bladderWeak/moderateb,cAflatoxinsHepatocellular carcinomaStrongBenzeneANLLStrongBis (chloromethyl)ether/chloromethyl methyletherLungModerate/strong1,3-ButadieneHaematolymphatic organsStrongDioxin (2,3,7,8-TCDD)All cancers combinedaSee

1	carcinomaStrongBenzeneANLLStrongBis (chloromethyl)ether/chloromethyl methyletherLungModerate/strong1,3-ButadieneHaematolymphatic organsStrongDioxin (2,3,7,8-TCDD)All cancers combinedaSee textd2,3,4,7,8-Pentachlorodibenzofuran⋅⋅See texta,d3,3’,4,4’,5-Pentachlorobiphenyl (PCB-126)⋅⋅See texta,dEthylene oxide⋅⋅StrongaFormaldehydeNasopharynxLeukemiagStrongModerateSulfur mustardLungStrongVinyl chlorideHepatic angiosarcoma, hepatocellular carcinomaStrongIron and steel foundingLungWeak/moderateIsopropyl alcohol manufacture using strong acidsNasal cavityWeak/lack of dataMineral oilsSkinWeak/lack of dataOccupational exposure as a painterLung, urinary bladder, pleural mesotheliomaStrongcRubber-manufacturing industryLeukaemia, lymphoma,g urinary bladder, lung,g stomachgStrongcShale oilsSkinWeak/lack of dataStrong inorganic acid mistsLarynxWeak/lack of dataANLL = acute nonlymphocytic leukemia; ALL = acute lymphocytic leukemia; CLL = chronic lymphocytic leukemia; MM = multiple myeloma; NH =

1	of dataStrong inorganic acid mistsLarynxWeak/lack of dataANLL = acute nonlymphocytic leukemia; ALL = acute lymphocytic leukemia; CLL = chronic lymphocytic leukemia; MM = multiple myeloma; NH = non-Hodgkin lymphoma; STS = soft-tissue sarcoma.aAgents classified in Group 1 on the basis of mechanistic information.bWeak evidence in workers, but strong evidence for some chemicals in this industry.cDue to the diversity and complexity of these exposures, other mechanisms may also be relevant.dStrong evidence for an aryl hydrocarbon receptor (AhR)-mediated mechanism.eParticularly myeloid leukemia.fAfter maternal exposure (before or during pregnancy, or both).gNew epidemiological findings.Reproduced with permission from Baan R, Grosse Y, Straif K, et al. A review of human carcinogens–part F: chemical agents and related occupations, Lancet Oncol. 2009 Dec;10(12):1143-1144.Brunicardi_Ch10_p0305-p0354.indd 32722/02/19 2:14 PM 328BASIC CONSIDERATIONSPART ITable 10-6Selected viral

1	F: chemical agents and related occupations, Lancet Oncol. 2009 Dec;10(12):1143-1144.Brunicardi_Ch10_p0305-p0354.indd 32722/02/19 2:14 PM 328BASIC CONSIDERATIONSPART ITable 10-6Selected viral carcinogensaVIRUSPREDOMINANT TUMOR TYPEbEpstein-Barr virusBurkitt’s lymphoma Hodgkin’s disease Immunosuppression-related lymphoma Sinonasal angiocentric T-cell lymphoma Nasopharyngeal carcinomaHepatitis B virusHepatocellular carcinomaHepatitis C virusHepatocellular carcinomaHIV type 1Kaposi’s sarcoma Cervical cancer Non-Hodgkin’s lymphomaHuman herpes virus 8Kaposi’s sarcomaHuman papillomavirus 16 and 18 Cervical cancerVulvar and vaginal cancer Penile cancer Oropharyngeal cancer (commonly base of tongue and tonsil) Anal cancerHuman T-cell lymphotropic virusesAdult T-cell leukemia/lymphomaMerkel cell polyoma virusMerkel cell carcinomaaData based on information in the International Agency for Research on Cancer monographs.bOnly tumor types for which causal relationships are established are

1	cell polyoma virusMerkel cell carcinomaaData based on information in the International Agency for Research on Cancer monographs.bOnly tumor types for which causal relationships are established are listed. Other cancer types may be linked to the agents with a lower frequency or with insufficient data to prove causality.have been causally linked to human cancers (Table 10-6).88 It is estimated that 15% of all human tumors worldwide are caused by viruses.93Viruses may cause or increase the risk of malignancy through several mechanisms, including direct transformation, expression of oncogenes that interfere with cell-cycle check-points or DNA repair, expression of cytokines or other growth factors, and alteration of the immune system. Oncogenic viruses may be RNA or DNA viruses. Oncogenic RNA viruses are retro-viruses and contain a reverse transcriptase. After the viral infec-tion, the single-stranded RNA viral genome is transcribed into a double-stranded DNA copy, which is then

1	RNA viruses are retro-viruses and contain a reverse transcriptase. After the viral infec-tion, the single-stranded RNA viral genome is transcribed into a double-stranded DNA copy, which is then integrated into the chromosomal DNA of the cell. Retroviral infection of the cell is permanent; thus, integrated DNA sequences remain in the host chromosome. Oncogenic transforming retroviruses carry onco-genes derived from cellular genes. These cellular genes, referred to as proto-oncogenes, usually are involved in mitogenic signal-ing and growth control, and include protein kinases, G proteins, growth factors, and transcription factors (Table 10-7).93Integration of the provirus upstream of a proto-oncogene may produce chimeric virus-cell transcripts and recombination during the next round of replication that could lead to incorpo-ration of the cellular gene into the viral genome.93 Then again, many retroviruses do not possess oncogenes but can cause tumors in animals regardless. This occurs

1	that could lead to incorpo-ration of the cellular gene into the viral genome.93 Then again, many retroviruses do not possess oncogenes but can cause tumors in animals regardless. This occurs by integration of the provirus near a normal cellular proto-oncogene and activation of the expression of these genes by the strong promoter and enhancer sequences in the integrated viral sequence.Unlike the oncogenes of the RNA viruses, those of the DNA tumor viruses are viral, not cellular, in origin. These genes are required for viral replication using the host cell machinery. In permissive hosts, infection with an oncogenic DNA virus may result in a productive lytic infection, which leads to cell death and the release of newly formed viruses. In nonpermis-sive cells, the viral DNA can be integrated into the cellular chromosomal DNA, and some of the early viral genes can be synthesized persistently, which leads to transformation of cells to a neoplastic state. The binding of viral oncoproteins

1	into the cellular chromosomal DNA, and some of the early viral genes can be synthesized persistently, which leads to transformation of cells to a neoplastic state. The binding of viral oncoproteins to cellu-lar tumor-suppressor proteins p53 and Rb is fundamental to the carcinogenesis induced by most DNA viruses, although some target different cellular proteins.Like other types of carcinogenesis, viral carcinogenesis is a multistep process. Some retroviruses contain two cellular onco-genes, rather than one, in their genome and are more rapidly tumorigenic than single-gene transforming retroviruses, which emphasizes the cooperation between transforming genes. Further-more, some viruses encode genes that suppress or delay apoptosis.Although immunocompromised individuals are at ele-vated risk, most patients infected with oncogenic viruses do not develop cancer. When cancer does develop, it usually occurs sev-eral years after the viral infection. It is estimated, for example, that the risk

1	most patients infected with oncogenic viruses do not develop cancer. When cancer does develop, it usually occurs sev-eral years after the viral infection. It is estimated, for example, that the risk of hepatocellular carcinoma (HCC) among individu-als infected with hepatitis C virus is 1% to 3% after 30 years.94 There may be synergy between various environmental factors and viruses in carcinogenesis.Recognition of a viral origin for some tumors has led to the pursuit of vaccination as a preventive strategy. The use of childhood hepatitis B vaccination has already translated into a decrease in liver cancer incidence in the East Asia.5 Similarly, it is recognized that cervical cancer and its obligate precursors, cervical intraepithelial neoplasia grades 2 and 3, and adenocar-cinoma in situ, are caused by oncogenic human papillomavirus (HPV); administration of HPV vaccine to HPV-naive women, substantially reduces the incidence of HPV16/18-related cervi-cal precancers and cervical

1	situ, are caused by oncogenic human papillomavirus (HPV); administration of HPV vaccine to HPV-naive women, substantially reduces the incidence of HPV16/18-related cervi-cal precancers and cervical cancer.95 Studies suggest that HPV vaccination may also reduce oral HPV infections that are a risk factor for the development of oropharyngeal cancer.96 The American Cancer Society recommends routine HPV vaccina-tion for girls and boys starting at age 11 or 12. The vaccination series can be started as early as age 9. HPV vaccination is also recommended for females 13 to 26 years old and for males 13 to 21 years old who have not started the vaccines, or who have started but have not completed the series. Males 22 to 26 years old may also be vaccinated. HPV vaccination is also recom-mended up until age 26 for men who have sex with men and for people with weakened immune systems (including people with HIV infection), if they have not previously been vaccinated. It is important for patients to

1	age 26 for men who have sex with men and for people with weakened immune systems (including people with HIV infection), if they have not previously been vaccinated. It is important for patients to know that vaccination at older ages is less effective in lowering cancer risk.97CANCER RISK ASSESSMENTCancer risk assessment is an important part of the initial evalua-tion of any patient. A patient’s cancer risk not only is an impor-tant determinant of cancer screening recommendations but also Brunicardi_Ch10_p0305-p0354.indd 32822/02/19 2:14 PM 329ONCOLOGYCHAPTER 10Table 10-7Selected cellular oncogenes in retrovirusesONCOGENEVIRUS NAMEORIGINPROTEIN PRODUCTablAbelson murine leukemia virusMouseTyrosine kinasefesST feline sarcoma virusCatTyrosine kinasefpsFujinami sarcoma virusChickenTyrosine kinasesrcRous sarcoma virusChickenTyrosine kinaseerbBAvian erythroblastosis virusChickenEpidermal growth factor receptorfmsMcDonough feline sarcoma virusCatColony-stimulating factor

1	kinasesrcRous sarcoma virusChickenTyrosine kinaseerbBAvian erythroblastosis virusChickenEpidermal growth factor receptorfmsMcDonough feline sarcoma virusCatColony-stimulating factor receptorkitHardy-Zuckerman 4 feline sarcoma virusCatStem cell factor receptormilAvian myelocytoma virusChickenSerine/threonine kinasemosMoloney murine sarcoma virusMouseSerine/threonine kinaserafMurine sarcoma virus 3611MouseSerine/threonine kinasesisSimian sarcoma virusMonkeyPlatelet-derived growth factorH-rasHarvey murine sarcoma virusRatGDP/GTP bindingK-rasKirsten murine sarcoma virusRatGDP/GTP bindingerbAAvian erythroblastosis virusChickenTranscription factor (thyroid hormone receptor)etsAvian myeloblastosis virus E26ChickenTranscription factorfosFBJ osteosarcoma virusMouseTranscription factor (AP1 component)junAvian sarcoma virus 17ChickenTranscription factor (AP1 component)mybAvian myeloblastosis virusChickenTranscription factormycMC29 myelocytoma virusChickenTranscription factor (NF-κB family)AP1 =

1	sarcoma virus 17ChickenTranscription factor (AP1 component)mybAvian myeloblastosis virusChickenTranscription factormycMC29 myelocytoma virusChickenTranscription factor (NF-κB family)AP1 = activator protein 1; FBJ = Finkel-Biskis-Jinkins; GDP = guanosine diphosphate; GTP = guanosine triphosphate; NF-κB = nuclear factor κB.Data from Coffin JM, Hughes SH, Varmus HE: Retroviruses. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1997.may alter how aggressively an indeterminant finding will be pursued for diagnosis. A “probably benign” mammographic lesion, for example, defined as one with <2% probability of malignancy (American College of Radiology category III) is usually managed with a 6-month follow-up mammogram in a patient at baseline cancer risk, but obtaining a tissue diagnosis may be preferable in a patient at high risk for breast cancer.98Cancer risk assessment starts with taking a complete his-tory that includes history of environmental exposures to potential

1	diagnosis may be preferable in a patient at high risk for breast cancer.98Cancer risk assessment starts with taking a complete his-tory that includes history of environmental exposures to potential carcinogens and a detailed family history. Risk assessment for breast cancer, for example, includes obtaining a family history to determine whether another member of the family is known to carry a breast cancer susceptibility gene; whether there is famil-ial clustering of breast cancer, ovarian cancer, thyroid cancer, sarcoma, adrenocortical carcinoma, endometrial cancer, brain tumors, dermatologic manifestations, leukemia, or lymphoma; and whether the patient is from a population at increased risk, such as individuals of Ashkenazi Jewish descent. Patients who have a family history suggestive of a cancer susceptibility syn-drome such as hereditary breast-ovarian syndrome, Li-Fraumeni Syndrome, or Cowden’s Disease would benefit from genetic counseling and possibly genetic testing.There are

1	of a cancer susceptibility syn-drome such as hereditary breast-ovarian syndrome, Li-Fraumeni Syndrome, or Cowden’s Disease would benefit from genetic counseling and possibly genetic testing.There are several models that can estimate risk based on complex family histories and assist clinicians in estimat-ing breast cancer risk or the likelihood that a BRCA mutation is present, including the Claus model, Tyrer-Cuzick model, BRCAPRO model, and the Breast and Ovarian Analysis of Dis-ease Incidence and Carrier Estimation Algorithm (BOADICEA) model.99-102 Patients who do have a strong hereditary component of risk can be evaluated on the basis of their age, race, per-sonal history, and exposures. One of the most commonly used models for risk assessment in breast cancer is the Gail model.103 Gail and colleagues analyzed the data from 2852 breast cancer cases and 3146 controls from the Breast Cancer Detection and Demonstration Project, a mammography screening project conducted in the 1970s,

1	and colleagues analyzed the data from 2852 breast cancer cases and 3146 controls from the Breast Cancer Detection and Demonstration Project, a mammography screening project conducted in the 1970s, and developed a model for project-ing breast cancer incidence. The model uses risk factors such as an individual’s age, age at menarche, age at first live birth, number of first-degree relatives with breast cancer, number of previous breast biopsy specimens, and whether the biopsy speci-men results revealed atypical ductal hyperplasia (Table 10-8).103 This model has led to the development of a breast cancer risk assessment tool, which is available on the World Wide Web.104 This tool incorporates the risk factors used in the Gail model, as well as race and ethnicity, and allows a health professional to project a woman’s individualized estimated risk for invasive breast cancer over a 5-year period and over her lifetime (to age 90 years). Notably, these risk projections assume that the woman

1	to project a woman’s individualized estimated risk for invasive breast cancer over a 5-year period and over her lifetime (to age 90 years). Notably, these risk projections assume that the woman is undergoing regular clinical breast examinations and screen-ing mammograms. Also of note is that this program underes-timates the risk for women who have already had a diagnosis of invasive or noninvasive breast cancer and does not take into account specific genetic predispositions such as mutations in BRCA1 or BRCA2. However, risk assessment tools such as this have been validated and are now in widespread clinical use. Similar models are in development or are being validated for Brunicardi_Ch10_p0305-p0354.indd 32922/02/19 2:14 PM 330BASIC CONSIDERATIONSPART ITable 10-8Assessment of risk for invasive breast cancerRISK FACTORRELATIVE RISK (%)Age at menarche (years) >141.00 12–131.10 <121.21Age at first live birth (years) Patients with no first-degree relatives with

1	of risk for invasive breast cancerRISK FACTORRELATIVE RISK (%)Age at menarche (years) >141.00 12–131.10 <121.21Age at first live birth (years) Patients with no first-degree relatives with cancer <201.00 20–241.24 25–29 or nulliparous1.55 ≥301.93Patients with one first degree-relative with cancer <201.00 20–242.64 25–29 or nulliparous2.76 ≥302.83Patients with ≥2 first-degree relatives with cancer <206.80 20–245.78 25–29 or nulliparous4.91 ≥304.17Breast biopsies (number) Patients aged <50 y at counseling 01.00 11.70 ≥22.88Patients aged ≥50 y at counseling 01.00 11.27 ≥21.62Atypical hyperplasia No biopsies1.00 At least 1 biopsy, no atypical hyperplasia0.93 No atypical hyperplasia, hyperplasiastatus unknown for at least 1 biopsy1.00 Atypical hyperplasia in at least 1 biopsy1.82Modified with permission from Gail MH, Brinton LA, Byar DP, et al. Projecting individualized probabilities of developing breast cancer for white females who are being examined annually, J Natl Cancer

1	with permission from Gail MH, Brinton LA, Byar DP, et al. Projecting individualized probabilities of developing breast cancer for white females who are being examined annually, J Natl Cancer Inst. 1989 Dec 20;81(24):1879-1886.as presence or absence of single nucleotide polymorphisms which each may confer low or intermediate cancer risk. Risk models that include biologic as well as environmental factors may accurately predict cancer risk, providing better guidance as to which patients should undergo more intensive screening (e.g., screening with magnetic resonance imaging of the breast, computerized tomography screening of the lung), and should be considered for preventive strategies.CANCER SCREENINGEarly detection is the key to success in cancer therapy. Screen-ing for common cancers using relatively noninvasive tests is expected to lead to early diagnosis, allow more conservative surgical therapies with decreased morbidity, and potentially improve surgical cure rates and overall

1	using relatively noninvasive tests is expected to lead to early diagnosis, allow more conservative surgical therapies with decreased morbidity, and potentially improve surgical cure rates and overall survival rates. Key factors that influence screening guidelines are how prevalent the cancer is in the population, what risk is associated with the screening measure, and whether early diagnosis actually affects outcome. The value of a widespread screening measure is likely to go up with the prevalence of the cancer in a population, which often determines the age cutoffs for screening and explains why screening is done only for common cancers. The risks asso-ciated with the screening measure are a significant consider-ation, especially with more invasive screening measures such as colonoscopy. The consequences of a false-positive screening test result also need to be considered. For example, when 1000 screening mammograms are taken, only 2 to 4 new cases of cancer will be identified; this

1	The consequences of a false-positive screening test result also need to be considered. For example, when 1000 screening mammograms are taken, only 2 to 4 new cases of cancer will be identified; this number is slightly higher (6 to 10 prevalent cancers per 1000 mammograms) for initial screen-ing mammograms.106 However, as many as 10% of screening mammograms may be potentially suggestive of an abnormal-ity, which requires further imaging (i.e., a 10% recall rate). Of those women with abnormal mammogram findings, only 5% to 10% will be determined to have a breast cancer. Among women for whom biopsy specimen is recommended, 25% to 40% will have a breast cancer. A false-positive screening result is likely to induce significant emotional distress in patients, leads to unnecessary biopsy specimens, and has cost implications for the health care system.American Cancer Society guidelines for the early detec-tion of cancer are listed in Table 10-9.96 These guidelines are updated periodically to

1	and has cost implications for the health care system.American Cancer Society guidelines for the early detec-tion of cancer are listed in Table 10-9.96 These guidelines are updated periodically to incorporate emerging technologies and new data on the efficacy of screening measures. Besides the American Cancer Society, several other professional bodies make recommendations for screening. Although the screening guidelines differ somewhat, most organizations do not empha-size one screening strategy as superior to another, but all empha-size the importance of age-appropriate screening.Screening guidelines are developed for the general base-line-risk population. These guidelines need to be modified for patients who are at high risk. For example, more intensive colorectal cancer screening is recommended for individuals at increased risk because of a history of adenomatous polyps, a personal history of colorectal cancer, a family history of either colorectal cancer or colorectal adenomas

1	recommended for individuals at increased risk because of a history of adenomatous polyps, a personal history of colorectal cancer, a family history of either colorectal cancer or colorectal adenomas diagnosed in a first-degree relative before age 60 years, a personal history of inflam-matory bowel disease of significant duration, or a family history or genetic test result indicating FAP or HNPCC. For some dis-eases, in higher risk populations, both the screening modality and the screening intensity may be altered. For example, breast magnetic resonance imaging is recommended as an adjunct to other cancers. For example, a lung cancer risk prediction model, which includes age, sex, asbestos exposure history, and smoking history, has been found to predict risk of lung cancer.105 There is now growing interest in using each individuals genotype, such Brunicardi_Ch10_p0305-p0354.indd 33022/02/19 2:14 PM 331ONCOLOGYCHAPTER 10Table 10-9American Cancer Society recommendations for early

1	now growing interest in using each individuals genotype, such Brunicardi_Ch10_p0305-p0354.indd 33022/02/19 2:14 PM 331ONCOLOGYCHAPTER 10Table 10-9American Cancer Society recommendations for early detection of cancer in average-risk, asymptomatic individualsCANCER SITEPOPULATIONTEST OR PROCEDUREFREQUENCYBreastWomen age 40 and aboveMammographyWomen with an average risk of breast cancer should undergo regular screening mammography starting at age 45 years. Women age 45 to 54 years should be screened annually. Women 55 years and older should transition to biennial screening or have the opportunity to continue screening annually. Women should continue screening mammography as long as their overall health is good and they have a life expectancy of 10 years or longer. The ACS does not recommend clinical breast examination for breast cancer screening among average-risk women at any age.CervixWomen, age 21–65 yPap test and HPV DNA testCervical cancer screening should begin at age 21 y.

1	clinical breast examination for breast cancer screening among average-risk women at any age.CervixWomen, age 21–65 yPap test and HPV DNA testCervical cancer screening should begin at age 21 y. For women age 21–29 y, screening should be done every 3 y with conventional or liquid-based Pap tests. For women age 30–65 y, screening should be done every 5 y with both the HPV test and the Pap test (preferred), or every 3 y with the Pap test alone (acceptable). Women age >65 y who have had ≥3 consecutive negative Pap tests or ≥2 consecutive negative HPV and Pap tests within the last 10 y, with the most recent test occurring within the last 5 y, and women who have had a total hysterectomy should stop cervical cancer screening.ColorectalMen and women age ≥50 ygFOBT, or FIT, or sDNA with a high sensitivity for cancerAnnual, starting at age 50 y. FSIG, orEvery 5 y, starting at age 50 y. FSIG can be performed alone, or consideration can be given to combining FSIG performed every 5 y with a

1	sensitivity for cancerAnnual, starting at age 50 y. FSIG, orEvery 5 y, starting at age 50 y. FSIG can be performed alone, or consideration can be given to combining FSIG performed every 5 y with a highly sensitive guaiac-based FOBT or FIT performed annually. DCBE, orEvery 5 y, starting at age 50 y. ColonoscopyEvery 10 y, starting at age 50 y. CT colonographyEvery 5 yr, starting at age 50 y.EndometrialWomen, at menopause At the time of menopause, women at average risk should be informed about the risks and symptoms of endometrial cancer and strongly encouraged to report any unexpected bleeding or spotting to their physicians.LungCurrent or former smokers age 50–74 in good health with at least a 30 pack/year historyLDCTClinicians with access to high-volume, high-quality lung cancer screening and treatment centers should initiate a discussion about lung cancer screening with apparently healthy patients age 55–74 y who have at least a 30 pack-y smoking history, and who currently smoke

1	and treatment centers should initiate a discussion about lung cancer screening with apparently healthy patients age 55–74 y who have at least a 30 pack-y smoking history, and who currently smoke or have quit within the past 15 y. A process of informed and shared decision-making with a clinician related to the potential benefits, limitations, and harms associated with screening for lung cancer with LDCT should occur before any decision is made to initiate lung cancer screening. Smoking cessation counseling remains a high priority for clinical attention in discussions with current smokers, who should be informed of their continuing risk of lung cancer. Screening should not be viewed as an alternative to smoking cessation.(Continued)Brunicardi_Ch10_p0305-p0354.indd 33122/02/19 2:14 PM 332BASIC CONSIDERATIONSPART ITable 10-9American Cancer Society recommendations for early detection of cancer in average-risk, asymptomatic individualsCANCER SITEPOPULATIONTEST OR

1	2:14 PM 332BASIC CONSIDERATIONSPART ITable 10-9American Cancer Society recommendations for early detection of cancer in average-risk, asymptomatic individualsCANCER SITEPOPULATIONTEST OR PROCEDUREFREQUENCYProstateMen, age ≥50 yDRE and PSAMen who have at least a 10-y life expectancy should have an opportunity to make an informed decision with their health care provider about whether to be screened for prostate cancer, after receiving information about the potential benefits, risks, and uncertainties associated with prostate cancer screening. Prostate cancer screening should not occur without an informed decision-making process. Men at average risk should receive this information beginning at age 50 years. Men in higher risk groups should receive this information before age 50 years. Men should either receive this information directly from their health care providers or be referred to reliable and culturally appropriate sources. Patient decision aids are helpful in preparing men to

1	should either receive this information directly from their health care providers or be referred to reliable and culturally appropriate sources. Patient decision aids are helpful in preparing men to make a decision whether to be tested.Cancer-related checkupMen and women age ≥20 y On the occasion of a periodic health examination, the cancer-related checkup should include examination for cancers of the thyroid, testicles, ovaries, lymph nodes, oral cavity, and skin, as well as health counseling about tobacco, sun exposure, diet and nutrition, risk factors, sexual practices, and environmental and occupational exposures.ACS = American Cancer Society; BSE = breast self-examination; CBE = clinical breast examination; Pap = Papanicolaou; HPV = human papillomavirus; gFOBT = guaiac-based fecal occult blood test; FIT = fecal immunochemical test; sDNA, stool DNA; DRE = digital rectal examination; FSIG = flexible sigmoidoscopy; DCBE = double-contrast barium enema; CT = computed tomography; LDCT =

1	blood test; FIT = fecal immunochemical test; sDNA, stool DNA; DRE = digital rectal examination; FSIG = flexible sigmoidoscopy; DCBE = double-contrast barium enema; CT = computed tomography; LDCT = low-dose helical CT; PSA = prostate-specific antigen.Reproduced with permission from Smith RA, Brooks D, Cokkinides V, et al: Cancer screening in the United States, 2013: a review of current American Cancer Society guidelines, current issues in cancer screening, and new guidance on cervical cancer screening and lung cancer screening, CA Cancer J Clin. 2013 Mar-Apr;63(2):88-105.mammography for breast cancer screening in BRCA mutation carriers, first-degree relatives of carriers, and women with a life-time breast cancer risk of 20% to 25% or higher.107The National Lung Screening Trial demonstrated a 20% reduction in lung cancer deaths in adults age 55 to 74 years who were at high risk of lung cancer and randomized to low-dose helical computed tomography (LDCT) screening compared with screening

1	a 20% reduction in lung cancer deaths in adults age 55 to 74 years who were at high risk of lung cancer and randomized to low-dose helical computed tomography (LDCT) screening compared with screening with annual CXR.108 In 2013, the American Cancer Society updated their lung cancer screening recommendations to emphasize that clinicians with access to high-volume, high-quality lung cancer screening and treatment centers should ascertain the smoking history of their patients 55 to 74 years of age, and should discuss lung cancer screening with those who have at least a 30 pack per year smoking history, currently smoke, or have quit within the past 15 years, and who are in relatively good health.109 It is recommended that this discussion include the benefits, uncertainties, and harms associated with screening for lung cancer with LDCT.CANCER DIAGNOSISThe definitive diagnosis of solid tumors is obtained by perform-ing a biopsy specimen of the lesion. Biopsy findings determine the tumor

1	with screening for lung cancer with LDCT.CANCER DIAGNOSISThe definitive diagnosis of solid tumors is obtained by perform-ing a biopsy specimen of the lesion. Biopsy findings determine the tumor histology and grade and thus, assist in definitive ther-apeutic planning. Biopsy specimens of mucosal lesions usually are obtained endoscopically (e.g., via colonoscope, broncho-scope, or cystoscope). Lesions that are easily palpable, such as those of the skin, can either be excised or sampled by punch biopsy specimen. Deep-seated lesions can be localized with computed tomographic (CT) scan or ultrasound guidance for acquisition of a biopsy specimen.A sample of a lesion can be obtained with a needle or with an open incisional or excisional biopsy specimen. Fine-needle aspiration is easy and relatively safe, but has the disad-vantage of not giving information on tissue architecture. For example, fine-needle aspiration biopsy specimen of a breast mass can make the diagnosis of malignancy but

1	safe, but has the disad-vantage of not giving information on tissue architecture. For example, fine-needle aspiration biopsy specimen of a breast mass can make the diagnosis of malignancy but cannot dif-ferentiate between an invasive and noninvasive tumor. There-fore core-needle biopsy specimen is more advantageous when the histologic findings will affect the recommended therapy. Core biopsy specimen, like fine-needle aspiration, is relatively safe and can be performed either by direct palpation (e.g., a breast mass or a soft tissue mass) or can be guided by an imag-ing study (e.g., stereotactic core biopsy specimen of the breast). Core biopsy specimens, like fine-needle aspirations, have the disadvantage of introducing sampling error. For example, 19% to 44% of patients with a diagnosis of atypical ductal hyperpla-sia based on core biopsy specimen findings of a mammographic abnormality are found to have carcinoma upon excision of the entire lesion.110 It is crucial to ensure that

1	of atypical ductal hyperpla-sia based on core biopsy specimen findings of a mammographic abnormality are found to have carcinoma upon excision of the entire lesion.110 It is crucial to ensure that the histologic find-ings are consistent with the clinical scenario and to know the appropriate interpretation of each histologic finding. A needle biopsy specimen for which the report is inconsistent with the clinical scenario should be either repeated or followed by an open biopsy procedure.Open biopsy specimens have the advantage of providing more tissue for histologic evaluation and the disadvantage of being an operative procedure. Incisional biopsy specimens are (Continued)Brunicardi_Ch10_p0305-p0354.indd 33222/02/19 2:14 PM 333ONCOLOGYCHAPTER 10reserved for very large lesions in which a definitive diagnosis cannot be made by needle biopsy specimen. Excisional biopsy specimens are performed for lesions for which either core biopsy specimen is not possible or the results are

1	which a definitive diagnosis cannot be made by needle biopsy specimen. Excisional biopsy specimens are performed for lesions for which either core biopsy specimen is not possible or the results are nondiagnostic. Excisional biopsy specimens should be performed with curative intent, that is, by obtaining adequate tissue around the lesion to ensure negative surgical margins. Marking of the orientation of the margins by sutures or clips by the surgeon and inking of the specimen margins by the pathologist will allow for determina-tion of the surgical margins and will guide surgical reexcision if one or more of the margins are positive for microscopic tumor or are close. The biopsy specimen incision should be oriented to allow for excision of the biopsy specimen scar if repeat opera-tion is necessary. Furthermore, the biopsy specimen incision should directly overlie the area to be removed rather than tun-neling from another site, which runs the risk of contaminating a larger field.

1	is necessary. Furthermore, the biopsy specimen incision should directly overlie the area to be removed rather than tun-neling from another site, which runs the risk of contaminating a larger field. Meticulous hemostasis during a biopsy specimen is essential because a hematoma can lead to contamination of the tissue planes and can make subsequent follow-up with physical examinations much more challenging.In the case where an enlarged lymph node is being inves-tigated due to a suspicion of lymphoma, it will usually be nec-essary for the surgeon to remove an entire lymph node through an open biopsy in order to permit an analysis of lymph node architecture and provide sufficient tissue for molecular and flow cytometric analyses.CANCER STAGINGCancer staging is a system used to describe the anatomic extent of a malignant process in an individual patient. Staging sys-tems may incorporate relevant clinical prognostic factors such as tumor size, location, extent, grade, and dissemination to

1	extent of a malignant process in an individual patient. Staging sys-tems may incorporate relevant clinical prognostic factors such as tumor size, location, extent, grade, and dissemination to regional lymph nodes or distant sites. Accurate staging is essen-tial in designing an appropriate treatment regimen for an indi-vidual patient. Staging of the lymph node basin is considered a standard part of primary surgical therapy for most surgical procedures and is discussed later in this chapter. Cancer patients who are considered to be at high risk for distant metastasis usu-ally undergo a preoperative staging work-up. This involves a set of imaging studies of sites of preferential metastasis for a given cancer type. For a patient with breast cancer, for example, a staging work-up would include a chest radiograph, bone scan, and liver ultrasound, or CT scans to evaluate for lung, bone, and liver metastases, respectively. A distant staging work-up usually is performed only for patients

1	a chest radiograph, bone scan, and liver ultrasound, or CT scans to evaluate for lung, bone, and liver metastases, respectively. A distant staging work-up usually is performed only for patients likely to have metastasis based on the characteristics of the primary tumor; for example, a staging work-up for a patient with ductal carcinoma in situ of the breast or a small invasive breast tumor is likely to be low yield and not cost effective.Recently there also is increased usage of molecular imag-ing with positron emission tomography (PET) scanning, or PET/CT, for cancer staging. Most commonly PET scanning is performed with fluorine 18 incorporated into fluorodeoxy-glucose (FDG). FDG PET assesses the rate of glycolysis. FDG uptake is increased in most malignant tissues but also in benign pathologic conditions such as inflammatory disorders, trauma, infection, and granulomatous disease. PET/CT combines a PET scanner and an X-ray CT scanner in a single gantry, in order to acquire

1	pathologic conditions such as inflammatory disorders, trauma, infection, and granulomatous disease. PET/CT combines a PET scanner and an X-ray CT scanner in a single gantry, in order to acquire sequential images from both devices in the same ses-sion. These separate images are combined into a single coreg-istered image that gives information on the size and shape of abnormal masses in conjunction with their metabolic activity. It has been especially useful in the staging and management of lymphoma, lung cancer, and colorectal cancer. The role of PET in evaluating many other cancers is evolving, and additional molecular tracers, such as 3′-deoxy-3′ (18F)-fluorothymidine, used to assess proliferation, are being actively pursued.A PET scan can be useful in staging a cancer that poten-tially can be treated radically, such as small cell lung cancer. In the case of some cancers such as GIST, a PET scan can be used to establish baseline staging before commencing targeted therapy and

1	can be treated radically, such as small cell lung cancer. In the case of some cancers such as GIST, a PET scan can be used to establish baseline staging before commencing targeted therapy and assessing the overall response to therapy. Another use for PET scanning is the evaluation of an indeterminate lesion as in the case of a solitary pulmonary nodule that is suspected to be malignant in nature. In testicular cancer (seminoma) and lymphoma, this imaging modality has been shown to be effec-tive in assessing suspected disease recurrence, relapse, and/or residual disease. Finally, PET scans have been effective in guid-ing biopsies in the setting of mesothelioma. Standardization of staging systems is essential to allow comparison of results from different studies from different institutions and worldwide. The staging systems proposed by the American Joint Committee on Cancer (AJCC) and the Union Internationale Contre le Cancer (International Union Against Cancer, or UICC) are among the

1	and worldwide. The staging systems proposed by the American Joint Committee on Cancer (AJCC) and the Union Internationale Contre le Cancer (International Union Against Cancer, or UICC) are among the most widely accepted staging systems. Both the AJCC and the UICC have adopted a shared tumor, node, and metastasis (TNM) staging system that defines the cancer in terms of the anatomic extent of disease and is based on assessment of three components: the size of the primary tumor (T), the presence (or absence) and extent of nodal metastases (N), and the presence (or absence) and extent of distant metastases (M).The TNM staging applies only to tumors that have been microscopically confirmed to be malignant. Standard TNM staging (clinical and pathologic) is completed at initial diag-nosis. Clinical staging (cTNM or TNM) is based on informa-tion gained up until the initial definitive treatment. Pathologic staging (pTNM) includes clinical information and information obtained from pathologic

1	staging (cTNM or TNM) is based on informa-tion gained up until the initial definitive treatment. Pathologic staging (pTNM) includes clinical information and information obtained from pathologic examination of the resected primary tumor and regional lymph nodes. Tumor size following neo-adjuvant chemotherapy is designated as ypT, and should be based on the largest single focus of residual invasive cancer. Other classifications, such as staging at the time of retreatment for recurrence (rTNM) or autopsy staging (aTNM), should be clearly identified as such.The clinical measurement of tumor size (T) is the one judged to be the most accurate for each individual case based on physical examination and imaging studies. For example, in breast cancer the size of the tumor could be obtained from a physical examination, mammogram, or ultrasound, and the tumor size is based only on the invasive component.If even one lymph node is involved by tumor, the N com-ponent is at least N1. For many solid

1	physical examination, mammogram, or ultrasound, and the tumor size is based only on the invasive component.If even one lymph node is involved by tumor, the N com-ponent is at least N1. For many solid tumor types, simply the absence or presence of lymph node involvement is recorded, and the tumor is categorized either as N0 or N1. For other tumor types, the number of lymph nodes involved, the size of the lymph nodes or the lymph node metastasis, or the regional lymph node basin involved also has been shown to have prog-nostic value. In these cancers, the designations N1, N2, and N3 suggest an increasing abnormality of lymph nodes based on size, characteristics, and location. NX indicates that the lymph nodes cannot be fully assessed.Cases in which there is no distant metastasis are designated M0, cases in which one or more distant metastases are detected Brunicardi_Ch10_p0305-p0354.indd 33322/02/19 2:14 PM 334BASIC CONSIDERATIONSPART Iare designated M1, and cases in which the

1	M0, cases in which one or more distant metastases are detected Brunicardi_Ch10_p0305-p0354.indd 33322/02/19 2:14 PM 334BASIC CONSIDERATIONSPART Iare designated M1, and cases in which the presence of distant metastasis cannot be assessed are designated MX. In clinical practice, negative findings on clinical history and examination are sufficient to designate a case as M0. However, in clinical trials, routine follow-up often is performed to standardize the detection of distant metastases.The practice of dividing cancer cases into groups accord-ing to stage is based on the observation that the survival rates are higher for localized (lower-stage) tumors than for tumors that have extended beyond the organ of origin. Therefore, staging assists in selection of therapy, estimation of prognosis, evalu-ation of treatments, and exchange of information among treat-ment centers. Notably, the AJCC regularly updates its staging system to incorporate advances in prognostic technology to improve

1	evalu-ation of treatments, and exchange of information among treat-ment centers. Notably, the AJCC regularly updates its staging system to incorporate advances in prognostic technology to improve the predictive accuracy of the TNM system. There-fore, it is important to know which revision of a staging system is being used when evaluating studies.TUMOR MARKERSPrognostic and Predictive Tissue MarkersTumor markers are substances that can be detected in higher than normal amounts in the serum, urine, or tissues of patients with certain types of cancer. Tumor markers are produced either by the cancer cells themselves or by the body in a response to the cancer.Over the past decade, there has been an especially high interest in identifying tissue tumor markers that can be used as prognostic or predictive markers. Although the terms prognostic marker and predictive marker are sometimes used interchange-ably, the term prognostic marker generally is used to describe molecular markers that

1	or predictive markers. Although the terms prognostic marker and predictive marker are sometimes used interchange-ably, the term prognostic marker generally is used to describe molecular markers that predict disease-free survival, disease-specific survival, and overall survival, whereas the term predic-tive marker often is used in the context of predicting response to certain therapies.The goal is to identify prognostic markers that can give information on prognosis independent of other clinical charac-teristics and therefore can provide information to supplement the projections based on clinical presentation. This would allow practitioners to further classify patients as being at higher or lower risk within clinical subgroups and to identify patients who may benefit most from adjuvant therapy. For example, ideal prognostic tumor markers would be able to help determine which patients with node-negative breast cancer are at higher risk of relapse so that adjuvant systemic therapy could

1	For example, ideal prognostic tumor markers would be able to help determine which patients with node-negative breast cancer are at higher risk of relapse so that adjuvant systemic therapy could be given only to that group. However, although a large number of studies have identified potential novel prognostic markers, most have not been tested with enough vigor to be shown to be of clinical util-ity. In the 2017, American Society of Clinical Oncology (ASCO) guidelines, it was decided that level of uPA/PAI-1 measured by enzyme-linked immunosorbent assay could be used to determine prognosis in cases of newly diagnosed node-negative, hormone receptor positive breast cancer.111 In contrast, the data for many other markers, including Ki-67, p27, HER1/EGFR, and p53 were felt to be insufficient to support their use in the management of these breast cancer patients.111 Similarly, guidelines are avail-able for the management of patients with colorectal cancer and emphasize the processes that

1	to support their use in the management of these breast cancer patients.111 Similarly, guidelines are avail-able for the management of patients with colorectal cancer and emphasize the processes that need to be in place for accurate measurement of abnormalities in DNA mismatch repair genes as well as EGFR and BRAF mutational status.112Predictive markers are markers that can prospectively identify patients who will benefit from a certain therapy. For Low-riskgroupIntermediate-risk groupHigh-riskgroup005510101515202025253030353540404550Rate of distant recurrence at 10 y(% of patients)Recurrence scoreFigure 10-13. Distant recurrence as a continuous function of the recurrence score derived from tumor levels of expression of 21 genes. (Redrawn from Paik S, Shak S, Tang G, et al: A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer, N Engl J Med. 2004 Dec 30;351(27):2817-2826.)example in breast cancer, estrogen receptor (ER), and HER2 assessment can

1	assay to predict recurrence of tamoxifen-treated, node-negative breast cancer, N Engl J Med. 2004 Dec 30;351(27):2817-2826.)example in breast cancer, estrogen receptor (ER), and HER2 assessment can identify patients who can benefit from anti-estrogen therapies (e.g., tamoxifen) and anti-HER2 targeted therapies (e.g., trastuzumab), respectively, and the ASCO guide-lines recommend that these markers be routinely assessed.111 High-throughput techniques such as transcriptional profiling allow for assessment of the relative mRNA levels of thou-sands of genes simultaneously in a given tumor using micro-array technology. With the advent of such molecular profiling technologies, researchers have focused on identifying expres-sion profiles that are prognostic for different cancer types. For breast cancer, although many such multiparameter tests are under development, few have reached the large-scale valida-tion stage.113 In 2007, ASCO guidelines suggested that one of these, the Oncotype DX

1	cancer, although many such multiparameter tests are under development, few have reached the large-scale valida-tion stage.113 In 2007, ASCO guidelines suggested that one of these, the Oncotype DX assay, can be used to predict recurrence in women with node-negative, ER-positive breast cancer who are treated with tamoxifen.111 Oncotype DX is a quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) test that used paraffin-fixed tissue. A 21-gene recurrence score (RS) is generated based on the expression of 16 cancer genes and 5 reference genes. The levels of expression are used to derive an RS that ranges from 0 to 100, using a prospectively defined mathematical algorithm. This novel quantitative approach to the evaluation of the best-known molecular pathways in breast cancer has produced impressive results. Use of this multigene assay to predict recurrence was validated in the National Surgi-cal Adjuvant Breast and Bowel Project (NSABP) B-14 trial, in which ER-positive,

1	has produced impressive results. Use of this multigene assay to predict recurrence was validated in the National Surgi-cal Adjuvant Breast and Bowel Project (NSABP) B-14 trial, in which ER-positive, node-negative patients had received tamoxifen.114 By multivariate Cox proportional analysis, RS was found to be independently associated with recurrence risk, with a hazard ratio of 3.21 (95% confidence interval of 2.23 to 4.65, P <.001). The RS was indeed able to stratify patients by freedom from distant recurrence (Fig. 10-13).114 The Trial Assessing Individualized Options for Treatment for breast cancer (TAILORx) is evaluating the utility of Oncotype DX for predicting prognosis in patients with ER-positive, node-negative tumors and will focus on women with intermediate RS scores in whom the role of chemotherapy is unclear. Several other multigene predictors for breast cancer are available including MammaPrint, a gene expression profiling platform assessing a

1	RS scores in whom the role of chemotherapy is unclear. Several other multigene predictors for breast cancer are available including MammaPrint, a gene expression profiling platform assessing a Brunicardi_Ch10_p0305-p0354.indd 33422/02/19 2:14 PM 335ONCOLOGYCHAPTER 1070-gene transcriptional signature.115 This assay was approved by the Food and Drug Administration (FDA) in February 2007. The usefulness of this assay in making therapy-related decisions was tested prospectively in a large-scale study, the Microarray in Node-Negative Disease May Avoid Chemotherapy (MIND-ACT) trial. Among women with early-stage breast cancer who were determined to be at high clinical risk and low genomic risk for cancer recurrence, the receipt of no adjuvant chemotherapy on the basis of the 70-gene signature result led to a 5-year metas-tasis survival rate that was 1.5% lower than the rate in patients who had received chemotherapy. In light of these findings, 45% of women with breast cancer who are

1	result led to a 5-year metas-tasis survival rate that was 1.5% lower than the rate in patients who had received chemotherapy. In light of these findings, 45% of women with breast cancer who are considered to be at high clinical risk might not require adjuvant chemotherapy.116Multigene profiles to predict prognosis are in development or in validation phases for many other solid tumor types, includ-ing lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, and melanoma. Gene signatures and genomic alterations also are being studied for their ability to predict response to specific chemotherapy regimens or targeted therapies. Many of these multigene marker sets will likely be incorporated into clinical practice in the years to come.Serum MarkersSerum markers are under active investigation because they may allow early diagnosis of a new cancer or may be used to follow cancer response to therapy or monitor for recurrence. Unfor-tunately, identification of serum markers of

1	investigation because they may allow early diagnosis of a new cancer or may be used to follow cancer response to therapy or monitor for recurrence. Unfor-tunately, identification of serum markers of clinical value has been challenging. Many of the tumor markers proposed so far have had low sensitivities and specificities.113 Tumor marker levels may not be elevated in all patients with cancer, especially in the early stages, when a serum marker would be most use-ful for diagnosis. Therefore, when a tumor marker is used to monitor recurrence, it is important to be certain that the level of the tumor marker was elevated before primary therapy. More-over, tumor marker levels can be elevated in benign conditions. Many tumor markers are not specific for a certain type of cancer and can be elevated with more than one type of tumor. Since there may be significant laboratory variability, it is important to obtain serial results from the same laboratory. In spite of these many clinical

1	be elevated with more than one type of tumor. Since there may be significant laboratory variability, it is important to obtain serial results from the same laboratory. In spite of these many clinical limitations, several serum markers are in clinical use. A few of the commonly measured serum tumor markers are discussed in the following sections.Prostate-Specific Antigen. Prostate-specific antigen (PSA) is an androgen-regulated serine protease produced by the pros-tate epithelium. PSA is normally present in low concentrations in the blood of all adult males. PSA levels may be elevated in the blood of men with benign prostate conditions such as pros-tatitis and benign prostatic hyperplasia, as well as in men with prostate cancer. PSA levels have been shown to be useful in evaluating the effectiveness of prostate cancer treatment and monitoring for recurrence after therapy. In monitoring for recur-rence, a trend of increasing levels is considered more significant than a single absolute

1	of prostate cancer treatment and monitoring for recurrence after therapy. In monitoring for recur-rence, a trend of increasing levels is considered more significant than a single absolute elevated value.Although PSA has been widely used for prostate cancer screening, the utility of PSA screening remains controversial. There is concern that the number of men who avoid dying from prostate cancer due to screening is small, while the harms related to the treatment of screen-detected cancers, including inconti-nence and erectile dysfunction are at least moderate. In 2012, the U.S. Preventive Services Task Force concluded with moder-ate certainty that the harms of PSA testing outweigh the benefits and on that basis recommended against PSA-based screening for all men.117 In 2010, the American Cancer Society updated its guidelines for the early detection of prostate cancer to state that men who have at least a 10-year life expectancy should have an opportunity to make an informed decision

1	Cancer Society updated its guidelines for the early detection of prostate cancer to state that men who have at least a 10-year life expectancy should have an opportunity to make an informed decision with their health care provider about whether to be screened for prostate cancer with digital rectal exam and serum PSA, after receiving information about the benefits, risks, and uncertainties associated with pros-tate cancer screening118; this recommendation was reinforced in their most recent guidelines.96Carcinoembryonic Antigen. Carcinoembryonic antigen (CEA) is a glycoprotein found in the embryonic endodermal epithelium. Elevated CEA levels have been detected in patients with primary colorectal cancer as well as in patients with breast, lung, ovarian, prostate, liver, and pancreatic cancer. Levels of CEA also may be elevated in benign conditions, including diverticulitis, peptic ulcer disease, bronchitis, liver abscess, and alcoholic cirrhosis, especially in smokers and in elderly

1	Levels of CEA also may be elevated in benign conditions, including diverticulitis, peptic ulcer disease, bronchitis, liver abscess, and alcoholic cirrhosis, especially in smokers and in elderly persons.CEA measurement is most commonly used in the man-agement of colorectal cancer. However, the appropriate use of CEA testing in patients with colorectal cancer has been debated. Use of CEA level as a screening test for colorectal cancer is not recommended. CEA levels may be useful if obtained pre-operatively and postoperatively in patients with a diagnosis of colorectal cancer. Preoperative elevation of CEA level is an indicator of poor prognosis. However, the ASCO clinical prac-tice guidelines state that the data are insufficient to support the use of CEA to determine whether to give a patient adjuvant therapy; the data are stronger for the use of CEA for monitoring for postoperative recurrence.111 CEA measurement is the most cost-effective approach for detecting metastasis, with over

1	adjuvant therapy; the data are stronger for the use of CEA for monitoring for postoperative recurrence.111 CEA measurement is the most cost-effective approach for detecting metastasis, with over 60% of recurrences being detected first by an elevation in CEA level. Therefore, in cases in which the patient would be a candidate for resection of recurrent colorectal cancer or systemic therapy, the ASCO guidelines recommend that postoperative CEA testing be performed every 3 months in patients with stage II or III disease for at least 3 years.112 CEA is the marker of choice for monitor-ing metastatic colorectal cancer during systemic therapy.112Alpha-Fetoprotein. Alpha-fetoprotein (AFP) is a glycoprotein normally produced by a developing fetus. AFP levels decrease soon after birth in healthy adults. An elevated level of AFP sug-gests the presence of either primary liver cancer or a germ cell tumor of the ovary or testicle. Rarely, other types of cancer such as gastric are associated with

1	adults. An elevated level of AFP sug-gests the presence of either primary liver cancer or a germ cell tumor of the ovary or testicle. Rarely, other types of cancer such as gastric are associated with an elevated AFP level. Benign conditions that can cause elevations of AFP include cirrhosis, hepatic necrosis, acute hepatitis, chronic active hepatitis, ataxia-telangiectasia, Wiskott-Aldrich syndrome, and pregnancy.119The sensitivity of an elevated AFP level for detecting HCC is approximately 60%. AFP is considered to be sensitive and specific enough to be used for screening for HCC in high-risk populations. Current consensus recommendations are to screen healthy hepatitis B virus carriers with annual or semi-annual measurement of AFP level and to screen carriers with cirrhosis or chronic hepatitis and patients with cirrhosis of any etiology with twice-yearly measurement of AFP level and liver ultrasonography.120 Although AFP testing has been used widely for a long time, its efficacy in

1	and patients with cirrhosis of any etiology with twice-yearly measurement of AFP level and liver ultrasonography.120 Although AFP testing has been used widely for a long time, its efficacy in early diagnosis of HCC is limited. With improvements in imaging technology, a larger proportion of patients diagnosed with HCC are now AFP seronegative.Cancer Antigen 19-9. Cancer antigen 19-9 (CA 19-9) is a tumor-related antigen that was originally defined by a monoclo-nal antibody produced by a hybridoma prepared from murine Brunicardi_Ch10_p0305-p0354.indd 33522/02/19 2:14 PM 336BASIC CONSIDERATIONSPART Ispleen cells immunized with a human colorectal cancer cell line.112 The data are insufficient to recommend use of CA 19-9 for screening, diagnosis, surveillance, or monitoring of therapy for colon cancer.112 Based on the 2006 ASCO guidelines, there are also insufficient data to recommend use of CA 19-9 for screening, diagnosis, or determination of the operability of pan-creatic cancer.112

1	cancer.112 Based on the 2006 ASCO guidelines, there are also insufficient data to recommend use of CA 19-9 for screening, diagnosis, or determination of the operability of pan-creatic cancer.112 However, for patients with locally advanced or metastatic cancer receiving active therapy, CA 19-9 can be measured at the start of therapy and every 1 to 3 months while therapy is given; elevations in serial CA 19-9 levels may indi-cate progressive disease and should be confirmed by additional studies.112Cancer Antigen 15-3. Cancer antigen 15-3 (CA 15-3) is an epitope of a large membrane glycoprotein encoded by the MUC1 gene that tumor cells shed into the bloodstream. The CA 15-3 epitope is recognized by two monoclonal antibodies in a sandwich radioimmunoassay. CA 15-3 levels are most useful in following the course of treatment in women diagnosed with advanced breast cancer. CA 15-3 levels are infrequently elevated in early-stage breast cancer. CA 15-3 levels can be increased in benign

1	in following the course of treatment in women diagnosed with advanced breast cancer. CA 15-3 levels are infrequently elevated in early-stage breast cancer. CA 15-3 levels can be increased in benign conditions such as chronic hepatitis, tuberculosis, sar-coidosis, pelvic inflammatory disease, endometriosis, systemic lupus erythematosus, pregnancy, and lactation, and in other types of cancer such as lung, ovarian, endometrial, and GI cancers.The sensitivity of CA 15-3 is higher for metastatic disease, and in these cases studies have shown sensitivity to be between 54% and 87%, with specificity as high as 96%. This has led to interest in using CA 15-3 for monitoring patients with advanced breast cancer for recurrence. Elevated CA 15-3 levels have been reported before relapse in 54% of patients, with a lead time of 4.2 months. Therefore, detection of elevated CA 15-3 levels dur-ing follow-up should prompt evaluation for recurrent disease. However, 6% to 8% of patients without recurrence

1	with a lead time of 4.2 months. Therefore, detection of elevated CA 15-3 levels dur-ing follow-up should prompt evaluation for recurrent disease. However, 6% to 8% of patients without recurrence will have ele-vated CA 15-3 levels that require evaluation. Furthermore, moni-toring with the use of CA 15-3 levels has shown no demonstrated impact on survival. Therefore, the 2007 ASCO guidelines state that the routine use of CA 15-3 for screening, diagnosis, stag-ing, or surveillance of breast cancer is not recommended because available data are insufficient.111 For monitoring patients during active therapy, CA 15-3 can be used in conjunction with diag-nostic imaging and history and physical examination.111 In the absence of measurable disease, an increase may be interpreted to indicate treatment failure. However, caution is advised when interpreting rising levels in the first 4 to 6 weeks of therapy.111Cancer Antigen 27-29. The MUC-1 gene product in the serum may be quantitated by using

1	failure. However, caution is advised when interpreting rising levels in the first 4 to 6 weeks of therapy.111Cancer Antigen 27-29. The MUC-1 gene product in the serum may be quantitated by using radioimmunoassay with a monoclonal antibody against the cancer antigen 27-29 (CA 27-29). CA 27-29 levels can be elevated in breast cancer as well as in cancers of the colon, stomach, kidney, lung, ovary, pan-creas, uterus, and liver. First-trimester pregnancy, endometrio-sis, benign breast disease, kidney disease, and liver disease also may be associated with elevated CA 27-29 levels.CA 27-29 has been reported to have a sensitivity of 57%, a specificity of 98%, a positive predictive value of 83%, and a negative predictive value of 93% in detecting breast cancer recurrences.121 Although CA 27-29 has been found to predict recurrence an average of 5.3 months before other symptoms or tests, testing of CA 27-29 levels has not been demonstrated to affect disease-free and overall survival

1	CA 27-29 has been found to predict recurrence an average of 5.3 months before other symptoms or tests, testing of CA 27-29 levels has not been demonstrated to affect disease-free and overall survival rates.121,122 Therefore, the ASCO guidelines state that, as with CA 15-3, the routine use of CA 27-29 for screening, diagnosis, staging, or surveillance of breast cancer is not recommended because available data are insufficient.111 CA 27-29 levels can be used together with diag-nostic imaging and history and physical examination to monitor patients during active therapy.111 When no measurable disease is present, an increase in level may be considered to indicate treat-ment failure. However, rising levels in the first 4 to 6 weeks of therapy should be interpreted with caution.111Circulating Tumor CellsCirculating tumor cells (CTCs) are cells present in the blood that possess antigenic or genetic characteristics of a specific tumor type.111 One CTC detection methodology is capture and

1	Tumor CellsCirculating tumor cells (CTCs) are cells present in the blood that possess antigenic or genetic characteristics of a specific tumor type.111 One CTC detection methodology is capture and quantita-tion of CTCs with immunomagnetic beads coated with antibody specific for cell-surface, epithelial, or cancer antigens. Another methodology used to detect cancer cells in the peripheral blood is RT-PCR. It has been suggested that measurement of CTCs can be an effective tool for selecting patients who have a high risk of relapse and for monitoring efficacy of cancer therapy.CTCs have probably been most extensively studied in breast cancer.111 The most promising data come from the use of CTC measures in metastatic breast cancer. In a prospective multicenter trial, the number of CTCs (≥5 CTCs vs. <5 CTCs per 7.5 mL of whole blood) before treatment of metastatic breast cancer was an independent predictor of progression-free and overall survival rates.123 The presence of >5 CTCs after the

1	vs. <5 CTCs per 7.5 mL of whole blood) before treatment of metastatic breast cancer was an independent predictor of progression-free and overall survival rates.123 The presence of >5 CTCs after the first course of therapy predicted lack of response to treatment. This technology, known as CellSearch, has been approved by the FDA for clinical use. In a well-designed single institutional study, detection of one or more CTCs in stage I to III breast cancer patients was associated with both decreased progres-sion-free survival and overall survival.124 A large clinical trial was launched by the German SUCCESS study group to evalu-ate the prognostic significance of CTCs in early breast cancer. Blood samples were obtained from over 2000 average-to-high-risk nonmetastatic breast cancer patients before chemotherapy and nearly 1500 patients post chemotherapy and examined for CTCs. Women with detectable CTCs before chemotherapy had significantly worse disease-free and overall survival, and those

1	chemotherapy and nearly 1500 patients post chemotherapy and examined for CTCs. Women with detectable CTCs before chemotherapy had significantly worse disease-free and overall survival, and those with five or more CTCs had the highest relapse risk.125 How-ever, there is limited data to prove that the use of CTC testing leads to improved survival or improved quality of life; thus, the ASCO 2017 guidelines update did not recommend the use of CTC measurement in any clinical setting.111 The clinical util-ity of measuring CTC response to initial therapy is now being tested prospectively in multiple clinical trials. The ability to conduct comprehensive analyses of cancer genomes within indi-vidual cells is becoming a real possibility, although the clinical utility of such information is still evolving. In addition to CTC, it has become possible in recent years to conduct a so-called “liquid tumor biopsy” by measuring levels of circulating tumor DNA and also circulating microRNAs. These new

1	In addition to CTC, it has become possible in recent years to conduct a so-called “liquid tumor biopsy” by measuring levels of circulating tumor DNA and also circulating microRNAs. These new techniques are investigational but have the potential to provide prognos-tic and diagnostic information via their presumed correlation with tumor progression and the development of genomic altera-tions.126 The prognostic implications of detection of CTCs by RT-PCR have been intensively studied for melanoma. In the recent multicenter Sunbelt Melanoma Trial, serial RT-PCR was performed on peripheral blood samples using four markers—tyrosinase, melanoma antigen reacting to T cell (MART-1), melanoma antigen 3 (MAGE3), and gp 100—to detect occult Brunicardi_Ch10_p0305-p0354.indd 33622/02/19 2:14 PM 337ONCOLOGYCHAPTER 10melanoma cells in the bloodstream.127 Although there were no differences in survival rates among patients in whom at least one marker was detected and those in whom no markers were

1	10melanoma cells in the bloodstream.127 Although there were no differences in survival rates among patients in whom at least one marker was detected and those in whom no markers were detected, the disease-free survival and distant disease-free sur-vival rates were worse for patients in whom more than one marker was detected at any time during follow-up.127Bone Marrow MicrometastasesMicrometastatic disease in the bone marrow, also referred to as minimal residual disease, continues to be investigated as a potential prognostic marker. Bone marrow micrometastatic disease usually is detected by staining bone marrow aspirates with monoclonal antibodies to cytokeratin, but other method-ologies such as flow cytometry and RT-PCR are in use. Breast cancer patients with bone marrow micrometastasis have larger tumors, tumors with a higher histologic grade, more lymph node metastases, and more hormone receptor-negative tumors than patients without bone marrow micrometastasis. In 4700 patients with

1	larger tumors, tumors with a higher histologic grade, more lymph node metastases, and more hormone receptor-negative tumors than patients without bone marrow micrometastasis. In 4700 patients with stage I, II, or III breast cancer, micrometastasis was a sig-nificant prognostic factor associated with poor overall survival, breast cancer-specific survival, disease-free survival, and distant disease-free survival during a 10-year observation period.128 The American College of Surgeons Oncology Group Z0010 trial enrolled women with clinical T1 to T2N0M0 invasive breast carcinoma in a prospective observational study to determine the association between survival and metastases detected by immunochemical staining of bone marrow specimens from patients with early-stage breast cancer.129 Of 3413 bone mar-row specimens examined by immunocytochemistry, only 104 (3.0%) were positive for tumor. Bone marrow involvement was associated with a decreased overall survival, but this association was not

1	bone mar-row specimens examined by immunocytochemistry, only 104 (3.0%) were positive for tumor. Bone marrow involvement was associated with a decreased overall survival, but this association was not significant on multivariable analysis. The prognostic implication of bone marrow involvement continues to be stud-ied by the National Surgical Adjuvant Breast and Bowel Project.At this time, the routine use of bone marrow testing is not recommended.111 Ongoing clinical trials are evaluating the role of routine assessment of bone marrow status in the care of patients with early and advanced breast cancer. The utility of assessment of bone marrow micrometastasis has also been evaluated in other tumor types, including gastric, esophageal, colorectal, lung, cervical, and ovarian cancer.130SURGICAL APPROACHES TO CANCER THERAPYMultidisciplinary Approach to CancerAlthough surgery is an effective therapy for most solid tumors, patients who die from cancer usually die of metastatic disease.

1	APPROACHES TO CANCER THERAPYMultidisciplinary Approach to CancerAlthough surgery is an effective therapy for most solid tumors, patients who die from cancer usually die of metastatic disease. Therefore, to improve patient survival rates, a multimodality approach, including systemic therapy and radiation therapy, is key for most tumors. It is important that surgeons involved in cancer care not only know the techniques for performing a cancer operation but also know the alternatives to surgery and be well versed in reconstructive options. It is also crucial that the surgeon be familiar with the indications for and complications of preoperative and postoperative chemotherapy and radiation therapy. Although the surgeon may not be delivering these other therapies, as the first physician to see a patient with a cancer diagnosis, he or she is ultimately responsible for initiating the appropriate consultations. For this reason, the surgeon often is responsible for determining the most

1	to see a patient with a cancer diagnosis, he or she is ultimately responsible for initiating the appropriate consultations. For this reason, the surgeon often is responsible for determining the most appropriate adjuvant ther-apy for a given patient as well as the best sequence for therapy. In most instances, a multidisciplinary approach beginning at the patient’s initial presentation is likely to yield the best result.Surgical Management of Primary TumorsThe goal of surgical therapy for cancer is to achieve oncologic cure. A curative operation presupposes that the tumor is confined to the organ of origin or to the organ and the regional lymph node basin. Patients in whom the primary tumor is not resectable with negative surgical margins are considered to have inoperable disease. The operability of primary tumors is best determined before surgery with appropriate imaging studies that can define the extent of local-regional disease. For example, a preoperative thin-section CT scan is

1	of primary tumors is best determined before surgery with appropriate imaging studies that can define the extent of local-regional disease. For example, a preoperative thin-section CT scan is obtained to determine resectability of pancreatic cancer, which is based on the absence of extrapancre-atic disease, the absence of tumor extension to the superior mes-enteric artery and celiac axis, and a patent superior mesenteric vein-portal vein confluence.131 Disease involving multiple distant metastases is deemed inoperable because it is usually not curable with surgery of the primary tumor. Therefore, patients who are at high risk of having distant metastasis should undergo a staging work-up before surgery for the primary tumor. On occasion, pri-mary tumors are resected in these patients for palliative reasons, such as improving the quality of life by alleviating pain, infec-tion, or bleeding. An example of this is toilet mastectomies for large ulcerated breast tumors. Patients with limited

1	reasons, such as improving the quality of life by alleviating pain, infec-tion, or bleeding. An example of this is toilet mastectomies for large ulcerated breast tumors. Patients with limited metastases from a primary tumor on occasion are considered surgical candi-dates if the natural history of isolated distant metastases for that cancer type is favorable or the potential complications associated with leaving the primary tumor intact are significant.In the past, it was presumed that the more radical the sur-gery, the better the oncologic outcome would be. Over the past three decades, this has been recognized as not necessarily being true, which has led to more conservative operations, with wide local excisions replacing compartmental resections of sarcomas, and partial mastectomies, skin-sparing mastectomies, and breast-conserving therapies replacing radical mastectomies for breast cancer. The uniform goal for all successful oncologic operations seems to be achieving widely negative

1	mastectomies, and breast-conserving therapies replacing radical mastectomies for breast cancer. The uniform goal for all successful oncologic operations seems to be achieving widely negative margins with no evidence of macroscopic or microscopic tumor at the surgical margins. The importance of negative surgical margins for local tumor control and/or survival has been documented for many tumor types, including sarcoma, breast cancer, pancreatic cancer, and rectal cancer. Thus, it is clear that every effort should be made to achieve microscopically negative surgical margins. Inking of the margins, orientation of the specimen by the surgeon, and immediate gross evaluation of the margins by a pathologist using frozen-section analysis when necessary may assist in achiev-ing negative margins at the first operation. In the end, although radiation therapy and systemic therapy can assist in decreasing local recurrence rates in the setting of positive margins, adjuvant therapy cannot substitute

1	the first operation. In the end, although radiation therapy and systemic therapy can assist in decreasing local recurrence rates in the setting of positive margins, adjuvant therapy cannot substitute for adequate surgery.Although it is clear that the surgical gold standard is nega-tive surgical margins, the appropriate surgical margins for optimal local control are controversial for many cancer types. In contrast, in melanoma the optimal margin width for any tumor depth has been better defined, owing to the systematic study of this ques-tion in randomized clinical trials.132,133 Although such randomized studies may not be possible for all tumor types, it is important to determine optimal surgical margins for each cancer type so that adjuvant radiation and systemic therapy can be offered to patients deemed to be at increased risk for local treatment failure.Brunicardi_Ch10_p0305-p0354.indd 33722/02/19 2:14 PM 338BASIC CONSIDERATIONSPART IFigure 10-14. Lymphatic mapping and

1	to patients deemed to be at increased risk for local treatment failure.Brunicardi_Ch10_p0305-p0354.indd 33722/02/19 2:14 PM 338BASIC CONSIDERATIONSPART IFigure 10-14. Lymphatic mapping and sen-tinel lymph node biopsy specimen for breast cancer. A. Peritumoral injection of blue dye. B. Blue dye draining into the sentinel lymph node.Surgical Management of the Regional Lymph Node BasinMost neoplasms have the ability to metastasize via the lymphat-ics. Therefore, most oncologic operations have been designed to remove the primary tumor and draining lymphatics en bloc. This type of operative approach usually is undertaken when the lymph nodes draining the primary tumor site lie adjacent to the tumor bed, as is the case for colorectal cancers and gastric can-cers. For tumors in which the regional lymph node basin is not immediately adjacent to the tumor (e.g., melanomas), lymph node surgery can be performed through a separate incision. Unlike most carcinomas, soft tissue sarcomas rarely

1	lymph node basin is not immediately adjacent to the tumor (e.g., melanomas), lymph node surgery can be performed through a separate incision. Unlike most carcinomas, soft tissue sarcomas rarely metasta-size to the lymph nodes (<5%); therefore, lymph node surgery usually is not necessary.It is generally accepted that a formal lymphadenectomy is likely to minimize the risk of regional recurrence of most cancers. For example, the introduction of total mesorectal excision of rectal cancer has been associated with a large decline in local-regional recurrence, and this procedure has become the new standard of operative management.134 On the other hand, there have been two opposing views regarding the role of lymphadenectomy in survival of cancer patients. The traditional Halsted view states that lymphadenectomy is important for staging and survival. The opposing view coun-ters that cancer is systemic at inception and that lymphade-nectomy, although useful for staging, does not affect

1	that lymphadenectomy is important for staging and survival. The opposing view coun-ters that cancer is systemic at inception and that lymphade-nectomy, although useful for staging, does not affect survival. For most cancers, involvement of the lymph nodes is one of the most significant prognostic factors. Interestingly, in some studies removal of a larger number of lymph nodes has been found to be associated with an improved overall survival rate for many tumors, including breast cancer, colon cancer, and lung cancer. Although this seems to support the Halsted the-ory that more extensive lymphadenectomy yielding of nodes reduces the risk of regional recurrence, there may be alterna-tive explanations for the same finding. For example, the sur-geon who performs a more extensive lymphadenectomy may obtain wider margins around the tumor or even provide better overall care, such as ensuring that patients receive the appropri-ate adjuvant therapy or undergo a more thorough staging work-up.

1	may obtain wider margins around the tumor or even provide better overall care, such as ensuring that patients receive the appropri-ate adjuvant therapy or undergo a more thorough staging work-up. Alternatively, the pathologist may perform a more thorough examination, identifying more nodes and more accurately stag-ing the nodes. The effect of appropriate staging on survival is twofold. Patients with nodal metastases may be offered adjuvant therapy, which improves their survival chances. Further, the enhanced staging can improve perceived survival rates through a “Will Rogers effect.” Such a phenomenon is observed when moving an element from one set to another set raises the average value of both sets. When commenting on the 1930s migration of poor farmers from a dustbowl state to a more prosperous west-ern state, humorist Will Rogers quipped that this event raised the average intelligence of both states. Thus, identification of small metastases that had formerly been silent and

1	more prosperous west-ern state, humorist Will Rogers quipped that this event raised the average intelligence of both states. Thus, identification of small metastases that had formerly been silent and unidenti-fied leads to stage migration for these patients and thus to a perceived improvement in chances of survival for the higher stage. In addition, there is improved survival for the lower stage, which is now minus the patients with low volume nodal disease. Clearly the impact of lymphadenectomy on survival will con-tinue to be a topic of clinical research.Surgical management of the clinically negative regional lymph node basin has evolved with the introduction of lym-phatic mapping technology (Fig. 10-14).135 Lymphatic mapping and sentinel lymph node biopsy specimen were first reported in 1977 by Cabanas for penile cancer.136 Now, sentinel node biopsy specimen is the standard of care for the management of melanoma and breast cancer. The utility of sentinel node biopsy is being

1	in 1977 by Cabanas for penile cancer.136 Now, sentinel node biopsy specimen is the standard of care for the management of melanoma and breast cancer. The utility of sentinel node biopsy is being explored in other cancer types such as head and neck squamous cell cancer and vulvar cancer.The first node to receive drainage from the tumor site is termed the sentinel node. This node is the node most likely to contain metastases, if metastases to that regional lymph node basin are present. The goal of lymphatic mapping and senti-nel lymph node biopsy specimen is to identify and remove the lymph node most likely to contain metastases in the least inva-sive fashion. The practice of sentinel lymph node biopsy speci-men followed by regional lymph node dissection for selected patients with a positive sentinel lymph node avoids the morbid-ity of lymph node dissections in patients with negative nodes. An additional advantage of the sentinel lymph node technique is that it directs attention to a

1	sentinel lymph node avoids the morbid-ity of lymph node dissections in patients with negative nodes. An additional advantage of the sentinel lymph node technique is that it directs attention to a single node, which allows more careful analysis of the lymph node most likely to have a positive yield and increases the accuracy of nodal staging. Two criteria are used to assess the efficacy of a sentinel lymph node biopsy specimen: the sentinel lymph node identification rate and the false-negative rate. The sentinel lymph node identification rate Brunicardi_Ch10_p0305-p0354.indd 33822/02/19 2:14 PM 339ONCOLOGYCHAPTER 10is the proportion of patients in whom a sentinel lymph node was identified and removed among all patients undergoing an attempted sentinel lymph node biopsy specimen. The false-negative rate is the proportion of patients with regional lymph node metastases in whom the sentinel lymph node was found to be negative. False-negative biopsy specimen results may be due to

1	false-negative rate is the proportion of patients with regional lymph node metastases in whom the sentinel lymph node was found to be negative. False-negative biopsy specimen results may be due to identifying the wrong node or to missing the sentinel node (i.e., surgical error) or they may be due to the cancer cells’ establishing metastases not in the first node encountered but in a second-echelon node (i.e., biologic variation). Alternatively, false-negative biopsy specimen results may be due to inadequate histologic evaluation of the lymph node. The false-negative rates for sentinel lymph node biopsy specimen in study series range between 0% and 11%. Both increases in the identification rate and decreases in the false-negative rate have been observed as surgeons gain experience with the technique.Lymphatic mapping is performed by using isosulfan blue dye, technetium-labeled sulfur colloid or albumin, or a combi-nation of both techniques to detect sentinel nodes. The combina-tion of

1	technique.Lymphatic mapping is performed by using isosulfan blue dye, technetium-labeled sulfur colloid or albumin, or a combi-nation of both techniques to detect sentinel nodes. The combina-tion of blue dye and technetium has been reported to improve the capability of detecting sentinel lymph nodes. The nodal drainage pattern usually is determined with a preoperative lym-phoscintigram, and the “hot” and/or blue nodes are identified with the assistance of a gamma probe and careful nodal basin exploration. Careful manual palpation is a crucial part of the procedure to minimize the false-negative rate.The nodes are evaluated with serial sectioning, hematoxy-lin and eosin staining, and immunohistochemical analysis with S-100 protein and homatropine methylbromide staining for mel-anoma and cytokeratin staining for breast cancer. The utility of molecular techniques such as RT-PCR to assess the sentinel nodes is still being explored.Another area of active investigation is the prognostic

1	cytokeratin staining for breast cancer. The utility of molecular techniques such as RT-PCR to assess the sentinel nodes is still being explored.Another area of active investigation is the prognostic value of minimal nodal involvement. For example, in breast cancer, nodes with isolated tumor cell deposits of <0.2 mm are considered to be N0 by the sixth edition of the AJCC staging manual. However, some retrospective studies have suggested that even this amount of nodal disease burden has negative prognostic implications.137 Molecular ultrastaging with RT-PCR for patients with node-negative disease was assessed in a pro-spective multicenter trial and was found not to be prognostic in malignant melanoma.127 However, a recent meta-analysis of 22 studies enrolling 4019 patients found that PCR positivity was associated with worse overall and disease-free survival.138 Fur-ther study of the utility of ultrastaging of nodes in breast cancer, melanoma, and several other tumor types is

1	that PCR positivity was associated with worse overall and disease-free survival.138 Fur-ther study of the utility of ultrastaging of nodes in breast cancer, melanoma, and several other tumor types is ongoing.Until recently, in breast cancer management, when senti-nel node mapping revealed a positive sentinel node, this was fol-lowed by a completion axillary lymph node dissection. Results of the American College of Surgeons Oncology Group Z0011 trial, challenged this practice. ACOSOG Z11 was a phase 3 mul-ticenter noninferiority trial conducted to determine the effects of complete axillary lymph node dissection on survival of patients with sentinel lymph node metastasis of breast cancer.129 Patients were women with clinical T1-T2 invasive breast cancer, no pal-pable adenopathy, and 1 to 2 SLNs containing metastases identi-fied by frozen section, touch preparation, or hematoxylin-eosin staining on permanent section. All patients underwent breast-conserving surgery and tangential

1	1 to 2 SLNs containing metastases identi-fied by frozen section, touch preparation, or hematoxylin-eosin staining on permanent section. All patients underwent breast-conserving surgery and tangential whole-breast irradiation. Those with sentinel node metastases identified by sentinel node biopsy specimen were randomized to undergo axillary lymph node dissection or no further axillary treatment. At a median follow-up of 6.3 years, 5-year overall survival was 91.8% (95% confidence interval [CI], 89.1%–94.5%) with axillary lymph node dissection and 92.5% (95% CI, 90.0%–95.1%) with sen-tinel node alone. The 5-year disease-free survival was 82.2% (95% CI, 78.3%–86.3%) with axillary lymph node dissection, and 83.9% (95% CI, 80.2%–87.9%) with sentinel node alone. Thus, ACOSOGZ11 demonstrated that among breast cancer patients with limited sentinel node metastasis treated with breast conservation and systemic therapy, the use of sentinel node alone compared with axillary lymph node dissection

1	among breast cancer patients with limited sentinel node metastasis treated with breast conservation and systemic therapy, the use of sentinel node alone compared with axillary lymph node dissection did not result in inferior survival. This study challenges the traditional surgical dictum of regional management, and has led to a selec-tive utilization of completion axillary lymph node dissection in breast cancer patients undergoing breast conservation. The role of completion lymph node dissections in melanoma is under investigation. In the MSLT-II clinical trial, melanoma patients with sentinel-node metastases were randomized to immediate completion lymph-node dissection or nodal observation with ultrasonography. The primary end point of this study was mela-noma-specific survival. Immediate completion lymph-node dis-section led to increased regional disease control and provided prognostic information but did not increase melanoma-specific survival among patients with

1	survival. Immediate completion lymph-node dis-section led to increased regional disease control and provided prognostic information but did not increase melanoma-specific survival among patients with intermediate-thickness melanoma and sentinel-node metastases.139Surgical Management of Distant MetastasesThe treatment of a patient with distant metastases depends on the number and sites of metastases, the cancer type, the rate of tumor growth, the previous treatments delivered and the responses to these treatments, and the patient’s age, physical condition, and desires. Although once a tumor has metastasized it usually is not curable with surgical therapy, such therapy has resulted in cure in selected cases with isolated metastases to the liver, lung, or brain.Patient selection is the key to the success of surgical therapy for distant metastases. The cancer type is a major determinant in surgical decision making. A liver metastasis from a colon cancer is more likely to be an isolated

1	to the success of surgical therapy for distant metastases. The cancer type is a major determinant in surgical decision making. A liver metastasis from a colon cancer is more likely to be an isolated and thus resectable lesion than a liver metastasis from a pancreatic carcinoma. The growth rate of the tumor also plays an important role and can be determined in part by the disease-free interval and the time between treatment of the primary tumor and detection of the distant recurrence. Patients with longer disease-free intervals have a higher survival rate after surgical metastasectomy than those with a short disease-free interval. Similarly, patients who have synchronous metastases (metastases diagnosed at the initial cancer diagnosis) do worse after metastasectomy than patients who develop metachronous metastases (metastasis diagnosed after a disease-free interval). The natural history of metastatic disease is so poor for some tumors (e.g., pancreatic cancer) that there is no role at

1	metachronous metastases (metastasis diagnosed after a disease-free interval). The natural history of metastatic disease is so poor for some tumors (e.g., pancreatic cancer) that there is no role at this time for surgical metastasectomy. In cancers with a more favorable outlook, observation for several weeks or months, potentially with initial treatment with systemic therapy, can allow the sur-geon to monitor for metastases at other sites.In curative surgery for distant metastases, as with surgery for primary tumors, the goal is to resect the metastases with negative margins. In patients with hepatic metastases that are unresectable because their location near intrahepatic blood ves-sels precludes a margin-negative resection, or because they are multifocal or hepatic function is inadequate, tumor ablation with cryotherapy or radiofrequency ablation is an alternative.140,141 Curative resections or ablative procedures should be attempted Brunicardi_Ch10_p0305-p0354.indd 33922/02/19

1	tumor ablation with cryotherapy or radiofrequency ablation is an alternative.140,141 Curative resections or ablative procedures should be attempted Brunicardi_Ch10_p0305-p0354.indd 33922/02/19 2:14 PM 340BASIC CONSIDERATIONSPART Ionly if the lesions are accessible and the procedure can be per-formed safely.CHEMOTHERAPYClinical Use of ChemotherapyIn patients with documented distant metastatic disease, chemo-therapy is usually the primary modality of therapy. The goal of therapy in this setting is to decrease the tumor burden, thus prolonging survival. It is rare to achieve cure with chemother-apy for metastatic disease for most solid tumors. Chemotherapy administered to a patient who is at high risk for distant recur-rence but has no evidence of distant disease is referred to as adjuvant chemotherapy. The goal of adjuvant chemotherapy is eradication of micrometastatic disease, with the intent of decreasing relapse rates and improving survival rates.Adjuvant therapy can be

1	as adjuvant chemotherapy. The goal of adjuvant chemotherapy is eradication of micrometastatic disease, with the intent of decreasing relapse rates and improving survival rates.Adjuvant therapy can be administered after surgery (post-operative chemotherapy) or before surgery (preoperative che-motherapy, neoadjuvant chemotherapy, or induction therapy). A portion or all of the planned adjuvant chemotherapy can be administered before the surgical removal of the primary tumor. Preoperative chemotherapy has three potential advantages. The first is that preoperative regression of tumor can facilitate resection of tumors that were initially inoperable or allow more conservative surgery for patients whose cancer was operable to begin with. In the NSABP B-18 project, for example, women were randomly assigned to receive adjuvant doxorubicin and cyclophosphamide preoperatively or postoperatively. More patients treated before surgery than after surgery underwent breast-conserving surgery (68% vs.

1	assigned to receive adjuvant doxorubicin and cyclophosphamide preoperatively or postoperatively. More patients treated before surgery than after surgery underwent breast-conserving surgery (68% vs. 60%).142 The second advan-tage of preoperative chemotherapy is the treatment of microme-tastases without the delay of postoperative recovery. The third advantage is the ability to assess a cancer’s response to treat-ment clinically, after a number of courses of chemotherapy, and pathologically, after surgical resection. This is especially impor-tant if alternative treatment regimens are available to be offered to patients whose disease responded inadequately. Molecular characterization of the residual disease may also give insight into mechanisms of chemoresistance and possible therapeutic targets.There are some potential disadvantages to preoperative chemotherapy, however. Although disease progression while the patient is receiving preoperative chemotherapy is rare in

1	therapeutic targets.There are some potential disadvantages to preoperative chemotherapy, however. Although disease progression while the patient is receiving preoperative chemotherapy is rare in chemotherapy-sensitive tumors such as breast cancer, it is more frequent in relatively chemotherapy-resistant tumors such as sarcomas.143 Thus, patient selection is critical to ensure that the opportunity to treat disease surgically is not lost by giving pre-operative chemotherapy. Often, rates of postoperative wound infection, flap necrosis, and delays in postoperative adjuvant therapy do not differ between patients who are treated with preoperative chemotherapy and patients who are treated with surgery first. However, preoperative chemotherapy can intro-duce special challenges to tumor localization, margin analysis, lymphatic mapping, and pathologic staging.Response to chemotherapy is monitored clinically with imaging studies as well as physical examinations. Response usually is defined as

1	margin analysis, lymphatic mapping, and pathologic staging.Response to chemotherapy is monitored clinically with imaging studies as well as physical examinations. Response usually is defined as complete response, partial response, stable disease, or progression. Response generally is assessed using the Response Evaluation Criteria in Solid Tumors (RECIST) criteria.144 Objective tumor response assessment is critical because tumor response is used as a prospective endpoint in clinical trials and tumor response is a guide to clinicians regard-ing continuation of current therapy.Principles of ChemotherapyChemotherapy destroys cells by first-order kinetics, which means that with the administration of a drug a constant percent-age of cells is killed, not a constant number of cells. If a patient with 1012 tumor cells is treated with a dose that results in 99.9% cell kill (3-log cell kill), the tumor burden will be reduced from 1012 to 109 cells (or 1 kg to 1 g). If the patient is retreated

1	with 1012 tumor cells is treated with a dose that results in 99.9% cell kill (3-log cell kill), the tumor burden will be reduced from 1012 to 109 cells (or 1 kg to 1 g). If the patient is retreated with the same drug, which theoretically could result in another 3-log cell kill, the cells would decrease in number from 109 to 106 (1 g to 1 mg) rather than being eliminated totally.Chemotherapeutic agents can be classified according to the phase of the cell cycle during which they are effective. Cell-cycle phase-nonspecific agents (e.g., alkylating agents) have a linear dose-response curve, such that the fraction of cells killed increases with the dose of the drug.145 In contrast, the cell-cycle phase-specific drugs have a plateau with respect to cell killing ability, and cell kill will not increase with further increases in drug dose.Anticancer AgentsAlkylating Agents. Alkylating agents are cell-cycle– nonspecific agents, that is, they are able to kill cells in any phase of the cell

1	increase with further increases in drug dose.Anticancer AgentsAlkylating Agents. Alkylating agents are cell-cycle– nonspecific agents, that is, they are able to kill cells in any phase of the cell cycle. They act by cross-linking the two strands of the DNA helix or by causing other direct damage to the DNA. The damage to the DNA prevents cell division and, if severe enough, leads to apoptosis. The alkylating agents are composed of three main subgroups: classic alkylators, nitrosoureas, and miscellaneous DNA-binding agents (Table 10-10).Antitumor Antibiotics. Antitumor antibiotics are the prod-ucts of fermentation of microbial organisms. Like the alkylat-ing agents, these agents are cell-cycle nonspecific. Antitumor antibiotics damage the cell by interfering with DNA or RNA synthesis, although the exact mechanism of action may differ by agent.Antimetabolites. Antimetabolites are generally cell-cycle–specific agents that have their major activity during the S phase of the cell cycle and

1	the exact mechanism of action may differ by agent.Antimetabolites. Antimetabolites are generally cell-cycle–specific agents that have their major activity during the S phase of the cell cycle and have little effect on cells in G0. These drugs are most effective, therefore, in tumors that have a high growth fraction. Antimetabolites are structural analogues of naturally occurring metabolites involved in DNA and RNA synthesis. Therefore, they interfere with normal synthesis of nucleic acids by substituting for purines or pyrimidines in the metabolic path-way to inhibit critical enzymes in nucleic acid synthesis. The antimetabolites include folate antagonists, purine antagonists, and pyrimidine antagonists.Plant Alkaloids. Plant alkaloids are derived from plants such as the periwinkle plant, Vinca rosea (e.g., vincristine, a vinca alkaloid), or the root of American mandrake, Podophyllum peltatum (e.g., etoposide, a podophyllotoxin).145 Vinca alkaloids affect the cell by binding to

1	Vinca rosea (e.g., vincristine, a vinca alkaloid), or the root of American mandrake, Podophyllum peltatum (e.g., etoposide, a podophyllotoxin).145 Vinca alkaloids affect the cell by binding to tubulin in the S phase. This blocks microtubule polymerization, which results in impaired mitotic spindle formation in the M phase. Taxanes such as paclitaxel, on the other hand, cause excess polymerization and stability of microtubules, which blocks the cell cycle in mitosis. The epi-podophyllotoxins (e.g., etoposide) act to inhibit a DNA enzyme called topoisomerase II by stabilizing the DNA-topoisomerase II complex. This results in an inability to synthesize DNA, and thus the cell cycle is stopped in the G1 phase.145Brunicardi_Ch10_p0305-p0354.indd 34022/02/19 2:14 PM 341ONCOLOGYCHAPTER 10Combination ChemotherapyCombination chemotherapy may provide greater efficacy than single-agent therapy by three mechanisms: (a) it provides maxi-mum cell kill within the range of toxicity for each drug

1	ChemotherapyCombination chemotherapy may provide greater efficacy than single-agent therapy by three mechanisms: (a) it provides maxi-mum cell kill within the range of toxicity for each drug that can be tolerated by the host, (b) it offers a broader range of coverage of resistant cell lines in a heterogeneous population, and (c) it prevents or delays the emergence of drug-resistant cell lines.145 When combination regimens are devised, drugs known to be active as single agents usually are selected. Drugs with different mechanisms of action are combined to allow for additive or syn-ergistic effects. Combining cell-cycle–specific and cell-cycle–nonspecific agents may be especially advantageous. Drugs with differing dose-limiting toxic effects are combined to allow for each drug to be given at therapeutic doses. Drugs with differ-ent patterns of resistance are combined whenever possible to minimize cross-resistance. The treatment-free interval between cycles is kept to the shortest

1	at therapeutic doses. Drugs with differ-ent patterns of resistance are combined whenever possible to minimize cross-resistance. The treatment-free interval between cycles is kept to the shortest possible time that will allow for recovery of the most sensitive normal tissue.Drug ToxicityTumors are more susceptible than normal tissue to chemothera-peutic agents, in part because they have a higher proportion of dividing cells. Normal tissues with a high growth fraction, such as the bone marrow, oral and intestinal mucosa and hair follicles are sensitive to chemotherapeutic effects. Therefore, treatment with chemotherapeutic agents can produce toxic effects such as bone marrow suppression, stomatitis, ulceration of the GI tract, and alopecia. Toxic effects usually are graded from 0 to 4 on the basis of World Health Organization standard criteria.146 Significant drug toxicity may necessitate a dosage reduction. A toxic effect requiring a dose modification or change in dose intensity is

1	the basis of World Health Organization standard criteria.146 Significant drug toxicity may necessitate a dosage reduction. A toxic effect requiring a dose modification or change in dose intensity is referred to as a dose-limiting toxic effect. Because maintaining dose intensity is important to preserve as high a tumor cell kill as possible, several supportive strategies have been developed, such as administration of colony-stimulating factors and erythropoietin to treat poor bone marrow reserve and administration of cytoprotectants such as mesna and amifostine to prevent renal dysfunction. Some toxicities, such as neuropa-thy, are not as easily reversible, and their potential effects on lifestyle must be considered when evaluating a patient prior to the initiation of chemotherapy.Administration of ChemotherapyChemotherapy usually is administered systemically (IV, IM, SC, or PO). Systemic administration treats micrometastases at widespread sites and prevents systemic recurrence.

1	of ChemotherapyChemotherapy usually is administered systemically (IV, IM, SC, or PO). Systemic administration treats micrometastases at widespread sites and prevents systemic recurrence. However, it increases the drug’s toxicity to a wide range of organs through-out the body. One method to minimize systemic toxicity while enhancing target organ delivery of chemotherapy is regional administration of chemotherapy. Many of these approaches require surgical access, such as intrahepatic delivery of chemo-therapy for hepatic carcinomas or metastatic colorectal cancer using a hepatic artery infusion pump, limb perfusion for extrem-ity melanoma and sarcoma, and intraperitoneal hyperthermic Table 10-10Classification of chemotherapeutic agentsAlkylating agents Classic alkylating agents Busulfan Chlorambucil Cyclophosphamide Ifosfamide Mechlorethamine (nitrogen mustard) Melphalan Mitomycin C Triethylene thiophosphoramide (thiotepa) Nitrosoureas Carmustine (BCNU) Lomustine

1	(nitrogen mustard) Melphalan Mitomycin C Triethylene thiophosphoramide (thiotepa) Nitrosoureas Carmustine (BCNU) Lomustine (CCNU) Semustine (MeCCNU) Streptozocin Miscellaneous DNA-binding agents Carboplatin Cisplatin Dacarbazine (DTIC) Hexamethylmelamine ProcarbazineAntitumor antibiotics Bleomycin Dactinomycin (actinomycin D) Daunorubicin Doxorubicin Idarubicin Plicamycin (mithramycin)Antimetabolites Folate analogues Methotrexate Purine analogues Azathioprine Mercaptopurine Thioguanine Cladribine (2-chlorodeoxyadenosine) Fludarabine Pentostatin Pyrimidine analogues Capecitabine Cytarabine Floxuridine Gemcitabine Ribonucleotide reductase inhibitors Hydroxyurea Plant alkaloids Vinca alkaloids Vinblastine Vincristine Vindesine Vinorelbine Epipodophyllotoxins Etoposide Teniposide Taxanes Paclitaxel Docetaxel Miscellaneous agents Asparaginase Estramustine MitotaneBrunicardi_Ch10_p0305-p0354.indd

1	agents Asparaginase Estramustine MitotaneBrunicardi_Ch10_p0305-p0354.indd 34122/02/19 2:14 PM 342BASIC CONSIDERATIONSPART Iperfusion for pseudomyxoma peritonei. Alternately, percutane-ous access may be utilized, such as limb infusion with percuta-neously placed catheters.HORMONAL THERAPYSome tumors, most notably breast and prostate cancers, origi-nate from tissues whose growth is under hormonal control. The first attempts at hormonal therapy were through surgical ablation of the organ producing the hormones involved, such as oophorectomy for breast cancer. Currently, hormonal anti-cancer agents include androgens, antiandrogens, antiestrogens, estrogens, glucocorticoids, gonadotropin inhibitors, progestins, aromatase inhibitors, and somatostatin analogues. Hormones or hormone-like agents can be administered to inhibit tumor growth by blocking or antagonizing the naturally occurring sub-stance, such as with the estrogen antagonist tamoxifen. Other substances that block the

1	agents can be administered to inhibit tumor growth by blocking or antagonizing the naturally occurring sub-stance, such as with the estrogen antagonist tamoxifen. Other substances that block the synthesis of the natural hormone can be administered as alternatives. Aromatase inhibitors, for exam-ple, block the peripheral conversion of endogenous androgens to estrogens in postmenopausal women. Hormonal therapy pro-vides a highly tumor-specific form of therapy in sensitive tis-sues. In breast cancer, estrogen and progesterone receptor status is used to predict the success of hormonal therapy. Androgen receptor is also being pursued as a therapeutic target for breast cancer treatment.TARGETED THERAPYOver the past decade, increased understanding of cancer biol-ogy has fostered the emerging field of molecular therapeutics. The basic principle of molecular therapeutics is to exploit the molecular differences between normal cells and cancer cells to develop targeted therapies. Thus, targeted

1	of molecular therapeutics. The basic principle of molecular therapeutics is to exploit the molecular differences between normal cells and cancer cells to develop targeted therapies. Thus, targeted therapies usually are directed at the processes involved in tumor growth rather than directly targeting the tumor cells. The ideal molecular target would be exclusively expressed in the cancer cells, be the driv-ing force of the proliferation of the cancer cells, and be critical to their survival. A large number of molecular targets are cur-rently being explored, both preclinically and in clinical trials. The major groups of targeted therapy agents are inhibitors of growth factor receptors, inhibitors of intracellular signal trans-duction, cell-cycle inhibitors, apoptosis-based therapies, and anti-angiogenic compounds.Protein kinases have come to the forefront as attrac-tive therapeutic targets with the success of imatinib mesylate (Gleevec) in treating chronic myelogenous leukemia and GI

1	anti-angiogenic compounds.Protein kinases have come to the forefront as attrac-tive therapeutic targets with the success of imatinib mesylate (Gleevec) in treating chronic myelogenous leukemia and GI stromal tumors, and trastuzumab (Herceptin) in treating breast cancer, and vemurafanib in treating melanoma. These drugs work by targeting bcr-abland c-kit (imatinib) and HER2 and BRAF, respectively. For example, a phase 3 randomized trial demonstrated that, compared with dacarbazine, standard of care chemotherapy option for patients with metastatic melanoma with a V600E BRAF mutation, the BRAF inhibitor vemurafenib led to significantly higher response rates (48% vs. 5%).147 At 6 months, overall survival was 84% (95% CI, 78–89) in the vemurafenib group and 64% (95% CI, 56–73) in the dacarba-zine group. The hazard ratio for tumor progression in the vemu-rafenib group was 0.26 (95% CI, 0.20–0.33; P<0.001). The estimated median progression-free survival was 5.3 months in the vemurafenib

1	group. The hazard ratio for tumor progression in the vemu-rafenib group was 0.26 (95% CI, 0.20–0.33; P<0.001). The estimated median progression-free survival was 5.3 months in the vemurafenib group and 1.6 months in the dacarbazine group. This trial highlights the fact that in at least some tumor types targeted therapies that inhibit a genomic alteration that is a driver is likely to be more effective than an unselected thera-peutic option.Sequencing of the human genome has revealed approxi-mately 500 protein kinases. Several tyrosine kinases have been shown to have oncogenic properties and many other protein kinases have been shown to be aberrantly activated in cancer cells.93 Therefore, protein kinases involved in these aberrantly activated pathways are being aggressively pursued in molecular therapeutics. Potential targets like HER2 can be targeted via dif-ferent strategies, such as transcriptional downregulation, targeting of mRNA, RNA inhibition, antisense strategies, direct

1	molecular therapeutics. Potential targets like HER2 can be targeted via dif-ferent strategies, such as transcriptional downregulation, targeting of mRNA, RNA inhibition, antisense strategies, direct inhibition of protein activity, and induction of immunity against the protein. Most of the compounds in development are monoclonal antibod-ies like trastuzumab or small-molecule kinase inhibitors like ima-tinib or vemurafanib. Some other agents, such as sunitinib, are multitargeted kinase inhibitors. Selected FDA-approved targeted therapies are listed in Table 10-11. Many of the promising path-ways, such as the PI3K/Akt/mTOR pathway, are being pursued as therapeutic targets with several drugs in development, targeting different aspects of the pathway (Fig. 10-15).148Development of molecularly targeted agents for clinical use presents several unique challenges. Once an appropriate compound is identified and confirmed to have activity in pre-clinical testing, predictive markers for activity

1	targeted agents for clinical use presents several unique challenges. Once an appropriate compound is identified and confirmed to have activity in pre-clinical testing, predictive markers for activity in the preclinical setting must be defined. Expression of a target may not be suf-ficient to predict response because the pathway of interest may not be activated or critical to the cancer’s survival. Although in traditional phase 1 trials the goal is to identify the maximum tol-erated dosage, the maximum dosage of biologic agents may not be necessary to achieve the desired biologic effect. Thus, assays to verify modulation of the target need to be developed to deter-mine at what dosage the desired effect is achieved. When phases 2 and 3 clinical trials are initiated, biomarker modulation studies should be integrated into the trial to determine whether clinical response correlates with target modulation and thus to identify additional parameters that impact response. Rational dose

1	studies should be integrated into the trial to determine whether clinical response correlates with target modulation and thus to identify additional parameters that impact response. Rational dose selec-tion and limitation of study populations to patients most likely to respond to the molecular therapy as determined by predictive markers are most likely to lead to successful clinical transla-tion of a product. Finally, most biologic agents are cytostatic, not cytotoxic. Thus, rational combination therapy mixing new biologic agents with either established chemotherapeutic agents that have synergy or with other biologic agents is more likely to lead to cancer cures.IMMUNOTHERAPYThe aim of immunotherapy is to induce or potentiate inherent antitumor immunity that can destroy cancer cells. Central to the process of antitumor immunity is the ability of the immune sys-tem to recognize tumor-associated antigens present on human cancers and to direct cytotoxic responses through humoral or

1	Central to the process of antitumor immunity is the ability of the immune sys-tem to recognize tumor-associated antigens present on human cancers and to direct cytotoxic responses through humoral or T-cell–mediated immunity. Overall, T-cell–mediated immunity appears to have the greater potential of the two for eradicating tumor cells. T cells recognize antigens on the surfaces of target cells as small peptides presented by class I and class II MHC molecules.Several antitumor strategies are under investigation. One approach to antitumor immunity is nonspecific immunotherapy, which stimulates the immune system as a whole through admin-istration of bacterial agents or their products, such as bacille Brunicardi_Ch10_p0305-p0354.indd 34222/02/19 2:14 PM 343ONCOLOGYCHAPTER 10Table 10-11Selected FDA-approved targeted therapiesGENERIC NAMETRADE NAMETARGETFDA-APPROVED INDICATIONSAdo-trastuzumab emtansineKadcylaHER2Breast cancerAxitinibInlytaKIT, FDGFRβ,

1	343ONCOLOGYCHAPTER 10Table 10-11Selected FDA-approved targeted therapiesGENERIC NAMETRADE NAMETARGETFDA-APPROVED INDICATIONSAdo-trastuzumab emtansineKadcylaHER2Breast cancerAxitinibInlytaKIT, FDGFRβ, VEGFR1/2/3RCCBevacizumabAvastinVEGFColorectal cancer, lung cancer, glioblastoma, NSCLCRCCBortezomibVelcadeProteasomeMyelomaBosutinibBosulifABLCML (Philadelphia chromosome+)CabozantinibCometriqFLT3, KIT, MET, RET, VEGR2Medullary thyroid cancerCetuximabErbituxEGFRColorectal cancer (KRAS wild-type)Squamous cell cancer of the head and neckCrizotinibXalkoriALK (anaplastic lymphoma kinase) and ROS1 (c-ros oncogene 1) inhibitorNon-small cell lung carcinomaDabrafenibTafinolarBRAF V600E mutationMelanomaDasatinibSprycelABL, src family, KIT, EPHA2, PDGFR-βCMLErlotinibTarcevaEGFRNSCLC,Pancreatic cancerEverolimusAfinitormTORPNET,RCC,Breast cancer.Nonresectable subependymal giant cell astrocytoma associated with tuberous sclerosisGefitinibIressaEGFRNSCLC with known/previous benefit from gefitinib

1	cancer.Nonresectable subependymal giant cell astrocytoma associated with tuberous sclerosisGefitinibIressaEGFRNSCLC with known/previous benefit from gefitinib (limited approval)IbrutinibImbruvicaBruton’s Tyrosine KinaseChronic lymphocytic leukemiaImatinibGleevecKIT, ABL, PDGFRCML,GIST (KIT+),Dermatofibrosarcoma protuberansLapatinibTykerbEGFR and HER2Breast cancer (HER2+)NilotinibTasignaABLCML (Philadelphia chromosome+)PanitumumabVectibixEGFRColorectal cancer (KRAS wild type)PazopanibVotrientVEGFR, PDGFR, KITRCCPertuzumabPerjetaHER2Breast cancer (HER+)PonatinibIclusigABL, FGFR1-3, FLT3, VEGFR2CML, ALL (Philadelphia chromosome+)RegorafenibStivargaKIT, PDGFRβ, RAF, RET, VEGFR1/2/3Colorectal cancer, GISTSorafenibNexavarVEGFR, PDGFR, KIT, RAFHCCRCCSunitinibSutentVEGFR PDGFR KIT, Flt-3, RETGIST,RCC,PNETTemsirolimusToriselmTORRCCTrastuzumabHerceptinHER2Breast cancer (HER2+)Gastric cancer (HER2+)VandetanibCaprelsaEGFR, RET, VEGFR2Medullary thyroid cancerVemurafenibZelborafBRAFMelanoma (BRAF

1	cancer (HER2+)Gastric cancer (HER2+)VandetanibCaprelsaEGFR, RET, VEGFR2Medullary thyroid cancerVemurafenibZelborafBRAFMelanoma (BRAF V600E mutant)VorinostatZolinzaHistone deacetylasesCutaneous T-cell lymphomaCML = chronic myelogenous leukemia; EGFR = epidermal growth factor receptor; EPHA2 = ephrin A2; FDA = Food and Drug Administration; Flt-3 = fms-related tyrosine kinase 3; GIST = GI stromal tumor; HCC = hepatocellular cancer, HER2 = human epidermal growth factor receptor 2; mTOR = mammalian target of rapamycin; NSCLC = non-small cell lung cancer, PDGF = platelet-derived growth factor; PDGFR = platelet-derived growth factor receptor; PNET = pancreatic neuroendocrine tumor; RCC = renal cell carcinoma; RET = rearranged during transfection; VEGF = vascular endothelial growth factor; VEGFR = vascular endothelial growth factor receptor.Brunicardi_Ch10_p0305-p0354.indd 34322/02/19 2:14 PM 344BASIC

1	RET = rearranged during transfection; VEGF = vascular endothelial growth factor; VEGFR = vascular endothelial growth factor receptor.Brunicardi_Ch10_p0305-p0354.indd 34322/02/19 2:14 PM 344BASIC CONSIDERATIONSPART IGlucoseAminoAcidsIRS1PI3KPDK1PIP2PIP3ATPAMPKActivatorsMAP4K3AMPKRapalogsFKBP12AktTSC2TSC1PI3KInhibitorsAktInhibitorsPTENmTORC2ProctorRICTORmTORmLST8SIN1GSK3FOXOBADASK1GDPGTPRhebRhebmTORC1PRAS40mTORRAPTORmLST84EBP1S6KeIF4EPDCD4eEF3KS6eIF4BmTORKinase InhibitorsDual Pl3K/mTORKinase InhibitorsPPFigure 10-15. Targeting PI3K/Akt/mTOR signaling. This central pathway is altered in many tumor types and is being pursued as a therapeu-tic target through development of numerous pathway inhibitors targeting PI3K, Akt, mTOR, and dual inhibitors as well as several upstream and downstream regulators. (Reproduced with permission from McAuliffe PF, Meric-Bernstam F, Mills GB, et al: Deciphering the role of PI3K/Akt/mTOR pathway in breast cancer biology and pathogenesis, Clin Breast

1	regulators. (Reproduced with permission from McAuliffe PF, Meric-Bernstam F, Mills GB, et al: Deciphering the role of PI3K/Akt/mTOR pathway in breast cancer biology and pathogenesis, Clin Breast Cancer. 2010 Nov;10 Suppl 3:S59-S65.)Calmette-Guérin. This approach is thought to activate the effec-tors of antitumor response such as natural killer cells and macro-phages, as well as polyclonal lymphocytes.149 Another approach to nonspecific immunotherapy is systemic administration of cytokines such as interleukin-2, interferon-α, and interferon-γ. Interleukin-2 stimulates proliferation of cytotoxic T lympho-cytes and maturation of effectors such as natural killer cells into lymphokine-activated killer cells. Interferons, on the other hand, exert antitumor effects directly by inhibiting tumor cell prolif-eration and indirectly by activating host immune cells, includ-ing macrophages, dendritic cells, and natural killer cells, and by enhancing human leukocyte antigen (HLA) class I expression

1	prolif-eration and indirectly by activating host immune cells, includ-ing macrophages, dendritic cells, and natural killer cells, and by enhancing human leukocyte antigen (HLA) class I expression on tumor cells.149Antigen-specific immunotherapy can be active, as is achieved through antitumor vaccines, or passive. In pas-sive immunotherapy, antibodies to specific tumor-associated antigens can be produced by hybridoma technique and then administered to patients whose cancers express these antigens, inducing antibody-dependent cellular cytotoxicity.The early attempts at vaccination against cancers used allo-geneic cultured cancer cells, including irradiated cells, cell lysates, and shed antigens isolated from tissue culture supernatants. An alternate strategy is the use of autologous tumor vaccines. These have the potential advantage of being more likely to contain anti-gens relevant for the individual patient but have the disadvantage of requiring a large amount of tumor tissue for

1	vaccines. These have the potential advantage of being more likely to contain anti-gens relevant for the individual patient but have the disadvantage of requiring a large amount of tumor tissue for preparation, which restricts eligibility of patients for this modality. Strategies to enhance immunogenicity of tumor cells include the introduction of genes encoding cytokines or chemokines, and fusion of the tumor cells to allogeneic MHC class II-bearing cells.150 Alternatively, heat shock proteins derived from a patient’s tumor can be used because heat shock protein peptide complexes are readily taken up by dendritic cells for presentation to T cells.150Identification of tumor antigens has made it possible to perform antigen-specific vaccination. For example, in the case of melanoma, several antigens have been identified that can be recognized by both CD8+ cytotoxic T cells and CD4+ helper T cells, including MART-1, gp 100, MAGE1, tyrosinase, TRP-1, TRP-2, and NY-ESO-1.151 Antigens tested

1	have been identified that can be recognized by both CD8+ cytotoxic T cells and CD4+ helper T cells, including MART-1, gp 100, MAGE1, tyrosinase, TRP-1, TRP-2, and NY-ESO-1.151 Antigens tested usually are over-expressed or mutated in cancer cells. Tissue specificity and immunogenicity are important determinants in choosing an appropriate target. Vaccines directed at defined tumor antigens aim to combine selected tumor antigens and appropriate routes for delivering these antigens to the immune system to optimize antitumor immunity.152 Several different vaccination approaches have been studied, including tumor cell-based vaccines, pep-tide-based vaccines, recombinant virus-based vaccines, DNA-based vaccines, and dendritic cell vaccines.In adoptive transfer, antigen-specific effector cells (i.e., cytotoxic T lymphocytes) or antigen-nonspecific effector cells Brunicardi_Ch10_p0305-p0354.indd 34422/02/19 2:14 PM 345ONCOLOGYCHAPTER 10(i.e., natural killer cells) can be transferred to a

1	cytotoxic T lymphocytes) or antigen-nonspecific effector cells Brunicardi_Ch10_p0305-p0354.indd 34422/02/19 2:14 PM 345ONCOLOGYCHAPTER 10(i.e., natural killer cells) can be transferred to a patient. These effector cells can be obtained from the tumor (tumor-infiltrating lymphocytes) or the peripheral blood.Clinical experience in patients with metastatic disease has shown objective tumor responses to a variety of immunothera-peutic modalities. It is thought, however, that the immune sys-tem is overwhelmed with the tumor burden in this setting, and thus adjuvant therapy may be preferable, with immunotherapy reserved for decreasing tumor recurrences. Trials to date sug-gest that immunotherapy is a potentially useful approach in the adjuvant setting. How to best select patients for this approach and how to integrate immunotherapy with other therapies are not well understood for most cancer types.Tolerance to self-antigens expressed in tumors is a limi-tation in generating antitumor

1	approach and how to integrate immunotherapy with other therapies are not well understood for most cancer types.Tolerance to self-antigens expressed in tumors is a limi-tation in generating antitumor responses.153 Recently, several pathways that modulate tolerance and approaches to manipulat-ing these pathways have been identified: pathways that activate professional antigen-presenting cells such as Toll-like receptors, growth factors, and the CD40 pathway; cytokines to enhance immunoactivation; and pathways that inhibit T-cell inhibitory signals or block the activity of immune-suppressive regulatory T cells (Tregs).153A new and highly effective strategy to activate the T-cell arm of anticancer immunity is the use of monoclonal antibodies to block inhibitory signaling pathways employed by the immune system to prevent T cell over activation and the development of auto-immunity. CTLA-4 and PD-1 are two important inhibitory T-cell checkpoints that can be blocked with neutralizing

1	by the immune system to prevent T cell over activation and the development of auto-immunity. CTLA-4 and PD-1 are two important inhibitory T-cell checkpoints that can be blocked with neutralizing antibod-ies and result in an effective antigen-specific anti-tumor response.CTLA-4 is an inhibitory receptor expressed by activated T cells that belongs to the immunoglobulin superfamily. CTLA4 is related to the T-cell costimulatory receptor, CD28, and both are bound by CD80 and CD86 (also known as B7-1 and B7-2) which are expressed on antigen-presenting cells. CTLA-4 con-veys an inhibitory signal to the T cell, whereas engagement of CD28 with ligand sends a stimulatory signal. CTLA-4 is able to outcompete CD28 for CD80 and CD86 ligands and therefore is able to dominate immune signaling in the setting of antigen recognition. CTLA-4 is also expressed by regulatory T cells, which contributes to their ability to inhibit T-cell function.154 Programmed death ligand 1 (PD-L1) is a 40 kDa type 1

1	setting of antigen recognition. CTLA-4 is also expressed by regulatory T cells, which contributes to their ability to inhibit T-cell function.154 Programmed death ligand 1 (PD-L1) is a 40 kDa type 1 trans-membrane protein that is thought to play an important role in suppressing the immune system. PD-L1 binds to its receptor, PD-1, which is found on activated T cells. The PD1/PDL1 path-way is increasingly recognized as a key contributor to tumor-mediated immune suppression. The interaction between PD-1 leads to reduced proliferation, altered production of stimulatory cytokines, and reduced T-cell lytic activity. Thus, both anti-PD1 and anti-PD-L1 strategies are actively being pursued for cancer therapy.155The FDA-approved CTLA-4 blocking antibody ipi-limumab has shown efficacy in patients with metastatic mela-noma.156,157 Nivolumab and pembrolizumab are antibodies that target PD-1, whereas blockade of PD-L1 is accomplished with agents such as atezolizumab.158 Cancers for which

1	with metastatic mela-noma.156,157 Nivolumab and pembrolizumab are antibodies that target PD-1, whereas blockade of PD-L1 is accomplished with agents such as atezolizumab.158 Cancers for which checkpoint inhibitors have found utility include melanoma, renal cell car-cinoma, bladder carcinoma, squamous cell carcinoma of the head and neck, and carcinoma of the lung. These agents pro-duce durable shrinkage of advanced disease in 20% to 40% of patients, and combination strategies that employ checkpoint inhibitors with cytokines, vaccines, cellular therapies, and other targeted agents are under active investigation.GENE THERAPYGene therapy is being pursued as a possible approach to modify-ing the genetic program of cancer cells as well as treating meta-bolic diseases. The field of cancer gene therapy uses a variety of strategies, ranging from replacement of mutated or deleted tumor-suppressor genes to enhancement of immune responses to cancer cells.159 Indeed, in preclinical models,

1	gene therapy uses a variety of strategies, ranging from replacement of mutated or deleted tumor-suppressor genes to enhancement of immune responses to cancer cells.159 Indeed, in preclinical models, approaches such as replacement of tumor-suppressor genes leads to growth arrest or apoptosis. However, the translation of these findings into clinically useful tools presents special challenges.One of the main difficulties in getting gene therapy tech-nology from the laboratory to the clinic is the lack of a perfect delivery system. An ideal vector would be administered through a noninvasive route and would transduce all of the cancer cells and none of the normal cells. Furthermore, the ideal vector would have a high degree of activity, that is, it would produce an adequate amount of the desired gene product to achieve target cell kill. Unlike genetic diseases in which delivery of the gene of interest into only a portion of the cells may be sufficient to achieve clinical effect, cancer

1	gene product to achieve target cell kill. Unlike genetic diseases in which delivery of the gene of interest into only a portion of the cells may be sufficient to achieve clinical effect, cancer requires either that the therapeutic gene be delivered to all of the cancer cells or that a therapeutic effect be achieved on nontransfected cells as well as transfected cells through a bystander effect. However, treatment of a meta-bolic disease requires prolonged gene expression, whereas tran-sient expression may be sufficient for cancer therapy.Several vector systems are under study for gene ther-apy; however, none is considered ideal. One of the promising approaches to increase the number of tumor cells transduced is the use of a replication-competent virus like a parvovirus, human reovirus, or vesicular stomatitis virus that selectively replicates within malignant cells and lyses them more efficiently than it does normal cells. Another strategy for killing tumor cells with suicide genes

1	or vesicular stomatitis virus that selectively replicates within malignant cells and lyses them more efficiently than it does normal cells. Another strategy for killing tumor cells with suicide genes exploits tumor-specific expression elements, such as the MUC-1, PSA, CEA, or VEGF promoters, that can be used to achieve tissue-specific or tumor-specific expression of the desired gene.Because the goal in cancer therapy is to eradicate systemic disease, optimization of delivery systems is the key to success for gene therapy strategies. Gene therapy is likely to be most successful when combined with standard therapies, but it will provide the advantage of customization of therapy based on the molecular status of an individual’s tumor.MECHANISMS OF INTRINSIC AND ACQUIRED DRUG RESISTANCESeveral tumor factors influence tumor cell kill. Tumors are het-erogeneous, and, according to the Goldie-Coldman hypothesis, tumor cells are genetically unstable and tend to mutate to form different cell

1	tumor factors influence tumor cell kill. Tumors are het-erogeneous, and, according to the Goldie-Coldman hypothesis, tumor cells are genetically unstable and tend to mutate to form different cell clones. This has been used as an argument for giv-ing chemotherapy as soon as possible in treatment to reduce the likelihood that resistant clones will emerge. Tumor size is another important variable. Larger tumors may have greater het-erogeneity, although heterogeneity may also differ based on bio-logic subtype. Tumor growth may be described by a Gompertz curve, named after Benjamin Gompertz, which has the form of a sigmoid function. Gompertzian models have thus been used to describe changes in tumor cell numbers over time where growth is slowest at the start and end of a time period, but are quite rapid in the middle. Theoretically, for any tumor, there is a period of time where cancer cells grow rapidly (exponential growth Brunicardi_Ch10_p0305-p0354.indd 34522/02/19 2:14 PM 346BASIC

1	rapid in the middle. Theoretically, for any tumor, there is a period of time where cancer cells grow rapidly (exponential growth Brunicardi_Ch10_p0305-p0354.indd 34522/02/19 2:14 PM 346BASIC CONSIDERATIONSPART Iphase), and then the growth slows down owing to hypoxia and decreased nutrient supply. Because of the larger proportion of cells dividing, smaller tumors may be more chemosensitive.Multiple mechanisms of systemic therapy resistance have been identified (Table 10-12).160 Cells may exhibit reduced sen-sitivity to drugs by virtue of their cell-cycle distribution. For example, cells in the G0 phase are resistant to drugs active in the S phase. This phenomenon of “kinetic resistance” usually is temporary, and if the drug level can be maintained, all cells will eventually pass through the vulnerable phase of the cell cycle.145 Alternatively, tumor cells may exhibit “pharmacologic resistance,” in which the failure to kill cells is due to insuffi-cient drug concentration. This may

1	the vulnerable phase of the cell cycle.145 Alternatively, tumor cells may exhibit “pharmacologic resistance,” in which the failure to kill cells is due to insuffi-cient drug concentration. This may occur when tumor cells are located in sites where effective drug concentrations are difficult to achieve (such as the central nervous system) or can be due to enhanced metabolism of the drug after its administration, decreased conversion of the drug to its active form, or decrease in the intracellular drug level caused by increased removal of the drug from the cell associated with enhanced expression of P-glycoprotein (Pgp). Pgp is the protein product of multidrug resis-tance gene 1 and extrudes cytotoxic drugs at the expense of ATP hydrolysis. Other mechanisms of resistance include decreased affinity of the target enzyme for the drug, altered amount of the target enzyme, or enhanced repair of the drug-induced defect. For drug-sensitive cancers, another factor limiting optimal killing is

1	affinity of the target enzyme for the drug, altered amount of the target enzyme, or enhanced repair of the drug-induced defect. For drug-sensitive cancers, another factor limiting optimal killing is inadequate dosing. Relative dose intensity (RDI) is defined as the actual amount of a particular chemotherapy given over a specific time in relation to what was ordered and is usually expressed as a percentage. An RDI below 80% is considered suboptimal and may impact survival in the adjuvant setting.145Cancer cells demonstrate adaptive responses to targeted therapy, like activating alternate pathways of survival; thus, these alterations may blunt therapeutic efficacy. Cancer cells also acquire resistance upon prolonged treatment with targeted therapy through a variety of mechanisms. One mechanism is through the loss of the target. For example, this was observed in a study of patients with HER2-positive breast cancer patients who were treated with neoadjuvant trastuzumab-based

1	One mechanism is through the loss of the target. For example, this was observed in a study of patients with HER2-positive breast cancer patients who were treated with neoadjuvant trastuzumab-based chemo-therapy.161 Post neoadjuvant treatment, a third of the samples from patients who did not have a complete pathologic response displayed loss of the HER2 amplification that had been pres-ent in their pretreatment-biopsy specimens.161 Another means by which cancers develop resistance is the acquisition of addi-tional genomic aberrations. In lung cancer, a second mutation in EGFR (T790M) and MET amplification have been described as two main mechanisms of drug resistance to EGFR inhibi-tors erlotinib and gefinitib.162-164 Other mechanisms like novel genetic changes, including HER2 and EGFR amplification, PIK3CA mutations, and markers of epithelial-to-mesenchymal transition have also been reported in EGFR inhibitor resistant lung.165,166 Analysis of metastases from patients with colorectal

1	PIK3CA mutations, and markers of epithelial-to-mesenchymal transition have also been reported in EGFR inhibitor resistant lung.165,166 Analysis of metastases from patients with colorectal cancer who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% of the cases.167 These studies emphasize the utility of repeat tumor biopsy specimens at the time of relapse or progression to iden-tify mechanisms of resistance and best combinatorial therapies.RADIATION THERAPYPhysical Basis of Radiation TherapyIonizing radiation is energy strong enough to remove an orbital electron from an atom. This radiation can be electromagnetic, like a high-energy photon, or particulate, such as an electron, proton, neutron, or alpha particle. Radiation therapy is delivered primar-ily as high-energy photons (gamma rays and X-rays) and charged particles (electrons). Gamma rays are photons that are released from

1	neutron, or alpha particle. Radiation therapy is delivered primar-ily as high-energy photons (gamma rays and X-rays) and charged particles (electrons). Gamma rays are photons that are released from the nucleus of a radioactive atom. X-rays are photons that are created electronically, such as with a clinical linear accelerator. Currently, high-energy radiation is delivered to tumors primarily with linear accelerators. X-rays traverse the tissue, depositing the maximum dose beneath the surface, and thus spare the skin. Elec-trons are used to treat superficial skin lesions, superficial tumors, or surgical beds to a depth of 5 cm. Gamma rays typically are produced by radioactive sources used in brachytherapy.The dose of radiation absorbed correlates with the energy of the beam. The basic unit is the amount of energy absorbed per unit of mass (joules per kilogram) and is known as a gray (Gy). One gray is equivalent to 100 rads, the unit of radiation measurement used in the past.Biologic

1	is the amount of energy absorbed per unit of mass (joules per kilogram) and is known as a gray (Gy). One gray is equivalent to 100 rads, the unit of radiation measurement used in the past.Biologic Basis of Radiation TherapyRadiation deposition results in DNA damage manifested by singleand double-strand breaks in the sugar phosphate back-bone of the DNA molecule.168 Cross-linking between the DNA strands and chromosomal proteins also occurs. The mecha-nism of DNA damage differs by the type of radiation deliv-ered. Electromagnetic radiation is indirectly ionizing through the actions of short-lived hydroxyl radicals, which are pro-duced primarily by the ionization of cellular hydrogen perox-ide (H2O2).168 Protons and other heavy particles are directly ionizing and directly damage DNA.Table 10-12General mechanisms of drug resistanceCellular and biochemical mechanisms Decreased drug accumulation Decreased drug influx Increased drug efflux Altered intracellular trafficking of

1	DNA.Table 10-12General mechanisms of drug resistanceCellular and biochemical mechanisms Decreased drug accumulation Decreased drug influx Increased drug efflux Altered intracellular trafficking of drug Decreased drug activation Increased inactivation of drug or toxic intermediate Increased repair of drug-induced damage to: DNA Protein Membranes Alteration of drug targets (quantitatively or qualitatively) Alteration of cofactor or metabolite levels Alteration of gene expression DNA mutation, amplification, or deletion Altered transcription, posttranscription processing, or translation Altered stability of macromoleculesMechanisms relevant in vivo Pharmacologic and anatomic drug barriers (tumor sanctuaries) Host-drug interactions Increased drug inactivation by normal tissues Decreased drug activation by normal tissues Relative increase in normal tissue drug sensitivity (toxicity)Host-tumor interactionsReproduced with permission from Bast R, Kufe D, Pollock R: Cancer

1	drug activation by normal tissues Relative increase in normal tissue drug sensitivity (toxicity)Host-tumor interactionsReproduced with permission from Bast R, Kufe D, Pollock R: Cancer Medicine. Hamilton: BC Decker, Inc; 2000.Brunicardi_Ch10_p0305-p0354.indd 34622/02/19 2:14 PM 347ONCOLOGYCHAPTER 10Radiation damage is manifested primarily by the loss of cellular reproductive integrity. Most cell types do not show signs of radiation damage until they attempt to divide, so slowly proliferating tumors may persist for months and appear viable. Some cell types, however, undergo apoptosis.The extent of DNA damage after radiation exposure is dependent on several factors. The most important of these is cellular oxygen. Hypoxic cells are significantly less radiosensi-tive than aerated cells. The presence of oxygen is thought to pro-long the half-life of free radicals produced by the interaction of X-rays and cellular H2O2, and thus indirectly ionizing radiation is less efficacious in

1	The presence of oxygen is thought to pro-long the half-life of free radicals produced by the interaction of X-rays and cellular H2O2, and thus indirectly ionizing radiation is less efficacious in tumors with areas of hypoxia.168 In contrast, radiation damage from directly ionizing radiation is independent of cellular oxygen levels.The extent of DNA damage from indirectly ionizing radiation is dependent on the phase of the cell cycle. The most radiation-sensitive phases are G2 and M, whereas G1 and late S phases are less sensitive. Thus, irradiation of a population of tumor cells results in killing of a greater proportion of cells in G2 and M phases. However, delivery of radiation in divided doses, a concept referred to as fractionation, allows the surviv-ing G1 and S phase cells to progress to more sensitive phases, a process referred to as reassortment. In contrast to DNA dam-age after indirectly ionizing radiation, that after exposure to directly ionizing radiation is less dependent

1	to more sensitive phases, a process referred to as reassortment. In contrast to DNA dam-age after indirectly ionizing radiation, that after exposure to directly ionizing radiation is less dependent on the cell-cycle phase.169Several chemicals can modify the effects of ionizing radia-tion. These include hypoxic cell sensitizers such as metronida-zole and misonidazole, which mimic oxygen and increase cell kill of hypoxic cells.168 A second category of radiation sensitiz-ers are the thymidine analogues iododeoxyuridine and bromo-deoxyuridine. These molecules are incorporated into the DNA in place of thymidine and render the cells more susceptible to radiation damage; however, they are associated with consid-erable acute toxicity. Several other chemotherapeutic agents sensitize cells to radiation through various mechanisms, includ-ing 5-fluorouracil, actinomycin D, gemcitabine, paclitaxel, topotecan, doxorubicin, and vinorelbine.168 The development of novel radiosensitizers is an active

1	through various mechanisms, includ-ing 5-fluorouracil, actinomycin D, gemcitabine, paclitaxel, topotecan, doxorubicin, and vinorelbine.168 The development of novel radiosensitizers is an active area of research and mul-tiple small molecules as well as novel nanomaterials are under investigation.170Radiation Therapy PlanningRadiation therapy is delivered in a homogeneous dose to a well-defined region that includes tumor and/or surrounding tissue at risk for subclinical disease. The first step in planning is to define the target to be irradiated as well as the dose-limiting organs in the vicinity.171 Treatment planning includes evaluation of alter-native treatment techniques, which is done through a process referred to as simulation. Once the beam distribution that will best achieve homogeneous delivery to the target volume and minimize the dose to the normal tissue is determined, immo-bilization devices and markings or tattoos on the patient’s skin are used to ensure that each daily

1	delivery to the target volume and minimize the dose to the normal tissue is determined, immo-bilization devices and markings or tattoos on the patient’s skin are used to ensure that each daily treatment is given in the same way. Conventional fractionation is 1.8 to 2 Gy/d, administered 5 days each week for 3 to 7 weeks.Radiation therapy may be used as the primary modality for palliation in certain patients with metastatic disease, pri-marily patients with bony metastases. In these cases, radiation is recommended for symptomatic metastases only. However, lytic metastases in weight-bearing bones such as the femur, tibia, or humerus also are considered for irradiation. Another circumstance in which radiation therapy might be appropriate is spinal cord compression due to metastases to the vertebral body that extend posteriorly to the spinal canal.The goal of adjuvant radiation therapy is to decrease local-regional recurrence rates. Adjuvant radiation therapy can be given before surgery,

1	body that extend posteriorly to the spinal canal.The goal of adjuvant radiation therapy is to decrease local-regional recurrence rates. Adjuvant radiation therapy can be given before surgery, after surgery, or, in selected cases, during surgery. Preoperative radiation therapy has several advantages. It may minimize seeding of the tumor during surgery and it allows for smaller treatment fields because the operative bed has not been contaminated with tumor cells. Also, radiation therapy for inoperable tumors may achieve adequate reduction to make them operable. The disadvantages of preoperative therapy are an increased risk of postoperative wound healing problems and the difficulty in planning subsequent radiation therapy in patients who have positive surgical margins. If radiation therapy is given postoperatively, it is usually given 3 to 4 weeks after surgery to allow for wound healing. The advantage of postoperative radia-tion therapy is that the surgical specimen can be evaluated

1	is given postoperatively, it is usually given 3 to 4 weeks after surgery to allow for wound healing. The advantage of postoperative radia-tion therapy is that the surgical specimen can be evaluated histo-logically and radiation therapy can be reserved for patients who are most likely to benefit from it. Further, the radiation therapy can be modified on the basis of margin status. The disadvantages of postoperative radiation therapy are that the volume of nor-mal tissue requiring irradiation may be larger owing to surgical contamination of the tissue planes and that the tumor may be less sensitive to radiation owing to poor oxygenation. Postlapa-rotomy adhesions may decrease the mobility of the small bowel loops, increasing the risk for radiation injury in abdominal or pel-vic irradiation. Given the potential advantages and disadvantages of both approaches, the roles of preoperative and postoperative radiation therapy are being actively evaluated and compared for many cancer

1	Given the potential advantages and disadvantages of both approaches, the roles of preoperative and postoperative radiation therapy are being actively evaluated and compared for many cancer types.Another mode of postoperative radiation therapy is brachytherapy. In brachytherapy, unlike in external beam therapy, the radiation source is in contact with the tissue being irradiated. The radiation source may be cesium, gold, iridium, or radium. Brachytherapy is administered via temporary or per-manent delivery implants such as needles, seeds, or catheters. Temporary brachytherapy catheters are placed either during open surgery or percutaneously soon after surgery. The implants are loaded interstitially, and treatment usually is given postop-eratively for a short duration, such as 1 to 3 days. Although brachytherapy has the disadvantages of leaving scars at the cath-eter insertion site and requiring special facilities for inpatient brachytherapy, the advantage of patient convenience owing to

1	brachytherapy has the disadvantages of leaving scars at the cath-eter insertion site and requiring special facilities for inpatient brachytherapy, the advantage of patient convenience owing to the shorter treatment duration has made intracavitary treatment approaches popular for the treatment of breast cancer.Another short delivery approach is intraoperative radio-therapy (IORT), often used in combination with external beam therapy. The oncologic consequences of the limited treatment volume and duration associated with brachytherapy and IORT are not well understood. Accelerated partial breast irradiation with interstitial brachytherapy, intracavitary brachytherapy (MammoSite), IORT, and three-dimensional conformal external beam radiotherapy is being compared with whole breast irra-diation in an intergroup phase 3 trial (NSABP B-39/Radiation Therapy Oncology Group 0413). Several additional studies of adjuvant IORT also are ongoing internationally. There has also been increased interest

1	in an intergroup phase 3 trial (NSABP B-39/Radiation Therapy Oncology Group 0413). Several additional studies of adjuvant IORT also are ongoing internationally. There has also been increased interest in utilizing intensity-modulated radiation therapy (IMRT). IMRT is a complex technique for the delivery of radiation therapy preferentially to target structures while mini-mizing doses to adjacent normal critical structures.172 It is widely utilized for the treatment of a variety of tumor types, including Brunicardi_Ch10_p0305-p0354.indd 34722/02/19 2:14 PM 348BASIC CONSIDERATIONSPART Ithe central nervous system, head and neck, breast, prostate, gas-trointestinal tract, and gynecologic organs, as well as in patients where previous radiation therapy has been delivered. Stereotac-tic radiosurgery uses extremely accurate image-guidance and patient positioning to deliver a high dose of radiation to a small tumor with well-defined margins. In this manner, the dose of radiation being

1	radiosurgery uses extremely accurate image-guidance and patient positioning to deliver a high dose of radiation to a small tumor with well-defined margins. In this manner, the dose of radiation being applied to normal tissues can be minimized. It is most commonly used for the treatment of brain and spinal tumors. Protons are a charged particle that can be also used in external beam radiation therapy. Proton therapy employs a beam of protons as a means of delivering radiation to a tumor. In con-trast to photons, which deposit energy continuously during their passage through tissue, protons deposit a large amount of their energy near the end of their path (known as the Bragg peak) and release less energy along the way. Thus, proton therapy could theoretically reduce the exposure of normal tissue to radiation, allowing the delivery of higher doses of radiation to a tumor. It is thought that chemotherapy given concurrently with radiation improves survival rates. Chemotherapy before

1	tissue to radiation, allowing the delivery of higher doses of radiation to a tumor. It is thought that chemotherapy given concurrently with radiation improves survival rates. Chemotherapy before radiation has the advantage of reducing the tumor burden, which facilitates radia-tion therapy. On the other hand, some chemotherapy regimens, when given concurrently with radiation, may sensitize the cells to radiation therapy. Chemoradiation is being investigated in many tumor types, including rectal cancer, pancreatic cancer, and esophageal cancer.173-175 In a Cochrane review of six ran-domized controlled trials, it was demonstrated that in patients with T3/4 rectal cancer, chemoradiation was associated with a significantly lower local recurrence rate compared with radiation therapy alone (OR 0.56, 95% CI 0.42–0.75, P <0.0001) but was not associated with improved survival.173Side EffectsBoth tumor and normal tissue have radiation dose-response rela-tionships that can be plotted as a

1	(OR 0.56, 95% CI 0.42–0.75, P <0.0001) but was not associated with improved survival.173Side EffectsBoth tumor and normal tissue have radiation dose-response rela-tionships that can be plotted as a sigmoidal curve (Fig. 10-16).171 A minimum dose of radiation must be given before any response is seen. The response to radiation then increases slowly with an increase in dose. At a certain dose level the curves become exponential, with increases in tumor response and normal tissue toxicity with each incremental dose increase. The side effects of radiation therapy can be acute, occurring during or 2 to 3 weeks after therapy, or chronic, occurring weeks to years after therapy. The side effects depend on the tissue included in the target volume. Some of the major acute and chronic sequelae of radiation are summarized in Table 10-13.171,176 In addition to these effects, a small increase in the risk for secondary malignancies is attribut-able to radiation therapy.CANCER PREVENTIONThe truth of

1	are summarized in Table 10-13.171,176 In addition to these effects, a small increase in the risk for secondary malignancies is attribut-able to radiation therapy.CANCER PREVENTIONThe truth of the old axiom “An ounce of prevention is worth a pound of cure” is being increasingly recognized in oncology. Cancer prevention can be divided into three categories: (a) pri-mary prevention (i.e., prevention of initial cancers in healthy indi-viduals), (b) secondary prevention (i.e., prevention of cancer in individuals with premalignant conditions), and (c) tertiary pre-vention (i.e., prevention of second primary cancers in patients cured of their initial disease).The systemic or local administration of therapeutic agents to prevent the development of cancer, called chemoprevention, is being actively explored for several cancer types. In breast can-cer, the NSABP Breast Cancer Prevention Trial demonstrated that tamoxifen administration reduces the risk of breast cancer by one half and reduces the

1	explored for several cancer types. In breast can-cer, the NSABP Breast Cancer Prevention Trial demonstrated that tamoxifen administration reduces the risk of breast cancer by one half and reduces the risk of estrogen receptor-positive tumors by 69% in high-risk patients.177 Therefore, tamoxifen has been approved by the FDA for breast cancer chemoprevention. The subsequent NSABP P-2 trial demonstrated that raloxifene is as effective as tamoxifen in reducing the risk of invasive breast cancer and is associated with a lower risk of thromboembolic events and cataracts but a nonstatistically significant higher risk of noninvasive breast cancer; these findings led the FDA to approve raloxifene for prevention as well. Several other agents are also under investigation.178 Celecoxib (a cyclooxygenase 2 [COX-2] inhibitor) has been shown to reduce polyp number and polyp burden in patients with FAP, which led to its approval by the FDA for these patients. However, celecoxib is no longer widely

1	2 [COX-2] inhibitor) has been shown to reduce polyp number and polyp burden in patients with FAP, which led to its approval by the FDA for these patients. However, celecoxib is no longer widely used as a primary preventative treatment in this setting due to the association between COX-2 inhibitors and coronary artery disease. In head and neck cancer, 13-cis-retinoic acid has been shown both to reverse oral leukoplakia and to reduce sec-ond primary tumor development.179,180 However, a large phase 3 clinical trial that utilized low-dose 13-cis-retinoic acid in patients with early stage squamous cell carcinoma of the head and neck showed no significant difference in the incidence of tumor recurrence or the second primary tumors between the pla-cebo and chemoprevention arms.181 Thus, the chemoprevention trials completed so far have had mixed results. Much remains to be done over the next few years to improve outcomes and decrease therapy-related toxic effects. It is important for

1	the chemoprevention trials completed so far have had mixed results. Much remains to be done over the next few years to improve outcomes and decrease therapy-related toxic effects. It is important for sur-geons to be aware of these preventive options because they are likely to be involved in the diagnosis of premalignant and malig-nant conditions and will be the ones to counsel patients about their chemopreventive options.In selected circumstances, the risk of cancer is high enough to justify surgical prevention. These high-risk settings include hereditary cancer syndromes such as hereditary breast-ovarian cancer syndrome, hereditary diffuse gastric cancer, multiple endocrine neoplasia type 2, FAP, and hereditary non-polyposis colorectal cancer, as well as some nonhereditary Tumor controlComplicationsABDosePercentFigure 10-16. The probability of tumor control and of complica-tions at different radiation doses. A. At lower doses, the probability of complications is low, with a moderate

1	10-16. The probability of tumor control and of complica-tions at different radiation doses. A. At lower doses, the probability of complications is low, with a moderate chance of tumor control. B. Increasing the dose may gain a higher chance of tumor control at the price of significantly higher complication risks. (Reproduced with permission from Eisbruch A, Lichter AS. What a surgeon needs to know about radiation, Ann Surg Oncol. 1997 Sep;4(6):516-522.)Brunicardi_Ch10_p0305-p0354.indd 34822/02/19 2:14 PM 349ONCOLOGYCHAPTER 10conditions such as chronic ulcerative colitis. Most prophy-lactic surgeries are large ablative surgeries (e.g., bilateral risk-reducing mastectomy or total proctocolectomy). There-fore, it is important that the patient be completely informed about potential surgical complications as well as long-term lifestyle consequences. Further, the conservative options of close surveillance and chemoprevention need to be discussed. The patient’s cancer risk needs to be

1	complications as well as long-term lifestyle consequences. Further, the conservative options of close surveillance and chemoprevention need to be discussed. The patient’s cancer risk needs to be assessed accurately and implications for survival discussed. Ultimately, the decision to proceed with surgical prevention should be individualized and made with caution.TRENDS IN ONCOLOGYCancer Screening and DiagnosisIt is clear that the practice of oncology will change dramati-cally over the next few decades because our understanding of the molecular basis of cancer and available technologies are evolving rapidly. One of the critical changes expected is earlier detection of cancers. With improvements in available imaging modalities and development of newer functional imaging tech-niques, it is likely that many tumors will be detected at earlier, more curable stages in the near future.Another area of rapid development is the identification of serum markers. High-throughput technologies such as

1	that many tumors will be detected at earlier, more curable stages in the near future.Another area of rapid development is the identification of serum markers. High-throughput technologies such as matrix-assisted laser desorption ionization time-of-flight mass spec-troscopy and liquid chromatography ion-spray tandem mass spectroscopy have revolutionized the field of proteomics and are now being used to compare the serum protein profiles of patients with cancer with those of individuals without cancer. Identification of unique proteins as well as unique proteomic profiles for most cancer types is being pursued actively by many researchers and, if successful, could dramatically enhance our ability to detect cancers early.182DNA fragments that are derived from tumors and are circulating in the blood stream are referred to as circulating tumor DNA (ctDNA). Analysis of ctDNA can potentially pro-vide information on the entire tumor genome and has poten-tial clinical utility as a so-called

1	the blood stream are referred to as circulating tumor DNA (ctDNA). Analysis of ctDNA can potentially pro-vide information on the entire tumor genome and has poten-tial clinical utility as a so-called “liquid biopsy” when blood samples are obtained during important junctures of a clinical scenario. ctDNA may originate directly from the tumor or from circulating tumor cells, which refers to intact tumor cells that are shed from primary tumors and enter the bloodstream. The precise mechanism of ctDNA release has not been determined; however, there is evidence to show that the length of the DNA fragments are similar to those seen during the process of apop-tosis. ctDNA can be reliably procured from peripheral blood and analyzed via a number of advanced techniques, including next generation sequencing. The main advantages of using ctDNA in genomic studies is the ability to obtain information on the entire tumor genome, thus avoiding the difficulties of tumor heteroge-neity that are

1	sequencing. The main advantages of using ctDNA in genomic studies is the ability to obtain information on the entire tumor genome, thus avoiding the difficulties of tumor heteroge-neity that are encountered with needle biopsies, and the ability to obtain multiple samples with much less risk to the patient.181Surgical TherapyThe current trend in surgery is toward more conservative resec-tions. With earlier identification of tumors, more conservative operations may be possible. The goal, however, is always to remove the tumor en bloc with wide negative margins. Another interesting area being explored is the destruction of tumors by techniques such as radiofrequency ablation, cryoablation, and heat-producing technologies like lasers, microwaves, or focused ultrasound.The debate over how to manage the regional lymph node basins for certain cancer types continues. With an increasing understanding of the metastatic process, surgeons may be able to stratify patients on the basis of the

1	to manage the regional lymph node basins for certain cancer types continues. With an increasing understanding of the metastatic process, surgeons may be able to stratify patients on the basis of the likelihood that their disease will spread metastatically, based on the gene expression profile of their primary tumors, and offer regional therapy accordingly. There is also a growing interest in minimally invasive surgical treatments for a variety of cancer types.Systemic TherapyThe current trend in systemic therapy is toward individual-ized therapy. Therefore, the intent is to determine the under-lying biology of each tumor to tailor therapy accordingly. Genomic, transcriptional, and proteomic profiling approaches are being used to identify molecular signatures that correlate Table 10-13Local effects of radiationORGANACUTE CHANGESCHRONIC CHANGESSkinErythema, wet or dry desquamation, epilationTelangiectasia, subcutaneous fibrosis, ulcerationGI tractNausea, diarrhea, edema, ulceration,

1	effects of radiationORGANACUTE CHANGESCHRONIC CHANGESSkinErythema, wet or dry desquamation, epilationTelangiectasia, subcutaneous fibrosis, ulcerationGI tractNausea, diarrhea, edema, ulceration, hepatitisStricture, ulceration, perforation, hematocheziaKidney—Nephropathy, renal insufficiencyBladderDysuriaHematuria, ulceration, perforationGonadsSterilityAtrophy, ovarian failureHematopoietic tissueLymphopenia, neutropenia, thrombocytopeniaPancytopeniaBoneEpiphyseal growth arrestNecrosisLungPneumonitisPulmonary fibrosisHeart—Pericarditis, vascular damageUpper aerodigestive tractMucositis, xerostomia, anosmiaXerostomia, dental cariesEyeConjunctivitisCataract, keratitis, optic nerve atrophyNervous systemCerebral edemaNecrosis, myelitisBrunicardi_Ch10_p0305-p0354.indd 34922/02/19 2:14 PM 350BASIC CONSIDERATIONSPART Iwith response to certain agents. It is likely that in the near future all tumors can be tested and treatments individualized. Patients who will respond to conventional

1	PM 350BASIC CONSIDERATIONSPART Iwith response to certain agents. It is likely that in the near future all tumors can be tested and treatments individualized. Patients who will respond to conventional therapies can be treated with these regimens, whereas patients who will not respond will not, which spares them the toxicity. Instead, the latter patients can be offered novel therapies. Furthermore, with the advent of effec-tive immune-based therapies, it is likely that patients may be given treatments that can specifically target the alterations driv-ing tumor growth in combination with drugs that can enhance the anticancer immune response. Patients can be genotyped for critical alleles that may affect drug metabolism and thus, may influence the efficacy as well as the side effects of the drugs given. Finally, stratification of patients by gene expression pro-file for prognosis may assist in determining which patients are at higher risk of relapse so that patients whose tumors have less

1	given. Finally, stratification of patients by gene expression pro-file for prognosis may assist in determining which patients are at higher risk of relapse so that patients whose tumors have less aggressive biologic characteristics can be spared further therapy.REFERENCESEntries highlighted in bright blue are key references. 1. Siegel R, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin. 2017;67:7-30. 2. Ravdin PM, Cronin KA, Howlader N, et al. The decrease in breast-cancer incidence in 2003 in the United States. N Engl J Med. 2007;356:1670-1674. 3. Siegel R, Miller KD, Jemal A. Cancer statistics, 2016. CA: Cancer J Clin. 2016;66:7-30. 4. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM; International Agency for Research on Cancer. GLO-BOCAN 2008 v2.0, Cancer incidence and mortality world-wide. Available at: https://www.iarc.fr/en/media-centre/iarcnews/2010/globocan2008.php. Accessed July 1, 2018. 5. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics,

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1	K. Drug resistance and its clinical circum-vention. In: Bast R, Kufe D, Pollock R, eds. Cancer Medicine. Hamilton: B.C. Decker, Inc; 2000:539. 161. Mittendorf EA, Wu Y, Scaltriti M, et al. Loss of HER2 amplification following trastuzumab-based neoadjuvant systemic therapy and survival outcomes. Clin Cancer Res. 2009;15:7381-7388. 162. Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2005;2:e73. 163. Engelman JA, Zejnullahu K, Mitsudomi T, et al. MET amplifi-cation leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 2007;316:1039-1043.Brunicardi_Ch10_p0305-p0354.indd 35322/02/19 2:14 PM 354BASIC CONSIDERATIONSPART I 164. Bean J, Brennan C, Shih JY, et al. MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci USA.

1	J, Brennan C, Shih JY, et al. MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci USA. 2007;104:20932-20937. 165. Sequist LV, Waltman BA, Dias-Santagata D, et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci Transl Med. 2011;3:75ra26. 166. Takezawa K, Pirazzoli V, Arcila ME, et al. HER2 amplifica-tion: a potential mechanism of acquired resistance to EGFR inhibition in EGFR-mutant lung cancers that lack the second-site EGFRT790M mutation. Cancer Discov. 2012;2:922-933. 167. Misale S, Yaeger R, Hobor S, et al. Emergence of KRAS muta-tions and acquired resistance to anti-EGFR therapy in colorec-tal cancer. Nature. 2012;486:532-536. 168. Mundt A, Roeske J, Weichelbaum R. Principles of radiation oncology. In: Bast R, Kuff D, Pollock R, eds. Cancer Medi-cine. Hamilton: B.C. Decker Inc; 2000:465. 169. Raju MR, Carpenter SG. A heavy

1	A, Roeske J, Weichelbaum R. Principles of radiation oncology. In: Bast R, Kuff D, Pollock R, eds. Cancer Medi-cine. Hamilton: B.C. Decker Inc; 2000:465. 169. Raju MR, Carpenter SG. A heavy particle comparative study. Part IV: acute and late reactions. Br J Radiol. 1978;51:720-727. 170. Wang H, Mu X, He H, Zhang XD. Cancer radiosensitizers. Trends Pharmacol Sci. 2018;39:24-48. 171. Eisbruch A, Lichter AS. What a surgeon needs to know about radiation. Ann Surg Oncol. 1997;4:516-522. 172. Hartford AC, Galvin JM, Beyer DC, et al. American College of Radiology (ACR) and American Society for Radiation Oncology (ASTRO) Practice Guideline for Intensity-modulated Radiation Therapy (IMRT). Am J Clin Oncol. 2012;35:612-617. 173. McCarthy K, Pearson K, Fulton R, Hewitt J. Pre-operative chemoradiation for non-metastatic locally advanced rectal cancer. Cochrane Database Syst Rev. 2012;12:CD008368. 174. Evans DB, Varadhachary GR, Crane CH, et al. Preoperative gemcitabine-based chemoradiation for

1	for non-metastatic locally advanced rectal cancer. Cochrane Database Syst Rev. 2012;12:CD008368. 174. Evans DB, Varadhachary GR, Crane CH, et al. Preoperative gemcitabine-based chemoradiation for patients with resect-able adenocarcinoma of the pancreatic head. J Clin Oncol. 2008;26:3496-3502. 175. Courrech Staal EF, Aleman BM, Boot H, van Velthuysen ML, van Tinteren H, van Sandick JW. Systematic review of the ben-efits and risks of neoadjuvant chemoradiation for oesophageal cancer. Br J Surg. 2010;97:1482-1496. 176. Daly J, Bertagnolli M, DeCosse J. Oncology. In: Schwartz S, Spencer F, Galloway A, eds. Principles of Surgery. New York: McGraw-Hill; 1999:297. 177. Fisher B, Costantino JP, Wickerham DL, et al. Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J Natl Cancer Inst. 1998;90:1371-1388. 178. Vogel VG, Costantino JP, Wickerham DL, et al. Effects of tamox-ifen vs raloxifene on the risk of developing invasive

1	Breast and Bowel Project P-1 Study. J Natl Cancer Inst. 1998;90:1371-1388. 178. Vogel VG, Costantino JP, Wickerham DL, et al. Effects of tamox-ifen vs raloxifene on the risk of developing invasive breast cancer and other disease outcomes: the NSABP Study of Tamoxifen and Raloxifene (STAR) P-2 trial. JAMA. 2006;295:2727-2741. 179. Lippman SM, Batsakis JG, Toth BB, et al. Comparison of low-dose isotretinoin with beta carotene to prevent oral carcino-genesis. N Engl J Med. 1993;328:15-20. 180. Hong WK, Lippman SM, Itri LM, et al. Prevention of second primary tumors with isotretinoin in squamous-cell carcinoma of the head and neck. N Engl J Med. 1990;323:795-801. 181. Bettegowda C, Sausen M, Leary RJ, et al. Detection of circu-lating tumor DNA in earlyand late-stage human malignan-cies. Sci Transl Med. 2014;6:224ra24. 182. Sidransky D. Emerging molecular markers of cancer. Nat Rev Cancer. 2002;2:210-219. 183. Meric-Bernstam F, Hung MC. Advances in targeting human epi-dermal growth factor

1	Med. 2014;6:224ra24. 182. Sidransky D. Emerging molecular markers of cancer. Nat Rev Cancer. 2002;2:210-219. 183. Meric-Bernstam F, Hung MC. Advances in targeting human epi-dermal growth factor receptor-2 signaling for cancer therapy. Clin Cancer Res. 2006;12:6326-6330. 184. Pao W, Hutchinson KE. Chipping away at the lung cancer genome. Nat Med. 2012;18:349-351.Brunicardi_Ch10_p0305-p0354.indd 35422/02/19 2:14 PM patients awaiting a transplant and the limited number of organs available is one of the field’s biggest challenges (Fig. 11-1). In 2017 alone, according to the United Network for Organ Shar-ing (UNOS), about 115,000 patients in the United States were awaiting a transplant, yet the number of transplants performed approached only about 35,000 (Fig. 11-2).DEFINITIONSIn addition to being the overall name of this relatively new field of medicine, transplantation is the process of transferring an organ, tissue, or cell from one place to another. An organ transplant is a

1	to being the overall name of this relatively new field of medicine, transplantation is the process of transferring an organ, tissue, or cell from one place to another. An organ transplant is a surgical procedure in which a failing organ is replaced by a functioning one. The organ is transplanted either orthotopically (implanted in the same anatomic location in the recipient as it was in the donor) or heterotopically (implanted in TransplantationDavid L. Dunn, Angelika C. Gruessner, and Rainer W.G. Gruessner 11chapterBackground 355Definitions 355History 356Transplant Immunobiology 357Transplant Antigens 358Allorecognition and Lymphocyte Activation 358Clinical Rejection 358Hyperacute / 358Acute / 358Chronic / 358Clinical Immunosuppression 358Induction 359Depleting Antibodies / 359Nondepleting Antibodies / 359Maintenance 359Corticosteroids / 359Azathioprine / 360Mycophenolate Mofetil / 361Sirolimus / 361Cyclosporine / 362Tacrolimus / 362Belatacept / 362Humoral Rejection 362Rituximab /

1	Antibodies / 359Maintenance 359Corticosteroids / 359Azathioprine / 360Mycophenolate Mofetil / 361Sirolimus / 361Cyclosporine / 362Tacrolimus / 362Belatacept / 362Humoral Rejection 362Rituximab / 362Bortezomib / 363Eculizumab / 363Infections and Malignancies 363Infections / 363Malignancies / 364Organ Procurement and Preservation 364Deceased Donors / 364Living Donors / 366Organ Preservation / 366Kidney Transplantation 368Introduction / 368Pretransplant Evaluation / 368Medical Evaluation / 369Surgical Evaluation / 370Recipient Operation / 370Grafts With Multiple Renal Arteries / 372En Bloc Grafts / 372Perioperative Care / 373Results / 373Pancreas Transplantation 374Donor Operation / 374Back Table Preparation of the Pancreas Graft / 375Recipient Operation / 375Complications / 377Living Donor Pancreas Transplants / 377Results / 378Islet versus Pancreas Transplants / 378Islet Transplantation 378Liver Transplantation 379History / 379Indications / 380Recipient Selection /

1	/ 377Living Donor Pancreas Transplants / 377Results / 378Islet versus Pancreas Transplants / 378Islet Transplantation 378Liver Transplantation 379History / 379Indications / 380Recipient Selection / 381Contraindications / 382Surgical Procedure / 382Pediatric Transplants / 383Deceased Donor Split-Liver Transplants / 383Living Donor Transplants / 383Postoperative Care / 383Evaluation of Graft Function / 384Complications / 385Intestine and Multivisceral Transplantation 385Indications and Recipient Selection / 386Surgical Procedure / 386Postoperative Care / 388Heart and Lung Transplantation 388History / 388Heart Transplants / 389Lung Transplants /390Heart-Lung Transplants / 391Xenotransplants 391BACKGROUNDOrgan transplantation is a relatively novel field of medicine that has made significant progress since the second half of the 20th century. Advances in surgical technique and a better under-standing of immunology are the two main reasons that transplants have evolved from experimental

1	progress since the second half of the 20th century. Advances in surgical technique and a better under-standing of immunology are the two main reasons that transplants have evolved from experimental procedures, just several decades ago, to a widely accepted treatment today for patients with end-stage organ failure. Throughout the world, for a variety of indications, kidney, liver, pancreas, intestine, heart, and lung transplants are now the current standard of care.But the success of transplantation has created new chal-lenges. A better understanding of the pathophysiology of end-stage organ failure as well as advances in critical care medicine and in the treatment of various diseases led to expanding the cri-teria for, and decreasing the contraindications to, transplants. As a result, the discrepancy between the ever-growing number of 1Brunicardi_Ch11_p0355-p0396.indd 35501/03/19 6:53 PM 356Figure 11-1. Patients on the waiting list and the number of organ transplants performed,

1	discrepancy between the ever-growing number of 1Brunicardi_Ch11_p0355-p0396.indd 35501/03/19 6:53 PM 356Figure 11-1. Patients on the waiting list and the number of organ transplants performed, 2000 to 2009. (U.S. data from the Scientific Registry of Transplant Recipients Annual Report, http://srtr.org)Key Points1 The field of transplantation has made tremendous advances in the last 50 years, mainly due to refinements in surgical technique and development of effective immunosuppressive medications.2 Although immunosuppressive medications are essential for transplantation, they are associated with significant shortand long-term morbidity.3 Opportunistic infections can be significantly lowered by the use of appropriate antimicrobial agents.4 Kidney transplantation represents the treatment of choice for almost all patients with end-stage renal disease. The gap between demand (patients on the waiting list) and supply (available kidneys) continues to widen.5 Pancreas transplantation

1	of choice for almost all patients with end-stage renal disease. The gap between demand (patients on the waiting list) and supply (available kidneys) continues to widen.5 Pancreas transplantation represents the most reliable way to achieve euglycemia in patients with poorly controlled diabetes.6 The results of islet transplantation continue to improve but still trail those of pancreas transplantation.7 Liver transplantation has become the standard of care for many patients with end-stage liver failure and/or liver cancer.another anatomic location). Orthotopic transplants require the removal of the diseased organ (heart, lungs, liver, or intestine); in heterotopic transplants, the diseased organ is kept in place (kidney, pancreas).According to the degree of immunologic similarity between the donor and recipient, transplants are divided into three main categories: (a) an autotransplant is the transfer of cells, tissue, or an organ from one part of the body to another part in the same

1	the donor and recipient, transplants are divided into three main categories: (a) an autotransplant is the transfer of cells, tissue, or an organ from one part of the body to another part in the same person, so no immunosuppression is required; this type of transplant includes skin, artery or vein, bone, carti-lage, nerve, and islet cell transplants; (b) an allotransplant is the transfer of cells, tissue, or an organ from one person to another of the same species; with the exception of identical twins, the immune system of the recipient recognizes the donated organ as a foreign body, so immunosuppression is required in order to avoid rejection; and (c) a xenotransplant is the transfer of cells, tissue, or an organ from one organism to another of a different species. To date, animal-to-human transplants are still experi-mental procedures, given the very complex immunologic and infectious issues that have yet to be solved.HISTORYOver the centuries, many different references to

1	transplants are still experi-mental procedures, given the very complex immunologic and infectious issues that have yet to be solved.HISTORYOver the centuries, many different references to transplantation can be found in the world’s literature, yet transplantation as a recognized scientific and medical field began to emerge only in 2000200120022003200420052006200720082009010,00020,00030,00040,00050,00060,00070,00080,00090,000100,000110,000Number of patients# Waiting# Transplantedthe middle of the 20th century. Two major events led to the rise of transplantation.First, the surgical technique of the vascular anastomosis was developed by the French surgeon Alexis Carrel.1 This led to increased transplant activity, especially in animal models. Rus-sian surgeon Yu Yu Voronoy was the first to report a series of human-to-human kidney transplants in the 1940s.2 But the out-comes were dismal, mainly because of the lack of understanding of the underlying immunologic processes.Second, the

1	to report a series of human-to-human kidney transplants in the 1940s.2 But the out-comes were dismal, mainly because of the lack of understanding of the underlying immunologic processes.Second, the findings of British scientist Sir Peter B. Medawar in the 1940s were also key.3 In his work with skin grafts in animal models and in human burn patients, he learned that the immune system plays a crucial role in the failure of skin grafts. His research led to a better understanding of the immune system and is considered to be the birth of transplant immunobiology.The first human transplant with long-term success was performed by Joseph Murray in Boston, Massachusetts, in 1954.4 Because it was a living related kidney transplant between identical twins, no immunosuppression was required; the recipi-ent lived for another 8 years before he died of issues unrelated to the transplanted kidney. Other centers performed similar trans-plants and could reproduce similar good results.Ultimately,

1	recipi-ent lived for another 8 years before he died of issues unrelated to the transplanted kidney. Other centers performed similar trans-plants and could reproduce similar good results.Ultimately, attempts were made to perform kidney trans-plants between nonidentical individuals. For immunosup-pression, total-body radiation and an anticancer agent called Brunicardi_Ch11_p0355-p0396.indd 35601/03/19 6:53 PM 357TRANSPLANTATIONCHAPTER 11Figure 11-2. Patients on the waiting list and the number of organ transplants performed, 2009. KP = kidney and pancreas. (U.S. data from the Scientific Registry of Transplant Recipients Annual Report, http://srtr.org)TotalKidneyPancreas and KPLiverIntestineHeartLungsHeart/Lung010,00020,00030,00040,00050,00060,00070,00080,00090,000100,000110,000Number of patients# Waiting# Transplanted6-mercaptopurine were used, but given the profound toxicity of both those methods of immunosuppression, results were dis-couraging. A breakthrough was achieved in the

1	patients# Waiting# Transplanted6-mercaptopurine were used, but given the profound toxicity of both those methods of immunosuppression, results were dis-couraging. A breakthrough was achieved in the early 1960s with the introduction of maintenance immunosuppression through a combination of corticosteroids and a less toxic derivative of 6-mercaptopurine, azathioprine.5,6Increasing experience with kidney transplants and the better results achieved with maintenance immunosuppression paved the way for the era of nonrenal transplants (Table 11-1). In 1963, the first liver transplant was performed by Thomas Starzl in Denver, Colorado, and the first lung transplant was performed by James Hardy in Jackson, Mississippi. In 1966, the first pancreas transplant was performed by William Kelly and Richard Lillehei in Minneapolis, Minnesota. In 1967, the first successful heart transplant was performed by Christiaan Barnard in Cape Town, South Africa. The early years of trans-plantation were marked by

1	Lillehei in Minneapolis, Minnesota. In 1967, the first successful heart transplant was performed by Christiaan Barnard in Cape Town, South Africa. The early years of trans-plantation were marked by high mortality, mainly because of irreversible rejection. However, dramatic advances occurred with the further development of new forms of immunosup-pression. The groundbreaking event was the introduction of the first anti-T lymphocyte (T cell) drug, cyclosporine, in the early 1980s.7 Since then, with an even better understanding of immunologic processes, many other drugs have been introduced that target specific pathways that lead to rejection. As a result, rejection rates have decreased substantially, allowing a 1-year graft survival rate in excess of 80% in all types of transplants.Table 11-1Transplant historyORGANYEARSURGEONLOCATIONKidney1954Joseph E. MurrayBoston, MALiver1963Thomas E. StarzlDenver, COLung1963James D. HardyJackson, MSPancreas1966Richard C. LilleheiMinneapolis,

1	11-1Transplant historyORGANYEARSURGEONLOCATIONKidney1954Joseph E. MurrayBoston, MALiver1963Thomas E. StarzlDenver, COLung1963James D. HardyJackson, MSPancreas1966Richard C. LilleheiMinneapolis, MNHeart1967Christiaan N. BarnardCape Town, South AfricaSmall intestine1967Richard C. LilleheiMinneapolis, MNHeart/lung1981Bruce ReitzStanford, CAMultivisceral1989Thomas E. StarzlPittsburgh, PAThe gradual increase in the organ shortage led to inno-vative surgical techniques. For example, deceased donor split-liver transplants and living donor liver transplants have helped expand the liver donor pool. Similarly, living donor intestine and pancreas techniques have been developed. The evolution of donor nephrectomy from an open to a minimally invasive procedure (laparoscopic or robotic) has helped increase the pool of living kidney donors.TRANSPLANT IMMUNOBIOLOGYThe outcomes of early transplants were less than satisfactory. The limiting factor was the lack of understanding of immuno-logic

1	increase the pool of living kidney donors.TRANSPLANT IMMUNOBIOLOGYThe outcomes of early transplants were less than satisfactory. The limiting factor was the lack of understanding of immuno-logic processes, and irreversible rejection was the reason for graft loss in the vast majority of recipients. A better understand-ing of transplant immunobiology led to significant improve-ments in patient and graft survival rates.8,9 The immune system is designed as a defense system to protect the body from foreign pathogens, such as viruses, bacteria, and fungi, but it also acts to reject transplanted cells, tissues, and organs, recognizing them as foreign. It mediates other complex processes as well, such as the body’s response to trauma or to tumor growth. No matter what type of agent, the immune system recognizes it as foreign and triggers a strong response that is designed to either to eradi-cate pathogenic organisms or reject foreign cells or tissue.Brunicardi_Ch11_p0355-p0396.indd

1	system recognizes it as foreign and triggers a strong response that is designed to either to eradi-cate pathogenic organisms or reject foreign cells or tissue.Brunicardi_Ch11_p0355-p0396.indd 35701/03/19 6:53 PM 358BASIC CONSIDERATIONSPART ITRANSPLANT ANTIGENSTransplants between genetically nonidentical persons lead to recognition and rejection of the organ by the recipient’s immune system, if no intervention is undertaken. The main antigens responsible for this process are part of the major histocompat-ibility complex (MHC). In humans, these antigens make up the human leukocyte antigen (HLA) system. The antigen-encoding genes are located on chromosome 6. Two major classes of HLA antigens are recognized. They differ in their structure, function, and tissue distribution. Class I antigens (HLA-A, HLA-B, and HLA-C) are expressed by all nucleated cells. Class II antigens (HLA-DR, HLA-DP, and HLA-DQ) are expressed by antigen-presenting cells (APCs) such as B lymphocytes, dendritic

1	(HLA-A, HLA-B, and HLA-C) are expressed by all nucleated cells. Class II antigens (HLA-DR, HLA-DP, and HLA-DQ) are expressed by antigen-presenting cells (APCs) such as B lymphocytes, dendritic cells, macrophages, and other phagocytic cells.The principal function of HLA antigens is to present the fragments of foreign proteins to T lymphocytes. This leads to recognition and elimination of the foreign antigen with great specificity. HLA molecules play a crucial role in transplant recipients as well. They can trigger rejection of a graft via two different mechanisms. The most common mechanism is cellular rejection, in which the damage is caused by acti-vated T lymphocytes. The process of activation and prolifera-tion is triggered by exposure of T lymphocytes to the donor’s HLA molecules. The other mechanism is humoral rejection, in which the damage is mediated by circulating antibodies against the donor’s HLA molecules. The donor-specific anti-bodies can be present either pretransplant,

1	other mechanism is humoral rejection, in which the damage is mediated by circulating antibodies against the donor’s HLA molecules. The donor-specific anti-bodies can be present either pretransplant, due to previous exposure (because of a previous transplant, pregnancy, blood transfusion, or immunization), or posttransplant. After anti-body binding to the donor’s HLA molecules, the complement cascade is activated, leading to cellular lysis.ALLORECOGNITION AND LYMPHOCYTE ACTIVATIONThe immune system of each person is designed to discriminate between self and nonself cells and tissues. This process is called allorecognition, with T cells playing the crucial role. The recog-nition of foreign HLA antigens by the recipient’s T cells may occur by either a direct or an indirect pathway. Direct recogni-tion occurs when the recipient’s T cells are activated by direct interaction with the donor’s HLA molecules. Indirect recogni-tion occurs when the recipient’s T cells are activated by

1	Direct recogni-tion occurs when the recipient’s T cells are activated by direct interaction with the donor’s HLA molecules. Indirect recogni-tion occurs when the recipient’s T cells are activated by interac-tion with APCs that have processed and presented the foreign antigen. The foreign antigen can be shed from the graft into the circulation, or it can be identified by the APCs within the graft itself.Independent of the pathway of foreign HLA antigen presentation, the ensuing activation of T cells is similar and involves complex cell surface receptors and markers, i.e., the T-cell receptor (TCR) and an array of cluster differentiation markers (CDs). A two-signal model, T-cell activation begins with the engagement of the TCR/CD3 complex with the foreign molecule. This interaction causes transmission of the signal into the cell, namely signal 1. However, this signal alone is not suf-ficient to activate the T cell. An additional costimulatory signal is required, i.e., signal 2. Two

1	transmission of the signal into the cell, namely signal 1. However, this signal alone is not suf-ficient to activate the T cell. An additional costimulatory signal is required, i.e., signal 2. Two well-characterized costimula-tory interactions are the CD40/CD154 and B7/CD28 pathways. The “master switch” is turned on by the interaction of CD40 protein with APCs, along with the interaction of CD154 pro-tein with T cells; this ligation induces the upregulation of other costimulatory molecules. Transmission of signal 1 and signal 2 into the cell nucleus leads to upregulation of the transcrip-tion of genes for several cytokines, including the T-cell growth factor interleukin-2 (IL-2). In turn, IL-2 activates a number of pathways, leading to proliferation and differentiation of T cells. Rejection is the result of an attack of activated T cells on the transplanted organ.Although T-cell activation is the main culprit in rejection, B-cell activation and subsequent antibody production also play

1	is the result of an attack of activated T cells on the transplanted organ.Although T-cell activation is the main culprit in rejection, B-cell activation and subsequent antibody production also play a role. After the foreign HLA antigen is processed by B cells, it interacts with activated helper T cells, leading to differentiation of B cells into plasma cells and subsequently to their prolifera-tion and antibody production.CLINICAL REJECTIONGraft rejection is due to a complex interaction of different com-ponents of the immune system, including B and T lymphocytes, APCs, and cytokines. The end result is graft damage caused by inflammatory injury. According to its onset and pathogenesis, rejection is divided into three main types: hyperacute, acute, and chronic, and each is described in the following sections.HyperacuteHyperacute rejection, a very rapid type of rejection, results in irreversible damage and graft loss within minutes to hours after organ reperfusion. It is triggered by

1	following sections.HyperacuteHyperacute rejection, a very rapid type of rejection, results in irreversible damage and graft loss within minutes to hours after organ reperfusion. It is triggered by preformed antibodies against the donor’s HLA or ABO blood group antigens. These antibodies activate a series of events that result in diffuse intra-vascular coagulation, causing ischemic necrosis of the graft. Fortunately, pretransplant blood group typing and cross-matching (in which the donor’s cells are mixed with the recipient’s serum, and then the cells are observed for any destruction) have virtu-ally eliminated the incidence of hyperacute rejection.AcuteAcute rejection, the most common type of rejection, usually occurs within a few days or weeks posttransplant. According to the mechanism involved, it is further divided into cellular (T-cell–mediated) rejection, humoral (antibody-mediated) rejection, or a combination of both. The diagnosis is based on the results of biopsies of the

1	involved, it is further divided into cellular (T-cell–mediated) rejection, humoral (antibody-mediated) rejection, or a combination of both. The diagnosis is based on the results of biopsies of the transplanted organ, special immu-nologic stains, and laboratory tests (such as elevated creatinine levels in kidney transplant recipients, elevated liver test values in liver transplant recipients, and elevated levels of glucose, amylase, and lipase in pancreas transplant recipients).ChronicChronic rejection occurs slowly and usually is progressive. It can manifest within the first year posttransplant but most often takes place gradually over several years. The mechanisms are not well understood, but the pathologic changes eventually lead to tissue fibrosis and loss of graft function. As advances in immunosuppression have diminished the incidence of acute rejection, this form of rejection is becoming more common.CLINICAL IMMUNOSUPPRESSIONA successful transplant hinges upon a balance between

1	in immunosuppression have diminished the incidence of acute rejection, this form of rejection is becoming more common.CLINICAL IMMUNOSUPPRESSIONA successful transplant hinges upon a balance between the extent of the recipient’s immune response, the health and viabil-ity of the donor allograft, and pharmacologic immunosuppres-sion. Immunosuppressive regimens are critical to graft and Brunicardi_Ch11_p0355-p0396.indd 35801/03/19 6:53 PM 359TRANSPLANTATIONCHAPTER 11patient survival posttransplant. Immunosuppression has evolved from the use of azathioprine and steroids in the 1960s and 1970s to the development in the 1980s of cyclosporine, the latter which markedly increased allograft survival.10,11 The intro-duction of tacrolimus and mycophenolate mofetil (MMF) in the 1990s further advanced the field of transplantation, enabling a variety of combinations to be used for immunosuppression often “tailored” for each recipient (Table 11-2).Presently, immunosuppressants are used in

1	advanced the field of transplantation, enabling a variety of combinations to be used for immunosuppression often “tailored” for each recipient (Table 11-2).Presently, immunosuppressants are used in multidrug regimens aimed at increasing efficacy by targeting multiple pathways to lower the immune response and to decrease the toxicity of individual agents. Certain regimens may involve withdrawal, avoidance, or minimization of certain classes of drugs. Transplant centers generally institute their immunosup-pressive protocols based on experience, risk profiles, cost con-siderations, and outcomes. Immunosuppression is delivered in two phases: induction (starting immediately posttransplant, when the risk of rejection is highest) and maintenance (usu-ally starting within days posttransplant and usually continuing for the life of the graft and the recipient). Thus, the degree of immunosuppression is highest in the first 3 to 6 months posttransplant; during this time, prophylaxis against a

1	usually continuing for the life of the graft and the recipient). Thus, the degree of immunosuppression is highest in the first 3 to 6 months posttransplant; during this time, prophylaxis against a number of different bacterial, viral, or even antifungal opportunistic pathogens is administered.12,13A conventional immunosuppressive protocol might include (a) induction with anti-T-lymphocyte–depleting or nondepleting antibodies and (b) maintenance with calcineurin inhibitors, antiproliferative agents, and corticosteroids. Char-acteristics of the most common immunosuppressive agents are listed in Table 11-3.2Table 11-2Immunosuppressive drugs by groupingImmunophilin binders Calcineurin inhibitors Cyclosporine Tacrolimus Noninhibitors of calcineurin SirolimusAntimetabolites Inhibitors of de novo purine synthesis Azathioprine Mycophenolate mofetilBiologic immunosuppression Polyclonal antibodies Atgam Antithymocyte immunoglobulin Monoclonal

1	of de novo purine synthesis Azathioprine Mycophenolate mofetilBiologic immunosuppression Polyclonal antibodies Atgam Antithymocyte immunoglobulin Monoclonal antibodies Muromonab-CD3 Basiliximab Belatacept Alemtuzumab Rituximab Bortezomib EculizumabOther CorticosteroidsINDUCTIONInduction includes the use of depleting (polyclonal) antibodies or nondepleting antibodies within the first month posttransplant. Studies have shown that induction with antibody regimens may prevent acute rejection, potentially leading to improved graft survival and the use of less maintenance immunosuppression.Depleting AntibodiesRabbit antithymocyte globulin (Thymoglobulin) is a purified gamma globulin obtained by immunizing rabbits with human thymocytes. Atgam, which has largely been replaced by Thy-moglobulin, is a purified gamma globulin obtained by immu-nizing horses with human thymocytes. These agents contain antibodies to T cells and B lymphocytes (B cells), integrins, and other adhesion

1	is a purified gamma globulin obtained by immu-nizing horses with human thymocytes. These agents contain antibodies to T cells and B lymphocytes (B cells), integrins, and other adhesion molecules, thereby resulting in rapid depletion of peripheral lymphocytes. Typically, the total dose of Thymo-globulin is roughly 6 mg/kg, a dose that has been shown to con-fer adequate lymphocyte depletion and better allograft survival. Doses of 3 mg/kg may not effectively prevent acute rejection, but more doses and prolonged administration increase the risk of infection and the potential occurrence of lymphoma. Thymo-globulin administration causes a cytokine release syndrome, so premedications (acetaminophen and diphenhydramine) are usu-ally given. The principal side effects of Thymoglobulin include fever, chills, arthralgias, thrombocytopenia, leukopenia, and an increased incidence of a variety of infections.14,15Nondepleting AntibodiesBasiliximab (Simulect) is an anti-CD25 monoclonal antibody. The

1	chills, arthralgias, thrombocytopenia, leukopenia, and an increased incidence of a variety of infections.14,15Nondepleting AntibodiesBasiliximab (Simulect) is an anti-CD25 monoclonal antibody. The alpha subunit of the IL-2 receptor, also known as Tac or CD25, is found exclusively on activated T cells. Blockade of this component by this monoclonal antibody selectively pre-vents IL-2–induced T-cell activation. No lymphocyte depletion occurs with basiliximab; thus, it is not designed to be used to treat acute rejection. Its selectivity in blocking IL-2–mediated responses makes it a powerful induction agent without the added risks of infections, malignancies, or other major side effects. Currently, basiliximab is the only available anti-CD25 mono-clonal antibody approved for clinical use. Usually, it is followed by the use of calcineurin inhibitors, corticosteroids, and MMF as maintenance immunosuppression.16Alemtuzumab (Campath, Lemtrada), another anti-CD52 monoclonal antibody, was

1	Usually, it is followed by the use of calcineurin inhibitors, corticosteroids, and MMF as maintenance immunosuppression.16Alemtuzumab (Campath, Lemtrada), another anti-CD52 monoclonal antibody, was initially used to treat chronic lym-phocytic leukemia. The use of alemtuzumab has grown in the field of transplantation, given its profound lymphocyte-depleting effects. It causes cell death by complement-mediated cytoly-sis, antibody-mediated cytotoxicity, and apoptosis. One dose alone (30 mg) depletes 99% of lymphocytes. Monocyte recov-ery can be seen at 3 months posttransplant; B-cell recovery at 12 months; and T-cell recovery, albeit only to 50% of base-line, at 36 months. Alemtuzumab causes a significant cytokine release reaction and often requires premedications (steroids and antihistamines). Because of the long-lasting T-cell depletion, the risks of infection and posttransplant lymphoproliferative dis-order remain. Initially, alemtuzumab was available only through a limited

1	Because of the long-lasting T-cell depletion, the risks of infection and posttransplant lymphoproliferative dis-order remain. Initially, alemtuzumab was available only through a limited distribution program, but more recently has been stud-ied in a number of clinical trials.17,18MAINTENANCECorticosteroidsCorticosteroids have had a role in immunosuppression since the beginning of the field of transplantation. Despite numerous Brunicardi_Ch11_p0355-p0396.indd 35901/03/19 6:53 PM 360BASIC CONSIDERATIONSPART ITable 11-3Summary of the main immunosuppressive drugsDRUGMECHANISM OF ACTIONADVERSE EFFECTSCLINICAL USESDOSAGECyclosporine (CSA)Binds to cyclophilinInhibits calcineurin and IL-2 synthesisNephrotoxicityTremorHypertensionHirsutismImproved bioavailability of microemulsion formOral dose 5 mg/kg per day (given in two divided doses)Tacrolimus (FK506)Binds to FKBPInhibits calcineurin and IL-2 synthesisNephrotoxicityHypertensionNeurotoxicityGI toxicity (nausea, diarrhea)Improved patient

1	5 mg/kg per day (given in two divided doses)Tacrolimus (FK506)Binds to FKBPInhibits calcineurin and IL-2 synthesisNephrotoxicityHypertensionNeurotoxicityGI toxicity (nausea, diarrhea)Improved patient and graft survival in (liver) primary immunosuppression and rescue therapyUsed as mainstay of maintenance protocolsIV 0.015 mg/kg per day as continuous infusionPO 0.05 mg/kg per day (given every 12 h)Mycophenolate mofetilAntimetaboliteInhibits enzyme necessary for de novo purine synthesisLeukopeniaGI toxicityEffective for primary immunosuppression in combination with tacrolimus1 g bid POSirolimusInhibits lymphocyte effects driven by IL-2 receptorThrombocytopeniaIncreased serum cholesterol/LDLPoor wound healingMay allow early withdrawal of steroids and decreased calcineurin doses2–4 mg/d, adjusted to trough drug levelsCorticosteroidsMultiple actionsAnti-inflammatoryInhibits lymphokine productionCushingoid stateGlucose intoleranceOsteoporosisUsed in induction, maintenance, and treatment of

1	to trough drug levelsCorticosteroidsMultiple actionsAnti-inflammatoryInhibits lymphokine productionCushingoid stateGlucose intoleranceOsteoporosisUsed in induction, maintenance, and treatment of acute rejectionVaries from milligrams to several grams per dayMaintenance doses, 5–10 mg/dAzathioprineAntimetaboliteInterferes with DNA and RNA synthesisThrombocytopeniaNeutropeniaLiver dysfunctionUsed in maintenance protocols or if intolerance to mycophenolate mofetil1–3 mg/kg per day for maintenanceBelataceptT-cell blockerIncreased risk of bacterial infectionsNew drug for maintenance immunosuppression in renal transplants only5–10 mg/kg per day infusionFKBP = FK506-binding protein; GI = gastrointestinal; IL = interleukin; IV = intravenous; LDL = low-density lipoprotein; PO = oralattempts to limit or discontinue their use, they remain an inte-gral component of most immunosuppressive protocols, for both induction and maintenance. Moreover, they are often the first-line agents in the treatment

1	or discontinue their use, they remain an inte-gral component of most immunosuppressive protocols, for both induction and maintenance. Moreover, they are often the first-line agents in the treatment of acute rejection. Steroids bind to glucocorticoid-responsive elements in DNA that prevent the transcription of cytokine genes and cytokine receptors. In addition, steroids have an impact on lymphocyte depletion, on decreases in cell-mediated immunity, and on T-cell activation of many phases of rejection.Nonetheless, the numerous adverse effects of steroid therapy contribute significantly to morbidity in transplant recipients.19 Common side effects include acne, increased appetite and asso-ciated weight gain, mood changes, diabetes, hypertension, and impaired wound healing.One of the most common maintenance immunosuppres-sive regimens consists of triple-drug therapy: prednisone, a cal-cineurin inhibitor, and an antimetabolite. Large doses of steroids are usually given perioperatively and

1	maintenance immunosuppres-sive regimens consists of triple-drug therapy: prednisone, a cal-cineurin inhibitor, and an antimetabolite. Large doses of steroids are usually given perioperatively and in the immediate postop-erative period. Protocols vary by center, but the steroid dose is usually tapered to an adult dose of roughly 5 to 15 mg daily, or completely stopped at some point. Steroids are substrates for CYP3A4, CYP3A5, and P-glycoprotein pathways where drug interactions might need to be monitored.20,21AzathioprineAn antimetabolite, azathioprine (AZA) is converted to 6-mercaptopurine and inhibits both the de novo purine synthe-sis and salvage purine synthesis. AZA decreases T-lymphocyte activity and decreases antibody production. It has been used as a first-line agent in transplant recipients for more than 40 years, but it became an adjunctive agent after the introduction of cyclospo-rine. With the development of newer agents such as MMF, the use of AZA has decreased

1	transplant recipients for more than 40 years, but it became an adjunctive agent after the introduction of cyclospo-rine. With the development of newer agents such as MMF, the use of AZA has decreased significantly. However, it is preferred in recipients who are considering conceiving a child because MMF is teratogenic and can cause birth defects. Use of AZA remains an option for recipients who cannot tolerate the gastro-intestinal (GI) side effects of MMF.The most significant side effect of AZA, often dose-related, is bone marrow suppression. Leukopenia is often reversible with dose reduction or temporary cessation of the drug. Other significant side effects include hepatotoxicity, pancreatitis, neoplasia, anemia, and pulmonary fibrosis. Its most significant drug interaction is with allopurinol, which blocks AZA metabolism, increasing the risk of pancytope-nia. Recommendations are to not use AZA and allopurinol together, or if doing so is unavoidable, to decrease the dose of AZA by

1	which blocks AZA metabolism, increasing the risk of pancytope-nia. Recommendations are to not use AZA and allopurinol together, or if doing so is unavoidable, to decrease the dose of AZA by 75%.22Brunicardi_Ch11_p0355-p0396.indd 36001/03/19 6:53 PM 361TRANSPLANTATIONCHAPTER 11Mycophenolate MofetilApproved in May 1995 by the U.S. Food and Drug Admin-istration (FDA) for preventing acute rejection after kidney transplants, MMF has now been incorporated into routine maintenance regimens after many solid organ transplants. Mycophenolate is the prodrug of mycophenolate acid, derived from Penicillium fungi. Mycophenolate acid is an inhibitor of inosine monophosphate dehydrogenase (IMPDH) involved in the de novo pathway of purine synthesis.23 MMF is available in capsules (250 and 500 mg); the starting dose is 1 g twice daily. In hopes of decreasing the GI side effects, an enteric-coated formulation called Myfortic was developed; its benefits have not been clearly demonstrated in studies,

1	starting dose is 1 g twice daily. In hopes of decreasing the GI side effects, an enteric-coated formulation called Myfortic was developed; its benefits have not been clearly demonstrated in studies, but in some conversion studies patients did report less GI intolerance. The pharmacoki-netics of MMF are complex; mycophenolic acid (MPA) levels are not routinely performed at most transplant centers. Studies have shown that MPA levels and the incidence of rejection are not significantly correlated.24 The most common side effects of MMF are GI in nature, most commonly diarrhea, nausea, dys-pepsia, and bloating. Esophagitis and gastritis occur in roughly 5% of recipients and may represent a cytomegalovirus (CMV) Table 11-4Side effects and drug interactions of the main immunosuppressive drugs COMMON SIDE EFFECTSOTHER MEDICATIONS THAT INCREASE BLOOD LEVELSOTHER MEDICATIONS THAT DECREASE BLOOD LEVELSOTHER MEDICATIONS THAT POTENTIATE TOXICITYCyclosporine (CSA)Hypertension, nephrotoxicity,

1	SIDE EFFECTSOTHER MEDICATIONS THAT INCREASE BLOOD LEVELSOTHER MEDICATIONS THAT DECREASE BLOOD LEVELSOTHER MEDICATIONS THAT POTENTIATE TOXICITYCyclosporine (CSA)Hypertension, nephrotoxicity, hirsutism, neurotoxicity, gingival hyperplasia, hypomagnesemia, hyperkalemiaVerapamil, diltiazem, clarithromycin, azithromycin, erythromycin, azole antifungals, protease inhibitors, grapefruit juiceIsoniazid, carbamazepine, phenobarbital, phenytoin, rifampin, St. John’s wortNephrotoxicity: ganciclovir, aminoglycosides, NSAIDs, ACE-Is, and ARBsTacrolimus (FK506)Hypertension, nephrotoxicity, alopecia, hyperglycemia, neurotoxicity, hypomagnesemia, hyperkalemiaVerapamil, diltiazem, clarithromycin, azithromycin, erythromycin, azole antifungals, protease inhibitors, grapefruit juiceIsoniazid, carbamazepine, phenobarbital, phenytoin, rifampin, St. John’s wortNephrotoxicity: ganciclovir, aminoglycosides, NSAIDs, ACE-Is, and ARBsSirolimusThrombocytopenia and neutropenia, elevated cholesterol, extremity

1	phenobarbital, phenytoin, rifampin, St. John’s wortNephrotoxicity: ganciclovir, aminoglycosides, NSAIDs, ACE-Is, and ARBsSirolimusThrombocytopenia and neutropenia, elevated cholesterol, extremity edema, impaired wound healingVerapamil, diltiazem, clarithromycin, azithromycin, erythromycin, azole antifungals, protease inhibitors, grapefruit juiceIsoniazid, carbamazepine, phenobarbital, phenytoin, rifampin, St. John’s wort—Mycophenolate mofetilLeukopenia, thrombocytopenia, GI upset—Cholestyramine, antacidsBone marrow suppression: valganciclovir, ganciclovir, TMP-SMXCorticosteroidsHyperglycemia, osteoporosis, cataracts, myopathy, weight gain———AzathioprineLeukopenia, anemia, thrombocytopenia, neoplasia, hepatitis, cholestasis——Bone marrow suppression: allopurinol, sulfonamidesACE-I = angiotensin-converting enzyme inhibitor; ARB = angiotensin receptor blocker; NSAID = nonsteroidal anti-inflammatory drug; TMP-SMX = trimethoprim-sulfamethoxazoleor herpesvirus family infection. The other

1	enzyme inhibitor; ARB = angiotensin receptor blocker; NSAID = nonsteroidal anti-inflammatory drug; TMP-SMX = trimethoprim-sulfamethoxazoleor herpesvirus family infection. The other important side effects are leukopenia, anemia, and thrombocytopenia (Table 11-4). Leukopenia can sometimes be reversed by lowering the MMF dose and discontinuing other agents like valganciclovir. MMF does not have any significant drug interactions, but clinicians should be careful to avoid additive toxicities with other medica-tions that might lead to leukopenia and thrombocytopenia.SirolimusThe first mammalian target of rapamycin (mTOR) inhibitors to enter clinical use was sirolimus (Rapamune). A key regulatory kinase, mTOR changes cells from the G1 to S phase in the cell cycle, in response to proliferation signals provided by cytokines like IL-2. The mTOR inhibitors bind to FK506-binding pro-tein (FKBP), and the sirolimus-FKBP complex binds to mTOR. Sirolimus also inhibits proliferation of vascular smooth

1	provided by cytokines like IL-2. The mTOR inhibitors bind to FK506-binding pro-tein (FKBP), and the sirolimus-FKBP complex binds to mTOR. Sirolimus also inhibits proliferation of vascular smooth muscle cells, possibly easing the vasculopathy and progressive fibrosis that can affect allografts. Sirolimus is a substrate for CYP3A4/4 and has many significant drug interactions (see Table 11-4).To date, sirolimus has been used in a variety of com-binations for maintenance immunosuppression, alone or in Brunicardi_Ch11_p0355-p0396.indd 36101/03/19 6:53 PM 362BASIC CONSIDERATIONSPART ITable 11-5Drug interactions and side effects associated with calcineurin inhibitorsINTERACTIONSMEDICATIONSInhibition of metabolismClarithromycin, erythromycin, azole antifungals, diltiazem, verapamil, nicardipine, amiodarone, grapefruit juice, ritonavir, azithromycinInduction of metabolismNevirapine, rifampin, St. John’s wort, carbamazepine, phenobarbital, phenytoin, caspofunginHyperkalemiaPotassium-sparing

1	amiodarone, grapefruit juice, ritonavir, azithromycinInduction of metabolismNevirapine, rifampin, St. John’s wort, carbamazepine, phenobarbital, phenytoin, caspofunginHyperkalemiaPotassium-sparing diuretics, angiotensin-converting enzyme inhibitors (ACE-Is), angiotensin receptor blockers (ARBs), β-blockers, trimethoprim-sulfamethoxazoleNephrotoxicityNonsteroidal anti-inflammatory drugs, aminoglycosides, amphotericin, ACE-Is, ARBsconjunction with one of the calcineurin inhibitors. In such com-binations, sirolimus usually is used to help withdraw from, or completely avoid, the use of steroids. It also has been used as an alternative to tacrolimus or cyclosporine, in a calcineurin-sparing protocol. One of the most significant side effects of siro-limus is hypertriglyceridemia, a condition that may be resistant to statins and fibrates. Impaired wound healing (immediately posttransplant in particular), thrombocytopenia, leukopenia, and anemia also are associated with sirolimus, and these

1	may be resistant to statins and fibrates. Impaired wound healing (immediately posttransplant in particular), thrombocytopenia, leukopenia, and anemia also are associated with sirolimus, and these problems are exacerbated when it is used in combination with MMF.25,26CyclosporineThe introduction of cyclosporine in the early 1980s dramati-cally altered the field of transplantation by significantly improv-ing outcomes after kidney transplantation. Cyclosporine binds with its cytoplasmic receptor protein, cyclophilin, which sub-sequently inhibits the activity of calcineurin, thereby decreas-ing the expression of several critical T-cell activation genes, the most important being for IL-2. As a result, T-cell activation is suppressed.27Many formulations of cyclosporine exist, so it is important to know which one the transplant recipient is taking. Sandim-mune, an older, oil-based formulation, has poor bioavailabil-ity and variable absorption. The newer formulations, Gengraf and Neoral, are

1	to know which one the transplant recipient is taking. Sandim-mune, an older, oil-based formulation, has poor bioavailabil-ity and variable absorption. The newer formulations, Gengraf and Neoral, are microemulsified with improved bioavailability. Cyclosporine can be given intravenously or orally to maintain trough levels of 250 to 350 ng/mL for the first 3 months post-transplant; then it can be tapered to 150 to 250 ng/mL.28The metabolism of cyclosporine is via the cytochrome P450 system, resulting in many significant drug interactions (see Table 11-4). Calcineurin inhibitors are nephrotoxic and constrict the afferent arteriole in a dose-dependent, reversible manner (Table 11-5). They also can cause hyperkalemia and hypomagnesemia. Several neurologic complications, including headaches, tremor, and seizures, also have been reported.29Cyclosporine has several undesirable cosmetic effects, including hirsutism and gingival hyperplasia. It is associated with a higher incidence of

1	tremor, and seizures, also have been reported.29Cyclosporine has several undesirable cosmetic effects, including hirsutism and gingival hyperplasia. It is associated with a higher incidence of hypertension and hyperlipidemia than is tacrolimus.TacrolimusThe calcineurin inhibitor tacrolimus (Prograf) is now the back-bone of most immunosuppressive regimens. Tacrolimus acts by binding FKBPs, causing roughly 10 to 100 times more potent inhibition of IL-2 production than cyclosporine (which acts by binding cyclophilins). It can be given intravenously, orally, or sublingually to maintain trough levels of 8 to 12 ng/mL for the first 3 months posttransplant; then it can be tapered to 6 to 10 ng/mL. The metabolism of tacrolimus is via the cytochrome P450 system, resulting in many significant drug interactions (see Table 11-4).Tacrolimus causes a higher incidence of new-onset dia-betes posttransplant than does cyclosporine. Other side effects include alopecia, nephrotoxicity, neurotoxicity,

1	interactions (see Table 11-4).Tacrolimus causes a higher incidence of new-onset dia-betes posttransplant than does cyclosporine. Other side effects include alopecia, nephrotoxicity, neurotoxicity, hypertension, hyperkalemia, hypomagnesemia, and an increased incidence of certain types of infection.30BelataceptThe best-characterized pathway of T-cell costimulation includes CD28; its homologue, the cytotoxic T-lymphocyte–associated protein 4 (CTLA4); and their ligands, CD80 and CD86. Belatacept (also known as LEA29Y) was developed through two amino acid substitutions to abatacept (also known as CTLA4-Ig), a fusion protein consisting of the extracellular domain of CTLA4 and the Fc domain of immunoglobulin G (IgG). It is a high-avidity molecule with slower dissociation rates.Clinical trials have compared the use of belatacept vs. a standard cyclosporine protocol in recipients of living donor, deceased donor, and extended-criteria donor kidneys. Belata-cept was not inferior to cyclosporine

1	compared the use of belatacept vs. a standard cyclosporine protocol in recipients of living donor, deceased donor, and extended-criteria donor kidneys. Belata-cept was not inferior to cyclosporine in both patient and allograft survival rates, but was associated with a higher rate of biopsy-proven acute cellular rejection.In terms of adverse effects, belatacept differs from stan-dard calcineurin-based regimens because of an increased risk of posttransplant lymphoproliferative disorder (PTLD); the greatest risk is in recipients who are Epstein-Barr virus (EBV)-seronegative pretransplant. The FDA recommends the use of belatacept only in seropositive recipients. Studies in liver trans-plant recipients were halted early because of increased mortality rates.However, belatacept does have a lower incidence of car-diovascular risk factors including metabolic lipid disorders, hypertension, neurotoxicity, glucose abnormalities, and adverse cosmetic effects. Except for the increased risk of

1	lower incidence of car-diovascular risk factors including metabolic lipid disorders, hypertension, neurotoxicity, glucose abnormalities, and adverse cosmetic effects. Except for the increased risk of malignancy, the more favorable adverse effect profile of belatacept and its convenient monthly dosing schedule may make it an attrac-tive option for maintenance of immunosuppression, possibly improving compliance.31,32HUMORAL REJECTIONRituximabA chimeric anti-CD20 (anti-B cell) monoclonal antibody, ritux-imab is currently FDA approved for treating several types of lymphoma. The CD20 antigen is expressed early in the B-cell cycle but is absent on mature plasma cells. The variable region binds to CD20 through three different mechanisms: (a) antibody-dependent cell cytotoxicity, (b) complement-dependent cell killing, and (c) induction of apoptotic cell death. The use of Brunicardi_Ch11_p0355-p0396.indd 36201/03/19 6:53 PM 363TRANSPLANTATIONCHAPTER 11rituximab has grown to include the

1	cell killing, and (c) induction of apoptotic cell death. The use of Brunicardi_Ch11_p0355-p0396.indd 36201/03/19 6:53 PM 363TRANSPLANTATIONCHAPTER 11rituximab has grown to include the treatment of antibody-mediated rejection and use in desensitization protocols. Studies so far have been small, with rituximab usually used in conjunc-tion with plasmapheresis, steroids, and intravenous immuno-globulin (IVIG).33-35BortezomibA proteasome inhibitor, bortezomib is FDA approved for treat-ing multiple myeloma. It can directly target plasma cells. Tradi-tional treatments have been successful in removing antibodies, inhibiting antibody activity, or lowering antibody production; however, targeting mature antibody production in plasma cells has not met with success. Bortezomib has been shown to cause apoptosis of normal plasma cells, thereby decreasing alloan-tibody production in sensitized patients. Several case reports and series have described the use of bortezomib for the treat-ment of

1	cause apoptosis of normal plasma cells, thereby decreasing alloan-tibody production in sensitized patients. Several case reports and series have described the use of bortezomib for the treat-ment of antibody-mediated rejection and in desensitization protocols.34,36,37EculizumabA humanized monoclonal antibody with high affinity for C5, eculizumab is a first-in-class, FDA-approved agent for treat-ing paroxysmal nocturnal hemoglobinuria, hemolytic uremic syndrome, and generalized myasthenia gravis. It blocks the activation of the terminal complement cascade. Most antibody-mediated rejection episodes are associated with early comple-ment activation as evidenced on renal transplant biopsies by the presence of C4d+ staining of the peritubular capillaries. Given its highly selective mechanism of action, this agent is predicted to be useful to treat antibody-mediated rejection and to desensi-tize patients pretransplant. However, its serious adverse effects include an increased risk of

1	of action, this agent is predicted to be useful to treat antibody-mediated rejection and to desensi-tize patients pretransplant. However, its serious adverse effects include an increased risk of infections, especially due to encap-sulated bacteria such as Neisseria meningitidis. Patients should be immunized with meningococcal vaccine at least 2 weeks before the administration of eculizumab.34,38,39INFECTIONS AND MALIGNANCIESAdvances in immunosuppression have led to improved graft survival rates. However, the growing population of immuno-suppressed patients, in turn, has led to an increased incidence of opportunistic infections and malignancies. Such posttransplant complications have become important barriers to long-term disease-free survival.InfectionsTransplant recipients are predisposed to a variety of infections. Immunosuppression is the obvious reason. Moreover, such patients have already endured end-stage organ disease pre-transplant and then the stress of an invasive transplant

1	to a variety of infections. Immunosuppression is the obvious reason. Moreover, such patients have already endured end-stage organ disease pre-transplant and then the stress of an invasive transplant opera-tion. Posttransplant, they continue to have significant comorbid conditions.Early. Early infections (i.e., infections occurring within 1 month posttransplant) can be due to a wide spectrum of pathogens (bacterial, viral, and fungal). In the immediate postoperative period, recipients are significantly compromised from the stress of the operation, from induction immunosuppression, and often from initially impaired graft function. Infections during this period can be devastating.It is imperative to differentiate between medical and surgical infections. Surgical infections are the most common and require expedient surgical intervention. Typical examples include generalized peritonitis, intra-abdominal abscesses, and wound infections.In liver and pancreas recipients, surgical infections

1	and require expedient surgical intervention. Typical examples include generalized peritonitis, intra-abdominal abscesses, and wound infections.In liver and pancreas recipients, surgical infections are most severe. The incidence of intra-abdominal infections is decreasing, but they remain a significant problem: they are the second most common reason (after vascular thrombosis) for graft loss in pancreas recipients.Lengthy operations with significant blood loss, prolonged warm and cold ischemic times, and spillage of contaminated fluid (bile, urine, or bowel contents) predispose patients to intra-abdominal infections. Other prominent risk factors are the high level of induction immunosuppression immediately post-transplant and anastomotic leaks. Furthermore, pretrans-plant infections can reemerge or worsen.The signs and symptoms of intra-abdominal infections are those of peritonitis: fever, hypotension, ileus, and abdominal pain, although the latter can be masked by immunosuppres-sion.

1	or worsen.The signs and symptoms of intra-abdominal infections are those of peritonitis: fever, hypotension, ileus, and abdominal pain, although the latter can be masked by immunosuppres-sion. Treatment entails a prompt return to the operating room. Intra-abdominal infections are usually polymicrobial, involving several bacterial and fungal species. Common bacterial isolates include Escherichia coli, as well as Enterococcus, Klebsiella, and Pseudomonas species. Common fungal isolates are Candida albicans, Candida krusei, and Candida glabrata. Localized infections or abscesses can be treated with percutaneous drain-age and antibiotics.Medical infections include respiratory, urinary tract, and bloodstream infections. Medical treatment should also be aggressive, often including empiric antibiotics and antifungal medications even before culture results are available. Recipients of organs from infected donors should be treated per the results of donor culture speciation and the antibiotic

1	and antifungal medications even before culture results are available. Recipients of organs from infected donors should be treated per the results of donor culture speciation and the antibiotic sensitivity profile.Late. Late infections primarily are due to chronic immunosup-pression, specifically the depression of cell-mediated immu-nity that renders recipients susceptible to viruses, fungi, and parasites.Members of the herpesvirus group are the most common etiologic agents of viral infections posttransplantation, with her-pes simplex virus (HSV), CMV, and EBV being the most prom-inent. Pretransplant exposure to viruses may confer immunity. Recipients who are seronegative for HSV, CMV, and/or EBV have a higher incidence of posttransplant infections, especially if they receive donor allografts from seropositive donors.CMV is a latent infection that can be transmitted to sero-naive recipients by donor organs from seropositive individuals, can reactivate during immunosuppression, or both.

1	from seropositive donors.CMV is a latent infection that can be transmitted to sero-naive recipients by donor organs from seropositive individuals, can reactivate during immunosuppression, or both. Infections usually occur 3 to 6 months posttransplant or during treatment for rejection. The incidence of CMV has been greatly reduced with 12-week acyclovir prophylaxis.40 CMV infections range from an asymptomatic or mild flu-like syndrome to tissue-invasive disease resulting in pneumonitis, hepatitis, and GI ulcerations. Symptomatic infections and all tissue-invasive CMV disease should be treated with intravenous (IV) ganciclovir, a reduction in immunosuppression, or both, although successful treatment of mild to moderate rejection and concurrent mild to moderate CMV disease has been described.EBV infections range from a mild mononucleosis syn-drome to severe hepatitis and highly morbid PTLD. PTLD ranges from a localized tumor to a progressive, diffuse infiltration of various organs

1	infections range from a mild mononucleosis syn-drome to severe hepatitis and highly morbid PTLD. PTLD ranges from a localized tumor to a progressive, diffuse infiltration of various organs including the brain. It results from the prolifera-tion of EBV-positive B cells in immunosuppressed patients. The main risk factors are a high degree of immunosuppression and 3Brunicardi_Ch11_p0355-p0396.indd 36301/03/19 6:53 PM 364BASIC CONSIDERATIONSPART Ia predisposing EBV serostatus (seronaive recipient, seroposi-tive donor). Among patients with early lesions, the first line of treatment is to reduce immunosuppression. For those with more advanced PTLD, rituximab is used.After 6 months posttransplant, the risk of invasive fungal infections is closely associated with environmental exposures. Blastomyces dermatitidis grows in moist soil in the Midwest and Southeast regions of the United States. Diagnosis is con-firmed by biopsy; the preferred treatment is IV amphotericin B.Coccidioides immitis

1	dermatitidis grows in moist soil in the Midwest and Southeast regions of the United States. Diagnosis is con-firmed by biopsy; the preferred treatment is IV amphotericin B.Coccidioides immitis can cause invasive coccidioidomy-cosis after inhalation of aerosolized infectious particles. It is endemic in the Southwest, Northern Mexico, and various parts of Central and South America. This infection can be resilient and difficult to treat. The first line of treatment is high-dose amphotericin B.Histoplasma capsulatum is found in chicken, pidgeon, and bat droppings in the Ohio River and Mississippi River val-leys. Dissemination is commonplace; up to a quarter of patients have central nervous system (CNS) involvement. Treatment consists of prolonged (3 to 13 months) administration of oral itraconazole.Opportunistic infections with Aspergillus, Cryptococcus, Mucor, and Rhizopus species are rare but can cause serious infections. Patients with invasive Candida or Aspergillus infec-tions exhibit

1	infections with Aspergillus, Cryptococcus, Mucor, and Rhizopus species are rare but can cause serious infections. Patients with invasive Candida or Aspergillus infec-tions exhibit a 20% mortality rate. Prophylaxis with fluconazole has been shown to reduce invasive fungal infections in liver recipients.41Pneumocystis jiroveci (also known as PCP) is ubiqui-tous and can cause pulmonary disease in immunocompromised patients. However, trimethoprim-sulfamethoxazole (TMP-SMX) is effective prophylaxis against PCP, and daily, lifelong administration has virtually eliminated this infection among transplant recipients.MalignanciesChronic immunosuppression increases the risk of develop-ing certain types of malignancies. The most extensive data, from a cohort study involving more than 175,000 solid organ transplant recipients, showed that 10,656 of them developed malignancies. The standardized incidence ratio was 2:10 (as compared with the general population). Recipients had at least a fivefold

1	organ transplant recipients, showed that 10,656 of them developed malignancies. The standardized incidence ratio was 2:10 (as compared with the general population). Recipients had at least a fivefold increase (as compared with the general population) in these types of malignancies: Kaposi’s sarcoma, nonmelanoma skin cancer, non-Hodgkin’s lymphoma, and cancer of the liver, anus, vulva, and lip. In addition, recipients had a statistically significant increase (as compared with the general population) in melanoma, Hodgkin’s lymphoma, and cancer of the lung, kidney, colon, rectum, and pancreas.42ORGAN PROCUREMENT AND PRESERVATIONOrgan procurement is a key element in organ transplantation. Currently, over 100 organ procurement organizations (OPOs) exist in the United States, all members of the Organ Procure-ment and Transplantation Network (OPTN), which is a feder-ally mandated network created by and overseen by UNOS. Each OPO is responsible for evaluating and procuring deceased donor

1	the Organ Procure-ment and Transplantation Network (OPTN), which is a feder-ally mandated network created by and overseen by UNOS. Each OPO is responsible for evaluating and procuring deceased donor organs for transplantation in a specific geographic region. Hospitals receiving any type of federal reimbursement for their services (whether transplant-related or not) are required to report all deaths to their OPO in a timely manner. Each OPO then determines the medical suitability of the deceased for organ donation; requests consent for donation from family members; if consent is given, contacts the OPTN to analyze and identify potential recipients whose HLA antigens most closely match those of the donor; and arranges for the recovery and transport of any donated organs.Strategies to increase organ donation and utilization have been successfully implemented in the last 10 to 15 years. The nationwide “Organ Donation Breakthrough Collaborative,” sponsored by the U.S. Department of Health

1	organ donation and utilization have been successfully implemented in the last 10 to 15 years. The nationwide “Organ Donation Breakthrough Collaborative,” sponsored by the U.S. Department of Health and Human Ser-vices in 2003, brought the OPOs and transplant communities into a single concerted program to develop best practices guide-lines. However, a severe donor shortage remains. The number of living organ donors peaked in 2007 and has declined since.Alternative options include tissue engineering and stem cell research, but those fields are in their infancy in terms of producing fully functional and vascularized human organs. With the development of genetic “knockout” pigs, xenotrans-plantation still shows promise, but two problems in particular—immunologic barriers and xenosis (also known as zoonosis) of endogenous porcine retroviruses—have yet to be satisfactorily addressed.Today, the gap between patients waiting for organ trans-plants and the number of organs available continues to

1	as zoonosis) of endogenous porcine retroviruses—have yet to be satisfactorily addressed.Today, the gap between patients waiting for organ trans-plants and the number of organs available continues to widen. More than 118,000 patients are on the waiting list for solid organ transplants, but only 33,611 transplants were performed in 2016.Deceased DonorsMost transplants today utilize organs from deceased donors. Formerly, death was determined by the cessation of both cardiac and respiratory function.Donation After Brain Death. In 1968, the concept of “irre-versible coma” was introduced by an ad hoc committee report at Harvard Medical School; that concept was pivotal to the final acceptance, in 1981, of “brain death” as a legal definition in the United States. The legal language states that the declara-tion of brain death should be in accordance with acceptable medical standards but does not specify clinical methodology. It is customary for hospitals to establish their own policies to

1	the declara-tion of brain death should be in accordance with acceptable medical standards but does not specify clinical methodology. It is customary for hospitals to establish their own policies to declare brain death, according to their standards of care and local regulations.Typically, brain death is defined as the irreversible cessa-tion of brain function, including the brainstem. The presence of medical conditions that mimic brain death—such as drug over-dose, medication side effects, severe hypothermia, hypoglyce-mia, induced coma, and chronic vegetative state—need to be excluded. The latest evidence-based guideline on determining brain death in adults reaffirmed the validity of current clinical practice.43 Briefly, the clinical diagnosis of brain death consists of four essential steps: (a) establishment of the proximate cause of the neurologic insult; (b) clinical examinations to determine coma, absence of brainstem reflexes, and apnea; (c) utilization of ancillary tests, such

1	(a) establishment of the proximate cause of the neurologic insult; (b) clinical examinations to determine coma, absence of brainstem reflexes, and apnea; (c) utilization of ancillary tests, such as electroencephalography (EEG), cere-bral angiography, or nuclear scans, in patients who do not meet clinical criteria; and (d) appropriate documentation. A similar guideline on determining brain death in pediatric patients was recently developed.44Once the diagnosis of brain death has been established, the local OPO assumes the care of the potential donor and initiates the process of donor evaluation and organ donation, and the potential donor is screened for contraindications to donation. The medical history and social history are obtained from the available family members. A battery of tests, including serologic Brunicardi_Ch11_p0355-p0396.indd 36401/03/19 6:53 PM 365TRANSPLANTATIONCHAPTER 11or molecular detection of human immunodeficiency virus (HIV) and viral hepatitis, are

1	tests, including serologic Brunicardi_Ch11_p0355-p0396.indd 36401/03/19 6:53 PM 365TRANSPLANTATIONCHAPTER 11or molecular detection of human immunodeficiency virus (HIV) and viral hepatitis, are performed. The exact medical conditions that preclude donation vary; nonetheless, in the United States, infections and other medical conditions that determine eligibil-ity are dictated by UNOS bylaws and routinely reviewed and updated.The OPO focuses on preserving organ function and opti-mizing peripheral oxygen delivery until organ procurement commences.45 In all deceased donors, core temperature, sys-temic arterial blood pressure, arterial oxygen saturation, and urine output must be determined routinely and frequently. Arterial blood gases, serum electrolytes, blood urea nitrogen, serum creatinine, liver enzyme, hemoglobin, and coagulation tests need to be monitored regularly. In all brain-dead donors, elevated intracranial pressure triggers a compensatory catechol-amine response to

1	creatinine, liver enzyme, hemoglobin, and coagulation tests need to be monitored regularly. In all brain-dead donors, elevated intracranial pressure triggers a compensatory catechol-amine response to maintain cerebral profusion pressure. Isch-emic injury to the spinal cord and the sympathetic system may lead to a profound vasodilation. As a result, brain-dead donors frequently have severe hemodynamic and metabolic derange-ments, so aggressive monitoring and intervention are required to prevent loss of precious organs.Previous studies of deceased donor care focused on organ-specific resuscitation protocols that resulted in only marginal gains in the number of organs transplanted. The latest develop-ments center on multisystem protocols to increase the number of organs transplanted per donor (OTPD).46,47 The goals are to maintain a core temperature between 36.0°C and 37.5°C, a mean arterial pressure >70 mmHg or a systolic pressure >100 mmHg, and a hemoglobin level between 7 and 10

1	(OTPD).46,47 The goals are to maintain a core temperature between 36.0°C and 37.5°C, a mean arterial pressure >70 mmHg or a systolic pressure >100 mmHg, and a hemoglobin level between 7 and 10 g/dL; hormonal therapy and aggressive treatment of arrhythmias and metabolic derangements are also called for.47Surgical Technique. Procurement of multiple organs (heart, lungs, kidney, liver, pancreas, and/or small bowel), or multivis-ceral procurement, was first described by the Pittsburgh group in 1987.48 Since then, most centers have incorporated changes, especially with regard to the timing and location of dissection and flushing.49,50 The basic steps involve a long incision to provide wide exposure of all thoracic and abdominal organs (Fig. 11-3). A Cattell-Braasch maneuver (complete mobiliza-tion of the distal small bowel, right colon, and duodenum) is performed to allow for identification of the distal aorta, iliac Figure 11-3. Exposure for thoracic and abdominal organ

1	mobiliza-tion of the distal small bowel, right colon, and duodenum) is performed to allow for identification of the distal aorta, iliac Figure 11-3. Exposure for thoracic and abdominal organ procurement.bifurcation, and distal inferior vena cava (IVC). The infrare-nal aorta is the site for inserting the cannula that will allow for flushing of the organs with cold preservation solution. Some-times, division of the inferior mesenteric artery is necessary to facilitate the exposure of the distal aorta. The third portion of the duodenum is retracted cephalad to expose the root of the supe-rior mesenteric artery (SMA). Limited dissection is performed at the root of the SMA, which is encircled with a vessel loop to enable its temporary occlusion at the time of flushing, thus reducing the incidence of overperfusion injury to the pancreas.A large anomalous or replaced right hepatic artery typi-cally rises from the SMA, and this should be identified and preserved. Lateral to the SMA is the

1	of overperfusion injury to the pancreas.A large anomalous or replaced right hepatic artery typi-cally rises from the SMA, and this should be identified and preserved. Lateral to the SMA is the inferior mesenteric vein (IMV), which can be cannulated for portal flushing. Dissection of the hepatic hilum and the pancreas should be limited to the common hepatic artery (CHA), and branches of the CHA (e.g., splenic, left gastric, and gastroduodenal arteries) are exposed. The gastrohepatic ligament is carefully examined to preserve a large anomalous or replaced left hepatic artery, if present. The supraceliac aorta can be exposed by dividing the left triangular ligament of the liver and the gastrohepatic ligament.The common bile duct is transected at the superior mar-gin of the head of the pancreas. The gallbladder is incised and flushed with ice-cold saline to clear the bile and sludge. If the pancreas is to be procured, the duodenum is flushed with anti-microbial solution. Before the

1	pancreas. The gallbladder is incised and flushed with ice-cold saline to clear the bile and sludge. If the pancreas is to be procured, the duodenum is flushed with anti-microbial solution. Before the cannulation of the distal aorta, systemic heparinization (300 units/kg) is administered. The supraceliac aorta is clamped; cold preservation fluid is infused via the aortic (systemic) and IMV (portal) cannulas. The tho-racic organs, liver, pancreas, and kidneys are then removed.Donation After Cardiac Death. Given the severe shortage of donor organs, donation after cardiac death (DCD)—also known as donation by non–heart-beating donors (NHBDs)—was rein-troduced to the transplant community in the 1990s.51 The cat-egory of DCD (Maastricht classification) was initially proposed at an international workshop and is now widely adopted for organ procurement.52 Currently, most NHBDs in the United States meet Maastricht classification III; that is, they have suffered a devastating injury with no

1	workshop and is now widely adopted for organ procurement.52 Currently, most NHBDs in the United States meet Maastricht classification III; that is, they have suffered a devastating injury with no chance of a meaningful recovery but do not meet the criteria for brain death. After consent for dona-tion is obtained from the next of kin, the donor’s life support is removed. After the cessation of cardiac and respiratory function, organ procurement commences. DCD procurement protocols vary between states; religious and cultural differences need to be taken into consideration. The surgical team must be familiar with, and respect, the local protocol.With cardiac death (as opposed to brain death), warm ischemic injury to organs can occur during the period between circulatory cessation and rapid core cooling through perfusion of preservation solution. However, the difference in long-term outcomes is negligible for recipients of organs from either type of donor. Still, a significant percentage

1	cooling through perfusion of preservation solution. However, the difference in long-term outcomes is negligible for recipients of organs from either type of donor. Still, a significant percentage of liver grafts procured after cardiac death, especially those with more than 25 minutes of warm ischemic time, develop devastating ischemic cholan-giopathy and fail.53A new development to minimize ischemic injury to organs procured after cardiac death has been the application of extra-corporeal membrane oxygenation (ECMO). With ECMO, DCD differs in two key ways: (a) cannulation occurs before with-drawal of life support and (b) organs are perfused via ECMO with warm oxygenated blood after declaration of cardiac death. Brunicardi_Ch11_p0355-p0396.indd 36501/03/19 6:53 PM 366BASIC CONSIDERATIONSPART IThe initial experience with organs procured using ECMO has been encouraging.Surgical Technique. Surgeons who perform multiple organ retrieval should be familiar and experienced with the

1	initial experience with organs procured using ECMO has been encouraging.Surgical Technique. Surgeons who perform multiple organ retrieval should be familiar and experienced with the super-rapid technique described by the Pittsburgh group.54 Preferably, NHBDs undergo withdrawal of life support in the operating room after the surgical site is prepped and draped, as soon as the surgical team is ready. Alternatively, the NHBD is transported to the operating room after declaration of cardiac death.A midline incision is used to rapidly gain entry into the abdominal cavity. An assistant retracts the small bowel and the sigmoid colon laterally, so that the bifurcation of the aorta can be easily identified on the left side of the vertebral column. A short segment of the distal aorta is dissected out from the retro-peritoneum. A moist umbilical tape is passed around the aorta, which is used to secure a cannula. The distal aorta is clamped. Next, a cannula is passed cephalad through an aortotomy

1	from the retro-peritoneum. A moist umbilical tape is passed around the aorta, which is used to secure a cannula. The distal aorta is clamped. Next, a cannula is passed cephalad through an aortotomy and secured. Flushing with cold preservation solution is started at once, followed by cross-clamping the aorta proximally (thoracic aorta) and venting through the vena cava. The portal flush is then instituted.The rest of the procedure is similar to procurement after brain death, with two noticeable differences. First, to avoid injury to a large anomalous or replaced left hepatic artery, the gastrohepatic ligament and the left gastric artery are separated from the stomach at the lesser curvature. Second, to avoid injury to a large anomalous or replaced right hepatic artery, the SMA is examined before it is divided. If the pancreas is not procured, a common aortic patch encompassing both the SMA and the celiac artery can be procured with the liver.Living DonorsThe maxim of medical ethics is

1	before it is divided. If the pancreas is not procured, a common aortic patch encompassing both the SMA and the celiac artery can be procured with the liver.Living DonorsThe maxim of medical ethics is “primum non nocere” (first, do no harm), and for that reason, living organ donation pres-ents unique ethical and legal challenges. Performing potentially harmful operations to remove organs from healthy individuals seems, at first glance, to contradict that maxim. But in fact, the ethical framework of living organ donation rests on three guid-ing principles respected in all discussions of medical practice: beneficence to the recipient, nonmaleficence to the donor, and the donor’s right to autonomy.55 In order to achieve optimal outcomes (the common good), transplant professionals should focus on maximizing the benefits for the recipient and minimiz-ing the damage to the donor. The Uniform Anatomical Gift Act adopted by all states in the United States (with slight variations) provides the

1	on maximizing the benefits for the recipient and minimiz-ing the damage to the donor. The Uniform Anatomical Gift Act adopted by all states in the United States (with slight variations) provides the legal framework for competent adult living donors to decide whether or not to donate. It is the fiduciary duty of transplant professionals to explain the risks of organ donation. Any decision to donate should be uncoerced, and no entice-ments should be offered.The use of living donors offers numerous advantages for recipients in need. First and foremost is the availability of lifesaving organs for those who would otherwise succumb to the progression of their end-stage disease. In certain parts of the world, such as East Asia, the concept of brain death and the use of deceased donors conflict with the prevailing culture or religion. Even in countries where the use of deceased donors is accepted, the use of living donors may significantly shorten the waiting time for recipients. A shorter

1	with the prevailing culture or religion. Even in countries where the use of deceased donors is accepted, the use of living donors may significantly shorten the waiting time for recipients. A shorter waiting time gener-ally implies a healthier recipient—one whose body has not been ravaged by prolonged end-stage organ failure. Moreover, with the use of living donors, transplants are planned (rather than emergency) procedures, allowing for better preoperative preparation of the recipient. Receiving an organ from a closely matched relative may also have immunologic benefits. And long-term results may be superior with the use of living donors, as is certainly the case with kidney transplants.The major disadvantage is the risk to the living donor. Medically, there is no possibility of benefit to the donor, only the potential for harm. The risk of death associated with dona-tion depends on the organ being removed. For a nephrectomy, the estimated mortality risk is less than 0.05%; for a

1	to the donor, only the potential for harm. The risk of death associated with dona-tion depends on the organ being removed. For a nephrectomy, the estimated mortality risk is less than 0.05%; for a partial hepatectomy, about 0.2%. The risk of surgical and medical complications also depends on the procedure being performed. In addition, long-term complications may be associated with a partial loss of organ function after donation. The guiding prin-ciple should be minimization of risk to the donor. All potential risks must be carefully explained to the potential donor, and written informed consent must be obtained.56Surgical Technique. The kidney, the first organ to be trans-planted from living donors, is still the most common organ donated by these individuals. The donor’s left kidney is usually preferable because of the long vascular pedicle. Use of living donor kidneys with multiple renal arteries should be avoided in order to decrease the complexity of the vascular reconstruction and

1	preferable because of the long vascular pedicle. Use of living donor kidneys with multiple renal arteries should be avoided in order to decrease the complexity of the vascular reconstruction and to help avoid graft thrombosis. Most donor nephrectomies are now performed via minimally invasive techniques, that is, laparoscopically, whether hand-assisted or not. With laparo-scopic techniques, an intraperitoneal approach is most common: it involves mobilizing the colon, isolating the ureter and renal vessels, mobilizing the kidney, dividing the renal vessels and the distal ureter, and removing the kidney (Fig. 11-4). Extensive dissection around the ureter should be avoided, and the surgeon should strive to preserve as much length of the renal artery and vein as possible.Liver transplants with living donors are not as commonly performed, given the significantly higher rates of donor mor-tality and morbidity. Initially, only adult donors for pediatric recipients were selected, but now,

1	with living donors are not as commonly performed, given the significantly higher rates of donor mor-tality and morbidity. Initially, only adult donors for pediatric recipients were selected, but now, living donor liver transplants also involve adult donors for adult recipients. In dual graft living donor liver transplants, segmental grafts from two living donors augment the recipient’s graft size.57 The donor hepatectomy is similar to a major lobar hepatectomy, except that it is impor-tant to preserve the integrity of the vascular structure until graft resection (Fig. 11-5).Living donor transplants of organs other than the kidney and liver are fairly uncommon, but certain centers do perform such transplants. Living donor pancreas transplants involve per-forming a distal pancreatectomy, with the graft consisting of the body and tail of the pancreas; vascular inflow and outflow are provided by the splenic artery and splenic vein. Living donor intestinal transplants usually involve

1	with the graft consisting of the body and tail of the pancreas; vascular inflow and outflow are provided by the splenic artery and splenic vein. Living donor intestinal transplants usually involve removal of about 200 cm of the donor’s ileum, with inflow and outflow provided by the ileocolic vessels. Living donor lung transplants involve removal of one lobe of one lung from each of two donors; both grafts are then transplanted into the recipient.Organ PreservationThe development and continuing refinement of organ preser-vation methods have completely revolutionized the transplant field. Extending the time that organs can be safely stored after procurement has enabled better organ utilization and better recipient outcomes.58,59 Hypothermia and pharmacologic inhibi-tion are the two most frequent methods. Both slow—yet cannot Brunicardi_Ch11_p0355-p0396.indd 36601/03/19 6:53 PM 367TRANSPLANTATIONCHAPTER 11ABCDEFFigure 11-4. Laparoscopic left donor nephroureterectomy. A. Takedown of

1	methods. Both slow—yet cannot Brunicardi_Ch11_p0355-p0396.indd 36601/03/19 6:53 PM 367TRANSPLANTATIONCHAPTER 11ABCDEFFigure 11-4. Laparoscopic left donor nephroureterectomy. A. Takedown of splenic flexure of colon to expose the left renal hilum. B. Dissection of left ureter off the psoas muscle. C. Dissection of left renal vein and gonadal vein. Left ureter seen lateral to the dissection. D. Dissection of left renal artery. Lumbar veins clipped and divided. E. Endo-TA stapler transection of the left renal artery. F. Placement of ports and Pfannenstiel incision for the donor kidney extraction.ABFigure 11-5. Donor hepatectomy (right hepatectomy). A. The liver parenchymal transection line (c, the Cantlie line) marked with cautery. Right portal vein (p) and right hepatic artery (a) isolated. b = bile duct. Cystic duct was cannulated for intraoperative cholangiography. B. Exposure of hepatic veins after transection of the parenchyma. IVC = inferior vena cava; L = left hepatic vein; M

1	b = bile duct. Cystic duct was cannulated for intraoperative cholangiography. B. Exposure of hepatic veins after transection of the parenchyma. IVC = inferior vena cava; L = left hepatic vein; M = middle hepatic vein; R = right hepatic vein.Brunicardi_Ch11_p0355-p0396.indd 36701/03/19 6:54 PM 368BASIC CONSIDERATIONSPART Icompletely shut down—the removed organ’s metabolic activ-ity, so both have adverse effects, such as cellular swelling and degradation. Cold storage solutions were introduced to mitigate some of the adverse effects of hypothermia or pharmacologic inhibition alone. Such solutions help prevent cellular swelling and the loss of cellular potassium.One, and perhaps the most effective, preservation solu-tion was developed at the University of Wisconsin and remains in wide use.60 Its ingredients include lactobionate (which helps prevent cellular swelling and reperfusion injury), raffinose, and hydroxyethyl starch (which helps reduce swelling of endothe-lial cells,

1	wide use.60 Its ingredients include lactobionate (which helps prevent cellular swelling and reperfusion injury), raffinose, and hydroxyethyl starch (which helps reduce swelling of endothe-lial cells, thereby decreasing edema). Histidine-tryptophan-ketoglutarate solution is also currently in wide use.61Despite enhancements in preservation methods, the amount of time that an organ can be safely stored remains rel-atively short (hours, not days), particularly with organs from marginal donors. Among kidney recipients, delayed graft func-tion becomes significantly more frequent after cold ischemic times of more than 24 hours, necessitating temporary dialysis, which is associated with increased risks of graft loss and higher costs.62 Among liver recipients, primary nonfunction and bili-ary complications ensue after prolonged cold ischemic times. In the case of heart and lung recipients, ischemic times should be under 6 hours. All of those times assume the use of normal donors.There is

1	complications ensue after prolonged cold ischemic times. In the case of heart and lung recipients, ischemic times should be under 6 hours. All of those times assume the use of normal donors.There is revived interest in the use of the pulsatile perfu-sion pump, a kidney graft preservation method that has been available for more than 40 years.63 With the increasing shortage of available donor organs and the rise in the use of organs after cardiac death, the pulsatile perfusion pump is garnering renewed enthusiasm as an adjunct method of preservation, even for donor organs other than kidneys.64,65KIDNEY TRANSPLANTATIONIntroductionUllman reported the first attempted human kidney transplant in 1902.66 For the next 50 years, sporadic attempts all ended in either technical failure or in graft failure from rejection. Joseph Murray performed the first successful kidney transplant in 1954, an epochal event in the history of organ transplantation. In that first case, the immunologic barrier was

1	from rejection. Joseph Murray performed the first successful kidney transplant in 1954, an epochal event in the history of organ transplantation. In that first case, the immunologic barrier was circumvented by transplanting a kidney between identical twins.67 For his pivotal contribution, Murray shared the Nobel Prize in Physiology or Medicine in 1990 with E. Donnall Thomas for their discoveries concerning “organ and cell transplantation in the treatment of human disease.”The introduction of AZA (Imuran) in 1960 marked the beginning of a new era in kidney transplantation. With the com-bination of steroids and AZA for maintenance immunosuppres-sion, the 1-year graft survival rate with a living related donor kidney approached 80%; with a deceased donor kidney, the rate was 65%.68 In the ensuing years, major milestones included the introduction of more effective immunosuppressive medications with lower toxicity profiles, such as polyclonal antilymphocyte globulin in the 1970s,

1	In the ensuing years, major milestones included the introduction of more effective immunosuppressive medications with lower toxicity profiles, such as polyclonal antilymphocyte globulin in the 1970s, cyclosporine in the 1980s, tacrolimus in the 1990s, and biologics in the first decade of the 21st century, as previously mentioned.Parallel to the developments in medical science were the transplant community’s concerted efforts to improve use of healthcare resources. In the United States, the Social Security amendments of 1972 provided Medicare coverage for patients with end-stage renal disease (ESRD). The National Organ Transplant Act of 1984 initiated the process of creating what later became UNOS, an umbrella organization to ensure access to organs by patients in need, to enhance organ procurement and allocation, and to improve posttransplant outcomes. This infrastructure later became the blueprint for other countries to follow. As a result, organ transplantation is the most

1	organ procurement and allocation, and to improve posttransplant outcomes. This infrastructure later became the blueprint for other countries to follow. As a result, organ transplantation is the most transparent field of medicine. Data such as transplant center performance are readily available on public websites; penalties for violation of regulations and for underperformance often result in trans-plant programs being shut down.Today, a kidney transplant remains the most definitive and durable renal replacement therapy for patients with ESRD. It offers better survival and improved quality of life and is considerably more cost-effective than dialysis.69,70 According to the 2016 Scientific Registry of Transplant Recipients (SRTR) annual report, nearly 100,000 adult patients were on the kidney transplant waiting list, while nearly 20,000 patients underwent renal transplantation. Trends over the past decade indicated that living related transplants remained rela-tively stable, while the

1	transplant waiting list, while nearly 20,000 patients underwent renal transplantation. Trends over the past decade indicated that living related transplants remained rela-tively stable, while the number of deceased donor transplants rose. Posttransplant outcomes have continued to improve: in 2015, the 1-year graft survival rate with a living donor kidney was nearly 98%; with a deceased donor kidney, the rate was approximately 95.0%.71The advantages of a living donor kidney transplant include better posttransplant outcomes, avoidance of prolonged waiting time and dialysis, and the ability to coordinate the donor and recipient procedures in a timely fashion. Living donor kidney recipients enjoy better long-term outcomes, a low incidence of delayed graft function, and reduced risks of posttransplant complications. Furthermore, the elective nature of living donor kidney transplants provides unique opportunities for recipient desensitization treatment if the donor and recipient are

1	posttransplant complications. Furthermore, the elective nature of living donor kidney transplants provides unique opportunities for recipient desensitization treatment if the donor and recipient are ABOincompatible or if the HLA cross-match results are positive.Some of the challenges transplant professionals face today are closing the growing gap between supply and demand and thereby reducing the current prolonged waiting times; refining immunosuppressive medications to achieve better outcomes with reduced toxicity; and caring for patients who develop rejection, especially antibody-mediated rejection.Pretransplant EvaluationActive infection or the presence of a malignancy, active substance abuse, and poorly controlled psychiatric illness are the few abso-lute contraindications to a kidney transplant. Studies have demon-strated the overwhelming benefits of kidney transplants in terms of patient survival, quality of life, and cost-effectiveness, so most patients with ESRD are referred

1	transplant. Studies have demon-strated the overwhelming benefits of kidney transplants in terms of patient survival, quality of life, and cost-effectiveness, so most patients with ESRD are referred for consideration of a kidney transplant. However, to achieve optimal transplant outcomes, the many risks (such as the surgical stress to the cardiovascu-lar system, the development of infections or malignancies with long-term immunosuppression, and the psychosocial and finan-cial impacts on compliance) must be carefully balanced.Any problems detected during the evaluation of transplant candidates are communicated to their referring physician and/or to a specialist if advanced evaluation and treatment are needed, ultimately improving overall care. Essentially, the pretransplant evaluation is a multifaceted approach to patient education and disease management.Before the pretransplant medical evaluation begins, kidney transplant candidates are encouraged to attend a group meeting

1	is a multifaceted approach to patient education and disease management.Before the pretransplant medical evaluation begins, kidney transplant candidates are encouraged to attend a group meeting 4Brunicardi_Ch11_p0355-p0396.indd 36801/03/19 6:54 PM 369TRANSPLANTATIONCHAPTER 11focused on patient education. The meeting is coordinated by a transplant physician or surgeon. The intent is to familiarize patients with the pretransplant evaluation process and with per-tinent medical concepts and terms. In an open forum format, important decisions such as type of donor (living vs. deceased) are discussed. The group meeting empowers patients to fully participate in their care and serves as an impetus for a meaning-ful dialogue with healthcare professionals.Medical EvaluationCardiovascular Disease. Diabetes and hypertension are the leading causes of chronic renal disease. Concomitant cardiovas-cular disease (CVD) is a common finding in this population. An estimated 30% to 42% of deaths with a

1	and hypertension are the leading causes of chronic renal disease. Concomitant cardiovas-cular disease (CVD) is a common finding in this population. An estimated 30% to 42% of deaths with a functioning kidney graft are due to CVD.72,73 Therefore, assessment of the potential kid-ney transplant candidate’s cardiovascular status is an important part of the pretransplant evaluation.In fact, the American Heart Association and the American College of Cardiology Foundation recently published their expert consensus on CVD evaluation and management for solid organ transplant candidates.74 The process should focus on care-ful screening for the presence of significant cardiac conditions (e.g., angina, valvular disease, and arrhythmias) and for a prior history of congestive heart failure, coronary interventions, or valvular surgery. The perioperative risk assessment is based on patient symptoms and exercise tolerance. For all kidney trans-plant candidates, a resting 12-lead electrocardiogram (ECG)

1	or valvular surgery. The perioperative risk assessment is based on patient symptoms and exercise tolerance. For all kidney trans-plant candidates, a resting 12-lead electrocardiogram (ECG) should be obtained. In addition, in this population, the use of echocardiography to analyze left ventricular function and to assess for pulmonary hypertension is useful.Stress testing may be considered in patients with no active cardiac condition but with risk factors such as diabetes, hemo-dialysis for more than 1 year, left ventricular hypertrophy, age greater than 60 years, smoking, hypertension, and dyslipidemia. The utility of noninvasive stress testing (as compared with angiographic studies) for evaluating coronary artery disease is controversial; an additional prognostic marker is the troponin T (cTnT) level.Malignancies. Because of the long-term use of immunosup-pressive medications, transplant recipients are at increased risk for development of malignancies. Untreated and/or active

1	T (cTnT) level.Malignancies. Because of the long-term use of immunosup-pressive medications, transplant recipients are at increased risk for development of malignancies. Untreated and/or active malig-nancies are absolute contraindications to a transplant (with two exceptions: nonmelanocytic skin cancer and incidental renal cell cancer identified at the time of concurrent nephrectomy [i.e., for polycystic kidney disease] and renal transplantation). For most patients who have undergone treatment of low-grade tumors with a low risk of recurrence (e.g., completely locally excised low-grade squamous cell cancer of the skin, colon cancer in a polyp absent stalk invasion), a wait of at least 2 years after suc-cessful treatment is recommended before a kidney transplant can be considered. However, for certain types of tumors, espe-cially at advanced stages or those with a high risk of recurrence (e.g., melanoma, lymphoma, renal cell cancer, breast cancer, colon cancer), a delay of at least 5

1	for certain types of tumors, espe-cially at advanced stages or those with a high risk of recurrence (e.g., melanoma, lymphoma, renal cell cancer, breast cancer, colon cancer), a delay of at least 5 years is advisable. Accord-ing to the Israel Penn International Transplant Tumor Registry, tumor recurrence posttransplant is not infrequent: the recurrence rate is 67% in patients with multiple myeloma, 53% in nonme-lanocytic skin cancer, 29% in bladder cancer, and 23% in breast cancer.75Infections. A thorough history of infections and immuniza-tions should be obtained from transplant candidates, who need all recommended age-appropriate vaccinations according to the Centers for Disease Control and Prevention (CDC) guidelines. Ideally, vaccinations should be completed at least 4 to 6 weeks before the kidney transplant takes place. Immunosuppressive medications blunt the immune response and reduce the effec-tiveness of vaccinations; even more important, with attenuated vaccines,

1	6 weeks before the kidney transplant takes place. Immunosuppressive medications blunt the immune response and reduce the effec-tiveness of vaccinations; even more important, with attenuated vaccines, vaccine-derived infections could occur. If a splenec-tomy is anticipated (e.g., in recipients whose donor is ABO-incompatible or whose HLA cross-match results are positive), then they should be immunized against encapsulated organisms (such as Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae) well in advance of the splenectomy.Transplant candidates should undergo routine tuberculosis (TB) screening. According to the Centers for Disease Control (CDC), in 2016, 9272 TB cases were diagnosed in the United States with 68.5 percent of cases occurring in foreign-born persons.76 Serologic screening combined with a chest roentgenogram for fungal infections such as coccidioidomycosis or histoplasmosis, in patients who either have a history of those infections or are from

1	Serologic screening combined with a chest roentgenogram for fungal infections such as coccidioidomycosis or histoplasmosis, in patients who either have a history of those infections or are from an endemic area, are recommended. Chronic infections such as osteomyelitis or endocarditis must be fully treated; a suitable waiting period after successful treatment must occur, in order to ensure that relapse does not occur.Hepatitis can be caused by five different type of viruses, hepatitis virus A, B, C, D, and E, with the first three being the most common. Acute viral hepatitis is a contraindication to a kidney transplant; however, chronic viral hepatitis (most com-monly caused by hepatitis B [HBV] or C [HCV]) does not pre-clude a recipient from undergoing a kidney transplant. In such candidates, obtaining a liver biopsy is essential to assess the disease severity. Recipients infected with HBV should undergo antiviral treatment (e.g., lamivudine) to prevent reactivation and progression of

1	obtaining a liver biopsy is essential to assess the disease severity. Recipients infected with HBV should undergo antiviral treatment (e.g., lamivudine) to prevent reactivation and progression of liver disease. Note that HBV is a noncytopathic virus; the liver damage is the result of an immune-mediated process.77 Moreover, the presence of normal liver enzymes in patients with HBV antigenemia does not predict the severity of parenchymal damage.Transplant candidates with chronic HCV infection often have HCV-related glomerulonephritis. As with HBV infection, the clinical presentation and biochemical findings with HCV infection are often unreliable in predicting liver damage. In such patients who also exhibit evidence of cirrhosis, a combined liver-kidney transplant should be considered. In appropriate candidates, pretransplant antiviral treatment with interferon-α may be considered. However, after a kidney transplant, inter-feron treatment is not recommended because it is an

1	In appropriate candidates, pretransplant antiviral treatment with interferon-α may be considered. However, after a kidney transplant, inter-feron treatment is not recommended because it is an immunos-timulant, and thus HIV may precipitate graft rejection.Thanks to the excellent outcomes of highly active anti-retroviral therapy (HAART), infection with HIV is no longer considered a contraindication to a kidney transplant. Kidney transplant candidates with HIV must have an undetectable HIV viral load and a CD4 lymphocyte count greater than 200/mm3; in addition, they must not have developed any opportunistic infection in the previous year.78Latent viral infections such as CMV and EBV are of par-ticular interest in the field of transplantation, given the risks of reactivation posttransplant and their detrimental effects on graft and patient survival. Knowing the CMV and EBV sero-logic status of the recipient and donor helps transplant pro-fessionals gauge the risk of immunosuppressive

1	and their detrimental effects on graft and patient survival. Knowing the CMV and EBV sero-logic status of the recipient and donor helps transplant pro-fessionals gauge the risk of immunosuppressive regimens in relation to potential infection, thereby guiding plans for post-transplant antiviral prophylaxis treatment or, as noted earlier, Brunicardi_Ch11_p0355-p0396.indd 36901/03/19 6:54 PM 370BASIC CONSIDERATIONSPART Iavoiding transplants between a seropositive donor and a sero-naive recipient.Kidney Disease. The third most common cause of graft loss in kidney transplant recipients is recurrence of glomerular diseases such as focal segmental glomerulosclerosis (FSGS), immunoglobulin A (IgA) nephropathy, hemolytic uremic syn-drome, systemic lupus erythematosus, and membranoprolifera-tive glomerulonephritis. FSGS deserves special mention due to its frequent occurrence and dramatic presentation of early graft loss. An estimated 30% to 40% of FSGS patients develop recur-rent disease

1	glomerulonephritis. FSGS deserves special mention due to its frequent occurrence and dramatic presentation of early graft loss. An estimated 30% to 40% of FSGS patients develop recur-rent disease posttransplant; of those, up to half eventually lose their graft.79 In recipients with a history of FSGS, posttrans-plant nephrotic proteinuria should be promptly investigated; if diagnosis is confirmed by transplant kidney biopsy, rescue plas-mapheresis should be instituted at once. Adjuvant therapy with rituximab has been proposed.80Hypercoagulopathy. Kidney transplant candidates with a history of thrombotic events, repeated miscarriages, or a fam-ily history of thrombophilia should be screened for the fol-lowing coagulopathic disorders: activated protein C resistance ratio, factor V Leiden mutation, factor II 20210 gene mutation, antiphospholipid antibody, lupus anticoagulant, protein C or S deficiency, antithrombin III deficiency, and hyperhomocyste-inemia. In recipients at risk for

1	mutation, factor II 20210 gene mutation, antiphospholipid antibody, lupus anticoagulant, protein C or S deficiency, antithrombin III deficiency, and hyperhomocyste-inemia. In recipients at risk for hypercoagulopathy, pediatric kidney grafts and any kidney allografts with a complex vascular anatomy should be avoided.81 A perioperative anticoagulation protocol is recommended in this population.Surgical EvaluationUrologic Evaluation. Kidney transplant candidates (pediatric patients, in particular) with chronic kidney disease as a result of congenital or genitourinary abnormalities should undergo a thorough urologic evaluation. A voiding cystourethrogram and a complete lower urinary tract evaluation to rule out out-let obstruction are essential. Indications for a native nephrec-tomy include chronic pyelonephritis, large polycystic kidneys with loss of intra-abdominal domain, significant vesicoureteral reflux, or uncontrollable renovascular hypertension.Vascular Evaluation. The potential

1	chronic pyelonephritis, large polycystic kidneys with loss of intra-abdominal domain, significant vesicoureteral reflux, or uncontrollable renovascular hypertension.Vascular Evaluation. The potential implant sites for a kidney graft include the recipient’s iliac vessels and, less commonly, the aorta and vena cava. Careful physical examination often reveals significant central and/or peripheral vascular disease. Findings such as a pulsatile intra-abdominal mass, diminished or absent peripheral pulse, claudication, rest pain, and tissue loss in lower extremities should be further evaluated by abdomi-nal computed tomography scan or ultrasound, Doppler studies, and/or angiography. With the popularity of endovascular inter-ventions, transplant surgeons also should be familiar with such technology and obtain detailed anatomic studies of patients with vascular stents.Immunologic Evaluation. ABO blood typing and HLA typ-ing (HLA-A, -B, and -DR) are required before a kidney trans-plant. The

1	and obtain detailed anatomic studies of patients with vascular stents.Immunologic Evaluation. ABO blood typing and HLA typ-ing (HLA-A, -B, and -DR) are required before a kidney trans-plant. The method of screening for preformed antibodies against HLA antigens (because of prior transplants, blood transfusions, or pregnancies) continues to evolve. The panel-reactive anti-body (PRA) assay is a screening test that examines the ability of serum from a kidney transplant candidate to lyse lymphocytes from a panel of HLA-typed donors. A numeric value, expressed as a percentage, indicates the likelihood of a positive cross-match with a donor. A higher PRA level identifies patients at high risk for a positive cross-match and therefore serves as a surrogate marker to measure the difficulty of finding a suitable donor and the subsequent risk of graft rejection.An important development in anti-HLA antibody screen-ing is Luminex technology, using HLA-coated fluorescent microbeads and flow

1	of finding a suitable donor and the subsequent risk of graft rejection.An important development in anti-HLA antibody screen-ing is Luminex technology, using HLA-coated fluorescent microbeads and flow cytometry, which is considered the “gold standard.” This technology pinpoints donor-specific antibodies (DSAs) in the serum of a kidney transplant candidate with a high PRA level. Since all organ donors must undergo HLA typing, a negative cross-match for recipients with a high PRA level can be ensured by avoiding the selection of donors carrying unac-ceptable antigens (i.e., a virtual cross-match).82 Kidney trans-plant candidate data (including ABO blood types, HLA types, and DSAs) are entered into a nationwide central database to facilitate deceased donor kidney allocation, as described earlier.Psychosocial Evaluation. Psychiatric disorders have been recognized as important contributing factors to poor outcomes posttransplant. Patients with uncontrolled psychiatric disor-ders are at high

1	Evaluation. Psychiatric disorders have been recognized as important contributing factors to poor outcomes posttransplant. Patients with uncontrolled psychiatric disor-ders are at high risk for noncompliance with drug treatment, impaired cognitive function, and the development of substance abuse. A robust psychosocial evaluation is essential to ensure that transplant candidates understand the risks and benefits of the procedure and that they adhere to the lifetime immunosup-pressive medication regimen.Recipient OperationKidney allografts usually are transplanted heterotopically. The iliac fossa is recognized as the ideal position because of its prox-imity to the recipient’s bladder and iliac vessels.83,84Retroperitoneal allograft placement also allows easy access for percutaneous biopsies and interventions for ureteral complications. The right iliac fossa is the preferred site because of its easy access to the recipient’s iliac vessels. However, if a pancreas transplant is anticipated

1	and interventions for ureteral complications. The right iliac fossa is the preferred site because of its easy access to the recipient’s iliac vessels. However, if a pancreas transplant is anticipated in the future or if now failed kidney grafts have been placed at the right iliac fossa, then the left iliac fossa is used for implantation. The current surgical technique for kidney transplants was developed and popularized in the 1950s and 1960s and has changed little since.85A large-bore three-lumen urinary catheter is inserted after the recipient is anesthetized, and it is occluded with a clamp beneath the surgical drapes. Recipients whose native kidneys produce urine will naturally fill up the urinary bladder; those individuals whose kidneys do not will require insufflation of saline prior to creation of the ureteral anastomosis.Exposure of the operative field starts with a curvilinear skin incision, one to two finger widths above the midline pubic bone and the lateral edge of the

1	to creation of the ureteral anastomosis.Exposure of the operative field starts with a curvilinear skin incision, one to two finger widths above the midline pubic bone and the lateral edge of the rectus sheath. Superiorly, the extension of the incision depends on the recipient’s body habitus and the size of the donor kidney. The anterior rectus sheath is incised, medially to laterally, until the lateral edge of the rectus sheath is exposed. The posterior rectus sheath is missing below the arcuate line, thus providing direct access to the extraperito-neal space. The rectus muscle can be easily mobilized medially without being divided. The remainder of the fascial incision is along the lateral edge of the rectus sheath until the desired expo-sure is achieved (Fig. 11-6).The retroperitoneal space of the iliac fossa is entered by mobilizing the peritoneum medially. The inferior epigastric ves-sels, the round ligament (in females), and the spermatic cord and its vasculature (in males) are

1	space of the iliac fossa is entered by mobilizing the peritoneum medially. The inferior epigastric ves-sels, the round ligament (in females), and the spermatic cord and its vasculature (in males) are encountered in this space; the former two structures are divided, while the latter is retracted with a vascular loop. A self-retained retractor is used to expose Brunicardi_Ch11_p0355-p0396.indd 37001/03/19 6:54 PM 371TRANSPLANTATIONCHAPTER 11ABCABFigure 11-6. Incision and exposure for kidney transplant. A. Mark for the skin incision. B. Anterior rectus sheath incised obliquely. The abdominal muscle transected lateral to the rectus muscle. C. External iliac artery and vein dissected.Figure 11-7. Vascular anastomoses of kidney transplant. A. Arterial anastomosis: donor renal artery with Carrel patch to recipient external iliac artery, end-to-side. B. Venous anastomosis: donor renal vein with caval extension conduit to recipient external iliac vein, end-to-side.the surgical field. The

1	patch to recipient external iliac artery, end-to-side. B. Venous anastomosis: donor renal vein with caval extension conduit to recipient external iliac vein, end-to-side.the surgical field. The iliac vessels should be dissected with great care. To minimize the risk of lymphocele development postoperatively, dissection of the iliac artery should be limited; the intertwining lymphatics around the iliac vessels should be ligated. In general, the donor’s renal artery and vein are anasto-mosed to the recipient’s external iliac vessels in an end-to-side fashion (Fig. 11-7). In recipients with a severely calcified iliac artery, the internal iliac artery can be used as an alternative, and in select cases, an endarterectomy must be performed.After restoring the circulation to the donor’s kidney, urinary continuity can be established via several approaches. The approach chosen depends on such factors as the length of the donor ureter and a recipient history of bladder surgery, native

1	kidney, urinary continuity can be established via several approaches. The approach chosen depends on such factors as the length of the donor ureter and a recipient history of bladder surgery, native nephrectomy, or pelvic radiation. The two most com-mon procedures to restore urinary continuity are the Leadbetter-Politano and a modification of the Lich (e.g., extravesical) ureteroneocystostomy.During the former procedure, a large cystotomy is cre-ated in the dome of the bladder, and the donor ureter is brought through a lateral and somewhat inferior 1-cm submucosal tunnel into the bladder, the end of which is spatulated and then sewn in place without tension with interrupted absorbable sutures placed through the mucosa and submucosa on the inside of the bladder.An extravesical ureteroneocystostomy is performed by careful dissection of a 1-cm portion of the muscular layers on the anterolateral portion of the bladder until a “bubble” of mucosa is exposed. The donor ureter is spatulated

1	is performed by careful dissection of a 1-cm portion of the muscular layers on the anterolateral portion of the bladder until a “bubble” of mucosa is exposed. The donor ureter is spatulated in a diamond-shaped fashion, the bladder mucosa is incised, absorbable interrupted sutures are placed in four quadrants, and a mucosa-to-mucosa anastomosis is created using running absorbable sutures with a temporary ureteral stent in place of the first three-quarters of the anastomosis. The muscular lay-ers of the bladder are then carefully approximated over the anastomosis to prevent reflux.The decision to place a ureteral stent depends on the sur-geon, who must try to balance the risk of infectious compli-cations with the possible technical complications of a ureteral anastomosis, but in general, this is not required except during the rarely performed donor ureter to recipient ureter anastomo-sis or in the case of a pediatric kidney transplant. Fixation of the donor’s kidneys is not necessary,

1	is not required except during the rarely performed donor ureter to recipient ureter anastomo-sis or in the case of a pediatric kidney transplant. Fixation of the donor’s kidneys is not necessary, except in the case of small kidneys (usually from a pediatric donor) or en bloc kidneys.Brunicardi_Ch11_p0355-p0396.indd 37101/03/19 6:54 PM 372BASIC CONSIDERATIONSPART IFigure 11-8. Arterial and venous reconstruction. A. Two renal arteries combined into a single Carrel patch (arrow). Right renal vein exten-sion conduit constructed with stapled caval patch. IVC = inferior vena cava; R = right renal vein. B. Three renal arteries anastomosed to external iliac artery separately.ABABFigure 11-9. En bloc kidney transplant (3-month-old donor kidneys). A. En bloc kidneys benched. Vascular integrity tested with methylene blue (blue hue look of the kidneys). B. En bloc kidneys transplanted into a 62-year-old woman. Donor aorta anastomosed to recipient’s exter-nal iliac artery; donor cava, to

1	tested with methylene blue (blue hue look of the kidneys). B. En bloc kidneys transplanted into a 62-year-old woman. Donor aorta anastomosed to recipient’s exter-nal iliac artery; donor cava, to recipient’s external iliac vein.Grafts With Multiple Renal ArteriesIn 10% to 30% of donor kidneys, multiple renal arteries are encountered. Unless kidney transplant candidates have hyper-coagulopathy, grafts with multiple renal arteries fare as well as those with single vessels.86 Vascular reconstruction options include implanting the donor’s arteries separately, reconstruct-ing the multiple arteries into a common channel, or combining multiple arteries into a common Carrel patch (Fig. 11-8).En Bloc GraftsDebate persists about whether to implant kidneys obtained from young donors (<5 years or whose body weight is under 20 kg) as a single en bloc unit into one recipient or separately into two recipients. The underlying issues are the shortage of donor organs, the complexity of the surgical

1	whose body weight is under 20 kg) as a single en bloc unit into one recipient or separately into two recipients. The underlying issues are the shortage of donor organs, the complexity of the surgical procedure, the risks of graft thrombosis, ureteral complications, and long-term outcomes.In en bloc kidney transplants, the donor aorta and vena cava are used as the vascular inflow and outflow conduits. Therefore, reconstruction of the en bloc graft pretransplant is key to a successful transplant. The donor’s suprarenal vena cava and aorta are oversewn. The lumbar branches of the cava and aorta are ligated. Dissection around the renal hilum should be avoided. The orientation of the cava and aorta should be clearly marked, in order to avoid torsion of the anastomosis. If the color of the two kidneys looks different after reperfusion, repositioning should be attempted to rule out vascular torsion; fixation of the en bloc kidneys to the retroperitoneum is often necessary. The donor’s

1	two kidneys looks different after reperfusion, repositioning should be attempted to rule out vascular torsion; fixation of the en bloc kidneys to the retroperitoneum is often necessary. The donor’s ureters are implanted to the recipient’s bladder, either as two separate anastomoses or as a common patch (Fig. 11-9). Only a handful of centers have performed en bloc kidney transplants, but the long-term outcomes are encouraging.87,88Brunicardi_Ch11_p0355-p0396.indd 37201/03/19 6:54 PM 373TRANSPLANTATIONCHAPTER 11Perioperative CarePreoperatively, a thorough history and physical examination should be performed. Any changes in transplant candidates’ recent medical history should be investigated in great detail. In those recipients with a historically negative PRA level who have recently undergone blood transfusions, a prospective tis-sue cross-match is necessary to avoid graft rejection. Electrolyte panels should be checked. Emergency dialysis may be neces-sary for transplant candidates

1	blood transfusions, a prospective tis-sue cross-match is necessary to avoid graft rejection. Electrolyte panels should be checked. Emergency dialysis may be neces-sary for transplant candidates experiencing hyperkalemia or fluid overload.For dialysis-dependent transplant candidates, the catheter sites should be examined preoperatively to rule out infections. Vascular access for hemodialysis is essential to avoid compli-cations related to posttransplant acute tubular necrosis (ATN). Vascular evaluation is mandatory; any changes in results should be investigated by appropriate imaging studies.As is routine for other major surgical procedures, trans-plant candidates should preoperatively undergo a chest X-ray, a 12-lead ECG, blood typing, cross-match tests, and prophylaxis against surgical site infection (by administration of a nonneph-rotoxic antibiotic with activity against both common skin micro-flora and gram-negative pathogens); candidates should receive nothing to eat or

1	surgical site infection (by administration of a nonneph-rotoxic antibiotic with activity against both common skin micro-flora and gram-negative pathogens); candidates should receive nothing to eat or drink.Intraoperatively, transplant recipients should be kept well hydrated to avoid ATN and should receive heparin prior to vas-cular occlusion. Before reperfusion of the transplanted kidney, the desired central venous pressure should be maintained at around 10 mmHg, and the systolic blood pressure should be above 120 mmHg. In pediatric recipients of an adult graft, a superphysiologic condition may be necessary to avoid ATN or graft thrombosis. Mannitol often is administered before reper-fusion as a radical scavenger and diuretic agent, and a diuretic such as furosemide is administered as well.Postoperatively, the guiding principles for the care of kidney transplant recipients are the same as for other surgical patients. The crucial elements include hemodynamic stability and fluid and

1	well.Postoperatively, the guiding principles for the care of kidney transplant recipients are the same as for other surgical patients. The crucial elements include hemodynamic stability and fluid and electrolyte balance. To achieve a euvolemic state, the recipient’s urine output is replaced with either an equal or a reduced volume of IV fluid on an hourly basis, depending on the medical status. In recipients undergoing brisk dieresis, aggressive replacement of electrolytes (including calcium, mag-nesium, and potassium) may be necessary. In recipients expe-riencing ATN, fluid overload, or hyperkalemia, however, fluid restriction, treatment for hyperkalemia, and even hemodialysis may be necessary.Hypotension is an unusual event immediately posttrans-plant. The differential diagnoses include hypovolemia, vasodila-tion, and myocardial infarction with cardiac failure. Immediate action should be taken to avoid life-threatening complications. Posttransplant hypertension can be mediated by

1	hypovolemia, vasodila-tion, and myocardial infarction with cardiac failure. Immediate action should be taken to avoid life-threatening complications. Posttransplant hypertension can be mediated by catecholamines, fluid overload, or immunosuppressive agents.Postoperatively, urine output is used as a surrogate marker to monitor graft function. Among recipients whose native kid-neys produce significant amounts of urine, normal or increased urine output can be misleading; for them, serum blood urea nitrogen and creatinine levels are more reliable indicators of kidney graft function.Suddenly decreased or minimal urine output requires immediate attention. A change in volume status is the most common cause, but other culprits include blockage of the uri-nary catheter, urinary leak, vascular thrombosis, hypotension, drug-related nephrotoxicity, ATN, and rejection (all of which must be thoroughly investigated). Diagnostic studies such as Doppler ultrasound, nuclear renograms, or biopsies

1	thrombosis, hypotension, drug-related nephrotoxicity, ATN, and rejection (all of which must be thoroughly investigated). Diagnostic studies such as Doppler ultrasound, nuclear renograms, or biopsies should be considered.Postoperative bleeding is an uncommon event after a kid-ney transplant. Recipients on anticoagulation or antiplatelet treatments are at increased risk. Signs and symptoms (such as an expanding hematoma over the surgical site, increased pain over the graft, a falling hemoglobin level, hypotension, and tachycardia) should arouse suspicion of hemorrhage. Doppler ultrasound is useful to establish the underlying cause. Surgical exploration seldom is required because the accumulated hema-toma tamponades the bleed. Indications for surgical explora-tion include ongoing transfusion requirement, hemodynamic instability, and graft dysfunction from hematoma compression. For recipients on anticoagulation or antiplatelet treatments, the threshold for surgical exploration is lower.

1	requirement, hemodynamic instability, and graft dysfunction from hematoma compression. For recipients on anticoagulation or antiplatelet treatments, the threshold for surgical exploration is lower. Small unligated ves-sels at the donor’s renal hilum or recipient’s retroperitoneum are likely sources of bleeding.One of the most devastating postoperative complications in kidney recipients is graft thrombosis. It is rare, occurring in fewer than 1% of recipients. The recipient risk factors include a history of recipient hypercoagulopathy and severe peripheral vascular disease; donor-related risk factors include the use of en bloc or pediatric donor kidneys, procurement damage, techni-cal factors such as intimal dissection or torsion of vessels, and hyperacute rejection. Graft thrombosis usually occurs within the first several days posttransplant. Acute cessation of urine output in recipients with brittle posttransplant diuresis and the sudden onset of hematuria or graft pain should arouse

1	occurs within the first several days posttransplant. Acute cessation of urine output in recipients with brittle posttransplant diuresis and the sudden onset of hematuria or graft pain should arouse suspicion of graft thrombosis. Doppler ultrasound may help confirm the diagnosis. In cases of graft thrombosis, an urgent thrombectomy is indi-cated; however, it rarely results in graft salvage.Urologic complications are seen in up to 5% of recipi-ents. The cause is often related to ureteral ischemia, damage during procurement of the donor’s distal ureter, or technical errors. Symptoms of urine leak include fever, pain, swelling at the graft site, increased creatinine level, decreased urine output, and cutaneous urinary drainage. Diagnosis can be confirmed by a combination of ultrasound, nuclear renography, drainage of perinephric fluid collection, and comparison of serum and fluid creatinine levels. Depending on the location and volume of the urine leak, satisfactory results can be

1	nuclear renography, drainage of perinephric fluid collection, and comparison of serum and fluid creatinine levels. Depending on the location and volume of the urine leak, satisfactory results can be achieved by surgi-cal exploration and repair or by percutaneous placement of a nephrostomy and ureteral stenting.Early urinary obstruction can be due to edema, blood clots, torsion of the ureter, or compression from a hematoma. Late urinary obstruction is often related to ischemia. The appear-ance of hydronephrosis on ultrasound is a good initial indicator. Treatment includes percutaneous placement of a nephrostomy and ureteral stenting. If transluminal intervention fails, surgical intervention (such as ureteral reimplantation or a ureteropyelos-tomy) can be undertaken.ResultsA kidney transplant remains the most common solid organ transplant in the world today. With the introduction of induc-tion immunosuppressive therapy and ever-improving, less toxic immunosuppressive medications,

1	remains the most common solid organ transplant in the world today. With the introduction of induc-tion immunosuppressive therapy and ever-improving, less toxic immunosuppressive medications, posttransplant outcomes have become better and better. And, as noted above, posttransplant outcomes have continued to improve: in 2014 allograft and patient survival rates were well over 90%, and in 2015, the 1-year graft survival rate with a living donor kidney was nearly Brunicardi_Ch11_p0355-p0396.indd 37301/03/19 6:54 PM 374BASIC CONSIDERATIONSPART I98%; with a deceased donor kidney, the rate was approximately 95%.71,89The biggest improvements have been in the reduction of 1-year graft failure. With a deceased donor kidney, the 1-year graft failure rate dropped from approximately 20% in 1989 to less than 7% in 2009 to 4.8% in 2015; with a living donor kidney, the rate dropped from 8.5% in 1989 to less than 3% in 2015.89 Furthermore, steroid-free protocols90 and calcineurin-free

1	in 1989 to less than 7% in 2009 to 4.8% in 2015; with a living donor kidney, the rate dropped from 8.5% in 1989 to less than 3% in 2015.89 Furthermore, steroid-free protocols90 and calcineurin-free protocols91 have been validated and implemented in the last several decades, further reducing medication-related side effects and vastly improving the quality of life for tens of thousands of recipients.Currently, the most common cause of graft loss is recipi-ent death (usually from cardiovascular causes) with a function-ing graft. The second most common cause is chronic allograft nephropathy; characterized by a slow, unrelenting deterioration of graft function, it likely has multiple causes (both immuno-logic and nonimmunologic).92,93 The graft failure rate due to complications related to surgical technique has remained at about 1% to 2%.PANCREAS TRANSPLANTATIONA successful pancreas transplant currently is the only definitive long-term treatment for patients with insulin-dependent

1	to surgical technique has remained at about 1% to 2%.PANCREAS TRANSPLANTATIONA successful pancreas transplant currently is the only definitive long-term treatment for patients with insulin-dependent diabetes mellitus (IDDM) that (a) restores normal glucose hemostasis without exposing patients to the risk of severe hypoglycemia and (b) prevents, halts, or, in some cases, reverses the development or progression of secondary complications of diabetes.94Given its vast medical, social, and financial implications, diabetes mellitus is a huge burden to patients and to society as a whole. An estimated 10% to 15% of the U.S. population is affected by it; of all diabetic patients, 10% have early-onset dis-ease. In the United States, diabetes mellitus is the most common cause of end-stage kidney disease, blindness, impotence, major limb amputations, and coronary or peripheral vascular bypass procedures. It is one of the most common causes of death, along with myocardial infarction and stroke.

1	blindness, impotence, major limb amputations, and coronary or peripheral vascular bypass procedures. It is one of the most common causes of death, along with myocardial infarction and stroke. Diabetes significantly decreases not only the quality of life but also life expectancy.Despite improvements in exogenous insulin administra-tion (including the use of devices such as insulin pumps), wide fluctuations in glucose levels and the risk of hypoglycemic epi-sodes are common. The Diabetes Control and Complications Trial (DCCT) demonstrated in the late 1990s that intensive insulin therapy may slow the rate of secondary complications of diabetes—yet at the expense of (life-threatening) iatrogenic hypoglycemia. The annual mortality rate of patients with insu-lin-induced inadvertent hypoglycemia is estimated to be as high as 2% to 3%.Since the first pancreas transplant in December 1966, per-formed by William Kelly and Richard Lillehei at the Univer-sity of Minnesota, more than 25,000

1	is estimated to be as high as 2% to 3%.Since the first pancreas transplant in December 1966, per-formed by William Kelly and Richard Lillehei at the Univer-sity of Minnesota, more than 25,000 pancreas transplants in the United States and more than 10,000 pancreas transplants from all over the world have been reported to the International Pan-creas Transplant Registry (IPTR).94,95Pancreas transplants are performed in three recipient categories:• Simultaneous pancreas and kidney (SPK) transplant in diabetic and uremic patients. Almost 80% of pancreas trans-plants are performed in this category. The recipient is already 5obligated to lifelong immunosuppressive therapy, due to the need for a kidney transplant, so only the surgical risk of a pancreas transplant is added. A successful SPK transplant renders the recipient dialysis-free and insulin-independent.• Pancreas after kidney (PAK) transplant in diabetic and posturemic patients. Approximately 15% of all pancreas trans-plants fall

1	renders the recipient dialysis-free and insulin-independent.• Pancreas after kidney (PAK) transplant in diabetic and posturemic patients. Approximately 15% of all pancreas trans-plants fall into this category. These patients previously under-went a kidney transplant with either a living or deceased donor, but are candidates for a subsequent pancreas transplant because of poor glucose control or because of progression of secondary diabetic complications (which may include the development of diabetic nephropathy in the transplanted kidney).• Pancreas transplant alone (PTA) in nonuremic patients with brittle diabetes mellitus. Only about 5% of all pancreas trans-plants are in this category. These patients have not yet devel-oped advanced diabetic nephropathy, but their glucose levels are extremely labile despite best efforts of control. Because of the lifelong need for immunosuppressive therapy, the sur-gical risk has to be balanced with the medical risks of brittle diabetes (e.g.,

1	extremely labile despite best efforts of control. Because of the lifelong need for immunosuppressive therapy, the sur-gical risk has to be balanced with the medical risks of brittle diabetes (e.g., frequent episodes of hypoglycemia and hypo-glycemic unawareness).In SPK recipients, a plethora of literature exists that demonstrates significant improvements in secondary dia-betic complications (across all organ systems) posttransplant. Improvements have been reported in diabetic nephropathy, neu-ropathy (autonomic and peripheral), microand macrovascular disease, retinopathy, gastroparesis, and other secondary com-plications.96 Currently, more than 1000 pancreas transplants are performed annually in the United States, with the goal of confer-ring the following benefits: excellent glucose control (similar to that of a functioning native pancreas), prevention or improve-ment of secondary diabetic complications, and increased quality of life and life expectancy. In addition, pancreas

1	control (similar to that of a functioning native pancreas), prevention or improve-ment of secondary diabetic complications, and increased quality of life and life expectancy. In addition, pancreas transplants can be successfully performed in patients who have undergone a total pancreatectomy for benign disease (such as chronic pan-creatitis) to treat both endocrine and exocrine deficiency after surgery.97Donor OperationThe general criteria for selecting deceased donors for pancreas procurement are similar to those for other solid organs; a history of type 1 diabetes mellitus obviously is a contraindication. Rela-tive contraindications include previous pancreatic procedure(s), as well as pancreatic disorders, such as chronic pancreatitis and intraductal papillary mucinous neoplasm. Hyperglycemia in itself is not a contraindication to pancreas procurement because its cause in brain-dead donors usually is severe insulin resis-tance, which is rarely observed in recipients.In light of

1	in itself is not a contraindication to pancreas procurement because its cause in brain-dead donors usually is severe insulin resis-tance, which is rarely observed in recipients.In light of better anatomic understanding and improved surgical skills, all three abdominal organs that share a common blood supply (pancreas, liver, and intestine) can be procured at the same time and transplanted into three different recipi-ents (Fig. 11-10). During pancreas procurement, a “no-touch” technique of the gland is preferred; dissection of the pancreas is carried out in a way that avoids direct manipulation of the organ such that simultaneous procurement of the spleen, duodenum, and surrounding connective tissues occurs.In contrast to the liver and kidneys, the pancreas should not be extensively flushed at the end of the procurement. To minimize the amount of preservation fluid that reaches the pan-creas, the splenic artery and SMA can be temporarily clamped Brunicardi_Ch11_p0355-p0396.indd

1	at the end of the procurement. To minimize the amount of preservation fluid that reaches the pan-creas, the splenic artery and SMA can be temporarily clamped Brunicardi_Ch11_p0355-p0396.indd 37401/03/19 6:54 PM 375TRANSPLANTATIONCHAPTER 11MHVLHVRHVIPDAMCARCAFigure 11-10. Simultaneous pancreas, in situ split-liver, and intestine procurement. IPDA = inferior pancreaticoduodenal artery; LHV = left hepatic vein; MCA = middle cerebral artery; MHV = middle hepatic vein; RCA = right coro-nary artery; RHV= right hepatic vein. (Repro-duced from Gruessner RWG, Sutherland DER: Transplantation of the Pancreas. New York, NY: Springer, 2004.)at their origin from the aorta. Usually, the celiac axis with an aortic Carrel patch is retained with the liver. The splenic artery is divided close to its origin and is retained with the pancreas. The SMA is also procured with an aortic Carrel patch and is retained with the pancreas.In case of a replaced or aberrant right hepatic artery, this first branch

1	and is retained with the pancreas. The SMA is also procured with an aortic Carrel patch and is retained with the pancreas.In case of a replaced or aberrant right hepatic artery, this first branch off of the SMA is carefully dissected out from the posterior surface of the pancreas. A replaced or aberrant right hepatic artery does not transverse the pancreas and is not a con-traindication to combined pancreas and liver procurement. But with this anatomic variant, an aortic Carrel patch with the proxi-mal SMA and replaced or aberrant right hepatic artery remains with the liver; the distal SMA with the inferior pancreaticoduo-denal artery remains with the pancreas.In the relatively rare event that the liver is not procured, then neither the splenic nor the gastroduodenal arteries need to be divided at their respective takeoff; the donor’s celiac axis and the SMA are included on a common Carrel patch. This tech-nique allows a single arterial anastomosis to be performed in the recipient

1	divided at their respective takeoff; the donor’s celiac axis and the SMA are included on a common Carrel patch. This tech-nique allows a single arterial anastomosis to be performed in the recipient without reconstruction. At the end of the procurement, the pancreas is attached to the spleen, duodenum, and proximal jejunum, which is stapled at both ends.98Back Table Preparation of the Pancreas GraftBack table preparation of the pancreas graft consists of four steps: (a) removal of the spleen; (b) shortening, restapling, and/or suture reinforcement of the mesenteric root; (c) trimming of any excess distal and proximal duodenum, along with reinforce-ment of the proximal staple line; and (d) arterial reconstruction.Back table preparation is carried out in a basin filled with chilled preservation solution. The most common tech-nique to create a single arterial inflow to the pancreas graft is the “Y-graft” reconstruction, using a resected segment of the donor iliac artery bifurcation. In

1	solution. The most common tech-nique to create a single arterial inflow to the pancreas graft is the “Y-graft” reconstruction, using a resected segment of the donor iliac artery bifurcation. In this technique, the donor exter-nal iliac artery is anastomosed end-to-end to the donor SMA, and the donor internal iliac artery is anastomosed end-to-end to the splenic artery (Fig. 11-11). This procedure allows the donor common iliac artery to be anastomosed as a single vessel to the recipient’s common iliac artery. For venous outflow, the portal vein is kept relatively short, in order to avoid the risk of venous thrombosis by kinking or impingement.98Recipient OperationOver the years, different surgical techniques have been described for (a) the management of exocrine pancreatic secretions and (b) the type of venous drainage. For the secretions, the two most common techniques are drainage of the duodenal segment to the bladder (bladder drainage) or to the small bowel (enteric drain-age)

1	(b) the type of venous drainage. For the secretions, the two most common techniques are drainage of the duodenal segment to the bladder (bladder drainage) or to the small bowel (enteric drain-age) (Figs. 11-12 and 11-13). For venous drainage, systemic venous drainage is preferred over portal venous drainage.The pancreas graft is usually placed intra-abdominally and preferably on the right side because the iliac vessels are Brunicardi_Ch11_p0355-p0396.indd 37501/03/19 6:54 PM 376BASIC CONSIDERATIONSPART IFigure 11-11. Posterior view of the pancreas graft with Y-graft reconstruction. EIA = external iliac artery; IIA = internal iliac artery; SA = splenic artery; SMA = superior mesenteric artery. (Reproduced from Gruessner RWG, Sutherland DER: Transplanta-tion of the Pancreas. New York, NY: Springer, 2004.)Figure 11-12. Whole-organ transplant with systemic vein and bladder exocrine drainage. (Reproduced from Gruessner RWG, Sutherland DER: Transplantation of the Pancreas. New York, NY:

1	Springer, 2004.)Figure 11-12. Whole-organ transplant with systemic vein and bladder exocrine drainage. (Reproduced from Gruessner RWG, Sutherland DER: Transplantation of the Pancreas. New York, NY: Springer, 2004.)SAIIAEIASMASMAin a more shallow position on the right than on the left side; moreover, the vessels are already appropriately aligned for the vascular anastomoses (i.e., a lateral position for the com-mon iliac vein, a medial position for the common iliac artery). Venous and arterial anastomoses are performed end-to-side. After restoration of blood flow to the graft, hemostasis must be meticulously maintained. Because the donor portal vein pur-posely is kept short, ligation and transection of all of the recipi-ent’s internal iliac vein branches are frequently performed in order to prevent tension on the venous anastomosis. The pan-creas usually is placed with the pancreatic head and duodenum pointing caudally.Bladder drainage is performed using either a hand-sewn or a stapled

1	prevent tension on the venous anastomosis. The pan-creas usually is placed with the pancreatic head and duodenum pointing caudally.Bladder drainage is performed using either a hand-sewn or a stapled anastomosis in which the antimesenteric side of the donor duodenum is sewn to the superior portion of the dome of the bladder. The stapled technique requires that a circular cut-ting stapler be inserted through the open distal end of the donor duodenum, which is subsequently closed. Bladder drainage has two main advantages. First, rejection of the exocrine pancreas precedes rejection of the endocrine pancreas by 5 to 7 days. Amylase levels are measured routinely in the recipient’s urine. With bladder drainage, antirejection treatment can successfully be implemented when the recipient is still normoglycemic and only hypoamylasuric. In the absence of hyperglycemia, more than 90% of pancreas rejection episodes are reversible. Second, bladder drainage avoids the bacterial contamination that

1	normoglycemic and only hypoamylasuric. In the absence of hyperglycemia, more than 90% of pancreas rejection episodes are reversible. Second, bladder drainage avoids the bacterial contamination that occurs with enteric drainage. If an anastomotic leak occurs, it is easier to treat because the infection usually remains localized to the right lower quadrant.Enteric drainage is more physiologic and has advantages as well. The antimesenteric side of the donor’s duodenum is anastomosed to the antimesenteric portion of the recipient’s jejunum in a side-to-side fashion. The enteric anastomosis can also involve a defunctionalized Roux-en-Y loop, which mini-mizes the potential complications if an enteric leak occurs.98 Currently, in the United States, more than 80% of all pancreas transplants are performed with enteric drainage for the exocrine pancreatic secretions, and more than 90% employ systemic venous drainage.95Brunicardi_Ch11_p0355-p0396.indd 37601/03/19 6:54 PM

1	transplants are performed with enteric drainage for the exocrine pancreatic secretions, and more than 90% employ systemic venous drainage.95Brunicardi_Ch11_p0355-p0396.indd 37601/03/19 6:54 PM 377TRANSPLANTATIONCHAPTER 11Figure 11-13. Whole-organ transplant with systemic vein and enteric exocrine drainage. (Reproduced from Gruessner RWG, Sutherland DER: Transplantation of the Pancreas. New York, NY: Springer, 2004.)ComplicationsThe technical complication rate for pancreas transplants is higher than for any other solid organ transplant. Four fac-tors contribute to the high surgical complication rate99: (a) the nature of the organ itself with inherent organ-specific surgical complications (e.g., pancreatitis, abscesses, necrosis, fistulas, and pseudocysts) and its low blood flow (which significantly increases the risk of thrombosis, as compared with a kidney or liver transplant); (b) the risk of a leak or infection after con-necting two hollow viscera (the duodenum and either the

1	significantly increases the risk of thrombosis, as compared with a kidney or liver transplant); (b) the risk of a leak or infection after con-necting two hollow viscera (the duodenum and either the blad-der or small intestine); (c) the increased incidence of rejection episodes because the pancreas is one of the most immunogenic solid organs; and (d) the underlying disease of diabetes mellitus, predisposing patients not only to infections but also to cardio-vascular and other complications.The most common surgical complications are throm-bosis (an incidence of 5%–15%), intra-abdominal abscesses (5%–10%), and bleeding (6%–8%). Other pancreas-specific complications include graft pancreatitis (frequently due to pro-curement or reperfusion injury), pancreatic fistulas, and pan-creatic pseudocysts. Anastomotic leaks do not always require a graft pancreatectomy, but arterial pseudoaneurysms, arteriove-nous fistulas, and wound dehiscence may. Bleeding frequently requires

1	pan-creatic pseudocysts. Anastomotic leaks do not always require a graft pancreatectomy, but arterial pseudoaneurysms, arteriove-nous fistulas, and wound dehiscence may. Bleeding frequently requires relaparotomy.Thrombosis usually occurs within the first week posttrans-plant. It manifests as a sudden increase in insulin requirements or as a sharp drop in urinary amylase levels. Venous thrombosis, which is more common than arterial thrombosis, is associated with distinct clinical symptoms, including a swollen and tender graft, hematuria, lower extremity edema, and deep vein throm-bosis, the latter two occurring ipsilaterally. Arterial thrombosis is less symptomatic and may not initially cause pain; its diag-nosis is usually confirmed by Doppler ultrasonography. Surgi-cal exploration in recipients with thrombosis usually requires a graft pancreatectomy.With the advent of advanced interventional radiologic procedures to drain intra-abdominal abscesses, the reoperation rate has markedly

1	with thrombosis usually requires a graft pancreatectomy.With the advent of advanced interventional radiologic procedures to drain intra-abdominal abscesses, the reoperation rate has markedly decreased. Pancreas transplant recipients are usually kept on broad-spectrum antimicrobial agents for the first 7 days posttransplant.The most common nonsurgical complication posttrans-plant is rejection. The incidence of rejection is about 30% within the first year. The diagnosis is usually based on an increase in serum amylase and lipase levels and, in bladder-drained recipi-ents, a decrease in urinary amylase levels. A sustained drop in urinary amylase levels greater than 25% from baseline should prompt a pancreas graft biopsy to rule out rejection. In enteric-drained recipients, one must rely on serum amylase and lipase levels only. Other signs and symptoms of rejection include tenderness over the graft, unexplained fever, and hyperglyce-mia, which usually is a late finding; fewer than 5% of

1	serum amylase and lipase levels only. Other signs and symptoms of rejection include tenderness over the graft, unexplained fever, and hyperglyce-mia, which usually is a late finding; fewer than 5% of all rejec-tion episodes can be reversed in its presence. The diagnosis of rejection should be confirmed by a percutaneous pancreas graft biopsy.Other nonsurgical complications include infections with CMV, HCV, or extra-abdominal bacteria or fungi; malignan-cies, such as PTLD; and, rarely, graft-versus-host disease. For such complications, the diagnosis and treatment are similar to what is recommended after other solid organ transplants.Bladder-drained pancreas recipients may experience an array of unique urologic complications. Usually the result of the irritating nature of pancreatic enzymes on the urothelium in the bladder and urethra, these urologic complications can lead to cystitis, hematuria, and dysuria. With the loss of bicarbonate from pancreatic secretions, dehydration and

1	on the urothelium in the bladder and urethra, these urologic complications can lead to cystitis, hematuria, and dysuria. With the loss of bicarbonate from pancreatic secretions, dehydration and metabolic acidosis are not uncommon. Many of these complications are chronic, such that approximately 20% to 30% of all bladder-drained recipients require conversion to enteric drainage within the first 5 years posttransplant.100Living Donor Pancreas TransplantsPancreas transplants using living donors also can be performed safely and successfully in select donors and recipients. Since 1979, about 150 such transplants have been performed world-wide, with 1-year graft survival rates in excess of 85% over the last decade. A meticulous donor evaluation using standard criteria remains key to a low donor metabolic and surgical com-plication rate. The concept of procuring the distal pancreas from a living donor is based on the observation that patients with benign or malignant pancreatic disorders can

1	metabolic and surgical com-plication rate. The concept of procuring the distal pancreas from a living donor is based on the observation that patients with benign or malignant pancreatic disorders can undergo a distal hemipancreatectomy without any serious change in endocrine function.Living donor pancreas transplants are ideal for patients with an identical twin, but other relatives can be suitable donors as well. In particular, patients with high PRA levels should be considered for a living donor transplant.Living donor pancreas transplants decrease the number of deaths of diabetic patients on the waiting list, help overcome the organ shortage, reduce mortality and morbidity, and improve the quality of life for patients with debilitating side effects of diabetes. The use of living donors also reduces the risk of graft rejection, as compared with the use of deceased donors. Yet living donor pancreas transplants remain relatively rare, per-formed under very selective circumstances. In

1	also reduces the risk of graft rejection, as compared with the use of deceased donors. Yet living donor pancreas transplants remain relatively rare, per-formed under very selective circumstances. In terms of surgical technique, the donor splenic artery and vein are anastomosed to the recipient’s external iliac artery and vein in an end-to-side fashion, and exocrine drainage can occur via an anastomosis Brunicardi_Ch11_p0355-p0396.indd 37701/03/19 6:54 PM 378BASIC CONSIDERATIONSPART IFigure 11-14. Segmental transplant with systemic vein and blad-der exocrine drainage. The donor splenic artery and splenic vein are anastomosed end-to-side to the recipient’s external iliac artery and vein. The splenic artery anastomosis is lateral and proximal to the splenic vein anastomosis. A two-layer ductocystostomy is constructed. (Reproduced from Gruessner RWG, Sutherland DER: Transplantation of the Pancreas. New York, NY: Springer, 2004.)of the pancreatic duct and transected end of the pancreas

1	is constructed. (Reproduced from Gruessner RWG, Sutherland DER: Transplantation of the Pancreas. New York, NY: Springer, 2004.)of the pancreatic duct and transected end of the pancreas to the bladder or bowel101 (Fig. 11-14).ResultsAs of December 2010, more than 35,000 pancreas transplants had been reported to the IPTR: more than 25,000 transplants in the United States and more than 10,000 in other countries. According to IPTR data, recipient age at the time of the trans-plant has increased significantly, and so has the number of trans-plants for patients with type 2 diabetes. The trend over time has been toward stricter donor criteria, with a concentration on younger donors, preferably trauma victims, and on short pan-creas graft preservation time.Drainage techniques have changed over time, too: enteric drainage of exocrine pancreatic secretions is now predominant, in combination with systemic drainage of the venous effluent of the pancreas graft. Immunosuppressive protocols have

1	time, too: enteric drainage of exocrine pancreatic secretions is now predominant, in combination with systemic drainage of the venous effluent of the pancreas graft. Immunosuppressive protocols have devel-oped toward antibody induction therapy, followed by administra-tion of tacrolimus and MMF for maintenance. Steroid avoidance has increased over time in all three recipient categories.Between 2005 and 2009 and 2010 and 2014, the num-ber of U.S. pancreas transplants declined by over 20%, while the overall number of pancreas transplants performed outside the United States has increased. The decline in U.S. numbers is predominantly due to the decline in primary and secondary pancreas after kidney transplants (PAK). During the time period studied, the number of PAK transplants dropped by 50%. In contrast, the number of simultaneous pancreas/kidney trans-plants (SPK) declined by only 10%, and the number of pan-creas transplants alone (PTA) by 20%. Over 90% of pancreas transplants worldwide

1	In contrast, the number of simultaneous pancreas/kidney trans-plants (SPK) declined by only 10%, and the number of pan-creas transplants alone (PTA) by 20%. Over 90% of pancreas transplants worldwide were performed, with a simultaneous kidney transplant and excellent results. Transplant outcomes in SPK improved significantly because of a decrease in the rates of technical and immunologic graft loss. In 2010 to 2014 vs. 2005 to 2009, U.S. SPK transplant patient survival at 1 year posttransplant increased from 95.7% to 97.4%, pancreas graft function increased from 88.3% to 91.3%, and kidney func-tion increased from 93.6% to 95.5%. A significant improve-ment was also noted in PAK transplants. One-year patient survival increased from 96.4% to 97.9%, and pancreas graft function increased from 81.0% to 86.0%. PTA 1-year patient survival remained constant at 97%, and pancreas 1-year graft survival improved from 81.0% to 85.7%. IPTR data show sig-nificant improvements in patient survival and

1	81.0% to 86.0%. PTA 1-year patient survival remained constant at 97%, and pancreas 1-year graft survival improved from 81.0% to 85.7%. IPTR data show sig-nificant improvements in patient survival and pancreas graft function rates since the inception of UNOS, over a course of 24 years.92,95,99,102 Clearly, pancreas transplants now offer excel-lent outcomes for patients with IDDM.Islet versus Pancreas TransplantsPancreas transplants are frequently compared with islet trans-plants (vide infra), which are less invasive and, therefore, more appealing. It is important to emphasize that these two types of transplants are not mutually exclusive but rather complementary. The results of islet transplants have improved over the past decade, but overall islet graft function, specifically long-term function, still significantly trails overall pancreas graft function.103 Islet transplants involve pancreas procurement (as described earlier) and then separation of islets from the exocrine

1	function, still significantly trails overall pancreas graft function.103 Islet transplants involve pancreas procurement (as described earlier) and then separation of islets from the exocrine pan-creatic tissues using proteolytic enzymes (as described later). The human pancreas contains about one million islets, of which half are lost during the isolation process. About 10,000 islets per kilo-gram of body weight are needed to achieve insulin independence when transplanted into the liver. Frequently, one donor pancreas does not suffice; in fact, up to four donor pancreases have been used for one islet recipient.Because of the relatively disappointing long-term out-comes, insurance providers in the United States do not provide reimbursement for islet transplants. Transplant centers with both pancreas and islet transplant programs follow an algorithm that favors islet transplants in patients with a high surgical risk and pancreas transplants in patients with a low surgical risk. Although

1	pancreas and islet transplant programs follow an algorithm that favors islet transplants in patients with a high surgical risk and pancreas transplants in patients with a low surgical risk. Although solitary donor pancreases are not in short supply, only one donor pancreas is required for a successful pancreas transplant; in con-trast, two to four donor pancreases are commonly used for one islet recipient with less favorable long-term outcomes.Of note, the primary goal of current islet transplant trials is not insulin independence but rather a reduction in the inci-dence and severity of hypoglycemic events, a reduction in exog-enous insulin requirements, and an amelioration of hemoglobin A1c levels. Islet transplants rarely maintain long-term insulin independence. A recent study showed a higher rate of insulin independence in PTA recipients than in recipients of an islet transplant alone, despite the use of up to three donor pancreases in each of the islet recipients.104 Until islet

1	higher rate of insulin independence in PTA recipients than in recipients of an islet transplant alone, despite the use of up to three donor pancreases in each of the islet recipients.104 Until islet transplant results sig-nificantly improve and include long-term insulin independence, a pancreas transplant remains the treatment of choice for β-cell replacement therapy in patients with IDDM.ISLET TRANSPLANTATIONTransplanting islets of Langerhans isolated from deceased donor pancreases is an appealing option for patients with type 1 diabetes. An islet transplant involves the procurement of a 6Brunicardi_Ch11_p0355-p0396.indd 37801/03/19 6:54 PM 379TRANSPLANTATIONCHAPTER 11donor pancreas and its transportation to a specialized islet iso-lation facility, where the pancreas is enzymatically digested; then, the islets are purified from the rest of the digested pan-creas using density gradients. The purified islets are then cul-tured and evaluated for their identity, viability, and

1	digested; then, the islets are purified from the rest of the digested pan-creas using density gradients. The purified islets are then cul-tured and evaluated for their identity, viability, and potency, before being infused into the portal vein of a diabetic recipient. When the procedure is successful, these islet cells engraft into the recipient and secrete insulin, providing excellent moment-to-moment control of blood glucose, as is seen with a whole-pancreas transplant.A successful islet transplant offers advantages over exog-enous insulin injections—advantages that are similar to those of a whole-pancreas transplant. These advantages include restor-ing β-cell secretory capacity, improving glucose counterregula-tion, restoring hypoglycemia awareness, providing perfect or near-perfect glucose homeostasis, and, potentially, preventing secondary diabetic complications.Unlike a whole-pancreas transplant, an islet transplant does not involve a major surgical procedure with its associated

1	homeostasis, and, potentially, preventing secondary diabetic complications.Unlike a whole-pancreas transplant, an islet transplant does not involve a major surgical procedure with its associated mortality and morbidity. Instead, it can generally be performed as an outpatient procedure using percutaneous catheter-based therapy to cannulate a branch of the portal vein, with minimal recovery time for the recipient. Potential complications associ-ated with islet injection include portal hypertension, portal vein thrombosis, hepatic abscesses, and bacteremia. Theoretically, islet transplants could have wider application (as compared with current practice and with whole-pancreas transplants), given the significantly lower surgical risk, the relatively small tissue vol-ume transplanted, and the potential for islet immunomodula-tion or immunoisolation, which could minimize or eliminate the need for immunosuppression.The first reported attempt at an islet transplant was in 1893 by

1	and the potential for islet immunomodula-tion or immunoisolation, which could minimize or eliminate the need for immunosuppression.The first reported attempt at an islet transplant was in 1893 by Watson-Williams and Harsant: they transplanted a sheep’s minced pancreas into the subcutaneous tissue of a young boy with ketoacidosis.105 The discovery of insulin may have reduced interest in islet transplants as a treatment for diabetes, at least until the realization that insulin could not provide perfect glyce-mic control and that, therefore, patients ultimately suffered dev-astating secondary complications. Several milestones ensued: the first whole-pancreas transplants,106 early success with rodent islet transplants,107 and then, in the 1970s, human islet autotrans-plants after pancreatectomy, in order to address the intractable pain associated with chronic pancreatitis, by Sutherland, Najarian, and colleagues in Minnesota.108Until recently, attempts to extend those trailblazing

1	in order to address the intractable pain associated with chronic pancreatitis, by Sutherland, Najarian, and colleagues in Minnesota.108Until recently, attempts to extend those trailblazing find-ings of clinical islet autotransplants to clinical islet allotrans-plants in patients with type 1 diabetes met with generally very poor success. For example, in 1995, a report of the International Islet Transplant Registry indicated that of 270 recipients, only 5% were insulin-independent at 1 year posttransplant.In 2000, Shapiro and colleagues reported the results of the Edmonton protocol, which enabled consistent diabetes rever-sal and short-term (<1 year) insulin independence.109-111 The Edmonton protocol prescribed transplanting a large number of freshly isolated islets (>10,000 islet equivalents per kilogram body weight, typically requiring the use of two to four pan-creases) with a specialized “islet-sparing,” steroid-free immu-nosuppressive protocol consisting of low-dose tacrolimus,

1	per kilogram body weight, typically requiring the use of two to four pan-creases) with a specialized “islet-sparing,” steroid-free immu-nosuppressive protocol consisting of low-dose tacrolimus, sirolimus, and IL-2 receptor antibody induction. Those results were replicated at other experienced transplant centers,112,113 but the rates of long-term (>5 year) insulin independence remained poor, well below those of whole-pancreas transplants.114 Still, despite the low rates of long-term insulin independence, most islet recipients were C-peptide positive and retained hypoglycemia awareness, indicating residual islet function and benefit. In fact, at 9 years posttransplant, 15% remained insulin-independent, and 73% had hypoglycemia awareness and corrected hemoglo-bin A1c levels.115In the mid-2000s, new trials began with the goal of estab-lishing protocols that enable insulin independence, using islets from a single donor pancreas; the results were good, especially with strict donor and

1	new trials began with the goal of estab-lishing protocols that enable insulin independence, using islets from a single donor pancreas; the results were good, especially with strict donor and recipient selection.116,117 In the most expe-rienced centers, long-term rates of diabetes reversal are now about 50% at 5 years posttransplant. The reasons include refine-ments in pancreas preservation, islet isolation, and culture pro-tocols, as well as the use of newer induction immunosuppressive agent combinations, such as a T-cell–depleting antibody (anti-CD3 antibody, alemtuzumab, or antithymocyte globulin) and a tumor necrosis factor-alpha (TNF-α) inhibitor (etanercept or infliximab). Presumably, viable β-cell mass is now preserved, both preand posttransplant.116-120 Thus, islet transplant results are approaching those of whole-pancreas transplants; however, because islets from more than one pancreas are typically needed, those results cannot be directly compared with the results of

1	results are approaching those of whole-pancreas transplants; however, because islets from more than one pancreas are typically needed, those results cannot be directly compared with the results of whole-pancreas transplants.121,122In the United States, an islet transplant is still officially deemed an experimental procedure. In contrast, since 2001, it has been considered a standard of care and is fully reimbursed in Canada and, more recently, in the United Kingdom, Sweden, Switzerland, France, and Italy as well.118 The full potential of islet transplants remains to be realized, but the future is excit-ing. As the latest improvements in pancreas preservation, islet isolation and purification, islet culture, and islet immunoiso-lation are implemented clinically, the hope is that sustained insulin independence may become consistently possible with a single pancreas donor and without the need for systemic immunosuppression.LIVER TRANSPLANTATIONThe first attempts at liver transplants in

1	insulin independence may become consistently possible with a single pancreas donor and without the need for systemic immunosuppression.LIVER TRANSPLANTATIONThe first attempts at liver transplants in the late 1960s through the 1980s were largely experimental endeavors, with a 1-year survival rate of only 30%. But breakthroughs in immunosup-pression, surgical technique, organ preservation, anesthesia, and critical care have improved that rate to approximately 85% today. Liver transplants remain daunting, especially in the face of an organ shortage that results in sicker potential candidates. Unfortunately, the perioperative mortality rate and the 1-year mortality rate are among the highest of any surgical operation currently performed.HistoryThe first experimental liver transplants in dogs are often attrib-uted to C. Stuart Welch in 1955 and then Jack Cannon in 1956. However, current scholarship reveals that Vittorio Staudacher first described the technique in 1952.123 A series of

1	dogs are often attrib-uted to C. Stuart Welch in 1955 and then Jack Cannon in 1956. However, current scholarship reveals that Vittorio Staudacher first described the technique in 1952.123 A series of canine experiments followed, which refined the surgical technique to ensure perioperative survival.The next obstacle—immunologic rejection—was addressed by drug immunosuppression with AZA and predni-sone. The first human liver transplant trials started in 1963 with Thomas Starzl, but a series of deaths led to a voluntary morato-rium for 3.5 years. With the resumption of clinical transplants in 1967, Starzl performed the first successful liver transplant. Brunicardi_Ch11_p0355-p0396.indd 37901/03/19 6:54 PM 380BASIC CONSIDERATIONSPART IStill, for the next decade, survival rates were dismal: only 20% of the 170 liver transplant recipients in Starzl’s program at the University of Colorado survived more than 5 years.124Several innovations dramatically improved outcomes. The advent of

1	only 20% of the 170 liver transplant recipients in Starzl’s program at the University of Colorado survived more than 5 years.124Several innovations dramatically improved outcomes. The advent of better immunosuppressive drugs was instrumental. In 1978, cyclosporine was introduced clinically in England. It was soon combined with prednisone to great effect. The arrival of tacrolimus in the 1990s further improved graft survival.Technical advances were also significant. Donor procure-ment techniques and cold organ preservation protocols were standardized, and the recipient operation was also refined. Choledochocholedochostomy or choledochojejunostomy to a Roux-en-Y limb became standard and significantly decreased the frequency of biliary complications. Innovations, including living donor liver transplants and deceased donor split-liver transplants, enabled more pediatric recipients to be transplanted. Improvements in portosystemic shunting and perioperative criti-cal care also were

1	liver transplants and deceased donor split-liver transplants, enabled more pediatric recipients to be transplanted. Improvements in portosystemic shunting and perioperative criti-cal care also were contributory.IndicationsIn general, any form of irreversible liver disease is an indication for a liver transplant. Chronic alcoholic disease and HCV are the most common indications in the United States. An extensive list of acute and chronic diseases of the liver that are treatable by a liver transplant is provided in Table 11-6.Offering transplants to alcoholic patients has always drawn some opposition because of the perception of it being a self-inflicted illness, as well as concerns about recidivism and the recipient’s possible inability to maintain postoperative immunosuppression and care. Yet studies have shown that such patients have excellent outcomes and that liver transplants for Table 11-6Diseases amenable to treatment by a liver transplantAutoimmune liver diseases Autoimmune

1	Yet studies have shown that such patients have excellent outcomes and that liver transplants for Table 11-6Diseases amenable to treatment by a liver transplantAutoimmune liver diseases Autoimmune hepatitis Primary biliary cirrhosis Primary sclerosing cholangitisCongenital Biliary atresiaViral hepatitis Hepatitis B Hepatitis CAlcoholic liver diseaseMetabolic diseases α1-Antitrypsin deficiency Cystic fibrosis Hemochromatosis Tyrosinemia Wilson’s diseaseHepatic malignancy Hepatocellular carcinoma Neuroendocrine tumor metastatic to liverFulminant hepatic failureOther Alagille syndrome Cryptogenic cirrhosis Budd-Chiari syndrome Polycystic liver disease Amyloidosisthem are cost-effective.125-127 Because patients who drink 4 to 8 ounces of liquor daily for 10 to 15 years have an increased risk of developing cirrhosis, the general requirement for accep-tance as a transplant candidate is 6 months of abstinence. Fur-thermore, most transplant centers recommend rehabilitation and Alcoholics

1	of developing cirrhosis, the general requirement for accep-tance as a transplant candidate is 6 months of abstinence. Fur-thermore, most transplant centers recommend rehabilitation and Alcoholics Anonymous programs.Transplants for HCV have yielded worse outcomes than transplants for other diseases.128 The reason is the universal recurrence of the virus posttransplant. Viral levels reach pre-transplant levels as early as 72 hours posttransplant.129 The course of the viral infection is often accelerated posttransplant: 10% to 20% of recipients develop cirrhosis after just 5 years.130 Studies have suggested that use of older donors may increase the chance of aggressive recurrence.131 The best method to prevent recurrence would be to eradicate the infection pretransplant, but doing so is not always possible because patients with decom-pensated cirrhosis often cannot tolerate treatment. Once recur-rence occurs, treatment methods are limited. One study found that pegylated interferon and

1	always possible because patients with decom-pensated cirrhosis often cannot tolerate treatment. Once recur-rence occurs, treatment methods are limited. One study found that pegylated interferon and ribavirin therapy achieved a sus-tained viral response in 44% of patients.132A substantial number of patients undergo liver transplants for cholestatic disorders. Primary biliary cirrhosis, an autoim-mune disease, is characterized by damage to the intralobular bile ducts that progresses to liver cirrhosis. Trends toward earlier treatment may explain the slight decrease in liver transplants for this disorder.133 Posttransplant outcomes in patients with this disorder have been excellent, with many centers achieving 1-year survival rates of 90% to 95%. Recurrence is relatively uncommon: a large series reported a 30% recurrence rate at 10 years posttransplant.134The second most common cholestatic disorder among liver transplant candidates is primary sclerosing cholangitis. It is characterized

1	reported a 30% recurrence rate at 10 years posttransplant.134The second most common cholestatic disorder among liver transplant candidates is primary sclerosing cholangitis. It is characterized by inflammation and fibrosis of large intraand extrahepatic biliary ducts; 70% of such patients also have inflammatory bowel disease. Recurrent cholangitis is common and increases mortality rates beyond what would be expected on the basis of laboratory values. On behalf of such patients, appeals can often be made for priority in allocation to the UNOS regional review boards. Posttransplant outcomes for such patients have been excellent. Primary sclerosing cholangitis is a significant risk factor for cholangiocarcinoma, so annual screen-ings (including imaging and measurement of serum CA 19-9 levels) should be carried out. Recurrence is fairly uncommon: studies have reported a recurrence rate of up to 20% at 10 years posttransplant.135Progressive metabolic disorders also are treatable with liver

1	should be carried out. Recurrence is fairly uncommon: studies have reported a recurrence rate of up to 20% at 10 years posttransplant.135Progressive metabolic disorders also are treatable with liver transplants. Hemochromatosis, an inherited disorder, results in excessive intestinal iron absorption. Iron deposition can cause cirrhosis and severe cardiomyopathy. Careful cardiac evaluation is necessary pretransplant.Another metabolic disorder, α1-antitrypsin deficiency, is characterized by insufficient levels of a protease inhibitor, resulting in early-onset emphysema and cirrhosis. Careful pul-monary evaluation is necessary pretransplant.Wilson’s disease, an autosomal recessive disorder char-acterized by impaired cellular copper transport, leads to cop-per accumulation in the liver, brain, and cornea. Patients can develop significant neurologic complications and cirrhosis. Several reports suggest improvement of neurologic deficiencies posttransplant.136,137Transplants can also be

1	and cornea. Patients can develop significant neurologic complications and cirrhosis. Several reports suggest improvement of neurologic deficiencies posttransplant.136,137Transplants can also be performed in patients with hepatic malignancies, but only in accordance with strict criteria. Brunicardi_Ch11_p0355-p0396.indd 38001/03/19 6:54 PM 381TRANSPLANTATIONCHAPTER 11Hepatocellular carcinoma (HCC), a complication of cirrhosis, is the most common type of hepatic malignancy. Resection is the first line of treatment if possible, but often, cirrhosis is too advanced. If the tumor meets the Milan criteria, a liver trans-plant can be performed. These criteria were established by a landmark paper in 1996 showing that patients with a single tumor under 5 cm in diameter, or with three tumors under 3 cm in diameter, in the absence of vascular invasion, had a 4-year survival rate of 85%.138 Patients with such tumors receive excep-tion points, based on their UNOS region, allowing for a timely

1	3 cm in diameter, in the absence of vascular invasion, had a 4-year survival rate of 85%.138 Patients with such tumors receive excep-tion points, based on their UNOS region, allowing for a timely transplant before their tumors spread.Transplants for cholangiocarcinoma remains controver-sial but may be performed if the center has an experimental protocol in place that entails strict recipient selection. The use of a multimodality oncologic approach including neoadjuvant chemo radiotherapy with subsequent OLT achieves excellent results for patients with localized, regional lymph node-negative phCCA. Patient survival after OLT is comparable to the results of OLT for other causes.139Acute fulminant hepatic failure also is an indication for a liver transplant; in fact, such patients are the highest priority for the next available liver in their UNOS region. This devas-tating illness is defined by acute and severe liver injury with impaired synthetic function and encephalopathy in a person

1	priority for the next available liver in their UNOS region. This devas-tating illness is defined by acute and severe liver injury with impaired synthetic function and encephalopathy in a person who had normal liver function. It is often caused by acetaminophen overdose; acute fulminant viral hepatitis A, B, and E; other viral infections; drug toxicity; ingestion of Amanita mushrooms; acute fatty liver of pregnancy; or Wilson’s disease. A significant number of patients will recover with supportive care. The diffi-culty lies in predicting who will not recover and therefore would benefit from a liver transplant. The King’s College criteria were developed for this purpose: patients with acetaminophen-induced disease, a pH <7.3 or grade III/IV encephalopathy, a pro-thrombin time >100 seconds, and serum creatinine >3.4 mg/dL meet those criteria.140 Management of acute liver failure is very intensive. Such patients suffer from severe coagulopathy, hypoglycemia, lactic acidosis, and renal

1	and serum creatinine >3.4 mg/dL meet those criteria.140 Management of acute liver failure is very intensive. Such patients suffer from severe coagulopathy, hypoglycemia, lactic acidosis, and renal dysfunction. They are susceptible to infections, which are frequently overwhelming. Cerebral edema, a serious complication of acute liver failure, is a leading cause of death from brain herniation. Intracranial pressure monitoring and serial imaging are often necessary; if a patient develops irreversible brain damage, a transplant is not performed.Recipient SelectionThe diagnosis of cirrhosis itself is not an indication for a trans-plant. Patients may have compensated cirrhosis for years such that the traditional indication for a transplant is decompensated cirrhosis, manifested by hepatic encephalopathy, ascites, spon-taneous bacterial peritonitis, portal hypertensive bleeding, and hepatorenal syndrome (each described below).Hepatic encephalopathy is an altered neuropsychiatric state caused

1	ascites, spon-taneous bacterial peritonitis, portal hypertensive bleeding, and hepatorenal syndrome (each described below).Hepatic encephalopathy is an altered neuropsychiatric state caused by metabolic abnormalities resulting from liver failure. The early stages result in sleep disturbances and depression. As the liver disease progresses, patients can become somnolent and confused and, in the end stages, comatose. Ammonia is pro-duced by enterocytes from glutamine and from colonic bacterial catabolism, and the use of serum ammonia levels as a marker of encephalopathy is controversial because a variety of factors can influence levels. Hyperammonemia suggests worsening liver function and bypass of portal blood flow around the liver. GI bleeding and infection can exacerbate hepatic encephalopathy.Ascites (the accumulation of fluid in the abdominal cavity) that is caused by cirrhosis is a transudate with a high serum-ascites gradient (>1.1 g/dL). Associated with portal hypertension, it

1	(the accumulation of fluid in the abdominal cavity) that is caused by cirrhosis is a transudate with a high serum-ascites gradient (>1.1 g/dL). Associated with portal hypertension, it is treated initially with sodium restriction and diuretics. Refractory ascites necessitates large-volume paracentesis and eventually a transjugular intrahepatic portosystemic shunt (TIPS). Contrain-dications to TIPS placement include significant hepatic enceph-alopathy, advanced liver disease, congestive heart failure, renal insufficiency, and severe pulmonary hypertension.141Spontaneous bacterial peritonitis, an infection of the ascitic fluid without an evident intra-abdominal source, is char-acterized by fever, abdominal pain, and an ascitic fluid poly-morphonuclear count ≥250 cell/mm3 on paracentesis. The first line of empiric treatment is with a third-generation cephalospo-rin because the majority of cases are caused by aerobic gram-negative microbes such as E. coli, although Gram stain and culture

1	first line of empiric treatment is with a third-generation cephalospo-rin because the majority of cases are caused by aerobic gram-negative microbes such as E. coli, although Gram stain and culture results should be used to guide therapy.Portal hypertensive bleeding can be a devastating event for patients with cirrhosis. Each bleeding event carries a 30% mortality rate and accounts for a third of all deaths related to cirrhosis. Only 50% of bleeding events cease spontaneously, so treatment must be expedient. The initial medical treatment is with vasopressin and octreotide. The initial intervention is endoscopy with sclerotherapy and band ligation of bleeding varices. If those initial attempts fail, more aggressive treatment is necessary with a balloon tamponade (using a Sengstaken-Blakemore tube) and with emergent TIPS placement. The last line of treatment is emergency surgery to place a portosystemic shunt, transect the esophagus, or devascularize the gastroesoph-ageal junction

1	tube) and with emergent TIPS placement. The last line of treatment is emergency surgery to place a portosystemic shunt, transect the esophagus, or devascularize the gastroesoph-ageal junction (Sugiura procedure). Preventing variceal bleed-ing is essential and can be achieved, with some success, using β-blockers.Hepatorenal syndrome is a form of acute renal failure that develops as liver disease worsens. The etiology is unclear, but splanchnic vasodilation from portal hypertension and increased production of circulating vasodilators result in a decline in renal perfusion. Characterized by oliguria (<500 mL of urine/day) and low urine sodium levels (<10 mEq/L), hepatorenal syndrome is often reversed by a liver transplant, even after dialysis depen-dence. Pretransplant, other causes of renal failure need to be excluded, including ATN, drug nephrotoxicity, and chronic renal disease. The initial medical therapy includes octreotide, midodrine, and vasopressin analogs, but the syndrome often

1	failure need to be excluded, including ATN, drug nephrotoxicity, and chronic renal disease. The initial medical therapy includes octreotide, midodrine, and vasopressin analogs, but the syndrome often progresses to dialysis dependence.The Model for End-Stage Liver Disease (MELD) was originally developed to assess risk for TIPS placement.142 Later analysis revealed it to be an excellent model to predict survival among patients with cirrhosis, especially those on the waiting list for a liver transplant.143 In 2002, liver graft allocation was restructured to be based on the MELD score.Although the historic indication for a liver transplant is decompensated cirrhosis, a landmark analysis comparing wait-ing list mortality with posttransplant mortality established that a minimum MELD score of 18 is necessary to have a survival benefit posttransplant. A MELD score between 15 and 18 does not confer a survival advantage, but a transplant may be justified if the patient has significant morbidity

1	is necessary to have a survival benefit posttransplant. A MELD score between 15 and 18 does not confer a survival advantage, but a transplant may be justified if the patient has significant morbidity from cirrhosis.144Acute liver failure itself is an indication for a liver trans-plant. To qualify for Status 1 (first priority for a donor liver within the UNOS region), the transplant candidate must meet the following criteria: (a) onset of hepatic encephalopa-thy within 8 weeks after the first symptoms of liver disease; Brunicardi_Ch11_p0355-p0396.indd 38101/03/19 6:54 PM 382BASIC CONSIDERATIONSPART I(b) absence of preexisting liver disease; and (c) ventilator depen-dence, dialysis, or an international normalized ratio (INR) >2.0.ContraindicationsIn general terms, contraindications to a liver transplant include insufficient cardiopulmonary reserve, uncontrolled malignancy or infection, and refractory noncompliance. Older age is only a relative contraindication: carefully selected

1	a liver transplant include insufficient cardiopulmonary reserve, uncontrolled malignancy or infection, and refractory noncompliance. Older age is only a relative contraindication: carefully selected recipients over the age of 70 years can achieve satisfactory outcomes.145Patients with reduced cardiopulmonary reserve are unlikely to survive a liver transplant. Candidates should have a normal ejection fraction. If coronary arterial disease is pres-ent, they should undergo revascularization pretransplant. Severe chronic obstructive pulmonary disease (COPD) with oxygen dependence is a contraindication. Severe pulmonary hyper-tension with a mean pulmonary artery pressure greater than 35 mmHg that is refractory to medical therapy is also a contra-indication. Candidates with pulmonary hypertension should be evaluated with a right heart catheterization.For candidates with alcoholic liver disease, few reliable predictors of posttransplant relapse exist.146 Most centers require 6 months of

1	should be evaluated with a right heart catheterization.For candidates with alcoholic liver disease, few reliable predictors of posttransplant relapse exist.146 Most centers require 6 months of abstinence from drugs and alcohol. Insurance com-panies often make more stringent demands, including random drug screening and 1 year of abstinence.Uncontrolled infections pretransplant are a substantial risk posttransplant when the patient becomes significantly immuno-suppressed. Fungal and multidrug-resistant bacterial infections are relative contraindications. Some centers require an extended period of treatment and documented eradication pretransplant. HIV infection is a relative contraindication; some centers have strict protocols that exclude patients with a history of acquired immunodeficiency syndrome (AIDS)-related illnesses as well as those who are coinfected with HCV.Ideally, patients with a history of malignancy (with the exception of HCC) should be cured of the cancer pretransplant.

1	syndrome (AIDS)-related illnesses as well as those who are coinfected with HCV.Ideally, patients with a history of malignancy (with the exception of HCC) should be cured of the cancer pretransplant. In most cases, this means eradication, completion of curative therapy, and absence of recurrence over a certain period of time, which varies by the tumor type, but can be up to 5 years or lon-ger for aggressive tumors (see “Malignancies”).Surgical ProcedureA liver transplant is among the most extensive operations per-formed, and it can be associated with considerable blood loss. A bilateral subcostal incision with midline extension is used. Mechanical retraction spreads the rib cage to allow access. The ligamentous attachments of the liver are dissected free. The vascular structures are isolated, including the suprahepatic and infrahepatic vena cava, the portal vein, and hepatic artery (Fig. 11-15). The bile duct, portal structures, and vena cava are divided, completing the hepatectomy

1	including the suprahepatic and infrahepatic vena cava, the portal vein, and hepatic artery (Fig. 11-15). The bile duct, portal structures, and vena cava are divided, completing the hepatectomy (Fig. 11-16)—often the bloodiest and most difficult part of the operation, particularly in the presence of extensive varices and severe coagulopathy.After the liver is removed, the anhepatic phase begins. This phase is characterized by the absence of inferior vena caval return to the heart and by portal congestion due to clamp-ing of the portal vein. Significant hemodynamic instability and increased variceal bleeding can occur. Patients who are unable to tolerate this phase can be placed on venovenous bypass, with cannulas drawing blood from the IVC via the femoral vein and via the portal vein, returning it to the systemic circulation via the subclavian vein. Venovenous bypass itself can cause complica-tions, including air embolism, thromboembolism, and trauma to the cannulated vessels.Figure

1	it to the systemic circulation via the subclavian vein. Venovenous bypass itself can cause complica-tions, including air embolism, thromboembolism, and trauma to the cannulated vessels.Figure 11-15. Cirrhotic liver immobilized in preparation for com-plete hepatectomy.Figure 11-16. Isolation and division of the hilar structures to dis-eased liver-hepatic artery, portal vein, and common bile duct.The donor liver is placed in the orthotopic position. The suprahepatic vena caval anastomosis is performed first in an end-to-end fashion, followed by the infrahepatic vena caval and portal anastomosis, both also end-to-end. The liver is then reper-fused, often leading to a period of hemodynamic instability and cardiac arrhythmias due to the release of byproducts of ischemia from the donor liver. Coagulopathy also can worsen because of these byproducts as well as fibrinolysis.The arterial anastomosis between the donor common hepatic or celiac trunk is most often performed with the recipi-ent

1	Coagulopathy also can worsen because of these byproducts as well as fibrinolysis.The arterial anastomosis between the donor common hepatic or celiac trunk is most often performed with the recipi-ent CHA in an end-to-end fashion. Of course, many variations are possible. After arterial reperfusion, the bile duct anastomosis is performed between the donor and recipient common ducts, also in an end-to-end fashion. If necessary for technical reasons, the recipient common duct can be joined to a Roux-en-Y limb. Some surgeons choose to insert a T-tube or place internal stents in the common bile duct to protect the anastomosis.The piggyback technique is a common variation of the standard technique. The recipient’s IVC is preserved by care-fully dissecting off the posterior aspect of the liver. This added dissection is a disadvantage of this variation, often increas-ing hepatectomy time and blood loss. The recipient’s liver is Brunicardi_Ch11_p0355-p0396.indd 38201/03/19 6:55 PM

1	liver. This added dissection is a disadvantage of this variation, often increas-ing hepatectomy time and blood loss. The recipient’s liver is Brunicardi_Ch11_p0355-p0396.indd 38201/03/19 6:55 PM 383TRANSPLANTATIONCHAPTER 11removed by dividing it at the confluence of the hepatic veins. The preserved IVC is an advantage of this variation, allow-ing venous return from the lower body to the heart during the anhepatic phase and improving renal perfusion. No randomized studies, however, have demonstrated the superiority of the pig-gyback technique over the standard technique.Pediatric TransplantsOutcomes after pediatric liver transplants are among the best after any type of transplant, with a 1-year survival rate of 90%. The most common indication is biliary atresia. After diagno-sis is confirmed, a Kasai procedure is promptly carried out: a Roux-en-Y loop of bowel is directly anastomosed to the hilum of the liver. The Kasai procedure often allows time for the chil-dren to grow in size,

1	a Kasai procedure is promptly carried out: a Roux-en-Y loop of bowel is directly anastomosed to the hilum of the liver. The Kasai procedure often allows time for the chil-dren to grow in size, reducing the risk of a transplant when it is required, as it eventually is in 75% of such children.The other common indication for a pediatric liver trans-plant is a metabolic disorder, such as α1-antitrypsin deficiency, tyrosine metabolism deficiencies, and primary oxalosis. Since the MELD score was developed for adults, pediatric liver allo-cation is based on an analogous model, the Pediatric End-Stage Liver Disease (PELD) score, which incorporates bilirubin lev-els, INR, albumin levels, age, and growth failure.The surgical procedure is similar to the adult procedure. Graft implantation is more challenging, given the pediatric recipient’s smaller vascular structures. As a result, surgical complications are much more common in pediatric recipients. Hepatic artery thrombosis is about three times

1	given the pediatric recipient’s smaller vascular structures. As a result, surgical complications are much more common in pediatric recipients. Hepatic artery thrombosis is about three times more common. Donor size matching is very important in the pediatric popula-tion and often limits the donor pool for pediatric recipients. To address this issue, deceased donor split-liver transplants and liv-ing donor transplants (both described in the following sections) have been developed.Deceased Donor Split-Liver TransplantsA deceased donor allograft can be split into two grafts, most frequently into a left lateral segment for a child and an extended right segment for an adult (Fig. 11-17). It can be done in vivo (during the donor operation) or ex vivo (on the back table after the donor liver is removed). Both techniques have simi-lar outcomes. Increased morbidity is associated with splitting allografts, whether for adult or pediatric recipients; however, the technique is justified given the

1	removed). Both techniques have simi-lar outcomes. Increased morbidity is associated with splitting allografts, whether for adult or pediatric recipients; however, the technique is justified given the donor shortage and has been important for improving access to transplants for pediatric recipients.147Living Donor TransplantsDonation by an adult living donor to an adult recipient requires either the right or left lobe of the liver (Fig. 11-18). Donation by an adult living donor to a pediatric recipient requires the left lateral lobe (Fig. 11-19). Donor safety is paramount. The over-all donor mortality rate after donation was 0.4%, and the over-all complication rate was 40%, with multiple complications occurring in 19% of the patients. The rate of serious complica-tions resulting in lasting disability was 1.1%, with liver failure or death in 0.4%148 Careful donor selection is vital. Potential donors should be medically and psychologically healthy, their hepatic anatomy should be

1	lasting disability was 1.1%, with liver failure or death in 0.4%148 Careful donor selection is vital. Potential donors should be medically and psychologically healthy, their hepatic anatomy should be amenable to donation, and absolutely no coercion can occur. A separate donor team should serve as the donor advocate and thoroughly explain all risks.Careful recipient selection is essential. Transplant can-didates also must qualify for a deceased donor liver trans-plant because a significant number of living donor transplant DonorLeft hepatic veinLeft portal veinLeft hepatic arteryRecipientHepatic arteryPortal veinRoux limbFigure 11-17. Donor and recipient procedure for living donor liver transplant into a pediatric recipient.recipients will eventually require a retransplant. Transplant can-didates should be medically fit enough to withstand the rigors of the operation and of the postoperative course with a partial graft. An absolute contraindication is a critical illness: the limited

1	should be medically fit enough to withstand the rigors of the operation and of the postoperative course with a partial graft. An absolute contraindication is a critical illness: the limited suc-cess of such transplants does not justify the risks to the living donor. The obvious advantages of a living donor transplant are that it can be done expediently (avoiding the waiting list mortal-ity associated with candidates for a deceased donor transplant) and that it can be planned.Postoperative CareA liver transplant imposes significant trauma on the major organ systems. Immediately posttransplant, the first goal is to stabi-lize those systems. Acid-base equilibrium and hemodynamic stability are often difficult to maintain but are essential. Periods of hypotension can increase the risk of hepatic artery throm-bosis. Careful attention needs to be paid to ongoing bleeding. Appropriate hemoglobin levels should be maintained. Ongoing bleeding mandates a return trip to the operating room; the

1	artery throm-bosis. Careful attention needs to be paid to ongoing bleeding. Appropriate hemoglobin levels should be maintained. Ongoing bleeding mandates a return trip to the operating room; the rate of reoperation can be as high as 25% among high-risk patients. Transfusion of platelets and fresh frozen plaza must be done prudently because theoretically their administration can increase the risk of hepatic artery thrombosis. Graft function should be evaluated frequently; if it is impaired, an ultrasound is urgently required to assess for the presence of vascular complications.Brunicardi_Ch11_p0355-p0396.indd 38301/03/19 6:55 PM 384BASIC CONSIDERATIONSPART ICHARHVMPVMHVLHVLHVS4S2FLS3IVCIVCLHVRHVMHVLHDLHALPVMPVPHACBDR.P.V.R.P.A.RHDC.A.C.D.ABFigure 11-18. A. Hepatic transection completed for right lobe removal. CA = cystic artery; CBD = common bile duct; CD = cystic duct; FL = falciform ligament; IVC = inferior vena cava; LHD = left hepatic duct; LHV= left hepatic vein; MHV = middle

1	for right lobe removal. CA = cystic artery; CBD = common bile duct; CD = cystic duct; FL = falciform ligament; IVC = inferior vena cava; LHD = left hepatic duct; LHV= left hepatic vein; MHV = middle hepatic vein; MPV = main portal vein; PHA = proper hepatic artery; RHA = right hepatic artery; RHV = right hepatic vein; RPV = right portal vein; S2, S3, S4 = segments 2, 3, and 4. B. Implantation of the donor right lobe with the MHV. CHA = common hepatic artery. (Reproduced with permission from Gruessner RWG, Benedetti E: Living Donor Organ Transplantation. New York, NY: McGraw-Hill Education; 2008.)Evaluation of Graft FunctionEvaluation of the graft begins in the operating room. Its appearance overall, any swelling, and the quantity and quality of bile production after reperfusion can help assess function. In the intensive care unit, hemodynamic stability, correc-tion of coagulopathy, euglycemia, successful temperature regulation, clearance of lactic acid, and restoration of neuro-logic

1	function. In the intensive care unit, hemodynamic stability, correc-tion of coagulopathy, euglycemia, successful temperature regulation, clearance of lactic acid, and restoration of neuro-logic status are all signs of a functioning graft, even before the first set of liver function test results are obtained. Trans-aminases usually peak by postoperative day 2. An aspartate transaminase (AST) level greater than 2500 IU/L is sugges-tive of significant injury. Cholestasis usually peaks from Brunicardi_Ch11_p0355-p0396.indd 38401/03/19 6:55 PM 385TRANSPLANTATIONCHAPTER 11Figure 11-19. A. Hepatic transection completed for removal of left lateral segments (S2 and S3). Bile ducts to segments 2 and 3 divided; vascular structures still intact. B. Implantation of the donor left lobe. (Reproduced with permission from Gruessner RWG, Benedetti E: Living Donor Organ Transplantation. New York, NY: McGraw-Hill Education; 2008.)S2 + 3ABpostoperative day 7 to 12. The INR should improve shortly

1	with permission from Gruessner RWG, Benedetti E: Living Donor Organ Transplantation. New York, NY: McGraw-Hill Education; 2008.)S2 + 3ABpostoperative day 7 to 12. The INR should improve shortly after reperfusion.In 3% to 4% of patients undergoing a liver transplant, the graft does not function for any identifiable reason, a condition termed primary nonfunction; in such cases, a retransplant is the only option. Some studies suggest that a peak AST level of 5000 IU/L may be predictive of primary nonfunction.149-151 Factors associated with primary nonfunction include donor macroste-atosis, prolonged cold and warm ischemic times, and prolonged donor hospital stay.151ComplicationsVascular complications occur in about 8% to 12% of recipi-ents and include thrombosis, stenosis, and pseudoaneurysm formation.The most common vascular complication is hepatic artery thrombosis. Initial reviews suggest that its incidence is between 1.6% and 4%152; the mortality rate is 50%, even after defini-tive

1	formation.The most common vascular complication is hepatic artery thrombosis. Initial reviews suggest that its incidence is between 1.6% and 4%152; the mortality rate is 50%, even after defini-tive therapy.153 Early presentation can be quite dramatic, with fulminant hepatic necrosis, primary nonfunction, transamini-tis, or fever. Late presentation, however, can be asymptom-atic or subtle, with cholangitis, bile leak, mild transaminitis, hepatic abscesses, or failure to thrive. Diagnostic imaging with ultrasound has more than 90% sensitivity and specificity. If hepatic artery thrombosis is identified, urgent reexploration is needed. A thrombectomy or revision of an anastomosis may be successful, but with significant hepatic necrosis, a retransplant is necessary.Thrombosis of the portal vein is very uncommon. Signs of early thrombosis include liver dysfunction, ascites, and variceal bleeding. Upon diagnosis, an operative thrombectomy should be attempted.Biliary complications remain the

1	is very uncommon. Signs of early thrombosis include liver dysfunction, ascites, and variceal bleeding. Upon diagnosis, an operative thrombectomy should be attempted.Biliary complications remain the “Achilles’ heel” of liver transplantation, affecting 10% to 35% of these organ recipients. Signs include fever and abdominal pain, with bilious drainage from surgical drains. Diagnosis is made with cholangiography.Complications manifest themselves as leaks or strictures. Leaks require a reoperation and surgical correction, whereas strictures can most often be managed with radiologic or endo-scopic interventions. Two common reconstructions are cho-ledochostomy and choledochojejunostomy. Some centers also routinely use T-tube stents or internal stents. Consensus has not been reached as to which reconstruction technique is superior. Early infectious complications are often associated with initial graft function and pretransplant risk factors. Intra-abdominal infections should raise concerns of

1	technique is superior. Early infectious complications are often associated with initial graft function and pretransplant risk factors. Intra-abdominal infections should raise concerns of a possible bile leak. Fungal infections are often associated with poor graft function. Given the immunosuppressed and compromised state of liver recipi-ents, early infectious complications can be devastating.The types of opportunistic infections that occur in liver transplant recipients are similar to those that occur in other types of solid organ transplant recipients and are due to suppression of cell-mediated immunity by chronic immunosuppressive drug administration.Acute rejection occurs in approximately 20% of liver recipients. The first line of treatment is with a high dose of a corticosteroid, which is usually effective; if not, antilympho-cyte therapy is initiated. Rejection of the liver (unlike other transplanted organs) does not adversely affect patient or graft survival rates. Maintenance

1	is usually effective; if not, antilympho-cyte therapy is initiated. Rejection of the liver (unlike other transplanted organs) does not adversely affect patient or graft survival rates. Maintenance immunosuppression consists of a corticosteroid, tacrolimus, and mycophenolate.INTESTINE AND MULTIVISCERAL TRANSPLANTATIONAfter the introduction of long-term total parenteral nutrition (TPN) in the late 1970s and the early success of liver, kidney, and heart transplants, the first attempts at intestine transplants were made. Over the first two decades, the results were dismal. But the introduction of the immunosuppressive drug tacrolimus in the late 1980s led to significant improvement in graft and patient survival rates. Nonetheless, intestine transplants remain the least frequently performed of all transplants, with the lowest graft survival rates.The main obstacle is the high immunogenicity of the intestine, caused by its abundant lymphoid tissue. High lev-els of immunosuppression are

1	of all transplants, with the lowest graft survival rates.The main obstacle is the high immunogenicity of the intestine, caused by its abundant lymphoid tissue. High lev-els of immunosuppression are needed, yet the rejection rate is still high. The microbial colonization of the intestine confers the risk of translocation of pathogenic microorganisms into the recipient’s circulation, causing severe systemic infections. Through the first decade of the 21st century, the survival of patients on long-term TPN was superior to the survival of intes-tine transplant recipients, so a transplant was considered only as rescue therapy for patients with life-threatening TPN-related complications.Brunicardi_Ch11_p0355-p0396.indd 38501/03/19 6:55 PM 386BASIC CONSIDERATIONSPART IOver the last several years, improvements in surgical tech-niques, in perioperative and postoperative care, and particularly in immunosuppressive protocols have led to significantly bet-ter patient and graft survival rates

1	improvements in surgical tech-niques, in perioperative and postoperative care, and particularly in immunosuppressive protocols have led to significantly bet-ter patient and graft survival rates posttransplant.154 Recent data indicate that survival rates after an intestine transplant often are better than, or at least similar to, survival rates among patients receiving chronic TPN in the home setting with improved qual-ity of life in selected patients.155 Today, an intestine or multivis-ceral transplant is recognized as a feasible treatment.Indications and Recipient SelectionAn intestine transplant is indicated for patients with irreversible intestine failure in combination with TPN failure. The defini-tion of intestine failure does not specify the exact length of the remaining intestine. Intestine failure is typically multifactorial. Variables include what part of the small intestine is absent, whether or not the ileocecal valve is present, whether or not the patient underwent an

1	Intestine failure is typically multifactorial. Variables include what part of the small intestine is absent, whether or not the ileocecal valve is present, whether or not the patient underwent an ostomy, and how long the remaining colon is. TPN failure is defined as significant biochemical or pathologic evidence of liver injury, loss of central vein access with thrombosis of at least two central veins, frequent indwell-ing catheter infection or a single episode of fungal infection, and recurrent episodes of severe dehydration despite IV fluid supplementation.Indications for a transplant differ between the adult and pediatric population. The leading causes of intestine failure are summarized in Table 11-7. The disease involvement of organs other than the intestine dictates the extent of the operation required. Liver failure is often seen in patients on long-term TPN. If pathologic or biochemical evidence of severe liver damage is combined with signs of portal hypertension, then a

1	of the operation required. Liver failure is often seen in patients on long-term TPN. If pathologic or biochemical evidence of severe liver damage is combined with signs of portal hypertension, then a combined liver-intestine transplant is the treatment of choice. However, a multivisceral transplant (liver, pancreas, stomach, duodenum, and/or small intestine) might be necessary among children who suffer diffuse intestinal dysmotility syndromes and adults who develop diffuse portomesenteric thrombosis, extensive intra-abdominal desmoid disease encasing the main visceral vascular structures with concurrent short gut syndrome, or massive abdominal trauma.Table 11-7Leading causes of intestine failureCHILDRENADULTSGastroschisisVisceral ischemia secondary to SMA/SMV thrombosisMidgut volvulusCrohn’s diseaseIntestinal atresiaTraumaNecrotizing enterocolitisMesenteric desmoid tumorsMicrovillus involution diseaseRadiation enteritisHirschsprung’s diseaseMassive resection secondary to tumorsCrohn’s

1	diseaseIntestinal atresiaTraumaNecrotizing enterocolitisMesenteric desmoid tumorsMicrovillus involution diseaseRadiation enteritisHirschsprung’s diseaseMassive resection secondary to tumorsCrohn’s diseaseChronic intestinal pseudo-obstructionPseudo-obstructionAutoimmune enteropathySMA = superior mesenteric artery; SMV = superior mesenteric veinSurgical ProcedureFor both the donor and recipient surgery, the key decision is which organs will be transplanted.156 For an isolated intestine transplant, the blood supply is based on the arterial inflow from the SMA and on the venous outflow from the superior mesen-teric vein (SMV). Both vessels are isolated at the root of the mesentery.For a combined liver-intestine transplant, the blood supply is based on the arterial inflow from the celiac axis and SMA, which are procured en bloc with an aortic patch. The liver, duo-denum, pancreas, and small intestine—because of their close anatomic relationship—are procured en bloc. If the hepatoduo-denal

1	and SMA, which are procured en bloc with an aortic patch. The liver, duo-denum, pancreas, and small intestine—because of their close anatomic relationship—are procured en bloc. If the hepatoduo-denal ligament is left intact, no biliary reconstruction is neces-sary, which virtually eliminates the risk of postoperative biliary complications.157 Because the entire splanchnic system drains into the liver, venous drainage is achieved by anastomosis of the hepatic veins to the recipient’s vena cava.For both an isolated intestine transplant and a combined liver-intestine transplant, the proximal transection of the GI tract occurs at the first portion of the duodenum. For a multivisceral transplant, the stomach is part of the graft; hence, the transection of the GI tract occurs at the distal esophagus. Figures 11-20 to 11-22 show these three main types of transplants.The vast majority of intestine transplants use a deceased donor organ. However, advances in surgical techniques have made the

1	Figures 11-20 to 11-22 show these three main types of transplants.The vast majority of intestine transplants use a deceased donor organ. However, advances in surgical techniques have made the use of living donors a feasible alternative for either an isolated intestine transplant or a combined liver-intestine transplant. With a living donor, the donor operation is slightly different: for an isolated intestine transplant, 150 to 200 cm of the donor’s ileum, on a vascular pedicle comprising the ileocolic artery and vein, are used158 (Fig. 11-23); for a combined liver-intestine transplant, performed almost exclusively for pediatric recipients, segments II and III of the donor’s liver are used, in addition to the intestine (Fig. 11-24).Figure 11-20. Isolated intestine transplant.Brunicardi_Ch11_p0355-p0396.indd 38601/03/19 6:55 PM 387TRANSPLANTATIONCHAPTER 11Figure 11-21. Combined liver-intestine transplant.Figure 11-22. Multivisceral transplant.ABFigure 11-23. A. Donor operation.

1	38601/03/19 6:55 PM 387TRANSPLANTATIONCHAPTER 11Figure 11-21. Combined liver-intestine transplant.Figure 11-22. Multivisceral transplant.ABFigure 11-23. A. Donor operation. About 180 to 200 cm of distal ileum on a vascular pedicle comprising the ileocolic artery and vein are removed. B. Recipient operation. The donor’s ileocolic artery and vein (or the terminal branches of the donor’s superior mesenteric artery and vein) are anastomosed end-to-side to the recipient’s infrarenal aorta and vena cava. (Reproduced with permission from Gruessner RWG, Benedetti E: Living Donor Organ Transplantation. New York, NY: McGraw-Hill Education; 2008.)Brunicardi_Ch11_p0355-p0396.indd 38701/03/19 6:55 PM 388BASIC CONSIDERATIONSPART IFigure 11-24. Recipient operation. For a combined living donor liver-intestine transplant in a pediatric recipient, liver segments 2 and 3 are implanted in standard fashion (the donor’s left hepatic vein to the recipient’s vena cava, the donor’s left hepatic artery

1	transplant in a pediatric recipient, liver segments 2 and 3 are implanted in standard fashion (the donor’s left hepatic vein to the recipient’s vena cava, the donor’s left hepatic artery to the recipient’s proper or common hepatic artery, the donor’s left portal vein branch to the recipient’s portal vein trunk). The donor’s ileocolic artery and vein are anastomosed to the recipient’s infra-renal aorta and cava. In the recipient, a duodenum-to-donor ileum anastomosis and a distal Bishop-Coop ileostomy are constructed to reestablish bowel continuity. A very short Roux-en-Y loop (10 to 20 cm) is anastomosed to the donor’s bile duct(s). (Reproduced with permission from Gruessner RWG, Benedetti E: Living Donor Organ Transplantation. New York, NY: McGraw-Hill Education; 2008.)Similarly, the recipient operation also varies by the organs transplanted. Generally, the recipient’s infrarenal aorta is used to achieve the arterial inflow to the graft. For an isolated intestine transplant, venous

1	operation also varies by the organs transplanted. Generally, the recipient’s infrarenal aorta is used to achieve the arterial inflow to the graft. For an isolated intestine transplant, venous drainage is achieved via systemic or portomesenteric drainage; for a combined liver-intestine trans-plant or a multivisceral transplant, venous drainage is achieved via the hepatic veins. Systemic venous drainage, given its lesser technical difficulty, is preferred over portomesenteric drainage. The diversion of splanchnic flow into the systemic venous cir-culation can cause several metabolic abnormalities, but no hard evidence shows any negative impact clinically on the recipient.After the organs are perfused, the continuity of the recipi-ent’s GI tract is restored, which includes the placement of a gastrostomy or jejunostomy feeding tube and an ileostomy. In the early postoperative period, the ileostomy enables regular endoscopic surveillance and biopsy of the intestinal mucosa. Once the

1	of a gastrostomy or jejunostomy feeding tube and an ileostomy. In the early postoperative period, the ileostomy enables regular endoscopic surveillance and biopsy of the intestinal mucosa. Once the recipient recovers, the ileostomy can be taken down.The last, but often the most difficult, part of the recipi-ent operation is abdominal wall closure. It is especially challenging in intestine transplant recipients because they have usually undergone multiple previous procedures, resulting in many scars, ostomies, feeding tubes, and the loss of abdomi-nal domain. To provide sufficient coverage of the transplanted organs, the use of prosthetic mesh often is necessary.Postoperative CareInitial postoperative care for intestine transplant recipients does not significantly differ from that for other organ transplant recipients. In the intensive care unit, each recipient’s cardio-vascular, pulmonary, and renal function is closely monitored; aggressive resuscitation with fluid, electrolytes, and

1	organ transplant recipients. In the intensive care unit, each recipient’s cardio-vascular, pulmonary, and renal function is closely monitored; aggressive resuscitation with fluid, electrolytes, and blood prod-ucts is performed. Broad-spectrum antibiotics are an integral component of care.Of all solid organ transplants, intestine transplants have the highest rate of rejection. With intestine transplants, no sero-logic marker of rejection is available, so frequent biopsies and histologic evaluation of the intestinal mucosa are of utmost importance. Rejection leads to structural damage of the intes-tinal mucosa. Translocation of endoluminal pathogens into the circulation can cause systemic infections.Thanks to the introduction of new immunosuppressive protocols, the rejection rates and the overall patient and graft survival rates have improved significantly. Variations between the protocols exist, but the general concept is to induce immu-nosuppression with polyclonal T-cell antibody and

1	patient and graft survival rates have improved significantly. Variations between the protocols exist, but the general concept is to induce immu-nosuppression with polyclonal T-cell antibody and high doses of a corticosteroid, followed by maintenance doses of cortico-steroids and the calcineurin inhibitor tacrolimus.Immediately, posttransplant, recipients are maintained on TPN. Enteral nutrition is initiated as early as possible, but it is advanced very cautiously. It can take several weeks for the transplanted intestine to achieve structural integrity and func-tionality and for the recipient to tolerate the full strength of tube feeds.Despite all the recent advances, the complication rate posttransplant remains high. The most common complica-tions include intra-abdominal abscesses, enteric leaks, intra-abdominal sepsis, the need for a reoperation, graft thrombosis, life-threatening bleeding, and central line problems. Immuno-suppression-specific complications include rejection, PTLD,

1	leaks, intra-abdominal sepsis, the need for a reoperation, graft thrombosis, life-threatening bleeding, and central line problems. Immuno-suppression-specific complications include rejection, PTLD, graft-versus-host disease (GVHD), infections, and malignan-cies. Tailoring the recipient’s immunosuppression plays a critical role in preventing these complications: a low level of immunosuppression leads to graft rejection, but too much con-fers a high risk of infectious complications, PTLD, and, less commonly, GVHD—all of which are associated with a signifi-cantly increased risk of graft failure and mortality.The long-term results of intestine transplants have improved significantly, even though they still remain inferior to the results of other abdominal organ transplants.159,160HEART AND LUNG TRANSPLANTATIONHistoryThe first successful heterotopic heart transplant, in an animal model, was performed by Carrel and Guthrie in 1905.161 Subse-quent progress with cardiopulmonary bypass and

1	LUNG TRANSPLANTATIONHistoryThe first successful heterotopic heart transplant, in an animal model, was performed by Carrel and Guthrie in 1905.161 Subse-quent progress with cardiopulmonary bypass and immunologic modulation facilitated the first successful adult human heart transplant, performed by Christiaan Barnard in 1967 in Cape Town, South Africa.162 However, it was Norman Shumway at Stanford who persisted with heart transplants, in the face of disappointing patient outcomes at a number of early centers. Brunicardi_Ch11_p0355-p0396.indd 38801/03/19 6:55 PM 389TRANSPLANTATIONCHAPTER 11Thanks to the diligence of Shumway and colleagues in perfect-ing heart transplant techniques, along with the development, by Caves, of endomyocardial biopsy as a method of allograft rejection surveillance, human heart transplants began to reap-pear in the 1980s as a viable solution to end-stage heart failure. By 1981, the introduction of cyclosporin A finally created the necessary clinical

1	human heart transplants began to reap-pear in the 1980s as a viable solution to end-stage heart failure. By 1981, the introduction of cyclosporin A finally created the necessary clinical immunologic modulation necessary to make long-term survival of heart recipients a reality.161Lung transplants have a similar history. In the 1950s, Metras in France and Hardin and Kittle in the United States performed canine lung transplants, demonstrating that meticu-lous anastomotic technique could produce normal pulmonary pressures. Hardy performed the first human lung transplant in 1963, although the patient lived only 18 days. The first suc-cessful long-term lung transplant was performed in 1983 in Toronto. These early lung recipients, however, were plagued by infection, rejection, and, most significantly, bronchial anas-tomotic dehiscence. Cooper and colleagues soon determined that the high-dose corticosteroids used for immunosuppression were responsible for the frequent occurrence of

1	significantly, bronchial anas-tomotic dehiscence. Cooper and colleagues soon determined that the high-dose corticosteroids used for immunosuppression were responsible for the frequent occurrence of dehiscence. The combination of high-dose corticosteroids and ischemic donor bronchi was deadly to lung recipients. Cooper, Morgan, and colleagues showed that the bronchial anastomosis could be pro-tected by wrapping it with a vascular omental pedicle, which not only provided neovascularity but also offered a buttress against any partial dehiscence.163Once cyclosporine became available for lung recipients, corticosteroid doses could be quickly tapered and stopped; cyclosporine poses no danger to the integrity of the bronchial anastomosis. In fact, the introduction of cyclosporine allowed the success of the first combined heart-lung transplant at Stanford in 1981 (after unsuccessful attempts by Cooley in 1969, Lillehei in 1970, and Barnard in 1981, all of whom used only high-dose

1	the success of the first combined heart-lung transplant at Stanford in 1981 (after unsuccessful attempts by Cooley in 1969, Lillehei in 1970, and Barnard in 1981, all of whom used only high-dose corticosteroids for immunosuppression). The 1980s marked the start of the modern age of thoracic transplants.Heart TransplantsIndications. The most common diagnosis leading to a heart transplant is ischemic dilated cardiomyopathy, which stems from coronary artery disease, followed by idiopathic dilated myopathy and congenital heart disease. About 3000 patients are added to the waiting list each year.Evaluation. Pretransplant, both candidates and potential donors are evaluated to ensure their suitability for the procedure. Transplant candidates undergo echocardiography, right and left heart catheterization, evaluation for any undiagnosed malignan-cies, laboratory testing to assess the function of other organs (such as the liver, kidneys, and endocrine system), a dental examination, psychosocial

1	evaluation for any undiagnosed malignan-cies, laboratory testing to assess the function of other organs (such as the liver, kidneys, and endocrine system), a dental examination, psychosocial evaluation, and appropriate screen-ing (such as mammography, colonoscopy, and prostate-specific antigen testing). Once the evaluation is complete, the selec-tion committee determines, at a multidisciplinary conference, whether or not a heart transplant is needed and is likely to be successful. Transplant candidates who meet all of the center’s criteria are added to the waiting list, according to the UNOS criteria, which are based on health status.Once a potential deceased donor is identified, the surgeon reviews the status report and screening examination results. The donor is initially matched to the recipient per the recipient’s status on the UNOS waiting list, the size match, and the blood type. Results of the donor’s serologic testing, echocardiography, chest X-ray, hemodynamic testing, and

1	recipient per the recipient’s status on the UNOS waiting list, the size match, and the blood type. Results of the donor’s serologic testing, echocardiography, chest X-ray, hemodynamic testing, and possibly coronary artery evaluation are assessed, in order to determine whether or not the donor’s heart can withstand up to 4 hours of cold ischemic time during procurement, transport, and surgery.Procedure. Heart transplants are most often performed ortho-topically (Fig. 11-25). The recipient’s native heart is removed, leaving the superior vena cava, the IVC, the left atrial cuff, the aorta, and the pulmonary artery in situ, in order to allow for anastomosis of the donor’s heart. Usually the left atrial cuff is anastomosed first, providing left heart inflow. Right heart inflow is achieved using a bicaval technique, by directly sew-ing the donor’s superior vena cava and IVC to the recipient’s venae cavae or by creating an anastomosis of the right atrium to a right atrial cuff. The donor’s

1	a bicaval technique, by directly sew-ing the donor’s superior vena cava and IVC to the recipient’s venae cavae or by creating an anastomosis of the right atrium to a right atrial cuff. The donor’s main pulmonary artery is con-nected to the recipient’s pulmonary artery, and finally, the aortic anastomosis is completed (Fig. 11-26).Once the cross-clamp is removed, the heart is allowed to receive circulation from the recipient and begins to function normally. Inotropic support with isoproterenol, dobutamine, or epinephrine is often required for 3 to 5 days, in order to support recovery from the cold ischemia.164On rare occasions, a heterotopic or “piggyback” heart can be transplanted, leaving the native heart in place. But this sce-nario is becoming very uncommon with the increasing use of mechanical circulatory support for single-ventricle failure.Posttransplant Care. Patient survival rates for heart recipients differ slightly after primary transplants vs. retransplants. In 2016, 3209

1	mechanical circulatory support for single-ventricle failure.Posttransplant Care. Patient survival rates for heart recipients differ slightly after primary transplants vs. retransplants. In 2016, 3209 heart transplants were performed in the United States. New, active listings increased 57% since 2005. Overall 1-year survival for patients who underwent heart transplant in 2009 to 2011 was 90.1%, 3-year survival was 83.5%, and 5-year Figure 11-25. A donor’s heart brought forward for anastomosis.Brunicardi_Ch11_p0355-p0396.indd 38901/03/19 6:55 PM 390BASIC CONSIDERATIONSPART IFigure 11-26. Suture lines for bicaval anastomosis (a), biatrial anastomosis (b), aortic anastomosis (c), and pulmonary artery anas-tomosis (d).aacbdsurvival was 78.3%, and the most common cause of death within the first year after transplant was infection.165 An increasing number of heart recipients have now survived more than 15 to 20 years with their first graft, especially those with no significant history of

1	year after transplant was infection.165 An increasing number of heart recipients have now survived more than 15 to 20 years with their first graft, especially those with no significant history of either cellular or antibody-mediated rejection.Heart recipients must be monitored for both early and late complications. Early complications include primary graft dys-function, acute cellular or antibody-mediated rejection, right heart failure secondary to pulmonary hypertension, and infec-tion. Hemodynamic values are monitored to assess early graft function; pharmacologic and sometimes mechanical support is instituted if needed.The goal of immunosuppression is to prevent rejection, which is assessed by immunosuppressive levels and, early on, by endomyocardial biopsy. Both T-cell–mediated (cellular) and B-cell–mediated (antibody-mediated) rejection are moni-tored. Most of the immunosuppression used is aimed at T cells; however, if the recipient has many preformed antibodies or develops

1	and B-cell–mediated (antibody-mediated) rejection are moni-tored. Most of the immunosuppression used is aimed at T cells; however, if the recipient has many preformed antibodies or develops donor-specific antibodies, other strategies (such as plasmapheresis or rituximab) are used to reduce the antibody load. Immunosuppressive regimens can vary by center, but most often consist of three categories of medications: a calcineurin inhibitor (usually tacrolimus or cyclosporine), an antiprolifera-tive agent (MMF or AZA), and a corticosteroid (prednisone). Other immunosuppressive agents can be used, depending on the needs of individual recipients.Recipients are also assessed for any infections, with visual inspection of wound healing and with monitoring of the com-plete blood count and cultures as needed. Other common early sequelae include drug-induced nephrotoxicity, glucose intoler-ance, hypertension, hyperlipidemia, osteoporosis, malignancies, and biliary disease.Late complications

1	as needed. Other common early sequelae include drug-induced nephrotoxicity, glucose intoler-ance, hypertension, hyperlipidemia, osteoporosis, malignancies, and biliary disease.Late complications include acquired transplant vasculopa-thy, progressive renal failure, and, most commonly, malignan-cies, especially skin cancer and PTLD. Accelerated coronary artery disease is the third most common cause of death posttrans-plant (after infections and acute rejection) and the most common cause after the first year. Coronary artery disease can begin to develop as early as 1 year posttransplant. Its pathogenesis is unknown, but it is believed to be immunologic. Because of these late complications, most transplant centers continue to perform screening tests and recipient examinations at least annually after the first year.Lung TransplantsIndications. The indications for a lung transplant include congenital disease, emphysema, COPD, cystic fibrosis, idio-pathic pulmonary fibrosis, primary

1	annually after the first year.Lung TransplantsIndications. The indications for a lung transplant include congenital disease, emphysema, COPD, cystic fibrosis, idio-pathic pulmonary fibrosis, primary pulmonary hypertension, α1-antitrypsin deficiency, and the need for a retransplant after primary graft failure. Each year in the United States, about 1600 patients are added to the waiting list; nearly a third of them have COPD and/or emphysema. The next most common diagnosis among patients on the waiting list is cystic fibrosis. A lung allo-cation score (LAS) was instituted in 2005. The average lung transplant candidate requires oxygen (often 4 L/min or more at rest) and has an extensively compromised quality of life, as documented by the results of pulmonary function and 6-minute walk tests.Evaluation. Evaluation for a lung transplant is very similar to evaluation for a heart transplant, except that lung transplant candidates undergo more extensive pulmonary function testing, a 6-minute

1	for a lung transplant is very similar to evaluation for a heart transplant, except that lung transplant candidates undergo more extensive pulmonary function testing, a 6-minute walk test, chest computed tomography, ventilation-perfusion (V-Q) scanning, and arterial blood gas assessment. In addition, all lung transplant candidates must have adequate cardiac function and must meet psychosocial requirements.Potential lung donors are also screened for blood type and size match. Larger lungs are accepted for COPD patients; smaller lungs are chosen for the restricted chest cavity of fibrotic patients. Donors should have a partial pressure of oxygen in arterial blood (Pao2) value >300 mmHg on a fraction of inspired oxygen (Fio2) of 100% and a positive end-expiratory pressure (PEEP) value of 5. Ideally, donors will have normal chest X-ray results, but exceptions for isolated abnormalities that will not affect subsequent graft function can be made. Living donors can donate a single lobe to a

1	Ideally, donors will have normal chest X-ray results, but exceptions for isolated abnormalities that will not affect subsequent graft function can be made. Living donors can donate a single lobe to a smaller recipient, such as a child. Single-lung transplants are common in many centers and can serve to increase the availability of lungs for multiple recipients. Newer concepts, such as “lung in the box” extracorporeal lung perfusion and stem cell technologies, may further improve the availability of donor lungs by optimizing the use of otherwise marginal grafts.Procedure. Lung transplants can be done either as (a) single-lung transplants (to either side via thoracotomy) or as (b) sequential bilateral-lung transplants (via bilateral thoracotomies or via a single clamshell incision that divides the sternum; Fig. 11-27). They can be done absent extracorporeal mechanical cardiopul-monary perfusion (bypass), with the lung with the worst func-tion (as predicted by preoperative ventilation

1	the sternum; Fig. 11-27). They can be done absent extracorporeal mechanical cardiopul-monary perfusion (bypass), with the lung with the worst func-tion (as predicted by preoperative ventilation and perfusion scanning) transplanted first. Despite careful surgical technique and excellent anesthesia, the poor pulmonary reserve of some lung recipients may require the institution of cardiopulmonary bypass to complete the transplant. Bypass is initiated through the chest by direct cardiac cannulation or peripherally via the femoral vessels.Once the thoracotomy is made, a recipient pneumonec-tomy is performed with care, in order to avoid injury to the phrenic or recurrent laryngeal nerves. The pulmonary veins and main pulmonary artery are encircled outside the peri-cardium. At this point, once the main pulmonary vessels are occluded, the need for cardiopulmonary bypass can be assessed. Brunicardi_Ch11_p0355-p0396.indd 39001/03/19 6:55 PM 391TRANSPLANTATIONCHAPTER 11Figure

1	point, once the main pulmonary vessels are occluded, the need for cardiopulmonary bypass can be assessed. Brunicardi_Ch11_p0355-p0396.indd 39001/03/19 6:55 PM 391TRANSPLANTATIONCHAPTER 11Figure 11-27. Clamshell incision. Bronchial anastomosis with ligated pulmonary arteries and veins.Figure 11-28. Bronchial anastomosis.The vessels and bronchus are ligated; the donor’s lung is pre-pared and brought to the table wrapped in cold iced gauze, in order to extend the cold preservation time. The bronchial anas-tomosis (Fig. 11-28) is performed first and then covered with peribronchial tissue or pericardium. The pulmonary artery and, finally, the vein are anastomosed. The lung is then de-aired before the final anastomotic suture is tightened, with gentle lung insufflation. All clamps are removed, and the lung is aerated. At least two chest tubes are left in place. After the transplant is complete, a bronchoscopy is performed to clear the airway of blood and secretions.Posttransplant

1	removed, and the lung is aerated. At least two chest tubes are left in place. After the transplant is complete, a bronchoscopy is performed to clear the airway of blood and secretions.Posttransplant Care. Patient survival rates for lung recipients vary significantly after primary vs. redo transplants. After pri-mary transplants, the patient survival rates at 1, 3, and 5 years are 83%, 62%, and 46%, respectively; after retransplants, the rates are 64%, 38%, and 28%.Postoperative care of lung recipients can be very labor-intensive. These patients require meticulous ventilator manage-ment, in order to maintain Fio2 at a minimum and to keep Pao2 at 70 mmHg. Most patients are extubated within the first 24 to 48 hours. Recipients can require multiple bronchoscopies for both airway management and surveillance biopsies. Diuretics are used generously to counteract any positive fluid balance from the operation and to help with pulmonary recovery.Early complications include technical

1	and surveillance biopsies. Diuretics are used generously to counteract any positive fluid balance from the operation and to help with pulmonary recovery.Early complications include technical complications, graft dysfunction, infections, and rejection. Technical complications often involve stenosis of one or more anastomoses leading to graft dysfunction. Bronchoscopy, V-Q scanning, echocardiogra-phy, and radiologic imaging are useful in identifying the causes of graft dysfunction. In up to 20% of recipients, primary early graft dysfunction can occur with no obvious cause. Such dys-function may be due to some pathology from the donor, perhaps an unknown aspiration, infection, or contusion; or it could result from poor graft preservation at the time of organ procurement. In the intensive care unit, aggressive ventilator and pharmaco-logic management can help, but recipients can nonetheless prog-ress to the need for mechanical support in the form of ECMO. Infections are treated with

1	care unit, aggressive ventilator and pharmaco-logic management can help, but recipients can nonetheless prog-ress to the need for mechanical support in the form of ECMO. Infections are treated with appropriate antibiotics, which can be challenging in patients with cystic fibrosis and a history of multidrug-resistant organisms. Rejection is monitored by biop-sies and treated as needed.Late complications include airway complications, such as strictures and, rarely, dehiscence, bronchiolitis obliterans, and malignancies. Strictures are treated with bronchoscopic dilation and intervention. Bronchiolitis obliterans often is a sequela of chronic rejection, but can be due to aspiration, chronic infec-tions, or various other causes. In recipients with a progressive fall in their forced expiratory volume in 1 second (FEV1), bron-chiolitis obliterans is suspected. All recipients should be taught to perform microspirometry at home as a screening tool post-transplant. Biopsies are performed to

1	volume in 1 second (FEV1), bron-chiolitis obliterans is suspected. All recipients should be taught to perform microspirometry at home as a screening tool post-transplant. Biopsies are performed to confirm the diagnosis of any complication and, if possible, the cause. Despite aggres-sive screening and treatment, more than 50% of recipients will develop graft dysfunction. Most if not all of the sequelae of chronic immunosuppression that occur in lung transplant recipi-ents are similar to those occurring in other groups of solid organ transplants.Heart-Lung TransplantsEvery year in the United States, 30 to 50 patients are added to the list of patients waiting to receive a simultaneous heart-lung transplant. The most common diagnosis is idiopathic pulmo-nary fibrosis, followed by primary pulmonary hypertension. Heart-lung candidates are often younger than their single-organ counterparts. The patient survival rates at 1, 3, and 5 years are 66%, 48%, and 39%, respectively. Often, lung

1	pulmonary hypertension. Heart-lung candidates are often younger than their single-organ counterparts. The patient survival rates at 1, 3, and 5 years are 66%, 48%, and 39%, respectively. Often, lung complications ultimately lead to graft failure. The immunosuppression is the same as that for single thoracic organ recipients, with emphasis on weaning the patient off corticosteroids as early as possible.XENOTRANSPLANTSXenotransplants (i.e., cross-species transplants of organs, tis-sues, or cells) have immense, yet untapped, potential to solve the critical shortage of available grafts. A primary hurdle is the formidable immunologic barrier between species, especially Brunicardi_Ch11_p0355-p0396.indd 39101/03/19 6:55 PM 392BASIC CONSIDERATIONSPART Iwith vascularized whole organs.161-170 Other problems include the potential risk of transmitting infections (known as zoono-ses or xenoses) and the ethical problems of using animals for widespread human transplants, even though great

1	Other problems include the potential risk of transmitting infections (known as zoono-ses or xenoses) and the ethical problems of using animals for widespread human transplants, even though great progress has been made in the past few years in efforts to overcome these problems.166-172Pigs are generally accepted as the most likely donor spe-cies for xenotransplants into human beings.173 Pigs would also be easier to raise on a large-scale basis. Guidelines for raising pigs in specialized facilities designated as pathogen-free have been established; in anticipation of clinical trials, such facilities have already been created and populated.171,172The immunologic barrier in pig-to-human xenotrans-plants is highly complex, but generally involves four subtypes of rejection.166 The first is hyperacute rejection (HAR), which is mediated by the presence of natural (preformed) xenoantibod-ies in humans. These antibodies bind to antigens found mainly on the vascular endothelial cells of porcine

1	rejection (HAR), which is mediated by the presence of natural (preformed) xenoantibod-ies in humans. These antibodies bind to antigens found mainly on the vascular endothelial cells of porcine donor organs, lead-ing to complement activation, intravascular coagulation, and rapid graft ischemia soon after the transplant. The second sub-type is acute humoral xenograft rejection (AHXR), a delayed form of antibody-mediated rejection seen in pig-to-nonhuman-primate transplants after steps to prevent HAR—steps such as depletion of antipig antibodies or complement from nonhu-man primates’ serum. Alternative names for AHXR include acute vascular rejection or delayed xenograft rejection. The third subtype is an acute cellular rejection process (similar to the classic T-cell–mediated acute rejection seen in allograft recipients). The fourth subtype is chronic rejection in grafts that survive for more than a few weeks (similar to the chronic rejection seen in long-surviving allograft recipients,

1	seen in allograft recipients). The fourth subtype is chronic rejection in grafts that survive for more than a few weeks (similar to the chronic rejection seen in long-surviving allograft recipients, with fea-tures of chronic vasculopathy).Many different options are being tested to overcome this immunologic barrier, including the genetic engineering of pigs, the use of agents to inhibit platelet aggregation and complement activation, and the administration of powerful immunosuppres-sive drugs.166-173During the first decade of the 21st century, the field of whole-organ xenotransplantation progressed significantly, thanks to the increasing availability of genetically engineered pigs and new immunosuppressive protocols. At a recent sympo-sium organized by the International Xenotransplantation Asso-ciation, data presented demonstrated extended survival time of porcine solid organs in nonhuman primates: from about 30 days to an average of 60 days and even up to 250 days (depending on the

1	Asso-ciation, data presented demonstrated extended survival time of porcine solid organs in nonhuman primates: from about 30 days to an average of 60 days and even up to 250 days (depending on the model).166,169,174,175 However, clinical application is still limited by thrombotic microangiopathy and consumptive coagu-lopathy; novel methods to prevent those complications will be required for further progress.Cellular xenotransplants have made great strides and are currently in the early stages of clinical trials. Porcine islet xenotransplants are the most advanced form; five independent groups have now demonstrated survival and function of porcine islets in nonhuman primates for more than 100 days.166,175-181 For the clinical trials, cost-benefit models have been developed, and the regulatory framework has been established.170-172,178 One trial of particular interest involves transplanting encapsulated porcine islets without immunosuppression.179 Early results are encouraging. But the

1	framework has been established.170-172,178 One trial of particular interest involves transplanting encapsulated porcine islets without immunosuppression.179 Early results are encouraging. But the efficacy of that approach may be limited until further genetic engineering enables proper oxygenation and nourishment of islet grafts, thereby supporting their viabil-ity and function.The future of xenotransplantation is exciting. Continued active research will focus on further genetic engineering of pigs, newer immunosuppressive drugs, and tissue engineering approaches that will minimize or eliminate the need for immu-nosuppression. Given recent progress, routine clinical applica-tion of cellular xenotransplants is likely within the next decade.REFERENCESEntries highlighted in bright blue are key references. 1. Carrel A. The surgery of blood vessels, etc. B Johns Hopkins Hosp. 1907;18:18-28. 2. Hamilton D, Reid, WA. Yu Yu Voronoy and the first human kidney allograft. Surg Gynecol Obstet.

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1	antibodies in organ transplant recipients–triumphs and challenges of the solid phase bead assay. Front Immunol. 2016;7:570. Available at: https://doi .org/10.3389/fimmu.2016.00570. Accessed May 8, 2018. 83. Hume DM, Merrill JP, Miller BF, et al. Experiences with renal homotransplantation in the human: report of 9 cases. J Clin Invest. 1955;34(2):327-382. 84. Murray JE, Merrill JP, Harrison JH. Kidney transplan-tation between 7 pairs of identical twins. Ann Surg. 1958;148(3):343-359. 85. Starzl TE, Marchioro TL, Dickinson TC, et al. Technique of renal homotransplantation: experience with 42 cases. Arch Surg. 1964;89(1):87-104. 86. Ghazanfar A, Tavakoli A, Zaki MR, et al. The outcome of living donor renal transplants with multiple renal arteries; a large cohort study with a mean follow-up period of 10 years. Transplant Proc. 2010;42:1654. 87. Laurence JM, Sandroussi C, Lam VWT, et al. Utilization of small pediatric donor kidneys: a decision analysis. Transplantation.

1	a mean follow-up period of 10 years. Transplant Proc. 2010;42:1654. 87. Laurence JM, Sandroussi C, Lam VWT, et al. Utilization of small pediatric donor kidneys: a decision analysis. Transplantation. 2011;91(10):1110-1113. 88. Lau KK, Berg GM, Schjoneman YG, et al. Pediatric en bloc kidney transplantation into pediatric recipients. Pediatr Transplant. 2010;14(1):100-104. 89. Wang JH, Skeans MA, Israni AK. Current status of kidney transplant outcomes: dying to survive. Adv Chronic Kidney Dis. 2016;23(5):281-286. 90. Rizzari MD, Suszynski TM, Gillingham KJ, et al. Ten-year outcome after rapid discontinuation of prednisone in adult primary kidney transplantation. Clin J Am Soc Nephrol. 2012;7(3):494-503. 91. Vincenti F, Larsen CP, Alberu J, et al. Three-year outcomes from BENEFIT, a randomized, active-controlled, parallel-group study in adult kidney transplant recipients. Am J Transplant. 2012;12(1):210-217. 92. Massy ZA, Guijarro C, Wiederkehr MR, et al. Chronic renal allograft

1	randomized, active-controlled, parallel-group study in adult kidney transplant recipients. Am J Transplant. 2012;12(1):210-217. 92. Massy ZA, Guijarro C, Wiederkehr MR, et al. Chronic renal allograft rejection: immunologic and nonimmunologic risk factors. Kidney Int. 1996;49(2):518-524. 93. Traynor C, Jenkinson A, Williams Y, et al. Twenty-year sur-vivors of kidney transplantation. Am J Transplant. 2012; 12:3289. 94. Sutherland DER, Gruessner RWG. History of pancreas trans-plantation (Chapter 4). In: Gruessner RWG, Sutherland DER, eds. Transplantation of the Pancreas. New York: Springer-Verlag; 2004:39-68. 95. Gruessner AC, Gruessner RW. Pancreas transplantation of U.S. and non-U.S. cases from 2005 to 2014 as reported to the United Network for Organ Sharing (UNOS) and the International Pancreas Transplant Registry (IPTR). Rev Diabet Stud. 2016:13(1):35-58. 96. Fioretto P, Maurer M. Effects of pancreas transplantation on secondary complications of diabetes (Chapter 16). In: Gruess-ner

1	Transplant Registry (IPTR). Rev Diabet Stud. 2016:13(1):35-58. 96. Fioretto P, Maurer M. Effects of pancreas transplantation on secondary complications of diabetes (Chapter 16). In: Gruess-ner RWG, Sutherland DER, eds. Transplantation of the Pancreas. New York: Springer-Verlag; 2004:455-508. 97. Gruessner RW, Sutherland DE, Drangstveit MB, Kandaswamy R, Gruessner AC. Pancreas allotransplants in patients with a previous total pancreatectomy for chronic pancreatitis. J Am Coll Surg. 2008;206:458-465. 98. Benedetti E, Pierpaolo S. Surgical aspects of pancreas trans-plantation (Chapter 8). In: Gruessner RWG, Sutherland DER, eds. Transplantation of the Pancreas. New York: Springer-Verlag; 2004:111-178. 99. Sutherland DER, Zamir GA, Brayman KL. Transplantation of the pancreas. In: Cameron JL, Cameron AM, eds. Current Surgical Therapy. 10th ed. New York: Elsevier Saunders; 2011:460-471. 100. Leone AP, Christensen K. Postoperative management (Chapter 9). In: Gruessner RWG, Sutherland DER,

1	Cameron AM, eds. Current Surgical Therapy. 10th ed. New York: Elsevier Saunders; 2011:460-471. 100. Leone AP, Christensen K. Postoperative management (Chapter 9). In: Gruessner RWG, Sutherland DER, eds. Trans-plantation of the Pancreas. New York: Springer-Verlag; 2004: 179-266. 101. Pancreas transplantation (Section II). In: Gruessner RWG, Benedetti E, eds. Living Donor Organ Transplantation. New York: McGraw-Hill; 2008:369-437. 102. Gruessner AC, Sutherland DER, Gruessner RWG. Long-term outcome after pancreas transplantation. Curr Opin Transplant. 2012;17:100-105.Brunicardi_Ch11_p0355-p0396.indd 39401/03/19 6:55 PM 395TRANSPLANTATIONCHAPTER 11 103. Hering BJ, Clarke WR, Bridges ND, et al. Phase 3 trial of transplantation of human islets in type 1 diabetes compli-cated by severe hypoglycemia. Diabetes Care. 2016;39(7): 1230-1240. 104. Maffi P, Scavini M, Socci C, et al. Risks and benefits of trans-plantation in the cure of type 1 diabetes; whole pancreas ver-sus islet

1	severe hypoglycemia. Diabetes Care. 2016;39(7): 1230-1240. 104. Maffi P, Scavini M, Socci C, et al. Risks and benefits of trans-plantation in the cure of type 1 diabetes; whole pancreas ver-sus islet transplantation. A single center study. Rev Diabet Stud. 2011;8:44-50. 105. Williams PW. Notes on diabetes treated with extract and by grafts of sheep’s pancreas. Br Med J. 1894;1303-1304. 106. Kelly WD, Lillehei RC, Merkel FK, et al. Allotransplantation of the pancreas and duodenum along with the kidney in dia-betic nephropathy. Surgery. 1967;61(6):827-837. 107. Ballinger WF, Lacy PE. Transplantation of intact pancreatic islets in rats. Surgery. 1972;72:175-186. 108. Najarian JS, Sutherland DE, Baumgartner D, et al. Total or near total pancreatectomy and islet autotransplantation for treat-ment of chronic pancreatitis. Ann Surg. 1980;192(4):526-542. 109. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplanta-tion in seven patients with type 1 diabetes mellitus using a

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1	Organ Transplant. 2011; 16(6):627-631. 119. Bellin MD, Kandaswamy R, Parkey J, et al. Prolonged insulin independence after islet allotransplants in recipients with type 1 diabetes. Am J Transplant. 2008;8(11):2463-2470. 120. Bellin MD, Barton FB, Heitman A, et al. Potent induction immunotherapy promotes long-term insulin independence after islet transplantation in type 1 diabetes. Am J Transplant. 2012;12(6):1576-1583. 121. Rickels MR. Recovery of endocrine function after islet and pancreas transplantation. Curr Diabet Rep. 2012;12:587-596. 122. Barton FB, Rickels MR, Alejandro R, et al. Improvement in outcomes of clinical islet transplantation: 1999-2010. Diabetes Care. 2012;35(7):1436-1445. 123. Busuttil RW, De Carlis LG, Mihaylov PV, Gridelli B, Fassati LR, Starzl TE. The first report of orthotopic liver trans-plantation in the Western world. Am J Transplant. 2012;12(6): 1385-1387. 124. Starzl TE, Demetris AJ, Trucco M, et al. Cell migra-tion and chimerism after whole-organ

1	report of orthotopic liver trans-plantation in the Western world. Am J Transplant. 2012;12(6): 1385-1387. 124. Starzl TE, Demetris AJ, Trucco M, et al. Cell migra-tion and chimerism after whole-organ transplantation: the basis of graft acceptance. Hepatology. 1993;17(6): 1127-1152. 125. Cohen C, Benjamin M. Alcoholics and liver transplantation. The Ethics and Social Impact Committee of the Transplant and Health Policy Center. JAMA. 1991;265(10):1299-1301. 126. Lucey MR. Liver transplantation in patients with alcoholic liver disease. Liver Transplant. 2011;17(7):751-759. 127. Longworth L, Young T, Buxton MJ, et al. Midterm cost-effectiveness of the liver transplantation program of England and Wales for three disease groups. Liver Transplant. 2003; 9(12):1295-1307. 128. Charlton M, Ruppert K, Belle SH, et al. Long-term results and modeling to predict outcomes in recipients with HCV infec-tion: results of the NIDDK liver transplantation database. Liver Transplant.

1	M, Ruppert K, Belle SH, et al. Long-term results and modeling to predict outcomes in recipients with HCV infec-tion: results of the NIDDK liver transplantation database. Liver Transplant. 2004;10(9):1120-1130. 129. Garcia-Retortillo M, Forns X, Feliu A, et al. Hepatitis C virus kinetics during and immediately after liver transplantation. Hepatology. 2002;35(3):680-687. 130. Berenguer M, Prieto M, Rayon JM, et al. Natural history of clinically compensated hepatitis C virus-related graft cirrho-sis after liver transplantation. Hepatology. 2000;32(4 Pt 1): 852-858. 131. Lake JR, Shorr JS, Steffen BJ, Chu AH, Gordon RD, Wiesner RH. Differential effects of donor age in liver trans-plant recipients infected with hepatitis B, hepatitis C, and without viral hepatitis. Am J Transplant. 2005;5(3):549-557. 132. Neff GW, Montalbano M, O’Brien CB, et al. Treatment of established recurrent hepatitis C in liver-transplant recipients with pegylated interferon-alfa-2b and ribavirin therapy.

1	GW, Montalbano M, O’Brien CB, et al. Treatment of established recurrent hepatitis C in liver-transplant recipients with pegylated interferon-alfa-2b and ribavirin therapy. Transplantation. 2004;78(9):1303-1307. 133. Lee J, Belanger A, Doucette JT, Stanca C, Friedman S, Bach N. Transplantation trends in primary biliary cirrhosis. Clin Gas-troenterol Hepatol. 2007;5(11):1313-1315. 134. Liermann Garcia RF, Evangelista Garcia C, McMaster P, Neuberger J. Transplantation for primary biliary cirrhosis: ret-rospective analysis of 400 patients in a single center. Hepatol-ogy. 2001;33(1):22-27. 135. Campsen J, Zimmerman MA, Trotter JF, et al. Clinically recur-rent primary sclerosing cholangitis following liver transplanta-tion: a time course. Liver Transplant. 2008;14(2):181-185. 136. Schilsky ML, Scheinberg IH, Sternlieb I. Liver transplantation for Wilson’s disease: indications and outcome. Hepatology. 1994;19(3):583-587. 137. Medici V, Mirante VG, Fassati LR, et al. Liver transplantation for

1	IH, Sternlieb I. Liver transplantation for Wilson’s disease: indications and outcome. Hepatology. 1994;19(3):583-587. 137. Medici V, Mirante VG, Fassati LR, et al. Liver transplantation for Wilson’s disease: the burden of neurological and psychiat-ric disorders. Liver Transplant. 2005;11(9):1056-1063. 138. Mazzaferro V, Regalia E, Doci R, et al. Liver transplanta-tion for the treatment of small hepatocellular carcinomas in patients with cirrhosis. N Engl J Med. 1996;334(11):693-699. 139. Sapisochín G, Fernández de Sevilla E, Echeverri J, et al. Liver transplantation for cholangiocarcinoma: Current status and new insights. World J Hepatol. 2015;7(22):2396-2403. 140. O’Grady JG, Alexander GJ, Hayllar KM, Williams R. Early indicators of prognosis in fulminant hepatic failure. Gastroenterology. 1989;97(2):439-445. 141. Boyer TD, Haskal ZJ. The role of transjugular intrahepatic portosystemic shunt (TIPS) in the management of portal hypertension: update 2009. Hepatology.

1	Gastroenterology. 1989;97(2):439-445. 141. Boyer TD, Haskal ZJ. The role of transjugular intrahepatic portosystemic shunt (TIPS) in the management of portal hypertension: update 2009. Hepatology. 2010;51(1):306. 142. Kamath PS, Wiesner RH, Malinchoc M, et al. A model to predict survival in patients with end-stage liver disease. Hepatology. 2001;33(2):464-470. 143. Wiesner R, Edwards E, Freeman R, et al. Model for end-stage liver disease (MELD) and allocation of donor livers. Gastroenterology. 2003;124(1):91-96. 144. Merion RM, Schaubel DE, Dykstra DM, Freeman RB, Port FK, Wolfe RA. The survival benefit of liver transplantation. Am J Transplant. 2005;5(2):307-313. 145. Safdar K, Neff GW, Montalbano M, et al. Liver transplant for the septuagenarians: importance of patient selection. Transplant Proc. 2004;36(5):1445-1448.Brunicardi_Ch11_p0355-p0396.indd 39501/03/19 6:55 PM 396BASIC CONSIDERATIONSPART I 146. Bellamy CO, DiMartini AM, Ruppert K, et al. Liver transplan-tation for

1	Proc. 2004;36(5):1445-1448.Brunicardi_Ch11_p0355-p0396.indd 39501/03/19 6:55 PM 396BASIC CONSIDERATIONSPART I 146. Bellamy CO, DiMartini AM, Ruppert K, et al. Liver transplan-tation for alcoholic cirrhosis: long term follow-up and impact of disease recurrence. Transplantation. 2001;72(4):619-626. 147. Vagefi PA, Parekh J, Ascher NL, Roberts JP, Freise CE. Out-comes with split liver transplantation in 106 recipients: the University of California, San Francisco, experience from 1993 to 2010. Arch Surg. 2011;146(9):1052-1059. 148. Abecassis MM, Fisher RA, Olthoff KM, et al. Complications of living donor hepatic lobectomy—a comprehensive report. Am J Transplant 2012;12:1208-1217. 149. Bilzer M, Gerbes AL. Preservation injury of the liver: mech-anisms and novel therapeutic strategies. J Hepatol. 2000; 32(3):508-515. 150. Jaeschke H. Preservation injury: mechanisms, prevention, and consequences. J Hepatol. 1996;25(5):774-780. 151. Serracino-Inglott F, Habib NA, Mathie RT. Hepatic

1	J Hepatol. 2000; 32(3):508-515. 150. Jaeschke H. Preservation injury: mechanisms, prevention, and consequences. J Hepatol. 1996;25(5):774-780. 151. Serracino-Inglott F, Habib NA, Mathie RT. Hepatic ischemia-reperfusion injury. Am J Surg. 2001;181(2):160-166. 152. Drazan K, Shaked A, Olthoff KM, et al. Etiology and man-agement of symptomatic adult hepatic artery thrombo-sis after orthotopic liver transplantation (OLT). Am Surg. 1996;62(3):237-240. 153. Tzakis AG, Gordon RD, Shaw BW, Jr., Iwatsuki S, Starzl TE. Clinical presentation of hepatic artery thrombosis after liver transplantation in the cyclosporine era. Transplantation. 1985;40(6):667-671. 154. Grant D, Abu-Elmagd K, Reyes J, et al. 2003 report of the intestine transplant registry: a new era has dawned. Ann Surg. 2005;241(4):607-613. 155. Bharadwaj S, Tandon P, Gohel TD, et al. Current status of intestinal and multivisceral transplantation. Gastroenterol Rep. 2017;5(1):20-28. 156. Yersiz H, Renz JF, Hisatake GM, et al.

1	S, Tandon P, Gohel TD, et al. Current status of intestinal and multivisceral transplantation. Gastroenterol Rep. 2017;5(1):20-28. 156. Yersiz H, Renz JF, Hisatake GM, et al. Multivisceral and iso-lated intestinal procurement techniques. Liver Transplant. 2003;9(8):881-886. 157. Bueno J, Abu-Elmagd K, Mazariegos G, Madariaga J, Fung J, Reyes J. Composite liver–small bowel allografts with preser-vation of donor duodenum and hepatic biliary system in chil-dren. J Pediatr Surg. 2000;35(2):291-295; discussion 95-96. 158. Farmer DG, McDiarmid SV, Edelstein S, et al. Improved out-come after intestinal transplantation at a single institution over 12 years. Transplant Proc. 2004;36(2):303-304. 159. Tzakis AG, Kato T, Levi DM, et al. 100 multivisceral trans-plants at a single center. Ann Surg. 2005;242(4):480-490; dis-cussion 91-93. 160. Gruessner RWG, Sharp HL. Living related intestinal trans-plantation: first report of a standardized surgical tech-nique. Transplant.

1	center. Ann Surg. 2005;242(4):480-490; dis-cussion 91-93. 160. Gruessner RWG, Sharp HL. Living related intestinal trans-plantation: first report of a standardized surgical tech-nique. Transplant. 1997;64:1605-1607. 161. Kouchoukos NT, Blackstone EH, Doty DB. Heart failure. In: Kouchoukos NT, Blackstone EH, Doty DB, et al, eds. Cardiac Surgery. 3rd ed. New York: Kirklin/Barratt-Boyes; 2003:1725. 162. First human heart transplant. The History Channel website. Available at: https://www.history.com/this-day-in-history/first-human-heart-transplant. Accessed May 8, 2018. 163. Meyers BF, Patterson GA, Haverich A, Harringer W. Lung transplantation, heart-lung transplantation. In: Pearson FG, Cooper JD, Deslauriers J, et al, eds. Thoracic Surgery. 2nd ed. New York: Churchill-Livingston; 2002:1085-1131. 164. Costanzo MR, Dipchand A, Starling R, et al. The International Society of Heart and Lung Transplantation guidelines for care of heart transplant recipients. J Heart Lung Transplant.

1	MR, Dipchand A, Starling R, et al. The International Society of Heart and Lung Transplantation guidelines for care of heart transplant recipients. J Heart Lung Transplant. 2010;29(8):914-956. 165. Colvin M, Smith JM, Hadley N, et al. OPTN/SRTR 2016 annual data report: heart. Amer J Transplant. 2018;18(S1):291-362. 166. Esker B, Cooper DKC. Overcoming the barriers to xenotrans-plantation: prospects for the future. Exp Rev Clin Immunol. 2010;6(2):219-230. 167. Schuurman HJ. Xenotransplantation: from the lab to the clinic: Sunrise Symposium at the XXIII International Congress of the Transplantation Society, Vancouver, Canada, August 2010. Clin Transplant. 2011;25(4):E415-E421. 168. Dooldeniya MD, Warrens AN. Xenotransplantation: where are we today? J R Soc Med. 2003;96:111. 169. Thompson P, Badell IR, Lowe M, et al. Alternative immu-nomodulatory strategies for xenotransplantation: CD40/154 pathway-sparing regimens promote xenograft survival. Am J Transplant.

1	P, Badell IR, Lowe M, et al. Alternative immu-nomodulatory strategies for xenotransplantation: CD40/154 pathway-sparing regimens promote xenograft survival. Am J Transplant. 2012;12(7):1765-1775. 170. Hering BJ, Cooper DKC, Cozzi E, et al. The International Xenotransplantation Association consensus statement on con-ditions for undertaking clinical trials of porcine islet products in type I diabetes: executive summary. Xenotransplantation. 2009;16:196-202. 171. Schuurman HJ. Regulatory aspects of pig-to-human islet transplantation. Xenotransplantation. 2008;15(2):116-120. 172. Schuurman HJ. The International Xenotransplantation Associ-ation consensus statement on conditions for undertaking clini-cal trials of porcine islet products in type 1 diabetes—chapter 2: Source pigs. Xenotransplantation. 2009;16(4):215-222. 173. Meier RPH, Muller YD, Balaphas A, et al. Xenotransplanta-tion: back to the future? Transplant Int. 2017; doi: 10.1111/tri.13104. 174. Greenstein JL, Schuurman H-J. Solid

1	2009;16(4):215-222. 173. Meier RPH, Muller YD, Balaphas A, et al. Xenotransplanta-tion: back to the future? Transplant Int. 2017; doi: 10.1111/tri.13104. 174. Greenstein JL, Schuurman H-J. Solid organ xenotransplantation: progress, promise, and regulatory issues. J Comm Biotech. 2001;8:15-29. 175. Thompson P, Badell IR, Lowe M, et al. Islet xenotransplanta-tion using gal-deficient neonatal donors improves engraftment and function. Am J Transplant. 2011;11:2593-2602. 176. Rood PPM, Cooper DKC. Islet xenotransplantation: are we really ready for clinical trials? Am J Transplant. 2006; 6(6):1269-1274. 177. Mihalicz D, Rajotte R, Rayat G. Porcine islet xenotransplan-tation for the treatment of type I diabetes. In: Type I Diabetes: Pathogenesis, Genetics and Immunotherapy. New York: InTech; 2011:479-502. 178. Beckwith J, Nyman JA, Flanagan B, et al. A health-economic anal-ysis of porcine islet xenotransplantation. Xenotransplantation. 2010;17:233-242. 179. Elliot RB, Living Cell

1	2011:479-502. 178. Beckwith J, Nyman JA, Flanagan B, et al. A health-economic anal-ysis of porcine islet xenotransplantation. Xenotransplantation. 2010;17:233-242. 179. Elliot RB, Living Cell Technologies, Ltd. Towards xeno-transplantation of pig islets in the clinic. Curr Opin Organ Transplant. 2011;16(2):195-200. 180. Marigliano M, Bertera S, Grupillo M, et al. Pig-to-nonhuman primate pancreatic islet xenotransplantation: an overview. Curr Diab Rep. 2011;11(5):402-412. 181. Dufrane D, Gianello P. Pig islet for xenotransplantation in human: structural and physiological compatibility for human clinical application. Transplant Rev (Orlando). 2012;26:183-188.Brunicardi_Ch11_p0355-p0396.indd 39601/03/19 6:55 PM errors, observing that 34% of patients with health problems in the United States report experiencing medical, medication, or test errors—the highest rate of any nation, and an analysis sug-gests that the problem of medical care gone wrong, i.e., medical errors including

1	States report experiencing medical, medication, or test errors—the highest rate of any nation, and an analysis sug-gests that the problem of medical care gone wrong, i.e., medical errors including systems errors, may rank as the third leading cause of death in the United States.1Medical error is defined as an unintended act (either of omission or commission) or one that does not achieve its intended outcome, the failure of a planned action to be com-pleted as intended (an error of execution) or the use of a wrong plan to achieve an aim (an error of planning), and a deviation from the process of care, which may or may not cause harm to the patient. Medical error can occur at the individual provider level or at the system level. An expanding taxonomy is matur-ing to better categorize the types of factors and events that are avoidable. The role of error may be complex; error can some-times tragically end the life of a thriving person with a long life expectancy, or it can also accelerate

1	of factors and events that are avoidable. The role of error may be complex; error can some-times tragically end the life of a thriving person with a long life expectancy, or it can also accelerate an imminent death.The most commonly cited report on the incidence of deaths due to medical error, the 1999 Institute of Medicine (IOM) report, describes an incidence of 44,000 to 98,000 deaths annually.2 However, this estimate by the IOM was not based on primary research conducted by the IOM; rather, it was based on two older studies conducted in 1984 and 1992. Both studies were small and limited. In 2013, after compiling more recent evidence from multiple sources, James estimated an incidence range of 210,000 to 400,000 deaths a year associated with Quality, Patient Safety, Assessments of Care, and ComplicationsMartin A. Makary, Peter B. Angood, and Mark L. Shapiro 12chapterBackground 397Medical Care Gone Wrong / 397Unnecessary Medical Care / 398The Science of Patient Safety 399High

1	Care, and ComplicationsMartin A. Makary, Peter B. Angood, and Mark L. Shapiro 12chapterBackground 397Medical Care Gone Wrong / 397Unnecessary Medical Care / 398The Science of Patient Safety 399High Reliability Organizations / 399The Conceptual Model / 399Creating a Culture of Safety 400Assessing an Organization’s Safety Culture / 401Teamwork and Communication 402Measuring Teamwork / 402Communication Tools 402Operating Room Briefings (A Surgical Checklist) / 402Operating Room Debriefings / 403Sign Outs / 403Implementation / 403Comprehensive Unit-Based Safety Program 404Measuring Quality in Surgery 405Practice Pattern Measures / 405Agency for Healthcare Research and Quality Patient Safety Indicators / 406The Surgical Care Improvement Project Measures / 406National Surgical Quality Improvement Program / 407The Leapfrog Group / 408World Health Organization “Safe Surgery Saves Lives” Initiative / 408National Quality Forum / 408“Never Events” in Surgery 409Retained Surgical Items /

1	Program / 407The Leapfrog Group / 408World Health Organization “Safe Surgery Saves Lives” Initiative / 408National Quality Forum / 408“Never Events” in Surgery 409Retained Surgical Items / 409Surgical Counts / 410Wrong-Site Surgery / 411The Joint Commission Universal Protocol to Ensure Correct Surgery / 411Transparency in Healthcare 412Public Reporting and Patient Assessment of Care / 412Risk Management 413The Importance of Communication in Managing Risk / 413Complications 415Robotic Surgery / 415Complications in Minor Procedures / 415Organ System Complications / 418Wounds, Drains, and Infection / 424Nutritional and Metabolic Support Complications / 426Problems with Thermoregulation / 427BACKGROUNDPatient harm due to medical mistakes can be catastrophic, result-ing in high-profile consequences for the patient, surgeon, and institution. A single error can even destroy a surgeon’s career. While mistakes are inherent to human nature, it is becoming more recognized that many mistakes

1	for the patient, surgeon, and institution. A single error can even destroy a surgeon’s career. While mistakes are inherent to human nature, it is becoming more recognized that many mistakes are preventable.Patient safety is a science that promotes the use of evi-dence-based medicine and local wisdom to minimize the impact of human error on quality patient care. Wrong-site/wrong-procedure surgeries, retained sponges, unchecked blood transfusions, mismatched organ transplants, and overlooked allergies are all examples of potentially catastrophic events that can be prevented by implementing safer hospital systems. This chapter provides an overview of the modern-day field of patient safety by reviewing key measures of safety and quality, compo-nents of culture, interventions and tools, assessment methods, risk management strategies, and a selected review of common complications in surgery.Medical Care Gone WrongToday, there are more medications, diagnoses, procedures, and handoffs

1	assessment methods, risk management strategies, and a selected review of common complications in surgery.Medical Care Gone WrongToday, there are more medications, diagnoses, procedures, and handoffs performed than ever in the history of medicine. More-over, overtreatment is now an endemic problem in some areas of healthcare. With more medical care being delivered, there are naturally more opportunities for things to go wrong. In fact, harm may be associated with complexity. The Commonwealth Fund reported that the United States leads the world in medical 12Brunicardi_Ch12_p0397-p0432.indd 39720/02/19 3:57 PM 398Figure 12-1. Causes of death in the United States 2013. (Reproduced with permission from Makary MA, Daniel M. Medical error–the third leading cause of death in the US, BMJ. 2016 May 3;353:i2139.)Key Points1 Medical error ranks as the third leading cause of death in the United States when defined to include system errors.2 One form of medical error is unnecessary or excessive

1	May 3;353:i2139.)Key Points1 Medical error ranks as the third leading cause of death in the United States when defined to include system errors.2 One form of medical error is unnecessary or excessive medi-cal care, which represents 21% of medical care administered in the United States.3 New peer-comparison metrics evaluate appropriateness of surgical care by measuring a physician’s practice pattern among all the physician’s patients benchmarked to the phy-sician’s peers.4 Judicious opioid prescribing upon discharge after surgery is critical given the magnitude of the opioid crisis.5 The structure-process-outcome framework within the context of an organization’s culture helps to clarify how risks and hazards embedded within the organization’s structure may potentially lead to error and injure or harm patients.6 Poor communication contributes to approximately 60% of the sentinel events reported to The Joint Commission.7 Operating room briefings are team discussions of critical issues

1	or harm patients.6 Poor communication contributes to approximately 60% of the sentinel events reported to The Joint Commission.7 Operating room briefings are team discussions of critical issues and potential hazards that can improve the safety of the operation and have been shown to improve operating room culture and decrease operating room delays.8 National Quality Forum surgical “never events” include retained surgical items, wrong-site surgery, and death on the day of surgery of a normal healthy patient (American Soci-ety of Anesthesiologists Class 1).9 The most important determinant of malpractice claims against a surgeon is patient rapport, not undertesting.medical errors among hospital patients.3 Any point estimate in this range would rank the problem of dying from “medical care gone wrong” as the third leading cause of death in the United States. In caring for patients and considering the risks of tests and procedures done for borderline indications, it is important to

1	care gone wrong” as the third leading cause of death in the United States. In caring for patients and considering the risks of tests and procedures done for borderline indications, it is important to consider the magnitude of the problem of patients dying from the care they receive rather than from the disease or injury that brought them to care.Human error is inevitable. But while we cannot eliminate human error, we can better measure the problem to design safer systems mitigating its frequency, visibility, and consequences. Hospitals should consider the magnitude of the problem of med-ical error as a barrier to achieving safe, highly-reliable medical care. Investigating errors to learn from them can guide local changes to prevent future events. The strategy to rein in the endemic problem of death from medical care should include three areas: (a) make errors less frequent (by following prin-ciples that take human limitations into account); (b) make errors more visible when they occur

1	of death from medical care should include three areas: (a) make errors less frequent (by following prin-ciples that take human limitations into account); (b) make errors more visible when they occur so that their effects can be intercepted; and (c) have remedies at hand when errors affect a patient. This multitier approach necessitates guidance from reliable data.Unnecessary Medical CareIncreasingly preventable complications and complications from unnecessary procedures are considered to be forms of medical error. Unnecessary medical care accounts for an estimated $210 billion in excess spending each year, according to the National Academy of Medicine. The issue represents a significant oppor-tunity to make improve patient safety and lower healthcare Causes of death, US, 2013Based on our estimate,medical error is the3rd most commoncause of death in the USMedicalerror251 kCancer585 kHeartdisease611 kCOPD149 kAll causes2,597 kMotorvehicles34 kFirearms34 kSuicide41

1	US, 2013Based on our estimate,medical error is the3rd most commoncause of death in the USMedicalerror251 kCancer585 kHeartdisease611 kCOPD149 kAll causes2,597 kMotorvehicles34 kFirearms34 kSuicide41 kBrunicardi_Ch12_p0397-p0432.indd 39820/02/19 3:57 PM 399QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12costs. In a Johns Hopkins study, surveying over 2000 physicians in the United States, unnecessary medical care was reported to be common.4 On average, these authors reported that 21% of medical care is unnecessary. Breaking the problem down by type of medical care, the doctors reported that 22% of prescription medications, 25% of medical tests, and 11% of procedures are unnecessary. These perceptions by U.S. physi-cians validate previous estimates of the National Academy of Medicine that suggest that one-third of healthcare spending is wasteful and does not result in better health. Addressing avoid-able medical and surgical care is a topic gaining increasing

1	Academy of Medicine that suggest that one-third of healthcare spending is wasteful and does not result in better health. Addressing avoid-able medical and surgical care is a topic gaining increasing recog-nition in healthcare.One example of overtreatment in surgical care is opioid over-prescribing.5 In the United States in 2015 alone, clini-cians handed out 249 million opioid prescriptions, almost one for every American adult. And in 2016, the United States produced 14 billion opioid pills (40 for every American citizen). With the exception of pain specialists treating patients with pain syndromes, surgeons are the most common prescribers of opi-oids. Judicious opioid prescribing is important because of the addictive potential of these medications. Moreover, many patients can recover comfortably after hospital discharge with nonopioid or nonaddictive pain regimens.THE SCIENCE OF PATIENT SAFETYMedicine is considered a high-risk system with a high error rate, but these two

1	recover comfortably after hospital discharge with nonopioid or nonaddictive pain regimens.THE SCIENCE OF PATIENT SAFETYMedicine is considered a high-risk system with a high error rate, but these two characteristics are not always correlated. Other high-risk industries have managed to maintain an impeccably low error rate. For example, one of the highest risk systems in existence today, the U.S. Navy’s nuclear submarine program, has an unmatched safety record.Much of the credit for their safety record is due to the culture of the nuclear submarine program, with its insistence on individual ownership, responsibility, attention to detail, professionalism, moral integrity, and mutual respect.6 These characteristics have created the cultural context necessary for high-quality communications under high-risk, high-stress con-ditions. Each reactor operator is aware of what is going on at all times and is responsible for understanding the implications and possible consequences of any action.

1	high-risk, high-stress con-ditions. Each reactor operator is aware of what is going on at all times and is responsible for understanding the implications and possible consequences of any action. Communication flows freely between crewmen and officers, and information about any mistakes that occur are dispersed rapidly through the entire system so that other workers can learn how to prevent similar mistakes in the future.High Reliability OrganizationsThe nuclear submarine program is an example of an organi-zation that has achieved the distinction of being considered a “high reliability organization.” High reliability organization theory recognizes that there are certain high-risk industries and organizations that have achieved very low accident and error rates compared to what would be expected given the inherent risks involved in their daily operations. Other high reliability industries and organizations include aircraft carrier flight decks, nuclear power plants, and the Federal

1	expected given the inherent risks involved in their daily operations. Other high reliability industries and organizations include aircraft carrier flight decks, nuclear power plants, and the Federal Aviation Administration’s air traffic control system. In fact, one reason why nuclear power plants have such an excellent reliability record may be that their operators are often former naval submarine officers whose pre-vious experience and training within one highly reliable organi-zation are easily transferable to other organizations.7One of the assumptions underlying the science of high reliability organizations is that humans who operate and manage 34complex systems are themselves not sufficiently complex to sense and anticipate the problems generated by the system.7 This introduces another important idea undergirding the sci-ence of patient safety: the concept of normal accident theory. Instead of attributing accidents to individual error, this theory states that accidents are

1	another important idea undergirding the sci-ence of patient safety: the concept of normal accident theory. Instead of attributing accidents to individual error, this theory states that accidents are intrinsic to high-volume activities and even inevitable in some settings. Accidents should not be used merely to identify and punish the person at fault, but should be seen as a systems problem and addressed at a broader level. As Ruchlin states, even the “best people can make the worst errors as a result of latent conditions.”7High-risk systems, as defined by Perrow in 19848:• Have the potential to create a catastrophe, loosely defined as an event leading to loss of human or animal life, despoiling of the environment, or some other situation that gives rise to the sense of “dread.”• Are complex, in that they have large numbers of highly inter-dependent subsystems with many possible combinations that are nonlinear and poorly understood.• Are tightly coupled, so that any perturbation in the

1	in that they have large numbers of highly inter-dependent subsystems with many possible combinations that are nonlinear and poorly understood.• Are tightly coupled, so that any perturbation in the sys-tem is transmitted rapidly between subsystems with little attenuation.However, high reliability organization theory suggests that proper oversight of people, processes, and technology can handle complex and hazardous activities and keep error rates acceptably low.7 Studies of multiple high reliability organiza-tions show that they share the following common characteristics:• People are supportive of one another.• People trust one another.• People have friendly, open relationships emphasizing cred-ibility and attentiveness.• The work environment is resilient and emphasizes creativity and goal achievement, providing strong feelings of credibility and personal trust.Developing these characteristics is an important step toward achieving a low error rate in any organization. For this reason,

1	achievement, providing strong feelings of credibility and personal trust.Developing these characteristics is an important step toward achieving a low error rate in any organization. For this reason, safety culture is a measure used by hospitals nationwide to improve outcomes and is increasingly recognized as a metric of hospital quality.The Conceptual ModelThe Donabedian model of measuring quality identifies three main types of improvements: changes to organizational struc-ture, changes in organizational processes, and changes in outcomes.9 Structure refers to the physical and organiza-tional tools, equipment, and policies that improve safety. Struc-tural measures ask, “Do the right tools, equipment, and policies exist?” Process is the application of these tools, equipment, and policies/procedures to patients (good practices and evidence-based medicine). Process measures ask, “Are the right tools, policies, and equipment being used?” Outcome is the result on patients. Outcome

1	to patients (good practices and evidence-based medicine). Process measures ask, “Are the right tools, policies, and equipment being used?” Outcome is the result on patients. Outcome measures ask, “How often are patients harmed?” In this model, structure (how care is organized) plus process (what we do) influences patient outcomes (the results achieved).10The structure, process, and outcome components of qual-ity measurement all occur within the context of an organiza-tion’s overall culture. The local culture impacts all aspects of the delivery of care because it affects how front-line personnel 5Brunicardi_Ch12_p0397-p0432.indd 39920/02/19 3:57 PM 400BASIC CONSIDERATIONSPART ITable 12-1Types of medical errorAdverse event• Injury caused by medical management rather than the underlying condition of the patient• Prolongs hospitalization, produces a disability at discharge, or both• Classified as preventable or unpreventableNegligence• Care that falls below a recognized standard of

1	condition of the patient• Prolongs hospitalization, produces a disability at discharge, or both• Classified as preventable or unpreventableNegligence• Care that falls below a recognized standard of care• Standard of care is considered to be care a reasonable physician of similar knowledge, training, and experience would use in similar circumstancesNear miss• An error that does not result in patient harm• Analysis of near misses provides the opportunity to identify and remedy system failures before the occurrence of harmSentinel event• An unexpected occurrence involving death or serious physical or psychological injury• The injury involves loss of limb or function• This type of event requires immediate investigation and response• Other examples• Hemolytic transfusion reaction involving administration of blood or blood products having major blood group incompatibilities• Wrong-site, wrong-procedure, or wrong-patient surgery• A medication error or other treatment-related error resulting

1	of blood or blood products having major blood group incompatibilities• Wrong-site, wrong-procedure, or wrong-patient surgery• A medication error or other treatment-related error resulting in death• Unintentional retention of a foreign body in a patient after surgeryReproduced with permission from Makary M: General Surgery Review. Washington, DC: Ladner-Drysdale; 2008.Case 12-1 Systems change resulting from medical errorLibby Zion was an 18-year-old woman who died after being admitted to the New York Hospital with fever and agitation on the evening of October 4, 1984. Her father, Sidney Zion, a lawyer and columnist for the N.Y. Daily News, was convinced that his daughter’s death was due to inadequate staffing and overworked physicians at the hospital and was determined to bring about changes to prevent other patients from suffering as a result of the teaching hospital system. Due to his efforts to publicize the circumstances surrounding his daughter’s death, Manhattan District

1	changes to prevent other patients from suffering as a result of the teaching hospital system. Due to his efforts to publicize the circumstances surrounding his daughter’s death, Manhattan District Attorney Robert Morgenthau agreed to let a grand jury consider murder charges. Although the hospital was not indicted, in May 1986, a grand jury issued a report strongly criticizing “the supervision of interns and junior resi-dents at a hospital in NY County.”As a result, New York State Health Commissioner David Axelrod convened a panel of experts headed by Bertrand M. Bell, a primary care physician at Albert Einstein College of Medicine who had long been critical of the lack of supervision of physicians-in-training, to evaluate the training and supervision of doctors in New York State. The Bell Commission recom-mended that residents work no more than 80 hours per week and no more than 24 consecutive hours per shift, and that a senior physician needed to be physically present in the

1	The Bell Commission recom-mended that residents work no more than 80 hours per week and no more than 24 consecutive hours per shift, and that a senior physician needed to be physically present in the hospital at all times. These recommendations were adopted by New York State in 1989. In 2003, the Accreditation Council on Graduate Medi-cal Education followed by mandating that all residency training programs adhere to the reduced work hour schedule.understand and deliver safe patient care. In fact, culture (col-lective attitudes and beliefs of caregivers) is increasingly being recognized to be the fourth measurable component to the structure-process-outcome model. This recognition is based on growing evidence that local culture is linked to a variety of important clinical outcomes.10 For any new patient safety initiative to be deemed successful, any change in structure or process must lead to a corresponding positive change in patient outcomes.11CREATING A CULTURE OF SAFETYCulture is to

1	new patient safety initiative to be deemed successful, any change in structure or process must lead to a corresponding positive change in patient outcomes.11CREATING A CULTURE OF SAFETYCulture is to an organization what personality is to the individual—a hidden, yet unifying theme that provides meaning, direction, and mobilization.7 Organizations with effective safety cultures share a constant commitment to safety as a top-level priority that permeates the entire organization. These organizations fre-quently share the following characteristics12:• An acknowledgment of the high-risk, error-prone nature of an organization’s activities• A nonpunitive environment where individuals are able to report errors or close calls without fear of punishment or retaliation• An expectation of collaboration across ranks to seek solutions to vulnerabilities• A willingness on the part of the organization to direct resources to address safety concernsTraditional surgical culture stands almost in direct

1	across ranks to seek solutions to vulnerabilities• A willingness on the part of the organization to direct resources to address safety concernsTraditional surgical culture stands almost in direct oppo-sition to the values upheld by organizations with effective safety cultures for several reasons. Surgeons are less likely to acknowledge their propensity to make mistakes or to admit these mistakes to others.13 Surgeons tend to minimize the effect of stress on their ability to make decisions.14 The surgical cul-ture, especially in the operating room (OR), is traditionally rife with hierarchy. Intimidation of other OR personnel by sur-geons was historically accepted as the norm. This can prevent nurses and other OR staff from pointing out potential errors or Brunicardi_Ch12_p0397-p0432.indd 40020/02/19 3:57 PM 401QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12• I know the proper channels to direct questions regarding patient safety in this clinical area.• I

1	40020/02/19 3:57 PM 401QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12• I know the proper channels to direct questions regarding patient safety in this clinical area.• I receive appropriate feedback about my performance.• I would feel safe being treated here as a patient.• In this clinical area, it is not difficult to discuss mistakes.Although perceptions of teamwork climate can differ as a function of one’s role in the OR, perceptions of safety climate are relatively consistent across OR providers in a given hospital. Validated in over 500 hospitals, the SAQ is used to establish benchmark safety culture scores by healthcare worker type, department, and hospital. Using this survey, hospitals can com-pare culture between different types of healthcare workers within a department as well as culture between departments throughout the institution. Scores can be compared to those of other par-ticipating institutions to compare safety climates. This allows

1	within a department as well as culture between departments throughout the institution. Scores can be compared to those of other par-ticipating institutions to compare safety climates. This allows hospitals to participate with one another to implement programs to improve safety culture. In addition, scores are used to evaluate the effectiveness of safety interventions by comparing the SAQ safety climate scores after implementation to baseline scores.Strong teamwork is at the core of any effective organiza-tion and is a key element to ensuring patient safety in the OR. Teamwork is dependent on the underlying culture and patterns of communication. The ability for all team members, to “speak up” about patient safety concerns is one of the most important elements of creating a culture of patient safety.TEAMWORK AND COMMUNICATIONAccording to The Joint Commission, communication breakdown is one of the top three root causes of sentinel events such as wrong-site surgery (Fig. 12-2). Poor

1	patient safety.TEAMWORK AND COMMUNICATIONAccording to The Joint Commission, communication breakdown is one of the top three root causes of sentinel events such as wrong-site surgery (Fig. 12-2). Poor communication contributed to over 60% of sentinel events reported to The Joint Commission in 2011.17 Good communication is an essential component of teamwork and is especially important in the OR, one of the most complex work environments in healthcare.Within the realm of patient care, there are enormous amounts of information being exchanged between healthcare pro-viders on a daily basis. Much of this information, if prioritized correctly, has the potential to prevent unintended medical errors and serious harm to patients. The importance of good communi-cation in preventing medical errors is undeniable; however, it is difficult to achieve. The traditional surgical hierarchy can prevent OR personnel from sharing important patient data and expressing safety concerns. One perioperative

1	is undeniable; however, it is difficult to achieve. The traditional surgical hierarchy can prevent OR personnel from sharing important patient data and expressing safety concerns. One perioperative field study showed a 30% rate of communication failure in the OR, with 36% of these break-downs having a substantial impact on patient safety.18In addition to overcoming the cultural barrier to better teamwork and communication, the prospective study by Chris-tian and associates of patient safety in the OR demonstrated that the standard workflow of the OR itself presents many opportunities for the loss or degradation of critical informa-tion.19 Hand-offs of patient care from the OR to other locations or providers are particularly prone to information loss, which has been demonstrated in other clinical settings. Hand-offs and auxiliary tasks, such as surgical sponge and instrument counts, frequently take place during critical portions of the case and place competing demands on provider

1	clinical settings. Hand-offs and auxiliary tasks, such as surgical sponge and instrument counts, frequently take place during critical portions of the case and place competing demands on provider attention from primary patient-centered activities. Communication between the surgeon and pathologist also is vulnerable because the communication often occurs through secondary messengers such as nurses or technicians. This information loss can lead to delays, overuse of 6Case 12-2 High-profile sentinel eventOn December 3, 1994, Betsy Lehman, a Boston Globe health columnist, died as a result of receiving four times the intended dose of chemotherapy for breast cancer. Remarkably, 2 days later, Maureen Bateman, a teacher being treated for cancer, also received a chemotherapy overdose and suffered irrevers-ible heart damage. After investigating the medication errors, the prescribing doctor, three druggists, and 15 nurses were disciplined by state regulators. The hospital was sued by the two

1	irrevers-ible heart damage. After investigating the medication errors, the prescribing doctor, three druggists, and 15 nurses were disciplined by state regulators. The hospital was sued by the two women’s families and by one of the doctors disciplined.As a result of this widely publicized event, the Dana-Farber Cancer Institute invested more than $11 million to overhaul their safety programs, including providing new training for their employees and giving doctors more time to meet with patients. The hospital adopted a full disclosure policy so that patients would be informed anytime a mistake had affected their care. Dana-Farber also started a patient com-mittee providing advice and feedback on ways to improve care at the hospital.mistakes. Moreover, this culture is not limited to the OR. In the intensive care unit (ICU), when compared to physicians, nurses reported more difficulty speaking up, disagreements were not appropriately resolved, and decisions were made without ade-quate

1	In the intensive care unit (ICU), when compared to physicians, nurses reported more difficulty speaking up, disagreements were not appropriately resolved, and decisions were made without ade-quate input.15 In addition, the field of medicine strongly values professional autonomy, which frequently promotes individual-ism over cooperation, often to the detriment of patient care.16 Finally, patient safety, although often viewed as important, is seldom promoted from an organizational priority to an organiza-tional value. Organizations often do not feel the need to devote resources to overhauling their patient safety systems as long as they perceive their existing processes to be adequate. It often takes a high-profile sentinel event to motivate leaders to com-mit the necessary time and resources to improving patient safety within their organization, as exemplified by the Dana-Farber Institute in the aftermath of Betsy Lehman’s death (Case 12-2).Assessing an Organization’s Safety

1	resources to improving patient safety within their organization, as exemplified by the Dana-Farber Institute in the aftermath of Betsy Lehman’s death (Case 12-2).Assessing an Organization’s Safety CultureEfforts to foster cultural change within an organization with regard to patient safety have been limited in the past by the inability to measure the impact of any given intervention. How-ever, studies have shown that employee attitudes about culture are associated with error reduction behaviors in aviation and with patient outcomes in ICUs. The Safety Attitudes Question-naire (SAQ) is a validated survey instrument that can be used to measure culture in a healthcare setting.9 Adapted from two safety tools used in aviation, the Flight Management Attitudes Questionnaire and its predecessor, the Cockpit Management Attitudes Questionnaire, the SAQ consists of a series of ques-tions measuring six domains: teamwork climate, safety climate, job satisfaction, perception of management, stress

1	the Cockpit Management Attitudes Questionnaire, the SAQ consists of a series of ques-tions measuring six domains: teamwork climate, safety climate, job satisfaction, perception of management, stress recognition, and working conditions.The safety climate scale portion of the questionnaire con-sists of the following seven items:• I am encouraged by my colleagues to report any patient safety concerns I may have.• The culture in this clinical area makes it easy to learn from the mistakes of others.• Medical errors are handled appropriately in this clinical area.Brunicardi_Ch12_p0397-p0432.indd 40120/02/19 3:57 PM 402BASIC CONSIDERATIONSPART I0 20 40 60 80 Medication UseContinuum of CareCare PlanningOperative CareInformation ManagementPhysical EnvironmentAssessmentCommunicationLeadershipHuman FactorsPercent of Events (%)Figure 12-2. Root causes of sentinel events 2004 to 2012. (Data from The Joint Commission, 2012.)staff and resources, uncertainty in clinical decision making and

1	FactorsPercent of Events (%)Figure 12-2. Root causes of sentinel events 2004 to 2012. (Data from The Joint Commission, 2012.)staff and resources, uncertainty in clinical decision making and planning, and oversights in patient preparation.Measuring TeamworkResearch in commercial aviation has demonstrated a strong correlation between better teamwork and improved safety per-formance. Cockpit crew members’ reluctance to question a cap-tain’s judgment has been identified as a root cause of aviation accidents. Good attitudes about teamwork are associated with error-reduction behaviors in aviation, improved patient out-comes in ICUs, and decreased nurse turnover in the OR. It is also associated with higher job satisfaction ratings and less sick time taken from work.The SAQ can be used to measure teamwork and provide benchmarks for departments or hospitals seeking to measure and improve their teamwork climate.20 The SAQ teamwork scores are responsive to interventions that aim to improve

1	teamwork and provide benchmarks for departments or hospitals seeking to measure and improve their teamwork climate.20 The SAQ teamwork scores are responsive to interventions that aim to improve team-work among operating teams, such as the implementation of ICU checklists, executive walk rounds, and preoperative brief-ing team discussions. The communication and collaboration sections of the SAQ reflect OR caregiver views on teamwork and can be used to distinguish meaningful interventions from impractical and ineffective programs.In a survey of OR personnel across 60 hospitals, the SAQ identified substantial differences in the perception of team-work in the OR depending on one’s role. Physicians frequently rated the teamwork of others as good, while nurses at the same institutions perceived teamwork as poor (Fig. 12-3). Similar 100%87%Surgeon rates OR nurseOR nurse rates surgeon48%90%80%70%60%Percent rating quality of collaborationand communication as high or very

1	institutions perceived teamwork as poor (Fig. 12-3). Similar 100%87%Surgeon rates OR nurseOR nurse rates surgeon48%90%80%70%60%Percent rating quality of collaborationand communication as high or very high50%40%30%20%10%0%Figure 12-3. Differences in teamwork perceptions between sur-geons and operating room (OR) nurses. (Reproduced with permis-sion from Makary MA, Sexton JB, Freischlag JA, et al. Operating room teamwork among physicians and nurses: teamwork in the eye of the beholder, J Am Coll Surg. 2006 May;202(5):746-752.)discrepancies have been found in ICUs. These discrepancies can be attributed to differences in the communication skills that are valued by surgeons and nurses. For example, nurses describe good collaboration as having their input respected, while physicians describe good collaboration as having nurses who can anticipate their needs and follow instructions. Efforts to improve the communication that takes place between physicians and nurses can directly improve the

1	collaboration as having nurses who can anticipate their needs and follow instructions. Efforts to improve the communication that takes place between physicians and nurses can directly improve the perception of teamwork and collaboration by the OR team (Table 12-2). Empowering well-respected surgeons to promote principles of teamwork and communication can go a long way toward transforming attitu-dinal and behavioral changes in fellow physicians as well as other members of the surgical team. Surgeons are increasingly encouraging the respectful and timely voicing of concerns of OR personnel.COMMUNICATION TOOLSHigh reliability organizations such as aviation frequently use tools such as prompts, checks, standard operating protocols, and communication interventions such as team briefings and debriefings. These tools identify and mitigate hazards and allow an organization to complete tasks more efficiently. They also foster a culture of open communication and speaking up if a team member

1	debriefings. These tools identify and mitigate hazards and allow an organization to complete tasks more efficiently. They also foster a culture of open communication and speaking up if a team member senses a safety concern. Safety checks and standardized team discussions serve as prompts to help “engineer out” human error, providing quality assurance and improving information flow. They also can prevent errors related to omissions, which are more likely to occur when there is information overload, multiple steps in a process, repetitions in steps, and planned departures from routine processes, and when there are other interruptions and distractions present while the process is being executed. These same interventions have been shown to improve patient safety in ORs and ICUs.21,22Operating Room Briefings (A Surgical Checklist)Preoperative briefings and checklists, when used appropriately, help to facilitate transfer of information between team members (Table 12-3). A briefing, or

1	Room Briefings (A Surgical Checklist)Preoperative briefings and checklists, when used appropriately, help to facilitate transfer of information between team members (Table 12-3). A briefing, or checklist, is any preprocedure dis-cussion of requirements, needs, and special issues of the proce-dure. Briefings often are locally adapted to the specific needs of the specialty. They have been associated with an improved safety culture, including increased awareness of wrong-site/wrong-procedure errors, early reporting of equipment prob-lems, reduced operational costs and fewer unexpected delays. In one study, 30.9% of OR personnel reported a delay before the 7Brunicardi_Ch12_p0397-p0432.indd 40220/02/19 3:57 PM 403QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12Table 12-2Percentage of operating room caregivers reporting a high or very high level of collaboration with other members of the operating room teamCAREGIVER POSITION PERFORMING RATING CAREGIVER POSITION

1	12-2Percentage of operating room caregivers reporting a high or very high level of collaboration with other members of the operating room teamCAREGIVER POSITION PERFORMING RATING CAREGIVER POSITION BEING RATEDSURGEONANESTHESIOLOGISTNURSECRNASurgeon85848887Anesthesiologist70968992Nurse48638168CRNA58757693The best teamwork scores were recorded by anesthesiologists when they rated their teamwork with other anesthesiologists (“high” or “very high” 96% of the time). The lowest teamwork ratings were recorded by nurses when they rated their teamwork with surgeons (“high” or “very high” 48% of the time).CRNA = certified registered nurse anesthetist.Reproduced with permission from Makary MA, Sexton JB, Freischlag JA, et al. Operating room teamwork among physicians and nurses: teamwork in the eye of the beholder, J Am Coll Surg. 2006 May;202(5):746-752.Table 12-3Five-point operating room briefingWhat are the names and roles of the team members?Is the correct patient/procedure confirmed? (The

1	of the beholder, J Am Coll Surg. 2006 May;202(5):746-752.Table 12-3Five-point operating room briefingWhat are the names and roles of the team members?Is the correct patient/procedure confirmed? (The Joint Commission Universal Protocol [TIME-OUT])Have antibiotics been given? (if appropriate)What are the critical steps of the procedure?What are the potential problems for the case?Data from Makary MA, Mukherjee A, Sexton JB, et al. Operating room briefings and wrong-site surgery, J Am Coll Surg. 2007 Feb; 204(2):236-243.institution of OR briefings, and only 23.3% reported delays after briefings were instituted.23 OR briefings are increasingly being used to ensure evidence-based measures are used, such as the appropriate administration of preoperative antibiotics and deep vein thrombosis (DVT) prophylaxis. Briefings allow personnel to discuss potential problems, before they become a “near miss” or cause actual harm.Operating Room DebriefingsPostprocedural debriefings improve patient

1	(DVT) prophylaxis. Briefings allow personnel to discuss potential problems, before they become a “near miss” or cause actual harm.Operating Room DebriefingsPostprocedural debriefings improve patient safety by allowing for discussion and reflection on causes for errors and critical incidents that occurred during the case. Errors or critical inci-dents are regarded as learning opportunities rather than cause for punishment. During the debriefing, the team also can discuss what went well during the case and designate a point person to follow up on any proposed actions that result from the discus-sion. In addition, most debriefings include a verification of the sponge, needle, and instrument counts and confirmation of the correct labeling of the surgical specimen.Errors in surgical specimen labeling have not received as much attention as incorrect sponge or instrument counts as an indicator of the quality of communication in the OR. How-ever, an error in communication or during the

1	labeling have not received as much attention as incorrect sponge or instrument counts as an indicator of the quality of communication in the OR. How-ever, an error in communication or during the hand-off process increases the risk of mislabeling a surgical specimen before its arrival in a pathology laboratory. In one study, this type of identification error occurred in 4.3 per 1000 surgical specimens, which implies an annualized rate of occurrence of 182 misla-beled specimens per year (Fig. 12-5).24 Errors involving speci-men identification can result in delays in care, the need for an additional biopsy or therapy, failure to use appropriate therapy, or therapy administered to the wrong body site, side, or patient. These system failures can lead to significant harm to the patient, costs to the institution, and distrust by a community. Given the frequency of occurrence and the feasibility and validity of mea-suring them, mislabeled surgical specimens may serve as a use-ful indicator of

1	the institution, and distrust by a community. Given the frequency of occurrence and the feasibility and validity of mea-suring them, mislabeled surgical specimens may serve as a use-ful indicator of patient safety and should be included in any postprocedural debriefing checklist.Sign OutsIn healthcare, information frequently passes to covering provid-ers without prioritizing potential concerns. This makes sign outs a very vulnerable process of care, which can lead to catastrophic events.The term sign out can refer to either the verbal or written communication of patient information to familiarize oncoming physicians about patients who will be under their care. Sign outs should occur whenever a patient’s care setting or provider is changing. When performed well, sign outs help to ensure the transfer of pertinent information. However, previous studies have shown the hand-off process to be variable, unstructured, and prone to error. Common categories of communication fail-ure during sign

1	transfer of pertinent information. However, previous studies have shown the hand-off process to be variable, unstructured, and prone to error. Common categories of communication fail-ure during sign outs include content omissions, such as failure to mention active medical problems, and failures in the actual communication process, such as leaving illegible or unclear notes (Case 12-3).25 These failures lead to confusion and uncer-tainty by the covering physician during patient care decisions, resulting in the delivery of inefficient and suboptimal care.The use of more structured verbal communication such as the Situational Debriefing Model, otherwise known as SBAR (situation, background, assessment, and recommendation), used by the U.S. Navy, can be applied to healthcare to improve the communication of critical information in a timely and orderly fashion.25 In addition, all sign outs should begin with the state-ment, “In this patient, I am most concerned about . . .” to signal to the

1	of critical information in a timely and orderly fashion.25 In addition, all sign outs should begin with the state-ment, “In this patient, I am most concerned about . . .” to signal to the healthcare provider on the receiving end the most impor-tant safety concerns regarding that specific patient.ImplementationTools such as checklists, sign outs, briefings, and debrief-ings improve communication between healthcare providers and create a safer patient environment (Fig. 12-6). Although their use in healthcare is still highly variable, specialties that Brunicardi_Ch12_p0397-p0432.indd 40320/02/19 3:57 PM 404BASIC CONSIDERATIONSPART ISurgical Safety ChecklistHas the patient conÿrmed his/her identity, site, procedure, and consent?YesIs the site marked?YesNot applicableYesYesNoYesDifÿcult airway or aspiration risk?NoYes, and equipment/assistance availableRisk of >500ml blood loss (7ml/kg in children)?NoYes, and two IVs/central access and ˜uids plannedConÿrm all team members have

1	airway or aspiration risk?NoYes, and equipment/assistance availableRisk of >500ml blood loss (7ml/kg in children)?NoYes, and two IVs/central access and ˜uids plannedConÿrm all team members have introduced themselves by name and role.Conÿrm the patient’s name, procedure, and where the incision will be made.Has antibiotic prophylaxis been given within the last 60 minutes?YesNot applicableAnticipated Critical EventsTo Surgeon:What are the critical or non-routine steps?How long will the case take?What is the anticipated blood loss?To Anaesthetist:Are there any patient-speciÿc concerns?To Nursing Team:Has sterility (including indicator results) been conÿrmed?Are there equipment issues or any concerns?YesNurse Verbally Conÿrms:The name of the procedureCompletion of instrument, sponge andneedle countsSpecimen labelling (read specimen labelsaloud, including patient name)Whether there are any equipment problemsto be addressedWhat are the key concerns for recovery andmanagement of this patient?

1	labelling (read specimen labelsaloud, including patient name)Whether there are any equipment problemsto be addressedWhat are the key concerns for recovery andmanagement of this patient? This checklist is not intended to be comprehensive. Additions and modiÿcations to ÿt local practice are encouraged.(with at least nurse and anaesthetist)(with nurse, anaesthetist and surgeon)(with nurse, anaesthetist and surgeon)© WHO, 2009Before induction of anaesthesiaBefore skin incisionBefore patient leaves operating roomRevised 1 / 2009To Surgeon, Anaesthetist and Nurse:Is essential imaging displayed?Not applicableIs the anaesthesia machine and medicationcheck complete? Is the pulse oximeter on the patient andfunctioning?Known allergy? Does the patient have a: Figure 12-4. World Health Organization’s surgical safety checklist. (Reproduced with permission from World Health Organization Safe Surgery Saves Lives. Available at: http://www.who.int/patientsafety/safesurgery/en/. Accessed November 8,

1	surgical safety checklist. (Reproduced with permission from World Health Organization Safe Surgery Saves Lives. Available at: http://www.who.int/patientsafety/safesurgery/en/. Accessed November 8, 2012.)Specimennot labeledEmptycontainerIncorrectlateralityIncorrecttissue siteIncorrectpatientnameNo patientnameNo tissuesite0.90.80.70.60.50.40.30.20.10Incidence (per 1000 specimens)Error typeFigure 12-5. Incidence of identification errors observed per 1000 specimens (n = 21,351). (Reproduced with permission from Makary MA, Epstein J, Pronovost PJ, et al. Surgical specimen identification errors: a new mea-sure of quality in surgical care, Surgery. 2007 Apr;141(4):450-455.)have incorporated them, such as intensive care and anesthesia, have made impressive strides in patient safety. Currently, com-munication breakdowns, information loss, hand off, multiple competing tasks, and high workload are considered “annoy-ing but accepted features” of the perioperative environment.20 As physician

1	com-munication breakdowns, information loss, hand off, multiple competing tasks, and high workload are considered “annoy-ing but accepted features” of the perioperative environment.20 As physician attitudes toward errors, stress, and teamwork in medicine become more favorable toward the common goals of reducing error and improving teamwork and communication, medicine will likely achieve many of the milestones in safety that high-reliability industries such as aviation have already accomplished.COMPREHENSIVE UNIT-BASED SAFETY PROGRAMAs medical care and hospitals continue to expand, the care that is provided to patients is becoming more fragmented. This frag-mentation makes communication more difficult and opportuni-ties for medical errors more common. These problems require common sense solutions, often necessitating a change in the way that care is delivered on the local level. Unit-based meetings to discuss processes that are potentially dangerous for patients can quickly bring

1	solutions, often necessitating a change in the way that care is delivered on the local level. Unit-based meetings to discuss processes that are potentially dangerous for patients can quickly bring danger areas out into the open. These meetings Brunicardi_Ch12_p0397-p0432.indd 40420/02/19 3:57 PM 405QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12Case 12-3 Inadequate sign out leading to medical errorJosie King was an 18-month-old child who was admitted to Johns Hopkins Hospital in January of 2001 for firstand second-degree burns. She spent 10 days in the pediatric intensive care unit and was well on her way to recovery. She was transferred to an intermediate care floor with the expectation that she would be sent home in a few days.The following week, her central line was removed, but nurses would not allow Josie to drink anything by mouth. Around 1 pm the next day, a nurse came to Josie’s bedside with a syringe of methadone. Although Josie’s mother told the

1	line was removed, but nurses would not allow Josie to drink anything by mouth. Around 1 pm the next day, a nurse came to Josie’s bedside with a syringe of methadone. Although Josie’s mother told the nurse that there was no order for narcotics, the nurse insisted that the orders had been changed and administered the drug. Josie’s heart stopped, and her eyes became fixed. She was moved to the pediatric intensive care unit and placed on life support. Two days later, on February 22, 2001, she died from severe dehydration.After her death, Josie’s parents, Sorrel and Jay King, were motivated to work with leaders at Johns Hopkins to ensure that no other family would have to endure the death of a child due to medical error. They later funded the Josie King Patient Safety Program and an academic scholarship in the field of safety.A preoperativediscussionincreased myawareness of thesurgical site andside beingoperated on.01020304050Percent of respondents who agreed60708090100The surgical siteof

1	in the field of safety.A preoperativediscussionincreased myawareness of thesurgical site andside beingoperated on.01020304050Percent of respondents who agreed60708090100The surgical siteof the operationwas clear to mebefore theincision.Decision makingutilized inputfrom relevantpersonnel.Surgery andanesthesiaworked togetheras a well-coordinatedteam.Postbriefing PrebriefingFigure 12-6. Impact of operating room briefings on team-work and communication.should be held on a regular basis and bring together a multidisci-plinary team of physicians, nurses, technicians, social workers, and other staff who can each voice their concerns about safety hazards in their area. This enables all aspects of patient care to be addressed and improved continuously, thereby streamlining and improving patient care.26The implementation of the Comprehensive Unit-based Safety Program (CUSP) involves measurement of a unit’s safety culture prior to starting the program and inclusion of hospital management from

1	care.26The implementation of the Comprehensive Unit-based Safety Program (CUSP) involves measurement of a unit’s safety culture prior to starting the program and inclusion of hospital management from the start. Having management involved allows for more efficient allocation of resources and allows them to better understand the problems faced by front-line pro-viders. Once CUSP is in place, changes can be made using local wisdom to advance patient care.26 The impact of changes made using CUSP can be measured using both patient outcomes and safety culture data.Implementation of CUSP has been associated with improved patient outcomes, including decreased surgical site infections. In a 2-year study of colorectal patients, where the first year was pre-CUSP implementation and the second year was post-CUSP implementation, there was a 33% decrease in the surgical site infection rate after CUSP.27 In this study, the CUSP group met monthly and came up with a list of interven-tions based on

1	was post-CUSP implementation, there was a 33% decrease in the surgical site infection rate after CUSP.27 In this study, the CUSP group met monthly and came up with a list of interven-tions based on their experience with these cases, including stan-dardization of skin preparation and warming of patients in the preanesthesia area. This study showed that CUSP can be highly effective in ameliorating patient harm and improving patient care.MEASURING QUALITY IN SURGERYDespite the newfound focus on patient safety in surgery and the number of initiatives being undertaken by many organizations to improve their safety culture, there are few tools to actually measure whether these efforts are effective in reducing the num-ber of errors. Several agencies and private groups have devel-oped criteria to evaluate quality and safety within hospitals.Practice Pattern MeasuresNew quality measures in healthcare focus on the appropriate-ness of medical care.28 These appropriateness indicators are

1	to evaluate quality and safety within hospitals.Practice Pattern MeasuresNew quality measures in healthcare focus on the appropriate-ness of medical care.28 These appropriateness indicators are doctor-defined and specialty-specific so they are smart and fair. One of the first of these new appropriateness metrics is the aver-age number of tissue blocks a skin cancer (Mohs) surgeon will use to surgically remove a skin cancer. The American College of Mohs Surgeons formalized and endorsed the surgeon metric: average number of blocks a surgeon requires to remove a stan-dardized skin cancer. In a report describing the national distri-bution of surgeons by their mean number of blocks per case, the national average was found to be 1.7 blocks per surgeon. Statistical outlier surgeons had an average four or more blocks per patient. Boundaries of normal variation was determined by expert physician leaders to define an acceptable range and an Brunicardi_Ch12_p0397-p0432.indd 40520/02/19 3:57

1	four or more blocks per patient. Boundaries of normal variation was determined by expert physician leaders to define an acceptable range and an Brunicardi_Ch12_p0397-p0432.indd 40520/02/19 3:57 PM 406BASIC CONSIDERATIONSPART Iunacceptable range (greater than two standard deviations from the national norm). The American College of Mohs Surgeons sent letters to outliers, letting them know where they stand, and offered coaching and retraining help. The new Mohs surgery metric demonstrates the opportunity to reduce unwarranted clinical variation and lower healthcare costs by simply using clinical wisdom and the power of peer-comparison.Appropriateness measures approach quality differently than traditional quality measures and rely on expert physicians to define the metric and set boundaries of reasonable versus unsafe variation in an individual physician’s practice pattern rel-ative to his or her peers nationally. This concept is being applied to utilization rates of minimally

1	of reasonable versus unsafe variation in an individual physician’s practice pattern rel-ative to his or her peers nationally. This concept is being applied to utilization rates of minimally invasive surgery in candidate patients as well as rates of physical therapy utilization before elective spine surgery for chronic pain.Agency for Healthcare Research and Quality Patient Safety IndicatorsThe Agency for Healthcare Research and Quality (AHRQ) was created in 1989 as a Public Health Service agency in the Depart-ment of Health and Human Services. Its mission is to improve the quality, safety, efficiency, and effectiveness of healthcare for all Americans. Nearly 80% of the AHRQ’s budget is awarded as grants and contracts to researchers at universities and other research institutions across the country. The AHRQ sponsors and conducts research that provides evidence-based information on healthcare outcomes, quality, cost, use, and access. It has advocated the use of readily available

1	the country. The AHRQ sponsors and conducts research that provides evidence-based information on healthcare outcomes, quality, cost, use, and access. It has advocated the use of readily available hospital inpatient admin-istrative data to measure healthcare quality. The information helps healthcare decision makers make more informed decisions and improve the quality of healthcare services.29One of the major contributions of the AHRQ is a set of Patient Safety Indicators (PSIs), initially released in 2003 and revised in 2010. PSIs are a tool to help health system leaders identify potential adverse events occurring during hospitaliza-tion. Developed after a comprehensive literature review, analy-sis of International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes, review by a clini-cian panel, implementation of risk adjustment, and empirical analyses, these 27 indicators provide information on potential in-hospital complications and adverse events

1	codes, review by a clini-cian panel, implementation of risk adjustment, and empirical analyses, these 27 indicators provide information on potential in-hospital complications and adverse events following surger-ies, procedures, and childbirth (Table 12-4).Provider-level indicators provide a measure of the poten-tially preventable complications for patients who received their initial care and the complication of care within the same hos-pitalization. They include only those cases where a secondary diagnosis code flags a potentially preventable complication. Area-level indicators capture all cases of the potentially pre-ventable complications that occur in a given area (e.g., metro-politan area or county), either during their initial hospitalization or resulting in subsequent hospitalization.30Currently, PSIs are considered indicators, not definitive measures, of patient safety concerns. They can identify potential safety problems that merit further investigation. They also can be used

1	PSIs are considered indicators, not definitive measures, of patient safety concerns. They can identify potential safety problems that merit further investigation. They also can be used to better prioritize and evaluate local and national initiatives, and even as benchmarks for tracking progress in patient safety. In the future, further growth in electronic health data will make administrative data-based tools like the PSIs more useful.31The Surgical Care Improvement Project MeasuresThe Surgical Care Improvement Project (SCIP) was established in 2003 by a national partnership of organizations committed Table 12-4Agency for Healthcare Research and Quality patient safety indicatorsProvider-level patient safety indicators• Complications of anesthesia• Death in low mortality diagnosis-related groups• Decubitus ulcer• Failure to rescue• Foreign body left in during procedure• Iatrogenic pneumothorax• Selected infections due to medical care• Postoperative hip fracture• Postoperative

1	groups• Decubitus ulcer• Failure to rescue• Foreign body left in during procedure• Iatrogenic pneumothorax• Selected infections due to medical care• Postoperative hip fracture• Postoperative hemorrhage or hematoma• Postoperative physiologic and metabolic derangements• Postoperative respiratory failure• Postoperative pulmonary embolism or deep vein thrombosis• Postoperative sepsis• Postoperative wound dehiscence in abdominopelvic surgical patients• Accidental puncture and laceration• Transfusion reaction• Birth trauma—injury to neonate• Obstetric trauma—vaginal delivery with instrument• Obstetric trauma—vaginal delivery without instrument• Obstetric trauma—cesarean deliveryArea-level patient safety indicators• Foreign body left in during procedure• Iatrogenic pneumothorax• Selected infections due to medical care• Postoperative wound dehiscence in abdominopelvic surgical patients• Accidental puncture and laceration• Transfusion reaction• Postoperative hemorrhage or hematomaReproduced

1	due to medical care• Postoperative wound dehiscence in abdominopelvic surgical patients• Accidental puncture and laceration• Transfusion reaction• Postoperative hemorrhage or hematomaReproduced with permission from Agency for Healthcare Research and Quality. Patient Safety Indicators Overview. AHRQ Quality Indicators. Rockville, MD: Agency for Healthcare Research and Quality; February 2006. Available at: https://www.qualityindicators.ahrq.gov/. Accessed October 24, 2018.to improving surgical care by reducing surgical complications. The steering committee is comprised of groups such as the Centers for Medicare & Medicaid Services, the American Hos-pital Association, Centers for Disease Control and Prevention (CDC), Institute for Healthcare Improvement, The Joint Com-mission, and others.The incidence of postoperative complications ranges from 6% for patients undergoing noncardiac surgery to more than 30% for patients undergoing high-risk surgery. Common post-operative complications

1	incidence of postoperative complications ranges from 6% for patients undergoing noncardiac surgery to more than 30% for patients undergoing high-risk surgery. Common post-operative complications include surgical site infections (SSIs), myocardial infarction, postoperative pneumonia, and thrombo-embolic complications. Patients who experience postoperative complications have increased hospital length of stay (3 to 11 days longer than those without complications), increased hospital costs (ranging from $1398 for an infectious complication to $18,310 for a thromboembolic event), and increased mortality (median patient survival decreases by up to 69%).32Despite well-established evidence that many of these adverse events are preventable, failure to comply with standards Brunicardi_Ch12_p0397-p0432.indd 40620/02/19 3:57 PM 407QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12of care known to prevent them results in unnecessary harm to a large number of patients.

1	40620/02/19 3:57 PM 407QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12of care known to prevent them results in unnecessary harm to a large number of patients. SCIP has identified three broad areas within surgery where potential complications have a high inci-dence and cost and there is a significant opportunity for pre-vention: SSIs, venous thromboembolism, and adverse cardiac events. The SCIP measures aim to reduce the incidence of these events during the perioperative period by advocating the use of proven process and outcome measures. These process and outcome measures are detailed in Table 12-5.SSIs account for 14% to 16% of all hospital-acquired infections and are a common complication of care, occurring in 2% to 5% of patients after clean extra-abdominal operations and up to 20% of patients undergoing intra-abdominal procedures. By implementing steps to reduce SSIs, hospitals could recog-nize a savings of $3152 and reduction in extended length of

1	operations and up to 20% of patients undergoing intra-abdominal procedures. By implementing steps to reduce SSIs, hospitals could recog-nize a savings of $3152 and reduction in extended length of stay by 7 days on each patient developing an infection.33Adverse cardiac events occur in 2% to 5% of patients undergoing noncardiac surgery and as many as 34% of patients undergoing vascular surgery. Certain perioperative cardiac Table 12-5The Surgical Care Improvement Project measuresProcess of care performance measuresInfection• Prophylactic antibiotic received within 1 h before surgical incision• Prophylactic antibiotic selection for surgical patients• Prophylactic antibiotics discontinued within 24 h after surgery end time (48 h for cardiac patients)• Cardiac surgery patients with controlled 6 a.m. postoperative serum glucose• Surgery patients with appropriate hair removal• Colorectal surgery patients with immediate postoperative normothermiaVenous thromboembolism• Surgery patients with

1	6 a.m. postoperative serum glucose• Surgery patients with appropriate hair removal• Colorectal surgery patients with immediate postoperative normothermiaVenous thromboembolism• Surgery patients with recommended venous thromboembolism prophylaxis ordered• Surgery patients who received appropriate venous thromboembolism prophylaxis within 24 h before surgery to 24 h after surgeryCardiac events• Surgery patients on a β-blocker prior to arrival who received a β-blocker during the perioperative periodProposed outcome measuresInfection• Postoperative wound infection diagnosed during index hospitalizationVenous thromboembolism• Intraor postoperative pulmonary embolism diagnosed during index hospitalization and within 30 d of surgery• Intraor postoperative deep vein thrombosis diagnosed during index hospitalization and within 30 d of surgeryCardiac events• Intraor postoperative acute myocardial infarction diagnosed during index hospitalization and within 30 d of surgeryGlobal

1	during index hospitalization and within 30 d of surgeryCardiac events• Intraor postoperative acute myocardial infarction diagnosed during index hospitalization and within 30 d of surgeryGlobal measures• Mortality within 30 d of surgery• Readmission within 30 d of surgeryData from The Joint Commission, 2012.events, such as myocardial infarction, are associated with a mortality rate of 40% to 70% per event, prolonged hospitaliza-tion, and higher costs. Appropriately administered β-blockers reduce perioperative ischemia, especially in at-risk patients. It has been found that nearly half of the fatal cardiac events could be preventable with β-blocker therapy.33DVT occurs after approximately 25% of all major surgi-cal procedures performed without prophylaxis, and pulmonary embolism (PE) occurs after 7%. Despite the well-established efficacy and safety of preventive measures, studies show that prophylaxis often is underused or used inappropriately. Both low-dose unfractionated heparin and

1	after 7%. Despite the well-established efficacy and safety of preventive measures, studies show that prophylaxis often is underused or used inappropriately. Both low-dose unfractionated heparin and low molecular weight heparin have similar efficacy in DVT and PE prevention. Pro-phylaxis using low-dose unfractionated heparin has been shown to reduce the incidence of fatal PEs by 50%.33The SCIP effort provides an infrastructure and guidelines for data collection and quality improvement on a national scale. By achieving high levels of compliance with evidence-based practices to reduce SSIs, venous thromboembolism events, and perioperative cardiac complications, the potential number of lives saved in the Medicare patient population alone exceeds 13,000 annually.32National Surgical Quality Improvement ProgramThe National Surgical Quality Improvement Program (NSQIP) is a measurement program that allows hospitals to sample their rates of postoperative events and compare them to similar

1	Improvement ProgramThe National Surgical Quality Improvement Program (NSQIP) is a measurement program that allows hospitals to sample their rates of postoperative events and compare them to similar hos-pitals. Created by the Veterans Health Administration (VA) in 1991, NSQIP has been credited with measuring and improving morbidity and mortality outcomes at the VA, reducing 30-day mortality rate after major surgery by 31%, and 30-day postop-erative morbidity by 45% in its first decade.34 Beta testing at 18 non-VA sites from 2001 to 2004 demonstrated the feasibility and utility of the program in the private sector. The program was subsequently expanded to the private sector in 2004 when the American College of Surgeons endorsed the program and encouraged hospital participation to measure and evaluate out-comes on a large scale. A study of 118 hospitals participating in NSQIP between 2005 to 2007 showed that 82% of hospitals decreased their complication rates and there was a decrease in

1	evaluate out-comes on a large scale. A study of 118 hospitals participating in NSQIP between 2005 to 2007 showed that 82% of hospitals decreased their complication rates and there was a decrease in morbidity of 11% and mortality of 17% annually per hospital.35 Currently, over 400 private-sector U.S. hospitals participate in the program.NSQIP uses a risk-adjusted ratio of the observed to expected outcome (focusing primarily on 30-day morbidity and mortal-ity) to compare the performance of participating hospitals with their peers. The data the program has compiled also can be used to conduct observational studies using prospectively collected information on more than 1.5 million patients and operations. The expansion of NSQIP to the private sector has helped shift the focus from merely preventing the provider errors and sentinel events highlighted by the IOM publication “To Err Is Human” to the larger goal of preventing all adverse postoperative outcomes.Several insights about patient

1	the provider errors and sentinel events highlighted by the IOM publication “To Err Is Human” to the larger goal of preventing all adverse postoperative outcomes.Several insights about patient safety have arisen as a result of NSQIP. First, safety is indistinguishable from overall quality of surgical care and should not be addressed separately. Defin-ing quality in terms of keeping a patient safe from adverse out-comes allows the NSQIP data to be used to assess and improve quality of care by making improvements in patient safety. In other words, prevention of errors is synonymous with the reduc-tion of adverse outcomes and can be used as a reliable quality measure. Second, during an episode of surgical care, adverse Brunicardi_Ch12_p0397-p0432.indd 40720/02/19 3:57 PM 408BASIC CONSIDERATIONSPART Ioutcomes, and hence, patient safety, are primarily determined by the quality of the systems of care. Errors in hospitals with higher than expected observed to expected outcomes ratios are

1	and hence, patient safety, are primarily determined by the quality of the systems of care. Errors in hospitals with higher than expected observed to expected outcomes ratios are more likely to be from system errors than from provider incom-petence. This underscores the importance of adequate communi-cation, coordination, and teamwork in achieving quality surgical care. Finally, reliable comparative outcomes data are imperative for the identification of system problems. Risk-adjusted rates of adverse outcomes must be compared with those at peer institu-tions to appreciate more subtle system errors that lead to adverse outcomes to prompt changes in the quality of an institution’s processes and structures.The Leapfrog GroupOne of the largest efforts to standardize evidence-based med-icine in the United States is led by The Leapfrog Group, an alliance of large public and private healthcare purchasers rep-resenting more than 37 million individuals across the United States. This healthcare

1	the United States is led by The Leapfrog Group, an alliance of large public and private healthcare purchasers rep-resenting more than 37 million individuals across the United States. This healthcare consortium was founded in 2000 with the aim to exert their combined leverage toward improving nation-wide standards of healthcare quality, optimizing patient out-comes, and ultimately lowering healthcare costs. The Leapfrog Group’s strategy to achieve these goals is through providing patient referral, financial incentives, and public recognition for hospitals that practice or implement evidence-based healthcare standards.The healthcare quality and safety practices (leaps) that Leapfrog initially identified to measure healthcare standards were hospital use of computerized physician order entry systems, 24-hour ICU physician staffing, and evidence-based hospital referral (EBHR) standards for five high-risk operations.36 In 2010, after the National Quality Forum (NQF) released its updated

1	systems, 24-hour ICU physician staffing, and evidence-based hospital referral (EBHR) standards for five high-risk operations.36 In 2010, after the National Quality Forum (NQF) released its updated Safe Practices for Better Healthcare, Leapfrog added a safe practices leap, which includes eight practices from the NQF report.37Leapfrog collects data on these practices through adminis-tration of an ongoing, voluntary, web-based hospital quality and safety survey. This survey is conducted in 41 regions that cover over half of the U.S. population and 62% of all hospital beds in the country. In 2011, more than 1200 urban, suburban, and rural hospitals participated in the survey. Leapfrog asks for informa-tion on eight high-risk conditions or procedures, including coro-nary artery bypass graft, percutaneous coronary intervention, abdominal aortic aneurysm (AAA) repair, pancreatic resection, and esophagectomy. These procedures were chosen because evidence exists that adherence to certain

1	percutaneous coronary intervention, abdominal aortic aneurysm (AAA) repair, pancreatic resection, and esophagectomy. These procedures were chosen because evidence exists that adherence to certain process measures can dramatically improve the outcomes of these procedures. In addi-tion, more than 100 studies also have demonstrated that better results are obtained at high-volume hospitals when undergoing cardiovascular surgery, major cancer resections, and other high-risk procedures. Hospitals fulfilling the EBHR Safety Standard are expected to meet the hospital and surgeon volume criteria shown in Table 12-6. Hospitals that do not meet these criteria but adhere to the Leapfrog-endorsed process measures for coro-nary artery bypass graft surgery, percutaneous coronary inter-vention, AAA repair, and care for high-risk neonates, receive partial credit toward fulfilling the EBHR Safety Standard. Leap-frog purchasers work to recognize and reward hospitals that pro-vide care for their

1	repair, and care for high-risk neonates, receive partial credit toward fulfilling the EBHR Safety Standard. Leap-frog purchasers work to recognize and reward hospitals that pro-vide care for their enrollees who meet EBHR standards.35In a recent study, Brooke and associates analyzed whether achieving Leapfrog’s established evidence-based standards for AAA repair, including meeting targets for case volume and Table 12-6Recommended annual volumes: hospitals and surgeons1. Coronary artery bypass graft≥450/1002. Percutaneous coronary intervention≥400/753. Abdominal aortic aneurysm repair≥50/224. Aortic valve replacement≥120/225. Pancreatic resection≥11/26. Esophagectomy≥13/27. Bariatric surgery>100/20Reproduced with permission from The Leapfrog Group. Available at: http://www.leapfroggroup.org/.perioperative β-blocker usage, correlated with improved patient outcomes over time.36 After controlling for differences in hos-pital and patient characteristics, hospitals that implemented a policy

1	β-blocker usage, correlated with improved patient outcomes over time.36 After controlling for differences in hos-pital and patient characteristics, hospitals that implemented a policy for perioperative β-blocker usage had an estimated 51% reduction in mortality following open AAA repair cases. Among 111 California hospitals in which endovascular AAA repair was performed, in-hospital mortality was reduced by an estimated 61% over time among hospitals meeting Leapfrog case volume standards, although this result was not statistically significant. These results suggest that hospital compliance with Leapfrog standards for elective AAA repair is an effective means to help improve in-hospital mortality outcomes over time and support further efforts aimed at standardizing patient referral to hospi-tals that comply with evidence-based medicine standards for other surgical procedures.The newest effort of the Leapfrog group is to promote transparency of hospital outcomes using a safety

1	to hospi-tals that comply with evidence-based medicine standards for other surgical procedures.The newest effort of the Leapfrog group is to promote transparency of hospital outcomes using a safety scorecard. This information can be viewed at www.hospitalsafetygrade.org.World Health Organization “Safe Surgery Saves Lives” InitiativeIn October 2004, the WHO launched a global initiative to strengthen healthcare safety and monitoring systems by creat-ing the World Alliance for Patient Safety. As part of the group’s efforts to improve patient safety, the alliance implemented a series of safety campaigns that brought together experts in spe-cific problem areas through individual Global Patient Safety Challenges. The second Global Patient Safety Challenge focuses on improving the safety of surgical care. The main goal of the campaign, called Safe Surgery Saves Lives, is to reduce surgical deaths and complications through the universal adaptation of a comprehensive perioperative surgical

1	care. The main goal of the campaign, called Safe Surgery Saves Lives, is to reduce surgical deaths and complications through the universal adaptation of a comprehensive perioperative surgical safety checklist in ORs worldwide (Fig. 12-4). In addition to the checklist, the WHO defined a set of uniform measures for national and international surveillance of surgical care to better assess the quantity and quality of surgical care being delivered worldwide.38 At the pop-ulation level, metrics include the number of surgeon, anesthesia, and nurse providers per capita, the number of ORs per capita, and overall surgical case volumes and mortality rates. At the hospital level, metrics include safety improvement structures and a surgical “Apgar score,” a validated method of prognos-ticating patient outcomes based on intraoperative events (i.e., hypotension, tachycardia, blood loss).39National Quality ForumThe National Quality Forum (NQF) is a coalition of health-care organizations that has

1	outcomes based on intraoperative events (i.e., hypotension, tachycardia, blood loss).39National Quality ForumThe National Quality Forum (NQF) is a coalition of health-care organizations that has worked to develop and implement a national strategy for healthcare quality measurement and Brunicardi_Ch12_p0397-p0432.indd 40820/02/19 3:57 PM 409QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12reporting. Their mission is to improve the quality of American healthcare by setting national priorities and goals for perfor-mance improvement, endorsing national consensus standards for measuring and publicly reporting on performance, and pro-moting the attainment of national goals through education and outreach programs.One of the major contributions of the NQF is the develop-ment of a list of Serious Reportable Events, which are frequently referred to as “never events.”40 According to the NQF, “never events” are errors in medical care that are clearly identifiable,

1	of a list of Serious Reportable Events, which are frequently referred to as “never events.”40 According to the NQF, “never events” are errors in medical care that are clearly identifiable, preventable, and serious in their consequences for patients and that indicate a real problem in the safety and credibility of a healthcare facility. Examples of never events include surgery performed on the wrong body part; a foreign body left in a patient after surgery; a mismatched blood transfusion; a major medication error; a severe pressure ulcer acquired in the hospital; and preventable postoperative deaths. Criteria for inclusion as a never event are listed in Table 12-7. The event must be:• Unambiguous (i.e., the event must be clearly identifiable and measurable, and thus feasible to include in a reporting system);• Usually preventable, with the recognition that some events are not always avoidable, given the complexity of healthcare;• Serious, resulting in death or loss of a body part,

1	in a reporting system);• Usually preventable, with the recognition that some events are not always avoidable, given the complexity of healthcare;• Serious, resulting in death or loss of a body part, disability, or more than transient loss of a body function; and• Any one of the following:• Adverse, and/or• Indicative of a problem in a healthcare facility’s safety sys-tems, and/or• Important for public credibility or public accountability.These events are not a reasonable medical risk of under-going surgery that the patient must accept but medical errors that should never happen (Case 12-4). The occurrence of any of these events signals that an organization’s patient safety culture or processes have defects that need to be evaluated and cor-rected (Table 12-8).“NEVER EVENTS” IN SURGERYNever events are errors in medical care that are clearly identifi-able, preventable, and serious in their consequences for patients and that indicate a real problem in the safety and credibility of a

1	events are errors in medical care that are clearly identifi-able, preventable, and serious in their consequences for patients and that indicate a real problem in the safety and credibility of a healthcare facility.40 Despite widespread agreement that surgi-cal never events are preventable and despite several national and local programs being launched to decrease them, never 8Table 12-7Surgical “never events”• Surgery performed on the wrong body part• Surgery performed on the wrong patient• Wrong surgical procedure performed on a patient• Unintended retention of a foreign object in a patient after surgery or other procedure• Intraoperative or immediately postoperative death in an ASA Class 1 patientASA = American Society of Anesthesiologists.Reproduced with permission from Serious Reportable Events in Healthcare 2011 Update: A Consensus Report. Washington, DC: National Quality Forum; 2011.Case 12-4 Surgical “never event”In 2002, Mike Hurewitz, a reporter for The Times Union of Albany,

1	Events in Healthcare 2011 Update: A Consensus Report. Washington, DC: National Quality Forum; 2011.Case 12-4 Surgical “never event”In 2002, Mike Hurewitz, a reporter for The Times Union of Albany, suddenly began vomiting blood 3 days after donating part of his liver to his brother while recovering on a hospital floor in which 34 patients were being cared for by one first-year resident. He aspirated and died immediately with no other physician available to assist the overworked first-year resident.Recognized for its advances in the field of liver trans-plantation, at the time, Mount Sinai Hospital was performing more adult-to-adult live-donor operations than any other hos-pital in the country. But the program was shut down by this event. Mount Sinai was held accountable for inadequate care and was banned from performing any live-donor adult liver transplants for more than 1 year. Of the 92 complaints inves-tigated by the state, 75 were filed against the liver transplant unit, with 62

1	and was banned from performing any live-donor adult liver transplants for more than 1 year. Of the 92 complaints inves-tigated by the state, 75 were filed against the liver transplant unit, with 62 involving patient deaths. The state concluded that most of the 33 serious violations exhibited by the hospital occurred within the liver transplant unit.As a result of the investigation, Mount Sinai revamped many of the procedures within its transplant unit. Among the changes, first-year residents no longer staffed the transplant service, two healthcare practitioners physically present in the hospital oversaw the transplant unit at all times, and any page coming from the transplant unit had to be answered within 5 minutes of the initial call. In addition, nurses monitored patients’ vital signs more closely after surgery, transplant sur-geons were required to make postoperative visits to both organ donor and recipient, and each registered nurse was assigned to four patients, rather than six

1	more closely after surgery, transplant sur-geons were required to make postoperative visits to both organ donor and recipient, and each registered nurse was assigned to four patients, rather than six or seven. The death also led New York to become the first state to develop guidelines for treating live organ donors. Finally, Mike Hurewitz’s widow became a patient safety advocate, urging stricter controls on live donor programs.events are still a significant problem. A study from Mehtsun and colleagues showed that from October 1990 to October 2010, nationwide there were 9744 paid malpractice claims for never events. Of these, mortality was reported in 6.6%, permanent injury in 33%, and temporary injury in 59%. The cost of the never events totaled $1.3 billion. Also, of physicians who were named in a surgical never event claim, 12.4% were named in a future never events claim.41 Another study in 2010 by The Joint Commission found that wrong-site surgery occurs 40 times per week

1	who were named in a surgical never event claim, 12.4% were named in a future never events claim.41 Another study in 2010 by The Joint Commission found that wrong-site surgery occurs 40 times per week nationwide.42 Future directions for decreasing these prob-lems include public reporting of never events by hospitals to increase hospital accountability, more formal training in team-work, and CUSP programs in hospitals that have higher rates of never events to help elucidate the root cause.Retained Surgical ItemsA retained surgical item refers to any surgical item found to be inside a patient after he or she has left the OR, thus requiring a second operation to remove the item.43 Estimates of retained for-eign bodies in surgical procedures range from one case per 8000 to 18,000 operations, corresponding to one case or more each year for a typical large hospital or approximately 1500 cases per year in the United States.44 This estimate is based on an analy-sis of malpractice claims and is

1	to one case or more each year for a typical large hospital or approximately 1500 cases per year in the United States.44 This estimate is based on an analy-sis of malpractice claims and is likely to underestimate the true incidence. The risk of having a retained surgical item increases during emergency surgery, when there are unplanned changes in Brunicardi_Ch12_p0397-p0432.indd 40920/02/19 3:57 PM 410BASIC CONSIDERATIONSPART ITable 12-8Four patient events that advanced the modern field of patient safetyPATIENTINSTITUTIONYEAREVENTROOT CAUSEOUTCOMELibby ZionNew York Hospital, New York, NY1984Missed allergy to DemerolPhysician fatigueBell Commission shortened resident work hoursBetsy LehmanDana-Farber Cancer Institute, Boston, MA1994Chemotherapy overdoseLack of medication checks and triggersFired doctor, three pharmacists, 15 nurses; overhauled safety programJosie KingJohns Hopkins Hospital, Baltimore, MD2001Severe dehydrationPoor communicationIncreased safety research fundingMike

1	doctor, three pharmacists, 15 nurses; overhauled safety programJosie KingJohns Hopkins Hospital, Baltimore, MD2001Severe dehydrationPoor communicationIncreased safety research fundingMike HurewitzMt. Sinai Hospital, New York, NY2002Inadequate postoperative careInadequate supervisionTransplant program shut down until better patient safety safeguards implementedprocedure (due to new diagnoses encountered in the OR), and in patients with higher body mass index (Table 12-9).44The most common retained surgical item is a surgical sponge, but other items, such as surgical instruments and nee-dles, can also be inadvertently left inside a patient during an operation. Retained surgical sponges are commonly discovered as an incidental finding on a routine postoperative radiograph, but also have been discovered in patients presenting with a mass or abdominal pain. Patients with sponges that were originally left in an intracavitary position (such as inside the chest or abdomen) also can present

1	discovered in patients presenting with a mass or abdominal pain. Patients with sponges that were originally left in an intracavitary position (such as inside the chest or abdomen) also can present with complications such as abscess, erosion through the skin, fistula formation, bowel obstruction, hematuria, or the development of a new, tumor-like lesion.Retained surgical needles usually are discovered inciden-tally, and reports of retained needles are uncommon. Retained surgical needles have not been reported to cause injury in the same way that nonsurgical needles (e.g., sewing needles, hypo-dermic needles) have been reported to perforate bowel or lodge in vessels and migrate. However, there have been reports of chronic pelvic pain and ocular irritation caused by retained sur-gical needles. A study of plain abdominal radiographs in pigs has demonstrated that mediumto large-size needles can eas-ily be detected. The decision to remove these retained needles depends on symptoms and

1	A study of plain abdominal radiographs in pigs has demonstrated that mediumto large-size needles can eas-ily be detected. The decision to remove these retained needles depends on symptoms and patient preference. Needles smaller than 13 mm have been found to be undetectable on plain radio-graph in several studies, have not been shown to cause injury to vessels or visceral organs, and can probably be left alone.Although the actual incidence of retained surgical instru-ments is unknown, they are retained with far less frequency than surgical sponges. The initial presentation of a retained surgical instrument is most commonly pain in the surgical site or the Table 12-9Risk factors for retained surgical sponges• Emergency surgery• Unplanned changes in procedure• Patient with higher body mass index• Multiple surgeons involved in same operation• Multiple procedures performed on same patient• Involvement of multiple operating room nurses/staff members• Case duration covers multiple nursing

1	index• Multiple surgeons involved in same operation• Multiple procedures performed on same patient• Involvement of multiple operating room nurses/staff members• Case duration covers multiple nursing “shifts”sensation of a mass of fullness after a surgical procedure that leads to the discovery of a metallic object on a radiographic study. Commonly retained instruments include the malleable and “FISH” instrument that are used to protect the viscera when closing abdominal surgery.A retained surgical foreign body should be included in the differential diagnosis of any postoperative patient who presents with pain, infection, a palpable mass, or a radiopaque structure on imaging. The diagnosis can usually be made using a com-puted tomographic (CT) scan, and this is often the only test needed. If a retained surgical item is identified in the setting of an acute clinical presentation, the treatment usually is removal of the item. However, if the attempt to remove the retained sur-gical item

1	If a retained surgical item is identified in the setting of an acute clinical presentation, the treatment usually is removal of the item. However, if the attempt to remove the retained sur-gical item can potentially cause more harm than the item itself, as in the case of a needle or a small part of a surgical item, then removal is occasionally not recommended. Retained surgical sponges should always be removed.The American College of Surgeons and the Association of Perioperative Registered Nurses, in addition to The Joint Com-mission, have issued guidelines to try to prevent the occurrence of retained surgical items. Current recommendations include the use of standard counting procedures, performing a thorough wound exploration before closing a surgical site, and using only X-ray–detectable items in the surgical wound. These organiza-tions also strongly endorse the completion of a postoperative debriefing after every operation. An X-ray at the completion of an operation is encouraged

1	items in the surgical wound. These organiza-tions also strongly endorse the completion of a postoperative debriefing after every operation. An X-ray at the completion of an operation is encouraged if there is any concern for a foreign body based on confusion regarding the counts by even a single member of the OR team or in the presence of a risk factor.Surgical CountsThe benefit of performing surgical counts to prevent the occur-rence of retained surgical items is controversial. The increased risk of a retained surgical item during emergency surgery in the study by Gawande and colleagues appeared to be related to bypassing the surgical count in many of these cases.44 However, in another study, the “falsely correct count,” in which a count is performed and declared correct when it is actually incorrect, occurred in 21% to 100% of cases in which a retained surgical item was found.45 This type of count was the most common circumstance encountered in all retained surgical item cases,

1	incorrect, occurred in 21% to 100% of cases in which a retained surgical item was found.45 This type of count was the most common circumstance encountered in all retained surgical item cases, which suggests that performing a surgical count in and of itself does not prevent this error from taking place. The counting pro-tocol also imposes significant demands on the nursing staff and Brunicardi_Ch12_p0397-p0432.indd 41020/02/19 3:57 PM 411QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12distracts them from focusing on other primarily patient-centered tasks, often during critical portions of the case.19A retained surgical item can occur even in the presence of a known incorrect count. This event is usually a result of poor communication in which a surgeon will dismiss the incorrect count and/or fail to obtain a radiograph before the patient leaves the OR. Having stronger institutional policies in place in case of an incorrect count (such as requiring a

1	dismiss the incorrect count and/or fail to obtain a radiograph before the patient leaves the OR. Having stronger institutional policies in place in case of an incorrect count (such as requiring a mandatory radiograph while the patient is still in the OR) can avoid conflict among caregivers and mitigate the likelihood of a retained surgical item occurring as a result of a known incorrect count.Although there is no single tool to prevent all errors, the development of multiple lines of defense to prevent retained surgical items and universally standardizing and adhering to OR safety protocols by all members of the surgical team will help reduce the incidence of this never event.45 Surgeons should take the lead in the prevention of retained surgical items by avoid-ing the use of small or nonradiologically detectable sponges in large cavities, performing a thorough wound inspection before closing any surgical incision, and having a vested interest in the counting procedure performed by

1	detectable sponges in large cavities, performing a thorough wound inspection before closing any surgical incision, and having a vested interest in the counting procedure performed by nursing staff. The value of routine radiography to prevent a retained surgical item in emergency cases or when major procedures involving multiple surgical teams are being performed is becoming more apparent.The widely accepted legal doctrine when a foreign body is erroneously left in a patient is that the mere presence of the item in the plaintiff’s body indicates that the patient did not receive proper surgical care. The characteristics of the surgeon and their style, bedside manner, honesty, and confidence demonstrated in the management of the case can go a long way in averting a lawsuit or mitigating damages.Wrong-Site SurgeryWrong-site surgery is any surgical procedure performed on the wrong patient, wrong body part, wrong side of the body, or wrong level of a correctly identified anatomic site. It

1	SurgeryWrong-site surgery is any surgical procedure performed on the wrong patient, wrong body part, wrong side of the body, or wrong level of a correctly identified anatomic site. It is dif-ficult to determine the true incidence of wrong-site surgery for several reasons. First, there is no standard definition for what constitutes wrong-site surgery among various healthcare orga-nizations. Another factor is that wrong-site surgery is under-reported by healthcare providers. Finally, the total number of potential opportunities for each type of wrong-site error is unknown. However, various studies show incidences ranging from 1 in 112,994 cases to 1 in 15,500 cases.46 The Washington University School of Medicine suggests a rate of 1 in 17,000 operations, which adds up to approximately 4000 wrong-site surgeries in the United States each year. If these numbers are correct, wrong-site surgery is the third most frequent life-threat-ening medical error in the United States.47Several states

1	wrong-site surgeries in the United States each year. If these numbers are correct, wrong-site surgery is the third most frequent life-threat-ening medical error in the United States.47Several states now require mandatory reporting of all wrong-site surgery events, including near misses. These data provide some insight into the number of actual errors compared to the number of potential opportunities to perform wrong-site surgery. Of the 427 reports of wrong-site surgery submitted from June 2004 through December 2006 to the Pennsylvania Patient Safety Reporting System, more than 40% of the errors actually reached the patient, and nearly 20% involved comple-tion of a wrong-site procedure.46The risk of performing wrong-site surgery increases when there are multiple surgeons involved in the same operation or multiple procedures are performed on the same patient, espe-cially if the procedures are scheduled or performed on different areas of the body.47 Time pressure, emergency surgery,

1	same operation or multiple procedures are performed on the same patient, espe-cially if the procedures are scheduled or performed on different areas of the body.47 Time pressure, emergency surgery, abnor-mal patient anatomy, and morbid obesity are also thought to be risk factors. Communication errors are the root cause in more than 70% of the wrong-site surgeries reported to The Joint Com-mission.46 Other risk factors include receiving an incomplete preoperative assessment; having inadequate procedures in place to verify the correct surgical site; or having an organizational culture that lacks teamwork or reveres the surgeon as someone whose judgment should never be questioned.There is a one in four chance that surgeons who work on symmetric anatomic structures will be involved in a wrong-site error sometime during their careers.47 The specialties most com-monly involved in reporting wrong-site surgeries according to The Joint Commission are orthopedic/podiatric surgery (41%); general

1	error sometime during their careers.47 The specialties most com-monly involved in reporting wrong-site surgeries according to The Joint Commission are orthopedic/podiatric surgery (41%); general surgery (20%); neurosurgery (14%); urology (11%); and maxillofacial, cardiovascular, otolaryngology, and oph-thalmology (14%).46 Most errors involved symmetric anatomic structures: lower extremities (30%), head/neck (24%), and geni-tal/urinary/pelvic/groin (21%).42 Although orthopedic surgery is the most frequently involved, this may be due to the higher volume of cases performed as well as the increased opportu-nity for lateralization errors inherent in the specialty. In addi-tion, because the American Academy of Orthopaedic Surgeons has historically tried as a professional organization to reduce wrong-site operations, orthopedic surgeons may be more likely to report these events when they do occur.47The Joint Commission Universal Protocol to Ensure Correct SurgeryThe movement to eliminate

1	wrong-site operations, orthopedic surgeons may be more likely to report these events when they do occur.47The Joint Commission Universal Protocol to Ensure Correct SurgeryThe movement to eliminate wrong-site surgery began among professional orthopedic societies in the mid-1990s, when both the Canadian Orthopaedic Association and the American Acad-emy of Orthopaedic Surgeons issued position statements and embarked on educational campaigns to prevent the occurrence of wrong-site surgery within their specialty.47 Other organiza-tions that issued position statements advocating for the elimina-tion of wrong-site surgery include the North American Spine Society, the American Academy of Ophthalmology, the Asso-ciation of Perioperative Registered Nurses, and the American College of Surgeons. After issuing a review of wrong-site sur-gery in their Sentinel Event Alert in 1998, The Joint Commis-sion made the elimination of wrong-site surgery one of their first National Patient Safety Goals in

1	issuing a review of wrong-site sur-gery in their Sentinel Event Alert in 1998, The Joint Commis-sion made the elimination of wrong-site surgery one of their first National Patient Safety Goals in 2003 and adopted a uni-versal protocol for preventing wrong-site, wrong-procedure, and wrong-person surgery in 2004. The protocol has been endorsed by more than 50 professional associations and organizations.A preoperative “time-out” or “pause for the cause” to con-firm the patient, procedure, and site to be operated on before incision was recommended by The Joint Commission and is now mandatory for all ORs in the United States. Elements of the protocol include the following:• Verifying the patient’s identity• Marking the surgical site• Using a preoperative site verification process such as a checklist• Confirming the availability of appropriate documents and studies before the start of a procedure• Taking a brief time-out immediately before skin incision, in which all members of the surgical

1	the availability of appropriate documents and studies before the start of a procedure• Taking a brief time-out immediately before skin incision, in which all members of the surgical team actively communicate and provide oral verification of the patient’s identity, surgi-cal site, surgical procedure, administration of preoperative Brunicardi_Ch12_p0397-p0432.indd 41120/02/19 3:57 PM 412BASIC CONSIDERATIONSPART Imedications, and presence of appropriate medical records, imaging studies, and equipment• Monitoring compliance with protocol recommendationsFocusing on individual process components of the uni-versal protocol, such as surgical site marking or the time-out, is not enough to prevent wrong-site surgery. Over a 30-month period in Pennsylvania, 21 wrong-site errors occurred despite the proper use of time-out procedures, with 12 of these errors resulting in complete wrong-site procedures. During the same period, correct site markings failed to prevent another 16 wrong-site

1	the proper use of time-out procedures, with 12 of these errors resulting in complete wrong-site procedures. During the same period, correct site markings failed to prevent another 16 wrong-site surgeries, of which six were not recognized until after the procedure had been completed.47Site verification begins with the initial patient encounter by the surgeon, continues throughout the preoperative verifica-tion process and during multiple critical points in the OR, and requires the active participation of the entire operating team, especially the surgeon and anesthesia provider. Based on a recent review of malpractice claims, two-thirds of wrong-site operations could have been prevented by a site-verification protocol.48Despite the proliferation of wrong-site protocols in the last decade, their effectiveness is difficult to measure as the inci-dence of wrong-site surgery is too rare to measure as a rate. Interestingly, the number of sentinel events reported to The Joint Commission has

1	their effectiveness is difficult to measure as the inci-dence of wrong-site surgery is too rare to measure as a rate. Interestingly, the number of sentinel events reported to The Joint Commission has not changed significantly since the wide-spread implementation of the Universal Protocol in 2004.47 This could be due to an increase in reporting rather than an actual increase in the incidence of wrong-site surgery.The legal treatment of wrong-site surgery is similar to that of surgical items erroneously left in a patient: the mere fact that it occurred indicates that the patient did not receive proper surgical care. A malpractice claim may lead to a settlement or award on verdict in the sixor seven-figure range in 2011 U.S. dollars.41Ultimately, the occurrence of retained surgical items or wrong-site surgery is a reflection of the quality of professional communication between caregivers and the degree of teamwork among the members of the operating team. In addition to stan-dardizing

1	or wrong-site surgery is a reflection of the quality of professional communication between caregivers and the degree of teamwork among the members of the operating team. In addition to stan-dardizing procedures like the surgical count, instituting man-datory postoperative radiographs in the presence of a known miscount, and reforming the processes of patient identification and site verification, organizations should also strive to create a culture of safety, create independent and redundant checks for key processes, and create a system in which caregivers can learn from their mistakes (Table 12-10).49TRANSPARENCY IN HEALTHCAREDespite a large increase in data being collected about patient safety and harm, much of it is not available to the public or other hospitals. This lack of transparency allows some hos-pitals to continue to practice outdated medicine and, in some cases, puts patients at a higher risk of serious complications. In a study by Mark Chassin, the health commissioner of

1	allows some hos-pitals to continue to practice outdated medicine and, in some cases, puts patients at a higher risk of serious complications. In a study by Mark Chassin, the health commissioner of New York State, having hospitals publicly disclose their mortality rates for coronary artery bypass graft (CABG) procedures resulted in a 41% decline in mortality from CABGs statewide.50 In this study, when CABG mortality data were initially made public, there was a wide range in cardiac surgery-related mortality from 1% to 18%, depending on the hospital; the standard of care is 2%. The reasons for higher mortality in the poorly performing Table 12-10Best practices for operating room safety• Conduct The Joint Commission Universal Protocol (“time-out”) to prevent wrong-site surgery.• Perform an operating room briefing (checklist) to identify and mitigate hazards early.• Promote a culture of speaking up about safety concerns.• Use a screening X-ray to detect foreign bodies in high-risk

1	an operating room briefing (checklist) to identify and mitigate hazards early.• Promote a culture of speaking up about safety concerns.• Use a screening X-ray to detect foreign bodies in high-risk cases.• Begin patient sign-outs with the most likely immediate safety hazard.Data from Michaels RK, Makary MA, Dahab Y, et al. Achieving the National Quality Forum’s “Never Events”: Prevention of wrong site, wrong procedure, and wrong patient operations, Ann Surg. 2007 Apr;245(4):526-532.hospitals ranged from poor communication between care teams to one rogue surgeon operating when the surgeon should not have been. The consequence of making this data transparent was that the hospitals held multidisciplinary, CUSP-like meetings, where as a team they decided on the measures to implement for improvement. Through this, over the next year, most hospitals decreased their mortality rate to below 2%. Even the hospital that had an 18% mortality rate decreased it to 7% within 3 years and 1.7% over the

1	Through this, over the next year, most hospitals decreased their mortality rate to below 2%. Even the hospital that had an 18% mortality rate decreased it to 7% within 3 years and 1.7% over the next several years.Transparency in healthcare is becoming central to the healthcare quality discussion. A new SCIP core measure is pub-lishing practitioner performance, and all Leapfrog survey results are published online where other hospitals and the public can see them. Additionally, different large medical societies, includ-ing the Society for Thoracic Surgery (STS), are encouraging and rewarding practitioners and hospitals that are transparent with their outcomes. Making hospital outcomes transparent makes hospitals accountable to the public for their outcomes and, in the case of New York, caused a radical improvement in the quality of care provided to patients. It also empowers patients by mak-ing them better informed about which hospital they choose for their care, which will further

1	caused a radical improvement in the quality of care provided to patients. It also empowers patients by mak-ing them better informed about which hospital they choose for their care, which will further incentivize hospitals to improve.Public Reporting and Patient Assessment of CareThe epiphany moment in contemporary healthcare created by the Institute of Medicine report2 generated far-reaching effects. One important aspect has been development of a variety of ini-tiatives focused on the generation, endorsement, and reporting of numerous measures related to the safety and quality of health-care—primarily process and outcomes measures. However, the science of measure development is slow paced and, unfortu-nately, has difficulty evolving at the same pace of change as clinical medicine or healthcare delivery systems.Given the strong interest for improved knowledge and information by consumers of healthcare, the trend toward public reporting has rapidly gained momentum and outpaced

1	or healthcare delivery systems.Given the strong interest for improved knowledge and information by consumers of healthcare, the trend toward public reporting has rapidly gained momentum and outpaced report-ing from the measurement science community. This has sub-sequently created occasional confusion and uncertainty in the marketplace—simply because the generation of public reports are not necessarily always based upon solid scientific data or evidence. The resulting net effect can be creation of a prema-ture focus by organizations and providers on achieving success within influential public reporting venues (e.g., U.S. News Best Hospitals) and uncertainty by patients on what are optimal healthcare information resources.Brunicardi_Ch12_p0397-p0432.indd 41220/02/19 3:57 PM 413QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12Ideally centered on the public good, federal government sponsored healthcare payment plans are also focused upon mea-surement and

1	PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12Ideally centered on the public good, federal government sponsored healthcare payment plans are also focused upon mea-surement and reporting within the industry. One such initiative funded and overseen by the Agency for Healthcare Research and Quality (AHRQ) is the Hospital Consumer Assessment of Health-care Providers and Systems (HCAHPS) program.51 AHRQ works closely with a consortium of public and private research organi-zations to develop and maintain the HCAHPS surveys, but they do not administer any of the surveys to patients or require use of the surveys. The intent of the HCAHPS initiative is to provide a standardized survey instrument and data collection methodology for measuring patients’ perspectives on hospital care (Fig. 12-7).While many hospitals have collected information on patient satisfaction, prior to HCAHPS there was no national standard for collecting or publicly reporting patients’ perspec-tives of

1	(Fig. 12-7).While many hospitals have collected information on patient satisfaction, prior to HCAHPS there was no national standard for collecting or publicly reporting patients’ perspec-tives of care information that would enable valid comparisons to be made across all hospitals. Three broad goals have shaped the HCAHPS Survey. First, the survey is designed to produce comparable data on the patient’s perspective on care that allows objective and meaningful comparisons between hospitals on domains that are important to consumers. Second, public report-ing of the survey results is designed to create incentives for hospitals to improve their quality of care. Hospitals frequently distribute the results of HCAHPS surveys of individual ser-vices or physicians to incentivize corrective steps and improve patients’ perceptions of their care. Third, public reporting will serve to enhance public accountability in healthcare by increas-ing the transparency of the quality of hospital care

1	and improve patients’ perceptions of their care. Third, public reporting will serve to enhance public accountability in healthcare by increas-ing the transparency of the quality of hospital care provided in return for the public investment (www.hcahpsonline.org).HCAHPS scores are now directly tied to a hospital’s CMS reimbursement by federal law, and survey results account for 25% of the value-based purchasing score—directly impacting a hospital’s Medicare payments.52 In May 2005, the National Quality Forum (NQF), an organization established to standard-ize healthcare quality measurement and reporting, formally endorsed the HCAHPS Hospital Survey. The NQF endorsement represents the consensus of many healthcare providers, consumer groups, professional associations, purchasers, federal agencies, and research and quality organizations (www.qualityforum.org).While the American College of Surgeons and other profes-sional societies continue to develop and implement scientifically based

1	agencies, and research and quality organizations (www.qualityforum.org).While the American College of Surgeons and other profes-sional societies continue to develop and implement scientifically based healthcare measures, the escalating prevalence of less robust measures developed outside of scientific communities will also continue to expand. The patient population, provider organizations, payer organization, and the surgical community will necessarily need to find the balance of developing and uti-lizing valid patient-based information for decision-making.RISK MANAGEMENTBetween one-half to two-thirds of hospital-wide adverse events are attributable to surgical care. Most surgical errors occur in the OR and are technical in nature. Surgical complications and adverse outcomes have previously been linked to lack of surgeon specialization, low hospital volume, communication breakdowns, fatigue, surgical residents and trainees, and numer-ous other factors.53However, poor surgical outcomes

1	been linked to lack of surgeon specialization, low hospital volume, communication breakdowns, fatigue, surgical residents and trainees, and numer-ous other factors.53However, poor surgical outcomes are not necessarily cor-related with a surgeon’s level of experience in performing a cer-tain procedure. In one study, three-fourths of the technical errors that occurred in a review of malpractice claims data involved fully trained and experienced surgeons operating within their area of expertise, and 84% occurred in routine operations that do not require advanced training. Rather than surgeon expertise, these errors likely occurred due to situations complicated by patient comorbidity, complex anatomy, repeat surgery, or equip-ment problems (Table 12-11). Because these errors occurred during routine operations, previous suggestions to limit the per-formance of high-complexity operations using selective refer-ral, regionalization, or limitation of privileging may not actually be effective

1	routine operations, previous suggestions to limit the per-formance of high-complexity operations using selective refer-ral, regionalization, or limitation of privileging may not actually be effective in reducing the incidence of technical error among surgical patients.53In any event, although there has been much emphasis on reducing the prevalence of surgical technical errors as a way of improving surgical care, a technical error in the OR may not be the most important indicator of whether a surgeon will be sued by a patient. Recent studies point to the importance of a surgeon’s communication skills in averting malprac-tice litigation. In the American College of Surgeons’ Closed Claims Study, although intraoperative organ injuries occurred in 40% of patients, a surgical technical misadventure was the most deficient component of care in only 12% of patients. In fact, communication and practice pattern violations were the most common deficiency in care for one third of patients in the

1	was the most deficient component of care in only 12% of patients. In fact, communication and practice pattern violations were the most common deficiency in care for one third of patients in the Closed Claims Study who received the expected standard of surgical care.54The Importance of Communication in Managing RiskThe manner and tone in which a physician communicates is potentially more important to avoiding a malpractice claim than the actual content of the dialogue. For example, a physician relating to a patient in a “negative” manner may trigger litigious feelings when there is a bad result, whereas a physician relating in a “positive” manner may not. Expressions of dominance, in which the voice tone is deep, loud, moderately fast, unaccented, and clearly articulated, may communicate a lack of empathy and understanding for the patient, whereas concern or anxiety in the surgeon’s voice is often positively related to expressing concern and empathy. General and orthopedic surgeons

1	a lack of empathy and understanding for the patient, whereas concern or anxiety in the surgeon’s voice is often positively related to expressing concern and empathy. General and orthopedic surgeons whose tone of voice was judged to be more dominant were more likely to have been sued than those who sounded less dominant.55When significant medical errors do occur, physicians have an ethical and professional responsibility to immediately dis-close them to patients. Failure to disclose errors to patients undermines public confidence in medicine and can create legal liability related to fraud. Physicians’ fear of litigation represents a major barrier to error disclosure. However, when handled appropriately, immediate disclosure of errors frequently leads to improved patient rapport, improved satisfaction, and fewer malpractice claims.56 In fact, rapport is the most important factor in determining whether a lawsuit is filed against a physician.In 1987, the Department of Veterans Affairs

1	satisfaction, and fewer malpractice claims.56 In fact, rapport is the most important factor in determining whether a lawsuit is filed against a physician.In 1987, the Department of Veterans Affairs Hospital in Lexington, Kentucky, implemented the nation’s first formal apology and medical error full disclosure program, which called for the hospital and its doctors to work with patients and their families to settle a case. As a result, the hospital improved from having one of the highest malpractice claims totals in the VA system to being ranked among the lowest quartile of a com-parative group of similar hospitals for settlement and litiga-tion costs over a 7-year period. Its average payout in 2005 was $16,000 per settlement vs the national VA average of $98,000 per settlement, and only two lawsuits went to trial during a 9Brunicardi_Ch12_p0397-p0432.indd 41320/02/19 3:57 PM 414BASIC CONSIDERATIONSPART IYOUR CARE FROM DOCTORSDuring this hospital stay, how oftendid doctors treat you

1	lawsuits went to trial during a 9Brunicardi_Ch12_p0397-p0432.indd 41320/02/19 3:57 PM 414BASIC CONSIDERATIONSPART IYOUR CARE FROM DOCTORSDuring this hospital stay, how oftendid doctors treat you with courtesyand respect?5.NeverSometimesUsuallyAlways1234During this hospital stay, how oftendid doctors listen carefully to you?6.NeverSometimesUsuallyAlways1234During this hospital stay, how oftendid doctors explain things in a wayyou could understand?7.During this hospital stay, how oftenwere your room and bathroom keptclean?8.NeverSometimesUsuallyAlways1234THE HOSPITAL ENVIRONMENTNeverSometimesUsuallyAlways1234During this hospital stay, how oftenwas the area around your room quietat night?9.NeverSometimesUsuallyAlways1234YOUR EXPERIENCES IN THIS HOSPITALDuring this hospital stay, did youneed help from nurses or otherhospital staff in getting to thebathroom or in using a bedpan?10.YesNo If No, Go to Question 1212During this hospital stay, did youhave any pain?12.YesNo If No, Go to

1	youneed help from nurses or otherhospital staff in getting to thebathroom or in using a bedpan?10.YesNo If No, Go to Question 1212During this hospital stay, did youhave any pain?12.YesNo If No, Go to Question 1512NeverSometimesUsuallyAlways1234How often did you get help in gettingto the bathroom or in using a bedpanas soon as you wanted?11.NeverSometimesUsuallyAlways1234During this hospital stay, how oftendid hospital staff talk with you abouthow much pain you had?13.During this hospital stay, how oftendid hospital staff talk with you abouthow to treat your pain?14.NeverSometimesUsuallyAlways12342January 2018Table 12-11Common causes of lawsuits in surgery• Positional nerve injury• Common bile duct injury• Failure to diagnose or delayed diagnosis• Failure to treat, delayed treatment, or wrong treatment• Inadequate documentation• Inappropriate surgical indication• Failure to call a specialist• Cases resulting in amputation/limb lossFigure 12-7. Survey page from 2018 HCAHPS patient

1	or wrong treatment• Inadequate documentation• Inappropriate surgical indication• Failure to call a specialist• Cases resulting in amputation/limb lossFigure 12-7. Survey page from 2018 HCAHPS patient questionnaire. (Modified with permission from Hospital Consumer Assessment of Healthcare Providers and Systems (HCAHPS) https://www.hcahpsonline.org/en/survey-instruments/. Accessed October 24, 2018.)10-year period. As a result of the success of this program, the Department of Veteran Affairs expanded the program to all VA hospitals nationwide in October 2005. This model also was rep-licated at the University of Michigan Health System with simi-lar results. Its full-disclosure program cut the number of pending lawsuits by one half and reduced litigation costs per case from $65,000 to $35,000, saving the hospital approximately $2 mil-lion in defense litigation bills each year. In addition, University of Michigan’s doctors, patients, and lawyers are happier with this system. The cultural

1	saving the hospital approximately $2 mil-lion in defense litigation bills each year. In addition, University of Michigan’s doctors, patients, and lawyers are happier with this system. The cultural shift toward honesty and openness also has led to the improvement of systems and processes to reduce medical errors, especially repeat medical errors.57Brunicardi_Ch12_p0397-p0432.indd 41420/02/19 3:57 PM 415QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12With regard to risk management, the importance of good communication by surgeons and other care providers cannot be overemphasized. Whether alerting other members of the care team about a patient’s needs, openly discussing concerns the patient and/or family might have, or disclosing the cause of a medical error, open communication with all parties involved can reduce anger and mistrust of the medical system; the frequency, morbidity, and mortality of preventable adverse events; and the likelihood of

1	error, open communication with all parties involved can reduce anger and mistrust of the medical system; the frequency, morbidity, and mortality of preventable adverse events; and the likelihood of litigation.COMPLICATIONSDespite the increased focus on improving patient safety and minimizing medical errors, it is impossible to eliminate human error entirely. Individual errors in judgment or technique can cause minor or major complications during or after a surgical procedure. Although these types of errors may not be quantified as easily as wrong-site surgery or a retained surgical item, they can still lead to surgical complications that prolong the course of illness, lengthen hospital stay, and increase morbidity and mortality rates. In addition to technical and management errors, patient comorbidities also increase the risk of complications. The recognition and management of complications is a critical component of surgical care.Robotic SurgerySurgical advancements would not exist

1	comorbidities also increase the risk of complications. The recognition and management of complications is a critical component of surgical care.Robotic SurgerySurgical advancements would not exist without intellectual curi-osity, innovation, and technical developments; robotic surgery is a prime example of such an advance. With these advance-ments, however, errors and complications appear to be an inevi-table and recognized risk by institutions and stakeholders due to unforeseen risks inherent in the new technology and the failure or delay of achieving expertise with a new device or technology. Although the reward for adopting new advances may be noto-riety, increased patient referrals, improved patient satisfaction, decreased pain, and possibly decreased length of stay, the risks of adopting new technologies and methods become apparent only after widespread use.Multiple surgical specialties have begun or continue to develop their experiences using robotic surgery from general surgery

1	new technologies and methods become apparent only after widespread use.Multiple surgical specialties have begun or continue to develop their experiences using robotic surgery from general surgery procedures such as inguinal hernia repairs to pancreati-coduodenectomies to complex thoracic, urologic and ear, nose, and throat procedures. When robotic surgery goes awry, how-ever, the complications can be serious. The MAUDE (Manu-facturer and User Facility Device Experience) is an open access database where mandatory and voluntary adverse events are collected. As it relates to robotic surgery, some important infor-mation has been elucidated. Device failures (electrocautery, instrument malfunctions), make up roughly half of the com-plications. A retrospective study over the past 14 years in the United States documented over 10,000 robotic device-related complications that have occurred, of which 98% were reported by the manufacturers and distributers, and 2% were voluntary reports by

1	in the United States documented over 10,000 robotic device-related complications that have occurred, of which 98% were reported by the manufacturers and distributers, and 2% were voluntary reports by hospitals and physicians. The data revealed that 1535 adverse events (14.4%) led to significant negative patient expe-riences (1391 injuries and 144 deaths). Additionally, the abso-lute number of reports increased 32 times since 2006, while in the same time period, the number of cases performed has only increased tenfold.58 Despite the large number of reports con-tained within this database, the extent to which it is a true rep-resentation of the complications associated with robotic surgery is uncertain due to the lack of comprehensive and mandatory reporting.59Despite the numbers and trends reported from this data-base, few prospective, controlled trials exist that examine the risks and benefits of robotic surgery with those of open and laparoscopic surgery. The data from 5 to 10 years

1	reported from this data-base, few prospective, controlled trials exist that examine the risks and benefits of robotic surgery with those of open and laparoscopic surgery. The data from 5 to 10 years ago may also be misleading as the approved use of robotic surgery continues to expand to additional specialties. The more recent adoption of robotic surgery by specialties such as gynecologic surgery, for example, appears to be accompanied by a disproportionately high rate of morbidity and mortality in robotically assisted pro-cedures.59 Teaching institutions are producing a newer genera-tion of robotic surgeons who will continue important advances in surgery and identify those patients who would benefit most from this type of approach. The challenge for the surgical com-munity is to develop robust and effective training programs to allow trainees and practicing surgeons to acquire the skills nec-essary to perform robotic procedures with the highest degree of safety. This need replicates

1	robust and effective training programs to allow trainees and practicing surgeons to acquire the skills nec-essary to perform robotic procedures with the highest degree of safety. This need replicates the development of skill acquisition processes that reversed the high number of bile duct injuries after the introduction of laparoscopic cholecystectomy and sug-gests that validated curricula and the use of robotic simulation applications will be crucial to achieve these goals (see Chapter 53, Skills and Simulation).Complications in Minor ProceduresWhen performing procedures such as central line insertion or arterial line insertion, one should consider the necessity of the access, the use of less invasive or lower risk alternatives such as PICC line insertion instead of central line insertion, and non-invasive cardiac monitoring instead of arterial line insertion. While these alternatives may not be reliable substitutes in all patients, considering less invasive procedures can reduce the

1	and non-invasive cardiac monitoring instead of arterial line insertion. While these alternatives may not be reliable substitutes in all patients, considering less invasive procedures can reduce the problem of avoidable harm.Central Venous Access Catheters. Complications of central venous access catheters are common. Improvements in ultra-sound technology and mass education surrounding the use and techniques in ultrasonography have led to increased employ-ment and enthusiasm for its use in central venous catheter placement. Numerous institutions have mandated the use of ultrasound for placement of all central venous lines. In addition, many subclavian catheters have been alternatively placed at the internal jugular position due to a perceived benefit of decreasing the complication of pneumothorax. This theoretical benefit may be offset by an increase in line infections as the neck is a dif-ficult site to keep clean and the dressing intact. Steps to decrease complications

1	of pneumothorax. This theoretical benefit may be offset by an increase in line infections as the neck is a dif-ficult site to keep clean and the dressing intact. Steps to decrease complications include:• Ensure that central venous access is indicated.• Experienced personnel should insert the catheter or should supervise the insertion.• Use proper positioning and sterile technique.• Ultrasound is recommended for internal jugular vein insertion.• All central venous catheters should be assessed on a daily basis and should be exchanged only for specific indications (not as a matter of routine).• All central catheters should be removed as soon as possible.Common complications of central venous access include the following.Pneumothorax Occurrence rates from both subclavian and internal jugular vein approaches are 1% to 6%. Prevention requires proper positioning of the patient and correct insertion technique. A postprocedure chest X-ray is recommended to confirm the presence or absence of a

1	vein approaches are 1% to 6%. Prevention requires proper positioning of the patient and correct insertion technique. A postprocedure chest X-ray is recommended to confirm the presence or absence of a pneumothorax, regardless Brunicardi_Ch12_p0397-p0432.indd 41520/02/19 3:57 PM 416BASIC CONSIDERATIONSPART Iof whether a pneumothorax is suspected. Recent reports have questioned whether a chest X-ray is required when the line is placed and confirmed under ultrasound guidance. Pneumotho-rax rates are higher among inexperienced providers and under-weight patients but occur with experienced operators as well. If the patient is stable, and the pneumothorax is small (<15%), close expectant observation may be adequate. If the patient is symptomatic, a thoracostomy tube should be placed. Occasion-ally, pneumothorax will occur as late as 48 to 72 hours after central venous access attempts. This usually creates sufficient compromise that a tube thoracostomy is required.Arrhythmias Arrhythmias

1	pneumothorax will occur as late as 48 to 72 hours after central venous access attempts. This usually creates sufficient compromise that a tube thoracostomy is required.Arrhythmias Arrhythmias can result from myocardial irritabil-ity secondary to guidewire placement and usually resolve when the catheter or guidewire is withdrawn from the right heart. Pre-vention requires electrocardiogram (ECG) monitoring whenever possible during catheter insertion and rapid recognition when a new arrhythmia occurs.Arterial Puncture Inadvertent puncture or laceration of an adja-cent artery with bleeding can occur, but the majority will resolve with direct pressure on or near the arterial injury site. Rarely will angiography, stent placement, or surgery be required to repair the puncture site, but close observation and a chest X-ray are indi-cated. Ultrasound-guided insertion has not mitigated this com-plication, but it may decrease the incidence of arterial puncture. Ultrasound use has also been shown

1	and a chest X-ray are indi-cated. Ultrasound-guided insertion has not mitigated this com-plication, but it may decrease the incidence of arterial puncture. Ultrasound use has also been shown to decrease the number of attempts and the time it takes to complete insertion.Lost Guidewire A guidewire or catheter that inadvertently migrates further into the vascular space away from the insertion site can be readily retrieved with interventional angiography techniques. A prompt chest X-ray and close monitoring of the patient until retrieval are indicated.Air Embolus Although estimated to occur in only 0.2% to 1% of patients, an air embolism can be dramatic and fatal. If an embolus is suspected, the patient should immediately be placed into a left lateral decubitus Trendelenburg position so the entrapped air can be stabilized within the right ventricle. Aus-cultation over the precordium may reveal a “crunching” sound, but a portable chest X-ray will help confirm the diagnosis. Aspi-ration via

1	air can be stabilized within the right ventricle. Aus-cultation over the precordium may reveal a “crunching” sound, but a portable chest X-ray will help confirm the diagnosis. Aspi-ration via a central venous line accessing the heart may decrease the volume of gas in the right side of the heart and minimize the amount traversing into the pulmonary circulation. Subsequent recovery of intracardiac and intrapulmonary air may require open surgical or angiographic techniques. Treatment may prove futile if the air bolus is larger than 50 mL, however.Pulmonary Artery Rupture Flow-directed, pulmonary artery (Swan-Ganz) catheters can cause pulmonary artery rupture due to excessive advancement of the catheter into the pulmonary cir-culation. There usually is a sentinel bleed with coughing noted when a pulmonary artery catheter balloon is inflated, followed by uncontrolled hemoptysis. Reinflation of the catheter balloon is the initial step in management, followed by immediate airway intubation

1	a pulmonary artery catheter balloon is inflated, followed by uncontrolled hemoptysis. Reinflation of the catheter balloon is the initial step in management, followed by immediate airway intubation with mechanical ventilation, an urgent portable chest X-ray, and notification of the OR that an emergent thoracotomy may be required. If there is no further bleeding after the bal-loon is reinflated, the X-ray shows no significant consolidation of lung fields from ongoing bleeding, and the patient is easily ventilated, then a conservative nonoperative approach may be considered. However, more typically a pulmonary angiogram with angioembolization or vascular stenting is required. Hemo-dynamically unstable patients rarely survive because of the time needed to initiate and perform interventional procedures or a thoracotomy and to identify the ruptured branch of the pulmo-nary artery.Central Venous Line Infection The CDC reports mortal-ity rates of 12% to 25% when a central venous line

1	procedures or a thoracotomy and to identify the ruptured branch of the pulmo-nary artery.Central Venous Line Infection The CDC reports mortal-ity rates of 12% to 25% when a central venous line infection becomes systemic, with a cost of approximately $25,000 per episode.60-62 The CDC does not recommend routine central line changes, but when the clinical suspicion of infection is high, the site of venous access must be changed. Nearly 15% of hospital-ized patients will acquire central venous line sepsis. In many instances, once an infection is recognized as central line sepsis, removing the line is adequate. Staphylococcus aureus infections, however, present a unique problem because of the potential for metastatic seeding of bacterial emboli. The required treatment is 4 to 6 weeks of tailored antibiotic therapy. Using a check-list when inserting central venous catheters has been shown to significantly decrease rates of line infections.63 Following a checklist strategy and close

1	tailored antibiotic therapy. Using a check-list when inserting central venous catheters has been shown to significantly decrease rates of line infections.63 Following a checklist strategy and close monitoring of catheters has resulted in significant reductions in infection rates for numerous institu-tions, and many are now reporting zero annual infection rates.Arterial Lines. Arterial lines are placed to facilitate arterial blood gas sampling and hemodynamic monitoring. The use of ultrasound to assist in placement of these catheters has become commonplace and markedly reduces the number of attempts and time for insertion completion.Arterial access requires a sterile Seldinger technique, and a variety of arteries are used, including the radial, femoral, bra-chial, axillary, dorsalis pedis, or superficial temporal arteries. Although complications occur less than 1% of the time, they can be catastrophic. Complications include thrombosis, bleed-ing, hematoma, arterial spasm (nonthrombotic

1	or superficial temporal arteries. Although complications occur less than 1% of the time, they can be catastrophic. Complications include thrombosis, bleed-ing, hematoma, arterial spasm (nonthrombotic pulselessness), and infection. Thrombosis or embolization of an extremity arte-rial catheter can result in the loss of a digit, hand, or foot, and the risk is nearly the same for both femoral and radial cannula-tion. Thrombosis with distal tissue ischemia is treated with anti-coagulation, but occasionally surgical intervention is required. Pseudoaneurysms and arteriovenous fistulae can also occur.Endoscopy and Bronchoscopy. The principal risk of gastro-intestinal (GI) endoscopy is perforation. Perforations occur in 1 in 10,000 patients with endoscopy alone but have a higher incidence rate when biopsies are performed (up to 10%). This increased risk is due to complications of intubating a GI diver-ticulum (either esophageal or colonic) or from the presence of weakened or inflamed tissue in

1	biopsies are performed (up to 10%). This increased risk is due to complications of intubating a GI diver-ticulum (either esophageal or colonic) or from the presence of weakened or inflamed tissue in the intestinal wall (e.g., diver-ticulitis, glucocorticoid use, or inflammatory bowel disease).Patients will usually complain of diffuse abdominal pain shortly after the procedure and then progress with worsen-ing abdominal discomfort and peritonitis on examination. In obtunded or elderly patients, a change in clinical status may be delayed for 24 to 48 hours. Radiologic studies to look for free intraperitoneal air, retroperitoneal air, or a pneumothorax are diagnostic. Open or laparoscopic exploration locates the perfo-ration and allows repair and local decontamination of the sur-rounding tissues.The occasional patient who may be a candidate for nonop-erative management is one in whom perforation arises during an elective, bowel-prepped endoscopy and who does not have sig-nificant pain or

1	occasional patient who may be a candidate for nonop-erative management is one in whom perforation arises during an elective, bowel-prepped endoscopy and who does not have sig-nificant pain or clinical signs of infection. These patients must be closely observed in a monitored setting and must be on strict dietary restriction and broad-spectrum antibiotics.Complications of bronchoscopy include bronchial plug-ging, hypoxemia, pneumothorax, lobar collapse, and bleeding. Brunicardi_Ch12_p0397-p0432.indd 41620/02/19 3:57 PM 417QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12When diagnosed in a timely fashion, they are rarely life-threatening. Bleeding usually resolves spontaneously and rarely requires surgery but may require repeat endoscopy for thermocoagula-tion or fibrin glue application. The presence of a pneumothorax necessitates placement of a thoracostomy tube when significant deoxygenation occurs or the pulmonary mechanics are compro-mised. Lobar

1	or fibrin glue application. The presence of a pneumothorax necessitates placement of a thoracostomy tube when significant deoxygenation occurs or the pulmonary mechanics are compro-mised. Lobar collapse or mucous plugging usually responds to aggressive pulmonary toilet but occasionally requires repeat bronchoscopy. If biopsies have been performed, the risk for these complications increases.Tracheostomy. Tracheostomy facilitates weaning from a ventilator, may decrease length of ICU or hospital stay, and improves pulmonary toilet. Tracheostomies are performed open, percutaneously, with or without bronchoscopy, and with or without Doppler guidance. The advantages of percutaneous tracheostomy include efficiency and cost containment over open tracheostomy. A recent literature review examining early (<3–7 days) vs late (>14 days) tracheostomy after endotracheal intubation demonstrates little difference in outcomes but does demonstrate greater patient comfort in those patients with

1	early (<3–7 days) vs late (>14 days) tracheostomy after endotracheal intubation demonstrates little difference in outcomes but does demonstrate greater patient comfort in those patients with tra-cheostomy than those with an endotracheal tube. Complications and outcomes between the two different methods remain largely equivalent.Recent studies do not support obtaining a routine chest X-ray after percutaneous or open tracheostomy.64,65 However, significant lobar collapse can occur from copious tracheal secre-tions or mechanical obstruction. The most dramatic complica-tion of tracheostomy is tracheoinnominate artery fistula (TIAF) (Fig. 12-8).66,67 This occurs rarely (∼0.3%) but carries a 50% to 80% mortality rate. TIAFs can occur as early as 2 days or as late as 2 months after tracheostomy. A sentinel bleed occurs in 50% of TIAF cases, followed by a large-volume bleed. Should a TIAF be suspected, the patient should be transported imme-diately to the OR for fiberoptic evaluation. If

1	A sentinel bleed occurs in 50% of TIAF cases, followed by a large-volume bleed. Should a TIAF be suspected, the patient should be transported imme-diately to the OR for fiberoptic evaluation. If needed, remove the tracheostomy and place a finger through the tracheostomy site to apply direct pressure anteriorly for compression of the innominate artery while preparation for a more definitive approach is organized.Percutaneous Endogastrostomy. A misplaced percutane-ous endogastrostomy (PEG) tube may lead to intra-abdominal sepsis with peritonitis and/or an abdominal wall abscess with necrotizing fasciitis. As in other minor procedures, the initial placement technique must be fastidious to avoid complications. Figure 12-8. This illustration depicts improper positioning of the percutaneous needle. It is possible to access the innominate artery via the trachea, thus placing the patient at risk for early tracheoin-nominate artery fistula.XEndoscopic transillumination of the abdomen from

1	needle. It is possible to access the innominate artery via the trachea, thus placing the patient at risk for early tracheoin-nominate artery fistula.XEndoscopic transillumination of the abdomen from within the stomach has been proposed to decrease the risk for error, but this is without supporting evidence. Inadvertent colotomies, intraperitoneal placement of the tube and subsequent leakage of tube feeds with peritonitis, and abdominal wall abscesses require surgery to correct the complications and to replace the PEG with an alternate feeding tube, usually a jejunostomy.A dislodged or prematurely removed PEG tube should be replaced as early as possible after dislodgment because the gas-trostomy site closes rapidly. A contrast X-ray (sinogram) should be performed to confirm the tube’s intragastric position before feeding. If there is uncertainty of the tube location, conversion to an open tube placement procedure is required.Tube Thoracostomy. Chest tube insertion is performed for

1	intragastric position before feeding. If there is uncertainty of the tube location, conversion to an open tube placement procedure is required.Tube Thoracostomy. Chest tube insertion is performed for pneumothorax, hemothorax, pleural effusions, or empyema. In most patients, a chest tube can be easily placed with a combina-tion of local analgesia and light conscious sedation. Common complications include inadequate analgesia or sedation, incom-plete penetration of the pleura with formation of a subcutaneous tube track, lacerations to the lung or diaphragm, intraperitoneal placement of the tube through the diaphragm, and bleeding. Additional problems include slippage of the tube out of posi-tion or mechanical problems related to the drainage system. In patients with bullous disease, there can be significant intrapleu-ral scarring, and it can be easy to mistakenly place the chest tube into bullae. All of these complications can be avoided with proper initial insertion techniques, plus a

1	be significant intrapleu-ral scarring, and it can be easy to mistakenly place the chest tube into bullae. All of these complications can be avoided with proper initial insertion techniques, plus a daily review of the drainage system and follow-up radiographs. Tube removal can create a residual pneumothorax if the patient does not maintain positive intrapleural pressure by Valsalva maneuver during tube removal and dressing application.Complications of Angiography. Intramural dissection of a cannulated artery can lead to complications such as ischemic stroke from a carotid artery dissection or occlusion, mesenteric ischemia from dissection of the superior mesenteric artery, or a more innocuous finding of “blue toe syndrome” from a dissected artery in a peripheral limb. Invasive or noninvasive imaging studies confirm the suspected problem. The severity of ischemia and extent of dissection determine if anticoagulation therapy or urgent surgical exploration is indicated.Bleeding from a

1	imaging studies confirm the suspected problem. The severity of ischemia and extent of dissection determine if anticoagulation therapy or urgent surgical exploration is indicated.Bleeding from a vascular access site usually is obvious, but may not be visible when the blood loss is tracking into the retroperitoneal tissue planes after femoral artery cannulation. These patients can present with hemorrhagic shock; an abdomi-nopelvic CT scan delineates the extent of bleeding along the retroperitoneum. Initial management is direct compression at the access site and resuscitation as indicated. Urgent surgical exploration may be required to control the bleeding site and evacuate larger hematomas.Renal complications of angiography occur in 1% to 2% of patients. Contrast nephropathy is a temporary and prevent-able complication of radiologic studies such as CT, angiogra-phy, and/or venography. Intravenous (IV) hydration before and after the procedure is the most efficient method for preventing

1	and prevent-able complication of radiologic studies such as CT, angiogra-phy, and/or venography. Intravenous (IV) hydration before and after the procedure is the most efficient method for preventing contrast nephropathy. Nonionic contrast also may be of benefit in higher-risk patients. Close communication between provid-ers is often required to resolve the priorities in care as well as to balance the risks versus benefits of renal protection when managing patients in need of angiographic procedures.Complications of Biopsies. Lymph node biopsies have direct and indirect complications that include bleeding, infection, Brunicardi_Ch12_p0397-p0432.indd 41720/02/19 3:57 PM 418BASIC CONSIDERATIONSPART Ilymph leakage, and seromas. Measures to prevent direct com-plications include proper surgical hemostasis, proper skin prepa-ration, and a single preoperative dose of antibiotic to cover skin flora 30 to 60 minutes before incision. Bleeding at a biopsy site usually can be controlled with

1	hemostasis, proper skin prepa-ration, and a single preoperative dose of antibiotic to cover skin flora 30 to 60 minutes before incision. Bleeding at a biopsy site usually can be controlled with direct pressure. Infection at a biopsy site will appear 5 to 10 days postoperatively and may require opening of the wound to drain the infection. Seromas or lymphatic leaks resolve with aspiration of seromas and the application of pressure dressings but may require repeated treat-ments or even placement of a vacuum drain.Organ System ComplicationsNeurologic System. Neurologic complications that occur after surgery include motor or sensory deficits and mental sta-tus changes. Peripheral motor and sensory deficits are often due to neurapraxia secondary to improper positioning and/or pad-ding during operations. Treatment is largely clinical observa-tion, and the majority of deficits resolve spontaneously within 1 to 3 months.Direct injury to nerves during a surgical intervention is a well-known

1	operations. Treatment is largely clinical observa-tion, and the majority of deficits resolve spontaneously within 1 to 3 months.Direct injury to nerves during a surgical intervention is a well-known complication of several specific operations, includ-ing superficial parotidectomy (facial nerve), carotid endarterec-tomy (hypoglossal nerve), thyroidectomy (recurrent laryngeal nerve), prostatectomy (nervi erigentes), inguinal herniorrhaphy (ilioinguinal nerve), and mastectomy (long thoracic and thora-codorsal nerves). The nerve injury may be a stretch injury or an unintentionally severed nerve. In addition to loss of function, severed nerves can result in a painful neuroma that may require subsequent surgery.Mental status changes in the postoperative patient can have numerous causes (Table 12-12). Mental status changes must be continually assessed. A noncontrast CT scan should be used early to detect new or evolving intracranial causes.Atherosclerotic disease increases the risk for

1	12-12). Mental status changes must be continually assessed. A noncontrast CT scan should be used early to detect new or evolving intracranial causes.Atherosclerotic disease increases the risk for intraoperative and postoperative stroke (cerebrovascular accident). Postopera-tively, hypotension and hypoxemia are the most likely causes of a cerebrovascular accident. Neurologic consultation should be obtained immediately to confirm the diagnosis. Management is largely supportive and includes adequate intravascular volume replacement plus optimal oxygen delivery. Advents in inter-ventional radiology by radiologists and vascular and neurologic Table 12-12Common causes of mental status changesELECTROLYTE IMBALANCETOXINSTRAUMAMETABOLICMEDICATIONSSodiumEthanolClosed head injuryThyrotoxicosisAspirinMagnesiumMethanolPainAdrenal insufficiencyβ-BlockersCalciumVenoms and poisonsShockHypoxemiaNarcoticsInflammationEthylene glycolPsychiatricAcidosisAntiemeticsSepsisCarbon monoxideDementiaSevere

1	insufficiencyβ-BlockersCalciumVenoms and poisonsShockHypoxemiaNarcoticsInflammationEthylene glycolPsychiatricAcidosisAntiemeticsSepsisCarbon monoxideDementiaSevere anemiaMAOIsAIDS DepressionHyperammonemiaTCAsCerebral abscess ICU psychosisPoor glycemic controlAmphetaminesMeningitis SchizophreniaHypothermiaAntiarrhythmicsFever/hyperpyrexia HyperthermiaCorticosteroids, anabolic steroidsAIDS = acquired immunodeficiency syndrome; ICU = intensive care unit; MAOI = monoamine oxidase inhibitor; TCA = tricyclic antidepressant.surgeons have proven successful alternatives in patients requir-ing diagnostic and therapeutic care in the immediate and acute postoperative period. Catheter-directed therapy with anticoagu-lants such as the kinases and tissue plasminogen activator (tPA) has potential benefit in postoperative thrombosis where reopera-tion carries significant risk. In addition, endoluminal stents with drug-eluting stents (DESs) or non-DESs have been used with some degree of success. DESs

1	in postoperative thrombosis where reopera-tion carries significant risk. In addition, endoluminal stents with drug-eluting stents (DESs) or non-DESs have been used with some degree of success. DESs do require systemic antiplatelet therapy due to the alternative coagulation pathway. Duration of antiplatelet therapy of 1 year is routine.Eyes, Ears, and Nose. Corneal abrasions are unusual, but are due to inadequate protection of the eyes during anesthe-sia. Overlooked contact lenses in patients occasionally cause conjunctivitis.Persistent epistaxis can occur after nasogastric tube place-ment or removal, and nasal packing is the best treatment option if prolonged persistent direct pressure on the external nares fails. Anterior and posterior nasal gauze packing with balloon tam-ponade, angioembolization, and fibrin glue placement may be required in refractory cases. The use of antibiotics for posterior packing is controversial.External otitis and otitis media occasionally occur

1	angioembolization, and fibrin glue placement may be required in refractory cases. The use of antibiotics for posterior packing is controversial.External otitis and otitis media occasionally occur post-operatively. Patients complain of ear pain or decreased hearing, and treatment includes topical antibiotics and nasal deconges-tion for symptomatic improvement.Ototoxicity due to aminoglycoside administration occurs in up to 10% of patients and is often irreversible. Vancomycin-related ototoxicity occurs about 3% of the time when used alone, and as often as 6% when used with other ototoxic agents.68Vascular Problems of the Neck. Complications of carotid endarterectomy include central or regional neurologic defi-cits or bleeding with an expanding neck hematoma. An acute change in mental status or the presence of localized neurologic deficit requires an immediate return to the OR. An expanding hematoma may warrant emergent airway intubation and subse-quent transfer to the OR for control of

1	or the presence of localized neurologic deficit requires an immediate return to the OR. An expanding hematoma may warrant emergent airway intubation and subse-quent transfer to the OR for control of hemorrhage. Intraopera-tive anticoagulation with heparin during carotid surgery makes bleeding a postoperative risk. Other complications include arte-riovenous fistulae, pseudoaneurysms, and infection, all of which are treated surgically.Brunicardi_Ch12_p0397-p0432.indd 41820/02/19 3:57 PM 419QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12Intraoperative hypotension during manipulation of the carotid bifurcation can occur and is related to increased tone from baroreceptors that reflexively cause bradycardia. Should hypotension occur when manipulating the carotid bifurcation, an injection of 1% lidocaine solution around this structure should attenuate this reflexive response.The most common delayed complication following carotid endarterectomy remains myocardial

1	an injection of 1% lidocaine solution around this structure should attenuate this reflexive response.The most common delayed complication following carotid endarterectomy remains myocardial infarction. The possibility of a postoperative myocardial infarction should be considered as a cause of labile blood pressure and arrhythmias in high-risk patients.Thyroid and Parathyroid Glands. Surgery of the thyroid and parathyroid glands can result in hypocalcemia in the immedi-ate postoperative period. Manifestations include ECG changes (shortened P-R interval), muscle spasm (tetany, Chvostek’s sign, and Trousseau’s sign), paresthesias, and laryngospasm. Treatment includes calcium gluconate infusion and, if tetany ensues, chemical paralysis with intubation. Maintenance treat-ment is thyroid hormone replacement (after thyroidectomy) in addition to calcium carbonate and vitamin D.Recurrent laryngeal nerve (RLN) injury occurs in less than 5% of patients. Of those with injury, approximately 10%

1	replacement (after thyroidectomy) in addition to calcium carbonate and vitamin D.Recurrent laryngeal nerve (RLN) injury occurs in less than 5% of patients. Of those with injury, approximately 10% are permanent. Dissection near the inferior thyroid artery is a com-mon area for RLN injury. At the conclusion of the operation, if there is suspicion of an RLN injury, direct laryngoscopy is diag-nostic. The cord on the affected side will be in the paramedian position. With bilateral RLN injury, the chance of a successful extubation is poor. If paralysis of the cords is not permanent, function may return 1 to 2 months after injury. Permanent RLN injury can be treated by various techniques to stent the cords in a position of function.Superior laryngeal nerve injury is less debilitating, as the common symptom is loss of projection of the voice. The glottic aperture is asymmetrical on direct laryngoscopy, and manage-ment is limited to clinical observation.Respiratory System. Surgical

1	as the common symptom is loss of projection of the voice. The glottic aperture is asymmetrical on direct laryngoscopy, and manage-ment is limited to clinical observation.Respiratory System. Surgical complications that put the respiratory system in jeopardy are not confined to techni-cal errors. Malnutrition, inadequate pain control, inadequate mechanical ventilation, inadequate pulmonary toilet, and aspi-ration can cause serious pulmonary problems.Pneumothorax can occur from central line insertion during anesthesia or from a diaphragmatic injury during an abdomi-nal procedure. Hypotension, hypoxemia, and tracheal deviation away from the affected side may be present. A tension pneumo-thorax can cause complete cardiovascular collapse. Treatment is by needle thoracostomy, followed by tube thoracostomy. The chest tube is inserted at the fifth intercostal space in the anterior axillary line. The anterior chest wall is up to 1 cm thicker than the lateral chest wall, so needle decompression

1	The chest tube is inserted at the fifth intercostal space in the anterior axillary line. The anterior chest wall is up to 1 cm thicker than the lateral chest wall, so needle decompression is more effec-tive in the lateral position. Attempted prehospital needle decom-pression in the traditional anterior position results in only 50% needle entry into the thoracic cavity.Hemothoraces should be evacuated completely. Delay in evacuation of a hemothorax leaves the patient at risk for empy-ema and entrapped lung. If evacuation is incomplete with tube thoracostomy, video-assisted thoracoscopy or open evacuation and pleurodesis may be required.Pulmonary atelectasis results in a loss of functional resid-ual capacity (FRC) of the lung and can predispose to pneumo-nia. Poor pain control in the postoperative period contributes to poor inspiratory effort and collapse of the lower lobes in particular. The prevention of atelectasis is facilitated by sit-ting the patient up as much as possible, early

1	period contributes to poor inspiratory effort and collapse of the lower lobes in particular. The prevention of atelectasis is facilitated by sit-ting the patient up as much as possible, early ambulation, and adequate pain control. An increase in FRC by 700 mL or more can be accomplished by sitting patients up to greater than 45°. For mechanically ventilated patients, simply placing the head of the bed at 30° to 45° elevation and delivering adequate tidal volumes (8–10 mL/kg) improves pulmonary outcomes.69Patients with inadequate pulmonary toilet are at increased risk for bronchial plugging and lobar collapse. Patients with copious and tenacious secretions develop these plugs most often, but foreign bodies in the bronchus can be the cause of lobar collapse as well. The diagnosis of bronchial plugging is based on chest X-ray and clinical suspicion with acute pulmo-nary decompensation with increased work of breathing and hypoxemia. Fiberoptic bronchoscopy can be useful to clear mucous

1	plugging is based on chest X-ray and clinical suspicion with acute pulmo-nary decompensation with increased work of breathing and hypoxemia. Fiberoptic bronchoscopy can be useful to clear mucous plugs and secretions.Aspiration complications include pneumonitis and pneu-monia. The treatment of pneumonitis is similar to that for acute respiratory distress syndrome (see later in this section) and includes oxygenation with general supportive care. Antibiotics are not indicated. Hospitalized patients who develop aspiration pneumonitis have a mortality rate as high as 70% to 80%. Early, aggressive, and repeated bronchoscopy for suctioning of aspi-rated material from the tracheobronchial tree will help mini-mize the inflammatory reaction of pneumonitis and facilitate improved pulmonary toilet. Forced diuresis to overcome ana-sarca and over-resuscitation remains controversial and unsub-stantiated. Complications of forced diuresis include electrolyte disturbances, replacement of those

1	Forced diuresis to overcome ana-sarca and over-resuscitation remains controversial and unsub-stantiated. Complications of forced diuresis include electrolyte disturbances, replacement of those electrolytes, metabolic alka-losis, hypotension, and acute kidney injury.Pneumonia is the second most common nosocomial infec-tion and is the most common infection in ventilated patients. Ventilator-associated pneumonia (VAP) occurs in 15% to 40% of ventilated ICU patients, with a probability rate of 5% per day, up to 70% at 30 days. The 30-day mortality rate of nosocomial pneumonia can be as high as 40% and depends on the micro-organisms involved and the timeliness of initiating appropri-ate antimicrobials. Protocol-driven approaches for prevention and treatment of VAP are recognized as beneficial in managing these difficult infectious complications.Once the diagnosis of pneumonia is suspected (an abnormal chest X-ray, fever, productive cough with purulent sputum, and no other obvious fever

1	in managing these difficult infectious complications.Once the diagnosis of pneumonia is suspected (an abnormal chest X-ray, fever, productive cough with purulent sputum, and no other obvious fever sources), it is invariably necessary to ini-tially begin treatment with broad-spectrum antibiotics until proper identification, colony count (≥100,000 colony-forming units [CFU]), and sensitivity of the microorganisms are determined. The spectrum of antibiotic coverage should be narrowed as soon as the culture sensitivities are determined. Double-coverage anti-biotic strategy for the two pathogens, Pseudomonas and Acineto-bacter spp., may be appropriate if the local prevalence of these particularly virulent organisms is high. One of the most helpful tools in treating pneumonia and other infections is the tracking of a medical center’s antibiogram every 6 to 12 months.70Epidural analgesia decreases the risk of perioperative pneumonia. This method of pain control improves pulmonary toilet and

1	is the tracking of a medical center’s antibiogram every 6 to 12 months.70Epidural analgesia decreases the risk of perioperative pneumonia. This method of pain control improves pulmonary toilet and the early return of bowel function; both have a sig-nificant impact on the potential for aspiration and for acquir-ing pneumonia. The routine use of epidural analgesia results in a lower incidence of pneumonia than patient-controlled analgesia.71Acute lung injury (ALI) was a diagnosis applied to patients with similar findings to those with acute respiratory distress Brunicardi_Ch12_p0397-p0432.indd 41920/02/19 3:57 PM 420BASIC CONSIDERATIONSPART Isyndrome (ARDS). The Berlin definition of ARDS developed by the American-European Consensus Conference of 2012 not only simplifies the definition of ARDS but also eliminates the term ALI from critical care vernacular. ARDS is now classified by partial pressure of oxygen in arterial blood (Pao2)/fraction of inspired oxygen (Fio2) ratios as mild

1	of ARDS but also eliminates the term ALI from critical care vernacular. ARDS is now classified by partial pressure of oxygen in arterial blood (Pao2)/fraction of inspired oxygen (Fio2) ratios as mild (300–201 mmHg), moder-ate (200–101 mmHg), and severe (<100 mmHg). Elements of modification of the definition include the following: <7 days of onset; removal of pulmonary artery occlusion pressure; and clinical judgment for characterizing hydrostatic pulmonary edema is acceptable, unless risk factors for ARDS have been eliminated, in which case objective analysis is necessary.72-75The definition of ARDS traditionally included five crite-ria (Table 12-13). The multicenter ARDS Research Network (ARDSnet) research trial demonstrated improved clinical out-comes for ARDS patients ventilated at tidal volumes of only 5 to 7 mL/kg.76 This strategy is no longer prescribed solely for patients with ARDS but is also recommended for patients with normal pulmonary physiology who are intubated for

1	tidal volumes of only 5 to 7 mL/kg.76 This strategy is no longer prescribed solely for patients with ARDS but is also recommended for patients with normal pulmonary physiology who are intubated for reasons other than acute respiratory failure. The beneficial effects of positive end-expiratory pressure (PEEP) for ARDS were con-firmed in this study as well. The maintenance of PEEP during ventilatory support is determined based on blood gas analysis, pulmonary mechanics, and requirements for supplemental oxy-gen. As gas exchange improves with resolving ARDS, the initial step in decreasing ventilatory support should be to decrease the levels of supplemental oxygen first, and then to slowly bring the PEEP levels back down to minimal levels.77 This is done to minimize the potential for recurrent alveolar collapse and a worsening gas exchange.Not all patients can be weaned easily from mechanical ventilation. When the respiratory muscle energy demands are not balanced or there is an ongoing

1	alveolar collapse and a worsening gas exchange.Not all patients can be weaned easily from mechanical ventilation. When the respiratory muscle energy demands are not balanced or there is an ongoing active disease state external to the lungs, patients may require prolonged ventilatory sup-port. Protocol-driven ventilator weaning strategies are success-ful and have become part of the standard of care. The use of a weaning protocol for patients on mechanical ventilation greater than 48 hours reduces the incidence of VAP and the overall length of time on mechanical ventilation. Unfortunately, there is still no reliable way of predicting which patient will be suc-cessfully extubated after a weaning program, and the decision for extubation is based on a combination of clinical parameters and measured pulmonary mechanics.78 The Tobin Index (fre-quency [breaths per minute]/tidal volume [L]), also known as the rapid shallow breathing index, is perhaps the best negative predictive instrument.79

1	pulmonary mechanics.78 The Tobin Index (fre-quency [breaths per minute]/tidal volume [L]), also known as the rapid shallow breathing index, is perhaps the best negative predictive instrument.79 If the result equals less than 105, then Table 12-13Inclusion criteria for the acute respiratory distress syndromeAcute onsetPredisposing conditionPao2:Fio2 <200 (regardless of positive end-expiratory pressure)Bilateral infiltratesPulmonary artery occlusion pressure <18 mmHgNo clinical evidence of right heart failureFio2 = fraction of inspired oxygen; Pao2 = partial pressure of arterial oxygen.there is nearly a 70% chance the patient will pass extubation. If the score is greater than 105, the patient has an approximately 80% chance of failing extubation. Other parameters such as the negative inspiratory force, minute ventilation, and respiratory rate are used, but individually these have no better predictive value than the rapid shallow breathing index.80Malnutrition and poor nutritional

1	inspiratory force, minute ventilation, and respiratory rate are used, but individually these have no better predictive value than the rapid shallow breathing index.80Malnutrition and poor nutritional support may adversely affect the respiratory system. The respiratory quotient (RQ), or respiratory exchange ratio, is the ratio of the rate of carbon dioxide (CO2) produced to the rate of oxygen uptake (RQ = Vco2/V.O2). Lipids, carbohydrates, and protein have differing effects on CO2 production. Patients consuming a diet of mostly carbohydrates have an RQ of 1 or greater. The RQ for a diet of mostly lipids is closer to 0.7, and that for a diet of mostly protein is closer to 0.8. Ideally, an RQ of 0.75 to 0.85 suggests adequate balance and composition of nutrient intake. An excess of car-bohydrate may negatively affect ventilator weaning because of the abnormal RQ due to higher CO2 production and altered pul-monary gas exchange.Although not without risk, tracheostomy decreases the

1	may negatively affect ventilator weaning because of the abnormal RQ due to higher CO2 production and altered pul-monary gas exchange.Although not without risk, tracheostomy decreases the pulmonary dead space and provides for improved pulmonary toilet. When performed before the tenth day of ventilatory sup-port, tracheostomy may decrease the incidence of VAP, the overall length of ventilator time, and the number of ICU patient days.The occurrence of PE is probably underdiagnosed. Its eti-ology is thought to stem from DVT. This concept, however, has recently been questioned by Spaniolas et al.81 The diagno-sis of PE is made when a high degree of clinical suspicion for PE leads to imaging techniques such as ventilation–perfusion nuclear scans or CT pulmonary angiogram. Clinical findings include elevated central venous pressure, hypoxemia, shortness of breath, hypocarbia secondary to tachypnea, and right heart strain on ECG. Ventilation–perfusion nuclear scans are often indeterminate in

1	elevated central venous pressure, hypoxemia, shortness of breath, hypocarbia secondary to tachypnea, and right heart strain on ECG. Ventilation–perfusion nuclear scans are often indeterminate in patients who have an abnormal chest X-ray and are less sensitive than a CT angiogram or pulmonary angio-gram for diagnosing PE. The pulmonary angiogram remains the gold standard for diagnosing PE, but spiral CT angiogram has become an alternative method because of its relative ease of use and reasonable rates of diagnostic accuracy. For cases without clinical contraindications to therapeutic anticoagula-tion, patients should be empirically started on heparin infusion until the imaging studies are completed if the suspicion of a PE is high.Sequential compression devices on the lower extremities and low-dose subcutaneous heparin or low molecular weight heparinoid administration are routinely used to prevent DVT and, by inference, the risk of PE. Neurosurgical and orthopedic patients have higher

1	low-dose subcutaneous heparin or low molecular weight heparinoid administration are routinely used to prevent DVT and, by inference, the risk of PE. Neurosurgical and orthopedic patients have higher rates of PE, as do obese patients and those at prolonged bed rest.When anticoagulation is contraindicated, or when a known clot exists in the inferior vena cava (IVC), decreasing the risk for PE includes insertion of an IVC filter. The Greenfield filter has been most widely studied, and it has a failure rate of less than 4%. Newer devices include those with nitinol wire that expands with body temperature and retrievable filters. Retrievable filters, however, must be considered as permanent. In most studies, the actual retrievable rate only reached about 20%. Some studies recognize the benefit of automated reminders and diligence of outlying patient follow-up, where higher retrieval rates have been achieved.82 Patients with spinal cord injury and multiple long-bone or pelvic fractures

1	of automated reminders and diligence of outlying patient follow-up, where higher retrieval rates have been achieved.82 Patients with spinal cord injury and multiple long-bone or pelvic fractures frequently receive IVC filters, and Brunicardi_Ch12_p0397-p0432.indd 42020/02/19 3:57 PM 421QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12there appears to be a low, but not insignificant, long-term com-plication rate with their use. However, IVC filters do not prevent PEs that originate from DVTs of the upper extremities.Cardiac System. Arrhythmias are often seen preoperatively in elderly patients but may occur postoperatively in any age group. Atrial fibrillation is the most common arrhythmia83 and occurs between postoperative days 3 to 5 in high-risk patients. This is typically when patients begin to mobilize their intersti-tial fluid into the vascular fluid space. Contemporary evidence suggests that rate control is more important than rhythm con-trol for

1	This is typically when patients begin to mobilize their intersti-tial fluid into the vascular fluid space. Contemporary evidence suggests that rate control is more important than rhythm con-trol for atrial fibrillation.84,85 The first-line treatment includes β-blockade and/or calcium channel blockade. β-Blockade must be used judiciously because hypotension, as well as withdrawal from β-blockade with rebound hypertension, is possible. Cal-cium channel blockers are an option if β-blockers are not toler-ated by the patient, but caution must be exercised in those with a history of congestive heart failure. Although digoxin is still a standby medication, it has limitations due to the need for opti-mal dosing levels. Cardioversion may be required if patients become hemodynamically unstable and the rhythm cannot be controlled.Ventricular arrhythmias and other tachyarrhythmias may occur in surgical patients as well. Similar to atrial rhythm prob-lems, these are best controlled with

1	and the rhythm cannot be controlled.Ventricular arrhythmias and other tachyarrhythmias may occur in surgical patients as well. Similar to atrial rhythm prob-lems, these are best controlled with β-blockade, but the use of other antiarrhythmics or cardioversion may be required if patients become hemodynamically unstable.Cardiac ischemia is a cause of postoperative mortality. Acute myocardial infarction (AMI) can present insidiously, or it can be more dramatic with the classic presentation of short-ness of breath, severe angina, and sudden cardiogenic shock. The workup to rule out an AMI includes an ECG and cardiac enzyme measurements. The patient should be transferred to a monitored (telemetry) floor. Morphine, supplemental oxygen, nitroglycerine, and aspirin (MONA) are the initial therapeutic maneuvers for those being investigated for AMI.Gastrointestinal System. Surgery of the esophagus is poten-tially complicated because of its anatomic location and blood supply. Nutritional support

1	maneuvers for those being investigated for AMI.Gastrointestinal System. Surgery of the esophagus is poten-tially complicated because of its anatomic location and blood supply. Nutritional support strategies should be considered for esophageal resection patients to maximize the potential for sur-vival. The two primary types of esophageal resection performed are the transhiatal resection and the transthoracic (Ivor-Lewis) resection.86 The transhiatal resection has the advantage that a formal thoracotomy incision is avoided. However, dissection of the esophagus is blind, and anastomotic leaks occur more than with other resections. However, when a leak does occur, simple opening of the cervical incision and draining the leak is all that is usually required.The transthoracic Ivor-Lewis resection includes an esoph-ageal anastomosis performed in the chest near the level of the azygos vein. These have lower leak rates, but the leaks that do occur result in mediastinitis and can be difficult

1	includes an esoph-ageal anastomosis performed in the chest near the level of the azygos vein. These have lower leak rates, but the leaks that do occur result in mediastinitis and can be difficult to control. The reported mortality is about 50% with an anastomotic leak, and the overall mortality of the procedure is about 5%, which is similar to transhiatal resection.Postoperative ileus is related to dysfunction of the neural reflex axis of the intestine. Excessive narcotic use may delay return of bowel function. Epidural anesthesia results in better pain control, and there is an earlier return of bowel function and a shorter length of hospital stay. The limited use of naso-gastric tubes and the initiation of early postoperative feeding are associated with an earlier return of bowel function.87 The use of chewing gum and other oral stimulants to minimize ileus remains controversial.Pharmacologic agents commonly used to stimulate bowel function include metoclopramide and erythromycin.

1	The use of chewing gum and other oral stimulants to minimize ileus remains controversial.Pharmacologic agents commonly used to stimulate bowel function include metoclopramide and erythromycin. Metoclo-pramide’s action is limited to the stomach and duodenum, and it may help primarily with gastroparesis. Erythromycin is a motilin agonist that works throughout the stomach and bowel. Several studies demonstrate significant benefit from the administration of erythromycin in those suffering from an ileus.88 Alvimopan, a newer agent and a µ-opioid receptor antagonist, has shown some promise in many studies for earlier return of gut function and subsequent reduction in length of stay.89,90 Neostigmine has been used in refractory pan-ileus patients (Ogilvie’s syndrome) with some degree of success. It is recommended for patients receiving this type of therapy to be in a monitored unit.91Small bowel obstruction occurs in less than 1% of early postoperative patients. When it does occur, adhesions

1	It is recommended for patients receiving this type of therapy to be in a monitored unit.91Small bowel obstruction occurs in less than 1% of early postoperative patients. When it does occur, adhesions are usu-ally the cause. Internal and external hernias, technical errors, and infections or abscesses are also causative. Hyaluronidase is a mucolytic enzyme that degrades connective tissue, and the use of a methylcellulose form of hyaluronidase, Seprafilm®, has been shown to result in a 50% decrease in adhesion formation in some patients.92,93 This may translate into a lower occurrence of postoperative bowel obstruction, but has yet to be proven.Fistulae are the abnormal communication of one structure to an adjacent structure or compartment and are associated with extensive morbidity and mortality. Common causes for fistula formation are summarized in the mnemonic FRIENDS (Foreign body, Radiation, Ischemia/Inflammation/Infection, Epitheli-alization of a tract, Neoplasia, Distal

1	and mortality. Common causes for fistula formation are summarized in the mnemonic FRIENDS (Foreign body, Radiation, Ischemia/Inflammation/Infection, Epitheli-alization of a tract, Neoplasia, Distal obstruction, and Steroid use). Postoperatively, they are most often caused by infection or obstruction leading to an anastomotic leak. The cause of the fis-tula must be recognized early, and treatment may include non-operative management with observation and nutritional support, or a delayed operative management strategy that also includes nutritional support and wound care.Gastrointestinal (GI) bleeding can occur perioperatively (Table 12-14). Technical errors such as a poorly tied suture, a nonhemostatic staple line, or a missed injury can all lead to Table 12-14Common causes of upper and lower gastrointestinal (GI) hemorrhageUPPER GI BLEEDLOWER GI BLEEDErosive esophagitisAngiodysplasiaGastric varicesRadiation proctitisEsophageal varicesHemangiomaDieulafoy’s

1	causes of upper and lower gastrointestinal (GI) hemorrhageUPPER GI BLEEDLOWER GI BLEEDErosive esophagitisAngiodysplasiaGastric varicesRadiation proctitisEsophageal varicesHemangiomaDieulafoy’s lesionDiverticulosisAortoduodenal fistulaNeoplastic diseasesMallory-Weiss tearTraumaPeptic ulcer diseaseVasculitisTraumaHemorrhoidsNeoplastic diseaseAortoenteric fistulaIntussusceptionIschemic colitisInflammatory bowel diseasePostprocedure bleedingBrunicardi_Ch12_p0397-p0432.indd 42120/02/19 3:57 PM 422BASIC CONSIDERATIONSPART Ipostoperative intestinal bleeding.94,95 The source of bleeding is in the upper GI tract about 85% of the time and is usually detected and treated endoscopically. Surgical control of intesti-nal bleeding is required in up to 40% of patients.96When patients in the ICU have a major bleed from stress gastritis, the mortality risk is as high as 50%. It is important to keep the gastric pH greater than 4 to decrease the overall risk for stress gastritis in patients

1	ICU have a major bleed from stress gastritis, the mortality risk is as high as 50%. It is important to keep the gastric pH greater than 4 to decrease the overall risk for stress gastritis in patients mechanically ventilated for 48 hours or greater and patients who are coagulopathic.97 Proton pump inhibitors, H2-receptor antagonists, and intragastric antacid installation are all effective measures. However, patients who are not mechanically ventilated or who do not have a history of gastritis or peptic ulcer disease should not be placed on gastritis prophylaxis postoperatively because it carries a higher risk of causing pneumonia.Hepatobiliary-Pancreatic System. Complications involv-ing the hepatobiliary system are usually due to technical errors. Laparoscopic cholecystectomy has become the standard of care for cholecystectomy, but common bile duct injury remains a nemesis of this approach. Intraoperative cholangiography has not been shown to decrease the incidence of common bile duct

1	standard of care for cholecystectomy, but common bile duct injury remains a nemesis of this approach. Intraoperative cholangiography has not been shown to decrease the incidence of common bile duct injuries because the injury to the bile duct usually occurs before the cholangiogram.98,99 Early recognition and immediate repair of an injury are important because delayed bile duct leaks often require a more complex repair.Ischemic injury due to devascularization of the common bile duct has a delayed presentation days to weeks after an operation. Endoscopic retrograde cholangiopancreatography (ERCP) demonstrates a stenotic, smooth common bile duct, and liver function studies are elevated. The recommended treatment is a Roux-en-Y hepaticojejunostomy.A bile leak due to an unrecognized injury to the ducts may present after cholecystectomy as a biloma. These patients may present with abdominal pain and hyperbilirubinemia. The diag-nosis of a biliary leak can be confirmed by CT scan, ERCP, or

1	the ducts may present after cholecystectomy as a biloma. These patients may present with abdominal pain and hyperbilirubinemia. The diag-nosis of a biliary leak can be confirmed by CT scan, ERCP, or radionuclide scan. Once a leak is confirmed, a retrograde biliary stent and external drainage are the treatment of choice.Hyperbilirubinemia in the surgical patient can be a com-plex problem. Cholestasis makes up the majority of causes for hyperbilirubinemia, but other mechanisms of hyperbiliru-binemia include reabsorption of blood (e.g., hematoma from trauma), decreased bile excretion (e.g., sepsis), increased unconjugated bilirubin due to hemolysis, hyperthyroidism, and impaired excretion due to congenital abnormalities or acquired disease. Errors in surgery that cause hyperbilirubinemia largely involve missed or iatrogenic injuries.The presence of cirrhosis predisposes to postoperative complications. Abdominal or hepatobiliary surgery is problem-atic in the cirrhotic patient. Ascites

1	involve missed or iatrogenic injuries.The presence of cirrhosis predisposes to postoperative complications. Abdominal or hepatobiliary surgery is problem-atic in the cirrhotic patient. Ascites leak in the postoperative period can be an issue when any abdominal operation has been performed. Maintaining proper intravascular oncotic pressure in the immediate postoperative period can be difficult, and resus-citation should be maintained with crystalloid solutions. Pre-vention of renal failure and the management of the hepatorenal syndrome can be difficult, as the demands of fluid resuscitation and altered glomerular filtration become competitive. Spirono-lactone with other diuretic agents may be helpful in the post-operative care. These patients often have a labile course, and bleeding complications due to coagulopathy are common. The operative mortality in cirrhotic patients is 10% for Child class A, 30% for Child class B, and 82% for Child class C patients.100Pyogenic liver abscess

1	due to coagulopathy are common. The operative mortality in cirrhotic patients is 10% for Child class A, 30% for Child class B, and 82% for Child class C patients.100Pyogenic liver abscess occurs in less than 0.5% of adult admissions, due to retained necrotic liver tissue, occult intesti-nal perforations, benign or malignant hepatobiliary obstruction, sepsis, and hepatic arterial occlusion. The treatment is long-term antibiotics with percutaneous drainage of large abscesses.Pancreatitis can occur following injection of contrast dur-ing cholangiography and after endoscopic cholangiopancreatog-raphy (ERCP). These episodes range from a mild elevation in amylase and lipase with abdominal pain, to a fulminant course of pancreatitis with necrosis requiring surgical debridement. The incidence of post-ERCP pancreatitis has been shown to be reduced by the administration of rectal indomethacin.101 Stud-ies are underway to determine whether the prophylactic use of pancreatic duct stenting in

1	of post-ERCP pancreatitis has been shown to be reduced by the administration of rectal indomethacin.101 Stud-ies are underway to determine whether the prophylactic use of pancreatic duct stenting in patients at high risk for post-ERCP pancreatitis can be avoided with the use of rectal indomethacin.Traumatic injuries to the pancreas can occur during surgi-cal procedures on the kidneys, GI tract, and spleen most com-monly. Treatment involves serial CT scans and percutaneous drainage to manage infected fluid and abscess collections; ster-ile collections should not be drained because drain placement can introduce infection. A pancreatic fistula may respond to antisecretory therapy with a somatostatin analogue. Manage-ment of these fistulae initially includes ERCP with or without pancreatic stenting, percutaneous drainage of any fistula fluid collections, total parenteral nutrition (TPN) with bowel rest, and repeated CT scans. The majority of pancreatic fistulae will eventually heal

1	stenting, percutaneous drainage of any fistula fluid collections, total parenteral nutrition (TPN) with bowel rest, and repeated CT scans. The majority of pancreatic fistulae will eventually heal spontaneously.Renal System. Renal failure can be classified as prerenal failure, intrinsic renal failure, and postrenal failure. Postrenal failure, or obstructive renal failure, should always be consid-ered when low urine output (oliguria) or anuria occurs. The most common cause is a misplaced or clogged urinary catheter. Other, less common causes to consider are unintentional ligation or transection of ureters during a difficult surgical dissection (e.g., colon resection for diverticular disease) or a large retro-peritoneal hematoma (e.g., ruptured aortic aneurysm).Oliguria is initially evaluated by flushing the urinary cath-eter using sterile technique. Urine electrolytes should also be measured (Table 12-15). A hemoglobin and hematocrit level should be checked immediately. Patients in

1	by flushing the urinary cath-eter using sterile technique. Urine electrolytes should also be measured (Table 12-15). A hemoglobin and hematocrit level should be checked immediately. Patients in compensated shock from acute blood loss may manifest anemia and end-organ mal-perfusion as oliguria.Acute tubular necrosis (ATN) carries a mortality risk of 25% to 50% due to the many complications that can cause, or result from, this insult. When ATN is due to poor inflow (prer-enal failure), the remedy begins with IV administration of crys-talloid or colloid fluids as needed. If cardiac insufficiency is the problem, the optimization of vascular volume is achieved first, followed by inotropic agents, as needed. Intrinsic renal failure Table 12-15Urinary electrolytes associated with acute renal failure and their possible etiologies FENaOSMOLARITYURNaETIOLOGYPrerenal<1>500<20CHF, cirrhosisIntrinsic failure>1<350>40Sepsis, shockCHF = congestive heart failure; FENa = fractional excretion of

1	failure and their possible etiologies FENaOSMOLARITYURNaETIOLOGYPrerenal<1>500<20CHF, cirrhosisIntrinsic failure>1<350>40Sepsis, shockCHF = congestive heart failure; FENa = fractional excretion of sodium; URNa = urinary excretion of sodium.Brunicardi_Ch12_p0397-p0432.indd 42220/02/19 3:57 PM 423QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12and subsequent ATN are often the result of direct renal toxins. Aminoglycosides, vancomycin, and furosemide, among other commonly used agents, contribute directly to nephrotoxicity. Contrast-induced nephropathy usually leads to a subtle or tran-sient rise in creatinine. In patients who are volume depleted or have poor cardiac function, contrast nephropathy may perma-nently impair renal function.102-105The treatment of renal failure due to myoglobinuria has shifted away from the use of sodium bicarbonate for alkalinizing the urine, to merely maintaining brisk urine output of 100 mL per hour with crystalloid fluid

1	renal failure due to myoglobinuria has shifted away from the use of sodium bicarbonate for alkalinizing the urine, to merely maintaining brisk urine output of 100 mL per hour with crystalloid fluid infusion. Mannitol and furosemide are not recommended. Patients who do not respond to resusci-tation are at risk for needing renal replacement therapy. Fortu-nately, most of these patients eventually recover from their renal dysfunction.Musculoskeletal System. A compartment syndrome can develop in any compartment of the body. Compartment syn-drome of the extremities generally occurs after a closed fracture. The injury alone may predispose the patient to compartment syndrome, but aggressive fluid resuscitation can exacerbate the problem. Pain with passive motion is the hallmark of com-partment syndrome, and the anterior compartment of the leg is usually the first compartment to be involved. Confirmation of the diagnosis is obtained by direct pressure measurement of the individual

1	syndrome, and the anterior compartment of the leg is usually the first compartment to be involved. Confirmation of the diagnosis is obtained by direct pressure measurement of the individual compartments. If the pressures are greater than 20 to 25 mmHg in any of the compartments, then a four-compartment fasciotomy is considered. Compartment syndrome can be due to ischemia-reperfusion injury, after an ischemic time of 4 to 6 hours. Renal failure (due to myoglobinuria), tissue loss, and a permanent loss of function are possible results of untreated compartment syndrome.Decubitus ulcers are preventable complications of pro-longed bed rest due to traumatic paralysis, dementia, chemi-cal paralysis, or coma. Unfortunately, they are still occurring despite extensive research and clinical initiatives that demon-strate successful prevention strategies. Ischemic changes in the microcirculation of the skin can be significant after 2 hours of sustained pressure. Routine skin care and turning of

1	that demon-strate successful prevention strategies. Ischemic changes in the microcirculation of the skin can be significant after 2 hours of sustained pressure. Routine skin care and turning of the patient help ensure a reduction in skin ulceration. This can be labor intensive, and special mattresses and beds are available to help. The treatment of a decubitus ulcer in the noncoagulopathic patient is surgical debridement. Once the wound bed has a via-ble granulation base without an excess of fibrinous debris, a vacuum-assisted closure dressing can be applied. Wet to moist dressings with frequent dressing changes is the alternative and is labor intensive. Expensive topical enzyme preparations are also available. If the wounds fail to respond to these measures, soft tissue coverage by flap is considered.Contractures are the result of muscle disuse. Whether from trauma, amputation, or vascular insufficiency, contractures can be prevented by physical therapy and splinting. If not attended

1	considered.Contractures are the result of muscle disuse. Whether from trauma, amputation, or vascular insufficiency, contractures can be prevented by physical therapy and splinting. If not attended to early, contractures will prolong rehabilitation and may lead to further wounds and wound healing issues. Depending on the functional status of the patient, contracture releases may be required for long-term care.Hematologic System. The traditional transfusion guideline of maintaining the hematocrit level in all patients at greater than 30% is no longer valid. Only patients with symptomatic anemia, who have significant cardiac disease, or who are critically ill and require increased oxygen-carrying capacity to adequately perfuse end organs require higher levels of hemoglobin. Other than these select patients, the decision to transfuse should gener-ally not occur until the hemoglobin level falls to 7 mg/dL or the hematocrit reaches 21%.Transfusion reactions are common complications of

1	select patients, the decision to transfuse should gener-ally not occur until the hemoglobin level falls to 7 mg/dL or the hematocrit reaches 21%.Transfusion reactions are common complications of blood transfusion. These can be attenuated with a leukocyte filter, but not completely prevented. The manifestations of a transfusion reaction include simple fever, pruritus, chills, muscle rigidity, and renal failure due to myoglobinuria secondary to hemolysis. Discontinuing the transfusion and returning the blood products to the blood bank is an important first step, but administration of antihistamine and possibly steroids may be required to control the reaction symptoms. Severe transfusion reactions are rare but can be fatal.Infectious complications in blood transfusion range from cytomegalovirus transmission, which is benign in the nontrans-plant patient, to human immunodeficiency virus (HIV) infec-tion, to passage of the hepatitis viruses (Table 12-16).Patients on warfarin (Coumadin) who

1	transmission, which is benign in the nontrans-plant patient, to human immunodeficiency virus (HIV) infec-tion, to passage of the hepatitis viruses (Table 12-16).Patients on warfarin (Coumadin) who require surgery can have anticoagulation reversal by administration of fresh frozen plasma. Each unit of fresh frozen plasma contains 200 to 250 mL of plasma and includes one unit of coagulation factor per milliliter of plasma.Thrombocytopenia may require platelet transfusion for a platelet count less than 20,000/mL when invasive procedures are performed, or when platelet counts are low and ongoing bleed-ing from raw surface areas persists. One unit of platelets will increase the platelet count by 5000 to 7500 per mL in adults. It is important to delineate the cause of the low platelet count. Usually there is a self-limiting or reversible condition such as sepsis. Rarely, it is due to heparin-induced thrombocytopenia I and II. Complications of heparin-induced thrombocytopenia II can be

1	Usually there is a self-limiting or reversible condition such as sepsis. Rarely, it is due to heparin-induced thrombocytopenia I and II. Complications of heparin-induced thrombocytopenia II can be serious because of the diffuse thrombogenic nature of the disorder. Simple precautions to limit this hypercoagulable state include saline solution flushes instead of heparin solutions and limiting the use of heparin-coated catheters. The treatment is anticoagulation with synthetic agents such as argatroban.For patients with uncontrollable bleeding due to dissemi-nated intravascular coagulopathy (DIC), a potentially useful drug is factor VIIa, but its use should be judicious.106-109 Origi-nally used in hepatic trauma and obstetric emergencies, this agent was lifesaving in some circumstances. The CONTROL Trial,109 however, has largely decreased overuse of this agent because investigators demonstrated no benefit over simple fac-tor replacement in severely coagulopathic patients. Factor VIIa use

1	Trial,109 however, has largely decreased overuse of this agent because investigators demonstrated no benefit over simple fac-tor replacement in severely coagulopathic patients. Factor VIIa use may also be limited due to its potential thrombotic com-plications. For some situations, the combination of ongoing, Table 12-16Rate of viral transmission in blood product transfusionsaHIV1:1.9 millionHBVb1:137,000HCV1:1 millionaPost-nucleic acid amplification technology (1999). Earlier rates were erroneously reported higher due to lack of contemporary technology.bHBV is reported with prenucleic acid amplification technology. Statistical information is unavailable with postnucleic acid amplification technology at this writing.Note that bacterial transmission is 50 to 250 times higher than viral transmission per transfusion.HBV = hepatitis B virus; HCV = hepatitis C virus.Brunicardi_Ch12_p0397-p0432.indd 42320/02/19 3:57 PM 424BASIC CONSIDERATIONSPART Inonsurgical bleeding and renal failure

1	per transfusion.HBV = hepatitis B virus; HCV = hepatitis C virus.Brunicardi_Ch12_p0397-p0432.indd 42320/02/19 3:57 PM 424BASIC CONSIDERATIONSPART Inonsurgical bleeding and renal failure can occasionally be suc-cessfully treated with desmopressin.In addition to classic hemophilia, other inherited coagula-tion factor deficiencies can be difficult to manage in surgery. When required, transfusion of appropriate replacement products is coordinated with the regional blood bank center before sur-gery. Other blood dyscrasias seen by surgeons include hyper-coagulopathic patients. Those who carry congenital anomalies such as the most common factor V Leiden deficiency, as well as protein C and S deficiencies, are likely to form thromboses if inadequately anticoagulated, and these patients should be man-aged in consultation with a hematologist.Abdominal Compartment Syndrome. Multisystem trauma, thermal burns, retroperitoneal injuries, and surgery related to the retroperitoneum are the major

1	be man-aged in consultation with a hematologist.Abdominal Compartment Syndrome. Multisystem trauma, thermal burns, retroperitoneal injuries, and surgery related to the retroperitoneum are the major initial causative factors that may lead to abdominal compartment syndrome (ACS). Ruptured AAA, major pancreatic injury and resection, or multiple intes-tinal injuries are also examples of clinical situations in which a large volume of IV fluid resuscitation puts these patients at risk for intra-abdominal hypertension. Manifestations of ACS typically include progressive abdominal distention followed by increased peak airway ventilator pressures, oliguria followed by anuria, and an insidious development of intracranial hyperten-sion.110 These findings are related to elevation of the diaphragm and inadequate venous return from the vena cava or renal veins secondary to the transmitted pressure on the venous system.Measurement of abdominal pressures is easily accom-plished by transducing bladder

1	inadequate venous return from the vena cava or renal veins secondary to the transmitted pressure on the venous system.Measurement of abdominal pressures is easily accom-plished by transducing bladder pressures from the urinary catheter after instilling 100 mL of sterile saline into the urinary bladder.111 A pressure greater than 20 mmHg constitutes intra-abdominal hypertension, but the diagnosis of ACS requires intra-abdominal pressure greater than 25 to 30 mmHg, with at least one of the following: compromised respiratory mechan-ics and ventilation, oliguria or anuria, or increasing intracranial pressures.112-114The treatment of ACS is to open any recent abdominal incision to release the abdominal fascia or to open the fascia directly if no abdominal incision is present. Immediate improve-ment in mechanical ventilation pressures, intracranial pressures, and urine output is usually noted. When expectant management for ACS is considered in the OR, the abdominal fascia should be left

1	in mechanical ventilation pressures, intracranial pressures, and urine output is usually noted. When expectant management for ACS is considered in the OR, the abdominal fascia should be left open and covered under sterile conditions (e.g., a vac-uum-assisted open abdominal wound closure system) with plans made for a second-look operation and delayed fascial closure. Patients with intra-abdominal hypertension should be monitored closely with repeated examinations and measurements of blad-der pressure, so that any further deterioration is detected and operative management can be initiated. Left untreated, ACS may lead to multiple system end-organ dysfunction or failure and has a high mortality.Abdominal wall closure should be attempted every 48 to 72 hours until the fascia can be reapproximated. If the abdo-men cannot be closed within 5 to 7 days following release of the abdominal fascia, a large incisional hernia is the net result. A variety of surgical options have evolved for

1	If the abdo-men cannot be closed within 5 to 7 days following release of the abdominal fascia, a large incisional hernia is the net result. A variety of surgical options have evolved for prevention and closure of the resultant hernias, but no standard approach has yet evolved.Wounds, Drains, and InfectionWound (Surgical Site) Infection. No prospective, random-ized, double-blind, controlled studies exist that demonstrate antibiotics used beyond 24 hours in the perioperative period prevent infections. Prophylactic use of antibiotics should sim-ply not be continued beyond this time. Irrigation of the operative field and the surgical wound with saline solution has shown benefit in controlling wound inoculum.115 Irrigation with an antibiotic-based solution has not demonstrated significant ben-efit in controlling postoperative infection.Antibacterial-impregnated polyvinyl placed over the oper-ative wound area for the duration of the surgical procedure has not been shown to decrease the rate

1	in controlling postoperative infection.Antibacterial-impregnated polyvinyl placed over the oper-ative wound area for the duration of the surgical procedure has not been shown to decrease the rate of wound infection.116-120 Although skin preparation with 70% isopropyl alcohol has the best bactericidal effect, it is flammable and could be hazard-ous when electrocautery is used. The contemporary formulas of chlorhexidine gluconate with isopropyl alcohol remain more advantageous.121-123There is a difference between wound colonization and infection. Overtreating colonization is just as injurious as under-treating infection. The strict definition of wound (soft tissue) infection is more than 105 CFU per gram of tissue. This warrants expeditious and proper antibiotic/antifungal treatment.124 Often, however, clinical signs raise enough suspicion that the patient is treated before a confirmatory culture is undertaken. The clinical signs of wound infection include rubor, tumor, calor, and dolor

1	however, clinical signs raise enough suspicion that the patient is treated before a confirmatory culture is undertaken. The clinical signs of wound infection include rubor, tumor, calor, and dolor (redness, swelling, heat, and pain). Once the diagnosis of wound infection has been established, the most definitive treatment remains open drainage of the wound. The use of antibiotics for wound infection treatment should be limited.125-128One type of wound dressing/drainage system that has gained popularity is the vacuum-assisted closure dressing. The principle of the system is to decrease local wound edema and to promote healing through the application of a sterile dressing that is then covered and placed under controlled suction for a period of 2 to 4 days at a time. Although costly, the benefits are frequently dramatic and may offset the costs of nursing care, frequent dressing changes, and operative wound debridement.Drain Management. The four indications for applying a surgi-cal drain

1	are frequently dramatic and may offset the costs of nursing care, frequent dressing changes, and operative wound debridement.Drain Management. The four indications for applying a surgi-cal drain are:• To collapse surgical dead space in areas of redundant tissue (e.g., neck and axilla)• To provide focused drainage of an abscess or grossly infected surgical site• To provide early warning notice of a surgical leak (either bowel contents, secretions, urine, air, or blood)—the so-called sentinel drain• To control an established fistula leakOpen drains are often used for large contaminated wounds such as perirectal or perianal fistulas and subcutaneous abscess cavities. They prevent premature closure of an abscess cavity in a contaminated wound. More commonly, surgical sites are drained by closed suction drainage systems, but data do not sup-port closed suction drainage to “protect an anastomosis” or to “control a leak” when placed at the time of surgery. Closed suc-tion devices can exert a

1	suction drainage systems, but data do not sup-port closed suction drainage to “protect an anastomosis” or to “control a leak” when placed at the time of surgery. Closed suc-tion devices can exert a negative pressure of 70 to 170 mmHg at the level of the drain; therefore, the presence of this excess suction may call into question whether an anastomosis breaks down on its own or whether the drain creates a suction injury that promotes leakage (Fig. 12-9).129On the other hand, CTor ultrasound-guided placement of percutaneous drains is now the standard of care for abscesses, loculated infections, and other isolated fluid collections such Brunicardi_Ch12_p0397-p0432.indd 42420/02/19 3:57 PM 425QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12ABFigure 12-9 This illustration demonstrates typical intraoperative placement of closed suction devices in pancreatic or small bowel surgery, where there may be an anastomosis. At negative pressures of 70 to 170 mmHg, these

1	demonstrates typical intraoperative placement of closed suction devices in pancreatic or small bowel surgery, where there may be an anastomosis. At negative pressures of 70 to 170 mmHg, these devices may actually encourage anas-tomotic leaks and not prevent them or become clogged by them.as pancreatic leaks. The risk of surgery is far greater than the placement of an image-guided drain.The use of antibiotics when drains are in place is often unnecessary as the drain provides direct source control. Twenty-four to 48 hours of antibiotic use after drain placement is pro-phylactic, and after this period, only specific treatment of positive cultures should be performed to avoid increased drug resistance and superinfection.Urinary Catheters. Several complications of urinary cath-eters can occur that lead to an increased length of hospital stay and morbidity. In general, use of urinary catheters should be minimized and every opportunity to expeditiously remove them should be encouraged. If

1	that lead to an increased length of hospital stay and morbidity. In general, use of urinary catheters should be minimized and every opportunity to expeditiously remove them should be encouraged. If needed, it is recommended that the catheter be inserted its full length up to the hub and that urine flow is established before the balloon is inflated because mis-placement of the catheter in the urethra with premature inflation of the balloon can lead to tears and disruption of the urethra.Enlarged prostatic tissue can make catheter insertion dif-ficult, and a catheter coudé may be required. If this attempt is also unsuccessful, then a urologic consultation for endoscopic placement of the catheter may be required to prevent harm to the urethra. For patients with urethral strictures, filiform-tipped catheters and followers may be used, but these can potentially cause bladder injury. If endoscopic attempts fail, the patient may require a percutaneously placed suprapubic catheter to obtain

1	catheters and followers may be used, but these can potentially cause bladder injury. If endoscopic attempts fail, the patient may require a percutaneously placed suprapubic catheter to obtain decompression of the bladder. Follow-up investigations of these patients are recommended so definitive care of the ure-thral abnormalities can be pursued.The most frequent nosocomial infection is urinary tract infection (UTI). These infections are classified into compli-cated and uncomplicated forms. The uncomplicated type is a UTI that can be treated with outpatient antibiotic therapy. The complicated UTI usually involves a hospitalized patient with an indwelling catheter whose UTI is diagnosed as part of a fever workup. The interpretation of urine culture results of less than 100,000 CFU/mL is controversial. Before treating such a patient, one should change the catheter and then repeat the cul-ture to see if the catheter was simply colonized with organisms. Cultures with more than 100,000

1	controversial. Before treating such a patient, one should change the catheter and then repeat the cul-ture to see if the catheter was simply colonized with organisms. Cultures with more than 100,000 CFU/mL should be treated with the appropriate antibiotics and the catheter changed or removed as soon as possible. Undertreatment or misdiagnosis of a UTI can lead to urosepsis and septic shock.Recommendations are mixed on the proper way to treat Candida albicans fungal bladder infections. Continuous blad-der washings with fungicidal solution for 72 hours have been recommended, but this is not always effective. Replacement of the urinary catheter and a course of fluconazole are appropriate treatments, but some infectious disease specialists claim that C. albicans in the urine may serve as an indication of fungal infection elsewhere in the body. If this is the case, then screen-ing cultures for other sources of fungal infection should be per-formed whenever a fungal UTI is

1	may serve as an indication of fungal infection elsewhere in the body. If this is the case, then screen-ing cultures for other sources of fungal infection should be per-formed whenever a fungal UTI is found.Empyema. One of the most debilitating infections is an empyema, or infection of the pleural space. Frequently, an overwhelming pneumonia is the source of an empyema, but a retained hemothorax, systemic sepsis, esophageal perforation from any cause, and infections with a predilection for the lung (e.g., tuberculosis) are potential etiologies as well. The diag-nosis is confirmed by chest X-ray or CT scan, followed by aspiration of pleural fluid for bacteriologic analysis. Gram’s stain, lactate dehydrogenase, protein, pH, and cell count are obtained, and broad-spectrum antibiotics are initiated while the laboratory studies are performed. Once the specific organisms are confirmed, anti-infective agents are tailored appropriately. Placement of a thoracostomy tube is needed to evacuate

1	while the laboratory studies are performed. Once the specific organisms are confirmed, anti-infective agents are tailored appropriately. Placement of a thoracostomy tube is needed to evacuate and drain the infected pleural fluid, but depending on the specific nidus of infection, video-assisted thoracoscopy may also be Brunicardi_Ch12_p0397-p0432.indd 42520/02/19 3:57 PM 426BASIC CONSIDERATIONSPART Ihelpful for irrigation and drainage of the infection. Refractory empyemas require specialized surgical approaches.Abdominal Abscesses. Postsurgical intra-abdominal abscesses can present with vague complaints of intermittent abdominal pain, fever, leukocytosis, and a change in bowel habits. Depending on the type and timing of the original pro-cedure, the clinical assessment of these complaints is some-times difficult, and a CT scan is usually required. When a fluid collection within the peritoneal cavity is found on CT scan, antibiotics and percutaneous drainage of the collection is the

1	is some-times difficult, and a CT scan is usually required. When a fluid collection within the peritoneal cavity is found on CT scan, antibiotics and percutaneous drainage of the collection is the treatment of choice. Initial antibiotic treatment is usually with broad-spectrum antibiotics such as piperacillin-tazobactam or imipenem. Should the patient exhibit signs of peritonitis and/or have free air on X-ray or CT scan, then re-exploration should be considered.For patients who present primarily (i.e., not postopera-tively) with the clinical and radiologic findings of an abscess but are clinically stable, the etiology of the abscess must be determined. A plan for drainage of the abscess and decisions about further diagnostic studies with consideration of the tim-ing of any definitive surgery all need to be balanced. This can be a complex set of decisions, depending on the etiology (e.g., appendicitis or diverticulitis), but if the patient exhibits signs of peritonitis, urgent surgical

1	all need to be balanced. This can be a complex set of decisions, depending on the etiology (e.g., appendicitis or diverticulitis), but if the patient exhibits signs of peritonitis, urgent surgical exploration should be performed.Necrotizing Fasciitis. Postoperative infections that progress to the fulminant soft tissue infection known as necrotizing fas-ciitis are uncommon. Group A streptococcal (M types 1, 3, 12, and 28) soft tissue infections, as well as infections with Clos-tridium perfringens and C. septicum, carry a mortality of 30% to 70%. Septic shock can be present, and patients can become hypotensive less than 6 hours following inoculation. Manifesta-tions of a group A Streptococcus pyogenes infection in its most severe form include hypotension, renal insufficiency, coagu-lopathy, hepatic insufficiency, ARDS, tissue necrosis, and ery-thematous rash.These findings constitute a surgical emergency, and the mainstay of treatment remains wide debridement of the necrotic tissue to

1	hepatic insufficiency, ARDS, tissue necrosis, and ery-thematous rash.These findings constitute a surgical emergency, and the mainstay of treatment remains wide debridement of the necrotic tissue to the level of bleeding, viable tissue. A gray serous fluid at the level of the necrotic tissue is usually noted, and as the infection spreads, thrombosed blood vessels are noted along the tissue planes involved with the infection. Typically, the patient requires serial trips to the OR for wide debridement until the infection is under control. Antibiotics are an important adjunct to surgical debridement, and broad-spectrum coverage should be used because these infections may be polymicrobial (i.e., so-called mixed-synergistic infections). Streptococcus pyogenes is eradicated with penicillin, and it should still be used as the initial drug of choice.Systemic Inflammatory Response Syndrome, Sepsis, and Multiple-Organ Dysfunction Syndrome. The systemic inflammatory response syndrome (SIRS) and

1	it should still be used as the initial drug of choice.Systemic Inflammatory Response Syndrome, Sepsis, and Multiple-Organ Dysfunction Syndrome. The systemic inflammatory response syndrome (SIRS) and the multiple-organ dysfunction syndrome (MODS) carry significant mortal-ity risks (Table 12-17). Specific criteria have been established for the diagnosis of SIRS (Table 12-18), but two criteria are not required for the diagnosis of SIRS: lowered blood pressure and blood cultures positive for infection. SIRS is the result of proin-flammatory cytokines related to tissue malperfusion or injury. The dominant cytokines implicated in this process include interleukin (IL)-1, IL-6, and tissue necrosis factor (TNF). Other mediators include nitric oxide, inducible macrophage-type nitric oxide synthase, and prostaglandin I2.Table 12-17Mortality associated with patients exhibiting two or more criteria for systemic inflammatory response syndrome (SIRS)PROGNOSISMORTALITY (%)2 SIRS criteria53 SIRS

1	and prostaglandin I2.Table 12-17Mortality associated with patients exhibiting two or more criteria for systemic inflammatory response syndrome (SIRS)PROGNOSISMORTALITY (%)2 SIRS criteria53 SIRS criteria104 SIRS criteria15–20Table 12-18Inclusion criteria for the systemic inflammatory response syndromeTemperature >38°C or <36°C (>100.4°F or <96.8°F)Heart rate >90 beats/minRespiratory rate >20 breaths/min or Paco2 <32 mmHgWhite blood cell count <4000 or >12,000 cells/mm3 or >10% immature formsPaco2 = partial pressure of arterial carbon dioxide.Sepsis is categorized as sepsis, severe sepsis, and septic shock. Sepsis is SIRS plus infection. Severe sepsis is sepsis plus signs of cellular hypoperfusion or end-organ dysfunction. Septic shock is sepsis plus hypotension after adequate fluid resuscitation.MODS is the culmination of septic shock and multiple end-organ failure.130 Usually there is an inciting event (e.g., perforated sigmoid diverticulitis), and as the patient undergoes

1	resuscitation.MODS is the culmination of septic shock and multiple end-organ failure.130 Usually there is an inciting event (e.g., perforated sigmoid diverticulitis), and as the patient undergoes resuscitation, he or she develops cardiac hypokinesis and oli-guric or anuric renal failure, followed by the development of ARDS and eventually septic shock with death.The international Surviving Sepsis Campaign (www.sccm.org/Documents/SSC-Guidelines.pdf) continues to dem-onstrate the importance of early recognition and initiation of specific treatment guidelines for optimal management of sep-sis. Management of SIRS/MODS includes aggressive global resuscitation and support of end-organ perfusion, correction of the inciting etiology, control of infectious complications, and management of iatrogenic complications.131-133 Drotrecogin-α, or recombinant activated protein C, appears to specifically counteract the cytokine cascade of SIRS/MODS, but its use is still limited.134,135 Other adjuncts for

1	complications.131-133 Drotrecogin-α, or recombinant activated protein C, appears to specifically counteract the cytokine cascade of SIRS/MODS, but its use is still limited.134,135 Other adjuncts for supportive therapy include tight glucose control, low tidal volumes in ARDS, vasopressin in septic shock, and steroid replacement therapy.Nutritional and Metabolic Support ComplicationsNutrition-Related Complications. A basic principle is to use enteral feeding whenever possible, but complications can inter-vene such as aspiration, ileus, and to a lesser extent, sinusitis. There is no difference in aspiration rates when a small-caliber feeding tube is placed postpyloric or if it remains in the stom-ach. Patients who are fed via nasogastric tubes are at risk for aspiration pneumonia because these large-bore tubes stent open the gastroesophageal junction, creating the possibility of gas-tric reflux. The use of enteric and gastric feeding tubes obviates Brunicardi_Ch12_p0397-p0432.indd

1	these large-bore tubes stent open the gastroesophageal junction, creating the possibility of gas-tric reflux. The use of enteric and gastric feeding tubes obviates Brunicardi_Ch12_p0397-p0432.indd 42620/02/19 3:57 PM 427QUALITY, PATIENT SAFETY, ASSESSMENTS OF CARE, AND COMPLICATIONSCHAPTER 12complications of TPN, such as pneumothorax, line sepsis, upper extremity DVT, and the related expense. There is growing evi-dence to support the initiation of enteral feeding in the early postoperative period, before the return of bowel function, where it is usually well tolerated.In patients who have had any type of nasal intubation who are having high, unexplained fevers, sinusitis must be enter-tained as a diagnosis. CT scan of the sinuses is warranted, fol-lowed by aspiration of sinus contents so the organism(s) are appropriately treated.Patients who have not been enterally fed for prolonged periods secondary to multiple operations, those who have had enteral feeds interrupted for any

1	so the organism(s) are appropriately treated.Patients who have not been enterally fed for prolonged periods secondary to multiple operations, those who have had enteral feeds interrupted for any other reason, or those with poor enteral access are at risk for the refeeding syndrome, which is characterized by severe hypophosphatemia and respiratory fail-ure. Slow progression of the enteral feeding administration rate can avoid this complication.Common TPN problems are mostly related to electrolyte abnormalities that may develop. These electrolyte errors include deficits or excesses in sodium, potassium, calcium, magnesium, and phosphate. Acid-base abnormalities can also occur with the improper administration of acetate or bicarbonate solutions.The most common cause for hypernatremia in hospitalized patients is under-resuscitation, and, conversely, hyponatremia is most often caused by fluid overload. Treatment for hyponatre-mia is fluid restriction in mild or moderate cases and the

1	hospitalized patients is under-resuscitation, and, conversely, hyponatremia is most often caused by fluid overload. Treatment for hyponatre-mia is fluid restriction in mild or moderate cases and the admin-istration of hypertonic saline for severe cases. An overly rapid correction of the sodium abnormality may result in central pon-tine myelinolysis, which results in a severe neurologic deficit. Treatment for hyponatremic patients includes fluid restriction to correct the free water deficit by 50% in the first 24 hours. An overcorrection of hyponatremia can result in severe cerebral edema, a neurologic deficit, or seizures.Glycemic Control. In 2001, Van den Berghe and colleagues demonstrated that tight glycemic control by insulin infusion is associated with a 50% reduction in mortality in the critical care setting.136 This prospective, randomized, controlled trial of 1500 patients had two study arms: the intensive-control arm, where the serum glucose was maintained between 80 and 110

1	the critical care setting.136 This prospective, randomized, controlled trial of 1500 patients had two study arms: the intensive-control arm, where the serum glucose was maintained between 80 and 110 mg/dL with insulin infusion; and the control arm, where patients received an insulin infusion only if blood glucose was greater than 215 mg/dL, but serum glucose was then maintained at 180 to 200 mg/dL.The tight glycemic control group had an average serum glucose level of 103 mg/dL, and the average glucose level in the control group was 153 mg/dL. Hypoglycemic episodes (glu-cose <40 mg/dL) occurred in 39 patients in the tightly controlled group, while the control group had episodes in six patients. The overall mortality was reduced from 8% to 4.6%, but the mortal-ity of those patients whose ICU stay lasted longer than 5 days was reduced from 20% to 10%. Secondary findings included an improvement in overall morbidity, a decreased percentage of ventilator days, less renal impairment, and a

1	stay lasted longer than 5 days was reduced from 20% to 10%. Secondary findings included an improvement in overall morbidity, a decreased percentage of ventilator days, less renal impairment, and a lower incidence of bloodstream infections. These finding have been corrobo-rated by subsequent similar studies, and the principal benefit appears to be a greatly reduced incidence of nosocomial infec-tions and sepsis. It is not known whether the benefits are due to strict euglycemia, to the anabolic properties of insulin, or both, but the maintenance of strict euglycemia between 140 and 180 mg/dL appears to be a powerful therapeutic strategy.136-138 A number of studies followed this sentinel publication of tight glycemic control. The NICE-SUGAR139 and COIITSS140 trials revisited the Van den Berghe study and found that the glyce-mic goals found initially to improve outcomes in critically ill patients were now found to be associated with a higher mortality when glucose was kept below 180

1	Berghe study and found that the glyce-mic goals found initially to improve outcomes in critically ill patients were now found to be associated with a higher mortality when glucose was kept below 180 mg/dL, due to an increase in incidents of hypoglycemia. When targeted goals of 180 mg/dL are achieved, fewer occurrences of hypoglycemia have been docu-mented, and improved survivorship has been achieved. In addi-tion, some studies find no relationship between glycemic control and improved outcomes. Thus, glycemic control in the critically ill still remains unclear and elusive at best.141,142 Part of the dif-ficulty in achieving “tight glycemic control” is the necessity for frequent (every 1–2 hours) blood glucose determinations. When this is performed, glycemic control is enhanced and hypoglyce-mia is avoided.Metabolism-Related Complications. “Stress dose steroids” have been advocated for the perioperative treatment of patients on corticosteroid therapy, but recent studies strongly

1	is avoided.Metabolism-Related Complications. “Stress dose steroids” have been advocated for the perioperative treatment of patients on corticosteroid therapy, but recent studies strongly discour-age the use of supraphysiologic doses of steroids when patients are on low or maintenance doses (e.g., 5–15 mg) of prednisone daily. Parenteral glucocorticoid treatment need only replicate physiologic replacement steroids in the perioperative period. When patients are on steroid replacement doses equal to or greater than 20 mg per day of prednisone, it may be appropriate to administer additional glucocorticoid doses for no more than 2 perioperative days.143-145Adrenal insufficiency may be present in patients with a baseline serum cortisol less than 20 μg/dL. A rapid provocative test with synthetic adrenocorticotropic hormone may confirm the diagnosis. After a baseline serum cortisol level is drawn, 250 μg of cosyntropin is administered. At exactly 30 and 60 minutes follow-ing the dose of

1	adrenocorticotropic hormone may confirm the diagnosis. After a baseline serum cortisol level is drawn, 250 μg of cosyntropin is administered. At exactly 30 and 60 minutes follow-ing the dose of cosyntropin, serum cortisol levels are obtained. There should be an incremental increase in the cortisol level of between 7 and 10 μg/dL for each half hour. If the patient is below these levels, a diagnosis of adrenal insufficiency is made, and glucocorticoid and mineralocorticoid administration is then warranted. Mixed results are common, but the complication of performing major surgery on an adrenally insufficient patient is sudden or profound hypotension that is not responsive to fluid resuscitation.131Thyroid hormone abnormalities usually consist of previ-ously undiagnosed thyroid abnormalities. Hypothyroidism and the so-called sick-euthyroid syndrome are more commonly recognized in the critical care setting. When surgical patients are not progressing satisfactorily in the perioperative

1	Hypothyroidism and the so-called sick-euthyroid syndrome are more commonly recognized in the critical care setting. When surgical patients are not progressing satisfactorily in the perioperative period, screening for thyroid abnormalities should be performed. If the results show mild to moderate hypothyroidism, then thyroid replacement should begin immediately, and thyroid function studies should be monitored closely. All patients should be reas-sessed after the acute illness has subsided regarding the need for chronic thyroid replacement therapy.Problems with ThermoregulationHypothermia. Hypothermia is defined as a core tempera-ture less than 35°C (95°F) and is divided into subsets of mild (35°C–32°C [95°F–89.6°F]), moderate (32°C–28°C [89.6°F –82.4°F]), and severe (<28°C [<82.4°F]) hypothermia. Shiver-ing, the body’s attempt to reverse the effects of hypothermia, occurs between 37°C and 31°C (98.6°F and 87.8°F), but ceases at temperatures below 31°C (87.8°F). Patients who are

1	hypothermia. Shiver-ing, the body’s attempt to reverse the effects of hypothermia, occurs between 37°C and 31°C (98.6°F and 87.8°F), but ceases at temperatures below 31°C (87.8°F). Patients who are moder-ately hypothermic are at higher risk for complications than are those who are more profoundly hypothermic.Brunicardi_Ch12_p0397-p0432.indd 42720/02/19 3:57 PM 428BASIC CONSIDERATIONSPART IHypothermia creates a coagulopathy that is related to platelet and clotting cascade enzyme dysfunction. This triad of metabolic acidosis, coagulopathy, and hypothermia is com-monly found in long operative cases and in patients with blood dyscrasias. The enzymes that contribute to the clotting cascade and platelet activity are most efficient at normal body tempera-tures; therefore, all measures must be used to reduce heat loss intraoperatively.146The most common cardiac abnormality is the develop-ment of arrhythmias when body temperature drops below 35°C (95°F). Bradycardia occurs with

1	must be used to reduce heat loss intraoperatively.146The most common cardiac abnormality is the develop-ment of arrhythmias when body temperature drops below 35°C (95°F). Bradycardia occurs with temperatures below 30°C (86°F). It is well known that hypothermia may induce CO2 retention, resulting in respiratory acidosis. Renal dysfunction of hypothermia manifests itself as a paradoxic polyuria and is related to an increased glomerular filtration rate, as peripheral vascular constriction creates central shunting of blood. This is potentially perplexing in patients who are undergoing resuscita-tion for hemodynamic instability because the brisk urine output provides a false sense of an adequate intravascular fluid volume.Induced peripheral hypothermia for hyperpyrexia due to infection (not to include neurologic or cardiac disease) is likely deleterious and does not appear to be beneficial. Plac-ing cooling blankets on or under the patient or ice packs in the axillae or groin may be

1	to include neurologic or cardiac disease) is likely deleterious and does not appear to be beneficial. Plac-ing cooling blankets on or under the patient or ice packs in the axillae or groin may be effective in cooling the skin, and when this occurs, a subsequent feedback loop triggers the hypothalamus to raise the internally regulated set point, thus raising core temperature even higher. This paradoxical reac-tion may be why those who feel the need to treat a fever in the ICU by cooling the skin and arguably the core have worse outcomes. Cooling core temperatures can be achieved reli-ably with catheter-directed therapy with commercially avail-able devices. Whether this is a worthwhile practice or not may be controversial. Poor data exist in support of treating fevers lower than 42°C in any fashion.147-149Adult trauma patients who underwent induced hypother-mia had poor outcomes in a recent investigation, and thus, this remains a procedure to be avoided. In a similar vein, pediatric

1	any fashion.147-149Adult trauma patients who underwent induced hypother-mia had poor outcomes in a recent investigation, and thus, this remains a procedure to be avoided. In a similar vein, pediatric patients who were induced did not show any improvement, and therefore, induced hypothermia is not recommended. Compli-cations with induced hypothermia include, but are not limited to, hypokalemia, diuresis, DVT (due to catheter-related vein injury), arrhythmias, shivering, undiagnosed catheter-related bloodstream infection, and bacteremia.150-152Neurologic dysfunction is inconsistent in hypothermia, but a deterioration in reasoning and decision-making skills progresses as body temperature falls, and profound coma (and a flat electroencephalogram) occurs as the temperature drops below 30°C (86°F). The diagnosis of hypothermia is important, so accurate measurement techniques are required to get a true core temperature.Methods used to warm patients include warm air circu-lation over the

1	(86°F). The diagnosis of hypothermia is important, so accurate measurement techniques are required to get a true core temperature.Methods used to warm patients include warm air circu-lation over the patient and heated IV fluids, as well as more aggressive measures such as bilateral chest tubes with warm solution lavage, intraperitoneal rewarming lavage, and extra-corporeal membrane oxygenation. A rate of temperature rise of 2°C to 4°C (3.6°F–7.2°F) per hour is considered adequate, but the most common complication for nonbypass rewarming is arrhythmia with ventricular arrest.Hyperthermia. Hyperthermia is defined as a core temperature greater than 38.6°C (101.5°F) and has a host of etiologies (Table 12-19).147 Hyperthermia can be environmentally induced (e.g., summer heat with inability to dissipate heat or control exposure), iatrogenically induced (e.g., heat lamps and medica-tions), endocrine in origin (e.g., thyrotoxicosis), or neurologi-cally induced (i.e., hypothalamic

1	to dissipate heat or control exposure), iatrogenically induced (e.g., heat lamps and medica-tions), endocrine in origin (e.g., thyrotoxicosis), or neurologi-cally induced (i.e., hypothalamic dysfunction).Malignant hyperthermia occurs intraoperatively after exposure to agents such as succinylcholine and some halothane-based inhalational anesthetics. The presentation is dramatic, with rapid onset of increased temperature, rigors, and myoglo-binuria related to myonecrosis. Medications must be discontin-ued immediately and dantrolene administered (2.5 mg/kg every 5 minutes) until symptoms subside. Aggressive cooling meth-ods are also implemented, such as an alcohol bath, or packing in ice. In cases of severe malignant hyperthermia, the mortality rate is nearly 30%.153,154Thyrotoxicosis can occur after surgery due to undiagnosed Graves’ disease. Hallmarks of the syndrome include hyperther-mia (>40°C [104°F]), anxiety, copious diaphoresis, congestive heart failure (present in about one

1	occur after surgery due to undiagnosed Graves’ disease. Hallmarks of the syndrome include hyperther-mia (>40°C [104°F]), anxiety, copious diaphoresis, congestive heart failure (present in about one fourth of episodes), tachycar-dia (most commonly atrial fibrillation), and hypokalemia (in up to 50% of patients). The treatment of thyrotoxicosis includes glucocorticoids, propylthiouracil, β-blockade, and iodide (Lugol’s solution) delivered in an emergent fashion. As the name suggests, these patients are usually toxic and require sup-portive measures as well. Acetaminophen, the cooling modali-ties noted in the previous paragraph, and vasoactive agents often are indicated.REFERENCESEntries highlighted in bright blue are key references. 1. Makary MA, Daniel M. Medical error—the third lead-ing cause of death in the US. Brit Med J. 2016;353:i2139. doi:10.1136/bmj.i2139 2. Kohn KT, Corrigan JM, Donaldson MS. To Err Is Human: Building a Safer Health System. Washington, DC: National Academy

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1	Evans HL, Chong TW, et al. Effect of an inten-sive care unit rotating empiric antibiotic schedule on the devel-opment of hospital-acquired infections on the non-intensive care unit ward. Crit Care Med. 2004;32(1):53-60. 71. Horn SD, Wright HL, Couperus JJ, et al. Association between patient-controlled analgesia pump use and postoperative sur-gical site infection in intestinal surgery patients. Surg Infect (Larchmt). 2002;3(2):109-118. 72. Ferguson ND, Fan E, Camporota L, et al. The Berlin definition of ARDS: an expanded rationale, justification, and supplemen-tary material. Intensive Care Med. 2012;38(10):1573-1582. 73. Villar J, Kacmarek RM. The American-European Consensus Conference definition of the acute respiratory distress syn-drome is dead, long live positive end-expiratory pressure! Med Intensiva. 2012;36(8):571-575. 74. ARDS Definition Task Force, Ranieri VM, Rubenfeld GD, et al. Acute respiratory distress syndrome: the Berlin Defini-tion. JAMA.

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1	RR, Porter JM, Simon RJ, et al. Intra-abdominal hypertension after life-threatening penetrating abdominal trauma: prophylaxis, incidence, and clinical relevance to gastric mucosal pH and abdominal compartment syndrome. J Trauma. 1998;44(6):1016-1021. 113. Ivatury RR, Sugerman HJ, Peitzman AB. Abdominal com-partment syndrome: recognition and management. Adv Surg. 2001;35:251-269. 114. Saggi BH, Sugerman HJ, Ivatury RR, Bloomfield GL. Abdom-inal compartment syndrome. J Trauma. 1998;45(3):597-609. 115. Anglen J, Apostoles PS, Christensen G, Gainor B, Lane J. Removal of surface bacteria by irrigation. J Orthop Res. 1966;14:251-254. 116. Lewis DA, Leaper DJ, Speller DC. Prevention of bacterial colonization of wounds at operation: comparison of iodine-impregnated (“Ioban”) drapes with conventional methods. J Hosp Infect. 1984;5:431-437. 117. O’Rourke E, Runyan D, O’Leary J, Stern J. Contami-nated iodophor in the operating room. Am J Infect Control. 2003;31:255-256. 118. Ostrander RV,

1	methods. J Hosp Infect. 1984;5:431-437. 117. O’Rourke E, Runyan D, O’Leary J, Stern J. Contami-nated iodophor in the operating room. Am J Infect Control. 2003;31:255-256. 118. Ostrander RV, Brage ME, Botte MJ. Bacterial skin contamina-tion after surgical preparation in foot and ankle surgery. Clin Orthop. 2003;406:246-252. 119. Ghogawala Z, Furtado D. In vitro and in vivo bactericidal activities of 10%, 2.5%, and 1% povidone-iodine solution. Am J Hosp Pharm. 1990;47:1562-1566. 120. Anderson RL, Vess RW, Carr JH, Bond WW, Panlilio AL, Favero MS. Investigations of intrinsic Pseudomonas cepa-cia contamination in commercially manufactured povidone-iodine. Infect Control Hosp Epidemiol. 1991;12(5):297-302. 121. Birnbach DJ, Meadows W, Stein DJ, Murray O, Thys DM, Sor-dillo EM. Comparison of povidone iodine and DuraPrep, an iodophor-in-isopropyl alcohol solution, for skin disinfection prior to epidural catheter insertion in parturients. Anesthesiol-ogy. 2003;98(1):164-169. 122. Moen MD,

1	povidone iodine and DuraPrep, an iodophor-in-isopropyl alcohol solution, for skin disinfection prior to epidural catheter insertion in parturients. Anesthesiol-ogy. 2003;98(1):164-169. 122. Moen MD, Noone MG, Kirson I. Povidone-iodine spray technique versus traditional scrub-paint technique for pre-operative abdominal wall preparation. Am J Obstet Gynecol. 2002;187(6):1434-1436; discussion 1436-1437. 123. Strand CL, Wajsbort RR, Sturmann K. Effect of iodophor vs iodine tincture skin preparation on blood culture contamina-tion rate. JAMA. 1993;269(8):1004-1006. 124. Paterson DL, Ko WC, Von Gottberg A, et al. International pro-spective study of Klebsiella pneumoniae bacteremia: impli-cations of extended-spectrum beta-lactamase production in nosocomial infections. Ann Intern Med. 2004;140(1):26-32. 125. Wittmann DH, Schein M. Let us shorten antibiotic prophylaxis and therapy in surgery. Am J Surg. 1966;172(6A):26S-32S. 126. Dellinger EP. Duration of antibiotic treatment in surgical

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1	DA, Duggan J, Brady HR. Renal-dose (low-dose) dopa-mine for the treatment of sepsis-related and other forms of acute renal failure: ineffective and probably dangerous. Clin Exp Pharmacol Physiol Suppl. 1999;26:S23-S28. 131. Vincent JL, Abraham E, Annane D, Bernard G, Rivers E, Van de Berghe G. Reducing mortality in sepsis: new directions. Crit Care. 2002;6(suppl 3):S1-S18. 132. Malay MB, Ashton RC Jr, Landry DW, Townsend RN. Low-dose vasopressin in the treatment of vasodilatory septic shock. J Trauma. 1999;47:699-703; discussion 703-705. 133. Annane D, Sebille V, Charpentier C, et al. Effect of treat-ment with low doses of hydrocortisone and fludrocorti-sone on mortality in patients with septic shock. JAMA. 2002;288(7):862-871. 134. Dhainaut JF, Laterre PF, LaRosa SP, et al. The clinical evaluation committee in a large multicenter phase 3 trial of drotrecogin alfa (activated) in patients with severe sepsis (PROWESS): role, methodology, and results. Crit Care Med. 2003;31(9):2291-2301;

1	committee in a large multicenter phase 3 trial of drotrecogin alfa (activated) in patients with severe sepsis (PROWESS): role, methodology, and results. Crit Care Med. 2003;31(9):2291-2301; comment 2405. 135. Betancourt M, McKinnon PS, Massanari RM, Kanji S, Bach D, Devlin JW. An evaluation of the cost effectiveness of drotrecogin alfa (activated) relative to the number of organ system failures. Pharmacoeconomics. 2003;21:1331-1340. 136. Van den Berghe G, Wouters P, Weekers F, et al. Intensive insulin therapy in critically ill patients. N Engl J Med. 2001;345(19):1359-1367. 137. Finney SJ, Zekveld C, Elia A, Evans TW. Glucose con-trol and mortality in critically ill patients. JAMA. 2003;290(15):2041-2047. 138. Furnary AP, Gao G, Grunkemeier GL, et al. Continuous insu-lin infusion reduces mortality in patients with diabetes under-going coronary artery bypass grafting. J Thorac Cardiovasc Surg. 2003;125(5):1007-1021. 139. NICE-SUGAR Study Investigators, Finfer S, Chittock DR, et al.

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1	Physiologic Monitoring of the Surgical PatientAnthony R. Cyr and Louis H. Alarcon 13chapterINTRODUCTIONThe Latin verb monere, which means “to warn, or advise” is the origin for the English word monitor. In modern medical prac-tice, patients undergo monitoring to detect pathologic varia-tions in physiologic parameters, providing advanced warning of impending deterioration in the status of one or more organ systems. The intended goal of this endeavor is to allow the clini-cian to take appropriate actions in a timely fashion to prevent or ameliorate the physiologic derangement. Furthermore, physi-ologic monitoring is used not only to warn, but also to titrate therapeutic interventions, such as fluid resuscitation or the infu-sion of vasoactive or inotropic drugs. The intensive care unit (ICU) and operating room are the two locations where the most advanced monitoring capabilities are routinely employed in the care of critically ill patients.In the broadest sense, physiologic monitoring

1	and operating room are the two locations where the most advanced monitoring capabilities are routinely employed in the care of critically ill patients.In the broadest sense, physiologic monitoring encom-passes a spectrum of endeavors, ranging in complexity from the routine and intermittent measurement of the classic vital signs (i.e., temperature, heart rate, arterial blood pressure, and respira-tory rate) to the continuous recording of the oxidation state of cytochrome oxidase, the terminal element in the mitochondrial electron transport chain. The ability to assess clinically relevant parameters of tissue and organ status and employ this knowl-edge to improve patient outcomes represents the “holy grail” of critical care medicine. Unfortunately, consensus is often lacking regarding the most appropriate parameters to monitor in order to achieve this goal. Furthermore, making an inappropriate ther-apeutic decision due to inaccurate physiologic data or misinter-pretation of good data

1	most appropriate parameters to monitor in order to achieve this goal. Furthermore, making an inappropriate ther-apeutic decision due to inaccurate physiologic data or misinter-pretation of good data can lead to a worse outcome than having no data at all. Of the highest importance is the integration of physiologic data obtained from monitoring into a coherent and evidenced-based treatment plan. Current technologies available to assist the clinician in this endeavor are summarized in this chapter. Also presented is a brief look at emerging tech-niques that may soon enter into clinical practice.In essence, the goal of hemodynamic monitoring is to ensure that the flow of oxygenated blood through the microcir-culation is sufficient to support aerobic metabolism at the cel-lular level. In general, mammalian cells cannot store oxygen for subsequent use in oxidative metabolism, although a relatively tiny amount is stored in muscle tissue as oxidized myoglobin. Thus, aerobic synthesis of

1	general, mammalian cells cannot store oxygen for subsequent use in oxidative metabolism, although a relatively tiny amount is stored in muscle tissue as oxidized myoglobin. Thus, aerobic synthesis of adenosine triphosphate (ATP), the energy “currency” of cells, requires the continuous delivery of oxygen by diffusion from hemoglobin in red blood cells to the oxidative machinery within mitochondria. Delivery of oxygen to mitochondria may be insufficient for several reasons. For example, cardiac output, hemoglobin concentration of blood, or the oxygen content of arterial blood each can be inadequate 1Introduction 433Arterial Blood Pressure 434Noninvasive Measurement of Arterial Blood Pressure / 434Invasive Monitoring of Arterial Blood Pressure / 435Electrocardiographic Monitoring 435Algorithmic Integrative Monitoring 436Cardiac Output and Related Parameters 436Determinants of Cardiac Performance / 436Placement of the Pulmonary Artery Catheter 437Hemodynamic

1	Monitoring 435Algorithmic Integrative Monitoring 436Cardiac Output and Related Parameters 436Determinants of Cardiac Performance / 436Placement of the Pulmonary Artery Catheter 437Hemodynamic Measurements 438Measurement of Cardiac Output by Thermodilution / 439Mixed Venous Oximetry / 439Effect of Pulmonary Artery Catheterization on Outcome 440Minimally Invasive Alternatives to the Pulmonary Artery Catheter 442Transpulmonary Thermodilution / 442Doppler Ultrasonography / 443Impedance Cardiography / 443Pulse Contour Analysis / 443Partial Carbon Dioxide Rebreathing / 444Transesophageal Echocardiography / 444Assessing Preload Responsiveness / 444Near-Infrared Spectroscopic Measurement of Tissue Hemoglobin Oxygen Saturation / 444Respiratory Monitoring 445Arterial Blood Gases / 445Determinants of Oxygen Delivery / 445Peak and Plateau Airway Pressure / 446Pulse Oximetry / 446Pulse CO-Oximetry / 446Capnometry /447Renal Monitoring 447Urine Output / 447Bladder Pressure /

1	/ 445Determinants of Oxygen Delivery / 445Peak and Plateau Airway Pressure / 446Pulse Oximetry / 446Pulse CO-Oximetry / 446Capnometry /447Renal Monitoring 447Urine Output / 447Bladder Pressure / 447Neurologic Monitoring 447Intracranial Pressure / 447Electroencephalogram and Evoked Potentials / 448Transcranial Doppler Ultrasonography / 448Jugular Venous Oximetry / 448Transcranial Near-Infrared Spectroscopy / 449Brain Tissue Oxygen Tension / 449Conclusions 449Brunicardi_Ch13_p0433-p0452.indd 43322/02/19 2:20 PM 434Figure 13-1. Graphical representation of the relationship between oxygen utilization (VO2) and oxygen delivery (DO2). Under most normal physiologic conditions oxygen utilization does not depend on oxygen delivery, but below the critical value DO2crit oxygen utili-zation decreases linearly as a function of oxygen delivery, rendering tissues susceptible to ischemic injury.Key Points1 The delivery of modern critical care is predicated on the abil-ity to monitor a large

1	decreases linearly as a function of oxygen delivery, rendering tissues susceptible to ischemic injury.Key Points1 The delivery of modern critical care is predicated on the abil-ity to monitor a large number of physiologic variables and formulate evidenced-based therapeutic strategies to manage these variables. Technological advances in monitoring have at least a theoretical risk of exceeding our ability to under-stand the clinical implications of the derived information. This could result in the use of monitoring data to make inap-propriate clinical decisions. Therefore, the implementation of any new monitoring technology must take into account the relevance and accuracy of the data obtained, the risks to the patient, and the evidence supporting any intervention directed at correcting the detected abnormality.2 The routine use of invasive monitoring devices, specifically the pulmonary artery catheter, must be questioned in light of the available evidence that does not demonstrate a

1	detected abnormality.2 The routine use of invasive monitoring devices, specifically the pulmonary artery catheter, must be questioned in light of the available evidence that does not demonstrate a clear ben-efit to its widespread use in various populations of critically ill patients. The future of physiologic monitoring will be dominated by the application of noninvasive and highly accurate devices which guide evidenced-based therapy.for independent reasons. Alternatively, despite adequate cardiac output, perfusion of capillary networks can be impaired as a consequence of dysregulation of arteriolar tone, microvascular thrombosis, or obstruction of nutritive vessels by sequestered leukocytes or platelets. Hemodynamic monitoring that does not take into account all of these factors will portray an incomplete and perhaps misleading picture of cellular physiology.Under normal conditions when the supply of oxygen is plentiful, aerobic metabolism is determined by factors other than the

1	portray an incomplete and perhaps misleading picture of cellular physiology.Under normal conditions when the supply of oxygen is plentiful, aerobic metabolism is determined by factors other than the availability of oxygen. These factors include the hor-monal milieu and mechanical workload of contractile tissues. However, in pathologic circumstances when oxygen availabil-ity is inadequate, oxygen utilization (VO2) becomes dependent upon oxygen delivery (DO2). The relationship of VO2 to DO2 over a broad range of DO2 values is commonly represented as two intersecting straight lines (Fig. 13-1). In the region of higher DO2 values, the slope of the line is approximately equal to zero, indicating that VO2 is largely independent of DO2. In contrast, in the region of low DO2 values, the slope of the line is nonzero and positive, indicating that VO2 is supply-dependent. The region where the two lines intersect is called the point of critical oxy-gen delivery (DO2crit), and represents the

1	of the line is nonzero and positive, indicating that VO2 is supply-dependent. The region where the two lines intersect is called the point of critical oxy-gen delivery (DO2crit), and represents the transition from supplyindependent to supply-dependent oxygen uptake. Below a critical Oxygen delivery, DO2Tissue hypoxiaSupply-dependentoxygen consumptionSupply-independentoxygen consumptionTissue normoxiaOxygen utilization, VO2DO2critthreshold of oxygen delivery, increased oxygen extraction can-not compensate for the delivery deficit; hence, oxygen con-sumption begins to decrease. The slope of the supply-dependent region of the plot reflects the maximal oxygen extraction capa-bility of the vascular bed being evaluated.The subsequent sections will describe the techniques and utility of monitoring various physiologic parameters.ARTERIAL BLOOD PRESSUREThe pressure exerted by blood in the systemic arterial system, commonly referred to simply as “blood pressure,” is a cardinal parameter

1	various physiologic parameters.ARTERIAL BLOOD PRESSUREThe pressure exerted by blood in the systemic arterial system, commonly referred to simply as “blood pressure,” is a cardinal parameter measured as part of the hemodynamic monitoring of patients. Extremes in blood pressure are either intrinsically deleterious or are indicative of a serious perturbation in normal physiology. Arterial blood pressure is a complex function of both cardiac output and vascular input impedance. Thus, inex-perienced clinicians may assume that the presence of a normal blood pressure is evidence that cardiac output and tissue perfu-sion are adequate. This assumption is frequently incorrect and is the reason why some critically ill patients may benefit from forms of hemodynamic monitoring in addition to measurement of arterial pressure.Blood pressure can be determined directly by measuring the pressure within the arterial lumen or indirectly using a cuff around an extremity. When the equipment is properly set

1	of arterial pressure.Blood pressure can be determined directly by measuring the pressure within the arterial lumen or indirectly using a cuff around an extremity. When the equipment is properly set up and calibrated, direct intra-arterial monitoring of blood pressure pro-vides accurate and continuous data. Additionally, intra-arterial catheters provide a convenient way to obtain samples of blood for measurements of arterial blood gases and other laboratory studies. Despite these advantages, intra-arterial catheters are invasive devices and occasionally are associated with serious complications.Noninvasive Measurement of Arterial Blood PressureBoth manual and automated means for the noninvasive determi-nation of blood pressure use an inflatable sphygmomanometer cuff to increase pressure around an extremity and to detect the presence or absence of arterial pulsations. Several methods exist for this purpose. The time-honored approach is the auscultation of the Korotkoff sounds, which are

1	an extremity and to detect the presence or absence of arterial pulsations. Several methods exist for this purpose. The time-honored approach is the auscultation of the Korotkoff sounds, which are heard over an artery distal to the cuff as the cuff is deflated from a pressure higher than systolic pressure to one less than diastolic pressure. Systolic pressure is defined as the pressure in the cuff when tapping sounds are first audible. Diastolic pressure is the pressure in the cuff when audible pulsations first disappear.Brunicardi_Ch13_p0433-p0452.indd 43422/02/19 2:20 PM 435PHYSIOLOGIC MONITORING OF THE SURGICAL PATIENTCHAPTER 13Another means for pulse detection when measuring blood pressure noninvasively depends upon the detection of oscillations in the pressure within the bladder of the cuff. This approach is simple, and unlike auscultation, can be performed even in a noisy environment (e.g., a busy emergency depart-ment or medical helicopter). Unfortunately, this approach is

1	of the cuff. This approach is simple, and unlike auscultation, can be performed even in a noisy environment (e.g., a busy emergency depart-ment or medical helicopter). Unfortunately, this approach is neither accurate nor reliable. Other methods, however, can be used to reliably detect the reappearance of a pulse distal to the cuff and thereby estimate systolic blood pressure. Two excellent and widely available approaches for pulse detection are use of a Doppler stethoscope (reappearance of the pulse produces an audible amplified signal) or a pulse oximeter (reappearance of the pulse is indicated by flashing of a light-emitting diode).A number of automated devices are capable of repeti-tively measuring blood pressure noninvasively. Some of these devices measure pressure oscillations in the inflatable bladder encircling the extremity to detect arterial pulsations as pressure in the cuff is gradually lowered from greater than systolic to less than diastolic pressure. Other automated

1	the inflatable bladder encircling the extremity to detect arterial pulsations as pressure in the cuff is gradually lowered from greater than systolic to less than diastolic pressure. Other automated noninvasive devices use a piezoelectric crystal positioned over the brachial artery as a pulse detector. The accuracy of these devices is variable, and often dependent on the size mismatch between the arm cir-cumference and the cuff size.1 If the cuff is too narrow (relative to the extremity), the measured pressure will be artifactually elevated. Therefore, the width of the cuff should be approxi-mately 40% of its circumference.Another noninvasive approach for measuring blood pres-sure relies on a technique called photoplethysmography. This method is capable of providing continuous information, since systolic and diastolic blood pressures are recorded on a beat-to-beat basis. Photoplethysmography uses the transmission of infrared light to estimate the amount of hemoglobin (directly related

1	since systolic and diastolic blood pressures are recorded on a beat-to-beat basis. Photoplethysmography uses the transmission of infrared light to estimate the amount of hemoglobin (directly related to the volume of blood) in a finger placed under a servo-controlled inflatable cuff. A feedback loop controlled by a microprocessor continually adjusts the pressure in the cuff to maintain the blood volume of the finger constant. Under these conditions, the pressure in the cuff reflects the pressure in the digi-tal artery. The measurements obtained using photoplethysmog-raphy generally agree closely with those obtained by invasive monitoring of blood pressure.2 However, these readings may be less accurate in patients with hypotension or hypothermia.Invasive Monitoring of Arterial Blood PressureDirect and continuous monitoring of arterial pressure in criti-cally ill patients may be performed by using fluid-filled tubing to connect an intra-arterial catheter to an external strain-gauge

1	and continuous monitoring of arterial pressure in criti-cally ill patients may be performed by using fluid-filled tubing to connect an intra-arterial catheter to an external strain-gauge transducer. The signal generated by the transducer is electroni-cally amplified and displayed as a continuous waveform by an oscilloscope or computerized display. Digital values for systolic and diastolic pressure also are displayed. Mean pressure, calcu-lated by electronically averaging the amplitude of the pressure waveform, can also be displayed. The fidelity of the catheter-tubing-transducer system is determined by numerous factors, including the compliance of the tubing, the surface area of the transducer diaphragm, and the compliance of the diaphragm. If the system is underdamped, then the inertia of the system, which is a function of the mass of the fluid in the tubing and the mass of the diaphragm, causes overshoot of the points of maximum positive and negative displacement of the diaphragm

1	of the system, which is a function of the mass of the fluid in the tubing and the mass of the diaphragm, causes overshoot of the points of maximum positive and negative displacement of the diaphragm during sys-tole and diastole, respectively. Thus, in an underdamped system, systolic pressure will be overestimated and diastolic pressure will be underestimated. In an overdamped system, displacement of the diaphragm fails to track the rapidly changing pressure waveform, and systolic pressure will be underestimated and diastolic pressure will be overestimated. It is important to note that even in an underdamped or overdamped system, mean pres-sure will be accurately recorded, provided the system has been properly calibrated. For these reasons, when using direct mea-surement of intra-arterial pressure to monitor patients, clinicians should make clinical decisions based primarily on the measured mean arterial blood pressure.The radial artery at the wrist is the site most commonly used for

1	pressure to monitor patients, clinicians should make clinical decisions based primarily on the measured mean arterial blood pressure.The radial artery at the wrist is the site most commonly used for intra-arterial pressure monitoring. Other sites include the femoral and axillary artery. It is important to recognize, however, that measured arterial pressure is determined in part by the site where the pressure is monitored. Central (i.e., aortic) and peripheral (e.g., radial artery) pressures typically are dif-ferent as a result of the impedance and inductance of the arte-rial tree. Systolic pressures typically are higher and diastolic pressures are lower in the periphery, whereas mean pressure is approximately the same in the aorta and more distal sites.Distal ischemia is an uncommon complication of intra-arterial catheterization. The incidence of thrombosis is increased when larger-caliber catheters are employed and when catheters are left in place for an extended period of time. The

1	of intra-arterial catheterization. The incidence of thrombosis is increased when larger-caliber catheters are employed and when catheters are left in place for an extended period of time. The incidence of thrombosis can be minimized by using a 20-gauge (or smaller) catheter in the radial artery and removing the catheter as soon as feasible. The risk of distal ischemic injury can be reduced by ensuring that adequate collateral flow is present prior to catheter insertion. At the wrist, adequate collateral flow can be documented by performing a modified version of the Allen test, wherein the artery to be cannulated is digitally compressed while using a Doppler stethoscope to listen for perfusion in the palmar arch vessels.Another potential complication of intra-arterial monitor-ing is retrograde embolization of air bubbles or thrombi into the intracranial circulation. In order to minimize this risk care should be taken to avoid flushing arterial lines when air is pres-ent in the system,

1	embolization of air bubbles or thrombi into the intracranial circulation. In order to minimize this risk care should be taken to avoid flushing arterial lines when air is pres-ent in the system, and only small volumes of fluid (less than 5 mL) should be employed for this purpose. Catheter-related infections can occur with any intravascular monitoring device. However, catheter-related bloodstream infection is a relatively uncommon complication of intra-arterial lines used for monitor-ing, occurring in 0.4% to 0.7% of catheterizations.3 The inci-dence increases with longer duration of arterial catheterization.ELECTROCARDIOGRAPHIC MONITORINGThe electrocardiogram (ECG) records the electrical activity associated with cardiac contraction by detecting voltages on the body surface. A standard 3-lead ECG is obtained by placing electrodes that correspond to the left arm (LA), right arm (RA), and left leg (LL). The limb leads are defined as lead I (LA-RA), lead II (LL-RA), and lead III (LL-LA).

1	ECG is obtained by placing electrodes that correspond to the left arm (LA), right arm (RA), and left leg (LL). The limb leads are defined as lead I (LA-RA), lead II (LL-RA), and lead III (LL-LA). The ECG waveforms can be continuously displayed on a monitor, and the devices can be set to sound an alarm if an abnormality of rate or rhythm is detected. Continuous ECG monitoring is widely available and applied to critically ill and perioperative patients. Monitoring of the ECG waveform is essential in patients with acute coronary syndromes or blunt myocardial injury because dysrhythmias are the most common lethal complication. In patients with shock or sepsis, dysrhythmias can occur as a consequence of inadequate myocardial oxygen delivery or as a complication of vasoactive or inotropic drugs used to support blood pressure and cardiac Brunicardi_Ch13_p0433-p0452.indd 43522/02/19 2:20 PM 436BASIC CONSIDERATIONSPART Ioutput. Dysrhythmias can be detected by continuously moni-toring the

1	used to support blood pressure and cardiac Brunicardi_Ch13_p0433-p0452.indd 43522/02/19 2:20 PM 436BASIC CONSIDERATIONSPART Ioutput. Dysrhythmias can be detected by continuously moni-toring the ECG tracing, and timely intervention may prevent serious complications. With appropriate computing hardware and software, continuous ST-segment analysis also can be per-formed to detect ischemia or infarction.Additional information can be obtained from a 12-lead ECG, which is essential for patients with potential myocardial ischemia or to rule out cardiac complications in other acutely ill patients. Continuous monitoring of the 12-lead ECG may be beneficial in certain patient populations. In a study of 185 vas-cular surgical patients, continuous 12-lead ECG monitoring was able to detect transient myocardial ischemic episodes in 20.5% of the patients.4 This study demonstrated that the precordial lead V4, which is not routinely monitored on a standard 3-lead ECG, is the most sensitive for

1	myocardial ischemic episodes in 20.5% of the patients.4 This study demonstrated that the precordial lead V4, which is not routinely monitored on a standard 3-lead ECG, is the most sensitive for detecting perioperative ischemia and infarction. To detect 95% of the ischemic episodes, two or more precordial leads were necessary. Furthermore, in a pro-spective observational study, 51 peripheral artery vascular sur-gery patients underwent ambulatory continuous 12-lead ECG monitoring in the postoperative setting. Ischemic load, defined as the area under the curve defined by ischemic ST-segment deviation and ischemic time, was shown to predict perioperative myocardial infarction with an area under the receiver operating characteristics curve of 0.87. Notably, ischemia was asymptom-atic in 14 of the 17 identified patients, demonstrating value of this modality as a warning tool.5 Thus, continuous 12-lead ECG monitoring may provide greater sensitivity than 3-lead ECG for the detection of

1	14 of the 17 identified patients, demonstrating value of this modality as a warning tool.5 Thus, continuous 12-lead ECG monitoring may provide greater sensitivity than 3-lead ECG for the detection of perioperative myocardial ischemia, and may become standard for monitoring high-risk surgical patients.Currently, there is considerable interest in using comput-erized approaches to analyze ECG waveforms and patterns to uncover hidden information that can be used to predict sudden cardiac death or the development of serious dysrhythmias. ECG patterns of interest include repetitive changes in the morphol-ogy of the T-wave (T-wave alternans; TWA)6 and heart rate variability.7ALGORITHMIC INTEGRATIVE MONITORINGIntegrated monitoring systems employ software that integrates vital signs to produce a single-parameter index that allows early detection of physiologic perturbations. The input variables include noninvasive measurements of heart rate, respiratory rate, blood pressure, SpO2, and

1	a single-parameter index that allows early detection of physiologic perturbations. The input variables include noninvasive measurements of heart rate, respiratory rate, blood pressure, SpO2, and temperature. The software uses neural networking to develop a probabilistic model of normal-ity, previously developed from a representative sample patient training set. Variance from this data set is used to evaluate the probability that the patient-derived vital signs are within the normal range. An abnormal index can occur while no single vital sign parameter is outside the range of normal if their com-bined patterns are consistent with known instability patterns. Employing such an integrated monitoring system in step-down unit patients has been shown to be a sensitive method to detect early physiologic abnormalities that may precede hemodynamic instability.8 This subsequently was demonstrated to reduce the amount of overall patient instability by facilitating earlier iden-tification and

1	physiologic abnormalities that may precede hemodynamic instability.8 This subsequently was demonstrated to reduce the amount of overall patient instability by facilitating earlier iden-tification and appropriate intervention by the medical team.9The large expansion of the electronic medical record (EMR) is also driving the development of new algorithmic assessment tools for inpatient monitoring. The Rothman Index (RI) is a proprietary data analysis toolkit encompassing a total of 26 variables including vital signs, nursing assessments, laboratory test values, and cardiac rhythms and was developed to make use of the vast amount of data input into the EMR on a real-time basis to help provide a global assessment of patient status. In the initial derivation, Rothman and colleagues dem-onstrated concordance of the RI with the Modified Early Warning Score (MEWS) system, which is designed to alert medical teams to clinical deterioration that precedes cardiac or pulmonary arrest events.10

1	concordance of the RI with the Modified Early Warning Score (MEWS) system, which is designed to alert medical teams to clinical deterioration that precedes cardiac or pulmonary arrest events.10 Subsequent publications evaluated performance of the RI in predicting both readmission to surgical ICUs in the postoperative setting as well as for rapid response team activations.11-13 Although more work is required to evalu-ate the broad applicability of the RI and similar measures, the evidence to date is compelling. Furthermore, as EMR interfaces become more sophisticated, other real-time data analysis soft-ware packages will likely be developed that provide further insight into the care of postsurgical patients.CARDIAC OUTPUT AND RELATED PARAMETERSBedside catheterization of the pulmonary artery was introduced into clinical practice in the 1970s. Although the pulmonary artery catheter initially was used primarily to manage patients with cardiogenic shock and other acute cardiac diseases,

1	was introduced into clinical practice in the 1970s. Although the pulmonary artery catheter initially was used primarily to manage patients with cardiogenic shock and other acute cardiac diseases, indi-cations for this form of invasive hemodynamic monitoring gradually expanded to encompass a wide variety of clinical con-ditions. Clearly, many clinicians believe that information valu-able for the management of critically ill patients is afforded by having a pulmonary artery catheter (PAC) in place. However, unambiguous data in support of this view are scarce, and several studies suggest that bedside pulmonary artery catheterization may not benefit most critically ill patients and in fact may lead to some serious complications (see “Effect of Pulmonary Artery Catheterization on Outcome”).Determinants of Cardiac PerformanceCardiac performance requires the integration of multiple mechanical and physiologic parameters of both the heart itself and of the circulatory system through which

1	of Cardiac PerformanceCardiac performance requires the integration of multiple mechanical and physiologic parameters of both the heart itself and of the circulatory system through which blood flows. The following sections discuss some of these factors, including preload, contractility, and afterload. A brief review of some of the graphical tools for evaluating cardiac physiology is demon-strated in Fig. 13-2.Preload. Starling’s law of the heart states that the force of muscle contraction depends on the initial length of the cardiac fibers. Using terminology that derives from early experiments using isolated cardiac muscle preparations, preload is the stretch of ventricular myocardial tissue just prior to the next contrac-tion. Strictly speaking, preload is determined by end-diastolic volume (EDV). In practice, EDV is challenging to measure precisely during the cardiac cycle, and so clinicians utilize the end-diastolic pressure (EDP) as a reasonable surrogate. For the right ventricle,

1	(EDV). In practice, EDV is challenging to measure precisely during the cardiac cycle, and so clinicians utilize the end-diastolic pressure (EDP) as a reasonable surrogate. For the right ventricle, central venous pressure (CVP) approximates right ventricular EDP. For the left ventricle, pulmonary artery occlusion pressure (PAOP), which is measured by transiently inflating a balloon at the end of a pressure monitoring catheter positioned in a small branch of the pulmonary artery, approxi-mates left ventricular EDP. The presence of atrioventricular val-vular stenosis may alter this relationship.There are limits to the utilization of EDP as a surrogate for EDV when evaluating preload. For example, EDP is deter-mined not only by volume but also by the diastolic compliance of the ventricular chamber. Ventricular compliance is altered by Brunicardi_Ch13_p0433-p0452.indd 43622/02/19 2:20 PM 437PHYSIOLOGIC MONITORING OF THE SURGICAL PATIENTCHAPTER 13Figure 13-2 A-D. Left ventricular

1	chamber. Ventricular compliance is altered by Brunicardi_Ch13_p0433-p0452.indd 43622/02/19 2:20 PM 437PHYSIOLOGIC MONITORING OF THE SURGICAL PATIENTCHAPTER 13Figure 13-2 A-D. Left ventricular pressure-volume loops constructed for various clinically relevant scenarios. For further information refer to the text. A. Standard left ventricular pressure-volume loop, with stroke volume, end systolic volume, and end diastolic volume highlighted for reference. Note the directionality of the pressure-volume loop, which is not annotated in the other figures for clarity. B-D. Demonstration of the effect of changing preload (B), contractility (C), or afterload (D) on the pressure-volume relationships in the left ventricle. Note the differences in stroke volume for various conditions, as well as the end-systolic volume and pressures, as these represent clinically significant parameters that govern patient care.various pathologic conditions and pharmacologic agents. Fur-thermore, the

1	well as the end-systolic volume and pressures, as these represent clinically significant parameters that govern patient care.various pathologic conditions and pharmacologic agents. Fur-thermore, the relationship between EDP and true preload is not linear, but rather is exponential (Fig. 13-2A,B). This fact limits the utility of EDP as a surrogate marker at extremes of EDV.Contractility. Contractility is defined as the inotropic state of the myocardium. Contractility is said to increase when the force of ventricular contraction increases at constant preload and afterload. Clinically, contractility is difficult to quantify because virtually all of the available measures are dependent to a certain degree on preload and afterload. If pressure-volume loops are constructed for each cardiac cycle, small changes in preload and/or afterload will result in shifts of the point defining the end of systole. These end-systolic points on the pressure-versus-volume diagram describe a straight line,

1	small changes in preload and/or afterload will result in shifts of the point defining the end of systole. These end-systolic points on the pressure-versus-volume diagram describe a straight line, known as the end-systolic pressure-volume line. A steeper slope of this line indicates greater contractility, as illustrated in Fig. 13-2C.Afterload. Afterload is another term derived from in vitro experiments using isolated strips of cardiac muscle and is defined as the force resisting fiber shortening once systole begins. Defined specifically for the in vivo system, afterload is the resistance to the expulsion of blood from the heart chamber of interest, usually the left ventricle. Several factors comprise the in vivo correlate of ventricular afterload, including ven-tricular chamber geometry, intracavitary pressure generation, and the arterial impedance in the systemic circulation. Since these factors are difficult to assess clinically, afterload is com-monly approximated by calculating

1	intracavitary pressure generation, and the arterial impedance in the systemic circulation. Since these factors are difficult to assess clinically, afterload is com-monly approximated by calculating systemic vascular resistance (SVR), defined as mean arterial pressure (MAP) divided by car-diac output (Fig. 13-2D).PLACEMENT OF THE PULMONARY ARTERY CATHETERIn its simplest form, the PAC has four channels. One channel terminates in a balloon at the tip of the catheter. The proximal end of this channel is connected to a syringe to permit inflation of the balloon with air. Prior to insertion of the PAC, the integ-rity of the balloon should be verified by inflating it. In order to minimize the risk of vascular or ventricular perforation by the relatively inflexible catheter, it also is important to verify that the inflated balloon extends just beyond the tip of the device. A second channel in the catheter contains wires that are connected Brunicardi_Ch13_p0433-p0452.indd 43722/02/19 2:21

1	to verify that the inflated balloon extends just beyond the tip of the device. A second channel in the catheter contains wires that are connected Brunicardi_Ch13_p0433-p0452.indd 43722/02/19 2:21 PM 438BASIC CONSIDERATIONSPART Ito a thermistor located near the tip of the catheter. At the proxi-mal end of the PAC, the wires terminate in a fitting that permits connection to appropriate hardware for the calculation of car-diac output using the thermodilution technique. The final two channels are used for pressure monitoring and the injection of the thermal indicator for determinations of cardiac output. One of these channels terminates at the tip of the catheter; the other terminates 20 cm proximal to the tip.Placement of a PAC requires access to the central venous circulation. Such access can be obtained at a variety of sites, including the antecubital, femoral, jugular, and subclavian veins. Percutaneous placement through either the jugular or sub-clavian vein generally is

1	access can be obtained at a variety of sites, including the antecubital, femoral, jugular, and subclavian veins. Percutaneous placement through either the jugular or sub-clavian vein generally is preferred. Right internal jugular vein cannulation carries the lowest risk of complications, and the path of the catheter from this site into the right atrium is straight. In the event of inadvertent arterial puncture, local pressure is significantly more effective in controlling bleeding from the carotid artery as compared to the subclavian artery. Neverthe-less, it is more difficult to keep occlusive dressings in place on the neck than in the subclavian fossa. Furthermore, the anatomic landmarks in the subclavian position are quite constant, even in patients with anasarca or massive obesity; the subclavian vein is always attached to the deep (concave) surface of the clavicle. In contrast, the appropriate landmarks to guide jugular venous cannulation are sometimes difficult to discern in

1	the subclavian vein is always attached to the deep (concave) surface of the clavicle. In contrast, the appropriate landmarks to guide jugular venous cannulation are sometimes difficult to discern in obese or very edematous patients. However, ultrasonic guidance, which should be used routinely, has been shown to facilitate bedside jugular venipuncture.14Cannulation of the vein is normally performed percuta-neously, using the Seldinger technique. A small-bore needle is inserted through the skin and subcutaneous tissue into the vein. After documenting return of venous blood, a guidewire with a flexible tip is inserted through the needle into the vein, and the needle is withdrawn. A dilator/introducer sheath is passed over Figure 13-3. Representative pressure traces at different stages of insertion of the PAC. In the central venous circulation, the pressure remains low, with characteristic waves from atrial filling and tricuspid valve closing. Upon entry into the right ventricle, the

1	insertion of the PAC. In the central venous circulation, the pressure remains low, with characteristic waves from atrial filling and tricuspid valve closing. Upon entry into the right ventricle, the pressure increases sharply, with the broadest range between systole and diastole. When in the main pulmonary artery, the systolic pressure remains elevated to the same degree, but the diastolic pressure is now significantly elevated due to the closure of the pulmonic valve during the cardiac cycle. Upon further advancement with the balloon inflated, the pressure differences become smaller and the magnitude of the mean pressure drops, reflecting an estimate of the left atrial pressure.the wire, and the wire and the dilator are removed. The proxi-mal terminus of the distal port of the PAC is connected through low-compliance tubing to a strain-gauge transducer, and the tubing-catheter system is flushed with fluid. While constantly observing the pressure tracing on a monitor screen, the PAC is

1	through low-compliance tubing to a strain-gauge transducer, and the tubing-catheter system is flushed with fluid. While constantly observing the pressure tracing on a monitor screen, the PAC is advanced with the balloon deflated until respiratory excur-sions are observed. The balloon is then inflated, and the catheter advanced further, while monitoring pressures sequentially in the right atrium and right ventricle en route to the pulmonary artery. The pressure waveforms for the right atrium, right ventricle, and pulmonary artery are each characteristic (Fig. 13-3). The cath-eter is advanced out the pulmonary artery until a damped tracing indicative of the “wedged” position is obtained. The balloon is then deflated, taking care to ensure that a normal pulmonary arterial tracing is again observed on the monitor; leaving the balloon inflated can increase the risk of pulmonary infarction or perforation of the pulmonary artery. Unnecessary measurements of the pulmonary artery occlusion

1	observed on the monitor; leaving the balloon inflated can increase the risk of pulmonary infarction or perforation of the pulmonary artery. Unnecessary measurements of the pulmonary artery occlusion pressure are discouraged as rupture of the pulmonary artery may occur.HEMODYNAMIC MEASUREMENTSEven in its simplest embodiment, the PAC is capable of pro-viding clinicians with a remarkable amount of information about the hemodynamic status of patients. Additional informa-tion may be obtained if various modifications of the standard PAC are employed. By combining data obtained through use of the PAC with results obtained by other means (i.e., blood hemoglobin concentration and oxyhemoglobin saturation), derived estimates of systemic oxygen transport and utilization can be calculated. Direct and derived parameters obtainable by bedside pulmonary arterial catheterization, along with sev-eral associated approximate normal ranges, are summarized in Table 13-1.Brunicardi_Ch13_p0433-p0452.indd

1	derived parameters obtainable by bedside pulmonary arterial catheterization, along with sev-eral associated approximate normal ranges, are summarized in Table 13-1.Brunicardi_Ch13_p0433-p0452.indd 43822/02/19 2:21 PM 439PHYSIOLOGIC MONITORING OF THE SURGICAL PATIENTCHAPTER 13Table 13-1Directly measured and derived hemodynamic data obtainable by bedside pulmonary artery catheterization, with normal associated rangesPARAMETERNORMAL RANGECVP0–6 mmHgPAPVariesPAOP6–12 mmHgSv–O2 (intermittent or continuous)65%–70%QT (intermittent or continuous)4–6 L/minQT* (intermittent or continuous)2.5–3.5 L·min-1·m-2RVEF>55%SV40–80 mLSVR800–1400 dyne·sec·cm-5SVRI1500–2400 dyne·sec·cm-5·m-2PVR100–150 dyne·sec·cm-5PVRI200–400 dyne·sec·cm-5·m-2RVEDVVariableD.O2400–660 mL·min-1·m-2V–O2115–165 mL·min-1·m-2ERVariableQS/QTVariableCVP = mean central venous pressure; D.O2 = systemic oxygen delivery; ER = systemic oxygen extraction ratio; PAOP = pulmonary artery occlusion (wedge) pressure; PAP = pulmonary

1	= mean central venous pressure; D.O2 = systemic oxygen delivery; ER = systemic oxygen extraction ratio; PAOP = pulmonary artery occlusion (wedge) pressure; PAP = pulmonary artery pressure; PVR = pulmonary vascular resistance; PVRI = pulmonary vascular resistance index; QS/QT = fractional pulmonary venous admixture (shunt fraction); QT = cardiac output; QT* = cardiac output indexed to body surface area (cardiac index); RVEDV = right ventricular end-diastolic volume; RVEF = right ventricular ejection fraction; SV = stroke volume; SVI = stroke volume index; Sv–O2= fractional mixed venous (pulmonary artery) hemoglobin saturation; SVR = systemic vascular resistance; SVRI = systemic vascular resistance index; V–O2 = systemic oxygen utilization.Measurement of Cardiac Output by ThermodilutionBefore the development of the PAC, determining cardiac output (QT) at the bedside required careful measurements of oxygen consumption (Fick method) or spectrophotometric determina-tion of indocyanine

1	the development of the PAC, determining cardiac output (QT) at the bedside required careful measurements of oxygen consumption (Fick method) or spectrophotometric determina-tion of indocyanine green dye dilution curves. Measurements of QT using the thermodilution technique are simple and reason-ably accurate. The measurements can be performed repetitively, and the principle is straightforward. If a bolus of an indicator is rapidly and thoroughly mixed with a moving fluid upstream from a detector, then the concentration of the indicator at the detector will increase sharply and then exponentially diminish back to zero. The area under the resulting time-concentration curve is a function of the volume of indicator injected and the flow rate of the moving stream of fluid. Larger volumes of indi-cator result in greater areas under the curve, and faster flow rates of the mixing fluid result in smaller areas under the curve. When QT is measured by thermodilution, the indicator is heat and

1	indi-cator result in greater areas under the curve, and faster flow rates of the mixing fluid result in smaller areas under the curve. When QT is measured by thermodilution, the indicator is heat and the detector is a temperature-sensing thermistor at the distal end of the PAC. The relationship used for calculating QT is called the Stewart-Hamilton equation:QVKK(TT)T(t)dtT12BIB=−˛where V is the volume of the indicator injected, TB is the tem-perature of blood (i.e., core body temperature), TI is the tem-perature of the indicator, K1 is a constant that is the function of the specific heats of blood and the indicator, K2 is an empiri-cally derived constant that accounts for several factors (the dead space volume of the catheter, heat lost from the indicator as it traverses the catheter, and the injection rate of the indicator), and ∫TB(t)dt is the area under the time-temperature curve. In clinical practice, the Stewart-Hamilton equation is solved by a microprocessor.Determination of

1	and the injection rate of the indicator), and ∫TB(t)dt is the area under the time-temperature curve. In clinical practice, the Stewart-Hamilton equation is solved by a microprocessor.Determination of cardiac output by the thermodilution method is generally quite accurate, although it tends to system-atically overestimate QT at low values. Changes in blood tem-perature and QT during the respiratory cycle can influence the measurement. Therefore, results generally should be recorded as the mean of two or three determinations obtained at random points in the respiratory cycle. Using cold injectate widens the difference between TB and TI and thereby increases signal-to-noise ratio. Nevertheless, most authorities recommend using room temperature injectate (normal saline or 5% dextrose in water) to minimize errors resulting from warming of the fluid as it transferred from its reservoir to a syringe for injection.Technologic innovations have been introduced that per-mit continuous

1	in water) to minimize errors resulting from warming of the fluid as it transferred from its reservoir to a syringe for injection.Technologic innovations have been introduced that per-mit continuous measurement of QT by thermodilution. In this approach, thermal transients are not generated by injecting a bolus of a cold indicator, but rather by heating the blood with a tiny filament located on the PAC upstream from the thermistor. By correlating the amount of current supplied to the heating element with the downstream temperature of the blood, it is pos-sible to estimate the average blood flow across the filament and thereby calculate QT. Based upon the results of several studies, continuous determinations of QT using this approach agree well with data generated by conventional measurements using bolus injections of a cold indicator.15 Information is lacking regarding the clinical value of being able to monitor QT continuously.Mixed Venous OximetryThe Fick equation can be written

1	using bolus injections of a cold indicator.15 Information is lacking regarding the clinical value of being able to monitor QT continuously.Mixed Venous OximetryThe Fick equation can be written as222QVO(COCO)Tav=−where CaO2 is the content of oxygen in arterial blood and CvO2 is the content of oxygen in mixed venous blood. The oxygen content in both arterial and venous blood is a function of the hemoglobin concentration in the blood, the hemoglobin satura-tion, and the partial pressure of oxygen:CO1.36HgbSO1000.0031POa/v2a/v2a/v2=××˛˝˙ˆˇ˘+×CO1.36HgbSO100//av2av2=××˛˝˙ˆˇ˘where Sa/vO2 is the fractional saturation of hemoglobin in either arterial or venous blood, Hgb is the concentration of hemoglobin Brunicardi_Ch13_p0433-p0452.indd 43922/02/19 2:21 PM 440BASIC CONSIDERATIONSPART Iin blood, and Pa/vO2 is the partial pressure of oxygen in the arte-rial or venous blood. Under most circumstances the contribution of dissolved oxygen to both CaO2 and CvO2 is negligible, allow-ing the

1	blood, and Pa/vO2 is the partial pressure of oxygen in the arte-rial or venous blood. Under most circumstances the contribution of dissolved oxygen to both CaO2 and CvO2 is negligible, allow-ing the second portion of equation to be functionally eliminated (see previous equation). Given that, if the Fick equation is rear-ranged to the following:2COCOVOQv2a2T=−Oxygen saturation can replace oxygen content, yielding the final clinically valuable equation:(1.36)222SOSOVOQHgbvaT=−××where SVO2 is the fractional saturation of hemoglobin in mixed venous blood, SaO2 is the fractional saturation of hemoglobin in arterial blood, and Hgb is the concentration of hemoglobin in blood. Thus, it can be seen that SVO2 is a function of VO2 (i.e., metabolic rate), QT, SaO2, and Hgb. Accordingly, subnormal val-ues of SVO2 can be caused by a decrease in QT (due, for example, to heart failure or hypovolemia), a decrease in SaO2 (due, for example, to intrinsic pulmonary disease), a decrease in Hgb (i.e.,

1	val-ues of SVO2 can be caused by a decrease in QT (due, for example, to heart failure or hypovolemia), a decrease in SaO2 (due, for example, to intrinsic pulmonary disease), a decrease in Hgb (i.e., anemia), or an increase in metabolic rate (due, for example, to seizures or fever). With a conventional PAC, measurements of SVO2 require aspirating a sample of blood from the distal (i.e., pulmonary arterial) port of the catheter and injecting the sample into a blood gas analyzer. Therefore, for practical purposes, mea-surements of SVO2 can be performed only intermittently.By adding a fifth channel to the PAC, it is possible to mon-itor SVO2 continuously. The fifth channel contains two fiber-optic bundles, which are used to transmit and receive light of the appropriate wavelengths to permit measurements of hemoglobin saturation by reflectance spectrophotometry. Continuous SVO2 devices provide measurements of SVO2 that agree quite closely with those obtained by conventional analyses of

1	measurements of hemoglobin saturation by reflectance spectrophotometry. Continuous SVO2 devices provide measurements of SVO2 that agree quite closely with those obtained by conventional analyses of blood aspi-rated from the pulmonary artery. Despite the theoretical value of being able to monitor SVO2 continuously, data are lacking to show that this capability favorably improves outcomes. In a prospective, observational study of 3265 patients undergoing cardiac surgery with either a standard PAC or a PAC with con-tinuous SVO2 monitoring, the oximetric catheter was associated with fewer arterial blood gases and thermodilution cardiac out-put determinations but no difference in patient outcome.16 Since pulmonary artery catheters that permit continuous monitoring of SVO2 are more expensive than conventional PACs, the routine use of these devices cannot be recommended.The saturation of oxygen in the right atrium or superior vena cava (ScvO2) correlates closely with SvO2 over a wide range

1	conventional PACs, the routine use of these devices cannot be recommended.The saturation of oxygen in the right atrium or superior vena cava (ScvO2) correlates closely with SvO2 over a wide range of conditions,17 although the correlation between ScvO2 and SvO2 has been questioned.18 Since measurement of ScvO2 requires placement of a central venous catheter rather than a PAC, it is somewhat less invasive and easier to carry out. By using a cen-tral venous catheter equipped to permit fiber-optic monitoring of ScvO2, it may be possible to titrate the resuscitation of patients with shock using a less invasive device than the PAC.17,19 The Surviving Sepsis Campaign international guidelines for the management of severe sepsis and septic shock recommends that during the first 6 hours of resuscitation, the goals of initial resuscitation of sepsis-induced hypoperfusion should include all of the following: CVP 8 to 12 mm Hg, MAP ≥65 mm Hg, urine output ≥0.5 mL/kg per hour, and ScvO2 of 70% or

1	the goals of initial resuscitation of sepsis-induced hypoperfusion should include all of the following: CVP 8 to 12 mm Hg, MAP ≥65 mm Hg, urine output ≥0.5 mL/kg per hour, and ScvO2 of 70% or SvO2 65%.20EFFECT OF PULMONARY ARTERY CATHETERIZATION ON OUTCOMEDespite initial enthusiasm for using the PAC in the manage-ment of critically ill patients, several studies have failed to show improved outcomes with their use. Connors and col-leagues reported results of a major observational study evaluat-ing the value of pulmonary artery catheterization in critically ill patients.21 These researchers compared two groups of patients: those who did undergo placement of a PAC during their first 24 hours of ICU care and those who did not. The investiga-tors recognized that the value of their intended analysis was completely dependent on the robustness of their methodology for case-matching because sicker patients (i.e., those at greater risk of mortality based upon the severity of their illness) were

1	was completely dependent on the robustness of their methodology for case-matching because sicker patients (i.e., those at greater risk of mortality based upon the severity of their illness) were presumably more likely to undergo pulmonary artery catheter-ization. Accordingly, the authors used sophisticated statistical methods for generating a cohort of study (i.e., PAC) patients, each one having a paired control matched carefully for severity of illness. Connors and associates concluded that placement of a pulmonary artery catheter during the first 24 hours of stay in an ICU is associated with a significant increase in the risk of mortality, even when statistical methods are used to account for severity of illness.A number of prospective, randomized controlled trials of pulmonary artery catheterization are summarized in Table 13-2. The study by Pearson and associates was underpowered with only 226 patients enrolled.22 In addition, the attending anes-thesiologists were permitted to

1	catheterization are summarized in Table 13-2. The study by Pearson and associates was underpowered with only 226 patients enrolled.22 In addition, the attending anes-thesiologists were permitted to exclude patients from the CVP group at their discretion; thus randomization was compromised. The study by Tuman and coworkers was large (1094 patients were enrolled), but different anesthesiologists were assigned to the different groups.23 Furthermore, 39 patients in the CVP group underwent placement of a PAC because of hemodynamic complications. All of the individual single-institution studies of vascular surgery patients were relatively underpowered, and all excluded at least certain categories of patients (e.g., those with a history of recent myocardial infarction).24,25In the largest randomized controlled trial of the PAC, Sandham and associates randomized nearly 2000 American Society of Anesthesiologists (ASA) classes III and IV patients undergoing major thoracic, abdominal, or

1	randomized controlled trial of the PAC, Sandham and associates randomized nearly 2000 American Society of Anesthesiologists (ASA) classes III and IV patients undergoing major thoracic, abdominal, or orthopedic surgery to placement of a PAC or CVP catheter.26 In the patients assigned to receive a PAC, physiologic goal-directed therapy was imple-mented by protocol. There were no differences in mortality at 30 days, 6 months, or 12 months between the two groups, and ICU length of stay was similar. There was a significantly higher rate of pulmonary emboli in the PAC group (0.9% vs. 0%). This study has been criticized because most of the patients enrolled were not in the highest risk category.In the “PAC-Man” trial, a multicenter, randomized trial in 65 UK hospitals, over 1000 ICU patients were managed with or without a PAC.27 The specifics of the clinical management were then left up to the treating clinicians. There was no dif-ference in hospital mortality between the 2 groups (with PAC

1	managed with or without a PAC.27 The specifics of the clinical management were then left up to the treating clinicians. There was no dif-ference in hospital mortality between the 2 groups (with PAC 68% vs. without PAC 66%, P = 0.39). However, a 9.5% com-plication rate was associated with the insertion or use of the PAC, although none of these complications were fatal. Clearly, these were critically ill patients, as noted by the high hospital mortality rates. Supporters of the PAC may cite methodology problems with this study, such as loose inclusion criteria and the lack of a defined treatment protocol.Brunicardi_Ch13_p0433-p0452.indd 44022/02/19 2:21 PM 441PHYSIOLOGIC MONITORING OF THE SURGICAL PATIENTCHAPTER 13Table 13-2Summary of randomized, prospective clinical trials comparing pulmonary artery catheter (PAC) with central venous pressure (CVP) monitoringAUTHORSTUDY POPULATIONGROUPSOUTCOMESSTRENGTHS/WEAKNESSESPearson et al22“Low risk” patients undergoing cardiac or vascular

1	pulmonary artery catheter (PAC) with central venous pressure (CVP) monitoringAUTHORSTUDY POPULATIONGROUPSOUTCOMESSTRENGTHS/WEAKNESSESPearson et al22“Low risk” patients undergoing cardiac or vascular surgeryCVP catheter (group 1); PAC (group 2); PAC with continuous Sv–O2 readout (group 3)No differences among groups for mortality or length of ICU stay; significant differences in costs (group 1 < group 2 < group 3)Underpowered (266 total patients enrolled); compromised randomization protocolsTuman et al23Cardiac surgical patientsPAC; CVPNo differences between groups for mortality, length of ICU stay, or significant noncardiac complicationsLarge trial (1094 patients); different anesthesiologists for different groupsBender et al24Vascular surgery patientsPAC; CVPNo differences between groups for mortality, length of ICU stay, or length of hospital stayRelatively underpoweredValentine et al25Aortic surgery patientsPAC + hemodynamic optimization in ICU night before surgery; CVPNo

1	groups for mortality, length of ICU stay, or length of hospital stayRelatively underpoweredValentine et al25Aortic surgery patientsPAC + hemodynamic optimization in ICU night before surgery; CVPNo difference between groups for mortality or length of ICU stay; significantly higher incidence of postoperative complications in PAC groupRelatively underpoweredSandham et al26“High risk” major surgeryPAC; CVPNo differences between groups for mortality, length of ICU stay; increased incidence of pulmonary embolism in PAC groupLargest RCT of PAC utilization; commonly criticized for smaller number of highest risk category patientsHarvey S et al27PAC-Man TrialMedical and surgical ICU patientsPAC vs no PAC, with option for alternative CO measuring device in non-PAC groupNo difference in hospital mortality between the 2 groups, increased incidence of complications in the PAC groupLoose inclusion criteria with lack of a defined treatment protocol for use of PAC dataBinanay et al29ESCAPE

1	mortality between the 2 groups, increased incidence of complications in the PAC groupLoose inclusion criteria with lack of a defined treatment protocol for use of PAC dataBinanay et al29ESCAPE TrialPatients with CHFPAC vs no PACNo difference in hospital mortality between the groups, increased incidence of adverse events in the PAC groupNo formal treatment protocol for PAC-driven therapyWheeler et al30FACTT TrialPatients with ALIPAC vs CVC with a fluid and inotropic management protocolNo difference in ICU or hospital mortality, or incidence of organ failure between the groups; increased incidence of adverse events in the PAC group ALI = acute lung injury; CHF = congestive heart failure; CO = cardiac output; CVC = central venous catheter; ICU = intensive care unit; PAC = pulmonary artery catheter; Sv–O2 = fractional mixed venous (pulmonary artery) hemoglobin saturation.A meta-analysis of 13 randomized studies of the PAC that included over 5000 patients was published in 2005.28 A broad

1	Sv–O2 = fractional mixed venous (pulmonary artery) hemoglobin saturation.A meta-analysis of 13 randomized studies of the PAC that included over 5000 patients was published in 2005.28 A broad spectrum of critically ill patients was included in these hetero-geneous trials, and the hemodynamic goals and treatment strate-gies varied. While the use of the PAC was associated with an increased use of inotropes and vasodilators, there were no differ-ences in mortality or hospital length of stay between the patients managed with a PAC and those managed without a PAC.The ESCAPE trial (which was one of the studies included in the previous meta-analysis)29 evaluated 433 patients with severe or recurrent congestive heart failure (CHF) admitted to the ICU. Patients were randomized to management by clinical assessment and a PAC or clinical assessment without a PAC. The goal in both groups was resolution of CHF, with addi-tional PAC targets of a pulmonary capillary occlusion pressure of 15 mmHg and a

1	assessment and a PAC or clinical assessment without a PAC. The goal in both groups was resolution of CHF, with addi-tional PAC targets of a pulmonary capillary occlusion pressure of 15 mmHg and a right atrial pressure of 8 mmHg. There was no formal treatment protocol, but inotropic support was dis-couraged. Substantial reduction in symptoms, jugular venous pressure, and edema was noted in both groups. There was no significant difference in the primary end point of days alive and out of the hospital during the first 6 months, or hospital mortality (PAC 10% vs without PAC 9%). Adverse events Brunicardi_Ch13_p0433-p0452.indd 44122/02/19 2:21 PM 442BASIC CONSIDERATIONSPART Iwere more common among patients in the PAC group (21.9% vs 11.5%; P = 0.04).Finally, the Fluids and Catheters Treatment Trial (FACTT) conducted by the Acute Respiratory Distress Syndrome (ARDS) Clinical Trials Network was published in 2006.30 The risks and benefits of PAC compared with central venous catheters

1	Trial (FACTT) conducted by the Acute Respiratory Distress Syndrome (ARDS) Clinical Trials Network was published in 2006.30 The risks and benefits of PAC compared with central venous catheters (CVC) were evaluated in 1000 patients with acute lung injury. Patients were randomly assigned to receive either a PAC or a CVC to guide management for 7 days via an explicit protocol. Patients also were randomly assigned to a conservative or liberal fluid strategy in a 2 × 2 factorial design (outcomes based on the fluid management strategy were published separately). Mortality dur-ing the first 60 days was similar in the PAC and CVC groups (27% and 26%, respectively; P = .69). The duration of mechani-cal ventilation and ICU length of stay also were not influenced by the type of catheter used. The type of catheter employed did not affect the incidence of shock, respiratory or renal failure, ventilator settings, or requirement for hemodialysis or vaso-pressors. There was a 1% rate of crossover from

1	of catheter employed did not affect the incidence of shock, respiratory or renal failure, ventilator settings, or requirement for hemodialysis or vaso-pressors. There was a 1% rate of crossover from CVC-guided therapy to PAC-guided therapy. The catheter used did not affect the administration of fluids or diuretics, and the fluid balance was similar in the two groups. The PAC group had approxi-mately twice as many catheter-related adverse events (mainly arrhythmias).Few subjects in critical care medicine have historically gen-erated more emotional responses among experts in the field than the use of the PAC. As these studies demonstrate, it is not possible to show that therapy directed by use of the PAC saves lives when it is evaluated in a large population of patients. Certainly, given the available evidence, routine use of the PAC cannot be justified. Whether selective use of the device in a few relatively uncommon clinical situations is warranted or valuable remains a controversial

1	available evidence, routine use of the PAC cannot be justified. Whether selective use of the device in a few relatively uncommon clinical situations is warranted or valuable remains a controversial issue. Consequently, a marked decline in the use of the PAC from 5.66 per 1000 medical admissions in 1993 to 1.99 per 1000 medical admissions in 2004 has been seen.31 Based upon the results and exclusion criteria in these pro-spective randomized trials, reasonable criteria for perioperative monitoring without use of a PAC are presented in Table 13-3.One of the reasons for using a PAC to monitor critically ill patients is to optimize cardiac output and systemic oxygen delivery. Defining what constitutes the optimum cardiac out-put, however, has proven to be difficult. A number of random-ized trials evaluating the effect on outcome of goal-directed as compared to conventional hemodynamic resuscitation have 2Table 13-3Suggested criteria for perioperative monitoring without use of a pulmonary

1	evaluating the effect on outcome of goal-directed as compared to conventional hemodynamic resuscitation have 2Table 13-3Suggested criteria for perioperative monitoring without use of a pulmonary artery catheter in patients undergoing cardiac or major vascular surgical proceduresNo anticipated need for suprarenal or supraceliac aortic cross-clampingNo history of myocardial infarction during 3 months prior to operationNo history of poorly compensated congestive heart failureNo history of coronary artery bypass graft surgery during 6 weeks prior to operationNo history of ongoing symptomatic mitral or aortic valvular heart diseaseNo history of ongoing unstable angina pectorisbeen published. Some studies provide support for the notion that interventions designed to achieve supraphysiologic goals for DO2, VO2, and QT improve outcome.32,33 However, other pub-lished studies do not support this view, and a meta-analysis con-cluded that interventions designed to achieve supraphysiologic goals

1	for DO2, VO2, and QT improve outcome.32,33 However, other pub-lished studies do not support this view, and a meta-analysis con-cluded that interventions designed to achieve supraphysiologic goals for oxygen transport do not significantly reduce mortality rates in critically ill patients.34,35 At this time, supraphysiologic resuscitation of patients in shock cannot be endorsed.There is no simple explanation for the apparent lack of effectiveness of pulmonary artery catheterization, although sev-eral concurrent possibilities exist. First, even though bedside pulmonary artery catheterization is quite safe, the procedure is associated with a finite incidence of serious complications, including ventricular arrhythmias, catheter-related sepsis, cen-tral venous thrombosis, pulmonary arterial perforation, and pul-monary embolism.26 The adverse effects of these complications on outcome may equal or even outweigh any benefits associated with using a PAC to guide therapy. Second, the data

1	perforation, and pul-monary embolism.26 The adverse effects of these complications on outcome may equal or even outweigh any benefits associated with using a PAC to guide therapy. Second, the data generated by the PAC may be inaccurate, leading to inappropriate thera-peutic interventions. Third, the measurements, even if accurate, are often misinterpreted.36 Furthermore, the current state of understanding is primitive when it comes to deciding what is the best management for certain hemodynamic disturbances, par-ticularly those associated with sepsis or septic shock. Taking all of this into consideration, it may be that interventions prompted by measurements obtained with a PAC are actually harmful to patients. As a result, the marginal benefit now available by placing a PAC may be quite small. Less invasive modalities are available that may provide clinically useful hemodynamic information.It may be true that aggressive hemodynamic resusci-tation of patients, guided by various forms

1	small. Less invasive modalities are available that may provide clinically useful hemodynamic information.It may be true that aggressive hemodynamic resusci-tation of patients, guided by various forms of monitoring, is valuable only during certain critical periods, such as the first few hours after presentation with septic shock or during surgery. For example, Rivers and colleagues reported that survival of patients with septic shock is significantly improved when resus-citation in the emergency department is guided by a protocol that seeks to keep ScvO2 greater than 70%.19 Similarly, a study using an ultrasound-based device (see “Doppler Ultrasonogra-phy”) to assess cardiac filling and SV showed that maximizing SV intraoperatively results in fewer postoperative complications and shorter hospital length of stay.37MINIMALLY INVASIVE ALTERNATIVES TO THE PULMONARY ARTERY CATHETERBecause of the cost, risks, and questionable benefit associated with bedside pulmonary artery catheterization,

1	length of stay.37MINIMALLY INVASIVE ALTERNATIVES TO THE PULMONARY ARTERY CATHETERBecause of the cost, risks, and questionable benefit associated with bedside pulmonary artery catheterization, there has been interest in the development of practical means for less invasive monitoring of hemodynamic parameters. Several approaches have been developed that have achieved variable degrees of suc-cess. None of these methods render the standard thermodilution technique of the pulmonary artery catheter obsolete. However, these strategies may contribute to improvements in the hemody-namic monitoring of critically ill patients.Transpulmonary ThermodilutionIn the standard PAC thermodilution technique, measurements rely on the detection of temperature changes in a relatively small area from the injection port to the thermistor on the same catheter. In contrast, the transpulmonary thermodilution (TPTD) technique measures temperature changes from cold Brunicardi_Ch13_p0433-p0452.indd 44222/02/19

1	port to the thermistor on the same catheter. In contrast, the transpulmonary thermodilution (TPTD) technique measures temperature changes from cold Brunicardi_Ch13_p0433-p0452.indd 44222/02/19 2:21 PM 443PHYSIOLOGIC MONITORING OF THE SURGICAL PATIENTCHAPTER 13bolus solution injected centrally, then measured using an arte-rial thermistor on a special arterial line, generally placed in the femoral artery. Both standard PAC thermodilution and TPTD make use of the Stewart-Hamilton equation to subsequently cal-culate cardiac output. Studies have demonstrated that this tech-nique provides comparable estimates of cardiac output when compared to routine PAC thermodilution and can accurately detect changes in cardiac output as small as 12%.38 However, due to the large blood circuit between the central injection point and the thermistor, data can be challenging to interpret in cer-tain pathophysiologic conditions (e.g., in pulmonary edema, as excess lung water serves as a temperature sink).

1	central injection point and the thermistor, data can be challenging to interpret in cer-tain pathophysiologic conditions (e.g., in pulmonary edema, as excess lung water serves as a temperature sink). On the other hand, thoughtful application of TPTD data allows clinicians access to several additional variables that the traditional PAC does not provide, such as estimation of the global end-diastolic volume (GEDV) and the extravascular lung water volume (EVLW).38 While these variables are of scientific interest, they are not yet in wide clinical use, and further studies are required to determine their utility. However, TPTD does currently play a prominent in the real-time calibration of pulse contour analysis, described in greater detail later in this chapter.Doppler UltrasonographyWhen ultrasonic sound waves are reflected by moving erythro-cytes in the bloodstream, the frequency of the reflected signal is increased or decreased, depending on whether the cells are mov-ing toward or away

1	sound waves are reflected by moving erythro-cytes in the bloodstream, the frequency of the reflected signal is increased or decreased, depending on whether the cells are mov-ing toward or away from the ultrasonic source. This change in frequency is called the Doppler shift, and its magnitude is deter-mined by the velocity of the moving red blood cells. Therefore, measurements of the Doppler shift can be used to calculate red blood cell velocity. With knowledge of both the cross-sectional area of a vessel and the mean red blood cell velocity of the blood flowing through it, one can calculate blood flow rate. If the ves-sel in question is the aorta, then QT can be calculated as:QT = HR × A × ∫ V(t)dtwhere A is the cross-sectional area of the aorta and ∫V(t)dt is the red blood cell velocity integrated over the cardiac cycle.Two approaches have been developed for using Doppler ultrasonography to estimate QT. The first approach uses an ultrasonic transducer, which is manually positioned in

1	over the cardiac cycle.Two approaches have been developed for using Doppler ultrasonography to estimate QT. The first approach uses an ultrasonic transducer, which is manually positioned in the suprasternal notch and focused on the root of the aorta. Aortic cross-sectional area can be estimated using a nomogram, which factors in age, height, and weight, back-calculated if an indepen-dent measure of QT is available, or by using two-dimensional transthoracic or transesophageal ultrasonography. While this approach is completely noninvasive, it requires a highly-skilled operator in order to obtain meaningful results and is labor-intensive. Moreover, unless QT measured using thermodilution is used to back-calculate aortic diameter, accuracy using the suprasternal notch approach is not acceptable. Accordingly, the method is useful only for obtaining very intermittent estimates of QT, and it has not been widely adopted by clinicians.A more promising, albeit more invasive, approach has been

1	Accordingly, the method is useful only for obtaining very intermittent estimates of QT, and it has not been widely adopted by clinicians.A more promising, albeit more invasive, approach has been introduced. In this method blood flow velocity is con-tinuously monitored in the descending thoracic aorta using a continuous-wave Doppler transducer introduced into the esoph-agus. The probe is connected to a monitor which continuously displays the blood flow velocity profile in the descending aorta as well as the calculated QT. In order to maximize the accuracy of the device, the probe position must be adjusted to obtain the peak velocity in the aorta. In order to transform blood flow in the descending aorta into QT, a correction factor is applied that is based on the assumption that only 70% of the flow at the root of the aorta is still present in the descending thoracic aorta. Aortic cross-sectional area is estimated using a nomogram based on the patient’s age, weight, and height. Results

1	the flow at the root of the aorta is still present in the descending thoracic aorta. Aortic cross-sectional area is estimated using a nomogram based on the patient’s age, weight, and height. Results using these methods appear to be reasonably accurate across a broad spectrum of patients. A meta-analysis of the available data shows a good correlation between cardiac output estimates obtained by trans-esophageal Doppler and PAC in critically ill patients.39 The ultrasonic device also calculates a derived parameter termed flow time corrected (FTc), which is the systolic flow time in the descending aorta corrected for heart rate. FTc is a function of preload, contractility, and vascular input impedance. Although it is not a pure measure of preload, Doppler-based estimates of SV and FTc have been used successfully to guide volume resuscitation in high-risk surgical patients undergoing major operations.37Impedance CardiographyThe impedance to flow of alternating electrical current in

1	have been used successfully to guide volume resuscitation in high-risk surgical patients undergoing major operations.37Impedance CardiographyThe impedance to flow of alternating electrical current in regions of the body is commonly called bioimpedance. In the thorax, changes in the volume and velocity of blood in the tho-racic aorta lead to detectable changes in bioimpedance. The first derivative of the oscillating component of thoracic bio-impedance (dZ/dt) is linearly related to aortic blood flow. On the basis of this relationship, empirically derived formulas have been developed to estimate SV, and subsequently QT, nonin-vasively. This methodology is called impedance cardiography. The approach is attractive because it is noninvasive, provides a continuous readout of QT, and does not require extensive train-ing. Despite these advantages, measurements of QT obtained by impedance cardiography are not sufficiently reliable to be used for clinical decision making and have poor

1	not require extensive train-ing. Despite these advantages, measurements of QT obtained by impedance cardiography are not sufficiently reliable to be used for clinical decision making and have poor correlation with thermodilution.40Because of the limitations of bioimpedance devices, a newer approach for processing the impedance signal was devel-oped and commercialized. This approach is based on the recog-nition that the impedance signal has two components: amplitude and phase. Whereas the amplitude of the thoracic impedance signal is determined by all of the components of the thoracic cavity (bone, blood, muscle, and other soft tissues), phase shifts are determined entirely by pulsatile flow. The vast majority of pulsatile flow is related to blood moving within the aorta. There-fore, the “bioreactance” signal correlates closely with aortic flow, and cardiac output determined using this approach agrees closely with cardiac output measured using conventional indica-tor dilution

1	the “bioreactance” signal correlates closely with aortic flow, and cardiac output determined using this approach agrees closely with cardiac output measured using conventional indica-tor dilution techniques.41Pulse Contour AnalysisAnother method for determining cardiac output is an approach called pulse contour analysis for estimating SV on a beat-to-beat basis. The mechanical properties of the arterial tree and SV determine the shape of the arterial pulse waveform. The pulse contour method of estimating QT uses the arterial pressure waveform as an input for a model of the systemic circulation in order to determine beat-to-beat flow through the circulatory system. The parameters of resistance, compliance, and imped-ance are initially estimated based on the patient’s age and sex and can be subsequently refined by using a reference standard measurement of QT. The reference standard estimation of QT is obtained periodically using the indicator dilution approach by injecting the indicator

1	subsequently refined by using a reference standard measurement of QT. The reference standard estimation of QT is obtained periodically using the indicator dilution approach by injecting the indicator into a central venous catheter and Brunicardi_Ch13_p0433-p0452.indd 44322/02/19 2:21 PM 444BASIC CONSIDERATIONSPART Idetecting the transient increase in indicator concentration in the blood using an arterial catheter. In one commercially available embodiment of this approach, the lithium ion (Li+) is the indi-cator used for the periodic calibrations of the device. The lith-ium carbonate indicator can be injected into a peripheral vein, and the doses do not exert pharmacologically relevant effects in adult patients. The Li+ indicator dilution method has shown to be at least as reliable as other thermodilution methods over a broad range of CO in a variety of patients.41 In another com-mercially available system, a conventional bolus of cold fluid is used as the indicator for

1	as other thermodilution methods over a broad range of CO in a variety of patients.41 In another com-mercially available system, a conventional bolus of cold fluid is used as the indicator for calibration, via TPTD approaches as described previously. When the pulse contour analysis is com-bined with intermittent TPTD in this fashion, the continuous data provided by contour analysis is more precise than TPTD alone.38Measurements of QT based on pulse contour monitoring using these two approaches are comparable in accuracy to stan-dard pulmonary artery catheter (PAC)-thermodilution methods, but they are less invasive because transcardiac catheterization is not needed.42 Using online pressure waveform analysis, the computerized algorithms can calculate SV, QT, SVR, and an estimate of myocardial contractility, the rate of rise of the arte-rial systolic pressure (dP/dT). The use of pulse contour analy-sis has been applied using noninvasive photoplethysmographic measurements of arterial

1	contractility, the rate of rise of the arte-rial systolic pressure (dP/dT). The use of pulse contour analy-sis has been applied using noninvasive photoplethysmographic measurements of arterial pressure. However, the accuracy of this technique has been questioned, and its clinical utility remains to be determined.43One commercially available device that can be used for estimating cardiac output does not require external calibration. Instead, the relationship between pulse pressure and stroke vol-ume is determined using a proprietary algorithm that uses bio-metric data, such as age, gender and height, as inputs. Although this methodology is gaining fairly wide acceptance in critical care medicine, reported accuracy (in comparison to “gold stan-dard” approaches) is not very good.41Partial Carbon Dioxide RebreathingPartial carbon dioxide (CO2) rebreathing uses the Fick prin-ciple to estimate QT noninvasively. By intermittently altering the dead space within the ventilator circuit via a

1	Carbon Dioxide RebreathingPartial carbon dioxide (CO2) rebreathing uses the Fick prin-ciple to estimate QT noninvasively. By intermittently altering the dead space within the ventilator circuit via a rebreathing valve, changes in CO2 production (VCO2) and end-tidal CO2 (ETCO2) are used to determine cardiac output using a modified Fick equation:Q=VETTCOCO22˜˜Commercially available devices use this Fick principle to cal-culate QT using intermittent partial CO2 rebreathing through a disposable rebreathing loop. These devices consist of a CO2 sen-sor based on infrared light absorption, an airflow sensor, and a pulse oximeter. Changes in intrapulmonary shunt and hemody-namic instability impair the accuracy of QT estimated by partial CO2 rebreathing. Continuous inline pulse oximetry and inspired fraction of inspired O2 (Fio2) are used to estimate shunt fraction to correct QT.Some studies of the partial CO2 rebreathing approach sug-gest that this technique is not as accurate as

1	and inspired fraction of inspired O2 (Fio2) are used to estimate shunt fraction to correct QT.Some studies of the partial CO2 rebreathing approach sug-gest that this technique is not as accurate as thermodilution, the gold standard for measuring QT.42,44 However, other studies sug-gest that the partial CO2 rebreathing method for determination of QT compares favorably to measurements made using a PAC in critically ill patients.45Transesophageal EchocardiographyTransesophageal echocardiography (TEE) has made the transi-tion from operating room to intensive care unit. TEE requires that the patient be sedated and usually intubated for airway pro-tection. Using this powerful technology, global assessments of LV and RV function can be made, including determinations of ventricular volume, EF, and QT. Segmental wall motion abnor-malities, pericardial effusions, and tamponade can be readily identified with TEE. Doppler techniques allow estimation of atrial filling pressures. The technique is

1	QT. Segmental wall motion abnor-malities, pericardial effusions, and tamponade can be readily identified with TEE. Doppler techniques allow estimation of atrial filling pressures. The technique is somewhat cumbersome and requires considerable training and skill in order to obtain reliable results. Recently, a TEE probe has been introduced into practice that is small enough in diameter that it can be left in place for as long as 72 hours. While only limited data are cur-rently available with this probe, it seems like it will be a useful cardiac monitoring tool for use in selected, complex patients.Assessing Preload ResponsivenessAlthough pulse contour analysis or partial CO2 rebreathing may be able to provide estimates of SV and QT, these approaches alone can offer little or no information about the adequacy of preload. Thus, if QT is low, some other means must be employed to estimate preload. Many clinicians assess the adequacy of car-diac preload by determining CVP or PAOP. However,

1	about the adequacy of preload. Thus, if QT is low, some other means must be employed to estimate preload. Many clinicians assess the adequacy of car-diac preload by determining CVP or PAOP. However, neither CVP nor PAOP correlate well with the true parameter of inter-est, left ventricular end-diastolic volume (LVEDV).46 Extremely high or low CVP or PAOP results are informative, but readings in a large middle zone (i.e., 5 to 20 mmHg) are less useful. Fur-thermore, changes in CVP or PAOP fail to correlate well with changes in stroke volume.47,48 Echocardiography can be used to estimate LVEDV, but this approach is dependent on the skill and training of the individual using it, and isolated measure-ments of LVEDV fail to predict the hemodynamic response to alterations in preload.49When intrathoracic pressure increases during the appli-cation of positive airway pressure in mechanically ventilated patients, venous return decreases, and as a consequence, left ventricular stroke volume

1	intrathoracic pressure increases during the appli-cation of positive airway pressure in mechanically ventilated patients, venous return decreases, and as a consequence, left ventricular stroke volume (LVSV) also decreases. Therefore, pulse pressure variation (PPV) during a positive pressure episode can be used to predict the responsiveness of cardiac output to changes in preload.50,51 PPV is defined as the differ-ence between the maximal pulse pressure and the minimum pulse pressure divided by the average of these two pressures (Fig. 13-4). This approach has validated this by comparing PPV, CVP, PAOP, and systolic pressure variation as predictors of pre-load responsiveness in a cohort of critically ill patients. Patients were classified as being “preload responsive” if their cardiac index increased by at least 15% after rapid infusion of a standard volume of intravenous fluid.52 Receiver-operating characteristic (ROC) curves demonstrated that PPV was the best predictor of preload

1	increased by at least 15% after rapid infusion of a standard volume of intravenous fluid.52 Receiver-operating characteristic (ROC) curves demonstrated that PPV was the best predictor of preload responsiveness. Although atrial arrhythmias can inter-fere with the usefulness of this technique, PPV remains a useful approach for assessing preload responsiveness in most patients because of its simplicity and reliability.49Near-Infrared Spectroscopic Measurement of Tissue Hemoglobin Oxygen SaturationNear-infrared spectroscopy (NIRS) allows continuous, nonin-vasive measurement of tissue hemoglobin oxygen saturation (StO2) using near-infrared wave lengths of light (700–1000 nm). This technology is based on Beer’s law, which states that the transmission of light through a solution with a dissolved Brunicardi_Ch13_p0433-p0452.indd 44422/02/19 2:21 PM 445PHYSIOLOGIC MONITORING OF THE SURGICAL PATIENTCHAPTER 13Figure 13-4. Calculation of pulse pressure variation as it would appear on bedside

1	Brunicardi_Ch13_p0433-p0452.indd 44422/02/19 2:21 PM 445PHYSIOLOGIC MONITORING OF THE SURGICAL PATIENTCHAPTER 13Figure 13-4. Calculation of pulse pressure variation as it would appear on bedside monitor. This provides a helpful and rapid assessment of fluid responsiveness in the critically ill mechanically ventilated patient.PPmax + PPmin2PPV (%) =PPmax – PPmin× 100InspirationArterial blood pressure (mmHg)ExpirationInspirationInspirationExpirationTimePPminPPmaxsolute decreases exponentially as the concentration of the sol-ute increases. In mammalian tissue, three compounds change their absorption pattern when oxygenated: cytochrome aa3, myoglobin, and hemoglobin. Because of the distinct absorption spectra of oxyhemoglobin and deoxyhemoglobin, Beer’s law can be used to detect their relative concentrations within tissue. Thus, the relative concentrations of the types of hemoglobin can be determined by measuring the change in light inten-sity as it passes through the tissue. Since

1	concentrations within tissue. Thus, the relative concentrations of the types of hemoglobin can be determined by measuring the change in light inten-sity as it passes through the tissue. Since about 20% of blood volume is intra-arterial and the StO2 measurements are taken without regard to systole or diastole, spectroscopic measure-ments are primarily indicative of the venous oxyhemoglobin concentration.NIRS has been evaluated to assess the severity of traumatic shock in animal models and in trauma patients. Studies have shown that peripheral muscle StO2, as determined by NIRS, is as accurate as other endpoints of resuscitation (i.e., base deficit, mixed venous oxygen saturation) in a porcine model of hemor-rhagic shock.53 Continuously measured StO2 has been evaluated in blunt trauma patients as a predictor of the development of multiple organ dysfunction syndrome (MODS) and mortality.54 383 patients were prospectively studied at seven level I trauma centers. StO2 was monitored for 24

1	as a predictor of the development of multiple organ dysfunction syndrome (MODS) and mortality.54 383 patients were prospectively studied at seven level I trauma centers. StO2 was monitored for 24 hours after admission along with vital signs and other endpoints of resuscitation such as base deficit (BD). Minimum StO2 (using a minimum StO2 ≤75% as a cutoff) had a similar sensitivity and specificity in predicting the development of MODS as BD ≥6 mEq/L. StO2 and BD were also comparable in predicting mortality. Thus, NIRS-derived muscle StO2 measurements perform similarly to BD in identify-ing poor perfusion and predicting the development of MODS or death after severe torso trauma, yet have the additional advan-tages of being continuous and noninvasive. Ongoing prospec-tive studies will help determine the clinical utility of continuous monitoring of StO2 in clinical scenarios such as trauma, hemor-rhagic shock, sepsis, etc.RESPIRATORY MONITORINGThe ability to monitor various parameters of

1	the clinical utility of continuous monitoring of StO2 in clinical scenarios such as trauma, hemor-rhagic shock, sepsis, etc.RESPIRATORY MONITORINGThe ability to monitor various parameters of respiratory func-tion is of utmost importance in critically ill patients. Many of these patients require mechanical ventilation. Monitoring of their respiratory physiology is necessary to assess the adequacy of oxygenation and ventilation, guide weaning and liberation from mechanical ventilation, and detect adverse events associ-ated with respiratory failure and mechanical ventilation. These parameters include gas exchange, neuromuscular activity, respi-ratory mechanics, and patient effort.Arterial Blood GasesBlood gas analysis may provide useful information when caring for patients with respiratory failure. However, even in the absence of respiratory failure or the need for mechanical ventilation, blood gas determinations also can be valuable to detect alterations in acid-base balance due to low

1	However, even in the absence of respiratory failure or the need for mechanical ventilation, blood gas determinations also can be valuable to detect alterations in acid-base balance due to low QT, sepsis, renal failure, severe trauma, medication or drug overdose, or altered mental status. Arterial blood can be analyzed for pH, Po2, Pco2, HCO3– con-centration and calculated base deficit. When indicated, carboxy-hemoglobin and methemoglobin levels also can be measured. In recent years, efforts have been made to decrease the unnecessary use of arterial blood gas analysis. Serial arterial blood gas deter-minations are not necessary for routine weaning from mechanical ventilation in the majority of postoperative patients.Most bedside blood gas analyses still involve removal of an aliquot of blood from the patient, although continuous bedside arterial blood gas determinations are now possible without sam-pling via an indwelling arterial catheter that contains a biosensor. In studies

1	of blood from the patient, although continuous bedside arterial blood gas determinations are now possible without sam-pling via an indwelling arterial catheter that contains a biosensor. In studies comparing the accuracy of continuous arterial blood gas and pH monitoring with a conventional laboratory blood gas analyzer, excellent agreement between the two methods has been demonstrated.55 Continuous monitoring can reduce the volume of blood loss due to phlebotomy and dramatically decrease the time necessary to obtain blood gas results. Continuous monitor-ing, however, is expensive and is not widely employed.Determinants of Oxygen DeliveryThe primary goal of the cardiovascular and respiratory systems is to deliver oxygenated blood to the tissues. DO2 is dependent to a greater degree on the oxygen saturation of hemoglobin (Hgb) in arterial blood (Sao2) than on the partial pressure of oxygen in arterial blood (Pao2). DO2 also is dependent on QT and Hgb. As discussed earlier and

1	on the oxygen saturation of hemoglobin (Hgb) in arterial blood (Sao2) than on the partial pressure of oxygen in arterial blood (Pao2). DO2 also is dependent on QT and Hgb. As discussed earlier and illustrated mathematically by previous equations, the dissolved oxygen in blood makes only a negligible contribution to DO2. Sao2 in mechanically venti-lated patients depends on the mean airway pressure, the frac-tion of inspired oxygen (Fio2), and SvO2. Thus, when Sao2 is low, the clinician has only a limited number of ways to improve this parameter. The clinician can increase mean airway pres-sure by increasing positive-end expiratory pressure (PEEP) or inspiratory time. Fio2 can be increased to a maximum of 1.0 by decreasing the amount of room air mixed with the oxygen sup-plied to the ventilator. SvO2 can be increased by increasing Hgb Brunicardi_Ch13_p0433-p0452.indd 44522/02/19 2:21 PM 446BASIC CONSIDERATIONSPART Ior QT or decreasing oxygen utilization (e.g., by administering a

1	SvO2 can be increased by increasing Hgb Brunicardi_Ch13_p0433-p0452.indd 44522/02/19 2:21 PM 446BASIC CONSIDERATIONSPART Ior QT or decreasing oxygen utilization (e.g., by administering a muscle relaxant and sedation).Peak and Plateau Airway PressureAirway pressures are routinely monitored in mechanically ven-tilated patients. The peak airway pressure measured at the end of inspiration (Ppeak) is a function of the tidal volume, the resistance of the airways, lung/chest wall compliance, and peak inspiratory flow. The airway pressure measured at the end of inspiration when the inhaled volume is held in the lungs by briefly clos-ing the expiratory valve is termed the plateau airway pressure (Pplateau). As a static parameter, plateau airway pressure is indepen-dent of the airway resistance and peak airway flow and is related to the lung/chest wall compliance and delivered tidal volume. Mechanical ventilators monitor Ppeak with each breath and can be set to trigger an alarm if the

1	and peak airway flow and is related to the lung/chest wall compliance and delivered tidal volume. Mechanical ventilators monitor Ppeak with each breath and can be set to trigger an alarm if the Ppeak exceeds a predetermined thresh-old. Pplateau is not measured routinely with each delivered tidal vol-ume but rather is measured intermittently by setting the ventilator to close the exhalation circuit briefly at the end of inspiration and record the airway pressure when airflow is zero.If both Ppeak and Pplateau are increased (and tidal volume is not excessive), then the problem is a decrease in the compli-ance in the lung/chest wall unit. Common causes of this problem include pneumothorax, hemothorax, lobar atelectasis, pulmo-nary edema, pneumonia, acute respiratory distress syndrome (ARDS), active contraction of the chest wall or diaphragmatic muscles, abdominal distention, and intrinsic PEEP, such as occurs in patients with bronchospasm and insufficient expira-tory times. When Ppeak is

1	active contraction of the chest wall or diaphragmatic muscles, abdominal distention, and intrinsic PEEP, such as occurs in patients with bronchospasm and insufficient expira-tory times. When Ppeak is increased but Pplateau is relatively nor-mal, the primary problem is an increase in airway resistance, such as occurs with bronchospasm, use of a small-caliber endo-tracheal tube, or kinking or obstruction of the endotracheal tube. A low Ppeak also should trigger an alarm, as it suggests a discon-tinuity in the airway circuit involving the patient and the ventila-tor. These scenarios are outlined in Table 13-4.Ventilator-induced lung injury (VILI) is now an estab-lished clinical entity of great relevance to the care of critically ill patients. Excessive airway pressure and tidal volume adversely affect pulmonary and possibly systemic responses to critical illness. Subjecting the lung parenchyma to excessive pressure, known as barotrauma, can result in parenchymal lung injury, diffuse

1	affect pulmonary and possibly systemic responses to critical illness. Subjecting the lung parenchyma to excessive pressure, known as barotrauma, can result in parenchymal lung injury, diffuse alveolar damage similar to ARDS, and pneumothorax, and can impair venous return and therefore limit cardiac output. Lung-protective ventilation strategies have been developed to prevent the development of VILI and improve patient outcomes. Table 13-4Scenarios associated with different combinations of Ppeak and Pplateau in ventilated patientsCONDITIONPpeakPplateauDecreased compliance of the system (ARDS, abdominal distention, intrinsic PEEP)⇑⇑Increase in airway resistance (bronchospasm, endotracheal tube obstruction/kinking, or small-caliber endotracheal tube)⇑normalDisconnected circuit⇓⇓In a large, multicenter, randomized trial of patients with ARDS from a variety of etiologies, limiting plateau airway pressure to less than 30 cm H2O and tidal volume to less than 6 mL/kg of ideal body weight

1	multicenter, randomized trial of patients with ARDS from a variety of etiologies, limiting plateau airway pressure to less than 30 cm H2O and tidal volume to less than 6 mL/kg of ideal body weight reduced 28-day mortality by 22% relative to a ventilator strategy that used a tidal volume of 12 mL/kg.56 For this reason, monitoring of plateau pressure and using a low tidal volume strategy in patients with ARDS is now the standard of care. Recent data also suggest that a lung-protective ventila-tion strategy is associated with improved clinical outcomes in ventilated patients without ARDS.57 Importantly, this strategy also has been shown to have benefit for high-risk patients under-going general anesthesia for surgical procedures, leading to a reduced overall rate of pulmonary complications in the peri-operative period as well as a reduced length of stay following surgery.58Pulse OximetryThe pulse oximeter is a microprocessor-based device that inte-grates oximetry and plethysmography to

1	in the peri-operative period as well as a reduced length of stay following surgery.58Pulse OximetryThe pulse oximeter is a microprocessor-based device that inte-grates oximetry and plethysmography to provide continuous noninvasive monitoring of the oxygen saturation of arterial blood (Sao2). It is considered one of the most important and useful technologic advances in patient monitoring. Continuous, noninvasive monitoring of arterial oxygen saturation is pos-sible using light-emitting diodes and sensors placed on the skin. Pulse oximetry employs two wavelengths of light (i.e., 660 nm and 940 nm) to analyze the pulsatile component of blood flow between the light source and sensor. Because oxyhemoglobin and deoxyhemoglobin have different absorption spectra, differ-ential absorption of light at these two wavelengths can be used to calculate the fraction of oxygen saturation of hemoglobin. Under normal circumstances, the contributions of carboxyhe-moglobin and methemoglobin are minimal.

1	at these two wavelengths can be used to calculate the fraction of oxygen saturation of hemoglobin. Under normal circumstances, the contributions of carboxyhe-moglobin and methemoglobin are minimal. However, if car-boxyhemoglobin levels are elevated, the pulse oximeter will incorrectly interpret carboxyhemoglobin as oxyhemoglobin and the arterial saturation displayed will be falsely elevated. When the concentration of methemoglobin is markedly increased, the Sao2 will be displayed as 85%, regardless of the true arterial saturation.59 The accuracy of pulse oximetry begins to decline at Sao2 values less than 92% and tends to be unreliable for values less than 85%.60Several studies have assessed the frequency of arterial oxygen desaturation in hospitalized patients and its effect on outcome. Monitoring pulse oximetry in surgical patients is asso-ciated with a reduction in unrecognized deterioration, rescue events, and transfers to the ICU.61 Because of its clinical rel-evance, ease of

1	Monitoring pulse oximetry in surgical patients is asso-ciated with a reduction in unrecognized deterioration, rescue events, and transfers to the ICU.61 Because of its clinical rel-evance, ease of use, noninvasive nature, and cost-effectiveness, pulse oximetry has become a routine monitoring strategy in patients with respiratory disease, intubated patients, and those undergoing surgical intervention under sedation or general anes-thesia. Pulse oximetry is especially useful in the titration of Fio2 and PEEP for patients receiving mechanical ventilation, and during weaning from mechanical ventilation. The widespread use of pulse oximetry has decreased the need for arterial blood gas determinations in critically ill patients.Pulse CO-OximetryWhile simple pulse oximeters such as those described previ-ously are helpful for determination of the Sao2, extensions of the technology may prove valuable for determination of total hemoglobin concentration as well. Through the use of multiple

1	previ-ously are helpful for determination of the Sao2, extensions of the technology may prove valuable for determination of total hemoglobin concentration as well. Through the use of multiple additional wavelengths of light, clinicians can leverage the dif-ferent spectrophotometric properties of the multiple different Brunicardi_Ch13_p0433-p0452.indd 44622/02/19 2:21 PM 447PHYSIOLOGIC MONITORING OF THE SURGICAL PATIENTCHAPTER 13oxidative states of hemoglobin to get a complete readout of the total hemoglobin present in a given volume, leading to a noninvasive measurement of Hgb. These devices are referred to as pulse CO-Oximeters, as opposed to pulse oximeters, to dif-ferentiate that they are capable of measuring other hemoglobin moieties. Currently, there are two such devices that are com-mercially available for clinical use.Theoretically, the capacity to noninvasively measure Hgb concentration in real time would offer significant clinical ben-efit. These include obviating the

1	are com-mercially available for clinical use.Theoretically, the capacity to noninvasively measure Hgb concentration in real time would offer significant clinical ben-efit. These include obviating the need for serial blood draws, the early detection of potential postsurgical hemorrhage, and more judicious usage of blood transfusions. In practice, there are mul-tiple factors that currently affect the accuracy of the technique. Multiple studies have demonstrated that biases with noninvasive Hgb monitoring are inversely correlated with hemoglobin con-centration in a variety of monitoring scenarios; with decreasing hemoglobin values the noninvasive approaches tend to overes-timate the true Hgb.62-64 This poses a significant challenge for monitoring the critically ill patient, as frequently anemia is a common comorbid condition. On the other hand, if the continu-ous monitoring capacity afforded by these monitors can provide usable trend data, that may still provide clinical utility despite

1	is a common comorbid condition. On the other hand, if the continu-ous monitoring capacity afforded by these monitors can provide usable trend data, that may still provide clinical utility despite less accuracy at low hemoglobin levels. To date, there have been relatively few studies validating the trending capacity of noninvasive Hgb monitoring compared to serial blood draws, with limited agreement due to differences in analysis and study design.65 Further studies are required to evaluate the clinical utility of this potentially useful technology.CapnometryCapnometry is the measurement of carbon dioxide in the airway throughout the respiratory cycle. Capnometry is most commonly measured by infrared light absorption. CO2 absorbs infrared light at a peak wavelength of approximately 4.27 µm. Capnom-etry works by passing infrared light through a sample chamber to a detector on the opposite side. More infrared light passing through the sample chamber (i.e., less CO2) causes a larger

1	µm. Capnom-etry works by passing infrared light through a sample chamber to a detector on the opposite side. More infrared light passing through the sample chamber (i.e., less CO2) causes a larger sig-nal in the detector relative to the infrared light passing through a reference cell. Capnometric determination of the partial pressure of CO2 in end-tidal exhaled gas (Petco2) is used as a surrogate for the partial pressure of CO2 in arterial blood (Paco2) during mechanical ventilation. In healthy subjects, Petco2 is about 1 to 5 mmHg less than Paco2.66 Thus, Petco2 can be used to estimate Paco2 without the need for blood gas determination. However, changes in Petco2 may not correlate with changes in Paco2 dur-ing a number of pathologic conditions.Capnography allows the confirmation of endotracheal intubation and continuous assessment of ventilation, integrity of the airway, operation of the ventilator, and cardiopulmonary function. Capnometers are configured with either an inline

1	endotracheal intubation and continuous assessment of ventilation, integrity of the airway, operation of the ventilator, and cardiopulmonary function. Capnometers are configured with either an inline sen-sor or a sidestream sensor. The sidestream systems are lighter and easy to use, but the thin tubing that samples the gas from the ventilator circuit can become clogged with secretions or condensed water, preventing accurate measurements. The inline devices are bulky and heavier but are less likely to become clogged. Continuous monitoring with capnography has become routine during surgery under general anesthesia and for some intensive care patients. A number of situations can be promptly detected with continuous capnography. A sudden reduction in Petco2 suggests either obstruction of the sam-pling tubing with water or secretions, or a catastrophic event such as loss of the airway, airway disconnection or obstruction, ventilator malfunction, or a marked decrease in QT. If the airway

1	sam-pling tubing with water or secretions, or a catastrophic event such as loss of the airway, airway disconnection or obstruction, ventilator malfunction, or a marked decrease in QT. If the airway is connected and patent and the ventilator is functioning prop-erly, then a sudden decrease in Petco2 should prompt efforts to rule out cardiac arrest, massive pulmonary embolism, or cardio-genic shock. Petco2 can be persistently low during hyperven-tilation or with an increase in dead space such as occurs with pulmonary embolization (even in the absence of a change in QT). Causes of an increase in Petco2 include reduced minute ventilation or increased metabolic rate.RENAL MONITORINGUrine OutputBladder catheterization with an indwelling catheter allows the monitoring of urine output, usually recorded hourly by the nurs-ing staff. With a patent Foley catheter, urine output is a gross indicator of renal perfusion. The generally accepted normal urine output is 0.5 mL/kg per hour for adults and

1	hourly by the nurs-ing staff. With a patent Foley catheter, urine output is a gross indicator of renal perfusion. The generally accepted normal urine output is 0.5 mL/kg per hour for adults and 1 to 2 mL/kg per hour for neonates and infants. Oliguria may reflect inadequate renal artery perfusion due to hypotension, hypovolemia, or low QT. Low urine flow also can be a sign of intrinsic renal dysfunc-tion. It is important to recognize that normal urine output does not exclude the possibility of impending renal failure.Bladder PressureThe triad of oliguria, elevated peak airway pressures, and ele-vated intra-abdominal pressure is known as abdominal com-partment syndrome (ACS). This syndrome, first described in patients after repair of ruptured abdominal aortic aneurysm, is associated with interstitial edema of the abdominal organs, resulting in elevated intra-abdominal pressure (IAP). When IAP exceeds venous or capillary pressures, perfusion of the kidneys and other intra-abdominal

1	interstitial edema of the abdominal organs, resulting in elevated intra-abdominal pressure (IAP). When IAP exceeds venous or capillary pressures, perfusion of the kidneys and other intra-abdominal viscera is impaired. Oligu-ria is a cardinal sign. While the diagnosis of ACS is a clinical one, measuring IAP is useful to confirm the diagnosis. Ideally, a catheter inserted into the peritoneal cavity could measure IAP to substantiate the diagnosis. In practice, transurethral bladder pressure measurement reflects IAP and is most often used to confirm the presence of ACS. After instilling 50 to 100 mL of sterile saline into the bladder via a Foley catheter, the tubing is connected to a transducing system to measure bladder pressure in the supine position at end-expiration.Intra-abdominal hypertension is defined as an IAP ≥12 mmHg recorded on three standard measurements conducted 4 to 6 hours apart and is separated into several grades. The diag-nosis of ACS is the presence of an IAP ≥20

1	is defined as an IAP ≥12 mmHg recorded on three standard measurements conducted 4 to 6 hours apart and is separated into several grades. The diag-nosis of ACS is the presence of an IAP ≥20 mmHg recorded by three measurements 1 to 6 hours apart, along with new onset of organ dysfunction (Table 13-5).67-69 Less commonly, gastric or inferior vena cava pressures can be monitored with appropriate catheters to detect elevated intra-abdominal pressures.NEUROLOGIC MONITORINGIntracranial PressureBecause the brain is rigidly confined within the bony skull, cere-bral edema or mass lesions increase intracranial pressure (ICP). Monitoring of ICP is currently recommended in patients with severe traumatic brain injury (TBI), defined as a Glasgow Coma Scale (GCS) score less than or equal to 8 with an abnormal computed tomography (CT) scan, and in patients with severe TBI and a normal CT scan if two or more of the following are present: age >40 years, unilateral or bilateral motor posturing,

1	an abnormal computed tomography (CT) scan, and in patients with severe TBI and a normal CT scan if two or more of the following are present: age >40 years, unilateral or bilateral motor posturing, Brunicardi_Ch13_p0433-p0452.indd 44722/02/19 2:21 PM 448BASIC CONSIDERATIONSPART ITable 13-5Bladder pressure measurements in the assessment of intra-abdominal hypertension or abdominal compartment syndromeRECORDED PRESSURE (mmHg)GRADE OF IAH OR ACS5–7NormalIn the absence of organ dysfunction:12–15Grade I IAH16–20Grade II IAH21–25Grade III IAH>25Grade IV IAHIn the presence of new onset organ dysfunction:>20ACSData from Kirkpatrick AW, Roberts DJ, De Waele J, et al. Intra-abdominal hypertension and the abdominal compartment syndrome: updated consensus definitions and clinical practice guidelines from the World Society of the Abdominal Compartment Syndrome, Intensive Care Med. 2013 Jul;39(7):1190-1206.or systolic blood pressure <90 mmHg.70 ICP monitoring also is indicated in patients with

1	from the World Society of the Abdominal Compartment Syndrome, Intensive Care Med. 2013 Jul;39(7):1190-1206.or systolic blood pressure <90 mmHg.70 ICP monitoring also is indicated in patients with acute subarachnoid hemorrhage with coma or neurologic deterioration, intracranial hemorrhage with intraventricular blood, ischemic middle cerebral artery stroke, fulminant hepatic failure with coma and cerebral edema on CT scan, and global cerebral ischemia or anoxia with cerebral edema on CT scan. The goal of ICP monitoring is to ensure that cerebral perfusion pressure (CPP) is adequate to support perfu-sion of the brain. CPP is equal to the difference between MAP and ICP: CPP = MAP – ICP.One type of ICP measuring device, the ventriculostomy catheter, consists of a fluid-filled catheter inserted into a cere-bral ventricle and connected to an external pressure transducer. This device permits measurement of ICP but also allows drain-age of cerebrospinal fluid (CSF) as a means to lower ICP and

1	into a cere-bral ventricle and connected to an external pressure transducer. This device permits measurement of ICP but also allows drain-age of cerebrospinal fluid (CSF) as a means to lower ICP and sample CSF for laboratory studies. Other devices locate the pressure transducer within the central nervous system and are used only to monitor ICP. These devices can be placed in the intraventricular, parenchymal, subdural, or epidural spaces. Ventriculostomy catheters are the accepted standard for moni-toring ICP in patients with TBI due to their accuracy, ability to drain CSF, and low complication rate. The associated com-plications include infection (5%), hemorrhage (1.1%), catheter malfunction or obstruction (6.3–10.5%), and malposition with injury to cerebral tissue.71The purpose of ICP monitoring is to detect and treat abnormal elevations of ICP that may be detrimental to cere-bral perfusion and function. In TBI patients, ICP greater than 20 mmHg is associated with unfavorable

1	ICP monitoring is to detect and treat abnormal elevations of ICP that may be detrimental to cere-bral perfusion and function. In TBI patients, ICP greater than 20 mmHg is associated with unfavorable outcomes.72 However, few studies have shown that treatment of elevated ICP improves clinical outcomes in human trauma patients. In a randomized, controlled, double-blind trial, Eisenberg and colleagues dem-onstrated that maintaining ICP less than 25 mmHg in patients without craniectomy and less than 15 mmHg in patients with craniectomy is associated with improved outcome.73 In patients with low CPP, therapeutic strategies to correct CPP can be directed at increasing MAP or decreasing ICP. While it has been recommended that CPP be maintained between 50 and 70 mmHg, the evidence to support this recommendation are not overly compelling.74 Furthermore, a retrospective cohort study of patients with severe TBI found that ICP/CPP-targeted neurointensive care was associated with prolonged

1	this recommendation are not overly compelling.74 Furthermore, a retrospective cohort study of patients with severe TBI found that ICP/CPP-targeted neurointensive care was associated with prolonged mechanical ventilation and increased therapeutic interventions, without evi-dence for improved outcome in patients who survive beyond 24 hours.75Electroencephalogram and Evoked PotentialsElectroencephalography offers the capacity to monitor global neurologic electrical activity, while evoked potential monitor-ing can assess pathways not detected by the conventional EEG. Continuous EEG (CEEG) monitoring in the intensive care unit permits ongoing evaluation of cerebral cortical activity. It is especially useful in obtunded and comatose patients. CEEG also is useful for monitoring of therapy for status epilepticus and detecting early changes associated with cerebral ischemia. CEEG can be used to adjust the level of sedation, especially if high-dose barbiturate therapy is being used to manage

1	status epilepticus and detecting early changes associated with cerebral ischemia. CEEG can be used to adjust the level of sedation, especially if high-dose barbiturate therapy is being used to manage elevated ICP. Somatosensory and brain stem evoked potentials are less affected by the administration of sedatives than is the EEG. Evoked potentials are useful for localizing brain stem lesions or proving the absence of such structural lesions in cases of metabolic or toxic coma. They also can provide prognostic data in posttraumatic coma.An advance in EEG monitoring is the use of the bispectral index (BIS) to titrate the level of sedative medications. While sedative drugs are usually titrated to the clinical neurologic examination, the BIS device has been used in the operating room to continuously monitor the depth of anesthesia. The BIS is an empiric measurement statistically derived from a data-base of over 5000 EEGs.76 The BIS is derived from bifrontal EEG recordings and analyzed for

1	monitor the depth of anesthesia. The BIS is an empiric measurement statistically derived from a data-base of over 5000 EEGs.76 The BIS is derived from bifrontal EEG recordings and analyzed for burst suppression ratio, rela-tive alpha to beta ratio, and bicoherence. Using a multivariate regression model, a linear numeric index (BIS) is calculated, ranging from 0 (isoelectric EEG) to 100 (fully awake). Its use has been associated with lower consumption of anesthet-ics during surgery and earlier awakening and faster recovery from anesthesia.77 The BIS also has been validated as a useful approach for monitoring the level of sedation for ICU patients, using the revised Sedation-Agitation Scale as a gold standard.78Transcranial Doppler UltrasonographyThis modality provides a noninvasive method for evaluating cerebral hemodynamics. Transcranial Doppler (TCD) measure-ments of middle and anterior cerebral artery blood flow velocity are useful for the diagnosis of cerebral vasospasm after

1	for evaluating cerebral hemodynamics. Transcranial Doppler (TCD) measure-ments of middle and anterior cerebral artery blood flow velocity are useful for the diagnosis of cerebral vasospasm after sub-arachnoid hemorrhage. Qureshi and associates demonstrated that an increase in the middle cerebral artery mean flow velocity as assessed by TCD is an independent predictor of symptom-atic vasospasm in a prospective study of patients with aneurys-mal subarachnoid hemorrhage.79 In addition, while some have proposed using TCD to estimate ICP, studies have shown that TCD is not a reliable method for estimating ICP and CPP and currently cannot be endorsed for this purpose.80 TCD also is useful to confirm the clinical examination for determining brain death in patients with confounding factors such as the presence of CNS depressants or metabolic encephalopathy.Jugular Venous OximetryWhen the arterial oxygen content, hemoglobin concentration, and the oxyhemoglobin dissociation curve are constant,

1	the presence of CNS depressants or metabolic encephalopathy.Jugular Venous OximetryWhen the arterial oxygen content, hemoglobin concentration, and the oxyhemoglobin dissociation curve are constant, changes in jugular venous oxygen saturation (Sjo2) reflect changes in the difference between cerebral oxygen delivery and demand. Brunicardi_Ch13_p0433-p0452.indd 44822/02/19 2:21 PM 449PHYSIOLOGIC MONITORING OF THE SURGICAL PATIENTCHAPTER 13Generally, a decrease in Sjo2 reflects cerebral hypoperfusion, whereas an increase in Sjo2 indicates the presence of hyperemia. Sjo2 monitoring cannot detect decreases in regional cerebral blood flow if overall perfusion is normal or above normal. This technique requires the placement of a catheter in the jugular bulb, usually via the internal jugular vein. Catheters that permit intermittent aspiration of jugular venous blood for analysis or continuous oximetry catheters are available.Low Sjo2 is associated with poor outcomes after TBI.81

1	jugular vein. Catheters that permit intermittent aspiration of jugular venous blood for analysis or continuous oximetry catheters are available.Low Sjo2 is associated with poor outcomes after TBI.81 Nevertheless, the value of monitoring Sjo2 remains unproven. If it is employed, it should not be the sole monitoring technique, but rather should be used in conjunction with ICP and CPP monitoring. By monitoring ICP, CPP, and Sjo2, early interven-tion with volume, vasopressors, and hyperventilation has been shown to prevent ischemic events in patients with TBI.82Transcranial Near-Infrared SpectroscopyTranscranial near-infrared spectroscopy (NIRS) is a noninvasive continuous monitoring method to determine cerebral oxygen-ation. It employs technology similar to that of pulse oximetry to determine the concentrations of oxyand deoxyhemoglobin with near-infrared light and sensors and takes advantage of the relative transparency of the skull to light in the near-infrared region of the spectrum.

1	the concentrations of oxyand deoxyhemoglobin with near-infrared light and sensors and takes advantage of the relative transparency of the skull to light in the near-infrared region of the spectrum. Continuous monitoring of cerebral per-fusion via transcranial NIRS may provide a method to detect early cerebral ischemia in patients with traumatic brain injury.83 Nevertheless, this form of monitoring remains largely a research tool at the present time.Recently, some authors have reported its use as a poten-tial triage tool for prehospital care in the management of TBI, as NIRS allows for rapid screening for intracranial hematoma. Two small EMS studies demonstrated that handheld NIRS devices may be feasible adjunct tools in this setting, particularly when CT scanners may not be readily available.84,85Brain Tissue Oxygen TensionWhile the standard of care for patients with severe TBI includes ICP and CPP monitoring, this strategy does not always prevent secondary brain injury. Growing

1	Tissue Oxygen TensionWhile the standard of care for patients with severe TBI includes ICP and CPP monitoring, this strategy does not always prevent secondary brain injury. Growing evidence suggests that moni-toring local brain tissue oxygen tension (PbtO2) may be a useful adjunct to ICP monitoring in these patients. Normal values for PbtO2 are 20 to 40 mmHg, and critical levels are 8 to 10 mmHg. A recent clinical study sought to determine whether the addi-tion of a PbtO2 monitor to guide therapy in severe traumatic brain injury was associated with improved patient outcomes.86 Twenty-eight patients with severe traumatic brain injury (GCS score ≤8) were enrolled in an observational study at a level I trauma center. These patients received invasive ICP and PbtO2 monitoring and were compared with 25 historical controls matched for age, injuries, and admission GCS score that had undergone ICP monitoring alone. Goals of therapy in both groups included maintaining an ICP <20 mmHg and a CPP

1	with 25 historical controls matched for age, injuries, and admission GCS score that had undergone ICP monitoring alone. Goals of therapy in both groups included maintaining an ICP <20 mmHg and a CPP >60 mmHg. Among patients with PbtO2 monitoring, therapy also was directed at maintaining PbtO2 >25 mmHg. The groups had similar mean daily ICP and CPP levels. The mortality rate in the historical controls treated with standard ICP and CPP management was 44%. Mortality was significantly lower in the patients who had therapy guided by PbtO2 monitoring in addition to ICP and CPP (25%; P <.05). The benefits of PbtO2 monitoring may include the early detection of brain tissue isch-emia despite normal ICP and CPP. In addition, PbtO2-guided management may reduce potential adverse effects associated with therapies to maintain ICP and CPP.CONCLUSIONSModern intensive care is predicated by the need and ability to continuously monitor a wide range of physiologic parameters. This capability has

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1	Med. 1999;27(8):1499-1504. 79. Qureshi AI, Sung GY, Razumovsky AY, Lane K, Straw RN, Ulatowski JA. Early identification of patients at risk for symp-tomatic vasospasm after aneurysmal subarachnoid hemorrhage. Crit Care Med. 2000;28(4):984-990. 80. Czosnyka M, Matta BF, Smielewski P, Kirkpatrick PJ, Pickard JD. Cerebral perfusion pressure in head-injured patients: a noninvasive assessment using transcranial Doppler ultrasonography. J Neurosurg. 1998;88(5):802-808. 81. Feldman Z, Robertson CS. Monitoring of cerebral hemody-namics with jugular bulb catheters. Crit Care Clin. 1997;13(1): 51-77. 82. Vigue B, Ract C, Benayed M, et al. Early SjvO2 monitoring in patients with severe brain trauma. Intensive Care Med. 1999;25(5):445-451. 83. Murkin JM, Arango M. Near-infrared spectroscopy as an index of brain and tissue oxygenation. Br J Anaesth. 2009;103(suppl 1):i3-i13. 84. Peters J, Van Wageningen B, Hoogerwerf N, Tan E. Near-infrared spectroscopy: a promising prehospital tool for

1	as an index of brain and tissue oxygenation. Br J Anaesth. 2009;103(suppl 1):i3-i13. 84. Peters J, Van Wageningen B, Hoogerwerf N, Tan E. Near-infrared spectroscopy: a promising prehospital tool for man-agement of traumatic brain injury. Prehosp Disaster Med. 2017;32(4):414-418. 85. Schober P, Bossers SM, Schwarte LA. Intracranial hematoma detection by near infrared spectroscopy in a helicopter emer-gency medical service: practical experience. Biomed Res Int. 2017;2017:1846830. 86. Stiefel MF, Spiotta A, Gracias VH, et al. Reduced mortality rate in patients with severe traumatic brain injury treated with brain tissue oxygen monitoring. J Neurosurg. 2005;103(5):805-811.Brunicardi_Ch13_p0433-p0452.indd 45122/02/19 2:21 PM

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1	Minimally Invasive Surgery, Robotics, Natural Orifice Transluminal Endoscopic Surgery, and Single-Incision Laparoscopic SurgeryDonn H. Spight, Blair A. Jobe, and John G. Hunter 14chapterINTRODUCTIONMinimally invasive surgery describes an area of surgery that crosses all traditional disciplines, from general surgery to neu-rosurgery. It is not a discipline unto itself, but more a philosophy of surgery, a way of thinking. Minimally invasive surgery is a means of performing major operations through small inci-sions, often using miniaturized, high-tech imaging sys-tems, to minimize the trauma of surgical exposure. Some believe that the term minimal access surgery more accurately describes the small incisions generally necessary to gain access to surgical sites in high-tech surgery, but John Wickham’s term minimally invasive surgery (MIS) is widely used because it describes the paradox of postmodern high-tech surgery—small holes, big operations.Robotic surgery today is practiced using a

1	Wickham’s term minimally invasive surgery (MIS) is widely used because it describes the paradox of postmodern high-tech surgery—small holes, big operations.Robotic surgery today is practiced using a single platform (Intuitive, Inc, Sunnyvale, CA) and should better be termed computer-enhanced surgery because the term robotics assumes autonomous action that is not a feature of the da Vinci robotic system. Instead, the da Vinci robot couples an ergonomic work-station that features stereoptic video imaging and intuitive micromanipulators (surgeon side) with a set of arms deliver-ing specialized laparoscopic instruments enhanced with more degrees of freedom than are allowed by laparoscopic surgery alone (patient side). A computer between the surgeon side and patient side removes surgical tremor and scales motion to allow 1precise microsurgery, which is helpful for microdissection and difficult anastomoses.Single-incision laparoscopic surgery (SILS), also called laparoendoscopic single-site

1	and scales motion to allow 1precise microsurgery, which is helpful for microdissection and difficult anastomoses.Single-incision laparoscopic surgery (SILS), also called laparoendoscopic single-site surgery (LESS), is a recent addi-tion to the armamentarium of the minimally invasive surgeon. As public awareness has grown, so too has its spread outside of larger institutions. SILS challenges the well-established paradigm of standard laparoscopic surgery by placing multiple trocars within the fascia at the umbilicus or through a single multichannel trocar at the umbilicus. The manipulation of tightly spaced instruments across the fulcrum of the abdomi-nal wall requires that the surgeon either operate in a crossed hands fashion or use specialized curved instruments to avoid clashing outside the body while working intra-abdominally. The primary advantage of SILS is the reduction to one surgical scar. Greater efficacy, safety, and cost savings have yet to be fully elucidated in the

1	the body while working intra-abdominally. The primary advantage of SILS is the reduction to one surgical scar. Greater efficacy, safety, and cost savings have yet to be fully elucidated in the increasing number of procedures that are being attempted in this manner. The advent of a robotic SILS platform now enables the computer reassignment of the surgeon’s hands, thus eliminating the difficult ergonomic challenges making the technique far more accessible.Natural orifice transluminal endoscopic surgery (NOTES) is an extension of interventional endoscopy. Using the mouth, anus, vagina, and urethra (natural orifices), flexible endoscopes are passed through the wall of the esophagus, stomach, colon, Introduction 453Historical Background 454Physiology and Pathophysiology of Minimally Invasive Surgery 455Laparoscopy / 455Thoracoscopy / 457Extracavitary Minimally Invasive Surgery / 457Anesthesia / 457The Minimally Invasive Team / 458Room Setup and the Minimally Invasive Suite /

1	Invasive Surgery 455Laparoscopy / 455Thoracoscopy / 457Extracavitary Minimally Invasive Surgery / 457Anesthesia / 457The Minimally Invasive Team / 458Room Setup and the Minimally Invasive Suite / 458Patient Positioning / 458General Principles of Access / 459Laparoscopic Access / 459Access for Subcutaneous and Extraperitoneal Surgery / 460Hand-Assisted Laparoscopic Access / 461Natural Orifice Transluminal Endoscopic Surgery Access / 461Single-Incision Laparoscopic Surgery Access / 462Port Placement / 462Imaging Systems / 463Energy Sources for Endoscopic and Endoluminal Surgery / 465Instrumentation / 467Robotic Surgery / 467Endoluminal and Endovascular Surgery / 469Natural Orifice Transluminal Endoscopic Surgery / 470Single-Incision Laparoscopic Surgery / 471Special Considerations 473Pediatric Laparoscopy / 473Laparoscopy During Pregnancy / 473Minimally Invasive Surgery and Cancer Treatment / 474Considerations in the Elderly and Infirm / 474Cirrhosis and Portal Hypertension /

1	Laparoscopy / 473Laparoscopy During Pregnancy / 473Minimally Invasive Surgery and Cancer Treatment / 474Considerations in the Elderly and Infirm / 474Cirrhosis and Portal Hypertension / 474Economics of Minimally Invasive Surgery / 474Education and Skill Acquisition / 474Telementoring / 475Innovation and Introduction of New Procedures / 475Brunicardi_Ch14_p0453-p0478.indd 45301/03/19 4:58 PM 454bladder, or vagina entering the mediastinum, the pleural space, or the peritoneal cavity. The advantage of this method of mini-mal access is principally the elimination of the scar associated with laparoscopy or thoracoscopy. Other advantages have yet to be elucidated, including pain reduction, need for hospitalization, and cost savings.HISTORICAL BACKGROUNDAlthough the term minimally invasive surgery is relatively recent, the history of its component parts is nearly 100 years old. What is considered the newest and most popular variety of MIS, laparoscopy, is in fact the oldest.

1	invasive surgery is relatively recent, the history of its component parts is nearly 100 years old. What is considered the newest and most popular variety of MIS, laparoscopy, is in fact the oldest. Primitive laparos-copy, placing a cystoscope within an inflated abdomen, was first performed by Kelling in 1901.1 Illumination of the abdomen required hot elements at the tip of the scope and was danger-ous. In the late 1950s, Hopkins described the rod lens, a method of transmitting light through a solid quartz rod with no heat and little light loss.1 Around the same time, thin quartz fibers were discovered to be capable of trapping light internally and conducting it around corners, opening the field of fiber optics and allowing the rapid development of flexible endoscopes.2,3 In the 1970s, the application of flexible endoscopy grew faster than that of rigid endoscopy except in a few fields such as gyne-cology and orthopedics.4 By the mid-1970s, rigid and flexible endoscopes made a rapid

1	application of flexible endoscopy grew faster than that of rigid endoscopy except in a few fields such as gyne-cology and orthopedics.4 By the mid-1970s, rigid and flexible endoscopes made a rapid transition from diagnostic instruments to therapeutic ones. The explosion of video-assisted surgery in the past 20 years was a result of the development of compact, high-resolution, charge-coupled devices (CCDs) that could be mounted on the internal end of flexible endoscopes or on the external end of a Hopkins telescope. Coupled with bright light sources, fiber-optic cables, and high-definition video monitors, the videoendoscope has changed our understanding of surgical anatomy and reshaped surgical practice.Flexible endoscopic imaging started in the 1960s with the first bundling of many quartz fibers into bundles, one for illu-mination and one for imaging. The earliest upper endoscopes revolutionized the diagnosis and treatment of gastroesophageal reflux and peptic ulcer disease and made

1	fibers into bundles, one for illu-mination and one for imaging. The earliest upper endoscopes revolutionized the diagnosis and treatment of gastroesophageal reflux and peptic ulcer disease and made possible early detec-tion of upper and lower gastrointestinal (GI) cancer at a stage that could be cured. The first endoscopic surgical procedure was the colonoscopic polypectomy, developed by Shinya and Wolfe, two surgeons from New York City. The percutane-ous endoscopic gastrostomy (PEG) invented by Gauderer and Ponsky may have been the first NOTES procedure, reported in 1981.5 Endoscopic pancreatic pseudocyst drainage is thought to be the next NOTES procedure developed; however, there was little energy and money put into the development of NOTES until a number of gastroenterologists claimed the ability to remove the gallbladder with a flexible endoscope, using a transgastric technique. With this pronouncement, the surgical community took notice and seized the momentum for NOTES

1	claimed the ability to remove the gallbladder with a flexible endoscope, using a transgastric technique. With this pronouncement, the surgical community took notice and seized the momentum for NOTES research and development. Today most intra-abdominal NOTES procedures remain within the realm of research or incorporate a hybrid laparoscopic technique outside of highly specialized centers. Clinically the transvaginal approach has been studied the most extensively. Evaluation of 551 female patients from the German NOTES registry has shown conversion and compli-cation rates similar to conventional laparoscopic surgery for cholecystectomy and appendectomy procedures.6 Endoscopic mucosal resection (EMR) of early-stage esophageal and gastric lesions has revolutionized the management of these malignan-cies. The peroral endoscopic myotomy (POEM) procedure for achalasia is showing clinical efficacy and gaining popularity.As the race to minimize the size and increase the function-ality of

1	malignan-cies. The peroral endoscopic myotomy (POEM) procedure for achalasia is showing clinical efficacy and gaining popularity.As the race to minimize the size and increase the function-ality of laparoscopic instruments progressed, the notion of using fewer access points to accomplish the same operations resulted in the development of single-incision laparoscopic surgery (SILS), synonymously termed laparoendoscopic single-site surgery (LESS). Viewed as a progression of laparoscopic surgery, SILS has recently garnered greater enthusiasm over its transvisceral NOTES counterpart.7 Currently the single-incision technique is used regularly across a wide variety of surgical areas including general, urologic, gynecologic, colorectal, and bariatric surgery.8 Although optical imaging produced the majority of MIS pro-cedures, other (traditionally radiologic) imaging technologies allowed the development of innovative procedures in the 1970s. Fluoroscopic imaging allowed the adoption of

1	the majority of MIS pro-cedures, other (traditionally radiologic) imaging technologies allowed the development of innovative procedures in the 1970s. Fluoroscopic imaging allowed the adoption of percutaneous vas-cular procedures, the most revolutionary of which was balloon angioplasty. Balloon-based procedures spread into all fields of medicine used to open up clogged lumens with minimal access. Stents were then developed that were used in many disciplines to keep the newly ballooned segment open. The culmination of fluoroscopic balloon and stent proficiency is exemplified by the transvenous intrahepatic portosystemic shunt and by the aortic stent graft, which has nearly replaced open elective abdominal aortic aneurysm repair.MIS procedures using ultrasound imaging have been limited to fairly crude exercises, such as fragmenting kidney stones and freezing liver tumors, because of the relatively low Key Points1 Minimally invasive surgery describes a philosophical approach to surgery in

1	crude exercises, such as fragmenting kidney stones and freezing liver tumors, because of the relatively low Key Points1 Minimally invasive surgery describes a philosophical approach to surgery in which access trauma is minimized without compromising the quality of the surgical procedure.2 The carbon dioxide pneumoperitoneum used for laparoscopy induces some unique pathophysiologic consequences.3 Robotic surgery has been most valuable in the performance of minimally invasive urologic, gynecologic, colorectal, and complex abdominal wall reconstruction procedures.4 Natural orifice transluminal endoscopic surgery represents an opportunity to perform truly scar-free surgery.5 Single-incision laparoscopic surgery reduces the amount of abdominal wall trauma but presents unique challenges to the traditional tenets of laparoscopic ergonomics.6 Laparoscopy during pregnancy is best performed in the sec-ond trimester and is safe if appropriate monitoring is performed.7 Laparoscopic surgery for

1	traditional tenets of laparoscopic ergonomics.6 Laparoscopy during pregnancy is best performed in the sec-ond trimester and is safe if appropriate monitoring is performed.7 Laparoscopic surgery for cancer is also appropriate if good tissue handling techniques are maintained.8 Training for laparoscopy requires practice outside of the operating room in a simulation laboratory.Brunicardi_Ch14_p0453-p0478.indd 45401/03/19 4:58 PM 455MINIMALLY INVASIVE SURGERYCHAPTER 14resolution of ultrasound devices. Newer, high-resolution ultra-sound methods with high-frequency crystals may act as a guide while performing minimally invasive resections of individual layers of the intestinal wall.Axial imaging, such as computed tomography (CT), has allowed the development of an area of MIS that often is not recognized because it requires only a CT scanner and a long needle. CT-guided drainage of abdominal fluid collections and percutaneous biopsy of abnormal tissues are minimally invasive means of

1	not recognized because it requires only a CT scanner and a long needle. CT-guided drainage of abdominal fluid collections and percutaneous biopsy of abnormal tissues are minimally invasive means of performing procedures that previously required a celi-otomy. CT-guided percutaneous radiofrequency (RF) ablation has emerged as a useful treatment for primary and metastatic liver tumors. This procedure also is performed laparoscopically under ultrasound guidance.9A powerful, noninvasive method of imaging that will allow the development of the least invasive—and potentially noninvasive—surgery is magnetic resonance imaging (MRI). MRI is an extremely valuable diagnostic tool, but it is only slowly coming to be of therapeutic value. One obstacle to the use of MRI for MIS is that image production and refreshment of the image as a procedure progresses are slow. Another is that all instrumentation must be nonmetallic when working with the powerful magnets of an MRI scanner. Moreover, MRI

1	and refreshment of the image as a procedure progresses are slow. Another is that all instrumentation must be nonmetallic when working with the powerful magnets of an MRI scanner. Moreover, MRI magnets are bulky and limit the surgeon’s access to the patient. Open magnets have been developed that allow the surgeon to stand between two large MRI coils, obtaining access to the portion of the patient being scanned. The advantage of MRI, in addition to the superb images produced, is that there is no radiation expo-sure to patient or surgeon. Some neurosurgeons are accumu-lating experience using MRI to perform frameless stereotactic surgery.Robotic surgery has been dreamed about for some time, and many science fiction–like devices have been developed over the years to provide mechanical assistance for the surgeon. The first computer-assisted robot was designed to accurately drill femoral shaft bone for wobble-free placement of hip prostheses. Although the concept was appealing, the robot

1	for the surgeon. The first computer-assisted robot was designed to accurately drill femoral shaft bone for wobble-free placement of hip prostheses. Although the concept was appealing, the robot proved no better than a skilled orthopedic surgeon and was a good deal slower. Following this, the first and only two commercially successful robots for laparoscopic surgery were developed in California. Computer Motion, founded by Yulun Wang in Santa Barbara, used National Science Foundation funds to create a mechanical arm, the Aesop robot, which held and moved the laparoscope with voice, foot, or hand control. In Northern California, a master-slave system first developed for surgery on the multina-tional space station by Philip Green was purchased by Fred Moll and Lonnie Smith, and then reengineered with the surgeon in mind to create a remarkably intuitive computer-enhanced surgi-cal platform. The company, Intuitive Surgical, was aptly named, and their primary product, the da Vinci robot, is

1	with the surgeon in mind to create a remarkably intuitive computer-enhanced surgi-cal platform. The company, Intuitive Surgical, was aptly named, and their primary product, the da Vinci robot, is currently the only major robotic platform on the market, although competi-tors are rapidly emerging in the horizon. Although eschewed by many experienced laparoscopists, the da Vinci achieved a toehold among many skilled surgeons who found that the robot could facilitate MIS procedures that were difficult with standard laparoscopic procedures. The latest iteration of the da Vinci Xi platform released in 2014 features high-defini-tion, three-dimensional vision and a dual-console capability allowing greater visualization, assistance, and instruction capa-bilities. Additionally, the new overhead boom design facilitates anatomical access from virtually any position enabling complex multiquadrant surgeries.PHYSIOLOGY AND PATHOPHYSIOLOGY OF MINIMALLY INVASIVE SURGERYEven with the least invasive of

1	design facilitates anatomical access from virtually any position enabling complex multiquadrant surgeries.PHYSIOLOGY AND PATHOPHYSIOLOGY OF MINIMALLY INVASIVE SURGERYEven with the least invasive of the MIS procedures, physiologic changes occur. Many minimally invasive procedures require minimal or no sedation, and there are few adverse consequences to the cardiovascular, endocrinologic, or immunologic systems. The least invasive of such procedures include stereotactic biopsy of breast lesions and flexible GI endoscopy. Minimally invasive procedures that require general anesthesia have a greater physi-ologic impact because of the anesthetic agent, the incision (even if small), and the induced pneumoperitoneum.LaparoscopyThe unique feature of laparoscopic surgery is the need to lift the abdominal wall from the abdominal organs. Two methods have been devised for achieving this.10 The first, used by most sur-geons, is a pneumoperitoneum. Throughout the early 20th century, intraperitoneal

1	wall from the abdominal organs. Two methods have been devised for achieving this.10 The first, used by most sur-geons, is a pneumoperitoneum. Throughout the early 20th century, intraperitoneal visualization was achieved by inflating the abdominal cavity with air, using a sphygmomanometer bulb.11 The problem with using air insufflation is that nitrogen is poorly soluble in blood and is slowly absorbed across the peritoneal surfaces. Air pneumoperitoneum was believed to be more pain-ful than nitrous oxide (N2O) pneumoperitoneum, but less pain-ful than carbon dioxide (CO2) pneumoperitoneum. Subsequently, CO2 and N2O were used for inflating the abdomen. N2O had the advantage of being physiologically inert and rap-idly absorbed. It also provided better analgesia for laparoscopy performed under local anesthesia when compared with CO2 or air.12 Despite initial concerns that N2O would not suppress combustion, controlled clinical trials have established its safety within the peritoneal

1	under local anesthesia when compared with CO2 or air.12 Despite initial concerns that N2O would not suppress combustion, controlled clinical trials have established its safety within the peritoneal cavity.13 In addition, N2O has been shown to reduce the intraoperative end-tidal CO2 and minute ventila-tion required to maintain homeostasis when compared to CO2 pneumoperitoneum.13 The effect of N2O on tumor biology and the development of port site metastasis are unknown. As such, caution should be exercised when performing laparoscopic can-cer surgery with this agent. Finally, the safety of N2O pneumo-peritoneum in pregnancy has yet to be elucidated.The physiologic effects of CO2 pneumoperitoneum can be divided into two areas: (a) gas-specific effects and (b) pressure-specific effects (Fig. 14-1). CO2 is rapidly absorbed across the peritoneal membrane into the circulation. In the circulation, 2Local effectsPeritoneal distentionVagal reactionElevated diaphragmAltered venous

1	effects (Fig. 14-1). CO2 is rapidly absorbed across the peritoneal membrane into the circulation. In the circulation, 2Local effectsPeritoneal distentionVagal reactionElevated diaphragmAltered venous returnPainSystemic effectsHypercarbiaAcidosisIncreased afterloadIncreased catecholaminesMyocardial stressCO2Figure 14-1. Carbon dioxide gas insufflated into the peritoneal cavity has both local and systemic effects that cause a complex set of hemodynamic and metabolic alterations. (Reproduced with permission from Hunter JG: Bailliere’s Clinical Gastroen-terology Laparoscopic Surgery. London/Philadelphia: Bailliere Tindall; 1993.)Brunicardi_Ch14_p0453-p0478.indd 45501/03/19 4:58 PM 456BASIC CONSIDERATIONSPART ICO2 creates a respiratory acidosis by the generation of carbonic acid.14 Body buffers, the largest reserve of which lies in bone, absorb CO2 (up to 120 L) and minimize the development of hypercarbia or respiratory acidosis during brief endoscopic pro-cedures.14 Once the body

1	buffers, the largest reserve of which lies in bone, absorb CO2 (up to 120 L) and minimize the development of hypercarbia or respiratory acidosis during brief endoscopic pro-cedures.14 Once the body buffers are saturated, respiratory aci-dosis develops rapidly, and the respiratory system assumes the burden of keeping up with the absorption of CO2 and its release from these buffers.In patients with normal respiratory function, this is not difficult; the anesthesiologist increases the ventilatory rate or vital capacity on the ventilator. If the respiratory rate required exceeds 20 breaths per minute, there may be less efficient gas exchange and increasing hypercarbia.15 Conversely, if vital capacity is increased substantially, there is a greater opportunity for barotrauma and greater respiratory motion–induced disrup-tion of the upper abdominal operative field. In some situations, it is advisable to evacuate the pneumoperitoneum or reduce the intra-abdominal pressure to allow time for

1	motion–induced disrup-tion of the upper abdominal operative field. In some situations, it is advisable to evacuate the pneumoperitoneum or reduce the intra-abdominal pressure to allow time for the anesthesiologist to adjust for hypercarbia.16 Although mild respiratory acidosis probably is an insignificant problem, more severe respiratory acidosis leading to cardiac arrhythmias has been reported.17 Hypercarbia also causes tachycardia and increased systemic vascular resistance, which elevates blood pressure and increases myocardial oxygen demand.14,17The pressure effects of the pneumoperitoneum on cardio-vascular physiology also have been studied. In the hypovolemic individual, excessive pressure on the inferior vena cava and a reverse Trendelenburg position with loss of lower extremity muscle tone may cause decreased venous return and decreased cardiac output.14,18 This is not seen in the normovolemic patient. The most common arrhythmia created by laparoscopy is brady-cardia. A rapid

1	tone may cause decreased venous return and decreased cardiac output.14,18 This is not seen in the normovolemic patient. The most common arrhythmia created by laparoscopy is brady-cardia. A rapid stretch of the peritoneal membrane often causes a vagovagal response with bradycardia and, occasionally, hypo-tension.19 The appropriate management of this event is desuf-flation of the abdomen, administration of vagolytic agents (e.g., atropine), and adequate volume replacement.20With the increased intra-abdominal pressure compressing the inferior vena cava, there is diminished venous return from the lower extremities. This has been well documented in the patient placed in the reverse Trendelenburg position for upper abdominal operations. Venous engorgement and decreased venous return promote venous thrombosis.21,22 Many series of advanced laparoscopic procedures in which deep venous thrombosis (DVT) prophylaxis was not used demonstrate the frequency of pulmonary embolus. This usually is an

1	thrombosis.21,22 Many series of advanced laparoscopic procedures in which deep venous thrombosis (DVT) prophylaxis was not used demonstrate the frequency of pulmonary embolus. This usually is an avoidable complication with the use of sequential compression stockings, subcutaneous heparin, or low molecular weight heparin.20,23 In short-duration laparoscopic procedures, such as appendectomy, hernia repair, or cholecystectomy, the risk of DVT may not be sufficient to warrant extensive DVT prophylaxis.The increased pressure of the pneumoperitoneum is trans-mitted directly across the paralyzed diaphragm to the thoracic cavity, creating increased central venous pressure and increased filling pressures of the right and left sides of the heart. If the intra-abdominal pressures are kept under 20 mmHg, the car-diac output usually is well maintained.22-24 The direct effect of the pneumoperitoneum on increasing intrathoracic pressure increases peak inspiratory pressure, pressure across the chest

1	the car-diac output usually is well maintained.22-24 The direct effect of the pneumoperitoneum on increasing intrathoracic pressure increases peak inspiratory pressure, pressure across the chest wall, and also, the likelihood of barotrauma. Despite these concerns, disruption of blebs and consequent pneumothoraces are rare after uncomplicated laparoscopic surgery.24 Pneumo-thoraces occurring with laparoscopic esophageal surgery may be very significant. The pathophysiology and management are discussed at the end of this section. Increased intra-abdominal pressure decreases renal blood flow, glomerular filtration rate, and urine output. These effects may be mediated by direct pressure on the kidney and the renal vein.25,26 The secondary effect of decreased renal blood flow is to increase plasma renin release, thereby increasing sodium retention. Increased circu-lating antidiuretic hormone levels also are found during the pneumoperitoneum, increasing free water reabsorption in the distal

1	renin release, thereby increasing sodium retention. Increased circu-lating antidiuretic hormone levels also are found during the pneumoperitoneum, increasing free water reabsorption in the distal tubules.27 Although the effects of the pneumoperitoneum on renal blood flow are immediately reversible, the hormonally mediated changes such as elevated antidiuretic hormone levels decrease urine output for up to 1 hour after the procedure has ended. Intraoperative oliguria is common during laparoscopy, but the urine output is not a reflection of intravascular volume status; intravenous (IV) fluid administration during an uncom-plicated laparoscopic procedure should not be linked to urine output. Because insensible fluid losses through the open abdo-men are eliminated with laparoscopy, the need for supplemen-tal fluid during a laparoscopic surgical procedure should only keep up with venous pooling in the lower limbs, third-space losses into the bowel, and blood loss, which is generally less

1	for supplemen-tal fluid during a laparoscopic surgical procedure should only keep up with venous pooling in the lower limbs, third-space losses into the bowel, and blood loss, which is generally less than occurs with an equivalent open operation.The hemodynamic and metabolic consequences of pneu-moperitoneum are well tolerated by healthy individuals for a prolonged period and by most individuals for at least a short period. Difficulties can occur when a patient with compromised cardiovascular function is subjected to a long laparoscopic pro-cedure. It is during these procedures that alternative approaches should be considered or insufflation pressure reduced. Alterna-tive gases that have been suggested for laparoscopy include the inert gases helium, neon, and argon. These gases are appeal-ing because they cause no metabolic effects, but are poorly soluble in blood (unlike CO2 and N2O) and are prone to create gas emboli if the gas has direct access to the venous system.22 Gas emboli

1	because they cause no metabolic effects, but are poorly soluble in blood (unlike CO2 and N2O) and are prone to create gas emboli if the gas has direct access to the venous system.22 Gas emboli are rare but serious complications of laparoscopic surgery.23,28 They should be suspected if hypotension develops during insufflation. Diagnosis may be made by listening (with an esophageal stethoscope) for the characteristic “mill wheel” murmur. The treatment of gas embolism is to place the patient in a left lateral decubitus position with the head down to trap the gas in the apex of the right ventricle.23 A rapidly placed central venous catheter then can be used to aspirate the gas out of the right ventricle.In some situations, minimally invasive abdominal surgery can be performed without insufflation. This is possible with the assistance of an abdominal lift device that can be placed through a 10to 12-mm trocar at the umbilicus.29 These devices have the advantage of creating little

1	insufflation. This is possible with the assistance of an abdominal lift device that can be placed through a 10to 12-mm trocar at the umbilicus.29 These devices have the advantage of creating little physiologic derangement, but they are bulky and intrusive. The exposure and working room offered by lift devices also are inferior to those accomplished by pneumoperitoneum. Lifting the anterior abdominal wall reduces space available laterally and thereby displaces the bowel medi-ally and anteriorly into the operative field. A pneumoperi-toneum, with its well-distributed intra-abdominal pressure, provides better exposure. Abdominal lift devices also cause more postoperative pain, but they do allow the performance of MIS with standard (nonlaparoscopic) surgical instruments.Endocrine responses to laparoscopic surgery are not always intuitive. Serum cortisol levels after laparoscopic opera-tions are often higher than after the equivalent operation per-formed through an open incision.30 The

1	to laparoscopic surgery are not always intuitive. Serum cortisol levels after laparoscopic opera-tions are often higher than after the equivalent operation per-formed through an open incision.30 The greatest difference Brunicardi_Ch14_p0453-p0478.indd 45601/03/19 4:58 PM 457MINIMALLY INVASIVE SURGERYCHAPTER 14between the endocrine response of open and laparoscopic sur-gery is the more rapid equilibration of most stress-mediated hormone levels after laparoscopic surgery. Immune suppression also is less after laparoscopy than after open surgery. There is a trend toward more rapid normalization of cytokine levels after a laparoscopic procedure than after the equivalent procedure performed by celiotomy.31Transhiatal mobilization of the distal esophagus is com-monly performed as a component of many laparoscopic upper abdominal procedures. If there is compromise of the mediastinal pleura with resultant CO2 pneumothorax, the defect should be enlarged so as to prevent a tension

1	a component of many laparoscopic upper abdominal procedures. If there is compromise of the mediastinal pleura with resultant CO2 pneumothorax, the defect should be enlarged so as to prevent a tension pneumothorax. Even with such a strategy, tension pneumothorax may develop, as medi-astinal structures may seal the hole during inspiration, allowing the chest to fill during expiration. In addition to enlargement of the hole, a thoracostomy tube (chest tube) should be placed across the breach into the abdomen with intra-abdominal pres-sures reduced below 8 mmHg, or a standard chest tube may be placed. When a pneumothorax occurs with laparoscopic Nissen fundoplication or Heller myotomy, it is preferable to place an 18-French red rubber catheter with multiple side holes cut out of the distal end across the defect. At the end of the procedure, the distal end of the tube is pulled out a 10-mm port site (as the port is removed), and the pneumothorax is evacuated to a primitive water seal using

1	across the defect. At the end of the procedure, the distal end of the tube is pulled out a 10-mm port site (as the port is removed), and the pneumothorax is evacuated to a primitive water seal using a bowl of sterile water or saline. During laparo-scopic esophagectomy, it is preferable to leave a standard chest tube, as residual intra-abdominal fluid will tend to be siphoned through the defect postoperatively if the tube is removed at the end of the case.ThoracoscopyThe physiology of thoracic MIS (thoracoscopy) is different from that of laparoscopy. Because of the bony confines of the thorax, it is unnecessary to use positive pressure when working in the thorax.32 The disadvantages of positive pressure in the chest include decreased venous return, mediastinal shift, and the need to keep a firm seal at all trocar sites. Without positive pressure, it is necessary to place a double-lumen endotracheal tube so that the ipsilateral lung can be deflated when the opera-tion starts. By

1	keep a firm seal at all trocar sites. Without positive pressure, it is necessary to place a double-lumen endotracheal tube so that the ipsilateral lung can be deflated when the opera-tion starts. By collapsing the ipsilateral lung, working space within the thorax is obtained. Because insufflation is unneces-sary in thoracoscopic surgery, it can be beneficial to use stan-dard instruments via extended port sites in conjunction with thoracoscopic instruments. This approach is particularly useful when performing advanced procedures such as thoracoscopic anatomic pulmonary resection.Extracavitary Minimally Invasive SurgeryMany MIS procedures create working spaces in extrathoracic and extraperitoneal locations. Laparoscopic inguinal her-nia repair usually is performed in the anterior extraperitoneal Retzius space.33,34 Laparoscopic nephrectomy often is per-formed with retroperitoneal laparoscopy. Endoscopic retro-peritoneal approaches to pancreatic necrosectomy have seen some limited use.35

1	Retzius space.33,34 Laparoscopic nephrectomy often is per-formed with retroperitoneal laparoscopy. Endoscopic retro-peritoneal approaches to pancreatic necrosectomy have seen some limited use.35 Lower extremity vascular procedures and plastic surgical endoscopic procedures require the development of working space in unconventional planes, often at the level of the fascia, sometimes below the fascia, and occasionally in nonanatomic regions.36 Some of these techniques use insuffla-tion of gas, but many use balloon inflation to develop the space, followed by low-pressure gas insufflation or lift devices to maintain the space (Fig. 14-2). These techniques produce fewer and less severe adverse physiologic consequences than does the ABCFigure 14-2. Balloons are used to create extra-anatomic working spaces. In this example (A through C), a balloon is introduced into the space between the posterior rectus sheath and the rectus abdom-inal muscle. The balloon is inflated in the preperitoneal

1	spaces. In this example (A through C), a balloon is introduced into the space between the posterior rectus sheath and the rectus abdom-inal muscle. The balloon is inflated in the preperitoneal space to create working room for extraperitoneal endoscopic hernia repair.pneumoperitoneum, but the insufflation of carbon dioxide into extraperitoneal locations can spread widely, causing subcutane-ous emphysema and metabolic acidosis.AnesthesiaProper anesthesia management during laparoscopic surgery requires a thorough knowledge of the pathophysiology of the CO2 pneumoperitoneum.20 The laparoscopic surgeon can influ-ence cardiovascular performance by reducing or removing the CO2 pneumoperitoneum. Insensible fluid losses are negligible, and therefore, IV fluid administration should not exceed that necessary to maintain circulating volume. MIS procedures are often outpatient procedures, so short-acting anesthetic agents are preferable. Because the factors that require hospitaliza-tion after

1	necessary to maintain circulating volume. MIS procedures are often outpatient procedures, so short-acting anesthetic agents are preferable. Because the factors that require hospitaliza-tion after laparoscopic procedures include the management of nausea, pain, and urinary retention, the anesthesiologist should minimize the use of agents that provoke these conditions and maximize the use of medications that prevent such problems. Critical to the anesthesia management of these patients is the use of nonnarcotic analgesics (e.g., ketorolac) when hemosta-sis allows it and the liberal use of antiemetic agents, including ondansetron and steroids.The Minimally Invasive TeamFrom the beginning, the tremendous success of MIS was founded on the understanding that a team approach was Brunicardi_Ch14_p0453-p0478.indd 45701/03/19 4:58 PM 458BASIC CONSIDERATIONSPART ITable 14-1Laparoscopic surgical proceduresBASICADVANCEDAppendectomyNissen fundoplicationLymph node dissectionCholecystectomyHeller

1	45701/03/19 4:58 PM 458BASIC CONSIDERATIONSPART ITable 14-1Laparoscopic surgical proceduresBASICADVANCEDAppendectomyNissen fundoplicationLymph node dissectionCholecystectomyHeller myotomyRoboticsInguinal hernia repairParaesophageal herniaBariatricEnteral accessGastrectomyComplex abdominal wall reconstruction Lysis of adhesionsEsophagectomy Bile duct explorationHepatectomy ColectomyPancreatectomy SplenectomyProstatectomy AdrenalectomyHysterectomy Nephrectomy Figure 14-3. An example of a typical minimally invasive surgery suite. All core equipment is located on easily movable consoles.necessary. The many laparoscopic procedures performed daily range from basic to advanced complexity, and require that the surgical team have an intimate understanding of the operative conduct (Table 14-1). Minimally invasive procedures require complicated and fragile equipment that demands constant main-tenance. In addition, multiple intraoperative adjustments to the equipment, camera, insufflator,

1	Minimally invasive procedures require complicated and fragile equipment that demands constant main-tenance. In addition, multiple intraoperative adjustments to the equipment, camera, insufflator, monitors, and patient/surgeon position are made during these procedures. As such, a coordi-nated team approach is mandated to ensure patient safety and excellent outcomes. More and more, flexible endoscopes are used to guide or provide quality control for laparoscopic pro-cedures. As NOTES, SILS, and robotic surgery become more common, hybrid procedures (laparoscopy and endoscopy) and complicated robotics cases will require a nursing staff capable of maintaining flexible endoscopes and understanding the oper-ation of sophisticated technology.A typical MIS team may consist of a laparoscopic surgeon and an operating room (OR) nurse with an interest in laparo-scopic and endoscopic surgery. Adding dedicated assistants and circulating staff with an intimate knowledge of the equipment will add to

1	and an operating room (OR) nurse with an interest in laparo-scopic and endoscopic surgery. Adding dedicated assistants and circulating staff with an intimate knowledge of the equipment will add to and enhance team competency. Studies have dem-onstrated that having a designated laparoscopic team increases the efficiency and safety of laparoscopic surgery, which is trans-lated into a benefit for the patient and the hospital.37Room Setup and the Minimally Invasive SuiteNearly all MIS, whether using fluoroscopic, ultrasound, or opti-cal imaging, incorporates a video monitor as a guide. Occasion-ally, two images are necessary to adequately guide the operation, as in procedures such as endoscopic retrograde cholangiopan-creatography, laparoscopic common bile duct exploration, and laparoscopic ultrasonography. When two images are necessary, the images should be displayed on two adjacent video monitors or projected on a single screen with a picture-in-picture effect. The video monitor(s)

1	ultrasonography. When two images are necessary, the images should be displayed on two adjacent video monitors or projected on a single screen with a picture-in-picture effect. The video monitor(s) should be set across the operating table from the surgeon. The patient should be interposed between the surgeon and the video monitor; ideally, the operative field also lies between the surgeon and the monitor. In pelviscopic sur-gery, it is best to place the video monitor at the patient’s feet, and in laparoscopic cholecystectomy, the monitor is placed at the 10 o’clock position (relative to the patient) while the surgeon stands on the patient’s left at the 4 o’clock position. The insuf-flating and patient-monitoring equipment ideally also is placed across the table from the surgeon so that the insufflating pres-sure and the patient’s vital signs and end-tidal CO2 tension can be monitored.The development of the minimally invasive surgical suite has been a tremendous contribution to the

1	the insufflating pres-sure and the patient’s vital signs and end-tidal CO2 tension can be monitored.The development of the minimally invasive surgical suite has been a tremendous contribution to the field of laparoscopy in that it has facilitated the performance of advanced proce-dures and techniques (Fig. 14-3). By having the core equipment (monitors, insufflators, and imaging equipment) located within mobile, ceiling-mounted consoles, the surgery team is able to accommodate and make small adjustments rapidly and con-tinuously throughout the procedure. The specifically designed minimally invasive surgical suite serves to decrease equipment and cable disorganization, ease the movements of operative per-sonnel around the room, improve ergonomics, and facilitate the use of advanced imaging equipment such as laparoscopic ultra-sound.38 Although having a minimally invasive surgical suite available is very useful, it is not essential to successfully carry out advanced laparoscopic

1	imaging equipment such as laparoscopic ultra-sound.38 Although having a minimally invasive surgical suite available is very useful, it is not essential to successfully carry out advanced laparoscopic procedures.Patient PositioningPatients usually are placed in the supine position for laparo-scopic surgery. When the operative field is the gastroesophageal junction or the left lobe of the liver, it is easiest to operate from between the legs. The legs may be elevated in Allen stirrups or abducted on leg boards to achieve this position. When pel-vic procedures are performed, it usually is necessary to place the legs in Allen stirrups to gain access to the perineum. A lat-eral decubitus position with the table flexed provides the best access to the retroperitoneum when performing nephrectomy or adrenalectomy. For laparoscopic splenectomy, a 45° tilt of the patient provides excellent access to the lesser sac and the lateral peritoneal attachments to the spleen. For thoracoscopic surgery,

1	or adrenalectomy. For laparoscopic splenectomy, a 45° tilt of the patient provides excellent access to the lesser sac and the lateral peritoneal attachments to the spleen. For thoracoscopic surgery, the patient is placed in the lateral position with table flexion to open the intercostal spaces and the distance between the iliac crest and costal margin (Fig. 14-4). Additional con-sideration must be made in robotic operations to position the Brunicardi_Ch14_p0453-p0478.indd 45801/03/19 4:58 PM 459MINIMALLY INVASIVE SURGERYCHAPTER 14Figure 14-4. Proper padding and protection of pressure points is an essential consideration in laparoscopic and thoracoscopic approaches. In preparation for thoracoscopy, this patient is placed in left lateral decubitus position with the table flexed, which serves to open the intercostal spaces and increase the distance between the iliac crest and the inferior costal margin.patient appropriately before starting. Clashing of the robotic arms with

1	which serves to open the intercostal spaces and increase the distance between the iliac crest and the inferior costal margin.patient appropriately before starting. Clashing of the robotic arms with surrounding equipment or each other can occur if not positioned correctly. This is more common in predecessors of the da Vinci Xi platform. Unless an operative table with inte-grated table motion is available, once the robot is docked to the patient the bed cannot be moved without undocking.When the patient’s knees are to be bent for extended peri-ods or the patient is going to be placed in a reverse Trendelen-burg position for more than a few minutes, DVT prophylaxis should be used. Sequential compression devices should be placed on the lower extremities during laparoscopic procedures to increase venous return and provides inhibition of thrombo-plastin activation.General Principles of AccessThe most natural ports of access for MIS and NOTES are the anatomic portals of entry and exit. The

1	venous return and provides inhibition of thrombo-plastin activation.General Principles of AccessThe most natural ports of access for MIS and NOTES are the anatomic portals of entry and exit. The nares, mouth, anus, vagina, and urethra are used to access the respiratory, GI, and urinary systems. The advantage of using these points of access is that no incision is required. The disadvantages lie in the long distances between the orifice and the region of interest. For NOTES procedures, the vagina may serve as point of access, entering the abdomen via the posterior cul-de-sac of the pelvis. Similarly, the peritoneal cavity may be reached through the side wall of the stomach or colon.Access to the vascular system may be accomplished under local anesthesia by cutting down and exposing the desired vessel, usually in the groin. Increasingly, vascular access is obtained with percutaneous techniques using a small incision, a needle, and a guidewire, over which are passed a variety of

1	the desired vessel, usually in the groin. Increasingly, vascular access is obtained with percutaneous techniques using a small incision, a needle, and a guidewire, over which are passed a variety of different-sized access devices. This approach, known as the Seldinger technique, is most frequently used by general sur-geons for placement of Hickman catheters, but it also is used to gain access to the arterial and venous system for performance of minimally invasive procedures. Guidewire-assisted, Seldinger-type techniques also are helpful for gaining access to the gut for procedures such as PEG, for gaining access to the biliary system through the liver, and for gaining access to the upper urinary tract.In thoracoscopic surgery, the access technique is similar to that used for placement of a chest tube. In these procedures, general anesthesia and single lung ventilation are essential. A small incision is made over the top of a rib and, under direct vision, carried down through the

1	of a chest tube. In these procedures, general anesthesia and single lung ventilation are essential. A small incision is made over the top of a rib and, under direct vision, carried down through the pleura. The lung is collapsed, and a trocar is inserted across the chest wall to allow access with a telescope. Once the lung is completely collapsed, subse-quent access may be obtained with direct puncture, viewing all entry sites through the videoendoscope. Because insufflation of the chest is unnecessary, simple ports that keep the small inci-sions open are all that is required to allow repeated access to the thorax.Laparoscopic AccessThe requirements for laparoscopy are more involved because the creation of a pneumoperitoneum requires that instruments of access (trocars) contain valves to maintain abdominal inflation.Two methods are used for establishing abdominal access during laparoscopic procedures.39,40 The first, direct puncture laparoscopy, begins with the elevation of the relaxed

1	abdominal inflation.Two methods are used for establishing abdominal access during laparoscopic procedures.39,40 The first, direct puncture laparoscopy, begins with the elevation of the relaxed abdominal wall with two towel clips or a well-placed hand. A small inci-sion is made in the umbilicus, and a specialized spring-loaded (Veress) needle is placed in the abdominal cavity (Fig. 14-5). Figure 14-5. A. Tip of spring loaded (Veress) needle. B. Veress needle held at its serrated collar with a thumb and forefinger. At the umbilicus, the abdominal wall is grasped with fingers or penetrating towel clip to elevate the abdominal wall away from the underlying structures.ABBrunicardi_Ch14_p0453-p0478.indd 45901/03/19 4:58 PM 460BASIC CONSIDERATIONSPART IFigure 14-6. It is essential to be able to interpret the insufflator pressure readings and flow rates. These readings indicate proper intraperitoneal placement of the Veress needle.Figure 14-7. The open laparoscopy technique involves

1	able to interpret the insufflator pressure readings and flow rates. These readings indicate proper intraperitoneal placement of the Veress needle.Figure 14-7. The open laparoscopy technique involves identifica-tion and incision of the peritoneum, followed by the placement of a specialized trocar with a conical sleeve to maintain a gas seal. Spe-cialized wings on the trocar are attached to sutures placed through the fascia to prevent loss of the gas seal.With the Veress needle, two distinct pops are felt as the surgeon passes the needle through the abdominal wall fascia and the peritoneum. The umbilicus usually is selected as the preferred point of access because, in this location, the abdominal wall is quite thin, even in obese patients. The abdomen is inflated with a pressure-limited insufflator. CO2 gas usually is used, with maximal pressures in the range of 14 to 15 mmHg. During the process of insufflation, it is essential that the surgeon observe the pressure and flow readings on

1	CO2 gas usually is used, with maximal pressures in the range of 14 to 15 mmHg. During the process of insufflation, it is essential that the surgeon observe the pressure and flow readings on the monitor to confirm an intraperitoneal location of the Veress needle tip (Fig. 14-6). Laparoscopic surgery can be performed under local anesthesia, but general anesthesia is preferable. Under local anesthesia, N2O is used as the insufflating agent, and insufflation is stopped after 2 L of gas is insufflated or when a pressure of 10 mmHg is reached.After peritoneal insufflation, direct access to the abdomen is obtained with a 5or 10-mm trocar. This can be performed through a radially dilating sheath placed over the Veress needle or an optical viewing trocar. In the latter technique, a camera is placed inside of a clear pyramidal trocar. Direct puncture entry is observed as the trocar is passed through the abdominal wall. The critical issues for safe direct-puncture laparoscopy include the use of

1	inside of a clear pyramidal trocar. Direct puncture entry is observed as the trocar is passed through the abdominal wall. The critical issues for safe direct-puncture laparoscopy include the use of a vented stylet for the trocar, or a trocar with a safety shield or dilating tip. An optical viewing trocar can be used without prior insufflation; however, proper recognition of the abdominal wall layers is critical to avoid entry into the mes-entery or underlying structures. In all direct puncture entry the trocar must be pointed away from the sacral promontory and the great vessels.41 Patient position should be surveyed before trocar placement to ensure a proper trajectory.Occasionally, the direct peritoneal access (Hasson) tech-nique is advisable.42 With this technique, the surgeon makes a small incision just below the umbilicus and under direct vision locates the abdominal fascia. Two Kocher clamps are placed on the fascia, and with curved Mayo scissors, a small incision is made

1	a small incision just below the umbilicus and under direct vision locates the abdominal fascia. Two Kocher clamps are placed on the fascia, and with curved Mayo scissors, a small incision is made through the fascia and underlying peritoneum. A fin-ger is placed into the abdomen to make sure that there is no adherent bowel. A sturdy suture is placed on each side of the fascia and secured to the wings of a specialized trocar, which is then passed directly into the abdominal cavity (Fig. 14-7). Rapid insufflation can make up for some of the time lost with the initial dissection. This technique is preferable for the abdo-men of patients who have undergone previous operations in which small bowel may be adherent to the undersurface of the abdominal wound. The close adherence of bowel to the perito-neum in the previously operated abdomen does not eliminate the possibility of intestinal injury but should make great vessel injury extremely unlikely. Because of the difficulties in visual-izing

1	in the previously operated abdomen does not eliminate the possibility of intestinal injury but should make great vessel injury extremely unlikely. Because of the difficulties in visual-izing the abdominal region immediately adjacent to the primary trocar, it is recommended that the telescope be passed through a secondary trocar to inspect the site of initial abdominal access.40 Secondary punctures are made with 5and 10-mm trocars. For safe access to the abdominal cavity, it is critical to visualize all sites of trocar entry.41,42 At the completion of the operation, all trocars are removed under direct vision, and the insertion sites are inspected for bleeding. If bleeding occurs, direct pres-sure with an instrument from another trocar site or balloon tamponade with a Foley catheter placed through the trocar site generally stops the bleeding within 3 to 5 minutes. When this is not successful, a full-thickness abdominal wall suture has been used successfully to tamponade trocar site

1	through the trocar site generally stops the bleeding within 3 to 5 minutes. When this is not successful, a full-thickness abdominal wall suture has been used successfully to tamponade trocar site bleeding.It is generally agreed that 5-mm trocars need no site sutur-ing. Ten-millimeter trocars placed off the midline, through a radially dilating sheath or above the transverse mesocolon do not typically require repair. Conversely, if the fascia has been dilated to allow the passage of the gallbladder or other organ, it should be repaired at the fascial level with interrupted sutures. The port site may be closed with suture delivery systems simi-lar to crochet needles enabling mass closure of the abdominal wall. This is especially helpful in obese patients where direct fascial closure may be challenging, through a small skin inci-sion. Failure to close lower abdominal trocar sites that are 10 mm in diameter or larger can lead to an incarcerated hernia.Access for Subcutaneous and

1	may be challenging, through a small skin inci-sion. Failure to close lower abdominal trocar sites that are 10 mm in diameter or larger can lead to an incarcerated hernia.Access for Subcutaneous and Extraperitoneal SurgeryThere are two methods for gaining access to nonanatomic spaces. For retroperitoneal locations, balloon dissection is effec-tive. This access technique is appropriate for the extraperitoneal repair of inguinal hernias and for retroperitoneal surgery for adrenalectomy, nephrectomy, lumbar discectomy, pancreatic necrosectomy, or para-aortic lymph node dissection.43,44 The Brunicardi_Ch14_p0453-p0478.indd 46001/03/19 4:58 PM 461MINIMALLY INVASIVE SURGERYCHAPTER 14initial access to the extraperitoneal space is performed in a way similar to direct puncture laparoscopy, except that the last layer (the peritoneum) is not traversed. Once the transversalis fascia has been punctured, a specialized trocar with a balloon on the end is introduced. The balloon is inflated in

1	that the last layer (the peritoneum) is not traversed. Once the transversalis fascia has been punctured, a specialized trocar with a balloon on the end is introduced. The balloon is inflated in the extraperitoneal space to create a working chamber. The balloon then is deflated, and a Hasson trocar is placed. An insufflation pressure of 10 mmHg usually is adequate to keep the extraperitoneal space open for dissection and will limit subcutaneous emphysema. Higher gas pressures force CO2 into the soft tissues and may contribute to hypercarbia. Extraperitoneal endosurgery provides less working space than laparoscopy but eliminates the possibil-ity of intestinal injury, intestinal adhesion, herniation at the tro-car sites, and ileus. These issues are important for laparoscopic hernia repair because extraperitoneal approaches prevent the small bowel from sticking to the prosthetic mesh.34Subcutaneous surgery has been most widely used in car-diac, vascular, and plastic surgery.36 In cardiac

1	extraperitoneal approaches prevent the small bowel from sticking to the prosthetic mesh.34Subcutaneous surgery has been most widely used in car-diac, vascular, and plastic surgery.36 In cardiac surgery, subcu-taneous access has been used for saphenous vein harvesting, and in vascular surgery for ligation of subfascial perforating veins (Linton procedure). With minimally invasive techniques, the entire saphenous vein above the knee may be harvested through a single incision (Fig. 14-8).45,46 Once the saphenous vein is located, a long retractor that holds a 5-mm laparoscope allows the coaxial dissection of the vein and coagulation or clipping of Figure 14-8. With two small incisions, virtually the entire saphe-nous vein can be harvested for bypass grafting.each side branch. A small incision above the knee also can be used to ligate perforating veins in the lower leg.Subcutaneous access also is used for plastic surgery pro-cedures.46 Minimally invasive approaches are especially well

1	above the knee also can be used to ligate perforating veins in the lower leg.Subcutaneous access also is used for plastic surgery pro-cedures.46 Minimally invasive approaches are especially well suited to cosmetic surgery, in which attempts are made to hide the incision. It is easier to hide several 5-mm incisions than one long incision. The technique of blunt dissection along fascial planes combined with lighted retractors and endoscope-holding retractors is most successful for extensive subcutaneous surgery. Some prefer gas insufflation of these soft tissue planes. The pri-mary disadvantage of soft tissue insufflation is that subcutane-ous emphysema can be created.Hand-Assisted Laparoscopic AccessHand-assisted laparoscopic surgery is thought to combine the tactile advantages of open surgery with the minimal access of laparoscopy and thoracoscopy. This approach commonly is used to assist with difficult cases before conversion to celiotomy is necessary. Additionally, hand-assisted

1	surgery with the minimal access of laparoscopy and thoracoscopy. This approach commonly is used to assist with difficult cases before conversion to celiotomy is necessary. Additionally, hand-assisted laparoscopic surgery is used to help surgeons negotiate the steep learning curve associ-ated with advanced laparoscopic procedures.47 This technology uses an entryway for the hand that preserves the pneumoperi-toneum and enables laparoscopic visualization in combination with the use of minimally invasive instruments (Fig. 14-9). For-mal investigation of this modality has been limited primarily to case reports and small series and has focused primarily on solid organ and colon surgery.Intraperitoneal, intrathoracic, and retroperitoneal access for robotic surgery adheres to the principles of laparoscopic and thoracoscopic access; however, the port size for the primary puncture is 12 mm to allow placement of the stereo laparoscope. Remaining trocars are 8 mm.Natural Orifice Transluminal

1	laparoscopic and thoracoscopic access; however, the port size for the primary puncture is 12 mm to allow placement of the stereo laparoscope. Remaining trocars are 8 mm.Natural Orifice Transluminal Endoscopic Surgery AccessMultiple studies have shown safety in the performance of NOTES procedures. Transvaginal, transvesicle, transanal, transcolonic, transgastric, and transoral approaches have all been attempted with varying success. The ease of decontamina-tion, entry, and closure of these structures create variable chal-lenges. The transvaginal approach for resection of the uterus has been employed for many years by gynecologists and has been modified by laparoscopists with great success. Extraction of the gallbladder, kidney, bladder, large bowel, and stomach can be Figure 14-9. This is an example of hand-assisted laparoscopic surgery during left colectomy. The surgeon uses a hand to provide retraction and counter tension during mobilization of the colon from its retroperitoneal

1	is an example of hand-assisted laparoscopic surgery during left colectomy. The surgeon uses a hand to provide retraction and counter tension during mobilization of the colon from its retroperitoneal attachments, as well as during division of the mesocolon. This technique is particularly useful in the region of the transverse colon.Brunicardi_Ch14_p0453-p0478.indd 46101/03/19 4:58 PM 462BASIC CONSIDERATIONSPART IFigure 14-10. Submucosal tunnel technique for transesophageal mediastinoscopy. (Reproduced with permission from Khashab MA, Kalloo AN. NOTES: current status and new horizons, Gastroenterology. 2012 Apr;142(4):704-710.e1.)performed via the vagina. The esophagus can be traversed to enter the mediastinum. Leaving the orifice or organ of entry with an endoscope requires the use of an endoscopic needle knife followed by submucosal tunneling or direct puncture and balloon dilation (Fig. 14-10). Closure has been performed using endoscopic clips or sutures with advanced endoscopic

1	an endoscopic needle knife followed by submucosal tunneling or direct puncture and balloon dilation (Fig. 14-10). Closure has been performed using endoscopic clips or sutures with advanced endoscopic platforms.Single-Incision Laparoscopic Surgery AccessThere is no standardized approach for SILS, and access tech-niques vary by surgeon preference. Traditionally, a single skin incision is made directly through the umbilical scar ranging from 1 to 3 cm. Through this single incision, multiple low-profile trocars can be placed separately into the fascia to allow insufflation, camera, and working instruments. The advantage of this technique is that conventional laparoscopic tools can be employed. The disadvantage becomes apparent when an extrac-tion site is needed. A variety of specialized multilumen trocars are on the market that can be placed through the umbilical ring48 (Fig. 14-11A,B). The advantages of these devices include faster access, improved safety, minimization of air leaks, and

1	trocars are on the market that can be placed through the umbilical ring48 (Fig. 14-11A,B). The advantages of these devices include faster access, improved safety, minimization of air leaks, and plat-form-derived instrument triangulation. The major disadvantage is cost.Port PlacementTrocars for the surgeon’s left and right hand should be placed at least 10 cm apart. For most operations, it is possible to orient ABCDEthe telescope between these two trocars and slightly back from them. The ideal trocar orientation creates an equilateral triangle between the surgeon’s right hand, left hand, and the telescope, with 10 to 15 cm on each leg. If one imagines the target of the operation (e.g., the gallbladder or gastroesophageal junc-tion) oriented at the apex of a second equilateral triangle built on the first, these four points of reference create a diamond (Fig. 14-12). The surgeon stands behind the telescope, which provides optimal ergonomic orientation but frequently requires that a

1	built on the first, these four points of reference create a diamond (Fig. 14-12). The surgeon stands behind the telescope, which provides optimal ergonomic orientation but frequently requires that a camera operator (or mechanical camera holder) reach between the surgeon’s hands to guide the telescope. SILS is challenging for even the experienced laparoscopist because it violates most of the aforementioned ergonomic principles. Hav-ing only a single point of entry into the abdominal cavity creates an inherently crowded port and hand position. The inability to space trocars severely limits the ability to triangulate the leftand right-hand instruments. As a result, the surgeon must often work in a crossed hands fashion (Fig. 14-13). Additionally, the axis of the camera view is often in line with the working instru-ments, making visualization difficult without a deflectable tip laparoscope.The position of the operating table should permit the sur-geon to work with both elbows in at the

1	with the working instru-ments, making visualization difficult without a deflectable tip laparoscope.The position of the operating table should permit the sur-geon to work with both elbows in at the sides, with arms bent 90° at the elbow.49 It usually is necessary to alter the operating table position with left or right tilt with the patient in the Tren-delenburg or reverse Trendelenburg position, depending on the operative field.50,51Brunicardi_Ch14_p0453-p0478.indd 46201/03/19 4:58 PM 463MINIMALLY INVASIVE SURGERYCHAPTER 14Figure 14-11. A. Specialized multilumen trocars can facilitate instrument placement. B. For single-incision laparoscopic surgery, multiple fascial punctures can be performed via a single skin incision. (Reproduced with permission from The Johns Hopkins University School of Medicine, Baltimore, MD; 2014. Illustration by Corinne Sandone.)Multiple trocarsthrough singleskin incision Single portaccommodatesmultiple trocarsABTHE DIAMOND OF SUCCESS"Home

1	University School of Medicine, Baltimore, MD; 2014. Illustration by Corinne Sandone.)Multiple trocarsthrough singleskin incision Single portaccommodatesmultiple trocarsABTHE DIAMOND OF SUCCESS"Home plate"(telescope)"First base"(R hand)"Third base"(L hand)"Second base"(hiatal hernia)15 cmFigure 14-12. The diamond configuration created by placing the telescope between the left and the right hand, recessed from the target by about 15 cm. The distance between the left and the right hand is also ideally 10 to 15 cm. In this “baseball diamond” con-figuration, the surgical target occupies the second base position.Figure 14-13. The single point of abdominal entry for trocars often requires that the surgeon work in a crossed hands fashion. (Reproduced with permission from The Johns Hopkins University School of Medi-cine, Baltimore, MD; 2014. Illustration by Corinne Sandone.)Imaging SystemsTwo methods of videoendoscopic imaging are widely used. Both methods use a camera with a charge-coupled

1	School of Medi-cine, Baltimore, MD; 2014. Illustration by Corinne Sandone.)Imaging SystemsTwo methods of videoendoscopic imaging are widely used. Both methods use a camera with a charge-coupled device (CCD), which is an array of photosensitive sensor elements (pixels) that convert the incoming light intensity to an electric charge. The electric charge is subsequently converted into a color image.52With videoendoscopy, the CCD chip is placed on the inter-nal end of a long, flexible endoscope. With older flexible endo-scopes, thin quartz fibers are packed together in a bundle, and the CCD camera is mounted on the external end of the endoscope. Most standard GI endoscopes have the CCD chip at the distal end, but small, delicate choledochoscopes and nephroscopes are equipped with fiber-optic bundles.53 Distally mounted CCD chips have been developed for laparoscopy but remain very expensive and therefore have not become as widely used.Video cameras come in two basic designs. Nearly all

1	bundles.53 Distally mounted CCD chips have been developed for laparoscopy but remain very expensive and therefore have not become as widely used.Video cameras come in two basic designs. Nearly all lapa-roscopic cameras contain a red, green, and blue input, and are identical to the color cameras used for television production.52 An additional feature of many video cameras is digital enhance-ment. Digital enhancement detects edges, areas where there are drastic color or light changes between two adjacent pixels.54 By enhancing this difference, the image appears sharper and surgi-cal resolution is improved. New laparoscopic cameras contain a high-definition (HD) chip, which increases the lines of resolu-tion from 480 to 1080 lines. To enjoy the benefit of the clarity of HD video imaging, HD monitors also are necessary.Priorities in a video imaging system for MIS are illumina-tion first, resolution second, and color third. Without the first two attributes, video surgery is unsafe.

1	HD monitors also are necessary.Priorities in a video imaging system for MIS are illumina-tion first, resolution second, and color third. Without the first two attributes, video surgery is unsafe. Illumination and resolu-tion are as dependent on the telescope, light source, and light cable as on the video camera used. Imaging for laparoscopy, thoracoscopy, and subcutaneous surgery uses a rigid metal telescope, usually 30 cm in length. Longer telescopes are avail-able for obese patients and for reaching the mediastinum and deep in the pelvis from a periumbilical entry site. The standard Brunicardi_Ch14_p0453-p0478.indd 46301/03/19 4:58 PM 464BASIC CONSIDERATIONSPART IFigure 14-14. The laparoscope tips come in a variety of angled configurations. All laparoscopes have a 70° field of view. A 30°-angled scope enables the surgeon to view this field at a 30° angle to the long axis of the scope.Figure 14-15. The Hopkins rod lens telescope includes a series of optical rods that effectively

1	A 30°-angled scope enables the surgeon to view this field at a 30° angle to the long axis of the scope.Figure 14-15. The Hopkins rod lens telescope includes a series of optical rods that effectively transmit light to the eyepiece. The video camera is placed on the eyepiece to provide the working image. The image is only as clear as the weakest link in the image chain. CCD = charge-coupled device. (Reproduced with permission from Toouli JG, Gossot D, Hunter JG: Endosurgery. New York/London: Churchill-Livingstone/Elsevier; 1996.)telescope contains a series of quartz optical rods and focusing lenses.55 Telescopes vary in size from 2 to 12 mm in diameter. Because light transmission is dependent on the cross-sectional area of the quartz rod, when the diameter of a rod/lens system is doubled, the illumination is quadrupled. Little illumination is needed in highly reflective, small spaces such as the knee, and a very small telescope will suffice. When working in the abdomi-nal cavity,

1	the illumination is quadrupled. Little illumination is needed in highly reflective, small spaces such as the knee, and a very small telescope will suffice. When working in the abdomi-nal cavity, especially if blood is present, the full illumination of a 10-mm telescope usually is necessary.Rigid telescopes may have a flat or angled end. The flat end provides a straight view (0°), and the angled end provides an oblique view (30° or 45°).52 Angled telescopes allow greater flexibility in viewing a wider operative field through a single trocar site (Fig. 14-14A); rotating an angled telescope changes LampLight sourceCameracontrollerCameraobjectivelensRelayedimageIlluminationlight guideImage formedby objective lensObservationpositionAdaption opticObjectivelens sectionRelaylens sectionEyepiecelens sectionFocus ringCCD chipMonitorCondensor lensLight guide cablethe field of view. The use of an angled telescope has distinct advantages for most videoendoscopic procedures, particularly in

1	sectionFocus ringCCD chipMonitorCondensor lensLight guide cablethe field of view. The use of an angled telescope has distinct advantages for most videoendoscopic procedures, particularly in visualizing the common bile duct during laparoscopic cho-lecystectomy or visualizing the posterior esophagus or the tip of the spleen during laparoscopic fundoplication. Flexible tip laparoscopes offer even greater optical freedom.Light is delivered to the endoscope through a fiber-optic light cable. These light cables are highly inefficient, losing >90% of the light delivered from the light source. Extremely bright light sources (300 watts) are necessary to provide ade-quate illumination for laparoscopic surgery.The quality of the videoendoscopic image is only as good as the weakest component in the imaging chain (Fig. 14-15). Therefore, it is important to use a video monitor that has a reso-lution equal to or greater than the camera being used.55 Resolu-tion is the ability of the optical system

1	imaging chain (Fig. 14-15). Therefore, it is important to use a video monitor that has a reso-lution equal to or greater than the camera being used.55 Resolu-tion is the ability of the optical system to distinguish between line pairs. The larger the number of line pairs per millimeter, the sharper and more detailed the image. Most high-resolution monitors have up to 700 horizontal lines. HD television can deliver up to eight times more resolution than standard moni-tors; when combined with digital enhancement, a very sharp and well-defined image can be achieved.52,55 A heads-up display is a high-resolution liquid crystal monitor that is built into eyewear worn by the surgeon.56 This technology allows the surgeon to view the endoscopic image and operative field simultaneously. The proposed advantages of heads-up display include a high-resolution monocular image, which affords the surgeon mobility and reduces vertigo and eyestrain. However, this technology has not yet been widely

1	advantages of heads-up display include a high-resolution monocular image, which affords the surgeon mobility and reduces vertigo and eyestrain. However, this technology has not yet been widely adopted.Interest in three-dimensional (3-D) laparoscopy has waxed and waned. 3-D laparoscopy provides the additional depth of field that is lost with two-dimensional endosurgery and improves performance of novice laparoscopists performing complex tasks of dexterity, including suturing and knot tying.57 The advantages of 3-D systems are less obvious to experienced Brunicardi_Ch14_p0453-p0478.indd 46401/03/19 4:58 PM 465MINIMALLY INVASIVE SURGERYCHAPTER 14laparoscopists. Additionally, because 3-D systems require the flickering of two similar images, which are resolved with spe-cial glasses, the images’ edges become fuzzy and resolution is lost. The optical accommodation necessary to rectify these slightly differing images is tiring and may induce headaches when one uses these systems for a

1	images’ edges become fuzzy and resolution is lost. The optical accommodation necessary to rectify these slightly differing images is tiring and may induce headaches when one uses these systems for a long period of time. The da Vinci robot uses a specialized laparoscope with two optical bundles on opposite sides of the telescope. A specialized bin-ocular eyepiece receives input from two CCD chips, each cap-turing the image from one of the two quartz rod lens systems, thereby creating true 3-D imaging without needing to employ active or passive technologies that have made 3-D laparoscopy so disappointing.Single-incision laparoscopy presents new challenges to visualization of the operative field. In the traditional laparo-scope, the light source enters the scope at a 90° angle. That position coupled with a bulky scope handle creates crowding in an already limited space. Additionally, because the scope and instruments enter the abdomen at the same point, an adequate perspective is often

1	coupled with a bulky scope handle creates crowding in an already limited space. Additionally, because the scope and instruments enter the abdomen at the same point, an adequate perspective is often unobtainable even with a 30° scope. The advent of increased length laparoscopes with lighting coming from the end and a deflectable tip now allows the surgeon to recreate a sense of internal triangulation with little compromise externally. The ability to move the shaft of the scope off line while maintaining the same image provides a greater degree of freedom for the working ports.Energy Sources for Endoscopic and Endoluminal SurgeryMany MIS procedures use conventional energy sources, but the benefits of bloodless surgery to maintain optimal visualization have spawned new ways of applying energy. The most common energy source is RF electrosurgery using an alternating current with a frequency of 500,000 cycles/s (Hz). Tissue heating pro-gresses through the well-known phases of coagulation

1	The most common energy source is RF electrosurgery using an alternating current with a frequency of 500,000 cycles/s (Hz). Tissue heating pro-gresses through the well-known phases of coagulation (60°C [140°F]), vaporization and desiccation (100°C [212°F]), and carbonization (>200°C [392°F]).58The two most common methods of delivering RF electro-surgery are with monopolar and bipolar electrodes. With mono-polar electrosurgery, a remote ground plate on the patient’s leg or back receives the flow of electrons that originate at a point source, the surgical electrode. A fine-tipped electrode causes a high current density at the site of application and rapid tissue heating. Monopolar electrosurgery is inexpensive and easy to modulate to achieve different tissue effects.59 A short-duration, high-voltage discharge of current (coagulation current) provides extremely rapid tissue heating. Lower-voltage, higher-wattage current (cutting current) is better for tissue desiccation and vaporization.

1	discharge of current (coagulation current) provides extremely rapid tissue heating. Lower-voltage, higher-wattage current (cutting current) is better for tissue desiccation and vaporization. When the surgeon desires tissue division with the least amount of thermal injury and least coagulation necrosis, a cutting current is used.With bipolar electrosurgery, the electrons flow between two adjacent electrodes. The tissue between the two electrodes is heated and desiccated. There is little opportunity for tissue cutting when bipolar current is used alone, but the ability to coapt the electrodes across a vessel provides the best method of small-vessel coagulation without thermal injury to adjacent tissues.60 Advanced laparoscopic device manufacturers have leveraged the ability to selectively use bipolar energy and combined it with compressive force and a controllable blade to create a number of highly functional dissection and vessel-sealing tools (Fig. 14-16).Figure 14-16. Examples of

1	use bipolar energy and combined it with compressive force and a controllable blade to create a number of highly functional dissection and vessel-sealing tools (Fig. 14-16).Figure 14-16. Examples of advanced bipolar devices. The flow of electrons passes from one electrode to the other heating and desic-cating tissue. A controllable blade travels the length of the jaw to divide intervening tissue.To avoid thermal injury to adjacent structures, the lapa-roscopic field of view must include all uninsulated portions of the electrosurgical electrode. In addition, the integrity of the insulation must be maintained and assured. Capacitive coupling occurs when a plastic trocar insulates the abdominal wall from the current; in turn, the current is bled off of a metal sleeve or laparoscope into the viscera54 (Fig. 14-17A). This may result in thermal necrosis and a delayed fecal fistula. Another potential mechanism for unrecognized visceral injury may occur with the direct coupling of current to

1	viscera54 (Fig. 14-17A). This may result in thermal necrosis and a delayed fecal fistula. Another potential mechanism for unrecognized visceral injury may occur with the direct coupling of current to the laparoscope and adjacent bowel58 (Fig. 14-17B).Another method of delivering RF electrosurgery is argon beam coagulation. This is a type of monopolar electrosurgery in which a uniform field of electrons is distributed across a tissue surface by the use of a jet of argon gas. The argon gas jet distrib-utes electrons more evenly across the surface than does spray electrofulguration. This technology has its greatest application for coagulation of diffusely bleeding surfaces such as the cut edge of liver or spleen. It is of less value in laparoscopic proce-dures because the increased intra-abdominal pressures created by the argon gas jet can increase the chances of a gas embolus. It is paramount to vent the ports and closely monitor insufflation pressure when using this source of energy

1	pressures created by the argon gas jet can increase the chances of a gas embolus. It is paramount to vent the ports and closely monitor insufflation pressure when using this source of energy within the context of laparoscopy.With endoscopic endoluminal surgery, RF alternating cur-rent in the form of a monopolar circuit represents the mainstay for procedures such as snare polypectomy, sphincterotomy, lower esophageal sphincter ablation, and biopsy.61,62 A ground-ing (return) electrode is necessary for this form of energy. Bipo-lar electrocoagulation is used primarily for thermal hemostasis. The electrosurgical generator is activated by a foot pedal so the endoscopist may keep both hands free during the endoscopic procedure.Gas, liquid, and solid-state lasers have been available for medical application since the mid-1960s.63 The CO2 laser (wavelength 10.6 µm) is most appropriately used for cutting Brunicardi_Ch14_p0453-p0478.indd 46501/03/19 4:58 PM 466BASIC

1	available for medical application since the mid-1960s.63 The CO2 laser (wavelength 10.6 µm) is most appropriately used for cutting Brunicardi_Ch14_p0453-p0478.indd 46501/03/19 4:58 PM 466BASIC CONSIDERATIONSPART IFigure 14-17. A. Capacitive coupling occurs as a result of high current density bleeding from a port sleeve or laparoscope into adjacent bowel. B. Direct coupling occurs when current is transmitted directly from the electrode to a metal instrument or laparoscope, and then into adjacent tissue. (Reproduced with permission from Hunter JG, Sackier JM: Minimally Invasive Surgery. New York, NY: McGraw-Hill Education; 1993.)Figure 14-18. This graph shows the absorption of light by various tissue compounds (water, melanin, and oxyhemoglobin) as a func-tion of the wavelength of the light. The nadir of the oxyhemoglo-bin and melanin curves is close to 1064 nm, the wavelength of the neodymium yttrium-aluminum garnet laser. (Reproduced with per-mission from Hunter JG, Sackier JM:

1	light. The nadir of the oxyhemoglo-bin and melanin curves is close to 1064 nm, the wavelength of the neodymium yttrium-aluminum garnet laser. (Reproduced with per-mission from Hunter JG, Sackier JM: Minimally Invasive Surgery. New York, NY: McGraw-Hill Education; 1993.)Conduction through ungrounded telescopeCannulaPlastic cannulaTelescopeBCapacitive coupled fault conditionCapacitivelycoupled energyto metalcannulaPlastic collarover metaltrocarAand superficial ablation of tissues. It is most helpful in locations unreachable with a scalpel such as excision of vocal cord granu-lomas. The CO2 laser beam must be delivered with a series of mirrors and is therefore somewhat cumbersome to use. The next most popular laser is the neodymium yttrium-aluminum garnet (Nd:YAG) laser. Nd:YAG laser light is 1.064 µm (1064 nm) in wavelength. It is in the near-infrared portion of the spectrum and, like CO2 laser light, is invisible to the naked eye. A unique feature of the Nd:YAG laser is that 1064-nm

1	is 1.064 µm (1064 nm) in wavelength. It is in the near-infrared portion of the spectrum and, like CO2 laser light, is invisible to the naked eye. A unique feature of the Nd:YAG laser is that 1064-nm light is poorly absorbed by most tissue pigments and therefore travels deep into tissue.64 Deep tissue penetration provides deep tissue heating (Fig. 14-18). For this reason, the Nd:YAG laser is capable of the greatest amount of tissue destruction with a single application.63 Absorption coefficientWavelength (nm)10610510410310210110–110–211001000 10,000UV Visible InfaredHbO2H2OH2O1064 nmMelanin Such capabilities make it the ideal laser for destruction of large fungating tumors of the rectosigmoid, tracheobronchial tree, or esophagus. A disadvantage is that the deep tissue heating may cause perforation of a hollow viscus.When it is desirable to coagulate flat lesions in the cecum, a different laser should be chosen. The frequency-doubled Nd:YAG laser, also known as the

1	heating may cause perforation of a hollow viscus.When it is desirable to coagulate flat lesions in the cecum, a different laser should be chosen. The frequency-doubled Nd:YAG laser, also known as the KTP laser (potassium thionyl phosphate crystal is used to double the Nd:YAG frequency), pro-vides 532-nm light. This is in the green portion of the spectrum, and at this wavelength, selective absorption by red pigments in tissue (such as hemangiomas and arteriovenous malformations) is optimal. The depth of tissue heating is intermediate, between those of the CO2 and the Nd:YAG lasers. Coagulation (without vaporization) of superficial vascular lesions can be obtained without intestinal perforation.64In flexible GI endoscopy, the CO2 and Nd:YAG lasers have largely been replaced by heater probes and endoluminal stents. The heater probe is a metal ball that is heated to a tem-perature (60–100°C [140°–212°F]) that allows coagulation of bleeding lesions without perforation.Photodynamic therapy

1	and endoluminal stents. The heater probe is a metal ball that is heated to a tem-perature (60–100°C [140°–212°F]) that allows coagulation of bleeding lesions without perforation.Photodynamic therapy is a palliative treatment for obstruct-ing cancers of the GI tract.65 Patients are given an IV dose of porfimer sodium, which is a photosensitizing agent that is taken up by malignant cells. Two days after administration, the drug is endoscopically activated using a laser. The activated porfimer sodium generates oxygen free radicals, which kill the tumor cells. The tumor is later endoscopically debrided. The use of this modality for definitive treatment of early cancers is limited.A unique application of laser technology provides extremely rapid discharge (<10–6 s) of large amounts of energy (>103 volts). These high-energy lasers, of which the pulsed dye laser has seen the most clinical use, allow the conversion of light energy to mechanical disruptive energy in the form of a shock wave.

1	(>103 volts). These high-energy lasers, of which the pulsed dye laser has seen the most clinical use, allow the conversion of light energy to mechanical disruptive energy in the form of a shock wave. Such energy can be delivered through a quartz fiber, and with rapid repetitive discharges, can provide sufficient shock-wave energy to fragment kidney stones and gallstones.66 Shock waves also may be created with miniature electric spark-plug discharge systems known as electrohydraulic lithotriptors. These devices Brunicardi_Ch14_p0453-p0478.indd 46601/03/19 4:58 PM 467MINIMALLY INVASIVE SURGERYCHAPTER 14also are inserted through thin probes for endoscopic application. Lasers have the advantage of pigment selectivity, but electrohy-draulic lithotriptors are more popular because they are substan-tially less expensive and are more compact.Methods of producing shock waves or heat with ultrasonic energy are also of interest. Extracorporeal shockwave lithotripsy creates focused shock waves

1	less expensive and are more compact.Methods of producing shock waves or heat with ultrasonic energy are also of interest. Extracorporeal shockwave lithotripsy creates focused shock waves that intensify as the focal point of the discharge is approached. When the focal point is within the body, large amounts of energy are capable of fragmenting stones. Slightly different configurations of this energy can be used to provide focused internal heating of tissues. Potential applications of this technology include the ability to noninvasively produce sufficient internal heating to destroy tissue without an incision.A third means of using ultrasonic energy is to create rap-idly oscillating instruments that are capable of heating tissue with friction; this technology represents a major step forward in energy technology.67 An example of its application is the lapa-roscopic coagulation shears device (Harmonic Scalpel), which is capable of coagulating and dividing blood vessels by first occluding

1	in energy technology.67 An example of its application is the lapa-roscopic coagulation shears device (Harmonic Scalpel), which is capable of coagulating and dividing blood vessels by first occluding them and then providing sufficient heat to weld the blood vessel walls together and to divide the vessel (Fig. 14-19). This nonelectric method of coagulating and dividing tissue with a minimal amount of collateral damage has facilitated the performance of numerous endosurgical procedures.68 It is espe-cially useful in the control of bleeding from medium-sized ves-sels that are too big to manage with monopolar electrocautery. The ability to clamp tissue between an active blade and passive blade allows annealing of tissues followed by cutting.InstrumentationHand instruments for MIS usually are duplications of conven-tional surgical instruments made longer, thinner, and smaller at the tip. It is important to remember that when grasping tissue with laparoscopic instruments, a greater force is

1	of conven-tional surgical instruments made longer, thinner, and smaller at the tip. It is important to remember that when grasping tissue with laparoscopic instruments, a greater force is applied over a smaller surface area, which increases the risk for perforation or injury.69Certain conventional instruments such as scissors are easy to reproduce with a diameter of 3 to 5 mm and a length of 20 to 45 cm, but other instruments such as forceps and clamps can-not provide remote access. Different configurations of grasp-ers were developed to replace the various configurations of surgical forceps and clamps. Standard hand instruments are 5 mm in diameter and 30 cm in length, but smaller and shorter hand instruments are now available for pediatric surgery, for microlaparoscopic surgery, and for arthroscopic procedures.69 A unique laparoscopic hand instrument is the monopolar electrical hook. This device usually is configured with a suction and irriga-tion apparatus to eliminate smoke and

1	arthroscopic procedures.69 A unique laparoscopic hand instrument is the monopolar electrical hook. This device usually is configured with a suction and irriga-tion apparatus to eliminate smoke and blood from the operative Figure 14-19. Ultrasonic shear. When closed vibration of black (active blade) against white (passive blade) cuts and cauterizes intervening tissue.field. The monopolar hook allows tenting of tissue over a bare metal wire with subsequent coagulation and division of the tissue.Instrumentation for NOTES is still evolving, but many long micrograspers, microscissors, electrocautery adapters, suturing devices, clip appliers, and visceral closure devices are in design and application. These instruments often require an entirely different endoscopic platform requiring manipula-tion by a surgeon and assistant to accomplish complex maneu-vers. Techniques such as mucosotomy, hydrodissection, and clip application require specialized training. The sheer size of the

1	manipula-tion by a surgeon and assistant to accomplish complex maneu-vers. Techniques such as mucosotomy, hydrodissection, and clip application require specialized training. The sheer size of the instrumentation often requires an overtube to allow easy exchange throughout the procedure. Instrumentation for SILS seeks to restore the surgeon’s ability to triangulate the left and right hands through variation in length, mechanical articulation, or curved design. Additionally, a lower profile camera head helps reduce the instrument crowding that occurs at the single point of abdominal entry.Robotic SurgeryThe term robot defines a device that has been programmed to perform specific tasks in place of those usually performed by people. The devices that have earned the title “surgical robots” would be more aptly termed computer-enhanced surgical devices, as they are controlled entirely by the surgeon for the purpose of improving performance. The first computer-assisted surgical device was the

1	be more aptly termed computer-enhanced surgical devices, as they are controlled entirely by the surgeon for the purpose of improving performance. The first computer-assisted surgical device was the laparoscopic camera holder (Aesop, Computer Motion, Goleta, CA), which enabled the surgeon to maneuver the laparoscope either with a hand control, foot con-trol, or voice activation. Randomized studies with such camera holders demonstrated a reduction in operative time, steadier image, and a reduction in the number of required laparoscope cleanings.70 This device had the advantage of eliminating the need for a human camera holder, which served to free valuable OR personnel for other duties. This technology has now been eclipsed by simpler systems using passive positioning of the camera with a mechanical arm, but the benefits of a steadier image and fewer members of the OR team remain.The major revolution in robotic surgery was the develop-ment of a master-slave surgical platform that

1	a mechanical arm, but the benefits of a steadier image and fewer members of the OR team remain.The major revolution in robotic surgery was the develop-ment of a master-slave surgical platform that returned the wrist to laparoscopic surgery and improved manual dexterity by developing an ergonomically comfortable work station, with 3-D imaging, tremor elimination, and scaling of movement (e.g., large, gross hand movements can be scaled down to allow suturing with microsurgical precision) (Fig. 14-20). The most recent iteration of the robotic platform features a second surgi-cal console enabling greater assisting and teaching opportuni-ties. The surgeon is physically separated from the operating table, and the working arms of the device are placed over the patient (Fig. 14-21). An assistant remains at the bedside and changes the instruments as needed, providing retraction as needed to facilitate the procedure. The robotic platform (da Vinci, Intuitive Surgical, Sunnyvale, CA) was

1	remains at the bedside and changes the instruments as needed, providing retraction as needed to facilitate the procedure. The robotic platform (da Vinci, Intuitive Surgical, Sunnyvale, CA) was initially greeted with some skepticism by expert laparoscopists, as it was difficult to prove additional value for operations performed with the da Vinci robot. Not only were the operations longer and the equip-ment more expensive, but additional quality could not be dem-onstrated. Two randomized controlled trials compared robotic and conventional laparoscopic approaches to Nissen fundoplica-tion.71,72 In both of these trials, the operative time was longer for robotic surgery, and there was no difference in ultimate outcome. Similar results were achieved for laparoscopic cholecystec-tomy.73 Nevertheless, the increased dexterity provided by the da Brunicardi_Ch14_p0453-p0478.indd 46701/03/19 4:58 PM 468BASIC CONSIDERATIONSPART IFigure 14-21. Room setup and position of surgeon and assistant

1	the increased dexterity provided by the da Brunicardi_Ch14_p0453-p0478.indd 46701/03/19 4:58 PM 468BASIC CONSIDERATIONSPART IFigure 14-21. Room setup and position of surgeon and assistant for robotic surgery. (© 2013 Intuitive Surgical, Inc. Reprinted with permission.)Vinci robot convinced many surgeons and health administrators that robotic platforms were worthy of investment, for marketing purposes if for no other reason. The success story for computer-enhanced surgery with the da Vinci started with cardiac surgery and migrated to the pelvis. Mitral valve surgery, performed with right thoracoscopic access, became one of the more popular procedures performed with the robot.74To date, a myriad of publications have demonstrated suc-cess performing procedures from thyroidectomies to colec-tomies with total mesorectal excision. Almost any procedure performed laparoscopically has been attempted robotically, although true advantage is demonstrated only very sparingly. In most cases,

1	with total mesorectal excision. Almost any procedure performed laparoscopically has been attempted robotically, although true advantage is demonstrated only very sparingly. In most cases, increased cost and operative time challenge the notion of “better.”The tidal wave of enthusiasm for robotic surgery came when most minimally invasive urologists declared robotic prostatectomy to be preferable to laparoscopic and open pros-tatectomy.75 The great advantage—it would appear—of robotic prostatectomy is the ability to visualize and spare the pelvic nerves responsible for erectile function. In addition, the cre-ation of the neocystourethrotomy, following prostatectomy, was greatly facilitated by needle holders and graspers with a wrist in them. Female pelvic surgery with the da Vinci robot is also reaching wide appeal. The magnified imaging provided makes this approach ideal for microsurgical tasks such as reanastomo-sis of the Fallopian tubes. In general surgery, there is emerging 3Figure

1	reaching wide appeal. The magnified imaging provided makes this approach ideal for microsurgical tasks such as reanastomo-sis of the Fallopian tubes. In general surgery, there is emerging 3Figure 14-20. Robotic instruments and hand controls. The sur-geon is in a sitting position, and the arms and wrists are in an ergo-nomic and relaxed position.Brunicardi_Ch14_p0453-p0478.indd 46801/03/19 4:58 PM 469MINIMALLY INVASIVE SURGERYCHAPTER 14popularity for the use of the robotic platform for revisional bar-iatric surgery and complex abdominal wall reconstruction. The ability to close the defect before placement of mesh in ventral hernia repairs or to perform complex transversus abdominus release herniorrhaphy is revolutionizing MIS hernia repair.The final frontier for computer-enhanced surgery is the promise of telesurgery, in which the surgeon is a great distance from the patient (e.g., combat or space). This application has rarely been used, as the safety provided by having the surgeon

1	is the promise of telesurgery, in which the surgeon is a great distance from the patient (e.g., combat or space). This application has rarely been used, as the safety provided by having the surgeon at bedside cannot be sacrificed to prove the concept. However, remote laparoscopic cholecystectomy has been performed when a team of surgeons located in New York performed a cholecys-tectomy on a patient located in France.76Endoluminal and Endovascular SurgeryThe fields of vascular surgery, interventional radiology, neu-roradiology, gastroenterology, general surgery, pulmonology, and urology all encounter clinical scenarios that require the urgent restoration of luminal patency. Based on this need, fun-damental techniques have been pioneered that are applicable to all specialties and virtually every organ system. As a result, all minimally invasive surgical procedures, from coronary artery angioplasty to palliation of pancreatic malignancy, involve the use of access devices, catheters,

1	every organ system. As a result, all minimally invasive surgical procedures, from coronary artery angioplasty to palliation of pancreatic malignancy, involve the use of access devices, catheters, guidewires, balloon dilators, stents, and other devices (e.g., lasers, atherectomy catheters) that are capable of opening up the occluded biologic cylinder77 (Table 14-2). Endoluminal balloon dilators may be inserted through an endoscope, or they may be fluoroscopically guided. Balloon dilators all have low compliance—that is, the balloons do not stretch as the pressure within the balloon is increased. The high pressures achievable in the balloon create radial expansion of the narrowed vessel or orifice, usually disrupting the atherosclerotic plaque, the fibrotic stricture, or the muscular band (e.g., esophageal achalasia).78Once the dilation has been attained, it is frequently ben-eficial to hold the lumen open with a stent.79 Stenting is particu-larly valuable in treating malignant lesions

1	esophageal achalasia).78Once the dilation has been attained, it is frequently ben-eficial to hold the lumen open with a stent.79 Stenting is particu-larly valuable in treating malignant lesions and atherosclerotic Figure 14-22. The deployment of a metal stent across an isolated vessel stenosis is illustrated. (Reproduced with permission from Hunter JG, Sackier JM, eds. Minimally Invasive Surgery. New York: McGraw-Hill; 1993:235.)GuidewireBalloonSheathBalloon with stentStent expandedStent in placeTable 14-2Modalities and techniques of restoring luminal patencyMODALITYTECHNIQUECore outPhotodynamic therapyLaserCoagulationEndoscopic biopsy forcepsChemicalUltrasoundFractureUltrasoundEndoscopic biopsyBalloonDilateBalloonBougieAngioplastyEndoscopeBypassTransvenous intrahepatic portosystemic shuntSurgical (synthetic or autologous conduit)StentSelf-expanding metal stentPlastic stentocclusions or aneurysmal disease (Fig. 14-22). Stenting is also of value to seal leaky cylinders, including

1	shuntSurgical (synthetic or autologous conduit)StentSelf-expanding metal stentPlastic stentocclusions or aneurysmal disease (Fig. 14-22). Stenting is also of value to seal leaky cylinders, including aortic dissections, traumatic vascular injuries, leaking GI anastomoses, and fistu-las. Stenting usually is not applicable for long-term manage-ment of benign GI strictures except in patients with limited life expectancy (Fig. 14-23).79–81A variety of stents are available that are divided into six basic categories: plastic stents, metal stents, drug-eluting stents (to decrease fibrovascular hyperplasia), covered metal stents, anchored stent grafts, and removable covered plastic stents80 (Fig. 14-24). Plastic stents came first and are used widely as endoprostheses for temporary bypass of obstructions in the biliary or urinary systems. Metal stents generally are delivered over a balloon and expanded with the balloon to the desired size. These metal stents usually are made of titanium or

1	in the biliary or urinary systems. Metal stents generally are delivered over a balloon and expanded with the balloon to the desired size. These metal stents usually are made of titanium or niti-nol and are still used in coronary stenting. A chemotherapeutic agent was added to coronary stents several years ago to decrease endothelial proliferation. These drug-eluting stents provide greater long-term patency but require long-term anticoagula-tion with antiplatelet agents to prevent thrombosis.82 Coated metal stents are used to prevent tissue ingrowth. Ingrowth may Brunicardi_Ch14_p0453-p0478.indd 46901/03/19 4:59 PM 470BASIC CONSIDERATIONSPART IFigure 14-23. This is an esophagram in a patient with severe dys-phagia secondary to advanced esophageal cancer (A) before and (B) after placement of a covered self-expanding metal stent.ABFigure 14-24. Covered self-expanding metal stents. These devices can be placed fluoroscopically or endoscopically.be an advantage in preventing stent

1	of a covered self-expanding metal stent.ABFigure 14-24. Covered self-expanding metal stents. These devices can be placed fluoroscopically or endoscopically.be an advantage in preventing stent migration, but such tissue ingrowth may occlude the lumen and cause obstruction anew. This is a particular problem when stents are used for palliation of GI malignant growth and may be a problem for the long-term use of stents in vascular disease. Filling the interstices with Silastic or other materials may prevent tumor ingrowth but also makes stent migration more likely. In an effort to minimize stent migration, stents have been incorporated with hooks and barbs at the proximal end of the stent to anchor it to the wall of the vessel. Endovascular stenting of aortic aneurysms has nearly replaced open surgery for this condition. Lastly, self-expanding plastic stents have been developed as temporary devices to be used in the GI tract to close internal fistulas and bridge leaking

1	replaced open surgery for this condition. Lastly, self-expanding plastic stents have been developed as temporary devices to be used in the GI tract to close internal fistulas and bridge leaking anastomoses.Natural Orifice Transluminal Endoscopic SurgeryThe use of the flexible endoscope to enter the GI, urinary, or reproductive tracts and then traverse the wall of the structure to enter the peritoneal cavity, the mediastinum, or the chest has strong appeal to patients wishing to avoid scars and pain caused by abdominal wall trauma. In truth, transluminal surgery has been performed in the stomach for a long time, either from the inside out (e.g., percutaneous, PEG, and transgastric pseudocyst drainage) or from the outside in (e.g., laparoscopic-assisted intragastric tumor resection). The catalyz-ing events for NOTES were the demonstration that a porcine gallbladder could be removed with a flexible endoscope passed through the wall of the stomach and then removed through the mouth and

1	catalyz-ing events for NOTES were the demonstration that a porcine gallbladder could be removed with a flexible endoscope passed through the wall of the stomach and then removed through the mouth and the demonstration in a series of 10 human cases from India of the ability to perform transgastric appendectomy. Since that time, a great deal of money has been invested by endo-scopic and MIS companies to help surgeons and gastroenterolo-gists explore this new territory. Systemic inflammatory markers such as C-reactive protein, tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 have been shown to be similar in transgastric and transcolonic NOTES when compared to laparoscopy in por-cine models.83 Concerns about the safety of transluminal access and limitations in equipment remain the greatest barriers to expansion. To date, the most headline-grabbing procedures have been the transvaginal and transgastric removal of the gallbladder84-86 (Fig. 14-25). To ensure safety, all human cases

1	barriers to expansion. To date, the most headline-grabbing procedures have been the transvaginal and transgastric removal of the gallbladder84-86 (Fig. 14-25). To ensure safety, all human cases thus far have involved laparoscopic assistance to aid in retrac-tion and ensure adequate closure of the stomach or vagina. To date, thousands of transvaginal and transgastric procedures have been performed internationally, with two large registries dem-onstrating noninferiority to conventional laparoscopy.87 The fact that the vast majority of these procedures are being done trans-vaginally creates an obvious limitation in applicability.The rapid growth of endoscopic technology catalyzed by NOTES has already spun off new technologies capable of performing a wide variety of endoscopic surgical procedures from EMR, to ablation of Barrett’s esophagus, to creation of competent antireflux valves in patients with gastroesophageal reflux disease.Peroral esophageal myotomy (POEM) has shown promise as a

1	from EMR, to ablation of Barrett’s esophagus, to creation of competent antireflux valves in patients with gastroesophageal reflux disease.Peroral esophageal myotomy (POEM) has shown promise as a NOTES treatment for esophageal achalasia.88 In this proce-dure, a 1.5to 2-cm mucosotomy is created within the anterior esophagus 10 cm proximal to the gastroesophageal junction. A submucosal tunnel is then created using a combination of elec-trocautery, hydrodissection, and carbon dioxide insufflation. The scope is advanced beyond the gastroesophageal junction, and a circular myotomy is performed avoiding disruption of the longitudinal fibers. The mucosotomy is then closed using endo-scopic clips (Fig. 14-26). Over 1000 clinical POEM cases have been performed worldwide. Data from expert NOTES surgeons suggest that this selective myotomy avoids abdominal trauma 4Brunicardi_Ch14_p0453-p0478.indd 47001/03/19 4:59 PM 471MINIMALLY INVASIVE SURGERYCHAPTER 14Figure 14-25. Transgastric

1	NOTES surgeons suggest that this selective myotomy avoids abdominal trauma 4Brunicardi_Ch14_p0453-p0478.indd 47001/03/19 4:59 PM 471MINIMALLY INVASIVE SURGERYCHAPTER 14Figure 14-25. Transgastric cholecystectomy using natural orifice transluminal endoscopic surgery technology and one to three laparoscopic ports has been performed occasionally in several locations around the world. (Reproduced with permission from The Johns Hopkins University School of Medicine, Baltimore, MD; 2007. Illustration by Jennifer Fairman.)and minimally disrupts the normal anatomic characteristics of the gastroesophageal junction while providing significant relief of symptoms.89 Randomized clinical trials and long-term follow-up need to be performed to further evaluate efficacy.Although this application is still considered experimen-tal, there is little doubt that when equivalent operations can be performed with less pain, fewer scars, and less disability, patients will flock to it. NOTES procedures are

1	considered experimen-tal, there is little doubt that when equivalent operations can be performed with less pain, fewer scars, and less disability, patients will flock to it. NOTES procedures are associated with an increased mental workload and significant learning curve for even experienced surgical endoscopists. Surgeons should engage only when they can perform these procedures with the safety and efficacy demanded by our profession.Single-Incision Laparoscopic SurgeryAs a surgical technique, SILS seems to be a natural progression from conventional laparoscopic surgery. As surgeons sought to reduce the number and size of abdominal wall trocars and NOTES procedures necessitated laparoscopic surveillance, the idea of a hybridization took off. An incision in the umbilicus, a preexisting scar, is thought to be less painful, have fewer wound complications, lead to quicker return to activity, and have a bet-ter cosmetic appearance than conventional laparoscopy. Per-haps one of the earliest

1	is thought to be less painful, have fewer wound complications, lead to quicker return to activity, and have a bet-ter cosmetic appearance than conventional laparoscopy. Per-haps one of the earliest examples of SILS is the application of laparoscopic instrumentation to resect lesions in the rectum or sigmoid colon. Using the anus as the portal of entry, transanal endoscopic microsurgery (TEMS) employs a specialized mul-tichannel trocar to reach lesions located 8 to 18 cm away from the anal verge (Fig. 14-27).More deformable versions of these complex trocars have been developed with features to allow insufflation and be ame-nable to maintaining a seal within the natural orifice of the umbili-cus (see Fig. 14-11). Ports typically contain three or four channels. The latter often affords the ability to place a dedicated retractor.There are many challenges faced by the operating surgeon in SILS procedures. These include crowded trocar placement, a lack of triangulation of leftand

1	the ability to place a dedicated retractor.There are many challenges faced by the operating surgeon in SILS procedures. These include crowded trocar placement, a lack of triangulation of leftand right-hand instruments, fre-quent crossing or clashing of instruments, limited visualiza-tion, and limited retraction ability. These challenges are mitigated by surgeon’s experience and the development of specialized instruments. Articulating or curved instruments of varying lengths and an extended length can improve working space. Curved instruments are typically reusable and offer less clutter than their more sophisticated counterparts, providing some cost reduction (Fig. 14-28). A low-profile HD scope with or without a deflect-able tip can improve visualization greatly. Even with such instru-mentation, the learning curve is very steep, particularly when the surgeon is forced to work in a cross-handed technique. The accomplished SILS surgeon will possess a tool bag of innovative

1	instru-mentation, the learning curve is very steep, particularly when the surgeon is forced to work in a cross-handed technique. The accomplished SILS surgeon will possess a tool bag of innovative 5Brunicardi_Ch14_p0453-p0478.indd 47101/03/19 4:59 PM 472BASIC CONSIDERATIONSPART IFigure 14-28. Example of curved instruments used in single-incision laparoscopic surgery. (© 2013 Intuitive Surgical, Inc. Reprinted with permission.)Figure 14-26. A. Peroral endoscopic esophageal myotomy for the treatment of achalasia. (Reproduced with permission from Inoue H, Minami H, Kobayashi Y, et al. Peroral endoscopic myot-omy (POEM) for esophageal achalasia, Endoscopy. 2010 Apr; 42(4):265-271.) B. Serial images showing overtube in submuco-sal tunnel, using needle knife to divide circular muscle fibers of esophagus, and closure of myotomy with clips. (Reproduced with permission from Rieder E, Dunst CM, Kastenmeier AS, et al: Devel-opment and technique of per oral endoscopic myotomy (POEM) for

1	of esophagus, and closure of myotomy with clips. (Reproduced with permission from Rieder E, Dunst CM, Kastenmeier AS, et al: Devel-opment and technique of per oral endoscopic myotomy (POEM) for achala, Eur Surg 2011 June;43(3):140–145.) ABFigure 14-27. Transanal endoscopic microsurgery scope. (Repro-duced with permission from The Johns Hopkins University School of Medicine, Baltimore, MD; 2014. Illustration by Corinne Sandone.)strategies to retract structures like the gallbladder away from the operative field. These tricks may range from the use of percutane-ous needlescopic instruments to the application of transfascial sutures. Expert consensus recommendations for efficient SILS are shown in Tables 14-3 and 14-4.8 When performing SILS proce-dures, it is imperative to follow proven tenets of operative con-duct such as visualizing the “critical view” of safety in a laparoscopic cholecystectomy. As safety should always be the paramount concern, the addition of extra trocars or

1	tenets of operative con-duct such as visualizing the “critical view” of safety in a laparoscopic cholecystectomy. As safety should always be the paramount concern, the addition of extra trocars or conversion to traditional laparoscopy should not be considered a failure.Contraindications include those true of traditional lapa-roscopy. Relative contraindications include previous surgery and high body mass index (BMI). Patients with a high BMI or central obesity can pose a challenge because the umbilicus may be located far from operative target. Size and morphology of the target organ should always be considered when doing SILS.Many studies have demonstrated equivalency to standard laparoscopic procedures regarding intraoperative and postop-erative complications. However, it is questionable what the full benefit of the dramatic reduction in ergonomics and the increase in complexity provide beyond an improved cosmetic appear-ance. This is in large part due to the already improved benefits

1	the full benefit of the dramatic reduction in ergonomics and the increase in complexity provide beyond an improved cosmetic appear-ance. This is in large part due to the already improved benefits of laparoscopic surgery.A meta-analysis performed by Ahmed and colleagues in 2010 found the conversion rate from SILS to conventional lapa-roscopy to be 0% to 24% for cholecystectomies, 0% to 41% Table 14-3Expert panel recommendations for accomplishing single-incision laparoscopic surgery efficientlyMultichannel port preferably to be placed intraumbilically, but an extraumbilical approach can be used in certain casesExtra ports should be used where there is a clinical needWhen applicable, sutures can be useful for added retractionClosure should be accomplished using sutures of absorbable material placed either continuously or interruptedSkin should be closed with absorbable sutures or glueReproduced with permission from Ahmed I, Cianco F, Ferrar V, et al. Current status of single-incision

1	placed either continuously or interruptedSkin should be closed with absorbable sutures or glueReproduced with permission from Ahmed I, Cianco F, Ferrar V, et al. Current status of single-incision laparoscopic surgery: European experts’ views, Surg Laparosc Endosc Percutan Tech. 2012 Jun;22(3):194-199.Brunicardi_Ch14_p0453-p0478.indd 47201/03/19 4:59 PM 473MINIMALLY INVASIVE SURGERYCHAPTER 14Figure 14-29. A and B. Robotic single-incision surgery platform. (©2013 Intuitive Surgical, Inc. Reprinted with permission.)ABTable 14-4Expert panel recommendations for single-incision laparoscopic surgery equipment and instrumentationRECOMMENDED EQUIPMENT/INSTRUMENTATIONBENEFIT TO SURGEONSlimline instruments with low-profile designReduces internal and external clashingVaried-length instrumentsReduces extracorporeal clashingLonger instrumentsAdvantageous for reaching the surgical fieldArticulating (or prebent) instrumentsRestore triangulationSmall-diameter, low-profile angle scopeReduces

1	extracorporeal clashingLonger instrumentsAdvantageous for reaching the surgical fieldArticulating (or prebent) instrumentsRestore triangulationSmall-diameter, low-profile angle scopeReduces clashing by providing additional spaceHigh-definition cameraAchieves high-quality images for intraoperative visualizationReproduced with permission from Ahmed I, Cianco F, Ferrar V, et al. Current status of single-incision laparoscopic surgery: European experts’ views, Surg Laparosc Endosc Percutan Tech. 2012 Jun;22(3):194-199.for appendectomies, and 0% to 33% for nephrectomies.90 The most common complications were intra-abdominal abscesses and wound infections. Existing and emerging robotics platforms may provide the bridge necessary to bypass the significant tech-nical skills learning curve required to operate through a single site (Fig. 14-29).SPECIAL CONSIDERATIONSPediatric LaparoscopyThe advantages of MIS in children may be more significant than in the adult population. MIS in the adolescent

1	to operate through a single site (Fig. 14-29).SPECIAL CONSIDERATIONSPediatric LaparoscopyThe advantages of MIS in children may be more significant than in the adult population. MIS in the adolescent is little dif-ferent from that in the adult, and standard instrumentation and trocar positions usually can be used. However, laparoscopy in the infant and young child requires specialized instrumentation. The instruments are shorter (15–20 cm), and many are 3 mm in diameter rather than 5 mm. Because the abdomen of the child is much smaller than that of the adult, a 5-mm telescope pro-vides sufficient illumination for most operations. The develop-ment of 5-mm clippers and bipolar devices has obviated the need for 10-mm trocars in pediatric laparoscopy.91 Because the abdominal wall is much thinner in infants, a pneumoperitoneum pressure of 8 mmHg can provide adequate exposure. DVT is rare in children, so prophylaxis against thrombosis probably is unnecessary. A wide variety of pediatric

1	in infants, a pneumoperitoneum pressure of 8 mmHg can provide adequate exposure. DVT is rare in children, so prophylaxis against thrombosis probably is unnecessary. A wide variety of pediatric surgical procedures are frequently performed with MIS access, from pull-through procedures for colonic aganglionosis (Hirschsprung’s disease) to repair of congenital diaphragmatic hernias.92Laparoscopy During PregnancyConcerns about the safety of laparoscopic cholecystectomy or appendectomy in the pregnant patient have been thoroughly investigated and are readily managed. Access to the abdomen in the pregnant patient should take into consideration the height of the uterine fundus, which reaches the umbilicus at 20 weeks. In order not to damage the uterus or its blood supply, most surgeons feel that the open (Hasson) approach should be 6used in favor of direct puncture laparoscopy. The patient should be positioned slightly on the left side to avoid compression of the vena cava by the uterus.

1	the open (Hasson) approach should be 6used in favor of direct puncture laparoscopy. The patient should be positioned slightly on the left side to avoid compression of the vena cava by the uterus. Because pregnancy poses a risk for thromboembolism, sequential compression devices are essential for all procedures. Fetal acidosis induced by maternal hypercar-bia also has been raised as a concern. The arterial pH of the fetus follows the pH of the mother linearly; and therefore, fetal acido-sis may be prevented by avoiding a respiratory acidosis in the mother.93 The pneumoperitoneum pressure induced by laparos-copy is not a safety issue either as it has been proved that mid-pregnancy uterine contractions provide a much greater pressure in utero than a pneumoperitoneum of 15 mmHg. More than 100 cases of laparoscopic cholecystectomy in pregnancy have been reported with uniformly good results.94 The operation should be performed during the second trimester of pregnancy if possible.

1	than 100 cases of laparoscopic cholecystectomy in pregnancy have been reported with uniformly good results.94 The operation should be performed during the second trimester of pregnancy if possible. Protection of the fetus against intraoperative X-rays Brunicardi_Ch14_p0453-p0478.indd 47301/03/19 4:59 PM 474BASIC CONSIDERATIONSPART Iis imperative. Some believe it advisable to track fetal pulse rates with a transvaginal ultrasound probe; however, the significance of fetal tachycardia or bradycardia is a bit unclear in the second trimester of pregnancy. To be prudent, however, heart rate decelerations reversibly associated with pneumoperitoneum cre-ation might signal the need to convert to open cholecystectomy or appendectomy.Minimally Invasive Surgery and Cancer TreatmentMIS techniques have been used for many decades to provide palliation for the patient with an obstructive cancer. Laser treat-ment, intracavitary radiation, stenting, and dilation are outpa-tient techniques that

1	have been used for many decades to provide palliation for the patient with an obstructive cancer. Laser treat-ment, intracavitary radiation, stenting, and dilation are outpa-tient techniques that can be used to reestablish the continuity of an obstructed esophagus, bile duct, ureter, or airway. MIS techniques also have been used in the staging of cancer. Medias-tinoscopy is still used occasionally before thoracotomy to assess the status of the mediastinal lymph nodes. Laparoscopy also is used to assess the liver in patients being evaluated for pancre-atic, gastric, or hepatic resection. New technology and greater surgical skills allow for accurate minimally invasive staging of cancer.95 Occasionally, it is appropriate to perform pallia-tive measures (e.g., laparoscopic gastrojejunostomy to bypass a pancreatic cancer) at the time of diagnostic laparoscopy if diag-nostic findings preclude attempts at curative resection.Initially controversial, the role of MIS to provide a safe curative

1	bypass a pancreatic cancer) at the time of diagnostic laparoscopy if diag-nostic findings preclude attempts at curative resection.Initially controversial, the role of MIS to provide a safe curative treatment of cancer has proven to be no different from the principles of open surgery. All gross and microscopic tumor should be removed (an R0 resection), and an ade-quate lymphadenectomy should be performed to allow accurate staging. Generally, this number has been 10 to 15 lymph nodes, although there is still debate as to the value of more extensive lymphadenectomy. All of the major abdominal cancer opera-tions have been performed with laparoscopy. Of the three major cancer resections of GI cancer (liver lobe, pancreatic head, and esophagus), only esophagectomy is routinely performed by a fair number of centers.96,97 Laparoscopic hepatectomy has attracted a loyal following, and distal pancreatectomy frequently is performed with laparoscopic access. In Japan, laparoscopic-assisted

1	by a fair number of centers.96,97 Laparoscopic hepatectomy has attracted a loyal following, and distal pancreatectomy frequently is performed with laparoscopic access. In Japan, laparoscopic-assisted gastrectomy has become quite popular for early gastric cancer, an epidemic in Japan far exceeding that of colon cancer in North America and Northern Europe. The most common can-cer operation performed laparoscopically is segmental colec-tomy, which has proven itself safe and efficacious in a multicenter, controlled, randomized trial.98Considerations in the Elderly and InfirmLaparoscopic cholecystectomy has made possible the removal of a symptomatic gallbladder in many patients previously thought to be too elderly or too ill to undergo a laparotomy. Older patients are more likely to require conversion to lapa-rotomy because of disease chronicity.98Operations on these patients require close monitoring of anesthesia. The intraoperative management of these patients may be more difficult with

1	to lapa-rotomy because of disease chronicity.98Operations on these patients require close monitoring of anesthesia. The intraoperative management of these patients may be more difficult with laparoscopic access than with open access. The advantage of MIS lies in what happens after the operation. Much of the morbidity of surgery in the elderly is a result of impaired mobility. In addition, pulmonary compli-cations, urinary tract sepsis, DVT, pulmonary embolism, con-gestive heart failure, and myocardial infarction often are the result of improper fluid management and decreased mobility. By allowing rapid and early mobilization, laparoscopic surgery 7has made possible the safe performance of procedures in the elderly and infirm.Cirrhosis and Portal HypertensionPatients with hepatic insufficiency pose a significant challenge for any type of surgical intervention.99 The ultimate surgical out-come in this population relates directly to the degree of under-lying hepatic dysfunction.100

1	pose a significant challenge for any type of surgical intervention.99 The ultimate surgical out-come in this population relates directly to the degree of under-lying hepatic dysfunction.100 Often, this group of patients has minimal reserve, and the stress of an operation will trigger com-plete hepatic failure or hepatorenal syndrome. These patients are at risk for major hemorrhage at all levels, including trocar insertion, operative dissection in a field of dilated veins, and secondary to an underlying coagulopathy. Additionally, ascitic leak from a port site may occur, leading to bacterial peritonitis. Therefore, a watertight port site closure should be carried out in all patients.It is essential that the surgeon be aware of the severity of hepatic cirrhosis as judged by a Model of End-Stage Liver Dis-ease (MELD) score or Child’s classification. Additionally, the presence of portal hypertension is a relative contraindication to laparoscopic surgery until the portal pressures are

1	Liver Dis-ease (MELD) score or Child’s classification. Additionally, the presence of portal hypertension is a relative contraindication to laparoscopic surgery until the portal pressures are reduced with portal decompression. For example, if a patient has an incarcer-ated umbilical hernia and ascites, a preoperative paracentesis or transjugular intrahepatic portosystemic shunt procedure in con-junction with aggressive diuresis may be considered. Because these patients commonly are intravascularly depleted, insuffla-tion pressures should be reduced to prevent a decrease in cardiac output, and minimal amounts of Na+-sparing IV fluids should be given.Economics of Minimally Invasive SurgeryMinimally invasive surgical procedures reduce the costs of sur-gery most when length of hospital stay can be shortened and return to work is quickened. For example, shorter hospital stays can be demonstrated in laparoscopic cholecystectomy, Nissen fundoplication, splenectomy, and adrenalectomy.

1	stay can be shortened and return to work is quickened. For example, shorter hospital stays can be demonstrated in laparoscopic cholecystectomy, Nissen fundoplication, splenectomy, and adrenalectomy. Procedures such as inguinal herniorrhaphy that are already performed as outpatient procedures are less likely to provide cost savings. Procedures that still require a 4to 7-day hospitalization, such as laparoscopy-assisted colectomy, are less likely to deliver a lower bottom line than their open surgery counterparts. None-theless, with responsible use of disposable instrumentation and a commitment to the most effective use of the inpatient setting, most laparoscopic procedures can be made less expensive than their conventional equivalents.Education and Skill AcquisitionHistorically, surgeons in training (residents, registrars, and fel-lows) acquired their skills in minimally invasive techniques through a series of operative experiences of graded complexity. This training occurred on

1	in training (residents, registrars, and fel-lows) acquired their skills in minimally invasive techniques through a series of operative experiences of graded complexity. This training occurred on patients. Although such a paradigm did not compromise patient safety, learning in the OR is costly. In addition, the recent worldwide constraint placed on resident work hours makes it attractive to teach laparoscopic skills out-side of the OR.Skills labs started at nearly every surgical training center in the 1990s with low fidelity box-type trainers. These were rudimentary simulated abdominal cavities with a video camera, monitor, trocars, laparoscopic instruments, and target models. These targets were often as simple as a pegboard and rubber rings, or a latex drain to practice suturing and knot tying. Virtual reality training devices present a unique opportunity to improve and enhance experiential learning in endoscopy and laparoscopy Brunicardi_Ch14_p0453-p0478.indd 47401/03/19 4:59 PM

1	tying. Virtual reality training devices present a unique opportunity to improve and enhance experiential learning in endoscopy and laparoscopy Brunicardi_Ch14_p0453-p0478.indd 47401/03/19 4:59 PM 475MINIMALLY INVASIVE SURGERYCHAPTER 14Figure 14-30. The progress of general sur-gery can be reflected by a series of performance curves. General anesthesia and sterile technique allowed the development of maximally inva-sive open surgery over the last 125 years. Video optics allowed the development of minimally invasive surgery over the last 25 years. Nonin-vasive (seamless) surgery will result when a yet undiscovered transformational event allows sur-gery to occur without an incision, and perhaps without anesthesia.PerformanceGeneral anesthesiasterile techniqueVideo optics?1880190019201940196019801985199019952000??Open surgeryLaparoscopic surgerySeamless surgeryProgress in surgeryfor all surgeons. This technology has the advantage of enabling objective measurement of psychomotor skills,

1	surgeryLaparoscopic surgerySeamless surgeryProgress in surgeryfor all surgeons. This technology has the advantage of enabling objective measurement of psychomotor skills, which can be used to determine progress in skill acquisition and, ultimately, techni-cal competency.101 Several of these devices have been validated as a means of measuring proficiency in skill performance. More importantly, training on virtual reality platforms has proven to translate to improved operative performance in randomized tri-als.102,103 Currently, surgical skills labs are mandatory for Resi-dency Review Committee credentialing. Successful completion of the Fundamentals of Laparoscopic Surgery (FLS) technical and cognitive examination became a mandatory prerequisite for the American Board of Surgery (ABS) qualification examination in general surgery in 2010. The Fundamentals of Endoscopic Surgery (FES) became a prerequisite to ABS qualification in 2015. In the future, institutions may require simulator

1	examination in general surgery in 2010. The Fundamentals of Endoscopic Surgery (FES) became a prerequisite to ABS qualification in 2015. In the future, institutions may require simulator training to document specific entrustable professional activities (EPA) related to laparoscopic procedures before privileging in the OR. A Fundamentals of Robotic Surgery (FRS) high stakes exam is on the horizon for future surgical trainees. The American Col-lege of Surgeons has taken a leadership position in accrediting skills labs across the world as American College of Surgeons–accredited educational institutes.TelementoringIn response to the Institute of Medicine’s call for the develop-ment of unique technologic solutions to deliver health care to rural and underserved areas, surgeons are beginning to explore the feasibility of telementoring. Teleconsultation or telemen-toring is two-way audio and visual communication between two geographically separated providers. This communication can take

1	to explore the feasibility of telementoring. Teleconsultation or telemen-toring is two-way audio and visual communication between two geographically separated providers. This communication can take place in the office setting or directly in the OR when complex scenarios are encountered. Although local commu-nication channels may limit its performance in rural areas, the technology is available and currently is being used, espe-cially in states and provinces with large geographically remote populations.103Innovation and Introduction of New ProceduresThe revolution in minimally invasive general surgery, which occurred in 1990, created ethical challenges for the profession. The problem was this: If competence is gained from experience, how was the surgeon to climb the competence curve (otherwise known as the learning curve) without injuring patients? If it was indeed impossible to achieve competence without making mis-takes along the way, how should one effectively communicate this to

1	known as the learning curve) without injuring patients? If it was indeed impossible to achieve competence without making mis-takes along the way, how should one effectively communicate this to patients such that they understand the weight of their decisions? Even more fundamentally important is determining the path that should be followed before one recruits the first patient for a new procedure.Although procedure development is fundamentally dif-ferent than drug development (i.e., there is great individual variation in the performance of procedures, but no difference between one tablet and the next), adherence to a process simi-lar to that used to develop a new drug is a reasonable path for a surgical innovator. At the outset, the surgeon must iden-tify the problem that is not solved with current surgical pro-cedures. For example, although the removal of a gallbladder through a Kocher incision is certainly effective, it creates a great deal of disability, pain, and scarification. As

1	current surgical pro-cedures. For example, although the removal of a gallbladder through a Kocher incision is certainly effective, it creates a great deal of disability, pain, and scarification. As a result of those issues, many patients with very symptomatic biliary colic delayed operation until life-threatening complications occurred. Clearly, there was a need for developing a less inva-sive approach (Fig. 14-30).Once the opportunity has been established, the next step involves a search through other disciplines for technologies and techniques that might be applied. Again, this is analogous to the drug industry, where secondary drug indications have often turned out to be more therapeutically important than the primary indication for drug development. The third step is in vivo stud-ies in the most appropriate animal model. These types of studies are controversial because of the resistance to animal experimen-tation, and yet without such studies, many humans would be injured or

1	in the most appropriate animal model. These types of studies are controversial because of the resistance to animal experimen-tation, and yet without such studies, many humans would be injured or killed during the developmental phase of medical drugs, devices, and techniques. These steps often are called the preclinical phase of procedure development.The decision as to when such procedures are ready to come out of the lab is a difficult one. Put simply, the proce-dure should be reproducible, provide the desired effect, and not have serious side effects. Once these three criteria are reached, the time for human application has arrived. Before the surgeon discusses the new procedure with patients, it is important to achieve full institutional support. Involvement of the medi-cal board, the chief of the medical staff, and the institutional review board is essential before commencing on a new proce-dure. These bodies are responsible for the use of safe, high-quality medical practices

1	the chief of the medical staff, and the institutional review board is essential before commencing on a new proce-dure. These bodies are responsible for the use of safe, high-quality medical practices within their institution, and they will demand that great caution and all possible safeguards are in place before proceeding.The dialogue with the patient who is to be first must be thorough, brutally honest, and well documented. The psychology Brunicardi_Ch14_p0453-p0478.indd 47501/03/19 4:59 PM 476BASIC CONSIDERATIONSPART Ithat allows a patient to decide to be first is quite interesting, and may, under certain circumstances, require psychiatric evalua-tion. Certainly, if a dying cancer patient has a chance with a new drug, this makes sense. Similarly, if the standard surgical procedure has a high attendant morbidity and the new procedure offers a substantially better outcome, the decision to be first is understandable. On the other hand, when the benefits of the new approach are

1	has a high attendant morbidity and the new procedure offers a substantially better outcome, the decision to be first is understandable. On the other hand, when the benefits of the new approach are small and the risks are largely unknown, a more complete psychological profile may be necessary before proceeding.For new surgical procedures, it generally is wise to assemble the best possible operative team, including a surgeon experienced with the old technique, and assistants who have participated in the earlier animal work. This initial team of experienced physicians and nurses should remain together until full competence with the procedure is attained. This may take 10 procedures, or it may take 50 procedures. The team will know that it has achieved competence when the majority of procedures take the same length of time and the team is relaxed and sure of the flow of the operation. This will complete phase I of the procedure development.In phase II, the efficacy of the procedure is

1	take the same length of time and the team is relaxed and sure of the flow of the operation. This will complete phase I of the procedure development.In phase II, the efficacy of the procedure is tested in a nonrandomized fashion. Ideally, the outcome of new techniques must be as good as or better than the procedure that is being replaced. This phase should occur at several medical centers to prove that good outcomes are achievable outside of the pioneer-ing institution. These same requirements may be applied to the introduction of new technology into the OR. The value equation requires that the additional measurable procedure quality exceeds the additional measurable cost to the patient or healthcare system. In phase III, a randomized trial pits the new procedure against the old.Once the competence curve has been climbed, it is appro-priate for the team to engage in the education of others. Dur-ing the ascension of the competence curve, other learners in the institution (i.e., surgical

1	curve has been climbed, it is appro-priate for the team to engage in the education of others. Dur-ing the ascension of the competence curve, other learners in the institution (i.e., surgical residents) may not have the opportunity to participate in the first case series. Although this may be dif-ficult for them, the best interest of the patient must be put before the education of the resident.The second stage of learning occurs when the new pro-cedure has proven its value and a handful of experts exist, but the majority of surgeons have not been trained to perform the new procedure. In this setting, it is relatively unethical for sur-geons to forge ahead with a new procedure in humans as if they had spent the same amount of time in intensive study that the first team did. The fact that one or several surgical teams were able to perform an operation does not ensure that all others with the same medical degrees can perform the operation with equal skill. It behooves the learners to

1	one or several surgical teams were able to perform an operation does not ensure that all others with the same medical degrees can perform the operation with equal skill. It behooves the learners to contact the experts and request their assistance to ensure an optimal outcome at the new center. Although it is important that the learners contact the experts, it is equally important that the experts be willing to share their experience with their fellow professionals. As well, the experts should provide feedback to the learners as to whether they feel the learners are equipped to forge ahead on their own. If not, further observation and assistance from the experts are required. Although this approach may sound obvious, it is fraught with difficulties. In many situations, ego, competitiveness, and mon-etary concerns have short-circuited this process and led to poor patient outcomes. To a large extent, MIS has recovered from the black eye it received early in development, when inadequately

1	and mon-etary concerns have short-circuited this process and led to poor patient outcomes. To a large extent, MIS has recovered from the black eye it received early in development, when inadequately trained surgeons caused an excessive number of significant complications.If innovative procedures and technologies are to be devel-oped and applied without the mistakes of the past, surgeons must be honest when they answer these questions: Is this procedure safe? Would I consider undergoing this procedure if I developed a surgical indication? Is the procedure as good as or better than the procedure it is replacing? Do I have the skills to apply this procedure safely and with equivalent results to the more expe-rienced surgeon? Answering these questions in the affirmative should be a professional obligation. A negative response should motivate the surgeon to seek an alternative procedure or outside assistance before subjecting a patient to the new procedure.REFERENCESEntries highlighted in

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1	et al. Mini-mally invasive esophagectomy: outcomes in 222 patients. Ann Surg. 2003;238(4):486-494; discussion 494. 97. Fleshman J, Sargent DJ, Green E, for the Clinical Out-comes of Surgical Therapy Study Group. Laparoscopic col-ectomy for cancer is not inferior to open surgery based on 5-year data from the COST Study Group trial. Ann Surg. 2007;246(4):655-662; discussion 662. 98. Fried GM, Clas D, Meakins JL. Minimally invasive surgery in the elderly patient. Surg Clin North Am. 1994;74(2):375-387. 99. Borman PC, Terblanche J. Subtotal cholecystectomy: for the difficult gallbladder in portal hypertension and cholecystitis. Surgery. 1985;98(1):1-6. 100. Litwin DWM, Pham Q. Laparoscopic surgery in the compli-cated patient. In: Eubanks WS, Swanstrom LJ, Soper NJ, eds. Mastery of Endoscopic and Laparoscopic Surgery. Philadelphia: Lippincott, Williams & Wilkins; 2000:57. 101. Gallagher AG, Smith CD, Bowers SP, et al. Psychomotor skills assessment in practicing surgeons experienced in

1	and Laparoscopic Surgery. Philadelphia: Lippincott, Williams & Wilkins; 2000:57. 101. Gallagher AG, Smith CD, Bowers SP, et al. Psychomotor skills assessment in practicing surgeons experienced in per-forming advanced laparoscopic procedures. J Am Coll Surg. 2003;197(3):479-488. 102. Seymour NE, Gallagher AG, Roman SA, et al. Virtual real-ity training improves operating room performance: results of a randomized, double-blinded study. Ann Surg. 2002;236(4): 458-463; discussion 463. 103. Anvari M. Telesurgery: remote knowledge translation in clinical surgery. World J Surg. 2007;31(8):1545-1550.Brunicardi_Ch14_p0453-p0478.indd 47801/03/19 4:59 PM

1	Molecular Biology, The Atomic Theory of Disease, and Precision SurgeryXin-Hua Feng, Xia Lin, Xinran Li, Juehua Yu, John Nemunaitis, and F. Charles Brunicardi 15chapterOVERVIEW OF MOLECULAR CELL BIOLOGYThe beginning of modern medicine can be traced back to centu-ries ago when physicians and scientists began studying human anatomy from cadavers in morgues and animal physiology fol-lowing hunting expeditions. Gradually, from the study of ani-mals and plants in greater detail and the discovery of microbes, scientific principles governing life led to the emergence of the biologic sciences. As biologic science developed and expanded, scientists and physicians began to utilize its principles to solve challenges of human diseases while continuing to explore the fundamentals of life in greater detail. With ever-evolving state-of-the-art scientific tools, our understanding of how cells, tis-sues, organs, and entire organisms function, down to the level of molecular and subatomic structure,

1	detail. With ever-evolving state-of-the-art scientific tools, our understanding of how cells, tis-sues, organs, and entire organisms function, down to the level of molecular and subatomic structure, has resulted in modern biology with an enormous impact on modern healthcare and the discovery of amazing treatments for disease at an exponential pace. Significant progress has been made in molecular studies of organ development, cell signaling, and gene regulation. The advent of recombinant DNA technology, polymerase chain reac-tion (PCR) techniques, and next-generation genomic sequenc-ing, which resulted in the sequencing of the human genome, holds the potential to have a transformational influence on healthcare and society this century by not only broadening our understanding of the pathophysiology of disease, but also by bringing about necessary changes in personalized medicine.Today’s practicing surgeons are becoming increasingly aware that many modern surgical procedures rely on the

1	of disease, but also by bringing about necessary changes in personalized medicine.Today’s practicing surgeons are becoming increasingly aware that many modern surgical procedures rely on the infor-mation gained through molecular research (i.e., precision surgery). Genomic information, such as deleterious BRCA and RET proto-oncogene mutations, is being used to help direct prophylactic procedures to remove potentially harmful tissues before they do damage to patients. Molecular engineering has led to cancer-specific gene therapy that could serve in the near future as a more effective adjunct to surgical debulking of tumors than radiation or chemotherapy, so surgeons will benefit from a clear introduction to how basic biochemical and biologic principles relate to the developing area of molecular biology. This chapter reviews the current information on modern molecular biology for the surgical community.Basic Concepts of Molecular ResearchThe modern era of molecular biology, which has

1	biology. This chapter reviews the current information on modern molecular biology for the surgical community.Basic Concepts of Molecular ResearchThe modern era of molecular biology, which has been mainly concerned with how genes govern cell activity, began in 1953 when James D. Watson and Francis H. C. Crick made one of the greatest scientific discoveries by deducing the double-helical structure of deoxyribonucleic acid (DNA).1,2 The year 2003 marked the 50th anniversary of this great discovery. In the same year, the Human Genome Project completed with sequencing approximately 20,000 to 25,000 genes and 3 billion base pairs in human DNA.3 Before 1953, one of the most mysterious aspects of biology was how genetic material was precisely duplicated from one generation to the next. Although DNA had been implicated as genetic material, it was the base-paired structure of DNA that provided a logical inter-pretation of how a double helix could “unzip” to make copies of itself. This DNA

1	DNA had been implicated as genetic material, it was the base-paired structure of DNA that provided a logical inter-pretation of how a double helix could “unzip” to make copies of itself. This DNA synthesis, termed replication, immediately gave rise to the notion that a template was involved in the trans-fer of information between generations, and thus confirmed the suspicion that DNA carried an organism’s hereditary information.Within cells, DNA is packed tightly into chromosomes. One important feature of DNA as genetic material is its abil-ity to encode important information for all of a cell’s functions (Fig. 15-1). Based on the principles of base complementarity, scientists also discovered how information in DNA is accurately transferred into the protein structure. DNA serves as a template for RNA synthesis, termed transcription, including messenger RNA (mRNA, or the protein-encoding RNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). mRNA carries the informa-tion from DNA to make

1	for RNA synthesis, termed transcription, including messenger RNA (mRNA, or the protein-encoding RNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). mRNA carries the informa-tion from DNA to make proteins, termed translation, with the assistance of rRNA and tRNA. Each of these steps is precisely 12Overview of Molecular Cell Biology 479Basic Concepts of Molecular Research /479Molecular Approaches to Surgical Research /480Fundamentals of Molecular and Cell Biology 480DNA and Heredity /480Gene Regulation /481Human Genome /485Cell Cycle and Apoptosis /486Signal Transduction Pathways /487Gene Therapy and Molecular Drugs in Cancer /490Stem Cell Research /492The Atomic Theory of Disease /493Technologies of Molecular and Cell Biology 493DNA Cloning /493Detection of Nucleic Acids and Proteins /494Cell Manipulations /499Genetic Manipulations /500Precision Medicine and Surgery /505Targeted Genome Editing Using the CRISPR-Cas9 System /506Brunicardi_Ch15_p0479-p0510.indd 47918/02/19

1	/494Cell Manipulations /499Genetic Manipulations /500Precision Medicine and Surgery /505Targeted Genome Editing Using the CRISPR-Cas9 System /506Brunicardi_Ch15_p0479-p0510.indd 47918/02/19 11:12 AM 480Figure 15-1. The flow of genetic information from DNA to pro-tein to cell functions. The process of transmission of genetic infor-mation from DNA to RNA is called transcription, and the process of transmission from RNA to protein is called translation. Proteins are the essential controlling components for cell structure, cell sig-naling, and metabolism. Genomics and proteomics are the study of the genetic composition of a living organism at the DNA and pro-tein level, respectively. The study of the relationship between genes and their cellular functions is called functional genomics.Key Points1 The biologic sciences have developed drastically in the last 66 years after the uncovering of DNA structure by Watson and Crick.2 The completion of the human genome sequence in 2003

1	Points1 The biologic sciences have developed drastically in the last 66 years after the uncovering of DNA structure by Watson and Crick.2 The completion of the human genome sequence in 2003 rep-resents a great milestone in modern science.3 The technology emerging from molecular and cellular biol-ogy has revolutionized the understanding of disease and will radically transform the practice of surgery.4 The use of genetically modified mouse models and cell lines using gene therapy and RNA interference therapy has greatly contributed to the understanding of the molecular basis for human diseases and targeted therapies.5 The sequencing of each individual’s genome has the poten-tial to improve the predication, prevention, and targeted treatment of disease, resulting in precision medicine and surgery.6 Fast-developing targeted genome editing tools like the CRISPR-Cas9 system greatly facilitate biomedical research in native conditions and have shown their potential in the treatment of genetic

1	targeted genome editing tools like the CRISPR-Cas9 system greatly facilitate biomedical research in native conditions and have shown their potential in the treatment of genetic diseases, including cancers, with a high degree of personalization and precision.7 The use of functional genomics and modern molecular anal-yses will facilitate the discovery of actionable genes to guide choice of care, including precision surgery.GenomicsProteomicsFunctionalgenomicsDNA RNAProteinsTranscriptionTranslationStructureMetabolismSignalingCell functionscontrolled in such a way that genes are properly expressed in each cell at a specific time and location. In recent years, new classes of noncoding RNAs (ncRNA), for example, microRNA (or miRNA), piwi-interacting RNA (or piRNA), and long inter-genic noncoding RNA (or lincRNA), have been identified. Although the number of ncRNAs encoded in the human genome is unknown and a lot of ncRNAs have not been validated for their functions, ncRNAs have been

1	RNA (or lincRNA), have been identified. Although the number of ncRNAs encoded in the human genome is unknown and a lot of ncRNAs have not been validated for their functions, ncRNAs have been associated to regulate gene expression through posttranscriptional gene regulation such as mRNA degradation or epigenetic regulation such as chromatin structure modification and DNA methylation induction.4 Con-sequently, the differential gene activity in a cell determines its actions, properties, and functions.Molecular Approaches to Surgical ResearchRapid advances in molecular and cellular biology over the past half century have revolutionized the understanding of disease and will radically transform the practice of surgery. In the future, molecular techniques will be increasingly applied to surgical disease and will lead to new strategies for the selection and implementation of operative therapy. Surgeons should be familiar with the fundamental principles of molecular and cel-lular biology so

1	disease and will lead to new strategies for the selection and implementation of operative therapy. Surgeons should be familiar with the fundamental principles of molecular and cel-lular biology so that emerging scientific breakthroughs can be translated into improved care of the surgical patient.The greatest advances in the field of molecular biology have been in the areas of analysis and manipulation of DNA.1 Since Watson and Crick’s discovery of DNA structure, an inten-sive effort has been made to unlock the deepest biologic secrets of DNA. Among the avalanche of technical advances, one dis-covery in particular has drastically changed the world of molec-ular biology: the uncovering of the enzymatic and microbiologic techniques that produce recombinant DNA. Recombinant DNA technology involves the enzymatic manipulation of DNA and, subsequently, the cloning of DNA. DNA molecules are cloned for a variety of purposes including safeguarding DNA samples, facilitating sequencing,

1	involves the enzymatic manipulation of DNA and, subsequently, the cloning of DNA. DNA molecules are cloned for a variety of purposes including safeguarding DNA samples, facilitating sequencing, generating probes, and expressing recombinant proteins in one or more host organisms. DNA can be produced by a number of means, including restricted diges-tion of an existing vector, PCR, and cDNA synthesis. As DNA cloning techniques have developed over the last quarter cen-tury, researchers have moved from studying DNA to studying the functions of proteins, and from cell and animal models to molecular therapies in humans. Expression of recombinant pro-teins provides a method for analyzing gene regulation, structure, and function. In recent years, the uses for recombinant proteins have expanded to include a variety of new applications, includ-ing gene therapy and biopharmaceuticals. The basic molecular approaches for modern surgical research include DNA cloning, cell manipulation, disease

1	to include a variety of new applications, includ-ing gene therapy and biopharmaceuticals. The basic molecular approaches for modern surgical research include DNA cloning, cell manipulation, disease modeling in animals, and clinical tri-als in human patients.FUNDAMENTALS OF MOLECULAR AND CELL BIOLOGYDNA and HeredityDNA forms a right-handed, double-helical structure that is composed of two antiparallel strands of unbranched polymeric deoxyribonucleotides linked by phosphodiester bonds between the 5′ carbon of one deoxyribose moiety to the 3′ carbon of the next (Fig. 15-2). DNA is composed of four types of deoxyribo-nucleotides: adenine (A), cytosine (C), guanine (G), and thy-mine (T). The nucleotides are joined together by phosphodiester bonds. In the double-helical structure deduced by Watson and Crick, the two strands of DNA are complementary to each other. Brunicardi_Ch15_p0479-p0510.indd 48018/02/19 11:12 AM 481MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION

1	Watson and Crick, the two strands of DNA are complementary to each other. Brunicardi_Ch15_p0479-p0510.indd 48018/02/19 11:12 AM 481MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15Building blocks of DNAGTGGCCTAAA5'3'3'5'ATCGATGGCCTAGGCTTA3'5'5'3'5'3'GGGCTADNA strandCAGGCCTTTASugarphosphatePhosphateSugarBaseDouble-stranded DNADNA double helixSugar-phosphatebackboneHydrogen-bondedpairs+NucleotideFigure 15-2. Schematic representation of a DNA molecule form-ing a double helix. DNA is made of four types of nucleotides, which are linked covalently into a DNA strand. A DNA molecule is composed of two DNA strands held together by hydrogen bonds between the pair bases. The arrowheads at the ends of the DNA strands indicate the polarities of the two strands, which run anti-parallel to each other in the DNA molecule. The diagram at the bottom left of the figure shows the DNA molecule straightened out. In reality, the DNA molecule is twisted into a double helix,

1	run anti-parallel to each other in the DNA molecule. The diagram at the bottom left of the figure shows the DNA molecule straightened out. In reality, the DNA molecule is twisted into a double helix, of which each turn of DNA is made up of 10.4 nucleotide pairs, as shown on the right.Because of size, shape, and chemical composition, A always pairs with T, and C with G, through the formation of hydrogen bonds between complementary bases that stabilize the double helix.Recognition of the hereditary transmission of genetic information is attributed to the Austrian monk, Gregor Mendel. His seminal work, ignored upon publication until its rediscovery in 1900, established the laws of segregation and of independent assortment. These two principles established the existence of paired elementary units of heredity and defined the statistical laws that govern them.5 DNA was isolated in 1869, and a number of important observations of the inherited basis of certain dis-eases were made in the early

1	of heredity and defined the statistical laws that govern them.5 DNA was isolated in 1869, and a number of important observations of the inherited basis of certain dis-eases were made in the early part of the 20th century. Although today it appears easy to understand how DNA replicates, before the 1950s the idea of DNA as the primary genetic material was not appreciated. The modern era of molecular biology began in 1944 with the demonstration that DNA was the substance that carried genetic information. The first experimental evidence that DNA was genetic material came from simple transforma-tion experiments conducted in the 1940s using Streptococcus pneumoniae. One strain of the bacteria could be converted into another by incubating it with DNA from the other, just as the treatment of the DNA with deoxyribonuclease would inactivate the transforming activity of the DNA. Similarly, in the early 1950s, before the discovery of the double-helical structure of DNA, the entry of viral DNA and

1	with deoxyribonuclease would inactivate the transforming activity of the DNA. Similarly, in the early 1950s, before the discovery of the double-helical structure of DNA, the entry of viral DNA and not the protein into the host bacterium was believed to be necessary to initiate infection by the bacterial virus or bacteriophage. Key historical events con-cerning genetics are outlined in Table 15-1.For cells to pass on the genetic material (DNA) to each progeny, the amount of DNA must be doubled. Watson and Crick recognized that the complementary base-pair structure of DNA implied the existence of a template-like mechanism for the copying of genetic material.1 The transfer of DNA material from the mother cell to daughter cells takes place during somatic cell division (also called mitosis). Before a cell divides, DNA must be precisely duplicated. During replication, the two strands of DNA separate, and each strand creates a new complementary strand by precise base-pair matching (Fig.

1	Before a cell divides, DNA must be precisely duplicated. During replication, the two strands of DNA separate, and each strand creates a new complementary strand by precise base-pair matching (Fig. 15-3). The two, new, double-stranded DNAs carry the same genetic information, which can then be passed on to two daughter cells. Proofread-ing mechanisms ensure that the replication process occurs in a highly accurate manner. The fidelity of DNA replication is absolutely crucial to maintaining the integrity of the genome from generation to generation. However, mistakes can still occur during this process, resulting in mutations, which may lead to a change of the DNA’s encoded protein and, consequently, a change of the cell’s behavior. The reliable dependence of many features of modern organisms on subtle changes in genome is linked to Mendelian inheritance and also contributes to the pro-cesses of Darwinian evolution. In addition, massive changes, so-called genetic instability, can occur in

1	on subtle changes in genome is linked to Mendelian inheritance and also contributes to the pro-cesses of Darwinian evolution. In addition, massive changes, so-called genetic instability, can occur in the genome of somatic cells such as cancer cells.Gene RegulationLiving cells have the necessary machinery to enzymatically transcribe DNA into RNA and translate the mRNA into pro-tein. This machinery accomplishes the two major steps required for gene expression in all organisms: transcription and trans-lation (Fig. 15-4). However, gene regulation is far more com-plex, particularly in eukaryotic organisms. For example, many gene transcripts must be spliced to remove the intervening sequences. The sequences that are spliced off are called introns, which appear to be useless, but in fact may carry some regula-tory information. The sequences that are joined together, and are eventually translated into protein, are called exons. Additional regulation of gene expression includes modification of

1	some regula-tory information. The sequences that are joined together, and are eventually translated into protein, are called exons. Additional regulation of gene expression includes modification of mRNA, control of mRNA stability, and its nuclear export into cytoplasm (where it is assembled into ribosomes for translation). After mRNA is translated into protein, the levels and functions of the proteins can be further regulated posttranslationally. However, the following sections will mainly focus on gene regulation at transcriptional and translational levels.Transcription. Transcription is the enzymatic process of RNA synthesis from DNA.6 In bacteria, a single RNA polymerase carries out all RNA synthesis, including that of mRNA, rRNA, Brunicardi_Ch15_p0479-p0510.indd 48118/02/19 11:12 AM 482BASIC CONSIDERATIONSPART IFigure 15-3. DNA replication. As the nucleotide A only pairs with T, and G with C, each strand of DNA can determine the nucleotide sequence in its complementary strand.

1	482BASIC CONSIDERATIONSPART IFigure 15-3. DNA replication. As the nucleotide A only pairs with T, and G with C, each strand of DNA can determine the nucleotide sequence in its complementary strand. In this way, double-helical DNA can be copied precisely.Table 15-1Historical events in genetics and molecular biologyYEARINVESTIGATOREVENT1865MendelLaws of genetics established1869MiescherDNA isolated1905GarrodHuman inborn errors of metabolism1913SturtevantLinear map of genes1927MullerX-rays cause inheritable genetic damage1928GriffithTransformation discovered1941Beadle and Tatum“One gene, one enzyme” concept1944Avery, MacLeod, McCartyDNA as material of heredity1950McKlintockExistence of transposons confirmed1953Watson and CrickDouble-helical structure of DNA1957Benzer and KornbergRecombination and DNA polymerase1966Nirenberg, Khorana, HolleyGenetic code determined1970Temin and BaltimoreReverse transcriptase1972Cohen, Boyer, BergRecombinant DNA technology1975SouthernTransfer of DNA

1	and DNA polymerase1966Nirenberg, Khorana, HolleyGenetic code determined1970Temin and BaltimoreReverse transcriptase1972Cohen, Boyer, BergRecombinant DNA technology1975SouthernTransfer of DNA fragments from sizing gel to nitrocellulose (Southern blot)1977Sanger, Maxim, GilbertDNA sequencing methods1982—GenBank database established1985MullisPolymerase chain reaction1986—Automated DNA sequencing1989CollinsCystic fibrosis gene identified by positional cloning and linkage analysis1990—Human Genome Project initiated1997Roslin InstituteMammalian cloning (Dolly)2001IHGSC and Celera GenomicsDraft versions of human genome sequence published2003—Human Genome Project completedIHGSC = International Human Genome Sequencing Consortium.GTGGCCTAAACAGGCCTTTACAGGCCTTTAGTGGCCTAAACAGGCCTTTAGTGGCCTAAAS strandS strandNew S strandNew S strandTemplate S strandTemplate S strand5'3'3'5'5'5'5'5'3'3'3'Parent DNA double helix (shown flat):3'DNA is a template for its own duplicationBrunicardi_Ch15_p0479-p0510.indd

1	strandNew S strandNew S strandTemplate S strandTemplate S strand5'3'3'5'5'5'5'5'3'3'3'Parent DNA double helix (shown flat):3'DNA is a template for its own duplicationBrunicardi_Ch15_p0479-p0510.indd 48218/02/19 11:12 AM 483MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15NucleusCytoplasmDNARNAtranscriptmRNAmRNAProteinActiveproteinmRNAturnoverProteinturnoverTranscriptionRNAtransportTranscriptionalcontrolPosttranscriptionalcontrolTranslationalcontrolPosttranslationalcontrolNuclear envelopeRNAdegradationProteindegradationPosttranslationalmodificationTranslationRNAprocessingFigure 15-4. Four major steps in the control of eukaryotic gene expression. Transcriptional and posttranscriptional control determine the level of messenger RNA (mRNA) that is available to make a protein, while translational and posttranslational control determine the final outcome of functional proteins. Note that posttranscriptional and posttranslational controls consist of several

1	make a protein, while translational and posttranslational control determine the final outcome of functional proteins. Note that posttranscriptional and posttranslational controls consist of several steps.and tRNA. Transcription often is coupled with translation in such a way that an mRNA molecule is completely accessible to ribosomes, and bacterial protein synthesis begins on an mRNA molecule even while it is still being synthesized. Therefore, a discussion of gene regulation with a look at the simpler prokary-otic system precedes that of the more complex transcription and posttranscriptional regulation of eukaryotic genes.Transcription in Bacteria Initiation of transcription in pro-karyotes begins with the recognition of DNA sequences by RNA polymerase. First, the bacterial RNA polymerase cata-lyzes RNA synthesis through loose binding to any region in the double-stranded DNA and then through specific binding to the promoter region with the assistance of accessory pro-teins called σ

1	cata-lyzes RNA synthesis through loose binding to any region in the double-stranded DNA and then through specific binding to the promoter region with the assistance of accessory pro-teins called σ factors (sigma factors). A promoter region is the DNA region upstream of the transcription initiation site. RNA polymerase binds tightly at the promoter sites and causes the double-stranded DNA structure to unwind. Consequently, few nucleotides can be base-paired with the DNA template to begin transcription. Once transcription begins, the σ factor is released. The growing RNA chain may begin to peel off as the chain elongates. This occurs in such a way that there are always about 10 to 12 nucleotides of the growing RNA chains that are base-paired with the DNA template.The bacterial promoter contains a region of about 40 bases that include two conserved elements called –35 region and –10 region. The numbering system begins at the initiation site, which is designated +1 position, and counts

1	a region of about 40 bases that include two conserved elements called –35 region and –10 region. The numbering system begins at the initiation site, which is designated +1 position, and counts backward (in nega-tive numbers) on the promoter and forward on the transcribed region. Although both regions on different promoters are not the same sequences, they are fairly conserved and very similar. This conservation provides the accurate and rapid initiation of transcription for most bacterial genes. It is also common in bac-teria that one promoter serves to transcribe a series of clustered genes, called an operon. A single transcribed mRNA contains a series of coding regions, each of which is later independently translated. In this way, the protein products are synthesized in a coordinated manner. Most of the time, these proteins are involved in the same metabolic pathway, thus demonstrating that the control by one operon is an efficient system. After ini-tiation of transcription, the

1	manner. Most of the time, these proteins are involved in the same metabolic pathway, thus demonstrating that the control by one operon is an efficient system. After ini-tiation of transcription, the polymerase moves along the DNA to elongate the chain of RNA, although at a certain point, it will stop. Each step of RNA synthesis, including initiation, elongation, and termination, will require the integral functions of RNA polymerase as well as the interactions of the poly-merase with regulatory proteins.Transcription in Eukaryotes Transcription mechanisms in eukaryotes differ from those in prokaryotes. The unique features of eukaryotic transcription are as follows: (a) Three separate RNA polymerases are involved in eukaryotes: RNA polymerase I transcribes the precursor of 5.8S, 18S, and 28S rRNAs; RNA polymerase II synthesizes the precursors of mRNA as well as microRNA; and RNA polymerase III makes tRNAs and 5S rRNAs. (b) In eukaryotes, the initial transcript is often the pre-cursor to

1	rRNAs; RNA polymerase II synthesizes the precursors of mRNA as well as microRNA; and RNA polymerase III makes tRNAs and 5S rRNAs. (b) In eukaryotes, the initial transcript is often the pre-cursor to final mRNAs, tRNAs, and rRNAs. The precursor is then modified and/or processed into its final functional form. RNA splicing is one type of processing to remove the noncoding introns (the region between coding exons) on an mRNA. (c) In contrast to bacterial DNA, eukaryotic DNA often is packaged with histone and nonhistone proteins into chromatins. Transcrip-tion will only occur when the chromatin structure changes in such a way that DNA is accessible to the polymerase. (d) RNA is made in the nucleus and transported into cytoplasm, where translation occurs. Therefore, unlike bacteria, eukaryotes undergo uncoupled transcription and translation.Eukaryotic gene transcription also involves the recogni-tion and binding of RNA polymerase to the promoter DNA. However, the interaction between the

1	undergo uncoupled transcription and translation.Eukaryotic gene transcription also involves the recogni-tion and binding of RNA polymerase to the promoter DNA. However, the interaction between the polymerase and DNA is far more complex in eukaryotes than in prokaryotes. Because the majority of studies have been focused on the regulation and functions of proteins, this chapter primarily focuses on how protein-encoding mRNA is made by RNA polymerase II.Translation. DNA directs the synthesis of RNA; RNA in turn directs the synthesis of proteins. Proteins are variable-length polypeptide polymers composed of various combinations of 20 different amino acids and are the working molecules of the cell. The process of decoding information on mRNA to synthesize proteins is called translation (see Fig. 15-1). Translation takes place in ribosomes composed of rRNA and ribosomal proteins. The numerous discoveries made during the 1950s made it easy to understand how DNA replication and transcription

1	15-1). Translation takes place in ribosomes composed of rRNA and ribosomal proteins. The numerous discoveries made during the 1950s made it easy to understand how DNA replication and transcription involve base-pairing between DNA and DNA or DNA and RNA. How-ever, at that time, it was still impossible to comprehend how mRNA transfers the information to the protein-synthesizing machinery. The genetic information on mRNA is composed of Brunicardi_Ch15_p0479-p0510.indd 48318/02/19 11:12 AM 484BASIC CONSIDERATIONSPART ITable 15-2The genetic codeSECOND BASE IN CODON U C A G First Base in CodonUUUUPhe[F]UCUSer[S]UAUTyr[Y]UGUCys[C]UThird Base in

1	48318/02/19 11:12 AM 484BASIC CONSIDERATIONSPART ITable 15-2The genetic codeSECOND BASE IN CODON U C A G First Base in CodonUUUUPhe[F]UCUSer[S]UAUTyr[Y]UGUCys[C]UThird Base in Codon UUCPhe[F]UCCSer[S]UACTyr[Y]UGCCys[C]C UUALeu[L]UCASer[S]UAASTOP—UGASTOP—A UUGLeu[L]UCGSer[S]UAGSTOP—UGGTrp[W]G CCUULeu[L]CCUPro[P]CAUHis[H]CGUArg[R]U CUCLeu[L]CCCPro[P]CACHis[H]CGCArg[R]C CUALeu[L]CCAPro[P]CAAGln[Q]CGAArg[R]A CUGLeu[L]CCGPro[P]CAGGln[Q]CGGArg[R]G AAUUIle[I]ACUThr[T]AAUAsn[N]AGUSer[S]U AUCIle[I]ACCThr[T]AACAsn[N]AGCSer[S]C AUAIle[I]ACAThr[T]AAALys[K]AGAArg[R]A AUGMet[M]ACGThr[T]AAGLys[K]AGGArg[R]G GGUUVal[V]GCUAla[A]GAUAsp[D]GGUGly[G]U GUCVal[V]GCCAla[A]GACAsp[D]GGCGly[G]C GUAVal[V]GCAAla[A]GAAGlu[E]GGAGly[G]A GUGVal[V]GCGAla[A]GAGGlu[E]GGGGly[G]G A = adenine; C = cytosine; G = guanine; U = uracil; Ala = alanine; Arg = arginine; Asn = asparagine; Asp = aspartic acid; Cys = cysteine; Glu = glutamic acid; Gln = glutamine; Gly = glycine; His = histidine;

1	C = cytosine; G = guanine; U = uracil; Ala = alanine; Arg = arginine; Asn = asparagine; Asp = aspartic acid; Cys = cysteine; Glu = glutamic acid; Gln = glutamine; Gly = glycine; His = histidine; Ile = isoleucine; Leu = leucine; Lys = lysine; Met = methionine; Phe = phenylalanine; Pro = proline; Ser = serine; Thr = threonine; Trp = tryptophan; Tyr = tyrosine; Val = valine. Letter in [ ] indicates single letter code for amino acid.arranged sequences of four bases that are transferred to the lin-ear arrangement of 20 amino acids on a protein. Amino acids are characterized by a central carbon unit linked to four side chains: an amino group (–NH2), a carboxy group (–COOH), a hydrogen, and a variable (–R) group. The amino acid chain is assembled via peptide bonds between the amino group of one amino acid and the carboxy group of the next. Because of this decoding, the information carried on mRNA relies on tRNA. Translation involves all three RNAs. The precise transfer of information from

1	amino acid and the carboxy group of the next. Because of this decoding, the information carried on mRNA relies on tRNA. Translation involves all three RNAs. The precise transfer of information from mRNA to protein is governed by genetic code, the set of rules by which codons are translated into an amino acid (Table 15-2). A codon, a triplet of three bases, codes for one amino acid. In this case, random combinations of the four bases form 4 × 4 × 4, or 64 codes. Because 64 codes are more than enough for 20 amino acids, most amino acids are coded by more than one codon. The start codon is AUG, which also corresponds to methionine; therefore, almost all proteins begin with this amino acid. The sequence of nucleotide triplets that follows the start codon signal is termed the reading frame. The codons on mRNA are sequentially recognized by tRNA adaptor proteins. Specific enzymes termed aminoacyl-tRNA synthetases link a specific amino acid to a specific tRNA. The translation of mRNA to

1	The codons on mRNA are sequentially recognized by tRNA adaptor proteins. Specific enzymes termed aminoacyl-tRNA synthetases link a specific amino acid to a specific tRNA. The translation of mRNA to protein requires the ribosomal complex to move step-wise along the mRNA until the initiator methionine sequence is identified. In concert with various protein initiator factors, the methionyl-tRNA is positioned on the mRNA and protein synthesis begins. Each new amino acid is added sequentially by the appropriate tRNA in conjunction with proteins called elongation factors. Protein synthesis proceeds in the amino-to-carboxy-terminus direction.The biologic versatility of proteins is astounding. Among many other functions, proteins serve as enzymes that catalyze critical biochemical reactions, carry signals to and from the extracellular environment, and mediate diverse signaling and regulatory functions in the intracellular environment. They also transport ions and various small molecules

1	carry signals to and from the extracellular environment, and mediate diverse signaling and regulatory functions in the intracellular environment. They also transport ions and various small molecules across plasma mem-branes. Proteins make up the key structural components of cells and the extracellular matrix and are responsible for cell motility. The unique functional properties of proteins are largely deter-mined by their structure (Fig. 15-5).Regulation of Gene Expression. The human organism is made up of a myriad of different cell types that, despite their vastly different characteristics, contain the same genetic mate-rial. This cellular diversity is controlled by the genome and is accomplished by tight regulation of gene expression. This leads to the synthesis and accumulation of different complements of RNA and, ultimately, to the proteins found in different cell types. For example, muscle and bone express different genes or the same genes at different times. Moreover, the

1	different complements of RNA and, ultimately, to the proteins found in different cell types. For example, muscle and bone express different genes or the same genes at different times. Moreover, the choice of which genes are expressed in a given cell at a given time depends on signals received from its environment. There are multiple levels at which gene expression can be controlled along the pathway from DNA to RNA to protein (see Fig. 15-4). Transcriptional control refers to the mechanism for regulating when and how often a gene is transcribed. Splicing of the primary RNA tran-script (RNA processing control) and selection of completed Brunicardi_Ch15_p0479-p0510.indd 48418/02/19 11:12 AM 485MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15Figure 15-5. Maturation of a functional protein. Although the lin-ear amino acid sequence of a protein often is shown, the function of a protein also is controlled by its correctly folded three-dimensional

1	15-5. Maturation of a functional protein. Although the lin-ear amino acid sequence of a protein often is shown, the function of a protein also is controlled by its correctly folded three-dimensional structure. In addition, many proteins also have covalent posttransla-tional modifications such as phosphorylation or noncovalent bind-ing to a small molecule or a protein.Unfolded inactive proteinFolded inactive protein Mature inactive proteinBinding proteinPosttranslationalmodification(e.g., phosphorylation)PCofactor bindingmRNAs for nuclear export (RNA transport control) represent additional potential regulatory steps. The mRNAs in the cyto-plasm can be selectively translated by ribosomes (translational control) or selectively stabilized or degraded (mRNA degrada-tion control). Finally, the resulting proteins can undergo selec-tive activation, inactivation, or compartmentalization (protein activity control).Because a large number of genes are regulated at the tran-scriptional level,

1	the resulting proteins can undergo selec-tive activation, inactivation, or compartmentalization (protein activity control).Because a large number of genes are regulated at the tran-scriptional level, regulation of gene transcripts (i.e., mRNA) often is referred to as gene regulation in a narrow definition. Each of the steps during transcription is properly regulated in eukaryotic cells. Because genes are differentially regulated from one another, one gene can be differentially regulated in differ-ent cell types or at different developmental stages. Therefore, gene regulation at the level of transcription is largely context dependent. However, there is a common scheme that applies to transcription at the molecular level (Fig. 15-6). Each gene promoter possesses unique sequences called TATA boxes that can be recognized and bound by a large complex containing RNA polymerase II, forming the basal transcription machinery. Usually located upstream of the TATA box (but sometimes lon-ger

1	TATA boxes that can be recognized and bound by a large complex containing RNA polymerase II, forming the basal transcription machinery. Usually located upstream of the TATA box (but sometimes lon-ger distances) are a number of regulatory sequences referred to as enhancers that are recognized by regulatory proteins called transcription factors. These transcription factors specifically bind to the enhancers, often in response to environmental or developmental cues, and cooperate with each other and with basal transcription factors to initiate transcription. Regulatory sequences that negatively regulate the initiation of transcription also are present on the promoter DNA. The transcription factors that bind to these sites are called repressors, in contrast to the activators that activate transcription. The molecular interactions Figure 15-6. Transcriptional control by RNA polymerase. DNA is packaged into a chromatin structure. TATA = the common sequence on the promoter recognized by TBP

1	The molecular interactions Figure 15-6. Transcriptional control by RNA polymerase. DNA is packaged into a chromatin structure. TATA = the common sequence on the promoter recognized by TBP and polymerase II holoenzyme; TBP = TATA-binding protein and associated factors; TF = hypothetical transcription factor; TFBS = transcription factor binding site; ball-shaped structures = nucleosomes. Coactivator or corepressor is a factor linking the TF with the Pol II complex.TFCoactivator orCorepressorPol IIHoloenzymeTBPTBPTATATFBSbetween transcription factors and promoter DNA, as well as between the cooperative transcription factors, are highly regu-lated and context-dependent. Specifically, the recruitment of transcription factors to the promoter DNA occurs in response to physiologic signals. A number of structural motifs in these DNA-binding transcription factors facilitate this recognition and interaction. These include the helix-turn-helix, the homeodo-main motif, the zinc finger, the leucine

1	of structural motifs in these DNA-binding transcription factors facilitate this recognition and interaction. These include the helix-turn-helix, the homeodo-main motif, the zinc finger, the leucine zipper, and the helix-loop-helix motifs.Human GenomeGenome is a collective term for all genes present in one organ-ism. The human genome contains DNA sequences of 3 billion base pairs, carried by 23 pairs of chromosomes. The human genome has an estimated 25,000 to 30,000 genes, and overall it is 99.9% identical in all people.7,8 Approximately 3 million locations where single-base DNA differences exist have been identified and termed single nucleotide polymorphisms. Single nucleotide polymorphisms may be critical determinants of human variation in disease susceptibility and responses to envi-ronmental factors.The completion of the human genome sequence in 2003 represented another great milestone in modern science. The Human Genome Project created the field of genomics, which is the study of

1	factors.The completion of the human genome sequence in 2003 represented another great milestone in modern science. The Human Genome Project created the field of genomics, which is the study of genetic material in detail (see Fig. 15-1). The medical field is building on the knowledge, resources, and technologies emanating from the human genome to further the understanding of the relationship of the genes and their muta-tions to human health and disease. This expansion of genomics into human health applications resulted in the field of genomic medicine.The emergence of genomics as a science will transform the practice of medicine and surgery in this century. This break-through has allowed scientists the opportunity to gain remarkable insights into the lives of humans. Ultimately, the goal is to use this information to develop new ways to treat, cure, or even prevent the thousands of diseases that afflict humankind. In the 21st century, work will begin to incorporate the information

1	is to use this information to develop new ways to treat, cure, or even prevent the thousands of diseases that afflict humankind. In the 21st century, work will begin to incorporate the information embedded in the human genome sequence into surgical practices. By doing so, the genomic information can be used for diagnosing and predicting disease and disease suscep-tibility. Diagnostic tests can be designed to detect errant genes in patients suspected of having particular diseases or of being at risk for developing them. Furthermore, exploration into the function of each human gene is now possible, which will shed 3Brunicardi_Ch15_p0479-p0510.indd 48518/02/19 11:12 AM 486BASIC CONSIDERATIONSPART Ilight on how faulty genes play a role in disease causation. This knowledge also makes possible the development of a new gen-eration of therapeutics based on genes. Drug design is being revolutionized as researchers create new classes of medicines based on a reasoned approach to the use of

1	the development of a new gen-eration of therapeutics based on genes. Drug design is being revolutionized as researchers create new classes of medicines based on a reasoned approach to the use of information on gene sequence and protein structure function rather than the tradi-tional trial-and-error method. Drugs targeted to specific sites in the body promise to have fewer side effects than many of today’s medicines. Finally, other applications of genomics will involve the transfer of genes to replace defective versions or the use of gene therapy to enhance normal functions such as immunity.Proteomics refers to the study of the structure and expression of proteins as well as the interactions among pro-teins encoded by a human genome (see Fig. 15-1).9 A num-ber of Internet-based repositories for protein sequences exist, including Swiss-Prot (www.expasy.ch). These databases allow comparisons of newly identified proteins with previously char-acterized sequences to allow prediction of

1	for protein sequences exist, including Swiss-Prot (www.expasy.ch). These databases allow comparisons of newly identified proteins with previously char-acterized sequences to allow prediction of similarities, identifi-cation of splice variants, and prediction of membrane topology and posttranslational modifications. Tools for proteomic profil-ing include two-dimensional gel electrophoresis, time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization, and protein microarrays. Structural proteomics aims to describe the three-dimensional structure of proteins that is critical to understanding function. Functional genomics seeks to assign a biochemical, physiologic, cell biologic, and/or developmental function to each predicted gene. An ever-increasing arsenal of approaches, including transgenic animals, RNA interference (RNAi), and various systematic mutational strategies, will allow dissection of functions associated with newly discovered genes. Although the potential

1	including transgenic animals, RNA interference (RNAi), and various systematic mutational strategies, will allow dissection of functions associated with newly discovered genes. Although the potential of this field of study is vast, it is in its early stages.It is anticipated that a genomic and proteomic approach to human disease will lead to a new understanding of pathogenesis that will aid in the development of effective strategies for early diagnosis and treatment.10 For example, identification of altered protein expression in organs, cells, subcellular structures, or protein complexes may lead to development of new biomark-ers for disease detection. Moreover, improved understanding of how protein structure determines function will allow rational identification of therapeutic targets, and thereby not only accel-erate drug development, but also lead to new strategies to evalu-ate therapeutic efficacy and potential toxicity.9Cell Cycle and ApoptosisEvery organism is composed of many

1	and thereby not only accel-erate drug development, but also lead to new strategies to evalu-ate therapeutic efficacy and potential toxicity.9Cell Cycle and ApoptosisEvery organism is composed of many different cell types at dif-ferent developmental stages. Some cell types continue to grow, while some cells stop growing after a developmental stage or resume growth after a break. For example, embryonic stem cells grow continuously, while nerve cells and striated muscle cells stop dividing after maturation. Cell cycle is the process for every cell including DNA replication and protein synthe-sis, DNA segregation in half, and package DNA and protein in two newly formed cells to enable passage of identical genetic information from one parental cell to two daughter cells. Thus, the cell cycle is the fundamental mechanism to maintain tissue homeostasis. A cell cycle comprises four periods: G1 (first gap phase before DNA synthesis), S (synthesis phase when DNA replication occurs), G2 (the gap

1	the fundamental mechanism to maintain tissue homeostasis. A cell cycle comprises four periods: G1 (first gap phase before DNA synthesis), S (synthesis phase when DNA replication occurs), G2 (the gap phase before mitosis), and M (mitosis, the phase when two daughter cells with identical DNA are generated) (Fig. 15-7). After a full cycle, the daughter Figure 15-7. The cell cycle and its control system. M is the mito-sis phase, when the nucleus and the cytoplasm divide; S is the phase when DNA is duplicated; G1 is the gap between M and S; G2 is the gap between S and M. A complex of cyclin and cyclin-dependent kinase (CDK) controls specific events of each phase. Without cyclin, CDK is inactive. Different cyclin/CDK complexes are shown around the cell cycle. A, B, D, and E stand for cyclin A, cyclin B, cyclin D, and cyclin E, respectively.B/CDK1A/CDK1A/CDK2E/CDK2D/CDK4D/CDK6G1G2SMMitosisDNA replicationcells enter G1 again, and when they receive appropriate signals, undergo another cycle,

1	cyclin B, cyclin D, and cyclin E, respectively.B/CDK1A/CDK1A/CDK2E/CDK2D/CDK4D/CDK6G1G2SMMitosisDNA replicationcells enter G1 again, and when they receive appropriate signals, undergo another cycle, and so on. The machinery that drives cell cycle progression is made up of a group of enzymes called cyclin-dependent kinases (CDKs). Cyclin expression fluctuates during the cell cycle, and cyclins are essential for CDK activi-ties and form complexes with CDK. The cyclin A/CDK1 and cyclin B/CDK1 drive the progression for the M phase, while cyclin A/CDK2 is the primary S phase complex. Early G1 cyclin D/CDK4/6 or late G1 cyclin E/CDK2 controls the G1-S transi-tion. There also are negative regulators for CDK termed CDK inhibitors, which inhibit the assembly or activity of the cyclin-CDK complex. Expression of cyclins and CDK inhibitors often is regulated by developmental and environmental factors.The cell cycle is connected with signal transduction path-ways as well as gene expression.

1	Expression of cyclins and CDK inhibitors often is regulated by developmental and environmental factors.The cell cycle is connected with signal transduction path-ways as well as gene expression. Although the S and M phases rarely are subjected to changes imposed by extracellular sig-nals, the G1 and G2 phases are the primary periods when cells decide whether or not to move on to the next phase. During the G1 phase, cells receive greenor red-light signals, S phase entry or G1 arrest, respectively. Growing cells proliferate only when supplied with appropriate mitogenic growth factors. Cells become committed to entry of the cell cycle only toward the end of G1. Mitogenic signals stimulate the activity of early G1 CDKs (e.g., cyclin D/CDK4) that inhibit the activity of pRb protein and activate the transcription factor called E2F to induce the expression of batteries of genes essential for G1-S progression. Meanwhile, cells also receive antiproliferative signals such as those from tumor

1	the transcription factor called E2F to induce the expression of batteries of genes essential for G1-S progression. Meanwhile, cells also receive antiproliferative signals such as those from tumor suppressors. These antiproliferative signals also act in the G1 phase to stop cells’ progress into the S phase by inducing CKI production. For example, when DNA is dam-aged, cells will repair the damage before entering the S phase. Therefore, G1 contains one of the most important checkpoints for cell cycle progression. If the analogy is made that CDK is to a cell as an engine is to a car, then cyclins and CKI are the gas pedal and brake, respectively. Accelerated proliferation or Brunicardi_Ch15_p0479-p0510.indd 48618/02/19 11:12 AM 487MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15improper cell cycle progression with damaged DNA would be disastrous. Genetic gain-of-function mutations in oncogenes (that often promote expression or activity of the

1	AND PRECISION SURGERYCHAPTER 15improper cell cycle progression with damaged DNA would be disastrous. Genetic gain-of-function mutations in oncogenes (that often promote expression or activity of the cyclin/CDK complex) or loss-of-function mutations in tumor suppressor (that stimulate production of CKI) are causal factors for malig-nant transformation.In addition to cell cycle control, cells use genetically pro-grammed mechanisms to kill cells. This cellular process, called apoptosis or programmed cell death, is essential for the mainte-nance of tissue homeostasis (Fig. 15-8).Normal tissues undergo proper apoptosis to remove unwanted cells, those that have completed their jobs or have been damaged or improperly proliferated. Apoptosis can be activated by many physiologic stimuli such as death receptor signals (e.g., Fas or cytokine tumor necrosis factor), growth fac-tor deprivation, DNA damage, and stress signals. Two major pathways control the biochemical mechanisms governing

1	as death receptor signals (e.g., Fas or cytokine tumor necrosis factor), growth fac-tor deprivation, DNA damage, and stress signals. Two major pathways control the biochemical mechanisms governing apop-tosis: the death receptor and mitochondrial. However, recent advances in apoptosis research suggest an interconnection of the two pathways. What is central to the apoptotic machinery is the activation of a cascade of proteinases called caspases. Similar to CDK in the cell cycle, activities and expression of caspases are well controlled by positive and negative regulators. The complex machinery of apoptosis must be tightly controlled. Perturbations of this process can cause neoplastic transforma-tion or other diseases.Signal Transduction PathwaysGene expression in a genome is controlled in a temporal and spatial manner, at least in part by signaling pathways.11 A sig-naling pathway generally begins at the cell surface and, after a signaling relay by a cascade of intracellular effectors,

1	temporal and spatial manner, at least in part by signaling pathways.11 A sig-naling pathway generally begins at the cell surface and, after a signaling relay by a cascade of intracellular effectors, ends up in the nucleus (Fig. 15-9). All cells have the ability to sense changes in their external environment. The bioactive substances to which cells can respond are many and include proteins, short peptides, amino acids, nucleotides/nucleosides, steroids, reti-noids, fatty acids, and dissolved gases. Some of these substances are lipophilic and thereby can cross the plasma membrane by NucleusDeath signal(e.g., TNF or Fas)DeathreceptorPlasmamembraneActivation ofcaspase cascadeCytochrome creleaseDeathreceptorsignalingpathwayMitochondrionNormal target cellApoptotic target cellFigure 15-8. A simplified view of the apop-tosis pathways. Extracellular death receptor pathways include the activation of Fas and tumor necrosis factor (TNF) receptors and consequent activation of the caspase path-way.

1	view of the apop-tosis pathways. Extracellular death receptor pathways include the activation of Fas and tumor necrosis factor (TNF) receptors and consequent activation of the caspase path-way. Intracellular death pathway indicates the release of cytochrome c from mitochon-dria, which also triggers the activation of the caspase cascade. During apoptosis, cells undergo DNA fragmentation and nuclear and cell membrane breakdown and are eventually digested by other cells.Ligand(e.g., growth factor)Cell-surfacereceptorPlasmamembraneNucleusGeneexpressionLigand(e.g., hormone)Signaling cascadeIntracellularreceptorFigure 15-9. Cell-surface and intracellular receptor pathways. Extracellular signaling pathway: Most growth factors and other hydrophilic signaling molecules are unable to move across the plasma membrane and directly activate cell-surface receptors such as G-protein–coupled receptors and enzyme-linked receptors. The receptor serves as the receiver and in turn activates the downstream

1	plasma membrane and directly activate cell-surface receptors such as G-protein–coupled receptors and enzyme-linked receptors. The receptor serves as the receiver and in turn activates the downstream signals in the cell. Intracellular signaling pathway: Hormones or other diffusible molecules enter the cell and bind to the intracel-lular receptor in the cytoplasm or in the nucleus. Either extracel-lular or intracellular signals often reach the nucleus to control gene expression.Brunicardi_Ch15_p0479-p0510.indd 48718/02/19 11:12 AM 488BASIC CONSIDERATIONSPART Idiffusion to bind to a specific target protein within the cyto-plasm (intracellular receptor). Other substances bind directly with a transmembrane protein (cell-surface receptor). Binding of ligand to receptor initiates a series of biochemical reactions (signal transduction) typically involving protein-protein inter-actions and the transfer of high-energy phosphate groups, lead-ing to various cellular end responses.Control and

1	biochemical reactions (signal transduction) typically involving protein-protein inter-actions and the transfer of high-energy phosphate groups, lead-ing to various cellular end responses.Control and specificity through simple protein-protein interactions—referred to as adhesive interactions—is a com-mon feature of signal transduction pathways in cells.12 Signaling also involves catalytic activities of signaling molecules, such as protein kinases/phosphatases, that modify the structures of key signaling proteins. Upon binding and/or modification by upstream signaling molecules, downstream effectors undergo a conformational (allosteric) change and, consequently, a change in function. The signal that originates at the cell surface and is relayed by the cytoplasmic proteins often ultimately reaches the transcriptional apparatus in the nucleus. It alters the DNA binding and activities of transcription factors that directly turn genes on or off in response to the stimuli. Abnormal

1	reaches the transcriptional apparatus in the nucleus. It alters the DNA binding and activities of transcription factors that directly turn genes on or off in response to the stimuli. Abnormal alterations in signaling activities and capacities in otherwise normal cells can lead to diseases such as cancer.Advances in biology in the last two decades have dramati-cally expanded the view on how cells are wired with signal-ing pathways. In a given cell, many signaling pathways operate simultaneously and crosstalk with one another. A cell gener-ally may react to a hormonal signal in a variety of ways: (a) by changing its metabolite or protein, (b) by generating an electric current, or (c) by contracting. Cells continually are subject to multiple input signals that simultaneously and sequentially acti-vate multiple receptorand non–receptor-mediated signal trans-duction pathways, which form a signaling network. Although the regulators responsible for cell behavior are rapidly identified as a

1	acti-vate multiple receptorand non–receptor-mediated signal trans-duction pathways, which form a signaling network. Although the regulators responsible for cell behavior are rapidly identified as a result of genomic and proteomic techniques, the specific functions of the individual proteins, how they assemble, and the networks that control cellular behavior remain to be defined. An increased understanding of cell regulatory pathways—and how they are disrupted in disease—will likely reveal common themes based on protein interaction domains that direct associa-tions of proteins with other polypeptides, phospholipids, nucleic acids, and other regulatory molecules. Advances in the under-standing of signaling networks will require methods of inves-tigation that move beyond traditional “linear” approaches into medical informatics and computational biology. The bewilder-ing biocomplexity of such networks mandates multidisciplinary and transdisciplinary research collaboration. The vast amount

1	approaches into medical informatics and computational biology. The bewilder-ing biocomplexity of such networks mandates multidisciplinary and transdisciplinary research collaboration. The vast amount of information that is rapidly emerging from genomic and pro-teomic data mining will require the development of new model-ing methodologies within the emerging disciplines of medical mathematics and physics.Signaling pathways often are grouped according to the properties of signaling receptors. Many hydrophobic signaling molecules are able to diffuse across plasma membranes and directly reach specific cytoplasmic targets. Steroid hormones, thyroid hormones, retinoids, and vitamin D are examples that exert their activity upon binding to structurally related recep-tor proteins that are members of the nuclear hormone receptor superfamily. Ligand binding induces a conformational change that enhances transcriptional activity of these receptors. Most extracellular signaling molecules interact

1	of the nuclear hormone receptor superfamily. Ligand binding induces a conformational change that enhances transcriptional activity of these receptors. Most extracellular signaling molecules interact with transmembrane protein receptors that couple ligand binding to intracellular sig-nals, leading to biologic actions.There are three major classes of cell-surface receptors: transmitter-gated ion channels, seven-transmembrane G-protein–coupled receptors (GPCRs), and enzyme-linked receptors. The superfamily of GPCRs is one of the largest families of proteins, representing over 800 genes of the human genome. Members of this superfamily share a characteristic seven-transmembrane configuration. The ligands for these receptors are diverse and include hormones, chemokines, neurotransmitters, protein-ases, inflammatory mediators, and even sensory signals such as odorants and photons. Most GPCRs signal through het-erotrimeric G proteins, which are guanine-nucleotide regula-tory complexes. Thus,

1	inflammatory mediators, and even sensory signals such as odorants and photons. Most GPCRs signal through het-erotrimeric G proteins, which are guanine-nucleotide regula-tory complexes. Thus, the receptor serves as the receiver, the G protein serves as the transducer, and the enzyme serves as the effector arm. Enzyme-linked receptors possess an extracellular ligand-recognition domain and a cytosolic domain that either has intrinsic enzymatic activity or directly links with an enzyme. Structurally, these receptors usually have only one transmembrane-spanning domain. Of at least five forms of enzyme-linked recep-tors classified by the nature of the enzyme activity to which they are coupled, the growth factor receptors such as tyrosine kinase receptor or serine/threonine kinase receptors mediate diverse cellular events including cell growth, differentiation, metabolism, and survival/apoptosis. Dysregulation (particularly mutations) of these receptors is thought to underlie conditions of

1	diverse cellular events including cell growth, differentiation, metabolism, and survival/apoptosis. Dysregulation (particularly mutations) of these receptors is thought to underlie conditions of abnormal cellular proliferation in the context of cancer. The following sections will further review two examples of growth factor signaling pathways and their connection with human diseases.Insulin Pathway and Diabetes.13 The discovery of insulin in the early 1920s is one of the most dramatic events in the treatment of human disease. Insulin is a peptide hormone that is secreted by the β-cell of the pancreas. Insulin is required for the growth and metabolism of most mammalian cells, which contain cell-surface insulin receptors (InsR). Insulin binding to InsR activates the kinase activity of InsR. InsR then adds phosphoryl groups, a process referred to as phosphorylation, and subsequently activates its immediate intracellular effector, called insulin receptor substrate (IRS). IRS plays a

1	InsR. InsR then adds phosphoryl groups, a process referred to as phosphorylation, and subsequently activates its immediate intracellular effector, called insulin receptor substrate (IRS). IRS plays a central role in coordinating the signaling of insulin by activating distinct sig-naling pathways, the PI3K-Akt pathway and MAPK pathway, both of which possess multiple protein kinases that can control transcription, protein synthesis, and glycolysis (Fig. 15-10).The primary physiologic role of insulin is in glucose homeostasis, which is accomplished through the stimulation of glucose uptake into insulin-sensitive tissues such as fat and skeletal muscle. Defects in insulin synthesis/secretion and/or responsiveness are major causal factors in diabetes, one of the leading causes of death and disability in the United States, affecting an estimated 16 million Americans. Type 2 diabetes accounts for about 90% of all cases of diabetes. Clustering of type 2 diabetes in certain families and ethnic

1	in the United States, affecting an estimated 16 million Americans. Type 2 diabetes accounts for about 90% of all cases of diabetes. Clustering of type 2 diabetes in certain families and ethnic populations points to a strong genetic background for the disease. More than 90% of affected individuals have insulin resistance, which develops when the body is no longer able to respond correctly to insu-lin circulating in the blood. Although relatively little is known about the biochemical basis of this metabolic disorder, it is clear that the insulin-signaling pathways malfunction in this disease. It is also known that genetic mutations in the InsR or IRS cause type 2 diabetes, although which one is not certain. The majority of type 2 diabetes cases may result from defects in downstream-signaling components in the insulin-signaling pathway. Brunicardi_Ch15_p0479-p0510.indd 48818/02/19 11:12 AM 489MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER

1	components in the insulin-signaling pathway. Brunicardi_Ch15_p0479-p0510.indd 48818/02/19 11:12 AM 489MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15PlasmamembraneNucleusInsulinreceptor(InsR)GeneexpressionMAPKcascadeLipid & glucosemetabolismCellsurvivalIRSInsulinAdaptorPI3KFigure 15-10. Insulin-signaling pathway. Insulin is a peptide growth factor that binds to and activates the heterotetrameric recep-tor complex (InsR). InsR possesses protein tyrosine kinase activity and is able to phosphorylate the downstream insulin receptor sub-strate (IRS). Phosphorylated IRS serves as a scaffold and controls the activation of multiple downstream pathways for gene expres-sion, cell survival, and glucose metabolism. Inactivation of the insulin pathway can lead to type 2 diabetes.Type 2 diabetes also is associated with declining β-cell func-tion, resulting in reduced insulin secretion; these pathways are under intense study. A full understanding of the basis of

1	2 diabetes.Type 2 diabetes also is associated with declining β-cell func-tion, resulting in reduced insulin secretion; these pathways are under intense study. A full understanding of the basis of insulin resistance is crucial for the development of new therapies for type 2 diabetes. Furthermore, apart from type 2 diabetes, insulin resistance is a central feature of several other common human disorders, including atherosclerosis and coronary artery disease, hypertension, and obesity.Transforming Growth Factor-a (TGF-a) Pathway and Cancers.14 Growth factor signaling controls cell growth, differ-entiation, and apoptosis. Although insulin and many mitogenic growth factors promote cell proliferation, some growth factors and hormones inhibit cell proliferation. TGF-β is one of them. The balance between mitogens and TGF-β plays an important role in controlling the proper pace of cell cycle progression. The growth inhibition function of TGF-β signaling in epithelial cells plays a major role

1	between mitogens and TGF-β plays an important role in controlling the proper pace of cell cycle progression. The growth inhibition function of TGF-β signaling in epithelial cells plays a major role in maintaining tissue homeostasis.The TGF-β superfamily comprises a large number of struc-turally related growth and differentiation factors that act through a receptor complex at the cell surface (Fig. 15-11). The com-plex consists of transmembrane serine/threonine kinases. The receptor signals through activation of heterotrimeric complexes of intracellular effectors called SMADs (which are contracted from homologous Caenorhabditis elegans Sma and Drosophila Mad, two evolutionarily conserved genes for TGF-β signaling). Upon phosphorylation by the receptors, SMAD complexes translocate into the nucleus, where they bind to gene promoters and cooperate with specific transcription factors to regulate the expression of genes that control cell proliferation and differen-tiation. For example,

1	the nucleus, where they bind to gene promoters and cooperate with specific transcription factors to regulate the expression of genes that control cell proliferation and differen-tiation. For example, TGF-β strongly induces the transcription PlasmamembraneTGF b receptorGeneexpressionNucleusSMADTGF bAnti-proliferationFigure 15-11. TGF-β signaling pathway. The TGF-β family has at least 29 members encoded in the human genome. They are also peptide growth factors. Each member binds to a heterotetrameric complex consisting of a distinct set of type I and type II recep-tors. TGF-β receptors are protein serine/threonine kinases and can phosphorylate the downstream substrates called SMAD proteins. Phosphorylated SMADs are directly transported into the nucleus, where they bind to the DNA and regulate gene expression that is responsible for inhibition of cell proliferation. Inactivation of the TGF-β pathway through genetic mutations in the TGF-β receptors or SMADs is frequent in human cancer,

1	gene expression that is responsible for inhibition of cell proliferation. Inactivation of the TGF-β pathway through genetic mutations in the TGF-β receptors or SMADs is frequent in human cancer, leading to the uncontrolled proliferation of cancer cells.of a gene called p15INK4B (a type of CKI) and, at the same time, reduces the expression of many oncogenes such as c-Myc. The outcome of the altered gene expression leads to the inhibition of cell cycle progression. Meanwhile, the strength and dura-tion of TGF-β signaling is fine-tuned by a variety of positive or negative modulators, including protein phosphatases. Therefore, controlled activation of TGF-β signaling is an intrinsic mecha-nism for cells to ensure controlled proliferation.Resistance to TGF-β’s anticancer action is one hall-mark of human cancer cells. TGF-β receptors and SMADs are identified as tumor suppressors. The TGF-β signaling circuit can be disrupted in a variety of ways and in different types of human tumors. Some

1	of human cancer cells. TGF-β receptors and SMADs are identified as tumor suppressors. The TGF-β signaling circuit can be disrupted in a variety of ways and in different types of human tumors. Some lose TGF-β responsiveness through downregulation or mutations of their TGF-β receptors. The cytoplasmic SMAD4 protein, which transduces signals from ligand-activated TGF-β receptors to downstream targets, may be eliminated through mutation of its encoding gene. The locus encoding cell cycle inhibitor p15INK4B may be deleted. Alterna-tively, the immediate downstream target of its actions, cyclin-dependent kinase 4 (CDK4), may become unresponsive to the inhibitory actions of p15INK4B because of mutations that block p15INK4B binding. The resulting cyclin D/CDK4 complexes con-stitutively inactivate tumor suppressor pRb by hyperphosphory-lation. Finally, functional pRb, the end target of this pathway, may be lost through mutation of its gene. For example, in pan-creatic and colorectal cancers,

1	tumor suppressor pRb by hyperphosphory-lation. Finally, functional pRb, the end target of this pathway, may be lost through mutation of its gene. For example, in pan-creatic and colorectal cancers, 100% of cells derived from these cancers carry genetic defects in the TGF-β signaling pathway. Therefore, the antiproliferative pathway converging onto pRb Brunicardi_Ch15_p0479-p0510.indd 48918/02/19 11:12 AM 490BASIC CONSIDERATIONSPART Iand the cell division cycle is, in one way or another, disrupted in a majority of human cancer cells. Besides cancer, dysregu-lation of TGF-β signaling also has been associated with other human diseases such as Marfan’s syndrome and thoracic aortic aneurysm.Gene Therapy and Molecular Drugs in CancerModern advances in the use of molecular biology to manipulate genomes have greatly contributed to the understanding of the molecular basis for how cells live, die, or differentiate. Given the fact that human diseases arise from improper changes in the

1	genomes have greatly contributed to the understanding of the molecular basis for how cells live, die, or differentiate. Given the fact that human diseases arise from improper changes in the genome, the continuous understanding of how the genome func-tions will make it possible to tailor medicine on an individual basis. Although significant hurdles remain, the course toward therapeutic application of molecular biology already has been mapped out by many proof-of-principle studies in the literature. In this section, cancer is used as an example to elaborate some therapeutic applications of molecular biology. Modern molecu-lar medicine includes gene therapy and molecular drugs that target genes or gene products within human cells.Cancer is a complex disease, involving uncontrolled growth and spread of tumor cells (Fig. 15-12). Cancer development depends on the acquisition and selection of specific character-istics that set the tumor cell apart from normal somatic cells. Cancer cells have

1	spread of tumor cells (Fig. 15-12). Cancer development depends on the acquisition and selection of specific character-istics that set the tumor cell apart from normal somatic cells. Cancer cells have defects in regulatory circuits that govern nor-mal cell proliferation and homeostasis. Many lines of evidence indicate that tumorigenesis in humans is a multistep process and that these steps reflect genetic alterations that drive the progres-sive transformation of normal human cells into highly malignant derivatives. The genomes of tumor cells are invariably altered at multiple sites, having suffered disruption through lesions as sub-tle as point mutations and as obvious as changes in chromosome complement. A succession of genetic changes, each conferring one or another type of growth advantage, leads to the progressive conversion of normal human cells into cancer cells.Cancer research in the past 20 years has generated a rich and complex body of knowledge, revealing cancer to be a

1	leads to the progressive conversion of normal human cells into cancer cells.Cancer research in the past 20 years has generated a rich and complex body of knowledge, revealing cancer to be a dis-ease involving dynamic changes in the genome. The causes of cancer include genetic predisposition, environmental influences, infectious agents, and aging. These transform normal cells into cancerous ones by derailing a wide spectrum of regulatory pathways including signal transduction pathways, cell cycle machinery, or apoptotic pathways.15,16 The early notion that cancer was caused by mutations in genes critical for the control of cell growth implied that genome stability is important for preventing oncogenesis. There are two classes of cancer genes in which alteration has been identified in human and animal cancer cells: oncogenes, with dominant gain-of-function muta-tions, and tumor suppressor genes, with recessive loss-of-function mutations. In normal cells, oncogenes promote cell growth by

1	and animal cancer cells: oncogenes, with dominant gain-of-function muta-tions, and tumor suppressor genes, with recessive loss-of-function mutations. In normal cells, oncogenes promote cell growth by activating cell cycle progression, whereas tumor suppres-sors counteract oncogenes’ functions. Therefore, the balance between oncogenes and tumor suppressors maintains a well-controlled state of cell growth.During the development of most types of human cancer, cancer cells can break away from primary tumor masses, invade adjacent tissues, and hence travel to distant sites where they form new colonies. This spreading process of tumor cells, called metastasis, is the cause of 90% of human cancer deaths. Meta-static cancer cells that enter the bloodstream can reach virtu-ally all tissues of the body. Bones are one of the most common places for these cells to settle and start growing again. Bone Figure 15-12. Tumor clonal evolution and metastasis. A tumor develops from mutant cells with

1	the body. Bones are one of the most common places for these cells to settle and start growing again. Bone Figure 15-12. Tumor clonal evolution and metastasis. A tumor develops from mutant cells with multiple genetic mutations. Through repeated alterations in the genome, mutant epithelial cells are able to develop into a cluster of cells (called a tumor clone) that proliferates in an uncontrollable fashion. Further changes in the tumor cells can transform the tumor cells into a population of cells that can enter the blood vessels and repopulate in a new location.Tumor cells escape fromblood vessel and proliferateto form metastatic tumorsBlood vesselTumor cells break looseand enter bloodstreamUncontrolled cellproliferationCell proliferationCell proliferationCell with multiplemutationsCell with two mutationsMutant epithelial cellNormal epithelial cellmetastasis is one of the most frequent causes of pain in people with cancer. It also can cause bones to break and create other symptoms and

1	two mutationsMutant epithelial cellNormal epithelial cellmetastasis is one of the most frequent causes of pain in people with cancer. It also can cause bones to break and create other symptoms and problems for patients.The progression in the knowledge of cancer biology has been accelerating in recent years. All of the scientific knowledge Brunicardi_Ch15_p0479-p0510.indd 49018/02/19 11:12 AM 491MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15acquired through hard work and discovery has made it possible for cancer treatment and prevention. As a result of explosive new discoveries, some modern treatments were developed. The success of these therapies, together with traditional treatments such as surgical procedures, is further underscored by the fact that in 2002 the cancer rate was reduced in the United States. Current approaches to the treatment of cancer involve killing cancer cells with toxic chemicals, radiation, or surgery. Alterna-tively,

1	that in 2002 the cancer rate was reduced in the United States. Current approaches to the treatment of cancer involve killing cancer cells with toxic chemicals, radiation, or surgery. Alterna-tively, several new biologicand gene-based therapies are aimed at enhancing the body’s natural defenses against invading can-cers. Understanding the biology of cancer cells has led to the development of designer therapies for cancer prevention and treatment. Gene therapy, immune system modulation, geneti-cally engineered antibodies, and molecularly designed chemical drugs are all promising fronts in the war against cancer.Immunotherapy. The growth of the body is controlled by many natural signals through complex signaling pathways. Some of these natural agents have been used in cancer treatment and have been proven effective for fighting several cancers through the clinical trial process. These naturally occurring biologic agents, such as interferons, interleukins, and other cytokines, can now be

1	been proven effective for fighting several cancers through the clinical trial process. These naturally occurring biologic agents, such as interferons, interleukins, and other cytokines, can now be produced in the laboratory. These agents, as well as the synthetic agents that mimic the natural signals, are given to patients to influence the natural immune response agents either by directly altering the cancer cell growth or by acting indirectly to help healthy cells control the cancer. One of the most exciting applications of immunotherapy has come from the identification of certain tumor targets called antigens and the aiming of an antibody at these targets. This was first used as a means of local-izing tumors in the body for diagnosis and was more recently used to attack cancer cells. Trastuzumab (Herceptin) is an exam-ple of such a drug.17 Trastuzumab is a monoclonal antibody that neutralizes the mitogenic activity of cell-surface growth fac-tor receptor HER-2, which is

1	cells. Trastuzumab (Herceptin) is an exam-ple of such a drug.17 Trastuzumab is a monoclonal antibody that neutralizes the mitogenic activity of cell-surface growth fac-tor receptor HER-2, which is overexpressed in approximately 25% of breast cancers. HER-2–overexpressing tumors tend to grow faster and generally are more likely to recur than tumors that do not overproduce HER-2. Trastuzumab is designed to attack cancer cells that overexpress HER-2 by slowing or pre-venting the growth of these cells, resulting in increased survival of HER-2–positive breast cancer patients. Another significant example is the administration of interleukin-2 (IL-2) to patients with metastatic melanoma or kidney cancer, which has been shown to mediate the durable regression of metastatic cancer. IL-2, a cytokine produced by human helper T lymphocytes, has a wide range of immune regulatory effects, including the expansion of lymphocytes following activation by a specific antigen. Although IL-2 has no direct

1	produced by human helper T lymphocytes, has a wide range of immune regulatory effects, including the expansion of lymphocytes following activation by a specific antigen. Although IL-2 has no direct impact on cancer cells, the impact of IL-2 on cancers in vivo derives from its ability to expand lymphocytes with antitumor activity. The expanded lymphocyte pool enables recognition of the antigen on cancer cells. Thus, the molecular identification of cancer antigens has opened new possibilities for the development of effective immunotherapies for patients with cancer. Clinical studies using immunization with peptides derived from cancer antigens have shown that high levels of lymphocytes with antitumor activity can be produced in cancer-bearing patients. Highly avid antitu-mor lymphocytes can be isolated from immunized patients and grown in vitro for use in cell-transfer therapies.Chemotherapy. The primary function of anticancer chemicals is to block different steps involved in cell

1	can be isolated from immunized patients and grown in vitro for use in cell-transfer therapies.Chemotherapy. The primary function of anticancer chemicals is to block different steps involved in cell growth and replica-tion. These chemicals often block a critical chemical reaction in a signal transduction pathway or during DNA replication or gene expression. For example, STI571, also known as Gleevec, is one of the first molecularly targeted drugs based on the changes that cancer causes in cells.18 STI571 offers promise for the treatment of chronic myeloid leukemia (CML) and may soon surpass interferon-γ as the standard treatment for the disease. In CML, STI571 is targeted at the Bcr-Abl kinase, an activated oncogene product in CML (Fig. 15-13). Bcr-Abl is an overly activated protein kinase resulting from a specific genetic abnor-mality generated by chromosomal translocation that is found in the cells of patients with CML. STI571-mediated inhibi-tion of Bcr-Abl kinase activity not only

1	resulting from a specific genetic abnor-mality generated by chromosomal translocation that is found in the cells of patients with CML. STI571-mediated inhibi-tion of Bcr-Abl kinase activity not only prevents cell growth of Bcr-Abl–transformed leukemic cells, but also induces apoptosis. Clinically, the drug quickly corrects the blood cell abnormali-ties caused by the leukemia in a majority of patients, achiev-ing a complete disappearance of the leukemic blood cells and the return of normal blood cells. Additionally, the drug appears to have some effect on other cancers including certain brain tumors and gastrointestinal (GI) stromal tumors, a very rare type of stomach cancer.Gene Therapy. Gene therapy is an experimental treatment that involves genetically altering a patient’s own tumor cells or lym-phocytes (cells of the immune system, some of which can attack cancer cells). For years, the concept of gene therapy has held promise as a new, potentially potent weapon to attack cancer.

1	or lym-phocytes (cells of the immune system, some of which can attack cancer cells). For years, the concept of gene therapy has held promise as a new, potentially potent weapon to attack cancer. Although a rapid progression in the understanding of the molec-ular and clinical aspects of gene therapy has been witnessed in the past decade, gene therapy treatment has not yet been shown to be superior to standard treatments in humans.Inactive(In the absence of ATP)Overly activeUncontrolledcell proliferationBlockedcell proliferationBlocked activityATPSubstrateBcr-AblkinasePO4TyrSTI571SubstrateBcr-AblkinaseTyrSubstrateBcr-AblkinaseTyrFigure 15-13. Mechanism of STI571 as a molecular drug. Bcr-Abl is an overly activated oncogene product resulting from a specific genetic abnormality generated by chromo-somal translocation that is found in cells of patients with chronic myeloid leukemia. Bcr-Abl is an activated protein kinase and thus requires adenosine triphosphate (ATP) to phosphorylate

1	by chromo-somal translocation that is found in cells of patients with chronic myeloid leukemia. Bcr-Abl is an activated protein kinase and thus requires adenosine triphosphate (ATP) to phosphorylate substrates, which in turn promote cell proliferation. STI571 is a small molecule that competes with the ATP-bind-ing site and thus blocks the transfer of phos-phoryl group to substrate. PO4 = phosphate; Tyr = tyrosine.Brunicardi_Ch15_p0479-p0510.indd 49118/02/19 11:12 AM 492BASIC CONSIDERATIONSPART ISeveral problems must be resolved to transform it into a clinically relevant form of therapy. The major issues that limit its translation to the clinic are improving the selectivity of tumor targeting, improving the delivery to the tumor, and the enhance-ment of the transduction rate of the cells of interest. In most gene therapy trials for malignant diseases, tumors can be accessed and directly injected (in situ gene therapy). In situ gene therapy also offers a better distribution of the

1	of interest. In most gene therapy trials for malignant diseases, tumors can be accessed and directly injected (in situ gene therapy). In situ gene therapy also offers a better distribution of the vector virus throughout the tumor. Finally, a combination of gene therapy strategies will be more effective than the use of a single gene therapy system. An important aspect of effective gene therapy involves the choice of appropriate genes for manipulation. Genes that promote the production of messenger chemicals or other immune-active sub-stances can be transferred into the patient’s cells. These include genes that inhibit cell cycle progression, induce apoptosis, enhance host immunity against cancer cells, block the ability of cancer cells to metastasize, and cause tumor cells to undergo suicide. Recent development of RNAi technology, which uses a loss-of-function approach to block gene functions, ensures a new wave of hopes for gene therapy. Nonetheless, gene therapy is still experimental

1	Recent development of RNAi technology, which uses a loss-of-function approach to block gene functions, ensures a new wave of hopes for gene therapy. Nonetheless, gene therapy is still experimental and is being studied in clinical trials for many different types of cancer. The mapping of genes respon-sible for human cancer is likely to provide new targets for gene therapy in the future. The preliminary results of gene therapy for cancer are encouraging, and as advancements are made in the understanding of the molecular biology of human cancer, the future of this rapidly developing field holds great potential for treating cancer.It is noteworthy that the use of multiple therapeutic meth-ods has proven more powerful than a single method. The use of chemotherapy after surgery to destroy the few remaining cancerous cells in the body is called adjuvant therapy. Adju-vant therapy was first tested and found to be effective in breast cancer. It was later adopted for use in other cancers. A

1	few remaining cancerous cells in the body is called adjuvant therapy. Adju-vant therapy was first tested and found to be effective in breast cancer. It was later adopted for use in other cancers. A major discovery in chemotherapy is the advantage of multiple che-motherapeutic agents (known as combination or cocktail chemotherapy) over single agents. Some types of fast-growing leukemias and lymphomas (tumors involving the cells of the bone marrow and lymph nodes) responded extremely well to combination chemotherapy, and clinical trials led to gradual improvement of the drug combinations used. Many of these tumors can be cured today by combination chemotherapy. As cancer cells carry multiple genetic defects, the use of combina-tion chemotherapy, immunotherapy, and gene therapies may be more effective in treating cancers.Stem Cell ResearchStem cell biology represents a cutting-edge scientific research field with potential clinical applications.19 It may have an enor-mous impact on human

1	effective in treating cancers.Stem Cell ResearchStem cell biology represents a cutting-edge scientific research field with potential clinical applications.19 It may have an enor-mous impact on human health by offering hope for curing human diseases such as diabetes mellitus, Parkinson’s disease, neuro-logic degeneration, and congenital heart disease. Stem cells are endowed with two remarkable properties (Fig. 15-14). First, stem cells can proliferate in an undifferentiated but pluripotent state and, as a result, can self-renew. Second, they have the abil-ity to differentiate into many specialized cell types. There are two groups of stem cells: embryonic stem (ES) cells and adult stem cells.Human ES cells (hESCs) are derived from early preim-plantation embryos called blastocysts (5 days postfertilization) and are capable of generating all differentiated germ layers in the body by chimera assays or 2-D/3-D differentiation in a Self-renewalStem

1	embryos called blastocysts (5 days postfertilization) and are capable of generating all differentiated germ layers in the body by chimera assays or 2-D/3-D differentiation in a Self-renewalStem cellDifferentiationTerminallydifferentiatedcellFigure 15-14. Stem cells. A stem cell is capable of self-renewal (unlimited cell cycle) and differentiation (becoming nondividing cells with specialized functions). Differentiating stem cells often undergo additional cell divisions before they become fully mature cells that carry out specific tissue functions.dish—ectoderm, mesoderm, and endoderm—and therefore are considered pluripotent. There are two pluripotent states associ-ated with hESCs, one of which is the classic culture with bFGF (basic fibroblast growth factor) and knock out replacer (KSR), termed as “primed” pluripotent state. More recently, “naive” hESC culture methods have been introduced based on mouse studies, by supplementing 2i inhibitors (MEK1 and GSK3β inhibitors) into the medium

1	as “primed” pluripotent state. More recently, “naive” hESC culture methods have been introduced based on mouse studies, by supplementing 2i inhibitors (MEK1 and GSK3β inhibitors) into the medium in addition to bFGF.Adult stem cells are present in and can be isolated from adult tissues. They often are tissue specific and only can gen-erate the cell types comprising a particular tissue in the body; therefore, they are considered multipotent. However, in some cases, they can transdifferentiate into cell types found in other tissues, called transdifferentiation. For example, hematopoietic stem cells are adult stem cells. They reside in bone marrow and are capable of generating all cell types of the blood and immune system. Another example is mesenchymal stem cells (MSCs), which is initially identified in the bone marrow (BM) to sup-port hematopoietic stem cell homeostasis. In addition to BM, MSCs are also present in adipose tissue, umbilical cord, pla-centa, amniotic fluid, dental pulp,

1	identified in the bone marrow (BM) to sup-port hematopoietic stem cell homeostasis. In addition to BM, MSCs are also present in adipose tissue, umbilical cord, pla-centa, amniotic fluid, dental pulp, skeletal muscle, tendons, and synovial, etc., and are reported to obtain the ability to differen-tiate into osteogenic, chondrogenic, and adipogenic lineages in vitro. Due to their characteristics of easy acquisition (from adi-pose tissue, for example), strong ex vivo proliferation, immune-modulatory function, and ability to migrate to damaged tissue, MSCs have been utilized in regenerative medicine.Stem cells can be grown in culture and be induced to dif-ferentiate into a particular cell type, either in vitro or in vivo. With the recent and continually increasing improvement in culturing stem cells, scientists are beginning to understand the molecular mechanisms of stem cell self-renewal and differentia-tion in response to environmental cues. It is believed that dis-covery of the signals

1	cells, scientists are beginning to understand the molecular mechanisms of stem cell self-renewal and differentia-tion in response to environmental cues. It is believed that dis-covery of the signals that control self-renewal vs differentiation will be extremely important for the therapeutic use of stem cells in treating disease. It is possible that success in the study of the changes in signal transduction pathways in stem cells will lead to the development of therapies to replace diseased or damaged cells in the body using stem cell derivatives. Recently, stem cell research has been transformed by the discovery from the Shinya Yamanaka group and the James Thomsen group, who have found that a simple genetic manipulation can reprogram adult differentiated cells back into pluripotent stem cells.20,21 This exciting discovery not only bypasses the ethical issues of using early embryos to generate ES cells, but also ensures a Brunicardi_Ch15_p0479-p0510.indd 49218/02/19 11:12 AM

1	stem cells.20,21 This exciting discovery not only bypasses the ethical issues of using early embryos to generate ES cells, but also ensures a Brunicardi_Ch15_p0479-p0510.indd 49218/02/19 11:12 AM 493MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15potentially limitless source of patient-specific stem cells for tis-sue engineering and regenerative medicine.The Atomic Theory of Disease22As early as the 5th century b.c., the ancient Greek Democritus first proposed that matter is composed of indivisible particles called “atoms.” In the 17th and 18th centuries, Isaac Newton described the expansion of gases as rushed atoms into empty space. The existence of atoms was doubted until the discovery of subatomic particles in the 20th century, which demonstrated that the atom was actually divisible into protons, neutrons, and electrons.Over 100 years after this discovery, direct impacts from utilizing subatomic particles were revealed. This began with the

1	that the atom was actually divisible into protons, neutrons, and electrons.Over 100 years after this discovery, direct impacts from utilizing subatomic particles were revealed. This began with the discovery of the X-ray. The most advanced and well-applied atomic technologies include accurate imaging such as X-ray computed tomography (CT) scan, magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), and positron emission tomography (PET). Additional applica-tions include radiation oncology, which utilizes ionizing parti-cles to treat malignant diseases by inducing double-strand DNA breaks resulting in programed cell death.The staggering advances in anatomy, physiology, and molecular biology over the past centuries have led us to our current state in which the atom is now the anatomy of the 21st century. As 99% of the body is composed of six elements (oxygen, carbon, hydrogen, nitrogen, calcium, and phosphorus), the next great advance in medicine will be

1	atom is now the anatomy of the 21st century. As 99% of the body is composed of six elements (oxygen, carbon, hydrogen, nitrogen, calcium, and phosphorus), the next great advance in medicine will be bridging the sub-atomic, molecular, and genomic levels by forming an atomic theory of disease, which states that alterations in the composi-tion of subatomic particles are the root cause of disease. The atomic theory of disease would include genetic alterations at the atomic/subatomic level that are akin to single nucleotide polymorphisms (SNPs), in which alleles for a gene differ on the exact nucleotide in a single location, which can change the ulti-mate protein structure. This can lead to subtle changes in func-tion or dramatic results that cause pathology. We hypothesize that on a subatomic level, there could potentially be polymor-phisms as well, in which there are subtle changes in the sea of subatomic particles. Isotopes, discovered 100 years ago, would fall into this category of

1	level, there could potentially be polymor-phisms as well, in which there are subtle changes in the sea of subatomic particles. Isotopes, discovered 100 years ago, would fall into this category of subatomic polymorphism, as they dif-fer in the number of neutrons present in the atom. Differences in other particles may not change the mass of the atom, but may alter some of the characteristics of the atom. This is where the basic human variations originate because an atomic polymerism would result in particular genetic change. Remarkably, somatic point mutations of KRAS and P53 are caused by a single proton shift in cytosine, which is known as tautomerization of cyto-sine. In turn, the tautomerized cytosine binds with adenine, and not guanine, thus leading to point mutations in KRAS and P53, which are well known driver mutations for many cancers. This is another example of alterations in subatomic particles that directly cause disease.A known example of a change in the subatomic milieu of

1	are well known driver mutations for many cancers. This is another example of alterations in subatomic particles that directly cause disease.A known example of a change in the subatomic milieu of an element leading to a disease process is that of methemo-globinemia, a disorder characterized by an overabundance of methemoglobin. Methemoglobin contains an oxidized form of iron (carrying an extra electron), as opposed to the reduced form in normal hemoglobin. This results in a shift in the oxygen-hemoglobin dissociation curve to the left, causing hypoxia. Met-hemoglobinemia can be congenital, due to a defect in an enzyme that normally reduces methemoglobin back to hemoglobin, or acquired, caused by breakdown products of drugs that can oxi-dize hemoglobin. Although there is less than 1% of methemo-globin normally present in human tissues, affecting local blood flow and inflammation through its effects on nitric oxide and heme, large quantities can lead to respiratory failure and

1	1% of methemo-globin normally present in human tissues, affecting local blood flow and inflammation through its effects on nitric oxide and heme, large quantities can lead to respiratory failure and death.Another example would be exposure to external energy such as radiation that leads to instability of nuclear genome. In Chernobyl and Fukushima, radiolabeled food was metabolized and incorporated into body cells and decay to emit gamma radi-ation, causing DNA damage. This radiation damage occurs pri-marily at a subatomic level from a radiobiologic point of view with a direct or indirect ionization of atoms. The clinical results depend on the tissue characteristics and the equilibrium between the damage applied to normal and diseased tissues.TECHNOLOGIES OF MOLECULAR AND CELL BIOLOGYDNA CloningSince the advent of recombinant DNA technology three decades ago, hundreds of thousands of genes have been identi-fied. Recombinant DNA technology is the technology that uses advanced enzymatic

1	the advent of recombinant DNA technology three decades ago, hundreds of thousands of genes have been identi-fied. Recombinant DNA technology is the technology that uses advanced enzymatic and microbiologic techniques to manipu-late DNA.23 Pure pieces of any DNA can be inserted into bac-teriophage DNA or other carrier DNA such as plasmids to produce recombinant DNA in bacteria. In this way, DNA can be reconstructed, amplified, and used to manipulate the functions of individual cells or even organisms. This technology, often referred to as DNA cloning, is the basis of all other DNA analy-sis methods. It is only with the awesome power of recombinant DNA technology that the completion of the Human Genome Project was possible. It also has led to the identification of the entire gene complements of organisms such as viruses, bacteria, worms, flies, and plants.Molecular cloning refers to the process of cloning a DNA fragment of interest into a DNA vector that ultimately is deliv-ered into

1	of organisms such as viruses, bacteria, worms, flies, and plants.Molecular cloning refers to the process of cloning a DNA fragment of interest into a DNA vector that ultimately is deliv-ered into bacterial or mammalian cells or tissues24,25 (Fig. 15-15). This represents a very basic technique that is widely used in almost all areas of biomedical research. DNA vectors often are called plasmids, which are extrachromosomal molecules of DNA that vary in size and can replicate and be transmitted from bacterial cell to cell. Plasmids can be propagated either in the cytoplasm or after insertion, as part of the bacterial chromosome in Escherichia coli. The process of molecular cloning involves several steps of manipulation of DNA. First, the vector plasmid DNA is cleaved with a restriction enzyme to create compatible ends with the foreign DNA fragment to be cloned. The vec-tor and the DNA fragment are then joined in vitro by a DNA ligase. Alternatively, DNA cloning can be simply done through

1	to create compatible ends with the foreign DNA fragment to be cloned. The vec-tor and the DNA fragment are then joined in vitro by a DNA ligase. Alternatively, DNA cloning can be simply done through the so-called Gateway Technology that allows for the rapid and efficient transfer of DNA fragments between different cloning vectors while maintaining reading frame and orientation, with-out the use of restriction endonucleases and DNA ligase. The technology, which is based on the site-specific recombination system of bacteriophage l, is simple, fast, robust, and automat-able and thus compatible for high-throughput DNA cloning.Finally, the ligation product or the Gateway reaction prod-uct is introduced into competent host bacteria; this procedure is called transformation, which can be done by either calcium/heat shock or electroporation. Precautions must be taken in every step of cloning to generate the desired DNA construct. Brunicardi_Ch15_p0479-p0510.indd 49318/02/19 11:12 AM

1	by either calcium/heat shock or electroporation. Precautions must be taken in every step of cloning to generate the desired DNA construct. Brunicardi_Ch15_p0479-p0510.indd 49318/02/19 11:12 AM 494BASIC CONSIDERATIONSPART IFigure 15-15. Generation of recombinant DNA. The vector is a circular DNA molecule that is capable of replicating in Escherichia coli cells. Insert DNA (often your favorite gene) is ligated to the vector after ends of both DNA are properly treated with restriction enzymes. Ligated DNA (i.e., the recombinant plasmid DNA) is then transformed into E. coli cells, where it replicates to produce recombinant progenies. E. coli cells carrying the recombinant plasmid can be propagated to yield large quantities of plasmid DNA.InsertDNA of interestDigest withrestriction enzymeVectorLigationRecombinantplasmidIntroduceinto E. coliE. coli containingrecombinant plasmidPropagationE. coli containingrecombinant plasmidThe vector must be correctly prepared to maximize the creation

1	E. coliE. coli containingrecombinant plasmidPropagationE. coli containingrecombinant plasmidThe vector must be correctly prepared to maximize the creation of recombinants; for example, it must be enzymatically treated to prevent self-ligation. Host bacteria must be made sufficiently competent to permit the entry of recombinant plasmids into cells. The selection of desired recombinant plasmid-bearing E. coli normally is achieved by the property of drug resistance conferred by the plasmid vectors. The plasmids encoding mark-ers provide specific resistance to (i.e., the ability to grow in the presence of) antibiotics such as ampicillin, kanamycin, and tetracycline. The foreign component in the plasmid vector can be a mammalian expression cassette, which can direct expres-sion of foreign genes in mammalian cells. The resulting plasmid vector can be amplified in E. coli to prepare large quantities of DNA for its subsequent applications such as transfection, gene therapy, transgenics, and

1	in mammalian cells. The resulting plasmid vector can be amplified in E. coli to prepare large quantities of DNA for its subsequent applications such as transfection, gene therapy, transgenics, and knockout mice.Detection of Nucleic Acids and ProteinsSouthern Blot Hybridization. Southern blotting refers to the technique of transferring DNA fragments from an electropho-resis gel to a membrane support and the subsequent analysis of the fragments by hybridization with a radioactively or chemi-luminescently labeled probe (Fig. 15-16).26 Southern blotting is named after E. M. Southern, who in 1975 first described the technique of DNA analysis. It enables reliable and efficient analysis of size-fractionated DNA fragments in an immobilized membrane support. Southern blotting is composed of several steps. It normally begins with the digestion of the DNA samples with appropriate restriction enzymes, which will discriminate wild-type and mutant DNA by size and the separation of DNA samples in an

1	steps. It normally begins with the digestion of the DNA samples with appropriate restriction enzymes, which will discriminate wild-type and mutant DNA by size and the separation of DNA samples in an agarose gel by electrophoresis with appropriate DNA size markers, called the DNA ladder. The DNA gel is stained with a dye, usually ethidium bromide, and photographed with a ruler laid alongside the gel so that band positions can later be identified on the membrane. The DNA gel then is treated so the DNA fragments are denatured (i.e., strand separation). The DNA then is transferred onto a nitrocellulose membrane by capillary diffusion or under electricity. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a radioactively or chemilumi-nescently labeled probe to be identified.The development of Southern transfer and the associated hybridization techniques made it possible for the first time to obtain information about the

1	chemilumi-nescently labeled probe to be identified.The development of Southern transfer and the associated hybridization techniques made it possible for the first time to obtain information about the physical organization of single and multicopy sequences in complex genomes. The later applica-tion of Southern blotting hybridization to the study of restriction fragment length polymorphisms opened up new possibilities DNA is digested withrestriction enzymes.DNA fragments are denaturedand separated by gelelectrophoresis.DNA fragments are transferredto a membrane filter.The filter is hybridized with a radioactive DNA probe.DNA fragment that is hybridized to the radioactive DNA is detected by autoradiography.Radioactive probeFigure 15-16. Southern blotting. Restriction enzymatic fragments of DNA are separated by agarose gel electrophoresis, transferred to a membrane filter, and then hybridized to a radioactive probe.such as genetic fingerprinting and prenatal diagnosis of genetic

1	of DNA are separated by agarose gel electrophoresis, transferred to a membrane filter, and then hybridized to a radioactive probe.such as genetic fingerprinting and prenatal diagnosis of genetic diseases.Northern Blot Hybridization. Northern blotting refers to the technique of size fractionation of RNA in a gel and the transfer-ring of an RNA sample to a solid support (membrane) in such a manner that the relative positions of the RNA molecules are maintained. The resulting membrane then is hybridized with a labeled probe complementary to the mRNA of interest. Signals generated from detection of the membrane can be used to deter-mine the size and abundance of the target RNA. In principle, Brunicardi_Ch15_p0479-p0510.indd 49418/02/19 11:12 AM 495MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15Northern blot hybridization is similar to Southern blot hybrid-ization (and hence its name), with the exception that RNA, not DNA, is on the membrane. Although

1	DISEASE, AND PRECISION SURGERYCHAPTER 15Northern blot hybridization is similar to Southern blot hybrid-ization (and hence its name), with the exception that RNA, not DNA, is on the membrane. Although reverse-transcriptase PCR has been used in many applications (described in the next sec-tion, “Polymerase Chain Reaction”), Northern analysis is the only method that provides information regarding mRNA size and has remained a standard method for detection and quantita-tion of mRNA. The process of Northern hybridization involves several steps, as does Southern hybridization, including elec-trophoresis of RNA samples in an agarose-formaldehyde gel, transfer to a membrane support, and hybridization to a radio-actively labeled DNA probe. Data from hybridization allow Etc.Double-strandedDNAHeat toseparatestrandsHybridization of primers+DNA polymerase+dATP+dGTP+dCTP+dTTP5'5'DNAfromsynthesisprimersStep 1Step 2Step 3First cycleSeparate DNA strandsand add primerDNAsynthesisRegion ofdouble-stranded

1	of primers+DNA polymerase+dATP+dGTP+dCTP+dTTP5'5'DNAfromsynthesisprimersStep 1Step 2Step 3First cycleSeparate DNA strandsand add primerDNAsynthesisRegion ofdouble-stranded DNA to beamplifiedDNA oligonucleotideprimersSeparate DNA strandsand annual primerDNAsynthesisSeparate DNA strandsand annual primerDNAsynthesisFirst cycleProducing two double-strandedDNA moleculesSecond cycleProducing four double-strandedDNA moleculesThird cycleProducing eight double-strandedDNA moleculesABFigure 15-17. Amplification of DNA using the polymerase chain reaction (PCR) technique. Knowledge of the DNA sequence to be ampli-fied is used to design two synthetic DNA oligonucleotides, each complementary to the sequence on one strand of the DNA double helix at opposite ends of the region to be amplified. These oligonucleotides serve as primers for in vitro DNA synthesis, which is performed by a DNA polymerase, and they determine the segment of the DNA that is amplified. A. PCR starts with a double-stranded DNA,

1	serve as primers for in vitro DNA synthesis, which is performed by a DNA polymerase, and they determine the segment of the DNA that is amplified. A. PCR starts with a double-stranded DNA, and each cycle of the reaction begins with a brief heat treatment to separate the two strands (Step 1). After strand separation, cooling of the DNA in the presence of a large excess of the two primer DNA oligonucleotides allows these primers to hybridize to complementary sequences in the two DNA strands (Step 2). This mixture is then incubated with DNA polymerase and the four deoxyribonucleoside triphosphates so that DNA is synthesized, starting from the two primers (Step 3). The entire cycle is then begun again by a heat treatment to separate the newly synthesized DNA strands. B. As the procedure is performed over and over again, the newly synthesized fragments serve as templates in their turn, and, within a few cycles, the predominant DNA is identical to the sequence bracketed by and including the

1	over and over again, the newly synthesized fragments serve as templates in their turn, and, within a few cycles, the predominant DNA is identical to the sequence bracketed by and including the two primers in the original template. Of the DNA put into the original reaction, only the sequence bracketed by the two primers is amplified because there are no primers attached anywhere else. In the example illustrated in B, three cycles of reaction produce 16 DNA chains, eight of which (boxed in brown) are the same length as and correspond exactly to one or the other strand of the original bracketed sequence shown at the far left; the other strands contain extra DNA downstream of the original sequence, which is replicated in the first few cycles. After three more cycles, 240 of the 256 DNA chains cor-respond exactly to the original bracketed sequence, and after several more cycles, essentially all of the DNA strands have this unique length. quantification of steady-state mRNA levels and, at

1	cor-respond exactly to the original bracketed sequence, and after several more cycles, essentially all of the DNA strands have this unique length. quantification of steady-state mRNA levels and, at the same time, provide information related to the presence, size, and integrity of discrete mRNA species. Thus, Northern blot analy-sis, also termed RNA gel blot analysis, commonly is used in molecular biology studies relating to gene expression.Polymerase Chain Reaction. PCR is an in vitro method for the polymerase-directed amplification of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the tar-get DNA (Fig. 15-17).27 One cycle of PCR reaction involves template denaturation, primer annealing, and the extension of Brunicardi_Ch15_p0479-p0510.indd 49518/02/19 11:12 AM 496BASIC CONSIDERATIONSPART Ithe annealed primers by DNA polymerase. Because the primer extension products synthesized in one cycle can serve

1	Brunicardi_Ch15_p0479-p0510.indd 49518/02/19 11:12 AM 496BASIC CONSIDERATIONSPART Ithe annealed primers by DNA polymerase. Because the primer extension products synthesized in one cycle can serve as a template in the next, the number of target DNA copies nearly doubles at each cycle. Thus, a repeated series of cycles result in the exponential accumulation of a specific fragment in which the termini are sharply defined by the 5′ ends of the primers. The introduction of the thermostable DNA polymerase (e.g., Taq polymerase) transforms the PCR into a simple and robust reaction. The reaction components (e.g., template, primers, Taq polymerase, 2′-deoxynucleoside 5′-triphosphates, and buffer) could all be assembled and the amplification reaction carried out by simply cycling the temperatures within the reaction tube. The specificity and yield in amplifying a particular DNA frag-ment by PCR reaction are affected by the proper setting of the reaction parameters (e.g., enzyme, primer, and

1	within the reaction tube. The specificity and yield in amplifying a particular DNA frag-ment by PCR reaction are affected by the proper setting of the reaction parameters (e.g., enzyme, primer, and Mg2+ concen-tration, as well as the temperature cycling profile). Modifying various PCR parameters to optimize the specificity of amplifi-cation yields more homogenous products, even in rare template reactions.The emergence of the PCR technique has dramatically altered the approach to both fundamental and applied bio-logic problems. The capability of amplifying a specific DNA fragment from a gene or the whole genome greatly advances the study of the gene and its function. It is simple, yet robust, speedy, and most of all, flexible. As a recombinant DNA tool, it underlies almost all of molecular biology. This revolution-ary technique enabled the modern methods for the isolation of genes, construction of a DNA vector, introduction of alterations into DNA, and quantitation of gene expression,

1	biology. This revolution-ary technique enabled the modern methods for the isolation of genes, construction of a DNA vector, introduction of alterations into DNA, and quantitation of gene expression, making it a fun-damental cornerstone of genetic and molecular analysis.Immunoblotting and Immunoprecipitation. Analyses of proteins are primarily carried out by antibody-directed immu-nologic techniques. For example, Western blotting, also called immunoblotting, is performed to detect protein levels in a popu-lation of cells or tissues, whereas immunoprecipitation is used to concentrate proteins from a larger pool. Using specific antibod-ies, microscopic analysis called immunofluorescence and immu-nohistochemistry is possible for the subcellular localization and expression of proteins in cells or tissues, respectively.Immunoblotting refers to the process of identifying a pro-tein from a mixture of proteins (Fig. 15-18). It consists of five steps: (a) sample preparation; (b) electrophoresis

1	or tissues, respectively.Immunoblotting refers to the process of identifying a pro-tein from a mixture of proteins (Fig. 15-18). It consists of five steps: (a) sample preparation; (b) electrophoresis (separation of a protein mixture by sodium dodecyl sulfate-polyacrylamide gel electrophoresis); (c) transfer (the electrophoretic transfer of proteins from gel onto membrane support [e.g., nitrocel-lulose, nylon, or polyvinylidene difluoride]); (d) staining (the subsequent immunodetection of target proteins with specific antibody); and (e) development (colorimetric, chemilumines-cent, and recently fluorescent visualization of the antibody-recognized protein). Thus, immunoblotting combines the resolution of gel electrophoresis with the specificity of immu-nochemical detection. Immunoblotting is a powerful tool used to determine a number of important characteristics of proteins. For example, immunoblotting analysis will determine the pres-ence and the quantity of a protein in a given

1	is a powerful tool used to determine a number of important characteristics of proteins. For example, immunoblotting analysis will determine the pres-ence and the quantity of a protein in a given cellular condition and its relative molecular weight. Immunoblotting also can be used to determine whether posttranslational modification such as phosphorylation has occurred on a protein. Importantly, through immunoblotting analysis, a comparison of the protein levels and modification states in normal vs diseased tissues is possible.Cell tissue lysates• Sample preparation• Gel electrophoresis Separation of proteins• Western transfer Transfer of proteins to membrane• Immunostaining Block membrane 1°/2° antibody staining• Development Colorimetric/chemiluminescence detection123456123456Figure 15-18. Immunoblotting. Proteins are prepared from cells or tissues, separated according to size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a membrane filter.

1	15-18. Immunoblotting. Proteins are prepared from cells or tissues, separated according to size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a membrane filter. Detection of a protein of interest can be done by sequential incubation with a primary antibody directed against the protein, and then with an enzyme-conjugated secondary antibody that rec-ognizes the primary antibody. Visualization of the protein is carried out by using colorimetric or luminescent substrates for the conju-gated enzyme.Immunoprecipitation, another widely used immunochemi-cal technique, is a method that uses antibody to enrich a pro-tein of interest and any other proteins that are associated with it (Fig. 15-19). The principle of the technique lies in the property of a strong and specific affinity between antibodies and their anti-gens to locate and pull down target proteins in solution. Once the antibody-antigen (target protein) complexes are formed in the solution, they are

1	specific affinity between antibodies and their anti-gens to locate and pull down target proteins in solution. Once the antibody-antigen (target protein) complexes are formed in the solution, they are collected and purified using small agarose beads with covalently attached protein A or protein G. Both protein A and protein G specifically interact with the antibodies, Brunicardi_Ch15_p0479-p0510.indd 49618/02/19 11:12 AM 497MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15Enriched YFP & YBPsWashSDS-PAGEAnti-YFP beadsAnti-YFP conjugated to beadsYour favorite protein (YFP)YFP-binding proteins (YBPs)Junk proteinsFigure 15-19. Immunoprecipitation. Proteins prepared from cells or tissues can be enriched using an antibody directed against them. The anti-body is first conjugated to agarose beads and then incubated with protein mixture. Due to the spe-cific high-affinity interaction between antibody and its antigen (the protein), the antigen-antibody complex

1	first conjugated to agarose beads and then incubated with protein mixture. Due to the spe-cific high-affinity interaction between antibody and its antigen (the protein), the antigen-antibody complex can be collected on beads by centrifuga-tion. The immunoprecipitated protein can then be analyzed by immunoblotting. Alternatively, if pro-teins are radiolabeled in cells or tissues, detection of immunoprecipitated proteins can be achieved by simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography.thus forming a large immobilized complex of antibody-antigen bound to beads. The purified protein can then be analyzed by a number of biochemical methods. When immunoprecipitation is combined with immunoblotting, it can be used for the sensitive detection of proteins in low concentrations, which would other-wise be difficult to detect. Moreover, combined immunoprecipi-tation and immunoblotting analysis is very efficient in analyzing the protein-protein

1	proteins in low concentrations, which would other-wise be difficult to detect. Moreover, combined immunoprecipi-tation and immunoblotting analysis is very efficient in analyzing the protein-protein interactions or determining the posttransla-tional modifications of proteins. In addition, immunoprecipi-tated proteins can be used as preparative steps for assays such as intrinsic or associated enzymatic activities. The success of immunoprecipitation is influenced by two major factors: (a) the abundance of the protein in the original preparation and (b) the specificity and affinity of the antibody for this protein.Recently, immunoprecipitation is even used to enrich modi-fied DNA (for example, 5-methylcytosine) for bisulfite sequenc-ing. Besides proteins of interest, specific antibodies can also be raised against specially modified DNA. Like the protein immuno-precipitation, modified DNA can be pulled down, taking advan-tage of the specificity and affinity of antibody to antigen.DNA

1	can also be raised against specially modified DNA. Like the protein immuno-precipitation, modified DNA can be pulled down, taking advan-tage of the specificity and affinity of antibody to antigen.DNA Microarray. Now that the human genome sequence is completed, the primary focus of biologists is rapidly shifting toward gaining an understanding of how genes function. One of the interesting findings about the human genome is that there are only approximately 25,000 to 30,000 protein-encoding genes. However, it is known that genes and their products function in a complicated and yet orchestrated fashion and that the surprisingly small number of genes from the genome sequence is sufficient to make a human being. Nonetheless, with the tens of thousands of genes present in the genome, traditional methods in molecular biology, which generally work on a one-gene-in-one-experiment basis, cannot generate the whole picture of genome function. In the past several years, a new technology called DNA

1	methods in molecular biology, which generally work on a one-gene-in-one-experiment basis, cannot generate the whole picture of genome function. In the past several years, a new technology called DNA microarray has attracted tremendous interest among biologists as well as cli-nicians. This technology promises to monitor the whole genome on a single chip so researchers can have a better picture of the interactions among thousands of genes simultaneously.DNA microarray, also called gene chip, DNA chip, and gene array, refers to large sets of probes of known sequences orderly arranged on a small chip, enabling many hybridization reactions to be carried out in parallel in a small device (Fig. 15-20).28 Like Southern and Northern hybridization, the underlying principle of this technology is the remarkable abil-ity of nucleic acids to form a duplex between two strands with complementary base sequences. DNA microarray provides a medium for matching known and unknown DNA samples based on

1	the remarkable abil-ity of nucleic acids to form a duplex between two strands with complementary base sequences. DNA microarray provides a medium for matching known and unknown DNA samples based on base-pairing rules and automating the process of identify-ing the unknowns. Microarrays require specialized robotics and imaging equipment that spot the samples on a glass or nylon substrate, carry out the hybridization, and analyze the data gener-ated. DNA microarrays containing different sets of genes from a variety of organisms are now commercially available, allowing biologists to simply purchase the chips and perform hybridiza-tion and data collection. The massive scale of microarray experi-ments requires the aid of computers. They are used during the capturing of the image of the hybridized target, the conversion of the image into usable measures of the extent of hybridization, and the interpretation of the extent of hybridization into a mean-ingful measure of the amount of the

1	target, the conversion of the image into usable measures of the extent of hybridization, and the interpretation of the extent of hybridization into a mean-ingful measure of the amount of the complementary sequence in the target. Some data-analysis packages are available commer-cially or can be found in the core facility of certain institutions.DNA microarray technology has produced many signifi-cant results in quite different areas of application. There are two major application forms for the technology: identifica-tion of sequence (gene/gene mutation) in multiple regions of a genome and determination of expression level (abundance) of large numbers of genes simultaneously. For example, analysis of genomic DNA detects amplifications and deletions found in human tumors. Differential gene expression analysis also has uncovered networks of genes differentially present in can-cers that cannot be distinguished by conventional means. Sig-nificantly, recent advancements in next-generation

1	expression analysis also has uncovered networks of genes differentially present in can-cers that cannot be distinguished by conventional means. Sig-nificantly, recent advancements in next-generation sequencing (e.g., Solexa and 454 technology) have demonstrated the preci-sion and speed to analyze gene expression in any genome.Next-Generation Sequencing.29,30 First-generation sequenc-ing, also termed Sanger’s sequencing, requires a single-stranded DNA template, a specific DNA primer, a DNA polymerase, nor-mal deoxynucleoside triphosphates (dNTPs), and modified di-deoxynucleotidetriphosphates (ddNTPs). In the process of DNA sequencing, DNA polymerase adds random dNTP or ddNTP Brunicardi_Ch15_p0479-p0510.indd 49718/02/19 11:12 AM 498BASIC CONSIDERATIONSPART ICell #1Cell #2mRNAcDNADNA microarrayDNA microarray datacDNAmRNAFigure 15-20. DNA microarrays. DNA microarrays, also referred to as gene chips, have arrayed oligonucleotides or com-plementary DNAs (cDNAs) corresponding to tens or

1	microarray datacDNAmRNAFigure 15-20. DNA microarrays. DNA microarrays, also referred to as gene chips, have arrayed oligonucleotides or com-plementary DNAs (cDNAs) corresponding to tens or hundreds of distinct genes. DNA microarray is used to comparatively analyze gene expression in different cells or tissues. Messenger RNAs (mRNAs) extracted from different sources are converted into cDNAs, which are then labeled with different fluorescent dyes. The two fluorescent cDNA probes are mixed and hybridized to the same DNA microarrays. The ratio of red to green fluorescence at each spot on the chip represents the relative expression of levels of that gene between two different cells. In the example shown in the figure, cDNA from cell #1 is labeled with red fluorescence and that from cell #2 is labeled with green fluorescence. On the micro-array, red spots demonstrate that the gene in the cell sample #1 is expressed at a higher level than the corresponding gene in cell sample #2. The green

1	labeled with green fluorescence. On the micro-array, red spots demonstrate that the gene in the cell sample #1 is expressed at a higher level than the corresponding gene in cell sample #2. The green spots indicate that the gene in the cell sam-ple #2 is expressed at a higher level than the corresponding gene in the cell sample #1. Yellow spots represent equal expression of the gene in both cell samples.after the primer. If ddNTP is incorporated at the end of the chain, it terminizes the reaction and results in DNA fragments of different sizes. The ddNTPs could be radioactively or fluo-rescently labeled for auto-machine detection. Usually, Sanger’s sequencing is able to read sequence below 1 kb with the quality deteriorating after 700 bp. The accuracy and success rate largely depends on the DNA polymerase used.Recombinant DNA technology greatly impacts the com-pletion of the Human Genome Project due to the invention of shotgun sequencing, which includes breaking the genome DNA into

1	the DNA polymerase used.Recombinant DNA technology greatly impacts the com-pletion of the Human Genome Project due to the invention of shotgun sequencing, which includes breaking the genome DNA into small pieces and randomly cloning those pieces into DNA vectors that are easily sequenced. Based on the overlapping sequence of each clone, computer analysis can be programmed to map and align the DNA sequence that will ultimately cover the whole human genome.Based on shotgun sequencing, as the sequencing technol-ogies advance, next-generation sequencing (NGS), also called second-generation sequencing, has become one of the most powerful tools to analyze DNA mutation, identify epigenetic modification, and profile gene expression or ncRNA expression.31 The next-generation sequencing process usually includes library construction, sequencing, and data analysis. There are three major NGS platforms: Roche 454, Life Technologies Ion Tor-rent, and Illumina Solexa. Take the Illumina

1	process usually includes library construction, sequencing, and data analysis. There are three major NGS platforms: Roche 454, Life Technologies Ion Tor-rent, and Illumina Solexa. Take the Illumina next-generation sequencing as an example: DNA are shared or digested into small pieces and then used to generate a DNA library with adapt-ers on both ends of each DNA piece. Then, the DNA library is diluted and loaded on a chamber of a slide, called a lane, for cluster amplification. Cycled fluorescent deoxyribonucleotide triphosphates (dNTPs) are then added to the chamber to enable DNA polymerization, resulting in different fluorescent emis-sion representing different dNTP reading on different clusters, into a microscope. The fluorescent signal is transformed into sequencing data that will be aligned and mapped to a standard genome database. The advantages of next-generation sequenc-ing include the following: no necessity of DNA cloning; fast and cost-effective; and a huge amount of data to

1	and mapped to a standard genome database. The advantages of next-generation sequenc-ing include the following: no necessity of DNA cloning; fast and cost-effective; and a huge amount of data to give good depth and accuracy of the sequence.Based on the applications, the most common next-generation sequencing technologies for whole-genome sequencing are whole-genome DNA sequencing, whole-genome bisulfite sequencing (BS-seq), RNA sequencing (RNA-seq), and chro-matin immunoprecipitation (ChIP) sequencing (ChIP-seq). Whole-genome DNA sequencing is purely to sequence the DNA sequence of a genome without any preprocessing of the DNA, reflecting any deletions, replications, and mutations within the genomic DNA. Given that the genomic information for human is enormous, to achieve great depth and accuracy the genome needs to be sequenced multiple times to reach statistic significance and pass quality control. Therefore, whole-genome DNA sequencing is still considered to be costly for diagnosis

1	the genome needs to be sequenced multiple times to reach statistic significance and pass quality control. Therefore, whole-genome DNA sequencing is still considered to be costly for diagnosis as well as for research purposes. Under clinical settings, one of the most applied DNA sequencing technologies is whole-exome sequencing, i.e., using techniques to capture and analyze exons in all coding genes, given that most known diseases are due to mutations in spliced-in regions of coding genes. Whole-exome sequencing is mostly used for detecting single nucleotide vari-ants and is less reliable in detecting insertion-deletion variants or small copy number variants.BS-seq is commonly used to identify DNA methyla-tion on the genome (5-methylcytosine [5mC]). The process always involves a bisulfite treatment of DNA before library Brunicardi_Ch15_p0479-p0510.indd 49818/02/19 11:12 AM 499MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15construction, during which

1	treatment of DNA before library Brunicardi_Ch15_p0479-p0510.indd 49818/02/19 11:12 AM 499MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15construction, during which the unmethylated cytosine will be transformed to a uracil, resulting in reading as a thymine in data output, whereas 5mC is protected and remains as cytosine in data output. Thus, 5mC and cytosine are distinguished this way. To perform whole-genome BS-seq, the library construc-tion starts with a decent amount of DNA followed by a complete conversion of unmethylated cytosine. Because it is also counted as whole-genome sequencing, a certain depth of data needs to be achieved for accurate and convincing results. To develop an affordable genome-wide DNA methylation analysis, the reduced representation bisulfite sequencing (RRBS) approach has been applied to enrich CpG-dense regions of the genome by digesting genomic DNA using a methylation-insensitive restric-tion enzyme, usually MspI. This

1	bisulfite sequencing (RRBS) approach has been applied to enrich CpG-dense regions of the genome by digesting genomic DNA using a methylation-insensitive restric-tion enzyme, usually MspI. This method covers a majority of promoters as well as some repetitive regions in the genome.RNA-seq is usually performed to analyze transcription for the same purpose as performing a microarray. However, RNA-seq is more accurate and provides more information such as splicing variants than traditional microarray. Usually, cDNA that is reversely transcribed from extracted RNA is used to generate libraries. Depending on the needs, mRNA and ncRNA can be enriched in different protocols for RNA extraction. Currently, techniques have been improved to perform RNA profiling on a single cell level. Single-cell RNA sequencing (scRNA-seq) allows expression analysis of individual cells in a population. One use is to examine heterogeneity in tumor cells. Although low copy number genes are usually poorly detected,

1	sequencing (scRNA-seq) allows expression analysis of individual cells in a population. One use is to examine heterogeneity in tumor cells. Although low copy number genes are usually poorly detected, scRNA-seq on a large number of cells can reveal rare cell subpopula-tions and uncommon RNA expression patterns among different subpopulations.ChIP-seq is always used to map the location of a DNA-binding protein in the genome. Prior to library construction, ChIP is performed to enrich DNA bound by the protein of interest (POI). First, POI and DNA are cross-linked before sonication. Then, a specific antibody is used to pull down POI and attached DNA fragments. After the protein and DNA are reverse cross-linked, DNA is purified to make the ChIP-seq library. If using an antibody against particular transcription fac-tor (TF), DNA sequences bound by this TF are pulled down CellisolationPrimaryculturePropagationTissuesampleProduction of recombinant proteinsAnalysis of gene

1	against particular transcription fac-tor (TF), DNA sequences bound by this TF are pulled down CellisolationPrimaryculturePropagationTissuesampleProduction of recombinant proteinsAnalysis of gene functionTransfectionwith DNAABFigure 15-21. Cell culture and transfection. A. Primary cells can be isolated from tissues and cultured in medium for a limited period of time. After genetic manipulations to over-come the cell aging process, primary cells can be immortalized into cell lines for long-term culture. B. DNA can be introduced into cells to produce recombinant gene products or to ana-lyze the biologic functions of the gene.and examined. The TF binding consensus sequences is then pre-dicted, and if the TF binds to a promoter region, it is likely that the gene using this promoter is regulated by this TF. If using an antibody against an epigenetic modification, the modified regions are marked up into the genome to facilitate identifica-tion of potential epigenetic regulating mechanisms.By

1	by this TF. If using an antibody against an epigenetic modification, the modified regions are marked up into the genome to facilitate identifica-tion of potential epigenetic regulating mechanisms.By using next-generation sequencing technology, any potential mutations in a patient can be scrutinized as well as any defects in epigenetic modification. By combining data from different kinds of sequencing (DNA-seq, RNA-seq, ChIP-seq), better understanding of mutation or transcription-caused diseases aligning with epigenetic regulation can be achieved, which will greatly facilitate the diagnosis of patients and per-sonalization of medicine in a fast and economic way by prevent-ing unnecessary medical costs and procedures.Third-generation sequencing has emerged rapidly at the research level to involve single molecule real-time sequencing (SMRT). Although first developed and marketed by Pacific Biosciences (Pac Bio), Roche is now leading this technology. Third-generation sequencing allows

1	involve single molecule real-time sequencing (SMRT). Although first developed and marketed by Pacific Biosciences (Pac Bio), Roche is now leading this technology. Third-generation sequencing allows amplification-free single-molecule sequencing with read length extension up to mega-bases and reduced sequencing coverage bias. It can be used to build the gap in the human genome (for example, low complex-ity regions), provide access to structural genomic variants, and simultaneously analyze genome-wide single-nucleotide methyl-ation. So far, clinical application and research is heavily depen-dent on NGS, especially standardizing and reducing the cost of post-NGS analysis. Third-generation sequencing is under fast development and has been adapted to aid and append NGS.Cell ManipulationsCell Culture. Cell culture has become one of the most power-ful tools in biomedical laboratories, as cultured cells are being used in a diversity of biologic fields ranging from biochemistry to molecular and

1	culture has become one of the most power-ful tools in biomedical laboratories, as cultured cells are being used in a diversity of biologic fields ranging from biochemistry to molecular and cellular biology.32 Through their ability to be maintained in vitro, cells can be manipulated by the introduc-tion of genes of interest (cell transfection) and be transferred into in vivo biologic receivers (cell transplantation) to study the biologic effect of the interested genes (Fig. 15-21). In common Brunicardi_Ch15_p0479-p0510.indd 49918/02/19 11:12 AM 500BASIC CONSIDERATIONSPART Ilaboratory settings, cells are cultured either as a monolayer (in which cells grow as one layer on culture dishes), considered 2-D, or in suspension or biomedical material skeleton such as hydrogel, considered 3-D.It is important to know the wealth of information concern-ing cell culturing before attempting the procedure. For example, conditions of culture will depend on the cell types to be cultured (e.g.,

1	is important to know the wealth of information concern-ing cell culturing before attempting the procedure. For example, conditions of culture will depend on the cell types to be cultured (e.g., origins of the cells such as epithelial or fibroblasts, or pri-mary vs immortalized/transformed cells). It is also necessary to use a cell type-specific culture medium that varies in combi-nation of growth factors and serum concentrations. If primary cells are derived from human patients or animals, some com-mercial resources have a variety of culture media available for testing. Generally, cells are manipulated in a sterile hood, and the working surfaces are wiped with 70% to 80% ethyl alcohol solution. Cultured cells are usually maintained in a humidified 5% carbon dioxide incubator at 37°C (98.6°F) to maintain a Ph value raging from 7.2 ∼ 7.4 and need to be examined daily under an inverted microscope to check for possible contamination and confluency (the area cells occupy on the dish). In

1	to maintain a Ph value raging from 7.2 ∼ 7.4 and need to be examined daily under an inverted microscope to check for possible contamination and confluency (the area cells occupy on the dish). In some cases, cells need to be maintained in hypoxia, and the oxygen input could be reduced to as low as 1%. As a general rule, cells should be fed with fresh medium every 2 to 3 days and split when they reach confluency. Depending on the growth rate of cells, the actual time and number of plates required to split cells in two varies from cell line to cell line. Splitting a monolayer requires the detachment of cells from plates by using a trypsin or colla-genase treatment, of which concentration and time period vary depending on cell lines. If cultured cells grow continuously in suspension, they are split or subcultured by dilution.Because cell lines may change their properties when cul-tured, it is not possible to maintain cell lines in culture indefi-nitely. Therefore, it is essential to store

1	split or subcultured by dilution.Because cell lines may change their properties when cul-tured, it is not possible to maintain cell lines in culture indefi-nitely. Therefore, it is essential to store cells at various time passages for future use. The common procedure to use is cryo-preservation. The solution for cryopreservation is usually fetal calf serum containing 10% dimethyl sulfoxide or glycerol, stored in liquid nitrogen (−196°C [−320.8°F]) for years of pres-ervation. However, the viability and health of cells when thawed will decrease over time even in liquid nitrogen.Cell Transfection. Cells are cultured for two reasons: to main-tain and to manipulate them (see Fig. 15-21). The transfer of foreign macromolecules, such as nucleic acid, into living cells provides an efficient method for studying a variety of cellular processes and functions at the molecular level. DNA transfec-tion has become an important tool for studying the regulation and function of genes. The cDNA to be

1	for studying a variety of cellular processes and functions at the molecular level. DNA transfec-tion has become an important tool for studying the regulation and function of genes. The cDNA to be expressed should be in a plasmid vector, behind an appropriate promoter working in mammalian cells (e.g., the constitutively active cytomegalo-virus promoter or inducible promoter). Depending on the cell type, many ways of introducing DNA into mammalian cells have been developed. Commonly used approaches include cal-cium phosphate, electroporation, liposome-mediated transfec-tion, the nonliposomal formulation, and the use of viral vectors. These methods have shown variable success when attempting to transfect a wide variety of cells. Transfection can be performed in the presence or absence of serum. It is suggested to test the transfection efficiency of cell lines of interest by comparing transfection with several different approaches. For a detailed transfection protocol, it is best to

1	serum. It is suggested to test the transfection efficiency of cell lines of interest by comparing transfection with several different approaches. For a detailed transfection protocol, it is best to follow the manufacturer’s instructions for the particular reagent. General considerations for a successful transfection depend on several parameters, such as the quality and quantity of DNA and cell culture (type of cell and growth phase). To minimize variations in both of these in transfection experiments, it is best to use cells that are healthy, proliferate well, and are plated at a constant density.Depending on the transfection method, DNA expression can be transient or stable. Using calcium phosphate and lipo-some-mediated transfection, after DNA is introduced into the cells it is normally maintained epitopically in cells and will be diluted while host cells undergo cell division. Therefore, func-tional assays should be performed 24 to 72 hours after transfec-tion, also termed

1	normally maintained epitopically in cells and will be diluted while host cells undergo cell division. Therefore, func-tional assays should be performed 24 to 72 hours after transfec-tion, also termed transient transfection. In many applications, it is important to study the long-term effects of DNA in cells by stable transfection. Thus, electroporation and viral vector are often used in these situations to enable integration of ectopic DNA into the host genome. Stable cell clones can be selected when plasmids carry an antibiotic-resistant marker. In the pres-ence of antibiotics, only those cells that continuously carry the antibiotic-resistant marker (after generations of cell division) can survive. One application of stable transfection is the gen-eration of transgenic or knockout mouse models, in which the transgene has to be integrated in the mouse genome in the ES cells, followed by microinjection of those transgenic ES cells into blastocysts to generate chimera mice. Stable cells

1	in which the transgene has to be integrated in the mouse genome in the ES cells, followed by microinjection of those transgenic ES cells into blastocysts to generate chimera mice. Stable cells also can be transplanted into host organs to test the effect of transgenic cells in vivo.Genetic ManipulationsUnderstanding how genes control the growth and differentia-tion of the mammalian organism has been the most challenging topic of modern research. It is essential for us to understand how genetic mutations and chemicals lead to the pathologic condi-tion of human bodies. The knowledge and ability to change the genetic program will inevitably make a great impact on society and have far-reaching effects on how we think of ourselves.The mouse has become firmly established as the primary experimental model for studying how genes control mammalian development. Genetically altered mice are powerful tools to study the function and regulation of genes as well as modeling human diseases.33 The gene

1	model for studying how genes control mammalian development. Genetically altered mice are powerful tools to study the function and regulation of genes as well as modeling human diseases.33 The gene function can be studied by cre-ating mutant mice through homologous recombination (gene knockout). A gene of interest (GOI) also can be intro-duced into the mouse (transgenic mouse) to study its effect on development or diseases. Because mouse models do not pre-cisely represent human biology, genetic manipulations of human somatic or ES cells provide a great means for the understanding of the molecular networks in human cells in addition to mouse models. In all cases, the gene to be manipulated must first be cloned. Gene cloning has been made easy by recombinant DNA technology and the availability of human and mouse genomes (see “Human Genome”). The following section briefly describes the technologies and the principles behind combining both mouse genetics and human cell culture to explore

1	of human and mouse genomes (see “Human Genome”). The following section briefly describes the technologies and the principles behind combining both mouse genetics and human cell culture to explore gene function and disease mechanisms.Transgenic Mice. During the past 20 years, DNA cloning and other techniques have allowed the introduction of new genetic material into the mouse germline. As early as 1980, the first genetic material was successfully introduced into the mouse germline by using pronuclear microinjection of DNA (Fig. 15-22). These animals, called transgenic, contain for-eign DNA within their genomes. In simple terms, a transgenic mouse is created by the microinjection of ectopic DNA into the one-celled mouse embryo to induce integration, allowing the 4Brunicardi_Ch15_p0479-p0510.indd 50018/02/19 11:12 AM 501MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15DNAPronucleusDNA microinjected intopronucleus of fertilized eggFoster

1	50018/02/19 11:12 AM 501MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15DNAPronucleusDNA microinjected intopronucleus of fertilized eggFoster mothercarryingmicroinjected DNATransgenicmouseFigure 15-22. Transgenic mouse tech-nology. DNA is microinjected into a pro-nucleus of a fertilized egg, which is then transplanted into a foster mother. The microinjected egg develops offspring mice. Incorporation of the injected DNA into offspring is indicated by the differ-ent coat color of offspring mice.efficient introduction of cloned genes into the following devel-oping mouse somatic tissues, as well as into the germline.Designs of a Transgene The transgenic technique has proven to be extremely important for basic investigations of gene regu-lation, creation of animal models of human disease, and genetic engineering of livestock. The design of a transgene construct is a simple task. Like constructs used in cell transfection, a simple transgene construct

1	of animal models of human disease, and genetic engineering of livestock. The design of a transgene construct is a simple task. Like constructs used in cell transfection, a simple transgene construct consists of a protein-encoding gene and a promoter that precedes it. The most common applications for the use of transgenic mice are similar to those in the cell culture system: (a) to study the functions of proteins encoded by the transgene, (b) to analyze the tissue-specific and developmental-stage–specific activity of a gene promoter, and (c) to generate reporter lines to facilitate biomedical studies. Examples of the first application include overexpression of oncogenes, growth factors, hormones, and other key regulatory genes, as well as genes of viral origins. Overexpression of the transgene normally represents gain-of-function mutations. The tissue distribution or expression of a transgene is determined primarily by cis-acting promoter enhancer elements within or in the immediate

1	normally represents gain-of-function mutations. The tissue distribution or expression of a transgene is determined primarily by cis-acting promoter enhancer elements within or in the immediate vicin-ity of the genes themselves. Thus, controlled expression of the transgene can be made possible by using an inducible or tissue-specific promoter. Furthermore, transgenic mice carry-ing dominant negative mutations of a regulatory gene also have been generated. For example, a truncated growth factor receptor that can bind to the ligand, but loses its catalytic activity when expressed in mice, can block the growth factor binding to the endogenous protein. In this way, the transgenic mice exhibit a loss of function of phenotype, possibly resembling the knockout of the endogenous gene. The second application of the trans-genic expression is to analyze the gene promoter of interest. The gene promoter of interest normally is fused to a reporter gene that encodes β-galactosidase (also called

1	application of the trans-genic expression is to analyze the gene promoter of interest. The gene promoter of interest normally is fused to a reporter gene that encodes β-galactosidase (also called LacZ), luciferase, or green fluorescence protein. Chemical staining of LacZ activity or detection of chemiluminescence/fluorescence can easily visu-alize the expression of the reporter gene. The third application originates from the second: when the activity of the promoter is known, a fluorescent reporter gene (such as GFP) will be driven by the tissue-specific promoter, therefore labeling a particular type of cells at a particular stage. This application is generally used to isolate a special cell type expressing the GFP reporter by fluorescence-activated cell sorting (FACS), as well as lineage-tracing experiments.Production of Transgenic Mice The success of generating transgenic mice is largely dependent on the proper quality and concentration of the DNA supplied for microinjection. For

1	experiments.Production of Transgenic Mice The success of generating transgenic mice is largely dependent on the proper quality and concentration of the DNA supplied for microinjection. For DNA to be microinjected into mouse embryos, it should be linear-ized by restriction digestion to increase the chance of proper transgene integration. Concentration of DNA should be accu-rately determined. Mice that develop from injected eggs often are termed founder mice.Genotyping of Transgenic Mice The screening of founder mice and the transgenic lines derived from the founders is accomplished by determining the integration of the injected gene into the genome. This normally is achieved by performing PCR or Southern blot analysis with a small amount of DNA extracted from the mouse tail. Once a given founder mouse is identified to be transgenic, it will be mated to begin establish-ing a transgenic line. Usually, for a given gene, more than one transgenic line is generated to assure that the

1	given founder mouse is identified to be transgenic, it will be mated to begin establish-ing a transgenic line. Usually, for a given gene, more than one transgenic line is generated to assure that the phenotype is due to transgene but not to the interruption of the gene where the transgene integrates into.Analysis of Phenotype of Transgenic Mice Phenotypes of transgenic mice are dictated by both the expression pattern and biologic functions of the transgene. Depending on the promoter and the transgene, phenotypes can be predictable or unpredict-able. Elucidation of the functions of the transgene-encoded pro-tein in vitro often offers some clue to what the protein might function to do in vivo. When a constitutively active promoter is used to drive the expression of transgenes, mice should express the gene in every tissue; however, this mouse model may not allow the identification and study of the earliest events in dis-ease pathogenesis. Ideally, the use of tissue-specific or induc-ible

1	the gene in every tissue; however, this mouse model may not allow the identification and study of the earliest events in dis-ease pathogenesis. Ideally, the use of tissue-specific or induc-ible promoter allows one to determine if the pathogenic protein leads to a reversible or irreversible disease process in a cell-autonomous manner. For example, rat insulin promoter can tar-get transgene expression exclusively in the β-cells of pancreatic islets. The phenotype of insulin promoter-mediated transgenic mice is projected to affect the function of human β-cells.Gene Knockout in Mice. The first recorded knockout mouse was created by Mario R. Capecchi, Sir Martin J. Evans, and Oliver Smithies in 1989. They were awarded the 2007 Nobel Prize in Physiology or Medicine. The isolation and genetic manipulation of mouse ES cells represents one of the most important milestones for modern genetic technologies.34 Sev-eral unique properties of ES cells, such as the pluripotency to differentiate into

1	of mouse ES cells represents one of the most important milestones for modern genetic technologies.34 Sev-eral unique properties of ES cells, such as the pluripotency to differentiate into all germ layers in an embryo, including the germline, make them an efficient vehicle to introduce genetic alterations in mice. An important breakthrough from this idea is to generate gene-targeted mutation in mice, first by introduc-ing the targeting vector into the ES cells, allowing selection Brunicardi_Ch15_p0479-p0510.indd 50118/02/19 11:12 AM 502BASIC CONSIDERATIONSPART IES cells growingin tissue cultureAltered versionof target geneconstructed bygenetic engineeringLet each cellgrow to forma colonyTest for the rarecolony in whichthe DNA fragmenthas replaced one copy of the normal geneES cells with one copy of target genereplaced by mutant geneInjectES cellsintoearly embryoFemale mouseMate and wait3 daysIsolatedearlyembryoEarly embryo partlyformed fromES cellsIntroduceearly embryo

1	cells with one copy of target genereplaced by mutant geneInjectES cellsintoearly embryoFemale mouseMate and wait3 daysIsolatedearlyembryoEarly embryo partlyformed fromES cellsIntroduceearly embryo intopseudopregnantmouseSomatic cellsof offspring tested forpresence ofaltered gene, and selected mice bredto test for genein germline cellsTransgenic mouse with one copy of target genereplaced by altered gene in germlineABBirthIntroduce a DNA fragmentcontaining altered geneinto many cellsFigure 15-23. Knockout mouse technology. Summary of the procedures used for making gene replacements in mice. In the first step (A), an altered version of the gene is introduced into cultured embryonic stem (ES) cells. Only a few rare ES cells will have their corresponding normal genes replaced by the altered gene through a homologous recombination event. Although the procedure is often laborious, these rare cells can be identified and cultured to produce many descendants, each of which carries an altered

1	gene through a homologous recombination event. Although the procedure is often laborious, these rare cells can be identified and cultured to produce many descendants, each of which carries an altered gene in place of one of its two normal corresponding genes. In the next step of the procedure (B), these altered ES cells are injected into a very early mouse embryo; the cells are incorporated into the growing embryo, and a mouse produced by such an embryo will contain some somatic cells that carry the altered gene. Some of these mice also will contain germline cells that contain the altered gene. When bred with a normal mouse, some of the progeny of these mice will contain the altered gene in all of their cells. If two such mice are in turn bred (not shown), some of the progeny will contain two altered genes (one on each chromosome) in all of their cells. If the original gene alteration completely inactivates the function of the gene, these mice are known as knockout mice. When such

1	two altered genes (one on each chromosome) in all of their cells. If the original gene alteration completely inactivates the function of the gene, these mice are known as knockout mice. When such mice are missing genes that function during development, they often die with specific defects long before they reach adulthood. These defects are carefully analyzed to help decipher the normal function of the missing gene. (Reproduced with permission from Alberts B, Johnson A, Lewis J, et al: Molecular Biology of the Cell, 6th ed. New York, NY: WW Norton Company; 2015.)for successful homologous recombination in a dish, then intro-ducing the selected ES clone into the blastocysts, and finally recovering animals bearing the mutant allele from the germline (Fig. 15-23). This not only makes mouse genetics a powerful approach to address important gene functions, but also identifies the mouse as a great system to model human disease.Targeting Vector The basic concept in building a target vec-tor to

1	a powerful approach to address important gene functions, but also identifies the mouse as a great system to model human disease.Targeting Vector The basic concept in building a target vec-tor to knock out a gene is to use two segments of homologous sequence to a GOI that flank a part of the gene essential for functions (e.g., the coding region). In the targeting vector, a pos-itive selectable marker (e.g., the neo gene) is placed between the homology arms. Upon the homologous recombination between the arms of the vector and the corresponding genomic regions of the GOI in ES cells, the positive selectable marker will replace the essential segment of the target gene, thus creating a null allele. In addition, a negative selectable marker also can Brunicardi_Ch15_p0479-p0510.indd 50218/02/19 11:12 AM 503MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15be used alone or in combination with the positive selectable marker, but must be placed outside of the

1	11:12 AM 503MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15be used alone or in combination with the positive selectable marker, but must be placed outside of the homologous arms to enrich for homologous recombination. To create a conditional knockout (i.e., gene knockout in a spatiotemporal fashion), site-specific recombinases such as the popular cre-loxP system are used. If the consensus loxP sequences that are recognized by cre recombinases are properly designed into targeting loci, con-trolled expression of the recombinase as a transgene can result in the site-specific recombination at the right time and in the right place (i.e., cell type or tissue). This method, often referred to as conditional knockout, is markedly useful to prevent devel-opmental compensations and to introduce null mutations in the adult mouse that would otherwise be lethal. To bring in addi-tional control to tissue-specific cre, an inducible cre could be adopted on top of the

1	and to introduce null mutations in the adult mouse that would otherwise be lethal. To bring in addi-tional control to tissue-specific cre, an inducible cre could be adopted on top of the tissue-specific promoter. The most popu-lar inducible cre includes CreER: CreER encodes a cre fused with estrogen receptor located to cytosol. With the signaling of tamoxifen, CreER is released and then translocates into the nucleus to induce the recombination of loxP. Therefore, the tim-ing of recombination could be precisely determined by control-ling the time of administering tamoxifen. Overall, this cre-loxP system allows for spatial and temporal control over transgene expression and takes advantage of inducers with minimal pleio-tropic effects.Introduction of the Targeting Vector into ES Cells ES cell lines can be obtained from other investigators or commer-cial sources or established from blastocyst-stage embryos. To maintain ES cells at their full developmental potential, opti-mal growth

1	cell lines can be obtained from other investigators or commer-cial sources or established from blastocyst-stage embryos. To maintain ES cells at their full developmental potential, opti-mal growth conditions should be provided in culture. If cul-ture conditions are inappropriate or inadequate, ES cells may acquire genetic lesions or alter their gene expression patterns and consequently decrease their pluripotency. Excellent proto-cols are available in public domains or in mouse facilities in most institutions.To alter the genome of ES cells, the targeting vector DNA then is transfected into ES cells. Electroporation is the most widely used and the most efficient transfection method for ES cells. Similar procedures for stable cell transfection are used for selecting ES cells that carry the targeting vector. High-quality, targeting-vector DNA free of contaminating chemicals is first linearized and then electroporated into ES cells. Stable ES cells are selected in the presence of a

1	the targeting vector. High-quality, targeting-vector DNA free of contaminating chemicals is first linearized and then electroporated into ES cells. Stable ES cells are selected in the presence of a positive selectable antibiotic drug. After a certain period of time and depending on the type of antibiotics, all sensitive cells die, and the resistant cells grow into individual colonies of the appropriate size for subcloning by picking. It is extremely important to minimize the time dur-ing which ES cells are in culture between selection and injection into blastocysts. Before injecting the ES cells, DNA is prepared from ES colonies to screen for positive ES cells that exhibit the correct integration or homologous recombination of the target-ing vector. Positive ES colonies are then expanded and used for creation of chimeras.Creation of the Chimera A chimeric organism is one in which cells originate from more than one embryo source. Here, chi-meric mice are denoted as those that contain

1	and used for creation of chimeras.Creation of the Chimera A chimeric organism is one in which cells originate from more than one embryo source. Here, chi-meric mice are denoted as those that contain some tissues from the ES cells with an altered genome. When these ES cells give rise to the lineage of the germ layer, the germ cells carrying the altered genome can be passed on to the offspring, thus creating the germline transmission from ES cells. There are two methods for introducing ES cells into preimplantation-stage embryos: injection and aggregation. The injection of embryonic cells directly into the cavity of blastocysts is one of the fundamen-tal methods for generating chimeras, but aggregation chimeras also have become an important alternative for transmitting the ES cell genome into mice. Since every tissue type of a chimera should contain cells from different origins, the mixture of rec-ognizable markers (e.g., coat color) that are specific to the donor mouse and the ES cells

1	mice. Since every tissue type of a chimera should contain cells from different origins, the mixture of rec-ognizable markers (e.g., coat color) that are specific to the donor mouse and the ES cells can be used to identify chimeric mice. However, most experimenters probably use existing mouse core facilities already established in some institutions or contract a commercial vendor for the creation of a chimera.Genotyping and Phenotyping of Knockout Animals The next step is to analyze whether germline transmission of tar-geted mutation occurs in mice. DNA from a small amount of tissue from offspring of the chimera is extracted and subjected to genomic PCR or Southern blot DNA hybridization. Positive mice (i.e., those with properly integrated targeting vector into the genome) will be used for the propagation of more knockout mice for phenotype analysis. When the knockout genes are cru-cial for early embryogenesis, mice often die in utero, an occur-rence called embryonic lethality. When

1	the propagation of more knockout mice for phenotype analysis. When the knockout genes are cru-cial for early embryogenesis, mice often die in utero, an occur-rence called embryonic lethality. When this happens, only the phenotype of the homozygous (both alleles ablated) knockout mouse embryos and the phenotype of the heterozygous (only one allele ablated) adult mice can be studied. Because most researchers are interested in the phenotype of adult mice, in par-ticular when using mice as disease models, it is recommended to create the conditional knockout using the cre-loxP system so that the GOI can be knocked out at will.To date, more than 5000 genes have been disrupted by homologous recombination and transmitted through the germline. The phenotypic studies of these mice provide ample information about the functions of these genes in growth and differentiation of organisms and during development of human diseases.RNA Interference. Although gene ablation in animal models provides an

1	about the functions of these genes in growth and differentiation of organisms and during development of human diseases.RNA Interference. Although gene ablation in animal models provides an important means to understand the in vivo functions of GOI, animal models may not adequately represent human biology. Alternatively, gene targeting can be used to knock out genes in human cells, including human ES cells. Gene targeting in human ES cells by homologous recombination has extremely low efficiency, although there are more new techniques emerg-ing aimed at increasing the targeting efficiency. A number of recent advances have made gene targeting in somatic cells as easy as in murine ES cells.33 However, gene targeting (knocking out both alleles) in somatic cells is a time-consuming process.Development of RNAi technology in the past few years has provided a more promising approach to understanding the biologic functions of human genes in human cells.35 RNAi is an ancient natural mechanism

1	of RNAi technology in the past few years has provided a more promising approach to understanding the biologic functions of human genes in human cells.35 RNAi is an ancient natural mechanism by which small, double-stranded RNA (dsRNA) acts as a guide for an enzyme complex that destroys complementary RNA and downregulates gene expres-sion in a sequence-specific manner. Although the mechanism by which dsRNA suppresses gene expression is not entirely understood, experimental data provide important insights. In nonmammalian systems such as Drosophila, it appears that longer dsRNA is processed into 21–23 nt dsRNA (called small interfering RNA or siRNA) by an enzyme called Dicer contain-ing RNase III motifs. The siRNA apparently then acts as a guide sequence within a multicomponent nuclease complex to target complementary mRNA for degradation. Because long dsRNA induces a potent antiviral response pathway in mammalian cells, short siRNAs are used to perform gene silencing experiments in

1	complex to target complementary mRNA for degradation. Because long dsRNA induces a potent antiviral response pathway in mammalian cells, short siRNAs are used to perform gene silencing experiments in mammalian cells (Fig. 15-24).Brunicardi_Ch15_p0479-p0510.indd 50318/02/19 11:12 AM 504BASIC CONSIDERATIONSPART IPol IIITTTTTshRNAsiRNADicerRISCUUUUUUmRNAm7GAAAAAAmRNA targeted by siRNAUUm7GAAAAAAmRNA cleaved and degradedm7GAAAAAAFigure 15-24. RNA interference in mammalian cells. Small interfering RNA (siRNA) can be produced from a polymerase III–driven expres-sion vector. Such a vector first synthesizes a 19–29 nt double-stranded (ds)RNA stem and a loop (labeled as shRNA in the figure), and then the RNase complex called Dicer processes the hairpin RNA into a small dsRNA (labeled as siRNA in the figure). siRNA can be chemically synthesized and directly introduced into the target cell. In the cell, through RNA-induced silencing complex (RISC), siRNA recognizes and degrades target

1	siRNA in the figure). siRNA can be chemically synthesized and directly introduced into the target cell. In the cell, through RNA-induced silencing complex (RISC), siRNA recognizes and degrades target messenger RNAs (mRNAs).For siRNA studies in mammalian cells, researchers have used two 21-mer RNAs with 19 complementary nucleotides and 3′ terminal noncomplementary dimers of thymidine or uri-dine. The antisense siRNA strand is fully complementary to the mRNA target sequence. Target sequences for an siRNA are identified visually or by software.The target 19 nucleotides should be compared to an appro-priate genome database to eliminate any sequences with sig-nificant homology to other genes. Those sequences that appear to be specific to the GOI are the potential siRNA target sites. A few of these target sites are selected for siRNA design. The antisense siRNA strand is the reverse complement of the target sequence. The sense strand of the siRNA is the same sequence as the target mRNA

1	of these target sites are selected for siRNA design. The antisense siRNA strand is the reverse complement of the target sequence. The sense strand of the siRNA is the same sequence as the target mRNA sequence. A deoxythymidine dimer is rou-tinely incorporated at the 3′ end of the sense strand siRNA, although it is unknown whether this noncomplementary dinu-cleotide is important for the activity of siRNAs.There are two ways to introduce siRNA to knock down gene expression in human cells:1. RNA transfection: siRNA can be made chemically or using an in vitro transcription method. Like DNA oligos, chemically synthesized siRNA oligos can be commercially ordered. However, synthetic siRNA is expensive, and sev-eral siRNAs may have to be tried before a particular gene is successfully silenced. In vitro transcription provides a more economic approach. Both short and long RNA can be synthesized using bacteriophage RNA polymerase T7, T3, or SP6. In the case of long dsRNAs, RNase such as

1	In vitro transcription provides a more economic approach. Both short and long RNA can be synthesized using bacteriophage RNA polymerase T7, T3, or SP6. In the case of long dsRNAs, RNase such as recom-binant Dicers will be used to process the long dsRNA into a mixture of 21–23 nt siRNA. siRNA oligos or mixtures can be transfected into a few characterized cell lines such as HeLa (human cervical carcinoma) and 293T cells (human kidney carcinoma). Transfection of siRNA directly into pri-mary cells may be difficult.2. DNA transfection: Expression vectors for expressing siRNA have been made using RNA polymerase III promoters such as U6 and H1. These promoters precisely transcribe a hairpin structure of dsRNA, which will be processed into siRNA in the cell (see Fig. 15-24). Therefore, properly designed DNA oligos corresponding to the desired siRNA will be inserted downstream of the U6 or H1 promoter. There are two advan-tages of the siRNA expression vectors over siRNA oligos. First, it is

1	designed DNA oligos corresponding to the desired siRNA will be inserted downstream of the U6 or H1 promoter. There are two advan-tages of the siRNA expression vectors over siRNA oligos. First, it is easier to transfect DNA into cells. Second, sta-ble populations of cells can be generated that maintain the long-term silencing of target genes. Furthermore, the siRNA expression cassette can be incorporated into a retroviral or adenoviral vector to provide a wide spectrum of applications in gene therapy.There has been a fast and fruitful development of RNAi tools for in vitro and in vivo use in mammals. These novel approaches, together with future developments, will be crucial to put RNAi technology to use for effective disease therapy or to exert the awesome power of mammalian genetics. Therefore, the applications of RNAi to human health are enormous. siRNA can be applied as a new tool for sequence-specific regulation of gene expression in functional genomics and biomedical studies. With

1	the applications of RNAi to human health are enormous. siRNA can be applied as a new tool for sequence-specific regulation of gene expression in functional genomics and biomedical studies. With the availability of the human genome sequences, RNAi approaches hold tremendous promise for unleashing the dor-mant potential of sequenced genomes.Practical applications of RNAi will possibly result in new therapeutic interventions. In 2002, the concept of using siRNA in battling infectious diseases and carcinogenesis was proven effective. These include notable successes in blocking repli-cation of viruses, such as HIV, hepatitis B virus, and hepati-tis C virus, in cultured cells using siRNA targeted at the viral genome or the human gene encoding viral receptors. RNAi has been shown to antagonize the effects of hepatitis C virus in mouse models. In cancers, silencing of oncogenes such as c-Myc or Ras can slow down the proliferation rate of cancer cells. Finally, siRNA also has potential

1	the effects of hepatitis C virus in mouse models. In cancers, silencing of oncogenes such as c-Myc or Ras can slow down the proliferation rate of cancer cells. Finally, siRNA also has potential applications for some dominant genetic disorders.The 21st century, already heralded as the “century of the gene,” carries great promise for alleviating suffering from dis-ease and improving human health. On the whole, completion of Brunicardi_Ch15_p0479-p0510.indd 50418/02/19 11:12 AM 505MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15the human genome blueprint, the promise of gene therapy and molecular therapies, and the existence of stem cells have cap-tured the imagination of the public and the biomedical commu-nity. Aside from their potential in curing human diseases, these emerging technologies also have provoked many political, eco-nomic, religious, and ethical discussions. As more is discerned about the technologic scientific advances, more attention

1	diseases, these emerging technologies also have provoked many political, eco-nomic, religious, and ethical discussions. As more is discerned about the technologic scientific advances, more attention must also be paid to concerns for their inherent risks and social impli-cations. It is important for surgeons to play a leadership role in the emergence of personalized medicine and surgery, as sur-geons have access to the diseased tissues. Surgeons should be establishing collaborations with the genomic and molecular sci-entists to develop genomic biobanks in order to study the genome and molecular signaling of the disease tissues that will help with an understanding of the underlying cause of an indi-vidual’s disease and ultimately lead to effective, targeted thera-pies. Surgeons must take this enormous opportunity to collaborate with basic and clinical scientists to develop the field of precision medicine and surgery this century.Bifunctional RNAi Technology.36 Over the last 20 years,

1	this enormous opportunity to collaborate with basic and clinical scientists to develop the field of precision medicine and surgery this century.Bifunctional RNAi Technology.36 Over the last 20 years, the field has worked to define oncogene and nononcogene addic-tion, discriminate between driver and passenger genes, and appreciate the complexity of complex, robust, network interac-tions. These insights have led to a preliminary understanding of therapeutically relevant sensitivity and resistance pathway signal patterns requiring multiple target modulation. However, this knowledge has not been effectively or reproducibly clini-cally translated. Clinical response is usually far greater when a combination of single-target molecular therapy is administered. However, it must also be realized that targeting two or more pathways may also increase the toxicity profile, particularly if target specificity is limited. When attempted, off-target toxic-ity has been demonstrated with combination

1	that targeting two or more pathways may also increase the toxicity profile, particularly if target specificity is limited. When attempted, off-target toxic-ity has been demonstrated with combination small-molecule therapy. In contrast, multitargeting bifunctional short hairpin (bi-shRNA) DNA vectors are designed to limit off-target effect given the high specificity for the genes they are designed to target.Exogenously applied hairpin constructs can be designed to be incorporated into cleavage-dependent RISC or cleav-age-independent RISC complexes, or both. The concept of a bifunctional shRNA37 is to increase knockdown efficiency without loss of sequence specificity by engaging both siRNA and miRNA-like (i.e., common biogenic pathway but comple-mentary to target sequence) RISCs, thereby concurrently acti-vating nucleolytic (Ago2-RISC) and nonnucleolytic (Ago1, 3, 4 ± Ago2-RISC) processes.38 Each bi-shRNA contains both a matched stem sequence to promote Ago2-mediated passenger strand

1	concurrently acti-vating nucleolytic (Ago2-RISC) and nonnucleolytic (Ago1, 3, 4 ± Ago2-RISC) processes.38 Each bi-shRNA contains both a matched stem sequence to promote Ago2-mediated passenger strand cleavage and a second partial mismatched stem sequence for cleavage-independent passenger strand departure. Thus, functionality of the effectors is set by programmed passenger strand guided RISC loading rather than Ago subset distribution in the cancer cell. Both component Ago2 and Ago (1, 2, 4 ± 3) RNAi moieties are fully complementary to the mRNA target sequence. Preliminary data indicate reduced “off-target effects” by shRNA compared with target-identical siRNAs. More than two mismatches in sequences within the target region drasti-cally reduce knockdown effect to undetectable levels (unpub-lished results). The design process involves in silico scanning of the entire human mRNA RefSeq database to avoid any potential sequence-related “off-target effects.” Published data also indicate

1	results). The design process involves in silico scanning of the entire human mRNA RefSeq database to avoid any potential sequence-related “off-target effects.” Published data also indicate persistent susceptibility to shRNA-mediated gene 5knockdown despite recent evidence of reduced Dicer expression in human cancer cells.39The first clinical experience with the bi-shRNA platform involved the ex vivo knockdown of furin, a Ca2+-dependent, nonredundant proprotein convertase that is essential for pro-teolytic maturational processing of immunosuppressive TGF-β isoforms (β1 and β2). An autologous whole-cell cancer vac-cine, FANG™ (furin-knockdown and GMCSF-augmented),40 was produced based on a dual function immunosensitization principle of augmenting tumor antigen expression, presentation, and processing via granulocyte-macrophage colony-stimulating factor (GMCSF) cytokine transgene expression and attenuating secretory immunosuppressive TGF-β. Harvested, autologous cancer cells are

1	and processing via granulocyte-macrophage colony-stimulating factor (GMCSF) cytokine transgene expression and attenuating secretory immunosuppressive TGF-β. Harvested, autologous cancer cells are transfected with the GMCSF/bi-shRNAfurin (FANG) expression plasmid via electroporation. A phase I clinical trial (BB-IND 14205) involving 52 cancer patients was recently completed. Results demonstrated better than 90% knockdown of the bi-shRNA target, furin, and better than 90% knockdown of furin-regulated proteins TGF-β1 and TGF-β2, thereby confirming the mechanistic expectation of this novel RNAi platform. Moreover, predicted extensive GMCSF expres-sion verified our ability to successfully construct multi-cassette vectors with good manufacturing practice techniques fulfilling Food and Drug Administration requirements for clinical testing.Twenty-seven patients received one or more vaccine dose, and 23 patients achieved stable disease as their best response. No toxic effect was identified.

1	requirements for clinical testing.Twenty-seven patients received one or more vaccine dose, and 23 patients achieved stable disease as their best response. No toxic effect was identified. Median survival of the FANG™-treated patients from time of procurement was 554 days and has not been reached from time of treatment. Expected survival of similar patients is historically less than 1 year. Sequential enzyme-linked immunosorbent spot (ELISPOT) analysis revealed a dramatic and significant increase in immune response from baseline to month 4 in half of the FANG™-treated patients. Comparison of survival between ELISPOT-positive and ELISPOT-negative patients demonstrated a statistically significant increase in survival from time of procurement (P = .045) and time of treatment (P = .025).These phase 1 study results demonstrated mechanism, safety, and effectiveness of the bi-shRNA technology and clini-cal functionality of a multitargeting (dual) DNA expression vec-tor. Further utilization of

1	1 study results demonstrated mechanism, safety, and effectiveness of the bi-shRNA technology and clini-cal functionality of a multitargeting (dual) DNA expression vec-tor. Further utilization of bi-shRNAi technology is under way clinically (targeting STMN1, a microtubule modulation criti-cal to cancer program) and preclinically targeting PDX141 (an oncogene-like transcription factor for pancreatic embryogenesis using nonviral nanoparticle delivery mechanisms).42Precision Medicine and Surgery43Genes determine our susceptibility to diseases and direct our body’s response to medicine. Because an individual’s genes dif-fer from those of another, the determination of each individual’s genome has the potential to improve the predication, prevention, and treatment of disease. Sequencing of individual genomes holds the key to realize this revolution called precision medicine and surgery. Next-generation sequencing, such as Illumina sequencing and 454 pyrosequencing technology, is promising to

1	genomes holds the key to realize this revolution called precision medicine and surgery. Next-generation sequencing, such as Illumina sequencing and 454 pyrosequencing technology, is promising to reduce the time and cost so that genome sequencing can be affordable within healthcare systems. The goal of precision medicine and surgery is to identify the gene variations in each individual and to target the specific gene variations causing the disease by choosing personalized treatments that effectively work in association with the individual’s genomic profile. The importance of surgeons in this transformational field of Brunicardi_Ch15_p0479-p0510.indd 50518/02/19 11:12 AM 506BASIC CONSIDERATIONSPART Ibiomedical science is that surgeons have access to the diseased tissues on a daily basis. Surgeons should partner with the genomic scientists to develop genomic biobanks in order to study the genome of the disease tissues and determine how this information can improve the outcomes of

1	Surgeons should partner with the genomic scientists to develop genomic biobanks in order to study the genome of the disease tissues and determine how this information can improve the outcomes of surgery, i.e., precision surgery. These discovery studies are rapidly leading to the uncovering of mutations and SNPs that are the underlying cause of an individual’s disease and ultimately lead to targeted thera-pies. Although precision medicine and surgery holds the poten-tial to revolutionize the practice of modern medicine, there currently exists a gap between our ability to sequence any given individual’s genome and how clinicians can apply this informa-tion to guide care. There is a rapidly growing list of single genes that are currently guiding care, and these genes are listed as type 1 precision genes. Examples of these genes are BRCA1, RET proto-oncogene, and CHD1 mutation, which guide potential use of mastectomy, thyroidectomy, and gastrec-tomy, respectively; however, the great

1	1 precision genes. Examples of these genes are BRCA1, RET proto-oncogene, and CHD1 mutation, which guide potential use of mastectomy, thyroidectomy, and gastrec-tomy, respectively; however, the great challenge before the sci-entific and medical community this century is to learn to use the entire genome to guide precision care.Targeted Genome Editing Using the CRISPR-Cas9 SystemConventional genetic manipulations have proven their value in biomedical research. Researchers today depend on the manipu-lation of genetic materials in cells or in animal models in almost every project they work on. These genetic manipulation tech-niques (see “Genetic Manipulations”), though sufficient for gen-eral research purposes, suffer from disadvantages. Transfection of target genes into cells is quick and specific, but nonnative. RNA interference (RNAi) is easy to perform and targets native genes, but RNAi never fully eliminates the target gene, and off-target effects are commonly seen using RNAi. Gene

1	but nonnative. RNA interference (RNAi) is easy to perform and targets native genes, but RNAi never fully eliminates the target gene, and off-target effects are commonly seen using RNAi. Gene knockout mice provide an ideal platform to study native genes with clean background, but conventional knockout methods are time-consuming and costly.An entirely new gene-editing method now known as the CRISPR-Cas9 system has emerged since 201344-46 and has quickly gained popularity among biologists for gene edit-ing. This new method is easy to perform, can work specifi-cally on the desired gene or DNA sequence, and can generate gene knockout, knock-in, point mutation, or the insertion of an epitope tag in almost any cell line or animal models with high efficiencies.CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.47 It is a region on the genomic DNA first discovered in the microbes as an adapted immune system against exogenous DNA. A typical CRISPR region contains a

1	Interspaced Short Palindromic Repeats.47 It is a region on the genomic DNA first discovered in the microbes as an adapted immune system against exogenous DNA. A typical CRISPR region contains a cluster of short (21–48 bp) DNA repeats (ranging from 2 to hundreds) interspaced by nonrepetitive sequences called spacers.47 Within a CRISPR region, while each spacer has its unique sequence, the sequence of the repeats is highly conserved. Several genes, called the CRISPR-associated (Cas) genes, are almost always found directly flanking the CRISPR region.Extensive studies in the past decade revealed the function of the CRISPR-cas system in DNA-interfering. When bacte-ria or archaea carrying the CRISPR-cas system are invaded by phage or plasmid DNA, a new spacer can be added to the CRISPR region with its sequence identical to the “proto-spacer,” a fragment of the invading DNA.47 It was found that the proto-spacer must be followed by a recognition sequence (NGG in the case of Cas9) called

1	with its sequence identical to the “proto-spacer,” a fragment of the invading DNA.47 It was found that the proto-spacer must be followed by a recognition sequence (NGG in the case of Cas9) called proto-spacer associated motif 67(PAM).46 With constitutive transcription, the CRISPR region is transcribed as mRNA, and cut by Cas proteins to generate RNA fragments minimally containing one spacer and parts of the repetitive sequence.47 This fragment associates with target DNA through Watson-Crick base-pairing and directs the cutting of target DNA by Cas proteins with nuclease activity.Several CRISPR-Cas systems have been characterized, with a variety of Cas proteins found in these systems. Cas9 in the type II system from Streptococcus pyogenes is the most commonly used Cas for gene editing.46,48 Cas9 contains one RuvC-like nuclease domain near its N-terminal and one HNH-like nuclease domain in the middle of the protein. The RuvC-like domain cuts the proto-spacer strand (the strand with

1	Cas9 contains one RuvC-like nuclease domain near its N-terminal and one HNH-like nuclease domain in the middle of the protein. The RuvC-like domain cuts the proto-spacer strand (the strand with PAM), and the HNH-like domain cuts the strand pairing with the spacer,49 resulting in a double-strand break (DSB) on the target DNA. It is known that Cas9 cuts a blunt end 3 bp 5’ to the PAM sequence.46 The specificity provided by the spacer, or so-called “guide RNA,” and the ability of Cas9 to cut double-stranded DNA means that this system can specifically target anywhere in a genome with a known sequence.CRISPR-Cas9–Guided Gene Editing. The CRISPR-Cas9 sys-tem was made suitable for gene editing in mammalian cell lines and in animal models through several years of optimization. The key concept for CRISPR-Cas9–mediated gene editing is to introduce DNA strand break and let the cell repair the break. Through the repair process, sequence deletions, insertions, and mutations can be applied to the

1	CRISPR-Cas9–mediated gene editing is to introduce DNA strand break and let the cell repair the break. Through the repair process, sequence deletions, insertions, and mutations can be applied to the target gene.Two DNA repair pathways are utilized in CRISPR-Cas9–mediated gene editing: the nonhomologous end-joining (NHEJ) pathway and the homology-directed repair (HDR).49 When a homologous repair template is unavailable, the NHEJ path-way joins the ends of the DSB together, usually with random insertion/deletion (indel) mutations. Such mutations within an open reading frame can cause frameshift and/or premature stop codons. When a repair template is available, however, the cell may choose HDR and repair the DSB through pairing with the template. The HDR pathway can introduce longer insertion/ deletion mutations than NHEJ and can specify the mutated sequence (by contrast, NHEJ creates random mutations). However, HDR is usually only active in dividing cells and is of a lower efficiency

1	deletion mutations than NHEJ and can specify the mutated sequence (by contrast, NHEJ creates random mutations). However, HDR is usually only active in dividing cells and is of a lower efficiency than NHEJ. Its efficiency also depends on gene loca-tion, cell type, and the repair template.50 Therefore, the choice of the pathway depends on the need of the outcome: to simply create a gene knockout, NHEJ is much simpler and highly effi-cient; to achieve precise gene editing (introduce a specific muta-tion, add or delete a specific sequence), HDR must be used. The two pathways have similar protocols, but they differ in certain details in experimental designs.Gene Editing Through NHEJ Tools for NHEJ-mediated gene editing can be incorporated onto one simple plasmid, including a single-guide RNA (sgRNA) sequence containing the guide RNA, a U6 promoter driving the expression of the sgRNA, and an expression cassette including codon-optimized Cas9 fused with nuclear localization sequences (NLS)

1	(sgRNA) sequence containing the guide RNA, a U6 promoter driving the expression of the sgRNA, and an expression cassette including codon-optimized Cas9 fused with nuclear localization sequences (NLS) and an optional selection marker (puromycin resistance or a GFP).46 NHEJ is usually used to knock out a target gene (Fig. 15-24). Generally, a 20 bp sequence preceding a PAM sequence (NGG for Cas9) is selected as the guide RNA sequence. This sequence can be chosen within the first couple of exons through running online search engines against the target genome to minimize off-target probabilities. The guide RNA sequence is then inserted into the Brunicardi_Ch15_p0479-p0510.indd 50618/02/19 11:12 AM 507MOLECULAR BIOLOGY, THE ATOMIC THEORY OF DISEASE, AND PRECISION SURGERYCHAPTER 15Figure 15-25. CRISPR-Cas9–mediated gene editing through NHEJ or HDR. The Cas9 protein cleaves target DNA through its RuvC-like and HNH-like domains, guided by the sgRNA. The cell repairs the double-strand

1	15-25. CRISPR-Cas9–mediated gene editing through NHEJ or HDR. The Cas9 protein cleaves target DNA through its RuvC-like and HNH-like domains, guided by the sgRNA. The cell repairs the double-strand break through either NHEJ or HDR. In NHEJ, the broken ends are recognized, bound, and tethered by end-binding protein complexes. The ends are then processed and ligated but may result in ran-dom insertion/deletion (experimentally, deletions are more commonly seen) mutations at the break site. The repair process in HDR, however, generates no random error due to the presence of a homologous repair template. During the repair, the broken strands find the homologous template and proceed with DNA synthesis using the template. This results in a repaired DNA that has the same sequence as the repair template. Therefore, point mutations, insertions, or deletions carried on the template will be inherited by the repaired DNA and thus achieve precise gene editing.designed site within the sgRNA, and the

1	Therefore, point mutations, insertions, or deletions carried on the template will be inherited by the repaired DNA and thus achieve precise gene editing.designed site within the sgRNA, and the constructed plasmid is transfected into target cells. Expressed Cas9 proteins would associate with expressed sgRNAs to mediate DSB directed by the guide RNA sequence. For cell lines, pure mutant clones can be generated by separating single colonies. Knockout mutations can be confirmed through PCR amplification of the target region and sequencing. If antibodies against the target protein are avail-able, western blotting can be used as a supplemental method to DNA sequencing results to confirm the knockout of the gene.Gene Editing Through HDR HDR requires a homologous tem-plate to repair the DSB. Therefore, apart from the guide RNA, another sequence homologous to the flanking regions of the DSB needs to be present (Fig. 15-25). The homologous sequence can carry insertions, deletions, and mutations

1	apart from the guide RNA, another sequence homologous to the flanking regions of the DSB needs to be present (Fig. 15-25). The homologous sequence can carry insertions, deletions, and mutations to replace the sequence at the DSB, thus achieving precise gene editing on the target. The homologous sequence can be introduced into the cell either as a template on a plasmid or as a single-stranded oligonucleotide.46 After the induction of the DSB, the homologous template pairs with the flanking regions of the DSB and serves as the template for the repair of the break site. HDR happens at lower efficiency than NHEJ and therefore is of lower success rate than NHEJ-mediated gene editing. Therefore, HDR is only recommended when precise mutations are desired. Fast screen of positive mutant clones can be achieved by incorporating restriction enzyme cutting sites within the homologous template.Reducing Off-Target Effects Using Cas9 Nickase The CRISPR-Cas9 system uses a 20 bp guide RNA for sequence

1	be achieved by incorporating restriction enzyme cutting sites within the homologous template.Reducing Off-Target Effects Using Cas9 Nickase The CRISPR-Cas9 system uses a 20 bp guide RNA for sequence recognition. Due to the similar length of recognition sequence to RNAi techniques, CRISPR-Cas9 system also suffers from off-target effects. Because CRISPR-Cas9 requires a PAM site directly following the guide RNA sequence, one way to reduce off-targets is to run through online databases against the genome of the target cell to select a target sequence with the least number of possible off-targets (CRISPR-Cas9 system tolerates no more than three mismatches). On the other hand, a mutant version of the Cas9 called Cas9 nickase can be used to minimize the risk of off-targets.As mentioned, Cas9 contains one RuvC-like and one HNH-like nuclease domain, each responsible for cutting one strand.49 The D10A mutant Cas9 (Cas9 nickase) lacks the activ-ity of the RuvC-like domain, leaving the

1	Cas9 contains one RuvC-like and one HNH-like nuclease domain, each responsible for cutting one strand.49 The D10A mutant Cas9 (Cas9 nickase) lacks the activ-ity of the RuvC-like domain, leaving the proto-spacer strand intact and a nick on the antisense strand.46 Using two prop-erly spaced (0–20 bp apart), oppositely oriented guide RNA, the Cas9 nickase will leave two single-strand nicks on both strands in close proximity, creating a double-strand break with 5’ overhangs on both strands. This leads to NHEJ or HDR at this breaking site, while other off-target single-strand nicks will be repaired without impact. This strategy doubles the number of base pairs required for site recognition, reducing off-target possibilities almost to zero (from about 1 off-target in 30 million bp to 1 in 1000 trillion bp).Application of the CRISPR-Cas9 System in Biomedical Sciences The biggest advantage of the CRISPR-Cas9 system is its ability to edit genes in almost any cell type and any ani-mal model

1	trillion bp).Application of the CRISPR-Cas9 System in Biomedical Sciences The biggest advantage of the CRISPR-Cas9 system is its ability to edit genes in almost any cell type and any ani-mal model with high efficiency and accuracy. Plus, it is easy to design and easy to use. Within a couple of years, success-ful gene editing in C. elegans,51 zebrafish,52 fruit fly,53 mouse,54 dogs,55 and even nonhuman primates56 was achieved. CRISPR-Cas9 was also reported to be successful in a variety of cell types including stem cells.Currently, CRISPR-Cas9 is most used for editing single genes, through gene knockout, gene mutation, or the addition of an epitope tag to a native gene, for functional characterization of the gene of interest. For example, oncogenes or tumor suppres-sor genes can be knocked out to identify the causative gene for a particular cancer type; point mutations in functional domains homologous templateprecise editingNHEJ5’3’5’3’5’3’5’3’5’3’3’5’3’5’3’5’3’5’3’5’3’Cas9 RuvC

1	out to identify the causative gene for a particular cancer type; point mutations in functional domains homologous templateprecise editingNHEJ5’3’5’3’5’3’5’3’5’3’3’5’3’5’3’5’3’5’3’5’3’Cas9 RuvC domainHNHdomainPAMsgRNA5’5’3’HDRRandom insertion/deletionBrunicardi_Ch15_p0479-p0510.indd 50718/02/19 11:12 AM 508BASIC CONSIDERATIONSPART Imay illustrate the mechanism of action of a protein; for proteins without available antibodies, epitope tags can be inserted onto the native gene for the detection of the native protein.Apart from single gene editing, CRISPR-Cas9 can be used for large scale loss-of-function gene screen. Multiple lentiviral guide RNA libraries have been established covering the whole human/mouse genome or particular subsets.Owing to its ability to bind to specific nucleotide sequences, CRISPR-Cas9 has also been used for non–gene-editing purposes. To achieve this, both the RuvC-like and the HNH-like domains are mutated to give a catalytically inactive Cas9 (dCas9).

1	sequences, CRISPR-Cas9 has also been used for non–gene-editing purposes. To achieve this, both the RuvC-like and the HNH-like domains are mutated to give a catalytically inactive Cas9 (dCas9). Directed by guide RNA, this dCas9 can bind to particu-lar genes to reversibly suppress or activate gene transcription by the fusion of transcription activators or suppressors with dCas9. Epigenetic modulators (e.g., DNA methylase) can also be fused with dCas9 to achieve controlled epigenetic modulations. More-over, dCas9 can also be fused with fluorescent markers such as GFP to track a particular DNA region in live cells.The most exciting potential application of the CRISPR-Cas9 system is perhaps the correction or modification of dis-ease-causing genes in human embryos or in human patients to eradicate disease-causing genes. However, extension of this application may lead to the creation of the so called “perfect human,” hence raising huge ethical concerns and controversies.57 Because of such

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1	genome editing and ethical considerations. Sci Eng Ethics. 2016;22(2):597-599. 58. Ma H, Marti-Gutierrez N, Park SW, et al. Correction of a pathogenic gene mutation in human embryos. Nature. 2017; 548(7668):413-419. 59. Ledford H. CRISPR fixes disease gene in viable human embryos. Nature. 2017;548(7665):13-14. 60. Ormond KE, Mortlock DP, Scholes DT, et al. Human germline genome editing. Am J Hum Genet. 2017;101(2):167-176.Brunicardi_Ch15_p0479-p0510.indd 50918/02/19 11:12 AM

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1	The Skin and Subcutaneous TissuePatrick Harbour and David H. Song 16chapterINTRODUCTIONThe skin is a complex organ encompassing the body’s surface and is continuous with the mucous membranes. Accounting for approximately 15% of total body weight, it is the largest organ in the human body. Enabled by an array of tissue and cell types, intact skin protects the body from external insults. However, the skin is also the source of a myriad of pathologies that include inflammatory disorders, mechanical and thermal injuries, infec-tious diseases, and benign and malignant tumors. The intrica-cies and complexities of this organ and associated pathologies are reasons the skin and subcutaneous tissue remain of great interest and require the attention of various surgical disciplines that include plastic surgery, dermatology, general surgery, and surgical oncology.ANATOMY AND HISTOLOGYBackgroundIt is important that surgeons understand completely the cutane-ous anatomy and its variability as they

1	surgery, dermatology, general surgery, and surgical oncology.ANATOMY AND HISTOLOGYBackgroundIt is important that surgeons understand completely the cutane-ous anatomy and its variability as they play an enormous role in patient health and satisfaction. The skin is made up of tissues derived from both the ectodermal and mesodermal germ cell layers.1 Three distinct tissue layers comprise the organ, and differ in composition based on location, age, sex, and ethnicity, among other variables. The outermost layer is the epidermis, which is predominantly characterized by a protective, highly keratinized layer of cells. The next layer is the dermis, which is made up of an organized collagen network to support the numerous epider-mal appendages, neurovascular structures, and supportive cells within the skin. The fatty layer below the dermis is collectively known as the hypodermis and functions in body processes of thermoregulation and energy storage, among others. These three distinct layers

1	the skin. The fatty layer below the dermis is collectively known as the hypodermis and functions in body processes of thermoregulation and energy storage, among others. These three distinct layers function together harmoniously and participate in numerous activities essential to life.2EpidermisThe epidermis is the outermost layer of the cutaneous tissue, and consists primarily of continually regenerating keratinocytes. The tissue is also stratified, forming four to five histologically distinct layers, depending on the location in the body. These layers are, from deep to superficial, the stratum basale, stratum spinosum, stratum granulosum, stratum lucidum and stratum corneum (Fig. 16-1). The different layers of the epidermis represent layers of keratinocytes at differing stages of their approximately thirty-day life cycle. A minority of other cell types are found in different layers of the epidermis as well. Some of these cells are permanent residents, while others are visitors from

1	approximately thirty-day life cycle. A minority of other cell types are found in different layers of the epidermis as well. Some of these cells are permanent residents, while others are visitors from other parts of the body. All the epidermal appendages, such as sweat glands and pilosebaceous follicles, are derived from this tissue. The thickness of the epidermis is quite variable with regard to location and age, ranging from 75 to 150 µm in thin skin (eyelids) to 0.4 to 1.5 mm in thick skin (palms and soles).2 The epidermis lacks any vascular Introduction513Anatomy and Histology513Background / 513Epidermis / 513Epidermal Components / 514Epidermal Appendages / 515Dermal Components / 516Cells / 516Cutaneous Vasculature / 516Cutaneous Innervation / 517Hypodermis / 517Inflammatory Conditions517Hidradenitis Suppurativa / 517Pyoderma Gangrenosum / 517Epidermal Necrolysis / 517Injuries518Radiation-Induced Injuries / 518Trauma-Induced Injuries / 519Caustic Injury / 520Thermal Injury /

1	Suppurativa / 517Pyoderma Gangrenosum / 517Epidermal Necrolysis / 517Injuries518Radiation-Induced Injuries / 518Trauma-Induced Injuries / 519Caustic Injury / 520Thermal Injury / 521Pressure Injury / 523Bioengineered Skin Substitutes524Bacterial Infections of the Skin and Subcutaneous Tissue524Introduction / 524Uncomplicated Skin Infections / 524Complicated Skin Infections / 524Actinomycosis / 526Viral Infections with Surgical Implications526Human Papillomavirus Infections / 526Cutaneous Manifestations of Human Immunodeficiency Virus / 527Benign Tumors527Hemangioma / 527Nevi / 527Cystic Lesions / 527Keratosis / 528Soft Tissue Tumors / 528Neural Tumors / 528Malignant Tumors528Basal Cell Carcinoma / 528Squamous Cell Carcinoma / 529Melanoma / 530Merkel Cell Carcinoma / 534Kaposi’s Sarcoma / 535Dermatofibrosarcoma Protuberans / 535Malignant Fibrous Histiocytoma (Undifferentiated Pleomorphic Sarcoma and Myxofibrosarcoma) / 535Angiosarcoma / 535Extramammary Paget’s Disease /

1	Sarcoma / 535Dermatofibrosarcoma Protuberans / 535Malignant Fibrous Histiocytoma (Undifferentiated Pleomorphic Sarcoma and Myxofibrosarcoma) / 535Angiosarcoma / 535Extramammary Paget’s Disease / 536Conclusion536Brunicardi_Ch16_p0511-p0540.indd 51319/02/19 3:08 PM 514Hair shaftStratum corneumPigment ligamentStratum germinativumStratum spinosumStratum basaleArrector pili muscleSebaceous glandHair folliclePapilla of hairBlood andlymph vesselsNerve ÿberSweatporeDermalpapillaSensory nerve ending for touchEpidermisDermisSubcutis(hypodermis)VeinArteryPaciniancorpuscleSweatglandFigure 16-1. Schematic representation of the skin and its appendages. Note that the root of the hair follicle may extend beneath the dermis into the subcutis.structures and obtains all nutrients from the dermal vasculature by diffusion.3Epidermal ComponentsKeratinocytes. Keratinocytes typically make up about 90% of the cells of the epidermis. These cells have four to five distinct stages in their life cycle, each

1	by diffusion.3Epidermal ComponentsKeratinocytes. Keratinocytes typically make up about 90% of the cells of the epidermis. These cells have four to five distinct stages in their life cycle, each visibly different under light microscopy. The stratum basale, or germinative layer, is a deep, single layer of asynchronous, continuously rep-licating cuboidal to columnar epithelial cells and is the 1beginning of the life cycle of the keratinocytes of the epidermis. This layer is bound to its basement membrane by complexes made of keratin filaments and anchoring structures called hemidesmosomes. They are bound to other keratinocytes by structures called desmosomes. High mitotic activity and thus large nuclei and basophilic staining characterize the stratum basale on light microscopy. This layer also lines the epidermal appendages that reside largely within the substance of the der-mis and later serves as a regenerative source of epithelium in the event of partial thickness wounds.Key

1	layer also lines the epidermal appendages that reside largely within the substance of the der-mis and later serves as a regenerative source of epithelium in the event of partial thickness wounds.Key Points1 The epidermis consists of continually regenerating strati-fied epithelium, and 90% of cells are ectodermally derived keratinocytes.2 Pilosebaceous units are lined by the germinal epithelium of the epidermis and thus serve as an important source of epidermal regeneration after partial-thickness injury or split-thickness skin graft.3 Dermal fibers are predominantly made of type I and III collagen in a 4:1 ratio. They are responsible for the mechanical resistance of skin.4 The drugs most commonly associated with epidermal necrolysis include aromatic anticonvulsants, sulfonamides, allopurinol, oxicams (nonsteroidal anti-inflammatory drugs), and nevirapine.5 In wounds being allowed to heal secondarily, negative pressure wound therapy can increase the rate of granula-tion tissue

1	allopurinol, oxicams (nonsteroidal anti-inflammatory drugs), and nevirapine.5 In wounds being allowed to heal secondarily, negative pressure wound therapy can increase the rate of granula-tion tissue formation.6 Staphylococcus aureus is the most common isolate of all skin infections. Impetigo, cellulitis, erysipelas, folliculitis, furuncles, and simple abscesses are examples of uncompli-cated infections, whereas deep-tissue infections, extensive cellulitis, necrotizing fasciitis, and myonecrosis are exam-ples of complicated infections.7 Hemangiomas arise from benign proliferation of endothe-lial cells surrounding blood-filled cavities. They most commonly present after birth, rapidly grow during the first year of life, and gradually involute in most cases.8 Basal cell carcinoma represents the most common tumor diagnosed in the United States, and the nodular variant is the most common subtype. The natural progression of basal cell carcinoma is one of local invasion rather than distant

1	the most common tumor diagnosed in the United States, and the nodular variant is the most common subtype. The natural progression of basal cell carcinoma is one of local invasion rather than distant metastasis.9 Squamous cell carcinoma is the second most common skin cancer, and typically arises from an actinic keratosis precur-sor. Primary treatment modalities are surgical excision and Mohs microsurgery. Cautery and ablation, cryotherapy, drug therapy, and radiation therapy are alternative treatments.10 Tumor thickness, ulceration, and mitotic rate are the most important prognostic indicators of survival in melanoma. Sentinel lymph node biopsy is often used to stage indi-viduals with biopsy-proven high risk melanoma and clini-cally node-negative disease.Brunicardi_Ch16_p0511-p0540.indd 51419/02/19 3:08 PM 515THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16The next layer is the stratum spinosum, or “spiny” layer. This layer is from five to fifteen cells in thickness and is so named due

1	51419/02/19 3:08 PM 515THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16The next layer is the stratum spinosum, or “spiny” layer. This layer is from five to fifteen cells in thickness and is so named due to the spinous appearance of the intercellular des-mosomal attachments under light microscopy. The production of keratin in this cell layer is responsible for their eosinophilic appearance on hematoxylin and eosin (H&E) staining.As the keratinocytes continue to migrate superficially, they begin to flatten and develop basophilic keratohyalin gran-ules. There are also structures called lamellar granules within these cells that contain the lipids and glycolipids that will ulti-mately undergo exocytosis to produce the lipid layer around the cells. It is in this layer that the keratinocytes manufacture many of the structures that will eventually serve to protect the skin and underlying tissues from environmental insult.4 At the super-ficial aspect of this layer, the keratinocytes begin to

1	many of the structures that will eventually serve to protect the skin and underlying tissues from environmental insult.4 At the super-ficial aspect of this layer, the keratinocytes begin to undergo programmed cell death, losing all cellular structures except for the keratin filaments and their associated proteins. In thick skin, such as that found on the palms and soles, there is a layer of flat, translucent keratinocytes called the stratum lucidum.The final stage of the keratinocyte life cycle results in the layer of the epidermis known as the stratum corneum, or cor-nified layer. The protein-rich, flattened keratinocytes are now anucleate and surrounded by a lipid-rich matrix. Together the cells and surrounding matrix of this layer serve to protect the tissue from mechanical, chemical, and bacterial disruption while preventing insensible water losses through the skin.4,5Langerhans Cells. Of the cells in the epidermis, 3% to 6% are immune cells known as Langerhans cells.6 Typically

1	and bacterial disruption while preventing insensible water losses through the skin.4,5Langerhans Cells. Of the cells in the epidermis, 3% to 6% are immune cells known as Langerhans cells.6 Typically found within the stratum spinosum, these mobile, dendritic cells inter-digitate between keratinocytes of the epidermis to create a dense network, sampling any antigens that attempt to pass through the cutaneous tissue. Through use of their characteristic rodor racket-shaped Birbeck granules, they take up antigens for pre-sentation to T-cells.7 These monocyte-derived cells represent a large part of the skin’s adaptive immunity. Because of the effec-tiveness of their antigen presentation, Langerhans cells could be utilized as vaccine vehicles in the future.8 The Langerhans cells are functionally impaired by UV radiation, specifically UVB radiation, and may play a role in the development of cutaneous malignancies after UV radiation exposure.9Melanocytes. Within the stratum basale are

1	impaired by UV radiation, specifically UVB radiation, and may play a role in the development of cutaneous malignancies after UV radiation exposure.9Melanocytes. Within the stratum basale are melanocytes, the cells responsible for production of the pigment melanin in the skin. These neural crest-derived cells are present in a density of four to ten keratinocytes per melanocytes, and about 500 to 2000 melanocytes per mm2 of cutaneous tissue. This density varies based on location in the body, but differences in skin pig-mentation are based on the activity of individual melanocytes and not the number of melanocytes. In darker-skinned ethnici-ties, melanocytes create and store melanosomes in keratinocytes at a higher rate, but still have a pale-staining cytoplasm on light microscopy. Hemidesmosomes also attach these cells to the basement membrane, but the intercellular desmosomal connec-tions are not present. The melanocytes interact with keratino-cytes of the stratum basale and spinosum

1	also attach these cells to the basement membrane, but the intercellular desmosomal connec-tions are not present. The melanocytes interact with keratino-cytes of the stratum basale and spinosum via long cytoplasmic extensions leading to invaginations in several keratinocytes. Tyrosinase is created and distributed into melanosomes, and these organelles travel along the dendritic processes to eventu-ally become phagocytized by keratinocytes and distributed in a supranuclear orientation. This umbrella-like cap then serves to protect the nuclear material from damage by radiation; this could explain why light-skinned ethnicities are more prone to the development of cutaneous malignancies.10,11 Melanocytes express the bcl-2 protein, S100 protein, and vimentin, which are important in the pathology and histologic diagnosis of disorders of melanocytes.Merkel Cells. Merkel cells are slow-adapting mechanorecep-tors of unclear origin essential for light touch sensation. Thus, they typically

1	and histologic diagnosis of disorders of melanocytes.Merkel Cells. Merkel cells are slow-adapting mechanorecep-tors of unclear origin essential for light touch sensation. Thus, they typically aggregate among basal keratinocytes of the skin in areas where light tactile sensation is warranted, such as the digits, lips, and bases of some hair follicles.12-14 They are joined to keratinocytes in the basal layer by desmosomes and have dense neurosecretory granules containing peptides. These neu-rosecretory granules allow communication with the CNS via afferent, unmyelinated nerve fibers that contact the basolateral portion of the cell via expanded terminal discs.3 The clinical significance of Merkel cells arises in the setting of Merkel cell carcinoma, a rare, but difficult-to-treat malignancy.Lymphocytes. Less than 1% of the cells in the epidermis are lymphocytes, and these are found primarily within the basal layer of keratinocytes. They typically express an effector memory T-cell

1	than 1% of the cells in the epidermis are lymphocytes, and these are found primarily within the basal layer of keratinocytes. They typically express an effector memory T-cell phenotype.15,16Toker Cells. Toker cells are found in the epidermis of the nip-ple in 10% of both males and females and were first described in 1970. While distinct from Paget’s cells, immunohistochemical studies have implicated them as a possible source of Paget’s disease of the nipple.17-20Epidermal AppendagesSweat Glands. Sweat glands, like other epidermal appendages, are derived from the embryologic ectoderm, but the bulk of their substance resides within the dermis. Their structure consists of a tubular-shaped exocrine gland and excretory duct. Eccrine sweat glands make up a majority of the sweat glands in the body and are extremely important to the process of thermoregu-lation. Solutes are released into the gland via exocytosis. They are present in greatest numbers on the palms, soles, axillae, and forehead.

1	and are extremely important to the process of thermoregu-lation. Solutes are released into the gland via exocytosis. They are present in greatest numbers on the palms, soles, axillae, and forehead. Collectively they produce approximately 10 L/d in an adult. These glands are the most effective means of temperature regulation in humans via evaporative heat loss.A second type of sweat gland, known as the apocrine sweat gland, is found around the axilla, anus, areola, eyelid, and external auditory canal. The cells in this gland undergo an excretion process that involves decapitation of part of the cell. These apocrine glands are typically activated by sex hormones and thus activate around the time of puberty. The secretion from apocrine glands is initially odorless, but bacteria in the region may cause an odor to develop. Pheromone production may have been a function of the apocrine glands, but this may now be vestigial. While eccrine sweat glands are activated by the cho-linergic system,

1	may cause an odor to develop. Pheromone production may have been a function of the apocrine glands, but this may now be vestigial. While eccrine sweat glands are activated by the cho-linergic system, apocrine glands are activated by the adrenergic system.There is also a third type of sweat gland called apoeccrine. This is similar to an apocrine gland but opens directly to the skin surface and does not present until puberty. 21 Both types of glands are surrounded by a layer of myoepithelial cells that can contract and assist in the excretion of glandular contents to the skin surface.Pilosebaceous Units. A pilosebaceous unit is a multicompo-nent unit made up of a hair follicle, sebaceous gland, an erector pili muscle, and a sensory organ. These units are responsible for the production of hair and sebum and are present almost entirely Brunicardi_Ch16_p0511-p0540.indd 51519/02/19 3:08 PM 516SPECIFIC CONSIDERATIONSPART IIthroughout the body, sparing the palms, soles, and mucosa. They

1	and sebum and are present almost entirely Brunicardi_Ch16_p0511-p0540.indd 51519/02/19 3:08 PM 516SPECIFIC CONSIDERATIONSPART IIthroughout the body, sparing the palms, soles, and mucosa. They are lined by the germinal epithelium of the epidermis and thus serve as an important source of epidermal regenera-tion after partial-thickness injury or split-thickness skin graft. The sebaceous glands secrete sebum into the follicle and skin via a duct. The lipid-secreting glands are largely influenced by androgens and become functionally active during puberty. They are present in greatest numbers on the face and scalp.Nails. The nails are keratinaceous structures overlying the dis-tal phalanges of the fingers and toes. The nail is made of three main parts. The proximal portion of the nail, continuous with the germinal nail matrix, is the nail root. The root is an adher-ence point for the nail. The nail plate is the portion of the nail that lies on top of the nail bed, the shape of which is

1	with the germinal nail matrix, is the nail root. The root is an adher-ence point for the nail. The nail plate is the portion of the nail that lies on top of the nail bed, the shape of which is determined by the underlying phalanx. The third part of the nail is the free edge, which overlies a thickened portion of epidermis known as the hyponychium. The nail functions to protect the distal digits and augment the function of the pulp of the digits as a source of counter-pressure.Dermal ComponentsArchitecture. The dermis is a mesoderm-derived tissue that protects and supports the epidermis while anchoring it to the underlying subcutaneous tissue. It consists primarily of three unique components: a fibrous structure, the ground substance that surrounds those fibers, and the cell population that is sup-ported by the dermis. In addition, the dermis houses the neuro-vasculature that supports the epidermis and facilitates interaction with the outward environment, as well as the epidermal

1	that is sup-ported by the dermis. In addition, the dermis houses the neuro-vasculature that supports the epidermis and facilitates interaction with the outward environment, as well as the epidermal append-ages previously described. The dermis varies in thickness based upon body region, thinnest in the eyelids and reaching a thick-ness of up to 4 mm on the back, and is composed of two distinct layers, the papillary layer and the reticular layer. The papillary layer is made up of papillae that interdigitate with the rete ridges of the deep portion of the epidermis. This structure increases the surface area between the dermis and epidermis, increasing the resistance to shear forces as well as facilitating greater diffusion of nutrients across the dermal-epidermal junction. The papil-lary layer is characterized by a greater density of cells, and the reticular layer is almost entirely made up of a coarse network of fibers and the ground substance that surrounds it.Fibers and Ground

1	layer is characterized by a greater density of cells, and the reticular layer is almost entirely made up of a coarse network of fibers and the ground substance that surrounds it.Fibers and Ground Substance. Ninety-eight percent of the dry weight of the dermis is made up of collagen, typically 80% to 90% type I collagen and 8% to 12% type III collagen. Collagen types IV and VII are also found in much smaller quantities in the dermo-epidermal junction. The structure of the fibers varies along the depth of the dermis. At the superficial part of the dermis, in the papillary layer, the collagen bundles are arranged more loosely and are primarily made up of type III collagen.22 Deeper in the reticular layer of the dermis, the col-lagen fibrils are larger in diameter and organized into interwo-ven bundles surrounded by elastic fibers all within the hydrated ground substance. In a healthy adult, these dermal fibers are in a constant state of breakdown and production, dictated by the activity

1	bundles surrounded by elastic fibers all within the hydrated ground substance. In a healthy adult, these dermal fibers are in a constant state of breakdown and production, dictated by the activity of matrix metalloproteases and fibroblasts, respectively. The activity of the MMPs is induced by UV radiation, thus lead-ing to increased degradation and disorganization of the collagen fibers, resulting in wrinkling and weakening of the dermis in sun-exposed areas.The retractile properties of skin are due in part to elas-tic fibers found throughout the dermis. These fibers, like the collagen fibers, are thinner and more perpendicularly oriented in the papillary dermis and become thicker and parallel in the reticular dermis. These elastic fibers are also produced by fibro-blasts, but they are unique in that they can stretch to twice their original length, and return to their original configuration. The elastic fibers are also in a constant state of turnover that can be negatively impacted by

1	in that they can stretch to twice their original length, and return to their original configuration. The elastic fibers are also in a constant state of turnover that can be negatively impacted by the effects of UV radiation.The fibrous network of the dermis lies within a hydrated amorphous ground substance made of a variety of proteoglycans and glycosaminoglycans, molecules that can contain up to 1000 times their weight in water. This ground substance facilitates the development of the structure of the dermis and cell migration within the dermis. It also assists in redistributing forces placed on the cutaneous tissues.CellsFibroblasts. Fibroblasts, like most cells in the dermis, are found in the loose, papillary layer, and are the fundamental cells of the dermis. They are responsible for producing all der-mal fibers and the ground substance within which those fibers reside. They are typically spindleor stellate-shaped and have a well-developed rough endoplasmic reticulum, typical of

1	producing all der-mal fibers and the ground substance within which those fibers reside. They are typically spindleor stellate-shaped and have a well-developed rough endoplasmic reticulum, typical of cells engaging in active protein production. The fibroblasts can also differentiate into myofibroblasts, cell types that harbor myofila-ments of smooth muscle actin and, more rarely, desmin, which help to decrease the surface area of the wound by contraction.23 Because of these fundamental functions of fibroblasts, they are the workhorses of wound healing, while macrophages are the orchestrators.Dermal Dendrocytes. Dermal dendrocytes are comprised of a variety of mesenchymal dendritic cells recognizable mainly by immunohistochemistry. They are responsible for antigen uptake and processing for presentation to the immune system, as well as the orchestration of processes involved in wound healing and tissue remodeling. They are typically found in the papillary dermis around vascular

1	for presentation to the immune system, as well as the orchestration of processes involved in wound healing and tissue remodeling. They are typically found in the papillary dermis around vascular structures as well as sweat glands and pilosebaceous units.Mast Cells. Mast cells are effector secretory cells of the immune system that are responsible for immediate type 1 hyper-sensitivity reactions. When primed with IgE antibodies, encoun-ter with a provoking antigen causes the release of histamine and cytokines, leading to vasodilation and dermatitis commonly seen in allergic reactions.Cutaneous VasculatureWhile the epidermis is void of any vasculature structures, the dermis has a rich supply of blood and nutrients supported by paired plexuses connected by a system of arteriovenous shunts. The superficial, subpapillary plexus is located between the papillary and reticular dermis and provides a vascular loop to every papilla of the papillary dermis.24 The deep dermal plexus is located at

1	superficial, subpapillary plexus is located between the papillary and reticular dermis and provides a vascular loop to every papilla of the papillary dermis.24 The deep dermal plexus is located at the junction of the reticular dermis and hypodermis, and it derives its blood supply from perforating arteries of larger vessels below the cutaneous tissues. The arteriovenous shunts connecting the two horizontal plexuses can divert blood flow to or away from the skin when necessary to conserve or release body heat, or to divert blood flow to vital organs when needed. Associated with the vascular loops of the dermal papillae are the blind-ended beginnings of lymphatic vessels, which serve to transport extravasated fluid and proteins from the soft tissues back into the venous circulatory system.23Brunicardi_Ch16_p0511-p0540.indd 51619/02/19 3:08 PM 517THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16Cutaneous InnervationThe skin is a highly specialized tool for interacting with our environment

1	51619/02/19 3:08 PM 517THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16Cutaneous InnervationThe skin is a highly specialized tool for interacting with our environment and, as such, carries a rich network of nervous tis-sue to facilitate this purpose. An afferent component made up of free nerve endings and specialized corpuscular receptors is responsible for conveying to our brain information about the environment, while numerous functions of the cutaneous tis-sues, such as AV-shunting, piloerection, and sweat secretion are controlled by the myelinated and unmyelinated fibers of an efferent component of the CNS.25HypodermisThe hypodermis, or subcutaneous tissue, is a richly vascularized loose connective tissue that separates and attaches the dermis to the underlying muscle and fascia. It is made up primarily of pockets of lipid-laden adipocytes separated by septae that contain cellular components similar to the dermis, neurovas-cular structures supplying the cutaneous tissue, and the

1	is made up primarily of pockets of lipid-laden adipocytes separated by septae that contain cellular components similar to the dermis, neurovas-cular structures supplying the cutaneous tissue, and the deepest parts of sweat glands.26 The hypodermis serves multiple func-tions—namely insulation, storage of energy, and protection from mechanical forces, allowing the skin to glide over the underlying tissues.INFLAMMATORY CONDITIONSHidradenitis SuppurativaHidradenitis suppurativa, also known as acne inversa, is a pain-ful skin condition typically affecting areas of the body bear-ing apocrine glands—typically the axillae, perineum, and the inframammary and inguinal folds. It is characterized by tender, deep nodules that can expand, coalesce, spontaneously drain, and form persistent sinus tracts in some cases leading to sig-nificant scarring and hyperkeratosis. There can be superimposed bacterial infection during episodic flares of the disease as well. In women, flares often occur

1	tracts in some cases leading to sig-nificant scarring and hyperkeratosis. There can be superimposed bacterial infection during episodic flares of the disease as well. In women, flares often occur premenstrually.Hidradenitis suppurativa typically affects females (female to male ratio of 3:1), most commonly during the third decade of life and has demonstrated associations with smoking and obesity.27 While the etiology of hidradenitis is incompletely understood, it is thought to be the consequence of a genetic pre-disposition exacerbated by environmental factors. About one-third of affected patients endorse a family history of the disease. A specific gene locus has not been identified, but mutations in the γ-secretase gene have been linked to the disease in some familial cases.28 The histologic progression of the disease is characterized by atrophy of the sebaceous gland, followed by inflammation of the pilosebaceous unit from both the innate and adaptive immune systems, causing

1	histologic progression of the disease is characterized by atrophy of the sebaceous gland, followed by inflammation of the pilosebaceous unit from both the innate and adaptive immune systems, causing hyperkeratosis and eventual granuloma forma-tion.29 Some studies have shown involvement of the IL12-IL23 pathway and TNF-α, supporting the theory that the disease is at least in part caused by an inflammatory disorder.30,31The diagnosis of hidradenitis is clinical, and the presenta-tion is most commonly categorized by the Hurley classification system, divided into three stages. Single or multiple nodules or abscesses without any sinus tracts or scarring would be classi-fied as stage 1 disease. As abscesses recur and sinus tracts and scarring form, the disease is classified as Hurley stage 2. Stage 3 is the most advanced stage, with diffuse disease and intercon-nected sinus tracts and abscesses.Treatment is typically based on Hurley staging, with topi-cal and systemic antibiotics

1	2. Stage 3 is the most advanced stage, with diffuse disease and intercon-nected sinus tracts and abscesses.Treatment is typically based on Hurley staging, with topi-cal and systemic antibiotics (typically clindamycin) being used for stage I and II disease,32 while radical excision, laser treat-ment, and biologic agents are reserved for more advanced stage II and III disease.33-36 Even with complete surgical resection, recurrence rates are still high, reaching up to 50% in the infra-mammary and inguino-perineal regions. Because of increased risks of recurrence with primary closure, it is preferable to pur-sue other methods of wound closure, like split-thickness skin grafting, local or regional flaps, and healing by secondary inten-tion. Topical antimicrobial creams should be used during the healing process.Pyoderma GangrenosumPyoderma gangrenosum is an uncommon inflammatory con-dition of the skin characterized by the development of sterile pustules which progress to painful,

1	during the healing process.Pyoderma GangrenosumPyoderma gangrenosum is an uncommon inflammatory con-dition of the skin characterized by the development of sterile pustules which progress to painful, ulcerating lesions with purple borders. This disease is typically diagnosed between the ages of 40 and 60 years and has a slightly higher prevalence in females. Although the exact etiology is currently unknown, it typically arises in individuals with a hematologic malignancy or inflammatory disorder, such as inflammatory bowel disease or rheumatoid arthritis. The most commonly affected sites are the legs, but lesions can occur anywhere. Extracutaneous mani-festations are also possible, and it can affect mucosal tissue and solid organs. While the initial pathology is sterile, it can easily become secondarily infected. The diagnosis of this condition is based upon history and clinical presentation after the exclu-sion of infectious etiologies. There are five distinct types of pyoderma

1	become secondarily infected. The diagnosis of this condition is based upon history and clinical presentation after the exclu-sion of infectious etiologies. There are five distinct types of pyoderma gangrenosum described: vegetative, pustular, peris-tomal, ulcerative, and bullous. The pathogenesis of this disease is incompletely understood, but it is thought to be a genetic predisposition that is triggered by an environmental influence. An inciting cutaneous injury can often be identified preceding the ulceration. Histopathologic studies have demonstrated sig-nificantly elevated levels of inflammatory cytokines, as well as neutrophils exhibiting aberrant chemotactic signaling.37-39 Treat-ment of pyoderma gangrenosum generally involves treatment of the underlying disorder (i.e., management of Crohn’s disease) as well as systemic anti-inflammatory medications such as steroids or immunosuppressants like calcineurin inhibitors. Patients with Crohn’s disease and PG treated with infliximab

1	of Crohn’s disease) as well as systemic anti-inflammatory medications such as steroids or immunosuppressants like calcineurin inhibitors. Patients with Crohn’s disease and PG treated with infliximab (tumor necrosis factor [TNF]-α inhibitor) and etanercept (TNF-α antagonist) had a marked improvement in their PG.40,41 In cases of peri-stomal pyoderma gangrenosum, topical calcineurin inhibitors have been shown to be useful.42 Concurrent treatment with sys-temic and topical antimicrobials, as well as local wound care, including the debridement of purulent exudate and devitalized tissue, is also beneficial. Surgical therapy without proper sys-temic treatment will generally result in recurrent disease. Final wound closure can be achieved with primary closure or grafts.Epidermal NecrolysisEpidermal necrolysis (EN) is a rare mucocutaneous disorder characterized by cutaneous destruction at the dermoepidermal junction. EN is commonly referred to as either Stevens-Johnson syndrome (SJS) or toxic

1	necrolysis (EN) is a rare mucocutaneous disorder characterized by cutaneous destruction at the dermoepidermal junction. EN is commonly referred to as either Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) depending on the extent of skin involvement present. SJS refers to cases in which <10% of total body surface area is involved, while cases with >30% involvement are considered TEN, with an SJS-TEN overlap syndrome referring to all cases in between. These two disorders are now considered to be the same clinical entity that vary simply on the extent of cutaneous involvement. Erythema multiforme was once considered as part of the clinical subgroup Brunicardi_Ch16_p0511-p0540.indd 51719/02/19 3:08 PM 518SPECIFIC CONSIDERATIONSPART IIFigure 16-2. Blisters on the forearm of a patient several days after exposure to vancomycin. Note the clear antishear dressing and the dark silver-impregnated antimicrobial dressing (Acticoat).encompassing SJS and TEN, but it is now

1	of a patient several days after exposure to vancomycin. Note the clear antishear dressing and the dark silver-impregnated antimicrobial dressing (Acticoat).encompassing SJS and TEN, but it is now thought to be a sepa-rate entity related to herpetic or Mycoplasma infections.The clinical presentation usually occurs within 8 weeks of initiation of a new drug treatment and is characterized by a macular rash beginning in the face and trunk and progressing to the extremities within hours to days. A positive Nikolsky sign is often present, in which lateral pressure on the skin causes separation of the epidermis from the dermis. (Fig. 16-2). The macular rashes then begin to blister and coalesce, forming bul-lae that eventually burst, leaving partial thickness wounds with exposed dermis. Mucous membrane involvement is seen in 90% of cases and can involve the oral, genital, and ocular mucosa, as well as the respiratory and gastrointestinal tracts. The cutaneous manifestations can also be

1	membrane involvement is seen in 90% of cases and can involve the oral, genital, and ocular mucosa, as well as the respiratory and gastrointestinal tracts. The cutaneous manifestations can also be associated with high fever and pain. It is important to distinguish EN from infectious etiologies like staphylococcal scalded skin syndrome due to their similar clini-cal presentation.While the etiology is not entirely clear, it is well docu-mented to be a reaction to various drugs. While over 100 drugs have been implicated as the inciting agent of EN,43,44 there are a handful of high-risk drugs that account for a majority of the cases.45 The drugs most commonly associated with EN include aromatic anticonvulsants, sulfonamides, allopurinol, oxi-cams (nonsteroidal anti-inflammatory drugs), and nevirap-ine. The pathophysiology is also incompletely understood, but it has generally been accepted that it involves cell-mediated cytotoxicity targeted at keratinocytes and the cytokine-induced

1	and nevirap-ine. The pathophysiology is also incompletely understood, but it has generally been accepted that it involves cell-mediated cytotoxicity targeted at keratinocytes and the cytokine-induced expression of “death-receptors” like Fas-L. Recently, studies have demonstrated greatly increased concentrations of granuly-sin, an apoptotic protein secreted by cytotoxic T cells, within EN lesions, and thus this protein may be implicated in the patho-genesis of EN.46 A genetic component may also exist, and genetic testing before carbamazepine treatment is recommended in people of Han Chinese ancestry to exclude carriers of HLA-B1502.47The prognosis of EN is generally related to the surface area affected and secondary complications of extensive cutane-ous damage, like secondary infections and loss of hemodynamic stability due to increased insensible losses and third spacing of fluid. Modern burnand ICU-care has decreased mortality 4significantly.48 The first principle of management of EN

1	loss of hemodynamic stability due to increased insensible losses and third spacing of fluid. Modern burnand ICU-care has decreased mortality 4significantly.48 The first principle of management of EN is dis-continuation of the offending agent, and in drugs with short half-lives, this can significantly increase chances of survival.49 Other management principles include maintenance of euvolemia, early enteral feeding, and measures to reduce risk of infection. This includes surgical debridement of devitalized tissue, the use of topical antibiotics or antimicrobial dressings, nonadherent dress-ings, or temporary biologic or synthetic grafts until the underly-ing dermis can reepithelialize. The cornea should regularly be inspected with a Wood’s lamp to evaluate for corneal sloughing. The use of systemic corticosteroids in the acute setting is con-troversial as there have been mixed results. Some studies have shown a slowed disease progression when corticosteroid therapy was administered

1	of systemic corticosteroids in the acute setting is con-troversial as there have been mixed results. Some studies have shown a slowed disease progression when corticosteroid therapy was administered early,50 while others showed increased rates of sepsis and overall mortality with no effect on disease progression. IVIG has also been used in an effort to inhibit the Fas-L cytotoxic pathway, with some mixed results. A 2007 meta-analysis of nine IVIG trials concluded that high-dose IVIG improves survival,51 while a large retrospective analysis in 2013 concluded that there was no mortality benefit.52 Other agents, like cyclosporine A, plasmapheresis and anti-TNF-α have been studied with mixed results.48 Recent guidelines out of the United Kingdom confirm that there is still no treatment with clearly demonstrated benefit in the management of EN.53 The cutaneous manifestations of EN generally progress for 7 to 10 days, while reepithelialization gen-erally occurs over 3

1	no treatment with clearly demonstrated benefit in the management of EN.53 The cutaneous manifestations of EN generally progress for 7 to 10 days, while reepithelialization gen-erally occurs over 3 weeks.INJURIESRadiation-Induced InjuriesRadiation injuries can result from exposure to electromag-netic radiation from industrial/occupation applications or, more commonly, from environmental exposure and medical treatments. This is especially true in the continually evolv-ing role of radiation therapy in the multidisciplinary approach to oncologic disease and other skin conditions. In addition to treatment for lymphomas, head and neck squamous cell car-cinomas, and prostate adenocarcinoma, it is often an adjuvant or neoadjuvant component of the surgical treatment of rectal, breast, esophageal, and cervical cancers. Although the new modalities and principles of radiation therapy have allowed for more precise administration of this therapy, there is still collateral damage in the cutaneous

1	and cervical cancers. Although the new modalities and principles of radiation therapy have allowed for more precise administration of this therapy, there is still collateral damage in the cutaneous and visceral tissues sur-rounding the treatment site.Environmental sources of radiation damage are typi-cally from UV radiation. UVC rays are filtered by the ozone layer, so the only UV rays that humans typically encounter are UVA (320–400 nm) and UVB (290–320 nm).54 The amount of exposure to UV radiation is dependent on seasonal, temporal, geographic and environmental variables. Ninety-five percent of the UV rays that reach the earth’s surface are UVA rays. This radiation is less energetic (longer wavelength) than UVB rays and affects the cutaneous tissues differently. UVA waves pen-etrate deeper into the tissues, with 20% to 30% reaching the deep dermis. UVB rays are mostly absorbed in the epidermis, with 70% reaching the stratum corneum, 20% reaching the deep epidermis, and only 10%

1	deeper into the tissues, with 20% to 30% reaching the deep dermis. UVB rays are mostly absorbed in the epidermis, with 70% reaching the stratum corneum, 20% reaching the deep epidermis, and only 10% reaching the papillary dermis. Major chromophores in the cutaneous tissue include nucleic acids, aro-matic amino acids, and melanin.The short-term effects of solar radiation include erythema and pigmentation. The resultant erythema peaks at 6 to 24 hours Brunicardi_Ch16_p0511-p0540.indd 51819/02/19 3:08 PM 519THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16after exposure. The pigmentation occurs differently for UVA and UVB rays. Pigmentation occurs because of photooxidation of melanin by UVA radiation. Partial fading of this pigment change occurs within an hour after exposure, but with higher and repeated doses of UVA, stable residual pigmentation is observed. UVB waves induce neomelanization, increasing the total amount of melanin in the epidermal tissues and resulting in an effect that is

1	repeated doses of UVA, stable residual pigmentation is observed. UVB waves induce neomelanization, increasing the total amount of melanin in the epidermal tissues and resulting in an effect that is observable 72 hours after exposure. The increase in melanin as a result of UVB exposure serves as a protective mechanism to defend the nuclei of the basal keratinocytes from further radiation-induced damage by absorbing the high-energy radiation in future exposures. Long-term effects of exposure to UV radiation can lead to chronic skin changes, such as irregular pigmentation, melasma, postinflammatory pigmentation, and actinic lentigines (sun spots). Lysozyme, an enzyme secreted by cells of the immune system, typically inhibits the activity of collagenase and elastase, playing a role in turnover of the elas-tin and collagen network of the dermis. Long-term exposure to UV radiation increases the activity of lysozyme, thus impairing the natural turnover of these fibers, resulting in a

1	turnover of the elas-tin and collagen network of the dermis. Long-term exposure to UV radiation increases the activity of lysozyme, thus impairing the natural turnover of these fibers, resulting in a disorganized accumulation of elastin, and an increase in the ratio of type III to type I collagen. This results in loss of firmness and resilience of the skin, leading to wrinkles and an aged appearance.The other major source of radiation injury that a surgeon will likely encounter is from therapeutic radiation. The vari-ous forms of radiation work to destroy the replicative potential of the target cells via damage to the nucleic acid structures in the cell. This is typically used to treat oncologic disease, but it can also be used to treat benign disease like eczema, psoria-sis, and keloid scarring at relatively low exposures. While this goal is accomplished, surrounding tissues are also affected and damaged. The most radiosensitive components of the cutane-ous tissue are the basal

1	scarring at relatively low exposures. While this goal is accomplished, surrounding tissues are also affected and damaged. The most radiosensitive components of the cutane-ous tissue are the basal keratinocytes, hair follicle stem cells, and melanocytes. Exposure to this intense radiation results in disorganized, uncontrolled cell death, leading to the release of reactive oxygen species and further damage and inflammation to the surrounding cellular network. Damage to the basal kera-tinocytes and fibroblasts hinders the replicative capacity of the epidermis and dermis, respectively.Acute skin changes to these structures manifest within weeks as erythema, edema, and alopecia. Permanent hyper-pigmentation, tightening, thickening, and fibrosis of the skin become apparent as the tissue attempts to heal. In severe radia-tion injury, there can be complete loss of the epidermis, resulting in partial-thickness wounds and fibrinous exudate. Reepitheli-alization typically occurs 14 days

1	attempts to heal. In severe radia-tion injury, there can be complete loss of the epidermis, resulting in partial-thickness wounds and fibrinous exudate. Reepitheli-alization typically occurs 14 days following initial injury, pro-vided other variables affecting wound healing are optimized (bacterial colonization, nutrition.) Long-term effects include compromise of the functional integrity of the skin secondary to thrombosis and necrosis of capillaries, hypovascularity, telangi-ectasia, ulceration, fibrosis, poor wound healing, and infection. These can present weeks to years after exposure.Treatment of minor radiation injury includes skin mois-turizers and local wound care when appropriate. Severe radia-tion injury may warrant surgical excision and reconstruction with free-tissue transfer from a part of the body unaffected by radiation.Trauma-Induced InjuriesMechanical Injury. Physical disruption of the skin can occur via numerous mechanisms. Treatment of the wound is depen-dent on the

1	a part of the body unaffected by radiation.Trauma-Induced InjuriesMechanical Injury. Physical disruption of the skin can occur via numerous mechanisms. Treatment of the wound is depen-dent on the size of the defect left behind by the insult, any exposed structures that remain in the wound bed, and the pres-ence of contaminating debris or infection. Clean, simple lacera-tions can be irrigated, debrided, and closed primarily. There is no systematic evidence to guide the optimal timing of closure within 24 hours,55 but many surgeons will close primarily within 6 hours of injury. Grossly contaminated or infected wounds should be allowed to heal by secondary intention or delayed primary closure.56 In wounds allowed to heal secondarily, nega-tive pressure wound therapy can increase the rate of granu-lation tissue formation.57 Tangential abrasions are treated similarly to burn wounds, with depth of injury dictating man-agement. Partial thickness injuries with preservation of the regenerative

1	tissue formation.57 Tangential abrasions are treated similarly to burn wounds, with depth of injury dictating man-agement. Partial thickness injuries with preservation of the regenerative pilosebaceous units can be allowed to heal on their own while maintaining a moist, antimicrobial wound environ-ment. Full thickness wounds may require reconstruction with splitor full-thickness skin grafting depending on the size of the defect and the need for future cosmesis and durability. In the setting of devitalization of full thickness tissue, the damaged tissue may be used as a full thickness graft, provided the wound is appropriately cleaned.Bite Wounds. Dog bites alone recently accounted for 4.5 million bites to humans in a single year. Bites from dogs, humans, and other animals can quickly lead to severe deep-tissue infections if not properly recognized and treated.58 The most com-mon location of bite wounds is the hand. This area is of particular importance, as the anatomy of the hand

1	lead to severe deep-tissue infections if not properly recognized and treated.58 The most com-mon location of bite wounds is the hand. This area is of particular importance, as the anatomy of the hand allows for rapid pro-gression of deep infection long relatively avascular structures and can lead to long term morbidity if not adequately treated.59 Bite bacteriology is influenced by normal mouth flora, as well as the content of the offending animal’s diet. Early presentation bite wounds yield polymicrobial cultures, while cultures from a late infection will typically exhibit one dominant pathogen. Common aerobic bacteria include Pasteurella multocida, Streptococcus, Staphylococcus, Neisseria, and Corynebacterium; anaerobic organisms include Fusobacterium, Porphyromonas, Prevotella, Propionibacterium, Bacteroides, and Peptostreptococcus. Capnocytophaga canimorsus bacteria after a dog bite are rare, and it appears that immunocompromised patients are most susceptible to this type of

1	Bacteroides, and Peptostreptococcus. Capnocytophaga canimorsus bacteria after a dog bite are rare, and it appears that immunocompromised patients are most susceptible to this type of infection and its complications. The bacterial load in dog bites is heavily influenced by the last meal of the animal, increasing with wet food and shorter time since the last meal60 (Fig. 16-3). Cat bite bacteriology is similar, with slightly higher prevalence of Pasturella species. Infections from Francisella tularensis (tularemia) and Yersinia pestis (human plague) have been reported.Bacteria colonizing human bites are those present on the skin or in the mouth. These include the gram-positive aerobic organisms Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus species, and anaerobes including Peptococ-cus species, Peptostreptococcus species, Bacteroides species, and Eikenella corrodens (facultative anaerobe). Human bites are characterized by a higher bacterial load (>105).

1	including Peptococ-cus species, Peptostreptococcus species, Bacteroides species, and Eikenella corrodens (facultative anaerobe). Human bites are characterized by a higher bacterial load (>105). Antibiotic prophylaxis after a human bite is recommended as it has been shown to significantly decrease the rate of infection.61 A course of 3 to 7 days of amoxicillin/clavulanate is typically used. Alter-natives are doxycycline or clindamycin with ciprofloxacin.There is controversy over the closure of bite wounds. Typically, in areas of aesthetic importance, the wound is thor-oughly irrigated and debrided and primarily closed with a short course of antibiotics and close follow-up to monitor for signs of infection. In areas that are less cosmetically sensitive and bites that look grossly contaminated or infected, the wounds 5Brunicardi_Ch16_p0511-p0540.indd 51919/02/19 3:08 PM 520SPECIFIC CONSIDERATIONSPART IIABCFigure 16-3. A. Dog bite to the face involving the lip. B. Primary multilayer

1	or infected, the wounds 5Brunicardi_Ch16_p0511-p0540.indd 51919/02/19 3:08 PM 520SPECIFIC CONSIDERATIONSPART IIABCFigure 16-3. A. Dog bite to the face involving the lip. B. Primary multilayer closure following debridement and irrigation. Closure was performed due to aesthetic and functional considerations. C. Follow up 1 week after injury following suture removal.are allowed to close secondarily. Special consideration should be paid to puncture wounds in areas like the hands, which have multiple small compartments. Some groups have found that as long as wounds are properly irrigated and cleansed with povidone iodine solution while a short course of antibiotics is prescribed, there is no difference in infection rates in dog bite wounds closed primarily.62Rabies in domestic animals in the United States is rare, and most cases are contracted from bat bites. In developing countries, dog bites remain the most common source of rabies. Management of this is beyond the scope of this

1	in the United States is rare, and most cases are contracted from bat bites. In developing countries, dog bites remain the most common source of rabies. Management of this is beyond the scope of this chapter.Caustic InjuryChemical burns make up to 10.7% of all burns but account for up to 30% of all burn-related deaths.63 The number of cases of industrial chemical burns is declining while chemical burns in the domestic setting is on the rise. The extent of tissue destruc-tion from a chemical burn is dependent on type of chemical agent, concentration, volume, and time of exposure, among other variables.Injuries from acidic solutions are typically not as severe as those from basic solutions. This is due to the mechanism of injury of each. Acidic injuries typically result in superficial eschar formation because the coagulative necrosis caused by acids limits tissue penetration. Acids can cause thermal injury in addition to the coagulative necrosis due to exothermic reactions. Without

1	formation because the coagulative necrosis caused by acids limits tissue penetration. Acids can cause thermal injury in addition to the coagulative necrosis due to exothermic reactions. Without treatment, acid injuries will progress to erythema and ulcers through the subcutaneous tissue. Injuries from basic solu-tions undergo liquefactive necrosis, unlike acids, and thus have no barrier preventing them from causing deeper tissue injury. Brunicardi_Ch16_p0511-p0540.indd 52019/02/19 3:08 PM 521THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16Figure 16-4. Self-inflicted alkali burn with cleaner fluid.(Fig. 16-4). Common examples of agents that often cause alka-line chemical burns are sodium hydroxide (drain decloggers and paint removers) and calcium hydroxide (cement).Treatment for acidic or alkaline chemical burns is first and foremost centered around dilution of the offending agent, typically using distilled water or saline for 30 minutes for acidic burns and 2 hours for alkaline

1	or alkaline chemical burns is first and foremost centered around dilution of the offending agent, typically using distilled water or saline for 30 minutes for acidic burns and 2 hours for alkaline injuries. Attempting to neutralize the offending agent is typically discouraged, as it does not offer an advantage over dilution and the neutralization reaction could be exothermic, increasing the amount of tissue damage. After removal of the caustic agent, the burn is treated like other burns and is based on the depth of tissue injury. Topical antimicrobials and nonadherent dressings are used for partial-thickness wounds with surgical debridement and reconstruction if needed for full-thickness injuries. Liposuction and saline dilution have been used in cases were injury to deeper structures was suspected.64 Prophylactic use of antibiotics is generally avoided.There are several chemical agents that have specific treat-ments, including the use of calcium gluconate for hydrofluoric acid burns

1	suspected.64 Prophylactic use of antibiotics is generally avoided.There are several chemical agents that have specific treat-ments, including the use of calcium gluconate for hydrofluoric acid burns and polyethylene glycol for phenol burns. These types of treatments are specific to the offending agent and out-side of the scope of this chapter.One type of caustic injury that is commonly seen in the hos-pital is extravasation injury, especially in the setting of chemo-therapeutic administration. Extravasation is estimated to occur in 0.1% to 0.7% of all cytotoxic drug administrations. Like other chemical burns, extravasation injuries depend on properties of the offending agent, time of exposure, concentration, and volume of drug delivered to the tissues. Extravasation injuries typically cause little damage, but they can cause significant morbidity in those with thin skin, fragile veins, and poor tissue perfusion, like neonates and the critically ill. (Fig. 16-5).Initial presentation of

1	little damage, but they can cause significant morbidity in those with thin skin, fragile veins, and poor tissue perfusion, like neonates and the critically ill. (Fig. 16-5).Initial presentation of extravasation injuries usually involves swelling, pain, erythema, and blistering. It may take days or longer for the extent of tissue damage to demarcate. Thorough evaluation to rule out injury to deeper tissues should be conducted. The treatment for extravasation injuries is usu-ally conservative management with limb elevation, but saline aspiration with a liposuction cannula in an effort to dilute and remove the offending agent has been used soon after injury pre-sentation.65 Infiltration of specific antidotes directed toward the offending agent has been described, but it lacks the support of randomized controlled trials, and no consensus in treatment has been reached.66 It is best to avoid cold or warm compression because the impaired temperature regulation of the damaged tissue may lead

1	randomized controlled trials, and no consensus in treatment has been reached.66 It is best to avoid cold or warm compression because the impaired temperature regulation of the damaged tissue may lead to thermal injury. After the wound demarcates, full-thickness skin death should be surgically debrided and man-aged like other wounds based on depth of injury.Thermal InjuryThermal injury involves the damage or destruction of the soft tissue due to extremes of temperature, and the extent of injury is dependent on the degree temperature to which the tissue is exposed and the duration of exposure. The pathophysiology and management are discussed in detail in a separate chapter. Briefly, the management of thermal wounds is initially guided by the concept of three distinct zones of injury. The focus of thermal injury that has already undergone necrosis is known as the zone of coagulation. Well outside the zone of coagulation is the zone of hyperemia, which exhibits signs of inflammation but

1	of thermal injury that has already undergone necrosis is known as the zone of coagulation. Well outside the zone of coagulation is the zone of hyperemia, which exhibits signs of inflammation but Brunicardi_Ch16_p0511-p0540.indd 52119/02/19 3:08 PM 522SPECIFIC CONSIDERATIONSPART IIABCFigure 16-5. A. Potassium chloride intravenous infiltrate in a critically ill patient on multiple vasopressors. B. Following operative debride-ment to paratenon layer. C. Temporary coverage with Integra skin substitute.will likely remain viable. In between these two zones is a zone of stasis with questionable tissue viability, and it is this area at which proper burn care can salvage viable tissue and decrease the extent of injury67 (Fig. 16-6).The mechanisms of injury in hypothermic situation dif-fer. Direct cellular damage can occur as a result of the crys-tallization of intracellular and extracellular components with resultant dehydration of the cell and disruption of lipid protein

1	dif-fer. Direct cellular damage can occur as a result of the crys-tallization of intracellular and extracellular components with resultant dehydration of the cell and disruption of lipid protein Brunicardi_Ch16_p0511-p0540.indd 52219/02/19 3:08 PM 523THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16complexes. During rewarming, further damage occurs because of the shifts of fluid in response to melting ice. Indirect effects of hypothermic injury include microvascular thrombosis and tis-sue ischemia. This, together with subsequent edema and inflam-mation upon rewarming, propagates tissue injury even further.68 Even so, the standard treatment of frostbite injury begins with rapid rewarming to 40°C to 42°C. In addition, further treatment includes debridement of all devitalized tissue, hydrotherapy, elevation, topical antimicrobials, topical antithromboxanes (aloe vera), and systemic antiprostaglandins (aspirin).Pressure InjuryA problem that all surgeons will encounter very early in their

1	elevation, topical antimicrobials, topical antithromboxanes (aloe vera), and systemic antiprostaglandins (aspirin).Pressure InjuryA problem that all surgeons will encounter very early in their careers is pressure necrosis. The development of pressure ulcers is increasingly being regarded as a marker of quality of care, and strategies aimed at prevention have been the source of recent study. Pressure ulcers are known to affect the critically ill (22% to 49% of all critically ill patients are affected), but pressure sources can also affect the chronically bedor wheelchair-bound, patients undergoing surgical procedures, and those with Foley catheters, artificial airways, or other medical equipment (Fig. 16-7).Pressure ulcers can present in several ways depending on the stage at presentation. They are typically grouped into 4 stages: stage 1, nonblanching erythema over intact skin; stage 2, partial-thickness injury with blistering or exposed dermis; stage 3, full-thickness injury

1	They are typically grouped into 4 stages: stage 1, nonblanching erythema over intact skin; stage 2, partial-thickness injury with blistering or exposed dermis; stage 3, full-thickness injury extending down to, but not including, fascia and without undermining of adjacent tissue; and stage 4, full-thickness skin injury with destruction Figure 16-6. Scald burn of upper arm, back, and buttock. Pink areas are superficial partial-thickness burn, whereas whiter areas are deeper burns in the dermis.ABFigure 16-7. A. Pressure wound after removal of a poorly padded cast. Stage cannot be determined until debridement but is at least a grade 2 lesion. B. Decubitus ulcer of the sacral region, stage 4, to the tendinous and bone layers.or necrosis of muscle, bone, tendon, or joint capsule. Tissue destruction occurs most easily at bony prominences due to the inability to redistribute forces along a greater surface area. The average perfusion pressure of the microcirculation is about 30 mmHg, and

1	occurs most easily at bony prominences due to the inability to redistribute forces along a greater surface area. The average perfusion pressure of the microcirculation is about 30 mmHg, and pressures greater than that cause local tissue isch-emia. In animal models, pressure greater than twice the capillary perfusion pressure produces irreversible tissue necrosis in just 2 hours. The most common areas affected are the ischial tuber-osity (28%), greater trochanter (19%), sacrum (17%), and heel (9%). Tissue pressures can measure up to 300 mmHg in the ischial region during sitting and 150 mmHg over the sacrum while lying supine.69 Tissues with a higher metabolic demand are Brunicardi_Ch16_p0511-p0540.indd 52319/02/19 3:09 PM 524SPECIFIC CONSIDERATIONSPART IItypically susceptible to insult from tissue hypoperfusion more rapidly than tissues with a lower metabolic demand. Because of this, it is possible to have muscle necrosis beneath cutaneous tis-sue that has yet to develop signs of

1	from tissue hypoperfusion more rapidly than tissues with a lower metabolic demand. Because of this, it is possible to have muscle necrosis beneath cutaneous tis-sue that has yet to develop signs of irreversible damage.Management of pressure sores first and foremost involves avoidance of prolonged pressure to at-risk areas. Strategies typically employed are pressure-offloading hospital beds or assist devices, patient repositioning every 2 hours, early mobilization, prophylactic silicone dressings, and nurs-ing education.70 From a wound healing perspective, patients should be nutritionally optimized and surgically debrided as appropriate.71,72 The presence of stage III or IV pressure ulcers is not necessarily an indication for surgery, and fevers in a patient with chronic pressure ulcers are often from a urinary or pulmonary source.73-75 Goals of surgical intervention are drain-age of fluid collections, wide debridement of devitalized and scarred tissue, excision of pseudobursa,

1	are often from a urinary or pulmonary source.73-75 Goals of surgical intervention are drain-age of fluid collections, wide debridement of devitalized and scarred tissue, excision of pseudobursa, ostectomy of involved bones, hemostasis, and tension-free closure of dead space with well-vascularized tissue (muscle, musculocutaneous, or fasciocutaneous flaps). Stage 2 and 3 ulcers may be left to heal secondarily after debridement. Subatmospheric pressure wound therapy devices (vacuum-assisted closure) play a role in wound management by removing excess interstitial fluid, promoting capillary circulation, decreasing bacterial coloniza-tion, increasing vascularity and granulation tissue formation, and contributing to wound size reduction.57BIOENGINEERED SKIN SUBSTITUTESThe management of soft tissue defects is more commonly including the use of bioengineered skin substitutes. These products are typically derived from or designed to imitate dermal tissue, providing a regenerative matrix or

1	tissue defects is more commonly including the use of bioengineered skin substitutes. These products are typically derived from or designed to imitate dermal tissue, providing a regenerative matrix or stimulating autogenous dermal regeneration while protecting the underly-ing soft tissue and structures. There are generally four types of skin substitutes: (a) autografts, which are taken from the patient and placed over a soft tissue defect (split-thickness and full-thickness skin grafts); (b) allografts, which are taken from human organ donors; (c) xenografts, which are taken from members of other animal species; and (d) synthetic and semisynthetic biomaterials that are constructed de novo and may be combined with biologic materials.76 Acellular dermal matrices are one type of skin substitute and are used quite often for wound healing and support of soft tissue reconstruction. They are from allogenic or xenogeneic sources and are com-posed of collagen, elastin, laminin, and

1	substitute and are used quite often for wound healing and support of soft tissue reconstruction. They are from allogenic or xenogeneic sources and are com-posed of collagen, elastin, laminin, and glycosaminoglycans. Tissue incorporation generally occurs within 1 to 2 weeks.77 Dermal matrices have been shown to be an effective bridge to split-thickness skin grafting for wounds that have exposed nerves, vessels, tendons, bones, or cartilage.78 Bilayered matri-ces can also be used to promote dermal regeneration in acute or chronic wounds. These products can be temporary, needing to be removed prior to grafting, or permanent, integrating into the host tissue and being grafted directly.BACTERIAL INFECTIONS OF THE SKIN AND SUBCUTANEOUS TISSUEIntroductionIn 1998, the Food and Drug Administration (FDA) categorized infections of the skin and skin structures for the purpose of clini-cal trials. A revision of this categorization in 2010 excluded spe-cific diagnoses such as bite wounds, decubitus

1	(FDA) categorized infections of the skin and skin structures for the purpose of clini-cal trials. A revision of this categorization in 2010 excluded spe-cific diagnoses such as bite wounds, decubitus ulcers, diabetic foot ulcers, perirectal abscesses, and necrotizing fasciitis. The general division into “uncomplicated” and “complicated” skin infections can be applied to help guide management.79 The agent most commonly responsible for skin and soft tissue infections is S aureus and is isolated in 44% of spec-imens.80 Less common isolates include other gram-positive bacteria such as Enterococcus species (9%), β-hemolytic strep-tococci (4%), and coagulase-negative staphylococci (3%). S aureus is more commonly responsible for causing abscesses. Patients with an impaired immune system (diabetic, cirrhotic, or neutropenic patients) are at higher risk of infection from gram-negative species like Pseudomonas aeruginosa (11%), Esche-richia coli (7.2%), Enterobacter (5%), Klebsiella (4%), and

1	cirrhotic, or neutropenic patients) are at higher risk of infection from gram-negative species like Pseudomonas aeruginosa (11%), Esche-richia coli (7.2%), Enterobacter (5%), Klebsiella (4%), and Serratia (2%), among others.Uncomplicated Skin InfectionsUncomplicated infections involve relatively small surface area (<75 cm2) and bacterial invasion limited to the skin and its appendages. Impetigo, erysipelas, cellulitis, folliculitis, and simple abscess fall into this category. Impetigo is a superficial infection, typically of the face, that occurs most frequently in infants or children, resulting in honey-colored crusting. Erysip-elas is a cutaneous infection localized to the upper layers of the dermis, while cellulitis is a deeper infection, affecting the deeper dermis and subcutaneous tissue. Folliculitis describes inflammation of the hair follicle, and a furuncle describes a fol-licle with swelling and a collection of purulent material. These lesions can sometimes coalesce into a

1	tissue. Folliculitis describes inflammation of the hair follicle, and a furuncle describes a fol-licle with swelling and a collection of purulent material. These lesions can sometimes coalesce into a carbuncle, an abscess with multiple different draining sinus tracts.It is recommended to culture infectious lesions to help identify the causative agent, but treatment without these studies is reasonable in typical cases. Minor infections can be safely treated with topical antimicrobials like 2% mupirocin to pro-vide coverage for methicillin-resistant S aureus (MRSA). Fol-liculitis generally resolves with adequate hygiene and warm soaks. Furuncles, carbuncles and other simple abscesses can be incised, drained, and packed, typically without the use of systemic antibiotics. The decision to use systemic antibiotics after incision and drainage of abscess should be made based upon presence or absence of systemic inflammatory response syndrome (SIRS) criteria.81For nonpurulent, uncomplicated

1	systemic antibiotics after incision and drainage of abscess should be made based upon presence or absence of systemic inflammatory response syndrome (SIRS) criteria.81For nonpurulent, uncomplicated cellulitis in which there is no drainable collection, systemic antibiotic coverage for β-hemolytic streptococcus is recommended. If there is no improvement in 48 to 72 hours or worsening of symptoms, antibiotic coverage should be added for MRSA. Systemic therapy for purulent cellulitis, which includes cutaneous abscesses, should cover MRSA, and empiric coverage for streptococcus is likely unnecessary. Antibiotic coverage for streptococcus is generally accomplished with β-lactam antibi-otics like penicillins or first-generation cephalosporins. MRSA coverage is accomplished with clindamycin, trimethoprim-sulfamethoxazole, linezolid, and tetracyclines. Clindamycin, trimethoprim-sulfamethoxazole, linezolid, or tetracycline combined with a β-lactam can all be used for dual coverage of

1	trimethoprim-sulfamethoxazole, linezolid, and tetracyclines. Clindamycin, trimethoprim-sulfamethoxazole, linezolid, or tetracycline combined with a β-lactam can all be used for dual coverage of streptococcus and MRSA.Complicated Skin InfectionsComplicated skin infections include superficial cellulitis encompassing a large surface area (>75 cm2) or deeper infec-tions extending below the dermis. Necrotizing soft tissue infec-tions (NSTIs), including necrotizing fasciitis, can rapidly cause extensive morbidity and mortality, thus their prompt diagnosis and appropriate management is crucial. A thorough history and 6Brunicardi_Ch16_p0511-p0540.indd 52419/02/19 3:09 PM 525THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16exam should be performed to elicit information (e.g., history of trauma, diabetes mellitus, cirrhosis, neutropenia, bites, IV or subcutaneous drug abuse) as well as physical findings such as crepitus (gas-forming organism), fluctuance (abscess), purpura (sepsis in streptococcal

1	mellitus, cirrhosis, neutropenia, bites, IV or subcutaneous drug abuse) as well as physical findings such as crepitus (gas-forming organism), fluctuance (abscess), purpura (sepsis in streptococcal infections), bullae (streptococci, Vibrio vulnificus), lymphangitis, and signs of a systemic inflammatory response.Extensive cellulitis is managed in a similar fashion as simple cellulitis. Initial treatment consists of intravenous anti-biotics that cover β-hemolytic streptococcus, such as ceph-alosporins, with the addition of MRSA coverage if there is no improvement in symptoms. Vancomycin is typically the first choice for MRSA coverage, but this drug is inferior to β-lactams for coverage of MSSA. Alternative antibiotics that are typically effective against MRSA are linezolid, daptomy-cin, tigecycline, and telavancin. Clindamycin is approved for use against MRSA, but resistance rates are increasing, and its use is discouraged if institutional rates of clindamycin resis-tance are

1	tigecycline, and telavancin. Clindamycin is approved for use against MRSA, but resistance rates are increasing, and its use is discouraged if institutional rates of clindamycin resis-tance are >15%.81Necrotizing soft tissue infections occur 500 to 1500 times a year in the United States82 and are frequently asso-ciated with diabetes mellitus, intravenous drug abuse, obe-sity, alcohol abuse, immune suppression, and malnutrition.83 Because NSTIs can often present initially with nonspecific findings, the physician should always have a high index of suspicion when evaluating a patient. The threshold for surgi-cal exploration and debridement should be low, particularly in a weakened host. Occasionally an inciting event or point of entry can be identified, but in 20% to 50% of cases, the exact cause is unknown. These infections are associated with a high mortality, ranging from 25% to 40%, with higher rates in the truncal and perineal cases.NSTIs are classified based on anatomic site,

1	cause is unknown. These infections are associated with a high mortality, ranging from 25% to 40%, with higher rates in the truncal and perineal cases.NSTIs are classified based on anatomic site, involved tis-sues, and the offending organisms. NSTIs commonly originate at the genitalia, perineum (Fournier’s gangrene), and abdomi-nal wall. Subcutaneous tissue, fascia and muscle can all be affected. Necrotizing fasciitis involves infection of the fascia, and the infection can quickly travel along the easily separable, avascular planes. There are three types of NSTIs when clas-sified by the offending agent. The most common is type 1, which is caused by a polymicrobial source including gram-positive cocci, gram-negative rods, and anaerobic bacteria, specifically Clostridium perfringens and C septicum. Type 2 is caused by a monomicrobial source of β-hemolytic Strepto-coccus or Staphylococcus species, with MRSA contributing to the increasing number of community-acquired NSTIs.84 A his-tory of

1	Type 2 is caused by a monomicrobial source of β-hemolytic Strepto-coccus or Staphylococcus species, with MRSA contributing to the increasing number of community-acquired NSTIs.84 A his-tory of trauma is often elicited and can be associated with toxic shock syndrome. Type 3 is a rare but fulminant subset result-ing from a V vulnificus infection of traumatized skin exposed to a body of salt-water.In addition to signs of SIRS, patients can present with skin changes like erythema, bullae, necrosis, pain, and crepitus. (Fig. 16-8). They may exhibit signs of hemodynamic instability, and gas within the soft tissues on imaging is pathognomonic. Patients can present with a range of symptoms, from minimal skin change to frank necrosis, and the time of progression to fulminant disease varies in each patient. Laboratory values are nonspecific and resemble values seen in sepsis. There have been attempts at creating scoring systems to assist in the diagnosis of NSTI. One study in 2000 used the

1	each patient. Laboratory values are nonspecific and resemble values seen in sepsis. There have been attempts at creating scoring systems to assist in the diagnosis of NSTI. One study in 2000 used the criteria of a white blood cell count >15,400 and a serum sodium level <135 mmol/L. This test was found to have a negative predictive value of 99%, but a positive predictive value of only 26%.85 In 2004, six criteria ABFigure 16-8. A. Initial presentation of necrotizing soft issue infec-tion in an obese, diabetic patient. B. Following operative debride-ment to muscle layer.were used and referred to as the Laboratory Risk Indicator for Necrotizing Fasciitis, or LRINEC, and included C-reactive protein (CRP), white blood cell (WBC) count, hemoglobin, plasma sodium, creatinine, and glucose.86 A score of 8 or greater Brunicardi_Ch16_p0511-p0540.indd 52519/02/19 3:09 PM 526SPECIFIC CONSIDERATIONSPART IIsuggested a high probability of NSTI, 6 or 7 an intermediate probability, and <5 a low

1	score of 8 or greater Brunicardi_Ch16_p0511-p0540.indd 52519/02/19 3:09 PM 526SPECIFIC CONSIDERATIONSPART IIsuggested a high probability of NSTI, 6 or 7 an intermediate probability, and <5 a low probability. This test was internally validated and found to have a PPV of 92% and an NPV of 96%. However, some have criticized this study because of its small sample size and over-reliance on CRP, which can be elevated in multiple other conditions. Blood cultures are not always posi-tive, and tissue samples will demonstrate necrosis, white blood cell infiltration, thrombosis, angiitis, and microorganisms. The use of cross-sectional imaging in the diagnosis of NSTI is lim-ited, and it should not delay appropriate surgical treatment.Three principles form the foundation of the management of NSTIs: (a) source control with wide surgical debridement, (b) broad-spectrum intravenous antibiotics, and (c) supportive care and resuscitation. As soon as the diagnosis is clear or the sus-picion is

1	NSTIs: (a) source control with wide surgical debridement, (b) broad-spectrum intravenous antibiotics, and (c) supportive care and resuscitation. As soon as the diagnosis is clear or the sus-picion is high, the patient should be taken for operative explo-ration and debridement. Incisions should be made parallel to neurovascular structures and through the fascial plane, removing any purulent or devitalized tissue until viable, bleeding tissue is encountered. On inspection, the tissue will appear necrotic with dead muscle, thrombosed vessels, the classic “dishwater” fluid, and a positive finger test, in which the tissue layers can be easily separated from one another. In Fournier’s gangrene, one should aim to preserve the anal sphincter as well as the testicles (blood supply is independent of the overlying tissue and is usually not infected). Return to the OR should be planned for the next 24 to 48 hours to verify source control and the extent of damage. Broad spectrum antibiotic therapy

1	the overlying tissue and is usually not infected). Return to the OR should be planned for the next 24 to 48 hours to verify source control and the extent of damage. Broad spectrum antibiotic therapy should be initiated as soon as possible, with the intent of covering gram positives (including MRSA), gram negatives, and anaerobic organisms. The Infec-tious Diseases Society of America recommends initiating ther-apy with intravenous vancomycin and piperacillin/tazobactam, unless a monomicrobial agent is identified, in which case more directed therapy would be appropriate.81 Antibiotic therapy should continue until the patient requires no further debride-ment, is clinically improving, and has been afebrile for 48 to 72 hours.Adjuncts to surgery include topical antimicrobial creams, subatmospheric pressure wound dressings, and optimization of nutrition. Controversial topics include the role of hyperbaric oxygen87 (may inhibit infection by creating an oxidative burst, with anecdotally fewer

1	pressure wound dressings, and optimization of nutrition. Controversial topics include the role of hyperbaric oxygen87 (may inhibit infection by creating an oxidative burst, with anecdotally fewer debridements required and improved survival, but limited availability) and IVIG (may modulate the immune response to streptococcal superantigens). Wound clo-sure is performed once bacteriologic, metabolic, and nutritional balances are obtained.ActinomycosisActinomycetes is a genus of gram positive rods that inhabit the oropharynx, gastrointestinal tract, and female genital tract. The most commonly isolated species causing disease in humans is A isrealii. The cervicofacial form of Actinomycetes infection is the most common presentation, representing 55% of cases, and typically presenting as an acute pyogenic infection in the submandibular or paramandibular area. Patients can also exhibit chronic soft tissue swelling, fibrosis, and sinus discharge of sulfur granules.88 Demonstration of

1	an acute pyogenic infection in the submandibular or paramandibular area. Patients can also exhibit chronic soft tissue swelling, fibrosis, and sinus discharge of sulfur granules.88 Demonstration of gram-positive filamentous organisms and sulfur granules on histological examination is strongly supportive of a diagnosis of actinomycosis.89 These infections are typically treated with high doses of intravenous followed by oral penicillin therapy. Surgical treatment is uti-lized if there is extensive necrotic tissue, poor response to anti-biotics, or the need for tissue biopsy to rule out malignancy.VIRAL INFECTIONS WITH SURGICAL IMPLICATIONSHuman Papillomavirus InfectionsHuman papillomaviruses represent a group of over 100 iso-lated types of small DNA viruses of the Papovavirus fam-ily that is highly host-specific to humans.90 These viruses are transmitted via cutaneous contact with individuals who have clinical or subclinical infection and occur more fre-quently in immunocompromised

1	is highly host-specific to humans.90 These viruses are transmitted via cutaneous contact with individuals who have clinical or subclinical infection and occur more fre-quently in immunocompromised individuals. The viruses are responsible for the development of verrucae, or warts. These are histologically characterized by nonspecific findings of hyperkeratosis, papillomatosis, and acanthosis, as well as the hallmark koilocytes (clear halo around nucleus). Clinically, these generally arise as slow-growing papules on the skin or mucosal surfaces. Regression of HPV lesions is frequently an immune-mediated, spontaneous event that is exemplified by the persistent and extensive manifestation of this virus in the immune-compromised patient.The subtypes are generally grouped, based on their pre-sentation, as cutaneous or mucosal. Cutaneous types most com-monly affect the hands and fingers. Verruca vulgaris, or common warts, are caused by HPV types 1, 2, and 4, with a prevalence of up to 33% in

1	as cutaneous or mucosal. Cutaneous types most com-monly affect the hands and fingers. Verruca vulgaris, or common warts, are caused by HPV types 1, 2, and 4, with a prevalence of up to 33% in school children and 3.5% in adults, and a higher prevalence in the immunosuppressed population.91 Plantar and palmar warts (HPV-1 and -4) typically occur at points of pres-sure and are characterized by a keratotic plug surrounded by a hyperkeratotic ring with black dots (thrombosed capillaries) on the surface. Plane warts occur on the face, dorsum of hands, and shins. They are caused by HPV-3 and -10 and tend to be multiple, flat-topped lesions with a smooth surface and light brown color. Cutaneous warts typically regress spontaneously in the immunocompetent patient. Epidermodysplasia verruci-formis is a rare, autosomal recessive inherited genetic skin dis-order that confers increased susceptibility to certain types of HPV. This presents with difficult-to-treat and often widespread verrucae that

1	is a rare, autosomal recessive inherited genetic skin dis-order that confers increased susceptibility to certain types of HPV. This presents with difficult-to-treat and often widespread verrucae that carry a higher risk of malignant transformation (30%–50% risk of squamous cell carcinoma), especially when caused by HPV types 5 and 8.92 A similar clinical picture has been described in human immunodeficiency virus (HIV) and transplant patients.93,94Mucosal HPV types cause lesions in the mucosal or geni-tal areas and behave like sexually transmitted infections. The most common mucosal types are HPV-6, -11, -16, -18, -31 and -33. These lesions present as condylomata acuminata, genital or veneral warts, papules that occur on the perineum, external genitalia, anus, and can extend into the mucosal surfaces of the vagina, urethra and rectum. These lesions are at risk for malig-nant transformation, with types 6 and 11 conferring low risk, and types 16, 18, 31 and 33 conferring a high risk. The

1	surfaces of the vagina, urethra and rectum. These lesions are at risk for malig-nant transformation, with types 6 and 11 conferring low risk, and types 16, 18, 31 and 33 conferring a high risk. The recently developed quadrivalent HPV vaccine, targeting HPV types -6, -11, -16, and -18, is now available to both males and females age 9 to 26 and is associated with an up to 90% reduction of infections from those HPV types.95Treatment is aimed at physical destruction of the affected cells. Children often require no treatment as spontaneous regres-sion is common. In cases causing physical or emotional discom-fort, or in cases of immunocompromise or risk of transmission, treatment may be indicated. Cryotherapy using liquid nitrogen is an effective treatment for most warts, but care must be taken not to damage underlying structures.96 Topical preparations of salicylic acid, silver nitrate, and glutaraldehyde may also be Brunicardi_Ch16_p0511-p0540.indd 52619/02/19 3:09 PM 527THE SKIN AND

1	not to damage underlying structures.96 Topical preparations of salicylic acid, silver nitrate, and glutaraldehyde may also be Brunicardi_Ch16_p0511-p0540.indd 52619/02/19 3:09 PM 527THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16used. Treatment of recalcitrant lesions includes a variety of ther-apeutic options aimed at physically destroying the lesions by electrodessication, cryoablation, and pulsed dye laser therapy. Additional modalities such as H2-antagonists and zinc sulfate may have a role in augmenting the immune response and reduc-ing recurrence rates.Cutaneous Manifestations of Human Immunodeficiency VirusThe HIV-infected patient is significantly more susceptible to infectious and inflammatory skin conditions than the rest of the population.97 These skin disorders may be due to the HIV infection itself or from opportunistic infections secondary to immunosuppression. During early stages, nonspecific cutane-ous manifestations may occur. Acute retroviral syndrome occurs following

1	HIV infection itself or from opportunistic infections secondary to immunosuppression. During early stages, nonspecific cutane-ous manifestations may occur. Acute retroviral syndrome occurs following inoculation in one-half to two-thirds of patients, and 30% to 50% of these patients can present with an acute viral exanthem.98 This is usually a morbilliform rash affecting the face, trunk, and upper extremities. Other skin changes, as well as common skin disorders with atypical features, can occur, including recurrent varicella zoster, hyperkeratotic warts, and seborrheic dermatitis. Condylomata acuminate and verrucae appear early; however, their frequency and severity do not change with disease progression.Late-presenting cutaneous manifestations include chronic herpes simplex virus (HSV), cytomegalovirus, and, to a lesser extent, molluscum contagiousum, which is typically treatable with imiquimod. HSV is the most common viral infection in the patient with HIV, and is more likely to

1	cytomegalovirus, and, to a lesser extent, molluscum contagiousum, which is typically treatable with imiquimod. HSV is the most common viral infection in the patient with HIV, and is more likely to display atypical fea-tures and less likely to spontaneously resolve in these patients.99 Mycobacterial infections and mucocutaneous candidiasis also occur. Bacterial infections such as impetigo and folliculitis may be more persistent and widespread.Malignant lesions such as Kaposi’s sarcoma occur in less than 5% of HIV-infected patients in the United States, although the worldwide prevalence in acquired immunodeficiency syn-drome (AIDS) patients exceeds 30%. Kaposi’s sarcoma is a vas-cular neoplasm that can affect cutaneous and visceral tissues. While the rates of Kaposi’s sarcoma development have sharply declined since the widespread use of antiretroviral therapy, the rates of other cutaneous malignancies have remained stable. The risk of an HIV-infected patient developing a cutaneous

1	have sharply declined since the widespread use of antiretroviral therapy, the rates of other cutaneous malignancies have remained stable. The risk of an HIV-infected patient developing a cutaneous malig-nancy is about 5.7%, with basal cell carcinoma being the most common type encountered.100With regard to general surgical considerations in HIV patients, contributing related morbidities such as malnutrition, decreased CD4 count, and presence of opportunistic infection may result in delayed and attenuated wound healing capacity.101BENIGN TUMORSHemangiomaHemangiomas are benign vascular tumors that arise from the proliferation of endothelial cells that surround blood-filled cavities. They occur in about 4% of children by 1 year of age. Their natural history is typically presentation shortly after birth, a period of rapid growth during the first year, and then gradual involution over childhood in more than 90% of cases. These hemangiomas are generally managed nonsurgically prior to

1	after birth, a period of rapid growth during the first year, and then gradual involution over childhood in more than 90% of cases. These hemangiomas are generally managed nonsurgically prior to involution. Occasionally, during the rapid growth phase, the lesions can obstruct the airway, GI tract, vision, and musculo-skeletal function. In these cases, surgical resection is indicated prior to the involution phase. Hemangiomas can sometimes con-sume a large percentage of cardiac output, resulting in high-output heart failure or a consumptive coagulopathy, which may also necessitate resection. These lesions characteristically express the GLUT-1 glucose transporter protein, which is absent in cells of the normal cutaneous vasculature.102 First-line ther-apy for these infantile hemangiomas is propranolol, which causes cessation of growth and, in most cases, actual regression of the lesions.103,104 Systemic corticosteroids and interferon-α can impede tumor progression, and laser therapy has

1	which causes cessation of growth and, in most cases, actual regression of the lesions.103,104 Systemic corticosteroids and interferon-α can impede tumor progression, and laser therapy has been used as well. If tumors persist into adolescence leaving a cosmeti-cally undesirable defect, surgical resection may be considered. When surgical resection or debulking is considered, upfront selective embolization can help with planned resection.NeviNevi (singular, nevus) are areas of melanocytic hyperplasia or neoplasia. These collections can be found in the epidermis (junctional), partially in the dermis (compound), or completely within the dermis (dermal). They commonly develop in child-hood and young adulthood, and will sometimes spontaneously regress. Exposure to UV radiation is associated with increased density of these lesions.105 Nevi are typically symmetric and small. Congenital nevi are the result of abnormal development of melanocytes. The events leading to this abnormal develop-ment

1	increased density of these lesions.105 Nevi are typically symmetric and small. Congenital nevi are the result of abnormal development of melanocytes. The events leading to this abnormal develop-ment may also affect the surrounding cells, resulting in longer, darker hair. Congenital nevi are found in less than 1% of neo-nates, and when characterized as giant congenital nevi, they have up to a 5% chance of developing into a malignant mela-noma, and may do so even in the first years of childhood.106,107 Treatment, therefore, consists of surgical excision of the lesion as early as is feasible. For larger lesions, serial excision and tissue expansion may be required, with the goal of lesion exci-sion being maintenance of function and form while decreasing oncologic risk.Cystic LesionsCutaneous cysts are benign lesions that are characterized by overgrowth of epidermis towards the center of the lesion, resulting in keratin accumulation. Epidermoid cysts (often mistakenly referred to as

1	cysts are benign lesions that are characterized by overgrowth of epidermis towards the center of the lesion, resulting in keratin accumulation. Epidermoid cysts (often mistakenly referred to as sebaceous cysts) are classically the result of keratin-plugged pilosebaceous units. They commonly affect adult men and women, and present as a dermal or sub-cutaneous cyst with a single, keratin-plugged punctum at the skin surface, often at or above the upper chest and back. Epi-dermoid cysts are the most common cutaneous cyst and are histologically characterized by mature epidermis complete with granular layer. Another type of cystic lesion is known as a trichilemmal cyst. These cysts are derived from the outer sheath of hair follicles, and, in contrast to epidermoid cysts, lack a granular layer. They are almost always found on the scalp and more commonly in women. A third type of cutaneous cyst is a dermoid cyst. Dermoid cysts are congenital variants that occur as the result of persistent

1	They are almost always found on the scalp and more commonly in women. A third type of cutaneous cyst is a dermoid cyst. Dermoid cysts are congenital variants that occur as the result of persistent epithelium within embry-onic lines of fusion. They occur most commonly between the forehead and nose tip, and the most frequent site is the eye-brow. They can lie in the subcutaneous tissue or intracranially, and often communicate with the skin surface via a small fis-tula. These cystic structures contain epithelial tissue, hair, and a variety of epidermal appendages. Treatment for these cystic structures includes surgical excision with care taken to remove the cyst lining to prevent recurrence.7Brunicardi_Ch16_p0511-p0540.indd 52719/02/19 3:09 PM 528SPECIFIC CONSIDERATIONSPART IIKeratosisActinic Keratosis. Actinic keratoses are neoplasms of epi-dermal keratinocytes that represent a range in a spectrum of disease from sun damage to squamous cell carcinoma. They typically occur in

1	Keratosis. Actinic keratoses are neoplasms of epi-dermal keratinocytes that represent a range in a spectrum of disease from sun damage to squamous cell carcinoma. They typically occur in fair-skinned, elderly individuals in primarily sun-exposed areas, and UV radiation exposure is the greatest risk factor. There are multiple variants, and they can present as erythematous and scaly to hypertrophic, keratinized lesions. They can become symptomatic, causing bleeding, pruritis and pain. They can regress spontaneously, persist without change, and transform into invasive squamous cell carcinoma. It is estimated that approximately 10% of actinic keratoses will transform into invasive squamous cell carcinoma, and that pro-gression takes about 2 years on average.108 About 60% to 65% of squamous cell carcinomas are believed to originate from actinic keratoses. The presence of actinic keratoses also serves as a predictor of development of other squamous cell and basal cell carcinomas.109

1	cell carcinomas are believed to originate from actinic keratoses. The presence of actinic keratoses also serves as a predictor of development of other squamous cell and basal cell carcinomas.109 Treatment options are excision, fluorouracil, cautery and destruction, and dermabrasion.110,111Seborrheic Keratosis. Seborrheic keratoses are benign lesions of the epidermis that typically present as well-demarcated, “stuck on” appearing papules or plaques over elderly individu-als. Clonal expansion of keratinocytes and melanocytes make up the substance of these lesions. They carry no malignant potential and treatment is primarily for cosmetic purposes.Soft Tissue TumorsAcrochordons. Acrochordons, also known as skin tags, are benign, pedunculated lesions on the skin made up of epider-mal keratinocytes surrounding a collagenous core. Although they can become irritated or necrotic, their removal is generally cosmetic.Dermatofibromas. Dermatofibromas are benign cutaneous proliferations that

1	surrounding a collagenous core. Although they can become irritated or necrotic, their removal is generally cosmetic.Dermatofibromas. Dermatofibromas are benign cutaneous proliferations that appear most commonly on the lower extremi-ties of women. They appear as pink to brown papules that pucker or dimple in the center when the lesion is pinched. It remains unclear whether these lesions have a neoplastic etiology or if they are the result of minor trauma or infection.112 These lesions are typically asymptomatic, and treatment is only indicated for cosmetic concerns or when a histologic diagnosis is required. Surgical excision is the recommended treatment, although cryo-therapy and laser treatment may be used.113 In rare cases, a basal cell carcinoma may develop within a dermatofibroma.Lipomas. Lipomas are the most common subcutaneous neo-plasm and have no malignant potential.114 They present as a painless, slow-growing, mobile mass of the subcutaneous tissue. Usually less than 5 cm in

1	are the most common subcutaneous neo-plasm and have no malignant potential.114 They present as a painless, slow-growing, mobile mass of the subcutaneous tissue. Usually less than 5 cm in diameter, these neoplasms can reach much larger sizes. Lipomas are largely asymptomatic but may cause pain due to regional nerve deformation. Surgical resection is indicated in cases of local pain, mass effect, or cosmetically sensitive areas. The tumors are usually well circumscribed and amenable to surgical resection. Liposarcoma is a malignant fatty tumor that can mimic a lipoma, but is often deep-seated, rapidly growing, painful, and invasive. In these cases, cross-sectional imaging is recommended prior to any surgical resection.Neural TumorsNeuromas. Neuromas do not represent a true clonal prolifera-tion of neural tissue, but rather disordered growth of Schwann cells and nerve axons, often at the site of previous trauma. They can present within surgical scar lines or at the site of previous

1	of neural tissue, but rather disordered growth of Schwann cells and nerve axons, often at the site of previous trauma. They can present within surgical scar lines or at the site of previous trauma as flesh-colored papules or nodules and are typically painful.Schwannomas. A schwannoma is a benign proliferation of the Schwann cells of the peripheral nerve sheath, and can arise sporadically or in association with type 2 neurofibromatosis. It contains no axons, but may displace the affected nerve and cause pain along the distribution of the nerve.Neurofibromas. Neurofibromas, in contrast, are benign prolif-erations that are made up of all nerve elements, and arise as fleshy and nontender, sessile or pedunculated masses on the skin. They can arise sporadically or in association with type 1 neurofibroma-tosis, and in these cases, are associated with café-au-lait spots and Lisch nodules. They are often asymptomatic, but may be pruritic. The development of pain at the site of a previously

1	neurofibroma-tosis, and in these cases, are associated with café-au-lait spots and Lisch nodules. They are often asymptomatic, but may be pruritic. The development of pain at the site of a previously asymptomatic neurofibroma may indicate a rare malignant transformation and requires surgical excision and biopsy.MALIGNANT TUMORSBasal Cell CarcinomaBasal cell carcinoma (BCC) is the most common tumor diag-nosed in the United States, with an estimated one million new cases occurring each year. It represents 75% of non-melanoma skin cancers and 25% of all cancers diagnosed each year.115 BCC is seen slightly more commonly in males and indi-viduals over the age of 60, though the incidence in younger age groups is increasing. The primary risk factor for disease devel-opment is sun exposure (UVB rays more than UVA rays), par-ticularly during adolescence. The pathogenesis of BCC stems from mutations of genes involved in tumor suppression, often caused by ionizing radiation. The p53 tumor

1	(UVB rays more than UVA rays), par-ticularly during adolescence. The pathogenesis of BCC stems from mutations of genes involved in tumor suppression, often caused by ionizing radiation. The p53 tumor suppressor gene is defective in approximately 50% of cases.116 There is a latency period of 20 to 50 years.BCC tends to occur on sun-exposed areas of the skin, most commonly the nose and other parts of the face. A malignant lesion on the upper lip is almost always BCC, and BCC is the most common malignant eyelid tumor. Because of the photo-protective effect of melanin, dark-skinned individuals are far less commonly affected. Other risk factors for development of BCC include immune suppression, chemical exposure, and ion-izing radiation exposure. There are also genetic susceptibilities to development of BCC in conditions such as xeroderma pig-mentosa, unilateral basal cell nevus syndrome, and nevoid BCC syndrome.115 The natural history of BCC is typically one of local invasion rather than

1	of BCC in conditions such as xeroderma pig-mentosa, unilateral basal cell nevus syndrome, and nevoid BCC syndrome.115 The natural history of BCC is typically one of local invasion rather than distant metastasis, but untreated BCC can often result in significant morbidity.There are multiple variants of BCC, and presentation can range from red, flesh-colored, or white macule or papule, to nodules and ulcerated lesions. Growth patterns of these lesions can either be well-circumscribed or diffuse and the most com-mon types of BCC are nodular and micronodular, superficial spreading, and infiltrative.117 The most common subtype is the nodular variant, characterized by raised, pearly pink papules with telangiectasias and occasionally a depressed tumor center with raised borders giving the classic “rodent ulcer” appearance. Superficial spreading BCC is confined to the epidermis as a flat, pink, scaling or crusting lesion, often mistaken for eczema, actinic keratosis, fungal infection, or

1	“rodent ulcer” appearance. Superficial spreading BCC is confined to the epidermis as a flat, pink, scaling or crusting lesion, often mistaken for eczema, actinic keratosis, fungal infection, or psoriasis. This subtype typically appears on the trunk or extremities and the mean age of diagnosis is 57 years. The infiltrative form appears on the 8Brunicardi_Ch16_p0511-p0540.indd 52819/02/19 3:09 PM 529THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16head and neck in the late 60s, often at embryonic fusion lines,117 with an opaque yellow-white color that blends with surrounding skin and has no raised edges.118 The morpheaform subtype rep-resents 2% to 3% of all BCC and is the most aggressive subtype. It usually presents as an indurated macule or papule with the appearance of an enlarging scar. The clinical margins are often indistinct, and the rate of positive margins after excision is high. There is also a pigmented variant of BCC that can be difficult to distinguish from certain melanoma

1	The clinical margins are often indistinct, and the rate of positive margins after excision is high. There is also a pigmented variant of BCC that can be difficult to distinguish from certain melanoma subtypes.Treatment of BCC varies according to size, location, type, and highor low-risk. Treatment options include surgical exci-sion, medical, or destructive therapies. Surgical excision should include 4 mm margins for small, primary BCC on cosmetically sensitive areas, and 10 mm margins otherwise.119 Mohs micro-surgical excision is sequential horizontal excision and has been shown to be cost-effective and associated with low recurrence rates for BCC (1%).120,121 It is the treatment of choice for mor-pheaform or other BCC with aggressive features, poorly delin-eated margins, recurrent tumors, or cosmetically sensitive areas, especially in the midface. A common approach used by derma-tologists for very small (<2 mm) and low risk lesions is cau-tery and destruction, although it should be

1	or cosmetically sensitive areas, especially in the midface. A common approach used by derma-tologists for very small (<2 mm) and low risk lesions is cau-tery and destruction, although it should be kept in mind that the local cure rates can be operator and institution dependent. Other destructive techniques include cryosurgery and laser ablation. Radiation therapy can be used as adjuvant therapy following surgery, or as primary therapy in poor surgical candidates with low-risk lesions. The practitioner must be aware of the poten-tial consequences of radiation therapy, including poor cosmetic outcomes and future cancer risk.Superficial medical therapies are generally reserved for patients in whom surgical and radiation treatment is not an option. Topical imiquimod or 5-fluorouracil have been used for periods of 6 to 16 weeks for small, superficial BCC of the neck, trunk or extremities.122-126 Lastly, topical photodynamic therapy has shown some benefit in treatment of premalignant or

1	been used for periods of 6 to 16 weeks for small, superficial BCC of the neck, trunk or extremities.122-126 Lastly, topical photodynamic therapy has shown some benefit in treatment of premalignant or super-ficial low-risk lesions as well.Patients with BCC need to have regular follow-up with full skin examinations every 6 to 12 months. Sixty-six percent of recurrences develop within 3 years, and with a few excep-tions occurring decades after initial treatment, the remaining recur within 5 years of initial treatment.121,127 A second primary BCC may develop after treatment and, in 40% of cases, presents within the first 3 years after treatment.Squamous Cell CarcinomaSquamous cell carcinoma (SCC) is the second most common skin cancer and accounts for approximately 100,000 cases each year. The primary risk factor for the development of SCC is UV radiation exposure128; however, other risks include light Fitzpatrick skin type (I or II), environmental factors such as chemical agents, physical

1	primary risk factor for the development of SCC is UV radiation exposure128; however, other risks include light Fitzpatrick skin type (I or II), environmental factors such as chemical agents, physical agents (ionizing radiation), pso-ralen, HPV-16 and -18 infections, immunosuppression, smok-ing, chronic wounds, burn scars, and chronic dermatoses. Heritable risk factors include xeroderma pigmentosum, epider-molysis bullosa, and oculocutaneous albinism.SCC classically appears as a scaly or ulcerated papule or plaque, and bleeding of the lesion with minimal trauma is not uncommon, but pain is rare. It can exhibit in situ (confined to the epidermis) or invasive subtypes. The most common in situ variant of SCC is actinic keratosis, described previously in this chapter. Invasive squamous cell carcinomas may arise de novo, but more commonly evolve from these precursors. Another in 9Figure 16-9. Squamous cell carcinoma forming in a chronic wound.situ variant is known as Bowen disease. This is

1	may arise de novo, but more commonly evolve from these precursors. Another in 9Figure 16-9. Squamous cell carcinoma forming in a chronic wound.situ variant is known as Bowen disease. This is characterized by full-thickness epidermal dysplasia and clinically appears as a scaly, erythematous patch often with pigmentation and fis-suring. When it occurs on the glans penis, it is known as eryth-roplasia of Queyrat. Ten percent of these cases will eventually become invasive.129 Outside of these instances, most in situ cases grow slowly and do not progress to invasive disease.Invasive SCC is characterized by invasion through the basement membrane into the dermis of the skin. It usually arises from an actinic keratosis precursor, but de novo varieties do occur and are higher risk. De novo invasive SCC commonly occurs in organ transplant and immunocompromised patients, and has a metastatic rate as high as 14%.130 De novo invasive SCC arising in areas of chronic wounds or burn scars are known

1	SCC commonly occurs in organ transplant and immunocompromised patients, and has a metastatic rate as high as 14%.130 De novo invasive SCC arising in areas of chronic wounds or burn scars are known as Marjolin’s ulcers, and have a higher metastatic potential (Fig. 16-9). Keratoacanthoma is now being accepted as a sub-type of SCC that is characterized by a rapidly growing nodule with a central keratin plug.131 The natural history of invasive disease depends on location and inherent tumor characteristics. Clinical risk factors for recurrence include presentation with neurologic symptoms, immunosuppression, tumor with poorly defined borders, and tumor that arises at a site of prior radiation. Perineural involvement also has a poorer survival with increased local recurrence and lymph node metastasis. Grades of differen-tiation are based on the ratio of differentiated to undifferentiated cells, with a lower ratio associated with a greater metastatic and recurrent potential. Large (>2 cm)

1	Grades of differen-tiation are based on the ratio of differentiated to undifferentiated cells, with a lower ratio associated with a greater metastatic and recurrent potential. Large (>2 cm) lesions, depth of invasion >4 mm, rapid growth, and location on the ear, lips, nose, scalp, or genitals are all also indicators of worse prognosis.When feasible, wide surgical excision including subcuta-neous fat is the treatment of choice for SCC. Margins of 4 mm are recommended for low-risk lesions and 6 mm for high-risk lesions.128 Mohs microsurgical excision is indicated for posi-tive margins, recurrent tumors, sites where cosmesis or function preservation is critical, poorly differentiated tumors, invasive lesions, and verrucous tumors. Using this modality often results in lower recurrence rates.127,130 It has also found use in nail bed lesions and in those arising in a background of osteomyelitis. The role of lymph node dissection in the setting of SCC contin-ues to evolve. Lymphadenectomy is

1	It has also found use in nail bed lesions and in those arising in a background of osteomyelitis. The role of lymph node dissection in the setting of SCC contin-ues to evolve. Lymphadenectomy is indicated following fine-needle aspiration or core biopsy for clinically palpable lymph nodes or nodes detected on cross-sectional imaging. Nodes Brunicardi_Ch16_p0511-p0540.indd 52919/02/19 3:09 PM 530SPECIFIC CONSIDERATIONSPART IIshould also be removed from susceptible regional lymph node basins in patients with SCC in the setting of chronic wounds. Patients with parotid disease benefit from a superficial or total parotidectomy (with facial nerve preservation) and adjuvant radiotherapy. Sentinel lymph node dissection may be used in high risk cases with clinically negative nodal disease. Radiation therapy is typically reserved as primary therapy for those who are poor surgical candidates, and as adjuvant therapy after surgi-cal resection for large, high-risk tumors. When used as primary

1	therapy is typically reserved as primary therapy for those who are poor surgical candidates, and as adjuvant therapy after surgi-cal resection for large, high-risk tumors. When used as primary therapy, cure rates may approach 90%.121MelanomaBackground. In 2017, an estimated 87,110 new cases of melanoma were diagnosed, as well as 9730 melanoma-related deaths. The incidence of melanoma is rising faster than most other solid malignancies, and these numbers likely represent an underestimation given the many in situ and thin melanoma cases that are underreported. These tumors primarily arise from mela-nocytes at the epidermal-dermal junction but may also originate from mucosal surfaces of the oropharynx, nasopharynx, eyes, proximal esophagus, anorectum, and female genitalia. Mela-noma characteristically metastasizes quite often, and can travel to most other tissues in the body. This metastasis confers a poor prognosis in patients, with a median life span of 6 to 8 months after

1	characteristically metastasizes quite often, and can travel to most other tissues in the body. This metastasis confers a poor prognosis in patients, with a median life span of 6 to 8 months after diagnosis.132The most important risk factor for the development of melanoma is exposure to UV radiation. It was recently reported that greater than 10 tanning bed sessions by adolescents and young adults increased their relative risk of developing mela-noma twofold,133 and there is a positive association with inter-mittent childhood sunburns and melanoma development.134 There is also an association with residence at high altitudes or in close proximity to the equator. Both personal and family history of melanomas increase the risk of primary melanoma develop-ment. Individuals with dysplastic nevi have a 6% to10% overall lifetime risk of melanoma, with tumors arising from preexisting nevi or de novo. Individuals with familial atypical multiple-mole melanoma syndrome have numerous melanocytic

1	have a 6% to10% overall lifetime risk of melanoma, with tumors arising from preexisting nevi or de novo. Individuals with familial atypical multiple-mole melanoma syndrome have numerous melanocytic nevi and a greatly increased risk of cutaneous melanoma. Congenital nevi increase the risk for melanoma proportionally with size, and giant congenital nevi (generally considered >20 cm in diameter) are associated with a 5% to 8% lifetime risk. Melanoma development is strongly associated with the p16/CDK4,6/Rb and p14ARF/HMD2/p53 tumor suppressor pathways and the RAF-MEK-ERK and PI3K-Akt oncogenic pathways.135Clinical Presentation. The presentation of melanoma is com-monly used to determine subtype but often starts as a localized, radial growth phase followed by a more aggressive, vertical growth phase. Approximately 30% of melanoma lesions arise from a preexisting melanocytic nevus. The most common sub-type of melanoma is superficial spreading (Fig. 16-10). This accounts for 50% to 70% of

1	phase. Approximately 30% of melanoma lesions arise from a preexisting melanocytic nevus. The most common sub-type of melanoma is superficial spreading (Fig. 16-10). This accounts for 50% to 70% of melanomas and typically arises from a precursor melanocytic nevus. Nodular subtype accounts for 15% to 30% of melanomas, and typically arises de novo, most commonly in men and on the trunk (Figs. 16-11 and 16-12). This subtype is aggressive with an early vertical growth pat-tern and is often diagnosed at a later stage. Up to 5% of these lesions will lack melanin and can be mistaken for other cutane-ous lesions. Lentigo maligna represents 10% of melanoma cases and is a less aggressive subtype of melanoma in situ that typi-cally arises on sun-exposed areas of the head and neck. Acral Figure 16-10. Primary cutaneous melanoma seen in the scalp of a 61-year-old male.Figure 16-11. Nodular melanoma seen in the leg of a 55-year-old male.lentiginous melanoma accounts for 29% to 72% of melanomas in

1	cutaneous melanoma seen in the scalp of a 61-year-old male.Figure 16-11. Nodular melanoma seen in the leg of a 55-year-old male.lentiginous melanoma accounts for 29% to 72% of melanomas in dark-skinned individuals, is occasionally seen in Caucasians, and is found on palmar, plantar, and subungual surfaces. This subtype is not thought to be due to sun exposure.Melanoma most commonly manifests as cutaneous dis-ease, and clinical characteristics of malignant transformation are often remembered by the initialism ABCDE. These lesions are typically Asymmetric with irregular Borders, Color variations, a Diameter greater than 6 mm, and are undergoing some sort of Evolution or change. Other key clinical characteristics include a pigmented lesion that has enlarged, ulcerated, or bled. Amela-notic lesions appear as raised pink, purple, or flesh-colored skin papules and are often diagnosed late.Diagnosis and Staging. Workup should begin with a his-tory and physical exam. The entire skin should be

1	appear as raised pink, purple, or flesh-colored skin papules and are often diagnosed late.Diagnosis and Staging. Workup should begin with a his-tory and physical exam. The entire skin should be checked for synchronous primaries, satellite lesions, and in-transit metas-tases, and all nodal basins should be examined for lymphade-nopathy. Suspicious lesions should undergo excisional biopsy with 1to 3-mm margins; however, tumors that are large or are in a cosmetically or anatomically challenging area can be approached by incisional biopsy, including punch biopsy.136 Brunicardi_Ch16_p0511-p0540.indd 53019/02/19 3:09 PM 531THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16ABCFigure 16-12. A. AP view of advanced melanoma in a 59-year-old male. B. Lateral view C. After resection and reconstruction with skin grafting.Tissue specimen should include full thickness of the lesion and a small section of normal adjacent skin to aid the pathologist in diagnosis. Clinically suspicious lymph nodes should

1	skin grafting.Tissue specimen should include full thickness of the lesion and a small section of normal adjacent skin to aid the pathologist in diagnosis. Clinically suspicious lymph nodes should undergo fine-needle aspiration (FNA), as this has been shown to have a high sensitivity and specificity for detection of melanoma in large lymph nodes.136-139Melanoma is characterized according to the American Joint Committee on Cancer (AJCC) as localized disease (stage I and II), regional disease (stage III), or distant metastatic disease (stage IV). The Breslow tumor thickness replaced the Clark’s level as the most important prognostic indicator for melanoma stag-ing.132,140 The Breslow tumor thickness measures the depth of penetration of the lesions from the top of the granular layer of the epidermis into the dermal layer and is directly related to the risk of disease progression. Tumor ulceration, mitotic rate ≥1 per mm2, and metastasis are all also associated with worse prognosis. In the

1	into the dermal layer and is directly related to the risk of disease progression. Tumor ulceration, mitotic rate ≥1 per mm2, and metastasis are all also associated with worse prognosis. In the presence of regional node metastasis, the num-ber of nodes affected is the most important prognostic indicator. For stage IV disease, the site of metastasis is strongly associated with prognosis, and elevated lactate dehydrogenase (LDH) is associated with a worse prognosis.141There is no supportive evidence for chest X-ray or com-puted tomography (CT) in the staging of patients unless there is positive regional lymph node disease, although it can be used to work up specific signs and symptoms when metastatic disease is suspected.136 In patients with stage III or greater disease, there is a high risk for distant metastasis, and imaging is recommended for baseline staging. These patients should receive additional imaging that includes CT of the chest, abdomen, and pelvis; whole-body positon

1	risk for distant metastasis, and imaging is recommended for baseline staging. These patients should receive additional imaging that includes CT of the chest, abdomen, and pelvis; whole-body positon emission tomography (PET)-CT; or brain magnetic resonance imaging (MRI).136The sentinel lymph node biopsy (SLNB) technique for melanoma was introduced in 1992 and has become a corner-stone in the management of melanoma, although its role in man-agement continues to be refined. SLNB is a standard staging procedure to evaluate the regional nodes for patients with clini-cally node-negative malignant melanoma. Detecting subclinical nodal metastasis in may benefit from lymphadenectomy or adju-vant therapy. This technique identifies the first draining lymph node from the primary lesion and has shown excellent accuracy and significantly less morbidity compared to complete resection of nodal basins. It is almost always performed at the time of initial wide excision, as SLN mapping after lymphatic

1	excellent accuracy and significantly less morbidity compared to complete resection of nodal basins. It is almost always performed at the time of initial wide excision, as SLN mapping after lymphatic violation from surgical excision could decrease the accuracy of the test. Recently, the results of MSLT-1, an international, multicenter, phase III trial were published. This study randomized clinically node negative patients to either SLNB at the time of primary melanoma excision (and completion lymphadenectomy if posi-tive) or nodal basin monitoring (and delayed complete lymph-adenectomy for recurrent lymph node disease).142 The results of this study demonstrated that SLNB, with immediate lymphad-enectomy if positive, improved disease-free survival by 7% and 10% in patients with intermediate thickness (1.2–3.5 mm) and thick (>3.5 mm) lesions respectively. The use of SLNB in lesions <1.2 mm thick did not affect disease-free survival. SLNB should also be offered to thin lesions with

1	thickness (1.2–3.5 mm) and thick (>3.5 mm) lesions respectively. The use of SLNB in lesions <1.2 mm thick did not affect disease-free survival. SLNB should also be offered to thin lesions with high-risk features (thickness >0.75, ulceration, mitoses ≥1 per mm2.136 The SLNB involves preoperative lymphoscintigraphy with intradermal injections of technetium-sulfur colloid to delineate lymphatic drainage and intraoperative intradermal injection of 1 mL of isosulfan or methylene blue dye near the tumor or biopsy site. (Figs. 16-13 and 16-14). The radioactive tracer-dye combination allows the sentinel node to be identified in 98% of cases. An incision over the lymph node basin of interest allows nodes to be excised and studied with hematoxylin and eosin and immunohistochemistry (S100, HMB45, and MART-1/Melan-A) staining (Fig. 16-15). 10Brunicardi_Ch16_p0511-p0540.indd 53119/02/19 3:09 PM 532SPECIFIC CONSIDERATIONSPART IIABSentinellymph nodeInjection siteSurgical exposure of sentinel

1	and MART-1/Melan-A) staining (Fig. 16-15). 10Brunicardi_Ch16_p0511-p0540.indd 53119/02/19 3:09 PM 532SPECIFIC CONSIDERATIONSPART IIABSentinellymph nodeInjection siteSurgical exposure of sentinel lymph nodeAfferent lymphaticchannelsSentinellymph nodePrimary melanomaSentinellymphnodeInguinal nodesABCFLOWINJ SITEAxillaryNODEANTFLOWPOSTTymphoMelanoma Primary Injection SiteSubmanibular Lymph nodesPopliteal nodesFigure 16-13. After injection of radioactive technetium-99–labeled sulfur colloid tracer at the primary cutaneous melanoma site, sentinel lymph node basins are identified. A. Lymphoscintig-raphy of 67-year-old male with a malignant melanoma of the right heel; sentinel lymph nodes in both the right popliteal fossa and inguinal region. B. Lymphoscintigraphy of 52-year-old male with a malignant melanoma of the posterior right upper arm; sentinel lymph node in the right axillary region. C. Lymphoscintigraphy of 69-year-old male with a facial melanoma; sentinel lymph nodes in the

1	a malignant melanoma of the posterior right upper arm; sentinel lymph node in the right axillary region. C. Lymphoscintigraphy of 69-year-old male with a facial melanoma; sentinel lymph nodes in the submandibular region. ANT = anterior; INJ = injection; POST = posterior.Risks of this technique are uncommon but include skin necrosis near the site of injection, anaphylactic shock, lymphedema, sur-gical site infections, seromas, and hematomas.Surgical Management of the Primary Tumors and Lymph Nodes. The appropriate excision margin is based on primary tumor thickness. Several retrospective studies suggest that for melanoma in situ, 0.5 to 1 cm margins are sufficient.143-145 We believe that 1-cm margins should be obtained in anatomically fea-sible areas given the possibility of an incidental finding of a small invasive component in permanent sections. Several studies com-pared 1to 3-cm margins and 2to 5-cm margins in melanoma <2 mm thick, and 2to 4-cm margins in melanoma lesions 1 to

1	finding of a small invasive component in permanent sections. Several studies com-pared 1to 3-cm margins and 2to 5-cm margins in melanoma <2 mm thick, and 2to 4-cm margins in melanoma lesions 1 to 4 mm thick and found no difference. 146-149 A British trial suggested that there is a limit to how narrow margins can be for melanomas >2 mm thick by showing that 1-cm margins provide worse outcomes compared to 3-cm margins.150 Tumors <1 mm thick require 0.5 to 1 cm margins. Tumors 1 to 2 mm thick require 1 to 2 cm margins, and tumors >2 mm thick require 2-cm margins.Completion lymphadenectomy is commonly performed in cases of sentinel nodes with metastatic disease, but it has been shown that most of these nodal basins do not have addi-tional disease. Thus, many surgeons do not perform routine completion lymphadenectomy for positive nodes, and data from the MSLT-2 may provide guidance. It has been shown that those patients with nonsentinel lymph node positivity found on completion lymph

1	completion lymphadenectomy for positive nodes, and data from the MSLT-2 may provide guidance. It has been shown that those patients with nonsentinel lymph node positivity found on completion lymph node dissection after a positive SLN have higher rates of recurrence and lower rates of sur-vival. The therapeutic value, however, has not been clearly demonstrated. In patients with clinically positive lymph nodes but absent signs of distant metastasis on PET-CT, therapeu-tic lymph node dissection is associated with 5-year survival rates of 30% to 50%. In these cases, resection of the primary melanoma lesion and a completion lymphadenectomy should be performed.Individuals with face, anterior scalp, and ear prima-ries who have a positive SLNB should undergo a superficial parotidectomy in addition to a modified radical neck dissection. Figure 16-14. Technique of sentinel lymph node biopsy for cutaneous melanoma. A. After injection of radioactive technetium-99–labeled sulfur colloid tracer at

1	to a modified radical neck dissection. Figure 16-14. Technique of sentinel lymph node biopsy for cutaneous melanoma. A. After injection of radioactive technetium-99–labeled sulfur colloid tracer at a lower abdominal wall primary cutaneous melanoma site, B. sentinel lymph node basins are identified. (Reproduced with permission from Gershenwald JE, Ross MI: Sentinel-lymph-node biopsy for cutane-ous melanoma, N Engl J Med. 2011 May 5;364(18):1738-1745.)Brunicardi_Ch16_p0511-p0540.indd 53219/02/19 3:09 PM 533THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16ABFigure 16-15. Operation of sentinel lymph node biopsy for cutaneous melanoma. After preoperative injection of radioactive technetium-99–labeled sulfur colloid tracer and intraoperative injection of Lymphazurin blue dye around the primary melanoma excision site, the nodal basin of interest is identified. An incision is made directly overlying the lymph node basin in the posterior axillary space. The sentinel lymph nodes are identified and

1	excision site, the nodal basin of interest is identified. An incision is made directly overlying the lymph node basin in the posterior axillary space. The sentinel lymph nodes are identified and excised.Patients with positive sentinel nodes in the inguino-femoral nodal basin should undergo an inguino-femoral lymphadenec-tomy that includes removal of Cloquet’s node. If Cloquet’s node is positive or the patient has three or more nodes that contain melanoma metastases the probability of clinically occult posi-tive pelvic nodes is increased. The effect of ileo-obturator lymph node dissection on the survival of these patients is unknown.Surgery for Regional and Distant Metastasis. Nonmeta-static, in-transit disease should undergo excision to clear mar-gins when feasible. However, disease not amenable to complete excision derives benefit from isolated limb perfusion (ILP) and isolated limb infusion (ILI) (Fig. 16-16). These two modali-ties are used to treat regional disease, and their

1	not amenable to complete excision derives benefit from isolated limb perfusion (ILP) and isolated limb infusion (ILI) (Fig. 16-16). These two modali-ties are used to treat regional disease, and their purpose is to administer high doses of chemotherapy, commonly melphalan, to an affected limb while avoiding systemic drug toxicity. ILI was shown to provide a 31% response rate in one study, while hyperthermic ILP provided a 63% complete response rate in an independent study.151-154The most common sites of metastasis of melanoma are the lung and liver. These are followed by the brain, gastroin-testinal tract, distant skin, and subcutaneous tissue. A limited subset of patients with small-volume, limited distant metastases to the brain, gastrointestinal tract, or distant skin can be treated with surgical resection or directed radiation. Liver metastases are better dealt without surgical resection unless they arise from an ocular primary. Adjuvant therapy after resection of meta-static

1	with surgical resection or directed radiation. Liver metastases are better dealt without surgical resection unless they arise from an ocular primary. Adjuvant therapy after resection of meta-static lesions is not standard of care. However, there are ongo-ing clinical trials addressing whether drugs and vaccines will be beneficial in this setting.115 Surgery may provide palliation for patients with gastrointestinal obstruction, gastrointestinal hem-orrhage, and nongastrointestinal hemorrhage. Radiotherapy for symptomatic bony or brain metastases provides palliation in dif-fuse disease.Adjuvant and Palliative Therapies. Eastern Cooperative Oncology Group (ECOG) Trials 1684, 1690, and 1694 were prospective randomized controlled trials that demonstrated Overhead heaterHot air blanketVenouscatheterArterialcatheterPneumatictourniquetPumpchamber25cc SyringeWarmingcoilEsmarchbandageDrug inpre-warmedsalineFigure 16-16. Isolated limb infusion. Schematic of isolated limb infusion of lower

1	SyringeWarmingcoilEsmarchbandageDrug inpre-warmedsalineFigure 16-16. Isolated limb infusion. Schematic of isolated limb infusion of lower extremity. (Adapted with permis-sion from Testori A, Verhoef C, Kroon HM, et al: Treatment of melanoma metas-tases in a limb by isolated limb perfusion and isolated limb infusion, J Surg Oncol. 2011 Sep;104(4):397-404.)Brunicardi_Ch16_p0511-p0540.indd 53319/02/19 3:09 PM 534SPECIFIC CONSIDERATIONSPART IIdisease-free survival advantages in patients with melanoma >4 mm in thickness with or without lymph node involvement if they received adjuvant treatment with high-dose interferon (IFN).155-157 A European Organization for Research and Treat-ment of Cancer (EORTC) trial also showed recurrence-free survival benefit with pegylated IFN.158 It is important to note that IFN therapy is not well tolerated and the pooled analysis of these trials did not show an improvement in overall survival benefit.Most patients with melanoma will not be surgical

1	to note that IFN therapy is not well tolerated and the pooled analysis of these trials did not show an improvement in overall survival benefit.Most patients with melanoma will not be surgical candi-dates. Although medical options for melanoma have historically been poor, several recent studies have shown promise in drug therapy for metastatic melanoma. BRAF inhibitors (sorafenib), anti-PD1 antibodies, CTLA antibodies (ipilimumab), and high-dose interleukin-2 (IL-2) with and without vaccines have been shown in randomized studies to provide survival benefit in metastatic disease.159-165 Despite the excitement of recent drugs, surgery will likely play an adjunct role in treating individuals who develop resistance to these drugs over time.Special Circumstances. Special circumstances of note are melanoma in pregnant women, melanoma of unknown prima-ries, and noncutaneous melanomas. The prognosis of pregnant patients is similar to women who are not pregnant. Extrapo-lation of studies

1	note are melanoma in pregnant women, melanoma of unknown prima-ries, and noncutaneous melanomas. The prognosis of pregnant patients is similar to women who are not pregnant. Extrapo-lation of studies examining the SLNB technique in pregnant women with breast cancer suggests lymphoscintigraphy may be done safely during pregnancy without risk to the fetus (blue dye is contraindicated). General anesthesia should be avoided during the first trimester, and local anesthetics should be used during this time. It has been suggested by some that after excising the primary tumor during pregnancy, the SLNB may be performed after delivery.Unknown primary melanoma occurs in 2% to 5% of cases and most commonly occurs in the lymph nodes. In these cases, a thorough search for the primary lesion should be sought, includ-ing eliciting a history about prior skin lesions, skin procedures (e.g., curettage and electrodessication, excision, laser), and review of any prior “benign” pathology. The surgeon

1	be sought, includ-ing eliciting a history about prior skin lesions, skin procedures (e.g., curettage and electrodessication, excision, laser), and review of any prior “benign” pathology. The surgeon should be aware that melanoma is known to spontaneously regress because of an immune response. Melanoma of unknown pri-mary has survival rates comparable to melanoma diagnosed with a known primary of the same stage.The most common noncutaneous disease site is ocular melanoma, and treatment of this condition includes photocoag-ulation, partial resection, radiation, or enucleation.166-168 Ocular melanomas exclusively metastasize to the liver and not regional lymph nodes, and some patients benefit from liver resection. Melanoma of the mucous membranes most commonly presents in the oral cavity, oropharynx, nasopharynx, paranasal sinus, anus, rectum, and female genitalia. Patients with this presenta-tion have a worse prognosis (10% 5-year survival) than patients with cutaneous melanomas.

1	oropharynx, nasopharynx, paranasal sinus, anus, rectum, and female genitalia. Patients with this presenta-tion have a worse prognosis (10% 5-year survival) than patients with cutaneous melanomas. Management should be excision to negative margins, and radical resections should be avoided because the role of surgery is locoregional control, not cure. Generally speaking, lymph node dissection should be avoided because the benefit is unclear.Merkel Cell CarcinomaMerkel cell carcinoma (MCC) is an aggressive neuroendocrine tumor of the skin whose incidence has been rapidly increas-ing. Although it is a much rarer malignancy than melanoma, the prognosis is much worse, with a 5-year survival of 46%.169 Merkel cells are epidermal appendages involved in the sensation Figure 16-17. Merkel cell carcinoma seen just above the left knee in a 44-year-old female.of light touch, and along with Merkel cell carcinoma, are cyto-keratin-20 positive. This stain is now used to confirm the diag-nosis. Other

1	seen just above the left knee in a 44-year-old female.of light touch, and along with Merkel cell carcinoma, are cyto-keratin-20 positive. This stain is now used to confirm the diag-nosis. Other risk factors include age >65 years (the median age of diagnosis is 70 years), UV exposure, Merkel cell polyoma virus, and immunosuppression. MCC typically presents as a rapidly growing, flesh-colored to red or purple papule or plaque (Fig. 16-17). Regional nodes are involved in 30% of patients at diagnosis, and 50% will develop systemic disease (skin, lymph nodes, liver, lung, bone, and brain).170,171 There are no standard-ized diagnostic imaging studies for staging, but CT of the chest, abdomen, pelvis and octreotide scans may provide useful infor-mation when clinically indicated.After a thorough skin examination, treatment should begin by evaluating nodal basins. Patients without clinical nodal dis-ease should undergo an SLNB prior to wide local excision because studies suggest a benefit.172

1	skin examination, treatment should begin by evaluating nodal basins. Patients without clinical nodal dis-ease should undergo an SLNB prior to wide local excision because studies suggest a benefit.172 In patients with sentinel lymph nodes with metastatic disease, completion lymphad-enectomy and/or radiation therapy may follow, and in patients with node-negative disease, observation or radiation therapy should be considered.172 SLNB is important for staging and treatment, and the literature suggests that it predicts recurrenceand relapse-free survival. Elective lymph node dissection may decrease regional nodal recurrence and in-transit metastases. Patients with clinically positive nodes should have an FNA to confirm disease. If positive, a metastatic staging workup should follow, and, if negative, treatment of the primary and nodal basin as managed for sentinel lymph node-positive disease should be considered. A negative FNA and open biopsy-negative disease should be managed by

1	if negative, treatment of the primary and nodal basin as managed for sentinel lymph node-positive disease should be considered. A negative FNA and open biopsy-negative disease should be managed by treatment of the primary disease alone. Brunicardi_Ch16_p0511-p0540.indd 53419/02/19 3:09 PM 535THE SKIN AND SUBCUTANEOUS TISSUECHAPTER 16Patients with metastatic disease should be managed according to consensus from a multidisciplinary tumor board.Important surgical principles for excision of the primary lesion are to excise with wide margins down to fascia and com-plete circumferential and peripheral deep-margin assessment. Recommended management for margins is 1 to 3 cm, but there are no randomized trials defining these margins. Chemotherapy and adjuvant radiation are commonly used, but there are no data to support a specific regimen or that demonstrate a definitive survival benefit.Recurrence of MCC is common. One study of 95 patients showed a 47% recurrence, with 80% of recurrences

1	are no data to support a specific regimen or that demonstrate a definitive survival benefit.Recurrence of MCC is common. One study of 95 patients showed a 47% recurrence, with 80% of recurrences occurring within 2 years and 96% occurring within 5 years.173,174 Regional lymph node disease is common, and 70% of patients will have nodal spread within 2 years of disease presentation. Five-year overall survival of head and neck disease in surgically treated patients is between 40% and 68%.Kaposi’s SarcomaKaposi’s sarcoma is characterized by the proliferation and inflammation of endothelial-derived spindle cell lesions. There are five major forms of this angioproliferative disorder: classic (Mediterranean), African endemic, HIV-negative men having sex with men (MSM)-associated, and immunosuppression-associated. They are all driven by the human herpesvirus (HHV-8).175 Kaposi’s sarcoma is diagnosed after the fifth decade of life and predominantly found on the skin but can occur anywhere in

1	They are all driven by the human herpesvirus (HHV-8).175 Kaposi’s sarcoma is diagnosed after the fifth decade of life and predominantly found on the skin but can occur anywhere in the body. In North America, the Kaposi’s sarcoma herpes virus is transmitted via sexual and nonsexual routes and predominantly affects individuals with compromised immune systems such as those with HIV and transplant recipients on immune-suppressing medications. Clinically, Kaposi’s sarcoma appears as multifocal, rubbery blue-red nodules. Treatment of AIDS-associated Kaposi’s sarcoma is with antiviral therapy, and many patients experience a dramatic treatment response.176,177 Those individuals who do not respond and have limited muco-cutaneous disease may benefit from cryotherapy, photodynamic therapy, radiation therapy, intralesional injections, and topical therapy. Surgical biopsy is important for disease diagnosis, but given the high local recurrence and the fact that Kaposi’s sar-coma represents more

1	therapy, intralesional injections, and topical therapy. Surgical biopsy is important for disease diagnosis, but given the high local recurrence and the fact that Kaposi’s sar-coma represents more of a systemic rather than local disease, the benefit of surgery is limited and generally should not be pursued except for palliation.Dermatofibrosarcoma ProtuberansThis rare, low-grade sarcoma of fibroblast origin commonly afflicts individuals during their third decade of life. It has low distant metastatic potential, but it behaves aggressively locally with finger-like extensions. Tumor depth is the most important prognostic variable. Presentation is characteristically a slow-growing, asymptomatic, violaceous plaque involving the trunk, head, neck, or extremities (Fig. 16-18). Nearly all cases are posi-tive for CD34 and negative for factor XIIIa.178,179 Treatment is wide local excision with 3-cm margins down to deep underly-ing fascia or Mohs microsurgery in cosmetically sensitive areas

1	are posi-tive for CD34 and negative for factor XIIIa.178,179 Treatment is wide local excision with 3-cm margins down to deep underly-ing fascia or Mohs microsurgery in cosmetically sensitive areas where maximum tissue preservation will benefit.180 No nodal dissection is needed, and both approaches provide similar local control.181 Some clinicians have used radiation therapy and bio-logic agents (imatinib) as adjuvant therapy with some success in patients with advanced disease. Local recurrence occurs in 50% to 75% of cases, usually within 3 years of treatment. Thus, clini-cal follow-up is important. Recurrent tumors should be resected whenever possible.Figure 16-18. Dermatofibrosarcoma protuberans of the left flank.Malignant Fibrous Histiocytoma (Undifferentiated Pleomorphic Sarcoma and Myxofibrosarcoma)This uncommon, cutaneous, spindle-cell, soft tissue sarcoma occurs in the extremities, head, and neck of elderly patients. They present as solitary, soft to firm, skin-colored

1	and Myxofibrosarcoma)This uncommon, cutaneous, spindle-cell, soft tissue sarcoma occurs in the extremities, head, and neck of elderly patients. They present as solitary, soft to firm, skin-colored subcutane-ous nodules. Complete surgical resection is the treatment of choice, and adjuvant radiation therapy provides local control; patients with positive margins benefit most from this combina-tion. Nevertheless, patients undergoing complete gross resection will experience recurrence in 30% to 35% of cases.135 Up to 50% of patients may present with distant metastasis, and this is a contraindication to surgical resection.AngiosarcomaAngiosarcoma is an uncommon, aggressive cancer that arises from vascular endothelial cells and occurs in four variants, all of which have a poor prognosis.182 The 5-year survival estimate is 15%.183 The head and neck variant presents in individuals older than 40 years as an ill-defined red patch on the face or scalp, often with satellite lesions and distant

1	5-year survival estimate is 15%.183 The head and neck variant presents in individuals older than 40 years as an ill-defined red patch on the face or scalp, often with satellite lesions and distant metastasis, and has a median survival of 18 to 28 months. Lymphedema-associated angiosarcoma (Stewart-Treves) develops on an extremity ipsi-lateral to an axillary lymphadenectomy. It appears on the upper, medial arm as a violaceous plaque in an individual with nonpit-ting edema and has a poor survival. Radiation-induced angio-sarcoma occurs 4 to 25 years after radiation therapy for benign and malignant conditions. Finally, the epithelioid variant of angiosarcoma involves the lower extremities and also has a poor prognosis. Surgical excision with wide margins is the treatment Brunicardi_Ch16_p0511-p0540.indd 53519/02/19 3:09 PM 536SPECIFIC CONSIDERATIONSPART IIof choice for localized disease, but the rate of recurrence is high. Adjuvant radiation therapy can be considered in a

1	53519/02/19 3:09 PM 536SPECIFIC CONSIDERATIONSPART IIof choice for localized disease, but the rate of recurrence is high. Adjuvant radiation therapy can be considered in a multidisci-plinary fashion. Cases of extremity disease can be considered for amputation. For widely metastatic disease, chemotherapy and radiation may provide palliation, but these modalities do not prolong overall survival.115Extramammary Paget’s DiseaseThis rare adenocarcinoma of apocrine glands arises in axillary, perianal, and genital regions of men and women.184 Clinical pre-sentation is that of erythematous or nonpigmented plaques with an eczema-like appearance that often persist after failed treat-ment from other therapies. An important characteristic and one that the surgeon must be acutely aware of is the high incidence of concomitant other malignancies with this cutaneous disease. Forty percent of cases are associated with primary gastrointesti-nal and genitourinary malignancies, and a diligent search

1	incidence of concomitant other malignancies with this cutaneous disease. Forty percent of cases are associated with primary gastrointesti-nal and genitourinary malignancies, and a diligent search should be made after a diagnosis of extramammary Paget’s disease is made. Treatment is surgical resection with negative microscopic margins, and adjuvant radiation may provide additional locore-gional control.CONCLUSIONThe skin is the largest organ in the human body and is com-posed of three organized layers that are the source of numer-ous pathologies. Recognition and management of cutaneous and subcutaneous diseases require an astute clinician to opti-mize clinical outcomes. Improvements in drugs, therapies, and healthcare practices have helped recovery from skin injuries. Skin and subcutaneous diseases are often managed medically, although surgery frequently complements treatment. Benign tumors are surgical diseases, while malignant tumors are pri-marily treated surgically, and additional

1	diseases are often managed medically, although surgery frequently complements treatment. Benign tumors are surgical diseases, while malignant tumors are pri-marily treated surgically, and additional modalities including chemotherapy and radiation therapy are sometimes required. The management of melanoma is at an exciting phase, requiring the coordinated multidisciplinary care of medical oncologists, surgical oncologists, radiation oncologists, der-matopathologists, and plastic and reconstructive surgeons. The advent of new drug therapies will redefine the role of surgery in this disease in the coming years.REFERENCESEntries highlighted in bright blue are key references. 1. Kanitakis J. Anatomy, histology and immunohistochemistry of normal human skin. Eur J Dermatology. 2002;12(4):390-401. 2. Chug D, Hake A, Holbrook K. The structure and development of skin. In: Freedberg I, Eisen A, Wolff K, eds. Fitzpatrick’s Dermatology in General Medicine. 6th ed. New York: McGraw-Hill;

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1	A-N, Wang J, Lema B, Kraybill WG, Zeitouni NC, Kane JM 3rd. Wide excision or Mohs micrographic sur-gery for the treatment of primary dermatofibrosarcoma protu-berans. Am J Clin Oncol. 2009;33(3):1. 182. Requena L, Sangueza OP. Cutaneous vascular proliferations. Part III. Malignant neoplasms, other cutaneous neoplasms with significant vascular component, and disorders errone-ously considered as vascular neoplasms. J Am Acad Dermatol. 1998;38(2 pt 1):143-175. 183. Holden CA, Spittle MF, Jones EW. Angiosarcoma of the face and scalp, prognosis and treatment. Cancer. 1987;59(5):1046-1057. 184. Wagner G, Sachse MM. Extramammary Paget disease— clinical appearance, pathogenesis, management. JDDG J der Dtsch Dermatologischen Gesellschaft. 2011;9(6):448-454.Brunicardi_Ch16_p0511-p0540.indd 54019/02/19 3:09 PM

1	The BreastCatherine C. Parker, Senthil Damodaran, Kirby I. Bland, and Kelly K. Hunt 17chapterA BRIEF HISTORY OF BREAST CANCER THERAPYBreast cancer has captured the attention of surgeons throughout the ages. The Smith Surgical Papyrus (3000–2500 b.c.) is the earliest known document to refer to breast cancer. The cancer was in a man, but the description encompassed most of the common clinical features. In reference to this cancer, the author concluded, “There is no treatment.”1 There were few other historical references to breast cancer until the first century. In De Medicina, Celsus commented on the value of operations for early breast cancer: “None of these may be removed but the cacoethes (early cancer), the rest are irritated by every method of cure. The more violent the operations are, the more angry they grow.”2 In the second century, Galen inscribed his classical clinical observation: “We have often seen in the breast a tumor exactly resembling the animal the crab. Just as the

1	the more angry they grow.”2 In the second century, Galen inscribed his classical clinical observation: “We have often seen in the breast a tumor exactly resembling the animal the crab. Just as the crab has legs on both sides of his body, so in this disease the veins extending out from the unnatural growth take the shape of a crab’s legs. We have often cured this disease in its early stages, but after it has reached a large size, no one has cured it. In all operations we attempt to excise the tumor in a circle where it borders on the healthy tissue.”3The Galenic system of medicine ascribed cancers to an excess of black bile and concluded that excision of a local bodily outbreak could not cure the systemic imbalance. Theories espoused by Galen dominated medicine until the Renaissance. In 1652, Tulp introduced the idea that cancer was contagious when he reported an elderly woman and her housemaid who both developed breast cancer (N. Tulp, Observationes medi-cae 1652). This single

1	In 1652, Tulp introduced the idea that cancer was contagious when he reported an elderly woman and her housemaid who both developed breast cancer (N. Tulp, Observationes medi-cae 1652). This single incidence was accepted as conclusive A Brief History of Breast Cancer Therapy541Embryology and Functional Anatomy of the Breast543Embryology / 543Functional Anatomy / 544Physiology of the Breast547Breast Development and Function / 547Pregnancy, Lactation, and Senescence / 548Gynecomastia / 549Infectious and Inflammatory Disorders of the Breast550Bacterial Infection / 550Mycotic Infections / 550Hidradenitis Suppurativa / 550Mondor’s Disease / 550Common Benign Disorders and Diseases of the Breast551Aberrations of Normal Development and Involution / 551Pathology of Nonproliferative Disorders / 552Pathology of Proliferative Disorders Without Atypia / 553Pathology of Atypical Proliferative Diseases / 553Treatment of Selected Benign Breast Disorders and Diseases / 554Risk Factors for Breast

1	/ 552Pathology of Proliferative Disorders Without Atypia / 553Pathology of Atypical Proliferative Diseases / 553Treatment of Selected Benign Breast Disorders and Diseases / 554Risk Factors for Breast Cancer555Hormonal and Nonhormonal Risk Factors / 555Risk Assessment Models / 555Risk Management / 556BRCA Mutations / 558Epidemiology and Natural History of Breast Cancer561Epidemiology / 561Natural History / 562Histopathology of Breast Cancer563Carcinoma In Situ / 563Invasive Breast Carcinoma / 565Diagnosis of Breast Cancer567Examination / 567Imaging Techniques / 567Breast Biopsy / 574Breast Cancer Staging and Biomarkers575Breast Cancer Staging / 575Biomarkers / 575Overview of Breast Cancer Therapy580In Situ Breast Cancer (Stage 0) / 580Early Invasive Breast Cancer (Stage I, IIA, or IIB) / 582Advanced Local-Regional Breast Cancer (Stage IIIA or IIIB) / 585Internal Mammary Lymph Nodes / 587Distant Metastases (Stage IV) / 587Local-Regional Recurrence / 587Breast Cancer Prognosis /

1	IIB) / 582Advanced Local-Regional Breast Cancer (Stage IIIA or IIIB) / 585Internal Mammary Lymph Nodes / 587Distant Metastases (Stage IV) / 587Local-Regional Recurrence / 587Breast Cancer Prognosis / 587Surgical Techniques in Breast Cancer Therapy588Excisional Biopsy With Needle Localization / 588Sentinel Lymph Node Dissection / 590Breast Conservation / 591Mastectomy and Axillary Dissection / 591Modified Radical Mastectomy / 592Reconstruction of the Breast and Chest Wall / 593Nonsurgical Breast Cancer Therapies594Radiation Therapy / 594Chemotherapy Adjuvant / 594Antiestrogen Therapy / 597Ablative Endocrine Therapy / 598Anti-HER2 Therapy / 598Special Clinical Situations599Nipple Discharge / 599Axillary Lymph Node Metastases in the Setting of an Unknown Primary Cancer / 600Breast Cancer During Pregnancy / 600Male Breast Cancer / 600Phyllodes Tumors / 600Inflammatory Breast Carcinoma / 601Rare Breast Cancers / 602Brunicardi_Ch17_p0541-p0612.indd 54101/03/19 5:04 PM 542evidence

1	During Pregnancy / 600Male Breast Cancer / 600Phyllodes Tumors / 600Inflammatory Breast Carcinoma / 601Rare Breast Cancers / 602Brunicardi_Ch17_p0541-p0612.indd 54101/03/19 5:04 PM 542evidence and started an idea which persisted into the 20th century among some lay people. The majority of respected sur-geons considered operative intervention to be a futile and ill-advised endeavor. The Renaissance and the wars of the 16th and 17th centuries brought developments in surgery, particularly in anatomical understanding. However, there were no new theories espoused in relation to cancer. Beginning with Morgagni, surgi-cal resections were more frequently undertaken, including some early attempts at mastectomy and axillary dissection. The 17th century saw the start of the Age of Enlightenment, which lasted until the 19th century. In terms of medicine, this resulted in the abandonment of Galen’s humoral pathology, which was repudi-ated by Le Dran, and the subsequent rise in cellular

1	which lasted until the 19th century. In terms of medicine, this resulted in the abandonment of Galen’s humoral pathology, which was repudi-ated by Le Dran, and the subsequent rise in cellular pathology as espoused by Virchow. Le Dran stated that breast cancer was a local disease that spread by way of lymph vessels to axillary lymph nodes. When operating on a woman with breast cancer, he routinely removed any enlarged axillary lymph nodes.4In the 19th century, Moore, of the Middlesex Hospital, London, emphasized complete resection of the breast for cancer and stated that palpable axillary lymph nodes also should be removed.5 In a presentation before the British Medical Asso-ciation in 1877, Banks supported Moore’s concepts and advo-cated the resection of axillary lymph nodes even when palpable lymphadenopathy was not evident, recognizing that occult involvement of axillary lymph nodes was frequently present. In 1894, Halsted and Meyer reported their operations for treatment of breast

1	lymphadenopathy was not evident, recognizing that occult involvement of axillary lymph nodes was frequently present. In 1894, Halsted and Meyer reported their operations for treatment of breast cancer.6 By demonstrating superior local-regional con-trol rates after radical resection, these surgeons established radi-cal mastectomy as state-of-the-art treatment for that era. Halsted and Meyer advocated complete dissection of axillary lymph node levels I to III. Both routinely resected the long thoracic nerve and the thoracodorsal neurovascular bundle with the axil-lary contents. In 1943, Haagensen and Stout described the grave signs of breast cancer, which included: (a) edema of the skin of the breast, (b) skin ulceration, (c) chest wall fixation, (d) an axillary lymph node >2.5 cm in diameter, and (e) fixed axillary lymph nodes. Women with two or more signs had a 42% local recurrence rate and only a 2% 5-year disease-free survival rate.7 Based on these findings, they declared that women

1	and (e) fixed axillary lymph nodes. Women with two or more signs had a 42% local recurrence rate and only a 2% 5-year disease-free survival rate.7 Based on these findings, they declared that women with grave signs were beyond cure by radical surgery. In 1948, Patey and Dyson of the Middlesex Hospital, London, advocated a modi-fied radical mastectomy for the management of advanced oper-able breast cancer, explaining, “Until an effective general agent for treatment of carcinoma of the breast is developed, a high proportion of these cases are doomed to die.”8 Their technique included removal of the breast and axillary lymph nodes with preservation of the pectoralis major muscle. They showed that removal of the pectoralis minor muscle allowed access to and clearance of axillary lymph node levels I to III.During the 1970s, there was a transition from the Halsted radical mastectomy to the modified radical mastectomy as the surgical procedure most frequently used by American surgeons to

1	levels I to III.During the 1970s, there was a transition from the Halsted radical mastectomy to the modified radical mastectomy as the surgical procedure most frequently used by American surgeons to treat breast cancer. This transition acknowledged that: (a) fewer patients were presenting with advanced local disease with or without the grave signs described by Haagensen, (b) extirpation of the pectoralis major muscle was not essential for local-regional control in stages I and II breast cancer, and (c) neither the modified radical mastectomy nor the Halsted radi-cal mastectomy consistently achieved local-regional control of stage III breast cancer. Radiation therapy was incorporated into the management of advanced breast cancer and demonstrated improvements in local-regional control. The National Surgical Key Points1 The breast receives its principal blood supply from per-forating branches of the internal mammary artery, lateral branches of the posterior intercostal arteries, and

1	The National Surgical Key Points1 The breast receives its principal blood supply from per-forating branches of the internal mammary artery, lateral branches of the posterior intercostal arteries, and branches from the axillary artery, including the highest thoracic, lat-eral thoracic, and pectoral branches of the thoracoacromial artery.2 The axillary lymph nodes usually receive >75% of the lymph drainage from the breast, and the rest flows through the lymph vessels that accompany the perforating branches of the internal mammary artery and enters the parasternal (internal mammary) group of lymph nodes.3 Breast development and function are initiated by a variety of hormonal stimuli, with the major trophic effects being modulated by estrogen, progesterone, and prolactin.4 Benign breast disorders and diseases are related to the nor-mal processes of reproductive life and to involution, and there is a spectrum of breast conditions that ranges from normal to disorder to disease (aberrations

1	and diseases are related to the nor-mal processes of reproductive life and to involution, and there is a spectrum of breast conditions that ranges from normal to disorder to disease (aberrations of normal devel-opment and involution classification).5 To calculate breast cancer risk using the Gail model, a woman’s risk factors are translated into an overall risk score by multiplying her relative risks from several cat-egories. This risk score is then compared with an adjusted population risk of breast cancer to determine the wom-an’s individual risk. This model is not appropriate for use in women with a known BRCA1 or BRCA2 mutation or women with lobular or ductal carcinoma in situ.6 Routine use of screening mammography in women ≥50 years of age reduces mortality from breast cancer by 25%. Magnetic resonance imaging (MRI) screening is recom-mended in women with BRCA mutations and may be con-sidered in women with a greater than 20% to 25% lifetime risk of developing breast

1	by 25%. Magnetic resonance imaging (MRI) screening is recom-mended in women with BRCA mutations and may be con-sidered in women with a greater than 20% to 25% lifetime risk of developing breast cancer.7 Core-needle biopsy is the preferred method for diagnosis of palpable or nonpalpable breast abnormalities.8 When a diagnosis of breast cancer is made, the surgeon should determine the clinical stage, histologic characteris-tics, and appropriate biomarker levels before initiating local therapy.9 Sentinel node dissection is the preferred method for stag-ing of the regional lymph nodes in women with clinically node-negative invasive breast cancer. Axillary dissection may be avoided in women with one to two positive senti-nel nodes who are treated with breast conserving surgery, whole breast radiation, and systemic therapy.10 Local-regional and systemic therapy decisions for an indi-vidual patient with breast cancer are best made using a multidisciplinary treatment approach. The sequencing

1	and systemic therapy.10 Local-regional and systemic therapy decisions for an indi-vidual patient with breast cancer are best made using a multidisciplinary treatment approach. The sequencing of therapies is dependent on patient and tumor related factors including breast cancer subtype.Brunicardi_Ch17_p0541-p0612.indd 54201/03/19 5:04 PM 543THE BREASTCHAPTER 17Adjuvant Breast and Bowel Project (NSABP) conducted a ran-domized trial in the early 1970s to determine the impact of local and regional treatments on survival in operable breast cancer. In the B-04 trial, 1665 women were enrolled and stratified by clinical assessment of the axillary lymph nodes. The clinically node-negative women were randomized into three treatment groups: (a) Halsted radical mastectomy; (b) total mastectomy plus radiation therapy; and (c) total mastectomy alone. Clini-cally node-positive women were randomized to Halsted radical mastectomy or total mastectomy plus radiation therapy. This trial accrued

1	plus radiation therapy; and (c) total mastectomy alone. Clini-cally node-positive women were randomized to Halsted radical mastectomy or total mastectomy plus radiation therapy. This trial accrued patients between 1971 and 1974, an era that pre-dated widespread availability of effective systemic therapy for breast cancer and therefore reflect survival associated with local-regional therapy alone. There were no differences in survival between the three groups of node-negative women or between the two groups of node-positive women. These overall survival equivalence patterns persisted at 25 years of follow-up.9The next major advance in the surgical management of breast cancer was the development of breast conserving surgery. Breast conserving surgery and radium treatment was first reported by Geoffrey Keynes of St Bartholomew’s Hospital, London in the British Medical Journal in 1937.10 Several decades later, the NSABP launched the B-06 trial, a phase 3 study that randomized 1851

1	by Geoffrey Keynes of St Bartholomew’s Hospital, London in the British Medical Journal in 1937.10 Several decades later, the NSABP launched the B-06 trial, a phase 3 study that randomized 1851 patients to total mastec-tomy, lumpectomy alone, or lumpectomy with breast irradia-tion. The results showed no difference in disease-free, distant disease-free, and overall survival among the three groups; how-ever, the omission of radiation therapy resulted in significantly higher rates of ipsilateral breast tumor recurrence in those who received lumpectomy alone.11 The B-06 trial excluded patients who had palpable axillary lymph nodes, and those patients randomized to breast conserving surgery had frozen sections performed. If on frozen section the margins were involved, the surgeon proceeded to perform a mastectomy, but the patient was included in the analysis as having had a breast conserv-ing operation. Furthermore, in B-06, local in-breast recurrences were regarded as “nonevents” in terms

1	a mastectomy, but the patient was included in the analysis as having had a breast conserv-ing operation. Furthermore, in B-06, local in-breast recurrences were regarded as “nonevents” in terms of disease-free survival. Both the NSABP B-04 and B-06 trials were taken to refute the Halstedian concept that cancer spread throughout a region of the breast to lymphatics and then on to distant sites. Bernard Fisher proposed the “alternative hypothesis” that breast cancer was a systemic disease at diagnosis and that tumor cells had access to both the blood and lymphatic systems and that regional lymph nodes were a marker of systemic disease and not a barrier to the dissemination of cancer cells. He proposed that host factors were important in the development of metastasis and that varia-tions in the local-regional approach to breast cancer treatment were not likely to substantially impact survival. This idea was dominant for a number of years but has been challenged by the Early Breast Cancer

1	local-regional approach to breast cancer treatment were not likely to substantially impact survival. This idea was dominant for a number of years but has been challenged by the Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) overview analysis, which reported that “the avoidance of recur-rence in a conserved breast . . . avoids about one breast cancer death over the next 15 years for every four such recurrences avoided,”12 indicating that not all breast cancer is a systemic disease at presentation.During the 1970s, clinical trials were initiated to determine the value of systemic therapy in the postoperative setting as an adjuvant to surgery. The EBCTCG was established in 1985 to coordinate the meta-analysis of data from randomized clinical trials in order to examine the impact of adjuvant treatments for breast cancer on recurrence and mortality. The EBCTCG overview has demonstrated that anthracycline containing regimens are superior to cyclophosphamide, methotrexate, and

1	of adjuvant treatments for breast cancer on recurrence and mortality. The EBCTCG overview has demonstrated that anthracycline containing regimens are superior to cyclophosphamide, methotrexate, and 5-fluorouracil (CMF), and more recently, that the addition of a taxane to an anthracycline-based regimen reduces breast cancer mortality by one-third.11 The overview has also demonstrated that tamoxifen is of benefit only in patients with estrogen recep-tor (ER) positive breast cancer and that tamoxifen may decrease mortality from breast cancer by as much as 30%.13 Importantly, the EBCTCG data have shown that proportional reduction in risk was not significantly affected by standard clinical and pathologic factors such as tumor size, ER status, and nodal status.14 This underscores the importance of stratification of risk in determining adjuvant therapy decisions in order to minimize the toxicities of therapies in those unlikely to benefit, yet real-ize the substantial benefits gained in

1	of stratification of risk in determining adjuvant therapy decisions in order to minimize the toxicities of therapies in those unlikely to benefit, yet real-ize the substantial benefits gained in local-regional control and survival in those at higher risk.Many early randomized clinical trials considered all patients similarly in terms of treatment viewing breast cancer as more of a homogeneous disease. Breast cancer has traditionally been defined by pathologic determinants using conventional light microscopy and basic histologic techniques. In the 1980s, immunohistochemistry allowed assessment of the expression of individual tumor markers (most commonly proteins) while DNA was initially assessed in terms of its ploidy status. Sub-sequently, breast cancer specimens have been interrogated at the level of the DNA by labeling genes of interest and allow-ing fluorescent dyes to quantify the abundance of a particular gene and comparing a large number of genes simultaneously in a single

1	at the level of the DNA by labeling genes of interest and allow-ing fluorescent dyes to quantify the abundance of a particular gene and comparing a large number of genes simultaneously in a single breast cancer specimen. Gene expression arrays have shown that breast cancers cluster according to their intrinsic gene expression patterns into at least five intrinsic subtypes and these intrinsic subtypes correlate with breast cancer outcomes.15 Breast cancers are now classified by molecular subtypes and these are being used for risk stratification and decision making in terms of local-regional and systemic therapies.Currently, 50% of American women will consult a sur-geon regarding breast disease, 25% will undergo breast biopsy for diagnosis of an abnormality, and 12% will develop some variant of breast cancer. Considerable progress has been made in the integration of surgery, radiation therapy, and systemic therapy to control local-regional disease, enhance survival, and improve the

1	of breast cancer. Considerable progress has been made in the integration of surgery, radiation therapy, and systemic therapy to control local-regional disease, enhance survival, and improve the quality of life of breast cancer survivors. Surgeons are traditionally the first physician consulted for breast care, and it is critical for them to be well trained in all aspects of the breast from embryologic development, to growth and development, to benign and malignant disease processes. This will allow the greatest opportunity to achieve optimal outcomes for patients and their families.EMBRYOLOGY AND FUNCTIONAL ANATOMY OF THE BREASTEmbryologyAt the fifth or sixth week of fetal development, two ventral bands of thickened ectoderm (mammary ridges, milk lines) are evident in the embryo.16 In most mammals, paired breasts develop along these ridges, which extend from the base of the forelimb (future axilla) to the region of the hind limb (inguinal area). These ridges are not prominent in the

1	mammals, paired breasts develop along these ridges, which extend from the base of the forelimb (future axilla) to the region of the hind limb (inguinal area). These ridges are not prominent in the human embryo and disappear after a short time, except for small portions that may persist in the pectoral region. Accessory breasts (polymastia) or accessory nipples (polythelia) may Brunicardi_Ch17_p0541-p0612.indd 54301/03/19 5:04 PM 544SPECIFIC CONSIDERATIONSPART IIFigure 17-1. The mammary milk line. (Visual Art: © 2013. The University of Texas MD Anderson Cancer Center.)Figure 17-2. Anatomy of the breast. Tangential and cross-sectional (sagittal) views of the breast and associated chest wall. (Reproduced with permission from Bland KI, Copeland EMI: The Breast: Comprehensive Management of Benign and Malignant Diseases, 4th ed. Philadelphia, PA: Elsevier/Saunders; 2009.)occur along the milk line (Fig. 17-1) when normal regression fails. Each breast develops when an ingrowth of

1	of Benign and Malignant Diseases, 4th ed. Philadelphia, PA: Elsevier/Saunders; 2009.)occur along the milk line (Fig. 17-1) when normal regression fails. Each breast develops when an ingrowth of ectoderm forms a primary tissue bud in the mesenchyme. The primary bud, in turn, initiates the development of 15 to 20 secondary buds. Epithelial cords develop from the secondary buds and extend into the surrounding mesenchyme. Major (lactiferous) ducts develop, which open into a shallow mammary pit. Dur-ing infancy, a proliferation of mesenchyme transforms the mammary pit into a nipple. If there is failure of a pit to elevate above skin level, an inverted nipple results. This congenital malformation occurs in 4% of infants. At birth, the breasts are identical in males and females, demonstrating only the pres-ence of major ducts. Enlargement of the breast may be evi-dent, and a secretion, historically referred to as witch’s milk, may be produced. These transitory events occur in response to

1	only the pres-ence of major ducts. Enlargement of the breast may be evi-dent, and a secretion, historically referred to as witch’s milk, may be produced. These transitory events occur in response to maternal hormones that cross the placenta.The breast remains undeveloped in the female until puberty, when it enlarges in response to ovarian estrogen and progesterone, which initiate proliferation of the epithelial and connective tissue elements. However, the breasts remain incompletely developed until pregnancy occurs. Absence of the breast (amastia) is rare and results from an arrest in mam-mary ridge development that occurs during the sixth fetal week. Poland’s syndrome consists of hypoplasia or complete absence of the breast, costal cartilage and rib defects, hypoplasia of the subcutaneous tissues of the chest wall, and brachysyndactyly. Breast hypoplasia also may be iatrogenically induced before puberty by trauma, infection, or radiation therapy. Symmastia is a rare anomaly

1	tissues of the chest wall, and brachysyndactyly. Breast hypoplasia also may be iatrogenically induced before puberty by trauma, infection, or radiation therapy. Symmastia is a rare anomaly recognized as webbing between the breasts across the midline. Accessory nipples (polythelia) occur in <1% of infants and may be associated with abnormalities of the urinary and cardiovascular systems. Supernumerary breasts may occur in any configuration along the mammary milk line but most frequently occur between the normal nipple location and the symphysis pubis. Turner’s syndrome (ovarian agenesis and dysgenesis) and Fleischer’s syndrome (displacement of the nipples and bilateral renal hypoplasia) may have polymastia as a component. Accessory axillary breast tissue is uncommon and usually is bilateral.Functional AnatomyThe breast is composed of 15 to 20 lobes (Fig. 17-2), which are each composed of several lobules.17 Fibrous bands of con-nective tissue travel through the breast (Cooper’s

1	bilateral.Functional AnatomyThe breast is composed of 15 to 20 lobes (Fig. 17-2), which are each composed of several lobules.17 Fibrous bands of con-nective tissue travel through the breast (Cooper’s suspensory ligaments), insert perpendicularly into the dermis, and provide structural support. The mature female breast extends from the level of the second or third rib to the inframammary fold at the sixth or seventh rib. It extends transversely from the lateral border of the sternum to the anterior axillary line. The deep or posterior surface of the breast rests on the fascia of the pecto-ralis major, serratus anterior, and external oblique abdominal muscles, and the upper extent of the rectus sheath. The retro-mammary bursa may be identified on the posterior aspect of the breast between the investing fascia of the breast and the fascia of the pectoralis major muscles. The axillary tail of Spence extends laterally across the anterior axillary fold. The upper outer quad-rant of the

1	the investing fascia of the breast and the fascia of the pectoralis major muscles. The axillary tail of Spence extends laterally across the anterior axillary fold. The upper outer quad-rant of the breast contains a greater volume of tissue than do the other quadrants. The breast has a protuberant conical form. The base of the cone is roughly circular, measuring 10 to 12 cm in diameter. Considerable variations in the size, contour, and den-sity of the breast are evident among individuals. The nulliparous breast has a hemispheric configuration with distinct flattening above the nipple. With the hormonal stimulation that accom-panies pregnancy and lactation, the breast becomes larger and increases in volume and density, whereas with senescence, it assumes a flattened, flaccid, and more pendulous configuration with decreased volume.Nipple-Areola Complex. The epidermis of the nipple-are-ola complex is pigmented and is variably corrugated. During puberty, the pigment becomes darker and the

1	configuration with decreased volume.Nipple-Areola Complex. The epidermis of the nipple-are-ola complex is pigmented and is variably corrugated. During puberty, the pigment becomes darker and the nipple assumes an elevated configuration. Throughout pregnancy, the areola Brunicardi_Ch17_p0541-p0612.indd 54401/03/19 5:04 PM 545THE BREASTCHAPTER 17Figure 17-3. Inactive human breast (100x). The epithelium, which is primarily ductal, is embedded in loose connective tissue. Dense connective tissue surrounds the terminal duct lobular units (TDLU). (Used with permission from Dr. Sindhu Menon, Consultant Histo-pathologist and Dr. Rahul Deb, Consultant Histopathologist and Lead Breast Pathologist, Royal Derby Hospital, Derby, UK.)Figure 17-4. Active human breast: pregnancy and lactation (160x). The alveolar epithelium becomes conspicuous during the early pro-liferative period. The alveolus is surrounded by cellular connective tissue. (Used with permission from Dr. Sindhu Menon, Consultant

1	The alveolar epithelium becomes conspicuous during the early pro-liferative period. The alveolus is surrounded by cellular connective tissue. (Used with permission from Dr. Sindhu Menon, Consultant Histopathologist and Dr. Rahul Deb, Consultant Histopathologist and Lead Breast Pathologist, Royal Derby Hospital, Derby, UK.)enlarges and pigmentation is further enhanced. The areola con-tains sebaceous glands, sweat glands, and accessory glands, which produce small elevations on the surface of the areola (Montgomery’s tubercles). Smooth muscle bundle fibers, which lie circumferentially in the dense connective tissue and longi-tudinally along the major ducts, extend upward into the nipple, where they are responsible for the nipple erection that occurs with various sensory stimuli. The dermal papilla at the tip of the nipple contains numerous sensory nerve endings and Meiss-ner’s corpuscles. This rich sensory innervation is of functional importance because the sucking of the infant

1	dermal papilla at the tip of the nipple contains numerous sensory nerve endings and Meiss-ner’s corpuscles. This rich sensory innervation is of functional importance because the sucking of the infant initiates a chain of neurohumoral events that results in milk letdown.Inactive and Active Breast. Each lobe of the breast termi-nates in a major (lactiferous) duct (2–4 mm in diameter), which opens through a constricted orifice (0.4–0.7 mm in diameter) into the ampulla of the nipple (see Fig. 17-2). Immediately below the nipple-areola complex, each major duct has a dilated portion (lactiferous sinus), which is lined with stratified squa-mous epithelium. Major ducts are lined with two layers of cuboidal cells, whereas minor ducts are lined with a single layer of columnar or cuboidal cells. Myoepithelial cells of ectoder-mal origin reside between the epithelial cells in the basal lamina and contain myofibrils. In the inactive breast, the epithelium is sparse and consists primarily of ductal

1	cells of ectoder-mal origin reside between the epithelial cells in the basal lamina and contain myofibrils. In the inactive breast, the epithelium is sparse and consists primarily of ductal epithelium (Fig. 17-3). In the early phase of the menstrual cycle, minor ducts are cord-like with small lumina. With estrogen stimulation at the time of ovulation, alveolar epithelium increases in height, duct lumina become more prominent, and some secretions accumulate. When the hormonal stimulation decreases, the alveolar epithe-lium regresses.With pregnancy, the breast undergoes proliferative and developmental maturation. As the breast enlarges in response to hormonal stimulation, lymphocytes, plasma cells, and eosin-ophils accumulate within the connective tissues. The minor ducts branch and alveoli develop. Development of the alveoli is asymmetric, and variations in the degree of development may occur within a single lobule (Fig. 17-4). With parturition, enlargement of the breasts occurs via

1	develop. Development of the alveoli is asymmetric, and variations in the degree of development may occur within a single lobule (Fig. 17-4). With parturition, enlargement of the breasts occurs via hypertrophy of alveolar epithelium and accumulation of secretory products in the lumina of the minor ducts. Alveolar epithelium contains abundant endo-plasmic reticulum, large mitochondria, Golgi complexes, and dense lysosomes. Two distinct substances are produced by the alveolar epithelium: (a) the protein component of milk, which is synthesized in the endoplasmic reticulum (merocrine secretion); and (b) the lipid component of milk (apocrine secretion), which forms as free lipid droplets in the cytoplasm. Milk released in the first few days after parturition is called colostrum and has low lipid content but contains considerable quantities of anti-bodies. The lymphocytes and plasma cells that accumulate within the connective tissues of the breast are the source of the antibody component.

1	content but contains considerable quantities of anti-bodies. The lymphocytes and plasma cells that accumulate within the connective tissues of the breast are the source of the antibody component. With subsequent reduction in the number of these cells, the production of colostrum decreases and lipid-rich milk is released.Blood Supply, Innervation, and Lymphatics. The breast receives its principal blood supply from: (a) perforating branches of the internal mammary artery; (b) lateral branches of the poste-rior intercostal arteries; and (c) branches from the axillary artery, including the highest thoracic, lateral thoracic, and pectoral branches of the thoracoacromial artery (Fig. 17-5). The second, third, and fourth anterior intercostal perforators and branches of the internal mammary artery arborize in the breast as the medial mammary arteries. The lateral thoracic artery gives off branches to the serratus anterior, pectoralis major and pectoralis minor, and subscapularis muscles. It

1	arborize in the breast as the medial mammary arteries. The lateral thoracic artery gives off branches to the serratus anterior, pectoralis major and pectoralis minor, and subscapularis muscles. It also gives rise to lateral mammary branches. The veins of the breast and chest wall follow the course of the arteries, with venous drainage being toward the axilla. The three principal groups of veins are: (a) per-forating branches of the internal thoracic vein, (b) perforating branches of the posterior intercostal veins, and (c) tributaries of the axillary vein. Batson’s vertebral venous plexus, which invests the vertebrae and extends from the base of the skull to the sacrum, may provide a route for breast cancer metastases to the vertebrae, skull, pelvic bones, and central nervous system. Lymph vessels generally parallel the course of blood vessels.1Brunicardi_Ch17_p0541-p0612.indd 54501/03/19 5:04 PM 546SPECIFIC CONSIDERATIONSPART IIFigure 17-5. Arterial supply to the breast, axilla,

1	vessels generally parallel the course of blood vessels.1Brunicardi_Ch17_p0541-p0612.indd 54501/03/19 5:04 PM 546SPECIFIC CONSIDERATIONSPART IIFigure 17-5. Arterial supply to the breast, axilla, and chest wall. (Reproduced with permission from Bland KI, Copeland EMI: The Breast: Comprehensive Management of Benign and Malignant Diseases, 4th ed. Philadelphia, PA: Elsevier/Saunders; 2009.)Figure 17-6. Lymphatic pathways of the breast. Arrows indicate the direction of lymph flow. (Visual Art: © 2013. The University of Texas MD Anderson Cancer Center.)Figure 17-7. Axillary lymph node groups. Level I includes lymph nodes located lateral to the pectoralis minor muscle; level II includes lymph nodes located deep to the pectoralis minor; and level III includes lymph nodes located medial to the pectoralis minor. The axillary vein with its major tributaries and the supracla-vicular lymph node group are also illustrated. (Visual Art: © 2013.The University of Texas MD Anderson Cancer

1	to the pectoralis minor. The axillary vein with its major tributaries and the supracla-vicular lymph node group are also illustrated. (Visual Art: © 2013.The University of Texas MD Anderson Cancer Center.)Lateral cutaneous branches of the third through sixth inter-costal nerves provide sensory innervation of the breast (lateral mammary branches) and of the anterolateral chest wall. These branches exit the intercostal spaces between slips of the serratus anterior muscle. Cutaneous branches that arise from the cervical plexus, specifically the anterior branches of the supraclavicular nerve, supply a limited area of skin over the upper portion of the breast. The intercostobrachial nerve is the lateral cutane-ous branch of the second intercostal nerve and may be visual-ized during surgical dissection of the axilla. Resection of the intercostobrachial nerve causes loss of sensation over the medial aspect of the upper arm.The boundaries for lymph drainage of the axilla are not well

1	dissection of the axilla. Resection of the intercostobrachial nerve causes loss of sensation over the medial aspect of the upper arm.The boundaries for lymph drainage of the axilla are not well demarcated, and there is considerable variation in the posi-tion of the axillary lymph nodes. The six axillary lymph node groups recognized by surgeons (Figs. 17-6 and 17-7) are: (a) the axillary vein group (lateral), which consists of four to six lymph nodes that lie medial or posterior to the vein and receive most of the lymph drainage from the upper extremity; (b) the external mammary group (anterior or pectoral group), which consists of five to six lymph nodes that lie along the lower border of the pectoralis minor muscle contiguous with the lateral thoracic vessels and receive most of the lymph drainage from the lat-eral aspect of the breast; (c) the scapular group (posterior or subscapular), which consists of five to seven lymph nodes that lie along the posterior wall of the axilla at the

1	drainage from the lat-eral aspect of the breast; (c) the scapular group (posterior or subscapular), which consists of five to seven lymph nodes that lie along the posterior wall of the axilla at the lateral border of the scapula contiguous with the subscapular vessels and receive lymph drainage principally from the lower posterior neck, the posterior trunk, and the posterior shoulder; (d) the central group, which consists of three or four sets of lymph nodes that are embedded in the fat of the axilla lying immediately posterior to the pectoralis minor muscle and receive lymph drainage both from the axillary vein, external mammary, and scapular groups of lymph nodes, and directly from the breast; (e) the subcla-vicular group (apical), which consists of six to twelve sets of lymph nodes that lie posterior and superior to the upper bor-der of the pectoralis minor muscle and receive lymph drainage from all of the other groups of axillary lymph nodes; and (f) the interpectoral group

1	that lie posterior and superior to the upper bor-der of the pectoralis minor muscle and receive lymph drainage from all of the other groups of axillary lymph nodes; and (f) the interpectoral group (Rotter’s lymph nodes), which consists of one to four lymph nodes that are interposed between the pec-toralis major and pectoralis minor muscles and receive lymph drainage directly from the breast. The lymph fluid that passes Brunicardi_Ch17_p0541-p0612.indd 54601/03/19 5:04 PM 547THE BREASTCHAPTER 17through the interpectoral group of lymph nodes passes directly into the central and subclavicular groups.As indicated in Fig. 17-7, the lymph node groups are assigned levels according to their anatomic relationship to the pectoralis minor muscle. Lymph nodes located lateral to or below the lower border of the pectoralis minor muscle are referred to as level I lymph nodes, which include the axillary vein, external mammary, and scapular groups. Lymph nodes located superficial or deep to the

1	border of the pectoralis minor muscle are referred to as level I lymph nodes, which include the axillary vein, external mammary, and scapular groups. Lymph nodes located superficial or deep to the pectoralis minor muscle are referred to as level II lymph nodes, which include the central and interpectoral groups. Lymph nodes located medial to or above the upper border of the pectoralis minor muscle are referred to as level III lymph nodes, which consist of the subclavicular group. The plexus of lymph vessels in the breast arises in the interlobular connective tissue and in the walls of the lactiferous ducts and communicates with the subareolar plexus of lymph vessels. Efferent lymph vessels from the breast pass around the lateral edge of the pectoralis major muscle and pierce the clavipectoral fascia, ending in the external mammary (anterior, pectoral) group of lymph nodes. Some lymph vessels may travel directly to the subscapular (pos-terior, scapular) group of lymph nodes. From the

1	fascia, ending in the external mammary (anterior, pectoral) group of lymph nodes. Some lymph vessels may travel directly to the subscapular (pos-terior, scapular) group of lymph nodes. From the upper part of the breast, a few lymph vessels pass directly to the subclavicular (api-cal) group of lymph nodes. The axillary lymph nodes usually receive >75% of the lymph drainage from the breast. The rest is derived primarily from the medial aspect of the breast, flows through the lymph vessels that accompany the per-forating branches of the internal mammary artery, and enters the parasternal (internal mammary) group of lymph nodes.PHYSIOLOGY OF THE BREASTBreast Development and FunctionBreast development and function are initiated by a variety of hormonal stimuli, including estrogen, progesterone, prolactin, oxytocin, thyroid hormone, cortisol, and growth hormone.17,18 Estrogen, progesterone, and prolactin especially have profound trophic effects that are essential to normal breast

1	prolactin, oxytocin, thyroid hormone, cortisol, and growth hormone.17,18 Estrogen, progesterone, and prolactin especially have profound trophic effects that are essential to normal breast develop-ment and function. Estrogen initiates ductal development, whereas progesterone is responsible for differentiation of epithe-lium and for lobular development. Prolactin is the primary hor-monal stimulus for lactogenesis in late pregnancy and the postpartum period. It upregulates hormone receptors and stimu-lates epithelial development. Fig. 17-8 depicts the secretion of neurotrophic hormones from the hypothalamus, which is respon-sible for regulation of the secretion of the hormones that affect the breast tissues. The gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate the release of estrogen and progesterone from the ovaries. In turn, the release of LH and FSH from the basophilic cells of the anterior pituitary is regulated by the secretion of

1	(FSH) regulate the release of estrogen and progesterone from the ovaries. In turn, the release of LH and FSH from the basophilic cells of the anterior pituitary is regulated by the secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus. Positive and negative feedback effects of circulating estrogen and progesterone regulate the secretion of LH, FSH, and GnRH. These hormones are respon-sible for the development, function, and maintenance of breast tissues (Fig. 17-9A). In the female neonate, circulating estrogen and progesterone levels decrease after birth and remain low throughout childhood because of the sensitivity of 23GRFLH-RHDopamineOxy/ADHTRHCRF-Figure 17-8. Overview of the neuroendocrine con-trol of breast development and function. ADH = antidiuretic hormone; CRF = corticotropin-releasing factor; GRF = growth hormone releasing factor; LH-RH = luteinizing hormone–releasing hormone; Oxy = oxytocin; TRH = thyrotropin-releasing hor-mone. (Reproduced with

1	CRF = corticotropin-releasing factor; GRF = growth hormone releasing factor; LH-RH = luteinizing hormone–releasing hormone; Oxy = oxytocin; TRH = thyrotropin-releasing hor-mone. (Reproduced with permission from Bland KI, Copeland EMI: The Breast: Comprehensive Man-agement of Benign and Malignant Diseases, 4th ed. Philadelphia, PA: Elsevier/Saunders; 2009.)Brunicardi_Ch17_p0541-p0612.indd 54701/03/19 5:04 PM 548SPECIFIC CONSIDERATIONSPART IIABCDFigure 17-9. The breast at different physi-ologic stages. The central column contains three-dimensional depictions of microscopic structures. A. Adolescence. B. Pregnancy. C. Lactation. D. Senescence.the hypothalamic-pituitary axis to negative feedback from these hormones. With the onset of puberty, there is a decrease in the sensitivity of the hypothalamic-pituitary axis to negative feed-back and an increase in its sensitivity to positive feedback from estrogen. These physiologic events initiate an increase in GnRH, FSH, and LH secretion

1	hypothalamic-pituitary axis to negative feed-back and an increase in its sensitivity to positive feedback from estrogen. These physiologic events initiate an increase in GnRH, FSH, and LH secretion and ultimately an increase in estrogen and progesterone secretion by the ovaries, leading to establish-ment of the menstrual cycle. At the beginning of the menstrual cycle, there is an increase in the size and density of the breasts, which is followed by engorgement of the breast tissues and epi-thelial proliferation. With the onset of menstruation, the breast engorgement subsides and epithelial proliferation decreases.Pregnancy, Lactation, and SenescenceA dramatic increase in circulating ovarian and placental estro-gens and progestins is evident during pregnancy, which initiates striking alterations in the form and substance of the breast (see Fig. 17-9B).17-19 The breast enlarges as the ductal and lobular epithelium proliferates, the areolar skin darkens, and the acces-sory areolar glands

1	in the form and substance of the breast (see Fig. 17-9B).17-19 The breast enlarges as the ductal and lobular epithelium proliferates, the areolar skin darkens, and the acces-sory areolar glands (Montgomery’s glands) become prominent. In the first and second trimesters, the minor ducts branch and develop. During the third trimester, fat droplets accumulate in the alveolar epithelium, and colostrum fills the alveolar and duc-tal spaces. In late pregnancy, prolactin stimulates the synthesis of milk fats and proteins.After delivery of the placenta, circulating progesterone and estrogen levels decrease, permitting full expression of the lactogenic action of prolactin. Milk production and release are controlled by neural reflex arcs that originate in nerve endings of the nipple-areola complex. Maintenance of lactation requires regular stimulation of these neural reflexes, which results in prolactin secretion and milk letdown. Oxytocin release results from the auditory, visual, and olfactory

1	of lactation requires regular stimulation of these neural reflexes, which results in prolactin secretion and milk letdown. Oxytocin release results from the auditory, visual, and olfactory stimuli associated with nursing. Oxytocin initiates contraction of the myoepithelial cells, which results in compression of alveoli and expulsion of milk into the lactiferous sinuses. After weaning of the infant, prolactin and oxytocin release decreases. Dormant milk causes increased pressure within the ducts and alveoli, which results in atrophy of the epithelium (Fig. 17-9C). With menopause, there is a decrease in the secretion of estrogen and progesterone by Brunicardi_Ch17_p0541-p0612.indd 54801/03/19 5:04 PM 549THE BREASTCHAPTER 17Table 17-1Pathophysiologic mechanisms of gynecomastia I. Estrogen excess states A. Gonadal origin 1. True hermaphroditism 2. Gonadal stromal (nongerminal) neoplasms of the testis a. Leydig cell (interstitial) b. Sertoli cell c. Granulosa-theca cell 3. Germ cell

1	excess states A. Gonadal origin 1. True hermaphroditism 2. Gonadal stromal (nongerminal) neoplasms of the testis a. Leydig cell (interstitial) b. Sertoli cell c. Granulosa-theca cell 3. Germ cell tumors a. Choriocarcinoma b. Seminoma, teratoma c. Embryonal carcinoma B. Nontesticular tumors 1. Adrenal cortical neoplasms 2. Lung carcinoma 3. Hepatocellular carcinoma C. Endocrine disorders D. Diseases of the liver—nonalcoholic and alcoholic cirrhosis E. Nutrition alteration states II. Androgen deficiency states A. Senescence B. Hypoandrogenic states (hypogonadism) 1. Primary testicular failure a. Klinefelter’s syndrome (XXY) b. Reifenstein’s syndrome c. Rosewater-Gwinup-Hamwi familial gynecomastia d. Kallmann syndrome e. Kennedy’s disease with associated gynecomastia f. Eunuchoidal state (congenital anorchia) g. Hereditary defects of androgen biosynthesis h. Adrenocorticotropic hormone deficiency 2. Secondary testicular

1	disease with associated gynecomastia f. Eunuchoidal state (congenital anorchia) g. Hereditary defects of androgen biosynthesis h. Adrenocorticotropic hormone deficiency 2. Secondary testicular failure a. Trauma b. Orchitis c. Cryptorchidism d. Irradiation C. Renal failure III. Pharmacologic causes IV. Systemic diseases with idiopathic mechanismsthe ovaries and involution of the ducts and alveoli of the breast. The surrounding fibrous connective tissue increases in density, and breast tissues are replaced by adipose tissues (Fig. 17-9D).GynecomastiaGynecomastia refers to an enlarged breast in the male.20 Physi-ologic gynecomastia usually occurs during three phases of life: the neonatal period, adolescence, and senescence. Common to each of these phases is an excess of circulating estrogens in relation to circulating testosterone. Neonatal gynecomastia is caused by the action of placental estrogens on neonatal breast tissues, whereas in adolescence, there is an excess of estradiol

1	in relation to circulating testosterone. Neonatal gynecomastia is caused by the action of placental estrogens on neonatal breast tissues, whereas in adolescence, there is an excess of estradiol relative to testosterone, and with senescence, the circulating testosterone level falls, which results in relative hyperestrin-ism. In gynecomastia, the ductal structures of the male breast enlarge, elongate, and branch with a concomitant increase in epithelium. During puberty, the condition often is unilateral and typically occurs between ages 12 and 15 years. In contrast, senescent gynecomastia is usually bilateral. In the nonobese male, breast tissue measuring at least 2 cm in diameter must be present before a diagnosis of gynecomastia may be made. Mammography and ultrasonography are used to differentiate breast tissues. Dominant masses or areas of firmness, irregular-ity, and asymmetry suggest the possibility of a breast cancer, particularly in the older male. Gynecomastia generally does

1	breast tissues. Dominant masses or areas of firmness, irregular-ity, and asymmetry suggest the possibility of a breast cancer, particularly in the older male. Gynecomastia generally does not predispose the male breast to cancer. However, the hypoandro-genic state of Klinefelter’s syndrome (XXY), in which gyneco-mastia is usually evident, is associated with an increased risk of breast cancer. Gynecomastia is graded based on the degree of breast enlargement, the position of the nipple with reference to the inframammary fold, and the degree of breast ptosis and skin redundancy: Grade I—mild breast enlargement without skin redundancy; Grade IIa—moderate breast enlargement without skin redundancy; Grade IIb—moderate breast enlargement with skin redundancy; and Grade III—marked breast enlargement with skin redundancy and ptosis.Table 17-1 identifies the pathophysiologic mechanisms that may initiate gynecomastia: estrogen excess states; andro-gen deficiency states; pharmacologic causes; and

1	with skin redundancy and ptosis.Table 17-1 identifies the pathophysiologic mechanisms that may initiate gynecomastia: estrogen excess states; andro-gen deficiency states; pharmacologic causes; and idiopathic causes. Estrogen excess results from an increase in the secretion of estradiol by the testicles or by nontesticular tumors, nutri-tional alterations such as protein and fat deprivation, endocrine disorders (hyperthyroidism, hypothyroidism), and hepatic dis-ease (nonalcoholic and alcoholic cirrhosis). Refeeding gyne-comastia is related to the resumption of pituitary gonadotropin secretion after pituitary shutdown. Androgen deficiency may initiate gynecomastia. Concurrently occurring with decreased circulating testosterone levels is an elevated level of circulating testosterone-binding globulin, which results in a reduction of free testosterone. This senescent gynecomastia usually occurs in men age 50 to 70 years. Hypoandrogenic states can be from primary testicular failure or

1	globulin, which results in a reduction of free testosterone. This senescent gynecomastia usually occurs in men age 50 to 70 years. Hypoandrogenic states can be from primary testicular failure or secondary testicular failure. Kline-felter’s syndrome (XXY) is an example of primary testicular failure that is manifested by gynecomastia, hypergonadotropic hypogonadism, and azoospermia. Secondary testicular failure may result from trauma, orchitis, and cryptorchidism. Renal failure, regardless of cause, also may initiate gynecomastia.Pharmacologic causes of gynecomastia include drugs with estrogenic activity (digitalis, estrogens, anabolic steroids, marijuana) or drugs that enhance estrogen synthesis (human chorionic gonadotropin). Drugs that inhibit the action or syn-thesis of testosterone (cimetidine, ketoconazole, phenytoin, spironolactone, antineoplastic agents, diazepam) also have been implicated. Drugs such as reserpine, theophylline, verapamil, tricyclic antidepressants, and

1	(cimetidine, ketoconazole, phenytoin, spironolactone, antineoplastic agents, diazepam) also have been implicated. Drugs such as reserpine, theophylline, verapamil, tricyclic antidepressants, and furosemide induce gynecomastia through idiopathic mechanisms.When gynecomastia is caused by androgen deficiency, then testosterone administration may cause regression. When it is caused by medications, then these are discontinued if possi-ble. When endocrine defects are responsible, then these receive specific therapy. As soon as gynecomastia is progressive and does not respond to other treatments, surgical therapy is con-sidered. Techniques include local excision, liposuction or sub-cutaneous mastectomy. Attempts to reverse gynecomastia with danazol have been successful, but the androgenic side effects of the drug are considerable.Brunicardi_Ch17_p0541-p0612.indd 54901/03/19 5:04 PM 550SPECIFIC CONSIDERATIONSPART IIINFECTIOUS AND INFLAMMATORY DISORDERS OF THE BREASTInfections in the

1	effects of the drug are considerable.Brunicardi_Ch17_p0541-p0612.indd 54901/03/19 5:04 PM 550SPECIFIC CONSIDERATIONSPART IIINFECTIOUS AND INFLAMMATORY DISORDERS OF THE BREASTInfections in the postpartum period remain proportionately the most common time for breast infections to occur. Infections of the breast unrelated to lactation are proportionately less com-mon, however, are still a relatively common presentation to breast specialists. The latter are classified as intrinsic (second-ary to abnormalities in the breast) or extrinsic (secondary to an infection in an adjacent structure, e.g., skin, thoracic cavity) the most common being probably periductal mastitis and infected sebaceous cysts, respectively.Bacterial InfectionStaphylococcus aureus and Streptococcus species are the organisms most frequently recovered from nipple discharge from an infected breast.17 Typically breast abscesses are seen in staphylococcal infections and present with point tenderness, erythema, and

1	organisms most frequently recovered from nipple discharge from an infected breast.17 Typically breast abscesses are seen in staphylococcal infections and present with point tenderness, erythema, and hyperthermia. When these abscesses are related to lactation they usually occur within the first few weeks of breastfeeding. If there is progression of a staphylococcal infec-tion, this may result in subcutaneous, subareolar, interlobular (periductal), and retromammary abscesses (unicentric or multi-centric). Previously almost all breast abscesses were treated by operative incision and drainage, but now the initial approach is antibiotics and repeated aspiration of the abscess, usually ultra-sound-guided aspiration.21 Operative drainage is now reserved for those cases that do not resolve with repeated aspiration and antibiotic therapy or cases in which there is some other indica-tion for incision and drainage (e.g., thinning or necrosis of the overlying skin). Preoperative ultrasonography

1	repeated aspiration and antibiotic therapy or cases in which there is some other indica-tion for incision and drainage (e.g., thinning or necrosis of the overlying skin). Preoperative ultrasonography is effective in delineating the required extent of the drainage procedure. While staphylococcal infections tend to be more localized and may be situated deep in the breast tissues, streptococcal infections usually present with diffuse superficial involvement. They are treated with local wound care, including application of warm compresses, and the administration of IV antibiotics (penicillins or cephalosporins). Breast infections may be chronic, possibly with recurrent abscess formation. In this situation, cultures are performed to identify acid-fast bacilli, anaerobic and aerobic bacteria, and fungi. Uncommon organisms may be encountered, and long-term antibiotic therapy may be required.Biopsy of the abscess cavity wall should be considered at the time of incision and drainage to rule

1	and fungi. Uncommon organisms may be encountered, and long-term antibiotic therapy may be required.Biopsy of the abscess cavity wall should be considered at the time of incision and drainage to rule out underlying breast cancer in patients where antibiotics and drainage have been ineffective.Nowadays hospital-acquired puerperal infections of the breast are much less common, but nursing women who pres-ent with milk stasis or noninfectious inflammation may still develop this problem. Epidemic puerperal mastitis is initiated by highly virulent strains of methicillin-resistant S aureus that are transmitted via the suckling neonate and may result in sub-stantial morbidity and occasional mortality. Purulent fluid may be expressed from the nipple. In this circumstance, breastfeed-ing is stopped, antibiotics are started, and surgical therapy is initiated. Nonepidemic (sporadic) puerperal mastitis refers to involvement of the interlobular connective tissue of the breast by an infectious

1	antibiotics are started, and surgical therapy is initiated. Nonepidemic (sporadic) puerperal mastitis refers to involvement of the interlobular connective tissue of the breast by an infectious process. The patient develops nipple fissuring and milk stasis, which initiates a retrograde bacterial infection. Emptying of the breast using breast suction pumps shortens the duration of symptoms and reduces the incidence of recurrences. The addition of antibiotic therapy results in a satisfactory out-come in >95% of cases.Zuska’s disease, also called recurrent periductal mastitis, is a condition of recurrent retroareolar infections and abscesses.22,23 Smoking has been implicated as a risk factor for this condition.24,25 This syndrome is managed symptomatically by antibiotics coupled with incision and drainage as necessary. Attempts to obtain durable long-term control by wide debride-ment of chronically infected tissue and/or terminal duct resec-tion have been reported and can be curative, but

1	and drainage as necessary. Attempts to obtain durable long-term control by wide debride-ment of chronically infected tissue and/or terminal duct resec-tion have been reported and can be curative, but they can also be frustrated by postoperative infections.26Mycotic InfectionsFungal infections of the breast are rare and usually involve blas-tomycosis or sporotrichosis.27 Intraoral fungi that are inoculated into the breast tissue by the suckling infant initiate these infec-tions, which present as mammary abscesses in close proxim-ity to the nipple-areola complex. Pus mixed with blood may be expressed from sinus tracts. Antifungal agents can be adminis-tered for the treatment of systemic (noncutaneous) infections. This therapy generally eliminates the necessity of surgical inter-vention, but occasionally drainage of an abscess, or even partial mastectomy, may be necessary to eradicate a persistent fungal infection. Candida albicans affecting the skin of the breast presents as

1	but occasionally drainage of an abscess, or even partial mastectomy, may be necessary to eradicate a persistent fungal infection. Candida albicans affecting the skin of the breast presents as erythematous, scaly lesions of the inframammary or axillary folds. Scrapings from the lesions demonstrate fungal elements (filaments and binding cells). Therapy involves the removal of predisposing factors such as maceration and the topi-cal application of nystatin.Hidradenitis SuppurativaHidradenitis suppurativa of the nipple-areola complex or axilla is a chronic inflammatory condition that originates within the accessory areolar glands of Montgomery or within the axillary sebaceous glands.27 Women with chronic acne are predisposed to developing hidradenitis. When located in and about the nipple-areola complex, this disease may mimic other chronic inflammatory states, Paget’s disease of the nipple, or invasive breast cancer. Involvement of the axillary skin is often multifo-cal and contiguous.

1	complex, this disease may mimic other chronic inflammatory states, Paget’s disease of the nipple, or invasive breast cancer. Involvement of the axillary skin is often multifo-cal and contiguous. Antibiotic therapy with incision and drain-age of fluctuant areas is appropriate treatment. Excision of the involved areas may be required. Large areas of skin loss may necessitate coverage with advancement flaps or split-thickness skin grafts.Mondor’s DiseaseMondor’s disease is a variant of thrombophlebitis that involves the superficial veins of the anterior chest wall and breast.28 In 1939, Mondor described the condition as “string phlebitis,” a thrombosed vein presenting as a tender, cord-like structure.29 Frequently involved veins include the lateral thoracic vein, the thoracoepigastric vein, and, less commonly, the superficial epi-gastric vein. Typically, a woman presents with acute pain in the lateral aspect of the breast or the anterior chest wall. A ten-der, firm cord is found to

1	and, less commonly, the superficial epi-gastric vein. Typically, a woman presents with acute pain in the lateral aspect of the breast or the anterior chest wall. A ten-der, firm cord is found to follow the distribution of one of the major superficial veins. Rarely, the presentation is bilateral, and most women have no evidence of thrombophlebitis in other ana-tomic sites. This benign, self-limited disorder is not indicative of a cancer. When the diagnosis is uncertain, or when a mass is present near the tender cord, biopsy is indicated. Therapy for Mondor’s disease includes the liberal use of anti-inflammatory medications and application of warm compresses along the symptomatic vein. The process usually resolves within 4 to 6 weeks. When symptoms persist or are refractory to therapy, excision of the involved vein segment may be considered.Brunicardi_Ch17_p0541-p0612.indd 55001/03/19 5:04 PM 551THE BREASTCHAPTER 17COMMON BENIGN DISORDERS AND DISEASES OF THE BREASTBenign breast

1	of the involved vein segment may be considered.Brunicardi_Ch17_p0541-p0612.indd 55001/03/19 5:04 PM 551THE BREASTCHAPTER 17COMMON BENIGN DISORDERS AND DISEASES OF THE BREASTBenign breast disorders and diseases encompass a wide range of clinical and pathologic entities. Surgeons require an in-depth understanding of benign breast disorders and diseases so that clear explanations may be given to affected women, appropriate treat-ment is instituted, and unnecessary long-term follow up is avoided.Aberrations of Normal Development and InvolutionThe basic principles underlying the aberrations of normal devel-opment and involution (ANDI) classification of benign breast conditions are the following: (a) benign breast disorders and diseases are related to the normal processes of reproductive life and to involution; (b) there is a spectrum of breast conditions that ranges from normal to disorder to disease; and (c) the ANDI classification encompasses all aspects of the breast condition,

1	life and to involution; (b) there is a spectrum of breast conditions that ranges from normal to disorder to disease; and (c) the ANDI classification encompasses all aspects of the breast condition, including pathogenesis and the degree of abnormality.30 The horizontal component of Table 17-2 defines ANDI along a spectrum from normal, to mild abnormality (disorder), to severe abnormality (disease). The vertical component indi-cates the period during which the condition develops.Early Reproductive Years. Fibroadenomas are seen and pres-ent symptomatically predominantly in younger women age 15 to 25 years (Fig. 17-10).31 Fibroadenomas usually grow to 1 or 2 cm in diameter and then are stable but may grow to a larger size. Small fibroadenomas (≤1 cm in size) are considered nor-mal, whereas larger fibroadenomas (≤3 cm) are disorders, and giant fibroadenomas (>3 cm) are disease. Similarly, multiple fibroadenomas (more than five lesions in one breast) are very uncommon and are considered

1	larger fibroadenomas (≤3 cm) are disorders, and giant fibroadenomas (>3 cm) are disease. Similarly, multiple fibroadenomas (more than five lesions in one breast) are very uncommon and are considered disease. It is noted that with the introduction of mammographic screening, asymptomatic 4Table 17-2ANDI classification of benign breast disorders NORMALDISORDERDISEASEEarly reproductive years (age 15–25 y) Lobular developmentStromal developmentFibroadenomaAdolescent hypertrophyGiant fibroadenomaGigantomastia Nipple eversionNipple inversionSubareolar abscess Mammary duct fistulaLater reproductive years (age 25–40 y)Cyclical changes of menstruationCyclical mastalgiaIncapacitating mastalgia Nodularity Epithelial hyperplasia of pregnancyBloody nipple discharge Involution (age 35–55 y)Lobular involutionMacrocysts— Sclerosing lesions Duct involution DilatationDuct ectasiaPeriductal mastitis SclerosisNipple retraction— Epithelial turnoverEpithelial hyperplasiaEpithelial hyperplasia with

1	lesions Duct involution DilatationDuct ectasiaPeriductal mastitis SclerosisNipple retraction— Epithelial turnoverEpithelial hyperplasiaEpithelial hyperplasia with atypiaANDI = aberrations of normal development and involution.Reproduced with permission from Mansel RE, Webster D, Sweetland H: Hughes, Mansel & Webster’s Benign Disorders and Diseases of the Breast, 3rd ed. London: Elsevier/Saunders; 2009.Figure 17-10. Fibroadenoma (40x). These benign tumors are typi-cally well circumscribed and are comprised of both stromal and glandular elements. (Used with permission from Dr. Sindhu Menon, Consultant Histopathologist and Dr. Rahul Deb, Consultant Histopathologist and Lead Breast Pathologist, Royal Derby Hospital, Derby, UK.)fibroadenomas are sometimes found in an older screened popu-lation. The precise etiology of adolescent breast hypertrophy is unknown. A spectrum of changes from limited to massive stro-mal hyperplasia (gigantomastia) is seen. Nipple inversion is a disorder of

1	The precise etiology of adolescent breast hypertrophy is unknown. A spectrum of changes from limited to massive stro-mal hyperplasia (gigantomastia) is seen. Nipple inversion is a disorder of development of the major ducts, which prevents nor-mal protrusion of the nipple. Mammary duct fistulas arise when nipple inversion predisposes to major duct obstruction, leading to recurrent subareolar abscess and mammary duct fistula.Brunicardi_Ch17_p0541-p0612.indd 55101/03/19 5:04 PM 552SPECIFIC CONSIDERATIONSPART IIABFigure 17-11. A. Ductal epithelial hyperplasia. The irregular intra-cellular spaces and variable cell nuclei distinguish this process from carcinoma in situ. B. Lobular hyperplasia. The presence of alveo-lar lumina and incomplete distention distinguish this process from carcinoma in situ. (Used with permission from Dr. R.L. Hackett.)Table 17-3Cancer risk associated with benign breast disorders and in situ carcinoma of the breastABNORMALITYRELATIVE RISKNonproliferative lesions

1	in situ. (Used with permission from Dr. R.L. Hackett.)Table 17-3Cancer risk associated with benign breast disorders and in situ carcinoma of the breastABNORMALITYRELATIVE RISKNonproliferative lesions of the breastNo increased riskSclerosing adenosisNo increased riskIntraductal papillomaNo increased riskFlorid hyperplasia1.5 to 2-foldAtypical lobular hyperplasia4-foldAtypical ductal hyperplasia4-foldDuctal involvement by cells of atypical ductal hyperplasia7-foldLobular carcinoma in situ10-foldDuctal carcinoma in situ10-foldData from Dupont WD, Page DL. Risk factors for breast cancer in women with proliferative breast disease, N Engl J Med. 1985 Jan 17; 312(3):146-151.Table 17-4Classification of benign breast disordersNonproliferative disorders of the breast Cysts and apocrine metaplasia Duct ectasia Mild ductal epithelial hyperplasia Calcifications Fibroadenoma and related lesionsProliferative breast disorders without atypia Sclerosing adenosis Radial and complex sclerosing

1	ectasia Mild ductal epithelial hyperplasia Calcifications Fibroadenoma and related lesionsProliferative breast disorders without atypia Sclerosing adenosis Radial and complex sclerosing lesions Ductal epithelial hyperplasia Intraductal papillomasAtypical proliferative lesions Atypical lobular hyperplasia Atypical ductal hyperplasiaData from Godfrey SE: Is fibrocystic disease of the breast precancerous? Arch Pathol Lab Med. 1986 Nov;110(11):991.include ductal and lobular hyperplasia, both of which display some features of carcinoma in situ. Women with atypical ductal or lobular hyperplasia have a fourfold increase in breast cancer risk (Table 17-3).Pathology of Nonproliferative DisordersOf paramount importance for the optimal management of benign breast disorders and diseases is the histologic differentia-tion of benign, atypical, and malignant changes.32,33 Determin-ing the clinical significance of these changes is a problem that is compounded by inconsistent nomenclature. The

1	histologic differentia-tion of benign, atypical, and malignant changes.32,33 Determin-ing the clinical significance of these changes is a problem that is compounded by inconsistent nomenclature. The classifica-tion system originally developed by Page separates the various types of benign breast disorders and diseases into three clini-cally relevant groups: nonproliferative disorders, proliferative disorders without atypia, and proliferative disorders with atypia (Table 17-4). Nonproliferative disorders of the breast account for 70% of benign breast conditions and carry no increased risk Later Reproductive Years. Cyclical mastalgia and nodular-ity usually are associated with premenstrual enlargement of the breast and are regarded as normal. Cyclical pronounced mastal-gia and severe painful nodularity are viewed differently than are physiologic discomfort and lumpiness. Painful nodularity that persists for >1 week of the menstrual cycle is considered a disor-der. In epithelial

1	painful nodularity are viewed differently than are physiologic discomfort and lumpiness. Painful nodularity that persists for >1 week of the menstrual cycle is considered a disor-der. In epithelial hyperplasia of pregnancy, papillary projections sometimes give rise to bilateral bloody nipple discharge.Involution. Involution of lobular epithelium is dependent on the specialized stroma around it. However, an integrated invo-lution of breast stroma and epithelium is not always seen, and disorders of the process are common. When the stroma invo-lutes too quickly, alveoli remain and form microcysts, which are precursors of macrocysts. The macrocysts are common, often subclinical, and do not require specific treatment. Sclerosing adenosis is considered a disorder of both the proliferative and the involutional phases of the breast cycle. Duct ectasia (dilated ducts) and periductal mastitis are other important components of the ANDI classification. Periductal fibrosis is a sequela of

1	and the involutional phases of the breast cycle. Duct ectasia (dilated ducts) and periductal mastitis are other important components of the ANDI classification. Periductal fibrosis is a sequela of periductal mastitis and may result in nipple retraction. About 60% of women ≥70 years of age exhibit some degree of epi-thelial hyperplasia (Fig. 17-11). Atypical proliferative diseases Brunicardi_Ch17_p0541-p0612.indd 55201/03/19 5:04 PM 553THE BREASTCHAPTER 17for the development of breast cancer. This category includes cysts, duct ectasia, periductal mastitis, calcifications, fibroad-enomas, and related disorders.Breast macrocysts are an involutional disorder, have a high frequency of occurrence, and are often multiple. Duct ecta-sia is a clinical syndrome characterized by dilated subareolar ducts that are palpable and often associated with thick nipple discharge. Haagensen regarded duct ectasia as a primary event that led to stagnation of secretions, epithelial ulceration, and leakage

1	ducts that are palpable and often associated with thick nipple discharge. Haagensen regarded duct ectasia as a primary event that led to stagnation of secretions, epithelial ulceration, and leakage of duct secretions (containing chemically irritating fatty acids) into periductal tissue.34 This sequence was thought to pro-duce a local inflammatory process with periductal fibrosis and subsequent nipple retraction. An alternative theory considers periductal mastitis to be the primary process, which leads to weakening of the ducts and secondary dilatation. It is possible that both processes occur and together explain the wide spec-trum of problems seen, which include nipple discharge, nipple retraction, inflammatory masses, and abscesses.Calcium deposits are frequently encountered in the breast. Most are benign and are caused by cellular secretions and debris or by trauma and inflammation. Calcifications that are associated with cancer include microcalcifications, which vary in shape and

1	Most are benign and are caused by cellular secretions and debris or by trauma and inflammation. Calcifications that are associated with cancer include microcalcifications, which vary in shape and density and are <0.5 mm in size, and fine, linear calcifications, which may show branching. Fibroadenomas have abundant stroma with histologically normal cellular elements. They show hormonal dependence similar to that of normal breast lobules in that they lactate during pregnancy and invo-lute in the postmenopausal period. Adenomas of the breast are well circumscribed and are composed of benign epithelium with sparse stroma, which is the histologic feature that differentiates them from fibroadenomas. They may be divided into tubular adenomas and lactating adenomas. Tubular adenomas are seen in young nonpregnant women, whereas lactating adenomas are seen during pregnancy or during the postpartum period. Ham-artomas are discrete breast tumors that are usually 2 to 4 cm in diameter, firm, and

1	young nonpregnant women, whereas lactating adenomas are seen during pregnancy or during the postpartum period. Ham-artomas are discrete breast tumors that are usually 2 to 4 cm in diameter, firm, and sharply circumscribed. Adenolipomas con-sist of sharply circumscribed nodules of fatty tissue that contain normal breast lobules and ducts.Fibrocystic Disease. The term fibrocystic disease is nonspe-cific. Too frequently, it is used as a diagnostic term to describe symptoms, to rationalize the need for breast biopsy, and to explain biopsy results. Synonyms include fibrocystic changes, cystic mastopathy, chronic cystic disease, chronic cystic mas-titis, Schimmelbusch’s disease, mazoplasia, Cooper’s disease, Reclus’ disease, and fibroadenomatosis. Fibrocystic disease refers to a spectrum of histopathologic changes that are best diagnosed and treated specifically.Pathology of Proliferative Disorders Without AtypiaProliferative breast disorders without atypia include sclerosing adenosis,

1	histopathologic changes that are best diagnosed and treated specifically.Pathology of Proliferative Disorders Without AtypiaProliferative breast disorders without atypia include sclerosing adenosis, radial scars, complex sclerosing lesions, ductal epithe-lial hyperplasia, and intraductal papillomas.32,33 Sclerosing ade-nosis is prevalent during the childbearing and perimenopausal years and has no malignant potential. Histologic changes are both proliferative (ductal proliferation) and involutional (stro-mal fibrosis, epithelial regression). Sclerosing adenosis is char-acterized by distorted breast lobules and usually occurs in the context of multiple microcysts, but occasionally presents as a palpable mass. Benign calcifications are often associated with this disorder. Sclerosing adenosis can be managed by observa-tion as long as the imaging features and pathologic findings are concordant. Central sclerosis and various degrees of epithelial proliferation, apocrine metaplasia, and

1	can be managed by observa-tion as long as the imaging features and pathologic findings are concordant. Central sclerosis and various degrees of epithelial proliferation, apocrine metaplasia, and papilloma formation characterize radial scars and complex sclerosing lesions of the breast. Lesions up to 1 cm in diameter are called radial scars, whereas larger lesions are called complex sclerosing lesions. Radial scars originate at sites of terminal duct branching where the characteristic histologic changes radiate from a central area of fibrosis. All of the histologic features of a radial scar are seen in the larger complex sclerosing lesions, but there is a greater disturbance of structure with papilloma formation, apocrine metaplasia, and occasionally sclerosing adenosis. Distinguish-ing between a radial scar and invasive breast carcinoma can be challenging based on core-needle biopsy sampling. Often the imaging features of a radial scar (which can be quite similar to an invasive

1	between a radial scar and invasive breast carcinoma can be challenging based on core-needle biopsy sampling. Often the imaging features of a radial scar (which can be quite similar to an invasive cancer) will dictate the need for either a vacuum-assisted biopsy or surgical excision in order to exclude the pos-sibility of carcinoma.Mild ductal hyperplasia is characterized by the presence of three or four cell layers above the basement membrane. Moder-ate ductal hyperplasia is characterized by the presence of five or more cell layers above the basement membrane. Florid duc-tal epithelial hyperplasia occupies at least 70% of a minor duct lumen. It is found in >20% of breast tissue specimens, is either solid or papillary, and is associated with an increased cancer risk (see Table 17-3). Intraductal papillomas arise in the major ducts, usually in premenopausal women. They generally are <0.5 cm in diameter but may be as large as 5 cm. A common presenting symptom is nipple discharge, which

1	papillomas arise in the major ducts, usually in premenopausal women. They generally are <0.5 cm in diameter but may be as large as 5 cm. A common presenting symptom is nipple discharge, which may be serous or bloody. Grossly, intraductal papillomas are pinkish tan, fri-able, and usually attached to the wall of the involved duct by a stalk. They rarely undergo malignant transformation, and their presence does not increase a woman’s risk of developing breast cancer (unless accompanied by atypia). However, multiple intraductal papillomas, which occur in younger women and are less frequently associated with nipple discharge, are susceptible to malignant transformation.Pathology of Atypical Proliferative DiseasesThe atypical proliferative diseases have some of the features of carcinoma in situ but either lack a major defining feature of car-cinoma in situ or have the features in less than fully developed form.34 Atypical ductal hyperplasia (ADH) appears similar to low grade ductal

1	in situ but either lack a major defining feature of car-cinoma in situ or have the features in less than fully developed form.34 Atypical ductal hyperplasia (ADH) appears similar to low grade ductal carcinoma in situ (DCIS) histologically and is composed of monotonous round, cuboidal, or polygonal cells enclosed by basement membrane with rare mitoses. A lesion will be considered to be ADH if it is up to 2 or 3 mm in size but would be called DCIS if it is larger than 3 mm. The diagnosis can be difficult to establish with core-needle biopsy specimen alone and many cases will require excisional biopsy specimen for classification. Individuals with a diagnosis of ADH are at increased risk for development of breast cancer and should be counseled appropriately regarding risk reduction strategies.In 1978, Haagensen et al described lobular neoplasia, a spectrum of disorders ranging from atypical lobular hyperplasia to lobular carcinoma in situ (LCIS).35 Atypical lobular hyper-plasia (ALH)

1	1978, Haagensen et al described lobular neoplasia, a spectrum of disorders ranging from atypical lobular hyperplasia to lobular carcinoma in situ (LCIS).35 Atypical lobular hyper-plasia (ALH) results in minimal distention of lobular units with cells that are similar to those seen in LCIS. The diagnosis of LCIS is made when small monomorphic cells that distend the terminal ductal lobular unit are noted. In cases of LCIS, the acini are full and distended while the overall lobular architec-ture is maintained (Fig. 17-12). Classic LCIS is not associated with a specific mammographic or palpable abnormality but is Brunicardi_Ch17_p0541-p0612.indd 55301/03/19 5:04 PM 554SPECIFIC CONSIDERATIONSPART IIFigure 17-12. Lobular carcinoma in situ (100x). There are small monomorphic cells that distend the terminal duct lobular unit, with-out necrosis or mitoses. (Used with permission from Dr. Sindhu Menon, Consultant Histopathologist and Dr. Rahul Deb, Consul-tant Histopathologist and Lead Breast

1	the terminal duct lobular unit, with-out necrosis or mitoses. (Used with permission from Dr. Sindhu Menon, Consultant Histopathologist and Dr. Rahul Deb, Consul-tant Histopathologist and Lead Breast Pathologist, Royal Derby Hospital, Derby, UK.)an incidental finding noted on breast biopsy. There is a variant of LCIS that has been termed pleomorphic LCIS. In the case of pleomorphic LCIS, there can be calcifications or other suspi-cious mammographic changes that dictate the need for biopsy. Classic LCIS is not treated with excision as the patient is at risk for developing invasive breast cancer in either breast and therefore the patient is counseled regarding appropriate risk reduction strategies. Pleomorphic LCIS can be difficult to dis-tinguish from high-grade DCIS and there are some proponents who have suggested that patients with pleomorphic LCIS be managed similar to those with DCIS with attention to margins and consideration for radiation therapy in the setting of breast

1	some proponents who have suggested that patients with pleomorphic LCIS be managed similar to those with DCIS with attention to margins and consideration for radiation therapy in the setting of breast conserving treatment. The use of immunohistochemical stain-ing for E-cadherin can help to discriminate between LCIS and DCIS. In lobular neoplasias, such as ALH and LCIS, there is a lack of E-cadherin expression, whereas the majority of ductal lesions will demonstrate E-cadherin reactivity.Treatment of Selected Benign Breast Disorders and DiseasesCysts. Because needle biopsy of breast masses may produce artifacts that make mammography assessment more difficult, many multidisciplinary teams prefer to image breast masses before performing either fine-needle aspiration or core-needle biopsy.36,37 In practice, however, the first investigation of pal-pable breast masses may be a needle biopsy, which allows for the early diagnosis of cysts. A 21-gauge needle attached to a 10-mL syringe is

1	In practice, however, the first investigation of pal-pable breast masses may be a needle biopsy, which allows for the early diagnosis of cysts. A 21-gauge needle attached to a 10-mL syringe is placed directly into the mass, which is fixed by fingers of the nondominant hand. The volume of a typical cyst is 5 to 10 mL, but it may be 75 mL or more. If the fluid that is aspirated is not bloodstained, then the cyst is aspirated to dryness, the needle is removed, and the fluid is discarded because cytologic examination of such fluid is not cost effec-tive. After aspiration, the breast is carefully palpated to exclude a residual mass. In most cases, however, imaging has been per-formed prior to a needle being introduced into the breast, and indeed the majority of cysts are now aspirated under ultrasound guidance. If a mass was noted on initial ultrasound or there is a residual mass post aspiration, then a tissue specimen is obtained, usually by core biopsy. When cystic fluid is bloodstained,

1	guidance. If a mass was noted on initial ultrasound or there is a residual mass post aspiration, then a tissue specimen is obtained, usually by core biopsy. When cystic fluid is bloodstained, fluid can be sent for cytologic examination. A simple cyst is rarely of concern, but a complex cyst may be the result of an underlying malignancy. A pneumocystogram can be obtained by injecting air into the cyst and then obtaining a repeat mammogram. When this technique is used, the wall of the cyst cavity can be more carefully assessed for any irregularities.Fibroadenomas. Most fibroadenomas are self-limiting and many go undiagnosed, so a more conservative approach is reasonable. Careful ultrasound examination with core-needle biopsy will provide for an accurate diagnosis. Ultrasonogra-phy may reveal specific features that are pathognomonic for fibroadenoma, and in a young woman (e.g., under 25 years) where the risk of breast cancer is already very low a core-needle biopsy may not be necessary.

1	specific features that are pathognomonic for fibroadenoma, and in a young woman (e.g., under 25 years) where the risk of breast cancer is already very low a core-needle biopsy may not be necessary. In patients where biopsy is performed, the patient is counseled concerning the ultra-sound and biopsy results, and surgical excision of the fibroad-enoma may be avoided. Cryoablation and ultrasound-guided vacuum-assisted biopsy are approved treatments for fibroad-enomas of the breast, especially lesions <3 cm. Larger lesions are often still best treated by excision. With short-term follow-up, a significant percentage of fibroadenomas will decrease in size and will no longer be palpable.38 However, many will remain palpable, especially those larger than 2 cm.39 There-fore, women should be counseled that the options for treat-ment include surgical removal, cryoablation, vacuum assisted biopsy, or observation.Sclerosing Disorders. The clinical significance of scleros-ing adenosis lies in its

1	that the options for treat-ment include surgical removal, cryoablation, vacuum assisted biopsy, or observation.Sclerosing Disorders. The clinical significance of scleros-ing adenosis lies in its imitation of cancer. On physical exami-nation, it may be confused with cancer, by mammography, and at gross pathologic examination. Excisional biopsy and histologic examination are frequently necessary to exclude the diagnosis of cancer. The diagnostic work-up for radial scars and complex sclerosing lesions frequently involves stereo-tactic biopsy. It usually is not possible to differentiate these lesions with certainty from cancer by mammographic features, so a larger tissue biopsy is recommended either by way of vacuum-assisted biopsy or an open surgical excisional biopsy. The mammographic appearance of a radial scar or sclerosing adenosis (mass density with spiculated margins) will usually lead to an assessment that the results of a core-needle biopsy specimen showing benign disease are

1	of a radial scar or sclerosing adenosis (mass density with spiculated margins) will usually lead to an assessment that the results of a core-needle biopsy specimen showing benign disease are discordant with the radiographic findings.Periductal Mastitis. Painful and tender masses behind the nipple-areola complex are aspirated with a 21-gauge needle attached to a 10-mL syringe. Any fluid obtained is submitted for culture using a transport medium appropriate for the detec-tion of anaerobic organisms. In the absence of pus, women are started on a combination of antibiotics to cover polymicrobial infections while awaiting the results of culture. Antibiotics are then continued based on sensitivity tests. Many cases respond satisfactorily to antibiotics alone, but when considerable puru-lent material is present, repeated ultrasound guided aspiration is performed, and ultimately in a proportion of cases surgical treatment is required. Unlike puerperal abscesses, a subareo-lar abscess is

1	material is present, repeated ultrasound guided aspiration is performed, and ultimately in a proportion of cases surgical treatment is required. Unlike puerperal abscesses, a subareo-lar abscess is usually unilocular and often is associated with a single duct system. Ultrasound will accurately delineate its extent. In those cases that come to surgery, the surgeon may either undertake simple drainage with a view toward formal Brunicardi_Ch17_p0541-p0612.indd 55401/03/19 5:04 PM 555THE BREASTCHAPTER 17Table 17-5Treatment of recurrent subareolar sepsisSUITABLE FOR FISTULECTOMYSUITABLE FOR TOTAL DUCT EXCISIONSmall abscess localized to one segmentLarge abscess affecting >50% of the areolar circumferenceRecurrence involving the same segmentRecurrence involving a different segmentMild or no nipple inversionMarked nipple inversionPatient unconcerned about nipple inversionPatient requests correction of nipple inversionYounger patientOlder patientNo discharge from other ductsPurulent

1	or no nipple inversionMarked nipple inversionPatient unconcerned about nipple inversionPatient requests correction of nipple inversionYounger patientOlder patientNo discharge from other ductsPurulent discharge from other ductsNo prior fistulectomyRecurrence after fistulectomyModified with permission from Mansel RE, Webster DJT: Benign Disorders and Diseases of the Breast: Concepts and Clinical Management, 2nd ed. London: Elsevier/Saunders; 2000.surgery, should the problem recur, or proceed with definitive surgery. In a woman of childbearing age, simple drainage is preferred, but if there is an anaerobic infection, recurrent infection frequently develops. Recurrent abscess with fistula is a difficult problem. Treatment of periductal fistula was ini-tially recommended to be opening up of the fistulous track and allowing it to granulate.40 This approach may still be used, especially if the fistula is recurrent after previous attempts at fistulectomy. However, nowadays the preferred

1	of the fistulous track and allowing it to granulate.40 This approach may still be used, especially if the fistula is recurrent after previous attempts at fistulectomy. However, nowadays the preferred initial surgical treatment is by fistulectomy and primary closure with anti-biotic coverage.41 Excision of all the major ducts is an alter-native option depending on the circumstances (Table 17-5). When a localized periareolar abscess recurs at the previous site and a fistula is present, the preferred operation is fistulec-tomy, which has minimal complications and a high degree of success. However, when subareolar sepsis is diffused rather than localized to one segment or when more than one fistula is present, total duct excision is the most expeditious approach. The first circumstance is seen in young women with squamous metaplasia of a single duct, whereas the latter circumstance is seen in older women with multiple ectatic ducts. Age is not always a reliable guide, however, and fistula

1	in young women with squamous metaplasia of a single duct, whereas the latter circumstance is seen in older women with multiple ectatic ducts. Age is not always a reliable guide, however, and fistula excision is the preferred initial procedure for localized sepsis irrespective of age. Antibiotic therapy is useful for recurrent infection after fistula excision, and a 2to 4-week course is recommended before total duct excision.Nipple Inversion. More women request correction of con-genital nipple inversion than request correction for the nipple inversion that occurs secondary to duct ectasia. Although the results are usually satisfactory, women seeking correction for cosmetic reasons should always be made aware of the surgi-cal complications of altered nipple sensation, nipple necrosis, and postoperative fibrosis with nipple retraction. Because nipple inversion is a result of shortening of the subareolar ducts, a com-plete division of these ducts is necessary for permanent correc-tion of

1	fibrosis with nipple retraction. Because nipple inversion is a result of shortening of the subareolar ducts, a com-plete division of these ducts is necessary for permanent correc-tion of the disorder.RISK FACTORS FOR BREAST CANCERHormonal and Nonhormonal Risk FactorsIncreased exposure to estrogen is associated with an increased risk for developing breast cancer, whereas reducing exposure is thought to be protective.42-48 Correspondingly, factors that increase the number of menstrual cycles, such as early men-arche, nulliparity, and late menopause, are associated with increased risk. Moderate levels of exercise and a longer lacta-tion period, factors that decrease the total number of menstrual cycles, are protective. The terminal differentiation of breast epi-thelium associated with a full-term pregnancy is also protective, so older age at first live birth is associated with an increased risk of breast cancer. Finally, there is an association between obesity and increased breast cancer

1	pregnancy is also protective, so older age at first live birth is associated with an increased risk of breast cancer. Finally, there is an association between obesity and increased breast cancer risk. Because the major source of estrogen in postmenopausal women is the conversion of andro-stenedione to estrone by adipose tissue, obesity is associated with a long-term increase in estrogen exposure.Nonhormonal risk factors include radiation exposure. Young women who receive mantle radiation therapy for Hodg-kin’s lymphoma have a breast cancer risk that is 75 times greater than that of age-matched control subjects. Survivors of the atomic bomb blasts in Japan during World War II have a very high incidence of breast cancer, likely because of somatic muta-tions induced by the radiation exposure. In both circumstances, radiation exposure during adolescence, a period of active breast development, magnifies the deleterious effect. Studies also sug-gest that the risk of breast cancer increases

1	In both circumstances, radiation exposure during adolescence, a period of active breast development, magnifies the deleterious effect. Studies also sug-gest that the risk of breast cancer increases as the amount of alcohol a woman consumes increases.49 Alcohol consumption is known to increase serum levels of estradiol. Finally, evidence suggests that long-term consumption of foods with a high fat content contributes to an increased risk of breast cancer by increasing serum estrogen levels.Risk Assessment ModelsThe average lifetime risk of breast cancer for newborn U.S. women is 12%.50,51 The longer a woman lives without cancer, the lower her risk of developing breast cancer. Thus, a woman age 50 years has an 11% lifetime risk of developing breast cancer, and a woman age 70 years has a 7% lifetime risk of developing breast cancer. Because risk factors for breast cancer interact, evaluating the risk conferred by combinations of risk factors is difficult. There are several risk

1	has a 7% lifetime risk of developing breast cancer. Because risk factors for breast cancer interact, evaluating the risk conferred by combinations of risk factors is difficult. There are several risk assessment models available to predict the risk of breast cancer. From the Breast Cancer Detec-tion Demonstration Project, a mammography screening program conducted in the 1970s, Gail et al developed the model most frequently used in the United States, which incorporates age, age at menarche, age at first live birth, the number of breast biopsy specimens, any history of atypical hyperplasia, and number of first-degree relatives with breast cancer.52 It predicts the cumula-tive risk of breast cancer according to decade of life. To calculate breast cancer risk using the Gail model, a woman’s risk factors are translated into an overall risk score by multiplying her rela-tive risks from several categories (Table 17-6). This risk score is then compared to an adjusted population risk of breast

1	factors are translated into an overall risk score by multiplying her rela-tive risks from several categories (Table 17-6). This risk score is then compared to an adjusted population risk of breast cancer to determine a woman’s individual or absolute risk. The output is a 5-year risk and a lifetime risk of developing breast cancer. A software program incorporating the Gail model is available from the National Cancer Institute at http://bcra.nci.nih.gov/brc. This model was recently modified to more accu-rately assess risk in African American women.52,53 There have also been modifications that project individualized absolute 5Brunicardi_Ch17_p0541-p0612.indd 55501/03/19 5:04 PM 556SPECIFIC CONSIDERATIONSPART IITable 17-6Relative risk estimates for the Gail modelVARIABLERELATIVE RISKAge at menarche (years) ≥14 12–13 <12Number of biopsy specimens/history of benign breast disease, age <50 y 0 1 ≥2Number of biopsy specimens/history of benign breast disease, age ≥50 y 0 1 ≥2Age at first

1	at menarche (years) ≥14 12–13 <12Number of biopsy specimens/history of benign breast disease, age <50 y 0 1 ≥2Number of biopsy specimens/history of benign breast disease, age ≥50 y 0 1 ≥2Age at first live birth (years) <20 y Number of first-degree relatives with history of breast cancer 0 1 ≥2 20–24 y Number of first-degree relatives with history of breast cancer 0 1 ≥2 25–29 y Number of first-degree relatives with history of breast cancer 0 1 ≥2 ≥30 y Number of first-degree relatives with history of breast cancer 0 1 ≥21.001.101.211.001.702.881.021.271.621.002.616.801.242.685.781.552.764.911.932.834.17Reproduced with permission from Armstrong K, Eisen A, Weber B: Assessing the risk of breast cancer, N Engl J Med. 2000 Feb 24;342(8):564-571.invasive breast cancer risk for Asian and Pacific Island American women. The Gail model is the most widely used model in the United States. Gail and colleagues have also described a revised model that includes body weight and

1	for Asian and Pacific Island American women. The Gail model is the most widely used model in the United States. Gail and colleagues have also described a revised model that includes body weight and mammographic density but excludes age at menarche.54Claus et al, using data from the Cancer and Steroid Hor-mone Study, a case-control study of breast cancer, developed the other frequently used risk assessment model, which is based on assumptions about the prevalence of high-penetrance breast cancer susceptibility genes.55 Compared with the Gail model, the Claus model incorporates more information about family his-tory but excludes other risk factors. The Claus model provides individual estimates of breast cancer risk according to decade of life based on presence of firstand second-degree relatives with breast cancer and their age at diagnosis. Risk factors that are less consistently associated with breast cancer (diet, use of oral contraceptives, lactation) or are rare in the general

1	relatives with breast cancer and their age at diagnosis. Risk factors that are less consistently associated with breast cancer (diet, use of oral contraceptives, lactation) or are rare in the general population (radiation exposure) are not included in either the Gail or Claus risk assessment model. Other models have been proposed that account for mammographic breast density in assessing breast cancer risk.54,56Neither the Gail model nor the Claus model accounts for the risk associated with mutations in the breast cancer suscepti-bility genes BRCA1 and BRCA2 (described in detail in the fol-lowing section). The BRCAPRO model is a Mendelian model that calculates the probability that an individual is a carrier of a mutation in one of the breast cancer susceptibility genes based on their family history of breast and ovarian cancer.57 The prob-ability that an individual will develop breast or ovarian cancer is derived from this mutation probability based on age-specific incidence curves for

1	history of breast and ovarian cancer.57 The prob-ability that an individual will develop breast or ovarian cancer is derived from this mutation probability based on age-specific incidence curves for both mutation carriers and noncarriers.58 Use of the BRCAPRO model in the clinic is challenging since it requires input of all family history information regarding breast and ovarian cancer. The Tyrer-Cuzick model attempts to utilize both family history information and individual risk information. It uses the family history to calculate the probability that an individual carries a mutation in one of the breast cancer suscep-tibility genes, and then the risk is adjusted based on personal risk factors, including age at menarche, parity, age at first live birth, age at menopause, history of atypical hyperplasia or LCIS, height, and body mass index.59 Once a risk model has been uti-lized to assess breast cancer risk, this must be communicated to the individual and put into context with

1	atypical hyperplasia or LCIS, height, and body mass index.59 Once a risk model has been uti-lized to assess breast cancer risk, this must be communicated to the individual and put into context with competing risk and medical comorbidities. This information can then be used to discuss options that are available to the individual for manag-ing risk.Risk ManagementSeveral important medical decisions may be affected by a wom-an’s underlying risk of developing breast cancer.60-68 These deci-sions include when to use postmenopausal hormone replacement therapy, at what age to begin mammography screening or incor-porate magnetic resonance imaging (MRI) screening, when to use tamoxifen to prevent breast cancer, and when to perform prophylactic mastectomy to prevent breast cancer. Postmeno-pausal hormone replacement therapy was widely prescribed in the 1980s and 1990s because of its effectiveness in controlling the symptoms of estrogen deficiency, namely vasomotor symp-toms such as hot flashes,

1	replacement therapy was widely prescribed in the 1980s and 1990s because of its effectiveness in controlling the symptoms of estrogen deficiency, namely vasomotor symp-toms such as hot flashes, night sweats and their associated sleep deprivation, osteoporosis, and cognitive changes. Furthermore, these hormone supplements were thought to reduce coronary artery disease as well. Use of combined estrogen and progester-one became standard for women who had not undergone hyster-ectomy because unopposed estrogen increases the risk of uterine cancer. Concerns of prolonging a woman’s lifetime exposure to estrogen, coupled with conflicting data regarding the impact of these hormones on cardiovascular health, motivated the imple-mentation of large-scale phase 3 clinical trials to definitively evaluate the risks vs. benefits of postmenopausal hormone replacement therapy. The Women’s Health Initiative (WHI) was therefore designed by the National Institutes of Health as a series of clinical trials

1	the risks vs. benefits of postmenopausal hormone replacement therapy. The Women’s Health Initiative (WHI) was therefore designed by the National Institutes of Health as a series of clinical trials to study the effects of diet, nutritional supplements, and hormones on the risk of cancer, cardiovascular disease, and bone health in postmenopausal women. Findings from primary studies of postmenopausal hormone replacement therapy were released in 2002, demonstrating conclusively that Brunicardi_Ch17_p0541-p0612.indd 55601/03/19 5:04 PM 557THE BREASTCHAPTER 17breast cancer risk is threefold to fourfold higher after >4 years of use and there is no significant reduction in coronary artery or cerebrovascular risks. The Collaborative Group on Hormonal Factors in Breast Cancer combined and reanalyzed data from a number of studies totaling 52,705 women with breast cancer and 108,411 women without breast cancer. They found an increased risk of breast cancer with every use of estrogen

1	and reanalyzed data from a number of studies totaling 52,705 women with breast cancer and 108,411 women without breast cancer. They found an increased risk of breast cancer with every use of estrogen replacement therapy. They also reported increased risk among current users but not past users and risk increased with increasing duration of use of hormone replacement therapy.69 Cheblowski et al also reported from the WHI study that estrogen + progesterone increased the incidence of breast cancer.70 This was con-firmed by the Million Women study, which also showed that the increased risk was substantially greater for the combined estrogen + progesterone replacement therapy than other types of hormone replacement therapy.71Breast Cancer Screening. Routine use of screening mam-mography in women ≥50 years of age has been reported to reduce mortality from breast cancer by 25%.72 This reduc-tion comes at an acceptable economic cost. More recently, there has been debate over the potential

1	women ≥50 years of age has been reported to reduce mortality from breast cancer by 25%.72 This reduc-tion comes at an acceptable economic cost. More recently, there has been debate over the potential harms associated with breast screening.73 Controversy over the age to initiate screening mammography is evident in the current recommendations. The U.S. Preventive Services Task Force (USPSTF), the American Cancer Society (ACS), and the National Comprehensive Cancer Network (NCCN) are three organizations with differing recom-mendations for screening mammography in average risk women. The guidelines, however, similarly define high-risk women as those with personal history of breast cancer, history of chest radiation at young age, and confirmed or suspected genetic mutation known to increase risk for developing breast cancer. The USPSTF recommends biennial screening mammog-raphy for women age 50 to 74 years. The USPSTF applies these guidelines to asymptomatic women age >40 years who do not

1	for developing breast cancer. The USPSTF recommends biennial screening mammog-raphy for women age 50 to 74 years. The USPSTF applies these guidelines to asymptomatic women age >40 years who do not have a preexisting breast cancer or who were not previously diagnosed with a high-risk breast lesion, and who are not at high risk for breast cancer because of a known underlying genetic mutation or history of chest radiation at a young age.74-76 In October 2015, the ACS released updated guidelines stating average-risk women should start annual screening mammogra-phy at 45 years of age. Women age 45 to 54 years should be screened annually, and those 55 years and older should transi-tion to biennial screening or have the opportunity to continue annual screening. Women should have the opportunity to begin annual screening between the ages of 40 and 44 years and should continue screening as long as their overall health is good and have a life expectancy of 10 years or longer. The ACS does not

1	to begin annual screening between the ages of 40 and 44 years and should continue screening as long as their overall health is good and have a life expectancy of 10 years or longer. The ACS does not recommend clinical breast examination for breast cancer screening among average-risk women at any age.77 The NCCN recommends that average-risk women begin annual screening mammograms at ≥40 years of age, along with annual clinical breast exams and breast awareness.78The United Kingdom recently established an independent expert panel to review the published literature and estimate the benefits and harms associated with screening women >50 years of age in its national screening program.79 The expert panel estimated that an invitation to breast screening delivers about a 20% reduction in breast cancer mortality. At the same time, however, the panel estimated that in women invited to the screening, about 11% of the cancers diagnosed in their lifetime constitute overdiagnosis. Despite the

1	cancer mortality. At the same time, however, the panel estimated that in women invited to the screening, about 11% of the cancers diagnosed in their lifetime constitute overdiagnosis. Despite the overdiagnosis, the panel concluded that breast screening confers significant benefit and should continue. The use of screening mammography in women <50 years of age is more controversial for several reasons: (a) breast density is greater, and screening mammography is less likely to detect early breast cancer (i.e., reduced sensitivity); (b) screening mammography results in more false-positive test findings (i.e., reduced specificity), which results in unneces-sary biopsy specimens; and (c) younger women are less likely to have breast cancer (i.e., lower incidence), so fewer young women will benefit from screening.80,81 In the United States, on a population basis, however, the benefits of screening mam-mography in women between the ages of 40 and 49 years is still felt to outweigh the risks;

1	from screening.80,81 In the United States, on a population basis, however, the benefits of screening mam-mography in women between the ages of 40 and 49 years is still felt to outweigh the risks; although targeting mammography to women at higher risk of breast cancer improves the balance of risks and benefits and is the approach some health care sys-tems have taken. In one study of women age 40 to 49 years, an abnormal mammography finding was three times more likely to be cancer in a woman with a family history of breast cancer than in a woman without such a history. Furthermore, as noted previously in the section Risk Assessment Models, mounting data regarding mammographic breast density demonstrate an independent correlation with breast cancer risk. Incorporation of breast density measurements into breast cancer risk assess-ment models appears to be a promising strategy for increasing the accuracy of these tools. Unfortunately, widespread applica-tion of these modified models is

1	into breast cancer risk assess-ment models appears to be a promising strategy for increasing the accuracy of these tools. Unfortunately, widespread applica-tion of these modified models is hampered by inconsistencies in the reporting of mammographic density. Ultrasonography can also be used for breast cancer screening in women with dense breasts, but there is no data available that the additional cancers detected with this modality reduce mortality from breast cancer.Current recommendations by the United States Preventive Services Task Force are that women undergo biennial mammo-graphic screening between the ages of 50 and 74 years.77 The use of MRI for breast cancer screening is recommended by the ACS for women with a 20% to 25% or greater lifetime risk using risk assessment tools based mainly on family history, BRCA mutation carriers, those individuals who have a family member with a BRCA mutation who have not been tested themselves, individuals who received radiation to the chest

1	mainly on family history, BRCA mutation carriers, those individuals who have a family member with a BRCA mutation who have not been tested themselves, individuals who received radiation to the chest between the ages of 10 and 30 years, and those individuals with a history of Li-Fraumeni syndrome, Cowden syndrome, or Bannayan-Riley-Ruvalcaba syndrome or those who have a first-degree relative with one of these syndromes. MRI is an extremely sensitive screening tool that is not limited by the density of the breast tissue as mammography is; however, its specificity is moderate, leading to more false-positive events and the increased need for biopsy.Chemoprevention. Tamoxifen, a selective estrogen receptor modulator, was the first drug shown to reduce the incidence of breast cancer in healthy women. There have been four pro-spective studies published evaluating tamoxifen vs. placebo for reducing the incidence of invasive breast cancer for women at increased risk. The largest trial was the

1	women. There have been four pro-spective studies published evaluating tamoxifen vs. placebo for reducing the incidence of invasive breast cancer for women at increased risk. The largest trial was the Breast Cancer Preven-tion Trial (NSABP P-01), which randomly assigned >13,000 women with a 5-year Gail relative risk of breast cancer of 1.66% or higher or LCIS to receive tamoxifen or placebo. After a mean follow-up period of 4 years, the incidence of breast cancer was reduced by 49% in the group receiving tamoxifen.60 The decrease was evident only in ER-positive breast cancers with no significant change in ER-negative tumors. The Royal Marsden Hospital Tamoxifen Chemoprevention Trial,78 the Italian Tamox-ifen Prevention Trial,82 and the International Breast Cancer Intervention Study I (IBIS-I) trial all83 showed a reduction in 6Brunicardi_Ch17_p0541-p0612.indd 55701/03/19 5:04 PM 558SPECIFIC CONSIDERATIONSPART IIER-positive breast cancers with the use of tamoxifen compared with

1	trial all83 showed a reduction in 6Brunicardi_Ch17_p0541-p0612.indd 55701/03/19 5:04 PM 558SPECIFIC CONSIDERATIONSPART IIER-positive breast cancers with the use of tamoxifen compared with placebo. There was no effect on mortality; however, the trials were not powered to assess either breast cancer mortality or all-cause mortality events. The adverse events were similar in all four randomized trials, including an increased risk of endo-metrial cancer, thromboembolic events, cataract formation, and vasomotor disturbances in individuals receiving tamoxifen.Tamoxifen therapy currently is recommended only for women who have a Gail relative risk of 1.66% or higher, who are age 35 to 59, women over the age of 60, or women with a diagnosis of LCIS or atypical ductal or lobular hyperplasia. In addition, deep vein thrombosis occurs 1.6 times as often, pulmonary emboli 3.0 times as often, and endometrial cancer 2.5 times as often in women taking tamoxifen. The increased risk for endometrial

1	addition, deep vein thrombosis occurs 1.6 times as often, pulmonary emboli 3.0 times as often, and endometrial cancer 2.5 times as often in women taking tamoxifen. The increased risk for endometrial cancer is restricted to early stage cancers in postmenopausal women. Cataract surgery is required almost twice as often among women taking tamoxifen. Gail et al sub-sequently developed a model that accounts for underlying risk of breast cancer as well as comorbidities to determine the net risk-benefit ratio of tamoxifen use for chemoprevention.84The NSABP completed a second chemoprevention trial, designed to compare tamoxifen and raloxifene for breast cancer risk reduction in high-risk postmenopausal women. Raloxifene, another selective estrogen receptor modulator, was selected for the experimental arm in this follow-up prevention trial because its use in managing postmenopausal osteoporosis suggested that it might be even more effective at breast cancer risk reduc-tion, but without the

1	arm in this follow-up prevention trial because its use in managing postmenopausal osteoporosis suggested that it might be even more effective at breast cancer risk reduc-tion, but without the adverse effects of tamoxifen on the uterus. The P-2 trial, the Study of Tamoxifen and Raloxifene (known as the STAR trial), randomly assigned 19,747 postmenopausal women at high-risk for breast cancer to receive either tamoxi-fen or raloxifene. The initial report of the P-2 trial showed the two agents were nearly identical in their ability to reduce breast cancer risk, but raloxifene was associated with a more favor-able adverse event profile.85 An updated analysis revealed that raloxifene maintained 76% of the efficacy of tamoxifen in pre-vention of invasive breast cancer with a more favorable side effect profile. The risk of developing endometrial cancer was significantly higher with tamoxifen use at longer follow-up.86 Although tamoxifen has been shown to reduce the incidence of LCIS and DCIS,

1	profile. The risk of developing endometrial cancer was significantly higher with tamoxifen use at longer follow-up.86 Although tamoxifen has been shown to reduce the incidence of LCIS and DCIS, raloxifene did not have an effect on the frequency of these diagnoses.Aromatase inhibitors (AIs) have been shown to be more effective than tamoxifen in reducing the incidence of contra-lateral breast cancers in postmenopausal women receiving AIs for adjuvant treatment of invasive breast cancer. The MAP.3 trial was the first study to evaluate an AI as a chemopreventive agent in postmenopausal women at high risk for breast cancer. The trial randomized 4560 women to exemestane 25 mg daily vs. placebo for 5 years. After a median follow-up of 35 months, exemestane was shown to reduce invasive breast cancer inci-dence by 65%. Side effect profiles demonstrated more grade II or higher arthritis and hot flashes in patients taking exemestane.87 The IBIS II trial on the other hand, randomized 3864

1	cancer inci-dence by 65%. Side effect profiles demonstrated more grade II or higher arthritis and hot flashes in patients taking exemestane.87 The IBIS II trial on the other hand, randomized 3864 postmeno-pausal women to either anastrozole, a nonsteroidal aromatase inhibitor, vs. placebo with a further randomization to bisphospho-nate or not based on bone density.88,89 After a median follow-up of 5 years, anastrozole reduced the incidence of invasive breast cancer by about 50%. The trial also had an initial sub-study that looked at the effect of the aromatase inhibitor on cogni-tive function and reported no adverse effects.90 The American Society of Clinical Oncology recommends tamoxifen for chemoprevention in premenopausal or postmenopausal women and consideration for raloxifene or exemestane in postmeno-pausal women who are noted to be at increased risk of breast cancer.91,92 The discussion with an individual patient should include risk assessment and potential risks and benefits

1	in postmeno-pausal women who are noted to be at increased risk of breast cancer.91,92 The discussion with an individual patient should include risk assessment and potential risks and benefits with each agent.Risk-Reducing Surgery. A retrospective study of women at high risk for breast cancer found that prophylactic mastectomy reduced their risk by >90%.62 However, the effects of prophylac-tic mastectomy on the long-term quality of life are poorly quan-tified. A study involving women who were carriers of a breast cancer susceptibility gene (BRCA) mutation found that the ben-efit of prophylactic mastectomy differed substantially according to the breast cancer risk conferred by the mutations. For women with an estimated lifetime risk of 40%, prophylactic mastec-tomy added almost 3 years of life, whereas for women with an estimated lifetime risk of 85%, prophylactic mastectomy added >5 years of life.66 Domchek et al evaluated a cohort of BRCA1 and 2 mutation carriers who were followed

1	life, whereas for women with an estimated lifetime risk of 85%, prophylactic mastectomy added >5 years of life.66 Domchek et al evaluated a cohort of BRCA1 and 2 mutation carriers who were followed prospectively and reported on outcomes with risk-reducing surgery.93 They found that risk-reducing mastectomy was highly effective at preventing breast cancer in both BRCA1 and 2 mutation carriers. Risk-reducing salpingo-oophorectomy was highly effective at reducing the incidence of ovarian cancer and breast cancer in BRCA mutation carriers and was associated with a reduction in breast cancer-specific mortality, ovarian cancer-specific mor-tality, and all-cause mortality. While studies of bilateral pro-phylactic or risk-reducing mastectomy have reported dramatic reductions in breast cancer incidence among those without known BRCA mutations, there is little data to support a survival benefit. Another consideration is that while most patients are satisfied with their decision to pursue

1	incidence among those without known BRCA mutations, there is little data to support a survival benefit. Another consideration is that while most patients are satisfied with their decision to pursue risk-reducing surgery, some are dissatisfied with the cosmetic outcomes mostly due to reconstructive issues.BRCA MutationsBRCA1. Up to 5% of breast cancers are caused by inheritance of germline mutations such as BRCA1 and BRCA2, which are inherited in an autosomal dominant fashion with varying degrees of penetrance (Table 17-7).94-100 BRCA1 is located on chromosome arm 17q, spans a genomic region of approximately 100 kilobases (kb) of DNA, and contains 22 coding exons for 1863 amino acids. Both BRCA1 and BRCA2 function as tumor-suppressor genes, and for each gene, loss of both alleles is required for the initiation of cancer. Data accumulated since the isolation of the BRCA1 gene suggest a role in transcription, cell-cycle control, and DNA damage repair pathways. More than 500 sequence

1	for the initiation of cancer. Data accumulated since the isolation of the BRCA1 gene suggest a role in transcription, cell-cycle control, and DNA damage repair pathways. More than 500 sequence variations in BRCA1 have been identified. It now is known that germline mutations in BRCA1 represent a predisposing genetic factor in as many as 45% of hereditary breast cancers and in at least 80% of hereditary ovarian cancers. Female mutation carriers have been reported to have up to an 85% lifetime risk (for some families) for developing breast cancer and up to a 40% lifetime risk for developing ovarian cancer. The initial families reported had high penetrance and subsequently the average lifetime risk has been reported to lie between 60% and 70%. Breast cancer susceptibility in these families appears as an autosomal dominant trait with high pen-etrance. Approximately 50% of children of carriers inherit the trait. In general, BRCA1-associated breast cancers are invasive ductal carcinomas, are

1	as an autosomal dominant trait with high pen-etrance. Approximately 50% of children of carriers inherit the trait. In general, BRCA1-associated breast cancers are invasive ductal carcinomas, are poorly differentiated, are in the majority Brunicardi_Ch17_p0541-p0612.indd 55801/03/19 5:04 PM 559THE BREASTCHAPTER 17Table 17-7Incidence of sporadic, familial, and hereditary breast cancerSporadic breast cancer65%–75%Familial breast cancer20%–30%Hereditary breast cancer5%–10% BRCA1a45% BRCA235% p53a (Li-Fraumeni syndrome)1% STK11/LKB1a (Peutz-Jeghers syndrome)<1% PTENa (Cowden disease)<1% MSH2/MLH1a (Muir-Torre syndrome)<1% ATMa (Ataxia-telangiectasia)<1% Unknown20%aAffected gene.Data from Martin AM, Weber BL: Genetic and hormonal risk factors in breast cancer, J Natl Cancer Inst. 2000 Jul 19;92(14):1126-1135.hormone receptor negative, and have a triple receptor negative (immunohistochemical profile: ER-negative, PR-negative, and HER2-negative) or basal phenotype (based on gene

1	Jul 19;92(14):1126-1135.hormone receptor negative, and have a triple receptor negative (immunohistochemical profile: ER-negative, PR-negative, and HER2-negative) or basal phenotype (based on gene expression profiling). BRCA1-associated breast cancers have a number of distinguishing clinical features, such as an early age of onset compared with sporadic cases; a higher prevalence of bilateral breast cancer; and the presence of associated cancers in some affected individuals, specifically ovarian cancer and possibly colon and prostate cancers.Several founder mutations have been identified in BRCA1. The two most common mutations are 185delAG and 5382insC, which account for 10% of all the mutations seen in BRCA1. These two mutations occur at a 10-fold higher frequency in the Ashkenazi Jewish population than in non-Jewish Caucasians. The carrier frequency of the 185delAG mutation in the Ashkenazi Jewish population is 1% and, along with the 5382insC mutation, accounts for almost all BRCA1

1	population than in non-Jewish Caucasians. The carrier frequency of the 185delAG mutation in the Ashkenazi Jewish population is 1% and, along with the 5382insC mutation, accounts for almost all BRCA1 mutations in this population. Analysis of germline mutations in Jewish and non-Jewish women with early-onset breast cancer indicates that 20% of Jewish women who develop breast cancer before age 40 years carry the 185delAG mutation. There are founder BRCA1 mutations in other populations including, among others, Dutch, Polish, Finnish, and Russian populations.101-105BRCA2. BRCA2 is located on chromosome arm 13q and spans a genomic region of approximately 70 kb of DNA. The 11.2-kb coding region contains 26 coding exons.94-100 It encodes a pro-tein of 3418 amino acids. The BRCA2 gene bears no homology to any previously described gene, and the protein contains no previously defined functional domains. The biologic function of BRCA2 is not well defined, but like BRCA1, it is postulated to play

1	to any previously described gene, and the protein contains no previously defined functional domains. The biologic function of BRCA2 is not well defined, but like BRCA1, it is postulated to play a role in DNA damage response pathways. BRCA2 mes-senger RNA also is expressed at high levels in the late G1 and S phases of the cell cycle. The kinetics of BRCA2 protein regu-lation in the cell cycle is similar to that of BRCA1 protein, which suggests that these genes are coregulated. The mutational spec-trum of BRCA2 is not as well established as that of BRCA1. To date, >250 mutations have been found. The breast cancer risk for BRCA2 mutation carriers is close to 85%, and the life-time ovarian cancer risk, while lower than for BRCA1, is still estimated to be close to 20%. Breast cancer susceptibility in BRCA2 families is an autosomal dominant trait and has a high penetrance. Approximately 50% of children of carriers inherit the trait. Unlike male carriers of BRCA1 mutations, men with

1	in BRCA2 families is an autosomal dominant trait and has a high penetrance. Approximately 50% of children of carriers inherit the trait. Unlike male carriers of BRCA1 mutations, men with germline mutations in BRCA2 have an estimated breast cancer risk of 6%, which represents a 100-fold increase over the risk in the general male population. BRCA2-associated breast cancers are invasive ductal carcinomas, which are more likely to be well differentiated and to express hormone receptors than are BRCA1-associated breast cancers. BRCA2-associated breast cancer has a number of distinguishing clinical features, such as an early age of onset compared with sporadic cases, a higher prevalence of bilateral breast cancer, and the presence of associ-ated cancers in some affected individuals, specifically ovarian, colon, prostate, pancreatic, gallbladder, bile duct, and stomach cancers, as well as melanoma. A number of founder mutations have been identified in BRCA2. The 6174delT mutation is found

1	ovarian, colon, prostate, pancreatic, gallbladder, bile duct, and stomach cancers, as well as melanoma. A number of founder mutations have been identified in BRCA2. The 6174delT mutation is found in Ashkenazi Jews with a prevalence of 1.2% and accounts for 60% of ovarian cancer and 30% of early-onset breast cancer patients among Ashkenazi women.106 Another BRCA2 founder mutation, 999del5, is observed in Icelandic and Finnish popula-tions, while more recently 3036delACAA has been observed in a number of Spanish families.107-109Identification of BRCA Mutation Carriers. Identifying hereditary risk for breast cancer is a four-step process that includes: (a) obtaining a complete, multigenerational family history, (b) assessing the appropriateness of genetic testing for a particular patient, (c) counseling the patient, and (d) interpret-ing the results of testing.110 Genetic testing should not be offered in isolation, but only in conjunction with patient education and counseling, including

1	counseling the patient, and (d) interpret-ing the results of testing.110 Genetic testing should not be offered in isolation, but only in conjunction with patient education and counseling, including referral to a genetic counselor. Initial determinations include whether the individual is an appropriate candidate for genetic testing and whether genetic testing will be informative for personal and clinical decision-making. A thor-ough and accurate family history is essential to this process, and the maternal and paternal sides of the family are both assessed because 50% of the women with a BRCA mutation have inher-ited the mutation from their fathers. To help clinicians advise women about genetic testing, statistically based models that determine the probability that an individual carries a BRCA mutation have been developed. A method for calculating carrier probability that has been demonstrated to have acceptable per-formance (i.e., both in terms of calibration and discrimination) such

1	BRCA mutation have been developed. A method for calculating carrier probability that has been demonstrated to have acceptable per-formance (i.e., both in terms of calibration and discrimination) such as the Manchester scoring system and BODICEA should be used to offer referral to a specialist genetic clinic. A heredi-tary risk of breast cancer is considered if a family includes Ash-kenazi Jewish heritage; a first-degree relative with breast cancer before age 50; a history of ovarian cancer at any age in the patient or firstor second-degree relative with ovarian cancer; breast and ovarian cancer in the same individual; two or more firstor second-degree relatives with breast cancer at any age; patient or relative with bilateral breast cancer; and male breast cancer in a relative at any age.111 The threshold for genetic test-ing is lower in individuals who are members of ethnic groups in whom the mutation prevalence is increased.BRCA Mutation Testing. Appropriate counseling for the

1	age.111 The threshold for genetic test-ing is lower in individuals who are members of ethnic groups in whom the mutation prevalence is increased.BRCA Mutation Testing. Appropriate counseling for the individual being tested for a BRCA mutation is strongly rec-ommended, and documentation of informed consent is required.110,112 The test that is clinically available for analyzing BRCA mutations is gene sequence analysis. In a family with a history suggestive of hereditary breast cancer and no previously Brunicardi_Ch17_p0541-p0612.indd 55901/03/19 5:04 PM 560SPECIFIC CONSIDERATIONSPART IItested member, the most informative strategy is first to test an affected family member. This person undergoes complete sequence analysis of both the BRCA1 and BRCA2 genes. If a mutation is identified, relatives are usually tested only for that specific mutation. An individual of Ashkenazi Jewish ancestry is tested initially for the three specific mutations that account for hereditary breast and

1	relatives are usually tested only for that specific mutation. An individual of Ashkenazi Jewish ancestry is tested initially for the three specific mutations that account for hereditary breast and ovarian cancer in that population. If results of that test are negative, it may then be appropriate to fully analyze the BRCA1 and BRCA2 genes.A positive test result is one that discloses the presence of a BRCA mutation that interferes with translation or function of the BRCA protein. A woman who carries a deleterious mutation has a breast cancer risk of up to 85% (in some families) as well as a greatly increased risk of ovarian cancer. A negative test result is interpreted according to the individual’s personal and family history, especially whether a mutation has been previously iden-tified in the family, in which case the woman is generally tested only for that specific mutation. If the mutation is not present, the woman’s risk of breast or ovarian cancer may be no greater than that of

1	in the family, in which case the woman is generally tested only for that specific mutation. If the mutation is not present, the woman’s risk of breast or ovarian cancer may be no greater than that of the general population. In addition, no BRCA muta-tion can be passed on to the woman’s children. In the absence of a previously identified mutation, a negative test result in an affected individual generally indicates that a BRCA mutation is not responsible for the familial cancer. However, the possibil-ity remains of an unusual abnormality in one of these genes that cannot yet be identified through clinical testing. It also is possible that the familial cancer is indeed caused by an identifi-able BRCA mutation but that the individual tested had sporadic cancer, a situation known as phenocopy. This is especially pos-sible if the individual tested developed breast cancer close to the age of onset of the general population (age 60 years or older) rather than before age 50 years, as is

1	This is especially pos-sible if the individual tested developed breast cancer close to the age of onset of the general population (age 60 years or older) rather than before age 50 years, as is characteristic of BRCA mutation carriers. Overall, the false-negative rate for BRCA mutation testing is <5%. Some test results, especially when a single base-pair change (missense mutation) is identified, may be difficult to interpret. This is because single base-pair changes do not always result in a nonfunctional protein. Thus, missense mutations not located within critical functional domains, or those that cause only minimal changes in protein structure, may not be disease associated and are usually reported as indetermi-nate results. In communicating indeterminate results to women, care must be taken to relay the uncertain cancer risk associ-ated with this type of mutation and to emphasize that ongoing research might clarify its meaning. In addition, testing other family members with breast

1	taken to relay the uncertain cancer risk associ-ated with this type of mutation and to emphasize that ongoing research might clarify its meaning. In addition, testing other family members with breast cancer to determine if a genetic variant tracks with their breast cancer may provide clarification as to its significance. Indeterminate genetic variance currently accounts for 12% of the test results.Concern has been expressed that the identification of hereditary risk for breast cancer may interfere with access to affordable health insurance. This concern refers to discrimina-tion directed against an individual or family based solely on an apparent or perceived genetic variation from the normal human genotype. The Health Insurance Portability and Accountability Act of 1996 (HIPAA) made it illegal in the United States for group health plans to consider genetic information as a preexist-ing condition or to use it to deny or limit coverage. Most states also have passed laws that prevent

1	illegal in the United States for group health plans to consider genetic information as a preexist-ing condition or to use it to deny or limit coverage. Most states also have passed laws that prevent genetic discrimination in the provision of health insurance. In addition, individuals applying for health insurance are not required to report whether relatives have undergone genetic testing for cancer risk, only whether those relatives have actually been diagnosed with cancer. Currently, there is little documented evidence of genetic dis-crimination resulting from findings of available genetic tests.Cancer Prevention for BRCA Mutation Carriers. Risk man-agement strategies for BRCA1 and BRCA2 mutation carriers include the following:1. Risk-reducing mastectomy and reconstruction2. Risk-reducing salpingo-oophorectomy3. Intensive surveillance for breast and ovarian cancer4. ChemopreventionAlthough removal of breast tissue reduces the likeli-hood that BRCA1 and BRCA2 mutation carriers will

1	salpingo-oophorectomy3. Intensive surveillance for breast and ovarian cancer4. ChemopreventionAlthough removal of breast tissue reduces the likeli-hood that BRCA1 and BRCA2 mutation carriers will develop breast cancer, mastectomy does not remove all breast tissue, and women continue to be at risk because a germline muta-tion is present in any remaining breast tissue. For postmeno-pausal BRCA1 and BRCA2 mutation carriers who have not had a mastectomy, it may be advisable to avoid hormone replace-ment therapy because no data exist regarding the effect of the therapy on the penetrance of breast cancer susceptibility genes. Because breast cancers in BRCA mutation carriers have the same mammographic appearance as breast cancers in noncarri-ers, a screening mammogram is likely to be effective in BRCA mutation carriers, provided it is performed and interpreted by an experienced radiologist with a high level of suspicion. Pres-ent screening recommendations for BRCA mutation carriers who do

1	in BRCA mutation carriers, provided it is performed and interpreted by an experienced radiologist with a high level of suspicion. Pres-ent screening recommendations for BRCA mutation carriers who do not undergo risk-reducing mastectomy include clinical breast examination every 6 months and mammography every 12 months beginning at age 25 years because the risk of breast cancer in BRCA mutation carriers increases after age 30 years. Recent attention has been focused on the use of MRI for breast cancer screening in high-risk individuals and known BRCA mutation carriers. MRI appears to be more sensitive at detect-ing breast cancer in younger women with dense breasts.113 How-ever, as noted previously, MRI does lead to the detection of benign breast lesions that cannot easily be distinguished from malignancy, and these false-positive events can result in more interventions, including biopsy specimens. The current recom-mendations from the American Cancer Society are for annual MRI in women

1	malignancy, and these false-positive events can result in more interventions, including biopsy specimens. The current recom-mendations from the American Cancer Society are for annual MRI in women with a 20% to 25% or greater lifetime risk of developing breast cancer (mainly based on family history), women with a known BRCA1 or BRCA2 mutation, those who have a first-degree relative with a BRCA1 or BRCA2 mutation and have not had genetic testing themselves, women who were treated with radiation therapy to the chest between the ages of 10 and 30 years, and those who have Li-Fraumeni syndrome, Cowden syndrome, or Bannayan-Riley-Ruvalcaba syndrome, or a first-degree relative with one of these syndromes.75,114 Despite a 49% reduction in the overall incidence of breast cancer and a 69% reduction in the incidence of estrogen receptor positive tumors in high-risk women taking tamoxifen reported in the NSABP P1 trial, there is insufficient evidence to recommend the use of tamoxifen uniformly

1	in the incidence of estrogen receptor positive tumors in high-risk women taking tamoxifen reported in the NSABP P1 trial, there is insufficient evidence to recommend the use of tamoxifen uniformly for BRCA1 mutation carriers.60 Cancers arising in BRCA1 mutation carriers are usually high grade and are most often hormone receptor negative. Approxi-mately 66% of BRCA1-associated DCIS lesions are estrogen receptor negative, which suggests early acquisition of the hor-mone-independent phenotype. In the NSABP P1 trial there was a 62% reduction in the incidence of breast cancer in BRCA2 carriers, similar to the overall reduction seen in the P1 trial. In contrast, there was no reduction seen in breast cancer incidence in BRCA1 carriers who started tamoxifen in P1 age 35 years or Brunicardi_Ch17_p0541-p0612.indd 56001/03/19 5:04 PM 561THE BREASTCHAPTER 17older.115 Tamoxifen appears to be more effective at preventing estrogen receptor-positive breast cancers.The risk of ovarian cancer in

1	56001/03/19 5:04 PM 561THE BREASTCHAPTER 17older.115 Tamoxifen appears to be more effective at preventing estrogen receptor-positive breast cancers.The risk of ovarian cancer in BRCA1 and BRCA2 muta-tion carriers ranges from 20% to 40%, which is 10 times higher than that in the general population. Risk-reducing salpingo-oophorectomy is a reasonable prevention option in mutation carriers. In women with a documented BRCA1 or BRCA2 mutation, consideration for bilateral risk-reducing salpingo-oophorectomy should be between the ages of 35 and 40 years at the completion of childbearing. Removing the ovaries reduces the risk of ovarian cancer and breast cancer when per-formed in premenopausal BRCA mutation carriers. Hormone replacement therapy is discussed with the patient at the time of oophorectomy. The Cancer Genetics Studies Consortium recommends yearly transvaginal ultrasound timed to avoid ovulation and annual measurement of serum cancer antigen 125 levels beginning at age 25 years

1	The Cancer Genetics Studies Consortium recommends yearly transvaginal ultrasound timed to avoid ovulation and annual measurement of serum cancer antigen 125 levels beginning at age 25 years as the best screening modalities for ovarian carcinoma in BRCA mutation carriers who have opted to defer risk-reducing surgery.PALB2 (partner and localizer of BRCA2) has recently been characterized as a potential high-risk gene for breast cancer. PALB2 allows nuclear localization of BRCA2 and provides a scaffold for the BRCA1–PALB2–BRCA2 complex. Analysis by Antoniou et al has suggested that the risk of breast cancer for PALB2 mutation carriers is as high as that of BRCA2 mutation carriers.116 The absolute risk of breast cancer for PALB2 female mutation carriers by 70 years of age ranged from 33% (95% CI, 25–44) for those with no family history of breast cancer to 58% (95% CI, 50–66) for those with two or more first-degree relatives with breast cancer at 50 years of age. The risk of breast cancer

1	CI, 25–44) for those with no family history of breast cancer to 58% (95% CI, 50–66) for those with two or more first-degree relatives with breast cancer at 50 years of age. The risk of breast cancer for female PALB2 mutation carriers, depending on the age, was about five to nine times as high compared with the gen-eral population. While screening with mammogram along with MRI has been suggested for PALB2 mutation carriers starting at age 30 with consideration of risk-reducing mastectomy, there is currently insufficient evidence regarding the risk of ovarian cancer and its management.Other hereditary syndromes associated with an increased risk of breast cancer include Cowden disease (PTEN mutations, in which cancers of the thyroid, GI tract, and benign skin and subcutaneous nodules are also seen), Li-Fraumeni syndrome (TP53 mutations, also associated with sarcomas, lymphomas, and adrenocortical tumors), hereditary diffuse gastric cancer syndrome (CDH1 mutations, associated with diffuse

1	seen), Li-Fraumeni syndrome (TP53 mutations, also associated with sarcomas, lymphomas, and adrenocortical tumors), hereditary diffuse gastric cancer syndrome (CDH1 mutations, associated with diffuse gastric cancer and lobular breast cancers), and syndromes of breast and melanoma. With the discovery of additional genes related to breast cancer susceptibility, panel testing is available for a number of genes in addition to BRCA1 and BRCA2. The inter-pretation of results is complex and is best done with a genetic counselor.EPIDEMIOLOGY AND NATURAL HISTORY OF BREAST CANCEREpidemiologyBreast cancer is the most common site-specific cancer in women and is the leading cause of death from cancer for women age 20 to 59 years. Based on Surveillance, Epidemiology, and End Results registries (SEER) data, 266,120 new cases were esti-mated in 2018 with 40,920 estimated deaths attributed to breast cancers.117 It accounts for 30% of all newly diagnosed cancers in women and is responsible for 14% of

1	data, 266,120 new cases were esti-mated in 2018 with 40,920 estimated deaths attributed to breast cancers.117 It accounts for 30% of all newly diagnosed cancers in women and is responsible for 14% of the cancer-related deaths in women.Breast cancer was the leading cause of cancer-related mortality in women until 1987, when it was surpassed by lung cancer. In the 1970s, the probability that a woman in the United States would develop breast cancer at some point in her lifetime was estimated at 1 in 13; in 1980 it was 1 in 11; and in 2004 it was 1 in 8. Cancer registries in Connecticut and upper New York State document that the age-adjusted incidence of new breast cancer cases had steadily increased since the mid-1940s. The incidence in the United States, based on data from nine SEER registries, has been decreasing by 23% per year since 2000. The increase had been approximately 1% per year from 1973 to 1980, and there was an additional increase in inci-dence of 4% between 1980 and 1987,

1	has been decreasing by 23% per year since 2000. The increase had been approximately 1% per year from 1973 to 1980, and there was an additional increase in inci-dence of 4% between 1980 and 1987, which was characterized by frequent detection of small primary cancers. The increase in breast cancer incidence occurred primarily in women age ≥55 years and paralleled a marked increase in the percentage of older women who had mammograms taken. At the same time, incidence rates for regional metastatic disease dropped and breast cancer mortality declined. From 1960 to 1963, 5-year overall survival rates for breast cancer were 63% and 46% in white and African American women, respectively, whereas the rates for 1981 to 1983 were 78% and 64%, respectively. For 2002 to 2008 rates were 92% and 78%, respectively.There is a 10-fold variation in breast cancer incidence among different countries worldwide. Cyprus and Malta have the highest age-adjusted mortality for breast cancer (29.6 per 100,000

1	respectively.There is a 10-fold variation in breast cancer incidence among different countries worldwide. Cyprus and Malta have the highest age-adjusted mortality for breast cancer (29.6 per 100,000 population), whereas Haiti has the lowest (2.0 deaths per 100,000 population). The United States has an age-adjusted mortality for breast cancer of 19.0 cases per 100,000 population. Women living in less industrialized nations tend to have a lower incidence of breast cancer than women living in industrialized countries, although Japan is an exception. In the United States, Mormons, Seventh Day Adventists, American Indians, Alaska natives, Hispanic/Latina Americans, and Japanese and Filipino women living in Hawaii have a below-average incidence of breast cancer, whereas nuns (due to nulliparity) and Ashkenazi Jewish women have an above-average incidence.The incidence rates of breast cancer increased in most countries through the 1990s. Since the estimates for 1990, there was an overall

1	and Ashkenazi Jewish women have an above-average incidence.The incidence rates of breast cancer increased in most countries through the 1990s. Since the estimates for 1990, there was an overall increase in incidence rates of approximately 0.5% annually. It was predicted that there would be approxi-mately 1.4 million new cases in 2010. The cancer registries in China have noted annual increases in incidence of up to 3% to 4%, and in eastern Asia, increases are similar.Data from the SEER program reveal declines in breast cancer incidence over the past decade, and this is widely attrib-uted to decreased use of hormone replacement therapy as a con-sequence of the Women’s Health Initiative reports.118Breast cancer burden has well-defined variations by geog-raphy, regional lifestyle, and racial or ethnic background.119 In general, both breast cancer incidence and mortality are rela-tively lower among the female populations of Asia and Africa, relatively underdeveloped nations, and nations

1	or ethnic background.119 In general, both breast cancer incidence and mortality are rela-tively lower among the female populations of Asia and Africa, relatively underdeveloped nations, and nations that have not adopted Westernized reproductive and dietary patterns. In contrast, European and North American women and women from heavily industrialized or Westernized countries have a substantially higher breast cancer burden. These international patterns are mirrored in breast cancer incidence and mortality rates observed for the racially, ethnically, and culturally diverse population of the United States.120Brunicardi_Ch17_p0541-p0612.indd 56101/03/19 5:04 PM 562SPECIFIC CONSIDERATIONSPART II10090807060504030201083%68%54%41%Middlesex Hospital 1805-1933 (250 cases)86%66%44%28%18%9%3.6%2%0.8%1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Median survival 2.7 years Natural survivalSurvival untreated casesDuration of life from onset of symptoms

1	2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Median survival 2.7 years Natural survivalSurvival untreated casesDuration of life from onset of symptoms (years)% SurvivalFigure 17-13. Survival of women with untreated breast cancer compared with natural survival. (Reproduced with permission from Bloom HJG, Richardson WW, Harries EJ: Natural history of untreated breast cancer (1805-1933). Comparison of untreated and treated cases according to histological grade of malignancy, Br Med J. 1962 Jul 28;2(5299):213-221.)Although often related, the factors that influence breast cancer incidence may differ from those that affect mortality. Incidence rates are lower among populations that are heavily weighted with women who begin childbearing at young ages and who have multiple full-term pregnancies followed by pro-longed lactation. These are features that characterize many underdeveloped nations and also many eastern nations. Breast cancer mortality rates

1	who have multiple full-term pregnancies followed by pro-longed lactation. These are features that characterize many underdeveloped nations and also many eastern nations. Breast cancer mortality rates should be lower in populations that have a lower incidence, but the mortality burden will simultaneously be adversely affected by the absence of effective mammographic screening programs for early detection and diminished access to multidisciplinary cancer treatment programs. These features are likely to account for much of the disproportionate mortal-ity risks that are seen in underdeveloped nations. Similar fac-tors probably account for differences in breast cancer burden observed among the various racial and ethnic groups within the United States. Interestingly, breast cancer incidence and mortality rates rise among secondand third-generation Asian Americans as they adopt Western lifestyles.Disparities in breast cancer survival among subsets of the American population are generating

1	mortality rates rise among secondand third-generation Asian Americans as they adopt Western lifestyles.Disparities in breast cancer survival among subsets of the American population are generating increased publicity because they are closely linked to disparities in socioeconomic status. Poverty rates and proportions of the population that lack health care insurance are two to three times higher among minority racial and ethnic groups such as African Americans and His-panic/Latino Americans. These socioeconomic disadvantages create barriers to effective breast cancer screening and result in delayed breast cancer diagnosis, advanced stage distribu-tion, inadequacies in comprehensive treatment, and, ultimately, increased mortality rates. Furthermore, the rapid growth in the Hispanic population is accompanied by increasing problems in health education because of linguistic barriers between physi-cians and recently immigrated, non–English-speaking patients. Recent studies also are

1	population is accompanied by increasing problems in health education because of linguistic barriers between physi-cians and recently immigrated, non–English-speaking patients. Recent studies also are documenting inequities in the treatments delivered to minority breast cancer patients, such as increased rates of failure to provide systemic therapy, use of sentinel lymph node dissection, and breast reconstruction. Some of the treatment delivery disparities are related to inadequately con-trolled comorbidities (such as hypertension and diabetes), which are more prevalent in minority populations. However, some studies that adjust for these factors report persistent and unex-plained unevenness in treatment recommendations. It is clear that breast cancer disparities associated with racial or ethnic background have a multifactorial cause, and improvements in outcome will require correction of many public health problems at both the patient and provider levels.Advances in the ability to

1	or ethnic background have a multifactorial cause, and improvements in outcome will require correction of many public health problems at both the patient and provider levels.Advances in the ability to characterize breast cancer sub-types and the genetics of the disease are now provoking specula-tion regarding possible hereditary influences on breast cancer risk that are related to racial or ethnic ancestry.121 These questions become particularly compelling when one looks at disparities in breast cancer burden between African Americans and Cau-casians. Lifetime risk of breast cancer is lower for African Americans, yet a paradoxically increased breast cancer mortal-ity risk also is seen. African Americans also have a younger age distribution for breast cancer; among women <45 years of age, breast cancer incidence is highest among African Americans compared to other subsets of the American population. Lastly and most provocatively, African American women of all ages have notably higher

1	breast cancer incidence is highest among African Americans compared to other subsets of the American population. Lastly and most provocatively, African American women of all ages have notably higher incidence rates for estrogen receptor-negative tumors. These same patterns of disease are seen in con-temporary female populations of western, sub-Saharan Africa, who are likely to share ancestry with African American women as a consequence of the Colonial-era slave trade. Interestingly, male breast cancer also is seen with increased frequency among both African Americans and Africans.Natural HistoryBloom and colleagues described the natural history of breast cancer based on the records of 250 women with untreated breast cancers who were cared for on charity wards in the Middlesex Hospital, London, between 1805 and 1933. The median survival of this population was 2.7 years after initial diagnosis (Fig. 17-13).122 The 5and 10-year survival rates for these women were 18.0% and 3.6%,

1	London, between 1805 and 1933. The median survival of this population was 2.7 years after initial diagnosis (Fig. 17-13).122 The 5and 10-year survival rates for these women were 18.0% and 3.6%, respectively. Only 0.8% survived for 15 years or longer. Autopsy data confirmed that 95% of these women died of breast cancer, whereas the remaining 5% died of other causes. Almost 75% of the women developed ulcer-ation of the breast during the course of the disease. The longest surviving patient died in the 19th year after diagnosis.Primary Breast Cancer. More than 80% of breast cancers show productive fibrosis that involves the epithelial and stro-mal tissues. With growth of the cancer and invasion of the surrounding breast tissues, the accompanying desmoplastic response entraps and shortens Cooper’s suspensory ligaments to produce a characteristic skin retraction. Localized edema (peau d’orange) develops when drainage of lymph fluid from the skin is disrupted. With continued growth, cancer

1	suspensory ligaments to produce a characteristic skin retraction. Localized edema (peau d’orange) develops when drainage of lymph fluid from the skin is disrupted. With continued growth, cancer cells invade the skin, and eventually ulceration occurs. As new areas of skin are invaded, small satellite nodules appear near the primary ulceration. The size of the primary breast cancer correlates with disease-free and overall survival, but there is a close associa-tion between cancer size and axillary lymph node involvement (Fig. 17-14). In general, up to 20% of breast cancer recurrences are local-regional, >60% are distant, and 20% are both local-regional and distant.Brunicardi_Ch17_p0541-p0612.indd 56201/03/19 5:04 PM 563THE BREASTCHAPTER 17xxxxxxxxxx**********Diameter (cm)0.980.950.900.800.700.600.500.400.300.20Proportion of patients with metastases10100Volume (ml)2345676891011100908070605040302010Percent survivors31529717363653126317714265321234909214425N + >3 (183)N + (381)N + 1

1	of patients with metastases10100Volume (ml)2345676891011100908070605040302010Percent survivors31529717363653126317714265321234909214425N + >3 (183)N + (381)N + 1 (198)N (335)Whole series (716)241068Years after mastectomyABFigure 17-14. A. Overall survival for women with breast cancer according to axillary lymph node status. The time periods are years after radical mastectomy. (Reproduced with permission from Vala-gussa P, Bonadonna G, Veronesi U, et al: Patterns of relapse and survival following radical mastectomy. Analysis of 716 consecutive patients, Cancer. 1978 Mar;41(3):1170-1178.) B. Risk of metasta-ses according to breast cancer volume and diameter. (Reproduced with permission from Koscielny S, Tubiana M, Lê MG, et al: Breast cancer: Relationship between the size of the primary tumour and the probability of metastatic dissemination, Br J Cancer. 1984 Jun;49(6):709-715.)Axillary Lymph Node Metastases. As the size of the pri-mary breast cancer increases, some cancer cells are

1	tumour and the probability of metastatic dissemination, Br J Cancer. 1984 Jun;49(6):709-715.)Axillary Lymph Node Metastases. As the size of the pri-mary breast cancer increases, some cancer cells are shed into cellular spaces and transported via the lymphatic network of the breast to the regional lymph nodes, especially the axillary lymph nodes. Lymph nodes that contain metastatic cancer are at first ill-defined and soft but become firm or hard with con-tinued growth of the metastatic cancer. Eventually the lymph nodes adhere to each other and form a conglomerate mass. Cancer cells may grow through the lymph node capsule and fix to contiguous structures in the axilla, including the chest wall. Typically, axillary lymph nodes are involved sequentially from the low (level I) to the central (level II) to the apical (level III) lymph node groups. Approximately 95% of the women who die of breast cancer have distant metastases, and traditionally the most important prognostic correlate of

1	(level II) to the apical (level III) lymph node groups. Approximately 95% of the women who die of breast cancer have distant metastases, and traditionally the most important prognostic correlate of disease-free and over-all survival was axillary lymph node status (see Fig. 17-14A). Women with node-negative disease had less than a 30% risk of recurrence, compared with as much as a 75% risk for women with node-positive disease.Distant Metastases. At approximately the 20th cell dou-bling, breast cancers acquire their own blood supply (neovas-cularization). Thereafter, cancer cells may be shed directly into the systemic venous blood to seed the pulmonary circulation via the axillary and intercostal veins or the vertebral column via Batson’s plexus of veins, which courses the length of the vertebral column. These cells are scavenged by natural killer lymphocytes and macrophages. Successful implantation of metastatic foci from breast cancer predictably occurs after the primary cancer

1	vertebral column. These cells are scavenged by natural killer lymphocytes and macrophages. Successful implantation of metastatic foci from breast cancer predictably occurs after the primary cancer exceeds 0.5 cm in diameter, which corresponds to the 27th cell doubling. For 10 years after initial treatment, distant metastases are the most common cause of death in breast cancer patients. For this reason, conclusive results cannot be derived from breast cancer trials until at least 5 to 10 years have elapsed. Although 60% of the women who develop distant metastases will do so within 60 months of treatment, metastases may become evident as late as 20 to 30 years after treatment of the primary cancer.123 Patients with estrogen receptor nega-tive breast cancers are proportionately more likely to develop recurrence in the first 3 to 5 years, whereas those with estrogen receptor positive tumors have a risk of developing recurrence, which drops off more slowly beyond 5 years than is seen with

1	develop recurrence in the first 3 to 5 years, whereas those with estrogen receptor positive tumors have a risk of developing recurrence, which drops off more slowly beyond 5 years than is seen with ER-negative tumors.124 Recently, a report showed that tumor size and nodal status remain powerful predictors of late recur-rences compared to more recently developed tools such as the immunohistochemical score (IHC4) and two gene expression profile tests (Recurrence Score and PAM50).125 Common sites of involvement, in order of frequency, are bone, lung, pleura, soft tissues, and liver. Brain metastases are less frequent over-all, although with the advent of adjuvant systemic therapies it has been reported that CNS disease may be seen earlier.126,127 There are also reports of factors that are associated with the risk of developing brain metastases.128 For example, they are more likely to be seen in patients with triple receptor negative breast cancer (ER-negative, PR-negative, and

1	are associated with the risk of developing brain metastases.128 For example, they are more likely to be seen in patients with triple receptor negative breast cancer (ER-negative, PR-negative, and HER2-negative) or patients with HER2-positive breast cancer who have received chemotherapy and HER2-directed therapies.HISTOPATHOLOGY OF BREAST CANCERCarcinoma In SituCancer cells are in situ or invasive depending on whether or not they invade through the basement membrane.129,130 Broders’s original description of in situ breast cancer stressed the absence of invasion of cells into the surrounding stroma and their confine-ment within natural ductal and alveolar boundaries.129 Because areas of invasion may be minute, the accurate diagnosis of in situ cancer necessitates the analysis of multiple microscopic sec-tions to exclude invasion. In 1941, Foote and Stewart published Brunicardi_Ch17_p0541-p0612.indd 56301/03/19 5:04 PM 564SPECIFIC CONSIDERATIONSPART IITable 17-8Salient

1	of multiple microscopic sec-tions to exclude invasion. In 1941, Foote and Stewart published Brunicardi_Ch17_p0541-p0612.indd 56301/03/19 5:04 PM 564SPECIFIC CONSIDERATIONSPART IITable 17-8Salient characteristics of in situ ductal (DCIS) and lobular (LCIS) carcinoma of the breast LCISDCISAge (years)44–4754–58Incidencea2%–5%5%–10%Clinical signsNoneMass, pain, nipple dischargeMammographic signsNoneMicrocalcificationsPremenopausal2/31/3Incidence of synchronous invasive carcinoma5%2%–46%Multicentricity60%–90%40%–80%Bilaterality50%–70%10%–20%Axillary metastasis1%1%–2%Subsequent carcinomas: Incidence25%–35%25%–70% LateralityBilateralIpsilateral Interval to diagnosis15–20 y5–10 y Histologic typeDuctalDuctalaIn biopsy specimens of mammographically detected breast lesions.Reproduced with permission from Bland KI, Copeland ED: The Breast: Comprehensive Management of Benign and Malignant Diseases, 2nd ed. Philadelphia, PA: Elsesvier/Saunders; 1998.Table 17-9Classification of breast ductal

1	from Bland KI, Copeland ED: The Breast: Comprehensive Management of Benign and Malignant Diseases, 2nd ed. Philadelphia, PA: Elsesvier/Saunders; 1998.Table 17-9Classification of breast ductal carcinoma in situ (DCIS)HISTOLOGIC SUBTYPE DETERMINING CHARACTERISTICSDCIS GRADE NUCLEAR GRADENECROSISComedoHighExtensiveHighIntermediateaIntermediateFocal or absentIntermediateNoncomedobLowAbsentLowaOften a mixture of noncomedo patterns.bSolid, cribriform, papillary, or focal micropapillary.Adapted with permission from Koo JS, Kim MJ, Kim EK, et al: Comparison of immunohistochemical staining in breast papillary neoplasms of cytokeratin 5/6 and p63 in core needle biopsies and surgical excisions, Appl Immunohistochem Mol Morphol. 2012 Mar;20(2):108-115.a landmark description of LCIS, which distinguished it from DCIS.130 In the late 1960s, Gallagher and Martin published their study of whole-breast sections and described a stepwise progres-sion from benign breast tissue to in situ cancer and

1	it from DCIS.130 In the late 1960s, Gallagher and Martin published their study of whole-breast sections and described a stepwise progres-sion from benign breast tissue to in situ cancer and subsequently to invasive cancer. Before the widespread use of mammography, diagnosis of breast cancer was by physical examination. At that time, in situ cancers constituted <6% of all breast cancers, and LCIS was more frequently diagnosed than DCIS by a ratio of >2:1. However, when screening mammography became popular, a 14-fold increase in the incidence of in situ cancer (45%) was demonstrated, and DCIS was more frequently diagnosed than LCIS by a ratio of >2:1. Table 17-8 lists the clinical and patho-logic characteristics of DCIS and LCIS. Multicentricity refers to the occurrence of a second breast cancer outside the breast quadrant of the primary cancer (or at least 4 cm away), whereas multifocality refers to the occurrence of a second cancer within the same breast quadrant as the primary cancer

1	outside the breast quadrant of the primary cancer (or at least 4 cm away), whereas multifocality refers to the occurrence of a second cancer within the same breast quadrant as the primary cancer (or within 4 cm of it). Multicentricity occurs in 60% to 90% of women with LCIS, whereas the rate of multicentricity for DCIS is reported to be 40% to 80%. LCIS occurs bilaterally in 50% to 70% of cases, whereas DCIS occurs bilaterally in 10% to 20% of cases.Lobular Carcinoma In Situ. LCIS originates from the termi-nal duct lobular units and develops only in the female breast. It is characterized by distention and distortion of the terminal duct lobular units by cells that are large but maintain a normal nuclear to cytoplasmic ratio. Cytoplasmic mucoid globules are a distinctive cellular feature. LCIS may be observed in breast tissues that contain microcalcifications, but the calcifications associated with LCIS typically occur in adjacent tissues. This neighborhood calcification is a feature

1	LCIS may be observed in breast tissues that contain microcalcifications, but the calcifications associated with LCIS typically occur in adjacent tissues. This neighborhood calcification is a feature that is unique to LCIS and contributes to its diagnosis. The frequency of LCIS in the general population cannot be reliably determined because it usu-ally presents as an incidental finding. The average age at diag-nosis is 45 years, which is approximately 15 to 25 years younger than the age at diagnosis for invasive breast cancer. LCIS has a distinct racial predilection, occurring 12 times more frequently in white women than in African-American women. Invasive breast cancer develops in 25% to 35% of women with LCIS. Invasive cancer may develop in either breast, regardless of which breast harbored the initial focus of LCIS, and is detected synchronously with LCIS in 5% of cases. In women with a his-tory of LCIS, up to 65% of subsequent invasive cancers are duc-tal, not lobular, in origin.

1	the initial focus of LCIS, and is detected synchronously with LCIS in 5% of cases. In women with a his-tory of LCIS, up to 65% of subsequent invasive cancers are duc-tal, not lobular, in origin. For these reasons, LCIS is regarded as a marker of increased risk for invasive breast cancer rather than as an anatomic precursor. Individuals should be counseled regarding their risk of developing breast cancer and appropriate risk reduction strategies, including observation with screening, chemoprevention, and risk-reducing bilateral mastectomy.Ductal Carcinoma In Situ. Although DCIS is predominantly seen in the female breast, it accounts for 5% of male breast cancers. Published series suggest a detection frequency of 7% in all biopsy tissue specimens. The term intraductal carcinoma is frequently applied to DCIS, which carries a high risk for progres-sion to an invasive cancer. Histologically, DCIS is characterized by a proliferation of the epithelium that lines the minor ducts, resulting in

1	applied to DCIS, which carries a high risk for progres-sion to an invasive cancer. Histologically, DCIS is characterized by a proliferation of the epithelium that lines the minor ducts, resulting in papillary growths within the duct lumina. Early in their development, the cancer cells do not show pleomorphism, mitoses, or atypia, which leads to difficulty in distinguishing early DCIS from benign hyperplasia. The papillary growths (papillary growth pattern) eventually coalesce and fill the duct lumina so that only scattered, rounded spaces remain between the clumps of atypical cancer cells, which show hyperchroma-sia and loss of polarity (cribriform growth pattern). Eventually pleomorphic cancer cells with frequent mitotic figures obliterate the lumina and distend the ducts (solid growth pattern). With continued growth, these cells outstrip their blood supply and become necrotic (comedo growth pattern). Calcium deposition occurs in the areas of necrosis and is a common feature seen on

1	With continued growth, these cells outstrip their blood supply and become necrotic (comedo growth pattern). Calcium deposition occurs in the areas of necrosis and is a common feature seen on mammography. DCIS is now frequently classified based on nuclear grade and the presence of necrosis (Table 17-9). Based Brunicardi_Ch17_p0541-p0612.indd 56401/03/19 5:04 PM 565THE BREASTCHAPTER 17on multiple consensus meetings, grading of DCIS has been rec-ommended. Although there is no universal agreement on clas-sification, most systems endorse the use of cytologic grade and presence or absence of necrosis.131The risk for invasive breast cancer is increased nearly fivefold in women with DCIS.132 The invasive cancers are observed in the ipsilateral breast, usually in the same quadrant as the DCIS that was originally detected, which suggests that DCIS is an anatomic precursor of invasive ductal carcinoma (Fig. 17-15A and B).Invasive Breast CarcinomaInvasive breast cancers have been described as

1	was originally detected, which suggests that DCIS is an anatomic precursor of invasive ductal carcinoma (Fig. 17-15A and B).Invasive Breast CarcinomaInvasive breast cancers have been described as lobular or duc-tal in origin.128-131 Early classifications used the term lobular to describe invasive cancers that were associated with LCIS, whereas all other invasive cancers were referred to as ductal. Current histologic classifications recognize special types of breast cancers (10% of total cases), which are defined by spe-cific histologic features. To qualify as a special-type cancer, at least 90% of the cancer must contain the defining histologic features. About 80% of invasive breast cancers are described as invasive ductal carcinoma of no special type (NST). These can-cers generally have a worse prognosis than special-type cancers. Foote and Stewart originally proposed the following classifica-tion for invasive breast cancer130:1. Paget’s disease of the nipple2. Invasive ductal

1	have a worse prognosis than special-type cancers. Foote and Stewart originally proposed the following classifica-tion for invasive breast cancer130:1. Paget’s disease of the nipple2. Invasive ductal carcinoma—Adenocarcinoma with produc-tive fibrosis (scirrhous, simplex, NST), 80%3. Medullary carcinoma, 4%4. Mucinous (colloid) carcinoma, 2%5. Papillary carcinoma, 2%6. Tubular carcinoma, 2%7. Invasive lobular carcinoma, 10%8. Rare cancers (adenoid cystic, squamous cell, apocrine)Paget’s disease of the nipple was described in 1874. It fre-quently presents as a chronic, eczematous eruption of the nipple, which may be subtle but may progress to an ulcerated, weeping lesion. Paget’s disease usually is associated with extensive DCIS and may be associated with an invasive cancer. A palpable mass may or may not be present. A nipple biopsy specimen will show a population of cells that are identical to the underlying DCIS cells (pagetoid features or pagetoid change). Pathognomonic of this cancer

1	or may not be present. A nipple biopsy specimen will show a population of cells that are identical to the underlying DCIS cells (pagetoid features or pagetoid change). Pathognomonic of this cancer is the presence of large, pale, vacuolated cells (Paget cells) in the rete pegs of the epithelium. Paget’s disease may be confused with superficial spreading melanoma. Differ-entiation from pagetoid intraepithelial melanoma is based on the presence of S-100 antigen immunostaining in melanoma and carcinoembryonic antigen immunostaining in Paget’s disease. Surgical therapy for Paget’s disease may involve lumpectomy or mastectomy, depending on the extent of involvement of the nipple-areolar complex and the presence of DCIS or invasive cancer in the underlying breast parenchyma.Invasive ductal carcinoma of the breast with productive fibrosis (scirrhous, simplex, NST) accounts for 80% of breast cancers and presents with macroscopic or microscopic axillary lymph node metastases in up to 25% of

1	of the breast with productive fibrosis (scirrhous, simplex, NST) accounts for 80% of breast cancers and presents with macroscopic or microscopic axillary lymph node metastases in up to 25% of screen-detected cases and up to 60% of symptomatic cases. This cancer occurs most frequently in perimenopausal or postmenopausal women in the fifth to sixth decades of life as a solitary, firm mass. It has poorly defined margins, and its cut surfaces show a central stellate con-figuration with chalky white or yellow streaks extending into surrounding breast tissues. The cancer cells often are arranged in small clusters, and there is a broad spectrum of histologic types with variable cellular and nuclear grades (Fig. 17-16A and B). In a large patient series from the SEER database, 75% of ductal cancers showed estrogen receptor expression.133Medullary carcinoma is a special-type breast cancer; it accounts for 4% of all invasive breast cancers and is a fre-quent phenotype of BRCA1 hereditary breast

1	showed estrogen receptor expression.133Medullary carcinoma is a special-type breast cancer; it accounts for 4% of all invasive breast cancers and is a fre-quent phenotype of BRCA1 hereditary breast cancer. Grossly, the cancer is soft and hemorrhagic. A rapid increase in size may occur secondary to necrosis and hemorrhage. On physi-cal examination, it is bulky and often positioned deep within the breast. Bilaterality is reported in 20% of cases. Medullary carcinoma is characterized microscopically by: (a) a dense lym-phoreticular infiltrate composed predominantly of lymphocytes and plasma cells; (b) large pleomorphic nuclei that are poorly ABFigure 17-15. Ductal carcinoma in situ (DCIS). A. Craniocau-dal mammographic view shows a poorly defined mass containing microcalcifications. (Used with permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screening, Royal Derby Hospital, Derby, UK.) B. Histopathologic preparation of the sur-gical specimen confirms DCIS with

1	permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screening, Royal Derby Hospital, Derby, UK.) B. Histopathologic preparation of the sur-gical specimen confirms DCIS with necrosis (100x). (Used with permission from Dr. Sindhu Menon, Consultant Histopathologist and Dr. Rahul Deb, Consultant Histopathologist and Lead Breast Pathologist, Royal Derby Hospital, Derby, UK.)Brunicardi_Ch17_p0541-p0612.indd 56501/03/19 5:04 PM 566SPECIFIC CONSIDERATIONSPART IIABFigure 17-16. Invasive ductal carcinoma with productive fibrosis (scirrhous, simplex, no special type) A. 100x. B. 200x. (Used with permis-sion from Dr. Sindhu Menon, Consultant Histopathologist and Dr. Rahul Deb, Consultant Histopathologist and Lead Breast Pathologist, Royal Derby Hospital, Derby, UK.)Figure 17-17. Lobular carcinoma (100×). Uniform, relatively small lobular carcinoma cells are seen arranged in a single-file orientation (“Indian file”). (Used with permission from Dr. Sindhu Menon,

1	17-17. Lobular carcinoma (100×). Uniform, relatively small lobular carcinoma cells are seen arranged in a single-file orientation (“Indian file”). (Used with permission from Dr. Sindhu Menon, Consultant Histopathologist and Dr. Rahul Deb, Consul-tant Histopathologist and Lead Breast Pathologist, Royal Derby Hospital, Derby, UK.)differentiated and show active mitosis; and (c) a sheet-like growth pattern with minimal or absent ductal or alveolar dif-ferentiation. Approximately 50% of these cancers are associated with DCIS, which characteristically is present at the periphery of the cancer, and <10% demonstrate hormone receptors. In rare circumstances, mesenchymal metaplasia or anaplasia is noted. Because of the intense lymphocyte response associated with the cancer, benign or hyperplastic enlargement of the lymph nodes of the axilla may contribute to erroneous clinical staging. Women with this cancer have a better 5-year survival rate than those with NST or invasive lobular

1	enlargement of the lymph nodes of the axilla may contribute to erroneous clinical staging. Women with this cancer have a better 5-year survival rate than those with NST or invasive lobular carcinoma.Mucinous carcinoma (colloid carcinoma), another spe-cial-type breast cancer, accounts for 2% of all invasive breast cancers and typically presents in the older population as a bulky tumor. This cancer is defined by extracellular pools of mucin, which surround aggregates of low-grade cancer cells. The cut surface of this cancer is glistening and gelatinous in quality. Fibrosis is variable, and when abundant it imparts a firm consis-tency to the cancer. Over 90% of mucinous carcinomas display hormone receptors.133 Lymph node metastases occur in 33% of cases, and 5and 10-year survival rates are 73% and 59%, respectively. Because of the mucinous component, cancer cells may not be evident in all microscopic sections, and analysis of multiple sections is essential to confirm the diagnosis of a

1	73% and 59%, respectively. Because of the mucinous component, cancer cells may not be evident in all microscopic sections, and analysis of multiple sections is essential to confirm the diagnosis of a mucinous carcinoma.Papillary carcinoma is a special-type cancer of the breast that accounts for 2% of all invasive breast cancers. It generally presents in the seventh decade of life and occurs in a dispropor-tionate number of nonwhite women. Typically, papillary car-cinomas are small and rarely attain a size of 3 cm in diameter. These cancers are defined by papillae with fibrovascular stalks and multilayered epithelium. In a large series from the SEER database 87% of papillary cancers have been reported to express estrogen receptor.133 McDivitt and colleagues noted that these tumors showed a low frequency of axillary lymph node metas-tases and had 5and 10-year survival rates similar to those for mucinous and tubular carcinoma.134Tubular carcinoma is another special-type breast cancer and

1	low frequency of axillary lymph node metas-tases and had 5and 10-year survival rates similar to those for mucinous and tubular carcinoma.134Tubular carcinoma is another special-type breast cancer and accounts for 2% of all invasive breast cancers. It is reported in as many as 20% of women whose cancers are diagnosed by mammographic screening and usually is diagnosed in the perimenopausal or early menopausal periods. Under low-power magnification, a haphazard array of small, randomly arranged tubular elements is seen. In a large SEER database 94% of tubular cancers were reported to express estrogen receptor.133 Approximately 10% of women with tubular carcinoma or with invasive cribriform carcinoma, a special-type cancer closely related to tubular carcinoma, will develop axillary lymph node metastases. However, the presence of metastatic disease in one or two axillary lymph nodes does not adversely affect survival. Distant metastases are rare in tubular carcinoma and invasive cribriform

1	metastases. However, the presence of metastatic disease in one or two axillary lymph nodes does not adversely affect survival. Distant metastases are rare in tubular carcinoma and invasive cribriform carcinoma. Long-term survival approaches 100%.Invasive lobular carcinoma accounts for 10% of breast cancers. The histopathologic features of this cancer include small cells with rounded nuclei, inconspicuous nucleoli, and scant cytoplasm (Fig. 17-17). Special stains may confirm the Brunicardi_Ch17_p0541-p0612.indd 56601/03/19 5:04 PM 567THE BREASTCHAPTER 17presence of intracytoplasmic mucin, which may displace the nucleus (signet-ring cell carcinoma). At presentation, invasive lobular carcinoma varies from clinically inapparent carcinomas to those that replace the entire breast with a poorly defined mass. It is frequently multifocal, multicentric, and bilateral. Because of its insidious growth pattern and subtle mammographic fea-tures, invasive lobular carcinoma may be difficult to

1	defined mass. It is frequently multifocal, multicentric, and bilateral. Because of its insidious growth pattern and subtle mammographic fea-tures, invasive lobular carcinoma may be difficult to detect. Over 90% of lobular cancers express estrogen receptor.133DIAGNOSIS OF BREAST CANCERIn ∼30% of cases, the woman discovers a lump in her breast. Other less frequent presenting signs and symptoms of breast cancer include: (a) breast enlargement or asymmetry; (b) nipple changes, retraction, or discharge; (c) ulceration or erythema of the skin of the breast; (d) an axillary mass; and (e) musculoskel-etal discomfort. However, up to 50% of women presenting with breast complaints have no physical signs of breast pathology. Breast pain usually is associated with benign disease.Misdiagnosed breast cancer accounts for the greatest num-ber of malpractice claims for errors in diagnosis and for the largest number of paid claims. Litigation often involves younger women, whose physical examination and

1	accounts for the greatest num-ber of malpractice claims for errors in diagnosis and for the largest number of paid claims. Litigation often involves younger women, whose physical examination and mammogram may be misleading. If a young woman (≤45 years) presents with a palpable breast mass and equivocal mammographic findings, ultrasound examination and biopsy are used to avoid a delay in diagnosis.ExaminationInspection. The clinician inspects the woman’s breast with her arms by her side (Fig. 17-18A), with her arms straight up in the air (Fig. 17-18B), and with her hands on her hips (with and without pectoral muscle contraction).135,136 Symmetry, size, and shape of the breast are recorded, as well as any evidence of edema (peau d’orange), nipple or skin retraction, or erythema. With the arms extended forward and in a sitting position, the woman leans forward to accentuate any skin retraction.Figure 17-18. Examination of the breast. A. Inspection of the breast with arms at sides. B.

1	arms extended forward and in a sitting position, the woman leans forward to accentuate any skin retraction.Figure 17-18. Examination of the breast. A. Inspection of the breast with arms at sides. B. Inspection of the breast with arms raised. C. Palpation of the breast with the patient supine. D. Palpa-tion of the axilla.Palpation. As part of the physical examination, the breast is carefully palpated. With the patient in the supine position (see Fig. 17-18C) the clinician gently palpates the breasts, making certain to examine all quadrants of the breast from the sternum laterally to the latissimus dorsi muscle and from the clavicle inferiorly to the upper rectus sheath. The examination is per-formed with the palmar aspects of the fingers, avoiding a grasp-ing or pinching motion. The breast may be cupped or molded in the examiner’s hands to check for retraction. A systematic search for lymphadenopathy then is performed. Figure 17-18D shows the position of the patient for examination of

1	may be cupped or molded in the examiner’s hands to check for retraction. A systematic search for lymphadenopathy then is performed. Figure 17-18D shows the position of the patient for examination of the axilla. By supporting the upper arm and elbow, the examiner stabi-lizes the shoulder girdle. Using gentle palpation, the clinician assesses all three levels of possible axillary lymphadenopathy. Careful palpation of supraclavicular and parasternal sites also is performed. A diagram of the chest and contiguous lymph node sites is useful for recording location, size, consistency, shape, mobility, fixation, and other characteristics of any palpable breast mass or lymphadenopathy (Fig. 17-19).Imaging TechniquesMammography. Mammography has been used in North Amer-ica since the 1960s, and the techniques used continue to be mod-ified and improved to enhance image quality.137-140 Conventional mammography delivers a radiation dose of 0.1 cGy per study. By comparison, chest radiography delivers

1	used continue to be mod-ified and improved to enhance image quality.137-140 Conventional mammography delivers a radiation dose of 0.1 cGy per study. By comparison, chest radiography delivers 25% of this dose. However, there is no increased breast cancer risk associated with the radiation dose delivered with screening mammography. Screening mammography is used to detect unexpected breast cancer in asymptomatic women. In this regard, it supplements history taking and physical examination. With screening mam-mography, two views of the breast are obtained: the craniocau-dal (CC) view (Fig. 17-20A,B) and the mediolateral oblique (MLO) view (Fig. 17-20C,D). The MLO view images the great-est volume of breast tissue, including the upper outer quadrant and the axillary tail of Spence. Compared with the MLO view, the CC view provides better visualization of the medial aspect of the breast and permits greater breast compression. Diagnos-tic mammography is used to evaluate women with abnormal

1	with the MLO view, the CC view provides better visualization of the medial aspect of the breast and permits greater breast compression. Diagnos-tic mammography is used to evaluate women with abnormal Figure 17-19. A breast examination record. Brunicardi_Ch17_p0541-p0612.indd 56701/03/19 5:04 PM 568SPECIFIC CONSIDERATIONSPART IIABCDFigure 17-20. A-D. Mammogram of a premenopausal breast with a dense fibroglandular pattern. E-H. Mammogram of a postmenopausal breast with a sparse fibroglandular pattern. (Used with permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screening, Royal Derby Hospital, Derby, UK.)Brunicardi_Ch17_p0541-p0612.indd 56801/03/19 5:04 PM 569THE BREASTCHAPTER 17EFGHFigure 17-20. (Continued)findings such as a breast mass or nipple discharge. In addition to the MLO and CC views, a diagnostic examination may use views that better define the nature of any abnormalities, such as the 90° lateral and spot compression views. The 90° lateral

1	In addition to the MLO and CC views, a diagnostic examination may use views that better define the nature of any abnormalities, such as the 90° lateral and spot compression views. The 90° lateral view is used along with the CC view to triangulate the exact location of an abnormality. Spot compression may be done in any pro-jection by using a small compression device, which is placed directly over a mammographic abnormality that is obscured by overlying tissues (Fig. 17-21C). The compression device mini-mizes motion artifact, improves definition, separates overlying tissues, and decreases the radiation dose needed to penetrate the breast. Magnification techniques (×1.5) often are combined with spot compression to better resolve calcifications and the margins of masses. Mammography also is used to guide inter-ventional procedures, including needle localization and needle biopsy.Brunicardi_Ch17_p0541-p0612.indd 56901/03/19 5:04 PM 570SPECIFIC CONSIDERATIONSPART IIABCFigure

1	also is used to guide inter-ventional procedures, including needle localization and needle biopsy.Brunicardi_Ch17_p0541-p0612.indd 56901/03/19 5:04 PM 570SPECIFIC CONSIDERATIONSPART IIABCFigure 17-21. Mammogram revealing a small, spiculated mass in the right breast A. A small, spiculated mass is seen in the right breast with skin tethering (CC view). B. Mass seen on oblique view of the right breast. C. Spot compression mammography view of the cancer seen in A and B. The spiculated margins of the cancer are accentuated by compression. (Used with permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screening, Royal Derby Hospital, Derby, UK.)Specific mammographic features that suggest a diagnosis of breast cancer include a solid mass with or without stellate features, asymmetric thickening of breast tissues, and clustered microcalcifications. The presence of fine, stippled calcium in and around a suspicious lesion is suggestive of breast cancer and occurs in

1	asymmetric thickening of breast tissues, and clustered microcalcifications. The presence of fine, stippled calcium in and around a suspicious lesion is suggestive of breast cancer and occurs in as many as 50% of nonpalpable cancers. These microcalcifications are an especially important sign of cancer in younger women, in whom it may be the only mammographic abnormality. The clinical impetus for screening mammogra-phy came from the Health Insurance Plan study and the Breast Cancer Detection Demonstration Project, which demonstrated a 33% reduction in mortality for women after72 screening mam-mography. Mammography was more accurate than clinical examination for the detection of early breast cancers, providing a true-positive rate of 90%. Only 20% of women with nonpal-pable cancers had axillary lymph node metastases, compared with 50% of women with palpable cancers.141 Current guide-lines of the National Comprehensive Cancer Network suggest that normal-risk women ≥20 years of age should

1	lymph node metastases, compared with 50% of women with palpable cancers.141 Current guide-lines of the National Comprehensive Cancer Network suggest that normal-risk women ≥20 years of age should have a breast examination at least every 3 years. Starting at age 40 years, breast examinations should be performed yearly, and a yearly mammogram should be taken.142 Screening mammography in women ≥50 years of age has been noted to reduce breast cancer mortality by 20% to 25%.72,79 With the increased discussion about the potential harms associated with breast screening, the United Kingdom recently established an independent expert panel to review the published literature and estimate the ben-efits and harms associated with its national screening program for women age >50 years. The panel estimated that in women invited to screening, about 11% of the cancers diagnosed in their lifetime constitute overdiagnosis. Despite this overdiagno-sis, the panel concluded that breast screening programs

1	that in women invited to screening, about 11% of the cancers diagnosed in their lifetime constitute overdiagnosis. Despite this overdiagno-sis, the panel concluded that breast screening programs confer significant benefit and should continue. The use of screening mammography in women <50 years of age is more controversial for previously noted reasons: (a) reduced sensitivity, (b) reduced specificity, and (c) lower incidence of breast cancer. Because of the combination of these three reasons, targeting mammography screening to women <50 years of age, who are at higher risk of breast cancer, improves the balance of risks and benefits and is the approach some health care systems have taken. There are now a number of risk assessment models—as described earlier in this chapter—that can be used to estimate a younger woman’s risk of developing breast cancer and that help assess the risks and benefits of regular screening.Screen film mammography has replaced xeromam-mography because it

1	used to estimate a younger woman’s risk of developing breast cancer and that help assess the risks and benefits of regular screening.Screen film mammography has replaced xeromam-mography because it requires a lower dose of radiation and provides similar image quality. Digital mammography was developed to allow the observer to manipulate the degree of contrast in the image. This is especially useful in women with dense breasts and women <50 years of age. Recently, investigators directly compared digital vs. screen film mam-mography in a prospective (DMIST) trial that enrolled over 42,000 women.143 The investigators found that digital and screen film mammography had similar accuracy; however, digital mammography was more accurate in women <50 years of age, women with mammographically dense breasts, and premenopausal or perimenopausal women. The use of digital breast tomosynthesis with 3D images has been introduced as an alternative to standard 2D mammography imaging that is limited by

1	breasts, and premenopausal or perimenopausal women. The use of digital breast tomosynthesis with 3D images has been introduced as an alternative to standard 2D mammography imaging that is limited by superimposition of breast parenchyma and breast density.144,145 The STORM trial reported that in 7,292 women screened, 3D mammography had a higher cancer detection rate and fewer false-positive recalls than the standard 2D imaging.146,147 Randomized controlled trials are planned to fur-ther study tomosynthesis and its role in breast cancer screen-ing. Standard two-dimensional mammography has limitations, Brunicardi_Ch17_p0541-p0612.indd 57001/03/19 5:04 PM 571THE BREASTCHAPTER 17such as the parenchymal density or superimposition of breast tissue, which obscures cancers or causes normal structures to appear suspicious reducing the sensitivity of mammography and increasing the false-positive rates. Digital breast tomo-synthesis is a technology developed to assist with overcom-ing these

1	to appear suspicious reducing the sensitivity of mammography and increasing the false-positive rates. Digital breast tomo-synthesis is a technology developed to assist with overcom-ing these limitations. In digital breast tomosynthesis, multiple projection images are reconstructed to allow visual review of thin breast sections, each reconstructed slice as thin as 0.5 mm, which provides better characterization of noncalcified lesions. These multiple projection exposures are obtained by a digi-tal detector from a mammography X-ray source that moves through a limited arc angle while the breast is compressed. Then these projection image data sets are reconstructed using specific algorithms, which provide the clinical reader a series of images through the entire breast.148In 2011, tomosynthesis was approved by the U.S. Food and Drug Administration (FDA) to be used in combination with standard digital mammography for breast cancer screening. The total radiation dose when tomosynthesis is

1	was approved by the U.S. Food and Drug Administration (FDA) to be used in combination with standard digital mammography for breast cancer screening. The total radiation dose when tomosynthesis is added is about twice the current dose of digital mammography alone but remains below the limits set by the FDA.149The STORM-2 trial reported that synthetic 2D-3D mammography yields similar breast cancer detection as dual-acquisition 2D-3D mammography with the advantage of reduc-ing radiation exposure.150Contrast-enhanced digital mammography (CEDM) was also approved by the FDA in 2001, which utilizes an iodinated contrast material and modified digital mammography units for imaging.148 CEDM has been shown to be feasible and detects breast cancers at a rate similar to MRI, which has potential to offer an alternative to MRI.151 The advantages of CEDM over MRI are that the use of compression limits motion, there is decrease in cost, decrease in exam time, and there is an option for patients who

1	an alternative to MRI.151 The advantages of CEDM over MRI are that the use of compression limits motion, there is decrease in cost, decrease in exam time, and there is an option for patients who are unable to tolerate MRI or who due to vari-ous reasons cannot have MRI due to incompatibility, such as the presence of a pacemaker or tissue expanders.148,152Ductography. The primary indication for ductography is nipple discharge, particularly when the fluid contains blood. Radiopaque contrast media is injected into one or more of the major ducts, and mammography is performed. A duct is gen-tly enlarged with a dilator, and then a small, blunt cannula is inserted under sterile conditions into the nipple ampulla. With the patient in a supine position, 0.1 to 0.2 mL of dilute con-trast media is injected, and CC and MLO mammographic views are obtained without compression. Intraductal papillomas are seen as small filling defects surrounded by contrast media (Fig. 17-22). Cancers may appear as

1	injected, and CC and MLO mammographic views are obtained without compression. Intraductal papillomas are seen as small filling defects surrounded by contrast media (Fig. 17-22). Cancers may appear as irregular masses or as mul-tiple intraluminal filling defects.Ultrasonography. Second only to mammography in fre-quency of use for breast imaging, ultrasonography is an impor-tant method of resolving equivocal mammographic findings, defining cystic masses, and demonstrating the echogenic qual-ities of specific solid abnormalities. On ultrasound examina-tion, breast cysts are well circumscribed, with smooth margins and an echo-free center (Fig. 17-23). Benign breast masses usually show smooth contours, round or oval shapes, weak internal echoes, and well-defined anterior and posterior mar-gins (Fig. 17-24). Breast cancer characteristically has irregular walls (Fig. 17-25) but may have smooth margins with acous-tic enhancement. Ultrasonography is used to guide fine-needle aspiration biopsy,

1	(Fig. 17-24). Breast cancer characteristically has irregular walls (Fig. 17-25) but may have smooth margins with acous-tic enhancement. Ultrasonography is used to guide fine-needle aspiration biopsy, core-needle biopsy, and needle localization ABFigure 17-22. Ductogram. Craniocaudal (A) and mediolateral oblique (B) mammographic views demonstrate a mass (arrows) posterior to the nipple and outlined by contrast, which also fills the proximal ductal structures. (Used with permission from B. Steinbach.)of breast lesions. Its findings are highly reproducible, and it has a high patient acceptance rate, but it does not reliably detect lesions that are ≤1 cm in diameter. Ultrasonography can also be utilized to image the regional lymph nodes in patients with breast cancer (Fig. 17-26). The sensitivity of examination for the status of axillary nodes ranges from 35% to 82% and specificity ranges from 73% to 97%. The features of a lymph node involved with cancer include cortical thickening,

1	of examination for the status of axillary nodes ranges from 35% to 82% and specificity ranges from 73% to 97%. The features of a lymph node involved with cancer include cortical thickening, change in shape of the node to more circular appearance, size larger Brunicardi_Ch17_p0541-p0612.indd 57101/03/19 5:05 PM 572SPECIFIC CONSIDERATIONSPART IIABFigure 17-24. Ultrasonography images of benign breast tumors. A. Fibroadenoma. B. Intraductal papilloma (see arrow). (Used with permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screening, Royal Derby Hospital, Derby, UK.)ABCFigure 17-23. Breast cyst. A. Simple cyst. Ultrasound image of the mass shows it to be anechoic with a well-defined back wall, characteristic of a cyst. B. Complex solid and cystic mass. C. Complex solid and cystic mass characteristic of intracystic papillary tumor. (Used with permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screening, Royal Derby Hospital, Derby,

1	solid and cystic mass characteristic of intracystic papillary tumor. (Used with permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screening, Royal Derby Hospital, Derby, UK.)than 10 mm, absence of a fatty hilum and hypoechoic internal echoes.153Magnetic Resonance Imaging. In the process of evaluating magnetic resonance imaging (MRI) as a means of character-izing mammographic abnormalities, additional breast lesions have been detected. However, in the circumstance of negative findings on both mammography and physical examination, the probability of a breast cancer being diagnosed by MRI is extremely low. There is current interest in the use of MRI to screen the breasts of high-risk women and of women with a newly diagnosed breast cancer. In the first case, women who have a strong family history of breast cancer or who carry known genetic mutations require screening at an early age because mammographic evaluation is limited due to the increased breast density

1	who have a strong family history of breast cancer or who carry known genetic mutations require screening at an early age because mammographic evaluation is limited due to the increased breast density in younger women. In the second case, an MRI study of the contralateral breast in women with a known breast cancer has shown a contralateral breast cancer in 5.7% of these women (Fig. 17-27). MRI can also detect additional tumors in the index breast (multifocal or multicen-tric disease) that may be missed on routine breast imaging and this may alter surgical decision making (Fig. 17-28). In fact, MRI has been advocated by some for routine use in surgical treatment planning based on the fact that additional disease can be identified with this advanced imaging modality and the Brunicardi_Ch17_p0541-p0612.indd 57201/03/19 5:05 PM 573THE BREASTCHAPTER 17Figure 17-25. Ultrasonography images of malignant breast lesions. A. 25 mm irregular mass. B. Ultrasound 30 mm mass anterior to an

1	57201/03/19 5:05 PM 573THE BREASTCHAPTER 17Figure 17-25. Ultrasonography images of malignant breast lesions. A. 25 mm irregular mass. B. Ultrasound 30 mm mass anterior to an implant. C. Ultrasound breast cancer with calcification. D. Ultrasound shows a 9 mm spiculated mass (see arrow) with attenuation. (Used with permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screening, Royal Derby Hospital, Derby, UK.)extent of disease may be more accurately assessed. A random-ized trial performed in the United Kingdom (COMICE trial) that enrolled 1623 women did not show a decrease in rates of reoperation in those women randomized to undergo MRI in addition to mammography and ultrasonography (19%) com-pared to those undergoing standard breast imaging without MRI (19%).154 Houssami and colleagues performed a meta-analysis including two randomized trials and seven compara-tive cohort studies to examine the effect of preoperative MRI compared to standard preoperative

1	Houssami and colleagues performed a meta-analysis including two randomized trials and seven compara-tive cohort studies to examine the effect of preoperative MRI compared to standard preoperative evaluation on surgical out-comes.155 They reported that the use of MRI was associated with increased mastectomy rates. This is problematic because there is no evidence that the additional disease detected by MRI is of clinical or biologic significance, particularly in light of the low local-regional failure rates currently reported in patients undergoing breast conserving surgery who receive whole breast irradiation and systemic therapies. There is an ongoing trial in the Alliance for Clinical Trials in Oncology that is randomizing patients to preoperative MRI vs. standard imaging to assess the impact of MRI on local regional recur-rence rates in patients with triple receptor negative and HER2 positive breast cancers.The use of dedicated breast coils is mandatory in the MRI imaging of the

1	impact of MRI on local regional recur-rence rates in patients with triple receptor negative and HER2 positive breast cancers.The use of dedicated breast coils is mandatory in the MRI imaging of the breast. A BIRADS lexicon is assigned to each examination and an abnormality noted on MRI that is not seen on mammography requires a focused ultrasound examination for further assessment. If the abnormality is not seen on corre-sponding mammogram or ultrasound, then MRI-guided biopsy is necessary. Some clinical scenarios where MRI may be use-ful include the evaluation of a patient who presents with nodal metastasis from breast cancer without an identifiable primary tumor; to assess response to therapy in the setting of neoadjuvant ABCDBrunicardi_Ch17_p0541-p0612.indd 57301/03/19 5:05 PM 574SPECIFIC CONSIDERATIONSPART IIFigure 17-26. Ultrasonography images of lymph nodes. A. Nor-mal axillary lymph node (see arrows). B. Indeterminate axillary lymph node. C. Malignant appearing axillary

1	CONSIDERATIONSPART IIFigure 17-26. Ultrasonography images of lymph nodes. A. Nor-mal axillary lymph node (see arrows). B. Indeterminate axillary lymph node. C. Malignant appearing axillary lymph node. (Used with permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screening, Royal Derby Hospital, Derby, UK.)ABCsystemic treatment; to select patients for partial breast irradia-tion techniques; and evaluation of the treated breast for tumor recurrence.Breast BiopsyNonpalpable Lesions. Image-guided breast biopsy specimens are frequently required to diagnose nonpalpable lesions.156 Ultrasound localization techniques are used when a mass is present, whereas stereotactic techniques are used when no mass is present (microcalcifications or architectural distortion only). The combination of diagnostic mammography, ultrasound or stereotactic localization, and fine-needle aspiration (FNA) biopsy achieves almost 100% accuracy in the preoperative diag-nosis of breast cancer.

1	combination of diagnostic mammography, ultrasound or stereotactic localization, and fine-needle aspiration (FNA) biopsy achieves almost 100% accuracy in the preoperative diag-nosis of breast cancer. However, although FNA biopsy permits cytologic evaluation, core-needle permits the analysis of breast tissue architecture and allows the pathologist to determine whether invasive cancer is present. This permits the surgeon and patient to discuss the specific management of a breast cancer before therapy begins. Core-needle biopsy is preferred over open biopsy for nonpalpable breast lesions because a single sur-gical procedure can be planned based on the results of the core biopsy. The advantages of core-needle biopsy include a low complication rate, minimal scarring, and a lower cost compared with excisional breast biopsy.Palpable Lesions. FNA or core biopsy of a palpable breast mass can usually be performed in an outpatient setting.157 A 1.5-in, 22-gauge needle attached to a 10-mL syringe

1	excisional breast biopsy.Palpable Lesions. FNA or core biopsy of a palpable breast mass can usually be performed in an outpatient setting.157 A 1.5-in, 22-gauge needle attached to a 10-mL syringe or a 14-gauge core biopsy needle is used. For FNA, use of a syringe holder 7Figure 17-27. MRI examination revealing contralateral breast cancer (see arrows) in a patient diag-nosed with unilateral breast cancer on mammography (two arrows). (Used with permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screening, Royal Derby Hospital, Derby, UK.)Brunicardi_Ch17_p0541-p0612.indd 57401/03/19 5:05 PM 575THE BREASTCHAPTER 17enables the surgeon performing the FNA biopsy to control the syringe and needle with one hand while positioning the breast mass with the opposite hand. After the needle is placed in the mass, suction is applied while the needle is moved back and forth within the mass. Once cellular material is seen at the hub of the needle, the suction is released

1	After the needle is placed in the mass, suction is applied while the needle is moved back and forth within the mass. Once cellular material is seen at the hub of the needle, the suction is released and the needle is with-drawn. The cellular material is then expressed onto microscope slides. Both air-dried and 95% ethanol–fixed microscopic sec-tions are prepared for analysis. When a breast mass is clinically and mammographically suspicious, the sensitivity and specific-ity of FNA biopsy approaches 100%. Core-needle biopsy of palpable breast masses is performed using a 14-gauge needle, such as the Tru-Cut needle. Automated devices also are avail-able. Vacuum-assisted core biopsy devices (with 8–10 gauge needles) are commonly utilized with image guidance where between 4 and 12 samples can be acquired at different posi-tions within a mass, area of architectural distortion or micro-calcifications. If the target lesion was microcalcifications, the specimen should be radiographed to confirm

1	acquired at different posi-tions within a mass, area of architectural distortion or micro-calcifications. If the target lesion was microcalcifications, the specimen should be radiographed to confirm appropriate sam-pling. A radiopaque marker should be placed at the site of the biopsy to mark the area for future intervention. In some cases the entire lesion is removed with the biopsy technique and clip placement allows for accurate targeting of the site for surgi-cal resection. Tissue specimens are placed in formalin and then processed to paraffin blocks. Although the false-negative rate for core-needle biopsy specimens is very low, a tissue speci-men that does not show breast cancer cannot conclusively rule out that diagnosis because a sampling error may have occurred. The clinical, radiographic, and pathologic findings should be in concordance. If the biopsy findings do not concur with the clinical and radiographic findings, the multidisciplinary team (including clinician,

1	radiographic, and pathologic findings should be in concordance. If the biopsy findings do not concur with the clinical and radiographic findings, the multidisciplinary team (including clinician, radiologist, and pathologist) should review the findings and decide whether or not to recommend an image-guided or open biopsy to be certain that the target lesion has been adequately sampled for diagnosis.BREAST CANCER STAGING AND BIOMARKERSBreast Cancer StagingThe clinical stage of breast cancer is determined primarily through physical examination of the skin, breast tissue, and regional lymph nodes (axillary, supraclavicular, and internal mammary).158 However, clinical determination of axillary lymph node metastases has an accuracy of only 33%. Ultrasound (US) is more sensitive than physical examination alone in determining axillary lymph node involvement during preliminary staging of breast carcinoma. FNA or core biopsy of sonographically inde-terminate or suspicious lymph nodes can

1	examination alone in determining axillary lymph node involvement during preliminary staging of breast carcinoma. FNA or core biopsy of sonographically inde-terminate or suspicious lymph nodes can provide a more defini-tive diagnosis than US alone.153,159 Pathologic stage combines the findings from pathologic examination of the resected pri-mary breast cancer and axillary or other regional lymph nodes. Fisher and colleagues found that accurate predictions regarding the occurrence of distant metastases were possible after resec-tion and pathologic analysis of 10 or more levels I and II axillary lymph nodes.160 A frequently used staging system is the TNM (tumor, nodes, and metastasis) system. The American Joint Committee on Cancer (AJCC) has recently modified the TNM system for breast cancer to include both anatomic and biologic factors161 (Tables 17-10 and 17-11). Koscielny and colleagues demonstrated that tumor size correlates with the presence of axillary lymph node metastases (see

1	to include both anatomic and biologic factors161 (Tables 17-10 and 17-11). Koscielny and colleagues demonstrated that tumor size correlates with the presence of axillary lymph node metastases (see Fig. 17-14B). Others have shown an association between tumor size, axillary lymph node metastases, and disease-free survival. One of the most important predictors of 10and 20-year survival rates in breast cancer is the number of axillary lymph nodes involved with metastatic disease. Routine biopsy of internal mammary lymph nodes is not generally performed; however, it has been reported that in the context of a “triple node” biopsy approach either the internal mammary node or a low axillary node when positive alone carried the same prognostic weight. When both nodes were positive, the prognosis declined to the level associated with apical node positivity. A double node biopsy of the low axil-lary node and either the apical or the internal mammary node gave the same maximum prognostic

1	declined to the level associated with apical node positivity. A double node biopsy of the low axil-lary node and either the apical or the internal mammary node gave the same maximum prognostic information as a triple node biopsy.162 With the advent of sentinel lymph node dissection and the use of preoperative lymphoscintigraphy for localization of the sentinel nodes, surgeons have again begun to biopsy the internal mammary nodes but in a more targeted manner. The 8th edition of the AJCC staging system does allow for staging based on findings from the internal mammary sentinel nodes.163 Drainage to the internal mammary nodes is more frequent with central and medial quadrant cancers. Clinical or pathologic evi-dence of metastatic spread to supraclavicular lymph nodes is no longer considered stage IV disease, but routine scalene or supraclavicular lymph node biopsy is not indicated.BiomarkersBreast cancer biomarkers are of several types. Risk factor biomarkers are those associated with

1	stage IV disease, but routine scalene or supraclavicular lymph node biopsy is not indicated.BiomarkersBreast cancer biomarkers are of several types. Risk factor biomarkers are those associated with increased cancer risk.164-168 These include familial clustering and inherited germline abnormalities, proliferative breast disease with atypia, and mammographic density. Exposure biomarkers are a subset of risk factors that include measures of carcinogen exposure such as DNA adducts. Surrogate endpoint biomarkers are biologic alterations in tissue that occur between cancer initiation and development. These biomarkers are used as endpoints in short-term chemoprevention trials and include histologic changes, indices of proliferation, and genetic alterations leading to cancer. Prognostic biomarkers provide information regarding Figure 17-28. MRI imaging of the breast reveal-ing multifocal tumors not detected with standard breast imaging. (Used with permission from Dr. Anne Turnbull, Consultant

1	provide information regarding Figure 17-28. MRI imaging of the breast reveal-ing multifocal tumors not detected with standard breast imaging. (Used with permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screening, Royal Derby Hospital, Derby, UK.)Brunicardi_Ch17_p0541-p0612.indd 57501/03/19 5:05 PM 576SPECIFIC CONSIDERATIONSPART IITable 17-10TNM staging system for breast cancerPrimary tumor (T)The T classification of the primary tumor is the same regardless of whether it is based on clinical or pathologic criteria, or both. Size should be measured to the nearest millimeter. If the tumor size is slightly less than or greater than a cutoff for a given T classification, it is recommended that the size be rounded to the millimeter reading that is closest to the cutoff. For example, a reported size of 1.1 mm is reported as 1 mm, or a size of 2.01 cm is reported as 2.0 cm. Designation should be made with the subscript “c” or “p” modifier to indicate whether

1	cutoff. For example, a reported size of 1.1 mm is reported as 1 mm, or a size of 2.01 cm is reported as 2.0 cm. Designation should be made with the subscript “c” or “p” modifier to indicate whether the T classification was determined by clinical (physical examination or radiologic) or pathologic measurements, respectively. In general, pathologic determination should take precedence over clinical determination of T size.TXT0Tis (DCIS)*Tis (Paget)T1 T1mi T1a T1b T1cT2T3T4 T4a T4b T4c T4dPrimary tumor cannot be assessedNo evidence of primary tumorDuctal carcinoma in situPaget disease of the nipple NOT associated with invasive carcinoma and/or carcinoma in situ (DCIS) in the underlying breast parenchyma. Carcinomas in the breast parenchyma associated with Paget disease are categorized based on the size and characteristics of the parenchymal disease, although the presence of Paget disease should still be noted.Tumor ≤20 mm in greatest dimensionTumor ≤1 mm in greatest dimensionTumor >1 mm

1	on the size and characteristics of the parenchymal disease, although the presence of Paget disease should still be noted.Tumor ≤20 mm in greatest dimensionTumor ≤1 mm in greatest dimensionTumor >1 mm but ≤5 mm in greatest dimension (round any measurement >l.0–1.9 mm to 2 mm).Tumor >5 mim but ≤10 mm in greatest dimensionTumor >10 mm but ≤20 mm in greatest dimensionTumor >20 mm but ≤50 mm in greatest dimensionTumor >50 mm in greatest dimensionTumor of any size with direct extension to the chest wall and/or to the skin (ulceration or macroscopic nodules); invasion of the dermis alone does not qualify as T4Extension to the chest wall; invasion or adherence to pectoralis muscle in the absence of invasion of chest wall structures does not qualify as T4Ulceration and/or ipsilateral macroscopic satellite nodules and/or edema (including peau d’orange) of the skin that does not meet the criteria for inflammatory carcinomaBoth T4a and T4b are presentInflammatory carcinoma (see section “Rules for

1	nodules and/or edema (including peau d’orange) of the skin that does not meet the criteria for inflammatory carcinomaBoth T4a and T4b are presentInflammatory carcinoma (see section “Rules for Classification”)*Note: Lobular carcinoma in situ (LCIS) is a benign entity and is removed from TNM staging in the AJCC Cancer Staging Manual, 8th edition.Regional lymph nodes—Clinical (N)cNX*cN0cN1 cN1mi**cN2 cN2a cN2bcN3 cN3a cN3b cN3cRegional lymph nodes cannot be assessed (e.g., previously removed)No regional lymph node metastases (by imaging or clinical examination)Metastases to movable ipsilateral Level I, II axillary lymph node(s)Micrometastases (approximately 200 cells, larger than 0.2 mm, but none larger than 2.0 mm)Metastases in ipsilateral Level I, II axillary lymph nodes that are clinically fixed or matted;or in ipsilateral internal mammary nodes in the absence of axillary lymph node metastasesMetastases in ipsilateral Level I, II axillary lymph nodes fixed to one another (matted) or

1	fixed or matted;or in ipsilateral internal mammary nodes in the absence of axillary lymph node metastasesMetastases in ipsilateral Level I, II axillary lymph nodes fixed to one another (matted) or to other structuresMetastases only in ipsilateral internal mammary nodes in the absence of axillary lymph node metastasesMetastases in ipsilateral infraclavicular (Level III axillary) lymph node(s) with or without Level I, II axillary lymph node involvement;or in ipsilateral internal mammary lymph node(s) with Level I, II axillary lymph node metastases;or metastases in ipsilateral supraclavicular lymph node(s) with or without axillary or internal mammary lymph node involvementMetastases in ipsilateral infraclavicular lymph node(s)Metastases in ipsilateral internal mammary lymph node(s) and axillary lymph node(s)Metastases in ipsilateral supraclavicular lymph node(s)Note: (sn) and (f) suffixes should be added to the N category to denote confirmation of metastasis by sentinel node biopsy or

1	lymph node(s)Metastases in ipsilateral supraclavicular lymph node(s)Note: (sn) and (f) suffixes should be added to the N category to denote confirmation of metastasis by sentinel node biopsy or fine needle aspiration/core needle biopsy respectively.*the cNX category is used sparingly in cases where regional lymph nodes have previously been surgically removed or where there is no documentation of physical examination of the axilla.**cN1mi is rarely used but may be appropriate in cases where sentinel node biopsy is performed before tumor resection, most likely to occur in cases treated with neoadjuvant therapy.(Continued)Brunicardi_Ch17_p0541-p0612.indd 57601/03/19 5:05 PM 577THE BREASTCHAPTER 17Table 17-10TNM staging system for breast cancerRegional lymph nodes—Pathologic (pN)pNXpN0 pN0(i+) pN0(mol+)pN1 pN1mi pN1a pN1b pN1cRegional lymph nodes cannot be assessed (e.g., not removed for pathological study or previously removed)No regional lymph node metastasis identified or ITCs

1	lymph nodes cannot be assessed (e.g., not removed for pathological study or previously removed)No regional lymph node metastasis identified or ITCs onlyITCs only (malignant cell clusters no larger than 0.2 mm) in regional lymph node(s)Positive molecular findings by reverse transcriptase polymerase chain reaction (RT-PCR); no ITCs detectedMicrometastases; or metastases in 1–3 axillary lymph nodes; and/or clinically negative internal mammary nodes with micrometastases or macrometastases by sentinel lymph node biopsyMicrometastases (approximately 200 cells, larger than 0.2 mm, but none larger than 2.0 mm)Metastases in 1–3 axillary lymph nodes, at least one metastasis larger than 2.0 mmMetastases in ipsilateral internal mammary sentinel nodes, excluding ITCspN1a and pNlb combinedpN2 pN2a pN2bpN3 pN3a pN3b pN3cMetastases in 4–9 axillary lymph nodes; or positive ipsilateral internal mammary lymph nodes by imaging in the absence of axillary lymph node metastasesMetastases in 4–9 axillary

1	in 4–9 axillary lymph nodes; or positive ipsilateral internal mammary lymph nodes by imaging in the absence of axillary lymph node metastasesMetastases in 4–9 axillary lymph nodes (at least one tumor deposit larger than 2.0 mm)Metastases in clinically detected internal mammary lymph nodes with or without microscopic confirmation; with pathologically negative axillary nodesMetastases in 10 or more axillary lymph nodes;or in infraclavicular (Level III axillary) lymph nodes;or positive ipsilateral internal mammary lymph nodes by imaging in the presence of one or more positive Level I, II axillary lymph nodes; or in more than three axillary lymph nodes and micrometastases or macrometastases by sentinel lymph node biopsy in clinically negative ipsilateral internal mammary lymph nodes; or in ipsilateral supraclavicular lymph nodesMetastases in 10 or more axillary lymph nodes (at least one tumor deposit larger than 2.0 mm); or metastases to the infraclavicular (Level III axillary lymph)

1	ipsilateral supraclavicular lymph nodesMetastases in 10 or more axillary lymph nodes (at least one tumor deposit larger than 2.0 mm); or metastases to the infraclavicular (Level III axillary lymph) nodespNla or pN2a in the presence of cN2b (positive internal mammary nodes by imaging); or pN2a in the presence of pNlbMetastases in ipsilateral supraclavicular lymph nodesNote: (sn) and (f) suffixes should be added to the N category to denote confirmation of metastasis by sentinel node biopsy or FNA/core needle biopsy respectively, with NO further resection of nodes.Distant metastasis (M)M0 cM0(i+)cM1pM1No clinical or radiographic evidence of distant metastases*No clinical or radiographic evidence of distant metastases in the presence of tumor cells or deposits no larger than 0.2 mm detected microscopically or by molecular techniques in circulating blood, bone marrow, or other nonregional nodal tissue in a patient without symptoms or signs of metastasesDistant metastases detected by

1	microscopically or by molecular techniques in circulating blood, bone marrow, or other nonregional nodal tissue in a patient without symptoms or signs of metastasesDistant metastases detected by clinical and radiographic meansAny histologically proven metastases in distant organs; or if in non-regional nodes, metastases greater than 0.2 mmUsed with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.(Continued)cancer outcome irrespective of therapy, whereas predictive bio-markers provide information regarding response to therapy.169 Candidate prognostic and predictive biomarkers and biologic targets for breast cancer include (a) the steroid hormone recep-tor pathway; (b) growth factors and growth factor receptors such as human epidermal growth factor receptor 2 (HER2)/neu, epidermal growth factor receptor (EGFR), transforming growth factor, platelet-derived growth factor, and the

1	and growth factor receptors such as human epidermal growth factor receptor 2 (HER2)/neu, epidermal growth factor receptor (EGFR), transforming growth factor, platelet-derived growth factor, and the insulin-like growth factor family; (c) indices of proliferation such as proliferating cell nuclear antigen (PCNA) and Ki-67; (d) indi-ces of angiogenesis such as vascular endothelial growth factor (VEGF) and the angiogenesis index; (e) the mammalian target of rapamycin (mTOR) signaling pathway; (f) tumor-suppressor genes such as p53; (g) the cell cycle, cyclins, and cyclin-depen-dent kinases; (h) the proteasome; (i) the COX-2 enzyme; (j) the peroxisome proliferator-activated receptors (PPARs); and (k) indices of apoptosis and apoptosis modulators such as bcl-2 and the bax:bcl-2 ratio.Steroid Hormone Receptor Pathway. Hormones play an important role in the development and progression of breast cancer. Estrogens, estrogen metabolites, and other steroid hor-mones such as progesterone all have

1	Receptor Pathway. Hormones play an important role in the development and progression of breast cancer. Estrogens, estrogen metabolites, and other steroid hor-mones such as progesterone all have been shown to have an effect. Breast cancer risk is related to estrogen exposure over time. In postmenopausal women, hormone replacement therapy consisting of estrogen plus progesterone increases the risk of breast cancer by 26% compared to placebo.70 Patients with hor-mone receptor-positive tumors survive two to three times longer after a diagnosis of metastatic disease than do patients with hor-mone receptor-negative tumors. Patients with tumors negative for both estrogen receptors and progesterone receptors are not considered candidates for hormonal therapy. Tumors positive Brunicardi_Ch17_p0541-p0612.indd 57701/03/19 5:05 PM 578SPECIFIC CONSIDERATIONSPART IITable 17-11TNM stage groupingsWhen T is...And N is...And M is...Then the stage group

1	therapy. Tumors positive Brunicardi_Ch17_p0541-p0612.indd 57701/03/19 5:05 PM 578SPECIFIC CONSIDERATIONSPART IITable 17-11TNM stage groupingsWhen T is...And N is...And M is...Then the stage group is...TisN0M00T1N0M0IAT0N1miM0IBT1N1miM0IBT0N1M0IIAT1N1M0IIAT2N0M0IIAT2N1M0IIBT3N0M0IIBT0N2M0IIIAT1N2M0IIIAT2N2M0IIIAT3N1M0IIIAT3N2M0IIIAT4N0M0IIIBT4N1M0IIIBT4N2M0IIIBAny TN3M0IIICAny TAny NM1IVNotes:1. T1 includes Tl mi.2. T0 and T1 tumors with nodal micrometastases (N1mi) are staged as Stage IB.3. T2, T3, and T4 tumors with nodal micrometastases (N1mi) are staged using the N1 category.4. M0 includes M0(i+).5. The designation pM0 is not valid; any M0 is clinical.6. If a patient presents with M1 disease prior to neoadjuvant systemic therapy, the stage is Stage IV and remains Stage IV regardless of response to neoadjuvant therapy.7. Stage designation may be changed if postsurgical imaging studies reveal the presence of distant metastases, provided the studies are per-formed within 4 months

1	of response to neoadjuvant therapy.7. Stage designation may be changed if postsurgical imaging studies reveal the presence of distant metastases, provided the studies are per-formed within 4 months of diagnosis in the absence of disease progres-sion, and provided the patient has not received neoadjuvant therapy.8. Staging following neoadjuvant therapy is denoted with a “yc” or “yp” prefix to the T and N classification. There is no anatomic stage group assigned if there is a complete pathological response (pCR) to neoad-juvant therapy, for example, ypT0ypN0cM0.Used with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.for estrogen or progesterone receptors have a higher response rate to endocrine therapy than tumors that do not express estro-gen or progesterone receptors. The determination of estrogen and progesterone receptor status used to require biochemical evaluation of fresh

1	endocrine therapy than tumors that do not express estro-gen or progesterone receptors. The determination of estrogen and progesterone receptor status used to require biochemical evaluation of fresh tumor tissue. Today, however, estrogen and progesterone receptor status can be measured in archived tis-sue using immunohistochemical techniques. Hormone receptor status also can be measured in specimens obtained with fine-needle aspiration biopsy or core-needle biopsy, and this can help guide treatment planning. Testing for estrogen and progesterone receptors should be performed on all primary invasive breast cancer specimens. The tumor hormone receptor status should be ascertained for both premenopausal and postmenopausal patients to identify patients who are most likely to benefit from endocrine therapy.Growth Factor Receptors and Growth Factors. Overexpres-sion of EGFR in breast cancer correlates with estrogen recep-tor–negative status and with p53 overexpression.170-172 Similarly,

1	therapy.Growth Factor Receptors and Growth Factors. Overexpres-sion of EGFR in breast cancer correlates with estrogen recep-tor–negative status and with p53 overexpression.170-172 Similarly, increased immunohistochemical membrane staining for the HER2 growth factor receptor in breast cancer is associated with mutated TP53, Ki67 overexpression, and estrogen receptor–negative status. HER2 is a member of the ErbB family of growth factor receptors in which ligand binding results in recep-tor homodimerization and tyrosine phosphorylation by tyrosine kinase domains within the receptor. Tyrosine phosphorylation is followed by signal transduction, which results in changes in cell behavior. An important property of this family of receptors is that ligand binding to one receptor type also may result in heterodimerization between two different receptor types that are coexpressed; this leads to transphosphorylation and transactiva-tion of both receptors in the complex (transmodulation). In this

1	in heterodimerization between two different receptor types that are coexpressed; this leads to transphosphorylation and transactiva-tion of both receptors in the complex (transmodulation). In this context, the lack of a specific ligand for the HER2/neu receptor suggests that HER2/neu may function solely as a co-receptor, modulating signaling by other EGFR family members. HER2/neu is both an important prognostic factor and a predictive fac-tor in breast cancer.173 When overexpressed in breast cancer, HER2/neu promotes enhanced growth and proliferation, and increases invasive and metastatic capabilities. Clinical studies have shown that patients with HER2/neu–overexpressing breast cancer have poorly differentiated tumors with high prolifera-tion rates, positive lymph nodes, decreased hormone receptor expression, and an increased risk of recurrence and death due to breast cancer.173-177 Routine testing of the primary tumor specimen for HER2/neu expression should be performed on all

1	receptor expression, and an increased risk of recurrence and death due to breast cancer.173-177 Routine testing of the primary tumor specimen for HER2/neu expression should be performed on all invasive breast cancers. This can be done with immunohis-tochemical analysis to evaluate for overexpression of the cell-surface receptor at the protein level or by using fluorescence in situ hybridization to evaluate for gene amplification. While HER2/ERBB2 activation can also be assessed based on mRNA expression and reverse transcription polymerase chain reaction (RT-PCR) (Oncotype Dx, Genomic Health), this approach is not recommended for clinical decision-making because of the high false negative rate.178 Patients whose tumors show HER2 ampli-fication or HER2/neu protein overexpression are candidates for anti-HER2/neu therapy. Trastuzumab (Herceptin) is a recombi-nant humanized monoclonal antibody directed against HER2. Randomized clinical trials have demonstrated that single-agent trastuzumab

1	for anti-HER2/neu therapy. Trastuzumab (Herceptin) is a recombi-nant humanized monoclonal antibody directed against HER2. Randomized clinical trials have demonstrated that single-agent trastuzumab therapy is well tolerated and active in the treatment of women with HER2/neu–overexpressing metastatic breast cancer.179 Subsequent adjuvant trials demonstrated that trastu-zumab also was highly effective in the treatment of women with early-stage breast cancer when used in combination with che-motherapy.180-182 Patients who received trastuzumab in combina-tion with chemotherapy had between a 40% and 50% reduction in the risk of breast cancer recurrence and approximately a one-third reduction in breast cancer mortality compared with those who received chemotherapy alone.181,183-185Indices of Proliferation. PCNA is a nuclear protein asso-ciated with a DNA polymerase whose expression increases in phase G1 of the cell cycle, reaches its maximum at the G1/S inter-face, and then decreases through

1	is a nuclear protein asso-ciated with a DNA polymerase whose expression increases in phase G1 of the cell cycle, reaches its maximum at the G1/S inter-face, and then decreases through G2.186-189 Immunohistochemical staining for PCNA outlines the proliferating compartments in Brunicardi_Ch17_p0541-p0612.indd 57801/03/19 5:05 PM 579THE BREASTCHAPTER 17breast tissue. Good correlation is noted between PCNA expres-sion and (a) cell-cycle distributions seen on flow cytometry based on DNA content, and (b) uptake of bromodeoxyuridine and the proliferation-associated Ki67 antigen. Individual prolif-eration markers are associated with slightly different phases of the cell cycle and are not equivalent. PCNA and Ki67 expression are positively correlated with p53 overexpression, high S-phase fraction, aneuploidy, high mitotic index, and high histologic grade in human breast cancer specimens, and are negatively cor-related with estrogen receptor content. Ki67 was included with three other

1	fraction, aneuploidy, high mitotic index, and high histologic grade in human breast cancer specimens, and are negatively cor-related with estrogen receptor content. Ki67 was included with three other widely measured breast cancer markers (ER, PR, and HER2) into a panel of four immunohistochemical makers (IHC4), which together provided similar prognostic informa-tion to that in the 21 Gene Recurrence Score (Oncotype DX, Genomic Health).190 While there has been significant interest in using Ki67 as a biomarker, and while the IHC4 panel would be much less expensive than the 21 Gene Recurrence Score, there remain issues regarding reproducibility across laboratories.Indices of Angiogenesis. Angiogenesis is necessary for the growth and invasiveness of breast cancer and promotes cancer progression through several different mechanisms, including delivery of oxygen and nutrients and the secretion of growth-promoting cytokines by endothelial cells.191,192 VEGF induces its effect by binding to

1	through several different mechanisms, including delivery of oxygen and nutrients and the secretion of growth-promoting cytokines by endothelial cells.191,192 VEGF induces its effect by binding to transmembrane tyrosine kinase recep-tors. Overexpression of VEGF in invasive breast cancer is cor-related with increased microvessel density and recurrence in node-negative breast cancer. An angiogenesis index has been developed in which microvessel density (CD31 expression) is combined with expression of thrombospondin (a negative modulator of angiogenesis) and p53 expression. Both VEGF expression and the angiogenesis index may have prognostic and predictive significance in breast cancer.193 Bevacizumab (a monoclonal antibody to VEGF) was approved by the FDA for use in metastatic breast cancer in combination with pacli-taxel chemotherapy. This approval was based on results from a phase 3 trial by the Eastern Cooperative Oncology Group. The group’s E2100 trial showed that when bevacizumab was

1	combination with pacli-taxel chemotherapy. This approval was based on results from a phase 3 trial by the Eastern Cooperative Oncology Group. The group’s E2100 trial showed that when bevacizumab was added to paclitaxel chemotherapy, median progression-free survival increased to 11.3 months from the 5.8 months seen in patients who received paclitaxel alone.194 The results were not repro-duced in other trials, and the indication for the drug was revoked by the FDA in 2011.Indices of Apoptosis. Alterations in programmed cell death (apoptosis), which may be triggered by p53-dependent or p53-independent factors, may be important prognostic and pre-dictive biomarkers in breast cancer.195-197 Bcl-2 family proteins appear to regulate a step in the evolutionarily conserved pathway for apoptosis, with some members functioning as inhibitors of apoptosis and others as promoters of apoptosis. Bcl-2 is the only oncogene that acts by inhibiting apoptosis rather than by directly increasing cellular

1	with some members functioning as inhibitors of apoptosis and others as promoters of apoptosis. Bcl-2 is the only oncogene that acts by inhibiting apoptosis rather than by directly increasing cellular proliferation. The death-signal protein bax is induced by genotoxic stress and growth factor deprivation in the presence of wild-type (normal) p53 and/or AP-1/fos. The bax to bcl-2 ratio and the resulting formation of either bax-baxhomodimers, which stimulate apoptosis, or bax–bcl-2 het-erodimers, which inhibit apoptosis, represent an intracellular regulatory mechanism with prognostic and predictive implica-tions. In breast cancer, overexpression of bcl-2 and a decrease in the bax to bcl-2 ratio correlate with high histologic grade, the presence of axillary lymph node metastases, and reduced disease-free and overall survival rates. Similarly, decreased bax expression correlates with axillary lymph node metastases, a poor response to chemotherapy, and decreased overall survival.The

1	disease-free and overall survival rates. Similarly, decreased bax expression correlates with axillary lymph node metastases, a poor response to chemotherapy, and decreased overall survival.The remaining biomarkers and biologic targets listed ear-lier are still in preclinical testing, and clinical trials are evaluat-ing their importance in breast cancer for both prognostic and predictive purposes.Coexpression of Biomarkers. Selection of optimal therapy for breast cancer requires both an accurate assessment of prog-nosis and an accurate prediction of response to therapy. The breast cancer markers that are most important in determining therapy are estrogen receptor, progesterone receptor, and HER2/neu. Clinicians evaluate clinical and pathologic staging and the expression of estrogen receptor, progesterone receptor, and HER2/neu in the primary tumor to assess prognosis and assign therapy. Adjuvant! Online (http://www.adjuvantonline.com) is one of a number of programs available to

1	receptor, progesterone receptor, and HER2/neu in the primary tumor to assess prognosis and assign therapy. Adjuvant! Online (http://www.adjuvantonline.com) is one of a number of programs available to clinicians that incor-porates clinical and pathologic factors for an individual patient and calculates risk of recurrence and death due to breast cancer and then provides an assessment of the reduction in risk of recurrence that would be expected with the use of combination chemotherapy, endocrine therapy, or both of these. Adjuvant! Online was developed using information from the SEER data-base, the EBCTCG overview analyses, and results from other individual published trials.198 The website is updated and modi-fied as new information becomes available. Clinicopathologic factors are used to separate breast cancer patients into broad prognostic groups, and treatment decisions are made on this basis (Table 17-12). Other indices and programs that are vali-dated and used include the

1	used to separate breast cancer patients into broad prognostic groups, and treatment decisions are made on this basis (Table 17-12). Other indices and programs that are vali-dated and used include the Nottingham Prognostic Index, and PREDICT.199-201 When an approach, which combines prognostic factors is used, up to 70% of early breast cancer patients receive adjuvant chemotherapy that is either unnecessary or ineffective. As described earlier, a wide variety of biomarkers have been shown to individually predict prognosis and response to therapy, but they do not improve the accuracy of either the assessment of prognosis or the prediction of response to therapy.As knowledge regarding cellular, biochemical, and molec-ular biomarkers for breast cancer have improved, prognostic indices have been developed that combine the predictive power Table 17-12Traditional prognostic and predictive factors for invasive breast cancerTUMOR FACTORSHOST FACTORSNodal statusAgeTumor sizeMenopausal

1	have been developed that combine the predictive power Table 17-12Traditional prognostic and predictive factors for invasive breast cancerTUMOR FACTORSHOST FACTORSNodal statusAgeTumor sizeMenopausal statusHistologic/nuclear gradeFamily historyLymphatic/vascular invasionPrevious breast cancerPathologic stageImmunosuppressionHormone receptor statusNutritionDNA content (ploidy, S-phase fraction)Prior chemotherapyExtent of intraductal componentPrior radiation therapyHER2/neu expression Modified with permission from Ellis N: Inherited Cancer Syndromes. New York, NY: Springer-Verlag; 2004.Brunicardi_Ch17_p0541-p0612.indd 57901/03/19 5:05 PM 580SPECIFIC CONSIDERATIONSPART IITable 17-13Diagnostic studies for breast cancer patients CANCER STAGE 0IIIIIIIVHistory & physicalXXXXXComplete blood count, platelet count XXXLiver function tests and alkaline phosphatase level XXXChest radiograph XXXBilateral diagnostic mammograms, ultrasound as indicatedXXXXXHormone receptor statusXXXXXHER2/neu

1	platelet count XXXLiver function tests and alkaline phosphatase level XXXChest radiograph XXXBilateral diagnostic mammograms, ultrasound as indicatedXXXXXHormone receptor statusXXXXXHER2/neu expression XXXXBone scan XXAbdominal (without or without pelvis) computed tomographic scan or ultrasound or magnetic resonance imaging XXAbdominal imaging and bone scanning are indicated for evaluation of symptoms or abnormal laboratory test results at any presenting stage.Data from NCCN Practice Guidelines in Oncology. Fort Washington, PA: National Comprehensive Cancer Network, 2006.of several individual biomarkers with the relevant clinicopatho-logic factors.Recent technological advances have enabled implemen-tation of high throughput gene expression assays in clinical practice.202 These assays enable detailed stratification of breast cancer patients for assessment of prognosis and for predic-tion of response to therapy. The Oncotype DX is a 21-gene RT-PCR–based assay that has been

1	assays enable detailed stratification of breast cancer patients for assessment of prognosis and for predic-tion of response to therapy. The Oncotype DX is a 21-gene RT-PCR–based assay that has been approved for use in newly diagnosed patients with node-negative, ER-positive breast cancer.203 A recurrence score is generated, and those patients with high recurrence scores are likely to benefit from che-motherapy, whereas those with low recurrence scores benefit most from endocrine therapy and may not require chemother-apy. Results from the Trial Assessing Individualized Options for Treatment for breast cancer (TAILORx), designed to pro-spectively validate the use of 21-gene expression assay, have shown that patients with low recurrence score (0 to 10) have a low rate of local-regional and distant recurrence (98.7%) and very good overall survival at 5 years (98%) with endocrine therapy alone without chemotherapy.204 This study has ran-domly assigned patients with an intermediate

1	and distant recurrence (98.7%) and very good overall survival at 5 years (98%) with endocrine therapy alone without chemotherapy.204 This study has ran-domly assigned patients with an intermediate recurrence score (11 to 25) to endocrine therapy alone or to chemotherapy fol-lowed by endocrine therapy.Additionally, retrospective analysis has shown that the 21-gene recurrence score can be used in postmenopausal patients with ER-positive tumors and 1 to 3 involved axillary lymph nodes to predict the benefit of chemotherapy in addition to endocrine therapy.205 Knowledge of the recurrence score has been shown to alter treatment recommendations by oncologists, and patients likewise change their decision to undergo treatment based on the risk of recurrence.206 The MammaPrint assay uses a 70-gene expression profile to assess the risk of distant metas-tasis. Mammaprint is FDA approved for use in stage-1 or stage-2, node negative, ER-positive or ER-negative breast cancers to identify patients

1	expression profile to assess the risk of distant metas-tasis. Mammaprint is FDA approved for use in stage-1 or stage-2, node negative, ER-positive or ER-negative breast cancers to identify patients with high or low risk of recurrence. Although fresh tissue was initially required to perform the assay, it has since been adapted for use in paraffin-embedded tissue sam-ples. The prospective RASTER study reported that patients classified as low risk based on MammaPrint had a 97% distant recurrence-free interval at five years.207 Results of the prospec-tive MINDACT (MicroarrayInNode negative and 1–3 positive lymph node Disease may Avoid ChemoTherapy) trial were recently reported.208 The study was designed to assess whether the 70-gene expression assay would help avoid chemotherapy in patients who are considered clinically high risk but categorized as low genomic risk based on the assay. A 5-year rate of distant metastasis-free survival of more than 92% was identified as the cutoff for the

1	who are considered clinically high risk but categorized as low genomic risk based on the assay. A 5-year rate of distant metastasis-free survival of more than 92% was identified as the cutoff for the benefit of chemotherapy. At 5 years, the rate of survival without distant metastasis in patients with high clinical risk and low genomic risk was 94.7%, meeting the criteria for noninferiority. However, the rate of disease-free survival and overall survival was higher with chemotherapy in the intention to treat population.OVERVIEW OF BREAST CANCER THERAPYBefore diagnostic biopsy, the surgeon must consider the possi-bility that a suspicious mass or mammographic finding may be a breast cancer. Once a diagnosis of breast cancer is made, the type of therapy offered to a breast cancer patient is determined by the stage of the disease, the biologic subtype, and the general health status of the individual. Laboratory tests and imaging studies are performed based on the initial stage as presented

1	by the stage of the disease, the biologic subtype, and the general health status of the individual. Laboratory tests and imaging studies are performed based on the initial stage as presented in Table 17-13. Before therapy is initiated, the patient and the sur-geon must share a clear perspective on the planned course of treatment. Before initiating local therapy, the surgeon should determine the clinical stage, histologic characteristics, and appropriate biomarker levels.In Situ Breast Cancer (Stage 0)Both LCIS and DCIS may be difficult to distinguish from atypical hyperplasia or from cancers with early invasion.60,209-214 Expert pathologic review is required in all cases. Bilateral mammography is performed to determine the extent of the in situ cancer and to exclude a second cancer. Because LCIS is considered a marker for increased risk rather than an inevitable precursor of invasive disease, the current treatment options for LCIS include observation, chemoprevention, and bilateral

1	LCIS is considered a marker for increased risk rather than an inevitable precursor of invasive disease, the current treatment options for LCIS include observation, chemoprevention, and bilateral total mastectomy. The goal of treatment is to prevent or detect at an early stage the invasive cancer that subsequently develops in 25% to 35% of these women. There is no benefit to excis-ing LCIS because the disease diffusely involves both breasts in many cases and the risk of developing invasive cancer is equal for both breasts. The use of tamoxifen as a risk-reduction strat-egy should be considered in women with a diagnosis of LCIS.Women with DCIS and evidence of extensive disease (>4 cm of disease or disease in more than one quadrant) usu-ally require mastectomy (Fig. 17-29). For women with lim-ited disease, lumpectomy and radiation therapy are generally recommended. For nonpalpable DCIS, needle localization or other image-guided techniques are used to guide the surgical resection.

1	with lim-ited disease, lumpectomy and radiation therapy are generally recommended. For nonpalpable DCIS, needle localization or other image-guided techniques are used to guide the surgical resection. Specimen mammography is performed to ensure that all visible evidence of cancer is excised. Adjuvant tamoxi-fen therapy is considered for DCIS patients with ER-positive 8Brunicardi_Ch17_p0541-p0612.indd 58001/03/19 5:05 PM 581THE BREASTCHAPTER 17ABFigure 17-29. Extensive DCIS seen on mammography. A. Exten-sive calcifications are seen throughout the breast on this cranial caudal view. B. Magnification view of calcifications. Due to the extent of the disease the patient is not a good candidate for breast conserving surgery. (Used with permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screening, Royal Derby Hospital, Derby, UK.)disease. The gold standard against which breast conservation therapy for DCIS is evaluated is mastectomy. Women treated with mastectomy

1	of Breast Screening, Royal Derby Hospital, Derby, UK.)disease. The gold standard against which breast conservation therapy for DCIS is evaluated is mastectomy. Women treated with mastectomy have local recurrence and mortality rates of <2%. There is no randomized trial comparing mastectomy vs. breast conserving surgery, and none of the randomized trials of breast-conserving surgery with or without radiotherapy for DCIS were powered to show a difference in mortality. Women treated with lumpectomy and adjuvant radiation therapy in the initial clinical trials were noted to have a local recurrence rate that is increased compared to mastectomy. About 45% of these recurrences will be invasive cancer when radiation therapy is not used. The B-17 trial was conducted by the NSABP to assess the need for radiation in patients treated with breast conserv-ing surgery for DCIS.215 Patients were randomly assigned to lumpectomy with radiation or lumpectomy alone, and after a mean follow-up time of 90

1	for radiation in patients treated with breast conserv-ing surgery for DCIS.215 Patients were randomly assigned to lumpectomy with radiation or lumpectomy alone, and after a mean follow-up time of 90 months rates of both ipsilateral noninvasive and invasive recurrences were significantly lower in patients who received radiation. However, in the B-17 trial the margins were not prospectively assessed, and it is estimated that up to half of the patients may have had tumor at the mar-gin of resection. The benefit of the addition of radiation over breast-conserving surgery alone for DCIS has also been dem-onstrated in several other randomized trials where margins were prospectively assessed including the European Organization for Research and Treatment of Cancer (EORTC) protocol 10853; the United Kingdom, Australia, New Zealand DCIS Trial; and the Swedish Trial.209,216-218 In 2016, the Society of Surgi-cal Oncology (SSO), American Society for Radiation Oncol-ogy (ASTRO), and the American

1	Kingdom, Australia, New Zealand DCIS Trial; and the Swedish Trial.209,216-218 In 2016, the Society of Surgi-cal Oncology (SSO), American Society for Radiation Oncol-ogy (ASTRO), and the American Society of Clinical Oncology (ASCO) established consensus guidelines on margins for patients with DCIS undergoing breast-conserving surgery.219 Based on a multidisciplinary consensus panel using a meta-analysis of margin width and ipsilateral breast tumor recur-rence, a 2-mm margin was determined as adequate width for DCIS for patients undergoing breast-conserving surgery with whole-breast radiation therapy.219Despite the data from randomized trials showing a benefit in all patient subgroups with the addition of radiation in DCIS, there has been an interest in trying to define a subset where radiation could be avoided in order to minimize the cost and inconvenience associated with radiation. In addition, there have been several studies published where patients were treated with excision alone

1	could be avoided in order to minimize the cost and inconvenience associated with radiation. In addition, there have been several studies published where patients were treated with excision alone and never developed invasive breast cancer even at 25 years of follow-up. Silverstein and colleagues were pro-ponents of avoiding radiation therapy in selected patients with DCIS who have widely negative margins after surgery.213 They reported that when greater than 10-mm margins were achieved, there was no additional benefit from radiation therapy. When margins were between 1 and 10 mm, there was a relative risk of local recurrence of 1.49, compared to 2.54 for those with margins less than 1 mm. These data suggested that appropri-ately selected patients with DCIS might not require postopera-tive radiation therapy.The Eastern Cooperative Oncology Group (ECOG) initi-ated a prospective registry trial (ECOG 5194) to identify those patients who could safely undergo breast-conserving surgery

1	radiation therapy.The Eastern Cooperative Oncology Group (ECOG) initi-ated a prospective registry trial (ECOG 5194) to identify those patients who could safely undergo breast-conserving surgery without radiation.222 Eligible patients were those with low or intermediate grade DCIS measuring 2.5 cm or less who had negative margins of at least 3 mm and those with high-grade DCIS who had tumors measuring 1 cm or less with a negative margin of at least 3 mm. At a median follow-up of 6.2 years, patients with low or intermediate grade DCIS had an in-breast Brunicardi_Ch17_p0541-p0612.indd 58101/03/19 5:05 PM 582SPECIFIC CONSIDERATIONSPART IIrecurrence rate of 6.1% while those with high-grade DCIS had a recurrence rate of 15.3%. Approximately 4% of patients developed a contralateral breast cancer during follow-up in both the low/intermediate and high-grade groups. This study identi-fied an acceptable recurrence rate for those patients with low or intermediate grade DCIS treated with

1	cancer during follow-up in both the low/intermediate and high-grade groups. This study identi-fied an acceptable recurrence rate for those patients with low or intermediate grade DCIS treated with excision alone with a margin of at least 3 mm. In contrast, patients with high-grade DCIS had an unacceptably high local recurrence rate.The Radiation Therapy Oncology Group (RTOG) initi-ated the 9804 trial for patients with “good risk” DCIS and randomized them to lumpectomy vs. lumpectomy with whole breast irradiation. Eligible patients were those with unicentric, low or intermediate grade DCIS measuring 2.5 cm or less with a margin of 3 mm or greater. The trial was closed early due to slow accrual; however, the results for 585 patients were recently reported with a median follow-up of 6.46 years.223,224 The local recurrence rate at 5 years was 0.4% for patients ran-domized to receive radiation and 3.2% for those who did not receive radiation.Solin et al utilized samples from the ECOG 5194

1	The local recurrence rate at 5 years was 0.4% for patients ran-domized to receive radiation and 3.2% for those who did not receive radiation.Solin et al utilized samples from the ECOG 5194 trial to develop a quantitative multigene RT-PCR assay for predict-ing recurrence risk in patients with DCIS treated with surgery alone.201 They were able to define low, intermediate, and high risk groups using a DCIS Score. The DCIS Score was able to quantify the risk of recurrence in the breast for both DCIS and invasive events. This tool has recently been evaluated in another dataset and appears to be a promising tool for clinical use.225 When selecting therapy for patients with DCIS, one must con-sider clinical and pathologic factors, including tumor size, grade, mammographic appearance, and patient preference. There is no single correct surgical treatment, and many patients will require extensive counseling in order to make a decision regarding sur-gical therapy. The role of axillary staging in

1	preference. There is no single correct surgical treatment, and many patients will require extensive counseling in order to make a decision regarding sur-gical therapy. The role of axillary staging in patients with DCIS is limited. One consideration is for patients undergoing mastec-tomy. Since most lesions are currently diagnosed with needle core biopsy, there is about a 20% incidence of invasive breast cancer on final pathologic assessment of the primary tumor. Since it is not feasible to perform sentinel node dissection after mastectomy, most surgeons will recommend the use of sentinel node dissection at the time of mastectomy for DCIS.Results from the NSABP B-24 trial reported a signifi-cant reduction in local recurrence after 5 years of tamoxifen in women with ER-positive DCIS. Based on this finding, some guidelines have advocated that all patients (women with ER-positive DCIS without contraindications to tamoxifen therapy) should be offered tamoxifen following surgery and

1	Based on this finding, some guidelines have advocated that all patients (women with ER-positive DCIS without contraindications to tamoxifen therapy) should be offered tamoxifen following surgery and radiation therapy for a duration of 5 years. The B-24 trial revealed a sig-nificant reduction in recurrence with adjuvant tamoxifen therapy for patients with DCIS; however, the results were not initially assessed based on ER status.226 There were 1804 women with DCIS randomized to lumpectomy and radiation with or without tamoxifen. The rate of breast cancer events was significantly lower in those who received tamoxifen at a median follow-up of 74 months (8.2% vs. 13.4%, P = 0.0009). Subsequently, Allred and colleagues evaluated 41% of patients with DCIS in the NSABP B-24 trial to determine the effect of tamoxifen based on ER status measured in the primary tumor.203 They found that 76% of women had DCIS that was ER-positive and these women had a greater reduction in ipsilateral breast tumor

1	effect of tamoxifen based on ER status measured in the primary tumor.203 They found that 76% of women had DCIS that was ER-positive and these women had a greater reduction in ipsilateral breast tumor recur-rence with tamoxifen than did patients with ER-negative DCIS (11% vs. 5.2%, P <0.001). However, it should be noted that 15% of patients in B-24 had tumor at the resection margins. For these patients, tamoxifen could be viewed as treating what, by the current standard, would be viewed as inadequate local exci-sion of the primary tumor.Early Invasive Breast Cancer (Stage I, IIA, or IIB)There have been six prospective randomized trials comparing breast-conserving surgery to mastectomy in early stage breast cancer, and all have shown equivalent survival rates regardless of the surgical treatment type. One caveat, however, is that the majority of studies had a restriction of tumor size; most were either 2 cm or 2.5 cm, while the NSABP B-06 trial was 4 cm, and the NCI trial was up to 5

1	type. One caveat, however, is that the majority of studies had a restriction of tumor size; most were either 2 cm or 2.5 cm, while the NSABP B-06 trial was 4 cm, and the NCI trial was up to 5 cm. NSABP B-06, which is the largest of all the breast conservation trials, compared total mastectomy to lumpectomy with or without radiation therapy in the treatment of women with stages I and II breast cancer.227-233 After 5and 8-year follow-up periods, the disease-free (DFS), distant dis-ease-free, and overall survival (OS) rates for lumpectomy with or without radiation therapy were similar to those observed after total mastectomy. However, the incidence of ipsilateral breast cancer recurrence was higher in the group not receiving radia-tion therapy. These findings supported the use of lumpectomy and radiation therapy in the treatment of stages I and II breast cancer and this has since become the preferred method of treat-ment for women with early stage breast cancer who have uni-focal disease

1	radiation therapy in the treatment of stages I and II breast cancer and this has since become the preferred method of treat-ment for women with early stage breast cancer who have uni-focal disease and who are not known BRCA mutation carriers. Reanalysis of the B-06 study results was undertaken after 20 years of follow-up and confirmed that there was no differ-ence in disease-free survival rates after total mastectomy or after lumpectomy with or without adjuvant radiation therapy. The in-breast recurrence rate was substantially higher in the lumpec-tomy alone group (39.2%) compared with the lumpectomy plus adjuvant radiation therapy group (14.3%), confirming the importance of radiation therapy in the management of patients with invasive disease. However, it should be noted that there were several criteria in the B-06 study. There was a specific lymphadenopathy exclusion criteria. Secondly, all patients ran-domized to breast-conserving surgery had a frozen section, and if the margins

1	several criteria in the B-06 study. There was a specific lymphadenopathy exclusion criteria. Secondly, all patients ran-domized to breast-conserving surgery had a frozen section, and if the margins were involved, they were converted to mastec-tomy but were included in the analysis as having had a breast-conserving operation (on the basis of intention to treat). Finally, in the breast-conserving group recurrences in the treated breast were considered as a “nonevent.”Data from all of the randomized trials where breast con-servation was performed with or without radiation therapy have been examined by the EBCTCG.12 At 15 years of follow-up, the absolute reduction in mortality with the use of radiation therapy after lumpectomy was 5.1% in node-negative patients and 7.1% in node-positive patients. These data support the concept that the addition of radiation not only improves local control but also has an impact on survival. Similar to DCIS, clinicians have sought to identify subgroups of

1	These data support the concept that the addition of radiation not only improves local control but also has an impact on survival. Similar to DCIS, clinicians have sought to identify subgroups of patients who may not benefit from the addition of radiation therapy, particularly older patients who may have a shorter life expectancy due to medical comor-bidities. Randomized trials have shown that in selected patients with small, ER-positive, low-grade tumors, lumpectomy alone without radiation therapy may be appropriate.211,212 The Cancer and Leukemia Group B (CALGB) C9343 trial enrolled women over the age of 70 with T1N0 breast cancer and randomized them to lumpectomy with or without radiation therapy. All patients received adjuvant tamoxifen.233a At 5 years, although Brunicardi_Ch17_p0541-p0612.indd 58201/03/19 5:05 PM 583THE BREASTCHAPTER 17there were fewer local recurrences with radiation (1% vs. 4%, P <0.001), there were no differences in DFS and OS. While long-term follow-up at

1	58201/03/19 5:05 PM 583THE BREASTCHAPTER 17there were fewer local recurrences with radiation (1% vs. 4%, P <0.001), there were no differences in DFS and OS. While long-term follow-up at 10 years showed fewer local recurrences with radiation (2% vs. 10%), there were no significant differ-ences in time to distant metastasis, breast cancer–specific sur-vival, or OS between the two groups. A trial similar to CALGB C9343 was conducted in Canada where they enrolled women age 50 years and older and randomized them to lumpectomy with or without radiation. Mean age was 68 years, and 80% of women had ER-positive tumors. Again, local recurrence rates were lower in women who received radiation (0.6% vs. 7.7%, P <0.001); however, at a median follow-up of 5.6 years, there were no differences in DFS or OS. The PRIME-2 study enrolled women age 65 years or older with ER-positive, node-negative, up to 3 cm breast cancers, who had undergone breast-conserving surgery and were candidates for adjuvant

1	or OS. The PRIME-2 study enrolled women age 65 years or older with ER-positive, node-negative, up to 3 cm breast cancers, who had undergone breast-conserving surgery and were candidates for adjuvant endocrine treatment. They were assigned to receive whole-breast irradiation or no treatment. After a median follow-up of 5 years, ipsilateral breast tumor recurrence was 1.3% with radiation vs. 41% in those assigned to no radiotherapy. However, no differences in distant metastases, contralateral breast cancers, or overall survival were noted between the groups.234 These studies suggest that radia-tion can be avoided in select older patients with ER-positive, early-stage breast cancer.Accelerated partial breast irradiation (APBI) is also an option for carefully selected patients with DCIS and early-stage breast cancer. Since the majority of recurrences after breast conservation occur in or adjacent to the tumor bed, there has been interest in limiting the radiation to the area of the

1	and early-stage breast cancer. Since the majority of recurrences after breast conservation occur in or adjacent to the tumor bed, there has been interest in limiting the radiation to the area of the primary tumor bed with a margin of normal tissue. APBI is delivered in an abbreviated fashion (twice daily for 5 days) and at a lower total dose compared with the standard course of 5 to 6 weeks of radiation (50 Gy with or without a boost) in the case of whole breast irradiation. Proponents have suggested that this shortened course of treatment may increase the feasibility of breast con-servation for some women and may improve radiation therapy compliance. The RTOG 04-13/NSABP B-39 trial is a random-ized comparison of whole breast irradiation to APBI in women with early stage breast cancer. The trial has completed accrual, and it will likely be several years before data are mature to report outcomes between the two radiation treatment strategies. TARGIT is another study that randomized

1	The trial has completed accrual, and it will likely be several years before data are mature to report outcomes between the two radiation treatment strategies. TARGIT is another study that randomized 3451 patients in 33 centers in over 10 countries to intraoperative breast irradiation (IORT) or external beam radiotherapy (EBRT). The prelimi-nary results were reported in 2012: with a median follow-up of 2.4 years, use of IORT had a recurrence rate of 3.3% vs. 1.3% with EBRT, a 2% increased recurrence risk.235,236 ASTRO developed guidelines for the use of APBI outside of clinical trials based on data reported from published studies.237,238 The ASTRO guidelines describe patients “suitable” for APBI to include women age 60 years or older with a unifocal, T1, ER-positive tumor with no lymphovascular invasion and margins of at least 2 mm. They describe a group where there is uncer-tainty about the appropriateness of APBI (“cautionary” group) to include patients with invasive lobular

1	invasion and margins of at least 2 mm. They describe a group where there is uncer-tainty about the appropriateness of APBI (“cautionary” group) to include patients with invasive lobular histology, a tumor size of 2.1 cm to 3 cm, ER-negative disease, focal lymphovascular invasion, or margins less than 2 mm. Finally, a group felt to be “unsuitable” for APBI includes those with T3 or T4 disease, ER-negative disease, multifocality, multicentricity, extensive LVI, or positive margins.Currently, mastectomy with axillary staging and breast conserving surgery with axillary staging and radiation therapy are considered equivalent treatments for patients with stages I and II breast cancer. Breast conservation is considered for all patients because of the important cosmetic advantages and equivalent survival outcomes; however, this approach is not advised in women who are known BRCA mutation carriers due to the high lifetime risk for development of additional breast cancers. Relative

1	equivalent survival outcomes; however, this approach is not advised in women who are known BRCA mutation carriers due to the high lifetime risk for development of additional breast cancers. Relative contraindications to breast conserva-tion therapy include (a) prior radiation therapy to the breast or chest wall, (b) persistently positive surgical margins after reex-cision, (c) multicentric disease, and (d) scleroderma or lupus erythematosus.For most patients with early-stage disease, reconstruc-tion can be performed immediately at the time of mastectomy. Immediate reconstruction allows for skin-sparing, thus optimiz-ing cosmetic outcomes. Skin-sparing mastectomy with immedi-ate reconstruction has been popularized over the past decade as reports of low local-regional failure rates have been reported and reconstructive techniques have advanced. There is a grow-ing interest in the use of nipple-areolar sparing mastectomy with reports suggesting the oncologic safety of this approach in

1	reported and reconstructive techniques have advanced. There is a grow-ing interest in the use of nipple-areolar sparing mastectomy with reports suggesting the oncologic safety of this approach in early stage breast cancer. Patients who are planned for postmastec-tomy radiation therapy may not be ideal candidates for nipple-sparing mastectomy because of the effects of radiation on the preserved nipple. In addition to providing optimal cosmesis from preservation of the skin and/or the nipple-areolar complex, immediate reconstruction allows patients to wake up with a breast mound, which provides some psychological benefit for the patient. Immediate reconstruction is also more economical as both the extirpative and reconstructive surgery are combined in one operation.Immediate reconstruction can be performed using implants or autologous tissue; tissue flaps commonly used include the transverse rectus abdominis myocutaneous flap, deep inferior epigastric perforator flap, and latissimus

1	can be performed using implants or autologous tissue; tissue flaps commonly used include the transverse rectus abdominis myocutaneous flap, deep inferior epigastric perforator flap, and latissimus dorsi flap (with or without an implant). If postmastectomy radiation therapy is needed, a tissue expander can be placed at the time of mastec-tomy to save the shape of the breast and reduce the amount of skin replacement needed at the time of definitive reconstruc-tion. The expander can be deflated at the initiation of radiation therapy to allow for irradiation of the chest wall and regional nodal basins. Removal of the tissue expander and definitive reconstruction, usually with autologous tissue, can proceed 6 months to 1 year after completion of radiation therapy.Axillary lymph node status has traditionally been an important determinant in staging and prognosis for women with early stage breast cancer. Historically, axillary lymph node dis-section (ALND) was utilized for axillary staging

1	traditionally been an important determinant in staging and prognosis for women with early stage breast cancer. Historically, axillary lymph node dis-section (ALND) was utilized for axillary staging and regional control by removing involved lymph nodes. Randomized tri-als evaluating immediate ALND over ALND performed in a delayed fashion once clinically palpable axillary disease became evident have not shown any detriment in survival.9,239 With increased mammographic screening and detection of smaller, node-negative breast cancers, it became clear that routine use of ALND for axillary staging was not necessary in up to 75% percent of women with operable breast cancer presenting with a negative axilla at the time of screening. Lymphatic mapping and sentinel lymph node (SLN) dissection were initially devel-oped for assessment of patients with clinically node-negative melanoma. Given the changing landscape of newly diagnosed breast cancer patients with a clinically node-negative axilla,

1	initially devel-oped for assessment of patients with clinically node-negative melanoma. Given the changing landscape of newly diagnosed breast cancer patients with a clinically node-negative axilla, sur-geons quickly began to explore the utility of SLN dissection as a replacement for ALND in axillary staging.Brunicardi_Ch17_p0541-p0612.indd 58301/03/19 5:05 PM 584SPECIFIC CONSIDERATIONSPART IIIn the early 1990s, David Krag at the University of Vermont began performing SLN dissection with injection of a radioisotope in the primary tumor site and localizing the SLN node with a handheld gamma probe.240 He was able to identify a SLN in 18 of 22 patients examined, and the SLN was posi-tive in all 7 patients with positive lymph nodes. Giuliano and colleagues initiated a pilot study in 1991 to examine the use of SLN dissection using blue dye in patients with clinically nega-tive nodes. They reported successful identification of a SLN in 114 (65.5%) of 174 patients, and in 109 (95.6%),

1	to examine the use of SLN dissection using blue dye in patients with clinically nega-tive nodes. They reported successful identification of a SLN in 114 (65.5%) of 174 patients, and in 109 (95.6%), the SLN accurately predicted the status of the axillary nodes.241,242 These studies along with initial work by Doug Reintgen and Charles Cox at the Moffitt Cancer Center and Umberto Veronesi and his colleagues at the European Institute of Oncology in Milan led the way toward validation of the technique in large single institution and multicenter studies.Following validation of the technique of SLN dissection for staging of the axilla by multiple centers, randomized tri-als were initiated in order to determine if SLN dissection could replace ALND in the contemporary management of breast cancer patients. The ALMANAC trial randomized 1031 patients with primary operable breast cancer to SLN dissection vs. standard axillary surgery. The incidence of lymphedema and sensory loss for the SLN group

1	The ALMANAC trial randomized 1031 patients with primary operable breast cancer to SLN dissection vs. standard axillary surgery. The incidence of lymphedema and sensory loss for the SLN group was significantly lower than with the standard axillary treatment. At 12 months, drain usage, length of hospital stay, and time to resumption of normal day-to-day activities after surgery were also statistically significantly lower in the SLN group.221The NSABP B-32 trial compared clinically node-negative patients undergoing SLN dissection followed by ALND with patients undergoing SLN dissection with ALND only if a SLN was positive for metastatic disease.243 A total of 5611 patients were randomized with a SLN identification rate of 97% and a false-negative rate of 9.7%. A total of 26% of these clini-cally node-negative patients had a positive SLN. Over 60% of patients with positive SLNs had no additional positive lymph nodes within the ALND specimen. The B-32 trial and other randomized trials

1	node-negative patients had a positive SLN. Over 60% of patients with positive SLNs had no additional positive lymph nodes within the ALND specimen. The B-32 trial and other randomized trials demonstrated no difference in DFS, OS, and local-regional recurrence rates between patients with negative SLNs who had SLN dissection alone compared with those who underwent ALND.244,245 Most important, patients who had SLN dissection alone were found to have decreased morbidity (arm swelling and range of motion) and improved quality of life vs. patients who underwent ALND.245,246The American College of Surgeons Oncology Group (ACOSOG) initiated the Z0010 and Z0011 trials in order to evaluate the incidence and prognostic significance of occult metastases identified in the bone marrow and SLNs (Z0010) of early-stage clinically node-negative patients and to evaluate the utility of ALND in patients with clinical T1-2, N0 breast cancer with 1 or 2 positive SLNs for patients treated with

1	SLNs (Z0010) of early-stage clinically node-negative patients and to evaluate the utility of ALND in patients with clinical T1-2, N0 breast cancer with 1 or 2 positive SLNs for patients treated with breast-conserving surgery and whole breast irradiation (WBI) (Z0011).247,248The Z0010 study enrolled 5539 patients with clinical T1-2 breast cancer planned for breast conserving surgery and WBI.247 Of these patients, 24% proved to have positive SLNs based on standard pathologic assessment, and of the negative SLNs sub-jected to immunohistochemical staining for cytokeratin, 10.5% proved to have occult metastasis. Of the patients who had bone marrow aspiration, 3.0% had immunohistochemically detected tumor cells in the bone marrow. Although the presence of dis-ease in the bone marrow identified a population at high risk for recurrence, neither immunohistochemical detection of disease in the SLNs or the bone marrow was statistically significant on multivariable analysis with clinicopathologic

1	population at high risk for recurrence, neither immunohistochemical detection of disease in the SLNs or the bone marrow was statistically significant on multivariable analysis with clinicopathologic and treatment factors included. The investigators concluded that routine use of immunohistochemistry to detect occult disease in SLNs is not warranted.The Z0011 trial was a companion study to Z0010 and was designed to study the role of completion ALND on survival in women with positive SLNs. Patients were not eligible if they received neoadjuvant chemotherapy or neoadjuvant hormonal therapy or if their treatment plan included mastectomy, lumpec-tomy without radiation, or lumpectomy with APBI. WBI was to be administered using standard tangential fields without specific treatment of the axilla or additional fields targeting other nodal basins. Patients with 1 or 2 positive SLNs were randomized to completion ALND or no further surgery. Adjuvant systemic therapy recommendations were left to

1	or additional fields targeting other nodal basins. Patients with 1 or 2 positive SLNs were randomized to completion ALND or no further surgery. Adjuvant systemic therapy recommendations were left to the treating clinicians. After median follow-up of 6.3 years, there was no difference between patients randomized to ALND and those randomized to no further surgery (SLN only) in terms of OS (91.9% and 92.5%, respectively; P = 0.25) or DFS (82.2% and 83.8%, respectively; P = 0.14). The low local regional failure rates and similar survival outcomes were recently reported with 10-year follow-up.249,250The morbidity of SLN dissection alone vs. SLN dissec-tion with completion ALND has been reported by the ACOSOG investigators.251,252 Immediate effects of SLN dissection in the Z0010 trial included wound infection in 1%, axillary seroma in 7.1%, and axillary hematoma in 1.4%.251 At 6 months following surgery, axillary paresthesias were noted in 8.6% of patients, decreased range of motion in the

1	wound infection in 1%, axillary seroma in 7.1%, and axillary hematoma in 1.4%.251 At 6 months following surgery, axillary paresthesias were noted in 8.6% of patients, decreased range of motion in the upper extremity was reported in 3.8%, and 6.9% of patients had a change in the arm circum-ference of >2 cm on the ipsilateral side, which was reported as lymphedema. Younger patients were more likely to report paresthesias, whereas increasing age and body mass index were more predictive of lymphedema. When adverse surgical effects were examined in the Z0011 trial, patients undergoing SLN dissection with ALND had more wound infections, seromas, and paresthesias than those women undergoing SLN dissec-tion alone. Lymphedema at 1 year after surgery was reported by 13% in the SLN plus ALND group but only 2% in the SLN dissection alone group. Arm circumference measurements were greater at 1 year in patients undergoing SLN dissection plus ALND, but the difference between study groups was not

1	but only 2% in the SLN dissection alone group. Arm circumference measurements were greater at 1 year in patients undergoing SLN dissection plus ALND, but the difference between study groups was not statisti-cally significant.252 This supports the results published from the ALMANAC trial.Prior to the publication of ACOSOG Z0011, completion ALND was standard of care for patients with positive SLNs. Since the reporting of ACOSOG Z0011, the National Com-prehensive Cancer Network (NCCN) guidelines now state that there was no OS difference for patients with 1 or 2 positive SLNs treated with breast-conserving surgery who underwent completion ALND vs. those who had no further axillary sur-gery. In addition, the American Society of Breast Surgeons issued a consensus statement supporting omission of ALND for patients who meet Z0011 criteria.253 The results of ACOSOG Z0011 have revolutionized management of the axilla and changed practice such that selected patients with axillary metas-tasis can

1	ALND for patients who meet Z0011 criteria.253 The results of ACOSOG Z0011 have revolutionized management of the axilla and changed practice such that selected patients with axillary metas-tasis can now avoid ALND if they have clinical and pathologic features similar to those patients enrolled on Z0011. However, there have been some concerns raised about the Z0011 study that include the fact that the study only recruited about half of Brunicardi_Ch17_p0541-p0612.indd 58401/03/19 5:05 PM 585THE BREASTCHAPTER 17the intended patients and that there was no standardization of whether or not patients received irradiation to the low axilla when the radiation oncologist irradiated the breast. These issues have thus far limited the uptake of the results of Z0011 by some centers.The International Breast Cancer Study Group (IBCSG) 23-01 trial was similar in design to Z0011 but enrolled only patients with micrometastases in the SLNs. Patients with SLN micrometastases were randomized to ALND

1	Breast Cancer Study Group (IBCSG) 23-01 trial was similar in design to Z0011 but enrolled only patients with micrometastases in the SLNs. Patients with SLN micrometastases were randomized to ALND vs. no further sur-gery. Unlike Z0011, the 23-01 trial did not exclude patients treated with mastectomy. Approximately 9% of patients ran-domized to each study arm underwent mastectomy. The inves-tigators published the primary and secondary endpoints of the trial showing no differences in OS or local-regional recurrence between the study arms.254 However, as with the Z0011 trial, some concerns have been raised regarding the 23-01 study. For example, in the statistics on the primary endpoint, local recur-rence included contralateral breast cancer and other tumor types as events. No hypothesis was presented as to why the differ-ence in axillary surgery should impact on either of these events. Including these events therefore reduced the power of the study to show a statistical difference

1	was presented as to why the differ-ence in axillary surgery should impact on either of these events. Including these events therefore reduced the power of the study to show a statistical difference between treatment arms. There is also concern that the study appears underpowered to show a meaningful difference in overall survival.Most pathology laboratories perform a more detailed anal-ysis of the SLN than is routinely done for axillary nodes recov-ered from a levels I and II dissection. This can include examining thin sections of the node with step sectioning at multiple levels through the paraffin blocks or performing immunohistochemi-cal staining of the SLN for cytokeratin or a combination of these techniques. The results of ACOSOG Z0010 and NSABP B-32 showed no clinically meaningful difference in survival based on detection of occult metastases in the SLNs using immu-nohistochemical staining and do not support the routine use in SLN processing. The type of intraoperative

1	difference in survival based on detection of occult metastases in the SLNs using immu-nohistochemical staining and do not support the routine use in SLN processing. The type of intraoperative assessment of SLNs also varies for different clinicians and pathology labo-ratories. Some centers prefer to use touch preparation cyto-logic analysis of the SLNs, whereas others use frozen-section analysis, and the sensitivity and specificity of these assays vary considerably. The GeneSearch Breast Lymph Node Assay is a real-time reverse-transcriptase polymerase chain reaction assay that detects breast tumor cell metastasis in lymph nodes through the identification of the gene expression markers mammaglobin and cytokeratin 19. These markers are present in higher lev-els in breast tissue and not in nodal tissue (cell type-specific messenger RNA). The GeneSearch breast lymph node assay generates expression data for genes of interest, which are then evaluated against predetermined criteria to

1	in nodal tissue (cell type-specific messenger RNA). The GeneSearch breast lymph node assay generates expression data for genes of interest, which are then evaluated against predetermined criteria to provide a qualitative (positive/negative) result. The assay is designed to detect foci that correspond to metastases that are seen with examination by standard hematoxylin and eosin staining and measure >0.2 mm. The GeneSearch assay results have been compared with per-manent-section histologic analysis and frozen-section analy-sis of sentinel nodes in a prospective trial, and the assay was approved by the FDA for the intraoperative assessment of senti-nel nodes.255 When a positive node is identified intraoperatively by touch preparation, frozen-section analysis, or GeneSearch assay, the surgeon can proceed with immediate ALND. With the findings of ACOSOG Z0011 that there is not a survival ben-efit to the use of ALND in selected patients, many surgeons have abandoned the intraoperative

1	can proceed with immediate ALND. With the findings of ACOSOG Z0011 that there is not a survival ben-efit to the use of ALND in selected patients, many surgeons have abandoned the intraoperative evaluation of SLNs. There are a number of nomograms and predictive models designed to determine which patients with a positive SLN are at risk for har-boring additional positive non-SLNs in the axilla. These tools can be helpful in determining the likelihood of additional disease in the axilla and may be used clinically to counsel patients.256In patients who present with axillary lymphadenopa-thy that is confirmed to be metastatic disease on FNA or core biopsy, SLN dissection is not necessary, and patients can pro-ceed directly to ALND or be considered for preoperative sys-temic therapy (see “Neoadjuvant [Preoperative] Chemotherapy” under “Nonsurgical Breast Cancer Therapies”). Initially there was controversy about the suitability of SLN dissection in women with larger primary tumors (T3) and

1	[Preoperative] Chemotherapy” under “Nonsurgical Breast Cancer Therapies”). Initially there was controversy about the suitability of SLN dissection in women with larger primary tumors (T3) and those treated with neoadjuvant chemotherapy. The American Society of Clini-cal Oncology has included SLN dissection is its guidelines as appropriate for axillary staging in these patients.257,258 If an SLN cannot be identified, then ALND is generally performed for appropriate staging. However, this is not universally accepted, and there are as yet no randomized studies that have assessed how a patient with a locally advanced cancer at presentation should be treated if SLN dissection reveals no metastases or micrometastases after neoadjuvant therapy.The ASCO guidelines suggest that adjuvant chemo-therapy should be considered for patients with positive lymph nodes, ER-negative disease, HER2-positive disease, Adju-vant! Online mortality greater than 10%, grade 3 node-neg-ative tumors >5 mm,

1	should be considered for patients with positive lymph nodes, ER-negative disease, HER2-positive disease, Adju-vant! Online mortality greater than 10%, grade 3 node-neg-ative tumors >5 mm, triple-negative tumors, lymphovascular invasion, or estimated distant relapse risk of greater than 15% at 10 years based on the 21 gene recurrence score assay.259 Adjuvant endocrine therapy is considered for women with hormone receptor-positive cancers, and an aromatase inhibi-tor is recommended if the patient is postmenopausal. HER2/neu status is determined for all patients with newly diagnosed invasive breast cancer and when positive, should be used to guide systemic therapy recommendations. The FDA approved trastuzumab in November 2006 for use as part of a treatment regimen containing doxorubicin, cyclophosphamide, and pacli-taxel for treatment of HER2/neu-positive, node-positive breast cancer.181,183 Subsequently, the BCIRG 006 study reported that giving trastuzumab concurrently with docetaxel

1	and pacli-taxel for treatment of HER2/neu-positive, node-positive breast cancer.181,183 Subsequently, the BCIRG 006 study reported that giving trastuzumab concurrently with docetaxel and carbopla-tin appeared as effective as giving trastuzumab following an anthracycline containing regimen.182,185 In addition to trastu-zumab, pertuzumab has also recently been FDA approved for adjuvant use in patients with HER2 amplified breast cancers with high risk of recurrence.Advanced Local-Regional Breast Cancer (Stage IIIA or IIIB)Women with stage IIIA and IIIB breast cancer have advanced local-regional breast cancer but have no clinically detected distant metastases (Fig. 17-30).260 In an effort to provide opti-mal local-regional disease-free survival as well as distant dis-ease-free survival for these women, surgery is integrated with radiation therapy and chemotherapy (Fig. 17-31). However, it should be noted that these patients have an increased risk of distant metastasis that is often

1	for these women, surgery is integrated with radiation therapy and chemotherapy (Fig. 17-31). However, it should be noted that these patients have an increased risk of distant metastasis that is often highlighted by radiological evidence when staging PET or CT and bone scans are per-formed. Thus, the paradigm for small screen detected cancers where cure can be expected in >90% of patients, often by local treatment alone, is not appropriate for patients with locally advanced disease.Preoperative (also known as neoadjuvant) chemotherapy should be considered in the initial management of patients with Brunicardi_Ch17_p0541-p0612.indd 58501/03/19 5:05 PM 586SPECIFIC CONSIDERATIONSPART IIFigure 17-30. Locally advanced breast cancer. A. Mammography of the right breast reveals a large tumor with enlarged axillary lymph nodes. B. Imaging of the left breast is normal. (Used with permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screening, Royal Derby Hospital,

1	with enlarged axillary lymph nodes. B. Imaging of the left breast is normal. (Used with permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screening, Royal Derby Hospital, Derby, UK.)Figure 17-31. Treatment pathways for stage IIIA and stage IIIB breast cancer.locally advanced stage III breast cancer, especially those with estrogen receptor negative tumors. Chemotherapy is used to maximize distant disease-free survival, whereas radiation ther-apy is used to maximize local-regional control and disease-free survival.In selected patients with stage IIIA cancer, preoperative chemotherapy can reduce the size of the primary cancer and permit breast-conserving surgery. Investigators from the MD Anderson Cancer Center reported that low local-regional fail-ure rates could be achieved in selected patients with stage III disease treated with preoperative chemotherapy followed by breast-conserving surgery and radiation.261 The 5-year actuarial ipsilateral breast tumor

1	be achieved in selected patients with stage III disease treated with preoperative chemotherapy followed by breast-conserving surgery and radiation.261 The 5-year actuarial ipsilateral breast tumor recurrence-free survival rates in this study were 95%. They noted that the ipsilateral breast tumor recurrence rates increased when patients had clinical N2 or N3 disease, >2 cm of residual disease in the breast at surgery, a pattern of multifocal residual disease in the breast at surgery, and lymphovascular space invasion in the primary tumor. This study demonstrated that breast-conserving surgery can be used for appropriately selected patients with locally advanced breast cancer who achieve a good response with preoperative che-motherapy. However, the Oxford overview of all randomized studies of neoadjuvant therapy (vs. adjuvant therapy) reported a hazard ratio of 1.5 (i.e., 50% increase) in local recurrence rates. ABBrunicardi_Ch17_p0541-p0612.indd 58601/03/19 5:05 PM 587THE

1	studies of neoadjuvant therapy (vs. adjuvant therapy) reported a hazard ratio of 1.5 (i.e., 50% increase) in local recurrence rates. ABBrunicardi_Ch17_p0541-p0612.indd 58601/03/19 5:05 PM 587THE BREASTCHAPTER 17A meta-analysis reported a hazard ratio of 1.3.262 These stud-ies included some patients treated with radiation therapy alone without resection of the primary tumor bed, which results in higher local failure rates. These findings are important in view of the previous findings that the avoidance of recurrence in a conserved breast avoids about one breast cancer death over the next 15 years for every four such recurrences avoided.12 The German Breast Cancer Group recently reported their local recurrence rate in 5535 patients in seven studies. With a median of 46 months (range 1–127) follow-up the local recurrence rates ranged from 7.6% to 19.5% for T1-T4 tumors and from 6.4% to 17.9% for N0-N3 tumors treated with neoadjuvant therapy.238 For patients with stage IIIA disease

1	follow-up the local recurrence rates ranged from 7.6% to 19.5% for T1-T4 tumors and from 6.4% to 17.9% for N0-N3 tumors treated with neoadjuvant therapy.238 For patients with stage IIIA disease who experience minimal response to chemotherapy and for patients with stage IIIB breast cancer, preoperative chemotherapy can decrease the local-regional cancer burden enough to permit subsequent modified radical mastectomy to establish local-regional con-trol. In both stages IIIA and IIIB disease, surgery is followed by adjuvant radiation therapy. However there is a small percent-age of patients who experience progression of disease during neoadjuvant therapy, and therefore the surgeon should review patients with the oncologist at regular points during the neoad-juvant regimen.For selected clinically indolent, ER-positive, locally advanced tumors, primary endocrine therapy may be considered, especially if the patient has other comorbid conditions. A series of 195 patients with ER-positive,

1	indolent, ER-positive, locally advanced tumors, primary endocrine therapy may be considered, especially if the patient has other comorbid conditions. A series of 195 patients with ER-positive, locally advanced breast cancer treated by endocrine therapy—median age 69 years, median tumor size 6 cm, median follow-up 61 months—reported a 5-year overall survival of 76%, a breast cancer–specific sur-vival of 86%, and a metastasis-free survival of 77%. The median time to an alternative treatment was 48 months.263 Given that this was a 20-year series, the number of such patients is small but should be considered when the clinician is discussing treat-ment options. Results from the ACOSOG Z1031 trial suggest that neoadjuvant endocrine therapy is a good option for tumor downstaging in patients with strongly ER-positive tumors. The preoperative endocrine prognostic index (PEPI score) can be calculated based on pathologic findings from surgery following neoadjuvant endocrine therapy. This can

1	strongly ER-positive tumors. The preoperative endocrine prognostic index (PEPI score) can be calculated based on pathologic findings from surgery following neoadjuvant endocrine therapy. This can help guide decision-making regarding the need for systemic chemotherapy in this patient population.264,265Internal Mammary Lymph NodesMetastatic disease to internal mammary lymph nodes may be occult, may be evident on chest radiograph or CT scan, or may present as a painless parasternal mass with or without skin involvement. There is no consensus regarding the need for internal mammary lymph node radiation therapy in women who are at increased risk for occult involvement (cancers involving the medial aspect of the breast, axillary lymph node involve-ment) but who show no signs of internal mammary lymph node involvement. Systemic chemotherapy and radiation therapy are indicated in the treatment of grossly involved internal mammary lymph nodes.Distant Metastases (Stage IV)Treatment for stage IV

1	lymph node involvement. Systemic chemotherapy and radiation therapy are indicated in the treatment of grossly involved internal mammary lymph nodes.Distant Metastases (Stage IV)Treatment for stage IV breast cancer is not curative but may prolong survival and enhance a woman’s quality of life.266 Endocrine therapies that are associated with minimal toxicity are preferred to cytotoxic chemotherapy in ER-positive disease. Appropriate candidates for initial endocrine therapy include women with hormone receptor-positive cancers who do not have immediately life threatening disease (or “visceral crisis”). This includes not only women with bone or soft tissue metastases but also women with limited visceral metastases. Symptoms per se (e.g., breathlessness) are not in themselves an indication for chemotherapy. For example, breathlessness due to a pleural effusion can be treated with percutaneous drainage, and if the breathlessness is relieved, the patient should be commenced on endocrine

1	for chemotherapy. For example, breathlessness due to a pleural effusion can be treated with percutaneous drainage, and if the breathlessness is relieved, the patient should be commenced on endocrine therapy; if the breathlessness is due to lymphangitic spread, then chemotherapy would be the treatment of choice. The same approach should be taken to other symptoms such as pain. Systemic chemotherapy is indicated for women with hormone receptor-negative cancers, “visceral crisis,” and hormone-refractory metastases. Women with stage IV breast cancer may develop anatomically localized problems that will benefit from individualized surgical or radiation treatment, such as brain metastases, pleural effusion, pericardial effusion, biliary obstruction, ureteral obstruction, impending or existing pathologic fracture of a long bone, spinal cord compression, and painful bone or soft tissue metastases. Bisphosphonates or anti-RANKL (receptor activator of nuclear factor kappa-B ligand) agent,

1	pathologic fracture of a long bone, spinal cord compression, and painful bone or soft tissue metastases. Bisphosphonates or anti-RANKL (receptor activator of nuclear factor kappa-B ligand) agent, denosumab, which may be given in addition to chemo-therapy or endocrine therapy, should be considered in women with bone metastases. Whether to perform surgical resection of the local-regional disease in women with stage IV breast cancer has been debated after several reports have suggested that women who undergo resection of the primary tumor have improved survival over those who do not. Khan and associates used the National Cancer Data Base to identify patterns of treat-ment in women with metastatic breast cancer and found that those who had surgical resection with negative margins had a better prognosis than those women who did not have surgical therapy.267 Gnerlich et al reported similar findings using the SEER database, and there have been several reports subsequent to this study from

1	prognosis than those women who did not have surgical therapy.267 Gnerlich et al reported similar findings using the SEER database, and there have been several reports subsequent to this study from single institutions that have confirmed these findings.268 Some have suggested that the finding of improved survival is due to selection bias and that local therapy should be reserved for palliation of symptoms. A randomized trial through ECOG (E2108) was designed to address this question.269 The surgical management of patients with stage IV disease should be addressed by obtaining multidisciplinary input and by con-sidering the treatment goals of each individual patient and the patient’s treating physicians.Local-Regional RecurrenceWomen with local-regional recurrence of breast cancer may be separated into two groups: those who have had mastec-tomy and those who have had lumpectomy. Women treated previously with mastectomy undergo surgical resection of the local-regional recurrence and

1	separated into two groups: those who have had mastec-tomy and those who have had lumpectomy. Women treated previously with mastectomy undergo surgical resection of the local-regional recurrence and appropriate reconstruction. Chemotherapy and antiestrogen therapy are considered, and adjuvant radiation therapy is given if the chest wall has not pre-viously received radiation therapy or if the radiation oncologist feels that given the time from previous treatment there is scope for further radiation therapy, particularly if this is palliative. Women treated previously with a breast-conservation procedure undergo a mastectomy and appropriate reconstruction. Chemo-therapy and antiestrogen therapy are considered depending of the hormone receptor status and HER2 status of the tumor.Breast Cancer PrognosisSurvival rates for women diagnosed with breast cancer in the United States can be obtained from the SEER Program of the Brunicardi_Ch17_p0541-p0612.indd 58701/03/19 5:05 PM 588SPECIFIC

1	PrognosisSurvival rates for women diagnosed with breast cancer in the United States can be obtained from the SEER Program of the Brunicardi_Ch17_p0541-p0612.indd 58701/03/19 5:05 PM 588SPECIFIC CONSIDERATIONSPART IIABFigure 17-32. Lesion to be targeted for excisional biopsy. A. Craniocaudal view of the left breast demonstrating 2 lesions (arrows) to be targeted for needle localization and excision. B. Oblique view demonstrating target lesions. (Used with permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screening, Royal Derby Hospital, Derby, UK.)National Cancer Institute. Data have been collected since 1973 and are updated at regular intervals. The overall 5-year rela-tive survival for breast cancer patients from the time period of 2003 to 2009 from 18 SEER geographic areas was 89.2%.270 The 5-year relative survival by race was reported to be 90.4% for white women and 78.7% for black women. The 5-year sur-vival rate for patients with localized disease

1	geographic areas was 89.2%.270 The 5-year relative survival by race was reported to be 90.4% for white women and 78.7% for black women. The 5-year sur-vival rate for patients with localized disease (61% of patients) is 98.6%; for patients with regional disease (32% of patients), 84.4%; and for patients with distant metastatic disease (5% of patients), 24.3%. Breast cancer survival has significantly increased over the past two decades due to improvements in screening and local and systemic therapies. Data from the American College of Surgeons National Cancer Data Base can also be accessed; this data reports survival based on stage of disease at presentation using the AJCC staging system.SURGICAL TECHNIQUES IN BREAST CANCER THERAPYExcisional Biopsy With Needle LocalizationExcisional biopsy implies complete removal of a breast lesion with a margin of normal-appearing breast tissue. In the past, surgeons would obtain prior consent from the patient, allow-ing mastectomy if the initial

1	implies complete removal of a breast lesion with a margin of normal-appearing breast tissue. In the past, surgeons would obtain prior consent from the patient, allow-ing mastectomy if the initial biopsy results confirmed cancer. Today it is important to consider the options for local therapy (lumpectomy vs. mastectomy with or without reconstruction) and the need for nodal assessment with SLN dissection. Needle-core biopsy is the preferred diagnostic method, and excisional biopsy should be reserved for those cases in which the needle biopsy results are discordant with the imaging findings or clini-cal examination (Fig. 17-32). In general, circumareolar incisions can be used to access lesions that are subareolar or within a short distance of the nipple-areolar complex. Elsewhere in the breast, incisions can be placed along the lines of tension in the skin that are generally concentric with the nipple-areola complex. In the lower half of the breast, the use of radial incisions typically

1	incisions can be placed along the lines of tension in the skin that are generally concentric with the nipple-areola complex. In the lower half of the breast, the use of radial incisions typically provides the best outcome. When the tumor is quite distant from the central breast, the biopsy incision can be excised separately from the primary mastectomy incision, should a mastectomy be required. Radial incisions in the upper half of the breast are not recommended because of possible scar contracture resulting in displacement of the ipsilateral nipple-areola complex. Similarly, curvilinear incisions in the lower half of the breast may displace the nipple-areolar complex downward.After excision of a suspicious breast lesion, the specimen should be X-rayed to confirm that the lesion has been excised with appropriate margins. The biopsy tissue specimen is ori-entated for the pathologist using sutures, clips, or dyes. Addi-tional margins (superior, inferior, medial, lateral, superficial, and

1	with appropriate margins. The biopsy tissue specimen is ori-entated for the pathologist using sutures, clips, or dyes. Addi-tional margins (superior, inferior, medial, lateral, superficial, and deep) may be taken from the surgical bed if the specimen X-ray shows the lesion is close to one or more margins. Some surgeons also take additional shavings from the margins as one approach to confirm complete excision of the suspicious lesion. Electrocautery or absorbable ligatures are used to achieve wound hemostasis. Cosmesis may be facilitated by approxima-tion of the surgical defect using 3-0 absorbable sutures. A run-ning subcuticular closure of the skin using 4-0 or 5-0 absorbable monofilament sutures is performed. Wound drainage is usually not required.Excisional biopsy with needle or seed localization requires a preoperative visit to the mammography suite for placement of a localization wire or a radioactive or magnetic seed that can be detected intraoperatively with a handheld probe.

1	localization requires a preoperative visit to the mammography suite for placement of a localization wire or a radioactive or magnetic seed that can be detected intraoperatively with a handheld probe. The lesion can also be targeted by sonography in the imaging suite or in the operating room. The lesion to be excised is accurately localized by mammography, and the tip of a thin wire hook or a seed is positioned close to the lesion (Fig. 17-33). Using the wire hook as a guide, or detection of the seed with a handheld probe, the surgeon subsequently excises the suspicious breast lesion while removing a margin of normal-appearing breast tissue. Before the patient leaves the operating room, specimen radiography is performed to confirm complete excision of the suspicious lesion (Fig. 17-34).Brunicardi_Ch17_p0541-p0612.indd 58801/03/19 5:05 PM 589THE BREASTCHAPTER 17Figure 17-33. Wire localization procedure. Mammographic images of hookwire in place targeting lesions for excision in the

1	58801/03/19 5:05 PM 589THE BREASTCHAPTER 17Figure 17-33. Wire localization procedure. Mammographic images of hookwire in place targeting lesions for excision in the left breast (A) and the right breast (B). (Used with permission from Dr. Anne Turnbull, Consultant Radiologist/Director of Breast Screen-ing, Royal Derby Hospital, Derby, UK.)Figure 17-34. Specimen mammography. Specimen mam-mograms demonstrating excision of targeted (A) density, (B) calcifications, and (C) spiculated mass seen on preoperative imaging. (Used with permission from Dr. Anne Turnbull, Con-sultant Radiologist/Director of Breast Screening, Royal Derby Hospital, Derby, UK.)ABCABBrunicardi_Ch17_p0541-p0612.indd 58901/03/19 5:05 PM 590SPECIFIC CONSIDERATIONSPART IISentinel Lymph Node DissectionSentinel lymph node (SLN) dissection is primarily used to assess the regional lymph nodes in women with early breast cancers who are clinically node-negative by physical examina-tion and imaging studies.271-279 This

1	(SLN) dissection is primarily used to assess the regional lymph nodes in women with early breast cancers who are clinically node-negative by physical examina-tion and imaging studies.271-279 This method also is accurate in women with larger tumors (T3 N0), but nearly 75% of these women will prove to have axillary lymph node metastases on histologic examination, and wherever possible it is better to identify them preoperatively as this will allow a definitive procedure for known axillary disease. SLN dissection has also been reported to be accurate for staging of the axilla after chemotherapy in women with clinically node-negative dis-ease at initial presentation.280,281 Tan et al in a review and meta-analysis of 449 cases of SLN biopsy in clinically lymph node-negative disease reported a sensitivity of 93%, giving a false negative rate of 7% with a negative predictive value of 94% and an overall accuracy of 95%.282 Clinical situations where SLN dissection is not recommended include

1	a sensitivity of 93%, giving a false negative rate of 7% with a negative predictive value of 94% and an overall accuracy of 95%.282 Clinical situations where SLN dissection is not recommended include patients with inflammatory breast cancers, those with biopsy proven metasta-sis, DCIS without mastectomy, or prior axillary surgery. Although limited data are available, SLN dissection appears to be safe in pregnancy when performed with radioisotope alone.Evidence from large prospective studies suggests that the combination of intraoperative gamma probe detection of radioactive colloid and intraoperative visualization of blue dye (isosulfan blue dye or methylene blue) is more accurate for identification of SLNs than the use of either agent alone. Some surgeons use preoperative lymphoscintigraphy, although it is not required for identification of the SLNs. On the day before surgery, or the day of surgery, the radioactive colloid is injected either in the breast parenchyma around the

1	although it is not required for identification of the SLNs. On the day before surgery, or the day of surgery, the radioactive colloid is injected either in the breast parenchyma around the primary tumor or prior biopsy site, into the subareolar region, or subdermally in proximity to the primary tumor site. With a 25-gauge needle, 0.5 mCi of 0.2-μm technetium 99m–labeled sulfur colloid is injected for same-day surgery, or a higher dose of 2.5 mCi of technetium-labeled sulfur colloid is administered when the isotope is to be injected on the day before surgery. Subdermal injections are given in proximity to the cancer site or in the subareolar location. Later, in the operating room, 3 to 5 mL of blue dye is injected either in the breast parenchyma or in the subareolar location. It is not recommended that the blue dye be used in a subdermal injection because this can result in tattoo-ing of the skin (isosulfan blue dye) or skin necrosis (methylene blue). For nonpalpable cancers, the

1	recommended that the blue dye be used in a subdermal injection because this can result in tattoo-ing of the skin (isosulfan blue dye) or skin necrosis (methylene blue). For nonpalpable cancers, the injection of the technetium-labeled sulfur colloid solution can be guided by ultrasound or by mammographic guidance. In women who have undergone previous excisional biopsy, the injections are made in the breast parenchyma around the biopsy cavity but not into the cavity itself. Women are told preoperatively that the isosulfan blue dye injection will cause a change in the color of their urine and that there is a very small risk of allergic reaction to the dye (1 in 10,000). Anaphylactic reactions have been documented, and some groups administer a regimen of antihistamine, steroids, and a histamine H-2 receptor antagonist preoperatively as a prophylactic regimen to prevent allergic reactions. The use of radioactive colloid is safe, and radiation exposure is very low. Sentinel node dissection

1	H-2 receptor antagonist preoperatively as a prophylactic regimen to prevent allergic reactions. The use of radioactive colloid is safe, and radiation exposure is very low. Sentinel node dissection can be performed in pregnancy with the radioactive colloid without the use of blue dye.A hand-held gamma counter is used to transcutaneously identify the location of the SLN. This can help to guide place-ment of the incision. A 3to 4-cm incision is made in line with that used for an axillary dissection, which is a curved transverse 9incision in the lower axilla just below the hairline. After dis-secting through the subcutaneous tissue, the surgeon dissects through the axillary fascia, being mindful to identify blue lym-phatic channels. Following these channels can lead directly to the SLN and limit the amount of dissection through the axillary tissues. The gamma probe is used to facilitate the dissection and to pinpoint the location of the SLN. As the dissection continues, the signal from

1	limit the amount of dissection through the axillary tissues. The gamma probe is used to facilitate the dissection and to pinpoint the location of the SLN. As the dissection continues, the signal from the probe increases in intensity as the SLN is approached. The SLN also is identified by visualization of blue dye in the afferent lymph vessel and in the lymph node itself. Before the SLN is removed, a 10-second in vivo radioactivity count is obtained. After removal of the SLN, a 10-second ex vivo radioactive count is obtained, and the node is then sent to the pathology laboratory for either permanentor frozen-section analysis. The lowest false-negative rates for SLN dissection have been obtained when all blue lymph nodes and all lymph nodes with counts >10% of the 10-second ex vivo count of the SLN are harvested (“10% rule”). Based on this, the gamma counter is used before closing the axillary wound to measure residual radioactivity in the surgical bed. A search is made for additional

1	the SLN are harvested (“10% rule”). Based on this, the gamma counter is used before closing the axillary wound to measure residual radioactivity in the surgical bed. A search is made for additional SLNs if the counts remain high. This procedure is repeated until residual radioactivity in the surgical bed is less than 10% of the 10-second ex vivo count of the most radioac-tive SLN and all blue nodes have been removed. Studies have demonstrated that 98% of all positive SLNs will be recovered with the removal of four SLNs; therefore, it is not necessary to remove greater than four SLNs for accurate staging of the axilla.Results from the NSABP B-32 trial showed that the false-negative rate for SLN dissection is influenced by tumor loca-tion, type of diagnostic biopsy, and number of SLNs removed at surgery.243 The authors reported that tumors located in the lateral breast were more likely to have a false-negative SLN. This may be explained by difficulty in discriminating the hot spot in

1	at surgery.243 The authors reported that tumors located in the lateral breast were more likely to have a false-negative SLN. This may be explained by difficulty in discriminating the hot spot in the axilla when the radioisotope has been injected at the primary tumor site in the lateral breast. Those patients who had undergone an excisional biopsy before the SLN procedure were significantly more likely to have a false-negative SLN. This report further confirms that surgeons should use needle biopsy for diagnosis whenever possible and reserve excisional biopsy for the rare situations in which needle biopsy findings are non-diagnostic or discordant. Finally, removal of a larger number of SLNs at surgery appears to reduce the false-negative rate. In B-32, the false-negative rate was reduced from 17.7% to 10% when two SLNs were recovered and to 6.9% when three SLNs were removed. Yi and associates reported that the number of SLNs that need to be removed for accurate staging is influenced by

1	17.7% to 10% when two SLNs were recovered and to 6.9% when three SLNs were removed. Yi and associates reported that the number of SLNs that need to be removed for accurate staging is influenced by individual patient and primary tumor factors.283In the B-32 trial, SLNs were identified outside the levels I and II axillary nodes in 1.4% of cases. This was significantly influenced by the site of radioisotope injection. When a subareo-lar or periareolar injection site was used, there were no instances of SLNs identified outside the level I or II axilla, compared with a rate of 20% when a peritumoral injection was used. This sup-ports the overall concept that the SLN is the first site of drain-age from the lymphatic vessels of the primary tumor. Although many patients will have similar drainage patterns from injec-tions given at the primary tumor site and at the subareolar plexus, some patients will have extra-axillary drainage, either alone or in combination with axillary node drainage,

1	patterns from injec-tions given at the primary tumor site and at the subareolar plexus, some patients will have extra-axillary drainage, either alone or in combination with axillary node drainage, and this is best assessed with a peritumoral injection of the radioiso-tope. Kong et al reported that internal mammary node drain-age on preoperative lymphoscintigraphy was associated with Brunicardi_Ch17_p0541-p0612.indd 59001/03/19 5:05 PM 591THE BREASTCHAPTER 17worse distant disease-free survival in early-stage breast cancer patients.284Breast ConservationBreast conservation involves resection of the primary breast cancer with a margin of normal-appearing breast tissue, adju-vant radiation therapy, and assessment of regional lymph node status.285,286 Resection of the primary breast cancer is alterna-tively called segmental mastectomy, lumpectomy, partial mas-tectomy, wide local excision, and tylectomy. For many women with stage I or II breast cancer, breast-conserving therapy (BCT) is

1	is alterna-tively called segmental mastectomy, lumpectomy, partial mas-tectomy, wide local excision, and tylectomy. For many women with stage I or II breast cancer, breast-conserving therapy (BCT) is preferable to total mastectomy because BCT produces survival rates equivalent to those after total mastectomy while preserv-ing the breast.287 Six prospective randomized trials have shown that overall and disease-free survival rates are similar with BCT and mastectomy; however, three of the studies showed higher local-regional failure rates in patients undergoing BCT. In two of these studies, there were no clear criteria for histologically negative margins.285-287 Data from the EBCTCG meta-analysis revealed that the addition of radiation reduces recurrence by half and improves survival at year 15 by about a sixth.288 When all of this information is taken together, BCT is considered to be oncologically equivalent to mastectomy.In addition to being equivalent to mastectomy in terms of

1	year 15 by about a sixth.288 When all of this information is taken together, BCT is considered to be oncologically equivalent to mastectomy.In addition to being equivalent to mastectomy in terms of oncologic safety, BCT appears to offer advantages over mas-tectomy with regard to quality of life and aesthetic outcomes. BCT allows for preservation of breast shape and skin as well as preservation of sensation, and it provides an overall psychologic advantage associated with breast preservation.Breast conservation surgery is currently the standard treat-ment for women with stage 0, I, or II invasive breast cancer. Women with DCIS require only resection of the primary cancer and adjuvant radiation therapy without assessment of regional lymph nodes. When a lumpectomy is performed, a curvilinear incision lying concentric to the nipple-areola complex is made in the skin overlying the breast cancer when the tumor is in the upper aspect of the breast. Radial incisions are preferred when the

1	incision lying concentric to the nipple-areola complex is made in the skin overlying the breast cancer when the tumor is in the upper aspect of the breast. Radial incisions are preferred when the tumor is in the lower aspect of the breast. Skin excision is not necessary unless there is direct involvement of the overlying skin by the primary tumor. The breast cancer is removed with an envelope of normal-appearing breast tissue that is adequate to achieve a cancer-free margin. Significant controversy has existed on the appropriate margin width for BCT.260 However, recently the SSO and ASTRO developed a consensus statement, supported by data from a systematic review data, encouraging “no tumor on ink” to be the standard definition of a negative margin for invasive stages I and II breast cancer in patients who undergo breast conserving surgery with whole-breast irradiation. The meta-analysis found that increasing the margin width does not affect local recurrence rates as long as the inked

1	in patients who undergo breast conserving surgery with whole-breast irradiation. The meta-analysis found that increasing the margin width does not affect local recurrence rates as long as the inked or transected margin is microscopically negative.289-292 Specimen X-ray should routinely be performed to confirm the lesion has been excised. Specimen orientation is performed by the surgeon. Additional margins from the surgical bed are taken as needed to provide a histologically negative margin. Requests for determination of ER, PR, and HER2 status are conveyed to the pathologist.It is the surgeon’s responsibility to ensure complete removal of cancer in the breast. Ensuring surgical margins that are free of breast cancer will minimize the chances of local recurrence and will enhance cure rates. If negative margins are not obtainable with reexcision, mastectomy is required. SLN is performed before removal of the primary breast tumor. When indicated, intraoperative assessment of the sentinel

1	If negative margins are not obtainable with reexcision, mastectomy is required. SLN is performed before removal of the primary breast tumor. When indicated, intraoperative assessment of the sentinel node can proceed while the segmental mastectomy is being performed.The use of oncoplastic surgery can be entertained at the time of segmental mastectomy or at a later time to improve the overall aesthetic outcome. The use of oncoplastic techniques range from a simple reshaping of breast tissue to local tissue rearrangement to the use of pedicled flaps or breast reduction techniques. The overall goal is to achieve the best possible aes-thetic result. In determining which patients are candidates for oncoplastic breast surgery, several factors should be considered, including the extent of the resection of breast tissue necessary to achieve negative margins, the location of the primary tumor within the breast, and the size of the patient’s breast and body habitus. Oncoplastic techniques are of

1	of breast tissue necessary to achieve negative margins, the location of the primary tumor within the breast, and the size of the patient’s breast and body habitus. Oncoplastic techniques are of prime consideration when (a) a significant area of breast skin will need to be resected with the specimen to achieve negative margins; (b) a large vol-ume of breast parenchyma will be resected resulting in a signifi-cant defect; (c) the tumor is located between the nipple and the inframammary fold, an area often associated with unfavorable cosmetic outcomes; or (d) excision of the tumor and closure of the breast may result in malpositioning of the nipple.Mastectomy and Axillary DissectionA skin-sparing mastectomy removes all breast tissue, the nipple-areola complex, and scars from any prior biopsy pro-cedures.293,294 There is a recurrence rate of less than 6% to 8%, comparable to the long-term recurrence rates reported with stan-dard mastectomy, when skin-sparing mastectomy is used for patients

1	There is a recurrence rate of less than 6% to 8%, comparable to the long-term recurrence rates reported with stan-dard mastectomy, when skin-sparing mastectomy is used for patients with Tis to T3 cancers. A total (simple) mastectomy without skin sparing removes all breast tissue, the nipple-areola complex, and skin. An extended simple mastectomy removes all breast tissue, the nipple-areola complex, skin, and the level I axillary lymph nodes. A modified radical (“Patey”) mastectomy removes all breast tissue, the nipple-areola complex, skin, and the levels I, II, and III axillary lymph nodes; the pectoralis minor that was divided and removed by Patey may be simply divided, giving improved access to level III nodes, and then left in situ, or occasionally the axillary clearance can be performed with-out dividing pectoralis minor. The Halsted radical mastectomy removes all breast tissue and skin, the nipple-areola complex, the pectoralis major and pectoralis minor muscles, and the levels

1	with-out dividing pectoralis minor. The Halsted radical mastectomy removes all breast tissue and skin, the nipple-areola complex, the pectoralis major and pectoralis minor muscles, and the levels I, II, and III axillary lymph nodes. The use of systemic che-motherapy and hormonal therapy as well as adjuvant radiation therapy for breast cancer have nearly eliminated the need for the radical mastectomy.Nipple-areolar sparing mastectomy has been popularized over the last decade especially for risk-reducing mastectomy in high risk women. For those patients with a cancer diagno-sis, many consider the following factors for eligibility: tumor located more than 2 to 3 cm from the border of the areola, smaller breast size, minimal ptosis, no prior breast surgeries with periareolar incisions, body mass index less than 40 kg/m2, no active tobacco use, no prior breast irradiation, and no evi-dence of collagen vascular disease.For a variety of biologic, economic, and psychosocial rea-sons, some

1	mass index less than 40 kg/m2, no active tobacco use, no prior breast irradiation, and no evi-dence of collagen vascular disease.For a variety of biologic, economic, and psychosocial rea-sons, some women desire mastectomy rather than breast con-servation. Women who are less concerned about cosmesis may view mastectomy as the most expeditious and desirable thera-peutic option because it avoids the cost and inconvenience of radiation therapy. Some women whose primary breast cancers cannot be excised with a reasonable cosmetic result or those who have extensive microcalcifications are best treated with Brunicardi_Ch17_p0541-p0612.indd 59101/03/19 5:05 PM 592SPECIFIC CONSIDERATIONSPART IImastectomy. Similarly, women with large cancers that occupy the subareolar and central portions of the breast and women with multicentric primary cancers also undergo mastectomy.Modified Radical MastectomyA modified radical mastectomy preserves the pectoralis major muscle with removal of levels I, II,

1	breast and women with multicentric primary cancers also undergo mastectomy.Modified Radical MastectomyA modified radical mastectomy preserves the pectoralis major muscle with removal of levels I, II, and III (apical) axillary lymph nodes.293 The operation was first described by David Patey, a surgeon at St Bartholomew’s Hospital London, who reported a series of cases where he had removed the pectoralis minor muscle allowing complete dissection of the level III axil-lary lymph nodes while preserving the pectoralis major and the lateral pectoral nerve. A modified radical mastectomy permits preservation of the medial (anterior thoracic) pectoral nerve, which courses in the lateral neurovascular bundle of the axilla and usually penetrates the pectoralis minor to supply the lateral border of the pectoralis major. Anatomic boundaries of the mod-ified radical mastectomy are the anterior margin of the latissi-mus dorsi muscle laterally, the midline of the sternum medially, the subclavius

1	of the pectoralis major. Anatomic boundaries of the mod-ified radical mastectomy are the anterior margin of the latissi-mus dorsi muscle laterally, the midline of the sternum medially, the subclavius muscle superiorly, and the caudal extension of the breast 2 to 3 cm inferior to the inframammary fold inferiorly. Skin-flap thickness varies with body habitus but ideally is 7 to 8 mm inclusive of skin and telasubcutanea (Fig. 17-35). Once the skin flaps are fully developed, the fascia of the pectoralis major muscle and the overlying breast tissue are elevated off the underlying musculature, which allows for the complete removal of the breast (Fig. 17-36).Subsequently, an axillary lymph node dissection is per-formed. The most lateral extent of the axillary vein is identified, and the areolar tissue of the lateral axillary space is elevated as the vein is cleared on its anterior and inferior surfaces. The areo-lar tissues at the junction of the axillary vein and the anterior edge of the

1	tissue of the lateral axillary space is elevated as the vein is cleared on its anterior and inferior surfaces. The areo-lar tissues at the junction of the axillary vein and the anterior edge of the latissimus dorsi muscle, which include the lateral and subscapular lymph node groups (level I), are cleared. Care is taken to preserve the thoracodorsal neurovascular bundle. The dissection then continues medially with clearance of the central axillary lymph node group (level II). The long thoracic nerve of Bell is identified and preserved as it travels in the investing fascia of the serratus anterior muscle. Every effort is made to preserve this nerve because permanent disability with a winged scapula and shoulder weakness will follow denervation of the serratus anterior muscle. Patey divided the pectoralis minor and removed it to allow access right up to the apex of the axilla. The pectoralis minor muscle is usually divided at the tendinous portion near its insertion onto the coracoid

1	the pectoralis minor and removed it to allow access right up to the apex of the axilla. The pectoralis minor muscle is usually divided at the tendinous portion near its insertion onto the coracoid process (Fig. 17-37 inset), which allows dissection of the axillary vein medially to the costoclavicular (Halsted’s) ligament. Finally, the breast and axillary contents are removed from the surgical bed and are sent for pathologic assessment. In his modified radical mastectomy, Patey removed the pectoralis minor muscle. Many surgeons now divide only the tendon of the pectoralis minor muscle at its insertion onto the coracoid process while leaving the rest of the muscle intact, which still provides good access to the apex of the axilla.Figure 17-35. Modified radical mastectomy: eleva-tion of skin flaps. Skin flaps are 7 to 8 mm in thick-ness, inclusive of the skin and telasubcutanea. (Visual Art: © 2013. The University of Texas MD Anderson Cancer Center.)Figure 17-36. Modified radical

1	of skin flaps. Skin flaps are 7 to 8 mm in thick-ness, inclusive of the skin and telasubcutanea. (Visual Art: © 2013. The University of Texas MD Anderson Cancer Center.)Figure 17-36. Modified radical mastectomy after resection of breast tissue. The pectoralis major muscle is cleared of its fascia as the overlying breast is elevated. The latissimus dorsi muscle is the lateral boundary of the dissection. (Visual Art: © 2013. The University of Texas MD Anderson Cancer Center.)Brunicardi_Ch17_p0541-p0612.indd 59201/03/19 5:05 PM 593THE BREASTCHAPTER 17Seromas beneath the skin flaps or in the axilla represent the most frequent complication of mastectomy and axillary lymph node dissection, reportedly occurring in as many as 30% of cases. The use of closed-system suction drainage reduces the incidence of this complication. Catheters are retained in the wound until drainage diminishes to <30 mL per day. Wound infections occur infrequently after a mastectomy, and the majority are a result

1	incidence of this complication. Catheters are retained in the wound until drainage diminishes to <30 mL per day. Wound infections occur infrequently after a mastectomy, and the majority are a result of skin-flap necrosis. Cultures of speci-mens taken from the infected wound for aerobic and anaerobic organisms, debridement, and antibiotic therapy are effective management. Moderate or severe hemorrhage in the postop-erative period is rare and is best managed with early wound exploration for control of hemorrhage and reestablishment of closed-system suction drainage. The incidence of functionally significant lymphedema after a modified radical mastectomy is approximately 20% but can be as high as 50% to 60% when postoperative radiation is employed. Extensive axillary lymph node dissection, the delivery of radiation therapy, the presence of pathologic lymph nodes, and obesity are predisposing factors. Patients should be referred to physical therapy at the earliest signs of lymphedema to

1	the delivery of radiation therapy, the presence of pathologic lymph nodes, and obesity are predisposing factors. Patients should be referred to physical therapy at the earliest signs of lymphedema to prevent progression to the later stages. The use of individually fitted compressive sleeves and complex decongestive therapy may be necessary.Reconstruction of the Breast and Chest WallThe goals of reconstructive surgery after a mastectomy for breast cancer are wound closure and breast reconstruction, which is either immediate or delayed.295 In most cases, wound closure after mastectomy is accomplished with simple approximation of the wound edges. However, if a more radical removal of skin and subcutaneous tissue is necessary, a pedicled myocutane-ous flap from the latissimus dorsi muscle is generally the best approach for wound coverage. A skin graft provides functional coverage that will tolerate adjuvant radiation therapy; however, this is not preferred because poor graft adherence may

1	the best approach for wound coverage. A skin graft provides functional coverage that will tolerate adjuvant radiation therapy; however, this is not preferred because poor graft adherence may delay delivery of radiation therapy. Breast reconstruction after risk-reducing mastectomy or after mastectomy for early-stage breast cancer may be performed at the same time as the mastectomy. This allows for a skin-sparing mastectomy to be performed, which offers the best overall cosmetic outcomes. Reconstruc-tion can proceed with an expander/implant reconstruction or with autologous tissue such as a pedicled myocutaneous flap or a free flap using microvascular techniques. In patients with locally advanced breast cancer, reconstruction is often delayed until after completion of adjuvant radiation therapy to ensure that local-regional control of disease is obtained. The expected use of postmastectomy radiotherapy should also be considered as a reason for delayed reconstruction as radiotherapy to a

1	to ensure that local-regional control of disease is obtained. The expected use of postmastectomy radiotherapy should also be considered as a reason for delayed reconstruction as radiotherapy to a reconstructed breast has been reported to result in inferior cos-metic outcomes. Consideration can be made for placement of a tissue expander to allow for skin-sparing, but this should be discussed with the radiation oncologist and other members of the treatment team. If chest wall coverage is needed to replace a large skin or soft tissue defect, many different types of myo-cutaneous flaps are employed, but the latissimus dorsi and the rectus abdominis myocutaneous flaps are most frequently used. The latissimus dorsi myocutaneous flap consists of a skin paddle based on the underlying latissimus dorsi muscle, which Figure 17-37. Modified radical mastectomy (Patey): axillary lymph node dissection. The dissection proceeds from lateral to medial, with complete visualization of the anterior and

1	dorsi muscle, which Figure 17-37. Modified radical mastectomy (Patey): axillary lymph node dissection. The dissection proceeds from lateral to medial, with complete visualization of the anterior and inferior aspects of the axillary vein. Loose areolar tissue at the junction of the axillary vein and the anterior margin of the latissimus dorsi muscle is swept inferomedially inclusive of the lateral (axillary) lymph node group (level I). Care is taken to preserve the thoracodorsal artery, vein, and nerve in the deep axillary space. The lateral lymph node group is resected in continuity with the subscapular lymph node group (level I) and the external mammary lymph node group (level I). Dissection anterior to the axillary vein allows removal of the central lymph node group (level II) and the apical (subclavicular) lymph node group (level III). The superomedial limit of this dissection is the clavipectoral fascia (Halsted’s ligament). Inset depicts division of the insertion of the

1	and the apical (subclavicular) lymph node group (level III). The superomedial limit of this dissection is the clavipectoral fascia (Halsted’s ligament). Inset depicts division of the insertion of the pectoralis minor muscle at the coracoid process. The surgeon’s finger shields the underlying brachial plexus. (Reproduced with permission from Bland KI, Copeland EMI: The Breast: Comprehensive Management of Benign and Malignant Diseases, 4th ed. Philadelphia, PA: Elsevier/Saunders; 2009.)Brunicardi_Ch17_p0541-p0612.indd 59301/03/19 5:05 PM 594SPECIFIC CONSIDERATIONSPART IIis supplied by the thoracodorsal artery with contributions from the posterior intercostal arteries. A transverse rectus abdominis myocutaneous (TRAM) flap consists of a skin paddle based on the underlying rectus abdominis muscle, which is supplied by vessels from the deep inferior epigastric artery. The free TRAM flap uses microvascular anastomoses to establish blood supply to the flap. When the bony chest wall is

1	muscle, which is supplied by vessels from the deep inferior epigastric artery. The free TRAM flap uses microvascular anastomoses to establish blood supply to the flap. When the bony chest wall is involved with cancer, resection of a portion of the bony chest wall is indicated. If only one or two ribs are resected and soft tissue coverage is pro-vided, reconstruction of the bony defect is usually not necessary because scar tissue will stabilize the chest wall. If more than two ribs are sacrificed, it is advisable to stabilize the chest wall with prosthetic material, which is then covered with soft tissue by using a latissimus dorsi or TRAM flap.NONSURGICAL BREAST CANCER THERAPIESRadiation TherapyRadiation therapy is used for all stages of breast cancer depending on whether the patient is undergoing BCT or mas-tectomy.296-302 Adjuvant radiation for patients with DCIS and early-stage breast cancer have been described previously in this chapter. Those women treated with mastectomy who

1	undergoing BCT or mas-tectomy.296-302 Adjuvant radiation for patients with DCIS and early-stage breast cancer have been described previously in this chapter. Those women treated with mastectomy who have cancer at the surgical margins are at sufficiently high risk for local recurrence to warrant the use of adjuvant radiation therapy to the chest wall postoperatively. Women with metastatic disease involving four or more axillary lymph nodes and premeno-pausal women with metastatic disease involving one to three lymph nodes also are at increased risk for recurrence and are candidates for the use of chest wall and supraclavicular lymph node radiation therapy. In advanced local-regional breast can-cer (stage IIIA or IIIB), women are at high risk for recurrent disease after surgical therapy, and adjuvant radiation therapy is used to reduce the risk of recurrence. Current recommenda-tions for stages IIIA and IIIB breast cancer are (a) adjuvant radiation therapy to the breast and

1	therapy, and adjuvant radiation therapy is used to reduce the risk of recurrence. Current recommenda-tions for stages IIIA and IIIB breast cancer are (a) adjuvant radiation therapy to the breast and supraclavicular lymph nodes after neoadjuvant chemotherapy and segmental mastectomy with or without axillary lymph node dissection, (b) adjuvant radiation therapy to the chest wall and supraclavicular lymph nodes after neoadjuvant chemotherapy and mastectomy with or without axillary lymph node dissection, and (c) adjuvant radiation therapy to the chest wall and supraclavicular lymph nodes after segmental mastectomy or mastectomy with axillary lymph node dissection and adjuvant chemotherapy. Data from the EBCTCG has shown improvements in local-regional con-trol and survival in patients treated with mastectomy and post-mastectomy radiation therapy for one to three positive axillary lymph nodes.303 This data is based on clinical trials from the era of axillary lymph node dissection for

1	with mastectomy and post-mastectomy radiation therapy for one to three positive axillary lymph nodes.303 This data is based on clinical trials from the era of axillary lymph node dissection for staging prior to the routine use of sentinel lymph node dissection. It is likely that the vol-ume of disease in the earlier trials was greater overall than what is currently seen in patients who have small volume metastases detected at sentinel node dissection. It is important to include all multidisciplinary team members (medical oncology, plastic surgery, radiation oncology, and surgical oncology) regarding the risks and benefits of postmastectomy radiation therapy in patients with one to three positive nodes.The use of partial breast irradiation (APBI) for patients treated with breast-conserving surgery has also been previously described. APBI can be delivered via brachytherapy, external beam radiation therapy using 3D conformal radiation, or inten-sity-modulated radiation therapy. Although

1	surgery has also been previously described. APBI can be delivered via brachytherapy, external beam radiation therapy using 3D conformal radiation, or inten-sity-modulated radiation therapy. Although initial results are Table 17-14Adjuvant chemotherapy regimens for breast cancerHER-2 NEGATIVEHER-2 POSITIVEPreferred Dose dense AC → Paclitaxel every 2 weeksDose dense AC → Paclitaxel weeklyTC (T = docetaxel)Other RegimensCMFAC → Docetaxel every 3 weeksAC → Paclitaxel weeklyTAC (T = docetaxel)AC → T + trastuzumab +/pertuzumab (T = paclitaxel)TCH (docetaxel, carboplatin, trastuzumab +/pertuzumab)Other RegimensAC → T + trastuzumab +/pertuzumab (T = docetaxel)Docetaxel + cyclophosphamide + trastuzumabFEC → Docetaxel + trastuzumab + pertuzumabFEC → Paclitaxel + trastuzumab + pertuzumabPaclitaxel + trastuzumabPaclitaxel + trastuzumab + pertuzumab → FECDocetaxel + trastuzumab + pertuzumab → FECA = Adriamycin (doxorubicin); C = cyclophosphamide; E = epirubicin; F = 5-fluorouracil; M =

1	+ trastuzumabPaclitaxel + trastuzumab + pertuzumab → FECDocetaxel + trastuzumab + pertuzumab → FECA = Adriamycin (doxorubicin); C = cyclophosphamide; E = epirubicin; F = 5-fluorouracil; M = methotrexate; T = Taxane (docetaxel or paclitaxel); → = followed by.Data from NCCN Practice Guidelines in Oncology. Fort Washington, PA: National Comprehensive Cancer Network, 2006.promising in highly selected low-risk populations, use of APBI should be based on current guidelines or offered in the setting of a prospective trial.304Chemotherapy AdjuvantChemotherapy. The Early Breast Cancer Trialists’ Collabora-tive Group overview analysis of adjuvant chemotherapy demon-strated reductions in the odds of recurrence and death in women ≤70 years of age with stage I, IIA, or IIB breast cancer.123,305-309 For those ≥70 years of age, the lack of definitive clinical trial data regarding adjuvant chemotherapy prevented definitive rec-ommendations. Adjuvant chemotherapy is of minimal benefit to women with

1	For those ≥70 years of age, the lack of definitive clinical trial data regarding adjuvant chemotherapy prevented definitive rec-ommendations. Adjuvant chemotherapy is of minimal benefit to women with negative nodes and cancers ≤0.5 cm in size and is not recommended. Women with negative nodes and cancers 0.6 to 1.0 cm are divided into those with a low risk of recurrence and those with unfavorable prognostic features that portend a higher risk of recurrence and a need for adjuvant chemotherapy. Adverse prognostic factors include blood vessel or lymph ves-sel invasion, high nuclear grade, high histologic grade, HER2/neu overexpression, and negative hormone receptor status. American Society of Clinical Oncology guidelines suggest that adjuvant chemotherapy should be considered for patients with positive lymph nodes, HER2-positive disease, Adjuvant! Online mortality greater than 10%, grade 3 lymph node negative tumors >5 mm, triple-negative tumors, lympho-vascular invasion, or estimated

1	with positive lymph nodes, HER2-positive disease, Adjuvant! Online mortality greater than 10%, grade 3 lymph node negative tumors >5 mm, triple-negative tumors, lympho-vascular invasion, or estimated distant relapse risk of greater than 15% at 10 years based on 21 gene recurrence score.259 Adjuvant chemotherapy is recommended by the NCCN guidelines for women with these unfavorable prognostic features. Table 17-14 lists the frequently used chemotherapy regimens for breast cancer.For women with hormone receptor-negative cancers that are >1 cm in size, adjuvant chemotherapy is appropriate. Brunicardi_Ch17_p0541-p0612.indd 59401/03/19 5:05 PM 595THE BREASTCHAPTER 17However, women with node-negative hormone receptor–positive cancers and T1 tumors are candidates for antiestrogen therapy with or without chemotherapy. Assessment of overall risk using known prognostic factors or additional testing such as the 21-gene recurrence score assay can help to guide deci-sion making regarding

1	with or without chemotherapy. Assessment of overall risk using known prognostic factors or additional testing such as the 21-gene recurrence score assay can help to guide deci-sion making regarding chemotherapy in patients with node-negative, ER-positive breast cancer. For special-type cancers (tubular, mucinous, medullary, etc), which are usually strongly estrogen receptor positive, adjuvant antiestrogen therapy should be advised for cancers >1 cm. For women with node-positive tumors or with a special-type cancer that is >3 cm, the use of chemotherapy is appropriate; those with hormone receptor-positive tumors should receive antiestrogen therapy.For stage IIIA breast cancer, preoperative chemotherapy with an anthracycline and taxane-containing regimen followed by either a modified radical mastectomy or segmental mastec-tomy with axillary dissection followed by adjuvant radiation therapy should be considered, especially for estrogen receptor negative disease. While the same regimen

1	mastectomy or segmental mastec-tomy with axillary dissection followed by adjuvant radiation therapy should be considered, especially for estrogen receptor negative disease. While the same regimen may be considered for estrogen receptor positive disease, it is known that these tumors respond less well to chemotherapy with <10% pCR rate overall and <3% pCR rate for lobular cancers. Other options such as neoadjuvant endocrine therapy followed by local-regional treatment or in some cases primary endocrine therapy may be considered depending on other tumor characteristics and the patient’s comorbid conditions and preference.Neoadjuvant (Preoperative) Chemotherapy. In the early 1970s, the National Cancer Institute in Milan, Italy, initiated two prospective randomized multimodality clinical trials for women with T3 or T4 breast cancer.310 The best results were achieved when surgery was interposed between chemotherapy courses, with 82% local-regional control and 25% having a 5-year

1	trials for women with T3 or T4 breast cancer.310 The best results were achieved when surgery was interposed between chemotherapy courses, with 82% local-regional control and 25% having a 5-year dis-ease-free survival. The NSABP B-18 trial evaluated the role of neoadjuvant chemotherapy in women with operable stages II and III breast cancer.206 Women entered into this study were randomly assigned to receive either surgery followed by che-motherapy or neoadjuvant chemotherapy followed by surgery. There was no difference in the 5-year disease-free survival rates for the two groups, but after neoadjuvant chemotherapy there was an increase in the number of lumpectomies performed and a decreased incidence of node positivity. It was suggested that neoadjuvant chemotherapy be considered for the initial manage-ment of breast cancers judged too large for initial lumpectomy.Several prospective clinical trials have evaluated the neo-adjuvant approach, and two meta-analyses have been performed,

1	initial manage-ment of breast cancers judged too large for initial lumpectomy.Several prospective clinical trials have evaluated the neo-adjuvant approach, and two meta-analyses have been performed, each showing that neoadjuvant vs. adjuvant chemotherapy are equivalent in terms of OS.262,311 These analyses also evaluated local-regional recurrence (LRR) and found that there was an increase in LRR rates for patients receiving neoadjuvant chemo-therapy when radiation therapy was used alone without surgery after completion of chemotherapy. Mittendorf and colleagues evaluated a contemporary series of almost 3000 patients treated with breast conserving surgery and radiation therapy who received either neoadjuvant or adjuvant chemotherapy for breast cancer.312 They found that the risk of LRR was driven by bio-logic factors and disease stage and was not impacted by the timing of chemotherapy delivery. These data high-light the importance of the multidisciplinary management of patients with

1	driven by bio-logic factors and disease stage and was not impacted by the timing of chemotherapy delivery. These data high-light the importance of the multidisciplinary management of patients with breast cancer in achieving the best outcomes.The use of neoadjuvant chemotherapy offers the oppor-tunity to observe the response of the intact primary tumor 10and any regional nodal metastases to a specific chemotherapy regimen.279 For patients whose tumors remain stable in size or even progress with the initial neoadjuvant chemotherapy regi-men, a new regimen may be considered that uses another class of agents, although there is no randomized data confirming this will improve outcome.After treatment with neoadjuvant chemotherapy, patients are assessed for clinical and pathologic response to the regimen. Patients whose tumors achieve a pathologic complete response to neoadjuvant chemotherapy have been shown to have statisti-cally improved survival outcomes to those of patients whose tumors

1	regimen. Patients whose tumors achieve a pathologic complete response to neoadjuvant chemotherapy have been shown to have statisti-cally improved survival outcomes to those of patients whose tumors demonstrate only a partial response, remain stable, or progress on treatment. Researchers at MD Anderson Cancer Center have shown that residual cancer burden (RCB)—categorized into four classes, RCB-0 or pathologic complete response, RCB-1, RCB-2, and RCB-3—is predictive of 10-year relapse-free survival with neoadjuvant chemotherapy in triple negative, ER-positive, and HER2-positive tumors.313 Patients who experience progression of disease during neoadjuvant che-motherapy have the poorest survival.314,315 This means that while patients who achieve a pCR will have a better prognosis based on their response to neoadjuvant chemotherapy. Equally other patients will have a poorer prognosis compared to when they started neoadjuvant therapy based on the nonresponse to treat-ment. Consequently,

1	their response to neoadjuvant chemotherapy. Equally other patients will have a poorer prognosis compared to when they started neoadjuvant therapy based on the nonresponse to treat-ment. Consequently, the FDA has supported the use of the neo-adjuvant platform and pathologic response rates as an endpoint for mechanism of accelerated approval for new agents in high risk early stage breast cancer, though the short-term endpoints (i.e., pCR) have not been shown to correlate with long-term out-comes (i.e., disease free survival and overall survival).Current NCCN recommendations for treatment of oper-able advanced local-regional breast cancer are neoadjuvant chemotherapy with an anthracycline-containing or taxane-containing regimen or both, followed by mastectomy or lumpec-tomy with axillary lymph node dissection if necessary, followed by adjuvant radiation therapy. For patients with HER2-positive breast cancer, trastuzumab and pertuzumab can be combined with chemotherapy in the preoperative

1	node dissection if necessary, followed by adjuvant radiation therapy. For patients with HER2-positive breast cancer, trastuzumab and pertuzumab can be combined with chemotherapy in the preoperative setting to increase patho-logic complete response rates. For inoperable stage IIIA and for stage IIIB breast cancer, neoadjuvant chemotherapy is used to decrease the local-regional cancer burden. This may then permit subsequent modified radical or radical mastectomy, which is followed by adjuvant radiation therapy.Nodal Evaluation in Patients Receiving Neoadjuvant Chemotherapy. The management of the axilla after neoadjuvant chemotherapy has not been specifically addressed in randomized trials. Standard practice has been to perform an axillary lymph node dissection after chemotherapy or to perform a sentinel lymph node dissection before chemotherapy for nodal staging before chemotherapy is initiated. A number of small single-institution studies, one multicenter study, and a recent

1	to perform a sentinel lymph node dissection before chemotherapy for nodal staging before chemotherapy is initiated. A number of small single-institution studies, one multicenter study, and a recent meta-analysis have explored the use of SLN dissection at the completion of chemo-therapy. The published results from these studies have demon-strated the feasibility of SLN dissection in breast cancer patients after neoadjuvant chemotherapy. A review of 14 studies with 818 patients showed a false negative rate of 11% with an overall accu-racy of 94%.280,281,316 While SLN dissection has been accepted for assessment of the axilla in the clinically node-negative axilla after neoadjuvant chemotherapy, clinicians have been slower to adopt this approach for axillary staging after chemotherapy in patients who started with initial node-positive disease. Several clinical Brunicardi_Ch17_p0541-p0612.indd 59501/03/19 5:05 PM 596SPECIFIC CONSIDERATIONSPART IItrials have been performed to evaluate

1	who started with initial node-positive disease. Several clinical Brunicardi_Ch17_p0541-p0612.indd 59501/03/19 5:05 PM 596SPECIFIC CONSIDERATIONSPART IItrials have been performed to evaluate the accuracy of SLN dis-section in patients with documented axillary metastases at initial presentation, including ACOSOG Z1071, SENTINA, and SN FNAC. ACOSOG Z1071 (Alliance) analyzed women with clini-cal T0-T4, N1-N2, M0 breast cancer who underwent both SLN surgery and axillary lymph node dissection (ALND).317 The pri-mary endpoint was the false-negative rate (FNR) of SLN surgery after chemotherapy with clinically node-positive disease with a prespecified endpoint of 10% considered to be an acceptable rate. However, the FNR was found to be 12.6%, though it was lower when dual-agent mapping technique was used and at least three or more SLNs removed.317 The SENTINA and SN FNAC trials had findings similar to Z1071. The results from Z1071 were further analyzed to determine if a clip was placed in

1	was used and at least three or more SLNs removed.317 The SENTINA and SN FNAC trials had findings similar to Z1071. The results from Z1071 were further analyzed to determine if a clip was placed in the positive node at initial diagnosis and if the clipped node location at surgery (SLN or ALND) was evaluated. Indeed, this showed that identification of the clipped node during the surgical procedure further decreased the FNR.318 The results from the ACOSOG Z1071 (Alliance) trial, in cases presenting with cN1 disease and at least two SLN resec-tions and clipped node was within the SLN specimen, showed that the FNR was 6.8%.318 Caudle et al at MD Anderson Cancer Center performed a prospective study of patients with biopsy-confirmed nodal metastases with a clip placed in the biopsy-proven lymph node, who were treated with neoadjuvant chemotherapy; at the time of surgery these patients underwent SLN dissection with targeting and removal of the clipped node (targeted axillary dis-section

1	node, who were treated with neoadjuvant chemotherapy; at the time of surgery these patients underwent SLN dissection with targeting and removal of the clipped node (targeted axillary dis-section [TAD]).319 TAD includes SLN surgery and selective local-ization and removal of the clipped node, with the goal to determine if pathologic changes in the clipped node accurately reflect the status of the nodal basin, and proposing that TAD improves the FNR compared to SLN surgery alone.319 In patients undergoing SLN surgery and ALND (n = 118), the FNR was 10.1% (95% CI, 4.2–19.8), and adding evaluation of the clipped node reduced the FNR to 1.4% (95% CI, 0.03–7.3; P = .03). TAD followed by ALND was performed in 85 patients, with an FNR of 2.0% (1 of 50; 95% CI, 0.05–10.7).319 Although the use of dual tracer tech-nique, retrieval of three or more SLNs, and TAD improve axillary staging after neoadjuvant chemotherapy, there is no long-term data about the oncologic safety of omitting ALND in

1	of dual tracer tech-nique, retrieval of three or more SLNs, and TAD improve axillary staging after neoadjuvant chemotherapy, there is no long-term data about the oncologic safety of omitting ALND in patients who con-vert from cN1 to cN0 disease at this time.Neoadjuvant Endocrine Therapy. While initially used in elderly women who were deemed poor candidates for surgery or cytotoxic chemotherapy, neoadjuvant endocrine therapy is being increasingly evaluated in clinical trials. As clinicians have gained experience with neoadjuvant treatment strategies, it is now clear from examination of predictors of complete pathologic response that ER-positive tumors do not shrink in response to chemotherapy as readily as ER-negative tumors.320 Indeed, the pCR rate in ER-negative tumors is approximately three times that of ER-positive tumors. Fisher et al examined the results of the NSABP B-14 and B-20 trials and found that, as age increased, women obtained less benefit from chemo-therapy. They

1	three times that of ER-positive tumors. Fisher et al examined the results of the NSABP B-14 and B-20 trials and found that, as age increased, women obtained less benefit from chemo-therapy. They recommended that factors214 including tumor estrogen receptor concentration, nuclear grade, histologic grade, tumor type, and markers of proliferation should be considered in these patients before choosing between the use of chemotherapy and hormonal therapy. If in fact the tumor is estrogen-receptor rich, these patients may benefit more from endocrine therapy in the neoadjuvant setting than they might if they received stan-dard chemotherapy. Neoadjuvant endocrine therapy has been shown to shrink tumors, enabling breast-conserving surgery in women with hormone receptor-positive disease who otherwise would have to be treated with mastectomy, although long-term recurrence rates have not been reported.265 The IMPACT trial evaluated neoadjuvant use of tamoxifen or anastrozole or both in

1	otherwise would have to be treated with mastectomy, although long-term recurrence rates have not been reported.265 The IMPACT trial evaluated neoadjuvant use of tamoxifen or anastrozole or both in combination in postmenopausal women with ER-positive operable or locally advanced breast cancer.321 While there were no significant differences in objective tumor response among tamoxifen, anastrozole, or a combination of the two, in patients who were initially deemed as mastectomy candidates, only 31% had breast-conserving surgery with tamoxifen, whereas 44% underwent breast-conserving surgery with anastrozole. Invasive lobular cancers in particular have been shown to respond poorly to neoadjuvant chemotherapy and may have better response to neoadjuvant endocrine therapy.322-324 A meta-analysis evaluating the response rate and rate of breast conservation surgery with the use of neoadjuvant endocrine therapy compared to combi-nation chemotherapy was recently reported. This meta-analysis

1	evaluating the response rate and rate of breast conservation surgery with the use of neoadjuvant endocrine therapy compared to combi-nation chemotherapy was recently reported. This meta-analysis included nearly 3500 patients across 20 studies.325 Interestingly, aromatase inhibitors had a similar response, and breast conserva-tion rates in comparison with combination chemotherapy albeit with lower toxicity suggest that neoadjuvant endocrine therapy is an appropriate alternative in ER-positive breast cancers. However, the incidence of complete pathological response was low (<10%) with both approaches. Also, aromatase inhibitors were associated with significantly higher response and breast conservation rates compared with tamoxifen. The ALTER-NATE (Alternate Approaches for Clinical Stage II or III Estro-gen Receptor Positive Breast Cancer Neoadjuvant Treatment in Postmenopausal Women) trial is currently evaluating neo-adjuvant endocrine therapy with fulvestrant or anastrozole or in

1	II or III Estro-gen Receptor Positive Breast Cancer Neoadjuvant Treatment in Postmenopausal Women) trial is currently evaluating neo-adjuvant endocrine therapy with fulvestrant or anastrozole or in combination.Increasing knowledge of secondary resistance mecha-nisms to endocrine therapy and cross talk between ER and the PI3K/Akt/mTOR pathway have led to the evaluation of PI3K pathway inhibitors in combination with endocrine therapy. Post-menopausal women with ER-positive early breast cancers were treated with letrozole or letrozole in combination with everoli-mus, a mTOR inhibitor, in a randomized, phase 2 clinical trial. Clinical response and antiproliferative response, characterized by reduction in Ki67, was superior in the combination arm, sug-gesting that everolimus can increase efficacy of neoadjuvant letrozole.326 The LORLEI study is evaluating the use of taselisib, a PI3K inhibitor in combination with letrozole compared with letrozole alone. With the approval of CDK 4/6

1	efficacy of neoadjuvant letrozole.326 The LORLEI study is evaluating the use of taselisib, a PI3K inhibitor in combination with letrozole compared with letrozole alone. With the approval of CDK 4/6 inhibitors in the metastatic setting, clinical trials are evaluating the use of CDK inhibitors in combination with neoadjuvant endocrine therapy. Neoadjuvant anastrozole in combination with palbociclib, a CDK4/6 inhibitor, has been shown to significantly reduce Ki67, suggesting that CDK4/6 inhibition can increase the efficacy of neoadjuvant endocrine therapy.With the use of neoadjuvant chemotherapy or endocrine therapy, observation of the response of the intact tumor and/or nodal metastases to a specific regimen could ultimately help to define which patients will benefit from specific therapies in the adjuvant setting. In adjuvant trials the primary endpoint is typi-cally survival, whereas in neoadjuvant trials the endpoints have more often been clinical or pathologic response rates. There

1	in the adjuvant setting. In adjuvant trials the primary endpoint is typi-cally survival, whereas in neoadjuvant trials the endpoints have more often been clinical or pathologic response rates. There are a number of clinical trials underway comparing neoadjuvant chemotherapy and endocrine therapy regimens with pretreat-ment and posttreatment biopsy samples obtained from the pri-mary tumors in all of the participants. These samples are being subjected to intensive genomic and proteomic analyses that may Brunicardi_Ch17_p0541-p0612.indd 59601/03/19 5:05 PM 597THE BREASTCHAPTER 17help to define a more personalized or individualized approach to breast cancer treatment in the future.Antiestrogen TherapyTamoxifen. Within the cytosol of breast cancer cells are spe-cific proteins (receptors) that bind and transfer steroid moieties into the cell nucleus to exert specific hormonal effects.308,327-331 The most widely studied hormone receptors are the estrogen receptor and progesterone

1	that bind and transfer steroid moieties into the cell nucleus to exert specific hormonal effects.308,327-331 The most widely studied hormone receptors are the estrogen receptor and progesterone receptor. Hormone receptors are detectable in >90% of well-differentiated ductal and lobular invasive cancers. Although the receptor status may remain the same between the primary cancer and metastatic disease in the same patient in the majority of cases, there are instances where the status is changed in the metastatic focus; therefore, biopsy of newly diagnosed metastatic disease should be considered for assessment of hormone receptor and HER2 status.After binding to estrogen receptors in the cytosol, tamoxi-fen blocks the uptake of estrogen by breast tissue. Clini-cal responses to antiestrogen are evident in >60% of women with hormone receptor-positive breast cancers but in <10% of women with hormone receptor-negative breast cancers. A meta-analysis by the Early Breast Cancer Trialists’

1	are evident in >60% of women with hormone receptor-positive breast cancers but in <10% of women with hormone receptor-negative breast cancers. A meta-analysis by the Early Breast Cancer Trialists’ Collabora-tive Group showed that adjuvant therapy with tamoxifen for 5 years reduced breast cancer mortality by about a third through the first 15 years of follow-up.14 This mortality benefit contin-ues to be statistically significant in the second and third 5-year periods (i.e., years 5–9 and 10–15) when the patients are no longer receiving endocrine treatment—the so-called carry-over effect. The analysis also showed a 39% reduction in the risk of cancer in the contralateral breast. The antiestrogens do have defined toxicity, including bone pain, hot flashes, nausea, vom-iting, and fluid retention. Thrombotic events occur in <3% of treated women. Cataract surgery is more frequently performed in patients receiving tamoxifen. The Stockholm trial showed that 5 years of tamoxifen was associated

1	Thrombotic events occur in <3% of treated women. Cataract surgery is more frequently performed in patients receiving tamoxifen. The Stockholm trial showed that 5 years of tamoxifen was associated with a significant reduction in locoregional recurrences and distant metastasis in postmenopausal women with ER-positive breast cancer.332 However, an increase in endometrial cancers was observed with long-term tamoxifen use. The NSABP B14 trial evaluated 10 years of tamoxifen compared to 5 years.333 However, the study was terminated based on interim analyses indicating no addi-tional benefit from tamoxifen beyond 5 years. The ATLAS trial also evaluated the use of tamoxifen for 5 years vs. 10 years in nearly 13,000 women across the world. This study showed that continuing tamoxifen for 10 years vs. 5 years produced a significant reduction in recurrence and mortality.334 Interestingly, the benefit was not seen in the second 5 years (i.e., years 5–9) while the patients were on treatment, but

1	5 years produced a significant reduction in recurrence and mortality.334 Interestingly, the benefit was not seen in the second 5 years (i.e., years 5–9) while the patients were on treatment, but it was seen from years 10 to 15. One reason the NSABP B14 study was led to conclude that 10 years of tamoxifen was not beneficial was that the follow-up time was shorter. Results of the ATLAS study were also corroborated by the aTTom study. Similarly, extended adjuvant therapy with letrozole after 5 years of tamoxifen was shown to improve disease-free survival without improvement in overall survival except in node-positive patients.335Tamoxifen therapy is also considered for women with DCIS that is found to be ER-positive. The goals of such ther-apy are to decrease the risk of an ipsilateral recurrence after breast conservation therapy for DCIS and to decrease the risk of a primary invasive breast cancer or a contralateral breast cancer event. Consequently, tamoxifen is not recommended for

1	after breast conservation therapy for DCIS and to decrease the risk of a primary invasive breast cancer or a contralateral breast cancer event. Consequently, tamoxifen is not recommended for patients who have had bilateral mastectomies with ER-positive DCIS. With the use of aromatase inhibitors in postmenopausal women, use of adjuvant tamoxifen has increasingly been limited to premenopausal women.Aromatase Inhibitors. In postmenopausal women, aromatase inhibitors are now considered first-line therapy in the adjuvant setting. Currently, three third-generation aromatase inhibitors are approved for clinical use: the reversible nonsteroidal inhibitors anastrozole and letrozole and the irreversible steroidal inhibitor exemestane. While all the aromatase inhibitors have been shown to have similar efficacy with a similar spectrum of adverse effects, the Early Breast Cancer Trialists’ Collaborative Group meta-analyses of 31,920 postmenopausal women with ER-positive early breast cancers

1	similar efficacy with a similar spectrum of adverse effects, the Early Breast Cancer Trialists’ Collaborative Group meta-analyses of 31,920 postmenopausal women with ER-positive early breast cancers treated with tamoxifen or aroma-tase inhibitors demonstrated that 5 years of aromatase inhibitors reduced the rate of recurrence by 30% and 10-year breast cancer mortality by about 15% compared to 5 years of tamoxifen.336-339 The NSABP B42 study evaluated whether an additional 5 years of letrozole improved disease-free survival in postmenopausal women who have completed 5 years of tamoxifen or an aromatase inhibitor. After a median follow-up of 6.9 years, while extended letrozole significantly improved breast cancer-free interval, no improvement in disease-free survival, the primary endpoint, was observed. Recently, the results of the MA-17R study, designed to assess the efficacy of adjuvant letrozole for 10 years, were reported.340 Similar to NSABP B42, extended letrozole improved

1	was observed. Recently, the results of the MA-17R study, designed to assess the efficacy of adjuvant letrozole for 10 years, were reported.340 Similar to NSABP B42, extended letrozole improved disease-free survival without significant improvement in overall survival. Patients who are node-positive, have received adjuvant chemotherapy, with prior receipt of tamoxifen are likely to ben-efit from long-term use of an aromatase inhibitor.The aromatase inhibitors are less likely than tamoxifen to cause endometrial cancer but do lead to changes in bone mineral density that may result in osteoporosis and an increased rate of fractures in postmenopausal women. The risk of osteoporosis can be averted by treatment with bisphosphonates. Joint pains are a side effect that affects a significant number of patients. Node-negative and node-positive breast cancer patients whose tumors express hormone receptors should be considered for endocrine therapy in the adjuvant setting. Women with hormone

1	of patients. Node-negative and node-positive breast cancer patients whose tumors express hormone receptors should be considered for endocrine therapy in the adjuvant setting. Women with hormone receptor–positive cancers achieve significant reduction in risk of recurrence of breast cancer and mortality from breast cancer through the use of endocrine therapies.For postmenopausal women with ER-positive, HER2-negative, metastatic breast cancer, available endocrine thera-pies include nonsteroidal aromatase inhibitors (anastrozole and letrozole); steroidal aromatase inhibitors (exemestane); serum ER modulators (tamoxifen or toremifene); ER down-regulators (fulvestrant); progestin (megestrol acetate); androgens (fluoxymesterone); and high-dose estrogen (ethinyl estradiol). A third generation nonsteroidal aromatase inhibitor or palbo-ciclib, the CDK 4/6 inhibitor, in combination with letrozole may be considered as a treatment option for first-line therapy. Activation of CDK4/CDK6 cell cycle

1	aromatase inhibitor or palbo-ciclib, the CDK 4/6 inhibitor, in combination with letrozole may be considered as a treatment option for first-line therapy. Activation of CDK4/CDK6 cell cycle signaling axis has been implicated in mediating endocrine resistance. Consequently, PALOMA-1 evaluated the safety and efficacy of palbociclib in combination with letrozole vs. letrozole alone as first-line treat-ment for patients with ER-positive, HER2-negative advanced breast cancer. Median progression-free survival (PFS) was doubled with the combination compared to letrozole alone (20.2 months vs. 10.2 months for the letrozole).341 Based on this, the FDA approved palbociclib in combination with letrozole for the treatment of postmenopausal women with ER-positive, HER2-negative advanced breast cancer as initial treatment. The Brunicardi_Ch17_p0541-p0612.indd 59701/03/19 5:05 PM 598SPECIFIC CONSIDERATIONSPART IIbenefit of palbociclib in combination with letrozole was sub-sequently confirmed in

1	as initial treatment. The Brunicardi_Ch17_p0541-p0612.indd 59701/03/19 5:05 PM 598SPECIFIC CONSIDERATIONSPART IIbenefit of palbociclib in combination with letrozole was sub-sequently confirmed in a phase 3 trial (PFS 24.8 months vs. 14.5 months for letrozole).342 Two additional CDK4/6 inhibitors, ribociclib and abemaciclib, have been approved for use in com-bination with endocrine therapy for patients with hormone receptor–positive advanced breast cancer.On the other hand, PALOMA-3 compared the combina-tion of palbociclib and fulvestrant to fulvestrant alone in preor postmenopausal ER-positive, HER2-negative metastatic breast cancer patients, whose disease progressed on prior endocrine therapy. Premenopausal women also received the GNRH ago-nist, goserelin. The median PFS was 9.2 months for the combi-nation compared to 3.8 months with fulvestrant alone.343 Thus, fulvestrant with palbociclib is a potential option for women with metastatic breast cancer who have progressed on prior

1	for the combi-nation compared to 3.8 months with fulvestrant alone.343 Thus, fulvestrant with palbociclib is a potential option for women with metastatic breast cancer who have progressed on prior endo-crine therapy. Additionally, abemaciclib in combination with fulvestrant or as single agent is approved for use in ER-posi-tive advanced breast cancers previously treated with endocrine therapy.In premenopausal women with stage IV ER-positive breast cancer without previous exposure to endocrine therapy, initial treatment with tamoxifen or ovarian suppression/ablation plus aromatase inhibitor with or without CDK4/6 inhibitors are reasonable options.Activation of the PI3K/mammalian target of rapamycin (mTOR) signal transduction pathway has also been implicated in secondary resistance to estrogen targeting. BOLERO-2 eval-uated the use of exemestane in combination with everolimus in postmenopausal women with ER-positive tumors who had progressed or recurred on a nonsteroidal aromatase

1	estrogen targeting. BOLERO-2 eval-uated the use of exemestane in combination with everolimus in postmenopausal women with ER-positive tumors who had progressed or recurred on a nonsteroidal aromatase inhibitor.344 An improvement in PFS was observed with combination com-pared to exemestane alone (11 vs. 4.1 months) leading to FDA approval. Similar improvement in PFS was observed with a combination of tamoxifen and everolimus.345 However, a phase 3 trial of letrozole in combination with temsirolimus, an mTOR inhibitor, did not show any improvement in PFS in aromatase inhibitor–naive metastatic postmenopausal women.346 Trials evaluating the adjuvant use of mTOR inhibitors and CDK 4/6 inhibitors are currently in progress.Women whose tumors respond to an endocrine therapy with either shrinkage of their breast cancer (objective response) or long-term stabilization of disease (stable disease) are con-sidered to represent “clinical benefit” and should receive addi-tional endocrine therapy at

1	of their breast cancer (objective response) or long-term stabilization of disease (stable disease) are con-sidered to represent “clinical benefit” and should receive addi-tional endocrine therapy at the time of progression because their chances of a further response remain high.294-296 Patients whose tumors progress de novo on an endocrine agent have a low rate of clinical benefit (<20%) to subsequent endocrine therapy; the choice of endocrine or chemotherapy should be considered based on the disease site and extent as well as the patient’s general condition and treatment preference.294The adjuvant use of aromatase inhibitors and recent advances in tumor genome sequencing technologies have enabled the identification of secondary ESR1 mutations.347,348 These mutations, typically present in the ligand binding domains, lead to ligand-independent activation of the receptor, mediate resistance to aromatase inhibitors, and are associated with shorter survival.349 Reported incidence of these

1	the ligand binding domains, lead to ligand-independent activation of the receptor, mediate resistance to aromatase inhibitors, and are associated with shorter survival.349 Reported incidence of these mutations are variable (20%–30%) based on prior exposure to aroma-tase inhibitors and are uncommon in primary breast cancers. Clinical trials evaluating novel selective estrogen receptor degraders with potential activity against these mutations are in progress.Ablative Endocrine TherapyIn the past, adrenalectomy and/or hypophysectomy were the pri-mary endocrine modalities used to treat metastatic breast cancer, but today these approaches are seldom used. In women who are premenopausal at diagnosis, ovarian ablation can be accomplished by oophorectomy or ovarian radiation. Ovarian suppression can be accomplished by the use of gonadotrophin-hormone releasing hormone agonists, such as goserelin or leuprolide. Evaluation of the combination of goserelin with tamoxifen vs.

1	Ovarian suppression can be accomplished by the use of gonadotrophin-hormone releasing hormone agonists, such as goserelin or leuprolide. Evaluation of the combination of goserelin with tamoxifen vs. cyclophospha-mide/methotrexate/fluorouracil chemotherapy in premenopausal ER-positive early-stage breast cancers showed that relapse-free survival was superior with endocrine therapy combination, with a similar trend in overall survival.350 Data from the SOFT and TEXT trials on adjuvant endocrine therapy show that exemes-tane plus ovarian suppression significantly reduces recurrences as compared with tamoxifen plus ovarian suppression.351,352 In these trials, ovarian suppression was achieved with the use of the gonadotropin-releasing hormone agonist triptorelin, oopho-rectomy, or ovarian irradiation. The disease-free survival was 89% in the tamoxifen plus ovarian suppression group, while it was 93% in exemestane plus ovarian suppression group; how-ever, there was no significant

1	irradiation. The disease-free survival was 89% in the tamoxifen plus ovarian suppression group, while it was 93% in exemestane plus ovarian suppression group; how-ever, there was no significant differences in overall survival. In the SOFT trial, while tamoxifen plus ovarian suppression was not superior to tamoxifen alone in terms of disease-free survival, improved outcomes were observed in ovarian suppression in women with a high risk of recurrence. In women who received no adjuvant chemotherapy, no meaningful benefit was obtained with ovarian suppression. Thus, ovarian suppression in combi-nation with an aromatase inhibitor can be considered in select premenopausal women with high-risk features (age <40 years, positive lymph nodes) who warranted adjuvant chemotherapy.Anti-HER2 TherapyThe determination of tumor HER-2 expression or gene ampli-fication for all newly diagnosed patients with breast cancer is now recommended.353-356 It is used to assist in the selection of adjuvant

1	determination of tumor HER-2 expression or gene ampli-fication for all newly diagnosed patients with breast cancer is now recommended.353-356 It is used to assist in the selection of adjuvant chemotherapy in both node-negative and node-positive patients. Trastuzumab was initially approved for the treatment of HER2/neu-positive breast cancer in patients with metastatic disease. Once efficacy was demonstrated for patients with metastatic disease, the NSABP and the North Central Cancer Treatment Group conducted phase 3 trials that evaluated the impact of adjuvant trastuzumab therapy in patients with early-stage breast cancer. After approval from the FDA, these groups amended their adjuvant trastuzumab trials (B-31 and N9831, respectively), to provide for a joint efficacy analysis. The first joint interim efficacy analysis demonstrated an improvement in 3-year disease-free survival from 75% in the control arm to 87% in the trastuzumab arm (hazard ratio = 0.48, P <.0001). There was an

1	joint interim efficacy analysis demonstrated an improvement in 3-year disease-free survival from 75% in the control arm to 87% in the trastuzumab arm (hazard ratio = 0.48, P <.0001). There was an accompanying 33% reduction in mortality in the patients who received trastuzumab (hazard ratio = 0.67, P = 0.015). The magnitude of reduction in hazard for disease-free survival events crossed prespecified early reporting boundaries, so the data-monitoring committees for both groups recommended that randomized accrual to the trials be ended, and the results were subsequently published.181While anthracycline-based adjuvant chemotherapy was considered preferable in HER2-positive breast cancer, the BCIRG 006 compared the use of anthracycline with taxane and trastuzumab (AC-TH) versus taxane, carboplatin chemotherapy with trastuzumab (TCH).182 With 10 years of follow-up, no statistical significance with regard to disease-free and overall Brunicardi_Ch17_p0541-p0612.indd 59801/03/19 5:05 PM

1	chemotherapy with trastuzumab (TCH).182 With 10 years of follow-up, no statistical significance with regard to disease-free and overall Brunicardi_Ch17_p0541-p0612.indd 59801/03/19 5:05 PM 599THE BREASTCHAPTER 17survival was observed for anthracycline-based chemotherapy. While anthracycline chemotherapy was numerically superior, this was accompanied by an increase in the incidence of leu-kemia and congestive heart failure. A year of adjuvant trastu-zumab is considered standard of care. Two years of adjuvant trastuzumab has been shown to be more effective, although it is associated with more toxicity than 1 year of trastuzumab.357 On the other hand, the PHARE trial examined 6 months vs. stan-dard 12 months of trastuzumab. After 3.5 years of follow-up, the study failed to demonstrate that 6 months was noninferior com-pared to the standard therapy.358 Patients with HER2-positive tumors benefit if trastuzumab is added to taxane chemotherapy. Because of overlapping cardiotoxicities,

1	that 6 months was noninferior com-pared to the standard therapy.358 Patients with HER2-positive tumors benefit if trastuzumab is added to taxane chemotherapy. Because of overlapping cardiotoxicities, trastuzumab is not usu-ally given concurrently with anthracyclines.Buzdar and colleagues reported the results of a random-ized neoadjuvant trial of trastuzumab in combination with sequential paclitaxel followed by FEC-75 (5-fluorouracil, epi-rubicin, cyclophosphamide) vs. the same chemotherapy regimen without trastuzumab in 42 women with early-stage operable breast cancer. The pathologic complete response rates in this trial increased from 25% to 66.7% when chemotherapy was given concurrently with trastuzumab.301 A subsequent report that included additional patients treated with concurrent chemo-therapy and trastuzumab further confirmed the high pathologic complete response rates and continued to show that cardiac function was preserved.302While novel agents have been approved for the

1	chemo-therapy and trastuzumab further confirmed the high pathologic complete response rates and continued to show that cardiac function was preserved.302While novel agents have been approved for the treatment of women with metastatic HER2-positive breast cancers, cur-rently trastuzumab is the only HER2-targeted agent approved for use in the adjuvant setting. Lapatinib is a dual tyrosine kinase inhibitor that targets both HER2 and EGFR. It was approved for use with capecitabine in patients with HER2-positive meta-static disease. Adjuvant lapatinib was shown to be inferior to trastuzumab, and the combination of lapatinib with trastuzumab did yield a significant improvement in disease-free survival compared to trastuzumab alone. Ado-trastuzumab emtansine (T-DM1) is approved for HER2-positive metastatic breast cancer patients who have previously received trastuzumab and a taxane either separately or in combination. T-DM1 is an antibody drug conjugate that incorporates the HER2 targeted

1	metastatic breast cancer patients who have previously received trastuzumab and a taxane either separately or in combination. T-DM1 is an antibody drug conjugate that incorporates the HER2 targeted activity of trastuzumab with the cytotoxic activity of DM1, a microtubule inhibitory agent leading to apoptosis.359Pertuzumab is a humanized monoclonal antibody that binds at a different epitope of the HER2 extracellular domain (subdomain II) and prevents dimerization of HER2 with other members of the family, primarily HER3. In the metastatic setting, it is approved in combination with trastuzumab and docetaxel for patients with metastatic HER2-positive breast cancer who have not received prior HER2-targeted therapy or chemotherapy for metastatic disease.360 In the neoadjuvant setting, pertuzumab is approved in combination with trastu-zumab and docetaxel in HER2-positive, early stage breast cancers that are greater than 2 cm or node-positive. However, this approval is based on improvement in

1	is approved in combination with trastu-zumab and docetaxel in HER2-positive, early stage breast cancers that are greater than 2 cm or node-positive. However, this approval is based on improvement in pathologic complete response rate, and not data based on improvement in event free or overall survival.361,362 In the NeoSphere trial, neoadjuvant use of pertuzumab with trastuzumab and docetaxel led to nearly a 17% increase in pathologic complete response in the breast (P = .0141).361 While in the TRYPHAENA study, pathologic complete responses ranging from 57% to 66% were observed with neoadjuvant pertuzumab and trastuzumab combination given with anthracycline-containing or nonanthracycline-containing chemotherapy.362 With the use of dual antibody therapy, cur-rently there is significant interest in identifying patients who can avoid chemotherapy and potentially be treated with HER2-targeted agents alone. The NeoSphere study showed 27% pathologic complete response in HER2-positive,

1	interest in identifying patients who can avoid chemotherapy and potentially be treated with HER2-targeted agents alone. The NeoSphere study showed 27% pathologic complete response in HER2-positive, ER-negative, breast cancer patients treated with pertuzumab and trastuzumab alone. Pertuzumab was recently FDA approved in combination with trastuzumab and chemotherapy in the adjuvant setting in HER2 amplified breast cancers with high risk of recurrence. Approval is based on APHINITY trial showing that the addition of pertuzumab improved invasive disease free survival (7.1%) compared to placebo (8.7%) (HR 0.82, 95% CI: 0.67, 1.00; p = 0.047). Overall survival data is not mature.The ExteNET study evaluated the use of neratinib, an irreversible inhibitor of EGFR, HER2, and HER4, in HER2-positive early stage patients who have completed adjuvant trastuzumab. A year of neratinib after completion of chemo-therapy and trastuzumab-based adjuvant therapy significantly improved 2-year disease-free

1	early stage patients who have completed adjuvant trastuzumab. A year of neratinib after completion of chemo-therapy and trastuzumab-based adjuvant therapy significantly improved 2-year disease-free survival, the primary endpoint.363 After two years, invasive disease free survival was 94.2% in patients treated with neratinib compared with 91.9% in those receiving placebo (HR 0.66; 95% CI: 0.49, 0.90, p = 0.008) leading to FDA approval for HER2 amplified breast cancers following a year of adjuvant trastuzumab.In addition to amplifications or copy number alterations, activating mutations or single nucleotide variants in HER2 have been described (2%).364 Typically observed in ER-positive breast cancers, a higher prevalence of HER2 mutations have been reported in invasive lobular carcinomas, particularly in the pleomorphic subtype.365 These mutations, usually exclusive with HER2 amplification, are observed in kinase or extracellular domains and predict for responses or resistance to

1	particularly in the pleomorphic subtype.365 These mutations, usually exclusive with HER2 amplification, are observed in kinase or extracellular domains and predict for responses or resistance to HER2-targeting agents.366,367 A phase 2 trial of neratinib in HER2-mutated meta-static breast cancers showed a clinical benefit rate of 36% with one complete response and one partial response in a heavily pre-treated population. A clinical trial evaluating the combination of neratinib with fulvestrant, in HER2-mutated, ER-positive breast cancers, is in progress.SPECIAL CLINICAL SITUATIONSNipple DischargeUnilateral Nipple Discharge. Nipple discharge is a finding that can be seen in a number of clinical situations. It may be suggestive of cancer if it is spontaneous, unilateral, localized to a single duct, present in women ≥40 years of age, bloody, or associated with a mass. A trigger point on the breast may be present so that pressure around the nipple-areolar complex induces discharge from a

1	duct, present in women ≥40 years of age, bloody, or associated with a mass. A trigger point on the breast may be present so that pressure around the nipple-areolar complex induces discharge from a single duct. In this circumstance, mammography and ultrasound are indicated for further evalu-ation. A ductogram also can be useful and is performed by can-nulating a single discharging duct with a small nylon catheter or needle and injecting 1.0 mL of water-soluble contrast solu-tion. Nipple discharge associated with a cancer may be clear, bloody, or serous. Testing for the presence of hemoglobin is helpful, but hemoglobin may also be detected when nipple dis-charge is secondary to an intraductal papilloma or duct ecta-sia. Definitive diagnosis depends on excisional biopsy of the offending duct and any associated mass lesion. A 3.0 lacrimal duct probe can be used to identify the duct that requires exci-sion. Another approach is to inject methylene blue dye within

1	of the offending duct and any associated mass lesion. A 3.0 lacrimal duct probe can be used to identify the duct that requires exci-sion. Another approach is to inject methylene blue dye within Brunicardi_Ch17_p0541-p0612.indd 59901/03/19 5:05 PM 600SPECIFIC CONSIDERATIONSPART IIthe duct after ductography. The nipple must be sealed with collodion or a similar material so that the blue dye does not discharge through the nipple but remains within the distended duct facilitating its localization. Localization with a wire or seed is performed when there is an associated mass that lies >2.0 to 3.0 cm from the nipple.Bilateral Nipple Discharge. Nipple discharge is suggestive of a benign condition if it is bilateral and multiductal in origin, occurs in women ≤39 years of age, or is milky or blue-green. Prolactin-secreting pituitary adenomas are responsible for bilat-eral nipple discharge in <2% of cases. If serum prolactin levels are repeatedly elevated, plain radiographs of the

1	or blue-green. Prolactin-secreting pituitary adenomas are responsible for bilat-eral nipple discharge in <2% of cases. If serum prolactin levels are repeatedly elevated, plain radiographs of the sellaturcica are indicated, and thin section CT scan is required. Optical nerve compression, visual field loss, and infertility are associated with large pituitary adenomas.Axillary Lymph Node Metastases in the Setting of an Unknown Primary CancerA woman who presents with an axillary lymph node metasta-sis that is consistent with a breast cancer metastasis has a 90% probability of harboring an occult breast cancer.303 However, axillary lymphadenopathy is the initial presenting sign in only 1% of breast cancer patients. Fine-needle aspiration biopsy or core-needle biopsy can be used to establish the diagnosis when an enlarged axillary lymph node is identified. When metastatic cancer is found, immunohistochemical analysis may classify the cancer as epithelial, melanocytic, or lymphoid in origin.

1	diagnosis when an enlarged axillary lymph node is identified. When metastatic cancer is found, immunohistochemical analysis may classify the cancer as epithelial, melanocytic, or lymphoid in origin. The presence of hormone receptors (estrogen or progesterone receptors) suggests metastasis from a breast cancer but is not diagnostic. The search for a primary cancer includes careful examination of the thyroid, breast, and pelvis, including the rectum. The breast should be examined with diagnostic mam-mography, ultrasonography, and MRI to evaluate for an occult primary lesion. Further radiologic and laboratory studies should include chest radiography and liver function studies. Additional imaging of the chest, abdomen, and skeleton may be indicated if the extent of nodal involvement is consistent with stage III breast cancer. Suspicious findings on mammography, ultra-sonography, or MRI necessitate breast biopsy. When a breast cancer is found, treatment consists of an axillary lymph node

1	with stage III breast cancer. Suspicious findings on mammography, ultra-sonography, or MRI necessitate breast biopsy. When a breast cancer is found, treatment consists of an axillary lymph node dissection with a mastectomy or preservation of the breast fol-lowed by whole-breast radiation therapy. Chemotherapy and endocrine therapy should be considered.Breast Cancer During PregnancyBreast cancer occurs in 1 of every 3000 pregnant women, and axillary lymph node metastases are present in up to 75% of these women.368 The average age of the pregnant woman with breast cancer is 34 years. Fewer than 25% of the breast nodules developing during pregnancy and lactation will be cancerous. Ultrasonography and needle biopsy specimens are used in the diagnosis of these nodules. Mammography is rarely indicated because of its decreased sensitivity during pregnancy and lac-tation; however, the fetus can be shielded if mammography is needed. Approximately 30% of the benign conditions encoun-tered will

1	because of its decreased sensitivity during pregnancy and lac-tation; however, the fetus can be shielded if mammography is needed. Approximately 30% of the benign conditions encoun-tered will be unique to pregnancy and lactation (galactoceles, lobular hyperplasia, lactating adenoma, and mastitis or abscess). Once a breast cancer is diagnosed, complete blood count, chest radiography (with shielding of the abdomen), and liver function studies are performed.Because of the potential deleterious effects of radiation therapy on the fetus, radiation cannot be considered until the fetus is delivered. A modified radical mastectomy can be per-formed during the first and second trimesters of pregnancy, even though there is an increased risk of spontaneous abortion after first-trimester anesthesia. During the third trimester, lumpec-tomy with axillary node dissection can be considered if adju-vant radiation therapy is deferred until after delivery. Lactation is suppressed. Chemotherapy

1	During the third trimester, lumpec-tomy with axillary node dissection can be considered if adju-vant radiation therapy is deferred until after delivery. Lactation is suppressed. Chemotherapy administered during the first tri-mester carries a risk of spontaneous abortion and a 12% risk of birth defects. There is no evidence of teratogenicity resulting from administration of chemotherapeutic agents in the second and third trimesters. For this reason, many clinicians now con-sider the optimal strategy to be delivery of chemotherapy in the second and third trimesters as a neoadjuvant approach, which allows local therapy decisions to be made after the delivery of the baby. Pregnant women with breast cancer often present at a later stage of disease because breast tissue changes that occur in the hormone-rich environment of pregnancy obscure early cancers. However, pregnant women with breast cancer have a prognosis, stage by stage, that is similar to that of nonpregnant women with breast

1	the hormone-rich environment of pregnancy obscure early cancers. However, pregnant women with breast cancer have a prognosis, stage by stage, that is similar to that of nonpregnant women with breast cancer.Male Breast CancerFewer than 1% of all breast cancers occur in men.369,370 The inci-dence appears to be highest among North Americans and the British, in whom breast cancer constitutes as much as 1.5% of all male cancers. Jewish and African-American men have the highest incidence. Male breast cancer is preceded by gyneco-mastia in 20% of men. It is associated with radiation exposure, estrogen therapy, testicular feminizing syndromes, and Kline-felter’s syndrome (XXY). Breast cancer is rarely seen in young males and has a peak incidence in the sixth decade of life. A firm, nontender mass in the male breast requires investigation. Skin or chest wall fixation is particularly worrisome.DCIS makes up <15% of male breast cancer, whereas infil-trating ductal carcinoma makes up >85%.

1	mass in the male breast requires investigation. Skin or chest wall fixation is particularly worrisome.DCIS makes up <15% of male breast cancer, whereas infil-trating ductal carcinoma makes up >85%. Special-type cancers, including infiltrating lobular carcinoma, have occasionally been reported. Male breast cancer is staged in the same way as female breast cancer, and stage by stage, men with breast cancer have the same survival rate as women. Overall, men do worse because of the more advanced stage of their cancer (stage II, III or IV) at the time of diagnosis. The treatment of male breast cancer is surgi-cal, with the most common procedure being a modified radical mastectomy. SLN dissection has been shown to be feasible and accurate for nodal assessment in men presenting with a clinically node-negative axilla. Adjuvant radiation therapy is appropriate in cases in which there is a high risk for local-regional recurrence. Approximately 80% of male breast cancers are hormone

1	a clinically node-negative axilla. Adjuvant radiation therapy is appropriate in cases in which there is a high risk for local-regional recurrence. Approximately 80% of male breast cancers are hormone recep-tor–positive, and adjuvant tamoxifen is considered. Systemic che-motherapy is considered for men with hormone receptor-negative cancers and for men with large primary tumors, multiple positive nodes, and locally advanced disease.Phyllodes TumorsThe nomenclature, presentation, and diagnosis of phyllodes tumors (including cystosarcoma phyllodes) have posed many problems for surgeons.371 These tumors are classified as benign, borderline, or malignant. Borderline tumors have a greater potential for local recurrence.Mammographic evidence of calcifications and morpho-logic evidence of necrosis do not distinguish between benign, borderline, and malignant phyllodes tumors. Consequently, it is difficult to differentiate benign phyllodes tumors from the Brunicardi_Ch17_p0541-p0612.indd

1	do not distinguish between benign, borderline, and malignant phyllodes tumors. Consequently, it is difficult to differentiate benign phyllodes tumors from the Brunicardi_Ch17_p0541-p0612.indd 60001/03/19 5:05 PM 601THE BREASTCHAPTER 17malignant variant and from fibroadenomas. Phyllodes tumors are usually sharply demarcated from the surrounding breast tissue, which is compressed and distorted. Connective tissue composes the bulk of these tumors, which have mixed gelati-nous, solid, and cystic areas. Cystic areas represent sites of infarction and necrosis. These gross alterations give the gross cut tumor surface its classical leaf-like (phyllodes) appearance. The stroma of a phyllodes tumor generally has greater cellular activity than that of a fibroadenoma. After microdissection to harvest clusters of stromal cells from fibroadenomas and from phyllodes tumors, molecular biology techniques have shown the stromal cells of fibroadenomas to be either polyclonal or mono-clonal (derived

1	clusters of stromal cells from fibroadenomas and from phyllodes tumors, molecular biology techniques have shown the stromal cells of fibroadenomas to be either polyclonal or mono-clonal (derived from a single progenitor cell), whereas those of phyllodes tumors are always monoclonal.Most malignant phyllodes tumors (Fig. 17-38) contain liposarcomatous or rhabdomyosarcomatous elements rather than fibrosarcomatous elements. Evaluation of the number of mitoses and the presence or absence of invasive foci at the tumor mar-gins may help to identify a malignant tumor. Small phyllodes tumors are excised with a margin of normal-appearing breast tissue. When the diagnosis of a phyllodes tumor with suspicious ABFigure 17-38. A. Malignant phyllodes tumor (cystosarcoma-phyllodes). B. Histologic features of a malignant phyllodes tumor (hematoxylin and eosin stain, ×100).malignant elements is made, reexcision of the biopsy specimen site to ensure complete excision of the tumor with a 1-cm mar-gin of

1	of a malignant phyllodes tumor (hematoxylin and eosin stain, ×100).malignant elements is made, reexcision of the biopsy specimen site to ensure complete excision of the tumor with a 1-cm mar-gin of normal-appearing breast tissue is indicated. Large phyl-lodes tumors may require mastectomy. Axillary dissection is not recommended because axillary lymph node metastases rarely occur.Inflammatory Breast CarcinomaInflammatory breast carcinoma (stage IIIB) accounts for <3% of breast cancers. This cancer is characterized by the skin changes of brawny induration, erythema with a raised edge, and edema (peau d’orange).372 Permeation of the dermal lymph vessels by cancer cells is seen in skin biopsy specimens. There may be an associated breast mass (Fig. 17-39). The clinical differentia-tion of inflammatory breast cancer may be extremely difficult, especially when a locally advanced scirrhous carcinoma invades dermal lymph vessels in the skin to produce peau d’orange and lymphangitis (Table

1	inflammatory breast cancer may be extremely difficult, especially when a locally advanced scirrhous carcinoma invades dermal lymph vessels in the skin to produce peau d’orange and lymphangitis (Table 17-15). Inflammatory breast cancer also may be mistaken for a bacterial infection of the breast. More than 75% of women who have inflammatory breast cancer present with palpable axillary lymphadenopathy, and distant metastases also are frequently present. A PET-CT scan should be considered at the time of diagnosis to rule out concurrent metastatic disease. A report of the SEER program described distant metastases at diagnosis in 25% of white women with inflammatory breast carcinoma.Surgery alone and surgery with adjuvant radiation therapy have produced disappointing results in women with inflamma-tory breast cancer. However, neoadjuvant chemotherapy with an anthracycline-containing regimen may affect dramatic regres-sions in up to 75% of cases. Tumors should be assessed for HER2 and

1	inflamma-tory breast cancer. However, neoadjuvant chemotherapy with an anthracycline-containing regimen may affect dramatic regres-sions in up to 75% of cases. Tumors should be assessed for HER2 and hormone receptors with treatment dictated based on receptor status. Modified radical mastectomy is performed after demonstrated response to systemic therapy to remove residual cancer from the chest wall and axilla. Adjuvant chemotherapy may be indicated depending on final pathologic assessment of the breast and regional nodes. Finally, the chest wall and the Figure 17-39. Inflammatory breast carcinoma. Stage IIIB cancer of the breast with erythema, skin edema (peau d’orange), nipple retraction, and satellite skin nodules.Brunicardi_Ch17_p0541-p0612.indd 60101/03/19 5:06 PM 602SPECIFIC CONSIDERATIONSPART IIsupraclavicular, internal mammary, and axillary lymph node basins receive adjuvant radiation therapy. This multimodal approach results in 5-year survival rates that approach 30%.

1	CONSIDERATIONSPART IIsupraclavicular, internal mammary, and axillary lymph node basins receive adjuvant radiation therapy. This multimodal approach results in 5-year survival rates that approach 30%. Patients with inflammatory breast cancer should be encouraged to participate in clinical trials.Rare Breast CancersSquamous Cell (Epidermoid) Carcinoma. Squamous cell (epidermoid) carcinoma is a rare cancer that arises from metaplasia within the duct system and generally is devoid of distinctive clinical or radiographic characteristics.373 Regional metastases occur in 25% of patients, whereas distant metastases are rare.Adenoid Cystic Carcinoma. Adenoid cystic carcinoma is very rare, accounting for <0.1% of all breast cancers. It is typically indistinguishable from adenoid cystic carcinoma arising in sali-vary tissues. These cancers are generally 1 to 3 cm in diameter at presentation and are well circumscribed. Axillary lymph node metastases are rare, but deaths from pulmonary metastases

1	in sali-vary tissues. These cancers are generally 1 to 3 cm in diameter at presentation and are well circumscribed. Axillary lymph node metastases are rare, but deaths from pulmonary metastases have been reported.Apocrine Carcinomas. Apocrine carcinomas are well-differentiated cancers that have rounded vesicular nuclei and prominent nucleoli. There is a very low mitotic rate and little variation in cellular features. However, apocrine carcinomas may display an aggressive growth pattern.Sarcomas. Sarcomas of the breast are histologically similar to soft tissue sarcomas at other anatomic sites. This diverse group includes fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, malignant schwannoma, rhab-domyosarcoma, osteogenic sarcoma, and chondrosarcoma. The clinical presentation is typically that of a large, painless breast mass with rapid growth. Diagnosis is by core-needle biopsy or by open incisional biopsy. Sarcomas are graded based on cellular-ity, degree of

1	is typically that of a large, painless breast mass with rapid growth. Diagnosis is by core-needle biopsy or by open incisional biopsy. Sarcomas are graded based on cellular-ity, degree of differentiation, nuclear atypia, and mitotic activity. Primary treatment is wide local excision, which may necessitate mastectomy. Axillary dissection is not indicated unless there is biopsy proven lymph node involvement. Angiosarcomas are classified as de novo, as postradiation, or as arising in associa-tion with postmastectomy lymphedema. In 1948, Stewart and Treves described lymphangiosarcoma of the upper extremity in women with ipsilateral lymphedema after radical mastectomy.374 Angiosarcoma is now the preferred name. The average interval between modified radical or radical mastectomy and the devel-opment of an angiosarcoma is 7 to 10 years. Sixty percent of women developing this cancer have a history of adjuvant radia-tion therapy. Forequarter amputation may be necessary to palli-ate the

1	devel-opment of an angiosarcoma is 7 to 10 years. Sixty percent of women developing this cancer have a history of adjuvant radia-tion therapy. Forequarter amputation may be necessary to palli-ate the ulcerative complications and advanced lymphedema.Lymphomas. Primary lymphomas of the breast are rare, and there are two distinct clinicopathologic variants. One type occurs in women ≤39 years of age, is frequently bilateral, and has the histologic features of Burkitt’s lymphoma. The second type is seen in women ≥40 years of age and is usually of the B-cell type. Breast involvement by Hodgkin’s lymphoma has been reported. An occult breast lymphoma may be diagnosed after detection of palpable axillary lymphadenopathy. Treatment depends on the stage of disease. Lumpectomy or mastectomy may be required. Axillary dissection for clearance of disease may be necessary. Recurrent or progressive local-regional disease is best man-aged by chemotherapy and radiation therapy. The prognosis is

1	be required. Axillary dissection for clearance of disease may be necessary. Recurrent or progressive local-regional disease is best man-aged by chemotherapy and radiation therapy. The prognosis is favorable, with 5and 10-year survival rates of 74% and 51%, respectively. More recently anaplastic large cell lymphoma has been described in association with breast implants for cosmetic or reconstructive purposes. This disease is treated with complete excision of the implant capsule with any associated soft tissue mass. More advanced cases may require systemic therapy and radiation treatment.REFERENCESEntries highlighted in bright blue are key references. 1. Breasted JH. The Edwin Smith Surgical Papyrus. University of Chicago Press, 1930;405. 2. Celsus AC. De Medicina (ed Loeb Classical Library Ed). Cambridge: Harvard University Press; 1935;131. 3. Beenken SW. History of the therapy of breast cancer. In: Copeland BA, ed. The Breast: Comprehensive Manage-ment of Benign and Malignant

1	Library Ed). Cambridge: Harvard University Press; 1935;131. 3. Beenken SW. History of the therapy of breast cancer. In: Copeland BA, ed. The Breast: Comprehensive Manage-ment of Benign and Malignant Disorder. Philadelphia: Saunders;2004;5. 4. Le Dran F. Mémoire avec une précis de plusieurs observa-tions sur le. Mem Acad Roy Chir Paris. 1757;3:1. 5. Moore C. On the influence of inadequate operations on the theory of cancer. R Med Chir Soc. 1867;1:244. 6. Halsted WS I. The results of operations for the cure of cancer of the breast performed at the Johns Hopkins Hospital from June, 1889, to January, 1894. Ann Surg. 1894;20:497-555. 7. Haagensen CD, Stout AP. Carcinoma of the breast. II-criteria of operability. Ann Surg. 1943;118:1032-1051. 8. Patey DH, Dyson WH. The prognosis of carcinoma of the breast in relation to the type of operation performed. Br J Cancer. 1948;2:7-13. 9. Fisher B, Jeong JH, Anderson S, et al. Twenty-five-year follow-up of a randomized trial comparing radical

1	of the breast in relation to the type of operation performed. Br J Cancer. 1948;2:7-13. 9. Fisher B, Jeong JH, Anderson S, et al. Twenty-five-year follow-up of a randomized trial comparing radical mas-tectomy, total mastectomy, and total mastectomy followed by irradiation. N Engl J Med. 2002;347:567-575. 10. Keynes G. Conservative treatment of cancer of the breast. Br Med J. 1937;2(3):643-666. 11. Fisher B, Anderson S, Bryant J, et al. Twenty-year follow-up of a randomized trial comparing total mastectomy, lumpec-tomy, and lumpectomy plus irradiation for the treatment of invasive breast cancer. N Engl J Med. 2002;347:1233-1241.Table 17-15Inflammatory vs. noninflammatory breast cancerINFLAMMATORYNONINFLAMMATORYDermal lymph vessel invasion is present with or without inflammatory changes.Inflammatory changes are present without dermal lymph vessel invasion.Cancer is not sharply delineated.Cancer is better delineated.Erythema and edema frequently involve >33% of the skin over the

1	changes are present without dermal lymph vessel invasion.Cancer is not sharply delineated.Cancer is better delineated.Erythema and edema frequently involve >33% of the skin over the breast.Erythema is usually confined to the lesion, and edema is less extensive.Lymph node involvement is present in >75% of cases.Lymph nodes are involved in approximately 50% of the cases.Distant metastases are more common at the initial presentation (25% of cases).Distant metastases are less common at presentation. Modified with permission from Bland KI, Copeland ED: The Breast: Comprehensive Management of Benign and Malignant Diseases, 2nd ed. Philadelphia, PA: Elsesvier/Saunders; 1998.Brunicardi_Ch17_p0541-p0612.indd 60201/03/19 5:06 PM 603THE BREASTCHAPTER 17 12. Clarke M, Collins R, Darby S, et al. Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: an overview of the randomised trials. Lancet.

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1	phase 3 randomised controlled trial. Lancet Oncol. 2016;17(4):425-439. 343a. Goetz, M. P., et al. (2017). MONARCH 3: Abemaciclib As Initial Therapy for Advanced Breast Cancer. J Clin Oncol. 35(32): 3638-3646. 343b. Sledge, G. W., Jr., et al. (2017). MONARCH 2: Abemaci-clib in Combination With Fulvestrant in Women With HR+/HER2Advanced Breast Cancer Who Had Progressed While Receiving Endocrine Therapy. J Clin Oncol. 35(25): 2875-2884. 344. Baselga J, Campone M, Piccart M, et al. Everolimus in postmenopausal hormone-receptor-positive advanced breast cancer. N Engl J Med. 2012;366:520-529. 345. Bachelot T, Bourgier C, Cropet C, et al. Randomized phase II trial of everolimus in combination with tamoxifen in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer with prior exposure to aromatase inhibitors: a GINECO study. J Clin Oncol. 2012;30(22):2718-2724. 346. Wolff AC, Lazar AA, Bondarenko I, et al. Randomized phase III

1	metastatic breast cancer with prior exposure to aromatase inhibitors: a GINECO study. J Clin Oncol. 2012;30(22):2718-2724. 346. Wolff AC, Lazar AA, Bondarenko I, et al. Randomized phase III placebo-controlled trial of letrozole plus oral tem-sirolimus as first-line endocrine therapy in postmenopausal women with locally advanced or metastatic breast cancer. J Clin Oncol. 2013;31(2):195-202. 347. Schiavon G, Hrebien S, Garcia-Murillas I, et al. Analysis of ESR1 mutation in circulating tumor DNA demonstrates evo-lution during therapy for metastatic breast cancer. Sci Transl Med. 2015;7(313):313ra182. 348. Robinson DR, Wu YM, Vats P, et al. Activating ESR1 muta-tions in hormone-resistant metastatic breast cancer. Nat Genet. 2013;45(12):1446-1451. 349. Toy W, Weir H, Razavi P, et al. Activating ESR1 mutations differentially affect the efficacy of ER antagonists. Cancer Discov. 2017;7(3):277-287. 350. Jakesz R, Hausmaninger H, Kubista E, et al. Random-ized adjuvant trial of tamoxifen and

1	ESR1 mutations differentially affect the efficacy of ER antagonists. Cancer Discov. 2017;7(3):277-287. 350. Jakesz R, Hausmaninger H, Kubista E, et al. Random-ized adjuvant trial of tamoxifen and goserelin versus Brunicardi_Ch17_p0541-p0612.indd 61101/03/19 5:06 PM 612SPECIFIC CONSIDERATIONSPART IIcyclophosphamide, methotrexate, and fluorouracil: evidence for the superiority of treatment with endocrine blockade in pre-menopausal patients with hormone-responsive breast cancer—Austrian Breast and Colorectal Cancer Study Group Trial 5. J Clin Oncol. 2002;20(24):4621-4627. 351. Francis PA, Regan MM, Fleming GF, et al. Adjuvant ovar-ian suppression in premenopausal breast cancer. N Engl J Med. 2015;372(5):436-446. 352. Pagani O, Regan MM, Walley BA, et al. Adjuvant exemes-tane with ovarian suppression in premenopausal breast cancer. N Engl J Med. 2014;371:107-118. 353. Early Breast Cancer Trialists’ Collaborative Group (EBCTCG). Effects of chemotherapy and hormonal ther-apy for early

1	suppression in premenopausal breast cancer. N Engl J Med. 2014;371:107-118. 353. Early Breast Cancer Trialists’ Collaborative Group (EBCTCG). Effects of chemotherapy and hormonal ther-apy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet. 2005;365:1687-1717. 354. Paik S, Bryant J, Tan-Chiu E, et al. Real-world performance of HER2 testing—National Surgical Adjuvant Breast and Bowel Project experience. J Natl Cancer Inst. 2002;94:852-854. 355. Press MF, Slamon DJ, Flom KJ, et al. Evaluation of HER-2/neu gene amplification and overexpression: comparison of frequently used assay methods in a molecularly char-acterized cohort of breast cancer specimens. J Clin Oncol. 2002;20:3095-3105. 356. Volpi A, De Paola F, Nanni O, et al. Prognostic significance of biologic markers in node-negative breast cancer patients: a prospective study. Breast Cancer Res Treat. 63:181-192. 357. Goldhirsch A, Gelber RD, Piccart-Gebhart MJ, et al. 2 years

1	significance of biologic markers in node-negative breast cancer patients: a prospective study. Breast Cancer Res Treat. 63:181-192. 357. Goldhirsch A, Gelber RD, Piccart-Gebhart MJ, et al. 2 years versus 1 year of adjuvant trastuzumab for HER2-positive breast cancer (HERA): an open-label, randomised controlled trial. Lancet. 2013;382(9897):1021-1028. 358. Pivot X, Romieu G, Debled M, et al. 6 months versus 12 months of adjuvant trastuzumab for patients with HER2-positive early breast cancer (PHARE): a randomised phase 3 trial. Lancet Oncol. 2013;14(8):741-748. 359. Verma S, Miles D, Gianni L, et al. Trastuzumab emtansine for HER2-positive advanced breast cancer. N Engl J Med. 2012;367:1783-1791. 360. Baselga J, Cortés J, Kim SB, et al. Pertuzumab plus trastu-zumab plus docetaxel for metastatic breast cancer. N Engl J Med. 2012;366(2):109-119. 361. Gianni L, Pienkowski T, Im YH, et al. Efficacy and safety of neoadjuvant pertuzumab and trastuzumab in women with locally advanced,

1	metastatic breast cancer. N Engl J Med. 2012;366(2):109-119. 361. Gianni L, Pienkowski T, Im YH, et al. Efficacy and safety of neoadjuvant pertuzumab and trastuzumab in women with locally advanced, inflammatory, or early HER2-positive breast cancer (NeoSphere): a randomised multi-centre, open-label, phase 2 trial. Lancet Oncol. 2012;13(1): 25-32. 362. Schneeweiss A, Chia S, Hickish T, et al. Pertuzumab plus trastuzumab in combination with standard neoad-juvant anthracycline-containing and anthracycline-free chemotherapy regimens in patients with HER2-positive early breast cancer: a randomized phase II cardiac safety study (TRYPHAENA). Ann Oncol. 2013;24(9):2278-2284.362a. Von Minckwitz, G., et al. (2017). Adjuvant Pertuzumab and Trastuzumab in Early HER2-Positive Breast Cancer. N Engl J Med. 377(2): 122-131. 363. Chan A, Delaloge S, Holmes FA, et al. Neratinib after trastuzumab-based adjuvant therapy in patients with HER2-positive breast cancer (ExteNET): a multicentre, randomised,

1	Med. 377(2): 122-131. 363. Chan A, Delaloge S, Holmes FA, et al. Neratinib after trastuzumab-based adjuvant therapy in patients with HER2-positive breast cancer (ExteNET): a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol. 2016;17(3):367-377. 364. Bose R, Kavuri SM, Searleman AC, et al. Activating HER2 mutations in HER2 gene amplification negative breast cancer. Cancer Discov. 2013;3(2):224-237. 365. Lien HC, Chen YL, Juang YL, Jeng YM. Frequent alterations of HER2 through mutation, amplification, or overexpression in pleomorphic lobular carcinoma of the breast. Breast Can-cer Res Treat. 2015;150:447-455. 366. Ben-Baruch NE, Bose R, Kavuri SM, Ma CX, Ellis MJ. HER2-mutated breast cancer responds to treatment with single-agent neratinib, a second-generation HER2/EGFR tyrosine kinase inhibitor. J Natl Compr Canc Netw. 2015;13:1061-1064. 367. Gandhi L, Bahleda R, Tolaney SM, et al. Phase I study of neratinib in combination with temsirolimus in

1	HER2/EGFR tyrosine kinase inhibitor. J Natl Compr Canc Netw. 2015;13:1061-1064. 367. Gandhi L, Bahleda R, Tolaney SM, et al. Phase I study of neratinib in combination with temsirolimus in patients with human epidermal growth factor receptor 2-dependent and other solid tumors. J Clin Oncol. 2014;32(2):68-75. 368. Robinson DS, Sundaram M, et al. Carcinoma of the breast in pregnancy and lactation. In: Bland KI, Copeland EMI, eds. The Breast: Comprehensive Management of Benign and Malignant Diseases. Philadelphia: WB Saunders; 1998:1433. 369. Giordano SH, Buzdar AU, Hortobagyi GN: Breast cancer in men. Ann Intern Med. 2002;137:678-687. 370. Wilhelm MC. Cancer of the male breast. In: Bland KI, Cope-land EMI, eds. The Breast: Comprehensive Management of Benign and Malignant Diseases. Philadelphia: WB Saunders; 1998:1416. 371. Khan SA, Badve S. Phyllodes tumors of the breast. Curr Treat Options Oncol. 2001;2:139-147. 372. Chittoor SR, Swain SM. Locally advanced breast cancer: Role of medical

1	WB Saunders; 1998:1416. 371. Khan SA, Badve S. Phyllodes tumors of the breast. Curr Treat Options Oncol. 2001;2:139-147. 372. Chittoor SR, Swain SM. Locally advanced breast cancer: Role of medical oncology. In: Bland KI, Copeland EMI, eds. The Breast: Comprehensive Management of Benign and Malignant Diseases. Philadelphia: WB Saunders; 1998:1403. 373. Mies C. Mammary sarcoma and lymphoma. In: Bland KI, Copeland EMI, eds. The Breast: Comprehensive Management of Benign and Malignant Diseases. Philadelphia: WB Saunders; 1998:307. 374. Stewart FW, Treves N. Lymphangiosarcoma in postmas-tectomy lymphedema; a report of six cases in elephantiasis chirurgica. Cancer. 1948;1:64-81.Brunicardi_Ch17_p0541-p0612.indd 61201/03/19 5:06 PM

1	Disorders of the Head and NeckAntoine Eskander, Stephen Y. Kang, Michael S. Harris, Bradley A. Otto, Oliver Adunka, Randal S. Weber, and Theodoros N. Teknos 18chapterCOMPLEX ANATOMY AND FUNCTIONThe anatomy of the head and neck is complex because of the proximity of vital structures such as framework, nerves, and arteries. Functionally, these structures afford most of the human senses: vision, taste, smell, and hearing. Even more fundamental, the upper aerodigestive tract is critical for breathing, speech, and swallowing. Otolaryngology—head and neck surgery is the field that predominantly deals with disorders of the head and neck; however, a multidisciplinary approach is required to achieve optimal outcomes. The multidisciplinary team can include audi-ology, speech language pathology, allergy/immunology, neurol-ogy, neurosurgery, radiation, and medical oncology. This chapter aims to provide an overview of the most common diseases pre-senting to and treated by the otolaryngologist—head

1	neurol-ogy, neurosurgery, radiation, and medical oncology. This chapter aims to provide an overview of the most common diseases pre-senting to and treated by the otolaryngologist—head and neck surgeon. It reviews benign conditions, trauma, malignancies, reconstruction, tracheotomy, and rehabilitation.BENIGN CONDITIONS OF THE HEAD AND NECKOtologyInfectious. Infectious processes of the ear may be consid-ered by their location (external, middle, or inner ear), their time course (acute or chronic), and the presence of complications. The external ear or pinna consists of a cartilaginous frame-work, perichondrium, and a relatively thin layer of skin. Ery-sipelas (St Anthony’s Fire) or impetigo are causes of external ear infection affecting the dermis or hypodermis of the auricle, typically caused by Streptococcus pyogenes or Staphylococcus aureus, respectively, that may be encountered posttraumatically or related to ear piercing. Treatment is oral antibiotic therapy targeting these

1	caused by Streptococcus pyogenes or Staphylococcus aureus, respectively, that may be encountered posttraumatically or related to ear piercing. Treatment is oral antibiotic therapy targeting these organisms. History and clinical features such as presence of bullae and golden crusting distinguish erysipelas and impetigo from other benign entities causing erythema and edema of the auricle, such as relapsing polychondritis, which is typically diffuse, lobule-sparing, and steroid-responsive.Acute otitis externa, often referred to as “swimmer’s ear,” denotes infection of the skin of the external auditory canal.1 Typically, the pathology is incited by moisture within the canal leading to skin maceration and pruritus. Subsequent trauma to the canal skin by scratching (i.e., instrumentation with a cot-ton swab or fingernail), erodes the normally protective skin/cerumen barrier. Hearing aid use and comorbid dermatologic conditions such as eczema or other forms of dermatitis may similarly serve

1	cot-ton swab or fingernail), erodes the normally protective skin/cerumen barrier. Hearing aid use and comorbid dermatologic conditions such as eczema or other forms of dermatitis may similarly serve as predisposing factors. The milieu of the exter-nal ear canal—dark, warm, humid—is ideal for rapid microbial proliferation. The most common offending organism is Pseu-domonas aeruginosa, although other bacteria and fungi may also be involved. Symptoms and signs of otitis externa include itching during the initial phases and pain with marked swelling of the canal soft tissues as the infection progresses. Treatment involves removal of debris under otomicroscopy and applica-tion of appropriate ototopical antimicrobials, such as neomycin/polymyxin or quinolone-containing eardrops. The topical ste-roid component of these drops (e.g., hydrocortisone or dexa-methasone) addresses swelling and, as a result, decreases the often intense pain associated with this infection. In cases of marked ear

1	ste-roid component of these drops (e.g., hydrocortisone or dexa-methasone) addresses swelling and, as a result, decreases the often intense pain associated with this infection. In cases of marked ear canal edema, the use of an otowick is required to facilitate delivery of ototopical medication medially into the ear canal. Fungal infections may call for the addition of 2% acetic acid to reestablish the premorbid pH balance. Patients with otitis externa should also be instructed to keep the ear dry. Systemic antibiotics are reserved for those with severe infections, diabet-ics, and immunosuppression.Complex Anatomy and Function 613Benign Conditions of the Head and Neck 613Otology / 613Sinonasal Inflammatory Disease / 617Pharyngeal and Adenotonsillar Disease / 622Benign Conditions of the Larynx / 624Vascular Lesions / 626Trauma of the Head and Neck 627Soft Tissue / 627Facial Fractures / 628Temporal Bone Fractures / 629Tumors of the Head and Neck 629Etiology and Epidemiology /

1	of the Larynx / 624Vascular Lesions / 626Trauma of the Head and Neck 627Soft Tissue / 627Facial Fractures / 628Temporal Bone Fractures / 629Tumors of the Head and Neck 629Etiology and Epidemiology / 630Anatomy and Histopathology / 630Second Primary Tumors in the Head and Neck / 631Staging / 632Upper Aerodigestive Tract / 632Nose and Paranasal Sinuses / 643Nasopharynx / 644Ear and Temporal Bone / 645Neck / 646Salivary Gland Tumors / 650Reconstruction 651Local Flaps and Skin Grafts / 651Regional Flaps / 651Free Tissue Transfer / 651Tracheotomy 652Indications and Timing / 652Technique and Complications / 652Speech with Tracheotomy and Decannulation / 653Long Term Management and Rehabilitation 654Palliative Care / 654Follow-Up Care / 654Brunicardi_Ch18_p0613-p0660.indd 61301/03/19 5:22 PM 614Figure 18-1. Acute otitis media.Malignant otitis externa, a fulminant necrotizing infec-tion of the soft tissues of the external ear canal combined with osteomyelitis of the temporal bone, is

1	PM 614Figure 18-1. Acute otitis media.Malignant otitis externa, a fulminant necrotizing infec-tion of the soft tissues of the external ear canal combined with osteomyelitis of the temporal bone, is a potentially life-threatening form of otitis externa seen most commonly among elderly patients with insulin-dependent diabetes mellitus or immunodeficiency.2,3 The classic physical finding is granulation tissue along the floor of the external auditory canal near the bony cartilaginous junction. Symptoms include persistent otalgia for longer than one month and purulent otorrhea. Biopsy is called for in order to exclude malignancy. Computed tomography (CT) and magnetic resonance imaging (MRI) define the extension of disease. Technetium 99-m scans are useful in gauging extend of bony involvement in early disease. Gallium-67 scans are valu-able for monitoring disease during the course of treatment and for determining duration of antibiotic therapy. These patients require aggressive medical

1	in early disease. Gallium-67 scans are valu-able for monitoring disease during the course of treatment and for determining duration of antibiotic therapy. These patients require aggressive medical therapy including ototopical and IV antibiotics targeting Pseudomonas. Other gram-negative bacteria and fungi are occasionally implicated, necessitating culturedirected therapy. Patients who do not respond to medical management require surgical debridement. This condition may progress to involvement of the adjacent skull base and soft tissues, meningitis, brain abscess, and death.Acute otitis media (AOM) typically implies a bacterial infec-tion of the middle ear.4 This diagnosis accounts for 25% of pedi-atric antibiotic prescriptions and is the most common bacterial infection of childhood. Most cases occur before 2 years of age and are secondary to immaturity of the Eustachian tube. Well-recog-nized contributing factors include upper respiratory viral infection and daycare attendance, as

1	cases occur before 2 years of age and are secondary to immaturity of the Eustachian tube. Well-recog-nized contributing factors include upper respiratory viral infection and daycare attendance, as well as craniofacial conditions affect-ing Eustachian tube function, such as cleft palate.It is important to distinguish between acute otitis media and otitis media with effusion (OME). The later denotes unin-fected serous fluid accumulation within the middle ear space. In children not already considered “at risk” for developmen-tal difficulties, OME is generally observed for resolution for a period of 3 months.5 Age-appropriate hearing testing should be performed when OME persists for ≥3 months or at any time when language delay, learning problems, or a significant hear-ing loss is suspected. In the absence of these factors, the child with OME should be reexamined at 3to 6-month intervals until the effusion is no longer present or until significant hear-ing loss is identified or structural

1	the absence of these factors, the child with OME should be reexamined at 3to 6-month intervals until the effusion is no longer present or until significant hear-ing loss is identified or structural abnormalities of the eardrum or middle ear are suspected. When hearing, speech, or structural concerns exist, myringotomy with tympanostomy tube place-ment is indicated.Signs and symptoms of infectious otitis media occurring for <3 weeks denote AOM. In this phase, otalgia and fever are the most common symptoms and physical exam reveals a bulging, opaque tympanic membrane (Fig. 18-1). If the process lasts 3 to 8 weeks, it is deemed subacute. Chronic otitis media, lasting more than 8 weeks, usually results from an unresolved acute otitis media. The most common organisms responsible are Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis.In order to minimize antibiotic resistance and obviate complications of antimicrobial therapy such as allergic reaction and diarrhea,

1	pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis.In order to minimize antibiotic resistance and obviate complications of antimicrobial therapy such as allergic reaction and diarrhea, guidelines have been established for the treatment of AOM.6,7 Pain associated with AOM should be recognized and treated with oral analgesics. In children older than 6 months who are not otherwise considered “high risk” for complications (e.g., immunocompromised, previous cochlear implantation, developmental anomalies of the inner ear) with symptoms con-sistent with unilateral AOM without otorrhea, an initial period of observation is offered. If initial observation is selected by the physician and family, a mechanism for reexamination in 48 to 72 hours to evaluate for clinical improvement must be in place. When these criteria are not met, or clinical improvement is not observed within 48 to 72 hours, oral antibiotics are begun. First-line therapy is high-dose amoxicillin or

1	must be in place. When these criteria are not met, or clinical improvement is not observed within 48 to 72 hours, oral antibiotics are begun. First-line therapy is high-dose amoxicillin or amoxicillin-clavulanate, for β-lactamase coverage. Chronic otitis media is frequently Key Points1 One of the most common benign head and neck disorders includes sinonasal inflammatory disease which can present as acute or chronic rhinosinusitis.2 Acute adeno-tonsillitis is a major cause of morbidity in children and adenotonsillectomy can significantly improve symptoms of both sleep disordered breathing and of symp-toms during acute infections.3 Squamous cell carcinoma comprises >90% of all of the malignant pathology of the mucosal lining of the upper aerodigestive tract.4 The ideal treatment protocol for these cancers varies by subsite, stage, patient comorbidity, and center preference/experience. Early stage disease is treated with unimodality and late stage disease is treated with multiple

1	for these cancers varies by subsite, stage, patient comorbidity, and center preference/experience. Early stage disease is treated with unimodality and late stage disease is treated with multiple modalities in the form of primary surgery with adjuvant radiotherapy or primary concurrent chemoradiotherapy.5 Free flap reconstruction of head and neck defects is integral to help improve patient-reported quality of life and to re-establish form and function.Brunicardi_Ch18_p0613-p0660.indd 61401/03/19 5:22 PM 615DISORDERS OF THE HEAD AND NECKCHAPTER 18treated with myringotomy and tube placement (Fig. 18-2). This treatment is indicated for frequent acute episodes and in the set-ting of COME as discussed previously. The purpose of this pro-cedure is to remove the effusion and provide a route for middle ear ventilation. Episodes of AOM following tube placement are still possible. Myringotomy tubes, however, allow for preven-tion of painful tympanic membrane distension, risk of perfora-tion

1	middle ear ventilation. Episodes of AOM following tube placement are still possible. Myringotomy tubes, however, allow for preven-tion of painful tympanic membrane distension, risk of perfora-tion and other complications, and permit delivery of ototopicals into the middle ear space, in most cases obviating the need for systemic antibiotic therapy.Spontaneous tympanic membrane perforation during acute otitis media provides for drainage of purulent fluid and middle ear ventilation and frequently results in immediate resolution of severe pain. In the majority of cases, these perforations will heal spontaneously after the infection has resolved.8 Chronic otitis media, however, may be associated with nonhealing tympanic membrane perforations. Patients may have persistent otorrhea, which is treated with topical drops. Preparations containing ami-noglycoside are avoided because this class of drugs is toxic to the inner ear. Solutions containing alcohol or acetic acid may be irritating or

1	treated with topical drops. Preparations containing ami-noglycoside are avoided because this class of drugs is toxic to the inner ear. Solutions containing alcohol or acetic acid may be irritating or caustic to the middle ear and are also avoided in the setting of a perforation. Nonhealing perforation requires surgical closure (tympanoplasty) after medical treatment of any residual acute infection.Chronic inflammatory changes from otitis media intersect with and share common etiological factors with cholesteatoma. Cholesteatoma is an epidermoid cyst of the middle ear and/or mastoid cavity that develops as result of Eustachian tube dysfunction. While several theories exist regarding causes of cholesteatoma, most cholesteatoma arises from squamous epi-thelium drawn into the middle ear via retraction pockets, most commonly in the pars flaccida.9 Squamous epithelium may also migrate into the middle ear via a perforation. Chronic mastoid-itis that fails medical management or is associated

1	pockets, most commonly in the pars flaccida.9 Squamous epithelium may also migrate into the middle ear via a perforation. Chronic mastoid-itis that fails medical management or is associated with cho-lesteatoma is treated by mastoidectomy. Chronic inflammation and destruction of middle ear structures by osteolytic enzymes of cholesteatoma matrix may also be associated with erosion of the ossicular chain, which can be reconstructed with various prostheses or autologous ossicular replacement techniques.Complications of otitis media with or without cholestea-toma may be grouped into two categories: intratemporal (oto-logic) and intracranial.10 Fortunately, complications are rare in the antibiotic era, but mounting antibiotic resistance necessitates an increased awareness of these conditions. Intratemporal com-plications include acute coalescent mastoiditis, petrositis, facial nerve paralysis, and labyrinthitis. In acute coalescing mastoid-itis, destruction of the bony lamellae by an acute

1	com-plications include acute coalescent mastoiditis, petrositis, facial nerve paralysis, and labyrinthitis. In acute coalescing mastoid-itis, destruction of the bony lamellae by an acute purulent pro-cess results in severe pain, fever, and fluctuance behind the ear. The mastoid air cells coalesce into one common space filled with pus. Mastoid infection may also spread to the petrous apex, causing retro-orbital pain and sixth-nerve palsy. These diagno-ses are confirmed by computed tomographic scan. Facial nerve paralysis may also occur secondary to an acute inflammatory process in the middle ear or mastoid.11Intratemporal complications of otitis media are managed by myringotomy tube placement in addition to appropriate IV antibiotics. In acute coalescent mastoiditis and petrositis, mas-toidectomy is also performed as necessary to drain purulent foci. Labyrinthitis refers to inflammation of the inner ear. Most cases are idiopathic or are secondary to viral infections of the

1	mas-toidectomy is also performed as necessary to drain purulent foci. Labyrinthitis refers to inflammation of the inner ear. Most cases are idiopathic or are secondary to viral infections of the endolymphatic space. The patient experiences vertigo together with sensorineural hearing loss, and symptoms may smolder over several weeks. Labyrinthitis associated with middle ear infection may be serous or suppurative. In the former case, bac-terial products and/or inflammatory mediators transudate into the inner ear via the round window membrane, establishing an inflammatory process therein. Total recovery is eventually pos-sible after the middle ear is adequately treated.Suppurative labyrinthitis, however, is a much more toxic condition in which the acute purulent bacterial infection extends into the inner ear and causes marked destruction of the sensory hair cells and neurons of the eighth-nerve ganglion. This con-dition may be a harbinger for meningitis and must be treated rapidly. The

1	into the inner ear and causes marked destruction of the sensory hair cells and neurons of the eighth-nerve ganglion. This con-dition may be a harbinger for meningitis and must be treated rapidly. The goal of management of inner ear infection, which occurs secondary to middle ear infection, is to “sterilize” the middle ear space with antibiotics and the placement of a myr-ingotomy tube.The most common intracranial complication of otitis media is meningitis. Otologic meningitis in children is most commonly associated with an H. influenzae type B infection. Other intra-cranial complications include epidural abscess, subdural abscess, brain abscess, otitic hydrocephalus, and sigmoid sinus thrombo-phlebitis. In these cases, the otogenic source must be urgently treated with antibiotics and myringotomy tube placement. Mas-toidectomy and neurosurgical consultation may be necessary.Facial Nerve Disorders. Bell’s palsy is the most common etiology of facial nerve weakness/paralysis and is

1	myringotomy tube placement. Mas-toidectomy and neurosurgical consultation may be necessary.Facial Nerve Disorders. Bell’s palsy is the most common etiology of facial nerve weakness/paralysis and is clinically dis-tinct from that occurring as a complication of otitis media in that the otologic exam is normal.12 Bell’s palsy is rapid, unilat-eral and, historically, considered idiopathic. It is now accepted, however, that the majority of these cases represent a viral neu-ropathy caused by herpes simplex. It is critical that clinicians distinguish Bell’s palsy from other causes of facial weakness/palsy. Alternative diagnoses are suggested by weakness/paraly-sis that arise gradually (rather than <72 hours), is bilateral, is accompanied by other neurological deficits, or does not show some recovery within 2 to 3 weeks and complete recovery at 3 to 4 months. Treatment includes oral steroids plus antiviral ther-apy (i.e., valacyclovir). Complete recovery is the norm, but it does not occur

1	within 2 to 3 weeks and complete recovery at 3 to 4 months. Treatment includes oral steroids plus antiviral ther-apy (i.e., valacyclovir). Complete recovery is the norm, but it does not occur universally, and selected cases may benefit from surgical decompression of the nerve within its bony canal. Elec-trophysiologic testing has been used to identify those patients in whom surgery might be indicated.13 The procedure involves decompression of the nerve via exposure in the mastoid and middle cranial fossa.Figure 18-2. Myringotomy and tube.Brunicardi_Ch18_p0613-p0660.indd 61501/03/19 5:22 PM 616SPECIFIC CONSIDERATIONSPART IIVaricella zoster virus may also cause facial nerve paraly-sis when the virus reactivates from dormancy in the nerve. This condition, known as Ramsay Hunt syndrome, is characterized by severe otalgia followed by the eruption of vesicles of the external ear and the soft palate. Treatment is similar to Bell’s palsy, but full recovery is only seen in approximately

1	is characterized by severe otalgia followed by the eruption of vesicles of the external ear and the soft palate. Treatment is similar to Bell’s palsy, but full recovery is only seen in approximately two-thirds of cases.Traumatic facial nerve injuries may occur secondary to accidental trauma or surgical injury. Iatrogenic facial nerve trauma most often occurs during mastoidectomy, most com-monly to the vertical segment of the nerve.14 Detailed knowl-edge of facial nerve anatomy and adjunctive use of nerve integrity monitoring systems are imperative in this context. When the facial nerve is injured during an operative procedure, it is explored. Injury to >50% of the neural diameter of the facial nerve is addressed either with primary reanastomosis or recon-structed with the use a nerve graft. Complete recovery of nerve function is uncommon in these cases.Lesions of the Internal Auditory Canal and Cerebello-pontine Angle. The most common lesion affecting the inter-nal auditory canal

1	graft. Complete recovery of nerve function is uncommon in these cases.Lesions of the Internal Auditory Canal and Cerebello-pontine Angle. The most common lesion affecting the inter-nal auditory canal (IAC) and the cerebellopontine angle (CPA) is vestibular schwannoma (formerly referred to as “acoustic neuroma”). Less commonly encountered lesions of the IAC and CPA include meningioma and epidermoid tumors. Vestibular schwannomas are benign tumors that comprise 60% to 92% of all CPA lesions and 6% to 10% of intracranial tumors. They demon-strate an average growth rate of 1 to 2 mm per year.15 Vestibular schwannomas are most commonly unilateral and sporadic; bilat-eral tumors are the hallmark of neurofibromatosis type 2 (NF2), an autosomal dominant condition linked to mutation of a tumor suppressor gene mapped to chromosome 22. The most common presenting symptoms of vestibular schwannoma are asymmetric sensorineural hearing loss and speech perception deficits often out of proportion to

1	gene mapped to chromosome 22. The most common presenting symptoms of vestibular schwannoma are asymmetric sensorineural hearing loss and speech perception deficits often out of proportion to degree of hearing loss indicated by audiom-etry. Unilateral tinnitus is also frequently reported. Disequilib-rium or, less commonly, episodic vertigo may be present. Facial nerve weakness or paralysis is rare. Larger tumors may feature facial numbness and loss of the cornea reflex from compression of the trigeminal nerve. Very large lesions can lead to brainstem compression, obstructive hydrocephalus, and death.Gadolinium-enhancement on T1-weighted MRI is the gold standard for diagnosis and detects even very small tumors (Fig. 18-3) The conventional armamentarium for vestibular Figure 18-3. A. Axial T1 magnetic resonance imaging (MRI) post-contrast showing left cerebellopontine angle tumor with avid gadolinium enhancement. Minimal internal auditory canal involvement is noted. B. Axial T2 MRI

1	T1 magnetic resonance imaging (MRI) post-contrast showing left cerebellopontine angle tumor with avid gadolinium enhancement. Minimal internal auditory canal involvement is noted. B. Axial T2 MRI showing left cerebellopontine angle tumor with thin cerebrospinal fluid cleft between tumor and brainstem/cerebellum. C. Axial T1 MRI post-contrast showing left cerebellopontine angle tumor with avid gadolinium enhancement. The lesion is confined to the internal auditory canal with minimal cerebellopontine angle involvement. D. Intraoperative phono during microsurgical resection via translabyrinthine approach. Black arrow indicates cochlear nerve.ABCDBrunicardi_Ch18_p0613-p0660.indd 61601/03/19 5:22 PM 617DISORDERS OF THE HEAD AND NECKCHAPTER 18schwannoma includes observation, microsurgical resection, and stereotactic radiation.16 Management of patients with ves-tibular schwannomas involves weighing a multitude of vari-ables particular to the tumor (location, size, growth pattern), the

1	resection, and stereotactic radiation.16 Management of patients with ves-tibular schwannomas involves weighing a multitude of vari-ables particular to the tumor (location, size, growth pattern), the patient (age, overall health, individual wishes), and the inter-action between tumor and patient (symptoms currently expe-rienced, symptoms likely to develop with lesion progression, degree of residual hearing). For patients who have hearing that may still benefit from acoustic amplification using a hearing aid, either a retrosigmoid or a middle fossa approach may be offered, depending on tumor location, size, patient preference, and provider experience. For patients without serviceable hear-ing preoperatively, a translabyrinthine approach is most com-monly offered.Sinonasal Inflammatory DiseaseRhinosinusitis. Rhinosinusitis is defined as symptomatic inflammation of the nasal cavity and paranasal sinuses. Rhi-nosinusitis is preferred over sinusitis because sinusitis almost always is

1	DiseaseRhinosinusitis. Rhinosinusitis is defined as symptomatic inflammation of the nasal cavity and paranasal sinuses. Rhi-nosinusitis is preferred over sinusitis because sinusitis almost always is accompanied by inflammation of the contiguous nasal mucosa. Rhinosinusitis is a significant health burden, affect-ing nearly 12% of the population.17 Rhinosinusitis is the fifth most common diagnosis responsible for antibiotic prescription and accounts for more than 20% of all antibiotics prescribed to adults. Rhinosinusitis may be broadly classified based on duration of symptomatology. Symptoms lasting <4 weeks may be classified as acute rhinosinusitis (ARS), while symptoms lasting >12 weeks may be classified as chronic rhinosinusitis (CRS). Rhinosinusitis lasting between 4 and 12 weeks has his-torically been defined as “subacute,” although the current clini-cal practice guideline published by the American Academy of Otolaryngology—Head and Neck Surgery does not distinguish rhinosinusitis

1	been defined as “subacute,” although the current clini-cal practice guideline published by the American Academy of Otolaryngology—Head and Neck Surgery does not distinguish rhinosinusitis in this time frame, noting that this group likely represents crossover symptoms from one of the other two sub-classes. Hence, the decision on how to manage this group of patients must be individualized.18 Because common conditions such as atypical migraine headache, laryngopharyngeal reflux, and allergic rhinitis frequently mimic rhinosinusitis, diagno-sis of rhinosinusitis is based not only on symptomatic criteria but also on objective evaluation with either imaging and/or endoscopy.Acute Rhinosinusitis. Acute rhinosinusitis most commonly occurs in the setting of a viral upper respiratory tract infection (URI). Although it is believed that acute bacterial rhinosinusitis (ABRS) typically follows a viral URI, it has been estimated that only up to 2% of viral URIs lead to ABRS.19 The most common

1	(URI). Although it is believed that acute bacterial rhinosinusitis (ABRS) typically follows a viral URI, it has been estimated that only up to 2% of viral URIs lead to ABRS.19 The most common viruses involved in ARS include rhinovirus, influenza virus, and parainfluenza virus. It is not known whether the viral URI precedes or only occurs along with ABRS. Regardless, viral infection leads to mucosal edema with sinus ostium obstruction, mucus stasis, tissue hypoxia, ciliary dysfunction, and epithelial damage, which may enhance bacterial adherence.20 Other con-ditions that may contribute to ABRS should be investigated, especially in the setting of recurrent ABRS. Such conditions include foreign body, sinus fungal ball (with bacterial secondary infection), and periapical dental disease (Figs. 18-4 and 18-5).The symptomatic criteria used to define ABRS include up to 4 weeks of purulent nasal drainage accompanied by nasal obstruction, facial pain with pressure and fullness, or both.18

1	18-4 and 18-5).The symptomatic criteria used to define ABRS include up to 4 weeks of purulent nasal drainage accompanied by nasal obstruction, facial pain with pressure and fullness, or both.18 ABFigure 18-4. A. Right periapical abscess (arrow) leading to acute bacterial rhinosinusitis. B. Follow-up scan of the same patients after administration of antibiotics demonstrating resolution of the sinonasal inflammatory changes. Therapy subsequently directed at the offending tooth will prevent recurrent symptoms.Figure 18-5. Computed tomography scan demonstrating a fungal ball of the right maxillary sinus, characterized by heterogeneous opacification of the sinus.Brunicardi_Ch18_p0613-p0660.indd 61701/03/19 5:22 PM 618SPECIFIC CONSIDERATIONSPART IIOther historical factors that may predict the development of ABRS include persistence of symptoms beyond 10 days, or worsening of symptoms, following initial improvement, within 10 days (“double worsening”). Although routine head and neck

1	the development of ABRS include persistence of symptoms beyond 10 days, or worsening of symptoms, following initial improvement, within 10 days (“double worsening”). Although routine head and neck examination may identify anteriorly or posteriorly draining purulent secretions, the utilization of a rigid endoscope may improve diagnostic sensitivity and may also facilitate culture acquisition (Fig. 18-6).The management of ABRS is heavily dependent on anti-biotics, either culture-directed or empirically chosen to cover the most common isolates of ABRS, including S pneumoniae, H influenza, and M catarrhalis. Nosocomial ABRS more com-monly involves P aeruginosa or S aureus. Methicillin-resistant S aureus (MRSA) has been isolated with increasing frequency.20 Other treatments include topical and systemic decongestants, nasal saline spray, topical nasal steroids, and oral steroids in selected cases. In the acute setting, surgery is reserved for com-plications or pending complications, which

1	systemic decongestants, nasal saline spray, topical nasal steroids, and oral steroids in selected cases. In the acute setting, surgery is reserved for com-plications or pending complications, which may include exten-sion to the eye (orbital cellulitis or abscess) or the intracranial space (meningitis or intracranial abscess).Chronic Rhinosinusitis. Chronic rhinosinusitis (CRS) is characterized by symptomatic inflammation of the nose and paranasal sinuses lasting over 12 weeks. CRS has been clini-cally classified into two main groups: those with CRS with nasal polyps (CRSwNP) tend to exhibit a Th2-biased inflammatory profile, and those with CRS without nasal polyps (CRSsNP) tend to exhibit a Th1-biased profile. Although the etiology of CRS is unclear and the development of the clinical subtypes may be distinct, there exists significant overlap not only in phys-iologic manifestations but also in symptomatology. Hence, the sinonasal cavities of patients with both subtypes of CRS tend to

1	may be distinct, there exists significant overlap not only in phys-iologic manifestations but also in symptomatology. Hence, the sinonasal cavities of patients with both subtypes of CRS tend to exhibit mucosal edema, ostial obstruction, ciliary dysfunction, and an abhorrent inflammatory milieu.Two of the following symptomatic criteria must be pres-ent to diagnose CRS: purulent nasal drainage, nasal obstruc-tion, facial pain-pressure-fullness, and decreased sense of smell. These patients may also experience acute exacerbation, generally signified by an escalation of symptoms. Frequently, this is due to bacterial infection. However, patients with acute exacerbation of CRS may be distinguished from patients with recurrent acute bacterial rhinosinusitis (four or more episodes of ABRS per year) through baseline comparison: patients with CRS are symptomatic, even while at baseline, while patients with recurrent acute bacterial sinusitis are normal at baseline. As with ARS, the diagnosis of

1	through baseline comparison: patients with CRS are symptomatic, even while at baseline, while patients with recurrent acute bacterial sinusitis are normal at baseline. As with ARS, the diagnosis of CRS requires objective confirmation utilizing either nasal endoscopy, CT scans, or, less commonly, MRI.Nasal endoscopy is a critical element of the diagnosis of CRS. Abnormalities that may confirm the diagnosis of CRS include• Purulent mucus in the middle meatus or anterior ethmoid region• Edema in the middle meatus or ethmoid region• Polyps in nasal cavity or the middle meatusIn addition to establishing the diagnosis, nasal endoscopy can be valuable in antibiotic selection by facilitating specific culture acquisition. Furthermore, simple polypectomy or ste-roid injection can be performed under topical anesthesia in the appropriate clinical setting.Imaging is also an important clinical tool in the diagnosis of CRS. In general, CT is the modality of choice for diagno-sis and management of

1	topical anesthesia in the appropriate clinical setting.Imaging is also an important clinical tool in the diagnosis of CRS. In general, CT is the modality of choice for diagno-sis and management of CRS. Usual diagnostic criteria include mucosal thickening, sinus opacification, and bony remodeling (erosion or hyperostosis). It should be underscored, however, that CT scan is not the positive gold standard because many asymptomatic patients will demonstrate findings on a sinus CT scan, and many patients with presumed sinusitis will have negative findings.19 CT scan has excellent negative predic-tive value when performed in the setting of active symptoms. Thus, if a patient complains of rhinosinusitis-like symptoms but has no specific physical (endoscopic) findings, and the scan Figure 18-6. Nasal endoscopy is commonly performed in the clinic setting to aid in the diagnosis and management of rhinosinusitis.Brunicardi_Ch18_p0613-p0660.indd 61801/03/19 5:22 PM 619DISORDERS OF THE HEAD

1	Nasal endoscopy is commonly performed in the clinic setting to aid in the diagnosis and management of rhinosinusitis.Brunicardi_Ch18_p0613-p0660.indd 61801/03/19 5:22 PM 619DISORDERS OF THE HEAD AND NECKCHAPTER 18Figure 18-7. Point-of-care computed tomography system. All components can be fit within an 8′ × 10′ room in an outpatient office setting.Figure 18-8. Triplanar imaging revealing proximity to critical structures such as the orbital wall and skull base. This can be used for diag-nosis of sinus opacification as well as stereotactic intraoperative navigation, where endoscope view (lower right) can be radiologically cor-related with location in the three cardinal planes. This case reflects classic allergic fungal sinusitis where the opacified sinuses are filled with heterogeneous whitish material on computed tomography images. Polyps in the ethmoid cavity are seen on the endoscope image.is negative, other diagnoses (e.g., allergic rhinitis, migraine headache, tension

1	whitish material on computed tomography images. Polyps in the ethmoid cavity are seen on the endoscope image.is negative, other diagnoses (e.g., allergic rhinitis, migraine headache, tension headaches, and laryngopharyngeal reflux) should be sought. This has led to the utility of point-of-care CT (POC-CT) scan that can be performed in the physician’s office. POC-CT utilizes cone beam technology,21 which acquires the equivalent of >100 axial slices in approximately 1 minute at an effective resolution of 0.3 mm or less. The equipment occupies a room of 8’ × 10’ and can thus be accommodated in almost any office setting (Fig. 18-7). Perhaps most important, the radiation dosing for even the most sophisticated protocol is 0.17 mSv, which is <10% the dose of a conventional head CT and equivalent to approximately 20 days of background radia-tion. One theoretical shortcoming of this technology is that it does not permit soft tissue imaging. This is seldom a concern in sinonasal evaluation, as

1	to approximately 20 days of background radia-tion. One theoretical shortcoming of this technology is that it does not permit soft tissue imaging. This is seldom a concern in sinonasal evaluation, as this is typically undertaken in bone windows. The acquired data are immediately formatted into triplanar (axial, sagittal, coronal) reconstructions and is also compatible with devices used for intraoperative stereotactic navigation, which can be used to confirm relationships between the disease process, medial orbital wall, and skull base during surgery (Figs. 18-8 and 18-9).Medical management of CRS is heavily dependent on topical intranasal therapy. The reasons for this lie not only in established effectiveness but also in tolerability and safety—the chronic nature of CRS generally lends to requisite long-term medication administration despite other measures such as surgery. Nasal irrigation and topical nasal steroids are commonplace in the management of CRSwNP and CRSsNP. Oral steroids

1	requisite long-term medication administration despite other measures such as surgery. Nasal irrigation and topical nasal steroids are commonplace in the management of CRSwNP and CRSsNP. Oral steroids have demonstrated effectiveness in patients with CRSwNP, although the role in CRSsNP is less clear. Although otolaryngologists commonly utilize antibiotics in the man-agement of CRS, indications and administration practices are not uniform. Oral antibiotic therapy given for short duration (<4 weeks) is generally useful in the management of acute exac-erbation related to bacterial infection. Long-term utilization of antibiotics may be necessary in the setting of chronic infection or osteomyelitis. Additionally, long-term macrolide administra-tion may be utilized for anti-inflammatory effects in the appro-priate clinical setting.In most cases, patients considering endoscopic sinus surgery (ESS) for CRS should have significant residual Brunicardi_Ch18_p0613-p0660.indd 61901/03/19 5:22 PM

1	the appro-priate clinical setting.In most cases, patients considering endoscopic sinus surgery (ESS) for CRS should have significant residual Brunicardi_Ch18_p0613-p0660.indd 61901/03/19 5:22 PM 620SPECIFIC CONSIDERATIONSPART IIsymptomatology despite medical therapy. However, there cur-rently exists no consensus regarding what constitutes a “maxi-mum” course of medical therapy. It should be noted that unless there is suspicion of neoplasm or pending complication of rhinosinusitis, the decision to proceed with surgery is highly individualized. This is because surgery for uncomplicated CRS is elective, and patients who “fail” medical management will exhibit significant variability in symptoms, physical signs, and CT findings. Furthermore, ESS is not necessarily curative—the intent of ESS is to remove the symptoms related to CRS rather than cure the underlying condition itself.Surgery is typically preformed endoscopically where the goals are to remove polyps, enlarge or remove

1	of ESS is to remove the symptoms related to CRS rather than cure the underlying condition itself.Surgery is typically preformed endoscopically where the goals are to remove polyps, enlarge or remove obstruct-ing tissue surrounding the natural sinus ostia (Fig. 18-10), and remove chronically infected bone and mucosa to promote both ventilation and drainage of the sinus cavities. Inspissated mucin or pus is drained and cultured. Eventual resolution of the chronic inflammatory process can be attained with a com-bination of meticulous surgery and directed medical therapy, although the patient must understand that surgery may not alter the underlying immunologic pathophysiology. In cases where resection of inflammatory tissue and polyps are not required, recent trends have also included use of angioplasty-type balloons to dilate sinus ostia. The exact role for this tech-nology is unclear, but it appears to have promise in outpatient office management of patients with focal or limited

1	of angioplasty-type balloons to dilate sinus ostia. The exact role for this tech-nology is unclear, but it appears to have promise in outpatient office management of patients with focal or limited obstruc-tive pathology.Endoscopic Skull Base Surgery. Over the past three decades, the development and expansion of multidisciplinary skull base teams has become somewhat commonplace at large academic institutions. Facilitated mainly by growing cooperation between otolaryngologists and neurosurgeons, a variety of approaches that utilize the sinonasal corridor to treat a plethora of patho-logic processes of the anterior skull base have been developed.Technological advances in endoscopy, instrumentation, and imaging have also facilitated the development of endo-scopic endonasal approaches (EEAs), allowing team members to work simultaneously while maintaining optimal visualization of the relevant anatomy and freedom of movement within the corridor. Although historically the sphenoid sinus has

1	allowing team members to work simultaneously while maintaining optimal visualization of the relevant anatomy and freedom of movement within the corridor. Although historically the sphenoid sinus has been the common access route in the management of sellar pathology, a series of modular approaches of varied complexity have been developed that have broadened the reach of EEAs to address lesions at virtually all comportments of the ventral skull base, from the crista galli to the anterior arch of C2.22One of the key tenets of the EEA is that the sinonasal cor-ridor presents the most prudent and safest path to the lesion of interest. Accordingly, the EEA is generally chosen for lesions adjacent to the skull base, without intervening brain parenchyma, cranial nerves, major vessels, or other important anatomical structures. Currently, EEAs are utilized to treat a significant number of pathologic process involving the skull base, including: cerebrospinal fluid leaks, encephaloceles,

1	other important anatomical structures. Currently, EEAs are utilized to treat a significant number of pathologic process involving the skull base, including: cerebrospinal fluid leaks, encephaloceles, meningoceles, pseudomeningoceles, benign intracranial tumors (Fig. 18-11), benign sinonasal tumors, malignant sinonasal tumors, and inflammatory or traumatic conditions leading to compression at the craniovertebral junction. Although EEAs tend to be considered “minimally invasive,” the corridor created in the sinonasal cavity is nonetheless comprehensive enough to Figure 18-9. Sphenoid sinus fungal ball. The sinus has been opened revealing cheesy material during this intraoperative endoscopic view (lower right). The crosshairs stereotactically confirm location within the sphenoid sinus radiologically in the cardinal planes.Brunicardi_Ch18_p0613-p0660.indd 62001/03/19 5:22 PM 621DISORDERS OF THE HEAD AND NECKCHAPTER 18ABFigure 18-10. A. Endoscopic view of the right nasal cavity

1	radiologically in the cardinal planes.Brunicardi_Ch18_p0613-p0660.indd 62001/03/19 5:22 PM 621DISORDERS OF THE HEAD AND NECKCHAPTER 18ABFigure 18-10. A. Endoscopic view of the right nasal cavity demonstrating the uncinate process (U), ethmoid bulla (EB), middle turbinate (MT), inferior turbinate (IT), and nasal septum (S). B. Endoscopic view of a microdebrider being used to widen the right maxillary sinus ostium.ABCDFigure 18-11. Preoperative coronal (A) and sagittal (B) magnetic resonance images of a large olfactory groove meningioma removed using endoscopic endonasal approach. Postoperative coronal (C) and sagittal (D) images demonstrating removal of the tumor. The skull base can be reconstructed using local flaps (most commonly a nasoseptal flap pedicled on the posterior nasal artery).Brunicardi_Ch18_p0613-p0660.indd 62101/03/19 5:23 PM 622SPECIFIC CONSIDERATIONSPART IIprovide maximal freedom of movement for the critical compo-nent of the case (i.e., tumor resection near

1	62101/03/19 5:23 PM 622SPECIFIC CONSIDERATIONSPART IIprovide maximal freedom of movement for the critical compo-nent of the case (i.e., tumor resection near vital structures). Once the corridor is created by the otolaryngologist, the neurosurgeon joins, and a two-person, threeto four-hand technique is utilized to address the lesion of interest and reconstruct the skull base (Fig. 18-12).Despite the relatively confined aperture provided by the nostrils, even large tumors can be removed using EEAs, albeit via piecemeal removal. For malignant tumors, this has required a philosophical shift whereby en bloc resection of the entire tumor is replaced by piecemeal removal of the bulk of the tumor followed by complete resection of the pedicle with sufficient margins. Outcomes utilizing EEAs for resection of malignant tumors, when chosen appropriately, parallel those of traditional open approaches. However, EEAs are not favored over tradi-tional approaches when oncological principles would

1	resection of malignant tumors, when chosen appropriately, parallel those of traditional open approaches. However, EEAs are not favored over tradi-tional approaches when oncological principles would otherwise need to be violated.Pharyngeal and Adenotonsillar DiseaseWaldeyer’s ring consists of the palatine tonsils between the anterior and posterior tonsillar pillars, the lingual tonsils (lym-phoid tissue in the base of tongue), and the adenoid located in the nasopharynx. These four main sites of Waldeyer’s ring are connected by other minor lymphoid tissue along the posterior and lateral pharyngeal wall completing the ring. These are all considered mucosa-associated lymphoid tissue (MALT). These tissues react to inflammatory disease, infection, trauma, acid reflux, and radiotherapy. Even the vibratory effects of chronic snoring have been implicated in the development of adenoton-sillar disease. Inflammation of these tissues can lead to referred pain through cranial nerves IX and X to the

1	vibratory effects of chronic snoring have been implicated in the development of adenoton-sillar disease. Inflammation of these tissues can lead to referred pain through cranial nerves IX and X to the throat and ear. Adenotonsillar tissue does not have any afferent lymphatics and receives antigen presentation directly, with appropriate produc-tion of memory cells. However, there is no clear immune com-promise after removal.Figure 18-12. Two-surgeon, threeto four-hand technique uti-lized in endoscopic endonasal surgery.Microbiology and Complications. Adenotonsillar infections present with three temporal patterns: acute, recurrent acute, and chronic. Acute infection is typically viral in origin but second-ary bacterial invasion may initiate chronic disease. Viruses do not cause chronic infections; however, Epstein-Barr Virus (EBV) can cause significant hypertrophy. Systemic EBV infection, also known as mononucleosis, can mimic bacterial pharyngitis, but the progression of signs and

1	infections; however, Epstein-Barr Virus (EBV) can cause significant hypertrophy. Systemic EBV infection, also known as mononucleosis, can mimic bacterial pharyngitis, but the progression of signs and symptoms demonstrates lymphade-nopathy, splenomegaly, and hepatitis. This can be diagnosed on bloodwork (heterophile antibody or atypical lymphocytes). The most common bacterial causes of acute tonsillitis are group A β-hemolytic streptococcus species (GABHS) and S pneumoniae.23 If GABHS is confirmed, then antibiotic therapy is warranted in the pediatric population to decrease the risk (3%) of developing rheu-matic fever. A positive test for GABHS historically meant a throat swab with culture and sensitivity; however, rapid antigen assays have been demonstrated to be reasonably sensitive and specific (85% and 95%, respectively), thus largely replacing cultures.24 If the rapid assay is negative, then a culture is warranted. The remainder of the bacteriology for adenotonsillar disease is

1	specific (85% and 95%, respectively), thus largely replacing cultures.24 If the rapid assay is negative, then a culture is warranted. The remainder of the bacteriology for adenotonsillar disease is similar to otitis media and sinusitis, which includes H influenzae and M catarrhalis. Atypical infections include Corynebacterium diph-theria, Neisseria gonorrhoeae, and Chlamydia trachomatis.Complications of GABHS pharyngitis, typically from S pyogenes, can be systematic and include poststreptococcal glomerulonephritis, scarlet fever, and rheumatic fever. Anti-biotic therapy does not decrease the incidence of glomerulo-nephritis. Scarlet fever, caused by blood-borne streptococcal toxins, causes a strawberry tongue and a punctate rash on the trunk that spreads distally while sparing the palms and soles. Peritonsillar abscess is also a common complication that is treated in an ambulatory setting through a transoral approach after appropriate topicalization and local anesthetic. Deep neck

1	and soles. Peritonsillar abscess is also a common complication that is treated in an ambulatory setting through a transoral approach after appropriate topicalization and local anesthetic. Deep neck space infections are rare from pharyngitis but can occur from odontogenic and salivary gland infections. These typically require a transcervical approach for incision and drainage.Adenoids and Adenoidectomy. Acute adenoiditis typically presents with purulent rhinorrhea, nasal obstruction, and fever and can be associated with otitis media, particularly in the pedi-atric population. Recurrent acute adenoiditis is defined as four or more acute infections in a 6-month period, but in an adult, this may be difficult to distinguish from recurrent acute sinus-itis, and endoscopy with or without imaging of the sinuses may be warranted to distinguish between the two diagnoses. Chronic adenoiditis presents with persistent nasal discharge, halitosis, chronic congestion, and postnasal drip. In children,

1	of the sinuses may be warranted to distinguish between the two diagnoses. Chronic adenoiditis presents with persistent nasal discharge, halitosis, chronic congestion, and postnasal drip. In children, obstructive adenoid hyperplasia often requires surgical intervention to help relieve obstructive symptoms such as snoring, obligate mouth breathing, and hyponasal voice.The management of adenoid disease is slightly different than that for tonsillar disease. Chronic infection can be treated with antibiotics, although this often does not lead to a full reso-lution of symptoms. If the adenoid bed appears hyperplastic on lateral X-ray imaging or endoscopy, a 2-month trial of nasal steroids may be helpful. Adenoidectomy is indicated for recur-rent and chronic infections that have failed conservative man-agement. These infections are not limited to the adenoid bed but also involve the sinuses and the middle year. Adenoidectomy with a myringotomy and ventilation tube placement is benefi-cial for

1	man-agement. These infections are not limited to the adenoid bed but also involve the sinuses and the middle year. Adenoidectomy with a myringotomy and ventilation tube placement is benefi-cial for recurrent or chronic otitis media in children because the Brunicardi_Ch18_p0613-p0660.indd 62201/03/19 5:23 PM 623DISORDERS OF THE HEAD AND NECKCHAPTER 18adenoid functions as a reservoir for bacteria that can enter the middle ear through the Eustachian tube.25Adenoidectomy is also the first line of surgical manage-ment for children with chronic sinusitis because the adenoid can obstruct mucociliary clearance from the sinonasal tract into the choana and ultimately into the pharynx. Patients with obstruc-tive systems attributable to the adenoids and suspected benign or malignant neoplasms of the adenoid bed are also candidates. However, the procedure is contraindicated in patients with vel-opalatine insufficiency (VPI) and in patients with a cleft pal-ate. Prior to adenoidectomy, patients

1	adenoid bed are also candidates. However, the procedure is contraindicated in patients with vel-opalatine insufficiency (VPI) and in patients with a cleft pal-ate. Prior to adenoidectomy, patients should be examined for a submucous cleft, a lack of midline muscular tissue of the soft palate. Clinical signs of this include a bifid uvula, a translucent portion of the muscular diastasis of the soft palate (zona pel-lucida), and a palpable notched hard palate.26 A number of dif-ferent methods can be used to perform an adenoidectomy: cold steel, suction coagulator, microdebrider, and coblation. Adenoid regrowth and bleeding rates are both low, and no study has been able to demonstrate the superiority of one technique over the other for either outcome.27,28 Adenoidectomy is not without complications though, beyond VPI and bleeding, halitosis and adenoid bed regrowth (∼1%) are common complications. Rare complications include torticollis secondary to inflammation of the prevertebral fascia,

1	though, beyond VPI and bleeding, halitosis and adenoid bed regrowth (∼1%) are common complications. Rare complications include torticollis secondary to inflammation of the prevertebral fascia, nasopharyngeal stenosis, and cervi-cal spine subluxation, which is more common in patients with Down syndrome.Tonsils and Tonsillectomy Patients with acute tonsillitis present with sore throat, fever, dysphagia, and tender cervi-cal nodes with erythematous or exudative tonsils. The Centor Criteria is used to identify the likelihood of bacterial infection in adult patients complaining of sore throat in the emergency department or walk-in clinic, a point is given for each of the following: fever, tonsillar exudate, lymphadenopathy, and lack of cough.29-31 A score of 0 to 1 warrants no treatment, a score of 2 to 3 warrants GABHS testing, and a score of 4 warrants initiation of antibiotic therapy. First-line treatment is with peni-cillin or a cephalosporin; however, in those with an allergy, a

1	a score of 2 to 3 warrants GABHS testing, and a score of 4 warrants initiation of antibiotic therapy. First-line treatment is with peni-cillin or a cephalosporin; however, in those with an allergy, a macrolide can be considered. Documentation of recurrent acute infections should include a temperature (>38.3oC), cervical adenopathy, tonsillar exudate, and a positive test for GABHS. According to the American Academy of Otolaryngology—Head and Neck Surgery (AAO-HNS) clinical practice guideline on tonsillectomy in children, tonsillectomy is indicated when chil-dren have more than 7 documented episodes per year, 5 epi-sodes per year in the past 2 years, or 3 episodes per year in the past 3 years.23 Tonsillectomy can still be considered in children who do not meet these criteria if they have multiple antibiotic allergies or intolerances, have a history of peritonsillar abscess after the acute inflammation has resolved, or have PFAPA (peri-odic fever, aphthous stomatitis, pharyngitis, and

1	multiple antibiotic allergies or intolerances, have a history of peritonsillar abscess after the acute inflammation has resolved, or have PFAPA (peri-odic fever, aphthous stomatitis, pharyngitis, and adenitis). A peritonsillar abscess is an infection of the peritonsillar salivary gland (Weber’s gland), located between the tonsil capsule and the muscles of the tonsillar fossa. In selected cases of active peritonsillar abscess, tonsillectomy is required in the acute set-ting to treat systemic toxicity or impending airway compromise. Multiple techniques have been described, including electrocau-tery, sharp dissection, laser, and radiofrequency ablation. There is no consensus as to the best method.Sleep Disordered Breathing and Adenotonsillar Disease. Patients with sleep-disordered breathing (SDB) and tonsil-lar hypertrophy may also benefit from tonsillectomy if they have growth retardation, poor school performance, enuresis, or behavioral problems. The benefits may be accentuated in

1	(SDB) and tonsil-lar hypertrophy may also benefit from tonsillectomy if they have growth retardation, poor school performance, enuresis, or behavioral problems. The benefits may be accentuated in children with abnormal polysomnography; however, DB may require further treatment after tonsillectomy when it is multifac-torial. Clinical documentation of tonsillar grade/size is based on the percentage of the transverse oropharyngeal space measured between the anterior tonsillar pillars: grade 1+ <25%; grade 2+ 25% to 49%; grade 3+ 50% to 74%; grade 4+ ≥75% or more sometimes referred to as “kissing tonsils.”32 Tonsillectomy is effective for control of SDB in 60% to 70% of patients with tonsillar hypertrophy, although this much lower (10%–25%) in obese children, and it is therefore not curative in obese chil-dren but may improve some of their symptoms nonetheless. In patients with Down syndrome, obesity, craniofacial abnormali-ties, neuromuscular disorders, sickle cell disease, or

1	curative in obese chil-dren but may improve some of their symptoms nonetheless. In patients with Down syndrome, obesity, craniofacial abnormali-ties, neuromuscular disorders, sickle cell disease, or mucopoly-saccharidoses, polysomnography (PSG) should be performed prior to tonsillectomy.33 When the need for surgery is uncertain or when there is a discordance between tonsillar size on physi-cal examination and the reported severity of SDB, physicians should advocate for PSG prior to tonsillectomy. Tonsillectomy, usually with adenoidectomy if the adenoids are enlarged, is often performed on an outpatient basis unless the patient has documented or strongly suspected obstructive sleep apnea (OSA), is <3 years of age, or has severe OSA (in children, an apnea-hypopnea index ≥10 or more, oxygen saturation <80%, or both). Other reasons for admission include a home >1 hour from a hospital, patients with craniofacial abnormalities, or any other medical issue. There is strong evidence to suggest

1	<80%, or both). Other reasons for admission include a home >1 hour from a hospital, patients with craniofacial abnormalities, or any other medical issue. There is strong evidence to suggest the routine administration of a single intraoperative dose of IV dexametha-sone in children undergoing tonsillectomy, though antibiotics should not be administered or prescribed perioperatively in children. The complications from tonsillectomy include peri-operative bleeding (3%–5%), airway obstruction, death, and readmission from postoperative dysphagia leading to dehydra-tion.34 It is recommended that surgeons calculate and quote their own primary and secondary posttonsillectomy hemorrhage rates yearly.23 A rare but serious complication in patients with obstructive adenotonsillar disease post adenotonsillectomy is postobstructive pulmonary edema syndrome, which presents with decreased oxygen saturation and frothy, blood-tinged oral secretions. Patients usually recover with reintubation, positive

1	is postobstructive pulmonary edema syndrome, which presents with decreased oxygen saturation and frothy, blood-tinged oral secretions. Patients usually recover with reintubation, positive pressure, diuresis, and supportive care.Multilevel Sleep Surgery. SDB surgery is often multilevel and is not limited to adenotonsillar disease. Patients with nasal obstruction may benefit from septoplasty and trubinate reduc-tion, although in the adult population this is most commonly used to allow patients to tolerate their OSA appliances. Simi-larly, patients with significant lingual tonsillar hypertrophy and a large base of tongue may benefit from a base of tongue reduction, tongue base advancement, or geniohyoidopexy. A base of tongue reduction alone does not often provide enough apnea-hypopnea index reduction (30%–60%) for resolution of symptoms and is fraught with a high morbidity rate.35 Rarely, maxillomandibular advance is required to open up the retrolin-gual space. In patients with life

1	reduction (30%–60%) for resolution of symptoms and is fraught with a high morbidity rate.35 Rarely, maxillomandibular advance is required to open up the retrolin-gual space. In patients with life threatening symptoms (right heart failure/cor pulmonale, oxygen saturation <70%, comorbid cardiopulmonary disease) who have failed other measures, the only “cure” for OSA is a tracheotomy.Other Tonsillar Pathology. Unilateral tonsillar hypertrophy is mostly likely benign but can also be the result of Mycobac-terium tuberculosis, atypical mycobacterium, fungi, or Actino-myces. With the epidemic rise in incidence of oropharyngeal Brunicardi_Ch18_p0613-p0660.indd 62301/03/19 5:23 PM 624SPECIFIC CONSIDERATIONSPART IIcancers, neoplasms (squamous cell carcinoma and lymphoma) have increasingly also presented as tonsillar asymmetry.36 Man-agement of these lesions is dependent on the pretest probability of malignancy and the type of malignancy. If squamous cell car-cinoma is suspected, then a

1	presented as tonsillar asymmetry.36 Man-agement of these lesions is dependent on the pretest probability of malignancy and the type of malignancy. If squamous cell car-cinoma is suspected, then a biopsy alone is sufficient so as to not impact the possibility of other future surgical interventions such as transoral robotic surgery. If lymphoma or a nonmalignant pathology is suspected, tonsillectomy is often recommended for diagnostic and therapeutic reasons, and the specimen should be sent fresh to pathology for a lymphoma protocol workup, bacte-rial and fungal culture, and gram stain. Pharyngitis may also be seen in immune-mediated conditions such as erythema multi-forme, bullous pemphigoid, and pemphigus vulgaris.Benign Conditions of the LarynxHoarseness is the most common presenting symptom for patients with a voice complaint. Other complaints include breathiness, weakness/hypophonia, aphonia, and pitch breaks. Voice disor-ders affect a large range of patient ages, occupations, and

1	for patients with a voice complaint. Other complaints include breathiness, weakness/hypophonia, aphonia, and pitch breaks. Voice disor-ders affect a large range of patient ages, occupations, and socio-economic statuses and affect both genders equally. They can be associated with dysphagia, globus sensation, laryngopharyngeal reflux (LPR) disease and, rarely, airway obstruction.37 Smoking can both cause and aggravate preexisting benign laryngeal con-ditions and raises the suspicion of malignancy often requiring a biopsy to exclude this diagnosis.Any discussion of laryngeal disorders should start with a review of the anatomy of the vocal cords (Fig. 18-13). The true vocal cords are formed from stratified squamous epithelium, beneath which is the superficial lamina propria (in Reinke’s space). Beneath this is the ligament that includes the middle and deep lamina propria. Beneath this ligament is the muscular layer that includes the thyroarytenoid muscle or vocalis. The cover-body theory

1	Beneath this is the ligament that includes the middle and deep lamina propria. Beneath this ligament is the muscular layer that includes the thyroarytenoid muscle or vocalis. The cover-body theory describes the freely mobile cover (mucosa and Reinke’s space) over the more rigid body (vocal ligament and vocalis).38Membranous vocal cord lesions have been notoriously dif-ficult to classify reliably; however, increased availability of vid-eostroboscopic examination and standardized definitions have improved the classification of these lesions.39 These lesions are usually mid cord because that is the site of maximal lateral displacement and amplitude. Vocal fold nodules are typically bilateral, fairly symmetric, and with normal or mild impairment of the mucosal wave, and they almost always resolve with voice therapy. A vocal fold polyp is more often unilateral than bilat-eral, is exophytic, and is associated with unorganized gelatinous debris in the subepithelial space. These can be

1	resolve with voice therapy. A vocal fold polyp is more often unilateral than bilat-eral, is exophytic, and is associated with unorganized gelatinous debris in the subepithelial space. These can be hemorrhagic as is often seen in males secondary to capillary rupture within the mucosa by shearing forces during voice abuse. Hemorrhagic polyps are seen more often in patients on anticoagulants. These lesions usually fail conservative measures (voice rest, voice therapy, smoking cessation, and reflux management) usually requiring micorlaryngeal surgery to remove the lesion while preserving normal mucosa. Vocal fold cyst is an encapsulated lesion within the subepithelial or ligamentous space and is asso-ciated with reduced mucosal wave. It typically does not resolve with voice therapy. These lesions require microlaryngeal sur-gery for complete removal of the cyst while preserving the over-lying mucosa, and this surgery can be performed with cold steel or carbon dioxide (CO2) laser. A fibrous

1	require microlaryngeal sur-gery for complete removal of the cyst while preserving the over-lying mucosa, and this surgery can be performed with cold steel or carbon dioxide (CO2) laser. A fibrous mass of the vocal fold is amorphous fibrous material within the subepithelial space or EpiglottisEpitheliumLayers oflamina propriaSuperficialIntermediateDeepVocalisHyoid boneCushion ofepiglottisThyroidcartilageFalse vocal cordLaryngealsinusTrue vocalcordThyroarytenoidmuscleCricoid cartilageAryteno-epiglottideanfoldFigure 18-13. Coronal view of the larynx demonstrate the supraglottic, glottic and subglottis (LEFT) and the layers of the true vocal cord (RIGHT).Brunicardi_Ch18_p0613-p0660.indd 62401/03/19 5:23 PM 625DISORDERS OF THE HEAD AND NECKCHAPTER 18ligament often associated with reduced mucosal wave, and it also does not resolve with voice therapy.Reinke’s edema is characterized by edema in the superfi-cial lamina propria of the vocal cord. Edema is thought to arise from injury to the

1	wave, and it also does not resolve with voice therapy.Reinke’s edema is characterized by edema in the superfi-cial lamina propria of the vocal cord. Edema is thought to arise from injury to the capillaries that exist in this layer, with sub-sequent extravasation of fluid. The etiology is multifactorial: smoking, LPR, hypothyroidism, and vocal misuse.40 This pathol-ogy is more common in women (because they present early due to a deep vocal pitch change in their voice) and heavy smokers. The physical examination findings are typically bilateral. Sur-gery typically involves microlaryngoscopy with removal of the gelatinous debris in Reinke’s space with trimming of the excess mucosa. However, smoking cessation and surgery do not fully reverse the structural abnormalities due to the presence of pos-sible structure alterations in fibroblasts caused by the toxicity of cigarette components, resulting in uncontrolled production of fibrous matrix in the lamina propria, thus preventing complete

1	pos-sible structure alterations in fibroblasts caused by the toxicity of cigarette components, resulting in uncontrolled production of fibrous matrix in the lamina propria, thus preventing complete vocal recovery.41Laryngeal granulomas typically occur in the posterior lar-ynx on the arytenoid mucosa (Fig. 18-14). These lesions are typically multifactorial: chronic throat clearing, phonotrauma, endotracheal intubation, compensatory supraglottic squeeze from vocal fold paralysis, and LPR.42 The majority of these lesions (82%) disappear within 48 weeks with conservative measures such as voice therapy, vocal rest, oral steroids, inhaled steroids, and proton pump inhibitors.42 Botulinum toxin of thy-roarytenoid and lateral cricoarytenoid muscles can be used as first-line treatment in patients who prefer a chemically activated voice rest regiment.42 LPR appears to be the most important contributing factor,42 and when aggressive conservative and medical therapy has failed, a Nissen

1	patients who prefer a chemically activated voice rest regiment.42 LPR appears to be the most important contributing factor,42 and when aggressive conservative and medical therapy has failed, a Nissen fundoplication may be indicated. Surgery is rarely required for patients with laryngeal granulomas because it does not address the underlying etiol-ogy and is frequently associated with recurrence. Nonetheless, excision is sometimes required in patients with airway obstruc-tion or the suspicion of malignancy. Careful preservation of the arytenoid perichondrium intraoperatively is required to assist with reepithelialization and to decrease the risk of recurrence postoperatively.Recurrent respiratory papillomatosis (RRP) is pathophysi-ologically associated with human papillomavirus (HPV) within the mucosa of the upper aerodigestive tract. The glottis and supra-glottis are the two most common involved subsites. HPV 6 and 11 are the most often implicated types; however, LPR and herpes simplex

1	the mucosa of the upper aerodigestive tract. The glottis and supra-glottis are the two most common involved subsites. HPV 6 and 11 are the most often implicated types; however, LPR and herpes simplex virus (HSV) type-2 are risk factors of adult-onset RRP.43 The disorder typically presents in early childhood (juvenile-onset RR; JoRRP) secondary to HPV acquisition during vaginal deliv-ery; however, children born by caesarean section are also at risk for the disease. JoRRP usually resolves around puberty but can progress into adulthood. Adult-onset RRP is less severe and is more likely to involve extralaryngeal subsites. There is no cure for RRP. Surgery excision is used to improve voice and airway symptoms in a palliative fashion. Surgical excision in the operat-ing room involves microlaryngoscopy with the use of the laser (CO2 for bulky disease or KTP for more superficial disease) or the use of a microdebrider. The microdebrider has been dem-onstrated to have superior voice outcomes in

1	with the use of the laser (CO2 for bulky disease or KTP for more superficial disease) or the use of a microdebrider. The microdebrider has been dem-onstrated to have superior voice outcomes in JoRRP; however, CO2 laser is the most commonly used operative ablative tech-nique used in adults.44 Recent advances have made it possible to treat a select group of adult RRP patients in the office using the KTP laser, typically for those with a lower disease burden.45 Several adjuvant treatments are used to increase the intersurgical interval, including intralesional cidofovir injection, oral indole-3-carbinol, oral methotrexate, and retinoic acid. In addition to preventing RRP in some patients, the HPV vaccine has also been demonstrated to increase the intersurgical interval in the most aggressive JoRRP patients.46,47Leukoplakia is a white patch seen on mucosa that can be wiped off on physical examination. This can be seen anywhere in the upper aerodigestive tract. In the larynx, this is

1	JoRRP patients.46,47Leukoplakia is a white patch seen on mucosa that can be wiped off on physical examination. This can be seen anywhere in the upper aerodigestive tract. In the larynx, this is typically seen on the superior surface of the true vocal cords and may represent squamous hyperplasia, dysplasia, and/or carcinoma with an associated risk of malignant transformation of 1% to 3% in hyperplastic lesions and 10% to 30% in dysplastic lesions. Lesions that are not overtly suspicious for malignancy, particularly in patients without a strong smoking or alcohol history, can be managed conservatively (increased hydration, elimination of poor vocal habits, phonotrauma, and manage-ment of LPR) for 1 month before reevaluation with fiberoptic laryngoscopy. Any lesions that progress, persist, or recur could have microlaryngoscopy with complete excision. Similarly, because erythroplasia and ulceration are more suggestive of malignancy, these lesions also require an excisional biopsy in the

1	or recur could have microlaryngoscopy with complete excision. Similarly, because erythroplasia and ulceration are more suggestive of malignancy, these lesions also require an excisional biopsy in the operating room.The most common cause of unilateral vocal cord paresis is iatrogenic in origin, following surgery to the thyroid, parathy-roid, carotid, spine through an anterior approach,48 or cardiotho-racic structures.49 It is therefore very important that all patients undergoing thyroid surgery receive preoperative visualization of the larynx, usually in the form of fiberoptic nasolaryngos-copy, although an indirect mirror exam can be used if adequate visualization is possible.50 Postthyroidectomy visualization may also be required to document normal vocal cord move-ment. Less common causes include malignancy of structures near the recurrent laryngeal nerve (RLN) from the skull base jugular foramen to the mediastinum. In the pediatric population, there can be neurologic causes, the

1	include malignancy of structures near the recurrent laryngeal nerve (RLN) from the skull base jugular foramen to the mediastinum. In the pediatric population, there can be neurologic causes, the most common of which is the Arnold-Chiari malformation.51 Overall, the left vocal cord is more commonly involved secondary to the longer course of the RLN on that side. Other rare etiologies include trauma, intu-bation injury, atypical infections, and neurotoxic medications. Patients typically present with a weak breathy voice and may have aspiration secondary to diminished supraglottic sensa-tion if the proximal vagal nerve or superior laryngeal nerve is involved. RLN injury is also associated with delayed relaxation Figure 18-14. Laryngeal granuloma.Brunicardi_Ch18_p0613-p0660.indd 62501/03/19 5:23 PM 626SPECIFIC CONSIDERATIONSPART IIof the cricopharyngeus muscle that can lead to dysphagia and decreased sensation in the hypopharynx, which can cause pool-ing of secretions. In children,

1	5:23 PM 626SPECIFIC CONSIDERATIONSPART IIof the cricopharyngeus muscle that can lead to dysphagia and decreased sensation in the hypopharynx, which can cause pool-ing of secretions. In children, stridor, weak cry, and airway com-promise may be presenting symptoms, whereas in adults this is rarely the case unless there is bilateral vocal cord paralysis. When an obvious cause is not identified after a thorough history and physical examination including fiberoptic nasolaryngos-copy, then a more comprehensive workup is required. A workup should not include autoimmune serology as a screen because this is low yield, but this can be included if there is a suspicion of autoimmune disorders. Imaging, in the form of a CT scan, is the mainstay of the workup and should include the skull base to the mediastinum. Repeat imaging is beneficial in this population within a 2-year period because many patients have undiagnosed small malignancies as the primary cause of their paralysis that are too

1	the mediastinum. Repeat imaging is beneficial in this population within a 2-year period because many patients have undiagnosed small malignancies as the primary cause of their paralysis that are too small to detect on initial imaging.52 Laryngeal electro-myography can assist with identifying whether the paresis is a result of a paralysis or cricoarytenoid joint fixation/disloca-tion. It can also help prognosticate a paralysis. This is, however, rarely used in practice. Despite an extensive workup, 20% to 35% of cases are idiopathic.The management of bilateral vocal cord paralysis almost always requires a tracheotomy because the cords are left in a paramedian position leaving a slit light glottic aperture. If the paralysis is permanent, then a cordectomy with or without ary-tenoidectomy can be used to open up the airway in an attempt to eventually decannulate the patient. However, this has obvi-ous implications for voice with a weak and breathing voice. Many patients with a unilateral

1	be used to open up the airway in an attempt to eventually decannulate the patient. However, this has obvi-ous implications for voice with a weak and breathing voice. Many patients with a unilateral paralysis compensate when the cord is in the paramedian position using supraglottic structure and the contralateral cord on their own or with speech therapy. However, in patients with a less than adequate voice-related quality of life, four techniques have been used to surgically manage patients with a unilateral vocal cord paralysis: injection laryngoplasty, medialization thyroplasty, arytenoid adduction, and laryngeal reinnervation. Injection laryngoplasty involves injecting a temporary filler medial to the vocalis into the liga-ment at the posterior and midmembranous vocal cord. This can be performed in the office or in the operating room, depend-ing on the comfort of the surgeon and patient characteristics. Materials used include autologous (fat, collagen) or alloplastic

1	This can be performed in the office or in the operating room, depend-ing on the comfort of the surgeon and patient characteristics. Materials used include autologous (fat, collagen) or alloplastic (hydroxyapatite, hyaluronic acid, micronized cadaveric human collagen) compounds. Early medialization is recommended in patients with mediastinal and thoracic malignancies because it is safe and has been shown to improve quality of life in a palli-ative setting.53 Teflon is historic and is no longer used because of its granulomatous side effects on the larynx. A more per-manent medialization can be performed using a medialization thyroplasty, during which a small window is created in the inferolateral aspect of the thyroid cartilage and a submucosal-carved silastic block is placed in the operating room with the patient under neurolept anesthetic so that vocalization and flex-ible laryngoscopic visualization of the larynx can be improved (Fig. 18-15). In some cases, this is not enough of a

1	room with the patient under neurolept anesthetic so that vocalization and flex-ible laryngoscopic visualization of the larynx can be improved (Fig. 18-15). In some cases, this is not enough of a medialization due to a large posterior glottic chink, and an arytenoid adduction is required to provide better closure of the posterior glottis and supraglottis with ensuing improved vocal outcomes. This is a technically challenging procedure that is rarely required, but in select patients it is associated with significant improvements in voice. Lastly, laryngeal reinnervation, typically with the ansa cervicalis that supplies motor function to the strap muscles, can also be performed. This is the best approach in patients who have had a recurrent laryngeal nerve severed during a central or upper mediastinal neck procedure because it is in the field.54 Multiple studies demonstrate favorable outcomes; however, no significant differences between treatment arms has been demon-strated based on

1	mediastinal neck procedure because it is in the field.54 Multiple studies demonstrate favorable outcomes; however, no significant differences between treatment arms has been demon-strated based on perceptual, acoustic, quality of life, and laryn-goscopic outcomes.55Vascular LesionsVascular lesions can be broadly classified into two groups: hem-angiomas and vascular malformations.56Hemangiomas. Hemangiomas are the most common vascular lesion present in infancy and early childhood. Infantile heman-giomas present largely within the first few weeks of life. Initially they proliferate (2 weeks to 1 year), and then they begin to invo-lute (1–7 years) until they have fully involuted, leaving the child with redundant skin, scar, or a fatty lesion. Children with large facial infantile hemangiomas benefit from regular neurological examinations and brain MRI to rule out PHACES syndrome (Posterior fossa malformations, Hemangiomas, Arterial lesions, Cardiac abnormalities/aortic coarctation, Eye

1	benefit from regular neurological examinations and brain MRI to rule out PHACES syndrome (Posterior fossa malformations, Hemangiomas, Arterial lesions, Cardiac abnormalities/aortic coarctation, Eye abnormalities). Only 10% of these lesions require early intervention because of impairment of vision or swallowing, or airway compromise. Early intervention can include medical management, such as systemic steroids, intralesional steroids, intralesional interferon α-2a, or photocoagulation therapy, and surgical management, including excision with CO2 laser/microdebrider and tracheot-omy. Systemic steroids assist with rapidly proliferating lesions until the child reaches approximately one year of age; however, it is associated with growth retardation and immune suppres-sion. Intralesional interferon α-2a has been largely abandoned because it is a daily subcutaneous injection and is associated Figure 18-15. Hand carved silastic block for thyroplasty.Brunicardi_Ch18_p0613-p0660.indd

1	interferon α-2a has been largely abandoned because it is a daily subcutaneous injection and is associated Figure 18-15. Hand carved silastic block for thyroplasty.Brunicardi_Ch18_p0613-p0660.indd 62601/03/19 5:23 PM 627DISORDERS OF THE HEAD AND NECKCHAPTER 18with significant neurological side effects, including spastic diplegia. Photocoagulation therapy with either the flashlamp-pumped pulsed-dye laser (FPDL), the potassium titanyl phos-phate (KTP) laser, or the neodymium yttrium-aluminum garnet (Nd:YAG) laser, is repeated every 4 to 6 weeks until the lesion disappears. A randomized trial recently demonstrated that pro-pranolol was effective at a dose of 3mg/kg per day for 5 months in the treatment of infantile hemangioma with a very acceptable and low side-effect profile.57 Other groups have had success at discontinuing propranolol at 1 year of age with excellent out-comes.58 For patients who do not require early intervention, the lesion is observed every 3 months for involution

1	have had success at discontinuing propranolol at 1 year of age with excellent out-comes.58 For patients who do not require early intervention, the lesion is observed every 3 months for involution after the pro-liferative phase has ended. Surgery is considered if regression has not occurred by 5 years of age because the cosmetic result is less likely to be satisfactory.Congenital hemangiomas differ from infantile heman-giomas in that they reach their maximal size at birth and do not have a proliferative phase. There are two subtypes: rapidly involuting (RICH), which typically disappears by 1 of age with minimal fatty appearance upon resolution, and noninvoluting (NICH). The management is similar to infantile hemangiomas with the exception that medical management is not typically necessary.Vascular Malformations. Vascular malformations, in contrast to infantile hemangioma, are always present at birth, although they may not be apparent for a few months. Although they do not have a

1	Malformations. Vascular malformations, in contrast to infantile hemangioma, are always present at birth, although they may not be apparent for a few months. Although they do not have a proliferative phase, they grow with the patient, have hormonal growth spurts and do not involute.59 Vascular mal-formations can be classified as low flow (capillary, venous, lymphatic, and mixed), which comprise approximately two-thirds of all vascular malformations, or high flow (arteria and arteriovenous).Capillary malformations arise from the cutaneous super-ficial plexus and are made up of capillary and postcapillary venules with a pink, red, or purple macular-papular appearance. Venous malformations arise from dilated vascular channels lined by normal endothelium; therefore, they are soft, compress-ible, and nonpulsatile. If they are superficial, they will increase in size with Valsalva or dependent positioning. They can grow suddenly with trauma or in association with hormonal changes. Lymphatic

1	and nonpulsatile. If they are superficial, they will increase in size with Valsalva or dependent positioning. They can grow suddenly with trauma or in association with hormonal changes. Lymphatic malformations typically present at birth with the majority (90%) being identified by 2 years of age. They can be macrocystic (>2 cm), microcystic (≤2 cm), or a combina-tion. They are most commonly found in the head and neck, particularly on the neck, and on physical examination they are soft and doughy with normal overlying skin. Infrahyoid lesions tend to be macrocystic, well circumscribed, and discrete and can be totally excised, whereas suprahyoid lesions are typically microcystic, infiltrative, and excision is usually incomplete. On MRI, the best imaging modality for this malformation, a sep-tated mass with low-intensity signal on T1 and high-intensity signal on T2 is noted. They grow slowly with the patient but can have a sudden increase in size with hemorrhage or infection. Rarely, they

1	mass with low-intensity signal on T1 and high-intensity signal on T2 is noted. They grow slowly with the patient but can have a sudden increase in size with hemorrhage or infection. Rarely, they cause airway compromise, feeding difficulties, and failure to thrive.Treatment of vascular malformations is based on depth, size, and growth pattern. Capillary malformations are typically treated with the pulsed dye laser (585 nm). Venous lesions can be treated with the KTP laser (532 nm) or the Nd:YAG laser (1064 nm), sclerotherapy, and, in select cases, complete surgi-cal excision is possible. Arteriovenous malformations are rare but typically require surgical excision with negative margins often after embolization. Lymphatic malformations are typically treated at least in part with surgical excision, although this is less successful for microcystic lesions. OK-432 is lyophilized low virulence S pyogenes cultured in penicillin. It is used as a sclerotherapy agent for lymphatic malformations

1	although this is less successful for microcystic lesions. OK-432 is lyophilized low virulence S pyogenes cultured in penicillin. It is used as a sclerotherapy agent for lymphatic malformations and has a 94% response rate in macrocystic lesions, a 63% response rate in mixed macromicrocystic lesions, and no response in micro-cystic lesions.60TRAUMA OF THE HEAD AND NECKSoft TissueSoft tissue trauma of the head and neck is managed with the same general surgical principles as any other body subsite with a few particularities. Most lacerations can be closed primarily if there is not soft tissue loss; even some devitalized soft tis-sue should be preserved because of the excellent blood sup-ply to head and neck tissue that allows it to recover at a higher rate. Thus, minimal debridement is usually required. Thor-ough irrigation to remove foreign bodies and clean the tissue is required. This is followed by a careful layered closure. On the face, the deep layers are usually closed with a 3-0 or

1	required. Thor-ough irrigation to remove foreign bodies and clean the tissue is required. This is followed by a careful layered closure. On the face, the deep layers are usually closed with a 3-0 or 4-0 Vicryl/Polysorb after a minimal amount of undermining, and interrupted 5-0 or 6-0 Prolene or Nylon is used for the skin. These sutures are removed at 5 days on the face. Antibiotics are reserved for through-and-through mucosal lacerations, con-taminated wounds, bite injuries, and when delayed closure is performed (>72 hours). The chosen antibiotic should cover S aureus. Patients are instructed to avoid sunlight because this can cause pigmentary abnormalities in the suture line as it heals and matures over the first year.Eyelid lacerations are closed in layers with careful reap-proximation of the orbicularis oculi as a separate layer. Another important layer to reapproximate separately is the gray line (con-junctival margin) so as to avoid height mismatch or lid notching. Lip injuries

1	of the orbicularis oculi as a separate layer. Another important layer to reapproximate separately is the gray line (con-junctival margin) so as to avoid height mismatch or lid notching. Lip injuries follow the same principle with a three-layer closure involving the orbicularis oris, which is the strength layer, fol-lowed by careful reapproximation of the vermillion border to avoid a step-deformity (Fig. 18-16). Of course, a mucosal layer closure may also be required for through-and-through defects. Rarely, locoregional flaps or grafts are required for closure when greater than one-fourth of the eyelid width or one-third of the lip width is missing. Auricular hematoma is managed with prompt incision and drainage followed by bolstering technique; anteriorly and posteriorly placed dental pledgets secured with through-and-through sutures. These are to remain in place for at least 4 days to prevent reaccumulation of the hematoma and to prevent a cauliflower ear deformity. Auricular

1	pledgets secured with through-and-through sutures. These are to remain in place for at least 4 days to prevent reaccumulation of the hematoma and to prevent a cauliflower ear deformity. Auricular lacerations are typically closed primarily with perichondrial sutures to preserve the precarious cartilage blood supply followed by a primary clo-sure of the skin, making sure to cover the cartilage to prevent chondritis. Given the rich vascular supply to the face and neck, many soft-tissue components that appear devitalized will indeed survive, and therefore minimal debridement of devitalized tissue is required.Facial lacerations resulting in facial nerve injury are not explored if they are anterior to a vertical line dropped from the lateral cantus as there is excellent collateral innervation in the anterior midface. Posterior to this line, the nerve should be repaired, primarily if possible, using 8-0 to 10-0 monofila-ment suture to approximate the epineurium under the operative

1	in the anterior midface. Posterior to this line, the nerve should be repaired, primarily if possible, using 8-0 to 10-0 monofila-ment suture to approximate the epineurium under the operative Brunicardi_Ch18_p0613-p0660.indd 62701/03/19 5:23 PM 628SPECIFIC CONSIDERATIONSPART IImicroscope. If primary reapproximation is not possible due to a missing segment, cable nerve grafts can be performed using the sural nerve or the greater auricular nerve. If the buccal branch is injured, this raises suspicion regarding injury to the parotid duct, which lies along an imaginary line drawn from the tragus to the midline upper lip. The duct should be repaired over a 22-gauge stent or marsupialized into the oral cavity.Facial FracturesThe most common facial fracture involves the mandible. Fig. 18-17 demonstrates the most common sites of fracture, which include the condyle (36%), body (35%), and angle (20%). In most cases, more than one site is involved due to reciprocating forces. The vector

1	demonstrates the most common sites of fracture, which include the condyle (36%), body (35%), and angle (20%). In most cases, more than one site is involved due to reciprocating forces. The vector forces from the muscles of mastication, vertical from the masseter and horizontal from the pterygoid muscles, can cause a fracture to be favorable or unfavorable depending on the angle of the fracture line. After taking a history and performing a physical examination, imaging is performed in the form of a Panorex or a CT scan. Where closed reduction can be achieved, patients are placed in maxillomandibular fixation (MMF) with arch bars applied via circumdental wiring, and these are left in place for 4 to 6 weeks depending on patient factors and the fracture location. In elderly patients, this is kept in for 6 to 8 weeks. In children and patients with condylar fractures only 2 to 3 weeks is required, and this is important to prevent condylar ankylosis. During this time, patients are placed on

1	in for 6 to 8 weeks. In children and patients with condylar fractures only 2 to 3 weeks is required, and this is important to prevent condylar ankylosis. During this time, patients are placed on a liquid diet and are provided with wire cutters in case of aspiration or airway emergency. Open reduction and fixation is indicated in patients with open, comminuted, displaced, or unfavorable fractures. In these patients, MMF is usually only temporary with a soft diet starting almost immediately in the postoperative setting. Because the MMF is temporary with rigid fixation, it is per-formed usually using the 4-point fixation technique, where the maxilla and mandible are held in occlusion by wires attached to intraoral cortical bone screws, with two screws above and below the occlusal line anteriorly. This is a benefit of open reduction and internal fixation because prolonged MMF is associated with gingival and dental disease, as well as with significant weight loss and malnutrition, during

1	This is a benefit of open reduction and internal fixation because prolonged MMF is associated with gingival and dental disease, as well as with significant weight loss and malnutrition, during the fixation period. After fixation, the fracture is exposed, more commonly from a transcervical compared to a transoral approach. Care is made not to injure the marginal mandibular branch of the facial nerve during this exposure. A rigid, locking, load-bearing mandibular plate is used. In edentulous patients, determining the baseline occlusion is of less significance because dentures may be refashioned once healing is complete.Midface fractures are rarely isolated and include multiple subsites. However, isolated zygoma fractures are typically dis-placed inferior inferiorly and medially with disruption of the suture lines between the temporal, frontal, and maxillary bones and the zygoma. If multiple zygoma fractures are present or if the zygomatic arch is significantly displaced, a coronal

1	of the suture lines between the temporal, frontal, and maxillary bones and the zygoma. If multiple zygoma fractures are present or if the zygomatic arch is significantly displaced, a coronal incision is required to perform the reduction and fixation. However, if it is an isolated depressed fracture, a Gilles reduction can be achieved inferiorly (transorally) or superiorly (along temporalis muscle). The pathophysiology of orbital blow-out fractures is (a) hydraulic from increased intraocular pressure or (b) buckling from direct bone conduction. This requires surgical intervention if there is a defect of >2 cm2 or >50% of the floor with herniation.61 A forced duction test, where the muscular attachment of the inferior oblique is grasped with forceps and manipulated to determine passive ocular mobility, is performed to ensure that there is not inferior rectus entrapment. If there is entrapment, this would also result in diploplia with upward gaze. Blowout fractures demonstrating

1	ocular mobility, is performed to ensure that there is not inferior rectus entrapment. If there is entrapment, this would also result in diploplia with upward gaze. Blowout fractures demonstrating significant entrapment or enophthal-mos are treated by orbital exploration and reinforcement of the floor with titanium mesh, hydroxyapatite, or split calvarial bone grafts. Sometimes, the anterior maxillary bone that has been fractured and is accessed in the process of repairing other factures can also be used.62There are three classic patterns of more extensive mid-face fractures: Le Fort I, II, and III. However, fractures rarely follow this exact pattern, and the two sides of the face may have different Le Fort fractures. Nonetheless, a full under-standing of midface buttresses is central in understanding these fractures (Fig. 18-18). There are three vertical buttresses: the nasofrontal-maxillary, the frontozygomaticomaxillary, and Key stitchFigure 18-16. Approximation of the vermilion

1	understanding these fractures (Fig. 18-18). There are three vertical buttresses: the nasofrontal-maxillary, the frontozygomaticomaxillary, and Key stitchFigure 18-16. Approximation of the vermilion border is the key step in the repair of lip lacerations.3%3%36%2%20%21%14%Figure 18-17. Sites of common mandible fractures.Brunicardi_Ch18_p0613-p0660.indd 62801/03/19 5:23 PM 629DISORDERS OF THE HEAD AND NECKCHAPTER 18pterygomaxillary. There are five horizontal buttresses: the fron-tal bone, nasal bones, upper alveolus, zygomatic arches, and the infraorbital region.63 Signs of midface fractures include subcon-junctival hemorrhage, ocular signs/symptoms, malocclusion, facial asymmetry, midface hypoesthesia (V2), hematoma, and a mobile maxillary complex. Transverse maxillary alveolus frac-tures above the teeth are Le Fort I fractures, which may result in a mobile hard palate. When this fracture extends superiorly to include the nasofrontal buttress, medial orbital wall, and even as

1	above the teeth are Le Fort I fractures, which may result in a mobile hard palate. When this fracture extends superiorly to include the nasofrontal buttress, medial orbital wall, and even as high as the infraorbital rim and zygomaticomaxillary articula-tion laterally, it is considered a Le Fort II. Mobility includes the palate, nasal dorsum, which is separated from the upper face, and the inferomedial aspect of the orbital rim. When the frac-ture disrupts the frontozygomaticomaxillary, frontomaxillary, and frontonasal suture line, there craniofacial disjunction, a Le Fort III fracture. Of note, all of the Le Fort fractures involve the pterygoid plates posteriorly (Fig. 18-19).Temporal Bone FracturesTemporal bone fractures occur in approximately one fifth of skull fractures. Temporal bone fractures were previously clas-sified as longitudinal or transverse describing the path along the temporal bone of the fracture line, but this has been largely replaced by the more relevant otic

1	bone fractures were previously clas-sified as longitudinal or transverse describing the path along the temporal bone of the fracture line, but this has been largely replaced by the more relevant otic capsule sparing or involv-ing classification given that most fractures are oblique.64 Otic capsule sparing fractures present with conductive hearing loss, ossicular injury, bloody otorrhea, and labyrinthine concussion.65 The facial nerve is rarely injured nor cerebrospinal fluid (CSF) leak common with this fracture pattern. However, in patients with otic capsule involving temporal bone fractures, typically caused by occipitomastoid impact, sensorineural hearing loss, vestibular dysfunction, facial nerve paralysis, and CSF leak are far more common.65 Regardless of the fracture pattern, when CSF leak is suspected, it usually resolves with conservative measures including bed rest, elevation of the head of the bed, stool softeners, and avoiding sneezing or straining. In some cases, a CSF

1	CSF leak is suspected, it usually resolves with conservative measures including bed rest, elevation of the head of the bed, stool softeners, and avoiding sneezing or straining. In some cases, a CSF drain can be placed if there is a delay in spontane-ous resolution. Rarely will surgical repair be required. Unlike CSF leaks with temporal bone fractures, the facial nerve needs to be assessed and managed urgently. An incomplete or delayed facial nerve paralysis almost always resolves spontaneously with conservative measures, including oral steroids. An imme-diate complete paralysis that does not recover within 1 week should be prognosticated to consider nerve decompression. Electroneurography (ENoG), EMG, and nerve stimulation tests have been used to help determine which patients with delayed-onset complete paralysis will benefit from surgical decompres-sion. The finding of >90% degeneration more than 72 hours after the onset of complete paralysis is considered an indica-tion for

1	delayed-onset complete paralysis will benefit from surgical decompres-sion. The finding of >90% degeneration more than 72 hours after the onset of complete paralysis is considered an indica-tion for surgery.66 A nerve excitability test, where thresholds are increased to elicit visible muscle contraction on each side, can indicate advanced degeneration when there is a difference of >3.0 to 3.5 mA between sides. Whether surgical intervention is indicated or not for facial nerve paresis, it is crucial to pro-tect the eye because a corneal drying and abrasion can lead to blindness in the abscess of eye closure and a blink reflex. This requires application of ocular lubricant at night with the eye taped shut, frequent artificial tears application while awake, and a humidity chapter.67TUMORS OF THE HEAD AND NECKSquamous cell carcinoma (SCC) comprises >90% of all of the malignant pathology of the mucosal lining of the upper aerodi-gestive tract. Naturally, a discussion of tumors of the head

1	THE HEAD AND NECKSquamous cell carcinoma (SCC) comprises >90% of all of the malignant pathology of the mucosal lining of the upper aerodi-gestive tract. Naturally, a discussion of tumors of the head and neck typically focuses on this pathology presenting from the lips and oral cavity to the larynx and hypopharynx. Management of these tumors requires a systematic approach.The ideal treatment protocol varies by subsite, stage, patient comorbidity, and center preference/experience. Given the relative rarity of these tumors, multidisciplinary management is of the utmost importance to provide the patient with a balanced perspective. This can be performed in the form of a multidisciplinary clinic where radiation and surgical oncologists simultaneously see the patient or through a tumor board where a new patient’s history, physical examination findings, imaging, and prior pathology Frontal barLateralzygomatico-maxillarybuttressesMedial nasomaxillary buttressesFigure 18-18. Major buttresses

1	a new patient’s history, physical examination findings, imaging, and prior pathology Frontal barLateralzygomatico-maxillarybuttressesMedial nasomaxillary buttressesFigure 18-18. Major buttresses of the midface.IIIIIIFigure 18-19. Classic Le Fort fracture patterns.Brunicardi_Ch18_p0613-p0660.indd 62901/03/19 5:23 PM 630SPECIFIC CONSIDERATIONSPART IIspecimens are reviewed. This encourages discussion from multiple points of view concerning the most appropriate treatment options available. In addition to radiation and surgical oncology, medical oncology, dentistry, speech language pathologists, radiologists, and pathologists contribute to the decision-making in this patient population. Some of the greatest advances in head and neck oncology over the last several decades include the development of standardized organ preservation protocols, advances in free flap reconstruction with microvascular techniques, and vaccinations. The future of head and neck oncology is bright with advances

1	of standardized organ preservation protocols, advances in free flap reconstruction with microvascular techniques, and vaccinations. The future of head and neck oncology is bright with advances in molecular biology, immunotherapy, and preventative methods with vaccination. These have the potential of significantly decreasing incidence rates and improving survival and quality of life for those with the disease.Etiology and EpidemiologyThe main etiological factors associated with head and neck cancers are tobacco products and alcohol. Overall, there has been a decline in incidence of head and neck cancers of the oral cavity and larynx/hypopharynx subsites,68 likely related to public health campaigns and government taxation policies as it relates to cigarette consumption.69 Similarly, the incidence of head and neck cancer between countries varies widely and is strongly associated with the incidence of cigarette smok-ing. Cigarette smoking triples the likelihood of developing an oral

1	incidence of head and neck cancer between countries varies widely and is strongly associated with the incidence of cigarette smok-ing. Cigarette smoking triples the likelihood of developing an oral cavity cancer, while the addition of alcohol synergistically increases the likelihood by 10to 15-fold.70 The risk increases as the number of years smoking and number of cigarettes smoked per day increases. Individuals who both smoke (two packs per day) and drink (four units of alcohol per day) had a 35-fold increased risk for the development of a carcinoma compared to controls.71The preoperative and perioperative periods are excellent opportunities for head and neck oncologists to pursue a smok-ing cessation intervention. Continued smoking after completion of treatment is associated with a 3to 4-fold increased risk of developing a second primary or recurrent tumor.72-74 A study assessing patients diagnosed with a new head and neck cancer demonstrated that of the patients that were smoking

1	4-fold increased risk of developing a second primary or recurrent tumor.72-74 A study assessing patients diagnosed with a new head and neck cancer demonstrated that of the patients that were smoking at diagno-sis, only 54% were able to quit, highlighting the difficulty this population has with smoking cessation.75Betel nut/quid chewing, which is a product of the areca catechu tree, is endemic to some parts of Asia and India, and in these regions oral cavity cancer is one of the most common can-cers.76,77 Betel nut when chewed acts as a mild stimulant similar to that of coffee but can be associated with submucous fibrosis that adds an additional challenge in the management of patients who present with a concurrent oral cavity cancer.77 These prod-ucts are associated with particular subsites secondary to direct contact (e.g., buccal mucosa) as well as subsites with depen-dent saliva drainage (e.g., floor of mouth, mandibular alveolus, and wet lip). Reverse smoking, where the lighted

1	secondary to direct contact (e.g., buccal mucosa) as well as subsites with depen-dent saliva drainage (e.g., floor of mouth, mandibular alveolus, and wet lip). Reverse smoking, where the lighted portion of the tobacco product is placed within the mouth during inhalation is also associated with oral cavity cancer, specifically hard palate carcinoma. The risk for this cancer is 47 times greater in patients that exhibit this behavior compared to nonsmokers.78In Europe and North America there has been an increas-ing interest in decriminalizing marijuana smoking. There is a strong correlation between this activity and head and neck can-cers (OR 2.5; 95% CI 1.1–6.6) when compared to nonusers.79 Furthermore, there is a dose-response relationship that is stron-ger in young patients (55 years of age or less). Ultraviolet light VermilionBuccal mucosaHard palateSoft palateRetromolar trigoneCircumvallate papillaeLower gingivaPalatine raphePalatine tonsilFigure 18-20. Oral cavity

1	(55 years of age or less). Ultraviolet light VermilionBuccal mucosaHard palateSoft palateRetromolar trigoneCircumvallate papillaeLower gingivaPalatine raphePalatine tonsilFigure 18-20. Oral cavity landmarks.exposure is associated with cutaneous malignancies of the head and neck as well as lip cancer. The lower lip is at a higher risk due to its increased anterior-posterior projection, and the major-ity of squamous cell carcinomas of the lip arise along the ver-milion border of the lower lip. Immunocompromised patients, particularly those who have received solid organ and bone mar-row transplants are at an increased risk of head and neck can-cers.80 Similarly, HIV-infected patients have a higher incidence of head and neck cancers, and despite aggressive treatment have poorer results compared to HIV-negative patients.81,82 Other conditions associated with oral cancer include Plummer-Vinson syndrome (iron-deficiency anemia, dysphagia, glossitis, cheilo-sis, and esophageal webs),

1	compared to HIV-negative patients.81,82 Other conditions associated with oral cancer include Plummer-Vinson syndrome (iron-deficiency anemia, dysphagia, glossitis, cheilo-sis, and esophageal webs), dyskeratosis congenita,83,84 Bloom’s syndrome,85,86 and Fanconi anemia.87HPV is a double stranded DNA virus that is transmitted through sexual contact. Over the last two decades, this virus, specifically the 16 and 18 subtypes,88 has been associated with an epidemic rise in oropharyngeal squamous cell carcinoma.89,90 The p16 protein is a surrogate for HPV positivity. HPV status in oropharynx cancer has prognostic and therefore treatment-related implications.91,92Anatomy and HistopathologyThe upper aerodigestive tract is divided into several distinct sites that include the oral cavity, pharynx, larynx, and nasal cav-ity/paranasal sinuses. Each of these sites has separate subsites as alluded to earlier with specific etiological, pathological, prog-nostic, and treatment-related peculiarities.

1	larynx, and nasal cav-ity/paranasal sinuses. Each of these sites has separate subsites as alluded to earlier with specific etiological, pathological, prog-nostic, and treatment-related peculiarities. Locoregional tumor spread is determined by weaknesses in the framework, fascial planes, and the course of neurovascular and lymphatic channels.The oral cavity extends from the vermilion border of the lip to the hard-palate/soft-palate junction superiorly, to circumval-late papillae inferiorly, and to the anterior tonsillar pillars later-ally. It is divided into eight subsites including the (a) mucosal lip, (b) the mandibular alveolus, (c) floor of mouth, (d) tongue (ante-rior two-thirds), (e) buccal mucosa, (f) retromolar trigone, (g) maxillary alveolus, and (e) hard palate (Fig. 18-20). Advanced oral cavity cancer can present with mandibular and/or maxillary invasion requiring resection, at least in part, of these structures. Oral cavity cancers typically metastasize to the submental,

1	oral cavity cancer can present with mandibular and/or maxillary invasion requiring resection, at least in part, of these structures. Oral cavity cancers typically metastasize to the submental, sub-mandibular, and upper jugular lymph nodes (levels I-III).Brunicardi_Ch18_p0613-p0660.indd 63001/03/19 5:23 PM 631DISORDERS OF THE HEAD AND NECKCHAPTER 18The pharynx is divided into three regions: nasopharynx, oropharynx, and hypopharynx (Fig. 18-21). The nasopharynx extends from the posterior nasal septum and choana to the skull base and includes the fossa of Rosenmüller and torus tubarius of the Eustachian tubes laterally. The inferior margin of the nasopharynx is the superior surface of the soft palate. In adults, the adenoids are typically absent secondary to invo-lution during late adolescence, but these can be seen in some adults in the posterior aspect of this subsite. Isolated posterior triangle (level V) lymphadenopathy in an adult should be con-sidered nasopharyngeal carcinoma

1	but these can be seen in some adults in the posterior aspect of this subsite. Isolated posterior triangle (level V) lymphadenopathy in an adult should be con-sidered nasopharyngeal carcinoma (NPC) until proven other-wise. Due to its midline location, bilateral regional metastatic spread is common in nasopharyngeal carcinoma. Given the epi-demic rise oropharyngeal cancers, isolated level V adenopathy in an adult may also represent oropharyngeal cancer, although cancers at this site typically drain to the upper and lower cervi-cal nodes (levels II–IV) as well as the retropharyngeal nodes. The oropharynx has a number of subsites including the tonsillar region, base of tongue, soft palate, and posterolateral pharyn-geal walls. The hypopharynx extends from the vallecula to the lower border of the cricoid posterior and lateral the larynx. It includes several subsites as well including the pyriform fossa, the postcricoid space, and the posterior pharyngeal wall. Lym-phatic drainage is to the

1	cricoid posterior and lateral the larynx. It includes several subsites as well including the pyriform fossa, the postcricoid space, and the posterior pharyngeal wall. Lym-phatic drainage is to the mid and lower cervical nodes (levels III–IV); however, usually the upper cervical nodes (level II) are addressed at the same time for tumors at this site.The larynx is divided into three regions: the supraglottis, glottis, and subglottis (Fig. 18-22). The supraglottis includes sev-eral subsites: the epiglottis, false vocal cords, medial surface of the aryepiglottic folds, and the upper half of the laryngeal ventri-cles. The glottic larynx includes the true vocal cords, the anterior and posterior commissure, and the lower half of the laryngeal ventricles. The subglottis extends from below the true vocal SoftpalateHardpalateUvulaNasopharynxOropharynxLaryngopharynxPalatinetonsilsLingualtonsilsEpiglottisOesophagusTracheaLarynxHyoid boneFigure 18-21. Sagittal view of the head and neck

1	the true vocal SoftpalateHardpalateUvulaNasopharynxOropharynxLaryngopharynxPalatinetonsilsLingualtonsilsEpiglottisOesophagusTracheaLarynxHyoid boneFigure 18-21. Sagittal view of the head and neck demonstrating the distinction between the nasopharynx, oropharynx and larynx/hypopharynx including the boundaries of each.SupraglottisGlottisHyoid boneLarynxSubglottisCricoidcartilageArytenoidcartilageFalse cordVocal cordPre-epiglotticspaceThyroid cartilageVentricle of MorganiFigure 18-22. Sagittal view of the larynx with the divisions of the supraglottis, glottis, and subglottis demonstrated.cords to the superior cricoid border from within. The supraglottis has a high rate of bilateral metastatic spread secondary to its rich lymphatic drainage, whereas isolated glottic cancers rarely have lymphatic spread. Laryngeal cancers, in addition to having the propensity for lymphatic spread, particularly in advanced cases, can have preepiglottic and paraglottic invasion as well as inva-sion of the

1	spread. Laryngeal cancers, in addition to having the propensity for lymphatic spread, particularly in advanced cases, can have preepiglottic and paraglottic invasion as well as inva-sion of the laryngeal framework (thyroid and cricoid cartilage). Furthermore, glottic and subglottic lesions, in addition to poten-tial spread to the upper and lower cervical nodes (levels II–IV), have the propensity for spread to the central neck (level VI) in the paralaryngeal and paratracheal region.Second Primary Tumors in the Head and NeckPatients with head and neck squamous cell carcinoma (HNSCC) are at increased risk for the development of a second primary malignancy (SPM), which is defined as a second malignancy that presents either simultaneously or after the diagnosis of an index tumor. A synchronous SPM is diagnosed simultaneously or within 6 months of the index tumor, while a metachronous SPM is diagnosed >6 months after the index tumor. SPMs need to be distinguished from local recurrences or

1	SPM is diagnosed simultaneously or within 6 months of the index tumor, while a metachronous SPM is diagnosed >6 months after the index tumor. SPMs need to be distinguished from local recurrences or metastasis of the primary tumor. The incidence of SPM ranges from 2% to 7% per year,93-95 and this risk remains constant from the time of initial diagnosis throughout the lifetime of the patient.93 Sec-ond primary malignancies represent the second leading cause of death in patients with HNSCC.96 One-quarter to one-third of deaths in these patients are attributable to SPM,96-98 highlight-ing the importance of SPM in the successful management of HNSCC.The classic criteria for defining second primary malig-nancy (SPM) were proposed by Warren and Gates and are: (a) histologic confirmation of malignancy in both the index and secondary tumors; (b) two malignancies that are anatomically Brunicardi_Ch18_p0613-p0660.indd 63101/03/19 5:23 PM 632SPECIFIC CONSIDERATIONSPART IIseparated by normal

1	in both the index and secondary tumors; (b) two malignancies that are anatomically Brunicardi_Ch18_p0613-p0660.indd 63101/03/19 5:23 PM 632SPECIFIC CONSIDERATIONSPART IIseparated by normal mucosa; and (c) the possibility of the SPM being a metastasis from the index tumor must be excluded. Most investigators use these criteria to define an SPM. However, dis-agreement exists regarding the application of the second and third criteria. For example, when both tumors appear in the same anatomic subsite, there is no agreement on the distance that should exist between the tumors, with some investigators favoring 1.5 cm99 and others requiring 2 cm.100 Furthermore, when the tumors occur in the same anatomic subsite, some investigators add that the SPM must present at least three years after the diagnosis of the index tumor,100 while others require that the SPM present at least five years after the index tumor.101 Others suggest that molecular analysis is required to classify a tumor as an

1	diagnosis of the index tumor,100 while others require that the SPM present at least five years after the index tumor.101 Others suggest that molecular analysis is required to classify a tumor as an SPM.102Treatment of SPMs of the upper aerodigestive tract is site specific. In general, the SPM should be treated as a sep-arate entity, in the same manner as a primary index tumor at the anatomic subsite. In many cases, particularly in metachro-nous SPMs, patients have already received a full complement of treatment, including primary or adjuvant radiation and/or chemoradiation treatment. In these cases, surgical treatment of the SPM is often indicated when feasible. Reirradiation is an option in carefully selected cases when salvage surgery is not possible. Proper patient selection for reirradiation is criti-cal, and only patients with minimal comorbidity and toxicity of previous radiation treatment should be considered.103 Patients at high risk for local recurrence after salvage surgery

1	is criti-cal, and only patients with minimal comorbidity and toxicity of previous radiation treatment should be considered.103 Patients at high risk for local recurrence after salvage surgery may benefit from increased locoregional control from adjuvant reirradiation, although there is no survival advantage compared with salvage surgery alone.103 Survival in patients with SPM depends upon the stage and location of the primary site of the SPM. Patients with SPM arising in the head and neck have significantly improved survival when compared with patients with SPM aris-ing in the lung and esophagus.104StagingStaging for upper aerodigestive tract malignancies is defined by the American Joint Committee on Cancer and follows the TNM (primary tumor, regional nodal metastases, distant metastasis) staging format which was recently updated in the 8th edition in 2017.105 The T stage for each subsite incorporates relevant anatomy; for instance, T3 lesions of the glottis are associated with vocal

1	staging format which was recently updated in the 8th edition in 2017.105 The T stage for each subsite incorporates relevant anatomy; for instance, T3 lesions of the glottis are associated with vocal cord immobility. Recent changes have incorporated HPV/P16 status for oropharynx cancer (Tables 18-1 and 18-2) and depth of invasion for oral cavity cancers (Table 18-3).The N classification for head and neck sites is nearly uni-form for all sites (Tables 18-4 and 18-5) except for the nasophar-ynx and for HPV-associated (p16-positive) oropharynx cancer. Recent changes have also incorporated extracapsular extension into this nodal staging to improve the discrimination and prog-nostication of the classification.Upper Aerodigestive TractThere are similarities in the initial assessment and manage-ment of all patients with a newly diagnosed upper aerodiges-tive tract malignancy. The frequently reviewed clinical practice guidelines (National Comprehensive Cancer Network; NCCN) provide valuable

1	of all patients with a newly diagnosed upper aerodiges-tive tract malignancy. The frequently reviewed clinical practice guidelines (National Comprehensive Cancer Network; NCCN) provide valuable information by site and stage with regard to workup and management and should be used to direct care.106 After a thorough history that should include assessment of the previously discussed risk factors, a comprehensive physical examination should follow. A full head and neck examination including inspection and palpation is critical for nearly all head and neck cancers. Oral cavity and oropharyngeal cancers should be palpated when possible to provide additional tactile informa-tion regarding depth of invasion, mobility, and invasion into adjacent structures. A cranial nerve (CN) examination with a focus on the assessment of trigeminal (V2/V3) parasthesia/Table 18-1Clinical and pathologic T category for HPV-associated (p16-positive) oropharyngeal cancerT CATEGORYT CRITERIAT0No primary

1	with a focus on the assessment of trigeminal (V2/V3) parasthesia/Table 18-1Clinical and pathologic T category for HPV-associated (p16-positive) oropharyngeal cancerT CATEGORYT CRITERIAT0No primary identifiedT1Tumor 2 cm or smaller in greatest dimensionT2Tumor larger than 2 cm but not larger than 4 cm in greatest dimensionT3Tumor larger than 4 cm in greatest dimension or extension to lingual surface of epiglottisT4Moderately advanced local diseaseTumor invades the larynx, extrinsic muscle of tongue, medial pterygoid, hard palate, or mandible or beyond**Mucosal extension to lingual surface of epiglottis from primary tumors of the base of the tongue and vallecula does not constitute invasion of the larynx.Used with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Table 18-2Clinical and pathologic T category for non–HPV-associated (p16-negative) oropharyngeal cancerT CATEGORYT

1	Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Table 18-2Clinical and pathologic T category for non–HPV-associated (p16-negative) oropharyngeal cancerT CATEGORYT CRITERIATXPrimary tumor cannot be assessedTisCarcinoma in situT1Tumor 2 cm or smaller in greatest dimensionT2Tumor larger than 2 cm but not larger than 4 cm in greatest dimensionT3Tumor larger than 4 cm in greatest dimension or extension to lingual surface of epiglottisT4Moderately advanced or very advanced local disease T4aModerately advanced local diseaseTumor invades the larynx, extrinsic muscle of tongue, medial pterygoid, hard palate, or mandible* T4bVery advanced local diseaseTumor invades lateral pterygoid muscle, pterygoid plates, lateral nasopharynx, or skull base or encases carotid artery*Mucosal extension to lingual surface of epiglottis from primary tumors of the base of the tongue and vallecula does not constitute invasion of the larynx.Used with the permission of the

1	artery*Mucosal extension to lingual surface of epiglottis from primary tumors of the base of the tongue and vallecula does not constitute invasion of the larynx.Used with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Brunicardi_Ch18_p0613-p0660.indd 63201/03/19 5:23 PM 633DISORDERS OF THE HEAD AND NECKCHAPTER 18anesthesia, CN VII, CN XI, and CN XII function. Flexible fiber-optic nasolaryngoscopy should be carried out to better charac-terize tumor extent, assess vocal cord mobility in laryngeal cancers, assess airway patency, and rule out any synchronous second primary tumors, as previously discussed.Investigations should include a diagnostic laryngoscopy and esophagoscopy to rule out second primaries and obtain tis-sue of any concerning lesions. A pathologic specimen is nearly always required before initiation of treatment. A metastatic work up including a CT of the neck

1	rule out second primaries and obtain tis-sue of any concerning lesions. A pathologic specimen is nearly always required before initiation of treatment. A metastatic work up including a CT of the neck and chest with contrast is indicated in all patients with a newly diagnosed head and neck cancer. In certain jurisdictions, a positron emission tomography (PET)-CT is used to rule out any distant metastases; however, this approach does lead to a high false positive rate.107Patients are then assessed in a multidisciplinary manner with radiation and surgical oncology. A dental evaluation is initiated before treatment because many patients undergoing primary or adjuvant radiotherapy require dental extraction to decrease the risk of osteoradionecrosis in the posttreatment period. Assessment by speech language pathology in the pre-operative period is imperative in all patients, but it is especially important in patients with laryngeal/hypopharyngeal pathology because speech and swallowing

1	speech language pathology in the pre-operative period is imperative in all patients, but it is especially important in patients with laryngeal/hypopharyngeal pathology because speech and swallowing dysfunction needs to be charac-terized and often helps drive management. Smoking cessation is initiated as early as possible.Lip. The lips starting at the vermillion border represent a tran-sition between external skin to internal mucosa. The sphincter function of the lip is created by activation of the circumferen-tial musculature of the orbicularis oris, a critical structure in lip form and function. Lip cancers are most common in men and are often seen in those with fairer complexions. In addition to tobacco use and immunosuppression, UV exposure is an addi-tional important risk factor unique to this head and neck subsite. The majority (>90%) of lip cancers present on the lower lip due to its increased protrusion and increased sun exposure.108 Although the vast majority of lip cancers

1	to this head and neck subsite. The majority (>90%) of lip cancers present on the lower lip due to its increased protrusion and increased sun exposure.108 Although the vast majority of lip cancers are SCC, other cuta-neous malignancies such as basal cell carcinoma and malignant melanoma are not uncommon at this subsite.Basal cell carcinoma presents more frequently on the upper lip than lower.Negative prognostic factors for lip cancers include peri-neural invasion, invasion into bone (maxilla or mandible), upper Table 18-3Clinical and pathologic T category for oral cavity cancerT CATEGORYT CRITERIATXPrimary tumor cannot be assessedTisCarcinoma in situT1Tumor ≤2 cm, ≤5 mm depth of invasion (DOI)DOI is depth of invasion and not tumor thickness.T2Tumor ≤2 cm, DOI >5 mm and ≤10 mmor tumor >2 cm but ≤4 cm, and DOI ≤10 mmT3Tumor >4 cmor any tumor with DOI >10 mm but ≤20 mmT4Moderately advanced or very advanced local disease T4aModerately advanced local diseaseTumor invades adjacent structures

1	but ≤4 cm, and DOI ≤10 mmT3Tumor >4 cmor any tumor with DOI >10 mm but ≤20 mmT4Moderately advanced or very advanced local disease T4aModerately advanced local diseaseTumor invades adjacent structures only (e.g., through cortical bone of the mandible or maxilla, or involves the maxillary sinus or skin of the face) or extensive tumor with bilateral tongue involvement and/or DOI >20 mm.Note: Superficial erosion of bone/tooth socket (alone) by a gingival primary is not sufficient to classify a tumor as T4. T4bVery advanced local diseaseTumor invades masticator space, pterygoid plates, or skull base and/or encases the internal carotid arteryUsed with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Table 18-4Clinical N category for non–HPV-associated (p16-negative) oropharyngeal cancerN CATEGORYN CRITERIANXRegional lymph nodes cannot be assessedN0No regional lymph node

1	Springer New York, 2017.Table 18-4Clinical N category for non–HPV-associated (p16-negative) oropharyngeal cancerN CATEGORYN CRITERIANXRegional lymph nodes cannot be assessedN0No regional lymph node metastasisN1Metastasis in a single ipsilateral lymph node, 3 cm or smaller in greatest dimension and ENE(-)N2Metastasis in a single ipsilateral node larger than 3 cm but not larger than 6 cm in greatest dimension and ENE(-); or metastases in multiple ipsilateral lymph nodes, none larger than 6 cm in greatest dimension and ENE(-); or in bilateral or contralateral lymph nodes, none larger than 6 cm in greatest dimension and ENE(-) N2aMetastasis in a single ipsilateral node larger than 3 cm but not larger than 6 cm in greatest dimension and ENE(-) N2bMetastasis in multiple ipsilateral nodes, none larger than 6 cm in greatest dimension and ENE(-) N2cMetastasis in bilateral or contralateral lymph nodes, none larger than 6 cm in greatest dimension and ENE(-)N3Metastasis in a lymph node larger

1	larger than 6 cm in greatest dimension and ENE(-) N2cMetastasis in bilateral or contralateral lymph nodes, none larger than 6 cm in greatest dimension and ENE(-)N3Metastasis in a lymph node larger than 6 cm in greatest dimension and ENE(-); or metastasis in any node(s) and clinically overt ENE(+) N3aMetastasis in a lymph node larger than 6 cm in greatest dimension and ENE(-) N3bMetastasis in any node(s) and clinically overt ENE(+)ENE = extranodal extension.Used with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Brunicardi_Ch18_p0613-p0660.indd 63301/03/19 5:23 PM 634SPECIFIC CONSIDERATIONSPART IIlip or oral commissure involvement, positive regional metasta-sis, and young age at diagnosis.The primary management of lip cancer is a surgical resection of the primary site with an adequate margin (1 cm). This provides margin analysis and additional pathologic information that can

1	diagnosis.The primary management of lip cancer is a surgical resection of the primary site with an adequate margin (1 cm). This provides margin analysis and additional pathologic information that can help stratify which patients may benefit from adjuvant treatment. The primary regional nodal drainage basin for lip cancers is the submandibular, submental, and perifacial nodes (level I), and metastases occur in <10% of patients with a higher incidence in those with upper lip cancers.109 When there are clinical evident notes, a neck dissection is indicated. Otherwise, in the clinically and radiographically negative neck observation is acceptable.109 Unfortunately, many lip cancers are not appropriately staged, and advanced regional failure is not infrequently seen. Adjuvant (postoperative) radiotherapy is indicated in patients with close (<5 mm) or positive margins, lymph node metastases, tumors with perineural invasion, and in thick (>4 mm) tumors.110 The overall 10-year survival rate

1	radiotherapy is indicated in patients with close (<5 mm) or positive margins, lymph node metastases, tumors with perineural invasion, and in thick (>4 mm) tumors.110 The overall 10-year survival rate is 84% to 92% for early stage disease but drops precipitously (11%–28%) for advanced stage disease predicted by regional and distant metastases.111The goals of lip reconstruction include providing oral competence, maintaining dynamic function, and achieving acceptable cosmesis, while avoiding severe microstomia. The proportion of the lip excised and whether the defect involves the oral commissure determines the reconstructive options. Regardless of the reconstructive technique, realignment of the vermilion border and reapproximation of the orbicularis oris are critical steps to a successful outcome. Defects of less than one-third of the lip are closed primarily, while defects between one-third and two-thirds of the lip borrow tissue from surrounding regions, mainly the upper lip and cheek

1	Defects of less than one-third of the lip are closed primarily, while defects between one-third and two-thirds of the lip borrow tissue from surrounding regions, mainly the upper lip and cheek to recreate the lip. This can be accomplished using an Abbe (lip switch) (Fig. 18-23) or Karapandzic flap (Fig. 18-24), if the commissure is preserved, or an Estlander flap (lip switch) if the commissure is resected. If there is insufficient lip tissue, rectangular excisions can be closed using upper Burrow’s triangles in combination with bilateral advancement flaps made possible by mental crease relaxing incisions; this technique is called Bernard-Burrow (Fig. 18-25).112 When more than two-thirds of the lip is excised, the Karapandzic can still be used when the defect is up to 80% as this provides a sensate lip with sphincter-like function; however, microstomia becomes a serious concern, and larger defects require free flap reconstruction. This typically does not achieve sphincter function even

1	sensate lip with sphincter-like function; however, microstomia becomes a serious concern, and larger defects require free flap reconstruction. This typically does not achieve sphincter function even when a sling is used. Microstomia can be a problem in patients that are edentulous who then cannot insert their dentures and in the dentulous who may not be able to get dental work performed with significant negative impact on their dental health.Oral Cavity. As previously mentioned, the oral cavity is com-posed of several sites. The anatomy of each subsite can uniquely impact the aggressiveness of disease, the function after resec-tion, and the surgical approach. We therefore in this next section briefly review each subsite with a focus on the relevant anatomy and treatment options.The preferred approach to management of these tumors is a surgical resection with adequate (1 cm) surgical margins with management of the regional nodal basin. In general, tumors of the oral cavity metastasize

1	approach to management of these tumors is a surgical resection with adequate (1 cm) surgical margins with management of the regional nodal basin. In general, tumors of the oral cavity metastasize to the submandibular, submental, and upper cervical nodes and are almost always treated with a supra-omohyoid neck dissection at the time of primary resection with a few rare exceptions (T1 oral tongue lesions that have less than 4 mm depth of invasion). In the “Neck” section of this chapter, we will discuss this in more detail. Adjuvant radiotherapy is indicated in patients with close margins, regional lymphade-nopathy, advanced stage tumors (T3/T4), perineural invasion, and lymphovascular invasion, while adjuvant chemoradiother-apy is reserved for those with positive margins or extracapsular invasion.113,114Oral Tongue The oral tongue is a muscular structure composed of intrinsic (longitudinal, vertical, and transverse muscle fibers) and extrinsic (genioglossus, hyoglossus, styloglossus,

1	invasion.113,114Oral Tongue The oral tongue is a muscular structure composed of intrinsic (longitudinal, vertical, and transverse muscle fibers) and extrinsic (genioglossus, hyoglossus, styloglossus, and pala-toglossus) muscles separated by a midline raphe and has overly-ing nonkeratinizing squamous epithelium. The posterior limit of the oral tongue is the circumvallate papillae beyond which the oropharynx begins while the ventral portion is contiguous with the anterior floor of mouth.Table 18-5Clinical N category for oral cavity, larynx, and hypopharynx cancerN CATEGORYN CRITERIANXRegional lymph nodes cannot be assessedN0No regional lymph node metastasisN1Metastasis in a single ipsilateral lymph node, 3 cm or smaller in greatest dimension ENE(-)N2Metastasis in a single ipsilateral node larger than 3 cm but not larger than 6 cm in greatest dimension and ENE(-); or metastases in multiple ipsilateral lymph nodes, none larger than 6 cm in greatest dimension and ENE(-); or in bilateral

1	larger than 3 cm but not larger than 6 cm in greatest dimension and ENE(-); or metastases in multiple ipsilateral lymph nodes, none larger than 6 cm in greatest dimension and ENE(-); or in bilateral or contralateral lymph nodes, none larger than 6 cm in greatest dimension, and ENE(-) N2aMetastasis in a single ipsilateral node larger than 3 cm but not larger than 6 cm in greatest dimension, and ENE(-) N2bMetastasis in multiple ipsilateral nodes, none larger than 6 cm in greatest dimension, and ENE(-) N2cMetastasis in bilateral or contralateral lymph nodes, none larger than 6 cm in greatest dimension, and ENE(-)N3Metastasis in a lymph node larger than 6 cm in greatest dimension and ENE(-); or metastasis in any node(s) and clinically overt ENE(+) N3aMetastasis in a lymph node larger than 6 cm in greatest dimension and ENE(-) N3bMetastasis in any node(s) and clinically overt ENE(+)ENE = extranodal extension.Used with the permission of the American College of Surgeons. Amin MB, Edge SB,

1	cm in greatest dimension and ENE(-) N3bMetastasis in any node(s) and clinically overt ENE(+)ENE = extranodal extension.Used with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Brunicardi_Ch18_p0613-p0660.indd 63401/03/19 5:23 PM 635DISORDERS OF THE HEAD AND NECKCHAPTER 18Tumors of the tongue typically start along the epithelial surface and can be endophytic or exophytic with or without ulceration (Fig. 18-26) and are typically seen on the lateral and ventral surfaces of the tongue. Lesions on the dorsal aspect of the tongue, particularly along the midline, are less likely to be malignant. What is seen on the surface is typically the tip of the iceberg, and palpation can provide further information regarding the depth of invasion of the tumor. These tumors can be extensive, and when they cross the midline and start to involve the base of tongue an extensive surgical resection

1	information regarding the depth of invasion of the tumor. These tumors can be extensive, and when they cross the midline and start to involve the base of tongue an extensive surgical resection including a total glossectomy may be required. However, most tumors present at an early stage due to significant pain, otal-gia, voice change secondary to difficulties with articulation, and dysphagia, which may lead to weight loss. On history and physical examination, ipsilateral paresthesias and deviation of the tongue protrusion with fasciculations or atrophy may indicate lingual nerve and hypoglossal nerve tumor invasion respectively (Fig. 18-27).Early lesions (T1–T2) can be closed primarily, allowed to heal by secondary intention, or reconstructed with a split thickness ACBDFigure 18-23. Estlander flap. A. Intra-operative image of lower lip squamous cell carcinoma with buccal and cutaneous extension pre-excision; B. Intra-operative defect and Estlander flap design. C. Immediate

1	Estlander flap. A. Intra-operative image of lower lip squamous cell carcinoma with buccal and cutaneous extension pre-excision; B. Intra-operative defect and Estlander flap design. C. Immediate post-operative flap. D. One year post-operative image.ABCFigure 18-24. A-C. Karapandzic labiaplasty for lower lip carcinoma.Brunicardi_Ch18_p0613-p0660.indd 63501/03/19 5:23 PM 636SPECIFIC CONSIDERATIONSPART IIskin graft after partial glossectomy. This procedure allows rea-sonable speech and swallowing function as long as there is not significant tethering in the floor of the mouth if this has been resected. Articulation is determined by premaxillary contact of the tongue, and dental appliances can be used in the postoperative setting to improve this. Tongue protrusion and lateral movement predicts a patient’s ability to swallow, and this is less difficult to repair secondarily. Therefore, many patients, even with small tongue cancers that require significant floor of mouth resection,

1	predicts a patient’s ability to swallow, and this is less difficult to repair secondarily. Therefore, many patients, even with small tongue cancers that require significant floor of mouth resection, receive soft pliable fasciocutaneous free flap reconstruction to improve these functional outcomes.115 Advanced lesions that require a more radical resection require free flaps, which obliter-ate the oral cavity dead space while creating bulk in the posterior oropharynx to improve the pharyngeal swallowing phase.116ABFigure 18-25. Bernard burrow flap reconstruction for a total lower lip defect involving upper and lip advancement rotation flap and cheek advancement.Figure 18-26. Oral tongue squamous cell carcinoma.ABSubmandibular glandDigastric m.(anterior belly)Myohyoid m.Stylopharyngeus,stylohyoid andstyloglossus mm.Digastric muscle(posterior belly)Styloid processHypoglossal n.Middleconstrictor m.External carotid a.Hyoid boneHyoglossus m.Lingual n.Deep lingual a.Dorsal lingual

1	andstyloglossus mm.Digastric muscle(posterior belly)Styloid processHypoglossal n.Middleconstrictor m.External carotid a.Hyoid boneHyoglossus m.Lingual n.Deep lingual a.Dorsal lingual a.Genioglossus m.Geniohyoid m.Sublingual a.Lingual n.Hyoid boneHypoglossal n.Figure 18-27. A and B. Anatomy of the floor of mouth and submandibular space. a. = artery; m. = muscle; n. = nerve.Brunicardi_Ch18_p0613-p0660.indd 63601/03/19 5:24 PM 637DISORDERS OF THE HEAD AND NECKCHAPTER 18Floor of Mouth The floor of mouth is a mucosal-covered semilu-nar area that extends from the anterior tonsillar pillar posteriorly to the frenulum anteriorly, and from the inner surface of the mandible to the ventral surface of the oral tongue. The ostia of the submax-illary and sublingual glands are contained in the anterior floor of mouth. The muscular floor of mouth is composed of the sling-like genioglossus, mylohyoid, and hyoglossus muscles, which serve as a barrier to the spread of disease. Invasion into these

1	floor of mouth. The muscular floor of mouth is composed of the sling-like genioglossus, mylohyoid, and hyoglossus muscles, which serve as a barrier to the spread of disease. Invasion into these muscles can result in decreased tongue mobility and poor articulation.The floor of mouth begins just below the lingual surface of the mandibular alveolus and ends at the ventral tongue where the frenulum connects the floor of mouth to the tongue along the mid-line and at the anterior tonsillar pillars posteriorly. Just deep to the floor of mouth mucosa is the submandibular (Wharton’s) duct and sublingual minor salivary glands followed by the genio-glossus, hyoglossus, and mylohyoid muscles. Direct invasion of these structures is not uncommon and can result in direct spread to the sublingual and submandibular spaces as well as decreased tongue mobility, leading to articulation complaints. The lingual nerve (a branch of V3) provides sensory innerva-tion to this subsite and is in close proximity

1	spaces as well as decreased tongue mobility, leading to articulation complaints. The lingual nerve (a branch of V3) provides sensory innerva-tion to this subsite and is in close proximity to it, often requir-ing resection of this structure. The contiguity of the floor of mouth mucosa with the lingual surface of the mandible can lead to mandibular invasion. This needs to be carefully examined bimanually on physical examination and using imaging (CT, MRI, or Panorex) because a marginal or segmental mandibu-lectomy may be required to excise these tumors (Fig. 18-28). If the lesion is not fixed to the mandibular cortex on physical examination, then a mandible-sparing procedure is feasible.117 Extension to the sublingual and submandibular ducts and spaces requires that the neck dissection specimen be removed en bloc with the primary tumor. Invasion of the intrinsic tongue muscu-lature requires a partial glossectomy. In our experience, except for the smallest (T1) very superficial floor of

1	be removed en bloc with the primary tumor. Invasion of the intrinsic tongue muscu-lature requires a partial glossectomy. In our experience, except for the smallest (T1) very superficial floor of mouth lesions, cancers at this subsite nearly always require a reconstructive procedure to separate the floor of mouth from the neck and to avoid tethering of the tongue using a pliable fasciocutaneous flap. If a segmental resection is performed, the vascularized osteocutaneous free flap is used. Given the anterior location of this tumor, a lip-splitting incision is rarely used unless resection of lip and chin skin is required as part of the resection in a select group of T4a tumors with through-and-through involvement.Mandibular Alveolus and Gingiva The alveolar mucosa overlies the bone of the mandible and extends from the gin-givobuccal sulcus to the mucosa of the floor of mouth to the second and third molar, which is the anterior border of the ret-romolar trigone subsite. Treatment of these

1	and extends from the gin-givobuccal sulcus to the mucosa of the floor of mouth to the second and third molar, which is the anterior border of the ret-romolar trigone subsite. Treatment of these lesions requires at the very least marginal resection of the mandibular bone given the proximity and early invasion of the periosteum in this region. A marginal resection is acceptable if there is only very early bony invasion (Fig. 18-29). If the inferior alveolar canal or the medullary cavity is invaded on physical examination or preoperative imaging, a negative locoregional prognostic fac-tor, a segmental resection is recommended with appropriate reconstruction.118,119Retromolar Trigone The retromolar trigone (RMT) is bor-dered medially by the anterior tonsillar pillar, anteriorly by the ABIncisionTissue excisedFigure 18-28. A and B. Differences in the transoral resection of a floor of mouth and alveolar ridge lesion.Brunicardi_Ch18_p0613-p0660.indd 63701/03/19 5:24 PM 638SPECIFIC

1	excisedFigure 18-28. A and B. Differences in the transoral resection of a floor of mouth and alveolar ridge lesion.Brunicardi_Ch18_p0613-p0660.indd 63701/03/19 5:24 PM 638SPECIFIC CONSIDERATIONSPART IIsecond or third molar, posteriorly by the maxillary tuberosity, inferiorly by the posterior mandibular alveolus, superiorly by the coronoid process of the mandible, and laterally by the buc-cal mucosa. Negative margin resection often requires a mar-ginal shave mandibulectomy, even when there is no evidence of mandibular cortical invasion, because of the close proxim-ity to the mandibular periosteum. This is typically achieved through a transoral approach while carefully protecting the lips and cheek.120 Extension to adjacent subsites including the buccal mucosa, maxillary tuberosity, floor of mouth, and posterolateral tongue often requires these structures be resected as part of the margin. Trismus at this and other subsites is an advanced indica-tion of involvement of the muscles

1	of mouth, and posterolateral tongue often requires these structures be resected as part of the margin. Trismus at this and other subsites is an advanced indica-tion of involvement of the muscles of mastication in the masti-cator space, which can extend to the skull base. These tumors are aggressive. Infiltration into the masticator space and bony invasion (maxilla more often than mandible) significantly wors-ens the prognosis.121Buccal Mucosa The buccal mucosa includes all of the mucosal lining from the inner surface of the lips to the line of attachment of mucosa of the alveolar ridges and pterygomandibular raphe. The mucosa includes the parotid (Stenson’s) duct opening adja-cent to the first and second maxillary molars. An understanding of the layers of the cheek from medial to lateral is important because these layers are very closely adherent to the buccal mucosa. Therefore, tumors in this region have a high propensity for early deep invasion and early lymphatic spread. The layers

1	important because these layers are very closely adherent to the buccal mucosa. Therefore, tumors in this region have a high propensity for early deep invasion and early lymphatic spread. The layers of the cheek from medial to lateral are: (a) buccal mucosa, (b) pharyngobasilar fascia, (c) buccinator muscle, (d) buccopha-ryngeal fascia, (e) buccinator fat pad, (f) masseter muscle, (g) muscles of facial expression and the superficial muscular apo-neurotic system (SMAS), (h) subcutaneous tissue, and (i) facial skin. It is not uncommon for tumors with deep invasion into the cheek to require a through-and-through resection. Reconstruc-tion aimed at providing both an internal and external lining may be accomplished with a folded fasciocutaneous free flap or a combination of a local flap for the external component and a free flap for the internal component. Marginal bone resection is often required in tumors that extend to the mandibular or maxil-lary alveolus.Maxillary Alveolus and Hard

1	the external component and a free flap for the internal component. Marginal bone resection is often required in tumors that extend to the mandibular or maxil-lary alveolus.Maxillary Alveolus and Hard Palate The hard palate and maxillary alveolus have classically been considered two sepa-rate subsites, but due to their anatomic contiguity and the simi-larities in their oncologic outcomes these two subsites should be discussed together.122 The junction between the hard palate and soft palate is the posterior border, while the maxillary tuberos-ity is the posterolateral border separating the retromolar trigone from the maxillary alveolus. The periosteum is at this subsite is closely adherent to the mucosa, and as such, superficial lesions require resection of the bone to achieve a clear margin. An infrastructure maxillectomy may be required for larger lesions involving the palate or maxillary antrum. The greater palatine nerve and foramen can be a pathway for neuropathic spread, and it

1	An infrastructure maxillectomy may be required for larger lesions involving the palate or maxillary antrum. The greater palatine nerve and foramen can be a pathway for neuropathic spread, and it is important to identify perineural invasion on these tumors in the biopsy specimen.Although SCC continues to be the primary malignant pathology at this subsite, minor salivary gland tumors such as adenoid cystic carcinoma, mucoepidermoid carcinoma, and adenocarcinoma can also present in this location. Minor sali-vary gland tumors tend to arise at the junction of the hard and soft palate.Nonmalignant pathology includes necrotizing sialometa-plasia, which appears as a butterfly-shaped ulcer on the hard palate that otherwise looks like a neoplasm. Treatment is symp-tomatic as these lesions typical disappear with time; however, a biopsy is warranted to confirm the diagnosis. A torus palatini is a benign bony outgrowth seen on midline of the hard palate. This does not require biopsy to confirm the

1	with time; however, a biopsy is warranted to confirm the diagnosis. A torus palatini is a benign bony outgrowth seen on midline of the hard palate. This does not require biopsy to confirm the diagnosis and only requires treatment to relieve symptoms.Reconstruction of the maxillectomy defect depends on a number of variables, including patient preference, dentition, patient comorbidity, and extent of defect. A partial palatectomy or partial infrastructure palatectomy can often be reconstructed with a dental obturator or a soft tissue flap alone to separate the oral cavity from the nasal cavity and maxillary sinus. More extensive suprastructure maxillectomies can be reconstructed with a free flap composed only of soft tissue, although this will leave the patient with a significant malar asymmetry over an osseous free flap. The layered fibular free flap and the scapular tip have been recently popularized to reconstruct more extensive orbitomaxillary reconstruction.123,124 Supporting the

1	over an osseous free flap. The layered fibular free flap and the scapular tip have been recently popularized to reconstruct more extensive orbitomaxillary reconstruction.123,124 Supporting the orbital floor when it is resected is critical in supporting the orbital contents and avoiding eventual diploplia because there can be a drop in these contents when they are not supported.Oropharynx The borders of the oropharynx start at the soft pal-ate superiorly, the hyoid (vallecular root) inferiorly, the anterior tonsillar pillar anterolaterally, and the cricumvallate papilla at the junction between the anterior two-thirds and posterior third of the tongue. There are five subsites in the oropharynx: the tonsillar region that includes the anterior and posterior tonsillar pillars, the soft palate, the posterior pharyngeal wall, the lateral pharyngeal wall, and the base of tongue. Tumors at this subsite can have direct extension laterally in the parapharyngeal space, posteriorly into the

1	the posterior pharyngeal wall, the lateral pharyngeal wall, and the base of tongue. Tumors at this subsite can have direct extension laterally in the parapharyngeal space, posteriorly into the retropharyngeal space, anteriorly into the oral cavity, superiorly into the nasopharynx, or inferiorly into Figure 18-29. Anterior mandibulotomy with mandibular swing to approach a posterior lesion.Brunicardi_Ch18_p0613-p0660.indd 63801/03/19 5:24 PM 639DISORDERS OF THE HEAD AND NECKCHAPTER 18the supraglottic larynx. Laterally, through the superior con-strictor, invasion of the jugular vein, carotid artery, and cranial nerves IX to XII, as well as the sympathetic chain, is possible. The pharyngobasilar fascia (resectable) deep to the constrictor muscles is a natural barrier from invasion into the prevertebral fascia (unresectable). The ascending ramus of the mandible can be involved when tumors invade the medial pterygoid muscle.Although SCC is the predominant pathology, minor sali-vary

1	the prevertebral fascia (unresectable). The ascending ramus of the mandible can be involved when tumors invade the medial pterygoid muscle.Although SCC is the predominant pathology, minor sali-vary gland tumors can present as submucosal lesions in the soft palate or tongue base, and lymphoma can present in the tonsils as an asymmetric enlargement, underlying the importance of a tissue diagnosis before treatment.Oropharyngeal cancers, other than those on the soft palate or tonsils, are often not obvious on oral cavity exam inspection; therefore, a high degree of suspicion should exist in patients with a muffled voice as would be experienced in tongue base tumors, patients with dysphagia and weight loss, or referred otalgia from the tympanic branches of CN IX and X. Trismus may indicate advanced disease with pterygoid involvement. As previously mentioned, because of the epidemic rise in incidence of oropharyngeal cancers, secondary to HPV-associated tumors, and the high regional

1	advanced disease with pterygoid involvement. As previously mentioned, because of the epidemic rise in incidence of oropharyngeal cancers, secondary to HPV-associated tumors, and the high regional metastatic rate for these tumors, the pre-senting symptom is often a nontender cervical lymphadenopa-thy, which should be investigated with a fine-needle aspiration (FNA) biopsy. Approximately 50% of patients have metastases at the time of diagnosis. Bilateral metastases are common in patients with soft palate and base of tongue tumors. Treatment of the neck should include the upper jugulodigastric nodes to which these tumors most commonly metastasize to, followed by levels II, IV, V, and the retropharyngeal lymph nodes.A discussion about oropharyngeal cancer cannot be had without discussing the important prognostic information pro-vided by the HPV status of these tumors. The incidence of oro-pharyngeal squamous cell carcinoma has increased significantly over the last four decades secondary

1	important prognostic information pro-vided by the HPV status of these tumors. The incidence of oro-pharyngeal squamous cell carcinoma has increased significantly over the last four decades secondary to HPV-16 related develop-ment of this tumor.125 HPV infection can induce the production of two viral oncoproteins, E6 and E7, which inactivate tumor suppressors p53 and Rb leading to tumor promotion.126 HPV-positive tumors are more common in younger male patients and are associated with a history of a higher lifetime number of sexual partners and oral sex.127 Ang et al demonstrated that oropharyngeal cancers can be stratified on overall survival into low risk (HPV-positive tumors in patients with ≤10 pack years of smoking or >10 pack years of smoking but N0-N2a), intermediate risk (HPV-positive tumors with >10 pack years of smoking and N2b-N3 or HPV-negative tumors in patients with ≤10 pack years of smoking and T2-T3 tumors), and high risk (HPV-negative tumors in patients with ≤10 pack

1	tumors with >10 pack years of smoking and N2b-N3 or HPV-negative tumors in patients with ≤10 pack years of smoking and T2-T3 tumors), and high risk (HPV-negative tumors in patients with ≤10 pack years of smok-ing and T4 tumors or HPV-negative tumors in patients with >10 pack years of smoking).92 The rate of distant metastases in the HPV-positive and HPV-negative tumors does not differ, and therefore the survival benefit in the HPV-positive group is due to improved locoregional control.Management of squamous cell cancers of this region includes single modality (surgery or radiotherapy alone) treat-ment for early stage disease (stage I/II) and multimodality treatment for advanced stage (stage III/IV) disease (surgery followed by postoperative radiotherapy or concurrent chemora-diotherapy).106 Historically, from 1971 to 2000, oropharyngeal cancers, at the time mostly HPV-negative, were treated hetero-geneously with surgery followed by radiotherapy or primary radiotherapy similar survival

1	Historically, from 1971 to 2000, oropharyngeal cancers, at the time mostly HPV-negative, were treated hetero-geneously with surgery followed by radiotherapy or primary radiotherapy similar survival until Parsons et al demonstrated in a meta-analysis similar survival rates between the two treatment groups with improved locoregional control in the radiation-alone group and much higher complication rates in the surgery group (32% severe complications, 3.5% mortality) compared to the radiotherapy group (3.8% severe complications, 0.4% mortal-ity).128 For this reason, for many years, advanced-stage tumors were treated with primary concurrent chemoradiotherapy. How-ever, this is now a moving target given the excellent results in early and some intermediate-stage HPV-positive disease regardless of treatment. More recently, there has been a push to study de-escalation, particularly in the aforementioned low and intermediate risk groups given the excellent survival rates. The standard of care,

1	treatment. More recently, there has been a push to study de-escalation, particularly in the aforementioned low and intermediate risk groups given the excellent survival rates. The standard of care, regardless of HPV status, for advanced tumors (T3/T4 or N2b-N3 or evidence of gross ECE) continues to be concurrent chemoradiotherapy.129The high complication and mortality rate in the surgi-cal group analyzed by Parsons et al was associated not just with HPV-negative tumors but also with open resections for advanced tumors that necessitated a lip-splitting mandibulotomy approach. More recently, particularly for early stage tumors (T1, T2, N0-N2a), there has been a push towards minimally invasive transoral robotic surgery (TORS) using the da Vinci Surgical System. Oncologic outcomes are similar between surgery and radiotherapy in this group, and TORS has been demonstrated to be cost-effective in this setting.130-132 Functional outcomes related to swallowing (G-tube dependency) and airway

1	between surgery and radiotherapy in this group, and TORS has been demonstrated to be cost-effective in this setting.130-132 Functional outcomes related to swallowing (G-tube dependency) and airway (tra-cheotomy dependency) are also similar between the groups.130 These outcomes are heavily dependent on the surgeon’s abil-ity to achieve negative margins, which can be challenging, and on good preoperative predictive value of imaging to stage the neck, given that advanced nodal disease, particularly with ECE, continues to benefit from adjuvant chemoradiotherapy. Positive margins or ECE ultimately leads to adjuvant chemoradiother-apy. This results in triple modality treatment with its associated higher morbidity. Therefore, clinical recommendations based on these favorable early retrospective poorly controlled studies with small sample sizes is not yet possible. Meanwhile, clinical trial evidence is pending to help elucidate in which settings and patients this new approach may be

1	poorly controlled studies with small sample sizes is not yet possible. Meanwhile, clinical trial evidence is pending to help elucidate in which settings and patients this new approach may be beneficial.133Extensive oropharyngeal cancers that fail concurrent chemoradiotherapy are treated with resection. If the mandible is involved, a marginal mandibulectomy or segmental man-dibulectomy may be required depending on the extent of bony invasion. Tongue base resection may necessitate total glossec-tomy depending on the contralateral extent of the tumor and the ability to save the lingual artery and to a lesser extent the hypo-glossal nerve on that side. When the larynx is preserved many patients, if careful reconstruction is performed, 90% of patients can be decannulated and have acceptable voice outcomes.134 However, it is not uncommon to have to perform a total laryn-gectomy at the same time as the total glossectomy for tumors with supraglottic extent, and this is associated with poor

1	voice outcomes.134 However, it is not uncommon to have to perform a total laryn-gectomy at the same time as the total glossectomy for tumors with supraglottic extent, and this is associated with poor quality of life. Generally, these patients also have poorer survival.135-137The primary goal of oropharyngeal reconstruction is swal-lowing rehabilitation. For soft palate defects, palatal obturators may assist in providing a seal between the nasopharynx and the posterior pharyngeal wall. The modified Gehanno technique sutures the posterior wall of the remaining soft palate to the remaining incised pharyngeal mucosa to close off the ipsilateral hemi-nasopharyngeal port.138,139 A flap can then be inset overly-ing this defect, which has effectively separated the nasopharynx from the oropharynx. This prevents nasal regurgitation of air Brunicardi_Ch18_p0613-p0660.indd 63901/03/19 5:24 PM 640SPECIFIC CONSIDERATIONSPART IIand liquids, therefore impacting both speech and swallowing.

1	This prevents nasal regurgitation of air Brunicardi_Ch18_p0613-p0660.indd 63901/03/19 5:24 PM 640SPECIFIC CONSIDERATIONSPART IIand liquids, therefore impacting both speech and swallowing. Similarly, total glossectomy reconstruction has several goals, including filling the oral cavity dead space, allowing the neo-tongue to reach the premaxilla to assist with articulation, and, most importantly, creating posterior bulk to allow the base of tongue to touch the posterior pharyngeal wall, which assists with the pharyngeal phase of swallowing. This is often achieved with a large rectus abdominis or anterolateral thigh free flap.138 If the neotongue does not successfully touch the premaxilla and hard palate and speech is impeded, a palatal obturator can be used to bring down the palate and achieve better contact.Hypopharynx and Cervical Esophagus The hypopharynx, which extends from the vallecular to the lower border of the cricoid cartilage (Fig. 18-30), has three subsites; the pyriform

1	better contact.Hypopharynx and Cervical Esophagus The hypopharynx, which extends from the vallecular to the lower border of the cricoid cartilage (Fig. 18-30), has three subsites; the pyriform sinuses, the lateral and posterior pharyngeal walls, and the post cricoid space. SCC of the hypopharynx typically presents with progressive dysphagia, first to solids then to liquids, fol-lowed by weight loss. Similar to oropharyngeal tumors, patients can also present with voice change, referred otalgia or a neck mass. Rarely, when the larynx is involved, patients may pres-ent with stridor and airway compromise necessitating an urgent tracheotomy.Unfortunately, there is significant delay in diagnosis of patients with hypopharyngeal cancer and late presentation is common.140 Routine physical examination will not typically detect the tumor. Fiberoptic nasolaryngoscopy is important in assessing the extent of the tumor and laryngeal function. Vocal cord paralysis is a poor prognostic factor and

1	will not typically detect the tumor. Fiberoptic nasolaryngoscopy is important in assessing the extent of the tumor and laryngeal function. Vocal cord paralysis is a poor prognostic factor and indicates fixation of the cricoarytenoid joint from direct extension of the tumor or recurrent laryngeal nerve invasion. A Valsalva maneuver dur-ing laryngoscopy allows for a better evaluation of the opened pyriform sinuses and postcricoid space. Functional endoscopic evaluation of swallowing (FEES) can be useful to assess laryn-geal penetration and aspiration, but a modified barium swal-low (MBS) is better at assessing inferior extent of the disease, multifocality within the esophagus, and aspiration. A thorough metastatic workup is required, with special attention paid to paratracheal and upper mediastinal metastases.This site has the poorest survival outcomes of all head and neck subsites. There is no difference in survival when surgery is used as the primary modality of treatment followed by

1	metastases.This site has the poorest survival outcomes of all head and neck subsites. There is no difference in survival when surgery is used as the primary modality of treatment followed by radio-therapy or chemoradiotherapy compared to primary radiother-apy or concurrent chemoradiotherapy followed by surgery.141 Concurrent chemoradiotherapy appears to be the modality of choice for laryngeal preservation; however, when surgical sal-vage is required, there is a low cure rate and increased wound complications.142 Early T1 lesions without clinical or radio-graphic evidence of adenopathy can be treated with primary radiotherapy, but this is relatively rare for this subsite due to a high rate of adenopathy and an advanced T stage at presentation.Surgical resection, typically in the salvage setting, involves a total laryngopharyngectomy typically with a circumferential defect or a very small strip of mucosa preserved in continuity with the cervical esophagus. A total thyroidectomy and

1	setting, involves a total laryngopharyngectomy typically with a circumferential defect or a very small strip of mucosa preserved in continuity with the cervical esophagus. A total thyroidectomy and cen-tral neck dissection (level VI) is simultaneously performed and removed en bloc with the specimen. Bilateral neck dissection of levels II to IV is indicated. Careful dissection of the central neck, and in some cases the upper mediastinum (level VII), is required to clear regional disease, and this is critical in prevent-ing a peristomal recurrence.Given the circumferential or near circumferential defect, reconstruction is required to prevent saliva from accumulating in the wound and to create a neopharynx. A pedicled pectoralis major flap sutured to the prevertebral fascia has been described, but advances in free flap reconstruction has popularized a num-ber of fasciocutaneous flaps for reconstruction of this defect, namely the radial forearm flap and the anterolateral thigh free

1	but advances in free flap reconstruction has popularized a num-ber of fasciocutaneous flaps for reconstruction of this defect, namely the radial forearm flap and the anterolateral thigh free flap.143-146 When total laryngopharyngoesophagectomy is required, a gastric pull-up may be performed for the pharyngeal reconstruction.Larynx Laryngeal carcinoma typical presents with a progres-sive voice complaint in a long-time smoker (Fig. 18-31). A thorough understanding of laryngeal anatomy is critical in the proper diagnosis, staging, and treatment of laryngeal cancers. The larynx is divided into the supraglottis, glottis, and subglottis as previously described (Fig. 18-32). The larynx starts superi-orly at the epiglottis and ends inferiorly at the inferior border of the cricoid cartilage of the larynx span from the epiglottis supe-riorly to the cricoid cartilage inferiorly. Laterally, it is separated from the hypopharynx by the aryepiglottic folds.The supraglottis includes all of the

1	of the larynx span from the epiglottis supe-riorly to the cricoid cartilage inferiorly. Laterally, it is separated from the hypopharynx by the aryepiglottic folds.The supraglottis includes all of the laryngeal structures above the inferior half of the ventricle, and this includes the upper half of the ventricle, the false vocal cords, the arytenoids, the aryepiglottic folds, and the epiglottis. The membranes and cartilages of the larynx act as barriers to laryngeal spread: the thyroid and cricoid cartilage, conus elasticus, the quandrangular membrane, the ventricle, the hyoepiglottic ligament, thyrohyoid membrane, and cricothyroid membrane. Although the majority of tumors of the larynx are SCC, minor salivary glands, and their associated malignancies, can be found in the supraglot-tis and subglottis. Other rarer pathologies include granular cell EpiglottisNasopharynxOropharynxEustachiantube orificeSoft palateHyoid boneLarynxHypopharynxPalatine tonsilAdenoidThyroid

1	supraglot-tis and subglottis. Other rarer pathologies include granular cell EpiglottisNasopharynxOropharynxEustachiantube orificeSoft palateHyoid boneLarynxHypopharynxPalatine tonsilAdenoidThyroid glandCricoidcartilageFigure 18-30. Relationship of nasopharynx, oropharynx, and hypopharynx.Brunicardi_Ch18_p0613-p0660.indd 64001/03/19 5:24 PM 641DISORDERS OF THE HEAD AND NECKCHAPTER 18tumors and laryngeal framework tumors, typically arising from the cricoid, such as chondroma and chondrosarcoma.The larynx functions to (a) phonate, (b) protect the air-way during swallowing, and (c) maintain airway patency. This is a fine balance. For instance, if the glottic aperture is enlarged and/or supraglottic structures are excised, phonation and air-way protection suffer while airway patency is improved. It is therefore not surprising that patients with laryngeal tumors can present with dysphonia (hot potato voice in supraglottic tumors and hoarseness in glottic tumors), dysphagia, and airway

1	It is therefore not surprising that patients with laryngeal tumors can present with dysphonia (hot potato voice in supraglottic tumors and hoarseness in glottic tumors), dysphagia, and airway con-cerns. These patients can also present with dysphagia, weight loss, referred otalgia, and a neck mass. Vocal cord fixation can be a result of a mass effect from large obstructing masses, sec-ondary to direct extension into the paraglottic space or through direct invasion of the cricoarytenoid joint involving either the muscle or the recurrent laryngeal nerve (RLN). Although sub-glottic tumors represent <1% of laryngeal cancers, they can also present with vocal cord paralysis and/or airway compromise.Direct laryngoscopy is beneficial in the assessment of laryngeal tumors to assess the local extent of tumor spread. This is particularly important in assessing vallecula and base of tongue as there can be direct extension to the oropharynx. Simi-larly, glottic cancers can have subglottic

1	extent of tumor spread. This is particularly important in assessing vallecula and base of tongue as there can be direct extension to the oropharynx. Simi-larly, glottic cancers can have subglottic extension, which neces-sitates a wider radiation field and/or a more extensive resection. Esophagoscopy and bronchoscopy are also recommended to assess second primary tumors. Furthermore, when a laryngec-tomy is planned, the direct laryngoscopy provides information about the best possible site of entry into the pharynx. Entry can be achieved through (a) a suprahyoid pharyngotomy, (b) ) lat-eral pharyngotomy (lateral to the thyroid cartilage), or (c) infe-riorly through a postcricoid or hypopharyngeal pharyngotomy.Appropriate preoperative staging with a CT scan with contrast is critical in assessing cervical lymphadenopathy and extralaryngeal spread. Erosion or invasion of the thyroid and cri-coid cartilage can significantly impact outcomes and treatment as can extension into the

1	in assessing cervical lymphadenopathy and extralaryngeal spread. Erosion or invasion of the thyroid and cri-coid cartilage can significantly impact outcomes and treatment as can extension into the preepiglottic or paraglottic spaces. The supraglottic and subglottic sites are lymphatic rich, and bilateral lymphadenopathy is not uncommon, whereas the glottic site has relatively poor lymphatic drainage (1%–4% regional metasta-sis for isolated larynx cancer). The supraglottis drains through the neurovascular bundle to the thyrohyoid membrane, mainly draining to the upper and lateral cervical nodes (levels II–IV), whereas the glottis and subglottis drain through the cricothyroid membrane and can have spread to the prelaryngeal (Delphian nodes), paratracheal, and lower cervical nodes (levels IV and VI), although in these cases we still treat levels II to IV surgi-cally because of the significant occult nodes in this region.The primary management of laryngeal cancer depends on a variety of

1	and VI), although in these cases we still treat levels II to IV surgi-cally because of the significant occult nodes in this region.The primary management of laryngeal cancer depends on a variety of factors, including tumor extent, patient comorbidi-ties, and surgeon/center experience. In general, similar to other subsites, early-stage disease can be treated with single modality treatment (surgery or radiotherapy) while advanced stage dis-ease is treated with at least two modalities, typically either sur-gery followed by radiotherapy (with or without chemotherapy) or concurrent chemoradiotherapy. Supraglottic and subglottic lesions are typically treated with primary concurrent chemo-radiotherapy in an attempt to preserve the organ; however, in patients where the primary functions of the larynx are not being fulfilled preoperatively (tracheotomy– and gastrostomy tube–dependent), primary surgical management with a total lar-yngectomy (Fig. 18-33) can be considered. The original trials

1	larynx are not being fulfilled preoperatively (tracheotomy– and gastrostomy tube–dependent), primary surgical management with a total lar-yngectomy (Fig. 18-33) can be considered. The original trials that popularized organ preservation techniques with concurrent chemoradiotherapy either excluded or had a very small sample size of large (T4) tumors.147,148 Similarly, advanced glottic can-cers (T3/T4a), even when there is no evidence of nodal disease or supraglottic tumors of all stages, have superior survival out-comes when surgery is used as the primary treatment modality.149,150 This is particularly true for tumors that extend beyond the endolarynx or with cartilage destruction, for which total Figure 18-31. Endoscopic view of a laryngeal squamous carcinoma.Figure 18-32. Total laryngectomy specimen featuring a locally invasive advanced stage glottic squamous carcinoma.Brunicardi_Ch18_p0613-p0660.indd 64101/03/19 5:24 PM 642SPECIFIC CONSIDERATIONSPART IIlaryngectomy followed

1	specimen featuring a locally invasive advanced stage glottic squamous carcinoma.Brunicardi_Ch18_p0613-p0660.indd 64101/03/19 5:24 PM 642SPECIFIC CONSIDERATIONSPART IIlaryngectomy followed by postoperative radiotherapy continues to be the standard of care. When primary chemoradiotherapy is used, surgical salvage is available if there is treatment failure or recurrent disease.The early glottic and supraglottic lesions can be safely treated with CO2 laser transoral microlaryngoscopic resection with excellent oncologic outcomes and laryngeal preservation rates.151,152 Patients with limited involvement of the arytenoid or anterior commissure are the best candidates for a good posttreat-ment vocal quality result with this approach. One of the benefits of this approach is that it does not burn any bridges to more inva-sive treatment. Often, multiple procedures are required to control the disease. Nonetheless, for early stage cancers of the glottis and the supraglottis, radiation therapy

1	any bridges to more inva-sive treatment. Often, multiple procedures are required to control the disease. Nonetheless, for early stage cancers of the glottis and the supraglottis, radiation therapy is equally as effective as surgery in controlling disease with excellent voice outcomes.Laryngeal Preservation Techniques Beyond CO2 laser tran-soral microlaryngoscopic resection for the most early of lesions, more advanced open laryngeal preservation techniques have been developed for the resection of select, moderately advanced supraglottic and glottic tumors. These techniques can be divided into vertical and horizontal partial laryngeal procedures.Vertical partial larygnectomy (VPL) (Fig. 18-34) involves a midline thyrotomy followed by dissection of the inner peri-chondrium off of the thyroid cartilage with resection of the entire true cord and a portion of the false cords, followed by reconstruction with pedicle strap muscles and bipedicled outer perichondrial flaps. A temporoparietal

1	cartilage with resection of the entire true cord and a portion of the false cords, followed by reconstruction with pedicle strap muscles and bipedicled outer perichondrial flaps. A temporoparietal fascial free flap has also been used to reconstruct these defects with excellent voice outcomes.153 This can be extended to include a frontal verti-cal VPL where the excision crosses the midline as far laterally as to leave only the posterior commissure and one functional cricoarytenoid unit. This procedure is best reserved for recurrent glottic T1/T2 lesions involving only one vocal cord (although anterior commissure involvement is not a contraindication), <5 mm sublottic extension, with a mobile cord, and no cricoid cartilage or extralaryngeal extension. This technique leads to excellent locoregional control with improvements in voice related quality of life with advanced reconstructive techniques.153Supraglottic and supracricoid partial laryngectomies are horizontally oriented

1	locoregional control with improvements in voice related quality of life with advanced reconstructive techniques.153Supraglottic and supracricoid partial laryngectomies are horizontally oriented resections. In a supraglottic laryngectomy, a laryngectomy is performed below the hyoid and includes the upper portion of the thyroid cartilage while preserving a lower portion approximately the height of the cricoid cartilage. This is reserved for lesions not involving the vocal cords, false cords, or the arytenoids. Cartilage invasion and extensive base of tongue involvement are contraindications. Most lesions amenable for resection using this procedure are typically small enough that a laser or TORS procedure is adequate for resection, and there-fore this procedure is rarely performed. For T3 glottic lesions without preepiglottic space or cricoarytenoid joint involvement, a supracricoid laryngectomy with a cricohyoidopexy or crico-hyoidoepiglottopexy (CHEP) are options. A single

1	For T3 glottic lesions without preepiglottic space or cricoarytenoid joint involvement, a supracricoid laryngectomy with a cricohyoidopexy or crico-hyoidoepiglottopexy (CHEP) are options. A single cricoaryte-noid unit is preserved to allow for phonation through apposition with the remnant epiglottis or base of tongue. The procedure is associated with excellent oncologic outcomes, tracheostomy decannulation rates, and swallowing function.154 Phonation is reasonable after this procedure but can be characterized as breathy and coarse. Many surgeons prefer not to decannulate patients until the patient has had a significant period of time with good oral intake to allow for pulmonary toilet given the high initial rate of aspiration with this procedure.All partial laryngeal procedures are associated with a high risk of aspiration. Therefore, patients should have excellent pul-monary reserve through pulmonary function tests. When this is not possible, a simple measure includes whether

1	associated with a high risk of aspiration. Therefore, patients should have excellent pul-monary reserve through pulmonary function tests. When this is not possible, a simple measure includes whether patients can climb two flights of stairs without stopping.PerichondriumUnilaterallesionThyroidcartilageFigure 18-33. Example of the resection of a vertical partial laryn-gectomy for an early stage glottic carcinoma.Angle of mandibleOhngren'slineMaxillarysinusMedial canthusFigure 18-34. Example of the Ohngren’s line and the relationship to the maxilla.Brunicardi_Ch18_p0613-p0660.indd 64201/03/19 5:24 PM 643DISORDERS OF THE HEAD AND NECKCHAPTER 18Speech and Swallowing Rehabilitation Speech and lan-guage pathology (SLP) assessment is critical in the manage-ment of patients with laryngeal and hypopharyngeal cancer. It is a critical part of the preoperative assessment and counseling and postoperative therapy. In the elderly larynx cancer popula-tion, Starmer et al demonstrated that SLP

1	and hypopharyngeal cancer. It is a critical part of the preoperative assessment and counseling and postoperative therapy. In the elderly larynx cancer popula-tion, Starmer et al demonstrated that SLP care is underutilized and is largely reserved for select patients in anticipation of total laryngectomy or after the onset of impaired airway and swal-lowing function. SLP care was, however, strongly associated with improved outcomes (lower rates of dysphagia, stricture, weight loss, and pneumonia).155SLP often discusses with the patient speech rehabilita-tion options after total laryngectomy, which include esophageal speech, tracheoesophageal puncture, and use of an electrolar-ynx. Esophageal speech is produced by actively swallowing and releasing air from the esophagus, resulting in vibrations of the esophageal walls and pharynx that can then be articulated into words. This requires a very motivated patient, and unfor-tunately, <20% of postlaryngectomy patients develop fluent esophageal

1	of the esophageal walls and pharynx that can then be articulated into words. This requires a very motivated patient, and unfor-tunately, <20% of postlaryngectomy patients develop fluent esophageal speech.The electrolarynx is a device that creates vibratory elec-tric type sounds when held against the neck or cheek that the patient can articulate into speech. This device is typically used in the postoperative inpatient setting, but it can also be used by patients who are not able to create esophageal speech.The ultimate speech rehabilitation for patients with laryn-gectomy is a tracheoesophageal puncture (TEP) with insertion of a voice prosthesis. This prosthesis is a one-way valve that allows air from the trachea to enter the upper esophagus while preventing retrograde passage of food or saliva into the trachea. Patients who undergo placement of a tracheoesophageal punc-ture have a success rate of >90% in achieving functional speech. Many surgeons do not like to place a TEP at the time

1	into the trachea. Patients who undergo placement of a tracheoesophageal punc-ture have a success rate of >90% in achieving functional speech. Many surgeons do not like to place a TEP at the time of the primary laryngectomy, particularly in the salvage setting after radiotherapy due to wound complication concerns. However, primary and secondary TEP patients experience similarly high complication rates, and the extent of the pharyngeal reconstruc-tion rather than preoperative exposure to radiotherapy appear to be more important factors in selection of TEP timing.156 Free flap patients used their TEP more commonly for primary com-munication after secondary versus primary TEP.Postoperative swallowing rehabilitation is another impor-tant task performed by SLPs. Modified barium swallows where the consistency and amount of food provided is varied to mini-mize aspiration can be critical particularly in the management of patients with partial laryngeal procedures. This is performed under

1	the consistency and amount of food provided is varied to mini-mize aspiration can be critical particularly in the management of patients with partial laryngeal procedures. This is performed under fluorosocopy in the radiology suite to allow for the assess-ment of all phases of swallowing. A more limited examination in FEES utilizes the fiberoptic nasolaryngoscope to visualize the larynx during swallow and directly visualize whether there is any laryngeal penetration.Unknown Primary Tumors Patients with cervical nodal metas-tases confirmed to be carcinoma without clinical or radiologic evidence of an upper aerodigestive tract primary tumor are referred to as having carcinoma of unknown primary (CUP). CUP comprise 2% to 5% of all head and neck cancers, although the true incidence is probably lower given advances in surgical visualization and radiological imaging to identify the primary site.157-159 Recently, there has been a rise in CUP likely related to the increase in HPV-associated

1	lower given advances in surgical visualization and radiological imaging to identify the primary site.157-159 Recently, there has been a rise in CUP likely related to the increase in HPV-associated oropharyngeal cancer, although CUP could also be from a primary thyroid or skin malignancy.160 After a thorough history and physical examination including fiberoptic nasolaryngoscopy, an FNA biopsy is used to confirm carcinoma in the cervical metastases. This is preferred over an open biopsy to avoid the risk of tumor spillage, challeng-ing revision surgery secondary to disruption of fascial planes, and increased risk of recurrence and distant metastases.161 If the primary is not identified on physical examination, patients should undergo a PET-CT scan. A recent systematic review of 7 studies (246 patients) demonstrates an overall sensitivity of 44% and specificity of 97% with this technique, which can often detect tumors >1 cm in size.162 This should be followed by thorough diagnostic

1	(246 patients) demonstrates an overall sensitivity of 44% and specificity of 97% with this technique, which can often detect tumors >1 cm in size.162 This should be followed by thorough diagnostic operative endoscopy (nasopharyngos-copy, direct laryngoscopy, esophagoscopy, and bronchoscopy). Operative manipulation of the tissues in the upper aerodiges-tive tract specifically with biopsy may lead to false positive results on the PET-CT scan, and therefore PET-CT should be performed before endoscopy. Furthermore, having the PET-CT results prior to operative endoscopy allows the surgeon to focus on specific high-risk sites for biopsy, particularly as it relates to the base of tongue.163 When the primary site is not evident, bilat-eral tonsillectomies and bilateral base of tongue biopsies can be performed to try to identify the primary site. Patients in whom a primary is identified proceed to receive appropriate treatment, and if radiotherapy is part of this treatment regimen, a more

1	can be performed to try to identify the primary site. Patients in whom a primary is identified proceed to receive appropriate treatment, and if radiotherapy is part of this treatment regimen, a more limited radiation field is administered, highlighting the impor-tance of identifying a primary site. When the primary site is not identified, primary chemoradiotherapy is advocated, treating all of the mucosal sources of the upper aerodigestive tract at risk (from nasopharynx to hypopharynx) and the cervical regional basin bilaterally. For patients with advanced neck disease (N2a or greater) or with persistent lymphadenopathy after radiation, a neck dissection may be necessary. In the preradiation setting, a neck dissection is preferred over radiotherapy for patients with N1 disease, according to the NCCN guidelines, because some of these patients will be upstaged, ECE is not accurately diagnosed on imaging alone, and because some patients without ECE and a pathologically N1 node benefit

1	to the NCCN guidelines, because some of these patients will be upstaged, ECE is not accurately diagnosed on imaging alone, and because some patients without ECE and a pathologically N1 node benefit from radiation alone without chemotherapy.106,164 The additional prognostic information pro-vided by a neck dissection can significantly impact treatment algorithms and is also associated with lower morbidity com-pared to postoperative neck dissection.Nose and Paranasal SinusesCancers of the nasal cavity and paranasal sinuses are exceed-ingly rare, and pathology in this anatomic subsite is dominated by infectious and inflammatory sources as previously discussed in the “Sinonasal Inflammatory Disease” section of this chapter. Malignant pathology at this site is often diagnosed after failed repeated treatment of suspected benign inflammatory sinona-sal pathology. Concerning preoperative imaging findings (uni-lateral disease; extensive disease; bony, orbital or intracranial invasion) and

1	repeated treatment of suspected benign inflammatory sinona-sal pathology. Concerning preoperative imaging findings (uni-lateral disease; extensive disease; bony, orbital or intracranial invasion) and unusual clinical features may raise concerns about malignancy, and in these cases referral to a tertiary head and neck oncology center is preferred. A concerning history is one that involves a slow progression and worsening of symptoms, which may include nasal obstruction, facial pain, headache, epistaxis, and facial numbness. Most tumors at this site pres-ent with advanced stage given the inevitable delay in diagnosis. Numbness in the V2 distribution suggests invasion of pterygo-palatine fossa, and V3 distribution numbness can be an indi-cation of extension to the infratemporal fossa and skull base invasion to foramen ovale. Proptosis, epiphora, diploplia, and change in vision (typically starting with loss of color vision) are Brunicardi_Ch18_p0613-p0660.indd 64301/03/19 5:24 PM

1	skull base invasion to foramen ovale. Proptosis, epiphora, diploplia, and change in vision (typically starting with loss of color vision) are Brunicardi_Ch18_p0613-p0660.indd 64301/03/19 5:24 PM 644SPECIFIC CONSIDERATIONSPART IIall signs of advanced orbital invasion. Maxillary sinus tumors, the most common site for cancers of this site, can be prognos-ticated simply using Ohgren’s line (Fig. 18-35), an imaginary line from medial canthus to the angle of the mandible, which divides maxillary sinus into anterior-inferior and posterior-superior parts. Tumors from the anterior-inferior are more prognostically favorable.Although the most common pathology at this site continues to be squamous cell carcinoma, a brief discussion of other histo-pathology is warranted given significant variety, prognostic, and treatment-related differences between these at this subsite. Benign pathology at this site includes inverted papilloma, hemangiomas, hemangiopericytomas, angiofibromas, minor salivary

1	prognostic, and treatment-related differences between these at this subsite. Benign pathology at this site includes inverted papilloma, hemangiomas, hemangiopericytomas, angiofibromas, minor salivary tumors, and benign fibrous histiocytomas. Fibro-osseous and osseous lesions, such as fibrous dysplasias, ossifying fibromas, osteo-mas, and myxomas, can also arise in this region. Additionally, encephaloceles and meningo-encephaloceles with herniation of intracranial content into the nasal cavity can present as sinonasal lesions; therefore, imaging, typically with an MRI, is warranted before biopsy of any sinonasal mass to prevent an iatrogenic CSF leak. In the evaluation of sinonasal malignant pathology, both CT and MRI are required because they provide complimentary information. MRI provides improved skull base, intracranial, and orbital invasion assessment, while CT provides better assessment of bony anatomy and invasion.Beyond squamous cell carcinoma, the next two most com-mon

1	provides improved skull base, intracranial, and orbital invasion assessment, while CT provides better assessment of bony anatomy and invasion.Beyond squamous cell carcinoma, the next two most com-mon malignancies at this site include adenoid cystic carcinoma and adenocarcinoma. Other pathologies include sinonasal undif-ferentiated carcinoma (SNUC), mucosal melanoma, lymphoma, esthesioneuroblastoma (previously known as olfactory neuro-blastoma), rhabdomyosarcoma, and angiosarcoma. Unlike other head and neck cancers, metastases to the regional lymphatic basis are extremely rare, and rarely will patients require or receive pri-mary or adjuvant treatment to the neck unless there is clinical or radiographic evidence of neck disease (approximately 15%).165The standard treatment for malignant tumors of the para-nasal sinuses is driven by the primary pathology; however, for most pathology, including SCC, the standard of care includes surgical resection followed by adjuvant radiotherapy.166

1	tumors of the para-nasal sinuses is driven by the primary pathology; however, for most pathology, including SCC, the standard of care includes surgical resection followed by adjuvant radiotherapy.166 Advances in EEAs has led to a shift in management of these tumors with minimally invasive approaches that are associated with significantly lower complication and morbidity rates with comparable oncologic outcomes.167,168 Open approaches are, however, indicated when there is tumor abutting the anterior wall of the frontal sinus, anterior extension into nasal bones, anterior maxillary wall invasion, facial skin or soft tissue inva-sion, dural involvement above the orbit or periorbital invasion, tumors with significant inratemporal fossa invasion, and exten-sion into the oral cavity, including the hard palate or the floor of the maxillary sinus. Many tumors can be treated with an endo-scopic approach such a medial maxillectomy when the tumor arises from the medial wall of the maxilla.

1	the hard palate or the floor of the maxillary sinus. Many tumors can be treated with an endo-scopic approach such a medial maxillectomy when the tumor arises from the medial wall of the maxilla. Multidisciplinary assessment and treatment should include a skull base tumor board discussion with a head and neck oncologist/surgeon, a neurosurgeon, opthalmologist including oculoplastic surgeons, prosthodontists, and reconstructive surgeons. Preoperative embolization within 24 hours of tumor excision can be useful for vascular tumors.Extent of surgery and prognosis is dependent on the tumor location and extension. For tumors limited to the hard palate and lower maxillary sinus, an infrastructure maxillectomy is sufficient. A total maxillectomy without removal of the orbital floor may be warranted for more extensive tumors limited to the maxillary sinus. When the orbital periosteum is not invaded but tumor abuts this region, removal of the orbital floor with appro-priate reconstruction is

1	for more extensive tumors limited to the maxillary sinus. When the orbital periosteum is not invaded but tumor abuts this region, removal of the orbital floor with appro-priate reconstruction is warranted. When there is invasion of periorbita, an orbital exenteration is warranted for most pathol-ogy. Tumors originating in the ethmoid sinuses may require excision of the cribriform plate and repair of subsequent skull base defect if the tumor originates or invades through the bony skull base. This is performed through an anterior craniofacial resection, where a neurosurgeon performs a frontal craniotomy for exposure of the anterior cranial fossa floor, while the head and neck surgeon performs a transfacial or endoscopic resection of the inferior bony and soft tissue structures. This approach often requires resection of dura and a dural repair to achieve negative margins. A less extensive surgery including a sphe-noethmoidectomy or medial maxillectomy can be entertained for smaller

1	often requires resection of dura and a dural repair to achieve negative margins. A less extensive surgery including a sphe-noethmoidectomy or medial maxillectomy can be entertained for smaller tumors originating in the lateral nasal wall through endoscopic or open approaches.Tumors are deemed to be unresectable if both optic nerves are involved, if there is carotid artery invasion, or if there is extensive intracranial extension. Chemotherapy has a limited application in the management of tumors at this subsite with two exceptions: rhabdomyosarcoma, which is primarily treated with chemotherapy followed by radiation therapy with surgery reserved for the salvage setting, and SNUC, where triple modal-ity treatment is required given tumor aggressiveness. Chemo-therapy in this setting may help to reduce the tumor bulk and allow for orbital preservation.NasopharynxThe anatomic borders of the nasopharyx are superiorly the adenoid patch, superolaterally the fossa of Rosenmüller and the

1	help to reduce the tumor bulk and allow for orbital preservation.NasopharynxThe anatomic borders of the nasopharyx are superiorly the adenoid patch, superolaterally the fossa of Rosenmüller and the Eustachian tube orifices (torus tubarius), inferiorly the plane of the hard palate from the choana, anteriorly the posterior nasal cavity, and posteriorly the posterior pharyngeal wall. Malignant Subtotal temporalbone resectionTotal temporalbone resectionLateraltemporalbone resectionFigure 18-35. Examples of resection specimens for lateral tem-poral bone resection, subtotal temporal bone resection, and total temporal bone resection.Brunicardi_Ch18_p0613-p0660.indd 64401/03/19 5:24 PM 645DISORDERS OF THE HEAD AND NECKCHAPTER 18tumors of the nasopharynx are typically well differentiated or lymphoepithelial SCC. However, other tumors can present in this region including lymphoma, chordoma, chondroma, nasopharyngeal cyst (Tornwaldt’s cyst), angiofibroma, minor salivary gland tumor,

1	or lymphoepithelial SCC. However, other tumors can present in this region including lymphoma, chordoma, chondroma, nasopharyngeal cyst (Tornwaldt’s cyst), angiofibroma, minor salivary gland tumor, paraganglioma, rhabdomyosarcoma, extramedullary plasmacytoma, and, rarely, sarcoma.Unlike other head and neck cancers, the nasopharynx site has unique ethnic and geographic predilection, namely, a higher incidence in southern China, Africa, Alaska, and in Green-land Eskimos. EBV is also more commonly seen in patients with NPC, and EBV titers are helpful in following treatment response.As previously discussed, a posterior (level V) neck mass should be considered NPC until proven otherwise. Other signs and symptoms include nasal obstruction, epistaxis, unilateral serous otitis media in an adult, and otalgia. Advanced disease can present with cranial neuropathies, particularly of the cranial nerves, which run in the cavernous sinus (CN V1, V2, III, IV, VI). Bilateral regional disease spread is

1	otalgia. Advanced disease can present with cranial neuropathies, particularly of the cranial nerves, which run in the cavernous sinus (CN V1, V2, III, IV, VI). Bilateral regional disease spread is common, and the lym-phatic level involved include the posterior neck (level V), as well as the upper (level II) cervical nodes and retropharyngeal nodes. Distant metastatic disease is present in 5% of patients at diagnosis, highlighting the importance of a thorough staging workup.Staging includes a thorough physical examination using either a flexible or rigid endoscope to assess the mucosal extent of the disease. CT and MRI are complimentary as in the assess-ment of nasal cavity and paranasal sinus tumors with CT provid-ing better assessment of bony invasion and the MRI providing better soft tissue delineation, skull base invasion, and perineural spread with cranial nerve enhancement. Multimodality therapy with chemoradiotherapy is superior to radiotherapy alone in the management of

1	soft tissue delineation, skull base invasion, and perineural spread with cranial nerve enhancement. Multimodality therapy with chemoradiotherapy is superior to radiotherapy alone in the management of nasopharyngeal carcinoma.169 Recurrent tumors are treated typically with reirradiation; however, there has been recent success with surgical salvage procedures, particular in those patients in which a negative margin can be achieved.170When resection is contemplated for recurrent nasopharyn-geal carcinoma or for low grade tumors such as some minor salivary gland tumors, a number of surgical approaches can be utilized for resection. These include endoscopic, transpalatal, transfacial via a maxillary swing procedure, and transcervical. In many cases, a combination of these techniques is required to achieve a negative margin. The transcervical approach pro-vides the added benefit of early access and control of the carotid artery. For benign and low-grade tumors, advances in EEA have made use

1	to achieve a negative margin. The transcervical approach pro-vides the added benefit of early access and control of the carotid artery. For benign and low-grade tumors, advances in EEA have made use of the open approaches less common.Ear and Temporal BoneTemporal bone and ear tumors are rare account for <0.5% of all head and neck cancers. Subsites in this head and neck site from lateral to medial include the pinna (external ear), external auditory canal, middle ear, mastoid, and petrous portion of the temporal bone. Although the typical pathology at this site is squamous cell carcinoma, minor salivary gland tumors such as adenocarcinoma and adenoid cystic carcinoma can also present here. Given that the ear is in the high-risk region for aggressive skin cancers due to its unique exposure to ultraviolet light, cuta-neous malignancies such as basal cell carcinoma and melanoma can also present here. In the pediatric population, soft tissue sar-comas, most commonly rhabdomyosarcoma, can

1	to ultraviolet light, cuta-neous malignancies such as basal cell carcinoma and melanoma can also present here. In the pediatric population, soft tissue sar-comas, most commonly rhabdomyosarcoma, can present at this site. These tumors typically present with an advanced stage,171 and resection with clear margins and functional preservation is challenging because of the close proximity of vital structures, namely the facial nerve and the external auditory canal.172 Tumors involving the petrous apex or intracranial structures may present with headache and palsies of CN V and VI as well.Patients can present with ulceration, granulation, or bleed-ings from the external ear and auditory canal. This is often mistaken for an infectious or inflammatory process given the rarity of malignancy at this subsite; however, persistent granu-lation tissue in the ear should be biopsied and imaged to rule out malignancy. Patients can then present with otorrhea, otal-gia, hearing loss, vertigo, and facial

1	subsite; however, persistent granu-lation tissue in the ear should be biopsied and imaged to rule out malignancy. Patients can then present with otorrhea, otal-gia, hearing loss, vertigo, and facial nerve paralysis. Appropri-ate imaging with CT and MRI is often required to appropriately delineate the lesion and stage and assist with the appropriate management plan.Cutaneous malignancies of the pinna and tragus can usu-ally be locally excised. However, at this subsite, spread into the perichondrium and cartilage can lead to rapid spread long that tissue plane. The importance of negative margins cannot be overstated at this subsite. Mohs microsurgery has been advo-cated for select tumors at this subsite for this reason; however, some tumors are so extensive that a total auriculectomy provides the best oncologic and cosmetic result. When there is exten-sion of tumor to the bony cartilaginous EAC junction, spread to parotid, temporomandibular joint, and skull base is possible. Advanced

1	the best oncologic and cosmetic result. When there is exten-sion of tumor to the bony cartilaginous EAC junction, spread to parotid, temporomandibular joint, and skull base is possible. Advanced tumors anterior to a vertical line along the EAC from a sagittal view benefit from a parotidectomy as well as a suprao-mohyoid neck dissection (levels I–III), whereas those behind this line benefit from a posterolateral neck dissection (levels II–V). As with other cutaneous malignancies, adjuvant radio-therapy is indicated for positive margins, perineural spread, or multiple involved lymph nodes.Tumors involving the EAC and middle ear require differ-ent management, including a sleeve resection of the external auditory canal, a lateral temporal bone resection, or a subtotal temporal bone resection (Fig. 18-36). A sleeve resection of the EAC skin and cartilage is rarely enough to achieve negative margins with the exception of some basal cell carcinomas of the skin overlying the cartilaginous

1	(Fig. 18-36). A sleeve resection of the EAC skin and cartilage is rarely enough to achieve negative margins with the exception of some basal cell carcinomas of the skin overlying the cartilaginous EAC. For more extensive IIIIIIVIIVVFigure 18-36. Levels of the neck denoting lymph node bearing regions.Brunicardi_Ch18_p0613-p0660.indd 64501/03/19 5:24 PM 646SPECIFIC CONSIDERATIONSPART IItumors and more aggressive pathology, a lateral temporal bone resection may be required removing the cartilaginous and bony external auditory canal as well as the middle ear en bloc.173 A subtotal temporal bone resection also removes the inner ear and facial nerve as part of the resection and is indicated when the tumor extends into the middle ear and a deeper resection margin is required. Both of these procedures are followed by postopera-tive radiotherapy, which provides improved locoregional con-trol.173 The neck is managed in a similar fashion to pinna and external auditory canal malignancies

1	procedures are followed by postopera-tive radiotherapy, which provides improved locoregional con-trol.173 The neck is managed in a similar fashion to pinna and external auditory canal malignancies typically requiring a supra-omohyoid (levels I–III) neck dissection. Survival outcomes are poor with a 5-year overall survival of <40%.174 Important pre-dictors of disease free survival include margin status, perineu-ral invasion, and regional lymphatic spread; the most important of these on multivariate analysis being lymphatic spread of disease.171Lateral temporal bone resections often require reconstruc-tion to close the wound, provide bulk, and vascularize tissue. If dura is encountered and even resected, a watertight dural closure is required to prevent a CSF leak and meningitis. Vascularized tissue has the added benefit of preparing the surgical bed for postoperative radiotherapy. These defects can be reconstructed with regional pedicled flaps (e.g., submental flap) or free flaps. The

1	tissue has the added benefit of preparing the surgical bed for postoperative radiotherapy. These defects can be reconstructed with regional pedicled flaps (e.g., submental flap) or free flaps. The most common free flaps used are the anterolateral thigh, although depending on body habitus and the depth of the defect, the radial forearm, lateral arm, and rectus abdominus may also be used.175 The deformity resulting from a total auriculectomy is often not reconstructed primarily, but an auricular prosthesis can be designed for further rehabilitation. Facial nerve reconstruc-tion when sacrifice is required is typically performed with cable grafts from the proximal facial nerve to select distal facial nerve branches. Because of the long distance between the proximal and distal branches, facial movement is typically delayed 6 to 12 months. However, if the masseteric nerve is connected through a cable graft to select distal facial nerve branches (typically the zygomatic branch), a shorter

1	movement is typically delayed 6 to 12 months. However, if the masseteric nerve is connected through a cable graft to select distal facial nerve branches (typically the zygomatic branch), a shorter cable graft is required, and facial movement can be achieved earlier. A variety of other static and dynamic procedures can be provided secondarily. The most important of these procedures are related to preserving eye clo-sure to avoid corneal abrasions or desiccation, which can ulti-mately lead to blindness. In the immediate postoperative period, taping of the eyelids and generous application of eye lubrication is required to prevent exposure keratitis. Upper lid gold weight implants, lower lid shortening procedures, and tarsorrhaphy can be performed secondarily to assist with eye closure.NeckAn undiagnosed neck mass needs to be carefully evaluated and worked up so as to not interfere with the definitive management of the patient and future treatment options. The patient’s age, social

1	undiagnosed neck mass needs to be carefully evaluated and worked up so as to not interfere with the definitive management of the patient and future treatment options. The patient’s age, social history, including alcohol and smoking history, preced-ing illness history, and synchronous upper aerodigestive tract physical examination findings can significantly impact the dif-ferential diagnosis and the investigation to work up a neck mass. A thorough history and head and neck examination, including fiberoptic nasolaryngoscopy, are therefore paramount to com-plete evaluation. With regard to age, in children, a neck mass is far more likely to be congenital, inflammatory, or infectious, whereas in adults, neck masses >2 cm have a >80% probability of being malignant. Typically, the first investigation is an FNA biopsy, which can be performed with ultrasound or CT guid-ance when the mass is not easily palpable or largely cystic with a small solid component. Imaging is critical in

1	investigation is an FNA biopsy, which can be performed with ultrasound or CT guid-ance when the mass is not easily palpable or largely cystic with a small solid component. Imaging is critical in characterizing the neck mass, particularly assessing the borders, consistency, and location which then impacts the differential diagnosis. For instance, a cystic neck mass can be a branchial cleft cyst or a regional metastasis from an oropharynx cancer or metastatic papillary thyroid cancer. Therefore, the imaging findings also significantly impact the differential diagnosis.When the imaging and FNA does not provide adequate information for a diagnosis, a core biopsy can be considered, particularly if the diagnosis of lymphoma is suspected and an open biopsy wants to be avoided. For a suspected carcinoma, an open biopsy may be required; however, in that case, the incision needs to be planned such that the procedure can be converted to a neck dissection, and a frozen section can be sent. If the

1	an open biopsy may be required; however, in that case, the incision needs to be planned such that the procedure can be converted to a neck dissection, and a frozen section can be sent. If the diagnosis of squamous cell carcinoma is confirmed on frozen section, then a neck dissection should be performed to further prognosticate the disease. In the case of lymphoma, biopsy does not need to remove the entire lymphoma, particularly if there is an added risk of injuring normal anatomical structures.Patterns of Lymph Node Metastasis. The lymphatic drain-age into the neck is divided into seven levels with standardized reporting within and across specialties, particularly as radiolo-gists, pathologists, surgeons, radiation oncologists, and radiolo-gists share the findings176,177 (Fig. 18-37). The levels include• Level I—the submental and submandibular nodes• Level Ia—the submental nodes; medial to the anterior belly of the digastric muscle bilaterally, symphysis of mandible superiorly, and

1	levels include• Level I—the submental and submandibular nodes• Level Ia—the submental nodes; medial to the anterior belly of the digastric muscle bilaterally, symphysis of mandible superiorly, and hyoid inferiorly; this level does not have any laterality as it includes both right and left sides• Level Ib—the submandibular nodes and gland; posterior to the anterior belly of digastric, anterior to the posterior belly of digastric, and inferior to the body of the mandibleFigure 18-37. Shaded region indicates the region included in a supraomohyoid neck dissection.Brunicardi_Ch18_p0613-p0660.indd 64601/03/19 5:24 PM 647DISORDERS OF THE HEAD AND NECKCHAPTER 18• Level IIa—upper jugular chain nodes; anterior to the poste-rior border of the sternocleidomastoid (SCM) muscle, poste-rior to the posterior aspect of the posterior belly of digastric, superior to the level of the hyoid, inferior to spinal accessory nerve (CN XI)• Level IIb—submuscular recess; superior to spinal accessory nerve

1	posterior aspect of the posterior belly of digastric, superior to the level of the hyoid, inferior to spinal accessory nerve (CN XI)• Level IIb—submuscular recess; superior to spinal accessory nerve to the level of the skull base• Level III—middle jugular chain nodes; inferior to the hyoid, superior to the level of the cricoid, deep to SCM muscle from posterior border of the muscle to the strap muscles medially• Level IV—lower jugular chain nodes; inferior to the level of the cricoid, superior to the clavicle, deep to SCM muscle from posterior border of the muscle to the strap muscles medially• Level V—posterior triangle nodes• Level Va—lateral to the posterior aspect of the SCM muscle, inferior and medial to splenius capitis and trapezius, superior to the spinal accessory nerve• Level Vb—lateral to the posterior aspect of SCM muscle, medial to trapezius, inferior to the spinal accessory nerve, superior to the clavicle• Level VI—anterior compartment nodes; inferior to the hyoid,

1	Vb—lateral to the posterior aspect of SCM muscle, medial to trapezius, inferior to the spinal accessory nerve, superior to the clavicle• Level VI—anterior compartment nodes; inferior to the hyoid, superior to suprasternal notch, medial to the lateral extent of the strap muscles bilaterally• Level VII—paratracheal nodes; inferior to the suprasternal notch in the upper mediastinumThere is a well-established pattern of regional spread from upper aerodigestive tract primary tumors.178 Lesions of the lip and oral cavity typically metastasize to levels I to III and skip metastases to the lower basin (levels III–IV) without involve-ment of the upper level (levels I–II). Oropharyngeal, laryngeal, and hypopharyngeal tumors most commonly spread to the lat-eral neck (levels II–IV). It is rare for any of these tumors to have isolated regional metastases to level V; however, naso-pharyngeal, thyroid, and head and neck malignant melanoma can metastasize to this level. Other sites for metastasis

1	any of these tumors to have isolated regional metastases to level V; however, naso-pharyngeal, thyroid, and head and neck malignant melanoma can metastasize to this level. Other sites for metastasis include the retropharyngeal nodes (oropharyngeal, nasopharyngeal, and hypopharyngeal tumors), paratracheal and level VII nodes (thyroid, hypopharynx, and cervical esophageal tumors), and pretracheal (Delphian) nodes (thyroid and advanced glottic tumors with subglottic extension).Historically, a radical neck dissection (RND) was per-formed for all upper aerodigestive tract malignancies with sac-rifice of the SCM, internal jugular vein (IJV), and accessory nerve (CN XI) and removal of all lymphatic level (levels I–V). This was because cervical metastasis decreased the 5-year over-all survival rate by approximately 50%. However, growing evi-dence demonstrated that this was not necessary, and now a neck dissection is only recommended for upper aerodigestive tract malignancies when the risk of

1	by approximately 50%. However, growing evi-dence demonstrated that this was not necessary, and now a neck dissection is only recommended for upper aerodigestive tract malignancies when the risk of occult disease is >20% in the clinically negative neck.179 When the neck is clinically positive, the level discussed in the previous paragraph for each site are excised with every attempt to preserve the SCM, IJV, and CN XI (selective neck dissection; SND). When there is direct exten-sion of the tumor or extralymphatic spread into these structures, sacrifice may be necessary in a modified radical neck dissection (MRND). The RND has been largely abandoned because the SND and MRND have been demonstrated to be equally effec-tive when it comes to oncologic outcomes with far improved functional outcomes.180,181SND has become the standard of care for most patients who are clinically node negative (cN0) and in those with limited cN1 disease. Patients with oral cavity cancer typically receive a

1	has become the standard of care for most patients who are clinically node negative (cN0) and in those with limited cN1 disease. Patients with oral cavity cancer typically receive a supraomohyoid (Fig. 18-38) neck dissection (levels I–III). Many surgeons will include a portion of level IV just below the omohyoid muscle given the rate of skip metastases previously discussed. Approximately 80% of patients with oral cavity can-cer present cN0; however, the rate of occult metastatic disease is approximately 30% and differs by subsite.182 This rate is further impacted by tumor thickness at the tongue subsite, with tumors 4 mm or thicker having a higher rate of occult disease.183 A recent prospective, randomized trial demonstrated the oncologic benefit of an elective neck dissection in cN0 oral cavity patients regardless of tumor thickness over an observation followed by therapeutic neck dissection in those with regional failures.184 An additional role of SND is as a staging tool to

1	oral cavity patients regardless of tumor thickness over an observation followed by therapeutic neck dissection in those with regional failures.184 An additional role of SND is as a staging tool to determine the need for postoperative radiation therapy. The lateral (Fig. 18-39) neck dissection (levels II–IV) is typically used in laryngeal and hypo-pharyngeal cancers. The posterolateral (Fig. 18-40 neck dissec-tion (levels II–V) is typically recommended in thyroid cancers, although recent evidence has demonstrated that a partial level V dissection may be all that is necessary for equivalent outcomes to a full level II to V neck dissection.176,185,186Despite advances in the surgical management of neck dis-ease, in clinically advanced nodal disease (with the exception of uncomplicated N1 disease), an MRND remains the treatment of choice. When the neck disease is advanced with extrano-dal extension (ENE), perineural invasion (PNI), lymphovas-cular invasion (LVI), and the presence of

1	disease), an MRND remains the treatment of choice. When the neck disease is advanced with extrano-dal extension (ENE), perineural invasion (PNI), lymphovas-cular invasion (LVI), and the presence of multiple involved nodes, postoperative radiotherapy improves locoregional con-trol.103 If there is a positive margin or ENE, then the addition of adjuvant chemotherapy to radiotherapy provides a survival benefit.113,187,188In patients receiving primary radiotherapy with advanced N stage disease (N2a or greater) or only a partial response to Figure 18-38. Shaded region indicates the region included in a lateral neck dissection.Brunicardi_Ch18_p0613-p0660.indd 64701/03/19 5:24 PM 648SPECIFIC CONSIDERATIONSPART IItreatment, a planned postradiotherapy neck dissection can be performed 6 to 8 weeks after completion of radiotherapy. This is to consolidate the treatment and provide prognostic information.Tumor factors that preclude surgery include prevertebral fascia invasion, skull base

1	8 weeks after completion of radiotherapy. This is to consolidate the treatment and provide prognostic information.Tumor factors that preclude surgery include prevertebral fascia invasion, skull base invasion, and >270o circumferential encasement of the internal carotid artery. These factors are asso-ciated with very poor 5-year survival (<20%). In such cases, sac-rifice of the carotid is not indicated given the risk of stroke and death. Surgical debulking is also not associated with improved survival. However, there is a role for neoadjuvant chemother-apy, and in those that respond and if the disease becomes resect-able, survival benefit has been demonstrated.189 Recurrent neck metastasis after radiotherapy to the neck or a comprehensive neck dissection is associated with very poor survival.190Parapharyngeal Space Masses. The parapharyngeal space is a potential inverted pyramidal space bordered superiorly at the skull base along the sphenoid and inferiorly at the greater cornu of the

1	Space Masses. The parapharyngeal space is a potential inverted pyramidal space bordered superiorly at the skull base along the sphenoid and inferiorly at the greater cornu of the hyoid. Medially it is bordered by the buccopha-ryngeal fascia covering the superior constrictor, anteriorly the pterygomandibular raphe, posteriorly the prevertebral fascia, and laterally by the deep surface of the parotid gland and ramus of the mandible. The differential diagnosis for parapharyngeal masses is very much dependent on the anatomy and contents of this space which is divided into the preand poststyloid spaces by the tensor-styloid fascia. This fascia attaches the tensor veli palatini muscle to the styloid. The contents of the prestyloid parapharyngeal space include fat, the deep lobe of the parotid, and lymph nodes, and branches of V3 (lingual, inferior alveo-lus, and auriculotemporal nerves), whereas the contents of the poststyloid space including cranial nerves IX to XII, the inter-nal jugular

1	and lymph nodes, and branches of V3 (lingual, inferior alveo-lus, and auriculotemporal nerves), whereas the contents of the poststyloid space including cranial nerves IX to XII, the inter-nal jugular vein, the internal carotid artery, and the sympathetic chain. Nearly half of all parapharyngeal masses are of parotid origin, while 20% to 25% are of neurogenic origin, such as paragangliomas (glomus vagale, carotid body tumor), schwan-nomas, and neurofibromas. Lymphatic origin masses such as lymphoma and lymph node metastases represent 15% of tumors at this subsite. Therefore, most prestyloid lesions are considered of salivary gland origin, whereas poststyloid lesions are typi-cally vascular or neurogenic.Tumors of the parapharyngeal space can displace the lat-eral pharyngeal wall medially into the oropharynx (Fig. 18-41) and can thus cause obstructive sleep apnea, voice change, and dysphagia in addition to cranial neuropathies, Horner’s syn-drome, or vascular compression. In addition to

1	the oropharynx (Fig. 18-41) and can thus cause obstructive sleep apnea, voice change, and dysphagia in addition to cranial neuropathies, Horner’s syn-drome, or vascular compression. In addition to CT and MRI, poststyloid lesions should be investigated with a 24-hour uri-nary catecholamine collection because some paragangliomas are functional and this should be managed preoperatively.Surgical access to these tumors can be performed using a purely transcervical approach with the excision of the subman-dibular gland for access. A transfacial or transparotid approach can be used as an adjunct for certain tumors by removing the parotid gland. This ensures identification of the facial nerve Figure 18-39. Shaded region indicates the region included in a posterolateral neck dissection.ParotidglandStylomandibularligamentFigure 18-40. Parapharyngeal mass—prestyloid with prominent oropharyngeal presentation typical of a dumbbell tumor.Brunicardi_Ch18_p0613-p0660.indd 64801/03/19 5:24 PM

1	18-40. Parapharyngeal mass—prestyloid with prominent oropharyngeal presentation typical of a dumbbell tumor.Brunicardi_Ch18_p0613-p0660.indd 64801/03/19 5:24 PM 649DISORDERS OF THE HEAD AND NECKCHAPTER 18prior to removal of the mass, which is just deep to it. Rarely, a transmandibular approach is required by performing a midline or parasymphyseal mandibulotomy with a lateral swing. Tran-soral approaches have been described, but they are not recom-mended and are largely contraindicated due to poor exposure and control of the associated vasculature.Benign Neck Masses. Many benign neck masses require surgical intervention for diagnostic, cosmetic, and symptom-atic relief. This is particularly true for lesions that are prone to recurrent infections, especially in the pediatric population. Such masses include thyroglossal duct cyst, branchial cleft cyst, lymphangioma (cystic hygroma), hemangioma, and der-moid cyst. Lymphangioma and hemangioma were previously discussed and will not be

1	Such masses include thyroglossal duct cyst, branchial cleft cyst, lymphangioma (cystic hygroma), hemangioma, and der-moid cyst. Lymphangioma and hemangioma were previously discussed and will not be discussed in this section.During fetal growth, the thyroid gland descends along a tract from the foramen cecum at the base of tongue into the ante-rior low neck. A vestigial remainder of this tract is called a thy-roglossal duct cyst, which typically presents as a subcutaneous swelling near the hyoid in the midline or slightly paramedian. Patients may complain of recurrent infections of this mass after an upper respiratory tract infection. Investigations include thy-roid function tests and a neck and thyroid ultrasound to confirm that the patient has thyroid tissue in the lower neck . Treatment involves removal of the cyst, the tract, and the central portion of the hyoid (Sistrunk procedure), often with a small portion of the base of tongue if the tract extends above the hyoid.During fetal

1	involves removal of the cyst, the tract, and the central portion of the hyoid (Sistrunk procedure), often with a small portion of the base of tongue if the tract extends above the hyoid.During fetal growth, the branchial cleft apparatus may persist, forming a branchial cleft remnant (cyst, sinus, or tract), numbered to their corresponding embryologic branchial cleft. First branchial cleft anomalies parallel the EAC (Work Type I; preauricular) or go through the parotid gland ending at the bony-cartilaginous EAC junction (Work Type II; angle of the mandible). Second branchial anomalies (Fig. 18-42), the most common type, start at the anterior border of the SCM and head toward the tonsillar fossa traveling deep to second arch struc-tures (CN VII and external carotid artery) and superficial to third arch structures (stylopharyngeus, IX, and internal carotid artery). Third and fourth branchial anomalies are difficult to dis-tinguish clinically and frequently open into the pyriform sinus

1	to third arch structures (stylopharyngeus, IX, and internal carotid artery). Third and fourth branchial anomalies are difficult to dis-tinguish clinically and frequently open into the pyriform sinus often presenting with recurrent thyroid infections.191 These anomalies ascend posterior the internal carotid artery and deep to CN IX but superficial to CN XI and XII. Dermoid cysts tend to present as midline masses and represent trapped epithelium originating from the embryonic closure of the midline. These can be reliably diagnosed and distinguished from thyroglossal duct cysts using an ultrasound predictive model.192Cervical Fascial Planes. The fascial planes often predict the pathway and extent of infectious spread in the neck and are there-fore clinically important. The deep fascial layers of the neck Figure 18-41. Computed tomography scan demonstrating a branchial cleft cyst with operative specimen.Facial n.Anterior facial v.Retromandibular v.Temporal branchFrontal branchPosterior

1	of the neck Figure 18-41. Computed tomography scan demonstrating a branchial cleft cyst with operative specimen.Facial n.Anterior facial v.Retromandibular v.Temporal branchFrontal branchPosterior bellyof digastric m.StylomastoidforamenCervicalbranchMasseter m.Zygomatic branchParotid ductBuccalbranchMandibularbranchFigure 18-42. Example of a tumor in the parotid with the pattern of the facial nerve and associated anatomy. m. = muscle; n. = nerve; v. = vein.Brunicardi_Ch18_p0613-p0660.indd 64901/03/19 5:24 PM 650SPECIFIC CONSIDERATIONSPART IIinclude three separate layers: the superficial deep (investing) layer, the pretracheal (visceral) layer, and the prevertebral layer. The investing layer forms a cone around the neck and surrounds the SCM muscle and the anterior and posterior neck. It spans from the mandible to the clavicle and manubrium. The visceral layer surrounds the trachea, thyroid, and esophagus and blends laterally with the carotid sheath extending inferiorly to the

1	It spans from the mandible to the clavicle and manubrium. The visceral layer surrounds the trachea, thyroid, and esophagus and blends laterally with the carotid sheath extending inferiorly to the upper mediastinum. Between this layer and the prevertebral fascia is the retropharyngeal space. The prevertebral fascia covers the pre-vertebral musculature and space and extends down to the tho-racic vertebra and diaphragm. Infections of the prevertebral space between this fascia and the prevertebral musculature are considered to be in the prevertebral space and can extend all the way down to the sacrum. Therefore, neck infections can extend to the mediasti-num or beyond and need to be treated aggressively.Salivary Gland TumorsPrimary malignant tumors of the salivary glands are relatively rare and account for <2% of all head and neck malignancies. As previously mentioned, minor salivary gland malignancies can present anywhere in the upper aerodigestive tract, particularly on the palate;

1	and account for <2% of all head and neck malignancies. As previously mentioned, minor salivary gland malignancies can present anywhere in the upper aerodigestive tract, particularly on the palate; however, the major salivary glands are the parotid, submandibular, and sublingual glands. The majority of tumors (80%) arise in the parotid gland (Fig. 18-44); however, 80% of these are benign, most commonly, pleomorphic adenomas (benign mixed tumors). As the salivary gland gets smaller, the proportion of tumors that are malignant increases; 50% of sub-mandibular/sublingual tumors and 80% of minor salivary gland tumors are malignant.Patients typically present with a mass because these tumors are well circumscribed and slow growing. However, certain signs and symptoms, such as pain, paresthesia, facial nerve weakness, or rapid growth, raise the concern for malig-nancy. If there is facial nerve weakness (10%–15% of cases), this usually represents tumor invading the facial nerve. Sub-mandibular

1	nerve weakness, or rapid growth, raise the concern for malig-nancy. If there is facial nerve weakness (10%–15% of cases), this usually represents tumor invading the facial nerve. Sub-mandibular and sublingual tumors present with a mass or swell-ing in the neck or floor of the mouth, respectively. Tumors in this region can invade the lingual nerve leading to tongue par-esthesia or the hypoglossal nerve invasion leading to paralysis. The close proximity to the mandible and tongue necessitates a thorough bimanual palpation to assess for fixation to these structures.The decision to dissect the neck in parotid cancers is fraught with uncertainty. However, parotid malignancies, par-ticularly carcinomas, have a propensity for regional lymphatic spread, first to the intraand periglandular nodes followed by the upper cervical chain (levels I–III). Occult nodal metastases are present in 30% of cases and are predicted by intraor peri-glandular nodes, high-risk histology (high histological

1	followed by the upper cervical chain (levels I–III). Occult nodal metastases are present in 30% of cases and are predicted by intraor peri-glandular nodes, high-risk histology (high histological grade), and extraparotid extension.193 Patients with advanced tumor stage (T3/T4a), perineural invasion, high risk histology, or clin-ically involved adenopathy should have their neck dissected. Submandibular gland cancers metastasize to the submental (Ia) and submandibular triangle lymph nodes followed by the upper cervical chain (levels II–III). Extraglandular extension and regional metastases are poor prognostic factors.Following a thorough history and physical examination, an FNA biopsy should be performed to provide an accurate preoperative diagnosis in 70% to 80% of cases when reviewed by an experienced cytopathologist. If the biopsy is nondiag-nostic, a repeat biopsy should be performed under image-guidance, typically with an ultrasound. An open or incisional biopsy should be avoided

1	an experienced cytopathologist. If the biopsy is nondiag-nostic, a repeat biopsy should be performed under image-guidance, typically with an ultrasound. An open or incisional biopsy should be avoided because of the risk of tumor spill-age and cutaneous spread. Also, this approach is fraught with risk to the facial nerve. Salivary gland tumors are worked up with appropriate imaging, typically with an MRI because of the increased soft tissue definition. FNA and imaging results are critical in guiding the surgeon to the extent of surgery. The minimal extent of surgery for salivary gland tumors is a superficial parotidectomy, removing all of the salivary gland tissue superficial to CN VII, which is meticulously dissected during this procedure.The final histopathologic diagnosis in salivary gland tumors can be challenging. Nonetheless, there is a well-outlined histological classification used by pathologists.194 Benign and malignant tumors of the salivary glands are divided into

1	gland tumors can be challenging. Nonetheless, there is a well-outlined histological classification used by pathologists.194 Benign and malignant tumors of the salivary glands are divided into epi-thelial, nonepithelial, and metastatic neoplasms. Benign epithe-lial tumors are most commonly pleomorphic adenoma (85%), monomorphic adenoma, Warthin’s tumor (papillary cystad-enoma lymphomatosum), oncocytoma, or sebaceous neoplasm. Nonepithelial benign lesions include lipoma and hemangioma. Treatment of benign neoplasms is surgical excision for diag-nostic and therapeutic purposes. The parotid superficial lobe is usually dissected off of the facial nerve, which is preserved. For pleomorphic adenoma, an extracapsular dissection is favored over enucleation due to tumor pseudopods, incomplete excision, and a higher risk of tumor spillage, all of which are associated with higher recurrence rates.195 Recurrence is associated with a high degree of morbidity.Malignant epithelial tumors range in

1	and a higher risk of tumor spillage, all of which are associated with higher recurrence rates.195 Recurrence is associated with a high degree of morbidity.Malignant epithelial tumors range in aggressiveness based on tumor histology, grade, perineural invasion, and regional metastases. Mucoepidermoid carcinoma is the most common primary malignancy of the salivary glands and can be high grade (more epidermoid) or low grade (more mucinous). High grade mucoepidermoid carcinoma can be hard to differentiated from squamous cell carcinoma, particularly on FNA. Adenoid cystic is the second most common primary salivary gland malignancy and has three histological subtypes: tubular, cribriform, and solid. Higher grade/risk tumors have a higher degree of solid differentiation.194 Adenoid cystic cancers are known for peri-neural invasion and late recurrences and distant metastases. Car-cinoma ex pleomorphic adenoma is an aggressive malignancy that arises from a preexisting benign mixed tumor

1	cancers are known for peri-neural invasion and late recurrences and distant metastases. Car-cinoma ex pleomorphic adenoma is an aggressive malignancy that arises from a preexisting benign mixed tumor highlighting the importance of removing these benign masses before malig-nant transformation.Surgical excision remains the standard of care, typi-cally with facial nerve preservation unless the nerve is directly invaded by tumor. For tumors that extend beyond the superficial lobe, nerve branches can be splayed, and a total parotid can be performed by removing parotid tissue deep to the nerve while preserving the integrity and function of the nerve. Whenever possible, the nerve is preserved even if microscopic disease is left on the nerve, so long as gross tumor is not left behind (i.e., the nerve is not encased). If this is not possible or if the nerve is not working preoperatively, nerve sacrifice is usually recommended.Elective neck dissection is warranted in high-grade muco-epidermoid

1	is not encased). If this is not possible or if the nerve is not working preoperatively, nerve sacrifice is usually recommended.Elective neck dissection is warranted in high-grade muco-epidermoid carcinomas and other high-risk pathology and grade where the risk of occult disease is greater than 15% to 20%. Therapeutic neck dissection is recommended in patients with clinically or radiographically evident disease. Postoperative radiotherapy is indicated in patients with perineural invasion, advanced local disease (T4a), extraglandular disease including regional metastases, and high-grade histology.Brunicardi_Ch18_p0613-p0660.indd 65001/03/19 5:24 PM 651DISORDERS OF THE HEAD AND NECKCHAPTER 18RECONSTRUCTIONLocal Flaps and Skin GraftsLocal flaps are commonly used for cutaneous reconstruction in the head and neck. Local flaps are most commonly utilized for reconstruction after Mohs micrographic surgery for cutaneous malignancy, or for reconstruction of melanoma defects. Skin grafts are

1	in the head and neck. Local flaps are most commonly utilized for reconstruction after Mohs micrographic surgery for cutaneous malignancy, or for reconstruction of melanoma defects. Skin grafts are also commonly used for reconstruction of scalp defects after surgical resection of cutaneous malignancies. Skin grafts may also be utilized in the oral cavity for resurfacing of super-ficial defects of the tongue, floor of mouth, and buccal mucosa.Regional FlapsThree regional flaps deserve mention as potential flaps for head and neck reconstruction. The first is the pectoralis major myo-cutaneous flap, based upon the thoracoacromial artery.196 This flap may be used as a primary option for hypopharyngeal recon-struction after total laryngectomy. This flap may also be utilized to protect the great vessels from becoming exposed, or as a sal-vage reconstructive procedure should the great vessels become exposed. Another commonly utilized regional flap is the sub-mental flap, based upon the

1	great vessels from becoming exposed, or as a sal-vage reconstructive procedure should the great vessels become exposed. Another commonly utilized regional flap is the sub-mental flap, based upon the submental vessel branches of the facial artery. This flap may be utilized for intraoral reconstruc-tion and/or parotid and temporal bone reconstruction.197 Care must be taken during the neck dissection in order to preserve the submental vessels that supply this flap. Finally, the supraclavic-ular flap is based upon the supraclavicular artery, arising from the transverse cervical artery.198 This is a thin, fasciocutaneous flap that is commonly used for external neck and facial recon-struction in which thin tissue is desired.Free Tissue TransferThe majority of major defects of the head and neck require free tissue transfer for optimal reconstruction.199 A full discussion of head and neck reconstructive microsurgery is beyond the scope of this chapter; however, a brief overview of free tissue

1	free tissue transfer for optimal reconstruction.199 A full discussion of head and neck reconstructive microsurgery is beyond the scope of this chapter; however, a brief overview of free tissue transfer is provided in this section. Free tissue transfer allows the sur-geon to transplant tissue from a wide array of donor sites, each of which have distinct advantages.200 For example, for floor of mouth reconstruction, where thin tissue is desired, the surgeon may select the radial forearm as the donor site. On the other hand, when presented with a total glossectomy defect, where thick tissue is desired for adequate volume reconstruction, the rectus may be the optimal donor site. Considering osseous defects, for reconstruction of a segmental mandible defect with minimal soft tissue deficit, the fibula osseocutaneous free tis-sue transfer may be the optimal choice.201 On the other hand, reconstruction of an osseous mandible defect with a large muco-sal and external soft tissue deficit may

1	fibula osseocutaneous free tis-sue transfer may be the optimal choice.201 On the other hand, reconstruction of an osseous mandible defect with a large muco-sal and external soft tissue deficit may be best served by the scapula donor site, where vascularized bone can be combined with a large skin paddle, and an additional latissimus dorsi myocutaneous free tissue transfer, if needed.202 The ability to harvest tissue from multiple donor sites is critical to obtain-ing the optimal reconstructive result. Table 18-6 lists the com-monly utilized donor sites and their reconstructive advantages and disadvantages.Table 18-6Free tissue transfer donor sites for head and neck reconstructionFLAPBLOOD SUPPLYCHARACTERISTICSCOMMON DEFECTSRadial forearmRadial arteryThin, pliable, long pediclePartial and hemiglossectomy, floor of mouth, buccal defectsAnterolateral thighDescending branch of lateral femoral circumflex arteryThicker adipose than radial forearm, can have myocutaneous (most common) or

1	hemiglossectomy, floor of mouth, buccal defectsAnterolateral thighDescending branch of lateral femoral circumflex arteryThicker adipose than radial forearm, can have myocutaneous (most common) or septocutaneous perforatorsHypopharynx, external neck/facial skin, extended hemiglossectomy/total glossectomyLateral armPosterior radial collateral arteryOutstanding color match for facial skin, resists ptosis, diminutive pedicleParotid, temporal bone, external face and neck skinRectusDeep inferior epigastric arteryThick adipose tissue for large volume defects, long pedicle, poor external skin color matchTotal glossectomy, skull baseLatissimus dorsiThoracodorsal arteryLarge surface area of muscle, requires semi-lateral position, can be difficult for two-team harvestExtensive scalp and skull base defectsFibula osseocutaneousPeroneal arteryExcellent bone stock and length, long pedicle, thin skin paddleSegmental mandible and maxillaScapula osseocutaneousCircumflex scapular arteryLess bone

1	base defectsFibula osseocutaneousPeroneal arteryExcellent bone stock and length, long pedicle, thin skin paddleSegmental mandible and maxillaScapula osseocutaneousCircumflex scapular arteryLess bone length compared to fibula, large scapular or parascapular skin paddles ideal for large composite defectsSegmental mandible and maxilla defects with extensive soft tissue componentsRadial forearm osseocutaneousRadial arteryLong pedicle, diminutive bone stockPartial mandible defects, orbitIliac crestDeep circumflex iliac arteryUp to 16 cm of bone available, limited soft tissue, significant donor site morbiditySegmental mandible defects with small intraoral component and large external skin componentBrunicardi_Ch18_p0613-p0660.indd 65101/03/19 5:24 PM 652SPECIFIC CONSIDERATIONSPART IIFigure 18-43 shows a prototypical hemiglossectomy defect from a T2 N0 oral tongue cancer that was reconstructed with a rectangle template radial forearm free tissue transfer.203 The radial forearm free tissue

1	shows a prototypical hemiglossectomy defect from a T2 N0 oral tongue cancer that was reconstructed with a rectangle template radial forearm free tissue transfer.203 The radial forearm free tissue transfer provides thin, pliable tis-sue, with a long pedicle, and is a staple for hemiglossectomy and partial glossectomy reconstruction.Figure 18-44 shows a composite mandible defect from a T4a N0 mandibular alveolus cancer, after segmental mandibu-lectomy, reconstructed with a fibula osseocutaneous free tissue transfer.204 The 2.5-mm titanium reconstruction plate was bent to a mandible model. A template of the osseous defect is made and transferred to the fibula, and wedge ostectomies are made in the bone so that it can be snug fit into the bone defect.Figure 18-45 shows a palate defect after an infrastructure maxillectomy for a T2 N0 maxillary alveolus cancer. The defect resulted in direct communication with the buccal space, nasal cavity, and maxillary sinus. A radial forearm free tissue

1	infrastructure maxillectomy for a T2 N0 maxillary alveolus cancer. The defect resulted in direct communication with the buccal space, nasal cavity, and maxillary sinus. A radial forearm free tissue transfer was utilized to achieve oronasal separation.TRACHEOTOMYIndications and TimingThe most common cause for tracheotomy is prolonged intuba-tion typically in critically ill intensive care unit patients. Pro-longed intubation increases the risk of laryngeal and subglottic injury, which may lead to stenosis. In the critically ill patient, it has been hypothesized that early tracheotomy may improve inpatient survival and decreased intensive care unit length of stay while increasing patient comfort. However, a large ran-domized clinical trial demonstrated no benefit from early tra-cheotomy on shortor long-term survival and other important secondary outcomes.205 Furthermore, clinicians are poor pre-dictors of which patients require extended ventilatory support. Another study demonstrated no

1	long-term survival and other important secondary outcomes.205 Furthermore, clinicians are poor pre-dictors of which patients require extended ventilatory support. Another study demonstrated no evidence that early tracheos-tomy reduced mortality, duration of mechanical ventilation, intensive care unit stay, or ventilatory associated pneumonia.206 It did, however, provide a shorter duration of sedation. Beyond prolonged intubation, tracheotomy is also indicated in patients who require frequent pulmonary toilet, in patients with neu-rologic deficits that impair protective airway reflexes, and in head and neck upper aerodigestive tract surgery as a temporary airway in the perioperative period to bypass airway obstruction.Technique and ComplicationsThe procedure can be performed using an open or a percuta-neous technique. Complications of tracheostomy include pneu-mothorax, tracheal stenosis, wound infection/stomatitis with large-vessel erosion, and failure to close after decannulation. A

1	or a percuta-neous technique. Complications of tracheostomy include pneu-mothorax, tracheal stenosis, wound infection/stomatitis with large-vessel erosion, and failure to close after decannulation. A meta-analysis of 15 randomized studies assessing nearly 1000 patients demonstrated no difference between the open and percutaneous techniques, although there was a trend toward fewer complications in the percutaneous approach.207 The per-cutaneous approach was also found to be cheaper and had the added benefit of being performed at the bedside outside of the operating room. A Cochrane review on the topic lower wound infection/stomatitis and unfavorable scarring rates with the per-cutaneous approach.208 Mortality and serious adverse events did not differ between the two techniques.The use of cricothyroidotomy, typically in the emergency setting, is inferior to a tracheotomy due to higher incidence of vocal cord dysfunction and subglottic stenosis. There-fore, soon after a cricothyroidotomy

1	typically in the emergency setting, is inferior to a tracheotomy due to higher incidence of vocal cord dysfunction and subglottic stenosis. There-fore, soon after a cricothyroidotomy is performed, a formal Figure 18-43. A. Defect after left hemiglossectomy for T2 N0 oral tongue squamous cell carcinoma. B. Radial forearm free tissue transfer harvested for reconstruction. C. Inset of the radial forearm free tissue transfer.ABCBrunicardi_Ch18_p0613-p0660.indd 65201/03/19 5:25 PM 653DISORDERS OF THE HEAD AND NECKCHAPTER 18Figure 18-45. A. Palate defect after infrastructure maxillectomy for T2 N0 squamous cell carcinoma of the maxillary alveolus. B. Inset of radial forearm free tissue transfer. C. Six month postop-erative result, with complete oronasal separation and return to full, preoperative levels of speech and swallowing.tracheotomy should be used with decannulation of the crico-thyroidotomy site. Most tracheostomies are not permanent and can be reversed simply by removing the

1	levels of speech and swallowing.tracheotomy should be used with decannulation of the crico-thyroidotomy site. Most tracheostomies are not permanent and can be reversed simply by removing the tube and applying a pressure dressing. The stoma usually spontaneously heals within 2 to 3 weeks.Speech with Tracheotomy and DecannulationWhen a large cuffed tracheostomy is initially placed, speech is not possible, particularly when the cuff is up. However, when the tube is downsized to a cuffless tracheostomy tube, ABCFigure 18-44. A. Segmental mandible defect after composite resec-tion for T4a N0 squamous cell carcinoma of the mandibular alveolus. B. Fibula free tissue transfer harvested for reconstruction and template for wedge ostectomy. C. Inset of fibula free tissue transfer.ABCBrunicardi_Ch18_p0613-p0660.indd 65301/03/19 5:25 PM 654SPECIFIC CONSIDERATIONSPART IIintermittent finger occlusion or placement of Passy-Muir valve can allow the patient to voice while still bypassing the upper

1	65301/03/19 5:25 PM 654SPECIFIC CONSIDERATIONSPART IIintermittent finger occlusion or placement of Passy-Muir valve can allow the patient to voice while still bypassing the upper airway obstruction in inspiration. Prior to decannulation, the patient has to tolerate capping for 24 to 48 hours, but this period can be extended in patients with concerns for pulmonary toilet and an inability to clear secretions.LONG TERM MANAGEMENT AND REHABILITATIONPalliative CareFor patients with unresectable disease (greater than 180o of encasement around the carotid artery, prevertebral fascia inva-sion, and skull base invasion) or distant metastases, palliative care options exist. The NCCN guidelines recommend clinical trials for patients in this category because there is not a single accepted regimen for patients with incurable disease but the goal of treatment is to control symptoms and maintain quality of life while minimizing the side effects of treatment.106 This may include a combination of

1	for patients with incurable disease but the goal of treatment is to control symptoms and maintain quality of life while minimizing the side effects of treatment.106 This may include a combination of radiotherapy, usually in a hypofrac-tionated pattern with high dose per fraction regimen, chemother-apy, or simply pain management. A recent trial demonstrated the utility of immunotherapy, specifically, Nivolumab, in the management of recurrent unresectable head and neck cancer, showing a higher response rate (13.3%) compared to standard therapy (5.8%) with lower treatment-related adverse events (13.1% vs. 35.1%, respectively).209 From a surgical perspective, some patients require tracheostomy or gastrostomy tube place-ment to manage airway compromise and dysphagia, respec-tively. Palliative care facilities and hospice care allow patients to retain dignity when they have a limited short-term outlook.Follow-Up CarePatients diagnosed and treated for a head and neck tumor require follow-up

1	care facilities and hospice care allow patients to retain dignity when they have a limited short-term outlook.Follow-Up CarePatients diagnosed and treated for a head and neck tumor require follow-up care aimed at monitoring for recurrence and the side effects of therapy. The NCCN guidelines recommend follow-up assessment every 3 months for the first year after treatment, every 4 months during the following year, and then every 6 months until year 4, with an annual follow-up at 5 years post treatment and thereafter.106 This regimen is not well followed in North America, and further investigation is required to assess why this might be and to improve adherence rates.210 Follow-up should consist of a thorough history to assess for any emerg-ing symptoms such as pain, otalgia, or dysphagia as these are often the first sign of a recurrence. Assessment by speech lan-guage pathology and a dietician is often beneficial to ascertain swallowing function and nutritional intake, respectively.

1	as these are often the first sign of a recurrence. Assessment by speech lan-guage pathology and a dietician is often beneficial to ascertain swallowing function and nutritional intake, respectively. Some patients require dilation or reinsertion of a gastrostomy tube if they develop pharyngeal strictures and are unable to maintain their weight. The history should be followed with a thorough head and neck examination, including fiberoptic nasolaryg-noscopy, because of the significant risk of developing a sec-ond primary in the upper aerodigestive tract.93 Patients should have their thyroid stimulating hormone (TSH) checked once a year, especially in those that have radiation as they may develop hypothyroidism at an earlier age than the general population. Shoulder dysfunction after neck dissection with extensive accessory nerve dissection or in patients who have had a scapu-lar system free flap should be managed with physiotherapy to minimize the long-term effects and improve function.

1	with extensive accessory nerve dissection or in patients who have had a scapu-lar system free flap should be managed with physiotherapy to minimize the long-term effects and improve function. Chronic pain can occur in head and neck cancer patients, and this is often assessed and managed by a pain specialist. Ongoing dental evaluation is needed in some patients to treat caries and prevent osteoradionecrosis.REFERENCESEntries highlighted in bright blue are key references. 1. Hajioff D, MacKeith S. Otitis externa. BMJ Clin Evid. 2015;2015:0510. 2. Sylvester MJ, Sanghvi S, Patel VM, Eloy JA, Ying YM. Malignant otitis externa hospitalizations: analysis of patient characteristics. Laryngoscope. 2017;127(10):2328-2336. 3. Carfrae MJ, Kesser BW. Malignant otitis externa. Otolaryngol Clin North Am. 2008;41(3):537-549, viii-ix. 4. Venekamp RP, Damoiseaux RA, Schilder AG. Acute otitis media in children. Am Fam Physician. 2017;95(2):109-110. 5. Rosenfeld RM, Shin JJ, Schwartz SR, et al. Clinical

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1	Chest Wall, Lung, Mediastinum, and PleuraKatie S. Nason, Rose B. Ganim, and James D. Luketich 19chapterTRACHEAAnatomyThe trachea is composed of cartilaginous and membranous por-tions, beginning with the cricoid cartilage, the first complete cartilaginous ring of the airway. The cricoid cartilage consists of an anterior arch and a posterior broad-based plate. Articulat-ing with the posterior cricoid plate are the arytenoid cartilages. The vocal cords originate from the arytenoid cartilages and then attach to the thyroid cartilage. The subglottic space, the nar-rowest part of the trachea with an internal diameter of approxi-mately 2 cm, begins at the inferior surface of the vocal cords and extends to the first tracheal ring. The remainder of the distal trachea is 10.0 to 13.0 cm long, consists of 18 to 22 rings, and has an internal diameter of 2.3 cm (Fig. 19-1).1Bronchoscopically, the tracheal rings are visible as C-shaped hyaline cartilaginous structures that provide rigid-ity to the

1	of 18 to 22 rings, and has an internal diameter of 2.3 cm (Fig. 19-1).1Bronchoscopically, the tracheal rings are visible as C-shaped hyaline cartilaginous structures that provide rigid-ity to the anterior and lateral tracheal walls. The open ends of the C-rings are connected by the trachealis smooth muscle and encased in a dense band of connective tissue called perichon-drium. The first tracheal ring is attached directly to the cricoid cartilage; there are approximately two rings for every 1 cm of tracheal length.The tracheal blood supply, which includes the inferior thy-roid, subclavian, supreme intercostal, internal thoracic, innomi-nate, and superior and middle bronchial arteries, enters the airway near the junction of the membranous and cartilaginous portions (Fig. 19-2). Each arterial branch supplies a segment of 1.0 to 2.0 cm, thereby limiting circumferential mobilization to that same distance. The vessels are interconnected along the lat-eral surface of the trachea by an

1	branch supplies a segment of 1.0 to 2.0 cm, thereby limiting circumferential mobilization to that same distance. The vessels are interconnected along the lat-eral surface of the trachea by an important longitudinal vascular anastomosis that feeds transverse segmental vessels to the soft tissues between the cartilages.Tracheal InjuryTracheal injury can result from a variety of causes, includ-ing inhalation of smoke or toxic fumes, aspiration of liquids or solid objects, endotracheal intubation, blunt and penetrating trauma, and iatrogenic injury during operative procedures. Early diagnosis is critical to avoid subsequent complications, includ-ing respiratory infection and tracheal stenosis. Management of smoke or toxic fume inhalation and liquid aspiration is com-monly supportive; use of antibiotics, respiratory support, and airway clearance with flexible bronchoscopy is dictated by the patient’s condition. In rare circumstances, extracorporeal mem-brane oxygenation is required if

1	of antibiotics, respiratory support, and airway clearance with flexible bronchoscopy is dictated by the patient’s condition. In rare circumstances, extracorporeal mem-brane oxygenation is required if there is associated injury to the more distal airways and lung parenchyma.Despite ubiquitous use of high-volume–low-pressure cuffs, overinflation of the endotracheal cuff is the most common cause of injury secondary to endotracheal intubation. High cuff pressures can cause ischemia of the contiguous airway wall in as short as 4 hours. Prolonged overinflation can lead to scarring Trachea661Anatomy / 661Tracheal Injury / 661Tracheal Fistulas / 664Tracheal Neoplasms / 665Lung667Anatomy / 667Normal Lung Histology / 668Preinvasive Lesions / 669Invasive or Malignant Lesions / 670Lung Cancer Epidemiology / 673Screening for Lung Cancer in High-Risk Populations / 675Solitary Pulmonary Nodule / 677Metastatic Lesions to the Lung / 678Primary Lung Cancer-Associated Signs and Symptoms / 680Lung

1	/ 673Screening for Lung Cancer in High-Risk Populations / 675Solitary Pulmonary Nodule / 677Metastatic Lesions to the Lung / 678Primary Lung Cancer-Associated Signs and Symptoms / 680Lung Cancer Management / 683Lung Cancer Treatment / 693Options for Thoracic Surgical Approaches / 701Postoperative Care / 703Postoperative Complications / 705Spontaneous Pneumothorax / 705Pulmonary Infections / 706Massive Hemoptysis / 717End-Stage Lung Disease / 719Chest Wall720Chest Wall Mass / 720Benign Chest Wall Neoplasms / 722Primary Malignant Chest Wall Tumors / 723Other Tumors of the Chest Wall / 725Chest Wall Reconstruction / 726Mediastinum726Anatomy and Pathologic Entities / 726History and Physical Examination / 727Imaging and Serum Markers / 727Diagnostic Nonsurgical Biopsies of the Mediastinum / 729Surgical Biopsies and Resection of Mediastinal Masses / 730Mediastinal Neoplasms / 730Mediastinal Cysts / 735Mediastinitis / 735Pleura and Pleural Space736Anatomy / 736Pleural Effusion /

1	/ 729Surgical Biopsies and Resection of Mediastinal Masses / 730Mediastinal Neoplasms / 730Mediastinal Cysts / 735Mediastinitis / 735Pleura and Pleural Space736Anatomy / 736Pleural Effusion / 736Access and Drainage of Pleural Fluid Collections / 736Malignant Pleural Effusion / 739Empyema / 740Chylothorax / 741Tumors of the Pleura / 743Brunicardi_Ch19_p0661-p0750.indd 66101/03/19 7:00 PM 662Key Points1 Lung cancer continues to be a highly lethal and extremely common cancer, with 57% of patients presenting with dis-tant metastasis and 5-year survival of 18%. Lung cancer incidence is second only to the incidence of prostate cancer in men and breast cancer in women, with 222,500 esti-mated new cases in 2017. Squamous cell carcinoma and adenocarcinoma of the lung are the most common sub-types and are rarely found in the absence of a smoking history. Nonsmokers who live with smokers have a 24% increased risk of lung cancer compared to nonsmokers who do not live with smokers.2 A

1	sub-types and are rarely found in the absence of a smoking history. Nonsmokers who live with smokers have a 24% increased risk of lung cancer compared to nonsmokers who do not live with smokers.2 A multidisciplinary approach to evaluation of NSCLC, with standardized criteria and terminology for diagnosis in cytologic and small biopsy specimens, and routine molec-ular testing for known mutations, such as EGFR mutations and EML4-ALK fusion oncogenes is now recommended for the evaluation and management of lung nodules due to major advances in targeted therapy. Adequate tissue acquisition at the time of diagnostic workup is critical and facilitates patient care while minimizing the number of procedures to which the patient is subjected.3 The terms bronchioloalveolar carcinoma and mixed subtype adenocarcinoma have been eliminated from the classification of lung adenocarcinoma as a result of increased understanding of important clinical, radiologic, pathologic, and genetic differences

1	adenocarcinoma have been eliminated from the classification of lung adenocarcinoma as a result of increased understanding of important clinical, radiologic, pathologic, and genetic differences between mucinous and nonmucinous adenocarcinomas. The classification system delineates a stepwise pathologic progression, from AAH to invasive adenocarcinoma based on the predominant histo-logic growth patterns.4 The U.S. Preventive Services Task Force now recommends annual screening for lung cancer with low-dose computed tomography screening in high risk patients. Annual screen-ing averted 14% of lung cancer deaths when applied to a population of asymptomatic adults age 55 to 80 years who have a 30 pack-year smoking history and are either currently smoking or have quit within the past 15 years. Patients should be healthy enough to tolerate curative treat-ment, specifically surgery per guidelines, and screening should be discontinued once the patient has not smoked for 15 years or develops a

1	Patients should be healthy enough to tolerate curative treat-ment, specifically surgery per guidelines, and screening should be discontinued once the patient has not smoked for 15 years or develops a life-limiting health condition, becomes unable to tolerate lung surgery, or is unwilling to undergo curative lung resection. With this approach, it is expected that 50% of diagnosed cancers will be early stage. Screening of patients age 50 years or older with a 20 pack-year or greater history and additional risk factors (as determined by the Tammemagi lung cancer risk calculator or other validated risk scores) that increase the risk of lung cancer to 1.3% or greater should also be considered as part of lung cancer screening programs. In all cases, patient–physician shared decision-making should be undertaken, with a discussion of the risks and benefits of screening.5 Assessment of patient risk before thoracic resection is based on clinical judgment and systematic assessment of

1	should be undertaken, with a discussion of the risks and benefits of screening.5 Assessment of patient risk before thoracic resection is based on clinical judgment and systematic assessment of cardiopulmonary status using established algorithms.6 Maximum oxygen consumption (v. o2max) values provide important additional information in those patients with severely impaired Dlco and forced expiratory volume in 1 second. Values of <10 mL/kg per minute generally pro-hibit any major pulmonary resection because the mortality in patients with these levels is 26% compared with only 8.3% in patients whose v. o2max is ≥10 mL/kg per minute; values of >15 mL/kg per minute generally indicate the patient’s ability to tolerate pneumonectomy.7 Tumor ablative strategies are viable alternatives to surgical resection for early stage lung cancer in inoperable patients. While premature, ablative techniques may ultimately be shown to have efficacy equivalent to lobectomy for the pri-mary treatment of very

1	resection for early stage lung cancer in inoperable patients. While premature, ablative techniques may ultimately be shown to have efficacy equivalent to lobectomy for the pri-mary treatment of very small peripheral early-stage lung cancers and become primary therapy, even in operable patients, although limited resection with wedge (at least 2 cm margin and at least 1:1 tumor/margin ratio) and seg-mentectomy provide better margins of treatment and nodal sampling ensures occult nodal metastasis are identified. Multidisciplinary collaboration among thoracic surgery, interventional radiology/pulmonology, and radiation oncol-ogy is required to ensure that development of these ablative techniques occurs through properly designed and well-con-trolled prospective studies and will ensure that patients receive the best available therapy, regardless of whether it is surgical resection or ablative therapy.8 The term non–small cell lung carcinoma (NSCLC) includes many tumor cell types, including

1	receive the best available therapy, regardless of whether it is surgical resection or ablative therapy.8 The term non–small cell lung carcinoma (NSCLC) includes many tumor cell types, including large cell, squa-mous cell, and adenocarcinoma. The approach to diagno-sis and management and the terminology used in describing these tumors are evolving rapidly. In particular, the evaluation and management of adenocarcinoma of the lung has shifted dramatically and firm establishment of NSCLC cell type prior to chemotherapy for advanced stage lung cancer is essential.9 Increasing evidence suggests a significant role for gastro-esophageal reflux disease in the pathogenesis of chronic lung diseases such as bronchiectasis and idiopathic pulmo-nary fibrosis, and it may also contribute to bronchiolitis obliterans syndrome in lung transplant patients.10 Treatment of pulmonary aspergillosis/aspergilloma is indi-vidualized. Following colonization of a lung cavity or area of bronchiectasis, fungal

1	obliterans syndrome in lung transplant patients.10 Treatment of pulmonary aspergillosis/aspergilloma is indi-vidualized. Following colonization of a lung cavity or area of bronchiectasis, fungal growth within the cavity appears as an irregular cavitary lining, progressing over time as a late finding in chronic pulmonary aspergillosis to a fungal ball called an aspergilloma. Asymptomatic patients can be observed without any additional therapy. Similarly, mild hemoptysis, which is not life-threatening, can be managed with medical therapy, including antifungals and cough suppressants. Oral triazole therapy is now considered the standard of care for chronic, cavitary pulmonary aspergil-losis. Massive hemoptysis had traditionally been an indi-cation for urgent or emergent operative intervention. However, with the advancement of endovascular tech-niques, bronchial artery embolization in select centers with experience in these techniques has been effective.11 In patients with malignant

1	However, with the advancement of endovascular tech-niques, bronchial artery embolization in select centers with experience in these techniques has been effective.11 In patients with malignant pleural effusion, poor expan-sion of the lung (because of entrapment by tumor or adhe-sions) generally predicts a poor result with pleurodesis and is the primary indication for placement of indwelling pleu-ral catheters. These catheters have dramatically changed the management of end-stage cancer treatment because they substantially shorten the amount of time patients spend in the hospital during their final weeks of life.Brunicardi_Ch19_p0661-p0750.indd 66201/03/19 7:00 PM

1	CHAPTER 19663CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAEpiglottisAryepiglottic m.Transverse, obliquearytenoid mm.Lateralcricoarytenoid m.Posteriorcricoarytenoid m.Thyroid cartilage facetRecurrentlaryngeal n.Internallaryngeal n.Thyroepiglottic m.Thyroarytenoid m.Cricothyroid m.(cut)Inferiorthyroid a.Branch from internal thoracic a. Superior bronchial a.Middle bronchial a.1Lateral longitudinalanastomosis32Figure 19-2. Arterial blood supply to the larynx and upper trachea. a. = artery.Figure 19-1. Anatomy of the larynx and upper trachea. m. = muscle; n. = nerve.and stenosis; full-thickness injury can result in fistulae between the innominate artery anteriorly and the esophagus posteriorly. Avoidance requires careful cuff management to keep pressures as low as possible; in circumstances of prolonged ventilatory support and high airway pressure, cuff pressure monitoring (to maintain pressures <20 mmHg) is advisable.Historically, clinically significant tracheal stenosis after

1	of prolonged ventilatory support and high airway pressure, cuff pressure monitoring (to maintain pressures <20 mmHg) is advisable.Historically, clinically significant tracheal stenosis after tracheostomy occurred in 3% to 12% of cases, with severe stenosis in 1% to 2%.2 With the use of low-pressure cuffs, the estimated incidence has decreased to 4.9 cases per million patients per year. Intubation-related risk factors include pro-longed intubation; high tracheostomy through the first tracheal ring or cricothyroid membrane; transverse rather than vertical incision on the trachea; oversized tracheostomy tube; prior tra-cheostomy or intubation; and traumatic intubation. Stenosis is also more common in older patients, in women, after radiation, or after excessive corticosteroid therapy, and in the setting of concomitant diseases such as autoimmune disorders, severe reflux disease, or obstructive sleep apnea and the setting of severe respiratory failure. However, even a properly placed

1	in the setting of concomitant diseases such as autoimmune disorders, severe reflux disease, or obstructive sleep apnea and the setting of severe respiratory failure. However, even a properly placed tra-cheostomy can lead to tracheal stenosis because of scarring and local injury. Mild ulceration and stenosis are frequently seen after tracheostomy removal. Use of the smallest tracheostomy tube possible, rapid downsizing, and a vertical tracheal incision minimize the risk for posttracheostomy stenosis.Stridor and dyspnea on exertion are the primary symp-toms of tracheal stenosis. In the setting of postintubation injury, a significant portion of the cartilaginous structural support to the airway is destroyed by regional ischemic necrosis; during healing, a weblike fibrous growth develops and narrows the airway (Fig. 19-3). In contrast, stenosis caused by tracheostomy is most commonly due to an excess of granulation tissue forma-tion around the tracheal stoma site. Time to onset of

1	and narrows the airway (Fig. 19-3). In contrast, stenosis caused by tracheostomy is most commonly due to an excess of granulation tissue forma-tion around the tracheal stoma site. Time to onset of symptoms after extubation or tracheostomy decannulation usually ranges from 2 to 12 weeks, but symptoms can appear immediately or as long as 1 to 2 years later. Frequently, patients are misdiag-nosed as having asthma or bronchitis, and treatment for such illnesses can persist for some time before the correct diagnosis is discovered. Generally, symptom intensity is related to the degree of stenosis and to the patient’s underlying pulmonary disease.Brunicardi_Ch19_p0661-p0750.indd 66301/03/19 7:00 PM 664SPECIFIC CONSIDERATIONSPART IIFigure 19-3. Diagram of the principal postintubation lesions. A. A circumferential lesion at the cuff site after the use of an endotracheal tube. B. Potential lesions after the use of tracheostomy tubes. Anterolateral stenosis can be seen at the stomal level.

1	A. A circumferential lesion at the cuff site after the use of an endotracheal tube. B. Potential lesions after the use of tracheostomy tubes. Anterolateral stenosis can be seen at the stomal level. Circumferential stenosis can be seen at the cuff level (lower than with an endotracheal tube). The segment in between is often inflamed and malacotic. C. Damage to the subglottic larynx. D. Tracheoesophageal fistula occurring at the level of the tracheostomy cuff; circumferential damage is usual at this level. E. Tracheoinnominate artery fistula. (Adapted with permission from Grillo H. Surgical treatment of postintubation tracheal injuries. J Thorac Cardiovasc Surg. 1979 Dec;78(6):860-875.)Acute Management. A comprehensive bronchoscopic evalua-tion is critical in the initial phase of evaluation. Stenosis length, location, distance between the vocal cords and proximal steno-sis, and distance from the distal aspect to the major carina must be documented. In patients with severe stenosis and

1	Stenosis length, location, distance between the vocal cords and proximal steno-sis, and distance from the distal aspect to the major carina must be documented. In patients with severe stenosis and respiratory compromise, rigid bronchoscopy can be used to dilate the steno-sis; this provides immediate relief of the airway obstruction and facilitates thorough evaluation of the stenosis. Rarely, if ever, is tracheostomy necessary.Most intubation injuries are located in the upper third of the trachea and can be accessed for resection through a col-lar incision. Resection typically involves 2 to 4 cm of trachea for benign stenosis. It is critical to fully resect all inflamed and scarred tissue. However, a primary anastomosis can still be per-formed without undue tension, even if up to one half of the tra-chea requires resection.2 Ideally, the patient is extubated in the operating room or shortly thereafter. For patients in whom tra-cheal resection is not possible, such as patients with

1	of the tra-chea requires resection.2 Ideally, the patient is extubated in the operating room or shortly thereafter. For patients in whom tra-cheal resection is not possible, such as patients with significant comorbidities or with an excessively long stenosis, endotracheal stenting, typically silicone T-tubes, can provide palliation. Wire mesh stents should not be used, given their known propensity to erode through the wall of the airway. Balloon dilation, laser ablation, and tracheoplasty have also been described, although the efficacy is marginal.Tracheal replacement is evolving as an option for manage-ment of tracheal stenosis as bioengineering techniques for decel-lularizing donor trachea have been developed. This removes all antigens against which the recipient immune system might react and enables use of the donor trachea scaffolding without risk of rejection. Following decellularization, the donor tracheal scaf-folding is seeded with recipient chondrocytes, to restore tracheal

1	and enables use of the donor trachea scaffolding without risk of rejection. Following decellularization, the donor tracheal scaf-folding is seeded with recipient chondrocytes, to restore tracheal rigidity, and with recipient epithelial cells, to recreate the inner epithelial lining. Several case reports of successful allogeneic tracheal transplantation have been published. The technique continues to be limited to a few highly specialized centers, due, in part, to the scarcity of donor trachea and the need for tissue bioengineering expertise as well as the lack of established effi-cacy for the approach. Current efforts are focused on creation of biosynthetic scaffolding that can be used instead of donor trachea. This would substantially increase the availability of the tracheal replacement material and enable widespread use of the technique, but early results have been contested, including three case reports called into question as containing multiple data fab-rications and

1	replacement material and enable widespread use of the technique, but early results have been contested, including three case reports called into question as containing multiple data fab-rications and omissions.Tracheal FistulasTracheoinnominate Artery Fistula. Tracheoinnominate artery fistula has two main causes: low placement of a trache-ostomy and hyperinflation of the tracheal cuff. Tracheostomy placement should be through the second to fourth tracheal rings without reference to the location of the sternal notch. When placed below the fourth tracheal ring, the inner curve of the tracheostomy cannula will be positioned to exert pressure on the posterior aspect of the innominate artery, leading to arterial ero-sion. Similarly, the tracheal cuff, when hyperinflated, will cause ischemic injury to the anterior airway and subsequent erosion into the artery. Most cuff-induced fistulas will develop within 2 weeks after placement of the tracheostomy.Clinically, tracheoinnominate artery

1	injury to the anterior airway and subsequent erosion into the artery. Most cuff-induced fistulas will develop within 2 weeks after placement of the tracheostomy.Clinically, tracheoinnominate artery fistulas present with bleeding. A premonitory hemorrhage often occurs and, although it is usually not massive, must not be ignored or simply attrib-uted to general airway irritation or wound bleeding. With sig-nificant bleeding, the tracheostomy cuff can be hyperinflated to temporarily occlude the arterial injury. If such an effort is unsuccessful, the tracheostomy incision should be immediately opened widely and a finger inserted to compress the artery Brunicardi_Ch19_p0661-p0750.indd 66401/03/19 7:00 PM

1	CHAPTER 19665CHEST WALL, LUNG, MEDIASTINUM, AND PLEURA1Cuffhyperinflation2Digital control3 BronchoscopiccompressionOrotracheal tubeready in place if neededOrotrachealtube replacingtracheostomytubeForward pressureapplied withbronchoscopeFigure 19-4. Steps in the emergency management of a tracheoinnominate artery fistula.against the manubrium (Fig. 19-4). The patient can then be orally intubated, and the airway suctioned free of blood. Emer-gent surgical resection of the involved segment of artery is per-formed, usually without reconstruction.Tracheoesophageal Fistula. Tracheoesophageal fistu-las (TEFs) occur primarily in patients receiving prolonged mechanical ventilator support concomitant with an indwelling nasogastric tube.4 Cuff compression of the membranous trachea against the nasogastric tube leads to airway and esophageal injury and fistula development. Clinically, airway suctioning reveals saliva, gastric contents, or tube feedings. Gastric insuf-flation, secondary to positive

1	tube leads to airway and esophageal injury and fistula development. Clinically, airway suctioning reveals saliva, gastric contents, or tube feedings. Gastric insuf-flation, secondary to positive pressure ventilation, can occur. Bronchoscopy is diagnostic; with the bronchoscope inserted, the endotracheal tube is withdrawn, and the fistula at the cuff site is exposed. Alternatively, esophagoscopy demonstrates the cuff of the endotracheal tube in the esophagus.Treatment, first and foremost, requires removing tubes from the esophagus and weaning the patient from the ventila-tor. The cuff of the endotracheal tube should be placed below the fistula, avoiding overinflation. To minimize aspiration, a gastrostomy tube should be placed for gastric decompression (to prevent reflux) and a jejunostomy tube for feeding. If aspira-tion persists, esophageal diversion with cervical esophagostomy can be performed. Once weaned from the ventilator, tracheal resection and primary anastomosis, repair of

1	tube for feeding. If aspira-tion persists, esophageal diversion with cervical esophagostomy can be performed. Once weaned from the ventilator, tracheal resection and primary anastomosis, repair of the esophageal defect, and interposition of a muscle flap between the trachea and esophagus can be performed (Fig. 19-5).5Tracheal NeoplasmsAlthough extremely rare, the most common primary tracheal neoplasms are squamous cell carcinomas (related to smoking) and adenoid cystic carcinomas. Clinically, tracheal tumors pres-ent with cough, dyspnea, hemoptysis, stridor, or symptoms of invasion of contiguous structures (such as the recurrent laryn-geal nerve or the esophagus). The most common radiologic finding of tracheal malignancy is tracheal stenosis, but it is found in only 50% of cases. With tumors other than squamous cell carcinomas, symptoms may persist for months because of slow tumor growth rates. Stage of presentation is advanced, with approximately 50% of patients presenting with stage

1	other than squamous cell carcinomas, symptoms may persist for months because of slow tumor growth rates. Stage of presentation is advanced, with approximately 50% of patients presenting with stage IV disease. Five-year survival for all tracheal neoplasms is 40% but falls to 15% for those with stage IV disease.6Squamous cell carcinomas often present with regional lymph node metastases and are frequently unresectable at pre-sentation. Their biologic behavior is similar to that of squamous cell carcinoma of the lung. Adenoid cystic carcinomas, a type of Brunicardi_Ch19_p0661-p0750.indd 66501/03/19 7:00 PM 666SPECIFIC CONSIDERATIONSPART IIACBDCricoidSternohyoid m.EsophagusFigure 19-5. Single-stage operation for clo-sure of a tracheoesophageal fistula and tra-cheal resection. A. The fistula is divided, and the trachea is transected below the level of damage. B. The fistula is closed on the tra-cheal side in a single layer and the esopha-geal side in a double layer. The damaged trachea

1	is divided, and the trachea is transected below the level of damage. B. The fistula is closed on the tra-cheal side in a single layer and the esopha-geal side in a double layer. The damaged trachea segment is resected. C. A pedicled muscle flap, such as the sternohyoid muscle, is used to buttress the esophageal repair. D. View of completed tracheal anastomosis. m. = muscle.salivary gland tumor, are generally slow growing, spread sub-mucosally, and tend to infiltrate along nerve sheaths and within the tracheal wall. Although indolent in nature, adenoid cystic carcinomas are malignant and can spread to regional lymph nodes, lung, and bone. Squamous cell carcinoma and adenoid cystic carcinomas represent approximately 65% of all tracheal neoplasms. The remaining 35% is comprised of small cell car-cinomas, mucoepidermoid carcinomas, adenocarcinomas, lym-phomas, and others.7Therapy. Evaluation and treatment of patients with tracheal tumors should include neck and chest computed tomography

1	car-cinomas, mucoepidermoid carcinomas, adenocarcinomas, lym-phomas, and others.7Therapy. Evaluation and treatment of patients with tracheal tumors should include neck and chest computed tomography (CT) and rigid bronchoscopy. Rigid bronchoscopy permits gen-eral assessment of the airway and tumor; it also allows debride-ment or laser ablation of the tumor to provide relief of dyspnea. If the tumor is judged to be completely resectable, primary resection and anastomosis is the treatment of choice for these tumors (Fig. 19-6). Up to 50% of the length of the trachea can be resected with primary anastomosis. In most tracheal resections, anterolateral tracheal mobilization and suturing of the chin to the sternum for 7 days are done routinely. Use of laryngeal and hilar release is determined at the time of surgery, based on the surgeon’s judgment of the degree of tension present. For longer resections, specialized maneuvers are necessary such as laryn-geal release and right hilar release to

1	the time of surgery, based on the surgeon’s judgment of the degree of tension present. For longer resections, specialized maneuvers are necessary such as laryn-geal release and right hilar release to minimize tension on the anastomosis.Postoperative mortality, which occurs in up to 10% of patients, is associated with the length of tracheal resection, use of laryngeal release, the type of resection, and the histologic type of the cancer. Factors associated with improved long-term survival include complete resection and use of radiation as adjuvant therapy in the setting of incomplete resection.8 Due to their radiosensitivity, radiotherapy is frequently given postop-eratively after resection of both adenoid cystic carcinomas and squamous cell carcinomas.9 A dose of 50 Gy or greater is usual. Nodal positivity does not seem to be associated with worse sur-vival. Survival at 5 and 10 years is much better for adenoid Brunicardi_Ch19_p0661-p0750.indd 66601/03/19 7:00 PM

1	CHAPTER 19667CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAHigh-index of suspicion(cough, dyspnea, hemoptysis,stridor, and hoarseness)Complete staging: computedtomography/PETscan/mediastinoscopyFlexible/rigid bronchoscopyTumor resectablePerformance statusadequate for surgeryDebridement and/orlaser ablationPrinciples of tracheal resection• May resect up to 50% of tracheal length• Anterolateral mobilization only• Suture head in forward flexion for 7 days• Laryngeal and hilar release as needed for relief of tensionRadiotherapy 50 Gy (±chemotherapy)(primary treatment or postoperatively)Tumor unresectable1) Probable grossly positive tracheal resection margin2) Metastatic disease3) Length of resection precludes safe reconstruction4) Invasion of unresectable adjacent organsPoor performancestatuscystic (73% and 57%, respectively) than for tracheal cancers (47% and 36%, respectively; P <.05). For patients with unre-sectable tumors, radiation may be given as the primary therapy to improve local

1	(73% and 57%, respectively) than for tracheal cancers (47% and 36%, respectively; P <.05). For patients with unre-sectable tumors, radiation may be given as the primary therapy to improve local control, but it is rarely curative. For recurrent airway compromise, stenting or laser therapies should be con-sidered as part of the treatment algorithm.LUNGAnatomySegmental Anatomy. The segmental bronchial and vascular anatomy of the lungs allows subsegmental and segmental resec-tions, if the clinical situation requires or if lung tissue can be preserved10 (Fig. 19-7). Note the continuity of the pulmonary parenchyma between adjacent segments of each lobe.Lymphatic Drainage. Lymph nodes that drain the lungs are divided into two groups according to the tumor-node-metastasis (TNM) staging system for lung cancer: the pulmonary lymph nodes (N1) and the mediastinal nodes (N2) (Fig. 19-8).The N1 lymph nodes constitute the following: (a) intrapul-monary or segmental nodes that lie at points of

1	for lung cancer: the pulmonary lymph nodes (N1) and the mediastinal nodes (N2) (Fig. 19-8).The N1 lymph nodes constitute the following: (a) intrapul-monary or segmental nodes that lie at points of division of seg-mental bronchi or in the bifurcations of the pulmonary artery; (b) lobar nodes that lie along the upper, middle, and lower lobe bronchi; (c) interlobar nodes located in the angles formed by the main bronchi bifurcating into the lobar bronchi; and (d) hilar nodes along the main bronchi. The interlobar lymph nodes lie in the depths of the interlobar fissure on each side and constitute a lymphatic sump for each lung, referred to as the lymphatic sump of Borrie; all of the pulmonary lobes of the corresponding lung drain into this group of nodes (Fig. 19-9). On the right, the nodes of the lymphatic sump lie around the bronchus inter-medius (bounded above by the right upper lobe bronchus and below by the middle lobe and superior segmental bronchi). On the left, the lymphatic sump

1	the lymphatic sump lie around the bronchus inter-medius (bounded above by the right upper lobe bronchus and below by the middle lobe and superior segmental bronchi). On the left, the lymphatic sump is confined to the interlobar fis-sure, with the lymph nodes in the angle between the lingular and lower lobe bronchi. These nodes are always in close proximity to pulmonary arterial branches and typically must be carefully dissected to identify the pulmonary arterial segments for divi-sion during lung resection.The N2 lymph nodes consist of four main groups. (a) The anterior mediastinal nodes are located in association with the upper surface of the pericardium, the phrenic nerves, the liga-mentum arteriosum, and the left aspect of the innominate vein. (b) The posterior mediastinal group includes paraesophageal lymph nodes within the inferior pulmonary ligament and, more superiorly, between the esophagus and trachea near the arch of the azygos vein. (c) The tracheobronchial lymph nodes are

1	paraesophageal lymph nodes within the inferior pulmonary ligament and, more superiorly, between the esophagus and trachea near the arch of the azygos vein. (c) The tracheobronchial lymph nodes are made up of three subgroups that are located near the bifurcation of the trachea. These include the subcarinal nodes, which lie in the obtuse angle between the trachea and each main stem bronchus, and the nodes that lay anterior to the lower end of the trachea. (d) Paratracheal lymph nodes are located in proximity to the trachea in the superior mediastinum. Those on the right side form a chain with the tracheobronchial nodes inferiorly and with some of the deep cervical nodes above (scalene lymph nodes).Lymphatic drainage to the mediastinal lymph nodes from the right lung is ipsilateral, except for occasional bilateral drain-age to the superior mediastinum. In contrast, in the left lung, particularly the left lower lobe, lymphatic drainage occurs with equal frequency to ipsilateral and

1	for occasional bilateral drain-age to the superior mediastinum. In contrast, in the left lung, particularly the left lower lobe, lymphatic drainage occurs with equal frequency to ipsilateral and contralateral superior medi-astinal nodes.Figure 19-6. Algorithm for eval-uation and treatment of tracheal neoplasm. PET = positron emis-sion tomography.Brunicardi_Ch19_p0661-p0750.indd 66701/03/19 7:00 PM 668SPECIFIC CONSIDERATIONSPART IIRight lung and bronchiLeft lung and bronchi11112222333333444445555556666668888888999999101010101010107771+21+2Segments 1. Apical2. Posterior3. Anterior4. Lateral5. Medial6. Superior7. Medial Basal *8. Anterior Basal9. Lateral Basal10. Posterior Basal * Medial basal (7) not present in left lungFigure 19-7. Segmental anatomy of the lungs and bronchi.3p3a6AoPA512LBrachiocephalicartery2R4RAo4LAzygos vein10R711R12,13,14R8R9R9L8LPA11L10L12,13,14LFigure 19-8. The location of regional lymph node stations for lung cancer.Figure 19-9. The lymphatic sump of

1	vein10R711R12,13,14R8R9R9L8LPA11L10L12,13,14LFigure 19-8. The location of regional lymph node stations for lung cancer.Figure 19-9. The lymphatic sump of Borrie includes the groups of lymph nodes that receive lymphatic drainage from all pulmonary lobes of the corresponding lung.Normal Lung HistologyThe lung can be conveniently viewed as two linked compo-nents: the tracheobronchial tree (or conducting airways com-ponent) and the alveolar spaces (or gas exchange component). The tracheobronchial tree consists of approximately 23 airway divisions to the level of the alveoli. It includes the main bronchi, lobar bronchi, segmental bronchi (to designated bronchopulmo-nary segments), and terminal bronchioles (i.e., the smallest air-ways still lined by bronchial epithelium and without alveoli). The tracheobronchial tree is normally lined by pseudostratified ciliated columnar cells and mucous (or goblet) cells, which both derive from basal cells (Fig. 19-10). Ciliated cells predominate. Goblet

1	tracheobronchial tree is normally lined by pseudostratified ciliated columnar cells and mucous (or goblet) cells, which both derive from basal cells (Fig. 19-10). Ciliated cells predominate. Goblet cells, which release mucus, can significantly increase in number in acute bronchial injury, such as exposure to cigarette smoke. The normal bronchial epithelium also contains bron-chial submucosal glands, which are mixed salivary-type glands Brunicardi_Ch19_p0661-p0750.indd 66801/03/19 7:00 PM

1	CHAPTER 19669CHEST WALL, LUNG, MEDIASTINUM, AND PLEURABAFigure 19-10. Normal lung histology. A. Pseudostratified ciliated columnar cells and mucous cells normally line the tracheobronchial tree. B. A Kulchitsky cell is depicted (arrow).containing mucous cells, serous cells, and neuroendocrine cells called Kulchitsky cells, which are also found within the surface epithelium. The bronchial submucosal glands can give rise to salivary gland–type tumors, including mucoepidermoid carci-nomas and adenoid cystic carcinomas.Two cell types, called type I and type II pneumocytes, make up the alveolar epithelium. Type I pneumocytes com-prise 40% of the total number of alveolar epithelial cells, but cover 95% of the surface area of the alveolar wall. These cells are not capable of regeneration because they have no mitotic potential. Type II pneumocytes cover only 3% of the alveo-lar surface, but comprise 60% of the alveolar epithelial cells. In addition, clusters of neuroendocrine cells are seen

1	they have no mitotic potential. Type II pneumocytes cover only 3% of the alveo-lar surface, but comprise 60% of the alveolar epithelial cells. In addition, clusters of neuroendocrine cells are seen in the alveolar spaces.Preinvasive LesionsThe term “precancerous” does not mean that an inevitable pro-gression to invasive carcinoma will occur, but such lesions, particularly those with high-grade dysplasia,11,12 do constitute a clear marker for potential development of invasive cancer. Three precancerous lesions of the respiratory tract are currently recognized.1. Squamous dysplasia and carcinoma in situ. Cigarette smoke can induce a transformation of the tracheobron-chial pseudostratified epithelium to metaplastic squamous mucosa, with subsequent evolution to dysplasia as cellu-lar abnormalities accumulate. Dysplastic changes include altered cellular polarity and increased cell size, number of cell layers, nuclear-to-cytoplasmic ratio, and number of mitoses. Gradations are considered

1	accumulate. Dysplastic changes include altered cellular polarity and increased cell size, number of cell layers, nuclear-to-cytoplasmic ratio, and number of mitoses. Gradations are considered mild, moderate, or severe. Carcinoma in situ represents carcinoma still con-fined by the basement membrane.2. Atypical adenomatous hyperplasia (AAH). AAH is a lesion smaller than 5.0 mm, comprising epithelial cells lining the alveoli that are similar to type II pneumocytes. Histologically, AAH is similar to adenocarcinoma in situ; it represents the beginning stage of a stepwise evolution to adenocarcinoma in situ and then to adenocarcinoma. With the availability of thin-section CT, it is possible to detect Brunicardi_Ch19_p0661-p0750.indd 66901/03/19 7:00 PM 670SPECIFIC CONSIDERATIONSPART IIpreinvasive adenocarcinoma lesions as early as AAH. These lesions can be multiple, are typically small (5 mm or less), and have a ground-glass appearance.3. Diffuse idiopathic pulmonary neuroendocrine cell

1	adenocarcinoma lesions as early as AAH. These lesions can be multiple, are typically small (5 mm or less), and have a ground-glass appearance.3. Diffuse idiopathic pulmonary neuroendocrine cell hyper-plasia. This rare lesion represents a diffuse proliferation of neuroendocrine cells, but without invasion of the basement membrane. It can exist as a diffuse increase in the number of single neuroendocrine cells, or as small lesions less than 5.0 mm in diameter. Lesions over 5.0 mm in size or that breach the basement membrane are carcinoid tumors.Invasive or Malignant LesionsThe pathologic diagnosis of lung cancer is currently based on light microscopic criteria and is broadly divided into two main groups: non–small cell lung carcinoma and neuroendocrine tumors.13 Immunohistochemical staining and electron micros-copy are used as adjuncts in diagnosis, particularly in the assess-ment of potential neuroendocrine tumors.Non–Small Cell Lung Carcinoma. The term non–small cell lung carcinoma

1	and electron micros-copy are used as adjuncts in diagnosis, particularly in the assess-ment of potential neuroendocrine tumors.Non–Small Cell Lung Carcinoma. The term non–small cell lung carcinoma (NSCLC) includes many tumor cell types, including large cell, squamous cell, and adenocarcinoma. His-torically, these subtypes were considered to be a uniform group based on limited understanding of the distinct clinical behaviors of the subtypes as well as the fact that there were few treat-ment options available. With increasing understanding of the molecular biology underlying these tumor subtypes, however, the approach to diagnosis and management and the terminology used in describing these tumors are evolving rapidly.Adenocarcinoma The incidence of adenocarcinoma has increased over the last several decades, and it is now the most common lung cancer, accounting for 30% of lung cancers in Table 19-1Difference between invasive mucinous adenocarcinoma and nonmucinous adenocarcinoma in

1	last several decades, and it is now the most common lung cancer, accounting for 30% of lung cancers in Table 19-1Difference between invasive mucinous adenocarcinoma and nonmucinous adenocarcinoma in situ/minimally invasive adenocarcinoma/lepidic predominant adenocarcinoma INVASIVE MUCINOUS ADENOCARCINOMA (FORMERLY MUCINOUS BAC)NONMUCINOUS AIS/MIA/LPA (FORMERLY NONMUCINOUS BAC)Female49/84 (58%)52,120-123101/140 (72%)52,120-123Smoker39/87 (45%)52,120-122,12475/164 (46%)52,120-122,124Radiographic appearanceMajority consolidation; air bronchogram125Majority ground-glass attenuation23,56,58,103,129-134 Frequent multifocal and multilobar presentation56,125-128 Cell typeMucin-filled, columnar, and/or goblet50-52,125,135Type II pneumocyte and/or Clara cell50-52,125,135Phenotype CK7Mostly positive (∼88%)a54,55,136-139Positive (∼98%)a54,55,136-139 CK20Positive (∼54%)a54,55,136-139Negative (∼5%)a54,55,136-139 TTF-1Mostly negative (∼17%)a54,55,120,137-139Positive

1	positive (∼88%)a54,55,136-139Positive (∼98%)a54,55,136-139 CK20Positive (∼54%)a54,55,136-139Negative (∼5%)a54,55,136-139 TTF-1Mostly negative (∼17%)a54,55,120,137-139Positive (∼67%)a54,55,120,137-139Genotype KRAS mutationFrequent (∼76%)a55,94,121,127,140-144Some (∼13%)a55,121,127,140-144 EGFR mutationAlmost none (∼3)a55,121,127,140-142Frequent (∼45%)a55,121,127,140-142Note: aNumbers represent the percentage of cases that are reported to be positive.Abbreviations: BAC = bronchioloalveolar carcinoma; AIS = adenocarcinoma in situ; MIA = minimally invasive adenocarcinoma; LPA = lepidic predominant adenocarcinoma; EGFR = epidermal growth factor receptor; TTF = thyroid transcription factor.Reproduced with permission from Travis WD, Brambilla E, Noguchi M, et al: International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma, J Thorac Oncol. 2011 Feb;6(2):244-285.male

1	for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma, J Thorac Oncol. 2011 Feb;6(2):244-285.male smokers and 40% of lung cancers in female smokers. Adenocarcinoma is the histologic subtype for 80% and 60% of lung cancers in nonsmoking females and males, respectively. It occurs more frequently in females than in males. It is the most frequent histologic subtype in women, patients who are under 45 years of age, and Asian populations.14Histologic Subtyping of Adenocarcinoma. Increasing under-standing of lung adenocarcinoma, such as important clinical, radiologic, pathologic, and genetic differences between mucinous and nonmucinous adenocarcinomas, prompted multiple changes in the classification system in 2011.15 Based on consensus, the international working group proposed a multidisciplinary approach, with standardized criteria and terminology for diagno-sis in cytologic and small biopsy

1	system in 2011.15 Based on consensus, the international working group proposed a multidisciplinary approach, with standardized criteria and terminology for diagno-sis in cytologic and small biopsy specimens, and routine molecu-lar testing for known mutations, such as EGFR and KRAS mutations (Table 19-1). The new classification system delineated a stepwise pathologic progression, from AAH to invasive adenocarcinoma based on the predominant histologic growth patterns; the terms bronchioloalveolar carcinoma and mixed subtype adenocarcinoma were eliminated in favor of more biologically driven classification (Table 19-2).1. Adenocarcinoma in situ (AIS). AISs are small (≤3 cm) sol-itary adenocarcinomas that have pure lepidic growth; lepidic growth is characterized by tumor growth within the alveolar spaces. These lesions are not invasive into the stroma, vas-cular system, or pleura and do not have papillary or micro-papillary patterns or intra-alveolar tumor cells. They are very rarely

1	alveolar spaces. These lesions are not invasive into the stroma, vas-cular system, or pleura and do not have papillary or micro-papillary patterns or intra-alveolar tumor cells. They are very rarely mucinous, consisting of type II pneumocytes or Clara cells. These patients are expected to have 100% dis-ease-specific survival with complete surgical resection. On CT scan, AIS can appear as a pure ground-glass neoplasm, 123Brunicardi_Ch19_p0661-p0750.indd 67001/03/19 7:00 PM

1	CHAPTER 19671CHEST WALL, LUNG, MEDIASTINUM, AND PLEURATable 19-2New classification system for lung adenocarcinomaPreinvasive lesions Atypical adenomatous hyperplasia Adenocarcinoma in situ (≤3 cm formerly BAC) Nonmucinous Mucinous Mixed mucinous/nonmucinousMinimally invasive adenocarcinoma (≤3 cm lepidic predominant tumor with ≤5 mm invasion) Nonmucinous Mucinous Mixed mucinous/nonmucinousInvasive adenocarcinoma Lepidic predominant (formerly nonmucinous BAC pattern, with >5 mm invasion) Acinar predominant Papillary predominant Micropapillary predominant Solid predominant with mucin productionVariants of invasive adenocarcinoma Invasive mucinous adenocarcinoma (formerly mucinous BAC) Colloid Fetal (low and high grade) EntericAbbreviations: BAC = bronchioloalveolar carcinoma; IASLC = International Association for the Study of Lung Cancer; ATS = American Thoracic Society; ERS = European Respiratory Society.Reproduced with permission from Travis WD, Brambilla E, Noguchi M, et al:

1	International Association for the Study of Lung Cancer; ATS = American Thoracic Society; ERS = European Respiratory Society.Reproduced with permission from Travis WD, Brambilla E, Noguchi M, et al: International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma, J Thorac Oncol. 2011 Feb;6(2):244-285.but occasionally it will present as part of a solid or part-solid nodule. Mucinous AIS is more likely to appear solid or to have the appearance of consolidation. As with AAH, the lesions can be single or multiple; the ground-glass changes in AIS, however, tend to have a higher attenuation com-pared to AAH. The 8th edition American Joint Committee on Cancer (AJCC) staging manual t-stage for AIS is tumor in situ (Tis).2. Minimally invasive adenocarcinoma (MIA). In the same size solitary lesion, if less than 5 mm of invasion are noted within a predominantly lepidic growth

1	t-stage for AIS is tumor in situ (Tis).2. Minimally invasive adenocarcinoma (MIA). In the same size solitary lesion, if less than 5 mm of invasion are noted within a predominantly lepidic growth pattern, the lesion is termed minimally invasive adenocarcinoma (MIA) to indicate a patient group with near 100% survival when the lesion is completely resected. This differentiates patients with AIS, but recognizes the fact that the presence of invasion becomes prognostically significant when the size of the invasive com-ponent reaches 5 mm or greater in size.16 If multiple areas of microscopic invasion are found within the lepidic growth, the size of the largest invasive area, measured in the larg-est dimension, is used; this area must be ≤5 mm to be con-sidered MIA. As with AIS, MIA is very rarely mucinous. The invasive component histologically is acinar, papillary, micropapillary, and/or solid and shows tumor cells infil-trating into the surrounding myofibroblastic stroma. On CT scan, the

1	mucinous. The invasive component histologically is acinar, papillary, micropapillary, and/or solid and shows tumor cells infil-trating into the surrounding myofibroblastic stroma. On CT scan, the appearance of MIA is often a part-solid nodule (≤5 mm) with a predominant ground-glass component, but can be highly variable. The 8th edition American Joint Committee on Cancer (AJCC) staging manual t-stage for MIA is T1mi.3. Lepidic predominant adenocarcinoma (LPA). If lympho-vascular invasion, pleural invasion, tumor necrosis, or more than 5 mm of invasion are noted in a lesion that has lepidic growth as its predominant component, MIA is excluded, the lesion is called lepidic predominant adenocarcinoma (LPA), and the size of the invasive component is recorded for the T stage.4. Invasive adenocarcinoma. The new classification system now recommends classifying invasive adenocarcinoma by the most predominant subtype after histologic evaluation of the resection specimen. To determine the

1	The new classification system now recommends classifying invasive adenocarcinoma by the most predominant subtype after histologic evaluation of the resection specimen. To determine the predominant subtype, histologic sections are evaluated, and the patterns are determined, in 5% increments, throughout the speci-men. This semiquantitative method encourages the viewer to identify and quantify all patterns present, rather than focus-ing on a single pattern. In the pathology report, the tumor is classified by the predominant pattern, with percentages of the subtypes also reported (Fig. 19-11).Subtypes include:a. Lepidic predominantb. Acinar predominantc. Papillary predominantd. Micropapillary predominante. Solid predominantAdenocarcinoma is often peripherally located and frequently discovered incidentally on routine chest radiographs, unlike squamous cell cancers. When symptoms occur, they are due to pleural or chest wall invasion (pleuritic or chest wall pain) or pleural seeding with

1	incidentally on routine chest radiographs, unlike squamous cell cancers. When symptoms occur, they are due to pleural or chest wall invasion (pleuritic or chest wall pain) or pleural seeding with malignant pleural effusion. Invasive adenocarcinoma is usually solid by CT scan, but can also be part-solid and even a ground-glass nodule. Occasionally, a lobar ground-glass opacification may be present, which is often associated with significant respiratory compromise and can be mistaken for lobar pneumonia. Bubble-like or cystic lucency on CT scan in small (≤2 cm) adenocarcinomas or extensive associated ground-glass components correlate with slow growth and well-differentiated tumors and a more favor-able prognosis. Intratumoral air bronchograms are usually indicative of well-differentiated tumor, whereas spiculations that are coarse and thick (≥2 mm) portend vascular invasion and nodal metastasis and are associated with decreased sur-vival following complete surgical resection. Pleural

1	tumor, whereas spiculations that are coarse and thick (≥2 mm) portend vascular invasion and nodal metastasis and are associated with decreased sur-vival following complete surgical resection. Pleural retraction is also a poor prognostic indicator.5. Additional histologic variants include colloid adenocarci-noma (formerly mucinous cystadenocarcinoma), fetal ade-nocarcinoma, and enteric adenocarcinoma. Clear cell and signet ring cell types are no longer considered to be distinct subtypes as they are found in association with most of the five dominant histologic patterns (lepidic, acinar, papillary, micropapillary, and solid). However, they are still notable, as they can signal clinically relevant molecular changes, such as the presence of the EML4-ALK fusion gene in solid tumors with signet ring features.Squamous Cell Carcinoma Representing 30% to 40% of lung cancers, squamous cell carcinoma is the most frequent cancer in men and highly correlated with cigarette smoking. They arise

1	ring features.Squamous Cell Carcinoma Representing 30% to 40% of lung cancers, squamous cell carcinoma is the most frequent cancer in men and highly correlated with cigarette smoking. They arise primarily in the main, lobar, or first segmental bronchi, which are collectively referred to as the central airways. Symptoms of airway irritation or obstruction are common, and include cough, Brunicardi_Ch19_p0661-p0750.indd 67101/03/19 7:00 PM 672SPECIFIC CONSIDERATIONSPART IIFigure 19-11. Major histologic patterns of invasive adenocarcinoma. A. Lepidic predominant pattern with mostly lepidic growth (right) and a smaller area of invasive acinar adenocarcinoma (left). B. Lepidic pattern consists of a proliferation type II pneumo-cytes and Clara cells along the surface alveolar walls. C. Area of invasive acinar adenocarcinoma (same tumor as in A and B). D. Acinar adenocarcinoma con-sists of round to oval-shaped malignant glands invad-ing a fibrous stroma. E. Papillary adenocarcinoma

1	Area of invasive acinar adenocarcinoma (same tumor as in A and B). D. Acinar adenocarcinoma con-sists of round to oval-shaped malignant glands invad-ing a fibrous stroma. E. Papillary adenocarcinoma consists of malignant cuboidal to columnar tumor cells growing on the surface of fibrovascular cores. F. Micropapillary adenocarcinoma consists of small papillary clusters of glandular cells growing within this airspace, most of which do not show fibrovascular cores. G. Solid adenocarcinoma with mucin consist-ing of sheets of tumor cells with abundant cytoplasm and mostly vesicular nuclei with several conspicuous nucleoli. No acinar, papillary, or lepidic patterns are seen, but multiple cells have intracytoplasmic baso-philic globules that suggest intracytoplasmic mucin. H. Solid adenocarcinoma with mucin. Numerous intracytoplasmic droplets of mucin are highlighted with this diastase-periodic acid Schiff stain. (Repro-duced with permission from Travis WD, Brambilla E, Noguchi M, et al:

1	with mucin. Numerous intracytoplasmic droplets of mucin are highlighted with this diastase-periodic acid Schiff stain. (Repro-duced with permission from Travis WD, Brambilla E, Noguchi M, et al: International association for the study of lung cancer/american thoracic society/european respiratory society international multidisci-plinary classification of lung adenocarcinoma, J Thorac Oncol. 2011 Feb;6(2):244-285.)hemoptysis, wheezing (due to high-grade airway obstruction), dyspnea (due to bronchial obstruction with or without postob-structive atelectasis), and pneumonia (caused by airway obstruc-tion with secretion retention and atelectasis).Occasionally a more peripherally based squamous cell carcinoma will develop in a tuberculosis scar or in the wall of a bronchiectatic cavity. Histologically, cells develop a pattern of clusters with intracellular bridges and keratin pearls. Central necrosis is frequent and may lead to the radiographic findings of a cavity (possibly with an

1	Histologically, cells develop a pattern of clusters with intracellular bridges and keratin pearls. Central necrosis is frequent and may lead to the radiographic findings of a cavity (possibly with an air-fluid level). Such cavities may become infected, with resultant abscess formation.Large Cell Carcinoma Large cell carcinoma accounts for 10% to 20% of lung cancers and may be located centrally or periph-erally. These tumors have cell diameters of 30 to 50 µm, which are often admixed with various other malignant cell types. Large cell carcinoma can be confused with a large cell variant of neu-roendocrine carcinoma, but can be differentiated by special immunohistochemical stains.Salivary Gland–Type Neoplasms. Salivary-type submucosal bronchial glands throughout the tracheobronchial tree can give rise to tumors that are histologically identical to those seen in the salivary glands. The two most common are adenoid cystic carcinoma and mucoepidermoid carcinoma. Both occur cen-trally due to

1	give rise to tumors that are histologically identical to those seen in the salivary glands. The two most common are adenoid cystic carcinoma and mucoepidermoid carcinoma. Both occur cen-trally due to their site of origin. Adenoid cystic carcinoma is a slow-growing tumor that is locally and systemically invasive, growing submucosally and infiltrating along perineural sheaths. Mucoepidermoid carcinoma consists of squamous and mucous cells and is graded as low or high grade, depending on mitotic rate and degree of necrosis.Neuroendocrine Neoplasms. Neuroendocrine lung tumors are classified into neuroendocrine hyperplasia and three grades of neuroendocrine carcinoma (NEC). Immunohistochemical staining for neuroendocrine markers (including chromogranins, synaptophysin, CD57, and neuron-specific enolase) is essential to accurate diagnosis.17Grade I NEC (classic or typical carcinoid) is a low-grade NEC; 80% arise in the central airway epithelium and occur primarily in younger patients.

1	enolase) is essential to accurate diagnosis.17Grade I NEC (classic or typical carcinoid) is a low-grade NEC; 80% arise in the central airway epithelium and occur primarily in younger patients. Because it is a central lesion, hemoptysis, with or without airway obstruction and pneumonia is the most common presentation. Histologically, tumor cells Brunicardi_Ch19_p0661-p0750.indd 67201/03/19 7:00 PM

1	CHAPTER 19673CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAare arranged in cords and clusters with a rich vascular stroma, which can lead to life-threatening hemorrhage with even simple bronchoscopic biopsy maneuvers. Regional lymph node metas-tases are seen in 15% of patients, but systemic spread and death from Grade I NEC is rare.Grade II NECs (atypical carcinoid) have a much higher malignant potential and, unlike grade I NEC, are etiologically linked to cigarette smoking and more likely to be peripherally located. Histologic findings may include areas of necrosis, nuclear pleomorphism, and higher mitotic rates. Lymph node metastases are found in 30% to 50% of patients. At diagnosis, 25% of patients already have remote metastases.Grade III NEC large cell–type tumors occur primarily in heavy smokers and in the mid to peripheral lung fields. Often large with central necrosis and a high mitotic rate, their neuro-endocrine nature is revealed by positive immunohistochemical staining for at

1	smokers and in the mid to peripheral lung fields. Often large with central necrosis and a high mitotic rate, their neuro-endocrine nature is revealed by positive immunohistochemical staining for at least one neuroendocrine marker.Grade IV NEC (small cell lung carcinoma [SCLC]) is the most malignant NEC and accounts for 25% of all lung cancers; these NECs often have early, widespread metastases. These cancers also arise primarily in the central airways. As with squamous cell cancers, symptoms include cough, hemoptysis, wheezing (due to high-grade airway obstruction), dyspnea (due to bronchial obstruction with or without postobstructive atel-ectasis), and pneumonia (caused by airway obstruction with secretion retention and atelectasis). Evaluation includes expert pathology review and comprehensive evaluation for metastatic disease. Three groups of grade IV NEC are recognized: pure small cell carcinoma (previously referred to as oat cell carci-noma), small cell carcinoma with a large

1	evaluation for metastatic disease. Three groups of grade IV NEC are recognized: pure small cell carcinoma (previously referred to as oat cell carci-noma), small cell carcinoma with a large cell component, and combined (mixed) tumors.Grade IV NECs consist of smaller cells (diameter 10 to 20 µm) with little cytoplasm and very dark nuclei; they can be difficult to distinguish from lymphoproliferative lesions and atypical carcinoid tumors. Histologically, a high mitotic rate with easily visualized multiple mitoses and areas of extensive necrosis are characteristic. Importantly, very small bronchoscopic biopsies can distinguish NSCLC from SCLC, but crush artifact may make NSCLC appear similar to SCLC. If uncertainty exists, special immunohistochemical stains or rebiopsy (or both) will be necessary. These tumors are the leading producer of paraneoplastic syndromes.Lung Cancer EpidemiologyLung cancer is the leading cancer killer and second most frequently diagnosed cancer in the United

1	necessary. These tumors are the leading producer of paraneoplastic syndromes.Lung Cancer EpidemiologyLung cancer is the leading cancer killer and second most frequently diagnosed cancer in the United States, account-ing for 26% of all cancer deaths in 2017—more than cancers of the breast, prostate, ovary, and colon and rectum com-bined (Fig. 19-12).18 Lung cancer incidence continues to decline, though at twice the rate for men compared to women (Fig. 19-13A, B). It is encouraging, however, that the average annual death rate declined by 3.5% per year for men and 2% per year for women from 2010 to 2014, representing a 43% decline in mortality for men and a 17% decline for women from 1990 MalesFemalesBreastLung & bronchusColon & rectumUterine corpusNon-Hodgkin lymphomaThyroidMelanoma of the skinPancreasLeukemiaKidney & renal pelvisAll Sites252,710105,51064,01061,38042,47034,94032,16025,84025,70023,380852,63030%12%8%7%5%4%4%3%3%3%100%MalesFemalesLung & bronchusProstateColon &

1	of the skinPancreasLeukemiaKidney & renal pelvisAll Sites252,710105,51064,01061,38042,47034,94032,16025,84025,70023,380852,63030%12%8%7%5%4%4%3%3%3%100%MalesFemalesLung & bronchusProstateColon & rectumPancreasLiver & intrahepatic bile ductLeukemiaEsophagusUrinary bladderNon-Hodgkin lymphomaBrain & other nervous systemAll SitesLung & bronchusBreastColon & rectumPancreasOvaryLeukemiaNon-Hodgkin lymphomaUterine corpusLiver & intrahepatic bile ductBrain & other nervous systemAll Sites71,28040,61023,11020,79014,08010,92010,2009,3108,6907,080282,50025%14%8%7%5%4%4%3%3%3%100%84,59027,15026,73022,30019,61014,30012,72012,24011,4509,620318,42027%9%8%7%6%4%4%4%4%3%100%ProstateLung & bronchusColon & rectumUrinary bladderMelanoma of the skinNon-Hodgkin lymphomaKidney & renal pelvisOral cavity & pharynxLeukemiaLiver & intrahepatic bile ductAll Sites161,360116,99071,42060,49052,17040,61040,08036,29035,72029,200836,15019%14%9%7%6%5%5%4%4%3%100%Estimated new casesEstimated deathsFigure 19-12. Leading

1	& intrahepatic bile ductAll Sites161,360116,99071,42060,49052,17040,61040,08036,29035,72029,200836,15019%14%9%7%6%5%5%4%4%3%100%Estimated new casesEstimated deathsFigure 19-12. Leading new cancer cases and deaths: 2017 estimates. *Excludes basal and squamous cell skin cancers and in situ carcinomas except urinary bladder. (Reproduced with permission from Siegel RL, Miller KD, Jemal A: Cancer Statistics, 2017, CA Cancer J Clin. 2017 Jan;67(1):7-30.)Brunicardi_Ch19_p0661-p0750.indd 67301/03/19 7:00 PM 674SPECIFIC CONSIDERATIONSPART IIFigure 19-13. Trends in death rates by sex for select cancers, United States, 1930 to 2014. A. Males. B. Female rates are age-adjusted to the 2000 U.S. standard population. Due to improvements in International Classification of Diseases (ICD) coding over time, numerator data for cancers of the lung and bronchus, colon and rectum, liver, and uterus differ from the contemporary time period. For example, rates for lung and bronchus include pleura, trachea,

1	numerator data for cancers of the lung and bronchus, colon and rectum, liver, and uterus differ from the contemporary time period. For example, rates for lung and bronchus include pleura, trachea, mediastinum, and other respiratory organs. (Adapted with permission from Siegel RL, Miller KD, Jemal A: Cancer Statistics, 2017, CA Cancer J Clin. 2017 Jan;67(1):7-30.)0102030405060708090100Pancreas†StomachLung and BronchusColon and RectumProstateLiver†LeukemiaTrends in Age-Adjusted Cancer Death Rates* by Site, Males, US, 1930-2014*Per 100,000, age adjusted to the 2000 US standard population.†Mortality rates for pancreatic and liver cancers are increasing.1930193419381942194619501954195819621966197019741978198219861990199419982002200620102014Trends in Age-Adjusted Cancer Death Rates* by Site, Females, US, 1930-2014*Per 100,000, age adjusted to the 2000 US standard population.†Uterus refers to uterine cervix and uterine corpus combined.‡Mortality rates for liver cancer are

1	Rates* by Site, Females, US, 1930-2014*Per 100,000, age adjusted to the 2000 US standard population.†Uterus refers to uterine cervix and uterine corpus combined.‡Mortality rates for liver cancer are increasing.010203040505152535451930193419381942194619501954195819621966197019741978198219861990199419982002200620102014StomachColon and RectumPancreas‡Lung and BronchusBreastUterus†Liver‡Brunicardi_Ch19_p0661-p0750.indd 67401/03/19 7:00 PM

1	CHAPTER 19675CHEST WALL, LUNG, MEDIASTINUM, AND PLEURATable 19-3Relative risk of lung cancer in smokersSMOKING CATEGORYRELATIVE RISKNever smoked1.0Currently smoke15.8–16.3Formerly smoked Years of abstinence 1–95.9–19.5 10–192.0–6.1 >201.9–3.7Data from Samet JM. Health benefits of smoking cessation, Clin Chest Med. 1991;12(4):669-679.to 2014.18 Unfortunately, most patients are still diagnosed at an advanced stage of disease (22% with regional metastasis and 57% with distant metastasis), so therapy is rarely curative.18Prognostic markers for lung cancer survival include female sex (5-year survival of 18.3% for women vs. 13.8% for men), younger age (5-year survival of 22.8% for those <45 years vs. 13.7% for those >65 years), and white race (5-year survival of 16.1% for whites vs. 12.2% for blacks). When access to advanced medical care is unrestricted, as for the military pop-ulation, the racial difference in survival disappears, suggesting that, at least in part, differences in

1	for blacks). When access to advanced medical care is unrestricted, as for the military pop-ulation, the racial difference in survival disappears, suggesting that, at least in part, differences in survival may be explained by less access to advanced medical care and later diagnosis.19Risk Factors for Lung Cancer. Cigarette smoking is the leading preventable cause of cancer death, accounting for 29% of the popu-lation attributable fraction in 2010, and is implicated as a causal factor in approximately 90% of lung cancers in men and nearly 80% in women. Two lung cancer types—squamous cell and small cell carcinoma—are extraordinarily rare in the absence of cigarette smoking. The risk of developing lung cancer escalates with the number of cigarettes smoked, the number of years of smoking, and the use of unfiltered cigarettes. Conversely, the risk of lung cancer declines with smoking cessation, but never drops to that of never smokers, regardless of the length of abstinence (Table 19-3).20

1	the use of unfiltered cigarettes. Conversely, the risk of lung cancer declines with smoking cessation, but never drops to that of never smokers, regardless of the length of abstinence (Table 19-3).20 Radon exposure accounts for the vast majority of the remaining cancers. Approximately 25% of all lung cancers worldwide and 53% of cancers in women are not related to smoking, and most of them (62%) are adenocarcinomas. Table 19-4 summarizes the existing data regarding the etiology of lung cancer in nonsmokers.21Nearly 3500 deaths from lung cancer each year are attrib-utable to secondhand (environmental) smoke exposure, which confers an excess risk for lung cancer of 24% when a non-smoker lives with a smoker.22 Risk is conferred by exposure to any burning tobacco, including cigars. The amount of second-hand exposure from one large cigar is equivalent to the exposure from 21 cigarettes. As with active smoking, risk of developing lung cancer increases with longer duration and higher level

1	of second-hand exposure from one large cigar is equivalent to the exposure from 21 cigarettes. As with active smoking, risk of developing lung cancer increases with longer duration and higher level of exposure to environmental tobacco.Over 7000 chemicals have been identified in tobacco smoke, and more than 70 of the compounds are known to be carcinogens. The main chemical carcinogens are polycyclic aromatic hydrocarbons, which are actively or passively inhaled in the tobacco smoke and absorbed; these compounds are acti-vated by specific enzymes and become mutagenic, bind to mac-romolecules such as deoxyribonucleic acid (DNA), and induce genetic mutations. In treating any patient with a previous smok-ing history, it is important to remember that there has been field cancerization of the entire aerodigestive tract. The patient’s risk is increased for cancers of the oral cavity, pharynx, larynx, tra-cheobronchial tree and lung, and esophagus. In examining such patients, a detailed

1	the entire aerodigestive tract. The patient’s risk is increased for cancers of the oral cavity, pharynx, larynx, tra-cheobronchial tree and lung, and esophagus. In examining such patients, a detailed history and physical examination of these organ systems must be performed.Other causes of lung cancer include exposure to a num-ber of industrial compounds, including asbestos, arsenic, and chromium compounds. In fact, the combination of asbestos and cigarette smoke exposure has a multiplicative effect on risk. Pre-existing lung disease confers an increased risk of lung cancer—up to 13%—for individuals who have never smoked. Patients with chronic obstructive pulmonary disease are at higher risk for lung cancer than would be predicted based on smoking risk alone. Patients with secondary scar formation related to a his-tory of tuberculosis or other lung infections also have a higher risk of primary lung carcinoma. This increase is thought to be related to poor clearance of inhaled

1	scar formation related to a his-tory of tuberculosis or other lung infections also have a higher risk of primary lung carcinoma. This increase is thought to be related to poor clearance of inhaled carcinogens and/or to the effects of chronic inflammation.Screening for Lung Cancer in High-Risk PopulationsIn 2002, the National Lung Screening Trial (NLST) was launched to determine whether screening with CT in high-risk populations would reduce mortality from lung cancer. The study randomized 53,353 eligible patients age 55 to 74 years to either three annual low-dose helical CT scans (LDCT; aka spiral CT) or posteroante-rior view chest radiograph. Patients were eligible for the trial if they had a greater than 30 pack-year history of cigarette smoking; had smoked within the past 15 years if a former smoker; had no prior history of lung cancer; had no history of other life-threatening cancers in the prior 5 years; did not have symptoms suggestive of an undiagnosed lung cancer (such as

1	if a former smoker; had no prior history of lung cancer; had no history of other life-threatening cancers in the prior 5 years; did not have symptoms suggestive of an undiagnosed lung cancer (such as hemoptysis or weight loss); and had not had a chest CT scan in the prior 18 months. Accrual to the study was excellent, and the primary endpoint of a 20% rela-tive reduction in mortality was achieved in 2010. An absolute risk reduction of lung cancer death of four per 1000 individuals screened by LDCT was realized. Interestingly, all-cause mortality was also reduced by nearly 7% in the LDCT group, further empha-sizing the impact of lung cancer on the mortality of smokers and former smokers.23 The U.S. Preventive Services Task Force (USPSTF) now recommends annual screening for lung cancer with low-dose computed tomography screening in high risk patients. Annual screening averted 14% of lung cancer deaths when applied to a population of asymptomatic adults age 55 to 80 years who have a 30

1	low-dose computed tomography screening in high risk patients. Annual screening averted 14% of lung cancer deaths when applied to a population of asymptomatic adults age 55 to 80 years who have a 30 pack-year smoking history and are either currently smoking or have quit within the past 15 years. Patients should be healthy enough to tolerate curative treatment, specifically surgery per guidelines, and screening should be dis-continued once the patient has not smoked for 15 years or develops a life-limiting health condition, becomes unable to tolerate lung surgery or is unwilling to undergo curative lung resection. Require-ments for coverage differ between Medicare and private insurers, with private insurers following the USPSTF guidelines, while Medicare uses age 55 to 77 years with the same smoking history but does not define comorbid conditions. Use of standardized reporting with criteria for lung nodule identification and classifica-tion is required by the Center for Medicaid &

1	same smoking history but does not define comorbid conditions. Use of standardized reporting with criteria for lung nodule identification and classifica-tion is required by the Center for Medicaid & Medicare Services (CMS) but is only recommended by private insurers. Shared deci-sion-making is also required by Medicare, but it is only recom-mended by private insurers. Medicare also differs from private 4Brunicardi_Ch19_p0661-p0750.indd 67501/03/19 7:00 PM 676SPECIFIC CONSIDERATIONSPART IITable 19-4Summary of selected studies of risk factors for lung cancer in individuals who never smokedRISK FACTORRISK ESTIMATE (95% CI)COMMENTSREFERENCEEnvironmental tobacco smoke1.19 (90% CI: 1.04–1.35)Meta-analysis of 11 U.S. studies of spousal exposure (females only)2251.21 (1.13–1.30)Meta-analysis of 44 case-control studies worldwide of spousal exposure2261.22 (1.13–1.33)Meta-analysis of 25 studies worldwide of workplace exposure2261.24 (1.18–1.29)Meta-analysis of 22 studies worldwide of

1	of 44 case-control studies worldwide of spousal exposure2261.22 (1.13–1.33)Meta-analysis of 25 studies worldwide of workplace exposure2261.24 (1.18–1.29)Meta-analysis of 22 studies worldwide of workplace exposure227Residential radon8.4% (3.0%–15.8%) per 100 Bq m3 increase in measured radonMeta-analysis of 13 European studies22811% (0%–28%) per 100 Bq m3Meta-analysis of 7 North American studies229Cooking oil vapors2.12 (1.81–2.47)Meta-analysis of 7 studies from China and Taiwan (females who never smoked)230Indoor coal and wood burning2.66 (1.39–5.07)Meta-analysis of 7 studies from China and Taiwan (both sexes)2301.22 (1.04–1.44)Large case-control study (2861 cases and 3118 controls) from Eastern and Central Europe (both sexes)2312.5 (1.5–3.6)Large case-control study (1205 cases and 1541 controls) from Canada (significant for women only)232Genetic factors: family history, CYP1A1 Ile462Val polymorphism, XRCC1 variants1.51 (1.11–2.06)Meta-analysis of 28 case-control, 17 cohort, and 7 twin

1	from Canada (significant for women only)232Genetic factors: family history, CYP1A1 Ile462Val polymorphism, XRCC1 variants1.51 (1.11–2.06)Meta-analysis of 28 case-control, 17 cohort, and 7 twin studies2332.99 (1.51–5.91)Meta-analysis of 14 case-control studies of Caucasian never smokers2342.04 (1.17–3.54)Meta-analysis of 21 case-control studies of Caucasian and Asian never smokers (significant for Caucasians only)235No associationMeta-analysis of 13 case-control studies236No association overall; reduced risk 0.65 (0.46–0.83) with Arg194Trp polymorphism and 0.56 (0.36–0.86) with Arg280His for heavy smokersLarge case-control study from Europe (2188 cases and 2198 controls)237Increased risk for never smokers 1.3 (1.0–1.8) and decreased risk for heavy smokers 0.5 (0.3–1.0) with Arg299GlnLarge case-control study from the United States (1091 cases and 1240 controls)238Viral factors: HPV 16 and 1810.12 (3.88–26.4) for never smoking women >60 yCase-control study (141 cases, 60 controls) from

1	study from the United States (1091 cases and 1240 controls)238Viral factors: HPV 16 and 1810.12 (3.88–26.4) for never smoking women >60 yCase-control study (141 cases, 60 controls) from Taiwan of never smoking women239Abbreviations: Bq = becquerels; CI = confidence interval; CYP1A1 = cytochrome P450 enzyme 1A1; HPV = human papilloma virus.Reproduced with permission from Sun S, Schiller JH, Gazdar AF: Lung cancer in never smokers—a different disease, Nat Rev Cancer. 2007 Oct; 7(10):778-790.insurers in that it requires accreditation of the imaging center and submission of all low-dose CT data to a CMS-approved national registry, among other specifications. With this approach, it is expected that 50% of diagnosed cancers will be early stage. Screening of patients age 50 years or older with a 20 pack-year or greater history and additional risk factors (as determined by the Tammemagi lung cancer risk calculator or other validated risk scores) that increase the risk of lung cancer to 1.3%

1	a 20 pack-year or greater history and additional risk factors (as determined by the Tammemagi lung cancer risk calculator or other validated risk scores) that increase the risk of lung cancer to 1.3% or greater should also be considered as part of lung cancer screening pro-grams. In all cases, patient–physician shared decision-making should be undertaken, with a discussion of the risks and benefits of screening. It is important to note that there was a 7% false-positive rate in NLST trial, which can lead to patient anxiety, invasive testing, and potentially morbid procedures to further evaluate the finding. The impact of these issues on patient quality of life and cost-effectiveness requires further study, particularly as screening programs are implemented outside of the controlled set-ting of a clinical trial. It is also critical that regulatory guidelines for patient eligibility, frequency of screening, interpretation of the scans, processes for further evaluation and management of

1	of a clinical trial. It is also critical that regulatory guidelines for patient eligibility, frequency of screening, interpretation of the scans, processes for further evaluation and management of posi-tive findings, and dose of radiation are well established and well accepted to ensure the generalizability of the results for patients who will be screened in the general medical community rather than in the specialized centers that performed the trial.Brunicardi_Ch19_p0661-p0750.indd 67601/03/19 7:00 PM

1	CHAPTER 19677CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAABCDFigure 19-14. Spiral computed tomography scan showing normal transverse chest anatomy at four levels. A. At the level of the tracheal bifurcation, the aorticopulmonary window can be seen. B. The origin of the left pulmonary artery can be seen at a level 1 cm inferior to A. C. The origin and course of the right pulmonary artery can be seen at this next most cephalad level. The left upper lobe bronchus can be seen at its origin from the left main bronchus. D. Cardiac chambers and pulmonary veins are seen in the lower thorax. AA = ascending aorta; APW = aorticopulmonary window; DA = descending aorta; LA = left ventricle; LMB = left main bronchus; LPA = left pulmonary artery; MPA = main pulmonary artery; RA = right atrium; RPA = right pulmonary artery; RV = right ventricle; SVC = superior vena cava; T = trachea.Solitary Pulmonary NoduleA solitary pulmonary nodule is typically described as a single, well-circumscribed, spherical

1	pulmonary artery; RV = right ventricle; SVC = superior vena cava; T = trachea.Solitary Pulmonary NoduleA solitary pulmonary nodule is typically described as a single, well-circumscribed, spherical lesion that is 3 cm or less cm in diameter and completely surrounded by normal aerated lung parenchyma.24 Lung atelectasis, hilar enlargement, and pleural effusion are absent. The majority are detected incidentally on chest radiographs (CXRs) or CT scans obtained for some other purpose. About 150,000 solitary nodules are found inciden-tally each year, with increasing numbers as low-dose computed tomography screening in high-risk populations is adopted. The clinical significance of such a lesion depends on whether or not it represents a malignancy.The differential diagnosis of a solitary pulmonary nodule should include a broad variety of congenital, neoplastic, inflam-matory, vascular, and traumatic disorders. The probability of cancer in a solitary pulmonary nodule increases if the patient

1	nodule should include a broad variety of congenital, neoplastic, inflam-matory, vascular, and traumatic disorders. The probability of cancer in a solitary pulmonary nodule increases if the patient has a history of smoking (50% or higher for smokers compared to 20% to 40% in never smokers). It is also more likely to be malignant if it is symptomatic or the patient is older, male, or has had occupational exposures.Solitary pulmonary nodules were defined by findings on CXR, but with the increased sensitivity of low-dose screening CT, up to 50% of solitary lesions are found to be associated with multiple (one to six) other, usually subcentimeter, nodules. In the Early Lung Cancer Action project, almost 7% of healthy volunteers were found to have between one and three nodules, and 25% had up to six nodules. CT scanning is necessary to characterize nodule number, location, size, margin morphol-ogy, calcification pattern, and growth rate.25 Spiral (helical) CT allows continuous scanning as

1	to six nodules. CT scanning is necessary to characterize nodule number, location, size, margin morphol-ogy, calcification pattern, and growth rate.25 Spiral (helical) CT allows continuous scanning as the patient is moved through a scanning gantry, allowing the entire thorax to be imaged during a single breath hold (Fig. 19-14). Compared to conventional CT, this provides a superior image quality, because motion artifacts are eliminated, and improves detection of pulmonary nodules and central airway abnormalities.26 The shorter acquisition time of spiral CT also allows for consistent contrast filling of the great vessels, resulting in markedly improved visualization of pathologic states and anatomic variation contiguous to vascular structures. In addition, three-dimensional spiral CT images can be reconstructed for enhanced visualization of spatial anatomic relationships.27 Thin sections (1 to 2 mm collimation) at 1-cm intervals should be used to evaluate pulmonary parenchyma and

1	images can be reconstructed for enhanced visualization of spatial anatomic relationships.27 Thin sections (1 to 2 mm collimation) at 1-cm intervals should be used to evaluate pulmonary parenchyma and peripheral bronchi. If the goal is to find any pulmonary metas-tases, thin sections at intervals of 5 to 7 mm collimation are recommended. For assessing the trachea and central bronchi, collimation of 3 to 5 mm is recommended. Providing accurate clinical history and data is of paramount importance to obtaining appropriate imaging.CT findings characteristic of benign lesions include small size, calcification within the nodule, and stability over time. Brunicardi_Ch19_p0661-p0750.indd 67701/03/19 7:00 PM 678SPECIFIC CONSIDERATIONSPART IIABCFigure 19-15. Computed tomography scan images of solitary pulmonary nodules. A. The corona radiata sign demonstrated by a solitary nodule. Multiple fine striations extend perpendicularly from the surface of the nodule like the spokes of a wheel. B. A

1	pulmonary nodules. A. The corona radiata sign demonstrated by a solitary nodule. Multiple fine striations extend perpendicularly from the surface of the nodule like the spokes of a wheel. B. A biopsy-proven adenocarcinoma demonstrating spiculation. C. A lesion with a scalloped border, an indeterminate finding suggesting an intermediate probability for malignancy.Table 19-5Actuarial survival data from the International Registry of Lung MetastasesSURVIVALCOMPLETE RESECTION (%)INCOMPLETE RESECTION (%)5 years361310 years26715 years22—Four patterns of benign calcification are common: diffuse, solid, central, and laminated or “popcorn.” Granulomatous infections such as tuberculosis can demonstrate the first three patterns, whereas the popcorn pattern is most common in hamartomas. In areas of endemic granulomatous disease, differentiating benign versus malignant can be challenging. Infectious granulomas arising from a variety of organisms account for 70% to 80% of this type of benign

1	of endemic granulomatous disease, differentiating benign versus malignant can be challenging. Infectious granulomas arising from a variety of organisms account for 70% to 80% of this type of benign solitary nodules; hamartomas are the next most common single cause, accounting for about 10%.CT findings characteristic of malignancy include growth over time; increasing density on CT scan (40% to 50% of partial solid lesions are malignant compared to only 15% of subcenti-meter solid or nonsolid nodules); size >3 cm; irregular, lobu-lated, or spiculated edges; and the finding of the corona radiata sign (consisting of fine linear strands extending 4 to 5 mm out-ward and appearing spiculated on radiographs) (Fig. 19-15). Calcification that is stippled, amorphous, or eccentric is usually associated with cancer.Growth over time is an important characteristic for differentiating benign and malignant lesions. Lung cancers have volume-doubling times from 20 to 400 days; lesions with shorter

1	with cancer.Growth over time is an important characteristic for differentiating benign and malignant lesions. Lung cancers have volume-doubling times from 20 to 400 days; lesions with shorter doubling times are likely due to infection, and longer doubling times suggest benign tumors, but can represent slower-growing lung cancer. Positron emission tomography (PET) scan-ning can differentiate benign from malignant nodules28; most lung tumors have increased signatures of glucose uptake, as compared with healthy tissues, and thus glucose metabolism can be measured using radio-labeled 18F-fluorodeoxyglucose (FDG). Meta-analysis estimates 97% sensitivity and 78% spec-ificity for predicting malignancy in a nodule. False-negative results can occur (especially in patients who have AIS, MIA, or LPA, carcinoids, and tumors <1 cm in diameter), as well as false-positive results (because of confusion with other infectious or inflammatory processes).Metastatic Lesions to the LungThe cause of a new

1	carcinoids, and tumors <1 cm in diameter), as well as false-positive results (because of confusion with other infectious or inflammatory processes).Metastatic Lesions to the LungThe cause of a new pulmonary nodule(s) in a patient with a previous malignancy can be difficult to discern.29 Features sug-gestive of metastatic disease are multiplicity; smooth, round borders on CT scan; and temporal proximity to the original pri-mary lesion. One must always entertain the possibility that a single new lesion is a primary lung cancer. The probability of a new primary cancer vs. metastasis in patients presenting with solitary lesions depends on the type of initial neoplasm. The highest likelihood of a new primary lung cancer is in patients with a history of uterine (74%), bladder (89%), lung (92%), and head and neck (94%) carcinomas.Surgical resection of pulmonary metastases has a role in properly selected patients.30 The best data regarding outcomes of resection of pulmonary metastases come

1	and head and neck (94%) carcinomas.Surgical resection of pulmonary metastases has a role in properly selected patients.30 The best data regarding outcomes of resection of pulmonary metastases come from the Interna-tional Registry of Lung Metastases (IRLM). The registry was established in 1991 by 18 thoracic surgery departments in Europe, the United States, and Canada and included data on 5206 patients. About 88% of patients underwent complete resection. Survival analysis at 5, 10, and 15 years (grouping all primary tumor types) was performed (Table 19-5). Multivariate analysis showed a better prognosis for patients with germ cell tumors, osteosarcomas, a disease-free interval over 36 months, and a single metastasis.31 Depicted in Fig. 19-16, survival after metastasectomy in a variety of cancers is optimal when meta-static disease is resectable, solitary, and identified 36 or more months after initial treatment. When any or all of these optimal characteristics are absent, survival

1	of cancers is optimal when meta-static disease is resectable, solitary, and identified 36 or more months after initial treatment. When any or all of these optimal characteristics are absent, survival progressively declines.Brunicardi_Ch19_p0661-p0750.indd 67801/03/19 7:00 PM

1	CHAPTER 19679CHEST WALL, LUNG, MEDIASTINUM, AND PLEURA10080604020002448All Sites7296120Soft Tissue Sarcomas100806040200024487296120Breast Cancer100806040200024487296120Osteosarcoma1001234806040200024487296120Colon Cancer100806040200024487296120Melanoma100806040200024487296120ABCDEFFigure 19-16. The actuarial survival after metastasectomy is depicted for patients with various tumor types (A-F) further categorized into four groups according to resectabil-ity, solitary or multiple, the inter-val between primary resection and metastesectomy, and a combination of factors known in our work and in others, as follows: (1) resectable, soli-tary, and disease-free interval (DFI) greater than or equal to 36 months; (2) resectable, solitary, and DFI 36+ months; (3) resectable, multiple metastases, and DFI <36 months; and (4) unresectable. (Reproduced with permission from Pastorino U: The development of an international registry, J Thorac Oncol. 2010 Jun; 5(6 Suppl 2):S196-S197.)The general

1	and DFI <36 months; and (4) unresectable. (Reproduced with permission from Pastorino U: The development of an international registry, J Thorac Oncol. 2010 Jun; 5(6 Suppl 2):S196-S197.)The general principles of patient selection for metasta-sectomy are listed in Table 19-6. The technical aim of pulmo-nary metastasectomy is complete resection of all macroscopic tumors. In addition, any involved adjacent structures should be resected en bloc (i.e., chest wall, diaphragm, and pericardium). Multiple lesions and/or hilar lesions may require lobectomy. Pneumonectomy is rarely justified or employed.Pulmonary metastasectomy can be approached through a thoracotomy or via video-assisted thoracic surgery (VATS) techniques. McCormack and colleagues reported their expe-rience at Memorial Sloan-Kettering in a prospective study of 18 patients who presented with no more than two pulmonary metastatic lesions and underwent VATS resection.32 A thora-cotomy was performed during the same operation; if

1	in a prospective study of 18 patients who presented with no more than two pulmonary metastatic lesions and underwent VATS resection.32 A thora-cotomy was performed during the same operation; if palpation Brunicardi_Ch19_p0661-p0750.indd 67901/03/19 7:00 PM 680SPECIFIC CONSIDERATIONSPART IITable 19-7Clinical presentation of lung cancerCATEGORYSYMPTOMCAUSEPulmonary symptomsCoughBronchus irritation or compressionDyspneaAirway obstruction or compressionWheezing>50% airway obstructionHemoptysisTumor erosion or irritationPneumoniaAirway obstructionNonpulmonary thoracic symptoms Pleuritic painParietal pleural irritation or invasionLocal chest wall painRib and/or muscle involvementRadicular chest painIntercostal nerve involvementPancoast’s syndromeStellate ganglion, chest wall, brachial plexus involvementHoarsenessRecurrent laryngeal nerve involvementSwelling of head and armsBulky involved mediastinal lymph nodes Medially based right upper lobe tumorTable 19-6General principles governing

1	involvementHoarsenessRecurrent laryngeal nerve involvementSwelling of head and armsBulky involved mediastinal lymph nodes Medially based right upper lobe tumorTable 19-6General principles governing appropriate selection of patients for pulmonary metastasectomy1. Primary tumor must already be controlled.2. Patient must be able to tolerate general anesthesia, potential single-lung ventilation, and the planned pulmonary resection.3. Metastases must be completely resectable based on computed tomographic imaging.4. There is no evidence of extrapulmonary tumor burden.5. Alternative superior therapy must not be available.identified any additional lesions, they were resected. The study concluded that the probability that a metastatic lesion will be missed by VATS excision is 56%. Patients in the Memorial study were evaluated before the advent of spiral CT scanning, however, and it remains controversial whether metastasis resec-tion should be performed via VATS. Proponents of VATS argue that

1	Memorial study were evaluated before the advent of spiral CT scanning, however, and it remains controversial whether metastasis resec-tion should be performed via VATS. Proponents of VATS argue that the resolution of spiral CT scanning is so superior that prior studies using standard CT scanners are no longer relevant. Indeed, a recent study suggested that only 18% of malignant nodules would be missed using a VATS approach in the current era while another study from the United Kingdom found equiv-alent outcomes with regard to missed lesions and pulmonary progression comparing open and VATS approaches. To date, no prospective study using spiral CT scan has been performed to resolve this clinical dilemma.Primary Lung Cancer-Associated Signs and SymptomsLung cancer displays one of the most diverse presentation pat-terns of all human maladies (Table 19-7). The wide range of symptoms and signs is related to (a) histologic features, which often help determine the anatomic site of origin in

1	presentation pat-terns of all human maladies (Table 19-7). The wide range of symptoms and signs is related to (a) histologic features, which often help determine the anatomic site of origin in the lung; (b) the specific tumor location in the lung and its relationship to surrounding structures; (c) biologic features and the pro-duction of a variety of paraneoplastic syndromes; and (d) the presence or absence of metastatic disease. Symptoms related to the local intrathoracic effect of the primary tumor can be conveniently divided into two groups: pulmonary and nonpul-monary thoracic.Pulmonary Symptoms. Pulmonary symptoms result from the direct effect of the tumor on the bronchus or lung tissue. Symptoms (in order of frequency) include cough (secondary to irritation or compression of a bronchus), dyspnea (usually due to central airway obstruction or compression, with or without atelectasis), wheezing (with narrowing of a central airway of >50%), hemoptysis (typically, blood streaking of

1	dyspnea (usually due to central airway obstruction or compression, with or without atelectasis), wheezing (with narrowing of a central airway of >50%), hemoptysis (typically, blood streaking of mucus that is rarely massive; indicates a central airway location), pneu-monia (usually due to airway obstruction by the tumor), and lung abscess (due to necrosis and cavitation, with subsequent infection).Nonpulmonary Thoracic Symptoms. Nonpulmonary tho-racic symptoms result from invasion of the primary tumor directly into a contiguous structure (e.g., chest wall, diaphragm, pericardium, phrenic nerve, recurrent laryngeal nerve, superior vena cava, and esophagus), or from mechanical compression of a structure (e.g., esophagus or superior vena cava) by enlarged tumor-bearing lymph nodes.Peripherally located tumors (often adenocarcinomas) extending through the visceral pleura lead to irritation or growth into the parietal pleura and potentially to continued growth into the chest wall structures.

1	located tumors (often adenocarcinomas) extending through the visceral pleura lead to irritation or growth into the parietal pleura and potentially to continued growth into the chest wall structures. Three types of symptoms, depend-ing on the extent of chest wall involvement, are possible: (a) pleuritic pain, from noninvasive contact of the parietal pleura with inflammatory irritation or direct parietal pleural invasion; (b) localized chest wall pain, from deeper invasion and involvement of the rib and/or intercostal muscles; and (c) radicular pain, from involvement of the intercostal nerve(s). Radicular pain may be mistaken for renal colic in the case of tumors invading the inferoposterior chest wall.Other specific nonpulmonary thoracic symptoms include:1. Pancoast’s syndrome. Tumors originating in the supe-rior sulcus (posterior apex) elicit: apical chest wall and/or shoulder pain (from involvement of the first rib and chest wall); Horner’s syndrome (unilateral enophthal-mos,

1	originating in the supe-rior sulcus (posterior apex) elicit: apical chest wall and/or shoulder pain (from involvement of the first rib and chest wall); Horner’s syndrome (unilateral enophthal-mos, ptosis, miosis, and facial anhidrosis from invasion of the stellate sympathetic ganglion); and radicular arm pain (from invasion of T1, and occasionally C8, brachial plexus nerve roots).2. Phrenic nerve palsy. The phrenic nerve traverses the hemi-thorax along the mediastinum, parallel and posterior to the superior vena cava and anterior to the pulmonary hilum. Tumors at the medial lung surface or anterior hilum can directly invade the nerve; symptoms include shoulder pain (referred), hiccups, and dyspnea with exertion because of Brunicardi_Ch19_p0661-p0750.indd 68001/03/19 7:00 PM

1	CHAPTER 19681CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAdiaphragm paralysis. Radiographically, unilateral diaphragm elevation on chest radiograph is present; the diagnosis is confirmed by fluoroscopic examination of the diaphragm with paradoxical motion with breathing and sniffing (the “sniff” test).3. Recurrent laryngeal nerve palsy. Recurrent laryngeal nerve (RLN) involvement most commonly occurs on the left side, given the hilar location of the left RLN as it passes under the aortic arch. Paralysis results from: (a) invasion of the vagus nerve above the aortic arch by a medially based left upper lobe tumor; or (b) direct invasion of the RLN by hilar tumor and/or hilar or aortopulmonary lymph node metastases. Symptoms include voice change, often referred to as hoarseness, but more typically a loss of tone associ-ated with a breathy quality, and coughing, particularly when drinking liquids.4. Superior vena cava (SVC) syndrome. As a result of bulky enlargement of involved mediastinal

1	a loss of tone associ-ated with a breathy quality, and coughing, particularly when drinking liquids.4. Superior vena cava (SVC) syndrome. As a result of bulky enlargement of involved mediastinal lymph nodes com-pressing or a medially based right upper lobe tumor invad-ing the SVC, SVC syndrome symptoms include variable degrees of swelling of the head, neck, and arms; headache; and conjunctival edema. It is seen most commonly with NEC grade IV (small cell) lung cancer.5. Pericardial tamponade. Pericardial effusions (benign or malignant), associated with increasing levels of dyspnea and/or arrhythmias, and pericardial tamponade occur with direct pericardial invasion. Diagnosis requires a high index of suspicion in the setting of a medially based tumor with symptoms of dyspnea and is confirmed by CT scan or echocardiography.6. Back pain. Results from direct invasion of a vertebral body and is often localized and severe. If the neural foramina are involved, radicular pain may also be

1	by CT scan or echocardiography.6. Back pain. Results from direct invasion of a vertebral body and is often localized and severe. If the neural foramina are involved, radicular pain may also be present.7. Other local symptoms. Dysphagia is usually secondary to external esophageal compression by enlarged lymph nodes involved with metastatic disease, usually with lower lobe tumors. Finally, dyspnea, pleural effusion, or referred shoulder pain can result from invasion of the diaphragm by a tumor at the base of a lower lobe.Associated Paraneoplastic Syndromes. All lung cancer his-tologies are capable of producing a variety of paraneoplastic syndromes, most often from systemic release of tumor-derived biologically active materials (Table 19-8). Paraneoplastic syn-dromes may produce symptoms even before any local symp-toms are produced by the primary tumor, thereby aiding in early diagnosis. Their presence does not influence resectabil-ity or treatment options. Symptoms often abate with

1	even before any local symp-toms are produced by the primary tumor, thereby aiding in early diagnosis. Their presence does not influence resectabil-ity or treatment options. Symptoms often abate with success-ful treatment; paraneoplastic symptom recurrence may herald tumor recurrence. The majority of such syndromes are associ-ated with grade IV NEC (small cell carcinoma), including many endocrinopathies.1. Hypertrophic pulmonary osteoarthropathy (HPO). Often severely debilitating, symptoms of HPO may antedate the diagnosis of cancer by months. Clinically, ankle, feet, fore-arm, and hand tenderness and swelling are characteristic, resulting from periostitis of the fibula, tibia, radius, meta-carpals, and metatarsals. Clubbing of the digits may occur in up to 30% of patients with grade IV NEC (Fig. 19-17). Plain radiographs show periosteal inflammation and elevation, while bone scans demonstrate intense but Table 19-8Paraneoplastic syndromes in patients with lung

1	with grade IV NEC (Fig. 19-17). Plain radiographs show periosteal inflammation and elevation, while bone scans demonstrate intense but Table 19-8Paraneoplastic syndromes in patients with lung cancerEndocrineHypercalcemia (ectopic parathyroid hormone)Cushing’s syndromeSyndrome of inappropriate secretion of antidiuretic hormoneCarcinoid syndromeGynecomastiaHypercalcitoninemiaElevated growth hormone levelElevated levels of prolactin, follicle-stimulating hormone, luteinizing hormoneHypoglycemiaHyperthyroidismNeurologicEncephalopathySubacute cerebellar degenerationProgressive multifocal leukoencephalopathyPeripheral neuropathyPolymyositisAutonomic neuropathyEaton-Lambert syndromeOptic neuritisSkeletalClubbingPulmonary hypertrophic osteoarthropathyHematologicAnemiaLeukemoid reactionsThrombocytosisThrombocytopeniaEosinophiliaPure red cell aplasiaLeukoerythroblastosisDisseminated intravascular coagulationCutaneousHyperkeratosisDermatomyositisAcanthosis nigricansHyperpigmentationErythema

1	red cell aplasiaLeukoerythroblastosisDisseminated intravascular coagulationCutaneousHyperkeratosisDermatomyositisAcanthosis nigricansHyperpigmentationErythema gyratum repensHypertrichosis lanuginosa acquistaOtherNephrotic syndromeHypouricemiaSecretion of vasoactive intestinal peptide with diarrheaHyperamylasemiaAnorexia or cachexiasymmetric uptake in the long bones. Aspirin or nonsteroidal anti-inflammatory agents provide temporary relief; treat-ment requires successful tumor eradication.2. Hypercalcemia. Up to 10% of patients with lung cancer will have hypercalcemia, most often due to metastatic disease. Ectopic parathyroid hormone secretion by the tumor, most often squamous cell carcinoma, is causative in up to 15%, however, and should be suspected if metastatic bone disease is not present. Symptoms of hypercalcemia include leth-argy, depressed level of consciousness, nausea, vomiting, Brunicardi_Ch19_p0661-p0750.indd 68101/03/19 7:00 PM 682SPECIFIC

1	bone disease is not present. Symptoms of hypercalcemia include leth-argy, depressed level of consciousness, nausea, vomiting, Brunicardi_Ch19_p0661-p0750.indd 68101/03/19 7:00 PM 682SPECIFIC CONSIDERATIONSPART IIABCFigure 19-17. Hypertrophic pulmonary osteoarthropathy associated with small cell carcinoma. A. Painful clubbing of the fingers. B. Painful clubbing of the toes (close-up). C. The arrows point to new bone formation on the femur.and dehydration. Following complete tumor eradication, the calcium level will normalize. Unfortunately, tumor recur-rence is extremely common and may manifest as recurrent hypercalcemia.3. Hyponatremia. Characterized by confusion, lethargy, and possible seizures, hyponatremia can result from the inappro-priate secretion of antidiuretic hormone from the tumor into the systemic circulation (syndrome of inappropriate secretion of antidiuretic hormone [SIADH]) in 10% to 45% of patients with grade IV NEC (small cell). It is diagnosed by the pres-ence

1	tumor into the systemic circulation (syndrome of inappropriate secretion of antidiuretic hormone [SIADH]) in 10% to 45% of patients with grade IV NEC (small cell). It is diagnosed by the pres-ence of hyponatremia, low serum osmolality, and high urinary sodium and osmolality. Another cause of hyponatremia can be the ectopic secretion of atrial natriuretic peptide (ANP).4. Cushing’s syndrome. Autonomous tumor production of an adrenocorticotropic hormone (ACTH)-like molecule leads to rapid serum elevation of ACTH and subsequent severe hypokalemia, metabolic alkalosis, and hyperglycemia. Symptoms are primarily related to the metabolic changes while the physical signs of Cushing’s syndrome (e.g., trun-cal obesity, buffalo hump, striae) are unusual due to the rapidity of ACTH elevation. Diagnosis is made by dem-onstrating hypokalemia (<3.0 mmol/L); nonsuppressible elevated plasma cortisol levels that lack the normal diurnal variation; elevated blood ACTH levels; or elevated urinary

1	Diagnosis is made by dem-onstrating hypokalemia (<3.0 mmol/L); nonsuppressible elevated plasma cortisol levels that lack the normal diurnal variation; elevated blood ACTH levels; or elevated urinary 17-hydroxycorticosteroids, all of which are not suppressible by administration of exogenous dexamethasone. Immuno-reactive ACTH is present in nearly all extracts of SCLC, and a high percentage of patients with SCLC have elevated ACTH levels by radioimmunoassay, yet fewer than 5% have symptoms of Cushing’s syndrome.5. Peripheral and central neuropathies. Unlike other para-neoplastic syndromes, which are usually due to ectopic secretion of an active substance, these syndromes are felt to be immune mediated. Cancer cells are thought to secrete antigens normally expressed only by the nervous system, generating antibodies leading either to interference with neurologic function or to immune neurologic destruction. Up to 16% of lung cancer patients have neuromuscular dis-ability, and, of these,

1	generating antibodies leading either to interference with neurologic function or to immune neurologic destruction. Up to 16% of lung cancer patients have neuromuscular dis-ability, and, of these, half have grade IV NEC (small cell) and 25% have squamous cell carcinomas. In patients with neurologic or muscular symptoms, central nervous system (CNS) metastases must be ruled out with CT or magnetic resonance imaging (MRI) of the head. Other metastatic dis-ease leading to disability must also be excluded.6. Lambert-Eaton syndrome. This myasthenia-like syndrome is caused by tumor secretion of immunoglobulin G (IgG) antibodies targeting voltage-gated calcium channels, which causes a neuromuscular conduction defect by decreasing the amount of acetylcholine released from presynaptic sites at the motor end plate. Symptoms, including gait abnormali-ties from proximal muscle weakness and impaired coordi-nation, may actually precede radiographic evidence of the tumor. Therapy is directed at the

1	motor end plate. Symptoms, including gait abnormali-ties from proximal muscle weakness and impaired coordi-nation, may actually precede radiographic evidence of the tumor. Therapy is directed at the primary tumor with resec-tion, radiation, and/or chemotherapy. Many patients have dramatic improvement after successful therapy. For patients with refractory symptoms, treatment consists of guanidine Brunicardi_Ch19_p0661-p0750.indd 68201/03/19 7:00 PM

1	CHAPTER 19683CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAhydrochloride, immunosuppressive agents such as predni-sone and azathioprine, and occasionally plasma exchange. Unlike with myasthenia gravis patients, neostigmine is usu-ally ineffective.Symptoms Associated with Metastatic Lung Cancer. Lung cancer metastasizes most commonly to the CNS, vertebral bod-ies, bone, liver, adrenal glands, lungs, skin, and soft tissues. CNS metastases are present at diagnosis in 10% of patients; another 10% to 15% will develop CNS metastases following diagnosis. Focal symptoms, including headache, nausea, vom-iting, seizures, hemiplegia, and dysarthria, are common. Lung cancer is the most common cause of spinal cord compression, either by primary tumor invasion of an intervertebral foramen or direct extension of vertebral metastases. Bony metastases are identified in 25% of lung cancer patients. They are primar-ily lytic and produce pain locally; thus, any new and localized skeletal symptoms must be

1	of vertebral metastases. Bony metastases are identified in 25% of lung cancer patients. They are primar-ily lytic and produce pain locally; thus, any new and localized skeletal symptoms must be evaluated radiographically. Liver metastases and adrenal metastases are typically asymptomatic and usually discovered by routine CT scan. Adrenal metastasis may lead to adrenal hypofunction. Skin and soft tissue metas-tases occur in 8% of patients dying of lung cancer and gener-ally present as painless subcutaneous or intramuscular masses. Occasionally, tumor erodes through overlying skin; excision may then be necessary for both mental and physical palliation.Nonspecific Cancer-Related Symptoms. Lung cancer often produces a variety of nonspecific symptoms such as anorexia, weight loss, fatigue, and malaise and their presence raises con-cern for metastatic disease.Lung Cancer ManagementRole of Histologic Diagnosis and Molecular Testing. Establishing a clear histologic diagnosis early in the

1	and malaise and their presence raises con-cern for metastatic disease.Lung Cancer ManagementRole of Histologic Diagnosis and Molecular Testing. Establishing a clear histologic diagnosis early in the evaluation and management of lung cancer is critical to effective treat-ment. Molecular signatures are also key determinants of treat-ment algorithms for adenocarcinoma and will likely become important for squamous cell carcinoma as well. Currently, differentiation between adenocarcinoma and squamous cell carcinoma in cytologic specimens or small biopsy specimens is imperative in patients with advanced stage disease, as treat-ment with pemetrexed or bevacizumab-based chemotherapy is associated with improved progression-free survival in patients with adenocarcinoma but not squamous cell cancer. Further-more, life-threatening hemorrhage has occurred in patients with squamous cell carcinoma who were treated with bevacizumab. Finally, EGFR mutation predicts response to EGFR tumor kinase

1	Further-more, life-threatening hemorrhage has occurred in patients with squamous cell carcinoma who were treated with bevacizumab. Finally, EGFR mutation predicts response to EGFR tumor kinase inhibitors and is now recommended as first-line therapy in advanced adenocarcinoma. Because adequate tissue is required for histologic assessment and molecular testing, each institution should have a clear, multidisciplinary approach to patient evalu-ation, tissue acquisition, tissue handling/processing, and tissue analysis (Fig. 19-18). In many cases, tumor morphology differ-entiates adenocarcinoma from the other histologic subtypes. If no clear morphology can be identified, then additional testing for one immunohistochemistry marker for adenocarcinoma and one for squamous cell carcinoma will usually enable differentiation. Immunohistochemistry for neuroendocrine markers is reserved for lesions exhibiting neuroendocrine morphology. Additional molecular testing should be performed on all

1	usually enable differentiation. Immunohistochemistry for neuroendocrine markers is reserved for lesions exhibiting neuroendocrine morphology. Additional molecular testing should be performed on all adenocarcinoma specimens for known predictive and prognostic tumor mark-ers (e.g., EGFR, KRAS, and EML4-ALK fusion gene). Ideally, use of tissue sections and cell block material is limited to the minimum necessary at each decision point. This emphasizes the importance of a multidisciplinary approach; surgeons and radiologists must work in direct cooperation with the cytopa-thologist to ensure that tissue samples are adequate for morpho-logic diagnosis as well as providing sufficient cellular material to enable molecular testing. With adoption of endobronchial and endoscopic ultrasound, electromagnetic navigational bronchos-copy, VATS, and even transthoracic image-guided fine-needle and core-needle biopsy, surgeons are increasingly involved in the acquisition of diagnostic tissue for

1	navigational bronchos-copy, VATS, and even transthoracic image-guided fine-needle and core-needle biopsy, surgeons are increasingly involved in the acquisition of diagnostic tissue for primary, metastatic, and recurrent intrathoracic disease, and a thorough understanding of key issues is necessary to ensure optimal treatment and patient outcomes.Patient Evaluation. Pretreatment evaluation encompasses three areas: diagnosis and assessment of the primary tumor, assessment for metastatic disease, and determination of func-tional status (the patient’s ability to tolerate the prescribed treatment regimen). A discrete approach to each area allows the surgeon to systematically evaluate the patient, perform accurate clinical staging, and determine the patient’s functional suitabil-ity for therapy (Table 19-9).Assessment of the Primary Tumor Primary tumor assessment begins with directed history questions regarding the presence or absence of pulmonary, nonpulmonary, thoracic, and

1	for therapy (Table 19-9).Assessment of the Primary Tumor Primary tumor assessment begins with directed history questions regarding the presence or absence of pulmonary, nonpulmonary, thoracic, and paraneo-plastic symptoms. Because patients often present to the surgeon with a CXR or CT scan demonstrating the lesion, tumor location can guide the history and physical examination.A routine chest CT scan should be performed; this should include intravenous contrast to enable assessment of the pri-mary tumor, delineation of mediastinal lymph nodes relative to normal mediastinal structures, and the tumor’s relationship to surrounding and contiguous structures. Recommendations for treatment and options for obtaining tissue diagnosis require a thorough understanding and assessment of CT findings.Concern for contiguous invasion of adjacent structures is often raised in response to a combination of symptom history, location of the primary tumor, and CT imaging. It is common to see the primary

1	for contiguous invasion of adjacent structures is often raised in response to a combination of symptom history, location of the primary tumor, and CT imaging. It is common to see the primary tumor abutting the chest wall without clear radiographic evidence of rib destruction. In this circumstance, localized pain can indicate parietal pleural, rib, or intercostal nerve involvement. Similar observations apply to tumors abut-ting the recurrent laryngeal nerve, phrenic nerve, diaphragm, vertebral bodies, and chest apex. Thoracotomy should not be denied because of presumptive evidence of invasion of the chest wall, vertebral body, or mediastinal structures; proof of invasion may require thoracoscopy or even thoracotomy.MRI of pulmonary lesions and mediastinal nodes, over-all, offers no real advantages over CT scanning. It is an excel-lent modality, however, for defining a tumor’s relationship to a major vessel, given its excellent imaging of vascular structures. This is especially true if

1	over CT scanning. It is an excel-lent modality, however, for defining a tumor’s relationship to a major vessel, given its excellent imaging of vascular structures. This is especially true if the use of iodine contrast material is contraindicated. Thus, use of MRI in lung cancer patients is reserved for those with contrast allergies or suspected mediasti-nal, vascular, or vertebral body invasion.Options for Tissue Acquisition The surgeon must have an evidence-based algorithm for diagnosis and treatment of a pul-monary nodule and masses (Fig. 19-19).24 Depending on nodule size, bronchial tree proximity, and the population prevalence of lung cancer, bronchoscopy has a 20% to 80% sensitivity for detecting neoplastic processes within a pulmonary lesion. Brunicardi_Ch19_p0661-p0750.indd 68301/03/19 7:00 PM 684SPECIFIC CONSIDERATIONSPART IISTEP 1STEP 2POSITIVE BIOPSY (FOB,TBBx, Core, SLBx)POSITIVE CYTOLOGY(effusion, aspirate, washings,brushings)Histology: Lepidic, papillary, and/oracinar

1	7:00 PM 684SPECIFIC CONSIDERATIONSPART IISTEP 1STEP 2POSITIVE BIOPSY (FOB,TBBx, Core, SLBx)POSITIVE CYTOLOGY(effusion, aspirate, washings,brushings)Histology: Lepidic, papillary, and/oracinar architecture(s)Cytology: 3-D arrangements, delicatefoamy/vacuolated (translucent)cytoplasm,Fine nuclear chromatin and oftenprominent nucleoliNuclei are often eccentrically situatedClassic morphology:ADCADC markerand/orMucin +ve;SQCCmarker –ve(or weak insame cells)NSCLC, favor ADCNE morphology, large cells,NE IHC+No clear ADC orSQCC morphology:NSCLC-NOSNSCLC, favor SQCCSQCC marker +veADC marker –ve/orMucin –veApply ancillary panel ofOne SQCC and one ADC marker+/OR MucinIHC –ve andMucin –veNSCLC NOSADC marker or Mucin +ve;as well as SQCC marker +vein different cellsMolecular analysis:e.g., EGFR mutation†NSCLC, NOS,possibleadenosquamous caIf tumor tissue inadequate for molecular testing,discuss need for further sampling — back to Step 1NE morphology, small cells, nonucleoli, NE IHC+, TTF-1

1	mutation†NSCLC, NOS,possibleadenosquamous caIf tumor tissue inadequate for molecular testing,discuss need for further sampling — back to Step 1NE morphology, small cells, nonucleoli, NE IHC+, TTF-1 +/–,CK+Keratinization, pearlsand/or intercellular bridgesNSCLC,LCNECClassic Morphology:SQCCSCLCSTEP 3Figure 19-18. Algorithm for adenocarcinoma diagnosis in small biopsies and/or cytology. Step 1: When positive biopsies (fiberoptic bronchos-copy [FOB], transbronchial [TBBx], core, or surgical lung biopsy [SLBx]) or cytology (effusion, aspirate, washings, and brushings) show clear adenocarcinoma (ADC) or squamous cell carcinoma (SQCC) morphology, the diagnosis can be firmly established. If there is neuroendocrine (NE) morphology, the tumor may be classified as small cell carcinoma (SCLC) or non–small cell lung carcinoma (NSCLC), probably large cell neuroendocrine carcinoma (LCNEC) according to standard criteria (+ = positive, – = negative, and ± = positive or negative). If there is no clear

1	cell lung carcinoma (NSCLC), probably large cell neuroendocrine carcinoma (LCNEC) according to standard criteria (+ = positive, – = negative, and ± = positive or negative). If there is no clear ADC or SQCC morphology, the tumor is regarded as NSCLC—not otherwise specified (NOS). Step 2: NSCLC-NOS can be further classified based on (a) immunohistochemical stains, (b) mucin (DPAS or mucicarmine) stains, or (c) molecular data. If the stains all favor ADC-positive ADC marker(s) (i.e., TTF-1 and/or mucin positive) with negative SQCC markers, then the tumor is classified as NSCLC, favor ADC. If SQCC markers (i.e., p63 and/or CK5/6) are positive with negative ADC markers, the tumor is classified as NSCLC, favor SQCC. If the ADC and SQCC markers are both strongly positive in different populations of tumor cells, the tumor is classified as NSCLC-NOS, with a comment it may represent adenosquamous carcinoma. If all markers are negative, the tumor is classified as NSCLC-NOS. †EGFR mutation

1	of tumor cells, the tumor is classified as NSCLC-NOS, with a comment it may represent adenosquamous carcinoma. If all markers are negative, the tumor is classified as NSCLC-NOS. †EGFR mutation testing should be performed in (1) classic ADC, (2) NSCLC, favor ADC, (3) NSCLC-NOS, and (4) NSCLC-NOS, possible adenosquamous carcinoma. In NSCLC-NOS, if EGFR mutation is positive, the tumor is more likely to be ADC than SQCC. Step 3: If clinical management requires a more specific diagnosis than NSCLC-NOS, additional biopsies may be indicated. CD = cluster designation; CK = cytokeratin; DPAS = diastase-periodic acid Schiff; DPAS +ve = periodic-acid Schiff with diastase; EGFR = epidermal growth factor receptor; IHC = immunohistochemistry; NB = of note; TTF-1 = thyroid transcription factor-1; –ve = negative; +ve = positive. (Reproduced with permission from Travis WD, Brambilla E, Noguchi M, et al: Diagnosis of lung cancer in small biopsies and cytology: implications of the 2011 International

1	= negative; +ve = positive. (Reproduced with permission from Travis WD, Brambilla E, Noguchi M, et al: Diagnosis of lung cancer in small biopsies and cytology: implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification, Arch Pathol Lab Med. 2013 May;137(5):668-684.)Brunicardi_Ch19_p0661-p0750.indd 68401/03/19 7:00 PM

1	CHAPTER 19685CHEST WALL, LUNG, MEDIASTINUM, AND PLEURATable 19-9Evaluation of patients with lung cancer PRIMARY TUMORMETASTATIC DISEASEFUNCTIONAL ASSESSMENTHistoryPulmonaryWeight lossAbility to walk up two flights of stairsNonpulmonary thoracicMalaiseAbility to walk on a flat surface indefinitelyParaneoplastic New bone painNeurologic signs or symptomsSkin lesionsPhysical examination VoiceSupraclavicular node palpationAccessory muscle usage Skin examinationAir flow by auscultation Neurologic examinationForce of coughRadiographic examinationChest CTChest CT, PETChest CT: tumor anatomy, atelectasisTissue analysisBronchoscopyBone scan, head MRI, abdominal CTQuantitative perfusion scanTransthoracic needle aspiration and biopsy Bronchoscopic lymph node FNAEndoscopic ultrasoundMediastinoscopyBiopsy of suspected metastasisOtherThoracoscopy—Pulmonary function tests (FEV1, Dlco, O2 consumption)Abbreviations: CT = computed tomography; Dlco = carbon monoxide diffusion capacity; FEV1 = forced

1	of suspected metastasisOtherThoracoscopy—Pulmonary function tests (FEV1, Dlco, O2 consumption)Abbreviations: CT = computed tomography; Dlco = carbon monoxide diffusion capacity; FEV1 = forced expiratory volume in 1 second; FNA = fine-needle aspiration; MRI = magnetic resonance imaging; O2 = oxygen; PET = positron emission tomography.Diagnostic tissue from bronchoscopy can be obtained by one of four methods:1. Brushings and washings for cytology2. Direct forceps biopsy of a visualized lesion3. Endobronchial ultrasound-guided fine-needle aspiration (FNA) of an externally compressing lesion without visual-ized endobronchial tumor4. Transbronchial biopsy with fluoroscopy to guide forceps to the lesion or electromagnetic navigational bronchoscopyElectromagnetic navigation bronchoscopy is a recent addi-tion to the surgeon’s armamentarium for transbronchial biopsy of peripheral lung lesions. Using electromagnetic markers that create a three-dimensional image and align the recorded CT images

1	addi-tion to the surgeon’s armamentarium for transbronchial biopsy of peripheral lung lesions. Using electromagnetic markers that create a three-dimensional image and align the recorded CT images to the patient’s true anatomy, a transbronchial catheter is advanced, and brushings, FNA, cup biopsy, and washings can be performed. Diagnostic yield using electromagnetic naviga-tion bronchoscopy as an adjunct to standard bronchoscopy is reported as high as 80%. The approach can also be used for placement of fiducial markers for subsequent stereotactic body radiation therapy and for tattooing the perilesional region to guide subsequent video-assisted thoracoscopic resection.Pneumothorax rates are approximately 1% to 3.5%.For peripheral lesions (roughly the outer half of the lung), transbronchial biopsy is performed first, followed by brushings and washings. This improves diagnostic yield by disrupting the lesion with the biopsy forceps and mobilizing additional cells. For central lesions,

1	biopsy is performed first, followed by brushings and washings. This improves diagnostic yield by disrupting the lesion with the biopsy forceps and mobilizing additional cells. For central lesions, direct forceps biopsy is often possible. For central lesions with external airway compression but no visible endobronchial lesions, endobronchial ultrasound (EBUS) is highly accurate and safe for transbronchial biopsies of both the primary tumor (when it abuts the central airways) as well as the mediastinal lymph nodes.33Image-guided transthoracic FNA (ultrasound or CT FNA) biopsy can accurately diagnose appropriately selected peripheral pulmonary lesions in up to 95% of patients. Three biopsy results are possible after image-guided biopsy procedures: malignant, a specific benign process, or indeterminate. Because falsenegative rates range from 3% to 29%, further diagnostic efforts are warranted in the absence of a specific benign diagnosis (such as granulomatous inflammation or hamartoma)

1	Because falsenegative rates range from 3% to 29%, further diagnostic efforts are warranted in the absence of a specific benign diagnosis (such as granulomatous inflammation or hamartoma) because malig-nancy is not ruled out.34 The primary complication is pneumo-thorax in as many as 30% of cases. Intrapulmonary bleeding occurs, but it rarely causes clinically significant hemoptysis or respiratory compromise.Some groups advocate use of video-assisted thoracoscopic biopsy as the first option for diagnosis, citing superior diagnostic accuracy and low surgical risk. With VATS, the nodule can be excised with a wedge or segmental resection, if less than 3 cm, or a core-needle biopsy can be performed under direct vision for larger lesions. VATS can also provide valuable staging informa-tion, including sampling/dissection of mediastinal lymph nodes and assessing whether the primary tumor has invaded a contigu-ous structure (such as the chest wall or mediastinum).Lesions most suitable for VATS

1	sampling/dissection of mediastinal lymph nodes and assessing whether the primary tumor has invaded a contigu-ous structure (such as the chest wall or mediastinum).Lesions most suitable for VATS are those that are located in the outer one-third of the lung. The surgeon should avoid direct manipulation of the nodule or violation of the visceral pleura overlying the nodule. In addition, the excised nodule must be extracted from the chest within a bag to prevent seeding of the chest wall. If the patient’s pulmonary reserve is adequate, the surgeon can proceed to lobectomy (either VATS or open) after frozen section diagnosis.Brunicardi_Ch19_p0661-p0750.indd 68501/03/19 7:00 PM 686SPECIFIC CONSIDERATIONSPART IIA thoracotomy is occasionally necessary to diagnose and stage a primary tumor. Although this occurs rarely, two circum-stances may require such an approach: (a) a deep-seated lesion that yielded an indeterminate needle biopsy result or that could not be biopsied for technical

1	Although this occurs rarely, two circum-stances may require such an approach: (a) a deep-seated lesion that yielded an indeterminate needle biopsy result or that could not be biopsied for technical reasons; or (b) inability to determine inva-sion of a mediastinal structure by any method short of palpation. In the circumstance of a deep-seated lesion without a diagnosis, tissue can be obtained via thoracotomy using FNA, core-needle biopsy, or excisional biopsy. Intraoperative frozen-section analysis is required; if the open biopsy frozen-section result is indetermi-nate, a lobectomy may be necessary in extremely rare situations. If a pneumonectomy is required to remove the lesion, a tissue diag-nosis of cancer must be made before proceeding.Assessment for Metastatic Disease Approximately 40% of patients with newly diagnosed lung cancer present with distant metastasis. The presence of lymph node or systemic metastases may imply inoperability. As with the primary tumor, assess-ment for

1	40% of patients with newly diagnosed lung cancer present with distant metastasis. The presence of lymph node or systemic metastases may imply inoperability. As with the primary tumor, assess-ment for the presence of metastatic disease should begin with the history and physical examination, focusing on new bone pain, neurologic symptoms, and new skin lesions. In addition, constitutional symptoms (e.g., anorexia, malaise, and unin-tentional weight loss of >5% of body weight) suggest either a large tumor burden or the presence of metastases. Physical examination focuses on overall appearance, noting any evi-dence of weight loss such as redundant skin or muscle wasting, and a complete examination of the head and neck, including NegativetestsPositivetestsNoNoNew SPN (8 mm to 30 mm)identified on CXR orCT scanBenign calcificationpresent or 2-year stabilitydemonstrated?Surgical risk acceptable?Assess clinicalprobability of cancer Low probabilityof cancer(<5%)Intermediateprobability of

1	on CXR orCT scanBenign calcificationpresent or 2-year stabilitydemonstrated?Surgical risk acceptable?Assess clinicalprobability of cancer Low probabilityof cancer(<5%)Intermediateprobability of cancer(>5%–60%)High probabilityof cancer(>60%)Establish diagnosis bybiopsy when possible.Consider XRT or monitorfor symptoms andpalliate as necessarySerial high-resolutionCT at 3, 6, 12 and24 monthsAdditional testing• PET imaging, if available• Contrast-enhanced CT, depending on institutional expertise• Transthoracic fine-needle aspiration biopsy, if nodule is peripherally located• Bronchoscopy, if airbronchogram present or if operator has expertise with newer guided techniques Video-assistedthoracoscopic surgery:examination of a frozensection, followed byresection if nodule ismalignantYesYesNo further interventionrequired except forpatients with pure groundglass opacities, in whomlonger annual follow-upshould be consideredFigure 19-19. Recommended management algorithm for patients with

1	further interventionrequired except forpatients with pure groundglass opacities, in whomlonger annual follow-upshould be consideredFigure 19-19. Recommended management algorithm for patients with solitary pulmonary nodules (SPNs) measuring 8 mm to 30 mm in diameter. CT = computed tomography; CXR = chest radiograph; PET = positron emission tomography; XRT = radiotherapy. (Adapted with permission from Gould MK, Fletcher J, Iannettoni MD, et al: Evaluation of patients with pulmonary nodules: when is it lung cancer?: ACCP evidence-based clinical practice guidelines (2nd edition), Chest. 2007 Sep;132(3 Suppl):108S-130S.24)Brunicardi_Ch19_p0661-p0750.indd 68601/03/19 7:01 PM

1	CHAPTER 19687CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAevaluation of cervical and supraclavicular lymph nodes and the oropharynx. This is particularly true for patients with a sig-nificant tobacco history. The skin should be thoroughly exam-ined. Routine laboratory studies include serum levels of hepatic enzymes (e.g., serum glutamic oxaloacetic transaminase and alkaline phosphatase), and serum calcium (to detect bone metas-tases or the ectopic parathyroid syndrome). Elevation of either hepatic enzymes or serum calcium levels typically occurs with extensive metastases.Mediastinal Lymph Nodes. Chest CT scanning facilitates assessment of mediastinal and hilar nodes for enlargement. How-ever, a positive CT result (i.e., nodal diameter >1.0 cm) predicts actual metastatic involvement in only about 70% of lung cancer patients. Thus, up to 30% of such nodes are enlarged from non-cancerous reactive causes (e.g., inflammation due to atelectasis or pneumonia secondary to the tumor). Patients

1	about 70% of lung cancer patients. Thus, up to 30% of such nodes are enlarged from non-cancerous reactive causes (e.g., inflammation due to atelectasis or pneumonia secondary to the tumor). Patients should not be denied an attempt at curative resection just because of a posi-tive CT result for mediastinal lymph node enlargement; any CT finding of metastatic nodal involvement must be confirmed his-tologically. The negative predictive value of normal-appearing lymph nodes by CT (lymph nodes <1.0 cm) is better than the positive predictive value of a suspicious-appearing lymph node, particularly with small squamous cell tumors. With normal-size lymph nodes and a T1 tumor, the false-negative rate is less than 10%, leading many surgeons to omit mediastinoscopy. How-ever, the false-negative rate increases to nearly 30% with cen-trally located and T3 tumors. It has also been demonstrated that T1 adenocarcinomas or large cell carcinomas have a higher rate of early micrometastasis. Therefore,

1	increases to nearly 30% with cen-trally located and T3 tumors. It has also been demonstrated that T1 adenocarcinomas or large cell carcinomas have a higher rate of early micrometastasis. Therefore, all such patients should undergo mediastinoscopy.Mediastinal lymph node staging by PET scanning appears to have greater accuracy than CT scanning. PET staging of mediastinal lymph nodes has been evaluated in two meta-analyses. The overall sensitivity for mediastinal lymph node metastasis was 79% (95% confidence interval [CI] 76%–82%), with a specificity of 91% (95% CI 89%–93%) and an accuracy of 92% (95% CI 90%–94%).35In comparing PET with CT scans in patients who also underwent lymph node biopsies, PET had a sensitivity of 88% and a specificity of 91%, whereas CT scanning had a sensitivity of 63% and a specificity of 76%. Combining CT and PET scan-ning may lead to even greater accuracy.36 In one study of CT, PET, and mediastinoscopy in 68 patients with potentially opera-ble NSCLC, CT

1	of 63% and a specificity of 76%. Combining CT and PET scan-ning may lead to even greater accuracy.36 In one study of CT, PET, and mediastinoscopy in 68 patients with potentially opera-ble NSCLC, CT correctly identified the nodal stage in 40 patients (59%). It understaged the tumor in 12 patients and overstaged it in 16 patients. PET correctly identified the nodal stage in 59 patients (87%). It understaged the tumor in five patients and overstaged it in four. For detecting N2 and N3 disease, the combination of PET and CT scanning yielded a sensitivity, specificity, and accuracy of 93%, 95%, and 94%, respectively. CT scan alone yielded 75%, 63%, and 68%, respectively. Studies examining combined PET-CT consistently show improved accuracy compared to PET or CT alone; accuracy for PET-CT nodal positivity confirmed by medi-astinoscopy is approximately 75%, with a negative predictive value of approximately 90%. Right upper lobe lesions were more likely to have occult N2 disease than other

1	positivity confirmed by medi-astinoscopy is approximately 75%, with a negative predictive value of approximately 90%. Right upper lobe lesions were more likely to have occult N2 disease than other lobes of the lung.37-40 PET-positive mediastinal lymph nodes require histologic verifica-tion of node positivity, either by EBUS-guided FNA or mediasti-noscopy, to minimize the risk of undertreatment, assuming node positivity without histologic confirmation relegates the patient to, at a minimum, induction chemotherapy. If there is a suggestion of TABLE 19-10Techniques for invasive mediastinal stagingEndoscopicEndobronchial ultrasound with transbronchial needle aspirationEndoscopic ultrasound with needle aspirationTransbronchial needle aspirationComputed tomography–guided transthoracic needle aspirationSurgicalVideo-assisted mediastinoscopyTranscervical extended mediastinal lymphadenectomy (TEMLA)Video-assisted mediastinal lymphadenectomy (VAMLA)Thoracoscopic transthoracic

1	needle aspirationSurgicalVideo-assisted mediastinoscopyTranscervical extended mediastinal lymphadenectomy (TEMLA)Video-assisted mediastinal lymphadenectomy (VAMLA)Thoracoscopic transthoracic lymphadenectomyIndications for invasive mediastinal staging in lung cancer1. Radiographically enlarged mediastinal lymph nodes2. Centrally located tumors3. N1 nodal enlargement4. Tumor size >3 cm5. Peripheral clinical stage I tumor with nonenlarged but FDG-avid mediastinal lymph nodesIndications for prethoracotomy/thoracoscopy biopsy of stations 5 and 6 lymph nodes1. Criteria for invasive staging met and other mediastinal lymph node stations are negative (assuming patient would have induction therapy if any nodal station positive)2. Enrollment criteria for induction therapy protocol require pathologic confirmation of N2 disease3. Computed tomography scan shows evidence of bulky nodal metastasis or extracapsular spread that could prevent complete resection4. Tissue diagnosis of a hilar mass or of

1	confirmation of N2 disease3. Computed tomography scan shows evidence of bulky nodal metastasis or extracapsular spread that could prevent complete resection4. Tissue diagnosis of a hilar mass or of lymph nodes causing recurrent laryngeal nerve paralysis is neededN3 disease, the patient would be incorrectly staged as having IIIB disease and would not be considered a candidate for potentially curative surgical resection.It is important for surgeons who are managing patients with lung cancer to have a clear algorithm for invasive medias-tinal staging. In general, invasive staging is underutilized, plac-ing many patients at risk for overor understaging and, thus, inappropriate treatment. An absolute indication for obtaining a tissue diagnosis is mediastinal lymph node enlargement greater than 1.0 cm by CT scan. There are several options for invasive mediastinal staging (Table 19-10):1. Endobronchial Ultrasound (EBUS)-guided transbron-chial needle aspiration. Less invasive than

1	than 1.0 cm by CT scan. There are several options for invasive mediastinal staging (Table 19-10):1. Endobronchial Ultrasound (EBUS)-guided transbron-chial needle aspiration. Less invasive than mediasti-noscopy, EBUS enables image-guided transtracheal and transbronchial FNA cytologic samples from hilar masses and lymph nodes from level 4R and 4L, level 7, level 10, and level 11. Rapid onsite pathologic evaluation with expert cytopathology evaluation greatly increases the diagnostic accuracy of the procedure; importantly, the intraoperative evaluation will confirm whether the target lesion is being sampled and greatly facilitates acquisition of satisfactory samples for determining the morphologic diagnosis as well as sufficient material for cell block for immunohistochemistry Brunicardi_Ch19_p0661-p0750.indd 68701/03/19 7:01 PM 688SPECIFIC CONSIDERATIONSPART IIFigure 19-20. Cervical mediastinoscopy. Paratracheal and sub-carinal lymph node tissues (within the pretracheal space) can

1	68701/03/19 7:01 PM 688SPECIFIC CONSIDERATIONSPART IIFigure 19-20. Cervical mediastinoscopy. Paratracheal and sub-carinal lymph node tissues (within the pretracheal space) can be sampled using a mediastinoscope introduced through a suprasternal skin incision.and molecular testing. EBUS does not allow assessment of level 3, 5, or 6 nodal stations.2. Endoscopic ultrasound (EUS). EUS can accurately visual-ize mediastinal paratracheal lymph nodes (stations 4R, 7, and 4L), paraesophageal (station 8) and inferior pulmonary ligament (station 9) lymph nodes and visualize primary lung lesions contiguous with or near the esophagus (see Fig. 19-8). Using FNA or core-needle biopsy, samples of lymph nodes or primary lesions can be obtained. Diagnos-tic yield is improved with intraoperative cytologic evalua-tion, which can be performed with the cytopathologist in the operating room. Limitations of EUS include the inability to visualize the anterior (pretracheal) mediastinum; thus, EUS does not

1	evalua-tion, which can be performed with the cytopathologist in the operating room. Limitations of EUS include the inability to visualize the anterior (pretracheal) mediastinum; thus, EUS does not replace mediastinoscopy/EBUS for complete medi-astinal nodal staging. However, it may not be necessary to perform mediastinoscopy if findings on EUS are positive for N2 nodal disease, particularly if more than one station is found to harbor metastases.3. Cervical video-assisted mediastinoscopy. Mediastinos-copy provides tissue sampling of all paratracheal and sub-carinal lymph nodes and permits visual determination of the presence of extracapsular extension of nodal metastasis (Fig. 19-20). With complex hilar or right paratracheal primary tumors, it allows direct biopsies and assessment of invasion into the mediastinum. Mediastinoscopy is recommended for centrally located tumors, T2 and T3 primary tumors, and occasionally for T1 adenocarcinomas or large cell carcinomas (due to their higher

1	into the mediastinum. Mediastinoscopy is recommended for centrally located tumors, T2 and T3 primary tumors, and occasionally for T1 adenocarcinomas or large cell carcinomas (due to their higher rate of metastatic spread). Some surgeons perform mediastinoscopy in all lung cancer patients because of the poor survival associated with surgi-cal resection of N2 disease.4. It is important to note that EBUS or EUS can be used for initial diagnosis in enlarged lymph nodes, but the predictive value of a negative EBUS in a patient with radiographically suspicious mediastinal disease is not sufficient to accurately guide treatment. At the authors’ institutions, it is standard to begin mediastinal lymph node staging with EBUS-guided FNA of clinically suspicious mediastinal lymphadenopathy. If intraoperative rapid onsite cytologic evaluation is nega-tive, mediastinoscopy is performed in the same operative setting to ensure accurate mediastinal staging. However, if the FNA is positive,

1	If intraoperative rapid onsite cytologic evaluation is nega-tive, mediastinoscopy is performed in the same operative setting to ensure accurate mediastinal staging. However, if the FNA is positive, mediastinoscopy is not performed, and the patient is referred to medical oncology for induc-tion therapy; avoiding a pretreatment mediastinoscopy in this manner facilitates the safe performance of a postinduc-tion mediastinoscopy for restaging of the mediastinum in patients who respond favorably to induction therapy.5. Left video-assisted thoracoscopic lymph node sampling may be needed for patients with left upper lobe tumors who have localized regional spread to stations 5 and 6 lymph nodes, without mediastinal paratracheal involvement (see Fig. 19-8). If there is a low index of suspicion for nodal metastasis, the patient can be schedule for VATS biopsy and lobectomy under the same anesthesia; the procedure begins by sampling the level 5 and 6 nodes for frozen section, and if the nodes are

1	metastasis, the patient can be schedule for VATS biopsy and lobectomy under the same anesthesia; the procedure begins by sampling the level 5 and 6 nodes for frozen section, and if the nodes are negative, the anatomic lung resection is performed. If the index of suspicion is high, the VATS biopsy is performed as a separate procedure. Cervical mediastinoscopy should precede VATS biopsy, even if patients have normal para-tracheal lymph nodes. Additional diagnostic evaluation of the lymph nodes in stations 5 and 6 may be unnecessary if the mediastinal lymph nodes are proven to be benign with biopsy during cervical mediastinoscopy and the preoperative CT scan suggests complete respectability of the tumor. There are, however, several indications for prethoracotomy biopsy of stations 5 and 6 lymph nodes, which are listed in Table 19-10. It is particularly important to prove that mediastinal lymph nodes are pathologically involved and not just radio-graphically suspicious for nodal

1	and 6 lymph nodes, which are listed in Table 19-10. It is particularly important to prove that mediastinal lymph nodes are pathologically involved and not just radio-graphically suspicious for nodal metastasis prior to deciding that the patient is not a candidate for resection.Pleural Effusion. The presence of pleural effusion on radio-graphic imaging should not be assumed to be malignant. Pleural effusion may be secondary to atelectasis or consolidation (seen with central tumors), cardiac dysfunction, or may be a reac-tive effusion. When associated with a peripherally based tumor abutting the visceral or parietal pleural surface, probability of being malignant is higher. If this is the only site concerning for metastatic disease, pathologic confirmation is mandatory. It is reasonable to start with thoracentesis, but cytology reveals malignant cells in only 50% of malignant effusions on initial thoracentesis; negative cytology 5 times is needed to have 95% certainty of a benign

1	to start with thoracentesis, but cytology reveals malignant cells in only 50% of malignant effusions on initial thoracentesis; negative cytology 5 times is needed to have 95% certainty of a benign process. Thoracoscopy may be needed to rule out pleural metastases in select patients and is usually per-formed as a separate staging procedure, often with subsequent mediastinoscopy if thoracoscopy is negative for metastasis.Distant Metastases. Currently, chest CT and PET are rou-tine in the evaluation of patients with lung cancer. Integrated PET-CT scanners have become standard and have substan-tially improved accuracy of detection and localization of lymph node and distant metastases, as compared with independently performed PET and CT scans (Fig. 19-21). This technology overcomes the imprecise information on the exact location of focal abnormalities seen on PET and has become the standard imaging modality for lung cancer. Compared to routine chest or abdominal CT and bone scans, PET

1	information on the exact location of focal abnormalities seen on PET and has become the standard imaging modality for lung cancer. Compared to routine chest or abdominal CT and bone scans, PET scanning detects 10% to 15% more distant metastases, but should be confirmed with MRI and/or biopsies if the patient otherwise has early-stage dis-ease. Brain MRI should be performed when the suspicion or risk of brain metastases is increased, such as in patients with Brunicardi_Ch19_p0661-p0750.indd 68801/03/19 7:01 PM

1	CHAPTER 19689CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAABCFigure 19-21. Imaging of non–small cell lung cancer by integrated positron emission tomography (PET)-computed tomography (CT) scan. A. CT of the chest showing a tumor in the left upper lobe. B. PET scan of the chest at the identical cross-sectional level. C. Coregistered PET-CT scan clearly showing tumor invasion (con-firmed intraoperatively). (Adapted with permission from Lardinois D, Weder W, Hany TF, et al. Staging of non-small-cell lung can-cer with integrated positron-emission tomography and computed tomography, N Engl J Med. 2003 Jun 19;348(25):2500-2507.)clinical stage III disease. In the absence of neurologic symp-toms or signs, the probability of a negative head CT scan is 95%. Liver abnormalities that are not clearly simple cysts or hemangiomas and adrenal enlargement, nodules, or masses are further evaluated by MRI scanning and, occasionally, by needle biopsy. Adrenal adenomas have a high lipid content (secondary to

1	cysts or hemangiomas and adrenal enlargement, nodules, or masses are further evaluated by MRI scanning and, occasionally, by needle biopsy. Adrenal adenomas have a high lipid content (secondary to steroid production), but metastases and most primary adrenal malignancies contain little if any lipid; thus, MRI is usually able to distinguish the two.Tumor, Node, and Metastasis: Lung Cancer Staging The staging of any tumor is an attempt to estimate the extent of dis-ease and determine the patient’s prognosis; in a given patient, tumors are typically classified into a clinical stage and a patho-logic stage. Clinical staging includes history and physical examination, radiographic test results, and diagnostic biopsy information. Therapeutic plans are generated based on clinical stage. After surgical resection of tumor and lymph nodes, post-operative pathologic stage (pTNM) is determined, providing further prognostic information.The staging of solid epithelial tumors is based on the TNM

1	surgical resection of tumor and lymph nodes, post-operative pathologic stage (pTNM) is determined, providing further prognostic information.The staging of solid epithelial tumors is based on the TNM staging system. The primary tumor “T” status provides infor-mation about tumor size and relationship to surrounding struc-tures; the “N” status provides information about regional lymph nodes; and the “M” status provides information about the pres-ence or absence of metastatic disease. The designation of lymph nodes as N1, N2, or N3 requires familiarity with the lymph node mapping system41 (see Fig. 19-8). Based on clearly delineated anatomic boundaries, accurate and reproducible localization of thoracic lymph nodes is possible, facilitating detailed nodal staging for individual patients and standardization of nodal assessment between surgeons.Pathologic staging criteria are based on the predicted sur-vival relative to each combination of tumor, node, and metas-tasis status. In 2018, the

1	standardization of nodal assessment between surgeons.Pathologic staging criteria are based on the predicted sur-vival relative to each combination of tumor, node, and metas-tasis status. In 2018, the AJCC eighth edition incorporated multiple changes into the staging system for NSCLC based on analysis of survival predictors from 77,156 lung cancer patients worldwide. Table 19-11a shows the clinical and pathologic cri-teria changes implemented and each of the TNM descriptors currently used in staging NSCLC (Table 19-11b) and the over-all stage classifications (Table 19-11c). T-staging is markedly changed, including T category designation for each centimeter in size up to 5 cm, as well as size of the invasive component in lepidic growth tumors. Visceral pleural invasion increases T-stage to T2 for patients with tumors ≤3 cm in size, and syn-chronous primary tumors have an added T suffix (m) in tumor staging. Metastatic disease has also been subdivided into intra-thoracic, single-site

1	to T2 for patients with tumors ≤3 cm in size, and syn-chronous primary tumors have an added T suffix (m) in tumor staging. Metastatic disease has also been subdivided into intra-thoracic, single-site extrathoracic, and multiple extrathoracic metastasis. In addition to the TNM stage, it is recommended that histologic grade, lymphovascular invasion, adequacy of resec-tion margins and mediastinal dissection, tumor mutation status, treatment, and residual tumor after treatment also be recorded into cancer registries to facilitate evaluation of these potential predictors in future analysis of staging criteria.Staging for small cell lung cancer (SCLC) is typically based on the extent of disease. SCLC presenting with bulky locoregional disease confined to the ipsilateral hemithorax, with no evidence for distant metastatic disease, is termed “limited” SCLC. Limited disease must be treatable within a tolerable field of radiation. Using AJCC descriptors, this includes any T stage, any N stage,

1	for distant metastatic disease, is termed “limited” SCLC. Limited disease must be treatable within a tolerable field of radiation. Using AJCC descriptors, this includes any T stage, any N stage, without metastatic disease (M0). The only excep-tion is when multiple lung nodules are widely spread throughout the ipsilateral lung in the same hemithorax; in these patients, the size of the involved area would preclude a “safe” radiation field. In contrast, in “disseminated” disease, tumor is beyond the ipsi-lateral hemithorax or widely spread within the ipsilateral lung and to distant sites. Metastases to the pleura and pericardium, with resultant effusions, are considered disseminated disease. Brunicardi_Ch19_p0661-p0750.indd 68901/03/19 7:01 PM 690SPECIFIC CONSIDERATIONSPART IITable 19-11aChanges in Descriptors for non-small cell lung cancer comparing the 7th and 8th editions of the American Joint Committee on Cancer Staging ManualDESCRIPTORSEVENTH EDITIONEIGHTH EDITIONT component0 cm

1	in Descriptors for non-small cell lung cancer comparing the 7th and 8th editions of the American Joint Committee on Cancer Staging ManualDESCRIPTORSEVENTH EDITIONEIGHTH EDITIONT component0 cm (pure lepidic adenocarcinoma ≤3 cm total size)T1a if ≤2 cm; T1b if >2–3 cmTis (AIS)≤0.5 cm invasive size (lepidic predominant adenocarcinoma ≤3 cm total size)T1a if ≤2 cm; T1b if >2–3 cmT1mi≤1 cmT1aT1a>1–2 cmT1aT1b>2–3 cmT1bT1c>3–4 cmT2aT2a>4–5 cmT2aT2b>5–7 cmT2bT3>7 cmT3T4Bronchus <2 cm from carinaT3T2Total atelectasis/pneumonitisT3T2Invasion of diaphragmT3T4Invasion of mediastinal pleuraT3-N componentNo assessment, no involvement, or involvement of regional lymph nodesNX, N0, N1, N2, N3No changeM componentMetastases within the thoracic cavityM1aM1aSingle extrathoracic metastasisM1bM1bMultiple extrathoracic metastasesM1bM1cAbbreviations: AIS, adenocarcinoma in situ; mi, minimally invasive adenocarcinoma; Tis, tumor in situ.Modified with permission from Rami-Porta R, Asamura H, Travis WD, et al:

1	metastasesM1bM1cAbbreviations: AIS, adenocarcinoma in situ; mi, minimally invasive adenocarcinoma; Tis, tumor in situ.Modified with permission from Rami-Porta R, Asamura H, Travis WD, et al: Lung cancer — major changes in the American Joint Committee on Cancer eighth edition cancer staging manual, A Cancer J Clin. 2017 Mar;67(2):138-155.(Continued)Table 19-11bAmerican Joint Committee on Cancer Lung Cancer Staging Eight EditionTPRIMARY TUMORTXPrimary tumor cannot be assessed, or tumor proven by the presence of malignant cells in sputum or bronchial washings but not visualized by imaging or bronchoscopyT0No evidence of primary tumorTisCarcinoma in situSquamous cell carcinoma in situ (SCIS)Adenocarcinoma in situ (AIS); adenocarcinoma with pure lepidic pattern, ≤3 cm in greatest dimensionT1Tumor ≤3 cm in greatest dimension, surrounded by lung or visceral pleura, without bronchoscopic evidence of invasion more proximal than the lobar bronchus (i.e., not in the main bronchus) T1miMinimally

1	≤3 cm in greatest dimension, surrounded by lung or visceral pleura, without bronchoscopic evidence of invasion more proximal than the lobar bronchus (i.e., not in the main bronchus) T1miMinimally invasive adenocarcinoma: adenocarcinoma (≤3 cm in greatest dimension) with a predominantly lepidic pattern and ≤5 mm invasion in greatest dimension T1aTumor ≤1 cm in greatest dimension. A superficial, spreading tumor of any size whose invasive component is limited to the bronchial wall and may extend proximal to the main bronchus also is classified as T1a, but these tumors are uncommon. T1bTumor >1 cm but ≤2 cm in greatest dimension T1cTumor >2 cm but ≤3 cm in greatest dimensionT2Tumor >3 cm but ≤5 cm or having any of the following features:• Involves the main bronchus regardless of distance to the carina, but without involvement of the carina• Invades visceral pleura (PL1 or PL2)• Associated with atelectasis or obstructive pneumonitis that extends to the hilar region, involving part or all

1	the carina, but without involvement of the carina• Invades visceral pleura (PL1 or PL2)• Associated with atelectasis or obstructive pneumonitis that extends to the hilar region, involving part or all of the lungT2 tumors with these features are classified as T2a if ≤4 cm or if the size cannot be determined and T2b if >4 cm but ≤5 cm.Brunicardi_Ch19_p0661-p0750.indd 69001/03/19 7:01 PM

1	CHAPTER 19691CHEST WALL, LUNG, MEDIASTINUM, AND PLEURATable 19-11cStage Group in the 8th AJCC Tumor, Node, Metastasis staging systemN0N1N2N3T1/M0T1aIA1IIBIIIAIIIBT1bIA2IIBIIIAIIIBT1cIA3IIBIIIAIIIBT2/M0T2aIBIIBIIIAIIIBT2bIIAIIBIIIAIIIBT3/M0IIBIIIAIIIBIIICT4/M0IIIAIIIAIIIBIIICTX/M1M1aIVAIVAIVAIVAM1bIVAIVAIVAIVAM1cIVBIVBIVBIVBUsed with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Table 19-11bAmerican Joint Committee on Cancer Lung Cancer Staging Eight Edition T2aTumor >3 cm but ≤4 cm in greatest dimension T2bTumor >4 cm but ≤5 cm in greatest dimensionT3Tumor >5 cm but ≤7 cm in greatest dimension or directly invading any of the following: parietal pleura (PL3), chest wall (including superior sulcus tumors), phrenic nerve, parietal pericardium; or separate tumor nodule(s) in the same lobe as the primaryT4Tumor >7 cm or tumor of any size invading one or more of the following:

1	superior sulcus tumors), phrenic nerve, parietal pericardium; or separate tumor nodule(s) in the same lobe as the primaryT4Tumor >7 cm or tumor of any size invading one or more of the following: diaphragm, mediastinum, heart, great vessels, trachea, recurrent laryngeal nerve, esophagus, vertebral body, or carina; separate tumor nodule(s) in an ipsilateral lobe different from that of the primaryNREGIONAL LYMPH NODENXRegional lymph nodes cannot be assessedN0No regional lymph node metastasisN1Metastasis in ipsilateral peribronchial and/or ipsilateral hilar lymph nodes and intrapulmonary nodes, including involvement by direct extensionN2Metastasis in ipsilateral mediastinal and/or subcarinal lymph node(s)N3Metastasis in contralateral mediastinal, contralateral hilar, ipsilateral or contralateral scalene, or supraclavicular lymph node(s)MDISTANT METASTASISM0No distant metastasisM1Distant metastasis M1aSeparate tumor nodule(s) in a contralateral lobe; tumor with pleural or pericardial

1	scalene, or supraclavicular lymph node(s)MDISTANT METASTASISM0No distant metastasisM1Distant metastasis M1aSeparate tumor nodule(s) in a contralateral lobe; tumor with pleural or pericardial nodules or malignant pleural or pericardial effusion. Most pleural (pericardial) effusions with lung cancer are a result of the tumor. In a few patients, however, multiple microscopic examinations of pleural (pericardial) fluid are negative for tumor, and the fluid is nonbloody and not an exudate. If these elements and clinical judgment dictate that the effusion is not related to the tumor, the effusion should be excluded as a staging descriptor. M1bSingle extrathoracic metastasis in a single organ (including involvement of a single nonregional node) M1cMultiple extrathoracic metastases in a single organ or in multiple organsUsed with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Metastases

1	or in multiple organsUsed with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Metastases to brain, bone, bone marrow, and the pleural and pericardial spaces are common.Assessment of Functional Status Patients with potentially resectable tumors require careful assessment of their functional status and ability to tolerate either lobectomy or pneumonectomy. The surgeon should first estimate the likelihood of pneumonec-tomy, lobectomy, or possibly sleeve resection, based on the CT images. A sequential process of evaluation then unfolds.5A patient’s history is the most important tool for gauging risk. Specific questions regarding performance status should be routinely asked. If the patient can walk on a flat surface indefi-nitely, without oxygen and without having to stop and rest sec-ondary to dyspnea, he will be very likely to tolerate lobectomy. If the patient can walk up two

1	can walk on a flat surface indefi-nitely, without oxygen and without having to stop and rest sec-ondary to dyspnea, he will be very likely to tolerate lobectomy. If the patient can walk up two flights of stairs (up two stan-dard levels), without having to stop and rest secondary to dys-pnea, she will likely tolerate pneumonectomy. Finally, nearly (Continued)Brunicardi_Ch19_p0661-p0750.indd 69101/03/19 7:01 PM 692SPECIFIC CONSIDERATIONSPART IIPercent mortalityppoDLCO%504030201002030405060708090100Figure 19-22. Operative mortality after major pulmonary resec-tion for non–small cell lung cancer (334 patients) as a function of percent predicted postoperative carbon monoxide diffusion capacity (ppoDlco%). Solid line indicates logistic regression model; dashed lines indicate 95% confidence limits. (Reproduced with permission from Wang J, Olak J, Ferguson MK: Diffusing capacity predicts operative mortality but not long-term survival after resection for lung cancer, J Thorac Cardiovasc

1	limits. (Reproduced with permission from Wang J, Olak J, Ferguson MK: Diffusing capacity predicts operative mortality but not long-term survival after resection for lung cancer, J Thorac Cardiovasc Surg. 1999 Mar;117(3):581-586.)all patients, except those with carbon dioxide (CO2) retention on arterial blood gas analysis, will be able to tolerate periods of single-lung ventilation and wedge resection. Formal assess-ment of cardiac fitness is mandatory; use of risk scores such as the Thoracic Revised Cardiac Risk Index developed by Dr. Brunelli and colleagues provides useful prognostic infor-mation for postresection survival from early-stage lung cancer.Current smoking status and sputum production are also pertinent. Current smokers and patients with a greater than 60 pack-year history of smoking have a significantly increased risk of postoperative pulmonary complications; heavy smokers are 2.5 times more likely to develop pulmonary complications and three times more likely to

1	of smoking have a significantly increased risk of postoperative pulmonary complications; heavy smokers are 2.5 times more likely to develop pulmonary complications and three times more likely to develop pneumonia compared to patients with a ≤60 pack-year history (odds ratio [OR] 2.54; 95% CI 1.28–5.04; P = .0008). Impaired exchange of CO2 is also pre-dictive of increased risk, independent of the smoking history. For every 10% decline in percent carbon monoxide diffusion capac-ity (%Dlco), the risk of any pulmonary complication increased by 42% (OR 1.42; 95% CI 1.16–1.75; P = .008).42 Risk reduc-tion requires smoking cessation at least 8 weeks preoperatively, a requirement that is often not feasible in a cancer patient. Nev-ertheless, abstinence for at least 2 weeks before surgery should be encouraged. Smoking cessation on the day of surgery leads to increased sputum production and potential secretion reten-tion postoperatively, and some authors have reported increased rates of

1	be encouraged. Smoking cessation on the day of surgery leads to increased sputum production and potential secretion reten-tion postoperatively, and some authors have reported increased rates of pulmonary complications in this group.43 Patients with chronic daily sputum production will have more problems post-operatively with retention and atelectasis; they are also at higher risk for pneumonia. Sputum culture, antibiotic administration, and bronchodilators may be warranted preoperatively.Pulmonary function studies are routinely performed when any resection greater than a wedge resection will be performed. Of all the measurements available, the two most valuable are forced expiratory volume in 1 second (FEV1) and carbon mon-oxide diffusion capacity (Dlco). General guidelines for the use of FEV1 in assessing the patient’s ability to tolerate pulmonary resection are as follows: greater than 2.0 L can tolerate pneumo-nectomy, and greater than 1.5 L can tolerate lobectomy. It must be

1	of FEV1 in assessing the patient’s ability to tolerate pulmonary resection are as follows: greater than 2.0 L can tolerate pneumo-nectomy, and greater than 1.5 L can tolerate lobectomy. It must be emphasized that these are guidelines only. It is also important to note that the raw value is often imprecise because normal values are reported as “percent predicted” based on corrections made for age, height, and gender. For example, a raw FEV1 value of 1.3 L in a 62-year-old, 75-inch (190-cm) male has a percent predicted value of 30% (because the normal expected value is 4.31 L); in a 62-year-old, 62-inch female, the predicted value is 59% (normal expected value 2.21 L). The male patient is at high risk for lobectomy, while the female could potentially tolerate pneumonectomy.To calculate the predicted postoperative value for FEV1 or Dlco, the percent predicted value of FEV1 or Dlco is mul-tiplied by the fraction of remaining lung after the proposed sur-gery. For example, with a planned

1	predicted postoperative value for FEV1 or Dlco, the percent predicted value of FEV1 or Dlco is mul-tiplied by the fraction of remaining lung after the proposed sur-gery. For example, with a planned right upper lobectomy, a total of three segments will be removed. Therefore, three of a total 20 segments will leave the patient with (20 – 3/20) × 100 = 85% of their original lung capacity. In the two patients mentioned ear-lier, the man will have a predicted postoperative FEV1 of 30% × 0.85 = 25%, whereas the woman will have a predicted postop-erative FEV1 of 50%. Percent predicted value of less than 50% for either FEV1 or Dlco correlates with risk for postoperative complications, particularly pulmonary complications; the risk of complications increases in a stepwise fashion for each 10% decline. Figure 19-22 shows the relationship between predicted postoperative Dlco and estimated operative mortality.Quantitative perfusion scanning is used in select circum-stances to help estimate the

1	Figure 19-22 shows the relationship between predicted postoperative Dlco and estimated operative mortality.Quantitative perfusion scanning is used in select circum-stances to help estimate the functional contribution of a lobe or whole lung. Such perfusion scanning is most useful when the impact of a tumor on pulmonary physiology is difficult to discern. With complete collapse of a lobe or whole lung, the impact is apparent, and perfusion scanning is usually unneces-sary. Figure 19-23 shows a tumor with significant right main stem airway obstruction with associated atelectasis and volume loss of the right lung. At presentation, the patient was dyspneic with ambulation, and the FEV1 was 1.38 L. Six months prior, this patient could walk up two flights of stairs without dyspnea. The surgeon can anticipate that the patient will tolerate pneumo-nectomy because the lung is already not functioning due to main stem airway obstruction, and may, in fact, be contributing to a shunt. However,

1	can anticipate that the patient will tolerate pneumo-nectomy because the lung is already not functioning due to main stem airway obstruction, and may, in fact, be contributing to a shunt. However, with centrally located tumors associated with partial obstruction of a lobar or main bronchus or of the pulmo-nary artery, perfusion scanning may be valuable in predicting the postoperative result of resection. For example, if the quan-titative perfusion to the right lung is measured to be 21% (nor-mal is 55%) and the patient’s percent predicted FEV1 is 60%, the predicted postoperative FEV1 after a right pneumonectomy would be 60% × 0.79 = 47%, indicating the ability to tolerate pneumonectomy. If the perfusion value is 55%, the predicted postoperative value would be 27%, and pneumonectomy would pose a significantly higher risk.It is not uncommon to encounter patients with significant reductions in their percent predicted FEV1 and Dlco whose his-tory shows a functional status that is

1	would pose a significantly higher risk.It is not uncommon to encounter patients with significant reductions in their percent predicted FEV1 and Dlco whose his-tory shows a functional status that is inconsistent with the pul-monary function tests. In these circumstances, exercise testing that yields maximal oxygen consumption (v. o2max ) has emerged as a valuable decision-making technique to help patients with abnormal FEV1 and Dlco (Table 19-12). Values <10 mL/kg/min are associated with a 26% mortality after major pulmonary Brunicardi_Ch19_p0661-p0750.indd 69201/03/19 7:01 PM

1	CHAPTER 19693CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAFigure 19-23. Chest computed tomography scan of an obstructing right main stem lung tumor. Arrow indicates location of right main bron-chus. The right lung volume is much less than the left lung volume.Table 19-12Relation between maximum oxygen consumption (v. o2max) as determined by preoperative exercise testing and perioperative mortalitySTUDYDEATHS/TOTALv. o2max 10–15 mL/kg per minute Smith et al1961/6 (33%) Bechard and Wetstein1970/15 (0%) Olsen et al1981/14 (7.1%) Walsh et al1991/5 (20%) Bolliger et al2002/17 (11.7%) Markos et al2011/11 (9.1%) Wang et al2020/12 (0%) Win et al2032/16 (12.5%) Total8/96 (8.3%)v. o2max <10 mL/kg per minute Bechard and Wetstein1972/7 (29%) Olsen et al1983/11 (27%) Holden et al2042/4 (50%) Markos et al2010/5 (0%) Total7/27 (26%)Reproduced with permission from Colice GL, Shafazand S, Griffin JP: Physiologic evaluation of the patient with lung cancer being considered for resectional surgery: ACCP

1	al2010/5 (0%) Total7/27 (26%)Reproduced with permission from Colice GL, Shafazand S, Griffin JP: Physiologic evaluation of the patient with lung cancer being considered for resectional surgery: ACCP evidenced-based clinical practice guidelines (2nd edition), Chest. 2007 Sep;132(3 Suppl):161S-177S.resection compared to only 8.3% with v. o2max ≥10 mL/kg/min. Values >15 mL/kg/min generally indicate the patient’s ability to tolerate pneumonectomy.The risk assessment of a patient is an amalgam of clinical judgment and data that must be integrated with the experienced clinician’s sense of the patient and with the patient’s attitude 6toward the disease and toward life. Figure 19-24 provides a useful algorithm for determining suitability for lung resection.44Lung Cancer TreatmentGrade IV NEC (Small Cell) Lung Carcinoma. In rare cir-cumstances where SCLC presents as an isolated lung lesion, lobectomy followed by chemotherapy is warranted after sur-gical mediastinal staging has confirmed the

1	Cell) Lung Carcinoma. In rare cir-cumstances where SCLC presents as an isolated lung lesion, lobectomy followed by chemotherapy is warranted after sur-gical mediastinal staging has confirmed the absence of N2 disease. Often, ultrasound-guided FNA provides a definitive positive diagnosis and more invasive approaches are not needed. However, less than 5% are stage I, and there is no benefit from surgical resection for more advanced-stage disease; treatment is chemotherapy with or without radiation therapy depending on the extent of disease and the patient performance status.Early-Stage Non–Small Cell Lung Cancer. Early-stage disease includes T1 and T2 tumors (with or without N1 nodal involvement) and T3 tumors (without N1 nodal involvement). This group represents a small but increasing proportion of the total number of patients diagnosed with lung cancer each year (approximately 16% of an estimated 222,500 patients in 2017).18 Surgical resection is the current standard, ideally

1	proportion of the total number of patients diagnosed with lung cancer each year (approximately 16% of an estimated 222,500 patients in 2017).18 Surgical resection is the current standard, ideally accomplished by video-assisted lobectomy or pneumonectomy, depending on the tumor location.Despite the term “early-stage,” the overall 5-year sur-vival rate for all localized lung cancer is 55% and only 26% when regional metastasis was present between 2004 and 2009.45 Median survival for untreated patients with stage IA NSCLC is 14 months, and 5-year survival rate is 22%.46 After surgical resection of postoperative pathologic stage IA disease, 5-year survival is better than with no treatment, but still only 67%.41 Survival declines with higher stages. Advanced age at diagno-sis, male sex, low socioeconomic status, nonsurgical treatment, and poor histologic grade are associated with increased mortal-ity risk on multivariate analysis.45Depending on tumor size and location, lobectomy, sleeve

1	socioeconomic status, nonsurgical treatment, and poor histologic grade are associated with increased mortal-ity risk on multivariate analysis.45Depending on tumor size and location, lobectomy, sleeve lobectomy, and occasionally pneumonectomy, with mediastinal lymph node dissection or sampling, are appropriate for patients with clinical early-stage disease. Sleeve resection is performed for tumors located at airway bifurcations when an adequate bronchial Brunicardi_Ch19_p0661-p0750.indd 69301/03/19 7:01 PM 694SPECIFIC CONSIDERATIONSPART IIVO2max > 15 ml/kg/minPerform spirometryUnexplained dyspneaor diffuse parenchymaldisease on CXR/CT?Perform CPETEstimate %ppoFEV1 and %ppoDLCOIncreased riskVO2max>15 mL/kg/minVO2max10 to 15 mL/kg/minVO2max<10 mL/kg/minFEV1 >1.5 L lobectomyFEV1 >2 L pneumonectomyFEV1 >80% predictedFEV1 <1.5 L lobectomyFEV1 <2 L pneumonectomyFEV1 <80% predictedYesNoDLCO >80%predictedDLCO <80%predicted%ppo FEV1 and%ppo DLCO >40%ppo FEV1 or%ppo DLCO <40%ppo FEV1 <30

1	>80% predictedFEV1 <1.5 L lobectomyFEV1 <2 L pneumonectomyFEV1 <80% predictedYesNoDLCO >80%predictedDLCO <80%predicted%ppo FEV1 and%ppo DLCO >40%ppo FEV1 or%ppo DLCO <40%ppo FEV1 <30 or%ppo FEV1 x%ppo DLCO <1650Measure DLCOAverage riskIncreased riskFigure 19-24. Algorithm for preoperative evaluation of pulmonary function and reserve prior to resectional lung surgery. CPET = cardiopulmonary exercise test; CT = computed tomographic scan; CXR = chest radiograph; Dlco = carbon monoxide diffusion capacity; FEV1 = forced expiratory volume in 1 second; %ppo = percent predicted postoperative lung function; V.o2max = maximum oxygen consumption. (Modified with permission from Colice GL, Shafazand S, Griffin JP: Physiologic evaluation of the patient with lung cancer being considered for resectional surgery: ACCP evidenced-based clinical practice guidelines (2nd edition), Chest. 2007 Sep;132(3 Suppl):161S-177S.44)Brunicardi_Ch19_p0661-p0750.indd 69401/03/19 7:01 PM

1	CHAPTER 19695CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAmargin cannot be obtained by standard lobectomy. Pneumonec-tomy is rarely performed; primary indications for pneumonec-tomy in early-stage disease include large central tumors involving the distal main stem bronchus and inability to completely resect involved N1 lymph nodes. The latter circumstance occurs with bulky adenopathy or with extracapsular nodal spread.Management of Early-Stage Lung Cancer in the High-Risk Patient Lobectomy may not be an option for some patients with early-stage disease, due to poor cardiopulmonary function or other comorbid illnesses. The ultimate decision that a patient is inoperable, both with regard to the patient’s ability to tolerate surgery and the likelihood of successful resection, should be accepted only after evaluation by an expert surgeon. Surgeons with limited expertise, when faced with a complicated patient, should refer the patient to a high-volume center for further eval-uation if they are

1	after evaluation by an expert surgeon. Surgeons with limited expertise, when faced with a complicated patient, should refer the patient to a high-volume center for further eval-uation if they are unable to offer the patient surgical resection in their own center.Rationale for Limited Resection in Early-Stage Lung Cancer. Limited resection, defined as segmentectomy or wedge resec-tion, is a viable option for achieving local control in high-risk patients. Historically, limited resection with wedge or segmen-tectomy has been considered a compromise operation due to unacceptably high rates of local recurrence and concerns for worse survival.47,48 Subsequent meta-analysis of the literature shows that the difference in death rate is likely negligible49 (Table 19-13). The high rates of local recurrence demonstrated by Ginsberg and others, however, remain a significant concern and continue to restrict the use of limited resection for early-stage lung cancer to the high-risk patient.With the

1	demonstrated by Ginsberg and others, however, remain a significant concern and continue to restrict the use of limited resection for early-stage lung cancer to the high-risk patient.With the recent publication of a 20% reduction in lung cancer mortality with screening CT scans in high-risk popula-tions, the topic of limited resection is again the subject of inten-sive review. Studies investigating anatomic segmentectomy (or extended wedge resection) with hilar and mediastinal lymph node dissection suggest that close attention to the ratio of surgi-cal margin to tumor diameter and a careful assessment of the lymph nodes substantially reduce local recurrence.50-52 Recur-rence rates were 6.2%, comparable to rates associated with lobectomy, when the margin-to-tumor diameter ratio exceeded 1, compared to 25% if the margin-to-tumor diameter ratio was less than 1.50 In most centers, this requires use of a thora-cotomy, although increasing experience with VATS in highvolume centers shows that

1	to 25% if the margin-to-tumor diameter ratio was less than 1.50 In most centers, this requires use of a thora-cotomy, although increasing experience with VATS in highvolume centers shows that limited resection is safe and feasi-ble, with perioperative adverse outcomes that are comparable to lobectomy.52-55Rationale for Tumor Ablation in the Management of Primary Lung Cancer. Limited resection, by definition, requires that the patient has sufficient cardiopulmonary reserve to undergo a general anesthesia and loss of at least one pulmonary seg-ment. For the high-risk or nonoperable patient, as determined by experience pulmonary surgeons, tumor ablation techniques have been developed for treatment of early-stage lung cancers.Current limitations of this approach include the absence of nodal staging, lack of tissue for molecular profiling, chemo-resistance, or sensitivity testing, concerns about definitions of locoregional recurrence, and a lack of uniformity across cen-ters. Surgeons

1	staging, lack of tissue for molecular profiling, chemo-resistance, or sensitivity testing, concerns about definitions of locoregional recurrence, and a lack of uniformity across cen-ters. Surgeons typically define locoregional recurrence as tumor growth within the operative field, including resectable lymph nodes, whereas local recurrence after ablation is most com-monly defined as tumor growth within the field of treatment. Despite the fact that in-transit or lymph node metastases are present in up to 27% of clinically stage I NSCLCs at resection, any tumor growth outside the field of ablative treatment is not be considered treatment failure.56Despite these limitations, tumor ablative strategies are increasingly proposed as viable alternatives to surgical resec-tion, even in potentially operable patients.57-62 While prema-ture, ablative techniques may ultimately be shown to have efficacy equivalent to lobectomy for the primary treatment of very small peripheral early-stage lung

1	operable patients.57-62 While prema-ture, ablative techniques may ultimately be shown to have efficacy equivalent to lobectomy for the primary treatment of very small peripheral early-stage lung cancers. Multidisci-plinary collaboration between thoracic surgery, interventional radiology/pulmonology, and radiation oncology is required to ensure that development of these ablative techniques occurs through properly designed and well-controlled prospective studies and will ensure that patients receive the best available therapy, regardless of whether it is surgical resection or ablative therapy.The two most commonly applied ablation techniques are radiofrequency ablation and stereotactic body radiotherapy.1. Radiofrequency ablation. Radiofrequency ablation is per-formed using either monopolar or bipolar delivery of elec-trical current to electrodes placed within the tumor tissue. In lung tumors, the electrodes are typically inserted into the tumor mass under CT guidance. An electrical

1	or bipolar delivery of elec-trical current to electrodes placed within the tumor tissue. In lung tumors, the electrodes are typically inserted into the tumor mass under CT guidance. An electrical current is delivered; the current is converted by means of friction into heat, which quickly leads to immediate and irreparable tissue destruction in the tissue surrounding the electrode. The efficacy of radiofrequency ablation for controlling the primary tumor and improving survival in poor operative candidates (either due to significant comorbid diseases precluding general anesthesia or poor pulmonary function excluding lung resection) is safe and feasible for peripheral lung nodules. In tumors <3.5 cm, the rate of radiographic resolution of tumor is up to 80%, and cancer-specific sur-vival at 2 years was approximately 90%, indicating excel-lent local control of the primary site.57-59,63 It has become the preferred modality for small peripheral tumors over standard external-beam radiation

1	2 years was approximately 90%, indicating excel-lent local control of the primary site.57-59,63 It has become the preferred modality for small peripheral tumors over standard external-beam radiation in centers where the tech-nique is available. Radiofrequency ablation is an excellent modality for the patient at risk for adverse outcomes with pulmonary resec-tion or for patients who refuse surgery, and surgeons should have an algorithm for determining which patients are optimal for this modality64-69 (see Fig. 19-24). Target lesions larger than 5 cm, tumor abutting the hilum, associated malignant pleural or pericardial effusion, greater than three lesions in one lung, and the presence of pulmonary hypertension are all contraindications to radiofrequency ablation.64 Proximity to a large vessel is a contraindication not only due to the risk of massive bleeding, but also because large blood vessels act as a heat sink and lethal cellular temperatures are less likely to be achieved. For

1	vessel is a contraindication not only due to the risk of massive bleeding, but also because large blood vessels act as a heat sink and lethal cellular temperatures are less likely to be achieved. For these patients, stereotactic body radiotherapy may provide local tumor control with less risk of major complications. Combination therapy with either external-beam radiation or stereotactic body radiotherapy is also under investigation.2. Stereotactic body radiotherapy. Stereotactic body radiotherapy applies highly focused, high-intensity, three-dimensional conformal radiation to the target lesion over a few sessions. Tumor motion quantification and image guidance technologies have significantly improved the delivery of radiation with high levels of precision 7Brunicardi_Ch19_p0661-p0750.indd 69501/03/19 7:01 PM 696SPECIFIC CONSIDERATIONSPART IITable 19-13New classification system for lung adenocarcinomaSTUDY OR SUBGROUPlog [HAZARD RATIO]SESEGMENTECTOMY TOTALLOBECTOMY

1	69501/03/19 7:01 PM 696SPECIFIC CONSIDERATIONSPART IITable 19-13New classification system for lung adenocarcinomaSTUDY OR SUBGROUPlog [HAZARD RATIO]SESEGMENTECTOMY TOTALLOBECTOMY TOTALWEIGHTHAZARD RATIO IV, RANDOM, 95% CIHAZARD RATIO IV, RANDOM, 95% CIBando 2002–0,3150,3495741323,3%0,73 [0,37,1,45]Favours Segmentectomy0.005200100.11Favours LobectomyCheng 2012–0,210,2908132324,8%0,81 [0,46, 1,43]Dai 20160,3320,21044240115209,2%1,39 [0,92, 2,11]Ginsberg 19950,3680,21571221258,7%1,44 [0,95, 2,21]Hamatake 20120,19890,280632775,1%1,22 [0,70, 2,11]Iwasaki 2007–0,083380,5076531551,6%0,92 [0,34, 2,49]Keenan 20040,1310,4158541472,3%1,14 [0,50, 2,58]Kilic 2009–0,23570,21683106788,6%0,79 [0,52, 1,21]Kodama 19970,10110,573246771,2%1,11 [0,36, 3,40]Koike 20030,07730,4385741592,1%1,08 [0,46, 2,55]Landreneau 20140,460,24821021176,6%1,58 [0,97, 2,58]Martin–Ucar 2005–1,04350,707117170,8%0,35 [0,09, 1,41]Nakamura 20110,2391382890,4%1,27 [0,18, 9,02]Okada 2001–0,11650,7168681040,8%0,89 [0,22,

1	2,55]Landreneau 20140,460,24821021176,6%1,58 [0,97, 2,58]Martin–Ucar 2005–1,04350,707117170,8%0,35 [0,09, 1,41]Nakamura 20110,2391382890,4%1,27 [0,18, 9,02]Okada 2001–0,11650,7168681040,8%0,89 [0,22, 3,63]Okumura 2007–0,16320,3595672733,1%0,85 [0,42, 1,72]Razi 2016–0,020,256911910516,1%0,98 [0,59, 1,62]Read 19900,31480,41841131312,3%1,37 [0,60, 3,11]Sienel 20071,04020,91581491500,5%2,83 [0,47,17,03]Soukiasian 20120,28521,6735561780,1%1,33 [0,05, 35,35]Sugi 2010–0,02020,2543956,5%0,98 [0,60, 1,60]Tsutani 2014–0,71330,3087983834,3%0,49 [0,27, 0,90]Warren 19940,54810,5661031,6%1,73 [0,65, 4,61]Watanabe 2005–0,61620,8979620570,5%0,54 [0,09, 3,14]Yamashita 20120,198860,73469901240,8%1,22 [0,29, 5,15]Yamato 20080,30010,40561532772,5%1,35 [0,61 , 2,99]Yendamuri 20110,2390,2756216225995,3%1,27 [0,74, 2,18]Zhong 2012–0,16250,19388398110,8%0,85 [0,58, 1,24]Total (95% CI)611118431100,0%1,04 [0,92, 1,18]Heterogeneity: Tau2 = 0,00; Chi2 = 25,04, df = 26 (P = 0,52); I2 = 0%Test for overall effect:

1	2,18]Zhong 2012–0,16250,19388398110,8%0,85 [0,58, 1,24]Total (95% CI)611118431100,0%1,04 [0,92, 1,18]Heterogeneity: Tau2 = 0,00; Chi2 = 25,04, df = 26 (P = 0,52); I2 = 0%Test for overall effect: Z = 0,68 (P = 0,50)Forest plot of HR for overall survival impact of operative approach (segmentectomy versus lobectomy) of stage I NSCLC patients. The pooled HR displayed in this figure when compared with segmentectomy suggested that there was not a significant benefit of lobectomy on HR of stage I patients (7-21) (HR 1.04; 95% CI, 0.92–1.18, P = 0.50) (22–33).Abbreviations: HR = hazard ratio; NSCLC = non–small cell lung cancer; CI = confidence interval; df = degree of freedom; SE = standard error.Reproduced with permission from Bedetti B, Bertolaccini L, Rocco R, et al: Segmentectomy versus lobectomy for stage I non-small cell lung cancer: a systematic review and meta-analysis, J Thorac Dis. 2017 Jun; 9(6):1615-1623.Brunicardi_Ch19_p0661-p0750.indd 69601/03/19 7:01 PM

1	CHAPTER 19697CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAto the target lesion. This accuracy is important because the lung is extremely sensitive to radiation injury and the majority of patients with early-stage lung cancer who are currently considered candidates for ablative therapy have marginal lung function; excessive injury to normal surrounding lung tissue is not desirable. Importantly, these techniques allow the safe delivery of up to 66 Gy of radiation to the target tumors without exceeding the maximum-tolerated dose.62-70 A phase II North American multicenter study recently demonstrated the safety and efficacy of this approach in 59 nonoperable patients.62 Patients with biopsy-proven, node-negative peripheral NSCLCs less than 5 cm in diameter (T1 or T2) were treated with stereotactic body radiotherapy after they were deemed inoperable, based on coexisting medical conditions, by a thoracic surgeon and/or pulmonolo-gist. Primary tumor control was excellent; at 3 years, 97.6% were

1	body radiotherapy after they were deemed inoperable, based on coexisting medical conditions, by a thoracic surgeon and/or pulmonolo-gist. Primary tumor control was excellent; at 3 years, 97.6% were deemed to have primary tumor control by the authors, and 90.6% had local control. However, it is important to note that primary tumor failure was defined specifically as at least a 20% increase in the longest diam-eter of the gross tumor volume by CT scan and evidence of tumor viability either by biopsy confirming carcinoma or by demonstration of FDG avidity on PET scan. For viability to be confirmed with PET scan, the uptake was required to be of similar intensity to the pretreatment staging PET scan. Failure beyond a 1.5to 2-cm margin around the primary tumor volume was considered local failure. Failure in regional node basins was seen in two patients. When compared to locoregional control rates of approximately 6.5% with limited resection, the 3-year locoregional recurrence was higher at

1	Failure in regional node basins was seen in two patients. When compared to locoregional control rates of approximately 6.5% with limited resection, the 3-year locoregional recurrence was higher at 12.8%.3. Patient selection for stereotactic body radiotherapy, as with limited resection and radiofrequency ablation, is impor-tant. Because the radiation field is so precise, patients with severe emphysema and chronic obstructive pulmonary dis-ease can be safely treated without significant concern for worsening lung function. However, patients with central tumors near the mediastinum and hilum have increased incidence of significant hypoxia, hemoptysis, atelecta-sis, pneumonitis, and reduced pulmonary function.70 In the multicenter trial detailed earlier, treated tumors were required to be greater than 2 cm from the proximal bron-chial tree in all directions (which they defined as the distal 2 cm of the trachea, carina, and named major lobar bronchi up to their first

1	required to be greater than 2 cm from the proximal bron-chial tree in all directions (which they defined as the distal 2 cm of the trachea, carina, and named major lobar bronchi up to their first bifurcation).62Rationale for Chemotherapy in the Management of Early-Stage NSCLC. The role of chemotherapy in early-stage (stages I and II) NSCLC is evolving, with several pro-spective phase 2 studies having shown a potential benefit.71,72 Initial concerns that induction chemotherapy may result in increased perioperative morbidity or mortality appear to be unwarranted, as the incidence of perioperative morbidity and mortality is not different between the two groups, except in patients undergoing right-sided pneumonectomy after induc-tion chemotherapy.73 As shown in Table 19-14, an absolute survival benefit of 4% to 7% can be realized using induction for all stages of lung cancer, and in situations where use of adjuvant chemotherapy is anticipated, induction chemother-apy is an acceptable

1	benefit of 4% to 7% can be realized using induction for all stages of lung cancer, and in situations where use of adjuvant chemotherapy is anticipated, induction chemother-apy is an acceptable alternative.Table 19-14Five-year stage-specific survival after induction chemotherapy followed by surgerySTAGE5-YEAR SURVIVAL (%)ABSOLUTE BENEFIT (%)NEW 5-YEAR SURVIVAL (%)IA75479IB55661IIA50757IIB40747IIIA15–356–721–42IIIB5–103–58–15Reproduced with permission from Burdett SS, Stewart LA, Rydzewska L: Chemotherapy and surgery versus surgery alone in non-small cell lung cancer, Cochrane Database Syst Rev. 2007 Jul 18;(3):CD006157.National Comprehensive Cancer Network guidelines cur-rently recommend observation for T1a (≤1 cm), T1b (>1–2 cm), and T1c (>2–3 cm), node-negative, completely resected NSCLCs (T1abcN0M0). For patients with larger tumors (T2a tumor >3–4 cm; T2b tumor >4–5 cm) that are node-negative, it is recommended that chemotherapy be considered in high-risk patients, ideally in

1	NSCLCs (T1abcN0M0). For patients with larger tumors (T2a tumor >3–4 cm; T2b tumor >4–5 cm) that are node-negative, it is recommended that chemotherapy be considered in high-risk patients, ideally in the setting of a clinical trial. High-risk tumor characteristics include poorly differentiated tumors, moderately to poorly differentiated lung neuroendocrine tumors, vascular invasion, resection limited to wedge resection only, tumors >4 cm in size, visceral pleural involvement, and when lymph node sampling at the time of resection was incomplete (Nx).Evaluation and Management of Locally Advanced NSCLC. Five-year relative survival in patients with locore-gional disease is 28%, but there is significant heterogeneity within the group. Stage III disease includes patients with small tumors that have metastasized to the mediastinal lymph nodes as well as large tumors (>7 cm), and tumor invading unresectable structures or the major carina with no nodal metastasis at all. Patients with

1	that have metastasized to the mediastinal lymph nodes as well as large tumors (>7 cm), and tumor invading unresectable structures or the major carina with no nodal metastasis at all. Patients with clinically evident N2 disease (i.e., bulky adenopa-thy present on CT scan or mediastinoscopy, with lymph nodes often replaced by tumor) have a 5-year survival rate of 5% to 10% with surgery alone. In contrast, patients with microscopic N2 disease discovered incidentally in one lymph node station after surgical resection have a 5-year survival rate that may be as high as 30%. As a result, many surgeons and oncologists dif-ferentiate between microscopic and bulky N2 lymphadenopathy and the number of involved N2 nodal stations in determining whether to proceed with resection following induction therapy. It is generally accepted that surgical resection is appropriate for patients with a single-station metastasis with a single lymph node smaller than 3 cm, although randomized trials specifically

1	It is generally accepted that surgical resection is appropriate for patients with a single-station metastasis with a single lymph node smaller than 3 cm, although randomized trials specifically investigating resection following induction therapy for patients with single-station microscopic disease have not yet been performed.Histologic confirmation of N2 nodal metastases is impera-tive; false-positive findings on PET scan are unacceptably high, and reliance on this modality will lead to significant undertreat-ment of patients with earlier stage cancers. This is particularly true in regions with high incidence of granulomatous diseases. Brunicardi_Ch19_p0661-p0750.indd 69701/03/19 7:01 PM 698SPECIFIC CONSIDERATIONSPART IIWhen N2 nodes are found, incidentally, to harbor metastasis at the time of planned anatomic lung resection, the decision to proceed with resection varies depending on surgeon preference; it is acceptable to either proceed with anatomic resection and mediastinal

1	the time of planned anatomic lung resection, the decision to proceed with resection varies depending on surgeon preference; it is acceptable to either proceed with anatomic resection and mediastinal lymph node sampling/dissection or to stop the pro-cedure, refer the patient for induction therapy, and reevaluate for resection after induction therapy is completed.When histologically confirmed metastases are found dur-ing preoperative staging evaluation, patients should be referred for induction chemotherapy; patients in whom the mediastinal nodes are sterilized by induction therapy have a better prognosis, and surgical resection is generally warranted as part of a mul-timodal approach. As with preinduction evaluation, histologic confirmation of persistent N2 disease after induction therapy is imperative; patients should not be denied surgical resection fol-lowing induction chemotherapy based on radiographic evidence for N2 disease because the survival for resected NSCLC is

1	therapy is imperative; patients should not be denied surgical resection fol-lowing induction chemotherapy based on radiographic evidence for N2 disease because the survival for resected NSCLC is sig-nificantly better than with definitive chemotherapy.Surgery in T4 and Stage IV Disease Surgery is occasion-ally appropriate for highly selected patients with tumors invad-ing the SVC, carinal or vertebral body involvement, or satellite nodules in the same lobe (T3, N0-1, M0) or in T4, N0-1 tumors with limited invasion into the mediastinum, heart, great vessels, trachea, recurrent laryngeal nerve, esophagus, vertebral body, diaphragm or carina through direct extension. Surgery gener-ally does not have a role in the care of patients with any tumor with N3 disease or T4 tumors with N2 disease. Survival rates remain extremely low for these patients. Similarly, the treat-ment of patients with stage IV disease is chemotherapy. How-ever, on occasion, patients with a single site of metastasis are

1	rates remain extremely low for these patients. Similarly, the treat-ment of patients with stage IV disease is chemotherapy. How-ever, on occasion, patients with a single site of metastasis are encountered, particularly with adenocarcinomas presenting with a solitary brain metastasis. In this highly select group, 5-year survival rates of 10% to 15% can be achieved with surgical exci-sion of the brain metastasis and the primary tumor, assuming it is early stage.Surgery for Management of Pancoast’s Tumor Carcinoma arising in the extreme apex of the chest with associated arm and shoulder pain, atrophy of the muscles of the hand, and Horner’s syndrome presents a unique challenge to the surgeon. Any tumor of the superior sulcus, including tumors without evi-dence for involvement of the neurovascular bundle, is now com-monly known as Pancoast’s tumors, after Henry Pancoast who described the syndrome in 1932. The designation is reserved for tumors involving the parietal pleura or deeper

1	bundle, is now com-monly known as Pancoast’s tumors, after Henry Pancoast who described the syndrome in 1932. The designation is reserved for tumors involving the parietal pleura or deeper structures overly-ing the first rib. Chest wall involvement at or below the second rib is not a Pancoast’s tumor.74 Treatment is multidisciplinary; due to the location of the tumor and involvement of the neu-rovascular bundle that supplies the ipsilateral extremity, pre-serving postoperative function of the extremity is critical. For this reason, resection should only be performed in patients who are proven negative for mediastinal lymph node involvement. Survival with N2 positive nodes is poor, and the morbidity and mortality associated with surgical resection are high. If bulky lymphadenopathy is present, EBUSor EUS-guided FNA may prove nodal involvement. However, a negative FNA is not suf-ficient for proving the absence of mediastinal involvement and should be followed by mediastinoscopy to

1	present, EBUSor EUS-guided FNA may prove nodal involvement. However, a negative FNA is not suf-ficient for proving the absence of mediastinal involvement and should be followed by mediastinoscopy to ensure accurate and complete evaluation of the mediastinum.Because Pancoast’s tumors have high rates of local recur-rence and incomplete resection, induction chemoradiotherapy fol-lowed by surgery is recommended. This treatment regimen was well tolerated in a study performed by the Southwest Oncology Group, with 95% of patients completing induction treatment. Com-plete resection was achieved in 76%. Five-year survival was 44% overall and 54% when complete resection was achieved. Disease progression with this regimen was predominantly at distant sites, with the brain being the most common.75 The current treatment algorithm for Pancoast’s tumors is presented in Fig. 19-25.Surgical excision is performed via thoracotomy with en bloc resection of the chest wall and vascular structures and

1	The current treatment algorithm for Pancoast’s tumors is presented in Fig. 19-25.Surgical excision is performed via thoracotomy with en bloc resection of the chest wall and vascular structures and ana-tomic lobectomy. A portion of the lower trunk of the brachial plexus and the stellate ganglion are also typically resected. With chest wall involvement, en bloc chest wall resection, along with lobectomy, is performed, with or without chest wall reconstruction.For small rib resections or those posterior to the scapula, chest wall reconstruction is usually unnecessary. Larger defects (two rib segments or more) are usually reconstructed with Gore-Tex to provide chest wall contour and stability. En bloc resection is also used for other locally advanced tumors (T3) with direct invasion of the adjacent chest wall, diaphragm, or pericardium. If a large portion of the pericardium is removed, reconstruction with thin Gore-Tex membrane will be required to prevent cardiac herniation and venous

1	adjacent chest wall, diaphragm, or pericardium. If a large portion of the pericardium is removed, reconstruction with thin Gore-Tex membrane will be required to prevent cardiac herniation and venous obstruction.Preoperative (Induction) Chemotherapy for NSCLC The use of chemotherapy before anatomic surgical resection has a number of potential advantages:1. The tumor’s blood supply is still intact, allowing better chemotherapy delivery and avoiding tumor cell hypoxia (in any residual microscopic tumor remaining postoperatively), which would increase radioresistance.2. The primary tumor may be downstaged, enhancing resectability.3. Patients are better able to tolerate chemotherapy before sur-gery and are more likely to complete the prescribed regimen than after surgery.4. It functions as an in vivo test of the primary tumor’s sensi-tivity to chemotherapy.5. Response to chemotherapy can be monitored and used to guide decisions about additional therapy.6. Systemic micrometastases are

1	an in vivo test of the primary tumor’s sensi-tivity to chemotherapy.5. Response to chemotherapy can be monitored and used to guide decisions about additional therapy.6. Systemic micrometastases are treated.7. It identifies patients with progressive disease/nonresponders and spares them a pulmonary resection.Potential disadvantages include:1. There is a possible increase in the perioperative complica-tion rate in patients requiring right pneumonectomy after induction chemotherapy.2. While the patient is receiving chemotherapy, potentially curative resection is delayed; if the patient does not respond, this delay could result in tumor spread.In stage IIIA N2 disease, the response rates to induction chemotherapy are high, in the range of 70%. The treatment is generally safe, as it does not cause a significant increase in peri-operative morbidity. Two randomized trials have now compared surgery alone for patients with N2 disease to preoperative che-motherapy followed by surgery. Both

1	cause a significant increase in peri-operative morbidity. Two randomized trials have now compared surgery alone for patients with N2 disease to preoperative che-motherapy followed by surgery. Both trials were stopped before complete accrual because of a significant increase in survival for the chemotherapy arm.The initially observed survival differences have been maintained for up to 3 years and beyond (5-year data not shown). Given these results, induction chemotherapy with Brunicardi_Ch19_p0661-p0750.indd 69801/03/19 7:01 PM

1	CHAPTER 19699CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAConfirm T3–4, N0-1 M0 NSCLCNo evidence for metastatic or N2 nodal diseaseTumor progression orpoor performance statusDefinitivechemoradiotherapyMetastatic disease or N2nodal diseaseConcurrent induction chemotherapy (Cisplatin/Etoposide)And radiotherapy: 45 Gy over 5 weeksCT chest/upper abdomenMRI/MRA of vessels/brachialplexusMediastinoscopyBrain CT or MRI andPET scanReassessment performance score, physiologic reserve,tumor responseRadiographic evaluation: CT scans of thechest, upper abdomen, and brain. PET scan for metastasesAdditional chemotherapyas toleratedAssess performance status:performance score, cardiopulmonary reserve,renal function and neurologic functionInitial evaluation, biopsy and stagingThoracotomy, en bloc chest wall resection, lobectomy, chest wall with reconstructionTumor stable/regression; good toexcellent performance statusPoor performance statusGood to excellent performance statusFigure 19-25. Treatment

1	wall resection, lobectomy, chest wall with reconstructionTumor stable/regression; good toexcellent performance statusPoor performance statusGood to excellent performance statusFigure 19-25. Treatment algo-rithm for Pancoast’s tumors. CT = computed tomography; MRA = magnetic resonance angiography; MRI = magnetic resonance imaging; NSCLC = non–small cell lung cancer; PET = positron emission tomography.cisplatin-based regimen (two to three cycles) has become stan-dard for patients with N2 disease. Table 19-15 summarizes the findings of a systematic review and meta-analysis reporting response rates, progression-free survival, and overall survival after induction chemotherapy followed by surgical resection.Postoperative (Adjuvant) Chemotherapy for NSCLC Post-operative adjuvant chemotherapy was previously thought to confer no benefit based on multiple prospective randomized tri-als, in part because patients who had undergone thoracotomy and lung resection had difficulty tolerating the

1	was previously thought to confer no benefit based on multiple prospective randomized tri-als, in part because patients who had undergone thoracotomy and lung resection had difficulty tolerating the adjuvant regi-mens. More recently, however, newer, more effective agents have shown promise, and adjuvant therapy is better tolerated after minimally invasive lung resection (i.e., VATS or robotic anatomic resection). Targeted therapies, which have been shown to be beneficial in advanced-stage lung cancer, are of particular interest.Any patient with nodal metastasis (N1 or N2) or with T3 tumors (defined as tumors >5 to ≤7 cm or separate tumor in same lobe or direct invasion of chest wall [includes parietal pleura and superior sulcus]/parietal pericardium/phrenic nerve) should receive adjuvant chemotherapy if they are able to toler-ate the regimen. In the situation where the margins of resec-tion are positive, re-resection is recommended. If not possible, concurrent chemoradiation is

1	chemotherapy if they are able to toler-ate the regimen. In the situation where the margins of resec-tion are positive, re-resection is recommended. If not possible, concurrent chemoradiation is recommended for macroscopic residual tumor and sequential chemoradiation for microscopic residual tumor.Definitive Nonsurgical Treatment for NSCLC. Recent advances in targeted therapies have changed the management of advanced NSCLC from a generalized, platinum-based approach to one in which molecular analysis and targeted, personalized therapies are now standard of care. It is now mandatory that the pathologist clearly differentiate between squamous cell carcinoma and adenocarcinoma because the therapeutic options are different and use of bevacizumab, while beneficial in patients with adenocarcinoma, has been found to cause exces-sive pulmonary hemorrhage in patients with squamous histol-ogy. For the surgeon, this requirement translates into a much more aggressive approach to tissue diagnosis.

1	has been found to cause exces-sive pulmonary hemorrhage in patients with squamous histol-ogy. For the surgeon, this requirement translates into a much more aggressive approach to tissue diagnosis. At our institution, the cytopathologist provides onsite rapid assessment of the fine-needle aspirate to determine whether tumor cells are present and confirm that sufficient tumor cells are present to enable molecu-lar testing. This has increased the number of passes performed during an EBUS-guided FNA or during CT-guided aspiration of a pulmonary or intrathoracic lesion; typically, an additional two to three passes are made for cell block material after confirming the presence of tumor cells in the target area. When insufficient cells are obtained for molecular testing, despite having a diag-nosis, additional sampling is warranted; this is mandatory in patients with adenocarcinoma and likely to become necessary for other non–small cell histologic types as advances in targeted therapies

1	additional sampling is warranted; this is mandatory in patients with adenocarcinoma and likely to become necessary for other non–small cell histologic types as advances in targeted therapies become available for clinical use. Acquiring adequate tissue for diagnosis may require mediastinoscopy or VATS; close communication between the oncologist, surgeon, patholo-gist, and patient is needed to ensure that the benefits to the patient clearly outweigh the risks and that results obtained through more aggressive diagnostic measures are needed to direct subsequent care.8Brunicardi_Ch19_p0661-p0750.indd 69901/03/19 7:01 PM 700SPECIFIC CONSIDERATIONSPART IITable 19-15Selected randomized trials of neoadjuvant chemotherapy for stage III non–small cell lung cancerTRIAL (REFERENCE)NO. OF PATIENTS (STAGE III)CHEMOTHERAPYRESPONSE RATE (%)PCR (%)COMPLETE RESECTIONPFSOS5-YEAR SURVIVALRosell et al8560 (60)MitomycinIfosfamideCisplatin60485%12 vs. 5 mo (DFS; P = .006)22 vs. 10 mo (P = .005)16% vs.

1	(STAGE III)CHEMOTHERAPYRESPONSE RATE (%)PCR (%)COMPLETE RESECTIONPFSOS5-YEAR SURVIVALRosell et al8560 (60)MitomycinIfosfamideCisplatin60485%12 vs. 5 mo (DFS; P = .006)22 vs. 10 mo (P = .005)16% vs. 0%Roth et al9060 (60)Cyclophosphamide EtoposideCisplatin35NR39% vs. 31%Not reached vs. 9 mo (P = .006)64 vs. 11 mo (P = .008)56% vs. 15%aPass et al9127 (27)EtoposideCisplatin62885% vs. 86%12.7 vs. 5.8 mo (P = .083)28.7 vs. 15.6 mo (P = .095)NRNagai et al9262 (62)CisplatinVindesine28065% vs. 77%NR17 vs. 16 mo (P = .5274)10% vs. 22%Gilligan et al93519 (80)Platinum basedb49482% vs. 80%NR54 vs. 55 mo (P = .86)44% vs. 45%Depierre et al94355 (167)MitomycinIfosfamideCisplatin641192% vs. 86%26.7 vs. 12.9 mo (P = .033)37 vs. 26 mo (P = .15)43.9% vs. 35.3%cPisters et al95354 (113)dCarboplatinPaclitaxel41NR94% vs. 89%33 vs. 21 mo (P = .07)75 vs. 46 mo (P = .19)50% vs. 43%Sorensen et al9690 (NR)PaclitaxelCarboplatin46079% vs. 70%NR34.4 vs. 22.5 mo (NS)36% vs. 24% (NS)Mattson et al97274

1	vs. 89%33 vs. 21 mo (P = .07)75 vs. 46 mo (P = .19)50% vs. 43%Sorensen et al9690 (NR)PaclitaxelCarboplatin46079% vs. 70%NR34.4 vs. 22.5 mo (NS)36% vs. 24% (NS)Mattson et al97274 (274)Docetaxel28NR77% vs. 76%e9 vs. 7.6 mo (NS)14.8 vs. 12.6 mo (NS)NRa3-year survival.bOptions included MVP (mitomycin C, vindesine, and platinum), MIC (mitomycin, ifosfamide, and cisplatin), NP (cisplatin and vinorelbine), PacCarbo (paclitaxel and carboplatin), GemCis (gemcitabine and cisplatin), and DocCarbo (docetaxel and carboplatin).c4-year survival.d113 patients (32%) were reported to have stage IIB or IIIA disease.e22 patients in the chemotherapy arm and 29 patients in the control arm had resectable disease.Abbreviations: DFS = disease-free survival; NR = not recorded; NS = not significant; OS = overall survival; pCR = pathologic complete response; PFS = progression-free survival.Reproduced with permission from Allen J, Jahanzeb M: Neoadjuvant chemotherapy in stage III NSCLC, J Natl Compr Canc Netw.

1	survival; pCR = pathologic complete response; PFS = progression-free survival.Reproduced with permission from Allen J, Jahanzeb M: Neoadjuvant chemotherapy in stage III NSCLC, J Natl Compr Canc Netw. 2008 Mar;6(3):285-293.Brunicardi_Ch19_p0661-p0750.indd 70001/03/19 7:01 PM

1	CHAPTER 19701CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAOnce a treatment plan has been devised, two strategies for delivery are available. “Sequential” chemoradiation involves full-dose systemic chemotherapy (i.e., cisplatin combined with a second agent) followed by standard radiotherapy (approxi-mately 60 Gy). The combination of chemotherapy followed by radiation has improved 5-year survival from 6% with radio-therapy alone to 17%.76 An alternative approach, referred to as “concurrent chemoradiation,” administers chemotherapy and radiation at the same time. Certain chemotherapeutic agents sen-sitize tumor cells to radiation and, thus, enhance the radiation effect. The advantages of this approach are improved primary tumor and locoregional lymph node control and elimination of the delay in administering radiotherapy that occurs with sequential treatment. A disadvantage, however, is the necessary reduction in chemotherapy dosage in order to diminish over-lapping toxicities; this can

1	in administering radiotherapy that occurs with sequential treatment. A disadvantage, however, is the necessary reduction in chemotherapy dosage in order to diminish over-lapping toxicities; this can potentially lead to undertreatment of systemic micrometastases. Randomized trials have shown a modest 5-year survival benefit as compared with chemotherapy. In a systematic review of 47 trials and six meta-analyses, an absolute survival benefit of 4% at 2 years was seen when con-current platinum-based chemoradiation was given compared to sequential radiation.77Definitive radiotherapy is predominantly used for pallia-tion of symptoms in patients with poor performance status; cure rates with radiation as a single modality in patients with N2 or N3 disease is less than 7%. Recent improvement has been seen with three-dimensional conformal radiotherapy and altered frac-tionation. Such poor results for patients with stage III lung cancer reflect the limitations of locoregional treatment in a

1	been seen with three-dimensional conformal radiotherapy and altered frac-tionation. Such poor results for patients with stage III lung cancer reflect the limitations of locoregional treatment in a disease where death results from systemic metastatic spread.Options for Thoracic Surgical ApproachesThoracic surgical approaches have changed over recent years with advancements in minimally invasive surgery. A surgeon trained in advanced minimally invasive techniques can now perform pleural-based, pulmonary and mediastinal procedures through multiple thoracoscopic ports without the need for a sub-stantial, rib-spreading incision. Subjective measures of quality of life after VATS, such as pain (Fig. 19-26) and perceived functional recovery, consistently and reproducibly favor VATS OpenVATSNoneMildModerateSeverep<0.001Figure 19-26. Pie chart comparison of pain control at 3 weeks after lobectomy by standard thoracotomy or video-assisted tho-racic surgery (VATS). The pie charts show that

1	19-26. Pie chart comparison of pain control at 3 weeks after lobectomy by standard thoracotomy or video-assisted tho-racic surgery (VATS). The pie charts show that patients undergo-ing VATS have significantly less pain (P <.01) as measured by the most potent analgesic still required: severe—schedule II nar-cotic; moderate—schedule III or lower narcotic; mild–nonsteroidal anti-inflammatory drug (NSAID) or acetaminophen. (Reproduced with permission from Demmy TL, Nwoque C. Is video-assisted thoracic surgery lobectomy better? Quality of life considerations, Ann Thorac Surg. 2008 Feb;85(2):S719-S728.)over thoracotomy. Objective measures such as functional sta-tus as measured by 6-minute walk, return to work, and ability to tolerate chemotherapy also favor VATS over thoracotomy. Finally, recovery of respiratory function occurs earlier in VATS patients. These findings are pronounced in patients with chronic obstructive pulmonary disease and in the elderly—populations whose quality of life

1	of respiratory function occurs earlier in VATS patients. These findings are pronounced in patients with chronic obstructive pulmonary disease and in the elderly—populations whose quality of life can be dramatically impacted by changes in their respiratory symptoms and function, thoracic pain, and physical performance. Table 19-16 provides a summary of pop-ulations that may benefit from VATS approaches.Video-Assisted (VATS)/Robotic-Assisted Thoraco-scopic Surgery (RATS). VATS/RATS has become the rec-ommended approach to diagnosis and treatment of pleural effusions, recurrent pneumothoraces, lung biopsies, lobec-tomy or segmental resection, resection of bronchogenic and mediastinal cysts, and intrathoracic esophageal mobilization for esophagectomy.78 These approaches are also utilized for pneumonectomy in some centers of excellence with very high Table 19-16Special circumstances under which lobectomy by video-assisted thoracic surgery may be preferableCONDITIONEXAMPLESPulmonary

1	for pneumonectomy in some centers of excellence with very high Table 19-16Special circumstances under which lobectomy by video-assisted thoracic surgery may be preferableCONDITIONEXAMPLESPulmonary compromisePoor FEV1/Dlco, heavy smoking, sleep apnea, recent pneumoniaCardiac dysfunctionCongestive heart failure, severe coronary artery disease, recent myocardial infarction, valvular diseaseExtrathoracic malignancySolitary brain metastasis from lung cancer, deep pulmonary metastases requiring lobectomyPoor physical performancePerformance status equivalent to a Zubrod score of 2 or 3, morbid obesityRheumatologic/orthopedic conditionSpinal disease, severe rheumatoid arthritis, severe kyphosis, lupus erythematosus, osteomyelitisAdvanced ageAge >70 yearsVascular problemsAneurysm, severe peripheral vascular diseaseRecent or impending major operationUrgent abdominal operation, joint replacement requiring use of crutches, need for contralateral thoracotomyPsychological/neurologic

1	peripheral vascular diseaseRecent or impending major operationUrgent abdominal operation, joint replacement requiring use of crutches, need for contralateral thoracotomyPsychological/neurologic conditionsSubstance abuse, poor command following, pain syndromesImmunosuppression/ impaired wound healingRecent transplantation, diabetesAbbreviations: Dlco = carbon monoxide diffusion capacity; FEV1 = forced expiratory volume in 1 second.Reproduced with permission from Demmy TL, Nwoque C. Is video-assisted thoracic surgery lobectomy better? Quality of life considerations, Ann Thorac Surg. 2008 Feb;85(2):S719-S728.Brunicardi_Ch19_p0661-p0750.indd 70101/03/19 7:01 PM 702SPECIFIC CONSIDERATIONSPART IIvolumes of VATS lung resection. VATS is performed via two to four incisions measuring 0.5 to 1.2 cm in length to allow insertion of the thoracoscope and instruments. An access incision, typically in the fourth or fifth intercostal space in the anterior axillary line, is used for dissection of

1	cm in length to allow insertion of the thoracoscope and instruments. An access incision, typically in the fourth or fifth intercostal space in the anterior axillary line, is used for dissection of the hilum during lung resection. The incision location varies accord-ing to the procedure. With respect to VATS lobectomy, port placement varies according to the lobe being resected and is highly variable among surgeons.79 The basic principle is to position the ports high enough on the thoracic cage to have access to the hilar structures. Endoscopic staplers are used to divide the major vascular structures and bronchus (Fig. 19-27). Robotic approaches are similarly tailored to the side and lobe under-going resection, with the entire operation performed using DissectBADCERetractRetractRetractRetractRetractViewViewViewViewHoldViewDissectFigure 19-27. Selected video-assisted thoracic surgery lobectomy maneuvers. All the maneuvers are shown with the patient positioned in the left lateral

1	19-27. Selected video-assisted thoracic surgery lobectomy maneuvers. All the maneuvers are shown with the patient positioned in the left lateral decubitus position. The same maneuvers can be performed in mirror image for left-sided work. A. Medial viewing and inferior holding of lung to allow dissection through the access incision. Example shows dissection of the apical hilum. B. Medial viewing and access holding of lung to allow stapling of hilar structures from below. Example shows division of the apical pulmonary artery trunk to the right upper lobe (upper lobe branch of vein divided and reflected away). C. Standard viewing and use of working port to dissect and divide structures while lung is retracted through access incision. Example shows use of stapler to divide pulmonary artery to right lower lobe. D. Standard viewing and use of working port to retract lung and access incision to dissect structures. This method is commonly used to dis-sect the pulmonary artery in the major

1	to right lower lobe. D. Standard viewing and use of working port to retract lung and access incision to dissect structures. This method is commonly used to dis-sect the pulmonary artery in the major fissure. Example shows inferior pulmonary vein after the pulmonary ligament was divided using this maneuver. E. Standard viewing and use of access incision to deliver stapler to divide fissures. Example shows division of the posterior fissure between the right lower lobe and the upper lobe.Brunicardi_Ch19_p0661-p0750.indd 70201/03/19 7:01 PM

1	CHAPTER 19703CHEST WALL, LUNG, MEDIASTINUM, AND PLEURATrapeziusLatissimus dorsidividedLatissimus dorsiBADCSerratus anterior5th rib6th ribRhomboidmajorScapularetractedIncisionTrapeziusrobotic arms, except for one assistant port through which spec-imens can be removed and suctioning and stapling performed (in early models; the newest robotic models have robotic sta-plers, allowing the surgeon to have complete control of the entire operation).Open Approaches to Thoracic Surgery. When a thoraco-scopic approach is not possible, an open approach, most fre-quently the posterolateral thoracotomy, is used to gain access to the intrathoracic space.80,81 The posterolateral thoracotomy incision can be used for most pulmonary resections, esopha-geal operations, and operations in the posterior mediastinum and vertebral column (Fig. 19-28). The anterolateral thora-cotomy has traditionally been used in trauma victims. This approach allows quick entry into the chest with the patient supine. In the

1	and vertebral column (Fig. 19-28). The anterolateral thora-cotomy has traditionally been used in trauma victims. This approach allows quick entry into the chest with the patient supine. In the face of hemodynamic instability, the lateral decubitus position significantly compromises control over the patient’s cardiopulmonary system and resuscitation efforts, whereas the supine position allows the anesthesiologist full access to the patient. A bilateral anterior thoracotomy incision with a transverse sternotomy (“clamshell” thoracotomy) is a standard operative approach to the heart and mediastinum in certain elective circumstances. It is the preferred incision for double-lung transplantation in many centers. A partial median sternotomy can also be added to an anterior thoracotomy (“trap-door” or “hemiclamshell” thoracotomy) for access to mediastinal structures. A hypesthetic nipple is a frequent com-plication of this approach. The median sternotomy incision allows exposure of anterior

1	or “hemiclamshell” thoracotomy) for access to mediastinal structures. A hypesthetic nipple is a frequent com-plication of this approach. The median sternotomy incision allows exposure of anterior mediastinal structures and is prin-cipally used for cardiac operations. Although the surgeon has access to both pleural cavities, incision into the pleural cavity can be avoided if entry is unnecessary (Fig. 19-29).Postoperative CareChest Tube Management. At the conclusion of most thoracic operations, the pleural cavity is drained with a chest tube(s). If the visceral pleura has not been violated and there is no concern Figure 19-28. The posterolateral thoracotomy incision. A. Skin incision from the anterior axillary line to the lower extent of the scapula tip. B and C. Division of the latissimus dorsi and shoulder girdle musculature. D. The pleural cavity is entered after dividing the intercostal muscles along the lower margin of the interspace, taking care not to injure the neurovascular

1	dorsi and shoulder girdle musculature. D. The pleural cavity is entered after dividing the intercostal muscles along the lower margin of the interspace, taking care not to injure the neurovascular bundle lying below each rib.Brunicardi_Ch19_p0661-p0750.indd 70301/03/19 7:01 PM 704SPECIFIC CONSIDERATIONSPART IIABThymusR. atrialappendageR. ventriclePreperitonealfatDiaphragmL. atrialappendageAortic archInnominate v.Pulmonary a.Figure 19-29. The median sternotomy incision. A. Skin incision from the suprasternal notch to the xiphoid process. B. Exposure of the pleural space. a. = artery; v. = vein.for pneumoor hemothorax (e.g., after VATS sympathectomy), a chest tube is unnecessary. After chest tube placement, the lung is reexpanded with positive-pressure ventilation. There are two reasons for the use of pleural tubes in this setting: first, the tube allows evacuation of air if an air leak is present; sec-ond, blood and pleural fluid can be drained, thereby preventing accumulation

1	for the use of pleural tubes in this setting: first, the tube allows evacuation of air if an air leak is present; sec-ond, blood and pleural fluid can be drained, thereby preventing accumulation within the pleural space that would compromise the patient’s respiratory status. The tube is removed when the air leak is resolved and when the volume of drainage decreases below an acceptable level over 24 hours.Historically, many surgeons have somewhat arbitrarily required less than 150 mL of drainage volume over 24 hours prior to removing a chest tube to minimize risk of reaccu-mulation. The pleural lymphatics, however, can absorb up to 0.40 mL/kg per hour in a healthy individual, which may be as much as 500 mL over a 24-hour period. In fact, studies have shown that pleural tubes can be removed after VATS lobec-tomy or thoracotomy with 24-hour drainage volumes as high as 400 mL, without subsequent development of pleural effusions.82 It is our current practice to remove chest tubes with

1	after VATS lobec-tomy or thoracotomy with 24-hour drainage volumes as high as 400 mL, without subsequent development of pleural effusions.82 It is our current practice to remove chest tubes with 24-hour outputs of 400 mL or less after lobectomy or lesser pulmonary resections. In settings where normal pleural fluid dynamics have been altered, such as malignant pleural effusion, pleural space infections or inflammation, and pleurodesis, strict adherence to a volume requirement before tube removal is appropriate (typi-cally 100 to 150 mL over 24 hours).For operations involving lung resection or parenchymal injury, suction levels of –20 cm H2O are routinely used to eradi-cate residual air spaces and to control postoperative parenchy-mal air leaks for the first 12 to 24 hours. The following day, however, the decision to continue suction or place the patient to water seal (off suction) must be made. Applying suction to an air leak has been shown to prolong the duration of the air leak and

1	however, the decision to continue suction or place the patient to water seal (off suction) must be made. Applying suction to an air leak has been shown to prolong the duration of the air leak and extend the time frame during which tube thoracostomy is needed.83 The main guidelines for the continued use of suction if an air leak is present depend on the expansion of the remain-ing lung as determined by CXR. If the lung is well expanded, the chest tube can remain to water seal drainage. If an und-rained pneumothorax is present on CXR, the chest tube and its attached tubing should be examined to ensure that the chest tube is patent and the attached tubing is not kinked or mechanically obstructed, such as occurs when the patient is lying on the tube. If the tube is a small caliber tube (pigtail catheter), it should be flushed with sterile saline through a three-way stopcock that has been cleaned with alcohol because these tubes tend to become clogged with fibrin. These catheters are also

1	catheter), it should be flushed with sterile saline through a three-way stopcock that has been cleaned with alcohol because these tubes tend to become clogged with fibrin. These catheters are also prone to kinking at the insertion site into the skin. Once the surgeon has con-firmed that the chest tube is patent, the patient is asked to volun-tarily cough or perform the Valsalva maneuver. This maneuver increases the intrathoracic pressure and will push air that is contained within the hemithorax out of the chest tube. During the voluntary cough, the fluid level in the water seal chamber should move up and down with the cough and with deep respira-tion, reflecting the pleural pressure changes occurring with these maneuvers. A stationary fluid level implies either a mechanical blockage (e.g., due to external tube compression or to a clot/debris within the tube) or pleurodesis. If bubbles pass through the water seal chamber, an air leak is presumed. If the leak is significant enough to

1	due to external tube compression or to a clot/debris within the tube) or pleurodesis. If bubbles pass through the water seal chamber, an air leak is presumed. If the leak is significant enough to induce atelectasis or collapse of the lung during use of water seal, suction should be used to achieve lung reexpansion.Pain Control. Good pain control after intrathoracic proce-dures is critical; it permits the patient to actively clear and manage secretions and promotes ambulation and a feeling of well-being. The most common techniques of pain management are epidural, paravertebral, and intravenous. Epidural catheters are commonly used, although we prefer to use paravertebral catheters in our center. Epidural catheters should be inserted at about the T6 level, roughly at the level of the scapular tip. Lower placement risks inadequate pain control, and higher placement may provoke hand and arm numbness. Typically, combinations of fentanyl at 0.3 µg/mL with either bupivacaine (0.125%) or

1	tip. Lower placement risks inadequate pain control, and higher placement may provoke hand and arm numbness. Typically, combinations of fentanyl at 0.3 µg/mL with either bupivacaine (0.125%) or ropivacaine (0.1%) are used. Ropivacaine has less cardiotoxicity than bupivacaine; thus, in the case of inadver-tent intravenous injection, the potential for refractory complete heart block is significantly less with ropivacaine. Paravertebral blocks can be placed using the same epidural catheter kit 2.5 cm lateral to the spinous process at T4 to T6. Combinations of narcotic and topical analgesia are then infused as with the epi-dural catheter.When properly placed, a well-managed epidural can provide outstanding pain control without significant systemic sedation.84 Thoracic epidurals do not commonly cause urinary retention, although a low thoracic epidural may block the sen-sory fibers to the bladder. Motor function, however, remains intact. In some patients who are having difficulty voiding,

1	cause urinary retention, although a low thoracic epidural may block the sen-sory fibers to the bladder. Motor function, however, remains intact. In some patients who are having difficulty voiding, it Brunicardi_Ch19_p0661-p0750.indd 70401/03/19 7:01 PM

1	CHAPTER 19705CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAmay be possible to avoid Foley catheterization by simply reminding the patient to void on a regular basis. In male patients with voiding difficulty prior to surgery, urinary catheterization may be required. In addition, the use of local anesthetics may cause sympathetic outflow blockade, leading to vasodilation and hypotension often requiring intravenous vasoconstrictors (an α-agonist such as phenylephrine) and/or fluid administration. In such circumstances, fluid administration for hypotension may be undesirable in pulmonary surgery patients, particularly after pneumonectomy. Paravertebral catheters provide equivalent pain control with less effect on hemodynamics.85 Recently, lipo-somal bupivacaine was introduced and has become the standard approach to pain management after thoracic surgery in several centers. The formulation allows for slow-release of bupivacaine for up to 72 hours after injection. Injected directly into the

1	standard approach to pain management after thoracic surgery in several centers. The formulation allows for slow-release of bupivacaine for up to 72 hours after injection. Injected directly into the inter-costal spaces immediately prior to chest closure, the formulation has shown great promise in several retrospective reports, with randomized trials not yet completed. One limiting factor in use of the formulation is the cost; future trials are needed, with cost-analysis, to determine whether the benefits in pain, reduction in narcotic use, shorter hospital stay and possibly decreased pul-monary complications justify the cost.Alternatively, intravenous narcotics via patient-controlled analgesia can be used, often in conjunction with ketorolac, gabapentin, and intravenous Tylenol. Dosing must be titrated to balance the degree of pain relief with the degree of seda-tion. Oversedated patients are as ominous as patients without adequate pain control because of the significant risk of

1	must be titrated to balance the degree of pain relief with the degree of seda-tion. Oversedated patients are as ominous as patients without adequate pain control because of the significant risk of secre-tion retention, atelectasis/pneumonia, and pulmonary aspiration, especially in elderly patients who should be carefully assessed for aspiration risk when ordered for dietary advancement. Proper pain control with intravenous narcotics requires a care-fully regulated balance of pain relief and sedation to maximize the benefits of pain control while minimizing these very real and potentially life-threatening complications.Whether on epidural, paravertebral, or intravenous pain control, the patient is typically transitioned to oral pain medica-tion on the third or fourth postoperative day. During both the parenteral and oral phase of pain management, a standardized regimen of stool softeners and laxatives is advisable in order to prevent severe constipation.Respiratory Care. The best

1	During both the parenteral and oral phase of pain management, a standardized regimen of stool softeners and laxatives is advisable in order to prevent severe constipation.Respiratory Care. The best respiratory care is achieved when the patient is able to deliver an effective cough to clear secre-tions and results from the commitment and proper training of all involved healthcare providers. The process begins preopera-tively, with clear instructions on using pillows (or other support techniques) over the wound and then applying pressure.Postoperatively, proper pain control without oversedation (as outlined earlier) is essential. Daily morning rounds should include a careful assessment of the patient’s pulmonary status, reminders to the patient and family about the importance of coughing and deep breathing, including use of adjunctive respi-ratory equipment if ordered, and mobilization of the patient. Early transition to a chair and to ambulation is the best respira-tory therapy and

1	and deep breathing, including use of adjunctive respi-ratory equipment if ordered, and mobilization of the patient. Early transition to a chair and to ambulation is the best respira-tory therapy and should be strongly encouraged. When avail-able, physical and/or cardiopulmonary rehabilitation services are vital additional members of the care team.Postoperative ComplicationsPostoperative complications after pulmonary resection range from minor to life-threatening. Strict attention to volume sta-tus, early and aggressive pulmonary toilet, and good pain control can reduce the risk of most complications, but does not completely eliminate them, even in centers of excellence. The most devastating complication after pulmonary resection is postpneumonectomy pulmonary edema, which occurs in 1% to 5% of patients undergoing pneumonectomy and more often after right compared to left pneumonectomy. Clinically, symp-toms of respiratory distress manifest hours to days after sur-gery.

1	occurs in 1% to 5% of patients undergoing pneumonectomy and more often after right compared to left pneumonectomy. Clinically, symp-toms of respiratory distress manifest hours to days after sur-gery. Radiographically, diffuse interstitial infiltration or frank alveolar edema is seen. The pathophysiologic causes are related increased permeability and filtration pressure and decreased lymphatic drainage from the affected lung. Judicious use of intravenous fluids perioperatively, including use of vasopres-sors rather than fluid boluses for hypotension intraand post-operatively, is critical to minimizing the risk of this syndrome. Treatment consists of ventilatory support, fluid restriction, and diuretics. Extracorporeal membrane oxygenation may be life-saving in centers where this option is available. The syndrome reportedly has a nearly 100% mortality rate despite aggressive therapy.Other postoperative complications include air leak and bronchopleural fistula. Although these are two

1	is available. The syndrome reportedly has a nearly 100% mortality rate despite aggressive therapy.Other postoperative complications include air leak and bronchopleural fistula. Although these are two very differ-ent problems, distinguishing between them may be difficult. Postoperative air leaks are common after pulmonary resection, particularly in patients with emphysematous lung, because the fibrosis and destroyed blood supply impairs healing of surface injuries. Prolonged air leaks (i.e., those lasting >5 days) may be treated by diminishing or discontinuing suction (if used), by continuing chest drainage, or by instilling a pleurodesis agent, usually doxycycline or talcum powder, which will cause pleurodesis of the lung within the chest cavity and minimize the possible collapse of the lung due to persistent air leak. This is useful only in patients in whom full lung expansion is achieved, either with suction or on water seal, as patients with a persistent pneumothorax on CXR will

1	lung due to persistent air leak. This is useful only in patients in whom full lung expansion is achieved, either with suction or on water seal, as patients with a persistent pneumothorax on CXR will not have adequate lung-to-parietal pleural apposition to achieve adequate pleurodesis.If the leak is moderate to large, a high index of suspicion for bronchopleural fistula from the resected bronchial stump should be maintained, particularly if the patient is immunocom-promised or had induction chemoand/or radiation therapy. If suspected, flexible bronchoscopy is performed to evaluate the bronchial stump. Management options include continued pro-longed chest tube drainage, reoperation, and reclosure (with stump reinforcement with intercostal or pedicled serratus mus-cle flap). If the fistula is very small (<4 mm), bronchoscopic fibrin glue application has been used successfully to seal the hole in some patients. Patients often have concomitant empy-ema, and open drainage may be

1	fistula is very small (<4 mm), bronchoscopic fibrin glue application has been used successfully to seal the hole in some patients. Patients often have concomitant empy-ema, and open drainage may be necessary.Spontaneous PneumothoraxSpontaneous pneumothorax is secondary to intrinsic abnormali-ties of the lung and can be classified as primary and secondary. Primary spontaneous pneumothorax is defined as a spontaneous pneumothorax without underlying lung disease. The most com-mon cause is rupture of an apical subpleural bleb. The cause of these blebs is unknown, but they occur more frequently in smokers and males, and they tend to predominate in young postadolescent males with a tall thin body habitus. Treatment is generally chest tube insertion with water seal. If a leak is present and persists for greater than 3 days, thoracoscopic man-agement (i.e., bleb resection with pleurodesis by talc or pleural abrasion) is performed. Recurrences or complete lung collapse with the first episode

1	for greater than 3 days, thoracoscopic man-agement (i.e., bleb resection with pleurodesis by talc or pleural abrasion) is performed. Recurrences or complete lung collapse with the first episode are generally indications for thoracoscopic Brunicardi_Ch19_p0661-p0750.indd 70501/03/19 7:01 PM 706SPECIFIC CONSIDERATIONSPART IIintervention.86 Additional indications for intervention on the first episode include occupational hazards such as air travel, deep-sea diving, or travel to remote locations. CT findings of multiple small bullae or a large bleb are associated with an increased risk of recurrent pneumothorax.87 Many surgeons are now using screening CT scan to recommend VATS bleb resection with pleurodesis for first-episode spontaneous pneumothorax.Secondary spontaneous pneumothorax occurs in the set-ting of underlying lung disease, such as emphysema (rupture of a bleb or bulla), cystic fibrosis, acquired immunodeficiency syndrome (AIDS), metastatic cancer (especially sarcoma),

1	occurs in the set-ting of underlying lung disease, such as emphysema (rupture of a bleb or bulla), cystic fibrosis, acquired immunodeficiency syndrome (AIDS), metastatic cancer (especially sarcoma), asthma, lung abscess, and occasionally lung cancer. Catame-nial pneumothorax, a rare but interesting cause of spontaneous pneumothorax in women in their second and third decades, occurs within 72 hours of the onset of menses and is possibil-ity related to endometriosis. Management of pneumothorax in these circumstances is similar to that of primary spontane-ous pneumothorax in that drainage and lung reexpansion are required. Additional therapy, however, is often tied to therapy of the specific disease process and may involve lung resection, thoracoscopic pleurectomy, or talc pleurodesis.Pulmonary InfectionsLung Abscess. A lung abscess is a localized area of pulmonary parenchymal necrosis caused by an infectious organism; tissue destruction results in a solitary or dominant cavity measuring

1	InfectionsLung Abscess. A lung abscess is a localized area of pulmonary parenchymal necrosis caused by an infectious organism; tissue destruction results in a solitary or dominant cavity measuring at least 2 cm in diameter. Less often, there may be multiple, smaller cavities (<2 cm). In that case, the infection is typically referred to as a necrotizing pneumonia. An abscess that is pres-ent for more than 6 weeks is considered chronic.Based on the etiology (Table 19-17), lung abscesses are further classified as primary or secondary. A primary lung abscess occurs, for example, in immunocompromised patients, as a result of highly virulent organisms inciting a necrotizing pulmonary infection, or in patients who have a predisposition to aspirate oropharyngeal or gastrointestinal secretions. A second-ary lung abscess occurs in patients with an underlying condition such as a partial bronchial obstruction, a lung infarct, or adjacent suppurative infections (subphrenic or hepatic

1	A second-ary lung abscess occurs in patients with an underlying condition such as a partial bronchial obstruction, a lung infarct, or adjacent suppurative infections (subphrenic or hepatic abscesses).88Pathogenesis Lung abscesses result when necrotizing micro-organisms infect the lower respiratory tract via inhalation of aerosolized particles, aspiration of oropharyngeal secretions, or hematogenous spread from distant sites. Direct extension from a contiguous site is less frequent. Most primary lung abscesses are suppurative bacterial infections secondary to aspiration. Risk factors for pulmonary aspiration include advanced age, conditions of impaired consciousness, suppressed cough reflex, dysfunctional esophageal motility, laryngopharygeal reflux disease, and centrally acting neurologic diseases (e.g., stroke). At the time of aspiration, the composition of the oropharyn-geal flora determines the etiologic organisms. With increasing use of proton pump inhibitors to suppress acid

1	diseases (e.g., stroke). At the time of aspiration, the composition of the oropharyn-geal flora determines the etiologic organisms. With increasing use of proton pump inhibitors to suppress acid secretion in the stomach, the oropharyngeal flora has shifted, and the risk of developing bacterial lung infections after an aspiration event has increased.89 Secondary lung abscesses occur most often distal to an obstructing bronchial carcinoma. Infected cysts or bullae are not considered true abscesses.Microbiology Normal oropharyngeal secretions contain many more Streptococcus species and more anaerobes (approxi-mately 1 × 108 organisms/mL) than aerobes (approximately 1 × 107 organisms/mL). Pneumonia that follows from Table 19-17Causes of lung abscess I. Primary A. Necrotizing pneumonia 1. Staphylococcus aureus, Klebsiella, Pseudomonas, Mycobacterium 2. Bacteroides, Fusobacterium, Actinomyces 3. Entamoeba, Echinococcus B. Aspiration pneumonia 1. Anesthesia 2. Stroke 3. Drugs or

1	aureus, Klebsiella, Pseudomonas, Mycobacterium 2. Bacteroides, Fusobacterium, Actinomyces 3. Entamoeba, Echinococcus B. Aspiration pneumonia 1. Anesthesia 2. Stroke 3. Drugs or alcohol C. Esophageal disease 1. Achalasia, Zenker’s diverticulum, gastroesophageal reflux D. Immunodeficiency 1. Cancer (and chemotherapy) 2. Diabetes 3. Organ transplantation 4. Steroid therapy 5. Malnutrition II. Secondary A. Bronchial obstruction 1. Neoplasm 2. Foreign body B. Systemic sepsis 1. Septic pulmonary emboli 2. Seeding of pulmonary infarct C. Complication of pulmonary trauma 1. Infection of hematoma or contusion 2. Contaminated foreign body or penetrating injury D. Direct extension from extraparenchymal infection 1. Pleural empyema 2. Mediastinal, hepatic, subphrenic abscessAdapted with permission from Schwartz SI, Fischer JE, Daly JM, et al: Principles of Surgery, 7th ed. New York, NY: McGraw-Hill Education; 1999.aspiration, with or without abscess development, is typically polymicrobial. An

1	from Schwartz SI, Fischer JE, Daly JM, et al: Principles of Surgery, 7th ed. New York, NY: McGraw-Hill Education; 1999.aspiration, with or without abscess development, is typically polymicrobial. An average of two to four isolates present in large numbers have been cultured from lung abscesses sam-pled percutaneously. Overall, at least 50% of these infections are caused by purely anaerobic bacteria, 25% are caused by mixed aerobes and anaerobes, and 25% or fewer are caused by aerobes only. In nosocomial pneumonia, 60% to 70% of the organisms are gram-negative bacteria. Immunosuppressed patients may develop abscesses because of the usual pathogens as well as less virulent and opportunistic organisms such as Salmonella species, Legionella species, Pneumocystis carinii, atypical mycobacteria, and fungi.Clinical Features and Diagnosis The typical presentation may include productive cough, fever (>38.9°C), chills, leuko-cytosis (>15,000 cells/mm3), weight loss, fatigue, malaise, pleu-ritic

1	and fungi.Clinical Features and Diagnosis The typical presentation may include productive cough, fever (>38.9°C), chills, leuko-cytosis (>15,000 cells/mm3), weight loss, fatigue, malaise, pleu-ritic chest pain, and dyspnea. Lung abscesses may also present in a more indolent fashion, with weeks to months of cough, mal-aise, weight loss, low-grade fever, night sweats, leukocytosis, and anemia. After aspiration pneumonia, 1 to 2 weeks typically elapse before cavitation occurs; 40% to 75% of such patients produce putrid, foul-smelling sputum. Severe complications Brunicardi_Ch19_p0661-p0750.indd 70601/03/19 7:01 PM

1	CHAPTER 19707CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAABCFigure 19-30. Lung abscess resulting from emesis and aspi-ration after an alcoholic binge. A. Chest X-ray showing an abscess cavity in the left upper lobe. B. A coronal tomogram highlights the thin wall of the abscess. C. Healing of the abscess cavity after 4 weeks of antibiotic therapy and pos-tural drainage.such as massive hemoptysis, endobronchial spread to other portions of the lungs, rupture into the pleural space and devel-opment of pyopneumothorax, or septic shock and respiratory failure are rare in the modern antibiotic era. The mortality rate is about 5% to 10%, except in the presence of immunosuppres-sion, where rates range from 9% to 28%.The CXR is the primary tool for diagnosing a lung abscess (Fig. 19-30). Its distinguishing characteristic is a density or mass with a relatively thin-walled cavity. An air-fluid level observed within the abscess indicates communication with the tracheobronchial tree. CT scan of the

1	characteristic is a density or mass with a relatively thin-walled cavity. An air-fluid level observed within the abscess indicates communication with the tracheobronchial tree. CT scan of the chest clarifies the diag-nosis when CXR is equivocal and identifies endobronchial obstruction and/or an associated mass and other pathologic anomalies. A cavitating lung carcinoma is frequently mistaken for a lung abscess. Differential diagnosis also includes loculated or interlobar empyema, infected lung cysts or bullae, tuberculo-sis, bronchiectasis, fungal infections, and noninfectious inflam-matory conditions (e.g., Wegener’s granulomatosis).Ideally, the specific etiologic organism is identified before antibiotic administration. Bronchoscopy, which is essential to Brunicardi_Ch19_p0661-p0750.indd 70701/03/19 7:01 PM 708SPECIFIC CONSIDERATIONSPART IIrule out endobronchial obstruction due to tumor or foreign body, is ideal for obtaining uncontaminated cultures using bronchoal-veolar lavage.

1	70701/03/19 7:01 PM 708SPECIFIC CONSIDERATIONSPART IIrule out endobronchial obstruction due to tumor or foreign body, is ideal for obtaining uncontaminated cultures using bronchoal-veolar lavage. Culture samples can also be obtained by percu-taneous, transthoracic FNA under ultrasound or CT guidance. Routine sputum cultures are often of limited usefulness because of contamination with upper respiratory tract flora.Actinomycosis and nocardiosis, although rare, are particu-larly virulent infections associated with lung abscess. Diagnosis can be difficult.90 Both frequently masquerade as other clinical syndromes; thus, it is important for the surgeon to keep these bacteria in mind when considering the differential diagnosis for cavitary lung lesions. Actinomyces, a normal oropharyngeal bacterium, causes extensive pulmonary damage. Actinomyco-sis lung infection typically begins as acute pneumonitis after pulmonary aspiration. The symptoms mimic pulmonary tuber-culosis, including

1	bacterium, causes extensive pulmonary damage. Actinomyco-sis lung infection typically begins as acute pneumonitis after pulmonary aspiration. The symptoms mimic pulmonary tuber-culosis, including chronic cough, night sweats, weight loss, and hemoptysis.Ongoing infection leads to chronic inflammation and fibrosis; cavitation occurs due to destruction of the pulmonary tissues. Without treatment, the infection continues to destroy surrounding structures, resulting in fistula formation to adja-cent structures, including the adjacent lung, interlobar fissures, pleural space, chest wall, and mediastinum. Actinomyces israelii is the most common of the species to cause disease. Nocardi-osis is also a rare opportunistic infection that usually occurs in an immunocompromised host (HIV or cancer patients) and causes both local and systemic suppurative infections. The most common site is pulmonary, caused by Nocardia asteroides in 90% of cases. Infection is slowly progressive, with weight loss,

1	and causes both local and systemic suppurative infections. The most common site is pulmonary, caused by Nocardia asteroides in 90% of cases. Infection is slowly progressive, with weight loss, fatigue, cough, and hemoptysis. An acute pulmonary infec-tion is common, with necrotizing pneumonia and cavitation or slowly enlarging pulmonary nodule. In some cases, empyema also develops.Management of Lung Abscess Systemic antibiotics directed against the causative organism represent the mainstay of therapy. The duration of antimicrobial therapy varies from 3 to 12 weeks for necrotizing pneumonia and lung abscess. It is likely best to treat until the cavity is resolved or until serial radiographs show significant improvement. Parenteral therapy is generally used until the patient is afebrile and able to demonstrate consistent enteral intake. Oral therapy can then be used to complete the course of therapy. For community-acquired infections second-ary to aspiration, likely pathogens are

1	and able to demonstrate consistent enteral intake. Oral therapy can then be used to complete the course of therapy. For community-acquired infections second-ary to aspiration, likely pathogens are oropharyngeal streptococci and anaerobes. Penicillin G, ampicillin, and amoxicillin are the main therapeutic agents, but a β-lactamase inhibitor or metro-nidazole should be added to cover the increasing prevalence of gram-negative anaerobes that produce β-lactamase. Clindamycin is also a primary therapeutic agent. For hospital-acquired infec-tions, Staphylococcus aureus and aerobic gram-negative bacilli are common organisms of the oropharyngeal flora. Piperacillin with a β-lactamase inhibitor (or equivalent alternatives) provide better coverage of likely pathogens.Surgical drainage of lung abscesses is uncommon since drainage usually occurs spontaneously via the tracheobron-chial tree. Indications for intervention are listed in Table 19-18. Drainage and resection may be required for

1	abscesses is uncommon since drainage usually occurs spontaneously via the tracheobron-chial tree. Indications for intervention are listed in Table 19-18. Drainage and resection may be required for actinomycosis and nocardiosis; diagnosis is often delayed because the bacteria are difficult to culture; invasion of the infection into surrounding structures is, therefore, common. Once identified, long-term antibiotics (months to years) are typically required along with drainage, debridement, and resection as needed. While penicil-lin derivatives are effective against most Actinomyces species, the infections are typically polymicrobial, and broad-spectrum parenteral antibiotics may be required. Nocardia species, in contrast, are highly variable; specific identification of the infect-ing species with antibiotic sensitivities is needed to direct appro-priate therapy. Evaluation for malignant spread, particularly to the brain, is also required in the management of nocardiosis, as systemic

1	with antibiotic sensitivities is needed to direct appro-priate therapy. Evaluation for malignant spread, particularly to the brain, is also required in the management of nocardiosis, as systemic dissemination occurs early and frequently.External drainage may be accomplished with tube tho-racostomy, percutaneous drainage, or surgical cavernostomy. The choice between tube thoracostomy versus radiographically guided catheter placement depends on the treating physician’s preference and institutional technical expertise in placing image-guided thoracostomy tubes. Surgical resection is required in fewer than 10% of lung abscess patients. Lobectomy is the preferred intervention for bleeding from a lung abscess or pyo-pneumothorax. An important intraoperative consideration is to protect the contralateral lung with a double-lumen tube, bron-chial blocker, or contralateral main stem intubation. Surgical treatment has a 90% success rate, with associated mortality of 1% to

1	is to protect the contralateral lung with a double-lumen tube, bron-chial blocker, or contralateral main stem intubation. Surgical treatment has a 90% success rate, with associated mortality of 1% to 13%.Bronchiectasis. Bronchiectasis is defined as a pathologic and permanent dilation of bronchi with bronchial wall thickening. This condition may be localized to certain bronchial segments, or it may be diffuse throughout the bronchial tree, typically affecting the medium-sized airways. Overall, this is a rare clini-cal entity in the United States with a prevalence of less than 1 in 10,000, although the incidence has increased in recent years and noncystic fibrosis–related bronchiectasis is now thought to affect 27.5 out of every 10,000 persons over age 75.Pathogenesis Development of bronchiectasis can be attributed to either congenital or acquired causes. The principal congeni-tal diseases that lead to bronchiectasis include cystic fibrosis, primary ciliary dyskinesia, and

1	of bronchiectasis can be attributed to either congenital or acquired causes. The principal congeni-tal diseases that lead to bronchiectasis include cystic fibrosis, primary ciliary dyskinesia, and immunoglobulin deficiencies (e.g., selective IgA deficiency). Congenital causes commonly produce a diffuse pattern of bronchial involvement. Acquired causes are categorized broadly as infectious and inflammatory. Bronchial obstruction from cancer, inhaled objects, extrinsic airway compression, or inspissated sputum promotes localized infection and subsequent medium airway destruction. Diffuse pneumonic processes from pathogens including necrotizing bacterial pneumonia, pertussis and measles pneumonia, severe influenza, or varicella pneumonia can lead to widespread bron-chiectasis. Chronic granulomatous disease, immunodeficiency disorders, and hypersensitivity disorders can also lead to diffuse bronchiectasis.Noninfectious causes of bronchiectasis include inhala-tion of toxic gases such as

1	disease, immunodeficiency disorders, and hypersensitivity disorders can also lead to diffuse bronchiectasis.Noninfectious causes of bronchiectasis include inhala-tion of toxic gases such as ammonia, which results in severe Table 19-18Indications for surgical drainage procedures for lung abscesses 1. Failure of medical therapy 2. Abscess under tension 3. Abscess increasing in size during appropriate treatment 4. Contralateral lung contamination 5. Abscess >4–6 cm in diameter 6. Necrotizing infection with multiple abscesses, hemoptysis, abscess rupture, or pyopneumothorax 7. Inability to exclude a cavitating carcinomaBrunicardi_Ch19_p0661-p0750.indd 70801/03/19 7:01 PM

1	CHAPTER 19709CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAFigure 19-31. Multiple cystic-type bronchiectatic cavities can be seen on a cut section of right lower lobe lung.and destructive airway inflammatory responses. Allergic bronchopulmonary aspergillosis, Sjögren’s syndrome, and α1-antitrypsin deficiency are some additional examples of presumed immunologic disorders that may be accompanied by bronchiectasis.In addition, recent studies have suggested an association between chronic gastroesophageal reflux disease, acid sup-pression, and nontuberculous mycobacterial infection with bronchiectasis.91,92 This interaction is thought to be related to chronic aspiration of colonized gastric secretions in the setting of acid suppression; while not proven to be causative, these findings suggest a role for gastroesophageal reflux dis-ease in the pathogenesis of bronchiectasis. The process shared by all causes of bronchiectasis is impairment of airway defenses or deficits in immunologic mechanisms,

1	for gastroesophageal reflux dis-ease in the pathogenesis of bronchiectasis. The process shared by all causes of bronchiectasis is impairment of airway defenses or deficits in immunologic mechanisms, which permit bacterial colonization and chronic infection. Common organisms include Haemophilus species (55%), Pseudomonas species (26%), and Streptococcus pneu-moniae (12%).93 Both the bacterial organisms and the inflamma-tory cells recruited to thwart the bacteria elaborate proteolytic and oxidative molecules, which progressively destroy the mus-cular and elastic components of the airway walls; those compo-nents are then replaced by fibrous tissue. Thus, chronic airway inflammation is the essential pathologic feature of bronchiec-tasis. The dilated airways are usually filled with thick purulent material; more distal airways are often occluded by secretions or obliterated by fibrous tissue. Bronchial wall vascularity increases, bronchial arteries become hypertrophied, and abnor-mal

1	purulent material; more distal airways are often occluded by secretions or obliterated by fibrous tissue. Bronchial wall vascularity increases, bronchial arteries become hypertrophied, and abnor-mal anastomoses form between the bronchial and pulmonary arterial circulation.There are three principal types of bronchiectasis, based on pathologic morphology: cylindrical—uniformly dilated bronchi; varicose—an irregular or beaded pattern of dilated bronchi; and saccular (cystic)—peripheral balloon-type bronchial dilation. The saccular type is the most common after bronchial obstruc-tion or infection (Fig. 19-31).Clinical Manifestations and Diagnosis Typical symptoms are a daily persistent cough and purulent sputum production; the quantity of daily sputum production (10 mL to >150 mL) corre-lates with disease extent and severity. Other patients may appear asymptomatic or have a dry nonproductive cough (“dry bronchi-ectasis”). These patients are prone to have involvement of the upper lobes.

1	with disease extent and severity. Other patients may appear asymptomatic or have a dry nonproductive cough (“dry bronchi-ectasis”). These patients are prone to have involvement of the upper lobes. The clinical course is characterized by progressive 9symptoms and respiratory impairment. Increasing resting and exertional dyspnea are the result of progressive airway obstruc-tion. Acute exacerbations may be triggered by viral or bacterial pathogens. Bleeding attributable to chronically inflamed, friable airway mucosa causes increasingly more frequent hemoptysis with disease progression. Massive bleeding may result from ero-sion of the hypertrophied bronchial arteries.Both mild and severe forms of bronchiectasis are read-ily demonstrated with chest CT scanning because it provides a highly detailed, cross-sectional view of bronchial architecture. CXRs, although less sensitive, may reveal characteristic signs of bronchiectasis such as lung hyperinflation, bronchiectatic cysts, and dilated,

1	cross-sectional view of bronchial architecture. CXRs, although less sensitive, may reveal characteristic signs of bronchiectasis such as lung hyperinflation, bronchiectatic cysts, and dilated, thick-walled bronchi forming train track–like pat-terns radiating from the lung hila. Sputum culture may identify characteristic pathogens.Sputum acid-fast bacillus smears/cultures should be performed to evaluate for nontuberculous mycobacteria, which is common in this setting. Spirometry provides assessment of the severity of airway obstruction and can be followed to track the course of disease. Management of Bronchiectasis Standard therapy includes optimizing airway clearance, use of bronchodilators to reverse any airflow limitation, and correction of reversible underly-ing causes whenever possible.94 Chest physiotherapy based on vibration, percussion, and postural drainage is widely accepted, although randomized trials demonstrating efficacy are lacking. Acute exacerbations should be treated

1	Chest physiotherapy based on vibration, percussion, and postural drainage is widely accepted, although randomized trials demonstrating efficacy are lacking. Acute exacerbations should be treated with a 2to 3-week course of broad-spectrum intravenous antibiotics tailored to culture and sensitivity profiles, followed by an oral regimen; this will result in a longer-lasting remission.Macrolide antibiotics have been shown to decrease sputum production, inhibit cytokine release, and inhibit neutrophil adhe-sion and formation of reactive oxygen species. They also inhibit migration of Pseudomonas, disrupt biofilm, and prevent release of virulence factors.95 While macrolide therapy does appear to be efficacious, it is important to remember that macrolides have sig-nificant activity against nontuberculous mycobacteria, and wide-spread prophylactic use for patients with bronchiectasis may lead to multidrug-resistant nontuberculous mycobacterial species. It has also been suggested that inhaled

1	mycobacteria, and wide-spread prophylactic use for patients with bronchiectasis may lead to multidrug-resistant nontuberculous mycobacterial species. It has also been suggested that inhaled antibiotics, such as tobramy-cin and colistin, improve rates of bacterial clearance and slow the decline in pulmonary function associated with bronchiectasis. Meta-analysis of 12 randomized trials involving 1154 patients showed efficacy of inhaled antibiotics compared to controls, but did not reduce risk of acute exacerbation. Quality of life, like-wise, was not improved and antibiotic-resistant Pseudomonas rates were similar. Importantly, inhaled antibiotics were associ-ated with a statistically significant reduction in forced expiratory volume in 1 second measures over time.96,97In addition to antibiotics, daily nebulized hypertonic saline appears to be effective. A recent randomized crossover study comparing lung function and quality of life has shown that 7% normal saline, compared to isotonic

1	daily nebulized hypertonic saline appears to be effective. A recent randomized crossover study comparing lung function and quality of life has shown that 7% normal saline, compared to isotonic saline, results in a statisti-cally significant 15% increase in FEV1 and an 11% increase in forced vital capacity (compared to 1.8% and 0.7%, respec-tively, with isotonic saline). Antibiotic use and emergency room utilization were significantly decreased; from this, hypertonic saline appears to be a reasonable adjunct to maintaining quality of life and decreasing exacerbations by reducing sputum vol-ume, improving mucociliary clearance, and slowing the decline in lung function.98 Studies supporting mucolytics such as DNase and Mucomyst for non–cystic fibrosis bronchiectasis have Brunicardi_Ch19_p0661-p0750.indd 70901/03/19 7:01 PM 710SPECIFIC CONSIDERATIONSPART IIshown either no change or a worsening of pulmonary status and require further study in the non–cystic fibrosis population.Surgical

1	70901/03/19 7:01 PM 710SPECIFIC CONSIDERATIONSPART IIshown either no change or a worsening of pulmonary status and require further study in the non–cystic fibrosis population.Surgical resection of a localized bronchiectatic segment or lobe, preserving as much functional lung as possible, may bene-fit patients with refractory symptoms while on maximal medical therapy. Multifocal disease must be excluded before any attempt at surgery; any uncorrectable predisposing factor (e.g., ciliary dyskinesia) must also be excluded. Patients with end-stage lung disease from bronchiectasis may be potential candidates for a bilateral lung transplant. Surgical resection is also indicated in patients with significant hemoptysis, although bronchial artery embolization is the preferred first option. Antireflux surgery may also prove beneficial in patients with chronic aspiration, but further studies are required. It is particularly important to recognize that antireflux surgery in patients with

1	Antireflux surgery may also prove beneficial in patients with chronic aspiration, but further studies are required. It is particularly important to recognize that antireflux surgery in patients with severe under-lying pulmonary dysfunction has higher risk for perioperative adverse outcomes than in the general population. It should be undertaken only by very experienced surgeons with direct involvement of the pulmonary medicine physicians to minimize postoperative pulmonary compromise.Mycobacterial Infections Epidemiology Tuberculosis is a widespread problem that affects nearly one-third of the world’s population. It is the ninth lead-ing cause of death worldwide and the leading infectious cause of death. The rate of death from tuberculosis has declined, from 1.7 million in 2000 to 1.3 million among HIV-negative people in 2016. There were 6.3 million new cases of tubercu-losis worldwide in 2016 according to the World Health Orga-nization (WHO); 56% are in the countries of India,

1	million among HIV-negative people in 2016. There were 6.3 million new cases of tubercu-losis worldwide in 2016 according to the World Health Orga-nization (WHO); 56% are in the countries of India, Indonesia, China, the Philippines, and Pakistan. Treatment success rate was 83%. Only 9257 new cases were reported to the WHO in the United States in 2016. HIV infection is the strongest risk fac-tor for developing active tuberculosis. The elderly, minorities, and recent immigrants are the most common populations to have clinical manifestations of infection, yet no age group, sex, or race is exempt from infection. In most large urban centers, reported cases of tuberculosis are more numerous among the homeless, prisoners, and drug-addicted populations. Immunocompromised patients additionally contribute to an increased incidence of tuber-culosis infection, often developing unusual systemic as well as pulmonary manifestations.99 As compared with past decades, presently surgical intervention is

1	to an increased incidence of tuber-culosis infection, often developing unusual systemic as well as pulmonary manifestations.99 As compared with past decades, presently surgical intervention is required more frequently in patients with multidrug-resistant or rifampicin-resistant (but isoniazid-susceptible) tuberculosis organisms (MDR/RR-TB) who do not respond to medical treatment and in selected patients with nontuberculous mycobacterial infections (NTM).Microbiology Mycobacterial species are obligate aerobes. They are primarily intracellular parasites with slow rates of growth. Their defining characteristic is the property of acid-fastness, which is the ability to withstand decolorization by an acidalcohol mixture after being stained. Mycobacterium tuberculosis is the highly virulent bacillus of this species that produces invasive infection among humans, principally pulmonary tuberculosis.100 Infections with M tuberculosis are primary when they are the first infection in a previously

1	of this species that produces invasive infection among humans, principally pulmonary tuberculosis.100 Infections with M tuberculosis are primary when they are the first infection in a previously unsensitized host and secondary or postprimary when reactivation of a previous infection occurs.Because of improper application of antimycobacterial drugs and multifactorial interactions, MDR-TB organisms, defined by their resistance to at least two of the first-line anti-mycobacterial drugs (isoniazid and rifampin), and rifampicin-resistant (but isoniazid-susceptible) (RR-TB), have emerged. According to the WHO Global Tuberculosis Report 2017, in 2014, there were 108 reported cases of TB from MDR/RR-TB organisms, with 78% of cases successfully treated. In addition, there is another rare variant termed extensively drug-resistant tuberculosis (XDR-TB). These organisms are resistant to iso-niazid and rifampin and have also developed resistance to either fluoroquinolones and injectable

1	variant termed extensively drug-resistant tuberculosis (XDR-TB). These organisms are resistant to iso-niazid and rifampin and have also developed resistance to either fluoroquinolones and injectable second-line drugs (e.g., capreomycin, amikacin, kanamycin), the two other classes of medications in the MDR-TB treatment regimen. In 2014, there were 109,680 MDRTB cases globally, with 6777 (6.2%) extensively drug resistant. Successful treatment was achieved in 54% of MDRTB and only 30% of XDR-TB. In 2016, it was estimated that MDR/RR was the responsible organism for more than 4% of new cases and nearly 20% of previously treated cases.The more important NTM organisms include Mycobacte-rium kansasii, M avium and M intracellulare complex (MAC), and M fortuitum. The highest incidence of M kansasii infection is in Midwestern U.S. cities among middle-aged men from good socioeconomic surroundings. MAC organisms are important infections in elderly and immunocompromised patient groups. M fortuitum

1	infection is in Midwestern U.S. cities among middle-aged men from good socioeconomic surroundings. MAC organisms are important infections in elderly and immunocompromised patient groups. M fortuitum infections are common complications of underly-ing severe debilitating disease. None of these organisms are as contagious as M tuberculosis.Pathogenesis and Pathology The main route of transmission is via airborne inhalation of viable mycobacteria. Three stages of primary infection have been described. In the first stage, alveolar macrophages become infected through ingesting the bacilli. In the second stage, from days 7 to 21, the patient typi-cally remains asymptomatic while the bacteria multiply within the infected macrophages. The third stage is characterized by the onset of cell-mediated immunity (CD4+ helper T cells) and delayed-type hypersensitivity. Activated macrophages acquire an increased capacity for bacterial killing. Macrophage death increases, resulting in the formation of a

1	(CD4+ helper T cells) and delayed-type hypersensitivity. Activated macrophages acquire an increased capacity for bacterial killing. Macrophage death increases, resulting in the formation of a granuloma, the charac-teristic lesion found on pathologic examination.Tuberculous granulomas are composed of blood-derived macrophages, degenerating macrophages or epithelioid cells, and multinucleated giant cells (fused macrophages with nuclei around the periphery; also known as Langerhans cells). The low oxygen content of this environment inhibits macrophage func-tion and bacillary growth, with subsequent central caseation as macrophage death occurs. A Ghon complex is a single, small lung lesion that is often the only remaining trace of a primary infection. The primary infection is usually located in the periph-eral portion of the middle zone of the lungs.Reactivation tuberculosis may occur after hydrolytic enzymes liquefy the caseum. Typically, the apical and posterior segments of the upper

1	in the periph-eral portion of the middle zone of the lungs.Reactivation tuberculosis may occur after hydrolytic enzymes liquefy the caseum. Typically, the apical and posterior segments of the upper lobes and the superior segments of the lower lobes are involved. Edema, hemorrhage, and mononuclear cell infiltration are also present. The tuberculous cavity may become secondarily infected with other bacteria, fungi, or yeasts, all of which may con-tribute to enhanced tissue destruction.The pathologic changes caused by NTM organisms are similar to those produced by M tuberculosis. M intracellulare complex infections commonly occur, not only in immunocom-promised patients but also in patients with previously damaged lungs. Caseous necrosis is uncommon and is characterized by clusters of tissue macrophages filled with mycobacteria. It has a poor granulomatous response and confinement of immune cell infiltration to the interstitium and alveolar walls. Cavitary dis-ease is infrequent,

1	tissue macrophages filled with mycobacteria. It has a poor granulomatous response and confinement of immune cell infiltration to the interstitium and alveolar walls. Cavitary dis-ease is infrequent, although nodules may be noted.Brunicardi_Ch19_p0661-p0750.indd 71001/03/19 7:01 PM

1	CHAPTER 19711CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAClinical Presentation and Diagnosis The clinical course of infection and the presentation of symptoms are influenced by many factors, including the site of primary infection, the stage of disease, and the degree of cell-mediated immunity. About 80% to 90% of tuberculosis patients present with clinical dis-ease in the lungs. In 85% to 90% of these patients, involution and healing occur, leading to a dormant phase that may last a lifetime. The only evidence of tuberculosis infection may be a positive skin reaction to tuberculin challenge or a Ghon com-plex observed on CXR. Within the first 2 years of primary infec-tion, reactivation may occur in up to 10% to 15% of infected patients. In 80%, reactivation occurs in the lungs; other reacti-vation sites include the lymph nodes, pleura, and the musculo-skeletal system.After primary infection, pulmonary tuberculosis is fre-quently asymptomatic. Systemic symptoms of low-grade fever,

1	reacti-vation sites include the lymph nodes, pleura, and the musculo-skeletal system.After primary infection, pulmonary tuberculosis is fre-quently asymptomatic. Systemic symptoms of low-grade fever, malaise, and weight loss are subtle and may go unnoticed. A productive cough may develop, usually after tubercle cavita-tion. Many radiographic patterns can be identified at this stage, including local exudative lesions, local fibrotic lesions, cavita-tion, bronchial wall involvement, acute tuberculous pneumonia, bronchiectasis, bronchostenosis, and tuberculous granulomas. Hemoptysis often develops from complications of disease such as bronchiectasis or erosion into vascular malformations associ-ated with cavitation.Extrapulmonary involvement is due to hematogenous or lymphatic spread from pulmonary lesions. Virtually any organ can become infected, giving rise to the protean manifestations of tuberculosis. The pleura, chest wall, and mediastinal organs may all be involved. More than

1	pulmonary lesions. Virtually any organ can become infected, giving rise to the protean manifestations of tuberculosis. The pleura, chest wall, and mediastinal organs may all be involved. More than one-third of immunocompro-mised patients have disseminated disease, with hepatomegaly, diarrhea, splenomegaly, and abdominal pain.The definitive diagnosis of tuberculosis requires identi-fication of the mycobacterium in a patient’s bodily fluids or involved tissues. Skin testing using purified protein deriva-tive is important for epidemiologic purposes and can help exclude infection in uncomplicated cases. For pulmonary tuberculosis, sputum examination is inexpensive and has a high diagnostic yield.Bronchoscopy with alveolar lavage may also be a useful diagnostic adjunct and has high diagnostic accuracy. Chest CT scan can delineate the extent of parenchymal disease.Management Medical therapy is the primary treatment of pul-monary tuberculosis and is often initiated before a mycobacte-rial

1	accuracy. Chest CT scan can delineate the extent of parenchymal disease.Management Medical therapy is the primary treatment of pul-monary tuberculosis and is often initiated before a mycobacte-rial pathogen is definitively identified. Combinations of two or more drugs are routinely used in order to minimize resistance, which inevitably develops with only single-agent therapy. A current treatment algorithm is outlined in Fig. 19-32. Gener-ally, therapy lasts about 26 weeks (2 months intensive therapy followed by 4 months continuation therapy). A 7-month con-tinuation phase should be considered for patients with cavitary pulmonary TB who remain positive on sputum culture after the 2-month intensive therapy, those patients who did not receive pyrazinamide during the intensive phase, HIV-positive patients who did not receive concomitant antiretroviral therapy, and patients treated with INH and rifapentine once weekly (not rec-ommended) who have persistent positive sputum after 2 months of

1	patients who did not receive concomitant antiretroviral therapy, and patients treated with INH and rifapentine once weekly (not rec-ommended) who have persistent positive sputum after 2 months of intensive therapy.In the United States, surgical intervention is most often required in order to treat patients with MDR/RR-TB organ-isms whose lungs have been destroyed and who have persis-tent thick-walled cavitation.101 The indications for surgery related to mycobacterial pulmonary infections are presented in Table 19-19. The governing principle of mycobacterial surgery is to remove all gross disease while preserving any uninvolved lung tissue. Scattered nodular disease may be left intact, given its low mycobacterial burden. Antimycobacterial medications should be given preoperatively (for about 3 months) and con-tinued postoperatively for 12 to 24 months. Overall, more than 90% of patients who were deemed good surgical candidates are cured when appropriate medical and surgical therapy is

1	3 months) and con-tinued postoperatively for 12 to 24 months. Overall, more than 90% of patients who were deemed good surgical candidates are cured when appropriate medical and surgical therapy is used.Pulmonary Fungal Infections. The incidence of fungal infections has increased significantly, with many new opportu-nistic fungi emerging. This increase is attributed to the growing population of immunocompromised patients (e.g., organ trans-plant recipients, cancer patients undergoing chemotherapy, HIV patients, and young and elderly patients) who are more likely to become infected with fungi.102 Clinically significant examples include species of Aspergillus, Cryptococcus, Candida, and Mucor. Other at-risk patient populations include those who are malnourished, severely debilitated, or diabetic or who have hematologic disorders.Patients receiving high-dose, intensive antibiotic therapies are also susceptible. There are, however, some fungi that are primary or true pathogens, able to

1	or who have hematologic disorders.Patients receiving high-dose, intensive antibiotic therapies are also susceptible. There are, however, some fungi that are primary or true pathogens, able to cause infections in other-wise healthy patients. Some endemic examples in the United States include species of Histoplasma, Coccidioides, and Blastomyces.103Direct identification of the organism in body exudates or tissues, preferably as growth in culture, provides definitive diag-nosis. Serologic testing to identify mycotic-specific antibodies may also be useful. Several new classes of antifungal agents have proven effective against many life-threatening fungi and are less toxic than older agents. In addition, thoracic surgery may be a useful therapeutic adjunct for patients with pulmonary mycoses.Aspergillosis The genus Aspergillus comprises over 150 spe-cies and is the most common cause of mortality due to inva-sive mycoses in the United States. It is typically acute in onset and

1	mycoses.Aspergillosis The genus Aspergillus comprises over 150 spe-cies and is the most common cause of mortality due to inva-sive mycoses in the United States. It is typically acute in onset and life-threatening and occurs in the setting of neutropenia, chronic steroid therapy, or cytotoxic chemotherapy. It can also occur in the general intensive care unit population of critically ill patients, including patients with underlying chronic obstruc-tive pulmonary disease (COPD), postoperative patients, patients with cirrhosis or alcoholism, and postinfluenza patients, with-out any of these factors present. The species most commonly responsible for clinical disease include A fumigatus, A flavus, A niger, and A terreus. Aspergillus is a saprophytic, filamentous fungus with septate hyphae. Spores (2.5 to 3 µm in diameter) are released and easily inhaled by susceptible patients; because the spores are microns in size, they are able to reach the distal bronchi and alveoli.Diagnosis of

1	Spores (2.5 to 3 µm in diameter) are released and easily inhaled by susceptible patients; because the spores are microns in size, they are able to reach the distal bronchi and alveoli.Diagnosis of aspergillosis requires one or more cavities on lung imaging, with or without fungal ball or nodules, micros-copy or culture positive for Aspergillus, or antibodies (precipi-tins) to Aspergillus on serum testing. The characteristics must have been present for 3 months. Aspergillus can manifest as one of three clinical syndromes: Aspergillus hypersensitivity lung disease, aspergilloma, or invasive pulmonary aspergil-losis. Overlap occurs between these syndromes, depending on the patient’s immune status.104 Aspergillus hypersensitiv-ity manifests as a productive cough, fever, wheezing, pulmo-nary infiltrates, eosinophilia, and elevation of IgE antibodies Brunicardi_Ch19_p0661-p0750.indd 71101/03/19 7:01 PM 712SPECIFIC

1	manifests as a productive cough, fever, wheezing, pulmo-nary infiltrates, eosinophilia, and elevation of IgE antibodies Brunicardi_Ch19_p0661-p0750.indd 71101/03/19 7:01 PM 712SPECIFIC CONSIDERATIONSPART IIINH/RIFINH/RIFINH/RIFINH/RIFINH/RIF/EMB*/PZA†0123469Time (months)INH/RPT‡§INH/RIF2-monthculture negativeHigh clinicalsuspicionfor activetuberculosis2-monthculture positiveCavitation on CXRorpositive AFB smearat 2 monthsNo cavitation on CXRandnegative AFB smearat 2 monthsNo cavitationCavitationFigure 19-32. Treatment algorithm for tuberculosis. Patients in whom tuberculosis is proven or strongly suspected should have treatment initiated with isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) for the initial 2 months. A repeat smear and cul-ture should be performed when 2 months of treatment has been completed. If cavities were seen on the initial chest radiograph (CXR) or the acid-fast bacillus (AFB) smear results are positive at completion of 2 months of

1	when 2 months of treatment has been completed. If cavities were seen on the initial chest radiograph (CXR) or the acid-fast bacillus (AFB) smear results are positive at completion of 2 months of treatment, the continuation phase of treatment should consist of INH and RIF daily or twice daily for 4 months to complete a total of 6 months of treatment. If cavitation was present on the initial CXR and the culture results at the time of completion of 2 months of therapy are positive, the continuation phase should be lengthened to 7 months (total of 9 months of treatment). If the patient has HIV infection and the CD4+ cell count is <100/µL, the continuation phase should consist of daily or three times weekly INH and RIF. In HIV-uninfected patients with no cavitation on CXR and negative results on AFB smears at completion of 2 months of treatment, the continuation phase may consist of either once weekly INH and rifapentine (RPT) or daily or twice weekly INH and RIF to complete a total of 6

1	on AFB smears at completion of 2 months of treatment, the continuation phase may consist of either once weekly INH and rifapentine (RPT) or daily or twice weekly INH and RIF to complete a total of 6 months of treatment (bottom). For patients receiving INH and RPT whose 2-month culture results are positive, treatment should be extended by an additional 3 months (total of 9 months). *EMB may be discontinued when results of drug susceptibility testing indicate no drug resistance. †PZA may be discontinued after it has been taken for 2 months (56 doses). ‡RPT should not be used in HIV-infected patients with tuberculosis or in patients with extrapulmonary tuberculosis. §Therapy should be extended to 9 months if results of 2-month culture are positive. (Reproduced with permission from Blumberg HM, Burman WJ, Chaisson RE, et al. American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America: treatment of tuberculosis, Am J Respir Crit Care Med.

1	HM, Burman WJ, Chaisson RE, et al. American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America: treatment of tuberculosis, Am J Respir Crit Care Med. 2003 Feb 15;167(4):603-662.)Table 19-19Indications for surgery to treat mycobacterial pulmonary infections 1. Complications resulting from previous thoracic surgery to treat tuberculosis 2. Failure of optimized medical therapy (e.g., progressive disease, lung gangrene, or intracavitary aspergillosis superinfection) 3. Need for tissue acquisition for definitive diagnosis 4. Complications of pulmonary scarring (e.g., massive hemoptysis, cavernomas, bronchiectasis, or bronchostenosis) 5. Extrapulmonary thoracic involvement 6. Pleural tuberculosis 7. Nontuberculous mycobacterial infectionto Aspergillus, whereas aspergilloma (fungal ball) is a matted sphere of hyphae, fibrin, and inflammatory cells that tends to colonize preexisting intrapulmonary cavities. Grossly, aspergil-loma appears as a

1	whereas aspergilloma (fungal ball) is a matted sphere of hyphae, fibrin, and inflammatory cells that tends to colonize preexisting intrapulmonary cavities. Grossly, aspergil-loma appears as a round or oval, friable, gray (or red, brown, or even yellow), necrotic-looking mass (Fig. 19-33). This form is the most common presentation of noninvasive pulmonary asper-gillosis. The most common symptoms are hemoptysis, chronic and productive cough, clubbing, malaise, or weight loss. CXR can suggest the diagnosis by the finding of a crescentic radiolu-cency above a rounded radiopaque lesion (Monad sign).The natural history varies greatly between patients and, therefore, treatment is individualized. Factors associated with poor prognosis include severe underlying pulmonary disease, growth in the number or size of the aspergilloma(s) during observation, immunosuppression or HIV infection, history of lung transplantation, chronic pulmonary sarcoidosis, and increasing Aspergillus-specific IgG

1	number or size of the aspergilloma(s) during observation, immunosuppression or HIV infection, history of lung transplantation, chronic pulmonary sarcoidosis, and increasing Aspergillus-specific IgG titers. 10Brunicardi_Ch19_p0661-p0750.indd 71201/03/19 7:01 PM

1	CHAPTER 19713CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAACBFigure 19-33. Pulmonary aspergilloma. A. The chest X-ray shows a solid mass within a cavity surrounded by a rim of air between the mass and cavity wall (Monad sign, arrows). B. A cut section shows the “fungus ball” occupying an old, fibrotic cavity. C. Histologic stain reveals characteristic Aspergillus hyphae invading the wall of the cavity.Asymptomatic patients can be observed without any additional therapy. Antifungals have limited utility due to the poor blood supply to the aspergilloma. Oral triazole therapy is now consid-ered the standard of care for chronic, cavitary pulmonary asper-gillosis. Hemoptysis is a harbinger of erosion of the disease into adjacent bronchial arteries and typically requires intervention. In the setting of very mild hemoptysis (e.g., blood-streaked spu-tum), cough suppression is warranted while further therapeutic evaluation is performed.Bronchial artery embolization is the first-line therapy for

1	of very mild hemoptysis (e.g., blood-streaked spu-tum), cough suppression is warranted while further therapeutic evaluation is performed.Bronchial artery embolization is the first-line therapy for massive hemoptysis and may be definitive therapy.105 This is particularly important to consider for patients with severely impaired pulmonary function who may not have sufficient reserve to tolerate even a very small pulmonary resection. Operative intervention may be required for recur-rent hemoptysis, particularly after bronchial artery emboli-zation, chronic cough with systemic symptoms, progressive infiltrate around the mycetoma, and a pulmonary mass of unknown cause.106When operative intervention is indicated, the surgeon must remain cognizant of the goals of the procedure. In the setting of simple aspergilloma, VATs wedge resection is pre-ferred. As this disease typically occurs in patients with signifi-cantly impaired pulmonary function, attempts should be made to excise all diseased

1	simple aspergilloma, VATs wedge resection is pre-ferred. As this disease typically occurs in patients with signifi-cantly impaired pulmonary function, attempts should be made to excise all diseased tissue with as limited a resection as pos-sible. Once resection is completed, the postresection space in the hemithorax should be obliterated with a pleural tent, pneu-moperitoneum, decortication of the remaining lung, intratho-racic rotation of a muscle or omental flap, or thoracoplasty. If completely resected for single aspergilloma, antifungal therapy is not needed. If multiple nodules are present or the disease is incompletely resected, however, antifungal therapy should be considered. Long-term follow-up is necessary, given that the recurrence rate after surgery is about 7%.Brunicardi_Ch19_p0661-p0750.indd 71301/03/19 7:01 PM 714SPECIFIC CONSIDERATIONSPART IIInvasive pulmonary aspergillosis typically affects immu-nocompromised patients who have dysfunctional cellular immu-nity,

1	71301/03/19 7:01 PM 714SPECIFIC CONSIDERATIONSPART IIInvasive pulmonary aspergillosis typically affects immu-nocompromised patients who have dysfunctional cellular immu-nity, namely defective polymorphonuclear leukocytes. Invasion of pulmonary parenchyma and blood vessels by a necrotizing bronchopneumonia may be complicated by thrombosis, hemor-rhage, and then dissemination. Patients present with fever that is nonresponsive to antibiotic therapy in the setting of neutro-penia. They may also have pleuritic chest pain, cough, dys-pnea, or hemoptysis. Characteristic signs on CT scan include the halo sign and cavitary lesions. Treatment with voriconazole must be prompt and aggressive, including reversal of neutrope-nia, if there is to be any chance for recovery. Mortality ranges from 93% to 100% in bone marrow transplant recipients, to approximately 38% in kidney transplant recipients, although this improves to approximately 60% at 12 weeks with antifun-gal therapy. Several other

1	93% to 100% in bone marrow transplant recipients, to approximately 38% in kidney transplant recipients, although this improves to approximately 60% at 12 weeks with antifun-gal therapy. Several other advances in diagnosis and treatment, including CT scans in high-risk populations and development of additional triazoles and echinocandins, have improved the early identification and response to therapy in this patient population. Additional treatment considerations include the use of hema-topoietic growth factors to minimize the neutropenic period, which contributes to uncontrolled disease. Surgical removal of the infectious nidus is advocated by some groups because medical treatment has such poor outcomes. Treatment continues until microbiologic clearance is achieved and clinical signs and radiographic imaging indicate resolution of disease. In addition, the patient should no longer be immunosuppressed. If continu-ation of immunosuppressive medications is required, antifungal therapy

1	radiographic imaging indicate resolution of disease. In addition, the patient should no longer be immunosuppressed. If continu-ation of immunosuppressive medications is required, antifungal therapy should also continue to prevent recurrence of invasive disease.Cryptococcosis Cryptococcosis is a subacute or chronic infec-tion caused by Cryptococcus neoformans, a round, budding yeast (5 to 20 µm in diameter) that is sometimes surrounded by a characteristic wide gelatinous capsule. Cryptococci are typically present in soil and dust contaminated by pigeon drop-pings. When inhaled, such droppings can cause a nonfatal dis-ease primarily affecting the pulmonary and central nervous systems. At present, cryptococcosis is one of the most common opportunistic infection in patients with HIV infection, affecting ∼3% of that population. Four basic pathologic patterns are seen in the lungs of infected patients: granulomas; granulomatous pneumonia; diffuse alveolar or interstitial involvement; and

1	affecting ∼3% of that population. Four basic pathologic patterns are seen in the lungs of infected patients: granulomas; granulomatous pneumonia; diffuse alveolar or interstitial involvement; and pro-liferation of fungi in alveoli and lung vasculature. Symptoms are nonspecific, as are the radiographic findings. Cryptococcus neoformans may be isolated from sputum, bronchial washings, percutaneous needle aspiration of the lung, or cerebrospinal fluid. If disease is suspected, serum cryptococcal antigen titers should be obtained; if positive or if the patient has persistent fever, evidence of progression, physiologic compromise, or dissemination, treatment should be promptly initiated. Accord-ing to the CDC, multiple antifungal agents are effective against C neoformans; asymptomatic infections, such as those identified through targeted screening, should be treated with fluconazole while severe lung infections require amphotericin B combined with flucytosine followed by fluconazole for

1	such as those identified through targeted screening, should be treated with fluconazole while severe lung infections require amphotericin B combined with flucytosine followed by fluconazole for an extended length of time. Duration of therapy is longer in patients who are immunocompromised.Candidiasis Candida organisms are oval, budding cells (with or without mycelial elements) that colonize the oropharynx of many healthy individuals. The fungi of this genus are com-mon hospital and laboratory contaminants. Usually, Candida albicans causes disease in the oral or bronchial mucosa, among other anatomic sites. Approximately 95% of all invasive Candida infections are caused by five species: C albicans, C tropicalis, C parapsilosis, C glabrata, and C krusei. The specific pathogen varies between patient populations and geo-graphic regions. Non–C albicans infections now constitute nearly 70% of all cases in the United States, with C glabrata leading the list. Resistance to fluconazole is

1	patient populations and geo-graphic regions. Non–C albicans infections now constitute nearly 70% of all cases in the United States, with C glabrata leading the list. Resistance to fluconazole is common in the non–C albicans species, either natural or developed in response to antifungal therapy, and the shift is likely related to the wide-spread use of this antifungal agent.107The incidence of Candida infections has increased and is no longer confined to immunocompromised patients. Increasing incidence of infection has been identified in patients with any of the following risk factors: critical illness of long duration; use of long-term antibiotics, particularly multiple; indwelling urinary or vascular catheter; gastrointestinal perforation; or burn wounds.108 With respect to the thorax, such patients commonly have candidal pneumonia, pulmonary abscess, esophagitis, and mediastinitis. Pulmonary candidal infections typically result in an acute or chronic granulomatous reaction. Because

1	patients commonly have candidal pneumonia, pulmonary abscess, esophagitis, and mediastinitis. Pulmonary candidal infections typically result in an acute or chronic granulomatous reaction. Because Candida can invade blood vessel walls and a variety of tissues, systemic or disseminated infections can occur, but are less common.Treatment for candidal infection includes both fungicidal and fungistatic agents. The fungicidal medications include poly-enes (amphotericin B deoxycholate [AmB-D] and various lipid-associated amphotericin B preparations) and the echinocandins (caspofungin, micafungin, and anidulafungin). Fungistatic drugs include the triazoles (fluconazole, itraconazole, voricon-azole, and posaconazole). The availability of multiple effective therapies allows for specific tailoring of treatment, including combination regimens, based on the patient’s ability to toler-ate associated toxicities, the microbiologic information for the specific candidal species, and the route of

1	of treatment, including combination regimens, based on the patient’s ability to toler-ate associated toxicities, the microbiologic information for the specific candidal species, and the route of administration. While demonstrated efficacy is similar, the triazoles and echinocan-dins appear to have fewer side effects and are better tolerated than the other classes of antifungal drugs. The current initial recommended regimen for adults with invasive candidiasis is an echinocandin.107In addition to prompt institution of antifungal therapy, it is advisable to remove all central venous catheters as soon as can be safely achieved. For fungemia, an eye examination should be performed. Treatment should continue for at least 2 weeks after the last positive blood culture. For patients with Candida medi-astinitis (which has a mortality rate of >50%), surgical inter-vention to debride all infected tissues is required, in addition to prolonged administration of antifungal drugs.Mucormycosis The

1	medi-astinitis (which has a mortality rate of >50%), surgical inter-vention to debride all infected tissues is required, in addition to prolonged administration of antifungal drugs.Mucormycosis The Mucor species, rare members of the class Zygomycetes, are responsible for rapidly fatal disease in immunocompromised patients. Other disease-causing spe-cies of the class Zygomycetes include Absidia, Rhizopus, and Mortierella.109 Characteristic of these fungi are nonsep-tate, branching hyphae that are difficult to culture. Infec-tion occurs via inhalation of spores. Immunocompromised patients, including patients with neutropenia, acidosis, dia-betes, and hematologic malignancy all predispose to clinical susceptibility. In the lungs, disease consists of blood vessel invasion, thrombosis, and infarction of infected organs.Tissue destruction is significant, along with cavitation and abscess formation. Initial treatment is to correct underly-ing risk factors and administer antifungal therapies,

1	of infected organs.Tissue destruction is significant, along with cavitation and abscess formation. Initial treatment is to correct underly-ing risk factors and administer antifungal therapies, although Brunicardi_Ch19_p0661-p0750.indd 71401/03/19 7:01 PM

1	CHAPTER 19715CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAthe optimal duration and optimal total dose are unknown. Lipid formulations of amphotericin B are recommended at this time. Surgical resection of any localized disease should be performed after initial medical treatment attempts fail.Primary Fungal Pathogens Histoplasma capsulatum Histoplasma capsulatum is a dimor-phic fungus existing in mycelial form in soil contaminated by fowl or bat excreta and in yeast form in human hosts. The most common of all fungal pulmonary infections, histoplasmosis, primarily affects the respiratory system after spores are inhaled. It is endemic in the Midwest and Mississippi River Valley of the United States, where about 500,000 new cases arise each year. In immunocompromised patients, the infection becomes systemic and more virulent; because cell-mediated immunity is impaired, uninhibited fungal proliferation occurs within pulmo-nary macrophages and then spreads. Acute forms of the disease present as

1	systemic and more virulent; because cell-mediated immunity is impaired, uninhibited fungal proliferation occurs within pulmo-nary macrophages and then spreads. Acute forms of the disease present as primary or disseminated pulmonary histoplasmosis; chronic forms present as pulmonary granulomas (histoplasmo-mas), chronic cavitary histoplasmosis, mediastinal granulomas, fibrosing mediastinitis, or bronchiolithiasis. Histoplasmosis is definitively diagnosed by fungal smear, culture, direct biopsy of infected tissues, or serologic testing.The clinical presentation depends on the inoculum size and on host factors.Symptoms of acute pulmonary histoplasmosis are fever, chills, headache, chest pain, musculoskeletal pain, and nonpro-ductive cough. CXRs may be normal or may show mediastinal lymphadenopathy and patchy parenchymal infiltrates. Most patients improve in a few weeks; mild to moderate disease can be treated with itraconazole. Amphotericin B is the treatment of choice if moderate

1	and patchy parenchymal infiltrates. Most patients improve in a few weeks; mild to moderate disease can be treated with itraconazole. Amphotericin B is the treatment of choice if moderate symptoms persist for 2 to 4 weeks or if the illness is extensive, including dyspnea and hypoxia, and if patients are immunosuppressed.110As the pulmonary infiltrates from acute histoplasmosis heal, consolidation into an asymptomatic solitary nodule or histoplasmoma may occur and is usually seen incidentally on radiographs as a coin-shaped lesion. Central and concentric calcification may occur; if so, no further treatment is required. Noncalcification of the lesion requires further diagnostic workup including chest CT scan, needle biopsy, or surgical excision to rule out a malignancy. Figure 19-34 demonstrates the differ-ences in pathologic findings between infections in normal and immunocompromised hosts.111When lymph nodes and pulmonary granulomas calcify over time, pressure atrophy on the bronchial

1	the differ-ences in pathologic findings between infections in normal and immunocompromised hosts.111When lymph nodes and pulmonary granulomas calcify over time, pressure atrophy on the bronchial wall may result in erosion and migration of the granulomatous mass into the bronchus, causing bronchiolithiasis. Typical symptoms include cough, hemoptysis, and dyspnea. Life-threatening complica-tions include massive hemoptysis or bronchoesophageal fistula. In addition to radiography, bronchoscopy should be performed to aid in diagnosis. Definitive treatment requires surgical exci-sion of the bronchial mass and repair of the airway and contigu-ous structures. Endobronchial debridement is not advised as this can result in massive, fatal bleeding.Fibrosing mediastinitis is an uncommon manifestation of histoplasmosis but can be fatal due to progressive distortion and compression of the major vessels and central airways.Diagnosis can be difficult, and symptoms may be present for extended periods,

1	of histoplasmosis but can be fatal due to progressive distortion and compression of the major vessels and central airways.Diagnosis can be difficult, and symptoms may be present for extended periods, even years, before the diagnosis is made. The differential diagnosis for the disease process includes granu-lomatous mediastinitis related to recent infection, malignancy, and chronic pulmonary thromboembolism. A trial of itracon-azole is worthwhile, although it is not proven to be effective. In cases where radiographic or physiologic improvement is achieved after a trial of 12 week of therapy, continuation of therapy is con-sidered for a full 12 months. In the majority of patients, how-ever, antifungal therapy has not been proven. There is no role for corticosteroids at this time or for antifibrotics. Occasionally, intravascular stents have been helpful for severe vascular compro-mise. Balloon dilatation and endobronchial silicone stents may be needed for airway compromise, although this

1	Occasionally, intravascular stents have been helpful for severe vascular compro-mise. Balloon dilatation and endobronchial silicone stents may be needed for airway compromise, although this should be directed by a surgeon with expertise in mediastinal and airway disease management.Chronic pulmonary histoplasmosis occurs in about 10% of patients who become symptomatic after infection. Most such patients have preexisting lung pathology, particularly emphy-sema, which becomes colonized, and subsequent pneumonitis and necrosis, cavity enlargement, new cavity formation, and pulmonary dissemination occur. Nonspecific symptoms, such as cough, sputum production, fever, weight loss, weakness, and hemoptysis are common. Chest radiography may reveal intrapulmonary cavitation and scarring. Occasionally, par-tial resolution of the inflammatory changes may be observed. Itraconazole provides effective therapy, but must be given for 12 to 24 months. It is superior to ketoconazole and fluconazole;

1	par-tial resolution of the inflammatory changes may be observed. Itraconazole provides effective therapy, but must be given for 12 to 24 months. It is superior to ketoconazole and fluconazole; these should only be used if itraconazole is not tolerated. Vori-conazole and posaconazole have been found to be useful for salvage therapy. Serum itraconazole levels should be monitored to ensure that the drug is being absorbed. Occasionally, lipid-associated amphotericin B is necessary for more severe infec-tions. Surgical excision should be considered in patients with adequate pulmonary reserve and localized, thick-walled cavities that have been unresponsive to antifungal therapy.Disseminated histoplasmosis occurs most frequently in patients who are severely immunocompromised, such as post-transplantation patients, patients with HIV, and patients using immunosuppressive medications. Presentation ranges from non-specific signs of fever, weight loss, and malaise, to shock, respi-ratory

1	patients, patients with HIV, and patients using immunosuppressive medications. Presentation ranges from non-specific signs of fever, weight loss, and malaise, to shock, respi-ratory distress, and multiorgan failure. Diagnosis can be made with a combination of Histoplasma urine antigen, serologic assay, and fungal culture and should be suspected in patients with the above symptoms in any endemic area, particularly if the patient is immunosuppressed.112 Any of the antifungal thera-pies can be used in treatment of disseminated histoplasmosis. Use of amphotericin B has decreased the mortality rate to less than 25% in this type of serious infection.Coccidioides immitis Coccidioides immitis is an endemic fun-gus found in soil and dust of the southwestern United States. Agricultural workers, military personnel, and other occupa-tions with extensive exposure to soil, especially in areas of endemic growth, are at highest risk, as are immunocompro-mised individuals.113 Spores (arthroconidia)

1	personnel, and other occupa-tions with extensive exposure to soil, especially in areas of endemic growth, are at highest risk, as are immunocompro-mised individuals.113 Spores (arthroconidia) are inhaled, swell into spherules, and subdivide into endospores, and subsequent infection develops. Diagnosis can be achieved through serum analysis for anticoccidioidal antibody, spherule identification in tissue, or by isolating the fungus in cultures from sputum, other body fluid, or tissue.Inhalation of the fungus causes pulmonary involvement in 95% of patients with symptomatic disease. Three main cat-egories of pulmonary involvement, based on the associated signs and symptoms, are possible: primary, complicated, and residual pulmonary coccidioidomycosis. Primary pulmonary Brunicardi_Ch19_p0661-p0750.indd 71501/03/19 7:01 PM 716SPECIFIC CONSIDERATIONSPART IIABCDEFFigure 19-34. Pathologic findings of infection in normal and immunocompromised hosts. Histopathologic preparations are shown

1	71501/03/19 7:01 PM 716SPECIFIC CONSIDERATIONSPART IIABCDEFFigure 19-34. Pathologic findings of infection in normal and immunocompromised hosts. Histopathologic preparations are shown contrast-ing acute diffuse pulmonary involvement in a lung segment of a normal host with a probable primary infection (A through D) with pulmonary granulomas from an immunocompromised patient who had an opportunistic reinfection with Histoplasma capsulatum (E, F). A. Diffuse interstitial pneumonitis in an adult (normal host) with recent heavy environmental exposure and subsequent development of progressive pulmonary disease. There is an inflammatory cell infiltrate primarily involving the interalveolar interstitial spaces but present within many alveolar spaces as well. The exudate consists mostly of mononuclear phagocytes, lymphocytes, and occasional plasma cells. Many of the alveolar walls are markedly thickened (hematoxylin and eosin stain [H&E], ×50). B. Another area from the same lung as in A

1	phagocytes, lymphocytes, and occasional plasma cells. Many of the alveolar walls are markedly thickened (hematoxylin and eosin stain [H&E], ×50). B. Another area from the same lung as in A showing focal vasculitis with an infiltrate of lymphocytes and macrophages (H&E, ×25). C. Relatively large alveolar macrophages packed with single and budding yeasts 2 to 4 µm in diameter (same lung as in A and B). The basophilic cytoplasm of these yeasts is retracted from their thin outer cell walls, leaving halo-like clear areas that can be confused with capsules (H&E, ×500). D. Intracellular and extracellular yeasts, 2 to 4 µm in diameter, some of which are single, budding, or in short chains (Gomori methenamine silver stain, ×500). E. Nonnecrotizing (sometimes called epithelioid cell or noncaseating) granuloma from a patient who had recently received chemotherapy for a germ cell tumor (different patient than in A through D). This lesion consists of a focal collection of macrophages (sometimes

1	granuloma from a patient who had recently received chemotherapy for a germ cell tumor (different patient than in A through D). This lesion consists of a focal collection of macrophages (sometimes referred to as histiocytes or epithelioid cells) plus lymphocytes and occasional plasma cells. A few multinucleated macrophages are present. A thin layer of fibroblasts circumscribes the lesion. Yeasts of H capsulatum, probably present within macrophages of this lesion at an earlier stage, were not identified in this granuloma or in any of several other nonnecrotizing granulomas within the specimen. Lesions of this type often undergo necrosis to become necrotiz-ing granulomas (H&E, ×50). F. Necrotizing (sometimes referred to as caseating) granuloma from the same lung as in E. This lesion has a necrotic center surrounded by macrophages, encapsulating fibroblasts, fibrous connective tissue in the periphery, and scattered lymphocytes. A prominent giant cell is present in the lower left of the

1	necrotic center surrounded by macrophages, encapsulating fibroblasts, fibrous connective tissue in the periphery, and scattered lymphocytes. A prominent giant cell is present in the lower left of the granuloma (at approximately 8 o’clock). Microorganisms are usually present only in relatively small numbers in these types of lesions. They are most frequently detected within the most central necrotic material in these granulomas (H&E, ×25). (Reproduced with permission from Hage CA, Wheat LJ, Loyd J, et al: Pulmonary histoplasmosis, Semin Respir Crit Care Med. 2008 Apr;29(2):151-165.)Brunicardi_Ch19_p0661-p0750.indd 71601/03/19 7:01 PM

1	CHAPTER 19717CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAcoccidioidomycosis occurs in about 40% of people who inhale spores. The other 60% will remain asymptomatic and develop life-long immunity. The constellation of symptoms of “valley fever,” including fever, chills, headache, erythema multiforme, erythema nodosum, polyarthralgias, nonproductive cough, and chest pain, and a CXR showing hilar and paratracheal adenopa-thy are highly suggestive of pulmonary coccidioidomycosis. In many patients, initial diagnosis is community-acquired pneumo-nia, and it is only when the patient fails to respond to appropriate antibiotic therapy that pulmonary coccidioidomycosis is consid-ered. The disease is self-limited in the majority of patients, and treatment is not required in these cases.Therapy should be considered for (a) patients with impaired cellular immunity; (b) comorbid illnesses that are adversely impacted by the infection, including chronic pulmo-nary dysfunction, renal failure, and

1	be considered for (a) patients with impaired cellular immunity; (b) comorbid illnesses that are adversely impacted by the infection, including chronic pulmo-nary dysfunction, renal failure, and congestive heart failure; and (c) when symptoms and radiographic findings persist for more than 6 to 8 weeks, at which time the disease is considered to be persistent coccidioidal pneumonia and occurs in approximately 1% of patients. Progression to caseous nodules, cavities, and calcified, fibrotic, or ossified lesions indicates complicated or residual stages of coccidioidomycosis.There are several relative indications for surgery in pulmo-nary coccidioidomycosis. A rapidly expanding (>4 cm) cavity that is close to the visceral pleura is a high risk for rupture into the pleural space and subsequent empyema. Other indications for operative intervention include life-threatening hemoptysis; hemoptysis that is persistent despite medical therapy; symptom-atic fungus ball; bronchopleural fistula;

1	empyema. Other indications for operative intervention include life-threatening hemoptysis; hemoptysis that is persistent despite medical therapy; symptom-atic fungus ball; bronchopleural fistula; cavitary lesions with persistent positive sputum; and pulmonary nodules that degen-erate over time. Finally, any nodule with signs that are concern-ing for malignancy should undergo further evaluation, including biopsy or resection, to determine the underlying etiology.Diagnosis of coccidioidomycosis is confirmed by histo-pathologic, mycologic, and serologic evaluation. Extrapulmo-nary disease may develop in approximately 0.5% of infected patients, with involvement of meninges, bones, joints, skin, or soft tissues. Immunocompromised patients are especially sus-ceptible to disseminated coccidioidomycosis, which carries a mortality rate over 40%. Treatment options for this disease vary depending on the severity of the disease as well as the stage. Amphotericin B deoxycholate or the triazoles

1	which carries a mortality rate over 40%. Treatment options for this disease vary depending on the severity of the disease as well as the stage. Amphotericin B deoxycholate or the triazoles continue to be the primary antifungal medications. If meningeal involvement is identified, fluconazole or itraconazole therapy is required for the remainder of the patient’s life. Intrathecal amphotericin B can also be administered in some cases.Blastomyces dermatitidis Blastomyces dermatitidis is a round, single-budding yeast with a characteristic thick, refrac-tile cell wall. It resides in the soil as a nonmotile spore called conidia. Exposure occurs when contaminated soil is disturbed and the conidia are aerosolized. The spore is inhaled and trans-forms into a yeast phase at body temperature.114 Infection is typically self-limited. A small minority of patients will develop chronic pulmonary infection or disseminated disease, includ-ing cutaneous, osteoarticular, and genitourinary involvement. B

1	is typically self-limited. A small minority of patients will develop chronic pulmonary infection or disseminated disease, includ-ing cutaneous, osteoarticular, and genitourinary involvement. B dermatitidis has a worldwide distribution; in the United States, it is endemic in the central states.115 With chronic infec-tion, the organism induces a granulomatous and pyogenic reac-tion with microabscesses and giant cells; caseation, cavitation, and fibrosis may also occur. Symptoms are nonspecific and con-sistent with chronic pneumonia in 60% to 90% of patients. They include cough, mucoid sputum production, chest pain, fever, malaise, weight loss, and, uncommonly, hemoptysis. In acute disease, radiographs are either completely negative or have nonspecific findings; in chronic disease, fibronodular lesions (with or without cavitation) similar to tuberculosis are noted. Pulmonary parenchymal abnormalities in the upper lobe(s) may be noted. Mass lesions similar to carcinoma are frequent, and

1	lesions (with or without cavitation) similar to tuberculosis are noted. Pulmonary parenchymal abnormalities in the upper lobe(s) may be noted. Mass lesions similar to carcinoma are frequent, and lung biopsy is frequently used. Over 50% of patients with chronic blastomycosis also have extrapulmonary manifesta-tions, but less than 10% of patients present with severe clinical manifestation.114Once a patient manifests symptoms of chronic blastomy-cosis, antifungal treatment is required to achieve resolution. Mortality approaches 60% if untreated.114 While controversial, a short course of triazole therapy (oral itraconazole 200 mg daily) for 6 months is the treatment of choice for most patients with mild to moderate forms of the disease. Because itraconazole has poor CNS penetration, the most common site of recurrence after apparently successful therapy is in the CNS. In the absence of therapy, close follow-up is warranted for evidence of progres-sion to chronic or extrapulmonary disease.

1	common site of recurrence after apparently successful therapy is in the CNS. In the absence of therapy, close follow-up is warranted for evidence of progres-sion to chronic or extrapulmonary disease. Amphotericin B is warranted for patients with severe or life-threatening disease, CNS involvement, disseminated disease, or extensive lung involvement and in immunocompromised patients. After ade-quate drug therapy, surgical resection of known cavitary lesions should be considered because viable organisms are known to persist in such lesions.Massive HemoptysisMassive hemoptysis is generally defined as expectoration of over 600 mL of blood within a 24-hour period. It is a medi-cal emergency associated with a mortality rate of 30% to 50%. Most clinicians would agree that losing over a liter of blood via the airway within 1 day is significant, yet use of an abso-lute volume criterion presents difficulties. First, it is difficult for the patient or caregivers to quantify the volume of blood

1	blood via the airway within 1 day is significant, yet use of an abso-lute volume criterion presents difficulties. First, it is difficult for the patient or caregivers to quantify the volume of blood being lost. Second, and most relevant, the rate of bleeding nec-essary to incite respiratory compromise is highly dependent on the individual’s prior respiratory status. For example, the loss of 100 mL of blood over 24 hours in a 40-year-old male with normal pulmonary function would be of little immediate con-sequence, because his normal cough would ensure his ability to clear the blood and secretions. In contrast, the same amount of bleeding in a 69-year-old male with severe COPD, chronic bronchitis, and an FEV1 of 1.1 L may be life-threatening.Anatomy. The lungs have two sources of blood supply: the pulmonary and bronchial arterial systems. The pulmonary sys-tem is a high-compliance, low-pressure system, and the walls of the pulmonary arteries are very thin and delicate. The bronchial

1	the pulmonary and bronchial arterial systems. The pulmonary sys-tem is a high-compliance, low-pressure system, and the walls of the pulmonary arteries are very thin and delicate. The bronchial arteries, part of the systemic circulation, have systemic pres-sures and thick walls; most branches originate from the proxi-mal thoracic aorta. Most cases of massive hemoptysis involve bleeding from the bronchial artery circulation or from the pul-monary circulation pathologically exposed to the high pres-sures of the bronchial circulation. In many cases of hemoptysis, particularly those due to inflammatory disorders, the bronchial arterial tree becomes hyperplastic and tortuous. The systemic pressures within these arteries, combined with a disease process within the airway and erosion, lead to bleeding.Causes. Significant hemoptysis has many causes, most com-monly including pulmonary, extrapulmonary, and iatrogenic. Table 19-20 summarizes the most common causes of hemopty-sis. Most are

1	to bleeding.Causes. Significant hemoptysis has many causes, most com-monly including pulmonary, extrapulmonary, and iatrogenic. Table 19-20 summarizes the most common causes of hemopty-sis. Most are secondary to inflammatory processes. Aneurysms Brunicardi_Ch19_p0661-p0750.indd 71701/03/19 7:01 PM 718SPECIFIC CONSIDERATIONSPART IITable 19-20Pulmonary and extrapulmonary causes of massive hemoptysisPULMONARYEXTRAPULMONARYIATROGENICPulmonary parenchymal diseaseBronchitisBronchiectasisTuberculosisLung abscessPneumoniaCavitary fungal infection (e.g., aspergilloma)Lung parasitic infection (ascariasis, schistosomiasis, paragonimiasis)Pulmonary neoplasmPulmonary infarction or embolismTraumaArteriovenous malformationPulmonary vasculitisPulmonary endometriosisWegener’s granulomatosisCystic fibrosisPulmonary hemosiderosisCongestive heart failureCoagulopathyMitral stenosisMedicationsIntrapulmonary catheterTable 19-21Treatment priorities in the management of massive hemoptysis 1. Achieve

1	fibrosisPulmonary hemosiderosisCongestive heart failureCoagulopathyMitral stenosisMedicationsIntrapulmonary catheterTable 19-21Treatment priorities in the management of massive hemoptysis 1. Achieve respiratory stabilization and prevent asphyxiation. 2. Localize the bleeding site. 3. Control the hemorrhage. 4. Determine the cause. 5. Definitively prevent recurrence.of the pulmonary artery (referred to as Rasmussen’s aneurysm) can develop within pulmonary cavities and can result in massive bleeding. Hemoptysis due to lung cancer is usually mild, result-ing in blood-streaked sputum. Massive hemoptysis in patients with lung cancer is typically caused by malignant invasion of pulmonary artery vessels by large central tumors. Although rare, it is often a terminal event.Management. Life-threatening hemoptysis is best managed by a multidisciplinary team of intensive care physicians, interven-tional radiologists, and thoracic surgeons. Treatment priorities begin with respiratory

1	hemoptysis is best managed by a multidisciplinary team of intensive care physicians, interven-tional radiologists, and thoracic surgeons. Treatment priorities begin with respiratory stabilization; intubation with isolation of the bleeding lung may be required to prevent asphyxiation. This can be done with main-stem intubation into the nonbleeding lung, endobronchial blockers into the bleeding lung, or double-lumen endotracheal intubation, depending on the urgency of the situation and the expertise of the providers. Once adequate ven-tilation has been achieved, the bleeding site should be localized; bronchoscopy can often provide direct visualization of blood coming from a specific area of the tracheobronchial anatomy. Control of the hemorrhage is then achieved endobronchially with laser or bronchial occlusion, endovascularly with bronchial and/or pulmonary artery embolization, or surgically with resec-tion of the involved area.116 The order of priorities in manage-ment is detailed in

1	bronchial occlusion, endovascularly with bronchial and/or pulmonary artery embolization, or surgically with resec-tion of the involved area.116 The order of priorities in manage-ment is detailed in Table 19-21.The clinically pragmatic definition of massive hemoptysis is a degree of bleeding that threatens respiratory stability. There-fore, clinical judgment of respiratory compromise is the first step in evaluating a patient.117,118 Two scenarios are possible: (a) bleeding is significant and persistent, but its rate allows a rapid but sequential diagnostic and therapeutic approach, and (b) bleeding is so rapid that emergency airway control and ther-apy are necessary.Scenario 1: Significant, Persistent, but Nonmassive Bleeding Although bleeding is brisk in scenario 1, the patient may be able to maintain clearance of the blood and secretions with his or her own respiratory reflexes. Immediate measures are admission to an intensive care unit; strict bed rest; Trendelenburg position-ing

1	to maintain clearance of the blood and secretions with his or her own respiratory reflexes. Immediate measures are admission to an intensive care unit; strict bed rest; Trendelenburg position-ing with the affected side down (if known); administration of humidified oxygen; cough suppression; monitoring of oxygen saturation and arterial blood gases; and insertion of large-bore intravenous catheters. Strict bed rest with sedation may lead to slowing or cessation of bleeding, and the judicious use of intravenous narcotics or other relaxants to mildly sedate the patient and diminish some of the reflexive airway activity is often necessary. Also recommended are administration of aero-solized adrenaline, intravenous antibiotic therapy if needed, and correction of abnormal blood coagulation study results. Finally, unless contraindicated, intravenous vasopressin (20 U over 15 minutes, followed by an infusion of 0.2 U/min) can be given.A CXR is the first test and often proves to be the most

1	results. Finally, unless contraindicated, intravenous vasopressin (20 U over 15 minutes, followed by an infusion of 0.2 U/min) can be given.A CXR is the first test and often proves to be the most revealing. Localized lesions may be seen, but the effects of blood soiling of other areas of the lungs may predominate, obscuring the area of pathology. Chest CT scan provides more detail and is nearly always performed if the patient is stable. Pathologic areas may be obscured by blood soiling.Flexible bronchoscopy is the next step in evaluating the patient’s condition. Some clinicians argue that rigid bronchos-copy should always be performed. However, if the patient is clinically stable and the ongoing bleeding is not imminently threatening, flexible bronchoscopy is appropriate. It allows diagnosis of airway abnormalities and will usually permit Brunicardi_Ch19_p0661-p0750.indd 71801/03/19 7:01 PM

1	CHAPTER 19719CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAlocalization of the bleeding site to either a lobe or even a seg-ment. The person performing the bronchoscopy must be pre-pared with excellent suction and must be able to perform saline lavage with a dilute solution of epinephrine.Most cases of massive hemoptysis arise from the bron-chial arterial tree; therefore, the next therapeutic option fre-quently is selective bronchial arteriography and embolization. Prearteriogram bronchoscopy is extremely useful to direct the angiographer. However, if bronchoscopy fails to localize the bleeding site, then bilateral bronchial arterio-grams can be performed. More recently, use of multidetec-tor CT angiography in patients with hemoptysis that is not immediately life-threatening has been shown to facilitate endovascular intervention; reformatting of the images in mul-tiple projections allows clear delineation of the pulmonary vascular anatomy.105 With this approach, abnormal bronchial and

1	to facilitate endovascular intervention; reformatting of the images in mul-tiple projections allows clear delineation of the pulmonary vascular anatomy.105 With this approach, abnormal bronchial and nonbronchial arteries can be visualized and subsequently targeted for therapeutic arterial embolization.119 Once the tar-geted arterial system has been embolized, immediate control and cessation of the hemoptysis is achieved in more than 80% of patients. If bleeding persists after bronchial artery emboli-zation, a pulmonary artery source should be suspected and a pulmonary angiogram performed at the same setting.Recurrence is seen in 30% to 60% of cases and is very common in the setting of invasive fungal infections such as aspergilloma. Recurrence after bronchial artery embolization is less common in the setting of malignancy and active tuberculo-sis but does occur and can ultimately result in patient death.120 Repeat embolization can be effective and is warranted for ini-tial management

1	in the setting of malignancy and active tuberculo-sis but does occur and can ultimately result in patient death.120 Repeat embolization can be effective and is warranted for ini-tial management of recurrent hemoptysis, but early surgical intervention should be considered, particularly in the setting of aspergilloma or other cavitary lesions.105If respiratory compromise is impending, orotracheal intu-bation should be performed. After intubation, flexible bronchos-copy should be performed to clear blood and secretions and to attempt localization of the bleeding site. Depending on the pos-sible causes of the bleeding, bronchial artery embolization or (if appropriate) surgery can be considered.Scenario 2: Significant, Persistent, and Massive Bleeding Life-threatening bleeding requires emergency airway control and preparation for potential surgery. Such patients are best cared for in an operating room equipped with rigid bronchos-copy. Immediate orotracheal intubation may be necessary to

1	airway control and preparation for potential surgery. Such patients are best cared for in an operating room equipped with rigid bronchos-copy. Immediate orotracheal intubation may be necessary to gain control of ventilation and suctioning. However, rapid trans-port to the operating room with rigid bronchoscopy should be facilitated. Rigid bronchoscopy allows adequate suctioning of bleeding with visualization of the bleeding site; the nonbleed-ing side can be cannulated with the rigid scope and the patient ventilated. After stabilization, ice-saline lavage of the bleeding site can then be performed (up to 1 L in 50-mL aliquots); bleed-ing stops in up to 90% of patients.121Alternatively, blockade of the main stem bronchus of the affected side can be accomplished with a double-lumen endo-tracheal tube, with a bronchial blocker, or by intubation of the nonaffected side by an uncut standard endotracheal tube. Place-ment of a double-lumen endotracheal tube is challenging in these

1	endo-tracheal tube, with a bronchial blocker, or by intubation of the nonaffected side by an uncut standard endotracheal tube. Place-ment of a double-lumen endotracheal tube is challenging in these circumstances, given the bleeding and secretions. Proper placement and suctioning may be difficult, and attempts could compromise the patient’s ventilation. The best option is to place a bronchial blocker in the affected bronchus with inflation. Endovascular embolization can be performed to stop the bleeding after control has been achieved with the bronchial blocker. The blocker is left in place for 24 hours; after 24 hours, the area is reexamined bronchoscopically.Surgical Intervention. In most patients, bleeding can be stopped, recovery can occur, and plans to definitively treat the underlying cause can be made. In scenario 1 (significant, persis-tent, but nonmassive bleeding), the patient may undergo further evaluation as an inpatient or outpatient. A chest CT scan and pulmonary function

1	cause can be made. In scenario 1 (significant, persis-tent, but nonmassive bleeding), the patient may undergo further evaluation as an inpatient or outpatient. A chest CT scan and pulmonary function studies should be obtained preoperatively. In scenario 2 (patients with significant, persistent, and massive bleeding), surgery, if appropriate, will usually be performed during the same hospitalization as the rigid bronchoscopy or main stem bronchus blockade. In a small number of patients (<10%), immediate surgery will be necessary due to the extent of bleeding. The bleeding site in these patients is localized using rigid bronchoscopy with immediate thoracotomy or sternotomy to follow.Surgical treatment is individualized according to the source of bleeding and the patient’s medical condition, prog-nosis, and pulmonary reserve. General indications for urgent surgery are presented in Table 19-22. In patients with significant cavitary disease or with fungus balls, the walls of the cavities

1	prog-nosis, and pulmonary reserve. General indications for urgent surgery are presented in Table 19-22. In patients with significant cavitary disease or with fungus balls, the walls of the cavities are eroded and necrotic; rebleeding will likely ensue. In addi-tion, bleeding from cavitary lesions may be due to pulmonary artery erosion, which requires surgery for control.End-Stage Lung DiseaseLung Volume Reduction Surgery. The ideal patient for lung volume reduction surgery (LVRS) has heterogeneous emphy-sema with apical predominance, meaning the worst emphyse-matous changes are in the apex (seen on chest CT scan) of the lungs. The physiologic lack of function of these areas is dem-onstrated by quantitative perfusion scan, which shows minimal or no perfusion. By surgically excising these nonfunctional areas, the volume of the lung is reduced, theoretically restor-ing respiratory mechanics. Diaphragm position and function are improved, and there may be an improvement in the dynamic

1	nonfunctional areas, the volume of the lung is reduced, theoretically restor-ing respiratory mechanics. Diaphragm position and function are improved, and there may be an improvement in the dynamic small airway collapse in the remaining lung.Operative mortality in the initial experience was 16.9%, with a 1-year mortality of 23%. In response, the National Emphysema Treatment Trial (NETT) performed a randomized trial of 1218 patients in a noncrossover design to medical versus surgical management after a 10-week pretreatment pulmonary rehabilitation program. Subgroup analysis demonstrated that in patients with the anatomic changes delineated by Cooper and colleagues, LVRS significantly improved exercise capacity, lung function, quality of life, and dyspnea compared to medi-cal therapy. After 2 years, functional improvements began to decline toward baseline. Similar parameters in medically treated patients steadily decline below baseline. LVRS was associated with increased short-term

1	2 years, functional improvements began to decline toward baseline. Similar parameters in medically treated patients steadily decline below baseline. LVRS was associated with increased short-term morbidity and mortality and did not confer a survival benefit over medical therapy.122Table 19-22General indications for urgent operative intervention for massive hemoptysis 1. Presence of a fungus ball 2. Presence of a lung abscess 3. Presence of significant cavitary disease 4. Failure to control the bleedingBrunicardi_Ch19_p0661-p0750.indd 71901/03/19 7:01 PM 720SPECIFIC CONSIDERATIONSPART IISurvival123BOS-free survival1.00.80.60.40.2Years posttransplantFigure 19-36. The survival rate after lung transplantation in the absence of bronchiolitis obliterans syndrome (BOS) at the University of Minnesota.Survival123Primary graft failure1.00.80.60.40.2No PGFPGFYears posttransplantFigure 19-37. The survival rate after lung transplantation at the University of Minnesota as a function of primary

1	graft failure1.00.80.60.40.2No PGFPGFYears posttransplantFigure 19-37. The survival rate after lung transplantation at the University of Minnesota as a function of primary graft failure (PGF).Lung Transplantation. The most common indications for lung transplant are COPD and idiopathic pulmonary fibrosis (IPF). Most patients with IPF and older patients with COPD are offered a single-lung transplant. Younger COPD patients and patients with α1-antitrypsin deficiency and severe hyperinflation of the native lungs are offered a bilateral-lung transplant. Most patients with primary pulmonary hypertension and almost all patients with cystic fibrosis are treated with a bilateral-lung transplant. A heart-lung transplant is reserved for patients with irreversible ventricular failure or uncorrectable congenital cardiac disease.Patients with COPD are considered for placement on the transplant waiting list when their FEV1 has fallen to below 25% of its predicted value. Patients with significant

1	congenital cardiac disease.Patients with COPD are considered for placement on the transplant waiting list when their FEV1 has fallen to below 25% of its predicted value. Patients with significant pulmonary hyper-tension should be listed earlier. IPF patients should be referred when their forced vital capacity has fallen to less than 60% or their Dlco has fallen to less than 50% of their predicted values.In the past, patients with primary pulmonary hypertension and New York Heart Association (NYHA) class III or IV symp-toms were listed for a lung transplant. However, treatment of such patients with intravenous prostacyclin and other pulmonary vasodilators has now markedly altered that strategy. Virtually all patients with primary pulmonary hypertension are now treated with intravenous epoprostenol. Several of these patients have experienced a marked improvement in their symptoms associ-ated with a decrease in their pulmonary arterial pressures and an increase in exercise capacity.

1	epoprostenol. Several of these patients have experienced a marked improvement in their symptoms associ-ated with a decrease in their pulmonary arterial pressures and an increase in exercise capacity. Listing of these patients is deferred until they develop NYHA class III or IV symptoms or until their mean pulmonary artery pressure rises above 75 mmHg.Medium-term and bronchiolitis obliterans syndrome (BOS)–free survival rates of patients who underwent a lung transplant at the University of Minnesota are shown in Figs. 19-35 and 19-36. The mortality of patients while waiting for transplants is about 10%. In an effort to expand the number of lung donors, many transplant groups have liberalized their criteria for donor selection. Still, the partial pressure of arterial oxygen (Pao2) should be greater than 300 mmHg on a fraction of inspired oxygen (Fio2) of 100%. In special circumstances, lungs may be used from donors with a smoking history; from donors older than 50 years of age; and from

1	greater than 300 mmHg on a fraction of inspired oxygen (Fio2) of 100%. In special circumstances, lungs may be used from donors with a smoking history; from donors older than 50 years of age; and from donors with positive Gram stains or infiltrates on CXR.123,124 The use of two living donors, each donating a single lower lobe, is another strategy for increasing the donor pool. Recipient outcomes are similar to those with cadaver donors in carefully selected patients.Survival123Overall survival1.00.80.60.40.2Years posttransplantFigure 19-35. The overall survival rate after lung transplantation at the University of Minnesota.Most of the early mortality after lung transplant is related to primary graft failure resulting from a severe ischemia-reper-fusion injury to the lung(s) (Fig. 19-37). Reperfusion injury is characterized radiographically by interstitial and alveolar edema and clinically by hypoxia and ventilation-perfusion mismatch. Donor neutrophils and recipient lymphocytes

1	Reperfusion injury is characterized radiographically by interstitial and alveolar edema and clinically by hypoxia and ventilation-perfusion mismatch. Donor neutrophils and recipient lymphocytes prob-ably play an important role in the pathogenesis of reperfu-sion injury. The most important impediment to longer-term survival after a lung transplant is the development of BOS, a manifestation of chronic rejection. Episodes of acute rejection are the major risk factors for developing BOS. Other injuries to the lung (including early reperfusion injury and chronic gas-troesophageal reflux disease) may also adversely affect long-term outcomes of patients.125,126CHEST WALLChest Wall MassClinical Approach. Surgeons confronted with a patient with a chest wall mass must be cognizant that their approach to diagno-sis and treatment has significant impact on the patient’s chances Brunicardi_Ch19_p0661-p0750.indd 72001/03/19 7:01 PM

1	CHAPTER 19721CHEST WALL, LUNG, MEDIASTINUM, AND PLEURADiagnosis is clear;a surgical resection is theprimary treatmentLesion <2.0 cmBenign Tumors Fibrous dysplasia Chondroma Osteochondroma Eosinophilic granulomaMalignant Tumors ChondrosarcomaWide surgical excisionCT or MRI or bothChest wall massFigure 19-38. Systematic approach for evaluating a chest wall mass when the clinical scenario is uncomplicated and initial imag-ing studies suggest a clear diagnosis. CT = computed tomography; MRI = magnetic resonance imaging.for long-term survival. All chest wall tumors should be consid-ered malignant until proven otherwise. It is critically important that the surgeon(s) be mindful of this tenet and well versed in the diagnostic and treatment principles for chest wall malignan-cies. These tenets must be applied from the initial biopsy, as the placement of the incision can impact significantly on the successful complete resection and reconstruction of the chest wall. Complete resection is

1	must be applied from the initial biopsy, as the placement of the incision can impact significantly on the successful complete resection and reconstruction of the chest wall. Complete resection is imperative if there is any hope for cure and/or long-term survival. A general approach is outlined in Figs. 19-38 and 19-39.Patients with chest wall tumors, regardless of etiology, typically complain of a slowly enlarging palpable mass (50% to 70%), chest wall pain (25% to 50%), or both. Interestingly, growing masses are often not noticed by the patient until they suffer a trauma to the area. Pain from a chest wall mass is typi-cally localized to the area of the tumor; it occurs more often and more intensely with malignant tumors, but it can also be present in up to one-third of patients with benign tumors. With Ewing’s sarcoma, fever and malaise may also be present. Benign chest wall tumors tend to occur in younger patients (average age 26 years), whereas malignant tumors tend to be found

1	tumors. With Ewing’s sarcoma, fever and malaise may also be present. Benign chest wall tumors tend to occur in younger patients (average age 26 years), whereas malignant tumors tend to be found in older patients (average age 40 years). Overall, between 50% and 80% of chest wall tumors are malignant.Evaluation and Management. Laboratory evaluations are useful in assessing chest wall masses for the following:1. Plasmacytoma. Serum protein electrophoresis demon-strates a single monoclonal spike, which is measuring the overproduction of one immunoglobulin from the malignant plasma cell clone.Chest wall massCT or MRI or bothDiagnosis is NOT clearPreoperative chemotherapyWide surgical excisionOsteosarcomaRhabdomyosarcomaNonrhabdomyosarcomaPNET/Ewing’s sarcomaNonrhabdosarcoma Fibrosarcoma Malignant fibrous histiocytoma Liposarcoma Synovial cell sarcoma DesmoidNeedle biopsyor incisional biopsyFigure 19-39. Systematic approach for evaluating a chest wall mass for which the diagnosis is

1	fibrous histiocytoma Liposarcoma Synovial cell sarcoma DesmoidNeedle biopsyor incisional biopsyFigure 19-39. Systematic approach for evaluating a chest wall mass for which the diagnosis is not unequivocal. A tissue diagnosis is critical for effective management of chest wall masses. CT = computed tomography; MRI = magnetic resonance imaging; PNET = primitive neuroectodermal tumor.2. Osteosarcoma. Alkaline phosphatase levels may be elevated.3. Ewing’s sarcoma. Erythrocyte sedimentation rates may be elevated.Radiography CXR may reveal rib destruction, calcification within the lesion, and if old films are available, a clue to growth rate. CT scanning, however, is necessary to determine the rela-tionship of the chest wall mass to contiguous structures (e.g., mediastinum, lung, soft tissues, and other skeletal elements), evaluate for pulmonary metastases, and assess for extraosseous bone formation and bone destruction, both typically seen with osteosarcoma.Because MRI provides

1	tissues, and other skeletal elements), evaluate for pulmonary metastases, and assess for extraosseous bone formation and bone destruction, both typically seen with osteosarcoma.Because MRI provides multiple planes of imaging (coronal, sagittal, and oblique), better definition of the relationship between tumor and muscle, and tumor and contiguous or nearby neurovascular structures or the spine, it is an important radio-graphic adjunct for preoperative planning. Compared to CT scan alone, MRI may further delineate tissue abnormalities, poten-tially enhancing the ability to distinguish benign from malignant sarcoma.Biopsy The first step in the management of all chest wall tumors is to obtain a tissue diagnosis. Inappropriate or mis-guided attempts at tissue diagnosis through casual open biopsy techniques have the potential (if the lesion is a sarcoma) to seed surrounding tissues and contiguous body cavities (e.g., the pleural space) with tumor cells, potentially compromising local

1	biopsy techniques have the potential (if the lesion is a sarcoma) to seed surrounding tissues and contiguous body cavities (e.g., the pleural space) with tumor cells, potentially compromising local Brunicardi_Ch19_p0661-p0750.indd 72101/03/19 7:01 PM 722SPECIFIC CONSIDERATIONSPART IItumor control and patient survival. Tissue diagnosis is accom-plished using one of three methods: needle biopsy (typically CT-guided, FNA, or a core biopsy), incisional biopsy, or exci-sional biopsy in limited and specific situations.1. Needle biopsy. Pathologists experienced with sarcomas can accurately diagnose approximately 90% of patients using FNA cytology. A needle biopsy (FNA or core) has the advantage of avoiding wound and body cavity contami-nation (a potential complication with an incisional biopsy).2. Incisional biopsy. If a needle biopsy is nondiagnostic, an incisional biopsy may be performed, with caveats. First, the skin incision must be placed directly over the mass and ori-ented to

1	biopsy).2. Incisional biopsy. If a needle biopsy is nondiagnostic, an incisional biopsy may be performed, with caveats. First, the skin incision must be placed directly over the mass and ori-ented to allow subsequent scar excision and skin flaps, and drains should be avoided. If the surgeon believes a hema-toma is likely to develop, a drain is useful for limiting soft tissue contamination by tumor cells. At the time of definitive surgical resection, the en bloc resection includes the biopsy scar and the drain tract along with the tumor.3. Excisional biopsy. Any lesion less than 2.0 cm can be excised as long as the resulting wound is small enough to close primarily. Otherwise, excisional biopsy is per-formed only when the initial diagnosis (based on radio-graphic evaluation) indicates that the lesion is benign or when the lesion has the classic appearance of a chondro-sarcoma (in which case, definitive surgical resection can be undertaken).Benign Chest Wall Neoplasms1. Chondroma.

1	that the lesion is benign or when the lesion has the classic appearance of a chondro-sarcoma (in which case, definitive surgical resection can be undertaken).Benign Chest Wall Neoplasms1. Chondroma. Chondromas, seen primarily in children and young adults, are one of the more common benign tumors of the chest wall. They usually occur at the costochondral junction anteriorly and may be confused with costochondri-tis, except that a painless mass is present. Radiographically, the lesion is lobulated and radiodense; it may have diffuse or focal calcifications; and it may displace the bony cortex without penetration. Chondromas may grow to huge sizes if left untreated. Treatment is surgical resection with a 2-cm margin. Large chondromas may harbor well-differentiated chondrosarcoma and should be managed with a 4-cm mar-gin to prevent local recurrence.1272. Fibrous dysplasia. As with chondromas, fibrous dysplasia most frequently occurs in young adults and may be associ-ated with trauma. Pain

1	managed with a 4-cm mar-gin to prevent local recurrence.1272. Fibrous dysplasia. As with chondromas, fibrous dysplasia most frequently occurs in young adults and may be associ-ated with trauma. Pain is an infrequent complaint, and the lesion is typically located in the posterolateral aspect of the rib cage. Radiographically, an expansile mass is present, with cortical thinning and no calcification. Local excision with a 2-cm margin is curative.3. Osteochondroma. Osteochondromas, often found inciden-tally as a solitary lesion on radiograph, are the most common benign bone tumor. Osteochondromas occur in the first two decades of life, and they arise at or near the growth plate of bones. Osteochondromas in the thorax arise from the rib cortex. They are one of several components to the autosomal dominant syndrome, hereditary multiple exostoses. When part of this syndrome, osteochondromas have a high rate of degeneration into chondrosarcomas. Any patient with heredi-tary multiple exostoses

1	dominant syndrome, hereditary multiple exostoses. When part of this syndrome, osteochondromas have a high rate of degeneration into chondrosarcomas. Any patient with heredi-tary multiple exostoses syndrome who develops new pain at the site of an osteochondroma or who notes gradual growth in the mass over time should be carefully evaluated for osteosarcoma. Local excision of a benign osteochondroma is sufficient. If malignancy is determined, wide excision is performed with a 4-cm margin.4. Eosinophilic granuloma. Eosinophilic granulomas are benign osteolytic lesions. Eosinophilic granulomas of the ribs can occur as solitary lesions or as part of a more gener-alized disease process of the lymphoreticular system termed Langerhans cell histiocytosis (LCH). In LCH, the involved tissue is infiltrated with large numbers of histiocytes (similar to Langerhans cells seen in skin and other epithelia), which are often organized as granulomas. The cause is unknown. Of all LCH bone lesions, 79% are

1	with large numbers of histiocytes (similar to Langerhans cells seen in skin and other epithelia), which are often organized as granulomas. The cause is unknown. Of all LCH bone lesions, 79% are solitary eosinophilic granulomas, 7% involve multiple eosinophilic granulomas, and 14% belong to other forms of more systemic LCH. Iso-lated single eosinophilic granulomas can occur in the ribs or skull, pelvis, mandible, humerus, and other sites. They are diagnosed primarily in children between the ages of 5 and 15 years. Because of the associated pain and tenderness, they may be confused with Ewing’s sarcoma or with an inflammatory process such as osteomyelitis. Healing may occur spontaneously, but the typical treatment is limited sur-gical resection with a 2-cm margin.5. Desmoid tumors. Soft tissue neoplasms arising from fas-cial or musculoaponeurotic structures, desmoid tumors con-sist of proliferations of benign-appearing fibroblastic cells, abundant collagen, and few mitoses. Desmoid

1	tissue neoplasms arising from fas-cial or musculoaponeurotic structures, desmoid tumors con-sist of proliferations of benign-appearing fibroblastic cells, abundant collagen, and few mitoses. Desmoid tumors pos-sess alterations in the adenomatous polyposis coli (APC)/β-catenin pathway. Cyclin D1 dysregulation is thought to play a significant role in their pathogenesis.128 Associations with other diseases and conditions are well documented, espe-cially those with similar alterations in the APC pathway, such as familial adenomatous polyposis (Gardner’s syn-drome). Other conditions with increased risk of desmoid tumor formation include increased estrogen states (preg-nancy) and trauma. Surgical incisions (abdominal and tho-rax) have been the site of desmoid development, either in or near the scar. Clinically, patients are usually in the third to fourth decade of life and have pain, a chest wall mass, or both. The tumor is usually fixed to the chest wall, but not to the over-lying skin.

1	scar. Clinically, patients are usually in the third to fourth decade of life and have pain, a chest wall mass, or both. The tumor is usually fixed to the chest wall, but not to the over-lying skin. There are no typical radiographic findings, but MRI may delineate muscle or soft tissue infiltration. Des-moid tumors do not metastasize, but they have a significant propensity to recur locally, with rates as high as 5% to 50%, sometimes despite complete initial resection with histologi-cally negative margins.129 Such locally aggressive behavior is secondary to microscopic tumor infiltration of muscle and surrounding soft tissues and prompts some to consider them a low-grade form of fibrosarcoma. Because the lesions have low cellularity and poor yield with FNA, an open incisional biopsy for lesions over 3 to 4 cm is often necessary, following the caveats listed ear-lier (see biopsy section). Surgery consists of wide local excision with a 2to 4-cm margin and intraoperative fro-zen section

1	over 3 to 4 cm is often necessary, following the caveats listed ear-lier (see biopsy section). Surgery consists of wide local excision with a 2to 4-cm margin and intraoperative fro-zen section assessment of resection margins. Typically, chest wall resection, including the involved rib(s) and one rib above and below the tumor with a 4to 5-cm margin of rib, is required. A margin of less than 1 cm results in much higher local recurrence rates. If a major neurovascu-lar structure would have to be sacrificed, leading to high morbidity, then a margin of less than 1 cm would have to suffice. Survival after wide local excision with negative margins is 90% at 10 years.130Brunicardi_Ch19_p0661-p0750.indd 72201/03/19 7:01 PM

1	CHAPTER 19723CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAPrimary Malignant Chest Wall TumorsMalignant tumors of the chest wall are either metastatic lesions from another primary tumor or sarcoma. Soft tissue sarcomas of the chest wall include fibrosarcomas, liposarcomas, malignant fibrous histiocytomas (MFHs), rhabdomyosarcomas, angiosar-comas, and other extremely rare lesions (Fig. 19-40). Despite the prevalence of localized disease, soft tissue sarcomas of the chest wall have significantly worse survival than similar tumors located on the extremities or the head and neck region. The fac-tors impacting on risk of death from soft tissue sarcomas of the chest wall are presented in Table 19-23. All sarcomas have a propensity to spread to the lungs.While many varieties of sarcoma exist, the primary features affecting prognosis are histologic grade and respon-siveness to chemotherapy (Table 19-24). Preoperative (neo-adjuvant) chemotherapy offers the ability to (a) assess tumor

1	exist, the primary features affecting prognosis are histologic grade and respon-siveness to chemotherapy (Table 19-24). Preoperative (neo-adjuvant) chemotherapy offers the ability to (a) assess tumor chemosensitivity by the degree of tumor size reduction and microscopic necrosis; (b) determine tumor sensitivity to spe-cific chemotherapeutic agents; and (c) improve resectability by reducing tumor size. Patients whose tumors are responsive to preoperative chemotherapy have a much better prognosis than those with a poor response. Information about tumor response to chemotherapy, the patient’s physiologic state and capacity to receive treatment, and metastatic disease status is used to determine optimal therapy. The initial treatment is either (a) preoperative chemotherapy (for patients with osteosarcoma, rhabdomyosarcoma, primitive neuroectoder-mal tumor, or Ewing’s sarcoma) followed by surgery and postoperative chemotherapy; (b) primary surgical resection and reconstruction (for

1	with osteosarcoma, rhabdomyosarcoma, primitive neuroectoder-mal tumor, or Ewing’s sarcoma) followed by surgery and postoperative chemotherapy; (b) primary surgical resection and reconstruction (for patients with nonmetastatic MFH, fibrosarcoma, liposarcoma, or synovial sarcoma); or (c) preoperative chemotherapy followed by surgical resection if indicated in patients presenting with metastatic soft tis-sue sarcomas. Contiguous involvement of underlying lung or other soft tissues or the presence of pulmonary metasta-ses does not preclude successful surgery. In fact, patients receiving surgical intervention have significantly better overall survival. Median survival with surgical resection is 25 months compared to 8 months without resection. Addi-tional prognostic variables that are important for long-term survival include tumor size, grade, stage, and negative re-resection margin.131 With the exception of rhabdomyosar-comas, the primary treatment of these lesions is wide surgical

1	for long-term survival include tumor size, grade, stage, and negative re-resection margin.131 With the exception of rhabdomyosar-comas, the primary treatment of these lesions is wide surgical resection with 4-cm margins and reconstruction.132The following is an overview of several chest wall sarcomas.1. Chondrosarcoma. Chondrosarcomas are the most common primary chest wall malignancy. As with chondromas, they usually arise anteriorly from the costochondral arches. CT scan shows a radiolucent lesion often with stippled calcifica-tions pathognomonic for chondrosarcomas (Fig. 19-41). The involved bony structures are also destroyed. Most chondro-sarcomas are slow-growing, low-grade tumors; these often painful masses can reach massive proportions.127 For this reason, any lesion in the anterior chest wall likely to be a low-grade chondrosarcoma should be treated with wide (4-cm) resection after metastatic disease to the lungs or bones is ruled out. Chondrosarcomas are not sensitive to

1	chest wall likely to be a low-grade chondrosarcoma should be treated with wide (4-cm) resection after metastatic disease to the lungs or bones is ruled out. Chondrosarcomas are not sensitive to radiation or chemo-therapy. Prognosis is determined by tumor grade and extent of resection. With a low-grade tumor and wide resection, patient survival at 5 to 10 years can be as high as 60% to 80%.2. Osteosarcoma. While osteosarcomas are the most com-mon bone malignancy, they represent only 10% to 15% of all malignant chest wall tumors.133,134 They primarily occur in young adults as rapidly enlarging, painful masses; how-ever, osteosarcomas can occur in older patients as well, Figure 19-40. Chest computed tomography scan showing a right chest wall tumor (arrow). Tissue diagnosis revealed that this mass was a leiomyosarcoma.Brunicardi_Ch19_p0661-p0750.indd 72301/03/19 7:01 PM 724SPECIFIC CONSIDERATIONSPART IITable 19-23Cox proportional hazards model for risk of death from soft tissue

1	this mass was a leiomyosarcoma.Brunicardi_Ch19_p0661-p0750.indd 72301/03/19 7:01 PM 724SPECIFIC CONSIDERATIONSPART IITable 19-23Cox proportional hazards model for risk of death from soft tissue sarcomaNHAZARD RATIO95% CIP VALUEGender Male Female39374113Reference group0.897Reference group0.843–0.955Reference group.001Age 50 years 51–70 years >70 years183730993114Reference group1.1311.538Reference group1.026–1.2471.395–1.697Reference group.013<.001Race Caucasian Non-Caucasian7152898Reference group1.212Reference group1.093–1.344Reference group<.001Histologic type Fibrosarcoma MFH Liposarcoma LMS/GIST489252915343498Reference group1.2810.8941.204Reference group1.097–1.4950.759–1.0541.033–1.403Reference group.002.182.018Location Head and neck Trunk Extremity Retroperitoneum57640542474946Reference group1.2551.0031.276Reference group1.096–1.4380.875–1.1511.093–1.489Reference group.001.960.002Stage Localized Regional Distant500617241320Reference group1.5752.897Reference

1	group1.2551.0031.276Reference group1.096–1.4380.875–1.1511.093–1.489Reference group.001.960.002Stage Localized Regional Distant500617241320Reference group1.5752.897Reference group1.458–1.7022.660–3.155Reference group<.001<.001Surgical treatment Yes No67541296Reference group1.562Reference group1.443–1.691Reference group<.001Radiation therapy Yes No21755875Reference group1.151Reference group1.070–1.239Reference group<.001Chemotherapy Yes No10626988Reference group0.909Reference group0.829–0.996Reference group.041Abbreviations: CI = confidence interval; GIST = gastrointestinal stromal tumor; LMS = leiomyosarcoma; MFH = malignant fibrous histiocytoma.Reproduced with permission from Gutierrez JC, Perez EA, Franceschi D, et al: Outcomes for soft-tissue sarcoma in 8249 cases from a large state cancer registry, J Surg Res. 2007;141(1):105-114.Table 19-24Classification of sarcomas by therapeutic responseTUMOR TYPECHEMOTHERAPY SENSITIVITYOsteosarcoma+Rhabdomyosarcoma+Primitive neuroectodermal

1	registry, J Surg Res. 2007;141(1):105-114.Table 19-24Classification of sarcomas by therapeutic responseTUMOR TYPECHEMOTHERAPY SENSITIVITYOsteosarcoma+Rhabdomyosarcoma+Primitive neuroectodermal tumor+Ewing’s sarcoma+Malignant fibrous histiocytoma±Fibrosarcoma±Liposarcoma±Synovial sarcoma±sometimes in association with previous radiation, Paget’s disease, or chemotherapy. Radiographically, the typical appearance consists of spicules of new periosteal bone formation producing a sunburst appearance. Osteosarcomas have a propensity to spread to the lungs, and up to one-third of patients present with metastatic disease. Osteosarcomas are potentially sensitive to chemotherapy. Currently, pre-operative chemotherapy is common. After chemotherapy, complete resection is performed with wide (4-cm) margins, followed by reconstruction. In patients presenting with lung metastases that are potentially amenable to surgical resection, induction chemotherapy may be given, followed by surgical resection

1	followed by reconstruction. In patients presenting with lung metastases that are potentially amenable to surgical resection, induction chemotherapy may be given, followed by surgical resection of the primary tumor and of the pul-monary metastases. Following surgical treatment of known disease, additional maintenance chemotherapy is usually recommended.Brunicardi_Ch19_p0661-p0750.indd 72401/03/19 7:01 PM

1	CHAPTER 19725CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAFigure 19-41. Chest computed tomography scan showing a right posterior lung tumor. In the appropriate clinical setting, stippled calcifica-tions (white streaks in right lung mass) are highly indicative of chondrosarcomas.3. Malignant fibrous histiocytoma. Originally thought to derive from histiocytes because of the microscopic appear-ance of cultured tumor cells, these tumors likely originate from the fibroblast. MFHs are generally the most common soft tissue sarcoma of late adult life, although they are rare on the chest wall. The typical age at presentation is between age 50 and 70 years. Presentation is pain, with or without a palpable mass. Radiographically, a mass is usually evident, with destruction of surrounding tissue and bone. Treatment is wide resection with a margin of 4 cm or more and recon-struction. Over two-thirds of patients suffer from distant metastasis or local recurrence.4. Liposarcoma. Liposarcomas make up 15%

1	Treatment is wide resection with a margin of 4 cm or more and recon-struction. Over two-thirds of patients suffer from distant metastasis or local recurrence.4. Liposarcoma. Liposarcomas make up 15% of chest wall sarcomas. Most liposarcomas are low-grade tumors that have a propensity to recur locally, given their infiltrative nature. They typically present as a painless mass. Treatment is wide resection and reconstruction. Intraoperative mar-gins should be evaluated (as with all sarcomas) and resec-tion continued, if feasible, until margins are negative. Local recurrence can be treated with reexcision, with occasional use of radiotherapy.5. Fibrosarcoma. Often presenting as a large, painful mass, these lesions are visible on plain radiograph or CT, with sur-rounding tissue destruction. Treatment is wide local excision with intraoperative frozen-section analysis of margins, fol-lowed by reconstruction. Local and systemic recurrence is frequent. Patient survival at 5 years is about 50%

1	is wide local excision with intraoperative frozen-section analysis of margins, fol-lowed by reconstruction. Local and systemic recurrence is frequent. Patient survival at 5 years is about 50% to 60%.6. Rhabdomyosarcoma. Rhabdomyosarcomas are rare tumors of the chest wall. Microscopically, they are a spindle cell tumor. The diagnosis often depends on immunohistochemi-cal staining for muscle markers. Rhabdomyosarcomas are sensitive to chemotherapy. Treatment consists of preopera-tive chemotherapy with subsequent surgical resection.Other Tumors of the Chest Wall1. Primitive neuroectodermal tumors (PNETs) and Ewing’s sarcoma. PNETs (neuroblastomas, ganglioneuroblastomas, and ganglioneuromas) derive from primordial neural crest cells that migrate from the mantle layer of the developing spinal cord. Histologically, PNETs and Ewing’s sarcomas are small, round cell tumors; both possess a translocation between the long arms of chromosomes 11 and 22 within their genetic makeup. They also share

1	cord. Histologically, PNETs and Ewing’s sarcomas are small, round cell tumors; both possess a translocation between the long arms of chromosomes 11 and 22 within their genetic makeup. They also share a consistent pattern of proto-oncogene expression and have been found to express the product of the MIC2 gene. Ewing’s sarcoma occurs in adolescents and young adults who present with progressive chest wall pain, but without the presence of a mass. Systemic symptoms of malaise and fever are often present. Laboratory studies reveal an ele-vated erythrocyte sedimentation rate and mild white blood cell elevation. Radiographically, the characteristic onion peel appearance is produced by multiple layers of perios-teum in the bone formation. Evidence of bony destruction is also common. The diagnosis can be made by a percutaneous needle biopsy or an incisional biopsy. These tumors have a strong propensity to metastasize to the lungs and skeleton; patient survival rates are thus only 50% or less

1	can be made by a percutaneous needle biopsy or an incisional biopsy. These tumors have a strong propensity to metastasize to the lungs and skeleton; patient survival rates are thus only 50% or less at 3 years. Increasing tumor size is associated with decreasing survival. Treatment has improved sig-nificantly and now consists of multiagent chemotherapy, radiation therapy, and surgery. Patients are typically treated preoperatively with chemotherapy and reevaluated with radiologic imaging. When residual disease is identified, sur-gical resection and reconstruction are performed followed by maintenance chemotherapy.2. Plasmacytoma. Solitary plasmacytomas of the chest wall are very rare, with approximately 25 to 30 cases per year in the United States.133 The typical presentation is pain with-out a palpable mass. Plain radiographs show an osteolytic lesion in the region of the pain. As with other chest wall tumors, a needle biopsy under CT guidance is performed for diagnosis.

1	is pain with-out a palpable mass. Plain radiographs show an osteolytic lesion in the region of the pain. As with other chest wall tumors, a needle biopsy under CT guidance is performed for diagnosis. Histologically, the lesion is identical to multiple myeloma, with sheets of plasma cells. It occurs at an aver-age age of 55 years. Evaluation for systemic myeloma is performed with bone marrow aspiration, testing of calcium levels, and measurement of urinary Bence Jones proteins. If the results of these studies are negative, then a solitary Brunicardi_Ch19_p0661-p0750.indd 72501/03/19 7:01 PM 726SPECIFIC CONSIDERATIONSPART IIplasmacytoma is diagnosed. Surgery is usually limited to a biopsy only, which may be excisional.134 Treatment consists of radiation with doses of 4000 to 5000 cGy. Up to 75% of patients develop systemic multiple myeloma with 10-year survival of approximately 20%.Chest Wall ReconstructionThe primary determinant of long-term freedom from recurrence and overall

1	Up to 75% of patients develop systemic multiple myeloma with 10-year survival of approximately 20%.Chest Wall ReconstructionThe primary determinant of long-term freedom from recurrence and overall survival is margin status; therefore, adequate mar-gins of normal tissue must be included in the en bloc resec-tion. En bloc resection should include involved ribs, sternum, superior sulcus, or spine if necessary; invasion of these struc-tures should not be considered a contraindication to surgery in an otherwise fit patient. The resection should include at least one normal adjacent rib above and below the tumor, with all intervening intercostal muscles and pleura. In addition, an en bloc resection of overlying chest wall muscles is often neces-sary, such as of the pectoralis minor or major, serratus anterior, or latissimus dorsi. When the periphery of the lung is involved with the neoplasm, it is appropriate to resect the adjacent part of the pulmonary lobe in continuity (Fig. 19-42).

1	serratus anterior, or latissimus dorsi. When the periphery of the lung is involved with the neoplasm, it is appropriate to resect the adjacent part of the pulmonary lobe in continuity (Fig. 19-42). Involvement of the sternum by a malignant tumor requires total resection of the sternum with the adjacent cartilage. Techniques for postop-erative respiratory support are now good enough that resection should not be compromised because of any concern about the patient’s ability to be adequately ventilated in the early postop-erative period.The extent of resection depends on the tumor’s location and on any involvement of contiguous structures. Laterally based lesions often require simple wide excision, with resec-tion of any contiguously involved lung, pleura, muscle, or skin. Anteriorly based lesions contiguous with the sternum require partial sternectomy. Primary malignant tumors of the sternum may require complete sternectomy. Posterior lesions involving the rib heads over their

1	based lesions contiguous with the sternum require partial sternectomy. Primary malignant tumors of the sternum may require complete sternectomy. Posterior lesions involving the rib heads over their articulations with the vertebral bodies may, depending on the extent of rib involvement, require partial en bloc vertebrectomy.Optimal management of larger tumors includes care-ful preoperative planning and execution of the surgery by the thoracic surgeon and an experienced plastic surgeon in order to ensure optimal physiologic and cosmetic results. With these measures, reconstruction at the same operation can be accomplished.135 Reconstruction of a large defect in the chest wall requires the use of some type of material to prevent lung herniation and to provide stability for the chest wall (see Fig. 19-42). Mild degrees of paradoxical motion are often well tolerated if the area of instability is relatively small. Historically, a wide variety of materials have been used to reestablish chest

1	Fig. 19-42). Mild degrees of paradoxical motion are often well tolerated if the area of instability is relatively small. Historically, a wide variety of materials have been used to reestablish chest wall stability, including rib autografts, steel struts, acrylic plates, and numerous synthetic meshes. The current preference is either a 2-mm polytetrafluoroethylene (Gore-Tex) patch or a double-layer polypropylene (Marlex) mesh sandwiched with methylmethacrylate. There are sev-eral properties that make Gore-Tex an excellent material for use in chest wall reconstruction: (a) it is impervious to fluid, which prevents pleural fluid from entering the chest wall and minimizes the formation of seromas, which can compromise the myocutaneous flap viability and provide a nidus for infec-tion; and (b) it provides excellent rigidity and stability when secured taut to the surrounding bony structure and, as a result, provides a firm platform for myocutaneous flap reconstruc-tion. Except for smaller

1	it provides excellent rigidity and stability when secured taut to the surrounding bony structure and, as a result, provides a firm platform for myocutaneous flap reconstruc-tion. Except for smaller lesions, tissue coverage requires the use of myocutaneous flaps (latissimus dorsi, serratus anterior, rectus abdominis, or pectoralis major muscles).136,137MEDIASTINUMAnatomy and Pathologic EntitiesThe mediastinum can be divided into compartments for classi-fication of anatomic components and disease processes, which, despite substantial overlap, facilitates understanding of general concepts of surgical interest. Several classification schemes exist, but for the purposes of this chapter, the three-compart-ment model is used (Fig. 19-43). The anterior compartment lies between the sternum and the anterior surface of the heart and great vessels. The visceral or middle compartment is located between the great vessels and the trachea. As the name implies, the posterior compartment lies posterior

1	anterior surface of the heart and great vessels. The visceral or middle compartment is located between the great vessels and the trachea. As the name implies, the posterior compartment lies posterior and includes the para-vertebral sulci, bilaterally, and the paraesophageal area.ABFigure 19-42. Principles of recon-struction after resection of a chest wall tumor (osteogenic sarcoma) are shown. A. En bloc resection of the involved chest wall, including nor-mal ribs above and below the tumor as well as pulmonary parenchyma, must be performed. The resected specimen is shown. B. A prosthesis has been sewn in place. In the lower third of the prosthesis, the line of diaphragm reattachment is seen. The skin defect was closed with a myo-cutaneous flap from the ipsilateral rectus muscle.Brunicardi_Ch19_p0661-p0750.indd 72601/03/19 7:01 PM

1	CHAPTER 19727CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAPosteriormediastinumAnterosuperiormediastinumMiddlemediastinumFigure 19-43. Anatomic division of the mediastinum.ThymusFigure 19-44. Normal appearance of the thymus gland in childhood. Ao = aorta; PA = pulmonary artery; VC = vena cava.The normal content of the anterior compartment includes the thymus gland or its remnant, the internal mammary artery and vein, lymph nodes, and fat. The thymus gland is large during childhood, occupying the entire anterior mediastinum (Fig. 19-44) but decreases in both thickness and length after adolescence and takes on a more fatty content, with only resid-ual islands of thymic cellular components (Fig. 19-45). The middle mediastinal compartment contains the pericardium and its contents, the ascending and transverse aorta, the superior and inferior venae cavae, the brachiocephalic artery and vein, the phrenic and upper vagus nerves, the trachea and main bronchi and corresponding lymph nodes, and the

1	transverse aorta, the superior and inferior venae cavae, the brachiocephalic artery and vein, the phrenic and upper vagus nerves, the trachea and main bronchi and corresponding lymph nodes, and the central portions of the pulmonary arteries and veins. The posterior compartment contains the descending aorta, esophagus, thoracic duct, azygos and hemiazygos veins, and lymph nodes. Numerous pathologic variants may be present in the various compartments, with much overlap. Table 19-25 includes the most common pathologic entities listed by compartment.138,139History and Physical ExaminationMediastinal pathology varies significantly by patient age. In children, neurogenic tumors of the posterior mediastinum are most common, followed by lymphoma, which is usually located in the anterior or middle compartment. Thymoma in child-hood is rare (Table 19-26). In adults, the most common tumors include neurogenic tumors of the posterior compartment, benign cysts occurring in any compartment, and

1	compartment. Thymoma in child-hood is rare (Table 19-26). In adults, the most common tumors include neurogenic tumors of the posterior compartment, benign cysts occurring in any compartment, and thymomas of the ante-rior mediastinum (Table 19-27). In both age groups, about 25% of mediastinal tumors are malignant. Pediatric tumors will be discussed in Chapter 39.Up to two-thirds of mediastinal tumors in adults are dis-covered as asymptomatic abnormalities on radiologic studies ordered for other problems, particularly now that screening CT examinations are more prevalent. When symptomatic, these tumors are significantly more likely to be malignant. Charac-teristics such as size, location, rate of growth, and associated inflammation are important factors that correlate with symp-toms. Large, bulky tumors, expanding cysts, and teratomas can cause compression of mediastinal structures, in particular the trachea, and lead to cough, dyspnea on exertion, or stridor. Chest pain or dyspnea may

1	bulky tumors, expanding cysts, and teratomas can cause compression of mediastinal structures, in particular the trachea, and lead to cough, dyspnea on exertion, or stridor. Chest pain or dyspnea may be reported secondary to associated pleural effusions, cardiac tamponade, or phrenic nerve involve-ment. Occasionally, a mediastinal mass near the aortopulmonary window may be identified in a workup for hoarseness because of left recurrent laryngeal nerve involvement. The patient in Fig. 19-46 presented with hoarseness due to nodal compression of the left recurrent laryngeal nerve from a primary lung cancer with metastases to the level 5 and 6 lymph nodes in the region of the aortopulmonary window.The history and physical examination in conjunction with the imaging findings may suggest a specific diagnosis (Table 19-28). In one series, systemic symptoms were present in 50% of patients with a mediastinal mass and a lymphoproliferative dis-order, as compared with only 29% of patients with

1	diagnosis (Table 19-28). In one series, systemic symptoms were present in 50% of patients with a mediastinal mass and a lymphoproliferative dis-order, as compared with only 29% of patients with other masses (such as thymic or neurogenic). Laboratory signs of inflamma-tion were also noted; the erythrocyte sedimentation rate and C-reactive protein levels were elevated and leukocytosis was present in 86% of patients with a lymphoproliferative disorder, as compared with only 58% of patients with other types of medi-astinal masses.Imaging and Serum MarkersChest CT or MRI is required to fully delineate the anatomy.140 A contrast-enhanced CT scan enables clear delineation of the Brunicardi_Ch19_p0661-p0750.indd 72701/03/19 7:01 PM 728SPECIFIC CONSIDERATIONSPART IITable 19-25Usual location of the common primary tumors and cysts of the mediastinumANTERIOR COMPARTMENTVISCERAL COMPARTMENTPARAVERTEBRAL SULCIThymomaEnterogenous cystNeurilemoma-schwannomaGerm cell

1	19-25Usual location of the common primary tumors and cysts of the mediastinumANTERIOR COMPARTMENTVISCERAL COMPARTMENTPARAVERTEBRAL SULCIThymomaEnterogenous cystNeurilemoma-schwannomaGerm cell tumorLymphomaNeurofibromaLymphomaPleuropericardial cystMalignant schwannomaLymphangiomaMediastinal granulomaGanglioneuromaHemangiomaLymphoid hamartomaGanglioneuroblastomaLipomaMesothelial cystNeuroblastomaFibromaNeuroenteric cystParagangliomaFibrosarcomaParagangliomaPheochromocytomaThymic cystPheochromocytomaFibrosarcomaParathyroid adenomaThoracic duct cystLymphomaReproduced with permission from Shields TW: Mediastinal Surgery. Philadelphia, PA: Lea & Febiger; 1991.Table 19-26Mediastinal tumors in childrenTUMOR TYPEPERCENTAGE OF TOTALLOCATIONNeurogenic tumors40PosteriorLymphomas18Anterior/middleCysts18AllGerm cell tumors11AnteriorMesenchymal tumors9AllThymomasRareAnteriorReproduced with permission from Silverman NA, Sabiston DC: Mediastinal masses, Surg Clin North Am. 1980

1	cell tumors11AnteriorMesenchymal tumors9AllThymomasRareAnteriorReproduced with permission from Silverman NA, Sabiston DC: Mediastinal masses, Surg Clin North Am. 1980 Aug;60(4):757-777.Figure 19-45. Computed tomography scan showing the normal appearance of an involuted thymus gland in an adult. Note the near-total fatty appearance of the gland with only tiny islands of soft tissue scattered within it (small arrows).soft tissue structures from the vasculature and is preferred over noncontrast studies. If there is concern for invasion of vas-cular structures or spinal involvement, MRI is more accurate than CT scan and provides important information regarding respectability.If an endocrine origin is suspected, several other imaging modalities are available (Table 19-29). Single-photon emission CT (SPECT) technology may be used to improve image contrast and give information on three-dimensional localization, largely replacing conventional two-dimensional nuclear imaging studies. If a

1	CT (SPECT) technology may be used to improve image contrast and give information on three-dimensional localization, largely replacing conventional two-dimensional nuclear imaging studies. If a thyroid origin is suspected, a thyroid scan using 131I or 123I can identify most intrathoracic goiters and identify the extent of functioning thyroid tissue. If indicated, the thyroid scan should precede other scans requiring iodine-containing contrast agents because they would subsequently interfere with iodine tracer uptake by thyroid tissue. If a pheochromo-cytoma or neuroblastoma is suspected, the octreotide scan or 123I-metaiodobenzylguanidine (MIBG) scans are helpful in diag-nosis and localization. The sestamibi scan may be useful for diagnosing and localizing a mediastinal parathyroid gland. PET is useful for distinguishing malignant from benign tumors and may help detect distant metastases in some patients. However, the role of routine PET imaging for staging surgically resectable

1	PET is useful for distinguishing malignant from benign tumors and may help detect distant metastases in some patients. However, the role of routine PET imaging for staging surgically resectable lesions of the mediastinum has not been established.The use of serum markers to evaluate a mediastinal mass can be invaluable in some patients. For example, nonsemino-matous and seminomatous germ cell tumors can frequently be diagnosed and often distinguished from one another by the lev-els of α-fetoprotein (AFP) and human chorionic gonadotropin (hCG). In over 90% of nonseminomatous germ cell tumors, either the AFP or the hCG level will be elevated. Results are close to 100% specific if the level of either AFP or hCG is greater than 500 ng/mL. Some centers institute chemotherapy based on this result alone, without biopsy confirmation of the diagnosis. In contrast, the AFP level in patients with mediastinal seminoma is always normal; only 10% will have elevated hCG, which is usually less than

1	alone, without biopsy confirmation of the diagnosis. In contrast, the AFP level in patients with mediastinal seminoma is always normal; only 10% will have elevated hCG, which is usually less than 100 ng/mL. Other serum markers, such as intact parathyroid hormone level for ectopic parathyroid Brunicardi_Ch19_p0661-p0750.indd 72801/03/19 7:01 PM

1	CHAPTER 19729CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAFigure 19-46. Computed tomography scan of a patient who pre-sented with hoarseness due to compression of the left recurrent laryngeal nerve caused by mediastinal lymph node metastases to the aortopulmonary window area (arrow) from a primary lung cancer.adenomas, may be useful for diagnosing and also for intraopera-tively confirming complete resection. After successful resection of a parathyroid adenoma, this hormone level should rapidly normalize.Diagnostic Nonsurgical Biopsies of the MediastinumThe treatment of up to 60% of patients with anterior mediastinal masses is ultimately nonsurgical, so it is essential to understand all options for obtaining adequate tissue for a definitive diagno-sis using the least invasive approach. CT-guided needle biopsy, EBUSand EUS-guided FNA, and even core-needle biopsy (either CT-guided and EUS-guided) have proven most useful for cytologic and tissue diagnosis of mediastinal masses and

1	CT-guided needle biopsy, EBUSand EUS-guided FNA, and even core-needle biopsy (either CT-guided and EUS-guided) have proven most useful for cytologic and tissue diagnosis of mediastinal masses and lymphadenopathy.When FNA and core-needle biopsy were combined, the accuracy was 98%, compared to 79% for each modality inde-pendently. In addition, core-needle biopsy changed the diagno-sis in nine cases that had been missed by FNA due to inadequate specimens. Finally, core-needle biopsy was better at diagnosis for benign diseases compared to FNA. Accessible nodal stations Table 19-27Mediastinal tumors in adultsTUMOR TYPEPERCENTAGE OF TOTALLOCATIONNeurogenic tumors21PosteriorCysts20AllThymomas19AnteriorLymphomas13Anterior/middleGerm cell tumors11AnteriorMesenchymal tumors7AllEndocrine tumors6Anterior/middleData from Shields TW: General Thoracic Surgery, 4th ed. Baltimore, MD: Lippincott Williams & Wilkins; 1994.include subcarinal (level 7), aortopulmonary (level 5), parae-sophageal (level 8),

1	from Shields TW: General Thoracic Surgery, 4th ed. Baltimore, MD: Lippincott Williams & Wilkins; 1994.include subcarinal (level 7), aortopulmonary (level 5), parae-sophageal (level 8), and inferior pulmonary ligament (level 9) as well as paratracheal (level 4).141 Technical expertise in these modalities should be pursued by thoracic and general surgeons.Historically, needle biopsies of anterior mediastinal masses were reportedly sensitive and specific for most carcinomatous tumors, but there were questions regarding accuracy for diagnos-ing lymphomas.142 However, advances in cytopathology as well as needle biopsy technology have substantially improved diag-nostic accuracy such that most centers are reporting yields rang-ing from 75% to 80% for the diagnosis of lymphoma as well. To achieve maximal diagnostic yield for mediastinal masses sug-gestive of a lymphoma, it is necessary to obtain multiple fine-needle aspirates, preferably with immediate onsite rapid cytologic analysis to

1	maximal diagnostic yield for mediastinal masses sug-gestive of a lymphoma, it is necessary to obtain multiple fine-needle aspirates, preferably with immediate onsite rapid cytologic analysis to confirm sampling of the target tissue and adequate cellularity. This also facilitates processing of the sample to ensure that proper studies for lymphoma, including flow cytometry, are obtained. If the needle biopsy is inconclusive, surgical biopsy can be performed.143,144 If the lesion is accessible by CT-guided or EUS-guided core-needle biopsy, intraoperative frozen section or immediate cytologic smear of a core biopsy can also be per-formed. Currently, core-needle biopsy with EBUS is not possible. The authors perform their own endobronchial, endoscopic, and CT-guided transbronchial and transthoracic biopsies, and in our experience, lack of cellularity in the aspirate is readily apparent. In general, plans to proceed with surgical biopsy are made in combination with the image-guided

1	transthoracic biopsies, and in our experience, lack of cellularity in the aspirate is readily apparent. In general, plans to proceed with surgical biopsy are made in combination with the image-guided aspiration and, as such, are performed in the same setting. This enables the authors to avoid a more invasive surgical procedure when FNA or core-needle biopsy is sufficient without contributing to delays in diagnosis by having multiple attempts from multiple providers (such as inter-ventional radiology and pulmonology) before involvement of the surgeon in the diagnostic workup.Table 19-28Signs and symptoms suggestive of various diagnoses in the setting of a mediastinal massDIAGNOSISHISTORY AND PHYSICAL FINDINGSCOMPARTMENT LOCATION OF MASSLymphomaNight sweats, weight loss, fatigue, extrathoracic adenopathy, elevated erythrocyte sedimentation rate or C-reactive protein level, leukocytosisAny compartmentThymoma with myasthenia gravisFluctuating weakness, early fatigue, ptosis,

1	extrathoracic adenopathy, elevated erythrocyte sedimentation rate or C-reactive protein level, leukocytosisAny compartmentThymoma with myasthenia gravisFluctuating weakness, early fatigue, ptosis, diplopiaAnteriorMediastinal granulomaDyspnea, wheezing, hemoptysisVisceral (middle)Germ cell tumorMale gender, young age, testicular mass, elevated levels of human chorionic gonadotropin and/or α-fetoproteinAnteriorBrunicardi_Ch19_p0661-p0750.indd 72901/03/19 7:01 PM 730SPECIFIC CONSIDERATIONSPART IITable 19-29Nuclear imaging relevant to the mediastinumRADIOPHARMACEUTICAL, RADIONUCLIDE, OR RADIOCHEMICALLABELDISEASE OF INTERESTIodine131I, 123IRetrosternal goiter, thyroid cancerMonoclonal antibodies111In, 99mTcNSCLC, colon and breast cancer, prostate cancer metastasesOctreotide111InAmine precursor uptake decarboxylation tumors: carcinoid, gastrinoma, insulinoma, small cell lung cancer, pheochromocytoma, glucagonoma, medullary thyroid carcinoma, paragangliomaGallium67GaLymphoma, NSCLC,

1	uptake decarboxylation tumors: carcinoid, gastrinoma, insulinoma, small cell lung cancer, pheochromocytoma, glucagonoma, medullary thyroid carcinoma, paragangliomaGallium67GaLymphoma, NSCLC, melanomaSestamibi99mTcMedullary thyroid carcinoma, nonfunctional papillary or follicular thyroid carcinoma, Hürthle cell thyroid carcinoma, parathyroid adenoma or carcinomaThallium201TlSee sestamibiMIBG131I, 123IPheochromocytoma, neuroblastoma; see also octreotideFluorodeoxyglucose18FGeneral oncologic imaging, breast and colon cancer, melanomaAbbreviations: MIGB = metaiodobenzylguanidine; NSCLC = non–small cell lung cancer.Reproduced with permission from Pearson FG, Cooper JD, Deslauriers J, et al: Thoracic Surgery, 2nd ed. New York, NY: Elsevier/Churchill Livingstone; 2002.Surgical Biopsies and Resection of Mediastinal MassesFor tumors of the mediastinum that are not amenable to an endo-scopic or CT-guided needle biopsy or that do not yield sufficient tissue for diagnosis, a surgical biopsy is

1	of Mediastinal MassesFor tumors of the mediastinum that are not amenable to an endo-scopic or CT-guided needle biopsy or that do not yield sufficient tissue for diagnosis, a surgical biopsy is indicated. The defini-tive approach to a surgical biopsy of the anterior mediastinum is through a median sternotomy. At the time of sternotomy, if the lesion is easily resectable, it should be completely removed. Given the invasiveness of the procedure and the inability in some patients to obtain a definitive diagnosis by frozen section, less invasive procedures are preferable if the lesion is large or if the CT scan or history suggests that surgery is not the best definitive treatment.Masses in the paratracheal region are easily biopsied by mediastinoscopy. For tumors of the anterior or posterior medi-astinum, a left or right VATS approach often allows safe and adequate surgical biopsies. In some patients, an anterior medi-astinotomy (i.e., Chamberlain procedure) may be ideal for an anterior

1	medi-astinum, a left or right VATS approach often allows safe and adequate surgical biopsies. In some patients, an anterior medi-astinotomy (i.e., Chamberlain procedure) may be ideal for an anterior tumor or a tumor with significant parasternal extension. Before a surgical biopsy is pursued, a discussion should be held with the pathologist regarding routine histologic assessment, spe-cial stains and markers, and requirements for lymphoma workup.Surgical resection using minimally invasive approaches, including video-assisted and robotic thoracoscopic surgery and transcervical, are now routine for the vast majority of middle and posterior tumors and for moderate sized (<5 to 6 cm) anterior mediastinal tumors.145-148 Outcomes comparing VATS to open thymectomy in patients with myasthenia gravis without thymoma were prospectively evaluated by Chang and colleagues in 2005, and no differences were seen in terms of response to therapy and recurrence of symptoms. Pain scores were

1	gravis without thymoma were prospectively evaluated by Chang and colleagues in 2005, and no differences were seen in terms of response to therapy and recurrence of symptoms. Pain scores were significantly better in the VATS approach.149 These reports and others support application of VATS for the majority of ante-rior mediastinal masses.Other minimally invasive approaches are under study. For example, good results have been reported using a cervical incision with a sternal retractor for thymus removal. The upward lift allows the surgeon reasonable access to the anterior medi-astinum and has proven adequate in some centers for definitive resection of the thymus gland for myasthenia gravis.150For larger anterior mediastinal masses or in centers where expertise in thoracoscopy is not available, median sternotomy and thoracotomy remain excellent options for resection of ante-rior mediastinal masses. Occasionally, a lateral thoracotomy with sternal extension (hemi-clamshell) provides

1	median sternotomy and thoracotomy remain excellent options for resection of ante-rior mediastinal masses. Occasionally, a lateral thoracotomy with sternal extension (hemi-clamshell) provides excellent exposure for extensive mediastinal tumors that have a lateral component.Most surgeons would agree that if a larger anterior medi-astinal tumor is seen or malignancy is suspected, a median ster-notomy with a more radical resection should be performed.Mediastinal NeoplasmsThymic Hyperplasia. Diffuse thymic hyperplasia was first described in children after successful chemotherapy for lym-phoma. It has now been described in adults and is referred to as “rebound thymic hyperplasia.”151 It is most frequently reported after chemotherapy for lymphoma or germ cell tumors. Ini-tially, atrophy of the thymic gland is seen with subsequent thymic gland enlargement, which can be dramatic. The usual time course for thymic hyperplasia is about 9 months after ces-sation of chemotherapy (range 2 weeks to

1	thymic gland is seen with subsequent thymic gland enlargement, which can be dramatic. The usual time course for thymic hyperplasia is about 9 months after ces-sation of chemotherapy (range 2 weeks to 12 months). Benign hyperplasia must be clearly distinguished from recurrent lym-phoma or germ cell tumors, which may be difficult since thymic hyperplasia is dramatic in some patients; careful follow-up with serial CT scans is the minimum requirement. The role of PET scanning is unclear. Thymic hyperplasia is a known cause of false-positive PET scans; in many patients, CT scan will show a triangular soft tissue density in the retrosternal space that has a characteristic bilobed anatomic appearance consistent with thymus gland.152 In addition, a low standardized uptake value of tracer on PET scan suggests a benign tumor.153 Biopsies may be required if the clinical index of suspicion is high.Brunicardi_Ch19_p0661-p0750.indd 73001/03/19 7:01 PM

1	CHAPTER 19731CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAThymoma. While it is the most frequently encountered neo-plasm of the anterior mediastinum in adults (seen most fre-quently between 40 and 60 years of age), thymoma is rare in children. Between 10% and 50% of patients with thymoma will have symptoms suggestive of myasthenia gravis or have circulating antibodies to acetylcholine receptor, but less than 10% of patients with myasthenia gravis have a thymoma. Most patients with thymoma are asymptomatic. Thymectomy leads to improvement or resolution of symptoms of myasthe-nia gravis in only about 25% of patients with thymomas. In contrast, in patients with myasthenia gravis and no thymoma, thymectomy results are superior: up to 50% of patients have a complete remission, and 90% improve. In 5% of patients with thymomas, other paraneoplastic syndromes, including red cell aplasia, hypogammaglobulinemia, systemic lupus erythema-tosus, Cushing’s syndrome, or SIADH, may be present. Large

1	In 5% of patients with thymomas, other paraneoplastic syndromes, including red cell aplasia, hypogammaglobulinemia, systemic lupus erythema-tosus, Cushing’s syndrome, or SIADH, may be present. Large thymic tumors may present with symptoms related to a mass effect, which may include cough, chest pain, dyspnea, or SVC syndrome.The diagnosis may be suspected based on CT scan and history, but imaging alone is not diagnostic. In most centers, the diagnosis is made after surgical resection because of the rela-tive difficulty of obtaining a needle biopsy and the likelihood that removal will ultimately be recommended. Biopsy should be avoided in cases where imaging is highly suggestive of thy-moma. In most patients, the distinction between lymphomas and thymomas can be made on CT scan since most lymphomas have marked lymphadenopathy and thymomas most frequently appear as a solitary encapsulated mass. PET scan may have a role in differentiating thymic cancer from thymoma, as thymic cancer

1	lymphomas have marked lymphadenopathy and thymomas most frequently appear as a solitary encapsulated mass. PET scan may have a role in differentiating thymic cancer from thymoma, as thymic cancer tends to be very FDG avid.153 In addition, PET scan may facilitate identification of low-risk and minimally invasive thy-moma; a standardized uptake value (SUV) <5 was associated with Masaoka stage I or II thymoma, whereas invasive thymoma and mediastinal lymphoma were more likely when the SUV was >5.154 In cases where the diagnosis is unclear, transmediastinal, not transpleural, CT-guided FNA biopsy has a diagnostic sen-sitivity of 87% and a specificity of 95% in specialized centers.The most commonly accepted staging system for thymoma is that of Masaoka.155 It is based on the presence or absence of gross or microscopic invasion of the capsule and of surrounding structures, as well as on the presence or absence of metastases (Table 19-30). Histologically, thymomas are characterized by a

1	of gross or microscopic invasion of the capsule and of surrounding structures, as well as on the presence or absence of metastases (Table 19-30). Histologically, thymomas are characterized by a mixture of epithelial cells and mature lymphocytes. Grossly, many thymomas remain well encapsulated. Even those with capsular invasion often lack histologic features of malignancy; they appear cytologically benign and identical to early-stage tumors. This lack of classic cellular features of malignancy is why most pathologists use the term “thymoma” or “invasive thymoma” rather than “malignant thymoma.” Thymic tumors with malignant cytologic features are classified separately and referred to as “thymic carcinoma.”The definitive treatment for thymoma is complete surgical removal; local recurrence rates and survival vary according to stage (Fig. 19-47). In centers with significant experience with VATS procedures, thymoma is not a contraindication to VATS approach, provided the principles of

1	rates and survival vary according to stage (Fig. 19-47). In centers with significant experience with VATS procedures, thymoma is not a contraindication to VATS approach, provided the principles of resection are adhered to, such as a complete resection without disrupting the capsule.156 Otherwise, resection is generally accomplished by median ster-notomy with extension to hemi-clamshell in more advanced cases. Even advanced tumors with local invasion of resectable structures such as the pericardium, SVC, or innominate vessels should be considered for resection with reconstruction.A multidisciplinary approach to nonresectable and more advanced lesions (stage ≥II) is mandatory to optimize patient care. The goal for surgical resection should be complete exci-sion of the mass with total thymectomy. All contiguous and noncontiguous disease is removed at the same setting; this may include resection of the pericardium or pleura, adjacent adher-ent lung, phrenic nerve, major vascular

1	thymectomy. All contiguous and noncontiguous disease is removed at the same setting; this may include resection of the pericardium or pleura, adjacent adher-ent lung, phrenic nerve, major vascular structures, and pleural metastasis. Bilateral phrenic nerve resection should be avoided, however, due to the major respiratory morbidity associated with bilateral paralyzed hemidiaphragms.The role of adjuvant or neoadjuvant therapies for advanced-stage tumors remains unclear. Traditionally, stage II thymomas have been treated by complete surgical resection fol-lowed by mediastinal radiation, but randomized trials have not been done. A recent retrospective review of a single-institution series of stage II thymoma patients showed no difference in survival or local recurrence after complete surgical resection alone, as compared with surgical resection with radiotherapy. Advanced thymomas have been shown to respond to platinum-based chemotherapy and to corticosteroids.157 One summary of

1	surgical resection alone, as compared with surgical resection with radiotherapy. Advanced thymomas have been shown to respond to platinum-based chemotherapy and to corticosteroids.157 One summary of chemotherapy trials showed an overall response rate of about 70%. Cisplatin/doxorubicin-based regimens appear to yield the best results. The combination radiotherapy and chemotherapy for local progression appears to prolong survival in some small series.158 Radiation therapy in surgically resected stage III thy-moma is likely beneficial in extending disease-specific survival; an analysis of the Surveillance, Epidemiology, and End Results (SEER) database identified 476 patients with stage III thymoma treated with primary surgery. Postoperative radiation was given Table 19-30Masaoka staging system for thymomaStage IEncapsulated tumor with no gross or microscopic evidence of capsular invasionStage IIGross capsular invasion or invasion into the mediastinal fat or pleura or microscopic capsular

1	for thymomaStage IEncapsulated tumor with no gross or microscopic evidence of capsular invasionStage IIGross capsular invasion or invasion into the mediastinal fat or pleura or microscopic capsular invasionStage IIIGross invasion into the pericardium, great vessels, or lungStage IVAPleural or pericardial disseminationStage IVBLymphogenous or hematogenous metastasis1.0.8.6.4.20Proportion surviving02010YearsP=.002Stage IStage IVStage IIIStage IIFigure 19-47. Stage-specific survival for thymomas.Brunicardi_Ch19_p0661-p0750.indd 73101/03/19 7:01 PM 732SPECIFIC CONSIDERATIONSPART IIFigure 19-48. Massive thymolipoma that was asymptomatic in an 18-year-old female.to 322 patients with a significant improvement in survival (127 months compared to 105 months, P = .038) despite the fact that these patients were more likely to have had debulking rather than curative resection. In multivariate analysis, disease-specific survival was better in the adjuvant radiation group.159 There-fore, it is

1	patients were more likely to have had debulking rather than curative resection. In multivariate analysis, disease-specific survival was better in the adjuvant radiation group.159 There-fore, it is imperative that all patients with thymomas undergo a thorough evaluation for potential resection. Current guidelines recommend radiation for patients with unresectable thymoma who have failed induction chemotherapy or for patients with incompletely resected invasive thymoma or thymic cancer. Planning the radiation ports requires input from the surgeon; it is important for the surgeon to carefully document areas of adherence between the thymoma and adjacent structures during the operation, with clips or other radiopaque markers placed to guide radiation therapy postoperatively. Extracapsular extension and positive surgical margins should be noted by the pathologist and correlated anatomically so that the surgeon and radiation oncologist can ensure appropriate radiation treatment.Thymic

1	extension and positive surgical margins should be noted by the pathologist and correlated anatomically so that the surgeon and radiation oncologist can ensure appropriate radiation treatment.Thymic Carcinoma. Thymic carcinomas are unequivocally malignant at the microscopic level. Suster and Rosai classified thymic carcinomas into low-grade and high-grade tumors.160 Low-grade tumors are well differentiated with squamous cell, mucoepidermoid, or basaloid features. High-grade thymic car-cinomas include those with lymphoepithelial, small cell neu-roendocrine, sarcomatoid, clear cell, and undifferentiated or anaplastic features. Care must be taken to differentiate thymic carcinoma from lung cancer metastatic to the thymus gland as the histologic features can be similar between the two. Com-pared with thymomas, they are a more heterogeneous group of malignancies with a propensity for early local invasion and widespread metastases. Malignant pleural and pericardial effu-sions occur

1	Com-pared with thymomas, they are a more heterogeneous group of malignancies with a propensity for early local invasion and widespread metastases. Malignant pleural and pericardial effu-sions occur frequently.Five-year survival rates are between 30% and 50%. Com-plete resection is occasionally curative and leads to improved survival, but most thymic carcinomas will recur and are refractory to chemotherapy.157 Management, therefore, depends on the completeness of the resection. Postoperative care includes radiation therapy, guided by residual gross disease or microscopically positive margins from the resection specimen. Chemotherapy may also be given, with carboplatin/paclitaxel recommended based on the best response rates with the least toxicity in clinical trials. The prognosis of patients with thymic cancer remains poor.Thymolipoma. Thymolipomas are rare benign tumors that may grow to a very large size prior to diagnosis. On CT scan, their appearance can be dramatic, with a

1	patients with thymic cancer remains poor.Thymolipoma. Thymolipomas are rare benign tumors that may grow to a very large size prior to diagnosis. On CT scan, their appearance can be dramatic, with a characteristic fat den-sity dotted by islands of soft tissue density representing islands of thymic tissue (Fig. 19-48). Thymolipomas are generally well-encapsulated, soft, and pliable masses that do not invade surrounding structures. Resection is recommended for large masses.Neurogenic Tumors. Most neurogenic tumors of the medi-astinum arise from the cells of the nerve sheath, from ganglion cells, or from the paraganglionic system (Table 19-31). The incidence, cell types, and risk of malignancy strongly correlate with patient age. Tumors of nerve sheath origin predominate in adults. Most present as asymptomatic incidental findings, and most are benign. In children and young adults, tumors of the autonomic ganglia predominate, with up to two-thirds being malignant.161Nerve Sheath

1	Most present as asymptomatic incidental findings, and most are benign. In children and young adults, tumors of the autonomic ganglia predominate, with up to two-thirds being malignant.161Nerve Sheath Tumors. Nerve sheath tumors account for 20% of all mediastinal tumors. More than 95% of nerve sheath tumors are benign neurilemomas or neurofibromas. Malignant neurosarcomas are much less common.Neurilemoma. Neurilemomas, also called schwannomas, arise from Schwann cells in intercostal nerves. They are firm, well encapsulated, and generally benign. Two characteristic histo-logic components are referred to as Antoni type A and Antoni type B regions. Antoni type A regions contain compact spindle Brunicardi_Ch19_p0661-p0750.indd 73201/03/19 7:01 PM

1	CHAPTER 19733CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAFigure 19-49. Magnetic resonance image of a neurogenic tumor with extension into the spinal canal via the foramen, giving a typical dumbbell appearance.Table 19-31Classification of neurogenic tumors of the mediastinumTUMOR ORIGINBENIGNMALIGNANTNerve sheathNeurilemoma, neurofibroma, melanotic schwannoma, granular cell tumorNeurofibrosarcomaGanglion cellGanglioneuromaGanglioneuroblastoma, neuroblastomaParaganglionic cellChemodectoma, pheochromocytomaMalignant chemodectoma, malignant pheochromocytomaReproduced with permission from Pearson FG, Cooper JD, Deslauriers J, et al: Thoracic Surgery, 2nd ed. New York, NY: Elsevier/Churchill Livingstone; 2002.cells with twisted nuclei and nuclear palisading. Antoni type B regions contain loose and myxoid connective tissue with hap-hazard cellular arrangement. These characteristics distinguish neurilemoma from malignant fibrosarcomatous tumors, which lack encapsulation and have no Antoni

1	and myxoid connective tissue with hap-hazard cellular arrangement. These characteristics distinguish neurilemoma from malignant fibrosarcomatous tumors, which lack encapsulation and have no Antoni features. If routine CT scan suggests extension of a neurilemoma into the intervertebral foramen, MRI is used to evaluate the extent of this “dumbbell” configuration (Fig. 19-49). Such a configuration may lead to cord compression and paralysis and requires a more complex surgical approach. Resection is recommended; VATS has been established as safe and effective for simple and, in experienced centers, even the more complex operations.162 It is reasonable to follow small, asymptomatic paravertebral tumors in older patients or in patients at high risk for surgery. In children, ganglioneuroblastomas or neuroblastomas are more common; therefore, all neurogenic tumors should be completely resected.Neurofibroma. Neurofibromas consist of both nerve sheath and nerve cells and account for up to 25%

1	or neuroblastomas are more common; therefore, all neurogenic tumors should be completely resected.Neurofibroma. Neurofibromas consist of both nerve sheath and nerve cells and account for up to 25% of nerve sheath tumors. Up to 40% of patients with mediastinal fibromas have general-ized neurofibromatosis (von Recklinghausen’s disease). About 70% of neurofibromas are benign, but malignant degeneration to neurofibrosarcoma occurs in 25% to 30% of patients.163 The risk of malignant degeneration increases with advancing age, von Recklinghausen’s disease, and exposure to previous radia-tion. Neurofibrosarcomas carry a poor prognosis because of rapid growth and aggressive local invasion along nerve bun-dles. Complete surgical resection is the mainstay of treatment. Adjuvant radiotherapy or chemotherapy does not confer a sig-nificant benefit, but may be added if complete resection is not possible.164 The 5-year survival rate is 53%, but it drops to 16% in patients with neurofibromatosis or

1	does not confer a sig-nificant benefit, but may be added if complete resection is not possible.164 The 5-year survival rate is 53%, but it drops to 16% in patients with neurofibromatosis or with large tumors (>5 cm).Ganglion Cell Tumors Ganglion cell tumors (ganglioneuro-mas, ganglioneuroblastomas, and neuroblastomas) arise from the sympathetic chain or from the adrenal medulla.1. Ganglioneuroma. Well-differentiated, benign tumors char-acterized histologically by well-differentiated ganglion cells with a background of Schwann cells, these are most often found incidentally in asymptomatic young adults. Diarrhea related to secretion of a vasoactive intestinal peptide has been described in some patients. These tumors have a pro-pensity for intraspinal canal extension, although they remain well-encapsulated; complete resection is curative, with a low risk of local recurrence.Brunicardi_Ch19_p0661-p0750.indd 73301/03/19 7:01 PM 734SPECIFIC CONSIDERATIONSPART II2. Ganglioneuroblastoma.

1	complete resection is curative, with a low risk of local recurrence.Brunicardi_Ch19_p0661-p0750.indd 73301/03/19 7:01 PM 734SPECIFIC CONSIDERATIONSPART II2. Ganglioneuroblastoma. Ganglioneuroblastomas contain a mixture of benign ganglion cells and malignant neuro-blasts. The distribution of these cells within the tumor is predictive of the clinical course. The nodular pattern has a high incidence of metastatic disease, whereas the diffuse pattern rarely metastasizes. Gross examination typically reveals encapsulated tumor; histologically, there are focal calcifications around regions of neuroblasts. Ganglioneu-roblastomas arise most frequently in infants and children <3 years old. The majority are resectable, with 80% 5-year survival.3. Neuroblastoma. Highly malignant, neuroblastomas are the most common extracranial solid malignancy of childhood. The primary site is intrathoracic malignancy in 14%; exten-sion into the spinal canal and osseous invasion commonly present. These

1	are the most common extracranial solid malignancy of childhood. The primary site is intrathoracic malignancy in 14%; exten-sion into the spinal canal and osseous invasion commonly present. These thoracic tumors are not as recalcitrant to chemotherapy and surgical resection as other chest malig-nancies; they are more likely to be resectable, with less inva-sion of surrounding organs. More than half occur in children under 2 years old; 90% arise within the first decade of life, and thus, these malignancies are discussed in more detail in Chapter 39.Paraganglionic Tumors. Paraganglionic tumors arising in the thoracic cavity include chemodectomas and pheochromo-cytomas. Only 10% of all pheochromocytomas are located in an extra-adrenal site. Intrathoracic pheochromocytomas are one of the rarest tumors.Approximately 10% of thoracic pheochromocytomas are malignant, a rate similar to that of adrenal tumors. The most common thoracic location is within the costovertebral sulcus, but

1	the rarest tumors.Approximately 10% of thoracic pheochromocytomas are malignant, a rate similar to that of adrenal tumors. The most common thoracic location is within the costovertebral sulcus, but paraganglionic tumors also arise within the visceral com-partment of the mediastinum. These catecholamine-producing lesions can lead to life-threatening hemodynamic problems, so complete removal is important. Diagnosis is generally confirmed by measuring elevated levels of urinary catecholamines and their metabolites. Localization is by CT scan, aided by MIBG scintigraphy. Preoperative care includes αand β-adrenergic blockade to prevent intraoperative malignant hypertension and arrhythmias. These tumors tend to be highly vascular and should be approached with care. Chemodectomas are rare tumors that may be located around the aortic arch, vagus nerves, or aor-ticosympathetics. They rarely secrete catecholamines and are malignant in up to 30% of patients.Lymphoma. Overall, lymphomas are the

1	that may be located around the aortic arch, vagus nerves, or aor-ticosympathetics. They rarely secrete catecholamines and are malignant in up to 30% of patients.Lymphoma. Overall, lymphomas are the most common malig-nancy of the mediastinum. In about 50% of patients who have both Hodgkin’s and non-Hodgkin’s lymphoma, the mediasti-num may be the primary site. The anterior compartment is most commonly involved, with occasional involvement of the mid-dle compartment and hilar nodes. The posterior compartment is rarely involved. Chemotherapy and/or radiation results in a cure rate of up to 90% for patients with early-stage Hodgkin’s disease and up to 60% with more advanced stages.Mediastinal Germ Cell Tumors. Germ cell tumors are uncom-mon neoplasms, but they are the most common malignancy in young men 15 to 35 years of age. Most germ cell tumors are gonadal in origin; primary mediastinal germ cell tumors com-prise less than 5% of all germ cell tumors and less than 1% of all mediastinal

1	young men 15 to 35 years of age. Most germ cell tumors are gonadal in origin; primary mediastinal germ cell tumors com-prise less than 5% of all germ cell tumors and less than 1% of all mediastinal tumors (usually occurring in the anterior com-partment). If a malignant mediastinal germ cell tumor is found, it is important to exclude a gonadal primary tumor. Primary mediastinal germ cell tumors (including teratomas, seminomas, and nonseminomatous malignant germ cell tumors) are a het-erogeneous group of benign and malignant neoplasms thought to originate from primitive pluripotent germ cells “misplaced” in the mediastinum during embryonic development. Previ-ously, most mediastinal germ cell tumors were thought to be metastatic. However, two lines of evidence suggest that many mediastinal germ cell tumors are primary, developing from plu-ripotent primordial germ cells in the mediastinum: (a) several autopsy series showed that patients with extragonadal sites of germ cell tumors,

1	germ cell tumors are primary, developing from plu-ripotent primordial germ cells in the mediastinum: (a) several autopsy series showed that patients with extragonadal sites of germ cell tumors, presumed previously to have originated from the gonads, had no evidence of an occult primary tumor or of any residual scar of the gonads, even after an exhaustive search; and (b) patients treated by surgery or radiation for their medi-astinal germ cell tumors had long-term survival with no late testicular recurrences.165About one-third of all primary mediastinal germ cell tumors are seminomatous. Two-thirds are nonseminomatous tumors or teratomas. Treatment and prognosis vary consider-ably within these two groups. Mature teratomas are benign and can generally be diagnosed by the characteristic CT findings of multilocular cystic tumors, encapsulated with combinations of fluid, soft tissue, calcium, and/or fat attenuation in the anterior compartment. FNA biopsy alone may be diagnostic for

1	CT findings of multilocular cystic tumors, encapsulated with combinations of fluid, soft tissue, calcium, and/or fat attenuation in the anterior compartment. FNA biopsy alone may be diagnostic for semi-nomas, usually with normal serum markers, including hCG and AFP. In 10% of seminomas, hCG levels may be slightly ele-vated. FNA findings, along with high hCG and AFP levels, can accurately diagnose nonseminomatous tumors. If the diagnosis remains uncertain after assessment of FNA findings and serum marker levels, then core-needle biopsies or surgical biopsies may be required. Thoracoscopy is the most frequent diagnostic surgical approach.1. Seminoma. Most patients with seminomas have advanced disease at the time of diagnosis and present with symptoms of local compression, including SVC syndrome, dyspnea, or chest discomfort. With advanced disease, the preferred treatment is combination cisplatin-based chemotherapy regimens with bleomycin and either etoposide or vinblas-tine. Complete

1	dyspnea, or chest discomfort. With advanced disease, the preferred treatment is combination cisplatin-based chemotherapy regimens with bleomycin and either etoposide or vinblas-tine. Complete responses have been reported in over 75% of patients treated with these regimens. Surgical resection may be curative for small asymptomatic seminomas that are found incidentally with screening CT scans. Surgical resection of residual masses after chemotherapy may be indicated.2. Nonseminomatous germ cell tumors. Nonseminomatous germ cell tumors include embryonal cell carcinomas, cho-riocarcinomas, endodermal sinus tumors, and mixed types. They are often bulky, irregular tumors of the anterior medi-astinum with areas of low attenuation on CT scan because of necrosis, hemorrhage, or cyst formation. Frequently, adjacent structures have been involved, with metastases to regional lymph nodes, pleura, and lungs. Lactate dehy-drogenase (LDH), AFP, and hCG levels are frequently elevated. Chemotherapy is

1	adjacent structures have been involved, with metastases to regional lymph nodes, pleura, and lungs. Lactate dehy-drogenase (LDH), AFP, and hCG levels are frequently elevated. Chemotherapy is the preferred treatment and includes combination therapy with cisplatin, bleomycin, and etoposide, followed by surgical resection of residual disease. With this regimen, survival is 67% at 2 years and 60% at 5 years. Surgical resection of residual masses is indicated, as it may guide further therapy. Up to 20% of residual masses contain additional tumors; in another 40%, mature teratomas; and the remaining 40%, fibrotic tissue. Brunicardi_Ch19_p0661-p0750.indd 73401/03/19 7:01 PM

1	CHAPTER 19735CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAIt is important to note that oxygen toxicity can occur in patients who have been exposed to bleomycin; high lev-els of oxygen supplementation in the perioperative setting should be avoided in these patients as respiratory failure and death can ensue.166 Factors independently predictive of survival after induction chemotherapy followed by resec-tion are elevated serum tumor markers after resection, postchemotherapy pathologic findings (complete necrosis vs. teratoma), and persistent germ cell or non–germ cell cancer in the pathologic specimen.1663. Teratoma. Teratomas are the most common type of medi-astinal germ cell tumors, accounting for 60% to 70% of mediastinal germ cell tumors. They contain two or three embryonic layers that may include teeth, skin, and hair (ectodermal), cartilage and bone (mesodermal), or bron-chial, intestinal, or pancreatic tissue (endodermal). Ther-apy for mature, benign teratomas is surgical resection,

1	include teeth, skin, and hair (ectodermal), cartilage and bone (mesodermal), or bron-chial, intestinal, or pancreatic tissue (endodermal). Ther-apy for mature, benign teratomas is surgical resection, which confers an excellent prognosis. Rarely, teratomas may contain a focus of carcinoma; these malignant tera-tomas (or teratocarcinomas) are locally aggressive. Often diagnosed at an unresectable stage, they respond poorly to chemotherapy and in a limited manner to radiotherapy; prognosis is uniformly poor.Mediastinal CystsBenign cysts account for up to 25% of mediastinal masses and are the most frequently occurring mass in the middle medias-tinal compartment. A CT scan showing characteristic features of near water density in a typical location is virtually 100% diagnostic.1671. Pericardial cyst. Usually asymptomatic and detected inci-dentally in the right costophrenic angle, pericardial cysts typically contain a clear fluid and are lined with a single layer of mesothelial cells. For

1	cyst. Usually asymptomatic and detected inci-dentally in the right costophrenic angle, pericardial cysts typically contain a clear fluid and are lined with a single layer of mesothelial cells. For most simple, asymptomatic pericardial cysts, observation alone is recommended. Sur-gical resection or aspiration may be indicated for complex cysts or large symptomatic cysts.2. Bronchogenic cyst. Developmental anomalies that occur during embryogenesis and occur as an abnormal budding of the foregut or tracheobronchial tree, bronchogenic cysts arise most often in the mediastinum just posterior to the carina or main stem bronchus. Approximately 15% occur within the pulmonary parenchyma. Thin-walled and lined with respiratory epithelium, they contain a protein-rich mucoid material and varying amounts of seromucous glands, smooth muscle, and cartilage. They may commu-nicate with the tracheobronchial tree. In adults, over half of all bronchogenic cysts are found incidentally during workup for an

1	of seromucous glands, smooth muscle, and cartilage. They may commu-nicate with the tracheobronchial tree. In adults, over half of all bronchogenic cysts are found incidentally during workup for an unrelated problem or during screening. The natural history of an incidentally diagnosed, asymptomatic bronchogenic cyst is unknown, but it is clear that many such cysts do not lead to clinical problems. In one study of young military personnel, 78% of all bronchogenic cysts found on routine CXRs were asymptomatic. However, in other reports with more comprehensive follow-up, up to 67% of adults with incidentally found bronchogenic cysts eventu-ally became symptomatic. Symptoms include chest pain, cough, dyspnea, and fever. If large (>6 cm) or symptomatic, resection is generally recommended since serious compli-cations may occur if the cyst becomes larger or infected. Complications include airway obstruction, infection, rup-ture, and, rarely, malignant transformation.168,169 Traditionally,

1	serious compli-cations may occur if the cyst becomes larger or infected. Complications include airway obstruction, infection, rup-ture, and, rarely, malignant transformation.168,169 Traditionally, complete removal of the cyst wall has been via posterolateral thoracotomy.170 Resection of infected cysts may be quite difficult because of dense adhesions; elective removal is often recommended before infection has a chance to occur. Thoracoscopic exploration and resec-tion are possible for small cysts with minimal adhesions. With increasing experience using video-assisted or robotic-assisted thoracoscopy, a greater proportion of these lesions are amenable to minimally invasive resection.3. Enteric cyst. Most clinicians agree that in contrast to bronchogenic cysts, esophageal cysts should be removed, regardless of the presence or absence of symptoms. Esopha-geal cysts have a propensity for serious complications sec-ondary to enlargement, leading to hemorrhage, infection, or perforation.

1	removed, regardless of the presence or absence of symptoms. Esopha-geal cysts have a propensity for serious complications sec-ondary to enlargement, leading to hemorrhage, infection, or perforation. Thus, surgical resection is the treatment of choice in both adults and children. As with bronchogenic cysts, experienced surgeons are approaching enteric cyst resections using minimally invasive techniques with great success.4. Thymic cyst. Generally asymptomatic, thymic cysts are often discovered incidentally. Simple cysts are of no con-sequence; however, the occasional cystic neoplasm must be ruled out. Cystic components occasionally are seen in patients with thymoma and Hodgkin’s disease.5. Ectopic endocrine glands. Up to 5% of all mediastinal masses are of thyroid origin; most are simple extensions of thyroid masses. Usually nontoxic, over 95% can be com-pletely resected through a cervical approach. True ectopic thyroid tissue of the mediastinum is rare. About 10% to 20% of abnormal

1	extensions of thyroid masses. Usually nontoxic, over 95% can be com-pletely resected through a cervical approach. True ectopic thyroid tissue of the mediastinum is rare. About 10% to 20% of abnormal parathyroid glands are found in the mediasti-num; most can be removed during exploration from a cervi-cal incision. In cases of true mediastinal parathyroid glands, thoracoscopic or open resection may be indicated. Location can generally be pinpointed by a combination of CT scan and Sestamibi scans.MediastinitisAcute Mediastinitis. Acute mediastinitis is a fulminant infec-tious process that spreads rapidly along the continuous fascial planes connecting the cervical and mediastinal compartments. Infections originate most commonly from esophageal perfora-tions, sternal infections, and oropharyngeal or neck infections, but a number of less common etiologic factors can lead to this deadly process (Table 19-32). Clinical signs and symptoms include fever, chest pain, dysphagia, respiratory

1	or neck infections, but a number of less common etiologic factors can lead to this deadly process (Table 19-32). Clinical signs and symptoms include fever, chest pain, dysphagia, respiratory distress, and cervical and upper thoracic subcutaneous crepitus. In severe cases, the clinical course can rapidly deteriorate to florid sepsis, hemodynamic instability, and death. Thus, a high index of sus-picion is required in the context of any infection with access to the mediastinal compartments.A chest CT scan illuminates the extent of spread and guides selection of the best approach to surgical drainage. Acute mediastinitis is a true surgical emergency; treatment must be instituted immediately and aimed at correcting the primary problem, such as the esophageal perforation or oro-pharyngeal abscess, and debridement and drainage of the spreading infectious process within the mediastinum, neck, pleura, and other tissue planes. Antibiotics, fluid resusci-tation, and other supportive measures are

1	and debridement and drainage of the spreading infectious process within the mediastinum, neck, pleura, and other tissue planes. Antibiotics, fluid resusci-tation, and other supportive measures are also important. Brunicardi_Ch19_p0661-p0750.indd 73501/03/19 7:01 PM 736SPECIFIC CONSIDERATIONSPART IITable 19-32Etiologic factors in acute mediastinitisEsophageal perforation Iatrogenic Balloon dilatation (for achalasia) Bougienage (for peptic stricture) Esophagoscopy Sclerotherapy (for variceal bleeding) Spontaneous Postemetic (Boerhaave’s syndrome) Straining during: Elimination Weight lifting Seizure Pregnancy Childbirth Ingestion of foreign bodies Trauma Blunt Penetrating Postsurgical Infection Anastomotic leak Erosion by cancerDeep sternotomy wound infectionOropharynx and neck infectionsLudwig’s anginaQuinsyRetropharyngeal abscessCellulitis and suppurative lymphadenitis of the neckInfections of the lung and pleuraSubphrenic abscessRib or vertebral

1	and neck infectionsLudwig’s anginaQuinsyRetropharyngeal abscessCellulitis and suppurative lymphadenitis of the neckInfections of the lung and pleuraSubphrenic abscessRib or vertebral osteomyelitisHematogenous or metastatic abscessReproduced with permission from Pearson FG, Cooper JD, Deslauriers J, et al: Thoracic Surgery, 2nd ed. New York, NY: Elsevier/Churchill Livingstone; 2002.Debridement may need to be repeated and other planes and cavities explored depending on the patient’s clinical status. Blood cell counts and serial CT scans may also be required. Persistent sepsis or collections on CT scan may require further radical surgical debridement.Chronic Mediastinitis. Sclerosing or fibrosing mediastinitis results from chronic mediastinal inflammation that originates in the lymph nodes, most frequently from granulomatous infec-tions such as histoplasmosis or tuberculosis. Chronic, low-grade inflammation leads to fibrosis and scarring, which can, in some patients, result in entrapment

1	frequently from granulomatous infec-tions such as histoplasmosis or tuberculosis. Chronic, low-grade inflammation leads to fibrosis and scarring, which can, in some patients, result in entrapment and compression of the low-pressure veins (including the SVC and innominate and azy-gos veins), the esophagus, and pulmonary arteries. There is no definitive treatment. Surgery is indicated only for diagnosis or in specific patients to relieve airway or esophageal obstruction or to achieve vascular reconstruction. Reports of palliative suc-cess with less invasive procedures (such as dilation and stenting of airways, the esophagus, or the SVC) are promising. In one series of 22 patients, ketoconazole was effective in controlling progression. In another series of 71 patients, 30% died from disease-associated complications during long-term follow-up. Chronic mediastinitis is similar to retroperitoneal fibrosis, scle-rosing cholangitis, and Riedel’s thyroiditis.PLEURA AND PLEURAL SPACEAnatomyEach

1	complications during long-term follow-up. Chronic mediastinitis is similar to retroperitoneal fibrosis, scle-rosing cholangitis, and Riedel’s thyroiditis.PLEURA AND PLEURAL SPACEAnatomyEach hemithorax has a mesothelial lining that invaginates at the hilum of each lung and continues on to cover each lung. The portion lining the bony rib cage, mediastinum, and dia-phragm is called the parietal pleura, whereas the portion encas-ing the lung is known as the visceral pleura. Between these two surfaces is the potential pleural space, which is normally occupied by a thin layer of lubricating pleural fluid. A network of somatic, sympathetic, and parasympathetic fibers innervates the parietal pleura. Irritation of the parietal surface by inflam-mation, tumor invasion, trauma, and other processes can lead to a sensation of chest wall pain. The visceral pleura have no somatic innervation.171,172Pleural EffusionPleural effusion refers to any significant collection of fluid within the pleural

1	can lead to a sensation of chest wall pain. The visceral pleura have no somatic innervation.171,172Pleural EffusionPleural effusion refers to any significant collection of fluid within the pleural space. Normally, between 5 and 10 L of fluid enters the pleural space each day by filtration through microves-sels supplying the parietal pleura (located mainly in the less dependent regions of the cavity). The net balance of pressures in these capillaries leads to fluid flow from the parietal pleural surface into the pleural space, and the net balance of forces in the pulmonary circulation leads to absorption through the vis-ceral pleura. Normally, 15 to 20 mL of pleural fluid is present at any given time. Any disturbance in these forces can lead to imbalance and accumulation of pleural fluid. Common patho-logic conditions in North America that lead to pleural effusion include congestive heart failure, bacterial pneumonia, malig-nancy, and pulmonary emboli (Table 19-33).173Access and

1	Common patho-logic conditions in North America that lead to pleural effusion include congestive heart failure, bacterial pneumonia, malig-nancy, and pulmonary emboli (Table 19-33).173Access and Drainage of Pleural Fluid CollectionsMost patients with pleural effusions of unknown cause should undergo thoracentesis with the following exceptions: effusions in the setting of congestive heart failure, hepatic failure or renal fail-ure, or small effusions associated with an improving pneumonia. If the clinical history suggests congestive heart failure as a cause, particularly in the setting of bilateral effusions, a trial of diuresis may be indicated (rather than thoracentesis). Up to 75% of effu-sions due to congestive heart failure resolve within 48 hours with diuresis alone. Similarly, thoracentesis can be avoided in patients with small effusions associated with resolving pneumonia. These patients typically present with cough, fever, leukocytosis, and uni-lateral infiltrate, and the

1	can be avoided in patients with small effusions associated with resolving pneumonia. These patients typically present with cough, fever, leukocytosis, and uni-lateral infiltrate, and the effusion is usually a result of a reactive, parapneumonic process. If the effusion is small and the patient responds to antibiotics, a diagnostic thoracentesis may be unneces-sary. If the effusion is large and compromising respiratory efforts, or if the patient has a persistent white blood cell count despite improving signs of pneumonia, an empyema of the pleural space must be considered. In these patients, early and aggressive drainage with chest tubes is required, possibly with surgical intervention.Once the decision is made to access a pleural effusion, the next step is to determine if a sample of the fluid or complete drainage of the pleural space is desired. This step is influenced by the clinical history, the type and amount of fluid present, the Brunicardi_Ch19_p0661-p0750.indd 73601/03/19

1	fluid or complete drainage of the pleural space is desired. This step is influenced by the clinical history, the type and amount of fluid present, the Brunicardi_Ch19_p0661-p0750.indd 73601/03/19 7:01 PM

1	CHAPTER 19737CHEST WALL, LUNG, MEDIASTINUM, AND PLEURATable 19-33Leading causes of pleural effusion in the United States, based on data from patients undergoing thoracentesisCAUSEANNUAL INCIDENCETRANSUDATEEXUDATECongestive heart failure500,000YesNoPneumonia300,000NoYesCancer200,000NoYesPulmonary embolus150,000SometimesSometimesViral disease100,000NoYesCoronary artery bypass surgery60,000NoYesCirrhosis with ascites50,000YesNoData from Light RW: Pleural diseases, 4th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2001.nature of the collection (such as free-flowing or loculated), the cause, and the likelihood of recurrence. For small, free-flowing effusions, an outpatient diagnostic and/or therapeutic thora-centesis with a relatively small-bore needle or catheter (14to 16-gauge) can be performed (Fig. 19-50). The appearance of the fluid is informative: clear straw-colored fluid is often transuda-tive; turbid or bloody fluid is often exudative.The site of entry for drainage of a

1	be performed (Fig. 19-50). The appearance of the fluid is informative: clear straw-colored fluid is often transuda-tive; turbid or bloody fluid is often exudative.The site of entry for drainage of a pleural effusion or pneumothorax may be based on the CXR alone if the effu-sion is demonstrated to be free-flowing. For free-flowing effu-sions, a low approach at the eighth or ninth intercostal space in the posterior midclavicular line facilitates complete drainage. If the effusion is loculated, CTor ultrasound-guided drainage may be indicated. If the goal is complete drainage, a small-bore pigtail catheter is inserted and connected to a closed drainage system with applied suction (typically –20 cm H2O). In general, the smallest-bore drainage catheter that will effec-tively drain the pleural space should be chosen. Smaller-diam-eter catheters significantly decrease the pain associated with the placement of chest tubes but are more prone to clogging and twisting.174,175 For clinical

1	space should be chosen. Smaller-diam-eter catheters significantly decrease the pain associated with the placement of chest tubes but are more prone to clogging and twisting.174,175 For clinical situations requiring biopsy or for potential interventions such as adhesiolysis or pleurodesis, minimally invasive surgery may be indicated, using a VATS approach.Figure 19-50. Techniques for aspiration and drainage of a pleural effusion. A. Needle aspiration. With careful appraisal of the x-ray findings, the best interspace is selected, and fluid is aspirated with a needle and syringe. Large volumes of fluid can be removed with a little patience and a large-bore needle. B. Chest tube insertion. After careful skin preparation, draping, and administration of local anesthesia, a short skin incision is made over the correct interspace. The incision is deepened into the intercostal muscles, and the pleura is penetrated (usually with a clamp). When any doubt exists about the status of the pleural

1	is made over the correct interspace. The incision is deepened into the intercostal muscles, and the pleura is penetrated (usually with a clamp). When any doubt exists about the status of the pleural space at the site of puncture, the wound is enlarged bluntly to admit a finger, which can be swept around the immediately adjacent pleural space to assess the situation and break down any adhesions. The tube is inserted, with the tip directed toward the optimal position suggested by the chest X-rays. In general, a high anterior tube is best for air (pneumothorax), and a low posterior tube is best for fluid. A 28F to 32F tube is adequate for most situations. A 36F tube is preferred for hemothorax or for a viscous empyema. Many surgeons prefer a very small tube (16F to 20F) for drainage of simple pneumothorax. C. The tube is connected to a water-seal drainage system. Suction is added, if necessary, to expand the lung; it usually will be required in a patient with a substantial air leak

1	simple pneumothorax. C. The tube is connected to a water-seal drainage system. Suction is added, if necessary, to expand the lung; it usually will be required in a patient with a substantial air leak (bronchopleural fistula).Brunicardi_Ch19_p0661-p0750.indd 73701/03/19 7:01 PM 738SPECIFIC CONSIDERATIONSPART IITable 19-34Differential diagnosis of pleural effusions I. Transudative pleural effusions A. Congestive heart failure B. Cirrhosis C. Nephrotic syndrome D. Superior vena caval obstruction E. Fontan procedure F. Urinothorax G. Peritoneal dialysis H. Glomerulonephritis I. Myxedema J. Cerebrospinal fluid leaks to pleura K. Hypoalbuminemia L. Pulmonary emboli M. Sarcoidosis II. Exudative pleural effusions A. Neoplastic diseases 1. Metastatic disease 2. Mesothelioma 3. Body cavity lymphoma 4. Pyothorax-associated lymphoma B. Infectious diseases 1. Tuberculosis 2. Other bacterial infections 3. Fungal infections 4. Parasitic infections 5. Viral infections C. Pulmonary

1	cavity lymphoma 4. Pyothorax-associated lymphoma B. Infectious diseases 1. Tuberculosis 2. Other bacterial infections 3. Fungal infections 4. Parasitic infections 5. Viral infections C. Pulmonary embolization D. Gastrointestinal disease 1. Pancreatic disease 2. Subphrenic abscess 3. Intrahepatic abscess 4. Intrasplenic abscess 5. Esophageal perforation 6. After abdominal surgery 7. Diaphragmatic hernia 8. Endoscopic variceal sclerosis 9. After liver transplantation E. Heart diseases 1. After coronary artery bypass graft surgery 2. Post-cardiac injury (Dressler’s) syndrome 3. Pericardial disease F. Obstetric and gynecologic diseases 1. Ovarian hyperstimulation syndrome 2. Fetal pleural effusion 3. Postpartum pleural effusion 4. Megis’ syndrome 5. Endometriosis G. Collagen vascular diseases 1. Rheumatoid pleuritis 2. Systemic lupus erythematosus 3. Drug-induced lupus 4. Immunoblastic lymphadenopathy 5. Sjögren’s syndrome 6. Familial Mediterranean fever 7. Churg-Strauss

1	vascular diseases 1. Rheumatoid pleuritis 2. Systemic lupus erythematosus 3. Drug-induced lupus 4. Immunoblastic lymphadenopathy 5. Sjögren’s syndrome 6. Familial Mediterranean fever 7. Churg-Strauss syndrome 8. Wegener’s granulomatosis H. Drug-induced pleural disease 1. Nitrofurantoin 2. Dantrolene 3. Methysergide 4. Ergot alkaloids 5. Amiodarone 6. Interleukin-2 7. Procarbazine 8. Methotrexate 9. Clozapine I. Miscellaneous diseases and conditions 1. Asbestos exposure 2. After lung transplantation 3. After bone marrow transplantation 4. Yellow nail syndrome 5. Sarcoidosis 6. Uremia 7. Trapped lung 8. Therapeutic radiation exposure 9. Drowning 10. Amyloidosis 11. Milk of calcium pleural effusion 12. Electrical burns 13. Extramedullary hematopoiesis 14. Rupture of mediastinal cyst 15. Acute respiratory distress syndrome 16. Whipple’s disease 17. Iatrogenic pleural effusions J. Hemothorax K. ChylothoraxReproduced with permission from Light RW: Pleural diseases, 5th ed. Philadelphia, PA:

1	respiratory distress syndrome 16. Whipple’s disease 17. Iatrogenic pleural effusions J. Hemothorax K. ChylothoraxReproduced with permission from Light RW: Pleural diseases, 5th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2007.Complications of Pleural Drainage. The most common complications of invasive pleural procedures are inadvertent injury to adjacent organs, including lung, with air leakage and pneumothorax; subdiaphragmatic entry and damage to the liver, spleen, or other intra-abdominal viscera; intercostal ves-sel injury with subsequent bleeding or larger vessel injury; and even cardiac puncture. Sometimes, bleeding may be the result of an underlying coagulopathy or anticoagulant therapy. Other technical complications include loss of a catheter, guidewire, or fragment in the pleural space and infections. Occasionally, rapid drainage of a large effusion can be followed by shortness of breath, clinical instability, and a phenomenon referred to as postexpansion pulmonary

1	pleural space and infections. Occasionally, rapid drainage of a large effusion can be followed by shortness of breath, clinical instability, and a phenomenon referred to as postexpansion pulmonary edema. For this reason, it is recom-mended to drain only up to 1500 mL initially. Most complica-tions can be avoided by consulting with a clinician experienced in pleural drainage techniques.Pleural Fluid Analysis. Pleural fluid collections are generally classified as transudates and exudates (Table 19-34). Transu-dates are protein-poor ultrafiltrates of plasma that result from alterations in the systemic hydrostatic pressures or colloid Brunicardi_Ch19_p0661-p0750.indd 73801/03/19 7:01 PM

1	CHAPTER 19739CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAosmotic pressures (for example, with congestive heart failure or cirrhosis). On gross visual inspection, a transudative effusion is generally clear or straw-colored. Exudates are protein-rich pleural fluid collections that generally result from inflamma-tion or pleural invasion by tumor. Grossly, they are often turbid, bloody, or purulent. Absent trauma, grossly bloody effusions are frequently malignant, but they may also occur in the setting of a pulmonary embolism or pneumonia.Transudates and exudates can be differentiated using Light’s criteria. An effusion is exudative if the pleural fluid-to-serum ratio of protein is greater than 0.5 and the LDH ratio is greater than 0.6 or the absolute pleural LDH level is greater than two-thirds of the normal upper limit for serum. If criteria suggest a transudate, a careful evaluation for congestive heart failure, cirrhosis, or conditions associated with transudates is under-taken. If

1	of the normal upper limit for serum. If criteria suggest a transudate, a careful evaluation for congestive heart failure, cirrhosis, or conditions associated with transudates is under-taken. If criteria suggest an exudate, further diagnostic studies may be helpful. If total and differential cell counts reveal a pre-dominance of neutrophils (>50% of cells), the effusion is likely associated with an acute inflammatory process (such as a para-pneumonic effusion or empyema, pulmonary embolus, or pan-creatitis). A predominance of mononuclear cells suggests a more chronic inflammatory process (such as cancer or tuberculosis). Gram stains and cultures should be obtained if possible, with inoculation into culture bottles at the bedside. Pleural fluid glu-cose levels are frequently decreased (<60 mg/dL) with complex parapneumonic effusions or malignant effusions. It is important to note that while the distinction between transudate and exudate can be diagnostically useful, the ultimate

1	(<60 mg/dL) with complex parapneumonic effusions or malignant effusions. It is important to note that while the distinction between transudate and exudate can be diagnostically useful, the ultimate decision for prolonged chest tube drainage or surgery depends on the effusion size and adequacy of drainage, presence of loculations, adequacy of lung reexpansion after drainage, and recurrence after initial drainage.A pleural effusion occurring in association with pleuritic chest pain, hemoptysis, or dyspnea out of proportion to the size of the effusion should raise concern for pulmonary embolism. These effusions may be transudative, but if an associated infarct near the pleural surface occurs, an exudate may be seen. If a pulmonary embolism is suspected in a postoperative patient, most clinicians would obtain a spiral CT scan. Alternatively, duplex ultrasonography of the lower extremities may yield a diagnosis of deep vein thrombosis, thereby indicating antico-agulant therapy and

1	clinicians would obtain a spiral CT scan. Alternatively, duplex ultrasonography of the lower extremities may yield a diagnosis of deep vein thrombosis, thereby indicating antico-agulant therapy and precluding the need for a specific diagnosis of pulmonary embolism. In some patients, a blood test for levels of d-dimer may be helpful; if a sensitive d-dimer blood test is negative, pulmonary embolism may be ruled out.Malignant Pleural EffusionMalignant pleural effusions may occur in association with a number of different malignancies, most commonly lung can-cer, breast cancer, and lymphomas, depending on the patient’s age and gender (Tables 19-35 and 19-36).176 Cytologic testing should be done on exudative effusions to rule out an associated malignancy; accuracy is 70% when associated with adenocarci-nomas, but it is less sensitive for mesotheliomas (<10%), squa-mous cell carcinomas (20%), or lymphomas (25% to 50%). If the diagnosis remains uncertain after drainage and fluid analy-sis,

1	adenocarci-nomas, but it is less sensitive for mesotheliomas (<10%), squa-mous cell carcinomas (20%), or lymphomas (25% to 50%). If the diagnosis remains uncertain after drainage and fluid analy-sis, thoracoscopy and direct biopsies are indicated.177,178 Malig-nant effusions are exudative and often tinged with blood. An effusion in the setting of a malignancy means a more advanced stage; mean survival ranges from 3 to 11 months, depending on the primary tumor location.Occasionally, effusions associated with a bronchogenic NSCLC are benign, and surgical resection may still be indicated.Table 19-35Primary organ site or neoplasm type in male patients with malignant pleural effusionsPRIMARY SITE OR TUMOR TYPENO. OF MALE PATIENTSPERCENTAGE OF MALE PATIENTSLung14049.1Lymphoma/leukemia6021.1Gastrointestinal tract207.0Genitourinary tract176.0Melanoma41.4Miscellaneous less common tumors103.5Primary site unknown3110.9Total285100.0Reproduced with permission from Johnston WW. The malignant

1	tract207.0Genitourinary tract176.0Melanoma41.4Miscellaneous less common tumors103.5Primary site unknown3110.9Total285100.0Reproduced with permission from Johnston WW. The malignant pleural effusion: a review of cytopathologic diagnoses of 584 specimens from 472 consecutive patients, Cancer. 1985 Aug 15;56(4):905-909.Table 19-36Primary organ site or neoplasm type in female patients with malignant pleural effusionsPRIMARY SITE OR TUMOR TYPENO. OF FEMALE PATIENTSPERCENTAGE OF FEMALE PATIENTSBreast7037.4Female genital tract3820.3Lung2815.0Lymphoma148.0Gastrointestinal tract84.3Melanoma63.2Urinary tract21.1Miscellaneous less common tumors31.6Primary site unknown179.1Total187100.0Reproduced with permission from Johnston WW. The malignant pleural effusion: a review of cytopathologic diagnoses of 584 specimens from 472 consecutive patients, Cancer. 1985 Aug 15;56(4):905-909.Effusion size and degree of associated dyspnea influence management. Symptomatic, moderate to large effusions should be

1	584 specimens from 472 consecutive patients, Cancer. 1985 Aug 15;56(4):905-909.Effusion size and degree of associated dyspnea influence management. Symptomatic, moderate to large effusions should be drained by tunneled indwelling pleural catheter, tube thora-costomy (chest tube or pigtail catheter) with subsequent instilla-tion of a sclerosing agent, or VATS with talc instillation. Management is based on patient preference, degree of known or anticipated lung reexpansion, and patient tolerance for operative intervention. Lung entrapment by tumor or adhesions limits reexpansion and generally predicts a poor result with pleurode-sis; it is the primary indication for placement of indwelling pleu-ral catheters. Patient preference is also considered, as is their life expectancy. Tunneled indwelling pleural catheters have dramati-cally changed the management of end-stage cancer treatment Brunicardi_Ch19_p0661-p0750.indd 73901/03/19 7:01 PM 740SPECIFIC CONSIDERATIONSPART IITable

1	indwelling pleural catheters have dramati-cally changed the management of end-stage cancer treatment Brunicardi_Ch19_p0661-p0750.indd 73901/03/19 7:01 PM 740SPECIFIC CONSIDERATIONSPART IITable 19-37Pathogenesis of empyemaContamination from a source contiguous to the pleural space (50%–60%) Lung Mediastinum Deep cervical area Chest wall and spine Subphrenic areaDirect inoculation of the pleural space (30%–40%) Minor thoracic interventions Postoperative infections Penetrating chest injuriesHematogenous infection of the pleural space from a distant site (<1%)Reproduced with permission from Pearson FG, Cooper JD, Deslauriers J, et al: Thoracic Surgery, 2nd ed. New York, NY: Elsevier/Churchill Livingstone; 2002.Outpatient referral for management of MPEPlacement ofindwelling pleuralcatheterLoculatedfluid/trapped lungFree-flowing with fulllung expansion Excellent performancestatus/long life expectancyVATS pleurodesisPoor performancestatus/short life expectancyPlacement of

1	pleuralcatheterLoculatedfluid/trapped lungFree-flowing with fulllung expansion Excellent performancestatus/long life expectancyVATS pleurodesisPoor performancestatus/short life expectancyPlacement of indwellingpleural catheterDecubitus films and/or CT scan Figure 19-51. Treatment decision algorithm for the management of malignant pleural effusion (MPE). CT = computed tomography; VATS = video-assisted tho-racic surgery.because they substantially shorten the amount of time patients spend in the hospital during their final weeks of life.179 If the lung is expected to fully expand and the patient has a longer life expectancy (e.g., malignant effusions in the setting of breast cancer), drainage with sclerosis is the preferred option. The choice of sclerosant includes mechanical pleurodesis or pleurectomy, talc, bleomycin, or doxycycline. Success rates range from 60% to 90% and are highest with talc. Typically, talc is administered as an aerosolized powder during video-assisted

1	or pleurectomy, talc, bleomycin, or doxycycline. Success rates range from 60% to 90% and are highest with talc. Typically, talc is administered as an aerosolized powder during video-assisted thoracoscopy, whereas doxycycline or a talc slurry is infused at the bedside through a previously placed pigtail catheter or larger bore chest tube. Figure 19-51 presents a decision algorithm for the management of malignant pleural effusion.EmpyemaThoracic empyema is defined by a purulent pleural effusion. Patients of all ages can develop empyema, but the frequency is increased in older or debilitated patients. Common associ-ated conditions include a pneumonic process in patients with pulmonary disorders and neoplasms, cardiac problems, diabe-tes mellitus, drug and alcohol abuse, neurologic impairments, postthoracotomy problems, and immunologic impairments. The mortality of empyema frequently depends on the degree of severity of the comorbidity; it may range from as low as 1% to over 40% in

1	postthoracotomy problems, and immunologic impairments. The mortality of empyema frequently depends on the degree of severity of the comorbidity; it may range from as low as 1% to over 40% in immunocompromised patients.Pathophysiology. The most common causes are parapneu-monic, but postsurgical, posttraumatic, and GI-associated (e.g., subphrenic or hepatic abscess, perforation of esophagus or other viscus) empyema is also common (Table 19-37). The spectrum of organisms involved in pneumonic processes that progress to empyema is changing. Pneumococci and staphylococci continue to be the most common, but gram-negative aerobic bacteria and anaerobes are becoming more prevalent. Cases involving myco-bacteria or fungi are rare. Multiple organisms may be found in up to 50% of patients. Cultures may be sterile, however, if antibiotics were initiated before the culture or if the culture pro-cess was not efficient. It is also fairly common for Pneumococ-cus to grow in blood cultures but not to

1	may be sterile, however, if antibiotics were initiated before the culture or if the culture pro-cess was not efficient. It is also fairly common for Pneumococ-cus to grow in blood cultures but not to grow in the pleural fluid cultures. The choice of antibiotics, therefore, is guided 11by the clinical scenario and not just the organisms found on culture. Broad-spectrum coverage may be required even when cultures do not grow out an organism or if a single organism is grown when the clinical picture is more consistent with a mul-tiorganism process. For example, a polymicrobial gram stain, particularly including yeast, is strongly suggestive of esopha-geal perforation. Common gram-negative organisms include Escherichia coli, Klebsiella, Pseudomonas, and Enterobacte-riaceae. Anaerobic organisms may be fastidious and difficult to document by culture and are associated with periodontal dis-eases (especially Streptococcus species), aspiration syndromes, alcoholism, general anesthesia, drug

1	may be fastidious and difficult to document by culture and are associated with periodontal dis-eases (especially Streptococcus species), aspiration syndromes, alcoholism, general anesthesia, drug abuse, or other functional associations with gastroesophageal reflux.Organisms gain entry into the pleural cavity through con-tiguous spread from pneumonia, lung abscess, liver abscess, or another, adjacent infectious processes. Organisms may also enter the pleural cavity by direct contamination from thora-centesis, thoracic surgical procedures, esophageal injuries, Brunicardi_Ch19_p0661-p0750.indd 74001/03/19 7:01 PM

1	CHAPTER 19741CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAor trauma. As organisms enter the pleural space, an influx of polymorphonuclear cells and fluid occurs, with subsequent release of inflammatory mediators and toxic oxygen radi-cals. These mechanisms lead to variable degrees of endothe-lial injury and capillary instability. This process overwhelms the normal pleural lymphatic drainage. This early effusion is watery and free-flowing in the pleural cavity. Thoracentesis at this stage yields fluid with a pH typically above 7.3, a glucose level greater than 60 mg/dL, and a low LDH level (<500 U/L). At this stage, the decision to use antibiotics alone or perform a repeat thoracentesis, chest tube drainage, thoracoscopy, or open thoracotomy depends on the amount of pleural fluid, its consis-tency, the clinical status of the patient, the degree of expansion of the lung after drainage, and the presence of loculated fluid in the pleural space (vs. free-flowing purulent fluid). Early in the

1	the clinical status of the patient, the degree of expansion of the lung after drainage, and the presence of loculated fluid in the pleural space (vs. free-flowing purulent fluid). Early in the parapneumonic process, when the purulent fluid is relatively thin, complete drainage with simple large-bore thoracentesis is possible. If complete lung expansion is obtained and the pneu-monic process is responding to antibiotics, no further drainage may be necessary. Pleural fluid with a pH lower than 7.2 and with a glucose level of less than 40 mg/dL means that a more aggressive approach to drainage should be pursued.The pleural fluid may become thick and loculated over the course of hours to days and may be associated with fibrinous adhesions (the fibrinopurulent stage). At this stage, chest tube insertion with closed-system drainage or drainage with thora-coscopy may be necessary to remove the fluid and adhesions and facilitate complete lung expansion.180 Further progression of the

1	tube insertion with closed-system drainage or drainage with thora-coscopy may be necessary to remove the fluid and adhesions and facilitate complete lung expansion.180 Further progression of the inflammatory process leads to the formation of a pleu-ral peel, which may be flimsy and easy to remove early on. However, as the process progresses, a thick pleural rind may develop, leaving a trapped lung; complete lung decortication by either thoracoscopy or thoracotomy would then be necessary.The use of intrapleural fibrinolytic therapy for manage-ment of empyema has been investigated in several large pro-spective trials. Intrapleural infusion of tissue plasminogen activator (t-PA) alone did not improve outcomes, whereas combined intrapleural t-PA and DNase was associated with a reduction in hospital stay of nearly 7 days, 77% fewer refer-rals for surgical intervention at 3 months, and more than double the reduction in the infected pleural fluid collection by CXR imaging.181 In this trial,

1	stay of nearly 7 days, 77% fewer refer-rals for surgical intervention at 3 months, and more than double the reduction in the infected pleural fluid collection by CXR imaging.181 In this trial, the medications were given twice daily by intrapleural injection; the dose was 5 mg for the DNase and 10 mg for t-PA. The chest drain was clamped for 1 hour after injection and released. This study suggests that the combination of fibrinolysis (t-PA) and cleavage of uncoiled DNA by DNase reduces fluid viscosity and facilitates pleural clearance.Management. If there is a residual space, persistent pleural infection is likely to occur. A persistent pleural space may be secondary to contracted, but intact, underlying lung; or it may be secondary to surgical lung resection. If the space is small and well drained by a chest tube, a conservative approach may be possible. This requires leaving the chest tubes in place and attached to closed-system drainage until symphysis of the vis-ceral and parietal

1	drained by a chest tube, a conservative approach may be possible. This requires leaving the chest tubes in place and attached to closed-system drainage until symphysis of the vis-ceral and parietal surfaces takes place. At this point, the chest tubes can be removed from suction; if the residual pleural space remains stable, the tubes can be cut and advanced out of the chest over the course of several weeks. If the patient is stable, tube removal can frequently be done in the outpatient setting, guided by the degree of drainage and the size of the residual space visualized on serial CT scans. Larger spaces may require open thoracotomy and decortication in an attempt to reexpand the lung to fill this residual space. If reexpansion has failed or appears too high risk, then open drainage, rib resection, and prolonged packing may be required, with delayed closure with muscle flaps or thoracoplasty.182 Most chronic pleural space problems can be avoided by early specialized thoracic

1	rib resection, and prolonged packing may be required, with delayed closure with muscle flaps or thoracoplasty.182 Most chronic pleural space problems can be avoided by early specialized thoracic surgi-cal consultation and complete drainage of empyema, allowing space obliteration by the reinflated lung.ChylothoraxChylothorax develops most commonly after surgical trauma to the thoracic duct or a major branch, but may be also associ-ated with a number of other conditions (Table 19-38).183 It is generally unilateral; for example, it may occur after dissection of the distal esophagus where the duct lies in close proximity to the esophagus as it enters the right chest from its origin in the Table 19-38Etiology of chylothoraxCongenital Atresia of thoracic duct Thoracic duct-pleural space fistula Birth traumaTraumatic and/or iatrogenic Blunt injury Penetrating injury Surgery Cervical Excision of lymph nodes Radical neck dissection Thoracic Correction of patent ductus

1	space fistula Birth traumaTraumatic and/or iatrogenic Blunt injury Penetrating injury Surgery Cervical Excision of lymph nodes Radical neck dissection Thoracic Correction of patent ductus arteriosus Correction of coarctation of the aorta Vascular procedure involving the origin of the left subclavian artery Esophagectomy Sympathectomy Resection of thoracic aneurysm Resection of mediastinal tumors Left pneumonectomy Abdominal Sympathectomy Radical lymph node dissection Diagnostic procedures Translumbar arteriography Subclavian vein catheterization Left-sided heart catheterizationNeoplasmsInfections Tuberculous lymphadenitis Nonspecific mediastinitis Ascending lymphangitis FilariasisMiscellaneous Venous thrombosis Left subclavian-jugular vein Superior vena cava Pulmonary lymphangiomatosisReproduced with permission from Sabiston DC, Spencer FC: Surgery of the Chest, 6th ed. Philadelphia, PA: Elsevier; 1995.Brunicardi_Ch19_p0661-p0750.indd 74101/03/19

1	cava Pulmonary lymphangiomatosisReproduced with permission from Sabiston DC, Spencer FC: Surgery of the Chest, 6th ed. Philadelphia, PA: Elsevier; 1995.Brunicardi_Ch19_p0661-p0750.indd 74101/03/19 7:01 PM 742SPECIFIC CONSIDERATIONSPART IIabdomen at the cisterna chyli (Fig. 19-52). If the mediastinal pleura are disrupted on both sides, bilateral chylothoraces may occur. Left-sided chylothoraces may develop after a left-sided neck dissection, especially in the region of the confluence of the subclavian and internal jugular veins. Chylothorax may also follow nonsurgical trauma, including penetrating or blunt inju-ries to the chest or neck area, central line placements, and other surgical misadventures. It may be seen in association with a variety of benign and malignant diseases that generally involve the lymphatic system of the mediastinum or neck.Pathophysiology. Most commonly, the thoracic duct origi-nates in the abdomen from the cisterna chyli, which is located in the midline,

1	involve the lymphatic system of the mediastinum or neck.Pathophysiology. Most commonly, the thoracic duct origi-nates in the abdomen from the cisterna chyli, which is located in the midline, near the level of the second lumbar vertebra. From this origin, the thoracic duct ascends into the chest through the aortic hiatus at the level of T10 to T12, and courses just to the right of the aorta (see Fig. 19-52). As the thoracic duct courses cephalad above the diaphragm, it most commonly remains in the right chest, lying just behind the esophagus, between the aorta and azygos vein. The duct continues superiorly, lying just to the right of the vertebral column. Then, at the fourth thoracic ver-tebra, it crosses behind the aorta and the aortic arch into the left posterior mediastinum and travels superiorly, staying near the esophagus and mediastinal pleura as it exits the thoracic inlet. As it exits the thoracic inlet, it passes to the left, just behind the carotid sheath and anterior to the

1	superiorly, staying near the esophagus and mediastinal pleura as it exits the thoracic inlet. As it exits the thoracic inlet, it passes to the left, just behind the carotid sheath and anterior to the inferior thyroid and vertebral bodies. Just medial to the anterior scalene muscle, it courses inferiorly and drains into the union of the internal jugular and subclavian veins. Given the extreme variability in the main duct and its branches, accumulation of chyle in the chest or flow from penetrating wounds may be seen after a variety of trau-matic and medical conditions.184The main function of the duct is to transport fat absorbed from the digestive system along with variable amounts of protein and lymphatic material (Table 19-39). Given the high volume of chyle that flows through the thoracic duct, signifi-cant injuries can cause leaks in excess of 2 L per day; if left untreated, protein, lymphocyte, and volume depletion can lead to serious metabolic effects and death. Thoracentesis is

1	duct, signifi-cant injuries can cause leaks in excess of 2 L per day; if left untreated, protein, lymphocyte, and volume depletion can lead to serious metabolic effects and death. Thoracentesis is usually grossly suggestive, revealing milky, nonpurulent pleural fluid. However, if the patient is taking nothing by mouth, the pleural fluid may not be grossly abnormal. Laboratory analysis of the pleural fluid shows the presence of chylomicrons, a high lym-phocyte count and high triglyceride levels. If the triglyceride level is greater than 110 mg/100 mL, a chylothorax is almost certainly present (a 99% accuracy rate). If the triglyceride level is less than 50 mg/mL, there is only a 5% chance of chylothorax. In many clinical situations, the accumulation of chyle may be slow because of minimal digestive fat flowing through the gas-trointestinal tract after major trauma or surgery, so the diagnosis may be more difficult to establish.Management. The treatment plan for any chylothorax depends

1	digestive fat flowing through the gas-trointestinal tract after major trauma or surgery, so the diagnosis may be more difficult to establish.Management. The treatment plan for any chylothorax depends on its cause, the amount of drainage, and the patient’s clinical status (Fig. 19-53). In general, most patients are treated with a short period of chest tube drainage, nothing by mouth (NPO) orders, total parenteral nutrition (TPN), and observation. In cen-ters with interventional radiology expertise, thoracic duct embo-lization as soon as possible after diagnosis should be considered. Thoracic d.Cisterna chyliFigure 19-52. Normal thoracic duct anatomy. The esophagus comes into close proximity with the thoracic duct as it enters the chest from its origin in the abdomen at the cisterna chyli.Table 19-39Composition of chyleCOMPONENTAMOUNT (PER 100 ML)Total fat0.4–5 g Total cholesterol65–220 mgTotal protein2.21–5.9 g Albumin1.1–4.1 g Globulin1.1–3.1 g Fibrinogen16–24 gSugars48–200

1	chyli.Table 19-39Composition of chyleCOMPONENTAMOUNT (PER 100 ML)Total fat0.4–5 g Total cholesterol65–220 mgTotal protein2.21–5.9 g Albumin1.1–4.1 g Globulin1.1–3.1 g Fibrinogen16–24 gSugars48–200 gElectrolytesSimilar to levels in plasmaCellular elements Lymphocytes400–6800/mm3 Erythrocytes50–600/mm3Antithrombin globulin>25% of plasma concentrationProthrombin>25% of plasma concentrationFibrinogen>25% of plasma concentrationReproduced with permission from Miller JI: Diagnosis and management of chylothorax, Chest Surg Clin N Am. 1996 Feb;6(1):139-148.Brunicardi_Ch19_p0661-p0750.indd 74201/03/19 7:02 PM

1	CHAPTER 19743CHEST WALL, LUNG, MEDIASTINUM, AND PLEURARadiationtherapyMedicallyunstablePleuralperitonealshuntDrainagepersists(Nonmalignant)Drainage persists(>500 mL/d)MalignantchylothoraxThoracentesisConfirm diagnosisMedicallystableThoracotomyChest tubeConservative management NPOChest tube to suctionCentral hyperalimentationWait 2 weeks*Drainage decrease(<250 mL/d)Continue1 weekThoracotomyDuct ligationMass ligationDecorticationPleurectomyDrainagestopsRemovechest tube Figure 19-53. Algorithm for the management of chylothorax. *If high output persists (>50 mL/d), early surgical ligation of the thoracic duct may be considered. NPO = nothing by mouth.Chest cavity drainage must be adequate to allow complete lung reexpansion. Somatostatin has been advocated by some authors, with variable results. If significant chyle drainage (>500 mL per day in an adult, >100 mL in an infant) continues despite TPN and good lung expansion, early surgical duct ligation or embo-lization is recommended (within

1	If significant chyle drainage (>500 mL per day in an adult, >100 mL in an infant) continues despite TPN and good lung expansion, early surgical duct ligation or embo-lization is recommended (within 4–7 days following diagnosis). Ligation can be approached best by right thoracotomy, and in some experienced centers, by right thoracoscopy. Chylothoraces due to malignant conditions often respond to radiation and/or chemotherapy and less commonly require surgical ligation. Significant nutritional and immunologic depletion results from untreated chylothorax; associated mortality is in excess of 50%. With early recognition and aggressive medical management as well as early surgical ligation or embolization for persistent leaks, the mortality rate of chylothorax is now less than 10%.Tumors of the PleuraMalignant mesothelioma is the most common type of primary tumor of the pleura, with approximately 3000 cases per year in the United States. Other, less common tumors include benign and

1	of the PleuraMalignant mesothelioma is the most common type of primary tumor of the pleura, with approximately 3000 cases per year in the United States. Other, less common tumors include benign and malignant fibrous tumors of the pleura, lipomas, and cysts.Malignant Mesothelioma. The only known risk factor for mesothelioma is exposure to asbestos, identified in over 50% of cases. Exposure is typically work-related in industries using asbestos in the manufacturing process, such as shipbuilding and brake pad linings. The risk extends to family members who are Brunicardi_Ch19_p0661-p0750.indd 74301/03/19 7:02 PM 744SPECIFIC CONSIDERATIONSPART IIexposed to the dust of the clothing or to the work environment. Asbestos exposure and smoking synergistically increase the risk for lung cancer, but smoking does not increase risk for malig-nant mesotheliomas. Male predominance is 2:1, and it occurs most commonly after the age of 40.Risk of developing mesothelioma after asbestos exposure

1	but smoking does not increase risk for malig-nant mesotheliomas. Male predominance is 2:1, and it occurs most commonly after the age of 40.Risk of developing mesothelioma after asbestos exposure differs depending on the physical characteristics of the asbestos and similar fibers (either serpentine or amphibole). The serpen-tine fibers are large and curly and are generally not able to travel beyond larger airways. However, the narrow, straight amphibole fibers, in particular the crocidolite fibers, may navigate distally into the pulmonary parenchyma and are most clearly associated with mesotheliomas. Like many carcinogens, the latency period between asbestos exposure and the development of mesothe-lioma is at least 20 years. The tumor generally is multicentric, with multiple pleural-based nodules coalescing to form sheets of tumor. This process initially involves the parietal pleura, gener-ally with early spread to the visceral surfaces and with a variable degree of invasion of

1	nodules coalescing to form sheets of tumor. This process initially involves the parietal pleura, gener-ally with early spread to the visceral surfaces and with a variable degree of invasion of surrounding structures. Autopsy studies have shown that most patients have distant metastases, but the natural history of the disease in untreated patients culminates in death due to local extension and effective strangulation of the lung.Clinical Presentation Most patients present with dyspnea and chest pain. Over 90% have a pleural effusion, but thora-centesis is diagnostic in less than 10% of patients. Frequently, a thoracoscopy or open pleural biopsy with special stains is required to differentiate mesotheliomas from adenocarcinomas (Table 19-40). Epithelial subtypes are associated with a more favorable prognosis, and long-term survival may be seen in rare patients with no treatment. Sarcomatous and mixed tumors share a more aggressive course.Management The treatment of malignant

1	a more favorable prognosis, and long-term survival may be seen in rare patients with no treatment. Sarcomatous and mixed tumors share a more aggressive course.Management The treatment of malignant mesotheliomas remains controversial. Treatment options include supportive care only, surgical resection, and multimodality approaches (using a combination of surgery, chemotherapy, and radia-tion therapy).186 Surgical approaches range from extrapleural pneumonectomy (removal of the lung, entire parietal pleural, ipsilateral pericardium, and hemidiaphragm with patch recon-struction), pleurectomy and decortication (removal of the vis-ceral and parietal pleura only), and palliative procedures such as talc pleurodesis and insertion of long-term tunneled indwelling pleural catheters.Table 19-40Differentiation of mesothelioma from adenocarcinoma MESOTHELIOMAADENOCARCINOMAImmunohistochemical results Carcinoembryonic antigenNegativePositive VimentinPositiveNegative Low molecular weight

1	19-40Differentiation of mesothelioma from adenocarcinoma MESOTHELIOMAADENOCARCINOMAImmunohistochemical results Carcinoembryonic antigenNegativePositive VimentinPositiveNegative Low molecular weight cytokeratinsPositiveNegativeElectron microscopic featuresLong, sinuous villiShort, straight villi with fuzzy glycocalyxFibrous Tumors of the Pleura. Fibrous tumors of the pleura are unrelated to asbestos exposure or malignant mesotheliomas. They generally occur as a single pedunculated mass arising from the visceral pleura but can occasionally arise from the parietal pleura. They can grow to be quite large, with most ranging from 5 to 10 cm and 100 to 400 g in size by the time they are dis-covered. Architecturally, the most common microscopic feature is the “patternless pattern.” This is characterized by randomly situated areas of hypercellularity, containing spindle cells with bland, vesicular, ovoidal nuclei and scarce cytoplasm, and hypo-cellularity, with fibrous connective tissue,

1	by randomly situated areas of hypercellularity, containing spindle cells with bland, vesicular, ovoidal nuclei and scarce cytoplasm, and hypo-cellularity, with fibrous connective tissue, hemorrhage, myxoid, or necrosis. They can also have an hemangiopericytoma-like appearance. The neoplastic cells are immunoreactive for CD34 and CD99 but negative for cytokeratins and desmin. Immuno-reactivity for Bcl-2 is variably positive.187They are frequently discovered incidentally on routine CXRs, without an associated pleural effusion. They occur with equal frequency in males and females and are most common in the sixth to seventh decade of life. Fibrous tumors of the pleura may be benign or malignant.188 Symptoms such as cough, chest pain, and dyspnea occur in 30% to 40% of patients but are found in 75% of patients with malignant tumors. Malignant tumors are differentiated from benign tumors based on high cellularity, more than four mitotic figures per 10 high-power fields, nuclear

1	are found in 75% of patients with malignant tumors. Malignant tumors are differentiated from benign tumors based on high cellularity, more than four mitotic figures per 10 high-power fields, nuclear pleomorphism, tumor necrosis, and hemorrhage. They are more likely to arise from the parietal pleura of the chest wall, dia-phragm, or mediastinum, or in the fissures or invaginating into the lung parenchyma. Hypoglycemia, associated pleural effu-sion, and hypertrophic pulmonary osteoarthropathy (clubbed digits, long bone ossifying periostitis, and arthritis) are associ-ated with these lesions in approximately 25% of patients. Less common are fever and hemoptysis.Symptoms resolve with surgical resection. Given the well-circumscribed and often pedunculated nature of fibrous tumors of the pleura, all benign lesions and approximately 50% of malignant lesions are cured by complete surgical resection.188 Incompletely resected malignant tumors may recur locally or metastasize, and more than 50%

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1	pulmonary resection with high out-put. J Thorac Cardiovasc Surg. 2008;135(2):269-273. 83. Cerfolio RJ, Bass C, Katholi CR. Prospective randomized trial compares suction versus water seal for air leaks. Ann Thorac Surg. 2001;71(5):1613-1617. 84. Bauer C, Hentz JG, Ducrocq X, et al. Lung function after lobectomy: a randomized, double-blinded trial compar-ing thoracic epidural ropivacaine/sufentanil and intravenous morphine for patient-controlled analgesia. Anesth Analg. 2007;105(1):238-244. 85. Casati A, Alessandrini P, Nuzzi M, et al. A prospective, randomized, blinded comparison between continuous Brunicardi_Ch19_p0661-p0750.indd 74601/03/19 7:02 PM

1	CHAPTER 19747CHEST WALL, LUNG, MEDIASTINUM, AND PLEURAthoracic paravertebral and epidural infusion of 0.2% ropi-vacaine after lung resection surgery. Eur J Anaesthesiol. 2006;23(12):999-1004. 86. Inderbitzi RG, Leiser A, Furrer M, Althaus U. Three years’ experience in video-assisted thoracic surgery (VATS) for spontaneous pneumothorax. J Thorac Cardiovasc Surg. 1994;107(6):1410-1415. 87. Warner BW, Bailey WW, Shipley RT. Value of computed tomography of the lung in the management of primary spon-taneous pneumothorax. Am J Surg. 1991;162(1):39-42. 88. Mansharamani N, Balachandran D, Delaney D, Zibrak JD, Silvestri RC, Koziel H. Lung abscess in adults: clinical com-parison of immunocompromised to non-immunocompromised patients. Respir Med. 2002;96(3):178-185. 89. Laheij RJ, Sturkenboom MC, Hassing RJ, Dieleman J, Stricker BH, Jansen JB. Risk of community-acquired pneu-monia and use of gastric acid-suppressive drugs. JAMA. 2004;292(16):1955-1960. 90. Conant EF, Wechsler RJ. Actinomycosis

1	RJ, Dieleman J, Stricker BH, Jansen JB. Risk of community-acquired pneu-monia and use of gastric acid-suppressive drugs. JAMA. 2004;292(16):1955-1960. 90. Conant EF, Wechsler RJ. Actinomycosis and nocardiosis of the lung. J Thorac Imaging. 1992;7(4):75-84. 91. Thomson RM, Armstrong JG, Looke DF. Gastroesopha-geal reflux disease, acid suppression, and Mycobacterium avium complex pulmonary disease. Chest. 2007;131(4): 1166-1172. 92. Koh WJ, Lee JH, Kwon YS, et al. Prevalence of gastroesopha-geal reflux disease in patients with nontuberculous mycobac-terial lung disease. Chest. 2007;131(6):1825-1830. 93. Angrill J, Agusti C, de Celis R, et al. Bacterial colonisation in patients with bronchiectasis: microbiological pattern and risk factors. Thorax. 2002;57(1):15-19. 94. Barker AF. Bronchiectasis. N Engl J Med. 2002;346(18):1383-1393. 95. Ilowite J, Spiegler P, Chawla S. Bronchiectasis: new find-ings in the pathogenesis and treatment of this disease. Curr Opin Infect Dis.

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1	Hutcheon MA, Waddell TK, Keshavjee SH. Marginal donor lungs: a reassessment. J Thorac Cardio-vasc Surg. 2002;123(3):421-427; discussion 427. 125. Palmer SM, Miralles AP, Howell DN, Brazer SR, Tapson VF, Davis RD. Gastroesophageal reflux as a reversible cause of allograft dysfunction after lung transplantation. Chest. 2000;118(4):1214-1217. 126. Dahlberg PS, Prekker ME, Hertz M, Thompson DJ, Park SJ. Recent trends in lung transplantation: the University of Min-nesota experience. Clin Transpl. 2002;243-251. 127. Somers J, Faber LP. Chondroma and chondrosarcoma. Semin Thorac Cardiovasc Surg. 1999;11(3):270-277. 128. Andino L, Cagle PT, Murer B, et al. Pleuropulmonary des-moid tumors: immunohistochemical comparison with solitary fibrous tumors and assessment of beta-catenin and cyclin D1 expression. Arch Pathol Lab Med. 2006;130(10):1503-1509. 129. Baliski CR, Temple WJ, Arthur K, Schachar NS. Desmoid tumors: a novel approach for local control. J Surg Oncol. 2002;80(2):96-99. 130. Abbas

1	Arch Pathol Lab Med. 2006;130(10):1503-1509. 129. Baliski CR, Temple WJ, Arthur K, Schachar NS. Desmoid tumors: a novel approach for local control. J Surg Oncol. 2002;80(2):96-99. 130. Abbas AE, Deschamps C, Cassivi SD, et al. Chest-wall des-moid tumors: results of surgical intervention. Ann Thorac Surg. 2004;78(4):1219-1223; discussion 1219-1223. 131. Gutierrez JC, Perez EA, Franceschi D, Moffat FL Jr, Livingstone AS, Koniaris LG. Outcomes for soft-tissue sarcoma in 8249 cases from a large state cancer registry. J Surg Res. 2007;141(1):105-114.Brunicardi_Ch19_p0661-p0750.indd 74701/03/19 7:02 PM 748SPECIFIC CONSIDERATIONSPART II 132. Walsh GL, Davis BM, Swisher SG, et al. A single-institutional, multidisciplinary approach to primary sarcomas involving the chest wall requiring full-thickness resections. J Thorac Car-diovasc Surg. 2001;121(1):48-60. 133. Liptay MJ, Fry WA. Malignant bone tumors of the chest wall. Semin Thorac Cardiovasc Surg. 1999;11(3):278-284. 134. Shah AA,

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1	of the medi-astinum. Diagn Cytopathol. 2007;35:705-709. 144. Pedote P, Gaudio F, Moschetta M, et al. CT-guided needle biopsy performed with modified coaxial technique in the diagnosis of malignant lymphomas. Radiol Med. 2010;115: 1292-1303. 145. Demmy TL, Krasna MJ. Multicenter VATS experience with mediastinal tumors. Ann Thorac Surg. 1998;66:187-192. 146. Bodner J, Wykypiel H, Greiner A, et al. Early experience with robot-assisted surgery for mediastinal masses. Ann Thorac Surg. 2004;78:259-265. 147. Yim AP. Video-assisted thoracoscopic resection of anterior mediastinal masses. Int Surg. 1996;81(4):350-353. 148. Weksler B, Tavares J. Robot-assisted thymectomy is superior to transsternal thymectomy. Surg Endosc. 2012;26:261-266. 149. Chang PC, Chou SH, Kao EL, et al. Bilateral video-assisted thoracoscopic thymectomy vs. extended transsternal thymec-tomy in myasthenia gravis: a prospective study. Eur Surg Res. 2005;37:199-203. 150. Meyers BF, Cooper JD. Transcervical thymectomy for

1	thoracoscopic thymectomy vs. extended transsternal thymec-tomy in myasthenia gravis: a prospective study. Eur Surg Res. 2005;37:199-203. 150. Meyers BF, Cooper JD. Transcervical thymectomy for myas-thenia gravis. Chest Surg Clin N Am. 2001;11(2):363-368. 151. Small EJ, Venook AP, Damon LE. Gallium-avid thymic hyper-plasia in an adult after chemotherapy for Hodgkin disease. Cancer. 1993;72(3):905-908. 152. Smith CS, Schoder H. Thymic extension in the superior medi-astinum in patients with thymic hyperplasia: potential cause of false-positive findings on 18F-FDG PET/CT. AJR Am J Roent-genol. 2007;188:1716-1721. 153. Quint LE. PET: other thoracic malignancies. Cancer Imaging. 2006;6:S82-S88. 154. Luzzi L, Campione A, Gorla A, et al. Role of fluorinefluo-rodeoxyglucose positron emission tomography/computed tomography in preoperative assessment of anterior mediastinal masses. Eur J Cardiothorac Surg. 2009;36:475-479. 155. Masaoka A, Monden Y, Nakahara K, Tanioka T. Follow-up study of

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1	Congenital Heart DiseaseRaghav Murthy, Tabitha G. Moe, Glen S. Van Arsdell, John J. Nigro, and Tara Karamlou20chapterINTRODUCTIONCongenital heart surgery is a dynamic and evolving field. The last 20 years have brought about rapid developments in technol-ogy, emphasis on a multidisciplinary approach to treatment, and a more thorough understanding of both the anatomy and patho-physiology of congenital heart disease, leading to the improved care of these challenging patients.These advancements have created a sustained paradigm shift in the field of congenital heart surgery. The traditional strategy of initial palliation followed by definitive correction at a later age, which had pervaded the thinking of most surgeons, began to evolve into emphasizing early repair. Defects such as hypoplastic left heart syndrome (HLHS) are now successfully managed with staged palliation, resulting in excellent survival outcomes for these children.The goal in most cases of congenital heart disease (CHD) is

1	left heart syndrome (HLHS) are now successfully managed with staged palliation, resulting in excellent survival outcomes for these children.The goal in most cases of congenital heart disease (CHD) is appropriate timing of complete repair. Rather than subdivid-ing lesions into cyanotic or noncyanotic lesions, a more appro-priate classification divides defects into three categories based on the feasibility of achieving complete repair: (a) defects that have no reasonable palliation and for which repair is the only option; (b) defects for which repair is not possible and for which palliation is the only option; and (c) defects that can either be repaired or palliated in infancy. It bears mentioning that all defects in the second category are those in which the appropriate anatomic components either are not present, as in hypoplastic left heart syndrome, or cannot be created from existing structures, i.e., unguarded tricuspid orifice.1Eight out of every 1000 live births will have some

1	either are not present, as in hypoplastic left heart syndrome, or cannot be created from existing structures, i.e., unguarded tricuspid orifice.1Eight out of every 1000 live births will have some form of CHD, most of which, however, are mild.1 In the United States nearly 40,000 infants are affected each year.2 As of 2010, it is estimated that there are about 2 million people living with CHD in the United States, and as of 2011 there are more adults (>18) than children.2 CHD is the most common birth defect and the most common cause of infant death related to birth defects, accounting for 28% of deaths due to birth defects in the first month of life. There are currently 127 centers in North America that perform congenital heart surgery. The Society for Thoracic Surgeons (STS) reports an overall national mortality of 3.1%.3DEFECTS AMENABLE TO COMPLETE REPAIRAtrial Septal DefectAn atrial septal defect (ASD) is defined as discontinuity of the interatrial septum that permits direct mixing

1	national mortality of 3.1%.3DEFECTS AMENABLE TO COMPLETE REPAIRAtrial Septal DefectAn atrial septal defect (ASD) is defined as discontinuity of the interatrial septum that permits direct mixing of blood between the systemic venous and pulmonary venous circulations.Embryology. The atrial and ventricular septa form between the third and sixth weeks of fetal development. After the paired heart tubes fuse into a single tube folded onto itself, the distal por-tion of the tube indents to form the roof of the common atrium. Near this portion of the roof, the septum primum originates and descends in a crescentic formation toward the atrioventricular (AV) junction. The ostium primum is situated superiorly to the crux of the heart at the atrioventricular junction. Prior to completion of endocardial cushion fusion with the septum pri-mum, a sequence of fenestrations appear that coalesce into the Introduction 751Defects Amenable to Complete Repair 751Atrial Septal Defect / 751Aortic Stenosis /

1	cushion fusion with the septum pri-mum, a sequence of fenestrations appear that coalesce into the Introduction 751Defects Amenable to Complete Repair 751Atrial Septal Defect / 751Aortic Stenosis / 755Patent Ductus Arteriosus / 759Aortic Coarctation / 761Truncus Arteriosus / 764Total Anomalous Pulmonary Venous Connection / 765Cor Triatriatum / 768Aortopulmonary Window / 769Vascular Rings and Pulmonary Artery Slings / 769Defects Requiring Palliation 770Tricuspid Atresia / 770Hypoplastic Left Heart Syndrome / 773Defects That May be Palliated or Repaired 777Ebstein’s Anomaly / 777Transposition of the Great Arteries / 780Double-Outlet Right Ventricle / 783Double-Outlet Right Ventricle With Noncommitted Ventricular Septal Defect / 783Double-Outlet Right Ventricle With Subaortic or Doubly Committed Ventricular Septal Defect Without Pulmonary Stenosis / 784Double-Outlet Right Ventricle With Subaortic or Doubly Committed Ventricular Septal Defect With Pulmonary Stenosis /

1	or Doubly Committed Ventricular Septal Defect Without Pulmonary Stenosis / 784Double-Outlet Right Ventricle With Subaortic or Doubly Committed Ventricular Septal Defect With Pulmonary Stenosis / 784Taussig–Bing Syndrome Without Pulmonary Stenosis / 784Taussig–Bing Syndrome With Pulmonary Stenosis / 784Tetralogy of Fallot / 784Ventricular Septal Defect / 786Atrioventricular Canal Defects / 789Interrupted Aortic Arch / 790Pediatric Mechanical Circulatory Support / 790Pediatric Heart Transplantation / 791Public Reporting and the STS Database in Congenital Heart Surgery / 792Future Directions / 793Brunicardi_Ch20_p0751-p0800.indd 75122/02/19 2:54 PM 752ostium secundum. During this coalescence, the septum secun-dum grows downward from the roof of the atrium, parallel to and to the right of the septum primum. The septum primum does not fuse, but creates an oblique pathway, called the foramen ovale, within the interatrial septum. After birth, the increase in left atrial pressure

1	right of the septum primum. The septum primum does not fuse, but creates an oblique pathway, called the foramen ovale, within the interatrial septum. After birth, the increase in left atrial pressure associated with an increase in SVR relative to PVR typically closes this pathway in approximately 80% of the population, obliterating the interatrial communication.Anatomy. ASDs can be classified into three different types (Fig. 20-1): (a) ostium secundum type defect (Fig. 20-1B,C) (deficiency of septum primum), which are the most prevalent subtype, comprising 80% of all ASDs; (b) ostium primum defects (Fig. 20-1A), which may also be described as partial or transitional AV canal defect; and (c) sinus venosus type defects, comprising approximately 5% to 10% of all ASDs.4Pathophysiology. ASDs result in an increase in pulmonary blood flow secondary to primarily left-to-right shunting through the defect. The direction of the intracardiac shunt is predomi-nantly determined by the compliance of

1	in an increase in pulmonary blood flow secondary to primarily left-to-right shunting through the defect. The direction of the intracardiac shunt is predomi-nantly determined by the compliance of the respective ventri-cles. In utero, the distensibility, or compliance, of the right and left ventricles is equal, but postnatally the left ventricle (LV) becomes less compliant than the right ventricle (RV). This shift occurs because the resistance of the downstream vascular beds changes after birth. The pulmonary vascular resistance falls with the infant’s first breath, decreasing RV pressure, whereas the systemic vascular resistance rises dramatically, increasing LV pressure. The increase in LV pressure promotes hypertrophy with a thicker muscle mass, which offers a greater resistance to diastolic filling than does the RV; thus, the majority of flow through the ASD occurs from left to right. The greater volume of blood returning to the right atrium causes volume overload in the RV, but

1	filling than does the RV; thus, the majority of flow through the ASD occurs from left to right. The greater volume of blood returning to the right atrium causes volume overload in the RV, but because of its lower muscle mass and low-resistance output, it easily distends to accommodate the increased volume.The long-term consequences of RV volume overload include hypertrophy with elevated RV end-diastolic pressure and a relative pulmonary stenosis across the pulmonary valve because it cannot accommodate the increased RV flow. Com-pliance gradually decreases as the right ventricular pressure approaches systemic pressure, and the size of the left-to-right shunt decreases. Patients at this stage have a balanced circula-tion and may deceptively appear less symptomatic.Key Points1 Congenital heart disease comprises a wide morphologic spec-trum. In general, lesions can be conceptualized as those that can be completely repaired, those that should be palliated, and those that can be either

1	heart disease comprises a wide morphologic spec-trum. In general, lesions can be conceptualized as those that can be completely repaired, those that should be palliated, and those that can be either repaired or palliated depending on particular patient and institutional characteristics.2 Percutaneous therapies for congenital heart disease are quickly becoming important adjuncts, and in some cases, alternatives, to standard surgical therapy. Important exam-ples include percutaneous closure of atrial and ventricular septal defects, the hybrid approach to hypoplastic left heart syndrome, radiofrequency perforation of the pulmonary valve, and percutaneous pulmonary valve placement. Further studies are necessary to establish criteria and current bench-marks for the safe integration of these novel approaches into the care of patients with congenital heart surgery.3 Patients with critical left ventricular outflow tract obstruc-tion, such as neonatal critical aortic stenosis, represent a

1	novel approaches into the care of patients with congenital heart surgery.3 Patients with critical left ventricular outflow tract obstruc-tion, such as neonatal critical aortic stenosis, represent a challenging population. It is critical that the correct decision (whether to pursue univentricular or biventricular repair) be made prior to the initial operation, as attrition when the incorrect decision is made is high. There are several pub-lished criteria (Congenital Heart Surgeons’ Society critical stenosis calculator) to help surgeons decide which strategy to pursue.4 Optimum strategy for repair of total anomalous pulmonary venous connection (TAPVC) remains a topic of some con-tention. Sutureless repair, formerly reserved for initial reste-nosis after conventional repair, has evolved in many centers to be the primary approach for high-risk patients. Defining whether sutureless repair should be considered in all patients with TAPVC will require further study.5 Vascular rings and

1	in many centers to be the primary approach for high-risk patients. Defining whether sutureless repair should be considered in all patients with TAPVC will require further study.5 Vascular rings and pulmonary artery slings often require multidisciplinary approaches for management. They can be associated with complete tracheal rings and tracheobronchomalacia.6 A recent prospective, randomized, multi-institutional trial sponsored by the National Institutes of Health, the Systemic Ventricle Reconstruction (SVR) trial, compared the out-comes of neonates with hypoplastic left heart syndrome hav-ing either a modified Blalock–Taussig shunt (MBTS) versus a right ventricle-to-pulmonary artery (RV-PA) shunt. The SVR trial demonstrated that transplantation-free survival 12 months after randomization was higher with the RV-PA shunt than with the MBTS. However, data collected over a mean follow-up period of 32 ± 11 months showed a nonsig-nificant difference in transplantation-free survival between

1	was higher with the RV-PA shunt than with the MBTS. However, data collected over a mean follow-up period of 32 ± 11 months showed a nonsig-nificant difference in transplantation-free survival between the two groups.7 Outcomes have improved substantially over time in congeni-tal heart surgery, and most complex lesions can be operated in early infancy. Neurologic protection, however, remains a key issue in the care of neonates undergoing surgery with cardiopulmonary bypass and deep hypothermic circulatory arrest. New monitoring devices and perioperative strategies are currently under investigation. Attention in the field has shifted from analyses of perioperative mortality, which for most lesions is under 10%, to longer-term outcomes, includ-ing quality of life and neurologic function.8 Pediatric mechanical circulatory support and heart transplan-tation is an upcoming and rapidly evolving component of congenital heart surgery. These are offering options for res-cue, palliation, and

1	mechanical circulatory support and heart transplan-tation is an upcoming and rapidly evolving component of congenital heart surgery. These are offering options for res-cue, palliation, and treatment of complex defects or children who were palliated and failing.9 Public reporting has become an integral part of this subspe-cialty. The Society of Thoracic Surgeons Congenital Heart Surgery Database (STS CHSD) remains the largest database in the world for congenital and pediatric heart surgery. Transparency in overall outcomes, mortality, and morbidity is allowing patients and their families an insight into the complexity of their diagnoses as well as the level of perfor-mance of different centers.Brunicardi_Ch20_p0751-p0800.indd 75222/02/19 2:54 PM 753CONGENITAL HEART DISEASECHAPTER 20ABCFigure 20-1. A. Echocardiogram of a patient with primum type artial septal defect (‘*’ points to the atrial septal defect). B. Echocardiogram of a large secundum type ASD (‘*’ points to the defect).

1	20-1. A. Echocardiogram of a patient with primum type artial septal defect (‘*’ points to the atrial septal defect). B. Echocardiogram of a large secundum type ASD (‘*’ points to the defect). C. Intra-operative picture during repair of atrial septal defect. A large fenestrated atrial septum is seen. Bicaval venous cannulation has been performed and a right atriotomy provides exposure to the atrial septum.Patients with large ASDs gradually develop progressive pulmonary vascular changes as a result of chronic overcircu-lation. The increased pulmonary vascular resistance in these patients leads to an equalization of left and right ventricular pressures, and their ratio of pulmonary (Qp) to systemic flow (Qs), Qp to Qs, will approach 1.5 This does not mean, however, that there is no intracardiac shunting, only that the ratio between the left-to-right component and the right-to-left component is equal.The ability of the RV to recover normal function is related to the duration of chronic

1	shunting, only that the ratio between the left-to-right component and the right-to-left component is equal.The ability of the RV to recover normal function is related to the duration of chronic overload because those undergoing ASD closure before age 10 years have a better likelihood of achieving normal RV volumes and function in the postopera-tive period.6The physiology of sinus venosus ASDs is similar to that discussed earlier, except that these are frequently accompanied by anomalous pulmonary venous drainage. This often results in significant hemodynamic derangements that accelerate the clinical course of these infants.The same increase in symptoms is true for those with ostium primum defects because the associated mitral insuffi-ciency from the “cleft” mitral valve can lead to more atrial vol-ume load and increased atrial level shunting.Diagnosis. Patients with ASDs upon auscultation may reveal prominence of the first heart sound with fixed splitting of the second heart sound.

1	atrial vol-ume load and increased atrial level shunting.Diagnosis. Patients with ASDs upon auscultation may reveal prominence of the first heart sound with fixed splitting of the second heart sound. This results from the relatively fixed left-to-right shunt throughout all phases of the cardiac cycle. A diastolic flow murmur indicating increased flow across the tricuspid valve may be discerned, and frequently, an ejection flow mur-mur can be heard across the pulmonary valve. A right ventricu-lar heave and increased intensity of the pulmonary component of the second heart sound indicates pulmonary hypertension.Chest radiographs in the patient with an ASD demonstrate increased pulmonary vascularity, with prominent hilar mark-ings and cardiomegaly. The electrocardiogram shows right axis deviation with an incomplete bundle-branch block. When right bundle-branch block is associated with a leftward or superior axis, an AV canal defect should be strongly suspected.Diagnosis is clarified by

1	with an incomplete bundle-branch block. When right bundle-branch block is associated with a leftward or superior axis, an AV canal defect should be strongly suspected.Diagnosis is clarified by two-dimensional echocardiogra-phy (Fig. 20-1A,C), and use of color-flow mapping facilitates an understanding of the physiologic derangements created by the defects. Older children and adults with unrepaired ASDs may present with stroke or systemic embolism from paradoxical embolism or atrial arrhythmias from dilation of the right atrium.Echocardiography also enables the clinician to estimate the amount of intracardiac shunting, and it can demonstrate the degree of mitral regurgitation in patients with ostium primum defects. With the addition of an agitated saline injection (bubble study), it can also assist in the detection of sinus venosus defects.The advent of two-dimensional echocardiography with color-flow Doppler has largely superseded the use of cardiac catheterization because the ASD can

1	assist in the detection of sinus venosus defects.The advent of two-dimensional echocardiography with color-flow Doppler has largely superseded the use of cardiac catheterization because the ASD can be well defined by echo-cardiography alone. However, in cases where the right ventric-ular systolic pressure is elevated, or patient is older than age 40 years, catheterization can quantify the degree of pulmonary hypertension because those with a fixed pulmonary vascular resistance greater than 12 U/mL may be considered inoperable.7 Cardiac catheterization also can be useful in that it provides data that enable the calculation of Qp and Qs so that the magnitude of the intracardiac shunt can be determined. The ratio (Qp to Qs) can then be used to determine whether closure is indicated in equivocal cases, because a ratio of Qp to Qs greater than 1.5:1 Brunicardi_Ch20_p0751-p0800.indd 75322/02/19 2:54 PM 754SPECIFIC CONSIDERATIONSPART IIis generally accepted as the threshold for surgical

1	cases, because a ratio of Qp to Qs greater than 1.5:1 Brunicardi_Ch20_p0751-p0800.indd 75322/02/19 2:54 PM 754SPECIFIC CONSIDERATIONSPART IIis generally accepted as the threshold for surgical intervention. Finally, in patients older than age 40 years, cardiac catheteriza-tion can be important to evaluate for the presence of coronary artery disease.In general, ASDs are closed when patients are between 4 and 5 years of age. Children of this size can usually be oper-ated on without the use of blood transfusion and have excellent outcomes. Patients who are symptomatic may require repair earlier, even in infancy. Some surgeons advocate routine repair in infants and children especially in cases where prematurity-related lung disease may accelerate damage to the pulmonary vascular bed, though this philosophy may not be widespread. In a review by Reddy and colleagues, 116 neonates weighing less than 2500 g who underwent repair of simple and complex cardiac defects with the use of

1	though this philosophy may not be widespread. In a review by Reddy and colleagues, 116 neonates weighing less than 2500 g who underwent repair of simple and complex cardiac defects with the use of cardiopulmonary bypass were found to have no intracerebral hemorrhages, no long-term neu-rologic sequelae, and a low operative mortality rate (10%). These results correlated with the length of cardiopulmonary bypass and the complexity of repair.8 These investigators also found an 80% actuarial survival at 1 year and, more importantly, that growth following complete repair was equivalent to weight-matched neonates free from cardiac defects.8Treatment. Simple secundum type ASDs can frequently be repaired via a transcatheter technique, and assessment for trans-catheter closure with TTE assessment is generally indicated prior to consideration of a surgical repair. The most common surgical approach requires standard cardiopulmonary bypass (CPB) tech-nique through a midline sternotomy approach.

1	is generally indicated prior to consideration of a surgical repair. The most common surgical approach requires standard cardiopulmonary bypass (CPB) tech-nique through a midline sternotomy approach. The details of the repair itself are generally straightforward. An oblique atriotomy is made, the position of the coronary sinus and all systemic and pulmonary veins are determined, and the rim of the defect is completely visualized. Closure of an ostium secundum defect is accomplished either by primary repair or by insertion of a patch that is sutured to the rim of the defect. The decision of whether patch closure is necessary can be determined by the size and shape of the defect as well as by the quality of the edges.The type of repair used for sinus venosus ASDs associated with partial anomalous pulmonary venous connection is dictated by the location of the anomalous pulmonary vein. If the anoma-lous veins connect to the atria or to the superior vena cava cau-dal to where the cava is

1	pulmonary venous connection is dictated by the location of the anomalous pulmonary vein. If the anoma-lous veins connect to the atria or to the superior vena cava cau-dal to where the cava is crossed by the right pulmonary artery, the ASD can be repaired by inserting a patch, with redirection of the pulmonary veins behind the patch to the left atrium. Care must be taken with this approach to avoid obstruction of the pulmonary veins or the superior vena cava, although usually the superior vena cava is dilated and provides ample room for patch insertion. If the anomalous vein connects to the superior vena cava cranial to the right pulmonary artery, an alternative technique, the Warden procedure, may be necessary. In this operation, the superior vena cava is transected cranial to the connection of the anomalous vein (usually the right superior pulmonary vein). The caudal end of the transected cava is over-sewn. The cranial end of the transected cava is anastomosed to the auricle of the

1	of the anomalous vein (usually the right superior pulmonary vein). The caudal end of the transected cava is over-sewn. The cranial end of the transected cava is anastomosed to the auricle of the right atrium. Inside the atrium, a patch is used to redirect pulmonary venous blood flow to the left atrium. In contrast to the repair for a defect where the pulmonary veins enter the right atrium or the superior vena cava below the right pulmonary artery, the patch covers the superior vena caval right atrial junction so that blood from the anomalous pulmonary vein that enters the cava is directed to the left atrium. Blood returning from the upper body enters the right atrium via the anastomosis between the superior vena cava and the right atrial appendage.Results and Complications of Surgical ASD Closure. Tra-ditional operative strategies, such as pericardial or synthetic patch closure, have been well established, with a low complica-tion rate and a mortality rate of zero among patients

1	ASD Closure. Tra-ditional operative strategies, such as pericardial or synthetic patch closure, have been well established, with a low complica-tion rate and a mortality rate of zero among patients without pulmonary hypertension.9 The most frequently reported imme-diate complications include postpericardiotomy syndrome and atrial arrhythmias. Beyond immediate postoperative outcomes, long-term outcomes following surgical closure (up to 20 years) document the low mortality rates and durability of functional status benefit. Importantly, however, atrial arrhythmias, par-ticularly atrial fibrillation, are not completely mitigated by closure and can occur in 10% to 40% of patients, especially in older patients (>40 years) or those with preexisting arrhyth-mias.10 Kutty and colleagues11 followed 300 patients from their institution, 152 of whom had surgical closure. Late mortality at 10 years was 3%, and functional health status had declined in only 15 patients during follow-up. Recently,

1	300 patients from their institution, 152 of whom had surgical closure. Late mortality at 10 years was 3%, and functional health status had declined in only 15 patients during follow-up. Recently, there have been an increasing number of reports regarding the results follow-ing surgical closure among elderly patients (>60 years of age), which demonstrate equivalent survival to younger patients, albeit with slightly higher complication rates.11-13 Hanninen and colleagues14 studied 68 patients between 68 and 86 years at their institution undergoing either surgical (n = 13) or device (n = 54) closure. Although the 23% incidence of major complications (including pneumothorax, heart failure, and pneumonia) was higher than that recently reported by Mascio et al15 using the Society of Thoracic Surgeons’ Congenital Database (20%) or a single-institution review by Hopkins et al16 (12%), there were no operative deaths among the elderly cohort. Moreover, after ASD closure, echocardiographic

1	Surgeons’ Congenital Database (20%) or a single-institution review by Hopkins et al16 (12%), there were no operative deaths among the elderly cohort. Moreover, after ASD closure, echocardiographic indices of right ventricular size and function were significantly improved from preoperative val-ues, and functional capacity as measured by standardized survey instruments was also significantly improved.New and Future Approaches to Traditional Surgical ASD Closure. Because of the uniformly excellent outcomes with traditional surgery, attention has shifted to improving the cos-metic result and minimizing hospital stay and convalescence. Multiple strategies have been described to achieve these aims, including the right submammary incision with anterior thora-cotomy, limited bilateral submammary incision with partial sternal split, and limited midline incision with partial sternal split. Some surgeons use either video-assisted thoracic surgery (VATS) in conjunction with the submammary and

1	incision with partial sternal split, and limited midline incision with partial sternal split. Some surgeons use either video-assisted thoracic surgery (VATS) in conjunction with the submammary and transxiphoid approaches to facilitate closure within a constricted operative field or totally endoscopic repair in selected patients.17-20 Use of robotics has also been reported in a small series of 12 adult patients by Argenziano and colleagues.18 The morbidity and mortality of all of these approaches are comparable to those of the traditional median sternotomy; however, each has technical drawbacks. Operative precision must be maintained with limited exposure in any minimally invasive technique. Extended CPB and aortic cross-clamp times, coupled with increased cost, may limit the utility of totally endoscopic or robotic-assisted ASD closure except at specific centers. Moreover, certain approaches have a specific patient population in whom they are most appli-cable. For example, the

1	of totally endoscopic or robotic-assisted ASD closure except at specific centers. Moreover, certain approaches have a specific patient population in whom they are most appli-cable. For example, the anterolateral thoracotomy should not be employed in prepubescent girls because it will interfere with breast development. Most totally endoscopic approaches are not feasible in very young patients because of the size of the tho-racoscopic ports. Despite these potential drawbacks, however, in carefully selected patients, minimally invasive techniques have demonstrated benefits. Luo and associates performed Brunicardi_Ch20_p0751-p0800.indd 75422/02/19 2:54 PM 755CONGENITAL HEART DISEASECHAPTER 20ABFigure 20-2. A. Picture of the Amplatzr device after open retrieval from the heart (dislodged during percutaenous catheter placement). B. Echocardiographic view of the septum after transcatheter closure of the atrial septal defect with an Amplatzar device.a prospective randomized study comparing

1	percutaenous catheter placement). B. Echocardiographic view of the septum after transcatheter closure of the atrial septal defect with an Amplatzar device.a prospective randomized study comparing ministernotomy (division of the upper sternum for aortic and pulmonary lesions and the lower sternum for septal lesions) to full sternotomy in 100 consecutive patients undergoing repair of septal lesions.19 The patients in the ministernotomy group had longer procedure times (by 15 to 20 minutes) but had less bleeding and shorter hospital stays. Consistent with these initiatives, conversion of “low-risk” patients undergoing minimally invasive ASD closure to an ambulatory population (discharge from hospital within 24 hours) has recently been described.21First performed in 1976, transcatheter closure of ASDs with the use of various occlusion devices is gaining widespread accep-tance.22 Certain types of ASDs, including patent foramen ovale, secundum defects, and some fenestrated secundum

1	closure of ASDs with the use of various occlusion devices is gaining widespread accep-tance.22 Certain types of ASDs, including patent foramen ovale, secundum defects, and some fenestrated secundum defects, are amenable to device closure, as long as particular ana-tomic criteria (e.g., an adequate superior and inferior rim for device seating and distance from the AV valve) are met. Since the introduction of percutaneous closure (Fig. 20-2A,B), there has been a dramatic rise in device closure prevalence to the point where device closure has supplanted surgical therapy as the domi-nant treatment modality for secundum ASD.23 A study from Karamlou et al23 found that ASD and patent foramen ovale clo-sures per capita increased dramatically from 1.08 per 100,000 population in 1988 to 2.59 per 100,000 population in 2005, an increase of 139%. When analyzed by closure type, surgical clo-sure increased by only 24% (from 0.86 per 100,000 population in 1988 to 1.07 per 100,000 in 2005), whereas

1	100,000 population in 2005, an increase of 139%. When analyzed by closure type, surgical clo-sure increased by only 24% (from 0.86 per 100,000 population in 1988 to 1.07 per 100,000 in 2005), whereas transcatheter closure increased by 3475% (from 0.04 per 100,000 population in 1988 to 1.43 per 100,000 in 2005). Importantly, this study determined that the paradigm shift favoring transcatheter closure has occurred mainly due to increased prevalence of closure in adults over age 40 years rather than an increase in closure in infants or children.Despite the simplicity of ASD repair, there are a myriad of options for patients and physicians who care for patients with CHD. The patient population that might benefit from closure (whether device or surgical) is likely to increase, challenging current ideas and treatment algorithms that optimize outcomes.2Aortic StenosisAnatomy and Classification. The spectrum of aortic valve abnormality represents the most common form of CHD, with the great

1	ideas and treatment algorithms that optimize outcomes.2Aortic StenosisAnatomy and Classification. The spectrum of aortic valve abnormality represents the most common form of CHD, with the great majority of patients being asymptomatic until midlife. Obstruction of the left ventricular outflow tract (LVOT) occurs at multiple levels: subvalvular, valvular, and supravalvular (Fig. 20-3A-D). The critically stenotic aortic valve in the neo-nate or infant is commonly unicommissural or bicommissural, with thickened, dysmorphic, and myxomatous leaflet tissue and a reduced cross-sectional area at the valve level. Associ-ated left-sided lesions are often present. In a review of 32 cases from the Children’s Hospital in Boston, 59% had unicommis-sural valves, and 40% had bicommissural valves.24 Associated lesions were frequent, occurring in 88% of patients, most com-monly patent ductus arteriosus, mitral regurgitation, and hypo-plastic LV. Endocardial fibroelastosis (EFE) also is common among

1	lesions were frequent, occurring in 88% of patients, most com-monly patent ductus arteriosus, mitral regurgitation, and hypo-plastic LV. Endocardial fibroelastosis (EFE) also is common among infants with critical aortic stenosis (AS). In this condi-tion, the LV is usually prohibitively hypoplastic and noncom-pliant, rendering these patients poor candidates for recruitment of the LV into the systemic circulation with techniques that can be utilized in those with more normal sized LVs. In some neonates with critical AS, a dilated LV with poor diastolic com-pliance rather than a hypertrophied LV is encountered.24Neonates with critical AS are a challenging population because one must make a decision about the suitability of the left-sided structures to support a biventricular circulation. There are recent approaches that include techniques, such as aortic valvotomy coupled with EFE resection and mitral valve inter-vention, that are directed at LV rehabilitation. The advent of fetal

1	There are recent approaches that include techniques, such as aortic valvotomy coupled with EFE resection and mitral valve inter-vention, that are directed at LV rehabilitation. The advent of fetal valvotomy for critical AS may also increase the number of infants who are candidates for biventricular repair.Pathophysiology. The unique intracardiac and extracardiac shunts present in fetal life allow even neonates with critical AS to survive. In utero, left ventricular hypertrophy and ischemia cause left atrial hypertension, which reduces the right-to-left flow across the foramen ovale. In severe cases, a reversal of Brunicardi_Ch20_p0751-p0800.indd 75522/02/19 2:54 PM 756SPECIFIC CONSIDERATIONSPART IIFigure 20-3. A. Congenital aortic valve stenosis, en fosse echocardiographic view of the stenotic bicuspid aortic valve. Parasternal long axis view of the same valve with a gradient of 60 mm of Hg (‘*’ points to the valve). B. Parasternal long axis ecocardiographic view of a patient with

1	stenotic bicuspid aortic valve. Parasternal long axis view of the same valve with a gradient of 60 mm of Hg (‘*’ points to the valve). B. Parasternal long axis ecocardiographic view of a patient with discrete subaortic membrane (‘*’ points to the membrane). C. Parasternal long axis ecocardiographic view of a patient with diffuse tunnel like subvalvar aortic stenosis with membrane. Doppler revealed a gradient of 81 mm of hg (‘*’ represents the area of diffuse narrowing). D. Appearance of supravalvar aortic stenosis on an aortogram performed in the cardiac catheterization lab (‘*’ points to the stenosis). E. Appearance after four patch reconstruction of the same patient shown in Figure 20.3 d. (Re-formatted images obtained from a CT angiogram).ABCDEBrunicardi_Ch20_p0751-p0800.indd 75622/02/19 2:54 PM 757CONGENITAL HEART DISEASECHAPTER 20flow may occur, causing right ventricular volume loading. The RV then provides the entire systemic output via the patent duc-tus arteriosus

1	75622/02/19 2:54 PM 757CONGENITAL HEART DISEASECHAPTER 20flow may occur, causing right ventricular volume loading. The RV then provides the entire systemic output via the patent duc-tus arteriosus (ductal-dependent systemic blood flow). Although cardiac output is maintained, the LV suffers continued damage as the intracavitary pressure precludes adequate coronary perfu-sion, resulting in LV infarction and subendocardial fibroelas-tosis. The presentation of the neonate with critical AS is then determined by the morphology of the LV and other left-sided heart structures, the degree of left ventricular dysfunction, and the completeness of the transition from a parallel circulation to an in-series circulation (i.e., on closure of the foramen ovale and the ductus arteriosus). Those infants with mild-to-moderate AS in whom LV function is preserved are asymptomatic at birth. The only abnormalities may be a systolic ejection murmur and electrocardiogram (ECG) evidence of left ventricular

1	with mild-to-moderate AS in whom LV function is preserved are asymptomatic at birth. The only abnormalities may be a systolic ejection murmur and electrocardiogram (ECG) evidence of left ventricular hypertro-phy. However, those neonates with severe AS and compromised LV function are unable to provide adequate cardiac output at birth and will present in circulatory collapse once the ductus closes, with dyspnea, tachypnea, irritability, narrowed pulse pressure, oliguria, and profound metabolic acidosis.24 If ductal patency is maintained, systemic perfusion will be provided by the RV via ductal flow, and cyanosis may be the only finding.Diagnosis. Neonates and infants with severe valvular AS may have a relatively nonspecific history of irritability and failure to thrive. Angina, if present, is usually manifested by episodic, inconsolable crying that coincides with feeding. As discussed previously, evidence of poor peripheral perfusion, such as extreme pallor, indicates severe LVOT

1	is usually manifested by episodic, inconsolable crying that coincides with feeding. As discussed previously, evidence of poor peripheral perfusion, such as extreme pallor, indicates severe LVOT obstruction. Differen-tial cyanosis is an uncommon finding, but it is present when enough antegrade flow occurs only to maintain normal upper body perfusion, while a large patent ductus arteriosus produces blue discoloration of the abdomen and legs.Physical findings include a systolic ejection murmur, although a quiet murmur may paradoxically indicate a more severe condition with reduced cardiac output. A systolic click correlates with a valvular etiology of obstruction. As LV dys-function progresses, evidence of congestive heart failure occurs.The chest radiograph is variable but may show dilatation of the aortic root, and the ECG often demonstrates LV hypertro-phy. Echocardiography with Doppler flow is extremely useful in establishing the diagnosis, as well as quantifying the transvalvular

1	of the aortic root, and the ECG often demonstrates LV hypertro-phy. Echocardiography with Doppler flow is extremely useful in establishing the diagnosis, as well as quantifying the transvalvular gradient. Furthermore, echocardiography can facilitate evaluation for the several associated defects that can be present in critical neonatal AS, including mitral stenosis, LV hypoplasia, LV endo-cardial fibroelastosis, subaortic stenosis, VSD, or coarctation. The presence of any or several of these defects has important impli-cations related to treatment options for these patients. Although cardiac catheterization is not routinely performed for diagnostic purposes, it can be invaluable as part of the treatment algorithm if the lesion is amenable to balloon valvotomy. Magnetic resonance imaging (MRI) is another very useful technique for assessing the adequacy of the left-sided structures and is increasingly utilized to determine candidacy for biventricular repairs.Treatment. As alluded to

1	(MRI) is another very useful technique for assessing the adequacy of the left-sided structures and is increasingly utilized to determine candidacy for biventricular repairs.Treatment. As alluded to previously, the first decision that must be made in the neonate with critical LVOT obstruction is whether the patient is a candidate for biventricular or univen-tricular repair. Central to this decision is assessment of the degree of hypoplasia of the LV and other left-sided structures. Alsoufi and colleagues25 have described a rational approach to the neonate with critical LVOT obstruction. The options vary depending on whether the infant follows a single or a 3biventricular pathway. The options for a single ventricle include the Norwood operation, a hybrid strategy (initial ductal stent and bilateral pulmonary artery bands followed by later completion of the Norwood operation) or heart transplantation. The options for a biventricular heart include balloon valvuloplasty, surgical

1	and bilateral pulmonary artery bands followed by later completion of the Norwood operation) or heart transplantation. The options for a biventricular heart include balloon valvuloplasty, surgical val-votomy, neonatal Ross operation, or a Yasui operation. Often valvotomy is accompanied by LV rehabilitation techniques, including EFE resection and mitral valve interventions. Fetal aortic valvotomy, which is now offered at specialized centers, is another promising strategy to decompress the LV in fetal life and potentially allow growth of the left-sided structures sufficient to permit a biventricular circulation. Regardless of whether the baby is triaged to a single or biventricular strategy, any infant with severe AS requires urgent intervention. Preoperative stabi-lization, however, has dramatically altered the clinical algorithm and outcomes for this patient population.25 The preoperative strategy begins with endotracheal intubation and inotropic sup-port. Prostaglandin infusion is

1	dramatically altered the clinical algorithm and outcomes for this patient population.25 The preoperative strategy begins with endotracheal intubation and inotropic sup-port. Prostaglandin infusion is initiated to maintain ductal patency, and confirmatory studies are performed prior to opera-tive intervention. Therapy is generally indicated in the presence of a transvalvular gradient of 50 mmHg with associated symp-toms including syncope, CHF, or angina, or if a gradient of 50 to 75 mmHg exists with concomitant ECG evidence of LV strain or ischemia. In the critically ill neonate, a gradient across the aortic valve may not be present because of poor LV function. However, the decision regarding treatment options must be based on a complete understanding of associated defects. For example, in the presence of a hypoplastic LV (left ventricular end-diastolic volume <20 mL/m2) or a markedly abnormal mitral valve, iso-lated aortic valvotomy should not be performed because studies have

1	in the presence of a hypoplastic LV (left ventricular end-diastolic volume <20 mL/m2) or a markedly abnormal mitral valve, iso-lated aortic valvotomy should not be performed because studies have demonstrated high mortality in this population following isolated valvotomy.26Patients who have an LV capable of providing systemic output are candidates for intervention to relieve AS, generally through balloon valvotomy. Occasionally, if catheter-based therapy is not an option, relief of valvular AS in infants and children can be accomplished with surgical valvotomy using standard techniques of CPB and direct exposure to the aortic valve. A transverse incision is made in the ascending aorta above the sinus of Valsalva, extending close to, but not into, the noncoronary sinus. Exposure is attained with placement of a retractor into the right coronary sinus. After inspection of the valve, the chosen commissure is incised to within 1 to 2 mm of the aortic wall (Fig. 20-4A,B).Balloon valvotomy

1	with placement of a retractor into the right coronary sinus. After inspection of the valve, the chosen commissure is incised to within 1 to 2 mm of the aortic wall (Fig. 20-4A,B).Balloon valvotomy performed in the catheterization lab is generally the procedure of choice for reduction of transvalvular gradients in symptomatic infants and children without signifi-cant aortic insufficiency. Balloon valvotomy provides relief of the valvular gradient and allows future surgical intervention (which is generally required in most patients when a larger prosthesis can be implanted) to be performed on an unscarred chest. An important issue when planning aortic valvotomy, whether percutaneously or via open surgical technique, is the risk of inducing hemodynamically significant aortic regurgita-tion. Induction of more than moderate aortic regurgitation is poorly tolerated in the infant with critical AS and may require an urgent procedure to replace or repair the aortic valve. Most often in these

1	Induction of more than moderate aortic regurgitation is poorly tolerated in the infant with critical AS and may require an urgent procedure to replace or repair the aortic valve. Most often in these patients, a Ross procedure represents the only real option as mechanical valve replacement in a neonate has exceptionally poor outcome.In general, catheter-based balloon valvotomy has supplanted open surgical valvotomy. The decision regarding Brunicardi_Ch20_p0751-p0800.indd 75722/02/19 2:54 PM 758SPECIFIC CONSIDERATIONSPART IIFigure 20-4. A. Intra-operative picture of a stenotic bicuspid aortic valve (as seen through an aortotomy). B. Intra-operative picture of the valve after a controlled valvotomy if performed. Note the forceps is across the opening of the aortic valve (‘*’ points to the valvotomy).the most appropriate method to use depends on several factors, including the available medical expertise, the patient’s overall status and hemodynamics, and the presence of associated

1	the valvotomy).the most appropriate method to use depends on several factors, including the available medical expertise, the patient’s overall status and hemodynamics, and the presence of associated cardiac defects requiring repair.25 Although evidence is emerging to the contrary, simple valvotomy, whether performed using percutaneous or open technique, is generally considered a palliative procedure. The goal is to relieve LVOT obstruction without producing clinically significant regurgitation, in order to allow sufficient annular growth for eventual aortic valve replacement. The reintervention rate is higher if balloon valvuloplasty is performed as the initial palliation (54%) compared to a surgical valvolomy (23%) as the latter is a more controlled division of the aortic commissure25 (Fig. 20-4C). The majority of infants who undergo aortic valvotomy will require further intervention on the aortic valve within 10 years following initial intervention.26Neonates with severely

1	(Fig. 20-4C). The majority of infants who undergo aortic valvotomy will require further intervention on the aortic valve within 10 years following initial intervention.26Neonates with severely hypoplastic LVs or significant LV endocardial fibroelastosis may not be candidates for biventricu-lar repair and are treated the same as infants with the hypoplas-tic left heart syndrome (HLHS), which is discussed later (see “Hypoplastic Left Heart Syndrome”).As mentioned previously, fetal intervention for the aortic valve has been described with the goal being to improve the growth of the left ventricle. The group at Boston Children’s Hospital have reported fairly favorable results in a small cohort.34Many surgeons previously avoided aortic valve replace-ment for AS in early childhood because the more commonly used mechanical valves would be outgrown and require replace-ment later and the obligatory anticoagulation for mechanical valves resulted in a substantial risk for complications. In

1	the more commonly used mechanical valves would be outgrown and require replace-ment later and the obligatory anticoagulation for mechanical valves resulted in a substantial risk for complications. In addi-tion, prosthetic valves have an incidence of bacterial endocardi-tis or perivalvular leak requiring reintervention.The use of allografts and the advent of the Ross procedure have largely obviated these issues and made early definitive cor-rection of critical AS a viable option.23,27,28 Donald Ross first described transposition of the pulmonary valve into the aortic position with allograft reconstruction of the pulmonary outflow tract in 1967.27 The result of this operation is a normal trileaf-let semilunar valve made of a patient’s native tissue with the potential for growth to adult size in the aortic position in place of the damaged aortic valve (Fig. 20-5). The Ross procedure has become a useful option for aortic valve replacement in children because it has improved durability and

1	in the aortic position in place of the damaged aortic valve (Fig. 20-5). The Ross procedure has become a useful option for aortic valve replacement in children because it has improved durability and can be performed with acceptable morbidity and mortality rates. The placement of a pulmonary conduit, which does not grow and becomes calci-fied and stenotic over time, does obligate the patient to rein-tervention (either surgically or using transcatheter techniques) to replace the RV-to-pulmonary artery conduit. Karamlou and colleagues29 have reviewed the outcomes and associated risk factors for aortic valve replacement in 160 children from the Hospital for Sick Children in Toronto. They found that younger age, lower operative weight, concomitant performance of aortic root replacement or reconstruction, and use of prosthesis type other than a pulmonary autograft were significant predictors of death, whereas the use of a bioprosthetic or allograft valve type and earlier year of operation

1	and use of prosthesis type other than a pulmonary autograft were significant predictors of death, whereas the use of a bioprosthetic or allograft valve type and earlier year of operation were identified as significant risk factors for repeated aortic valve replacement. Autograft use was associated with a blunted progression of the peak prosthetic valve gradient and a rapid decrease in the left ventricular end-diastolic dimension. In agreement with these findings, Lupinetti and Jones28 compared allograft aortic valve replacement with the Ross procedure and found a more significant transvalvular gradient reduction and regression of left ventricular hypertro-phy in those patients who underwent the Ross procedure. In some cases, the pulmonary valve may not be usable because of associated defects or congenital absence. These children are not candidates for the Ross procedure and can be treated with cryopreserved allografts (cadaveric human aortic valves) or prosthetic aortic valve

1	defects or congenital absence. These children are not candidates for the Ross procedure and can be treated with cryopreserved allografts (cadaveric human aortic valves) or prosthetic aortic valve replacement. At times, there may be a size discrepancy between the right ventricular outflow tract (RVOT) and the LVOT, especially in cases of severe critical AS in infancy. For these cases, the pulmonary autograft is placed in a manner that also provides enlargement of the aortic annulus (Ross/Konno).Subvalvular AS occurs beneath the aortic valve and may be classified as discrete or tunnel-like (diffuse). A thin, ABBrunicardi_Ch20_p0751-p0800.indd 75822/02/19 2:54 PM 759CONGENITAL HEART DISEASECHAPTER 20fibromuscular diaphragm immediately proximal to the aortic valve characterizes discrete subaortic stenosis. This diaphragm typically extends for 180o or more in a crescentic or circular fash-ion, often attaching to the mitral valve as well as the interven-tricular septum. The aortic valve

1	stenosis. This diaphragm typically extends for 180o or more in a crescentic or circular fash-ion, often attaching to the mitral valve as well as the interven-tricular septum. The aortic valve itself is usually normal in this condition, although the turbulence imparted by the subvalvular stenosis may affect leaflet morphology and valve competence.Diffuse subvalvular AS results in a long, tunnel-like obstruction that may extend to the left ventricular apex. In some individuals, there may be difficulty in distinguishing between hypertrophic cardiomyopathy and diffuse subaortic steno-sis. Operation for subvalvular AS is indicated with a gradient exceeding 30 mmHg, in the presence of aortic valve insuffi-ciency, or when symptoms indicating LVOT obstruction are present.30 Given that repair of isolated discrete subaortic ste-nosis can be done with low rates of morbidity and mortality, some surgeons advocate repair in all cases of discrete AS to avoid progression of the stenosis and the

1	of isolated discrete subaortic ste-nosis can be done with low rates of morbidity and mortality, some surgeons advocate repair in all cases of discrete AS to avoid progression of the stenosis and the development of aortic insufficiency, although more recent data demonstrate that sub-aortic resection should be delayed until the LV gradient exceeds 30 mmHg because most children with an initial LV gradient less than 30 mmHg have quiescent disease.31 Diffuse AS is a more complex lesion and often requires aortoventriculoplasty. Results are generally excellent, with operative mortality less than 5%.32Supravalvular AS occurs more rarely and also can be clas-sified into a discrete type, which produces an hourglass defor-mity of the aorta, and a diffuse form that can involve the entire arch and brachiocephalic arteries. The aortic valve leaflets are usually normal, but in some cases, the leaflets may adhere to the supravalvular stenosis, thereby narrowing the sinuses of Valsalva in diastole

1	brachiocephalic arteries. The aortic valve leaflets are usually normal, but in some cases, the leaflets may adhere to the supravalvular stenosis, thereby narrowing the sinuses of Valsalva in diastole and restricting coronary artery perfusion. In addition, accelerated intimal hyperplastic changes in the coronary arteries can be demonstrated in these patients because the proximal position of the coronary arteries subjects them to abnormally high perfusion pressures.The signs and symptoms of supravalvular AS are similar to other forms of LVOT obstruction. An asymptomatic murmur is the presenting manifestation in approximately half of these patients. Syncope, poor exercise tolerance, and angina may all occur with nearly equal frequency. Supravalvar AS is associated with Williams’ syndrome, a constellation of elfin facies, mental retardation, and hypercalcemia.33 Following routine evaluation, cardiac catheterization should be performed in order to delin-eate coronary anatomy, as well as to

1	of elfin facies, mental retardation, and hypercalcemia.33 Following routine evaluation, cardiac catheterization should be performed in order to delin-eate coronary anatomy, as well as to delineate the degree of obstruction. A gradient of 50 mmHg or greater is an indication for operation. However, the clinician must be cognizant of any coexistent lesions, most commonly pulmonic stenosis, which may add complexity to the repair.The localized form of supravalvular AS can be treated by creating an inverted Y-shaped aortotomy across the area of ste-nosis, straddling the right coronary artery. The obstructing shelf is then excised, and a pantaloon-shaped patch (Doty technique) or individual sinus patch enlargement (Brom technique) is used (Fig. 20-3E).The diffuse form of supravalvular stenosis is more vari-able (Fig. 20-6), and the particular operative approach must be tailored to each specific patient’s anatomy. In general, either an aortic endarterectomy with patch augmentation can be

1	is more vari-able (Fig. 20-6), and the particular operative approach must be tailored to each specific patient’s anatomy. In general, either an aortic endarterectomy with patch augmentation can be per-formed or if the narrowing extends past the aorta arch, a pros-thetic graft can be placed between the ascending and descending aorta. Operative results for discrete supravalvular AS are gen-erally good, with a hospital mortality of less than 1% and an actuarial survival rate exceeding 90% at 20 years.35 In contrast, however, the diffuse form is more hazardous to repair and car-ried a mortality of 15% in a recent series.35,36Patent Ductus ArteriosusAnatomy. The ductus arteriosus is derived from the sixth aor-tic arch and normally extends from the main or left pulmonary artery to the upper descending thoracic aorta, distal to the left subclavian artery. In the normal fetal cardiovascular system, ductal flow is considerable (approximately 60% of the com-bined ventricular output) and is

1	descending thoracic aorta, distal to the left subclavian artery. In the normal fetal cardiovascular system, ductal flow is considerable (approximately 60% of the com-bined ventricular output) and is directed exclusively from the pulmonary artery to the aorta. In infancy, the length of the duc-tus may vary from 2 to 8 mm, with a diameter of 4 to 12 mm.Locally produced and circulating prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2) induce active relaxation of the duc-tal musculature, maintaining maximal patency during the fetal period.38 At birth, increased pulmonary blood flow metabo-lizes these prostaglandin products, and absence of the placenta removes an important source of them, resulting in a marked decrease in these ductal-relaxing substances. In addition, release of histamines, catecholamines, bradykinin, and acetylcholine all promote ductal contraction. Despite all of these complex Figure 20-5. Appearance of the stenotic aortic valve during aortography performed in the

1	catecholamines, bradykinin, and acetylcholine all promote ductal contraction. Despite all of these complex Figure 20-5. Appearance of the stenotic aortic valve during aortography performed in the cardiac catheterization lab. (left). Balloon valvuloplasty being performed. (right). The ‘*’ points to the the “waist” created by the stenotic valve during dilation. (Used with permission from Kelly Rosso MD.)Brunicardi_Ch20_p0751-p0800.indd 75922/02/19 2:54 PM 760SPECIFIC CONSIDERATIONSPART IIFigure 20-6. Reformatted image obtained after CT angiography of a child with diffuse supravalvar aortic stenosis (‘*’ points to the transverse aortic arch).interactions, the rising oxygen tension in the fetal blood is the main stimulus causing smooth muscle contraction and ductal closure within 10 to 15 hours postnatally.39 Anatomic closure by fibrosis produces the ligamentum arteriosum connecting the pulmonary artery to the aorta.Delayed closure of the ductus is termed prolonged patency, whereas

1	15 hours postnatally.39 Anatomic closure by fibrosis produces the ligamentum arteriosum connecting the pulmonary artery to the aorta.Delayed closure of the ductus is termed prolonged patency, whereas failure of closure causes persistent patency, which may occur as an isolated lesion or in association with more complex congenital heart defects. In many of these infants with more complex congenital heart defects, either pulmonary or systemic perfusion may depend on ductal flow, and these infants may decompensate if exogenous PGE is not administered to maintain ductal patency.Natural History. The incidence of patent ductus arteriosus (PDA) is approximately 1 in every 2000 births; however, it increases dramatically with increasing prematurity.39 In some series, PDAs have been noted in 75% of infants of 28 to 30 weeks gestation. Persistent patency occurs more commonly in females, with a 2:1 ratio.40PDA is not a benign entity, although prolonged survival has been reported. The estimated

1	of infants of 28 to 30 weeks gestation. Persistent patency occurs more commonly in females, with a 2:1 ratio.40PDA is not a benign entity, although prolonged survival has been reported. The estimated death rate for infants with iso-lated, untreated PDA is approximately 30%.41 The leading cause of death is congestive heart failure, with respiratory infection as a secondary cause. Endocarditis is more likely to occur with a small ductus and is rarely fatal if aggressive antibiotic therapy is initiated early.Clinical Manifestations and Diagnosis. After birth, in an otherwise normal cardiovascular system, a PDA results in a left-to-right shunt that depends on both the size of the ductal lumen and its total length. As the pulmonary vascular resistance falls 8 to 10 weeks postnatally, the shunt will increase, and its flow will ultimately be determined by the relative resistances of the pulmonary and systemic circulations.The hemodynamic consequences of an unrestrictive duc-tal shunt are

1	will increase, and its flow will ultimately be determined by the relative resistances of the pulmonary and systemic circulations.The hemodynamic consequences of an unrestrictive duc-tal shunt are left ventricular volume overload with increased left atrial and pulmonary artery pressures and right ventricular strain from the augmented afterload. These changes result in increased sympathetic discharge, tachycardia, tachypnea, and ventricular hypertrophy. The diastolic shunt results in lower aortic diastolic pressure and increases the potential for myo-cardial ischemia and underperfusion of other systemic organs, while the increased pulmonary flow leads to increased work of breathing and decreased gas exchange. Unrestrictive ductal flow may lead to pulmonary hypertension within the first year of life. These changes will be significantly attenuated if the size of the ductus is only moderate, and they will be completely absent if the ductus is small.Physical examination of the afflicted

1	of life. These changes will be significantly attenuated if the size of the ductus is only moderate, and they will be completely absent if the ductus is small.Physical examination of the afflicted infant will reveal evi-dence of a hyperdynamic circulation with a widened pulse pres-sure and a hyperactive precordium. Auscultation demonstrates a systolic or continuous murmur, often termed a machinery mur-mur. Cyanosis is not present in uncomplicated isolated PDA.The chest radiograph may reveal increased pulmonary vascularity or cardiomegaly, and the ECG may show LV strain, left atrial enlargement, and possibly RV hypertrophy. Echocar-diogram with color mapping reliably demonstrates the patency of the ductus as well as estimates the shunt size. Cardiac cath-eterization is necessary only when pulmonary hypertension is suspected.Therapy. The presence of a persistent PDA is sufficient indica-tion for closure because of the increased mortality and risk of endocarditis.40 In older patients with

1	hypertension is suspected.Therapy. The presence of a persistent PDA is sufficient indica-tion for closure because of the increased mortality and risk of endocarditis.40 In older patients with pulmonary hypertension, closure may not improve symptoms and is associated with much higher mortality.In premature infants, aggressive intervention with indometh-acin or ibuprofen to achieve early closure of the PDA is beneficial unless contraindications such as necrotizing enterocolitis or renal insufficiency are present.41 Term infants, however, are gener-ally unresponsive to pharmacologic therapy with indomethacin, so mechanical closure must be undertaken once the diagnosis is established. This can be accomplished either surgically (Fig. 20-7) or with catheter-based therapy.15,42,43 Currently, transluminal placement of various occlusive devices, such as the Rashkind double-umbrella device or embolization with Gianturco coils, is in widespread use.42 However, there are a number of

1	transluminal placement of various occlusive devices, such as the Rashkind double-umbrella device or embolization with Gianturco coils, is in widespread use.42 However, there are a number of complications inherent with the use of percutaneous devices, such as thromboem-bolism, endocarditis, incomplete occlusion, vascular injury, and hemorrhage secondary to perforation.43 In addition, these tech-niques may not be applicable in very young infants because the peripheral vessels do not provide adequate access for the delivery devices. Attempts are being made to develop such devices for pre-mature infants with early successful results in study populations.44Surgical closure can be achieved via either open or video-assisted approaches. The open approach employs a muscle-sparing posterior lateral thoracotomy in the third or fourth intercostal space on the side of the aorta (generally the left). The lung is then retracted anteriorly. In the neonate, the PDA is singly ligated with a surgical

1	thoracotomy in the third or fourth intercostal space on the side of the aorta (generally the left). The lung is then retracted anteriorly. In the neonate, the PDA is singly ligated with a surgical clip or permanent suture. Care must be taken to avoid the recurrent laryngeal nerve, which courses around the PDA. The PDA can also be ligated via a median sternotomy; however, this approach is generally reserved for patients who have additional cardiac or great vessel lesions requiring repair. Occasionally, a short, broad ductus, in which the dimension of Brunicardi_Ch20_p0751-p0800.indd 76022/02/19 2:54 PM 761CONGENITAL HEART DISEASECHAPTER 20its width approaches that of its length, will be encountered. In this case, division between vascular clamps with oversewing of both ends is advisable (Fig. 20-8). In extreme cases, the use of CPB to decompress the large ductus during ligation is an option.Video-assisted thoracoscopic occlusion, using metal clips, also has been described, although

1	(Fig. 20-8). In extreme cases, the use of CPB to decompress the large ductus during ligation is an option.Video-assisted thoracoscopic occlusion, using metal clips, also has been described, although it offers few advantages over the standard surgical approach. Preterm newborns and children may do well with a surgical technique, while older patients (older than age 5 years) and those with smaller ducts (<3 mm) do well with coil occlusion. In fact, Moore and colleagues recently concluded from their series that coil occlusion is the procedure Figure 20-7. Chest x-ray before and after PDA ligation showing the dramatic improvement in the lung fields after ligation (arrow points to the clip used for PDA ligation).Figure 20-8. Surgical PDA ligation. A clip has been applied to occlude the ductus arteriosus. Note the relationship of the recurrent laryngeal nerve to the ductus arteriosus. (Used with permission from Kelly Rosso MD.)of choice for ducts smaller than 4 mm.45 Complete closure rates

1	arteriosus. Note the relationship of the recurrent laryngeal nerve to the ductus arteriosus. (Used with permission from Kelly Rosso MD.)of choice for ducts smaller than 4 mm.45 Complete closure rates using catheter-based techniques have steadily improved.Outcomes. In premature infants, the surgical mortality is very low, although the overall hospital death rate is significant as a consequence of other complications of prematurity. In older infants and children, mortality is less than 1%. Bleeding, chylo-thorax, vocal cord paralysis, and the need for reoperation occur infrequently. With the advent of muscle-sparing thoracotomy, the risk of subsequent arm dysfunction or breast abnormalities is virtually eliminated.46Aortic CoarctationAnatomy. Coarctation of the aorta (COA) is defined as a lumi-nal narrowing in the aorta that causes an obstruction to blood flow. This narrowing is most commonly located distal to the left subclavian artery. The embryologic origin of COA is a sub-ject of

1	lumi-nal narrowing in the aorta that causes an obstruction to blood flow. This narrowing is most commonly located distal to the left subclavian artery. The embryologic origin of COA is a sub-ject of some controversy. One theory holds that the obstructing shelf, which is largely composed of tissue found within the duc-tus, forms as the ductus involutes.47 The other theory holds that a diminished aortic isthmus develops secondary to decreased aortic flow in infants with enhanced ductal circulation.Extensive collateral circulation develops, predominantly involving the intercostals and mammary arteries as a direct result of aortic flow obstruction. This translates into the well-known finding of “rib-notching” on chest radiograph, as well as a prominent pulsation underneath the ribs.Other associated anomalies, such as ventricular septal defect, PDA, and ASD, may be seen with COA, but the most common is that of a bicuspid aortic valve, which can be demon-strated in 25% to 42% of

1	associated anomalies, such as ventricular septal defect, PDA, and ASD, may be seen with COA, but the most common is that of a bicuspid aortic valve, which can be demon-strated in 25% to 42% of cases.48Pathophysiology. Infants with COA develop symptoms con-sistent with left ventricular outflow obstruction, including pulmo-nary overcirculation and, later, biventricular failure. In addition, proximal systemic hypertension develops as a result of mechanical obstruction to ventricular ejection, as well as hypoperfusion-induced activation of the renin-angiotensin-aldosterone system. PAAoBrunicardi_Ch20_p0751-p0800.indd 76122/02/19 2:54 PM 762SPECIFIC CONSIDERATIONSPART IIFigure 20-9. Reformatted images obtained from CT angiography of a baby showing a descrete coarctation of the aorta (‘*’ points to the coarctation).ABFigure 20-10. A. Reformatted images obtained from a CT angio-gram of a child with discrete coarctation of the aorta (‘*’ points to the coarctation). B. Aortogram performed

1	to the coarctation).ABFigure 20-10. A. Reformatted images obtained from a CT angio-gram of a child with discrete coarctation of the aorta (‘*’ points to the coarctation). B. Aortogram performed in the cardiac catheteriza-tion lab after stenting the coarctation (‘*’ points to the stent).Interestingly, hypertension is often persistent after surgical correction despite complete amelioration of the mechanical obstruction and pressure gradient.49 It has been shown that early surgical correction may prevent the development of long-term hypertension, which undoubtedly contributes to many of the adverse sequelae of COA, including the development of circle of Willis aneurysms, aortic dissection and rupture, and an increased incidence of coronary arteriopathy with resulting myocardial infarction.50Diagnosis. COA is likely to become symptomatic either in the newborn period if other anomalies are present or in the late ado-lescent period with the onset of left ventricular failure.Physical

1	is likely to become symptomatic either in the newborn period if other anomalies are present or in the late ado-lescent period with the onset of left ventricular failure.Physical examination will demonstrate a hyperdynamic precordium with a harsh murmur localized to the left chest and back. Femoral pulses will be dramatically decreased when com-pared to upper extremity pulses, and differential cyanosis may be apparent until ductal closure.Echocardiography will reliably demonstrate the narrowed aortic segment, as well as define the pressure gradient across the stenotic segment. In addition, detailed information regarding other associated anomalies can be gleaned. Aortography (Fig. 20-9) is reserved for those cases in which the echocardiographic findings are equivocal. Cross-sectional imaging with computed tomogra-phy (CT) scan or MRI is also increasing to facilitate definition of arch anatomy (i.e., transverse arch hypoplasia), assess intracardiac volumes, and associated

1	imaging with computed tomogra-phy (CT) scan or MRI is also increasing to facilitate definition of arch anatomy (i.e., transverse arch hypoplasia), assess intracardiac volumes, and associated defects.Therapy. The routine management of hemodynamically sig-nificant COA in all age groups has traditionally been surgical. Transcatheter repairs (Fig. 20-10) are used with increasing frequency in older patients and those with recoarctation fol-lowing surgical repair. Balloon dilatation of native coarctation in neonates generally is avoided because of the high recoarc-tation rate. However, in infants who present with severely depressed LV function and a closed ductus arteriosus, initial decompression with balloon dilation of the COA followed by later surgical intervention may be useful. The most common surgical techniques in current use are resection with end-to-end anastomosis or extended end-to-end anastomosis, taking care to remove all residual ductal tissue.51,52 Extended end-to-end

1	most common surgical techniques in current use are resection with end-to-end anastomosis or extended end-to-end anastomosis, taking care to remove all residual ductal tissue.51,52 Extended end-to-end anastomosis (Fig. 20-11) may also allow the surgeon to treat transverse arch hypoplasia, which is commonly encoun-tered in infants with aortic coarctation.53,54 The subclavian flap Brunicardi_Ch20_p0751-p0800.indd 76222/02/19 2:55 PM 763CONGENITAL HEART DISEASECHAPTER 20aortoplasty is another repair, although it is used less frequently in the modern era because of the risk of late aneurysm formation and possible underdevelopment of the left upper extremity isch-emia.52 In this method, the left subclavian artery is transected and brought down over the coarcted segment as a vascular-ized patch. The main benefit of these techniques is that they do not involve the use of prosthetic materials, and evidence sug-gests that extended end-to-end anastomosis may promote arch growth, especially

1	The main benefit of these techniques is that they do not involve the use of prosthetic materials, and evidence sug-gests that extended end-to-end anastomosis may promote arch growth, especially in infants with the smallest initial aortic arch diameters.53Despite the benefits, however, extended end-to-end anas-tomosis may not be feasible when there is a long segment of coarctation or in the presence of previous surgery because suf-ficient mobilization of the aorta above and below the lesion may not be possible. In this instance, prosthetic materials, such as a patch aortoplasty, in which a prosthetic patch is used to enlarge the coarcted segment, or an interposition tube graft must be employed. One of the most important decisions in infants and neonates with COA and some degree of transverse arch hypoplasia is whether the lesion should be approached with a sternotomy or a thoracotomy. Cross-sectional imaging with CT scan can be extremely helpful in assessing the adequacy of the

1	arch hypoplasia is whether the lesion should be approached with a sternotomy or a thoracotomy. Cross-sectional imaging with CT scan can be extremely helpful in assessing the adequacy of the transverse arch and any associated abnormalities with branching that may complicate repair from the side.The most common complications after COA repair are late restenosis (Fig. 20-12) and aneurysm formation at the repair site.55-57 Aneurysm formation is particularly common after patch aortoplasty when using Dacron material. In a large series of 891 patients, aneurysms occurred in 5.4% of the total, with 89% occurring in the group who received Dacron-patch aortoplasty and only 8% occurring in those who received resection with primary end-to-end anastomosis.55 A further complication, although uncommon, is lower-body paralysis resulting from ischemic spinal cord injury during the repair. This dreaded outcome complicates 0.5% of all surgical repairs, but its incidence can be lessened with the use of

1	is lower-body paralysis resulting from ischemic spinal cord injury during the repair. This dreaded outcome complicates 0.5% of all surgical repairs, but its incidence can be lessened with the use of some form of distal perfusion, preferably left heart bypass with the use of femoral arterial or distal thoracic aorta for arterial inflow and Figure 20-11. Appearance of the aorta after resection of the seg-ment of coarctation and reconstruction with an extended end-to-end anastomosis. (Used with permission from Kelly Rosso MD.)Figure 20-12. Reformatted images obtained from a CT angiogram after recurrent coarctation repaired by an extra anatomic bypass (‘*’ points to the bypass graft).the femoral vein or left atrium for venous return.51 These tech-niques are generally reserved for older patients with complex coarctations that may need prolonged aortic cross clamp times for repair, often in the setting of large collateral vessels and/or previous surgery.58Hypertension is also well

1	patients with complex coarctations that may need prolonged aortic cross clamp times for repair, often in the setting of large collateral vessels and/or previous surgery.58Hypertension is also well recognized following repair of COA. Bouchart and colleagues reported that in a cohort of 35 hypertensive adults (mean age, 28 years) undergoing repair, despite a satisfactory anatomic outcome, only 23 patients were normotensive at a mean follow-up period of 165 months.56 Like-wise, Bhat and associates reported that in a series of 84 patients (mean age at repair, 29 years), 31% remained hypertensive at a mean follow-up of 5 years following surgery.57Although operative repair is still the gold standard, treat-ment of COA by catheter-based intervention has become more widespread for older children and adults. Both balloon dilata-tion and primary stent implantation have been used successfully. The most extensive study of the results of balloon angioplasty reported on 970 procedures: 422 native

1	and adults. Both balloon dilata-tion and primary stent implantation have been used successfully. The most extensive study of the results of balloon angioplasty reported on 970 procedures: 422 native and 548 recurrent COAs. Mean gradient reduction was 74% ± 24% for native and 70% ± 31% for recurrent COA.59 This demonstrated that catheter-based therapy could produce equally effective results both in recurrent and in primary COA, a finding with far-reaching implications in the new paradigm of multidisciplinary treatment algorithms for CHD. In the Valvuloplasty and Angioplasty of Congeni-tal Anomalies (VACA) report, higher preangioplasty gradient, earlier procedure date, older patient age, and the presence of recurrent COA were independent risk factors for suboptimal procedural outcome.5The gradient after balloon dilatation in most series is gen-erally acceptable. However, there is a significant minority of patients (0%–26%) for whom the procedural outcome is sub-optimal, with a

1	gradient after balloon dilatation in most series is gen-erally acceptable. However, there is a significant minority of patients (0%–26%) for whom the procedural outcome is sub-optimal, with a postprocedure gradient of 20 mmHg or greater. These patients may be ideal candidates for primary stent place-ment. Deaths from the procedure also are infrequent (<1% of cases), and the main major complication is aneurysm formation, PAAoBrunicardi_Ch20_p0751-p0800.indd 76322/02/19 2:55 PM 764SPECIFIC CONSIDERATIONSPART IIwhich occurs in 7% of patients.51 With stent implantation, many authors have demonstrated improved resolution of stenosis compared with balloon dilatation alone, yet the long-term com-plications on vessel wall compliance remain largely unknown because only mid-term data are widely available.In summary, children younger than age 6 months with native COA should be treated with surgical repair, while those requiring intervention at later ages may be ideal candidates for balloon

1	available.In summary, children younger than age 6 months with native COA should be treated with surgical repair, while those requiring intervention at later ages may be ideal candidates for balloon dilatation or primary stent implantation.51 Additionally, catheter-based therapy should be employed for those cases of restenosis following either surgical or primary endovascular management.Truncus ArteriosusAnatomy. Truncus arteriosus is a rare anomaly, compris-ing between 1% and 2% of all live born cases of CHD.60 It is characterized by a single great artery that arises from the heart, overrides the ventricular septum, and supplies the pulmonary, systemic, and coronary circulations.The two major classification systems are those of Collett and Edwards, described in 1949, and Van Praagh, described in 1965 (Fig. 20-13).61,62 The Collett and Edwards classification focuses mainly on the origin of the pulmonary arteries from the common arterial trunk, whereas the Van Praagh system is based on

1	in 1965 (Fig. 20-13).61,62 The Collett and Edwards classification focuses mainly on the origin of the pulmonary arteries from the common arterial trunk, whereas the Van Praagh system is based on the presence or absence of a VSD, the degree of formation of the aorticopulmonary septum, and the status of the aortic arch.During embryonic life, the truncus arteriosus normally begins to separate and spiral into a distinguishable anterior pul-monary artery and posterior aorta. Persistent truncus, therefore, represents an arrest in embryologic development at this stage.63 Other implicated events include twisting of the dividing trun-cus because of ventricular looping, subinfundibular atresia, and abnormal location of the semilunar valve anlages.64The neural crest may also play a crucial role in the normal formation of the great vessels, as experimental studies in chick embryos have shown that ablation of the neural crest results in persistent truncus arteriosus.65 The neural crest also

1	in the normal formation of the great vessels, as experimental studies in chick embryos have shown that ablation of the neural crest results in persistent truncus arteriosus.65 The neural crest also develops into the pharyngeal pouches that give rise to the thymus and parathyroids, which likely explains the prevalent association of truncus arteriosus and DiGeorge’s syndrome.66The annulus of the truncal valve usually straddles the ventricular septum in a “balanced” fashion; however, it is not unusual for it to be positioned predominantly over the RV, which increases the potential for LVOT obstruction following surgical repair. In the great majority of cases, the leaflets are thickened and deformed, which leads to valvular insufficiency. There are usually three leaflets (60%), but occasionally a bicus-pid (5%) or even a quadricuspid valve (25%) is present.61In truncus arteriosus, the pulmonary trunk bifurcates, with the left and right pulmonary arteries forming posteriorly and to the

1	a bicus-pid (5%) or even a quadricuspid valve (25%) is present.61In truncus arteriosus, the pulmonary trunk bifurcates, with the left and right pulmonary arteries forming posteriorly and to the left in most cases. The caliber of the pulmonary arterial branches is usually normal, with stenosis or diffuse hypoplasia occurring in rare instances.The coronary arteries may be normal; however, anomalies are not unusual and occur in 50% of cases.67 Many of these are relatively minor, although two variations are of particular importance because they have implications in the conduct of operative repair. The first is that the left coronary ostium may arise high in the sinus of Valsalva or even from the truncal tis-sue at the margin of the pulmonary artery tissue. This coronary artery can be injured during repair when the pulmonary arteries are removed from the trunk or when the resulting truncal defect is closed. The second is that the right coronary artery can give rise to an important

1	injured during repair when the pulmonary arteries are removed from the trunk or when the resulting truncal defect is closed. The second is that the right coronary artery can give rise to an important accessory anterior descending artery, which often passes across the RV in the exact location where the right ventriculotomy is commonly performed during repair.68Physiology and Diagnosis. The main pathophysiologic con-sequences of truncus arteriosus are (a) the obligatory mixing of systemic and pulmonary venous blood at the level of the ven-tricular septal defect (VSD) and truncal valve, which leads to arterial saturations near 85% and (b) the presence of a nonre-strictive left-to-right shunt, which occurs during both systole and diastole, the volume of which is determined by the relative resistances of the pulmonary and systemic circulations. Addi-tionally, truncal valve stenosis or regurgitation, the presence of important LVOT obstruction, and stenosis of pulmonary artery branches can

1	of the pulmonary and systemic circulations. Addi-tionally, truncal valve stenosis or regurgitation, the presence of important LVOT obstruction, and stenosis of pulmonary artery branches can further contribute to both pressure and volume-loading of the ventricles. The presence of these lesions often results in severe heart failure and cardiovascular instability early in life. Pulmonary vascular resistance may develop as early as 6 months of age, leading to poor results with late surgical correction.Patients with truncus arteriosus usually present in the neo-natal period, with signs and symptoms of congestive heart fail-ure and mild to moderate cyanosis. A pansystolic murmur may be noted at the left sternal border, and occasionally a diastolic murmur may be heard in the presence of truncal regurgitation.Chest radiography will be consistent with pulmonary over-circulation, and a right aortic arch can be appreciated 35% of the time. The thymus is prominent by its absence if associated

1	regurgitation.Chest radiography will be consistent with pulmonary over-circulation, and a right aortic arch can be appreciated 35% of the time. The thymus is prominent by its absence if associated with DiGeorge syndrome (Fig. 20-14). The ECG is usually non-specific, demonstrating normal sinus rhythm with biventricular hypertrophy.Echocardiography with Doppler color-flow or pulsed Doppler is diagnostic and usually provides sufficient informa-tion to determine the type of truncus arteriosus, the origin of the Figure 20-13. Collett & Edwards classification for Truncus arteriosus. (Used with permission from Kelly Rosso MD.)RPARPARPAType 1Type 2Type 3LPALPALPABrunicardi_Ch20_p0751-p0800.indd 76422/02/19 2:55 PM 765CONGENITAL HEART DISEASECHAPTER 20coronary arteries and their proximity to the pulmonary trunk, the character of the truncal valves, and the extent of truncal insuffi-ciency (Fig. 20-15). CT scan helps define the pulmonary arteries and the coronary anatomy (Fig. 20-16).

1	to the pulmonary trunk, the character of the truncal valves, and the extent of truncal insuffi-ciency (Fig. 20-15). CT scan helps define the pulmonary arteries and the coronary anatomy (Fig. 20-16). Cardiac catheterization can be helpful in cases where pulmonary hypertension is sus-pected or to further delineate coronary artery anomalies prior to repair.The presence of truncus is an indication for surgery. Repair should be undertaken in the neonatal period or as soon as the diagnosis is established.Repair. Truncus arteriosus was first managed with pulmonary artery banding as described by Armer and colleagues in 1961.69 However, this technique led to only marginal improvements in 1-year survival rates because ventricular failure inevitably occurred. In 1967, however, complete repair was accomplished by McGoon and his associates based on the experimental work of Rastelli, who introduced the idea that an extracardiac valved conduit could be used to restore ventricular-to-pulmonary artery

1	by McGoon and his associates based on the experimental work of Rastelli, who introduced the idea that an extracardiac valved conduit could be used to restore ventricular-to-pulmonary artery continuity.70 Over the next 20 years, improved survival rates led to uniform adoption of complete repair even in the youngest and smallest infants.71Surgical correction entails the use of CPB. Repair is completed by separation of the pulmonary arteries from the aorta, closure of the aortic defect (occasionally with a patch) to minimize coronary flow complications, placement of a valved cryopreserved allograft or jugular venous valved conduit (Con-tegra) to reconstruct the RVOT, and VSD closure. Important branch pulmonary arterial stenosis should be repaired at the time of complete repair and can usually be accomplished with longitudinal allograft patch arterioplasty. Severe truncal valve insufficiency occasionally requires truncal valve repair or even replacement, which can be accomplished with a

1	be accomplished with longitudinal allograft patch arterioplasty. Severe truncal valve insufficiency occasionally requires truncal valve repair or even replacement, which can be accomplished with a cryopreserved allograft.72Results. The results of complete repair of truncus have steadily improved. Ebert reported a 91% survival rate in his series of 77 patients who were younger than 6 months of age; later reports by others confirmed these findings and demonstrated that excel-lent results could be achieved in even smaller infants with com-plex-associated defects.71Newer extracardiac conduits also have been developed and used with success, which has widened the repertoire of the modern congenital heart surgeon and improved outcomes.72,73 Severe truncal regurgitation, interrupted aortic arch, coexistent coronary anomalies, chromosomal or genetic anomalies, and age younger than 100 days are risk factors associated with peri-operative death and poor outcome.Total Anomalous Pulmonary Venous

1	coexistent coronary anomalies, chromosomal or genetic anomalies, and age younger than 100 days are risk factors associated with peri-operative death and poor outcome.Total Anomalous Pulmonary Venous ConnectionTotal anomalous pulmonary venous connection (TAPVC) occurs in 1% to 2% of all cardiac malformations and is char-acterized by abnormal drainage of the pulmonary veins into the right heart, whether through connections into the right atrium or into its tributaries.74 Accordingly, the only mechanism by which oxygenated blood can return to the left heart is through an ASD, which is almost uniformly present with TAPVC.Figure 20-14. Chest x-ray of a baby with DiGeorge syndrome and truncus arteriosus. Note the absence of the thymic shadow in the superior mediastinum (‘*’).Figure 20-15. Echo appearance of a baby with Truncus Ateriosus. The ‘*’ represents the VSD, and the arrow points to the truncal valve.Figure 20-16. CT scan of a baby with Truncus Arteriosus Type 2. The ‘*’ mark the RPA

1	appearance of a baby with Truncus Ateriosus. The ‘*’ represents the VSD, and the arrow points to the truncal valve.Figure 20-16. CT scan of a baby with Truncus Arteriosus Type 2. The ‘*’ mark the RPA and the LPA. Note the stenosis at the origin of the LPA.Brunicardi_Ch20_p0751-p0800.indd 76522/02/19 2:55 PM 766SPECIFIC CONSIDERATIONSPART IIUnique to this lesion is the absence of a definitive form of palliation. Thus, TAPVC with concomitant obstruction (Fig. 20-17) represents one of the only true surgical emergen-cies across the entire spectrum of congenital heart surgery.Anatomy and Embryology. The lungs develop from an out-pouching of the foregut, and their venous plexus arises as part of the splanchnic venous system. TAPVC arises when the pul-monary vein evagination from the posterior surface of the left atrium fails to fuse with the pulmonary venous plexus surround-ing the lung buds. In place of the usual connection to the left atrium, at least one connection of the pulmonary

1	surface of the left atrium fails to fuse with the pulmonary venous plexus surround-ing the lung buds. In place of the usual connection to the left atrium, at least one connection of the pulmonary plexus to the splanchnic plexus persists. Accordingly, the pulmonary veins drain to the heart through a systemic vein.Darling and colleagues classified TAPVC (Fig. 20-18) according to the site or level of connection of the pulmonary veins to the systemic venous system75: type I (45%), anomalous connection at the supracardiac level; type II (25%), anomalous connection at the cardiac level; type III (25%), anomalous con-nection at the infracardiac level; and type IV (5%), anomalous connection at multiple levels.76 Within each category, further subdivisions can be implemented, depending on whether pul-monary venous obstruction exists. Obstruction to pulmonary venous drainage is a powerful predictor of adverse natural out-come and occurs most frequently with the infracardiac type, especially when

1	venous obstruction exists. Obstruction to pulmonary venous drainage is a powerful predictor of adverse natural out-come and occurs most frequently with the infracardiac type, especially when the pattern of infracardiac connection prevents the ductus venosus from bypassing the liver.77Pathophysiology and Diagnosis. Because both pulmonary and systemic venous blood returns to the right atrium in all forms of TAPVC, a right-to-left intracardiac shunt must be present in order for the afflicted infant to survive. This invariably occurs via a nonrestrictive patent foramen ovale. Because of this obliga-tory mixing, cyanosis is usually present, and its degree depends on the ratio of pulmonary to systemic blood flow. Decreased Figure 20-17. Infracardiac type of TAPVR. Note the stenosis (‘*’) of the descending vertical vein as it drains into the portal system.Figure 20-18. The various types of TAPVC as described by Darling and colleagues. (Used with permission from Nicholas Clarke MD.)pulmonary

1	descending vertical vein as it drains into the portal system.Figure 20-18. The various types of TAPVC as described by Darling and colleagues. (Used with permission from Nicholas Clarke MD.)pulmonary blood flow is a consequence of pulmonary venous obstruction, the presence of which is unlikely if the right ven-tricular pressure is less than 85% of systemic pressure.78The child with TAPVC may present with severe cyanosis and respiratory distress, necessitating urgent surgical interven-tion if a severe degree of pulmonary venous obstruction is pres-ent. However, in cases where there is no obstructive component, the clinical picture is usually one of pulmonary overcircula-tion, hepatomegaly, tachycardia, and tachypnea with feeding. In a child with serious obstruction, arterial blood gas analysis reveals severe hypoxemia (partial pressure of oxygen [Po2] < 20 mmHg), with metabolic acidosis.79Chest radiography (Fig. 20-19) will show normal heart size with generalized pulmonary edema.

1	analysis reveals severe hypoxemia (partial pressure of oxygen [Po2] < 20 mmHg), with metabolic acidosis.79Chest radiography (Fig. 20-19) will show normal heart size with generalized pulmonary edema. Two-dimensional echocardiography is very useful in establishing the diagnosis and also can assess ventricular septal position, which may be leftward secondary to small left ventricular volumes, as well as estimate the right ventricular pressure based on the height of the tricuspid regurgitant jet. Echocardiography can usually identify the pulmonary venous connections (types I to IV), and it is rarely necessary to perform other diagnostic tests.Cardiac catheterization is not recommended in these patients because the osmotic load from the intravenous contrast can exacerbate the degree of pulmonary edema.80 When cardiac catheterization is performed, equalization of oxygen saturations in all four heart chambers is a hallmark finding in this disease because the mixed blood returned to the right

1	edema.80 When cardiac catheterization is performed, equalization of oxygen saturations in all four heart chambers is a hallmark finding in this disease because the mixed blood returned to the right atrium gets dis-tributed throughout the heart.Therapy. Operative correction of TAPVC requires anastomo-sis of the common pulmonary venous channel to the left atrium, obliteration of the anomalous venous connection, and closure of the ASD.79,81IIIAIVCIVCSVCLPVLARARPVIVCRVLVRARVDVVVPVLVLASVCCPVIVCIIBIIISVCVVLPVCPVSVCRPRALARVLVCPVLALVRARVBrunicardi_Ch20_p0751-p0800.indd 76622/02/19 2:55 PM 767CONGENITAL HEART DISEASECHAPTER 20All types of TAPVC are approached through a median ster-notomy, and many surgeons use deep hypothermic circulatory arrest in order to achieve an accurate and widely patent anastomo-sis. The technique for supracardiac TAPVC includes early division of the vertical vein, retraction of the aorta and the superior vena cava laterally to expose the posterior aspect of the

1	anastomo-sis. The technique for supracardiac TAPVC includes early division of the vertical vein, retraction of the aorta and the superior vena cava laterally to expose the posterior aspect of the left atrium and the pulmonary venous confluence, and a side-to-side anastomosis between a long, horizontal biatrial incision and a longitudinal inci-sion within the pulmonary venous confluence. The ASD can then be closed with an autologous pericardial or synthetic patch.In patients with TAPVC to the coronary sinus without obstruction, a simple unroofing of the coronary sinus can be performed through a single right atriotomy with concomitant closure of the ASD. If pulmonary venous obstruction is pres-ent, the repair should include generous resection of roof of the coronary sinus.79Repair of infracardiac TAPVC entails ligation of the verti-cal vein at the diaphragm, followed by construction of a proximal, patulous longitudinal venotomy. This repair is usually performed by “rolling” the heart

1	TAPVC entails ligation of the verti-cal vein at the diaphragm, followed by construction of a proximal, patulous longitudinal venotomy. This repair is usually performed by “rolling” the heart toward the left, thus exposing the left atrium where it usually overlies the descending vertical vein.As originally described by Lacour-Gayet and colleagues at the Marie-Lannelongue Hospital, Paris, and Coles and col-leagues at The Hospital for Sick Children, Toronto, the suture-less technique was developed for patients with anastomotic stenosis occurring after TAPVC repair.80,81 After determining that favorable outcomes were possible using this technique, it is currently used in selected patients upon initial presentation of TAPVC.81 Incisions are made in the venous confluence. Based on the surgeon’s discretion, the incisions are extended into both upper and lower pulmonary veins separately if judged to be important for an unobstructed pathway. An atriopericardial anastomosis is created using the

1	discretion, the incisions are extended into both upper and lower pulmonary veins separately if judged to be important for an unobstructed pathway. An atriopericardial anastomosis is created using the pericardium adjacent to where the pulmonary veins enter the pericardium (Fig. 20-20). This anastomosis avoids direct contact with the incision site in the wall of the pulmonary veins and allows the free egress of blood from the lungs to the left atrium.The perioperative care of these infants is crucial because episodes of pulmonary hypertension can occur within the first 48 hours, which contribute significantly to mortality following repair. Muscle relaxants and narcotics should be administered during this period to maintain a constant state of anesthesia. Arterial partial pressure of carbon dioxide (Pco2) should be maintained at 30 mmHg with use of a volume ventilator, and the fraction of inspired oxygen (Fio2) should be increased to keep the pulmonary arterial pressure at less than

1	dioxide (Pco2) should be maintained at 30 mmHg with use of a volume ventilator, and the fraction of inspired oxygen (Fio2) should be increased to keep the pulmonary arterial pressure at less than two-thirds of the systemic pressure.Results. Results of TAPVC in infancy have markedly improved in recent years, with an operative mortality of 5% or less in some series.79-82 This improvement is probably multifac-torial, mainly as a consequence of early noninvasive diagnosis and aggressive perioperative management. The routine use of echocardiography; improvements in myocardial protection with specific attention to the RV; creation of a large, tension-free anastomosis with maximal use of the venous confluence and atrial tissue; use of a sutureless technique in selected cases; and prevention of pulmonary hypertensive events have likely played a major role in reducing operative mortality. The importance of risk factors for early mortality, such as venous obstruction at presentation, urgency of

1	hypertensive events have likely played a major role in reducing operative mortality. The importance of risk factors for early mortality, such as venous obstruction at presentation, urgency of operative repair, and infradiaphrag-matic anatomic type, has been debated.81,83Bando and colleagues84 made the controversial statement that both preoperative pulmonary venous obstruction and ana-tomic type had been neutralized as potential risk factors beyond calendar year 1991. Hyde et al82 similarly reported that connec-tion type was not related to outcome. However, a large single-institution report of 377 children with TAPVC by the author from the Hospital for Sick Children in Toronto85 found that, although outcomes had improved over time, patient anatomic factors were still important determinants of both survival and the need for subsequent reoperation. Risk factors for postrepair death were earlier operation year, younger age at repair, cardiac connection type, and postoperative pulmonary

1	of both survival and the need for subsequent reoperation. Risk factors for postrepair death were earlier operation year, younger age at repair, cardiac connection type, and postoperative pulmonary venous obstruc-tion. Risk-adjusted estimated 1-year survival for a patient repaired at birth with unfavorable morphology in 2006 was 37% (95% confidence interval [CI], 8%–80%) compared with 96% (95% CI, 91%–99%) for a patient with favorable morphology repaired at age 1 year. Freedom from reoperation was 82% ± 6% 4Figure 20-19. Chest x-ray of a newborn with obstructed infracar-diac type of TAPVR rescued by ECMO. Note the ECMO cannulas in the right neck (‘*’).InfracardiacTAPVCConventionalRepairSuturelessRepairFigure 20-20. Differences between conventional repair of total anomalous pulmonary venous connection (TAPVC) and sutureless repair of TAPVC. In the sutureless techniques, there are no sutures placed in the fragile veins themselves. Rather, the pericardial flaps are used to create a “well”

1	connection (TAPVC) and sutureless repair of TAPVC. In the sutureless techniques, there are no sutures placed in the fragile veins themselves. Rather, the pericardial flaps are used to create a “well” for the pulmonary venous return (bottom inset). Early and late extrinsic stenosis are thought to be reduced using this latter technique.Brunicardi_Ch20_p0751-p0800.indd 76722/02/19 2:55 PM 768SPECIFIC CONSIDERATIONSPART IIat 11 years after repair, with increased risk associated with mixed connection and postoperative pulmonary venous obstruction. A study from the Hospital for Sick Children, Toronto, showed a lower incidence of reoperation in the sutureless technique com-pared to conventional pulmonary venous confluence–left atrial anastomosis.86 However, there was no statistically significant difference suggesting similar results between the strategies. Although the sutureless technique appears to have favorable outcomes at primary repair for TAPVC, long-term follow-up is necessary to

1	difference suggesting similar results between the strategies. Although the sutureless technique appears to have favorable outcomes at primary repair for TAPVC, long-term follow-up is necessary to evaluate the occurrence of arrhythmias, such as complete heart block and atrial tachycardia, since an incision on the atrial septum and atrial wall is more invasive compared to the conventional technique.The most significant postoperative complication of TAPVC repair is pulmonary venous obstruction (Figure 20-21), which occurs 9% to 11% of the time, regardless of the surgi-cal technique employed. Mortality varies between 30% and 45%, and alternative catheter interventions do not offer defini-tive solutions.80 Recurrent pulmonary venous obstruction can be localized at the site of the pulmonary venous anastomosis (extrinsic), which usually can be cured with patch enlargement or balloon dilatation, or it may be secondary to endocardial thickening of the pulmonary venous ostia frequently

1	venous anastomosis (extrinsic), which usually can be cured with patch enlargement or balloon dilatation, or it may be secondary to endocardial thickening of the pulmonary venous ostia frequently resulting in diffuse pulmonary venous sclerosis (intrinsic), which car-ries a 66% mortality rate because few good solutions exist.77 More commonly, postrepair left ventricular dysfunction can occur as the noncompliant LV suddenly is required to handle an increased volume load from redirected pulmonary venous return. This can manifest as an increase in pulmonary artery pressure but is distinguishable from primary pulmonary hyper-tension (another possible postoperative complication following repair of TAPVC) from the elevated left atrial pressure and LV dysfunction along with echocardiographic evidence of poor LV contractility. In pulmonary hypertension, the left atrial pressure may be low, the LV may appear “underfilled” (by echocardiog-raphy), and the RV may appear dilated. In either case,

1	of poor LV contractility. In pulmonary hypertension, the left atrial pressure may be low, the LV may appear “underfilled” (by echocardiog-raphy), and the RV may appear dilated. In either case, postop-erative support for a few days with extracorporeal membrane oxygenation may be lifesaving, and TAPVC should be repaired in centers that have this capacity.Some investigators have speculated that preoperative pul-monary venous obstruction is associated with increased medial thickness within the pulmonary vasculature, which may predis-pose these infants to intrinsic pulmonary venous stenosis despite adequate pulmonary venous decompression.82 The majority of studies demonstrating that preoperative pulmonary venous obstruction is a predictor of subsequent need for reoperation to correct recurrent pulmonary venous obstruction lend credence to this notion.Cor TriatriatumAnatomy. Cor triatriatum is a rare congenital heart defect char-acterized by the presence of a fibromuscular diaphragm that

1	pulmonary venous obstruction lend credence to this notion.Cor TriatriatumAnatomy. Cor triatriatum is a rare congenital heart defect char-acterized by the presence of a fibromuscular diaphragm that par-titions the left atrium into two chambers: a superior chamber that receives drainage from the pulmonary veins, and an inferior chamber that communicates with the mitral valve and the LV (Fig. 20-22). An ASD frequently exists between the superior chamber and the right atrium, or, more rarely, between the right atrium and the inferior chamber.Pathophysiology and Diagnosis. Cor triatriatum results in obstruction of pulmonary venous return to the left atrium. The degree of obstruction is variable and depends on the size of fen-estrations present in the left atrial membrane, the size of the ASD, and the existence of other associated anomalies. If the communication between the superior and inferior chambers is <3 mm, patients usually are symptomatic during the first year of life. The afflicted

1	the existence of other associated anomalies. If the communication between the superior and inferior chambers is <3 mm, patients usually are symptomatic during the first year of life. The afflicted infant will present with the stigmata of low cardiac output and pulmonary venous hypertension, as well as congestive heart failure and poor feeding.Physical examination may demonstrate a loud pulmonary S2 sound and a right ventricular heave, as well as jugular venous distention and hepatomegaly. Chest radiography will show car-diomegaly and pulmonary vascular prominence, and the ECG will suggest right ventricular hypertrophy. Two-dimensional echocardiography provides a definitive diagnosis in most cases, with catheterization necessary only when echocardiographic evaluation is equivocal.Therapy. Operative treatment for cor triatriatum is fairly simple. CPB and cardioplegic arrest are used. A right atriotomy usually Figure 20-21. Angiogram showing the discrete stenosis (‘*’) of the right-sided

1	treatment for cor triatriatum is fairly simple. CPB and cardioplegic arrest are used. A right atriotomy usually Figure 20-21. Angiogram showing the discrete stenosis (‘*’) of the right-sided pulmonary veins after conventional repair for supra-cardiac type TAPVC.Figure 20-22. Echocardiogram (apical 4 chamber view) showing the discrete membrane (‘*’) in a patient with Cor triatritum.Brunicardi_Ch20_p0751-p0800.indd 76822/02/19 2:55 PM 769CONGENITAL HEART DISEASECHAPTER 20allows access to the left atrial membrane through the existing ASD because it is dilated secondary to communication with the pulmonary venous chamber. The membrane is then excised, tak-ing care not to injure the mitral valve or the interatrial septum, and the ASD is closed with a patch. Alternatively, if the right atrium is small, the membrane can be exposed through an inci-sion directly into the superior left atrial chamber, just anterior to the right pulmonary veins. Surgical results are uniformly excel-lent for

1	is small, the membrane can be exposed through an inci-sion directly into the superior left atrial chamber, just anterior to the right pulmonary veins. Surgical results are uniformly excel-lent for this defect, with survival approaching 100%.The utility of catheter-based intervention for this diagnosis remains controversial, although there have been some reports of successful balloon dilatation.87Aortopulmonary WindowEmbryology and Anatomy. Aortopulmonary window (APW) is a rare congenital lesion, occurring in about 0.2% of patients, characterized by incomplete development of the septum that normally divides the truncus into the aorta and the pulmonary artery88In the vast majority of cases, APW occurs as a single defect of minimal length, which begins a few millimeters above the semilunar valves on the left lateral wall of the aorta (Fig. 20-23). Coronary artery anomalies, such as aberrant origin of the right or left coronary artery from the main pulmonary artery, are occa-sionally

1	valves on the left lateral wall of the aorta (Fig. 20-23). Coronary artery anomalies, such as aberrant origin of the right or left coronary artery from the main pulmonary artery, are occa-sionally present.Pathophysiology and Diagnosis. The dominant pathophysi-ology of APW is that of a large left-to-right shunt with increased pulmonary flow and the early development of congestive heart failure. Like other lesions with left-to-right flow, the magnitude of the shunt is determined by both the size of the defect and the pulmonary vascular resistance.Infants with APW present with frequent respiratory tract infections, tachypnea with feeding, and failure to thrive. Cya-nosis usually is absent because these infants deteriorate prior to the onset of significant pulmonary hypertension. The rapid decline with this defect occurs because shunt flow continues during both phases of the cardiac cycle, which limits systemic perfusion and increases ventricular work.89The diagnosis of APW begins with

1	decline with this defect occurs because shunt flow continues during both phases of the cardiac cycle, which limits systemic perfusion and increases ventricular work.89The diagnosis of APW begins with the physical exami-nation, which may demonstrate a systolic flow murmur, a hyperdynamic precordium, and bounding peripheral pulses. The chest radiograph will show pulmonary overcirculation and cardiomegaly, and the ECG will usually demonstrate either left ventricular hypertrophy or biventricular hypertrophy. Echocar-diography (Fig. 20-24) can detect the defect and also provide information about associated anomalies. Retrograde aortogra-phy will confirm the diagnosis but is rarely necessary.Therapy. All infants with APW require surgical correction once the diagnosis is made. Repair is undertaken through a median sternotomy and the use of CPB. The pulmonary arteries are occluded once the distal aorta is cannulated, and a transaor-tic repair using a prosthetic patch for pulmonary artery

1	through a median sternotomy and the use of CPB. The pulmonary arteries are occluded once the distal aorta is cannulated, and a transaor-tic repair using a prosthetic patch for pulmonary artery closure is then carried out. The coronary ostia must be carefully visual-ized and included on the aortic side of the patch. Alternatively, a two-patch technique can be used, which may eliminate recurrent fistulas from suture line leaks that occasionally occur with the single-patch method.90Results. Results are generally excellent, with an operative mortality in most large series of less than 5%.Vascular Rings and Pulmonary Artery SlingsVascular rings constitute a group of disorders derived from anomalies that result from abnormal development of the aortic arches resulting in compression of the trachea or esophagus. The surgical management of vascular rings dates back to 1945 when Dr. Gross described the surgical management of a kid with double aortic arch.91 Most children present with symptoms

1	or esophagus. The surgical management of vascular rings dates back to 1945 when Dr. Gross described the surgical management of a kid with double aortic arch.91 Most children present with symptoms during the first few months of life. Vascular rings can be com-plete (e.g., double aortic arch, right aortic arch with left liga-ment) or partial (e.g., innominate artery compression syndrome, pulmonary artery sling).Anatomy. The embryologic basis of vascular rings involves the development of six pairs of aortic arches and the dorsal and ventral aortae. The development of a specific type of vascular ring depends of the deletion or preservation of a specific seg-ment of these structures. The persistence of the right and left fourth arches leads to the development of double aortic arch. Persistence of the fourth right aortic arch and the involution of the left fourth arch leads to the development of a right aor-tic arch system with various combinations of mirror imaging Figure 20-23. Cartoon

1	of the fourth right aortic arch and the involution of the left fourth arch leads to the development of a right aor-tic arch system with various combinations of mirror imaging Figure 20-23. Cartoon depicting the various types of aortopulmonary window. (Used with permission from Nicholas Clarke MD.)Figure 20-24. Echo demonstrating an aortopulmonary window (‘*’).Type IType IIType IIIBrunicardi_Ch20_p0751-p0800.indd 76922/02/19 2:55 PM 770SPECIFIC CONSIDERATIONSPART IIbranching, aberrant subclavian arteries or with a left-sided liga-mentum arterisum. When the developing left lung captures its blood supply from the right sixth arch caudad to the tracheo-bronchial tree, it leads to the development of a pulmonary artery sling. The left pulmonary artery arises from the right pulmonary artery and then wraps around the trachea and esophagus forming a “sling.”92 The pathophysiology of innominate artery compres-sion syndrome is not very well understood.Pathophysiology and Diagnosis. The

1	and then wraps around the trachea and esophagus forming a “sling.”92 The pathophysiology of innominate artery compres-sion syndrome is not very well understood.Pathophysiology and Diagnosis. The symptoms associated with vascular rings include respiratory distress, barking cough, stridor, apnea, dysphagia, and recurrent respiratory tract infec-tions. The diagnosis often requires a high index of suspicion. Minor respiratory tract infections may precipitate serious respi-ratory distress. The work up includes chest X-rays, echocardiog-raphy, bronchoscopy, CT scan (Fig. 20-25), MRI (Fig. 20-26), and, rarely, cardiac catheterization. Chest X-rays show the rela-tionship of the aortic arch to the trachea. Tracheal compression can be better evaluated using lateral films. Unilateral hyperinfla-tion of the lung is sometimes seen and is often associated with a pulmonary artery sling (Fig. 20-27). PA slings (Fig. 20-28) are often associated with complete tracheal rings necessitating a bronchoscopy

1	of the lung is sometimes seen and is often associated with a pulmonary artery sling (Fig. 20-27). PA slings (Fig. 20-28) are often associated with complete tracheal rings necessitating a bronchoscopy when this diagnosis is made (Fig. 20-29). Patients with dysphagia require a barium esophagogram as a part of their work-up (Fig. 20-30).Treatment. All symptomatic patients should undergo surgery. On close questioning nearly all patients are symptomatic.93 The treatment varies depending on the type of vascular ring. A left posterolateral thoracotomy provides good exposure to most types. A right thoracotomy is often used for innominate artery compression syndrome, and a median sternotomy often with cardiopulmonary bypass is used to treat pulmonary artery slings with or without associated complete tracheal rings. The out-comes and results for vascular rings are excellent (Fig. 20-31). Video-assisted thoracoscopic approaches have been developed for the management of these conditions.94-96 The

1	tracheal rings. The out-comes and results for vascular rings are excellent (Fig. 20-31). Video-assisted thoracoscopic approaches have been developed for the management of these conditions.94-96 The criticism often stated involves retraction of vascular structures into the medias-tinum and losing control of the stumps prior to definitve control leading to exsanguination.96DEFECTS REQUIRING PALLIATIONTricuspid AtresiaTricuspid atresia occurs in 2% to 3% of patients with CHD and is characterized by atresia of the tricuspid valve. This results in discontinuity between the right atrium and RV. The RV is generally hypoplastic, and left-heart filling is dependent on an ASD. Tricuspid atresia is the most common form of the single-ventricle complex, indicating that there is functionally only one ventricular chamber.Anatomy. As mentioned, tricuspid atresia results in a lack of communication between the right atrium and the RV, and in the 5Figure 20-25. CT angiogram showing the four artery sign

1	chamber.Anatomy. As mentioned, tricuspid atresia results in a lack of communication between the right atrium and the RV, and in the 5Figure 20-25. CT angiogram showing the four artery sign classic of double aortic arch.Figure 20-26. MRI showing a double aortic arch.Figure 20-27. Unilateral hyperinflation of the left lung associ-ated with a rare vascular ring: left ascending aorta and right sided descending aorta.Figure 20-28. CT angiogram showing a PA sling. Note the LPA wrapping around behind the trachea.Brunicardi_Ch20_p0751-p0800.indd 77022/02/19 2:55 PM 771CONGENITAL HEART DISEASECHAPTER 20majority of patients there is no identifiable valve tissue or rem-nant.98 The right atrium is generally enlarged and muscular, with a fibrofatty floor. An unrestrictive ASD is usually present. The LV is often enlarged as it receives both systemic and pulmonary blood flow, but the left AV valve is usually normal.The RV, however, is usually severely hypoplastic, and there is sometimes a VSD in

1	LV is often enlarged as it receives both systemic and pulmonary blood flow, but the left AV valve is usually normal.The RV, however, is usually severely hypoplastic, and there is sometimes a VSD in its trabeculated or infundibular portion. In many cases, the interventricular communication is a site of obstruction to pulmonary blood flow, but obstruction may also occur at the level of the outlet valve or in the subval-vular infundibulum.99 In most cases, pulmonary blood flow is dependent on the presence of a PDA, and there may be no flow into the pulmonary circulation except for this PDA.Tricuspid atresia is classified according to the relationship of the great vessels and by the degree of obstruction to pulmo-nary blood flow. Because of the rarity of tricuspid atresia with transposed great arteries, we will restrict our discussion to tri-cuspid atresia with normally related great vessels.Pathophysiology. The main pathophysiology in tricuspid atresia is that of a univentricular heart

1	arteries, we will restrict our discussion to tri-cuspid atresia with normally related great vessels.Pathophysiology. The main pathophysiology in tricuspid atresia is that of a univentricular heart of left ventricular morphology. That is, the LV must receive systemic blood via the interatrial communication and then distribute it to both the pulmonary circulation and the systemic circulation. Unless there is a VSD (as is found in some cases), pulmonary flow is dependent on the presence of a PDA. As the ductus begins to close shortly after birth, infants become intensely cyanotic. Reestablishing ductal patency (with PGE1) restores pulmonary blood flow and stabilizes patients for surgical intervention. Pulmonary hypertension is unusual in tricuspid atresia. However, occasional patients have a large VSD between the LV and the infundibular portion of the RV (just below the pulmonary valve). If there is no obstruction at the level of this VSD or at the valve, these infants may actually

1	a large VSD between the LV and the infundibular portion of the RV (just below the pulmonary valve). If there is no obstruction at the level of this VSD or at the valve, these infants may actually present with heart failure from excessive pulmonary blood flow. Regardless of whether these infants are “ductal-dependent” for pulmonary blood flow or have pulmonary blood flow provided across a VSD, they will be cyanotic since the obligatory right-to-left shunt at the atrial level will provide complete mixing of systemic and pulmonary venous return so that the LV ejects a hypoxemic mixture into the aorta.Diagnosis. The signs and symptoms of tricuspid atresia are dependent on the underlying anatomic variant, but most infants are cyanotic and hypoxic as a result of decreased pulmonary blood flow and the complete mixing at the atrial level. When pulmonary blood flow is provided through a VSD, there may be a prominent systolic murmur. Tricuspid atresia with pulmonary blood flow from a PDA may

1	and the complete mixing at the atrial level. When pulmonary blood flow is provided through a VSD, there may be a prominent systolic murmur. Tricuspid atresia with pulmonary blood flow from a PDA may present with the soft, continuous murmur of a PDA in conjunction with cyanosis.In the minority of patients with tricuspid atresia, symp-toms of congestive heart failure will predominate. This is often related to excessive flow across a VSD. The natural history of the muscular VSDs in these infants is that they will close and the congestive heart failure will dissipate and transform into cyano-sis with reduced pulmonary blood flow. Chest radiography will show decreased pulmonary vascularity. The ECG is strongly suggestive because uncharacteristic left axis deviation will be present, due to underdevelopment of the RV. Two-dimensional echocardiography readily confirms the diagnosis and the ana-tomic subtype. (Fig 20-32)Treatment. The treatment for tricuspid atresia in the earlier era of

1	underdevelopment of the RV. Two-dimensional echocardiography readily confirms the diagnosis and the ana-tomic subtype. (Fig 20-32)Treatment. The treatment for tricuspid atresia in the earlier era of palliation was aimed at correcting the defect in the pul-monary circulation. That is, patients with too much pulmonary flow received a pulmonary band, and those with insufficient flow received a systemic-to-pulmonary artery shunt. Systemic-to-pulmonary artery shunts, or Blalock–Taussig (BT) shunts, were first applied to patients with tricuspid atresia in the 1940s and 1950s.98 Likewise pulmonary artery banding was applied Figure 20-29. Rigid bronchoscopy showing complete tracheal rings in a the patient with pulmonary artery sling.Figure 20-30. Barium esophagogram showing posterior indenta-tion of the esophagus caused by a vascular ring (right aortic arch, aberrant left subclavian artery and left ligamentum).Brunicardi_Ch20_p0751-p0800.indd 77122/02/19 2:55 PM 772SPECIFIC

1	indenta-tion of the esophagus caused by a vascular ring (right aortic arch, aberrant left subclavian artery and left ligamentum).Brunicardi_Ch20_p0751-p0800.indd 77122/02/19 2:55 PM 772SPECIFIC CONSIDERATIONSPART IIto patients with tricuspid atresia and congestive failure in 1957. However, despite the initial relief of either cyanosis or conges-tive heart failure, long-term mortality was high, as the single ventricle was left unprotected from either volume or pressure overload.99Recognizing the inadequacies of the initial repairs, Glenn described the first successful cavopulmonary anastomosis, an end-to-side right pulmonary artery-to-superior vena cava shunt in 1958, and later modified this to allow flow to both pulmonary arteries.100 This end-to-side right pulmonary artery-to-superior vena cava anastomosis was known as the bidirectional Glenn, and it is the first stage to final Fontan repair in widespread use today (Fig. 20-33). The Fontan repair was a major advancement in the

1	vena cava anastomosis was known as the bidirectional Glenn, and it is the first stage to final Fontan repair in widespread use today (Fig. 20-33). The Fontan repair was a major advancement in the treatment of CHD, as it essentially bypassed the right heart and allowed separation of the pulmonary and systemic circulations. It was first performed by Fontan in 1971 and con-sisted of a classic Glenn anastomosis, ASD closure, and direct connection of the right atrium to the proximal end of the left pulmonary artery using an aortic homograft.101 The main pul-monary artery was ligated, and a homograft valve was inserted into the orifice of the inferior vena cava.Figure 20-32. Echo showing tricuspid atresia. The ‘*’ demonstrates the membranous tissue instead of the presence of a tricuspid valve.Figure 20-33. Angiogram showing a widely patent Glenn. The SVC (‘*’) is seen draining into the central pulmonary artery.Figure 20-31. Bronchoscopy before and after repair of a vascular ring: right

1	20-33. Angiogram showing a widely patent Glenn. The SVC (‘*’) is seen draining into the central pulmonary artery.Figure 20-31. Bronchoscopy before and after repair of a vascular ring: right arch, left descending aorta, and left ligament.Multiple modifications of this initial repair were per-formed over the next 20 years. One of the most important was the description by deLeval and colleagues of the creation of an interatrial lateral tunnel that allowed the inferior vena caval blood to be channeled exclusively to the superior vena cava.102 A total cavopulmonary connection could then be accomplished by dividing the superior vena cava and suturing the superior portion to the upper side of the right pulmonary artery and the inferior end to the augmented undersurface of the right pulmonary artery. Pulmonary flow then occurs passively, in a laminar fashion, driven by the central venous pressure. This repair became known as the modified Fontan operation.Another important modification, the

1	Pulmonary flow then occurs passively, in a laminar fashion, driven by the central venous pressure. This repair became known as the modified Fontan operation.Another important modification, the fenestrated Fontan repair, was introduced in 1988.103 In this procedure, a residual 20% to 30% right-to-left shunt is either created or left unre-paired at the time of cavopulmonary connection to help sustain systemic output in the face of transient elevations in the pulmo-nary vascular resistance postoperatively.103Brunicardi_Ch20_p0751-p0800.indd 77222/02/19 2:55 PM 773CONGENITAL HEART DISEASECHAPTER 20The last notable variation on the original Fontan repair uses an extracardiac prosthetic tube graft (Fig. 20-34), usually 18 to 20 mm in diameter, as the conduit directing inferior vena cava blood to the pulmonary arteries.105 This technique has the advantages of decreasing atrial geometric alterations by avoid-ing intra-atrial suture lines and improving flow dynamics in the systemic venous

1	to the pulmonary arteries.105 This technique has the advantages of decreasing atrial geometric alterations by avoid-ing intra-atrial suture lines and improving flow dynamics in the systemic venous pathway by maximizing laminar flow. Several investigators have shown a decrease in supraventricular arrhyth-mias, as well as an improvement in ventricular function, which may be secondary to decreased atrial tension and alleviation of chronic elevations in coronary sinus pressure.102,103One potential disadvantage of the extracardiac Fontan is that it delays performance of the Fontan in order to allow placement of a conduit of sufficient size. Despite these innova-tive approaches, the current strategy for operative management still relies on the idea of palliation. Patients are approached in a staged manner, to maximize their physiologic state so that they will survive to undergo a Fontan operation. The therapeu-tic strategy must begin in the neonatal period and should be directed toward

1	a staged manner, to maximize their physiologic state so that they will survive to undergo a Fontan operation. The therapeu-tic strategy must begin in the neonatal period and should be directed toward reducing the patient’s subsequent risk factors for a Fontan procedure. Accordingly, small systemic pulmonary shunts, which are usually performed through a median sternot-omy, should be constructed for palliation of ductus-dependent univentricular physiology. This can easily be replaced with a bidirectional Glenn shunt or hemi-Fontan operation at 6 months of life. In non–ductus-dependent univentricular physiology, the infant can be managed medically until primary construction of a bidirectional cavopulmonary anastomosis becomes feasible. This is possible in the majority of cases because the physiologi-cally elevated pulmonary vascular resistance prevents pulmo-nary overcirculation during the neonatal period.The Fontan is usually performed when the child is between 2 and 4 years of age, and

1	elevated pulmonary vascular resistance prevents pulmo-nary overcirculation during the neonatal period.The Fontan is usually performed when the child is between 2 and 4 years of age, and it is generally successful if the infant was staged properly, with a protected single ventricle, and there is adequate pulmonary artery growth. The pulmonary vascular resistance should be below 4 Wood units, and the ejection frac-tion should be more than 45% to ensure success.106 In patients with high pulmonary artery pressure, fenestration of the atrial baffle may be helpful because their pulmonary vascular resis-tance may preclude adequate cardiac output postoperatively.99,103Results. Recent reports of the Fontan procedure for tricuspid atresia have been encouraging, with an overall survival of 86% and an operative mortality of 2%.107 The main complications following repair are atrial arrhythmias, particularly atrial flutter; conduit obstruction requiring reoperation; protein-losing enter-opathy; and

1	operative mortality of 2%.107 The main complications following repair are atrial arrhythmias, particularly atrial flutter; conduit obstruction requiring reoperation; protein-losing enter-opathy; and decreased exercise tolerance.A prospective multi-institutional study from the Congeni-tal Heart Surgeons Society reported the outcomes of 150 neo-nates with tricuspid atresia and normally related great vessels.107 Five-year survival was 86%, and by the age of 2 years, 89% had undergone cavopulmonary anastomosis, and 75% of those surviving cavopulmonary anastomosis underwent Fontan opera-tion within 3 years. Competing risks methodology was used in this study to determine the rates of transition to end-states and their associated determinants (Fig. 20-35). Risk factors for death without cavopulmonary anastomosis in this study included the presence of mitral regurgitation and palliation with systemic-to-pulmonary artery shunts not originating from the innominate artery. Factors associated

1	anastomosis in this study included the presence of mitral regurgitation and palliation with systemic-to-pulmonary artery shunts not originating from the innominate artery. Factors associated with decreased transition rate to cavo-pulmonary anastomosis included patient variables (younger age at admission to a participating institution and noncardiac anom-alies) and procedural variables (larger systemic-to-pulmonary arterial shunt diameter and previous palliation).9Hypoplastic Left Heart SyndromeHLHS comprises a wide spectrum of cardiac malformations, including hypoplasia or atresia of the aortic and mitral valves and hypoplasia of the LV and ascending aorta.108 HLHS has a reported prevalence of 0.2 per 1000 live births and occurs twice as often in boys as in girls. Left untreated, HLHS is invari-ably fatal and is responsible for 25% of early cardiac deaths in neonates.109 However, the recent evolution of palliative surgical procedures has dramatically improved the outlook for patients

1	invari-ably fatal and is responsible for 25% of early cardiac deaths in neonates.109 However, the recent evolution of palliative surgical procedures has dramatically improved the outlook for patients with HLHS, and an improved understanding of anatomic and physiologic alterations has spurred advances in parallel arenas such as intrauterine diagnosis and fetal intervention, echocardio-graphic imaging, and neonatal critical care.Anatomy. As implied by its name, HLHS involves varying degrees of underdevelopment of left-sided structures (Fig. 20-36), including the LV and the aortic and mitral valves. Thus, HLHS can be classified into four anatomic subtypes based on the val-vular morphology: (a) aortic and mitral stenosis; (b) aortic and mitral atresia; (c) aortic atresia and mitral stenosis; and (d) AS and mitral atresia. Aortic atresia tends to be associated with more severe degrees of hypoplasia of the ascending aorta than does AS.Even in cases without frank aortic atresia, however, the

1	(d) AS and mitral atresia. Aortic atresia tends to be associated with more severe degrees of hypoplasia of the ascending aorta than does AS.Even in cases without frank aortic atresia, however, the aortic arch is generally hypoplastic and, in severe cases, may even be interrupted. There is an associated coarctation shelf in 80% of patients with HLHS, and the ductus itself is usually quite large, as is the main pulmonary artery.7The segmental pulmonary arteries, however, are small, secondary to reduced intrauterine pulmonary blood flow, which is itself a consequence of the left-sided outflow obstruction (Fig. 20-36). The left atrial cavity is generally smaller than nor-mal and is accentuated because of the leftward displacement of the septum primum. There is almost always an interatrial com-munication via the foramen ovale, which can be large, but more Figure 20-34. Angiogram in a patient with a fenestrated extra-cardiac fontan constructed with a 20 mm Gore-tex tube graft

1	com-munication via the foramen ovale, which can be large, but more Figure 20-34. Angiogram in a patient with a fenestrated extra-cardiac fontan constructed with a 20 mm Gore-tex tube graft (‘*’).Brunicardi_Ch20_p0751-p0800.indd 77322/02/19 2:55 PM 774SPECIFIC CONSIDERATIONSPART IIcommonly restricts right-to-left flow. In rare cases, there is no atrial-level communication, which can be lethal for these infants because there is no way for pulmonary venous return to cross over to the RV.Associated defects can occur with HLHS, and many of them have importance with respect to operative repair. For example, if a VSD is present, the LV can retain its normal size during development even in the presence of mitral atresia. This is because a right-to-left shunt through the defect impels growth of the LV.110 This introduces the feasibility of biventricular repair for this subset of patients.Although HLHS undoubtedly results from a complex interplay of developmental errors in the early stages

1	the LV.110 This introduces the feasibility of biventricular repair for this subset of patients.Although HLHS undoubtedly results from a complex interplay of developmental errors in the early stages of cardio-genesis, many investigators have hypothesized that the altered blood flow is responsible for the structural underdevelopment that characterizes HLHS. In other words, if the stimulus for nor-mal development of the ascending aorta from the primordial aortic sac is high-pressure systemic blood flow from the LV through the aortic valve, then an atretic or stenotic aortic valve, which impedes flow and leads to only low-pressure diastolic retrograde flow via the ductus, will change the developmental signals and result in hypoplasia of the downstream structures (Fig. 20-37). Normal growth and development of the LV and mitral valve can be secondarily affected, resulting in hypoplasia or atresia of these structures.108Pathophysiology and Diagnosis. In HLHS, pulmonary venous blood enters

1	and development of the LV and mitral valve can be secondarily affected, resulting in hypoplasia or atresia of these structures.108Pathophysiology and Diagnosis. In HLHS, pulmonary venous blood enters the left atrium, but atrial systole cannot propel blood across the stenotic or atretic mitral valve into the LV. Thus, the blood is shunted across the foramen ovale into the right atrium, where it contributes to volume loading of the RV. The end result is pulmonary venous hypertension from outflow obstruction at the level of the left atrium, as well as pulmonary overcirculation and right ventricular failure. As the pulmonary vascular resistance falls postnatally, the condition is exacerbated because right ventricular output is preferentially directed away from the systemic circulation, resulting in profound underperfu-sion of the coronary arteries and the vital organs. Closure of the ductus is incompatible with life in these neonates.Neonates with severe HLHS receive all pulmonary,

1	resulting in profound underperfu-sion of the coronary arteries and the vital organs. Closure of the ductus is incompatible with life in these neonates.Neonates with severe HLHS receive all pulmonary, sys-temic, and coronary blood flow from the RV. Generally, a child with HLHS will present with respiratory distress within the first day of life, and mild cyanosis may be noted. These infants must be rapidly triaged to a tertiary center, and echocardiography should be performed to confirm the diagnosis. Prostaglandin E1 must be administered to maintain ductal patency, and the Figure 20-36. Echo In a patient with HLHS. Note the extremely hypoplastic left ventricle (‘*’).0200.00.40.81.2Years from diagnosis1.62.0406080100Proportion (%) of patients in each stateBDCPA (2 year prevalence = 90%)Dead without BDCPA(2 year prevalence = 5%)Single-stage Fontan(2 year prevalence = 1%)Alive without BDCPA(2 year prevalence = 4%)Figure 20-35. Competing risks depiction of events after diagnosis in 150

1	without BDCPA(2 year prevalence = 5%)Single-stage Fontan(2 year prevalence = 1%)Alive without BDCPA(2 year prevalence = 4%)Figure 20-35. Competing risks depiction of events after diagnosis in 150 patients with tricuspid atresia. All patients began alive and thereafter migrated to one of four mutually exclusive end states (death, bidirectional cavopulmonary anastomosis [BDCPA], single-stage Fontan completion, or remaining alive without BDCPA) at time-dependent rates defined by the underlying hazard functions. At any point in time, the sum of propor-tions of children in each state is 100%. For example, estimated prevalences after 2 years from diagnosis are as follows: 89% BDCPA, 6% dead without BDCPA, 4% alive without BDCPA, and 1% single-stage Fontan completion. Solid lines represent parametric point estimates; dashed lines enclose 70% confidence intervals; circles with error bars represent nonparametric estimates; numbers in parentheses indicate the estimated propor-tion of patients

1	point estimates; dashed lines enclose 70% confidence intervals; circles with error bars represent nonparametric estimates; numbers in parentheses indicate the estimated propor-tion of patients in each state at 2 years from diagnosis. (Reproduced with permission from Karamlou T, Ashburn DA, Caldarone CA, et al: Matching procedure to morphology improves outcomes in neonates with tricuspid atresia, J Thorac Cardiovasc Surg. 2005 Dec;130(6):1503-1510.) Brunicardi_Ch20_p0751-p0800.indd 77422/02/19 2:55 PM 775CONGENITAL HEART DISEASECHAPTER 20ventilatory settings must be adjusted to avoid excessive oxygen-ation and increase carbon dioxide tension. These maneuvers will maintain pulmonary vascular resistance and promote improved systemic perfusion.5,7,108 Cardiac catheterization should gener-ally be avoided because it is not usually helpful and might result in injury to the ductus and compromised renal function second-ary to the osmotic dye load.Treatment. In 1983, Norwood and colleagues

1	be avoided because it is not usually helpful and might result in injury to the ductus and compromised renal function second-ary to the osmotic dye load.Treatment. In 1983, Norwood and colleagues described a two-stage palliative surgical procedure for relief of HLHS111 that was later modified to the currently used three-stage method of palliation.109 Stage 1 palliation, also known as the modified Norwood procedure (Fig. 20-38), bypasses the LV by creating a single outflow vessel, the neoaorta, which arises from the RV.The current technique of arch reconstruction involves completion of a connection between the pulmonary root, the native ascending aorta, and a piece of pulmonary homograft used to augment the diminutive native aorta. There are several modifications of this anastomosis, most notably the Damus-Kaye-Stansel (DKS) anastomosis, which involves dividing both the aorta and the pulmonary artery at the sinotubular junction. The proximal aorta is anastomosed to the proximal

1	most notably the Damus-Kaye-Stansel (DKS) anastomosis, which involves dividing both the aorta and the pulmonary artery at the sinotubular junction. The proximal aorta is anastomosed to the proximal pulmonary artery, creating a “double-barreled” outlet from the heart. This outlet is anastomosed to the distal aorta, which can be augmented with homograft material if there is an associated coarctation. At the completion of arch reconstruction, a 3.5or 4-mm shunt is placed from the innominate artery to the right pulmonary artery. The interatrial septum is then widely excised, thereby creating a large interatrial communication and prevent-ing pulmonary venous hypertension.The DKS connection, as described earlier, might avoid postoperative distortion of the tripartite connection in the neo-aorta, and thus decrease the risk of coronary insufficiency.112 It can be used when the aorta is 4 mm or larger. Unfortunately, in many infants with HLHS, especially if there is aortic atresia, the aorta

1	and thus decrease the risk of coronary insufficiency.112 It can be used when the aorta is 4 mm or larger. Unfortunately, in many infants with HLHS, especially if there is aortic atresia, the aorta is diminutive and often less than 2 mm in diameter. The alternate technique available to provide pulmonary blood flow instead of a shunt is a RV-PA conduit often referred to as a “Sano.” It is usually a 5 or 6 mm ribbed Gore-tex graft.113The postoperative management of infants following stage 1 palliation is complex because favorable outcomes depend on establishing a delicate balance between pulmonary and systemic perfusion. Recent literature suggests that these infants require adequate postoperative cardiac output in order to supply both the pulmonary and the systemic circulations and that the use of oxi-metric catheters to monitor mixed venous oxygen saturation (Svo2) aids clinicians in both the selection of inotropic agents and in ventilatory management.114 Introduction of a shunt between

1	of oxi-metric catheters to monitor mixed venous oxygen saturation (Svo2) aids clinicians in both the selection of inotropic agents and in ventilatory management.114 Introduction of a shunt between the RV and the pulmonary artery (Sano shunt) dimin-ishes the diastolic flow created by the modified BT shunt and may augment coronary perfusion, resulting in improved postop-erative cardiac function.113 A recent prospective, randomized, multi-institutional trial sponsored by the National Institutes of Health, the Systemic Ventricle Reconstruction (SVR) trial, com-pared the outcomes of neonates having either a modified Blalock–Taussig shunt (MBTS) or a Sano shunt.115 The SVR trial demonstrated that transplantation-free survival 12 months after randomization was higher with the Sano shunt than with the MBTS (74% vs. 64%, P = .01). However, the Sano shunt group had more unintended interventions (P = .003) and complications (P = .002). Right ventricular size and function at the age of 14 months

1	the MBTS (74% vs. 64%, P = .01). However, the Sano shunt group had more unintended interventions (P = .003) and complications (P = .002). Right ventricular size and function at the age of 14 months and the rate of nonfatal serious adverse events at the age of 12 months were similar in the two groups. Data collected over a mean (± standard deviation) follow-up period of 32 ± 11 months showed a nonsignificant difference in transplanta-tion-free survival between the two groups (P = .06).115Since the initial SVR publications in 2010, the 3-year and 6-year results have been analyzed. At 3 years, the com-bined death and cardiac transplantation rates for the RVPAS vs. MBTS groups were 33% vs. 39% (P = 0.14). When all available data were examined by Kaplan-Meier analysis (mean follow-up 4.4 ± 1.0 years), there was also no difference between groups (log rank P = 0.11). Overall, there were 100 deaths and 10 trans-plantations in the MBTS cohort and 86 deaths and 11 transplan-tations in the RVPAS

1	years), there was also no difference between groups (log rank P = 0.11). Overall, there were 100 deaths and 10 trans-plantations in the MBTS cohort and 86 deaths and 11 transplan-tations in the RVPAS group.116 At 6 years, although the point averages continued to reflect a difference favoring the RVPAS (combined death/transplantation rate, 36%) in comparison with the MBTS (41%), the number of subjects was not sufficient to 6Figure 20-37. Angiogram obtained in a patient with HLSH (AS/MS). Note the extremely diminutive ascending aorta (‘*’).PatchmBTSRPALPAFigure 20-38. Cartoon depicting the Norwood procedure. The anas-tomosis of the aortic and pulmonary valve annulus is not shown. The ascending aorta and hyplastic arch are reconstructed by patch augmentation. The pulmonary blood flow has been provided in this case by a mBTS. (Used with permission from Kelly Rosso MD.)Brunicardi_Ch20_p0751-p0800.indd 77522/02/19 2:55 PM 776SPECIFIC CONSIDERATIONSPART IIdemonstrate a statistically

1	been provided in this case by a mBTS. (Used with permission from Kelly Rosso MD.)Brunicardi_Ch20_p0751-p0800.indd 77522/02/19 2:55 PM 776SPECIFIC CONSIDERATIONSPART IIdemonstrate a statistically significant difference between the two groups (log rank P = 0.13). Similar to the 3-year results, RVPAS subjects had a higher incidence of any catheter inter-vention (0.38 vs. 0.23 interventions/patient-year, P <0.001), including balloon angioplasty (P = 0.014), stent (P = 0.009), and coiling (P <0.001).113,114 Currently, there remains an ongoing controversy regarding MBTS vs. RV-PA conduit as the source of pulmonary blood flow after the Norwood operation.119,120Although surgical palliation with the Norwood procedure is still the mainstay of therapy for infants with HLHS, a combined surgical and percutaneous option (hybrid procedure), which con-sists of bilateral pulmonary artery banding and placement of a ductal stent, has emerged as a promising alternative that obviates the need for CPB

1	percutaneous option (hybrid procedure), which con-sists of bilateral pulmonary artery banding and placement of a ductal stent, has emerged as a promising alternative that obviates the need for CPB in the fragile neonatal period.121,122 The hybrid procedure is performed in a “hybrid suite,” incorporating both advanced fluoroscopic imaging facilities combined with com-plete operating room capabilities. A 3or 3.5-mm PTFE tube graft is cut to a width of 3 to 4 mm and used as the bands on the branch pulmonary arteries, placed just distal to the main pulmo-nary artery. The ductal stent is then positioned in order to cover all ductal tissue and is deployed through a purse-string suture in the main pulmonary artery. A reverse systemic-to-pulmonary shunt is considered in patients with aortic atresia and preductal coarctation to improve coronary perfusion; however, a recent study demonstrated no difference in survival between those with and without the shunt.123 The hybrid procedure can also be

1	and preductal coarctation to improve coronary perfusion; however, a recent study demonstrated no difference in survival between those with and without the shunt.123 The hybrid procedure can also be used as a bridge to heart transplantation in those infants with severe AV valve regurgitation or otherwise unsuitable single-ventricle anatomy.124Following stage 1 palliation, the second surgical proce-dure is the creation of a bidirectional cavopulmonary shunt (Fig. 20-39) or hemi-Fontan, generally at 3 to 6 months of life when the pulmonary vascular resistance has decreased to nor-mal levels. This is the first step in separating the pulmonary and systemic circulations, and it decreases the volume load on the single ventricle. The existing innominate artery-to-pulmonary shunt (or RV-to-pulmonary shunt) or MBTS is eliminated dur-ing the same operation.The third stage of surgical palliation, known as the modi-fied Fontan procedure, completes the separation of the sys-temic and pulmonary

1	shunt) or MBTS is eliminated dur-ing the same operation.The third stage of surgical palliation, known as the modi-fied Fontan procedure, completes the separation of the sys-temic and pulmonary circulations and is performed between 18 months and 3 years of age, or when the patient experiences increased cyanosis (i.e., has outgrown the capacity to perfuse the systemic circulation with adequately oxygenated blood). This has traditionally required a lateral tunnel within the right atrium to direct blood from the inferior vena cava to the pulmo-nary artery, allowing further relief of the volume load on the RV and providing increased pulmonary blood flow to alleviate cyanosis. More recently, many favor using an extracardiac con-duit (e.g., 18to 20-mm tube graft) to connect the inferior vena cava to the pulmonary artery (Fig. 20-40).Not all patients with HLHS require this three-stage pallia-tive repair. Some infants afflicted with a milder form of HLHS, recently described as hypoplastic left

1	to the pulmonary artery (Fig. 20-40).Not all patients with HLHS require this three-stage pallia-tive repair. Some infants afflicted with a milder form of HLHS, recently described as hypoplastic left heart complex (HLHC), have aortic or mitral hypoplasia without intrinsic valve stenosis and antegrade flow in the ascending aorta. In this group, a two-ventricle repair can be achieved with reasonable outcome. Tch-ervenkov has published the results with 12 patients with HLHC who underwent biventricular repair at a mean age of 7 days.114 The operative technique consisted of a pulmonary homograft patch aortoplasty of the aortic arch and ascending aorta and closure of the interatrial and interventricular communications. The left heart was capable of sustaining systemic perfusion in 92% of patients, and early mortality was 15.4%. Four patients required reoperations to relieve LVOT obstruction, most com-monly between 12 and 39 months following repair. The group from Boston Children’s Hospital

1	and early mortality was 15.4%. Four patients required reoperations to relieve LVOT obstruction, most com-monly between 12 and 39 months following repair. The group from Boston Children’s Hospital has been very aggressive in left ventricular recruitment. These operations still carry a high burden of late death and several reoperations.Although the Norwood procedure is the most widely per-formed initial operation for HLHS, transplantation can be used as a first-line therapy and may be preferred when anatomic or physiologic considerations exist that preclude a favorable out-come with palliative repair. Significant tricuspid regurgitation, intractable pulmonary artery hypertension, or progressive right ventricular failure are cases where cardiac replacement may be advantageous. Widespread adaptation of transplantation as SVCLPAAtriumFigure 20-39. Cartoon depicting a bidirectional Glenn. (Used with permission from Kelly Rosso MD.)SVCGore-textube graftAtriumIVCFigure 20-40. Extra cardiac

1	of transplantation as SVCLPAAtriumFigure 20-39. Cartoon depicting a bidirectional Glenn. (Used with permission from Kelly Rosso MD.)SVCGore-textube graftAtriumIVCFigure 20-40. Extra cardiac fenestrated Fontan. ‘*’ shows the fen-estration. (Used with permission from Kelly Rosso MD.)Brunicardi_Ch20_p0751-p0800.indd 77622/02/19 2:55 PM 777CONGENITAL HEART DISEASECHAPTER 20first-line treatment for HLHS has been limited by improved Norwood survival rates as the operation and preand postop-erative management of the patient have evolved and by lim-ited organ availability. Organ availability should be considered prior to electing transplantation, as 24% of infants died awaiting transplantation in the largest series to date.126,127Results. Outcomes for HLHS are still significantly worse than those for other complex cardiac defects. However, with improvements in perioperative care and modifications in surgical technique, the survival following the Norwood proce-dure now exceeds 90% in

1	those for other complex cardiac defects. However, with improvements in perioperative care and modifications in surgical technique, the survival following the Norwood proce-dure now exceeds 90% in experienced centers.115-120 The out-come for low-birth-weight infants has improved, but low weight still remains a major predictor of adverse survival, especially when accompanied by significant tricuspid valve insufficiency, a restructive interatrial communication, poor RV function, or extracardiac or chromosomal anomalies.DEFECTS THAT MAY BE PALLIATED OR REPAIREDEbstein’s AnomalyAnatomy. This is a rare defect, occurring in less than 1% of CHD patients. The predominant maldevelopment in this lesion is the inferior displacement of the tricuspid valve into the RV, although Bove128 and others have emphasized the fact that Ebstein’s anomaly is primarily a defect in right ventricular morphology rather than an isolated defect in the tricuspid valve. The anterior leaflet is usually attached in its

1	emphasized the fact that Ebstein’s anomaly is primarily a defect in right ventricular morphology rather than an isolated defect in the tricuspid valve. The anterior leaflet is usually attached in its normal position to the annulus, but the septal and posterior leaflets are displaced toward the ventricle. This effectively divides the RV into two parts: the inlet portion (atrialized RV) and the outlet portion (true or trabeculated RV) (Fig. 20-41). The atrialized RV is usu-ally thin and dilated. Similarly, the tricuspid annulus and the right atrium are extremely dilated, and the tricuspid valve is usually regurgitant with a “sail-like” leaflet (Fig. 20-42). There is commonly an ASD present, which results in a right-to-left shunt at the atrial level. Occasionally, there is true anatomic pulmonary atresia or milder forms of RVOT obstruction.A Wolff-Parkinson-White (WPW) syndrome (Fig. 20-43) type of accessory pathway with associated preexcitation is pres-ent in 15% of

1	true anatomic pulmonary atresia or milder forms of RVOT obstruction.A Wolff-Parkinson-White (WPW) syndrome (Fig. 20-43) type of accessory pathway with associated preexcitation is pres-ent in 15% of patients.128Pathophysiology. Right ventricular dysfunction occurs in patients with Ebstein’s anomaly because of two basic mecha-nisms: the inflow obstruction at the level of the atrialized ven-tricle, which produces ineffective RV filling and contractile dysfunction. Inflow obstruction and tricuspid regurgitation, which is exacerbated by progressive annular dilatation, both produce ineffective RV filling. Contractile dysfunction of the RV is a result of a decrease in the number of myocardial fibers, as well as the discordant contraction of the large atrialized portion.The lack of forward flow at the right ventricular level may lead to physiologic or functional pulmonary atresia, and the infant is dependent on ductal patency for survival. All sys-temic venous return must be directed through

1	the right ventricular level may lead to physiologic or functional pulmonary atresia, and the infant is dependent on ductal patency for survival. All sys-temic venous return must be directed through an ASD to the left atrium, where it can be shunted through the ductus for gas exchange. However, the left ventricular function is usually compromised in infants with severe Ebstein’s anomaly as well because the enormous RV and the to-and-fro flow within the atrialized RV prevent adequate intracardiac mixing. Left ven-tricular function may also be severely compromised in Ebstein’s anomaly because the large RV causes left ventricular compres-sion (Fig. 20-44A,B).Diagnosis. There is a spectrum of clinical presentation in infants with Ebstein’s anomaly that mirrors the anatomic spec-trum of this anomaly. Some infants with less severe forms may present with a mild degree of cyanosis, whereas the onset of clinical symptoms in patients surviving childhood is gradual, with the average age of

1	anomaly. Some infants with less severe forms may present with a mild degree of cyanosis, whereas the onset of clinical symptoms in patients surviving childhood is gradual, with the average age of diagnosis in the mid-teens.However, the infant with severe atrialization and pulmo-nary stenosis will be both cyanotic and acidotic at birth. The chest radiograph may demonstrate the classic appearance, which 7Figure 20-41. Echo showing a patient with Ebsteins anomaly. Note the inferiorly displaced tricuspid valve (‘*’) and the atrialized por-tion of the RV (arrow).Figure 20-42. Echo in a patient with severe Ebsteins anomaly showing the large ‘sail like’ anterior leaflet (‘*’).Brunicardi_Ch20_p0751-p0800.indd 77722/02/19 2:56 PM 778SPECIFIC CONSIDERATIONSPART IIconsists of a globular “wall-to-wall” heart (Fig. 20-45), similar to that seen with pericardial effusion. The ECG may show right bundle-branch block and right axis deviation. WPW syndrome, as mentioned earlier, is a common finding

1	heart (Fig. 20-45), similar to that seen with pericardial effusion. The ECG may show right bundle-branch block and right axis deviation. WPW syndrome, as mentioned earlier, is a common finding in these patients. Echocardiography will confirm the diagnosis and provide criti-cal information including tricuspid valvular function, size of the atrialized portion of the RV, degree of pulmonary stenosis, and the atrial size.128The Great Ormond Street Score (GOSE) (Table 20-1),129 which consists of the area of the right atrium plus the area of the atrialized portion of the RV divided by the diastolic area of the remaining cardiac chambers, has been proposed as a useful prognostic tool to stratify neonates with Ebstein’s anomaly. A score of greater than 2 translates into uniformly fatal outcome. Electrophysiology study with radiofrequency ablation is indi-cated in patients with evidence of WPW syndrome or in children Figure 20-43. EKG of a newborn with Ebsteins anomaly and WPW syndrome. Note

1	Electrophysiology study with radiofrequency ablation is indi-cated in patients with evidence of WPW syndrome or in children Figure 20-43. EKG of a newborn with Ebsteins anomaly and WPW syndrome. Note the pre-excitation (arrow).ABFigure 20-44. A. Echo (short axis view) of a patient with severe Ebsteins anomaly showing the large RV (‘*’) and small LV (arrow) in diastole. B. Echo (short axis view) of a patient with severe Ebsteins anomaly showing the large RV (‘*’) and small ‘pancaked’ LV (arrow) in systole.Brunicardi_Ch20_p0751-p0800.indd 77822/02/19 2:56 PM 779CONGENITAL HEART DISEASECHAPTER 20with a history of supraventricular tachycardia, undefined wide-complex tachycardia, or syncope.Treatment. Surgery is indicated for symptomatic infants and for older children and adults with arrhythmias, progressive cya-nosis, or New York Heart Association class III or IV. How-ever, the operative repair may be different, depending on the patient’s age, because older children usually are

1	arrhythmias, progressive cya-nosis, or New York Heart Association class III or IV. How-ever, the operative repair may be different, depending on the patient’s age, because older children usually are candidates for a biventricular or one-and-a-half ventricle repair, whereas moder-ate survival has been reported for neonates, using a procedure that converts the anatomy to a single-ventricle physiology, as described by Starnes and coworkers.130The surgical approach in widespread use today for patients surviving infancy was described by Danielson and colleagues in 1992.128,131 This procedure entails excision of redundant right atrial tissue and patch closure of any associated ASD, plication of the atrialized portion of the ventricle with obliteration of the aneurysmal cavity, posterior tricuspid annuloplasty to narrow the tricuspid annulus, reconstruction of the tricuspid valve if the anterior leaflet is satisfactory, or replacement of the tricuspid valve if necessary.131 If the tricuspid

1	annuloplasty to narrow the tricuspid annulus, reconstruction of the tricuspid valve if the anterior leaflet is satisfactory, or replacement of the tricuspid valve if necessary.131 If the tricuspid valve is not amenable to reconstruction, valve replacement should be considered. Care must be taken when performing the posterior annuloplasty, or during the conduct of tricuspid valve replacement, to avoid the conduction system, because complete heart block can compli-cate this procedure. In addition, patients who demonstrated preoperative evidence of preexcitation should undergo electro-physiologic mapping and ablation.Neonatal Ebstein’s anomaly is a separate entity. Results with surgical correction have been poor, and many neonates are not candidates for operative repair as previously described. Surgical options for the symptomatic neonate include palliative procedures, the one-and-a-half ventricle repair, or conversion to single-ventricle physiology.132 Arguably, the most favorable

1	Surgical options for the symptomatic neonate include palliative procedures, the one-and-a-half ventricle repair, or conversion to single-ventricle physiology.132 Arguably, the most favorable out-comes in symptomatic neonatal Ebstein’s anomaly or repair in slightly older infants have been achieved using the right ventric-ular exclusion premise. This technique, known as the “Starnes” procedure (Fig. 20-46),130 uses a fenestrated patch to close the tricuspid valve orifice coupled with systemic-to-pulmonary artery shunt. The patch must be fenestrated to allow decom-pression of the RV in instances of anatomic pulmonary atresia. Although Knott-Craig and colleagues132 have described tricus-pid valve repair for the full spectrum of neonates and infants with excellent shortand mid-term results, these results have not been reproduced in other institutions.133 The one-and-a-half ventricle repair was first described by Billingsly and cowork-ers as an attempt to achieve a more physiologic

1	these results have not been reproduced in other institutions.133 The one-and-a-half ventricle repair was first described by Billingsly and cowork-ers as an attempt to achieve a more physiologic “pulsatile” pul-monary circulation in patients with a hypoplastic or dysplastic RV.134 This is accomplished by diverting the superior vena caval blood directly into the pulmonary arterial system by a bidirec-tional cavopulmonary shunt while recruiting the RV to propel the inferior vena caval blood directly to the pulmonary arteries via the RVOT. Thus, the hemodynamics of the one-and-a-half ventricle repair are characterized by separate systemic and pul-monary circulations in series. The systemic circulation is fully supported by a systemic ventricle, and the pulmonary circula-tion is supported by both the bidirectional Glenn shunt and the hypoplastic (pulmonary) ventricle. Proponents of this approach report a decreased right atrial pressure and a decrease in inferior vena cava hypertension,

1	both the bidirectional Glenn shunt and the hypoplastic (pulmonary) ventricle. Proponents of this approach report a decreased right atrial pressure and a decrease in inferior vena cava hypertension, which is theorized to be responsible for many of the dreaded complications of the Fontan circulation, including protein-losing encephalopathy, hepatic congestion, atrial arrhythmias, and systemic ventricular failure. In addition, the maintenance of pulsatile pulmonary blood flow, as opposed to continuous laminar flow as in the Fontan circulation, may be advantageous to the pulmonary microcirculation, although it has not been proven in any studies thus far.134,135 Certain criteria, most notably an adequate tricuspid valve Z score, as well as Figure 20-45. CXR in a newborn with severe Ebsteins anomaly showing a ‘wall-to-wall’ heart.Table 20-1The Great Ormond Street Score (GOSE)GOSE Score: Area of RA + aRA/Area of RV + LA + LVGOSE ScoreRatioMortality (%)1<0.5820.5–1.0831.1–1.41004>1.5100Figure

1	showing a ‘wall-to-wall’ heart.Table 20-1The Great Ormond Street Score (GOSE)GOSE Score: Area of RA + aRA/Area of RV + LA + LVGOSE ScoreRatioMortality (%)1<0.5820.5–1.0831.1–1.41004>1.5100Figure 20-46. Echo appearance after a Starnes operation. Note the jet of flow across the fenestration In the patch.Brunicardi_Ch20_p0751-p0800.indd 77922/02/19 2:56 PM 780SPECIFIC CONSIDERATIONSPART IIthe absence of severe pulmonary hypertension or concomitant defects requiring intricate intracardiac repair, should be satis-fied prior to electing the one-and-a-half ventricle approach.136 Patients who do not fulfill these criteria may be approached with a two-ventricle repair and atrial fenestration or a Fontan repair.In the infant with severe Ebstein’s anomaly, initial stabili-zation with prostaglandin to maintain ductal patency, mechanical ventilation, and correction of cyanosis is mandatory. Metabolic acidosis, if present from compromised systemic perfusion, must be aggressively treated with

1	to maintain ductal patency, mechanical ventilation, and correction of cyanosis is mandatory. Metabolic acidosis, if present from compromised systemic perfusion, must be aggressively treated with afterload reduction. Many of these infants will improve over 1 to 2 weeks as pulmonary vascu-lar resistance falls and they are able to improve antegrade flow into the pulmonary circulation through their abnormal RV and tricuspid valve. When stabilization and medical palliation fail, surgical management remains an option, although its success depends on numerous anatomic factors (e.g., adequacy of the tricuspid valve, RV, and pulmonary outflow tract), and surgery for symptomatic neonates with Ebstein’s anomaly carries a high risk. Knott-Craig and associates reported three cases where two-ventricle repair was undertaken by subtotal closure of the ASD, extensive resection of the right atrium, and vertical plication of the atrialized chamber.132 Five-year follow-up revealed all patients to be

1	repair was undertaken by subtotal closure of the ASD, extensive resection of the right atrium, and vertical plication of the atrialized chamber.132 Five-year follow-up revealed all patients to be asymptomatic and in sinus rhythm without medi-cations. Recently, they have reported on their 20-year experi-ence with treating 32 such neonates with an overall mortality of 40%. Surgical management of neonates with Ebstein’s anom-aly remains challenging. For neonates with Ebstein’s anomaly and anatomical pulmonary atresia, single-ventricle palliation is associated with lower early mortality compared with two-ventricle repair.132Results. In the neonatal period, the most common postopera-tive problem, whether after a simple palliative procedure such as a BT shunt or following a more extensive procedure such as attempted exclusion of the RV, has been low cardiac out-put. Supraventricular tachycardia also has been problematic postoperatively. Complete heart block necessitating pacemaker

1	procedure such as attempted exclusion of the RV, has been low cardiac out-put. Supraventricular tachycardia also has been problematic postoperatively. Complete heart block necessitating pacemaker implantation should be uncommon if the techniques described to avoid suturing between the coronary sinus and the tricuspid annulus are used.There are few published reports of outcomes, due to the rarity of this defect. However, based on the natural history of this condition, which is remarkably benign for the majority of older patients, the outlook should be excellent for patients who have survived the neonatal period.127,131,132,137Transposition of the Great ArteriesAnatomy. Complete transposition is characterized by connec-tion of the atria to their appropriate ventricles with inappropriate ventriculoarterial connections. Thus, the aorta arises anteriorly from the RV, while the pulmonary artery arises posteriorly from the LV. Van Praagh and coworkers introduced the term dextro-transposition

1	connections. Thus, the aorta arises anteriorly from the RV, while the pulmonary artery arises posteriorly from the LV. Van Praagh and coworkers introduced the term dextro-transposition of the great arteries (D-TGA) to describe this defect, whereas levo-transposition of the great arteries (L-TGA) describes a form of corrected transposition where there is concomitant AV discordance.138,139D-TGA requires an obligatory intracardiac mixing of blood, which usually occurs at both the atrial and the ventricu-lar levels or via a patent ductus. Significant coronary anomalies occur frequently in patients with D-TGA. The most common pattern, occurring in 68% of cases, is characterized by the left main coronary artery arising from the leftward coronary sinus, giving rise to the left anterior descending and circumflex arteries. The most common variant is for the circumflex coro-nary artery to arise as a branch from the right coronary artery instead of from the left coronary

1	left anterior descending and circumflex arteries. The most common variant is for the circumflex coro-nary artery to arise as a branch from the right coronary artery instead of from the left coronary artery.Pathophysiology. D-TGA results in parallel pulmonary and systemic circulations, with patient survival dependent on intracardiac mixing of blood. After birth, both ventricles are relatively noncompliant, and thus, infants initially have higher pulmonary flow due to the decreased downstream resistance. This causes left atrial enlargement and a left-to-right shunt via the patent foramen ovale.Postnatally, the LV does not hypertrophy because it is not subjected to systemic afterload. The lack of normal extrauter-ine left ventricular maturation has important implications for the timing of surgical repair because the LV must be converted to the systemic ventricle and be able to function against sys-temic vascular resistance. If complete repair is done within the first few weeks of life,

1	repair because the LV must be converted to the systemic ventricle and be able to function against sys-temic vascular resistance. If complete repair is done within the first few weeks of life, the LV usually adapts easily to systemic resistance since it is conditioned to high intrauterine pulmonary vascular resistance. After a few weeks of life, the LV that is conditioned to the decrease in pulmonary resistance that occurs when the lungs inflate after birth may have difficulty adapting to systemic vascular resistance without preoperative preparation or postoperative support. Novel techniques of LV “preparation” using a pulmonary arterial band have been used in cases where complete repair has been delayed (Fig. 20-47A,B).Clinical Manifestations and Diagnosis. Infants with D-TGA and an intact ventricular septum are usually cyanotic at birth, with an arterial Po2 between 25 and 40 mmHg. If duc-tal patency is not maintained, deterioration will be rapid with ensuing metabolic acidosis and

1	ventricular septum are usually cyanotic at birth, with an arterial Po2 between 25 and 40 mmHg. If duc-tal patency is not maintained, deterioration will be rapid with ensuing metabolic acidosis and death. Conversely, those infants with a coexisting VSD may be only mildly hypoxemic and may come to medical attention after 2 to 3 weeks, when the falling pulmonary vascular resistance leads to symptoms of congestive heart failure.The ECG will reveal right ventricular hypertrophy, and the chest radiograph will reveal the classic egg-shaped con-figuration. Definitive diagnosis is made by echocardiography, which reliably demonstrates ventriculoarterial discordance and any associated lesions. Cardiac catheterization is rarely nec-essary, except in infants requiring surgery after the neonatal period, to assess the suitability of the LV to support the sys-temic circulation. Limited catheterization, however, is useful for performance of atrial septostomy in neonates with inadequate intracardiac

1	to assess the suitability of the LV to support the sys-temic circulation. Limited catheterization, however, is useful for performance of atrial septostomy in neonates with inadequate intracardiac mixing.Surgical Repair. Blalock and Hanlon introduced the first operative intervention for D-TGA with the creation of an atrial septectomy to enhance intracardiac mixing.140 This initial proce-dure was feasible in the pre-CPB era, but carried a high mortal-ity rate. Later, Rashkind and Causo developed a catheter-based balloon septostomy, which largely obviated the need for open septectomy.42These early palliative maneuvers, however, met with lim-ited success, and it was not until the late 1950s, when Senning and Mustard developed the first “atrial repair,” that outcomes improved. The Senning operation consisted of rerouting venous flow at the atrial level by incising and realigning the atrial sep-tum over the pulmonary veins and using the right atrial free wall to create a pulmonary venous

1	consisted of rerouting venous flow at the atrial level by incising and realigning the atrial sep-tum over the pulmonary veins and using the right atrial free wall to create a pulmonary venous baffle (Fig. 20-48).141Although the Mustard repair (Fig. 20-49) was similar, it made use of either autologous pericardium or synthetic material to create the interatrial baffle.142 These atrial switch procedures Brunicardi_Ch20_p0751-p0800.indd 78022/02/19 2:56 PM 781CONGENITAL HEART DISEASECHAPTER 20ABFigure 20-47. A. Echocardiographic appearance of the LV (‘*’) prior to “LV training”. B. Echocardiographic appearance of the LV (‘*’) after “LV training” achieved by the application of a tight PA band and a mBTS.ACBDFigure 20-48. The Senning operation. A. The atrial septum is cut near the tricuspid valve, creating a flap attached posteriorly between the caval veins. B. The flap of atrial septum is sutured to the anterior lip of the orifices of the left pulmonary veins, effectively separating

1	valve, creating a flap attached posteriorly between the caval veins. B. The flap of atrial septum is sutured to the anterior lip of the orifices of the left pulmonary veins, effectively separating the pulmonary and systemic venous channels. C. The posterior edge of the right atrial incision is sutured to the remnant of the atrial septum, diverting the systemic venous channel to the mitral valve. D. The anterior edge of the right atrial incision (lengthened by short incisions at each corner) is sutured around the cava above and below to the lateral edge of the LA incision, completing the pulmonary channel and diversion of pulmonary venous blood to the tricuspid valve area. (Reproduced with permission from Mavroudis C, Backer CL: Pediatric Cardiac Surgery, 2nd ed. St. Louis, MO: Mosby; 1994.) Figure 20-49. Angiographic appearance of a Mustard type baffle repair for dTGA.resulted in a physiologic correction, but not an anatomic one, as the systemic circulation is still based on the RV.

1	Figure 20-49. Angiographic appearance of a Mustard type baffle repair for dTGA.resulted in a physiologic correction, but not an anatomic one, as the systemic circulation is still based on the RV. Still, survival rose to 95% in most centers by using an early balloon septostomy fol-lowed by an atrial switch procedure at 3 to 8 months of age.141,142Despite the improved early survival rates, long-term problems, such as superior vena cava or pulmonary venous obstruction, baffle leak, arrhythmias, tricuspid valve regurgita-tion, and right ventricular failure, prompted the development of the arterial switch procedure by Jatene in 1975.143 The arterial switch procedure involves the division of the aorta and the pul-monary artery, posterior translocation of the aorta (LeCompte maneuver), mobilization of the coronary arteries, placement of a pantaloon-shaped pericardial patch, and proper alignment of the coronary arteries on the neoaorta (Fig. 20-50).The most important consideration is the

1	of the coronary arteries, placement of a pantaloon-shaped pericardial patch, and proper alignment of the coronary arteries on the neoaorta (Fig. 20-50).The most important consideration is the timing of surgical repair because arterial switch should be performed within 2 weeks after birth, before the LV loses its ability to pump against sys-temic afterload. In patients presenting later than 2 weeks, the LV can be retrained with preliminary pulmonary artery banding Brunicardi_Ch20_p0751-p0800.indd 78122/02/19 2:56 PM 782SPECIFIC CONSIDERATIONSPART IIFigure 20-50. The Arterial Switch Operation. A. The maneuver of Lecompte (positioning the pulmo-nary artery anterior to the aorta) is shown with aortic cross-clamp repositioning to retract the pulmonary artery during the neoaortic reconstruction. A and B. After the coronary patches are rotated for an optimal lie, they are sutured to the linearly incised sinuses of Valsalva at the old pulmonary artery (neoaorta) (C). (Reproduced with

1	A and B. After the coronary patches are rotated for an optimal lie, they are sutured to the linearly incised sinuses of Valsalva at the old pulmonary artery (neoaorta) (C). (Reproduced with permission from Mavroudis C, Backer CL: Arterial Switch. Cardiac Surgery: State of the Art Review. Vol. 5, no. 1. Philadelphia, PA: Hanley & Belfus; 1991.) Figure 20-51. Angiographic appearance of the pulmonary arteries before and after balloon dilation. The RV pressures dropped from “systemic” to “1/2 systemic” after dilation.and aortopulmonary shunt followed by definitive repair. Alter-natively, the unprepared LV can be supported following arterial switch with a mechanical assist device for a few days while it recovers ability to manage systemic pressures. Echocardiogra-phy can be used to assess left ventricular performance and guide operative planning in these circumstances.The subset of patients who present with D-TGA compli-cated by LVOT obstruction and VSD may not be suitable for an arterial

1	ventricular performance and guide operative planning in these circumstances.The subset of patients who present with D-TGA compli-cated by LVOT obstruction and VSD may not be suitable for an arterial switch operation. The Rastelli operation, first performed in 1968, uses placement of an intracardiac baffle to direct left ventricular blood to the aorta and an extracardiac valved conduit to establish continuity between the RV and the pulmonary artery, which has led to successful outcomes in these complex patients.144Results. For patients with D-TGA, intact ventricular septum, and VSD, the arterial switch operation provides excellent long-term results with a mortality rate of less than 5%. Operative risk is increased when unfavorable coronary anatomic configu-rations are present or when augmentation of the aortic arch is required. The most common complication is supravalvular pul-monary stenosis, occurring 10% of the time, which may require ballooning or reoperation (Fig.

1	or when augmentation of the aortic arch is required. The most common complication is supravalvular pul-monary stenosis, occurring 10% of the time, which may require ballooning or reoperation (Fig. 20-51).145Results of the Rastelli operation have improved substan-tially, with an early mortality rate of 5%.146 Late mortality rate results were less favorable because conduit failure requiring reoperation, pacemaker insertion, or relief of LVOT obstruc-tion was frequent.Brunicardi_Ch20_p0751-p0800.indd 78222/02/19 2:56 PM 783CONGENITAL HEART DISEASECHAPTER 20Double-Outlet Right VentricleAnatomy. Double-outlet RV (DORV) accounts for 5% of CHD and exists when both the aorta and pulmonary artery arise wholly, or in large part, from the RV (Fig. 20-52). DORV encompasses a spectrum of malformations because the incom-plete shift of the aorta toward the LV is often associated with other abnormalities of cardiac development, such as ventricular looping and infundibular-truncal spiraling.147

1	because the incom-plete shift of the aorta toward the LV is often associated with other abnormalities of cardiac development, such as ventricular looping and infundibular-truncal spiraling.147 The vast majority of hearts exhibiting DORV have a concomitant VSD, which varies in its size and spatial association with the great vessels. The VSD is usually nonrestrictive and represents the only out-flow for the LV; its location relative to the great vessels dictates the dominant physiology of DORV, which can be analogous to that of a large isolated VSD, tetralogy of Fallot, or D-TGA. In 1972, Lev et al148 suggested considering DORV as a spectrum of hearts that “pass imperceptibly from tetralogy with VSD with overriding aorta into double-outlet right ventricle with subaor-tic VSD.” Thus, Lev and colleagues described a classification scheme for DORV based on the “commitment” of the VSD to either or both great arteries.148 The VSD can be subaortic, dou-bly committed, noncommitted, or

1	Lev and colleagues described a classification scheme for DORV based on the “commitment” of the VSD to either or both great arteries.148 The VSD can be subaortic, dou-bly committed, noncommitted, or subpulmonic.The subaortic type is the most common (47%) and occurs when the VSD is located directly beneath the aortic annulus. Doubly committed VSD (4%) is present when the VSD lies beneath both the aorta and the pulmonary artery, which are usually side-by-side in this lesion. The noncommitted VSD (26%) exists when the VSD is remote from the great vessels. The subset of DORV hearts with the VSD located beneath the pulmonary valve also are classified as the Taussig–Bing syn-drome (Fig. 20-53).149 This occurs in 23% of cases of DORV with VSD, and it occurs when the aorta rotates more anteriorly, with the pulmonary artery rotated more posteriorly.150Clinical Manifestations and Diagnosis. Patients with DORV typically present with one of the following three scenar-ios: (a) those with doubly

1	with the pulmonary artery rotated more posteriorly.150Clinical Manifestations and Diagnosis. Patients with DORV typically present with one of the following three scenar-ios: (a) those with doubly committed or subaortic VSD present with congestive heart failure and a high propensity for pulmo-nary hypertension, much like infants with a large single VSD; (b) those with a subaortic VSD and pulmonary stenosis present with cyanosis and hypoxia, much like infants with tetralogy of Fallot; and (c) those with subpulmonic VSD present with cya-nosis, much like those with D-TGA, because streaming directs desaturated systemic venous blood to the aorta and oxygenated blood to the pulmonary artery.140 Thus, the three critical factors influencing the clinical presentation and subsequent manage-ment of infants with DORV are the size and location of the VSD, the presence or absence of important RVOT obstruc-tion, and the presence of other anomalies (especially associ-ated hypoplasia of left-sided

1	of infants with DORV are the size and location of the VSD, the presence or absence of important RVOT obstruc-tion, and the presence of other anomalies (especially associ-ated hypoplasia of left-sided structures sometimes seen with subpulmonary VSD).Echocardiography is the mainstay of diagnosis and can also provide valuable information regarding the feasibility of biventricular repair. Specific anatomic questions that should be resolved to assist in surgical planning in addition to those mentioned earlier include the coronary anatomy (presence of a conal branch or left anterior descending from the right coronary coursing across the conus), the presence of additional muscular VSDs remote from either great vessel, and the distance between the tricuspid and pulmonary valve. Cardiac catheterization is rarely necessary in neonates or infants, except to determine the degree of pulmonary hypertension and to determine the effects of previous palliative procedures on the pulmonary arterial

1	is rarely necessary in neonates or infants, except to determine the degree of pulmonary hypertension and to determine the effects of previous palliative procedures on the pulmonary arterial anatomy.Therapy. The goals of corrective surgery are to relieve pul-monary stenosis, to provide separate and unobstructed outflow pathways from each ventricle to the correct great vessel, and to achieve separation of the systemic and pulmonary circulations.Double-Outlet Right Ventricle With Noncommitted Ventricular Septal DefectThe repair of hearts with DORV and noncommitted VSD can be accomplished by constructing an intraventricular tunnel con-necting the VSD to the aorta, closing the pulmonary artery, and placing a valved extracardiac conduit from the RV to the pulmonary artery. In patients without pulmonary stenosis who have intractable congestive failure, a pulmonary artery band can be placed in the first 6 months to control pulmonary artery Figure 20-53. Angiographic appearance of the aorta in

1	stenosis who have intractable congestive failure, a pulmonary artery band can be placed in the first 6 months to control pulmonary artery Figure 20-53. Angiographic appearance of the aorta in a patient with Taussig-Bing anomaly. Note the hypoplastic arch (‘*’).Figure 20-52. DORV, aortomitral discontinuity (‘*’), aorta mostly arising from RV (arrow).Brunicardi_Ch20_p0751-p0800.indd 78322/02/19 2:56 PM 784SPECIFIC CONSIDERATIONSPART IIovercirculation and prevent the development of pulmonary hypertension.Infants with pulmonary stenosis can be managed with a systemic-to-pulmonary shunt followed by biventricular repair as described by Belli and colleagues in 1999, or with a modi-fied Fontan.151 There is no consensus on the timing of repair, but recent literature suggests that repair within the first 6 months is associated with better outcome. However, in cases where an extracardiac-valved conduit is necessary, it is better to delay definitive repair until the child is 2 to 3 years of

1	the first 6 months is associated with better outcome. However, in cases where an extracardiac-valved conduit is necessary, it is better to delay definitive repair until the child is 2 to 3 years of age because this allows placement of a larger conduit and possibly reduces the number of future obligatory conduit replacements.147Double-Outlet Right Ventricle With Subaortic or Doubly Committed Ventricular Septal Defect Without Pulmonary StenosisThis group of patients can be treated by creating an intracardiac baffle that directs blood from the LV into the aorta. Enlargement of the VSD may be necessary to allow ample room for the baf-fle; this should be done anterosuperiorly to avoid injury to the conduction system that normally lies inferoposteriorly along the border of the VSD. In addition, other important considerations in constructing the LV outflow tunnel include the prominence of the conal septum, the attachments of the tricuspid valve to the conal septum, and the distance between

1	other important considerations in constructing the LV outflow tunnel include the prominence of the conal septum, the attachments of the tricuspid valve to the conal septum, and the distance between the tricuspid and pulmonary valves. In some instances, unfavorable anatomy may preclude placement of an adequate intracardiac baffle, neces-sitating single ventricle repair.Double-Outlet Right Ventricle With Subaortic or Doubly Committed Ventricular Septal Defect With Pulmonary StenosisRepair of this defect is similar to the above except that concomi-tant RVOT reconstruction must be performed in addition to the intracardiac tunnel. The RVOT augmentation can be accom-plished with the placement of a transannular patch or with place-ment of an extracardiac-valved conduit when an anomalous left anterior descending artery precludes use of a patch.Taussig–Bing Syndrome Without Pulmonary StenosisThese infants are best treated with a balloon septostomy dur-ing the neonatal period to improve

1	anterior descending artery precludes use of a patch.Taussig–Bing Syndrome Without Pulmonary StenosisThese infants are best treated with a balloon septostomy dur-ing the neonatal period to improve mixing, followed by VSD closure baffling LV egress to the pulmonary artery and an arte-rial switch operation. The Kawashima procedure,152 in which an intraventricular tunnel is used to baffle LV egress directly to the aorta, may alternatively be used when the aorta is more posterior or when there is associated pulmonary stenosis.Taussig–Bing Syndrome With Pulmonary StenosisThis defect may be treated with a variety of techniques, depend-ing on the specific anatomic details and the expertise of the treat-ment team. A Rastelli-type repair, which involves construction of an intraventricular tunnel through the existing VSD that con-nects the LV to both great vessels, followed by division of the pulmonary artery at its origin and insertion of a valved conduit from the RV to the distal pulmonary

1	through the existing VSD that con-nects the LV to both great vessels, followed by division of the pulmonary artery at its origin and insertion of a valved conduit from the RV to the distal pulmonary artery, can be performed.153 Alternatively, a Yasui procedure, which involves baffling the VSD to the pulmonary artery and creation of a DKS anastomo-sis between the pulmonary artery and the aorta with patch aug-mentation, can be accomplished concomitant with placement of an RV pulmonary artery conduit.154Results. The results of DORV repairs are generally favor-able, especially for the tetralogy-type DORV with subaortic VSD.150,155 However, more complex types of DORV, including noncommitted VSD and Taussig–Bing type, still carry impor-tant morbidity and mortality.150,151,155 Furthermore, repeated interventions for RVOT reconstruction or staged operations for patients triaged to single-ventricle pathways pose late hazards for patients surviving initial repair. A single-institution series

1	interventions for RVOT reconstruction or staged operations for patients triaged to single-ventricle pathways pose late hazards for patients surviving initial repair. A single-institution series evaluated 393 patients with DORV.150 The authors found that the need for reintervention approached 37% at 15 years follow-ing repair. Arterial switch operation, as opposed to Rastelli-type repair, was associated with an increased risk of early postrepair mortality, but mitigated against the risk of late death. Patients with hypoplastic left-sided structures and a nonsubaortic VSD may fare better with a single-ventricle repair.Tetralogy of FallotAnatomy. The original description of tetralogy of Fallot (TOF) by Ettienne Louis Fallot,156 as the name implies, included four abnormalities: a large perimembranous VSD adjacent to the tri-cuspid valve; an overriding aorta; a variable degree of RVOT obstruction, which might include hypoplasia and dysplasia of the pulmonary valve as well as obstruction at

1	VSD adjacent to the tri-cuspid valve; an overriding aorta; a variable degree of RVOT obstruction, which might include hypoplasia and dysplasia of the pulmonary valve as well as obstruction at the subvalvar and pulmonary artery level; and right ventricular hypertrophy. More recently, the Van Praagh et al157 pointed out that TOF could be more correctly termed monology of Fallot, since the four com-ponents are explained by the malposition of the infundibular sep-tum. When the infundibular septum is displaced anteriorly and leftward, the RVOT is narrowed and its anterior displacement results in failure of fusion of the ventricular septum between the arms of the trabeculo-septo-marginalis (Fig. 20-54).The morphology of TOF is markedly heterogeneous and includes an absent pulmonary valve, concomitant AV septal defects, and pulmonary atresia with major aortopulmonary collaterals. The present discussion will focus only on the so-called classic presentation of TOF without coexisting

1	concomitant AV septal defects, and pulmonary atresia with major aortopulmonary collaterals. The present discussion will focus only on the so-called classic presentation of TOF without coexisting intracardiac defects.Anomalous coronary artery patterns, related to either ori-gin or distribution, have been described in TOF.158 However, the most surgically important coronary anomaly occurs when AortaMPAVSDMultilevelpulmonary stenosisRVHFigure 20-54. Tetrology of Fallot. (Used with permission from Kelly Rosso MD.)Brunicardi_Ch20_p0751-p0800.indd 78422/02/19 2:56 PM 785CONGENITAL HEART DISEASECHAPTER 20the left anterior descending artery arises as a branch of the right coronary artery. This occurs in approximately 3% of cases of TOF and may preclude placement of a transannular patch, as the left anterior descending coronary artery crosses the RVOT at varying distances from the pulmonary valve annulus.159Pathophysiology and Clinical Presentation. The initial presentation of a child

1	the left anterior descending coronary artery crosses the RVOT at varying distances from the pulmonary valve annulus.159Pathophysiology and Clinical Presentation. The initial presentation of a child afflicted with TOF depends on the degree of RVOT obstruction. Children with cyanosis at birth usually have severe pulmonary annular hypoplasia with concomitant hypoplasia of the peripheral pulmonary arteries. Most children, however, present with mild cyanosis at birth, which then pro-gresses as the right ventricular hypertrophy further compromises the RVOT. Cyanosis usually becomes significant within the first 6 to 12 months of life, and the child may develop characteristic “tet” spells, which are periods of extreme hypoxemia. These spells are characterized by decreased pulmonary blood flow and an increase in systemic blood flow. They can be triggered by any stimulus that decreases systemic vascular resistance, such as fever, agitation, or vigorous physical activity. Cyanotic spells

1	and an increase in systemic blood flow. They can be triggered by any stimulus that decreases systemic vascular resistance, such as fever, agitation, or vigorous physical activity. Cyanotic spells increase in severity and frequency as the child grows, and older patients with uncorrected TOF may often squat, which increases peripheral vascular resistance and relieves the cyanosis.Evaluation in the older patient with TOF may demonstrate clubbing, polycythemia, hemoptysis, or brain abscesses. Chest radiography will demonstrate a boot-shaped heart (Fig. 20-55), and EKG will show the normal pattern of right ventricular hypertrophy. Echocardiography confirms the diagnosis because it demonstrates the position and nature of the VSD, defines the character of the RVOT obstruction, and often visualizes the branch pulmonary arteries and the proximal coronary arteries. Cardiac catheterization is rarely necessary and is actually risky in TOF since it can create spasm of the RVOT muscle and result in

1	the branch pulmonary arteries and the proximal coronary arteries. Cardiac catheterization is rarely necessary and is actually risky in TOF since it can create spasm of the RVOT muscle and result in a hypercyanotic episode (tet spell). Occasionally, aortogra-phy (Fig. 20-56) is necessary to delineate the coronary artery anatomy.Treatment. John Deanfield160 stated “…long follow-up inevi-tably means surgery in an earlier era: More recent surgery, at a younger age, with better preoperative, operative, and post-operative care, will improve long-term results. Data from the former (earlier) era will be overly pessimistic.” This statement is particularly pertinent as surgical correction of TOF has evolved from a staged approach of antecedent palliation in infancy fol-lowed by intracardiac repair to primary repair during the first few months of life without prior palliative surgery.However, systemic-to-pulmonary shunts, generally an MBTS, may still be preferred with an unstable neonate younger

1	primary repair during the first few months of life without prior palliative surgery.However, systemic-to-pulmonary shunts, generally an MBTS, may still be preferred with an unstable neonate younger than 3 months of age, when an extracardiac conduit is required because of an anomalous left anterior descending coronary artery, or when pulmonary atresia, significant branch pulmo-nary artery hypoplasia, or severe noncardiac anomalies coexist with TOF.Traditionally, TOF was repaired through a right ventricu-lotomy, providing excellent exposure for closure of the VSD and relief of the RVOT obstruction, but concerns that the resul-tant scar would significantly impair right ventricular function or lead to lethal arrhythmias led to the development of a transatrial approach. Transatrial repair, except in cases when the presence of diffuse RVOT hypoplasia requires insertion of a transannular patch, is now being increasingly advocated by many, although its superiority has not been conclusively

1	in cases when the presence of diffuse RVOT hypoplasia requires insertion of a transannular patch, is now being increasingly advocated by many, although its superiority has not been conclusively demonstrated.161The operative technique involves the use of CPB. All existing systemic-to-pulmonary arterial shunts, as well as the ductus arteriosus, are ligated. A right atriotomy is then made, and the anatomy of the VSD and the RVOT are assessed by retracting the tricuspid valve. The outflow tract obstruction is relieved by resecting the offending portion of the infundibular septum as well as any muscle trabeculations. If necessary, a pul-monary valvotomy or, alternatively, a longitudinal incision in the main pulmonary artery can be performed to improve expo-sure. The diameter of the pulmonary valve annulus is assessed by inserting Hegar dilators across the outflow tract; if the pul-monary artery/aorta diameter is less than 0.5, or the estimated RV/LV pressure is greater than 0.7, or the

1	valve annulus is assessed by inserting Hegar dilators across the outflow tract; if the pul-monary artery/aorta diameter is less than 0.5, or the estimated RV/LV pressure is greater than 0.7, or the size of the pulmo-nary valve is less than a Z score of −2.5, a transannular patch is inserted. Patch closure of the VSD is then accomplished, taking Figure 20-56. CT aortogram showing the large aorta often associated with conotruncal anomalies, rotated coronaries, and extremely hypoplastic main and branch pulmonary arteries in a patient with TOF.Figure 20-55. Chest x-ray showing a boot shaped heart in an infant with tetralogy of Fallot.Brunicardi_Ch20_p0751-p0800.indd 78522/02/19 2:56 PM 786SPECIFIC CONSIDERATIONSPART IIcare when placing sutures along the posteroinferior portion to avoid the conduction system.Results. Operative mortality for primary repair of TOF in infancy is less than 5% in most series.161 Previously reported risk factors such as transannular patch insertion or

1	the conduction system.Results. Operative mortality for primary repair of TOF in infancy is less than 5% in most series.161 Previously reported risk factors such as transannular patch insertion or younger age at time of repair have been eliminated secondary to improved intraoperative and postoperative care. According to the Society of Thoracic Surgeons Congenital Heart Surgery Database, dis-charge mortality from 3059 operations from 2002 to 2007 was 7.5% for initial palliation, 1.3% for primary repair, and 0.9% for staged repair, indicating similar outcomes for patients get-ting primary repair compared to staged repair.162 Nevertheless, for neonatal repair, discharge mortality increased to 6.2% with palliation and 7.8% with primary repair. This may be partly explained by a higher chance of postoperative complications in neonates.A major complication of repaired TOF is the develop-ment of pulmonary insufficiency, which subjects the RV to the adverse effects of acute and chronic volume

1	postoperative complications in neonates.A major complication of repaired TOF is the develop-ment of pulmonary insufficiency, which subjects the RV to the adverse effects of acute and chronic volume overload. This is especially problematic if residual lesions such as a VSD or peripheral pulmonary stenosis exist. Pulmonary valve regurgita-tion after repair of TOF is relatively well tolerated in the short term, partly because the hypertrophied RV usually adapts to the altered hemodynamic load.163 The detrimental effects of chronic pulmonary valve regurgitation are, however, numerous, and include progressive right ventricular dilatation and failure, tri-cuspid valve regurgitation, exercise intolerance, arrhythmia, and sudden death. Mechanoelectrical interaction, by which a dila-tated RV provides the substrate for electrical instability, might underlie the propensity toward ventricular arrhythmia.164 In sup-port of this contention, Gatzoulis and colleagues163,164 found that the risk of

1	the substrate for electrical instability, might underlie the propensity toward ventricular arrhythmia.164 In sup-port of this contention, Gatzoulis and colleagues163,164 found that the risk of symptomatic arrhythmia was high in patients with marked right ventricular enlargement and QRS prolongation on resting ECG of more than 180 ms. Karamlou et al have shown that similar structural and hemodynamic abnormalities, including a larger right atrial volume and right ventricular chamber size, are also related to atrial arrhythmias in patients following TOF repair.165 We found that prolongation of the QRS duration beyond a threshold of 160 ms increased the risk of atrial arrhythmias.165 Together, these data show that a similar mechanism could be responsible for both atrial and ventricular arrhythmias after repair in TOF patients.When significant deterioration of ventricular func-tion occurs, insertion of a pulmonary valve may be required, although this is rarely necessary in infants.

1	arrhythmias after repair in TOF patients.When significant deterioration of ventricular func-tion occurs, insertion of a pulmonary valve may be required, although this is rarely necessary in infants. Unfortunately, there are no universal criteria establishing the timing of pulmonary valve replacement. The current criteria for pulmonary valve replacement are the presence of two of the following criteria: RVEDD index >160 ml/m2, RVEDI >70 ml/m2, LVEDV index >65 ml/m2, RVEF <45%, RVOT aneurysm, and clinical symp-toms or signs, including syncope or VT.166 PVR can be achieved with minimal morbidity and mortality.167The alternative to surgical PVR is percutaneous pulmo-nary valve implantation. The Melody valve system (Fig. 20-57) is the most popular of such systems. Following risk adjustment, no significant differences were observed between surgical or transcatheter PVR. However, transcatheter PVR was associated with a shorter hospitalization. Hospitalization costs are similar for both

1	no significant differences were observed between surgical or transcatheter PVR. However, transcatheter PVR was associated with a shorter hospitalization. Hospitalization costs are similar for both procedures.168Arrhythmias are potentially the most serious late complication following TOF repair. In a multicenter cohort of 793 patients studied by Gatzoulis et al,164 a steady increase was documented in the prevalence of ventricular and atrial tachyarrhythmia and sudden cardiac death in the first 5 to 10 years after intracardiac repair. Clinical events were reported in 12% of patients at 35 years after repair. Prevalence of atrial arrhythmias from other studies, however, ranges from 1% to 11%,163,164 which is a reflection of the strong time dependence of arrhythmia onset.Underlying causes of arrhythmia following repair are complex and multifactorial, resulting in poorly defined opti-mum screening and treatment algorithms. Older repair age has been associated with an increased frequency of

1	following repair are complex and multifactorial, resulting in poorly defined opti-mum screening and treatment algorithms. Older repair age has been associated with an increased frequency of both atrial and ventricular arrhythmias. Impaired ventricular function second-ary to a protracted period of cyanosis before repair might con-tribute to the propensity for arrhythmia in older patients.Ventricular Septal DefectAnatomy. VSD refers to a hole between the LV and RV. These defects are common, comprising 20% to 30% of all cases of CHD, and may occur as an isolated lesion or as part of a more Figure 20-57. The Melody valve.Brunicardi_Ch20_p0751-p0800.indd 78622/02/19 2:56 PM 787CONGENITAL HEART DISEASECHAPTER 20complex malformation.169 VSDs vary in size from 3 to 4 mm to more than 3 cm and are classified into four types based on their location in the ventricular septum: perimembranous (or paramembranous, conoventricular), AV canal (inlet), outlet or supracristal, and muscular (Fig.

1	and are classified into four types based on their location in the ventricular septum: perimembranous (or paramembranous, conoventricular), AV canal (inlet), outlet or supracristal, and muscular (Fig. 20-58).Perimembranous VSDs are the most common type requir-ing surgical intervention, comprising approximately 80% of cases.169 These defects involve the membranous septum and include the malalignment defects seen in tetralogy of Fallot. In rare instances, the anterior and septal leaflets of the tricus-pid valve adhere to the edges of the perimembranous defect, forming a channel between the LV and the right atrium. These defects result in a large left-to-right shunt due to the large pres-sure differential between the two chambers.AV canal defects, also known as inlet defects, occur when part or all of the septum of the AV canal is absent. The VSD lies beneath the tricuspid valve and is limited upstream by the tricuspid annulus, without intervening muscle.The supracristal or outlet VSD

1	or all of the septum of the AV canal is absent. The VSD lies beneath the tricuspid valve and is limited upstream by the tricuspid annulus, without intervening muscle.The supracristal or outlet VSD results from a defect within the conal septum. Characteristically, these defects are limited upstream by the pulmonary valve and are otherwise surrounded by the muscle of the infundibular septum.Muscular VSDs are the most common type and may lie in four locations: anterior, midventricular, posterior, or apical. These are surrounded by muscle and can occur anywhere along the trabecular portion of the septum. The rare “Swiss-cheese” type of muscular VSD consists of multiple communications between the RV and LV, complicating operative repair.Pathophysiology and Clinical Presentation. The size of the VSD determines the initial pathophysiology of the disease. Large VSDs are classified as nonrestrictive and are at least equal in diameter to the aortic annulus. These defects allow free flow of

1	the VSD determines the initial pathophysiology of the disease. Large VSDs are classified as nonrestrictive and are at least equal in diameter to the aortic annulus. These defects allow free flow of blood from the LV to the RV, elevating right ventricular pres-sures to the same level as systemic pressure.Consequently, the pulmonary-to-systemic flow ratio (Qp to Qs) is inversely dependent on the ratio of pulmonary vas-cular resistance to systemic vascular resistance. Nonrestrictive VSDs produce a large increase in pulmonary blood flow, and the afflicted infant will present with symptoms of congestive heart failure. However, if untreated, these defects will cause pulmonary hypertension with a corresponding increase in pulmonary vascular resistance. This will lead to a reversal of flow (a right-to-left shunt), which is known as Eisenmenger’s syndrome.Small restrictive VSDs offer significant resistance to the passage of blood across the defect, and therefore right ventricu-lar pressure is

1	shunt), which is known as Eisenmenger’s syndrome.Small restrictive VSDs offer significant resistance to the passage of blood across the defect, and therefore right ventricu-lar pressure is either normal or only minimally elevated and the ratio of Qp to Qs rarely exceeds 1.5. These defects are generally asymptomatic because there are few physiologic consequences. However, there is a long-term risk of endocarditis because endo-cardial damage from the jet of blood through the defect may serve as a possible nidus for colonization (Fig. 20-59A,B).Diagnosis. The child with a large VSD will present with severe congestive heart failure and frequent respiratory tract infections. Children with Eisenmenger’s syndrome may be deceptively asymptomatic until frank cyanosis develops.The chest radiograph will show cardiomegaly and pulmo-nary overcirculation, and the ECG will show signs of left ven-tricular or biventricular hypertrophy. Echocardiography provides definitive diagnosis and can estimate

1	will show cardiomegaly and pulmo-nary overcirculation, and the ECG will show signs of left ven-tricular or biventricular hypertrophy. Echocardiography provides definitive diagnosis and can estimate the degree of shunting as well as pulmonary arterial pressures. Cardiac catheterization has MembranousMuscularInletSupracristalTVFigure 20-58. Types of VSD. (Used with permission from Kelly Rosso MD.)ABFigure 20-59. A. Severe TV and VSD endocarditis (‘*’) in a 4 yo untreated patient. B. Echocardiographic appearance of the same patient after patch repair(‘*’) of the VSD and complete exci-sion of the tricuspid valve.Brunicardi_Ch20_p0751-p0800.indd 78722/02/19 2:56 PM 788SPECIFIC CONSIDERATIONSPART IIlargely been supplanted by echocardiography, except in older children where measurement of pulmonary resistance is neces-sary prior to recommending closure of the defect.Treatment. VSDs may close or narrow spontaneously, and the probability of closure is inversely related to the age at which

1	resistance is neces-sary prior to recommending closure of the defect.Treatment. VSDs may close or narrow spontaneously, and the probability of closure is inversely related to the age at which the defect is observed. Thus, infants at 1 month of age have an 80% incidence of spontaneous closure, whereas a child at 12 months of age has only a 25% chance of closure.170 This has an important impact on operative decision-making because a small or moder-ate-size VSD may be observed for a period of time in the absence of symptoms. Large defects and those in severely symptomatic neonates should be repaired during infancy to relieve symptoms and because irreversible changes in pulmonary vascular resis-tance may develop during the first year of life.Repair of isolated VSDs requires the use of CPB with moderate hypothermia and cardioplegic arrest. The right atrial approach (Fig. 20-60) is preferable for most defects, except apical muscular defects, which often require a right ventricu-lotomy for

1	moderate hypothermia and cardioplegic arrest. The right atrial approach (Fig. 20-60) is preferable for most defects, except apical muscular defects, which often require a right ventricu-lotomy for adequate exposure. Supracristal defects may alter-natively be exposed via a pulmonary arteriotomy or through an incision in the RV immediately beneath the pulmonary valve (Fig. 20-61). Regardless of the type of defect present, a right atrial approach can be used initially to inspect the anatomy, as this may be abandoned should it offer inadequate exposure for repair. After careful inspection of the heart for any associated malformations, a patch repair is employed, taking care to avoid the conduction system. Routine use of intraoperative trans-esophageal echocardiography should be used to assess for any residual defect.Successful percutaneous device closure of VSDs using the Amplatzer device has been described.152 The device has demon-strated a 100% closure rate in a small series of patients

1	for any residual defect.Successful percutaneous device closure of VSDs using the Amplatzer device has been described.152 The device has demon-strated a 100% closure rate in a small series of patients with iso-lated or residual VSDs, or as a collaborative treatment strategy for the VSD component in more complex congenital lesions. Proponents of device closure argue that its use can decrease the complexity of surgical repair, avoid reoperation for a small residual lesion, or avoid the need for a ventriculotomy. The use of devices to close paramembranous defects can cause heart block because the defect is in close association to the conduction system (Fig 20-62).171 The procedure can be performed percuta-neously or through the per ventricular approach. Embolization of the device is an added risk.Multiple or “Swiss-cheese” VSDs represent a special case, and many cannot be repaired during infancy. In patients in whom definitive VSD closure cannot be accomplished, tem-porary placement of a

1	risk.Multiple or “Swiss-cheese” VSDs represent a special case, and many cannot be repaired during infancy. In patients in whom definitive VSD closure cannot be accomplished, tem-porary placement of a pulmonary artery band can be employed to control pulmonary flow. This allows time for spontaneous closure of many of the smaller defects, thus simplifying surgi-cal repair.172Some centers, however, have advocated early definitive repair of the Swiss-cheese septum, by using oversize patches, fibrin glue, and combined intraoperative device closure, as well as techniques to complete the repair transatrially.173Results. Even in very small infants, closure of VSDs can be safely performed with hospital mortality near 0%. The main risk factor remains the presence of other associated lesions, espe-cially when present in symptomatic neonates with large VSDs.Figure 20-60. Intra-op picture during a VSD closure performed by interrupted suture technique with patch closure.Figure

1	lesions, espe-cially when present in symptomatic neonates with large VSDs.Figure 20-60. Intra-op picture during a VSD closure performed by interrupted suture technique with patch closure.Figure 20-61. Echocardiographic appearance of a supracristal VSD (arrow). Note its location just beneath the pulmonary valve (‘*’).Brunicardi_Ch20_p0751-p0800.indd 78822/02/19 2:56 PM 789CONGENITAL HEART DISEASECHAPTER 20Atrioventricular Canal DefectsAnatomy. AV canal defects result from failure of fusion of the endocardial cushions in the central portion of the heart, caus-ing a lesion that involves the atrial and the ventricular septum, as well as the anterior mitral and septal tricuspid valve leaf-lets. Defects involving primarily the atrial septum are known as partial AV canal defects and frequently occur in conjunction with a cleft anterior mitral leaflet. Complete AV canal defects have a combined deficiency of the atrial and ventricular sep-tum associated with a common AV orifice rather than

1	occur in conjunction with a cleft anterior mitral leaflet. Complete AV canal defects have a combined deficiency of the atrial and ventricular sep-tum associated with a common AV orifice rather than separate tricuspid and mitral valves. The common AV valve generally has five leaflets, three lateral (free wall) and two bridging (septal) leaflets. The defect in the ventricular septum can lie either between the two bridging leaflets or beneath them. The relationship between the septal defect and the anterior bridging leaflet forms the basis of the Rastelli classification for complete AV canal defects (Fig. 20-63).174,175Pathophysiology and Diagnosis. Partial AV canal defects, in the absence of AV valvular regurgitation, frequently resemble isolated ASDs. Left-to-right shunting predominates as long as pulmonary vascular resistance remains low. However, 40% of patients with partial AV canal defects have moderate-to-severe valve incompetence, and progressive heart failure occurs early in

1	as long as pulmonary vascular resistance remains low. However, 40% of patients with partial AV canal defects have moderate-to-severe valve incompetence, and progressive heart failure occurs early in this patient population.175 Complete AV canal defects produce more severe pathophysiologic changes because the large intra-cardiac communication and significant AV valve regurgitation contribute to ventricular volume loading and pulmonary hyper-tension. Children with complete AV canal defects develop signs of congestive heart failure within the first few months of life.Physical examination may reveal a right ventricular heave and a systolic murmur. Children may also present with endo-carditis or paradoxical emboli as a result of the intracardiac communication. Chest radiography will be consistent with con-gestive heart failure, and the EKG demonstrates right ventricu-lar hypertrophy with a prolonged PR interval and is classically associated with left axis deviation.Two-dimensional

1	consistent with con-gestive heart failure, and the EKG demonstrates right ventricu-lar hypertrophy with a prolonged PR interval and is classically associated with left axis deviation.Two-dimensional echocardiography (Fig. 20-64) with color-flow mapping is confirmatory, but cardiac catheterization can be employed to define the status of the pulmonary vascula-ture, with a pulmonary vascular resistance greater than 12 Wood units indicating inoperability.Treatment. The management of patients with AV canal defects can be especially challenging. Timing of operation is individualized. Patients with partial defects can be electively repaired between 2 and 5 years of age, whereas complete AV canal defects should be repaired within the first year of life to prevent irreversible changes in the pulmonary circulation. Complete repair in infancy should be accomplished, with palliative procedures such as pulmonary artery banding reserved for only those infants with other complex lesions or who are

1	circulation. Complete repair in infancy should be accomplished, with palliative procedures such as pulmonary artery banding reserved for only those infants with other complex lesions or who are too ill to tolerate CPB.The operative technique requires the use of either continu-ous hypothermic CPB or, for small infants, deep hypothermic circulatory arrest. The heart is initially approached through an oblique right atriotomy, and the anatomy is carefully observed. In the case of a partial AV canal, the cleft in the mitral valve is repaired with interrupted sutures and the ASD is closed with a pericardial patch. Complete AV canal defects are repaired by patch closure of the VSD, separating the common AV valve into tricuspid and mitral components and suspending the neovalves from the top of the VSD patch and closing the ASD.Results. Partial AV canal defects have an excellent outcome, with a mortality rate of 0% to 2% in most series.175 Complete AV canal defects are associated with

1	of the VSD patch and closing the ASD.Results. Partial AV canal defects have an excellent outcome, with a mortality rate of 0% to 2% in most series.175 Complete AV canal defects are associated with anoperative mortality of 3% to 4%.176The most frequently encountered postoperative problems are complete heart block (1%–2%), right bundle-branch block (22%), arrhythmias (11%), RVOT obstruction (11%), and severe mitral regurgitation (13%–24%).175 The increasing use of intraoperative transesophageal echocardiography may positively Figure 20-62. Intraoperative picture at the time of removal of a percutaneously placed VSD device causing severe TR and complete heart block. Note the close association of the device to the tricuspid valve leaflet (arrow) and cordae.Type AType BType CFigure 20-63. Rastelli classification of complete AVSD. (Used with permission from Kelly Rosso MD.)Figure 20-64. Echo of an infant with complete AVSD. Note the prominent absence of the ‘crux’ (‘*’) of the heart in this

1	classification of complete AVSD. (Used with permission from Kelly Rosso MD.)Figure 20-64. Echo of an infant with complete AVSD. Note the prominent absence of the ‘crux’ (‘*’) of the heart in this defect.Brunicardi_Ch20_p0751-p0800.indd 78922/02/19 2:56 PM 790SPECIFIC CONSIDERATIONSPART IIinfluence outcomes, as the adequacy of repair can be assessed and treated without need for subsequent reoperation.174-175Interrupted Aortic ArchAnatomy. Interrupted aortic arch (IAA) is a rare defect, com-prising approximately 1% of all cases of CHD.177 It is defined as an absence of luminal continuity between the ascending and descending aorta and does not occur as an isolated defect in most cases because a VSD or PDA is usually present. IAA is classified based on the location of the interruption (Fig. 20-65 to Fig. 20-67).Clinical Manifestations and Diagnosis. Infants with IAA have ductal-dependent systemic blood flow and will develop profound metabolic acidosis and hemodynamic collapse upon

1	(Fig. 20-65 to Fig. 20-67).Clinical Manifestations and Diagnosis. Infants with IAA have ductal-dependent systemic blood flow and will develop profound metabolic acidosis and hemodynamic collapse upon ductal closure. In the rare instance of failed ductal closure, the diagnosis may be missed during infancy, and the child will pres-ent with symptoms of congestive heart failure from a persistent left-to-right shunt.Once definitive diagnosis is made in infants, usually with echocardiography, preparations are made for operative interven-tion, and prostaglandin E1 is infused to maintain ductal patency and correct acidosis. The infant’s hemodynamic status should Figure 20-66. CT angiogram of a Type A IAA.AoAoPAType AType BType CPAPAAoFigure 20-65. Types of IAA. (Used with permission from Nicholas Clarke MD.)Figure 20-67. MRI reconstruction of a Type B IAA.be optimized with mechanical ventilation and inotropic support. An effort should be made to increase pulmonary vascular resis-tance by

1	Clarke MD.)Figure 20-67. MRI reconstruction of a Type B IAA.be optimized with mechanical ventilation and inotropic support. An effort should be made to increase pulmonary vascular resis-tance by decreasing the fractional inspired oxygen and avoiding hyperventilation because this will preferentially direct blood into the systemic circulation.Treatment. Initial strategies for the management of IAA involved palliation though a left thoracotomy by using one of the arch vessels as a conduit to restore aortic continuity. Pulmo-nary artery banding can be simultaneously performed to limit left-to-right shunting because it is not feasible to repair the VSD or other intracardiac communications with this approach.However, complete one stage surgical repair in infants with IAA is now preferable. The operative technique involves use of a median sternotomy and CPB with short periods of cir-culatory arrest. Aortic arch reconstruction can be accomplished with either direct anastomosis or patch

1	The operative technique involves use of a median sternotomy and CPB with short periods of cir-culatory arrest. Aortic arch reconstruction can be accomplished with either direct anastomosis or patch aortoplasty followed by closure of the VSD.178In certain cases, the defect will involve hypoplasia of the left heart, precluding attempts at definitive repair. These infants should be managed with a Norwood procedure followed by a Fontan repair.Results. Outcomes in infants with IAA have improved sub-stantially over the last decades as a result of improved periop-erative care. Operative mortality is now less than 10% in most series.177,179 Some authors advocate the use of patch augmenta-tion of the aorta to ensure adequate relief of LVOT obstruction and to diminish anastomotic tension, thus reducing the subse-quent risk of restenosis and tracheobronchial compression.178Pediatric Mechanical Circulatory SupportMechanical circulatory support has become standard therapy for adults with end stage

1	the subse-quent risk of restenosis and tracheobronchial compression.178Pediatric Mechanical Circulatory SupportMechanical circulatory support has become standard therapy for adults with end stage heart failure. There has been a sig-nificant lag with development of similar devices for the pediatric population. This is probably related to the smaller mar-ket for these devices and the technical challenges associated with the anatomical constraints secondary to anatomy and size of the patients. Extracorporeal membrane oxygenation (ECMO) 8Brunicardi_Ch20_p0751-p0800.indd 79022/02/19 2:56 PM 791CONGENITAL HEART DISEASECHAPTER 20has been the mainstay of mechanical support in many centers for the pediatric population. The adaptation of other adult devices to the pediatric population las led to the slow but steady devel-opment of pediatric durable mechanical devices. The Berlin Heart EXCOR (Berlin Heart AG, Berlin, Germany) device was approved by the FDA in 2011 in the United States as a

1	to the slow but steady devel-opment of pediatric durable mechanical devices. The Berlin Heart EXCOR (Berlin Heart AG, Berlin, Germany) device was approved by the FDA in 2011 in the United States as a paracor-poreal device that can be used as a bridge to transplantation. This device has a 73% overall survival post implant at 12 months.97 Infection, stroke and bleeding remain significant morbidities associated with it. Young age and small body surface area still remain poor prognostic factors. In 2010, the National Heart, Lung, and Blood Institute launched the Pumps for Kids, Infants, and Neonates (PumpKIN) program to promote development of new devices with the goal of clinical use.ECMO remains the most commonly used form of mechan-ical support in the pediatric population in the United States. Per the ECLS Registry report released by the Extracorporeal Life Support Organization, as of January 2017, there were a total of 16,531 ECMO runs performed for cardiac causes, internation-ally.180

1	Per the ECLS Registry report released by the Extracorporeal Life Support Organization, as of January 2017, there were a total of 16,531 ECMO runs performed for cardiac causes, internation-ally.180 The survival to discharge is about 40% in the neona-tal population as opposed to 50% in the pediatric population. ECMO remains the only means of salvage for newborns and infants in many institutions. The biggest limitation remains the short duration it can be used. It is often used as a bridge to recovery and sometimes as a bridge to transplantation. The abil-ity to place small infants on ECMO with peripheral cannulation continues to make it a very attractive first line option.Ventricular assist devices can be either of the pulsatile or continuous types. The Berlin Heart EXCOR (Berlin Heart AG, Berlin, Germany) remains a classic example of a pulsa-tile device. The Impella 2.5 (Abiomed) (Fig. 20-68) has been used in the pediatric population as a temporary support device for recovering

1	AG, Berlin, Germany) remains a classic example of a pulsa-tile device. The Impella 2.5 (Abiomed) (Fig. 20-68) has been used in the pediatric population as a temporary support device for recovering myocarditis, during treatment of acute rejection after heart transplantation and high-risk interventions in frag-ile patients with marginal function.181,182 Other continuous flow devices available for the pediatric patient include the Heartmate II Figure 20-68. Impella 2.5 (Abiomed). (Reproduced with permis-sion from Abiomed. Danvers, MA.)Figure 20-69. Heartware HVAD. (HeartWare® HVAD (Heart-Ware Inc., Miami Lakes, FL.)and Heartmate III devices (Thoratec, Pleasanton, CA), DeBakey VAD Child (MicroMed Technology, Houston, TX), PediMag (Thoratec, Pleasanton, CA), Jarvik2015 and HeartWare HVAD (Fig 20-69) (HeartWare international Inc, Framingham, MA).183 The total artificial heart (SynCardia Systems Inc, Tuscon, Az, USA) is an implantable biventricular device that replaces both ventricles. With

1	20-69) (HeartWare international Inc, Framingham, MA).183 The total artificial heart (SynCardia Systems Inc, Tuscon, Az, USA) is an implantable biventricular device that replaces both ventricles. With the new introduction of the 50 ml pump, its popularity in the pediatric population has risen.Posttransplant survival of patients bridged with and with-out mechanical circulatory support (ventricular assist device or total artificial heart) at 5 years post transplant remains the same. However, patients bridged to transplant with ECMO have a sig-nificantly worse survival.184 All in all, the field of pediatric heart surgery is very exciting and rapidly expanding.Pediatric Heart TransplantationHeart transplantation is currently an accepted mode of therapy in infants and children. Annually, about 600 pediatric heart transplants are performed worldwide,184 about 400 of which are performed in the United States.185 The common indications for heart transplant in the pediatric population are

1	about 600 pediatric heart transplants are performed worldwide,184 about 400 of which are performed in the United States.185 The common indications for heart transplant in the pediatric population are congenital heart disease, dilated cardiomyopathy, retransplantation, and other rare indications (e.g., arrhythmogenic right ventricular dysplasia, cancer, muscular dystrophy, and restrictive cardio-myopathy). The most common congenital heart defect requir-ing transplantation remains hypoplastic left heart syndrome. Although in the past some centers have advocated primary heart transplantation for this lesion, the improved outcomes with surgical palliation have eliminated this as an option. The first year post transplant remains the greatest risk for mortality. The overall median survival is 20.7 years for infants, 18.2 years for children age 1 to 5 years, 14 years for age 6 to 10 years, and 12.7 years for those age 11 to 17 years.184 Males seem to have a modestly superior overall survival

1	years for infants, 18.2 years for children age 1 to 5 years, 14 years for age 6 to 10 years, and 12.7 years for those age 11 to 17 years.184 Males seem to have a modestly superior overall survival compared with females. The causes of mortality include cardiac allograft vasculopathy, acute Brunicardi_Ch20_p0751-p0800.indd 79122/02/19 2:57 PM 792SPECIFIC CONSIDERATIONSPART IIrejection, infections, and graft failures. In the current era, the expected 1-year survival rate is 80% to 90%, the 2-year survival rate is 80% to 85%, and the 5-year survival rate is approximately 70% to 80% in experienced centers.186 Interestingly, infants who undergo transplantation in the first month of life appear to have a survival advantage over infants who undergo transplantation during the remainder of the first year of life.The two main techniques for performing the implant of the heart are the right atrial technique developed by Lower and Shumway and the bicaval-left atrial technique described by

1	the first year of life.The two main techniques for performing the implant of the heart are the right atrial technique developed by Lower and Shumway and the bicaval-left atrial technique described by Sievers and associates.187 In the latter technique, implantation consists of five anastamoses performed using a running prolene suture. These include the left atrial cuff, aorta, pulmonary artery, and the superior and inferior vena cave. One of the cornerstones of postoperative management remains immunosuppression. The triple drug regimen remains popular, corticosteroids, calcineurin inhibitor (cyclosporine or tacrolimus), and an antiproliferative agent (azathioprine or mycophenolate mofetil). Endomyocardial biopsy and coronary angiography are performed at regular inter-vals to monitor rejection. The field of pediatric heart transplan-tation has made huge strides since the days of “Baby Fae.”188,189Public Reporting and the STS Database in Congenital Heart SurgeryThere has been a recent

1	The field of pediatric heart transplan-tation has made huge strides since the days of “Baby Fae.”188,189Public Reporting and the STS Database in Congenital Heart SurgeryThere has been a recent impetus in the filed of congenital and pediatric cardiac surgery toward public reporting of out-comes. The advantages of this include promoting patient autonomy, shows a commitment to quality improvement, and also serves as a free marketing tool. The Society of Thoracic Surgeons Congenital Heart Surgery Database (STS-CHSD), is the largest clinical database in the world for congenital and pedi-atric cardiac surgery. It was founded in 1994. It contains data of about 394,980 operations as of September 9, 2016.192 These data are the foundation for assessment of performance by benchmark and comparison of individual programmatic outcomes to national aggregate data, development and subsequent applica-tion of sophisticated risk adjustment models, quality improve-ment initiatives, research, voluntary

1	of individual programmatic outcomes to national aggregate data, development and subsequent applica-tion of sophisticated risk adjustment models, quality improve-ment initiatives, research, voluntary public reporting, development of reimbursement strategies, and governmental and regulatory collaborations.190 The database is currently in its 25th overall data harvest and records and represents data from 120 participants and 392 surgeons. Thus, this database has greater than 95% penetrance. STS CHSD public reporting started in January 2015, and participation is voluntary. Report-ing is restricted to the hospital level and involves a rolling 4-year analytic window of data. Public reporting is based on the STS CHSD Operative Mortality Risk Model. Developed in 2014, this risk model calculates the operative mortality rate of hospitals performing such surgery, adjusting for procedural and patient level factors. The overall mortality rate over a 4-year period and the operative mortality rate

1	the operative mortality rate of hospitals performing such surgery, adjusting for procedural and patient level factors. The overall mortality rate over a 4-year period and the operative mortality rate for each of the five STAT (Society of Thoracic Surgeons—European Association for Cardio-Thoracic Surgery) categories is reported. The STAT categories are a multi-institutional, validated complexity stratification tool. They range from a score of 1 to 5, and the risk of mortality increases with each category.190 In addition, the STS star rating system was introduced, and every institution is rated as one, two, or three stars. This system is based on the confidence limits of the O/E (observed to expected) overall mortality for the institu-tion (Fig. 20-70). One star equals higher than expected operative 9Rady Children’s Hospital San DiegoRady Children’s Hospital San Diego SurgeonsEnc Devaney, MDDaniel DiBardino, MDJohn Lambert, MDPeter Pastuszko, MDOverall Star RatingPopulation:

1	expected operative 9Rady Children’s Hospital San DiegoRady Children’s Hospital San Diego SurgeonsEnc Devaney, MDDaniel DiBardino, MDJohn Lambert, MDPeter Pastuszko, MDOverall Star RatingPopulation: Neonates,Infants, Children & AdultsOvarallSTAT Mortality Category 1STAT Mortality Category 2STAT Mortality Category 3STAT Mortality Category 4STAT Mortality Category 5#/Eligible28/11650/3067/3991/12817/2953/37Observed2.4%0.0%1.8%0.8%5.8%8.1%Expected3.0%0.6%1.5%2.1%6.7%14.7%OE (95% CI)0.79 (0.53, 1.14)0.00 (0.00, 2.16)1.20 (0.48, 2.45)0.37 (0.01, 2.05)0.87 (0.51, 1.36)0.55 (0.12, 1.49)Adj. Rate (95% CI)2.5 (1.6, 3.5)0.0 (0.0, 1.1)2.0 (0.8, 4.1)1.0 (0.0, 5.3)6.0 (3.5, 9.4)8.7 (1.8, 23.6)San DiegoCAWebsite: http://www.rchsd.org/programs-services/cardiologyOperative and Adjusted Operative Mortality, Last 4 Years (January 2012–December 2015)Figure 20-70. Program performance as currently reported by the STS-CHSD.Brunicardi_Ch20_p0751-p0800.indd 79222/02/19 2:57 PM 793CONGENITAL HEART

1	Last 4 Years (January 2012–December 2015)Figure 20-70. Program performance as currently reported by the STS-CHSD.Brunicardi_Ch20_p0751-p0800.indd 79222/02/19 2:57 PM 793CONGENITAL HEART DISEASECHAPTER 20mortality (the 95% confidence interval for their risk-adjusted O/E mortality ratio was entirely above the number 1), two stars equals the same as expected operative mortality (the 95% con-fidence interval for their risk-adjusted O/E mortality ratio over-lapped with the number 1), and three stars equals lower than expected operative mortality (the 95% confidence interval for their risk-adjusted O/E mortality ratio was entirely below the number 1). The Spring 2016 STS CHSD Feedback Report includes data from 117 participants in the STS-CHSD, including 14 one-star programs, 83 two-star programs, and 8 three-star programs. Twelve participants did not receive a star rating due to incomplete data.191 Public reporting increased from 23% to 57.6% (all three-star programs, 50 two-star and

1	and 8 three-star programs. Twelve participants did not receive a star rating due to incomplete data.191 Public reporting increased from 23% to 57.6% (all three-star programs, 50 two-star and three one-star programs). The online public reporting portal can be accessed at www.sts.org/congenital-public-reporting-module-search.There are several criticisms to the current methodology used for reporting. Important limitations of current publicly reported data (including the STS star rating system) will need to be addressed in future initiatives in order to completely engage parents of children with CHD and reassure providers that risk-adjustment models are optimized. There are four spe-cific areas that should be considered when making decisions how to improve this methodology: (a) While the mortality risk-adjustment model on which the star rating system is based is mature now, there are not comparable models that provide risk-adjusted morbidity (complication) rates. The assessment of the

1	risk-adjustment model on which the star rating system is based is mature now, there are not comparable models that provide risk-adjusted morbidity (complication) rates. The assessment of the quality of congenital heart disease care at different centers should include complication metrics and incorporate failure-to-rescue as an important discriminator; (b) the star rating system does not provide risk-adjusted outcomes for specific procedures or, more importantly, for specific diagnoses. This is mainly because of the exceptionally wide spectrum of diagnoses and procedures in pediatric cardiac surgery that preclude sufficiently large numbers in most procedure-specific categories; (c) the star rating system, although the “best” we have at present, may not be understood equally by all families. It will be critical to provide equivalent information to the large numbers of under-resourced and non–English-speaking families; (d) finally, the current adjusted mortality rate reported by the STS

1	It will be critical to provide equivalent information to the large numbers of under-resourced and non–English-speaking families; (d) finally, the current adjusted mortality rate reported by the STS is calculated from a statistical formula and refers to what the hospital’s mor-tality rate would be if the measured performance (in this case the mortality rate) were extrapolated to the overall case-mix or make-up of patients within the entire STS database. This is a critical point because a hospital’s case-mix is highly variable, and discrimination based on mortality is mostly related to out-comes of more complex procedures. In other words, if hospital A has excellent survival for less complex procedures and therefore performs very few highly complex procedures (i.e., choosing a case-mix consistent with its expertise), the application of an extrapolated mortality rate may not reflect the actual quality of care for that particular hospital. This issue is evident because the majority of

1	with its expertise), the application of an extrapolated mortality rate may not reflect the actual quality of care for that particular hospital. This issue is evident because the majority of experienced centers with arguably the highest complexity received a “middle star” rating of 2. This rating may reflect calibration issues with the current rating system, whereby centers are potentially penalized for high-complexity predominance.Fortunately, there are efforts to correct these deficien-cies. In 2016, the STS CHSD Task Force and STS Quality Measurement Task Force began to collaborate on an initiative to refine risk adjustment for chromosomal abnormalities, syn-dromes, and noncardiac congenital anatomic abnormalities and to then enhance the STS CHSD Mortality Risk Model with this additional information. Upon completion of this project, STS CHSD Task Force plans to collaborate with the STS Quality Measurement Task Force to study the relationship between vol-ume (programmatic volume and

1	information. Upon completion of this project, STS CHSD Task Force plans to collaborate with the STS Quality Measurement Task Force to study the relationship between vol-ume (programmatic volume and surgeon volume) and outcome using this enhanced STS CHSD Mortality Risk Model.192 Also, currently under development is a multidomain quality metric that incorporates mortality, morbidity, postoperative length of stay, and the occurrence of complications. As the largest con-genital and pediatric cardiac surgical clinical data registry in the world, containing data about nearly all pediatric cardiac operations performed in the United States, STS CHSD contains a truly representative sample of national aggregate data that is useful for multiple purposes.192Future DirectionsThe future of congenital heart surgery remains very bright and exciting. The development of novel technologies such as four-dimensional MRI flow studies (Fig. 20-71) and three-dimen-sional printing have offered this field

1	heart surgery remains very bright and exciting. The development of novel technologies such as four-dimensional MRI flow studies (Fig. 20-71) and three-dimen-sional printing have offered this field several new tools to help understand complex anatomy and pathophysiology. Three-dimensional printing of complex congenital heart defects has helped surgeons in preoperative planning by allowing transla-tion of two-dimensional cross-sectional imaging studies into a tangible and easily visualized model.193 The hollow nature of the human heart and the direct correlation of structure to disease in the congenital population allows this technology to be used in abundance in this field. Its utilization to train young surgeons is very appealing (Figs. 20-72 and 20-73).194,196 Current research in the field of genetics, device bioengineering and miniaturization, stem cell therapy, and fusion imaging technology is expected to further improve patient outcome.195,198 The improved outcomes and survival of

1	of genetics, device bioengineering and miniaturization, stem cell therapy, and fusion imaging technology is expected to further improve patient outcome.195,198 The improved outcomes and survival of these young and fragile patients with congeni-tal heart disease has led to the development of a complex new field termed adult congenital heart disease. The field of con-genital heart surgery is young and offers brilliant, motivated, and upcoming surgeons a very daunting challenge to better the future of these babies.Figure 20-71. 4D MRI flow study obtained in a complex single ventricle patient for the evaluation of persistent hypoxia.Brunicardi_Ch20_p0751-p0800.indd 79322/02/19 2:57 PM 794SPECIFIC CONSIDERATIONSPART IIFigure 20-72. 3D printed models of complex heart defects which were very helpful for preoperative surgical planning and patient education.ADEFBCMAPCAPulmonaryArteryAortaFigure 20-73. Example of Pre-Interventional Planning Using 3D Printed Models. Transthoracic

1	were very helpful for preoperative surgical planning and patient education.ADEFBCMAPCAPulmonaryArteryAortaFigure 20-73. Example of Pre-Interventional Planning Using 3D Printed Models. Transthoracic echocardiogram (A) confirms tetralogy of Fallot/pulmonary atresia/multiple aortopulmonary collateral arteries (MAPCAs) diagnosis. Three-dimensional (3D) reconstruction (B and C) illustrates spatial relationship of patient-specific geometry such as true pulmonary arteries (blue), aorta (red), and MAPCAs (green and yellow) for central aortopulmonary shunt placement and coil planning. Three-dimensional printing (D) provides absolute scaling for planning purposes, as well as patient/family education. Angiography (E and F) captured after central shunt and prior to placement of MAPCA embolization coils. (Reproduced with permission from Ryan JR, Moe TG, Richardson R, et al: A novel approach to neonatal management of tetralogy of Fallot, with pulmonary atresia, and multiple aortopulmonary

1	coils. (Reproduced with permission from Ryan JR, Moe TG, Richardson R, et al: A novel approach to neonatal management of tetralogy of Fallot, with pulmonary atresia, and multiple aortopulmonary collaterals, JACC Cardiovasc Imaging. 2015 Jan;8(1):103-104.)Brunicardi_Ch20_p0751-p0800.indd 79422/02/19 2:57 PM 795CONGENITAL HEART DISEASECHAPTER 20REFERENCESEntries highlighted in bright blue are key references. 1. American Heart Association. About congenital heart defects. Available at: http://www.heart.org/HEARTORG/Conditions/CongenitalHeartDefects/Congenital-Heart-Defects_UCM_001090_SubHomePage.jsp. Accessed May 18, 2018. 2. Congenital Heart Public Health Consortium. FAQ fact sheet. Available at: https://www.aap.org/en-us/Documents/chphc/chd_fact_sheet_long.pdf. Accessed May 18, 2018. 3. Society for Thoracic Surgeons. Congenital heart surgery pub-lic reporting. Available at: https://www.sts.org/congenital-public-reporting-module-search. Accessed May 18, 2018. 4. Kouchoukos NT,

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1	JACC Cardiovasc Imaging. 2015;8(1):103-104. 197. Ferro G, Murthy R, Sebastian VA, Guleserian KJ, Forbess JM. Single-center experience with the Senning Proce-dure in the Current Era. Semin Thorac Cardiovasc Surg. 2016;28(2):514-520. 198. Zuluaga MA, Burgos N, Mendelson AF, Taylor AM, Ourselin S. Voxelwise atlas rating for computer assisted diagnosis: Application to congenital heart diseases of the great arteries. Med Image Anal. 2015;26(1):185-194.Brunicardi_Ch20_p0751-p0800.indd 80022/02/19 2:57 PM

1	Acquired Heart DiseaseMatthew R. Schill, Ali J. Khiabani, Puja Kachroo, and Ralph J. Damiano Jr 21chapterCardiac Assessment 801Clinical Evaluation / 801History / 801Physical Examination / 803Cardiac Risk Assessment in Noncardiac Surgery Patients / 804Diagnostic Studies / 804Extracorporeal Perfusion 807History / 807Technique / 807Adverse Effects / 808Myocardial Protection / 808Coronary Artery Disease 808History / 808Etiology and Pathogenesis / 809Risk Factors and Prevention / 809Clinical Manifestations / 809Preoperative Evaluation / 809Coronary Artery Bypass Grafting 809Indications / 809Percutaneous Coronary Intervention vs. Coronary Artery Bypass Grafting / 810Summary / 811Operative Techniques and Results / 811New Developments / 814Valvular Heart Disease 814General Principles / 814Surgical Options / 815Mitral Valve Disease 817Mitral Stenosis / 817Mitral Regurgitation / 819Mitral Valve Operative Techniques and Results / 821Aortic Valve Disease 823Aortic Stenosis / 823Aortic

1	814Surgical Options / 815Mitral Valve Disease 817Mitral Stenosis / 817Mitral Regurgitation / 819Mitral Valve Operative Techniques and Results / 821Aortic Valve Disease 823Aortic Stenosis / 823Aortic Insufficiency / 825Aortic Valve Operative Techniques and Results / 828Tricuspid Valve Disease 829Tricuspid Stenosis and Insufficiency / 829Multivalve Disease / 831Surgical Therapy for the Failing Heart 831Epidemiology of Heart Failure / 831Etiology and Pathophysiology / 831CABG for Ischemic Cardiomyopathy / 831Secondary Mitral Regurgitation / 832Left Ventricular Aneurysmorrhaphy and Surgical Ventricular Restoration / 833Mechanical Circulatory Support / 835Right Ventricular Assist Devices and Biventricular Assist Devices / 837Total Artificial Heart / 837Surgery For Arrhythmias 837Atrial Fibrillation / 838Surgery for Pericardial Disease 839Acute Pericarditis / 839Relapsing Pericarditis / 840Chronic Constrictive Pericarditis / 840Cardiac Neoplasms 841Overview and General Clinical

1	Fibrillation / 838Surgery for Pericardial Disease 839Acute Pericarditis / 839Relapsing Pericarditis / 840Chronic Constrictive Pericarditis / 840Cardiac Neoplasms 841Overview and General Clinical Features / 841Myxoma / 842Other Benign Cardiac Tumors / 843Malignant Cardiac Tumors / 843Metastatic Cardiac Tumors / 843CARDIAC ASSESSMENTClinical EvaluationAs with any other field in medicine, the history and physical examination form the foundation for the evaluation of a patient with acquired heart disease requiring surgical intervention. Obtaining a complete history identifies comorbid conditions and assists in delineating the operative risks and prognosis after sur-gery. Physical examination reveals factors that may increase the complexity of surgery, such as previous surgery or the presence of peripheral arterial or cerebrovascular disease. These may influence the operative approach, but they also help guide the choice and sequence of diagnostic studies. A complete assess-ment of the

1	of peripheral arterial or cerebrovascular disease. These may influence the operative approach, but they also help guide the choice and sequence of diagnostic studies. A complete assess-ment of the patient allows the surgeon to make educated deci-sions regarding the optimal treatment strategy for the patient.HistorySymptoms suggestive of heart disease include: chest discomfort, fatigue, edema, dyspnea, palpitations, and syncope. Adequate definition of these symptoms calls for detailed history-taking, paying particular attention to onset, intensity, radiation, dura-tion, and exacerbating or alleviating factors. The demands on the heart are determined by its loading conditions and the metabolic state of the patient. Cardiac symptoms are commonly accentuated with physical exertion or postural changes.Angina pectoris is the hallmark of coronary artery disease (CAD), but may occur with other cardiac pathologies that result in ischemia from a mismatch between the supply of oxygen by the

1	changes.Angina pectoris is the hallmark of coronary artery disease (CAD), but may occur with other cardiac pathologies that result in ischemia from a mismatch between the supply of oxygen by the coronary circulation and the metabolic demand of the myo-cardium. Typically, angina is described as tightness, heaviness, or dull pain, frequently substernal, that lasts for a few minutes. This discomfort may radiate to the left arm, neck, mandible, or epigastrium. Angina is most often provoked by activities that increase metabolic demand on the heart such as exercise, eating, and states of intense emotion, and it is typically alleviated by rest or use of nitroglycerin. It is important to note that a signifi-cant number of patients with myocardial ischemia, particularly diabetics, females, and the elderly, may have “silent” angina or angina equivalents (dyspnea, diaphoresis, nausea, or fatigue). The overlap of these features with those of noncardiac etiologies Brunicardi_Ch21_p0801-p0852.indd

1	elderly, may have “silent” angina or angina equivalents (dyspnea, diaphoresis, nausea, or fatigue). The overlap of these features with those of noncardiac etiologies Brunicardi_Ch21_p0801-p0852.indd 80101/03/19 5:32 PM 802such as costochondritis, biliary colic, gastroesophageal reflux disease, diffuse esophageal spasm, and peptic ulcer disease, to name a few, can sometimes lead to misdiagnosis.Heart failure can occur from either left and/or right heart dysfunction, and respective symptoms arise from congestion of blood flow owing to an inadequate cardiac output. Left heart failure manifests as dyspnea, usually with exertion. Orthopnea, defined as dyspnea while lying flat, suggests worsened pulmo-nary congestion with increased venous return. Ascites, periph-eral edema, and hepatomegaly reflect congestion in the systemic venous circulation and are prominent features of right heart failure. Peripheral edema can occur in right heart failure second-ary to systemic venous congestion or

1	reflect congestion in the systemic venous circulation and are prominent features of right heart failure. Peripheral edema can occur in right heart failure second-ary to systemic venous congestion or in left heart failure due to salt and fluid retention as a result of impaired renal perfusion. Patients with chronic suboptimal perfusion and oxygenation can also have digital clubbing and cyanosis.It is difficult to implicate cardiac disease based solely on the presence of fatigue, which is a very nonspecific symptom. However, most cardiac pathologies do result in fatigue or exer-cise intolerance to some degree. It is important to differenti-ate fatigue from exertional dyspnea which some patients may describe as “fatigue.”Dyspnea is another common symptom. Although gen-erally a late symptom in patients with valvular heart disease or cardiomyopathy, it may be a relatively early complaint in some patients, particularly those with mitral stenosis. As stated previously, dyspnea is also an

1	in patients with valvular heart disease or cardiomyopathy, it may be a relatively early complaint in some patients, particularly those with mitral stenosis. As stated previously, dyspnea is also an anginal equivalent and may sig-nal a myocardial ischemic episode. Many primary pulmonary disorders feature dyspnea as their cardinal symptom and should be evaluated simultaneously as the physiology of the heart and lungs are intimately related and can have dramatic influences on one another.Patients typically describe palpitations as a “skipped beat” or “racing heart.” Depending on the clinical context, such as occasional premature atrial or ventricular beats in otherwise healthy individuals, these may be benign. Clinically significant arrhythmias, however, require thorough investigation. Atrial fibrillation is the most common arrhythmia and can occur alone or with other cardiac pathologies. It results in an irregular, and at times, rapid heartbeat. Concurrent symptoms such as angina,

1	Atrial fibrillation is the most common arrhythmia and can occur alone or with other cardiac pathologies. It results in an irregular, and at times, rapid heartbeat. Concurrent symptoms such as angina, lightheadedness, or syncope are particularly worrisome for life-threatening arrhythmias such as ventricular tachycardia or ventricular fibrillation, particularly in patients with preexisting heart failure.Syncope associated with heart disease results from an abrupt reduction in cerebral perfusion. Many of the potential cardiac etiologies are serious, including sinus node dysfunction, atrioventricular conduction abnormalities, malignant arrhyth-mias, aortic stenosis, and hypertrophic obstructive cardiomy-opathy. Noncardiac causes of syncope includes, but are not limited to, neurologic causes (e.g., transient ischemic attacks [TIAs]), orthostatic hypotension, vasavagal events, and carotid sinus hypersensitivity. Any episode of syncope warrants a thor-ough evaluation and search for the root

1	transient ischemic attacks [TIAs]), orthostatic hypotension, vasavagal events, and carotid sinus hypersensitivity. Any episode of syncope warrants a thor-ough evaluation and search for the root cause.1,2 In addition to a thorough inquiry regarding the aforementioned symptoms, it is important to obtain details about the patient’s medical and Key Points1 Although advances have been made in percutaneous coro-nary intervention techniques for coronary artery disease, survival is superior with coronary artery bypass grafting in patients with left main disease, multivessel disease, and in diabetic patients.2 Despite the theoretical advantages, the superiority of off-pump coronary artery bypass to conventional coronary artery bypass grafting has not been clearly established, and other factors likely dominate the overall outcome for either technique.3 Although mechanical valves offer enhanced durability over tissue valve prosthesis, they require permanent systemic anticoagulation therapy to

1	dominate the overall outcome for either technique.3 Although mechanical valves offer enhanced durability over tissue valve prosthesis, they require permanent systemic anticoagulation therapy to mitigate the risk of valve throm-bosis and thromboembolic sequelae and thus are associated with an increased risk of hemorrhagic complications.4 Mitral valve repair is recommended over mitral valve replacement in the majority of patients with severe chronic mitral regurgitation. The decision to proceed with mitral valve repair is based on the skill and experience of the sur-geon in performing the repair and on the pathology of mitral valve disease encountered at the time of operation. Trans-catheter mitral valve therapies are also becoming increas-ingly available as options to offer patients who are deemed high risk for surgical intervention for their mitral valve disease.5 Although open aortic valve replacement has traditionally been the only effective treatment in patients with severe

1	who are deemed high risk for surgical intervention for their mitral valve disease.5 Although open aortic valve replacement has traditionally been the only effective treatment in patients with severe cal-cific aortic stenosis, transcatheter aortic valve replacement is a developing technology that has proven beneficial for the treatment of aortic stenosis in seriously ill patients that had previously been deemed high risk or inoperable. It has also recently been approved for us in patients at moderate risk for surgical aortic valve replacement and is in clinical trials for low-risk patients.6 Mechanical circulatory support with newer generation con-tinuous flow left ventricular assist devices has proven to be durable and effective both as a bridge to transplant and as a means of destination therapy for patients who are not trans-plant candidates.7 Performing a biatrial Cox-Maze lesion set results in freedom from atrial fibrillation in approximately 90% of patients and is superior to

1	therapy for patients who are not trans-plant candidates.7 Performing a biatrial Cox-Maze lesion set results in freedom from atrial fibrillation in approximately 90% of patients and is superior to both catheter-ablation and more limited lesion sets for patients with persistent atrial fibrillation or enlarged left atria. Surgical ablation of atrial fibrillation is recom-mended for patients referred with concomitant valvular dis-ease and those who have previously failed or are poor candidates for catheter-based approaches.8 The preferred treatment for pericarditis depends on the underlying cause, although the disease typically follows a self-limited course and is best managed medically. Surgical pericardiectomy may have a role in treating relapsing peri-carditis and, more commonly, chronic constrictive pericarditis.9 Myxomas are the most common cardiac tumors, and, while benign, they should be promptly excised after diagnosis due to the risk of embolization, obstructive complications,

1	constrictive pericarditis.9 Myxomas are the most common cardiac tumors, and, while benign, they should be promptly excised after diagnosis due to the risk of embolization, obstructive complications, and arrhythmias.Brunicardi_Ch21_p0801-p0852.indd 80201/03/19 5:32 PM 803ACQUIRED HEART DISEASECHAPTER 21Table 21-1New York Heart Association (NYHA) functional classificationCLASSDESCRIPTIONIPhysical activity not limited by symptoms: fatigue, palpitations, or dyspnea.IIComfortable at rest. Slight limitation of physical activity. Fatigue, palpitations, or dyspnea with ordinary physical activity.IIIComfortable at rest. Marked limitation of physical activity. Fatigue, palpitations, or dyspnea with less than ordinary physical activity.IVInability to carry out any physical activity. Symptoms may be present at rest and increase with activity.Table 21-2Canadian Cardiovascular Society (CCS) angina classificationCLASSDESCRIPTIONIOrdinary physical activity (walking, climbing stairs) does not

1	may be present at rest and increase with activity.Table 21-2Canadian Cardiovascular Society (CCS) angina classificationCLASSDESCRIPTIONIOrdinary physical activity (walking, climbing stairs) does not cause angina. Angina occurs with strenuous, rapid, or prolonged exertion during work or recreation.IISlight limitation of ordinary activity. Angina occurs with climbing stairs rapidly, walking uphill in the wind, under emotional stress, in the cold, or after meals. Walking more than 2 blocks or climbing one flight of stairs causes angina.IIIMarked limitation of ordinary physical activity (climbing a flight of stairs or walking 1 to 2 blocks at a normal pace).IVInability to carry out any physical activity without discomfort. Angina may be present at rest.surgical history, family history, social habits (including alcohol and tobacco use), current medications, focused review of sys-tems, as well as an assessment of the patient’s functional status and frailty. Frailty is often defined as a

1	habits (including alcohol and tobacco use), current medications, focused review of sys-tems, as well as an assessment of the patient’s functional status and frailty. Frailty is often defined as a state of increased vul-nerability to adverse health outcomes. Clinicians can use frailty index calculators to assess a patient’s risk for adverse outcomes following cardiac intervention. Specific attention should also be directed to the patient’s comorbidities which not only sheds light on their general health but also helps delineate expected risks from surgery. A strong family history of coronary artery disease, myocardial infarction, hypertension, or diabetes is of particular importance as they increase the individual’s risk for having an adverse cardiac event.Functional Disability and Angina. With regard to heart failure, functional capacity is strongly correlated with mortality. The New York Heart Association (NYHA) functional class is a widely used classification system in categorizing

1	regard to heart failure, functional capacity is strongly correlated with mortality. The New York Heart Association (NYHA) functional class is a widely used classification system in categorizing patients based on their functional status (Table 21-1).3 The NYHA clas-sification has become one basis by which to compare patient populations in many studies. Although less commonly used, the Canadian Cardiovascular Society (CCS) angina classification is also used to incorporate anginal symptoms into the functional assessment for prognostic value (Table 21-2).Physical ExaminationThe physical examination is an invaluable tool in directing further diagnostic studies in the management of a patient with suspected heart disease. The astute clinician may be able to detect subtle signs that may further characterize the underlying pathology.The general appearance of a patient is important in the clinical assessment. A pale, diaphoretic, and obviously uncom-fortable patient is more likely to be in a

1	characterize the underlying pathology.The general appearance of a patient is important in the clinical assessment. A pale, diaphoretic, and obviously uncom-fortable patient is more likely to be in a clinically critical condition than one who is conversing comfortably with an unre-markable demeanor. In addition to basic vital signs, particular attention should be directed to the patient’s mental status as well as the color and temperature of the skin, as these may be reflec-tive of the general adequacy of perfusion. Overall frailty and dementia have also been shown to be predictors of operative and late mortality.4Palpation of the precordium may demonstrate devia-tion in the point of maximal impulse, indicative of ventricular hypertrophy, or parasternal heaves, seen in right ventricular overload. Auscultation should be performed in a quiet envi-ronment as critical murmurs, rubs, or gallops may be subtle. Murmurs are characterized by their location, timing, quality, and radiation. They

1	Auscultation should be performed in a quiet envi-ronment as critical murmurs, rubs, or gallops may be subtle. Murmurs are characterized by their location, timing, quality, and radiation. They are typically secondary to valvular or other structural pathology, and new findings require further investi-gation. A rub due to pericardial friction is indicative of pericar-ditis. A third heart sound (S3) is generated by the rapid filling of a stiff ventricle and can be normal in young patients, but when present in older adults, is indicative of diastolic dysfunction and is pathologic. Increased contribution of the atrial pump function to ventricular filling may manifest as a fourth heart sound (S4) and is also suggestive of ventricular dysfunction.Palpation of peripheral pulses is important not only to assess the adequacy of perfusion, but also the burden of coro-nary artery disease often correlates with the degree of peripheral arterial disease. The presence of a carotid bruit may indicate

1	only to assess the adequacy of perfusion, but also the burden of coro-nary artery disease often correlates with the degree of peripheral arterial disease. The presence of a carotid bruit may indicate clinically significant stenosis and may alter the course and tim-ing of treatment, especially if symptomatic.Heart failure will frequently have extracardiac manifesta-tions and examination of the other organ systems should not be neglected. For example, auscultation of the lung fields may reveal rales in patients with pulmonary edema. The use of acces-sory muscles of breathing may be present in patients with sig-nificant pleural effusions and volume overload. The presence of jugular venous distention and hepatosplenomegaly may signal right heart failure.Additionally, clinicians should know the manifestation of other cardiac pathologies including endocarditis and rheu-matic heart disease, although less commonly seen. Endocarditis is an inflammation of the endocardium, usually on the heart

1	manifestation of other cardiac pathologies including endocarditis and rheu-matic heart disease, although less commonly seen. Endocarditis is an inflammation of the endocardium, usually on the heart valves. A cardiac murmur is a common physical exam finding. Relatively uncommon but more specific clinical manifestations for infectious endocarditis are: Janeway lesions, which are nontender erythematous macules on the palms and soles; Osler nodes, which are described as tender subctanous nodules mostly on the pads of the fingers and toes; and Roth spots, which are exudative hemorrhagic lesions of the retina with pale centers.Brunicardi_Ch21_p0801-p0852.indd 80301/03/19 5:32 PM 804SPECIFIC CONSIDERATIONSPART IICardiac Risk Assessment in Noncardiac Surgery PatientsCardiovascular complications occur in approximately 3% of patients undergoing inpatient noncardiac surgery.5 The American College of Cardiology (ACC) and American Heart Association (AHA) have formed a joint task force to

1	occur in approximately 3% of patients undergoing inpatient noncardiac surgery.5 The American College of Cardiology (ACC) and American Heart Association (AHA) have formed a joint task force to publish a consensus statement, with periodic focused updates, on guide-lines and recommendations that were revised in 2017.6 The aim of these guidelines is to incorporate surgery-specific risks and patient characteristics to guide perioperative decision-making in the management of patient with valvular heart disease.Surgical procedures have been categorized based on cardiovascular risk into low risk, moderate risk, and vascu-lar procedures. Vascular procedures, likely due to both the nature of the procedures themselves as well as the associated cardiovascular pathology in many of these patients, carry the highest reported risk of cardiac events at more than 7%.5 Low risk procedures, including endoscopic procedures, superficial operations, cataract surgery, breast surgery, and ambulatory

1	carry the highest reported risk of cardiac events at more than 7%.5 Low risk procedures, including endoscopic procedures, superficial operations, cataract surgery, breast surgery, and ambulatory surgeries, have a risk generally less than 1%. Intermediate risk procedures include intraperitoneal and intrathoracic surgery, head and neck surgery, orthopedic procedures, and prostate surgery.5Patient characteristics can be classified by the status of the patient’s cardiac disease, comorbid conditions, and functional capacity. Patients are considered to be at major perioperative clinical risk if they have one or more of the following active car-diac conditions: acute coronary syndrome, decompensated heart failure, significant arrhythmias, or severe valvular heart disease. In these patients, intensive evaluation and treatment prior to sur-gery (unless emergent) is warranted, prior to proceeding with the noncardiac surgery.If the patient does not have any of the previously men-tioned active

1	evaluation and treatment prior to sur-gery (unless emergent) is warranted, prior to proceeding with the noncardiac surgery.If the patient does not have any of the previously men-tioned active cardiac conditions, the perioperative risk of major adverse cardiac events (MACE) should be estimated. Both the operation performed and the patient’s risk factors are predictive of MACE, and the ACC/AHA guidelines recommend the use of either the American College of Surgeons’ NSQIP risk calculator or the Revised Cardiac Risk Index for the estimation of patient-specific risk. Patients at low (<1%) risk or patients at elevated risk with functional capacity greater than or equal to 4 metabolic equivalents (METs), should proceed to surgery without further testing. It is reasonable to perform pharmacologic stress testing in patients with poor or unknown functional capacity if this test-ing will impact decision making or perioperative care. Patients with abnormal stress test results should undergo

1	stress testing in patients with poor or unknown functional capacity if this test-ing will impact decision making or perioperative care. Patients with abnormal stress test results should undergo confirmatory test such as coronary angiography, if indicated, before an elec-tive noncardiac surgery. The previous guidelines included inter-mediate and low cardiovascular risk profiles, but this has been replaced by cardiovascular risk factors in the update. These risk factors are: history of ischemic heart disease, history of prior or compensated heart failure, history of cerebrovascular dis-ease, diabetes mellitus, and renal insufficiency. Based on the number of present risk factors and the surgery-specific risk, the guidelines recommend pathways for further evaluation and risk management. The most recent guidelines from ACC/AHA were published in 2014 (Fig. 21-1).7 One important subgroup of patients at elevated risk are those who have recently undergone percutaneous coronary intervention. In

1	recent guidelines from ACC/AHA were published in 2014 (Fig. 21-1).7 One important subgroup of patients at elevated risk are those who have recently undergone percutaneous coronary intervention. In these patients, elective noncardiac surgery should be delayed until the risk of stent thrombosis decreases (30 days for bare metal stents and 180 to 365 days for drug-eluting stents), and dual antiplatelet therapy should be continued unless the risk of bleeding exceeds the risk of stent thrombosis.5Diagnostic StudiesElectrocardiogram and Chest X-ray. Electrocardiograms (ECGs) and chest X-rays are noninvasive diagnostic studies that provide invaluable information in the preoperative assess-ment of patients with cardiac pathology. ECGs can be useful in detecting old myocardial infarction, dilation or hypertrophy of the cardiac chambers, arrhythmias, and conduction abnormali-ties. A stress ECG requires a patient to exercise to a target heart rate and is used to help diagnose ischemic

1	dilation or hypertrophy of the cardiac chambers, arrhythmias, and conduction abnormali-ties. A stress ECG requires a patient to exercise to a target heart rate and is used to help diagnose ischemic pathologies that may not be evident at rest.A plain film of the chest can detect pulmonary pathol-ogy, sequelae of heart failure (e.g., pulmonary edema, cardiac enlargement, pleural effusions), as well as presence of hardware (e.g., prosthetic heart valves, sternal wires, pacemakers, and defibrillators).Echocardiography. Echocardiography utilizes reflected sound waves to image the heart. Transthoracic echocardiography (TTE) is used widely due to its noninvasive nature. It is the pri-mary diagnostic tool used to evaluate structural diseases of the heart, including valvular pathology, septal defects, cardiomyop-athies, and cardiac masses. Although more invasive, transesoph-ageal echocardiography (TEE) can provide more information and better definition of some valvular and structural

1	defects, cardiomyop-athies, and cardiac masses. Although more invasive, transesoph-ageal echocardiography (TEE) can provide more information and better definition of some valvular and structural abnormali-ties. It is particularly useful in identifying left atrial thrombi in patients with atrial fibrillation. Echocardiography is indispens-able in assessing surgical prostheses such as valves, leads, or mechanical circulatory support devices. These examinations can be performed with M-mode imaging (motion along a single line) as well as two-dimensional (2D) and three-dimensional (3D) imaging depending on the information required.Doppler technology has become a standard addition to assess changes in flow patterns across dysfunctional valves. Velocity measurements can be obtained to estimate pres-sure gradients across structures using the continuity equation. A common example would be the estimation of pulmonary arterial systolic pressure calculated from the regurgitant tricuspid jet

1	pres-sure gradients across structures using the continuity equation. A common example would be the estimation of pulmonary arterial systolic pressure calculated from the regurgitant tricuspid jet profile during right ventricular systole.Transthoracic echocardiography requires no sedation and is generally performed with the patient in a slight left lateral decubitus position. Standardized views are obtained with the ultrasound probe placed in the apical, parasternal, subcostal, and suprasternal positions. The apical four-chamber view is a use-ful window for visualizing all four cardiac chambers simultane-ously as well as the tricuspid and mitral valves. Other windows can be obtained to assess specific structures such as the individ-ual valve anatomy or myocardial wall segments. Dobutamine-stress echocardiography is a study similar in idea to the stress ECG that utilizes a pharmacologic agent to assess the patient for ischemia or stress-induced valvular abnormalities.Transesophageal

1	echocardiography is a study similar in idea to the stress ECG that utilizes a pharmacologic agent to assess the patient for ischemia or stress-induced valvular abnormalities.Transesophageal echocardiography, on the other hand, is performed using a special endoscope with an ultrasound probe mounted on its end that is introduced orally into the esophagus under sedation. Posterior structures such as the mitral valve and left atrium are particularly well visualized. TEEs are frequently used intraoperatively during cardiothoracic surgery to assess global cardiac function, integrity of valve repairs and replace-ments, intracavitary thrombus and/or air, and aortic athero-sclerosis or dissections that can have significant influences on operative strategy.Brunicardi_Ch21_p0801-p0852.indd 80401/03/19 5:32 PM 805ACQUIRED HEART DISEASECHAPTER 21Figure 21-1. Stepwise approach to perioperative cardiac assessment for coronary artery disease. (Reproduced with permission from Fleisher LA,

1	80401/03/19 5:32 PM 805ACQUIRED HEART DISEASECHAPTER 21Figure 21-1. Stepwise approach to perioperative cardiac assessment for coronary artery disease. (Reproduced with permission from Fleisher LA, Fleischmann KE, Auerbach AD, et al: 2014 ACC/AHA guideline on perioperative cardiovascular evaluation and management of patients undergoing noncardiac surgery: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines, Circulation. 2014 Dec 9;130(24):2215-2245.)EmergencyACS†(Step 2)YesYesNoNoModerate or greater(˜4 METs) functionalcapacityPoor OR unknownfunctional capacity(<4 METs):Will further testing impactdecision making ORperioperative care?(Step 6)No orunknownPatient scheduled for surgery withknown or risk factors for CAD*(Step 1)Clinical risk stratiÿcationand proceed to surgeryEvaluate and treataccording to GDMT†Estimated perioperative risk of MACEbased on combined clinical/surgical risk(Step 3)Low risk (<1%)(Step

1	1)Clinical risk stratiÿcationand proceed to surgeryEvaluate and treataccording to GDMT†Estimated perioperative risk of MACEbased on combined clinical/surgical risk(Step 3)Low risk (<1%)(Step 4)Proceed tosurgeryElevated risk(Step 5)No furthertesting(Class III:NB)Proceed to surgeryaccording to GDMT ORalternate strategies(noninvasive treatment,palliation)(Step 7)No furthertesting(Class lIa)No furthertesting(Class IIb)Pharmacologicstress testing(Class lIa)Coronaryrevascularizationaccording toexisting CPGs(Class I)Proceed tosurgeryYesIfnormalIfabnormalNoExcellent(>10 METs)Moderate/Good(˜4–10 METs)Brunicardi_Ch21_p0801-p0852.indd 80501/03/19 5:32 PM 806SPECIFIC CONSIDERATIONSPART IIThere are some more recent additions to the echocardio-graphic armamentarium that capitalize on the strengths of ultra-sound imaging. Three-dimensional TEE is playing an increasing role in the preoperative and intraoperative evaluation of patients with valvular heart disease and is especially helpful in

1	of ultra-sound imaging. Three-dimensional TEE is playing an increasing role in the preoperative and intraoperative evaluation of patients with valvular heart disease and is especially helpful in percuta-neous mitral intervention. Tissue Doppler imaging is based on principles akin to conventional Doppler echocardiography, but attention is directed to the myocardium itself as opposed to the motion of blood to quantify abnormalities in wall motion. Strain imaging with speckle-tracking echocardiography measures the actual deformation of the myocardium by following inhomoge-neities inherent to the myocardium and is a useful measure of myocardial function.Radionuclide Studies. Although ECGs are useful, inexpen-sive, and safe, baseline abnormalities in the ECG may limit its diagnostic capacity. In particular, ventricular rhythms, bundle-branch blocks, left ventricular hypertrophy, drug effects, and baseline ST-segment depressions can make stress ECGs dif-ficult to interpret for the presence

1	In particular, ventricular rhythms, bundle-branch blocks, left ventricular hypertrophy, drug effects, and baseline ST-segment depressions can make stress ECGs dif-ficult to interpret for the presence of myocardial ischemia. In this setting, myocardial perfusion imaging (MPI) using radionu-clides can be utilized. Thallium 201 (201Tl) was the initial radio-nuclide used for MPI, but due to its long half-life and relatively low photopeak, it has largely been replaced by technetium-99m (sestamibi and tetrofosmin) because of its more favorable char-acteristics. In the past, planar imaging with three separate 2D views of the heart were obtained. Currently, it is more common to have the images acquired by single-photon emission com-puted tomography (SPECT) technology, which detects emit-ted photons from 180° to 360° around the patient. The signals are then processed to reconstruct multiple slices that together provide a 3D image. The distribution of radionuclides depends on perfusion, and

1	from 180° to 360° around the patient. The signals are then processed to reconstruct multiple slices that together provide a 3D image. The distribution of radionuclides depends on perfusion, and therefore areas that show uptake at rest, but not during stress, are concerning for ischemia. The amount of uptake at both rest and stressed states is compared to assess ischemia and viability of the myocardium. Territories that do not show uptake at rest or during stress are likely to be nonvi-able scar. The sensitivity and specificity of exercise SPECT are 87% and 73%, respectively.8,9The image acquisition may also be gated to a simultane-ously obtained ECG to assess global ventricular function. The endocardial and epicardial borders (as delineated by radionu-clide uptake) are detected throughout the cardiac cycle and the ejection fraction, along with end-systolic and end-diastolic vol-umes, can be calculated. This study is also useful in revealing hypokinetic segments of the myocardium.One

1	the cardiac cycle and the ejection fraction, along with end-systolic and end-diastolic vol-umes, can be calculated. This study is also useful in revealing hypokinetic segments of the myocardium.One significant drawback of SPECT imaging is that it shows regional ischemia well, but it does not adequately detect global or “balanced” ischemia that can occur with diffuse CAD. Positron emission tomography (PET) has been used due to its ability to obtain absolute quantitative data on both myocardial perfusion and metabolism. Tracers used in PET scans can be divided into those that assess perfusion (oxygen-15, nitrogen-13, and rubidiuim-82) and those that assess metabolism (carbon-11 and fluorine-18). The specificity of PET in detecting CAD is better than SPECT at 86% due to its superior spatial resolution.10Magnetic Resonance Imaging. Magnetic resonance imaging (MRI) has a wide variety of uses in cardiac imaging depending on the pulse sequence and signal weighting. Cine-loop imaging of the

1	resolution.10Magnetic Resonance Imaging. Magnetic resonance imaging (MRI) has a wide variety of uses in cardiac imaging depending on the pulse sequence and signal weighting. Cine-loop imaging of the heart throughout the cardiac cycle can yield information on global chamber function, geometry, and valvular pathologies. The differential response of normal and ischemic myocardium to certain pulse sequences allows imaging of myocardial per-fusion using MRI. Use of contrast agents such as gadolinium can enhance scar tissue and are very useful in viability assess-ment. Myocardial strain imaging can also be performed taking advantage of radio-frequency tagging of the myocardium, which deforms with the tissue and can be followed throughout the car-diac cycle.Cardiac Catheterization. Cardiac catheterization involves access to the cardiac chambers, coronary arteries, and great vessels with a peripherally inserted catheter under fluoroscopic guidance. It is a versatile tool used to investigate

1	involves access to the cardiac chambers, coronary arteries, and great vessels with a peripherally inserted catheter under fluoroscopic guidance. It is a versatile tool used to investigate cardiac cham-ber pressures, valvular abnormalities, wall motion, and coronary artery anatomy. While some of these roles are being replaced by less invasive techniques mentioned previously, cardiac cath-eterization continues to be widely performed and is the gold standard for the assessment of coronary artery disease.11Left heart catheterization is performed by percutaneous access of the femoral, or radial, artery. Under fluoroscopic guid-ance, the catheter is threaded into the ascending aorta where a contrast aortogram may be performed. Coronary angiography requires manipulation of this catheter into the coronary ostia where contrast is directly injected. With advancement of the catheter retrograde through the aortic valve, left ventricular pressures can be obtained. This measurement is used to

1	the coronary ostia where contrast is directly injected. With advancement of the catheter retrograde through the aortic valve, left ventricular pressures can be obtained. This measurement is used to calcu-late direct pressure gradients across the aortic valve, in con-trast to echocardiography that indirectly measures pressure, and can be used to confirm severe aortic stenosis. Again, contrast injection into the left ventricle can be used to estimate ejection fraction and visualize hypokinetic segments of the myocardium. Inappropriate retrograde leakage of contrast may indicate insuf-ficiency of the aortic and/or mitral valves.Coronary angiography provides information on hemody-namically significant stenoses in the coronary circulation as well as an anatomical roadmap for surgeons to plan revascularization (Fig. 21-2A,B). A stenosis is considered to be significant if it narrows the lumen of the artery by 70% (or 50% in the case of left main coronary artery). Borderline lesions or

1	plan revascularization (Fig. 21-2A,B). A stenosis is considered to be significant if it narrows the lumen of the artery by 70% (or 50% in the case of left main coronary artery). Borderline lesions or complex lesions may be assessed by fractional flow reserve (FFR) or instant wave-free radio (iFR), which obviates the need of adenosine.12 This has been shown to very helpful in guiding revasculariza-tion strategies in recent clinical trials.13 Additional assessment can also be done using intravascular ultrasound (IVUS) inside the coronary circulation. There is some variability in the coro-nary arterial anatomy with the posterior descending artery being supplied by the right coronary artery in approximately 80% of patients (right dominant) or the left coronary artery in approxi-mately 15% of patients (left dominant). The remaining patients have a codominant circulation where the posterior descending artery is supplied by both the right and left coronaries.Right heart catheterization is

1	patients (left dominant). The remaining patients have a codominant circulation where the posterior descending artery is supplied by both the right and left coronaries.Right heart catheterization is performed by the introduc-tion of catheter through a peripheral vein that is advanced into the right side of the heart.14 Right-sided pressures and structures are assessed in a similar fashion as in the left heart. Extension of the catheter into the pulmonary artery allows measurement of pulmonary artery pressures as well as pulmonary capillary wedge pressure (reflecting left ventricular end diastolic pres-sure) with an occlusive balloon. In addition to these measure-ments, cardiac output can be measured using thermodilution or by the Fick method using oxygen saturations of blood sampled from the various locations during the procedure.Brunicardi_Ch21_p0801-p0852.indd 80601/03/19 5:32 PM 807ACQUIRED HEART DISEASECHAPTER 21Figure 21-2. Cardiac catheterization angiography. A. Stenosis of

1	the various locations during the procedure.Brunicardi_Ch21_p0801-p0852.indd 80601/03/19 5:32 PM 807ACQUIRED HEART DISEASECHAPTER 21Figure 21-2. Cardiac catheterization angiography. A. Stenosis of right coronary artery indicated by the arrow. B. Still image of a normal left ventriculogram.ABAn advantage of cardiac catheterization is that it offers an opportunity for interventional therapy of coronary artery disease, arrhythmias, valvular abnormalities, and other struc-tural defects of the heart. Cardiac catheterization is generally safe, but being an invasive procedure, it is associated with rare complications. The overall mortality is 0.11%, and total rate of major complications, including MI, stroke, arrhythmia, vascular injury, contrast reaction including allergic reaction and contrast-induced nephropathy, hemodynamic instability, and cardiac per-foration is usually <2%.15Cardiac Computed Tomography. Multislice computed tomography (CT) imaging can be used to assess the coronary

1	nephropathy, hemodynamic instability, and cardiac per-foration is usually <2%.15Cardiac Computed Tomography. Multislice computed tomography (CT) imaging can be used to assess the coronary vasculature. The coronary calcium score is an index devel-oped to quantify the degree of coronary atherosclerotic burden by measuring Hounsfield units in a noncontrast cardiac CT. Although this technique is quite sensitive for angiographic stenoses (>50%) it remains fairly nonspecific as calcification often precedes significant luminal narrowing.16 CT coronary angiography using intravenous contrast is also utilized clini-cally to assess coronary pathology and is particularly useful in the emergency room to perform a “triple rule-out” for acute coronary events, pulmonary embolism, and aortic dissection in patients who present with undifferentiated chest pain. LV ejec-tion fraction may be measured by this technique, and, together with the degree of coronary stenosis, it has been shown to have

1	in patients who present with undifferentiated chest pain. LV ejec-tion fraction may be measured by this technique, and, together with the degree of coronary stenosis, it has been shown to have incremental prognostic value for the presence of coronary artery disease and in the prediction of adverse coronary events.17EXTRACORPOREAL PERFUSIONHistoryPrior to the development of extracorporeal perfusion, heart sur-gery was rarely performed and was limited to brief periods of asystole and/or hypothermia. The need for obtaining a bloodless operating field, while maintaining perfusion of heart and other organs, was evident.John Gibbon’s motivation to develop a means for extra-corporeal perfusion came from a desire to safely open the pulmonary artery in a patient who suffered from a pulmonary embolus following a cholecystectomy. After numerous experi-mental iterations, Gibbon’s cardiopulmonary bypass machine was first used clinically in 1953 to repair an atrial septal defect in an 18-year-old

1	following a cholecystectomy. After numerous experi-mental iterations, Gibbon’s cardiopulmonary bypass machine was first used clinically in 1953 to repair an atrial septal defect in an 18-year-old woman.18 Although Gibbon is credited for its invention, the development of modern cardiopulmonary bypass (CPB) is a culmination of the work of many investi-gators throughout the world. The early bubble oxygenators have evolved into the currently used membrane oxygenators. The search for an ideal biocompatible material that minimizes the inflammatory cascade initiated by the contact of blood with the circuit components continues to this day.TechniqueThe basic CPB circuit consists of the venous cannulae, a venous reservoir, pump, oxygenator, filter, and the arterial cannula.Anticoagulation is required during CPB, and 300 to 400 units/kg of heparin are given to increase the activated clotting time (ACT) to greater than 450 seconds. Once an ade-quate level of anticoagulation is achieved,

1	required during CPB, and 300 to 400 units/kg of heparin are given to increase the activated clotting time (ACT) to greater than 450 seconds. Once an ade-quate level of anticoagulation is achieved, arterial cannulation is performed through a purse-string suture or through a side graft sewn onto the native artery. The distal ascending thoracic aorta is the most common site of cannulation. Other routinely utilized sites of cannulation include the femoral artery, the axillary artery, the innominate artery, or the distal aortic arch, and they are altered based on the indicated surgical repair and the pres-ence of native arterial disease. Venous cannulation is performed through purse-string sutures placed on the right atrium either for a single cannula or for two separate cannulae placed into the superior and inferior vena cava, respectively. Alternatively, the venous cannula may be inserted from the femoral vein and advanced into the right atrium and superior vena cava using TEE guidance.

1	superior and inferior vena cava, respectively. Alternatively, the venous cannula may be inserted from the femoral vein and advanced into the right atrium and superior vena cava using TEE guidance. This technique is frequently used in minimally invasive cardiac surgery.Effective communication between the surgeon, the anes-thesiologist, and the perfusionist is mandatory for effective Brunicardi_Ch21_p0801-p0852.indd 80701/03/19 5:32 PM 808SPECIFIC CONSIDERATIONSPART IIcardiopulmonary bypass. Once the appropriate cannulations and connections are complete, CPB is commenced. Venous return is initiated followed by arterial flow while monitoring systemic blood pressure. At normothermia, the flow required is approxi-mately 2.4 L/min/m2, but with hypothermia, oxygen consump-tion is reduced by 50% for every 10°C drop in temperature, and a flow of only 1 L/min/m2 is required at 18°C. Once the heart is decompressed and hemodynamics are acceptable, ventilation is stopped. The oxygenator is

1	by 50% for every 10°C drop in temperature, and a flow of only 1 L/min/m2 is required at 18°C. Once the heart is decompressed and hemodynamics are acceptable, ventilation is stopped. The oxygenator is adjusted to maintain a Pao2 of 150 mmHg and normocarbia. Blood can also be filtered and returned through vents that are placed in chambers of the heart (such as the left ventricle or pulmonary artery) or through the cardiotomy suction used to aspirate blood from the surgical field. When the cardiac procedure is complete, the patient is rewarmed, the lungs ventilated, and the heart defibrillated, if needed. The venous return to the CPB machine is gradually reduced allowing the heart to fill. The pump is also slowed while hemodynamics and global cardiac function are assessed with a TEE probe. Ino-tropic and vasopressor support may be used to augment cardiac function and treat hypotension. Once CPB has been weaned and stable hemodynamics achieved, the cannulae are removed. The heparin

1	Ino-tropic and vasopressor support may be used to augment cardiac function and treat hypotension. Once CPB has been weaned and stable hemodynamics achieved, the cannulae are removed. The heparin anticoagulation is reversed with 1 mg protamine per 100 units of heparin and hemostasis is achieved.19Adverse EffectsCardiopulmonary bypass has a number of deleterious effects as various intertwining processes result in derangements in hemo-stasis, an enhanced systemic inflammatory response, and end-organ function.Anticoagulation prior to the commencement of CPB is required as contact of blood with the artificial surfaces of the cir-cuit can initiate a thrombogenic cascade. Generation of thrombin plays a major role in both thrombotic and bleeding phenomena during CPB. The endothelium that normally regulates the fine balance between procoagulant and anticoagulant pathways is perturbed. Fibrinogen is consumed rapidly as thrombin converts fibrinogen to fibrin while fibrinolytic mechanisms

1	regulates the fine balance between procoagulant and anticoagulant pathways is perturbed. Fibrinogen is consumed rapidly as thrombin converts fibrinogen to fibrin while fibrinolytic mechanisms (initiated by the activated endothelium) degrade the fibrin macromolecules. Platelets are activated by the converging hemostatic pathways and are consumed.The response of the humoral and cellular immune systems partly overlap with the hemostatic pathways. The classic and alternative complement pathways are activated by CPB gen-erating powerful chemotaxic molecules and anaphylatoxins.20 Monocytes, platelets, and neutrophils are activated releasing acute inflammatory mediators and cytokines that persist even after conclusion of CPB.21 These inflammatory cells also pro-duce reactive oxidants that may have cytotoxic and cardiovas-cular effects such as vasodilation and hypotension.The large quantity of unfractionated heparin used during cardiac surgery predisposes patients to developing

1	that may have cytotoxic and cardiovas-cular effects such as vasodilation and hypotension.The large quantity of unfractionated heparin used during cardiac surgery predisposes patients to developing heparin-induced thrombocytopenia (HIT) with an incidence of 1% to 5%.22 Platelet factor-4 (PF4) is produced by platelets and avidly binds to heparin to form a heparin-PF4 complex that can be antigenic in some patients binding IgG. The IgG-heparin-PF4 complex can bind to platelets, which causes release of more PF4, perpetuating the process. The earliest sign is a sudden drop of more than 50% in the platelet count, usually seen from sev-eral hours to days after surgery. HIT can be confirmed with an enzyme-linked immunosorbent assay (ELISA) or serotonin release assay (SRA). Of the patients with HIT, 20% to 50% of patients develop thromboses in arterial or venous beds, designated as heparin-induced thrombocytopenia and thrombo-sis (HITT), which can be life-threatening.23 Treatment is

1	with HIT, 20% to 50% of patients develop thromboses in arterial or venous beds, designated as heparin-induced thrombocytopenia and thrombo-sis (HITT), which can be life-threatening.23 Treatment is anti-coagulation with nonheparin anticoagulant (e.g., argatroban, bivalirudin).The etiology of end-organ dysfunction resulting from extracorporeal circulation can mostly be categorized into one of three mechanisms: hypoperfusion, embolization, and whole-body inflammatory response. Although cardiac output and blood pressure are monitored carefully during CPB, they are surrogates for regional perfusion and cannot detect end-organ hypoperfusion directly. This can be a problem particularly with the cerebral, renal, and mesenteric circulations. With manip-ulation of diseased vessels and dysregulation of the native coagulation system, macroscopic and microscopic emboli are a concern. Activated cells and circulating cytotoxic products of the immune response may cause microvascular injury and edema

1	of the native coagulation system, macroscopic and microscopic emboli are a concern. Activated cells and circulating cytotoxic products of the immune response may cause microvascular injury and edema of other organs manifesting as neurocognitive deficits, respiratory failure, and renal injury.24Myocardial ProtectionDuring CPB, pharmacologic agents in cardioplegic solutions may be delivered into the coronary circulation to arrest the heart, allowing for a still operating target and improved myocardial protection. The most common cardioplegia consists of potas-sium-rich solutions that can be mixed with autologous blood and are delivered into the coronary circulation.25 Antegrade cardioplegia is delivered into the root of a cross-clamped aorta or directly into the individual coronary ostial using specialized catheters. A retrograde cardioplegia catheter is a balloon-cuffed catheter that is placed through the right atrium into the coro-nary sinus and is used to perfuse the coronary

1	using specialized catheters. A retrograde cardioplegia catheter is a balloon-cuffed catheter that is placed through the right atrium into the coro-nary sinus and is used to perfuse the coronary circulation in the opposite direction through the venous circulation. This has the advantage of more uniform distribution in patients with diffuse coronary artery disease and is not dependent on a competent aortic valve for delivery.There is continued debate regarding the best method (ante-grade vs. retrograde vs. both), type (crystalloid vs. blood), tem-perature (cold vs. warm vs. tepid), and interval (continuous vs. intermittent) of cardioplegia delivery. The optimal combination is beyond the scope of this text. However, most cardiac surgeons in the United States favor cold blood potassium cardioplegia.CORONARY ARTERY DISEASEHistoryAortocoronary bypass for myocardial ischemia was first pro-posed and performed in laboratory animals by Carrel in 1910.26 The Vineberg operation, one of the

1	cardioplegia.CORONARY ARTERY DISEASEHistoryAortocoronary bypass for myocardial ischemia was first pro-posed and performed in laboratory animals by Carrel in 1910.26 The Vineberg operation, one of the initial attempts at surgical revascularization of the myocardium, was introduced in 1951.27 This procedure involved implantation of the internal thoracic artery directly into the myocardium itself. While some patients were relieved of their anginal symptoms, this resulted in vir-tually no increase in coronary flow and was soon supplanted by methods to restore flow directly. Coronary endarterectomy was introduced by Longmire during this time period but had high rates of restenosis and occlusion.28 The use of vein patches to repair the arteriotomy sites was described by Senning in 1961.29 The first saphenous vein coronary artery bypass grafting (CABG) was performed by Sabiston in 1962,30 but was popular-ized by Favalaro and Sones in 1967.31 In 1968, the internal tho-racic artery was

1	The first saphenous vein coronary artery bypass grafting (CABG) was performed by Sabiston in 1962,30 but was popular-ized by Favalaro and Sones in 1967.31 In 1968, the internal tho-racic artery was introduced as a bypass conduit by Green, who used it to bypass the left anterior descending coronary artery.32Brunicardi_Ch21_p0801-p0852.indd 80801/03/19 5:32 PM 809ACQUIRED HEART DISEASECHAPTER 21Etiology and PathogenesisAtherosclerotic stenoses are the primary mechanism of CAD. The pathophysiologic process is initiated with vascular endo-thelial injury and is potentiated by inflammatory mechanisms, circulating lipids, toxins, and other vasoactive agents in the blood. Macrophages and platelets are attracted to this area of endothelial dysfunction inciting a local inflammatory response. During this process, macrophages infiltrate into the intimal lay-ers and accumulate cholesterol-containing low-density lipo-proteins. The growth factors secreted promote proliferation of smooth muscle

1	this process, macrophages infiltrate into the intimal lay-ers and accumulate cholesterol-containing low-density lipo-proteins. The growth factors secreted promote proliferation of smooth muscle cells within the intima and media of the arteries. Together with the accumulation of the lipid-laden macrophages, the smooth muscle hyperplasia results in an atheroma and subse-quent stenosis of the vessel. These atheromas have a fibrous cap that may rupture, exposing the underlying cells and extracellular matrix, which are very prothrombotic. Acute plaque rupture and thrombus formation is thought to be the main pathophysiologic mechanism responsible for acute coronary syndromes.33-35Risk Factors and PreventionPrior to the establishment of modern management strategies, the annual mortality rate from ischemic heart disease was 482 out of 100,000 persons.36 Since the peak of coronary heart dis-ease mortality in 1968, modern primary and secondary preven-tion strategies such as risk factor

1	rate from ischemic heart disease was 482 out of 100,000 persons.36 Since the peak of coronary heart dis-ease mortality in 1968, modern primary and secondary preven-tion strategies such as risk factor modification, percutaneous and surgical revascularization, use of medications (e.g., aspirin, HMG-CoA reductase inhibitors [statins], and β-blockers), has decreased mortality from coronary artery disease by 74%.36The major risk factors for atherosclerosis include advanced age, cigarette smoking, hypertension, dyslipidemias, sedentary lifestyle, obesity, and diabetes. Likely due to increased public awareness and aggressive medical management, these risk fac-tors (with the exception of glucose intolerance and obesity) have recently been on the decline.Current guidelines outlined in the AHA/ACC consensus statement summarize the secondary prevention recommendations.37 Class I recommendations include smoking cessation and avoid-ance of environmental tobacco exposure, blood pressure con-trol to

1	statement summarize the secondary prevention recommendations.37 Class I recommendations include smoking cessation and avoid-ance of environmental tobacco exposure, blood pressure con-trol to under 140/90 mmHg (under 130/80 mmHg in those with diabetes or chronic kidney disease), LDL cholesterol levels less than 100 mg/dL, aspirin therapy in all patients without contra-indications, a BMI target of less than 25 kg/m2, diabetes man-agement with target HbA1c <7%, and encouragement of daily moderate-intensity aerobic exercise. β-Blockers should be used in all patients with LV dysfunction and following MI, ACS, or revascularization, unless a specific contraindication is pres-ent. Renin-angiogensin-aldosterone system blockade in patients with hypertension, LV dysfunction, diabetes, or chronic kidney disease should also be considered.Clinical ManifestationsPatients with CAD may have a spectrum of presentations, including angina pectoris, myocardial infarction, ischemic heart failure,

1	kidney disease should also be considered.Clinical ManifestationsPatients with CAD may have a spectrum of presentations, including angina pectoris, myocardial infarction, ischemic heart failure, arrhythmias, and sudden death.Angina pectoris is the pain or discomfort caused by myo-cardial ischemia and is typically substernal and may radiate to the left upper extremity, neck, or epigastrium. The variety of presentations can make myocardial ischemia challenging to diagnose. Characteristics of chest pain that make myocardial ischemia less likely include pleuritic chest pain, pain reproduc-ible by movement or palpation, or brief episodes lasting only seconds. Typical angina is relieved by rest and/or use of sub-lingual nitroglycerin. Differential diagnoses to be considered include, but are not limited to, musculoskeletal pain, pulmonary disorders, esophageal spasm, pericarditis, aortic dissection, gas-troesophageal reflux, neuropathic pain, and anxiety.Myocardial infarction is a serious

1	limited to, musculoskeletal pain, pulmonary disorders, esophageal spasm, pericarditis, aortic dissection, gas-troesophageal reflux, neuropathic pain, and anxiety.Myocardial infarction is a serious consequence of CAD occurring when ischemia results in myocardial necrosis. This may be silent and need not be preceded by angina. Necrosis may result in disruption of the myocardial integrity leading to devastating conditions such as intracardiac shunts from ventric-ular septal defects, acute valvular regurgitation from rupture of necrotic papillary muscles, and cardiac aneurysms, which have the potential for fatal rupture.Ischemic insults from CAD may lead to congestive heart failure. The initial myocardial damage sets off a cascade of both local and systemic responses. Over time, these changes can cause deleterious myocardial loading and abnormal neuro-humoral responses that result in pathologic remodeling of the heart. Heart failure should be suspected in patients who present with

1	changes can cause deleterious myocardial loading and abnormal neuro-humoral responses that result in pathologic remodeling of the heart. Heart failure should be suspected in patients who present with dyspnea, orthopnea, fatigue, and edema.Arrhythmias may also be sequelae of CAD. Ischemic etiologies should be investigated in patients who present with new arrhythmias. CAD may result in arrhythmias following an acute MI or as the result of ultrastructural and electrophysi-ologic remodeling secondary to chronic ischemic heart disease. Ischemia of the electrical conduction system may be present in the form of new onset complete or partial atrioventricular conduction blocks.Preoperative EvaluationA focused history and physical examination is essential with par-ticular attention directed to the signs, symptoms, and clinical man-ifestations mentioned previously. The patient’s functional status is of importance not only because it is a component of preoperative risk assessment, but also

1	the signs, symptoms, and clinical man-ifestations mentioned previously. The patient’s functional status is of importance not only because it is a component of preoperative risk assessment, but also because quality of life improvement and symptomatic relief are both goals of surgical therapy.Coronary angiography is the primary diagnostic tool. The coronary anatomy and degrees of stenoses are delineated allow-ing for planning of surgical revascularization.Noninvasive diagnostic studies, in combination with pro-vocative maneuvers (exercise or pharmacologic agents) offer information regarding the functional significance of ischemic disease. A stress ECG is frequently used as a screening tool with 50% sensitivity and 90% specificity for coronary artery with a threshold of 1 mm of ST-segment depression.38 This test, how-ever, requires patients to achieve a certain elevation in their heart rate and is therefore not suitable for those that cannot achieve this goal. Furthermore, baseline ECG

1	This test, how-ever, requires patients to achieve a certain elevation in their heart rate and is therefore not suitable for those that cannot achieve this goal. Furthermore, baseline ECG abnormalities may render it impossible to detect typical ischemic changes with stress.Echocardiography and nuclear imaging may be performed under pharmacologic stress (with dobutamine or dipyridamole) to assess reversible ischemia and myocardial viability. Technetium-99m or thallium-201 perfusion scans and stress echocardiogra-phy are more sensitive than stress ECG.39 These studies also have the ability to assess global ventricular function in terms of left ventricle ejection fraction, which can be used to determine operative risk. Please refer to the diagnostic studies section for more details.CORONARY ARTERY BYPASS GRAFTINGIndicationsA joint committee established by the ACC/AHA have pub-lished guidelines for surgical revascularization (CABG) in Brunicardi_Ch21_p0801-p0852.indd 80901/03/19 5:32

1	ARTERY BYPASS GRAFTINGIndicationsA joint committee established by the ACC/AHA have pub-lished guidelines for surgical revascularization (CABG) in Brunicardi_Ch21_p0801-p0852.indd 80901/03/19 5:32 PM 810SPECIFIC CONSIDERATIONSPART IITable 21-3Algorithm set forth by ACC/AHA guidelines for preoperative cardiovascular evaluation before noncardiac surgery for patients who are scheduled for nonemergent, non-low risk surgery, no active cardiac disease, and less than 3 METsNUMBER OF RISK FACTORSaRECOMMENDATION0Proceed with planned surgery.1–2Control HR and proceed with planned surgery or pursue further testing if it will change management.3–5Pursue further testing if it will advance management.aRisk factors are history of ischemic heart disease, history of prior or compensated heart failure, history of cerebrovascular disease, diabetes mellitus, and renal insufficiency.Table 21-4Data from ACC/AHA guidelines for CABG in CAD to improve survivalANATOMYCLASS OF RECOMMENDATIONLEVEL OF

1	history of cerebrovascular disease, diabetes mellitus, and renal insufficiency.Table 21-4Data from ACC/AHA guidelines for CABG in CAD to improve survivalANATOMYCLASS OF RECOMMENDATIONLEVEL OF EVIDENCE• LMIB• 3-vessel +/− proximal LADIB• 2-vessel + proximal LADIB• 2-vessel – proximal LADIIa – with extensive ischemiaIIb – without extensive ischemiaBC• Multivessel disease with DMIIa (CABG preferred over PCI)B• Proximal LAD onlyIIa – with LITA for long-term benefitB• 1-vessel – proximal LADIII – HarmB• LV dysfunctionIIa – LVEF 35%–50%IIb – LVEF <35% without LM diseaseBB• Survivor of ischemia-mediated VTIBDM = Diabetes mellitus; LITA = Left internal thoracic/mammary artery; LM = Left main coronary artery; LV = left ventricle; VT = ventricular tachycardia. Class of recommendation: I – Benefit far outweighs risks and procedure should be performed; IIa – Benefit outweighs risks and procedure is considered to be reasonable; IIb – Potential benefits may exceed risks and procedure may be

1	far outweighs risks and procedure should be performed; IIa – Benefit outweighs risks and procedure is considered to be reasonable; IIb – Potential benefits may exceed risks and procedure may be considered; III – Procedure not helpful and may cause harm. Level of evidence: A – Strong; multiple supporting randomized controlled trials or meta-analyses, B – Limited; data based on a single randomized trial or nonrandomized trials, C – Very limited; based on expert consensus, case studies or standards of care.CAD. The indications, categorized by presentation and angio-graphic disease burden as well as by treatment intention (sur-vival improvement and symptom relief), are summarized later (Tables 21-3, 21-4, and 21-5).40,41Percutaneous Coronary Intervention vs. Coronary Artery Bypass GraftingThere have been multiple prospective, randomized, controlled trials as well as large retrospective studies looking at the com-parative effectiveness of percutaneous coronary interventions (PCI) and CABG.

1	have been multiple prospective, randomized, controlled trials as well as large retrospective studies looking at the com-parative effectiveness of percutaneous coronary interventions (PCI) and CABG. Some of the representative studies are sum-marized here.The New York State Study (2005). A retrospective review of 59,314 patients in a New York state registry with multivessel (two or more) coronary disease was performed. Of these, 37,212 patients received a CABG, and the remainder underwent a PCI. After adjusting by means of proportional-hazards methods, CABG was associated with higher adjusted rates of long-term survival than PCI.42Stent or Surgery Trial (2008). An international multicenter, randomized, controlled trial of 988 patients (n = 488 PCI, n = 500 CABG) with multivessel CAD was performed to compare revascularization strategies. The median follow-up was extended to 6 years, and a survival advantage persisted in the CABG group over the PCI group.43Synergy Between Percutaneous

1	performed to compare revascularization strategies. The median follow-up was extended to 6 years, and a survival advantage persisted in the CABG group over the PCI group.43Synergy Between Percutaneous Coronary Intervention With Taxus and Cardiac Surgery (SYNTAX Trial, 2009, updated 2013). Revascularization strategies, CABG vs. PCI, were compared in a 1:1 randomized prospective trial of 1800 patients with high-risk coronary artery disease (left-main or triple-vessel disease). Rates of requirement for repeat revascu-larization and major adverse cardiac or cerebrovascular events (MACCE) at 1 year were lower in the CABG patients (5.9% and 12.4%, respectively) compared to PCI patients (13.5% and 17.8%, respectively). No difference in mortality was seen between the groups at 1 year.44 At 5 years, the estimated rate of MACCE was 26.9% in the CABG group vs. 37.3% in the PCI group. No difference in all-cause mortality was seen. The dif-ference in MACCE between PCI and CABG was confined to

1	years, the estimated rate of MACCE was 26.9% in the CABG group vs. 37.3% in the PCI group. No difference in all-cause mortality was seen. The dif-ference in MACCE between PCI and CABG was confined to patients with intermediate or high SYNTAX scores.45The ACCF and STS Database Collaboration on the Com-parative Effectiveness of Revascularization Strategies (ASCERT Study, 2012). This study, performed by collabo-ration of the American College of Cardiology Foundation and the Society of Thoracic Surgeons, reviewed their respective national databases of patients over the age of 65 who had multi-vessel coronary disease (excluding those with left main disease). CABG was performed on 86,244 patients, and 103,549 under-went PCI. There was no difference in adjusted mortality at 1 year, but there was a significantly lower mortality with CABG than PCI at 4 years.46 Although CABG had higher cost, this was Brunicardi_Ch21_p0801-p0852.indd 81001/03/19 5:32 PM 811ACQUIRED HEART DISEASECHAPTER

1	was a significantly lower mortality with CABG than PCI at 4 years.46 Although CABG had higher cost, this was Brunicardi_Ch21_p0801-p0852.indd 81001/03/19 5:32 PM 811ACQUIRED HEART DISEASECHAPTER 21Table 21-5Data from ACC/AHA guidelines for CABG in CAD to improve symptomsANATOMY ASSOCIATED SYMPTOMSCLASS OF RECOMMENDATIONLEVEL OF EVIDENCE• Unacceptable angina with presence of ≥1 stenoses amenable to revascularization despite medical treatmentIA• Complex 3-vessel CAD +/− proximal LAD involvementIIa (CABG preferred over PCI)B• Unacceptable angina with presence of ≥1 stenoses amenable to revascularization but medical treatment is not possibleIIaC• Previous CABG with ≥1 stenoses associated with ischemia and angina despite medical treatmentIIbCmodest with an incremental cost-effectiveness ratio of $30,454 per quality-adjusted life year gained.47Everolimus-Eluting Stents or Bypass Surgery for Left Main Coronary Artery Disease (EXCEL Trial, 2016). This study randomized 1905 patients with

1	of $30,454 per quality-adjusted life year gained.47Everolimus-Eluting Stents or Bypass Surgery for Left Main Coronary Artery Disease (EXCEL Trial, 2016). This study randomized 1905 patients with left main coronary artery disease of low or intermediate complexity to PCI or CABG. At 3 years, no difference was observed in the primary endpoint of death, stroke, myocardial infarction or ischemia-driven revas-cularization. However, it should be noted that the PCI group exhibited a greater increase in these events between 30 days and three years than the CABG group (11.5% vs. 7.9%).48SummaryPCI technology has improved over time, and rates of periproce-dural adverse events have decreased significantly. Man-agement strategies must be tailored to the individual patient’s clinical status and context, but CABG maintains improved long-term outcome and remains the standard of care for patients with left-main, multivessel coronary artery disease and patients with diabetes. Recent and upcoming trials

1	but CABG maintains improved long-term outcome and remains the standard of care for patients with left-main, multivessel coronary artery disease and patients with diabetes. Recent and upcoming trials will con-tinue to clarify which patient populations benefit most from either revascularization strategy.Operative Techniques and ResultsBypass Conduit Selection. The most important criterion in conduit selection is graft patency. The conduit with the highest patency rate (98% at 5 years and 85%–90% at 10 years) is the internal thoracic artery, which is most commonly left attached proximally to the subclavian artery (although occasionally used as a free graft) and anastomosed distally to the target coronary artery.49,50 The use of both internal thoracic arteries has been shown to increase event-free survival in a number of studies.51,52The greater saphenous vein can be harvested using an open or endoscopic technique. In the open technique, the initial inci-sion is made along the course of

1	survival in a number of studies.51,52The greater saphenous vein can be harvested using an open or endoscopic technique. In the open technique, the initial inci-sion is made along the course of the vein on the medial aspect of the lower extremity. The vein is harvested with meticulous attention directed towards minimizing manipulation of the vein itself. The incision may be continuous or bridged in an attempt to decrease the size of the incision, but multiple bridged inci-sions may have the potential risk of increased conduit manipula-tion during harvest. Endoscopic harvest is performed by making a small incision just above and medial to the knee where the endoscope is inserted. Side branches are cauterized under endo-scopic visualization using bipolar electrocautery until dissec-tion is carried proximally until the required length of vein is mobilized. A proximal counterincision is then made to extract the venous conduit, which is prepared in the standard fashion.1The radial artery is

1	proximally until the required length of vein is mobilized. A proximal counterincision is then made to extract the venous conduit, which is prepared in the standard fashion.1The radial artery is another frequently used conduit. After confirmation of ulnar collateral flow to the hand by the clinical Allen’s test or a duplex ultrasound study, an incision is made from a point just proximal to the radial styloid process and end-ing just medial and distal to the biceps tendon on the nondomi-nant hand. With lateral retraction of the brachioradialis muscle, the radial artery is dissected sharply with care to avoid injury to the cutaneous nerves in this area and minimize manipulation of the artery itself. This artery can also be harvested using an endoscopic technique.Many studies have looked at the patency rates of the radial artery graft in comparison to the saphenous vein graft. Although some studies have resulted in equivocal data, general consen-sus favors the use of radial arterial

1	at the patency rates of the radial artery graft in comparison to the saphenous vein graft. Although some studies have resulted in equivocal data, general consen-sus favors the use of radial arterial grafts over vein grafts with 5-year patency rates of 98% and 86%, respectively.53,54From a historical perspective, the anterior circulation (left anterior descending artery) is generally bypassed using the internal thoracic artery, and the lateral (circumflex artery) or inferior (right coronary artery) territories are bypassed using a saphenous vein or radial artery graft. These conduits may be combined to form a composite Tor Y-graft, or sewn to multiple targets as sequential grafts. Since patency is best with arterial grafts, recent data have suggested that the best long-term results are achieved using a multiple or all-arterial revascularization strategy, particularly in patients >70 years of age and patients with diabetes.55-57 Other conduits such as the gastroepiploic arteries, lesser

1	using a multiple or all-arterial revascularization strategy, particularly in patients >70 years of age and patients with diabetes.55-57 Other conduits such as the gastroepiploic arteries, lesser saphenous veins, and cephalic veins have been described, but these are not widely used and will not be dis-cussed here.Conventional Coronary Artery Bypass Grafting. Tradi-tionally, CABGs are performed with the patient lying supine through a median sternotomy. Left internal thoracic artery and other conduit harvests are performed. After the patient is heparinized, cardiopulmonary bypass is initiated. The aorta is cross-clamped, and cardioplegia is delivered. Once adequate myocardial protection has been achieved, coronary arterioto-mies are made, and distal anastomoses are performed using polypropylene suture (Fig. 21-3A,B). The proximal anastomo-ses are then performed directly onto the ascending aorta or onto preexisting grafts. It is important to note that significant coro-nary stenoses can

1	suture (Fig. 21-3A,B). The proximal anastomo-ses are then performed directly onto the ascending aorta or onto preexisting grafts. It is important to note that significant coro-nary stenoses can cause differential distribution of cardioplegia and myocardial protection. It is therefore recommended to use retrograde cardioplegia or to revascularize the area with the most concern for ischemia first and give cardioplegia down the completed graft. The left internal thoracic artery to left anterior descending (LAD) graft is frequently performed last to avoid kinking or disruption of this important conduit. Once all grafts Brunicardi_Ch21_p0801-p0852.indd 81101/03/19 5:32 PM 812SPECIFIC CONSIDERATIONSPART IIFigure 21-3. Coronary artery bypass grafting. A. Intraoperative photograph of the distal anastomoses performed between the left internal thoracic artery and left anterior descending coronary artery with a continuous 8-0 suture. B. Fifteen-year follow-up coronary angiogram of a left

1	anastomoses performed between the left internal thoracic artery and left anterior descending coronary artery with a continuous 8-0 suture. B. Fifteen-year follow-up coronary angiogram of a left internal thoracic artery to left anterior descend-ing coronary artery bypass demonstrating a widely patent free of any significant atherosclerotic stenosis. Anastomotic site is shown by the arrow.Figure 21-4. Epicardial stabilizing device used during off-pump coronary anastomosis.ABare in place, the patient is weaned from bypass. During this time, the heart is monitored closely by direct visual inspection, transesophageal echocardiography. It is recommended that bypass graft patency be assessed with ultrasonic flow probes or Doppler to confirm flow and determine the pulsatility index (PI). These tools are utilized to detect abnormalities that may signify inadequate revascularization or technical problems with the bypass grafts. Upon confirmation of hemostasis, chest tubes are placed, the

1	tools are utilized to detect abnormalities that may signify inadequate revascularization or technical problems with the bypass grafts. Upon confirmation of hemostasis, chest tubes are placed, the sternum is approximated with sternal wires, and the incisions are closed.Conventional CABG Results. Several early randomized trials showed improved survival in patients who received a CABG as opposed to medical therapy.58-60 A propensity-matched study identified that CABG greatly benefited patients with LV dysfunction and left main stenosis >50% compared to medi-cal management.61 The Bypass Angioplasty Revascularization Investigation (BARI) trial demonstrated impressively superior results with CABG compared to PCI in terms of 5-year cardiac mortality (5.8% vs. 20.6%) in patients with diabetes in addi-tion to CAD.62 In a study examining the benefits of CABG over medical management for specific CAD distributions, survival was better in patients with proximal LAD stenoses, regardless of the

1	in addi-tion to CAD.62 In a study examining the benefits of CABG over medical management for specific CAD distributions, survival was better in patients with proximal LAD stenoses, regardless of the number of diseased vessels.63 In general, these studies show survival rates of over 90% at 5 years and approximately 75% at 10 years following CABG.The mortality and morbidity of the procedure itself has changed over time. Data from the Society of Thoracic Surgeons (STS) database accounts for 1,497,254 patients who under-went solitary CABG from 2000 to 2009. The mortality rate of CABGs has improved significantly from 2.4% in 2000 to 1.9% in 2009, despite the relatively constant predicted mortality rate of around 2.3%. In parallel with this, postoperative complication rates have also decreased: stroke (1.6%–1.2%), bleeding requir-ing reoperation (2.4%–2.2%), and deep sternal wound infection (0.59%–0.37%).64Off-pump Coronary Artery Bypass. To avoid the adverse consequences of cardiopulmonary

1	stroke (1.6%–1.2%), bleeding requir-ing reoperation (2.4%–2.2%), and deep sternal wound infection (0.59%–0.37%).64Off-pump Coronary Artery Bypass. To avoid the adverse consequences of cardiopulmonary bypass, off-pump coronary artery bypass (OPCAB) was developed and has been adopted in some centers over the past two decades.With OPCAB, the heart is left beating. Performing anas-tomoses on the beating heart requires the use of myocardial sta-bilization devices, which help portions of the epicardial surface to remain relatively immobile while the anastomoses are being performed (Fig. 21-4).Apical suction devices are used to aid in exposure, particu-larly of the lateral and inferior vessels. Many creative maneu-vers have been developed, including patient repositioning, opening the right pleural space to allow for cardiac displace-ment, and creation of a pericardial cradle to minimize compro-mise of cardiac function while exposing the various surfaces of the heart. Temporary proximal

1	pleural space to allow for cardiac displace-ment, and creation of a pericardial cradle to minimize compro-mise of cardiac function while exposing the various surfaces of the heart. Temporary proximal occlusion of the target coronary artery, or the use of an intracoronary shunt, are necessary to Brunicardi_Ch21_p0801-p0852.indd 81201/03/19 5:32 PM 813ACQUIRED HEART DISEASECHAPTER 21provide adequate exposure of the anastomosis. Occlusion causes temporary ischemia, and if not tolerated during a test occlusion, coronary shunts can be employed.OPCAB Results. The superiority of OPCAB over on-pump CABG remains a controversy despite the large body of literature on this topic. A pooled analysis of two random-ized trials, the Beating Heart Against Cardioplegic Arrest Studies (BHACAS 1 & 2), is one of several studies that have touted lower short-term mortality rates with the off-pump com-pared to the on-pump technique.65-67 Other studies, however, have demonstrated equivocal or contrary

1	is one of several studies that have touted lower short-term mortality rates with the off-pump com-pared to the on-pump technique.65-67 Other studies, however, have demonstrated equivocal or contrary results.68-70 Further-more, the prospective and much larger ROOBY (Randomized On/Off Bypass) trial showed increased rates of adverse cardiac events with OPCAB compared to conventional CABG.71 Despite the initial enthusiasm for the theoretical advantages of avoiding cardiopulmonary bypass, consistent benefits in clinical outcome have not been observed. There does seem to be a more or less uniform trend towards decreased perioperative blood product transfusions with OPCAB compared to on-pump CABG. In terms of other measures of early outcome, postoperative renal failure, stroke, and acute MI, the superiority of OPCAB has been unclear.69,72,73 A more recent Cochrane review by Moller et al did not demonstrate any significant benefit of off-pump compared with on-pump CABG regarding mortality,

1	superiority of OPCAB has been unclear.69,72,73 A more recent Cochrane review by Moller et al did not demonstrate any significant benefit of off-pump compared with on-pump CABG regarding mortality, stroke, or myocardial infarction. In contrast, better long-term survival in the group of patients undergoing on-pump CABG with the use of cardiopul-monary bypass and cardioplegic arrest was observed.74The higher cardiac morbidity in the ROOBY trial was associated with decreased 1-year angiographic patency rates.71 However, studies with contrasting findings exist, quoting equiv-alent rates of graft patency for OPCAB usage.75,76 The broad variety in results may be suggestive that other factors (e.g., surgeon skill, technical difficulty, patient factors) may be dom-inating the outcome rather than the use or avoidance of cardio-pulmonary bypass.77 After almost two decades, OPCAB has not been widely adopted and remains less than 20% of all CABG procedures in the United States.Minimally Invasive

1	the use or avoidance of cardio-pulmonary bypass.77 After almost two decades, OPCAB has not been widely adopted and remains less than 20% of all CABG procedures in the United States.Minimally Invasive Direct Coronary Artery Bypass. As an extension of the off-pump coronary revascularization technique, minimally invasive direct coronary artery bypass (MIDCAB) has been described. MIDCAB is performed using a left ante-rior mini-thoracotomy through which mobilization of the left internal thoracic and direct in situ anastomosis to the left ante-rior descending artery (or its diagonal branches) is performed. This technique is primarily applicable to single-vessel disease, although reports of multivessel revascularizations do exist.MIDCAB Results. A review of 411 patients undergoing MID-CAB quotes an operative mortality >1%. In this study, all patients received revascularization of the LAD only, regard-less of the number of diseased vessels. The 3-year mortality in patients with single-vessel

1	an operative mortality >1%. In this study, all patients received revascularization of the LAD only, regard-less of the number of diseased vessels. The 3-year mortality in patients with single-vessel disease following a MIDCAB was 3.1%, which was, not surprisingly, lower than those with multi-vessel disease (8.7%).78There is an inherent selection bias in retrospective reviews comparing MIDCAB to OPCAB or conventional CABG as MIDCAB patients tend to have less extensive disease. Because of this, there have been multiple randomized controlled trials looking at the efficacy of MIDCAB compared to PCI. A meta-analysis of 12 randomized prospective trials comparing PCI to MIDCAB revascularization of isolated proximal left anterior descending artery demonstrated comparable results in terms of 2mortality and MI but a lower revascularization requirement in the MIDCAB group.79A recent meta-analysis by Lee et al revealed CABG, as compared with PCI with DES, reduced long-term rates of the composite

1	and MI but a lower revascularization requirement in the MIDCAB group.79A recent meta-analysis by Lee et al revealed CABG, as compared with PCI with DES, reduced long-term rates of the composite of all-cause death, myocardial infarction, or stroke in patients with left main or multivessel CAD. Compared to PCI with DES, CABG was found to be superior in patients with multivessel CAD (P = 0.001), but no between-group differences in those with left main CAD (P = 0.427).80 Similar conclusions have been made by multiple other studies.Total Endoscopic Coronary Artery Bypass. With the advent of robotic surgical technology allowing stereoscopic visual-ization and increased instrument dexterity, total endoscopic coronary artery bypass (TECAB) has become possible. In July of 2004, the da Vinci robotic surgical system received FDA approval for use in coronary anastomoses. Extracorporeal cir-culation with peripheral cannulation has been used in earlier reports, but the development of mechanical

1	surgical system received FDA approval for use in coronary anastomoses. Extracorporeal cir-culation with peripheral cannulation has been used in earlier reports, but the development of mechanical stabilizers has pro-vided the ability to perform the internal thoracic artery harvest and coronary anastomosis off-pump with use of the robotic arms only. Several studies have looked at the feasibility of TECAB and have shown acceptable results, but this procedure has not been adopted by most surgeons because of its steep learning curve, longer operative times, and lack of demonstrable clinical benefit.81-83 Although the volume of robotic-assisted CABG is increasing, such procedures constituted <1% of all CABG pro-cedures performed in the United States in 2012.84Hybrid Coronary Revascularization. With the increasing collaboration between cardiothoracic surgeons and interven-tional cardiologists, hybrid coronary revascularization (HCR) combining a minimally invasive surgical technique (MIDCAB

1	the increasing collaboration between cardiothoracic surgeons and interven-tional cardiologists, hybrid coronary revascularization (HCR) combining a minimally invasive surgical technique (MIDCAB or TECAB) with PCI has become a reality. This capitalizes on a major advantage of both treatments, utilizing the durable left internal thoracic artery to left anterior descending coronary artery bypass graft while treating other stenoses with PCI, obvi-ating the need for a large surgical incision or cardiopulmonary bypass. HCR is not without its downsides as there are some concerns with this approach because aggressive anti-platelet therapy is required with PCI and may increase the hemorrhagic complications of surgical revascularization. A small study com-paring HCR to OPCAB showed comparable graft patency and decreased hospital stay with HCR without an increase in com-plication rates.85 There are, however, some studies that have reported increased rates of requirement for reintervention in

1	patency and decreased hospital stay with HCR without an increase in com-plication rates.85 There are, however, some studies that have reported increased rates of requirement for reintervention in patients undergoing HCR, and this requires further study.86,87 A recent multicenter prospective observational study showed equivalent outcomes between HCR and multivessel PCI.88 HCR has not gained widespread acceptance, and its clinical value remains a matter of debate.Transmyocardial Laser Revascularization. Despite the advancement of technology and revascularization strategies, patients with end-stage coronary artery disease may not be amenable to complete revascularization. Transmyocardial laser revascularization (TMR) relies on a CO2 or holmium:yttrium-aluminum-garnet (Ho:YAG) laser to create multiple transmu-ral channels (1 mm in diameter) through the myocardium. The initial concept was that these channels would serve as conduits for direct perfusion from the ventricle, but evidence

1	multiple transmu-ral channels (1 mm in diameter) through the myocardium. The initial concept was that these channels would serve as conduits for direct perfusion from the ventricle, but evidence suggests that the resultant angiogenesis is primarily responsible for the improved perfusion. A meta-analysis of seven randomized con-trolled trials comparing TMR to medical therapy for chronic Brunicardi_Ch21_p0801-p0852.indd 81301/03/19 5:32 PM 814SPECIFIC CONSIDERATIONSPART IITable 21-6Classification of cardiac murmursMURMURCONDITIONMECHANISM/ETIOLOGYSYSTOLIC MURMURSHolosystolic (pansystolic)VSDFlow between chambers that have widely different pressures throughout systoleMid-systolic (systolic ejection)High flow rate, MS, MR, TS, TIOften crescendo-decrescendo in configuration; occur as blood is ejected into the left and right ventricular outflow tractsEarly systolicEarly TI, acute MRLess commonMid to late systolicMR, MVPSoft to moderate high-pitched murmurs at the LV apex; often due to

1	is ejected into the left and right ventricular outflow tractsEarly systolicEarly TI, acute MRLess commonMid to late systolicMR, MVPSoft to moderate high-pitched murmurs at the LV apex; often due to apical tethering and malcoaptation of MV leaflets; an associated click indicates prolapse of the MV leafletsDIASTOLIC MURMURSEarly high-pitchedAI, PRGenerally decrescendo in configuration; occur when the associated ventricular pressure drops sufficiently below that of the outflow tractMid-diastolicMS, TS, PDA*, VSD*, ASD*Due to a relative disproportion between valve orifice size and diastolic blood flow volume; seen in normal MV and TV with increased diastolic blood flow associated with these conditions*PresystolicMS, TSOccur during the period of ventricular filling that follows atrial contraction (i.e., only occur in sinus rhythm)CONTINUOUS MURMURSPDAUncommon, due to shunts that persist through the end of systole and the some or all of diastoleAI = aortic insufficiency; ASD = atrial septal

1	(i.e., only occur in sinus rhythm)CONTINUOUS MURMURSPDAUncommon, due to shunts that persist through the end of systole and the some or all of diastoleAI = aortic insufficiency; ASD = atrial septal defect; MR = mitral regurgitation; MS = mitral stenosis; MVP = mitral valve prolapse; PDA = patent ductus arteriosus; PR = pulmonic regurgitation; TI = tricuspid insufficiency; TS = tricuspid stenosis; VSD = ventricular septal defect.angina has shown higher rates of angina improvement in the TMR but was not able to show a difference in mortality between the two groups.89TMR is also being used as an adjunct to CABG in the treatment of extensive CAD that is not amenable to surgical revascularization alone. In a study looking at the benefits of TMR in addition to CABG, Allen et al concluded that TMR decreases angina burden when added to CABG in patients who cannot be revascularized by CABG alone.90 The current STS guidelines support the consideration of TMR in patients with ischemic myocardial

1	decreases angina burden when added to CABG in patients who cannot be revascularized by CABG alone.90 The current STS guidelines support the consideration of TMR in patients with ischemic myocardial territories that cannot be revascularized by PCI or CABG.91 Because of equivocal late results at most centers, this therapeutic strategy has not gained widespread acceptance, and ACC/AHA guidelines give TMR a class IIb recommendation for treatment of refractory angina.39New DevelopmentsRegenerative Medicine and Tissue Engineering. Provoca-tive investigations are being performed on the level of signaling molecules, gene therapy, stem cells, and tissue engineering to regenerate or replace damaged tissue in patients with ischemic heart disease. Growth factors, such as fibroblast growth fac-tor (FGF) and vascular endothelial growth factor (VEGF), are receiving attention due to their ability to induce growth of new vessels. Although concerns regarding systemic administration of these pleiotropic

1	and vascular endothelial growth factor (VEGF), are receiving attention due to their ability to induce growth of new vessels. Although concerns regarding systemic administration of these pleiotropic signaling molecules exist, early placebo-controlled clinical trials have shown some promising results with administration of these agents.92,93 Adenoviral transfection of diseased tissue with transgenes for growth factors and tran-scription factors has been attempted with variable results.Research in tissue engineering has been directed at cre-ation of vascular conduits that are resistant to atherosclerosis. Stem cells have also been infused directly into the site of injury or in the generation of new tissue around a biodegradable scaffold.94 Despite their potential, these technologies are still in their infancy, and significant progress will be needed before more widespread clinical adoption.VALVULAR HEART DISEASEGeneral PrinciplesThe number of patients undergoing surgical management of

1	in their infancy, and significant progress will be needed before more widespread clinical adoption.VALVULAR HEART DISEASEGeneral PrinciplesThe number of patients undergoing surgical management of valvular heart disease has increased over the last decade, from a total of 26,547 isolated aortic or mitral valve procedures reported to the STS Adult Cardiac Surgery Database in 2006 to 45,253 such procedures in 2015.95 In 2016, valve procedures represented over 50% of the cases performed at our institution. Although congenital and inherited etiologies represent impor-tant clinical entities, age-associated and acquired conditions still represent the primary causes of valvular heart disease and are the focus of this section.The most common screening method for valvular heart disease is cardiac auscultation, with murmurs classified based primarily on their timing in the cardiac cycle, but also on their configuration, location and radiation, pitch, intensity, and dura-tion (Table 21-6).96

1	auscultation, with murmurs classified based primarily on their timing in the cardiac cycle, but also on their configuration, location and radiation, pitch, intensity, and dura-tion (Table 21-6).96 Although some systolic murmurs are related to normal physiologic increases in blood flow, some may indi-cate cardiac disease, such as valvular aortic stenosis (AS), that are important to diagnose, even when asymptomatic. Diastolic and continuous murmurs, on the other hand, are frequently Brunicardi_Ch21_p0801-p0852.indd 81401/03/19 5:32 PM 815ACQUIRED HEART DISEASECHAPTER 21Table 21-7Hemodynamic alterations in cardiac murmur intensityINTERVENTIONEFFECTRespirationRight-sided murmurs increase with inspiration. Left-sided murmurs increase with expiration.Valsalva maneuverMost murmurs decrease in length and intensity. The murmur of HCM becomes louder, and the murmur of MVP becomes louder and longer.ExerciseBenign flow murmurs and murmurs caused by stenotic valves become louder with isotonic

1	length and intensity. The murmur of HCM becomes louder, and the murmur of MVP becomes louder and longer.ExerciseBenign flow murmurs and murmurs caused by stenotic valves become louder with isotonic and isometric exercise. The murmurs of MR, VSD, and AI also increase with isometric exercise.Positional changesMost murmurs decrease with standing; the murmur of HCM becomes louder, and the murmur of MVP becomes louder and longer. Brisk squatting and passive leg raising increases most murmurs; the murmurs of HCM and MVP diminish.Postventricular premature beat or atrial fibrillationBenign flow murmurs and stenosis at the semilunar valves increase in intensity following a ventricular premature beat or a long cycle length in atrial fibrillation. Systolic murmurs of atrioventricular valve regurgitation do not change.Pharmacologic interventionsThe initial hypotensive phase following inhalation of amyl nitrate decreases the murmurs of MR, VSD, and AI, and increases the murmur of AS. The later

1	do not change.Pharmacologic interventionsThe initial hypotensive phase following inhalation of amyl nitrate decreases the murmurs of MR, VSD, and AI, and increases the murmur of AS. The later tachycardic phase following inhalation of amyl nitrate increases right-sided murmurs and the murmur of MS. The response in MVP is biphasic (softer then louder than control).Transient arterial occlusionTransient external compression of the upper extremity increases the murmurs of MR, VSD, and AI.AI = aortic insufficiency; AS = aortic stenosis; HCM = hypertrophic cardiomyopathy; MR = mitral regurgitation; MS = mitral stenosis; MVP = mitral valve prolapse; VSD = ventricular septal defect.pathologic in nature. Dynamic cardiac auscultation provides further evidence as to the significance and origin of many mur-murs (Table 21-7).96Although auscultation may provide initial evidence to the existence of valvular disease, associated signs and symptoms may help narrow the diagnosis. Abnormalities in the

1	many mur-murs (Table 21-7).96Although auscultation may provide initial evidence to the existence of valvular disease, associated signs and symptoms may help narrow the diagnosis. Abnormalities in the splitting of the heart sounds and additional heart sounds should be noted, as should the presence of pulmonary rales. Peripheral pulses should be checked for abnormal intensity or timing, and the presence of a jugular venous wave should be documented. Addi-tionally, symptoms of syncope, angina pectoris, heart failure, and peripheral thromboembolism are important and may help guide diagnosis and management.Several imaging examinations are also available to aid in the diagnosis and classification of various valvular disorders. Electrocardiograms may provide information regarding ven-tricular hypertrophy, atrial enlargement, arrhythmias, conduc-tion abnormalities, prior myocardial infarction, and evidence of active ischemia that would prompt further workup. Posteroan-terior and lateral chest

1	atrial enlargement, arrhythmias, conduc-tion abnormalities, prior myocardial infarction, and evidence of active ischemia that would prompt further workup. Posteroan-terior and lateral chest X-rays are also easy to obtain and may yield information regarding cardiac chamber size, pulmonary blood flow, pulmonary and systemic venous pressure, and cardiac calcifications. The gold standard for the evaluation of valvular heart disease is transthoracic echocardiography (TTE), which is helpful in the noninvasive evaluation of valve mor-phology and function, chamber size, wall thickness, ventricu-lar function, pulmonary and hepatic vein flow, and pulmonary artery pressures. Specialized examinations based on the specific findings of TTE examinations are discussed in the following sections.Regardless of the etiology, valvular heart disease can pro-duce a myriad of hemodynamic derangements. Left untreated, valvular stenosis and insufficiency can produce significant pressure and volume overload on

1	of the etiology, valvular heart disease can pro-duce a myriad of hemodynamic derangements. Left untreated, valvular stenosis and insufficiency can produce significant pressure and volume overload on the affected cardiac chamber, respectively, with mixed disease consequently causing mixed pathology. Although the heart can initially compensate for alter-ations in cardiac physiology, cardiac function eventually dete-riorates, leading to heart failure, decreased patient functional status, ventricular dysfunction, and eventually death. In order to optimize long-term survival, surgery or transcatheter therapeu-tics are recommended in various forms of valvular heart disease and in an increasing number of elderly and high-risk patients.Surgical OptionsAlthough valve repair is increasingly indicated, especially in patients with aortic, mitral or tricuspid insufficiency, valve replacement is appropriate in certain patient populations. Valve replacement can be accomplished with either mechanical

1	especially in patients with aortic, mitral or tricuspid insufficiency, valve replacement is appropriate in certain patient populations. Valve replacement can be accomplished with either mechanical or bio-logical prostheses, and the choice of valve depends on many patient-specific factors such as age, health status, and desire for future pregnancy. Preexisting indications or contraindications to anticoagulation therapy also influence the choice of mechanical versus tissue valve prosthesis.Current options for mechanical valve replacement include either tilting disc valves or bileaflet valves. Although mechanical valves are highly durable, they require perma-nent anticoagulation to mitigate the risk of valve thrombosis and thromboembolic sequelae.97 Due to the concordant risk of hem-orrhagic complications, patient characteristics such as debility, lifestyle, and contraindications to systemic anticoagulation therapy may preclude mechanical valve replacement. Moreover, young women who are

1	complications, patient characteristics such as debility, lifestyle, and contraindications to systemic anticoagulation therapy may preclude mechanical valve replacement. Moreover, young women who are planning future pregnancies cannot take warfarin due to its teratogenic potential. Conversely, patients with other indications for systemic anticoagulation, such as other risk factors for thromboembolism (i.e., atrial fibrillation), or the presence of a mechanical prosthetic valve in place in another position, may benefit from mechanical valve replace-ment. Current ACC/AHA guidelines recommend a shared-decision-making process between patient and physician when determining the choice of valve prosthesis, with the use of 3Brunicardi_Ch21_p0801-p0852.indd 81501/03/19 5:32 PM 816SPECIFIC CONSIDERATIONSPART IIFigure 21-5. SJM Regent mechanical heart valve. (SJM Regent and St. Jude Medical are trademarks of St. Jude Medical, LLC or its related companies. Reproduced with permission of St.

1	21-5. SJM Regent mechanical heart valve. (SJM Regent and St. Jude Medical are trademarks of St. Jude Medical, LLC or its related companies. Reproduced with permission of St. Jude Medical, ©2017. All rights reserved.)bioprosthetic valves in all patients who have a contraindication to lifelong anticoagulation or who are unwilling to receive it.98The potential to avoid the hazards of serious bleeding com-plications spurred the development of valve prostheses using biological materials, which obviate the need for systemic anti-coagulation therapy. As tissue valves are naturally less throm-bogenic, the attendant yearly risks of both thromboembolic and anticoagulation-related complications are considerably less than with mechanical valves.99 Consequently, tissue valve replace-ment is generally recommended for patients averse to systemic anticoagulation therapy, with potential concerns regarding com-pliance or follow-up while taking anticoagulant medications, and in the case of reoperation

1	recommended for patients averse to systemic anticoagulation therapy, with potential concerns regarding com-pliance or follow-up while taking anticoagulant medications, and in the case of reoperation for a thrombosed mechanical valve. However, biological valves are more prone to degenera-tion, especially when implanted in the mitral position, and in younger patients, and those in renal failure, on hemodialysis, or with hypercalcemia.99 Improved manufacturing methods have made currently available tissue valves more durable than previ-ous versions, and valve replacement with a biological prosthesis is generally preferred in patients without other indications for anticoagulation therapy who are >60 years of age for the aortic position and >70 years of age for the mitral position. The avail-ability of transcatheter aortic valves for re-replacement, has resulted in patients and surgeons preferring bioprosthetic aortic valves in patients even <60 years of age in many centers.Mechanical

1	of transcatheter aortic valves for re-replacement, has resulted in patients and surgeons preferring bioprosthetic aortic valves in patients even <60 years of age in many centers.Mechanical Valves. The first bileaflet mechanical valve was introduced in 1977. Bileaflet valves are comprised of two semi-circular leaflets that open and close, creating one central and two peripheral orifices (Fig. 21-5). Bileaflet mechanical valves have demonstrated excellent flow characteristics, low risk of late valve-related complications, including valve failure, and are currently the most commonly implanted type of mechanical valve prosthesis in the world.100Although mechanical valves necessitate systemic antico-agulation, careful monitoring of the International Normalized Ratio (INR) reduces the risk of thromboembolic events and hemorrhagic complications and improves overall survival.101 Patients undergoing mechanical aortic valve replacement gen-erally have a target INR of 2 to 3 times normal;

1	of thromboembolic events and hemorrhagic complications and improves overall survival.101 Patients undergoing mechanical aortic valve replacement gen-erally have a target INR of 2 to 3 times normal; however, after a randomized study, one of the mechanical aortic valves has been improved for an INR range of 1.5 to 2.0.102 Patients undergoing mechanical mitral valve replacement frequently have increased left atrial size, concomitant atrial fibrillation, and are at higher risk for thromboembolism that those undergoing aortic valve replacement and are thus recommended to have a target INR 2.5 to 3.5 times normal. When managed appropriately, the yearly risk of major bleeding is <1.4%. Patients with mechanical mitral valve prostheses have nearly twice the thromboembolic risk of those with mechanical aortic valve prostheses (1.3% vs. 0.8% per year).103Tissue Valves. A xenograft valve is one implanted from another species, such as porcine xenograft valves, or manufac-tured from tissue such as

1	aortic valve prostheses (1.3% vs. 0.8% per year).103Tissue Valves. A xenograft valve is one implanted from another species, such as porcine xenograft valves, or manufac-tured from tissue such as bovine pericardium. A variety of xeno-graft tissue valves exist and are primarily differentiated by the presence or absence of a mounting stent. Stented valves are the most commonly implanted, and the most popular valve in the United States is a stented bovine pericardial valve.104The more traditional stented valves are attached to a sew-ing ring, which decreases the technical complexity of valve replacement compared with stentless valves (Fig. 21-6). The chief disadvantage of stented tissue valves is a smaller effective orifice area, which increases the transvalvular gradient. This phenomenon is referred to as patient prosthetic mismatch. This effect is most pronounced in patients with small prosthetic valve areas, specifically <0.85 cm2 valve area per square meter body surface area and may

1	referred to as patient prosthetic mismatch. This effect is most pronounced in patients with small prosthetic valve areas, specifically <0.85 cm2 valve area per square meter body surface area and may affect survival, symptomatic improve-ment, and the hemodynamic response to exercise following surgery.105Stentless porcine xenograft valves were developed in order to minimize the limitations in flow characteristics seen in patients with small prosthetic valve areas and have demon-strated an increase in effective valve area of approximately 10% over stented xenografts of equivalent size.100 They can result in improved hemodynamics, both at rest and with exercise.106 The absence of a stent and sewing ring both increases the techni-cal complexity of valve replacement and takes advantage of the biologic mobility of the aortic valve apparatus. Though results with stentless valves seem promising, some stentless valves have been shown to have poor durability,107 and stentless valves have not

1	biologic mobility of the aortic valve apparatus. Though results with stentless valves seem promising, some stentless valves have been shown to have poor durability,107 and stentless valves have not been widely adopted due to the technical complexity of implantation.Recently, rapid deployment valves have been introduced to further decrease the complexity and time required for aortic valve replacement. Two of these valves are on the market in the United States. The Perceval valve by LivaNova is the only sutureless, stentless valve available.108 These rapid deployment valves have shown improved hemodynamics, particularly in patients with small annuli, and shorter implantation times. In some series, these benefits have been shown to reduce early morbidity.108Homografts. Homograft valves from human cadavers, also known as allografts, have been used for aortic valve replace-ment since the technique was originally described over 50 years ago.109 Since that time, homografts have typically

1	human cadavers, also known as allografts, have been used for aortic valve replace-ment since the technique was originally described over 50 years ago.109 Since that time, homografts have typically been used for aortic and pulmonary valve replacements and have been successfully harvested from brain dead organ donors and the Brunicardi_Ch21_p0801-p0852.indd 81601/03/19 5:32 PM 817ACQUIRED HEART DISEASECHAPTER 21Figure 21-6. Carpentier-Edwards PERIMOUNT Magna Ease stented porcine bioprosthesis. (Edwards Lifesciences LLC, Irvine, CA. Additionally, it should be noted that Edwards, Edwards Lifesciences, the stylized E logo, Carpentier-Edwards, Carpentier-Edwards Classic, Magna, Magna Ease, Magna Mitral Ease are trademarks of Edwards Lifesciences Corporation.)explanted hearts of heart transplant recipients. Following har-vest, these valves are sterilized using an antibiotic solution and subsequently stored in fixative or cryopreservation solution.Like other types of tissue valves, the

1	recipients. Following har-vest, these valves are sterilized using an antibiotic solution and subsequently stored in fixative or cryopreservation solution.Like other types of tissue valves, the risk of thrombo-embolic complications with homograft valves is low, and sys-temic anticoagulation therapy is not required. Additionally, the structure of homograft valves is naturally low-profile, allow-ing for larger effective valve orifices and lower postoperative transvalvular gradients compared with stented xenograft valves. Additionally, they have been shown to have some advantages in patients with endocarditis.110The major shortcoming of homograft valves is their lim-ited long-term durability due to tissue degeneration. Within one year of implantation, these valves undergo substantial loss of cellular components and subsequent structural compro-mise, which may ultimately lead to valve failure.111 Although enhanced preservation techniques has improved cellular viabil-ity, which approaches

1	cellular components and subsequent structural compro-mise, which may ultimately lead to valve failure.111 Although enhanced preservation techniques has improved cellular viabil-ity, which approaches the 15-year viability of xenograft valves, the limited availability of these valves and techniques has lim-ited the use of homograft tissue valves.Autografts. In 1967, Donald Ross described a procedure in which the diseased aortic valve was replaced using the patient’s native pulmonary valve as an autograft, which was in turn replaced with a homograft in the pulmonic position.112 The pro-cedure resulted in minimal transvalvular gradients and favor-able left ventricular mechanics, both at rest and during exercise. Known as the Ross procedure, this operation has been shown to be particularly beneficial in children, as the pulmonary trunk grows with the child and long-term anticoagulation is not required.113The late results of the Ross procedure are discussed later in this chapter. In

1	beneficial in children, as the pulmonary trunk grows with the child and long-term anticoagulation is not required.113The late results of the Ross procedure are discussed later in this chapter. In addition to potential concerns with durability, performance of the Ross procedure has also been limited by its technical complexity and the increased surgical risk associated with double valve replacement.Valve Repair. Valve repair offers a number of advantages over valve replacement, due in large part to the preservation of the patient’s native valvular and subvalvular apparatus. In mitral valve (MV) surgery, preservation of the mitral apparatus has been shown to lead to better postoperative left ventricular func-tion and survival.114,115 Additionally, as there is no implanted prosthesis, the patient avoids the risks of chronic anticoagula-tion, infection, thromboembolic complications, and prosthetic valve failure after surgery.In the case of MV repair, freedom from reoperation and

1	the patient avoids the risks of chronic anticoagula-tion, infection, thromboembolic complications, and prosthetic valve failure after surgery.In the case of MV repair, freedom from reoperation and valve-related complications has been excellent in certain patient populations, even at 20-year follow-up.116 It has also been dem-onstrated that patients undergoing MV surgery with moder-ate functional tricuspid regurgitation (TR) do not experience increased perioperative complication rates when a concomitant tricuspid valve (TV) repair is performed.117 Midterm results in this group are encouraging, with greater than 98% freedom from reoperation reported by some groups at 5 years, suggesting fur-ther indications for valve repair.Despite its advantages for the patient, valve repair is gen-erally more technically demanding than valve replacement and may occasionally fail. Both the suitability of the patient for valve repair and the skill and expertise of the surgeon perform-ing the operation

1	more technically demanding than valve replacement and may occasionally fail. Both the suitability of the patient for valve repair and the skill and expertise of the surgeon perform-ing the operation are important when considering valve repair in the individual patient.MITRAL VALVE DISEASEMitral StenosisEtiology. Acquired mitral stenosis (MS) is most often caused by rheumatic fever, with approximately 60% of patients with pure MS presenting with a clinical history of rheumatic heart disease.96 Rarely, other conditions can cause obstruction to fill-ing of the left ventricle (LV), mimicking MS. Acquired causes of MV stenosis include left atrial myxoma, prosthetic valve thrombosis, mucopolysaccharidosis, previous chest radiation, and severe annular calcification.Pathology. Although rheumatic heart disease is associated with a transmural pancarditis, pathological fibrosis of the valves Brunicardi_Ch21_p0801-p0852.indd 81701/03/19 5:32 PM 818SPECIFIC CONSIDERATIONSPART IIFigure

1	heart disease is associated with a transmural pancarditis, pathological fibrosis of the valves Brunicardi_Ch21_p0801-p0852.indd 81701/03/19 5:32 PM 818SPECIFIC CONSIDERATIONSPART IIFigure 21-7. Mitral stenosis. The thickened, fused leaflets of the diseased mitral valve are viewed through a left atriotomy. (Reproduced with permission from Centers for Disease Control and Prevention, Edwin P. Ewing, Jr.)Table 21-8Data from ACC/AHA guidelines for the classification of the severity of mitral valve disease in adultsMITRAL STENOSISINDICATORMILDMODERATESEVEREMean gradient (mmHg)a< 55–10>10Pulmonary artery systolic pressure (mmHg)< 3030–50> 50Valve area (cm2)>1.51.0–1.5< 1.0MITRAL REGURGITATIONQUALITATIVEMILDMODERATESEVEREAngiographic grade1+2+3+Color Doppler jet areaSmall, central jet (< 4 cm2 or < 20% left atrial area)More than mild criteria, but no severe criteria presentVena contracta width > 0.7 cm with large central jet (area > 40% of left atrial area) or with a wall-impinging jet

1	or < 20% left atrial area)More than mild criteria, but no severe criteria presentVena contracta width > 0.7 cm with large central jet (area > 40% of left atrial area) or with a wall-impinging jet of any size, swirling in left atriumDoppler vena contracta width (cm)< 0.30.3–0.69≥ 0.7QUANTITATIVE (CATH OR ECHO)Regurgitant volume (ml per beat)< 3030–59≥ 60Regurgitant fraction (%)< 3030–49≥ 50Regurgitant orifice area (cm2)0.20.2–0.39≥ 0.4ADDITIONAL ESSENTIAL CRITERIALeft atrial size EnlargedLeft ventricular size EnlargedaValve gradients are flow dependent and when used as estimates of severity of valve stenosis should be assessed with knowledge of cardiac output or forward flow across the valve.results primarily from the endocarditic process. The damage caused by endocardial inflammation and fibrosis is progressive, causing commissural fusion, subvalvular shortening of the chor-dae tendineae, and calcification of the valvular and subvalvu-lar apparatus.118 The resulting stenotic MV has

1	fibrosis is progressive, causing commissural fusion, subvalvular shortening of the chor-dae tendineae, and calcification of the valvular and subvalvu-lar apparatus.118 The resulting stenotic MV has a funnel shape, with doming of the leaflets, and a significantly narrowed orifice caused by interchordal and commissural fusion (Fig. 21-7). The degree of mitral stenosis should be determined preoperatively, as these pathological features may help determine the timing and type of intervention to perform.96Pathophysiology. As the normal MV area of 4.0 to 5.0 cm2 is reduced by the rheumatic process, blood can flow from the left atrium to the left ventricle only if it is propelled by an ever-increasing pressure gradient. This increased left atrial pressure causes left atrial enlargement and eventually pulmonary hyper-tension and decreased exercise tolerance. Patients with diastolic valve doming, usually accompanied by a history of rheumatic fever, are defined as having stage A MS, even with

1	pulmonary hyper-tension and decreased exercise tolerance. Patients with diastolic valve doming, usually accompanied by a history of rheumatic fever, are defined as having stage A MS, even with normal trans-mitral flow velocities. Stage B MS is defined by an increased transmitral flow velocity with mitral valve area greater than 1.5 cm2. This condition is associated with mild to moderate left atrial enlargement but normal pulmonary arterial pressure. Stage C MS consists of a severely stenotic valve (mitral valve area ≤1.5 cm2) without symptoms. This is frequently associated with commissural fusion and diastolic doming of the leaflets as well as a pulmonary arterial pressure of >30 mmHg. Stage D MS is defined as the aforementioned criteria with the onset of decreased exercise tolerance and/or dyspnea on exertion (Table 21-8).98The onset of symptoms is due to the evolution of patho-physiological processes, beginning with an elevation in left atrial pressure. The increased left atrial

1	dyspnea on exertion (Table 21-8).98The onset of symptoms is due to the evolution of patho-physiological processes, beginning with an elevation in left atrial pressure. The increased left atrial pressure is subsequently transmitted to the pulmonary venous system, causing pulmo-nary edema as the hydrostatic pressure in the vessels exceeds the plasma oncotic pressure. Decreased pulmonary venous compli-ance exacerbates the pulmonary venous hypertension, though a concomitant decrease in microvascular permeability may Brunicardi_Ch21_p0801-p0852.indd 81801/03/19 5:32 PM 819ACQUIRED HEART DISEASECHAPTER 21preclude pulmonary edema in the chronic setting.119 Patients may also develop pulmonary arterial hypertension, owing to vasoconstriction, intimal hyperplasia, and medial hypertrophy of the pulmonary arterioles in response to the increased pul-monary venous pressure. The secondary obstruction to flow caused by reactive pulmonary arterial hypertension may serve to protect against

1	of the pulmonary arterioles in response to the increased pul-monary venous pressure. The secondary obstruction to flow caused by reactive pulmonary arterial hypertension may serve to protect against pulmonary edema, but it also exacerbates the intractable decrease in cardiac output that develops as stenosis worsens.120Throughout the process, the left atrium becomes dilated and hypertrophied due to increased work in filling the ventricle against a fixed obstruction. Atrial fibrillation (AF) may develop, exacerbating the patient’s symptoms and increasing the risk of left atrial thrombus and subsequent embolization. Left ventricu-lar structure and function are typically preserved owing to the protective effect of the stenotic valve.Clinical Manifestations. The sudden opening of the thick-ened, nonpliable valve with left atrial contraction produces an opening snap, followed by a diastolic rumble caused by rapid entry of blood into the left ventricle. When diastole is complete, the MV

1	nonpliable valve with left atrial contraction produces an opening snap, followed by a diastolic rumble caused by rapid entry of blood into the left ventricle. When diastole is complete, the MV subsequently closes very rapidly, causing an increased first heart sound. The murmur, classically known as the aus-cultatory triad, is best heard at the apex. Associated mitral and tricuspid insufficiencies are heard as a pansystolic murmur radi-ating to the axilla and a systolic murmur at the xiphoid process, respectively.The first clinical signs of MS are those associated with pulmonary venous congestion, namely exertional dyspnea, decreased exercise capacity, orthopnea, and paroxysmal noctur-nal dyspnea. Hemoptysis and pulmonary edema may develop as the venous hypertension worsens. Advanced MS can also cause pulmonary arterial hypertension and subsequent right heart fail-ure, manifested as jugular venous distention, hepatomegaly, ascites, and lower extremity edema.2As mentioned previously,

1	can also cause pulmonary arterial hypertension and subsequent right heart fail-ure, manifested as jugular venous distention, hepatomegaly, ascites, and lower extremity edema.2As mentioned previously, atrial fibrillation may develop as left atrial pathology worsens, causing atrial stasis and subse-quent thromboembolism. Patients with MS may initially present with signs of arterial embolization, even rarely with angina from coronary occlusion.2Diagnostic Studies. All patients with a clinical history and physical exam suggestive of MS should have an electrocardio-gram (ECG) and chest X-ray. Abnormalities in the ECG may include atrial fibrillation, left atrial enlargement, or right axis deviation. Chest X-ray findings may include enlargement of the left atrium and pulmonary artery, creating a double contour behind the right atrial shadow and obliterating the normal con-cavity between the aorta and left ventricle. Findings consistent with pulmonary congestion may also be present.2The

1	a double contour behind the right atrial shadow and obliterating the normal con-cavity between the aorta and left ventricle. Findings consistent with pulmonary congestion may also be present.2The diagnostic tool of choice is TTE, which not only con-firms the diagnosis of MS, but also rules out other concomi-tant myocardial or valvular heart disease.121 Two-dimensional TTE can be used to calculate the MV area and to determine the morphology of the MV apparatus, including leaflet mobility and flexibility, leaflet thickness and calcification, subvalvular fusion, and the appearance of the commissures. Doppler TTE can also be used in combination with various equations to esti-mate the hemodynamic severity of MS in terms of the mean transmitral pressure gradient, the MV area, and the pulmonary artery systolic pressure.In most cases, further examinations are not necessary. A preoperative TEE may be utilized to rule out left atrial appendage thrombus, when the patient is being considered for

1	artery systolic pressure.In most cases, further examinations are not necessary. A preoperative TEE may be utilized to rule out left atrial appendage thrombus, when the patient is being considered for percutane-ous balloon mitral commissurotomy, or if the preoperative TTE is insufficient for diagnosis. Exercise TTE is indicated when resting TTE parameters are discordant with symptom sever-ity.122 Routine coronary angiography is usually performed prior to valve surgery,98 except in young patients (≤30 years of age) with no risk factor for coronary artery disease.Indications for Operation. Depending on the severity and the morphology of the diseased MV (see Table 21-8), balloon commissurotomy, surgical repair, or MV replacement may be indicated for the treatment of MS (Table 21-9).98Mitral RegurgitationEtiology. The most important cause of MR in the United States is myxomatous degenerative disease of the MV, which occurs in 2% to 3% of the population.123 Other important causes of MR

1	RegurgitationEtiology. The most important cause of MR in the United States is myxomatous degenerative disease of the MV, which occurs in 2% to 3% of the population.123 Other important causes of MR include rheumatic heart disease, infective endocarditis, ischemic heart disease, and dilated cardiomyopathies. Less frequently, MR can be caused by collagen vascular diseases, trauma, pre-vious chest radiation, hypereosinophilic syndrome, carcinoid disease, and exposure to certain drugs.96Pathology. The MV apparatus consists of the mitral leaflets, chordae tendineae, papillary muscles, and mitral annulus, and abnormalities in any one of these components has the potential to cause MR.124 The system for classifying MR proposed by Carpentier focuses on the functional and anatomic characteris-tics of the MV pathology and proposes three basic types of dis-eased valves based on the motion of the free edge of the leaflet relative to the plane of the mitral annulus.125In Type I MR, valvular

1	of the MV pathology and proposes three basic types of dis-eased valves based on the motion of the free edge of the leaflet relative to the plane of the mitral annulus.125In Type I MR, valvular insufficiency occurs secondary to annular dilatation or leaflet perforation, and normal leaflet motion is maintained. Type II MR is seen in patients with mitral valve prolapse and is due to prolapse of often thickened exces-sive leaflet tissue that gives the valve a “billowing” appearance, in addition to ruptured or elongated chordae tendineae caus-ing increased leaflet motion. Type III insufficiency, as seen in patients with rheumatic and ischemic heart disease, occurs from restricted leaflet motion, either during systole and diastole (Type IIIA) or during systole alone (Type IIIB).Pathophysiology. The basic pathophysiologic abnormality of MR is the retrograde flow of a portion of the LV stroke volume into the left atrium during systole due to an incompetent MV or dilated MV annulus.Acute

1	basic pathophysiologic abnormality of MR is the retrograde flow of a portion of the LV stroke volume into the left atrium during systole due to an incompetent MV or dilated MV annulus.Acute severe MR can result from ruptured chordae ten-dineae, a ruptured papillary muscle, or infective endocarditis and causes a sudden volume overload of both the left atrium and ventricle.96 Although an acute increase in preload provides a modest increase in overall stroke volume, the left atrium and ventricle are unable to fully accommodate the regurgitant vol-ume or maintain forward stroke volume in the acute setting due to a lack of remodeling.Chronic MR generally has a more indolent course, with increasing volume overload of the left atrium and ventricle as the effective regurgitant orifice size becomes larger. The result-ing increase in left atrial and ventricular volume initially allows for an increase in the total stroke volume by Starling’s law and accommodation of the regurgitant volume, thus

1	larger. The result-ing increase in left atrial and ventricular volume initially allows for an increase in the total stroke volume by Starling’s law and accommodation of the regurgitant volume, thus maintaining forward cardiac output and alleviating pulmonary congestion during the compensatory phase of chronic MR.126 However, as the left atrium becomes more dilated, the development of AF becomes more likely, disrupting atrioventricular synchrony and Brunicardi_Ch21_p0801-p0852.indd 81901/03/19 5:32 PM 820SPECIFIC CONSIDERATIONSPART IITable 21-9Data from ACC/AHA guidelines for MV surgery in specific clinical contextsCLINICAL SETTINGCLASS OF RECOMMENDATIONLEVEL OF EVIDENCEBalloon Valvotomy for Mitral Stenosis• Symptomatic patients (NYHA II, III, IV) with moderate or severe MS and favorable valve morphology, without left atrial thrombus or moderate to severe MR• Asymptomatic patients with moderate or severe MS, favorable valve morphology, and pulmonary hypertension (PASP > 50 mmHg at

1	valve morphology, without left atrial thrombus or moderate to severe MR• Asymptomatic patients with moderate or severe MS, favorable valve morphology, and pulmonary hypertension (PASP > 50 mmHg at rest, > 60 mmHg with exercise), without left atrial thrombus or moderate to severe MR• Symptomatic patients (NYHA III, IV) with moderate or severe MS and favorable valve morphology, who are high risk or not candidates for surgery• Asymptomatic patients with moderate or severe MS, favorable valve morphology, and new onset atrial fibrillation, without left atrial thrombus or moderate to severe MR• Symptomatic patients (NYHA II, III, IV) with MV area > 1.5 cm2 if there is evidence of hemodynamically significant MS (PASP > 60 mmHg, PAWP ≥ 25 mmHg, mean MV gradient >15 mmHg during exercise)• Symptomatic patients (NYHA III, IV) with moderate or severe MS and favorable valve morphology, as an alternative to surgery• Patients with mild MS• Patients with moderate to severe MR or left atrial

1	patients (NYHA III, IV) with moderate or severe MS and favorable valve morphology, as an alternative to surgery• Patients with mild MS• Patients with moderate to severe MR or left atrial thrombusIIIIaIIbIIbIIbIII – HarmIII – HarmACCCCCCCSurgery for Mitral Stenosisa• Symptomatic patients (NYHA III, IV) with moderate or severe MS when:Balloon valvotomy is unavailableBalloon valvotomy is contraindicated due to thrombus or MRValve morphology is not favorable for balloon valvotomy• Symptomatic patients with moderate to severe MS who also have moderate to severe MR• Mildly symptomatic patients (NYHA I, II) with severe MS and severe pulmonary hypertension (PASP > 60 mmHg)• Asymptomatic patients with moderate or severe MS and recurrent embolic events while receiving adequate anticoagulation, when the likelihood of successful MVr is high• MVr in the setting of mild MS• Closured commissurotomy in the setting of MVr; open commissurotomy should be performedIIIIaIIbIII – HarmIII –

1	when the likelihood of successful MVr is high• MVr in the setting of mild MS• Closured commissurotomy in the setting of MVr; open commissurotomy should be performedIIIIaIIbIII – HarmIII – HarmBCCCCCSurgery for Mitral Regurgitationa• Symptomatic patients with acute severe MR• Symptomatic patients (NYHA II, III, IV) with chronic severe MR without LV dysfunction (LVEF < 0.30) and/or end-systolic dimension > 55 mm• Asymptomatic patients with chronic severe MR and mild to moderate LV dysfunction (LVEF 0.30–0.60) and/or end-systolic dimension ≥ 40 mm• Asymptomatic patients with chronic severe MR and preserved LV function (LVEF > 0.60, end-systolic dimension < 40 mm), when the likelihood of successful MVr is > 90%• Asymptomatic patients with chronic severe MR, preserved LV function, and 1) New onset atrial fibrillation, 2) Pulmonary hypertension (PASP > 50 mmHg at rest, > 60 mmHg with exercise)• Symptomatic patients (NYHA III, IV) with chronic severe MR due to a primary abnormality of the

1	onset atrial fibrillation, 2) Pulmonary hypertension (PASP > 50 mmHg at rest, > 60 mmHg with exercise)• Symptomatic patients (NYHA III, IV) with chronic severe MR due to a primary abnormality of the mitral apparatus and severe LV dysfunction (LVEF < 0.30, end-systolic dimension > 55 mm), when the likelihood of successful MVr is high• Symptomatic patients (NYHA III, IV) with chronic severe MR secondary to severe LV dys-function (LVEF < 0.30) who remain symptomatic despite optimal medical management for heart failure, including biventricular pacing• Asymptomatic patients with MR and preserved LV function (LVEF > 0.60, end-systolic dimension < 40 mm), when the likelihood of successful repair is low• Isolated MV surgery in the setting of mild or moderate MRIIIIIaIIaIIaIIbIII – HarmIII – HarmBBBBCCCCCLV = left ventricular; LVEF = left ventricular ejection fraction; MR = mitral regurgitation; MS = mitral stenosis; MV = mitral valve; MVr = mitral valve repair; MVR = mitral valve

1	– HarmBBBBCCCCCLV = left ventricular; LVEF = left ventricular ejection fraction; MR = mitral regurgitation; MS = mitral stenosis; MV = mitral valve; MVr = mitral valve repair; MVR = mitral valve replacement; NYHA = New York Heart Association; PASP = pulmonary artery systolic pressure; PAWP = pulmonary artery wedge pressure; a = mitral valve repair should be performed when possible in this population.Brunicardi_Ch21_p0801-p0852.indd 82001/03/19 5:32 PM 821ACQUIRED HEART DISEASECHAPTER 21predisposing to thrombus formation. Additionally, chronic vol-ume overload may lead to LV contractile dysfunction, result-ing in impaired ejection and end-systolic volume increases. LV dilatation and elevated LV end-diastolic pressures may also worsen throughout the progression of MR, reducing cardiac output and causing congestion of the pulmonary vasculature. These changes herald LV decompensation and heart failure and often indicate irreversible myocardial injury.Clinical Manifestations. In cases

1	and causing congestion of the pulmonary vasculature. These changes herald LV decompensation and heart failure and often indicate irreversible myocardial injury.Clinical Manifestations. In cases of acute severe MR, patients are often symptomatic and present with pulmonary congestion and reduced forward stroke volume. In severe cases, patients may present with cardiogenic shock.2 Because the LV has not remodeled in the acute setting, a hyperdynamic apical impulse may not be present in the precordium. The typical sys-tolic murmur of MR may be holosystolic or absent, with a third heart sound and/or diastolic flow murmur being the only aus-cultatory findings.In cases of chronic MR, patients may remain asymptom-atic for long periods of time due to the compensatory mecha-nisms of the remodeled LV. However, once the LV begins to fail, patients become increasingly symptomatic from exertional dyspnea, decreased exercise capacity, orthopnea, and eventu-ally pulmonary hypertension and right heart

1	However, once the LV begins to fail, patients become increasingly symptomatic from exertional dyspnea, decreased exercise capacity, orthopnea, and eventu-ally pulmonary hypertension and right heart failure.2 Physical examination may demonstrate displacement of the LV apical impulse due to cardiac enlargement from chronic volume over-load, a third heart sound, or an early diastolic flow rumble. The characteristic auscultatory findings also include an apical sys-tolic murmur, which is variably transmitted to the axilla or the left sternal border, depending on the location of the pathology. As mentioned previously, patients may present with AF due to dilatation of the left atrium. Findings consistent with pulmonary hypertension frequently indicate late-stage disease.Diagnostic Studies. In the setting of acute heart failure, TTE should be performed and may demonstrate the anatomical loca-tion and severity of the MV pathology. However, TTE may underestimate lesion severity due to

1	the setting of acute heart failure, TTE should be performed and may demonstrate the anatomical loca-tion and severity of the MV pathology. However, TTE may underestimate lesion severity due to inadequate views of the color flow jet. In this case, severe MR should be suspected if hyperdy-namic systolic function of the LV is visualized, and TEE may be used to confirm the diagnosis and direct repair strategies.127In cases of chronic MR, ECG and chest X-ray are per-formed to assess rhythm status and the degree of pulmonary vascular congestion.96,98 An initial 2D and Doppler TTE should be performed for a baseline estimation of LV and left atrial size, LV systolic function, pulmonary artery pressure, MV morphol-ogy, and MR severity.128 A central color flow jet in the setting of a structurally normal MV on TTE suggests functional MR, which may be due to LV dilatation or tethering of the posterior leaflet in patients with ischemic heart disease. In the setting of organic MR (i.e., rheumatic

1	normal MV on TTE suggests functional MR, which may be due to LV dilatation or tethering of the posterior leaflet in patients with ischemic heart disease. In the setting of organic MR (i.e., rheumatic and degenerative MR), which is suggested by the presence of an eccentric color flow jet and morphological abnormalities in the MV apparatus on TTE, the presence of calcium in the annulus or leaflets, the redundancy of the leaflets, and the anatomy of the MV pathology should be assessed. Follow-up TTE is indicated on an annual or semian-nual basis in patients with asymptomatic moderate to severe MR in order to assess changes from baseline parameters and direct the timing of surgery. Any abrupt change in signs or symp-toms in a patient with chronic MR is also an indication for TTE examination.98Additional preoperative studies are variably indicated in certain patient populations. Preoperative TEE is indicated in patients with indications for surgery or poor windows on TTE in order to

1	preoperative studies are variably indicated in certain patient populations. Preoperative TEE is indicated in patients with indications for surgery or poor windows on TTE in order to determine the severity and anatomic basis of MR and to evaluate LV systolic function.96 Preoperative TEE is also indicated in cases when a discrepancy exists between a patient’s functional status and the severity of MR on TTE. It is helpful for preoperative planning when assessing the feasibility of repair in the individual patient. Exercise stress-echocardiography may also be useful to detect LV systolic dysfunction in well-compensated patients, who may not demonstrate a rise in the end-systolic dimension of the heart or a drop in ejection fraction on routine TTE.129 Coronary angiography should be performed prior to valve surgery in patients with evidence of ischemia, decreased LV systolic function, a history of coronary artery dis-ease or coronary risk factors, including postmenopausal status and age ≥40

1	to valve surgery in patients with evidence of ischemia, decreased LV systolic function, a history of coronary artery dis-ease or coronary risk factors, including postmenopausal status and age ≥40 in men and premenopausal women.98Indications for Operation. Based on the etiology, morphol-ogy, and severity of MR (see Table 21-8), MV repair, MV replacement with preservation of part or all of the mitral apparatus may be variably performed for the treatment of MR. As the intraoperative findings may dictate MV replacement whenever a MV repair is planned, current recommendations are for MV surgery in general (see Table 21-9).98Mitral Valve Operative Techniques and ResultsMitral valve surgery is performed on the arrested heart with the assistance of cardiopulmonary bypass. Traditionally, a median sternotomy incision has been used; however, the left atrium can also be approached via minimally invasive incisions, such as a right thoracotomy, or a fully endoscopic approach. The MV is commonly

1	sternotomy incision has been used; however, the left atrium can also be approached via minimally invasive incisions, such as a right thoracotomy, or a fully endoscopic approach. The MV is commonly exposed through a left atrial incision placed poste-rior and parallel to the intra-atrial groove or via a transseptal approach through the right atrium.Commissurotomy. Upon opening the left atrium, the MV is visualized and the left atrium is examined for thrombus. A nerve hook or right-angle clamp is subsequently introduced beneath the commissures and used to evaluate the MV apparatus for leaflet mobility, commissural fusion, and subvalvular chordal abnormalities. The commissure is then carefully incised in a slightly anterior direction 2 to 3 mm at a time, making sure with each extension of the incision that the chordae tendineae remain attached to the commissural leaflets. The commissurotomy is generally stopped 1 to 2 mm from the annulus where the leaf-let tissue thins, indicating the

1	the incision that the chordae tendineae remain attached to the commissural leaflets. The commissurotomy is generally stopped 1 to 2 mm from the annulus where the leaf-let tissue thins, indicating the transition to normal commissural tissue. The papillary muscles are subsequently examined and incised as necessary in order to maximize the mobility of the leaflets.After the commissurotomy is complete and the associated chordae tendineae and papillary muscles are mobilized, leaflet mobility is assessed. The anterior leaflet is grasped with forceps and brought through its complete range of motion. If subvalvu-lar restriction or leaflet rigidity is identified, further division or excision of fused chordae and debridement of calcium may be necessary. Occasionally, the leaflets can be debrided carefully to increase mobility. In rheumatic patients, the thickened leaf-lets can be thinned by careful dissection.130 Valve replacement may be more appropriate if extensive secondary mobilization is

1	to increase mobility. In rheumatic patients, the thickened leaf-lets can be thinned by careful dissection.130 Valve replacement may be more appropriate if extensive secondary mobilization is required. At the end of the procedure, competence of the valve is assessed with injection of cold saline into the ventricle.Open surgical commissurotomy has an operative risk of <1%, and has been shown to have good long-term results, with freedom from reoperation as high as 88.5%, 80.3%, and 78.7% at 10, 20, and 30 years, respectively.131 The incidence of 4Brunicardi_Ch21_p0801-p0852.indd 82101/03/19 5:32 PM 822SPECIFIC CONSIDERATIONSPART IIFigure 21-8. Mitral valve replacement. A St. Jude bileaflet mechanical valve is viewed through a left atriotomy.postoperative thromboembolic complications is generally <1% per patient-year, and the lack of required systemic anticoagula-tion precludes the development of hemorrhagic complications long term.132 In some institutions, balloon valvuloplasty has

1	generally <1% per patient-year, and the lack of required systemic anticoagula-tion precludes the development of hemorrhagic complications long term.132 In some institutions, balloon valvuloplasty has replaced commissurotomy.Mitral Valve Replacement. After exposing the valve, an inci-sion is made in the anterior mitral leaflet, in the midline. The chordal attachments are preserved if possible, with leaflet tis-sue being excised as needed. Attempts are made to preserve the annular and subvalvular apparatus when possible. If it is necessary to excise the anterior leaflet and chordae, the papil-lary muscles can be reattached to the annulus with PTFE suture. If possible, the posterior leaflet along with its associated sub-valvular structures are preserved. The annulus is subsequently sized, and an appropriate mitral prosthesis is implanted using pledgeted horizontal mattress sutures. The annular sutures may be placed from the atrial to the ventricular side, seating the valve

1	sized, and an appropriate mitral prosthesis is implanted using pledgeted horizontal mattress sutures. The annular sutures may be placed from the atrial to the ventricular side, seating the valve intra-annularly, or from the ventricular to the atrial side, seating the valve in a supra-annular position. When placing the mattress sutures, care must be taken to stay within the annular tissue, as excessively deep bites may cause injury to critical structures such as the circumflex coronary artery posterolaterally, the atrioventricular node anteromedially, or the aortic valve antero-laterally. The sutures are subsequently placed through the sew-ing ring, and the valve prosthesis is lowered onto the annulus, where it is secured (Fig. 21-8).The factors associated with increased operative risk for MV replacement include age, left ventricular systolic dys-function, emergent procedure status, NYHA functional status, previous cardiac surgery, associated coronary artery disease, and concomitant

1	MV replacement include age, left ventricular systolic dys-function, emergent procedure status, NYHA functional status, previous cardiac surgery, associated coronary artery disease, and concomitant disease in another valve. However, for most patients, MV replacement is associated with an operative mor-tality between 2% to 6%, and 65% to 70% 5-year survival.133,134 Although preservation of the mitral apparatus during MV replacement is important for subsequent left ventricular func-tion, there was no difference between complete and partial preservation with respect to 30-day and 5-year mortality.133 Mechanical valves are associated with increased durability compared to bioprosthetic valves, and they have demonstrated a freedom from reoperation of 98% vs. 79% at 15 years, respectively.135 Despite these findings, the choice of prosthetic valve depends on many factors and should be decided on a patient-by-patient basis.Mitral Valve Repair. There are many techniques available for MV repair

1	these findings, the choice of prosthetic valve depends on many factors and should be decided on a patient-by-patient basis.Mitral Valve Repair. There are many techniques available for MV repair that are variably used depending on the preoperative and intraoperative assessment of valvular pathology. On open-ing the atrium, the endocardium is examined for a jet lesion, a roughened area caused by a regurgitant jet striking the wall, in order to better localize the area of valvular insufficiency. The commissures are examined for evidence of prolapse, fusion, and malformation. The subvalvular apparatus and individual leaflets are subsequently examined, and areas of prolapse, restriction, fibrosis, and calcification are identified. Leaflet perforations are generally repaired primarily, or with a pericardial patch. The degree of annular dilation is also noted. The basic components of MV repair based on this assessment may include resection of the posterior and/or anterior leaflet, chordal

1	a pericardial patch. The degree of annular dilation is also noted. The basic components of MV repair based on this assessment may include resection of the posterior and/or anterior leaflet, chordal shortening, chordal transposition, artificial chordal replacement, and annuloplasty. Recent trends have been toward leaflet preservation.One of the mainstays of MV repair is triangular resection of the posterior leaflet. Excision of the diseased leaflet tissue extends down towards but generally not to the mitral annulus. After repair has been completed, valvular competency is evalu-ated by injecting saline into the ventricle with a bulb syringe and assessing leaflet mobility and apposition. If focal insufficiency is identified in other areas, additional procedures are performed.The anterior leaflet may be repaired via chordal shorten-ing, chordal transposition, artificial chordal replacement, and triangular resection of the anterior leaflet. Chordal shortening has generally been abandoned

1	may be repaired via chordal shorten-ing, chordal transposition, artificial chordal replacement, and triangular resection of the anterior leaflet. Chordal shortening has generally been abandoned in favor of chordal replacement. During chordal transposition, a resected portion of the posterior leaflet with attached chordae is transposed onto the prolapsed portion of the anterior leaflet to provide structural support, and this is followed by posterior leaflet repair, as described pre-viously. The procedure of artificial chordal replacement uses polytetrafluoroethylene sutures to attach the papillary muscle to the free edge of the prolapsing anterior leaflet. Triangular resection with primary repair of the anterior leaflet removes the prolapsing segment of the anterior MV leaflet, while preserv-ing adjacent chordal tissue, and may be especially helpful in patients with a ruptured chord or large amount of redundant anterior leaflet tissue.Annular dilation is generally corrected using a MV

1	adjacent chordal tissue, and may be especially helpful in patients with a ruptured chord or large amount of redundant anterior leaflet tissue.Annular dilation is generally corrected using a MV annu-loplasty device, such as a ring or partial band. Annuloplasty is known to improve the durability of all types of MV repair (Fig. 21-9).136 A number of devices are available and include rigid or semirigid rings that geometrically remodel the annulus, flexible rings or bands that restrict annular dilation while main-taining the physiologic sphincter motion of the annulus, and semirigid bands that provide a combination of annular remodel-ing and support of physiologic motion.Another technique known as the “double-orifice” or “edge-to-edge” repair was introduced by Alfieri in 1995, and it involves tacking the free edge of the anterior leaflet to the opposing free edge of the posterior leaflet.137 This procedure effectively gives the valve a double-orifice “bow tie” configura-tion, and it has

1	tacking the free edge of the anterior leaflet to the opposing free edge of the posterior leaflet.137 This procedure effectively gives the valve a double-orifice “bow tie” configura-tion, and it has been used as both a primary repair technique and an adjunct to other repair techniques, usually in cases of anterior leaflet pathology or Barlow’s disease. While some groups report excellent late results, its use remains controversial, and it is used mainly as a bail-out procedure of last resort in many centers.Brunicardi_Ch21_p0801-p0852.indd 82201/03/19 5:32 PM 823ACQUIRED HEART DISEASECHAPTER 21Figure 21-9. Mitral valve repair. The narrow arrow indicates the posterior leaflet repair, and the wide arrow indicates the ring annu-loplasty as viewed through a left atriotomy.Figure 21-10. Aortic stenosis. The aorta has been removed to demonstrate the thickened, fused aortic valve leaflets associated with rheumatic heart disease. (Reproduced with permission from Centers for Disease Control

1	stenosis. The aorta has been removed to demonstrate the thickened, fused aortic valve leaflets associated with rheumatic heart disease. (Reproduced with permission from Centers for Disease Control and Prevention, Edwin P. Ewing, Jr.)Due to the variety in operations and etiologies of MV dis-ease, there is heterogeneity in outcomes following MV repair. In general, the operative risk for elective, younger patients undergoing MV repair is <1%, and late results across a broad range of patients have demonstrated benefits in survival and valve-related complications, such as thromboembolic events, infective endocarditis, and anticoagulation-related hemorrhage, compared to MV replacement.116,138 Patients with MR due to degenerative disease have especially encouraging outcomes, demonstrating rates of survival and freedom from reoperation of >50% and >94% at 20 years, respectively.116 Historically, isolated anterior leaflet prolapse increased the risk of reopera-tion fivefold in this population.

1	survival and freedom from reoperation of >50% and >94% at 20 years, respectively.116 Historically, isolated anterior leaflet prolapse increased the risk of reopera-tion fivefold in this population. However, increasing experi-ence and the expanded use of chordal replacement has greatly improved these results in recent series.139 Independent predic-tors of mortality have included higher NYHA class, lower left ventricular ejection fraction, renal dysfunction, and age. Older patients have demonstrated slightly worse outcomes overall, with an operative mortality of approximately 4%, and a 10-year survival of 54% in patients ≥65 years of age. However, the superiority of repair over replacement persists even for patients >80 years of age.134Patients with rheumatic disease have demonstrated slightly worse outcomes, with one study showing significantly better freedom from operation at 10 years in patients with non-rheumatic MV disease (88% vs. 73%, P <0.005).140 Similarly, in patients with MR

1	worse outcomes, with one study showing significantly better freedom from operation at 10 years in patients with non-rheumatic MV disease (88% vs. 73%, P <0.005).140 Similarly, in patients with MR secondary to myocardial ischemia, there is growing recent evidence that mitral valve replacement may be significantly more durable than repair.141,142 Despite these differ-ences in outcomes, MV repair remains the procedure of choice for the majority of patients with amenable MV disease.Transcatheter Mitral Valve Repair and Replace-ment. Since the successful introduction of transcatheter aortic valve replacement, efforts have been made to translate the les-sons learned to treatment of the mitral valve. The first device to receive FDA approval for repair of severe mitral regurgitation due to degenerative mitral disease was the MitraClip (Abbott, Abbott Park, IL), introduced in 2003. This device allows a sur-geon or cardiologist to grasp the anterior and posterior leaflets of the mitral valve

1	mitral disease was the MitraClip (Abbott, Abbott Park, IL), introduced in 2003. This device allows a sur-geon or cardiologist to grasp the anterior and posterior leaflets of the mitral valve together, approximating the Alfieri double-orifice repair technique.143 Transcatheter mitral valve repair is now in clinical use in patients with chronic severe primary MR in whom surgery would be too great a risk, as judged by a heart team approach including a cardiologist skilled in structural heart intervention and an experienced mitral valve surgeon (see Table 21-9). In a recent randomized controlled trial comparing trans-catheter mitral valve repair to open surgical mitral valve repair, 20.1% (n = 37) of patients who received transcatheter mitral valve repair (n = 184) underwent second intervention with sur-gical mitral valve repair or replacement within 12 months com-pared with 2% (n = 2) of patients who underwent surgical repair (n = 95) (P <0.001).143,144 Surgical repair was associated

1	with sur-gical mitral valve repair or replacement within 12 months com-pared with 2% (n = 2) of patients who underwent surgical repair (n = 95) (P <0.001).143,144 Surgical repair was associated with higher rates of blood transfusion and mechanical ventilation >48 hours but had otherwise equivalent safety. Other transcatheter techniques, including transcatheter mitral valve replacement, have begun clinical trials. This is an area of active research; however, at present, open surgical repair or replacement remains the standard of care for most patients.AORTIC VALVE DISEASEAortic StenosisEtiology. The most common cause of adult aortic stenosis (AS) is calcification of a normal trileaflet or congenital bicuspid aortic valve, particularly in patients >70 years of age. Another important cause of AS is rheumatic heart disease, which is par-ticularly common in developing countries (Fig. 21-10).Pathology. Calcific aortic valve disease, also known as senile or degenerative disease, is an

1	of AS is rheumatic heart disease, which is par-ticularly common in developing countries (Fig. 21-10).Pathology. Calcific aortic valve disease, also known as senile or degenerative disease, is an age-related disorder characterized by lipid accumulation, proliferative and inflammatory changes, upregulation of angiotensin-converting enzyme activity, oxidative stress, and infiltration of macrophages and T lymphocytes.145 This process, which closely resembles atherosclerotic vascular calcification, initially results in bone formation within the base of the cusps, reducing leaflet motion. Calcification progresses to involve the leaflets, and eventually results in obstructive Brunicardi_Ch21_p0801-p0852.indd 82301/03/19 5:32 PM 824SPECIFIC CONSIDERATIONSPART IITable 21-10Data from ACC/AHA guidelines for the classification of the severity of aortic valve disease in adultsAORTIC STENOSISINDICATORMILDMODERATESEVEREJet velocity (m per s)< 303.0–4.0> 40Mean gradient (mmHg)a< 2525–40> 4.0Valve

1	for the classification of the severity of aortic valve disease in adultsAORTIC STENOSISINDICATORMILDMODERATESEVEREJet velocity (m per s)< 303.0–4.0> 40Mean gradient (mmHg)a< 2525–40> 4.0Valve area (cm2)> 1.51.0–1.5< 1.0Valve area index (cm2 per m2) < 0.6AORTIC REGURGITATIONQUALITATIVEMILDMODERATESEVEREAngiographic grade1+2+3–4+Color Doppler jet widthCentral jet, width < 25% of left ventricular outflow tractGreater than mild, but no signs of severe regurgitationCentral jet, width >65% of left ventricular outflow tractDoppler vena contracta width (cm)< 0.30.3–0.6> 0.6QUANTITATIVE (CATH OR ECHO)Regurgitant volume (ml per beat)< 3030–59≥ 60Regurgitant fraction (%)< 3030–49≥ 50Regurgitant orifice area (cm2)< 0.10.1–0.29≥ 0.3ADDITIONAL ESSENTIAL CRITERIALeft ventricular size EnlargedaValve gradients are flow dependent and when used as estimates of severity of valve stenosis should be assessed with knowledge of cardiac output or forward flow across the valve.disease, with a reduced

1	gradients are flow dependent and when used as estimates of severity of valve stenosis should be assessed with knowledge of cardiac output or forward flow across the valve.disease, with a reduced effective valve area without signs of leaflet fusion.Pathophysiology. In general, once moderate AS is present, the average rate of progression includes an increase in jet velocity of 0.3 m/s per year, an increase in mean pressure gradient of 7 mmHg per year, and a decrease in valve area of 0.1 cm2 per year (Table 21-10).96 In most adult patients with AS, obstruction develops gradually and includes a long latent period free from symptoms. During this time, the LV typically hypertrophies in response to systolic pressure overload, and normal intracavi-tary volume is maintained.146 Afterload, which is defined as left ventricular systolic wall stress, and thus ejection fraction remain normal early in this process as the increase in myocardial thickness is usually enough to counter the increased

1	is defined as left ventricular systolic wall stress, and thus ejection fraction remain normal early in this process as the increase in myocardial thickness is usually enough to counter the increased intracavi-tary systolic pressures. Patients without a typical hypertrophic response to systolic pressure overload or with a depressed con-tractile state of the myocardium do not follow the common clini-cal course, but they experience an early decrease in ejection fraction due to excessively increased afterload without a com-pensatory response.147Concentric LV hypertrophy without chamber dilatation eventually leads to increased end-diastolic pressures and dia-stolic dysfunction. Forceful atrial contraction in the face of elevated end-diastolic pressures becomes an important compo-nent of ventricular filling, even as mean left atrial and pulmo-nary venous pressures remain in the normal range. Disorders such as atrial fibrillation that disrupt atrial contraction can lead to clinical

1	of ventricular filling, even as mean left atrial and pulmo-nary venous pressures remain in the normal range. Disorders such as atrial fibrillation that disrupt atrial contraction can lead to clinical deterioration. Although systolic function is gener-ally preserved long into the natural history of the disease, left ventricular decompensation eventually occurs in the setting of longstanding increased afterload and is an indication for surgery even in the absence of other symptoms.Although concentric hypertrophy is a compensatory mech-anism to maintain ejection fraction in the face of high intracavi-tary pressures, the hypertrophied heart becomes increasingly vulnerable to ischemic injury. Coronary blood flow may become inadequate, despite the absence of epicardial coronary artery disease.148 Coronary vasodilation is mitigated by the hypertro-phied myocardium, and the hemodynamic stress of exercise or tachyarrhythmias can lead to subendocardial ischemia and further systolic or

1	Coronary vasodilation is mitigated by the hypertro-phied myocardium, and the hemodynamic stress of exercise or tachyarrhythmias can lead to subendocardial ischemia and further systolic or diastolic dysfunction. When ischemic insults occur, patients with ventricular hypertrophy experience larger infarcts and higher mortality rates than those without hypertrophy.149 In some patients, ventricular hypertrophy occurs in excess of what is needed to compensate for increased intra-cavitary pressures, creating a high-output state that is also asso-ciated with increased perioperative morbidity and mortality.150Clinical Manifestations. The characteristic auscultatory findings of AS include a harsh, crescendo-decrescendo systolic murmur at the right second or third intercostal space, often with radiation to the carotid arteries.2 As the disease progresses, aor-tic valve closure may follow pulmonic valve closure, causing paradoxical splitting of the second heart sound. Other physical findings

1	to the carotid arteries.2 As the disease progresses, aor-tic valve closure may follow pulmonic valve closure, causing paradoxical splitting of the second heart sound. Other physical findings associated with AS include an apical impulse com-monly described as a “prolonged heave,” and the presence of a narrow and sustained peripheral pulse, known as pulsus parvus et tardus.The classic symptoms of AS are exertional dyspnea, angina, and syncope.2 Although many patients are diagnosed prior to the onset of symptoms, the most common clinical Brunicardi_Ch21_p0801-p0852.indd 82401/03/19 5:32 PM 825ACQUIRED HEART DISEASECHAPTER 21presentation in patients with a known diagnosis of AS is wors-ening exertional dyspnea. Angina occurs in over half of patients with AS and is due to the increased oxygen demand of the hypertrophied myocardium in the setting of reduced oxygen supply secondary to coronary compression. Although some patients may have concomitant coronary disease, angina occurs

1	oxygen demand of the hypertrophied myocardium in the setting of reduced oxygen supply secondary to coronary compression. Although some patients may have concomitant coronary disease, angina occurs without significant epicardial coronary artery disease in half of all patients with AS.151 Syncope is most common during exer-tion, as systemic vasodilation in the setting of a fixed cardiac output causes decreased cerebral perfusion. However, at times, it may occur at rest secondary to paroxysmal atrial fibrillation and subsequent loss of atrial booster pump function. Late find-ings of AS include atrial fibrillation, pulmonary hypertension, systemic venous hypertension, and, rarely, sudden death.Diagnostic Studies. Evidence of LV hypertrophy is found in approximately 85% of patients with AS on routine ECG, though the correlation between the absolute electrocardiographic volt-ages in precordial leads and the severity of AS is poor.2 ECG also may demonstrate signs of left atrial enlargement

1	routine ECG, though the correlation between the absolute electrocardiographic volt-ages in precordial leads and the severity of AS is poor.2 ECG also may demonstrate signs of left atrial enlargement and vari-ous forms and degrees of atrioventricular or intraventricular block due to calcific infiltration of the conduction system. Rou-tine chest X-ray usually demonstrates a normal heart size, with rounding of the left ventricular border and apex. A late finding on chest X-ray is cardiac enlargement, or cardiomegaly, a sign of LV failure.Transthoracic echocardiography is indicated in all patients with a systolic murmur graded ≥2/6, a single second heart sound, or symptoms characteristic of AS.98 Initial TTE examinations are often diagnostic and provide an assessment of left ventricular size and function, the degree of left ventricular hypertrophy, the degree of valvular calcification, and the pres-ence of other associated valvular disease. Doppler evaluation should be performed to define

1	function, the degree of left ventricular hypertrophy, the degree of valvular calcification, and the pres-ence of other associated valvular disease. Doppler evaluation should be performed to define the maximum jet velocity, which is a useful measure for following disease severity and predict-ing clinical outcome.2 Additionally, color flow Doppler assesses the severity of the stenotic lesion by allowing calculation of the mean transvalvular pressure gradient and effective valve orifice area (see Table 21-10).98 Follow-up TTE is indicated depending on the severity of AS in order to assess changes from baseline parameters and direct the timing of surgery: yearly for severe AS; every 1 to 2 years for moderate AS; and every 3 to 5 years for mild AS. Any abrupt change in signs or symptoms in a patient with AS is an indication for TTE examination.Additional preoperative studies may be necessary in some patients. Rarely, when TTE images are suboptimal, TEE or fluo-roscopy may be indicated to

1	patient with AS is an indication for TTE examination.Additional preoperative studies may be necessary in some patients. Rarely, when TTE images are suboptimal, TEE or fluo-roscopy may be indicated to assess the degree of valve calcifica-tion and effective valve orifice area. As in other patients with valvular heart disease, coronary angiography should be per-formed prior to aortic valve surgery in most patients.98 Since the symptoms of AS often mimic those of ischemic heart disease, left heart catheterization and coronary angiography may be nec-essary at the initial evaluation in patients with AS. Stress echo-cardiography may also be useful in the asymptomatic patient with AS in order to elicit exercise-induced symptoms or abnor-mal blood pressure responses during exertion. It is also useful in the evaluation of low-gradient AS in patients with depressed LV function.98 However, exercise stress-echocardiography is con-traindicated in patients with ischemic heart disease.98 In patients

1	in the evaluation of low-gradient AS in patients with depressed LV function.98 However, exercise stress-echocardiography is con-traindicated in patients with ischemic heart disease.98 In patients with evidence of aortic root disease by TTE, chest computed tomography is useful in evaluating aortic dilatation at several anatomic levels and is necessary for clinical decision making and surgical planning.2Indications for Operation. Based on the severity of AS (see Table 21-10) and the predicted risk with surgical aortic valve replacement (SAVR) determined using the STS risk calculator, SAVR or transcatheter aortic valve replacement (TAVR) may be recommended for the treatment of AS (Table 21-11).98,152 As this field is rapidly evolving, attention to guideline updates and a multidisciplinary heart team approach to risk stratification and treatment selection are mandatory. In patients with severe calcific AS, AVR via either approach is the only effective treat-ment, though controversy exists

1	team approach to risk stratification and treatment selection are mandatory. In patients with severe calcific AS, AVR via either approach is the only effective treat-ment, though controversy exists as to the timing of intervention in asymptomatic patients. Balloon aortic valvuloplasty creates a modest hemodynamic effect and temporary symptom improve-ment in patients with calcific AS. However, the procedure has not been shown to affect long-term outcomes and is often used in hemodynamically unstable patients as a bridge to AVR.98Aortic InsufficiencyEtiology. The most common cause of isolated aortic insuffi-ciency (AI) in patients undergoing AVR is aortic root disease, and it represents over 50% of such patients in some studies.2 Other common causes of AI include congenital abnormalities of the aortic valve such as bicuspid aortic valve, calcific degenera-tion, rheumatic disease, infective endocarditis, systemic hyper-tension, myxomatous degeneration, dissection of the ascending aorta,

1	the aortic valve such as bicuspid aortic valve, calcific degenera-tion, rheumatic disease, infective endocarditis, systemic hyper-tension, myxomatous degeneration, dissection of the ascending aorta, and Marfan syndrome. Less common causes of AI include traumatic injuries to the aortic valve, ankylosing spondylitis, syphilitic aortitis, rheumatoid arthritis, osteogenesis imperfecta, giant cell aortitis, Ehlers-Danlos syndrome, Reiter’s syndrome, discrete subaortic stenosis, and ventricular septal defects with prolapse of an aortic cusp.96 Although most of these lesions produce chronic aortic insufficiency, rarely acute severe aortic regurgitation can result, often with devastating consequences.Pathology. Regardless of its cause, AI produces volume over-load with dilation and hypertrophy of the left ventricle and sub-sequent dilation of the MV annulus. Depending on the severity of AI, the left atrium may undergo dilation and hypertrophy as well. Frequently, the regurgitant jet causes

1	of the left ventricle and sub-sequent dilation of the MV annulus. Depending on the severity of AI, the left atrium may undergo dilation and hypertrophy as well. Frequently, the regurgitant jet causes endocardial lesions at the site of impact on the left ventricular wall.Diseases causing AI can be classified as primary disorders of the aortic valve leaflets and/or disorders involving the wall of the aortic root. Diseases causing dilation of the ascending aorta are a more common indication for AVR due to isolated AI, and they include disorders such as age-related (degenera-tive) aortic dilation, cystic medial necrosis of the aorta as is seen in Marfan syndrome, aortic dilation secondary to bicuspid valves, and aortic dissection, to name a few.153 In these disor-ders, the aortic annulus becomes dilated, causing separation of the valve leaflets and subsequent AI. The diseased aortic wall may dissect secondarily and further escalate regurgitation across the valve, and secondary thickening

1	dilated, causing separation of the valve leaflets and subsequent AI. The diseased aortic wall may dissect secondarily and further escalate regurgitation across the valve, and secondary thickening and shortening of the valve cusps may occur due to undue tension placed on the valvular apparatus by the dilated aortic root. As the disease progresses, the valve leaflets become too small to close the aortic orifice, causing further aortic insufficiency and exacerbating dilation of the ascending aorta.There are also many primary valvular diseases that cause AI, generally in association with AS. One such disorder is age-related calcific AS, which causes some degree of AI in up to 75% of patients.154 Infective endocarditis may involve the aortic valve apparatus and cause AI through direct destruction of the valve leaflets, perforation of a leaflet, or formation of vegetations that interfere with proper coaptation of the valve Brunicardi_Ch21_p0801-p0852.indd 82501/03/19 5:32 PM 826SPECIFIC

1	of the valve leaflets, perforation of a leaflet, or formation of vegetations that interfere with proper coaptation of the valve Brunicardi_Ch21_p0801-p0852.indd 82501/03/19 5:32 PM 826SPECIFIC CONSIDERATIONSPART IITable 21-11Data from ACC/AHA guidelines for AV surgery in specific clinical contextsCLINICAL SETTINGCLASS OF RECOMMENDATIONLEVEL OF EVIDENCEBalloon Valvotomy for Aortic Stenosis• Bridge to surgery in hemodynamically unstable patients with AS at high risk for AVRIIbC• Palliation in adult patients with AS, who are not candidates for AVRIIbC• Alternative to AVR in adult patients with ASIII – HarmBSurgery for Aortic Stenosis• Symptomatic patients with severe ASIB• Severe AS in the setting of1) Concomitant CABG2) Concomitant valvular or aortic surgery3) LV systolic dysfunction (LVEF <0.50)IC• Moderate AS in the setting of1) Concomitant CABG2) Concomitant valvular or aortic surgeryIIaB• Asymptomatic patients with severe AS and• Abnormal response to exercise• High likelihood of

1	<0.50)IC• Moderate AS in the setting of1) Concomitant CABG2) Concomitant valvular or aortic surgeryIIaB• Asymptomatic patients with severe AS and• Abnormal response to exercise• High likelihood of rapid progression• High likelihood of delay if surgery is withheld until time of symptom onset• Expected operative mortality ≤1.0%IIbC• Mild AS in patients undergoing CABG, when there is high likelihood of rapid progressionIIbC• AVR for prevention of sudden death in asymptomatic patients with AS without any of the findings aboveIII – HarmBSurgery for Aortic Insufficiency• Symptomatic patients with severe AIIB• Asymptomatic patients with chronic severe AI in the setting of1) Concomitant CABG2) Concomitant valvular or aortic surgery3) LV systolic dysfunction (LVEF ≤0.50)ICCB• Asymptomatic patients with severe AI, normal LV systolic function (LVEF >0.50), but severe LV dilatation (end-diastolic dimension >75 mm, end-systolic dimension >55 mm)IIaB• Moderate AI in the setting of1) Concomitant

1	with severe AI, normal LV systolic function (LVEF >0.50), but severe LV dilatation (end-diastolic dimension >75 mm, end-systolic dimension >55 mm)IIaB• Moderate AI in the setting of1) Concomitant CABG2) Concomitant surgery on the ascending aortaIIbC• Asymptomatic patients with severe AI, normal LV systolic function at rest (LVEF >0.50), and LV dilatation (end-diastolic dimension ≥70 mm, end-systolic dimension ≥50 mm) in the setting of1) Progressive LV dilatation2) Declining exercise tolerance3) Abnormal hemodynamic responses to exerciseIIbC• Asymptomatic patients with mild, moderate, or severe AI and normal LV systolic function (LVEF >0.50), when the degree of LV dilatation is not moderate or severe (end-diastolic dimension <70 mm, end-systolic dimension <50 mm)III – HarmBAS = aortic stenosis; AVR = aortic valve replacement; CABG = coronary artery bypass grafting; LV = left ventricular; LVEF = left ventricular ejection fraction; NYHA = New York Heart Association.cusps. Rheumatic

1	stenosis; AVR = aortic valve replacement; CABG = coronary artery bypass grafting; LV = left ventricular; LVEF = left ventricular ejection fraction; NYHA = New York Heart Association.cusps. Rheumatic disease causes fibrous infiltration of the valve cusps and subsequent retraction of the valve leaflets, inhibiting apposition of the cusps during diastole and producing a central regurgitant jet. Patients with large ventricular septal defects or membranous subaortic stenosis may develop progressive AI, owing to a Venturi effect that results in prolapse of the aortic valve leaflets.Pathophysiology. The basic pathophysiologic abnormality of AI is the retrograde flow of a portion of the LV stroke volume into the LV during diastole, producing volume overload.Acute severe AI results most commonly from infective endocarditis, acute aortic dissection, or trauma, and it causes a sudden volume overload of the left ventricle.54 Although an acute increase in preload provides a small increase in

1	from infective endocarditis, acute aortic dissection, or trauma, and it causes a sudden volume overload of the left ventricle.54 Although an acute increase in preload provides a small increase in overall Brunicardi_Ch21_p0801-p0852.indd 82601/03/19 5:32 PM 827ACQUIRED HEART DISEASECHAPTER 21stroke volume due to the Starling mechanism, the left ventricle often is unable to accommodate the large regurgitant volume and maintain forward stroke volume in the acute setting due to a lack of remodeling. Left ventricular end-diastolic and left atrial pressures increase dramatically as the LV is unable to develop compensatory chamber dilation. Although tachycardia develops as a compensatory mechanism to maintain forward flow, this attempt is often inadequate, and patients frequently present in heart failure and even cardiogenic shock. Moreover, subendo-cardial myocardial ischemia frequently develops as a result of decreased coronary diastolic perfusion pressures and increased LV

1	present in heart failure and even cardiogenic shock. Moreover, subendo-cardial myocardial ischemia frequently develops as a result of decreased coronary diastolic perfusion pressures and increased LV end-diastolic pressure, as well as increased myocardial oxy-gen demand due to acute dilation. In the setting of a chronic ventricular hypertrophy and preexisting diastolic dysfunction, the pressure-volume relationship is even more extreme, exacer-bating the hemodynamic derangements seen in acute AI.Chronic AI generally has a more indolent course, with vol-ume overload of the LV causing compensatory increases in left ventricular end-diastolic volume and chamber compliance as well as a combination of eccentric and concentric hypertrophy.155 Compensatory remodeling of the LV allows for accommodation of the regurgitant volume without a significant increase in fill-ing pressures and maintains the preload reserve of the cham-ber. Eccentric left ventricular hypertrophy develops, permitting

1	of the regurgitant volume without a significant increase in fill-ing pressures and maintains the preload reserve of the cham-ber. Eccentric left ventricular hypertrophy develops, permitting normal contractile performance across the enlarged chamber circumference and subsequent ejection of a larger total stroke volume in order to maintain forward flow, despite the regurgi-tant fraction.155,156 However, the enlarged chamber size results in an increase in systolic myocardial wall stress and causes further ventricular hypertrophy. As the disease progresses, recruitment of preload reserve and compensatory hypertrophy maintains ejection fraction within the normal range despite elevated after-load, causing many patients to remain asymptomatic throughout the compensatory phase.155,157Eventually, left ventricular compensatory mechanisms fail, and systolic dysfunction ensues. As the disease progresses, preload reserve may become exhausted, the hypertrophic response may become inadequate, and

1	left ventricular compensatory mechanisms fail, and systolic dysfunction ensues. As the disease progresses, preload reserve may become exhausted, the hypertrophic response may become inadequate, and impaired myocardial contractility may develop so that ejection fraction begins to decline.158 Although left ventricular systolic dysfunction related to excessive afterload is reversible early in the course, irrevers-ible damage occurs once chamber enlargement predominates as the primary cause of diminished myocardial contractility.Clinical Manifestations. In cases of acute severe AI, patients are symptomatic and invariably present with compensatory tachycardia, often associated with acute pulmonary congestion and cardiogenic shock.2 Because the left ventricular and aortic pressures often equalize before the end of diastole, the diastolic murmur of AI may be short and/or soft. The reduced systolic pressure may attenuate the increase in peripheral pulse pressure seen in chronic AI, and early

1	before the end of diastole, the diastolic murmur of AI may be short and/or soft. The reduced systolic pressure may attenuate the increase in peripheral pulse pressure seen in chronic AI, and early closing of the mitral valve due to elevated left ventricular end-diastolic pressures may diminish the intensity of the first heart sound in the acute setting.In patients with chronic AI, symptoms of heart fail-ure and myocardial ischemia develop after the compensatory phase.2 Patients gradually begin to complain of exertional dys-pnea, fatigue, orthopnea, and paroxysmal nocturnal dyspnea, often after significant myocardial dysfunction has developed. Angina is a common complaint late in the course, especially during sleep when heart rate slows and arterial diastolic pres-sure falls. Patients also may experience exertional angina secondary to diminished coronary perfusion in the setting of myocardial hypertrophy. Occasionally, the compensatory tachy-cardia that develops with chronic AI will

1	may experience exertional angina secondary to diminished coronary perfusion in the setting of myocardial hypertrophy. Occasionally, the compensatory tachy-cardia that develops with chronic AI will cause palpitations, and the increased pulse pressure will cause a sensation of pound-ing in the patient’s head. Peripherally, the widened pulse pres-sure causes a forceful, bounding, and quickly collapsing pulse known as Corrigan’s water-hammer pulse. Premature ventricu-lar contractions have been reported to cause particularly trou-bling symptoms, owing to the heave of the volume-loaded left ventricle during the postextrasystolic beat. The classic ausculta-tory finding associated with AI is a high-pitched decrescendo diastolic murmur heard best in the left third intercostal space; an associated S3 gallop is often indicative of late disease. The Austin Flint murmur has also been described, and it is heard as a middiastolic rumble at the apex that simulates mitral stenosis and occurs in severe

1	gallop is often indicative of late disease. The Austin Flint murmur has also been described, and it is heard as a middiastolic rumble at the apex that simulates mitral stenosis and occurs in severe AI when the regurgitant jet impedes mitral valve leaflet opening.Diagnostic Studies. In the acute setting, TTE should be per-formed to confirm the presence and severity of aortic regurgita-tion, the degree of pulmonary hypertension, and the cause of valvular dysfunction.98 When aortic dissection is suspected as the cause of acute AI, TEE or chest CT angiography may be substituted if more readily available.159,160 In these patients with confirmed aortic dissection, cardiac catheterization and coro-nary angiography are rarely indicated and can delay life-saving urgent surgical intervention.In cases of chronic AI, the ECG frequently demonstrates left axis deviation and, late in the course, intraventricular con-duction defects associated with left ventricular dysfunction. On chest X-ray, the

1	of chronic AI, the ECG frequently demonstrates left axis deviation and, late in the course, intraventricular con-duction defects associated with left ventricular dysfunction. On chest X-ray, the left ventricle enlarges predominantly in an infe-rior and leftward direction, causing marked increase in the long axis diameter of the heart, frequently with little or no change in the transverse diameter. The chest X-ray should be examined for aneurysmal dilation of the aorta.2 An initial TTE should be performed to confirm the diagnosis and severity of AI, assess the cause of AI (including valve morphology and aortic root size and morphology), and assess the degree of left ventricular hypertrophy, volume, and systolic function.98 Follow-up TTE is indicated on an annual or semiannual basis in patients with asymptomatic moderate to severe AI in order to assess changes from baseline parameters and direct the timing of surgery. Any abrupt change in signs or symptoms in a patient with chronic AI

1	with asymptomatic moderate to severe AI in order to assess changes from baseline parameters and direct the timing of surgery. Any abrupt change in signs or symptoms in a patient with chronic AI is an indication for TTE examination.Additional preoperative studies are indicated in certain patient populations.98 In patients with poor windows on TTE, TEE or MRI is indicated for initial and serial assessment of AI severity and left ventricular volume and function at rest. In symptomatic patients with chronic AI, it is reasonable to pro-ceed directly to TEE or cardiac catheterization if TTE examina-tions are inadequate. Exercise stress testing may be helpful for an assessment of functional capacity and symptomatic responses in patients with equivocal symptoms. Coronary angiography should be performed prior to valve surgery in most patients.98Indications for Operation. Based on the morphology and severity of valve dysfunction (see Table 21-10), AV repair or replacement may be performed for

1	prior to valve surgery in most patients.98Indications for Operation. Based on the morphology and severity of valve dysfunction (see Table 21-10), AV repair or replacement may be performed for the treatment of AI (see Table 21-11).96 Although the indications for AV repair and AV replacement do not differ, it is recommended that AV repair be performed only in those surgical centers that have developed the appropriate technical expertise, gained experience in patient selection, and which have demonstrated outcomes equivalent to those of valve replacement.Brunicardi_Ch21_p0801-p0852.indd 82701/03/19 5:32 PM 828SPECIFIC CONSIDERATIONSPART IIAortic Valve Operative Techniques and ResultsAortic valve surgery has traditionally been performed through a median sternotomy with the assistance of cardiopulmonary bypass. However, minimally invasive incisions for aortic valve surgery have been introduced, including mini-sternotomy and mini-thoracotomy approaches. After the aorta is cross-clamped,

1	bypass. However, minimally invasive incisions for aortic valve surgery have been introduced, including mini-sternotomy and mini-thoracotomy approaches. After the aorta is cross-clamped, cold blood cardioplegia is delivered antegrade through the aortic root and/or retrograde through the coronary sinus.Aortic Valve Replacement. During aortic valve replacement, an aortotomy is performed, extending medially from approxi-mately 1 to 2 cm above the right coronary artery and inferiorly into the noncoronary sinus. The valve is completely excised. The annulus is thoroughly debrided of calcium deposits. At this point, the annulus is sized and an appropriate prosthesis is selected. Pledgeted horizontal mattress sutures are then placed into the aortic valve annulus and subsequently through the sew-ing ring of the prosthetic valve, taking care to avoid damage to the coronary ostia, the conduction system, and the MV appara-tus. The annular sutures may be placed from below the annulus, seating the

1	ring of the prosthetic valve, taking care to avoid damage to the coronary ostia, the conduction system, and the MV appara-tus. The annular sutures may be placed from below the annulus, seating the valve supra-annularly, or from above the annulus for intra-annular placement (Fig. 21-11).The major components to increased operative risk associ-ated with surgical AVR include age, body surface area, diabetes, renal failure, hypertension, chronic lung disease, peripheral vas-cular disease, neurologic events, infectious endocarditis, previ-ous cardiac surgery, myocardial infarction, cardiogenic shock, NYHA functional status, and pulmonary hypertension. For most patients, the risk of operative mortality associated with AVR is 1% to 5%, and 5-year survival has been reported to be >80%, even in patients >70 years of age.138-161 The choice of valve is dependent on many patient-related factors, and it is accompa-nied by the attendant postoperative risks of decreased durability and thromboembolic

1	>70 years of age.138-161 The choice of valve is dependent on many patient-related factors, and it is accompa-nied by the attendant postoperative risks of decreased durability and thromboembolic vs. hemorrhagic complications for biologi-cal and mechanical valves, respectively.Aortic Valve Repair. Although aortic valve replacement is performed more commonly, AV repair may be recommended at centers of excellence.96For patients with aortoannular ectasia, AI is due to annular dilatation and distortion of the sinotubular junction. For these Figure 21-11. Aortic valve replacement. The stented porcine bio-prosthesis as viewed through an aortotomy.patients, competence of the aortic valve can be achieved by functionally repairing the annulus in a method analogous to homograft implantation. The aneurysmal portion of the aor-tic root is excised, and the aortic valve is reimplanted inside a tubular Dacron graft, with concomitant reimplantation of the coronary arteries. Alternatively, the

1	The aneurysmal portion of the aor-tic root is excised, and the aortic valve is reimplanted inside a tubular Dacron graft, with concomitant reimplantation of the coronary arteries. Alternatively, the aneurysmal tissue and supravalvular tissue can be excised in their entirety, with subse-quent implantation of the Dacron graft onto the superior aspect of the annulus and reimplantation of the coronary arteries.Valve-sparing root replacement for root and annular sta-bilization in patients with AI due to aortoannular ectasia has led to a more durable outcome than is seen with subcommis-sural annuloplasty or leaflet-related procedures alone. One study demonstrated equivalent overall survival between patients undergoing subcommissural annuloplasty or aortic valve repair without annuloplasty and patients undergoing valve-sparing root replacement at 6 years.162 However, patients who under-went valve-sparing root replacement had higher freedom from reoperation and aortic insufficiency >2+ (100%

1	undergoing valve-sparing root replacement at 6 years.162 However, patients who under-went valve-sparing root replacement had higher freedom from reoperation and aortic insufficiency >2+ (100% vs. 90%, P = 0.03; and 100% vs. 77%, P = 0.002, respectively) at midterm follow-up.162For patients with AI associated with redundant leaflet tis-sue, aortic valve repair may be accomplished with free margin plication or resuspension of the valve cusps, with or without triangular resection of the redundant segment. Excision of the diseased portion of the involved valve cusp improves symme-try of the valve leaflets, and annular plication of one or both commissures helps to ensure adequate coaptation. Generally, the free margins of the excised leaflets are reapproximated pri-marily, but in the absence of adequate cusp tissue, a triangular autologous or bovine pericardial patch may be used for cusp restoration.AV cusp repair with a free margin plication or resuspen-sion technique has demonstrated

1	of adequate cusp tissue, a triangular autologous or bovine pericardial patch may be used for cusp restoration.AV cusp repair with a free margin plication or resuspen-sion technique has demonstrated encouraging results in expert centers, both in patients with tricuspid and bicuspid aortic valves. Freedom from AV reoperation in patients with a tricus-pid AV has been reported to be 89% to 92% at 10 years, with a freedom from recurrent AI >2+ of 80% to 86% at the same time point. In patients with bicuspid aortic valves, who gener-ally represent a younger cohort of patients, 10-year survival has been reported at 94% following AV repair, with a freedom from AV reoperation of 81% at the same time point.163Ross Procedure. As mentioned previously, the Ross procedure involves replacing the diseased AV with the patient’s native pulmonary valve as an autograft, which is in turn replaced with a homograft in the pulmonic position.112 The autograft may be implanted in the aortic position directly

1	AV with the patient’s native pulmonary valve as an autograft, which is in turn replaced with a homograft in the pulmonic position.112 The autograft may be implanted in the aortic position directly with resuspension of the valve commissures, or in association with a root replacement, which requires reimplantation of the coronary ostia.The cylinder root replacement technique is most reproduc-ible and involves transecting the native aorta approximately 5 mm above the sinotubular ridge, with subsequent excision of the aortic valve leaflets and supra-annular tissue. The main pulmonary artery is transected at the bifurcation, and the right ventricular outflow tract is incised, allowing the pulmonary valve and artery to be removed en bloc from the outflow tract. The annulus of the pulmonary autograft is sewn to the native aortic annulus with continuous or interrupted sutures, and the coronary ostia are reimplanted into the pulmonary artery graft. The pulmonary valve and right ventricular

1	is sewn to the native aortic annulus with continuous or interrupted sutures, and the coronary ostia are reimplanted into the pulmonary artery graft. The pulmonary valve and right ventricular outflow tract are subsequently reconstructed using an aortic homograft.Brunicardi_Ch21_p0801-p0852.indd 82801/03/19 5:32 PM 829ACQUIRED HEART DISEASECHAPTER 21The primary benefit of the Ross procedure compared to traditional AV surgery is a low risk of thromboembolism with-out the need for systemic anticoagulation. Although patients undergoing the Ross procedure are generally younger, periop-erative mortality has been reported to be as low as 2.5% in this group, with an overall survival of 90% at 18-year follow-up.164 However, the long-term durability of the procedure is somewhat questionable. Although Ross reported a freedom from autograft replacement of 75% at 20 years, other groups have reported freedom from autograft reoperation and allograft reinterven-tion of 51% and 82%, respectively,

1	Ross reported a freedom from autograft replacement of 75% at 20 years, other groups have reported freedom from autograft reoperation and allograft reinterven-tion of 51% and 82%, respectively, at 18-year follow-up.164,165 Progressive aortic insufficiency has been described as a cause of late failure in these patients, as well as calcification of the pulmonary homograft and pulmonary stenosis.Transcatheter Aortic Valve Replacement. Transcatheter aortic valve replacement (TAVR) has proven beneficial for the treatment of AS in patients who are either moderate or high-risk candidates for conventional surgery. TAVR is now indicated for patients with severe AS with a STS score predicted risk of mortality of greater than or equal to 3%.152 Clinical trials in the low-risk population are currently underway.There are two types of transcatheter valves that are approved for commercial use: a balloon-expandable valve (Edwards) and a self-expandable valve (CoreValve). A trans-catheter valve may be

1	underway.There are two types of transcatheter valves that are approved for commercial use: a balloon-expandable valve (Edwards) and a self-expandable valve (CoreValve). A trans-catheter valve may be inserted via the femoral artery, the left subclavian artery, the ascending thoracic aorta via an upper mini-sternotomy, or LV apex via a small left anterior thoracot-omy. By far the most common route is transfemoral, making up the majority of TAVR in most centers. The principle of valves placed via these routes are to place the aortic prosthesis inside the patient’s native aortic valve. Rigorous preoperative plan-ning is needed to ensure adequate sizing of the valve as well as placement to ensure there is no risk of coronary occlusion or malalignment of the valve.A series of large, multicenter clinical trials have been per-formed investigating the role and safety of TAVR in patients with severe aortic stenosis requiring surgical treatment. The PARTNER I trial looked at mortality rate as

1	clinical trials have been per-formed investigating the role and safety of TAVR in patients with severe aortic stenosis requiring surgical treatment. The PARTNER I trial looked at mortality rate as the primary endpoint in patients with severe aortic stenosis who were not suitable can-didates for surgery (high-risk patients).166 TAVR, as compared with standard surgical treatment (SAVR), significantly reduced the rates of death from any cause (30.7% vs. 50.7%, at 1 year, P <0.001), the composite endpoint of death from any cause or repeat hospitalization (42.5% vs. 71.6%, P <0.001), and cardiac symptoms (25.2% vs. 58.0%, P <0.001), despite the higher inci-dence of major strokes (5.0% vs. 1.1%, P = 0.06) and major vas-cular events (16.2% vs. 1.1%, P <0.001).166 In the PARTNER II trial, 2032 intermediate-risk patients with severe aortic stenosis were randomly assigned to undergo either TAVR or SAVR.167 It was found that in intermediate-risk patients, TAVR was simi-lar to SAVR with respect

1	patients with severe aortic stenosis were randomly assigned to undergo either TAVR or SAVR.167 It was found that in intermediate-risk patients, TAVR was simi-lar to SAVR with respect to the primary end point of death or disabling stroke (P = 0.001 for noninferiority). TAVR resulted in larger aortic valve areas than did surgery and also resulted in lower rates of acute kidney injury, severe bleeding, and new-onset atrial fibrillation; surgery resulted in fewer major vascu-lar complications and less paravalvular aortic regurgitation.167 Similarly, the Surgical or Transcatheter Aortic-Valve Replace-ment in Intermediate-Risk patients trial (SURTAVI) evaluated the clinical outcomes in intermediate-risk patients with severe, symptomatic aortic stenosis in a randomized trial comparing TAVR (performed with the use of a self-expanding prosthesis) 5with surgical aortic-valve replacement.168 TAVR was found to be a noninferior (i.e., similar composite endpoint of death from any cause or disabling

1	with the use of a self-expanding prosthesis) 5with surgical aortic-valve replacement.168 TAVR was found to be a noninferior (i.e., similar composite endpoint of death from any cause or disabling stroke at 24 months) alternative to surgery in patients with severe aortic stenosis at intermediate surgical risk, with a different pattern of adverse events associ-ated with each procedure. Surgery was associated with higher rates of acute kidney injury, atrial fibrillation, and transfusion requirements, whereas TAVR had higher rates of residual aor-tic regurgitation and need for pacemaker implantation.168 The PARTNER III trial, currently under investigation, is designed to establish the safety and effectiveness of TAVR in patients with severe, calcific aortic stenosis who are at low operative risk for standard surgical aortic valve replacement (SAVR). Ongoing trials will continue to define the roles for SAVR and TAVR in the future.TRICUSPID VALVE DISEASETricuspid Stenosis and

1	operative risk for standard surgical aortic valve replacement (SAVR). Ongoing trials will continue to define the roles for SAVR and TAVR in the future.TRICUSPID VALVE DISEASETricuspid Stenosis and InsufficiencyEtiology. Acquired tricuspid valve (TV) disease can be clas-sified as either organic or functional and affects approximately 0.8% of the general population.169 Tricuspid stenosis (TS) is almost always a result of rheumatic heart disease or rarely endo-carditis. In the case of rheumatic disease, tricuspid stenosis with or without associated insufficiency is invariably associated with mitral valve disease. Other less common causes of obstruction to right atrial emptying include congenital tricuspid atresia, right atrial tumors, and endomyocardial fibrosis.Tricuspid insufficiency (TR), on the other hand, is most often a functional disease caused by secondary dilation of the tricuspid annulus due to pulmonary hypertension and/or right heart failure. This is most commonly caused by

1	on the other hand, is most often a functional disease caused by secondary dilation of the tricuspid annulus due to pulmonary hypertension and/or right heart failure. This is most commonly caused by MV disease. Conditions such as right ventricular infarction and pulmonic stenosis can also lead to increased right ventricular pressures and functional TR. The less common causes of organic TR, with or without associated stenosis, include endocarditis, carcinoid syndrome, radiation therapy, trauma such as repeated endomyo-cardial biopsy, and Marfan syndrome.Pathology. The changes associated with TS closely resemble those associated with MS, including fusion of the commissures.169 In the case of rheumatic disease, mixed TS and TR may result from fusion and shortening of the chordae tendineae, and fusion of the commissures, causing retraction of the valve leaflets. The right atrium is frequently dilated and thickened in chronic TS, and chronic obstruction to right ventricular filling often

1	and fusion of the commissures, causing retraction of the valve leaflets. The right atrium is frequently dilated and thickened in chronic TS, and chronic obstruction to right ventricular filling often produces signs of systemic venous congestion such as hepatomegaly and splenomegaly.2In most cases of TR, dilation and deformation of the tri-cuspid annulus is the most prominent feature; the valve leaflets oftentimes appear stretched but are otherwise pliable and nor-mal in appearance.2 When TR is caused by carcinoid syndrome, white fibrous carcinoid plaques are found on the ventricular sur-faces of the TV, causing the cusps to adhere to the underlying right ventricular wall and stenting the valve open.2Pathophysiology. The basic pathophysiologic abnormality of both TS and severe TR is elevated right atrial pressure, produc-ing systemic congestion and right heart failure. Severe TS is marked by a valve area <1.0 cm2, and severe TR is defined as a vena contracta width of >0.7 cm in

1	elevated right atrial pressure, produc-ing systemic congestion and right heart failure. Severe TS is marked by a valve area <1.0 cm2, and severe TR is defined as a vena contracta width of >0.7 cm in combination with sys-tolic flow reversal in the hepatic veins.98 However, in patients with TS, a diastolic pressure gradient of only 5 mmHg, or a TV Brunicardi_Ch21_p0801-p0852.indd 82901/03/19 5:32 PM 830SPECIFIC CONSIDERATIONSPART IITable 21-12Data from ACC/AHA guidelines for TV surgery in specific clinical contextsCLINICAL SETTINGCLASS OF RECOMMENDATIONLEVEL OF EVIDENCESurgery for Tricuspid Valve Disease• TVr for severe TI in patients with MV disease requiring MV surgeryIB• TVR or annuloplasty for severe symptomatic primary TIIIaC• TVR for severe TI secondary to diseased/abnormal TV leaflets not amenable to annuloplasty or TVrIIaC• Annuloplasty for less than severe TI in patients undergoing MV surgery in the setting of1) Pulmonary hypertension2) Tricuspid annular dilatationIIbC• TVR

1	not amenable to annuloplasty or TVrIIaC• Annuloplasty for less than severe TI in patients undergoing MV surgery in the setting of1) Pulmonary hypertension2) Tricuspid annular dilatationIIbC• TVR or annuloplasty is not indicated in asymptomatic patients with TI, a normal MV, and a PASP <60 mmHgIII – HarmC• TVR or annuloplasty is not indicated in patients with mild primary TIIII – HarmCMV = mitral valve; PASP = pulmonary artery systolic pressure; TI = tricuspid insufficiency; TV = tricuspid valve; TVr = tricuspid valve repair; TVR = tricuspid valve replacement.orifice <1.5 cm2, is frequently enough to cause jugular venous distention, organomegaly, and peripheral edema. In severe cases, cardiac output is compromised, especially during exercise when the fixed obstruction prevents an increase in forward flow. Patients with severe insufficiency and pulmonary hypertension experience similar hemodynamic derangements.Clinical Manifestations. Patients with TS and severe TR develop symptoms of

1	in forward flow. Patients with severe insufficiency and pulmonary hypertension experience similar hemodynamic derangements.Clinical Manifestations. Patients with TS and severe TR develop symptoms of right heart failure associated with chroni-cally elevated right atrial pressures.2 The classic clinical signs and symptoms of TS and severe TR are jugular venous disten-tion, hepatomegaly, splenomegaly, ascites, and lower extremity edema. Uncomfortable fluttering in the neck has been reported in patients with TV disease, and sensations of throbbing in the eyeballs and pulsatile varicose veins have been reported to occur, especially in patients with severe TR.The low cardiac output syndrome occasionally associated with TS and severe TR can cause fatigue, weakness, and exer-cise intolerance in these patients. In the absence of pulmonary hypertension, dyspnea is not a prominent feature of tricuspid disease. The auscultatory findings associated with TS include a presystolic and middiastolic

1	these patients. In the absence of pulmonary hypertension, dyspnea is not a prominent feature of tricuspid disease. The auscultatory findings associated with TS include a presystolic and middiastolic murmur characterized by a tri-cuspid opening snap that increases on inspiration. The lower left parasternal murmur of TR may be holosystolic or less than holosystolic, depending on the degree of regurgitation, may be associated with a middiastolic murmur in severe cases, and may increase on inspiration.Diagnostic Studies. In patients with TV disease, chest X-ray frequently demonstrates enlargement of the right atrium and ventricle. Patients with TS demonstrate an exaggerated a wave and a diminished rate of y descent in the jugular venous pulse, while patients with TR have abnormal systolic c and v waves.2 TTE examination should be performed in patients with TV disease in order to characterize the structure and motion of the TV, the size of the tricuspid annulus, and other cardiac

1	c and v waves.2 TTE examination should be performed in patients with TV disease in order to characterize the structure and motion of the TV, the size of the tricuspid annulus, and other cardiac abnormalities that may affect TV function.98 In patients with a pulmonary artery systolic pressure >55 mmHg, TI commonly occurs in the setting of structurally normal valves; however, structural derangement of the TV apparatus is frequently present if TR is documented with a pulmonary artery systolic pressure <40 mmHg. Doppler TTE allows estimations of the severity of TR, the right ventricular systolic pressure, and the TV diastolic gradient.Indications for Operation. As an isolated lesion, mild or moderate TV disease does not require surgical correction. How-ever, patients with severe TV disease should be considered for surgical intervention, especially in the setting of right ventricu-lar enlargement and impaired systolic function, as this improves life expectancy and the development of

1	should be considered for surgical intervention, especially in the setting of right ventricu-lar enlargement and impaired systolic function, as this improves life expectancy and the development of sequelae such as heart failure and atrial fibrillation.169 Depending on the patient’s clini-cal status and the cause of TV dysfunction, TV repair and TV replacement be variably recommended for the treatment of TV dysfunction (Table 21-12).98 In patients with TR, the valve can usually be repaired with modern techniques.Operative Techniques and Results. The TV can be approached through a median sternotomy, a right thoracotomy, or port-based techniques. Surgery is performed with the assis-tance of cardiopulmonary bypass and, though TV surgery is usually performed on the beating heart, a brief period of car-dioplegic arrest may be rarely needed to allow for complete inspection of the interatrial septum and to close any defects that may be present.TV repair may include a suture or ring

1	period of car-dioplegic arrest may be rarely needed to allow for complete inspection of the interatrial septum and to close any defects that may be present.TV repair may include a suture or ring annuloplasty as well as valvuloplasty, and multiple methods have been described.169 Historically, bicuspidization of the TV was accomplished by a figure-of-eight suture plication of the annulus of the posterior leaflet; however, this technique has been essentially replaced by suture or ring annuloplasty. Suture annuloplasty is gener-ally performed by placing pledgeted sutures along the base of the anterior and posterior leaflets, partially encircling the annulus. Ring annuloplasty can be accomplished by suturing the TV annulus to a variety of rigid or semirigid annuloplasty rings, which generally have an opening at the level of the anterosep-tal commissure to avoid passing the anchoring sutures near to the conduction system. Most surgeons favor ring over suture annuloplasty. In severe annular

1	have an opening at the level of the anterosep-tal commissure to avoid passing the anchoring sutures near to the conduction system. Most surgeons favor ring over suture annuloplasty. In severe annular dilatation, augmentation of the anterior leaflet with autologous pericardium has been used with some success. Tricuspid valvuloplasty is infrequently performed and may include commissurotomy, triangular leaflet resection, Brunicardi_Ch21_p0801-p0852.indd 83001/03/19 5:32 PM 831ACQUIRED HEART DISEASECHAPTER 21primary perforation repair, and traditional leaflet repair tech-niques such as chordal transfer, shortening, and replacement, papillary muscle plication, tricuspid leaflet augmentation, and the edge-to-edge repair technique used in MV prolapse.For patients with functional TV disease, TV repair is gen-erally preferred to replacement due to favorable results without the associated risks of thrombosis and the need for anticoagu-lation. In the setting of concomitant mitral valve

1	TV repair is gen-erally preferred to replacement due to favorable results without the associated risks of thrombosis and the need for anticoagu-lation. In the setting of concomitant mitral valve surgery, TV repair has not been associated with additional perioperative complications, and 5-year freedom from reoperation has been impressive at 98%. However, a subgroup of patients report late failure following TV repair, and this may be worse following suture annuloplasty compared with ring annuloplasty.117Prosthetic valve replacement may be necessary due to extensive leaflet destruction, as may be seen in patients with endocarditis, or marked annular dilatation not amenable to repair. In some cases, the valve prosthesis may be anchored directly to the leaflet tissue instead of the valve annulus, reduc-ing the risk of injury to the conduction system.169 If this tech-nique is used, it should be confirmed that the residual tissue does not interfere with the movement of the prosthetic

1	annulus, reduc-ing the risk of injury to the conduction system.169 If this tech-nique is used, it should be confirmed that the residual tissue does not interfere with the movement of the prosthetic leaflets after implantation. Pledgeted sutures should be used and may be placed on the ventricular or atrial side of the annulus.Outcomes data following TV replacement are difficult to interpret, as most reports are in patients with previous TV surgery and/or signs of severe right heart failure. Operative mortality has been >20% in some studies.169 One study of 87 patients undergoing TV replacement between 1994 and 2007 showed an in-hospital mortality of only 1.4%. The choice of prosthetic valve is also somewhat controversial. Though bio-prosthetic valves are more durable in the tricuspid than mitral or aortic positions, valve degeneration is an important cause of bioprosthetic valve dysfunction at reoperation. The ability to replace a degenerated tricuspid valve bioprosthesis with a

1	than mitral or aortic positions, valve degeneration is an important cause of bioprosthetic valve dysfunction at reoperation. The ability to replace a degenerated tricuspid valve bioprosthesis with a trans-catheter valve has led some surgeons to favor biological valves in this position. This is particularly true since the increased risk of valve thrombosis seen with mechanical valves mandates rig-orous systemic anticoagulation. Even with these precautions, mechanical tricuspid valves are associated with an increased risk of hemorrhagic and thrombotic complications. The choice of valve is usually decided on a case-by-case basis, and late out-comes have been similar with biological and mechanical valves in this position. In general, TV replacement may be a reasonable choice in select patients, though more data are needed regarding long-term outcomes in the modern era.Multivalve DiseasePathology involving multiple valves is relatively common and may result from diseases such as rheumatic

1	though more data are needed regarding long-term outcomes in the modern era.Multivalve DiseasePathology involving multiple valves is relatively common and may result from diseases such as rheumatic fever, calcific dis-ease, Marfan syndrome, and other connective tissue disorders. However, multivalve disease may also be caused by secondary valvular dysfunction due to a distal valvular lesion, as in the case of myxomatous degeneration of the mitral valve, resulting in pulmonary hypertension, dilation of the tricuspid annulus, and functional TR. If the primary pathology is corrected early in the disease course, these secondary functional changes may resolve without the need for intervention.In patients with multivalve disease, the clinical manifesta-tions may be dependent on the severity of each individual valve lesion, but this is not always the case.2 In patients with con-comitant mitral and tricuspid dysfunction, the prominent symp-toms of dyspnea, paroxysmal nocturnal dyspnea, and

1	each individual valve lesion, but this is not always the case.2 In patients with con-comitant mitral and tricuspid dysfunction, the prominent symp-toms of dyspnea, paroxysmal nocturnal dyspnea, and orthopnea commonly associated with MV dysfunction are sometimes diminished by associated TV dysfunction. Symptoms of multi-valve disease are most commonly masked when valvular abnor-malities are of approximately equal severity, highlighting the importance of careful examination of each valve both preopera-tively and in the operating room.Surgery for multivalve disease is associated with a higher perioperative mortality than single-valve procedures, and this risk is exacerbated by factors such as pulmonary artery hyper-tension, age, triple-valve procedures, concomitant coronary artery bypass grafting, previous heart surgery, renal insuffi-ciency, and diabetes.170 Failing to recognize significant con-comitant valvular dysfunction at the time of surgery is also associated with higher

1	grafting, previous heart surgery, renal insuffi-ciency, and diabetes.170 Failing to recognize significant con-comitant valvular dysfunction at the time of surgery is also associated with higher perioperative mortality. For this reason, patients suspected of having multivalve involvement should undergo full preoperative Doppler TTE or TEE evaluation and heart catheterization.98 In selected patients, procedures correcting multivalve disease demonstrate significant clinical improvement in symptoms and quality of life, as well as accept-able mortality and survival rates.170SURGICAL THERAPY FOR THE FAILING HEARTEpidemiology of Heart FailureHeart failure affects approximately 5 million patients in the United States, with >550,000 new cases diagnosed annually.171 The disorder is the primary reason for 12 to 15 million office visits and >1 million hospitalizations each year. Overall 1-year mortality is estimated to be around 25%, but this can increase to as high as 75% for patients with more

1	for 12 to 15 million office visits and >1 million hospitalizations each year. Overall 1-year mortality is estimated to be around 25%, but this can increase to as high as 75% for patients with more advanced heart failure (NYHA class IV).172 While heart transplantation remains the gold standard for the treatment of end stage disease, an increas-ing number of patients deteriorate while on the waiting list, and up to 30% die before transplantation.173 The total direct and indirect costs associated with the treatment of heart failure are estimated to be $32 billion, and this is projected to increase to $70 billion by 2030.174 Advances in the surgical management of heart failure over the two decades have pushed surgery for CHF into the mainstream. As a result, there is an increasing number of patients with lateor end-stage disease who are being consid-ered for surgical therapies.Etiology and PathophysiologyHeart failure can be classified as acute or chronic, genetic or acquired, left-sided

1	with lateor end-stage disease who are being consid-ered for surgical therapies.Etiology and PathophysiologyHeart failure can be classified as acute or chronic, genetic or acquired, left-sided and/or right-sided, and systolic and/or diastolic dysfunction. The underlying causes and treatments for each of these vary considerably. In the Framingham Heart Study, coronary artery disease accounted for 67% of heart fail-ure cases, valvular heart disease accounted for 10%, and 20% of cases were attributable to primary myocardial diseases, of which dilated cardiomyopathy predominated.175 In all cases, heart failure is a progressive disorder that through complex mechanisms of ventricular remodeling, altered hemodynamics, neurohumoral activation, cytokine overexpression, and vascu-lar and endothelial dysfunction either disrupts the ability of the myocardium to generate force or results in a loss of function-ing cardiac myocytes, thereby preventing normal myocardial contraction.CABG for Ischemic

1	dysfunction either disrupts the ability of the myocardium to generate force or results in a loss of function-ing cardiac myocytes, thereby preventing normal myocardial contraction.CABG for Ischemic CardiomyopathySurgical coronary revascularization is among the most com-monly performed procedures for CHF. CABG is beneficial as it protects from further myocardial infarction and/or malignant Brunicardi_Ch21_p0801-p0852.indd 83101/03/19 5:32 PM 832SPECIFIC CONSIDERATIONSPART IIventricular arrhythmias. It is most successful when treating hibernating as opposed to infarcted myocardium.While the majority of evidence supporting CABG for patients with ischemic cardiomyopathy comes from nonran-domized, retrospective studies, the prospective, randomized, multicenter international Surgical Treatment of Ischemic Heart Failure (STICH) trial compared CABG with medical therapy to medical therapy alone. Entry criteria included an EF ≤35% with CAD and anatomy suitable for CABG. No significant

1	of Ischemic Heart Failure (STICH) trial compared CABG with medical therapy to medical therapy alone. Entry criteria included an EF ≤35% with CAD and anatomy suitable for CABG. No significant difference was seen in overall mortality by study completion, but patients who underwent CABG did have fewer deaths or hospitalizations from cardiovascular causes (58% vs. 68%, P <0.001).176,177Myocardial viability testing has been shown by multiple studies to be pivotal in identifying patients that will have improved outcomes following CABG for ischemic cardiomyopathy.178,179 A meta-analysis performed by Allman et al demonstrated an 80% reduction in mortality in patients who underwent revas-cularization with viable myocardium compared to patients who received medical therapy alone (3.2% vs. 16%, P <0.0001). Most importantly, in this analysis, CABG had no benefit over medical therapy for patients without viable myocardium. A more recent study by Gerber et al prospectively compared CABG and medical

1	Most importantly, in this analysis, CABG had no benefit over medical therapy for patients without viable myocardium. A more recent study by Gerber et al prospectively compared CABG and medical therapy to medical therapy alone in 114 patients with CAD and low EF (24% ± 8%) who underwent viability testing using delayed-enhancement cardiac MRI.180 This study demon-strated worse 3-year survival in medically treated patients with dysfunctional but viable myocardium than in medically treated patients with nonviable myocardium (48% vs. 77%, P = 0.02). This corresponded with a 4.56 times increased hazard of death when medical treatment was selected over full revasculariza-tion. In contrast, survival after CABG was not significantly different whether myocardium was viable or not (88% vs. 71%, P = NS). These studies underscore both the importance of viable myocardium as well as the adverse consequences of not offering a patient with viability surgical intervention.Patients with ischemic

1	P = NS). These studies underscore both the importance of viable myocardium as well as the adverse consequences of not offering a patient with viability surgical intervention.Patients with ischemic cardiomyopathy are a heterogeneous group, and, as with any surgery, appropriate patient selection is central to success. In one retrospective study of 96 patients with ischemic cardiomyopathy (EF ≤25%), age and poor distal vessel quality were predictors of poor outcomes.181 Mortality in patients with poor vessel quality was 100%, compared with 90% when vessel quality was fair and 10% when it was good. Therefore, poor vessel quality should be considered a contraindication to surgical revascularization even in the presence of angina.LV size and LV dyssynchrony are also risk factors for adverse shortand intermediate-term outcomes. A LV end-diastolic dimension of >100 mL/m2 is associated with a signifi-cantly reduced 5-year survival following CABG (85% vs. 53%, P <0.05), as well as worse 5-year

1	intermediate-term outcomes. A LV end-diastolic dimension of >100 mL/m2 is associated with a signifi-cantly reduced 5-year survival following CABG (85% vs. 53%, P <0.05), as well as worse 5-year freedom from recurrent CHF (85% vs. 31%).182 Moreover, LV dyssynchrony has been shown to have a significant impact on mortality in patients undergo-ing moderateto high-risk revascularization and may compound risk in patients with nonviable myocardium.183 In patients with severe preoperative LV dyssynchrony, the 30-day mortality was 27% vs. 3% in patients without significant dyssynchrony (P <0.001). Similar differences were seen with the presence of postoperative LV dyssynchrony, and outcomes were worse when patients also had fewer segments of viable myocardium.Secondary Mitral RegurgitationSecondary mitral regurgitation describes MR that results from damage to the left ventricle as a result of either ischemia or dilated cardiomyopathy rather than from a problem with the valve itself.184

1	mitral regurgitation describes MR that results from damage to the left ventricle as a result of either ischemia or dilated cardiomyopathy rather than from a problem with the valve itself.184 Ischemic MR (IMR) typically results from sys-tolic restriction of the mitral leaflets due to tethering of the subvalvular apparatus. This occurs mainly from regional wall motion abnormalities in areas of the LV adjacent to papillary muscle attachments. Alterations in the size and shape of the mitral annulus and posterior displacement of the posteromedial papillary muscle, which occurs primarily after an inferoposterior MI, may also contribute. Additionally, functional MR (FMR) is caused by LV dilatation and increased sphericity, which displace the papillary muscles apically and radially, creating lateral forces on the valve that lead to increased retraction of the mitral leaflets by the chordae tendineae. LV dyssynchrony may also contribute to FMR through poor coordination of the contraction of

1	forces on the valve that lead to increased retraction of the mitral leaflets by the chordae tendineae. LV dyssynchrony may also contribute to FMR through poor coordination of the contraction of the septum and lateral walls, producing MR that may vary in intensity during the cardiac cycle. Functional MR is usually referred to as a Carpentier class I/IIIb lesion due to the presence of both annular dilatation (Carpentier type I) and systolic restriction of the mitral leaflets due to LV dysfunction (Carpentier type IIIb). Ultimately, increased regurgitation leads to increased preload, LV wall tension, and LV work load, all of which contribute to progressive dysfunction of the LV and worsening heart failure.Several observational and population-based studies have demonstrated a significant impact of secondary MR on long-term survival. Following MI, the 5-year survival rate dropped significantly from 61% in patients who did not have MR to 47% and 29% in patients with mild and moderate to

1	of secondary MR on long-term survival. Following MI, the 5-year survival rate dropped significantly from 61% in patients who did not have MR to 47% and 29% in patients with mild and moderate to severe MR, respectively.185 Similarly, in a series of 2057 patients with symptomatic heart failure and an LVEF <40%, the 5-year sur-vival rate for patients without MR was 54%, and it decreased to 40% in patients with moderate to severe secondary MR.186 Moreover, medical therapy and PCI have not reduced the impact of IMR on late mortality.187Although specific recommendations to intervene for secondary MR are controversial and have not been rigorously defined, guidelines are available (Table 21-13).96,188 Some sur-geons initially advocated performing only revascularization in cases of moderate ischemic MR with the idea that revasculariz-ing viable myocardium would lead to improvements in LV func-tion and effect reverse remodeling, ultimately contributing to a decrease in MR. While several

1	ischemic MR with the idea that revasculariz-ing viable myocardium would lead to improvements in LV func-tion and effect reverse remodeling, ultimately contributing to a decrease in MR. While several studies, including a recent large, multicenter, randomized, controlled trial, have shown that MR often persists following revascularization alone, the addition of a mitral valve annuloplasty in those studies did not improve long-term functional status or survival in patients with isch-emic MR.189-192 Nevertheless, other studies have shown that the persistence of MR after CABG is associated with a decreased survival rate and that CABG alone only has modest effects on reducing MR at 1 month follow-up.184 As a result, some centers continue to repair moderate MR in this patient population. Indi-cations for surgery in ischemic MR patients in the absence of revascularization options are even less well defined.For patients with functional MR, the goal of mitral valve surgery is to avoid or

1	for surgery in ischemic MR patients in the absence of revascularization options are even less well defined.For patients with functional MR, the goal of mitral valve surgery is to avoid or postpone transplantation in eligible patients. Mitral valve repair has been considered the procedure of choice when surgery is indicated for secondary MR. How-ever, in patients with severe ischemic MR, a recent randomized, multicenter trial showed improved late freedom from moderate or severe recurrent MR with mitral valve replacement compared to repair (2.3% vs. 32.6%, P <0.001).141 There was not a signifi-cantly higher mortality in the replacement group in this study.141 Brunicardi_Ch21_p0801-p0852.indd 83201/03/19 5:32 PM 833ACQUIRED HEART DISEASECHAPTER 21Table 21-13Data guidelines for surgical intervention for secondary mitral regurgitationCLINICAL SETTINGCLASS OF RECOMMENDATIONLEVEL OF EVIDENCEChronic Ischemic MR (ESC Guidelines)• Severe MR, LVEF >30%, undergoing CABGIC• Moderate MR,

1	intervention for secondary mitral regurgitationCLINICAL SETTINGCLASS OF RECOMMENDATIONLEVEL OF EVIDENCEChronic Ischemic MR (ESC Guidelines)• Severe MR, LVEF >30%, undergoing CABGIC• Moderate MR, undergoing CABG, if mitral repair is feasibleIIaC• Severe MR, symptomatic patients, LVEF <30%, candidate for revascularizationIIaC• Severe MR, LVEF >30%, no option for revascularization, refractory to optimal medical therapy, low comorbidityIIbCChronic Functional MR (ESC and ACC/AHA Guidelines)• Chronic severe MR due to LV dysfunction, EF <30%, persistent NYHA class III-IV, symptoms despite optimal medical therapyIIbCMR = mitral regurgitation; ESC = European Society of Cardiology; CABG = coronary artery bypass grafting; LVEF = left ventricular ejection fraction; LV = left ventricle; ACC = American College of Cardiology; AHA = American Heart Association; NYHA = New York Heart Association.In patients with poor LV function, dilated LV, and severe MR with significant leaflet tethering, we favor MV

1	College of Cardiology; AHA = American Heart Association; NYHA = New York Heart Association.In patients with poor LV function, dilated LV, and severe MR with significant leaflet tethering, we favor MV replacement with preservation of the subvalvular apparatus. Bioprosthetic valve is usually used due to the poor late survival in this group of patients. Currently, for patients undergoing repair recom-mendations are to use a semirigid or rigid annuloplasty ring to downsize the mitral annulus.184 There have also been various techniques proposed to correct the papillary muscle displace-ment, but most reports are single center, retrospective, and small. Mitral valve replacement with preservation of the sub-valvular apparatus is indicated when repair is not feasible due to severe tethering of the leaflets or massive LV dilation.Outcomes from surgery vary among centers and among patients in this heterogeneous group. Operative mortality ranges between 0% and 9% in most modern series.184

1	of the leaflets or massive LV dilation.Outcomes from surgery vary among centers and among patients in this heterogeneous group. Operative mortality ranges between 0% and 9% in most modern series.184 Generally speak-ing, mortality and recurrence rates are higher and long-term prognosis is worse compared to outcomes for primary MR. Recurrent MR is as high as 15% to 30% in some series, and 5-year mortality is between 44% and 48%.184,193,194 Some reduc-tions in left atrial dimension and LV reverse remodeling may be achieved.195Left Ventricular Aneurysmorrhaphy and Surgical Ventricular RestorationPathophysiology of Ventricular Aneurysms. A transmural infarction of approximately 5% to 10% of the myocardium may result in formation of an LV aneurysm as necrotic myocardium is replaced by fibrous tissue. This usually occurs 4 to 8 weeks following the infarct. In the last decade, prompt revasculariza-tion of the culprit artery by either surgical or interventional tech-niques generally results in

1	This usually occurs 4 to 8 weeks following the infarct. In the last decade, prompt revasculariza-tion of the culprit artery by either surgical or interventional tech-niques generally results in sparing of the subepicardial muscle while the subendocardial muscle remains necrotic.196 There-fore, it is not uncommon for the LV wall to show both living myocardium during thallium testing and an akinetic zone on echocardiogram or angiogram. It has been demonstrated that once more than 20% of the myocardium is necrosed there is irreversible progression to ventricular dilation and failure.197 Once heart failure develops after postinfarction remodeling, the 1-year mortality reaches 32% despite current therapies.198 The classic aneurysm is a 4 to 6 mm thick scar, which bulges out-ward in paradoxical motion as the LV contracts during systole. More than 80% develop in the anteroseptal and apical portions of the left ventricle as a result of left anterior descending artery occlusion. The rest are

1	motion as the LV contracts during systole. More than 80% develop in the anteroseptal and apical portions of the left ventricle as a result of left anterior descending artery occlusion. The rest are inferior in location and the result of cir-cumflex or right coronary occlusion.This patient population typically suffers from associated ventricular arrhythmias for several reasons. First, electrical dys-synchrony results from postinfarction remodeling, and triggers for ventricular arrhythmias typically occur in the scar border zone in patients with ischemic cardiomyopathy.199,200 Second, increased ventricular volume causes high wall stress and stretch, and stretch has been shown to be arrhythmogenic.201 Third, LV aneurysms represent an independent risk factor for SCD after MI.202 Surgical ventricular restoration (SVR) addresses each of these issues by removing the anatomic substrate during resection of the postinfarct scar and/or aneurysm, accomplishing volume reduction and mechanical

1	ventricular restoration (SVR) addresses each of these issues by removing the anatomic substrate during resection of the postinfarct scar and/or aneurysm, accomplishing volume reduction and mechanical resynchronization and relieving ischemia through complete revascularization and reduction in myocardial wall tension and oxygen demand.Clinical Presentation and Diagnosis. Symptoms of LV aneurysms include angina, CHF, ventricular arrhythmias, and, rarely, embolic phenomenon. Rupture is extremely uncommon. Patients generally present for coronary artery bypass or during evaluation of CHF or arrhythmias. While transthoracic echo-cardiography gives pertinent information regarding LV func-tion, size, mitral valve function, and the presence of thrombus, it is generally accepted that cardiac MRI is the best diagnostic modality to accurately identify areas of scar and viable tissue and to best define ventricular geometry.197Surgical Treatment and Results. In 1985, Vincent Dor described a surgical

1	best diagnostic modality to accurately identify areas of scar and viable tissue and to best define ventricular geometry.197Surgical Treatment and Results. In 1985, Vincent Dor described a surgical technique called the endoventricular cir-cular patch plasty that was intended to improve geometric reconstruction compared with the standard linear repair in LV aneurysm surgery. SVR is a somewhat broader term that arose from surgical repair of ventricular aneurysms and has now come to be applied to a group of surgical procedures designed to correct the effects of postinfarction ventricular remodeling. It is also sometimes referred to as surgical ventricular remodel-ing or reconstruction, surgical anterior ventricular endocardial reconstruction (SAVER), or the Dor procedure. SVR is specifi-cally intended to reduce the size and sphericity of the LV by Brunicardi_Ch21_p0801-p0852.indd 83301/03/19 5:32 PM 834SPECIFIC CONSIDERATIONSPART IIexcluding akinetic and dyskinetic areas, most often

1	intended to reduce the size and sphericity of the LV by Brunicardi_Ch21_p0801-p0852.indd 83301/03/19 5:32 PM 834SPECIFIC CONSIDERATIONSPART IIexcluding akinetic and dyskinetic areas, most often by using a circular patch inserted inside the ventricle on contractile myo-cardium (Fig. 21-12A,B).Candidates for SVR are typically patients who have had a remote anterior or anteroseptal myocardial infarction, signifi-cant ventricular enlargement with a significant area of akinetic or dyskinetic myocardium, a discrete aneurysm, a clinical pic-ture consistent with heart failure (LVEF <40%), retained func-tion of the basilar and lateral portions of the heart, and good right ventricular function. These patients should also be candi-dates for repair of any other concomitant cardiac disease. Dor currently emphasizes the importance of complete revasculariza-tion and repair of any mitral pathology at the time of operative SVR. In patients with spontaneous (13%) or inducible (25%) ventricular

1	currently emphasizes the importance of complete revasculariza-tion and repair of any mitral pathology at the time of operative SVR. In patients with spontaneous (13%) or inducible (25%) ventricular tachycardia (VT), it is additionally necessary to per-form nonguided endocardial resection and cryoablation encir-cling the resected area.197Results with this approach have been good in treating both heart failure and its sequelae, such as VT. In Dor’s series of 1150 patients, the operative mortality varied based on the LVEF, ranging from 1% (patients with EF >40%) to 13% (patients with EF <30%), and the 5-year survival approached 85%.197 Overall, more than 80% of survivors either stabilized or improved, and the quality of life was shown to improve significantly by 6 months after the Dor procedure.203 This is likely due in part to the fact that the Dor procedure restores LV geometry, resulting in a mean ejection fraction increase between 10% and 15%, with significant alleviation of

1	procedure.203 This is likely due in part to the fact that the Dor procedure restores LV geometry, resulting in a mean ejection fraction increase between 10% and 15%, with significant alleviation of symptoms.197,204-206 These data are rein-forced by the international RESTORE group, which examined SVR in a registry of 1198 postinfarction patients between 1998 and 2003.207 They found that 5-year overall freedom from hos-pital readmission for CHF was 78%. Moreover, 67% of patients had preoperative NYHA class III or IV symptoms, whereas 85% of patients were NYHA functional class I or II postoperatively.With respect to VT, Dor et al reported on 106 patients with ischemic ventricular arrhythmias that underwent recon-struction for postinfarction LV aneurysm and visually directed endocardiectomy plus or minus cryoablation and coronary revascularization.208 At a mean follow-up of 21.3 months, only 10.8% of patients had inducible VT, and no spontaneous VT was documented. Results from similar

1	plus or minus cryoablation and coronary revascularization.208 At a mean follow-up of 21.3 months, only 10.8% of patients had inducible VT, and no spontaneous VT was documented. Results from similar series have also been excellent,197,206,209 but the efficacy of left ventricular restoration alone has been controversial.210,211 Inferior results seen in some Figure 21-12. Surgical ventricular restoration of a ventricular aneurysm using the Dor procedure. A. The size and sphericity of the left ventricle are reduced by excluding akinetic and dyskinetic areas. B. Most often this is completed using a circular patch inserted inside the ventricle on contractile myocardium.series have been attributed to failure to perform endocardial resection and/or cryoablation at the border of the transitional zone, as well as differences in stimulation protocols and pos-sible inadequate volume reduction of the ventricle.A large, randomized, multicenter study, the STICH trial, concluded that adding SVR to

1	zone, as well as differences in stimulation protocols and pos-sible inadequate volume reduction of the ventricle.A large, randomized, multicenter study, the STICH trial, concluded that adding SVR to reduce ventricular volume to CABG does not improve symptoms or exercise tolerance and fails to lower death rate or cardiac rehospitalization compared to CABG alone.176 While this trial has some shortcomings, it has resulted in a marked decrease in referrals for this procedure. The main problem is that the LV volume was reduced by only 19% in the STICH trial, reflecting an inadequate repair as determined by the Surgery Therapy Committee, whose “acceptable STICH procedure” guideline required a 30% reduction at the 4-month postoperative cardiac MRI.212 Previous studies have reported an average reduction of end-systolic volume index (ESVI) of 40% with a range between 30% and 58%, suggesting that the STICH SVR procedure may have involved an inadequately small LV plication or limited

1	average reduction of end-systolic volume index (ESVI) of 40% with a range between 30% and 58%, suggesting that the STICH SVR procedure may have involved an inadequately small LV plication or limited intracavitary reconstruction.212 Moreover, this trial enrolled 13% of patients who had never had an MI and changed criteria such that enrollment required documented LV anterior wall dysfunction rather than demonstration of scar. This could have captured patients with hibernating myocardium that would recover following CABG alone. Dor subsequently pub-lished the results of 117 patients who would have been eligible for the STICH trial and demonstrated durable improvement in left ventricular function.213 However, this was a single-center, retrospective experience. Caution should be exercised so as not to broadly extrapolate the results of the STICH trial and inap-propriately deny appropriate patients effective treatment. This remains an area of controversy.With the recent advances in

1	so as not to broadly extrapolate the results of the STICH trial and inap-propriately deny appropriate patients effective treatment. This remains an area of controversy.With the recent advances in percutaneous interventions, the Parachute device has been trialed in human subjects. It is composed of a self-expanding nitinol frame covered with an impermeable fluoropolymer that is deployed into the LV apex walling off akinetic or dyskinetic segments of the LV. The PARACHUTE trial reported 3-year echocardiographic and clinical outcomes of patients with ischemic heart failure who underwent placement of the Parachute device as a feasibility and safety study. They demonstrated that of the 31 patients who received the device, there was improvement or maintenance of NYHA functional class in 85% as well as significant reduction of the LV EDV index.198Brunicardi_Ch21_p0801-p0852.indd 83401/03/19 5:32 PM 835ACQUIRED HEART DISEASECHAPTER 21Mechanical Circulatory SupportIntra-Aortic Balloon

1	as well as significant reduction of the LV EDV index.198Brunicardi_Ch21_p0801-p0852.indd 83401/03/19 5:32 PM 835ACQUIRED HEART DISEASECHAPTER 21Mechanical Circulatory SupportIntra-Aortic Balloon Pump. The intra-aortic balloon pump (IABP) is a commonly used device for mechanical circulatory support and has been in use since 1968. The device is inserted percutaneously through a peripheral artery into the thoracic aorta. The balloon is synchronized so that it inflates during dias-tole and deflates during systole, resulting in augmentation of diastolic perfusion of the coronary arteries and decreased after-load. Typically, this improves cardiac index and decreases both preload and myocardial oxygen consumption.Common indications for use of an IABP is cardiogenic shock during or following cardiac catheterization or cardiac sur-gery. It also is utilized for preoperative stabilization of high-risk patients with either severe coronary artery disease, LV dysfunc-tion, or refractory,

1	cardiac catheterization or cardiac sur-gery. It also is utilized for preoperative stabilization of high-risk patients with either severe coronary artery disease, LV dysfunc-tion, or refractory, unstable angina. Kang et al have reported that risk-adjusted mortality was significantly lower for selected high-risk patients undergoing open heart surgery when a pre-operative IABP was used.214 In 2012, Thiele et al reported their data following a randomized, prospective, multicenter clinical trial looking at the outcome of using intra-aortic balloon pump in the treatment of cardiogenic shock complicating acute myo-cardial infarction in patients who underwent early revasculariza-tion (by means of percutaneous coronary intervention or bypass surgery) (IABP-SHOCK II trial).215 IABP did not significantly reduce 30-day mortality in this group of patients. Addition-ally, they reported no significant differences in secondary end points, including the time to hemodynamic stabilization, the length of

1	reduce 30-day mortality in this group of patients. Addition-ally, they reported no significant differences in secondary end points, including the time to hemodynamic stabilization, the length of stay in the intensive care unit, serum lactate levels, the dose and duration of catecholamine therapy, renal function, and major complications including major bleeding and peripheral ischemia.215Generally, an IABP is used for a few days and the amount of support is weaned as the patient’s condition improves. Mor-bidity associated with device use is typically minimal; however, in one series of 911 patients undergoing CABG who received an IABP, there was a 12% incidence of minor or major vascu-lar complications, including an approximately 3% incidence of limb ischemia requiring thromboembolectomy. This is the most serious complication of IABP placement. To prevent this prob-lem, frequent lower extremity neurovascular checks are neces-sary while an IABP is in place.216Ventricular Assist Device

1	This is the most serious complication of IABP placement. To prevent this prob-lem, frequent lower extremity neurovascular checks are neces-sary while an IABP is in place.216Ventricular Assist Device Indications and Cannulation. Patients in need of ventricular assist devices (VADs) may have preexisting chronic heart failure, refractory ventricular arrhythmias, or acute heart failure following an MI, cardiopul-monary arrest, viral illness, pregnancy, or cardiotomy. Device therapy is intended to preserve end-organ perfusion and func-tion and may be categorized as shortor long-term support for the left heart, the right heart, or both. In general, VADs may be used rarely for support while the heart recovers (bridge to recovery, BTR), while the patient waits for a heart transplant (bridge to transplant, BTT) or increasingly more commonly to treat a chronic heart failure patient who is not a transplant candidate (destination therapy, DT). The Interagency Registry for Mechanically Assisted

1	transplant, BTT) or increasingly more commonly to treat a chronic heart failure patient who is not a transplant candidate (destination therapy, DT). The Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) database, a joint effort by the NHLBI, FDA, CMS, academia, and industry to prospectively track patient outcomes, reported that in 2015 and 2016 indications for device implantation were BTR (0.2%), BTT listed (26.4%), BTT likely (15.5%), BTT moderate (7.7%), BTT unlikely (2.5%), DT (49.2%), and res-cue therapy (0.5%).217 The percentage of patients receiving a VAD as destination therapy has markedly increased over the last decade as results and devices improved.Left ventricular assist devices (LVADs) provide support for the failing heart by unloading blood from the left ventricle and pumping it into the aorta. Cannulas may be inserted into the LV apex or the left atrium for inflow into the pump, and return is through an arterial cannula or graft sewn to

1	the left ventricle and pumping it into the aorta. Cannulas may be inserted into the LV apex or the left atrium for inflow into the pump, and return is through an arterial cannula or graft sewn to either the ascending or descending aorta. For right-sided devices, inflow drainage is most often from a cannula in the right atrium, and blood is returned through a graft sewn to the pulmonary artery or right ventricular outflow tract.Left Ventricular Assist Devices. The first generation LVADs were pulsatile devices. They provided adequate support for the heart but were limited by their large size and durabil-ity.218 More recently, continuous-flow LVADs based on rotary pump technology have been introduced. These devices are smaller, quieter, and durable enough for long term support. The two most commonly used devices today are the HeartMate II (Thoratec, Pleasanton, CA) and the HeartWare HVAD (Heart-Ware, Inc., Framingham, MA) (Figs. 21-13A,B and 21-14A,B). These devices differ in that the

1	commonly used devices today are the HeartMate II (Thoratec, Pleasanton, CA) and the HeartWare HVAD (Heart-Ware, Inc., Framingham, MA) (Figs. 21-13A,B and 21-14A,B). These devices differ in that the HeartMate II is implanted sub-diaphragmatically, whereas the smaller HeartWare HVAD is implanted within the pericardium. Frequently used short-term support devices include the Abiomed BVS 5000 (Abiomed, Inc., Danvers, MA) and the CentriMag (Thoratec), which are both extracorporeal pumps, as well as the Impella (Abiomed), which may be inserted percutaneously. These devices are com-monly used in either post-MI or postcardiotomy heart failure. They have the benefit of faster and easier insertion, making them ideal rescue devices and allowing time for patient transfer to a tertiary referral center, device weaning, transplantation, or transition to a permanent VAD as DT or BTT.Bridge to Recovery. The ideal clinical situation would be for all LVADs to be temporary with the goal of myocardial

1	device weaning, transplantation, or transition to a permanent VAD as DT or BTT.Bridge to Recovery. The ideal clinical situation would be for all LVADs to be temporary with the goal of myocardial recov-ery. However, as noted previously, this is rare with only 0.2% of devices in the most recent INTERMACS data placed with intent for bridge to recovery.217 The LVAD Working Group Recovery Study, a prospective multicenter trial investigating myocardial recovery in BTT patients, has shown significant improvements in left ventricular ejection fraction and significant reductions in left ventricular end-diastolic diameter following support with continuous flow pumps, but myocardial recovery resulting in device explantation was still only seen in six patients (9%).219 Current data suggest that significant reverse remodeling is more likely to occur in the young and those with myocarditis.220Nevertheless, some encouraging results have been reported using a combination of treatment modalities. In a

1	reverse remodeling is more likely to occur in the young and those with myocarditis.220Nevertheless, some encouraging results have been reported using a combination of treatment modalities. In a few small studies of patients with LVADs inserted for nonischemic cardiomyopathy, deliberate and aggressive medical therapy, including the β2-agonist clenbuterol, resulted in successful LVAD explantation in 69% to 73% of patients,221,222 but these results have been difficult to replicate. Moreover, early results from clinical trials using stem cell therapy to treat patients with ischemic cardiomyopathy suggest that stem cells may be another adjuvant treatment with potential to aid in myocardial recovery.223,224Bridge to Transplant. LVADs are used as a bridge to trans-plant in patients who are candidates for heart transplantation but are not predicted to survive the waiting list period due to sequelae of cardiac failure, including end-organ dysfunc-tion, rising pulmonary artery pressures,

1	candidates for heart transplantation but are not predicted to survive the waiting list period due to sequelae of cardiac failure, including end-organ dysfunc-tion, rising pulmonary artery pressures, escalating inotrope Brunicardi_Ch21_p0801-p0852.indd 83501/03/19 5:32 PM 836SPECIFIC CONSIDERATIONSPART IIABFigure 21-13. The HeartMate II LVAD viewed from the (A) outside and (B) inside. The device is an axial flow, rotary pump that produces no pulsatile action. The pump contains a magnet, and the rotor assembly functions by the electromotive force generated by the motor. The result is that blood is propelled from the inflow cannula to systemic circulation at flows up to 10 L/min. (HeartMate, HeartMate II and St. Jude Medical are trademarks of St. Jude Medical, LLC or its related companies. Reproduced with permission of St. Jude Medical, ©2017. All rights reserved.)ABFigure 21-14. The HeartWare HVAD system. A. Both the device controller and batteries are held in a wearable carrying

1	Reproduced with permission of St. Jude Medical, ©2017. All rights reserved.)ABFigure 21-14. The HeartWare HVAD system. A. Both the device controller and batteries are held in a wearable carrying case and con-nected to the ventricular assist device through the driveline. B. The main component is a centrifugal blood pump, called the HVAD, which is implanted within the pericardium. The only moving part in the device, the impeller, is suspended within the pump using magnets and thrust bearings. Similar to the HeartMate II, it can deliver a flow rate of up to 10 L/min. (Reproduced with permission of Medtronic, Inc.)requirements, malignant ventricular arrhythmias, and risk for sudden death. Due to the scarcity of donor organs, the improved survival seen with LVAD usage has resulted in more patients remaining alive while on the transplantation waiting list. It is currently estimated that 35% of patients who go on to receive a heart transplant have had a previous LVAD implantation, although

1	remaining alive while on the transplantation waiting list. It is currently estimated that 35% of patients who go on to receive a heart transplant have had a previous LVAD implantation, although at more aggressive tertiary care facilities this number may be as high as 75% to 90%.172The HeartMate II pump was evaluated as a BTT in an observational, prospective multicenter trial of 133 patients with persistent NYHA class IV heart failure despite optimal medical management who were status 1A or 1B on the transplant list.225 At 6 months, 100 patients (75%) had undergone transplantation, had cardiac recovery, or continued on mechanical support while remaining eligible for transplantation. There were significant improvements in both quality of life and functional status with device therapy. At 3 months, 81% of patients were in class I or II heart failure. Moreover, complications, including bleeding requir-ing reoperation, stroke, drive-line infection, and need for right ventricular assist

1	3 months, 81% of patients were in class I or II heart failure. Moreover, complications, including bleeding requir-ing reoperation, stroke, drive-line infection, and need for right ventricular assist device support, were significantly less frequent than with the previous generation HeartMate XVE.226 These data led to FDA approval of the HeartMate II as a BTT LVAD in 2008, and clinical use of the device increased dramatically. More recently, a multicenter, prospective trial compared the Heart-Ware HVAD to contemporaneously inserted devices for use as a BTT.227 This trial demonstrated noninferiority of the HeartWare HVAD, but in contrast to the 2007 trial, approximately 90% of patients in both groups were transplanted, explanted for recovery, or remained alive and eligible for transplant with LVAD support at 6 months. Most important, data suggest that patients bridged to transplant with an LVAD in the current era experience similar shortand long-term posttransplant survival and

1	with LVAD support at 6 months. Most important, data suggest that patients bridged to transplant with an LVAD in the current era experience similar shortand long-term posttransplant survival and complications and do not have a higher incidence of allosensitization compared to standard cardiac transplant patients.228,229Brunicardi_Ch21_p0801-p0852.indd 83601/03/19 5:32 PM 837ACQUIRED HEART DISEASECHAPTER 21Most recently, in 2017, Mehra et al reported their multi-center outcome (Mechanical Circulatory Support Therapy with HeartMate 3 trial—MOMENTUM 3) comparing the safety and effectiveness of centrifugal-flow pump (HeatMate 3) to the axial-flow pump (HeatMate II).230 Implantation of a fully mag-netically levitated centrifugal-flow pump (HeartMate 3) was associated with better outcomes at 6 months than was implanta-tion of an axial-flow pump (HeartMate II). The improved out-comes were primarily due to the lower rate of reoperation for pump malfunction. Additionally, no patients in the

1	6 months than was implanta-tion of an axial-flow pump (HeartMate II). The improved out-comes were primarily due to the lower rate of reoperation for pump malfunction. Additionally, no patients in the HeartMate 3 group were suspected or found to have pump thrombosis.230Destination Therapy. The Randomized Evaluation of Mechan-ical Assistance for Treatment of Congestive Heart Failure (REMATCH) trial was conducted to compare the efficacy of LVAD insertion against optimal medical management in patients with NYHA class IV heart failure. While the pulsatile devices used in this trial had high failure rates, poor durability, and high associated mortality, there was still a clear survival ben-efit in patients treated with LVADs. This led to the FDA approval of the first LVADs for destination therapy in 2002.218Subsequent trials have proven the increased efficacy of second-generation devices for DT. In one such landmark trial, patients with advanced heart failure who were ineligible for

1	therapy in 2002.218Subsequent trials have proven the increased efficacy of second-generation devices for DT. In one such landmark trial, patients with advanced heart failure who were ineligible for transplantation were randomized in a 2:1 ratio to either a Heart-Mate II or HeartMate XVE.231 While both groups showed sig-nificant improvements in functional capacity and quality of life, actuarial survival at 2 years was superior for HeartMate II patients (58% vs. 24%, P = 0.008) and adverse event rates were signifi-cantly lower. These data established the benefit of continuous flow LVADs over optimal medical management for end-stage heart failure, and led to FDA approval of the HeartMate II for DT in 2010. In certain populations, 2-year survival with the HeartMate II is now 80%.232 Several smaller third generation devices are in various stages of development or clinical trials. Some of these devices eliminate the drive line by using alter-native energy sources, thereby removing a

1	smaller third generation devices are in various stages of development or clinical trials. Some of these devices eliminate the drive line by using alter-native energy sources, thereby removing a significant nidus for device infections. Long-term outcomes with these devices are expected to continue to improve, approaching that of cardiac transplantation and providing a viable solution to organ short-age for many patients.232 In 2013, Slaughter et al reported their multicenter clinical trial outcome to evaluate the HeartWare Ventricular Assist Device (HVAD) system for BTT in patients with advance heart failure in the United States (ADVANCE BTT trial).233 The use of the HeartWare pump as a BTT continued to demonstrate a high 180-day survival rate (91%) despite a low rate of transplant. Adverse event rates were similar or better than those observed in historical BTT trials, despite longer exposure times due to longer survival and lower transplant rates.233Current eligibility criteria for

1	event rates were similar or better than those observed in historical BTT trials, despite longer exposure times due to longer survival and lower transplant rates.233Current eligibility criteria for mechanical support as des-tination therapy include (a) NYHA class III or IV heart failure despite guideline-directed medical therapy including cardiac resynchronization therapy if indicated; (b) peak oxygen con-sumption <12 mL/kg per min or failure to wean from continuous IV inotropes; (c) left ventricular ejection fraction <25%; and (d) presence of a contraindication for heart transplantation (i.e., age >65 years, irreversible pulmonary hypertension, chronic renal failure, insulin-dependent diabetes with end-organ dam-age, or other clinically significant comorbidities).172,234 Once a patient has an LVAD inserted as DT, close and intensive follow-up by a multidisciplinary heart failure team is required in order to optimize medical therapy, reduce device-related morbidity, and improve

1	has an LVAD inserted as DT, close and intensive follow-up by a multidisciplinary heart failure team is required in order to optimize medical therapy, reduce device-related morbidity, and improve survival.6It is also important to keep in mind that while some con-traindications to transplantation are irreversible, others can be modified. As such, approximately 10% of patients implanted with an initial strategy of destination therapy become BTT patients,172 and in some patients, the LVAD itself facilitates this transition. For example, an improvement in mean pulmonary vascular resistance was reported following implantation of the HeartMate II in patients with end-stage heart failure (2.1 vs. 3.6 Woods units, P <0.001).235 These data are also relevant to patients that receive LVADs as a bridge to decision.Right Ventricular Assist Devices and Biventricular Assist DevicesMost patients who present with advanced heart failure and a failing left ventricle also have some degree of right

1	a bridge to decision.Right Ventricular Assist Devices and Biventricular Assist DevicesMost patients who present with advanced heart failure and a failing left ventricle also have some degree of right ventricu-lar dysfunction, but the majority of these patients do well with only an LVAD. However, implantation of an LVAD may cause acute worsening of tricuspid regurgitation and exacerbations of right heart failure through leftward deviation of the intraven-tricular septum and as a result of the significant volume-loading and transfusion requirement that is often necessary to achieve adequate flows postoperatively. Overall, approximately 20% of HeartMate II BTT patients had persistent right ventricular failure (RVF) requiring either a subsequent RVAD (6%) or intravenous inotropic support for >14 days (14%), and these patients had significantly worse 6-month survival compared to those without RVF (71% vs. 89%, P <0.001).236 Typically, mechanical right-ventricular support is temporary with

1	>14 days (14%), and these patients had significantly worse 6-month survival compared to those without RVF (71% vs. 89%, P <0.001).236 Typically, mechanical right-ventricular support is temporary with intent to wean the device, and isolated right-ventricular assist devices are unusual.Biventricular support is most commonly indicated for acute cardiogenic shock after an MI or postcardiotomy heart failure. Biventricular support is temporary, although some patients may be successfully bridged to transplant or perma-nent left-sided assist devices. There is currently no destination therapy device for biventricular failure.Total Artificial HeartThe total artificial heart (TAH, SynCardia Systems, Tucson, AZ) is currently indicated as a bridge to transplant for patients in biventricular failure, particularly for those who are critically ill and too large for extracorporeal BiVAD support. Unlike ven-tricular assist devices, the TAH replaces the entire heart. The ven-tricles of the TAH are

1	particularly for those who are critically ill and too large for extracorporeal BiVAD support. Unlike ven-tricular assist devices, the TAH replaces the entire heart. The ven-tricles of the TAH are implanted orthotopically to the atrial cuffs on the ventricular side of the AV groove, and the outflow conduits are attached to the great vessels. This approach has the benefit of obviating the hemodynamic influence of pulmonary hyperten-sion, right heart failure, myocardial or valvular problems, cardiac arrhythmias, and inotropic agents.237 While this device has failed to reach its potential as a replacement for cardiac transplantation, the TAH has achieved favorable results as a BTT with a >70% survival in selected centers.238-240 However, at most centers results with the TAH have been suboptimal, and it is not frequently used. A total of 226 TAH implants between 2013 and 2016 were reported to INTERMACS database. One-year and 2-year sur-vival was reported at 52% and 37%, respectively.

1	and it is not frequently used. A total of 226 TAH implants between 2013 and 2016 were reported to INTERMACS database. One-year and 2-year sur-vival was reported at 52% and 37%, respectively. Compare to the previously reported survival (2006 through 2012), no significant improvement in survival was noted with time.217SURGERY FOR ARRHYTHMIASThe success of catheter-based ablation and implantable cardio-verter defibrillators (ICDs) has significantly diminished refer-rals for the surgical treatment of arrhythmias such as ventricular Brunicardi_Ch21_p0801-p0852.indd 83701/03/19 5:32 PM 838SPECIFIC CONSIDERATIONSPART IItachycardia, Wolff-Parkinson-White syndrome, atrial flutter, and atrioventricular nodal reentry. On the other hand, the intro-duction of surgical ablation modalities such as radiofrequency and cryothermal energy, has simplified the surgical treatment of atrial fibrillation and has led to an increase in the number of surgical procedures performed annually for AF, although

1	radiofrequency and cryothermal energy, has simplified the surgical treatment of atrial fibrillation and has led to an increase in the number of surgical procedures performed annually for AF, although this has plateaued in recent years.241,242Atrial FibrillationEpidemiology of Atrial Fibrillation. AF remains the most common arrhythmia in the world with an overall prevalence of 0.4% to 1% that increases to 8% in those older than 80 years old.243 The most serious complication of AF is thromboembo-lism with resultant stroke,244 but serious morbidity and mortality may also result from hemodynamic compromise due to loss of atrial contraction and exacerbations of CHF from atrioventricu-lar asynchrony and tachycardia-induced cardiomyopathy.Medical Management. Most patients are treated medically, but the shortcomings of pharmacological management have left an important role for interventional therapies. Antiarrhythmic medications have been limited by modest efficacy and sig-nificant

1	but the shortcomings of pharmacological management have left an important role for interventional therapies. Antiarrhythmic medications have been limited by modest efficacy and sig-nificant proarrhythmic and systemic toxicities.245 Conversely, rate control strategies leave the patient in AF, do not address the impaired hemodynamics or symptoms associated with this arrhythmia, and may render subsequent attempts at rhythm con-trol therapies less effective for younger patients who may suf-fer irreversible cardiac remodeling due to the prolonged period of time in AF. Additionally, AF is associated with a fivefold greater risk of ischemic stroke or systemic embolism compared with normal sinus rhythm. Annual risk of major bleeding in those on anticoagulation is estimated at 1.2%.246Restoration of normal sinus rhythm has several potential benefits over other strategies.247-249 These include improvement in atrial systolic function, which improves cardiac output and often symptoms of CHF;

1	of normal sinus rhythm has several potential benefits over other strategies.247-249 These include improvement in atrial systolic function, which improves cardiac output and often symptoms of CHF; lowered risks of stroke; potential free-dom from anticoagulation; and likely reversal of atrial structural and/or electrical remodeling.Indications for Surgical Management. Consensus guide-lines published by the Heart Rhythm Society state that surgical ablation for atrial fibrillation is indicated for (a) all symptom-atic AF patients undergoing other cardiac surgery; (b) selected asymptomatic AF patients undergoing cardiac surgery in which the ablation can be performed with minimal additional risk; and (c) symptomatic patients with lone AF who have failed medi-cal therapy and prefer a surgical approach, have failed one or more attempts at catheter ablation, or are poor candidates for catheter ablation.243 At our institution, relative indications for surgical ablation in patients with

1	approach, have failed one or more attempts at catheter ablation, or are poor candidates for catheter ablation.243 At our institution, relative indications for surgical ablation in patients with permanent AF that were not included in the consensus statement are (a) a contraindica-tion to long term anticoagulation for patients at high risk for stroke (CHADS2 score ≥2) and (b) a history of stroke while on therapeutic anticoagulation. Since the consensus statement was released, a multicenter, randomized, controlled trial of surgical ablation in patients undergoing mitral valve surgery showed a significant improvement in freedom from atrial fibrillation in patients receiving surgical ablation (63% vs. 29%, P <0.001).250 Controversially, this trial did not show a difference between left atrial and biatrial ablation; this may have been due to techni-cal issues with the operations.251 The STS has recently released guidelines for surgical ablation that give a Class I Level A rec-ommendation

1	and biatrial ablation; this may have been due to techni-cal issues with the operations.251 The STS has recently released guidelines for surgical ablation that give a Class I Level A rec-ommendation for concomitant surgical ablation at the time of mitral valve surgery and a Class I Level B-NR recommendation for concomitant surgical ablation at the time of AVR, CABG, or AVR-CABG.252The Cox-Maze IV Procedure. The first successful operation for atrial fibrillation, the Cox-Maze procedure, was introduced clinically in 1987 by James Cox. The procedure involved the completion of a maze-like pattern of surgical incisions across both the right and left atrial that were designed to interrupt the multiple macroreentrant circuits thought to be responsible for AF, while still allowing propagation of the sinus impulse, restoring atrioventricular synchrony, and preserving atrial trans-port function. While effective at eliminating AF and reducing the risk of thromboembolism, it was not widely

1	of the sinus impulse, restoring atrioventricular synchrony, and preserving atrial trans-port function. While effective at eliminating AF and reducing the risk of thromboembolism, it was not widely performed because it was technically difficult and significantly prolonged time on cardiopulmonary bypass. In 2002, the Cox-Maze IV, was introduced. The Cox-Maze IV uses a combination of bipo-lar radiofrequency (RF) ablation and cryoablation to effectively replace the majority of incisions that comprise the Cox-Maze III while significantly shortening cross-clamp time and reducing operative complexity.The Cox-Maze IV is performed on cardiopulmonary bypass through either a median sternotomy, often in combina-tion with other cardiac surgery or a right minithoracotomy.253,254 In most cases, the right atrial lesion set performed on the beat-ing heart, whereas the left atrial lesions are performed during cardioplegic arrest (Fig. 21-15).Results from the Cox-Maze IV procedure have been excellent.

1	atrial lesion set performed on the beat-ing heart, whereas the left atrial lesions are performed during cardioplegic arrest (Fig. 21-15).Results from the Cox-Maze IV procedure have been excellent. The Washington University group reported a series of 576 consecutive patients in 2015, demonstrating free-dom from atrial tachyarrhythmias in 92% of patients at 1 year and 73% of patients at 5 years postoperatively.255 Additionally, freedom from atrial tachyarrhythmias and antiarrhythmic drugs was 81% at 1 year and 61% at 5 years. A recent propensity-matched analysis showed that the addition of the Cox-Maze IV procedure to a routine cardiac surgery did not significantly increase postoperative morbidity or mortality and was associ-ated with improved late survival compared with patients with untreated AF and a similar survival to patients without a his-tory of AF.256 A propensity analysis has shown that results are similar between the traditional “cut-and-sew” maze (Cox-Maze III) and the

1	untreated AF and a similar survival to patients without a his-tory of AF.256 A propensity analysis has shown that results are similar between the traditional “cut-and-sew” maze (Cox-Maze III) and the Cox-Maze IV.257 This procedure is often successful in patients who are poor candidates for catheter-based ablation, such as those with large left atria and patients with long-standing persistent AF.The combination of surgical management of the left atrial appendage (LAA) and restoration of normal sinus rhythm after the Cox-Maze procedure significantly reduces stroke risk. It is our practice to stop warfarin at 3 months postoperatively in patients who are in normal sinus rhythm and without another indication for anticoagulation, regardless of CHA2DS2-VASc score. With this approach, the stroke rate following the Cox-Maze procedure off anticoagulation has been remarkably low (annual risk = 0.2%).258 In contrast, in one report the annual rate of intracranial hemorrhage in anticoagulated

1	rate following the Cox-Maze procedure off anticoagulation has been remarkably low (annual risk = 0.2%).258 In contrast, in one report the annual rate of intracranial hemorrhage in anticoagulated patients with AF was 0.9% per year, and the overall rate of major bleeding complications was 2.3% per year.259Left Atrial Lesion Sets. Some surgeons perform more lim-ited ablation procedures, such as isolated pulmonary vein isola-tion or lesion sets that are limited to the left side of the heart. This is done in order to further reduce the complexity of the pro-cedure and takes advantage of the fact that in most patients AF 7Brunicardi_Ch21_p0801-p0852.indd 83801/03/19 5:32 PM 839ACQUIRED HEART DISEASECHAPTER 21Figure 21-15. The Cox-Maze IV Lesion Set. A. The left atrial lesion set is comprised of right and left pulmonary vein isolation, connecting lesions between the left and right superior and inferior pulmonary veins, a lesion from the left atrial appendage excision site to the

1	comprised of right and left pulmonary vein isolation, connecting lesions between the left and right superior and inferior pulmonary veins, a lesion from the left atrial appendage excision site to the pulmonary vein, and a lesion to the mitral valve annulus. B. The right atrial lesion set consists of lines of ablation along the superior and inferior vena cavae, the free wall of the right atrium, and down to the tricuspid valve annulus. (Reproduced with permission from Weimar T, Bailey MS, Watanabe Y, et al: The Cox-maze IV procedure for lone atrial fibrillation: a single center experience in 100 consecutive patients, J Interv Card Electrophysiol. 2011 Jun;31(1):47-54.)ABoriginates from the pulmonary veins and posterior left atrium. However, there is seldom justification for limited lesion sets in experienced hands.While there is a high degree of variability in both the techniques and energy sources that have been attempted for left-sided atrial lesion sets, these procedures have all

1	sets in experienced hands.While there is a high degree of variability in both the techniques and energy sources that have been attempted for left-sided atrial lesion sets, these procedures have all incorpo-rated some subset of the left atrial lesion set of the Cox-Maze procedure. Pulmonary vein isolation is ubiquitously performed, and the LAA is often excised. Results differ greatly between series, including the recent CTSNet multicenter trial,250 but a meta-analysis of the published literature by Ad and colleagues revealed that a biatrial lesion set resulted in a significantly higher late freedom from AF compared with a left atrial lesion set alone (87% vs. 73%, P = 0.05).260 These results are not sur-prising, as our intraoperative mapping experience with such patients showed a distinct region of stable dominant frequency in the left atrium only 30% of the time.261 The dominant fre-quency was located in the right atrium 12% of the time and moved during the recording period in almost

1	region of stable dominant frequency in the left atrium only 30% of the time.261 The dominant fre-quency was located in the right atrium 12% of the time and moved during the recording period in almost half of all patients. It must also be kept in mind that recurrent right atrial flutter is a known complication of performing only the left atrial lesions. When it does occur, atrial flutter can be treated with catheter-based ablation; however, recurrent left atrial flutter can be very difficult to ablate.Pulmonary Vein Isolation. Pulmonary vein isolation (PVI) is an attractive therapeutic option because it can be performed off of cardiopulmonary bypass (CPB) through small or thora-coscopic incisions. The results of PVI have been variable and highly dependent on patient selection since outcomes are con-sistently worse in patients with longstanding persistent AF. In a study from Edgerton et al, only 56% of patients were free from AF at 6 months (35% off antiarrhythmic drugs), and with

1	are con-sistently worse in patients with longstanding persistent AF. In a study from Edgerton et al, only 56% of patients were free from AF at 6 months (35% off antiarrhythmic drugs), and with concomitant procedures, the success rate of PVI has been even lower.262 Several devices are available to close the LAA at the time of PVI. These include staplers and epicardial clips that can be placed without the need for CPB.263While surgical PVI has had poorer results than a Cox-Maze procedure, it has had superior results to catheter-based PVI. The Atrial Fibrillation Catheter Ablation Versus Surgi-cal Ablation Treatment (FAST) Trial, which was a two cen-ter, randomized clinical trial, compared catheter-based ablation to thoracoscopic PVI in patients with antiarrhythmic drug-refractory AF and either left atrial dilatation and hypertension or failed prior catheter-ablation.264 This study demonstrated that the 12-month freedom from AF and antiarrhythmic drugs was 37% for the catheter ablation

1	left atrial dilatation and hypertension or failed prior catheter-ablation.264 This study demonstrated that the 12-month freedom from AF and antiarrhythmic drugs was 37% for the catheter ablation group and 66% for the PVI group (P = 0.002).264SURGERY FOR PERICARDIAL DISEASEAcute PericarditisPericarditis is characterized by infiltration of the cellular and fibrous pericardium by inflammatory cells. The exact incidence and prevalence of pericarditis is unknown, but it is estimated that pericarditis is found in approximately 1% of autopsies and accounts for up to 5% of presentations of nonischemic chest pain.265,266 The etiologies of acute pericarditis are diverse and may result from primary pericardial disorders or occur sec-ondary to a systemic illness.267 In developed countries, 80% to 90% of cases are now considered idiopathic or related to a viral pathogen, but nonviral infection, autoimmune diseases, myocardial infarction, radiation, malignancy, endocrinopathy, myocarditis, aortic

1	of cases are now considered idiopathic or related to a viral pathogen, but nonviral infection, autoimmune diseases, myocardial infarction, radiation, malignancy, endocrinopathy, myocarditis, aortic dissection, uremia, trauma, pharmacologi-cal side effects, and previous cardiothoracic surgery must be included in the differential diagnosis. The relative incidences of peri-infarction pericarditis, which was once common, and post-cardiac injury syndrome have been dramatically reduced with the advent of thrombolytics and coronary angioplasty.267Clinical Presentation and Diagnosis. Diagnosis of acute pericarditis typically requires the identification of at least two of four cardinal features (Table 21-14). The presentation may be confused with several more common cardiopulmonary condi-tions, particularly myocardial infarction, making a careful his-tory and physical critical. Patients with pericarditis classically complain of sudden onset, retrosternal pain that may be pleuritic in nature.

1	myocardial infarction, making a careful his-tory and physical critical. Patients with pericarditis classically complain of sudden onset, retrosternal pain that may be pleuritic in nature. The pain may also be positional, with alleviation of pain when the patient is upright and leaning forward. Pain from pericarditis is typically sharp or stabbing, as opposed to the dull pain or pressure that is common with angina, and it typically does not crescendo. While both conditions cause pain that often radiates to the neck, arms, and shoulders, pericarditis pain may Brunicardi_Ch21_p0801-p0852.indd 83901/03/19 5:32 PM 840SPECIFIC CONSIDERATIONSPART IIuniquely radiate to the trapezius ridge due to innervation from the phrenic nerve.268-269The presence of a pericardial friction rub is pathogno-monic for pericarditis, but it tends to vary in intensity over time and may be absent in 15% to 65% of patients.265,268 As such, the sensitivity of this physical finding is dependent on the fre-quency

1	for pericarditis, but it tends to vary in intensity over time and may be absent in 15% to 65% of patients.265,268 As such, the sensitivity of this physical finding is dependent on the fre-quency and quality of auscultation. A pericardial friction rub is best heard at the left lower sternal border and is typically described as a high-pitched scratchy or squeaky sound with a triphasic cadence corresponding to the movement of the heart during atrial systole, ventricular systole, and early ventricular diastole. However, it may be monophasic or biphasic in up to 50% of patients.Electrocardiogram changes typically progress through four stages representing global subepicardial myocarditis and subsequent recovery. Pericarditis patients may have concave ST deflections with diffuse changes, spanning the leads of multiple coronary artery distributions, but ST segment abnormalities are absent in 20% to 40% of patients.270,271 Acute pericarditis should not result in the development of infarct

1	the leads of multiple coronary artery distributions, but ST segment abnormalities are absent in 20% to 40% of patients.270,271 Acute pericarditis should not result in the development of infarct patterns, such as Q waves or loss of R waves, and T-wave inversions from pericarditis tend to result later in the disease process after the ST segment has returned to baseline.Echocardiography is routinely performed in the evaluation of acute pericarditis. Its role is primarily to assess for a pericar-dial effusion. However, in a patient who can be demonstrated to have previously had normal cardiac function, it may be used to exclude segmental wall motion abnormalities that may suggest ischemia.The remaining workup should attempt to determine the underlying cause of the pericarditis and should be directed by the history and physical. Most inflammatory markers and labo-ratory tests are nonspecific, but C-reactive protein may be useful in predicting recurrence risks and in guiding the duration of

1	by the history and physical. Most inflammatory markers and labo-ratory tests are nonspecific, but C-reactive protein may be useful in predicting recurrence risks and in guiding the duration of anti-inflammatory medications.272 Rarely, other imaging modalities, such as CT scanning, pericardial biopsies, or pericardiocentesis may aid in diagnosis.Treatment. The preferred treatment depends on the underly-ing cause of the pericarditis. The disease usually follows a self-limited and benign course and can be successfully treated with a short course of nonsteroidal anti-inflammatory agents (NSAIDs). The addition of colchicine may be beneficial.273 Some patients may require judicious use of steroids or IV anti-biotics. In cases of purulent pyogenic pericarditis, surgical exploration and drainage are occasionally necessary. Rarely, accumulation of fluid in the pericardium may lead to tampon-ade, requiring prompt evacuation of the pericardial space. While pericardiocentesis will typically

1	are occasionally necessary. Rarely, accumulation of fluid in the pericardium may lead to tampon-ade, requiring prompt evacuation of the pericardial space. While pericardiocentesis will typically suffice, surgical drain-age may be required for thick, viscous, or clotted fluid or in patients with significant scarring from previous operations. More commonly, surgical intervention is required to manage recurrent disease.8Table 21-14Features of acute pericarditis• Pleuritic and positional, retrosternal chest pain• Pericardial friction rub• EKG changes: diffuse ST elevation and PR depression• Pericardial effusionEKG = Electrocardiogram.Relapsing PericarditisAs many as one-third of patients with acute pericarditis will develop at least one episode of relapse.267 While many of these patients can be treated medically during their initial relapse and do not experience further episodes, a subset of patients experi-ence chronic relapsing pericarditis that can significantly impact their quality of

1	treated medically during their initial relapse and do not experience further episodes, a subset of patients experi-ence chronic relapsing pericarditis that can significantly impact their quality of life. Recurrence may develop either from the original etiology or from an autoimmune process that occurs as a consequence of damage from the initial episode. Relapsing pericarditis normally responds to a longer course of NSAIDS ± colchicine. While steroids may induce rapid symptomatic response, their use should be limited to patients who have mul-tiple relapses and are unresponsive to first-line agents, as several studies have suggested that steroid administration may favor relapse.273,274Pericardiectomy may be considered a last resort treatment in patients with relapsing pericarditis who are severely symp-tomatic despite optimal medical management, are unable to tol-erate steroids, or have recurrence with tamponade. Evidence for this approach is lacking, as few studies have described

1	are severely symp-tomatic despite optimal medical management, are unable to tol-erate steroids, or have recurrence with tamponade. Evidence for this approach is lacking, as few studies have described pericar-diectomy in this population.275-277 The largest study and the only one to compare surgical treatment with medical management for patients with persistent relapsing pericarditis was a report of 184 patients from the Mayo Clinic.276 About 58 patients were identi-fied as having undergone a pericardiectomy after failed medi-cal treatment, whereas the remainder were treated with medical management only. Compared to medical treatment only, peri-cardiectomy resulted in significantly fewer relapses (8.6% vs. 28.6%, P = 0.009) at long term follow-up, as well as a nonsig-nificant trend towards less medication and corticosteroid usage. Of note, 80% of patients in the pericardiectomy group who had relapses reported significant improvements in their symptoms and had fewer relapses than before

1	less medication and corticosteroid usage. Of note, 80% of patients in the pericardiectomy group who had relapses reported significant improvements in their symptoms and had fewer relapses than before pericardiectomy. No periop-erative deaths were observed, and only two patients (3%) had major complications. Hence, at experienced centers pericardi-ectomy may be a safe and viable option in select patients with relapsing pericarditis.Chronic Constrictive PericarditisEtiology, Pathology, and Pathophysiology. Constrictive pericarditis can occur after any pericardial disease process but remains a rare outcome of recurrent pericarditis. It results when chronic pericardial scarring and fibrosis cause adhesion of the visceral and parietal layers and resultant obliteration of the pericardial space. While the pericardium is often grossly thickened with either focal or diffuse calcification in chronic disease, constriction may occur with normal pericardial thick-ness in approximately 20% of

1	While the pericardium is often grossly thickened with either focal or diffuse calcification in chronic disease, constriction may occur with normal pericardial thick-ness in approximately 20% of cases.267,278 In developed nations, idiopathic causes and cardiac surgery (accounting for almost 40% of cases in some series) are the predominant underlying eti-ologies, followed by mediastinal radiation, pyogenic infections (i.e., Staphylococcus), and other miscellaneous causes. Tuber-culosis is an additional common cause in immunosuppressed patients and in developing or underdeveloped countries.Clinically, pericardial constriction limits diastolic filling of the ventricles and mimics right heart failure since the right-sided chambers are more affected by a rise in filling pressures. Subsequent increases in central venous pressure result in the progressive development of hepatomegaly, ascites, peripheral edema, abdominal pain, dyspnea on exertion, anorexia, and nausea (in part due to hepatic

1	in central venous pressure result in the progressive development of hepatomegaly, ascites, peripheral edema, abdominal pain, dyspnea on exertion, anorexia, and nausea (in part due to hepatic and bowel congestion). In many patients, these symptoms develop insidiously over a course Brunicardi_Ch21_p0801-p0852.indd 84001/03/19 5:32 PM 841ACQUIRED HEART DISEASECHAPTER 21of years. Since many of these symptoms are similar to those seen in patients with restrictive cardiomyopathy, the distinc-tion between the two entities is difficult, but it remains critical because the treatment is completely different for restriction. The primary difference is that restrictive cardiomyopathy is defined by a nondilated ventricle with a rigid myocardium that causes a significant decrease in myocardial compliance, which is not seen in constrictive pericarditis.Clinical and Diagnostic Findings. Classic physical exam findings include jugular venous distention with Kussmaul’s sign, diminished cardiac apical

1	which is not seen in constrictive pericarditis.Clinical and Diagnostic Findings. Classic physical exam findings include jugular venous distention with Kussmaul’s sign, diminished cardiac apical impulses, peripheral edema, ascites, pulsatile liver, a pericardial knock, and, in advanced disease, signs of liver dysfunction, such as jaundice or cachexia. The “pericardial knock” is an early diastolic sound that reflects a sudden impediment to ventricular filling, similar to an S3 but of higher pitch.Several findings are characteristic on noninvasive and invasive testing. CVP is often elevated 15 to 20 mmHg or higher. ECG commonly demonstrates nonspecific low voltage QRS complexes and isolated repolarization abnormalities. Chest X-ray may demonstrate calcification of the pericardium, which is highly suggestive of constrictive pericarditis in patients with heart failure, but this is present in only 25% of cases.274 Cardiac CT or MRI (cMRI) typically demonstrate increased pericardial

1	is highly suggestive of constrictive pericarditis in patients with heart failure, but this is present in only 25% of cases.274 Cardiac CT or MRI (cMRI) typically demonstrate increased pericardial thickness (>4 mm) and calcification, dilation of the inferior vena cava, deformed ventricular contours, and flattening or leftward shift of the ventricular septum. Pericardial adhesions may also be seen on tagged cine MRI studies.As discussed, it is most important to distinguish peri-cardial constriction from restrictive cardiomyopathy, which is best done with either echocardiography or right heart catheter-ization. Findings favoring constriction on echocardiography include respiratory variation of ventricular septal motion and mitral inflow velocity, preserved or increased mitral annulus early diastolic filling velocity, and increased hepatic vein flow reversal with expiration.267,274 Cardiac catheterization will show increased atrial pressures, equalization of end-diastolic pressure and

1	diastolic filling velocity, and increased hepatic vein flow reversal with expiration.267,274 Cardiac catheterization will show increased atrial pressures, equalization of end-diastolic pressure and early ventricular diastolic filling with a subsequent plateau, called the “square-root sign.” Additional findings upon cath-eterization that would favor constriction include respiratory variation in ventricular filling and increased ventricular inter-dependence, manifest as a discordant change in the total area of the LV and RV systolic pressure curve with respiration.Surgical Treatment. Transient constrictive pericarditis may occur weeks to months after an initial injury and follows a self-limiting course of weeks to a few months. These patients are best treated with medical therapy alone. They often lack calci-fication of their pericardium, and the degree of late gadolinium enhancement of the pericardium on cardiac MRI has shown promise in predicting which patients may have resolution of

1	often lack calci-fication of their pericardium, and the degree of late gadolinium enhancement of the pericardium on cardiac MRI has shown promise in predicting which patients may have resolution of the process.279 Still, there is no ideal way to distinguish these patients from those who will develop chronic constrictive peri-carditis, which is permanent. Therefore, if a newly diagnosed patient is hemodynamically stable, it is recommended that con-servative management is attempted for 2 to 3 months prior to performing a pericardiectomy.278 Surgical therapy should not be delayed indefinitely, however, as results are improved when the operation is performed earlier in the course of the disease. A series of 938 patients undergoing pericardiectomy reported by the Mayo Clinic, 355 of whom underwent pericardiec-tomy for constrictive pericarditis, showed significantly lower survival in patients with constrictive pericarditis compared with patients with effusive/relapsing pericarditis.280

1	pericardiec-tomy for constrictive pericarditis, showed significantly lower survival in patients with constrictive pericarditis compared with patients with effusive/relapsing pericarditis.280 Patients with left ventricular systolic dysfunction or right ventricular dilata-tion are at increased risk of early mortality.281 Additional fac-tors that predict adverse long-term outcomes include older age and prior ionizing radiation, as well as cardiopulmonary and renal dysfunction.274,281 Surgery should therefore be approached cautiously in patients with advanced, “end-stage” constrictive pericarditis, mixed constrictive-restrictive disease (often from radiation), and significant myocardial or renal dysfunction, as those patients are at increased risk from surgery and may not experience improvement of symptoms.In order to minimize recurrence following pericardiec-tomy, complete pericardial resection is desirable. This is typi-cally performed through either a median sternotomy or left

1	of symptoms.In order to minimize recurrence following pericardiec-tomy, complete pericardial resection is desirable. This is typi-cally performed through either a median sternotomy or left anterolateral thoracotomy while on cardiopulmonary bypass. Radical pericardiectomy involves wide resection of the con-stricting pericardium from the anterior surface of the heart between the phrenic nerves and the diaphragmatic surface. Decortication of the right atrium and vena cavae is not univer-sally performed, but doing so improves the risk of persistent disease or relapse.282,283The extent of myocardial involvement may also affect long-term outcomes, and, thus, the depth of decortication is an important consideration.282 Even when an adequate pericardi-ectomy is performed, epicardial sclerosis can cause persistent hemodynamic instability or a delayed response to surgery. Scle-rotic epicardium is often thin and nearly transparent, but in cases of severe chronic constrictive pericarditis it can

1	cause persistent hemodynamic instability or a delayed response to surgery. Scle-rotic epicardium is often thin and nearly transparent, but in cases of severe chronic constrictive pericarditis it can be difficult to remove it without injury to the heart.Surgical Results. While most patients experience significant improvement in their symptoms following pericardiectomy, symptomatic relief may take several months. Since there is a significant perioperative morbidity and mortality, pericardiec-tomy is best performed by experienced surgeons at high-volume centers. Between 1970 and 1985, the operative mortality was reported to be 12%, but a lower mortality of approximately 4% to 8% was noted between 1977 and 2006 at several experienced centers.278,283-287Long-term survival is in part determined by etiology of the disease. In a report from the Cleveland Clinic, 7-year survival rates following pericardiectomy for idiopathic, postsurgical, and radiation-induced constrictive pericarditis were

1	by etiology of the disease. In a report from the Cleveland Clinic, 7-year survival rates following pericardiectomy for idiopathic, postsurgical, and radiation-induced constrictive pericarditis were 88%, 66%, and 27%, respectively.284 Results are worst for radiation-induced disease because ionizing radiation is often associated with myo-cardial injury as well as pericardial disease.Despite the risks, many patients experience significant benefits from surgical treatment. In one large series, 83% of patients were reported to be free of symptoms at last follow-up.287 This is in agreement with other studies that have shown a signif-icant improvement in NYHA functional status from class III/IV preoperatively to class I/II following pericardiectomy in >95% of patients.283,285-287CARDIAC NEOPLASMSOverview and General Clinical FeaturesCardiac neoplasms are rare, with an incidence ranging from 0.001% to 0.3% in autopsy studies and a 0.15% incidence in major echocardiographic series.288,289 In

1	and General Clinical FeaturesCardiac neoplasms are rare, with an incidence ranging from 0.001% to 0.3% in autopsy studies and a 0.15% incidence in major echocardiographic series.288,289 In one large autopsy series, 99.2% of cardiac tumors were metastatic in origin; however, Brunicardi_Ch21_p0801-p0852.indd 84101/03/19 5:32 PM 842SPECIFIC CONSIDERATIONSPART IIthese patients almost never present for surgical management as they usually have fatal diffuse metastatic disease.290 As a result, a majority of surgical series describe management of primary cardiac neoplasms. Benign cardiac tumors are most common and account for 75% of primary neoplasms. Approximately 50% of benign cardiac tumors are myxomas, with the remainder being papillary fibroelastomas, lipomas, rhabdomyomas, fibro-mas, hemangiomas, teratomas, lymphangiomas, and others, in order of decreasing frequency. Most malignant primary cardiac tumors are sarcomas (angiosarcoma, rhabdomyosarcoma, fibro-sarcoma, leiomyosarcoma,

1	hemangiomas, teratomas, lymphangiomas, and others, in order of decreasing frequency. Most malignant primary cardiac tumors are sarcomas (angiosarcoma, rhabdomyosarcoma, fibro-sarcoma, leiomyosarcoma, and liposarcoma), with a small inci-dence of malignant lymphomas.Clinical Presentation. The clinical presentation of cardiac neoplasms varies greatly depending on the location of the tumor, as well as its size, rate of growth, invasiveness, and fri-ability. While as many as 10% of patients are asymptomatic, most manifest some combination of symptoms from the classic triad resulting from blood flow obstruction, tumor embolization, and constitutional symptoms.291,292 Systemic manifestations of disease include fever, myalgias, chills, night sweats, weight loss, and fatigue and occur in up to one-third of patients.Obstruction of cardiac blood flow accounts for the major-ity of presenting symptoms.292 When the tumor is located in the left atrium, symptoms tend to mimic mitral valve disease

1	one-third of patients.Obstruction of cardiac blood flow accounts for the major-ity of presenting symptoms.292 When the tumor is located in the left atrium, symptoms tend to mimic mitral valve disease with dyspnea and pulmonary edema; although more severe presenta-tions with syncopal episodes, hypotension, and sudden cardiac death have been reported from temporary valve orifice occlu-sion. When the tumor is located in the right atrium, symptoms may mimic right heart failure and include hepatomegaly, asci-tes, and peripheral edema. Outflow tract obstruction is rare but may be caused by large ventricular tumors.293Tumor lysis and embolization may also lead to neurologic presentations such as stroke, retinal artery occlusion, or cere-bral aneurysms, particularly in the case of pedunculated tumors and those with frond-like projections.294 Embolic tumor cells are able to lodge and penetrate distant vessel walls via subintimal growth, which leads to weakening of the arterial wall and

1	tumors and those with frond-like projections.294 Embolic tumor cells are able to lodge and penetrate distant vessel walls via subintimal growth, which leads to weakening of the arterial wall and sub-sequent aneurysm formation. This has been documented as late as 5 years after successful primary myxoma resection.295 Alter-natively, embolic implants may metastasize and create space occupying lesions. While rare, myxomatous tumor emboli have also been identified in the coronary arteries, common iliac and femoral arteries, kidney, spleen, pancreas, and liver.294Certain clinical features may be helpful in distinguishing benign from malignant primary cardiac tumors.292 Malignant tumors, primarily sarcomas, do not demonstrate a gender pref-erence and tend to present after the fourth decade of life. They are often multifocal within the right atrium, and intramyocardial invasion can lead to refractory congestive heart failure, arrhyth-mias, hemopericardium, and ischemia. Conversely, benign

1	life. They are often multifocal within the right atrium, and intramyocardial invasion can lead to refractory congestive heart failure, arrhyth-mias, hemopericardium, and ischemia. Conversely, benign tumors, primarily myxomas, are typically unifocal in the left atrium, have a 3:1 female preference, and occur in younger patients. Arrhythmias and pericardial effusions are very rare in this population.Diagnosis and Characterization of Cardiac Masses. Trans-thoracic echocardiography is the mainstay imaging technique for the detection of cardiac tumors.292 However, echocardiogra-phy is limited by dependence on an acoustic window, subopti-mal visualization of extracardiac extension, and poor soft-tissue visualization. TEE is generally only beneficial for small local-ized tumors due to its limited field of view. cMRI is therefore the current standard for delineating the anatomical extent of the tumor and assessing the paracardiac space and great vessels. Advantages of cMRI over CT scans

1	field of view. cMRI is therefore the current standard for delineating the anatomical extent of the tumor and assessing the paracardiac space and great vessels. Advantages of cMRI over CT scans include better soft-tissue evaluation without the need for iodinated contrast and no expo-sure to ionizing radiation.It is important in the initial workup to distinguish a cardiac tumor from an intracardiac thrombus, which may be common in the atria of patients with AF and can mimic echocardiographic features of atrial myxomas. This determination is critical, as an atrial thrombus may be expected to resolve with anticoagula-tion, whereas a tumor requires surgical intervention. Moreover, anticoagulation can potentially increase the risk of peripheral embolization in patients with cardiac tumors. Delayed enhance-ment cMRI is the best modality to separate these two entities. cMRI may show vascularization, areas of necrosis, hemorrhage, or calcification in cardiac tumors that are not present in

1	Delayed enhance-ment cMRI is the best modality to separate these two entities. cMRI may show vascularization, areas of necrosis, hemorrhage, or calcification in cardiac tumors that are not present in thrombi.MyxomaPathology and Genetics. Cardiac myxomas are the most common cardiac tumor and are characterized by several distinguishing features. About 75% of the time, they arise from the interatrial septum near the fossa ovalis in the left atrium.296 Most others will develop in the right atrium, but, less commonly, they can arise from valvular surfaces and the walls of other cardiac chambers. Macroscopically, these tumors are pedunculated with a gelatinous consistency, and the surface may be smooth (65%), villous, or friable.291 Size varies greatly with these tumors and ranges from 1 to 15 cm in diameter. Internally, myxomas are heterogeneous and often contain hemorrhage, cysts, necrosis, or calcification. Histologically, these tumors contain cells that arise from a multipotent

1	1 to 15 cm in diameter. Internally, myxomas are heterogeneous and often contain hemorrhage, cysts, necrosis, or calcification. Histologically, these tumors contain cells that arise from a multipotent mesenchyme and are contained within a mucopolysaccharide stroma.297While the majority of myxomas occur spontaneously with the highest incidence in women aged 40 to 60 years old, approx-imately 7% of cases are familial as part of Carney complex.291 Carney complex is an autosomal dominant disorder character-ized by two or more of the following conditions: atrial and extra-cardiac myxomas, schwannomas, cutaneous lentiginosis, spotty pigmentation, myxoid fibroadenomas of the breast, endocrine overactivity (pituitary adenomas or primary adrenal hyperplasia with Cushing’s syndrome), and testicular tumors. Compared to sporadic myxomas, those that occur as part of Carney complex are more commonly found in the right atrium (37% vs. 18%) or one of the ventricles (25% vs. 0%), more often

1	testicular tumors. Compared to sporadic myxomas, those that occur as part of Carney complex are more commonly found in the right atrium (37% vs. 18%) or one of the ventricles (25% vs. 0%), more often multicentric (33% vs. 6%) and more likely to recur (20% vs. 3%).296 They also present earlier at an average age of 24 years old (range 4–48 years).Pathophysiology. Larger tumors are more likely to be asso-ciated with cardiovascular symptoms from obstruction, and embolic symptoms tend to occur from organized thrombi pres-ent on friable or villous tumors (Fig. 21-16). The relative fre-quencies of symptoms was illustrated by a series of 112 patients who reported cardiovascular symptoms (67%), most commonly resembling mitral valve obstruction; systemic embolization (29%); neurologic deficits (20%); and constitutional symptoms (34%).291 Similar incidences of symptoms have been reported in other large studies.Treatment. Cardiac myxomas should be promptly excised after diagnosis due to the

1	(20%); and constitutional symptoms (34%).291 Similar incidences of symptoms have been reported in other large studies.Treatment. Cardiac myxomas should be promptly excised after diagnosis due to the significant risk of embolization and cardiovascular complications, including sudden death. Resec-tion may be performed through either a median sternotomy or 9Brunicardi_Ch21_p0801-p0852.indd 84201/03/19 5:32 PM 843ACQUIRED HEART DISEASECHAPTER 21Figure 21-16. Massive left atrial myxoma. A. Intraoperative echocardiogram of a large left atrial mass, diagnosed preoperatively as a left atrial myxoma. The mass can be seen prolapsing through the mitral valve orifice causing intermittent symptoms of mitral stenosis. B. The resected specimen. The neck of the mass that was obstructing the mitral orifice is clearly delineated.a minimally invasive right thoracotomy while on cardiopulmo-nary bypass. Care is taken not to manipulate the tumor before cross clamping of the aorta in order to avoid

1	orifice is clearly delineated.a minimally invasive right thoracotomy while on cardiopulmo-nary bypass. Care is taken not to manipulate the tumor before cross clamping of the aorta in order to avoid embolization. Left atrial tumors may be approached through a standard left atriotomy.298 Exposure of large tumors attached to the interatrial septum may be facilitated by an additional parallel incision in the right atrium, but this is rarely necessary. An ideal resection encompasses both the tumor and a portion of the cardiac wall or interatrial septum to which it is attached. In order to prevent recurrence, a full thickness excision of the attachment site is preferred, but partial thickness excisions and cryoablation of the base have been performed with good late results.298 The defect created in the atrial septum can either be repaired primarily or with a small patch. Finally, patients with valvular involvement may require additional valvular reconstruction or replacement, and rare cases

1	in the atrial septum can either be repaired primarily or with a small patch. Finally, patients with valvular involvement may require additional valvular reconstruction or replacement, and rare cases of cardiac autotransplantation (with atrial recon-struction) or transplantation have been reported as strategies for complex cases of recurrent atrial myxoma.299,300Shortand long-term results following excision are excellent for benign cardiac myxomas. Operative mortality is low, and the probability of disease-free survival at 20 years has been reported to be as high as 92% for benign, sporadic myxomas.291,298 Risk of recurrence is significantly higher for familial cases. Other risk factors for recurrence include younger age, smaller tumor mass, and ventricular tumor location.301Other Benign Cardiac TumorsThere are several benign cardiac tumors apart from myxomas that are infrequent but have distinct pathophysiologic features.292 Papillary fibroelastomas are the second most common primary

1	TumorsThere are several benign cardiac tumors apart from myxomas that are infrequent but have distinct pathophysiologic features.292 Papillary fibroelastomas are the second most common primary cardiac tumor, representing approximately 8% of all cases. These tumors typically occur in more elderly patients; are small (<1 cm in diameter) sessile, pedunculated masses that arise from the mitral or aortic valves; and frequently result in embo-lization. Fibroelastomas can almost always be resected with preservation of the native valve leaflets, and cryoablation of the valve leaflet after resection can help prevent recurrence. Lipomas are encapsulated tumors that usually arise from the epicardium and remain asymptomatic in most patients. Heman-giomas, which may arise from any cardiac structure, including the pericardium, account for 2% of benign cardiac tumors, and atrioventricular node tumors, which often lead to sudden cardiac death from heart block and ventricular fibrillation, are

1	including the pericardium, account for 2% of benign cardiac tumors, and atrioventricular node tumors, which often lead to sudden cardiac death from heart block and ventricular fibrillation, are exceedingly rare.In children, rhabdomyomas are the most common pri-mary cardiac tumor, whereas fibromas are the most commonly resected cardiac tumor. Rhabdomyomas are myocardial hamar-tomas that are often multicentric in the ventricles. About 50% of cases are associated with tuberous sclerosis, and while resection is occasionally necessary, most disappear spontaneously. Fibro-mas are congenital lesions that one-third of the time are found in children younger than 1-year old. These tumors, conversely, are ordinarily solitary lesions found in the inner interventricu-lar septum, and they may present with heart failure, cyanosis, arrhythmias, syncopal episodes, chest pain, or sudden cardiac death.Malignant Cardiac TumorsPrimary cardiac malignancies are very rare, but when they occur they tend to

1	with heart failure, cyanosis, arrhythmias, syncopal episodes, chest pain, or sudden cardiac death.Malignant Cardiac TumorsPrimary cardiac malignancies are very rare, but when they occur they tend to have a right-sided predominance and frequently demonstrate extracardiac extension and involvement.292,302 Malignant cardiac tumors include intimal sarcoma, angiosar-coma, osteosarcoma, leiomyosarcoma, rhabdomyosarcoma, liposarcoma, and primary cardiac lymphomas. Intimal sarcoma is the most common subtype.303 Angiosarcomas are aggressive, rapidly invading adjacent structures, and 47% to 89% of patients present with lung, liver, or brain metastases by the time of diag-nosis. Leiomyosarcomas are sessile masses with a mucous appearance that are typically found in the posterior wall of the left atrium. Rhabdomyosarcomas are bulky (>10 cm in diam-eter) tumors that usually occur in children and do not have a predilection for any particular chamber. They frequently invade nearby cardiac structures

1	Rhabdomyosarcomas are bulky (>10 cm in diam-eter) tumors that usually occur in children and do not have a predilection for any particular chamber. They frequently invade nearby cardiac structures and are multicentric in 60% of cases. Finally, while not as frequent as secondary cardiac lymphomas, primary cardiac lymphomas are increasing in frequency due to lymphoproliferative disorders caused by Epstein-Barr virus in immunosuppressed patients. The absence of necrotic foci in lymphomas can be used to differentiate these tumors from car-diac sarcomas.Metastatic Cardiac TumorsCardiac metastases have been found in approximately 10% of autopsies performed for malignant disease.292 Secondary cardiac tumors, unlike primary tumors, are therefore relatively common. They may arise from direct extension of mediastinal tumors, hematological spread, intracavitary extension from the inferior vena cava or lymphatic extension, although the latter is the most common mechanism.While they can occur with

1	of mediastinal tumors, hematological spread, intracavitary extension from the inferior vena cava or lymphatic extension, although the latter is the most common mechanism.While they can occur with most any primary tumor, they are generally observed late in the course of disease. Malignant melanomas have a high potential for cardiac involvement, but Brunicardi_Ch21_p0801-p0852.indd 84301/03/19 5:32 PM 844SPECIFIC CONSIDERATIONSPART IIother soft tissue tumors such as lung cancer, breast cancer, sarcomas, renal carcinoma, esophageal cancer, hepatocellu-lar carcinoma, and thyroid cancer may all progress to cardiac involvement. Cardiac metastases may also develop from leuke-mia and lymphoma in 25% to 40% of cases.304Metastatic cardiac tumors are typically found in random locations, excluding the valvular tissue where lymphatics are absent, and they may be multifocal or diffusely extend along the epicardial surface. Signs of malignant cardiac involvement in cancer patients include

1	the valvular tissue where lymphatics are absent, and they may be multifocal or diffusely extend along the epicardial surface. Signs of malignant cardiac involvement in cancer patients include pericardial effusion or tamponade, tachyarrhythmias, and heart failure symptoms. Workup is simi-lar to other cardiac tumors. Treatment is generally with com-bined chemotherapy and radiation and is rarely effective.REFERENCESEntries highlighted in bright blue are key references. 1. Task Force for the Diagnosis and Management of Syncope; European Society of Cardiology (ESC); European Heart Rhythm Association (EHRA); et al. Guidelines for the diag-nosis and management of syncope (version 2009). Eur Heart J. 2009;30(21):2631-2671. 2. Braunwald E, Bonow RO. Braunwald’s Heart Disease: A Textbook of Cardiovascular Medicine, 9th ed. Philadel-phia: Saunders; 2012. 3. New York Heart Association. Diseases of the Heart and Blood Vessels: Nomenclature and Criteria for Diagnosis, 6th ed. Boston: Little,

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1	Thoracic Aneurysms and Aortic DissectionScott A. LeMaire, Ourania Preventza, and Joseph S. Coselli 22chapterANATOMY OF THE AORTAThe aorta consists of two major segments—the proximal aorta and the distal aorta—whose anatomic characteristics affect both the clinical manifestations of disease in these segments and the selection of treatment strategies for such disease (Fig. 22-1). The proximal aortic segment includes the ascending aorta and the transverse aortic arch. The ascending aorta begins at the aortic valve and ends at the origin of the innominate artery. The first portion of the ascending aorta is the aortic root, which includes the aortic valve annulus and the three sinuses of Valsalva; the coronary arteries originate from two of these sinuses. The aortic root joins the tubular portion of the ascending aorta at the sinotubular ridge. The transverse aortic arch is the area from which the brachio-cephalic branches arise. The distal aortic segment includes the descending thoracic

1	of the ascending aorta at the sinotubular ridge. The transverse aortic arch is the area from which the brachio-cephalic branches arise. The distal aortic segment includes the descending thoracic aorta and the abdominal aorta. The descending thoracic aorta begins distal to the origin of the left subclavian artery and extends to the diaphragmatic hia-tus, where it joins the abdominal aorta. The descending tho-racic aorta gives rise to multiple bronchial and esophageal branches, as well as to the segmental intercostal arteries, which provide circulation to the spinal cord.The volume of blood that flows through the thoracic aorta at high pressure is far greater than that found in any other vascular structure. For this reason, any condition that disrupts the integrity of the thoracic aorta, such as aortic dissection, aneurysm rupture, or traumatic injury, can have catastrophic consequences.Historically, open surgical repair of such conditions has been an intimidating undertaking associated

1	as aortic dissection, aneurysm rupture, or traumatic injury, can have catastrophic consequences.Historically, open surgical repair of such conditions has been an intimidating undertaking associated with significant morbidity and mortality. Strategies for protecting the brain and spinal cord during such repairs have become critical in preventing devastating complications. Endovascular therapy for such conditions in selected patients has become accepted practice, producing fewer adverse outcomes than traditional approaches.THORACIC AORTIC ANEURYSMSAortic aneurysm is defined as a permanent, localized dilatation of the aorta to a diameter that is at least 50% greater than is normal at that anatomic level.1 The annual incidence of thoracic aortic aneurysms is estimated to be 5.9 per 100,000 persons.2 The clinical manifestations, methods of treatment, and treatment results in patients with aortic aneurysms vary according to the cause and the aortic segment involved. Causes of thoracic

1	persons.2 The clinical manifestations, methods of treatment, and treatment results in patients with aortic aneurysms vary according to the cause and the aortic segment involved. Causes of thoracic aortic aneurysms include degenerative disease of the aortic wall, aor-tic dissection, aortitis, infection, and trauma. Aneurysms can be localized to a single aortic segment, or they can involve multiple segments. Thoracoabdominal aortic aneurysms, for example, involve both the descending thoracic aorta and the abdominal aorta. In the most extreme cases, the entire aorta is aneurysmal; this condition is often called mega-aorta.Aortic aneurysms can be either “true” or “false.” True aneurysms can take two forms: fusiform and saccular. Fusiform aneurysms are more common and can be described as sym-metrical dilatations of the aorta. Saccular aneurysms are local-ized outpouchings of the aorta. False aneurysms, also called pseudoaneurysms, are leaks in the aortic wall that are contained by the

1	dilatations of the aorta. Saccular aneurysms are local-ized outpouchings of the aorta. False aneurysms, also called pseudoaneurysms, are leaks in the aortic wall that are contained by the outer layer of the aorta and/or the periaortic tissue; they are caused by disruption of the aortic wall and lead blood to collect in pouches of fibrotic tissue.Aneurysms of the thoracic aorta consistently increase in size and eventually progress to cause serious complications. These include rupture, which usually is a fatal event. Therefore, aggressive treatment is indicated in all but the poorest surgical candidates. Small, asymptomatic thoracic aortic aneurysms can be followed, especially in high-surgical-risk patients, and can be treated surgically later if symptoms or complications develop, or if progressive enlargement occurs. Meticulous control of hyper-tension is the primary medical treatment for patients with small, asymptomatic aneurysms.Elective resection with graft replacement is indicated

1	enlargement occurs. Meticulous control of hyper-tension is the primary medical treatment for patients with small, asymptomatic aneurysms.Elective resection with graft replacement is indicated in asymptomatic patients with an aortic diameter of at least twice Anatomy of the Aorta853Thoracic Aortic Aneurysms853Causes and Pathogenesis / 854Clinical History / 857Clinical Manifestations / 857Diagnostic Evaluation / 858Treatment / 860Aortic Dissection876Pathology and Classification / 876Causes and Clinical History / 879Clinical Manifestations / 879Diagnostic Evaluation / 880Treatment / 881Outcomes885Repair of Proximal Aortic Aneurysms / 885Treatment of Acute Ascending Aortic Dissection / 888Repair of Distal Aortic Aneurysms / 888Treatment of Descending Thoracic Aortic Dissection / 888Conclusions889Acknowledgments889Brunicardi_Ch22_p0853-p0896.indd 85301/03/19 5:40 PM 854Figure 22-1. Illustration of normal thoracic aortic anatomy. The brachiocephalic vessels arise from the transverse

1	85301/03/19 5:40 PM 854Figure 22-1. Illustration of normal thoracic aortic anatomy. The brachiocephalic vessels arise from the transverse aortic arch and are used as anatomic landmarks to define the aortic regions. The ascending aorta is proximal to the innominate artery, whereas the descending aorta is distal to the left subclavian artery.normal in the involved segment (5 to 6 cm in most thoracic segments). Elective repair is contraindicated by extreme opera-tive risk due to severe coexisting cardiac or pulmonary dis-ease and by other conditions that limit life expectancy, such as malignancy. An emergency operation is performed for any patient in whom a ruptured aneurysm is suspected.Patients with thoracic aortic aneurysm often have coexisting aneurysms of other aortic segments. A common cause of death after repair of a thoracic aortic aneurysm is rupture of a different aortic aneurysm. Therefore, staged repair of multiple aortic seg-ments often is necessary. As with any major

1	common cause of death after repair of a thoracic aortic aneurysm is rupture of a different aortic aneurysm. Therefore, staged repair of multiple aortic seg-ments often is necessary. As with any major operation, careful pre-operative evaluation for coexisting disease and subsequent medical optimization are important for successful surgical treatment.An alternative to traditional open repair of a descending thoracic aortic aneurysm is endovascular stent grafting. Certain anatomic criteria for use—such as a landing zone that includes at least 2 cm of landing zone of healthy aortic tissue proximal and distal to the targeted aneurysm—are preferable, but not absolutely necessary. Although few data on long-term outcomes have recently been published, endovascular repair of descending thoracic aortic aneurysm has become an accepted practice that produces excellent midterm results.Causes and PathogenesisGeneral Considerations. The normal aorta derives its elastic-ity and tensile strength from

1	aneurysm has become an accepted practice that produces excellent midterm results.Causes and PathogenesisGeneral Considerations. The normal aorta derives its elastic-ity and tensile strength from the medial layer, which contains approximately 45 to 55 lamellae of elastin, collagen, smooth muscle cells, and ground substance. Elastin content is highest within the ascending aorta, as would be expected because of its compliant nature, and decreases distally into the descending and abdominal aorta. Maintenance of the aortic matrix involves complex interactions among smooth muscle cells, macrophages, proteases, and protease inhibitors. Any alteration in this delicate balance can lead to aortic disease.Thoracic aortic aneurysms have a variety of causes (Table 22-1). Although these disparate pathologic processes differ in biochemical and histologic terms, they share the final common pathway of progressive aortic expansion and eventual rupture.Hemodynamic factors clearly contribute to the

1	pathologic processes differ in biochemical and histologic terms, they share the final common pathway of progressive aortic expansion and eventual rupture.Hemodynamic factors clearly contribute to the process of aortic dilatation. The vicious cycle of increasing diameter and increasing wall tension, as characterized by Laplace’s law (tension = pressure × radius), is well established. Turbulent Key Points1 Assessing urgency of repair is essential to developing the appropriate management plan. Although emergent repair carries greater operative risk than does elective repair, any inappropriate delay of repair risks death.2 The clinical progression of an aortic aneurysm is continued expansion and eventual dissection or rupture. Hence, regular noninvasive imaging studies, as part of a lifelong surveil-lance plan, are necessary to ensure long-term patient health. Even small asymptomatic aneurysms should be routinely imaged to assess overall size and yearly rate of expansion.3 Endovascular

1	surveil-lance plan, are necessary to ensure long-term patient health. Even small asymptomatic aneurysms should be routinely imaged to assess overall size and yearly rate of expansion.3 Endovascular repair devices are approved for the treatment of descending thoracic aortic aneurysms, descending thoracic aortic dissections, aortic trauma, and penetrating aortic ulcer.4 Practice guidelines have been published to help standardize the decision-making process and select an appropriate surgi-cal intervention, as well as to standardize the use of imaging studies for patients with thoracic aortic disease.5 Ascending aortic aneurysms that are symptomatic or ≥5.5 cm in diameter should be repaired regardless of whether the patient has a bicuspid or tricuspid aortic valve. This threshold is lowered for patients with certain heritable disor-ders affecting the aorta and for patients with additional risk factors, such as rapid aortic expansion (≥0.5 cm per year) or a family history of

1	is lowered for patients with certain heritable disor-ders affecting the aorta and for patients with additional risk factors, such as rapid aortic expansion (≥0.5 cm per year) or a family history of dissection.6 Surgical repair involves the development of a patienttailored plan based on careful preoperative medical evalua-tion. When appropriate, optimizing a patient’s health status—to mitigate existing comorbidities—is important before surgical intervention.7 The development and use of surgical adjuncts like antegrade selective cerebral perfusion and cerebrospinal fluid drainage have significantly reduced the morbidity rates traditionally associated with complex aortic repair.8 Proximal aortic dissection is a life-threatening condition, and immediate operative repair is generally indicated, although definitive aortic repair may be delayed until after severe mal-perfusion has been treated.Brunicardi_Ch22_p0853-p0896.indd 85401/03/19 5:40 PM 855THORACIC ANEURYSMS AND AORTIC

1	indicated, although definitive aortic repair may be delayed until after severe mal-perfusion has been treated.Brunicardi_Ch22_p0853-p0896.indd 85401/03/19 5:40 PM 855THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22Table 22-1Causes of thoracic aortic aneurysmNonspecific medial degenerationAortic dissectionHeritable conditions Marfan syndrome Loeys-Dietz syndrome Ehlers-Danlos syndrome Turner syndrome Familial thoracic aortic aneurysm Aneurysms-osteoarthritis syndrome Congenital bicuspid aortic valve Bovine aortic archPoststenotic dilatationInfectionAortitis Takayasu arteritis Giant cell arteritis Rheumatoid aortitisTrauma (pseudoaneurysm)blood flow also is recognized as a factor. Poststenotic aortic dilatation, for example, occurs in some patients with aortic valve stenosis or coarctation of the descending thoracic aorta. Hemodynamic derangements, however, are only one piece of a complex puzzle.Atherosclerosis is commonly cited as a cause of thoracic aortic aneurysms. However,

1	of the descending thoracic aorta. Hemodynamic derangements, however, are only one piece of a complex puzzle.Atherosclerosis is commonly cited as a cause of thoracic aortic aneurysms. However, although atherosclerotic disease often is found in conjunction with aortic aneurysms, the notion that atherosclerosis is a distinct cause of aneurysm formation has been challenged. In most thoracic aortic aneurysms, ath-erosclerosis appears to be a coexisting process, rather than the underlying cause.Research into the pathogenesis of abdominal aortic aneurysms has focused on the molecular mechanisms of aortic wall degeneration and dilatation.3 For example, imbalances between proteolytic enzymes (e.g., matrix metalloproteinases) and their inhibitors contribute to abdominal aortic aneurysm formation. Building on these advances, current investigations are attempting to determine whether similar inflammatory and proteolytic mechanisms are involved in thoracic aortic disease, in hope of identifying

1	Building on these advances, current investigations are attempting to determine whether similar inflammatory and proteolytic mechanisms are involved in thoracic aortic disease, in hope of identifying potential molecular targets for pharmacologic therapy.Nonspecific Medial Degeneration. Nonspecific medial degeneration is the most common cause of thoracic aortic dis-ease. Histologic findings of mild medial degeneration, includ-ing fragmentation of elastic fibers and loss of smooth muscle cells, are expected in the aging aorta. However, an advanced, accelerated form of medial degeneration leads to progressive weakening of the aortic wall, aneurysm formation, and eventual dissection, rupture, or both. The underlying causes of medial degenerative disease remain poorly understood.Aortic Dissection. An aortic dissection usually begins as a tear in the inner aortic wall, which initiates a progressive sepa-ration of the medial layers and creates two channels within the aorta. This event

1	aortic dissection usually begins as a tear in the inner aortic wall, which initiates a progressive sepa-ration of the medial layers and creates two channels within the aorta. This event profoundly weakens the outer wall. As the most common catastrophe involving the aorta, dissection repre-sents a major, distinct cause of thoracic aortic aneurysms and is discussed in detail in the second half of this chapter.Heritable Conditions. Several heritable conditions cause thoracic aortic aneurysms. To better characterize these disorders, the National Institutes of Health (NIH) sponsored a longitudinal registry for individuals affected by genetically triggered thoracic aortic aneurysms and cardiovascular conditions (GenTAC) more than a decade ago.4 The registry enrollment includes adults and children in 13 clinical categories, including Marfan syndrome, Ehlers-Danlos syndrome, Loeys-Dietz syndrome, familial tho-racic aortic aneurysms and dissections, aneurysms-osteoarthritis syndrome, and

1	in 13 clinical categories, including Marfan syndrome, Ehlers-Danlos syndrome, Loeys-Dietz syndrome, familial tho-racic aortic aneurysms and dissections, aneurysms-osteoarthritis syndrome, and congenital bicuspid aortic valve.Marfan Syndrome Marfan syndrome is an autosomal domi-nant genetic disorder characterized by a specific connective tissue defect that leads to aneurysm formation. The phenotype of patients with Marfan syndrome typically includes a tall stat-ure, high palate, joint hypermobility, eye lens disorders, mitral valve prolapse, and aortic aneurysms. The aortic wall is weak-ened by fragmentation of elastic fibers and deposition of exten-sive amounts of mucopolysaccharides (a process previously called cystic medial degeneration or cystic medial necrosis). Patients with Marfan syndrome have a mutation in the fibrillin gene located on the long arm of chromosome 15. The tradi-tional view has been that abnormal fibrillin in the extracellular matrix decreases connective tissue

1	syndrome have a mutation in the fibrillin gene located on the long arm of chromosome 15. The tradi-tional view has been that abnormal fibrillin in the extracellular matrix decreases connective tissue strength in the aortic wall and produces abnormal elasticity, which predisposes the aorta to dilatation from wall tension caused by left ventricular ejec-tion impulses.5 More recent evidence, however, shows that the abnormal fibrillin causes degeneration of the aortic wall matrix by increasing the activity of transforming growth factor beta (TGF-β).6 Between 75% and 85% of patients with Marfan syn-drome have dilatation of the ascending aorta and annuloaortic ectasia (dilatation of the aortic sinuses and annulus).7 Marfan syndrome also is frequently associated with aortic dissection, and aortic complications are the most common cause of death among patients with Marfan syndrome.8Loeys-Dietz Syndrome Loeys-Dietz syndrome is phenotypi-cally distinct from Marfan syndrome. It is characterized

1	complications are the most common cause of death among patients with Marfan syndrome.8Loeys-Dietz Syndrome Loeys-Dietz syndrome is phenotypi-cally distinct from Marfan syndrome. It is characterized as an aneurysmal syndrome with widespread systemic involvement. Loeys-Dietz syndrome is an aggressive, autosomal dominant condition that is distinguished by the triad of arterial tortuosity and aneurysms, hypertelorism (widely spaced eyes), and bifid uvula or cleft palate. It is caused by heterozygous mutations in the genes encoding TGF-β receptors.9,10 Patients with Loeys-Dietz syndrome—including young children—are at increased risk of aortic rupture and aortic dissection; diameter-based thresholds of repair tend to be lower for patients with this syndrome than for patients with other heritable disorders.Ehlers-Danlos Syndrome Ehlers-Danlos syndrome includes a spectrum of inherited disorders of collagen synthesis. The sub-types represent differing defective steps of collagen production.

1	disorders.Ehlers-Danlos Syndrome Ehlers-Danlos syndrome includes a spectrum of inherited disorders of collagen synthesis. The sub-types represent differing defective steps of collagen production. Vascular type Ehlers-Danlos syndrome is characterized by an autosomal dominant defect in type III collagen synthesis, which can have life-threatening cardiovascular manifestations. Sponta-neous arterial rupture, usually involving the mesenteric vessels, is the most common cause of death in these patients. Thoracic aortic aneurysms and dissections are less commonly associated with Ehlers-Danlos syndrome, but when they do occur, they pose a particularly challenging surgical problem because of the reduced integrity of the aortic tissue.11 An Ehlers-Danlos variant of periventricular heterotopia associated with joint and skin hyperextensibility and aortic dilation has been described as being caused by mutations in the gene encoding filamin A Brunicardi_Ch22_p0853-p0896.indd 85501/03/19 5:40 PM

1	with joint and skin hyperextensibility and aortic dilation has been described as being caused by mutations in the gene encoding filamin A Brunicardi_Ch22_p0853-p0896.indd 85501/03/19 5:40 PM 856SPECIFIC CONSIDERATIONSPART II(FLNA), an actin-binding protein that links the smooth muscle cell contractile unit to the cell surface.12Familial Thoracic Aortic Aneurysm and Dissection Fami-lies without the heritable syndromes described earlier also can be affected by genetic conditions that cause thoracic aortic aneurysm. In fact, it is estimated that at least 20% of patients with thoracic aortic aneurysms and dissections have a genetic predisposition to them. The involved mutations are characterized by autosomal dominant inheritance with decreased penetrance and variable expression. The number of genes for which mutations have been identified as causes of familial thoracic aortic aneurysm and dissection is expanding rapidly; involved genes include those related to TGF-β receptors (TGFBR1

1	of genes for which mutations have been identified as causes of familial thoracic aortic aneurysm and dissection is expanding rapidly; involved genes include those related to TGF-β receptors (TGFBR1 and TGFBR2), TGF-β ligands (TGFB2 and TGFB3), myosin (MYH11 and MYLK), elastin (ELN), elastin microfibril interfacer 1 (EMLIN1), microfibril-associated glycoprotein 2 (MFAP5), fibrillin-2 (FBN2), fibulin-4 (FBLN4), lysyl oxidase (LOX), and α-smooth muscle cell actin (ACTA2).3,13-16 ACTA2 mutations are present in approximately 14% of families with familial thoracic aortic aneurysms and dissections.Aneurysms-Osteoarthritis Syndrome Aneurysmsosteoarthritis syndrome is an autosomal dominant disorder char-acterized by aortic and arterial aneurysms, arterial tortuosity, aor-tic dissection, mild craniofacial abnormalities, and early-onset osteoarthritis. Aneurysms-osteoarthritis syndrome is caused by mutations in the gene encoding SMAD3, a transcription factor for TGF-β. Affected patients have a

1	abnormalities, and early-onset osteoarthritis. Aneurysms-osteoarthritis syndrome is caused by mutations in the gene encoding SMAD3, a transcription factor for TGF-β. Affected patients have a high incidence of aortic dissection, which often occurs in a mildly dilated aorta and causes sudden death.17Congenital Bicuspid Aortic Valve Bicuspid aortic valve is the most common congenital malformation of the heart or great vessels, affecting up to 2% of Americans.18 Compared to patients with a normal, trileaflet aortic valve, patients with bicuspid aortic valve have an increased incidence of ascending aortic aneurysm formation and, often, a more rapid rate of aortic enlargement.19 The location of the fused leaflet, or raphe, may be predictive of aortic dilation and other abnormalities.20 Fifty to 70% of adults with bicuspid aortic valve, but without significant valve dysfunction, have echocardiographically detectable aortic dilatation.21,22 This dilatation usually is limited to the ascending

1	70% of adults with bicuspid aortic valve, but without significant valve dysfunction, have echocardiographically detectable aortic dilatation.21,22 This dilatation usually is limited to the ascending aorta and root.23 Dilation occasionally is found in the arch and only rarely in the descending or abdominal aorta. In addition, aortic dissection occurs 10 times more often in patients with bicuspid valves than in the general population.24 Recent findings suggest that aneurysms associated with bicuspid aortic valve have a fundamentally different pathobiologic cause than aneu-rysms that occur in patients with trileaflet valves.25Although the exact mechanism responsible for aneu-rysm formation in patients with bicuspid aortic valve remains unclear, evidence suggests that these patients have a congeni-tal connective tissue abnormality that predisposes the aorta to medial degeneration.25-31 For example, fibrillin-1 content is sig-nificantly lower and matrix metalloproteinase activity is

1	a congeni-tal connective tissue abnormality that predisposes the aorta to medial degeneration.25-31 For example, fibrillin-1 content is sig-nificantly lower and matrix metalloproteinase activity is signifi-cantly higher in the aortic media in patients with bicuspid aortic valve than in persons with a normal, tricuspid aortic valve.25-27 Further, the process of medial degeneration in patients with bicuspid aortic valve may be exacerbated by the presence of chronic turbulent flow through the deformed valve.Bovine Aortic Arch Bovine aortic arch—a common origin of the innominate and left common carotid arteries—has been con-sidered a normal anatomic variant. Studies from Yale University have identified a higher prevalence of bovine aortic arch in patients with thoracic aortic disease; an association was found between this anomaly and a generalized increase in aortic aneu-rysmal disease (without any predisposition to a particular aortic region). However, bovine aortic arch was not

1	an association was found between this anomaly and a generalized increase in aortic aneu-rysmal disease (without any predisposition to a particular aortic region). However, bovine aortic arch was not associated dis-tinctly with bicuspid aortic valve or aortic dissection, but with a higher mean aortic growth rate: 0.29 cm per year in patients with bovine aortic arch, compared with 0.09 cm per year in controls. Therefore, bovine aortic arch may be better character-ized as a precursor of aortic aneurysm than as a simple normal anatomic variant.32 Further studies are needed to delineate the underlying mechanism for this association.Infection. Primary infection of the aortic wall resulting in aneurysm formation is rare. Although these lesions are termed mycotic aneurysms, the responsible pathogens usually are bac-teria rather than fungi. Bacterial invasion of the aortic wall may result from bacterial endocarditis, endothelial trauma caused by an aortic jet lesion, or extension from an

1	usually are bac-teria rather than fungi. Bacterial invasion of the aortic wall may result from bacterial endocarditis, endothelial trauma caused by an aortic jet lesion, or extension from an infected laminar clot within a preexisting aneurysm. The most common causative organisms are Staphylococcus aureus, Staphylococcus epider-midis, Salmonella, and Streptococcus.33,34 Unlike most other causes of thoracic aortic aneurysms, which generally produce fusiform aneurysms, infection often produces saccular aneu-rysms located in areas of aortic tissue destroyed by the infec-tious process.Although syphilis was once the most common cause of ascending aortic aneurysms, the advent of effective antibiotic therapy has made syphilitic aneurysms a rarity in developed nations. In other parts of the world, however, syphilitic aneu-rysms remain a major cause of morbidity and mortality. The spi-rochete Treponema pallidum causes an obliterative endarteritis of the vasa vasorum that results in medial

1	however, syphilitic aneu-rysms remain a major cause of morbidity and mortality. The spi-rochete Treponema pallidum causes an obliterative endarteritis of the vasa vasorum that results in medial ischemia and loss of the elastic and muscular elements of the aortic wall. The ascend-ing aorta and arch are the most commonly involved areas. The emergence of HIV infection in the 1980s was associated with a substantial increase in the incidence of syphilis in both HIV-positive and HIV-negative patients. Because syphilitic aortitis often presents 10 to 30 years after the primary infection, the inci-dence of associated aneurysms may increase in the near future.Aortitis. In patients with preexisting degenerative thoracic aortic aneurysms, localized transmural inflammation and subse-quent fibrosis can develop. The dense aortic infiltrate responsible for the fibrosis consists of lymphocytes, plasma cells, and giant cells. The cause of the intense inflammatory reaction is unknown. Although the

1	can develop. The dense aortic infiltrate responsible for the fibrosis consists of lymphocytes, plasma cells, and giant cells. The cause of the intense inflammatory reaction is unknown. Although the severe inflammation is a superimposed problem rather than a primary cause, its onset within an aneurysm can further weaken the aortic wall and precipitate expansion.Systemic autoimmune disorders also cause thoracic aor-titis. Aortic Takayasu arteritis generally produces obstructive lesions related to severe intimal thickening, but associated medial necrosis can lead to aneurysm formation. In patients with giant cell arteritis (temporal arteritis), granulomatous inflam-mation may develop that involves the entire thickness of the aortic wall, causing intimal thickening and medial destruction. Rheumatoid aortitis is an uncommon systemic disease that is associated with rheumatoid arthritis and ankylosing spondylitis. The resulting medial inflammation and fibrosis can affect the aortic root,

1	Rheumatoid aortitis is an uncommon systemic disease that is associated with rheumatoid arthritis and ankylosing spondylitis. The resulting medial inflammation and fibrosis can affect the aortic root, causing annular dilatation, aortic valve regurgitation, and ascending aortic aneurysm formation.Pseudoaneurysms. Pseudoaneurysms of the thoracic aorta usually represent chronic leaks that are contained by surrounding Brunicardi_Ch22_p0853-p0896.indd 85601/03/19 5:40 PM 857THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22tissue and fibrosis. By definition, the wall of a pseudoaneurysm is not formed by intact aortic tissue; rather, the wall devel-ops from organized thrombus and associated fibrosis. Pseu-doaneurysms can arise from primary defects in the aortic wall (e.g., after trauma or contained aneurysm rupture) or from anas-tomotic or cannulation site leaks that occur after cardiovascular surgery. Anastomotic pseudoaneurysms can be caused by tech-nical problems or by deterioration

1	aneurysm rupture) or from anas-tomotic or cannulation site leaks that occur after cardiovascular surgery. Anastomotic pseudoaneurysms can be caused by tech-nical problems or by deterioration of the native aortic tissue, graft material, or suture. Commonly, they occur in patients with Marfan syndrome, Loeys-Dietz syndrome, or other heritable conditions that markedly weaken the vessel wall.35 Tissue dete-rioration usually is related to either progressive degenerative disease or infection. Improvements in sutures, graft materials, and surgical techniques have decreased the incidence of tho-racic aortic pseudoaneurysm. Should thoracic aortic pseudoan-eurysms occur, they typically require expeditious open surgical or catheter-based repair because they are associated with a high incidence of morbidity and rupture.Clinical HistoryTreatment decisions in cases of thoracic aortic aneurysm are guided by our current understanding of the clinical history of these aneurysms, which classically is

1	morbidity and rupture.Clinical HistoryTreatment decisions in cases of thoracic aortic aneurysm are guided by our current understanding of the clinical history of these aneurysms, which classically is characterized as progres-sive aortic dilatation and eventual dissection, rupture, or both. An analysis by Elefteriades of data from 1600 patients with thoracic aortic disease has helped quantify these well-recognized risks.36 Average expansion rates were 0.07 cm per year in ascending aortic aneurysms and 0.19 cm per year in descending thoracic aortic aneurysms. As expected, aortic diam-eter was a strong predictor of rupture, dissection, and mortality. For thoracic aortic aneurysms >6 cm in diameter, annual rates of catastrophic complications were 3.6% for rupture, 3.7% for dissection, and 10.8% for death. Critical “hinge-point” diame-ters, at which the incidence of expected complications signifi-cantly increased, were 6.0 cm for aneurysms of the ascending aorta and 7.0 cm for aneurysms of

1	for death. Critical “hinge-point” diame-ters, at which the incidence of expected complications signifi-cantly increased, were 6.0 cm for aneurysms of the ascending aorta and 7.0 cm for aneurysms of the descending thoracic aorta; the corresponding risks of rupture after reaching these diameters were 31% and 43%, respectively.37Certain types of aneurysms have an elevated propensity for expansion and rupture. For example, aneurysms in patients with Marfan or Loeys-Dietz syndrome tend to dilate at an accelerated rate and rupture or dissect at smaller diameters than sporadic, nonheritable aneurysms. Before the era of surgical treatment for aortic aneurysms, the aggressive form of aortic disease in Marfan patients resulted in an average life expectancy of 32 years, with aortic root complications causing the majority of deaths.38 Saccular aneurysms, which commonly are associated with aortic infection and typically affect only a discrete small section of the aorta, tend to grow more rapidly

1	causing the majority of deaths.38 Saccular aneurysms, which commonly are associated with aortic infection and typically affect only a discrete small section of the aorta, tend to grow more rapidly than fusiform aneurysms, which are associated with more widespread degen-erative changes and generally affect a larger section of the aorta.One common clinical scenario deserves special attention. A moderately dilated ascending aorta (i.e., 4 to 5 cm) often is encountered during aortic valve replacement or coronary artery bypass operations. The clinical history of these ectatic ascend-ing aortas has been defined by several studies. Michel and colleagues39 studied patients whose ascending aortic diameters were >4 cm at the time of aortic valve replacement; 25% of these patients required reoperation for ascending aortic replacement. Prenger and colleagues40 reported that aortic dissection occurred in 27% of patients who had aortic diameters of >5 cm at the time of aortic valve replacement.

1	for ascending aortic replacement. Prenger and colleagues40 reported that aortic dissection occurred in 27% of patients who had aortic diameters of >5 cm at the time of aortic valve replacement. Attention has been directed toward whether or not a mildly dilated aortic root should be replaced in patients with bicuspid aortic valve who are undergoing iso-lated valve replacement, and at what threshold to intervene. Although this is a controversial issue, many surgeons believe that the tendency toward late aortic dilatation in these patients war-rants aggressive treatment.41,42 According to a recent guidelines clarification,43 in patients with bicuspid aortic valve who are undergoing aortic valve replacement or repair, replacing the ascending aorta is reasonable when the diameter of the ascending aorta is greater than 4.5 cm (Class IIa, Level C recommendation).Clinical ManifestationsIn many patients with thoracic aortic aneurysms, the aneurysm is discovered incidentally when imaging

1	ascending aorta is greater than 4.5 cm (Class IIa, Level C recommendation).Clinical ManifestationsIn many patients with thoracic aortic aneurysms, the aneurysm is discovered incidentally when imaging studies are performed for unrelated reasons. Therefore, patients often are asymptom-atic at the time of diagnosis. However, thoracic aortic aneurysms that initially go undetected eventually create symptoms and signs that correspond with the segment of aorta that is involved. These aneurysms have a wide variety of manifestations, includ-ing compression or erosion of adjacent structures, aortic valve regurgitation, distal embolism, and rupture.Local Compression and Erosion. Initially, aneurysmal expan-sion and impingement on adjacent structures causes mild, chronic pain. The most common symptom in patients with ascending aor-tic aneurysms is anterior chest discomfort; the pain is frequently precordial in location but may radiate to the neck and jaw, mim-icking angina. Aneurysms of the

1	in patients with ascending aor-tic aneurysms is anterior chest discomfort; the pain is frequently precordial in location but may radiate to the neck and jaw, mim-icking angina. Aneurysms of the ascending aorta and transverse aortic arch can cause symptoms related to compression of the superior vena cava, the pulmonary artery, the airway, or the ster-num. Rarely, these aneurysms erode into the superior vena cava or right atrium, causing acute high-output failure. Expansion of the distal aortic arch can stretch the recurrent laryngeal nerve, which results in left vocal cord paralysis and hoarseness. Descending thoracic and thoracoabdominal aneurysms frequently cause back pain localized between the scapulae. When the aneurysm is larg-est in the region of the aortic hiatus, it may cause middle back and epigastric pain. Thoracic or lumbar vertebral body erosion typically causes severe, chronic back pain; extreme cases can present with spinal instability and neurologic deficits from spinal

1	back and epigastric pain. Thoracic or lumbar vertebral body erosion typically causes severe, chronic back pain; extreme cases can present with spinal instability and neurologic deficits from spinal cord compression. Although mycotic aneurysms have a peculiar propensity to destroy vertebral bodies, spinal erosion also occurs with degenerative aneurysms. Descending thoracic aortic aneu-rysms may cause varying degrees of airway obstruction, mani-festing as cough, wheezing, stridor, or pneumonitis. Pulmonary or airway erosion presents as hemoptysis. Compression and ero-sion of the esophagus cause dysphagia and hematemesis, respec-tively. Thoracoabdominal aortic aneurysms can cause duodenal obstruction or, if they erode through the bowel wall, gastrointes-tinal bleeding. Jaundice due to compression of the liver or porta hepatis is uncommon. Erosion into the inferior vena cava or iliac vein presents with an abdominal bruit, widened pulse pressure, edema, and heart failure.Aortic Valve

1	of the liver or porta hepatis is uncommon. Erosion into the inferior vena cava or iliac vein presents with an abdominal bruit, widened pulse pressure, edema, and heart failure.Aortic Valve Regurgitation. Ascending aortic aneurysms can cause displacement of the aortic valve commissures and annular dilatation. The resulting deformation of the aortic valve leads to progressively worsening aortic valve regurgitation. In response to the volume overload, the heart remodels and becomes increasingly dilated. Patients with this condition may present with progressive heart failure, a widened pulse pressure, and a diastolic murmur.Distal Embolization. Thoracic aortic aneurysms—particularly those involving the descending and thoracoabdominal aorta—are commonly lined with friable, atheromatous plaque and 1Brunicardi_Ch22_p0853-p0896.indd 85701/03/19 5:40 PM 858SPECIFIC CONSIDERATIONSPART IIFigure 22-2. Chest radiographs showing a calcified rim (arrows) in the aortic wall of a thoracoabdominal

1	and 1Brunicardi_Ch22_p0853-p0896.indd 85701/03/19 5:40 PM 858SPECIFIC CONSIDERATIONSPART IIFigure 22-2. Chest radiographs showing a calcified rim (arrows) in the aortic wall of a thoracoabdominal aortic aneurysm. A. Anteroposterior view. B. Lateral view.mural thrombus. This debris may embolize distally, caus-ing occlusion and thrombosis of the visceral, renal, or lower-extremity branches.Rupture. Patients with ruptured thoracic aortic aneurysms often experience sudden, severe pain in the anterior chest (ascending aorta), upper back or left chest (descending thoracic aorta), or left flank or abdomen (thoracoabdominal aorta). When ascending aortic aneurysms rupture, they usually bleed into the pericardial space, producing acute cardiac tamponade and death. Descending thoracic aortic aneurysms rupture into the pleural cavity, producing a combination of severe hemorrhagic shock and respiratory compromise. External rupture is extremely rare; saccular syphilitic aneurysms have been

1	aneurysms rupture into the pleural cavity, producing a combination of severe hemorrhagic shock and respiratory compromise. External rupture is extremely rare; saccular syphilitic aneurysms have been observed to rupture externally after eroding through the sternum.Diagnostic EvaluationDiagnosis and characterization of thoracic aneurysms require imaging studies, which also provide critical information that guides the selection of treatment options. Although the best choice of imaging technique for the thoracic and thoracoab-dominal aorta is somewhat institution-specific, varying with the availability of imaging equipment and expertise, efforts have been made to standardize key elements of image acquisition and reporting. Recent practice guidelines44 recommend that aortic imaging reports plainly state the location of aortic abnormalities (including calcification and the extent to which abnormalities extend into branch vessels), the maximum external aortic diam-eters (rather than

1	plainly state the location of aortic abnormalities (including calcification and the extent to which abnormalities extend into branch vessels), the maximum external aortic diam-eters (rather than internal, lumen-based diameters), internal fill-ing defects, and any evidence of rupture. Whenever possible, all results should be compared with those of prior imaging studies.Plain Radiography. Plain radiographs of the chest, abdomen, or spine often provide enough information to support the initial diag-nosis of thoracic aortic aneurysm. Ascending aortic aneurysms produce a convex shadow to the right of the cardiac silhouette. The anterior projection of an ascending aneurysm results in the loss of the retrosternal space in the lateral view. An aneurysm may be indistinguishable from elongation and tortuosity.45 Impor-tantly, chest radiographs (CXRs) may appear normal in patients with thoracic aortic disease and thus cannot exclude the diagnosis of aortic aneurysm. Aortic root aneurysms, for

1	tortuosity.45 Impor-tantly, chest radiographs (CXRs) may appear normal in patients with thoracic aortic disease and thus cannot exclude the diagnosis of aortic aneurysm. Aortic root aneurysms, for example, often are hidden within the cardiac silhouette. Plain CXRs may reveal convexity in the right superior mediastinum, loss of the retroster-nal space, or widening of the descending thoracic aortic shadow, which may be highlighted by a rim of calcification outlining the dilated aneurysmal aortic wall. Aortic calcification also may be seen in the upper abdomen on a standard radiograph made in the anteroposterior or lateral projection (Fig. 22-2). Once a thoracic aortic aneurysm is detected on plain radiographs, additional stud-ies are required to define the extent of aortic involvement.Echocardiography and Abdominal Ultrasonography. Ascending aortic aneurysms are commonly discovered during echocardiography in patients presenting with symptoms or signs of aortic valve regurgitation. Both

1	and Abdominal Ultrasonography. Ascending aortic aneurysms are commonly discovered during echocardiography in patients presenting with symptoms or signs of aortic valve regurgitation. Both transthoracic and transesophageal echocardiography provide excellent visualization of the ascending aorta, including the aortic root.46 Transesophageal echocardiography also allows visualization of the descending thoracic aorta but is not ideal for evaluating the transverse aortic arch (which is obscured by air in the tracheobronchial tree) or the upper abdominal aorta. Effective echocardiography requires considerable technical skill, both in obtaining adequate images and in interpreting them. This imaging modality has the added Brunicardi_Ch22_p0853-p0896.indd 85801/03/19 5:40 PM 859THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22Figure 22-3. Current practice guidelines44 seek to standardize the reporting of aortic diameters by indicating key locations of mea-surement. These include (1) the

1	AND AORTIC DISSECTIONCHAPTER 22Figure 22-3. Current practice guidelines44 seek to standardize the reporting of aortic diameters by indicating key locations of mea-surement. These include (1) the sinuses of Valsalva, (2) the sinotu-bular junction, (3) the mid-ascending aorta, (4) the proximal aortic arch at the origins of the innominate artery, (5) the mid-aortic arch, which is between the left common carotid and left subclavian arter-ies, (6) the proximal descending thoracic aorta, which begins at the isthmus (approximately 2 cm distal to the origins of the left subcla-vian artery), (7) the mid-descending thoracic artery, (8) the aorta at the diaphragm, and (9) the abdominal aorta at the origins of the celiac axis. (Used with permission of Baylor College of Medicine.)benefit of assessing cardiac function and revealing any other abnormalities that may be present. During ultrasound evaluation of a suspected infrarenal abdominal aortic aneurysm, if a definitive neck cannot be identified

1	cardiac function and revealing any other abnormalities that may be present. During ultrasound evaluation of a suspected infrarenal abdominal aortic aneurysm, if a definitive neck cannot be identified at the level of the renal arteries, the possibility of thoracoabdominal aortic involvement should be suspected and investigated by using other imaging modalities. Caution should be exercised while interpreting aneurysm dimensions from ultrasound imaging because intraluminal measurements are often reported, whereas external measurements are usually used in other imaging modalities.Computed Tomography. Computed tomographic (CT) scan-ning is widely available, provides visualization of the entire thoracic and abdominal aorta, and permits multiplanar and 3-dimensional aortic reconstructions. Consequently, CT is the most common—and arguably the most useful—imaging modal-ity for evaluating thoracic aortic aneurysms.47 In addition to establishing the diagnosis, CT provides information about an

1	CT is the most common—and arguably the most useful—imaging modal-ity for evaluating thoracic aortic aneurysms.47 In addition to establishing the diagnosis, CT provides information about an aneurysm’s location, extent, anatomic anomalies, and relation-ship to major branch vessels. CT is particularly useful in deter-mining the absolute diameter of the aorta, especially in the presence of laminated clot, and also detects aortic calcification. Contrast-enhanced CT provides information about the aortic lumen and can detect mural thrombus, aortic dissection, inflam-matory periaortic fibrosis, and mediastinal or retroperitoneal hematoma due to contained aortic rupture. To increase consis-tency and ensure uniform reporting, current practice guidelines suggest that measurements be taken perpendicular to blood flow and at standard anatomic locations44 (Fig. 22-3); this should reduce the likelihood of erroneous measurements, espe-cially during serial imaging surveillance .The major disadvantage

1	to blood flow and at standard anatomic locations44 (Fig. 22-3); this should reduce the likelihood of erroneous measurements, espe-cially during serial imaging surveillance .The major disadvantage of contrast-enhanced CT scanning is the possibility of contrast-induced acute renal failure in patients who are at risk (e.g., patients with preexisting renal disease or diabetes) even though the risk is smaller than was assumed in the past.48,49 If possible, surgery is performed at least 1 day after contrast administration to allow time to observe renal function and to permit diuresis. If renal insufficiency occurs or is worsened, elective surgery is postponed until renal function returns to normal or stabilizes.Magnetic Resonance Angiography. Magnetic resonance angiography (MRA) is becoming widely available and can facilitate visualization of the entire aorta. This modality pro-duces aortic images comparable to those produced by contrast-enhanced CT but does not necessitate exposure to

1	widely available and can facilitate visualization of the entire aorta. This modality pro-duces aortic images comparable to those produced by contrast-enhanced CT but does not necessitate exposure to ionizing radiation.50 In addition, MRA offers excellent visualization of branch-vessel details, and it is useful in detecting branch-vessel stenosis.51 However, MRA is limited by high expense and a sus-ceptibility to artifacts created by ferromagnetic materials, and gadolinium—the contrast agent for MRA—may be linked to nephrogenic systemic fibrosis and acute renal failure in patients with advanced renal insufficiency.52 Furthermore, the MRA environment is not appropriate for many critically ill patients, and unlike CT imaging, MRA imaging is suboptimal in patients with extensive aortic calcification.Invasive Aortography and Cardiac Catheterization. Although catheter-based contrast aortography was previously considered the gold standard for evaluating thoracic aortic dis-ease,

1	calcification.Invasive Aortography and Cardiac Catheterization. Although catheter-based contrast aortography was previously considered the gold standard for evaluating thoracic aortic dis-ease, cross-sectional imaging (i.e., CT and MRA) has largely replaced this modality. Technologic improvements have enabled CT and MRA to provide excellent aortic imaging while causing less morbidity than catheter-based studies do, so CT and 2MRA are now the primary modes for evaluating thoracic aortic disease. Today, the use of invasive aortography in patients with thoracic aortic disease is generally limited to those undergoing endovascular therapies or when other types of studies are con-traindicated or have not provided satisfactory results.Unlike standard aortography, cardiac catheterization con-tinues to play an important role in diagnosis and preoperative planning, especially in patients with ascending aortic involve-ment. Proximal aortography can reveal not only the status of the coronary

1	to play an important role in diagnosis and preoperative planning, especially in patients with ascending aortic involve-ment. Proximal aortography can reveal not only the status of the coronary arteries and left ventricular function but also the degree of aortic valve regurgitation, the extent of aortic root involvement, coronary ostial displacement, and the relationship of the aneurysm to the arch vessels.The value of the information one can obtain from catheter-based diagnostic studies should be weighed against Brunicardi_Ch22_p0853-p0896.indd 85901/03/19 5:40 PM 860SPECIFIC CONSIDERATIONSPART IIthe established limitations and potential complications of such studies. A key limitation of aortography is that it images only the lumen and may therefore underrepresent the size of large aneurysms that contain laminated thrombus. Manipulation of intraluminal catheters can result in embolization of laminated thrombus or atheromatous debris. Proximal aortography carries a 0.6% to 1.2%

1	aneurysms that contain laminated thrombus. Manipulation of intraluminal catheters can result in embolization of laminated thrombus or atheromatous debris. Proximal aortography carries a 0.6% to 1.2% risk of stroke. Other risks include allergic reac-tion to the contrast agent, iatrogenic aortic dissection, and bleed-ing at the arterial access site. In addition, the volumes of contrast agent required to adequately fill large aneurysms can cause sig-nificant renal toxicity. To minimize the risk of contrast nephrop-athy, patients receive periprocedural intravenous (IV) fluids for hydration, mannitol for diuresis, and acetylcysteine.53,54 As with contrast-enhanced CT, surgery is performed ≥1 day after angiography whenever possible to ensure that renal func-tion has stabilized or returned to baseline.TreatmentSelecting the Appropriate Treatment. Once a thoracic aor-tic aneurysm is detected, management begins with patient edu-cation, particularly if the patient is asymptomatic, because

1	to baseline.TreatmentSelecting the Appropriate Treatment. Once a thoracic aor-tic aneurysm is detected, management begins with patient edu-cation, particularly if the patient is asymptomatic, because aortic disease may progress rapidly and unexpectedly in some patients. A detailed medical history is collected, a physical examination is performed, and a systematic review of medical records is car-ried out to clearly assess the presence or absence of pertinent symptoms and signs, despite any initial denial of symptoms by the patient. Signs of heritable conditions such as Marfan syn-drome or Loeys-Dietz syndrome are thoroughly reviewed. If clinical criteria are met for a heritable condition, confirmatory laboratory tests are conducted. Patients with heritable disorders are best treated in a dedicated aortic clinic where they can be appropriately followed up. Surveillance imaging and aggressive blood pressure control are the mainstays of initial management for asymptomatic patients. When

1	dedicated aortic clinic where they can be appropriately followed up. Surveillance imaging and aggressive blood pressure control are the mainstays of initial management for asymptomatic patients. When patients become symptomatic or their aneurysms grow to meet certain size criteria, the patients become surgical candidates.Endovascular therapy has become an accepted treatment for descending thoracic aortic aneurysm.55,56 Its role in treating proximal aortic disease and thoracoabdominal aortic aneurysm remains experimental;55 nonetheless, endoluminal stenting is approved by the U.S. Food and Drug Administration for the treatment of isolated descending thoracic aortic aneurysm, and several different devices have been approved for the treatment of blunt aortic injury and penetrating aortic ulcer. In practice, however, the off-label application of aortic stent grafts is widespread and accounts for well over half their use57; endovascular approaches may be helpful in emergent aneurysm

1	ulcer. In practice, however, the off-label application of aortic stent grafts is widespread and accounts for well over half their use57; endovascular approaches may be helpful in emergent aneurysm repair, such as for patients with aortic rupture.58 Endovascular therapy has evolved to include hybrid repairs, which combine open “debranching” techniques (to reroute branching vessels) with endovascular aortic repair.59,60 Despite these advances, for the repair of aneurysms with proximal aortic involvement and of thoracoabdominal aortic aneurysms, open procedures remain the gold standard and preferred approach.Determination of the Extent and Severity of Disease. Cross-sectional imaging with reconstruction is critical when one is evaluating a thoracic aneurysm, determining treatment strategy, and planning necessary procedures. Note that patients with a thoracic aortic aneurysm may also have a second, remote aneurysm.2 In such cases, the more threatening lesion usually is addressed first.

1	and planning necessary procedures. Note that patients with a thoracic aortic aneurysm may also have a second, remote aneurysm.2 In such cases, the more threatening lesion usually is addressed first. In many patients, staged operative procedures are necessary for complete repair of extensive aneurysms involving the ascending aorta, transverse arch, and descending thoracic or thoracoabdominal aorta.61 When the descending segment is not disproportionately large (compared with the proximal aorta) and is not causing symptoms, the proximal aortic repair is carried out first. An important benefit of this approach is that it allows treatment of valvular and coronary artery occlusive disease at the first operation.Proximal aneurysms (proximal to the left subclavian artery) usually are addressed via a sternotomy approach. Aneu-rysms involving the descending thoracic aorta are evaluated in terms of criteria (described in the following section) for poten-tial endovascular repair; those unsuitable

1	a sternotomy approach. Aneu-rysms involving the descending thoracic aorta are evaluated in terms of criteria (described in the following section) for poten-tial endovascular repair; those unsuitable for an endovascular approach are repaired with open techniques through a left thora-cotomy. A CT scan can reveal detailed information about aortic calcification and luminal thrombus. These details are important in preventing embolization during surgical manipulation.Indications for Operation Thoracic aortic aneurysms are repaired to prevent fatal rupture. Therefore, on the basis of clini-cal history studies and other data, practice guidelines for tho-racic aortic disease43,44,62 recommend elective operation in asymptomatic patients when the diameter of an ascending aortic aneurysm is >5.5 cm, when the diameter of a descending thoracic aortic aneurysm is >6.0 cm, or when the rate of dilata-tion is >0.5 cm per year. In patients with heritable disorders such as Marfan and Loeys-Dietz

1	cm, when the diameter of a descending thoracic aortic aneurysm is >6.0 cm, or when the rate of dilata-tion is >0.5 cm per year. In patients with heritable disorders such as Marfan and Loeys-Dietz syndromes, the threshold for opera-tion is based on a smaller aortic diameter (5.0 cm for the ascend-ing aorta in patients with Marfan syndrome, 4.4 to 4.6 cm for the ascending aorta in patients with Loeys-Dietz syndrome, and <6.0 cm for the descending thoracic aorta in patients with either disorder). For women with heritable disorders who are consider-ing pregnancy, prophylactic aortic root replacement is considered because the risk of aortic dissection or rupture increases at an aortic diameter of 4.0 cm and greater. For patients with ascending aortic aneurysm and bicuspid aortic valve, repair is recommended if aortic diameter is 5.0 cm or greater and additional risk factors are present (e.g., family his-tory of dissection, expansion rate exceeding 0.5 cm per year), if aortic diameter is

1	is recommended if aortic diameter is 5.0 cm or greater and additional risk factors are present (e.g., family his-tory of dissection, expansion rate exceeding 0.5 cm per year), if aortic diameter is 5.5 cm or larger and no additional risk factors are present, or if aortic diameter exceeds 4.5 cm and the patient is undergoing aortic valve replacement or repair.43 For low-risk patients with chronic aortic dissection, descending thoracic repair is recommended at an aortic diameter of 5.5 cm or greater.The acuity of presentation is a major factor in decisions about the timing of surgical intervention. Many patients are asymptomatic at the time of presentation, so there is time for thorough preoperative evaluation and improvement of their cur-rent health status, such as through smoking cessation and other optimization programs. In contrast, patients who present with symptoms may need urgent operation. Symptomatic patients are at increased risk of rupture and warrant expe-ditious evaluation.

1	and other optimization programs. In contrast, patients who present with symptoms may need urgent operation. Symptomatic patients are at increased risk of rupture and warrant expe-ditious evaluation. The onset of new pain in patients with known aneurysms is especially concerning because it may herald sig-nificant expansion, leakage, or impending rupture. Emergent intervention is reserved for patients who present with aneurysm rupture or superimposed acute dissection.63Open Repair vs. Endovascular Repair As noted earlier, endo-vascular repair has become the standard approach for patients with isolated degenerative descending thoracic aortic aneurysm; in fact, practice guidelines recommend that endovascular repair be strongly considered for patients with descending thoracic aneurysm at an aortic diameter of 5.5 cm (which is slightly below the 6.0-cm threshold for open repair).44 For endovascular 3456Brunicardi_Ch22_p0853-p0896.indd 86001/03/19 5:40 PM 861THORACIC ANEURYSMS AND AORTIC

1	diameter of 5.5 cm (which is slightly below the 6.0-cm threshold for open repair).44 For endovascular 3456Brunicardi_Ch22_p0853-p0896.indd 86001/03/19 5:40 PM 861THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22repairs to produce optimal outcomes, several anatomic criteria must be met. For one, the proximal and distal neck diameters should fall within a range that will allow proper sealing. Also, the proximal and distal landing zones should ideally be at least 20 mm long so that an appropriate seal can be made. Note that the limiting structures proximally and distally are the brachio-cephalic vessels and celiac axis, respectively. Vascular access continues to be one of the most important determinants of suc-cessful deployment of the current endovascular devices. The femoral and iliac arteries have to be wide enough to accommo-date the sheaths used to deploy the stent grafts. As endovascular technology evolves, newer devices are using smaller sheaths (or are “sheathless”

1	and iliac arteries have to be wide enough to accommo-date the sheaths used to deploy the stent grafts. As endovascular technology evolves, newer devices are using smaller sheaths (or are “sheathless” self-deployed stent grafts) to accommodate smaller arteries. Tortuosity of the iliac vessels and abdominal aorta can make these procedures technically challenging. Occa-sionally, an 8or 10-mm polyester “side graft” is anastomosed to the iliac artery through a retroperitoneal incision if the femo-ral vessels are too small to access easily.Of note, attempts have been made to extend the use of endovascular therapy to aortic arch aneurysms and thoracoab-dominal aortic aneurysms. Although reports of purely endovas-cular repair of the aortic arch remain limited, Greenberg and colleagues64 have reported their experience with a large series of purely endovascular thoracoabdominal aortic repairs. Addition-ally, there have been numerous reports of small series of off-label, experimental hybrid

1	reported their experience with a large series of purely endovascular thoracoabdominal aortic repairs. Addition-ally, there have been numerous reports of small series of off-label, experimental hybrid procedures that involve debranching the aortic arch or the visceral vessels of the abdominal aorta, fol-lowed by endovascular exclusion of the aneurysm. The majority of hybrid approaches involve repairing the aortic arch.59,60 In its simplest form, hybrid arch repair involves an open bypass from the left subclavian to the left common carotid artery, which is followed by deliberate coverage of the origins of the left subcla-vian artery by the stent graft. In its most complex form, hybrid arch repair involves rerouting all of the brachiocephalic vessels, followed by proximal placement of the stent graft in the ascend-ing aorta and extending repair distally into the aortic arch and descending thoracic aorta.The patients who theoretically benefit the most from an endovascular approach are

1	stent graft in the ascend-ing aorta and extending repair distally into the aortic arch and descending thoracic aorta.The patients who theoretically benefit the most from an endovascular approach are those who are of advanced age or have significant comorbidities, as many of these patients face substantial risks when undergoing traditional open repair.65 For example, with regard to open repair of a descending thoracic aortic aneurysm, significant pulmonary morbidity can occur postoperatively; therefore, patients with borderline pulmonary reserve may better tolerate an endovascular procedure than a standard open repair. Patients with heritable syndromic condi-tions generally are not considered candidates for elective endo-vascular repair except in specific circumstances.66 Endovascular repair in patients with heritable syndromic conditions have pro-duced poor results, which are mainly due to progressive dilata-tion, stent graft migration, and endoleak.67,68Preoperative Assessment and

1	in patients with heritable syndromic conditions have pro-duced poor results, which are mainly due to progressive dilata-tion, stent graft migration, and endoleak.67,68Preoperative Assessment and Preparation. Given the impact of comorbid conditions on perioperative complications, a careful preoperative assessment of physiologic reserve is criti-cal in assessing operative risk. Therefore, most patients undergo a thorough evaluation—with emphasis on cardiac, pulmonary, and renal function—before undergoing elective surgery.69,70Cardiac Evaluation Coronary artery disease is common in patients with thoracic aortic aneurysm and is responsible for a substantial proportion of early and late postoperative deaths in such patients. Similarly, valvular disease and myocardial dysfunction have important implications when one is planning anesthetic management and surgical approaches for aortic repair. Transthoracic echocardiography is a satisfactory noninvasive method for evaluating both valvular and

1	implications when one is planning anesthetic management and surgical approaches for aortic repair. Transthoracic echocardiography is a satisfactory noninvasive method for evaluating both valvular and biventricular function. Dipyridamole-thallium myocardial scanning identifies regions of myocardium that have reversible ischemia, and this test is more practical than exercise testing in older patients with concomitant lower-extremity peripheral vascular disease. Cardiac catheteriza-tion and coronary arteriography are performed in patients who have evidence of coronary disease—as indicated by either the patient’s history or the results of noninvasive studies—or who have a left ventricular ejection fraction of ≤30%. If significant valvular or coronary artery disease is identified before a proxi-mal aortic operation, the disease can be addressed directly dur-ing the procedure. Patients who have asymptomatic distal aortic aneurysms and severe coronary occlusive disease undergo per-cutaneous

1	aortic operation, the disease can be addressed directly dur-ing the procedure. Patients who have asymptomatic distal aortic aneurysms and severe coronary occlusive disease undergo per-cutaneous transluminal angioplasty or surgical revascularization before the aneurysmal aortic segment is replaced.Pulmonary Evaluation Pulmonary function screening with arterial blood gas measurement and spirometry is routinely per-formed before thoracic aortic operations. Patients with a forced expiratory volume in 1 second of >1.0 L and a partial pressure of carbon dioxide of <45 mmHg are considered appropriate can-didates for open surgical repair. In suitable patients, borderline pulmonary function can be improved by implementing a regi-men that includes smoking cessation, weight loss, exercise, and treatment of bronchitis for a period of 1 to 3 months before surgery. Although surgery is not withheld from patients with symptomatic aortic aneurysms and poor pulmonary function, adjustments in operative

1	of bronchitis for a period of 1 to 3 months before surgery. Although surgery is not withheld from patients with symptomatic aortic aneurysms and poor pulmonary function, adjustments in operative technique should be made to maximize these patients’ chances of recovery. In such patients, preserving the left recurrent laryngeal nerve, the phrenic nerves, and dia-phragmatic function is particularly important.Renal Evaluation Renal function is assessed preoperatively by measuring serum electrolyte, blood urea nitrogen, and cre-atinine levels. Information about kidney size and perfusion can be obtained from the imaging studies used to evaluate the aorta.Obtaining accurate information about baseline renal function has important therapeutic and prognostic implications. For exam-ple, perfusion strategies and perioperative medications are adjusted according to renal function. Patients with severely impaired renal function frequently require at least temporary hemodialysis after surgery. These

1	and perioperative medications are adjusted according to renal function. Patients with severely impaired renal function frequently require at least temporary hemodialysis after surgery. These patients also have a mortality rate that is signifi-cantly higher than normal. Patients with thoracoabdominal aortic aneurysms and poor renal function secondary to severe proximal renal occlusive disease undergo renal artery endarterectomy, stent-ing, or bypass grafting during the aortic repair.Operative Repair Proximal Thoracic Aortic Aneurysms Open Repair Traditional open operations to repair proximal aortic aneurysms—which involve the ascending aorta, trans-verse aortic arch, or both—are performed through a midsternal incision and require cardiopulmonary bypass. The best choice of aortic replacement technique depends on the extent of the aneurysm and the condition of the aortic valve.71 The spectrum of operations (Fig. 22-4) ranges from simple graft replacement of the tubular portion of the

1	technique depends on the extent of the aneurysm and the condition of the aortic valve.71 The spectrum of operations (Fig. 22-4) ranges from simple graft replacement of the tubular portion of the ascending aorta only (Fig. 22-4A) to replacement of the ascending aorta and the proximal aortic arch (Fig. 22-4B) to graft replacement of the entire proximal aorta, including the aortic root, and reattachment of the coronary Brunicardi_Ch22_p0853-p0896.indd 86101/03/19 5:40 PM 862SPECIFIC CONSIDERATIONSPART IIABCEFGHIJDKFigure 22-4. Illustrations of proximal aortic repairs in which the native aortic root is left intact. A. Graft replacement of the tubular portion of the ascending aorta with the aortic arch left intact. B. Hemiarch beveled graft replacement, in which the ascending aorta and a portion of the lesser curvature of the aortic arch are replaced. C. A modified arch with additional graft replacement of the innominate artery. D. Patch repair of the aortic arch. E. Traditional total

1	of the lesser curvature of the aortic arch are replaced. C. A modified arch with additional graft replacement of the innominate artery. D. Patch repair of the aortic arch. E. Traditional total arch replacement using an island approach to reattach the brachiocephalic vessels. F. The branched graft approach, which replaces the brachiocephalic vessels by following their original anatomic location. G. The elephant trunk approach with a concomitant island brachiocephalic artery reattachment. Contemporary Y-graft arch repairs include (H) the single Y-graft approach, (I) the double Y-graft approach, (J) the elephant trunk approach with a single Y-graft, and (K) the elephant trunk approach with a double Y-graft.Brunicardi_Ch22_p0853-p0896.indd 86201/03/19 5:40 PM 863THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22arteries and brachiocephalic branches. The options for treating aortic valve disease, repairing aortic aneurysms, and maintain-ing perfusion during repair procedures each

1	AORTIC DISSECTIONCHAPTER 22arteries and brachiocephalic branches. The options for treating aortic valve disease, repairing aortic aneurysms, and maintain-ing perfusion during repair procedures each deserve detailed consideration (Table 22-2).Aortic Valve Disease and Root Aneurysms Many patients undergoing proximal aortic operations have aortic valve dis-ease that requires concomitant surgical correction. When such disease is present and the sinus segment is normal, separate repair or replacement of the aortic valve and graft replacement of the tubular segment of the ascending aorta are carried out. In such cases, mild to moderate valve regurgitation with annular dilatation can be addressed by plicating the annulus with mat-tress sutures placed below each commissure, thereby preserving the native valve. In patients with more severe valvular regur-gitation or with valvular stenosis, the valve is replaced with a stented biologic or mechanical prosthesis; mechanical prosthe-ses

1	the native valve. In patients with more severe valvular regur-gitation or with valvular stenosis, the valve is replaced with a stented biologic or mechanical prosthesis; mechanical prosthe-ses necessitate following a lifelong anticoagulation regimen. Separate replacement of the aortic valve and ascending aorta is Table 22-2Options for open surgical repair of proximal aortic aneurysmsOptions for treating aortic valve disease Aortic valve annuloplasty (annular plication) Aortic valve replacement (with mechanical or biologic prosthesis) Aortic root replacement Composite valve graft (with mechanical or biologic valve) Aortic homograft Stentless porcine root Pulmonary autograft (Ross procedure) Valve-sparing techniquesOptions for graft repair of the aortic aneurysm Patch aortoplasty Ascending replacement only Beveled hemiarch replacement Total arch replacement with reattachment of brachiocephalic branches (island technique) Elephant trunk technique with island reattachment Total arch

1	replacement only Beveled hemiarch replacement Total arch replacement with reattachment of brachiocephalic branches (island technique) Elephant trunk technique with island reattachment Total arch replacement with bypass grafts to the brachiocephalic branches (including Y-graft approaches) Elephant trunk technique with Y-graft approach Hybrid aortic arch repairs (including “frozen elephant trunk technique”)Perfusion options Standard cardiopulmonary bypass Profound hypothermic circulatory arrest without adjuncts Hypothermic circulatory arrest with adjuncts Retrograde cerebral perfusion Antegrade cerebral perfusion Balloon perfusion catheters Right axillary artery cannulation Innominate artery cannulationnot performed in patients with Marfan syndrome or Loeys-Dietz syndrome, because progressive dilatation of the remaining sinus segment eventually leads to complications that necessitate reop-eration. Therefore, patients with Marfan syndrome, Loeys-Dietz syndrome, or annuloaortic

1	progressive dilatation of the remaining sinus segment eventually leads to complications that necessitate reop-eration. Therefore, patients with Marfan syndrome, Loeys-Dietz syndrome, or annuloaortic ectasia require some form of aortic root replacement.72In many cases, the aortic root is replaced with a mechani-cal or biologic graft that has both a valve and an aortic conduit. Currently, the following graft options are commercially avail-able: composite valve grafts with a mechanical valve, which consist of a bileaflet mechanical valve attached to a polyester tube graft; composite valve grafts with a biological valve (avail-able in Europe only at this point); aortic root homografts, which are harvested from cadavers and cryopreserved73; and stentless porcine aortic root grafts.74,75 In the United States, because no biologic composite valve grafts are commercially available, another option for surgeons is to construct a bioprosthetic com-posite valve graft during the operation by

1	In the United States, because no biologic composite valve grafts are commercially available, another option for surgeons is to construct a bioprosthetic com-posite valve graft during the operation by suturing a stented tis-sue valve to a polyester tube graft.Although select patients may be offered the Ross procedure—in which the patient’s pulmonary artery root is excised and placed in the aortic position and then the right ven-tricular outflow tract is reconstructed by using a cryopreserved pulmonary homograft—this option is rarely used. This is largely because it is a technically demanding procedure, and there are concerns about the potential for autograft dilatation in patients with heritable conditions.76An additional option is valve-sparing aortic root replace-ment, which has evolved substantially during the past decade.77,78 The valve-sparing technique that is currently favored is called aortic root reimplantation and involves excising the aortic sinuses, attaching a prosthetic

1	substantially during the past decade.77,78 The valve-sparing technique that is currently favored is called aortic root reimplantation and involves excising the aortic sinuses, attaching a prosthetic graft to the patient’s annulus (Fig. 22-5), and resuspending the native aortic valve inside the graft. The superior hemodynamics of the native valve and the avoidance of anticoagulation are major advantages of the valve-sparing approach. Long-term results in carefully selected patients have been excellent.79 Although the durability of this procedure in patients with Marfan syndrome has been satisfac-tory in some centers, it remains uncertain whether long-term durability can be reliably achieved with this approach.78 Further, acceptable mid-term outcomes have been reported for patients with bicuspid aortic valve.80 Patients who have structural leaf-let deterioration or excessive annular dilatation are typically deemed unsuitable for valve-sparing repair.Regardless of the type of conduit

1	bicuspid aortic valve.80 Patients who have structural leaf-let deterioration or excessive annular dilatation are typically deemed unsuitable for valve-sparing repair.Regardless of the type of conduit used, aortic root replace-ment requires reattaching the coronary arteries to openings in the graft. In the original procedure described by Bentall and De Bono,81 this was accomplished by suturing the intact aortic wall surrounding each coronary artery to the openings in the graft. The aortic wall was then wrapped around the graft to establish hemostasis. However, this technique frequently pro-duced leaks at the coronary reattachment sites that eventually led to pseudoaneurysm formation. Cabrol’s modification, in which a separate, small tube graft is sutured to the coronary ostia and the main aortic graft, achieves tension-free coronary anas-tomoses, and reduces the risk of pseudoaneurysm formation.82 Kouchoukos’s button modification of the Bentall procedure is currently the most widely

1	aortic graft, achieves tension-free coronary anas-tomoses, and reduces the risk of pseudoaneurysm formation.82 Kouchoukos’s button modification of the Bentall procedure is currently the most widely used technique.83 The aneurysmal aorta is excised, and buttons of aortic wall are left surrounding both coronary arteries, which are then mobilized and sutured to the aortic graft (Fig. 22-6). The coronary suture lines may be reinforced with polytetrafluoroethylene felt or pericardium Brunicardi_Ch22_p0853-p0896.indd 86301/03/19 5:40 PM 864SPECIFIC CONSIDERATIONSPART IIAEIJKLFGHBCDFigure 22-5. Illustration of our current valve-sparing procedure for replacing the aortic root and ascending aorta for treatment of (A) aortic root aneurysm. B. The ascending aorta is opened after cardiopulmonary bypass and cardioplegic arrest are established and the distal ascending aorta is clamped. The diseased aortic tissue (including the sinuses of Valsalva) is excised. Buttons of surrounding tissue are

1	bypass and cardioplegic arrest are established and the distal ascending aorta is clamped. The diseased aortic tissue (including the sinuses of Valsalva) is excised. Buttons of surrounding tissue are used to mobi-lize the origins of the coronary arteries. C. A synthetic graft is sewn to the distal ascending aorta with continuous suture. D. After the distal anastomosis is completed, six sutures reinforced with Teflon pledgets are placed in the plane immediately below the aortic valve annulus. E. The subannular sutures are placed through the base of a synthetic aortic root graft, which is then is parachuted down around the valve. F. After the root graft is cut to an appropriate length, the valve commissures and leaflets are positioned within the graft. The annular sutures are then tied. G. Each of the three commissures is then secured near the top of the graft. H. The supra-annular aortic tissue is sewn to the graft in continuous fashion. I. The button surrounding the origin of the left

1	of the three commissures is then secured near the top of the graft. H. The supra-annular aortic tissue is sewn to the graft in continuous fashion. I. The button surrounding the origin of the left main coronary artery is sewn to an opening cut in the root graft. J. The two aortic grafts are sewn together with continuous suture. K. The button surrounding the origin of the right coronary artery is sewn to an opening cut in the root graft. L. The completed valve-sparing aortic root replacement and graft repair of the ascending aorta are shown. (Used with permission of Baylor College of Medicine.)to enhance hemostasis. When the coronary arteries cannot be mobilized adequately because of extremely large aneurysms or scarring from previous surgery, the Cabrol technique or a related modification can be used. Another option, originally described by Zubiate and Kay,84 is the construction of bypass grafts by using interposition saphenous vein or synthetic grafts.Aortic Arch Aneurysms Several

1	can be used. Another option, originally described by Zubiate and Kay,84 is the construction of bypass grafts by using interposition saphenous vein or synthetic grafts.Aortic Arch Aneurysms Several options are also available for handling aneurysms that extend into the transverse aortic arch Brunicardi_Ch22_p0853-p0896.indd 86401/03/19 5:40 PM 865THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22Figure 22-6. Illustration of the modified Bentall procedure for replacing the aortic root and ascending aorta. Commonly, the ascending aorta is replaced by a straight “tube” graft, and the aortic root, including the valve apparatus and the sinuses of Valsalva, is replaced by a mechanical composite valve graft with neosinuses to better mimic its native shape. The coronary arteries with buttons of surrounding aortic tissue have been mobilized and are being reat-tached to openings in the aortic graft. Shown at the proximal aspect of the innominate artery, the ligated remnant of an 8-mm graft

1	of surrounding aortic tissue have been mobilized and are being reat-tached to openings in the aortic graft. Shown at the proximal aspect of the innominate artery, the ligated remnant of an 8-mm graft was used to ease cannulation of the artery, which was used as inflow during cardiopulmonary bypass. (Used with permission of Baylor College of Medicine.)(see Fig. 22-4C-K).65 The surgical approach depends on the extent of involvement and the need for cardiac and cerebral pro-tection. Saccular aneurysms that arise from the lesser curvature of the distal transverse arch and that encompass <50% of the aortic circumference can be treated by patch graft aortoplasty; such aneurysms are particularly well suited for hybrid repair with arch debranching followed by exclusion of the aneurysm with an endovascular graft.85 For fusiform aneurysms, when the distal portion of the arch is a reasonable size, a single, beveled replacement of the lower curvature (hemiarch) is performed. More extensive arch

1	graft.85 For fusiform aneurysms, when the distal portion of the arch is a reasonable size, a single, beveled replacement of the lower curvature (hemiarch) is performed. More extensive arch aneurysms require total replacement involving a distal anastomosis to the proximal descending tho-racic aorta and separate reattachment of the brachiocephalic branches. The brachiocephalic vessels can be reattached to one or more openings made in the graft, or if these vessels are aneurysmal, they can be replaced with separate, smaller grafts. Alternatively, a Y-graft approach86,87 can be used to debranch the brachiocephalic vessels and move them forward, thereby permitting the distal anastomosis to be brought forward, which aids in hemostasis. When the aneurysm involves the entire arch and extends into the descending thoracic aorta, it is approached by using Borst’s elephant trunk technique of staged total arch replacement.88 The distal anastomosis is performed such that a portion of the graft is

1	the descending thoracic aorta, it is approached by using Borst’s elephant trunk technique of staged total arch replacement.88 The distal anastomosis is performed such that a portion of the graft is left suspended within the proximal descending thoracic aorta (Fig. 22-7). A collared graft can be used to accommodate any discrepancy in aortic diameter.86 During a subsequent operation, the suspended “trunk” is used to facilitate repair of the remaining descending thoracic or tho-racoabdominal aortic aneurysm by an endovascular technique or by open repair through a thoracotomy incision, depending on the extent of disease and other factors. The elephant trunk technique permits access to the distal portion of the graft during the second operation without the need for dissection around the distal transverse aortic arch; this reduces the risk of injuring the left recurrent laryngeal nerve, esophagus, and pulmonary artery if an open approach is used at the second stage. As described in the

1	transverse aortic arch; this reduces the risk of injuring the left recurrent laryngeal nerve, esophagus, and pulmonary artery if an open approach is used at the second stage. As described in the section on hybrid repair of arch aneurysms (see later), the elephant trunk can be completed by using a hybrid endovascular approach (Fig. 22-8) in certain settings. A newer technique that is currently under investigation involves using a graft compris-ing a conventional polyester proximal portion and a stent graft distal elephant trunk portion. This “frozen elephant trunk” tech-nique can enable treatment of the entire aortic pathology during a single procedure or can facilitate a subsequent endovascular procedure (Fig. 22-9).89-91Cardiopulmonary Bypass Perfusion Strategies Like the opera-tions themselves, perfusion strategies used during proximal aor-tic surgery depend on the extent of the repair. Aneurysms that are isolated to the ascending segment can be replaced by using standard

1	themselves, perfusion strategies used during proximal aor-tic surgery depend on the extent of the repair. Aneurysms that are isolated to the ascending segment can be replaced by using standard cardiopulmonary bypass and distal ascending aortic clamping. This provides constant perfusion of the brain and other vital organs during the repair. Aneurysms involving the transverse aortic arch, however, cannot be clamped during the repair, which necessitates the temporary withdrawal of cardio-pulmonary bypass support; this is called circulatory arrest. To protect the brain and other vital organs during the circulatory arrest period, hypothermia must be initiated before pump flow is stopped. However, the deep levels of hypothermia (below 20°C) that have been traditionally used in open arch repair are not without risk, and pure hypothermic circulatory arrest contin-ues to have substantial limitations. Importantly, although brief periods of total circulatory arrest generally are well tolerated

1	are not without risk, and pure hypothermic circulatory arrest contin-ues to have substantial limitations. Importantly, although brief periods of total circulatory arrest generally are well tolerated at cold temperatures, as the duration of circulatory arrest increases, the well-recognized risks of brain injury and death increase dra-matically. Additionally, deep levels of hypothermia are associ-ated with coagulopathy.Because of the inherent complexity of aortic arch repairs and their general tendency to require longer periods Brunicardi_Ch22_p0853-p0896.indd 86501/03/19 5:41 PM 866SPECIFIC CONSIDERATIONSPART IIADFGECBFigure 22-7. Illustration of a contemporary Y-graft approach to total arch replacement for aortic arch aneurysm. A. The proximal portions of the brachiocephalic arteries are exposed. B. The first two branches of the graft are sewn end-to-end to the transected left subclavian and left common carotid arteries. The proximal ends of the transected brachiocephalic arteries

1	exposed. B. The first two branches of the graft are sewn end-to-end to the transected left subclavian and left common carotid arteries. The proximal ends of the transected brachiocephalic arteries are ligated. C. A balloon-tipped perfusion cannula is placed inside the double Y-graft and used to deliver antegrade cerebral perfusion. After systemic circulatory arrest is initiated, the innominate artery is clamped, transected, and sewn to the distal end of the main graft. D. The proximal aspect of the Y-graft is clamped. This directs flow from the axillary artery to all three brachiocephalic arteries. The arch is then replaced with a collared elephant trunk graft. E. The distal anastomosis between the elephant trunk graft and the aorta is created between the innominate and left common carotid arteries. The collared graft accommodates any discrepancy in aortic diameter. F. The aortic graft is clamped, and a second limb from the arterial inflow tubing of the cardiopulmonary bypass circuit

1	arteries. The collared graft accommodates any discrepancy in aortic diameter. F. The aortic graft is clamped, and a second limb from the arterial inflow tubing of the cardiopulmonary bypass circuit is used to deliver systemic perfusion through a side-branch of the arch graft while the proximal por-tion of the ascending aorta is replaced. Once the proximal aortic anastomosis is completed, the main trunk of the double Y-graft is cut to an appropriate length, and the beveled end is then sewn to an oval opening created in the right anterolateral aspect of the ascending aortic graft, which completes the repair (G). (Modified with permission from LeMaire SA, Price MD, Parenti JL, et al. Early outcomes after aortic arch replacement by using the Y-graft technique, Ann Thorac Surg. 2011 Mar;91(3):700-707.)of hypothermic circulatory arrest, two cerebral perfusion strategies—retrograde cerebral perfusion (RCP) and antegrade cerebral perfusion (ACP)—were developed to supplement this process by

1	hypothermic circulatory arrest, two cerebral perfusion strategies—retrograde cerebral perfusion (RCP) and antegrade cerebral perfusion (ACP)—were developed to supplement this process by delivering cold, oxygenated blood to the brain and further reduce the risks associated with repair. Retrograde cerebral perfusion involves directing blood from the cardio-pulmonary bypass circuit into the brain through the superior vena cava.92 However, RCP is thought to be less beneficial than ACP,93 and although it may be helpful in the retrograde flush-ing of air and debris from the arch, many centers have stopped using RCP.In contrast, ACP delivers blood directly into the brachio-cephalic arteries to maintain cerebral flow. Although its use was cumbersome in the past, contemporary ACP techniques (Fig. 22-10) have been simplified and commonly involve can-nulating either the right axillary artery or the innominate artery and subsequent connection to the cardiopulmonary bypass circuit.94-96 Often, a

1	22-10) have been simplified and commonly involve can-nulating either the right axillary artery or the innominate artery and subsequent connection to the cardiopulmonary bypass circuit.94-96 Often, a small section of graft is used as a conduit to ease cannulation, but there remains a small procedure-related risk of brachial plexus or vascular injury. Upon initiation, cold blood is delivered into the brain via the right common carotid artery and, if bilateral ACP is desired, the left common carotid artery. Note that, with the unilateral ACP technique, blood flow to the left side of the brain requires collateral circulation, ideally through an intact circle of Willis.Methods to help determine the adequacy of unilateral ACP to deliver cerebral cross-circulation include preoperative imag-ing and intraoperative monitoring. A commonly used method Brunicardi_Ch22_p0853-p0896.indd 86601/03/19 5:41 PM 867THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22ABCFigure 22-8. Illustration of

1	and intraoperative monitoring. A commonly used method Brunicardi_Ch22_p0853-p0896.indd 86601/03/19 5:41 PM 867THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22ABCFigure 22-8. Illustration of Borst’s elephant trunk technique using a contemporary Y-graft approach. A. Stage 1: The proximal repair includes replacing the ascending aorta and entire arch, with Y-graft reattachment of the brachiocephalic vessels. The distal anastomosis is facilitated by using a collared elephant trunk graft to accommodate the larger diameter of the distal aorta. A section of the graft is left sus-pended within the proximal descending thoracic aorta. B. Stage 2: The distal repair uses the floating “trunk” for the proximal anastomosis. C. An alternate “hybrid” approach may be used in patients with less extensive distal aortic disease. Endovascular stent grafts are placed within the elephant trunk to complete the repair. (Used with permission of Baylor College of Medicine.)ABFigure 22-9. Illustration of a

1	distal aortic disease. Endovascular stent grafts are placed within the elephant trunk to complete the repair. (Used with permission of Baylor College of Medicine.)ABFigure 22-9. Illustration of a frozen elephant trunk repair, which is a hybrid approach to repair that combines open aortic replacement with endovascular aortic repair. A. Extensive aortic disease affects the proximal and distal aorta. B. Aortic repair is extended into the proximal por-tion of the descending thoracic aorta after the transverse aortic arch is fully replaced. (Used with permission of Baylor College of Medicine.)Brunicardi_Ch22_p0853-p0896.indd 86701/03/19 5:41 PM 868SPECIFIC CONSIDERATIONSPART IIFigure 22-10. Illustration of a contemporary technique for deliv-ering antegrade cerebral perfusion during aortic arch repair. A. A graft sewn to the right axillary artery or to the innominate artery (inset) is used to return oxygenated blood from the cardiopulmo-nary bypass circuit. B. After adequate hypothermia

1	repair. A. A graft sewn to the right axillary artery or to the innominate artery (inset) is used to return oxygenated blood from the cardiopulmo-nary bypass circuit. B. After adequate hypothermia is established, the innominate artery is occluded with a tourniquet (inset) so that flow is diverted to the right common carotid artery, which maintains cerebral circulation. (Used with permission from Baylor College of Medicine.)of intraoperative monitoring is brain near-infrared spectroscopy (NIRS), which measures cerebral oxygenation. If NIRS moni-toring indicates inadequate perfusion, an additional perfusion catheter can be inserted into the left common carotid artery to provide blood flow to the left side of the brain.Because ACP provides excellent brain protection, many surgeons now use more moderate levels of hypothermia (often between 22°C and 24°C) to decrease the risks associated with deep hypothermia.97 However, some authors have raised the ABconcern that reducing the degree of

1	more moderate levels of hypothermia (often between 22°C and 24°C) to decrease the risks associated with deep hypothermia.97 However, some authors have raised the ABconcern that reducing the degree of hypothermia increases the risk of ischemic complications involving the spinal cord, kid-neys, and other organs that are ischemic (without the benefit of deep hypothermia) during the systemic circulatory arrest period.98 Consequently, some groups supplement ACP with additional perfusion strategies that provide flow to the descend-ing aorta during arch repair.99,100Endovascular Repair Experience with purely endovascular treatment of proximal aortic disease remains limited and only investigational.101 The unique anatomy of the aortic arch and the need for uninterrupted cerebral perfusion pose difficult chal-lenges. There are reports of the use of “homemade” grafts to exclude arch aneurysms; however, these grafts are experimen-tal at this time. For example, in 1999, Inoue and colleagues102

1	difficult chal-lenges. There are reports of the use of “homemade” grafts to exclude arch aneurysms; however, these grafts are experimen-tal at this time. For example, in 1999, Inoue and colleagues102 reported placing a triple-branched stent graft in a patient with an aneurysm of the aortic arch. The three brachiocephalic branches were positioned by placing percutaneous wires in the right brachial, left carotid, and left brachial arteries. The patient underwent two subsequent procedures: surgical repair of a right brachial pseudoaneurysm and placement of a distal stent graft extension to control a major perigraft leak. Since then, efforts to employ endovascular techniques in the treatment of the proxi-mal aorta have been essentially limited to the use of approved devices for off-label indications, such as the exclusion of pseu-doaneurysms in the ascending aorta.Hybrid Repair In June 1991, the Ukrainian surgeon Nikolay Volodos and his colleagues performed the first hybrid aortic arch

1	such as the exclusion of pseu-doaneurysms in the ascending aorta.Hybrid Repair In June 1991, the Ukrainian surgeon Nikolay Volodos and his colleagues performed the first hybrid aortic arch repair103,104; 22 years later, Volodos reported that the patient was still alive.105 Unlike purely endovascular approaches, hybrid repairs of the aortic arch have entered the mainstream clinical arena, although they remain controversial. Hybrid arch repairs involve some form of “debranching” of the brachiocephalic vessels (which are not unlike Y-graft approaches), followed by endovascular exclusion of some or all of the aortic arch (Fig. 22-11). Although this technique has many variants, they often involve sewing a branched graft to the proximal ascend-ing aorta with the use of a partial aortic clamp. The branches of the graft are then sewn to the arch vessels. Once the arch is “debranched,” the arch aneurysm can be excluded with an endograft. Commonly, a zone 0 approach (Fig. 22-12) is under-taken

1	branches of the graft are then sewn to the arch vessels. Once the arch is “debranched,” the arch aneurysm can be excluded with an endograft. Commonly, a zone 0 approach (Fig. 22-12) is under-taken in which the proximal end of the endograft is secured within the ascending aorta. Other hybrid approaches aim to extend repair into the distal arch and descending thoracic aorta (see the following section). The arguments for using a hybrid approach to treat aortic arch aneurysm include the potential to avoid using cardiopulmonary bypass, circulatory arrest, and car-diac ischemia.59,60It is not yet clear whether hybrid repairs are as durable as traditional ones because little midor long-term data have been published, and there are very few comparative studies.65 Procedure-related risks include the risk of embolization and stroke due to wire and device manipulation within the aortic arch (this risk appears to be greatest in zone 0 repairs106), retrograde acute aortic dissection,107 type I

1	the risk of embolization and stroke due to wire and device manipulation within the aortic arch (this risk appears to be greatest in zone 0 repairs106), retrograde acute aortic dissection,107 type I endoleak,108 and paraplegia.27 Because iatrogenic retrograde dissection of the ascending aorta is a particularly lethal complication, special considerations, including careful blood pressure management and wire manipulation, are recommended to avoid this problem in patients who are undergoing hybrid arch zone 0 stent deployment.109 Notably, patients with an ascending aortic diameter greater than 4.2 cm may be more susceptible to retrograde dissection. In an effort to Brunicardi_Ch22_p0853-p0896.indd 86801/03/19 5:41 PM 869THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22Figure 22-11. Illustration of a “Zone 0” hybrid arch repair. A. A distal arch aneurysm is shown that extends into the proximal aspect of the descending thoracic aorta. B. The brachiocephalic vessels are debranched onto

1	of a “Zone 0” hybrid arch repair. A. A distal arch aneurysm is shown that extends into the proximal aspect of the descending thoracic aorta. B. The brachiocephalic vessels are debranched onto a Y-graft, and a separate graft is used as a conduit for ante-grade endovascular deployment of the stent graft. C. In the completed repair, the proximal landing zone of the endograft is within zone 0. (Used with permission from Baylor College of Medicine.)reduce the risk of iatrogenic dissection, some centers have begun to replace a small section of the ascending aorta with a standard polyester graft such that the endograft’s proximal landing zone comprises prosthetic material rather than native aortic tissue.107Distal Thoracic Aortic Aneurysms Open Repair In patients with descending thoracic or thoracoab-dominal aortic aneurysms, several aspects of treatment—includ-ing preoperative risk assessment, anesthetic management, choice of incision, and use of protective adjuncts—are dictated by the

1	thoracoab-dominal aortic aneurysms, several aspects of treatment—includ-ing preoperative risk assessment, anesthetic management, choice of incision, and use of protective adjuncts—are dictated by the overall extent of aortic involvement. By definition, descend-ing thoracic aortic aneurysms involve the portion of the aorta between the left subclavian artery and the diaphragm. Thora-coabdominal aneurysms can involve the entire thoracoabdominal aorta, from the origin of the left subclavian artery to the aortic bifurcation. Surgical repair of thoracoabdominal aortic aneu-rysms is categorized by the extent of aortic replacement accord-ing to the Crawford classification scheme (Fig. 22-13). Extent I thoracoabdominal aortic aneurysm repairs involve most of the descending thoracic aorta, usually beginning near the left sub-clavian artery, and extend down into the suprarenal abdominal aorta. Extent II repairs also begin near the left subclavian artery but extend distally into the infrarenal

1	beginning near the left sub-clavian artery, and extend down into the suprarenal abdominal aorta. Extent II repairs also begin near the left subclavian artery but extend distally into the infrarenal abdominal aorta, and they often reach the aortic bifurcation. Extent III repairs extend from the lower descending thoracic aorta (below the sixth rib) and into the abdomen. Extent IV repairs begin at the diaphragmatic hiatus and often involve the entire abdominal aorta.Descending thoracic aortic aneurysms not amenable to endovascular therapy are currently repaired through a left thoracotomy. In patients with thoracoabdominal aortic aneu-rysm, the thoracotomy is extended across the costal margin and into the abdomen.110 Using a double-lumen endobronchial tube allows selective ventilation of the right lung and deflation of the left lung. Transperitoneal exposure of the thoracoabdominal aorta is achieved by performing medial visceral rotation and circumferential division of the diaphragm.

1	the right lung and deflation of the left lung. Transperitoneal exposure of the thoracoabdominal aorta is achieved by performing medial visceral rotation and circumferential division of the diaphragm. During a period of aortic clamping, the diseased segment is replaced with a polyes-ter tube graft. Important branch arteries—including intercostal arteries and the celiac, superior mesenteric, and renal arteries—are reattached to openings made in the side of the graft. In patients with Marfan syndrome and other heritable conditions, separate (8and 10-mm) grafts to the visceral branches are often used to prevent subsequent “patch aneurysms” that can develop in residual aortic tissue.111 Visceral and renal artery occlusive dis-ease is commonly encountered during aneurysm repair; options for correcting branch-vessel stenosis include endarterectomy, direct arterial stenting, and bypass grafting.Clamping the descending thoracic aorta causes ischemia of the spinal cord and abdominal viscera.

1	branch-vessel stenosis include endarterectomy, direct arterial stenting, and bypass grafting.Clamping the descending thoracic aorta causes ischemia of the spinal cord and abdominal viscera. Clinically significant manifestations of hepatic, pancreatic, and bowel ischemia are relatively uncommon. However, both acute renal failure and spinal cord injury resulting in paraplegia or paraparesis remain major causes of morbidity and mortality after these operations. Therefore, several aspects of the operation are devoted to minimizing spinal and renal ischemia (Table 22-3). Our multimodal approach to spinal cord protection includes expeditious repair to minimize aortic clamping time, moderate systemic heparinization (1.0 mg/kg) to prevent small-vessel AB5-FrenchsheathMarked pigtailcatheterDeliverysheathStiffguidewireUndeployedendograftCBrunicardi_Ch22_p0853-p0896.indd 86901/03/19 5:41 PM 870SPECIFIC CONSIDERATIONSPART IIFigure 22-13. Illustration of the Crawford classification of

1	86901/03/19 5:41 PM 870SPECIFIC CONSIDERATIONSPART IIFigure 22-13. Illustration of the Crawford classification of thora-coabdominal aortic aneurysm repair, based on the extent of aortic replacement. (Used with permission from Baylor College of Medicine.)Right commoncarotid arteryInnominatearteryLeft commoncarotid arteryLeft subclavianarteryLanding Zone ClassificationsFigure 22-12. Illustration of the Criado landing zones, which are used to describe aortic anatomy during thoracic endovascular repair. The arch is the short segment that includes the origins of the three brachiocephalic arteries—the innominate artery, the left com-mon carotid artery, and the left subclavian artery. Zone 0 includes the ascending aorta and the origin of the innominate artery. Zone 1 includes the origin of the left common carotid artery. Zone 2 includes the left subclavian artery origin. Zone 3 is a short section of the aorta that comprises the 2 cm immediately distal to the origin of the left

1	of the left common carotid artery. Zone 2 includes the left subclavian artery origin. Zone 3 is a short section of the aorta that comprises the 2 cm immediately distal to the origin of the left subclavian artery, and zone 4 begins where zone 3 ends. (Used with permission from Baylor College of Medicine.)Table 22-3Current strategy for spinal cord and visceral protection during repair of distal thoracic aortic aneurysmsAll extents• Permissive mild hypothermia (32°C–34°C, nasopharyngeal)• Moderate heparinization (1 mg/kg)• Aggressive reattachment of segmental arteries, especially between T8 and L1• Sequential aortic clamping when possible• Perfusion of renal arteries with 4°C crystalloid solution when possibleCrawford extent I and II thoracoabdominal repairs• Cerebrospinal fluid drainage• Left heart bypass during proximal anastomosis• Selective perfusion of celiac axis and superior mesenteric artery during intercostal and visceral anastomosesthrombosis, mild permissive hypothermia (32°C

1	heart bypass during proximal anastomosis• Selective perfusion of celiac axis and superior mesenteric artery during intercostal and visceral anastomosesthrombosis, mild permissive hypothermia (32°C to 34°C [89.6°F to 93.2°F] nasopharyngeal temperature), and reattachment of segmental intercostal and lumbar arteries. As the aorta is replaced from proximal to distal, the aortic clamp is moved sequentially to lower positions along the graft to restore perfusion to newly reattached branch vessels. During extensive thoracoabdominal aortic repairs (i.e., Crawford extent I and II repairs), cerebrospinal fluid drainage is used to improve spinal perfusion by reducing cerebrospinal fluid pressure. Because the benefits of this adjunct have been confirmed in a randomized clinical trial,112 its use is recommended in current guidelines (Class I, Level B recommendation).44 During cerebral spinal fluid drainage, the cerebral spinal fluid pressure is closely monitored, and the amount of fluid that is

1	recommended in current guidelines (Class I, Level B recommendation).44 During cerebral spinal fluid drainage, the cerebral spinal fluid pressure is closely monitored, and the amount of fluid that is removed is carefully limited to avoid the devastating complication of intracranial hemorrhage.113 Motor evoked potentials are used by some groups to monitor the spinal cord throughout the operation.114,115 Left heart bypass, which provides perfusion of the distal aorta and its branches during the clamping period, is also used during extensive thoracoabdominal aortic repairs.116-118 Because left heart bypass unloads the heart, it is also useful in patients with poor cardiac reserve. Balloon perfusion cannulas connected to the left heart bypass circuit can be used to deliver blood directly to the celiac axis and superior mesenteric artery during their reattachment. The potential benefits of reducing hepatic and bowel ischemia include reduced risks of postoperative coagulopathy and

1	to the celiac axis and superior mesenteric artery during their reattachment. The potential benefits of reducing hepatic and bowel ischemia include reduced risks of postoperative coagulopathy and bacterial translocation, respectively. Whenever possible, renal protection is achieved by perfusing the kidneys with cold (4°C [39.2°F]) crystalloid. In a randomized clinical trial, reduced kidney temperature was found to be associated with renal protection, and the use of cold crystalloid independently predicted preserved renal function.119Hypothermic circulatory arrest can also be used dur-ing descending thoracic or thoracoabdominal aortic repairs.120 At our center, the primary indication for this approach is the inability to clamp the aorta because of rupture, extremely large aneurysm size, or extension of the aneurysm into the distal transverse aortic arch, or because a prior endovascular repair hinders clamping.67As discussed previously, complete repair of extensive aneurysm involving the

1	of the aneurysm into the distal transverse aortic arch, or because a prior endovascular repair hinders clamping.67As discussed previously, complete repair of extensive aneurysm involving the ascending aorta, transverse arch, and descending thoracic aorta generally requires staged open 7Brunicardi_Ch22_p0853-p0896.indd 87001/03/19 5:41 PM 871THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22Figure 22-14. Illustration of the reversed elephant trunk technique using a traditional “island” approach to total aortic arch replacement. A. Stage 1: The distal aorta is repaired through a left thoracoabdominal approach. The aneurysm is opened after the aorta is clamped between the left common carotid artery and the left subclavian artery, which is also clamped. Before the proximal anastomosis is performed, the end of the graft is partly invaginated to leave a “trunk” for the subsequent repair. Proximal intercostal arteries are oversewn. B. After the proximal suture line is completed, the

1	is performed, the end of the graft is partly invaginated to leave a “trunk” for the subsequent repair. Proximal intercostal arteries are oversewn. B. After the proximal suture line is completed, the clamps are repositioned to restore blood flow to the left subclavian artery. The repair is completed by reattaching patent intercostal arteries to an opening in the side of the graft and creating a beveled distal anastomosis at the level of the visceral branches. C. Stage 2: The proximal aorta is repaired through a median sternotomy. The aortic arch is opened under hypothermic circulatory arrest. The “trunk” is pulled out and used to replace the aortic arch and ascending aorta. This eliminates the need for a new distal anastomosis and sim-plifies the procedure. Circulatory arrest and operative time, along with their attendant risks, are reduced. D. The completed two-stage repair of the entire thoracic aorta. (Modified with permission from Coselli JS, LeMaire SA, Carter SA, et al: The

1	time, along with their attendant risks, are reduced. D. The completed two-stage repair of the entire thoracic aorta. (Modified with permission from Coselli JS, LeMaire SA, Carter SA, et al: The reversed elephant trunk technique used for treatment of complex aneurysms of the entire thoracic aorta, Ann Thorac Surg. 2005 Dec;80(6):2166-2172.)operations or a hybrid approach. In such procedures, when the descending or thoracoabdominal component is symptomatic (e.g., causes back pain or has ruptured) or is disproportionately large (compared with the ascending aorta), the distal segment is treated during the initial operation, and repair of the ascend-ing aorta and transverse aortic arch is performed as a second procedure. A reversed elephant trunk repair, in which a portion of the proximal end of the aortic graft is inverted down into the lumen, can be performed during the first operation; this tech-nique facilitates the second-stage repair of the ascending aorta and transverse aortic arch

1	of the aortic graft is inverted down into the lumen, can be performed during the first operation; this tech-nique facilitates the second-stage repair of the ascending aorta and transverse aortic arch (Fig. 22-14).121Although spinal cord ischemia and renal failure receive the most attention, several other complications warrant consid-eration. The most common complication of extensive repairs is pulmonary dysfunction. With aneurysms adjacent to the left subclavian artery, the vagus and left recurrent laryngeal nerves are often adherent to the aortic wall and thus are susceptible to injury. Vocal cord paralysis should be suspected in patients who have postoperative hoarseness, and the presence of nerve damage should be confirmed by endoscopic examination. Vocal cord paralysis can be treated effectively by direct cord medial-ization (type 1 thyroplasty).122 Injury to the esophagus during Brunicardi_Ch22_p0853-p0896.indd 87101/03/19 5:41 PM 872SPECIFIC CONSIDERATIONSPART IIFigure

1	effectively by direct cord medial-ization (type 1 thyroplasty).122 Injury to the esophagus during Brunicardi_Ch22_p0853-p0896.indd 87101/03/19 5:41 PM 872SPECIFIC CONSIDERATIONSPART IIFigure 22-15. Illustration of a thoracoabdominal aortic aneurysm repair in a patient with a patent left internal thoracic artery-to-left anterior descending coronary artery graft. The proximal anastomosis is being performed while the aorta is clamped between the left common carotid and subclavian arteries. Myo-cardial perfusion is maintained through the carotid-subclavian bypass graft. (Modifed with permission from Jones MM, Akay M, Murariu D, et al: Safe aortic arch clamping in patients with patent inter-nal thoracic artery grafts. Ann Thorac Surg. 2010 Apr;89(4):e31-e32.)the proximal anastomosis can have catastrophic consequences. Carefully separating the proximal descending thoracic aorta from the underlying esophagus before performing the proximal anastomosis minimizes the risk of a secondary

1	have catastrophic consequences. Carefully separating the proximal descending thoracic aorta from the underlying esophagus before performing the proximal anastomosis minimizes the risk of a secondary aortoesophageal fistula. In patients who have previously undergone coronary artery bypass with a left internal thoracic artery graft, clamp-ing proximal to the left subclavian artery can precipitate severe myocardial ischemia and cardiac arrest. When the need to clamp at this location is anticipated in these patients, a left common carotid-to-subclavian bypass is performed to prevent cardiac complications (Fig. 22-15).123Endovascular Repair Descending Thoracic Aortic Aneurysms Stent graft repair has become the standard treatment for patients with descending thoracic aortic aneurysm.55,56,124 Although aortic repair with a self-fixing endoprosthesis was reported by Volodos103,104 in the mid 1980s, it was the report by Parodi and associates125 of using endovascular stent grafting to repair

1	Although aortic repair with a self-fixing endoprosthesis was reported by Volodos103,104 in the mid 1980s, it was the report by Parodi and associates125 of using endovascular stent grafting to repair abdominal aortic aneurysm that launched widespread interest in developing this approach. Only 3 years after this seminal report was published, Dake and colleagues126 reported performing endovascular descending tho-racic aortic repair with “homemade” stent grafts in 13 patients.Guidelines for the use of endovascular repair in thoracic aortic disease have been published,44 and reporting standards to uniformly describe the endovascular repair process have been established.127 Although endografting was initially approved to treat degenerative descending thoracic aortic aneurysm, newer devices have been approved for use in treating various descending thoracic aortic pathologies, including blunt aortic injury, penetrating aortic ulcer (see following section), coarcta-tion, and dissection.

1	have been approved for use in treating various descending thoracic aortic pathologies, including blunt aortic injury, penetrating aortic ulcer (see following section), coarcta-tion, and dissection. Although the use of stent grafts in cases of aortic infection is not ideal, patients with a fistula or mycotic aneurysm are sometimes treated with endovascular devices as a bridge to open repair.In elderly patients with severe comorbidity and patients who have undergone previous complex thoracic aortic procedures, endovascular repair is a particularly attractive alternative to stan-dard open surgical procedures.128 Patients who undergo endo-vascular repair tend to have a lower incidence of intraoperative complications, a shorter length of stay, and a higher likelihood of being discharged to home than those who undergo open repair.129 As mentioned previously, appropriate patient selection depends on specific measurements taken from preoperative CT angiograms.To protect patients against

1	home than those who undergo open repair.129 As mentioned previously, appropriate patient selection depends on specific measurements taken from preoperative CT angiograms.To protect patients against spinal cord ischemia during endovascular repair of the descending thoracic aorta, the most important maneuver is to keep the mean arterial perfusion pres-sure between 90 and 110 mmHg after the endograft is deployed. In patients who have had previous open or endovascular abdom-inal aortic aneurysm repair, cerebrospinal fluid drainage is rec-ommended.130 The first step in the repair procedure is to obtain appropriate vascular access for the insertion of the thoracic stent graft. If the femoral artery will not accommodate the necessary Brunicardi_Ch22_p0853-p0896.indd 87201/03/19 5:41 PM 873THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22sheath, then an iliac artery is exposed. If necessary, a graft can be sewn to the iliac artery in an end-to-side fashion to facilitate the deployment of

1	ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22sheath, then an iliac artery is exposed. If necessary, a graft can be sewn to the iliac artery in an end-to-side fashion to facilitate the deployment of the endograft. After 5,000 to 10,000 units of heparin are administered, a guidewire and the delivery sheath are typically inserted into the access artery under fluoroscopic guidance; depending on which endovascular device is used, the stent graft can be advanced through a sheath or with no sheath. The endograft is then advanced into the aorta and suitably posi-tioned. Note that the best view of the distal arch and descending thoracic aorta is usually in the left anterior oblique position at an angle of approximately 40° to 50°. The device is then deployed, and the proximal and distal ends can be ballooned for better apposition of the stent graft to the aortic wall. An aortogram is then performed to rule out any endoleak, and protamine is administered. As an alternative to aortography,

1	can be ballooned for better apposition of the stent graft to the aortic wall. An aortogram is then performed to rule out any endoleak, and protamine is administered. As an alternative to aortography, intravascular ultrasonography (IVUS) can be used to identify the proximal and distal landing zones, and the entire procedure can be per-formed with minimal or no contrast.Although it is not uncommon to cover the left subcla-vian artery with the endograft to lengthen the proximal landing zone,131 findings suggest that the risk of spinal cord complica-tions is heightened when the subclavian artery is covered and not revascularized, presumably because of a loss of collateral circulation to the spinal cord.132 To prevent this complication, a carotid-to-subclavian bypass can be easily constructed to main-tain vertebral artery blood flow and minimize neurologic injury (Fig. 22-16).133,134 In addition, recent studies suggest that revas-cularization of the left subclavian artery is associated

1	to main-tain vertebral artery blood flow and minimize neurologic injury (Fig. 22-16).133,134 In addition, recent studies suggest that revas-cularization of the left subclavian artery is associated with lower stroke risk in patients in whom an endograft was deployed in Zone 2 and covered the left subclavian artery.135 In addition, new generations of stent grafts are being designed with side branches that can be placed within the left subclavian artery. This feature is particularly attractive if the proximal neck is short or if the patient has a patent left internal thoracic artery-to-left anterior descending coronary artery bypass. Indications for left subclavian artery revascularization include previous coro-nary artery bypass with patent internal thoracic artery, dominant left vertebral artery, aneurysm arising from the left subclavian artery, left arm arterio-venous fistula, and coverage of a long segment of the descending thoracic aorta.Elephant Trunk Completion In select patients,

1	aneurysm arising from the left subclavian artery, left arm arterio-venous fistula, and coverage of a long segment of the descending thoracic aorta.Elephant Trunk Completion In select patients, elephant trunk completion repairs can be done with an endovascular approach (see Fig. 22-8C), rather than by the traditional open operation through a thoracotomy.136 Recall that an elephant trunk is used when an aortic aneurysm extends from the distal arch to the descending thoracic aorta. An endograft can be deployed at the time of elephant trunk construction or during a separate, subse-quent procedure.97,107,137 When the stent is deployed in a retro-grade manner during a second-stage procedure, the procedure is facilitated by placing radiopaque markers at the end of the elephant trunk during the first-stage procedure. This allows the distal end of the trunk to be identified via fluoroscopy. A guide-wire can then be manipulated into the trunk and advanced into the ascending aorta to stabilize

1	first-stage procedure. This allows the distal end of the trunk to be identified via fluoroscopy. A guide-wire can then be manipulated into the trunk and advanced into the ascending aorta to stabilize it during stent deployment. Note that advancing a wire in retrograde fashion from the femoral artery into the elephant trunk can be challenging. Occasionally, the wire must be advanced in an antegrade fashion from a bra-chial artery. The frozen elephant trunk technique—in which a short stent graft is delivered antegrade inside the trunk—can be used to perform the entire repair in one stage or to facilitate the second stage.89,90Thoracoabdominal Aortic Aneurysms Although endovascular thoracoabdominal aortic aneurysm repair remains experimental, it has been shown to be feasible in a handful of specialized cen-ters. Endovascular thoracoabdominal aortic aneurysm repairs are quite complex, because at least one of the visceral arteries is incorporated into the repair. The number of visceral

1	of specialized cen-ters. Endovascular thoracoabdominal aortic aneurysm repairs are quite complex, because at least one of the visceral arteries is incorporated into the repair. The number of visceral branches that need to be addressed varies with the extent of aortic coverage.138 The types of stent grafts used include fenestrated grafts, reinforced fenestrated grafts, branched or cuffed grafts, modular combinations of grafts, and multilayer stents.139 Graft fenestrations and branch vessels are typically aligned by using inflatable angioplasty balloons. Procedure time is not insignifi-cant, nor is the amount of contrast medium required to obtain the highly detailed images needed to plan these procedures. In addi-tion, some of the stent grafts used in endovascular thoracoab-dominal aortic aneurysm repair are custom-made in advance and thus may take several weeks to obtain; therefore, their use is limited to cases of elective repair.107 In efforts to hasten repair and utilize

1	aortic aneurysm repair are custom-made in advance and thus may take several weeks to obtain; therefore, their use is limited to cases of elective repair.107 In efforts to hasten repair and utilize off-the-shelf devices, parallel graft approaches, which use a combination of largeand small-diameter stents, have been reported.140 And, although some centers now propose distal coverage of the celiac axis141 for extent I thoracoabdomi-nal aortic aneurysm repairs, this potentially risky approach is not widely used.It should be noted that, like open thoracoabdominal aortic aneurysm repair, endovascular repair carries risks of paraplegia, renal failure, stroke, and death, despite the apparent benefits of its being a less invasive procedure. Notably, reports from centers experienced in endovascular thoracoabdominal aortic repair primarily describe limited extent IV repairs.64 Although the technology is progressing rapidly, at present endovascular thoracoabdominal aortic aneurysm repair should

1	thoracoabdominal aortic repair primarily describe limited extent IV repairs.64 Although the technology is progressing rapidly, at present endovascular thoracoabdominal aortic aneurysm repair should be considered investigational.Hybrid Repair Extensive hybrid thoracoabdominal aortic aneurysm repair142,143 can be a life-saving option in patients at high surgical risk, such as those who have limited physiologic reserve, are of advanced age, or have significant comorbidities. Hybrid procedures use open surgical techniques to reroute blood supply to the visceral arteries so that their aortic origins can be covered by stent grafts without causing visceral ischemia (Fig. 22-17). Endovascular methods are then used (either as part of the same procedure or at a later stage) to repair the aor-tic aneurysm, often with simple tube stent grafts; such devices are more readily available than the customized, modular stent grafts deployed in strictly endovascular repairs. Overall, results for hybrid

1	aneurysm, often with simple tube stent grafts; such devices are more readily available than the customized, modular stent grafts deployed in strictly endovascular repairs. Overall, results for hybrid thoracoabdominal aortic aneurysm repair have been somewhat disappointing.144 However, a handful of centers report acceptable outcomes in high-risk patients, particularly when a staged hybrid approach is used.145Postoperative Considerations Open Procedures Aortic anastomoses are often extremely fragile during the early postoperative period. Even brief episodes of postoperative hypertension can disrupt suture lines and precipitate severe bleeding or pseudoaneurysm formation. Therefore, during the initial 24 to 48 hours, meticulous blood pressure control is maintained to protect the integrity of the anastomoses. Generally, we liberally use IV vasoactive agents to keep the mean arterial blood pressure between 80 and 90 mmHg. In patients with extremely friable aortic tissue, such as those

1	of the anastomoses. Generally, we liberally use IV vasoactive agents to keep the mean arterial blood pressure between 80 and 90 mmHg. In patients with extremely friable aortic tissue, such as those with Marfan syndrome, we lower the target range to 70 to 80 mmHg. It is a delicate balancing act because one must be Brunicardi_Ch22_p0853-p0896.indd 87301/03/19 5:41 PM 874SPECIFIC CONSIDERATIONSPART IIFigure 22-16. Illustration of a “Zone 2” hybrid repair of the proximal descending thoracic aorta. A. The preoperative representation of the aneurysm shows that establishing a 2-cm proximal landing zone for a stent graft will require covering the origin of the left subclavian artery. B. Through a supraclavicular approach, a bypass from the left common carotid artery to the left subclavian artery is performed to reroute circulation and create a landing zone for the stent graft. After the bypass is completed, the left subclavian artery is ligated proximal to the graft. C. In the completed

1	is performed to reroute circulation and create a landing zone for the stent graft. After the bypass is completed, the left subclavian artery is ligated proximal to the graft. C. In the completed hybrid repair, the aneurysm has been excluded successfully by a stent graft that covers the origin of the left subclavian artery, and the proximal landing zone of the endograft is within zone 2. Importantly, blood flow to the left vertebral artery and arm is preserved by the bypass graft. (Reproduced with permission from Bozinovski J, LeMaire SA, Weldon SA: Hybrid Repairs of the Distal Aortic Arch and Proximal Descending Thoracic Aorta, Oper Tech Thorac Cardiovasc Surg 2007;12(3):167-177.)VertebralarterySubclavianarteryPhrenicnerveAnterior scalenemuscle (divided)Internal thoracicarterySternocleidomastoidmuscle (divided)Commoncarotid arteryVagusnerveABCBrunicardi_Ch22_p0853-p0896.indd 87401/03/19 5:41 PM 875THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22Figure 22-17. Illustration of a

1	(divided)Commoncarotid arteryVagusnerveABCBrunicardi_Ch22_p0853-p0896.indd 87401/03/19 5:41 PM 875THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22Figure 22-17. Illustration of a “Zone 0” hybrid approach—which combines open and endovascular techniques—for repair of an extensive aortic aneurysm. Debranching the arch and thoracoab-dominal segments allows the use of a series of endovascular stent grafts to exclude the entire aneurysm. Note that the arterial inflow for the debranched visceral arteries comes from the left common iliac artery.Table 22-4Classification of and common treatment strategies for endoleakType I• Incomplete seal between stent graft and aorta at the proximal landing site (Type Ia), the distal landing site (Type Ib), or branch module, fenestration, or plug (Type Ic)• Early reintervention to improve seal or conversion to open surgeryType II• Retrograde perfusion of sac from excluded collateral arteries• Surveillance; as-needed occlusion with percutaneous or other

1	reintervention to improve seal or conversion to open surgeryType II• Retrograde perfusion of sac from excluded collateral arteries• Surveillance; as-needed occlusion with percutaneous or other interventionsType III• Incomplete seal between overlapping stent graft or module (Type IIIa), or tear in graft fabric (Type IIIb)• Early reintervention to cover or conversion to open surgeryType IV• Perfusion of sac due to porosity of material• Surveillance; as-needed reintervention to reline stent graftType V• Expansion of sac with no identifiable source• Surveillance; as-needed reintervention to reline stent graftmindful of spinal cord perfusion and avoid periods of relative hypotension while maintaining these low pressures.Endovascular Procedures Many of the complications are directly related to manipulation of the delivery system within the iliac arteries and aorta.146 Patients with small, calcified, tor-tuous iliofemoral arteries are at particularly high risk for life-threatening iliac

1	to manipulation of the delivery system within the iliac arteries and aorta.146 Patients with small, calcified, tor-tuous iliofemoral arteries are at particularly high risk for life-threatening iliac artery rupture. Although relatively uncommon, acute iatrogenic retrograde dissection into the aortic arch and ascending aorta is a life-threatening complication that neces-sitates emergency repair of the ascending aorta and aortic arch via sternotomy and cardiopulmonary bypass.109 The most important risk factors for this complication include incautious wire and catheter manipulation, aggressive proximal ballooning (especially in cases of acute descending thoracic aortic dissection), and hybrid arch repair in which the native ascending aorta is dilated (more than 4 cm). Retrograde proximal dissection con-verts a localized descending thoracic aortic aneurysm into an acute problem involving the entire thoracic aorta. Of note, ret-rograde aortic dissection may also occur several months after

1	con-verts a localized descending thoracic aortic aneurysm into an acute problem involving the entire thoracic aorta. Of note, ret-rograde aortic dissection may also occur several months after initial repair.147Another significant complication of descending thoracic aortic stent grafting is endoleak. An endoleak occurs when there is a persistent flow of blood (visible on radiologic imaging) into the aneurysm sac, and it may occur during the initial proce-dure or develop over time. Although endoleaks are a relatively common complication,148,149 they are not benign because they lead to continual pressurization of the sac, which can cause expansion or even rupture. These complications are categorized (Table 22-4) according to the site of the leak.127 Although all endoleaks may progress such that they can be considered life-threatening, type I and type III endoleaks generally necessitate early and aggressive intervention. Recently published reporting guidelines aid standardized

1	such that they can be considered life-threatening, type I and type III endoleaks generally necessitate early and aggressive intervention. Recently published reporting guidelines aid standardized reporting.127Other complications include stent graft misdeployment, device migration, endograft kinking or infolding, and stent graft infection, including fistula. Although not all complications related to stent grafts are fatal, endovascular repairs should be performed by expert teams qualified to address the variety of problems that may arise; some patients may need to have these devices removed and replaced with polyester grafts.67,68,150,151 Complications of endovascular repair are relatively common, so regularly scheduled radiologic imaging surveillance is of the utmost importance.Brunicardi_Ch22_p0853-p0896.indd 87501/03/19 5:41 PM 876SPECIFIC CONSIDERATIONSPART IIAORTIC DISSECTIONPathology and ClassificationAortic dissection, the most common catastrophic event involving the aorta,

1	87501/03/19 5:41 PM 876SPECIFIC CONSIDERATIONSPART IIAORTIC DISSECTIONPathology and ClassificationAortic dissection, the most common catastrophic event involving the aorta, is a progressive separation of the aortic wall layers that usually occurs after a tear forms in the intima and inner media. As the separation of the layers of the media propagates, two channels are typically formed (Fig. 22-18): the original lumen, which remains lined by the intima and which is called the true lumen, and the newly formed channel within the layers of the media, which is called the false lumen. The dissecting membrane separates the true and false lumens. Additional tears in the dis-secting membrane that allow communication between the two channels are called reentry sites. Although the separation of lay-ers primarily progresses distally along the length of the aorta, it can also proceed in a proximal direction; this process often is referred to as proximal extension or retrograde dissection.The

1	lay-ers primarily progresses distally along the length of the aorta, it can also proceed in a proximal direction; this process often is referred to as proximal extension or retrograde dissection.The extensive disruption of the aortic wall has severe anatomic consequences (Fig. 22-19). First, the outer wall of the false lumen is extremely thin, inflamed, and fragile, which makes it prone to expansion or rupture in the face of ongoing hemodynamic stress. Second, the expanding false lumen can compress the true lumen and cause malperfusion syndrome by interfering with blood flow in the aorta or any of its branch vessels, including the coronary, carotid, inter-costal, visceral, renal, and iliac arteries. Finally, when the separation of layers occurs within the aortic root, the aortic valve commissures can become unhinged, which results in acute valvular regurgitation. The clinical consequences of each of these sequelae are addressed in detail in the section on clinical

1	the aortic valve commissures can become unhinged, which results in acute valvular regurgitation. The clinical consequences of each of these sequelae are addressed in detail in the section on clinical manifestations.Dissection vs. Aneurysm. The relationship between dissec-tion and aneurysmal disease requires clarification. Dissection and aneurysm are separate entities, although they often coexist and are mutual risk factors. In some cases, dissection occurs in patients without aneurysms, and the subsequent progressive dilatation of the weakened outer aortic wall ultimately results in an aneurysm. On the other hand, in patients with degenera-tive aneurysms, the ongoing deterioration of the aortic wall can lead to a superimposed dissection. The overused term dissecting aneurysm should be reserved for this specific situation.Classification. For management purposes, aortic dissec-tions are classified according to their location and chronicity. Improvements in imaging have increasingly

1	reserved for this specific situation.Classification. For management purposes, aortic dissec-tions are classified according to their location and chronicity. Improvements in imaging have increasingly revealed variants of aortic dissection that probably represent different forms along the spectrum of this condition.Location To guide treatment, dissections are categorized according to their anatomic location and extent. The two tra-ditional classification schemes that remain in common use are the DeBakey and the Stanford classification systems (Fig. 22-20).152,153 In their current forms, both of these schemes describe the segments of aorta that are involved in the dissec-tion, rather than the site of the initial intimal tear. The main drawback of the Stanford classification system is that it does not distinguish between patients with isolated ascending aor-tic dissection and patients with dissection involving the entire aorta. Both types of patients would be classified as having type A

1	does not distinguish between patients with isolated ascending aor-tic dissection and patients with dissection involving the entire aorta. Both types of patients would be classified as having type A dissections, despite the fact that their treatment, follow-up, and prognosis are substantially different.Additional classification schemas include that by Borst and associates,154 in which the ascending and descending aorta are considered independently; the recent modification of the DeBakey classification by Tsagakis et al,155 which extends type II dissection into the aortic arch; and the Penn modification of the Stanford classification,156,157 which expands the classification to include the presence of tissue and global malperfusion. These modifications may help to better streamline the primary surgical intervention.Normal aortaAortic dissectionIntramural hematomaPenetrating aortic ulcerFigure 22-18. Illustration of longitudinal sections of the aortic wall and lumen. Blood flows freely

1	surgical intervention.Normal aortaAortic dissectionIntramural hematomaPenetrating aortic ulcerFigure 22-18. Illustration of longitudinal sections of the aortic wall and lumen. Blood flows freely downstream in normal aortic tissue. In classic aortic dissection, blood entering the media through a tear creates a false channel in the wall. Intramural hematomas arise when hemor-rhage from the vasa vasorum causes blood to collect within the media; the intima is intact. Penetrating aortic ulcers are deep atherosclerotic lesions that burrow into the aortic wall and allow blood to enter the media. In each of these conditions, the outer aortic wall is severely weakened and prone to rupture.Brunicardi_Ch22_p0853-p0896.indd 87601/03/19 5:42 PM 877THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22Figure 22-19. Illustration of the potential anatomic consequences of aortic dissection, with a mapped diagram of affected regions (inset). A. Ascending aortic rupture and cardiac tamponade. B.

1	22Figure 22-19. Illustration of the potential anatomic consequences of aortic dissection, with a mapped diagram of affected regions (inset). A. Ascending aortic rupture and cardiac tamponade. B. Disruption of coronary blood flow. C. Injury to the aortic valve causing regurgitation. D, E, and F. Compromised blood flow to branch vessels, causing ischemic complications. (Adapted with permission from Creager MA, Dzau VS, Loscalzo J: Vascular Medicine, 7th ed. Philadelphia, PA: Elsevier/Saunders; 2006.)Figure 22-20. Illustration of the classification schemes for aortic dissection based on which portions of the aorta are involved. Dissection can be confined to the ascending aorta (left) or the descending aorta (middle), or it can involve the entire aorta (right). (Used with permission from Baylor College of Medicine.)ABCDEFBrunicardi_Ch22_p0853-p0896.indd 87701/03/19 5:42 PM 878SPECIFIC CONSIDERATIONSPART IIContrast-enhanced CT scanTransfer to operatingroom, intubation,diagnostic

1	Baylor College of Medicine.)ABCDEFBrunicardi_Ch22_p0853-p0896.indd 87701/03/19 5:42 PM 878SPECIFIC CONSIDERATIONSPART IIContrast-enhanced CT scanTransfer to operatingroom, intubation,diagnostic TEESuspected acutedissectionAortic dissection?Ascending aorticdissection (Stanford A orDeBakey I or II)?Secondary diagnosticstudy (MRA, TEE, oraortography)Emergency operationEmergency operationTransfer to intensive carefor further stabilizationand diagnostic work-upTransfer to intensive care unitfor blood pressure control,anti-impulse therapy Emergency endovascular(fenestration, stent) oropen interventionYesYesYesYesYesYesNoNoNoNoNoNoFurther diagnosticwork-upHemodynamicallystable?Anti-impulse therapy(beta blockers),blood pressure controlAortic dissection?Complicated descendingaortic dissection (malperfusion, rupture)?Management of acute aortic dissectionAscending aorticdissection (Stanford A orDeBakey I or II)?Figure 22-21. Algorithm used to facilitate decisions regarding treatment of

1	(malperfusion, rupture)?Management of acute aortic dissectionAscending aorticdissection (Stanford A orDeBakey I or II)?Figure 22-21. Algorithm used to facilitate decisions regarding treatment of acute aortic dissection. CT = computed tomography; MRA = magnetic resonance angiography; TEE = transesophageal echocardiography.Regardless of which system is used, patients with isolated ascending aortic dissection usually undergo emergent opera-tion, as do patients with dissection involving both the ascend-ing and descending thoracic segments. Patients with isolated descending thoracic and abdominal aortic dissection are typi-cally treated medically, unless complications requiring surgery develop. Understanding the precise extent of dissection has become increasingly important as some aortic centers consider augmenting traditional ascending aortic repairs with endovascu-lar techniques to treat dissected distal aortic segments.158Chronicity Aortic dissection also is categorized according to

1	consider augmenting traditional ascending aortic repairs with endovascu-lar techniques to treat dissected distal aortic segments.158Chronicity Aortic dissection also is categorized according to the time elapsed since the initial tear. Dissection is considered acute within the first 14 days after the initial tear; after 14 days, the dissection is considered chronic. Although arbitrary, the dis-tinction between acute and chronic dissections has important implications, not only for decision making about perioperative management strategies and operative techniques, but also for evaluating surgical results. Figure 22-21 provides an algorithm for the management of acute aortic dissection. In light of the importance of acuity, Borst and associates154 have proposed a third phase—termed subacute—to describe the transition between the acute and chronic phases. The subacute period encompasses days 15 through 60 after the initial tear. Although this is past the traditional 14-day acute phase,

1	describe the transition between the acute and chronic phases. The subacute period encompasses days 15 through 60 after the initial tear. Although this is past the traditional 14-day acute phase, patients with subacute dissection continue to have extremely fragile aortic Brunicardi_Ch22_p0853-p0896.indd 87801/03/19 5:42 PM 879THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22tissue, which may complicate operative treatment and increase the risks associated with surgery. Recently, the International Registry of Acute Aortic Dissections (IRAD) investigators pro-posed a new classification system for characterizing the phases of aortic dissection: The period within 24 hours from the onset of symptoms was defined as hyperacute, the period between 2 and 7 days was defined as acute, the period between 8 and 30 days was defined as subacute, and the period beyond 30 days was defined as chronic.159Variants As noted earlier, advancements in noninvasive imag-ing of the aorta have revealed

1	between 8 and 30 days was defined as subacute, and the period beyond 30 days was defined as chronic.159Variants As noted earlier, advancements in noninvasive imag-ing of the aorta have revealed variants of aortic dissection (see Fig. 22-18). The recently introduced term acute aortic syn-drome encompasses classic aortic dissection and its variants. Other aortic syndromes, which were once thought to be rare, include intramural hematoma (IMH) and penetrating aortic ulcer (PAU). Although the issue is somewhat controversial, the current consensus is that, in most cases, these variants of dissec-tion should be treated identically to classic dissection.An IMH is a collection of blood within the aortic wall, without an intimal tear, that is believed to be due to rupture of the vasa vasorum within the media. The accumulation of blood can result in a secondary intimal tear that ultimately leads to a dissection.160 Because IMH and aortic dissection represent a continuum, it is possible that IMH

1	the media. The accumulation of blood can result in a secondary intimal tear that ultimately leads to a dissection.160 Because IMH and aortic dissection represent a continuum, it is possible that IMH is seen less frequently than aortic dissection because IMH rapidly progresses to true dissec-tion. The prevalence of IMH among patients with acute aortic syndromes is approximately 6%, and 16% progress to full dis-section.161 An IMH can be classified according to its location (i.e., ascending or descending) and should be treated analo-gously to classic dissection.162A PAU is essentially a disrupted atherosclerotic plaque that projects into the aortic wall and is associated with surround-ing hematoma. Eventually, the ulcer can penetrate the aortic wall, which leads to dissection or rupture. The rate of disease progression is higher than that of IMH alone.163Causes and Clinical HistoryAortic dissection is a lethal condition with a reported incidence of 3.5 per 100,000 in the United

1	The rate of disease progression is higher than that of IMH alone.163Causes and Clinical HistoryAortic dissection is a lethal condition with a reported incidence of 3.5 per 100,000 in the United States.164 Without appropriate mod-ern medical or surgical treatment, most patients (approximately 90%) die within 3 months of dissection, mostly from rupture.165,166Although several risk factors for aortic dissection have been identified, the specific causes remain unknown. Ultimately, any condition that weakens the aortic wall increases the risk of aortic dissection. Common general cardiovascular risk factors, such as smoking, hypertension, atherosclerosis, and hypercholesterolemia, are associated with aortic dissection. Patients with heritable forms of aortopathy, aortitis, bicuspid aortic valve, or preexisting medial degenerative disease are at risk for dissection, especially if they already have a thoracic aortic aneurysm.24 Aortic injury during cardiac catheterization, surgery, or

1	valve, or preexisting medial degenerative disease are at risk for dissection, especially if they already have a thoracic aortic aneurysm.24 Aortic injury during cardiac catheterization, surgery, or endovascular aortic repair is a common cause of iatrogenic dissection. Other conditions that are associated with aortic dissection include cocaine and amphetamine abuse,167 as well as severe emotional stress or extreme physical exertion such as during weightlifting.168 Advances in the understanding of the molecular mechanisms behind abdominal aortic aneu-rysms have prompted similar investigations of thoracic aortic dissection.169-171Clinical ManifestationsThe onset of dissection often is associated with severe chest or back pain, classically described as “tearing,” that migrates Table 22-5Anatomic complications of aortic dissection and their associated symptoms and signsANATOMIC MANIFESTATIONSYMPTOMS AND SIGNSAortic valve insufficiencyDyspneaMurmurPulmonary ralesShockCoronary

1	22-5Anatomic complications of aortic dissection and their associated symptoms and signsANATOMIC MANIFESTATIONSYMPTOMS AND SIGNSAortic valve insufficiencyDyspneaMurmurPulmonary ralesShockCoronary malperfusionChest pain with characteristics of anginaNausea/vomitingShockIschemic changes on electrocardiogramElevated cardiac enzymesPericardial tamponadeDyspneaJugular venous distensionPulsus paradoxusMuffled cardiac tonesShockLow-voltage electrocardiogramSubclavian or iliofemoral artery malperfusionCold, painful extremityExtremity sensory and motor deficitsPeripheral pulse deficitCarotid artery malperfusionSyncopeFocal neurologic deficit (transient or persistent)Carotid pulse deficitComaSpinal malperfusionParaplegiaIncontinenceMesenteric malperfusionNausea/vomitingAbdominal painRenal malperfusionOliguria or anuriaHematuriadistally as the dissection progresses along the length of the aorta. The location of the pain often indicates which aortic segments are involved. Pain in the anterior

1	or anuriaHematuriadistally as the dissection progresses along the length of the aorta. The location of the pain often indicates which aortic segments are involved. Pain in the anterior chest suggests involvement of the ascending aorta, whereas pain in the back and abdomen generally indicates involvement of the descending and thora-coabdominal aorta. Additional clinical sequelae of acute aortic dissection vary substantially and are best considered in terms of the dissection’s potential anatomic manifestations at each level of the aorta (see Fig. 22-19 and Table 22-5). Thus, potential complications of dissection of the aorta (and involved second-ary arteries) may include cardiac ischemia (coronary artery) or tamponade, stroke (brachiocephalic arteries), paraplegia or paraparesis (intercostal arteries), mesenteric ischemia (superior mesenteric artery), kidney failure (renal arteries), and limb isch-emia or loss of motor function (brachial or femoral arteries).Ascending aortic dissection

1	arteries), mesenteric ischemia (superior mesenteric artery), kidney failure (renal arteries), and limb isch-emia or loss of motor function (brachial or femoral arteries).Ascending aortic dissection can directly injure the aortic valve, causing regurgitation. The severity of the regurgitation varies with the degree of commissural disruption, which ranges from partial separation of only one commissure, producing mild Brunicardi_Ch22_p0853-p0896.indd 87901/03/19 5:42 PM 880SPECIFIC CONSIDERATIONSPART IIvalvular regurgitation, to full separation of all three commis-sures and complete prolapse of the valve into the left ventricle, producing severe acute heart failure. Patients with acute aortic valve regurgitation may report rapidly worsening dyspnea.Ascending dissections also can extend into the coronary arteries or shear the coronary ostia off of the true lumen, caus-ing acute coronary occlusion; when this occurs, it most often involves the right coronary artery. The sudden

1	extend into the coronary arteries or shear the coronary ostia off of the true lumen, caus-ing acute coronary occlusion; when this occurs, it most often involves the right coronary artery. The sudden disruption of coronary blood flow can cause a myocardial infarction. This presentation of acute myocardial ischemia can mask the pres-ence of aortic dissection, which results in delayed diagnosis and treatment.172The thin and inflamed outer wall of a dissected ascend-ing aorta often produces a serosanguineous pericardial effusion that can accumulate and cause tamponade. Suggestive signs include jugular venous distention, muffled heart tones, pulsus paradoxus, and low-voltage electrocardiogram (ECG) tracings. Free rupture into the pericardial space produces rapid tampon-ade and is generally fatal.As the dissection progresses, any branch vessel from the aorta can become involved, which results in compromised blood flow and ischemic complications (i.e., malperfusion). Therefore, depending on

1	the dissection progresses, any branch vessel from the aorta can become involved, which results in compromised blood flow and ischemic complications (i.e., malperfusion). Therefore, depending on which arteries are involved, the dissection can produce acute stroke, paraplegia, hepatic failure, bowel infarc-tion, renal failure, or a threatened ischemic limb.Diagnostic EvaluationBecause of the variations in severity and the wide variety of potential clinical manifestations, the diagnosis of acute aortic dissection can be challenging.173-175 Only 3 out of every 100,000 patients who present to an emergency department with acute chest, back, or abdominal pain are eventually diagnosed with aortic dissection. Not surprisingly, diagnostic delays are com-mon; delays beyond 24 hours after hospitalization occur in up to 39% of cases. Unfortunately, delays in diagnosis lead to delays in treatment, which can have disastrous consequences. The European Society of Cardiology Task Force on Aortic

1	occur in up to 39% of cases. Unfortunately, delays in diagnosis lead to delays in treatment, which can have disastrous consequences. The European Society of Cardiology Task Force on Aortic Dis-section stated, “The main challenge in managing acute aortic dissection is to suspect and thus diagnose the disease as early as possible.”173 A recent study by the IRAD investigators exam-ined the reasons for delayed diagnosis and found that diagnosis lagged in women, as well as in patients with atypical symptoms, such as fever or mild pain (rather than severe pain).172 A high index of suspicion is critical, particularly in younger, atypical patients, who may have heritable disorders or other, less com-mon risk factors.Most patients with acute aortic dissection (80% to 90%) experience severe pain in the chest, back, or abdomen.173-175 The pain usually occurs suddenly, has a sharp or tearing quality, and often migrates distally as the dissection progresses along the aorta. For classification

1	in the chest, back, or abdomen.173-175 The pain usually occurs suddenly, has a sharp or tearing quality, and often migrates distally as the dissection progresses along the aorta. For classification purposes (acute vs. subacute vs. chronic), the onset of pain is generally considered to represent the beginning of the dissection process. Most of the other com-mon symptoms either are nonspecific or are caused by the sec-ondary manifestations of dissection.A discrepancy between the extremities in pulse, blood pressure, or both is the classic physical finding in patients with aortic dissection. It often occurs because of changes in flow in the true and false lumens, and it does not necessarily indi-cate extension into an extremity branch vessel. Involvement of the aortic arch often creates differences between the right and left arms, whereas descending aortic dissection often causes differences between the upper and lower extremities. Like symptoms, most of the physical signs after

1	differences between the right and left arms, whereas descending aortic dissection often causes differences between the upper and lower extremities. Like symptoms, most of the physical signs after dissection are related to the secondary manifestations and therefore vary considerably (see Table 22-5). For example, signs of stroke or a threatened ischemic limb may dominate the physical findings in patients with carotid or iliac malperfusion, respectively.Unfortunately, laboratory studies are of little help in diag-nosing acute aortic dissection. There has been continued inter-est in using D-dimer level to aid in making this diagnosis.176 Several reports indicate that D-dimer is an extremely sensitive indicator of acute aortic dissection; elevated levels are found in approximately 97% of affected patients.177 Tests that are com-monly used to detect acute coronary events—including ECG and tests for serum markers of myocardial injury—deserve spe-cial consideration and need to be interpreted

1	patients.177 Tests that are com-monly used to detect acute coronary events—including ECG and tests for serum markers of myocardial injury—deserve spe-cial consideration and need to be interpreted carefully. Normal ECGs and serum marker levels in patients with acute chest pain should raise suspicion about the possibility of aortic dissection. It is important to remember that ECG changes and elevated serum marker levels associated with myocardial infarction do not exclude the diagnosis of aortic dissection because dissection can cause coronary malperfusion. Of note, abnormal ECGs have recently been shown to delay the diagnosis of aortic dissection, and the possibility of aortic dissection should not be prema-turely ruled out.172,178 Similarly, although CXRs may show a widened mediastinum or abnormal aortic contour, up to 16% of patients with dissection have a normal-appearing CXR.174 The value of the CXR for detecting aortic dissection is limited, with a sensitivity of 67% and a

1	or abnormal aortic contour, up to 16% of patients with dissection have a normal-appearing CXR.174 The value of the CXR for detecting aortic dissection is limited, with a sensitivity of 67% and a specificity of 86%.179Once the diagnosis of dissection is considered, the tho-racic aorta should be imaged with CT, MRA, or echocardiogra-phy. The accuracy of these noninvasive imaging tests has all but eliminated the need for diagnostic aortography in most patients with suspected aortic dissection. Currently, the diagnosis of aor-tic dissection is usually established with contrast-enhanced CT, which has a sensitivity of 98% and a specificity of 87%, and, most importantly, acquires images swiftly.180 The classic diag-nostic feature is a double-lumen aorta (Fig. 22-22). In addition, CT scans provide essential information about the segments of the aorta involved; the acuity of the dissection; aortic dilata-tion, including the presence of preexisting degenerative aneu-rysms; and the development

1	essential information about the segments of the aorta involved; the acuity of the dissection; aortic dilata-tion, including the presence of preexisting degenerative aneu-rysms; and the development of threatening sequelae, including pericardial effusion, early aortic rupture, and branch-vessel compromise. Although MRA also provides excellent imaging (with both a sensitivity and specificity of 98%), the MR suite is not well suited for critically ill patients. In patients who cannot undergo contrast-enhanced CT or MRA, transthoracic echocar-diography can be used to establish the diagnosis.Transesophageal echocardiography (TEE) is excellent for detecting dissection, aneurysm, and IMH in the ascending aorta. In appropriate hands, TEE has a demonstrated sensitivity and specificity as high as 98% and 95%, respectively.181 Further-more, TEE offers important information about ventricular func-tion and aortic valve competency. Finally, TEE is the diagnostic modality of choice for

1	as high as 98% and 95%, respectively.181 Further-more, TEE offers important information about ventricular func-tion and aortic valve competency. Finally, TEE is the diagnostic modality of choice for hemodynamically unstable patients in whom the diagnosis of ascending dissection is suspected; ide-ally, these patients should be taken to the operating room, where the TEE can be performed and, if the TEE is confirmatory, sur-gery can be started immediately.In selected patients with ascending aortic dissection (i.e., those who have evidence of preexisting coronary artery dis-ease), coronary angiography can be considered before surgery. Specific relative indications in these patients include a history Brunicardi_Ch22_p0853-p0896.indd 88001/03/19 5:42 PM 881THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22of angina or myocardial infarction, a recent myocardial per-fusion study with abnormal results, previous coronary artery bypass or angioplasty, and acute ischemic changes on ECG.

1	DISSECTIONCHAPTER 22of angina or myocardial infarction, a recent myocardial per-fusion study with abnormal results, previous coronary artery bypass or angioplasty, and acute ischemic changes on ECG. Contraindications include hemodynamic instability, aortic rupture, and pericardial effusion.182 In our practice, patients with acute aortic dissections rarely undergo coronary angiog-raphy. However, all patients presenting for elective repair of chronic ascending dissections have diagnostic coronary angio-grams taken.Of note, when malperfusion of the renal, visceral, or lower extremity arteries develops, the patient is usually treated in an angiography suite or hybrid operating room.158 Although the dissection usually is diagnosed on CT scan, these patients also undergo aortography, during which the mechanism of the malperfusion is ascertained and, if possible, corrected. Hence, catheter-based aortography may be obsolete as a diagnostic test for dissection, but it remains beneficial for

1	the mechanism of the malperfusion is ascertained and, if possible, corrected. Hence, catheter-based aortography may be obsolete as a diagnostic test for dissection, but it remains beneficial for patients with malperfusion.TreatmentInitial Assessment and Management. Regardless of the location of the dissection, the initial treatment is the same for all patients with suspected or confirmed acute aortic dissection (see Fig. 22-21). Furthermore, because of the potential for rup-ture before the diagnosis is confirmed, aggressive pharmaco-logic management is started once there is clinical suspicion of dissection, and this treatment is continued during the diagnostic evaluation. The goals of pharmacologic treatment are to stabi-lize the dissection and prevent rupture.Patients are monitored closely in an intensive care unit. Indwelling radial arterial catheters are used to monitor blood pressure and optimize titration of antihypertensive agents. Blood pressures in a malperfused limb can

1	closely in an intensive care unit. Indwelling radial arterial catheters are used to monitor blood pressure and optimize titration of antihypertensive agents. Blood pressures in a malperfused limb can underrepresent the central aortic pressure; therefore, blood pressure is measured in the arm with the better pulse. Central venous catheters assure reliable IV access for delivering vasoactive medications. Pul-monary artery catheters are reserved for patients with severe cardiopulmonary dysfunction.In addition to confirming the diagnosis of dissection and defining its acuity and extent, the initial evaluation focuses on determining whether any of several life-threatening compli-cations are present. Particular attention is paid to changes in neurologic status, peripheral pulses, and urine output. Serial laboratory studies—including arterial blood gas concentrations, complete blood cell count, prothrombin and partial thrombo-plastin times, and serum levels of electrolytes, creatinine, blood

1	Serial laboratory studies—including arterial blood gas concentrations, complete blood cell count, prothrombin and partial thrombo-plastin times, and serum levels of electrolytes, creatinine, blood urea nitrogen, and liver enzymes—are useful for detecting organ ischemia and optimizing management.The initial management strategy, commonly described as anti-impulse therapy or blood pressure control, focuses on reducing aortic wall stress, the force of left ventricular ejection, chronotropy, and the rate of change in blood pressure (dP/dT). Reductions in dP/dT are achieved by lowering both cardiac contractility and blood pressure. The drugs initially used to accomplish these goals include IV b-adrenergic blockers, direct vasodilators, calcium channel blockers, and angiotensinconverting enzyme inhibitors. These agents are used to achieve a heart rate between 60 and 80 bpm, a systolic blood pressure between 100 and 110 mmHg, and a mean arterial blood pres-sure between 60 and 75 mmHg. These

1	inhibitors. These agents are used to achieve a heart rate between 60 and 80 bpm, a systolic blood pressure between 100 and 110 mmHg, and a mean arterial blood pres-sure between 60 and 75 mmHg. These hemodynamic targets are maintained as long as urine output remains adequate and neuro-logic function is not impaired. Achieving adequate pain control with IV opiates, such as morphine and fentanyl, is important for maintaining acceptable blood pressure control.b-Antagonists are administered to all patients with acute aortic dissections unless there are strong contraindications, such as severe heart failure, bradyarrhythmia, high-grade atrioven-tricular conduction block, or bronchospastic disease. Esmolol Figure 22-22. Computed tomographic scans showing that the aorta has been separated into two channels—the true (T) and false (F) lumens—in two patients with different phases of aortic dissection. A. An acute DeBakey type I aortic dissection. The dissecting membrane appears wavy (arrows) in

1	channels—the true (T) and false (F) lumens—in two patients with different phases of aortic dissection. A. An acute DeBakey type I aortic dissection. The dissecting membrane appears wavy (arrows) in the early phase of dissection. Here, the true lumen of the proximal aorta can be seen to be extensively compressed. This may lead to malperfusion of the heart. B. A chronic DeBakey type III aortic dissection. In the chronic phase, the membrane appears straighter and less mobile (arrow) because it has stabilized over time. (Used with permission of Baylor College of Medicine.)Brunicardi_Ch22_p0853-p0896.indd 88101/03/19 5:42 PM 882SPECIFIC CONSIDERATIONSPART IIcan be useful in patients with bronchospastic disease because it is a cardioselective, ultra-fast-acting agent with a short half-life. Labetalol, which causes both nonselective b-blockade and postsynaptic α1-blockade, reduces systemic vascular resistance without impairing cardiac output. Doses of b-antagonists are titrated to

1	Labetalol, which causes both nonselective b-blockade and postsynaptic α1-blockade, reduces systemic vascular resistance without impairing cardiac output. Doses of b-antagonists are titrated to achieve a heart rate of 60 to 80 bpm. In patients who cannot receive b-antagonists, calcium channel blockers such as diltiazem are an effective alternative. Nitroprusside, a direct vasodilator, can be administered once b-blockade is adequate. When used alone, however, nitroprusside can cause reflex increases in heart rate and contractility, elevated dP/dT, and pro-gression of aortic dissection. Enalapril and other angiotensin-converting enzyme inhibitors are useful in patients with renal malperfusion. These drugs inhibit renin release, which may improve renal blood flow.Treatment of Ascending Aortic Dissection Acute Dissection Because of the risk of aortic rupture, acute ascending aortic dissection is usually considered an absolute indication for emergency surgical repair. However, specific

1	Dissection Acute Dissection Because of the risk of aortic rupture, acute ascending aortic dissection is usually considered an absolute indication for emergency surgical repair. However, specific patient groups may benefit from nonoperative manage-ment or delayed operation.183 Delayed repair may be con-sidered for patients who (a) present with severe acute stroke or mesenteric ischemia, (b) are elderly and have substantial comor-bidity, (c) are in stable condition and may benefit from transfer to specialized centers, or (d) have undergone a cardiac operation in the remote past. Regarding the last group, it is important that the previous operation not be too recent; dissections that occur during the first 3 weeks after cardiac surgery pose a high risk of rupture and tamponade, and such dissections warrant early operation.184In the absence of the aforementioned circumstances, most patients with acute ascending aortic dissection undergo emer-gent graft replacement of the ascending aorta.

1	warrant early operation.184In the absence of the aforementioned circumstances, most patients with acute ascending aortic dissection undergo emer-gent graft replacement of the ascending aorta. Operative repair is similar to that for aneurysm of the transverse aortic arch (pre-viously described) because hypothermic circulatory arrest is commonly used regardless of the extent of repair. Immediately before the operation begins, intraoperative TEE is commonly performed to further assess baseline myocardial and valvular function and, if necessary, to confirm the diagnosis. The opera-tion is performed via a median sternotomy with cardiopulmo-nary bypass and hypothermic circulatory arrest (Fig. 22-23). In preparation for circulatory arrest, cannulas are placed in the right axillary artery (to provide arterial inflow) and in the right atrium (to provide venous drainage).94 The innominate artery is some-times used for arterial inflow if it is not dissected.185 After an appropriate level of

1	provide arterial inflow) and in the right atrium (to provide venous drainage).94 The innominate artery is some-times used for arterial inflow if it is not dissected.185 After an appropriate level of cooling has been achieved (approximately 24°C), cardiopulmonary bypass is stopped, and the ascending aorta is opened. The innominate artery is then occluded with a clamp or snare, and flow from the axillary artery cannula is used to provide ACP.186 Currently as a default, we use bilateral ACP with a separate perfusion catheter in the left common carotid artery to ensure perfusion of the left side of the brain. This strat-egy of performing the distal anastomosis during a brief period of circulatory arrest, often termed open distal anastomosis, obvi-ates the need to place a clamp across the fragile aorta, avoiding further aortic damage. Also, it allows the surgeon to carefully inspect the aortic arch for intimal tears. Traditionally, the entire arch is replaced only if a primary intimal tear

1	aorta, avoiding further aortic damage. Also, it allows the surgeon to carefully inspect the aortic arch for intimal tears. Traditionally, the entire arch is replaced only if a primary intimal tear is located in the arch or if the arch is aneurysmal; most commonly, repair is lim-ited to replacement of the entire ascending aorta or to a bev-eled “hemiarch” repair.187 Conservative repair has been shown 8to increase the likelihood of early survival.188 The distal aortic cuff is prepared by tacking the inner and outer walls together and occasionally using a small amount of surgical adhesive to obliterate the false lumen and strengthen the tissue. A polyester tube graft is sutured to the distal aortic cuff. The anastomosis between the graft and the aorta is fashioned so that blood flow will be directed into the true lumen; this often alleviates distal malperfusion problems that were present preoperatively. After the distal anastomosis has been completed and adequately rein-forced, the graft

1	into the true lumen; this often alleviates distal malperfusion problems that were present preoperatively. After the distal anastomosis has been completed and adequately rein-forced, the graft is deaired and clamped, full cardiopulmonary bypass is resumed, rewarming is initiated, and the proximal por-tion of the repair is started. In the absence of conditions that generally necessitate aortic root replacement (i.e., annuloaortic ectasia or heritable disorders, particularly Marfan and Loeys-Dietz syndromes), aortic valve regurgitation can be corrected by resuspending the commissures onto the outer aortic wall.189 The proximal aortic cuff is prepared with tacking sutures and occasionally a small amount of surgical adhesive before the proximal aortic anastomosis is performed.In the majority of patients who undergo surgical repair of acute ascending dissection, the dissection persists distal to the site of the operative repair; the residually dissected aorta, which generally includes at

1	of patients who undergo surgical repair of acute ascending dissection, the dissection persists distal to the site of the operative repair; the residually dissected aorta, which generally includes at least a portion of the transverse aortic arch as well as a large portion of the distal aorta, is susceptible to dil-atation over time. Extensive dilatation of the arch or distal aorta develops in 25% to 40% of survivors190,191 and often necessitates further aortic repair. Additionally, long-term survival after acute proximal aortic dissection is generally poor, and rupture of the dilated distal aorta is a common cause of late death in these patients.188,190-192The challenges that survivors of acute proximal aortic dissec-tion commonly face over time have led to the development of alter-nate acute dissection strategies such as total arch replacement193 and hybrid arch strategies to extend proximal aortic repair into the distal aorta. The goal of hybrid arch approaches in acute dis-section

1	dissection strategies such as total arch replacement193 and hybrid arch strategies to extend proximal aortic repair into the distal aorta. The goal of hybrid arch approaches in acute dis-section is to thrombose the residual false lumen by compressing it with the radial force that is exerted by a stent graft placed in the true lumen, thereby facilitating remodeling and prevent-ing late aneurysm formation.194,195 However, in such repairs, the compressed false lumen may continue to be perfused in a ret-rograde fashion.In Europe, Japan, and elsewhere, one-piece hybrid pros-theses are now available that incorporate a polyester graft for the proximal repair and a stent graft component for the descending aorta. The device enables single-stage “frozen elephant trunk” repair of the ascending aorta, entire aortic arch, and proximal descending thoracic aorta.196 In the United States, such devices are not currently available, so this repair is commonly done by concomitantly deploying a

1	aorta, entire aortic arch, and proximal descending thoracic aorta.196 In the United States, such devices are not currently available, so this repair is commonly done by concomitantly deploying a commercially available stent graft in an antegrade fashion after fully replacing the ascending aorta and aortic arch. In some variations of this off-label approach, the stent graft is directly sutured to the distal aspect of the proximal open repair, whereas in others, there may be a gap of native tissue between the open and endovascular repair. Although this technique appears to be extensively used outside the United States, and with early and mid-term success,194,197-199 only a few U.S. reports describe its use.200-203 Emerging reports describe an enhanced risk of spinal cord ischemia, a risk that is not usually associated with open arch repair. This is prob-ably due to the extensive coverage of the intercostal vessels by the stent graft. Uncertainties in the frozen elephant trunk procedure

1	is not usually associated with open arch repair. This is prob-ably due to the extensive coverage of the intercostal vessels by the stent graft. Uncertainties in the frozen elephant trunk procedure need to be addressed before it becomes a standard Brunicardi_Ch22_p0853-p0896.indd 88201/03/19 5:42 PM 883THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22Figure 22-23. Illustration of proximal aortic repair for acute ascending aortic dissection. A. This repair requires a median sternotomy and cardiopulmonary bypass. The ascending aorta is opened during hypothermic circulatory arrest, while antegrade cerebral perfusion is delivered via an axillary artery graft (shown) or via an innominate artery graft, provided that the innominate artery is not dissected (see Fig. 22-10). B. The dissecting membrane is removed to expose the true lumen. C. An open distal anastomosis prevents clamp injury of the friable arch tis-sue and allows inspection of the arch lumen. A balloon perfusion catheter in

1	membrane is removed to expose the true lumen. C. An open distal anastomosis prevents clamp injury of the friable arch tis-sue and allows inspection of the arch lumen. A balloon perfusion catheter in the left common carotid artery ensures bilateral antegrade cerebral perfusion. If the origin of the dissection (i.e., intimal tear or disruption) does not extensively involve the greater curvature of the aortic arch, and if there is no evidence of a preexisting arch aneurysm, a beveled, hemiarch repair is carried out, preserving most of the greater curvature of the arch. The aorta is transected, beginning at the greater curvature immediately proximal to the origin of the innominate artery and extending distally toward the lesser curvature to the level of the left subclavian artery. Consequently, most of the transverse aortic arch, except for the dorsal segment containing the brachiocephalic arteries, is removed. An appropriately sized, sealed (with collagen or gelatin) polyester tube graft

1	most of the transverse aortic arch, except for the dorsal segment containing the brachiocephalic arteries, is removed. An appropriately sized, sealed (with collagen or gelatin) polyester tube graft is selected, and the beveled distal anastomosis is made with continuous 3-0 or 4-0 monofilament suture; the potential space between the true and false lumen can be obliterated with a small amount of surgical adhesive or by using a strip of Teflon felt. To improve hemostasis, the distal anastomosis can be reinforced by placing interrupted mattress sutures with felt pledgets. D. After cardiopulmonary bypass is resumed and a cross-clamp is applied to the hemiarch replacement graft, the aortic valve is assessed. Disrupted commissures are resuspended with pledgeted mattress sutures to restore valvular competence. E. The aorta is generally transected at the sinotubular junction, and a very small amount of surgical adhesive can be applied between the true and false lumens, or more commonly, the

1	competence. E. The aorta is generally transected at the sinotubular junction, and a very small amount of surgical adhesive can be applied between the true and false lumens, or more commonly, the false lumen within the proximal aortic stump is obliterated by inserting a semicircle of felt within the false lumen of the noncoronary sinus. The trimmed edges are brought together by using 6-0 polypropylene sutures. F. The proximal anastomosis is carried out at the sinotubular junction, incorporating the distal margin of the commissures. G. In patients with residual distal aortic dissection (such as in DeBakey type I aortic dissection), hemiarch repair can be extended with antegrade stent delivery to the descending thoracic aorta. (Used with permission of Baylor College of Medicine.)ACFGDEBBrunicardi_Ch22_p0853-p0896.indd 88301/03/19 5:42 PM 884SPECIFIC CONSIDERATIONSPART IIrecommendation for this subset of patients.204 Another alter-native employs separate grafts: a standard polyester

1	88301/03/19 5:42 PM 884SPECIFIC CONSIDERATIONSPART IIrecommendation for this subset of patients.204 Another alter-native employs separate grafts: a standard polyester graft to replace the ascending aorta and proximal hemiarch, and a stent graft delivered antegrade into the descending thoracic aorta (Fig. 22-23G). Although this procedure differs from a formal “frozen elephant trunk” repair in that it does not replace the entire arch, it is meant to achieve the same goal: promoting remodeling of the dissected descending aortic segment.Chronic Dissection Occasionally, patients with ascending aor-tic dissection present for repair in the chronic phase. In most respects, the operation is similar to that for acute dissection repair. One notable difference is that the tissue is stronger in chronic dissection than in acute dissection, which makes sutur-ing safer. In addition, the false lumen is not obliterated at the distal anastomosis; instead, the dissecting membrane is fenes-trated into

1	dissection than in acute dissection, which makes sutur-ing safer. In addition, the false lumen is not obliterated at the distal anastomosis; instead, the dissecting membrane is fenes-trated into the arch to assure perfusion of both lumens and to prevent postoperative malperfusion complications. Unlike operations for acute dissection, operations for chronic dissec-tion are often aggressive repairs that extend into the arch and root because the tissues are much less fragile.Treatment of Descending Aortic Dissection Nonoperative Management Nonoperative, pharmacologic management of acute descending aortic dissection results in lower morbidity and mortality rates than traditional open surgi-cal treatment does.174 The most common causes of death during nonoperative treatment are aortic rupture and end-organ malp-erfusion. Therefore, patients are continually reassessed for new complications. Serial CT scans are generally obtained during the index hospitalization—usually on day 2 or 3 and on

1	and end-organ malp-erfusion. Therefore, patients are continually reassessed for new complications. Serial CT scans are generally obtained during the index hospitalization—usually on day 2 or 3 and on day 8 or 9 of treatment—and compared with the initial scan to rule out significant aortic expansion.Once the patient’s condition has been stabilized, phar-macologic management is gradually shifted from IV to oral medications. Oral therapy, usually including a b-antagonist, is initiated when systolic pressure is consistently between 100 and 110 mmHg and the neurologic, renal, and cardiovascular systems are stable. Many patients can be discharged after their blood pressure is well controlled with oral agents and after serial CT scans confirm the absence of aortic expansion.Long-term pharmacologic therapy is important for patients with chronic aortic dissection. b-Blockers remain the drugs of choice.205 In a 20-year follow-up study, DeBakey and col-leagues206 found that inadequate blood

1	therapy is important for patients with chronic aortic dissection. b-Blockers remain the drugs of choice.205 In a 20-year follow-up study, DeBakey and col-leagues206 found that inadequate blood pressure control was associated with late aneurysm formation. Aneurysms developed in only 17% of patients with “good” blood pressure control, compared with 45% of patients with “poor” control.Aggressive imaging follow-up is recommended for all patients with chronic aortic dissection.207 Both contrast-enhanced CT and MRA scans provide excellent aortic imag-ing and facilitate serial comparisons to detect progressive aortic expansion. The first surveillance scan is obtained approximately 6 weeks after the onset of dissection. Subsequent scans are obtained at 3 to 6 months and then at 1 year after onset. If the aorta appears to be stable, imaging is obtained annually there-after. Scans are obtained more frequently in high-risk patients, such as those with Marfan or Loeys-Dietz syndrome, and in those

1	If the aorta appears to be stable, imaging is obtained annually there-after. Scans are obtained more frequently in high-risk patients, such as those with Marfan or Loeys-Dietz syndrome, and in those in whom significant aortic expansion is detected. For patients who have undergone graft repair of descending aor-tic dissection, annual CT or MRA scans are also obtained to detect false aneurysm formation or dilatation of unrepaired seg-ments of aorta. Early detection of worrisome changes allows timely, elective intervention before rupture or other complica-tions develop; rupture of the distal aorta is relatively common in patients with chronic aortic dissection and often results in death.192Indications for Open Surgery In the acute phase of descend-ing aortic dissection, open surgery has been traditionally reserved for patients who experience complications.208 Compli-cated acute distal aortic dissections are those with aortic rupture, increasing periaortic or pleural fluid volume, rapidly

1	traditionally reserved for patients who experience complications.208 Compli-cated acute distal aortic dissections are those with aortic rupture, increasing periaortic or pleural fluid volume, rapidly expanding aortic diameter, uncontrolled hypertension, and persistent pain despite adequate medical therapy and malperfusion. In general terms, emergency open operations were originally intended to prevent or repair rupture and relieve life-threatening ischemic manifestations. However, open operation is associated with high morbidity in such cases; now that stent graft technology is available, endovascular surgical intervention is recommended for patients with complicated acute distal aortic dissection.Acute dissection superimposed on a preexisting aneu-rysm is considered a life-threatening condition and is therefore another indication for operation. Finally, patients who have a history of noncompliance with medical therapy may ultimately benefit more from surgical intervention if they are

1	and is therefore another indication for operation. Finally, patients who have a history of noncompliance with medical therapy may ultimately benefit more from surgical intervention if they are otherwise reasonable operative candidates.In the chronic phase, the indications for open surgical inter-vention for aortic dissection are similar to those for degenerative thoracic aortic aneurysm, although a slightly lower threshold of repair is now recommended. Guidelines for thoracic aortic disease44 recommend elective operation in otherwise healthy patients when the affected segment has reached a diameter of 5.5 cm, especially in patients with heritable disorders. Rapid aortic enlargement and other factors that increase the likelihood of aortic rupture may also be considered.Endovascular Treatment Malperfusion Syndrome Endovascular therapy is routinely used in patients with descending aortic dissection complicated by visceral malperfusion.209 Abdominal malperfusion syn-drome often is fatal;

1	Syndrome Endovascular therapy is routinely used in patients with descending aortic dissection complicated by visceral malperfusion.209 Abdominal malperfusion syn-drome often is fatal; prompt identification of visceral ischemia and expedited treatment to restore hepatic, gastrointestinal, and renal perfusion are imperative for a positive outcome. As described in a later section, several open surgical techniques can be used to reestablish blood flow to compromised organs. However, in acute cases, open surgery is associated with poor outcomes. Therefore, endovascular intervention is the pre-ferred initial approach in such cases. In one endovascular tech-nique known as endovascular fenestration, a balloon is used to create a tear in the dissection flap, which allows blood to flow in both the true and false lumens. Although endovascular fen-estration was commonly used in the past, its use has declined in recent years as direct aortic and branch-vessel stenting tech-niques have evolved and

1	true and false lumens. Although endovascular fen-estration was commonly used in the past, its use has declined in recent years as direct aortic and branch-vessel stenting tech-niques have evolved and gained favor. Placing a stent graft in the true lumen of the aorta can resolve a “dynamic” malperfu-sion. Occasionally, a small stent must be placed directly in the lumen of a visceral or renal artery because the dissection has propagated into the branch, resulting in “static” malperfusion at the origin.209Iliofemoral malperfusion causing limb-threatening leg ischemia also can be treated via an endovascular approach. Limb malperfusion usually resolves after the endovascular repair of acute descending thoracic aortic dissection. If the malperfusion does not resolve, then a femoral-to-femoral arterial bypass graft is an effective option.Brunicardi_Ch22_p0853-p0896.indd 88401/03/19 5:42 PM 885THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22Acute Dissection Although surgery has been

1	arterial bypass graft is an effective option.Brunicardi_Ch22_p0853-p0896.indd 88401/03/19 5:42 PM 885THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22Acute Dissection Although surgery has been traditionally recommended for patients with complicated acute descending aortic dissection, many centers have shifted toward using endo-vascular stent grafts as the preferred approach in these cases because of the high morbidity associated with the open opera-tion. Evidence suggests that emergent endovascular repair in patients with true lumen collapse and complications such rupture or dynamic malperfusion may be lifesaving in these difficult-to-treat patients. However, these patients remain at risk of further complication or future reintervention. Although endovascular repair in patients with heritable aortic disorders is generally not recommended, this technique can be used as a bridge to later, definitive repair in such life-threatening circumstances.66Controversy exists regarding the

1	heritable aortic disorders is generally not recommended, this technique can be used as a bridge to later, definitive repair in such life-threatening circumstances.66Controversy exists regarding the use of endovascular stent grafts to treat uncomplicated acute descending dissection; some encouraging data have been published in the last couple of years.210 The goal of this treatment strategy is to use the stent graft to cover the intimal tear, seal the entry site of the dis-section, and eventually cause thrombosis of the false lumen to aid in aortic remodeling and reduce late aortic expansion. Such procedures take place in a hybrid operating room. After the true lumen is accessed through the femoral arteries, an aor-togram is taken, and the intimal tear is identified. Note that the diameter of the true lumen is measured on the preoperative contrast-enhanced CT scan. The use of IVUS is encouraged to help access the true lumen, verify navigation of the wire inside the true lumen, and

1	of the true lumen is measured on the preoperative contrast-enhanced CT scan. The use of IVUS is encouraged to help access the true lumen, verify navigation of the wire inside the true lumen, and confirm measurements. For these cases, a stent graft is selected with a diameter no more than 10% greater than that of the true lumen. Unlike stents deployed to treat most descending thoracic aortic aneurysms, stents deployed to treat descending thoracic aortic dissections must not be ballooned, because ballooning can cause a new intimal tear, retrograde dissection into the ascending aorta, or even aortic rupture. The ideal length of the descending thoracic aorta that should be cov-ered in patients with acute distal descending dissection remains unclear. Close monitoring with serial imaging is necessary after endovascular repair because the false lumen remains at risk for retrograde perfusion or pressurization.Chronic Dissection Endovascular treatment of chronic descend-ing aortic dissection

1	after endovascular repair because the false lumen remains at risk for retrograde perfusion or pressurization.Chronic Dissection Endovascular treatment of chronic descend-ing aortic dissection is supported by the 5-year data of the INSTEAD-XL trial, which showed that endovascular repair com-bined with optimal medical treatment was associated with slower disease progression and greater aorta-specific survival than opti-mal medical treatment alone.211 Importantly, patients in the trial had dissections in the early chronic phase, many within 10 to 12 weeks of onset and all within 1 year of onset. Endovascular repair of chronic dissection is particularly challenging because the rela-tive rigidity of the dissecting membrane—which increases over time during the chronic phase—and the presence of multiple reen-try sites make it difficult to exclude the false lumen.Penetrating Aortic Ulcer Patients with PAUs appear to be good candidates for endovascular intervention. Covering the focal

1	of multiple reen-try sites make it difficult to exclude the false lumen.Penetrating Aortic Ulcer Patients with PAUs appear to be good candidates for endovascular intervention. Covering the focal ulceration with a stent graft has been shown to be an effec-tive treatment.212 In a recent study by Patel and colleagues,213 endovascular repair of PAU was associated with better early outcomes than open repair. However, when PAU was associ-ated with adjacent hematoma within the aortic wall, rates of subsequent reintervention were increased.Open Repair Acute Dissection In patients with acute aortic dissection, open surgical repair of the descending thoracic or thoracoabdominal aorta has been traditionally associated with high morbidity and mortality.174 Therefore, surgery was generally only performed to prevent fatal rupture or to restore branch-vessel perfusion in patients with complicated dissection.208 With the evolution of endovascular technology, open repair has fallen out of favor in

1	to prevent fatal rupture or to restore branch-vessel perfusion in patients with complicated dissection.208 With the evolution of endovascular technology, open repair has fallen out of favor in recent years.Malperfusion Syndrome In patients with malperfusion, when an endovascular approach is unavailable or unsuccessful, open surgery is necessary. Lower-extremity ischemia can be readily addressed with surgical extra-anatomic revascularization techniques, such as femoral-to-femoral bypass grafting. In patients with abdominal organ ischemia, flow to the compromised bed must be reestab-lished swiftly. Although they are considered second-line therapies, multiple techniques are available, including graft replacement of the aorta (with flow redirected into the true lumen), open aortic fenestration, and visceral or renal artery bypass.Chronic Dissection A more aggressive replacement usually is performed during elective aortic repairs in patients with chronic dissection. In many regards, the

1	and visceral or renal artery bypass.Chronic Dissection A more aggressive replacement usually is performed during elective aortic repairs in patients with chronic dissection. In many regards, the operative approach used in these patients is identical to that used for descending thoracic and tho-racoabdominal aortic aneurysms, as described in the first half of this chapter (Fig. 22-24). One key difference is the need to excise as much dissecting membrane as possible to clearly identify the true and false lumens and to locate all important branch vessels. When the dissection extends into the visceral or renal arteries, the membrane can be fenestrated, or the false lumen can be obliter-ated with sutures or intraluminal stents. Asymmetric expansion of the false lumen can create wide separation of the renal arteries. This problem is addressed by reattaching the mobilized left renal artery to a separate opening in the graft or by performing a left renal artery bypass with a side graft.

1	of the renal arteries. This problem is addressed by reattaching the mobilized left renal artery to a separate opening in the graft or by performing a left renal artery bypass with a side graft. Wedges of dissecting mem-brane also are excised from the aorta adjacent to the proximal and distal anastomoses, which allows blood to flow through both true and false lumens. When placing the proximal clamp is not technically feasible, hypothermic circulatory arrest can be used to facilitate the proximal portion of the repair.OUTCOMESImprovements in anesthesia, surgical techniques, and periopera-tive care have led to substantial improvements in outcome after thoracic aortic aneurysm repair. When performed in specialized centers, these operations are associated with excellent survival rates and acceptable morbidity rates. The interpretation of out-comes data is complicated by site-specific variables, such as the number of years reported and whether data are taken from single-practice centers or

1	morbidity rates. The interpretation of out-comes data is complicated by site-specific variables, such as the number of years reported and whether data are taken from single-practice centers or from pooled, multicenter, or national registries, and by patient-specific variables, such as type of enrollment, urgency and extent of repair, concomitant proce-dures performed, and the presence of preexisting risk factors such as advanced age, previous cardiovascular repair, disease of any system or organ, or heritable conditions.Repair of Proximal Aortic AneurysmsRisks associated with the open repair of the proximal aorta vary by extent of repair and are greatest for repairs involving total arch replacement.71,214 All varieties of aortic root replacement have shown acceptable early mortality rates and few complica-tions. Two groups with 20 and 27 years’ experience with com-posite valve graft replacement reported early mortality rates of 5.6% and 1.9%, respectively; the more recent repairs had

1	and few complica-tions. Two groups with 20 and 27 years’ experience with com-posite valve graft replacement reported early mortality rates of 5.6% and 1.9%, respectively; the more recent repairs had better outcomes.215,216 Early mortality rates for stentless porcine Brunicardi_Ch22_p0853-p0896.indd 88501/03/19 5:42 PM 886SPECIFIC CONSIDERATIONSPART IIFigure 22-24. Illustration of distal aortic repair of a chronic dissection. A. Thoracoabdominal incision. B. Extent II thoracoabdominal aortic aneurysm resulting from chronic aortic dissection. The patient has previously undergone composite valve graft replacement of the aortic root and ascending aorta. After left heart bypass is initiated, the proximal portion of the aneurysm is isolated by placing clamps on the left sub-clavian artery, between the left common carotid and left subclavian arteries, and across the middle descending thoracic aorta. C. The isolated segment of aorta is opened by using electrocautery. D. The dissecting

1	between the left common carotid and left subclavian arteries, and across the middle descending thoracic aorta. C. The isolated segment of aorta is opened by using electrocautery. D. The dissecting membrane is excised, and bleeding intercostal arteries are oversewn. The aorta is prepared for proximal anastomosis by transecting it distal to the proximal clamp and separating this portion from the esophagus (not shown). E. The proximal anastomosis between the aorta and an appropriately sized polyester graft is completed with continuous polypro-pylene suture. F. After left heart bypass has been stopped and the distal aortic cannula has been removed, the proximal clamp is repositioned onto the graft, the other two clamps are removed, and the remainder of the aneurysm is opened. G. The rest of the dissecting membrane is excised, and the openings to the celiac, superior mesenteric, and renal arteries are identified. H. Selective visceral perfusion with oxygenated blood from the bypass circuit

1	dissecting membrane is excised, and the openings to the celiac, superior mesenteric, and renal arteries are identified. H. Selective visceral perfusion with oxygenated blood from the bypass circuit is delivered through balloon perfusion catheters placed in the celiac and superior mesenteric arterial ostia. Cold crystalloid is delivered to the renal arteries. The critical intercostal arteries are reattached to an opening cut in the graft. I. To minimize spinal cord ischemia, the proximal clamp is repositioned distal to the intercostal reattachment site. A second oval opening is fashioned in the graft adjacent to the visceral vessels. Selective perfusion of the visceral arteries continues during their reattachment to the graft. A separate anas-tomosis is often required to reattach the left renal artery. J. After the balloon perfusion catheters are removed and the visceral anastomosis is completed, the clamp is again moved distally, restoring blood flow to the celiac, renal, and superior

1	artery. J. After the balloon perfusion catheters are removed and the visceral anastomosis is completed, the clamp is again moved distally, restoring blood flow to the celiac, renal, and superior mesenteric arteries. The final anastomosis is created between the graft and the distal aorta. (Reproduced with permission from Creager MA, Dzau VS, Loscalzo J: Vascular Medicine, 7th ed. Philadelphia, PA: Elsevier/Saunders; 2006.)Left heartbypasscircuitCold renalperfusionsystemFalselumenABCDEtissue root replacements are also low, ranging from 3.6% to 6.0%.217-221 Early mortality rates for contemporary valve-sparing approaches to aortic root replacement are quite low (1%–2%) in experienced centers.77,78,80,222,223 Late survival rates after valve-sparing root procedures range from 97% to 99% at 5 years80,222,223 and approach 94% at 10 years.80Repairs incorporating the ascending aorta and aortic arch have acceptable outcomes; risk increases with patient-specific factors such as severe

1	99% at 5 years80,222,223 and approach 94% at 10 years.80Repairs incorporating the ascending aorta and aortic arch have acceptable outcomes; risk increases with patient-specific factors such as severe atherosclerosis224 or as larger sections of the aortic arch are incorporated into the repair.225,226 A revised surgical strategy—such as the use of hypothermic circulatory arrest—is often needed to avoid clamping atherosclerotic sections in the “porcelain” aorta. In Zingone and colleagues’ series224 of 64 patients who underwent replacement of atherosclerotic ascend-ing aorta, hypothermic circulatory arrest was used in 61 patients (95%). Even though these patients had substantial comorbidity and 83% underwent concomitant cardiac repairs, acceptable rates of early mortality (11%) and stroke (6%) were obtained. Other Brunicardi_Ch22_p0853-p0896.indd 88601/03/19 5:42 PM 887THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22Figure 22-24. (Continued)studies indicate that the enhanced risk of

1	were obtained. Other Brunicardi_Ch22_p0853-p0896.indd 88601/03/19 5:42 PM 887THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22Figure 22-24. (Continued)studies indicate that the enhanced risk of neurocognitive distur-bances in ascending repairs using circulatory arrest are not offset by lower rates of early mortality.227,228 Regarding extended proxi-mal repair, reported early mortality rates after traditional stage 1 elephant trunk repairs (primarily using island reattachment strate-gies) range from 2.3 to 13.9%.229-233Contemporary mortality rates for extensive proximal aortic repair have improved as new strategies and modified adjuncts have been adopted. For example, by adopting contem-porary approaches, we have reduced early mortality for stage 1 elephant trunk repairs from 12% to 2% in our patients.86,230 Similarly, in a report by Kazui and colleagues234 covering 20 years of experience and 472 consecutive patients who under-went aortic arch repair with selective ACP, operative

1	in our patients.86,230 Similarly, in a report by Kazui and colleagues234 covering 20 years of experience and 472 consecutive patients who under-went aortic arch repair with selective ACP, operative mortality was 16.0% for early repairs and 4.1% for more recent repairs. Other contemporary reports of the use of techniques such as moderate hypothermia and Y-graft approaches235-238 indicate similarly improved outcomes; early mortality ranges from 1% to 7%, stroke rates range from 1% to 6%, and no cases of para-plegia are reported. Although paraplegia has traditionally been an unusual and infrequent complication of aortic arch repair, it has been reported as a complication of “long” elephant trunk approaches239 and frozen elephant trunk approaches.240Because of the heterogeneity of hybrid arch approaches and the tendency to use these approaches in high-risk patients, results of hybrid arch repair are difficult to interpret. In a recent report from our group,65 among 319 consecutive

1	arch approaches and the tendency to use these approaches in high-risk patients, results of hybrid arch repair are difficult to interpret. In a recent report from our group,65 among 319 consecutive patients who underwent total arch replacement in the last 8.5 years, 274 patients had traditional open repair and 45 patients had hybrid zone 0 exclusion repairs. The rate of permanent adverse outcome (death, persistent neurologic deficit at dis-charge, or persistent hemodialysis at discharge) was not sig-nificantly different between the two groups. A higher overall stroke rate was noticed in the hybrid group, reinforcing the importance of catheter skills and careful wire manipulation. A meta-analysis conducted by Koullias and Wheatley241 of data from 15 studies with 463 patients found an average 30-day mortality rate of 8.3%; stroke, 4.4%; paraplegia, 3.9%; and endoleak, 9.2%. Of note, relatively few repairs (30%) were performed “off-pump,” and the majority of repairs used cardiopulmonary

1	30-day mortality rate of 8.3%; stroke, 4.4%; paraplegia, 3.9%; and endoleak, 9.2%. Of note, relatively few repairs (30%) were performed “off-pump,” and the majority of repairs used cardiopulmonary bypass or hypothermic circulatory arrest. Additionally, several reports of small series have documented increased risk of acute retrograde aortic dissection during hybrid arch repairs; rates range from 0% to 7.5%, and these patients face significant mortality risk (ranging from 33% to 100%) should this occur.108,109,242-244Brunicardi_Ch22_p0853-p0896.indd 88701/03/19 5:42 PM 888SPECIFIC CONSIDERATIONSPART IITreatment of Acute Ascending Aortic DissectionThe International Registry of Acute Aortic Dissection (IRAD) provides the most comprehensive data on contemporary out-comes in patients with acute aortic dissection. This registry was established in 1996 and has accumulated data from >7000 patients treated for acute aortic dissection at 51 centers in 12 countries. An IRAD analysis of data

1	acute aortic dissection. This registry was established in 1996 and has accumulated data from >7000 patients treated for acute aortic dissection at 51 centers in 12 countries. An IRAD analysis of data from 776 patients who underwent surgical repair of acute ascending aortic dissection revealed an in-hospital mortality rate of 23.8%.245 The investiga-tors identified several preoperative predictors of early mortality, including age >70 years, previous cardiac surgery, hypotension or shock at presentation, abrupt onset of symptoms, migrat-ing pain, cardiac tamponade, preoperative renal failure, pulse deficit, and evidence of myocardial ischemia or infarction on ECG.245,246 In a report from IRAD, in-hospital mortality after surgical treatment had decreased from 25% in 1995 to 18% in 2013.247 The German Registry for Acute Aortic Dissection (GERAADA) has collected data on more than 3300 patients from 56 centers since 2006. 248 In a report of 1436 patients with acute proximal dissection that

1	German Registry for Acute Aortic Dissection (GERAADA) has collected data on more than 3300 patients from 56 centers since 2006. 248 In a report of 1436 patients with acute proximal dissection that was surgically repaired using hypothermic circulatory arrest with or without unilateral and bilateral ACP, the early mortality rates ranged from 13.9% to 19.4%; the 628 patients with unilateral ACP had the lowest rate of early death.249 Operative mortality reported by North American centers varies from 5% to 17%; improvements in out-comes may be related to the implementation of protocol-based management and the assembly of thoracic aortic teams.203Repair of Distal Aortic AneurysmsEndovascular Repair of Descending Thoracic Aortic Aneurysms. In the earliest series of endovascular repairs of descending thoracic aortic aneurysms, mortality and morbidity were difficult to assess. Most of the reported series were small and included a large proportion of high-risk patients with sub-stantial

1	descending thoracic aortic aneurysms, mortality and morbidity were difficult to assess. Most of the reported series were small and included a large proportion of high-risk patients with sub-stantial comorbidity.250,251 Subsequent evidence from pivotal, nonrandomized trials that compared patients who underwent endograft exclusion with historical or concurrent patients who underwent open repair252-254 showed that the stent graft groups had significantly less morbidity and early mortality than the open repair groups, although in two of the trials, a nonsig-nificant between-group difference was observed in the rate of stroke.252,254 Five-year comparative data show that the two groups differed significantly in their aneurysm-related mortality rates (2.8% for endovascular patients and 11.7% for open repair patients) but not in their rates of all-cause mortality (which were 32% and 31%, respectively).255 Additional pivotal trial 5-year outcomes256 indicate the growing disparity between

1	for open repair patients) but not in their rates of all-cause mortality (which were 32% and 31%, respectively).255 Additional pivotal trial 5-year outcomes256 indicate the growing disparity between aneurysm-related (96.1%) and all-cause survival (58.5%) in patients with endovascular repair, leading some to comment on the possible futility of repair in many patients.257 Among 8967 patients iden-tified in the National Inpatient Sample database (8255 with open repair and 712 with endovascular repair), the odds of death were 46% lower among patients who underwent endovascular repair rather than open repair.56 The endovascular repair group also had lower odds of postoperative neurologic, cardiac, and respi-ratory complications.Open Repair of Descending Thoracic and Thoracoabdominal Aortic Aneurysms. Contemporary results of open repairs of descending thoracic aortic aneurysms, including those performed in select patients with chronic dissection, indicate that early mor-tality rates range

1	results of open repairs of descending thoracic aortic aneurysms, including those performed in select patients with chronic dissection, indicate that early mor-tality rates range from 4.1% to 8.0%, renal failure rates range from to 4.2% to 7.5%, and paraplegia rates range from 2.3% to 5.7%; stroke rates are generally lower, ranging from 1.8% to 2.1%.258-260 In our series, although the risk of paraplegia increased with the extent of repair, the risk of mortality was greatest for those under-going repair of the proximal two thirds of the descending aorta.258 As expected, stroke rates after distal aortic repairs were highest when the clamp site was near the left subclavian artery.Contemporary series of open thoracoabdominal aortic repairs show acceptable survival. Reported outcome rates range from 5% to 12% for early mortality, 3.8% to 9.5% for paraple-gia, 1.7% to 5.2% for stroke, and 6% to 12% for renal compli-cations.261-265 Many of these series summarize 10 to 20 years of surgical

1	from 5% to 12% for early mortality, 3.8% to 9.5% for paraple-gia, 1.7% to 5.2% for stroke, and 6% to 12% for renal compli-cations.261-265 Many of these series summarize 10 to 20 years of surgical experience,262-265 although some present a shorter but more contemporary experience.261 Even for complex tho-racoabdominal aortic repairs, such as stage 2 elephant trunk repairs, several centers report acceptable early mortality rates ranging from 0% to 10%.229-233 Worse outcomes are also docu-mented, as in a statewide, nonfederal analysis of data from 1010 patients whose early mortality rate was 25%. Of note, 40% of these patients were treated at centers averaging only one tho-racoabdominal aortic aneurysm repair per year.266 Cowan and colleagues,267 who examined the influence of familiarity with the procedure on rates of mortality and morbidity after tho-racoabdominal aortic aneurysm repair, reported that patients treated at low-volume centers fared less well. Replacing the entire

1	with the procedure on rates of mortality and morbidity after tho-racoabdominal aortic aneurysm repair, reported that patients treated at low-volume centers fared less well. Replacing the entire thoracoabdominal aorta (i.e., performing an extent II repair) carries the highest risk of death, bleeding, renal failure, and paraplegia.118,262,263 Early survival has been estimated at 79% at 2 years,268 and mid-term survival has been estimated at 63% at 5 years.265 In our recent report of 3309 repairs,269 the overall mortality rate was 7.5%, and the rate of operative death was higher in extent II and III repairs than in extent I and IV. Perma-nent paraplegia and paraparesis occurred in 2.9% and 2.4% of patients, respectively, and the incidence of paraplegia in patients 50 years of age or younger was only 1.1%. Estimated survival after repair was 84% ± 1% at 1 year, 64% ± 1% at 5 years, 37% ± 1% at 10 years, and 18% ± 1% at 15 years.Treatment of Descending Thoracic Aortic

1	of age or younger was only 1.1%. Estimated survival after repair was 84% ± 1% at 1 year, 64% ± 1% at 5 years, 37% ± 1% at 10 years, and 18% ± 1% at 15 years.Treatment of Descending Thoracic Aortic DissectionNonoperative Management. The in-hospital mortality rate is 8.7% for patients with acute descending aortic dissection who receive nonoperative treatment247; however, when IRAD stratified patients according to clinical presentation, the mor-tality rate for patients with uncomplicated dissection was less than 4%, whereas the mortality rate for patients with compli-cated dissection was more than 20%.174,270 The primary causes of death during nonoperative management are rupture, malper-fusion, and cardiac failure. Risk factors associated with treat-ment failure—defined as death or need for surgery—include an enlarged aorta, persistent hypertension despite maximal treatment, oliguria, and peripheral ischemia. Among patients who receive nonoperative treatment for descending aortic

1	for surgery—include an enlarged aorta, persistent hypertension despite maximal treatment, oliguria, and peripheral ischemia. Among patients who receive nonoperative treatment for descending aortic dis-section and who survive the acute period, approximately 90% remain alive 1 year later, and approximately 76% are alive 3 years later.271Endovascular Treatment. For patients with complicated acute descending thoracic aortic dissection, including rupture and malperfusion of the visceral or renal arteries, an endovas-cular approach is ideal. The Stanford group reported a 93% technical success rate for endovascular reperfusion of an isch-emic bed.272 Their experience with the use of first-generation stent grafts to treat acute complicated descending dissections Brunicardi_Ch22_p0853-p0896.indd 88801/03/19 5:42 PM 889THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22was also encouraging: Complete thrombosis of the false lumen occurred in 79% of patients. The early mortality rate was 16%,

1	88801/03/19 5:42 PM 889THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22was also encouraging: Complete thrombosis of the false lumen occurred in 79% of patients. The early mortality rate was 16%, comparable to that associated with open techniques.273 A meta-analysis of observational studies of endovascular repair, which included 248 patients with acute descending thoracic aortic dissection, found a 30-day mortality rate of 9.8%.274 When compared with early mortality rates obtained from IRAD data,174 this rate is substantially lower than the rate associated with open surgical treatment and is similar to the rate achieved with nonoperative management. However, patients with com-plicated acute descending dissection remain susceptible to late events; at 1 year, survival is approximately 70%, and reinterven-tion is needed in about 10% of survivors.275The ADSORB trial276 focused on patients with uncom-plicated acute descending thoracic aortic dissection. Patients were randomly assigned

1	and reinterven-tion is needed in about 10% of survivors.275The ADSORB trial276 focused on patients with uncom-plicated acute descending thoracic aortic dissection. Patients were randomly assigned to optimal medical therapy alone (n = 31) or endovascular repair plus optimal medical therapy (n = 30).277 The 1-year results showed aortic remodeling with false lumen thrombosis and reduced diameter in the group treated with endovascular repair.The INSTEAD-XL trial involved 140 patients with stable, early-chronic descending thoracic aortic dissection who were randomly assigned to either endovascular repair plus opti-mal medical treatment or optimal medical therapy alone.211 The eagerly anticipated 5-year data showed that endovascular repair was associated with greater survival and slower disease progression.CONCLUSIONSAortic aneurysm may present as localized or extensive dis-ease. The availability and development of adjuncts and endo-vascular techniques have supported the constant

1	disease progression.CONCLUSIONSAortic aneurysm may present as localized or extensive dis-ease. The availability and development of adjuncts and endo-vascular techniques have supported the constant evolution of surgical strategies to tackle these complex problems. Repair strategies range from isolated, totally endovascular aortic repair for descending thoracic aneurysms to extensive total aortic and staged replacements with a combination of both open and endovascular techniques. Regardless of the difficulty of accu-rately assessing the risks associated with aortic repair, surgical repair of the thoracoabdominal aorta clearly remains the most challenging aortic repair in terms of mortality and morbidity. Accordingly, replacing the entire thoracoabdominal aorta (i.e., performing an extent II repair) carries the highest risk of death, renal failure, and paraplegia.69,70,261,263,269ACKNOWLEDGMENTSThe authors wish to thank Susan Y. Green, MPH, and Stephen N. Palmer, PhD, ELS, for editorial

1	repair) carries the highest risk of death, renal failure, and paraplegia.69,70,261,263,269ACKNOWLEDGMENTSThe authors wish to thank Susan Y. Green, MPH, and Stephen N. Palmer, PhD, ELS, for editorial assistance; Scott A. Weldon, MA, CMI, and Carol P. Larson, CMI, for creating the illus-trations; and Kapil Sharma, MD, and Raja Gopaldas, MD, for their substantial contributions to the chapters published in the 9th and 10th editions of this book, on which this updated chapter is based.REFERENCESEntries highlighted in bright blue are key references. 1. Johnston KW, Rutherford RB, Tilson MD, et al. Suggested standards for reporting on arterial aneurysms. Subcommit-tee on Reporting Standards for Arterial Aneurysms, Ad Hoc Committee on Reporting Standards, Society for Vascular Surgery and North American Chapter, International Society for Cardiovascular Surgery. J Vasc Surg. 1991;13(3):452-458. 2. Bickerstaff LK, Pairolero PC, Hollier LH, et al. Thoracic aortic aneurysms: a population-based

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1	PM 895THORACIC ANEURYSMS AND AORTIC DISSECTIONCHAPTER 22with central aortic fenestration and renal artery stenting. J Vasc Surg. 2008;47(5):903-910. 210. Hughes GC. Management of acute type B aortic dissec-tion; ADSORB trial. J Thorac Cardiovasc Surg. 2015;149 (2 suppl):S158-S162. 211. Nienaber CA, Kische S, Rousseau H, et al. Endovascular repair of type B aortic dissection: long-term results of the randomized investigation of stent grafts in aortic dissec-tion trial. Circ Cardiovasc Interv. 2013;6(4):407-416. The eagerly anticipated report of a randomized trial comparing endovascular repair plus optimal medical treatment with opti-mal medical treatment alone for the treatment of early-chronic descending thoracic aortic dissection. 212. Demers P, Miller DC, Mitchell RS, et al. Stent-graft repair of penetrating atherosclerotic ulcers in the descending tho-racic aorta: mid-term results. Ann Thorac Surg. 2004;77(1): 81-86. 213. Patel HJ, Sood V, Williams DM, et al. Late outcomes with

1	repair of penetrating atherosclerotic ulcers in the descending tho-racic aorta: mid-term results. Ann Thorac Surg. 2004;77(1): 81-86. 213. Patel HJ, Sood V, Williams DM, et al. Late outcomes with repair of penetrating thoracic aortic ulcers: the merits of an endovascular approach. Ann Thorac Surg. 2012;94(2): 516-522. 214. Preventza O, Coselli JS, Garcia A, et al. Aortic root surgery with circulatory arrest: predictors of prolonged postoperative hospital stay. J Thorac Cardiovasc Surg. 2017;153(3):511-518. 215. Aomi S, Nakajima M, Nonoyama M, et al. Aortic root replace-ment using composite valve graft in patients with aortic valve disease and aneurysm of the ascending aorta: twenty years’ experience of late results. Artif Organs. 2002;26(5):467-473. 216. Kindo M, Billaud P, Gerelli S, et al. Twenty-seven-year experi-ence with composite valve graft replacement of the aortic root. J Heart Valve Dis. 2007;16(4):370-377. 217. David TE, Mohr FW, Bavaria JE, et al. Initial experience with

1	al. Twenty-seven-year experi-ence with composite valve graft replacement of the aortic root. J Heart Valve Dis. 2007;16(4):370-377. 217. David TE, Mohr FW, Bavaria JE, et al. Initial experience with the Toronto Root bioprosthesis. J Heart Valve Dis. 2004;13(2):248-251. 218. Gleason TG, David TE, Coselli JS, Hammon JW, Jr, Bavaria JE. St. Jude Medical Toronto biologic aortic root prosthe-sis: early FDA phase II IDE study results. Ann Thorac Surg. 2004;78(3):786-793. 219. Kincaid EH, Cordell AR, Hammon JW, Adair SM, Kon ND. Coronary insufficiency after stentless aortic root replacement: risk factors and solutions. Ann Thorac Surg. 2007;83(3):964-968. 220. Kon ND, Cordell AR, Adair SM, Dobbins JE, Kitzman DW. Aortic root replacement with the freestyle stent-less porcine aortic root bioprosthesis. Ann Thorac Surg. 1999;67(6):1609-1615. 221. Melina G, De Robertis F, Gaer JA, et al. Mid-term pattern of survival, hemodynamic performance and rate of complica-tions after Medtronic Freestyle

1	Ann Thorac Surg. 1999;67(6):1609-1615. 221. Melina G, De Robertis F, Gaer JA, et al. Mid-term pattern of survival, hemodynamic performance and rate of complica-tions after Medtronic Freestyle versus homograft full aortic root replacement: results from a prospective randomized trial. J Heart Valve Dis. 2004;13(6):972-975. 222. Badiu CC, Eichinger W, Bleiziffer S, et al. Should root replacement with aortic valve-sparing be offered to patients with bicuspid valves or severe aortic regurgitation? Eur J Car-diothorac Surg. 2010;38(5):515-522. 223. David TE, Maganti M, Armstrong S. Aortic root aneurysm: principles of repair and long-term follow-up. J Thorac Car-diovasc Surg. 2010;140(6 suppl):S14-S19. 224. Zingone B, Gatti G, Spina A, et al. Current role and outcomes of ascending aortic replacement for severe nonaneurysmal aor-tic atherosclerosis. Ann Thorac Surg. 2010;89(2):429-434. 225. Achneck HE, Rizzo JA, Tranquilli M, Elefteriades JA. Safety of thoracic aortic surgery in the present

1	for severe nonaneurysmal aor-tic atherosclerosis. Ann Thorac Surg. 2010;89(2):429-434. 225. Achneck HE, Rizzo JA, Tranquilli M, Elefteriades JA. Safety of thoracic aortic surgery in the present era. Ann Thorac Surg. 2007;84(4):1180-1185. 226. Estrera AL, Miller CC, III, Madisetty J, et al. Ascending and transverse aortic arch repair: the impact of glomerular filtra-tion rate on mortality. Ann Surg. 2008;247(3):524-529. 227. Fleck TM, Czerny M, Hutschala D, et al. The inci-dence of transient neurologic dysfunction after ascending aortic replacement with circulatory arrest. Ann Thorac Surg. 2003;76(4):1198-1202. 228. Immer FF, Barmettler H, Berdat PA, et al. Effects of deep hypo-thermic circulatory arrest on outcome after resection of ascend-ing aortic aneurysm. Ann Thorac Surg. 2002;74(2):422-425. 229. Heinemann MK, Buehner B, Jurmann MJ, Borst HG. Use of the “elephant trunk technique” in aortic surgery. Ann Thorac Surg. 1995;60(1):2-6. 230. LeMaire SA, Carter SA, Coselli JS. The

1	MK, Buehner B, Jurmann MJ, Borst HG. Use of the “elephant trunk technique” in aortic surgery. Ann Thorac Surg. 1995;60(1):2-6. 230. LeMaire SA, Carter SA, Coselli JS. The elephant trunk tech-nique for staged repair of complex aneurysms of the entire thoracic aorta. Ann Thorac Surg. 2006;81(5):1561-1569. 231. Safi HJ, Miller CC, III, Estrera AL, et al. Staged repair of extensive aortic aneurysms: long-term experience with the elephant trunk technique. Ann Surg. 2004;240(4):677-684. 232. Sundt TM, Moon MR, DeOliviera N, et al. Contempo-rary results of total aortic arch replacement. J Card Surg. 2004;19(3):235-239. 233. Svensson LG, Kim KH, Blackstone EH, et al. Elephant trunk procedure: newer indications and uses. Ann Thorac Surg. 2004;78(1):109-116. 234. Kazui T, Yamashita K, Washiyama N, et al. Aortic arch replacement using selective cerebral perfusion. Ann Thorac Surg. 2007;83(2):S796-798. 235. Bischoff MS, Brenner RM, Scheumann J, et al. Long-term outcome after aortic arch

1	N, et al. Aortic arch replacement using selective cerebral perfusion. Ann Thorac Surg. 2007;83(2):S796-798. 235. Bischoff MS, Brenner RM, Scheumann J, et al. Long-term outcome after aortic arch replacement with a trifurcated graft. J Thorac Cardiovasc Surg. 2010;140(6 suppl):S71-S76. 236. Iba Y, Minatoya K, Matsuda H, et al. Contemporary open aortic arch repair with selective cerebral perfusion in the era of endovascular aortic repair. J Thorac Cardiovasc Surg. 2013;145(3 suppl):S72-S77. 237. Thomas M, Li Z, Cook DJ, Greason KL, Sundt TM. Contem-porary results of open aortic arch surgery. J Thorac Cardio-vasc Surg. 2012;144(4):838-844. 238. Urbanski PP, Lenos A, Bougioukakis P, et al. Mild-tomoderate hypothermia in aortic arch surgery using circula-tory arrest: a change of paradigm? Eur J Cardiothorac Surg. 2012;41(1):185-191. 239. Kondoh H, Taniguchi K, Funatsu T, et al. Total arch replace-ment with long elephant trunk anastomosed at the base of the innominate artery: a single-centre

1	Cardiothorac Surg. 2012;41(1):185-191. 239. Kondoh H, Taniguchi K, Funatsu T, et al. Total arch replace-ment with long elephant trunk anastomosed at the base of the innominate artery: a single-centre longitudinal experience. Eur J Cardiothorac Surg. 2012;42(5):840-848. 240. Flores J, Kunihara T, Shiiya N, et al. Extensive deployment of the stented elephant trunk is associated with an increased risk of spinal cord injury. J Thorac Cardiovasc Surg. 2006;131(2): 336-342. 241. Koullias GJ, Wheatley GH, III. State-of-the-art of hybrid pro-cedures for the aortic arch: a meta-analysis. Ann Thorac Surg. 2010;90(2):689-697. 242. Antoniou GA, Mireskandari M, Bicknell CD, et al. Hybrid repair of the aortic arch in patients with extensive aortic dis-ease. Eur J Vasc Endovasc Surg. 2010;40(6):715-721. 243. Geisbusch P, Kotelis D, Muller-Eschner M, Hyhlik-Durr A, Bockler D. Complications after aortic arch hybrid repair. J Vasc Surg. 2011;53(4):935-941. 244. Czerny M, Weigang E, Sodeck G, et al.

1	P, Kotelis D, Muller-Eschner M, Hyhlik-Durr A, Bockler D. Complications after aortic arch hybrid repair. J Vasc Surg. 2011;53(4):935-941. 244. Czerny M, Weigang E, Sodeck G, et al. Targeting landing zone 0 by total arch rerouting and TEVAR: midterm results of a trans-continental registry. Ann Thorac Surg. 2012;94(1):84-89. 245. Trimarchi S, Eagle KA, Nienaber CA, et al. Role of age in acute type A aortic dissection outcome: report from the Inter-national Registry of Acute Aortic Dissection (IRAD). J Thorac Cardiovasc Surg. 2010;140(4):784-789. 246. Rampoldi V, Trimarchi S, Eagle KA, et al. Simple risk models to predict surgical mortality in acute type A aortic dissection: the International Registry of Acute Aortic Dissection score. Ann Thorac Surg. 2007;83(1):55-61. 247. Pape LA, Awais M, Woznicki EM, et al. Presentation, diag-nosis, and outcomes of acute aortic dissection: 17-year trends From the International Registry of Acute Aortic Dissection. Brunicardi_Ch22_p0853-p0896.indd

1	Woznicki EM, et al. Presentation, diag-nosis, and outcomes of acute aortic dissection: 17-year trends From the International Registry of Acute Aortic Dissection. Brunicardi_Ch22_p0853-p0896.indd 89501/03/19 5:42 PM 896SPECIFIC CONSIDERATIONSPART IIJ Am Coll Cardiol. 2015;66(4):350-358. A comprehensive analysis of outcomes in patients with acute aortic dissection from the largest existing international registry. 248. Boening A, Karck M, Conzelmann LO, et al. German Registry for Acute Aortic Dissection Type A: structure, results, and future perspectives. Thorac Cardiovasc Surg. 2017;65(2):77-84. 249. Kruger T, Weigang E, Hoffmann I, et al. Cerebral protection during surgery for acute aortic dissection type A: results of the German Registry for Acute Aortic Dissection Type A (GERAADA). Circulation. 2011;124(4):434-443. 250. Dake MD, Miller DC, Mitchell RS, et al. The “first genera-tion” of endovascular stent-grafts for patients with aneurysms of the descending thoracic aorta. J

1	Circulation. 2011;124(4):434-443. 250. Dake MD, Miller DC, Mitchell RS, et al. The “first genera-tion” of endovascular stent-grafts for patients with aneurysms of the descending thoracic aorta. J Thorac Cardiovasc Surg. 1998;116(5):689-703. 251. Demers P, Miller DC, Mitchell RS, et al. Midterm results of endovascular repair of descending thoracic aortic aneurysms with first-generation stent grafts. J Thorac Cardiovasc Surg. 2004;127(3):664-673. 252. Bavaria JE, Appoo JJ, Makaroun MS, et al. Endovascular stent grafting versus open surgical repair of descending thoracic aortic aneurysms in low-risk patients: a multicenter compara-tive trial. J Thorac Cardiovasc Surg. 2007;133(2):369-377. 253. Fairman RM, Criado F, Farber M, et al. Pivotal results of the Medtronic Vascular Talent Thoracic Stent Graft System: the VALOR trial. J Vasc Surg. 2008;48(3):546-554. 254. Matsumura JS, Cambria RP, Dake MD, et al. International controlled clinical trial of thoracic endovascular aneurysm repair with

1	Graft System: the VALOR trial. J Vasc Surg. 2008;48(3):546-554. 254. Matsumura JS, Cambria RP, Dake MD, et al. International controlled clinical trial of thoracic endovascular aneurysm repair with the Zenith TX2 endovascular graft: 1-year results. J Vasc Surg. 2008;47(2):247-257. 255. Makaroun MS, Dillavou ED, Wheatley GH, Cambria RP. Five-year results of endovascular treatment with the Gore TAG device compared with open repair of thoracic aortic aneurysms. J Vasc Surg. 2008;47(5):912-918. 256. Foley PJ, Criado FJ, Farber MA, et al. Results with the Talent thoracic stent graft in the VALOR trial. J Vasc Surg. 2012;56(5):1214-1221 e1221. 257. Miller DC. Through the looking glass: The first 20 years of thoracic aortic stent-grafting. J Thorac Cardiovasc Surg. 2013;145(3 suppl):S142-S148. 258. Coselli JS, LeMaire SA, Conklin LD, Adams GJ. Left heart bypass during descending thoracic aortic aneurysm repair does not reduce the incidence of paraplegia. Ann Thorac Surg.

1	suppl):S142-S148. 258. Coselli JS, LeMaire SA, Conklin LD, Adams GJ. Left heart bypass during descending thoracic aortic aneurysm repair does not reduce the incidence of paraplegia. Ann Thorac Surg. 2004;77(4):1298-1303. 259. Chiesa R, Tshomba Y, Civilini E, et al. Open repair of descend-ing thoracic aneurysms. HSR Proc Intensive Care Cardiovasc Anesth. 2010;2(3):177-190. 260. Estrera AL, Miller CC, III, Chen EP, et al. Descending thoracic aortic aneurysm repair: 12-year experience using distal aortic perfusion and cerebrospinal fluid drainage. Ann Thorac Surg. 2005;80(4):1290-1296. 261. LeMaire SA, Price MD, Green SY, Zarda S, Coselli JS. Results of open thoracoabdominal aortic aneurysm repair. Ann Cardiothorac Surg. 2012;1(3):286-292. 262. Chiesa R, Melissano G, Civilini E, et al. Ten years experience of thoracic and thoracoabdominal aortic aneurysm surgical repair: lessons learned. Ann Vasc Surg. 2004;18(5):514-520. 263. Coselli JS, Bozinovski J, LeMaire SA. Open surgical repair of

1	experience of thoracic and thoracoabdominal aortic aneurysm surgical repair: lessons learned. Ann Vasc Surg. 2004;18(5):514-520. 263. Coselli JS, Bozinovski J, LeMaire SA. Open surgical repair of 2286 thoracoabdominal aortic aneurysms. Ann Thorac Surg. 2007;83(2):S862-S864. 264. Conrad MF, Crawford RS, Davison JK, Cambria RP. Thoracoabdominal aneurysm repair: a 20-year perspective. Ann Thorac Surg. 2007;83(2):S856-861. 265. Schepens MA, Kelder JC, Morshuis WJ, et al. Long-term follow-up after thoracoabdominal aortic aneurysm repair. Ann Thorac Surg. 2007;83(2):S851-S855. 266. Rigberg DA, McGory ML, Zingmond DS, et al. Thirty-day mortality statistics underestimate the risk of repair of thora-coabdominal aortic aneurysms: a statewide experience. J Vasc Surg. 2006;43(2):217-222. 267. Cowan JA, Jr, Dimick JB, Henke PK, et al. Surgical treatment of intact thoracoabdominal aortic aneurysms in the United States: hospital and surgeon volume-related outcomes. J Vasc Surg.

1	JA, Jr, Dimick JB, Henke PK, et al. Surgical treatment of intact thoracoabdominal aortic aneurysms in the United States: hospital and surgeon volume-related outcomes. J Vasc Surg. 2003;37(6):1169-1174. 268. Wong DR, Parenti JL, Green SY, et al. Open repair of tho-racoabdominal aortic aneurysm in the modern surgical era: contemporary outcomes in 509 patients. J Am Coll Surg. 2011;212(4):569-579. 269. Coselli JS, LeMaire SA, Preventza O, et al. Outcomes of 3309 thoracoabdominal aortic aneurysm repairs. J Thorac Cardiovasc Surg. 2016;151(5):1323-1337. This report describes the largest experience with open repair of thoracoabdominal aortic aneurysm and focuses on factors associated with adverse outcomes. 270. Trimarchi S, Tolenaar JL, Tsai TT, et al. Influence of clini-cal presentation on the outcome of acute B aortic dissec-tion: evidences from IRAD. J Cardiovasc Surg (Torino). 2012;53(2):161-168. 271. Tsai TT, Fattori R, Trimarchi S, et al. Long-term survival in patients presenting

1	the outcome of acute B aortic dissec-tion: evidences from IRAD. J Cardiovasc Surg (Torino). 2012;53(2):161-168. 271. Tsai TT, Fattori R, Trimarchi S, et al. Long-term survival in patients presenting with type B acute aortic dissection: insights from the International Registry of Acute Aortic Dissection. Circulation. 2006;114(21):2226-2231. 272. Slonim SM, Miller DC, Mitchell RS, et al. Percutaneous balloon fenestration and stenting for life-threatening isch-emic complications in patients with acute aortic dissection. J Thorac Cardiovasc Surg. 1999;117(6):1118-1126. 273. Dake MD, Kato N, Mitchell RS, et al. Endovascular stent-graft placement for the treatment of acute aortic dissection. N Engl J Med. 1999;340(20):1546-1552. 274. Eggebrecht H, Nienaber CA, Neuhauser M, et al. Endovascu-lar stent-graft placement in aortic dissection: a meta-analysis. Eur Heart J. 2006;27(4):489-498. 275. White RA, Miller DC, Criado FJ, et al. Report on the results of thoracic endovascular aortic repair

1	stent-graft placement in aortic dissection: a meta-analysis. Eur Heart J. 2006;27(4):489-498. 275. White RA, Miller DC, Criado FJ, et al. Report on the results of thoracic endovascular aortic repair for acute, complicated, type B aortic dissection at 30 days and 1 year from a multidisciplinary subcommittee of the Soci-ety for Vascular Surgery Outcomes Committee. J Vasc Surg. 2011;53(4):1082-1090. 276. Brunkwall J, Lammer J, Verhoeven E, Taylor P. ADSORB: a study on the efficacy of endovascular grafting in uncom-plicated acute dissection of the descending aorta. Eur J Vasc Endovasc Surg. 2012;44(1):31-36. 277. Brunkwall J, Kasprzak P, Verhoeven E, et al. Endovascular repair of acute uncomplicated aortic type B dissection pro-motes aortic remodelling: 1 year results of the ADSORB trial. Eur J Vasc Endovasc Surg. 2014;48(3):285-291.Brunicardi_Ch22_p0853-p0896.indd 89601/03/19 5:42 PM

1	Arterial DiseasePeter H. Lin, Carlos F. Bechara, Changyi Chen, and Frank J. Veith 23chapterGeneral Approach to the Vascular Patient 898The Vascular History / 898The Vascular Physical Examination / 899Noninvasive Diagnostic Evaluation of the Vascular Patient / 899Radiologic Evaluation of the Vascular Patient / 900Preoperative Cardiac Evaluation / 903Basic Principles of Endovascular Therapy 903Needles and Access / 904Guidewires / 904Hemostatic Sheaths / 905Catheters / 905Angioplasty Balloons / 905Stents / 905Stent Grafts / 906Carotid Artery Disease 907Epidemiology and Etiology of Carotid Occlusive Disease / 907Clinical Manifestations of Cerebral Ischemia / 907Diagnostic Evaluation / 909Treatment of Carotid Occlusive Disease / 910Carotid Endarterectomy Versus Angioplasty and Stenting / 911Surgical Techniques of Carotid Endarterectomy / 912Techniques of Carotid Angioplasty and Stenting / 914Nonatherosclerotic Disease of the Carotid Artery / 916Abdominal Aortic Aneurysm 919Causes

1	/ 911Surgical Techniques of Carotid Endarterectomy / 912Techniques of Carotid Angioplasty and Stenting / 914Nonatherosclerotic Disease of the Carotid Artery / 916Abdominal Aortic Aneurysm 919Causes and Risk Factors / 919Natural History of Aortic Aneurysm / 920Clinical Manifestations / 920Relevant Anatomy / 920Diagnostic Evaluation / 921Surgical Repair of Abdominal Aortic Aneurysm / 921Endovascular Repair of Abdominal Aortic Aneurysm / 922Results From Clinical Studies Comparing Endovascular Versus Open Repair / 925Classification and Management of Endoleak / 927Mesenteric Artery Disease 928Anatomy and Pathophysiology / 928Types of Mesenteric Artery Occlusive Disease / 929Clinical Manifestations / 930Diagnostic Evaluation / 930Surgical Repair / 932Endovascular Treatment / 933Clinical Results of Interventions for Mesenteric Ischemia / 934Renal Artery Disease 935Etiology / 935Clinical Manifestations / 936Diagnostic Evaluation / 936Treatment Indications / 937Surgical Reconstruction /

1	of Interventions for Mesenteric Ischemia / 934Renal Artery Disease 935Etiology / 935Clinical Manifestations / 936Diagnostic Evaluation / 936Treatment Indications / 937Surgical Reconstruction / 938Clinical Results of Surgical Repair / 938Endovascular Treatment / 938Clinical Results of Endovascular Interventions / 939Aortoiliac Occlusive Disease 940Diagnostic Evaluation / 941Differential Diagnosis / 941Collateral Arterial Network / 941Disease Classification / 941General Treatment Considerations / 943Surgical Reconstruction of Aortoiliac Occlusive Disease / 944Complications of Surgical Aortoiliac Reconstruction / 946Endovascular Treatment for Aortic Disease / 947Endovascular Treatment for Iliac Artery Disease / 948Complications of Endovascular Aortoiliac Interventions / 949Clinical Results Comparing Surgical and Endovascular Treatment of Aortoiliac Disease / 949Lower Extremity Arterial Occlusive Disease 950Epidemiology / 950Diagnostic Evaluation / 950Differential Diagnosis /

1	Results Comparing Surgical and Endovascular Treatment of Aortoiliac Disease / 949Lower Extremity Arterial Occlusive Disease 950Epidemiology / 950Diagnostic Evaluation / 950Differential Diagnosis / 951Lower Extremity Occlusive Disease Classification / 951Etiology of Acute Limb Ischemia / 952Clinical Manifestations of Acute Limb Ischemia / 954Treatment Considerations for Acute Limb Ischemia / 955Endovascular Treatment / 955Surgical Treatment / 956Complications Related to Treatment for Acute Limb Ischemia / 956Clinical Manifestations of Chronic Limb Ischemia / 958Treatment Considerations for Chronic Limb Ischemia / 959Endovascular Treatment / 960Complications of Endovascular Interventions / 965Surgical Treatment for Chronic Limb Ischemia due to Femoropopliteal Disease / 966Complications of Surgical Reconstruction / 966Choice of Conduit for Infrainguinal Bypass Grafting / 967Clinical Results of Surgical and Endovascular Interventions for Femoropopliteal Occlusive Disease /

1	of Surgical Reconstruction / 966Choice of Conduit for Infrainguinal Bypass Grafting / 967Clinical Results of Surgical and Endovascular Interventions for Femoropopliteal Occlusive Disease / 968Nonatherosclerotic Disorders of Blood Vessels 969Giant Cell Arteritis (Temporal Arteritis) / 969Takayasu’s Arteritis / 969Ehlers-Danlos Syndrome / 970Marfan’s Syndrome / 970Pseudoxanthoma Elasticum / 970Kawasaki’s Disease / 971Inflammatory Arteritis and Vasculitis / 971Behçet’s Disease / 971Polyarteritis Nodosa / 971Radiation-Induced Arteritis / 972Raynaud’s Syndrome / 972Fibromuscular Dysplasia / 972Nonatherosclerotic Disease Affecting the Popliteal Artery Disease / 973Buerger’s Disease (Thromboangiitis Obliterans) / 974Brunicardi_Ch23_p0897-p0980.indd 89727/02/19 4:13 PM 898Table 23-1Pertinent elements in vascular history• History of stroke or transient ischemic attack• History of coronary artery disease, including previous myocardial infarction and angina• History of peripheral

1	elements in vascular history• History of stroke or transient ischemic attack• History of coronary artery disease, including previous myocardial infarction and angina• History of peripheral arterial disease• History of diabetes• History of hypertension• History of tobacco use• History of hyperlipidemiaGENERAL APPROACH TO THE VASCULAR PATIENTSince the vascular system involves every organ system in our body, the symptoms of vascular disease are as varied as those encountered in any medical specialty. Lack of adequate blood supply to target organs typically presents with pain, for exam-ple, calf pain with lower extremity claudication, postprandial abdominal pain from mesenteric ischemia, and arm pain with axillo-subclavian arterial occlusion. In contrast, stroke and tran-sient ischemic attack (TIA) are the presenting symptoms from middle cerebral embolization as a consequence of a stenosed internal carotid artery. The pain syndrome of arterial disease is usually divided clinically into

1	(TIA) are the presenting symptoms from middle cerebral embolization as a consequence of a stenosed internal carotid artery. The pain syndrome of arterial disease is usually divided clinically into acute and chronic types, with all shades of severity between the two extremes. Sudden onset of pain can indicate complete occlusion of a critical vessel, leading to more severe pain and critical ischemia in the target organ, resulting in lower limb gangrene or intestinal infarction. Chronic pain results from a slower, more progressive atheroscle-rotic occlusion, which can be totally or partially compensated by developing collateral vessels. Acute on chronic is another pain pattern in which a patient most likely has an underlying arterial stenosis that suddenly occludes, for example, the patient with a history of calf claudication who now presents with sudden, severe acute limb-threatening ischemia. The clinician should always try to understand and relate the clinical manifestations to the

1	with a history of calf claudication who now presents with sudden, severe acute limb-threatening ischemia. The clinician should always try to understand and relate the clinical manifestations to the underlying pathologic process.The Vascular HistoryAppropriate history should be focused based on the present-ing symptoms related to the vascular system (Table 23-1). Of particular importance in the previous medical history is noting prior vascular interventions (endovascular or open surgical), and all vascular patients should have inquiry made about their prior cardiac history and current cardiac symptoms. Approximately 30% of vascular patients will be diabetic. A history of prior and current smoking status should be noted.The patient with carotid disease in most cases is com-pletely asymptomatic, having been referred based on the find-ing of a cervical bruit or duplex finding of stenosis. Symptoms of carotid territory TIAs include transient monocular blindness (amaurosis), contralateral

1	having been referred based on the find-ing of a cervical bruit or duplex finding of stenosis. Symptoms of carotid territory TIAs include transient monocular blindness (amaurosis), contralateral weakness or numbness, and dys-phasia. Symptoms persisting longer than 24 hours constitute a stroke. In contrast, the patient with chronic mesenteric isch-emia is likely to present with postprandial abdominal pain and weight loss. The patient fears eating because of the pain, avoids food, and loses weight. It is very unlikely that a patient with abdominal pain who has not lost weight has chronic mesenteric ischemia.The patient with lower extremity pain on ambulation has intermittent claudication that occurs in certain muscle groups; for example, calf pain upon exercise usually reflects superficial femoral artery disease, while pain in the buttocks reflects iliac disease. In most cases, the pain manifests in one muscle group below the level of the affected artery, occurs only with exercise, and

1	femoral artery disease, while pain in the buttocks reflects iliac disease. In most cases, the pain manifests in one muscle group below the level of the affected artery, occurs only with exercise, and is relieved with rest only to recur at the same location, hence the term “window gazer’s disease.” Rest pain (a manifestation of severe underlying occlusive disease) is constant and occurs in the foot (not the muscle groups), typically at the metatarso-phalangeal junction, and is relieved by dependency. Often the Key Points1 Carotid intervention as a preventive strategy should be per-formed in patients with 60% or greater symptomatic internal carotid artery stenosis and those with 80% or greater asymp-tomatic internal carotid artery stenosis. Carotid intervention for asymptomatic stenosis between 60% and 79% remains controversial. The modality of carotid intervention—carotid endarterectomy versus carotid stenting—remains contro-versial; currently, carotid endarterectomy appears to be

1	between 60% and 79% remains controversial. The modality of carotid intervention—carotid endarterectomy versus carotid stenting—remains contro-versial; currently, carotid endarterectomy appears to be associated with lower stroke rate with long term durability, whereas carotid stenting is more suitable under certain chal-lenging anatomic or physiologic conditions.2 Abdominal aortic aneurysms should be repaired when the risk of rupture, determined mainly by aneurysm size, exceeds the risk of death due to perioperative complications or concurrent illness. Endovascular repair is associated with less perioperative morbidity and mortality compared with open reconstruction and is preferred in patients with suitable anatomic morphology for stent-graft placement.3 Treatment objectives for symptomatic mesenteric ischemia are to improve quality of life and prevent bowel infarc-tion. Endovascular intervention with stenting has similar treatment efficacy comparative with less perioperative

1	mesenteric ischemia are to improve quality of life and prevent bowel infarc-tion. Endovascular intervention with stenting has similar treatment efficacy comparative with less perioperative mor-bidity compared to open mesenteric bypass. Surgical recon-struction has a proven durability and patency rate compared with endovascular intervention.4 Aortoiliac occlusive disease can be treated with either endovascular means or open reconstruction, depending on patient risk stratification, occlusion characteristics, and symptomatology.5 Claudication is a marker of extensive atherosclerosis and is mainly managed with risk factor modification and pharma-cotherapy. Only 5% of patients with claudication will need intervention because of disabling extremity pain. The 5-year mortality of a patient with claudication approaches 30%. Patients with rest pain or tissue loss need expeditious evalu-ation and vascular reconstruction to ameliorate the severe extremity pain and prevent limb loss. Endovascular

1	approaches 30%. Patients with rest pain or tissue loss need expeditious evalu-ation and vascular reconstruction to ameliorate the severe extremity pain and prevent limb loss. Endovascular interven-tion is preferred as the first line of therapy for lower extrem-ity occlusive disease, whereas bypass reconstruction should be considered in failed endovascular therapy or long seg-ment femoropopliteal occlusive disease.Brunicardi_Ch23_p0897-p0980.indd 89827/02/19 4:13 PM 899ARTERIAL DISEASECHAPTER 23Table 23-2Grading scales for peripheral pulsesTRADITIONAL SCALEBASIC SCALE4+Normal2+Normal3+Slightly reduced1+Diminished2+Markedly reduced0Absent1+Barely palpable patient is prompted to sleep with their foot hanging off one side of the bed to increase the hydrostatic pressure.The Vascular Physical ExaminationSpecific vascular examination should include abdominal aortic palpation, carotid artery examination, and pulse examination of the lower extremity (femoral, popliteal, posterior tibial,

1	ExaminationSpecific vascular examination should include abdominal aortic palpation, carotid artery examination, and pulse examination of the lower extremity (femoral, popliteal, posterior tibial, and dorsalis pedis arteries). The abdomen should be palpated for an abdominal aortic aneurysm, detected as an expansile pulse above the level of the umbilicus. It should also be examined for the presence of bruits. Because the aorta typically divides at the level of the umbilicus, an aortic aneurysm is most frequently palpable in the epigastrium. In thin individuals, a normal aortic pulsation is palpable, while in obese patients, even large aor-tic aneurysms may not be detectable. Suspicion of a clinically enlarged aorta should lead to the performance of an ultrasound scan for a more accurate definition of aortic diameter.The carotids should be auscultated for the presence of bruits, although there is a higher correlation with coronary artery disease than underlying carotid stenosis. A bruit

1	of aortic diameter.The carotids should be auscultated for the presence of bruits, although there is a higher correlation with coronary artery disease than underlying carotid stenosis. A bruit at the angle of the mandible is a significant finding, leading to follow-up duplex scanning. The differential diagnosis is a transmitted murmur from a sclerotic or stenotic aortic valve. The carotid is palpable deep to the sternocleidomastoid muscle in the neck. Palpation, however, should be gentle and rarely yields clinically useful information.Upper extremity examination is necessary when an arte-riovenous graft is to be inserted in patients who have symptoms of arm pain with exercise. Thoracic outlet syndrome (TOS) can result in occlusion or aneurysm formation of the subclavian artery. Distal embolization is a manifestation of TOS; conse-quently, the fingers should be examined for signs of ischemia and ulceration. The axillary artery enters the limb below the middle of the clavicle, where it

1	is a manifestation of TOS; conse-quently, the fingers should be examined for signs of ischemia and ulceration. The axillary artery enters the limb below the middle of the clavicle, where it can be palpated in thin patients. It is usually easily palpable in the axilla and medial upper arm. The brachial artery is most easily located at the antecubital fossa immediately medial to the biceps tendon. The radial artery is palpable at the wrist anterior to the radius.For lower extremity vascular examination, the femoral pulse is usually palpable midway between the anterior supe-rior iliac spine and the pubic tubercle. The popliteal artery is palpated in the popliteal fossa with the knee flexed to 45° and the foot supported on the examination table to relax the calf muscles. Palpation of the popliteal artery is a bimanual tech-nique. Both thumbs are placed on the tibial tuberosity anteriorly, and the fingers are placed into the popliteal fossa between the two heads of the gastrocnemius

1	popliteal artery is a bimanual tech-nique. Both thumbs are placed on the tibial tuberosity anteriorly, and the fingers are placed into the popliteal fossa between the two heads of the gastrocnemius muscle. The popliteal artery is palpated by compressing it against the posterior aspect of the tibia just below the knee. The posterior tibial pulse is detected by palpation 2 cm posterior to the medial malleolus. The dorsa-lis pedis is detected 1 cm lateral to the hallucis longus extensor tendon, which dorsiflexes the great toe and is clearly visible on the dorsum of the foot. Pulses can be graded using either the traditional four-point scale or the basic two-point scale system (Table 23-2). The foot should also be carefully examined for pallor on elevation and rubor on dependency, as these findings are indicative of chronic ischemia. Note should also be made of nail changes and loss of hair. Ulceration and other findings specific to disease states are described in relevant sections later

1	are indicative of chronic ischemia. Note should also be made of nail changes and loss of hair. Ulceration and other findings specific to disease states are described in relevant sections later in this chapter.After reconstructive vascular surgery, the graft may be available for examination, depending on its type and course. The in situ lower extremity graft runs in the subcutaneous fat and can be palpated along most of its length. A change in pulse quality, aneurysmal enlargement, or a new bruit should be care-fully noted. Axillofemoral grafts, femoral-to-femoral grafts, and arteriovenous access grafts can usually be easily palpated as well.Noninvasive Diagnostic Evaluation of the Vascular PatientAnkle-Brachial Index. There is increasing interest in the use of the ankle-brachial index (ABI) to evaluate patients at risk for cardiovascular events. An ABI less than 0.9 correlates with increased risk of myocardial infarction and indicates significant, although perhaps asymptomatic,

1	(ABI) to evaluate patients at risk for cardiovascular events. An ABI less than 0.9 correlates with increased risk of myocardial infarction and indicates significant, although perhaps asymptomatic, underlying peripheral vascular disease. The ABI is determined in the following ways. Blood pressure is measured in both upper extremities using the high-est systolic blood pressure as the denominator for the ABI. The ankle pressure is determined by placing a blood pressure cuff above the ankle and measuring the return to flow of the posterior tibial and dorsalis pedis arteries using a pencil Doppler probe over each artery. The ratio of the systolic pressure in each ves-sel divided by the highest arm systolic pressure can be used to express the ABI in both the posterior tibial and dorsalis pedis arteries (Fig. 23-1). Normal is more than 1. Patients with clau-dication typically have an ABI in the 0.5 to 0.7 range, and those with rest pain are in the 0.3 to 0.5 range. Those with gangrene have

1	arteries (Fig. 23-1). Normal is more than 1. Patients with clau-dication typically have an ABI in the 0.5 to 0.7 range, and those with rest pain are in the 0.3 to 0.5 range. Those with gangrene have an ABI of less than 0.3. These ranges can vary depending on the degree of compressibility of the vessel. The test is less reliable in patients with heavily calcified vessels. Due to non-compressibility, some patients, such as diabetics and those with end-stage renal disease, may have ABI ≥1.40 and require addi-tional noninvasive diagnostic testing to evaluate for peripheral artery disease. Alternative tests include toe-brachial pressures, pulse volume recordings, transcutaneous oxygen measurements, or vascular imaging (duplex ultrasound).Segmental Limb Pressures. By placing serial blood pressure cuffs down the lower extremity and then measuring the pressure with a Doppler probe as flow returns to the artery below the cuff, it is possible to determine segmental pressures down the leg. This

1	cuffs down the lower extremity and then measuring the pressure with a Doppler probe as flow returns to the artery below the cuff, it is possible to determine segmental pressures down the leg. This data can then be used to infer the level of the occlu-sion. The systolic pressure at each level is expressed as a ratio, with the highest systolic pressure in the upper extremities as the denominator. Normal segmental pressures commonly show high thigh pressures 20 mmHg or greater in comparison to the brachial artery pressures. The low thigh pressure should be equivalent to brachial pressures. Subsequent pressures should fall by no more than 10 mmHg at each level. A pressure gradient of 20 mmHg between two subsequent levels is usually indica-tive of occlusive disease at that level. The most frequently used index is the ratio of the ankle pressure to the brachial pressure, Brunicardi_Ch23_p0897-p0980.indd 89927/02/19 4:13 PM 900SPECIFIC CONSIDERATIONSPART IIFigure 23-1. Calculating the

1	used index is the ratio of the ankle pressure to the brachial pressure, Brunicardi_Ch23_p0897-p0980.indd 89927/02/19 4:13 PM 900SPECIFIC CONSIDERATIONSPART IIFigure 23-1. Calculating the ankle-brachial index (ABI).Right ABI = ratio ofHigher of the right ankle systolic pressures (posterior tibial or dorsalis pedis)Higher arm systolic pressure (left or right arm)Left ABI = ratio ofHigher of the left ankle systolic pressures (posterior tibial or dorsalis pedis)Higher arm systolic pressure (left or right arm)the ABI. Normally, the ABI is greater than 1.0, and a value of less than 0.9 indicates some degree of arterial obstruction and has been shown to be correlated with an increased risk of coronary heart disease. Limitations of relying on segmental limb pressures include: (a) missing isolated moderate stenoses (usually iliac) that produce little or no pressure gradient at rest; (b) falsely elevated pressures in patients with diabetes and end-stage renal disease; and (c) the inability

1	moderate stenoses (usually iliac) that produce little or no pressure gradient at rest; (b) falsely elevated pressures in patients with diabetes and end-stage renal disease; and (c) the inability to differentiate between stenosis and occlusion.1 Patients with diabetes and end-stage renal disease have calcified vessels that are difficult to com-press, thus rendering this method inaccurate, due to recording of falsely elevated pressure readings. Noncompressible arter-ies yield ankle systolic pressures ≥250 mmHg and ABIs >1.40. In this situation, absolute toe and ankle pressures can be mea-sured to gauge critical limb ischemia. Ankle pressures less than 50 mmHg or toe pressures less than 30 mmHg are indica-tive of critical limb ischemia. The toe pressure is normally 30 mmHg less than the ankle pressure, and a toe-brachial index (TBI) <0.70 is abnormal. False-positive results with the TBI are unusual. The main limitation of this technique is that it may be impossible to measure pressures

1	pressure, and a toe-brachial index (TBI) <0.70 is abnormal. False-positive results with the TBI are unusual. The main limitation of this technique is that it may be impossible to measure pressures in the first and second toes due to preexisting ulceration.Pulse Volume Recording. In patients with noncompressible vessels, segmental plethysmography can be used to determine underlying arterial occlusive disease. Cuffs placed at different levels on the leg detect changes in blood volume and produce a pulse volume recording (PVR) when connected to a plethysmo-graph (Fig. 23-2). To obtain accurate PVR waveforms, the cuff is inflated to 60 to 65 mmHg, so as to detect volume changes without causing arterial occlusion. Pulse volume tracings are suggestive of proximal disease if the upstroke of the pulse is not brisk, the peak of the wave tracing is rounded, and there is disappearance of the dicrotic notch.Although isolated segmental limb pressures and PVR mea-surements are 85% accurate when

1	pulse is not brisk, the peak of the wave tracing is rounded, and there is disappearance of the dicrotic notch.Although isolated segmental limb pressures and PVR mea-surements are 85% accurate when compared with angiography in detecting and localizing significant atherosclerotic lesions, when used in combination, accuracy approaches 95%.2 For this reason, it is suggested that these two diagnostic modalities be used in combination when evaluating peripheral artery disease.Radiologic Evaluation of the Vascular PatientUltrasound. Ultrasound examinations are relatively time consuming, require experienced technicians, and may not visu-alize all arterial segments. Doppler waveform analysis can sug-gest atherosclerotic occlusive disease if the waveforms in the insonated arteries are biphasic, monophasic, or asymmetrical. B-mode ultrasonography provides black and white, real-time images. B-mode ultrasonography does not evaluate blood flow; thus, it cannot differentiate between fresh thrombus

1	or asymmetrical. B-mode ultrasonography provides black and white, real-time images. B-mode ultrasonography does not evaluate blood flow; thus, it cannot differentiate between fresh thrombus and flow-ing blood, which have the same echogenicity. Calcification in atherosclerotic plaques will cause acoustic shadowing. B-mode ultrasound probes cannot be sterilized. Use of the B-mode probe intraoperatively requires a sterile covering and gel to maintain an acoustic interface. Experience is needed to obtain and interpret images accurately. Duplex ultrasonography entails performance of B-mode imaging, spectral Doppler scanning, and color-flow duplex scanning. The caveat to performance of duplex ultraso-nography is meticulous technique by a certified vascular ultra-sound technician, so that the appropriate 60° Doppler angle is maintained during insonation with the ultrasound probe. Altera-tion of this angle can markedly alter waveform appearance and subsequent interpretation of velocity

1	the appropriate 60° Doppler angle is maintained during insonation with the ultrasound probe. Altera-tion of this angle can markedly alter waveform appearance and subsequent interpretation of velocity measurements. Direct imaging of intra-abdominal vessels with duplex ultrasound is less reliable because of the difficulty in visualizing the vessels through overlying bowel. These disadvantages currently limit the applicability of duplex scanning in the evaluation of aortoil-iac and infrapopliteal disease. A clinical study has shown that duplex ultrasonography had lower sensitivity in the calculation of infrapopliteal vessel stenosis in comparison to conventional digital subtraction or computed tomography angiography.3 Few surgeons rely solely on duplex ultrasonography for preopera-tive planning in lower extremity revascularizations; but with experience, lower extremity arteries can be insonated to deter-mine anatomy, and the functional significance of lesions can be determined by

1	planning in lower extremity revascularizations; but with experience, lower extremity arteries can be insonated to deter-mine anatomy, and the functional significance of lesions can be determined by calculation of degree of stenosis from velocity ratios. Duplex scanning is unable to evaluate recently implanted polytetrafluoroethylene (PTFE) and polyester (Dacron) grafts because they contain air, which prevents ultrasound penetration.Computed Tomography Angiography. Computed tomogra-phy angiography (CTA) is a noninvasive, contrast-dependent method for imaging the arterial system. It depends on intrave-nous infusion of iodine-based contrast agents. The patient is advanced through a rotating gantry, which images serial trans-verse slices. The contrast-filled vessels can be extracted from the slices and rendered in three-dimensional format (Fig. 23-3). Brunicardi_Ch23_p0897-p0980.indd 90027/02/19 4:13 PM 901ARTERIAL DISEASECHAPTER 23Figure 23-2. Typical report of peripheral vascular

1	slices and rendered in three-dimensional format (Fig. 23-3). Brunicardi_Ch23_p0897-p0980.indd 90027/02/19 4:13 PM 901ARTERIAL DISEASECHAPTER 23Figure 23-2. Typical report of peripheral vascular study with arterial segmental pressure measurement plus Doppler evaluation of the lower extremity.Femoral0.750.500.250.000.25Sup.femoral1.501.000.500.000.50Popliteal1.501.000.500.000.50Posteriortibial1.501.000.500.000.50Dorsalispedis0.750.500.250.000.250.75Femoral0.500.250.000.25Popliteal0.750.500.250.000.25Posteriortibial0.750.500.250.000.25Dorsalispedis0.750.500.250.000.25Sup.femoral0.750.500.250.250.00149Brachial0.66U. thighL. thighCalfAnkle-PTAnkle-DPToe0.770.741.011.051.011.050.700.730.660.620.59Indexes144RightLeftDoppler waveforms1 sec/div15715015710910599PT 98DT 92PT 151DT 11111488ABFigure 23-3. A multidetector computed tomography angiography with three-dimensional reconstruction of the iliofemoral arterial circulation in two patients with lower leg claudication. A. A 50-year-old man

1	23-3. A multidetector computed tomography angiography with three-dimensional reconstruction of the iliofemoral arterial circulation in two patients with lower leg claudication. A. A 50-year-old man with an occluded right superficial femoral artery (single long arrow) with reconstituted superficial femoral artery at the level of mid-thigh. Arterial calcifications (single short arrows) are present in the bilateral distal superficial femoral arteries. B. A 53-year-old man with occluded right common iliac artery (double arrows).The extracted images can also be rotated and viewed from sev-eral different directions during postacquisition image process-ing. This technology has been advanced as a consequence of aortic endografting. CTA provides images for postprocessing that can be used to display the aneurysm in a format that demon-strates thrombus, calcium, lumen, and the outer wall, and allows “fitting” of a proposed endograft into the aneurysm (Fig. 23-4). CTA is increasingly being used

1	the aneurysm in a format that demon-strates thrombus, calcium, lumen, and the outer wall, and allows “fitting” of a proposed endograft into the aneurysm (Fig. 23-4). CTA is increasingly being used to image the carotid bifurca-tion, and as computing power increases, the speed of image acquisition and resolution will continue to increase. The major limitations of multidetector CTA are use of contrast and pres-ence of artifacts caused by calcification and stents. CTA can Brunicardi_Ch23_p0897-p0980.indd 90127/02/19 4:13 PM 902SPECIFIC CONSIDERATIONSPART IIFigure 23-4. Three-dimensional computed tomog-raphy angiogram of an abdominal aortic aneurysm that displays various aneurysm components including thrombus, aortic calcification, blood circulation, and aneurysm wall.Figure 23-5. Magnetic resonance angiogram of aortic arch and carotid arteries. This study can provide a three-dimensional analysis of vascular structure such as aortic arch branches and carotid and vertebral

1	resonance angiogram of aortic arch and carotid arteries. This study can provide a three-dimensional analysis of vascular structure such as aortic arch branches and carotid and vertebral arteries.overestimate the degree of in-stent stenosis, while heavy calci-fication can limit the diagnostic accuracy of the method by caus-ing a “blooming artifact.”4 The artifacts can be overcome with alteration in image acquisition technique. There are no random-ized trials to document the superiority of multidetector CTA over traditional angiography, but there is emerging evidence to support the claim that multidetector CTA has sensitivity, speci-ficity, and accuracy that rival invasive angiography.4Magnetic Resonance Angiography. Magnetic resonance angiography (MRA) has the advantage of not requiring iodin-ated contrast agents to provide vessel opacification (Fig. 23-5). Gadolinium is used as a contrast agent for MRA studies, and because it is generally not nephrotoxic, it can be used in patients

1	iodin-ated contrast agents to provide vessel opacification (Fig. 23-5). Gadolinium is used as a contrast agent for MRA studies, and because it is generally not nephrotoxic, it can be used in patients with elevated creatinine. MRA is contraindicated in patients with pacemakers, defibrillators, spinal cord stimulators, intrace-rebral shunts, cochlear implants, and cranial clips. Patients with claustrophobia may require sedation to be able to complete the test. The presence of metallic stents causes artifacts and signal drop-out; however, these can be dealt with using alternations in image acquisition and processing. Nitinol stents produce mini-mal artifact. Compared to other modalities, MRA is relatively slow and expensive. However, due to its noninvasive nature and decreased nephrotoxicity, MRA is being used more frequently for imaging vasculature in various anatomic distributions.Diagnostic Angiography. Diagnostic angiography is consid-ered the gold standard in vascular imaging. In

1	MRA is being used more frequently for imaging vasculature in various anatomic distributions.Diagnostic Angiography. Diagnostic angiography is consid-ered the gold standard in vascular imaging. In many centers, its use is rapidly decreasing due to the development of noninvasive imaging modalities such as duplex arterial mapping, CTA, and MRA. Nevertheless, contrast angiography still remains in wide-spread use. The essential aspects of angiography are vascular access and catheter placement in the vascular bed that requires examination. The imaging system and the contrast agent are used to opacify the target vessel. Although in the past this func-tion has largely been delegated to the interventional radiology service, an increasing number of surgeons are performing this procedure and following the diagnostic imaging with immediate surgical or endovascular intervention. There are several consid-erations when relying on angiography for imaging.Approximately 70% of atherosclerotic plaques

1	the diagnostic imaging with immediate surgical or endovascular intervention. There are several consid-erations when relying on angiography for imaging.Approximately 70% of atherosclerotic plaques occur in an eccentric location within the blood vessel; therefore, images can be misleading when trying to evaluate stenoses because angiog-raphy is limited to a uniplanar “lumenogram.” With increased use of intravascular stent deployment, it has also been noted that assessment of stent apposition and stent position in rela-tion to surrounding branches may be inaccurate. Furthermore, angiography exposes the patient to the risks of both ionizing radiation and intravascular contrast. Nevertheless, contrast angi-ography remains the most common invasive method of vascular investigation for both diagnostic and therapeutic intervention. The angiogram usually provides the final information needed to decide whether or not to proceed with operation or endovascular interventions.Digital subtraction

1	and therapeutic intervention. The angiogram usually provides the final information needed to decide whether or not to proceed with operation or endovascular interventions.Digital subtraction angiography (DSA) offers some advan-tages over conventional cut-film angiography such as excellent visualization despite use of lower volumes of contrast media. In particular, when multilevel occlusive lesions limit the amount of contrast reaching distal vessels, supplemental use of digital sub-traction angiographic techniques may enhance visualization and definition of anatomy. Intra-arterial DSA uses a portable, axially Brunicardi_Ch23_p0897-p0980.indd 90227/02/19 4:13 PM 903ARTERIAL DISEASECHAPTER 23Figure 23-6. Digital subtraction angiography (DSA) provides excellent visualization of intravascular circulation with intra-arterial contrast administration. As depicted in this DSA study, multilevel lesions are demonstrated, which include a focal left iliac artery stenosis (large arrow), right

1	circulation with intra-arterial contrast administration. As depicted in this DSA study, multilevel lesions are demonstrated, which include a focal left iliac artery stenosis (large arrow), right superficial femoral occlusion (curved arrows), left superficial femoral stenosis (small arrow), and mul-tiple tibial artery stenoses (arrowheads).rotatable imaging device that can obtain views from different angles. DSA also allows for real-time video replay (Fig. 23-6). An entire extremity can be filmed with DSA using repeated injections of small amounts of contrast agent to obtain sequen-tial angiographic images, the so-called pulse-chase technique.Preoperative Cardiac EvaluationThe most important and most controversial aspect of preopera-tive evaluation in patients with atherosclerotic disease requiring surgical intervention is the detection and subsequent manage-ment of associated coronary artery disease. Several studies have documented the existence of significant coronary artery disease

1	requiring surgical intervention is the detection and subsequent manage-ment of associated coronary artery disease. Several studies have documented the existence of significant coronary artery disease in 40% to 50% or more of patients requiring peripheral vascular reconstructive procedures, 10% to 20% of whom may be relatively asymptomatic largely because of their inability to exercise.5 Myocardial infarction is responsible for the major-ity of both early and late postoperative deaths. Most available screening methods lack sensitivity and specificity to predict postoperative cardiac complications. There have been conflict-ing reports regarding the utility of preoperative dipyridamole-thallium nuclear imaging or dobutamine-echocardiography to stratify vascular patients in terms of perioperative cardiac mor-bidity and mortality. In nearly half of patients, thallium imaging proves to be unnecessary because cardiac risk can be predicted by clinical information alone.6 Even with coronary

1	cardiac mor-bidity and mortality. In nearly half of patients, thallium imaging proves to be unnecessary because cardiac risk can be predicted by clinical information alone.6 Even with coronary angiography, it is difficult to relate anatomic findings to functional signifi-cance and, hence, surgical risk. There are no data confirming that percutaneous coronary interventions or surgical revascu-larization prior to vascular surgical procedures impact mortality or incidence of myocardial infarctions. In fact, coronary angi-ography is associated with its own inherent risks, and patients undergoing coronary artery bypass grafting or coronary percuta-neous transluminal angioplasty (PTA) before needed aortoiliac reconstructions are subjected to the risks and complications of both procedures.The Coronary Artery Revascularization Prophylaxis (CARP) trial showed that coronary revascularization in patients with peripheral vascular disease and significant coronary artery disease, who are considered

1	Artery Revascularization Prophylaxis (CARP) trial showed that coronary revascularization in patients with peripheral vascular disease and significant coronary artery disease, who are considered high risk for perioperative com-plications, did not reduce overall mortality or perioperative myocardial infarction.7 Additionally, patients who underwent prophylactic coronary revascularization had significant delays prior to undergoing their vascular procedure and increased limb morbidity compared to patients who did not. Studies do support improvement in cardiovascular and overall prognosis with med-ical optimization of patients.8 Therefore, use of perioperative β-blockade, as well as use of antiplatelet medication, statins, and angiotensin-converting enzyme inhibitors, is encouraged in vascular patients.BASIC PRINCIPLES OF ENDOVASCULAR THERAPYCardiovascular disease remains a major cause of mortality in the developed world since the beginning of the 21st century. Although surgical

1	vascular patients.BASIC PRINCIPLES OF ENDOVASCULAR THERAPYCardiovascular disease remains a major cause of mortality in the developed world since the beginning of the 21st century. Although surgical revascularization has played a predominant role in the management of patients with vascular disease, the modern treatment paradigms have evolved significantly with increased emphasis of catheter-based percutaneous interven-tions over the past two decades. The increasing role of this mini-mally invasive vascular intervention is fueled by various factors, including rapid advances in imaging technology, reduced mor-bidity and mortality in endovascular interventions, and faster convalescence following percutaneous therapy when compared to traditional operations. There is little doubt that with continued device development and refined image-guided technology, endo-vascular intervention will provide improved clinical outcomes and play an even greater role in the treatment of vascular disease.The

1	device development and refined image-guided technology, endo-vascular intervention will provide improved clinical outcomes and play an even greater role in the treatment of vascular disease.The technique of percutaneous access for both the diagnos-tic and therapeutic management of vascular disease has resulted in tremendous changes in the practice of several subspecialties, including interventional radiology, invasive cardiology, and vas-cular surgery. The development of catheter and endoscopic instru-mentation allows the vascular surgeon to operate via an intraor extraluminal route. Endovascular techniques are now able to treat the full spectrum of vascular pathology, including stenoses and occlusions resulting from several etiologies, aneurysmal pathol-ogy, and traumatic lesions. Many of these procedures have only recently been developed and, as such, have not been investigated Brunicardi_Ch23_p0897-p0980.indd 90327/02/19 4:13 PM 904SPECIFIC CONSIDERATIONSPART IIFigure 23-7. A.

1	these procedures have only recently been developed and, as such, have not been investigated Brunicardi_Ch23_p0897-p0980.indd 90327/02/19 4:13 PM 904SPECIFIC CONSIDERATIONSPART IIFigure 23-7. A. Antegrade femoral artery access. The needle is inserted just below the inguinal ligament in the common femoral artery whereby the guidewire is inserted in the ipsilateral super-ficial femoral artery. B. Brachial artery approach. The needle is inserted in a retrograde fashion in the brachial artery just above the antecubital fossa, whereby the guidewire is next inserted in the brachial artery.in a manner that would enable an accurate comparison with the more traditional methods of open surgical intervention. Long-term follow-up for these procedures is frequently lacking; how-ever, because of the potential to treat patients with decreased mortality and morbidity, endovascular skills and techniques are being adopted into mainstream vascular surgery.Needles and AccessNeedles are used to achieve

1	potential to treat patients with decreased mortality and morbidity, endovascular skills and techniques are being adopted into mainstream vascular surgery.Needles and AccessNeedles are used to achieve percutaneous vascular access. The size of the needle will be dictated by the diameter of the guide-wire used. Most often, an 18-gauge needle is used, as it will accept a 0.035-inch guidewire. A 21-gauge micropuncture nee-dle will accept a 0.018-inch guidewire. The most popular access needle is the Seldinger needle, which can be used for singleand double-wall puncture techniques.Femoral arterial puncture is the most common site for access. The common femoral artery (CFA) is punctured over the medial third of the femoral head, which is landmarked using flu-oroscopy. The single-wall puncture technique requires a sharp, beveled needle tip and no central stylet. The anterior wall of the vessel is punctured with the bevel of the needle pointing up, and pulsatile back-bleeding indicates an

1	technique requires a sharp, beveled needle tip and no central stylet. The anterior wall of the vessel is punctured with the bevel of the needle pointing up, and pulsatile back-bleeding indicates an intraluminal position. This method is most useful for graft punctures, patients with abnor-mal clotting profiles, or if thrombolytic therapy is anticipated. Once the needle assumes an intraluminal position, verified by pulsatile back-bleeding, the guidewire may be advanced. This is always passed gently and under fluoroscopic guidance to avoid subintimal dissection or plaque disruption. Double-wall puncture techniques are performed with a blunt needle that has a remov-able inner cannula. The introducer needle punctures both walls of the artery and is withdrawn until bleeding is obtained to confirm intraluminal position prior to advancing a guidewire. There can be troublesome bleeding from the posterior arterial wall punc-ture; therefore, single puncture techniques are preferred.Retrograde

1	intraluminal position prior to advancing a guidewire. There can be troublesome bleeding from the posterior arterial wall punc-ture; therefore, single puncture techniques are preferred.Retrograde femoral access is the most common arte-rial access technique (Fig. 23-7). The advantages of this technique include the size and fixed position of the CFA, as well as the relative ease of compression against the femoral head at the end of the procedure. Care should be taken to avoid puncturing the external iliac artery above the inguinal ligament because this can result in retroperitoneal hemor-rhage secondary to ineffective compression of the puncture site. Likewise, puncturing too low, at or below the CFA bifurcation, can result in thrombosis or pseudoaneurysm for-mation of the superficial femoral artery (SFA) or profunda femoris artery (PFA). Antegrade femoral access is more dif-ficult than retrograde femoral access, and there is a greater tendency to puncture the SFA, but it is invaluable

1	artery (SFA) or profunda femoris artery (PFA). Antegrade femoral access is more dif-ficult than retrograde femoral access, and there is a greater tendency to puncture the SFA, but it is invaluable when the aortic bifurcation cannot be traversed or when devices are not long enough to reach a lesion from a contralateral femo-ral access approach. Occasionally, when the distal aorta or bilateral iliac arteries are inaccessible because of the extent of atherosclerotic lesions, scarring, or presence of bypass conduits, the brachial artery must be used to obtain access for diagnostic and therapeutic interventions. The left bra-chial artery is punctured because this avoids the origin of the carotid artery and thus decreases the risk of catheter-related emboli to the brain. The artery is accessed with a micropunc-ture needle just proximal to the antecubital crease. The use of brachial access is associated with a higher risk of thrombosis and nerve injuries than femoral

1	The artery is accessed with a micropunc-ture needle just proximal to the antecubital crease. The use of brachial access is associated with a higher risk of thrombosis and nerve injuries than femoral access.GuidewiresGuidewires are used to introduce, position, and exchange cath-eters. A guidewire generally has a flexible and stiff end. In general, only the flexible end of the guidewire is placed in the vessel. All guidewires are composed of a stiff inner core and an outer tightly coiled spring that allows a catheter to track over the guidewire. There are five essential characteristics of guidewires: size, length, stiffness, coating, and tip configuration.Guidewires come in different maximum transverse diam-eters, ranging from 0.011 to 0.038 inches. For most aortoiliac procedures, a 0.035-inch wire is most commonly used, whereas the smaller diameter 0.018-inch guidewires are reserved for selective small vessel angiography such as infrageniculate or carotid lesions. In addition to

1	wire is most commonly used, whereas the smaller diameter 0.018-inch guidewires are reserved for selective small vessel angiography such as infrageniculate or carotid lesions. In addition to diameter size, guidewires come in varying lengths, usually ranging from 180 to 260 cm in length. Increasing the length of the wire always makes it more difficult to handle and increases the risk of contamination. While per-forming a procedure, it is important to maintain the guidewire across the lesion until the completion arteriogram has been sat-isfactorily completed.The stiffness of the guidewire is also an important charac-teristic. Stiff wires allow for passage of large aortic stent graft devices without kinking. They are also useful when trying to perform sheath or catheter exchanges around a tortuous artery. An example of a stiff guidewire is the Amplatz wire. Hydro-philic coated guidewires, such as the Glidewire, have become invaluable tools for assisting in difficult catheterizations. The

1	artery. An example of a stiff guidewire is the Amplatz wire. Hydro-philic coated guidewires, such as the Glidewire, have become invaluable tools for assisting in difficult catheterizations. The coating is primed by bathing the guidewire in saline solution. The slippery nature of this guidewire along with its torque capa-bility significantly facilitate in difficult catheterizations. Guide-wires also come in various tip configurations. Angled tip wires like the angled Glidewire can be steered to manipulate a catheter across a tight stenosis or to select a specific branch of a vessel. The Rosen wire has a soft curled end, which makes it ideal for renal artery stenting. The soft curl of this wire prevents it from perforating small renal branch vessels.Brunicardi_Ch23_p0897-p0980.indd 90427/02/19 4:13 PM 905ARTERIAL DISEASECHAPTER 23Figure 23-8. All percutaneous endovascular procedures are per-formed through an introducer sheath (large arrow), which pro-vides an access conduit from

1	4:13 PM 905ARTERIAL DISEASECHAPTER 23Figure 23-8. All percutaneous endovascular procedures are per-formed through an introducer sheath (large arrow), which pro-vides an access conduit from skin to intravascular compartment. The sheath also acts to protect the vessel from injury as guidewires (small arrows) and catheters are introduced.Hemostatic SheathsThe hemostatic sheath is a device through which endovascular procedures are performed. The sheath acts to protect the vessel from injury as wires and catheters are introduced (Fig. 23-8). A one-way valve prevents bleeding through the sheath, and a side-port allows contrast or heparin flushes to be administered during the procedure. Sheaths are sized by their inner diameter. The most commonly used sheaths for percutaneous access have a 5to 9-French inner diameter, but with open surgical exposure of the CFA, sheaths as large as 26 French can be introduced. Sheaths also vary in length, and long sheaths are available so that interventions

1	9-French inner diameter, but with open surgical exposure of the CFA, sheaths as large as 26 French can be introduced. Sheaths also vary in length, and long sheaths are available so that interventions remote from the site of arterial access can be performed.CathetersA wide variety of catheters exist that differ primarily in the con-figuration of the tip. The multiple shapes permit access to ves-sels of varying dimensions and angulations. Catheters are used to perform angiography and protect the passage of balloons and stents, and they can be used to direct the guidewire through tight stenoses or tortuous vessels.Figure 23-9. A. An artery with luminal narrowing caused by plaque. B. A balloon angioplasty catheter is positioned within the diseased artery, which is inflated to enlarge the intravascular channel. C. The plaque is compressed with widened flow lumen as the result of balloon angioplasty.Angioplasty BalloonsAngioplasty balloons differ primarily in their length and diam-eter, as

1	channel. C. The plaque is compressed with widened flow lumen as the result of balloon angioplasty.Angioplasty BalloonsAngioplasty balloons differ primarily in their length and diam-eter, as well as the length of the catheter shaft. As balloon technology has advanced, lower profiles have been manufac-tured (i.e., the size that the balloon assumes upon deflation). Balloons are used to perform angioplasty on vascular stenoses, to deploy stents, and to assist with additional expansion after insertion of self-expanding stents (Fig. 23-9). Besides length and diameter, operators need to be familiar with several other balloon characteristics. Noncompliant and low-compliance balloons tend to be inflated to their preset diameter and offer greater dilating force at the site of stenosis. Low-compliance balloons are the mainstay for peripheral intervention. Lower profile balloons are less likely to get caught during passage through stents and are easier to pull out of sheaths. Under fluoroscopic

1	balloons are the mainstay for peripheral intervention. Lower profile balloons are less likely to get caught during passage through stents and are easier to pull out of sheaths. Under fluoroscopic guidance, balloon inflation is performed until the waist of the atherosclerotic lesion disappears and the balloon is at the full profile. The duration of balloon inflation and pres-sures used for the angioplasty depend on the indication for the intervention and the location and characteristics of the lesion being treated. Frequently, several inflations are required to achieve a full profile of the balloon. Occasionally, a lower pro-file balloon is needed to predilate the tight stenosis so that the selected balloon catheter can cross the lesion. After inflation, most balloons do not regain their preinflation diameter and assume a larger profile. Trackability, pushability, and cross-ability of the balloon should all be considered when choosing a particular balloon. Lastly, shoulder length is an

1	diameter and assume a larger profile. Trackability, pushability, and cross-ability of the balloon should all be considered when choosing a particular balloon. Lastly, shoulder length is an important characteristic to consider when selecting a balloon because of the potential to cause injury during performance of PTA in adjacent arterial segments. There is always risk of causing dis-section or rupture during PTA; thus, a completion angiogram is performed while the wire is still in place. Leaving the wire in place provides access for repeating the procedure, placing a stent or stent graft if warranted.StentsVascular stents are commonly used after an inadequate angio-plasty with dissection or elastic recoil of an arterial stenosis. They serve to buttress collapsible vessels and help prevent atherosclerotic restenosis. Appropriate indications for primary stenting of a lesion without an initial trial of angioplasty alone are evolving in manners that are dependent on the extent and site of

1	atherosclerotic restenosis. Appropriate indications for primary stenting of a lesion without an initial trial of angioplasty alone are evolving in manners that are dependent on the extent and site of the lesion. Stents are manufactured from a variety of metals including stainless steel, tantalum, cobalt-based alloy, Brunicardi_Ch23_p0897-p0980.indd 90527/02/19 4:13 PM 906SPECIFIC CONSIDERATIONSPART IIFigure 23-10. Self-expanding stents are made of tempered stainless steel or nitinol, an alloy of nickel and titanium, and are restrained when folded inside a delivery catheter. After being released from the restraining catheter, the self-expanding stents will expand to a final diameter that is determined by stent geometry, hoop strength, and vessel size.Figure 23-11. In a balloon-expandable stent, the stent is pre-mounted on a balloon catheter. The balloon stretches the stent members beyond their elastic limit. The stent is deployed by full balloon expansion. This type of stent has a

1	stent, the stent is pre-mounted on a balloon catheter. The balloon stretches the stent members beyond their elastic limit. The stent is deployed by full balloon expansion. This type of stent has a higher degree of crush resistance when compared to self-expanding stents, which is ideal for short-segment calcified ostial lesions.and nitinol. Vascular stents are classified into two basic categories: balloon-expandable stents and self-expanding stents.Self-expanding stents (Fig. 23-10) are deployed by retract-ing a restraining sheath and usually consist of Elgiloy (a cobalt, chromium, nickel alloy) or nitinol (a shape memory alloy com-posed of nickel and titanium), the latter of which will contract and assume a heat-treated shape above a transition temperature that depends on the composition of the alloy. Self-expanding stents will expand to a final diameter that is determined by stent geometry, hoop strength, and vessel size. The self-expanding stent is mounted on a central shaft and is

1	of the alloy. Self-expanding stents will expand to a final diameter that is determined by stent geometry, hoop strength, and vessel size. The self-expanding stent is mounted on a central shaft and is placed inside an outer sheath. It relies on a mechanical spring-like action to achieve expansion. With deployment of these stents, there is some degree of foreshortening that has to be taken into account when choosing the area of deployment. In this way, self-expanding stents are more difficult to place with absolute precision. There are several advantages related to self-expanding stents. Self-expanding stents generally come in longer lengths than balloon-expandable stents and are therefore used to treat long and tortuous lesions. Their ability to continually expand after deliv-ery allows them to accommodate adjacent vessels of different size. This makes these stents ideal for placement in the internal carotid artery. These stents are always oversized by 1 to 2 mm relative to the largest

1	to accommodate adjacent vessels of different size. This makes these stents ideal for placement in the internal carotid artery. These stents are always oversized by 1 to 2 mm relative to the largest diameter of normal vessel adjacent to the lesion in order to prevent immediate migration.Balloon-expandable stents are usually composed of stain-less steel, mounted on an angioplasty balloon, and deployed by balloon inflation (Fig. 23-11). They can be manually placed on a chosen balloon catheter or obtained premounted on a balloon catheter. The capacity of a balloon-expandable stent to shorten in length during deployment depends on both stent geometry and the final diameter to which the balloon is expanded. These stents are more rigid and are associated with a shorter time to complete endothelialization. They are often of limited flexibility and have a higher degree of crush resistance when compared to self-expanding stents. This makes them ideal for short-segment lesions, especially those

1	They are often of limited flexibility and have a higher degree of crush resistance when compared to self-expanding stents. This makes them ideal for short-segment lesions, especially those that involve the ostia such as proximal common iliac or renal artery stenosis.An important area of evolution in endovascular therapy in recent years is the development of drug-eluting stents (DES). These stents are usually composed of nitinol and have vari-ous anti-inflammatory drugs bonded to them. Over time, the stents release the drug into the surrounding arterial wall and help prevent restenosis. Numerous randomized controlled trials have proven their benefit in coronary arteries.9 Clinical studies have similarly proved early efficacy of DES in the treatment of peripheral arterial disease.10Stent GraftsThe combination of a metal stent covered with fabric gave birth to the first stent grafts. Covered stents have been designed with either a surrounding PTFE or polyester fabric and have been used

1	combination of a metal stent covered with fabric gave birth to the first stent grafts. Covered stents have been designed with either a surrounding PTFE or polyester fabric and have been used predominantly for treatment of traumatic vascular lesions, including arterial disruption and arteriovenous fistulas (Fig. 23-12). However, these devices may well find a growing role in treatment of iliac or femoral arterial occlusive disease as well as popliteal aneurysms.Endovascular aneurysm repair using the concept of stent grafts was initiated by Parodi in 1991.11 Since that time, a large number of endografts have been inserted under the auspice of clinical trials initially and now as Food and Drug Administra-tion (FDA)–approved devices. Currently there are more than Brunicardi_Ch23_p0897-p0980.indd 90627/02/19 4:13 PM 907ARTERIAL DISEASECHAPTER 23Figure 23-12. A stent graft is a metal stent covered with fabric that is com-monly used for aneurysm exclusion.eight FDA-approved endovascular

1	90627/02/19 4:13 PM 907ARTERIAL DISEASECHAPTER 23Figure 23-12. A stent graft is a metal stent covered with fabric that is com-monly used for aneurysm exclusion.eight FDA-approved endovascular devices for abdominal aortic aneurysm repair. In general, majority of these devices require that patients have an infrarenal aneurysm with at least a 15-mm proximal aortic neck below the renal arteries and not greater than 60° of angulation. For those patients with associated com-mon iliac artery aneurysmal disease, endovascular treatment can be achieved by initial coil embolization of the ipsilateral hypogastric artery with extension of the endovascular device into the external iliac artery. Newer generation devices with branched endograft can be deployed in the internal iliac artery while maintain in-line flow from the common iliac to exter-nal iliac artery to exclude the common iliac artery aneurysm. Recent clinical trials have demonstrated clinical efficacy of fenestrated aortic endograft

1	in-line flow from the common iliac to exter-nal iliac artery to exclude the common iliac artery aneurysm. Recent clinical trials have demonstrated clinical efficacy of fenestrated aortic endograft in treating aneurysm involving the visceral segment of the abdominal aorta.12 The FDA has simi-larly approved several thoracic endograft devices for the treat-ment of descending thoracic aortic aneurysm. Clinical studies have similarly demonstrated durability and efficacy of thoracic aortic devices in patients with traumatic aortic transections and aortic dissections.13CAROTID ARTERY DISEASEAtherosclerotic occlusive plaque is by far the most common pathology seen in the carotid artery bifurcation. Thirty percent to 60% of all ischemic strokes are related to atherosclerotic carotid bifurcation occlusive disease. In the following section, we first focus our discussion on the clinical presentation, diagnosis, and management—including medical therapy, surgical carotid end-arterectomy, and

1	occlusive disease. In the following section, we first focus our discussion on the clinical presentation, diagnosis, and management—including medical therapy, surgical carotid end-arterectomy, and stenting—of atherosclerotic carotid occlusive disease. In the second part of the section, we provide a review on other less common nonatherosclerotic diseases involving the extracranial carotid artery, including kink and coil, fibromuscu-lar dysplasia, arterial dissection, aneurysm, radiation arteritis, Takayasu’s arteritis, and carotid body tumor.Epidemiology and Etiology of Carotid Occlusive DiseaseApproximately 700,000 Americans suffer a new or recur-rent stroke each year.14 Eighty-five percent of all strokes are ischemic, and 15% are hemorrhagic. Hemorrhagic strokes are caused by head trauma or spontaneous disruption of intracere-bral blood vessels. Ischemic strokes are due to hypoperfusion from arterial occlusion or, less commonly, to decreased flow resulting from proximal arterial

1	or spontaneous disruption of intracere-bral blood vessels. Ischemic strokes are due to hypoperfusion from arterial occlusion or, less commonly, to decreased flow resulting from proximal arterial stenosis and poor collateral network. Common causes of ischemic strokes are cardiogenic emboli in 35%, carotid artery disease in 30%, lacunar in 10%, miscellaneous in 10%, and idiopathic in 15%.19 The term cere-brovascular accident is often used interchangeably to refer to an ischemic stroke. A transient ischemic attack (TIA) is defined as a temporary focal cerebral or retinal hypoperfusion state that resolves spontaneously within 24 hours after its onset. However, the majority of TIAs resolve within minutes, and longer-lasting neurologic deficits more likely represent a stroke. Recently, the term brain attack has been coined to refer to an acute stroke or TIA, denoting the condition as a medical emergency requiring immediate attention, similar to a heart attack.Stroke due to carotid

1	the term brain attack has been coined to refer to an acute stroke or TIA, denoting the condition as a medical emergency requiring immediate attention, similar to a heart attack.Stroke due to carotid bifurcation occlusive disease is usu-ally caused by atheroemboli (Fig. 23-13). The carotid bifurca-tion is an area of low flow velocity and low shear stress. As the blood circulates through the carotid bifurcation, there is separa-tion of flow into the low-resistance internal carotid artery and the high-resistance external carotid artery. Characteristically, atherosclerotic plaque forms in the outer wall opposite to the flow divider (Fig. 23-14). Atherosclerotic plaque formation is complex, beginning with intimal injury, platelet deposition, smooth muscle cell proliferation, and fibroplasia, and leading to subsequent luminal narrowing. With increasing degree of ste-nosis in the internal carotid artery, flow becomes more turbu-lent, and the risk of atheroembolization escalates. The severity

1	leading to subsequent luminal narrowing. With increasing degree of ste-nosis in the internal carotid artery, flow becomes more turbu-lent, and the risk of atheroembolization escalates. The severity of stenosis is commonly divided into three categories accord-ing to the luminal diameter reduction: mild (<50%), moderate (50–69%), and severe (70–99%). Severe carotid stenosis is a strong predictor for stroke.15 In turn, a prior history of neuro-logic symptoms (TIA or stroke) is an important determinant for recurrent ipsilateral stroke. The risk factors for the development of carotid artery bifurcation disease are similar to those causing atherosclerotic occlusive disease in other vascular beds. Increas-ing age, male gender, hypertension, tobacco smoking, diabetes mellitus, homocysteinemia, and hyperlipidemia are well-known predisposing factors for the development of atherosclerotic occlusive disease.Clinical Manifestations of Cerebral IschemiaTIA is a focal loss of neurologic function,

1	and hyperlipidemia are well-known predisposing factors for the development of atherosclerotic occlusive disease.Clinical Manifestations of Cerebral IschemiaTIA is a focal loss of neurologic function, lasting for less than 24 hours. Crescendo TIAs refer to a syndrome comprising repeated TIAs within a short period of time that is character-ized by complete neurologic recovery in between. At a minimum, the term should probably be reserved for those with either daily events or multiple resolving attacks within 24 hours. Brunicardi_Ch23_p0897-p0980.indd 90727/02/19 4:13 PM 908SPECIFIC CONSIDERATIONSPART IIFigure 23-14. A. The carotid bifurcation is an area of low flow velocity and low shear stress. As the blood circulates through the carotid bifurcation, there is separation of flow into the low-resistance internal carotid artery and the high-resistance external carotid artery. B. The carotid atherosclerotic plaque typi-cally forms in the outer wall opposite to the flow divider due in

1	low-resistance internal carotid artery and the high-resistance external carotid artery. B. The carotid atherosclerotic plaque typi-cally forms in the outer wall opposite to the flow divider due in part to the effect of the low shear stress region, which also creates a tran-sient reversal of flow during the cardiac cycle.InternalcarotidarteryEmboliUlcerPlaqueCommoncarotidarteryExternalcarotidarterySuperiorthyroidarteryFigure 23-13. Stroke due to carotid bifurcation occlusive disease is usually caused by atheroemboli arising from the internal carotid artery, which provides the majority of blood flow to the cerebral hemisphere. With increasing degree of stenosis in the carotid artery, flow becomes more turbulent, and the risk of atheroembolization escalates.BASectional viewLow-shearregionHigh-shearregionHemodynamic TIAs represent focal cerebral events that are aggravated by exercise or hemodynamic stress and typically occur after short bursts of physical activity, postprandially, or

1	TIAs represent focal cerebral events that are aggravated by exercise or hemodynamic stress and typically occur after short bursts of physical activity, postprandially, or after getting out of a hot bath. It is implied that these are due to severe extracranial disease and poor intracranial collateral recruitment. Reversible ischemic neurologic deficits refer to ischemic focal neurologic symptoms lasting longer than 24 hours but resolv-ing within 3 weeks. When a neurologic deficit lasts longer than 3 weeks, it is considered a completed stroke. Stroke in evolution refers to progressive worsening of the neurologic deficit, either linearly over a 24-hour period or interspersed with transient periods of stabilization and/or partial clinical improvement.Patients who suffer cerebrovascular accidents typically present with three categories of symptoms including ocular symptoms, sensory/motor deficit, and/or higher cortical dys-function. The common ocular symptoms associated with extra-cranial

1	typically present with three categories of symptoms including ocular symptoms, sensory/motor deficit, and/or higher cortical dys-function. The common ocular symptoms associated with extra-cranial carotid artery occlusive disease include amaurosis fugax and presence of Hollenhorst plaques. Amaurosis fugax, com-monly referred to as transient monocular blindness, is a tempo-rary loss of vision in one eye that patients typically describe as a window shutter coming down or grey shedding of the vision. This partial blindness usually lasts for a few minutes and then resolves. Most of these phenomena (>90%) are due to embolic occlusion of the main artery or the upper or lower divisions. Monocular blindness progressing over a 20-minute period sug-gests a migrainous etiology. Occasionally, the patient will recall no visual symptoms while the optician notes a yellowish plaque within the retinal vessels, which is also known as Hollenhorst plaque. These plaques are frequently derived from

1	the patient will recall no visual symptoms while the optician notes a yellowish plaque within the retinal vessels, which is also known as Hollenhorst plaque. These plaques are frequently derived from cholesterol embolization from the carotid bifurcation and warrant further investigation. Additionally, several ocular symptoms may be caused by microembolization from extracranial carotid dis-eases including monocular visual loss due to retinal artery or optic nerve ischemia, the ocular ischemia syndrome, and visual field deficits secondary to cortical infarction and ischemia of the optic tracts. Typical motor and/or sensory symptoms asso-ciated with cerebrovascular accidents are lateralized or focal neurologic deficits. Ischemic events tend to have an abrupt onset, with the severity of the insult being apparent from the onset and not usually associated with seizures or paresthesia. In contrast, they represent loss or diminution of neurologic func-tion. Furthermore, motor or sensory

1	the insult being apparent from the onset and not usually associated with seizures or paresthesia. In contrast, they represent loss or diminution of neurologic func-tion. Furthermore, motor or sensory deficits can be unilateral or Brunicardi_Ch23_p0897-p0980.indd 90827/02/19 4:14 PM 909ARTERIAL DISEASECHAPTER 23bilateral, with the upper and lower limbs being variably affected depending on the site of the cerebral lesion. The combination of a motor and sensory deficit in the same body territory is sug-gestive of a cortical thromboembolic event as opposed to lacu-nar lesions secondary to small vessel disease of the penetrating arterioles. However, a small proportion of the latter may present with a sensorimotor stroke secondary to small vessel occlusion within the posterior limb of the internal capsule. Pure sensory and pure motor strokes and those strokes where the weakness affects one limb only or does not involve the face are more typically seen with lacunar as opposed to cortical

1	capsule. Pure sensory and pure motor strokes and those strokes where the weakness affects one limb only or does not involve the face are more typically seen with lacunar as opposed to cortical infarction. A number of higher cortical functions, including speech and language disturbances, can be affected by thromboembolic phe-nomena from the carotid artery, with the most important clinical example for the dominant hemisphere being dysphasia or apha-sia and visuospatial neglect being an example of nondominant hemisphere injury.Diagnostic EvaluationDuplex ultrasonography is the most widely used screening tool to evaluate for atherosclerotic plaque and stenosis of the extracranial carotid artery. It is also commonly used to monitor patients serially for progression of disease or after intervention (carotid endarterectomy or angioplasty). Duplex ultrasound of the carotid artery combines B-mode gray-scale imaging and Doppler waveform analysis. Characterization of the carotid plaque on

1	(carotid endarterectomy or angioplasty). Duplex ultrasound of the carotid artery combines B-mode gray-scale imaging and Doppler waveform analysis. Characterization of the carotid plaque on gray-scale imaging provides useful information about its composition. However, there are currently no universal rec-ommendations that can be made based solely on the sonographic appearance of the plaque. On the other hand, criteria have been developed and well refined for grading the degree of carotid ste-nosis based primarily on Doppler-derived velocity waveforms.The external carotid artery has a high-resistance flow pat-tern with a sharp systolic peak and a small amount of flow in diastole. In contrast, a normal internal carotid artery will have a low-resistance flow pattern with a broad systolic peak and a large amount of flow during diastole. The flow pattern in the common carotid artery resembles that in the internal carotid artery, as 80% of the flow is directed to the internal carotid artery,

1	and a large amount of flow during diastole. The flow pattern in the common carotid artery resembles that in the internal carotid artery, as 80% of the flow is directed to the internal carotid artery, with waveforms that have broad systolic peaks and mod-erate amount of flow during diastole. Conventionally, velocity measurements are recorded in the common, external, carotid bulb, and the proximal, mid, and distal portions of the internal carotid artery. Characteristically, the peak systolic velocity is increased at the site of the vessel stenosis. The end-diastolic velocity is increased with greater degree of stenosis. In addition, stenosis of the internal carotid artery can lead to color shifts with color mosaics indicating a poststenotic turbulence. Dampening of the Doppler velocity waveforms is typically seen in areas distal to severe carotid stenosis where blood flow is reduced. It is well known that occlusion of the ipsilateral internal carotid artery can lead to a “falsely”

1	waveforms is typically seen in areas distal to severe carotid stenosis where blood flow is reduced. It is well known that occlusion of the ipsilateral internal carotid artery can lead to a “falsely” elevated velocity on the contralat-eral side due to an increase in compensatory blood flow. In the presence of a high-grade stenosis or occlusion of the internal carotid artery, the ipsilateral common carotid artery displays high flow resistance waveforms, similar to those seen in the external carotid artery. If there is a significant stenosis in the proximal common carotid artery, its waveforms may be damp-ened with low velocities.The Doppler grading systems of carotid stenosis were initially established by comparison to angiographic findings of disease. Studies have shown variability in the measurements of the duplex properties by different laboratories, as well as hetero-geneity in the patient population, study design, and techniques. One the most commonly used classifications was

1	the measurements of the duplex properties by different laboratories, as well as hetero-geneity in the patient population, study design, and techniques. One the most commonly used classifications was established at the University of Washington School of Medicine in Seattle, Washington.16 Diameter reduction of 50% to 79% is defined by peak systolic velocity greater than 125 cm/s with extensive spectral broadening. For stenosis in the range of 80% to 99%, the peak systolic velocity is greater than 125 cm/s, and peak diastolic velocity is greater than 140 cm/s. The ratio of inter-nal carotid to common carotid artery peak systolic velocity has also been part of various ultrasound diagnostic classifications. A ratio greater than 4 is a great predictor of angiographic ste-nosis of 70% to 99%. A multispecialty consensus panel has developed a set of criteria for grading carotid stenosis by duplex examination (Table 23-3).MRA is increasingly being used to evaluate for athero-sclerotic carotid

1	multispecialty consensus panel has developed a set of criteria for grading carotid stenosis by duplex examination (Table 23-3).MRA is increasingly being used to evaluate for athero-sclerotic carotid occlusive disease and intracranial circulation. MRA is noninvasive and does not require iodinated contrast agents. MRA uses phase contrast or time-of-flight, with either two-dimensional or three-dimensional data sets for greater accu-racy. Three-dimensional contrast-enhanced MRA allows data to be obtained in coronal and sagittal planes with improved image qualities due to shorter study time. In addition, the new MRA techniques allow for better reformation of images in vari-ous planes to allow better grading of stenosis. There have been numerous studies comparing the sensitivity and specificity of Table 23-3Carotid duplex ultrasound criteria for grading internal carotid artery stenosisDEGREE OF STENOSIS (%)ICA PSV (CM/S)ICA/CCA PSV RATIOICA EDV (CM/S)PLAQUE ESTIMATE

1	and specificity of Table 23-3Carotid duplex ultrasound criteria for grading internal carotid artery stenosisDEGREE OF STENOSIS (%)ICA PSV (CM/S)ICA/CCA PSV RATIOICA EDV (CM/S)PLAQUE ESTIMATE (%)aNormal<125<2.0<40None<50<125<2.0<40<5050–69125–2302.0–4.040–100≥50≥70 to less than near occlusion>230>4.0>100≥50Near occlusionHigh, low, or not detectedVariableVariableVisibleTotal occlusionNot detectedNot applicableNot detectedVisible, no lumenaPlaque estimate (diameter reduction) with gray-scale and color Doppler ultrasound.CCA = common carotid artery; EDV = end-diastolic velocity; ICA = internal carotid artery; PSV = peak systolic velocity.Brunicardi_Ch23_p0897-p0980.indd 90927/02/19 4:14 PM 910SPECIFIC CONSIDERATIONSPART IIFigure 23-15. A. Carotid computed tomography angiography is a valuable imaging modality that can provide a three-dimensional image reconstruction with high image resolution. A carotid artery occlusion is noted in the internal carotid artery B. The entire segment of

1	imaging modality that can provide a three-dimensional image reconstruction with high image resolution. A carotid artery occlusion is noted in the internal carotid artery B. The entire segment of extracranial carotid artery is visualized from the thoracic compartment to the base of skull.Figure 23-16. A carotid angiogram reveals an ulcerated carotid plaque (arrow) in the proximal internal carotid artery, which also resulted in a high-grade internal carotid artery stenosis.MRA imaging for carotid disease to duplex and selective con-trast angiography.17 Magnetic resonance imaging (MRI) of the brain is essential in the assessment of acute stroke patients. MRI with diffusion-weighted imaging can differentiate areas of acute ischemia, areas still at risk for ischemia (penumbra), and chronic cerebral ischemic changes. However, computed tomography (CT) imaging remains the most expeditious test in the evaluation of acute stroke patients to rule out intracerebral hemorrhage. Recently,

1	cerebral ischemic changes. However, computed tomography (CT) imaging remains the most expeditious test in the evaluation of acute stroke patients to rule out intracerebral hemorrhage. Recently, multidetector CTA has gained increasing popularity in the evaluation of carotid disease.18 This imaging modality can provide volume rendering, which allows rotation of the object with accurate anatomic structures from all angles (Fig. 23-15). The advantages of CTA over MRA include faster data acquisition time and better spatial resolution. However, grading of carotid stenosis by CTA requires further validation at the time of this writing before it can be widely applied.Historically, DSA has been the gold standard test to evalu-ate the extraand intracranial circulation (Fig. 23-16). This is an invasive procedure, typically performed via a transfemoral puncture, and involves selective imaging of the carotid and ver-tebral arteries using iodinated contrast. The risk of stroke during cerebral

1	invasive procedure, typically performed via a transfemoral puncture, and involves selective imaging of the carotid and ver-tebral arteries using iodinated contrast. The risk of stroke during cerebral angiography is generally reported at approximately 1% and is typically due to atheroembolization related to wire and catheter manipulation in the arch aorta or proximal branch vessels. Over the last few decades, however, the incidence of neurologic complications following angiography has been reduced, due to the use of improved guidewires and catheters, better resolution digital imaging, and increased experience. Local access compli-cations of angiography are infrequent and include development of hematoma, pseudoaneurysm, distal embolization, and acute vessel thrombosis. Currently, selective angiography is particu-larly used for patients with suspected intracranial disease and for patients in whom percutaneous revascularization is con-sidered. The techniques of carotid angioplasty and

1	angiography is particu-larly used for patients with suspected intracranial disease and for patients in whom percutaneous revascularization is con-sidered. The techniques of carotid angioplasty and stenting for carotid bifurcation occlusive disease are described in detail later in this chapter. We generally use CTA or MRA to get informa-tion about the aortic arch anatomy and presence of concomitant intracranial disease and collateral pathway in planning our strategy for carotid stenting or endarterectomy.Treatment of Carotid Occlusive DiseaseConventionally, patients with carotid bifurcation occlusive dis-ease are divided into two broad categories: patients without prior history of ipsilateral stroke or TIA (asymptomatic) and those with prior or current ipsilateral neurologic symptoms BA(symptomatic). It is estimated that 15% of all strokes are pre-ceded by a TIA. The 90-day risk of a stroke in a patient present-ing with a TIA is 3% to 17%.14 According to the Cardiovascular Health

1	BA(symptomatic). It is estimated that 15% of all strokes are pre-ceded by a TIA. The 90-day risk of a stroke in a patient present-ing with a TIA is 3% to 17%.14 According to the Cardiovascular Health Study, a longitudinal population-based study of coronary artery disease and stroke in men and women, the prevalence of TIA in men was 2.7% for ages of 65 and 69 and 3.6% for ages 75 to 79; the prevalence in women was 1.4% and 4.1%, respectively.24 There have been several studies reporting on the effectiveness of stroke prevention with medical treatment and carotid endarterectomy for symptomatic patients with moderate to severe carotid stenosis. Early and chronic aspirin therapy has been shown to reduce stroke recurrence rate in sev-eral large clinical trials.19Symptomatic Carotid Stenosis. Currently, most stroke neu-rologists prescribe both aspirin and clopidogrel for secondary 1Brunicardi_Ch23_p0897-p0980.indd 91027/02/19 4:14 PM 911ARTERIAL DISEASECHAPTER 23stroke prevention in

1	most stroke neu-rologists prescribe both aspirin and clopidogrel for secondary 1Brunicardi_Ch23_p0897-p0980.indd 91027/02/19 4:14 PM 911ARTERIAL DISEASECHAPTER 23stroke prevention in patients who have experienced a TIA or stroke.19 In patients with symptomatic carotid stenosis, the degree of stenosis appears to be the most important predic-tor in determining risk for an ipsilateral stroke. The risk of a recurrent ipsilateral stroke in patients with severe carotid ste-nosis approaches 40%. Two large multicenter randomized clinical trials, the European Carotid Surgery Trial (ECST) and the North American Symptomatic Carotid Endarterectomy Trial (NASCET), have both shown a significant risk reduction in stroke for patients with symptomatic high-grade stenosis (70–99%) undergoing carotid endarterectomy when compared to medical therapy alone.20,21 Although there has been much discussion regarding the different methodologies used in the measurement of carotid stenosis and calculation of

1	when compared to medical therapy alone.20,21 Although there has been much discussion regarding the different methodologies used in the measurement of carotid stenosis and calculation of the life-table data between the two studies, both of these studies had similar clinical results. The findings of these two landmark tri-als have also been reanalyzed in many subsequent publications. The main conclusions of the trials remain validated and widely acknowledged. Briefly, the NASCET study showed that for high-grade carotid stenosis, the cumulative risk of ipsilateral stroke was 26% in the medically treated group and 9% in the surgically treated group at 2 years. For patients with moderate carotid artery stenosis (50–69%), the benefit of carotid endar-terectomy is less but still favorable when compared to medical treatment alone; the 5-year fatal or nonfatal ipsilateral stroke rate was 16% in the surgically treated group versus 22% in the medically treated group.22 The risk of stroke was

1	compared to medical treatment alone; the 5-year fatal or nonfatal ipsilateral stroke rate was 16% in the surgically treated group versus 22% in the medically treated group.22 The risk of stroke was similar for the remaining group of symptomatic patients with less than 50% carotid stenosis, whether they had endarterectomy or medical treatment alone. The ECST reported similar stroke risk reduc-tion for patients with severe symptomatic carotid stenosis and no benefit in patients with mild stenosis when carotid endarter-ectomy was performed versus medical therapy.21The optimal timing of carotid intervention after acute stroke, however, remains debatable. Earlier studies showed an increased rate of postoperative stroke exacerbation and conver-sion of a bland to hemorrhagic infarction when carotid endarter-ectomy was carried out within 5 to 6 weeks after acute stroke. The dismal outcome reported in the early experience was likely related to poor patient selection. The rate of stroke

1	carotid endarter-ectomy was carried out within 5 to 6 weeks after acute stroke. The dismal outcome reported in the early experience was likely related to poor patient selection. The rate of stroke recurrence is not insignificant during the interval period and may be reduced with early intervention for symptomatic carotid stenosis. Con-temporary series have demonstrated acceptable low rates of perioperative complications in patients undergoing carotid end-arterectomy within 4 weeks after acute stroke.22 In a recent ret-rospective series, carotid artery stenting when performed early (<2 weeks) after the acute stroke was associated with higher mortality than when delayed (>2 weeks).23Asymptomatic Carotid Stenosis. Whereas there is univer-sal agreement that carotid revascularization (endarterectomy or stenting) is effective in secondary stroke prevention for patients with symptomatic moderate and severe carotid stenosis, the management of asymptomatic patients remains an important

1	or stenting) is effective in secondary stroke prevention for patients with symptomatic moderate and severe carotid stenosis, the management of asymptomatic patients remains an important controversy to be resolved. Generally, the detection of carotid stenosis in asymptomatic patients is related to the presence of a cervical bruit or based on screening duplex ultrasound findings. In one of the earlier observational studies, the authors showed that the annual occurrence rate of neurologic symptoms was 4% in a cohort of 167 patients with asymptomatic cervical bruits followed prospectively by serial carotid duplex scan.24 The mean annual rate of carotid stenosis progression to a greater than 50% stenosis was 8%. The presence of or progression to a greater than 80% stenosis correlated highly with either the development of a total occlusion of the internal carotid artery or new symptoms. The major risk factors associated with disease progression were cigarette smoking, diabetes mellitus, and

1	either the development of a total occlusion of the internal carotid artery or new symptoms. The major risk factors associated with disease progression were cigarette smoking, diabetes mellitus, and age. This study supported the contention that it is prudent to fol-low a conservative course in the management of asymptomatic patients presenting with a cervical bruit.One of the first randomized clinical trials on the treatment of asymptomatic carotid artery stenosis was the Asymptomatic Carotid Atherosclerosis Study (ACAS), which evaluated the benefits of medical management with antiplatelet therapy versus carotid endarterectomy.25 Over a 5-year period, the risk of ipsi-lateral stroke in individuals with a carotid artery stenosis greater than 60% was 5.1% in the surgical arm. On the other hand, the risk of ipsilateral stroke in patients treated with medical man-agement was 11%. Carotid endarterectomy produced a relative risk reduction of 53% over medical management alone. The results of

1	the risk of ipsilateral stroke in patients treated with medical man-agement was 11%. Carotid endarterectomy produced a relative risk reduction of 53% over medical management alone. The results of a larger randomized trial from Europe, the Asymptom-atic Carotid Surgery Trial (ACST), recently confirmed similar beneficial stroke risk reduction for patients with asymptomatic, greater than 70% carotid stenosis undergoing endarterectomy versus medical therapy.26 An important point derived from this latter trial was that even with improved medical therapy, includ-ing the addition of statin drugs and clopidogrel, medical ther-apy was still inferior to endarterectomy in the primary stroke prevention for patients with high-grade carotid artery stenosis. It is generally agreed that asymptomatic patients with severe carotid stenosis (80–99%) are at significantly increased risk for stroke and stand to benefit from either surgical or endovascular revascularization. However, revascularization for

1	with severe carotid stenosis (80–99%) are at significantly increased risk for stroke and stand to benefit from either surgical or endovascular revascularization. However, revascularization for asymptomatic patients with a less severe degree of stenosis (60–79%) remains controversial.Carotid Endarterectomy Versus Angioplasty and StentingCurrently, the argument is no longer whether medical therapy alone is inferior to surgical endarterectomy in stroke preven-tion for severe carotid stenosis. Rather, the debate now revolves around whether carotid angioplasty and stenting produce the same benefits demonstrated by carotid endarterectomy. Since carotid artery stenting was approved by the FDA for clinical application in 2004, this percutaneous procedure has become a treatment alternative in patients who are deemed “high risk” for endarterectomy (Table 23-4). In contrast to many endovascular peripheral arterial interventions, percutaneous carotid stent-ing represents a much more challenging

1	who are deemed “high risk” for endarterectomy (Table 23-4). In contrast to many endovascular peripheral arterial interventions, percutaneous carotid stent-ing represents a much more challenging procedure, because it requires complex catheter-based skills using the 0.014-inch guidewire system and distal protection device. Moreover, cur-rent carotid stent devices predominantly use the monorail guide-wire system, which requires more technical agility compared with the over-the-wire catheter system that is routinely used in peripheral interventions. This percutaneous intervention often requires balloon angioplasty and stent placement through a long carotid guiding sheath via a groin approach. Poor techni-cal skills can result in devastating treatment complications such as stroke, which can occur in part due to plaque embolization during the balloon angioplasty and stenting of the carotid artery. Because of these various procedural components that require high technical proficiency, many

1	in part due to plaque embolization during the balloon angioplasty and stenting of the carotid artery. Because of these various procedural components that require high technical proficiency, many early clinical investigations of carotid artery stenting, which included physicians with little or no carotid stenting experience, resulted in alarmingly poor Brunicardi_Ch23_p0897-p0980.indd 91127/02/19 4:14 PM 912SPECIFIC CONSIDERATIONSPART IITable 23-4Conditions qualifying patients as high surgical risk for carotid endarterectomyANATOMIC FACTORSPHYSIOLOGIC FACTORS• High carotid bifurcation (above C2 vertebral body)• Low common carotid artery (below clavicle)• Contralateral carotid occlusion• Restenosis of ipsilateral prior carotid endarterectomy• Previous neck irradiation• Prior radical neck dissection• Contralateral laryngeal nerve palsy• Presence of tracheostomy• Age ≥80 years• Left ventricular ejection fraction ≤30%• New York Heart Association class III/IV congestive heart

1	neck dissection• Contralateral laryngeal nerve palsy• Presence of tracheostomy• Age ≥80 years• Left ventricular ejection fraction ≤30%• New York Heart Association class III/IV congestive heart failure• Unstable angina: Canadian Cardiovascular Society class III/IV angina pectoris• Recent myocardial infarction• Clinically significant cardiac disease (congestive heart failure, abnormal stress test, or need for coronary revascularization)• Severe chronic obstructive pulmonary disease• End-stage renal disease on dialysisclinical outcomes. A Cochrane review noted that, before 2006, a total of 1269 patients had been studied in five randomized controlled trials comparing percutaneous carotid intervention and surgical carotid reconstruction.27 Taken together, these trials revealed that carotid artery stenting had a greater procedural risk of stroke and death when compared to carotid endarterectomy (odds ratio, 1.33; 95% confidence interval, 0.86–2.04). Addi-tionally, a greater incidence of

1	artery stenting had a greater procedural risk of stroke and death when compared to carotid endarterectomy (odds ratio, 1.33; 95% confidence interval, 0.86–2.04). Addi-tionally, a greater incidence of carotid restenosis was noted in the stenting group than the endarterectomy cohorts.However, the constant improvement of endovascular devices, procedural techniques, and adjunctive pharmacologic therapy will likely improve the treatment success of percutane-ous carotid intervention. Critical appraisals of several prospec-tive randomized trials comparing the efficacy of carotid stenting versus endarterectomy are available for review.28 Two recently published randomized controlled trial, the Carotid Revascu-larization Endarterectomy Versus Stent Trial (CREST) and the International Carotid Stenting Study (ICSS) have reported somewhat differing results.29 CREST compared the efficacy of carotid endarterectomy and carotid stenting in both symptom-atic and asymptomatic patients.30 Primary end

1	Study (ICSS) have reported somewhat differing results.29 CREST compared the efficacy of carotid endarterectomy and carotid stenting in both symptom-atic and asymptomatic patients.30 Primary end points included 30-day periprocedural composite death, stroke, myocardial infarction, or any ipsilateral stroke up to 4 years. CREST inves-tigators reported no difference between stenting (5.2%) and endarterectomy (4.5%) in terms of primary end point. When each variable was independently analyzed, there was a higher rate of stroke in the stenting group at 30 days (4.1% vs. 2.3%) and a higher rate of myocardial infarction in the endarterectomy group (2.3% vs. 1.1%). The ICSS was a multicenter, interna-tional, randomized controlled trial comparing carotid stenting versus endarterectomy in patients with symptomatic carotid ste-nosis.31 The risk of stroke, death, and myocardial infarction in the stenting group (8.5%) was significantly higher than in the surgical arm (5.2%). The finding that carotid

1	symptomatic carotid ste-nosis.31 The risk of stroke, death, and myocardial infarction in the stenting group (8.5%) was significantly higher than in the surgical arm (5.2%). The finding that carotid endarterectomy is safer than carotid stenting is also supported by the results of an MRI substudy, which showed significantly more new lesions by diffusion-weighted imaging in the carotid stenting than the carotid endarterectomy patients.All available randomized studies have provided some answers and raised some questions. Some ongoing clinical tri-als will undoubtedly provide more insights on the efficacy of carotid stenting in the near future. Currently, the Society for Vascular Surgeons recommends carotid endarterectomy as first-line treatment for most symptomatic patients with stenosis of 50% to 99% and asymptomatic patients with stenosis of 60% to 99%.32 The perioperative risk of stroke and death in asymp-tomatic patients must be below 3% to ensure benefit for the patient. Carotid

1	of 50% to 99% and asymptomatic patients with stenosis of 60% to 99%.32 The perioperative risk of stroke and death in asymp-tomatic patients must be below 3% to ensure benefit for the patient. Carotid artery stenting should be reserved for symptom-atic patients with stenosis of 50% to 99% at high risk for carotid endarterectomy for anatomic or medical reasons. Carotid artery stenting is not recommended for asymptomatic patients at this time. Asymptomatic patients at high risk for intervention or with a life expectancy of less than 3 years should be considered for medical management as the first-line therapy.Surgical Techniques of Carotid EndarterectomyAlthough carotid endarterectomy is one of the earliest vas-cular operations ever described and its techniques have been perfected in the last two decades, surgeons continue to debate many aspects of this procedure. For instance, there is no uni-versal agreement with regard to the best anesthetic of choice, the best intraoperative cerebral

1	two decades, surgeons continue to debate many aspects of this procedure. For instance, there is no uni-versal agreement with regard to the best anesthetic of choice, the best intraoperative cerebral monitoring, whether to “rou-tinely” shunt, open versus eversion endarterectomy, and patch versus primary closure. Various anesthetic options are avail-able for patient undergoing carotid endarterectomy including general, local, and regional anesthesia. Typically, the anesthe-sia of choice depends on the preference of the surgeon, anes-thesiologist, and patient. However, depending on the anesthetic given, the surgeon must decide whether intraoperative cerebral monitoring is necessary or intra-arterial carotid shunting will be used. In general, if the patient is awake, then his or her abilities to respond to commands during carotid clamp period determine the adequacy of collateral flow to the ipsilateral hemisphere. On the other hand, intraoperative electroencepha-logram (EEG) or

1	abilities to respond to commands during carotid clamp period determine the adequacy of collateral flow to the ipsilateral hemisphere. On the other hand, intraoperative electroencepha-logram (EEG) or transcranial power Doppler (TCD) has been used to monitor for adequacy of cerebral perfusion during the clamp period for patients undergoing surgery under general anesthesia. Focal ipsilateral decreases in amplitudes and slow-ing of EEG waves are indicative of cerebral ischemia. Simi-larly, a decrease to less than 50% of baseline velocity in the ipsilateral middle cerebral artery is a sign of cerebral ischemia. For patients with poor collateral flow exhibiting signs of cere-bral ischemia, intra-arterial carotid shunting with removal of the clamp will restore cerebral flow for the remaining part of the surgery. Stump pressures have been used to determine the need for intra-arterial carotid shunting. Some surgeons prefer to shunt all patients on a routine basis and do not use intraop-erative

1	of the surgery. Stump pressures have been used to determine the need for intra-arterial carotid shunting. Some surgeons prefer to shunt all patients on a routine basis and do not use intraop-erative cerebral monitoring.Brunicardi_Ch23_p0897-p0980.indd 91227/02/19 4:14 PM 913ARTERIAL DISEASECHAPTER 23Figure 23-17. To perform carotid endarterectomy, the patient’s neck is slightly hyperextended and turned to the contralateral side. An oblique incision is made along the anterior border of the ster-nocleidomastoid muscle centered on top of the carotid bifurcation.The patient’s neck is slightly hyperextended and turned to the contralateral side, with a roll placed between the shoulder blades. An oblique incision is made along the anterior border of the sternocleidomastoid muscle centered on top of the carotid bifurcation (Fig. 23-17). The platysma is divided completely. Typically, tributaries of the anterior jugular vein are ligated and divided. The dissection is carried medial to the

1	top of the carotid bifurcation (Fig. 23-17). The platysma is divided completely. Typically, tributaries of the anterior jugular vein are ligated and divided. The dissection is carried medial to the sternocleido-mastoid. The superior belly of the omohyoid muscle is usually encountered just anterior to the common carotid artery. This muscle can be divided. The carotid fascia is incised, and the common carotid artery is exposed. The common carotid artery is mobilized cephalad toward the bifurcation. The dissection of the carotid bifurcation can cause reactive bradycardia related to stimulation of the carotid body. This reflex can be blunted with injection of lidocaine 1% into the carotid body or reversed with administration of intravenous atropine. A useful landmark in the dissection of the carotid bifurcation is the common facial vein. This vein can be ligated and divided. Frequently the 12th cranial nerve (hypoglossal nerve) traverses the carotid bifurcation just behind the common

1	the carotid bifurcation is the common facial vein. This vein can be ligated and divided. Frequently the 12th cranial nerve (hypoglossal nerve) traverses the carotid bifurcation just behind the common facial vein. The external carotid artery is mobilized just enough to get a clamp across. Often, a branch of the external carotid artery crossing to the sternocleidomas-toid can be divided to allow further cephalad mobilization of the internal carotid artery. For high bifurcation, division of the posterior belly of the digastric muscle is helpful in establishing distal exposure of the internal carotid artery.Intravenous heparin sulfate (1 mg/kg) is routinely admin-istered just prior to carotid clamping. The internal carotid artery is clamped first using a soft noncrushing vascular clamp to prevent distal embolization. The external and common carotid arteries are clamped subsequently. A longitudinal arteriotomy is made in the distal common carotid artery and extended into the bulb and past

1	distal embolization. The external and common carotid arteries are clamped subsequently. A longitudinal arteriotomy is made in the distal common carotid artery and extended into the bulb and past the occlusive plaque into the normal part of the internal carotid artery. Endarterectomy is carried out to remove the occlusive plaque (Fig. 23-18). If necessary, a tem-porary shunt can be inserted from the common carotid artery to the internal carotid artery to maintain continuous antegrade cerebral blood flow (Fig. 23-19). Typically, a plane is teased out from the vessel wall, and the entire plaque is elevated and Figure 23-18. A. During carotid endarterectomy, vascular clamps are applied in the common carotid, external carotid, and internal carotid arteries. Carotid plaque is elevated from the carotid lumen. B. Carotid plaque is removed, and the arteriotomy is closed either primarily or with a patch angioplasty.Figure 23-19. A temporary carotid shunt is inserted from the com-mon carotid

1	carotid lumen. B. Carotid plaque is removed, and the arteriotomy is closed either primarily or with a patch angioplasty.Figure 23-19. A temporary carotid shunt is inserted from the com-mon carotid artery (long arrow) to the internal carotid artery (short arrow) during carotid endarterectomy to provide continuous ante-grade cerebral blood flow.removed. The distal transition line in the internal carotid artery where the plaque had been removed must be examined care-fully and should be smooth. Tacking sutures are placed when an intimal flap remains in this transition to ensure no obstruction to flow (Fig. 23-20). The occlusive plaque is usually removed from the origin of the external carotid artery using the ever-sion technique. The endarterectomized surface is then irrigated and any debris removed. A patch (autogenous saphenous vein, synthetic such as polyester, PTFE, or biologic material) is sewn to close the arteriotomy (Fig. 23-21). Whether patch closure is necessary in all patients

1	removed. A patch (autogenous saphenous vein, synthetic such as polyester, PTFE, or biologic material) is sewn to close the arteriotomy (Fig. 23-21). Whether patch closure is necessary in all patients and which patch is the best remain con-troversial. However, most surgeons agree that patch closure is Brunicardi_Ch23_p0897-p0980.indd 91327/02/19 4:14 PM 914SPECIFIC CONSIDERATIONSPART IIFigure 23-20. The distal transition line (left side of the picture) in the internal carotid artery where the plaque had been removed must be examined carefully and should be smooth. Tacking sutures (arrows) are placed when an intimal flap remains in this transition to ensure no obstruction to flow.Figure 23-21. A. An autologous or synthetic patch can be used to close the carotid arteriotomy incision, which maintains the luminal patency. B. A completion closure of carotid endarterectomy incision using a synthetic patch.BAindicated particularly for the small vessel (<7 mm). The ever-sion technique has

1	maintains the luminal patency. B. A completion closure of carotid endarterectomy incision using a synthetic patch.BAindicated particularly for the small vessel (<7 mm). The ever-sion technique has also been advocated for removing the plaque from the internal carotid artery. In the eversion technique, the internal carotid artery is transected at the bulb, the edges of the divided vessel are everted, and the occluding plaque is “peeled” off the vessel wall. The purported advantages of the eversion technique are no need for patch closure and a clear visualization of the distal transition area. Reported series have not shown a clear superiority of one technique over the others.33 Surgeons will likely continue to use the technique of their choice. Just prior to completion of the anastomosis to close the arteriotomy, we thoroughly flush the vessels of any potential debris. When the arteriotomy is closed, flow is restored to the external carotid artery first and to the internal carotid

1	to close the arteriotomy, we thoroughly flush the vessels of any potential debris. When the arteriotomy is closed, flow is restored to the external carotid artery first and to the internal carotid artery second. Intravenous protamine sulfate can be given to reverse the effect of heparin anticoagulation following carotid endarterectomy. The wound is closed in layers. After surgery, the patient’s neurologic con-dition is asserted in the operating room prior to transfer to the recovery area.Complications of Carotid Endarterectomy. Most patients tolerate carotid endarterectomy very well and typically are discharged home within 24 hours after surgery. Complica-tions after endarterectomy are infrequent but can be poten-tially life-threatening or disabling. Acute ipsilateral stroke is a dreaded complication following carotid endarterectomy. Cerebral ischemia can be due to either intraoperative or post-operative events. Embolizations from the occlusive plaque or prolonged cerebral ischemia

1	complication following carotid endarterectomy. Cerebral ischemia can be due to either intraoperative or post-operative events. Embolizations from the occlusive plaque or prolonged cerebral ischemia are potential causes of intraopera-tive stroke. The most common cause of postoperative stroke is due to embolization. Less frequently, acute carotid artery occlu-sion can cause acute postoperative stroke. This is usually due to carotid artery thrombosis related to closure of the arteriotomy, an occluding intimal flap, or distal carotid dissection. When patients experience acute symptoms of neurologic ischemia after endarterectomy, immediate intervention may be indicated. Carotid duplex scan can be done expeditiously to assess patency of the extracranial internal carotid artery. Reexploration is man-dated for acute carotid artery occlusion. Cerebral angiography can be useful if intracranial revascularization is considered.Local complications related to surgery include excessive bleeding and

1	man-dated for acute carotid artery occlusion. Cerebral angiography can be useful if intracranial revascularization is considered.Local complications related to surgery include excessive bleeding and cranial nerve palsies. Postoperative hematoma in the neck after carotid endarterectomy can lead to devastating airway compromise. Any expanding hematoma should be evac-uated and active bleeding stopped. Securing an airway is critical and can be extremely difficult in patients with large postopera-tive neck hematoma. The reported incidence of postoperative cranial nerve palsies after carotid endarterectomy varies from 1% to 30%.34 Well-recognized injuries involve the marginal mandibular, vagus, hypoglossal, superior laryngeal, and recur-rent laryngeal nerves. Often these are traction injuries but can also be due to severance of the respective nerves.Techniques of Carotid Angioplasty and StentingPercutaneous carotid artery stenting has become an accepted alternative treatment in the

1	but can also be due to severance of the respective nerves.Techniques of Carotid Angioplasty and StentingPercutaneous carotid artery stenting has become an accepted alternative treatment in the management of patients with carotid bifurcation disease (Fig. 23-22). The perceived advantages of percutaneous carotid revascularization are related to the mini-mal invasiveness of the procedure compared to surgery. There are anatomic conditions based on angiographic evaluation in which carotid artery stenting should be avoided due to increased procedure-related risks (Table 23-5). In preparation for carotid stenting, the patient should be given oral clopidogrel 3 days prior to the intervention if the patient was not already taking the drug. The procedure is done in either the operating room with angiographic capabilities or in a dedicated angiography room. The patient is placed in the supine position. The patient’s blood pressure and cardiac rhythm are closely monitored.To gain access to the

1	angiographic capabilities or in a dedicated angiography room. The patient is placed in the supine position. The patient’s blood pressure and cardiac rhythm are closely monitored.To gain access to the carotid artery, a retrograde trans-femoral approach is most commonly used as the access site for carotid intervention. Using the Seldinger technique, we insert a diagnostic 5or 6-French sheath in the CFA. A diagnostic arch Brunicardi_Ch23_p0897-p0980.indd 91427/02/19 4:14 PM 915ARTERIAL DISEASECHAPTER 23BAFigure 23-22. A. Carotid angiogram demonstrating a high-grade stenosis of the left internal carotid artery. B. Com-pletion angiogram demonstrating a sat-isfactory result following a carotid stent placement.Table 23-5Unfavorable carotid angiographic appearance in which carotid stenting should be avoided• Extensive carotid calcification• Polypoid or globular carotid lesions• Severe tortuosity of the common carotid artery• Long-segment stenoses (>2 cm in length)• Carotid artery

1	should be avoided• Extensive carotid calcification• Polypoid or globular carotid lesions• Severe tortuosity of the common carotid artery• Long-segment stenoses (>2 cm in length)• Carotid artery occlusion• Severe intraluminal thrombus (angiographic defects)• Extensive middle cerebral artery atherosclerosisaortogram is obtained. The carotid artery to be treated is then selected using a 5-French diagnostic catheter, and contrast is injected to show the carotid anatomy. It is important to assess the contralateral carotid artery, vertebrobasilar, and intracranial circulation if these are not known based on the preoperative noninvasive studies. Once the decision is made to proceed with carotid artery stenting, with the tip of the diagnostic catheter still in the common carotid artery, a 0.035-inch, 260-cm long stiff guidewire is placed in the ipsilateral external carotid artery. Anticoagulation with intravenous bivalirudin bolus (0.75 mg/kg) followed by an infusion rate of 2.5 mg/kg per

1	260-cm long stiff guidewire is placed in the ipsilateral external carotid artery. Anticoagulation with intravenous bivalirudin bolus (0.75 mg/kg) followed by an infusion rate of 2.5 mg/kg per hour for the remain-der of the procedure is routinely administered. Next the diagnos-tic catheter is withdrawn and a 90-cm, 6-French guiding sheath is advanced into the common carotid artery over the stiff glide wire. It is critical not to advance the sheath beyond the occlu-sive plaque in the carotid bulb. The stiff wire is then removed, and preparation is made to deploy the distal embolic protection device (EPD). Several distal EPDs are available (Table 23-6). The EPD device is carefully deployed beyond the target lesion. With regard to the carotid stents, there are several stents that have received approval from the FDA and are commercially Table 23-6Commonly used embolic protection devices (EPDs)MECHANISMNAME OF EPDPORE SIZE (μM)Distal balloon occlusionPercuSurge Guard Wire, Export catheter

1	approval from the FDA and are commercially Table 23-6Commonly used embolic protection devices (EPDs)MECHANISMNAME OF EPDPORE SIZE (μM)Distal balloon occlusionPercuSurge Guard Wire, Export catheter (Medtronic)NADistal filterAngioguard (Cordis)Accunet (Abbott)Emboshield (Abbott)FilterWire (Boston Scientific)SpiderRx (EV3)100150140110<100Flow reversalaParodi Neuro Protection (Gore)NAaClinical trial (EMPIRE) in United States.NA = not applicable.available for carotid revascularization (Table 23-7). All current carotid stents use the rapid-exchange monorail 0.014-inch plat-form. The size selection is typically based on the size of com-mon carotid artery. Predilatation using a 4-mm balloon may be necessary to allow passage of the stent delivery catheter. Once the stent is deployed across the occlusive plaque, postdilatation is usually performed using a ≤5.5-mm balloon. It is noteworthy that balloon dilation of the carotid bulb may lead to immediate bradycardia due to stimulation of the

1	occlusive plaque, postdilatation is usually performed using a ≤5.5-mm balloon. It is noteworthy that balloon dilation of the carotid bulb may lead to immediate bradycardia due to stimulation of the glossopharyngeal nerve. The EPD is then retrieved and the procedure is completed with removal of the sheath from the femoral artery. The puncture site is closed using available closure device or with manual compression. Throughout the procedure, the patient’s neuro-logic function is closely monitored. The bivalirudin infusion is Brunicardi_Ch23_p0897-p0980.indd 91527/02/19 4:14 PM 916SPECIFIC CONSIDERATIONSPART IITable 23-7Currently approved carotid stents in the United StatesNAME OF STENTMANUFACTURERCELL DESIGNTAPERED STENTDELIVERY SYSTEM SIZE (FRENCH)AcculinkAbbottOpenYes6ExactAbbottClosedYes6NexStentBoston ScientificClosedSelf-tapering5Protégé RXEV3OpenYes6Precise RXCordisOpenNo6ExponentMedtronicOpenNo6Figure 23-23. Excessive elongation of the carotid artery can result in carotid

1	ScientificClosedSelf-tapering5Protégé RXEV3OpenYes6Precise RXCordisOpenNo6ExponentMedtronicOpenNo6Figure 23-23. Excessive elongation of the carotid artery can result in carotid kinking (arrow), which can compromise cerebral blood flow and lead to cerebral ischemia.stopped and the patient is kept on clopidogrel (75 mg daily) for at least 1 month and aspirin indefinitely.Complications of Carotid Stenting. Although there have been no randomized trials comparing carotid stenting with and without EPD, the availability of EPDs appears to have reduced the risk of distal embolization and stroke. The results of the various clinical trials and registries of carotid stenting have been reported and compared. It is well known that distal embolization as detected by TCD is much more frequent with carotid stenting even with EPD, when compared with carotid endarterectomy. However, the clinical significance of the distal embolization detected by TCD is not clear because most are asymptomatic. Acute

1	stenting even with EPD, when compared with carotid endarterectomy. However, the clinical significance of the distal embolization detected by TCD is not clear because most are asymptomatic. Acute carotid stent thrombosis is rare. The incidence of in-stent carotid restenosis is not well known but is estimated at 10% to 30%. Duplex surveillance shows elevated peak systolic velocities within the stent after carotid stenting not infrequently. However, velocity criteria are being formulated to determine the severity of in-stent restenosis after carotid stenting by ultra-sound duplex.35 It appears that systolic velocities exceeding 300 to 400 cm/s would represent >70% to 80% restenosis. Bradycar-dia and hypotension occur in up to 20% of patients undergoing carotid stenting.36 Systemic administration of atropine is usually effective in reversing the bradycardia. Other technical compli-cations of carotid stenting are infrequent and include carotid artery dissection and access site

1	administration of atropine is usually effective in reversing the bradycardia. Other technical compli-cations of carotid stenting are infrequent and include carotid artery dissection and access site complications, such as groin hematoma, femoral artery pseudoaneurysm, distal embolization, and acute femoral artery thrombosis.Nonatherosclerotic Disease of the Carotid ArteryCarotid Coil and Kink. A carotid coil consists of an excessive elongation of the internal carotid artery producing tortuosity of the vessel (Fig. 23-23). Embryologically, the carotid artery is derived from the third aortic arch and dorsal aortic root and is uncoiled as the heart and great vessels descend into the medi-astinum. In children, carotid coils appear to be congenital in origin. In contrast, elongation and kinking of the carotid artery in adults are associated with the loss of elasticity and an abrupt angulation of the vessel. Kinking is more common in women than men. Cerebral ischemic symptoms caused by

1	of the carotid artery in adults are associated with the loss of elasticity and an abrupt angulation of the vessel. Kinking is more common in women than men. Cerebral ischemic symptoms caused by kinks of the carotid artery are similar to those from atherosclerotic carotid lesions but are more likely due to cerebral hypoperfusion than embolic episodes. Classically, sudden head rotation, flexion, or extension can accentuate the kink and provoke ischemic symp-toms. Most carotid kinks and coils are found incidentally on carotid duplex scan. However, interpretation of the Doppler fre-quency shifts and spectral analysis in tortuous carotid arteries can be difficult because of the uncertain angle of insonation. Cerebral angiography, with multiple views taken in neck flex-ion, extension, and rotation, is useful in the determination of the clinical significance of kinks and coils.Fibromuscular Dysplasia. Fibromuscular dysplasia (FMD) usually involves medium-sized arteries that are long and have

1	is useful in the determination of the clinical significance of kinks and coils.Fibromuscular Dysplasia. Fibromuscular dysplasia (FMD) usually involves medium-sized arteries that are long and have few branches (Fig. 23-24). Women in the fourth or fifth decade of life are more commonly affected than men. Hormonal effects on the vessel wall are thought to play a role in the pathogenesis of FMD. FMD of the carotid artery is commonly bilateral, and in about 20% of patients, the vertebral artery is also involved.37 Brunicardi_Ch23_p0897-p0980.indd 91627/02/19 4:14 PM 917ARTERIAL DISEASECHAPTER 23Figure 23-24. A carotid fibromuscular dysplasia with typical characteristics of multiple stenoses with intervening aneurysmal outpouching dilatations. The disease involves the media, with the smooth muscle being replaced by fibrous connective tissue.An intracranial saccular aneurysm of the carotid siphon or mid-dle cerebral artery can be identified in up to 50% of the patients with FMD. Four

1	muscle being replaced by fibrous connective tissue.An intracranial saccular aneurysm of the carotid siphon or mid-dle cerebral artery can be identified in up to 50% of the patients with FMD. Four histologic types of FMD have been described in the literature. The most common type is medial fibroplasia, which may present as a focal stenosis or multiple lesions with intervening aneurysmal outpouchings. The disease involves the media with the smooth muscle being replaced by fibrous con-nective tissue. Commonly, mural dilations and microaneurysms can be seen with this type of FMD. Medial hyperplasia is a rare type of FMD, with the media demonstrating excessive amounts of smooth muscle. Intimal fibroplasia accounts for 5% of all cases and occurs equally in both sexes. The media and adven-titia remain normal, and there is accumulation of subendothe-lial mesenchymal cells with a loose matrix of connective tissue causing a focal stenosis in adults. Finally, premedial dyspla-sia represents a

1	remain normal, and there is accumulation of subendothe-lial mesenchymal cells with a loose matrix of connective tissue causing a focal stenosis in adults. Finally, premedial dyspla-sia represents a type of FMD with elastic tissue accumulating between the media and adventitia. FMD can also involve the renal and external iliac arteries. It is estimated that approxi-mately 40% of patients with FMD present with a TIA due to embolization of platelet aggregates.37 DSA demonstrates the characteristic “string of beads” pattern, which represents alter-nating segments of stenosis and dilatation. The string of beads can also be shown noninvasively by CTA or MRA. FMD should be suspected when an increased velocity is detected across a stenotic segment without associated atherosclerotic changes on carotid duplex ultrasound. Antiplatelet medication is the gener-ally accepted therapy for asymptomatic lesions. Endovascular treatment is recommended for patients with documented lateral-izing symptoms.

1	duplex ultrasound. Antiplatelet medication is the gener-ally accepted therapy for asymptomatic lesions. Endovascular treatment is recommended for patients with documented lateral-izing symptoms. Surgical correction is rarely indicated.Carotid Artery Dissection. Dissection of the carotid artery accounts for approximately 20% of strokes in patients younger than 45 years of age. The etiology and pathogenesis of sponta-neous carotid artery dissection remain incompletely understood. Arterial dissection involves hemorrhage within the media, which can extend into the subadventitial and subintimal layers. When the dissection extends into the subadventitial space, there is an increased risk of aneurysm formation. Subintimal dissections can lead to intramural clot or thrombosis. Traumatic dissection is typically a result of hyperextension of the neck during blunt trauma, neck manipulation, strangulation, or penetrating injuries to the neck. Even in supposedly spontaneous cases, a history of

1	is typically a result of hyperextension of the neck during blunt trauma, neck manipulation, strangulation, or penetrating injuries to the neck. Even in supposedly spontaneous cases, a history of preceding unrecognized minor neck trauma is not uncommon. Connective disorders, such as Ehlers-Danlos syndrome, Marfan’s syndrome, α1-antitrypsin deficiency, or FMD, may predispose to carotid artery dissection. Iatrogenic dissections can also occur due to catheter manipulation or balloon angioplasty.Typical clinical features of carotid artery dissection include unilateral neck pain, headache, and ipsilateral Horner’s syndrome in up to 50% of patients, followed by manifestations of the cerebral or ocular ischemia and cranial nerve palsies. Neurologic deficits can result either because of hemodynamic failure (caused by luminal stenosis) or by an artery-to-artery thromboembolism. The ischemia may cause TIAs or infarctions, or both. Catheter angiography has been the method of choice to diagnose

1	failure (caused by luminal stenosis) or by an artery-to-artery thromboembolism. The ischemia may cause TIAs or infarctions, or both. Catheter angiography has been the method of choice to diagnose arterial dissections, but with the advent of duplex ultra-sonography, MRI/MRA, and CTA, most dissections can now be diagnosed using noninvasive imaging modalities (Fig. 23-25). The dissection typically starts in the internal carotid artery distal to the bulb. Uncommonly, the dissection can start in the com-mon carotid artery or is an extension of a more proximal aortic dissection. Medical therapy has been the accepted primary treat-ment of symptomatic of carotid artery dissection. Anticoagula-tion (heparin and warfarin) and antiplatelet therapy have been commonly used, although there have not been any randomized studies to evaluate their effectiveness. The prognosis depends on the severity of neurologic deficit but is generally good in extracranial dissections. The recurrence rate is low.

1	been any randomized studies to evaluate their effectiveness. The prognosis depends on the severity of neurologic deficit but is generally good in extracranial dissections. The recurrence rate is low. Therapeutic interventions have been reserved for recurrent TIAs or strokes or failure of medical treatment. Endovascular options include intra-arterial stenting, coiling of associated pseudoaneurysms, or, more recently, deployment of covered stents.Figure 23-25. Carotid ultrasound reveals a patient with a carotid artery dissection in which carotid flow is separated in the true flow lumen (long arrow) from the false lumen (short arrow).Brunicardi_Ch23_p0897-p0980.indd 91727/02/19 4:14 PM 918SPECIFIC CONSIDERATIONSPART IIFigure 23-26. A. An anteroposterior angiogram of the neck revealing a carotid artery aneurysm. B. A lateral projection of the carotid artery aneurysm. C. Following endovascular placement, the carotid artery aneurysm is successfully excluded.Carotid Artery

1	revealing a carotid artery aneurysm. B. A lateral projection of the carotid artery aneurysm. C. Following endovascular placement, the carotid artery aneurysm is successfully excluded.Carotid Artery Aneurysms. Carotid artery aneurysms are rare, encountered in less than 1% of all carotid operations (Fig. 23-26). The true carotid artery aneurysm is generally due to atherosclerosis or medial degeneration. The carotid bulb is involved in most carotid aneurysms, and bilaterality is present in 12% of the patients. Patients typically present with a pul-satile neck mass. The available data suggest that, untreated, these aneurysms lead to neurologic symptoms from emboliza-tion. Thrombosis and rupture of the carotid aneurysm are rare. Pseudoaneurysms of the carotid artery can result from injury or infection. Mycotic aneurysms often involved syphilis in the past, but they are now more commonly associated with peri-tonsillar abscesses caused by Staphylococcus aureus infection. FMD and spontaneous

1	Mycotic aneurysms often involved syphilis in the past, but they are now more commonly associated with peri-tonsillar abscesses caused by Staphylococcus aureus infection. FMD and spontaneous dissection of the carotid artery can lead to A B CA BFigure 23-27. A. A carotid body tumor (arrow) located adjacent to the carotid bulb. B. Following periad-ventitial dissection, the carotid body tumor is removed.the formation of true aneurysms or pseudoaneurysms. Whereas conventional surgery has been the primary mode of treatment in the past, carotid aneurysms are currently being treated more commonly using endovascular approaches.38Carotid Body Tumor. The carotid body originates from the third branchial arch and from neuroectodermal-derived neural crest lineage. The normal carotid body is located in the adven-titia or periadventitial tissue at the bifurcation of the common carotid artery

1	branchial arch and from neuroectodermal-derived neural crest lineage. The normal carotid body is located in the adven-titia or periadventitial tissue at the bifurcation of the common carotid artery (Fig. 23-27). The gland is innervated by the glos-sopharyngeal nerve. Its blood supply is derived predominantly from the external carotid artery but can also come from the ver-tebral artery. Carotid body tumor is a rare lesion of the neuroen-docrine system. Other glands of neural crest origin are seen in Brunicardi_Ch23_p0897-p0980.indd 91827/02/19 4:14 PM 919ARTERIAL DISEASECHAPTER 23the neck, parapharyngeal spaces, mediastinum, retroperitoneum, and adrenal medulla. Tumors involving these structures have been referred to as paraganglioma, glomus tumor, or chemo-dectoma. Approximately 5% to 7% of carotid body tumors are malignant. Although chronic hypoxemia has been invoked as a stimulus for hyperplasia of carotid body, approximately 35% of carotid body tumors are hereditary.39 The risk

1	7% of carotid body tumors are malignant. Although chronic hypoxemia has been invoked as a stimulus for hyperplasia of carotid body, approximately 35% of carotid body tumors are hereditary.39 The risk of malignancy is greatest in young patients with familial tumors.Symptoms related to the endocrine products of the carotid body tumor are rare. Patients usually present between the fifth and seventh decades of life with an asymptomatic lateral neck mass. The diagnosis of carotid body tumor requires confir-mation on imaging studies. Carotid duplex scan can localize the tumor to the carotid bifurcation, but CT or MRI is usually required to further delineate the relationship of the tumor to the adjacent structures. Classically, a carotid body tumor will widen the carotid bifurcation. The Shamblin classification describes the tumor extent: I, tumor is less than 5 cm and relatively free of vessel involvement; II, tumor is intimately involved but does not encase the vessel wall; and III, tumor

1	classification describes the tumor extent: I, tumor is less than 5 cm and relatively free of vessel involvement; II, tumor is intimately involved but does not encase the vessel wall; and III, tumor is intramural and encases the carotid vessels and adjacent nerves.40 With good-resolution CT and MRI, arteriography is usually not required. However, arteriography can provide an assessment of the vessel invasion and intracranial circulation and allows for preoperative embolization of the feeder vessels, which has been reported to reduce intraoperative blood loss. Surgical resection is the rec-ommended treatment for suspected carotid body tumor.Carotid Trauma. Blunt or penetrating trauma to the neck can cause injury to the carotid artery. Notwithstanding the massive bleeding from carotid artery transection, injury to the carotid artery can result in carotid dissection, thrombosis, or pseudoa-neurysm formation. Carotid duplex ultrasound can be useful to locate the site of injury in the

1	transection, injury to the carotid artery can result in carotid dissection, thrombosis, or pseudoa-neurysm formation. Carotid duplex ultrasound can be useful to locate the site of injury in the cervical segment of the carotid artery. Spiral CTA has become the modality of choice to detect extracranial carotid artery injury. Confirmation of carotid injury by contrast cerebral angiography remains the gold standard diagnostic test. Injuries to the cervical segment of the common and internal carotid arteries can be repaired surgically. Acute carotid artery thrombosis is usually treated medically with anti-coagulation if the patient is asymptomatic. Revascularization should be considered for patients presenting with ongoing cere-bral ischemia related to carotid artery thrombosis. Traumatic carotid artery dissection can cause cerebral ischemia due to thromboembolization, decreased flow, or thrombosis. Com-monly, the dissection involves the distal portion of the cervi-cal and petrous segment

1	artery dissection can cause cerebral ischemia due to thromboembolization, decreased flow, or thrombosis. Com-monly, the dissection involves the distal portion of the cervi-cal and petrous segment of the internal carotid artery. Medical management with antiplatelet or anticoagulation therapy is usu-ally adequate for uncomplicated traumatic carotid dissection. In patients with pseudoaneurysms of the carotid artery that are located in a segment that is out of surgical reach, the use of selective coil embolization of the pseudoaneurysm or exclu-sion of the pseudoaneurysm by a covered stent graft has been reported. Bare metal stent has been used with success in the treatment of traumatic carotid artery dissection.ABDOMINAL AORTIC ANEURYSMDespite more than 50,000 patients undergoing elective repair of abdominal aortic aneurysm (AAA) each year in the United States, approximately 15,000 patients die annually as a result of ruptured aneurysm, making it the 10th leading cause of death in men in

1	of abdominal aortic aneurysm (AAA) each year in the United States, approximately 15,000 patients die annually as a result of ruptured aneurysm, making it the 10th leading cause of death in men in this country.41 The incidence appears to be increasing, and this is due in part to improvements in diagnostic imaging and, more importantly, a growing elderly population. With early diagnosis and timely intervention, aneurysm rupture–related death is largely preventable. Conventional treatment of an AAA involves replacing the aneurysmal segment of the aorta with a prosthetic graft, with the operation performed through a large abdominal incision. Techniques for this open abdominal surgery have been refined, adapted, and extensively studied by vascular surgeons over the past four decades. Despite a well-documented low perioperative mortality rate of 2% to 3% in large academic institutions, the thought of undergoing an open abdominal aortic operation often provokes a sense of anxiety in many

1	a well-documented low perioperative mortality rate of 2% to 3% in large academic institutions, the thought of undergoing an open abdominal aortic operation often provokes a sense of anxiety in many patients due in part to the postoperative pain associated with the large abdominal incision as well as the long recovery time needed before the patient can return to normal physical activity.The most common location of aortic aneurysms is the infrarenal aorta. Endovascular stent graft placement represents a revolutionary and minimally invasive treatment for infrarenal AAAs that only requires 1 to 2 days of hospitalization, and the patient can return to normal physical activity within 1 week. The concept of using an endoluminal device in the management of vascular disease was first proposed by Dotter and colleagues, who successfully treated a patient with iliac occlusion using transluminal angioplasty in 1964.42 Nearly two decades later, Parodi and colleagues reported the first successful

1	Dotter and colleagues, who successfully treated a patient with iliac occlusion using transluminal angioplasty in 1964.42 Nearly two decades later, Parodi and colleagues reported the first successful endovascu-lar repair of AAA using a stent graft device.11 Since then, a variety of stent graft technologies have been developed to treat AAA. The rapid innovation of this new treatment modality has undoubtedly captured the attention of patients with aortic aneu-rysms as well as physicians who practice endovascular therapy. Physicians in general should be knowledgeable regarding avail-able treatment options of AAA in order to provide adequate evaluation and education to patients and their families. The pur-pose of this section is to outline the treatment options for AAAs, including conventional repair and endovascular approach. Advantages and potential complications of these treatments will also be addressed.Causes and Risk FactorsThe pathogenesis of aneurysmal disease of the aorta is

1	repair and endovascular approach. Advantages and potential complications of these treatments will also be addressed.Causes and Risk FactorsThe pathogenesis of aneurysmal disease of the aorta is complex and multifactorial. A degenerative process in the aortic wall is the most common cause of AAA development.43 Matrix metal-loproteinases (MMP), proteolytic enzymes, are found abun-dantly in the wall of AAA. Atherosclerotic disease, age, male sex, smoking history, family history, hypertension, coronary artery disease, and chronic obstructive pulmonary disease are associated with the development of AAA.44 Diabetes and black race have negative association with AAA. Other less common causes include inflammation, infection, and connective tissue disease. Inflammatory AAA accounts for 5% to 10% of all AAAs.43 In contrast to atherosclerotic AAA, the inflammatory variant is characterized pathologically by marked thickening of the aneurysm wall, fibrosis of the adjacent retroperitoneum, and rigid

1	all AAAs.43 In contrast to atherosclerotic AAA, the inflammatory variant is characterized pathologically by marked thickening of the aneurysm wall, fibrosis of the adjacent retroperitoneum, and rigid adherence of the adjacent structures to the anterior aneurysm wall. Male sex and smoking are even stronger risk factors in inflammatory AAA.45 Smoking cessation is the first step of medical therapy, followed by surgical repair. Infectious or mycotic AAA is rare but is associated with high mortality. Patients with connective tissue disorders such as Marfan’s syn-drome and Ehlers-Danlos syndrome tend to have more exten-sive and larger aneurysms at a younger age.43Brunicardi_Ch23_p0897-p0980.indd 91927/02/19 4:14 PM 920SPECIFIC CONSIDERATIONSPART IITable 23-8Annualized risk of rupture of abdominal aortic aneurysm (AAA) based on sizeDESCRIPTIONDIAMETER OF AORTA (CM)ESTIMATED ANNUAL RISK OF RUPTURE (%)ESTIMATED 5-YEAR RISK OF RUPTURE (%)aNormal aorta2–300 (unless AAA develops)Small

1	of abdominal aortic aneurysm (AAA) based on sizeDESCRIPTIONDIAMETER OF AORTA (CM)ESTIMATED ANNUAL RISK OF RUPTURE (%)ESTIMATED 5-YEAR RISK OF RUPTURE (%)aNormal aorta2–300 (unless AAA develops)Small AAA4–515–10Moderate AAA5–62–530–40Large AAA6–73–10>50Very large AAA>7>10Approaching 100aThe estimated 5-year risk is more than five times the estimated annual risk because over that 5 years, the AAA, if left untreated, will continue to grow in size.Natural History of Aortic AneurysmThe natural history of an AAA is to expand and rupture. AAA exhibits a “staccato” pattern of growth, where periods of relative quiescence may alternate with expansion. Therefore, although an individual pattern of growth cannot be predicted, average aggregate growth is approximately 3 to 4 mm/year. There is some evidence to suggest that larger aneurysms may expand faster than smaller aneurysms, but there is significant overlap between the ranges of growth rates at each strata of size.Rupture risk appears to be

1	to suggest that larger aneurysms may expand faster than smaller aneurysms, but there is significant overlap between the ranges of growth rates at each strata of size.Rupture risk appears to be directly related to aneurysm size as predicted by Laplace’s Law. Although more sophisti-cated methods of assessing rupture risk based on finite element analysis of wall stress are under active investigation, maximum transverse diameter remains the standard method of risk assess-ment for aneurysm rupture. In the past, AAA rupture risk has been overestimated. More recently, two landmark studies have served to better define the natural history of AAA.46,47 Based on best available evidence, the annualized risk of rupture is given in Table 23-8. The rupture risk is quite low below 5.5 cm and begins to rise exponentially thereafter. This size can serve as an appropriate threshold for recommending elective repair provided one’s surgical mortality is below 5%. For each size strata, however, women appear

1	exponentially thereafter. This size can serve as an appropriate threshold for recommending elective repair provided one’s surgical mortality is below 5%. For each size strata, however, women appear to be at higher risk for rup-ture than men, and a lower threshold of 4.5 to 5.0 cm may be reasonable in good-risk patients. Although data are less compel-ling, a pattern of rapid expansion of >0.5 cm within 6 months can be considered a relative indication for elective repair. Aneu-rysms that fall below these indications may safely be followed with CT or ultrasound at 6-month intervals, with long-term out-comes equivalent to earlier surgical repair. Interestingly, in the Aneurysm Detection and Management (ADAM) study, 80% of all AAAs that were followed in this manner eventually came to repair within 5 years.48Unless symptomatic or ruptured, AAA repair is a pro-phylactic repair. The rationale for recommending repair is predicated on the assumption that the risk of aneurysm rupture exceeds the

1	5 years.48Unless symptomatic or ruptured, AAA repair is a pro-phylactic repair. The rationale for recommending repair is predicated on the assumption that the risk of aneurysm rupture exceeds the combined risk of death from all other causes such as cardiopulmonary disease and cancer. On the other hand, our limitation in predicting timing and cause of death is underscored by the observation that over 25% of patients who were deemed unfit for surgical repair because of their comorbidities died from rupture of their aneurysms within 5 years.Clinical ManifestationsMost AAAs are asymptomatic and are usually found inciden-tally during workup for chronic back pain or kidney stones. Physical examination is neither sensitive nor specific except 2in thin patients. Large aneurysms may be missed in the obese, while normal aortic pulsations may be mistaken for an aneurysm in thin individuals. Rarely patients present with back pain and/or abdominal pain with a tender pulsatile mass. Patients with

1	the obese, while normal aortic pulsations may be mistaken for an aneurysm in thin individuals. Rarely patients present with back pain and/or abdominal pain with a tender pulsatile mass. Patients with these symptoms must be treated as a rupture until proven otherwise. If the patient is hemodynamically stable and the aneurysm is intact on a CT scan, the patient is admitted for blood pressure control with intravenous antihypertensive agents and undergoes repair usually within 12 to 24 hours or at least during the same hospitalization. In contrast, patients who are hemodynamically unstable with a history of acute back pain and/or syncope and a known unrepaired AAA or a pulsatile abdominal mass should be immediately taken to the operating room with a presumed diagnosis of a ruptured AAA.Overall mortality of AAA rupture is 71% to 77%, which includes all out-of-hospital and in-hospital deaths, as compared with 2% to 6% for elective open surgical repair.49 Nearly half of all patients with

1	mortality of AAA rupture is 71% to 77%, which includes all out-of-hospital and in-hospital deaths, as compared with 2% to 6% for elective open surgical repair.49 Nearly half of all patients with ruptured AAA will die before reaching the hospital. For the remainder, surgical mortality is 45% to 50% and has not substantially changed in the last 30 years.50Relevant AnatomyAn AAA is defined as a pathologic focal dilation of the aorta that is greater than 30 mm or 1.5 times the adjacent diameter of the normal aorta (Fig. 23-28). Male aortas tend to be larger than female aortas, and there is generalized growth of the aortic diameter with each decade of life. Ninety percent of AAAs are infrarenal in location and have a fusiform morphology. There is a higher predilection for juxtarenal and suprarenal AAAs in women compared with men. Concomitant common iliac and/or hypogastric artery aneurysms can be found in 20% to 25% of patients. Although the etiology of most aortic aneurysms is

1	and suprarenal AAAs in women compared with men. Concomitant common iliac and/or hypogastric artery aneurysms can be found in 20% to 25% of patients. Although the etiology of most aortic aneurysms is ath-erosclerotic, clinically significant peripheral occlusive disease is unusual and present in less than 10% of all cases.Although extravascular anatomy is important for open surgical repair of AAA, intravascular anatomy and aortoiliac morphology are important for endovascular repair. Pertinent anatomic dimensions include the diameter of the proximal non-dilated infrarenal aortic neck, which can range from 18 to 30 mm; common iliac artery, which can range from 8 to 16 mm; and external iliac arteries, which can range from 6 to 10 mm. Mor-phologically, the aortic neck can manifest complex angulation above and below the renal arteries due to combination of elon-gation and anterolateral displacement by the posterior bulge of the aneurysmal aorta. Furthermore, the shape of the proxi-mal neck

1	above and below the renal arteries due to combination of elon-gation and anterolateral displacement by the posterior bulge of the aneurysmal aorta. Furthermore, the shape of the proxi-mal neck is rarely tubular, but often is conical, reverse coni-cal, or barrel-shaped. Distally, the iliac arteries can have severe Brunicardi_Ch23_p0897-p0980.indd 92027/02/19 4:14 PM 921ARTERIAL DISEASECHAPTER 23Figure 23-28. An operative view of an infrarenal aortic aneurysm.tortuosity with multiple compound turns. Although significant from hemodynamic standpoint, severe iliac calcifications com-bined with extreme tortuosity can pose a formidable challenge during endovascular repair.Diagnostic EvaluationPreoperative evaluation should include routine history and physical exam with particular attention to (a) any symptoms referable to the aneurysm, which may impact the timing of repair; (b) history of pelvic surgery or radiation, in the event retroperitoneal exposure is required or interruption of

1	to (a) any symptoms referable to the aneurysm, which may impact the timing of repair; (b) history of pelvic surgery or radiation, in the event retroperitoneal exposure is required or interruption of hypogas-tric circulation is planned; (c) claudication suggestive of sig-nificant iliac occlusive disease; (d) lower extremity bypass or other femoral reconstructive procedures; and (e) chronic renal insufficiency or contrast allergy.Cross-sectional imaging is required for definitive evalu-ation of AAA. Although ultrasound is safe, widely available, relatively accurate, and inexpensive and thus the screening modality of choice, CT scan remains the gold standard for deter-mination of anatomic eligibility for endovascular repair. Size of AAA may differ up to 1 cm between CT and ultrasound, and during longitudinal follow-up, comparisons should be made between identical modalities. With modern multirow detector scanners, a timed-bolus intravenous contrast-enhanced, 2.5to 3.0-mm slice spiral CT

1	longitudinal follow-up, comparisons should be made between identical modalities. With modern multirow detector scanners, a timed-bolus intravenous contrast-enhanced, 2.5to 3.0-mm slice spiral CT of the chest, abdomen, and pelvis can be performed in less than 30 seconds with a single breath hold. Extremely high-resolution images are obtained with submilli-meter spatial resolution (Fig. 23-29). Proper window level and width (brightness and contrast) are important for discrimination among aortic wall, calcific plaque, thrombus, and lumen. The only major drawback to CT is the risk of contrast nephropathy in diabetics and in patients with renal insufficiency.The spiral technique further affords the ability for three-dimensional reconstruction. Three-dimensional reconstructions Figure 23-29. High resolution of image displaying an aortic aneu-rysm (arrow) can be achieved with multidetector computed tomog-raphy angiography.can yield important morphologic information that is critical to

1	resolution of image displaying an aortic aneu-rysm (arrow) can be achieved with multidetector computed tomog-raphy angiography.can yield important morphologic information that is critical to endovascular therapy. Using third-party software, these images can be viewed and manipulated on one’s desktop computer, and so-called “center-line” (transverse slices perpendicular to the central flow lumen of the aorta) diameter and length measure-ments obtained. Conventional angiography has a minimal role in the current management of AAA. Angiography is invasive with an increased risk of complications. Indications for angiog-raphy are isolated to concomitant iliac occlusive disease (pres-ent in <10% of patients with AAA) and unusual renovascular anatomy.Surgical Repair of Abdominal Aortic AneurysmGeneral anesthesia is necessary when performing a conventional open AAA repair. While a retroperitoneal incision is a well-accepted surgical approach, a midline transabdominal incision remains the more

1	anesthesia is necessary when performing a conventional open AAA repair. While a retroperitoneal incision is a well-accepted surgical approach, a midline transabdominal incision remains the more common approach for open aortic aneurysm operation. Since the abdominal incision can lead to significant pain and discomfort, an epidural catheter can be placed prior to the operation for postoperative analgesic infusion to provide pain control. Once the abdominal cavity is opened, the small intestines and transverse colon are retracted to expose the ret-roperitoneum overlying the AAA. The retroperitoneum is next divided, followed by isolation of both proximal and distal seg-ments of the AAA. Intravenous heparin (100 IU/kg) is given followed by clamping of the proximal and distal segments of the aneurysm. The aneurysm sac is open next, and a prosthetic graft is used to reconstruct the aorta. If the aneurysm only involves the abdominal aorta, a tube graft can be used to replace the aorta (Fig.

1	The aneurysm sac is open next, and a prosthetic graft is used to reconstruct the aorta. If the aneurysm only involves the abdominal aorta, a tube graft can be used to replace the aorta (Fig. 23-30). If the aneurysm extends distally to the iliac arter-ies, a prosthetic bifurcated graft is used for either an aorto-bi-iliac or aorto-bi-femoral bypass reconstruction (Fig. 23-31). The overlying aneurysm sac and the retroperitoneum are closed to cover the prosthetic bypass graft to minimize potential bowel contact to the graft. Small and large intestines are returned to the abdominal cavity followed by the closure of the abdominal fascia and skin.Brunicardi_Ch23_p0897-p0980.indd 92127/02/19 4:14 PM 922SPECIFIC CONSIDERATIONSPART IIABFigure 23-30. A. Schematic depiction of an aortic tube graft used to repair an aortic aneurysm. B. Intraoperative image of an aortic tube graft reconstruction.Advantages and Risks of Open Abdominal Aortic Aneu-rysm Repair. The main advantage of a

1	tube graft used to repair an aortic aneurysm. B. Intraoperative image of an aortic tube graft reconstruction.Advantages and Risks of Open Abdominal Aortic Aneu-rysm Repair. The main advantage of a conventional open repair is that the AAA is permanently eliminated because it is entirely replaced by a prosthetic aortic graft. The risk of aneurysm recurrence or delayed rupture no longer exists. As a result, long-term imaging surveillance is not needed with these patients. In contrast, the long-term efficacy of endovascular repair remains unclear. Consequently, long-term imaging sur-veillance is critical to ensure that the aortic aneurysm remains properly sealed by the stent graft. Other potential advantages of open repair include direct assessment of the circulatory integrity of the colon. If signs of colonic ischemia become evident after aortic bypass grafting, a concomitant mesenteric artery bypass can be performed to revascularize the colonic circulation. In addition, open repair

1	If signs of colonic ischemia become evident after aortic bypass grafting, a concomitant mesenteric artery bypass can be performed to revascularize the colonic circulation. In addition, open repair permits the surgeons to explore for other abdominal pathologies, such as gastrointestinal tumors, liver mass, or cholelithiasis.As for the risks associated with open repair, cardiac com-plications, in the form of either myocardial infarction or arrhyth-mias, remain the most common morbidity, with an incidence between 2% and 6%.51 Another significant complication is renal failure or transient renal insufficiency as a result of peri-operative hypotension, atheromatous embolization, inadvertent injury to the ureter, preoperative contrast-induced nephropathy, or suprarenal aortic clamping. Although the incidence of renal failure is less than 2% in elective aneurysm repair, it can occur in more than 20% of patients after repair of a ruptured AAA.49Ischemic colitis is a devastating potential

1	the incidence of renal failure is less than 2% in elective aneurysm repair, it can occur in more than 20% of patients after repair of a ruptured AAA.49Ischemic colitis is a devastating potential complication after open repair. The likelihood of such a complication is highest in those who had a prior colon resection and undergo repair of a ruptured AAA, due to the loss of collateral blood supply to the rectosigmoid colon. It is estimated that 5% of patients who undergo elective aneurysm repair will develop partial-thickness ischemic colitis but without significant clini-cal sequelae.52 However, if the partial-thickness ischemia pro-gresses to full-thickness gangrene and peritonitis, mortality can be as high as 90%.52The incidence of prosthetic graft infection ranges between 1% and 4% after open repair.53 It is more common in those who undergo repair of a ruptured AAA. If the prosthetic graft is not fully covered by the aneurysm sac or retroperitoneum, intestinal adhesion with

1	4% after open repair.53 It is more common in those who undergo repair of a ruptured AAA. If the prosthetic graft is not fully covered by the aneurysm sac or retroperitoneum, intestinal adhesion with subsequent bowel erosion may occur, resulting in an aortoenteric fistula. The predominant sign of such a com-plication is massive hematemesis, and it typically occurs years after the operation. Despite these potential complications, how-ever, the majority of patients who undergo successful elective open repair have an uneventful recovery.Endovascular Repair of Abdominal Aortic AneurysmOver a decade has passed since the first report of human implan-tation of a homemade stent graft for endovascular repair of an AAA by Parodi in 1991.11 Several prospective clinical trials across different devices and analysis of large Medicare adminis-trative databases and meta-analyses of published literature have consistently demonstrated significantly decreased operative time, blood loss, hospital length

1	and analysis of large Medicare adminis-trative databases and meta-analyses of published literature have consistently demonstrated significantly decreased operative time, blood loss, hospital length of stay, and overall periopera-tive morbidity and mortality of endovascular repair compared Brunicardi_Ch23_p0897-p0980.indd 92227/02/19 4:14 PM 923ARTERIAL DISEASECHAPTER 23Figure 23-31. Intraoperative view of a bifurcated graft used to repair an aortic aneurysm.with open surgical repair. For patients who are at increased risk for surgery because of age or comorbidity, endovascular repair is a superior minimally invasive alternative.The principle of endovascular repair of AAA involves the implantation of an aortic stent graft that is fixed proximally and distally to nonaneurysmal aortoiliac segment and thereby endoluminally excluding the aneurysm from the aortic circu-lation (Fig. 23-32). Unlike open surgical repair, the aneurysm BAFigure 23-32. A. An aortogram dem-onstrating a large

1	segment and thereby endoluminally excluding the aneurysm from the aortic circu-lation (Fig. 23-32). Unlike open surgical repair, the aneurysm BAFigure 23-32. A. An aortogram dem-onstrating a large infrarenal abdominal aortic aneurysm. B. Following endovas-cular stent graft implantation, the aortic aneurysm is successfully excluded.sac is not resected, which is subjected for potential aneurysm expansion or even rupture. Importantly, aortic branches, such as lumbar arteries or the inferior mesenteric artery (IMA), are occluded, which can lead to persistent aneurysm pressurization and aneurysm expansion. Currently, there are more than fifteen different endovascular devices approved for clinical use for infrarenal aortic aneurysm implantation throughout the world. Despite some differences in physical appearance, mechanical properties, and endograft materials, these endovascular devices will be discussed collectively for this chapter. Most of these devices are modular devices consisting of

1	appearance, mechanical properties, and endograft materials, these endovascular devices will be discussed collectively for this chapter. Most of these devices are modular devices consisting of a primary device or main body and one or two iliac limbs that insert into the main body to complete the repair. Depending on the device, there are varying degrees of flexibility in the choice of iliac limbs that can be matched to the main body, which can impact the customiz-ability for a particular anatomy.A severe limitation of the endovascular repair devices is the need for adequate proximal neck to achieve a durable seal-ing zone. Several techniques have been proposed to overcome this limitation. These include fenestrated or branched endografts and the “chimney,” “snorkel,” and “periscope” techniques. The fenestrated stent grafts rely on precise alignment between the fen-estration and the corresponding visceral artery.12,54 Multiple clinical trials using customized fenestrated stent graft for

1	The fenestrated stent grafts rely on precise alignment between the fen-estration and the corresponding visceral artery.12,54 Multiple clinical trials using customized fenestrated stent graft for the treatment of short-necked and juxtarenal aortic aneurysm repair have shown promising shortand mid-term results.54,55 How-ever, fenestrated stent graft generally requires device customiza-tion which is accessible only to high volume tertiary institutions, and not widely available to all hospital facilities. Alternatively, some centers have reported good results with intraoperative surgeon-modified endograft to create fenestrations for the treat-ment of complex aortic aneurysms in high-risk patients.56 Fur-ther development of the fenestrated techniques also opens the way for endovascular treatment of suprarenal and thoracoab-dominal aneurysm.57 The review of literature showed that open surgery remains a safe and effective treatment option for good-risk patients with juxtarenal aortic

1	treatment of suprarenal and thoracoab-dominal aneurysm.57 The review of literature showed that open surgery remains a safe and effective treatment option for good-risk patients with juxtarenal aortic aneurysm.58 Fenestrated endovascular repair is associated with low mortality and com-pares favorably with open surgery in terms of morbidity, espe-cially renal function impairment and cardiac complications.59Brunicardi_Ch23_p0897-p0980.indd 92327/02/19 4:14 PM 924SPECIFIC CONSIDERATIONSPART IIPatient Selection for Endovascular Aortic Aneurysm Repair. Anatomic eligibility for endovascular repair is mainly based on three areas: the proximal aortic neck, common iliac arteries, and external iliac and common femoral arteries, which relate to the proximal and distal landing zones or fixation sites and the access vessels, respectively. The requirements for the proximal aortic neck are a diameter of 18 to 28 mm and a mini-mum length of 15 mm (Table 23-9). Usually, multiple measure-ments of

1	sites and the access vessels, respectively. The requirements for the proximal aortic neck are a diameter of 18 to 28 mm and a mini-mum length of 15 mm (Table 23-9). Usually, multiple measure-ments of the diameter are taken along the length of the neck to assess its shape. All diameter measurements are mid-wall to mid-wall of the vessel. Secondary considerations include mural calcifications (<50% circumference), luminal thrombus (<50% circumference), and angulation (<45°). Presence of a significant amount of any one of these secondary features in combination with a relatively short proximal neck may compromise success-ful shortand long-term fixation of the stent graft and exclusion of the aneurysm. The usual distal landing zone is the common iliac artery. The external iliac artery may serve as an alternate site when the ipsilateral common iliac artery is aneurysmal or ectatic. The treatable diameters of common iliac arteries range from 8 to 20 mm, and there should be at least 20 mm of

1	as an alternate site when the ipsilateral common iliac artery is aneurysmal or ectatic. The treatable diameters of common iliac arteries range from 8 to 20 mm, and there should be at least 20 mm of patent artery of uniform diameter to allow adequate fixation. Finally, at least one of two common femoral and external iliac arteries must be at least 7 mm in diameter in order to safely introduce the main delivery sheath. Slightly smaller iliac diameters may be tolerated depending on the specific device and in the absence of severe tortuosity and calcific disease. Difficult access is one of the main causes of increased procedural time and intraopera-tive complications. Using these criteria, approximately 60% of all AAAs are anatomic candidates for endovascular repair.The next step in the preoperative planning is device selec-tion. Typically, the proximal diameter of the main device is oversized by 10% to 20% of the nominal diameter of the aor-tic neck. Distally, the iliac limbs are

1	preoperative planning is device selec-tion. Typically, the proximal diameter of the main device is oversized by 10% to 20% of the nominal diameter of the aor-tic neck. Distally, the iliac limbs are oversized by 1 to 4 mm depending on the individual device’s instructions for use. The biggest challenge to proper device selection remains determin-ing the optimal length from the renal arteries to the hypogastric arteries. Despite availability of sophisticated three-dimensional reconstructions, the exact path that a device will take from the proximal aortic neck to the distal iliac arteries is difficult to pre-dict. It is dependent on a host of factors related to the mechanical properties of the stent graft and the morphology of the aortoiliac flow lumen. “Plumb-line” measurements of axial CT images can be quite inaccurate, typically grossly underestimating the Table 23-9Ideal characteristics of an aneurysm for endovascular abdominal aortic aneurysm repairNeck length (mm)>15Neck diameter

1	images can be quite inaccurate, typically grossly underestimating the Table 23-9Ideal characteristics of an aneurysm for endovascular abdominal aortic aneurysm repairNeck length (mm)>15Neck diameter (mm)>18, <32Aortic Neck angle (degrees)<60Neck mural calcification (% circumference)<50Neck luminal thrombus (% circumference)<50Common iliac artery diameter (mm)Between 8 and 20Common iliac artery length (mm)>20External iliac artery diameter (mm)>7length, whereas center-line measurements usually overestimate the length. Angiographic measurements using a marker catheter are invasive, require contrast and radiation exposure, and are also inaccurate because they fail to account for the stiffness of the stent graft. The consequences of not choosing the correct length of the device include inadvertent coverage of the hypo-gastric artery if too long and the need for additional devices if too short.Advantages and Risks of Endovascular Repair. The obvi-ous advantage of an endovascular AAA

1	inadvertent coverage of the hypo-gastric artery if too long and the need for additional devices if too short.Advantages and Risks of Endovascular Repair. The obvi-ous advantage of an endovascular AAA repair is its minimally invasive nature. Typically, patients who undergo this procedure stay in the hospital for only 1 to 3 days, in contrast to the 5to 10-day stay required after conventional open surgical repair. In our institution, patients who have had an endovascular repair are routinely transferred to a general vascular ward from the post-anesthesia recovery unit, avoiding admission to a more costly intensive care unit.Because an abdominal incision is not necessary in endo-vascular repair, the procedure is particularly beneficial in patients with severe pulmonary disease, such as chronic obstruc-tive pulmonary disease or emphysema. Patients can sustain ade-quate breathing in the postoperative period, avoiding respiratory complications or prolonged mechanical ventilation. Because

1	obstruc-tive pulmonary disease or emphysema. Patients can sustain ade-quate breathing in the postoperative period, avoiding respiratory complications or prolonged mechanical ventilation. Because the abdominal cavity has not been entered, the risk of gastro-intestinal complications, such as ileus, ventral hernia, or bowel obstruction due to intestinal adhesion, is also greatly reduced. Moreover, regional or epidural anesthesia can be used, avoid-ing the risks associated with general anesthesia in patients with severe cardiopulmonary dysfunction.Despite its many advantages, endovascular repair does have potential complications. Since the stent graft device is attached endoluminally within the abdominal aorta, an endoleak due to incomplete stent graft exclusion of the aneurysm can occur. With this type of leak, blood flow persists outside the lumen of the endoluminal graft but within an aneurysm sac. A meta-analysis of 1118 patients who underwent successful endo-vascular repair found an

1	this type of leak, blood flow persists outside the lumen of the endoluminal graft but within an aneurysm sac. A meta-analysis of 1118 patients who underwent successful endo-vascular repair found an endoleak incidence of 24%.60 Although a small endoleak usually poses little clinical significance because it will typically become thrombosed spontaneously, a large or persistent endoleak may lead to continuous aneurysm perfusion and ultimately to aneurysm rupture. The rupture rate following an endovascular AAA repair has been reported to be less than 0.8%.61Stent graft iliac limb dysfunction resulting in thrombosis has been reported following endovascular repair. One possible cause is aneurysm remodeling, resulting in a shortening in the aortic length, which can cause the stent graft to kink. Alterna-tively, progression of an underlying iliac atherosclerotic lesion may cause compression of the iliac limb and ultimately result in graft-limb occlusion. Treatment options include thrombolysis

1	Alterna-tively, progression of an underlying iliac atherosclerotic lesion may cause compression of the iliac limb and ultimately result in graft-limb occlusion. Treatment options include thrombolysis or graft thrombectomy to determine the underlying cause and possibly additional stent graft placement. Renal artery occlusion may occur due to improper stent graft positioning or migration.62 Graft limb separation or dislocation has also been reported.62In patients with AAA and concurrent iliac artery aneu-rysms who undergo preoperative coil embolization of the inter-nal iliac artery, 20% to 45% experience symptoms of pelvic ischemia.63 These symptoms may include buttock claudication, impotence, gluteal skin sloughing, and colonic ischemia. Other complications pertaining to endovascular repair relate to the access site and include groin hematoma and wound infection. Brunicardi_Ch23_p0897-p0980.indd 92427/02/19 4:14 PM 925ARTERIAL DISEASECHAPTER 23Occasionally, the stent graft device

1	relate to the access site and include groin hematoma and wound infection. Brunicardi_Ch23_p0897-p0980.indd 92427/02/19 4:14 PM 925ARTERIAL DISEASECHAPTER 23Occasionally, the stent graft device can malfunction by either failing to deploy or dislodging during the deployment proce-dure. If the device cannot be salvaged or rescued endoluminally, open surgical repair of the aneurysm may be necessary.Technical Considerations of Endovascular Aortic Aneu-rysm Repair. Although endovascular AAA repair may be performed in any venue with appropriate digital fluoroscopic imaging capability, due to the need for absolute sterility and aseptic technique, it is most safely performed in a surgical suite. The patient is prepped and draped just as in open AAA repair. Patients with renal insufficiency should be started on periopera-tive oral N-acetylcysteine (Mucomyst) and sodium bicarbonate infusion to reduce the risk of contrast nephropathy. A variety of anesthetic options may be used. Regional

1	should be started on periopera-tive oral N-acetylcysteine (Mucomyst) and sodium bicarbonate infusion to reduce the risk of contrast nephropathy. A variety of anesthetic options may be used. Regional anesthesia may be appropriate for patients with pulmonary disease. There are reports of success with local anesthetics alone, as the incisions are typically smaller than a typical open inguinal hernia repair.64Groin access for endovascular aortic aneurysm repair can be achieved by either surgical cutdown for femoral artery expo-sure or percutaneous approach using “preclose” technique with the Perclose suture-mediated vascular closure device (Abbott Perclose, Redwood City, CA). Review of reported series on this percutaneous technique suggest a technical success rate of 95% for medium-size sheaths ranging from 12 to 16 French, and 87% success for 18to 24-French sizes.65 Once femoral artery access is obtained followed by introducer sheath placement, initial soft-tipped starter guidewires are

1	ranging from 12 to 16 French, and 87% success for 18to 24-French sizes.65 Once femoral artery access is obtained followed by introducer sheath placement, initial soft-tipped starter guidewires are exchanged for stiff guidewires that are advanced to the thoracic arch. Intravenous heparin at 80 IU/kg are administered, and the activated clotting time is maintained at 200 to 250 seconds. These guidewires provide the necessary support for the subsequent introduction of the large-diameter delivery catheters and devices. In the absence of special anatomic considerations, the primary device is inserted through the right side and the contralateral iliac limb is inserted through the left side. After administration of heparin, the deliv-ery catheter or the introducer sheath is advanced to the L1-L2 vertebral space, which typically marks the location of the renal arteries. An angiographic catheter is advanced from the contra-lateral femoral artery to the same level.A road-mapping aortogram is

1	vertebral space, which typically marks the location of the renal arteries. An angiographic catheter is advanced from the contra-lateral femoral artery to the same level.A road-mapping aortogram is obtained to localize the renal arteries. The primary device is rotated to the desired ori-entation and deployed immediate below the lowest renal artery (Fig. 23-33). The angiographic catheter is replaced with a direc-tional catheter and an angled guidewire, and the opening for the contralateral limb on the main device is cannulated. Intrastent passage of the guidewire is confirmed, and the angled guidewire is replaced with a stiff guidewire. The contralateral iliac limb is inserted into the docking opening of the primary device and deployed. A completion angiogram is performed looking for patency of the renal and hypogastric arteries, the device limbs, proximal and distal fixation, and endoleak. Adjunctive interven-tions including additional devices, balloons, and bare stents are performed

1	of the renal and hypogastric arteries, the device limbs, proximal and distal fixation, and endoleak. Adjunctive interven-tions including additional devices, balloons, and bare stents are performed as needed. The procedure is concluded with routine repairs of the femoral arteries and closure of the groin incisions. The patients recover in the recovery room for 2 to 4 hours and admitted to the general care floor. Although in the past, patients were admitted to the intensive care unit, this is rarely needed. Most patients can be started on a regular diet that evening and discharged the next morning.Surveillance Following Endovascular Aortic Aneurysm Repair. Life-long follow-up is essential to the long-term ADCBFigure 23-33. A. During an endovascular aortic aneurysm repair, the main endograft device is inserted through a femoral artery approach. B. The device is deployed in the aorta just below the renal arteries. C. A contralateral iliac endograft device is inserted through a

1	endograft device is inserted through a femoral artery approach. B. The device is deployed in the aorta just below the renal arteries. C. A contralateral iliac endograft device is inserted through a contralateral gate opening, which is next deployed. D. Completion of deployment of the endograft device should fully exclude an aortic aneurysm while preserving flow of the renal and hypogastric arteries.success after endovascular AAA repair. Indeed, one may go so far as to say that absence of appropriate follow-up is tantamount to not having had a repair at all. A triple-phase (noncontrast, contrast, and delayed) spiral CT scan and a four-view (antero-posterior, lateral, and two obliques) abdominal X-ray should be obtained within the first month. Subsequent imaging can be obtained at 6-month intervals in the first 1 to 2 years and yearly thereafter. After the first 6 months, patients who cannot travel easily may obtain their studies locally and submit them for review. The CT scan is for

1	intervals in the first 1 to 2 years and yearly thereafter. After the first 6 months, patients who cannot travel easily may obtain their studies locally and submit them for review. The CT scan is for detection of endoleaks, subtle proxi-mal migrations, and changes in aneurysm size. The abdominal X-ray gives a “birds-eye” view of the overall morphology of the stent graft. Subtle changes in conformation of the iliac limbs relative to each other and/or the spine can provide early signs of impending component separation or loss of fixation. Further, stent fractures and/or suture breaks that can compromise long-term device integrity can sometimes only be detected on a plain film and not on a CT scan.Results From Clinical Studies Comparing Endovascular Versus Open RepairThe primary success rate after endovascular repair of AAA has been reported to be as high as 95%.41 The less invasive nature Brunicardi_Ch23_p0897-p0980.indd 92527/02/19 4:14 PM 926SPECIFIC CONSIDERATIONSPART IIof this

1	after endovascular repair of AAA has been reported to be as high as 95%.41 The less invasive nature Brunicardi_Ch23_p0897-p0980.indd 92527/02/19 4:14 PM 926SPECIFIC CONSIDERATIONSPART IIof this procedure is appealing to many physicians and patients. In addition, virtually all reports indicate a decreased blood loss, transfusion requirements, and length of intensive care unit and hospital stay for endovascular repair of AAAs compared with the standard surgical approach.47 With the advent of bifurcated grafts and improved delivery systems in the future, the only real limitation will be cost. When evaluating the literature for results from clinical series, it is important to look at a comparison of endoluminal versus open repair and device-specific outcome and cost analysis studies.Early reports on results with endovascular repair were often flawed due to selection biases. This is because from its inception, endovascular repair has been used mostly in patients who are at higher risk

1	reports on results with endovascular repair were often flawed due to selection biases. This is because from its inception, endovascular repair has been used mostly in patients who are at higher risk for open repair. At the same time, only patients with favorable anatomy including less tortuosity and the presence of a suitable infrarenal neck were considered for endovascular repair. Randomization is also difficult because most patients who anatomically qualify for endovascular repair would withdraw from the study if randomized to open repair. Consequently, there are very few randomized controlled tri-als that have compared outcomes in patients with similar risk factors and anatomy who are eligible for both types of repair. Two such European trials have recently published short-term outcome data that are unbiased in design.The DREAM trial is a multicenter randomized trial that compared open versus endovascular repair among a group of 345 patients at 28 European centers using multiple

1	data that are unbiased in design.The DREAM trial is a multicenter randomized trial that compared open versus endovascular repair among a group of 345 patients at 28 European centers using multiple different devices including Gore, AneuRx, and Zenith.66 Patients were included only if they were considered to be candidates for both types of repairs. The operative mortality rate was 4.6% in the operative group versus 1.2% in the endoluminal group at 30 days. When looking at the combined rate of operative mor-tality and severe complications, there was an incidence of 9.8% in the open repair group versus 4.7% in the endoluminal group. The difference here was largely due to the higher frequency of pulmonary complications seen in the open group. There was a higher incidence of graft-related complications in the endolu-minal group. There was no difference in the nonvascular local complication rate among the two groups. The Endovascular Repair-1 (EVAR-1) trial is also a multicenter randomized

1	in the endolu-minal group. There was no difference in the nonvascular local complication rate among the two groups. The Endovascular Repair-1 (EVAR-1) trial is also a multicenter randomized trial that compared open to endoluminal repair.67 This study was con-ducted on 1082 patients at 34 centers in the United Kingdom using all available devices. Short-term mortality at 30 days was 4.7% in the open group and 1.7% in the endoluminal group. The in-hospital mortality rate was also increased in the open when compared to the endoluminal group (6.2% vs. 2.1%). As expected, the secondary intervention rate was higher in the endoluminal group (9.8% vs. 5.8%). Complication rates were not reported in the EVAR-1 trial. Criticisms can be applied to both of these trials. Patients had to be eligible for either type of repair in order to be included in the study. Consequently, these findings cannot be generalized to patients who are too sick to undergo open surgery or to patients whose anatomy

1	for either type of repair in order to be included in the study. Consequently, these findings cannot be generalized to patients who are too sick to undergo open surgery or to patients whose anatomy precludes them from undergoing endovascular repair.The Open Versus Endovascular Repair (OVER) Veter-ans Affairs Cooperative Study Group randomly assigned 881 patients with asymptomatic AAAs who were candidates for both procedures to either endovascular repair (n = 444) or open repair (n = 437) and followed them for up to 9 years.68 Reduction in perioperative mortality with endovascular repair was sustained at 3 years but not thereafter. There was no difference in pri-mary outcome of all-cause mortality. Endovascular repair and open repair resulted in similar long-term survival. Six aneurysm ruptures were confirmed in the endovascular repair group ver-sus none in the open repair group. Rupture after endovascular repair remains a concern. A significant interaction was observed between age and

1	were confirmed in the endovascular repair group ver-sus none in the open repair group. Rupture after endovascular repair remains a concern. A significant interaction was observed between age and type of treatment. Endovascular repair led to increased long-term survival among younger patients but not among older patients, for whom a greater benefit from the endo-vascular approach had been expected.Device-Specific Outcome. Matsumura and associates com-pared endoluminal versus open repair using the Excluder device.69 In their review, they demonstrated a 30-day mortality rate of 1% along with endoleak rates of 17% and 20% at 1and 2-year intervals, respectively. The limb narrowing, limb migra-tion, and trunk migration were all 1% at 2 years. There were no deployment failures or early conversions. There was an annual 7% reintervention rate. Aneurysm growth was demonstrated in 14% of patients at 2 years. The Zenith device by Cook has been studied by Greenberg and associates, who compared

1	There was an annual 7% reintervention rate. Aneurysm growth was demonstrated in 14% of patients at 2 years. The Zenith device by Cook has been studied by Greenberg and associates, who compared standard surgical repair with endoluminal repair in low-risk patients and endoluminal repair in high-risk patients.70 They reported a 30-day mortality rate of 3.5%, which was equal to the open group. The endoleak rates were 7.4% and 5.4% at 1and 2-year intervals, respectively. There was a 5.3% migration of 5 mm at 1 year. Freedom from rupture was 100% in the low-risk group and 98.9% in the high-risk endoluminal group at 2 years. Expe-rience with the AneuRx device has been reported by Zarins.71 In this 4-year review, they found a 30-day mortality rate of 2.8%. Endoleak rate at 4 years was 13.9%, aneurysm enlargement was 11.5%, and stent graft migration was 9.5%. Freedom from rup-ture was noted to be 98.4% at 4 years. Criado and associates have reported on their 1-year experience with the Talent

1	enlargement was 11.5%, and stent graft migration was 9.5%. Freedom from rup-ture was noted to be 98.4% at 4 years. Criado and associates have reported on their 1-year experience with the Talent LPS device by Medtronic.72 They report a 30-day mortality rate of 0.8%. Endoleak rate was 10%. Three deployment failures were noted, and freedom from rupture was 100%. Aneurysm growth and migration rates were divided into three different neck size groups. Patients with a wide neck (>26 mm) had a 3% growth and migration rate. Narrow-neck patients (<26 mm) had a 1% growth rate and a 2% migration neck. Interestingly, short-neck patients (<15 mm) had no aneurysm growths and a 2% migra-tion rate.Cost Analysis. The current climate of cost containment and limited reimbursement for healthcare services mandates a criti-cal analysis of the economic impact of any new medical technol-ogy on the market. The in-hospital costs for both endovascular and open repair include graft cost, operating room fees,

1	mandates a criti-cal analysis of the economic impact of any new medical technol-ogy on the market. The in-hospital costs for both endovascular and open repair include graft cost, operating room fees, radiol-ogy, pharmacy, ancillary care, intensive care unit charges, and floor charges. Despite the improved morbidity and mortality rates, several early studies have reported no cost benefit with the application of endovascular repair.73,74 The limiting factor appears to be the cost of the device. Despite commercializa-tion of endovascular repair, the device costs are still in the range of $5000 to $6000 with no signs of abating. A report by Angle and associated further corroborates previous studies.75 In their review, despite decreased hospital and intensive care unit stays and utilization of pharmacy and respiratory services, cost of endovascular repair was 1.74 times greater than the stan-dard surgical approach. In addition, these cost analysis stud-ies are centered on in-hospital costs

1	pharmacy and respiratory services, cost of endovascular repair was 1.74 times greater than the stan-dard surgical approach. In addition, these cost analysis stud-ies are centered on in-hospital costs and do not even begin to address secondary costs such as postoperative surveillance that is required with endovascular repair. In the OVER trial, endo-vascular repair was found to be a cost-effective alternative to Brunicardi_Ch23_p0897-p0980.indd 92627/02/19 4:14 PM 927ARTERIAL DISEASECHAPTER 23open repair in the U.S. Veterans Affairs healthcare system for at least the first 2 years.76 The primary outcomes were mean total healthcare cost per life-year and per quality-adjusted life-year. There were no differences found in survival, quality of life, and costs after 2 years between the endovascular and the open group. Although graft costs were higher in the endovas-cular group, length of stay was shorter, resulting in lower cost of AAA repair hospitalization in the endovascular group.

1	and the open group. Although graft costs were higher in the endovas-cular group, length of stay was shorter, resulting in lower cost of AAA repair hospitalization in the endovascular group. Costs remained lower after 2 years in the endovascular group, but the difference was no longer significant.Classification and Management of EndoleakAn endoleak is an extravasation of contrast outside the stent graft and within the aneurysm sac (Fig. 23-34). It can be pres-ent in up to 20% to 30% of all endovascular AAA repairs in the early postoperative period.77,78 In general, over half of these endoleaks will resolve spontaneously during the first 6 months, resulting in a 10% incidence of chronic endoleaks in all cases beyond the first year of follow-up. Endoleaks can be detected using conventional angiography, contrast CT (Fig. 23-35), MRA, and color-flow duplex ultrasound. Although there is no recognized gold standard, in practice, angiography is consid-ered the least sensitive but most

1	angiography, contrast CT (Fig. 23-35), MRA, and color-flow duplex ultrasound. Although there is no recognized gold standard, in practice, angiography is consid-ered the least sensitive but most specific for characterizing the Type I endoleak Type II endoleak Type III endoleak Type IV endoleakFigure 23-34. The four types of endoleak include the following: type I endoleak = attachment site leak; type II endoleak = side branch leak caused by lumbar or side branches; type III endoleak = endograft junctional leak due to overlapping device components; and type IV endoleak = endograft fabric or porosity leak.Figure 23-35. A computed tomography scan demonstrating an endoleak (small arrow) as evidenced by contrast flow outside the aortic endograft (long arrow).source of the endoleak, whereas the CT scan is the most sensi-tive but least specific. Widespread availability and reliability that is relatively independent of technique have made the CT

1	arrow).source of the endoleak, whereas the CT scan is the most sensi-tive but least specific. Widespread availability and reliability that is relatively independent of technique have made the CT scan the de facto standard imaging modality for postoperative surveillance. Conversely, routine use of duplex ultrasound and MRA has been limited by the lack of proper equipment and local expertise. On the other hand, investigational techniques such as time-resolved MRA may provide greater sensitivity and specificity than either angiography or CT in the future.Four types of endoleaks have been described (Table 23-10). Type I endoleak refers to fixation-related leaks that occur at the proximal or distal attachment sites. These represent less than 5% of all endoleaks and are seen as an early blush of contrast into the aneurysm sac from the proximal or distal ends of the device during completion angiography.77,78 Although seen as marker of poor patient selection or inadequate repair, over 80% of

1	contrast into the aneurysm sac from the proximal or distal ends of the device during completion angiography.77,78 Although seen as marker of poor patient selection or inadequate repair, over 80% of these leaks spontaneously seal in the first 6 months. Persistent type I endoleaks, on the other hand, require prompt treatment. Type II endoleak refers to retrograde flow originating from a lumbar, inferior mesenteric, accessory renal, or hypogastric artery. They are the most common type of endoleak, accounting for 20% to 30% of all cases, and about half resolve spontaneously. On angi-ography, they are seen as a late filling of the aneurysm sac from a branch vessel(s). Type II endoleaks carry a relatively benign natural history and do not merit intervention unless associated with aneurysm growth. Type III endoleaks refer to failure of device integrity or component separation from modular sys-tems. If detected intraoperatively or in the early perioperative period, it is usually from

1	growth. Type III endoleaks refer to failure of device integrity or component separation from modular sys-tems. If detected intraoperatively or in the early perioperative period, it is usually from inadequate overlap between two stent Table 23-10Endoleak classificationCLASSIFICATIONDESCRIPTIONType I endoleakAttachment site leakType II endoleakSide branch leak caused by lumbar or inferior mesenteric arteriesType III endoleakJunctional leak (of overlapping endograft components) and graft fabric defectType IV endoleakEndograft fabric porosity leakBrunicardi_Ch23_p0897-p0980.indd 92727/02/19 4:14 PM 928SPECIFIC CONSIDERATIONSPART IIgrafts, whereas in the late period, the endoleak may be from a fabric tear or junctional separation from conformational changes of the aneurysm. Regardless of the etiology or timing, these should be promptly repaired. Finally, type IV endoleak refers to the diffuse, early blush seen during completion angiography due to graft porosity and/or suture holes of

1	the etiology or timing, these should be promptly repaired. Finally, type IV endoleak refers to the diffuse, early blush seen during completion angiography due to graft porosity and/or suture holes of some Dacron-based devices. It does not have any clinical significance and usually cannot be seen after 48 hours and heparin reversal. Endoleaks that are initially considered type IV but persist become type III endoleaks by definition because this indicates a more significant material defect than simple porosity or a suture hole.Endotension Following Endovascular Aortic Aneurysm Repair. In approximately 5% of cases after an apparently suc-cessful endovascular repair, the aneurysm continues to grow without any demonstrable endoleak.79,80 This phenomenon has been described as endotension. Although it was initially thought that an endoleak was really present but simply not detected, case have been reported where the aneurysm has been surgically opened and the contents were completely devoid

1	it was initially thought that an endoleak was really present but simply not detected, case have been reported where the aneurysm has been surgically opened and the contents were completely devoid of any blood and no extravasation could be found. The mechanism of con-tinued pressurization of the aneurysm sac following successful exclusion from the arterial circulation remains unsolved at this time. One putative mechanism has been linked to a transuda-tive process related to certain expanded PTFE graft materials.81 More importantly, however, the natural history of these enlarg-ing aneurysms without endoleaks is unknown, but to date, there has been no evidence to suggest that they carry an increased risk of rupture. Conservatively speaking, until further long-term data become available, if the patient is a suitable surgical risk, elec-tive open conversion should be considered.Secondary Interventions Following Endovascular Aortic Aneurysm Repair. There is approximately 10% to 15% per year

1	patient is a suitable surgical risk, elec-tive open conversion should be considered.Secondary Interventions Following Endovascular Aortic Aneurysm Repair. There is approximately 10% to 15% per year risk of secondary interventions following endovascular AAA repair.62 These procedures are critical in the long-term success of the primary procedure in prevention of aneurysm rup-ture and aneurysm-related death. These secondary procedures, in order of frequency, include proximal or distal extender place-ment for migrations, highly selective or translumbar emboliza-tion for type II endoleaks, direct surgical or laparoscopic branch vessel ligations, bridging cuffs for component separations, and late open surgical conversions.Multiple large series have reported that an annual rupture rate of approximately 1% to 1.5% per year after endovascular repair.82,83 The EUROSTAR registry reports a rupture rate of 2.3% over 15.4 months in patients with an endoleak, compared with 0.3% in those

1	of approximately 1% to 1.5% per year after endovascular repair.82,83 The EUROSTAR registry reports a rupture rate of 2.3% over 15.4 months in patients with an endoleak, compared with 0.3% in those without.82,83 Various causes of late ruptures have been reported in the literature, although presence of a per-sistent endoleak with aneurysm enlargement remains a com-mon culprit for this complication. It has been shown that even successfully excluded aneurysms can lead to the development of attachment-site leaks and device failure, caused in part by aneurysm remodeling resulting in stent migration or kinking. Mehta and colleagues reported that 63% of delayed AAA rup-tures after endovascular repair were caused by type I endoleaks with endograft migration, 11% by type I without migration, 19% by type II, and the rest of unknown type.84Treatment of rupture may be open conversion or endo-vascular stent graft placement. May and associates reported a mortality rate of 43% in those patients who

1	type II, and the rest of unknown type.84Treatment of rupture may be open conversion or endo-vascular stent graft placement. May and associates reported a mortality rate of 43% in those patients who underwent open conversion.85 Emergent endovascular repair should be consid-ered in these patients since it is potentially much faster and less likely to cause physiologic stress than open conversion. Several reports have shown that endovascular repair can be performed successfully in patients previously treated with endoluminal prostheses.80,86MESENTERIC ARTERY DISEASEVascular occlusive disease of the mesenteric vessels is a rela-tively uncommon but potentially devastating condition that gen-erally presents in patients over 60 years of age, is three times more frequent in women, and has been recognized as an entity since 1936.87 The incidence of such a disease is low and repre-sents 2% of the revascularization operations for atheromatous lesions. The most common cause of mesenteric ischemia

1	as an entity since 1936.87 The incidence of such a disease is low and repre-sents 2% of the revascularization operations for atheromatous lesions. The most common cause of mesenteric ischemia is atherosclerotic vascular disease. Autopsy studies have dem-onstrated splanchnic atherosclerosis in 35% to 70% of cases.88 Other etiologies exist and include FMD, panarteritis nodosa, arteritis, and celiac artery compression from a median arcuate ligament, but they are unusual and have an incidence of one in nine compared with that of atherosclerosis.Chronic mesenteric ischemia is related to a lack of blood supply in the splanchnic region and is caused by disease in one or more visceral arteries: the celiac trunk, the superior mesen-teric artery, and the IMA. Mesenteric ischemia is thought to occur when two of the three visceral vessels are affected with severe stenosis or occlusion; however, in as many as 9% of cases, only a single vessel is involved (SMA in 5% and celiac trunk in 4% of

1	occur when two of the three visceral vessels are affected with severe stenosis or occlusion; however, in as many as 9% of cases, only a single vessel is involved (SMA in 5% and celiac trunk in 4% of cases).89 This disease process may evolve in a chronic fashion, as in the case of progressive luminal oblitera-tion due to atherosclerosis. On the other hand, mesenteric isch-emia can occur suddenly, as in the case of thromboembolism. Despite recent progress in perioperative management and bet-ter understanding of pathophysiology, mesenteric ischemia is considered one of the most catastrophic vascular disorders with mortality rates ranging from 50% to 75%. Delays in diagnosis and treatment are the main contributing factors in its high mor-tality. It is estimated that mesenteric ischemia accounts for 1 in every 1000 hospital admissions in this country. The prevalence is rising due in part to the increased awareness of this disease, the advanced age of the population, and the significant

1	for 1 in every 1000 hospital admissions in this country. The prevalence is rising due in part to the increased awareness of this disease, the advanced age of the population, and the significant comor-bidity of these elderly patients. Early recognition and prompt treatment before the onset of irreversible intestinal ischemia are essential to improve the outcome.Anatomy and PathophysiologyMesenteric arterial circulation is remarkable for its rich collat-eral network. Three main mesenteric arteries provide the arterial perfusion to the gastrointestinal system: the celiac artery (CA), the superior mesenteric artery (SMA), and the IMA. In gen-eral, the CA provides arterial circulation to the foregut (distal esophagus to duodenum), hepatobiliary system, and spleen; the SMA supplies the midgut (jejunum to mid-colon); and the IMA supplies the hindgut (mid-colon to rectum). The CA and SMA arise from the ventral surface of the infradiaphragmatic supra-renal abdominal aorta, whereas the IMA

1	(jejunum to mid-colon); and the IMA supplies the hindgut (mid-colon to rectum). The CA and SMA arise from the ventral surface of the infradiaphragmatic supra-renal abdominal aorta, whereas the IMA originates from the left lateral portion of the infrarenal aorta. These anatomic origins in relation to the aorta are important when a mesenteric angio-gram is performed to determine the luminal patency. In order to fully visualize the origins of the CA and SMA, it is necessary to perform both an anteroposterior and a lateral projection of the aorta since most arterial occlusive lesions occur in the proximal segments of these mesenteric trunks.Brunicardi_Ch23_p0897-p0980.indd 92827/02/19 4:14 PM 929ARTERIAL DISEASECHAPTER 23Because of the abundant collateral flow between these mesenteric arteries, progressive diminution of flow in one or even two of the main mesenteric trunks is usually tolerated, provided that uninvolved mesenteric branches can enlarge over time to provide sufficient

1	progressive diminution of flow in one or even two of the main mesenteric trunks is usually tolerated, provided that uninvolved mesenteric branches can enlarge over time to provide sufficient compensatory collateral flow. In contrast, acute occlusion of a main mesenteric trunk may result in profound ischemia due to lack of sufficient collateral flow. Collateral networks between the CA and the SMA exist primarily through the superior and inferior pancreaticoduodenal arteries. The IMA may provide collateral arterial flow to the SMA through the marginal artery of Drummond, the arc of Riolan, and other unnamed retroperitoneal collateral vessels termed meandering mesenteric arteries (Fig. 23-36). Lastly, collateral visceral vessels may provide important arterial flow to the IMA and the hindgut through the hypogastric arteries and the hemorrhoidal arterial network.Regulation of mesenteric blood flow is largely modulated by both hormonal and neural stimuli, which characteristically regulate

1	through the hypogastric arteries and the hemorrhoidal arterial network.Regulation of mesenteric blood flow is largely modulated by both hormonal and neural stimuli, which characteristically regulate systemic blood flow. In addition, the mesenteric cir-culation responds to the gastrointestinal contents. Hormonal regulation is mediated by splanchnic vasodilators, such as nitric oxide, glucagon, and vasoactive intestinal peptide. Certain intrinsic vasoconstrictors, such as vasopressin, can diminish the mesenteric blood flow. On the other hand, neural regulation is provided by the extensive visceral autonomic innervation.Clinical manifestation of mesenteric ischemia is pre-dominantly postprandial abdominal pain, which signifies that the increased oxygen demand of digestion is not met by the gastrointestinal collateral circulation. The postprandial pain fre-quently occurs in the mid-abdomen, suggesting that the diver-sion of blood flow from the SMA to supply the stomach impairs perfusion

1	gastrointestinal collateral circulation. The postprandial pain fre-quently occurs in the mid-abdomen, suggesting that the diver-sion of blood flow from the SMA to supply the stomach impairs perfusion to the small bowel. This leads to transient anaerobic metabolism and acidosis. Persistent or profound mesenteric ischemia will lead to mucosal compromise with release of intra-cellular contents and by-products of anaerobic metabolism to Figure 23-36. An aortogram showing a prominent collateral ves-sel, which is the arc of Riolan (arrow) in a patient with an inferior mesenteric artery (IMA) occlusion. This vessel network provides collateral flow between the superior mesenteric artery and IMA.the splanchnic and systemic circulation. Injured bowel mucosa allows unimpeded influx of toxic substances from the bowel lumen with systemic consequences. If full-thickness necrosis occurs in the bowel wall, intestinal perforation ensues, which will lead to peritonitis. Concomitant atherosclerotic

1	from the bowel lumen with systemic consequences. If full-thickness necrosis occurs in the bowel wall, intestinal perforation ensues, which will lead to peritonitis. Concomitant atherosclerotic disease in cardiac or systemic circulation frequently compounds the diag-nostic and therapeutic complexity of mesenteric ischemia.Types of Mesenteric Artery Occlusive DiseaseThere are three major mechanisms of visceral ischemia involv-ing the mesenteric arteries: (a) acute mesenteric ischemia, which can be either embolic or thrombotic in origin; (b) chronic mes-enteric ischemia; and (c) nonocclusive mesenteric ischemia. Despite the variability of these syndromes, a common anatomic pathology is involved in these processes. The superior mesen-teric artery (SMA) is the most commonly involved vessel in acute mesenteric ischemia. Acute thrombosis occurs in patients with underlying mesenteric atherosclerosis, which typically involves the origin of the mesenteric arteries while sparing the collateral

1	acute mesenteric ischemia. Acute thrombosis occurs in patients with underlying mesenteric atherosclerosis, which typically involves the origin of the mesenteric arteries while sparing the collateral branches. In acute embolic mesenteric ischemia, the emboli typically originate from a cardiac source and frequently occur in patients with atrial fibrillation or following myocar-dial infarction (Figs. 23-37 and 23-38). Nonocclusive mesen-teric ischemia is characterized by a low flow state in otherwise normal mesenteric arteries and most frequently occurs in criti-cally ill patients on vasopressors. Finally, chronic mesenteric ischemia is a functional consequence of a long-standing ath-erosclerotic process that typically involves at least two of the three main mesenteric vessels. The gradual development of the occlusive process allows the development of collateral ves-sels that prevent the manifestations of acute ischemia, but are not sufficient to meet the high postprandial intestinal

1	development of the occlusive process allows the development of collateral ves-sels that prevent the manifestations of acute ischemia, but are not sufficient to meet the high postprandial intestinal oxygen requirements, giving rise to the classical symptoms of postpran-dial abdominal pain and the resultant food fear.Several less common syndromes of visceral ischemia involving the mesenteric arteries can also cause serious debili-tation. Chronic mesenteric ischemic symptoms can occur due Figure 23-37. An anteroposterior view of a selective superior mesenteric artery angiogram shows an abrupt cutoff of the middle colic artery, which was caused by emboli (arrow) due to atrial fibrillation.Brunicardi_Ch23_p0897-p0980.indd 92927/02/19 4:14 PM 930SPECIFIC CONSIDERATIONSPART IIFigure 23-38. A lateral mesenteric angiogram showing an abrupt cutoff of the proximal superior mesenteric artery (SMA), which is consistent with SMA embolism (arrow).to extrinsic compression of the celiac artery by

1	lateral mesenteric angiogram showing an abrupt cutoff of the proximal superior mesenteric artery (SMA), which is consistent with SMA embolism (arrow).to extrinsic compression of the celiac artery by the diaphragm, which is termed median arcuate ligament syndrome or celiac artery compression syndrome. Acute visceral ischemia may occur following an aortic operation, due to ligation of the IMA in the absence of adequate collateral vessels. Furthermore, acute visceral ischemia may develop in aortic dissection, which involves the mesenteric arteries, or after coarctation repair. Finally, other unusual causes of ischemia include mesenteric arteritis, radiation arteritis, and cholesterol emboli.Clinical ManifestationsAbdominal pain out of proportion to physical findings is the classic presentation in patients with acute mesenteric ischemia and occurs following an embolic or thrombotic ischemic event of the SMA. Other manifestations include sudden onset of abdominal cramps in patients with

1	in patients with acute mesenteric ischemia and occurs following an embolic or thrombotic ischemic event of the SMA. Other manifestations include sudden onset of abdominal cramps in patients with underlying cardiac or ath-erosclerotic disease, often associated with bloody diarrhea, as a result of mucosal sloughing secondary to ischemia. Fever, nausea, vomiting, and abdominal distention are some common but nonspecific manifestations. Diffuse abdominal tenderness, rebound, and rigidity are late signs and usually indicate bowel infarction and necrosis.Clinical manifestations of chronic mesenteric ischemia are more subtle due to the extensive collateral development. How-ever, when intestinal blood flow is unable to meet the physio-logic gastrointestinal demands, mesenteric insufficiency ensues. The classical symptoms include postprandial abdominal pain, food fear, and weight loss. Persistent nausea and occasionally diarrhea may coexist. Diagnosis remains challenging, and most of the

1	ensues. The classical symptoms include postprandial abdominal pain, food fear, and weight loss. Persistent nausea and occasionally diarrhea may coexist. Diagnosis remains challenging, and most of the patients will undergo an extensive and expensive gastro-intestinal tract workup for the above symptoms prior to referral to a vascular service.The typical patient who develops nonocclusive mesenteric ischemia is an elderly patient who has multiple comorbidities, such as congestive heart failure, acute myocardial infarction with cardiogenic shock, hypovolemic or hemorrhagic shock, sepsis, pancreatitis, and administration of digitalis or vasocon-strictor agents such as epinephrine. Abdominal pain is only present in approximately 70% of these patients. When present, the pain is usually severe but may vary in location, character, and intensity. In the absence of abdominal pain, progressive abdominal distention with acidosis may be an early sign of isch-emia and impending bowel

1	severe but may vary in location, character, and intensity. In the absence of abdominal pain, progressive abdominal distention with acidosis may be an early sign of isch-emia and impending bowel infarction.Abdominal pain due to narrowing of the origin of the CA may occur as a result of extrinsic compression or impingement by the median arcuate ligament (Fig. 23-39). This condition is known as celiac artery compression syndrome or median arcuate ligament syndrome. Angiographically, there is CA compression that augments with deep expiration and poststenotic dilatation. The celiac artery compression syndrome has been implicated in some variants of chronic mesenteric ischemia. Most patients are young females between 20 and 40 years of age. Abdominal symptoms are nonspecific, but the pain is localized in the upper abdomen, which may be precipitated by meals.Diagnostic EvaluationThe differential diagnosis of acute mesenteric ischemia includes other causes of severe abdominal pain of acute

1	localized in the upper abdomen, which may be precipitated by meals.Diagnostic EvaluationThe differential diagnosis of acute mesenteric ischemia includes other causes of severe abdominal pain of acute onset, such as perforated viscus, intestinal obstruction, pancreatitis, cholecys-titis, and nephrolithiasis. Laboratory evaluation is neither sensitive nor specific in distinguishing these various diagnoses. Figure 23-39. A lateral projection of the magnetic resonance angi-ography of the aorta showing a chronic compression of the celiac artery by the median arcuate ligament (arrow).Brunicardi_Ch23_p0897-p0980.indd 93027/02/19 4:14 PM 931ARTERIAL DISEASECHAPTER 23In the setting of mesenteric ischemia, complete blood count may reveal hemoconcentration and leukocytosis. Metabolic acidosis develops as a result of anaerobic metabolism. Elevated serum amylase may indicate a diagnosis of pancreatitis but is also com-mon in the setting of intestinal infarction. Finally, increased lac-tate

1	develops as a result of anaerobic metabolism. Elevated serum amylase may indicate a diagnosis of pancreatitis but is also com-mon in the setting of intestinal infarction. Finally, increased lac-tate levels, hyperkalemia, and azotemia may occur in the late stages of mesenteric ischemia.Plain abdominal radiographs may provide helpful infor-mation to exclude other causes of abdominal pain such as intes-tinal obstruction, perforation, or volvulus, which may exhibit symptoms mimicking intestinal ischemia. Pneumoperitoneum, pneumatosis intestinalis, and gas in the portal vein may indicate infarcted bowel. In contrast, radiographic appearance of an ady-namic ileus with a gasless abdomen is the most common finding in patients with acute mesenteric ischemia.Upper endoscopy, colonoscopy, or barium radiogra-phy does not provide any useful information when evaluating acute mesenteric ischemia. Moreover, barium enema is con-traindicated if the diagnosis of mesenteric ischemia is being considered.

1	radiogra-phy does not provide any useful information when evaluating acute mesenteric ischemia. Moreover, barium enema is con-traindicated if the diagnosis of mesenteric ischemia is being considered. The intraluminal barium can obscure accurate visu-alization of mesenteric circulation during angiography. In addi-tion, intraperitoneal leakage of barium can occur in the setting of intestinal perforation, which can lead to added therapeutic challenges during mesenteric revascularization.Diagnosis of chronic mesenteric ischemia can be more challenging. Usually prior to the evaluation by a vascular ser-vice, the patients have undergone an extensive workup for the symptoms of chronic abdominal pain, weight loss, and anorexia. Rarely, the vascular surgeon is the first to encounter a patient with the above symptoms. In this situation, it is advisable to keep in mind that mesenteric ischemia is a rare entity and that a full diagnostic workup that should include CT scan of the abdo-men and

1	with the above symptoms. In this situation, it is advisable to keep in mind that mesenteric ischemia is a rare entity and that a full diagnostic workup that should include CT scan of the abdo-men and evaluation by gastroenterologist should be performed. Mesenteric occlusive disease may coexist with malignancy, and symptoms of mesenteric vessel stenosis may be the result of extrinsic compression by a tumor.Duplex ultrasonography is a valuable noninvasive means of assessing the patency of the mesenteric vessels. Moneta and associates evaluated the use of duplex ultrasound in the diag-nosis of mesenteric occlusive disease in a blinded prospective study.90,91 A peak systolic velocity in the SMA >275 cm/s dem-onstrated a sensitivity of 92%, specificity of 96%, and overall accuracy of 96% for detecting >70% stenosis. The same authors found sensitivity and specificity of 87% and 82%, respectively, with an accuracy of 82% in predicting >70% celiac trunk ste-nosis. Duplex has been successfully

1	>70% stenosis. The same authors found sensitivity and specificity of 87% and 82%, respectively, with an accuracy of 82% in predicting >70% celiac trunk ste-nosis. Duplex has been successfully used for follow-up after open surgical reconstruction or endovascular treatment of the mesenteric vessels to assess recurrence of the disease. Finally, spiral CT with three-dimensional reconstruction (Fig. 23-40) and MRA (Fig. 23-41) have been promising in providing clear radiographic assessment of the mesenteric vessels.The definitive diagnosis of mesenteric vascular disease is made by biplanar mesenteric arteriography, which should be performed promptly in any patient with suspected mesenteric occlusion. It typically shows occlusion or near-occlusion of the CA and SMA at or near their origins from the aorta. In most cases, the IMA has been previously occluded secondary to dif-fuse infrarenal aortic atherosclerosis. The differentiation of the different types of mesenteric arterial occlusion may

1	the aorta. In most cases, the IMA has been previously occluded secondary to dif-fuse infrarenal aortic atherosclerosis. The differentiation of the different types of mesenteric arterial occlusion may be sug-gested with biplanar mesenteric arteriogram. Mesenteric emboli typically lodge at the orifice of the middle colic artery, which creates a “meniscus sign” with an abrupt cutoff of a normal Figure 23-40. Computed tomography angiogram of the abdomen with three-dimensional reconstruction provides a clear view of the celiac artery, superior mesenteric artery (SMA), and inferior mes-enteric artery (IMA).Figure 23-41. A cross-sectional view of a magnetic resonance angiogram provides a clear view of the luminal patency of the supe-rior mesenteric artery.proximal SMA several centimeters from its origin on the aorta. Mesenteric thrombosis, in contrast, occurs at the most proximal SMA, which tapers off at 1 to 2 cm from its origin. In the case of chronic mesenteric occlusion, the appearance

1	origin on the aorta. Mesenteric thrombosis, in contrast, occurs at the most proximal SMA, which tapers off at 1 to 2 cm from its origin. In the case of chronic mesenteric occlusion, the appearance of collateral circulation is typically present. Nonocclusive mesenteric isch-emia produces an arteriographic image of segmental mesenteric vasospasm with a relatively normal-appearing main SMA trunk (Fig. 23-42).Mesenteric arteriography can also play a therapeutic role. Once the diagnosis of nonocclusive mesenteric ischemia is Brunicardi_Ch23_p0897-p0980.indd 93127/02/19 4:14 PM 932SPECIFIC CONSIDERATIONSPART IIFigure 23-42. Mesenteric arteriogram showing nonocclusive mes-enteric ischemia as evidenced by diffuse spasm of intestinal arcades with poor filling of intramural vessels.made on the arteriogram, an infusion catheter can be placed at the SMA orifice, and vasodilating agents, such as papaverine, can be administered intra-arterially. The papaverine infusion may be continued

1	on the arteriogram, an infusion catheter can be placed at the SMA orifice, and vasodilating agents, such as papaverine, can be administered intra-arterially. The papaverine infusion may be continued postoperatively to treat persistent vasospasm, a common occurrence following mesenteric reperfusion. Trans-catheter thrombolytic therapy has little role in the management of thrombotic mesenteric occlusion. Although thrombolytic agents may transiently recannulate the occluded vessels, the underlying occlusive lesions require definitive treatment. Fur-thermore, thrombolytic therapy typically requires a prolonged period of time to restore perfusion, during which the intestinal viability will be difficult to assess.A word of caution would be appropriate here regarding patients with typical history of chronic intestinal angina who present with an acute abdomen and classical findings of peri-toneal irritation. Arteriography is the gold standard for the diagnosis of mesenteric occlusive disease;

1	of chronic intestinal angina who present with an acute abdomen and classical findings of peri-toneal irritation. Arteriography is the gold standard for the diagnosis of mesenteric occlusive disease; however, it can be a time-consuming diagnostic modality. In this group of patients, immediate exploration for assessment of intestinal viability and vascular reconstruction is the best choice.Surgical RepairAcute Embolic Mesenteric Ischemia. Initial management of patients with acute mesenteric ischemia includes fluid resus-citation and systemic anticoagulation with heparin to prevent further thrombus propagation. Significant metabolic acidosis not responding to fluid resuscitation should be corrected with sodium bicarbonate. A central venous catheter, peripheral arte-rial catheter, and Foley catheter should be placed for hemody-namic status monitoring. Appropriate antibiotics are given prior to surgical exploration. The operative management of acute mesenteric ischemia is dictated by the

1	catheter should be placed for hemody-namic status monitoring. Appropriate antibiotics are given prior to surgical exploration. The operative management of acute mesenteric ischemia is dictated by the cause of the occlusion. It is helpful to obtain a preoperative mesenteric arteriogram to confirm the diagnosis and to plan appropriate treatment options. However, the diagnosis of mesenteric ischemia frequently can-not be established prior to surgical exploration, and therefore, patients in a moribund condition with acute abdominal symp-toms should undergo immediate surgical exploration, avoiding the delay required to perform an arteriogram.The primary goal of surgical treatment in embolic mes-enteric ischemia is to restore arterial perfusion with removal of the embolus from the vessel. The abdomen is explored through a midline incision, which often reveals variable degrees of intestinal ischemia from the mid-jejunum to the ascending or transverse colon. The transverse colon is lifted

1	abdomen is explored through a midline incision, which often reveals variable degrees of intestinal ischemia from the mid-jejunum to the ascending or transverse colon. The transverse colon is lifted superiorly, and the small intestine is reflected toward the right upper quad-rant. The SMA is approached at the root of the small bowel mesentery, usually as it emerges from beneath the pancreas to cross over the junction of the third and fourth portions of the duodenum. Alternatively, the SMA can be approached by incising the retroperitoneum lateral to the fourth portion of the duodenum, which is rotated medially to expose the SMA. Once the proximal SMA is identified and controlled with vascular clamps, a transverse arteriotomy is made to extract the embolus, using standard balloon embolectomy catheters. In the event the embolus has lodged more distally, exposure of the distal SMA may be obtained in the root of the small bowel mesentery by isolating individual jejunal and ileal branches to

1	catheters. In the event the embolus has lodged more distally, exposure of the distal SMA may be obtained in the root of the small bowel mesentery by isolating individual jejunal and ileal branches to allow a more comprehensive thromboembolectomy. Following the restora-tion of SMA flow, an assessment of intestinal viability must be made, and nonviable bowel must be resected. Several methods have been described to evaluate the viability of the intestine, which include intraoperative intravenous fluorescein injection and inspection with a Wood’s lamp, and Doppler assessment of antimesenteric intestinal arterial pulsations. A second-look pro-cedure should be considered in many patients and is performed 24 to 48 hours following embolectomy. The goal of the proce-dure is reassessment of the extent of bowel viability, which may not be obvious immediately following the initial embolectomy. If nonviable intestine is evident in the second-look procedure, additional bowel resections should be

1	of bowel viability, which may not be obvious immediately following the initial embolectomy. If nonviable intestine is evident in the second-look procedure, additional bowel resections should be performed at that time.Acute Thrombotic Mesenteric Ischemia. Thrombotic mesenteric ischemia usually involves a severely atheroscle-rotic vessel, typically the proximal CA and SMA. Therefore, these patients require a reconstructive procedure to the SMA to bypass the proximal occlusive lesion and restore adequate mesenteric flow. The saphenous vein is the graft material of choice, and prosthetic materials should be avoided in patients with nonviable bowel, due to the risk of bacterial contamina-tion if resection of necrotic intestine is performed. The bypass graft may originate from either the aorta or iliac artery. Advan-tages from using the supraceliac infradiaphragmatic aorta as opposed to the infrarenal aorta as the inflow vessel include a smoother graft configuration with less chance of

1	or iliac artery. Advan-tages from using the supraceliac infradiaphragmatic aorta as opposed to the infrarenal aorta as the inflow vessel include a smoother graft configuration with less chance of kinking and the absence of atherosclerotic disease in the supraceliac aortic segment. Exposure of the supraceliac aorta is technically more challenging and time consuming than that of the iliac artery, which unless calcified is an appropriate inflow. Patency rates are similar regardless of inflow vessel choice.92Chronic Mesenteric Ischemia. The therapeutic goal in patients with chronic mesenteric ischemia is to revascularize mesenteric circulation and prevent the development of bowel infarction. Mesenteric occlusive disease can be treated successfully by either transaortic endarterectomy or mesenteric artery bypass. Brunicardi_Ch23_p0897-p0980.indd 93227/02/19 4:14 PM 933ARTERIAL DISEASECHAPTER 23Transaortic endarterectomy is indicated for ostial lesions of pat-ent CA and SMA. A left

1	artery bypass. Brunicardi_Ch23_p0897-p0980.indd 93227/02/19 4:14 PM 933ARTERIAL DISEASECHAPTER 23Transaortic endarterectomy is indicated for ostial lesions of pat-ent CA and SMA. A left medial rotation is performed, and the aorta and the mesenteric branches are exposed. A lat-eral aortotomy is performed encompassing both the CA and SMA orifices. The visceral arteries must be adequately mobi-lized so that the termination site of endarterectomy can be visu-alized. Otherwise, an intimal flap may develop, which can lead to early thrombosis or distal embolization.For occlusive lesions located 1 to 2 cm distal to the mes-enteric origin, mesenteric artery bypass should be performed. Multiple mesenteric arteries are typically involved in chronic mesenteric ischemia, and both the CA and SMA should be revascularized whenever possible. In general, bypass grafting may be performed either antegrade from the supraceliac aorta or retrograde from either the infrarenal aorta or iliac artery. Both

1	be revascularized whenever possible. In general, bypass grafting may be performed either antegrade from the supraceliac aorta or retrograde from either the infrarenal aorta or iliac artery. Both autogenous saphenous vein grafts and prosthetic grafts have been used with satisfactory and equivalent success. An antegrade bypass also can be performed using a small-caliber bifurcated graft from the supraceliac aorta to both the CA and SMA, which yields an excellent long-term result.93Celiac Artery Compression Syndrome. The decision to intervene in patients with CA compression syndrome should be based on both an appropriate symptom complex and the finding of celiac artery compression in the absence of other findings to explain the symptoms. The treatment goal is to release the ligamentous structure that compresses the proximal CA and to correct any persistent stricture by bypass grafting. Some sur-geons advocate careful celiac plexus sympathectomy in addition to arcuate ligament

1	structure that compresses the proximal CA and to correct any persistent stricture by bypass grafting. Some sur-geons advocate careful celiac plexus sympathectomy in addition to arcuate ligament decompression to ensure good treatment outcome.102 The patient should be cautioned that relief of the celiac compression cannot be guaranteed to relieve the symp-toms. In a number of reports on endovascular management of chronic mesenteric ischemia, the presence of CA compres-sion syndrome has been identified as a major factor of techni-cal failure and recurrence. Therefore, angioplasty and stenting should not be undertaken if extrinsic compression of the CA by the median arcuate ligament is suspected based on preop-erative imaging studies. Open surgical treatment should be performed instead. A study that analyzed the outcome of lapa-roscopic and open median arcuate ligament release cases in the literature showed both approaches to be effective in symptom relief (85%), with no difference in

1	study that analyzed the outcome of lapa-roscopic and open median arcuate ligament release cases in the literature showed both approaches to be effective in symptom relief (85%), with no difference in late symptom recurrence rate (6.8% in the open group and 5.7% in the laparoscopic group).94Endovascular TreatmentChronic Mesenteric Ischemia. Endovascular treatment of mesenteric artery stenosis or short segment occlusion by bal-loon dilatation or stent placement represents a less invasive therapeutic alternative to open surgical intervention, particu-larly in patients whose medical comorbidities place them at a high operative risk category. Endovascular therapy is also suited in patients with recurrent disease or anastomotic steno-sis following previous open mesenteric revascularization. Pro-phylactic mesenteric revascularization is rarely performed in the asymptomatic patient undergoing an aortic procedure for other indications. However, the natural history of untreated chronic

1	Pro-phylactic mesenteric revascularization is rarely performed in the asymptomatic patient undergoing an aortic procedure for other indications. However, the natural history of untreated chronic mesenteric ischemia may justify revascularization in some minimally symptomatic or asymptomatic patients if the operative risks are acceptable, since the first clinical presenta-tion may be acute intestinal ischemia in as many as 50% of the patients, with a mortality rate that ranges from 15% to 70%.95 3This is particularly true when the SMA is involved. Mesenteric angioplasty and stenting is particularly suitable for this patient subgroup given its low morbidity and mortality. Because of the limited experience with stent use in mesenteric vessels, appro-priate indications for primary stent placement have not been clearly defined. Guidelines generally include calcified ostial stenoses, high-grade eccentric stenoses, chronic occlusions, and significant residual stenosis >30% or the presence of

1	have not been clearly defined. Guidelines generally include calcified ostial stenoses, high-grade eccentric stenoses, chronic occlusions, and significant residual stenosis >30% or the presence of dissection after angioplasty. Restenosis after PTA is also an indication for stent placement.96Acute Mesenteric Ischemia. Catheter-directed thrombo-lytic therapy is a potentially useful treatment modality for acute mesenteric ischemia, which can be initiated with intra-arterial delivery of thrombolytic agent into the mesenteric thrombus at the time of diagnostic angiography. Various thrombolytic medi-cations, including urokinase (Abbokinase; Abbott Laboratory, North Chicago, IL) or recombinant tissue plasminogen acti-vator (Activase; Genentech, South San Francisco, CA), have been reported to be successful in a small series of case reports. Catheter-directed thrombolytic therapy has a higher probabil-ity of restoring mesenteric blood flow success when performed within 12 hours of symptom

1	be successful in a small series of case reports. Catheter-directed thrombolytic therapy has a higher probabil-ity of restoring mesenteric blood flow success when performed within 12 hours of symptom onset. Successful resolution of a mesenteric thrombus will facilitate the identification of the underlying mesenteric occlusive disease process. As a result, subsequent operative mesenteric revascularization or mesen-teric balloon angioplasty and stenting may be performed elec-tively to correct the mesenteric stenosis. There are two main drawbacks with regard to thrombolytic therapy in mesenteric ischemia. Percutaneous catheter-directed thrombolysis does not allow the possibility to inspect the potentially ischemic intes-tine following restoration of the mesenteric flow. Additionally, a prolonged period of time may be necessary in order to achieve successful catheter-directed thrombolysis, due in part to serial angiographic surveillance to document thrombus resolution. An incomplete or

1	period of time may be necessary in order to achieve successful catheter-directed thrombolysis, due in part to serial angiographic surveillance to document thrombus resolution. An incomplete or unsuccessful thrombolysis may lead to delayed operative revascularization, which may further necessitate bowel resection for irreversible intestinal necrosis. Therefore, catheter-directed thrombolytic therapy for acute mesenteric ischemia should only be considered in selected patients under a closely scrutinized clinical protocol.Nonocclusive Mesenteric Ischemia. The treatment of non-occlusive mesenteric ischemia is primarily pharmacologic with selective mesenteric arterial catheterization followed by infu-sion of vasodilatory agents, such as tolazoline or papaverine. Once the diagnosis is made on the mesenteric arteriography (see Fig. 23-42), intra-arterial papaverine is given at a dose of 30 to 60 mg/h. This must be coupled with the cessation of other vaso-constricting agents. Concomitant

1	on the mesenteric arteriography (see Fig. 23-42), intra-arterial papaverine is given at a dose of 30 to 60 mg/h. This must be coupled with the cessation of other vaso-constricting agents. Concomitant intravenous heparin should be administered to prevent thrombosis in the cannulated vessels. Treatment strategy thereafter is dependent on the patient’s clini-cal response to the vasodilator therapy. If abdominal symptoms improve, mesenteric arteriography should be repeated to docu-ment the resolution of vasospasm. The patient’s hemodynamic status must be carefully monitored during papaverine infusion as significant hypotension can develop in the event that the infu-sion catheter migrates into the aorta, which can lead to systemic circulation of papaverine. Surgical exploration is indicated if the patient develops signs of continued bowel ischemia or infarction as evidenced by rebound tenderness or involuntary guarding. In these circumstances, papaverine infusion should be contin-ued

1	if the patient develops signs of continued bowel ischemia or infarction as evidenced by rebound tenderness or involuntary guarding. In these circumstances, papaverine infusion should be contin-ued intraoperatively and postoperatively. The operating room Brunicardi_Ch23_p0897-p0980.indd 93327/02/19 4:14 PM 934SPECIFIC CONSIDERATIONSPART IIshould be kept as warm as possible, and warm irrigation fluid and laparotomy pads should be used to prevent further intestinal vasoconstriction during exploration.Techniques of Endovascular Interventions. To perform endovascular mesenteric revascularization, intraluminal access is performed via a femoral or brachial artery approach. Once an introducer sheath is placed in the femoral artery, an anteroposte-rior and lateral aortogram just below the level of the diaphragm is obtained with a pigtail catheter to identify the origin of the CA and SMA. Initial catheterization of the mesenteric artery can be performed using a variety of selective angled

1	of the diaphragm is obtained with a pigtail catheter to identify the origin of the CA and SMA. Initial catheterization of the mesenteric artery can be performed using a variety of selective angled catheters, which include the RDC, Cobra-2, Simmons I (Boston Scientific/Meditech, Natick, MA), or SOS Omni catheter (Angiodynam-ics, Queensbury, NY). Once the mesenteric artery is cannulated, systemic heparin (5000 IU) is administered intravenously. A selective mesenteric angiogram is then performed to identify the diseased segment, which is followed by the placement of a 0.035-inch or less traumatic 0.014to 0.018-inch guidewire to cross the stenotic lesion. Once the guidewire is placed across the stenosis, the catheter is carefully advanced over the guide-wire across the lesion. In the event that the mesenteric artery is severely angulated as it arises from the aorta, a second stiffer guidewire (Amplatz or Rosen Guidewire, Boston Scientific) may be exchanged through the catheter to

1	event that the mesenteric artery is severely angulated as it arises from the aorta, a second stiffer guidewire (Amplatz or Rosen Guidewire, Boston Scientific) may be exchanged through the catheter to facilitate the place-ment of a 6-French guiding sheath (Pinnacle, Boston Scientific).With the image intensifier angled in a lateral position to fully visualize the proximal mesenteric segment, a balloon angioplasty is advanced over the guidewire through the guiding sheath and positioned across the stenosis. The balloon diameter should be chosen based on the vessel size of the adjacent nor-mal mesenteric vessel. Once balloon angioplasty is completed, a postangioplasty angiogram is necessary to document the proce-dural result. Radiographic evidence of either residual stenosis or mesenteric artery dissection constitutes suboptimal angioplasty results that warrants mesenteric stent placement. Moreover, ath-erosclerotic involvement of the proximal mesenteric artery or vessel orifice should be

1	dissection constitutes suboptimal angioplasty results that warrants mesenteric stent placement. Moreover, ath-erosclerotic involvement of the proximal mesenteric artery or vessel orifice should be treated with balloon-expandable stent placement. These stents can be placed over a low-profile 0.014or 0.018-inch guidewire system. It is preferable to deliver the balloon-mounted stent through a guiding sheath, which is posi-tioned just proximal to the mesenteric orifice while the balloon-mounted stent is advanced across the stenosis. The stent is next deployed by expanding the angioplasty balloon to its designated inflation pressure. The balloon is then deflated and carefully withdrawn through the guiding sheath.Completion angiogram is performed by hand injecting a small volume of contrast though the guiding sheath. It is criti-cal to maintain the guidewire access until satisfactory comple-tion angiogram is obtained. If the completion angiogram reveals suboptimal radiographic results, such

1	the guiding sheath. It is criti-cal to maintain the guidewire access until satisfactory comple-tion angiogram is obtained. If the completion angiogram reveals suboptimal radiographic results, such as residual stenosis or dis-section, additional catheter-based intervention can be performed through the same guidewire. These interventions may include repeat balloon angioplasty for residual stenosis or additional stent placement for mesenteric artery dissection. During the procedure, intra-arterial infusion of papaverine or nitroglycerine can be used to decrease vasospasm. Administration of antiplatelet agents is also recommended for at least 6 months or even indefinitely if other risk factors of cardiovascular disease are present.Complications of Endovascular Treatment. Complica-tions are not common and rarely become life threatening. These include access site thrombosis, hematomas, and infection. Dissection can occur during PTA and is managed with place-ment of a stent. Balloon-mounted

1	common and rarely become life threatening. These include access site thrombosis, hematomas, and infection. Dissection can occur during PTA and is managed with place-ment of a stent. Balloon-mounted stents are preferred over the self-expanding ones because of the higher radial force and the more precise placement. Distal embolization has also been reported, but it never resulted in acute intestinal ischemia, likely due to the rich network of collaterals already developed.Clinical Results of Interventions for Mesenteric IschemiaThe first successful percutaneous angioplasty of the SMA was reported in 1980.97 Since 1995, multiple series and scattered case reports have reported results from endovascular manage-ment of mesenteric occlusive disease.92,96 A literature review by AbuRahma and colleagues in 2003 showed that endovascu-lar intervention had an overall technical success rate of 91%, early and late pain relief rates of 84% and 71%, respectively, and 30-day morbidity and mortality

1	in 2003 showed that endovascu-lar intervention had an overall technical success rate of 91%, early and late pain relief rates of 84% and 71%, respectively, and 30-day morbidity and mortality rates of 16.4% and 4.3%, respectively. The average patency was 63% during an average 26-month follow-up.98In our review of the literature from published series since 1995, restenosis developed in 22% of patients during 24.5 months of average follow-up.92 The long-term clinical relief without reintervention was 82%. Among the patients who experienced a technical failure, 15 were ultimately diagnosed with median arcuate ligament syndrome and underwent suc-cessful surgical treatment, an observation that emphasizes the need for careful patient selection. Interestingly, the addition of selective stenting after PTA that was started in 1998, while it slightly increases the technical success rate, is not correlated with any substantial overall clinical benefit or improved long-term patency rates.In

1	after PTA that was started in 1998, while it slightly increases the technical success rate, is not correlated with any substantial overall clinical benefit or improved long-term patency rates.In contrast to endovascular treatment, open surgical tech-niques have achieved an immediate clinical success rate that approaches 100%, a surgical mortality rate of 0% to 17%, and an operative morbidity rate that ranges from 19% to 54% in a number of different series.89,93,99 AbuRahma and colleagues reported their experience of endovascular interventions of 22 patients with symptomatic mesenteric ischemia due to either SMA or CA stenosis.98 They noted an excellent initial technical and clinical success rates, which were 96% (23 of 24 patients) and 95% (21 of 22 patients), respectively, with no periopera-tive mortality or major morbidity. During a mean follow-up of 26 months (range, 1–54 months), the primary late clinical suc-cess rate was 61%, and freedom from recurrent stenosis was 30%. The

1	mortality or major morbidity. During a mean follow-up of 26 months (range, 1–54 months), the primary late clinical suc-cess rate was 61%, and freedom from recurrent stenosis was 30%. The freedom from recurrent stenosis rates at 1, 2, 3, and 4 years were 65%, 47%, 39%, and 13%, respectively. The authors concluded that mesenteric stenting, which provides excellent early results, is associated with a relative high inci-dence of late restenosis.Several studies have attempted to compare the endovas-cular with the standard open surgical approach.89,100 The results of the open surgery appear to be more durable, but it tends to be associated with higher morbidity and mortality rates and an overall longer hospital stay. In one study that compared the clin-ical outcome of open revascularization with percutaneous stent-ing for patients with chronic mesenteric ischemia, 28 patients underwent endovascular treatment and 85 patients underwent open mesenteric bypass grafting.99 With both patient

1	with percutaneous stent-ing for patients with chronic mesenteric ischemia, 28 patients underwent endovascular treatment and 85 patients underwent open mesenteric bypass grafting.99 With both patient cohorts having similar baseline comorbidities and symptom duration, there was no difference in early in-hospital complication or mortality rates. Moreover, both groups had similar 3-year cumu-lative recurrent stenosis and mortality rates. However, patients Brunicardi_Ch23_p0897-p0980.indd 93427/02/19 4:14 PM 935ARTERIAL DISEASECHAPTER 23treated with mesenteric stenting had a significantly higher inci-dence of recurrent symptoms. The authors concluded that opera-tive mesenteric revascularization should be offered to patients with low surgical risk.Based on the above results one could argue that mesen-teric angioplasty and stenting demonstrate an inferior technical and clinical success rate. Long-term patency rates appear to also be superior with the open technique. There is a general

1	that mesen-teric angioplasty and stenting demonstrate an inferior technical and clinical success rate. Long-term patency rates appear to also be superior with the open technique. There is a general con-sensus, however, that the endovascular approach is associated with lower morbidity and mortality rates and is therefore more suitable for high-risk patients. One should also keep in mind that practices representing standard of care for stent placement today were absent in the early era of endovascular experience. These include perioperative heparinization and short-term anti-platelet therapy, use of stents with higher radial force, routine use of postoperative surveillance with arterial duplex and early reintervention to prevent a high-grade stenosis from progressing to occlusion, and placement of drug-eluting stents. One such example is a recent nonrandomized study to compare the out-comes of mesenteric angioplasty using covered stents or bare metal stents in patients undergoing

1	placement of drug-eluting stents. One such example is a recent nonrandomized study to compare the out-comes of mesenteric angioplasty using covered stents or bare metal stents in patients undergoing primary or reintervention for chronic mesenteric ischemia. The study showed that covered stents are associated with less restenosis (18% vs. 47%), symp-tom recurrence (18% vs. 50%), and reintervention (9% vs. 44%) at 24 months and better primary patency at 3 years (92% vs. 52%) than bare metal stents in the primary intervention group.101 Similar results were found in the reintervention group as well.RENAL ARTERY DISEASEObstructive lesions of the renal artery can produce hypertension, resulting in a condition known as renovascular hypertension, which is the most common form of hypertension amenable to therapeutic intervention, and affects 5% to 10% of all hyperten-sive patients in the United States.102 Patients with renovascular hypertension are at an increased risk for irreversible

1	amenable to therapeutic intervention, and affects 5% to 10% of all hyperten-sive patients in the United States.102 Patients with renovascular hypertension are at an increased risk for irreversible end-organ dysfunction, including permanent kidney damage, if inadequate pharmacologic therapies are used to control the blood pressure. The majority of patients with renal artery obstructive disease have vascular lesions of either atherosclerotic disease or fibro-dysplasia involving the renal arteries. The proximal portion of the renal artery represents the most common location for the development of atherosclerotic disease. It is well established that renal artery intervention, either by surgical or endovascular revascularization, provides an effective treatment for controlling renovascular hypertension as well as preserving renal function. The decision for intervention is complex and needs to consider a variety of anatomic, physiologic, and clinical features, unique for the individual

1	hypertension as well as preserving renal function. The decision for intervention is complex and needs to consider a variety of anatomic, physiologic, and clinical features, unique for the individual patient.EtiologyApproximately 80% of all renal artery occlusive lesions are caused by atherosclerosis, which typically involves a short seg-ment of the renal artery ostia and represents spillover disease from a severely atheromatous aorta (Fig. 23-43).102 Atheroscle-rotic lesions are bilateral in two thirds of patients. Individuals with this disease commonly present during the sixth decade of life. Men are affected twice as frequently as women. Atheroscle-rotic lesions in other territories such as the coronary, mesenteric, cerebrovascular, and peripheral arterial circulation are common. When a unilateral lesion is present, the disease process equally affects the right and left renal arteries.Figure 23-43. Occlusive disease of the renal artery typically involves the renal ostium (arrow) as

1	unilateral lesion is present, the disease process equally affects the right and left renal arteries.Figure 23-43. Occlusive disease of the renal artery typically involves the renal ostium (arrow) as a spillover plaque extension from aortic atherosclerosis.The second most common cause of renal artery stenosis is FMD, which accounts for 20% of cases and is most frequently encountered in young, often multiparous women. FMD of the renal artery represents a heterogeneous group of lesions that can produce histopathologic changes in the intima, media, or adventitia. The most common variety consists of medial fibro-plasia, in which thickened fibromuscular ridges alternate with attenuated media producing the classic angiographic “string of beads” appearance (Figs. 23-44 and 23-45). The cause of medial fibroplasia remains unclear. Most common theories involve a modification of arterial smooth muscle cells in response to Figure 23-44. Abdominal aortogram reveals a left renal artery fibromuscular

1	fibroplasia remains unclear. Most common theories involve a modification of arterial smooth muscle cells in response to Figure 23-44. Abdominal aortogram reveals a left renal artery fibromuscular dysplasia (arrows) with a characteristic “string of beads” appearance.Brunicardi_Ch23_p0897-p0980.indd 93527/02/19 4:14 PM 936SPECIFIC CONSIDERATIONSPART IIFigure 23-45. Magnetic resonance angiography of the abdominal aorta reveals the presence of a left renal artery fibromuscular dys-plasia (arrows).estrogenic stimuli during the reproductive years, unusual trac-tion forces on affected vessels, and mural ischemia from impair-ment of vasa vasorum blood flow. Fibromuscular hyperplasia usually affects the distal two thirds of the main renal artery, and the right renal artery is affected more frequently than the left. Other less common causes of renal artery stenosis include renal artery aneurysm (compressing the adjacent normal renal artery), arteriovenous malformations, neurofibromatosis,

1	than the left. Other less common causes of renal artery stenosis include renal artery aneurysm (compressing the adjacent normal renal artery), arteriovenous malformations, neurofibromatosis, renal artery dissections, renal artery trauma, Takayasu’s arteritis, and renal arteriovenous fistula.Clinical ManifestationsRenovascular hypertension is the most common sequela of renal artery occlusive disease. Its prevalence varies from 2% in patients with diastolic blood pressure greater than 100 mmHg to almost 30% in those with diastolic blood pressure over 125 mmHg.103 Clinical features that may indicate the presence of renovascular hypertension include the following: (a) systolic and diastolic upper abdominal bruits; (b) diastolic hypertension of greater than 115 mmHg; (c) rapid onset of hypertension after the age of 50 years; (d) a sudden worsening of mild to moder-ate essential hypertension; (e) hypertension that is difficult to control with three or more antihypertensives; (f)

1	of hypertension after the age of 50 years; (d) a sudden worsening of mild to moder-ate essential hypertension; (e) hypertension that is difficult to control with three or more antihypertensives; (f) development of renal insufficiency after angiotensin-converting enzyme inhibi-tors; and (g) development of hypertension during childhood.103All patients with significant hypertension, especially ele-vated diastolic blood pressure, must be considered as suspect for renovascular disease. Young adults with hypertension have a great deal to gain by avoiding lifelong treatment if renovascular hypertension is diagnosed and corrected. Appropriate diagnostic studies and intervention must be timely instituted to detect the possibility of renovascular hypertension in patients with pri-mary hypertension who present for clinical evaluation.Diagnostic EvaluationThe diagnostic requisites for renovascular hypertension include both hypertension and renal artery stenosis. Impairment of the renal function

1	who present for clinical evaluation.Diagnostic EvaluationThe diagnostic requisites for renovascular hypertension include both hypertension and renal artery stenosis. Impairment of the renal function may coexist, although the occurrence of renal insufficiency prior to the development of hypertension is uncommon. Nearly all diagnostic studies for renovascular hypertension evaluate either the anatomic stenosis or renal parenchymal dysfunction attributed to the stenosis. The follow-ing section provides an overview of the strengths and limitations of the most common tests used in the diagnostic evaluation of the patient with suspected renovascular hypertension prior to intervention.Captopril renal scanning is a functional study that assesses renal perfusion before and after administration of the angioten-sin-converting enzyme inhibitor captopril. Captopril inhibits the secretion of angiotensin II. Through this mechanism, it reduces the efferent arteriole vasoconstriction and, as a result,

1	angioten-sin-converting enzyme inhibitor captopril. Captopril inhibits the secretion of angiotensin II. Through this mechanism, it reduces the efferent arteriole vasoconstriction and, as a result, the glo-merular filtration rate (GFR). The test consists of a baseline renal scan and a second renal scan after captopril administra-tion. A positive result indicates that captopril administration (a) increases the time to peak activity to more than 11 minutes or (b) the GFR ratio between sides increases to greater than 1.5:1 compared to a normal baseline scan. Significant parenchymal disease limits the reliability of this study.Renal artery duplex ultrasonography is a noninvasive test of assessing renal artery stenosis both by visualization of the vessel and measurement of the effect of stenosis on blood flow velocity and waveforms. The presence of a severe renal artery stenosis correlates with peak systolic velocities of greater than 180 cm/s and the ratio of these velocities to those in

1	on blood flow velocity and waveforms. The presence of a severe renal artery stenosis correlates with peak systolic velocities of greater than 180 cm/s and the ratio of these velocities to those in the aorta of greater than 3.5 (Table 23-11). Renal artery duplex is a techni-cally demanding exam, requiring a substantial amount of opera-tor expertise. In addition, the presence of bowel gas and obesity make the exam difficult to perform and interpret. However, in experienced hands and with appropriate patient selection, it can be a high-yield exam and is typically the initial screening test for patients with suspected renal artery occlusive disease.Selective catheterization of the renal vein via a femoral vein approach for assessing renin activity is a more invasive test of detecting the physiologic sequelae of renal artery stenosis. If unilateral disease is present, the affected kidney should secrete high levels of renin while the contralateral kidney should have low renin production. A

1	sequelae of renal artery stenosis. If unilateral disease is present, the affected kidney should secrete high levels of renin while the contralateral kidney should have low renin production. A ratio between the two kidneys, or the renal vein renin ratio (RVRR), of greater than 1.5 is indica-tive of functionally important renovascular hypertension, and it also predicts a favorable response from renovascular revascu-larization. Since this study assesses the ratio between the two kidneys, it is not useful in patients with bilateral disease because both kidneys may secrete abnormally elevated renin levels.The renal:systemic renin index (RSRI) is calculated by subtracting systemic renin activity from individual renal vein renin activity and dividing the remainder by systemic renin activity. This value represents the contribution of each kidney to renin production. In the absence of renal artery stenosis, the Table 23-11Renal duplex diagnostic criteriaRENAL ARTERY DIAMETER REDUCTIONRENAL

1	value represents the contribution of each kidney to renin production. In the absence of renal artery stenosis, the Table 23-11Renal duplex diagnostic criteriaRENAL ARTERY DIAMETER REDUCTIONRENAL ARTERY PSVRARNormal<180 cm/s<3.5<60%≥180 cm/s<3.5≥60%≥180 cm/s≥3.5OcclusionNo signalNo signalPSV = peak systolic velocity; RAR = renal-to-aortic ratio.Brunicardi_Ch23_p0897-p0980.indd 93627/02/19 4:14 PM 937ARTERIAL DISEASECHAPTER 23renal vein renin activity from each kidney is typically 24% or 0.24 higher than the systemic level. As the result, the total of both kidneys’ renin activity is usually 48% greater than the sys-temic activity, a value that represents a steady state of renal renin activity. The RSRI of the affected kidney in patients with renovascular hypertension is greater than 0.24. In the case of unilateral renal artery stenosis with normal contralateral kid-ney, the increase in ipsilateral renin release is normally balanced by suppression of the contralateral kidney renin

1	In the case of unilateral renal artery stenosis with normal contralateral kid-ney, the increase in ipsilateral renin release is normally balanced by suppression of the contralateral kidney renin production, which results in a drop in its RSRI to less than 0.24. Bilateral renal artery disease may negate the contralateral compensatory response, and the autonomous release of renin from both dis-eased kidneys may result in the sum of the individual RSRIs to be considerably greater than 0.48. The prognostic value of RSRI remains limited in that approximately 10% of patients with favorable clinical response following renovascular revas-cularization do not exhibit contralateral renin suppression. As a result, the use of RSRI must be applied with caution in the management of patients with renovascular hypertension.MRA with intravenous gadolinium contrast enhancement has been increasingly used for renal artery imaging because of its ability to provide high-resolution images (Figs. 23-46 and

1	hypertension.MRA with intravenous gadolinium contrast enhancement has been increasingly used for renal artery imaging because of its ability to provide high-resolution images (Figs. 23-46 and 23-47) while using a minimally nephrotoxic agent. Flow void may be inaccurately interpreted as occlusion or stenosis in MRA. Therefore, unless the quality of the image analysis soft-ware is superior, MRA should be interpreted with caution and used in conjunction with other modalities prior to making plans for operative or endovascular treatment.DSA remains the gold standard to assess renal artery occlusive disease. A flush aortogram is performed first so that any accessory renal arteries can be detected and the origins of all the renal arteries are adequately displayed. The presence of collateral vessels circumventing a renal artery stenosis strongly supports the hemodynamic importance of the stenosis. A pres-sure gradient of 10 mmHg or greater is necessary for collateral Figure 23-46. Magnetic

1	circumventing a renal artery stenosis strongly supports the hemodynamic importance of the stenosis. A pres-sure gradient of 10 mmHg or greater is necessary for collateral Figure 23-46. Magnetic resonance angiography of the abdominal aorta reveals bilateral normal renal arteries.Figure 23-47. Magnetic resonance angiography of the abdominal aorta reveals bilateral ostial renal artery stenosis (arrows).vessel development, which is also associated with activation of the renin-angiotensin cascade.Treatment IndicationsThe therapeutic goals in patients with renovascular disease include: (a) improved blood pressure control, in order to prevent end-organ damage on systems such as the cerebral, coronary, pulmonary, and peripheral circulations; and (b) preservation and possibly improvement of the renal function (Table 23-12).Table 23-12Indications for renal artery revascularizationAngiography Criteria• Documented renal artery stenosis (>70% diameter reduction)• Fibromuscular dysplasia

1	of the renal function (Table 23-12).Table 23-12Indications for renal artery revascularizationAngiography Criteria• Documented renal artery stenosis (>70% diameter reduction)• Fibromuscular dysplasia lesion• Pressure gradient >20 mmHg• Affected/unaffected kidney renin ratio >1.5 to 1Clinical Criteria• Refractory or rapidly progressive hypertension• Hypertension associated with flash pulmonary edema without coronary artery disease• Rapidly progressive deterioration in renal function• Intolerance to antihypertensive medications• Chronic renal insufficiency related to bilateral renal artery occlusive disease or stenosis to a solitary functioning kidney• Dialysis-dependent renal failure in a patient with renal artery stenosis but without another definite cause of end-stage renal disease• Recurrent congestive heart failure or flash pulmonary edema not attributable to active coronary ischemiaBrunicardi_Ch23_p0897-p0980.indd 93727/02/19 4:14 PM 938SPECIFIC CONSIDERATIONSPART IIThe

1	congestive heart failure or flash pulmonary edema not attributable to active coronary ischemiaBrunicardi_Ch23_p0897-p0980.indd 93727/02/19 4:14 PM 938SPECIFIC CONSIDERATIONSPART IIThe indications for endovascular treatment for renal artery occlusive disease include 70% or greater stenosis of one or both renal arteries and at least one of the following clinical criteria:• Inability to adequately control hypertension despite appropri-ate antihypertensive regimen.• Chronic renal insufficiency related to bilateral renal artery occlusive disease or stenosis to a solitary functioning kidney.• Dialysis-dependent renal failure in a patient with renal artery stenosis but without another definite cause of end-stage renal disease.• Recurrent congestive heart failure or flash pulmonary edema not attributable to active coronary ischemia.Prior to 1990, the most common treatment modality in patients with renal artery occlusive disease is surgical revascu-larization, with either renal artery

1	not attributable to active coronary ischemia.Prior to 1990, the most common treatment modality in patients with renal artery occlusive disease is surgical revascu-larization, with either renal artery bypass grafting or renal artery endarterectomy. The advancement of endovascular therapy in the past decade has led to various minimally invasive treatment strategies such as renal artery balloon angioplasty or stenting to control hypertension or to preserve renal function.Surgical ReconstructionThe typical approach for surgical renal artery revasculariza-tion involves a midline xiphoid-to-pubis incision. The posterior peritoneum is incised, and the duodenum is mobilized to the right, starting at the ligament of Treitz. The left renal hilum can be exposed by extending the retroperitoneal dissection to the left along the avascular plane along the inferior border of the pancreas. Mobilization of the left renal vein is essential in these cases and can be achieved by dividing the gonadal,

1	to the left along the avascular plane along the inferior border of the pancreas. Mobilization of the left renal vein is essential in these cases and can be achieved by dividing the gonadal, ilio-lumbar, and adrenal veins. The proximal portion of the right renal artery can be exposed through the base of the mesentery by retraction of the left renal vein cephalad and the vena cava to the right. Accessing the most distal portion of the right renal artery requires a Kocher maneuver and duodenal mobiliza-tion. Another approach useful for treating bilateral renal artery lesions involves mobilization of the entire small bowel and the right colon, with a dissection that starts at the ligament of Treitz and proceeds toward the cecum and then along the line of Todd in the right paracolic gutter. Simultaneous dissection along the inferior border of the pancreas provides additional visualization of the left renal artery. Finally, division of the diaphragmatic crura that encircle the suprarenal

1	Simultaneous dissection along the inferior border of the pancreas provides additional visualization of the left renal artery. Finally, division of the diaphragmatic crura that encircle the suprarenal aorta may sometimes be neces-sary to achieve suprarenal clamping.Types of Surgical Reconstruction. Aortorenal bypass is the most frequently performed reconstruction of ostial occlu-sive renal artery disease. After proximal and distal control is obtained, an elliptical segment of the aorta is excised, and the proximal anastomosis is performed in end-to-side fashion. Autologous vein is the preferred conduit. If the vein is not suit-able, then prosthetic material can be used. An end-to-end anas-tomosis is then performed between the conduit of choice and the renal artery using either a 6-0 or 7-0 polypropylene suture. The length of the arteriotomy needs to be at least three times the diameter of the renal artery to prevent anastomotic restenosis. In the event that the surgeon plans to perform

1	polypropylene suture. The length of the arteriotomy needs to be at least three times the diameter of the renal artery to prevent anastomotic restenosis. In the event that the surgeon plans to perform a side-to-side anasto-mosis between the conduit and the renal artery, this is performed first, and the aortic anastomosis follows.Endarterectomy, either transrenal or transaortic, is an alter-native to bypass for short ostial lesions or in patients with mul-tiple renal arteries. The transrenal endarterectomy is performed with a transverse longitudinal incision on the aorta that extends into the diseased renal artery. After plaque removal, the arteri-otomy is closed with a prosthetic patch. Transaortic endarter-ectomy is well suited for patients with multiple renal arteries and short ostial lesions. The aorta is opened longitudinally and aortic sleeve endarterectomy is performed, followed by ever-sion endarterectomy of the renal arteries. Adequate mobiliza-tion of the renal arteries is

1	lesions. The aorta is opened longitudinally and aortic sleeve endarterectomy is performed, followed by ever-sion endarterectomy of the renal arteries. Adequate mobiliza-tion of the renal arteries is essential for a safe and complete endarterectomy.Hepatorenal and splenorenal bypass are alternative options of revascularization for patients who might not tolerate aortic clamping or for those with calcified aorta that precludes ade-quate control. For hepatorenal bypass, a right subcostal inci-sion is used, and the hepatic artery is exposed with an incision in the lesser omentum. A Kocher maneuver is performed, the right renal vein is identified and mobilized, and the right renal artery is identified and controlled posteriorly to the vein. Greater saphenous vein is the conduit of choice. The anastomosis is per-formed end-to-side with the common hepatic artery, and end-to-end with the renal artery anterior to the inferior vena cava. The splenorenal bypass is performed via a left subcostal

1	anastomosis is per-formed end-to-side with the common hepatic artery, and end-to-end with the renal artery anterior to the inferior vena cava. The splenorenal bypass is performed via a left subcostal inci-sion. The splenic artery is mobilized from the lesser sac, brought through a retropancreatic plane, and anastomosed end-to-end to the renal artery.Reimplantation of the renal artery is an attractive option of reconstruction in children or in adults with ostial lesions. A redundant renal artery is a prerequisite for the procedure. After mobilization, the artery is transected and spatulated, eversion endarterectomy is performed if necessary, and an end-to-side anastomosis with the aorta is created.Clinical Results of Surgical RepairResults reflect the need for performance of renal artery bypass in high-volume and experienced centers. In a review from a large tertiary center, 92% of the patients with nonatherosclerotic vascular disease had improvement in hypertension, but only 43% were

1	in high-volume and experienced centers. In a review from a large tertiary center, 92% of the patients with nonatherosclerotic vascular disease had improvement in hypertension, but only 43% were completely cured and taken off antihypertensives.104 Patients younger than age 45 fair better, with a cure rate of 68% and improvement rate of 32%. In patients with atherosclerotic renal artery disease, the cure rate was even smaller (12%), and the overall response to hypertension rate was 85%. The opera-tive mortality rates were 3.1% and 0% in the atherosclerotic and nonatherosclerotic groups, respectively.Renal function improvement occurs within the first week of the operation in approximately two-thirds of patients. A pro-gressive decrease in the GFR is seen after this initial improve-ment, but the rate of decrease is less compared with patients who did not respond at all to operative intervention. Up to three-quarters of patients were permanently removed from dialysis in a large series.105

1	the rate of decrease is less compared with patients who did not respond at all to operative intervention. Up to three-quarters of patients were permanently removed from dialysis in a large series.105 Favorable response of renal function to revas-cularization improves overall survival.Endovascular TreatmentEndovascular treatment of renal artery occlusive disease was first introduced by Grüntzig who successfully dilated a renal artery stenosis using a balloon catheter technique. This tech-nique requires passage of a guidewire under fluoroscopic control typically from a femoral artery approach to across the stenosis in the renal artery. A balloon dilating catheter is passed over the guidewire and positioned within the area of stenosis and inflated to produce a controlled disruption of the arterial wall. Alternatively, a balloon-mounted expandable stent can be used Brunicardi_Ch23_p0897-p0980.indd 93827/02/19 4:14 PM 939ARTERIAL DISEASECHAPTER 23to primarily dilate the renal artery

1	arterial wall. Alternatively, a balloon-mounted expandable stent can be used Brunicardi_Ch23_p0897-p0980.indd 93827/02/19 4:14 PM 939ARTERIAL DISEASECHAPTER 23to primarily dilate the renal artery stenosis. Completion angiog-raphy is usually performed to assess the immediate results. The technical aspect of an endovascular renal artery revasculariza-tion is discussed in the following section.Techniques of Renal Artery Angioplasty and Stenting. Access to the renal artery for endovascular intervention is typi-cally performed via a femoral artery approach, although a bra-chial artery approach can be considered in the event of severe aortoiliac occlusive disease, aortoiliac aneurysm, or severe cau-dal renal artery angulation. Once an introducer sheath is placed in the femoral artery, an aortogram is performed with a pigtail catheter placed in the suprarenal aorta. Additional oblique views are frequently necessary to more precisely visualize the orifice of the stenosed renal artery and

1	is performed with a pigtail catheter placed in the suprarenal aorta. Additional oblique views are frequently necessary to more precisely visualize the orifice of the stenosed renal artery and thoroughly assess the presence of accessory renal arteries. Noniodinated contrast agents, such as carbon dioxide and gadolinium, can be used in endovascular renal intervention in patients with renal dysfunction or history of allergic reaction.After systemic heparinization, catheterization of the renal artery can be performed using a variety of selective angled cath-eters, including the RDC, Cobra-2, Simmons I, or SOS Omni catheter. A selective renal angiogram is then performed to con-firm position, and the lesion is crossed with either 0.035-inch or a 0.018to 0.014-inch guidewires. It is important to main-tain the distal wire position without movement in the tertiary renal branches during guiding sheath placement to reduce the possibility of parenchymal perforation and spasm. A guiding sheath or

1	main-tain the distal wire position without movement in the tertiary renal branches during guiding sheath placement to reduce the possibility of parenchymal perforation and spasm. A guiding sheath or a guiding catheter is then advanced at the orifice of the renal artery and provides a secure access for balloon and stent deployment.Balloon angioplasty is performed with a balloon sized to the diameter of the normal renal artery adjacent to the stenosis. Choosing a balloon with diameter 4 mm is a reasonable first choice. The luminal diameter of the renal artery can be further assessed by comparing it to the fully inflated balloon. Such a comparison may provide a reference guide to determine whether renal artery dilatation with a larger diameter angioplasty balloon is necessary.Once balloon angioplasty of the renal artery is completed, an angiogram is performed to document the procedural result. Radiographic evidence of either residual stenosis or renal artery dissection constitutes

1	angioplasty of the renal artery is completed, an angiogram is performed to document the procedural result. Radiographic evidence of either residual stenosis or renal artery dissection constitutes suboptimal angioplasty results, which warrants an immediate renal artery stent placement. Moreover, atherosclerotic involvement of the very proximal renal artery that involves the vessel orifice typically requires stent place-ment. A balloon-expandable stent is typically used and is positioned in such a way that it protrudes into the aorta by 1 to 2 mm. The size of the stent is determined by the size of the renal artery, taking into account a desirable 10% to 20% over-sizing. After the stent deployment, the angiogram is repeated, and upon a satisfactory result, the devices are withdrawn. It is critical to maintain the guidewire access across the renal lesion until satisfactory completion angiogram is obtained. Spasm of the branches of the renal artery will usually respond to nitro-glycerin

1	critical to maintain the guidewire access across the renal lesion until satisfactory completion angiogram is obtained. Spasm of the branches of the renal artery will usually respond to nitro-glycerin 100 to 200 μg administered through the guiding sheath directly into the renal artery.While endovascular therapy of renal artery occlusive disease is considerably less invasive than conventional renal artery bypass operation, complications relating to this treat-ment modality can occur. In a study in which Guzman and colleagues compared the complications following renal artery angioplasty and surgical revascularization, the authors noted that major complication rates following endovascular and sur-gical treatment were 17% and 31%, respectively.106 In contrast, significantly greater minor complications were associated with the endovascular cohort, with a minor complication rate of 48% compared with 7% in the surgical group. In a prospective randomized study that compared the clinical

1	complications were associated with the endovascular cohort, with a minor complication rate of 48% compared with 7% in the surgical group. In a prospective randomized study that compared the clinical outcome of renal artery balloon angioplasty versus stenting for renal ostial ath-erosclerotic lesion, comparable complications rates were found in the two groups (39% vs. 43%, respectively). However, the incidence of restenosis at 6 months was significantly higher in the balloon angioplasty cohort than the stenting group (48% vs. 14%, respectively). This study underscores the clinical superior-ity of renal stenting compared to renal balloon angioplasty alone in patients with ostial stenosis.107Deterioration in renal function, albeit transient, is a com-mon complication following endovascular renal artery inter-vention. This is most likely the combined result of the use of iodinated contrast and the occurrence of renal parenchymal embolism due to wire and catheter manipulation. In most

1	renal artery inter-vention. This is most likely the combined result of the use of iodinated contrast and the occurrence of renal parenchymal embolism due to wire and catheter manipulation. In most cases, this is a temporary problem, as supportive care with adequate fluid hydration is sufficient to reverse the renal dysfunction. However, transient hemodialysis may become necessary in approximately 1% of patients. Other complications include vas-cular access complications (bleeding, hematoma, femoral nerve injury, arteriovenous fistula, and pseudoaneurysm), target ves-sel dissection, perinephric hematoma, early postoperative renal artery thrombosis, and extremity atheroembolism from throm-bus in the aorta or the iliac arteries.Clinical Results of Endovascular InterventionsPercutaneous Transluminal Balloon Angioplasty. FMD of the renal artery is the most common treatment indication for percutaneous transluminal balloon angioplasty. Patients with symptomatic FMD such as hypertension or

1	Balloon Angioplasty. FMD of the renal artery is the most common treatment indication for percutaneous transluminal balloon angioplasty. Patients with symptomatic FMD such as hypertension or renal insufficiency usually respond well to renal artery balloon angioplasty alone. In contrast, balloon angioplasty generally is not an effective treat-ment for patients with renal artery stenosis or proximal occlusive disease of the renal artery, due to the high incidence of restenosis with balloon angioplasty alone. In the latter group of patients, primary stent placement is the preferred endovascular treatment. The long-term benefit of renal artery balloon angioplasty in patients with FMD was reported by Surowiec and colleagues.108 They followed 14 patients who underwent 19 interventions on 18 renal artery segments. The technical success rate of balloon angioplasty for FMD was 95%. Primary patency rates were 81%, 69%, 69%, and 69% at 2, 4, 6, and 8 years, respectively. Assisted primary patency

1	artery segments. The technical success rate of balloon angioplasty for FMD was 95%. Primary patency rates were 81%, 69%, 69%, and 69% at 2, 4, 6, and 8 years, respectively. Assisted primary patency rates were 87%, 87%, 87%, and 87% at 2, 4, 6, and 8 years, respectively. The restenosis rate was 25% at 8 years. Clinical benefit, as defined by either improved or cured hypertension, was found in 79% of patients overall, with two-thirds of patients having maintained this benefit at 8 years. The authors concluded that balloon angioplasty is highly effective in symptomatic FMD with excellent durable functional benefits.The utility of balloon angioplasty alone in the treatment of renovascular hypertension appears to be limited. van Jaars-veld and associates performed a prospective study in which patients with renal artery stenosis were randomized to either drug therapy or balloon angioplasty treatment.109 A total of 106 patients with 50% diameter stenosis or greater plus hyperten-sion or

1	patients with renal artery stenosis were randomized to either drug therapy or balloon angioplasty treatment.109 A total of 106 patients with 50% diameter stenosis or greater plus hyperten-sion or renal insufficiency were randomized in the study. At 3 months, there was no difference in the degree to which blood Brunicardi_Ch23_p0897-p0980.indd 93927/02/19 4:14 PM 940SPECIFIC CONSIDERATIONSPART IIpressure was controlled between the two groups. However, the degree and dose of antihypertensive medications were slightly lowered in the balloon angioplasty group. The above advantage of the angioplasty group completely disappeared at 12 months, making the authors conclude that in the treatment of patients with hypertension and renal artery stenosis, percutaneous trans-luminal balloon angioplasty alone offers minimal advantage over antihypertensive drug therapy.Renal Artery Stenting. Endovascular stent placement is the treatment of choice for patients with symptomatic or high-grade renal

1	alone offers minimal advantage over antihypertensive drug therapy.Renal Artery Stenting. Endovascular stent placement is the treatment of choice for patients with symptomatic or high-grade renal artery occlusive disease (Fig. 23-48). This is due in part to the high incidence of restenosis with balloon angioplasty alone, particularly in the setting of ostial stenosis. Renal artery stenting is also indicated for renal artery dissection caused by balloon angioplasty or other catheter-based interventions. Numerous studies have clearly demonstrated the clinical efficacy of renal artery stenting when compared to balloon angioplasty alone in patients with high-grade renal artery stenosis.White and colleagues conducted a study to evaluate the role of renal artery stenting in patients with poorly controlled hyper-tension and renal artery lesions that did not respond well to bal-loon angioplasty alone.110 The technical success of the procedure was 99%. The mean blood pressure values were 173 ±

1	controlled hyper-tension and renal artery lesions that did not respond well to bal-loon angioplasty alone.110 The technical success of the procedure was 99%. The mean blood pressure values were 173 ± 25/88 ± 17 mmHg prior to stent implantation and 146 ± 20/77 ± 12 mmHg 6 months after renal artery stenting (P <0.01). Angiographic follow-up with 67 patients (mean 8.7 ± 5 months) demonstrated that restenosis, as defined by 50% or greater luminal narrowing, occurred in 15 patients (19%). The study concluded that renal artery stenting is a highly effective treatment for renovascular hypertension, with a low angiographic restenosis rate. In another similar study, Blum and colleagues prospectively performed renal artery stenting in 68 patients (74 lesions) with ostial renal artery stenosis and suboptimal balloon angioplasty.111 Patients were fol-lowed for a mean of 27 months with measurements of blood pres-sure and serum creatinine, duplex sonography, and intra-arterial angiography.

1	and suboptimal balloon angioplasty.111 Patients were fol-lowed for a mean of 27 months with measurements of blood pres-sure and serum creatinine, duplex sonography, and intra-arterial angiography. Five-year patency was 84.5% (mean follow-up, 27 months). Restenosis occurred in 8 of 74 arteries (11%), but after reintervention, the secondary 5-year patency rate was 92.4%. Hypertension was cured or improved in 78% of patients. The authors concluded that primary stent placement is an effec-tive treatment for renal artery stenosis involving the ostium.BAFigure 23-48. Renal artery stenting. A. Focal lesion in the renal artery (arrow). B. Poststenting angiogram reveals a satisfactory result following a renal artery stenting placement (arrow).The clinical utility of renal artery stenting in renal func-tion preservation was analyzed by several studies, which mea-sured serial serum creatinine levels to determine the response of renal function following endovascular intervention.112,113 In a

1	func-tion preservation was analyzed by several studies, which mea-sured serial serum creatinine levels to determine the response of renal function following endovascular intervention.112,113 In a study reported by Harden and colleagues who performed 33 renal artery stenting procedures in 32 patients with renal insuf-ficiency, they noted that renal function improved or stabilized in 22 patients (69%).113 In a similar study, Watson and associates evaluated the effect of renal artery stenting on renal function by comparing the slopes of the regression lines derived from the reciprocal of serum creatinine versus time.112 A total of 61 renal stenting procedures were performed in 33 patients, and the authors found that after stent placement, the slopes of the reciprocal of the serum creatinine (1/Scr) were positive in 18 patients and less negative in 7 patients. The study concluded that in patients with chronic renal insufficiency due to obstructive renal artery stenosis, renal artery

1	(1/Scr) were positive in 18 patients and less negative in 7 patients. The study concluded that in patients with chronic renal insufficiency due to obstructive renal artery stenosis, renal artery stenting is effective in improv-ing or stabilizing renal function.The clinical outcome of several large clinical studies of renal artery stenting in the treatment of renovascular hyperten-sion or chronic renal insufficiency is shown in Table 23-13. These studies uniformly demonstrated an excellent technical success rate with low incidence of restenosis or procedural-related complications. A similar analysis was reported by Leer-touwer and colleagues who performed a meta-analysis of 14 studies comparing patients with renal arterial stent placement to those who underwent balloon angioplasty alone for renal arterial stenosis.114 The study found that stent placement proved highly successful, with an initial technical success of 98%. The overall cure rate for hypertension was 20%, whereas

1	alone for renal arterial stenosis.114 The study found that stent placement proved highly successful, with an initial technical success of 98%. The overall cure rate for hypertension was 20%, whereas hypertension was improved in 49%. Renal function improved in 30% of patients and stabilized in 38% of patients. The restenosis rate at follow-up of 6 to 29 months was 17%. Renal stenting resulted in a higher technical success rate and a lower restenosis rate when compared to balloon angioplasty alone.AORTOILIAC OCCLUSIVE DISEASEThe distal abdominal aorta and the iliac arteries are common sites affected by atherosclerosis. The symptoms and natural history of the atherosclerotic process affecting the aortoiliac Brunicardi_Ch23_p0897-p0980.indd 94027/02/19 4:14 PM 941ARTERIAL DISEASECHAPTER 23arterial segment are influenced by the disease distribution and extent. Atherosclerotic plaques may cause clinical symptoms by restricting blood flow due to luminal obstruction or by embo-lizing

1	23arterial segment are influenced by the disease distribution and extent. Atherosclerotic plaques may cause clinical symptoms by restricting blood flow due to luminal obstruction or by embo-lizing atherosclerotic debris to the lower extremity circulation. If the aortoiliac plaques reach sufficient mass and impinge on the arterial lumen, obstruction of blood flow to lower extremi-ties occurs. Various risk factors exist that can lead to the devel-opment of aortoiliac occlusive disease. Recognition of these factors and understanding of this disease entity will enable phy-sicians to prescribe the appropriate treatment strategy, which may alleviate symptoms and improve quality of life.Diagnostic EvaluationOn clinical examination patients often have weakened femoral pulses and a reduced ABI. Verification of iliac occlusive dis-ease is usually made by color duplex scanning, which reveals either a peak systolic velocity ratio ≥2.5 at the site of stenosis and or a monophasic waveform.

1	ABI. Verification of iliac occlusive dis-ease is usually made by color duplex scanning, which reveals either a peak systolic velocity ratio ≥2.5 at the site of stenosis and or a monophasic waveform. Noninvasive tests such as pulse volume recordings (PVRs) of the lower extremity with estima-tion of the thigh-brachial pressure index may be suggestive of aortoiliac disease. MRA and multidetector CTA are increasingly being used to determine the extent and type of obstruction. DSA offers the interventionalist the benefit of making a diagnosis and the option of performing an endovascular treatment in a single session. Angiography provides important information regard-ing distal arterial runoff vessels as well as the patency of the PFA. Presence of pelvic and groin collaterals is important to provide crucial collateral flow in maintaining lower limb viabil-ity. It must be emphasized, however, that patients should be subjected to angiography only if their symptoms warrant surgi-cal

1	to provide crucial collateral flow in maintaining lower limb viabil-ity. It must be emphasized, however, that patients should be subjected to angiography only if their symptoms warrant surgi-cal intervention.Differential DiagnosisDegenerative hip or spine disease, lumbar disk herniation, spinal stenosis, diabetic neuropathy, and other neuromuscular problems can produce symptoms that may be mistaken for vas-cular claudication. Such cases can be distinguished from true claudication by the fact that the discomfort from neuromuscular problems is often relieved by sitting or lying down, as opposed to cessation of ambulation. In addition, complaints that are Table 23-13Clinical outcome of renal artery stent placement in the treatment of renovascular hypertension and renal insufficiencyAUTHORYEARPATIENT NO.TECHNICAL SUCCESS (%)FOLLOW-UP (MONTHS)RENAL INSUFFICIENCY (%)RENOVASCULAR HYPERTENSION (%)COMPLICATION (%)RESTENOSIS

1	of renovascular hypertension and renal insufficiencyAUTHORYEARPATIENT NO.TECHNICAL SUCCESS (%)FOLLOW-UP (MONTHS)RENAL INSUFFICIENCY (%)RENOVASCULAR HYPERTENSION (%)COMPLICATION (%)RESTENOSIS (%)STABLEIMPROVEDCUREDIMPROVEDIannone1301996 63 991045364351314Harden1261997 32100 63434N/AN/A 313Blum1241997 6810027N/AN/A1662 011White1231997100 99 6N/A20N/AN/A 219Shannon1321998 21100 92943N/AN/A 90Rundback1311998 45 9417N/AN/AN/AN/A 925Dorros128199816310048N/AN/A35111N/AHenry1291999210 9925N/A291961 39Bush1272001 73 8920213813611216N/A = not applicable.experienced upon standing suggest nonvascular causes. When confusion persists, the use of noninvasive vascular laboratory testing modalities, including treadmill exercise, can help estab-lish the diagnosis.Collateral Arterial NetworkThe principal collateral pathways in severe aortoiliac artery occlusive disease or chronic aortic occlusion that may provide blood flow distal to the aortoiliac lesion include: (a) the superior mesenteric artery to

1	pathways in severe aortoiliac artery occlusive disease or chronic aortic occlusion that may provide blood flow distal to the aortoiliac lesion include: (a) the superior mesenteric artery to the distal IMA via its superior hemorrhoidal branch to the middle and inferior hemorrhoidals to the internal iliac artery; (b) the lumbar arteries to the superior gluteal artery to the internal iliac system; (c) the lumbar arteries to the lateral and deep circumflex arteries to the CFA; and (d) Winslow’s pathway from the subclavian to the superior epigastric artery to the inferior epigastric artery to the external iliac arteries at the groin (Fig. 23-49). In general, treatment indications for aor-toiliac artery occlusive disease include disabling claudication, ischemic rest pain, nonhealing lower extremity tissue wound, and lower extremity microembolization that arises from aor-toiliac lesions.Disease ClassificationBased on the atherosclerotic disease pattern, aortoiliac occlusive disease can be

1	tissue wound, and lower extremity microembolization that arises from aor-toiliac lesions.Disease ClassificationBased on the atherosclerotic disease pattern, aortoiliac occlusive disease can be classified into three types (Fig. 23-50). Type I aortoiliac disease, which occurs in 5% to 10% of patients, is confined to the distal abdominal aorta and common iliac vessels (Fig. 23-51). Due to the localized nature of this type of aortic obstruction and formation of collateral blood flow around the occluded segment, limb-threatening symptoms are rare in the absence of more distal disease (Fig. 23-52). This type of aor-toiliac occlusive disease occurs in a relatively younger group of patients (in their mid-50s), compared with patients who have more femoropopliteal disease. Patients with a type I disease pat-tern have a lower incidence of hypertension and diabetes but a significant frequency of abnormal blood lipid levels, particu-larly type IV hyperlipoproteinemia. Symptoms typically consist of

1	pat-tern have a lower incidence of hypertension and diabetes but a significant frequency of abnormal blood lipid levels, particu-larly type IV hyperlipoproteinemia. Symptoms typically consist of bilateral thigh or buttock claudication and fatigue. Men report diminished penile tumescence and may have complete loss of erectile function. These symptoms in the absence of femoral Brunicardi_Ch23_p0897-p0980.indd 94127/02/19 4:14 PM 942SPECIFIC CONSIDERATIONSPART IIFigure 23-50. Aortoiliac disease can be classified into three types. Type I represents focal disease affecting the distal aorta and proxi-mal common iliac artery. Type II represents diffuse aortoiliac dis-ease above the inguinal ligament. Type III represents multisegment occlusive diseases involving aortoiliac and infrainguinal arterial vessels.Figure 23-51. Type I aortoiliac disease is confined to the distal abdominal aorta (long arrow) or proximal common iliac arteries. Due to the localized nature of this type of aortic

1	arterial vessels.Figure 23-51. Type I aortoiliac disease is confined to the distal abdominal aorta (long arrow) or proximal common iliac arteries. Due to the localized nature of this type of aortic obstruction and formation of collateral blood flow around the occluded segment (short arrows), limb-threatening symptoms are rare in the absence of more distal disease.Figure 23-49. Pertinent collateral pathways are developed in the event of chronic severe aortoiliac occlusive disease. As illustrated in this multidetector computed tomography angiography, these col-laterals include epigastric arteries (large white arrows), an enlarged inferior mesenteric artery (arrowhead), and enlarged lumbar arter-ies (black arrows).Type IType IIType IIIpulses constitute Leriche’s syndrome. Rest pain is unusual with isolated aortoiliac disease unless distal disease coexists. Occa-sionally patients report a prolonged history of thigh and buttock claudication that recently becomes more severe. It is likely

1	with isolated aortoiliac disease unless distal disease coexists. Occa-sionally patients report a prolonged history of thigh and buttock claudication that recently becomes more severe. It is likely that this group has underlying aortoiliac disease that has progressed to acute occlusion of the terminal aorta. Others may present with “trash foot,” which represents microembolization into the distal vascular bed (Fig. 23-53). Type II aortoiliac disease rep-resents a more diffuse atherosclerotic progression that involves predominately the abdominal aorta with disease extension into the common iliac artery. This disease pattern affects approxi-mately 25% patients with aortoiliac occlusive disease. Type III aortoiliac occlusive disease, which affects approximately 65% of patients with aortoiliac occlusive disease, is widespread dis-ease that is seen above and below the inguinal ligament (Fig. 23-54). Patients with “multilevel” disease are older, more com-monly male (with a male-to-female

1	occlusive disease, is widespread dis-ease that is seen above and below the inguinal ligament (Fig. 23-54). Patients with “multilevel” disease are older, more com-monly male (with a male-to-female ratio of 6:1), and much more likely to have diabetes, hypertension, and associated atheroscle-rotic disease involving cerebral, coronary, and visceral arter-ies. Progression of the occlusive process is more likely in these patients than in those with localized aortoiliac disease. For these reasons, most patients with a type III pattern tend to present with symptoms of advanced ischemia and require revasculariza-tion for limb salvage rather than for claudication. These patients have a decreased 10-year life expectancy when compared to patients with localized aortoiliac disease.The most commonly used classification system of iliac lesions has been set forth by the TransAtlantic Inter-Society Consensus (TASC) group with recommended treatment options. This lesion classification categorizes the

1	used classification system of iliac lesions has been set forth by the TransAtlantic Inter-Society Consensus (TASC) group with recommended treatment options. This lesion classification categorizes the extent of atheroscle-rosis and has suggested a therapeutic approach based on this classification (Table 23-14 and Fig. 23-55).1 According to this consensus document, endovascular therapy is the treatment of choice for type A lesions, and surgery is the treatment of choice for type D lesions. Endovascular treatment is the preferred treat-ment for type B lesions, and surgery is the preferred treatment for good-risk patients with type C lesions. In comparison to the 2000 TASC document, the commission has not only made allowances for treatment of more extensive lesions, but it also takes into account the continuing evolution of endovascular technology and the skills of individual interventionalists when stating that the patient’s comorbidities, fully informed patient preference, and the local

1	the continuing evolution of endovascular technology and the skills of individual interventionalists when stating that the patient’s comorbidities, fully informed patient preference, and the local operator’s long-term success rates Brunicardi_Ch23_p0897-p0980.indd 94227/02/19 4:14 PM 943ARTERIAL DISEASECHAPTER 23Figure 23-52. A. Multidetector computed tomography angiography of the aortoiliac artery circulation in a 63-year-old male with buttock claudication. B. Three-dimensional image reconstruction shows intra-arterial calcification of the aorta (large arrow) and right common iliac artery (small arrow). This is consistent with type I aortoiliac occlusive disease.Figure 23-53. Atherosclerotic disease involving the aortoiliac segment can result in microembolization of the lower leg circulation, resulting in trash foot or digital gangrene of toes.must be considered when making treatment decisions for type B and type C lesions.1,115General Treatment ConsiderationsThere is no effective

1	resulting in trash foot or digital gangrene of toes.must be considered when making treatment decisions for type B and type C lesions.1,115General Treatment ConsiderationsThere is no effective medical therapy for the management of aortoiliac disease, but control of risk factors may help slow pro-gression of atherosclerosis. Patients should have hypertension, hyperlipidemia, and diabetes mellitus controlled. They should be advised to stop smoking. Most patients are empirically placed on antiplatelet therapy. A graduated exercise program may improve walking efficiency, endothelial function, and metabolic adaptations in skeletal muscle, but, there is usually minimal improvement in patients with aortoiliac disease who are treated with these measures. Failure to respond to exercise and/or drug therapy should prompt consideration for limb revas-cularization. Patients with buttock claudication and reduced or absent femoral pulses who fail to respond to exercise and drug therapy should be

1	therapy should prompt consideration for limb revas-cularization. Patients with buttock claudication and reduced or absent femoral pulses who fail to respond to exercise and drug therapy should be considered for revascularization because they are less likely than patients with more distal lesions to improve without concomitant surgical or endovascular intervention.Brunicardi_Ch23_p0897-p0980.indd 94327/02/19 4:14 PM 944SPECIFIC CONSIDERATIONSPART IIFigure 23-54. Type III aortoiliac occlusive disease is a mul-tilevel disease pattern that affects the aortoiliac segment as well as infrainguinal femoropopliteal vessels. Most patients with this disease pattern tend to present with symptoms of advanced isch-emia and require revascularization for limb salvage rather than for claudication.Table 23-14TASC classification of aortoiliac occlusive lesionsType A lesions• Unilateral or bilateral stenoses of CIA• Unilateral or bilateral single short (≤3 cm) stenosis of EIAType B lesions• Short (≤3

1	23-14TASC classification of aortoiliac occlusive lesionsType A lesions• Unilateral or bilateral stenoses of CIA• Unilateral or bilateral single short (≤3 cm) stenosis of EIAType B lesions• Short (≤3 cm) stenosis of infrarenal aorta• Unilateral CIA occlusion• Single or multiple stenosis totaling 3–10 cm involving the EIA not extending into the CFA• Unilateral EIA occlusion not involving the origins of internal iliac artery or CFAType C lesions• Bilateral CIA occlusions• Bilateral EIA stenoses 3–10 cm long not extending into the CFA• Unilateral EIA stenosis extending into the CFA• Unilateral EIA occlusion that involves the origins of internal iliac artery and/or CFA• Heavily calcified unilateral EIA occlusion with or without involvement of origins of internal iliac artery and/or CFAType D lesions• Infrarenal aortoiliac occlusion• Diffuse disease involving the aorta and both iliac arteries requiring treatment• Diffuse multiple stenoses involving the unilateral CIA, EIA, and

1	CFAType D lesions• Infrarenal aortoiliac occlusion• Diffuse disease involving the aorta and both iliac arteries requiring treatment• Diffuse multiple stenoses involving the unilateral CIA, EIA, and CFA• Unilateral occlusions of both CIA and EIA• Bilateral occlusions of EIA• Iliac stenoses in patients with AAA requiring treatment and not amenable to endograft placement or other lesions requiring open aortic or iliac surgeryAAA = abdominal aortic aneurysm; CFA = common femoral artery; CIA = common iliac artery; EIA = external iliac artery.Type A lesionsType B lesionsType D lesionsType C lesionsFigure 23-55. Schematic depiction of the TransAtlantic Inter-Society Consensus classification of aortoiliac occlusive lesions.Surgical Reconstruction of Aortoiliac Occlusive DiseaseAortobifemoral Bypass. Surgical options for treatment of aortoiliac occlusive diseases consist of various configurations of aortobifemoral bypass grafting, various types of extra-anatomic bypass grafts, and aortoiliac

1	options for treatment of aortoiliac occlusive diseases consist of various configurations of aortobifemoral bypass grafting, various types of extra-anatomic bypass grafts, and aortoiliac endarterectomy. The procedure performed is determined by several factors, including ana-tomic distribution of the disease, clinical condition of the patient, and personal preference of the surgeon.In most cases, aortobifemoral bypass is performed because patients usually have disease in both iliac systems. Although one side may be more severely affected than the other, progression does occur, and bilateral bypass does not complicate the procedure or add to the physiologic stress of the operation. Aortobifemoral bypass reliably relieves symp-toms, has excellent long-term patency (approximately 70–80% at 10 years), and can be completed with a tolerable periopera-tive mortality (2–3%).Technical Considerations for Aortobifemoral Bypass Both femoral arteries are initially exposed to ensure that they are

1	10 years), and can be completed with a tolerable periopera-tive mortality (2–3%).Technical Considerations for Aortobifemoral Bypass Both femoral arteries are initially exposed to ensure that they are ade-quate for the distal anastomoses. The abdomen is then opened in the midline, the small intestine is retracted to the right, and the posterior peritoneum overlying the aorta is incised. A retro-peritoneal approach may be selected as an alternative in certain situations. This approach involves making a left flank incision and displacing the peritoneum and its contents to the right. Such an approach is contraindicated if the right renal artery is acutely occluded, since visualization from the left flank is very poor. Tunneling of a graft to the right femoral artery is also more 4difficult from a retroperitoneal approach, but can be achieved. The retroperitoneal approach has been reputed to be better toler-ated than midline laparotomy for patients with multiple previ-ous abdominal

1	from a retroperitoneal approach, but can be achieved. The retroperitoneal approach has been reputed to be better toler-ated than midline laparotomy for patients with multiple previ-ous abdominal operations and with severe pulmonary disease. Further proposed advantages of the retroperitoneal approach include less gastrointestinal disturbance, decreased third space Brunicardi_Ch23_p0897-p0980.indd 94427/02/19 4:14 PM 945ARTERIAL DISEASECHAPTER 23fluid losses, and ease with which the pararenal aorta can be accessed. There are randomized reports, however, that support and refute the superiority of this approach. A collagen-impreg-nated, knitted Dacron graft is used to perform the proximal aortic anastomosis, which can then be made in either an end-to-end or end-to-side fashion using 3-0 polypropylene suture. The proximal anastomosis should be made as close as possible to the renal arteries to decrease the incidence of restenosis from pro-gression of the atherosclerotic occlusive

1	3-0 polypropylene suture. The proximal anastomosis should be made as close as possible to the renal arteries to decrease the incidence of restenosis from pro-gression of the atherosclerotic occlusive process in the future.An end-to-end proximal aortic anastomosis is necessary in patients with an aortic aneurysm or complete aortic occlu-sion extending up to the renal arteries (Fig. 23-56). Although in theory the end-to-end configuration allows for less turbulence and less chance of competitive flow with still patent host iliac vessels, there have not been consistent results to substantiate differences in patency between end-to-end and end-to-side grafts. Relative indications for an end-to-side proximal aortic anastomosis include the presence of large aberrant renal arter-ies, an unusually large IMA with poor back-bleeding suggesting inadequate collateralization, and/or occlusive disease involv-ing bilateral external iliac arteries. Under such circumstances, end-to-end bypass from the

1	large IMA with poor back-bleeding suggesting inadequate collateralization, and/or occlusive disease involv-ing bilateral external iliac arteries. Under such circumstances, end-to-end bypass from the proximal aorta to the femoral level devascularizes the pelvic region because there is no antegrade or retrograde flow in the occluded external iliac arteries to supply the hypogastric arteries. As a result of the pelvic devasculariza-tion, there is an increased incidence of impotence, postoperative colon ischemia, buttock ischemia, and paraplegia secondary to spinal cord ischemia despite the presence of excellent femoral and distal pulses.An end-to-side proximal aortic anastomosis can be associ-ated with certain disadvantages, which include the potential for distal embolization when applying a partially occlusive aortic clamp (Fig. 23-57). Furthermore, the distal aorta often pro-ceeds to total occlusion after an end-to-side anastomosis. There Figure 23-56. In an end-to-end proximal aortic

1	partially occlusive aortic clamp (Fig. 23-57). Furthermore, the distal aorta often pro-ceeds to total occlusion after an end-to-side anastomosis. There Figure 23-56. In an end-to-end proximal aortic anastomosis, the aorta is divided in half. The proximal end of the aorta is anasto-mosed to the end of a prosthetic graft, while the distal divided aortic stump is oversewn.Figure 23-57. In an end-to-side aortic anastomosis, the end of a prosthetic graft is connected to the side of an aortic incision.may also be a higher incidence of aortoenteric fistula follow-ing construction of end-to-side proximal anastomoses because the anterior projection makes subsequent tissue coverage and reperitonealization of the graft more difficult. The limbs of the graft are tunneled through the retroperitoneum to the groin, where an end-to-side anastomosis is fashioned between the graft and the bifurcation of the CFA using 5-0 polypropylene suture. Endarterectomy or patch angioplasty of the profunda femo-ris

1	the groin, where an end-to-side anastomosis is fashioned between the graft and the bifurcation of the CFA using 5-0 polypropylene suture. Endarterectomy or patch angioplasty of the profunda femo-ris may be required concurrently. Once the anastomoses have been fashioned and the graft thoroughly flushed, the clamps are removed and the surgeon carefully controls the degree of aortic occlusion until full flow is reestablished. During this period, the patient must be carefully monitored for hypotension. Declamp-ing hypotension is a complication of sudden restoration of aortic flow, particularly following prolonged occlusion. Once flow has been reestablished, the peritoneum is carefully reapproximated over the prosthesis to prevent fistulization into the intestine.Despite the presence of multilevel disease in most patients, a properly performed aortobifemoral operation can provide arterial inflow and alleviate claudication symptoms in 70% to 80% of patients; however, 10% to 15% of patients

1	disease in most patients, a properly performed aortobifemoral operation can provide arterial inflow and alleviate claudication symptoms in 70% to 80% of patients; however, 10% to 15% of patients will require simultaneous outflow reconstruction to address distal ischemia and facilitate limb salvage. The advantage of concomitant distal revascularization is avoidance of reoperation in a scarred groin. As a rule, if the profunda femoris can accept a 4-mm probe and if a No. 3 Fogarty embolectomy catheter can be passed distally for 20 cm or more, the PFA will be sufficient for outflow, and concomitant distal revascularization is not necessary.Aortic Endarterectomy. Aortoiliac endarterectomy is rarely performed because it is associated with greater blood loss and greater sexual dysfunction and is more difficult to perform. Long-term patency is comparable with aortobifemoral grafting, and thus it remains a reasonable option in cases in which the risk of infection of a graft is excessive

1	and is more difficult to perform. Long-term patency is comparable with aortobifemoral grafting, and thus it remains a reasonable option in cases in which the risk of infection of a graft is excessive because it involves no pros-thetic tissue. Aortoiliac endarterectomy was useful when disease was localized to either the aorta or common iliac arteries; how-ever, at present, aortoiliac PTA, stents, and other catheter-based therapies have become first-line treatment in this scenario. End-arterectomy should not be performed if the aorta is aneurysmal because of continued aneurysmal degeneration of the endarter-ectomized segment. If there is total occlusion of the aorta to the level of the renal arteries, aortic transection several centime-ters below the renal arteries with thrombectomy of the aortic cuff followed by graft insertion is easier and more expeditious when compared to endarterectomy. Involvement of the external iliac artery makes aortic endarterectomy more difficult to com-plete

1	cuff followed by graft insertion is easier and more expeditious when compared to endarterectomy. Involvement of the external iliac artery makes aortic endarterectomy more difficult to com-plete because of decreased vessel diameter, increased length, and exposure issues. The ability to establish an appropriate Brunicardi_Ch23_p0897-p0980.indd 94527/02/19 4:14 PM 946SPECIFIC CONSIDERATIONSPART IIendarterectomy plane is compromised due to the muscular and inherently adherent nature of the media in this location. There is a higher incidence of early thrombosis and late failure with extended aortoiliofemoral endarterectomy when compared to bypass grafting as a result of recurrent stenosis.Axillofemoral Bypass. An axillofemoral bypass is an extra-anatomic reconstruction that derives arterial inflow from the axillary artery to the femoral artery. This is a treatment option for patients with medical comorbidities that prohibit an abdominal vascular reconstruction. It may be performed

1	inflow from the axillary artery to the femoral artery. This is a treatment option for patients with medical comorbidities that prohibit an abdominal vascular reconstruction. It may be performed under local anesthesia and is used for limb salvage. Extra-anatomic bypasses have lower patency when compared to aortobifemo-ral and, therefore, are seldom recommended for claudication. Before performing this operation, the surgeon should check pulses and blood pressure in both arms to ensure that there is no obvious disease affecting flow through the axillary system. Angiography of the axillosubclavian vasculature is not neces-sary, but can be helpful if performed at the time of aortography. The axillary artery is exposed below the clavicle, and a 6to 8-mm externally reinforced PTFE graft is tunneled subcutane-ously down the lateral chest wall and lateral abdomen to the groin. It is anastomosed ipsilaterally at the CFA bifurcation into the SFA and PFA. A femorofemoral crossover graft using a

1	subcutane-ously down the lateral chest wall and lateral abdomen to the groin. It is anastomosed ipsilaterally at the CFA bifurcation into the SFA and PFA. A femorofemoral crossover graft using a 6to 8-mm externally reinforced PTFE graft is then used to revascularize the opposite extremity if necessary. Reported patency rates over 5 years vary from 30% to 80%.116 Paradoxi-cally, although it is a less complex procedure than aortofemoral grafting, the mortality rate is higher (10%), reflecting the com-promised medical status of these patients.Iliofemoral Bypass. One option for patients with unilateral occlusion of the distal common iliac or external iliac arteries is iliofemoral grafting (Fig. 23-58). Long-term patency is compa-rable to aortounifemoral bypass, and because the procedure can be performed using a retroperitoneal approach without clamping the aorta, the perioperative mortality is less.116Femorofemoral Bypass. A femorofemoral bypass is another option for patients with

1	can be performed using a retroperitoneal approach without clamping the aorta, the perioperative mortality is less.116Femorofemoral Bypass. A femorofemoral bypass is another option for patients with unilateral stenosis or occlusion of the common or external iliac artery who have rest pain, tissue loss, BAFigure 23-58. A. Skin markings showing the incisions of an ilio-femoral bypass. B. A prosthetic bypass graft is used for an iliofemo-ral artery bypass in which the proximal anastomosis is connected to the common iliac artery (long arrow) while the distal anastomosis is connected to the common femoral artery (short arrow).or intractable claudication. The primary (assisted) patency at 5 years is reported to be 60% to 70%, and although this is inferior when compared to aortofemoral bypass, there are physi-ologic benefits, especially for patients with multiple comorbidi-ties because it is not necessary to cross-clamp the aorta.117 There are no studies supporting the superiority of

1	there are physi-ologic benefits, especially for patients with multiple comorbidi-ties because it is not necessary to cross-clamp the aorta.117 There are no studies supporting the superiority of unsupported or exter-nally supported PTFE over Dacron for choice of conduit. The fear of the recipient extremity stealing blood from the extremity ipsilateral to the donor limb is not realized unless the donor iliac artery and donor outflow arteries are diseased. Depending on the skills of the interventionalist or surgeon, many iliac lesions classified as TASC B, C, or D can now be addressed using an endovascular approach, thus obviating the need to perform a femorofemoral bypass. Additionally, femorofemoral bypass can be used as an adjuvant procedure after iliac inflow has been optimized with endovascular methods.Obturator Bypass. An obturator bypass is used to recon-struct arterial anatomy in patients with groin sepsis resulting from prior prosthetic grafting, intra-arterial drug abuse, groin

1	methods.Obturator Bypass. An obturator bypass is used to recon-struct arterial anatomy in patients with groin sepsis resulting from prior prosthetic grafting, intra-arterial drug abuse, groin neoplasm, or damage from prior groin irradiation. This bypass can originate from the common iliac artery, external iliac artery, or uninvolved limb of an aortobifemoral bypass. A conduit of Dacron, PTFE, or autologous vein is tunneled through the anteromedial portion of the obturator membrane to the distal superficial femoral artery or popliteal artery. The obturator membrane must be divided sharply so as avoid injury to adja-cent structures, and care must be taken to identify the obturator artery and nerve that pass posterolaterally. After the bypass is completed and the wounds isolated, the infected area is entered, the involved arteries are debrided to healthy tissue, and vascu-larized muscle flaps are mobilized to cover the ligated ends. There have been varied results in terms of patency and

1	area is entered, the involved arteries are debrided to healthy tissue, and vascu-larized muscle flaps are mobilized to cover the ligated ends. There have been varied results in terms of patency and limb salvage for obturator bypass. Some authors have reported 57% 5-year patency and 77% 5-year limb salvage rates, whereas others have shown a high rate of reinfection and low patency requiring reintervention.118,119Thoracofemoral Bypass. The indications for thoracofemoral bypass are (a) multiple prior surgeries with a failed infrarenal aortic reconstruction and (b) infected aortic prosthesis. This pro-cedure is more physiologically demanding than other extra-ana-tomic reconstructions because the patient must not only tolerate clamping the descending thoracic aorta but also performance of a left thoracotomy. The graft is tunneled to the left CFA from the left thorax posterior to the left kidney in the anterior axillary line using a small incision in the periphery of the diaphragm and an

1	left thoracotomy. The graft is tunneled to the left CFA from the left thorax posterior to the left kidney in the anterior axillary line using a small incision in the periphery of the diaphragm and an incision in the left inguinal ligament to gain access to the extraperitoneal space from below. The right limb is tunneled in the space of Retzius in an attempt to decrease kinking that is more likely to occur with subcutaneous, suprapubic tunneling. Thoracofemoral bypass has long-term patency comparable to aortofemoral bypass.Complications of Surgical Aortoiliac ReconstructionWith current surgical techniques and conduits, early postopera-tive hemorrhage is unusual and occurs in 1% to 2%, which is usually the result of technical oversight or coagulation abnor-mality.120 Acute limb ischemia occurring after aortoiliac surgery may be the result of acute thrombosis or distal thromboembo-lism. The surgeon can prevent thromboembolic events by (a) avoiding excessive manipulation of the aorta, (b)

1	after aortoiliac surgery may be the result of acute thrombosis or distal thromboembo-lism. The surgeon can prevent thromboembolic events by (a) avoiding excessive manipulation of the aorta, (b) ensuring Brunicardi_Ch23_p0897-p0980.indd 94627/02/19 4:14 PM 947ARTERIAL DISEASECHAPTER 23adequate systemic heparinization, (c) judicious placement of vascular clamps, and (d) thorough flushing prior to restoring blood flow. Acute thrombosis of an aortofemoral graft limb in the early perioperative period occurs in 1% to 3% of patients.120 Thrombectomy of the graft limb is performed through a trans-verse opening in the hood of the graft at the femoral anastomo-sis. With this approach, it is possible to inspect the interior of the anastomosis and pass embolectomy catheters distally to clear the superficial femoral and profunda arteries. Various complica-tions may be encountered following aortoiliac or aortobifemoral reconstruction (Table 23-15).Intestinal ischemia following aortic

1	clear the superficial femoral and profunda arteries. Various complica-tions may be encountered following aortoiliac or aortobifemoral reconstruction (Table 23-15).Intestinal ischemia following aortic reconstruction occurs in approximately 2% of cases; however, with colonoscopy mucosal ischemia, which is a milder form, is seen more fre-quently. The surgeon can identify patients who require con-comitant revascularization of the IMA, hypogastric arteries, or mesenteric arteries by examining the preoperative arteriogram for the presence of associated occlusive lesions in the celiac axis, the superior mesenteric arteries, or both. Likewise, patients with a patent and enlarged IMA or a history of prior colonic resections will benefit from IMA reimplantation.In a comprehensive review of 747 patients who had aor-toiliac operations for occlusive disease, secondary operations for late complications such as reocclusion, pseudoaneurysms, and infection were necessary in 21% over a 22-year

1	747 patients who had aor-toiliac operations for occlusive disease, secondary operations for late complications such as reocclusion, pseudoaneurysms, and infection were necessary in 21% over a 22-year period.121 The most frequent late complication is graft thrombosis. Limb occlusion occurs in 5% to 10% of patients within 5 years of the index operation and in 15% to 30% of patients ≥10 years after the index operation. Anastomotic pseudoaneurysms occur in 1% and 5% of femoral anastomoses in patients with aortofemo-ral grafts. Predisposing factors to pseudoaneurysm formation Table 23-15Perioperative complications of aortobifemoral bypass graftingMedical Complications• Perioperative myocardial infarction• Respiratory failure• Ischemia-induced renal failure• Bleeding from intravenous heparinization• StrokeProcedure-Related ComplicationsEarly• Declamping shock• Graft thrombosis• Retroperitoneal bleeding• Groin hematoma• Bowel ischemia/infarction• Peripheral embolization• Erectile

1	heparinization• StrokeProcedure-Related ComplicationsEarly• Declamping shock• Graft thrombosis• Retroperitoneal bleeding• Groin hematoma• Bowel ischemia/infarction• Peripheral embolization• Erectile dysfunction• Lymphatic leak• Chylous ascites• ParaplegiaLate• Graft infection• Anastomotic pseudoaneurysm• Aortoenteric fistula• Aortourinary fistula• Graft thrombosisinclude progression of degenerative changes within the host artery, excessive tension at the anastomosis, and infection.121 Due to the associated risks of thrombosis, distal emboliza-tion, infection, and rupture, anastomotic aneurysms should be repaired expeditiously.Infection following aortoiliac reconstruction is a devastat-ing complication that occurs in 1% of cases. Femoral anastomo-ses of aortofemoral reconstructions and axillofemoral bypasses are prone to infection. Use of prophylactic antibiotics and metic-ulous surgical technique are vital in preventing contamination of the graft at the time of implantation. If

1	axillofemoral bypasses are prone to infection. Use of prophylactic antibiotics and metic-ulous surgical technique are vital in preventing contamination of the graft at the time of implantation. If infection appears local-ized to a single groin, graft preservation and local measures such as antibiotic irrigation, aggressive debridement, and soft tissue coverage with rotational muscle flaps may prove successful. Most patients with infected aortoiliofemoral reconstructions usually require graft excision and revascularization via remote uncontaminated routes or the use of in situ replacement to clear the infective process and maintain limb viability. Aortoenteric fistula and associated gastrointestinal hemorrhage are devas-tating complications, with a 50% incidence of death or limb loss. The incidence of aortoenteric fistula formation appears to be higher after an end-to-side proximal anastomosis because it is more difficult to cover the prosthesis with viable tissue and avoid contact

1	incidence of aortoenteric fistula formation appears to be higher after an end-to-side proximal anastomosis because it is more difficult to cover the prosthesis with viable tissue and avoid contact with the gastrointestinal tract with this configu-ration.121 Treatment of aortoenteric fistula requires resection of all prosthetic material, closure of the infrarenal abdominal aorta, repair of the gastrointestinal tract, and revascularization by means of an extra-anatomic graft.Endovascular Treatment for Aortic DiseaseAlthough aortofemoral bypass surgery has excellent long-term patency and can be performed with low mortality rates, there are patients who are unable to withstand the physiologic stress of longer open procedures performed under general anesthe-sia, which require aortic cross-clamping and which are associ-ated with greater blood loss. These patients are more suited to endovascular interventions despite the decreased durability and requirement for more frequent

1	cross-clamping and which are associ-ated with greater blood loss. These patients are more suited to endovascular interventions despite the decreased durability and requirement for more frequent reinterventions.Focal Aortic Stenosis. The endovascular technique used to treat infrarenal aortic stenoses is similar to that used for iliac artery disease. Bilateral CFA access is established followed by insertion of a 6-French sheath. The lesion is crossed using a hydrophilic wire and a supporting selective catheter and then changed for a stiffer guidewire. A self-expanding nitinol stent or a balloon-expandable stent mounted on a larger-caliber angio-plasty balloon is implanted followed by adequate postdilation. At the physician’s discretion, “kissing” stents, simultaneous bilateral proximal iliac stents, are deployed if the lesion is in the distal aorta in the proximity of the aortic bifurcation. The role of covered stents such as cuffs made for endoluminal AAA repair has not been rigorously

1	stents, are deployed if the lesion is in the distal aorta in the proximity of the aortic bifurcation. The role of covered stents such as cuffs made for endoluminal AAA repair has not been rigorously studied. The aortic diameter should be sized with a calibrated catheter during the angiography or by preintervention CT scanning to avoid undersizing. Balloon size will range from 12 to 18 mm in most cases. A single stent is generally sufficient in most cases. Concentric aortic stenosis may encroach upon the IMA, and coverage of this vessel may be unavoidable. Care should be taken to use low inflation pres-sures (5 mmHg) to minimize the risk of aortic rupture. Patient complaints of back or abdominal pain during balloon infla-tion should be taken seriously as they may suggest impending rupture. In case of a calcified small-caliber, hypoplastic aorta (≤12 mm, typically in female patients), it is recommended to Brunicardi_Ch23_p0897-p0980.indd 94727/02/19 4:14 PM 948SPECIFIC

1	rupture. In case of a calcified small-caliber, hypoplastic aorta (≤12 mm, typically in female patients), it is recommended to Brunicardi_Ch23_p0897-p0980.indd 94727/02/19 4:14 PM 948SPECIFIC CONSIDERATIONSPART IIuse smaller diameter stents. To achieve clinical improvement, these patients can be recanalized to an aortic diameter of 8 or 9 mm. Distal embolization is one of the potential complications of endovascular treatment for aortic stenoses. Full hepariniza-tion, meticulous technique during wire and catheter manipula-tions, and primary stenting reduce the risk of this complication. Since calcified aortic stenoses are prone to rupture during dila-tion, it is recommended to be cognizant of the extent of the calcification with preoperative CT scans. In case of aortic rup-ture, as long as wire access has been maintained, an occlusion balloon can be inflated proximal to the disrupted segment to achieve hemostasis, and the rupture can be covered with a stent graft or repaired with

1	as wire access has been maintained, an occlusion balloon can be inflated proximal to the disrupted segment to achieve hemostasis, and the rupture can be covered with a stent graft or repaired with open surgery.Occlusive Lesions of the Aortic Bifurcation. Occlusive lesions are treated with the kissing balloon technique to avoid dislodging aortic plaque. Two angioplasty balloons of equal size are positioned across the ostia of the common iliac arteries, using a retrograde approach, and inflated. Simultaneous bal-loon dilatation at the origins of both common iliac arteries is advocated, even in the presence of unilateral lesion, to protect the contralateral common iliac artery from dissection or plaque embolization. Calcified lesions that typically occur at the aor-tic bifurcation are not amenable to balloon dilatation and fre-quently require that a distal aortic reconstruction be performed using “kissing stents.” Fears that the proximal ends of the stents that extend into the distal

1	amenable to balloon dilatation and fre-quently require that a distal aortic reconstruction be performed using “kissing stents.” Fears that the proximal ends of the stents that extend into the distal aorta will become a nidus for throm-bus formation or cause hemolysis have not been realized. The results are difficult to interpret because these bifurcation lesions are usually included in studies with iliac artery lesions. Patency rates for aortic bifurcation PTA range from 76% to 92% at 3 years. The largest series reported to date includes 79 patients with aortic bifurcation lesions. The cumulative clinical success rate at a mean of 4 years was 93%.122 Stents have also been used to reconstruct the aortic bifurcation with high success rates. The kissing stent technique is well suited for orificial lesions. Technical success with kissing stents at the aortic bifurcation has been reported to be 95% to 100%.123 In the largest series reported, the primary patency at 3 years was

1	for orificial lesions. Technical success with kissing stents at the aortic bifurcation has been reported to be 95% to 100%.123 In the largest series reported, the primary patency at 3 years was 79%.124Endovascular Treatment for Iliac Artery DiseasePercutaneous Transluminal Angioplasty. PTA is most use-ful in the treatment of isolated iliac stenoses of less than 4 cm in length. When used for stenoses rather than occlusion, a 2-year patency of 86% can be achieved.125 The complication rate is approximately 2%, consisting of distal embolization, medial dissection, and acute thrombosis.Technical Considerations for Iliac Interventions Crossing a high-grade stenosis or occlusion can be challenging in the iliac arteries. It is vital to image the lesion well because multiple views and use of the image intensifier will frequently uncover the anatomic reason for the difficulty. Frequently, the difficulty is the result of vessel tortuosity that cannot be appreciated on the original view. Use of

1	image intensifier will frequently uncover the anatomic reason for the difficulty. Frequently, the difficulty is the result of vessel tortuosity that cannot be appreciated on the original view. Use of an angled hydrophilic guidewire and an angled catheter can provide steering and add extra support for the wire trying to cross the lesion. Patience, persistence, and periodic reimaging will facilitate the crossing of a lesion in the great majority of cases. Guidewire traversal must be achieved for performance of endovascular iliac intervention. Over 90% of iliac occlusions can be passed with simple guidewire tech-niques. The preferred approach for recanalizing a common iliac artery occlusion is retrograde passage of devices from an ipsi-lateral CFA puncture because, in this manner, distance to the lesion is short and access is straighter. A stenosis is normally crossed using a combination of a soft-tip 0.035-inch guidewire (i.e., Bentson-type wire) or hydrophilic wire and a 5-French

1	to the lesion is short and access is straighter. A stenosis is normally crossed using a combination of a soft-tip 0.035-inch guidewire (i.e., Bentson-type wire) or hydrophilic wire and a 5-French straight or selective catheter. One of the hazards of retrograde recanalization is that the guidewire stays in a subintimal loca-tion and cannot be redirected into the true lumen at the aortic bifurcation. There are several approaches that can be used to achieve reentry of total chronic occlusions. Specialized cath-eters allow passage of a needle and guidewire across the intima distal to the occlusion. Intravascular ultrasound can be used for true lumen reentry under fluoroscopic guidance. Another method of achieving true lumen reentry involves performing the recanalization from an antegrade contralateral CFA approach. A 4-French Berenstein catheter (Cordis Corp., Miami Lakes, FL) is used to probe the occlusion. The lesion can be crossed in most instances (5–20% failure rate) with a

1	contralateral CFA approach. A 4-French Berenstein catheter (Cordis Corp., Miami Lakes, FL) is used to probe the occlusion. The lesion can be crossed in most instances (5–20% failure rate) with a hydrophilic guidewire or occasionally with its stiffer back end. As soon as the guidewire has crossed the obstruction and lies within the ipsilateral exter-nal iliac artery lumen, it is snared and partially pulled out of the ipsilateral CFA. A short catheter is then inserted in a retrograde fashion over the wire end into the abdominal aorta proximal to the lesion. The hydrophilic guidewire is then exchanged for a stiffer Amplatz (Boston Scientific, Natick, MA) guidewire to facilitate iliac stenting.Obtaining arterial access when there are absent femoral pulsations is aided by the use of ultrasound guidance and “road-map” imaging software, which is available on modern angio-graphic equipment. When the lesion is successfully crossed, balloons of an appropriate size and length are selected for

1	guidance and “road-map” imaging software, which is available on modern angio-graphic equipment. When the lesion is successfully crossed, balloons of an appropriate size and length are selected for the angioplasty. Most common iliac arteries will accommodate 8to 10-mm diameter balloons, whereas most external iliac arteries will accommodate 6to 8-mm diameter balloons. Inflation is performed with caution, especially if there is heavy calcifica-tion, and should be guided by patient discomfort, pressure gauge readings, and changes in balloon outline.If guidewire traversal is straightforward, consideration should be given to the presence of an acute thrombosis that may benefit from catheter-directed thrombolysis. If guidewire traversal is challenging, it is unlikely that catheter-directed thrombolysis will be beneficial. Stents should be placed after inadequate angioplasty. Stents are warranted when there is a greater than 30% residual stenosis, when there is a flow-limiting dissection, or

1	will be beneficial. Stents should be placed after inadequate angioplasty. Stents are warranted when there is a greater than 30% residual stenosis, when there is a flow-limiting dissection, or when there is a pressure gradient of ≥5 mmHg across the treated segment. Placement of stents can precipitate distal embolization in up to 10%, especially if lesions are friable and vulnerable to manipulation. Routine primary stent place-ment is not recommended because it has not been found to be superior to selective stenting in terms of outcomes or cost.Primary Stenting Versus Selective Stenting in Iliac Arteries. Primary stenting rather than selective stenting should be considered for longer iliac lesions and for all TASC C and D lesions. The primary patency rates at 1, 2, and 3 years were 96%, 90%, and 72%, respectively, for longer lesions (>5 cm) that were primarily stented versus 46%, 46%, and 28%, respectively, with selective stenting.126 Primary stenting is generally advocated for chronic

1	and 72%, respectively, for longer lesions (>5 cm) that were primarily stented versus 46%, 46%, and 28%, respectively, with selective stenting.126 Primary stenting is generally advocated for chronic iliac artery occlusions, recur-rent stenosis after previous iliac PTA, and complex stenoses with eccentric, calcified, ulcerated plaques or plaques with spontaneous dissection. All of these lesions are prone to distal Brunicardi_Ch23_p0897-p0980.indd 94827/02/19 4:14 PM 949ARTERIAL DISEASECHAPTER 23embolization during manipulation of wires and angioplasty balloons. Distal embolization with isolated PTA is not com-mon for uncomplicated lesions, but can occur in up to 24% of cases, when treating ulcerated plaques, aortoiliac bifurca-tion lesions, or iliac occlusions. It is believed that direct stent placement without predilation significantly reduces the risk of distal embolization by trapping potentially embologenic material between the arterial wall and the stent mesh. While PTA has

1	stent placement without predilation significantly reduces the risk of distal embolization by trapping potentially embologenic material between the arterial wall and the stent mesh. While PTA has demonstrated excellent results in focal stenoses of the abdominal aorta and iliacs, primary stenting in these locations is safe, improves patency rates, reduces the degree of resteno-sis when compared with PTA alone, and decreases the risk of distal embolization. Additional potential advantages of direct stenting include shorter procedural time and less radiation exposure. The Dutch Iliac Stent Trial has provided evidence that refutes the superiority of primary stenting over angio-plasty alone.151 Most interventionalists continue to perform angioplasty first and stent selectively for inadequate results. The approach to aortoiliac stenting is intuitive. Individual judgment and experience are important in the decision-making process, and there are lesions with unstable morphology such as long

1	results. The approach to aortoiliac stenting is intuitive. Individual judgment and experience are important in the decision-making process, and there are lesions with unstable morphology such as long occlusions, ulceration, and dissection that warrant pri-mary stenting.Stent Graft Placement for Aortoiliac Interventions. Stent grafts have been used to treat complex iliac lesions in an attempt to exclude these sources of embolization. A recent report sug-gested that the use of stent grafts was beneficial for TASC C and D lesions.127 Bosiers and colleagues published a series of 91 limbs with diseased iliacs that they treated with 107 stent grafts. They reported successful deployment in all patients with-out distal embolization or vessel rupture and a primary patency rate of 91.1% at 1 year.128 The authors commented about their concerns of causing embolization during placement of the stent grafts and recommended that once an occlusion was traversed with the guidewire, to gently predilate

1	The authors commented about their concerns of causing embolization during placement of the stent grafts and recommended that once an occlusion was traversed with the guidewire, to gently predilate with a 5-mm balloon, followed by smooth stent graft insertion into the newly created channel. The role of stent grafts in aortoiliac occlusive disease has not been fully elucidated yet.Complications of Endovascular Aortoiliac InterventionsIliac artery angioplasty is associated with a 2% to 4% major complication rate and 4% to 15% minor complication rate. Many of these minor complications are related to the arterial puncture site. The most frequent complications relate to access site cannulation. Hemorrhage can range from the more common access site hematoma to the rarer retroperitoneal and intraperi-toneal hemorrhage. Distal embolization occurs in 2% to 10% of iliac PTA and stenting procedures.120 Percutaneous catheter aspiration should be the initial treatment for calf vessel embo-lization,

1	hemorrhage. Distal embolization occurs in 2% to 10% of iliac PTA and stenting procedures.120 Percutaneous catheter aspiration should be the initial treatment for calf vessel embo-lization, but, for larger emboli, such as those that lodge in the profunda femoris or common femoral arteries, surgical embo-lectomy may be required because the embolic material contains atherosclerotic plaque, which is not amenable to transcatheter aspiration or catheter-directed thrombolysis. The incidence of pseudoaneurysm formation at the puncture site is 0.5%. The treatment of choice for pseudoaneurysms >2 cm in diameter is percutaneous thrombin injection under ultrasound guidance. Arterial rupture may complicate the procedure in 0.3% of cases. Tamponade of the ruptured artery with an occlusion balloon should be performed, and a covered stent should be placed. In case of failure, surgical treatment is required.Clinical Results Comparing Surgical and Endovascular Treatment of Aortoiliac DiseaseThe

1	should be performed, and a covered stent should be placed. In case of failure, surgical treatment is required.Clinical Results Comparing Surgical and Endovascular Treatment of Aortoiliac DiseaseThe mortality risk of aortobifemoral bypass in patients with iso-lated, localized aortoiliac disease is relatively low, whereas for patients with concomitant atherosclerosis in coronary, carotid, and visceral vessels, mortality and morbidity are higher. For this reason, the cumulative long-term survival rate for patients receiving aortoiliac reconstruction remains 10 to 15 years less than anticipated for a normal ageand sex-matched population. Twenty-five percent to 30% of patients with concomitant ath-erosclerosis in other vascular distributions are dead within 5 years, and 50% to 60% will have died by 10 years.129Compared with conventional aortobifemoral bypass, common iliac angioplasty was shown to have a 10% to 20% lower overall patency rate. It should be noted that these results were

1	died by 10 years.129Compared with conventional aortobifemoral bypass, common iliac angioplasty was shown to have a 10% to 20% lower overall patency rate. It should be noted that these results were reported in early trials that used older generations of endovascular equipment. With continued progress and newer angioplasty balloons and stenting practices, more comparable outcomes are being reported. Review of the literature confirms that there is an 85% to 90% graft patency rate at 5 years and a 70% to 75% graft patency rate at 10 years after aortobifemo-ral reconstruction.127 Due in part to factors including continued refinements in anesthetic management, intraoperative monitor-ing, and postoperative intensive care, low perioperative mortal-ity rates for aortobifemoral bypass can be achieved commonly in today’s clinical practice. The most recent systematic review and meta-analysis of 5358 patients who underwent direct open bypass or endovascular treatment for aortoiliac occlusive

1	commonly in today’s clinical practice. The most recent systematic review and meta-analysis of 5358 patients who underwent direct open bypass or endovascular treatment for aortoiliac occlusive dis-ease demonstrated superior durability for open bypass, although with longer length of stay and increased risk for complications and mortality, when compared to the endovascular approach.130 In this study, poor preoperative runoff was greater in the open bypass group (50.0% vs. 24.6%). Mean length of hospital stay was 13 days for open bypass versus 4 days for endovascular treatment procedures. The open bypass group experienced more complications (18.0% vs. 13.4%) and greater 30-day mortality (2.6% vs. 0.7%). At 1, 3, and 5 years, pooled primary patency rates were greater in the open bypass group (94.8% vs. 86.0%, 86.0% vs. 80.0%, and 82.7% vs. 71.4%, respectively); the same was true for secondary patency (95.7% vs. 90.0%, 91.5% vs. 86.5%, and 91.0% vs. 82.5%, respectively).Despite its lower

1	(94.8% vs. 86.0%, 86.0% vs. 80.0%, and 82.7% vs. 71.4%, respectively); the same was true for secondary patency (95.7% vs. 90.0%, 91.5% vs. 86.5%, and 91.0% vs. 82.5%, respectively).Despite its lower long-term success, common iliac angio-plasty is a useful procedure in patients with focal disease and mild symptoms in whom a major surgical revascularization is not justified. Angioplasty of the iliac vessels can be a useful adjunct to distal surgical bypass as well, increasing the success of dis-tal revascularization and eliminating the risks associated with aortoiliac bypass. Thus, with long-term patency less than, but comparable to, open surgical bypass, and with more favorable morbidity rates, iliac angioplasty has become a well-accepted modality of treatment for iliac occlusive disease. Ideal iliac angioplasty lesions are nonocclusive and short. Patency after intervention is better when lesions occur in larger diameter ves-sels, when stenoses rather than occlusions are treated, when

1	Ideal iliac angioplasty lesions are nonocclusive and short. Patency after intervention is better when lesions occur in larger diameter ves-sels, when stenoses rather than occlusions are treated, when run-off vessels are patent, and when the indication for intervention is lifestyle-limiting claudication rather than critical limb ischemia.Becker and colleagues estimated a 5-year patency rate of 72% in an analysis of 2697 cases of iliac angioplasty and noted a better patency (79%) in claudicants.131 Less favorable results are obtained with long stenoses, external iliac stenoses, and tan-dem lesions. The reported technical and initial clinical success of balloon angioplasty in iliac artery stenoses exceeds 90% in 5Brunicardi_Ch23_p0897-p0980.indd 94927/02/19 4:14 PM 950SPECIFIC CONSIDERATIONSPART IImost series, and the 5-year patency rates range from 54% to 92%. The reported technical and initial clinical success of bal-loon angioplasty in iliac artery occlusions ranges from 78% to

1	series, and the 5-year patency rates range from 54% to 92%. The reported technical and initial clinical success of bal-loon angioplasty in iliac artery occlusions ranges from 78% to 98%, and the 3-year patency rates range from 48% to 85%.131,132Factors reported to affect the patency of aortoiliac endovas-cular interventions adversely include quality of runoff vessels, severity of ischemia, and length of diseased segments treated. Likewise, as vessel diameter and flow rates change, so do suc-cess rates after angioplasty. It was reported in the literature that location of the lesion at the external iliac artery adversely affects both primary and assisted-primary patency. Following angio-plasty of the common iliac artery, patency rates were 81% and 52% at 1 and 6 years, respectively; whereas, after external iliac artery angioplasty, they were 74% and 48% at 1 and 4 years, respectively.133 Although some literature supports location of the lesion in the external iliac artery as a factor

1	after external iliac artery angioplasty, they were 74% and 48% at 1 and 4 years, respectively.133 Although some literature supports location of the lesion in the external iliac artery as a factor that adversely affects both primary and assisted-primary patency, this has not been a universal finding. Female patients are also reported to have lower patency rates than males following iliac PTA, with or without stent placement in the external iliac artery.134Stenting of the iliac arteries provides a durable and curative treatment, with a 3-year patency rate of 41% to 92% for stenosis and a 3-year patency rate of 64% to 85% and 4-year patency rate of 54% to 78% for occlusions.132 A meta-analysis of 2116 patients by Bosch and Hunink showed that aortoiliac stenting resulted in a 39% improvement in long-term patency compared to balloon angioplasty, despite the fact that complication rates and 30-day mortality rates did not differ significantly.135 Park and colleagues presented long-term

1	in long-term patency compared to balloon angioplasty, despite the fact that complication rates and 30-day mortality rates did not differ significantly.135 Park and colleagues presented long-term follow-up results in a cohort of patients with all four TASC types of iliac lesions. The authors presented primary patency rates of 87%, 83%, 61%, and 49% at 3, 5, 7, and 10 years, respectively, after the index intervention.136 Leville and colleagues achieved primary and secondary patency rates of 76% and 90%, respectively, after 3 years, in a cohort of patients who received stents for iliac occlusions.137 The authors postulated that endovascular treatment for iliac occlusive dis-ease should be extended to type C and D lesions, because they observed no detectable differences between the four TASC clas-sifications in terms of primary and secondary patency rates.137 They concluded that presence of TASC C and D lesions should not preclude endovascular treatment and believe that endovas-cular

1	clas-sifications in terms of primary and secondary patency rates.137 They concluded that presence of TASC C and D lesions should not preclude endovascular treatment and believe that endovas-cular attempts should be exhausted before open surgical repair of iliac occlusions is attempted because of the decreased periop-erative morbidity and good midterm durability.Not all results have been in favor of stenting, and at present, universal primary stenting cannot be recommended. Although stents are often used to improve the outcome of PTA, there is no general consensus that stenting should be mandatory in all iliac lesions. Complex, ulcerated iliac lesions with high embologenic potential or recanalized chronic iliac occlusions may be an exception. In the Dutch Iliac Stent Trial, primary stenting did not prove to be superior to iliac angioplasty and selective stenting.138 The researchers in this prospective ran-domized multicenter study concluded that balloon angioplasty with selective

1	did not prove to be superior to iliac angioplasty and selective stenting.138 The researchers in this prospective ran-domized multicenter study concluded that balloon angioplasty with selective stenting had comparable 2-year patency rates with primary stenting (77% and 78%, respectively). It must be noted, however, that it was necessary to stent 43% of the patients in the PTA treatment group due to unsatisfactory angioplasty results. The 5-year outcomes between the two groups were also similar, with 82% and 80% of the treated iliac segments remaining free of the need for new revascularization procedures after a mean follow-up of 5.6 ± 1.3 years.138LOWER EXTREMITY ARTERIAL OCCLUSIVE DISEASEThe symptoms of lower extremity occlusive disease are classi-fied into two large categories: acute limb ischemia (ALI) and chronic limb ischemia (CLI). Ninety percent of acute ischemia cases are either thrombotic or embolic. Frequently, sudden onset of limb-threatening ischemia may be the result of

1	ischemia (ALI) and chronic limb ischemia (CLI). Ninety percent of acute ischemia cases are either thrombotic or embolic. Frequently, sudden onset of limb-threatening ischemia may be the result of acute exacerbation of the preexisting atherosclerotic disease. Chronic ischemia is largely due to atherosclerotic changes of the lower extremity that manifest from asymptomatic to limb-threatening gangrene. As the population ages, the prevalence of chronic occlusive disease of the lower extremity is increasing, and it significantly influences lifestyle, morbidity, and mortality. In addition, multiple comorbid conditions increase risks of surgical procedures. Endovascular interventions become an important alternative in treating lower extremity occlusive disease. How-ever, despite rapidly evolving endovascular technology, lower extremity endovascular intervention continues to be one of the most controversial areas of endovascular therapy.EpidemiologyIn a detailed review of the literature,

1	endovascular technology, lower extremity endovascular intervention continues to be one of the most controversial areas of endovascular therapy.EpidemiologyIn a detailed review of the literature, McDaniel and Cronenwett concluded that claudication occurred in 1.8% of patients under 60 years of age, 3.7% of patients between 60 and 70 years of age, and 5.2% of patients over 70 years of age.139 Leng and his colleagues scanned 784 subjects using ultrasound in a random sample of men and women age 56 to 77 years. Of the subjects who were scanned, 64% demonstrated atherosclerotic plaque.140 However, a large number of patients had occlusive disease with-out significant symptoms. In a study by Schroll and Munck, only 19% of patients with peripheral vascular disease were symptomatic.141 Using ABIs, Stoffers and colleagues scanned 3171 individuals between the ages of 45 and 75 and identified that 6.9% of patients had ABIs <0.95, only 22% of whom had symptoms.142 In addition, they demonstrated

1	Stoffers and colleagues scanned 3171 individuals between the ages of 45 and 75 and identified that 6.9% of patients had ABIs <0.95, only 22% of whom had symptoms.142 In addition, they demonstrated that concomitant cardiovascular and cerebrovascular diseases were three to four times higher among the group with asymptomatic peripheral vascular diseases than those without peripheral vascular disease. Furthermore, they confirmed that 68% of all peripheral arterial obstructive diseases were unknown to the primary care physi-cian, and this group mainly represented less advanced cases of atherosclerosis. However, among patients with an ABI ratio <0.75, 42% were unknown to the primary physicians.Diagnostic EvaluationThe diagnosis of lower extremity occlusive disease is often made based on a focused history and physical examination and confirmed by the imaging studies. A well-performed physi-cal examination often reveals the site of lesions by detecting changes in pulses, temperature, and

1	history and physical examination and confirmed by the imaging studies. A well-performed physi-cal examination often reveals the site of lesions by detecting changes in pulses, temperature, and appearances. The bedside ABIs using blood pressure cuff also aid in diagnosis. Various clinical signs and symptoms are useful to differentiate condi-tions of viable, threatened, and irreversible limb ischemia caused by arterial insufficiency (Table 23-16).Noninvasive studies are important in documenting the severity of occlusive disease objectively. Ultrasound Dopplers measuring ABIs and segmental pressures are widely used in North America and Europe. Normal ABI is greater than 1.0. In patients with claudication, ABIs decrease to 0.5 to 0.9 and to even lower levels in patients with rest pain or tissue loss. Seg-mental pressures are helpful in identifying the level of involve-ment. Decrease in segmental pressure between two segments indicates significant disease. Ultrasound duplex scans are used

1	loss. Seg-mental pressures are helpful in identifying the level of involve-ment. Decrease in segmental pressure between two segments indicates significant disease. Ultrasound duplex scans are used Brunicardi_Ch23_p0897-p0980.indd 95027/02/19 4:14 PM 951ARTERIAL DISEASECHAPTER 23Table 23-16Signs and symptoms of acute limb ischemiaDESCRIPTIONCATEGORYVIABLETHREATENEDIRREVERSIBLEClinical descriptionNot immediately threatenedSalvageable if promptly treatedMajor tissue loss, amputation unavoidableCapillary returnIntactIntact, slowAbsent (marbling)Muscle weaknessNoneMild, partialProfound, paralysis (rigor)Sensory lossNoneMild, incompleteProfound anestheticArteriovenous Doppler findingAudibleInaudible or audibleInaudibleto identify the site of lesion by revealing flow disturbance and velocity changes. A meta-analysis of 71 studies by Koelemay and associates confirmed that duplex scanning is accurate for assessing arterial occlusive disease in patients suffering from claudication or

1	changes. A meta-analysis of 71 studies by Koelemay and associates confirmed that duplex scanning is accurate for assessing arterial occlusive disease in patients suffering from claudication or critical ischemia with an accumulative sensitiv-ity of 80% and specificity of over 95%.143 Adding an ultrasound contrast agent further increases the sensitivity and specificity of ultrasound technology. Other noninvasive imaging technolo-gies, such as MRA and CTA, are rapidly evolving and gaining popularity in the diagnosis of lower extremity occlusive disease (Figs. 23-59 and 23-60).Contrast angiography remains the gold standard imaging study. Using contrast angiography, interventionists can locate and size the anatomic significant lesions and measure the pres-sure gradient across the lesion, as well as plan for potential inter-vention. Angiography is, however, semi-invasive and should be confined to patients for whom surgical or percutaneous inter-vention is contemplated. Patients with

1	as well as plan for potential inter-vention. Angiography is, however, semi-invasive and should be confined to patients for whom surgical or percutaneous inter-vention is contemplated. Patients with borderline renal function may need to have alternate contrast agents, such as gadolinium or carbon dioxide, to avoid contrast-induced nephrotoxicity.Differential DiagnosisArterial insufficiency frequently leads to muscle ischemic pain involving the lower extremity muscles, particularly during exer-cise. Intermittent claudication is pain affecting the calf and, less commonly, the thigh and buttock that is induced by exercise and relieved by rest. Symptom severity varies from mild to severe. Intermittent claudication occurs as a result of muscle ischemia during exercise caused by obstruction to arterial flow. Regarding the differential diagnosis of intermittent claudication, there are a variety of neurologic, musculoskeletal, and venous conditions that may produce symptoms of calf pain (Table

1	flow. Regarding the differential diagnosis of intermittent claudication, there are a variety of neurologic, musculoskeletal, and venous conditions that may produce symptoms of calf pain (Table 23-17). Addi-tionally, various nonatherosclerotic conditions can also cause symptoms consistent with intermittent lower extremity claudi-cation (Table 23-18). Nocturnal calf muscle spasms or night cramps are not indicative of arterial disease. They are common but are difficult to diagnose with certainty. Foot ulceration is not always the result of arterial insufficiency. Ischemic ulcers occur on the toes or lateral side of the foot and are painful. By com-parison, venous ulcers, which are also common, occur above the medial malleolus, usually in an area with the skin changes of lipodermatosclerosis, and cause mild discomfort. Neuropathic ulcers are usually found on weight-bearing surfaces, have thick calluses, and are pain free. Ulcers may be the result of more than one etiology. Rest pain must

1	and cause mild discomfort. Neuropathic ulcers are usually found on weight-bearing surfaces, have thick calluses, and are pain free. Ulcers may be the result of more than one etiology. Rest pain must be distinguished from periph-eral neuropathy, which is prevalent in diabetic patients. Patients with diabetic neuropathy tend to have decreased vibration and Figure 23-59. High-resolution computed tomography angiogra-phy of a patient with normal right lower extremity arterial circu-lation. Distal occlusive disease is noted in the left tibial arteries (arrow).position sense and decreased reflexes. Spinal stenosis causes pain that is exacerbated with standing and back extension.Lower Extremity Occlusive Disease ClassificationLower extremity occlusive disease may range from exhibiting no symptoms to limb-threatening gangrene. There are two major classifications developed based on the clinical presentations.Brunicardi_Ch23_p0897-p0980.indd 95127/02/19 4:15 PM 952SPECIFIC

1	no symptoms to limb-threatening gangrene. There are two major classifications developed based on the clinical presentations.Brunicardi_Ch23_p0897-p0980.indd 95127/02/19 4:15 PM 952SPECIFIC CONSIDERATIONSPART IIFigure 23-60. Multidetector computed tomography angiography of a patient with an (A) infrapopliteal arterial circulation and (B) pedal arterial circulation. The high spatial resolution and image quality of these images shows three patent infrapopliteal runoff vessels and patent pedal vessels at the foot level.BAThe Fontaine classification uses four stages: Fontaine I is the stage when patients are asymptomatic; Fontaine II is when they have mild (IIa) or severe (IIb) claudication; Fon-taine III is when they have ischemic rest pain; and Fontaine IV is when patients suffer tissue loss, such as ulceration or gangrene (Table 23-19).144The Rutherford classification has four grades (0–III) and seven categories (0–6). Asymptomatic patients are classi-fied into category 0;

1	tissue loss, such as ulceration or gangrene (Table 23-19).144The Rutherford classification has four grades (0–III) and seven categories (0–6). Asymptomatic patients are classi-fied into category 0; claudicants are stratified into grade I and divided into three categories based on the severity of the symp-toms; patients with rest pain belong to grade II and category 4; and patients with tissue loss are classified into grade III and categories 5 and 6 based on the significance of the tissue loss.2 These clinical classifications help to establish uniform standards in evaluating and reporting the results of diagnostic measure-ments and therapeutic interventions (Table 23-19).The most clinically useful classification of lower extrem-ity atherosclerotic disease should be based on the morphologic character of the lesions. The TASC taskforce published a guide-line separating lower extremity arterial diseases into femoropop-liteal and infrapopliteal lesions (Table 23-20). This guideline is

1	character of the lesions. The TASC taskforce published a guide-line separating lower extremity arterial diseases into femoropop-liteal and infrapopliteal lesions (Table 23-20). This guideline is particularly useful in determining intervention strategies based on the disease classifications. Based on the guideline, femoro-popliteal lesions are divided into four types: A, B, C, and D. Type A lesions are single focal lesions less than 3 cm in length and do not involve the origins of the SFA or the distal popliteal artery. Type B lesions are single lesions 3 to 5 cm in length not involving the distal popliteal artery or multiple or heavily calci-fied lesions less than 3 cm in length. Type C lesions are multiple stenoses or occlusions greater than 15 cm in length or recurrent stenoses or occlusions that need treatment after two endovascu-lar interventions. Type D lesions are those with complete occlu-sion of CFA, SFA, or popliteal artery.115In a similar fashion, infrapopliteal arterial

1	that need treatment after two endovascu-lar interventions. Type D lesions are those with complete occlu-sion of CFA, SFA, or popliteal artery.115In a similar fashion, infrapopliteal arterial diseases are classified into four types based on TASC guideline (Fig. 23-61). Type A lesions are single lesions less than 1 cm in length not involving the trifurcation. Type B lesions are multiple lesions less than 1 cm in length or single lesions shorter than 1 cm involving the trifurcation. Type C lesions are lesions that exten-sively involve trifurcation or 1to 4-cm stenotic or 1to 2-cm occlusive lesions. Type D lesions are occlusions longer than 2 cm or diffuse lesions.115Etiology of Acute Limb IschemiaALI is defined as sudden loss of limb perfusion, and the term is applicable up to 2 weeks after an initiating event. While the instances of acute leg ischemia caused by emboli have decreased due to more effective treatment of rheumatic fever and atrial fibrillation, the incidence of thrombotic

1	an initiating event. While the instances of acute leg ischemia caused by emboli have decreased due to more effective treatment of rheumatic fever and atrial fibrillation, the incidence of thrombotic acute leg ischemia has increased. Even with the extensive use of newer endovascular techniques including thrombolysis, most published series report a 10% to 30% 30-day amputation rate.115 The short-term mortal-ity of patients presenting with acute ischemia is 15% to 20%. The most common etiologies of ALI include embolism, native vessel thrombosis, reconstruction thrombosis, trauma, and com-plications of peripheral aneurysm. Most cases of lower extrem-ity ALI are the result of thrombosis of a prosthetic conduit. This stems from increased use of prosthetic conduits to address CLI.Presenting symptoms in ALI are pain and loss of sensory or motor function. The abruptness and time of onset of the pain, its location and intensity, and change in severity over time should all be taken into

1	symptoms in ALI are pain and loss of sensory or motor function. The abruptness and time of onset of the pain, its location and intensity, and change in severity over time should all be taken into consideration. The duration and inten-sity of the pain and presence of motor or sensory changes are very important in clinical decision making and urgency of revas-cularization. Thrombolysis may be less effective for thrombosis of ≥2 weeks in duration compared with acute thrombosis.145Arterial Embolism. The heart is the most common source of distal emboli, which accounts for more than 90% of peripheral arterial embolic events. Atrial fibrillation is the most common source. Sudden cardioversion results in the dilated noncon-tractile atrial appendage regaining contractile activity, which can dislodge the contained thrombus. Other cardiac sources include mural thrombus overlying a myocardial infarction or thrombus forming within a dilated left ventricular aneurysm. Mural thrombi can also develop

1	the contained thrombus. Other cardiac sources include mural thrombus overlying a myocardial infarction or thrombus forming within a dilated left ventricular aneurysm. Mural thrombi can also develop within a ventricle dilated by Brunicardi_Ch23_p0897-p0980.indd 95227/02/19 4:15 PM 953ARTERIAL DISEASECHAPTER 23Table 23-17Differential diagnosis of intermittent claudicationCONDITIONLOCATION OF PAIN OR DISCOMFORTCHARACTERISTIC DISCOMFORTONSET RELATIVE TO EXERCISEEFFECT OF RESTEFFECT OF BODY POSITIONOTHER CHARACTERISTICSIntermittent claudication (calf)Calf musclesCramping painAfter same degree of exerciseQuickly relievedNoneReproducibleChronic compartment syndromeCalf musclesTight, bursting painAfter much exercise (e.g., jogging)Subsides very slowlyRelief speeded by elevationTypically heavy-muscled athletesVenous claudicationEntire leg, but usually worse in thigh and groinTight, bursting painAfter walkingSubsides slowlyRelief speeded by elevationHistory of iliofemoral deep venous

1	heavy-muscled athletesVenous claudicationEntire leg, but usually worse in thigh and groinTight, bursting painAfter walkingSubsides slowlyRelief speeded by elevationHistory of iliofemoral deep venous thrombosis, signs of venous congestion, edemaNerve root compression (e.g., herniated disk)Radiates down leg, usually posteriorlySharp lancinating painSoon, if not immediately after onsetNot quickly relieved (also often present at rest)Relief may be aided by adjusting back positionHistory of back problemsSymptomatic Baker’s cystBehind knee, down calfSwelling, soreness, tendernessWith exercisePresent at restNoneNot intermittentIntermittent claudication (hip, thigh, buttock)Hip, thigh, buttocksAching discomfort, weaknessAfter same degree of exerciseQuickly relievedNoneReproducibleHip arthritisHip, thigh, buttocksAching discomfortAfter variable degree of exerciseNot quickly relieved (and may be present at rest)More comfortable sitting, weight taken off legsVariable, may relate to activity

1	thigh, buttocksAching discomfortAfter variable degree of exerciseNot quickly relieved (and may be present at rest)More comfortable sitting, weight taken off legsVariable, may relate to activity level, weather changesSpinal cord compressionHip, thigh, buttocks (follows dermatome)Weakness more than painAfter walking or standing for same length of timeRelieved by stopping only if position changedRelief by lumbar spine flexion (sitting or stooping forward) pressureFrequent history of back problems, provoked by increased intra-abdominal pressureIntermittent claudication (foot)Foot, archSevere deep pain and numbnessAfter same degree of exerciseQuickly relievedNoneReproducibleArthritic, inflammatory processFoot, archAching painAfter variable degree of exerciseNot quickly relieved (and may be present at rest)May be relieved by not bearing weightVariable, may relate to activity levelBrunicardi_Ch23_p0897-p0980.indd 95327/02/19 4:15 PM 954SPECIFIC CONSIDERATIONSPART IITable

1	(and may be present at rest)May be relieved by not bearing weightVariable, may relate to activity levelBrunicardi_Ch23_p0897-p0980.indd 95327/02/19 4:15 PM 954SPECIFIC CONSIDERATIONSPART IITable 23-18Nonatherosclerotic causes of intermittent claudication• Aortic coarctation• Arterial fibrodysplasia• Iliac syndrome of the cyclist• Peripheral emboli• Persistent sciatic artery• Popliteal aneurysm• Popliteal cyst• Popliteal entrapment• Primary vascular tumors• Pseudoxanthoma elasticum• Remote trauma or radiation injury• Takayasu’s disease• Thromboangiitis obliteransTable 23-19Classification of peripheral arterial disease based on the Fontaine and Rutherford classificationsFONTAINE CLASSIFICATIONRUTHERFORD CLASSIFICATIONSTAGECLINICALGRADECATEGORYCLINICALIAsymptomatic00AsymptomaticIIaMild claudicationI1Mild claudicationIIbModerate to severe claudicationI2Moderate claudication I3Severe claudicationIIIIschemic rest painII4Ischemic rest painIVUlceration or gangreneIII5Minor tissue

1	claudicationI1Mild claudicationIIbModerate to severe claudicationI2Moderate claudication I3Severe claudicationIIIIschemic rest painII4Ischemic rest painIVUlceration or gangreneIII5Minor tissue loss III6Major tissue losscardiomyopathy. Emboli that arise from a ventricular aneurysm or from a dilated cardiomyopathy can be very large and can lodge at the aortic bifurcation (saddle embolus), thus rendering both legs ischemic. Diseased valves are another source of distal embolization. Historically, this occurred as a result of rheumatic heart disease. Currently, subacute endocarditis and acute bacte-rial endocarditis are the more common causes. Infected emboli can seed the recipient vessel wall, creating mycotic aneurysms.An electrocardiogram (ECG) will diagnose atrial fibrilla-tion. A transthoracic or transesophageal echocardiogram should be performed looking for a cardiac source. It is important to seek other sources of the embolus using CT scanning of the descending thoracic and

1	transthoracic or transesophageal echocardiogram should be performed looking for a cardiac source. It is important to seek other sources of the embolus using CT scanning of the descending thoracic and abdominal aorta. More unusual sources include mural thrombus from an aortic aneurysm, and occa-sionally, idiopathic arterial-to-arterial thrombus occurs, usually from thrombus that has formed in an atherosclerotic aortic arch or descending thoracic aorta. The presence of mobile plaque on transesophageal echocardiography is suggestive of this source.Paradoxical embolus occurs when a patient has a patent foramen ovale and an embolus from a deep venous thrombosis crosses through the atrial defect into the left side of the heart and passes into the peripheral circulation. This is diagnosed using a bubble echocardiography, in which air bubbles introduced into the venous circulation can be seen traversing the septal defect.Arterial Thrombosis. Thrombosis can occur in native arteries and in

1	a bubble echocardiography, in which air bubbles introduced into the venous circulation can be seen traversing the septal defect.Arterial Thrombosis. Thrombosis can occur in native arteries and in arterial reconstructions. Patients with thrombosed arte-rial segments often have an underlying atherosclerotic lesion at the site of thrombosis or aneurysmal degeneration with mural thrombosis. It is important to obtain a history, determine risk factors for atherosclerosis and hypercoagulable status, and examine the contralateral extremity for circulatory problems. Patients with thrombosis of prior arterial reconstructions have limb incisions from previous surgery, and graft occlusion can be confirmed with duplex imaging.Clinical Manifestations of Acute Limb IschemiaAcute lower extremity ischemia manifests with the “five Ps”: pain, pallor, paresthesias, paralysis, and pulselessness, to which some add a sixth “P”—poikilothermia or “perishing cold.” Pain is the usual symptom that causes a

1	manifests with the “five Ps”: pain, pallor, paresthesias, paralysis, and pulselessness, to which some add a sixth “P”—poikilothermia or “perishing cold.” Pain is the usual symptom that causes a patient to present to the emergency room. The most common location for an embolus to lodge in the leg is at the common femoral bifurcation. Typically, a patient will complain of foot and calf pain. Pulses are absent, and there may be diminution of sensation. Inability to move the affected muscle group is a sign of very severe ischemia and necessitates urgent revascularization. During evaluation of the affected extremity, it is important to compare findings with the contralateral limb. Clinical evaluation is extremely important in determining the etiology and location of the obstruction. One of the most important pieces of information to obtain is whether the patient has had prior vascular procedures or if there is a history of lower extremity claudication. Either of these features suggests

1	the most important pieces of information to obtain is whether the patient has had prior vascular procedures or if there is a history of lower extremity claudication. Either of these features suggests preexisting vascular disease, renders revasculariza-tion more complicated, and usually mandates angiography to permit surgical planning. On the contrary, in a patient with no history suggestive of prior vascular disease, the etiology is most likely embolic, and simple thrombectomy is more likely to be successful.Absent bilateral femoral pulses in a patient with bilateral lower extremity ischemia is most likely due to saddle embolus to the aortic bifurcation. A palpable femoral pulse and absent popliteal and distal pulses may either be due to distal common femoral embolus (the pulse being palpable above the level of occlusion) or embolus to the superficial femoral or popliteal arteries. Typically, emboli lodge at arterial bifurcations where they are trapped due to sudden reductions in

1	palpable above the level of occlusion) or embolus to the superficial femoral or popliteal arteries. Typically, emboli lodge at arterial bifurcations where they are trapped due to sudden reductions in arterial diameter. A popliteal trifurcation embolus will present with calf ischemia and absent pedal pulses, possibly with a popliteal pulse present. The finding of palpable contralateral pulses and the absence of ipsilateral pulses in the acutely ischemic leg are suggestive of an embolus, irrespective of presence of Doppler signals. Brunicardi_Ch23_p0897-p0980.indd 95427/02/19 4:15 PM 955ARTERIAL DISEASECHAPTER 23Table 23-20TransAtlantic Inter-Society Consensus classification of femoral popliteal occlusive lesionsType A lesions• Single stenosis ≤10 cm in length• Single occlusion ≤5 cm in lengthType B lesions• Multiple lesions (stenoses or occlusions), each ≤5 cm• Single stenosis or occlusion ≤15 cm not involving the infrageniculate popliteal artery• Single or multiple lesions in the

1	lengthType B lesions• Multiple lesions (stenoses or occlusions), each ≤5 cm• Single stenosis or occlusion ≤15 cm not involving the infrageniculate popliteal artery• Single or multiple lesions in the absence of continuous tibial vessels to improve inflow for a distal bypass• Heavily calcified occlusion ≤5 cm in length• Single popliteal stenosisType C lesions• Multiple stenoses or occlusions totaling >15 cm with or without heavy calcification• Recurrent stenoses or occlusions that need treatment after two endovascular interventionsType D lesions• Chronic total occlusions of CFA or SFA (>20 cm, involving the popliteal artery)• Chronic total occlusion of popliteal artery and proximal trifurcation vesselsCFA = common femoral artery; SFA = superficial femoral artery.Type A lesionsType B lesionsType C lesionsType D lesionsFigure 23-61. Schematic depiction of TransAtlantic Inter-Society Consensus classification of femoral popliteal occlusive lesions.Arteriography is not mandatory in patients

1	C lesionsType D lesionsFigure 23-61. Schematic depiction of TransAtlantic Inter-Society Consensus classification of femoral popliteal occlusive lesions.Arteriography is not mandatory in patients without antecedent history suggestive of vascular disease; nevertheless, all patients should be positioned on the operating room table in such a way that fluoroscopic access to the entire inflow and outflow tract is possible if necessary.The main question to be answered by the history and phys-ical examination is the severity of the ALI, which is the major consideration in early management decisions. Patients with ALI should be evaluated in a fashion that considers the severity and duration of ischemia at the time of presentation. Ideally, all patients with acute ischemia should be investigated with imag-ing, especially if there is an antecedent vascular reconstruction; however, the clinical condition and access to resources must guide further investigations. Unnecessary delays can result in

1	imag-ing, especially if there is an antecedent vascular reconstruction; however, the clinical condition and access to resources must guide further investigations. Unnecessary delays can result in amputation. Arteriography, if it can be performed in a timely fashion, is an excellent modality for localizing obstructions and deciding which type of intervention (endovascular, embolec-tomy, or bypass) patients will benefit more from. One of the goals of treatment for ALI is to prevent thrombus propagation; therefore, expedient anticoagulation with heparin is indicated as soon as the diagnosis is suspected.Treatment Considerations for Acute Limb IschemiaIn the absence of any significant contraindication, the patient with an ischemic lower extremity should be immediately anti-coagulated. This will prevent propagation of the clot into unaf-fected vascular beds. Intravenous fluid should be started and a Foley catheter inserted to monitor urine output. Baseline labs should be obtained and

1	will prevent propagation of the clot into unaf-fected vascular beds. Intravenous fluid should be started and a Foley catheter inserted to monitor urine output. Baseline labs should be obtained and creatinine levels noted. A hypercoagula-ble workup should be performed prior to initiation of heparin if there is sufficient suspicion. According to results from random-ized trials, there is no clear superiority for thrombolysis over surgery in terms of 30-day limb salvage or mortality. Access to each treatment option is a major issue in the decision-making process, as time is often critical. National registry data from the United States reveal that surgery is used threeto five-fold more frequently than thrombolysis. Three randomized studies have investigated the role of catheter-directed thrombolytic therapy in the treatment of ALI.145Endovascular TreatmentThe potential to reduce mortality and morbidity while achiev-ing limb salvage is the impetus that makes thrombolysis prefer-able to open

1	therapy in the treatment of ALI.145Endovascular TreatmentThe potential to reduce mortality and morbidity while achiev-ing limb salvage is the impetus that makes thrombolysis prefer-able to open surgery as first-line treatment in patients with ALI (classes I and IIa). Advantages of thrombolytic therapy over balloon embolectomy include the reduced endothelial trauma and potential for more gradual and complete clot lysis in branch vessels usually too small to access by embolectomy balloons. It is hoped that the more gradual clot dissolution with throm-bolysis may decrease the incidence of reperfusion injury that is encountered after open surgical procedures where rapid return of blood flow may precipitate compartment syndrome. Skeletal muscle tissue appears to be most vulnerable to ischemia. Patho-physiologic studies reveal that irreversible damage to muscle tissue starts after 3 hours of ischemia and is nearly complete at 6 hours. Progressive microvascular damage appears to follow

1	Patho-physiologic studies reveal that irreversible damage to muscle tissue starts after 3 hours of ischemia and is nearly complete at 6 hours. Progressive microvascular damage appears to follow rather than precede skeletal muscle tissue damage. The more severe the cellular damage, the greater are the microvascu-lar changes. When the musculature and microvasculature are severely damaged, amputation rather than attempts at revascu-larization may be the most prudent course to prevent wash-out of toxic by-product from the ischemic limb into the systemic circulation. The mortality rate associated with reperfusion syn-drome is high because of the development of concomitant adult respiratory distress syndrome, shock, disseminated intravascular coagulation, and renal failure.Patients with small-vessel occlusion are poor candidates for surgery because they lack distal target vessels to use for bypass. These patients should be offered a trial of thromboly-sis, unless they have contraindications

1	occlusion are poor candidates for surgery because they lack distal target vessels to use for bypass. These patients should be offered a trial of thromboly-sis, unless they have contraindications to thrombolysis or their Brunicardi_Ch23_p0897-p0980.indd 95527/02/19 4:15 PM 956SPECIFIC CONSIDERATIONSPART IIischemia is so severe that the time needed to achieve adequate lysis is considered too long. The major contraindications of thrombolysis are recent stroke, intracranial primary malignancy, brain metastases, or intracranial surgical intervention. Relative contraindications for performance of thrombolysis include renal insufficiency, allergy to contrast material, cardiac thrombus, diabetic retinopathy, coagulopathy, and recent arterial puncture or surgery (Table 23-21).Advances in clot removal techniques with percutaneous mechanical thrombectomy and thromboaspiration may extend the applicability of this intervention to patients with more advanced degrees of ALI (class IIb) and

1	clot removal techniques with percutaneous mechanical thrombectomy and thromboaspiration may extend the applicability of this intervention to patients with more advanced degrees of ALI (class IIb) and contraindications to thrombolysis. Several thrombectomy devices have received FDA approval for acute lower extremity arterial thrombosis. The utility of these thrombectomy devices is that they can be used as standalone therapy when there are contraindications for thrombolytic therapy. Additionally, these thrombectomy devices can be used in conjunction with thrombolytic agents, for pharmacomechanical thrombectomy, to enhance clot lysis and to limit the doses and time required for thrombolysis.145Surgical TreatmentEmbolectomy. When a decision is made to proceed with open surgical intervention, the abdomen, contralateral groin, and entire lower extremity are prepped in the field. The groin is opened through a vertical incision, exposing the CFA and its bifurcation. Frequently, the location

1	the abdomen, contralateral groin, and entire lower extremity are prepped in the field. The groin is opened through a vertical incision, exposing the CFA and its bifurcation. Frequently, the location of the embolus at the femo-ral bifurcation is readily apparent by the presence of a palpable proximal femoral pulse, which disappears distally. The artery is clamped and opened transversely over the bifurcation. Throm-bus is extracted by passing a Fogarty balloon embolectomy catheter. Good back-bleeding and antegrade bleeding suggest that the entire clot has been removed. Embolic material often forms a cast of the vessel and is sent for culture and histologic examination. Completion angiography is advisable to ascertain Table 23-21Contraindications to thrombolytic therapyAbsolute contraindicationsEstablished cerebrovascular events (including transient ischemic attack) within last 2 monthsActive bleeding diathesisRecent (<10 days) gastrointestinal bleedingNeurosurgery (intracranial or

1	cerebrovascular events (including transient ischemic attack) within last 2 monthsActive bleeding diathesisRecent (<10 days) gastrointestinal bleedingNeurosurgery (intracranial or spinal) within last 3 monthsIntracranial trauma within last 3 monthsIntracranial malignancy or metastasisRelative major contraindicationsCardiopulmonary resuscitation within last 10 daysMajor nonvascular surgery or trauma within last 10 daysUncontrolled hypertension (>180 mmHg systolic or >110 mmHg diastolic)Puncture of noncompressible vesselIntracranial tumorRecent eye surgeryMinor contraindicationsHepatic failure, particularly with coagulopathyBacterial endocarditisPregnancyDiabetic hemorrhagic retinopathythe adequacy of clot removal. The artery is then closed and the patient fully anticoagulated.When an embolus lodges in the popliteal artery, in most cases it can be extracted via a femoral incision using the tech-niques previously described. A femoral approach is preferred because the larger diameter of

1	lodges in the popliteal artery, in most cases it can be extracted via a femoral incision using the tech-niques previously described. A femoral approach is preferred because the larger diameter of the femoral artery results in decreased likelihood of arterial compromise when the arte-riotomy is closed. The disadvantage with using the femoral approach for embolectomy is the greater difficulty involved in directing the embolectomy catheter into each of the infrapopli-teal arteries. Use of fluoroscopic imaging and an over-the-wire thrombectomy catheter can overcome this problem. Alterna-tively, use of a separate incision to expose the popliteal bifurca-tion may be necessary to achieve a complete thrombectomy.A more complex situation arises when a patient has antecedent peripheral vascular disease and in situ thrombosis develops on top of preexisting atheroma because, frequently, embolectomy catheters will not pass through these occlusions. Similarly, when a bypass graft fails, it is

1	disease and in situ thrombosis develops on top of preexisting atheroma because, frequently, embolectomy catheters will not pass through these occlusions. Similarly, when a bypass graft fails, it is usually due to progres-sion of atheroma proximal or distal to the graft anastomoses or to intrinsic stenoses that develop within a vein graft. In these scenarios, expeditious angiography is useful to determine the extent of the occlusion, to search for inflow and distal outflow vessels, and to decide whether thrombolysis or surgery will be the better intervention. Although the surgeon’s preference tends to dictate the approach selected, the decision is based on the presence or absence of good target vessels and availability of a suitable bypass conduit. If there are good distal vessels and the saphenous vein is suitable, surgical bypass is recommended because it is fast, durable, and reliable. In the absence of a good distal target and saphenous vein, or in a patient at high risk for

1	the saphenous vein is suitable, surgical bypass is recommended because it is fast, durable, and reliable. In the absence of a good distal target and saphenous vein, or in a patient at high risk for surgery, lysis is recommended.Bypass Graft Thrombectomy. Bypass thrombectomy is more likely to succeed with prosthetic bypasses. Bypass graft revision or replacement is more appropriate for acute vein graft failures because they are less likely to respond to thrombolysis and require some type of revision, such as valve lysis, interposition, or extension. Thrombectomy of autogenous grafts is prone to failure unless an anatomic cause for failure such as a retained valve or unligated side branch is found and corrected. The per-formance of a fasciotomy to circumvent reperfusion injury/com-partment syndrome is an important consideration.Complications Related to Treatment for Acute Limb IschemiaAdverse events related to catheter-directed thrombolysis are primarily related to bleeding

1	syndrome is an important consideration.Complications Related to Treatment for Acute Limb IschemiaAdverse events related to catheter-directed thrombolysis are primarily related to bleeding complications. The overall risk of hemorrhagic stroke from a thrombolysis procedure has been reported to be 1% to 2.3%, with 50% of hemorrhagic compli-cations occurring during the thrombolytic procedure.146 Hema-toma at the vascular puncture site has been reported in 12% to 17% of cases. Gastrointestinal bleeding is reported in 5% to 10% of cases. Hematuria following thrombolysis is uncom-mon and should prompt a search for urinary tumors. Hemor-rhage requiring transfusion can occur in approximately 25% of patients undergoing thrombolysis. Lytic agents are absolutely contraindicated in patients with intracranial surgery, intracra-nial hemorrhage within the last 3 months, or any active bleed-ing. Most bleeding complications occur at the arterial puncture sites, but concealed retroperitoneal bleeding

1	surgery, intracra-nial hemorrhage within the last 3 months, or any active bleed-ing. Most bleeding complications occur at the arterial puncture sites, but concealed retroperitoneal bleeding is possible. The most feared complication that patients can sustain is intrace-rebral hemorrhage. Older patients may be more susceptible Brunicardi_Ch23_p0897-p0980.indd 95627/02/19 4:15 PM 957ARTERIAL DISEASECHAPTER 23to this complication, and thus many interventionalists are extremely reticent to use thrombolysis in patients older than 80 years of age.Patients who are treated for acute ischemia are susceptible to two major complications following revascularization: reper-fusion and compartment syndromes. Other procedure-related complications include arterial rethrombosis, recurrent emboli-zation, and arterial injuries secondary to the balloon catheter manipulations.Reperfusion of the ischemic limb is variable in its physi-ologic effects and directly relates to the severity and extent of the

1	and arterial injuries secondary to the balloon catheter manipulations.Reperfusion of the ischemic limb is variable in its physi-ologic effects and directly relates to the severity and extent of the ischemia. Patients with a saddle embolus of the aortic bifurcation and severely ischemic limbs may develop the full-blown “reperfusion syndrome,” whereas patients with minimal muscle ischemia who are reperfused in a timely fashion essen-tially develop no effects. Many patients with ALI have severe underlying cardiac disease and are unable to tolerate even short ischemic periods. Complications occurring after revasculariza-tion of the lower extremity and causes of recurrent thrombosis are listed in Table 23-22.Compartment syndrome occurs after prolonged ischemia is followed by reperfusion. The capillaries leak fluid into the interstitial space in the muscles, which are enclosed within a nondistensible fascial envelope. When the pressure inside the compartment exceeds the capillary perfusion

1	leak fluid into the interstitial space in the muscles, which are enclosed within a nondistensible fascial envelope. When the pressure inside the compartment exceeds the capillary perfusion pressure, nutrient flow ceases and progressive ischemia occurs, even in the pres-ence of peripheral pulses. Consequently, every patient who has sustained an ischemic event and is reperfused is monitored for compartment syndrome, which is characterized by excessive Table 23-22Complications of arterial revascularizationCompartment syndromeIschemic neuropathyMuscle necrosisRecurrent thrombosisLower leg swellingReperfusion syndrome Hypotension Hyperkalemia Myoglobinuria Renal failureTable 23-23Fascial compartments of the lower leg ANTERIOR COMPARTMENTLATERAL COMPARTMENTSUPERFICIAL POSTERIOR COMPARTMENTDEEP POSTERIOR COMPARTMENTMusclesTibialis anteriorExtensor digitorum longusPeroneus tertiusExtensor hallucis longusExtensor digitorum brevisExtensor hallucis brevisPeroneus longusPeroneus

1	COMPARTMENTDEEP POSTERIOR COMPARTMENTMusclesTibialis anteriorExtensor digitorum longusPeroneus tertiusExtensor hallucis longusExtensor digitorum brevisExtensor hallucis brevisPeroneus longusPeroneus brevisGastrocnemiusPlantarisSoleusTibialis posteriorFlexor digitorum longusFlexor hallucis longusArteryAnterior tibial arteryAnterior and posterior tibial branches of the popliteal arteryPosterior tibial arteryPeroneal arteryNerveDeep peroneal nerveSuperficial peroneal nerve Tibial nervepain in the compartment, pain on passive stretching of the com-partment, and sensory loss due to nerve compression of the nerves coursing through the compartment (Table 23-23 and Fig. 23-62). The most commonly affected compartment is the anterior compartment in the leg. Numbness in the web space between the first and second toes is diagnostic due to com-pression of the deep peroneal nerve. Compartment pressure is measured by inserting an arterial line into the compartment and recording the pressure.

1	the first and second toes is diagnostic due to com-pression of the deep peroneal nerve. Compartment pressure is measured by inserting an arterial line into the compartment and recording the pressure. Although controversial, pressures greater than 20 mmHg are an indication for fasciotomy. Com-partment pressures are relieved in the leg by medial and lateral incisions. Through the medial incision, long openings are then made in the fascia of the superficial and deep posterior com-partments. Through the lateral incision, the anterior and pero-neal compartments are opened. Both skin and fascial incisions should be of adequate length to ensure full compartment decom-pression. Laboratory evidence of rhabdomyolysis is seen in 20% of cases. The myoglobin from damaged muscle precipitates in kidney tubules and causes acute tubular necrosis. Alkalinization of urine increases the solubility of myoglobin, thus preventing Tibia AnteriorcompartmentLateralcompartmentFibulaDeep

1	in kidney tubules and causes acute tubular necrosis. Alkalinization of urine increases the solubility of myoglobin, thus preventing Tibia AnteriorcompartmentLateralcompartmentFibulaDeep posteriorcompartmentSuperficial posterior compartmentFigure 23-62. Schematic illustration of fascial compartments of the lower extremity.Brunicardi_Ch23_p0897-p0980.indd 95727/02/19 4:15 PM 958SPECIFIC CONSIDERATIONSPART IIit from crystallizing in the tubules. In addition to alkalinization, therapy consists of forced saline diuresis and removal of the source of dead muscle that is releasing the myoglobin.Clinical Manifestations of Chronic Limb IschemiaThe term CLI is reserved for patients with objectively proven arterial occlusive disease and symptoms lasting for more than 2 weeks. Symptoms include rest pain and tissue loss, such as ulceration or gangrene (Table 23-24). The diagnosis should be corroborated with noninvasive diagnostic tests, such as the ABI, toe pressures, and transcutaneous

1	rest pain and tissue loss, such as ulceration or gangrene (Table 23-24). The diagnosis should be corroborated with noninvasive diagnostic tests, such as the ABI, toe pressures, and transcutaneous oxygen measurements. Ischemic rest pain most commonly occurs below an ankle pres-sure of 50 mmHg or a toe pressure less than 30 mmHg. Ulcers are not always of an ischemic etiology (Table 23-25). In many instances, there are other etiologic factors (traumatic, venous, or neuropathic) that are contributory, but it is underlying peripheral arterial disease that may be responsible for delayed or absent healing (Fig. 23-63). Healing of ulcers requires an inflammatory response and greater perfusion than is required to support intact skin and underlying tissues. As a result, the ankle and toe pres-sure levels needed for healing are higher than the pressures seen with ischemic rest pain. For patients with ulcers or gangrene, the presence of CLI is suggested by an ankle pressure less than 70 mmHg or a

1	needed for healing are higher than the pressures seen with ischemic rest pain. For patients with ulcers or gangrene, the presence of CLI is suggested by an ankle pressure less than 70 mmHg or a toe systolic pressure less than 50 mmHg.147 It is important to understand that there is no definite consensus regarding the vascular hemodynamic parameters required to make the diagnosis of CLI.One of the most common sites for occlusive disease is in the distal SFA as it passes deep through the adductor canal. It may be that the entrapment by the adductor hiatus prevents the compensatory dilation that occurs in atherosclerotic vessels. Stenoses, which develop here, progress to occlusion of the dis-tal third of the SFA (Fig. 23-64). When distal SFA occlusion develops slowly, it may be totally asymptomatic because of development of collaterals from the proximal SFA, or the PFA can bypass the occlusion and reconstitute the popliteal artery. Table 23-24Clinical categories of chronic limb

1	asymptomatic because of development of collaterals from the proximal SFA, or the PFA can bypass the occlusion and reconstitute the popliteal artery. Table 23-24Clinical categories of chronic limb ischemiaGRADECATEGORYCLINICAL DESCRIPTIONOBJECTIVE CRITERIA00Asymptomatic—no hemodynamically significant occlusive diseaseNormal treadmill or reactive hyperemia test 1Mild claudicationAble to complete treadmill exercisea; AP after exercise >50 mmHg but at least 20 mmHg lower than resting valueI2Moderate claudicationBetween categories 1 and 3 3Severe claudicationCannot complete standard treadmill exercisea and AP after exercise <50 mmHgIIb4Ischemic rest painResting AP <40 mmHg, flat or barely pulsatile ankle or metatarsal PVR; TP <30 mmHgIIIb5Minor tissue loss—nonhealing ulcer, focal gangrene with diffuse pedal ischemiaResting AP <60 mmHg, ankle or metatarsal PVR flat or barely pulsatile; TP <40 mmHg 6Major tissue loss—extending above TM level, functional foot no longer salvageableSame as

1	with diffuse pedal ischemiaResting AP <60 mmHg, ankle or metatarsal PVR flat or barely pulsatile; TP <40 mmHg 6Major tissue loss—extending above TM level, functional foot no longer salvageableSame as category 5aFive minutes at 2 miles per hour on a 12% incline of treadmill exercise.bGrades II and III, categories 4, 5, and 6, are encompassed by the term chronic critical ischemia.AP = ankle pressure; PVR = pulse volume recording; TM = transmetatarsal; TP = toe pressure.Table 23-25Symptoms and signs of neuropathic ulcer versus ischemic ulcerNEUROPATHIC ULCERISCHEMIC ULCERPainlessPainfulNormal pulsesAbsent pulsesRegular margins, typically punched-out appearanceIrregular marginOften located on plantar surface of footCommonly located on toes, glabrous marginsPresence of callusesCalluses absent or infrequentLoss of sensation, reflexes, and vibrationVariable sensory findingsIncreased in blood flow (arteriovenous shunting)Decreased in blood flowDilated veinsCollapsed veinsDry, warm footCold

1	or infrequentLoss of sensation, reflexes, and vibrationVariable sensory findingsIncreased in blood flow (arteriovenous shunting)Decreased in blood flowDilated veinsCollapsed veinsDry, warm footCold footBony deformitiesNo bony deformitiesRed or hyperemic in appearancePale and cyanotic in appearanceSymptom development is a function of the extent of occlusion, adequacy of collaterals, and the activity level of the patients.Presenting symptoms of femoropopliteal occlusive dis-ease are broadly classified into two types: limb-threatening and non–limb-threatening ischemia. Claudication is non– limb-threatening, while rest pain, ulceration, and gangrene are limb-threatening and warrant urgent intervention. Occlusive disease of the femoral artery may be isolated or occur in con-junction with multilevel disease that involves both the aortoil-iac segment and the tibial vessels. Symptoms in patients with Brunicardi_Ch23_p0897-p0980.indd 95827/02/19 4:15 PM 959ARTERIAL DISEASECHAPTER

1	multilevel disease that involves both the aortoil-iac segment and the tibial vessels. Symptoms in patients with Brunicardi_Ch23_p0897-p0980.indd 95827/02/19 4:15 PM 959ARTERIAL DISEASECHAPTER 23BAFigure 23-63. A. A neuropathic ulcer is characterized by a punched-out appearance with loss of sensation in the surrounding skin. The foot may be warm to touch, and pulses may be present in the distal pedal arteries. B. An ischemic ulcer is characterized by a gangrenous skin change in the foot or toes. The foot is usually cold to touch with absent pedal pulses. The foot is painful to touch with decreased distal capil-lary refills.Figure 23-64. Computed tomography angiogram of a patient with an occluded left superficial femoral artery (single long arrow) with reconstituted superficial femoral artery at the level of mid-thigh. Diffuse arterial calcifications (double small arrows) are noted in the mid and distal left superficial femoral arteries.multilevel disease are more severe than in

1	artery at the level of mid-thigh. Diffuse arterial calcifications (double small arrows) are noted in the mid and distal left superficial femoral arteries.multilevel disease are more severe than in those with single-level disease. Pain from isolated SFA and popliteal occlusion typically manifests as calf claudication. Cramping pain develops in the calf on ambulation, occurs at a reproducible distance, and is relieved by rest. Activities such as climbing stairs or going uphill also exacerbate the pain. Many patients report worsen-ing symptoms during cold weather. It is important to evaluate whether the symptoms are progressive or static. In greater than 70% of patients, the disease is stable, particularly with risk fac-tor modification.Progression of the underlying atherosclerotic process is more likely to occur in patients with diabetes, those who con-tinue to smoke, and those who fail to modify their atherosclerotic risk factors. In comparison, rest pain is constant, and usually

1	is more likely to occur in patients with diabetes, those who con-tinue to smoke, and those who fail to modify their atherosclerotic risk factors. In comparison, rest pain is constant, and usually occurs in the forefoot across the metatarsophalangeal joint. It is worse at night and requires placing the foot in a dependent posi-tion to improve symptoms. Patients may report that they either sleep in a chair or hang the foot off the side of the bed. The pain is severe and relentless, even with narcotics. Ischemic ulcer-ation most commonly involves the toes. Any toe can be affected. Occasionally ulcers develop on the dorsum of the foot. Ulcer-ation can occur in atypical positions in an ischemic foot from trauma such as friction from poorly fitting shoes. Injury to a foot with borderline ischemia can convert an otherwise stable situ-ation into one that is limb-threatening. The initial development of gangrene commonly involves the digits. As with all vascular patients, it is important to

1	can convert an otherwise stable situ-ation into one that is limb-threatening. The initial development of gangrene commonly involves the digits. As with all vascular patients, it is important to evaluate their risk factors, intercurrent cardiac diseases, and any prior vascular interventions.Treatment Considerations for Chronic Limb IschemiaPatients with vascular diseases frequently have complicated medical comorbidities. Careful patient evaluation and selection should be performed for any peripheral arterial vascular proce-dure. The fundamental principle is to assess not only the surgi-cal risk from the peripheral arterial system but also the global nature of the atherosclerotic process. Full cardiac evaluations are often necessary due to the high incidence of concomitant atherosclerotic coronary artery disease, resulting in a high risk for ischemic events. Hertzer and associates reviewed coronary angiographies on 1000 patients undergoing elective vascu-lar procedure and identified

1	coronary artery disease, resulting in a high risk for ischemic events. Hertzer and associates reviewed coronary angiographies on 1000 patients undergoing elective vascu-lar procedure and identified 25% of concomitant correctable coronary artery disease, including 21% in patients undergoing elective peripheral vascular intervention.5 Conte and associates analyzed their 20-year experience in 1642 open lower extremity reconstructive surgeries and concluded that patients requiring lower extremity reconstruction presented an increasingly com-plex medical and surgical challenge compared with the previous decade in a tertiary practice setting.148 With aging of the popu-lation, a growing number of vascular patients have prohibitive medical comorbidities and are deemed high-risk for open sur-gical repair. Endovascular intervention provides an attractive alternative.As for open surgical repair, the clinical indications for endovascular intervention of lower extremity peripheral arte-rial

1	repair. Endovascular intervention provides an attractive alternative.As for open surgical repair, the clinical indications for endovascular intervention of lower extremity peripheral arte-rial diseases include lifestyle-limiting claudication, ischemic rest pain, and tissue loss or gangrene. Importantly, endovas-cular procedures should be performed by a competent vas-cular interventionist who understands the vascular disease process and is familiar with a variety of endovascular tech-niques. In addition, certain lesions may not be amendable Brunicardi_Ch23_p0897-p0980.indd 95927/02/19 4:15 PM 960SPECIFIC CONSIDERATIONSPART IIto endovascular treatment or may be associated with poor outcomes, such as long segment occlusion, heavily calcified lesion, orifice lesion, or lesions that cannot be traversed by a guidewire. Proper selection of patients and techniques is criti-cal in achieving good long-term outcome.Endovascular intervention for lower extremity occlusive disease is

1	cannot be traversed by a guidewire. Proper selection of patients and techniques is criti-cal in achieving good long-term outcome.Endovascular intervention for lower extremity occlusive disease is continuously evolving. Success and patency rates of endovascular intervention are closely related to the anatomic and morphologic characteristics of the treated lesions. The TASC work group made recommendations on the intervention strate-gies of lower extremity arterial diseases based on the morpho-logic characteristics. Based on TASC guidelines, endovascular treatment is recommended for type A lesions, open surgery is recommended for type D lesions, and no recommendations were made for types B and C lesions. However, with rapid advance-ment in endovascular technologies, there are increased numbers of lesions amendable to endovascular interventions.There is less literature support for infrapopliteal endo-vascular intervention due to higher complication and lower success rates. The treatment

1	of lesions amendable to endovascular interventions.There is less literature support for infrapopliteal endo-vascular intervention due to higher complication and lower success rates. The treatment is restricted for patients with limb-threatening ischemia who lack surgical alternatives. However, with further advancement of endovascular technology and the development of new devices, endovascular intervention is becoming an integral part of treatment (Table 23-26). By itself or combined with open technique, percutaneous intervention plays an important role in therapeutic options for lower extrem-ity occlusive disease. As described by TASC guidelines, four criteria should be measured to evaluate the clinical success of the treatment: improvement in walking distance, symptomatic improvement, quality of life, and overall graft patency. These criteria should all be carefully weighed and evaluated for each individual prior to endovascular therapy.Table 23-26Summary of endovascular treatment

1	quality of life, and overall graft patency. These criteria should all be carefully weighed and evaluated for each individual prior to endovascular therapy.Table 23-26Summary of endovascular treatment strategies using device-based infrapopliteal interventionINTERVENTIONADVANTAGESDISADVANTAGESAngioplasty• Easy to use• Broad range of applications• Failure in long lesions, calcified lesions, and disease at multiple levelsBalloon-expandable stent• Overcomes arterial recoil from angioplasty• Useful in treatment of flow-limiting dissection• Crushability can lead to restenosis• Poor distal runoff can result in stent thrombosis• Limited dataSelf-expanding stent• Vessel conformability and wall apposition prevent kinking and crushing of stent• Limited sizes• Limited data; multicenter trials under wayBioabsorbable stent• Overcomes arterial recoil from angioplasty• Absorbed long term to prevent risk of stent thrombosis• Limited data; multicenter trials under wayCryoplasty• Reduces the risk of

1	wayBioabsorbable stent• Overcomes arterial recoil from angioplasty• Absorbed long term to prevent risk of stent thrombosis• Limited data; multicenter trials under wayCryoplasty• Reduces the risk of flow-limiting dissection, therefore reducing the need for stent implantation• Short-term results of a multicenter trial are promising; however, long-term data are limitedCutting balloon• Useful in anastomotic segments of bypass grafts and in-stent restenosis where “watermelon seeding” can prevent adequate expansion of plaque• Limited dataMechanical atherectomy• Allows for debulking of plaque without the need for stent implantation in most cases• Allows for removal of plaque for histologic analysis• Limited use in areas of heavy calcification• No large, randomized, prospective trial comparing this technique to angioplasty and stentingLaser• Useful in acute thrombotic and chronic total occlusions• Minimal data in infrapopliteal arteries• Need adjunctive treatment with angioplasty, stenting,

1	technique to angioplasty and stentingLaser• Useful in acute thrombotic and chronic total occlusions• Minimal data in infrapopliteal arteries• Need adjunctive treatment with angioplasty, stenting, or atherectomyEndovascular TreatmentTechnical Considerations. A sterile field is required in either an operating room or an angiography suite with image capabil-ity. The most common and safest access site is CFA via either a retrograde or an antegrade approach. For diagnostic angiogra-phy, arterial access should be contralateral to the symptomatic sides. For therapeutic procedures, location of the lesion and the anatomic structures of the arterial tree determine the puncture site. To avoid puncturing the iliac artery or SFA, the femoral head is located under the fluoroscopy and used as the guide for the level of needle entry. In addition, there are several useful techniques to help access a pulseless CFA including ultrasound-guided puncture, using a micropuncture kit, and targeting

1	the guide for the level of needle entry. In addition, there are several useful techniques to help access a pulseless CFA including ultrasound-guided puncture, using a micropuncture kit, and targeting cal-cification in a calcified vessel. The antegrade approach may be challenging, particularly in obese patients. Meticulous technique is crucial in preventing complications, and a bony landmark can be used as guidance to ensure CFA puncture.Traversing the lesion with a wire is the most critical part of the procedure. Typically, 0.035-inch guidewires are used for femoropopliteal lesions, and 0.014or 0.018-inch guidewires are used for infrapopliteal access. Hydrophilic-coated wires, such as Glidewires, are useful in navigating through tight ste-nosis or occlusion. An angled-tip wire with a torque device may be helpful in crossing an eccentric lesion, and a shaped selective catheter is frequently used to help manipulate the wire across the lesion. The soft and floppy end of the wire is

1	torque device may be helpful in crossing an eccentric lesion, and a shaped selective catheter is frequently used to help manipulate the wire across the lesion. The soft and floppy end of the wire is care-fully advanced crossing the lesion under fluoroscopy, and gentle force is applied while manipulating the wire. Once the lesion us traversed, one needs to pay particular attention on the tip of the wire to ensure a secure wire access and avoid vessel wall perforation or dissection.Brunicardi_Ch23_p0897-p0980.indd 96027/02/19 4:15 PM 961ARTERIAL DISEASECHAPTER 23Once the access to the diseased vessel is secured and the wire has successfully traversed the lesion, several treatment modalities can be used either alone or in conjunction with oth-ers, including angioplasty, stent or stent graft placement, and atherectomy. The available angioplasty techniques are balloon angioplasty, cryoplasty, subintimal angioplasty, and cutting bal-loon; the most commonly used atherectomy techniques

1	graft placement, and atherectomy. The available angioplasty techniques are balloon angioplasty, cryoplasty, subintimal angioplasty, and cutting bal-loon; the most commonly used atherectomy techniques include percutaneous atherectomy catheter and laser atherectomy device.Systemic anticoagulation should be maintained routinely during lower extremity arterial interventions to minimize the risk of pericatheter thrombosis. Unfractionated heparin is the most commonly used agent, administered using a weight-based formula. We typically use 80 to 100 mg/kg initial bolus for therapeutic procedure to achieve an activated clotting time above 250 seconds upon catheter insertion and administer a sub-sequent 1000 units for each additional hour of the procedure. Newer agents, such as low molecular weight heparin, platelet IIb/IIIa inhibitors, direct thrombin inhibitors, or recombinant hirudin, have been available and can be used either alone or in conjunction with heparin, particularly in patients

1	heparin, platelet IIb/IIIa inhibitors, direct thrombin inhibitors, or recombinant hirudin, have been available and can be used either alone or in conjunction with heparin, particularly in patients who are sensi-tive to unfractionated heparin. After the procedure, all patients are placed on antiplatelet therapy, such as aspirin. Additional antiplatelet agents, such as clopidogrel (Plavix), are given to selected patients with stent placement for at least 6 weeks after lower extremity interventions unless otherwise contraindicated.Percutaneous Transluminal Balloon Angioplasty. After the lesion is crossed with a wire, an appropriated balloon angio-plasty catheter is selected and tracked along the wire to traverse the lesion. The length of the selected catheter should be slightly longer than the lesion, and the diameter should be equal to the adjacent normal vessel. The balloon tends to be approximately 10% to 20% oversized. The radiopaque markers of the balloon catheter are placed so that

1	lesion, and the diameter should be equal to the adjacent normal vessel. The balloon tends to be approximately 10% to 20% oversized. The radiopaque markers of the balloon catheter are placed so that they will straddle the lesion. Then, the balloon is inflated with saline and contrast mixture to allow visualization of the insufflation process under the fluoroscopy (Fig. 23-65). The patient may experience mild pain, which is not uncommon. However, severe pain can be indicative of ves-sel rupture, dissection, or other complications. An angiography is crucial in confirming the intraluminal location of the catheter and absence of contrast extravasation. The inflation is continued until the waist of the atherosclerotic lesion is disappeared and the balloon is at the full profile. Frequently, several inflations are required to achieve a full profile of the balloon (Fig. 23-66). Occasionally, a lower profile balloon is needed to predilate the tight stenosis so that the selected balloon

1	several inflations are required to achieve a full profile of the balloon (Fig. 23-66). Occasionally, a lower profile balloon is needed to predilate the tight stenosis so that the selected balloon catheter can cross the lesion.Besides length and diameter, the operators need to be familiar with several balloon characters. Noncompliant and low-compliant balloons tend to be inflated to their preset diam-eter and offer greater dilating force at the site of stenosis. Low-compliant balloons are the mainstay for peripheral intervention. A balloon with a low profile is used to minimize complications at the entry site and for crossing the tight lesions. Upon infla-tion, most balloons do not rewrap to their preinflation diameter and assume larger profiles. Furthermore, trackability, pushabil-ity, and crossability of the balloon should be considered when choosing a particular type of balloon. Lastly, shoulder length is an important characteristic when performing PTA to avoid injury to the

1	and crossability of the balloon should be considered when choosing a particular type of balloon. Lastly, shoulder length is an important characteristic when performing PTA to avoid injury to the adjacent arterial segments. After PTA, a comple-tion angiogram is performed while the wire is still in place. Leaving the wire in place provides access for repeating the pro-cedure if the result is unsatisfactory.PTA is an established and effective therapy for select patients with lower extremity occlusive diseases. Studies have shown that PTA of femoropopliteal segment achieved over 90% technical success rate and 38% to 58% 5-year primary patency rates.149,150 However, efficacy of PTA is highly dependent on anatomic selection and patient condition. PTA of lesions longer than 7 to 10 cm offers limited patency, whereas PTA of shorter lesions, such as those less than 3 cm, has fairly good results. Lofberg and associates performed 127 femoropopliteal PTA Figure 23-65. A. Angiogram demonstrating a

1	patency, whereas PTA of shorter lesions, such as those less than 3 cm, has fairly good results. Lofberg and associates performed 127 femoropopliteal PTA Figure 23-65. A. Angiogram demonstrating a focal stenosis in the superficial femoral artery (arrow). B. This lesion was treated with a bal-loon angioplasty catheter that inflated a dilating balloon and expanded the flow lumen. C. Completion angiogram demonstrating satisfactory radiographic result.Brunicardi_Ch23_p0897-p0980.indd 96127/02/19 4:15 PM 962SPECIFIC CONSIDERATIONSPART IIFigure 23-66. A. Angiogram demonstrating a segmental occlusion in the distal superficial femoral artery (single arrow). B. This lesion was treated with cryoplasty, which lowered the balloon catheter temperature to a temporary freezing state during the balloon angioplasty procedure (double arrows). C. Completion angiogram demonstrated satisfactory result with no evidence of vessel dissection.procedures and reported a primary 5-year success rate of 12% in

1	angioplasty procedure (double arrows). C. Completion angiogram demonstrated satisfactory result with no evidence of vessel dissection.procedures and reported a primary 5-year success rate of 12% in limbs with occlusion longer than 5 cm versus 32% in limbs with occlusion less than 5 cm in length.151 Occlusive lesions have much worse initial technical success rates than stenotic lesions. Concentric lesions respond better to PTA than eccentric lesions, and heavy calcifications have a negative impact on success rates. A meta-analysis by Hunink and associates showed that adjusted 5-year primary patencies after angioplasty of femoropopliteal lesions varied from 12% to 68%, with the best results being for patients with claudication and stenotic lesions.152 Distal runoff is another powerful predictor of long-term success. Johnston analyzed 254 consecutive patients who underwent femoral and popliteal PTA and reported a 5-year patency rate of 53% for ste-notic lesions and 36% for occlusive

1	of long-term success. Johnston analyzed 254 consecutive patients who underwent femoral and popliteal PTA and reported a 5-year patency rate of 53% for ste-notic lesions and 36% for occlusive lesions in patients with good runoff versus a 5-year patency rate of 31% for stenotic lesions and 16% for occlusive lesions in patients with poor runoff.149 Literature reviews showed that 5-year patency rates varied from 27% to 67% based on runoff status.152Due to limited success with infrapopliteal PTA, the indi-cation for infrapopliteal PTA is stringent and reserved for limb salvage. Current patency rates from infrapopliteal PTA can be improved further by proper patient selection, ensuring straight-line flow to the foot in at least one tibial vessel, and close patient surveillance for early reintervention. Possible future advances, including the use of drug-eluting stents, cutting balloons, and atherectomy devices, are being investigated to improve clini-cal outcomes following endovascular

1	Possible future advances, including the use of drug-eluting stents, cutting balloons, and atherectomy devices, are being investigated to improve clini-cal outcomes following endovascular interventions on the tibial arteries. Varty and associates reported a 1-year limb salvage rate of 77% in patients with critical ischemia who underwent infrapopliteal PTA.153 In patients with favorable anatomies, the 2-year limb salvage rate after infrapopliteal PTA is expected to exceed 80%.Subintimal Angioplasty. The technique of subintimal angio-plasty was first described in 1987 when successful establish-ment of flow was made by accidental creation of a subintimal channel during treatment of a long popliteal artery occlusion. Subintimal angioplasty is recommended for chronic occlusion, long segment of lesion, and heavily calcified lesions. In addi-tion, this technique is applicable for vessels with diffuse dis-ease and for vessels that had previously failed an intraluminal approach, when it is

1	of lesion, and heavily calcified lesions. In addi-tion, this technique is applicable for vessels with diffuse dis-ease and for vessels that had previously failed an intraluminal approach, when it is difficult to negotiate the wire across the entire diseased segment without dissection.The principle of this technique is to bypass the occlusion by deliberately creating a subintimal dissection plan commenc-ing proximal to the lesion and continuing in the subintimal space before retry into the true lumen distal to the lesion. The occluded lumen is recanalized through the subintimal plan. Sub-intimal angioplasty can be performed through either an ipsilat-eral antegrade or contralateral retrograde approach using the CFA approach. If selecting contralateral CFA puncture, a long guiding sheath is placed across the aortic bifurcation to provide access for the femoropopliteal and infrapopliteal vessels. The subintimal dissection is initiated at the origin of an occlusion by directing the tip of

1	placed across the aortic bifurcation to provide access for the femoropopliteal and infrapopliteal vessels. The subintimal dissection is initiated at the origin of an occlusion by directing the tip of an angled guide wire, usually an angled hydrophilic wire, such as a Glidewire. A supporting catheter is used to guide the tip of the guidewire away from the impor-tant collaterals. When the wire is advanced, a loop is naturally formed at the tip of the guidewire. Once the subintimal plan is entered, the wire tends to move freely in dissection space. Sub-intimal location of the wire and the catheter can be confirmed by injecting a small amount of diluted contrast. At this point, the wire and the catheter are then advanced along the subintimal plan until the occlusion segment is passed. A loss of resistance is often encountered as the guidewire reenters the true lumen distal to the occlusion. Recanalization is confirmed by advanc-ing the catheter over the guidewire beyond the point of

1	loss of resistance is often encountered as the guidewire reenters the true lumen distal to the occlusion. Recanalization is confirmed by advanc-ing the catheter over the guidewire beyond the point of reen-try and obtaining an angiogram. This is followed by a balloon angioplasty. To confirm the patency following balloon dilata-tion, a completion angiogram is performed prior to withdraw-ing the catheter and wire. If flow is impaired, repeat balloon Brunicardi_Ch23_p0897-p0980.indd 96227/02/19 4:15 PM 963ARTERIAL DISEASECHAPTER 23dilatation may be necessary. Frequently, a stent is required to maintain a patent lumen and treat residual stenosis if more than 30% luminal reduction is confirmed on completion angiogram.Multiple studies have demonstrated the efficacy of subin-timal angioplasty. Bolia and colleagues reported their extensive experiences on subintimal angioplasty for treating long-segment occlusions of infrainguinal vessels.154 They achieved a techni-cal success rate of over

1	Bolia and colleagues reported their extensive experiences on subintimal angioplasty for treating long-segment occlusions of infrainguinal vessels.154 They achieved a techni-cal success rate of over 80% for both femoropopliteal and tibial arteries. One-year patency rates varied from 53% for infrapopli-teal vessels to 71% for femoropopliteal segments. Limb salvage rates reached over 80% at 12 months. They also reported that the factors influencing patency are smoking, number of runoff vessels, and occlusion length. Studies by other groups showed similar results.155 Treiman and colleagues treated 25 patients with 6to 18-cm femoropopliteal occlusion and achieved a technical success rate of 92% and a 13-month primary patency rate of 92%,156 whereas Lipsitz and associates reported a techni-cal success rate of 87% in 39 treated patients and a 12-month cumulative patency rate of 74%.155 In addition, Ingle and asso-ciates reported a technical success rate of 87% in 67 patients with

1	techni-cal success rate of 87% in 39 treated patients and a 12-month cumulative patency rate of 74%.155 In addition, Ingle and asso-ciates reported a technical success rate of 87% in 67 patients with femoropopliteal lesions and a 36-month limb salvage rate of 94%.157 As demonstrated herein, although technical success rates are similar in most series, the patency rates vary widely in different studies. Patient selection, anatomic character, and lesion locations may account for the wide range of outcomes.Stent Placement. Although suggested by Dotter during the late 1960s, the use of an endoluminal stent was not pursued until the limitations of PTA were widely recognized. There are sev-eral situations where stent placement is appealing. The primary indication is the potential salvage of an unacceptable angio-plasty result. Stent placement is typically used when residual stenosis after PTA is 30% or greater. An endoluminal stent is also used for dissection, perforation, and other PTA

1	an unacceptable angio-plasty result. Stent placement is typically used when residual stenosis after PTA is 30% or greater. An endoluminal stent is also used for dissection, perforation, and other PTA complica-tions. Primary stent placement has become a viable alternative for treating ulcerative lesions that may potentially be the source for embolization. Primary stent is also used to treat occlusive lesions that have a tendency for reocclusion and distal emboliza-tion after PTA. In addition, an endoluminal stent is potentially beneficial for early restenosis after PTA. Drug-eluting stents are currently under investigation in the United States and may be promising in decreasing restenosis rates.Although technical success rates are high, published series on femoropopliteal artery stents show that patency rates are comparable to PTA alone, with primary patency rates vary-ing from 18% to 72% at 3 years.158 Gray and associates stented 58 limbs after suboptimal PTA for long SFA lesions and

1	that patency rates are comparable to PTA alone, with primary patency rates vary-ing from 18% to 72% at 3 years.158 Gray and associates stented 58 limbs after suboptimal PTA for long SFA lesions and dem-onstrated a 1-year primary patency rate of 22%.159 However, Mewissen treated 137 limbs using self-expanding SMART niti-nol stents in patients with TASC A, B, and C femoropopliteal lesions and reported a 1-year primary patency of 76% and a 24-month primary patency rate of 60%.160 Appropriate patient selection and the anatomic characteristics of the lesions are cru-cial in the success of treatment outcomes. Additionally, stent characteristics may contribute to the patency rate.Several clinical studies have demonstrated the significant improvements of the new generation of nitinol stents for the SFA lesions: the German Multicenter Experience, the Mewis-sen trial, the BLASTER Trial, and the SIROCCO trial.161 The German Multicenter Experience was a retrospective review of 111 SFA stenting

1	the SFA lesions: the German Multicenter Experience, the Mewis-sen trial, the BLASTER Trial, and the SIROCCO trial.161 The German Multicenter Experience was a retrospective review of 111 SFA stenting procedures and predicted that the 6-month patency rate for SMART stents was 82% versus 37% for the Wallstent. The BLASTER (Bilateral Lower Arterial Stenting Employing Reopro) Trial evaluated the feasibility of using nitinol stents with and without intravenous abciximab for the treatment of femoral artery disease, and the preliminary results showed a 1-year clinical patency rate of 83%.162Furthermore, the drug-eluting stent, which proved effec-tive in decreasing restenosis in coronary intervention, may offer another promising alternative in lower extremity diseases. The drug released over a period of time interferes with smooth mus-cle cell proliferation, the main cellular element and source of extracellular matrix–producing restenosis. The first drug-eluting stent clinical trial used

1	a period of time interferes with smooth mus-cle cell proliferation, the main cellular element and source of extracellular matrix–producing restenosis. The first drug-eluting stent clinical trial used Cordis Cypher SMART stents coated with sirolimus (SIROCCO trial).163 The SIROCCO results showed binary in-lesion restenosis rates of 0% in the sirolimus-eluting group versus 23.5% in the noneluting group at 6-month follow-up angiography. The PaRADISE (Preventing Amputa-tions Using Drug-Eluting Stents) Trial investigated the efficacy and safety of using balloon-expandable drug-eluting stents to prevent amputations in patients with below-the-knee critical limb ischemia.164 One hundred six patients (118 limbs) were treated with drug-eluting stents in this prospective, nonrandom-ized trial. There were 228 drug-eluting stents implanted (83% Cypher [Cordis, Johnson & Johnson, Warren, NJ], 17% Taxus [Boston Scientific, Maple Grove, MN]). The average length treated was 60 mm. The 3-year

1	There were 228 drug-eluting stents implanted (83% Cypher [Cordis, Johnson & Johnson, Warren, NJ], 17% Taxus [Boston Scientific, Maple Grove, MN]). The average length treated was 60 mm. The 3-year cumulative incidence of amputa-tion was 6%, the survival rate was 71%, and the amputation-free survival rate was 68%. Only 12% of patients who died had a preceding major amputation. Rutherford category, age, creati-nine level, and dialysis were predictors of death but not amputa-tion. Target limb revascularization occurred in 15% of patients.Stent Graft. The concept of endoluminal bypass using stent graft in treating atherosclerotic SFA disease has been enter-tained. A stent graft is placed percutaneously across a long segment or multiple segments of lesions and is used to create a femoropopliteal bypass. Theoretically, endobypass has the potential of being as successful as surgical bypass graft by relin-ing the vessel wall in its anatomic position without the nega-tive impact of anastomosis.

1	bypass. Theoretically, endobypass has the potential of being as successful as surgical bypass graft by relin-ing the vessel wall in its anatomic position without the nega-tive impact of anastomosis. Stent grafts can be divided into two categories: unsupported and fully supported. The unsupported grafts consist of segments of bypass graft, such as PTFE, with an expandable stent at one or both ends. The unsupported grafts are flexible with a low profile, but prone to external compres-sion. The supported stent grafts consist of a metallic skeleton covered with graft fabric. The presence of a dense metal skele-ton promotes an extensive inflammatory response and increases the risk of thrombosis. There is no FDA-approved stent graft for peripheral intervention. However, Viabahn (WL Gore & Associates, Flagstaff, AZ) is the most commonly used device in the United States and is composed of an ultra-thin PTFE graft externally supported by self-expanding nitinol meshwork. The Viabahn device has

1	Flagstaff, AZ) is the most commonly used device in the United States and is composed of an ultra-thin PTFE graft externally supported by self-expanding nitinol meshwork. The Viabahn device has a specific delivery mechanism by pulling back the attached string, which results in proximal-to-distal delivery of the endoprosthesis.Although it is an intriguing concept, data on endobypass results are limited, and the graft thrombosis rate is high. Addi-tionally, covering major collateral vessels can potentially jeop-ardize the viability of the limb if stent graft occlusion occurs. Bauermeister treated 35 patients with Hemobahn and reported a 28.6% occlusion rate at an average 7-month follow-up.165 Kedora and colleagues recently conducted a prospective, randomized study comparing covered PTFE/nitinol self-expanding stent grafts with prosthetic above-the-knee femoropopliteal bypass. Fifty limbs were randomized into each group. Primary patency Brunicardi_Ch23_p0897-p0980.indd 96327/02/19

1	self-expanding stent grafts with prosthetic above-the-knee femoropopliteal bypass. Fifty limbs were randomized into each group. Primary patency Brunicardi_Ch23_p0897-p0980.indd 96327/02/19 4:15 PM 964SPECIFIC CONSIDERATIONSPART IIat 1 year was approximately 74% for both cohorts, with a mean follow-up of 18 months. The covered nitinol/PTFE stent graft in the SFA had a 1-year patency comparable to surgical bypass, with a significantly shorter hospital stay (0.9 vs. 3.1 days).166 A recent randomized prospective study comparing the treatment of SFA occlusive disease percutaneously with an expanded PTFE (ePTFE)/nitinol self-expanding stent graft (stent graft) versus surgical femoral to above-knee popliteal artery bypass with synthetic graft material showed no difference between the two groups with respect to primary or secondary patency rates at 48 months.167 Mean total lesion length of the treated arterial seg-ment in the stent graft group was 25.6 cm. The stent graft group

1	two groups with respect to primary or secondary patency rates at 48 months.167 Mean total lesion length of the treated arterial seg-ment in the stent graft group was 25.6 cm. The stent graft group demonstrated a primary patency of 72%, 63%, 63%, and 59% with a secondary patency of 83%, 74%, 74%, and 74% at 12, 24, 36, and 48 months, respectively. The surgical femoral-popliteal group demonstrated a primary patency of 76%, 63%, 63%, and 58% with a secondary patency of 86%, 76%, 76%, and 71% at 12, 24, 36, and 48 months, respectively. The authors concluded that ePTFE/nitinol self-expanding stent graft placement can be offered as an alternative to treatment of the SFA segment for revascularization when prosthetic bypass is being considered or when autologous conduit is unavailable.Atherectomy. The basic principle of atherectomy is to remove the atheroma from obstructed arterial vessels. The currently available atherectomy devices can be generally categorized into directional,

1	basic principle of atherectomy is to remove the atheroma from obstructed arterial vessels. The currently available atherectomy devices can be generally categorized into directional, nondirectional, orbital, and rotational types based on their mechanism. A few examples of FDA-approved atherectomy devices are Simpson AtheroCath (DVI, Redwood City, CA), Transluminal Extraction Catheter (Interventional Technologies, San Diego, CA), Thoratec recanalization arterial catheter (Thoratec, Pleasanton, CA), Auth Rotablator (Heart Technologies, Redmond, WA), SilverHawk system (FoxHollow Technologies, Redwood City, CA), Jetstream atherectomy sys-tem (Bayer, Indianola, PA), Diamondback 360° orbital atherec-tomy device (Cardiovascular Systems, Inc, St. Paul, MN), and Rotablator system (Boston Scientific Corporation, Natick, MA). These devices either cut and remove or pulverize the atheroma plaques.The Simpson AtheroCath has a directional cutting ele-ment that is exposed to one-third of the

1	Corporation, Natick, MA). These devices either cut and remove or pulverize the atheroma plaques.The Simpson AtheroCath has a directional cutting ele-ment that is exposed to one-third of the circumference of the arterial wall. The atheroma protruding into the window is excised and pushed into the collection chamber. The Translu-minal Extraction Catheter has an over-the-wire nondirectional cutter mounted on the distal end of a torque tube. The excised atheroma is simultaneously removed by aspiration through the torque tube. The Thoratec recanalization arterial catheter is a nondirectional, noncoaxial, atheroablative device. The rotating cam tip pulverizes the atheromatous lesion into minute particles. The Auth Rotablator is a nondirectional, coaxial, atheroablative device with a metal burr embedded with fine diamond chips. SilverHawk device is a monorail catheter designed to overcome the drawbacks of a directional atherectomy catheter. The work-ing end consists of a hinged housing unit

1	embedded with fine diamond chips. SilverHawk device is a monorail catheter designed to overcome the drawbacks of a directional atherectomy catheter. The work-ing end consists of a hinged housing unit containing a carbide cutting blade. The blade is activated from the motor drive unit, and the catheter is then advanced through the length of the lesion. Once each pass is completed, the cutter then packs the tissue into the distal end of the nosecone to maximize collec-tion capacity. The SilverHawk can then either be removed or torqued to treat a different quadrant in the same lesion or other lesions. Jetstream atherectomy system is a rotating, aspirat-ing catheter with tip sizes of 1.6 and 1.8 mm for tibial arteries, and an expandable catheter with a tip size ranging from 2.1 to 3.4 mm for active removal of atherosclerotic debris and throm-bus. The Diamondback 360° orbital atherectomy device uses a drive shaft with an eccentrically mounted, diamond-coated crown to create an orbital

1	for active removal of atherosclerotic debris and throm-bus. The Diamondback 360° orbital atherectomy device uses a drive shaft with an eccentrically mounted, diamond-coated crown to create an orbital spin. As the speed of the crown increases from centrifugal force, it sands wider spaces, thereby providing variability in its working range. It can create a lumen that is >1.75 times the crossing profile depending on the size of the grit and the eccentricity of the offset. The greater the speed of the crown, the larger is the arc of debulking and, ultimately, the resultant lumen size. A constant flow of saline solution is delivered by a roller pump that lubricates the device and helps to flush the debris. The Rotablator system high-speed rotational device uses calcium ablation to achieve larger lumens. It has been used for more than 20 years to treat challenging, calcified coronary artery disease. The diamond-coated burr is designed to preferentially engage calcium and modify lesion

1	larger lumens. It has been used for more than 20 years to treat challenging, calcified coronary artery disease. The diamond-coated burr is designed to preferentially engage calcium and modify lesion compliance.Despite the promising early technical and clinical success, the midand long-term results have been disappointing due to high incidence of restenosis. A multicenter clinical registry of plaque atherectomy in patients with femoropopliteal occlusive disease showed potential clinical efficacy of this technology, as the 6and 12-month rates of survival free of target lesion revascularization were 90% and 80%, respectively.168 Impor-tantly, nearly three-quarters (73%) of patients treated with plaque excision modality did not require adjunctive endovas-cular therapy as infrainguinal stenting was necessary in only 6.3% of lesions. Results from the TALON registry support the role of plaque excision in selected patients with lower extremity arterial disease.Recent technologic advances have

1	was necessary in only 6.3% of lesions. Results from the TALON registry support the role of plaque excision in selected patients with lower extremity arterial disease.Recent technologic advances have made it possible to increase the spectrum of treatable peripheral arterial lesions with high acute procedure success rates. Recently presented data from multiple registries have shown some promising results in terms of short-term primary patency rates and freedom from unplanned major amputation.169 Randomized clinical trials, which may provide conclusions on the effectiveness of these procedures, are expected.Laser Atherectomy. Since laser atherectomy was reported in the 1960s, a variety of innovative approaches have been devel-oped trying to overcome the limitation of laser angioplasty. Recent developments in Excimer laser technology have led to increased optimism regarding the ability to safely deliver laser energy. Excimer laser atherectomy approved by the FDA for peripheral artery

1	developments in Excimer laser technology have led to increased optimism regarding the ability to safely deliver laser energy. Excimer laser atherectomy approved by the FDA for peripheral artery intervention employs precision laser energy control (shallow tissue penetration) and safer wavelengths (ultraviolet as opposed to the infrared spectra in older laser technology), which decrease perforation and thermal injury to the treated vessels.A laser atherectomy catheter, with diameters varying from 0.9 to 2.5 mm, is tracked over the guidewire to the desired tar-get. Once activated, the Excimer laser uses ultraviolet energy to ablate the lesion and create a nonthrombogenic arterial lumen. This lumen is further dilated by an angioplasty balloon. Because the Excimer laser can potentially reduce the rate of distal embo-lization by evaporating the lesion, it may be used as an adjunct tool for ostial lesions and lesions that can be traversed by a wire but not an angioplasty balloon

1	reduce the rate of distal embo-lization by evaporating the lesion, it may be used as an adjunct tool for ostial lesions and lesions that can be traversed by a wire but not an angioplasty balloon catheter.Several studies regarding the use of Excimer laser atherectomy combined with balloon angioplasty on lower extremity occlusive disease have shown promising clinical outcomes.199,200 The Peripheral Excimer Laser Angioplasty (PELA) trial involved 318 patients with chronic SFA occlusion Brunicardi_Ch23_p0897-p0980.indd 96427/02/19 4:15 PM 965ARTERIAL DISEASECHAPTER 23and achieved a technical success rate of 83.2%, a 1-year pri-mary patency rate of 33.6%, and an assisted-primary patency rate of 65%.170 Steinkamp and colleagues treated 127 patients with long-segment popliteal artery occlusion using laser ather-ectomy followed by balloon angioplasty and reported a 3-year primary patency rate of 22%.171 A multicenter clinical trial, the Laser Angioplasty for Critical Limb Ischemia (LACI)

1	using laser ather-ectomy followed by balloon angioplasty and reported a 3-year primary patency rate of 22%.171 A multicenter clinical trial, the Laser Angioplasty for Critical Limb Ischemia (LACI) trial, sup-ports the efficacy of this treatment modality in selected patients, with 6-month primary patency and clinical improvement rates of 33% and 89%, respectively.172 The technology and devices continue to evolve. With the TurboBooster and Turbo-Tandem technologies (Spectranetics Corporation, Colorado Springs, CO), the efficacy of plaque reduction was reported to be sig-nificantly improved in the CliRpath Excimer Laser System to Enlarge Lumen Openings (CELLO) study.173 The CELLO study was a single-arm, prospective registry trial conducted at 17 investigational sites in the United States to evaluate the safety and efficacy of a modified laser catheter designed for the endovascular treatment of peripheral artery disease affecting the SFA and proximal popliteal artery. Laser ablation

1	to evaluate the safety and efficacy of a modified laser catheter designed for the endovascular treatment of peripheral artery disease affecting the SFA and proximal popliteal artery. Laser ablation reduced percent diameter stenosis from 77% to 21% after adjunctive therapy with balloon angioplasty or balloon angioplasty with stenting; 12.3% patients did not receive postlaser adjunctive therapy. Patency rates were 59% and 54% at 6 and 12 months, respectively. Target lesion revascularization was not required in 76.9% of CELLO participants within the 1-year follow-up.Complications of Endovascular InterventionsAngioplasty-Related Complications. Complications related to PTA vary widely and include dissection, rupture, emboliza-tion, pseudoaneurysms, restenosis, hematoma, and acute occlu-sion secondary to thrombosis, vasospasm, or intimal injury. Clark and associates analyzed the data from 205 patients in the SCVIR Transluminal Angioplasty and Revascularization (STAR) registry and reported a

1	to thrombosis, vasospasm, or intimal injury. Clark and associates analyzed the data from 205 patients in the SCVIR Transluminal Angioplasty and Revascularization (STAR) registry and reported a complication rate of 7.3% for patients undergoing femoropopliteal angioplasty.174 Minor complications accounted for 75% of the cases, including distal emboli (41.7%), puncture site hematomas (41.7%), contained vessel rupture (8.3%), and vagal reactions (8.3%). In another study, Axisa and colleagues reported an overall rate of significant complica-tions for patients undergoing PTA of the lower extremities of 4.2%, including retroperitoneal bleeding (0.2%), false aneurysm (0.2%), ALI (1.5%), and vessel perforation (1.7%).175Complications limiting the application of subintimal angioplasty are parallel to those of PTA. A study investigating the use of subintimal angioplasty in 65 patients with SFA occlu-sion found that complications developed in 15% of patients.176 These complications included

1	to those of PTA. A study investigating the use of subintimal angioplasty in 65 patients with SFA occlu-sion found that complications developed in 15% of patients.176 These complications included significant stenosis (44%), SFA rupture (6%), distal embolization (3%), retroperitoneal hemor-rhage (1.5%), and pseudoaneurysm (1.5%). Additional compli-cations reported included perforation, thrombosis, dissection, and extensions beyond the planned reentry site.177 Importantly, damage to significant collateral vessels may occur in 1% to 1.5% of patients who undergo subintimal angioplasty. If a suc-cessful channel is not achieved in this situation, the patient may have a compromised distal circulation that necessitates dis-tal bypass. Cryoplasty is a modified form of angioplasty, and long-term results on lower extremity intervention are not yet available. Fava and associates treated 15 patients with femo-ropopliteal disease and had a 13% complication rate involving guidewire dissection and

1	results on lower extremity intervention are not yet available. Fava and associates treated 15 patients with femo-ropopliteal disease and had a 13% complication rate involving guidewire dissection and PTA-induced dissection of a tandem lesion remote to the cryoplasty zone.178Endoluminal Stent– and Stent Graft–Related Complica-tions. In addition to the aforementioned complications with angioplasty, endoluminal stent is associated with the risk of stent fraction and deformity. The adductor canal has nonlami-nar flow dynamics, especially with walking. The forces exerted on the SFA include torsion, compression, extension, and flex-ion. These forces exert significant stress on the SFA and stents. In addition, the lower extremity is subject to external trauma, which further increases the risk of stent deformity and fracture (Fig. 23-67). The SIROCCO study showed that stent fracture, although not associated with clinical symptoms, occurs in 18.2% of the procedures involving both drug-eluting

1	stent deformity and fracture (Fig. 23-67). The SIROCCO study showed that stent fracture, although not associated with clinical symptoms, occurs in 18.2% of the procedures involving both drug-eluting stents and control stents.163Stent grafts may present the additional complication of covering important collaterals, which results in compromised distal circulation. A prospective study evaluating Hemo-bahn stent grafts in the treatment of femoropopliteal arterial Figure 23-67. Due to various geometric forces, including torsion, compression, extension, and flexion, exerted on the superficial fem-oral artery (SFA), stent fracture (arrows) is a known complication following SFA stent placement.Brunicardi_Ch23_p0897-p0980.indd 96527/02/19 4:15 PM 966SPECIFIC CONSIDERATIONSPART IIocclusions demonstrated a 23% immediate complication rate including distal embolization (7.7%), groin hematoma (13.5%), and arteriovenous fistula (1.9%).179Atherectomy-Related Complications. Overall complication

1	a 23% immediate complication rate including distal embolization (7.7%), groin hematoma (13.5%), and arteriovenous fistula (1.9%).179Atherectomy-Related Complications. Overall complication rates associated with atherectomy range from 15.4% to 42.8%, including spasm, thrombosis, dissection, perforation, distal emboli, no reflow, and hematoma.180,181 Jahnke and associates conducted a prospective study evaluating high-speed rotational atherectomy in 15 patients with infrapopliteal occlusive disease. They yielded a 94% technical success rate, but success was complicated by vessel rupture (5%), distal embolization (5%), and arterial spasm (5%).179 Although Excimer laser atherectomy reduces embolic events by evaporating the lesion, embolization still remains a problematic complication. Studies show that dis-tal embolic events occur in 3% to 4% of procedures and perfora-tion occurs in 2.2% to 4.3% of cases.170,171 Other complications associated with laser atherectomy therapy include acute

1	show that dis-tal embolic events occur in 3% to 4% of procedures and perfora-tion occurs in 2.2% to 4.3% of cases.170,171 Other complications associated with laser atherectomy therapy include acute throm-bosis, vasospasm, direct vessel injury, and dissection.170,171Surgical Treatment for Chronic Limb Ischemia due to Femoropopliteal DiseaseEndarterectomy. Endarterectomy has a limited, albeit impor-tant role in lower extremity occlusive disease. It is most fre-quently used when there is disease in the CFA or involving the PFA. In this procedure, the surgeon opens the diseased segment longitudinally and develops a cleavage plane within the media that is developed proximally and distally. This permits the inner layer containing the atheroma to be excised. Great care must be taken at the distal end of the endarterectomy to either ensure a smooth transition or tack down the distal endpoint to prevent the flow from elevating a potentially occlusive atheromatous flap. Currently, there is

1	end of the endarterectomy to either ensure a smooth transition or tack down the distal endpoint to prevent the flow from elevating a potentially occlusive atheromatous flap. Currently, there is essentially no role for long open endar-terectomy in the treatment of SFA stenoses or occlusions. The high incidence of restenosis is what limits utility of endarterec-tomy in this location. Short-segment stenoses are more appro-priately treated with balloon angioplasty. Endarterectomy using a catheter-based approach (e.g., Moll endarterectomy device) supplemented with stent grafting or stenting across the endpoint of the endarterectomy is currently being reevaluated; however, no long-term data are available.Bypass Grafting. Bypass grafting remains the primary inter-vention for lower extremity occlusive disease. The type of bypass and the type of conduit are important variables to con-sider. Patients with occlusive disease limited to the SFA, who have at least 4 cm (ideally 10 cm) of normal

1	disease. The type of bypass and the type of conduit are important variables to con-sider. Patients with occlusive disease limited to the SFA, who have at least 4 cm (ideally 10 cm) of normal popliteal artery reconstituted above the knee joint, and with at least one continu-ous vessel to the foot can be treated with an above-knee femo-ropopliteal bypass graft. Despite the fact that in this above-knee location, the differential patencies between prosthetic (PTFE) and vein graft are comparable, undoubtedly, it remains ideal to use a saphenous vein as the bypass conduit if possible. Saving the vein for future coronary artery bypass or distal leg bypass grafting has been shown to be a flawed argument. One must also consider that the consequences to the vascular outflow after a thrombosed prosthetic are worse than after a thrombosed vein graft.When the disease extends to involve the popliteal artery or the tibial vessels, the surgeon must select an appropriate out-flow vessel to perform a

1	are worse than after a thrombosed vein graft.When the disease extends to involve the popliteal artery or the tibial vessels, the surgeon must select an appropriate out-flow vessel to perform a bypass. Suitable outflow vessels are defined as uninterrupted flow channels beyond the anastomosis into the foot. Listed in order of descending preference, they are as follows: above-knee popliteal artery, below-knee popli-teal artery, posterior tibial artery, anterior tibial artery, and peroneal artery. In patients with diabetes, it is frequently the peroneal artery that is spared. Although it has no direct flow into the foot, collateralization to the posterior tibial and anterior tibial arteries makes it an appropriate outflow vessel. There is no objective evidence to preferentially select tibial over peroneal arteries if they are vessels of equal caliber and quality. The dor-salis pedis, which is the continuation of the anterior tibial in the foot, is frequently spared from atherosclerotic

1	over peroneal arteries if they are vessels of equal caliber and quality. The dor-salis pedis, which is the continuation of the anterior tibial in the foot, is frequently spared from atherosclerotic disease and can be used as a target for distal bypasses. Patency is affected by the length of the bypass (longer bypasses have reduced patency), quality of the recipient artery, extent of runoff to the foot, and quality of the conduit (saphenous vein/graft). Five-year assisted patency rate for infrapopliteal venous bypasses is 60%. Venous conduits have also been shown to be suitable for bypasses to plantar arteries. In this location, venous conduits have a 3-year limb salvage rate of 84% and a 3-year secondary patency rate of 74%.1 A meta-analysis suggests unsatisfactory results when PTFE-coated grafts are used to bypass to infrapopliteal arteries. In this location, prosthetic grafts have a 5-year primary patency rate of 30.5%.182 Additionally, due to distal embolization and compromise of

1	grafts are used to bypass to infrapopliteal arteries. In this location, prosthetic grafts have a 5-year primary patency rate of 30.5%.182 Additionally, due to distal embolization and compromise of outflow vessels, prosthetic graft occlusion may have more severe consequences than vein graft occlusion.Two techniques are used for distal bypass grafting: reversed saphenous vein grafting and in situ saphenous vein grafting. There is no difference in outcomes (patency or limb salvage) between these techniques. In the former, the vein is excised in its entirety from the leg using open or endoscopic vein harvest, reversed to render the valves nonfunctional, and tunneled from the CFA inflow to the distal target vessels. End-to-side anastomoses are then created.Several adjunctive techniques have been used to try to improve the patency of bypass grafts to tibial arteries. Cre-ation of an arteriovenous fistula at the distal anastomosis is one option, but it has not been shown to improve

1	have been used to try to improve the patency of bypass grafts to tibial arteries. Cre-ation of an arteriovenous fistula at the distal anastomosis is one option, but it has not been shown to improve patency.183 Another method involves creating varying configurations of vein cuffs or patches at the distal anastomosis in an attempt to streamline the flow and to reduce the likelihood of neointimal hyperplasia. Results with this approach are more promising, especially when done to improve patency of a below-the-knee prosthetic; how-ever, there are no definitive comparative trials that support the superiority of one configuration over another.Amputation. Primary amputation is defined as an amputation that is performed without a prior attempt at surgical or endovas-cular revascularization. It is rarely necessary in patients who, as a result of neglect, present with class III ALI. Primary ampu-tation may play a role in patients with critical limb ischemia who are deemed nonambulatory because

1	rarely necessary in patients who, as a result of neglect, present with class III ALI. Primary ampu-tation may play a role in patients with critical limb ischemia who are deemed nonambulatory because of knee contractures, debilitating strokes, or dementia.Complications of Surgical ReconstructionVein Graft Stenoses. Fifteen percent of vein grafts will develop intrinsic stenoses within the first 18 months follow-ing implantation. Consequently, patients with vein grafts were entered into duplex surveillance protocols (scans every 3 months) to detect elevated (>300 cm/s) or abnormally low (<45 cm/s) graft velocities early. Stenoses greater than 50%, especially if associated with changes in ABI, should be repaired to prevent graft thrombosis. Repair usually entails patch angio-plasty or short-segment venous interposition, but PTA/stenting Brunicardi_Ch23_p0897-p0980.indd 96627/02/19 4:15 PM 967ARTERIAL DISEASECHAPTER 23is an option for short, focal lesions. Grafts with stenoses that are

1	venous interposition, but PTA/stenting Brunicardi_Ch23_p0897-p0980.indd 96627/02/19 4:15 PM 967ARTERIAL DISEASECHAPTER 23is an option for short, focal lesions. Grafts with stenoses that are identified and repaired prior to thrombosis have assisted-primary patency identical to primary patency, whereas a throm-bosed autogenous bypass has limited longevity resulting from ischemic injury to the vein wall. Secondary patency is mark-edly inferior to primary assisted patency. The recommenda-tion for routine duplex ultrasound surveillance of autogenous infrainguinal bypasses was recently brought into question by a randomized controlled trial that demonstrated no cost benefit or quality-of-life improvement in patients with femoropopliteal venous bypasses after 18 months.184 Many surgeons continue with programs of vein graft surveillance, as has been suggested in older trials, awaiting further confirmation of the findings from the more recent study. When intervening in a failing

1	continue with programs of vein graft surveillance, as has been suggested in older trials, awaiting further confirmation of the findings from the more recent study. When intervening in a failing infraingui-nal bypass, the original indication for surgery is an important consideration. Limb salvage rates for occluded grafts are better if the indication for the original bypass was claudication rather than rest pain or tissue loss. An acutely occluded infrainguinal graft (≤30 postoperative days) has a 25% limb salvage rate.185Limb Swelling. Limb swelling is common following revascu-larization and usually returns to baseline within 2 to 3 months. The etiology is multifactorial with lymphatic interruption, inter-stitial edema, and disruption of venous drainage all contributing. Limb swelling tends to worsen with repeat revascularization (see Table 23-22).Wound Infection. Since the most common inflow vessel for distal bypass is the CFA, groin infection is common and occurs in 7% of cases.186

1	to worsen with repeat revascularization (see Table 23-22).Wound Infection. Since the most common inflow vessel for distal bypass is the CFA, groin infection is common and occurs in 7% of cases.186 When an autogenous conduit such as the saphenous vein is used, most infections can be managed with local wound care because the infection involves the subcutane-ous tissue or skin rather than infection of the actual vein. When a prosthetic graft has been used, management of graft infection is a major undertaking. Infection of a lower extremity prosthetic bypass graft is associated with a significant amputation rate because of the tendency for graft thrombosis and anastomotic disruption. Prosthetic graft infections cannot be eradicated with antibiotics and mandate graft excision and complex revascular-ization using a vein if available.Choice of Conduit for Infrainguinal Bypass GraftingAutogenous Vein. The autogenous vein is superior to pros-thetic conduits for all infrainguinal bypasses,

1	using a vein if available.Choice of Conduit for Infrainguinal Bypass GraftingAutogenous Vein. The autogenous vein is superior to pros-thetic conduits for all infrainguinal bypasses, even in the above-knee position. This preference is applicable not only for the initial bypass but also for reoperative cases. For long bypasses, the ipsilateral great saphenous vein, contralateral great saphe-nous vein, small saphenous vein, arm vein, and spliced vein are used in decreasing order of preference. If only a short segment of vein is missing, the SFA can be endarterectomized and the proximal anastomosis performed distally to decrease the length of the conduit and to avoid harvesting and splicing additional vein. When the great saphenous vein is not available and a rela-tively short bypass is necessary, the arm vein or small saphe-nous vein is effective. The small saphenous vein is of particular utility when a posterior approach is used. If a longer bypass with vein is necessary, the arm vein

1	the arm vein or small saphe-nous vein is effective. The small saphenous vein is of particular utility when a posterior approach is used. If a longer bypass with vein is necessary, the arm vein is preferable because it is less awkward to harvest. Another conduit alternative is to har-vest the upper arm basilic, median cubital, and cephalic veins in continuity, while incising valves in the basilic segment and using the cephalic segment in reversed configuration to provide a relatively long, unspliced autogenous conduit.187Cryopreserved Grafts. Cryopreserved grafts are usually cadaveric arteries or veins that have been subjected to rate-controlled freezing with dimethyl sulfoxide (DMSO) and other cryopreservants. Cryopreserved vein grafts are more expensive than prosthetic grafts and are more prone to failure. The endo-thelial lining is lost as part of the freezing process, making these grafts prone to early thrombosis. Cryopreserved grafts are also prone to aneurysmal degeneration.

1	more prone to failure. The endo-thelial lining is lost as part of the freezing process, making these grafts prone to early thrombosis. Cryopreserved grafts are also prone to aneurysmal degeneration. Despite the fact that these grafts have not performed as well as prosthetic bypasses and autogenous vein bypasses in clinical practice, they can still play a role when revascularization is required following removal of infected prosthetic bypass grafts, especially when the autog-enous vein is unavailable to create a new bypass through clean tissue planes.Human Umbilical Vein. Human umbilical vein (HUV) is less commonly used than PTFE because it is thicker and more cum-bersome to handle and because of concerns about aneurysmal degeneration. HUV allografts are stabilized with glutaralde-hyde and do not have viable cells or antigenic reactivity. These grafts have poor handling characteristics and require extra care when suturing because of an outer Dacron mesh wrapping that is used to

1	and do not have viable cells or antigenic reactivity. These grafts have poor handling characteristics and require extra care when suturing because of an outer Dacron mesh wrapping that is used to decrease aneurysmal degeneration. Dardik and col-leagues have reported favorable results after using HUV and an adjunctive distal arteriovenous fistula.188 One trial compar-ing HUV with PTFE and saphenous vein showed that HUV was better than PTFE but worse than saphenous vein in terms of 5-year patency in the above-knee location.189 In a system-atic review, HUV appears to perform better than cryopre-served veins in terms of 1-year graft patency in infrainguinal revascularization.190Prosthetic Conduits and Adjunctive Modifications. If a vein is truly unavailable, PTFE or Dacron is the best option for above-knee bypass. The addition of rings to PTFE did not con-fer benefit in a single prospective, randomized clinical trial.191 For infrageniculate prosthetic bypasses, use of a vein patch, cuff,

1	for above-knee bypass. The addition of rings to PTFE did not con-fer benefit in a single prospective, randomized clinical trial.191 For infrageniculate prosthetic bypasses, use of a vein patch, cuff, or other venous anastomotic modification can improve patency (52% patency at 2 years for PTFE with vein cuff vs. 29% for PTFE with no cuff) and also improve limb salvage (84% vs. 62%).192Although prosthetic grafts are quickly available, easy to handle, and do not require extensive dissection to harvest, their propensity to undergo thrombosis and develop neointimal hyperplasia makes them a less favorable alternative when com-pared to vein. In a recent review of vein and prosthetic above-knee femoropopliteal bypasses, the 5-year primary patency rates were reported to be 74% and 39%, respectively.193 Outcomes were even worse for below-knee prosthetic bypasses. Unfortu-nately, the use of autologous venous conduits is not possible in as many as 30% of patients. The great saphenous vein may be

1	Outcomes were even worse for below-knee prosthetic bypasses. Unfortu-nately, the use of autologous venous conduits is not possible in as many as 30% of patients. The great saphenous vein may be unsuitable because of small size and poor quality or unavailable due to prior harvest.Methods to improve prosthetic graft performance have consisted of altering the geometry at the distal anastomosis to get the benefit obtained with vein cuffs (Distaflo; Bard Peripheral Vascular, Tempe, AZ) and covalently bonding agents onto the luminal surface with anticoagulant, anti-inflammatory, and anti-proliferative characteristics (Propaten; Gore, Flagstaff, AZ). One randomized trial that compared precuffed PTFE versus PTFE with a vein cuff enrolled 104 patients at 10 centers. Of 89 patients, 47 were randomized to precuffed PTFE bypasses and 44 were randomized to bypasses with a vein cuff.194 At 1 and Brunicardi_Ch23_p0897-p0980.indd 96727/02/19 4:15 PM 968SPECIFIC CONSIDERATIONSPART II2 years,

1	to precuffed PTFE bypasses and 44 were randomized to bypasses with a vein cuff.194 At 1 and Brunicardi_Ch23_p0897-p0980.indd 96727/02/19 4:15 PM 968SPECIFIC CONSIDERATIONSPART II2 years, primary patency rates were 52% and 49% in the pre-cuffed group and 62% and 44% in the vein cuffed group, respec-tively. At 1 and 2 years, the limb salvage rates were 72% and 65% in the precuffed group and 75% and 62% in the vein cuffed group, respectively. Although numbers are small and follow-up short, the midterm analysis revealed that Distaflo precuffed grafts and PTFE grafts with vein cuff had similar results. The authors concluded that a precuffed graft was a reasonable alternative for infragenicular reconstruction in the absence of saphenous vein.194 Other authors have been less optimistic and question whether there is any benefit derived from geometri-cally altering prosthetic conduits.195Another approach for improving outcomes when using prosthetic for bypass grafts involves bonding

1	and question whether there is any benefit derived from geometri-cally altering prosthetic conduits.195Another approach for improving outcomes when using prosthetic for bypass grafts involves bonding anticoagulants to the conduit. The Gore Propaten graft has heparin bonded onto the luminal surface of the PTFE graft using Carmeda BioActive Surface (CBAS) technology, which immobilizes the heparin molecule with a single covalent bond that does not alter its anti-coagulant properties.196 The heparin binding does not alter the microstructure and handling characteristics of the PTFE. A pro-spective, randomized trial by Devine and colleagues suggested that heparin-bonded Dacron or PTFE was superior to plain PTFE for above-knee popliteal bypasses. The 3-year primary patency rate for the heparin-bonded grafts was 55% compared with 42% for PTFE (P <0.044). Both of these patency rates are inferior to great saphenous vein grafts; however, if the improved results with heparin bonding continue to be

1	grafts was 55% compared with 42% for PTFE (P <0.044). Both of these patency rates are inferior to great saphenous vein grafts; however, if the improved results with heparin bonding continue to be substantiated, then heparin-bonded prosthetic grafts will become the preferred con-duit for above-knee bypass in the absence of suitable vein.197 A recent review of available studies with this graft showed an 80% 1-year patency rate for below-knee bypasses.198 Randomized controlled clinical trials with more patients and longer follow-up are necessary to validate whether the Propaten vascular graft is superior to other prosthetics and whether it is comparable to autogenous vein for below-knee interventions.Clinical Results of Surgical and Endovascular Interventions for Femoropopliteal Occlusive DiseaseBalloon angioplasty of the femoropopliteal vessels has not enjoyed the degree of success seen with iliac angioplasty. Patency in this region is dependent on whether the patient pres-ents with

1	DiseaseBalloon angioplasty of the femoropopliteal vessels has not enjoyed the degree of success seen with iliac angioplasty. Patency in this region is dependent on whether the patient pres-ents with claudication versus limb-threatening ischemia, the sta-tus of the distal runoff vessels, and lesion morphology. Initial technical success for femoropopliteal angioplasty is seen in 80% to 90% of cases, with failure to cross a lesion occurring in 7% of stenoses and 18% of occlusive lesions. Studies have shown that PTA of the femoropopliteal segment achieved a greater than 90% technical success rate and had a 38% to 58% 5-year pri-mary patency rate.152,199 PTA of lesions longer than 7 to 10 cm results in compromised patency, whereas PTA of shorter lesions (<3 cm) gives fairly good results. Lofberg and colleagues per-formed 127 femoropopliteal PTA procedures and reported a primary patency rate at 5-year follow-up of 12% in limbs with occlusion longer than 5 cm versus 32% in limbs with

1	Lofberg and colleagues per-formed 127 femoropopliteal PTA procedures and reported a primary patency rate at 5-year follow-up of 12% in limbs with occlusion longer than 5 cm versus 32% in limbs with occlusion less than 5 cm in length.151 Occlusive lesions have much worse initial technical success rates than stenotic lesions. Concentric lesions respond better to PTA than eccentric lesions, and heavy calcifications have a negative impact on success rates. Distal runoff is another powerful predictor of long-term success.Johnston analyzed 254 consecutive patients who under-went femoropopliteal PTA and reported a 5-year patency rate of 53% for stenotic lesions and 36% for occlusive lesions in patients with good runoff versus a 5-year patency rate of 31% for stenotic lesions and 16% for occlusive lesions in patients with poor runoff.149 A meta-analysis by Hunink and colleagues showed that adjusted 5-year primary patencies after angioplasty of femoropopliteal lesions varied from 12% to 68%,

1	lesions in patients with poor runoff.149 A meta-analysis by Hunink and colleagues showed that adjusted 5-year primary patencies after angioplasty of femoropopliteal lesions varied from 12% to 68%, with the best results occurring in patients with claudication and stenotic lesions.152 Although the initial technical success is better for stenoses than occlusions, long-term patency rates for stenoses and short occlusions have been variable, and there have been conflicting results regarding the efficacy of stent use. Early pub-lished series that examined efficacy of femoropopliteal artery stents showed patency rates that were comparable to standalone PTA, with primary patency rates varying from 18% to 72% at 3 years.158 Patient selection and the anatomic character of the lesions may play important roles in the outcomes. Additionally, stent characteristics may contribute to the patency rate. Several recent clinical studies have demonstrated significant improve-ments in patency when the

1	roles in the outcomes. Additionally, stent characteristics may contribute to the patency rate. Several recent clinical studies have demonstrated significant improve-ments in patency when the newer generations of nitinol stents are used to treat SFA lesions.160,200Mewissen treated 137 lower limbs in 122 patients with CLI, secondary to TASC A (n = 12) or TASC B or C (n = 125) lesions in the SFA. Patients were treated with Cordis SMART self-expanding nitinol stents. Binary restenosis (>50%) was measured by standard duplex velocity criteria at various postint-ervention intervals. Primary stent patency, defined as absence of binary restenosis in this study, was calculated by life-table methods from the time of intervention. The mean lesion length was 12.2 cm (range, 4–28 cm). The technical success was 98%. Mean follow-up was 302 days. The primary stent patency rates were 92%, 76%, 66%, and 60% at 6, 12, 18, and 24 months, respectively.160 Ferreira and colleagues treated 59 patients who had

1	was 98%. Mean follow-up was 302 days. The primary stent patency rates were 92%, 76%, 66%, and 60% at 6, 12, 18, and 24 months, respectively.160 Ferreira and colleagues treated 59 patients who had 74 femoropopliteal lesions (60% TASC D) with Zilver nitinol self-expanding stents (Cook, Bloomington, IN). Mean recanalization length was 19 cm (range, 3–53 cm). Mean fol-low-up time was 2.4 years (range, 3 days–4.8 years). Kaplan-Meier estimates for primary patency rates were 90%, 78%, 74%, 69%, and 69% at 1, 2, 3, 4, and 4.8 years, respectively.201There is general agreement that for suboptimal PTA of an SFA lesion, stent placement is indicated, but a recent random-ized trial by Schillinger and associates suggests that primary stenting results in lower restenosis rates than PTA and selective stenting. Restenosis rates at 2 years were 45.7% versus 69.2% in favor of primary stenting compared with PTA and optional secondary stenting using an intent-to-treat analysis (P = 0.031). Consistently,

1	Restenosis rates at 2 years were 45.7% versus 69.2% in favor of primary stenting compared with PTA and optional secondary stenting using an intent-to-treat analysis (P = 0.031). Consistently, stenting, both primary and selective, was supe-rior to standalone PTA with respect to the occurrence of reste-nosis (49.2% vs. 74.3%; P = 0.028) by a treatment-received analysis.202Nitinol bare metal stents that are designed specifically for below-knee interventions are showing very encouraging results. Bosiers and colleagues reported their 12-month results using the commercially available non–drug-eluting Xpert (Abbott Vascular, Santa Clara, CA) nitinol stent system in below-knee arterial interventions.203 They had a 12-month primary patency rate of 76.3% and a limb salvage rate of 95.9%. They fol-lowed patients for 12 months and performed angiography with quantitative vessel analysis on the 73% of patients available. Angiography revealed a binary restenosis rate (>50%) of only 20.5%, which is

1	patients for 12 months and performed angiography with quantitative vessel analysis on the 73% of patients available. Angiography revealed a binary restenosis rate (>50%) of only 20.5%, which is comparable to well-accepted coronary drug-eluting stent study outcomes. The authors attributed this opti-mal performance to the maintenance of flow dynamics because Brunicardi_Ch23_p0897-p0980.indd 96827/02/19 4:15 PM 969ARTERIAL DISEASECHAPTER 23the stent was specifically designed for use in small vessels. Kickuth and colleagues also have obtained good results using the Xpert stent. After stent placement, the primary cumulative patency rate at 6 months for the study group of 35 patients was 82%. The sustained clinical improvement rate as evidenced by improved ABI was 80%, and freedom from major amputation was 100% at the 6-month follow-up. The rate of major compli-cations was 17%.204Wolf and colleagues published a multicenter, prospec-tive randomized trial comparing PTA with bypass in 263

1	amputation was 100% at the 6-month follow-up. The rate of major compli-cations was 17%.204Wolf and colleagues published a multicenter, prospec-tive randomized trial comparing PTA with bypass in 263 men who had iliac, femoral, or popliteal artery obstruction.205 In 56 patients, cumulative 1-year primary patency rate was 43% after PTA and 82% after bypass surgery, demonstrating that for long SFA stenoses or occlusions, surgery is better than PTA. Another recent randomized study (BASIL trial) of 452 patients with CLI demonstrated no difference in amputation-free survival at 6 months between surgery and PTA/stenting.206 The authors com-mented that surgery was somewhat more expensive and rec-ommended that endovascular intervention should be used as first-line therapy especially in medically unfit patients. They did conclude that at the 2-year follow-up, healthy patients with-out medical comorbidities derived greater benefit from surgery because it was associated with decreased need for

1	unfit patients. They did conclude that at the 2-year follow-up, healthy patients with-out medical comorbidities derived greater benefit from surgery because it was associated with decreased need for reintervention and had a decreased hazard ratio in terms of all-cause mortality. The recently published randomized prospective study compar-ing the treatment of SFA occlusive disease percutaneously with an ePTFE/nitinol self-expanding stent graft versus surgical fem-oral to above-knee popliteal artery bypass with synthetic graft material showed no difference between the two groups with respect to primary or secondary patency rate at four years.167 This finding suggests that ePTFE/nitinol self-expanding stent graft placement can be offered as an alternative to treatment of the SFA segment for revascularization when prosthetic bypass is being considered or when autologous conduit is unavailable. Using the 2000 TASC definitions and a Markov state transi-tion model decision analysis, Nolan and

1	when prosthetic bypass is being considered or when autologous conduit is unavailable. Using the 2000 TASC definitions and a Markov state transi-tion model decision analysis, Nolan and colleagues showed that PTA/stenting surpasses bypass efficacy for TASC C lesions if PTA/stenting primary patency is >32% at 5 years, patient age is >80 years, and/or greater saphenous vein bypass operative mortality is >6%.207NONATHEROSCLEROTIC DISORDERS OF BLOOD VESSELSThe majority of cases of peripheral vascular disease that are seen by vascular surgeons are attributable to underlying athero-sclerosis. Nonatherosclerotic disease states that result in arterial pathology are less commonly encountered, but are nonetheless important, as they are potentially treatable lesions that may mimic atherosclerotic lesions and result in vascular insuffi-ciency (see Table 23-18). A thorough knowledge of these rare disease states is important for the practicing vascular surgeon in order to make medical

1	lesions and result in vascular insuffi-ciency (see Table 23-18). A thorough knowledge of these rare disease states is important for the practicing vascular surgeon in order to make medical recommendations and provide appropri-ate surgical treatment.Giant Cell Arteritis (Temporal Arteritis)Giant cell arteritis is also known as temporal arteritis, which is a systemic chronic inflammatory vascular disease with many characteristics similar to those of Takayasu’s disease. The histo-logic and pathologic changes and laboratory findings are similar. Patients tend to be white women over the age of 50 years, with a high incidence in Scandinavia and women of Northern European descent. Genetic factors may play a role in disease pathogenesis, with a human leukocyte antigen (HLA) variant having been identified. Differences exist between Takayasu’s and giant cell arteritis in terms of presentation, disease location, and therapeutic efficacy. The inflammatory process typically involves the aorta and

1	Differences exist between Takayasu’s and giant cell arteritis in terms of presentation, disease location, and therapeutic efficacy. The inflammatory process typically involves the aorta and its extracranial branches, of which the superficial temporal artery is specifically affected.The clinical syndrome begins with a prodromal phase of constitutional symptoms, including headache, fever, malaise, and myalgias. The patients may be initially diagnosed with coexisting polymyalgia rheumatica; an HLA-related associa-tion may exist between the two diseases. As a result of vascular narrowing and end-organ ischemia, complications may occur such as visual alterations, including blindness and mural weak-ness, resulting in acute aortic dissection that may be devastating. Ischemic optic neuritis resulting in partial or complete blindness occurs in up to 40% of patients and is considered a medical emergency. Cerebral symptoms occur when the disease process extends to the carotid arteries. Jaw

1	resulting in partial or complete blindness occurs in up to 40% of patients and is considered a medical emergency. Cerebral symptoms occur when the disease process extends to the carotid arteries. Jaw claudication and temporal artery tenderness may be experienced. Aortic lesions are usually asymptomatic until later stages and consist of thoracic aneu-rysms and aortic dissections.The diagnostic gold standard is a temporal artery biopsy, which will show the classic histologic findings of multinucle-ated giant cells with a dense perivascular inflammatory infil-trate. Treatment regimens are centered on corticosteroids, and giant cell arteritis tends to rapidly respond. Remission rates are high, and treatment tends to have a beneficial and preventative effect on the development of subsequent vascular complications.Takayasu’s ArteritisTakayasu’s arteritis is a rare but well-recognized chronic inflam-matory arteritis affecting large vessels, predominantly the aorta and its main branches

1	vascular complications.Takayasu’s ArteritisTakayasu’s arteritis is a rare but well-recognized chronic inflam-matory arteritis affecting large vessels, predominantly the aorta and its main branches (Table 23-27). Chronic vessel inflamma-tion leads to wall thickening, fibrosis, stenosis, and thrombus formation. Symptoms are related to end-organ ischemia. The acute inflammation can destroy the arterial media and lead to aneurysm formation. This rare autoimmune disease occurs pre-dominantly in women between the ages of 10 and 40 years who are of Asian descent. Genetic studies have demonstrated a high frequency of HLA haplotypes in patients from Japan and Mexico, suggesting increased susceptibility to developing the disease in patients with certain alleles. However, these associations have not been seen in North America. Vascular inflammation leads Table 23-27Angiographic classification of Takayasu’s arteritisTYPEVESSEL INVOLVEMENTType IBranches from the aortic archType IIaAscending aorta,

1	been seen in North America. Vascular inflammation leads Table 23-27Angiographic classification of Takayasu’s arteritisTYPEVESSEL INVOLVEMENTType IBranches from the aortic archType IIaAscending aorta, aortic arch and its branchesType IIbAscending aorta, aortic arch and its branches, thoracic descending aortaType IIIThoracic descending aorta, abdominal aorta, and/or renal arteriesType IVAbdominal aorta and/or renal arteriesType VCombined features of types IIb and IVInvolvement of the coronary or pulmonary arteries is designated as C (+) or P (+), respectively.Brunicardi_Ch23_p0897-p0980.indd 96927/02/19 4:15 PM 970SPECIFIC CONSIDERATIONSPART IIto arterial wall thickening, stenosis, and eventually, fibrosis and thrombus formation. The pathologic changes produce stenosis, dilation, aneurysm formation, and/or occlusion.The clinical course of Takayasu’s arteritis begins with a “prepulseless” phase in which the patient demonstrates constitu-tional symptoms. These include fever, anorexia,

1	formation, and/or occlusion.The clinical course of Takayasu’s arteritis begins with a “prepulseless” phase in which the patient demonstrates constitu-tional symptoms. These include fever, anorexia, weight loss, gen-eral malaise, arthralgias, and malnutrition. As the inflammation progresses and stenoses develop, more characteristic features of the disease become evident. During the chronic phase, the dis-ease is inactive or “burned out.” It is during this latter stage that patients most frequently present with bruits and vascular insuf-ficiency according to the arterial bed involved. Laboratory data may show elevations in erythrocyte sedimentation rate, C-reactive protein, and white blood cell count, or conversely, anemia may predominate. Characteristic clinical features during the second phase vary according to the involved vascular bed and include hypertension reflecting renal artery stenosis, retinopathy, aortic regurgitation, cerebrovascular symptoms, angina and con-gestive heart

1	phase vary according to the involved vascular bed and include hypertension reflecting renal artery stenosis, retinopathy, aortic regurgitation, cerebrovascular symptoms, angina and con-gestive heart failure, abdominal pain or gastrointestinal bleeding, pulmonary hypertension, or extremity claudication.The gold standard for diagnosis remains angiography showing narrowing or occlusion of the entire aorta or its pri-mary branches, or focal or segmental changes in large arteries in the upper or lower extremities. Six types of Takayasu’s arteritis exist and are graded in terms of severity: type I, affecting the aorta and arch vessels; type IIa, affecting the ascending aorta, aortic arch, and branches; type IIb, affecting the ascending aorta, aortic arch and branches, and thoracic descending aorta; type III, affecting the thoracic descending aorta, abdominal aorta, and/or renal arteries; type IV, affecting the abdominal aorta and/or renal arteries; and type V, with combined features of

1	aorta; type III, affecting the thoracic descending aorta, abdominal aorta, and/or renal arteries; type IV, affecting the abdominal aorta and/or renal arteries; and type V, with combined features of types IIb and IV.208Treatment consists of steroid therapy initially, with cyto-toxic agents used in patients who do not achieve remission. Surgical treatment is performed only in advanced stages, and bypass needs to be delayed during active phases of inflam-mation. There is no role for endarterectomy, and synthetic or autogenous bypass grafts need to be placed onto disease-free segments of vessels. For focal lesions, there have been reports of success with angioplasty.Ehlers-Danlos SyndromeEhlers-Danlos syndrome is one of the more significant inheritable disorders affecting the connective tissue, along with Marfan’s syndrome. This syndrome represents a heterogenous group of connective tissue disorders (types I through IV) that were first described in 1682 by van Meekeren.238 It is an

1	tissue, along with Marfan’s syndrome. This syndrome represents a heterogenous group of connective tissue disorders (types I through IV) that were first described in 1682 by van Meekeren.238 It is an autosomal domi-nant disorder affecting approximately 1 in 5000 persons that is characterized by skin elasticity, joint hypermobility, tissue fragility, multiple ecchymoses, and subcutaneous pseudotu-mors. Ehlers-Danlos syndrome is a disorder of fibrillar collagen metabolism with identifiable, specific defects that have been found in the collagen biosynthetic pathway that produce clini-cally distinct forms of this disease. Ten different phenotypes have been described, each with variable modes of inheritance and biochemical defects. Of the four basic types of collagen found in the body, the predominant type in blood vessels is type III. Within the vessel wall, type III collagen contributes to structural integrity and tensile strength and plays a role in platelet aggregation and thrombus

1	predominant type in blood vessels is type III. Within the vessel wall, type III collagen contributes to structural integrity and tensile strength and plays a role in platelet aggregation and thrombus formation.Of the three types of Ehlers-Danlos syndrome that have arterial complications, type IV represents 5% of cases and is the one most likely to be seen by a vascular surgeon. These patients synthesize abnormal type III collagen (mutation COL3A1) and represent 5% of all cases.209 Affected individuals do not show the typical skin and joint manifestations, and thus typically pres-ent for diagnosis when a major vascular catastrophe occurs. In a review of 36 patients with this disorder, Cikrit and colleagues reported a 44% mortality rate from major hemorrhage prior to any surgical intervention.210 In the 20 patients who underwent 29 vascular procedures, there was a 29% mortality rate. Arterial rupture, aneurysm formation, and acute aortic dissection may occur in any major artery, with

1	In the 20 patients who underwent 29 vascular procedures, there was a 29% mortality rate. Arterial rupture, aneurysm formation, and acute aortic dissection may occur in any major artery, with the most frequent site of rupture being the abdominal cavity. Repair is problematic because the vessel wall is soft and sutures pull through the fragile tissue. Ligation may be the only option in many circumstances.Marfan’s SyndromeAnother heterogeneous heritable disorder of connective tissue, Marfan’s syndrome is characterized by abnormal musculo-skeletal, ocular, and cardiovascular features first described by Antoine Marfan in 1896.211 The inborn error of metabolism in this syndrome has been localized to the long arm of chromo-some 15 (15q21.3). Defects occur in fibrillin, a basic protein in the microfibrillar apparatus that serves as a backbone for elas-tin, which is one of the main extracellular structural proteins in blood vessels. This is an autosomal dominant gene with high penetrance;

1	microfibrillar apparatus that serves as a backbone for elas-tin, which is one of the main extracellular structural proteins in blood vessels. This is an autosomal dominant gene with high penetrance; however, approximately 15% to 20% of cases are secondary to new spontaneous mutations.Classic recognizable features of Marfan’s syndrome include tall stature, long limbs (dolichostenomelia), long fingers (arachnodactyly), joint hyperextensibility, chest wall deformi-ties, and scoliosis. Ocular manifestations are flattened corneas, lens subluxation, and myopia. Ninety-five percent of patients have cardiovascular involvement, which may include ascend-ing aortic dilatation, mitral valve prolapse, valvular regurgita-tion, and aortic dissection. Skin, central nervous system, and pulmonary features may be present as well. Aortic root dilata-tion will generally occur in all patients. This may not be evident on standard chest radiograph until dilatation has resulted in an ascending aortic

1	may be present as well. Aortic root dilata-tion will generally occur in all patients. This may not be evident on standard chest radiograph until dilatation has resulted in an ascending aortic aneurysm, aortic valve regurgitation, or dis-section. Left untreated, the cardiovascular complications are devastating and reduce the life expectancy to about 40 years for men and slightly higher for women. Death is usually attrib-utable to life-threatening complications of aortic regurgitation, dissection, and rupture after the ascending aorta has dilated to 6 cm or more.Aggressive medical management with β-adrenergic block-ing agents and other blood pressure–lowering regimens is cru-cial to treatment. Surgical intervention entails replacement of the aortic root with a composite valve graft (e.g., Bentall pro-cedure).212 Prophylactic operative repair is indicated for an aneurysm greater than 5.5 cm, with an acceptable perioperative mortality of less than 5%.Pseudoxanthoma ElasticumPseudoxanthoma

1	pro-cedure).212 Prophylactic operative repair is indicated for an aneurysm greater than 5.5 cm, with an acceptable perioperative mortality of less than 5%.Pseudoxanthoma ElasticumPseudoxanthoma elasticum is a rare inherited disorder of con-nective tissue that is characterized by an unbalanced elastic fiber metabolism and synthesis, resulting in fragmentation and calci-fication of the fibers. Clinical manifestations occur in the skin, ocular, gastrointestinal, and cardiovascular systems. Character-istic skin lesions are seen in the axilla, antecubital and popliteal fossae, and groin. The yellow, xanthoma-like papules occur in redundant folds of skin and are said to resemble plucked chicken skin. The inheritance pattern includes both autosomal dominant Brunicardi_Ch23_p0897-p0980.indd 97027/02/19 4:15 PM 971ARTERIAL DISEASECHAPTER 23and recessive types and has a prevalence of 1 in 160,000 indi-viduals.213 The ATP-binding cassette subfamily C member 6 (ABCC6) gene has been

1	97027/02/19 4:15 PM 971ARTERIAL DISEASECHAPTER 23and recessive types and has a prevalence of 1 in 160,000 indi-viduals.213 The ATP-binding cassette subfamily C member 6 (ABCC6) gene has been demonstrated to be responsible, and 43 mutations have been identified, all of which lead to calcification of the internal elastic laminae of medium-sized vessel walls.214Cardiovascular features are common and include prema-ture coronary artery disease, cerebrovascular disease, renovas-cular hypertension, diminished peripheral pulses, and restrictive cardiomyopathy. Symptom onset typically occurs in the second decade of life, with onset at an average age of 13 years. Patients should be counseled to reduce potential contributing factors for atherosclerosis such as tobacco use and high cholesterol levels. Calcium intake should be restricted in adolescents, as a positive correlation has been found between disease severity and cal-cium intake. Surgical management involves standard vascular

1	levels. Calcium intake should be restricted in adolescents, as a positive correlation has been found between disease severity and cal-cium intake. Surgical management involves standard vascular techniques, with the exception that arterial conduits should not be employed in cardiac bypass.Kawasaki’s DiseaseKawasaki’s disease was first described in 1967, as a mucocu-taneous lymph node syndrome occurring in young children. In most studies, more than half the patients are younger than 2 years of age, with a higher prevalence in boys.215 Although originally described in Japan, the disease is found worldwide. An infec-tious agent may be causative; however, no specific agent has been identified. Immune activation with the contribution of cytokines, elastases, growth factors, and metalloproteinases is believed to be a mechanism for inflammation and aneurysm for-mation. Coronary artery aneurysms, the hallmark of the disease, histologically demonstrate a panarteritis with fibrinoid necro-sis.

1	is believed to be a mechanism for inflammation and aneurysm for-mation. Coronary artery aneurysms, the hallmark of the disease, histologically demonstrate a panarteritis with fibrinoid necro-sis. Coronary arteriography may show occlusions, recanaliza-tion, and localized stenosis, in addition to multiple aneurysms. A variety of constitutional symptoms and signs resulting from systemic vasculitis are present in the acute phase of the illness.Medical therapy for Kawasaki’s disease clearly decreases the manifestations of coronary artery involvement. Intravenous gamma globulin and aspirin therapy are most successful if begun within the first 10 days of illness. Up to 20% of untreated patients will develop coronary arterial lesions. A long-term, low-dose aspirin therapy regimen is usually recommended.Inflammatory Arteritis and VasculitisChronic inflammatory arteritis and vasculitis (i.e., inflamma-tory changes within veins as well as arteries) include a spec-trum of disease processes caused

1	Arteritis and VasculitisChronic inflammatory arteritis and vasculitis (i.e., inflamma-tory changes within veins as well as arteries) include a spec-trum of disease processes caused by immunologic mechanisms. These terms signify a necrotizing transmural inflammation of the vessel wall associated with antigen-antibody immune com-plex deposition within the endothelium. These conditions show pronounced cellular infiltration in the adventitia, thickened inti-mal fibrosis, and organized thrombus. These disease processes may clinically mimic atherosclerosis, and most are treated by corticosteroid therapy or chemotherapeutic agents. Even so, it is important to recognize distinguishing characteristics of each disease in order to establish the course of treatment and long-term prognosis. A classification system of systemic vasculitis by vessel size is shown in Table 23-28.Behçet’s DiseaseBehçet’s disease is a rare syndrome characterized by oral and genital ulcerations and ocular inflammation,

1	system of systemic vasculitis by vessel size is shown in Table 23-28.Behçet’s DiseaseBehçet’s disease is a rare syndrome characterized by oral and genital ulcerations and ocular inflammation, affecting males in Japan and the Mediterranean. An HLA linkage has been found, indicating a genetic component to the etiology.216 Vascular involvement is seen in 7% to 38% of patients and is localized Table 23-28Classification of vasculitis based on vessel involvementLarge-Vessel VasculitisTakayasu’s arteritisGiant cell arteritisBehçet’s diseaseMedium-Vessel VasculitisPolyarteritis nodosaKawasaki’s diseaseBuerger’s diseaseSmall-Vessel VasculitisHypersensitivity angiitisto the abdominal aorta, femoral artery, and pulmonary artery. Vascular lesions may also include venous complications such as deep venous thrombosis or superficial thrombophlebitis. Arterial aneurysmal degeneration can occur; however, this is an uncommon, albeit potentially devastating, complication. Mul-tiple true aneurysms and

1	venous thrombosis or superficial thrombophlebitis. Arterial aneurysmal degeneration can occur; however, this is an uncommon, albeit potentially devastating, complication. Mul-tiple true aneurysms and pseudoaneurysms may develop, and rupture of an aortic aneurysm is the major cause of death in patients with Behçet’s disease.217Histologically, degeneration of the vasa vasorum with surrounding perivascular lymphocyte infiltration is seen, along with thickening of the elastic laminae around the tunica media. Aneurysm formation is believed to be associated with a loss of the nutrient flow and elastic component of the vessels, lead-ing to progressive dilatation. Multiple aneurysms are relatively common, with a reported occurrence of 36% in affected Japanese patients.218 Furthermore, pseudoaneurysm formation after surgical bypass is common at anastomotic suture lines due to the vascular wall fragility and medial destruction. Systemic therapy with corticosteroids and immunosuppressive agents

1	formation after surgical bypass is common at anastomotic suture lines due to the vascular wall fragility and medial destruction. Systemic therapy with corticosteroids and immunosuppressive agents may diminish symptoms related to the inflammatory process; however, they have no effect on the rate of disease progression and arterial degeneration.Polyarteritis NodosaPolyarteritis nodosa (PAN) is another systemic inflammatory disease process, which is characterized by a necrotizing inflam-mation of medium-sized or small arteries that spares the small-est blood vessels (i.e., arterioles and capillaries). This disease predominantly affects men over women by a 2 to 1 ratio. PAN develops subacutely, with constitutional symptoms that last for weeks to months. Intermittent, low-grade fevers, malaise, weight loss, and myalgias are common presenting symptoms. As medium-sized vessels lie within the deep dermis, cutane-ous manifestations occur in the form of livedo reticularis, nod-ules,

1	malaise, weight loss, and myalgias are common presenting symptoms. As medium-sized vessels lie within the deep dermis, cutane-ous manifestations occur in the form of livedo reticularis, nod-ules, ulcerations, and digital ischemia.218 Skin biopsies of these lesions may be sufficient for diagnosis. Inflammation may be seen histologically, with pleomorphic cellular infiltrates and segmental transmural necrosis leading to aneurysm formation.Neuritis from nerve infarction occurs in 60% of patients, and gastrointestinal complications occur in up to 50%.219 Addi-tionally, renal involvement is found in 40% and manifests as microaneurysms within the kidney or segmental infarctions. Cardiac disease is a rare finding except at autopsy, where thickened, diseased coronary arteries may be seen, as well as patchy myocardial necrosis. Patients may succumb to renal fail-ure, intestinal hemorrhage, or perforation. End-organ ischemia from vascular occlusion or aneurysm rupture can be disastrous

1	as well as patchy myocardial necrosis. Patients may succumb to renal fail-ure, intestinal hemorrhage, or perforation. End-organ ischemia from vascular occlusion or aneurysm rupture can be disastrous Brunicardi_Ch23_p0897-p0980.indd 97127/02/19 4:15 PM 972SPECIFIC CONSIDERATIONSPART IIcomplications with high mortality rates. The mainstay of treat-ment is steroid and cytotoxic agent therapy. Up to 50% of patients with active PAN will experience remission with high dosing.Radiation-Induced ArteritisRadiation-induced arteritis results from progressive stenosis due to endothelial damage that leads to cellular proliferation and fibrosis. These are well-described complications of combined irradiation and chemotherapy for the treatment of head and neck malignancy. Arterial lesions are known complications of radia-tion and are similar to those found in atherosclerotic occlusive disease. A history of therapeutic irradiation to the neck can complicate the management of carotid artery

1	known complications of radia-tion and are similar to those found in atherosclerotic occlusive disease. A history of therapeutic irradiation to the neck can complicate the management of carotid artery occlusive disease. Radiation-induced damage to blood vessels has been well stud-ied. The small capillaries and sinusoids are most susceptible to radiation effects, as endothelial cells are the most radiosensi-tive cells. The radiation effects on the mediumand large-sized arteries include myointimal proliferation, with or without lipid deposits, and thrombosis. Characteristically, irregular spindle-shaped cells are seen replacing the normal endothelial cells in the healing phase. Occlusive lesions develop in the irradiated carotid arteries and are either the result of vessel wall fibrosis or, more commonly, due to accelerated atherosclerosis. Neuro-logic complications related to radiation-induced carotid artery disease are similar to those due to nonirradiated atherosclerotic occlusive

1	more commonly, due to accelerated atherosclerosis. Neuro-logic complications related to radiation-induced carotid artery disease are similar to those due to nonirradiated atherosclerotic occlusive disease.Rupture of the carotid artery has been reported following neck irradiation and is likely related to local wound compli-cation and superimposed infection. The diagnosis of radiation arteritis is based on the clinical history and confirmation of the occlusive lesion by duplex ultrasound, MRA, CTA, or sub-traction angiography. Irradiated lesions can be confined to the irradiated segment of the internal carotid artery with the remain-ing part of the vessel spared of disease. Characteristically, the radiation-induced atherosclerotic lesion does not involve the carotid bulb, unlike the nonradiated atherosclerotic lesions. The indications for intervention in radiation-induced carotid lesions are the same as previously discussed for atherosclerotic carotid occlusive lesions. However,

1	nonradiated atherosclerotic lesions. The indications for intervention in radiation-induced carotid lesions are the same as previously discussed for atherosclerotic carotid occlusive lesions. However, asymptomatic irradiated carotid artery lesions should be considered for intervention because they can be more prone to progression and develop-ment of neurologic complications. Endovascular treatment with carotid angioplasty/stenting has become the treatment of choice for radiation-induced lesions, although surgical endarterectomy and bypass have been shown to be safe. The rate of recurrent stenosis is higher in radiation-induced carotid lesions, whether stented or surgically treated.Raynaud’s SyndromeFirst described in 1862 by Maurice Reynaud, the term Raynaud’s syndrome applies to a heterogeneous symptom array associated with peripheral vasospasm, more commonly occurring in the upper extremities. The characteristically intermittent vasospasm classically follows exposure to various

1	symptom array associated with peripheral vasospasm, more commonly occurring in the upper extremities. The characteristically intermittent vasospasm classically follows exposure to various stimuli, including cold temperatures, tobacco, or emotional stress. Formerly, a distinc-tion was made between Raynaud’s “disease” and Raynaud’s “phenomenon” for describing a benign disease occurring in isolation or a more severe disease secondary to another under-lying disorder, respectively. However, many patients develop collagen vascular disorders at some point after the onset of vaso-spastic symptoms; progression to a connective tissue disorder ranges from 11% to 65% in reported series.220 Therefore, the term Raynaud’s syndrome is now used to encompass both the primary and secondary conditions.Characteristic color changes occur in response to the arterio-lar vasospasm, ranging from intense pallor to cyanosis to redness as the vasospasm occurs. The digital vessels then relax, even-tually leading

1	color changes occur in response to the arterio-lar vasospasm, ranging from intense pallor to cyanosis to redness as the vasospasm occurs. The digital vessels then relax, even-tually leading to reactive hyperemia. The majority of patients are young women less than 40 years of age. Up to 70% to 90% of reported patients are women, although many patients with only mild symptoms may never present for treatment.220 Geo-graphic regions with cooler, damp climates such as the Pacific Northwest and Scandinavian countries have a higher reported prevalence of the syndrome. Certain occupational groups, such as those who use vibrating tools, may be more predisposed to Raynaud’s syndrome or digital ischemia. The exact patho-physiologic mechanism behind the development of such severe vasospasm remains elusive, and much attention has focused on increased levels of α2-adrenergic receptors and their hypersen-sitivity in patients with Raynaud’s syndrome, as well as abnor-malities in the thermoregulatory

1	and much attention has focused on increased levels of α2-adrenergic receptors and their hypersen-sitivity in patients with Raynaud’s syndrome, as well as abnor-malities in the thermoregulatory response, which is governed by the sympathetic nervous system.The diagnosis of severe vasospasm may be made using noninvasive measurements in the vascular laboratory. Angiog-raphy is usually reserved for those who have digital ulceration and in whom an embolic or obstructive cause is believed to be present and potentially surgically correctable. Different changes in digital blood pressure will occur in patients with Raynaud’s syndrome. Normal individuals will show only a slight decrease in digital blood pressure in response to external cold stimuli, whereas those with Raynaud’s syndrome will show a similar curve until a critical temperature is reached. It is at this point that arterial closure acutely occurs.There is no cure for Raynaud’s syndrome; thus, all treat-ments mainly palliate symptoms

1	similar curve until a critical temperature is reached. It is at this point that arterial closure acutely occurs.There is no cure for Raynaud’s syndrome; thus, all treat-ments mainly palliate symptoms and decrease the severity and perhaps frequency of attacks. Conservative measures predomi-nate, including the wearing of gloves, use of electric or chemi-cally activated hand warmers, avoiding occupational exposure to vibratory tools, abstinence from tobacco, and relocating to a warmer, dryer climate. The majority (90%) of patients will respond to avoidance of cold and other stimuli. The remaining 10% of patients with more persistent or severe syndromes can be treated with a variety of vasodilatory drugs, albeit with only a 30% to 60% response rate. Calcium channel–blocking agents such as diltiazem and nifedipine are the drugs of choice. The selective serotonin reuptake inhibitor fluoxetine has been shown to reduce the frequency and duration of vasospastic episodes. Intravenous infusions

1	and nifedipine are the drugs of choice. The selective serotonin reuptake inhibitor fluoxetine has been shown to reduce the frequency and duration of vasospastic episodes. Intravenous infusions of prostaglandins have been reserved for nonresponders with severe symptoms.Surgical therapy is limited to debridement of digital ulcer-ations and amputation of gangrenous digits, which are rare complications. Upper extremity sympathectomy may provide relief in 60% to 70% of patients; however, the results are short-lived with a gradual recurrence of symptoms in 60% of patients within 10 years.221Fibromuscular DysplasiaFMD is a vasculopathy of uncertain etiology that is character-ized by segmental arterial involvement. Histologically, fibrous tissue proliferation, smooth muscle cell hyperplasia, and elastic fiber destruction alternate with mural thinning. The characteris-tic beaded appearance of FMD is due to areas of medial thinning alternating with areas of stenosis. The most commonly affected

1	elastic fiber destruction alternate with mural thinning. The characteris-tic beaded appearance of FMD is due to areas of medial thinning alternating with areas of stenosis. The most commonly affected Brunicardi_Ch23_p0897-p0980.indd 97227/02/19 4:15 PM 973ARTERIAL DISEASECHAPTER 23are medium-sized arteries, including the internal carotid, renal, vertebral, subclavian, mesenteric, and iliac arteries. The inter-nal carotid artery is the second most common site of involve-ment after the renal arteries. FMD occurs most frequently in women (90%) and is recognized at approximately 55 years of age.222 Only 10% of patients with FMD will have complications attributable to the disease. Pathologically, FMD is a heterog-enous group of four distinct types of lesions that are subgrouped based on the predominant site of involvement within the vessel wall. Of the four types (medial fibroplasia, intimal fibroplasia, medial hyperplasia, and perimedial dysplasia), medial fibropla-sia is the most

1	the predominant site of involvement within the vessel wall. Of the four types (medial fibroplasia, intimal fibroplasia, medial hyperplasia, and perimedial dysplasia), medial fibropla-sia is the most common pathologic type, affecting the internal carotid artery (ICA) and the renal artery, and occurring in 85% of reported cases.223The two main clinical syndromes associated with FMD are TIAs from disease in the internal carotid artery and hyper-tension from renal artery involvement. Symptoms produced by FMD are generally secondary to associated arterial stenosis and are clinically indistinguishable from those caused by atheroscle-rotic disease. Often, asymptomatic disease is found incidentally on conventional angiographic studies being performed for other reasons. Within the internal carotid artery, FMD lesions tend to be located higher in the extracranial segment than with athero-sclerotic lesions and may not be readily demonstrated by duplex scan.Clinically, symptoms are due to

1	carotid artery, FMD lesions tend to be located higher in the extracranial segment than with athero-sclerotic lesions and may not be readily demonstrated by duplex scan.Clinically, symptoms are due to encroachment on the ves-sel lumen and a reduction in flow. Additionally, thrombi may form in areas of mural dilatation from a stagnation of flow, lead-ing to distal embolization. Surgical treatment has been favored for symptomatic patients with angiographically proven disease. Due to the distal location of FMD lesions in the extracranial carotid artery, resection and repair are not usually feasible. Instead, graduated luminal dilatation under direct vision has been used successfully in patients, with antiplatelet therapy continued postoperatively. PTA has been used effectively in patients with FMD-induced hypertension. Several series have documented a high technical success rate, with recurrence rates of 8% to 23% at more than 1 year.223 However, the therapeutic effect of blood pressure

1	FMD-induced hypertension. Several series have documented a high technical success rate, with recurrence rates of 8% to 23% at more than 1 year.223 However, the therapeutic effect of blood pressure control may continue to be observed despite restenosis. Surgical reconstruction of the renal arteries for FMD has good long-term results and is recommended for recurrent lesions after angioplasty. Open balloon angioplasty of the ICA has been described, which allows for precise fluo-roscopic guidance, rather than blind dilatation with calibrated metal probes, and back-bleeding after dilatation to eliminate cerebral embolization. Distal neuroprotective devices may allow this procedure to be performed completely percutane-ously, thereby lessening the threat of cerebral emboli.Nonatherosclerotic Disease Affecting the Popliteal Artery DiseaseThere are three distinct nonatherosclerotic disease entities that may result in lower extremity claudication that predominantly occur in 40to 50-year-old

1	Affecting the Popliteal Artery DiseaseThere are three distinct nonatherosclerotic disease entities that may result in lower extremity claudication that predominantly occur in 40to 50-year-old men. Adventitial cystic disease, pop-liteal artery entrapment syndrome, and Buerger’s disease should be considered in any young patients presenting with intermittent claudication.Adventitial Cystic Disease of the Popliteal Artery. The first successful operative repair of popliteal artery occlusion caused by a cyst arising from the adventitia was reported in 1954 by Ejrup and Hierton.224 Adventitial cystic disease is a rare arterial condition occurring at an incidence of 0.1%, usually in the popliteal artery. This disease affects men in a ratio of approximately 5:1 and appears predominantly in the fourth and fifth decades. The incidence is approximately 1 in 1200 cases of claudication or 1 in 1000 peripheral arteriograms. The predominance of reported cases is found in Japan and Europe. However,

1	and fifth decades. The incidence is approximately 1 in 1200 cases of claudication or 1 in 1000 peripheral arteriograms. The predominance of reported cases is found in Japan and Europe. However, this disease may affect other vascular sites, such as the femoral, external iliac, radial, ulnar, and brachial arteries. Besides claudication as a symptom, this diagnosis should be considered in young patients who have a mass in a nonaxial ves-sel in proximity to a related joint. These synovial-like, mucin-filled cysts reside in the subadventitial layer of the vessel wall and have a similar macroscopic appearance to ganglion cysts. Despite this similarity and suggestion of a joint origin for these lesions, histochemical markers have failed to link the cystic lin-ing to synovium.Patients presenting at a young age with bilateral lower extremity claudication and minimal risk factors for atheroma formation should be evaluated for adventitial cystic disease, as well as the other two

1	presenting at a young age with bilateral lower extremity claudication and minimal risk factors for atheroma formation should be evaluated for adventitial cystic disease, as well as the other two nonatherosclerotic vascular lesions described here. Because of luminal encroachment and com-pression, peripheral pulses may be present in the limb when extended, but then can disappear during knee joint flexion. Noninvasive studies may suggest arterial stenosis with ele-vated velocities. Color-flow duplex scanning followed by T2-weighted MRI now appears to be the best diagnostic choice. Angiography will demonstrate a smooth, well-defined, crescent-shaped filling defect, the classic “scimitar” sign.224 There may be associated calcification in the cyst wall and no other evidence of atherosclerotic occlusive disease.Various therapeutic methods have been described for the treatment of adventitial cystic disease. The recommended treat-ments are excision of the cyst with the cystic wall,

1	occlusive disease.Various therapeutic methods have been described for the treatment of adventitial cystic disease. The recommended treat-ments are excision of the cyst with the cystic wall, enucleation, or simple aspiration when the artery is stenotic. Retention of the cystic lining leads to continued secretion of the cystic fluid and recurrent lesions. In 30% of patients who have an occluded artery, resection of the affected artery, followed by an interposi-tion graft using autogenous saphenous vein, is recommended.Popliteal Artery Entrapment Syndrome. Love and col-leagues first coined the term popliteal artery entrapment in 1965 to describe a syndrome combining muscular involvement with arterial ischemia occurring behind the knee, with the suc-cessful surgical repair having taken place 6 years earlier.225 This is a rare disorder with an estimated prevalence of 0.16% that occurs with a male-to-female ratio of 15:1. Five types of anatomic entrapment have been defined, according to the

1	years earlier.225 This is a rare disorder with an estimated prevalence of 0.16% that occurs with a male-to-female ratio of 15:1. Five types of anatomic entrapment have been defined, according to the posi-tion of the medial head of the gastrocnemius muscle, abnormal muscle slips or tendinous bands, or the course of the popliteal artery itself (Table 23-29). Concomitant popliteal vein impinge-ment occurs in up to 30% of cases. Twenty-five percent of cases are bilateral.The typical patient presents with swelling and claudica-tion of isolated calf muscle groups following vigorous physi-cal activity. Various differential diagnoses must be considered when encountering patients with symptoms and signs sugges-tive of popliteal artery entrapment syndrome (Table 23-30). In a large series of 240 patients, the median age for surgical treat-ment was 28.5 years.226 Noninvasive studies with ABIs should be performed with the knee extended and the foot in a neutral, forced plantar, and dorsiflexed

1	patients, the median age for surgical treat-ment was 28.5 years.226 Noninvasive studies with ABIs should be performed with the knee extended and the foot in a neutral, forced plantar, and dorsiflexed position. A drop in pressure of 50% or greater or dampening of the plethysmographic wave-forms in plantar or dorsiflexion is a classic finding. Contraction Brunicardi_Ch23_p0897-p0980.indd 97327/02/19 4:15 PM 974SPECIFIC CONSIDERATIONSPART IITable 23-29Classification of popliteal entrapment syndromeTYPEDESCRIPTIONIPopliteal artery is displaced medially around a normal medial head of the gastrocnemiusIIMedial head of gastrocnemius, which arises lateral to popliteal arteryIIIPopliteal artery is compressed by an accessory slip of muscle from medial head of gastrocnemiusIVEntrapment by a deeper popliteus muscleVAny of the above plus popliteal vein entrapmentVIFunctional entrapmentTable 23-30Differential diagnosis for popliteal entrapment syndromeVascular EtiologiesAtherosclerosisBuerger’s

1	popliteus muscleVAny of the above plus popliteal vein entrapmentVIFunctional entrapmentTable 23-30Differential diagnosis for popliteal entrapment syndromeVascular EtiologiesAtherosclerosisBuerger’s diseaseTraumaPopliteal aneurysmAdventitial cystic diseaseExtrinsic compressionCardiac embolismDeep vein thrombosisVenous entrapmentMusculoskeletal EtiologiesGastrocnemius or soleus strainPeriostitisCompartment syndromeStress fracturesTibialis posterior tendonitisMuscular anomaliesGeneral Neurologic EtiologiesSpinal stenosisof the gastrocnemius should compress the entrapped popliteal artery. The sudden onset of signs and symptoms of acute isch-emia with absent distal pulses is consistent with popliteal artery occlusion secondary to entrapment. Other conditions resulting from entrapment are thrombus formation with distal emboli or popliteal aneurysmal degeneration. Although CT and MRI have been employed, angiography remains the most widely used test. Angiography performed with the foot in a

1	formation with distal emboli or popliteal aneurysmal degeneration. Although CT and MRI have been employed, angiography remains the most widely used test. Angiography performed with the foot in a neutral position may demonstrate classical medial deviation of the popliteal artery or normal anatomic positioning. Coexisting abnormalities may include stenosis, luminal irregularity, delayed flow, aneurysm, or complete occlusion. Diagnostic accuracy is increased with the use of ankle stress view-active plantar flexion and passive dorsiflexion.The treatment of popliteal artery entrapment consists of surgical decompression of the impinged artery with possible arterial reconstruction. Division of the anomalous musculoten-dinous insertion site with or without saphenous vein interposi-tion grafting to bypass the damaged arterial segment has been described to be the procedure of choice. The natural history of entrapment is progressive arterial degeneration leading to com-plete arterial thrombosis.

1	bypass the damaged arterial segment has been described to be the procedure of choice. The natural history of entrapment is progressive arterial degeneration leading to com-plete arterial thrombosis. In such instances, thrombolytic ther-apy is needed with subsequent release of the functional arterial impairment. Lysis will improve distal runoff and may improve limb-salvage and bypass patency rates.Buerger’s Disease (Thromboangiitis Obliterans)Buerger’s disease, also known as thromboangiitis obliterans, is a progressive nonatherosclerotic segmental inflammatory disease that most often affects smalland medium-sized arteries, veins, and nerves of the upper and lower extremities. The clinical and pathologic findings of this disease entity were published in 1908 by Leo Buerger in a description of 11 amputated limbs.227 The typical age range for occurrence is 20 to 50 years, and the dis-order is more frequently found in males who smoke. The upper extremities may be involved, and a migratory

1	of 11 amputated limbs.227 The typical age range for occurrence is 20 to 50 years, and the dis-order is more frequently found in males who smoke. The upper extremities may be involved, and a migratory superficial phlebi-tis may be present in up to 16% of patients, thus indicating a sys-temic inflammatory response. In young adults presenting to the Mayo Clinic (1953–1981) with lower limb ischemia, Buerger’s disease was diagnosed in 24%.228 Conversely, the diagnosis was made in 9% of patients with ischemic finger ulcerations. The cause of thromboangiitis obliterans is unknown; however, use of or exposure to tobacco is essential to both the diagnosis and progression of the disease.Pathologically, thrombosis occurs in smallto medium-sized arteries and veins with associated dense polymorphonu-clear leukocyte aggregation, microabscesses, and multinucleated giant cells. The chronic phase of the disease shows a decrease in the hypercellularity and frequent recanalization of the vessel lumen.

1	leukocyte aggregation, microabscesses, and multinucleated giant cells. The chronic phase of the disease shows a decrease in the hypercellularity and frequent recanalization of the vessel lumen. End-stage lesions demonstrate organized thrombus and blood vessel fibrosis. Although the disease is common in Asia, North American males do not appear to have any particular pre-disposition, as the diagnosis is made in less than 1% of patients with severe limb ischemia.Buerger’s disease typically presents in young male smok-ers, with symptoms beginning prior to age 40. Patients initially present with foot, leg, arm, or hand claudication, which may be mistaken for joint or neuromuscular problems. Progression of the disease leads to calf claudication and eventually ischemic rest pain and ulcerations on the toes, feet, or fingers. A complete history should exclude diabetes, hyperlipidemia, or autoimmune disease as possible etiologies for the occlusive lesions. Because it is likely that multiple

1	on the toes, feet, or fingers. A complete history should exclude diabetes, hyperlipidemia, or autoimmune disease as possible etiologies for the occlusive lesions. Because it is likely that multiple limbs are involved, angiography should be performed of all four limbs. Even if symptoms are not yet present in a limb, angiographic findings may be demonstrated. Characteristic angiographic findings show disease confinement to the distal circulation, usually infrapopliteal and distal to the brachial artery. The occlusions are segmental and show “skip” lesions with extensive collateralization, the so-called corkscrew collaterals.The treatment of thromboangiitis obliterans revolves around strict smoking cessation. In patients who are able to abstain, disease remission is impressive, and amputation avoid-ance is increased. In the experience reported from the Oregon Health Sciences Center, no disease progression with associated tissue loss occurred after discontinuation of tobacco. The role of

1	avoid-ance is increased. In the experience reported from the Oregon Health Sciences Center, no disease progression with associated tissue loss occurred after discontinuation of tobacco. The role of surgical intervention is minimal in Buerger’s disease, as there is often no acceptable target vessel for bypass. Furthermore, autog-enous vein conduits are limited secondary to coexisting migra-tory thrombophlebitis. Mills and associates reported their results Brunicardi_Ch23_p0897-p0980.indd 97427/02/19 4:15 PM 975ARTERIAL DISEASECHAPTER 23of 31% limb loss in 26 patients over 15 years, thus authenti-cating the virulence of Buerger’s disease involving the lower extremities.229 In addition, others have described a significant discrepancy in limb loss in patients who continued to smoke versus those who discontinued tobacco use (67% vs. 35%).REFERENCESEntries highlighted in bright blue are key references. 1. Norgren L, Hiatt WR, Dormandy JA, et al. Inter-society consensus for the

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1	BH, Sullivan TM, Childs MB, Young JR, Olin JW. High incidence of restenosis/reocclusion of stents in the percutaneous treatment of long-segment superficial femo-ral artery disease after suboptimal angioplasty. J Vasc Surg. 1997;25(1):74-83. 160. Mewissen MW. Self-expanding nitinol stents in the femoro-popliteal segment: technique and mid-term results. Tech Vasc Interv Radiol. 2004;7(1):2-5. 161. Laird JR. Interventional options in SFA. Endovascular Today. 2004;(supp):9-12. 162. Ansel GM, Silver MJ, Botti CF Jr, et al. Functional and clini-cal outcomes of nitinol stenting with and without abciximab for complex superficial femoral artery disease: a randomized trial. Catheter Cardiovasc Interv. 2006;67(2):288-297. 163. Duda SH, Poerner TC, Wiesinger B, et al. Drug-eluting stents: potential applications for peripheral arterial occlusive disease. J Vasc Interv Radiol. 2003;14(3):291-301. 164. Feiring AJ, Krahn M, Nelson L, Wesolowski A, Eastwood D, Szabo A. Preventing leg amputations in

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1	16. 167. McQuade K, Gable D, Pearl G, Theune B, Black S. Four-year randomized prospective comparison of percutaneous ePTFE/nitinol self-expanding stent graft versus prosthetic femoral-popliteal bypass in the treatment of superficial femoral artery occlusive disease. J Vasc Surg. 2010;52(3):584-590; discus-sion 590-581, 591 e581-591 e587. 168. Ramaiah V, Gammon R, Kiesz S, et al. Midterm outcomes from the TALON Registry: treating peripherals with SilverHawk: outcomes collection. J Endovasc Ther. 2006;13(5):592-602. 169. Franzone A, Ferrone M, Carotenuto G, et al. The role of ather-ectomy in the treatment of lower extremity peripheral artery disease. BMC Surg. 2012;12(suppl 1):S13. 170. Scheinert D, Laird JR Jr, Schroder M, Steinkamp H, Balzer JO, Biamino G. Excimer laser-assisted recanalization of long, chronic superficial femoral artery occlusions. J Endovasc Ther. 2001;8(2):156-166. 171. Steinkamp HJ, Rademaker J, Wissgott C, et al. Percutane-ous transluminal laser angioplasty versus

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1	arterial obstruc-tions: midterm results of a prospective trial. J Vasc Interv Radiol. 2003;14(1):41-51. 180. Grubnic S, Heenan SD, Buckenham TM, Belli AM. Evalu-ation of the pullback atherectomy catheter in the treatment of lower limb vascular disease. Cardiovasc Intervent Radiol. 1996;19(3):152-159. 181. Savader SJ, Venbrux AC, Mitchell SE, et al. Percutaneous transluminal atherectomy of the superficial femoral and pop-liteal arteries: long-term results in 48 patients. Cardiovasc Intervent Radiol. 1994;17(6):312-318. 182. Albers M, Battistella VM, Romiti M, Rodrigues AA, Pereira CA. Meta-analysis of polytetrafluoroethylene bypass grafts to infrapopliteal arteries. J Vasc Surg. 2003;37(6):1263-1269. 183. Hamsho A, Nott D, Harris PL. Prospective randomised trial of distal arteriovenous fistula as an adjunct to femoro-infrap-opliteal PTFE bypass. Eur J Vasc Endovasc Surg. 1999;17(3): 197-201. 184. Davies AH, Hawdon AJ, Sydes MR, Thompson SG. Is duplex surveillance of value after leg

1	fistula as an adjunct to femoro-infrap-opliteal PTFE bypass. Eur J Vasc Endovasc Surg. 1999;17(3): 197-201. 184. Davies AH, Hawdon AJ, Sydes MR, Thompson SG. Is duplex surveillance of value after leg vein bypass grafting? Princi-pal results of the Vein Graft Surveillance Randomised Trial (VGST). Circulation. 2005;112(13):1985-1991. 185. Baldwin ZK, Pearce BJ, Curi MA, et al. Limb salvage after infrainguinal bypass graft failure. J Vasc Surg. 2004;39(5): 951-957. 186. Stone PA, Flaherty SK, Aburahma AF, et al. Factors affect-ing perioperative mortality and wound-related complications following major lower extremity amputations. Ann Vasc Surg. 2006;20(2):209-216. 187. Holzenbein TJ, Pomposelli FB Jr, Miller A, et al. The upper arm basilic-cephalic loop for distal bypass grafting: techni-cal considerations and follow-up. J Vasc Surg. 1995;21(4): 586-592; discussion 592-594. 188. Dardik H, Wengerter K, Qin F, et al. Comparative decades of experience with glutaraldehyde-tanned human

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1	of infrainguinal vascular allografts. J Vasc Surg. 2006;44(3):518-524. 191. Gupta SK, Veith FJ, Kram HB, Wengerter KR. Prospective, randomized comparison of ringed and nonringed polytetra-fluoroethylene femoropopliteal bypass grafts: a preliminary report. J Vasc Surg. 1991;13(1):163-172. 192. Stonebridge PA, Prescott RJ, Ruckley CV. Randomized trial comparing infrainguinal polytetrafluoroethylene bypass graft-ing with and without vein interposition cuff at the distal anas-tomosis. The Joint Vascular Research Group. J Vasc Surg. 1997;26(4):543-550. 193. Klinkert P, van Dijk PJ, Breslau PJ. Polytetrafluoroethylene femorotibial bypass grafting: 5-year patency and limb salvage. Ann Vasc Surg. 2003;17(5):486-491. 194. Panneton JM, Hollier LH, Hofer JM. Multicenter randomized prospective trial comparing a pre-cuffed polytetrafluoroethyl-ene graft to a vein cuffed polytetrafluoroethylene graft for infra-genicular arterial bypass. Ann Vasc Surg. 2004;18(2):199-206. 195. Bellosta R, Luzzani L,

1	a pre-cuffed polytetrafluoroethyl-ene graft to a vein cuffed polytetrafluoroethylene graft for infra-genicular arterial bypass. Ann Vasc Surg. 2004;18(2):199-206. 195. Bellosta R, Luzzani L, Carugati C, Melloni C, Sarcina A. Which distal anastomosis should be used in PTFE femoro-tibial bypass? J Cardiovasc Surg (Torino). 2005;46(5):499-503. 196. Begovac PC, Thomson RC, Fisher JL, Hughson A, Gallhagen A. Improvements in GORE-TEX vascular graft performance by Carmeda BioActive surface heparin immobilization. Eur J Vasc Endovasc Surg. 2003;25(5):432-437. 197. Devine C, Hons B, McCollum C. Heparin-bonded Dacron or polytetrafluoroethylene for femoropopliteal bypass grafting: a multicenter trial. J Vasc Surg. 2001;33(3):533-539. 198. Walluscheck KP, Bierkandt S, Brandt M, Cremer J. Infrain-guinal ePTFE vascular graft with bioactive surface heparin bonding. First clinical results. J Cardiovasc Surg (Torino). 2005;46(4):425-430. 199. Hunink MG, Donaldson MC, Meyerovitz MF, et al. Risks and

1	ePTFE vascular graft with bioactive surface heparin bonding. First clinical results. J Cardiovasc Surg (Torino). 2005;46(4):425-430. 199. Hunink MG, Donaldson MC, Meyerovitz MF, et al. Risks and benefits of femoropopliteal percutaneous balloon angioplasty. J Vasc Surg. 1993;17(1):183-192; discussion 192-194. 200. Dua A, Koprowski S, Upchurch G, Lee CJ, Desai SS. Pro-gressive shortfall in open aneurysm experience for vascular surgery trainees with the impact of fenestrated and branched endovascular technology. J Vasc Surg. 2017;65(1):257-261. 201. Ferreira M, Lanziotti L, Monteiro M, et al. Superficial femo-ral artery recanalization with self-expanding nitinol stents: long-term follow-up results. Eur J Vasc Endovasc Surg. 2007; 34(6):702-708. 202. Schillinger M, Sabeti S, Dick P, et al. Sustained benefit at 2 years of primary femoropopliteal stenting compared with balloon angioplasty with optional stenting. Circulation. 2007;115(21):2745-2749. 203. Bosiers M, Deloose K, Verbist J,

1	Sustained benefit at 2 years of primary femoropopliteal stenting compared with balloon angioplasty with optional stenting. Circulation. 2007;115(21):2745-2749. 203. Bosiers M, Deloose K, Verbist J, Peeters P. Nitinol stenting for treatment of “below-the-knee” critical limb ischemia: 1-year angiographic outcome after Xpert stent implantation. J Car-diovasc Surg (Torino). 2007;48(4):455-461. 204. Kickuth R, Keo HH, Triller J, Ludwig K, Do DD. Initial clinical experience with the 4-F self-expanding XPERT stent system for infrapopliteal treatment of patients with severe claudication and critical limb ischemia. J Vasc Interv Radiol. 2007;18(6):703-708. 205. Wolf GL, Wilson SE, Cross AP, Deupree RH, Stason WB. Sur-gery or balloon angioplasty for peripheral vascular disease: a randomized clinical trial. Principal investigators and their Associates of Veterans Administration Cooperative Study Number 199. J Vasc Interv Radiol. 1993;4(5):639-648. 206. Adam DJ, Beard JD, Cleveland T, et al.

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1	Venous and Lymphatic DiseaseAtish Chopra, Timothy K. Liem, and Gregory L. Moneta 24chapterVENOUS ANATOMYVeins are part of a dynamic and complex system that returns low-nutrient deoxygenated blood to the heart. Venous blood flow is dependent on multiple factors such as gravity, venous valves, the cardiac and respiratory cycles, blood volume, and the calf muscle and feet pumps. Alterations in the intricate balance of these factors can result in venous pathology.Structure of VeinsVeins are thin-walled, highly distensible, and collapsible. Their structure specifically supports the primary functions of veins to transport blood toward the heart and serve as a reservoir to prevent intravascular volume overload.The venous intima is composed of a nonthrombogenic endothelium with an underlying basement membrane and an elastic lamina. The endothelium produces endothelium-derived relaxing factors such as nitric oxide and prostacy-clin, which help maintain a nonthrombogenic surface through

1	basement membrane and an elastic lamina. The endothelium produces endothelium-derived relaxing factors such as nitric oxide and prostacy-clin, which help maintain a nonthrombogenic surface through inhibition of platelet aggregation and promotion of platelet disaggregation.1 The capacitance function of veins is facili-tated by circumferential rings of elastic tissue, and smooth muscle located in the media of the vein allows for changes in vein caliber with minimal changes in venous pressure. The adventitia is most prominent in large veins and consists of collagen, elastic fibers, and fibroblasts. When a vein is maxi-mally distended, its diameter may be several times greater than that in the supine position.In the axial veins, unidirectional blood flow is achieved with multiple venous valves. The inferior vena cava (IVC), common iliac veins, portal venous system, and cranial sinuses are valveless. In the axial veins, valves are more numerous distally in the extremities than proximally.

1	The inferior vena cava (IVC), common iliac veins, portal venous system, and cranial sinuses are valveless. In the axial veins, valves are more numerous distally in the extremities than proximally. Each valve consists of two thin cusps of a fine connective tissue skeleton covered by endothelium. Venous valves close in response to cephalad-to-caudal blood flow at a velocity of at least 30 cm/s.2Lower Extremity VeinsLower extremity veins are divided into superficial, deep, and perforating veins. The superficial venous system lies above the uppermost fascial layer of the leg and thigh and consists of the great saphenous vein (GSV) and small saphenous vein (SSV) and their tributaries. The GSV originates from the dorsal pedal venous arch and courses cephalad and medially, anterior to the medial malleolus, entering the common femoral vein approxi-mately 4 cm inferior and lateral to the pubic tubercle. The saphe-nous nerve accompanies the GSV medially from the ankle to the level of the knee

1	malleolus, entering the common femoral vein approxi-mately 4 cm inferior and lateral to the pubic tubercle. The saphe-nous nerve accompanies the GSV medially from the ankle to the level of the knee and supplies cutaneous sensation to the medial leg and ankle. The SSV originates laterally from the dorsal pedal venous arch and courses cephalad in the posterior calf. Most often, it penetrates the popliteal fossa, between the medial and lateral heads of the gastrocnemius muscle, to join the popliteal vein. The termination of the SSV may be quite vari-able, however, with a proximal extension of the SSV (the vein of Giacomini) connecting with the deep femoral vein or GSV. The sural nerve accompanies the SSV laterally along its course and supplies cutaneous sensation to the lateral malleolar region.The deep veins follow the course of major arteries in the extremities. In the lower leg, paired veins parallel the course of the anterior tibial, posterior tibial, and peroneal arteries, to join

1	deep veins follow the course of major arteries in the extremities. In the lower leg, paired veins parallel the course of the anterior tibial, posterior tibial, and peroneal arteries, to join behind the knee forming the popliteal vein. Venous bridges con-nect the paired axial tibial veins in the lower leg. The popliteal vein continues through the adductor hiatus to become the femo-ral vein. In the proximal thigh, the femoral vein joins with the deep femoral vein to form the common femoral vein, becoming the external iliac vein at the inguinal ligament.Multiple perforator veins traverse the deep fascia to connect the superficial and deep venous systems. Potentially clinically important perforator veins are the posterior tibial and paratibial perforators (formerly known as the Cockett and Boyd perforators, respectively). The posterior tibial perforator veins drain the medial lower leg and are relatively constant. They Venous Anatomy981Structure of Veins / 981Lower Extremity Veins /

1	Boyd perforators, respectively). The posterior tibial perforator veins drain the medial lower leg and are relatively constant. They Venous Anatomy981Structure of Veins / 981Lower Extremity Veins / 981Upper Extremity Veins / 982Evaluation of the Venous System982Clinical Evaluation / 982Venous Thromboembolism984Epidemiology / 984Risk Factors / 984Diagnosis / 986Treatment / 987Prophylaxis / 992Other Venous Thrombotic Disorders994Superficial Vein Thrombophlebitis / 994Upper Extremity Vein Thrombosis / 995Mesenteric Vein Thrombosis / 996Varicose Veins996Chronic Venous Insufficiency997Evaluation of Venous Insufficiency / 997Lymphedema1001Pathophysiology / 1001Clinical Diagnosis / 1001Radiologic Diagnosis / 1002Management / 1002Summary1003Brunicardi_Ch24_p0981-p1008.indd 98122/02/19 3:00 PM 982connect the posterior accessory GSV (formerly known as the posterior arch vein, a tributary to the GSV) and the posterior tibial vein. They may become varicose or incompetent in venous

1	3:00 PM 982connect the posterior accessory GSV (formerly known as the posterior arch vein, a tributary to the GSV) and the posterior tibial vein. They may become varicose or incompetent in venous insufficiency states. The posterior accessory GSV has relevance as it represents a connection of the three ankle perforating veins, which are likely of particular importance in the development of a venous stasis ulcers. The paratibial perforator veins connect the GSV to the deep veins approximately 10 cm below the knee and 1 to 2 cm medial to the tibia. Additional perforators in the thigh are known as the perforators of the femoral canal (also known as Hunter’s and Dodd’s perforators).Venous sinuses are thin-walled, large veins located within the substance of the soleus and gastrocnemius muscles. These sinuses are valveless and are linked by valved, small venous channels that prevent reflux. A large amount of blood can be stored in the venous sinuses before draining into the posterior

1	These sinuses are valveless and are linked by valved, small venous channels that prevent reflux. A large amount of blood can be stored in the venous sinuses before draining into the posterior tibial and peroneal veins. With each contraction of the calf muscle bed, blood is pumped out through the venous channels into the main conduit veins to return to the heart.Upper Extremity VeinsAs in the lower extremity, there are deep and superficial veins in the upper extremity. Deep digital veins form the palmar venous arches of the hand and empty into the paired radial and ulnar veins. These follow the named arteries in the arm and are known as the venae comitantes. They become the brachial veins most often near the antecubital fossa and then combine to contrib-ute to forming the axillary vein. Superficial veins of the upper extremity are the cephalic and basilic veins and their tributaries. The cephalic vein originates at the lateral wrist and courses over the lateral ventral surface of the

1	veins of the upper extremity are the cephalic and basilic veins and their tributaries. The cephalic vein originates at the lateral wrist and courses over the lateral ventral surface of the forearm. In the upper arm, the cephalic vein terminates in the infraclavicular fossa, piercing the clavipectoral fascia to empty into the axillary vein. The basilic vein runs medially along the forearm and penetrates the deep fascia as it courses past the elbow in the upper arm. It then joins with the deep brachial veins to become the axil-lary vein, a landmark for identification of the axillary vein. The median antecubital vein joins the cephalic and the basilic veins on the ventral surface of the elbow.The axillary vein becomes the subclavian vein at the lat-eral border of the first rib. At the medial border of the scalenus anterior muscle, the subclavian vein joins with the internal jugu-lar vein to become the brachiocephalic vein, with the subclavian vein coursing anterior to the scalenus

1	medial border of the scalenus anterior muscle, the subclavian vein joins with the internal jugu-lar vein to become the brachiocephalic vein, with the subclavian vein coursing anterior to the scalenus anterior muscle. The left and right brachiocephalic veins join to become the superior vena cava, which empties into the right atrium.EVALUATION OF THE VENOUS SYSTEMClinical EvaluationEvaluation of the venous system begins with a detailed history and physical examination. Risk factors for acute and chronic venous disease are identified. They include increased age, his-tory of venous thromboembolism (VTE), malignancy, trauma and spinal cord injury, hospitalization and immobilization, obe-sity, nephrotic syndrome, pregnancy, recent postpartum state, oral contraceptive use or hormone replacement therapy, vari-cose veins, and hypercoagulable states, as well as the postopera-tive state. Venous pathology is often, but not always, associated with visible or palpable signs that can be identified

1	therapy, vari-cose veins, and hypercoagulable states, as well as the postopera-tive state. Venous pathology is often, but not always, associated with visible or palpable signs that can be identified during the physical examination. There is variation among individuals in the prominence of superficial veins when the person is standing (Fig. 24-1). The superficial veins of a lean athletic person, even when normal, will appear large and easily visualized, but these veins will be far less obvious in the obese individual. Signs of superficial venous abnormalities are listed in Table 24-1. Key Points1 Thrombolytic therapy, surgical thrombectomy, and place-ment of inferior vena cava filters are adjunctive treatments that may be indicated in patients with extensive and compli-cated venous thromboembolism.2 Deep vein thrombosis (DVT) and pulmonary embolism are well-recognized complications after major abdominal and orthopedic procedures. The risk is further increased in patients with

1	thromboembolism.2 Deep vein thrombosis (DVT) and pulmonary embolism are well-recognized complications after major abdominal and orthopedic procedures. The risk is further increased in patients with malignancy and a history of venous thrombo-embolism. Options for DVT prophylaxis include intermit-tent pneumatic compression, use of graduated compression stockings, and administration of low-dose unfractionated heparin, low molecular weight heparin, fondaparinux, and vitamin K antagonists. Direct thrombin inhibitors and factor Xa inhibitors are approved for prophylactic use only for orthopedic procedures and for recurrent VTE. However, prophylaxis should be stratified based on the patient’s level of risk.3 In patients with established DVT, unfractionated heparin, low molecular weight heparin, fondaparinux, and some factor Xa inhibitors are options for initial antithrombotic therapy. Vitamin-K antagonists, direct thrombin inhibitors, and factor Xa inhibitors are utilized for long-term

1	fondaparinux, and some factor Xa inhibitors are options for initial antithrombotic therapy. Vitamin-K antagonists, direct thrombin inhibitors, and factor Xa inhibitors are utilized for long-term anticoagulation. The duration and type of long-term anticoagulation should be stratified based on the provoked or unprovoked nature of the DVT, the location of the DVT, previous occurrence of DVT, and presence of concomitant malignancy.4 High ligation and stripping, endovenous laser, or radiofre-quency ablation and sclerotherapy are effective therapies for patients with saphenous vein valvular insufficiency. Con-comitant varicose veins may be managed with compression therapy, sclerotherapy, and phlebectomy. New nonthermal ablative techniques, including the combination of sclero-therapy with endoluminal mechanical injury as well as injec-tion of cyanoacrylate, show early promising results.5 The mainstay of treatment for chronic venous insufficiency is compression therapy. Sclerotherapy,

1	endoluminal mechanical injury as well as injec-tion of cyanoacrylate, show early promising results.5 The mainstay of treatment for chronic venous insufficiency is compression therapy. Sclerotherapy, perforator vein liga-tion, and venous reconstruction or ablative techniques may be indicated in patients in whom conservative management fails or as a means to decrease ulcer recurrence.6 Lymphedema is categorized as congenital, primary (with early or delayed onset), or secondary. The goals of treatment are to minimize edema and prevent infection. Lymphatic massage, sequential pneumatic compression, use of com-pression garments, and limb elevation are effective forms of therapy.Brunicardi_Ch24_p0981-p1008.indd 98222/02/19 3:00 PM 983VENOUS AND LYMPHATIC DISEASECHAPTER 24Figure 24-1. Varicose vein demonstrating evidence of chronic venous insufficiency.Figure 24-2. Characteristic hyperpigmentation of chronic venous insufficiency.Table 24-1Possible signs of superficial venous

1	24-1. Varicose vein demonstrating evidence of chronic venous insufficiency.Figure 24-2. Characteristic hyperpigmentation of chronic venous insufficiency.Table 24-1Possible signs of superficial venous abnormalitiesTortuosityVaricosityVenous sacculeDistended subdermal venules (corona phlebectatica)Distended intradermal venules (spider angiomata)Warmth, erythema, tenderness (superficial thrombophlebitis)The deep veins cannot be directly assessed clinically, and abnormalities within them can only be inferred indirectly from changes found on clinical examination.Chronic venous insufficiency (CVI) may lead to character-istic changes in the skin and subcutaneous tissues in the affected limb. CVI results from incompetence of venous valves, venous obstruction, or both. Most CVI involves venous reflux, and severe CVI often reflects a combination of reflux and venous obstruction. It is important to remember that although CVI originates with abnormalities of the veins, the target organ of CVI is

1	and severe CVI often reflects a combination of reflux and venous obstruction. It is important to remember that although CVI originates with abnormalities of the veins, the target organ of CVI is the skin, and the underlying physiologic and biochemical mechanisms leading to the cutaneous abnormalities associated with CVI are poorly understood. A typical leg affected by CVI will be edematous, with edema increasing over the course of the day. The leg may also be indurated and pigmented with eczema and dermatitis. These changes are associated with excessive proteinaceous capillary exudate. Deposition of a pericapil-lary fibrin cuff may limit nutritional exchange. In addition, an increase in white blood cell trapping within the skin microcir-culation in CVI patients may lead to microvascular congestion and thrombosis. Subsequently, white blood cells may migrate into the interstitium and release necrotizing lysosomal enzymes, potentially leading to tissue destruction and eventual

1	congestion and thrombosis. Subsequently, white blood cells may migrate into the interstitium and release necrotizing lysosomal enzymes, potentially leading to tissue destruction and eventual ulceration.Fibrosis can eventually develop from impaired nutrition, chronic inflammation, and fat necrosis (lipodermatosclerosis). Hemosiderin deposition due to the extravasation of red cells and subsequent lysis in the skin contributes to the characteristic pigmentation of chronic venous disease (Fig. 24-2). Ulceration can develop with longstanding venous hypertension and is associated with alterations in microcirculatory and cutaneous lymphatic anatomy and function. The most common location of venous ulceration is approximately 3 cm proximal to the medial malleolus, frequently referred to the “gaiter” region (Fig. 24-3).Trendelenburg’s test is a clinical test, historically impor-tant but now rarely used, that can help determine whether incompetent valves are present and in which of the three

1	region (Fig. 24-3).Trendelenburg’s test is a clinical test, historically impor-tant but now rarely used, that can help determine whether incompetent valves are present and in which of the three venous systems (superficial, deep, or perforator) the valves are abnormal. There are two components to this test. First, with the patient supine, the leg is elevated 45° to empty the veins, and the GSV is occluded with the examiner’s hand or with a rub-ber tourniquet. Then, with the GSV still occluded, the patient stands, and the superficial veins are observed for blood filling. The compression on the GSV is released, and the superficial veins are observed for filling with blood. A positive result is the sudden filling of veins with standing while the GSV remains occluded, indicating incompetent perforator and deep veins. Additionally, the GSV valves are incompetent if rapid filling is noted following release of compression. A negative result, indicating no clinically relevant venous reflux, is

1	and deep veins. Additionally, the GSV valves are incompetent if rapid filling is noted following release of compression. A negative result, indicating no clinically relevant venous reflux, is the gradual filling of the veins from arterial inflow. Interpretation of the findings of Trendelenburg’s test is subjective, and therefore, it has largely been supplanted by the more objective noninvasive vascular laboratory tests to localize sites of venous reflux.Noninvasive Evaluation. Before the development of vascu-lar ultrasound, noninvasive techniques to evaluate the venous system were based on plethysmographic techniques. Although a variety of plethysmographic techniques are used in the Brunicardi_Ch24_p0981-p1008.indd 98322/02/19 3:01 PM 984SPECIFIC CONSIDERATIONSPART IIFigure 24-3. Venous ulceration located proximal to the medial malleolus.evaluation of both acute and chronic venous disease, they are all based on the detection of volume changes in the limb in response to blood

1	ulceration located proximal to the medial malleolus.evaluation of both acute and chronic venous disease, they are all based on the detection of volume changes in the limb in response to blood flow.Duplex ultrasonography (DUS) augmented by color flow imaging is now the most important noninvasive diagnos-tic method in the evaluation of the venous system. DUS has become standard for the detection of infrainguinal deep vein thrombosis (DVT), with near 100% sensitivity and specificity in symptomatic patients.3 It is also the preferred method of evalu-ation for upper extremity venous thrombosis and is useful in the evaluation of CVI by documenting the presence of valvular reflux and venous obstruction. Overlying bowel gas and large body habitus many times make DUS less applicable to evalu-ation of intra-abdominal veins. Magnetic resonance venogra-phy (MRV) and computed tomography (CT) venography are alternative noninvasive techniques for evaluation of pelvic and intra-abdominal

1	evalu-ation of intra-abdominal veins. Magnetic resonance venogra-phy (MRV) and computed tomography (CT) venography are alternative noninvasive techniques for evaluation of pelvic and intra-abdominal veins.Invasive Evaluation. Improved accuracy of noninvasive techniques for diagnostic purposes has made the use of invasive procedures more selective. Both venography and intravascular ultrasound (IVUS) are used as adjuncts to percutaneous or open surgical treatment of venous disorders. When planning endo-vascular or open surgical treatment, venography may be used to identify areas of obstruction in infrainguinal, intra-abdominal, and upper extremity veins as well as reflux in intra-abdominal and infrainguinal veins. IVUS, with access generally via the common femoral vein, is used primarily to assess for occlusive lesions of the iliac veins and appears more sensitive than venog-raphy in detecting iliac vein obstruction.4Complications of venography include pain, thrombosis, or hematoma at

1	assess for occlusive lesions of the iliac veins and appears more sensitive than venog-raphy in detecting iliac vein obstruction.4Complications of venography include pain, thrombosis, or hematoma at the puncture site. Pain is lower with nonionic low-osmolality contrast media than with conventional contrast agents (with 18% vs. 44% of patients experiencing discomfort, respectively).5 Systemic effects of iodinated contrast media include allergic reaction and risk of renal failure. Postvenogra-phy venous thrombosis occurs distal to the venous puncture site in 1% to 9% of patients undergoing venography secondary to intimal damage from the intravenous (IV) contrast agent.5 Com-plications and limitations of IVUS are related to complications at the access site and cost of the catheters.VENOUS THROMBOEMBOLISMEpidemiologyDespite increased awareness and use of prophylactic modalities, DVT or pulmonary embolism (PE), venous thromboembolism (VTE), remain important preventable sources of morbidity

1	increased awareness and use of prophylactic modalities, DVT or pulmonary embolism (PE), venous thromboembolism (VTE), remain important preventable sources of morbidity and mortality, especially in the surgical patient. The incidence of VTE is approximately 100 per 100,000 people per year in the general population, with 20% of the diagnoses made within 3 months of a surgical procedure. Of the symptomatic patients, one-third will present with PE and two-thirds with DVT.6,7 The estimated number of cases of VTE may well be over 600,000 per year in the United States, making it a major U.S. health problem.8 Furthermore, death occurs in 6% of DVT and 12% of PE cases within 1 month of diagnosis, although not all deaths are directly secondary to VTE, with many related to the under-lying problem leading to the VTE event.6 However, not only does VTE pose a veritable threat to life, it also places patients at higher risk for recurrence and post-VTE sequelae such as pul-monary hypertension and

1	leading to the VTE event.6 However, not only does VTE pose a veritable threat to life, it also places patients at higher risk for recurrence and post-VTE sequelae such as pul-monary hypertension and postthrombotic syndrome, with 4% and up to 30% incidence, respectively.9-11Risk FactorsThree broadly stated conditions, first described by Rudolf Vir-chow in 1862, contribute to VTE formation: stasis of blood flow, endothelial damage, and hypercoagulability. Of these risk factors, relative hypercoagulability appears most impor-tant in cases of spontaneous VTE, or so-called idiopathic VTE, whereas stasis and endothelial damage likely play a greater role in secondary VTE, or so-called provoked VTE, occurring in association with transient risk factors such as immobiliza-tion, surgical procedures, and trauma. Identifiable risk factors for VTE generally relate to one of the conditions described by Virchow. Often more than one risk factor is present contribut-ing in an exponential, rather than

1	and trauma. Identifiable risk factors for VTE generally relate to one of the conditions described by Virchow. Often more than one risk factor is present contribut-ing in an exponential, rather than additive, manner. Specific risk factors for VTE are listed in Table 24-2.The more common acquired VTE risk factors include older age (>40 years), hospitalization and immobilization, hor-mone replacement and oral contraceptive therapy, pregnancy and the recently postpartum state, prior VTE, malignancy, major surgery, obesity, nephrotic syndrome, trauma and spinal cord injury, long-haul travel (>6 hours), varicose veins, antiphospho-lipid syndrome, myeloproliferative disorders, and polycythemia. Heritable risk factors include male sex, factor V Leiden muta-tion; prothrombin 20210A gene variant; antithrombin, protein C, and protein S deficiencies; and dysfibrinogenemias. In some patients, the cause of the thrombophilia may have both a heri-table and an acquired component. These mixed causes

1	antithrombin, protein C, and protein S deficiencies; and dysfibrinogenemias. In some patients, the cause of the thrombophilia may have both a heri-table and an acquired component. These mixed causes include homocysteinemia; factors VII, VIII, IX, and XI elevation; hyperfibrinogenemia; and activated protein C resistance in the Brunicardi_Ch24_p0981-p1008.indd 98422/02/19 3:01 PM 985VENOUS AND LYMPHATIC DISEASECHAPTER 24Table 24-2Risk factors for venous thromboembolismAcquiredAdvanced ageHospitalization/immobilizationHormone replacement therapy and oral contraceptive usePregnancy and puerperiumPrior venous thromboembolismMalignancyMajor surgeryObesityNephrotic syndromeTrauma or spinal cord injuryLong-haul travel (>6 hours)Varicose veinsAntiphospholipid antibody syndromeMyeloproliferative diseasePolycythemiaInheritedFactor V LeidenProthrombin 20210AAntithrombin deficiencyProtein C deficiencyProtein S deficiencyFactor XI elevationDysfibrinogenemiaMixed EtiologyHomocysteinemiaFactors

1	diseasePolycythemiaInheritedFactor V LeidenProthrombin 20210AAntithrombin deficiencyProtein C deficiencyProtein S deficiencyFactor XI elevationDysfibrinogenemiaMixed EtiologyHomocysteinemiaFactors VII, VIII, IX, XI elevationHyperfibrinogenemiaActivated protein C resistance without factor V Leidenabsence of factor V Leiden.12 There may be a synergistic effect when particular multiple inherited and acquired risk factors are present in the same patient.Other patient-specific factors associated with venous thrombosis include the traditional cardiovascular risk factors of obesity, hypertension, and diabetes. VTE is more common in whites and African Americans than Asians and Native Americans.13,14 Certain gene variants (single nucleotide polymorphisms) are also associated with a mildly increased risk for VTE, and their presence may interact with other risk factors to increase the overall risk for venous thrombosis.15Anatomic factors may also contribute to development of DVT. At the site

1	risk for VTE, and their presence may interact with other risk factors to increase the overall risk for venous thrombosis.15Anatomic factors may also contribute to development of DVT. At the site where the right iliac artery crosses over the left iliac vein, the left iliac vein may become chronically compressed predisposing to iliofemoral venous thrombosis, so-called May-Thurner syndrome. External compression of major veins by masses of various types can also lead to venous thrombosis.Many cases of VTE are potentially preventable. Accord-ingly, in current clinical practice, preoperative VTE risk assess-ment is becoming increasingly common to identify patients at moderate and high risk. Scoring systems have been developed that take into account the number of VTE risk factors in an individual patient. These risk stratification scores, such as the Rogers score16 and Caprini score,17 provide individual patient risk stratification and recommendations for prophylactic anti-coagulation. The

1	patient. These risk stratification scores, such as the Rogers score16 and Caprini score,17 provide individual patient risk stratification and recommendations for prophylactic anti-coagulation. The ninth edition of the American College of Chest Physicians (ACCP) Guidelines for Prevention of VTE in Non-Orthopedic Surgical Patients acknowledges both the Rogers and Caprini scores and provides recommendations for VTE prophylaxis (Table 24-3). Orthopedic surgical patients are generally excluded from risk assessment scores because of the disproportionately increased risk of VTE in orthopedic surgery compared with the general and abdominopelvic surgery population.Table 24-3Thromboembolism risk and recommended thromboprophylaxis in surgical patientsLEVEL OF RISKAPPROXIMATE DVT RISK WITHOUT THROMBOPROPHYLAXIS (%)SUGGESTED THROMBOPROPHYLAXIS OPTIONSVery low risk General or abdominopelvic surgery<0.5% (Rogers score <7; Caprini score 0)No specific thromboprophylaxisEarly ambulationLow

1	THROMBOPROPHYLAXIS (%)SUGGESTED THROMBOPROPHYLAXIS OPTIONSVery low risk General or abdominopelvic surgery<0.5% (Rogers score <7; Caprini score 0)No specific thromboprophylaxisEarly ambulationLow risk General or abdominopelvic surgery∼1.5% (Rogers score 7–10; Caprini score 1–2)Mechanical prophylaxisModerate risk General or abdominopelvic surgery∼3.0% (Rogers score >10; Caprini score 3–4)LMWH (at recommended doses), LDUH, or mechanical prophylaxisHigh bleeding risk Mechanical prophylaxisHigh risk General or abdominopelvic surgery∼6% (Caprini score ≥5)LMWH (at recommended doses), fondaparinux and mechanical prophylaxisHigh bleeding risk General or abdominopelvic surgery for cancer Mechanical thromboprophylaxisExtended-duration LMWH (4 weeks)DVT = deep vein thrombosis; INR = international normalized ratio; LDUH = low-dose unfractionated heparin; LMWH = low molecular weight heparin; VTE = venous thromboembolism.Data from Gould MK, Garcia DA, Wren SM, et al: Prevention of VTE in

1	normalized ratio; LDUH = low-dose unfractionated heparin; LMWH = low molecular weight heparin; VTE = venous thromboembolism.Data from Gould MK, Garcia DA, Wren SM, et al: Prevention of VTE in nonorthopedic surgical patients: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines, Chest. 2012 Feb;141(2 Suppl):e227S-e277S.Brunicardi_Ch24_p0981-p1008.indd 98522/02/19 3:01 PM 986SPECIFIC CONSIDERATIONSPART IIFigure 24-4. Phlegmasia cerulea dolens of the left leg. Note the bluish discoloration.Figure 24-5. Duplex ultrasound scan of a normal femoral vein with phasic flow signals.DiagnosisClinical Evaluation. Early in the course of DVT development, venous thrombosis is thought to begin in an area of relative stasis, such as a soleal sinus vein or immediately downstream of the cusps of a venous valve in an axial calf vein. Isolated proxi-mal DVT without tibial vein thrombosis is unusual. Early in the

1	stasis, such as a soleal sinus vein or immediately downstream of the cusps of a venous valve in an axial calf vein. Isolated proxi-mal DVT without tibial vein thrombosis is unusual. Early in the course of a DVT, there may be no or few clinical findings such as pain or swelling. Even extensive DVT may sometimes be present without signs or symptoms if the patient is nonambula-tory or bedbound. History and physical examination are notori-ously unreliable in the diagnosis of DVT. In addition, symptoms and signs often associated with DVT, such as extremity pain and/or swelling, are nonspecific. In large studies, DVT has been found by venography or DUS in ≤50% of patients in whom it was clinically suspected.18,19 Objective studies are therefore required to confirm a diagnosis of VTE or to exclude the pres-ence of VTE.Clinical symptoms may worsen as DVT propagates and involves the major proximal deep veins. Extensive DVT of the major axial deep venous channels of the lower extremity with

1	the pres-ence of VTE.Clinical symptoms may worsen as DVT propagates and involves the major proximal deep veins. Extensive DVT of the major axial deep venous channels of the lower extremity with rel-ative sparing of collateral veins causes a condition called phleg-masia cerulea dolens (Fig. 24-4). This condition is characterized by pain and pitting edema with associated cyanosis. When the thrombosis extends to the collateral veins, massive fluid seques-tration and more significant edema ensue, resulting in a condition known as phlegmasia alba dolens.20 The affected extremity in phlegmasia alba dolens is extremely painful and edematous and pale secondary to arterial insufficiency from dramatically ele-vated below lower knee compartment pressures. Both phlegmasia cerulean dolens and phlegmasia alba dolens can be complicated by venous gangrene and the need for amputation.Vascular Lab and Radiologic Evaluation Duplex Ultrasound DUS is now the most commonly per-formed test for the detection

1	alba dolens can be complicated by venous gangrene and the need for amputation.Vascular Lab and Radiologic Evaluation Duplex Ultrasound DUS is now the most commonly per-formed test for the detection of infrainguinal DVT, both above and below the knee, and has a sensitivity and specificity of >95% in symptomatic patients.3 DUS refers to the combina-tion of real-time B-mode ultrasound with compression and flow augmentation amneuvres combined with pulsed Doppler capa-bility. For VTE detection, color flow imaging is an additional extremely useful adjunct in the evaluation of possible axial calf vein DVT and evaluation of intra-abdominal veins. DUS provides the ability to noninvasively visualize venous anatomy, detect occluded and partially occluded venous segments, and demonstrate physiologic flow characteristics.In the supine patient, normal lower extremity venous flow is phasic (Fig. 24-5), decreasing with inspiration in response to increased intra-abdominal pressure with the descent of

1	flow characteristics.In the supine patient, normal lower extremity venous flow is phasic (Fig. 24-5), decreasing with inspiration in response to increased intra-abdominal pressure with the descent of the diaphragm and then increasing with expiration as the diaphragm rises and intra-abdominal pressure decreases. When the patient is upright, the decrease in intra-abdominal pressure with expira-tion cannot overcome the hydrostatic column of pressure exist-ing between the right atrium and the calf. Muscular contractions of the calf, along with the one-way venous valves, are then required to promote venous return to the heart. Flow also can be increased by leg elevation or compression and decreased by sud-den elevation of intra-abdominal pressure (Valsalva maneuver). In a venous DUS examination performed with the patient supine, spontaneous flow, variation of flow with respiration, and response of flow to Valsalva maneuver are all assessed. From the common femoral through the popliteal

1	performed with the patient supine, spontaneous flow, variation of flow with respiration, and response of flow to Valsalva maneuver are all assessed. From the common femoral through the popliteal vein, the primary method of detecting DVT with ultrasound is demonstration of the lack of compressibility of the vein with probe pressure on B-mode imaging. Normally, in transverse section, the vein walls should coapt with pressure. Lack of coaptation indicates throm-bus. Axial calf vein thrombi are often best detected by abnor-malities in color flow imaging as compressibility is difficult in the calf.The examination begins at the ankle and continues proxi-mally to the groin. Each vein is visualized, and the flow signal is assessed with distal and proximal compression. Lower extremity DVT can be diagnosed by any of the following DUS findings: lack of spontaneous flow (Fig. 24-6), inability to compress the vein (Fig. 24-7), absence of color filling of the lumen by color flow DUS, loss of

1	be diagnosed by any of the following DUS findings: lack of spontaneous flow (Fig. 24-6), inability to compress the vein (Fig. 24-7), absence of color filling of the lumen by color flow DUS, loss of respiratory flow variation, and venous disten-tion. Again, lack of venous compression on B-mode imaging is the primary diagnostic variable. Several studies comparing B-mode ultrasound to venography for the detection of femo-ropopliteal DVT in patients clinically suspected to have DVT report sensitivities of >91% and specificities of >97%.21,22 The ability of DUS to assess isolated calf vein DVT varies greatly, with sensitivities ranging from 50% to 93% and specificities approaching 100%.23,24Impedance Plethysmography Impedance plethysmography (IPG) was the primary noninvasive method of diagnosing DVT before the widespread use of DUS but is infrequently used today. Changes in electrical resistance resulting from lower extremity blood volume changes are quantified. IPG is less accurate than

1	DVT before the widespread use of DUS but is infrequently used today. Changes in electrical resistance resulting from lower extremity blood volume changes are quantified. IPG is less accurate than DUS for the detection of proximal DVT, with 83% sensitivity in symptomatic patients. It is a poor detector of calf vein DVT.25Brunicardi_Ch24_p0981-p1008.indd 98622/02/19 3:01 PM 987VENOUS AND LYMPHATIC DISEASECHAPTER 24Figure 24-6. Duplex ultrasound of a femoral vein containing thrombus demonstrating no flow within the femoral vein.No compressionCompressionR FVPR FVPFigure 24-7. B-mode ultrasound of the femoral vein in crosssection. The femoral vein does not collapse with external compres-sion (arrows).Figure 24-8. Venogram showing a filling defect in the popliteal vein (arrows).Iodine-125 Fibrinogen Uptake Iodine-125 fibrinogen uptake (FUT) is a seldom-used technique that involves IV administra-tion of radioactive fibrinogen and monitoring for increased uptake in fibrin clots. An

1	Fibrinogen Uptake Iodine-125 fibrinogen uptake (FUT) is a seldom-used technique that involves IV administra-tion of radioactive fibrinogen and monitoring for increased uptake in fibrin clots. An increase of 20% or more in one area of a limb indicates an area of thrombus. FUT can detect DVT in the calf, but high background radiation from the pelvis and the urinary tract limits its ability to detect proximal DVT. It also cannot be used in an extremity that has recently undergone surgery or has active inflammation. In a prospective study, FUT had a sensitivity of 73% and specificity of 71% for identification of DVT in a group of symptomatic and asymptomatic patients.26 Currently, FUT is primarily a research tool of historic interest.Venography Venography is the gold standard to which other diagnostic modalities are compared. A small catheter is placed in a dorsal foot vein with injection of a radiopaque contrast agent. Radiographs are obtained in at least two projections. A positive

1	diagnostic modalities are compared. A small catheter is placed in a dorsal foot vein with injection of a radiopaque contrast agent. Radiographs are obtained in at least two projections. A positive study result is failure to fill the deep system with passage of the contrast medium into the superficial system or demonstration of discrete filling defects (Fig. 24-8). A normal study result virtually excludes the presence of DVT. In a study of 160 patients with a normal venogram followed for 3 months, only two patients (1.3%) subsequently developed DVT, and no patients experienced symptoms of PE.27 Venography is not routinely used in clinical practice because of its invasiveness and complication risk. It is still, however, sometimes used in research studies evaluating DVT prophylaxis.TreatmentOnce the diagnosis of VTE has been made, antithrombotic ther-apy should be initiated promptly. If clinical suspicion for VTE is high, it may be prudent to start treatment before the diagnosis is

1	the diagnosis of VTE has been made, antithrombotic ther-apy should be initiated promptly. If clinical suspicion for VTE is high, it may be prudent to start treatment before the diagnosis is objectively confirmed. The goals of VTE treatment are the pre-vention of mortality and morbidity associated with PE and the prevention of the postthrombotic syndrome (PTS). Treatment regimens may include antithrombotic therapy, temporary or permanent vena cava filter placement, catheter-directed or systemic thrombolytic therapy, and operative thrombectomy.Antithrombotic Therapy. Most often, antithrombotic therapy for VTE is initiated with IV or subcutaneous (SC) unfraction-ated heparin or SC low molecular weight heparin. Fondaparinux, a synthetic pentasaccharide, is sometimes also used as an alter-native to heparin to initiate therapy. An oral vitamin K antago-nist, usually sodium warfarin, is begun shortly after initiation of IV or SC therapy. Either SC or IV therapy is continued until effective

1	to heparin to initiate therapy. An oral vitamin K antago-nist, usually sodium warfarin, is begun shortly after initiation of IV or SC therapy. Either SC or IV therapy is continued until effective oral anticoagulation with warfarin is achieved as indi-cated by an international normalized ratio (INR) ≥2 for 24 hours. A minimum of 5 days of heparin or fondaparinux therapy is 1Brunicardi_Ch24_p0981-p1008.indd 98722/02/19 3:01 PM 988SPECIFIC CONSIDERATIONSPART IIrecommended.28 Recently, several oral anticoagulants that function by either directly inhibiting thrombin or inhibit-ing factor Xa have additionally been approved by the United States Food and Drug Administration (FDA) for both treatment and prophylaxis for VTE. A principle advantage is they do not require monitoring of laboratory parameters for use.Unfractionated heparin (UFH) binds to antithrombin via a specific 18-saccharide sequence. This increases antithrombin activity over a thousandfold. The antithrombin-heparin complex

1	parameters for use.Unfractionated heparin (UFH) binds to antithrombin via a specific 18-saccharide sequence. This increases antithrombin activity over a thousandfold. The antithrombin-heparin complex primarily inhibits factor IIa (thrombin) and factor Xa and, to a lesser degree, factors IXa, XIa, and XIIa of the coagulation cascade. In addition, UFH also binds to tissue factor pathway inhibitor, which inhibits the conversion of factors X to Xa, and factors IX to IXa. Finally, UFH catalyzes the inhibition of thrombin by heparin cofactor II via a mechanism independent of antithrombin.UFH therapy is most commonly administered with an initial IV bolus of 80 units/kg. Weight-based UFH dosages have been shown to be more effective than standard fixed boluses in rapidly achieving therapeutic levels.29 The initial bolus is followed by a continuous IV drip at 18 units/kg per hour. The half-life of IV UFH ranges from 45 to 90 minutes and is dose dependent. The level of antithrombotic therapy

1	The initial bolus is followed by a continuous IV drip at 18 units/kg per hour. The half-life of IV UFH ranges from 45 to 90 minutes and is dose dependent. The level of antithrombotic therapy should be moni-tored every 6 hours using the activated partial thromboplastin time (aPTT), with the goal range of 1.5 to 2.5 times control values. This should correspond with plasma heparin anti-Xa activity levels of 0.3 to 0.7 IU/mL.Initial anticoagulation with UFH may also be administered SC, although this route is less commonly used. Adjusted-dose therapeutic SC UFH is initiated with 17,500 units, followed by 250 units/kg twice daily, and dosing is adjusted to an aPTT goal range similar to that for IV UFH. Fixed-dose unmonitored SC UFH is started with a bolus of 333 units/kg, followed by 250 units/kg twice daily.30Hemorrhage is the primary complication of UFH therapy. The rate of major hemorrhage (fatal, intracranial, retroperitoneal, or requiring transfusion of >2 units of packed red blood

1	twice daily.30Hemorrhage is the primary complication of UFH therapy. The rate of major hemorrhage (fatal, intracranial, retroperitoneal, or requiring transfusion of >2 units of packed red blood cells) is approximately 5% in hospitalized patients undergoing UFH therapy (1% in medical patients and 8% in surgical patients).30 For patients with UFH-related bleeding complications, cessation of UFH is required, and anticoagulation may be reversed with protamine sulfate. Protamine sulfate binds to UFH and forms an inactive salt compound. Each milligram of protamine neutral-izes 90 to 115 units of heparin, and the dosage should not exceed 50 mg IV over any 10-minute period. Side effects of protamine sulfate include hypotension, pulmonary edema, and anaphylaxis. Patients with prior exposure to protamine-containing insulin (NPH) and patients with allergy to fish may have an increased risk of hypersensitivity, although no direct relationship has been established. Protamine administration should

1	insulin (NPH) and patients with allergy to fish may have an increased risk of hypersensitivity, although no direct relationship has been established. Protamine administration should be performed judi-ciously and terminated if any side effects occur.In addition to hemorrhage, heparin also has other, unique, complications. Heparin-induced thrombocytopenia (HIT) results from heparin-associated antiplatelet antibodies (HAAbs) directed against platelet factor 4 complexed with heparin.31 HIT occurs in 1% to 5% of patients treated with heparin.32,33 In patients with repeat heparin exposure (such as vascular surgery patients), the incidence of HAAbs may be as high as 21%.34 HIT occurs most frequently in the second week of therapy and may lead to disastrous venous or arterial thrombotic complications. Therefore, platelet counts should be monitored periodically in patients receiving continuous heparin therapy.HIT is diagnosed based on previous exposure to heparin, platelet count less than

1	Therefore, platelet counts should be monitored periodically in patients receiving continuous heparin therapy.HIT is diagnosed based on previous exposure to heparin, platelet count less than 100,000, and/or platelet count decline of 50% following exposure. All heparin must be stopped and alter-native anticoagulation initiated immediately to avoid thrombotic complications, which may approach 50% over the subsequent 30 days in affected individuals.35Another complication of prolonged high-dose heparin therapy is osteopenia. Heparin-induced osteopenia results from impairment of bone formation and enhancement of bone resorp-tion by heparin.Low molecular weight heparins (LMWHs) are derived from the depolymerization of porcine UFH. Like UFH, LMWHs bind to antithrombin via a specific pentasaccharide sequence to expose an active site for the neutralization of fac-tor Xa. However, LMWHs have fewer additional saccharide units. This results in less inactivation of thrombin (factor IIa). In

1	sequence to expose an active site for the neutralization of fac-tor Xa. However, LMWHs have fewer additional saccharide units. This results in less inactivation of thrombin (factor IIa). In comparison to UFH, LMWHs have increased bioavailability (>90% after SC injection), longer half-lives (approximately 4 to 6 hours), and more predictable elimination rates.Most patients treated with weight-based onceor twice-daily SC LMWH injections do not require laboratory monitoring for anticoagulant effect, a distinct advantage over continuous IV infusions of UFH. Patients who do require monitoring include those with significant renal insufficiency, pediatric patients, obese patients greater than 120 kg, and pregnant patients. Moni-toring may be performed using anti-Xa activity assays. The ther-apeutic anti-Xa goal range depends on the type of LMWH and the frequency of dosing. There are numerous LMWHs avail-able, and the various preparations differ in their anti-Xa and anti-IIa activities.

1	anti-Xa goal range depends on the type of LMWH and the frequency of dosing. There are numerous LMWHs avail-able, and the various preparations differ in their anti-Xa and anti-IIa activities. Treatment dosing for one LMWH, therefore, cannot be extrapolated for use with another. The anticoagulant effect of LMWHs may be partially reversed (approximately 60%) with protamine sulfate.Numerous well-designed trials comparing SC LMWH with IV and SC UFH for the treatment of DVT have been critically evaluated in several meta-analyses and demonstrate a decrease in thrombotic complications, bleeding, and mortality with LMWHs.36-38 LMWHs also are associated with a decreased rate of HAAb formation and HIT (<2%) compared with UFH (at least in prophylactic doses).30 However, patients with estab-lished HIT also should not receive LMWHs because there is cross-reactivity between the drugs.39A major benefit of LMWHs is that it allows outpatient treatment of VTE.40,41 In a randomized study comparing IV UFH

1	also should not receive LMWHs because there is cross-reactivity between the drugs.39A major benefit of LMWHs is that it allows outpatient treatment of VTE.40,41 In a randomized study comparing IV UFH and the LMWH nadroparin calcium,40 there was no sig-nificant difference in recurrent thromboembolism (8.6% for UFH vs. 6.9% for LMWH) or major bleeding complications (2.0% for UFH vs. 0.5% for LMWH). There was, however, a 67% reduction in mean days in the hospital for the LMWH group.Fondaparinux currently is a synthetic pentasaccharide that has been approved by the FDA for the initial treatment of DVT and PE. Its five-polysaccharide sequence binds and activates antithrombin, causing specific inhibition of factor Xa. In two large noninferiority trials, fondaparinux was compared with the LMWH enoxaparin for the initial treatment of DVT and with IV UFH for the initial treatment of PE.42,43 The rates of recurrent VTE ranged from 3.8% to 5%, with rates of major bleeding of 2% to 2.6%, for all

1	for the initial treatment of DVT and with IV UFH for the initial treatment of PE.42,43 The rates of recurrent VTE ranged from 3.8% to 5%, with rates of major bleeding of 2% to 2.6%, for all treatment arms. The drug is administered SC once daily with a weight-based dosing protocol: 5 mg, 7.5 mg, or 10 mg for patients weighing <50 kg, 50 to 100 kg, or >100 kg, respectively. The half-life of fondaparinux is approximately 2Brunicardi_Ch24_p0981-p1008.indd 98822/02/19 3:01 PM 989VENOUS AND LYMPHATIC DISEASECHAPTER 2417 hours in patients with normal renal function. There are rare case reports of fondaparinux-induced thrombocytopenia.44Direct thrombin inhibitors (DTIs) include parental forms with recombinant hirudin, argatroban, and bivalirudin, as well as an oral agent, dabigatran. These antithrombotic agents bind to thrombin, inhibiting the conversion of fibrinogen to fibrin as well as thrombin-induced platelet activation. These actions are independent of antithrombin. The parental

1	antithrombotic agents bind to thrombin, inhibiting the conversion of fibrinogen to fibrin as well as thrombin-induced platelet activation. These actions are independent of antithrombin. The parental DTIs should be reserved for (a) patients in whom there is a high clinical suspi-cion or confirmation of HIT, and (b) patients who have a his-tory of HIT or test positive for heparin-associated antibodies whereas dabigatran can be used as an alternative to Warfarin when INR monitoring is difficult or impractical. In patients with established HIT, DTIs should be administered for at least 7 days, or until the platelet count normalizes. Warfarin may then be introduced slowly, overlapping therapy with a DTI for at least 5 days, or dabigatran may be continued instead of Warfarin.45Bivalirudin is approved primarily for patients with or without HIT who undergo percutaneous coronary intervention and is rarely used outside of that setting.Argatroban is indicated for the prophylaxis and treatment of

1	primarily for patients with or without HIT who undergo percutaneous coronary intervention and is rarely used outside of that setting.Argatroban is indicated for the prophylaxis and treatment of thrombosis in HIT. It also is approved for patients with, or at risk for, HIT undergoing percutaneous coronary intervention. Antithrombotic prophylaxis and therapy are initiated with a con-tinuous IV infusion of 2 µg/kg per minute, without the need for a bolus. The half-life ranges from 39 to 51 minutes, and the dosage is adjusted to maintain an aPTT of 1.5 to 3 times normal. Large ini-tial boluses and higher rates of continuous infusion are reserved for patients with coronary artery thrombosis and myocardial infarc-tion. In these patients, therapy is monitored using the activated clotting time. Argatroban is metabolized and excreted by the liver; therefore, dosage adjustments are needed in patients with hepatic impairment. There is no reversal agent for argatroban.The oral agent, dabigatran,

1	is metabolized and excreted by the liver; therefore, dosage adjustments are needed in patients with hepatic impairment. There is no reversal agent for argatroban.The oral agent, dabigatran, is US FDA-approved since 2014 for the treatment of VTE and prophylaxis for recurrent VTE. Additionally, limited approval was obtained in 2015 for prophylaxis of VTE after hip replacement surgery. It is admin-istered as a prodrug, dabigatran etexilate, that is converted to the active form, dabigatran, in the liver. The half-life ranges from 12 to 17 hours; it is therefore administered once daily for prophylaxis and twice daily for VTE therapy. Absorption is not dietary dependent, and no drug level monitoring is required. Dabigatran is metabolized in the kidney, and dose adjustment is required for renal insufficiency. Data on use in obese patients is limited; therefore, use is not recommended for patients with a body mass index ≥40 kg/m2 or ≥120 kg.46 Dyspepsia is a com-mon side effect that may limit

1	Data on use in obese patients is limited; therefore, use is not recommended for patients with a body mass index ≥40 kg/m2 or ≥120 kg.46 Dyspepsia is a com-mon side effect that may limit use in some patients.47 Dabigatran may be reversed with idarucizumab in emergent situations.48 It is contraindicated in patients with mechanical heart valves.Direct factor Xa inhibitors, which are comprised of the oral agents rivaroxaban, apixiban, and edoxaban, are FDA approved for treatment in VTE and prophylaxis for recurrent VTE. Additionally, rivaroxoban and apixiban are approved by the FDA for VTE prophylaxis following knee and hip replace-ment surgery. These medications function by inactivating cir-culating and thrombus-bound factor Xa. They are metabolized in the kidney (25–35%) and in the liver; therefore, use is not rec-ommended in patients with renal insufficiency (creatinine clear-ance <30 mL/min for rivaroxaban, or <15 mL/min for apixiban and edoxaban) or severe hepatic insufficiency. As

1	therefore, use is not rec-ommended in patients with renal insufficiency (creatinine clear-ance <30 mL/min for rivaroxaban, or <15 mL/min for apixiban and edoxaban) or severe hepatic insufficiency. As with the oral DTI, dabigatran, data on use in obese patients is limited; there-fore, use is not recommended in these patients. Additionally, these agents are contraindicated in pregnancy. There are no specific reversal agents available for direct factor Xa inhibi-tors. For severe cases of hemorrhage, indirect partial reversal may be achieved with use of prothrombin complex concentrate administration.49Rixaroxaban has a half-life of 7 to 17 hours. Therapy does not require monitoring. Prophylactic dosing is 10 mg once daily, and therapeutic dosing is 15 mg twice daily for 21 days, fol-lowed by 20 mg once daily thereafter.Apixiban has a half-life of 5 to 9 hours. Therapy does not require monitoring, and there are no dietary restrictions. If monitoring is desired in situations of bleeding or

1	20 mg once daily thereafter.Apixiban has a half-life of 5 to 9 hours. Therapy does not require monitoring, and there are no dietary restrictions. If monitoring is desired in situations of bleeding or concern for subor supratherapeutic dosing, serum anti-Xa levels can be obtained. Prophylactic dosing is 2.5 mg twice daily, and thera-peutic dosing is 10 mg daily for 7 days, followed by 5 mg twice daily thereafter.Edoxaban has a half-life of 10 to 14 hours. Therapy does not require monitoring. Typical dosing is 60 mg once daily, and 30 mg once daily if creatinine clearance ranges from 15 to 50 mL/min, or body weight ≤60 kg.Vitamin K antagonists, which include warfarin and other coumarin derivatives, are the traditional mainstay of long-term antithrombotic therapy in patients with VTE. Warfarin inhib-its the γ-carboxylation of vitamin K–dependent procoagulants (factors II, VII, IX, and X) and anticoagulants (proteins C and S), resulting in formation of less functional proteins.

1	Warfarin inhib-its the γ-carboxylation of vitamin K–dependent procoagulants (factors II, VII, IX, and X) and anticoagulants (proteins C and S), resulting in formation of less functional proteins. Warfarin usu-ally requires several days to achieve full effect because normal circulating coagulation proteins must first undergo their normal degradation. Factors X and II have the longest half-lives, in the range of 36 and 72 hours, respectively. A steady-state concen-tration of warfarin is usually not reached for 4 to 5 days.Warfarin therapy is monitored by measuring the INR, calculated using the following equation:INR = (patient prothrombin time/laboratorynormal prothrombin time)ISIwhere ISI is the international sensitivity index. The ISI describes the strength of the thromboplastin that is added to activate the extrinsic coagulation pathway. The therapeutic target INR range is usually 2.0 to 3.0, but the response to warfarin is variable and depends on liver function, diet, age, and

1	is added to activate the extrinsic coagulation pathway. The therapeutic target INR range is usually 2.0 to 3.0, but the response to warfarin is variable and depends on liver function, diet, age, and concomitant medica-tions. In patients receiving anticoagulation therapy without con-comitant thrombolysis or venous thrombectomy, the vitamin K antagonist may be started on the same day as the initial paren-teral anticoagulant, usually at doses ranging from 5 to 10 mg. Smaller initial doses may be needed in older and malnourished patients, in those with liver disease or congestive heart failure, and in those who have recently undergone major surgery.49The recommended duration of warfarin antithrombotic therapy is stratified based on whether the DVT was provoked or unprovoked, whether it was the first or a recurrent episode, where the DVT is located, and whether malignancy or thrombophilia is present. Current ACCP recommendations for duration of warfarin therapy are summarized in Table

1	the first or a recurrent episode, where the DVT is located, and whether malignancy or thrombophilia is present. Current ACCP recommendations for duration of warfarin therapy are summarized in Table 24-4.In patients with proximal DVT, several randomized clini-cal trials have demonstrated that shorter-term antithrombotic therapy (4 to 6 weeks) is associated with a higher rate of VTE recurrence than 3 to 6 months of anticoagulation.50-52 In these trials, most of the patients with transient risk factors had a low rate of recurrent VTE, and most recurrences were in patients with continuing risk factors. The ACCP recommendation, there-fore, is that 3 months of anticoagulation are sufficient to prevent 3Brunicardi_Ch24_p0981-p1008.indd 98922/02/19 3:01 PM 990SPECIFIC CONSIDERATIONSPART IITable 24-4Summary of American College of Chest Physicians recommendations regarding duration of long-term antithrombotic therapy for deep vein thrombosis (DVT)CLINICAL SUBGROUPANTITHROMBOTIC TREATMENT

1	24-4Summary of American College of Chest Physicians recommendations regarding duration of long-term antithrombotic therapy for deep vein thrombosis (DVT)CLINICAL SUBGROUPANTITHROMBOTIC TREATMENT DURATIONFirst episode DVT/transient risk/surgeryVKA or LMWH for 3 monthsFirst episode DVT/unprovokedVKA or LMWH for 3 monthsConsider for long-term therapy if:• Proximal DVT• Minimal bleeding risk• Stable coagulation monitoringDistal DVT/unprovoked• Symptomatic• Asymptomatic and no risk factors for progressionVKA for 3 monthsSerial imaging in 2 weeks, if progression VKA for 3 monthsSecond episode DVT/unprovokedDVT and cancerVKA for extended therapyLMWH for extended therapy over VKALMWH = low molecular weight heparin; VKA = vitamin K antagonist.Data from Kearon C, Akl EA, Comerota AJ, et al: Antithrombotic therapy for VTE disease: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines, Chest. 2012 Feb;141(2

1	therapy for VTE disease: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines, Chest. 2012 Feb;141(2 Suppl): e419S-e496S.recurrent VTE in patients with DVT occurring around the time of a transient risk factor (e.g., hospitalization or orthopedic or major general surgery).In contrast to patients with thrombosis related to transient risk factors, patients with unprovoked VTE are much more likely to develop recurrence (rates as high as 40% at 10 years). In this latter group of patients, numerous clinical trials have compared 3 to 6 months of anticoagulation therapy with extended-duration warfarin therapy, both at low intensity (INR of 1.5 to 2.0) and at conventional intensity (INR of 2.0 to 3.0).53-55 In patients with idiopathic DVT, extended-duration antithrombotic therapy is associated with a relative reduction in the rate of recurrent VTE by 75% to >90%. In addition, conventional-intensity

1	In patients with idiopathic DVT, extended-duration antithrombotic therapy is associated with a relative reduction in the rate of recurrent VTE by 75% to >90%. In addition, conventional-intensity warfarin reduces the risk even further compared with low-intensity war-farin (0.7 events per 100 person-years vs. 1.9 events per 100 person-years) without an increase in bleeding complications.56In patients with VTE in association with a hypercoagulable condition, the optimal duration of anticoagulation therapy is influ-enced more by the clinical circumstances at the time of the VTE (idiopathic vs. secondary) than by the actual presence or absence of the more common thrombophilic conditions. In patients with VTE related to malignancy, increasing evidence suggests that longer-term therapy with LMWH (up to 6 months) is associated with a lower VTE recurrence than treatment using conventional vitamin K antagonists.57,58 The primary complication of warfarin therapy is hemorrhage, and the risk is

1	(up to 6 months) is associated with a lower VTE recurrence than treatment using conventional vitamin K antagonists.57,58 The primary complication of warfarin therapy is hemorrhage, and the risk is related to the magnitude of INR prolongation. Depending on the INR and the presence of bleeding, warfarin anticoagulation may be reversed by (a) omit-ting or decreasing subsequent dosages, (b) administering oral or parenteral vitamin K, or (c) administering fresh frozen plasma, prothrombin complex concentrate, or recombinant factor VIIa.49Warfarin therapy rarely may be associated with the devel-opment of skin necrosis and limb gangrene. These conditions occur more commonly in women (4:1), and the most commonly affected areas are the breast, buttocks, and thighs. This com-plication, which usually occurs in the first days of therapy, is occasionally, but not exclusively, associated with protein C or S deficiency and malignancy. Patients who require continued anticoagulation may restart

1	usually occurs in the first days of therapy, is occasionally, but not exclusively, associated with protein C or S deficiency and malignancy. Patients who require continued anticoagulation may restart low-dose warfarin (2 mg) while receiving concomitant therapeutic heparin. The warfarin dosage is then gradually increased over a 1to 2-week period.49Systemic and Catheter-Directed Thrombolysis. Patients with extensive proximal, iliofemoral DVT may benefit from systemic thrombolysis or catheter-directed thrombolysis (CDT). CDT appears to be more effective (see later in chapter) and potentially reduces acute congestive lower extremity symp-toms more rapidly than anticoagulation alone and decreases the development of PTS.Several thrombolytic agents are available, including strep-tokinase, urokinase, alteplase (recombinant tissue plasminogen activator), reteplase, and tenecteplase. All share the ability to con-vert plasminogen to plasmin, which leads to the degradation of fibrin. They differ

1	alteplase (recombinant tissue plasminogen activator), reteplase, and tenecteplase. All share the ability to con-vert plasminogen to plasmin, which leads to the degradation of fibrin. They differ with regard to their half-lives, their potential for inducing fibrinogenolysis (generalized lytic state), their poten-tial for antigenicity, and their FDA-approved indications for use.Streptokinase is purified from β-hemolytic Streptococcus and is approved for the treatment of acute myocardial infarction, PE, DVT, arterial thromboembolism, and occluded central lines and arteriovenous shunts. It is not specific for fibrin-bound plas-minogen, however, and its use is limited by its significant rates of antigenicity. Fevers and shivering occur in 1% to 4% of patients.Urokinase is derived from human neonatal kidney cells grown in tissue culture. Currently, it is only approved for lysis of massive PE or PE associated with unstable hemodynamics.Alteplase, reteplase, and tenecteplase all are

1	human neonatal kidney cells grown in tissue culture. Currently, it is only approved for lysis of massive PE or PE associated with unstable hemodynamics.Alteplase, reteplase, and tenecteplase all are recombinant variants of tissue plasminogen activator. Alteplase is indicated for the treatment of acute myocardial infarction, acute ischemic stroke, and acute massive PE. However, it often is used for CDT of DVT. Reteplase and tenecteplase are indicated only for the treatment of acute myocardial infarction.Systemic thrombolysis was evaluated in numerous older prospective and randomized clinical trials, and its efficacy was summarized in a recent Cochrane Review.59 In 12 studies involving over 700 patients, systemic thrombolysis was associ-ated with significantly more clot lysis (relative risk [RR] 0.24 to 0.37) and significantly less PTS (RR 0.66). However, venous function was not significantly improved. In addition, more bleeding complications occurred (RR 1.73).In an effort to minimize

1	[RR] 0.24 to 0.37) and significantly less PTS (RR 0.66). However, venous function was not significantly improved. In addition, more bleeding complications occurred (RR 1.73).In an effort to minimize bleeding complications and increase efficacy, CDT techniques were developed for the treat-ment of symptomatic primarily iliofemoral DVT. With catheterdirected therapy, venous access may be achieved through percutaneous catheterization of the ipsilateral popliteal vein, retrograde catheterization through the contralateral femoral vein, or retrograde cannulation from the internal jugular vein. Multi–side-hole infusion catheters, with or without infusion wires, are used to deliver the lytic agent directly into the throm-bus. Lytic agents may be administered alone or, now more commonly, in combination with catheter-based methods to Brunicardi_Ch24_p0981-p1008.indd 99022/02/19 3:01 PM 991VENOUS AND LYMPHATIC DISEASECHAPTER 24ABFigure 24-9. Preoperative computed tomography imaging and

1	with catheter-based methods to Brunicardi_Ch24_p0981-p1008.indd 99022/02/19 3:01 PM 991VENOUS AND LYMPHATIC DISEASECHAPTER 24ABFigure 24-9. Preoperative computed tomography imaging and intraoperative photo demonstrating erosion of IVC filter through the IVC wall.physically break up the clot—so-called pharmacomechanical thrombolysis. One commonly used device to perform phar-macomechanical thrombolysis is the AngioJet, which utilizes pulsed injection of thrombolytic via a percutaneously inserted catheter followed by active aspiration to remove the thrombus.The efficacy of CDT for the treatment of symptomatic iliofemoral DVT has been reported previously in a large, multi-center, randomized control trial. Two-hundred and nine patients with proximal DVT identified within 21 days of onset of symp-toms were assigned to conventional anticoagulant therapy vs. conventional anticoagulant therapy plus CDT. In the CDT group, placement of a venous stent was permitted for any identified iliac

1	of symp-toms were assigned to conventional anticoagulant therapy vs. conventional anticoagulant therapy plus CDT. In the CDT group, placement of a venous stent was permitted for any identified iliac vein stenotic lesion. At 6 months, iliac vein patency was signifi-cantly improved in the thrombolysis group (65.9% vs. 47.4%). At 2 years, in the CDT group, there was an absolute risk reduc-tion of nearly 15% for development of PTS, translating to a num-ber needed to treat of seven patients to prevent one case of PTS.60 However, these results were, in part, contradictory to the results reported the more recent Acute Venous Thrombosis: Throm-bus Removal with Adjunctive Catheter-Directed Thrombolysis (ATTRACT) trial, a prospective, randomized, multicenter trial evaluating nearly 700 patients comparing anticoagulant use with CDT in patients with acute femoropopliteal, and/or iliac vein DVT.61 However, the purpose of this trial was to see if indica-tions for CDT should be extended for isolated

1	anticoagulant use with CDT in patients with acute femoropopliteal, and/or iliac vein DVT.61 However, the purpose of this trial was to see if indica-tions for CDT should be extended for isolated femoropopliteal DVT (43% of the patients in this study) and therefore, was under-powered to compare treatment efficacy for iliofemoral DVT, which known to have a higher risk of PTS. The study found that PTS occurred with equal frequency in the two groups (47% vs. 48%, P = NS), but patients who were treated with pharmacom-echanical CDT plus anticoagulation were less likely to develop moderate-to-severe PTS than those treated with anticoagulation alone (18% vs. 24%, P = .003). There was no difference between the two groups in quality of life. There was an increase in both overall hemorrhage (4.5% vs. 1.7%) and major hemorrhage (1.7% vs 0.3%) with CDT but no fatal or intracranial hemor-rhage in either cohort. Taken in combination, the findings from these trials support selective use of CDT with

1	1.7%) and major hemorrhage (1.7% vs 0.3%) with CDT but no fatal or intracranial hemor-rhage in either cohort. Taken in combination, the findings from these trials support selective use of CDT with anticoagulation in young patients with acute iliofemoral DVT and anticoagulation alone in the remaining patient with DVT.There are contraindications to thrombolytic therapy. Absolute contraindications include prior history of ischemic or hemorrhagic stroke within 3 months, head trauma within 3 months, neurologic surgery within 6 months, known intra-cranial neoplasm, internal bleeding within 6 weeks, active or known bleeding disorder, traumatic cardiopulmonary resuscita-tion within 3 weeks or suspected aortic dissection. Fortunately, serious remote bleeding is uncommon, and intracranial hem-orrhage rarely occurs. The majority of bleeding complications are limited to the venous access site. Symptomatic pulmonary embolism occurs uncommonly and is very rarely fatal.Inferior Vena Caval

1	hem-orrhage rarely occurs. The majority of bleeding complications are limited to the venous access site. Symptomatic pulmonary embolism occurs uncommonly and is very rarely fatal.Inferior Vena Caval Filters. Since the introduction of the Kimray-Greenfield filter in the United States in 1973, numerous vena caval filters have been developed. Although the designs are variable, they all are designed to prevent pulmonary emboli, while allowing continuation of venous blood flow through the IVC. Early filters were placed surgically through the femoral vein. Currently, less invasive techniques allow percutaneous filter placement through a femoral vein, internal jugular vein, or a peripheral vein under fluoroscopic or ultrasound guidance.Placement of an IVC filter is indicated for patients who have manifestations of lower extremity VTE and absolute contraindications to anticoagulation, those that have a bleeding complication from anticoagulation therapy of acute VTE, or those who develop

1	have manifestations of lower extremity VTE and absolute contraindications to anticoagulation, those that have a bleeding complication from anticoagulation therapy of acute VTE, or those who develop recurrent DVT or PE despite adequate anticoagula-tion therapy and for patients with severe pulmonary hypertension.When possible, anticoagulation therapy should be contin-ued in patients with vena cava filters. The duration of antico-agulation is determined by the underlying VTE and not by the presence of the IVC filter itself. Practically speaking, however, many patients who require an IVC filter for recurrent VTE are the same ones who would benefit most from indefinite antico-agulation. In patients who are not able to receive anticoagulants due to recent surgery or trauma, the clinician should continually reassess if anticoagulation may be started safely at a later date.Placement of permanent IVC filters has been evaluated as an adjunct to routine anticoagulation in patients with proximal

1	continually reassess if anticoagulation may be started safely at a later date.Placement of permanent IVC filters has been evaluated as an adjunct to routine anticoagulation in patients with proximal DVT.62 Routine IVC filter placement has not been shown to prolong early or late survival in patients with proximal DVT but did decrease the rate of PE (HR, 0.22; 95% CI, 0.05–0.90); however, there is an increased rate of recurrent DVT in patients with IVC filters (HR, 1.87; 95% CI, 1.10–3.20).IVC filters are associated with acute and late complica-tions. Acute complications include thrombosis or bleeding at the insertion site and misplacement of the filter. Late complications include thrombosis of the IVC, DVT, breaking, migration, or ero-sion of the filter through the IVC (Fig. 24-9). The rate of fatal complications is <0.12%.63 As a result of the increasing number Brunicardi_Ch24_p0981-p1008.indd 99122/02/19 3:01 PM 992SPECIFIC CONSIDERATIONSPART IIFigure 24-10. Autopsy specimen

1	rate of fatal complications is <0.12%.63 As a result of the increasing number Brunicardi_Ch24_p0981-p1008.indd 99122/02/19 3:01 PM 992SPECIFIC CONSIDERATIONSPART IIFigure 24-10. Autopsy specimen showing a massive pulmonary embolism.of reported complications with IVC filters, the FDA issued a warning in 2010 recommending removal of IVC filters as soon as they are no longer needed.64 This was followed by an update in 2014 where the recommendation was made to remove IVC filters within 29 and 54 days after implantation based upon a mathemati-cal model that suggested an increased risk-to-benefit ratio at this time point.65In some patients, the need for an IVC filter may be self-limited. Such patients can be treated with so-called removable IVC filters. Depending on the device, removable IVC filters are potentially removable by percutaneous endovascular tech-niques for up to several months after their initial implantation, assuming the filter is no longer required and does not have

1	IVC filters are potentially removable by percutaneous endovascular tech-niques for up to several months after their initial implantation, assuming the filter is no longer required and does not have large amounts of trapped thrombi. IVC filters that have been in place for an extended period of time may require adjunctive techniques, including laser-assisted removal or open surgical removal when they are embedded within the vena cava. All tem-porary IVC filters are approved for permanent implantation, and many so-called temporary filters end up as permanent devices with all the potential complications of permanent IVC filters.Operative Venous Thrombectomy. In patients with acute iliofemoral DVT, surgical therapy is generally reserved for patients who worsen with anticoagulation therapy and those with phlegmasia cerulea dolens and impending venous gan-grene. If the patient has phlegmasia cerulea dolens, a fasciotomy of the calf compartments is first performed. In iliofemoral DVT, a

1	and those with phlegmasia cerulea dolens and impending venous gan-grene. If the patient has phlegmasia cerulea dolens, a fasciotomy of the calf compartments is first performed. In iliofemoral DVT, a longitudinal venotomy is made in the common femoral vein, and a venous balloon embolectomy catheter is passed through the thrombus into the IVC and pulled back several times until no further thrombus can be extracted. The distal thrombus in the leg is removed by manual pressure beginning in the foot. This is accomplished by application of a tight rubber elastic wrap beginning at the foot and extending to the thigh. If the thrombus in the femoral vein is old and cannot be extracted, the vein may be ligated. For a thrombus that extends into the IVC, the IVC is exposed transperitoneally and controlled below the renal veins. The IVC is opened, and the thrombus is removed by gentle mas-sage. An intraoperative completion venogram determines if any residual thrombus or stenosis is present. If a

1	below the renal veins. The IVC is opened, and the thrombus is removed by gentle mas-sage. An intraoperative completion venogram determines if any residual thrombus or stenosis is present. If a residual iliac vein stenosis is present, intraoperative angioplasty and stenting can be performed. In most cases, an arteriovenous fistula is then created by anastomosing the great saphenous vein (GSV) end to side with the superficial femoral artery in an effort to main-tain patency of the thrombectomized iliofemoral venous seg-ment. Heparin is administered postoperatively for several days. Warfarin anticoagulation is maintained for at least 6 months after thrombectomy. Complications of iliofemoral thrombec-tomy include PE in up to 20% of patients67 and death in <1% of patients.68One study followed 77 limbs for a mean of 8.5 years after thrombectomy for acute iliofemoral DVT. In limbs with suc-cessful thrombectomy, valvular competence in the thrombecto-mized venous segment was 80% at 5 years and

1	limbs for a mean of 8.5 years after thrombectomy for acute iliofemoral DVT. In limbs with suc-cessful thrombectomy, valvular competence in the thrombecto-mized venous segment was 80% at 5 years and 56% at 10 years. More than 90% of patients had minimal or no symptoms of PTS. There were 12 (16%) early thrombectomy failures. Patients were required to wear compression stockings for at least 1 year after thrombectomy.69Survival rates for surgical pulmonary embolectomy have improved over the past 20 years with the addition of cardio-pulmonary bypass. Emergency pulmonary embolectomy for acute PE is rarely indicated. Patients with preterminal massive PE (Fig. 24-10) for whom thrombolysis has failed or who have contraindications to thrombolytics may be candidates for this procedure. Open pulmonary artery embolectomy is performed through a posterolateral thoracotomy with direct visualization of the pulmonary arteries. Mortality rates range between 20% and 40%.70-72Percutaneous catheter-based

1	artery embolectomy is performed through a posterolateral thoracotomy with direct visualization of the pulmonary arteries. Mortality rates range between 20% and 40%.70-72Percutaneous catheter-based techniques for removal of a PE involve mechanical thrombus fragmentation or embolec-tomy using suction devices. Mechanical clot fragmentation is followed by CDT. Results of catheter-based fragmentation are based on small case series. In a study in which a fragmentation device was used in 10 patients with acute massive PE, frag-mentation was successful in 7 patients with a mortality rate of 20%.73 Transvenous catheter pulmonary suction embolectomy has also been performed for acute massive PE with a reported 76% successful extraction rate and a 30-day survival of 70%.74ProphylaxisPatients who undergo major general surgical, gynecologic, urologic, and neurosurgical procedures without thrombopro-phylaxis have a significant incidence of perioperative DVT. An estimated one-third of the 150,000 to

1	major general surgical, gynecologic, urologic, and neurosurgical procedures without thrombopro-phylaxis have a significant incidence of perioperative DVT. An estimated one-third of the 150,000 to 200,000 VTE-related deaths per year in the United States occur following surgery.75 The goal of prophylaxis is to reduce the mortality and morbidity associated with VTE. The first manifestation of VTE may be a life-threatening PE (Fig. 24-11), and as indicated earlier, clinical evaluation to detect DVT before PE is unreliable.Effective methods of VTE prophylaxis involve the use of one or more pharmacologic or mechanical modalities. Cur-rently available pharmacologic agents include low-dose UFH, LMWH, synthetic pentasaccharides, and vitamin K antago-nists. Mechanical methods include intermittent pneumatic com-pression (IPC) and graduated compression stockings. There is insufficient evidence to consider aspirin alone as adequate DVT prophylaxis. Methods of prophylaxis vary with regard to

1	pneumatic com-pression (IPC) and graduated compression stockings. There is insufficient evidence to consider aspirin alone as adequate DVT prophylaxis. Methods of prophylaxis vary with regard to efficacy, and the 2012 ACCP Clinical Practice Guidelines strat-ify their uses according to the patient’s level of VTE risk, bleed-ing risk, and the values and preferences of individual patients (see Table 24-3).Venous Thromboembolism Prophylaxis in Nonorthopedic Surgery. The risk for VTE associated with a surgical procedure depends on the type of operation, type of anesthesia, duration of surgery, and other risk factors, such as patient age, presence of cancer, prior VTE, obesity, presence of infection, and known thrombophilic disorders. VTE risk can be stratified according to Brunicardi_Ch24_p0981-p1008.indd 99222/02/19 3:01 PM 993VENOUS AND LYMPHATIC DISEASECHAPTER 24Figure 24-11. Computed tomography angiogram showing mul-tiple pulmonary embolisms (arrows). (Used with permission from Dr.

1	99222/02/19 3:01 PM 993VENOUS AND LYMPHATIC DISEASECHAPTER 24Figure 24-11. Computed tomography angiogram showing mul-tiple pulmonary embolisms (arrows). (Used with permission from Dr. Scott Ambruster.)Table 24-5Risk assessment model from the Patient Safety in Surgery StudyRISK FACTORRISK SCORE POINTSOperation type other than endocrine Respiratory and hernia Thoracoabdominal aneurysm, embolectomy/thrombectomy, venous reconstruction, and endovascular repair Aneurysm Mouth, palate Stomach, intestines Integument Hernia9744432ASA, physical status classification 3,4, or 5 2 Female sex211Work RVU >17 10–1732Two points for each of these conditions Disseminated cancer Chemotherapy for malignancy within 30 days of operation Preoperative serum sodium >145 mmol/L Transfusion >4 units packed RBCs in 72 hours before operation Ventilator dependent2One point for each of these conditions Wound class (clean/contaminated) Preoperative hematocrit ≤38% Preoperative bilirubin >1 mg/dL Dyspnea Albumin

1	in 72 hours before operation Ventilator dependent2One point for each of these conditions Wound class (clean/contaminated) Preoperative hematocrit ≤38% Preoperative bilirubin >1 mg/dL Dyspnea Albumin ≤3.5 mg/dL Emergency1Zero points for each of these conditions ASA physical class of 1 Work RVU <10 Male sex0ASA = American Society of Anesthesiologists; RBCs = red blood cells; RVU = relative value unit.Reproduced with permission from Rogers SO Jr, Kilaru RK, Hosokawa P, et al: Multivariable predictors of postoperative venous thromboembolic events after general and vascular surgery: results from the patient safety in surgery study, J Am Coll Surg. 2007 Jun;204(6):1211-1221.the previously mentioned risk assessment models, the Caprini score and Rogers score. These risk assessment models are included in the prophylaxis guidelines for nonorthopedic sur-gery (Tables 24-5 and 24-6). A composite score is created using assigned values for each risk factor. The cumulative score for each patient is

1	in the prophylaxis guidelines for nonorthopedic sur-gery (Tables 24-5 and 24-6). A composite score is created using assigned values for each risk factor. The cumulative score for each patient is then used to predict thrombosis risk and provide recommendations regarding VTE prophylaxis.Patients at very low risk (<0.5%; Rogers score <7; Caprini score 0) who undergo general or abdominopelvic procedures do not require pharmacologic or mechanical pro-phylaxis; however, early ambulation is required. Patients at low risk (<1.5%; Rogers score 7–10; Caprini score 1–2) should receive mechanical prophylaxis. Patients at moderate risk (3%; Rogers score >10; Caprini score 3–4) should receive LMWH at recommended doses, low-dose UFH, or mechanical pro-phylaxis. Patients at high risk (6%; Caprini score ≥5) should receive LMWH at recommended doses or low-dose UFH and mechanical prophylaxis. Thromboprophylaxis should con-tinue until discharge, except in select high-risk patients with malignancy in whom

1	should receive LMWH at recommended doses or low-dose UFH and mechanical prophylaxis. Thromboprophylaxis should con-tinue until discharge, except in select high-risk patients with malignancy in whom extended-duration prophylaxis (up to 4–6 weeks) may be beneficial. Patients with significant risk for bleeding should receive mechanical prophylaxis until this risk subsides.75Overall, low-dose UFH and LMWH reduce the risk for symptomatic and asymptomatic VTE by 60% to 70%. The risks for bleeding differ, depending on the dosage. Lower dosages of LMWH appear to be associated with less bleeding risk than low-dose UFH, but the latter produces less bleeding risk than higher prophylactic dosages of LMWH.76 Other advantages of LMWH include once-daily dosing protocols and a lower rate of heparin-associated antibody formation.Fondaparinux has been compared with the LMWH dalte-parin in patients who undergo high-risk major abdominal sur-gery. It also has been compared with IPC alone in patients

1	antibody formation.Fondaparinux has been compared with the LMWH dalte-parin in patients who undergo high-risk major abdominal sur-gery. It also has been compared with IPC alone in patients undergoing non–high-risk abdominal surgery.77,78 Fondaparinux demonstrated rates of VTE prevention, bleeding complications, and mortality similar to those of LMWH. It was more beneficial than IPC alone in reducing VTE but with a higher rate of bleed-ing (1.6% vs. 0.2%).Prophylactic insertion of IVC filters has been suggested for VTE prophylaxis in high-risk trauma patients, bariatric surgical patients, and some patients with malignancy who have contraindications for LMWH therapy.79 A 5-year study of prophylactic IVC filter placement in 132 trauma patients at high risk of PE (head injury, spinal cord injury, pelvic or Brunicardi_Ch24_p0981-p1008.indd 99322/02/19 3:01 PM 994SPECIFIC CONSIDERATIONSPART IITable 24-6Caprini risk assessment model1 POINT2 POINTS3 POINTS5 POINTSAge 41–60Age 61–74Age

1	injury, pelvic or Brunicardi_Ch24_p0981-p1008.indd 99322/02/19 3:01 PM 994SPECIFIC CONSIDERATIONSPART IITable 24-6Caprini risk assessment model1 POINT2 POINTS3 POINTS5 POINTSAge 41–60Age 61–74Age ≥75Stroke (<1 month)Minor surgeryArthroscopic surgeryHistory of VTEElective arthroplastyBMI >25 kg/m2Major open surgery (> 45 minutes)Family history of VTEHip, pelvis, or leg fractureSwollen legsLaparoscopic surgery (> 45 minutes)Factor V LeidenAcute spinal cord injury (<1 month)Varicose veinsMalignancyProthrombin 20210A Pregnancy or postpartumConfined to bed (>72 hours)Lupus anticoagulant History of unexplained or recurrent spontaneous abortionImmobilizing plaster castAnticardiolipin antibody Oral contraceptives of hormone replacementCentral venous accessElevated serum homocysteine Sepsis (<1 month) Heparin-induced thrombocytopenia Serious lung disease, including pneumonia (<1 month) Other congenital or acquired thrombophilia Abnormal pulmonary function test Acute myocardial

1	(<1 month) Heparin-induced thrombocytopenia Serious lung disease, including pneumonia (<1 month) Other congenital or acquired thrombophilia Abnormal pulmonary function test Acute myocardial infarction Congestive heart failure History of inflammatory bowel disease Medical patient at bed rest BMI = body mass index; VTE = venous thromboembolism.Data from Bahl V, Hu HM, Henke PK, et al: A validation study of retrospective venous thromboembolism risk scoring method, Ann Surg. 2010 Feb; 251(2):344-350.long bone fractures) reported a 0% incidence of symptom-atic PE in patients with a correctly positioned IVC filter.80 In 47 patients with a malpositioned IVC filter (strut malposition or filter tilt), there was a 6.3% incidence of symptomatic PE with three deaths. DVT occurred at the insertion site in 3.1% of the patients. IVC patency was 97.1% at 3 years.Fatal and nonfatal PE can still occur in patients with vena cava interruption. As noted earlier, long-term complications

1	the insertion site in 3.1% of the patients. IVC patency was 97.1% at 3 years.Fatal and nonfatal PE can still occur in patients with vena cava interruption. As noted earlier, long-term complications associated with permanent IVC filters include IVC thrombo-sis and DVT. Currently, the ACCP recommends IVC filters be placed only if a proximal DVT is present and anticoagula-tion therapy is contraindicated. Placement of an IVC filter in the setting of severe pulmonary embolism development while anticoagulated remains controversial. IVC filter insertion is not recommended for primary prophylaxis.75Removable IVC filters may be placed in patients with a temporarily increased risk of PE.81 The best patient groups for retrievable filter placement may include young trauma patients with transient immobility, patients undergoing surgi-cal procedures associated with a high risk of PE, and patients with hypercoagulable states who cannot receive anticoagula-tion therapy for a short period of time.

1	patients undergoing surgi-cal procedures associated with a high risk of PE, and patients with hypercoagulable states who cannot receive anticoagula-tion therapy for a short period of time. Careful follow-up is required to assure all potentially removable filters are in fact removed.OTHER VENOUS THROMBOTIC DISORDERSSuperficial Vein ThrombophlebitisSuperficial vein thrombophlebitis (SVT) most commonly occurs in varicose veins but can occur in normal veins. When SVT recurs at variable sites in normal superficial veins, thrombophlebitis migrans, it may signify a hidden visceral malignancy or a systemic disorder such as a blood dyscrasia and/or a collagen vascular disease. SVT also frequently occurs as a complication of indwelling catheters, with or without asso-ciated extravasation of injected material. Upper extremity vein thrombosis has been reported to occur in 38% of patients with peripherally inserted central catheters; 57% of these developed in the cephalic vein (Fig. 24-12).82

1	material. Upper extremity vein thrombosis has been reported to occur in 38% of patients with peripherally inserted central catheters; 57% of these developed in the cephalic vein (Fig. 24-12).82 Suppurative SVT may occur in veins with indwelling catheters and may be associated with generalized sepsis.Clinical signs of SVT include redness, warmth, and ten-derness along the distribution of the affected veins, often asso-ciated with a palpable cord. Patients with suppurative SVT may have fever and leukocytosis. DUS should be performed in patients with signs and symptoms of acute SVT to confirm the diagnosis and to determine if any associated DVT is present. Concomitant lower extremity DVT may be present in 5% to 40% of patients with SVT; most occur in patients with greater Brunicardi_Ch24_p0981-p1008.indd 99422/02/19 3:01 PM 995VENOUS AND LYMPHATIC DISEASECHAPTER 24Figure 24-12. Duplex ultrasound of a brachial vein containing thrombus and percutaneously inserted central catheter

1	99422/02/19 3:01 PM 995VENOUS AND LYMPHATIC DISEASECHAPTER 24Figure 24-12. Duplex ultrasound of a brachial vein containing thrombus and percutaneously inserted central catheter (PICC).Figure 24-13. Upper extremity venogram showing stenosis of the right subclavian vein at the first rib (arrow).saphenous vein SVT within 1 cm of the saphenofemoral junction. A follow-up DUS should be performed in 5 to 7 days in patients with SVT in the proximal GSV but without deep vein involvement. Approximately 10% to 20% of patients with SVT involving the proximal GSV experience progression to deep venous involvement within 1 week.83,84Treatment of SVT is quite variable. A Cochrane Review reported that LMWHs and nonsteroidal anti-inflammatory drugs both reduce the rate of SVT extension or recurrence.85 Addi-tionally, a multicenter, randomized, blinded trial comparing use of fondaparinux to placebo in lower extremity SVT found use of fondaparinux reduced the rate of VTE formation by 85%, though the

1	Addi-tionally, a multicenter, randomized, blinded trial comparing use of fondaparinux to placebo in lower extremity SVT found use of fondaparinux reduced the rate of VTE formation by 85%, though the incidence of VTE formation was low in both groups (0.2% vs. 1.3%, P <.001). The number needed to treat to prevent one episode of VTE was 88 patients. There was no difference in mortality between the two arms of the trial.85Topical medications appear to improve local symptoms. Surgical treatment, combined with the use of graduated com-pression stockings, is associated with a lower rate of VTE and SVT progression.86 The treatment is individualized and depends on the location of the thrombus and the severity of symptoms. In patients with SVT not within 1 cm of the saphenofemoral junction, treatment consists of compression and administra-tion of an anti-inflammatory medication such as indomethacin. In patients with suppurative SVT, antibiotics and removal of any existing indwelling catheters

1	consists of compression and administra-tion of an anti-inflammatory medication such as indomethacin. In patients with suppurative SVT, antibiotics and removal of any existing indwelling catheters are mandatory. Excision of the vein may be necessary but is usually reserved for patients with systemic symptoms or when excision of the involved vein is straightforward. If the SVT extends proximally to within 1 cm of the saphenofemoral junction, extension into the com-mon femoral vein is more likely to occur. In these patients, anticoagulation therapy for 6 weeks and GSV ligation appear equally effective in preventing thrombus extension into the deep venous system.87,88Upper Extremity Vein ThrombosisAxillary-subclavian venous thrombosis (ASVT) is classified into two forms. Primary ASVT occurs in only a small minority of all patients with ASVT. In the primary form, no clear cause for the thrombosis is readily identifiable at initial evaluation. Patients with primary ASVT often give a

1	in only a small minority of all patients with ASVT. In the primary form, no clear cause for the thrombosis is readily identifiable at initial evaluation. Patients with primary ASVT often give a history of performing prolonged, repetitive motion activities, which results in damage to the subclavian vein, usually where it passes between the head of the clavicle and the first rib in association with the subclavius muscle. This condition is also known as venous thoracic outlet syndrome, effort thrombosis, and Paget-Schroetter syndrome. Secondary ASVT is more common and is associated with an easily identified cause such as an indwelling catheter or a hyper-coagulable state. Over 30% of patients with tunneled subclavian vein access devices develop ASVT.89A patient with ASVT may be asymptomatic or may pres-ent with varying degrees of upper extremity edema, tenderness, and conspicuous superficial venous enlargement. DUS can be performed initially to confirm the diagnosis, but limitations to

1	or may pres-ent with varying degrees of upper extremity edema, tenderness, and conspicuous superficial venous enlargement. DUS can be performed initially to confirm the diagnosis, but limitations to the exam by the clavicle and collateralization can lead to a false-negative study. Venography is recommended when there is nonconcordance between the duplex study and clinical suspi-cion. Anticoagulation therapy should be initiated once ASVT is diagnosed to prevent PE and decrease symptoms.Treatment of patients with primary upper extremity venous thrombosis is controversial because the natural history of the disease may vary from minimal to no symptoms to significant symptoms with vigorous upper extremity activities. In recent years, patients presenting with acute symptomatic primary ASVT are often considered candidates for CDT therapy with the goal of minimizing long-term symptoms of venous congestion. Venography is performed through a catheter placed using an ultrasound-guided

1	ASVT are often considered candidates for CDT therapy with the goal of minimizing long-term symptoms of venous congestion. Venography is performed through a catheter placed using an ultrasound-guided percutaneous basilic vein approach to docu-ment the extent of the thrombus (Fig. 24-13). A guidewire is traversed through the thrombus, and a catheter is placed within the thrombus. Typically, tissue plasminogen activator is admin-istered through a multi–side-hole infusion catheter. Various catheter-based mechanical techniques may also be employed to speed thrombus removal. Heparin is administered concurrently with the thrombolytic infusion. After completion of thrombo-lytic therapy, a follow-up venogram is obtained. Correctable anatomic abnormalities may then be considered for treatment. Adjuvant procedures after thrombolytic therapy may include Brunicardi_Ch24_p0981-p1008.indd 99522/02/19 3:01 PM 996SPECIFIC CONSIDERATIONSPART IIcervical or first rib resection for thoracic outlet

1	procedures after thrombolytic therapy may include Brunicardi_Ch24_p0981-p1008.indd 99522/02/19 3:01 PM 996SPECIFIC CONSIDERATIONSPART IIcervical or first rib resection for thoracic outlet abnormalities, scalenectomy, surgical venous reconstruction, and balloon angioplasty of residual venous stenosis.90 The ACCP guidelines recommend the same intensity and duration of anticoagulant therapy in patients who undergo thrombolysis as in patients who are treated with anticoagulation alone.Mesenteric Vein ThrombosisFive percent to 15% of cases of acute mesenteric ischemia occur as a result of mesenteric vein thrombosis (MVT). Mortality rates in patients with MVT may approach 50%.91 The usual present-ing symptom is nonspecific abdominal pain and distention, often accompanied by nausea, vomiting, and diarrhea.92 Perito-neal signs, suggesting intestinal infarction, are present in fewer than half of MVT patients. MVT is more common in patients with a hypercoagulable states, malignancy, and

1	and diarrhea.92 Perito-neal signs, suggesting intestinal infarction, are present in fewer than half of MVT patients. MVT is more common in patients with a hypercoagulable states, malignancy, and cirrhosis. MVT also occurs as a rare complication of laparoscopic surgery.92,93Most cases of MVT are diagnosed with contrast-enhanced CT scanning or magnetic resonance imaging (MRI) in the course of an evaluation for abdominal pain. The sensitivity and speci-ficity for CT and MRI approach 100% and 98%, respectively.94 Ultrasound can also be used and has reported sensitivity and specificity of 93% and 99%, respectively.Patients with MVT are treated with fluid resuscitation, heparin anticoagulation, and bowel rest. Once the patient’s clinical status improves, oral intake can be carefully started. The patient is transitioned to oral anticoagulation over 3 to 4 days and, depending on the etiology of the MVT, continued for 3 to 6 months or indefinitely. Most patients with MVT can be treated

1	The patient is transitioned to oral anticoagulation over 3 to 4 days and, depending on the etiology of the MVT, continued for 3 to 6 months or indefinitely. Most patients with MVT can be treated nonoperatively, but urgent laparotomy is indicated in patients with peritoneal findings. Broad-spectrum antibiotics are admin-istered perioperatively. Operative findings consist of edema and cyanotic discoloration of the mesentery and bowel wall. In more advanced cases, thrombus involves the distal mesenteric veins. The arterial supply to the involved bowel is usually intact. Nonviable bowel is resected, and primary anastomosis can be performed. If the viability of the remaining bowel is in question, a second-look operation is performed within 24 to 48 hours.VARICOSE VEINSVaricose veins are common and are present in at least 10% of the general population.95 The findings of varicose veins may include dilated and tortuous veins, telangiectasias, and fine reticular varicosities. Risk factors for

1	and are present in at least 10% of the general population.95 The findings of varicose veins may include dilated and tortuous veins, telangiectasias, and fine reticular varicosities. Risk factors for varicose veins include obesity, female sex, inactivity, and family history.96 Varicose veins can be classified as primary or secondary. Primary vari-cose veins result from intrinsic abnormalities of the venous wall, whereas secondary varicose veins are associated with deep and/or superficial venous insufficiency.Patients with varicose veins may complain of unsightly appearance, aching, heaviness, pruritus, and early fatigue of the affected leg. These symptoms worsen with prolonged standing and sitting and are relieved by elevation of the leg above the level of the heart. A mild amount of edema is often present. More severe signs include thrombophlebitis, hyperpigmentation, lipodermato-sclerosis, ulceration, and bleeding from attenuated vein clusters.An important component of treatment for

1	is often present. More severe signs include thrombophlebitis, hyperpigmentation, lipodermato-sclerosis, ulceration, and bleeding from attenuated vein clusters.An important component of treatment for patients with vari-cose veins is the use of elastic compression stockings. Patients may be prescribed elastic stockings with compression ranging from 20 to 30, 30 to 40, or even 40 to 50 mmHg. Stockings range in length from knee high to waist high, and they should cover the symptomatic varices. Elastic compression provides sufficient relief of symptoms in many symptomatic patients.Cosmetic concerns may lead to intervention. Addition-ally, interventions are warranted in patients whose symptoms worsen or are unrelieved despite compression therapy or who have lipodermatosclerosis or venous ulcer. Randomized trials of symptomatic patients with varicose veins have demonstrated improved quality of life with interventional treatment. Interven-tional management includes injection sclerotherapy,

1	Randomized trials of symptomatic patients with varicose veins have demonstrated improved quality of life with interventional treatment. Interven-tional management includes injection sclerotherapy, thermal ablation, surgical therapy, or a combination of these techniques. Injection sclerotherapy alone can be successful in varicose veins and in telangiectatic vessels. A recent multicenter, randomized trial compared foam sclerotherapy versus placebo for symp-tomatic varicose veins found significant symptom relief and improved cosmetic appearance with sclerotherapy.97 Sclero-therapy acts by destroying the venous endothelium. Scleros-ing agents include hypertonic saline, sodium tetradecyl sulfate, and polidocanol. Concentrations of 11.7% to 23.4% hypertonic saline, 0.125% to 0.250% sodium tetradecyl sulfate, and 0.5% polidocanol are used for telangiectasias. Larger varicose veins require higher concentrations: 23.4% hypertonic saline, 0.50% to 1% sodium tetradecyl sulfate, and 0.75% to 1.0%

1	sulfate, and 0.5% polidocanol are used for telangiectasias. Larger varicose veins require higher concentrations: 23.4% hypertonic saline, 0.50% to 1% sodium tetradecyl sulfate, and 0.75% to 1.0% polidocanol.92 Elastic bandages are wrapped around the leg after injection and worn continuously for 3 to 5 days to produce apposition of the inflamed vein walls and prevent thrombus formation. After the bandages are removed, elastic compression stockings should be worn for a minimum of 2 weeks. Complications from sclero-therapy include allergic reaction, local hyperpigmentation, thrombophlebitis, DVT, and possible skin necrosis.Newer devices combine sclerotherapy with catheter-based mechanical endoluminal injury to achieve nonthermal ablation.98 Additional nonthermal, nonsclerosant ablative techniques using proprietary adhesive formulations with cyanoacrylate are current being evaluated and have demonstrated promising early results.99Patients with symptomatic GSV or SSV reflux may be treated

1	using proprietary adhesive formulations with cyanoacrylate are current being evaluated and have demonstrated promising early results.99Patients with symptomatic GSV or SSV reflux may be treated with endovenous ablation techniques or surgical removal of the affected vein. Endovenous laser and radiofrequency abla-tion (RFA) techniques have gained in popularity in the past several years. Such techniques are generally associ-ated with equally effective but more rapid postprocedure recov-ery than traditional open surgical stripping of the GSV.With either endoluminal technique, the distal thigh or proximal calf GSV is punctured with a 21-gauge needle under ultrasound guidance. A sheath is placed over a guidewire, and the laser fiber or RFA catheter is advanced until it is near to, but not at, the saphenofemoral junction. Tumescent anesthetic is administered around the GSV, and the vein is treated as the catheter is withdrawn. Endovenous laser treatment and RFA result in durable ablation of

1	saphenofemoral junction. Tumescent anesthetic is administered around the GSV, and the vein is treated as the catheter is withdrawn. Endovenous laser treatment and RFA result in durable ablation of the GSV, with rates of varicose vein recurrence and clinical severity scores comparable to those seen with open surgery.100,101 Risks of endovenous ablation include DVT, ecchymosis, and saphenous nerve injury.Saphenous vein ligation and stripping may still be the pre-ferred therapy for patients with GSVs of very large diameter (>2 cm). Surgical removal of the GSV usually is performed via small incisions placed medially in the groin and just below the knee. The GSV is removed using a blunt tip catheter or an invagi-nation pin stripper. Complications associated with GSV stripping include ecchymosis, hematoma, lymphocele, DVT, infection, and saphenous nerve injury. GSV stripping is associated with a lower rate of recurrence of varicose veins and a better quality of life than saphenofemoral

1	hematoma, lymphocele, DVT, infection, and saphenous nerve injury. GSV stripping is associated with a lower rate of recurrence of varicose veins and a better quality of life than saphenofemoral junction ligation alone.4Brunicardi_Ch24_p0981-p1008.indd 99622/02/19 3:01 PM 997VENOUS AND LYMPHATIC DISEASECHAPTER 24Figure 24-14. Removal of varicose veins via stab avulsions.Larger varicose veins are best treated by surgical exci-sion using the “stab avulsion” technique. Stab avulsions are performed by making 2-mm incisions directly over branch varicosities, and the varicosity is dissected from the surrounding subcutaneous tissue as far proximally and distally as possible through the small incisions (Fig. 24-14). In most cases the vein is simply avulsed with no attempt at ligation. Bleeding is easily controlled with leg elevation, manual compression, and prepro-cedure tumescent anesthesia.CHRONIC VENOUS INSUFFICIENCYChronic venous insufficiency (CVI) affects an estimated 600,000 people

1	is easily controlled with leg elevation, manual compression, and prepro-cedure tumescent anesthesia.CHRONIC VENOUS INSUFFICIENCYChronic venous insufficiency (CVI) affects an estimated 600,000 people in the United States.102 Patients complain of leg fatigue, discomfort, and heaviness. Signs of CVI may include varicose veins, pigmentation, lipodermatosclerosis, and venous ulceration. Importantly, severe CVI is not necessarily associ-ated with varicose veins. Chronic venous ulcers carry significant negative physical, financial, and psychological implications. A quality-of-life study reported that 65% of patients with chronic leg ulcers had severe pain, 81% had decreased mobility, and 100% experienced a negative impact of their disease on their work capacity.103 The socioeconomic impact of chronic venous leg ulcers is staggering, with an estimated 2 million workdays lost per year.102 The annual healthcare cost in the United States to treat CVI is estimated at $1 billion.105CVI can be

1	chronic venous leg ulcers is staggering, with an estimated 2 million workdays lost per year.102 The annual healthcare cost in the United States to treat CVI is estimated at $1 billion.105CVI can be primary or secondary. Primary CVI results from intrinsic abnormalities of the vein wall, whereas second-ary CVI, so-called postthrombotic syndrome (PTS), occurs as a result of DVT. The signs and symptoms of CVI can therefore be attributed to venous reflux, venous obstruction, calf muscle pump dysfunction, or a combination of these factors, as well as loss of venous wall elasticity.106 In the majority of patients with CVI, the most important factor appears to be venous reflux, although most severe cases tend to have an obstructive etiology as well. Venous reflux results from abnormalities of the venous valve. Primary valvular reflux or incompetence is diagnosed when there is no known underlying cause of valvular dysfunction. Secondary valvular reflux is diagnosed when an identifiable cause

1	venous valve. Primary valvular reflux or incompetence is diagnosed when there is no known underlying cause of valvular dysfunction. Secondary valvular reflux is diagnosed when an identifiable cause is present. The most frequent secondary cause is DVT.Evaluation of Venous InsufficiencyEarly diagnostic studies to evaluate CVI required invasive mea-surements of ambulatory venous pressure (AVP) and venous recovery time (VRT). To measure AVP and VRT, a needle is inserted into a dorsal foot vein and connected to a pressure transducer. The patient is asked to perform 10 tiptoe exercises. Initially there is often a slight upward deflection of pressure with the onset of exercise followed by a decline in pressure with each subsequent tiptoe maneuver. After approximately 10 tiptoes, the measured pressure stabilizes and reflects a bal-ance of venous inflow and outflow. The pressure at this point is the AVP, which is measured in millimeters of mercury. The patient is then asked to stop exercising

1	stabilizes and reflects a bal-ance of venous inflow and outflow. The pressure at this point is the AVP, which is measured in millimeters of mercury. The patient is then asked to stop exercising to allow the vein to fill with return of the venous pressure to baseline. The time required for the venous pressure to return from the AVP level to 90% of baseline pressure is referred to as the VRT. A normal VRT typically ranges from 20 to 60 seconds. Values less than 20 seconds indicate significant reflux with increasing severity. To distinguish between superficial and deep venous reflux, a thigh tourniquet can be placed inflated to 50 mmHg to eliminate influence of the superficial venous. A VRT that remains below 20 seconds after tourniquet inflation suggests both superficial and deep venous reflux. Elevations of AVP indicate venous hypertension. The magnitude of AVP reflects the severity of CVI. There is an 80% incidence of venous ulceration in patients with an AVP of >80

1	deep venous reflux. Elevations of AVP indicate venous hypertension. The magnitude of AVP reflects the severity of CVI. There is an 80% incidence of venous ulceration in patients with an AVP of >80 mmHg.107Plethysmography. Noninvasive plethysmography has been used to evaluate CVI. Venous photoplethysmography indirectly evaluates venous function through the use of infrared light. A light-emitting diode is placed just above the medial malleolus, and the patient then performs a series of tiptoe maneuvers. Pho-toplethysmography provides a measurement of VRT. In limbs with CVI, VRT is shortened compared with that in a normal limb. AVP and VRT are only measures of the overall venous function of a lower extremity venous system. They cannot local-ize the site of reflux or evaluate the function of the calf pump.Air plethysmography is a theoretically attractive but not widely used method to assess calf pump function, venous reflux, and overall lower extremity venous function.108 An air-filled

1	the calf pump.Air plethysmography is a theoretically attractive but not widely used method to assess calf pump function, venous reflux, and overall lower extremity venous function.108 An air-filled plastic pressure bladder is placed on the calf to detect volume changes in the leg during a standard set of maneuvers. The patient is first supine, and then the leg is elevated and the minimum volume of venous blood recorded. The patient is then asked to assume an upright position with the examined leg non-weight bearing. The venous volume of the examined leg is determined when the volume curve flattens. The venous fill-ing index (VFI), a measure of reflux, is calculated by dividing the maximum venous volume by the time required to achieve maximum venous volume. Next, the patient performs a single tiptoe maneuver, and the ejection fraction (EF) is determined. The EF is the volume change between the recorded volume before and after the tiptoe maneuver and is a measure of calf pump function.

1	tiptoe maneuver, and the ejection fraction (EF) is determined. The EF is the volume change between the recorded volume before and after the tiptoe maneuver and is a measure of calf pump function. At this point, the veins of the leg are allowed to refill. The patient then performs 10 tiptoe maneuvers, and the residual volume fraction is calculated by dividing the venous volume in the leg after 10 tiptoe exercises by the venous volume present before the exercises. The residual volume fraction is a reflection of overall venous function. Theoretically, patients with increased VFIs and normal EFs (indicating the presence of reflux with normal calf pump function) would benefit from anti-reflux surgery, whereas patients with normal VFIs and dimin-ished EFs would not.Venous Duplex Ultrasound. Venous DUS has become the gold standard for evaluation of venous function largely supplanting venographic and plethysmographic techniques. Brunicardi_Ch24_p0981-p1008.indd 99722/02/19 3:01 PM

1	DUS has become the gold standard for evaluation of venous function largely supplanting venographic and plethysmographic techniques. Brunicardi_Ch24_p0981-p1008.indd 99722/02/19 3:01 PM 998SPECIFIC CONSIDERATIONSPART IIFigure 24-15. Evaluation of a patient with chronic venous insuf-ficiency with duplex ultrasonography.Figure 24-16. Multilayered dressing for treatment of chronic venous insufficiency.The principle advantage of DUS is that it can be used to evalu-ate reflux in individual venous segments targeting abnormal areas for treatment. The examination has been validated when performed with the patient in the standing position and the examined leg non-weight bearing. Pneumatic pressure cuffs of appropriate size are placed around the thigh, calf, and fore-foot. The ultrasound transducer is positioned over the venous segment to be examined, just proximal to the pneumatic cuff (Fig. 24-15). The cuff is then inflated to a standard pressure for 3 seconds and then rapidly deflated.

1	is positioned over the venous segment to be examined, just proximal to the pneumatic cuff (Fig. 24-15). The cuff is then inflated to a standard pressure for 3 seconds and then rapidly deflated. Ninety-five percent of normal venous valves close within 0.5 second.109 The presence of reflux for >0.5 second is considered abnormal. Typically, the common femoral, femoral, popliteal, and posterior tibial veins, as well as the GSV and SSV, are evaluated in a complete examination.Nonoperative Treatment of Chronic Venous Insufficiency Compression Therapy. Compression therapy is the mainstay of CVI management. Compression can be achieved using a vari-ety of techniques, including elastic compression stockings, paste gauze boots (Unna’s boots), multilayer elastic wraps or dressings (Fig. 24-16), and pneumatic compression devices. Nonelastic compression bandages generally achieve higher and more prolonged degrees of compression than elastic compression bandages. The exact mechanism by which

1	and pneumatic compression devices. Nonelastic compression bandages generally achieve higher and more prolonged degrees of compression than elastic compression bandages. The exact mechanism by which compression therapy can improve CVI remains uncertain. An improvement in skin and 5subcutaneous tissue microcirculatory hemodynamics as well as a direct effect on subcutaneous pressure have been hypothesized as the mechanisms of efficacy of compression therapy.110 Addition-ally, compression therapy has demonstrated quantifiable differ-ences in ulcer healing with decreases in matrix metalloproteins and inflammatory cytokines.111,112 Clinically, routine use of elastic and nonelastic bandages reduces lower extremity edema in patients with CVI. In addition, supine perimalleolar subcutaneous pressure has been demonstrated to increase with elastic compression.111 With edema reduction, cutaneous metabolism may improve due to enhanced diffusion of oxygen and other nutrients to the cellular elements

1	has been demonstrated to increase with elastic compression.111 With edema reduction, cutaneous metabolism may improve due to enhanced diffusion of oxygen and other nutrients to the cellular elements of skin and subcutaneous tissues. Increases in subcutaneous tissue pressure with elastic compres-sion bandages may counteract transcapillary Starling forces, which favor leakage of fluid out of the capillary.Before the initiation of therapy for CVI, patients must be educated about their chronic disease and the need to comply with their treatment plan to heal ulcers and prevent recurrence. A definitive diagnosis of venous ulceration must be made before treatment is initiated. A detailed history should be obtained from a patient presenting with lower extremity ulcerations, includ-ing medications used and associated medical conditions that may promote lower extremity ulceration. Arterial insufficiency is assessed by physical examination or noninvasive studies. In addition, systemic conditions

1	used and associated medical conditions that may promote lower extremity ulceration. Arterial insufficiency is assessed by physical examination or noninvasive studies. In addition, systemic conditions that affect wound healing and leg edema, such as diabetes mellitus, immunosuppression, mal-nutrition, and congestive heart failure, should be improved as much as possible.Compression therapy is most commonly achieved with graduated elastic compression stockings. Elastic compression stockings are available in various compositions, strengths, and lengths, and can be customized for a particular patient. The ben-efits of elastic compression stocking therapy for the treatment of CVI and healing of ulcerations have been well documented.114-117 In a retrospective study involving 113 venous ulcer patients,115 the use of below-knee, 30to 40-mmHg elastic compression stockings, after edema and cellulitis were first resolved if pres-ent, resulted in 93% healing. Complete ulcer healing occurred in 99

1	the use of below-knee, 30to 40-mmHg elastic compression stockings, after edema and cellulitis were first resolved if pres-ent, resulted in 93% healing. Complete ulcer healing occurred in 99 of 102 patients (97%) who were compliant with stocking use vs. 6 of 11 patients (55%) who were noncompliant (P <.0001). The mean time to ulcer healing was 5 months. The rate of ulcer recurrence was lower in patients who were compliant with their compression therapy. By life table analysis, ulcer recurrence was 29% at 5 years for compliant patients and 100% at 3 years for noncompliant patients.In addition to promoting ulcer healing, elastic compres-sion therapy can also improve quality of life in patients with CVI. In one prospective study,118 112 patients with CVI docu-mented by DUS were administered a questionnaire to quantify the symptoms of swelling, pain, skin discoloration, cosmesis, activity tolerance, depression, and sleep alterations. Patients were treated with 30to 40-mmHg elastic

1	a questionnaire to quantify the symptoms of swelling, pain, skin discoloration, cosmesis, activity tolerance, depression, and sleep alterations. Patients were treated with 30to 40-mmHg elastic compression stock-ings. There were overall improvements in symptom severity scores at 1 month after initiation of treatment. Further improve-ments were noted at 16 months after treatment.Patient compliance with compression therapy is crucial in treating CVI and especially venous leg ulcers. Many patients are initially intolerant of compression in areas of hypersensitiv-ity adjacent to an active ulcer or at sites of previously healed ulcers. They may also have difficulty applying elastic stockings. To improve compliance, patients should be instructed to wear their stockings initially only as long as it is easily tolerated and Brunicardi_Ch24_p0981-p1008.indd 99822/02/19 3:01 PM 999VENOUS AND LYMPHATIC DISEASECHAPTER 24Figure 24-17. Elastic compression device with Velcro to facilitate

1	long as it is easily tolerated and Brunicardi_Ch24_p0981-p1008.indd 99822/02/19 3:01 PM 999VENOUS AND LYMPHATIC DISEASECHAPTER 24Figure 24-17. Elastic compression device with Velcro to facilitate treatment of chronic venous insufficiency.then gradually to increase the amount of time the stockings are worn. Alternatively, patients can be fitted with lower-strength stockings initially followed by introduction of higher-strength stockings over a period of several weeks. Many commercially available devices, such as silk inner toe liners, adjustable elas-tic compression with Velcro, stockings with zippered sides (Fig. 24-17), and metal fitting aids (Fig. 24-18), are available to assist patients in applying elastic stockings. However, despite all these available adjuncts, many patients remain noncompliant with elastic compression therapy.Another method of compression was developed by the German dermatologist Paul Gerson Unna. Unna’s boot has been used for many years to treat venous

1	remain noncompliant with elastic compression therapy.Another method of compression was developed by the German dermatologist Paul Gerson Unna. Unna’s boot has been used for many years to treat venous ulcers and is available in many versions. A typical Unna’s boot consists of a three-layer dressing and requires application by trained personnel. A rolled gauze bandage impregnated with calamine, zinc oxide, glyc-erin, sorbitol, gelatin, and magnesium aluminum silicate is first applied with graded compression from the forefoot to just below the knee. The next layer consists of a 10-cm-wide continuous gauze dressing followed by an outer layer of elastic wrap, also applied with graded compression. The bandage becomes stiff after drying, and the rigidity may aid in preventing edema for-mation. Unna’s boot is changed weekly or sooner if the patient experiences significant drainage and soiling of the dressing.Once applied, Unna’s boot requires minimal patient involvement and provides

1	Unna’s boot is changed weekly or sooner if the patient experiences significant drainage and soiling of the dressing.Once applied, Unna’s boot requires minimal patient involvement and provides continuous compression and topical therapy. However, Unna’s boot has several disadvantages. It Figure 24-18. Metal fitting aid to assist in placement of elastic compression stockings.is bulky and can be uncomfortable, which may affect patient compliance. In addition, the ulcer cannot be monitored after the boot is applied, the technique is labor intensive, and the degree of compression provided is operator dependent. Occasionally, patients may also develop contact dermatitis to the components of Unna’s boot.The efficacy of Unna’s dressing has been studied. A ret-rospective 15-year survey encompassing 998 patients with one or more venous ulcers treated weekly with Unna’s dressing119 reported that 73% of ulcers healed in patients who returned for more than one treatment. The median time to healing

1	998 patients with one or more venous ulcers treated weekly with Unna’s dressing119 reported that 73% of ulcers healed in patients who returned for more than one treatment. The median time to healing for individual ulcers was 9 weeks. Unna’s dressing has been com-pared to other forms of treatment. A randomized, prospective study comparing Unna’s boot to polyurethane foam dressing in 36 patients with venous ulcers demonstrated superior healing over 12 months in patients treated with Unna’s boot (94.7% vs. 41.2%).120 A recent Cochrane Review of 39 randomized controlled trials demonstrated that compression increases ulcer healing rates compared with no compression, multicomponent systems are more effective than single-component systems, and multicomponent systems that include an elastic bandage are more effective than those composed mainly of inelastic constituents.121Other forms of compression dressing available to treat CVI include multilayered dressings and legging orthoses. The

1	bandage are more effective than those composed mainly of inelastic constituents.121Other forms of compression dressing available to treat CVI include multilayered dressings and legging orthoses. The purported advantages of multilayered dressings include main-tenance of compression for a longer period of time, more even distribution of compression, and better absorption of wound Brunicardi_Ch24_p0981-p1008.indd 99922/02/19 3:01 PM 1000SPECIFIC CONSIDERATIONSPART IIFigure 24-19. Apligraf skin graft material supplied as a disk on an agarose gel nutrient medium.Figure 24-20. Trocar placement for subfascial endoscopic perfo-rator vein surgery. (Used with permission from Dr. Pankaj Patel.)exudates. However, the efficacy of multilayered dressings depends on the wrapping technique of healthcare personnel. A commercially available legging orthosis consisting of multiple adjustable loop-and-hook closure compression bands provides compression similar to that of Unna’s boot and can be applied

1	personnel. A commercially available legging orthosis consisting of multiple adjustable loop-and-hook closure compression bands provides compression similar to that of Unna’s boot and can be applied daily by the patient.122Skin Substitutes. Several types of skin substitutes are com-mercially available or under clinical study in the United States.105 Bioengineered skin ranges in composition from acellular skin substitutes to partial living skin substitutes. Their mechanism of action in healing venous ulcers is uncertain; however, they may serve as delivery vehicles for various growth factors and cytokines important in wound healing.Apligraf is a commercially available bilayered living skin construct that closely approximates human skin for use in the treatment of venous ulcers. It contains a protective stratum corneum and a keratinocyte-containing epidermis overlying a dermis consisting of dermal fibroblasts in a collagen matrix.122 Apligraf is between 0.5 mm and 1.0 mm thick and is

1	a protective stratum corneum and a keratinocyte-containing epidermis overlying a dermis consisting of dermal fibroblasts in a collagen matrix.122 Apligraf is between 0.5 mm and 1.0 mm thick and is supplied as a disk of living tissue on an agarose gel nutrient medium. It must be used within 5 days of release from the manufacturer123 (Fig. 24-19). The disk is easily handled and applied and con-forms to irregularly contoured ulcer beds though it is very costly.A prospective randomized study comparing multilayer compression therapy alone to treatment with Apligraf in addi-tion to multilayered compression therapy has been performed to assess the efficacy of Apligraf in the treatment of venous ulcers.118 More patients treated with Apligraf had ulcer healing at 6 months (63% vs. 49%, P = .02). The median time to com-plete ulcer closure was significantly shorter in patients treated with Apligraf (61 days vs. 181 days, P = .003). The ulcers that showed the greatest benefit with the living skin

1	median time to com-plete ulcer closure was significantly shorter in patients treated with Apligraf (61 days vs. 181 days, P = .003). The ulcers that showed the greatest benefit with the living skin construct were ones that were large and deep (>1000 mm2) or were longstand-ing (>6 months). No evidence of rejection or sensitization has been reported in response to Apligraf application.Surgical/Interventional Treatment of Chronic Venous Insufficiency Perforator Vein Ligation. Incompetence of the perforating veins connecting the superficial and deep venous systems of the lower extremities has been implicated in the development of venous ulcers. The classic open tech-nique described by Linton in 1938 for perforator vein ligation has a high incidence of wound complications and has largely been abandoned.124 A minimally invasive technique termed subfascial endoscopic perforator vein surgery (SEPS) evolved with improvement of endoscopic equipment.DUS is performed preoperatively in patients

1	abandoned.124 A minimally invasive technique termed subfascial endoscopic perforator vein surgery (SEPS) evolved with improvement of endoscopic equipment.DUS is performed preoperatively in patients undergoing SEPS to document deep venous competence and to identify per-forating veins in the posterior compartment. The patient is posi-tioned on the operating table with the affected leg elevated at 45° to 60°. An Esmarch bandage and a thigh tourniquet are used to exsanguinate the limb. The knee is then flexed, and two small incisions are made in the proximal medial leg away from areas of maximal induration at the ankle. Laparoscopic trocars are then positioned, and the subfascial dissection is performed with a combination of blunt and sharp dissection. Carbon dioxide is then used to insufflate the subfascial space. The thigh tourni-quet is inflated to prevent air embolism. The perforators are then identified and doubly clipped and divided. After completion of the procedure, the leg is

1	the subfascial space. The thigh tourni-quet is inflated to prevent air embolism. The perforators are then identified and doubly clipped and divided. After completion of the procedure, the leg is wrapped in a compression bandage for 5 days postoperatively.The efficacy of SEPS as a stand-alone procedure in treatment of venous insufficiency is controversial and unproven. In a report from a large North American registry of 146 patients undergoing SEPS125 (Fig. 24-20), healing was achieved in 88% of ulcers (75 of 85) at 1 year. Adjunctive procedures, primarily superficial vein stripping, were performed in 72% of patients. Ulcer recurrence was predicted to be 16% at 1 year and 28% at 2 years by life table analysis. These results are similar to those achieved in some studies with compression therapy alone. A review of several studies from 2003 to 2011 demonstrated, when taken in aggregate, that 2059 limbs with 896 ulcers underwent SEPS and concomitant saphenous vein ablation (70%) with a 0%

1	alone. A review of several studies from 2003 to 2011 demonstrated, when taken in aggregate, that 2059 limbs with 896 ulcers underwent SEPS and concomitant saphenous vein ablation (70%) with a 0% to 16% complication rate and achieved ulcer healing in 90% of patients.126 There has been a multicenter, prospective, European trial performed in patients with venous ulcers to evaluate the efficacy of SEPS. Post hoc analysis suggested possible benefit for SEPS in certain categories of patients with venous ulcer. Overall, however, primary analysis of the study’s end points indicated no advantage to SEPS in addition to superficial venous surgery and compression in the healing of venous ulcers.127 The technique appears to have fallen out of favor in most institutions with injection sclerotherapy preferred due to ease of use.Superficial Venous Surgery. Currently it is accepted that superficial venous surgery in addition to compression therapy has a role in the treatment of patients with venous

1	due to ease of use.Superficial Venous Surgery. Currently it is accepted that superficial venous surgery in addition to compression therapy has a role in the treatment of patients with venous ulcer. The ESCHAR trial was a randomized prospective trial performed in the United Kingdom to evaluate the combination of superficial Brunicardi_Ch24_p0981-p1008.indd 100022/02/19 3:01 PM 1001VENOUS AND LYMPHATIC DISEASECHAPTER 24Figure 24-21. Patient with severe longstanding lymphedema.venous surgery and compression vs. compression alone in the treatment of venous ulcer. Superficial venous surgery had no additive effect to compression alone in the healing of a venous ulcer but significantly reduced venous ulcer recurrence at 4 years. Based on the results of this trial, it is reasonable to offer ablation or removal of the GSV in addition to compression therapy in patients with abnormal saphenous veins and signs and/or symptoms of severe CVI.128Deep Venous Valvular Reconstruction. In the

1	ablation or removal of the GSV in addition to compression therapy in patients with abnormal saphenous veins and signs and/or symptoms of severe CVI.128Deep Venous Valvular Reconstruction. In the absence of sig-nificant deep vein valvular incompetence, saphenous vein stripping and possibly perforator vein ligation can be effective in the treat-ment of CVI. However, in patients with a combination of superfi-cial and deep vein valvular incompetence, the addition of deep vein valvular reconstruction theoretically may improve ulcer healing.130 Numerous techniques of deep vein valve correction have been reported. These techniques consist of repair of existing valves, transplant of venous segments from the arm, transposition of an incompetent vein onto an adjacent competent vein, and implanta-tion of cryopreserved vein segments including competent valves.Successful long-term outcomes of 60% to 80% have been reported for venous valve reconstructions by internal suture repair.129,130 However,

1	of cryopreserved vein segments including competent valves.Successful long-term outcomes of 60% to 80% have been reported for venous valve reconstructions by internal suture repair.129,130 However, among patients who initially had ulcer-ation, 40% to 50% still had persistence or recurrence of ulcers in the long term.127,129Valve transplantation involves replacement of a segment of incompetent femoral vein or popliteal vein with a segment of axillary or brachial vein with competent valves. Early results are similar to those for venous valve reconstruction.129,130 How-ever, in the long term, the transplanted venous segments tend to develop incompetence, intimal hyperplasia, and cusp sinus thrombosis with long-term outcomes that are poorer than those for venous valve reconstructions. The outcomes for venous transposition are similar to those for valve transplantation.Currently, reconstruction techniques for deep venous insufficiency and associated CVI are rarely performed.Venous

1	The outcomes for venous transposition are similar to those for valve transplantation.Currently, reconstruction techniques for deep venous insufficiency and associated CVI are rarely performed.Venous Stenting. Currently there is great interest in the role of venous stents in the treatment of CVI. Stenotic lesions of the iliac veins, primarily documented with IVUS, are being reported in a very high percentage of patients with edema, lipo-dermatosclerosis, or ulceration secondary to venous disease. It appears possible to percutaneously place stents in the iliac veins with near 100% technical success and excellent patency of the stent out to 4 years. Retrospective case series suggest favorable effects on ulcer healing, symptoms of CVI, and quality of life in patients with CVI. The role of venous stenting as an indepen-dent procedure in the treatment of patients with CVI remains an area of active investigation.131LYMPHEDEMAPathophysiologyLymphedema is extremity swelling that results from a

1	stenting as an indepen-dent procedure in the treatment of patients with CVI remains an area of active investigation.131LYMPHEDEMAPathophysiologyLymphedema is extremity swelling that results from a reduction in lymphatic transport and accumulation of lymph within the interstitial space. It is caused by anatomic and or physiologic abnormalities such as lymphatic hypoplasia, functional insuf-ficiency, or absence of lymphatic valves.The original classification system, described by Allen, is based on the cause of the lymphedema. Primary lymphedema is further subdivided into congenital lymphedema, lymph-edema praecox, and lymphedema tarda. Congenital lymph-edema may involve a single lower extremity, multiple limbs, the genitalia, or the face. The edema typically develops before 2 years 6of age and may be associated with specific hereditary syndromes (Turner syndrome, Milroy syndrome, Klippel-Trénaunay-Weber syndrome). Lymphedema praecox is the most common form of primary lymphedema,

1	age and may be associated with specific hereditary syndromes (Turner syndrome, Milroy syndrome, Klippel-Trénaunay-Weber syndrome). Lymphedema praecox is the most common form of primary lymphedema, accounting for 94% of cases. Lymphedema praecox is far more common in women, with the gender ratio favoring women 10:1. The onset is during childhood or the teen-age years, and the swelling involves the foot and calf. Lymph-edema tarda is uncommon, accounting for <10% of cases of primary lymphedema. The onset of edema is after 35 years of age.Secondary lymphedema is far more common than primary lymphedema. Secondary lymphedema develops as a result of lymphatic obstruction or disruption. Axillary node dissection leading to lymphedema of the arm is the most common cause of secondary lymphedema in the United States. Other causes of secondary lymphedema include radiation therapy, trauma, infection, and malignancy. Globally, filariasis (an infection caused by Wuchereria bancrofti, Brugia malayi,

1	the United States. Other causes of secondary lymphedema include radiation therapy, trauma, infection, and malignancy. Globally, filariasis (an infection caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori) and environmental exposure to minerals in volcanic soil resulting in podoconiosis in barefoot populations are the most common causes of secondary lymphedema.Clinical DiagnosisIn most patients, the diagnosis of lymphedema can be made based on the history and physical examination alone. Patients commonly complain of heaviness and fatigue in the affected extremity. The limb size increases throughout the day and decreases to some extent, usually minimally, over the course of the night when the patient is recumbent. The limb, however, never completely nor-malizes. In the lower extremity, the swelling classically involves the dorsum of the foot, and the toes have a squared-off appear-ance. In advanced cases, hyperkeratosis of the skin develops, and fluid weeps from

1	lower extremity, the swelling classically involves the dorsum of the foot, and the toes have a squared-off appear-ance. In advanced cases, hyperkeratosis of the skin develops, and fluid weeps from lymph-filled vesicles (Fig. 24-21).Brunicardi_Ch24_p0981-p1008.indd 100122/02/19 3:01 PM 1002SPECIFIC CONSIDERATIONSPART IIFigure 24-22. Lymphoscintigraphy of the lower extremity.Figure 24-23. Normal lymphangiogram of the pelvis.Recurrent cellulitis is a common complication of lymph-edema. Repeated infection results in further lymphatic damage, worsening existing disease. The clinical presentation of cellu-litis ranges from subtle erythema and worsening of edema to a rapidly progressive soft tissue infection with systemic toxicity.Many medical conditions can cause edema. If the symp-toms are mild, distinguishing lymphedema from other causes of leg swelling can be difficult. Venous insufficiency is often confused with lymphedema. However, patients with advanced venous insufficiency

1	are mild, distinguishing lymphedema from other causes of leg swelling can be difficult. Venous insufficiency is often confused with lymphedema. However, patients with advanced venous insufficiency typically have lipodermatosclerosis in the gaiter region, skin ulceration, and/or varicose veins. Bilateral pitting edema is typically associated with congestive heart fail-ure, renal failure, or a hypoproteinemic state.Radiologic DiagnosisDuplex Ultrasound. When a patient is evaluated for edema, it is often difficult to distinguish the early stages of lymphedema from venous insufficiency. DUS of the venous system can determine if there is concomitant venous thrombosis or venous reflux, perhaps contributing to extremity edema. The diagnostic modalities discussed in the following sections have limited use in clinical practice. They are invasive and tedious and rarely change the management of a patient with lymphedema. Most physicians rely on the patient’s history and physical examina-tion

1	limited use in clinical practice. They are invasive and tedious and rarely change the management of a patient with lymphedema. Most physicians rely on the patient’s history and physical examina-tion alone to make the diagnosis of lymphedema.Lymphoscintigraphy. Lymphoscintigraphy has become the most commonly, but still overall uncommonly, used diagnostic test to identify lymphatic abnormalities. It has largely replaced lymphangiography. A radiolabeled sulfur colloid (technetium 99 m sulfur colloid) is injected into the subdermal, interdigital region of the affected limb. The lymphatic transport is moni-tored with a whole-body gamma camera, and major lymphatics and nodes can be visualized (Fig. 24-22). In normal individu-als, tracer activity may be detected in the inguinal region within 15 to 60 minutes. Within 3 hours, uptake should be present in the pelvic and abdominal lymph nodes. In patients with lymph-edema, various patterns may be seen on lymphoscintigraphy. There may be delayed

1	to 60 minutes. Within 3 hours, uptake should be present in the pelvic and abdominal lymph nodes. In patients with lymph-edema, various patterns may be seen on lymphoscintigraphy. There may be delayed or absent transport to the inguinal nodes. Increased cutaneous collaterals may be seen with obstruction of the primary axial channels. There may also be localized regions of reduced uptake in patients with prior node dissection or radia-tion therapy.Lymphangiography. Radiologic lymphangiography is per-formed by first visualizing the lymphatics by injecting colored dye into the hand or foot. The visualized lymphatic segment is exposed through a small incision and cannulated with a 27to 30-gauge needle. An oil-based dye is then injected slowly into the lymphatics over several hours. The lym-phatic channels and nodes are then visualized with traditional radiographs (Figs. 24-23 and 24-24). Lymphangiography is reserved for patients with lymphangiectasia or lymphatic fis-tulas, and patients

1	channels and nodes are then visualized with traditional radiographs (Figs. 24-23 and 24-24). Lymphangiography is reserved for patients with lymphangiectasia or lymphatic fis-tulas, and patients who are being considered for microvascular reconstruction.ManagementAn important aspect of the management of lymphedema is patient understanding that there is no cure for lymphedema. The primary goals of treatment are to minimize swelling and to prevent recurrent infections. Controlling the chronic limb swelling can improve discomfort, heaviness, and tightness, and potentially reduce the progression of disease.132Brunicardi_Ch24_p0981-p1008.indd 100222/02/19 3:01 PM 1003VENOUS AND LYMPHATIC DISEASECHAPTER 24Figure 24-24. Normal lymphangiogram of the thigh and lower leg.Compression Garments. Graded compression stockings are widely used in the treatment of lymphedema. The stockings reduce the amount of swelling in the involved extremity by decreasing edema accumulation while the extremity is

1	compression stockings are widely used in the treatment of lymphedema. The stockings reduce the amount of swelling in the involved extremity by decreasing edema accumulation while the extremity is dependent. When worn daily, compression stockings have been associated with long-term maintenance of reduced limb circumference.134 They may also protect the tissues against chronically elevated intrinsic pressures, which lead to thickening of the skin and sub-cutaneous tissue.135 Compression stockings also offer a degree of protection against external trauma that may lead to cellulitis.The amount of compression required for controlling lymphedema ranges from 20 to 60 mmHg and varies among patients. The stockings can be custom made or prefabricated and are available in aboveand below-knee lengths. The stock-ings should be worn during waking hours. The garments should be replaced approximately every 6 months when they lose elasticity.Bedrest and Leg Elevation. Elevation is an important aspect

1	The stock-ings should be worn during waking hours. The garments should be replaced approximately every 6 months when they lose elasticity.Bedrest and Leg Elevation. Elevation is an important aspect of controlling lower extremity swelling and is often the first recommended intervention. However, continuous elevation throughout the day can interfere with quality of life more than lymphedema itself. Elevation is an adjunct to lymphedema ther-apy but is not the mainstay of treatment.Intermittent Pneumatic Compression Therapy. The use of IPC with a single-chamber or multichamber pump temporar-ily reduces edema and provides another adjunct to the use of compression stockings. These devices have been shown to be effective in reducing limb volume; however, use of compres-sion stockings is necessary to maintain the volume reduction when the patient is no longer supine because fluid transport is not associated with the transport of macromolecules (proteins) from the tissue. Typically, IPC is

1	to maintain the volume reduction when the patient is no longer supine because fluid transport is not associated with the transport of macromolecules (proteins) from the tissue. Typically, IPC is used for 4 to 6 hours per day at home when the patient is supine, with pressure ranges between 30 and 60 mmHg demonstrated to be most effective.135Lymphatic Massage. Manual lymphatic drainage is a form of massage developed by Vodder136 that is directed at reduc-ing edema. In combination with the use of compression stock-ings, manual lymphatic drainage is associated with a long-term reduction in edema and fewer infections per patient per year.137Antibiotic Therapy. Patients with lymphedema are at increased risk of developing cellulitis in the affected extremity due to microscopic breakdown in the skin barrier either second-ary to swelling or unrecognized and untreated tinea pedis. Recur-rent infection can damage the lymphatics, aggravating the edema and increasing the risk for subsequent

1	the skin barrier either second-ary to swelling or unrecognized and untreated tinea pedis. Recur-rent infection can damage the lymphatics, aggravating the edema and increasing the risk for subsequent infection. Staphylococcus and β-hemolytic Streptococcus are the most common organisms causing soft tissue infection. Aggressive antibiotic therapy and elevation with compression are recommended at the earliest signs or symptoms of cellulitis. The drug of choice is penicil-lin or a cephalosporin active against Streptococcus for 5 days. In patients with recurrent cellulitis despite methods to reduced edema, treatment with monthly intramuscular injections of ben-zathine penicillin 1.2 MU, twice-daily erythromycin 250 mg, or penicillin V 1 g daily has proven effective at suppression.138Surgery. A variety of surgical procedures have been devised for the treatment of lymphedema. Surgical treatment involves either excision of extra tissue139 or anastomosis of a lymphatic vessel to another

1	variety of surgical procedures have been devised for the treatment of lymphedema. Surgical treatment involves either excision of extra tissue139 or anastomosis of a lymphatic vessel to another lymphatic or vein.140 In excisional procedures, part or all of the edematous tissue is removed. This does not improve lymphatic drainage but debulks redundant tissue. The microsurgical procedures involve the creation of a lymphati-colymphatic or lymphaticovenous anastomosis, which theo-retically improves lymphatic drainage. No long-term follow-up data are available for these interventions, and therefore opera-tive therapy for lymphedema is not well accepted worldwide. Furthermore, operative intervention has the potential to further obliterate lymphatic channels, worsening the edema.141SUMMARYLymphedema is a chronic condition caused by ineffective lymphatic transport, which results in edema and skin damage. Lymphedema is not curable, but the symptoms and long-term effects can be controlled with a

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1	FN, Dannenberg AL, Abbott RD, et al. The epidemiol-ogy of varicose veins: the Framingham Study. Am J Prev Med. 1988;4:96-101. 97. Gibson K, Kabnick L, Varithena 013 Investigator G. A mul-ticenter, randomized, placebo-controlled study to evaluate the efficacy and safety of Varithena(R) (polidocanol endove-nous microfoam 1%) for symptomatic, visible varicose veins with saphenofemoral junction incompetence. Phlebology. 2017;32(3):185-193. 98. Witte ME, Reijnen MM, de Vries JP, Zeebregts CJ. Mechano-chemical Endovenous Occlusion of Varicose Veins Using the ClariVein(R) Device. Surg Technol Int. 2015;26:219-225. 99. Proebstle TM, Alm J, Dimitri S, et al. The European multi-center cohort study on cyanoacrylate embolization of refluxing great saphenous veins. J Vasc Surg Venous Lymphat Disord. 2015;3(1):2-7. 100. Lurie F, Creton D, Eklof B, et al. Prospective randomized study of endovenous radiofrequency obliteration (closure) versus ligation and vein stripping (EVOLVeS): two-year follow-up.

1	F, Creton D, Eklof B, et al. Prospective randomized study of endovenous radiofrequency obliteration (closure) versus ligation and vein stripping (EVOLVeS): two-year follow-up. Eur J Vasc Endovasc Surg. 2005;29:67-73. 101. Darwood RJ, Theivacumar N, Dellagrammaticas D, et al. Ran-domized clinical trial comparing endovenous laser ablation with surgery for the treatment of primary great saphenous varicose veins. Br J Surg. 2008;95:294-301. 102. Falanga V. Venous ulceration. J Dermatol Surg Oncol. 1993;19:764-771. 103. Phillips T, Stanton B, Provan A, et al. A study of the impact of leg ulcers on quality of life: financial, social, and psychologic implications. J Am Acad Dermatol. 1994;31:49-53. 104. Skin Substitute Consensus Development Panel. Nonoperative management of venous ulcers: evolving role of skin substi-tutes. Vasc Surg. 1999;33:197. 105. Abenhaim L, Kurz X. The VEINES study (VEnous Insuffi-ciency Epidemiologic and Economic Study): an international cohort study on chronic

1	role of skin substi-tutes. Vasc Surg. 1999;33:197. 105. Abenhaim L, Kurz X. The VEINES study (VEnous Insuffi-ciency Epidemiologic and Economic Study): an international cohort study on chronic venous disorders of the leg. VEINES Group. Angiology. 1997;48:59-66. 106. Clarke H, Smith SR, Vasdekis SN, et al. Role of venous elasticity in the development of varicose veins. Br J Surg. 1989;76:577-580. 107. Nicolaides AN, Hussein MK, Szendro G, et al. The relation of venous ulceration with ambulatory venous pressure measure-ments. J Vasc Surg. 1993;17:414-419. 108. Christopoulos DG, Nicolaides AN, Szendro G, et al. Air-pleth-ysmography and the effect of elastic compression on venous hemodynamics of the leg. J Vasc Surg. 1987;5:148-159. 109. van Bemmelen PS, Bedford G, Beach K, et al. Quantitative segmental evaluation of venous valvular reflux with duplex ultrasound scanning. J Vasc Surg. 1989;10:425-431. 110. Nehler MR, Porter JM. The lower extremity venous system. Part II: the

1	Quantitative segmental evaluation of venous valvular reflux with duplex ultrasound scanning. J Vasc Surg. 1989;10:425-431. 110. Nehler MR, Porter JM. The lower extremity venous system. Part II: the pathophysiology of chronic venous insufficiency. Perspect Vasc Surg. 1992;5:81-97. 111. Beidler SK, Douillet CD, Berndt DF, et al. Multiplexed analy-sis of matrix metalloproteinases in leg ulcer tissue of patients with chronic venous insufficiency before and after compres-sion therapy. Wound Repair Regen. 2008;16:642-648. 112. Beidler SK, Douillet CD, Berndt DF, et al. Inflammatory cyto-kine levels in chronic venous insufficiency ulcer tissue before and after compression therapy. J Vasc Surg. 2009;49:1013-1020. 113. Nehler MR, Moneta GL, Woodard DM, et al. Perimalleolar subcutaneous tissue pressure effects of elastic compression stockings. J Vasc Surg. 1993;18:783-788. 114. Dinn E. Treatment of venous ulceration by injection sclero-therapy and compression hosiery: a 5-year study.

1	pressure effects of elastic compression stockings. J Vasc Surg. 1993;18:783-788. 114. Dinn E. Treatment of venous ulceration by injection sclero-therapy and compression hosiery: a 5-year study. Phlebology. 1992;7:23-26. 115. Mayberry JC, Moneta GL, Taylor LM, Jr, et al. Fifteen-year results of ambulatory compression therapy for chronic venous ulcers. Surgery. 1991;109(5):575-581. This review highlights the impact of compression therapy in venous ulcer therapy. 116. Falanga V, Margolis D, Alvarez O, et al. Rapid healing of venous ulcers and lack of clinical rejection with an allergenic cultured human skin equivalent. Human Skin Equivalent Investigators Group. Arch Dermatol. 1998;134:293-300. 117. Phillips TJ. New skin for old: developments in biological skin substitutes [editorial; comment]. Arch Dermatol. 1998;134:344. 118. Motykie GD, Caprini JA, Arcelus JI, et al. Evaluation of therapeutic compression stockings in the treatment of chronic venous insufficiency. Dermatol Surg.

1	comment]. Arch Dermatol. 1998;134:344. 118. Motykie GD, Caprini JA, Arcelus JI, et al. Evaluation of therapeutic compression stockings in the treatment of chronic venous insufficiency. Dermatol Surg. 1999;25:116-120. 119. Lippmann HI, Fishman LM, Farrar RH, et al. Edema control in the management of disabling chronic venous insufficiency. Arch Phys Med Rehabil. 1994;75:436-441. 120. Rubin JR, Alexander J, Plecha EJ, et al. Unna’s boot vs. polyurethane foam dressings for the treatment of venous ulceration. A randomized prospective study. Arch Surg. 1990;125:489-490. 121. O’Meara S, Cullum NA, Nelson EA. Compression for venous leg ulcers. Cochrane Database Syst Rev. 2009;1:CD000265. 122. Vernick SH, Shapiro D, Shaw FD. Legging orthosis for venous and lymphatic insufficiency. Arch Phys Med Rehabil. 1987;68:459-461.Brunicardi_Ch24_p0981-p1008.indd 100622/02/19 3:01 PM 1007VENOUS AND LYMPHATIC DISEASECHAPTER 24 123. Sibbald RG. Apligraf living skin equivalent for healing venous and

1	Rehabil. 1987;68:459-461.Brunicardi_Ch24_p0981-p1008.indd 100622/02/19 3:01 PM 1007VENOUS AND LYMPHATIC DISEASECHAPTER 24 123. Sibbald RG. Apligraf living skin equivalent for healing venous and chronic wounds. J Cutan Med Surg. 1998;3(suppl 1): S1-24-8. 124. Linton R. The communicating veins of the lower leg and the operative technique for their ligation. Ann Surg. 1938;107:582-593. 125. Gloviczki P, Bergan JJ, Rhodes JM, Canton LG, Harmsen S, Ilstrup DM. Mid-term results of endoscopic perforator vein interruption for chronic venous insufficiency: lessons learned from the North American Subfascial Endoscopic Perforator Surgery Registry. The North American Study Group. J Vasc Surg. 1999;29(3):489-502. This study reviews registry outcomes following subfacial endoscopic perforator surgery performed across 17 centers in North America. 126. Vashist MG, Malik V, Singhal N. Role of subfascial endo-scopic perforator surgery (SEPS) in management of perfora-tor incompetence in varicose

1	performed across 17 centers in North America. 126. Vashist MG, Malik V, Singhal N. Role of subfascial endo-scopic perforator surgery (SEPS) in management of perfora-tor incompetence in varicose veins: a prospective randomized study. India J Surg. 2014;76(2):117-123. 127. van Gent WB, Hop WC, van Praag MC, et al. Conservative versus surgical treatment of venous leg ulcers: a prospective randomized, multicenter trial. J Vasc Surg. 2006;44:563-571. 128. Gohel MS, Barwell JR, Taylor M, et al. Long term results of compression therapy alone versus compression plus surgery in chronic venous ulceration (ESCHAR): randomized controlled trial. BMJ. 2007;335:83. 129. Sottiurai VS. Surgical correction of recurrent venous ulcer. J Cardiovasc Surg. 1991;32:104-109. 130. Raju S, Fredericks R. Valve reconstruction procedures for non-obstructive venous insufficiency: rationale, techniques, and results in 107 procedures with twoto eight-year follow-up. J Vasc Surg. 1988;7:301-310. 131. Raju S, Darcey

1	procedures for non-obstructive venous insufficiency: rationale, techniques, and results in 107 procedures with twoto eight-year follow-up. J Vasc Surg. 1988;7:301-310. 131. Raju S, Darcey R, Neglen P. Unexpected major role for venous stenting in deep reflux disease. J Vasc Surg. 2010;51:401-408. 132. Masuda EM, Kistner RL. Long-term results of venous valve reconstruction: a fourto twenty-one-year follow-up. J Vasc Surg. 1994;19:391-403. 133. Rockson SG, Miller LT, Senie R, et al. American Cancer Soci-ety Lymphedema Workshop. Workgroup III: diagnosis and management of lymphedema. Cancer. 1998;83:2882-2885. 134. Yasuhara H, Shigematsu H, Muto T. A study of the advan-tages of elastic stockings for leg lymphedema. Int Angiol. 1996;15:272-277. 135. Feldman JL, Stout NL, Wanchai A, et al. Intermittent pneu-matic compression therapy: a systematic review. Lymphology. 2012;45:13-25. 136. Vodder E. Le Drainage Lymphatique, une Novelle Méthode Thérapeutique. Paris: Santé pour tous;

1	et al. Intermittent pneu-matic compression therapy: a systematic review. Lymphology. 2012;45:13-25. 136. Vodder E. Le Drainage Lymphatique, une Novelle Méthode Thérapeutique. Paris: Santé pour tous; 1936. 137. Ko DS, Lerner R, Klose G, et al. Effective treatment of lymph-edema of the extremities. Arch Surg. 1998;133:452-458. 138. Stevens DL, Bisno AL, Chambers HF, et al. Practice guide-lines for the diagnosis and management of skin and soft tissue infections. Clin Infect Dis. 2005;41:1373-1406. 139. Miller TA, Wyatt LE, Rudkin GH. Staged skin and subcutane-ous excision for lymphedema: a favorable report of long-term results. Plast Reconstr Surg. 1998;102:1486-1501. 140. Baumeister RG, Siuda S. Treatment of lymphedema by micro-surgical lymphatic grafting: what is proved? Plast Reconstr Surg. 1990;85:64-74. 141. Bernas MJ, Witte CL, Witte MH. The diagnosis and treatment of peripheral lymphedema: draft revision of the 1995 Con-sensus Document of the International Society of Lymphology

1	1990;85:64-74. 141. Bernas MJ, Witte CL, Witte MH. The diagnosis and treatment of peripheral lymphedema: draft revision of the 1995 Con-sensus Document of the International Society of Lymphology Executive Committee for discussion at the September 3–7, 2001, XVIII International Congress of Lymphology in Genoa, Italy. Lymphology. 2001;34:84-91.Brunicardi_Ch24_p0981-p1008.indd 100722/02/19 3:01 PM

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1	SURGICAL ANATOMYThe esophagus is a muscular tube that starts as the continu-ation of the pharynx and ends as the cardia of the stomach. When the head is in a normal anatomic position, the transi-tion from pharynx to esophagus occurs at the lower border of the sixth cervical vertebra. Topographically this corresponds to the cricoid cartilage anteriorly and the palpable transverse process of the sixth cervical vertebra laterally (Fig. 25-1). The esophagus is firmly attached at its upper end to the cricoid cartilage and at its lower end to the diaphragm; during swal-lowing, the proximal points of fixation move craniad the dis-tance of one cervical vertebral body.The esophagus lies in the midline, with a deviation to the left in the lower portion of the neck and upper portion of the thorax, and returns to the midline in the midportion of the tho-rax near the bifurcation of the trachea (Fig. 25-2). In the lower portion of the thorax, the esophagus again deviates to the left and anteriorly

1	returns to the midline in the midportion of the tho-rax near the bifurcation of the trachea (Fig. 25-2). In the lower portion of the thorax, the esophagus again deviates to the left and anteriorly to pass through the diaphragmatic hiatus.Esophagus and Diaphragmatic HerniaBlair A. Jobe, John G. Hunter, and David I. Watson 25chapterSurgical Anatomy1009Physiology1015Swallowing Mechanism / 1015Physiologic Reflux / 1017Assessment of Esophageal Function1018Tests to Detect Structural Abnormalities / 1018Tests to Detect Functional Abnormalities / 1019Videoand Cineradiography / 1028Tests to Detect Increased Exposure to Gastric Juice / 1028Tests of Duodenogastric Function / 1030Gastroesophageal Reflux Disease1031The Human Antireflux Mechanism and the Pathophysiology of Gastroesophageal Reflux Disease / 1032Complications Associated With Gastroesophageal Reflux Disease / 1033Metaplastic (Barrett’s Esophagus) and Neoplastic (Adenocarcinoma) Complications / 1035Respiratory Complications /

1	Disease / 1032Complications Associated With Gastroesophageal Reflux Disease / 1033Metaplastic (Barrett’s Esophagus) and Neoplastic (Adenocarcinoma) Complications / 1035Respiratory Complications / 1035Surgical Therapy for Gastroesophageal Reflux Disease / 1038Primary Antireflux Repairs / 1040Giant Diaphragmatic (Hiatal) Hernias1045Incidence and Etiology / 1045Clinical Manifestations / 1047Diagnosis / 1047Pathophysiology / 1048Treatment / 1048Diaphragmatic Repair / 1048The Short Esophagus and PEH / 1049Results / 1049Schatzki’s Ring1049Scleroderma1050Eosinophilic Esophagitis1051Symptoms / 1051Signs / 1051Pathology / 1051Treatment / 1051Motility Disorders of the Pharynx and Esophagus1052Clinical Manifestations / 1052Motility Disorders of the Pharynx and Upper Esophagus—Transit Dysphagia / 1052Diagnostic Assessment of the Cricopharyngeal Segment / 1052Motility Disorders of the Esophageal Body and Lower Esophageal Sphincter / 1055Operations for Esophageal Motor Disorders and

1	/ 1052Diagnostic Assessment of the Cricopharyngeal Segment / 1052Motility Disorders of the Esophageal Body and Lower Esophageal Sphincter / 1055Operations for Esophageal Motor Disorders and Diverticula1060Long Esophageal Myotomy for Motor Disorders of the Esophageal Body / 1060Myotomy of the Lower Esophageal Sphincter (Heller Myotomy) / 1063Open Esophageal Myotomy / 1065Laparoscopic Cardiomyotomy / 1065Per Oral Endoscopic Myotomy (POEM) / 1065Outcome Assessment of the Therapy for Achalasia / 1065Esophageal Resection for End-Stage Motor Disorders of the Esophagus / 1068Carcinoma of the Esophagus1068Clinical Manifestations / 1068General Approach to Esophageal Cancer / 1069Staging of Esophageal Cancer / 1069Clinical Approach to Carcinoma of the Esophagus and Cardia / 1070Palliation of Esophageal Cancer / 1074Surgical Treatment / 1074Comparative Studies of Esophagectomy Technique / 1077Alternative Therapies / 1077Sarcoma of the Esophagus1078Benign Tumors and Cysts1080Leiomyoma /

1	of Esophageal Cancer / 1074Surgical Treatment / 1074Comparative Studies of Esophagectomy Technique / 1077Alternative Therapies / 1077Sarcoma of the Esophagus1078Benign Tumors and Cysts1080Leiomyoma / 1081Esophageal Cyst / 1083Esophageal Perforation1083Diagnosis / 1083Management / 1084Mallory-Weiss Syndrome1085Caustic Injury1086Pathology / 1086Clinical Manifestations / 1086Treatment / 1086Acquired Fistula1088Techniques of Esophageal Reconstruction1089Partial Esophageal Resection / 1089Reconstruction After Total Esophagectomy / 1089Composite Reconstruction / 1090Vagal Sparing Esophagectomy With Colon Interposition / 1090Brunicardi_Ch25_p1009-p1098.indd 100901/03/19 6:01 PM 1010abcdeA BKey Points1 Benign esophageal disease is common and is best evaluated with thorough physiologic testing (high resolution esopha-geal motility, 24-hour ambulatory pH measurement, and/or esophageal impedance testing) and anatomic testing (esoph-agoscopy, video esophagography, and/or computed

1	testing (high resolution esopha-geal motility, 24-hour ambulatory pH measurement, and/or esophageal impedance testing) and anatomic testing (esoph-agoscopy, video esophagography, and/or computed tomog-raphy [CT] scanning).2 Gastroesophageal reflux disease (GERD) is the most com-mon disease of the gastrointestinal tract for which patients seek medical therapy. When GERD symptoms (heartburn, regurgitation, chest pain, and/or supraesophageal symptoms) are troublesome despite adequately dosed PPI, surgical cor-rection may be indicated.3 Barrett’s esophagus is the transformation of the distal esoph-ageal epithelium from squamous to a specialized columnar epithelium capable of further neoplastic progression. The detection of Barrett’s esophagus on endoscopy and biopsy increases the future risk of cancer by >40x compared to indi-viduals without Barrett’s esophagus.4 Giant hiatal hernia, otherwise known as paraesophageal her-nia, should be repaired when symptomatic or associated with iron

1	of cancer by >40x compared to indi-viduals without Barrett’s esophagus.4 Giant hiatal hernia, otherwise known as paraesophageal her-nia, should be repaired when symptomatic or associated with iron deficiency anemia. Laparoscopic hiatal hernia repair with fundoplication is the most common approach to repair.5 Achalasia is the most common primary esophageal motor disorder. It is characterized by an absence of peristalsis and a hypertensive nonrelaxing lower esophageal sphincter. It is best treated with laparoscopic Heller myotomy and partial fundoplication.6 Most esophageal cancer presents with dysphagia, at which time it has invaded the muscularis of the esophagus and is often associated with lymph node metastases. The preferred treatment at this stage is multimodality therapy with chemo-radiation therapy followed by open or minimally invasive esophagectomy.Figure 25-1. A. Topographic relationships of the cervical esophagus: (a) hyoid bone, (b) thyroid cartilage, (c) cricoid cartilage,

1	therapy followed by open or minimally invasive esophagectomy.Figure 25-1. A. Topographic relationships of the cervical esophagus: (a) hyoid bone, (b) thyroid cartilage, (c) cricoid cartilage, (d) thyroid gland, (e) sternoclavicular. B. Lateral radio-graphic appearance with landmarks identified as labeled in A. The location of C6 is also included (f). (Reproduced with permission from Shields TW: General Thoracic Surgery, 3rd ed. Philadelphia, PA: Lea & Febiger; 1989.)Three normal areas of esophageal narrowing are evident on the barium esophagogram or during esophagoscopy. The uppermost narrowing is located at the entrance into the esopha-gus and is caused by the cricopharyngeal muscle. Its luminal diameter is 1.5 cm, and it is the narrowest point of the esopha-gus. The middle narrowing is due to an indentation of the ante-rior and left lateral esophageal wall caused by the crossing of the left main stem bronchus and aortic arch. The luminal diameter at this point is 1.6 cm. The

1	is due to an indentation of the ante-rior and left lateral esophageal wall caused by the crossing of the left main stem bronchus and aortic arch. The luminal diameter at this point is 1.6 cm. The lowermost narrowing is at the hiatus of the diaphragm and is caused by the gastroesophageal sphincter mechanism. The luminal diameter at this point varies somewhat, depending on the distention of the esophagus by the passage of food, but has been measured at 1.6 to 1.9 cm. These normal constrictions tend to hold up swallowed foreign objects, and the overlying mucosa is subject to injury by swallowed corrosive liquids due to their slow passage through these areas.Figure 25-3 shows the average distance in centimeters measured during endoscopic examination between the incisor teeth and the cricopharyngeus, aortic arch, and cardia of the stomach. Manometrically, the length of the esophagus between the lower border of the cricopharyngeus and upper border of the lower sphincter varies according to

1	aortic arch, and cardia of the stomach. Manometrically, the length of the esophagus between the lower border of the cricopharyngeus and upper border of the lower sphincter varies according to the height of the individual.Brunicardi_Ch25_p1009-p1098.indd 101001/03/19 6:01 PM 1011ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25ABFigure 25-2. Barium esophagogram. A. Posterior-anterior view. White arrow shows deviation to left. Black arrow shows return to midline. B. Lateral view. Black arrow shows anterior deviation. (Reproduced with permission from Shields TW: General Thoracic Surgery, 3rd ed. Philadelphia, PA: Lea & Febiger; 1989.)Pharynx24–26cmUpper sphincter(C6)40cm38cmLower sphincter(T11)15cm14cmAortic arch(T4)25cm 23cmIncisor teethFigure 25-3. Important clinical endoscopic measurements of the esophagus in adults. (Reproduced with permission from Shields TW: General Thoracic Surgery, 3rd ed. Philadelphia, PA: Lea & Febiger; 1989.) Superior pharyngeal constrictor m.Middle pharyngeal

1	of the esophagus in adults. (Reproduced with permission from Shields TW: General Thoracic Surgery, 3rd ed. Philadelphia, PA: Lea & Febiger; 1989.) Superior pharyngeal constrictor m.Middle pharyngeal constrictor m.Inferior pharyngeal constrictor m.Cricopharyngeus m.EsophagusBAFigure 25-4. External muscles of the pharynx. A. Posterolateral view. B. Posterior view. Dotted line represents usual site of myotomy. (Reproduced with permission from Shields TW: General Thoracic Surgery, 3rd ed. Philadelphia, PA: Lea & Febiger; 1989.)The pharyngeal musculature consists of three broad, flat, overlapping fan-shaped constrictors (Fig. 25-4). The opening of the esophagus is collared by the cricopharyngeal muscle, which arises from both sides of the cricoid cartilage of the lar-ynx and forms a continuous transverse muscle band without an interruption by a median raphe. The fibers of this muscle Brunicardi_Ch25_p1009-p1098.indd 101101/03/19 6:02 PM 1012SPECIFIC CONSIDERATIONSPART IIblend

1	transverse muscle band without an interruption by a median raphe. The fibers of this muscle Brunicardi_Ch25_p1009-p1098.indd 101101/03/19 6:02 PM 1012SPECIFIC CONSIDERATIONSPART IIblend inseparably with those of the inferior pharyngeal constric-tor above and the inner circular muscle fibers of the esophagus below. Some investigators believe that the cricopharyngeus is part of the inferior constrictor; that is, that the inferior constric-tor has two parts, an upper or retrothyroid portion having diago-nal fibers, and a lower or retrocricoid portion having transverse fibers. Keith in 1910 showed that these two parts of the same muscle serve totally different functions. The retrocricoid portion serves as the upper sphincter of the esophagus and relaxes when the retrothyroid portion contracts, to force the swallowed bolus from the pharynx into the esophagus.The cervical portion of the esophagus is approximately 5 cm long and descends between the trachea and the vertebral column, from

1	to force the swallowed bolus from the pharynx into the esophagus.The cervical portion of the esophagus is approximately 5 cm long and descends between the trachea and the vertebral column, from the level of the sixth cervical vertebra to the level of the interspace between the first and second thoracic verte-brae posteriorly, or the level of the suprasternal notch anteriorly. The recurrent laryngeal nerves lie in the right and left grooves between the trachea and the esophagus. The left recurrent nerve lies somewhat closer to the esophagus than the right, owing to the slight deviation of the esophagus to the left, and the more lateral course of the right recurrent nerve around the right sub-clavian artery. Laterally, on the left and right sides of the cervi-cal esophagus are the carotid sheaths and the lobes of the thyroid gland.The thoracic portion of the esophagus is approximately 20 cm long. It starts at the thoracic inlet. In the upper portion of the thorax, it is in intimate

1	sheaths and the lobes of the thyroid gland.The thoracic portion of the esophagus is approximately 20 cm long. It starts at the thoracic inlet. In the upper portion of the thorax, it is in intimate relationship with the posterior wall of the trachea and the prevertebral fascia. Just above the tracheal bifurcation, the esophagus passes to the right of the aorta. This anatomic positioning can cause a notch indentation in its left lateral wall on a barium swallow radiogram. Immediately below this notch, the esophagus crosses both the bifurcation of the trachea and the left main stem bronchus, owing to the slight deviation of the terminal portion of the trachea to the right by the aorta (Fig. 25-5). From there down, the esophagus passes over the posterior surface of the subcarinal lymph nodes (LNs), and then descends over the pericardium of the left atrium to reach the diaphragmatic hiatus (Fig. 25-6). From the bifurcation of the trachea downward, both the vagal nerves and the esophageal

1	(LNs), and then descends over the pericardium of the left atrium to reach the diaphragmatic hiatus (Fig. 25-6). From the bifurcation of the trachea downward, both the vagal nerves and the esophageal nerve plexus lie on the muscular wall of the esophagus.Dorsally, the thoracic esophagus follows the curvature of the spine and remains in close contact with the vertebral bod-ies. From the eighth thoracic vertebra downward, the esopha-gus moves vertically away from the spine to pass through the hiatus of the diaphragm. The thoracic duct passes through the hiatus of the diaphragm on the anterior surface of the verte-bral column behind the aorta and under the right crus. In the thorax, the thoracic duct lies dorsal to the esophagus between the azygos vein on the right and the descending thoracic aorta on the left.The abdominal portion of the esophagus is approximately 2 cm long and includes a portion of the lower esophageal sphincter (LES). It starts as the esophagus passes through the

1	aorta on the left.The abdominal portion of the esophagus is approximately 2 cm long and includes a portion of the lower esophageal sphincter (LES). It starts as the esophagus passes through the diaphragmatic hiatus and is surrounded by the phrenoesopha-geal membrane, a fibroelastic ligament arising from the subdia-phragmatic fascia as a continuation of the transversalis fascia lining the abdomen (Fig. 25-7). The upper leaf of the membrane attaches itself in a circumferential fashion around the esopha-gus, about 1 to 2 cm above the level of the hiatus. These fibers blend in with the elastic-containing adventitia of the abdominal esophagus and the cardia of the stomach. This portion of the esophagus is subjected to the positive-pressure environment of the abdomen.The musculature of the esophagus can be divided into an outer longitudinal and an inner circular layer. The upper 2 to 6 cm of the esophagus contains only striated muscle fibers. From then on, smooth muscle fibers gradually

1	can be divided into an outer longitudinal and an inner circular layer. The upper 2 to 6 cm of the esophagus contains only striated muscle fibers. From then on, smooth muscle fibers gradually become more abundant. Most clinically significant esophageal motility dis-orders involve only the smooth muscle in the lower two-thirds of the esophagus. When a long surgical esophageal myotomy is indicated, the incision needs to extend only this distance.The longitudinal muscle fibers originate from a crico-esophageal tendon arising from the dorsal upper edge of the anteriorly located cricoid cartilage. The two bundles of mus-cle diverge and meet in the midline on the posterior wall of the esophagus about 3 cm below the cricoid (see Fig. 25-4). From this point on, the entire circumference of the esophagus is cAThymusPericardiumSuperior vena cavaTracheal carinaRight main stembronchusEsophagusAscending aortaLeft main stem bronchusBottom of aortic archDescendingaortaIVBaebdFigure 25-5. A.

1	the esophagus is cAThymusPericardiumSuperior vena cavaTracheal carinaRight main stembronchusEsophagusAscending aortaLeft main stem bronchusBottom of aortic archDescendingaortaIVBaebdFigure 25-5. A. Cross-section of the thorax at the level of the tracheal bifurcation. B. Computed tomographic scan at same level viewed from above: (a) ascending aorta, (b) descending aorta, (c) tracheal carina, (d) esophagus, (e) pulmonary artery. (Reproduced with permission from Shields TW: General Thoracic Surgery, 3rd ed. Philadelphia, PA: Lea & Febiger; 1989.)Brunicardi_Ch25_p1009-p1098.indd 101201/03/19 6:02 PM 1013ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25BAPericardiumRight ventricleRight atriumPericardiumPleuraVIIPleuraAortaEsophagusLeft atriumLeft ventriclefdecabgFigure 25-6. A. Cross-section of the thorax at the midleft atrial level. B. Computed tomographic scan at same level viewed from above: (a) aorta, (b) esophagus, (c) left atrium, (d) right atrium, (e) left ventricle, (f) right

1	of the thorax at the midleft atrial level. B. Computed tomographic scan at same level viewed from above: (a) aorta, (b) esophagus, (c) left atrium, (d) right atrium, (e) left ventricle, (f) right ventricle, (g) pulmonary vein. (Reproduced with permission from Shields TW: General Thoracic Surgery, 3rd ed. Philadelphia, PA: Lea & Febiger; 1989.)Phreno-esophageal membrane(Ascending leaf)ParietalperitoneumVisceralperitoneumDiaphragmPara-esophageal fat padPhreno-esophageal membrane(Descending leaf)Figure 25-7. Attachments and structure of the phrenoesophageal membrane. Transversalis fascia lies just above the parietal peri-toneum. (Reproduced with permission from Shields TW: General Thoracic Surgery, 3rd ed. Philadelphia, PA: Lea & Febiger; 1989.)covered by a layer of longitudinal muscle fibers. This configura-tion of the longitudinal muscle fibers around the most proximal part of the esophagus leaves a V-shaped area in the posterior wall covered only with circular muscle fibers.

1	fibers. This configura-tion of the longitudinal muscle fibers around the most proximal part of the esophagus leaves a V-shaped area in the posterior wall covered only with circular muscle fibers. Contraction of the longitudinal muscle fibers shortens the esophagus. The cir-cular muscle layer of the esophagus is thicker than the outer longitudinal layer. In situ, the geometry of the circular muscle is helical and makes the peristalsis of the esophagus assume a wormlike drive, as opposed to segmental and sequential squeez-ing. As a consequence, severe motor abnormalities of the esoph-agus assume a corkscrew-like pattern on the barium swallow radiogram.The cervical portion of the esophagus receives its main blood supply from the inferior thyroid artery. The thoracic por-tion receives its blood supply from the bronchial arteries, with 75% of individuals having one right-sided and two left-sided branches. Two esophageal branches arise directly from the aorta. The abdominal portion of the

1	supply from the bronchial arteries, with 75% of individuals having one right-sided and two left-sided branches. Two esophageal branches arise directly from the aorta. The abdominal portion of the esophagus receives its blood supply from the ascending branch of the left gastric artery and from inferior phrenic arteries (Fig. 25-8). On entering the wall of the esophagus, the arteries assume a T-shaped division to form a longitudinal plexus, giving rise to an intramural vascular network in the muscular and submucosal layers. As a conse-quence, the esophagus can be mobilized from the stomach to the level of the aortic arch without fear of devascularization and ischemic necrosis. Caution, however, should be exercised as to the extent of esophageal mobilization in patients who have had a previous thyroidectomy with ligation of the inferior thyroid arteries proximal to the origin of the esophageal branches.Blood from the capillaries of the esophagus flows into a submucosal venous plexus, and

1	thyroidectomy with ligation of the inferior thyroid arteries proximal to the origin of the esophageal branches.Blood from the capillaries of the esophagus flows into a submucosal venous plexus, and then into a periesophageal Left gastric arteryRight bronchialartery Inferior thyroid arterySuperior leftbronchial arteryInferior leftbronchial arteryAortic esophagealarteriesAscending branches ofleft gastric artery Esophageal branchFigure 25-8. Arterial blood supply of the esophagus. (Reproduced with permission from Shields TW: General Thoracic Surgery, 3rd ed. Philadelphia, PA: Lea & Febiger; 1989.)Brunicardi_Ch25_p1009-p1098.indd 101301/03/19 6:02 PM 1014SPECIFIC CONSIDERATIONSPART IIInferior thyroid veinsAccessory azygous veinHemiazygous veinShort gastric veinsSplenic veinSuperior mesenteric vein Portal vein Coronary vein Azygous vein Figure 25-9. Venous drainage of the esophagus. (Reproduced with permission from Shields TW: General Thoracic

1	veinSuperior mesenteric vein Portal vein Coronary vein Azygous vein Figure 25-9. Venous drainage of the esophagus. (Reproduced with permission from Shields TW: General Thoracic Surgery, 3rd ed. Philadelphia, PA: Lea & Febiger; 1989.)Left vagus nerveLeft recurrentlaryngeal nerveThoracic chainLeft or anteriorvagal trunkRight or posterior vagal trunkAnterior esophagealplexusRight recurrentlaryngeal nerveRight vagus nerveRecurrent laryngealnervesFigure 25-10. Innervation of the esophagus. (Reproduced with permission from Shields TW: General Thoracic Surgery, 3rd ed. Philadelphia, PA: Lea & Febiger; 1989.)Internal jugularnodesParatrachealnodesSubcarinal nodesInferior paraesophagealnodesParahiatal nodes Splenic arterynodesCeliac artery nodes Hepatic artery nodesLeft gastric artery nodesPulmonary hilarnodesSuperiorparaesophageal nodesFigure 25-11. Lymphatic drainage of the esophagus. (Reproduced with permission from DeMeester TR, Barlow AP. Surgery and cur-rent

1	gastric artery nodesPulmonary hilarnodesSuperiorparaesophageal nodesFigure 25-11. Lymphatic drainage of the esophagus. (Reproduced with permission from DeMeester TR, Barlow AP. Surgery and cur-rent management for cancer of the esophagus and cardia: Part I, Curr Probl Surg. 1988 Jul;25(7):475-531.)venous plexus from which the esophageal veins originate. In the cervical region, the esophageal veins empty into the inferior thy-roid vein; in the thoracic region, they empty into the bronchial, azygos, or hemiazygos veins; and in the abdominal region, they empty into the coronary vein (Fig. 25-9). The submucosal venous networks of the esophagus and stomach are in continuity with each other, and, in patients with portal venous obstruction, this communication functions as a collateral pathway for portal blood to enter the superior vena cava via the azygos vein.The parasympathetic innervation of the pharynx and esophagus is provided mainly by the vagus nerves. The con-strictor muscles of the

1	portal blood to enter the superior vena cava via the azygos vein.The parasympathetic innervation of the pharynx and esophagus is provided mainly by the vagus nerves. The con-strictor muscles of the pharynx receive branches from the pharyngeal plexus, which is on the posterior lateral surface of the middle constrictor muscle, and is formed by pharyngeal branches of the vagus nerves with a small contribution from cra-nial nerves IX and XI (Fig. 25-10). The cricopharyngeal sphinc-ter and the cervical portion of the esophagus receive branches from both recurrent laryngeal nerves, which originate from the vagus nerves—the right recurrent nerve at the lower margin of the subclavian artery and the left at the lower margin of the aortic arch. They are slung dorsally around these vessels and ascend in the groove between the esophagus and trachea, giving branches to each. Damage to these nerves interferes not only with the function of the vocal cords but also with the function of the

1	and ascend in the groove between the esophagus and trachea, giving branches to each. Damage to these nerves interferes not only with the function of the vocal cords but also with the function of the cricopharyngeal sphincter and the motility of the cervical esophagus, predisposing the individual to pulmonary aspiration on swallowing.Afferent visceral sensory pain fibers from the esophagus end without synapse in the first four segments of the thoracic spinal cord, using a combination of sympathetic and vagal path-ways. These pathways are also occupied by afferent visceral sensory fibers from the heart; hence, both organs have similar symptomatology.The lymphatics located in the submucosa of the esopha-gus are so dense and interconnected that they constitute a single plexus (Fig. 25-11). There are more lymph vessels than blood capillaries in the submucosa. Lymph flow in the submucosal plexus runs in a longitudinal direction, and, on injection of a contrast medium, the longitudinal

1	There are more lymph vessels than blood capillaries in the submucosa. Lymph flow in the submucosal plexus runs in a longitudinal direction, and, on injection of a contrast medium, the longitudinal spread is seen to be about six times that of the transverse spread. In the upper two-thirds of the esophagus, the lymphatic flow is mostly cephalad, and, in the lower third, caudad. In the thoracic portion of the esophagus, Brunicardi_Ch25_p1009-p1098.indd 101401/03/19 6:02 PM 1015ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25the submucosal lymph plexus extends over a long distance in a longitudinal direction before penetrating the muscle layer to enter lymph vessels in the adventitia. As a consequence of this nonsegmental lymph drainage, a primary tumor can extend for a considerable length superiorly or inferiorly in the submucosal plexus. Consequently, free tumor cells can follow the submu-cosal lymphatic plexus in either direction for a long distance before they pass through the

1	superiorly or inferiorly in the submucosal plexus. Consequently, free tumor cells can follow the submu-cosal lymphatic plexus in either direction for a long distance before they pass through the muscularis and on into the regional LNs. The cervical esophagus has more direct segmental lymph drainage into the regional nodes, and, as a result, lesions in this portion of the esophagus have less submucosal extension and a more regionalized lymphatic spread.The efferent lymphatics from the cervical esophagus drain into the paratracheal and deep cervical LNs, and those from the upper thoracic esophagus empty mainly into the paratracheal LNs. Efferent lymphatics from the lower thoracic esophagus drain into the subcarinal nodes and nodes in the inferior pulmo-nary ligaments. The superior gastric nodes receive lymph not only from the abdominal portion of the esophagus, but also from the adjacent lower thoracic segment.PHYSIOLOGYSwallowing MechanismThe act of alimentation requires the passage of

1	receive lymph not only from the abdominal portion of the esophagus, but also from the adjacent lower thoracic segment.PHYSIOLOGYSwallowing MechanismThe act of alimentation requires the passage of food and drink from the mouth into the stomach. One-third of this distance con-sists of the mouth and hypopharynx, and two-thirds is made up by the esophagus. To comprehend the mechanics of alimenta-tion, it is useful to visualize the gullet as a mechanical model in which the tongue and pharynx function as a piston pump with three valves, and the body of the esophagus and cardia function as a worm-drive pump with a single valve. The three valves in the pharyngeal cylinder are the soft palate, epiglottis, and cricopharyngeus. The valve of the esophageal pump is the LES. Failure of the valves or the pumps leads to abnormali-ties in swallowing—that is, difficulty in food propulsion from mouth to stomach—or regurgitation of gastric contents into the esophagus or pharynx.Food is taken into the

1	the pumps leads to abnormali-ties in swallowing—that is, difficulty in food propulsion from mouth to stomach—or regurgitation of gastric contents into the esophagus or pharynx.Food is taken into the mouth in a variety of bite sizes, where it is broken up, mixed with saliva, and lubricated. Once initiated, swallowing is entirely a reflex act. When food is ready for swallowing, the tongue, acting like a piston, moves the bolus into the posterior oropharynx and forces it into the hypopharynx (Fig. 25-12). Concomitantly with the posterior movement of the tongue, the soft palate is elevated, thereby closing the passage between the oropharynx and nasopharynx. This partitioning prevents pressure generated in the oropharynx from being dissipated through the nose. When the soft palate is paralyzed, for example, after a cerebrovascular accident, food is commonly regurgitated into the nasopharynx. During swal-lowing, the hyoid bone moves upward and anteriorly, elevating the larynx and opening

1	for example, after a cerebrovascular accident, food is commonly regurgitated into the nasopharynx. During swal-lowing, the hyoid bone moves upward and anteriorly, elevating the larynx and opening the retrolaryngeal space, bringing the epiglottis under the tongue (see Fig. 25-12). The backward tilt of the epiglottis covers the opening of the larynx to prevent aspi-ration. The entire pharyngeal part of swallowing occurs within 1.5 seconds.During swallowing, the pressure in the hypopharynx rises abruptly, to at least 60 mmHg, due to the backward movement of the tongue and contraction of the posterior pharyngeal con-strictors. A sizable pressure difference develops between the hypopharyngeal pressure and the less-than-atmospheric mid-esophageal or intrathoracic pressure (Fig. 25-13). This pressure 1. Elevation of tongue2. Posterior movement of tongue3. Elevation of soft palate4. Elevation of hyoid5. Elevation of larynx6. Tilting of epiglottis123456Figure 25-12. Sequence of events during

1	1. Elevation of tongue2. Posterior movement of tongue3. Elevation of soft palate4. Elevation of hyoid5. Elevation of larynx6. Tilting of epiglottis123456Figure 25-12. Sequence of events during the oropharyngeal phase of swallowing. (Reproduced with permission from Zuidema GD, Orringer MB: Shackelford’s Surgery of the Alimentary Tract, 3rd ed. Vol 1. Philadelphia, PA: Elsevier/Saunders; 1991.)gradient speeds the movement of food from the hypopharynx into the esophagus when the cricopharyngeus or upper esopha-geal sphincter relaxes. The bolus is both propelled by peristaltic contraction of the posterior pharyngeal constrictors and sucked into the thoracic esophagus. Critical to receiving the bolus is the compliance of the cervical esophagus; when compliance is lost due to muscle pathology, dysphagia can result. The upper esophageal sphincter closes within 0.5 seconds of the initiation of the swallow, with the immediate closing pressure reaching Pressure (mm Hg)% Esophagus

1	pathology, dysphagia can result. The upper esophageal sphincter closes within 0.5 seconds of the initiation of the swallow, with the immediate closing pressure reaching Pressure (mm Hg)% Esophagus length100–10–505101520253035408060Upright position40200DESGECPAirFigure 25-13. Resting pressure profile of the foregut showing the pressure differential between the atmospheric pharyngeal pressure (P) and the less-than-atmospheric midesophageal pressure (E) and greater-than-atmospheric intragastric pressure (G), with the inter-posed high-pressure zones of the cricopharyngeus (C) and distal esophageal sphincter (DES). The necessity for relaxation of the cri-copharyngeus and DES pressure to move a bolus into the stomach is apparent. Esophageal work occurs when a bolus is pushed from the midesophageal area (E), with a pressure less than atmospheric, into the stomach, which has a pressure greater than atmospheric (G). (Reproduced with permission from Waters PF, DeMeester TR: Foregut motor

1	area (E), with a pressure less than atmospheric, into the stomach, which has a pressure greater than atmospheric (G). (Reproduced with permission from Waters PF, DeMeester TR: Foregut motor disorders and their surgical managemen, Med Clin North Am. 1981 Nov;65(6):1235-1268.)Brunicardi_Ch25_p1009-p1098.indd 101501/03/19 6:02 PM 1016SPECIFIC CONSIDERATIONSPART II0102030405060mmHgSwallowSeconds01020304050SecondsSeconds01020304050Seconds01020304050Seconds01020304050StomachHigh pressure zoneEsophageal bodyCricopharyngeusPharynxFigure 25-14. Intraluminal esophageal pressures in response to swallowing. (Reproduced with permission from Waters PF, DeMeester TR: Foregut motor disorders and their surgical man-agemen, Med Clin North Am. 1981 Nov;65(6):1235-1268.)approximately twice the resting level of 30 mmHg. The postre-laxation contraction continues down the esophagus as a peri-staltic wave (Fig. 25-14). The high closing pressure and the initiation of the peristaltic wave prevents reflux

1	of 30 mmHg. The postre-laxation contraction continues down the esophagus as a peri-staltic wave (Fig. 25-14). The high closing pressure and the initiation of the peristaltic wave prevents reflux of the bolus from the esophagus back into the pharynx. After the peristaltic wave has passed farther down the esophagus, the pressure in the upper esophageal sphincter returns to its resting level.Swallowing can be started at will, or it can be reflexively elicited by the stimulation of areas in the mouth and pharynx, among them the anterior and posterior tonsillar pillars or the posterior lateral walls of the hypopharynx. The afferent sen-sory nerves of the pharynx are the glossopharyngeal nerves and the superior laryngeal branches of the vagus nerves. Once aroused by stimuli entering via these nerves, the swallowing center in the medulla coordinates the complete act of swallow-ing by discharging impulses through cranial nerves V, VII, X, XI, and XII, as well as the motor neurons of C1 to C3.

1	the swallowing center in the medulla coordinates the complete act of swallow-ing by discharging impulses through cranial nerves V, VII, X, XI, and XII, as well as the motor neurons of C1 to C3. Dis-charges through these nerves occur in a rather specific pattern and last for approximately 0.5 seconds. Little is known about the organization of the swallowing center, except that it can trigger swallowing after a variety of different inputs, but the response is always a rigidly ordered pattern of outflow. Following a cere-brovascular accident, this coordinated outflow may be altered, causing mild to severe abnormalities of swallowing. In more severe injury, swallowing can be grossly disrupted, leading to repetitive aspiration.The striated muscles of the cricopharyngeus and the upper one-third of the esophagus are activated by efferent motor fibers distributed through the vagus nerve and its recurrent laryngeal branches. The integrity of innervation is required for the cri-copharyngeus to

1	of the esophagus are activated by efferent motor fibers distributed through the vagus nerve and its recurrent laryngeal branches. The integrity of innervation is required for the cri-copharyngeus to relax in coordination with the pharyngeal contraction, and resume its resting tone once a bolus has entered the upper esophagus. Operative damage to the innervation can interfere with laryngeal, cricopharyngeal, and upper esophageal function, and predispose the patient to aspiration.The pharyngeal activity in swallowing initiates the esoph-ageal phase. The body of the esophagus functions as a worm-drive propulsive pump due to the helical arrangement of its circular muscles, and it is responsible for transferring a bolus of food into the stomach. The esophageal phases of swallow-ing represent esophageal work done during alimentation, in that food is moved into the stomach from a negative-pressure environment of –6 mmHg intrathoracic pressure, to a positive-pressure environment of 6 mmHg

1	esophageal work done during alimentation, in that food is moved into the stomach from a negative-pressure environment of –6 mmHg intrathoracic pressure, to a positive-pressure environment of 6 mmHg intra-abdominal pressure, or over a gradient of 12 mmHg (see Fig. 25-13). Effective and coordinated smooth muscle function in the lower one-third of the esophagus is therefore important in pumping the food across this gradient.The peristaltic wave generates an occlusive pressure vary-ing from 30 to 120 mmHg (see Fig. 25-14). The wave rises to a peak in 1 second, lasts at the peak for about 0.5 seconds, and then subsides in about 1.5 seconds. The whole course of the rise and fall of occlusive pressure may occupy one point in the esophagus for 3 to 5 seconds. The peak of a primary peri-staltic contraction initiated by a swallow (primary peristalsis) moves down the esophagus at 2 to 4 cm/s and reaches the distal esophagus about 9 seconds after swallowing starts. Consecutive swallows produce

1	contraction initiated by a swallow (primary peristalsis) moves down the esophagus at 2 to 4 cm/s and reaches the distal esophagus about 9 seconds after swallowing starts. Consecutive swallows produce similar primary peristaltic waves, but when the act of swallowing is rapidly repeated, the esophagus remains relaxed and the peristaltic wave occurs only after the last move-ment of the pharynx. Progress of the wave in the esophagus is caused by sequential activation of its muscles, initiated by effer-ent vagal nerve fibers arising in the swallowing center.Continuity of the esophageal muscle is not necessary for sequential activation if the nerves are intact. If the muscles, but not the nerves, are cut across, the pressure wave begins dis-tally below the cut as it dies out at the proximal end above the cut. This allows a sleeve resection of the esophagus to be done without destroying its normal function. Afferent impulses from receptors within the esophageal wall are not essential for

1	end above the cut. This allows a sleeve resection of the esophagus to be done without destroying its normal function. Afferent impulses from receptors within the esophageal wall are not essential for prog-ress of the coordinated wave. Afferent nerves, however, do go to the swallowing center from the esophagus because if the esoph-agus is distended at any point, a contraction wave begins with a forceful closure of the upper esophageal sphincter and sweeps down the esophagus. This secondary contraction occurs without any movements of the mouth or pharynx. Secondary peristalsis can occur as an independent local reflex to clear the esophagus of ingested material left behind after the passage of the primary wave. Current studies suggest that secondary peristalsis is not as common as once thought.Despite the powerful occlusive pressure, the propulsive force of the esophagus is relatively feeble. If a subject attempts to swallow a bolus attached by a string to a counterweight, the maximum

1	the powerful occlusive pressure, the propulsive force of the esophagus is relatively feeble. If a subject attempts to swallow a bolus attached by a string to a counterweight, the maximum weight that can be overcome is 5 to 10 g. Orderly contractions of the muscular wall and anchoring of the esopha-gus at its inferior end are necessary for efficient aboral propul-sion to occur. Loss of the inferior anchor, as occurs with a large hiatal hernia, can lead to inefficient propulsion.The LES provides a pressure barrier between the esopha-gus and stomach and acts as the valve on the worm-drive pump of the esophageal body. Although an anatomically distinct LES has been difficult to identify, microdissection studies show that, in humans, the sphincter-like function is related to the Brunicardi_Ch25_p1009-p1098.indd 101601/03/19 6:02 PM 1017ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Gastro-esophagealmuscular ringObliquefibersGreater curvaturewall thicknessLesser curvaturewall

1	101601/03/19 6:02 PM 1017ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Gastro-esophagealmuscular ringObliquefibersGreater curvaturewall thicknessLesser curvaturewall thicknessAnterior wall thicknessPhreno-esophagealmembraneSemi-circularfibers50-0-20--50-0 mm-20-50-0 mm-20Figure 25-15. Wall thickness and orientation of fibers on micro-dissection of the cardia. At the junction of the esophageal tube and gastric pouch, there is an oblique muscular ring composed of an increased muscle mass inside the inner muscular layer. On the lesser curve side of the cardia, the muscle fibers of the inner layer are oriented transversely and form semicircular muscle clasps. On the greater curve side of the cardia, these muscle fibers form oblique loops that encircle the distal end of the cardia and gastric fundus. Both the semicircular muscle clasps and the oblique fibers of the fundus contract in a circular manner to close the cardia. (Reproduced with permission from Glenn WWL: Thoracic and

1	gastric fundus. Both the semicircular muscle clasps and the oblique fibers of the fundus contract in a circular manner to close the cardia. (Reproduced with permission from Glenn WWL: Thoracic and Cardiovascular Surgery, 4th ed. Norwalk, CT: Appleton-Century-Crofts; 1983.)architecture of the muscle fibers at the junction of the esoph-ageal tube with the gastric pouch (Fig. 25-15). The sphincter actively remains closed to prevent reflux of gastric contents into the esophagus and opens by a relaxation that coincides with a pharyngeal swallow (see Fig. 25-14). The LES pressure returns to its resting level after the peristaltic wave has passed through the esophagus. Consequently, reflux of gastric juice that may occur through the open valve during a swallow is cleared back into the stomach.If the pharyngeal swallow does not initiate a peristaltic con-traction, then the coincident relaxation of the LES is unguarded and reflux of gastric juice can occur. This may be an explanation for the

1	the pharyngeal swallow does not initiate a peristaltic con-traction, then the coincident relaxation of the LES is unguarded and reflux of gastric juice can occur. This may be an explanation for the observation of spontaneous lower esophageal relaxation, thought by some to be a causative factor in gastroesophageal reflux disease (GERD). The power of the worm-drive pump of the esophageal body is insufficient to force open a valve that does not relax. In dogs, a bilateral cervical parasympathetic blockade abolishes the relaxation of the LES that occurs with pharyngeal swallowing or distention of the esophagus. Conse-quently, vagal function appears to be important in coordinating the relaxation of the LES with esophageal contraction.The antireflux mechanism in human beings is composed of three components: a mechanically effective LES, efficient esophageal clearance, and an adequately functioning gastric reservoir. A defect of any one of these three components can lead to increased

1	of three components: a mechanically effective LES, efficient esophageal clearance, and an adequately functioning gastric reservoir. A defect of any one of these three components can lead to increased esophageal exposure to gastric juice and the development of mucosal injury.Physiologic RefluxOn 24-hour esophageal pH monitoring, healthy individuals have occasional episodes of gastroesophageal reflux. This physi-ologic reflux is more common when awake and in the upright position than during sleep in the supine position. When reflux of gastric juice occurs, normal subjects rapidly clear the acid gastric juice from the esophagus regardless of their position.There are several explanations for the observation that physiologic reflux in normal subjects is more common when they are awake and in the upright position than during sleep in the supine position. First, reflux episodes occur in healthy vol-unteers primarily during transient losses of the gastroesophageal barrier, which may be due to

1	upright position than during sleep in the supine position. First, reflux episodes occur in healthy vol-unteers primarily during transient losses of the gastroesophageal barrier, which may be due to a relaxation of the LES or intra-gastric pressure overcoming sphincter pressure. Gastric juice can also reflux when a swallow-induced relaxation of the LES is not protected by an oncoming peristaltic wave. The average frequency of these “unguarded moments” or of transient losses of the gastroesophageal barrier is far less while asleep and in the supine position than while awake and in the upright posi-tion. Consequently, there are fewer opportunities for reflux to occur in the supine position. Second, in the upright position, there is a 12-mmHg pressure gradient between the resting, posi-tive intra-abdominal pressure measured in the stomach and the most negative intrathoracic pressure measured in the esophagus at midthoracic level. This gradient favors the flow of gastric juice up into the

1	pressure measured in the stomach and the most negative intrathoracic pressure measured in the esophagus at midthoracic level. This gradient favors the flow of gastric juice up into the thoracic esophagus when upright. The gradi-ent diminishes in the supine position. Third, the LES pressure in normal subjects is significantly higher in the supine posi-tion than in the upright position. This is due to the apposition of the hydrostatic pressure of the abdomen to the abdominal portion of the sphincter when supine. In the upright position, the abdominal pressure surrounding the sphincter is negative compared with atmospheric pressure, and, as expected, the abdominal pressure gradually increases the more caudally it is measured. This pressure gradient tends to move the gastric con-tents toward the cardia and encourages the occurrence of reflux into the esophagus when the individual is upright. In contrast, in the supine position, the gastroesophageal pressure gradient diminishes, and the

1	the cardia and encourages the occurrence of reflux into the esophagus when the individual is upright. In contrast, in the supine position, the gastroesophageal pressure gradient diminishes, and the abdominal hydrostatic pressure under the diaphragm increases, causing an increase in sphincter pressure and a more competent cardia.The LES has intrinsic myogenic tone, which is modu-lated by neural and hormonal mechanisms. α-Adrenergic neu-rotransmitters or β-blockers stimulate the LES, and α-blockers and β-stimulants decrease its pressure. It is not clear to what extent cholinergic nerve activity controls LES pressure. The vagus nerve carries both excitatory and inhibitory fibers to the esophagus and sphincter. The hormones gastrin and motilin have been shown to increase LES pressure; and cholecystokinin, estrogen, glucagon, progesterone, somatostatin, and secretin decrease LES pressure. The peptides bombesin, l-enkephalin, and substance P increase LES pressure; and calcitonin

1	and cholecystokinin, estrogen, glucagon, progesterone, somatostatin, and secretin decrease LES pressure. The peptides bombesin, l-enkephalin, and substance P increase LES pressure; and calcitonin gene-related peptide, gastric inhibitory peptide, neuropeptide Y, and vasoactive intestinal polypeptide decrease LES pressure. Some pharmacologic agents such as antacids, cholinergics, agonists, domperidone, metoclopramide, and prostaglandin F2 are known to increase LES pressure; and anticholinergics, barbiturates, cal-cium channel blockers, caffeine, diazepam, dopamine, meperi-dine, prostaglandin E1 and E2, and theophylline decrease LES pressure. Peppermint, chocolate, coffee, ethanol, and fat are all associated with decreased LES pressure and may be responsible for esophageal symptoms after a sumptuous meal.Brunicardi_Ch25_p1009-p1098.indd 101701/03/19 6:02 PM 1018SPECIFIC CONSIDERATIONSPART IIASSESSMENT OF ESOPHAGEAL FUNCTIONA thorough understanding of the patient’s underlying anatomic

1	meal.Brunicardi_Ch25_p1009-p1098.indd 101701/03/19 6:02 PM 1018SPECIFIC CONSIDERATIONSPART IIASSESSMENT OF ESOPHAGEAL FUNCTIONA thorough understanding of the patient’s underlying anatomic and functional deficits before making therapeutic decisions is fundamental to the successful treatment of esophageal disease. The diagnostic tests, as presently used, may be divided into four broad groups: (a) tests to detect structural abnormalities of the esophagus; (b) tests to detect functional abnormalities of the esophagus; (c) tests to detect increased esophageal expo-sure to gastric juice; and (d) tests of duodenogastric function as they relate to esophageal disease.Tests to Detect Structural AbnormalitiesEndoscopic Evaluation. The first diagnostic test in patients with suspected esophageal disease is usually upper gastrointesti-nal endoscopy. This allows assessment and biopsy of the mucosa of the stomach and the esophagus, as well as the diagnosis and assessment of obstructing lesions in

1	is usually upper gastrointesti-nal endoscopy. This allows assessment and biopsy of the mucosa of the stomach and the esophagus, as well as the diagnosis and assessment of obstructing lesions in the upper gastrointestinal tract. In any patient complaining of dysphagia, esophagoscopy is indicated, even in the face of a normal radiographic study.For the initial endoscopic assessment, the flexible fiber-optic esophagoscope is the instrument of choice because of its technical ease, patient acceptance, and the ability to simultane-ously assess the stomach and duodenum. Rigid endoscopy is now only rarely required, mainly for the disimpaction of diffi-cult foreign bodies impacted in the esophagus, and few individ-uals now have the skill set and experience to use this equipment.When GERD is the suspected diagnosis, particular atten-tion should be paid to detecting the presence of esophagitis and Barrett’s columnar-lined esophagus (CLE). When endoscopic esophagitis is seen, severity and the

1	suspected diagnosis, particular atten-tion should be paid to detecting the presence of esophagitis and Barrett’s columnar-lined esophagus (CLE). When endoscopic esophagitis is seen, severity and the length of esophagitis involved are recorded. Whilst many different grading systems have been proposed, the commonest system now in use is the Los Angeles (LA) grading system. In this system, mild esopha-gitis is classified LA grade A or B—one or more erosions lim-ited to the mucosal fold(s) and either less than or greater than 5 mm in longitudinal extent respectively (Fig. 25-16). More severe esophagitis is classified LA grade C or D. In grade C, erosions extend over the mucosal folds but over less than three-quarters of the esophageal circumference; in grade D, confluent erosions extend across more than three-quarters of the esopha-geal circumference. In addition to these grades, more severe damage can lead to the formation of a stricture. A stricture’s severity can be assessed by the

1	more than three-quarters of the esopha-geal circumference. In addition to these grades, more severe damage can lead to the formation of a stricture. A stricture’s severity can be assessed by the ease of passing a standard endo-scope. When a stricture is observed, the severity of the esopha-gitis above it should be recorded. The absence of esophagitis above a stricture suggests the possibility of a chemical-induced injury or a neoplasm as a cause. The latter should always be considered and is ruled out only by evaluation of a tissue biopsy of adequate size. It should be remembered that gastroesophageal reflux is not always associated with visible mucosal abnormali-ties, and patients can experience significant reflux symptoms, despite an apparently normal endoscopy examination.Barrett’s esophagus (BE) is a condition in which the tubu-lar esophagus is lined with columnar epithelium, as opposed to the normal squamous epithelium (see Fig. 25-16). Histologi-cally, it appears as intestinal

1	(BE) is a condition in which the tubu-lar esophagus is lined with columnar epithelium, as opposed to the normal squamous epithelium (see Fig. 25-16). Histologi-cally, it appears as intestinal metaplasia (IM). It is suspected at endoscopy when there is difficulty in visualizing the squamoco-lumnar junction at its normal location, and by the appearance of a redder, salmon-colored mucosa in the lower esophagus, with a clearly visible line of demarcation at the top of the Barrett’s esophagus segment. Its presence is confirmed by biopsy. Mul-tiple biopsy specimens should be taken in a cephalad direction to confirm the presence of IM, and to evaluate the Barrett’s epi-thelium for dysplastic changes. BE is susceptible to ulceration, bleeding, stricture formation, and, most important, malignant degeneration. The earliest sign of the latter is high grade dys-plasia or intramucosal adenocarcinoma (see Fig. 25-16). These dysplastic changes have a patchy distribution, so a minimum of four biopsy

1	The earliest sign of the latter is high grade dys-plasia or intramucosal adenocarcinoma (see Fig. 25-16). These dysplastic changes have a patchy distribution, so a minimum of four biopsy samples spaced 2 cm apart should be taken from the Barrett’s-lined portion of the esophagus. Changes seen in one biopsy are significant. Nishimaki has determined that the tumors occur in an area of specialized columnar epithelium near the squamocolumnar junction in 85% of patients, and within 2 cm of the squamocolumnar junction in virtually all patients. Particular attention should be focused on this area in patients suspected of harboring a carcinoma.Abnormalities of the gastroesophageal flap valve can be visualized by retroflexion of the endoscope. Hill has graded the appearance of the gastroesophageal valve from I to IV according to the degree of unfolding or deterioration of the normal valve architecture (Fig. 25-17). The appearance of the valve correlates with the presence of increased esophageal

1	from I to IV according to the degree of unfolding or deterioration of the normal valve architecture (Fig. 25-17). The appearance of the valve correlates with the presence of increased esophageal acid exposure, occur-ring predominantly in patients with grade III and IV valves.A hiatal hernia is endoscopically confirmed by finding a pouch lined with gastric rugal folds lying 2 cm or more above the margins of the diaphragmatic crura, identified by having the patient sniff. A hernia is best demonstrated with the stomach fully insufflated and the gastroesophageal junction observed with a retroflexed endoscope. A prominent sliding hiatal hernia frequently is associated with increased esophageal exposure to gastric juice. When a paraesophageal hernia (PEH) is observed, particular attention is taken to exclude gastric (Cameron’s) ulcers or gastritis within the pouch. The intragastric retroflex or J maneuver is important in evaluating the full circumference of the mucosal lining of the

1	is taken to exclude gastric (Cameron’s) ulcers or gastritis within the pouch. The intragastric retroflex or J maneuver is important in evaluating the full circumference of the mucosal lining of the herniated stomach.When an esophageal diverticulum is seen, it should be carefully explored with the flexible endoscope to exclude ulceration or neoplasia. When a submucosal mass is identified, biopsy specimens are usually not performed. At the time of sur-gical resection, a submucosal leiomyoma or reduplication cyst can generally be dissected away from the intact mucosa, but if a biopsy sample is taken, the mucosa may become fixed to the underlying abnormality. This complicates the surgical dissec-tion by increasing the risk of mucosal perforation. Endoscopic ultrasound provides a better method for evaluating these lesions.Radiographic Evaluation. Barium swallow evaluation is under-taken selectively to assess anatomy and motility. The anatomy of large hiatal hernias is more clearly

1	method for evaluating these lesions.Radiographic Evaluation. Barium swallow evaluation is under-taken selectively to assess anatomy and motility. The anatomy of large hiatal hernias is more clearly demonstrated by contrast radi-ology than endoscopy, and the presence of coordinated esopha-geal peristalsis can be determined by observing several individual swallows of barium traversing the entire length of the organ, with the patient in the horizontal position. Hiatal hernias are best demonstrated with the patient prone because the increased intra-abdominal pressure produced in this position promotes displace-ment of the esophagogastric junction above the diaphragm. To detect lower esophageal narrowing, such as rings and strictures, fully distended views of the esophagogastric region are crucial. The density of the barium used to study the esophagus can poten-tially affect the accuracy of the examination. Esophageal disorders shown clearly by a full-column technique include

1	are crucial. The density of the barium used to study the esophagus can poten-tially affect the accuracy of the examination. Esophageal disorders shown clearly by a full-column technique include circumferential carcinomas, peptic strictures, large esophageal ulcers, and hia-tal hernias. A small hiatal hernia is usually not associated with significant symptoms or illness, and its presence is an irrelevant finding unless the hiatal hernia is large (Fig. 25-18) or the hernia 1Brunicardi_Ch25_p1009-p1098.indd 101801/03/19 6:02 PM 1019ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Figure 25-16. Complications of reflux disease as seen on endoscopy. A. Linear erosions of LA grade B esophagitis. B. Uncomplicated Barrett’s mucosa. C. High-grade dysplasia in Barrett’s mucosa. D. Early adenocarcinoma arising in Barrett’s mucosa.is of the paraesophageal variety. Lesions extrinsic but adjacent to the esophagus can be reliably detected by the full-column tech-nique if they contact the distended

1	arising in Barrett’s mucosa.is of the paraesophageal variety. Lesions extrinsic but adjacent to the esophagus can be reliably detected by the full-column tech-nique if they contact the distended esophageal wall. Conversely, a number of important disorders may go undetected if this is the sole technique used to examine the esophagus. These include small esophageal neoplasms, mild esophagitis, and esophageal varices. Thus, the full-column technique should be supplemented with mucosal relief or double-contrast films to enhance detection of these smaller or more subtle lesions.Motion-recording techniques greatly aid in evaluating functional disorders of the pharyngoesophageal and esophageal phases of swallowing. The technique and indications for cineand videoradiography will be discussed in the section entitled “Videoand Cineradiography,” as they are more useful to evalu-ate function and seldom used to detect structural abnormalities.The radiographic assessment of the esophagus is not

1	the section entitled “Videoand Cineradiography,” as they are more useful to evalu-ate function and seldom used to detect structural abnormalities.The radiographic assessment of the esophagus is not com-plete unless the entire stomach and duodenum have been examined. A gastric or duodenal ulcer, partially obstructing gastric neoplasm, or scarred duodenum and pylorus may contribute significantly to symptoms otherwise attributable to an esophageal abnormality.When a patient’s complaints include dysphagia and no obstructing lesion is seen on the barium swallow, it is useful to have the patient swallow a barium-impregnated marshmallow, a barium-soaked piece of bread, or a hamburger mixed with bar-ium. This test may bring out a functional disturbance in esopha-geal transport that can be missed when liquid barium is used.Tests to Detect Functional AbnormalitiesIn many patients with symptoms of an esophageal disorder, standard radiographic and endoscopic evaluation fails to dem-onstrate a

1	when liquid barium is used.Tests to Detect Functional AbnormalitiesIn many patients with symptoms of an esophageal disorder, standard radiographic and endoscopic evaluation fails to dem-onstrate a structural abnormality. In these situations, esophageal function tests are necessary to identify a functional disorder.Esophageal Motility. Esophageal motility is a widely used technique to examine the motor function of the esophagus and ABCDBrunicardi_Ch25_p1009-p1098.indd 101901/03/19 6:02 PM 1020SPECIFIC CONSIDERATIONSPART IIBACFigure 25-17. A. Grade I flap valve appearance. Note the ridge of tissue that is closely approximated to the shaft of the retroflexed endoscope. It extends 3 to 4 cm along the lesser curve. B. Grade II flap valve appearance. The ridge is slightly less well defined than in grade I and it opens rarely with respiration and closes promptly. C. Grade III flap valve appearance. The ridge is barely present, and there is often failure to close around the endoscope. It

1	than in grade I and it opens rarely with respiration and closes promptly. C. Grade III flap valve appearance. The ridge is barely present, and there is often failure to close around the endoscope. It is nearly always accompanied by a hiatal hernia. D. Grade IV flap valve appearance. There is no muscular ridge at all. The gastroesophageal valve stays open all the time, and squamous epithelium can often be seen from the retroflexed position. A hiatal hernia is always present. (Reproduced with permission from Hill LD, Kozarek RA, Kraemer SJ, et al: The gastroesophageal flap valve: in vitro and in vivo observations, Gastrointest Endosc. 1996 Nov;44(5):541-547.)Brunicardi_Ch25_p1009-p1098.indd 102001/03/19 6:02 PM 1021ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Figure 25-18. Radiogram of an intrathoracic stomach. This is the end stage of a large hiatal hernia, regardless of its initial classification.RIP = Respiratory inversion pointRIP43424140393837 cmOverall lengthPressure10

1	of an intrathoracic stomach. This is the end stage of a large hiatal hernia, regardless of its initial classification.RIP = Respiratory inversion pointRIP43424140393837 cmOverall lengthPressure10 secEsophagealbaselinepressureAbdominal lengthGastricbaselinepressureFigure 25-19. Manometric pressure profile of the lower esophageal sphincter. The distances are measured from the nares. (Reproduced with permission from Zaninotto G, DeMeester TR, Schwizer W, et al: The lower esophageal sphincter in health and disease, Am J Surg. 1988 Jan;155(1):104-11.)DFigure 25-17. (Continued )its sphincters. The esophageal motility study (EMS) is indicated whenever a motor abnormality of the esophagus is suspected on the basis of complaints of dysphagia, odynophagia, or noncar-diac chest pain, and the barium swallow or endoscopy does not show a clear structural abnormality. EMS is particularly neces-sary to confirm the diagnosis of specific primary esophageal motility disorders (i.e., achalasia, diffuse

1	swallow or endoscopy does not show a clear structural abnormality. EMS is particularly neces-sary to confirm the diagnosis of specific primary esophageal motility disorders (i.e., achalasia, diffuse esophageal spasm [DES], nutcracker esophagus, and hypertensive LES). It also identifies nonspecific esophageal motility abnormalities and motility disorders secondary to systemic disease such as sclero-derma, dermatomyositis, polymyositis, or mixed connective tis-sue disease. In patients with symptomatic GERD, manometry of the esophageal body can identify a mechanically defective LES and evaluate the adequacy of esophageal peristalsis and contraction amplitude. EMS has become an essential tool in the preoperative evaluation of patients before antireflux surgery, guiding selection of the appropriate procedure based upon the patient’s underlying esophageal function and excluding patients with achalasia who can be misdiagnosed with gastroesophageal reflux when clinical and endoscopic

1	appropriate procedure based upon the patient’s underlying esophageal function and excluding patients with achalasia who can be misdiagnosed with gastroesophageal reflux when clinical and endoscopic parameters alone are used for diagnosis.EMS is performed using electronic, pressure-sensitive transducers located within the catheter, or water-perfused cath-eters with lateral side holes attached to transducers outside the body. The traditional water perfused catheter has largely been replaced by high resolution motility (HRM), but knowledge of traditional methods of assessing esophageal motility is helpful for understanding esophageal physiology.As the pressure-sensitive station is brought across the gas-troesophageal junction (GEJ), a rise in pressure above the gas-tric baseline signals the beginning of the LES. The respiratory inversion point is identified when the positive excursions that occur in the abdominal cavity with breathing change to negative deflections in the thorax. The

1	beginning of the LES. The respiratory inversion point is identified when the positive excursions that occur in the abdominal cavity with breathing change to negative deflections in the thorax. The respiratory inversion point serves as a reference point at which the amplitude of LES pressure and the length of the sphincter exposed to abdominal pressure are measured. As the pressure-sensitive station is withdrawn into the body of the esophagus, the upper border of the LES is identified by the drop in pressure to the esophageal baseline. From these measurements, the pressure, abdominal length, and overall length of the sphincter are determined (Fig. 25-19). To Brunicardi_Ch25_p1009-p1098.indd 102101/03/19 6:02 PM 1022SPECIFIC CONSIDERATIONSPART IILALPLPARPRRA25050Figure 25-20. Radial configuration of the lower esophageal sphincter. A = anterior; L = left; LA = left anterior; LP = left pos-terior; P = posterior; R = right; RA = right anterior; RP = right pos-terior. (Reproduced with

1	of the lower esophageal sphincter. A = anterior; L = left; LA = left anterior; LP = left pos-terior; P = posterior; R = right; RA = right anterior; RP = right pos-terior. (Reproduced with permission from Winans CS: Manometric asymmetry of the lower-esophageal high-pressure zone, Am J Dig Dis. 1977 Apr;22(4):348-354.)Table 25-1Normal manometric values of the distal esophageal sphincter, n = 50 MEDIAN PERCENTILE2.597.5Pressure (mmHg)135.827.7Overall length (cm)3.62.15.6Abdominal length (cm)20.94.7 MEANMEAN – 2 SDMEAN + 2 SDPressure (mmHg)13.8 ± 4.64.623.0Overall length (cm)3.7 ± 0.82.15.3Abdominal length (cm)2.2 ± 0.80.63.8SD = standard deviation.Reproduced with permission from Moody FG, Carey LC, Jones RS, et al: Surgical Treatment of Digestive Disease. Chicago, IL: Year Book Medical; 1990.account for the asymmetry of the sphincter (Fig. 25-20), the pressure profile is repeated with each of the five radially ori-ented transducers, and the average values for sphincter pressure above

1	for the asymmetry of the sphincter (Fig. 25-20), the pressure profile is repeated with each of the five radially ori-ented transducers, and the average values for sphincter pressure above gastric baseline, overall sphincter length, and abdominal length of the sphincter are calculated.Table 25-1 shows the values for these parameters in 50 normal volunteers without subjective or objective evidence of a foregut disorder. A mechanically defective sphincter is identified by having one or more of the following characteristics: an average LES pressure of <6 mmHg, an average length exposed to the positive-pressure environment in the abdomen of 1 cm or less, and/or an average overall sphincter length of 2 cm or less.High-Resolution Manometry. Esophageal manometry was introduced into clinical practice in the 1970s and, until recently, has changed little. In 1991, Ray Clouse introduced the concept of improving conventional manometry by increasing the number of recording sites and adding a

1	practice in the 1970s and, until recently, has changed little. In 1991, Ray Clouse introduced the concept of improving conventional manometry by increasing the number of recording sites and adding a three-dimensional assessment. This “high-resolution manometry” is a variant of the conventional manometry in which multiple, circumferential recording sites are used, in essence creating a “map” of the esophagus and its sphincters. High-resolution catheters contain 36 miniaturized pressure sensors positioned every centimeter along the length of the catheter. The vast amount of data generated by these sensors is then processed and presented in traditional linear plots or as a visually enhanced spatiotemporal video tracing that is readily interpreted. The function of the esophageal body is assessed with 10 to 15 wet swallows. Amplitude, duration, and morphology of contractions following each swallow are visually displayed (Fig. 25-21).The relationship of the esophageal contractions following

1	with 10 to 15 wet swallows. Amplitude, duration, and morphology of contractions following each swallow are visually displayed (Fig. 25-21).The relationship of the esophageal contractions following a swallow is classified as peristaltic or simultaneous. The data are used to identify motor disorders of the esophagus.The position, length, and function of the lower esopha-geal sphincter (LES) are demonstrated by a high-pressure zone that should relax at the inception of swallowing and contract after the water or solid bolus passes through the LES. Simul-taneous acquisition of data for the upper esophageal sphinc-ter, esophageal body, LES, and gastric pressure minimizes the movement artifacts and study time associated with conven-tional esophageal manometry. This technology significantly enhances esophageal diagnostics, bringing it into the realm of “image”-based studies. High-resolution manometry may allow the identification of focal motor abnormalities previ-ously overlooked. It has

1	esophageal diagnostics, bringing it into the realm of “image”-based studies. High-resolution manometry may allow the identification of focal motor abnormalities previ-ously overlooked. It has enhanced the ability to predict bolus propagation and increased sensitivity in the measurement of pressure gradients.Esophageal Impedance. Newer technology introduced into the clinical realm a decade ago allows measurement of esophageal function and gastroesophageal reflux in a way that was previously not possible. An intraluminal electrical imped-ance catheter is used to measure GI function. Impedance is the ratio of voltage to current, and is a measure of the electrical conductivity of a hollow organ and its contents. Intraluminal electrical impedance is inversely proportional to the electrical conductivity of the luminal contents and the cross-sectional area of the lumen. Air has a very low electrical conductivity and, therefore, high impedance. Saliva and food cause an imped-ance decrease

1	conductivity of the luminal contents and the cross-sectional area of the lumen. Air has a very low electrical conductivity and, therefore, high impedance. Saliva and food cause an imped-ance decrease because of their increased conductivity. Luminal dilatation results in a decrease in impedance, whereas luminal contraction yields an impedance increase. Investigators have established the impedance waveform characteristics that define esophageal bolus transport. This allows for the characterization of both esophageal function, via quantification of bolus trans-port, and gastroesophageal reflux (Fig. 25-22). The probe mea-sures impedance between adjacent electrodes, with measuring segments located at 2, 4, 6, 8, 14, and 16 cm from the distal tip. An extremely low electric current of 0.00025 μW is transmitted across the electrodes at a frequency of 1 to 2 kHz and is limited Brunicardi_Ch25_p1009-p1098.indd 102201/03/19 6:02 PM 1023ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Figure

1	μW is transmitted across the electrodes at a frequency of 1 to 2 kHz and is limited Brunicardi_Ch25_p1009-p1098.indd 102201/03/19 6:02 PM 1023ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Figure 25-21A. Normal high-resolution manometry motility study. Pressure measurements are recorded with color coding (red = high; blue = low). LES = lower esophageal sphincter; PIP = pressure inversion point; UES = upper esophageal sphincter.UES19.0LES41.840.343.7Gastric 46.2PIP42.3EsophagusPharynxStomachBrunicardi_Ch25_p1009-p1098.indd 102301/03/19 6:02 PM 1024SPECIFIC CONSIDERATIONSPART IIFigure 25-21B. High-resolution manometry motility study in patient with mechanically defective lower esophageal sphincter. Note the absence of lower esophageal sphincter tone. Pressure measure-ments are recorded with color coding (red = high; blue = low). LES = lower esophageal sphincter; PIP = pressure inversion point; UES = upper esophageal

1	esophageal sphincter tone. Pressure measure-ments are recorded with color coding (red = high; blue = low). LES = lower esophageal sphincter; PIP = pressure inversion point; UES = upper esophageal sphincter.EsophagusStomachPharynxUES20.8LES41.9PIP41.841.342.7Gastric 50.3Brunicardi_Ch25_p1009-p1098.indd 102401/03/19 6:02 PM 1025ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Figure 25-21C. High-resolution manometry motility study in patient with deficient esophageal body peristalsis. Note the very weak peristalsis in the lower two-thirds of the esophagus. Pressure measurements are recorded with color coding (red = high; blue = low). LES = lower esophageal sphincter; PIP = pressure inversion point; UES = upper esophageal sphincter.EsophagusPharynxUES18.740.944.6Gastric 47.5LES42.2PIP42.3StomachBrunicardi_Ch25_p1009-p1098.indd 102501/03/19 6:02 PM 1026SPECIFIC CONSIDERATIONSPART IIFigure 25-21D. High-resolution manometry motility study in patient with achalasia. Note the complete

1	102501/03/19 6:02 PM 1026SPECIFIC CONSIDERATIONSPART IIFigure 25-21D. High-resolution manometry motility study in patient with achalasia. Note the complete absence of esophageal body peristalsis, and the lack of relaxation of the lower esophageal sphincter. Pressure measurements are recorded with color coding (red = high; blue = low). LES = lower esophageal sphincter; PIP = pressure inversion point; UES = upper esophageal sphincter.EsophagusUES18.0Gastric 48.542.745.7LES43.8PIP44.1StomachPharynxBrunicardi_Ch25_p1009-p1098.indd 102601/03/19 6:03 PM 1027ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Figure 25-21E. High-resolution manometry motility study in patient with diffuse esophageal spasm. Note the very high amplitude contractions in the esophageal body. Pressure measurements are recorded with color coding (red = high; blue = low). LES = lower esophageal sphincter; PIP = pressure inversion point; UES = upper esophageal sphincter.Gastric

1	body. Pressure measurements are recorded with color coding (red = high; blue = low). LES = lower esophageal sphincter; PIP = pressure inversion point; UES = upper esophageal sphincter.Gastric 51.745.6PharynxEsophagusLES47.4PIP47.1UES20.349.7StomachBrunicardi_Ch25_p1009-p1098.indd 102701/03/19 6:03 PM 1028SPECIFIC CONSIDERATIONSPART IIpH siteImpedence site17cm15cm9cm7cm5cmDistance above LESDistance above LES5cmLES3cmFigure 25-22. Esophageal impedance probe measures electrical resistance between evenly spaced electrodes. LES = lower esopha-geal sphincter.to 8 μA. This is below the stimulation threshold for nerves and muscles and is three orders of magnitude below the thresh-old of cardiac stimulation. A standard pH electrode is located 5 cm from the distal tip so that the acidic or nonacidic nature of refluxate can be correlated with the number of reflux events.Esophageal impedance has been validated as an appropri-ate method for the evaluation of GI function and is used

1	or nonacidic nature of refluxate can be correlated with the number of reflux events.Esophageal impedance has been validated as an appropri-ate method for the evaluation of GI function and is used selec-tively for the diagnosis of gastroesophageal reflux. It has been compared to cineradiography showing that impedance waves correspond well with actual bolus transport illustrated by radi-ography. Bolus entry, transit, and exit can be clearly identified by impedance changes in the corresponding measuring seg-ments. Studies comparing standard esophageal manometry with impedance measurements in healthy volunteers have shown that esophageal impedance correlates with peristaltic wave progres-sion and bolus length.Twenty-four-hour pH monitoring, the historical gold stan-dard for diagnosing and quantifying gastroesophageal reflux, has some significant limitations. With 24-hour ambulatory pH testing, reflux is defined as a drop in the pH below 4, which effectively “blinds” the test to reflux

1	quantifying gastroesophageal reflux, has some significant limitations. With 24-hour ambulatory pH testing, reflux is defined as a drop in the pH below 4, which effectively “blinds” the test to reflux occurring at higher pH values. Furthermore, in patients with persistent symptoms on proton pump inhibitor (PPI) therapy, pH monitoring has lim-ited use as it can only detect abnormal acid reflux (pH <4), the occurrence of which has been altered by the antisecretory medi-cation. Given that PPI antisecretory therapy is highly effective in neutralizing gastric acid, the question of whether persistent symptoms are a result of persistent acid reflux, nonacid reflux, or are not reflux related becomes a key issue in surgical decision making. Until recently, this differentiation could not be made. Detection of both acid and nonacid reflux has potential to define these populations of patients and thus improve patient selection for antireflux surgery. Multichannel intraluminal impedance technology

1	of both acid and nonacid reflux has potential to define these populations of patients and thus improve patient selection for antireflux surgery. Multichannel intraluminal impedance technology allows the measurement of both acid and nonacid reflux, with potential to enhance diagnostic accuracy.Using this technology, Balaji and colleagues showed that most gastroesophageal reflux remains despite acid suppression. Impedance pH may be particularly useful in evaluating patients with persistent symptoms despite PPI treatment, patients with respiratory symptoms, and postoperative patients who are hav-ing symptoms that are elusive to diagnosis.Esophageal Transit Scintigraphy. The esophageal transit of a 10-mL water bolus containing technetium-99m (99mTc) sulfur colloid can be recorded with a gamma camera. Using this tech-nique, delayed bolus transit has been shown in patients with a variety of esophageal motor disorders, including achalasia, scleroderma, DES, and nutcracker esophagus.Videoand

1	camera. Using this tech-nique, delayed bolus transit has been shown in patients with a variety of esophageal motor disorders, including achalasia, scleroderma, DES, and nutcracker esophagus.Videoand CineradiographyHigh-speed cinematic or video recording of radiographic studies allows re-evaluation by reviewing the studies at various speeds. This technique is more useful than manometry in the evaluation of the pharyngeal phase of swallowing. Observations suggesting oropharyngeal or cricopharyngeal dysfunction include misdirec-tion of barium into the trachea or nasopharynx, prominence of the cricopharyngeal muscle, a Zenker’s diverticulum, a narrow pharyngoesophageal segment, and stasis of the contrast medium in the valleculae or hypopharyngeal recesses (Fig. 25-23). These findings are usually not specific, but rather common manifesta-tions of neuromuscular disorders affecting the pharyngoesoph-ageal area. Studies using liquid barium, barium-impregnated solids, or radiopaque pills aid

1	not specific, but rather common manifesta-tions of neuromuscular disorders affecting the pharyngoesoph-ageal area. Studies using liquid barium, barium-impregnated solids, or radiopaque pills aid the evaluation of normal and abnormal motility in the esophageal body. Loss of the normal stripping wave or segmentation of the barium column with the patient in the recumbent position correlates with abnormal motility of the esophageal body. In addition, structural abnor-malities such as small diverticula, webs, and minimal extrin-sic impressions of the esophagus may be recognized only with motion-recording techniques. The simultaneous computerized capture of videofluoroscopic images and manometric tracings is now available and is referred to as manofluorography. Mano-fluorographic studies allow precise correlation of the anatomic events, such as opening of the upper esophageal sphincter, with manometric observations, such as sphincter relaxation. Mano-fluorography, although not widely

1	allow precise correlation of the anatomic events, such as opening of the upper esophageal sphincter, with manometric observations, such as sphincter relaxation. Mano-fluorography, although not widely available, is presently the best means available to evaluate complex functional abnormalities.Tests to Detect Increased Exposure to Gastric JuiceTwenty-Four-Hour Ambulatory pH Monitoring. The most direct method of measuring increased esophageal exposure to gas-tric juice is by an indwelling pH electrode, or, more recently, via a radiotelemetric pH monitoring capsule that can be clipped to the esophageal mucosa. The latter consists of an antimony pH elec-trode fitted inside a small, capsule-shaped device accompanied by a battery and electronics that allow 48-hour monitoring and transmission of the pH data via transcutaneous radio telemetry to a waist-mounted data logger. The device can be introduced either transorally or transnasally, and it can be clipped to the esophageal mucosa using

1	of the pH data via transcutaneous radio telemetry to a waist-mounted data logger. The device can be introduced either transorally or transnasally, and it can be clipped to the esophageal mucosa using endoscopic fastening techniques. It passes sponta-neously within 1 to 2 weeks. Prolonged monitoring of esophageal pH is performed by placing the pH probe or telemetry capsule 5 cm above the manometrically measured upper border of the dis-tal sphincter for 24 hours. It measures the actual time the esopha-geal mucosa is exposed to gastric juice, measures the ability of the esophagus to clear refluxed acid, and correlates esophageal acid exposure with the patient’s symptoms. A 24to 48-hour period is necessary so that measurements can be made over one or two complete circadian cycles. This allows measuring the effect of physiologic activity, such as eating or sleeping, on the reflux of gastric juice into the esophagus (Fig. 25-24).Brunicardi_Ch25_p1009-p1098.indd 102801/03/19 6:03 PM

1	measuring the effect of physiologic activity, such as eating or sleeping, on the reflux of gastric juice into the esophagus (Fig. 25-24).Brunicardi_Ch25_p1009-p1098.indd 102801/03/19 6:03 PM 1029ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25mpmppH8642mppH8642pH8642sp06:0000:0022:0002:0004:0022:0016:0014:0018:0020:0014:0008:0006:0010:0012:00Figure 25-24. Strip chart display of a 24-hour esophageal pH monitoring study in a patient with increased esophageal acid expo-sure. mp = meal period; sp = supine period. (Reproduced with per-mission from Zuidema GD, Orringer MB: Shackelford’s Surgery of the Alimentary Tract, 3rd ed. Vol 1. Philadelphia, PA: Elsevier/Saunders; 1991.)BATable 25-2Normal values for esophageal exposure to pH <4 (n = 50)COMPONENTMEANSD95%Total time1.511.364.45Upright time2.342.348.42Supine time0.631.03.45No. of episodes19.0012.7646.90No. >5 min0.841.183.45Longest episode6.747.8519.80SD = standard deviation.Reproduced with permission from Moody FG, Carey LC, Jones RS,

1	time0.631.03.45No. of episodes19.0012.7646.90No. >5 min0.841.183.45Longest episode6.747.8519.80SD = standard deviation.Reproduced with permission from Moody FG, Carey LC, Jones RS, et al: Surgical Treatment of Digestive Disease. Chicago, IL: Year Book Medical; 1990.Figure 25-23. Esophagograms from a patient with cricopharyngeal achalasia. A. Anteropos-terior film showing retention of the contrast medium at the level of the vallecula and piriform recesses, with no barium passing into the esopha-gus. B. Lateral film, taken opposite the C5–C6 vertebrae, showing posterior indentation of the cricopharyngeus, retention in the hypopharynx, and tracheal aspiration. (Reproduced with per-mission from DeMeester TR, Matthews H: Inter-national Trends in General Thoracic Surgery. Vol 3. Benign Esophageal Disease. St. Louis, Mo: Mosby; 1987.)The 24-hour esophageal pH monitoring should not be con-sidered a test for reflux, but rather a measurement of the esopha-geal exposure to gastric juice. The

1	Disease. St. Louis, Mo: Mosby; 1987.)The 24-hour esophageal pH monitoring should not be con-sidered a test for reflux, but rather a measurement of the esopha-geal exposure to gastric juice. The measurement is expressed by the time the esophageal pH was below a given threshold during the 24-hour period (Table 25-3). This single assess-ment, although concise, does not reflect how the exposure has occurred; that is, did it occur in a few long episodes or several short episodes? Consequently, two other assessments are neces-sary: the frequency of the reflux episodes and their duration.The units used to express esophageal exposure to gastric juice are: (a) cumulative time the esophageal pH is below a cho-sen threshold, expressed as the percentage of the total, upright, and supine monitored time; (b) frequency of reflux episodes below a chosen threshold, expressed as number of episodes per 24 hours; and (c) duration of the episodes, expressed as the number of episodes >5 minutes per 24

1	time; (b) frequency of reflux episodes below a chosen threshold, expressed as number of episodes per 24 hours; and (c) duration of the episodes, expressed as the number of episodes >5 minutes per 24 hours, and the time in minutes of the longest episode recorded. Table 25-2 shows the normal values for these components of the 24-hour record at the whole-number pH threshold derived from 50 normal asymptom-atic subjects. The upper limits of normal were established at the 95th percentile. Most centers use pH 4 as the threshold.Based on these studies and extensive clinical experience, 48-hour esophageal pH monitoring is considered to be the gold standard for the diagnosis of GERD.The Bravo pH Capsule (Medtronics, Minneapolis, MN) measures pH levels in the esophagus and transmits continuous Brunicardi_Ch25_p1009-p1098.indd 102901/03/19 6:03 PM 1030SPECIFIC CONSIDERATIONSPART II210:0012:0014:0016:0018:0047pH218:0020:0022:0000:0002:0047202:0004:0006:0008:0010:0047pH probe5 cmabove5

1	Brunicardi_Ch25_p1009-p1098.indd 102901/03/19 6:03 PM 1030SPECIFIC CONSIDERATIONSPART II210:0012:0014:0016:0018:0047pH218:0020:0022:0000:0002:0047202:0004:0006:0008:0010:0047pH probe5 cmabove5 cmbelowBACombined 24-hourgastric and esophagealpH monitoringFigure 25-25. A. Combined esophageal and gastric pH monitoring showing position of probes in relation to the lower esophageal sphincter. B. Combined ambulatory esophageal (upper tracing) and gastric (lower tracing) pH monitoring showing duodenogastric reflux (arrows) with propagation of the alkaline juice into the esophagus of a patient with complicated Barrett’s esophagus. The gastric tracing (lower) is taken from a probe lying 5 cm below the upper esophageal sphincter. The esophageal tracing (upper) is taken from a probe lying 5 cm above the lower esophageal sphincter. Note that in only a small proportion of time does duodenogastric reflux move the pH of the esophagus above the threshold of 7, causing the iceberg effect.

1	5 cm above the lower esophageal sphincter. Note that in only a small proportion of time does duodenogastric reflux move the pH of the esophagus above the threshold of 7, causing the iceberg effect. (Reproduced with permission from Zuidema GD, Orringer MB: Shackelford’s Surgery of the Alimentary Tract, 3rd ed. Vol 1. Philadelphia, PA: Elsevier/Saunders; 1991.)Table 25-3Normal composite score for various pH thresholds: upper level of normal valuepH THRESHOLD95TH PERCENTILE<114.2<217.37<314.10<414.72<515.76<612.76>714.90>88.50Reproduced with permission from Moody FG, Carey LC, Jones RS, et al: Surgical Treatment of Digestive Disease. Chicago, IL: Year Book Medical; 1990.esophageal pH readings to a receiver worn on the patient’s belt or waistband (Fig. 25-25). Symptoms that the patient experi-ences are recorded in a diary and/or by pressing buttons on the receiver unit. Generally, 48 hours of pH data are measured with this probe. A recent study has shown that the addition of a second

1	are recorded in a diary and/or by pressing buttons on the receiver unit. Generally, 48 hours of pH data are measured with this probe. A recent study has shown that the addition of a second day of pH monitoring increased the sensitivity of pH measurement by 22%. The capsule eventually detaches and passes through the digestive tract in 5 to 7 days.Radiographic Detection of Gastroesophageal Reflux. The definition of radiographic gastroesophageal reflux varies depend-ing on whether reflux is spontaneous or induced by various maneu-vers. In only about 40% of patients with classic symptoms of GERD is spontaneous reflux (i.e., reflux of barium from the stom-ach into the esophagus with the patient in the upright position) observed by the radiologist. In most patients who show spon-taneous reflux on radiography, the diagnosis of increased esophageal acid exposure is confirmed by 24-hour esophageal pH monitoring. Therefore, the radiographic demonstration of sponta-neous regurgitation of barium

1	on radiography, the diagnosis of increased esophageal acid exposure is confirmed by 24-hour esophageal pH monitoring. Therefore, the radiographic demonstration of sponta-neous regurgitation of barium into the esophagus in the upright position is a reliable indicator that reflux is present. However, fail-ure to see this does not indicate the absence of disease, and for this reason this test is rarely used for clinical diagnosis.Tests of Duodenogastric FunctionEsophageal disorders are frequently associated with abnormali-ties of duodenogastric function. Abnormalities of the gastric res-ervoir or increased gastric acid secretion can be responsible for increased esophageal exposure to gastric juice. Reflux of alka-line duodenal juice, including bile salts, pancreatic enzymes, and bicarbonate, is thought to have a role in the pathogenesis of esophagitis and complicated Barrett’s esophagus. Furthermore, functional disorders of the esophagus are often not confined to

1	and bicarbonate, is thought to have a role in the pathogenesis of esophagitis and complicated Barrett’s esophagus. Furthermore, functional disorders of the esophagus are often not confined to 2Brunicardi_Ch25_p1009-p1098.indd 103001/03/19 6:03 PM 1031ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25the esophagus alone, but are associated with functional disor-ders of the rest of the foregut (i.e., stomach and duodenum). Tests of duodenogastric function that are helpful to investigate esophageal symptoms include gastric emptying studies, gastric acid analysis, and cholescintigraphy (for the diagnosis of patho-logic duodenogastric and/or duodenogastroesophageal reflux).Gastric Emptying Study. Gastric emptying studies are performed with radionuclide-labeled meals. Emptying of solids and liquids can be assessed simultaneously when both phases are marked with different tracers. After ingestion of a labeled standard meal, gamma camera images of the stomach are obtained at 5to 15-minute

1	liquids can be assessed simultaneously when both phases are marked with different tracers. After ingestion of a labeled standard meal, gamma camera images of the stomach are obtained at 5to 15-minute inter-vals for 2 to 4 hours. After correction for decay, the counts in the gastric area are plotted as the percentage of total counts at the start of the imaging. The resulting emptying curve can be compared with data obtained in normal volunteers. In general, normal subjects will empty 59% of a meal within 90 minutes. Although delayed gas-tric emptying is often associated with gastroesophageal reflux, in general delayed emptying does not correlate with a poorer clinical outcome after antireflux surgery, and it should not be considered a contraindication to surgical treatment.GASTROESOPHAGEAL REFLUX DISEASEGERD was not recognized as a significant clinical problem until the mid-1930s and was not identified as a precipitating cause for esophagitis until after World War II. In the early 21st

1	REFLUX DISEASEGERD was not recognized as a significant clinical problem until the mid-1930s and was not identified as a precipitating cause for esophagitis until after World War II. In the early 21st century, it has grown to be a very common problem and now accounts for a majority of esophageal pathology. It is recognized as a chronic disease, and when medical therapy is required, it is often lifelong treatment. Recent efforts at the development of various endoscopic antireflux interventions, although innovative, have not been successful in consistently controlling gastroesophageal reflux. Antireflux surgery is an effective and long-term therapy and is the only treatment that is able to restore the gastroesopha-geal barrier. Despite the common prevalence of GERD, it can be one of the most challenging diagnostic and therapeutic problems in clinical medicine. A contributing factor to this is the lack of a universally accepted definition of the disease.The most simplistic approach is to

1	challenging diagnostic and therapeutic problems in clinical medicine. A contributing factor to this is the lack of a universally accepted definition of the disease.The most simplistic approach is to define the disease by its symptoms. However, symptoms thought to be indicative of GERD, such as heartburn or acid regurgitation, are very com-mon in the general population and many individuals consider them to be normal and do not seek medical attention. Even when excessive, these symptoms are not specific for gastroesophageal reflux. They can be caused by other diseases such as achalasia, DES, esophageal carcinoma, pyloric stenosis, cholelithiasis, gastritis, gastric or duodenal ulcer, and coronary artery disease.A thorough, structured evaluation of the patient’s symptoms is essential before any therapy, particularly any form of esopha-geal surgery. The presence and severity of both typical symp-toms of heartburn, regurgitation, and dysphagia, and atypical symptoms of cough, hoarseness,

1	any therapy, particularly any form of esopha-geal surgery. The presence and severity of both typical symp-toms of heartburn, regurgitation, and dysphagia, and atypical symptoms of cough, hoarseness, chest pain, asthma, and aspira-tion should be discussed with the patient in detail. Many of these atypical symptoms may not be esophageal related and hence will not improve and may even worsen with antireflux surgery.Heartburn is generally defined as a substernal burning-type discomfort, beginning in the epigastrium and radiating upward. It is often aggravated by meals, spicy or fatty foods, chocolate, alcohol, and coffee and can be worse in the supine position. It is commonly, although not universally, relieved by antacid or antisecretory medications. Epidemiologic studies have shown that heartburn occurs monthly in as many as 40% Table 25-4American Gastroenterologic Association Gallup poll on nighttime gastroesophageal reflux disease symptoms• 50 million Americans have nighttime

1	that heartburn occurs monthly in as many as 40% Table 25-4American Gastroenterologic Association Gallup poll on nighttime gastroesophageal reflux disease symptoms• 50 million Americans have nighttime heartburn at least 1/wk• 80% of heartburn sufferers had nocturnal symptoms—65% both day & night• 63% report that it affects their ability to sleep and impacts their work the next day• 72% are on prescription medications• Nearly half (45%) report that current remedies do not relieve all symptomsto 50% of the Western population. The occurrence of heartburn at night and its effect on quality of life have recently been high-lighted by a Gallup poll conducted by the American Gastroen-terologic Society (Table 25-4).Regurgitation, the effortless return of acid or bitter gastric contents into the chest, pharynx, or mouth, is highly suggestive of foregut pathology. It is often particularly severe at night when supine or when bending over and can be secondary to either an incompetent or obstructed

1	pharynx, or mouth, is highly suggestive of foregut pathology. It is often particularly severe at night when supine or when bending over and can be secondary to either an incompetent or obstructed GEJ. With the latter, as in achalasia, the regurgitant is often bland, as if food was put into a blender. When questioned, most patients can distinguish the two. It is the regurgitation of gastric contents that may result in associated pulmonary symptoms, including cough, hoarseness, asthma, and recurrent pneumonia. Bronchospasm can be precipitated by esophageal acidification and cough by either acid stimulation or distention of the esophagus.Dysphagia, or difficulty swallowing, is a relatively non-specific term but arguably the most specific symptom of foregut disease. It can be a sign of underlying malignancy and should be aggressively investigated until a diagnosis is established. Dyspha-gia refers to the sensation of difficulty in the passage of food from the mouth to the stomach and can

1	malignancy and should be aggressively investigated until a diagnosis is established. Dyspha-gia refers to the sensation of difficulty in the passage of food from the mouth to the stomach and can be divided into oropharyngeal and esophageal etiologies. Oropharyngeal dysphagia is charac-terized by difficulty transferring food out of the mouth into the esophagus, nasal regurgitation, and/or aspiration. Esophageal dys-phagia refers to the sensation of food sticking in the lower chest or epigastrium. This may or may not be accompanied by pain (ody-nophagia) that will be relieved by the passage of the bolus.Chest pain, although commonly and appropriately attrib-uted to cardiac disease, is frequently secondary to esophageal pathology as well. Nearly 50% of patients with severe chest pain, normal cardiac function, and normal coronary arterio-grams have positive 24-hour pH studies, implicating gastro-esophageal reflux as the underlying etiology. Exercise-induced gastroesophageal reflux is well

1	cardiac function, and normal coronary arterio-grams have positive 24-hour pH studies, implicating gastro-esophageal reflux as the underlying etiology. Exercise-induced gastroesophageal reflux is well known to occur, and may result in exertional chest pain similar to angina. It can be quite diffi-cult, if not impossible, to distinguish between the two etiologies, particularly on clinical grounds alone. Nevens and colleagues evaluated the ability of experienced cardiologists to differentiate pain of cardiac vs. esophageal origin. Of 248 patients initially seen by cardiologists, 185 were thought to have typical angina, and 63 were thought to have atypical chest pain. Forty-eight (26%) of those thought to have classic angina had normal coro-nary angiograms, and 16 of the 63 with atypical pain had abnor-mal angiogram. Thus, the cardiologists’ clinical impression was wrong 25% of the time. Finally, Pope and associates investi-gated the ultimate diagnosis in 10,689 patients presenting to an

1	had abnor-mal angiogram. Thus, the cardiologists’ clinical impression was wrong 25% of the time. Finally, Pope and associates investi-gated the ultimate diagnosis in 10,689 patients presenting to an Brunicardi_Ch25_p1009-p1098.indd 103101/03/19 6:03 PM 1032SPECIFIC CONSIDERATIONSPART IITable 25-5Normal manometric values of the distal esophageal sphincter, n = 50PARAMETERMEDIAN VALUE2.5TH PERCENTILE97.5TH PERCENTILEPressure (mmHg)135.827.7Overall length (cm)3.62.15.6Abdominal length (cm)20.94.7emergency department with acute chest pain. Approximately 17% were found to have acute ischemia, 6% had stable angina, 21% had other cardiac causes, and 55% had noncardiac causes. The investigators concluded that the majority of people present-ing to the emergency department with chest pain do not have an underlying cardiac etiology for their symptoms. Chest pain pre-cipitated by meals, occurring at night while supine, nonradiat-ing, responsive to antacid medication, or accompanied by other

1	have an underlying cardiac etiology for their symptoms. Chest pain pre-cipitated by meals, occurring at night while supine, nonradiat-ing, responsive to antacid medication, or accompanied by other symptoms suggesting esophageal disease such as dysphagia or regurgitation should trigger the thought of possible esophageal origin. Furthermore, the distinction between heartburn and chest pain is also difficult and largely dependent upon the individual patient. One person’s heartburn is another’s chest pain.The precise mechanisms accounting for the generation of symptoms secondary to esophageal pathology remain unclear. Considerable insight has been acquired, however. Investiga-tions into the effect of luminal content, esophageal distention and muscular function, neural pathways, and brain localization have provided a basic understanding of the stimuli responsible for symptom generation. It is also clear that the visceroneural pathways of the foregut are complexly intertwined with that of

1	have provided a basic understanding of the stimuli responsible for symptom generation. It is also clear that the visceroneural pathways of the foregut are complexly intertwined with that of the tracheobronchial tree and heart. This fact accounts for the common overlap of clinical presentations with diverse disease processes in upper GI, cardiac, and pulmonary systems.The Human Antireflux Mechanism and the Pathophysiology of Gastroesophageal Reflux DiseaseThere is a high-pressure zone located at the esophagogastric junc-tion in humans. Although this is typically referred to as the lower esophageal “sphincter,” there are no distinct anatomical land-marks that define its beginning and end. Architecturally speak-ing, there is a specialized thickening in this region that is made up of the collar sling musculature and the clasp fibers. The collar sling is located on the greater curvature side of the junction, and the clasp fibers are located on the lesser curvature side. These muscles

1	collar sling musculature and the clasp fibers. The collar sling is located on the greater curvature side of the junction, and the clasp fibers are located on the lesser curvature side. These muscles remain in tonic opposition until the act of swallowing, whereupon receptive relaxation occurs allowing passage of a food bolus into the stomach. In addition, the LES will also open when the gastric fundus is distended with gas and liquid, thus resulting in an unfolding of the valve and enabling venting of gas (a belch). Whether physiologic or pathologic, the common denominator for most episodes of gastroesophageal reflux is the loss of the high-pressure zone and thus a decrease in the resistance it imparts to the retrograde flow of gastric juice into the esophageal body.The Lower Esophageal Sphincter. As defined by esophageal manometry, there are three characteristics of the LES that work in unison to maintain its barrier function. These characteristics include the resting LES pressure,

1	Sphincter. As defined by esophageal manometry, there are three characteristics of the LES that work in unison to maintain its barrier function. These characteristics include the resting LES pressure, its overall length, and the intra-abdominal length that is exposed to the positive pressure environment of the abdomen (Table 25-5). The resistance to gastroesophageal reflux is a function of both the resting LES pressure and length over which this pressure is exerted. Thus, as the sphincter becomes shorter, a higher pressure will be required in order to prevent a given amount of reflux (Fig. 25-26). Much like the neck of a balloon as it is inflated, as the stomach fills and distends, sphincter length decreases. Therefore, if the over-all length of the sphincter is permanently short from repeated distention of the fundus secondary to large volume meals, then with minimal episodes of gastric distention and pressure, there will be insufficient sphincter length for the barrier to remain

1	repeated distention of the fundus secondary to large volume meals, then with minimal episodes of gastric distention and pressure, there will be insufficient sphincter length for the barrier to remain competent, and reflux will occur.LES length (cm)LES pressure (mmHg)60012CompetentIncompetent345121824Figure 25-26. As the esophageal sphincter becomes shorter, increased pressure is necessary to maintain competence. LES = lower esophageal sphincter.A third characteristic of the LES that impacts its ability to prevent reflux is its position about the diaphragm. It is important that a portion of the total length of the LES be exposed to the effects of an intra-abdominal pressure. That is, during periods of elevated intra-abdominal pressure, the resistance of the barrier would be overcome if pressure were not applied equally to both the LES and stomach simultaneously. Thus, in the presence of a hiatal hernia, the sphincter resides entirely within the chest cavity and cannot respond to an

1	pressure were not applied equally to both the LES and stomach simultaneously. Thus, in the presence of a hiatal hernia, the sphincter resides entirely within the chest cavity and cannot respond to an increase in intra-abdominal pressure because the pinch valve mechanism is lost and gastro-esophageal reflux is more liable to occur.Therefore, a permanently defective sphincter is defined by one or more of the following characteristics: an LES with a mean resting pressure of less than 6 mmHg, an overall sphincter length of <2 cm, and intra-abdominal sphincter length of <1 cm. Compared to normal subjects without GERD these values are below the 2.5 percentile for each parameter. The most com-mon cause of a defective sphincter is an inadequate abdominal length.Once the sphincter is permanently defective, this condi-tion is irreversible, and although esophageal mucosal injury may be healed with antisecretory medication, reflux will continue to occur. Additionally, the presence of a

1	defective, this condi-tion is irreversible, and although esophageal mucosal injury may be healed with antisecretory medication, reflux will continue to occur. Additionally, the presence of a defective LES may be associated with reduced esophageal body function and thus decrease clearance times of refluxed material. In addition, the progressive loss of effective esophageal clearance may predis-pose the patient to severe mucosal injury, volume regurgitation, aspiration, and pulmonary injury. Reflux may occur in the face of a normal LES resting pressure. This condition is usually due to a functional problem of gastric emptying or excessive air swallowing. These conditions may lead to gastric disten-tion, increased intra-gastric pressure, a resultant shortening or Brunicardi_Ch25_p1009-p1098.indd 103201/03/19 6:03 PM 1033ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Table 25-6Complications of gastroesophageal reflux disease: 150 consecutive cases with proven gastroesophageal reflux

1	103201/03/19 6:03 PM 1033ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Table 25-6Complications of gastroesophageal reflux disease: 150 consecutive cases with proven gastroesophageal reflux disease (24-hour esophageal pH monitoring endoscopy, and motility)COMPLICATIONNO.STRUCTURALLY NORMAL SPHINCTER (%)STRUCTURALLY DEFECTIVE SPHINCTER (%)None595842Erosive esophagitis472377aStricture191189Barrett’s esophagus250100Total150 aGrade more severe with defective cardia.Reproduced with permission from Moody FG, Carey LC, Jones RS, et al: Surgical Treatment of Digestive Disease. Chicago, IL: Year Book Medical; 1990.unfolding of the LES, and subsequent reflux. The mechanism by which gastric distention contributes to LES unfolding pro-vides a mechanical explanation for “transient LES relaxation.” It is thought that with repeated gastric distention secondary to large meal volume or chronic air swallowing, there is repeated unfolding of the LES and subsequent attenuation of the collar sling

1	It is thought that with repeated gastric distention secondary to large meal volume or chronic air swallowing, there is repeated unfolding of the LES and subsequent attenuation of the collar sling musculature. It is at this point that the physiologic and nor-mal mechanism of gastric venting is replaced with pathologic and severe postprandial reflux disease. In addition, patients with GERD will increase the frequency of swallowing in an effort to neutralize the refluxed acid with their saliva (pH 7.0). This phe-nomenon leads to increased air swallowing and further gastric distention, thus compounding the problem. Therefore, GERD may have its origins in the stomach secondary to gastric disten-tion due to overeating/drinking, air swallowing, or consump-tion of carbonated liquids, and this may be further compounded by the ingestion of fatty meals, which result in delayed gastric emptying.Relationship Between Hiatal Hernia and Gastroesopha-geal Reflux Disease. As the collar sling

1	this may be further compounded by the ingestion of fatty meals, which result in delayed gastric emptying.Relationship Between Hiatal Hernia and Gastroesopha-geal Reflux Disease. As the collar sling musculature and clasp fibers become attenuated with repeated gastric distention, the esophagogastric junction begins to assume an “upside down funnel” appearance, with progressive opening of the acute angle of His. This in turn may result in attenuation and stretching of the phrenoesophageal ligament, with subsequent enlargement of the hiatal opening and axial herniation. There is a high degree of correlation between reflux threshold and the degree of hiatal herniation (Fig. 25-27).Summary. It is believed that GERD has its origins within the stomach. Distention of the fundus occurs because of overeat-ing and delayed gastric emptying secondary to a high-fat diet. The resultant distention causes “unrolling” of the sphincter by the expanding fundus, and this subsequently exposes the squa-mous

1	and delayed gastric emptying secondary to a high-fat diet. The resultant distention causes “unrolling” of the sphincter by the expanding fundus, and this subsequently exposes the squa-mous epithelium in the region of the distal LES to gastric juice. Repeated exposure results in inflammation and the development of columnar epithelium at the cardia. This is the initial step of the development of carditis and explains why in early disease esophagitis is mild and commonly limited to the very distal aspect of the esophagus. The patient attempts to compensate for Yield pressure (mmHg)04No hernia< 3 cm hernia3 cm hernia81216202428323640Figure 25-27. Yield pressure of the lower esophageal sphincter decreases as hiatal hernia size increases.this by increased swallowing, allowing the saliva to neutralize the refluxed gastric juice and thus, alleviate the discomfort induced by the reflux event. The increased swallowing results in aeropha-gia, bloating, and belching. This in turn creates a

1	neutralize the refluxed gastric juice and thus, alleviate the discomfort induced by the reflux event. The increased swallowing results in aeropha-gia, bloating, and belching. This in turn creates a vicious cycle of increased gastric distention and thus further exposure and repeti-tive injury to the distal esophagus. The development of carditis explains the complaint of epigastric pain often experienced by patients with early reflux disease. Additionally, this process can lead to a fibrotic mucosal ring located at the squamocolumnar junction, which is termed a “Schatzki ring” and which may result in dysphagia. This inflammatory process may extend into muscu-laris propria and thus result in a progressive loss in the length and pressure of the LES. This explanation for the pathophysiology of GERD is supported by the observation that severe esophagitis is almost always associated with a defective LES.Complications Associated With Gastroesophageal Reflux DiseaseThe complications of

1	of GERD is supported by the observation that severe esophagitis is almost always associated with a defective LES.Complications Associated With Gastroesophageal Reflux DiseaseThe complications of gastroesophageal reflux disease may result from the direct injurious effects of gastric fluid on the mucosa, larynx, or respiratory epithelium. Complications due to repetitive reflux are esophagitis, stricture, and BE; repetitive aspiration may lead to progressive pulmonary fibrosis. The severity of the complications is directly related to the prevalence of a structurally defective sphincter (Table 25-6). The observation that a structurally defective sphincter occurs in 42% of patients without complications (most of whom have one or two components failed) suggests that disease may be confined to the sphincter due to compensation by a vigorously contracting esophageal body. Eventually, all three components of the sphincter fail, allowing unrestricted reflux of gastric juice into the esophagus

1	the sphincter due to compensation by a vigorously contracting esophageal body. Eventually, all three components of the sphincter fail, allowing unrestricted reflux of gastric juice into the esophagus and overwhelming its normal clearance mechanisms. This leads to esophageal mucosal injury with progressive deterioration of esophageal contractility, as is commonly seen in patients with strictures and BE. The loss of esophageal clearance increases the potential for regurgitation into the pharynx with aspiration.Brunicardi_Ch25_p1009-p1098.indd 103301/03/19 6:03 PM 1034SPECIFIC CONSIDERATIONSPART II70Prevalence%Gastric reflux(n = 22)Mixed reflux(n = 31)6050403020100A20151050% TimepH<4BpH4–7pH>7Figure 25-29. A. Prevalence of reflux types in 53 patients with gastroesophageal reflux disease. B. Esophageal luminal pH dur-ing bilirubin exposure. (Reproduced with permission from Kauer WK, Peters JH, DeMeester TR, etal: Mixed reflux of gastric and duodenal juices is more harmful to the

1	B. Esophageal luminal pH dur-ing bilirubin exposure. (Reproduced with permission from Kauer WK, Peters JH, DeMeester TR, etal: Mixed reflux of gastric and duodenal juices is more harmful to the esophagus than gastric juice alone. The need for surgical therapy re-emphasized, Ann Surg. 1995 Oct;222(4):525-531.)350300250200150100500123pH4567891018:00Time06:00Bile acid conc. umol/l0Figure 25-28. Sample bile acid concentration and esophageal pH plotted against time to obtain detailed profiles; in this case showing both significant bile acid (vertical bars) and acid (linear plot) reflux. (Reproduced with permission from Nehra D, Watt P, Pye JK, et al. Automated oesophageal reflux sampler: a new device used to moni-tor bile acid reflux in patients with gastroesophageal reflux disease, J Med Eng Technol. 1997 Jan-Feb;21(1):1-9.)The potential injurious components that reflux into the esophagus include gastric secretions such as acid and pepsin, as well as biliary and pancreatic secretions that

1	1997 Jan-Feb;21(1):1-9.)The potential injurious components that reflux into the esophagus include gastric secretions such as acid and pepsin, as well as biliary and pancreatic secretions that regurgitate from the duodenum into the stomach. There is a considerable body of experimental evidence to indicate that maximal epithelial injury occurs during exposure to bile salts combined with acid and pepsin. These studies have shown that while acid alone does minimal damage to the esophageal mucosa, the combination of acid and pepsin is highly deleterious. Similarly, the reflux of duodenal juice alone does little damage to the mucosa, although the combination of duodenal juice and gastric acid is particu-larly noxious.Complications of gastroesophageal reflux such as esopha-gitis, stricture, and Barrett’s metaplasia occur in the presence of two predisposing factors: a mechanically defective LES and an increased esophageal exposure to fluid containing duodenal content that includes bile and

1	Barrett’s metaplasia occur in the presence of two predisposing factors: a mechanically defective LES and an increased esophageal exposure to fluid containing duodenal content that includes bile and pancreatic juice. The duodenal origin of esophageal contents in patients with an increased exposure to a pH >7 has previously been confirmed by esopha-geal aspiration studies (Fig. 25-28). Studies have clarified and expanded these observations by measuring esophageal bilirubin exposure over a 24-hour period as a marker for the presence of duodenal juice. Direct measurement of esophageal bilirubin exposure as a marker for duodenal juice has shown that 58% of patients with GERD have increased esophageal exposure to duodenal juice and that this exposure occurs most commonly when the esophageal pH is between 4 and 7 (Fig. 25-29). These earlier studies have been confirmed by other studies that mea-sure volume reflux using impedance technology (Fig. 25-30).If reflux of gastric juice is allowed to

1	between 4 and 7 (Fig. 25-29). These earlier studies have been confirmed by other studies that mea-sure volume reflux using impedance technology (Fig. 25-30).If reflux of gastric juice is allowed to persist and sustained or repetitive esophageal injury occurs, two sequelae can result. First, a luminal stricture can develop from submucosal and even-tually intramural fibrosis. Second, the tubular esophagus may become replaced with columnar epithelium. The columnar epi-thelium is resistant to acid and is associated with the alleviation of the complaint of heartburn. This columnar epithelium often becomes intestinalized, identified histologically by the presence 100Prevalence of patients with increased bilirubin806040200Normalsubjectsn = 25No mucosalinjuryn = 16Erosiveesophagitisn = 10Barrett’sesophagusn = 27Figure 25-30. Prevalence of abnormal esophageal bilirubin expo-sure in healthy subjects and in patients with gastroesophageal reflux disease with varied degrees of mucosal injury. (*P

1	= 27Figure 25-30. Prevalence of abnormal esophageal bilirubin expo-sure in healthy subjects and in patients with gastroesophageal reflux disease with varied degrees of mucosal injury. (*P <.03 vs. all other groups; **P <.03 vs. healthy subjects.) (Reproduced with permis-sion from Kauer WK, Peters JH, DeMeester TR, et al: Mixed reflux of gastric and duodenal juices is more harmful to the esophagus than gastric juice alone. The need for surgical therapy re-emphasized, Ann Surg. 1995 Oct;222(4):525-531.)Brunicardi_Ch25_p1009-p1098.indd 103401/03/19 6:03 PM 1035ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25of goblet cells. This specialized IM is currently required for the diagnosis of BE. Endoscopically, BE can be quiescent or associ-ated with complications of esophagitis, stricture, Barrett’s ulcer-ation, and dysplasia. The complications associated with BE may be due to the continuous irritation from refluxed duodenogastric juice. This continued injury is pH dependent and may be

1	Barrett’s ulcer-ation, and dysplasia. The complications associated with BE may be due to the continuous irritation from refluxed duodenogastric juice. This continued injury is pH dependent and may be modi-fied by medical therapy. The incidence of metaplastic Barrett’s epithelium becoming dysplastic and progressing to adenocarci-noma is approximately 0.2% to 0.5% per year.An esophageal stricture can be associated with severe esophagitis or BE. In the latter situation, it occurs at the site of maximal inflammatory injury (i.e., the columnar-squamous epi-thelial interface). Patients who have a stricture in the absence of Barrett’s esophagus should have the presence of gastroesopha-geal reflux documented before the presence of the stricture is ascribed to reflux esophagitis. In patients with normal acid exposure and no endoscopic or CT evidence of cancer, the stric-ture may be a result of a drug-induced chemical injury, the latter resulting from the lodgment of a capsule or tablet in the

1	normal acid exposure and no endoscopic or CT evidence of cancer, the stric-ture may be a result of a drug-induced chemical injury, the latter resulting from the lodgment of a capsule or tablet in the distal esophagus. In such patients, dilation usually corrects the prob-lem of dysphagia. It is also possible for drug-induced injuries to occur in patients who have underlying esophagitis and a distal esophageal stricture secondary to gastroesophageal reflux. In this situation, a long, string-like stricture progressively devel-ops as a result of repetitive caustic injury from capsule or tablet lodgment on top of an initial reflux stricture. These strictures are often resistant to dilation. The incidence of this problem has lessened since the introduction of proton pump inhibitor medication.Metaplastic (Barrett’s Esophagus) and Neoplastic (Adenocarcinoma) ComplicationsThe condition whereby the tubular esophagus is lined with columnar epithelium rather than squamous epithelium was first

1	(Barrett’s Esophagus) and Neoplastic (Adenocarcinoma) ComplicationsThe condition whereby the tubular esophagus is lined with columnar epithelium rather than squamous epithelium was first described by Norman Barrett in 1950. He incorrectly believed it to be congenital in origin. It is now realized that it is an acquired abnormality, occurs in 10% to 15% of patients with GERD, and represents the end stage of the natural history of this disease. It is also distinctly different from the congenital condition in which islands of gastric fundic epithelium are found in the upper half of the esophagus.The definition of BE has evolved considerably over the past decade. Traditionally, BE was identified by the presence of columnar mucosa extending at least 3 cm into the esophagus. It is now recognized that the specialized, intestinal-type epi-thelium, or intestinal metaplasia (IM) found in the Barrett’s mucosa, is the only tissue predisposed to malignant degenera-tion. Consequently, the

1	recognized that the specialized, intestinal-type epi-thelium, or intestinal metaplasia (IM) found in the Barrett’s mucosa, is the only tissue predisposed to malignant degenera-tion. Consequently, the diagnosis of BE is presently made given any length of endoscopically identifiable columnar mucosa that proves, on biopsy, to show IM. Although long segments of columnar mucosa without IM do occur, they are uncommon and might be congenital in origin.The hallmark of IM is the presence of intestinal goblet cells. There is a high prevalence of biopsy-demonstrated IM at the cardia, on the gastric side of the squamocolumnar junction, in the absence of endoscopic evidence of a CLE. Evidence is accumulating that these patches of what appears to be Barrett’s in the cardia have a similar malignant potential as in the longer segments, and are precursors for carcinoma of the cardia.The long-term relief of symptoms remains the primary rea-son for performing antireflux surgery in patients with BE.

1	potential as in the longer segments, and are precursors for carcinoma of the cardia.The long-term relief of symptoms remains the primary rea-son for performing antireflux surgery in patients with BE. Heal-ing of esophageal mucosal injury and the prevention of disease progression are important secondary goals. In this regard, patients with BE are no different than the broader population of patients with gastroesophageal reflux. They should be con-sidered for antireflux surgery when patient data suggest severe disease or predict the need for long-term medical management. Most patients with BE are symptomatic. Although it has been argued that some patients with BE may not have symptoms, careful history taking will reveal the presence of symptoms in most, if not all, patients.Patients with BE have a spectrum of disease ranging from visually identifiable but short segments, to long segments of classic BE. In general, however, they represent a relatively severe stage of gastroesophageal

1	BE have a spectrum of disease ranging from visually identifiable but short segments, to long segments of classic BE. In general, however, they represent a relatively severe stage of gastroesophageal reflux, usually with markedly increased esophageal acid exposure, deficient LES characteris-tics, poor esophageal body function, and a high prevalence of duodenogastroesophageal reflux. Gastric hypersecretion occurs in 44% of patients. Most will require long-term PPI therapy for relief of symptoms and control of coexistent esophageal muco-sal injury. Given such profound deficits in esophageal physi-ology, antireflux surgery is an excellent means of long-term control of reflux symptoms for most patients with BE.The typical complications in BE include ulceration in the columnar-lined segment, stricture formation, and a dysplasia-cancer sequence. Barrett’s ulceration is unlike the erosive ulceration of reflux esophagitis in that it more closely resem-bles peptic ulceration in the stomach or

1	formation, and a dysplasia-cancer sequence. Barrett’s ulceration is unlike the erosive ulceration of reflux esophagitis in that it more closely resem-bles peptic ulceration in the stomach or duodenum, and has the same propensity to bleed, penetrate, or perforate. Fortunately, this complication occurs very rarely. The strictures found in BE occur at the squamocolumnar junction, and they are typically higher than peptic strictures in the absence of BE. Ulceration and stricture in association with BE were commonly reported before 1975, but with the advent of potent acid suppression medication, they have become less common. In contrast, the complication of adenocarcinoma developing in Barrett’s mucosa has become more common. Adenocarcinoma developing in Bar-rett’s mucosa was considered a rare tumor before 1975. Today, it occurs at approximately 0.2% to 0.5% per year of follow-up, which represents a risk 40 times that of the general popula-tion. Most, if not all, cases of adenocarcinoma of

1	tumor before 1975. Today, it occurs at approximately 0.2% to 0.5% per year of follow-up, which represents a risk 40 times that of the general popula-tion. Most, if not all, cases of adenocarcinoma of the esophagus arise in Barrett’s epithelium (Fig. 25-31). About one-third of all patients with BE present with malignancy.The long-term risk of progression to dysplasia and ade-nocarcinoma, although not the driving force behind the deci-sion to perform antireflux surgery, is a significant concern for both patient and physician. Although to date, there have been no prospective randomized studies documenting that antireflux surgery has an effect on the risk of progression to dysplasia and carcinoma, complete control of reflux of gastric juice into the esophagus is clearly a desirable goal.Respiratory ComplicationsA significant proportion of patients with GERD will have associated respiratory symptoms. These patients may have laryngopharyngeal reflux-type symptoms, adult-onset asthma, or

1	ComplicationsA significant proportion of patients with GERD will have associated respiratory symptoms. These patients may have laryngopharyngeal reflux-type symptoms, adult-onset asthma, or even idiopathic pulmonary fibrosis. These symptoms and organ injury may occur in isolation or in conjunction with typi-cal reflux symptoms such as heartburn and regurgitation. Sev-eral studies have demonstrated that up to 50% of patients with asthma have either endoscopically evident esophagitis or abnor-mal distal esophageal acid exposure. These findings support a causal relationship between GERD and aerodigestive symptoms and complications in a proportion of patients.3Brunicardi_Ch25_p1009-p1098.indd 103501/03/19 6:03 PM 1036SPECIFIC CONSIDERATIONSPART IIABFigure 25-31. Photomicrographs. A. Barrett’s epithelium with severe dysplasia. (×200.) Note nuclear irregularity, stratification, and loss of polarity. B. Barrett’s epithelium with intramucosal carcinoma. (×66.) Note malignant cells in the

1	epithelium with severe dysplasia. (×200.) Note nuclear irregularity, stratification, and loss of polarity. B. Barrett’s epithelium with intramucosal carcinoma. (×66.) Note malignant cells in the mucosa (upper arrow), but not invading the muscularis mucosae (bottom arrow). (Reproduced with permission from Zuidema GD, Orringer MB: Shackelford’s Surgery of the Alimentary Tract, 3rd ed. Vol 1. Philadelphia, PA: Elsevier/Saunders; 1991.)Etiology of Reflux-Induced Respiratory Symptoms. There are two mechanisms that have been proposed as the cause of reflux-induced respiratory symptoms. The reflux theory sug-gests that these symptoms are the direct result of laryngopha-ryngeal exposure and aspiration of gastric contents. The reflex theory suggests that the vagal-mediated afferent fibers result in bronchoconstriction during episodes of distal esophageal acidification. The evidence supporting a mechanism of direct exposure to the aerodigestive system is based in clinical studies that have

1	in bronchoconstriction during episodes of distal esophageal acidification. The evidence supporting a mechanism of direct exposure to the aerodigestive system is based in clinical studies that have documented a strong correlation between idiopathic pulmonary fibrosis and hiatal hernia. In addition, the presence of GERD was demonstrated to be highly associated with several pulmonary diseases in a recent Department of Veteran Affairs multivariate analysis. Next, with ambulatory pH testing, acid exposure within the proximal esophagus is more frequently identified in patients with gastroesophageal reflux and respi-ratory symptoms than in patients who have gastroesophageal reflux symptoms alone. These findings are supported by scinti-graphic studies, which have demonstrated aspiration of ingested radioisotope in patients with both gastroesophageal reflux and pulmonary symptoms. In animal studies, tracheal instillation of acid has been demonstrated to profoundly increase airway resis-tance.

1	radioisotope in patients with both gastroesophageal reflux and pulmonary symptoms. In animal studies, tracheal instillation of acid has been demonstrated to profoundly increase airway resis-tance. Finally, in patients who have undergone multichannel intraluminal impedance testing with a catheter configured to detect laryngopharyngeal reflux, a correlation between proxi-mal fluid movement and laryngopharyngeal symptoms, such as cough, can be demonstrated.The reflex mechanism is supported by the bronchocon-striction that occurs with the infusion of acid into the distal esophagus. There is a shared embryologic origin of the tracheo-esophageal tract and vagus nerve, and this reflex is thought to be an afferent fiber–mediated reflex that protects the aerodigestive system from the aspiration of refluxate. In patients with respira-tory symptoms and documented gastroesophageal reflux with-out proximal esophageal acid exposure, pulmonary symptoms will often times significantly improve or

1	of refluxate. In patients with respira-tory symptoms and documented gastroesophageal reflux with-out proximal esophageal acid exposure, pulmonary symptoms will often times significantly improve or completely resolve after undergoing laparoscopic fundoplication. It is likely that both of the proposed mechanisms work simultaneously to cause these symptoms in the face of GERD.The most difficult clinical challenge in formulating a treat-ment plan for reflux-associated respiratory symptoms resides in establishing the diagnosis. Although the diagnosis may be straightforward in patients with predominately typical reflux symptoms and secondary respiratory complaints, a substan-tial number of patients will have respiratory symptoms that dominate the clinical scenario. Typical gastroesophageal reflux Brunicardi_Ch25_p1009-p1098.indd 103601/03/19 6:03 PM 1037ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25symptoms, such as heartburn and regurgitation, may often be completely absent only to be

1	Brunicardi_Ch25_p1009-p1098.indd 103601/03/19 6:03 PM 1037ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25symptoms, such as heartburn and regurgitation, may often be completely absent only to be uncovered with objective esopha-geal physiology testing. Traditionally, the diagnosis of reflux-induced respiratory injury is established using ambulatory dual probe pH monitoring, with one probe positioned within the dis-tal esophagus and the other at a proximal location. Proximal probe positioning has included multiple locations such as the trachea, pharynx, and proximal esophagus. Although ambu-latory esophageal pH monitoring allows a direct correlation between esophageal acidification and respiratory symptoms, sensitivity of this testing modality is poor, and the temporal rela-tionship between laryngeal or pulmonary symptoms and reflux events is complex. In addition, as the refluxed gastric fluid trav-els proximally, it may be neutralized by saliva and therefore go undetected with pH

1	laryngeal or pulmonary symptoms and reflux events is complex. In addition, as the refluxed gastric fluid trav-els proximally, it may be neutralized by saliva and therefore go undetected with pH monitoring. Impedance testing may also be used to detect the movement of fluid throughout the entire esophageal column regardless of pH content.Treatment. Once the diagnosis is established, treatment may be initiated with either PPI therapy or antireflux surgery. A trial of high-dose PPI therapy may help establish that reflux is partly or completely responsible for the respiratory symptoms. It is important to note that the persistence of symptoms in the face of aggressive PPI treatment does not necessarily rule out reflux as a possible cofactor or sole etiology.Although there is probably some element of a placebo effect, relief of respiratory symptoms can be anticipated in up to 50% of patients with reflux-induced asthma treated with anti-secretory medications. However, when examined

1	some element of a placebo effect, relief of respiratory symptoms can be anticipated in up to 50% of patients with reflux-induced asthma treated with anti-secretory medications. However, when examined objectively, <15% of patients can be expected to have improvement in their pulmonary function with medical therapy. In properly selected patients, antireflux surgery improves respiratory symptoms in nearly 90% of children and 70% of adults with asthma and reflux disease. Improvements in pulmonary function can be demonstrated in around 30% of patients. Uncontrolled studies of the two forms of therapy (PPI and surgery) and the evidence from the two randomized controlled trials of medical vs. sur-gical therapy indicate that surgical valve reconstruction is the most effective therapy for reflux-induced asthma. The superi-ority of the surgery over PPI is most noticeable in the supine position, which corresponds with the nadir of PPI blood levels and resultant acid breakthrough and is the time

1	asthma. The superi-ority of the surgery over PPI is most noticeable in the supine position, which corresponds with the nadir of PPI blood levels and resultant acid breakthrough and is the time in the circadian cycle when asthma symptoms are at their worst.In asthmatic patients with an esophageal motility disorder, performing an antireflux operation will not prevent the regur-gitation and possible aspiration of swallowed liquid or food “upstream” to the valve reconstruction. It is critical that esopha-geal body function be considered prior to surgical intervention in this patient population.Medical Therapy for Gastroesophageal Reflux Disease. With the widespread availability of over-the-counter antisecre-tory medications, most patients with mild or moderate symp-toms will carry self-medication. When initially identified with mild symptoms of uncomplicated GERD, patients can be placed on 12 weeks of simple antacids before diagnostic testing is initi-ated. This approach may successfully

1	When initially identified with mild symptoms of uncomplicated GERD, patients can be placed on 12 weeks of simple antacids before diagnostic testing is initi-ated. This approach may successfully and completely resolve the symptoms. Patients should be counseled to elevate the head of the bed; avoid tight-fitting clothing; eat small, frequent meals; avoid eating the nighttime meal immediately prior to bedtime; and avoid alcohol, coffee, chocolate, and peppermint, which are known to reduce resting LES pressure and may aggravate symptoms.Used in combination with simple antacids, alginic acid may augment the relief of symptoms by creating a physical bar-rier to reflux, as well as by acid reduction. Alginic acid reacts with sodium bicarbonate in the presence of saliva to form a highly viscous solution that floats like a raft on the surface of the gastric contents. When reflux occurs, this protective layer is refluxed into the esophagus, and acts as a protective barrier against the noxious

1	solution that floats like a raft on the surface of the gastric contents. When reflux occurs, this protective layer is refluxed into the esophagus, and acts as a protective barrier against the noxious gastric contents. Medications to promote gastric emptying, such as metoclopramide or domperidone, are beneficial in early disease but of little value in more severe disease.In patients with persistent symptoms, the mainstay of medical therapy is acid suppression. High-dosage regimens of hydrogen potassium PPIs, such as omeprazole (up to 40 mg/d), can reduce gastric acidity by as much as 80% to 90%. This usu-ally heals mild esophagitis. In severe esophagitis, healing may occur in only one-half of the patients. In patients who reflux a combination of gastric and duodenal juice, acid-suppression therapy may give relief of symptoms, while still allowing mixed reflux to occur. This can allow persistent mucosal damage in an asymptomatic patient. Unfortunately, within 6 months of discontinuation

1	therapy may give relief of symptoms, while still allowing mixed reflux to occur. This can allow persistent mucosal damage in an asymptomatic patient. Unfortunately, within 6 months of discontinuation of any form of medical therapy for GERD, 80% of patients have a recurrence of symptoms, and 40% of individuals with daily GERD eventually develop symptoms that “breakthrough” adequately dosed PPIs. Once initiated, most patients with GERD will require lifelong treatment with PPIs, both to relieve symptoms and to control any coexistent esophagitis or stricture. Although control of symptoms has his-torically served as the endpoint of therapy, the wisdom of this approach has recently been questioned, particularly in patients with BE. Evidence suggesting that reflux control may prevent the development of adenocarcinoma and lead to regression of dysplastic and nondysplastic Barrett’s segments has led many to consider control of reflux, and not symptom control, a better therapeutic endpoint.

1	of adenocarcinoma and lead to regression of dysplastic and nondysplastic Barrett’s segments has led many to consider control of reflux, and not symptom control, a better therapeutic endpoint. However, this hypothesis remains contro-versial. It should be noted that complete control of reflux using PPIs can be difficult, as has been highlighted by studies of acid breakthrough while on PPI therapy and of persistent reflux fol-lowing antireflux surgery. Castell, Triadafilopoulos, and others have shown that 40% to 80% of patients with BE continue to have abnormal esophageal acid exposure despite up to 20 mg twice daily of PPIs. Ablation trials have shown that mean doses of 56 mg of omeprazole were necessary to normalize 24-hour esophageal pH studies. It is likely that antireflux surgery results in more reproducible and reliable elimination of reflux of both acid and duodenal contents, although long-term outcome studies suggest that as many as 25% of postfundoplication patients will have

1	in more reproducible and reliable elimination of reflux of both acid and duodenal contents, although long-term outcome studies suggest that as many as 25% of postfundoplication patients will have persistent pathologic esophageal acid exposure confirmed by positive 24-hour pH studies.Suggested Therapeutic Approach. Traditionally a stepwise approach is used for the treatment of GERD. First-line therapy entails antisecretory medication, usually PPIs, in most patients. Failure of medication to adequately control GERD symptoms suggests either that the patient may have relatively severe dis-ease or a non-GERD cause for his or her symptoms. Endoscopic examination at this stage of the patient’s evaluation is recom-mended and will provide the opportunity to assess the degree of mucosal injury and presence of BE. Treatment options for these patients entails either long term PPI use vs. antireflux surgery. Laparoscopic antireflux surgery in these patients achieves long-term control of symptoms

1	presence of BE. Treatment options for these patients entails either long term PPI use vs. antireflux surgery. Laparoscopic antireflux surgery in these patients achieves long-term control of symptoms in 85% to 90%. The measurement Brunicardi_Ch25_p1009-p1098.indd 103701/03/19 6:03 PM 1038SPECIFIC CONSIDERATIONSPART IIof esophageal acid exposure via 24-hour pH should be under-taken when patients are considered for surgery. The status of the LES and esophageal body function with esophageal manom-etry should also be performed at this stage. These studies will serve to establish the diagnosis and assess esophageal body dysfunction.Surgical Therapy for Gastroesophageal Reflux DiseaseSelection of Patients for Surgery. Studies of the natural history of GERD indicate that most patients have a relatively benign form of the disease that is responsive to lifestyle changes and dietary and medical therapy and do not need surgical treat-ment. Approximately 25% to 50% of the patients with GERD

1	a relatively benign form of the disease that is responsive to lifestyle changes and dietary and medical therapy and do not need surgical treat-ment. Approximately 25% to 50% of the patients with GERD have persistent or progressive disease, and it is this patient pop-ulation that is best suited to surgical therapy. In the past, the presence of esophagitis and a structurally defective LES were the primary indications for surgical treatment, and many inter-nists and surgeons were reluctant to recommend operative pro-cedures in their absence. However, one should not be deterred from considering antireflux surgery in a symptomatic patient with or without esophagitis or a defective sphincter, provided the disease process has been objectively documented by 24-hour pH monitoring. This is particularly true in patients who have become dependent upon therapy with PPIs, or require increasing doses to control their symptoms. It is important to note that a good response to medical therapy in this

1	true in patients who have become dependent upon therapy with PPIs, or require increasing doses to control their symptoms. It is important to note that a good response to medical therapy in this group of patients pre-dicts an excellent outcome following antireflux surgery.In general, the key indications for antireflux surgery are (a) objectively proven gastroesophageal reflux disease, and (b) typical symptoms of gastroesophageal reflux disease (heartburn and/or regurgitation) despite adequate medical management, or (c) a younger patient unwilling to take lifelong medication. In addition, a structurally defective LES can also predict which patients are more likely to fail with medical therapy. Patients with normal sphincter pressures tend to remain well controlled with medical therapy, whereas patients with a structurally defec-tive LES may not respond as well to medical therapy, and often develop recurrent symptoms within 1 to 2 years of beginning therapy. Such patients should be

1	whereas patients with a structurally defec-tive LES may not respond as well to medical therapy, and often develop recurrent symptoms within 1 to 2 years of beginning therapy. Such patients should be considered for an antireflux operation, regardless of the presence or absence of endoscopic esophagitis.Young patients with documented reflux disease with or without a defective LES are also excellent candidates for anti-reflux surgery. They usually will require long-term medical therapy for control of their symptoms, and some will go on to develop complications of the disease. An analysis of the cost of therapy based on data from the Veterans Administration Coop-erative trial indicates that surgery has a cost advantage over medical therapy in patients <49 years of age.Severe endoscopic esophagitis in a symptomatic patient with a structurally defective LES is also an indication for early surgical therapy. These patients are prone to breakthrough of their symptoms while receiving medical

1	in a symptomatic patient with a structurally defective LES is also an indication for early surgical therapy. These patients are prone to breakthrough of their symptoms while receiving medical therapy. Symptoms and mucosal injury can be controlled in such patients, but careful monitoring is required, and increasing dosages of PPIs are nec-essary. In everyday clinical practice, however, such treatment can be both difficult and impractical, and, in such cases, antire-flux surgery can be considered early, especially if PPI therapy is problematic.The development of a stricture in a patient represents a fail-ure of medical therapy, and it is also an indication for a surgical antireflux procedure. In addition, strictures are often associated with a structurally defective sphincter and loss of esophageal contractility. Before proceeding with surgical treatment, malig-nancy and a drug-related etiology of the stricture should be excluded, and the stricture should be progressively dilated up to

1	contractility. Before proceeding with surgical treatment, malig-nancy and a drug-related etiology of the stricture should be excluded, and the stricture should be progressively dilated up to a 50 to 60F bougie. When the stricture is fully dilated, the relief of dysphagia is evaluated, and esophageal manometry is performed to determine the adequacy of peristalsis in the distal esophagus. If dysphagia is relieved and the amplitude of esopha-geal contractions is adequate, an antireflux procedure should be performed; if there is a global loss of esophageal contractility, caution should be exercised in performing an antireflux proce-dure with a complete fundoplication, and a partial fundoplica-tion should be considered.Barrett’s CLE is commonly associated with a severe structural defect of the LES and often poor contractility of the esophageal body. Patients with BE are at risk of the development of an adenocarcinoma. Whilst surgeons would like to think that an antireflux procedure can

1	the LES and often poor contractility of the esophageal body. Patients with BE are at risk of the development of an adenocarcinoma. Whilst surgeons would like to think that an antireflux procedure can reduce the risk of progression to cancer, the evidence supporting this is relatively weak, and for now Barrett’s esophagus should be considered to be evidence that the patient has gastroesophageal reflux, and progression to antireflux surgery is indicated for the treatment of reflux symptoms, not cancer progression. If, however, high grade dysplasia or intramucosal carcinoma is found on mucosal biopsy specimens, treatment should then be directed at the BE and the lesion, using either evaluation endoscopic ablation, endoscopic resection, or esophageal resection.The majority of patients requiring treatment for reflux have a relatively mild form of disease and will respond to antise-cretory medications. Patients with more severe forms of disease, particularly those who develop persistent or

1	treatment for reflux have a relatively mild form of disease and will respond to antise-cretory medications. Patients with more severe forms of disease, particularly those who develop persistent or progressive disease, should be considered for definitive therapy. Laparoscopic fun-doplication will provide a long-term cure in the majority of these patients, with minimal discomfort and an early return to normal activity.Preoperative Evaluation. Before proceeding with an antire-flux operation, several factors should be evaluated. The clinical symptoms should be consistent with the diagnosis of gastro-esophageal reflux. Patients presenting with the typical symp-toms of heartburn and/or regurgitation which have responded, at least partly, to PPI therapy, will generally do well following surgery, whereas patients with atypical symptoms have a less predictable response. Reflux should also be objectively con-firmed by either the presence of ulcerative esophagitis or an abnormal 24-hour pH

1	whereas patients with atypical symptoms have a less predictable response. Reflux should also be objectively con-firmed by either the presence of ulcerative esophagitis or an abnormal 24-hour pH study.The propulsive force of the body of the esophagus should be evaluated by esophageal manometry to determine if it has sufficient power to propel a bolus of food through a newly reconstructed valve. Patients with normal peristaltic contrac-tions can be considered for a 360° Nissen fundoplication or a partial fundoplication, depending on patient and surgeon pref-erences. When peristalsis is absent, a partial fundoplication is probably the procedure of choice, but only if achalasia has been ruled out.Hiatal anatomy should also be assessed. In patients with smaller hiatal hernias, endoscopy evaluation usually provides sufficient information. However, when patients present with a very large hiatus hernia or for revision surgery after previous antireflux surgery, contrast radiology provides

1	usually provides sufficient information. However, when patients present with a very large hiatus hernia or for revision surgery after previous antireflux surgery, contrast radiology provides better anatomical information. The concept of anatomic shortening of the esoph-agus is controversial, with divergent opinions held about how Brunicardi_Ch25_p1009-p1098.indd 103801/03/19 6:03 PM 1039ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25DistentionFigure 25-32. A graphic illustration of the shortening of the lower esophageal sphincter that occurs as the sphincter is “taken up” by the cardia as the stomach distends.common this problem is. Believers claim that anatomic short-ening of the esophagus compromises the ability of the surgeon to perform an adequate repair without tension and that this can lead to an increased incidence of breakdown or thoracic displace-ment of the repair. Some of those who hold this view claim that esophageal shortening is present when a barium swallow X-ray

1	this can lead to an increased incidence of breakdown or thoracic displace-ment of the repair. Some of those who hold this view claim that esophageal shortening is present when a barium swallow X-ray identifies a sliding hiatal hernia that will not reduce in the upright position or that measures more than 5 cm in length at endoscopy. When such identification is made, these surgeons usually add a gastroplasty to the antireflux procedure. Others claim that esoph-ageal shortening is overdiagnosed and rarely seen, and that the morbidity of adding a gastroplasty outweighs any benefits. These surgeons would recommend a standard antireflux procedure in all patients undergoing primary surgery.Principles of Surgical Therapy. The primary goal of anti-reflux surgery is to safely create a new antireflux valve at the gastroesophageal junction, while preserving the patient’s abil-ity to swallow normally and to belch to relieve gaseous disten-tion. Regardless of the choice of the procedure, this goal

1	valve at the gastroesophageal junction, while preserving the patient’s abil-ity to swallow normally and to belch to relieve gaseous disten-tion. Regardless of the choice of the procedure, this goal can be achieved if attention is paid to some basic principles when reconstructing the antireflux mechanism. First, the operation should create a flap valve which prevents regurgitation of gas-tric contents into the esophagus. This will result in an increase in the pressure of the distal esophageal sphincter region. Follow-ing a Nissen fundoplication the expected increase is to a level twice the resting gastric pressure (i.e., 12 mmHg for a gastric pressure of 6 mmHg). The extent of the pressure rise is often less following a partial fundoplication, although with all types of fundoplication the length of the reconstructed valve should be at least 3 cm. This not only augments sphincter characteristics in patients in whom they are reduced before surgery but also prevents unfolding of a normal

1	length of the reconstructed valve should be at least 3 cm. This not only augments sphincter characteristics in patients in whom they are reduced before surgery but also prevents unfolding of a normal sphincter in response to gastric distention (Fig. 25-32). Preoperative and postoperative esopha-geal manometry measurements have shown that the resting sphincter pressure and the overall sphincter length can be surgi-cally augmented over preoperative values, and that the change in the former is a function of the degree of gastric wrap around the esophagus (Fig. 25-33). However, the aim of any fundopli-cation is to create a loose wrap and to maintain the position of the gastric fundus close to the distal intra-abdominal esophagus, in a flap valve arrangement. The efficacy of this relies on the close relationship between the fundus and the esophagus, not the “tightness” of the wrap.Second, the operation should place an adequate length of the distal esophageal sphincter in the

1	relies on the close relationship between the fundus and the esophagus, not the “tightness” of the wrap.Second, the operation should place an adequate length of the distal esophageal sphincter in the positive-pressure 051015˜ P mmHg 20240Degree of wrapY = 4.63 + .023 (x)P < .01BelseyHillN=15NissenN=15N=15360Figure 25-33. The relationship between the augmentation of sphincter pressure over preoperative pressure (ΔP) and the degree of gastric fundic wrap in three different antireflux procedures. (Repro-duced with permission from O’Sullivan GC, DeMeester TR, Joels-son BE, et al: Interaction of lower esophageal sphincter pressure and length of sphincter in the abdomen as determinants of gastro-esophageal competence, Am J Surg. 1982 Jan;143(1):40-47.)environment of the abdomen by a method that ensures its response to changes in intra-abdominal pressure. The permanent restoration of 2 or more cm of abdominal esophagus ensures the preservation of the relationship between the fundus and the

1	ensures its response to changes in intra-abdominal pressure. The permanent restoration of 2 or more cm of abdominal esophagus ensures the preservation of the relationship between the fundus and the esophagus. All of the popular antireflux procedures increase the length of the sphincter exposed to abdominal pressure by an average of at least 1 cm.Third, the operation should allow the reconstructed car-dia to relax on deglutition. In normal swallowing, a vagally mediated relaxation of the distal esophageal sphincter and the gastric fundus occurs. The relaxation lasts for approximately 10 seconds and is followed by a rapid recovery to the former tonicity. To ensure relaxation of the sphincter, three factors are important: (a) Only the fundus of the stomach should be used to buttress the sphincter, because it is known to relax in con-cert with the sphincter; (b) the gastric wrap should be properly placed around the sphincter and not incorporate a portion of the stomach or be placed around

1	because it is known to relax in con-cert with the sphincter; (b) the gastric wrap should be properly placed around the sphincter and not incorporate a portion of the stomach or be placed around the stomach itself, because the body of the stomach does not relax with swallowing; and (c) damage to the vagal nerves during dissection of the thoracic esophagus should be avoided because it may result in failure of the sphincter to relax.Fourth, the fundoplication should not increase the resis-tance of the relaxed sphincter to a level that exceeds the peri-staltic power of the body of the esophagus. The resistance of the relaxed sphincter depends on the degree, length, and diameter of the gastric fundic wrap, and on the variation in intra-abdominal pressure. A 360° gastric wrap should be no longer than 2 cm and constructed over a large (50 to 60F) bougie. This will ensure that the relaxed sphincter will have an adequate diameter with minimal resistance. A bougie is not necessary when

1	no longer than 2 cm and constructed over a large (50 to 60F) bougie. This will ensure that the relaxed sphincter will have an adequate diameter with minimal resistance. A bougie is not necessary when construct-ing a partial wrap.Fifth, the operation should ensure that the fundoplication can be placed in the abdomen without undue tension and main-tained there by approximating the crura of the diaphragm above the repair. Leaving the fundoplication in the thorax converts a sliding hernia into a PEH, with all the complications associ-ated with that condition. Maintaining the repair in the abdomen Brunicardi_Ch25_p1009-p1098.indd 103901/03/19 6:03 PM 1040SPECIFIC CONSIDERATIONSPART IIunder tension predisposes to an increased incidence of recur-rence. How common this problem is encountered is disputed, with some surgeons advocating lengthening the esophagus by gastroplasty and constructing a partial fundoplication, and oth-ers claiming that this issue is now rarely encountered.Procedure

1	disputed, with some surgeons advocating lengthening the esophagus by gastroplasty and constructing a partial fundoplication, and oth-ers claiming that this issue is now rarely encountered.Procedure Selection. A laparoscopic approach is now used routinely in all patients undergoing primary antireflux surgery. Some surgeons advocate the use of a single antireflux procedure for all patients, whereas others advocate a tailored approach. Advocates of the laparoscopic Nissen fundoplication as the pro-cedure of choice for a primary antireflux repair would generally apply this procedure in all patients with normal or near normal esophageal motility, and they would reserve a partial fundopli-cation for use in individuals with poor esophageal body motility. Others, based on the good longer-term outcomes now reported following partial fundoplication procedures, advocate the rou-tine application of a partial fundoplication procedure, thereby avoiding any concerns about constructing a

1	outcomes now reported following partial fundoplication procedures, advocate the rou-tine application of a partial fundoplication procedure, thereby avoiding any concerns about constructing a fundoplication in individuals with poor esophageal motility.Experience and randomized studies have shown that both the Nissen fundoplication and various partial fundoplication procedures are all effective and durable antireflux repairs that generate an excellent outcome in approximately 90% of patients at longer-term follow-up.Primary Antireflux RepairsNissen Fundoplication. The most common antireflux proce-dure is the Nissen fundoplication. In the past, this procedure has been performed through an open abdominal or a chest incision, but with the development of laparoscopic approaches primary antireflux surgery is now routinely undertaken using the laparo-scope. Rudolph Nissen described this procedure as a 360° fun-doplication around the lower esophagus for a distance of 4 to 5 cm, without

1	surgery is now routinely undertaken using the laparo-scope. Rudolph Nissen described this procedure as a 360° fun-doplication around the lower esophagus for a distance of 4 to 5 cm, without division of the short gastric blood vessels. Although this provided good control of reflux, it was associated with a number of side effects that have encouraged modifica-tions of the procedure as originally described. These include using only the gastric fundus to envelop the esophagus in a fash-ion analogous to a Witzel jejunostomy, sizing the fundoplication with a large (50 to 60F) bougie, limiting the length of the fun-doplication to 1 to 2 cm, and dividing the short gastric vessels. The essential elements necessary for the performance of a trans-abdominal fundoplication are common to both the laparoscopic and open procedures and include the following:1. Hiatal dissection and preservation of both vagi along their entire length2. Circumferential esophageal mobilization3. Hiatal closure, usually

1	and open procedures and include the following:1. Hiatal dissection and preservation of both vagi along their entire length2. Circumferential esophageal mobilization3. Hiatal closure, usually posterior to the esophagus4. Creation of a short and floppy fundoplication over an esoph-ageal dilatorIn addition, many surgeons also routinely divide the short gastric blood vessels, although this step is not universally applied, and the results of several randomized trials have failed to show that this step yields any benefit.The laparoscopic approach to fundoplication has now replaced the open abdominal Nissen fundoplication as the pro-cedure of choice. Five ports are usually used (Fig. 25-34), and dissection is begun by incising the gastrohepatic omentum above and below the hepatic branch of the anterior vagus nerve, which is usually preserved. The circumference of the diaphragmatic L R Figure 25-34. Patient positioning and trocar placement for lap-aroscopic antireflux surgery. The patient

1	anterior vagus nerve, which is usually preserved. The circumference of the diaphragmatic L R Figure 25-34. Patient positioning and trocar placement for lap-aroscopic antireflux surgery. The patient is placed with the head elevated approximately 30° in the modified lithotomy position. The surgeon stands between the patient’s legs, and the procedure is completed using five abdominal access ports.hiatus is dissected and the esophagus is mobilized by careful dis-section of the anterior and posterior soft tissues within the hiatus. The esophagus is held anterior and to the left and the hiatal pillars are approximated with interrupted nonabsorbable sutures, starting posteriorly and working anteriorly. A tension-free fundoplication should be constructed. This can usually be achieved either with or without division of the short gastric blood vessels, accord-ing to surgeon preference. If the vessels are divided, the upper one-third of the greater curvature is mobilized by sequentially

1	with or without division of the short gastric blood vessels, accord-ing to surgeon preference. If the vessels are divided, the upper one-third of the greater curvature is mobilized by sequentially dissecting and dividing these vessels, commencing distally and working proximally. Following complete fundal mobilization, the posterior wall of the fundus is brought behind the esophagus to the right side, and the anterior wall of the fundus is brought anterior to the esophagus. The fundic lips are manipulated to allow the fundus to envelop the esophagus without twisting. A 50 to 60F bougie is passed to properly size the fundoplication, and it is sutured using nonabsorbable sutures. Some surgeons use a single U-stitch of 2-0 polypropylene buttressed with felt pledgets (Fig. 25-35), and others use 2-4 interrupted sutures.Brunicardi_Ch25_p1009-p1098.indd 104001/03/19 6:03 PM 1041ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Posterior Partial Fundoplication. Partial fundoplications were

1	use 2-4 interrupted sutures.Brunicardi_Ch25_p1009-p1098.indd 104001/03/19 6:03 PM 1041ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Posterior Partial Fundoplication. Partial fundoplications were developed as an alternative to the Nissen procedure in an attempt to minimize the risk of postfundoplication side effects, such as dysphagia, inability to belch, and flatulence. The commonest approach has been a posterior partial or Toupet fundoplication. Some surgeons use this type of procedure for all patients present-ing for antireflux surgery, whereas others apply a tailored approach in which a partial fundoplication is constructed in patients with impaired esophageal motility, in which the propulsive force of the esophagus is thought to be insufficient to overcome the outflow obstruction of a complete fundoplication. The Toupet posterior partial fundoplication consists of a 270° gastric fundoplication around the distal 4 cm of esophagus (Fig. 25-36). It is usually stabilized by

1	of a complete fundoplication. The Toupet posterior partial fundoplication consists of a 270° gastric fundoplication around the distal 4 cm of esophagus (Fig. 25-36). It is usually stabilized by anchoring the wrap posteriorly to the hiatal rim.Anterior Partial Fundoplication. An alternative approach to partial fundoplication is to construct an anterior partial fundopli-cation. Following posterior hiatal repair, the anterior fundus is rolled over the front of the esophagus and sutured to the hiatal rim and the esophageal wall. Division of the short gastric vessels Figure 25-35. A. Laparoscopic Nissen fundoplication is performed with a five-trocar technique. B. The liver retractor is affixed to a mechani-cal arm to hold it in place throughout the operation. C. After division of the gastrohepatic omentum above the hepatic branch of the vagus (pars flaccida), the surgeon places a blunt atraumatic grasper beneath the phrenoesophageal ligament. D. After completion of the crural closure, an

1	omentum above the hepatic branch of the vagus (pars flaccida), the surgeon places a blunt atraumatic grasper beneath the phrenoesophageal ligament. D. After completion of the crural closure, an atraumatic grasper is placed right to left behind the gastroesophageal junction. The grasper is withdrawn, pulling the posterior aspect of the gastric fundus behind the esophagus. E. Once the suture positions are chosen, the first stitch (2-0 silk, 20 cm long) is introduced through the 10-mm trocar, and the needle is passed first through the left limb of the fundus, then the esophagus (2.5 cm above the gastroesophageal junction), then through the right limb of the fundus. F. Final position of the fundoplication.Brunicardi_Ch25_p1009-p1098.indd 104101/03/19 6:03 PM 1042SPECIFIC CONSIDERATIONSPART IIFigure 25-36. Completed laparoscopic posterior partial (Toupet) fundoplication. The fundoplication does not cover the anterior sur-face of the esophagus, and it is stabilized by suturing the

1	25-36. Completed laparoscopic posterior partial (Toupet) fundoplication. The fundoplication does not cover the anterior sur-face of the esophagus, and it is stabilized by suturing the fundus to the side of the esophagus, and posteriorly to the right hiatal pillar.is never needed when constructing this type of fundoplication. Various degrees of anterior partial fundoplication have been described—90°, 120°, 180°. The anterior 180° partial fundopli-cation (Fig. 25-37) provides a more robust fundoplication and achieves an excellent longer-term outcome in approximately 90% of patients at follow-up of at least 10 years. With this procedure, the fundus and esophagus are sutured to the right side of the hiatal rim to create a flap valve at the gastroesophageal junction and to stabilize a 3 to 4 cm length of intra-abdominal esophagus.Collis Gastroplasty. When a shortened esophagus is encoun-tered, many surgeons choose to add an esophageal lengthening procedure before fundoplication, to reduce

1	length of intra-abdominal esophagus.Collis Gastroplasty. When a shortened esophagus is encoun-tered, many surgeons choose to add an esophageal lengthening procedure before fundoplication, to reduce the tension on the gastroesophageal junction, believing this will minimize the risk of failure due to postoperative hiatus hernia. The commonest approach to this is the Collis gastroplasty. This entails using a stapler to divide the cardia and upper stomach, parallel to the lesser curvature of Figure 25-37. Completed laparoscopic anterior 180° partial fun-doplication. The fundoplication fully covers the anterior surface of the esophagus, and it is stabilized by suturing the fundus to the right side of the esophagus, and to the right hiatal pillar. Unlike the Nissen procedure, the fundus is not pulled behind the esophagus.the stomach, thereby creating a gastric tube in continuity with the esophagus, and effectively lengthening the esophagus by several centimeters. Laparoscopic techniques for

1	behind the esophagus.the stomach, thereby creating a gastric tube in continuity with the esophagus, and effectively lengthening the esophagus by several centimeters. Laparoscopic techniques for Collis gastroplasty have been described (Fig. 25-38). Following gastroplasty a fundoplica-tion is constructed, with the highest suture is placed on the native esophagus when constructing a Nissen fundoplication. Not all sur-geons choose to undertake a Collis procedure, however, as there is controversy about the actual incidence of the shortened esophagus and widely divergent views are held about how often this prob-lem is encountered. In addition, some surgeons have questioned the wisdom of creating an amotile tube of gastric wall, which can secrete acid, and then placing a Nissen fundoplication below this.Outcome After Fundoplication. Studies of long-term outcome following both open and laparoscopic fundoplication document the ability of laparoscopic fundoplication to relieve typical reflux

1	below this.Outcome After Fundoplication. Studies of long-term outcome following both open and laparoscopic fundoplication document the ability of laparoscopic fundoplication to relieve typical reflux symptoms (heartburn, regurgitation, and dysphagia) in more than Figure 25-35. (Continued )Brunicardi_Ch25_p1009-p1098.indd 104201/03/19 6:03 PM 1043ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Figure 25-38. A. After removal of the fat pad and release of tension on the Penrose drain, the gastroesophageal junction (GES) retracts to the level of the hiatus. The interior end of the staple line is marked 2/5 cm below the angle of His. B. The first horizontal firing of the stapler occurs by maximally articulating the stapler to the left, aiming toward the previously marked spot adjacent to the dilator. C. The vertical staple line is created by a single firing of the GIA placed parallel and flush against the 48F dilator. D. The highest Nissen fundoplication suture is placed on the native

1	dilator. C. The vertical staple line is created by a single firing of the GIA placed parallel and flush against the 48F dilator. D. The highest Nissen fundoplication suture is placed on the native esophagus, and the second suture tucks in the apex of the staple line.90% of patients at follow-up intervals averaging 2 to 3 years and 80% to 90% of patients 5 years or more following surgery. This includes evidence-based reviews of antireflux surgery, pro-spective randomized trials comparing antireflux surgery to PPI therapy and open to laparoscopic fundoplication and analysis of U.S. national trends in use and outcomes. Postoperative pH stud-ies indicate that more than 90% of patients will normalize their pH tracings. The results of laparoscopic fundoplication compare favorably with those of the “modern” era of open fundoplica-tion. They also indicate the less predictable outcome of atypical reflux symptoms (cough, asthma, laryngitis) after surgery, being relieved in only two-thirds of

1	the “modern” era of open fundoplica-tion. They also indicate the less predictable outcome of atypical reflux symptoms (cough, asthma, laryngitis) after surgery, being relieved in only two-thirds of patients.The goal of surgical treatment for GERD is to relieve the symptoms of reflux by reestablishing the gastroesophageal barrier. The challenge is to accomplish this without inducing dysphagia or other untoward side effects. Dysphagia, existing before surgery, usually improves following laparoscopic fun-doplication. Temporary dysphagia is common after surgery and generally resolves within 3 months, but it can take up to 12 months in some individuals, and dysphagia sufficient to require ongoing dietary modification persists in up to 5% of individuals following Nissen fundoplication. Other side effects common to antireflux surgery include the inability to belch and vomit and increased flatulence. Most patients cannot vomit through an intact wrap, though this is rarely clinically

1	Other side effects common to antireflux surgery include the inability to belch and vomit and increased flatulence. Most patients cannot vomit through an intact wrap, though this is rarely clinically relevant. Most patients are unable to belch gas from the stomach in the first 3 to 6 months after fundoplication, but 80% to 90% regain the ability to belch normally beyond the first 12 months of fol-low-up. Hyperflatulence is a common and noticeable problem, likely related to increased air swallowing that is present in most patients with reflux disease, aggravated by the inability to belch in some patients.Brunicardi_Ch25_p1009-p1098.indd 104301/03/19 6:03 PM 1044SPECIFIC CONSIDERATIONSPART IIRandomized Controlled Trials Addressing Surgical Technique Division of the Short Gastric Blood Vessels Originally, Nissen’s description of a total fundoplication entailed a 360° fundoplication during which the short gastric blood vessels were left intact. However, with reports of troublesome

1	Vessels Originally, Nissen’s description of a total fundoplication entailed a 360° fundoplication during which the short gastric blood vessels were left intact. However, with reports of troublesome postoperative dysphagia, division of these vessels—to achieve full fundal mobilization and thereby ensure a loose fundoplication—was promoted and has entered common practice. The evidence sup-porting dividing these vessels has been based on the outcomes from uncontrolled case series of patients undergoing Nissen fundoplication either with vs. without division of the short gas-tric vessels. However, the results from these studies have been conflicting, with different proponents reporting good results irrespective of whether these vessels have been divided or not. To address this issue, six randomized trials that enrolled a total of 438 patients have been reported. None of these trials demon-strated any differences for the postoperative dysphagia or recur-rent gastro-esophageal reflux.

1	randomized trials that enrolled a total of 438 patients have been reported. None of these trials demon-strated any differences for the postoperative dysphagia or recur-rent gastro-esophageal reflux. However, in the three largest of the six trials an increased incidence of flatulence and bloating symptoms, as well as greater difficulty with belching, was seen in patients in whom the short gastric vessels were divided.A recent meta-analysis from Engstrom et al, generated by combining the raw data from Australian and Swedish trials, eval-uated a larger cohort of 201 patients, with 12 years of follow-up in 170, and also confirmed equivalent reflux control but found more abdominal bloating after division of the short gastric ves-sels. Overall, these trials fail to support the belief that dividing the short gastric vessels improves any outcome following Nissen fun-doplication. The trials actually suggest that dividing the vessels increases the complexity of the procedure and leads to a

1	the short gastric vessels improves any outcome following Nissen fun-doplication. The trials actually suggest that dividing the vessels increases the complexity of the procedure and leads to a poorer outcome due to the increase in bloating symptoms.Nissen vs. Posterior Partial Fundoplication Eleven randomized trials have compared Nissen vs. posterior partial fundoplication. Some of the trials contributed little to the pool of evidence, as they are either small or underpowered, and failed to show significant outcome differences. The larger trials, however, have consistently demonstrated equivalent reflux control, but they also show a reduced incidence of wind-related side-effects (flatulence, bloating, and inability to belch) following posterior partial fundoplication procedures, although less dysphagia fol-lowing a posterior fundoplication was only demonstrated in 2 of the 11 trials. Lundell et al reported the outcomes of Nissen vs. Toupet partial fundoplication in a trial that

1	less dysphagia fol-lowing a posterior fundoplication was only demonstrated in 2 of the 11 trials. Lundell et al reported the outcomes of Nissen vs. Toupet partial fundoplication in a trial that enrolled 137 patients with reported follow-up to 18 years. Reflux control and dyspha-gia symptoms were similar, but flatulence was commoner after Nissen fundoplication at some medium-term follow-up time points, and revision surgery was more common following Nissen fundoplication, mainly to correct postoperative paraoesophageal herniation. At 18 years follow-up, success rates of more than 80% were reported for both procedures, as well as no significant differences in the incidence of side effects. The data from this trial suggested that the mechanical side effects following Nis-sen fundoplication progressively improve with very long-term follow-up. Strate et al reported 2-year follow-up in a trial that enrolled 200 patients. Approximately 85% of each group was satisfied with the clinical

1	progressively improve with very long-term follow-up. Strate et al reported 2-year follow-up in a trial that enrolled 200 patients. Approximately 85% of each group was satisfied with the clinical outcome, but dysphagia was signifi-cantly more common following Nissen fundoplication (19 vs. 8 patients).Other trials (Guérin et al–140 patients, Booth et al–127, Khan et al–121, Shaw et al–100) also report similar reflux control within the first few years of follow-up. Only Booth et al demonstrated less dysphagia following posterior fundoplica-tion. Subgroup analysis in 3 trials (Booth, Shaw, Zornig) did not reveal differences between patients with vs. without poor pre-operative oesophageal motility. Overall these trials suggest that some side-effects, mainly wind-related issues, are less common following posterior partial fundoplication. However, the hypoth-esis that dysphagia is less of a problem following posterior par-tial fundoplication has only been substantiated in 2 of 11

1	less common following posterior partial fundoplication. However, the hypoth-esis that dysphagia is less of a problem following posterior par-tial fundoplication has only been substantiated in 2 of 11 trials.Nissen vs. Anterior Fundoplication Six trials have evaluated Nissen vs. anterior partial fundoplication variants. Four have assessed Nissen vs. anterior 180° partial fundoplication (Watson et al–107 patients, Baigrie et al–161, Cao et al–100, Raue et al–64). These trials all demonstrated equivalent reflux control, but less dysphagia and less wind-related side effects after anterior 180° partial fundoplication at up to 5 years follow-up. Only the study from Watson et al has reported follow-up to 10 years, and at late follow-up in their trial there were no significant outcome differences for the two procedures, with equivalent control of reflux, and no differences for side effects due to a progressive decline in dysphagia as follow-up extended beyond 5 years.Two trials compared

1	for the two procedures, with equivalent control of reflux, and no differences for side effects due to a progressive decline in dysphagia as follow-up extended beyond 5 years.Two trials compared laparoscopic anterior 90° partial fundoplication vs. Nissen fundoplication (Watson et al–112 patients, Spence et al–79). In both of these trials, side-effects were less common following anterior 90° fundoplication, but this was offset by a slightly higher incidence of recurrent reflux at up to 5 years follow-up. Satisfaction with the overall outcome was similar for both fundoplication variants.Anterior vs. Posterior Partial Fundoplication Two ran-domized trials have directly compared anterior vs. posterior partial fundoplication. Hagedorn et al randomized 95 patients to undergo either Toupet vs. anterior 120° partial fundoplica-tion, and Khan et al enrolled 103 patients to anterior 180° vs. posterior partial fundoplication. Both studies demonstrated bet-ter reflux control, offset by more side

1	120° partial fundoplica-tion, and Khan et al enrolled 103 patients to anterior 180° vs. posterior partial fundoplication. Both studies demonstrated bet-ter reflux control, offset by more side effects following posterior partial fundoplication. The anterior 120° partial fundoplication performed by Hagedorn et al was similar to the anterior 90° vari-ant described above. However, the outcomes following this pro-cedure were much worse in this trial than the outcomes in other studies, with the average exposure time to acid (pH <4%–5.6%) following anterior fundoplication in their study unusually high compared to other studies. Khan et al only reported 6 months follow-up, and longer-term outcomes are awaited before draw-ing firm conclusions. The overall results from all eight trials that included an anterior fundoplication variant suggest that this type of fundoplication achieves satisfactory reflux control, with less dysphagia and other side-effects, yielding a good overall outcome.

1	included an anterior fundoplication variant suggest that this type of fundoplication achieves satisfactory reflux control, with less dysphagia and other side-effects, yielding a good overall outcome. However, the reduced incidence of troublesome side-effects is traded off against a higher risk of recurrent reflux.Outcome of Antireflux Surgery in Patients With Barrett’s Esophagus. Few studies have focused on the alleviation of symp-toms after antireflux surgery in patients with BE (Table 25-7). Those that are available document excellent to good results in 72% to 95% of patients at 5 years following surgery. Several nonrandomized studies have compared medical and surgical therapy and report better outcomes after antireflux surgery. Par-rilla and colleagues reported the only randomized trial to evaluate this issue. They enrolled 101 patients over 18 years, and median follow-up was 6 years. Medical therapy consisted of 20 mg of omeprazole (PPI) twice daily since 1992 in all medically

1	to evaluate this issue. They enrolled 101 patients over 18 years, and median follow-up was 6 years. Medical therapy consisted of 20 mg of omeprazole (PPI) twice daily since 1992 in all medically treated patients, and surgical therapy consisted of an open Nissen Brunicardi_Ch25_p1009-p1098.indd 104401/03/19 6:03 PM 1045ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Table 25-7Symptomatic outcome of surgical therapy for Barrett’s esophagusAUTHORYEARNO. OF PATIENTS% EXCELLENT TO GOOD RESPONSEMEAN FOLLOW-UP, YEARSStarnes19848752Williamson199037923DeMeester199035773McDonald199611382.26.5Ortiz19963290.65fundoplication. The symptomatic outcome in the two groups was nearly identical, although esophagitis and/or stricture persisted in 20% of the medically treated patients, compared to only 3% to 7% of patients following antireflux surgery. About 15% of patients had abnormal acid exposure after surgery. Although pH data were not routinely collected in patients on PPI therapy, in the subgroup

1	7% of patients following antireflux surgery. About 15% of patients had abnormal acid exposure after surgery. Although pH data were not routinely collected in patients on PPI therapy, in the subgroup of 12 patients that did have 24-hour monitoring on treat-ment, 3 of 12 (25%) had persistently high esophageal acid expo-sure, and most (75%) had persistently high bilirubin exposure.The common belief that Barrett’s epithelium cannot be reversed by antireflux surgery may not be correct. Within the control arm of a randomized trial of ablation vs. surveillance, Bright and associates identified approximately 50% regression in the length of Barrett’s esophagus in 20 patients within the control arm of a randomized trial of ablation vs. surveillance.Current data indicate that patients with BE should remain in an endoscopic surveillance program following antireflux surgery. Biopsy specimens should be reviewed by a patholo-gist with expertise in the field. If low-grade dysplasia is con-firmed,

1	remain in an endoscopic surveillance program following antireflux surgery. Biopsy specimens should be reviewed by a patholo-gist with expertise in the field. If low-grade dysplasia is con-firmed, biopsy specimens should be repeated after 12 weeks of high-dose acid suppression therapy. If high-grade dysplasia or intramucosal cancer is evident on more than one biopsy speci-men, then treatment is escalated. Treatment options include endoscopic mucosal resection, endoscopic ablation of the BE, or esophageal resection. Esophageal resection is advisable when an invasive cancer (stage T1b or deeper) is present, or for mul-tifocal long segment BE in younger and fit patients in whom endoscopic treatments are unlikely to be adequate. Endoscopic mucosal resection allows smaller intramucosal tumors to be removed with clear pathology margins, and it can be used as a “big biopsy” to obtain better pathological staging, and even to excise shorter segments of BE in a piecemeal fashion. Ablation,

1	to be removed with clear pathology margins, and it can be used as a “big biopsy” to obtain better pathological staging, and even to excise shorter segments of BE in a piecemeal fashion. Ablation, commonly using radiofrequency ablation, has been shown at short-term follow-up in a randomized trial to reduce the rate of progression from high grade dysplasia to invasive cancer by approximately 50%. However, following any endoscopic treatment, patients need to continue with close endoscopic sur-veillance as recurrence can occur and the longer-term outcome following these treatments remains uncertain. Early detection and treatment have been shown to decrease the mortality rate from esophageal cancer in these patients.If the dysplasia is reported as lower grade or indetermi-nant, then inflammatory change that is often confused with dysplasia should be suppressed by a course of acid suppression therapy in high doses for 2 to 3 months, followed by rebiopsy of the Barrett’s segment.Reoperation

1	change that is often confused with dysplasia should be suppressed by a course of acid suppression therapy in high doses for 2 to 3 months, followed by rebiopsy of the Barrett’s segment.Reoperation for Failed Antireflux Repairs. Failure of an antireflux procedure occurs when, after the repair, the patient is unable to swallow normally, experiences upper abdominal dis-comfort during and after meals, or has recurrence or persistence of reflux symptoms. The assessment of these symptoms and the selection of patients who need further surgery are challenging problems. Functional assessment of patients who have recur-rent, persistent, or emergent new symptoms following a primary antireflux repair is critical to identifying the cause of the failure. Analysis of patients requiring reoperation after a previous anti-reflux procedure shows that placement of the wrap around the stomach is the most frequent cause for failure after open proce-dures, while herniation of the repair into the chest is

1	a previous anti-reflux procedure shows that placement of the wrap around the stomach is the most frequent cause for failure after open proce-dures, while herniation of the repair into the chest is the most frequent cause of failure after a laparoscopic procedure. Partial or complete breakdown of the fundoplication and construction of a too-tight a fundoplication or overnarrowing the esophageal hiatus occurs with both open and closed procedures.Patients who have recurrence of heartburn and regurgitation without dysphagia and have good esophageal motility are most amenable to reoperation, and they can be expected to have an excellent outcome. When dysphagia is the cause of failure, the sit-uation can be more difficult to manage. If the dysphagia occurred immediately following the repair, it is usually due to a technical failure, most commonly a misplaced fundoplication around the upper stomach, or overnarrowing of the esophageal diaphragmatic hiatus and reoperation is usually

1	repair, it is usually due to a technical failure, most commonly a misplaced fundoplication around the upper stomach, or overnarrowing of the esophageal diaphragmatic hiatus and reoperation is usually satisfactory. When dysphagia is associated with poor motility and multiple previous repairs, fur-ther revision fundoplication is unlikely to be successful, and in otherwise fit patients it is appropriate to seriously consider esopha-geal resection. With each reoperation, the esophagus is damaged further, and the chance of preserving function is decreased. Also, blood supply is reduced, and ischemic necrosis of the esophagus can occur after several previous mobilizations.GIANT DIAPHRAGMATIC (HIATAL) HERNIASWith the advent of clinical radiology, it became evident that a diaphragmatic hernia was a relatively common abnormality and was not always accompanied by symptoms. Three types of esophageal hiatal hernia were identified: (a) the sliding hernia, type I, characterized by an upward

1	was a relatively common abnormality and was not always accompanied by symptoms. Three types of esophageal hiatal hernia were identified: (a) the sliding hernia, type I, characterized by an upward dislocation of the cardia in the posterior mediastinum (Fig. 25-39A); (b) the roll-ing or PEH, type II, characterized by an upward dislocation of the gastric fundus alongside a normally positioned cardia (Fig. 25-39B); and (c) the combined sliding-rolling or mixed hernia, type III, characterized by an upward dislocation of both the cardia and the gastric fundus (Fig. 25-39C). The end stage of type I and type II hernias occurs when the whole stomach migrates up into the chest by rotating 180° around its longitu-dinal axis, with the cardia and pylorus as fixed points. In this situation, the abnormality is usually referred to as an intratho-racic stomach (Fig. 25-39D). In some taxonomies, a type IV hiatal hernia is declared when an additional organ, usually the colon, herniates as well. Types

1	is usually referred to as an intratho-racic stomach (Fig. 25-39D). In some taxonomies, a type IV hiatal hernia is declared when an additional organ, usually the colon, herniates as well. Types II–IV hiatal hernias are also referred to as paraesophageal hernia (PEH), as a portion of the stomach is situated adjacent to the esophagus, above the gastroesophageal junction.Incidence and EtiologyThe true incidence of a hiatal hernia is difficult to determine because of the absence of symptoms in a large number of patients who are subsequently shown to have a hernia. When radiographic examinations are done in response to GI symptoms, Brunicardi_Ch25_p1009-p1098.indd 104501/03/19 6:03 PM 1046SPECIFIC CONSIDERATIONSPART IICDBAFigure 25-39. A. Radiogram of a type I (sliding) hiatal hernia. B. Radiogram of a type II (rolling or paraesophageal) hernia. C. Radiogram of a type III (combined sliding-rolling or mixed) hernia. D. Radiogram of an intrathoracic stomach. This is the end stage of a

1	Radiogram of a type II (rolling or paraesophageal) hernia. C. Radiogram of a type III (combined sliding-rolling or mixed) hernia. D. Radiogram of an intrathoracic stomach. This is the end stage of a large hiatal hernia regardless of its initial classification. Note that the stomach has rotated 180° around its longitudinal axis, with the cardia and pylorus as fixed points. (Reproduced with permission from Nyhus LM, Condon RE: Hernia, 3rd ed. Philadelphia, PA: Lippincott Williams & Wilkins; 1989.)Brunicardi_Ch25_p1009-p1098.indd 104601/03/19 6:03 PM 1047ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25the incidence of a sliding hiatal hernia is seven times higher than that of a PEH. The PEH is also known as the giant hiatal hernia. Over time the pressure gradient between the abdomen and chest enlarges the hiatal hernia. In many cases the type 1 sliding hernia will evolve into a type III mixed hernia. Type II hernias are quite rare. The age distribution of patients with PEHs is

1	and chest enlarges the hiatal hernia. In many cases the type 1 sliding hernia will evolve into a type III mixed hernia. Type II hernias are quite rare. The age distribution of patients with PEHs is significantly different from that observed in sliding hiatal hernias. The median age of the former is 61 years old; of the latter, 48 years old. PEHs are more likely to occur in women by a ratio of 4:1.Structural deterioration of the phrenoesophageal mem-brane over time may explain the higher incidence of hiatal her-nias in the older age group. These changes involve thinning of the upper fascial layer of the phrenoesophageal membrane (i.e., the supradiaphragmatic continuation of the endothoracic fascia) and loss of elasticity in the lower fascial layer (i.e., the infra-diaphragmatic continuation of the transversalis fascia). Conse-quently, the phrenoesophageal membrane yields to stretching in the cranial direction due to the persistent intra-abdominal pres-sure and the tug of esophageal

1	of the transversalis fascia). Conse-quently, the phrenoesophageal membrane yields to stretching in the cranial direction due to the persistent intra-abdominal pres-sure and the tug of esophageal shortening on swallowing. Inter-estingly, the stretching and thinning occurs more anteriorly and posteriorly, with fixation of the left crus of the diaphragm to the stomach at the 3 o’clock position, as viewed from the foot. This creates an anterior and posterior hernia sac, the latter of which is often filled with epiphrenic and retroperitoneal fat. These obser-vations point to the conclusion that the development of a hiatal hernia is an age-related phenomenon secondary to repetitive upward stretching of the phrenoesophageal membrane.Clinical ManifestationsThe clinical presentation of a giant hiatal (paraesophageal) her-nia differs from that of a sliding hernia. There is usually a higher prevalence of symptoms of dysphagia and postprandial fullness with PEHs, but the typical symptoms of

1	hiatal (paraesophageal) her-nia differs from that of a sliding hernia. There is usually a higher prevalence of symptoms of dysphagia and postprandial fullness with PEHs, but the typical symptoms of heartburn and regurgi-tation present in sliding hiatal hernias can also occur. Both are caused by gastroesophageal reflux secondary to an underlying mechanical deficiency of the cardia. The symptoms of dysphagia and postprandial fullness in patients with a PEH are explained by the compression of the adjacent esophagus by a distended cardia, or twisting of the GEJ by the torsion of the stomach that occurs as it becomes progressively displaced in the chest. The postprandial fullness or retrosternal chest pain is a thought to be a result of distension of the stomach with gas or food in the hiatal hernia. Many patients with sliding hernias and reflux symptoms will lose the reflux symptoms when the hernia evolves into the paraesophageal variety. This can be explained by the recreation of the

1	hernia. Many patients with sliding hernias and reflux symptoms will lose the reflux symptoms when the hernia evolves into the paraesophageal variety. This can be explained by the recreation of the cardiophrenic angle when the stomach herniates along-side the GEJ or becomes twisted in the sac. Repair of the hernia without addressing the reflux can create extremely bothersome heartburn. Respiratory complications are frequently associated with a PEH and consist of dyspnea and recurrent pneumonia from aspiration. New research demonstrates that the cause of dyspnea in the presence of a giant PEH is more likely to be left atrial compression, decreasing cardiac output, than a restrictive pulmonary effect, as has been hypothesized for many years.Approximately one-third of patients with a PEH are found to be anemic, which is due to recurrent bleeding from ulceration of the gastric mucosa in the herniated portion of the stomach, even if ulcerations are not detected at the time of endoscopy. The

1	to be anemic, which is due to recurrent bleeding from ulceration of the gastric mucosa in the herniated portion of the stomach, even if ulcerations are not detected at the time of endoscopy. The association of anemia and PEH is best proven by fixing the hernia. Anemia is corrected in >90% of patients with this condition. With time, more and more stomach migrates into the chest and can cause intermittent foregut obstruction due to the rotation that has occurred. In contrast, many patients with PEH are asymptomatic or complain of minor symptoms. However, the presence of a PEH can be life-threatening in that the hernia can lead to sudden catastrophic events, such as excessive bleed-ing or volvulus with acute gastric obstruction or infarction. With mild dilatation of the stomach, the gastric blood supply can be markedly reduced, causing gastric ischemia, ulceration, perfora-tion, and sepsis. The probability of incarceration/strangulation is not well known, although recent studies suggest

1	blood supply can be markedly reduced, causing gastric ischemia, ulceration, perfora-tion, and sepsis. The probability of incarceration/strangulation is not well known, although recent studies suggest that the lifetime risk is less than 5%, making this concern an insufficient concern for routine repair of the asymptomatic PEH.The symptoms of sliding hiatal hernias are usually due to functional abnormalities associated with gastroesophageal reflux and include heartburn, regurgitation, and dysphagia. These patients have a mechanically defective LES, giving rise to the reflux of gastric juice into the esophagus and the symp-toms of heartburn and regurgitation. The symptom of dysphagia occurs from the presence of mucosal edema, Schatzki’s ring, stricture, or the inability to organize peristaltic activity in the body of the esophagus as a consequence of the disease.There is a group of patients with sliding hiatal hernias not associated with reflux disease who have dysphagia without any

1	activity in the body of the esophagus as a consequence of the disease.There is a group of patients with sliding hiatal hernias not associated with reflux disease who have dysphagia without any obvious endoscopic or manometric explanation. Video barium radiograms have shown that the cause of dysphagia in these patients is an obstruction of the swallowed bolus by diaphrag-matic impingement on the herniated stomach. Manometrically, this is reflected by a double-humped high-pressure zone at the GEJ. The first pressure rise is due to diaphragmatic impinge-ment on the herniated stomach, and the second is due to the true distal esophageal sphincter. These patients usually have a mechanically competent sphincter, but the impingement of the diaphragm on the stomach can result in propelling the contents of the supradiaphragmatic portion of the stomach up into the esophagus and pharynx, resulting in complaints of pharyngeal regurgitation and aspiration. Consequently, this abnormality is often

1	of the supradiaphragmatic portion of the stomach up into the esophagus and pharynx, resulting in complaints of pharyngeal regurgitation and aspiration. Consequently, this abnormality is often confused with typical GERD. Surgical reduction of the hernia results in relief of the dysphagia in 91% of patients.DiagnosisA chest X-ray with the patient in the upright position can diag-nose a hiatal hernia if it shows an air-fluid level behind the car-diac shadow. This is usually caused by a PEH or an intrathoracic stomach. The accuracy of the upper GI barium study in detect-ing a paraesophageal hiatal hernia is greater than for a sliding hernia because the latter can often spontaneously reduce. The paraesophageal hiatal hernia is a permanent herniation of the stomach into the thoracic cavity, so a barium swallow provides the diagnosis in virtually every case. Attention should be focused on the position of the GEJ, when seen, to differentiate it from a type II hernia (see Fig. 25-39B and C).

1	a barium swallow provides the diagnosis in virtually every case. Attention should be focused on the position of the GEJ, when seen, to differentiate it from a type II hernia (see Fig. 25-39B and C). Fiber-optic esophagos-copy is useful in the diagnosis and classification of a hiatal hernia because the scope can be retroflexed. In this position, a sliding hiatal hernia can be identified by noting a gastric pouch lined with rugal folds extending above the impression caused by the crura of the diaphragm, or measuring at least 2 cm between the crura, identified by having the patient sniff, and the squamoco-lumnar junction on withdrawal of the scope (Fig. 25-40). A PEH is identified on retroversion of the scope by noting a separate orifice adjacent to the GEJ into which gastric rugal folds ascend. A sliding-rolling or mixed hernia can be identified by noting a gastric pouch lined with rugal folds above the diaphragm, with the GEJ entering about midway up the side of the

1	rugal folds ascend. A sliding-rolling or mixed hernia can be identified by noting a gastric pouch lined with rugal folds above the diaphragm, with the GEJ entering about midway up the side of the pouch.Brunicardi_Ch25_p1009-p1098.indd 104701/03/19 6:03 PM 1048SPECIFIC CONSIDERATIONSPART IIFigure 25-40. Endoscopic view through a retroflexed fiber-optic gastroscope showing the shaft of the scope (arrow) coming down through a sliding hernia. Note the gastric rugal folds extending above the impression caused by the crura of the diaphragm. (Repro-duced with permission from Nyhus LM, Condon RE: Hernia, 3rd ed. Philadelphia, PA: Lippincott Williams & Wilkins; 1989.)PathophysiologyPhysiologic testing with 24-hour esophageal pH monitoring has shown increased esophageal exposure to acid gastric juice in 60% of the patients with a paraesophageal hiatal hernia, com-pared with the observed 71% incidence in patients with a sliding hiatal hernia. It is now recognized that paraesophageal hiatal

1	juice in 60% of the patients with a paraesophageal hiatal hernia, com-pared with the observed 71% incidence in patients with a sliding hiatal hernia. It is now recognized that paraesophageal hiatal her-nia can be associated with pathologic gastroesophageal reflux.Physiologic studies have also shown that the competency of the cardia depends on an interrelationship between distal esophageal sphincter pressure, the length of the sphincter that is exposed to the positive-pressure environment of the abdomen, and the overall length of the sphincter. A deficiency in any one of these manometric characteristics of the sphincter is associated with incompetency of the cardia regardless of whether a hernia is present. Patients with a PEH who have an incompetent cardia have been shown to have a distal esophageal sphincter with nor-mal pressure, but a shortened overall length and displacement outside the positive-pressure environment of the abdomen. One might expect esophageal body function to be

1	esophageal sphincter with nor-mal pressure, but a shortened overall length and displacement outside the positive-pressure environment of the abdomen. One might expect esophageal body function to be diminished with the esophagus “accordioned” up into the chest. Surprisingly, esophageal peristalsis in patients with PEH is normal in 88%.TreatmentThe treatment of paraesophageal hiatal hernia is largely surgi-cal. Controversial aspects include: (a) indications for repair, (b) diaphragmatic repair, (c) role of fundoplication, and (d) exis-tence and treatment of the short esophagus.Indications and Surgical Approach. The presence of a paraesophageal hiatal hernia has traditionally been consid-ered an indication for surgical repair. This recommendation is largely based upon two clinical observations. First, retrospec-tive studies have shown a significant incidence of catastrophic, life-threatening complications of bleeding, infarction, and per-foration in patients being followed with known

1	First, retrospec-tive studies have shown a significant incidence of catastrophic, life-threatening complications of bleeding, infarction, and per-foration in patients being followed with known paraesophageal herniation. Second, emergency repair carries a high mortality. In the classic report of Skinner and Belsey, six of 21 patients with a PEH, treated medically because of minimal symptoms, died from the complications of strangulation, perforation, exsangui-nating hemorrhage, or acute dilatation of the herniated intratho-racic stomach. For the most part, these catastrophes occurred without warning. Others have reported similar findings.Recent studies suggest that catastrophic complications may be somewhat less common. Allen and colleagues followed 23 patients for a median of 78 months with only four patients pro-gressively worsening. There was a single mortality secondary to aspiration that occurred during a barium swallow examination to investigate progressive symptoms. Although

1	with only four patients pro-gressively worsening. There was a single mortality secondary to aspiration that occurred during a barium swallow examination to investigate progressive symptoms. Although emergency repairs had a median hospital stay of 48 days compared to a stay of 9 days in those having elective repair, there were only three cases of gastric strangulation in 735 patient-years of follow-up.If surgery is delayed and repair is done on an emergency basis, operative mortality is high, compared to <1% for an elec-tive repair. With this in mind, patients with a PEH are generally counseled to have elective repair of their hernia, particularly if they are symptomatic. Watchful waiting of asymptomatic PEHs may be an acceptable option.The surgical approach to repair of a paraesophageal hiatal hernia may be either transabdominal (laparoscopic or open) or transthoracic. Each has its advantages and disadvantages. A transthoracic approach facilitates complete esophageal mobi-lization

1	hiatal hernia may be either transabdominal (laparoscopic or open) or transthoracic. Each has its advantages and disadvantages. A transthoracic approach facilitates complete esophageal mobi-lization but is rarely used because the access trauma and postopera-tive pain are significantly greater than a laparoscopic approach.The transabdominal approach facilitates reduction of the volvulus that is often associated with PEHs. Although some degree of esophageal mobilization can be accomplished tran-shiatally, complete mobilization to the aortic arch is difficult or impossible without risk of injury to the vagal nerves.Laparoscopic repair of PEH would appear to have become the standard approach. Laparoscopic repair of a pure type II, or mixed type III PEH is an order of magnitude more difficult than a standard laparoscopic Nissen fundoplication. Most would rec-ommend that these procedures are best avoided until the surgeon has accumulated considerable experience with laparoscopic antireflux

1	than a standard laparoscopic Nissen fundoplication. Most would rec-ommend that these procedures are best avoided until the surgeon has accumulated considerable experience with laparoscopic antireflux surgery. There are several reasons for this. First, the vertical and horizontal volvulus of the stomach often associated with PEHs makes identification of the anatomy, in particular the location of the esophagus, difficult. Second, dissection of a large PEH sac may result in significant bleeding if the surgeon deviates from the correct plane of dissection between the peri-toneal sac and the endothoracic fascia. Finally, redundant tissue present at the GEJ following dissection of the sac frustrates the creation of a fundoplication. This tissue, which includes the epi-phrenic fat pad and hernia sac should be removed at the time of PEH repair. Mindful of these difficulties, and given appropriate experience, patients with PEH may be approached laparoscopi-cally, with expectation of success in

1	sac should be removed at the time of PEH repair. Mindful of these difficulties, and given appropriate experience, patients with PEH may be approached laparoscopi-cally, with expectation of success in the majority.Diaphragmatic RepairIt has been shown that PEH repair has a relatively high incidence of recurrence (10–40%) when the crura is closed primarily with permanent suture. Techniques to reduce hernia recurrence con-tinue to evolve. Most surgeons believe that recurrence may be reduced with the use of synthetic or biologic mesh to reinforce the standard crural closure. Randomized controlled studies have 4Brunicardi_Ch25_p1009-p1098.indd 104801/03/19 6:04 PM 1049ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25demonstrated a reduction in PEH recurrence rate when mesh was used. Nonabsorbable synthetic mesh must be used carefully and not in a keyhole fashion at the hiatus because of a potential risk of esophagus or gastric erosion and mesh infection. Bio-logic mesh (acellular porcine

1	synthetic mesh must be used carefully and not in a keyhole fashion at the hiatus because of a potential risk of esophagus or gastric erosion and mesh infection. Bio-logic mesh (acellular porcine dermis, acellular human dermis, porcine small intestinal submucosa) has become more widely used, but these meshes are significantly more expensive than synthetic mesh, and the only randomized study supporting bio-logic mesh usage failed to demonstrate superiority over suture alone after 5 years of rigorous follow-up.Role of Fundoplication in Giant Hiatal Hernia Repair. Controversy remains as to whether to perform an antireflux procedure at all, in selected cases only, or in all patients. Most advocate the routine addition of an antireflux procedure follow-ing repair of the hernia defect. There are several reasons for this. Physiologic testing with 24-hour esophageal pH monitoring has shown increased esophageal exposure to acid gastric juice in 60% to 70% of patients with a paraesophageal

1	are several reasons for this. Physiologic testing with 24-hour esophageal pH monitoring has shown increased esophageal exposure to acid gastric juice in 60% to 70% of patients with a paraesophageal hiatal hernia, nearly identical to the observed 71% incidence in patients with a sliding hiatal hernia. Furthermore, there is no relation between the symptoms experienced by the patient with a PEH and the competency of the cardia. Finally, dissection of the gastro-esophageal esophagus may lead to postoperative reflux despite a negative preoperative pH score.The Short Esophagus and PEHGiant PEH can be associated with a short esophagus in up to 5% to 20% of patients as a result of chronic cephalad displacement of the GEJ. The presence of a short esophagus increases the dif-ficulty of laparoscopic PEH repair. Approximately 10% to 20% of surgical failures with PEH repair is due to the lack of recogni-tion of a short esophagus. Preoperative results of barium swallow and

1	of laparoscopic PEH repair. Approximately 10% to 20% of surgical failures with PEH repair is due to the lack of recogni-tion of a short esophagus. Preoperative results of barium swallow and esophagogastroduodenoscopy may provide an indication of short esophagus, but no combination of preoperative clinical vari-ables reliably predict the presence of short esophagus, defined as the failure to achieve 2.5 cm of intra-abdominal esophagus with standard mediastinal dissection techniques. Hence, the diagno-sis of this entity continues to be made definitively only in the operating room. Collis gastroplasty achieves esophageal length-ening by creation of a neoesophagus using the gastric cardia. The totally laparoscopic approach to the short esophagus has evolved from a method using an end-to-end anastomosis circular stapler to the current approach that uses a linear stapler creating a sta-pled wedge gastroplasty. Elements of importance in fashioning the fundoplication after Collis gastroplasty

1	circular stapler to the current approach that uses a linear stapler creating a sta-pled wedge gastroplasty. Elements of importance in fashioning the fundoplication after Collis gastroplasty include placement of the initial suture of the fundoplication on the esophagus, immedi-ately above the GEJ to ensure that acid-secreting (gastric) mucosa does not reside above the fundoplication. A second element that ensures safety and avoids wrap deformation is to place the gastric portion of the staple line against the neoesophagus, such that the tip of the gastric staple line sits adjacent to the middle suture of the fundoplication on the right side of the esophagus.ResultsMost outcome studies report relief of symptoms following sur-gical repair of PEHs in more than 90% of patients. The current literature suggests that laparoscopic repair of a paraesophageal hiatal hernia can be successful. Most authors report symptom-atic improvement in 80% to 90% of patients, and <10% to 15% prevalence of

1	literature suggests that laparoscopic repair of a paraesophageal hiatal hernia can be successful. Most authors report symptom-atic improvement in 80% to 90% of patients, and <10% to 15% prevalence of recurrent symptomatic hernia. However, the problem of recurrent asymptomatic or minimally symp-tomatic hernia following PEH repair, open or laparoscopic, is Figure 25-41. Barium esophagogram showing Schatzki’s ring (i.e., a thin circumferential ring in the distal esophagus at the squa-mocolumnar junction). Below the ring is a hiatal hernia.becoming increasingly appreciated. Recurrent hiatal hernia is the most common cause of anatomic failure following laparoscopic Nissen fundoplication done for GERD (5–10%), but this risk is compounded for the giant hernia where radiologic recurrence is detected in 25% to 40% of patients. It appears that optimal results with open or laparoscopic giant hiatal hernia repair should include options for mesh buttressing of hiatal closure and selec-tive

1	detected in 25% to 40% of patients. It appears that optimal results with open or laparoscopic giant hiatal hernia repair should include options for mesh buttressing of hiatal closure and selec-tive esophageal lengthening with one of the many techniques developed for the creation of a Collis gastroplasty. Despite this high incidence of radiologic recurrence, and the surgical pursuit of a remedy, it must be reinforced that asymptomatic recurrent hernias, like primary PEH, do not need to be repaired. The risk of incarceration, strangulation, or obstruction is minimal.SCHATZKI’S RINGSchatzki’s ring is a thin submucosal circumferential ring in the lower esophagus at the squamocolumnar junction, often associ-ated with a hiatal hernia. Its significance and pathogenesis are unclear (Fig. 25-41). The ring was first noted by Templeton, but Schatzki and Gary defined it as a distinct entity in 1953. Its prevalence varies from 0.2% to 14% in the general population, depending on the technique of

1	The ring was first noted by Templeton, but Schatzki and Gary defined it as a distinct entity in 1953. Its prevalence varies from 0.2% to 14% in the general population, depending on the technique of diagnosis and the criteria used. Stiennon believed the ring to be a pleat of mucosa formed by infolding of redundant esophageal mucosa due to shortening of the esophagus. Others believe the ring to be congenital, and still others suggest it is an early stricture resulting from inflamma-tion of the esophageal mucosa caused by chronic reflux.Schatzki’s ring is a distinct clinical entity having different symptoms, upper GI function studies, and response to treatment compared with patients with a hiatal hernia, but without a ring. Twenty-four-hour esophageal pH monitoring has shown that patients with a Schatzki’s ring have a lower incidence of reflux than hiatal hernia controls. They also have better LES function. This, together with the presence of a ring, could represent a pro-tective

1	with a Schatzki’s ring have a lower incidence of reflux than hiatal hernia controls. They also have better LES function. This, together with the presence of a ring, could represent a pro-tective mechanism to prevent gastroesophageal reflux.Brunicardi_Ch25_p1009-p1098.indd 104901/03/19 6:04 PM 1050SPECIFIC CONSIDERATIONSPART IISymptoms associated with Schatzki’s ring are brief epi-sodes of dysphagia during hurried ingestion of solid foods. Its treatment has varied from dilation alone to dilation with antire-flux measures, antireflux procedure alone, incision, and even excision of the ring. Little is known about the natural progres-sion of Schatzki’s rings. Using radiologic techniques, Chen and colleagues showed progressive stenosis of rings in 59% of patients, whereas Schatzki found that the rings decreased in diameter in 29% of patients and remained unchanged in the rest.Symptoms in patients with a ring are caused more by the presence of the ring than by gastroesophageal reflux.

1	the rings decreased in diameter in 29% of patients and remained unchanged in the rest.Symptoms in patients with a ring are caused more by the presence of the ring than by gastroesophageal reflux. Most patients with a ring but without proven reflux respond to one dilation, while most patients with proven reflux require repeated dilations. In this regard, the majority of Schatzki’s ring patients without proven reflux have a history of ingestion of drugs known to be damaging to the esophageal mucosa. Bonavina and associates have suggested drug-induced injury as the cause of stenosis in patients with a ring, but without a history of reflux. Because rings also occur in patients with proven reflux, it is likely that gastroesophageal reflux also plays a part. This is supported by the fact that there is less drug ingestion in the history of these patients. Schatzki’s ring is prob-ably an acquired lesion that can lead to stenosis from chemical-induced injury by pill lodgment in the distal

1	there is less drug ingestion in the history of these patients. Schatzki’s ring is prob-ably an acquired lesion that can lead to stenosis from chemical-induced injury by pill lodgment in the distal esophagus, or from reflux-induced injury to the lower esophageal mucosa.The best form of treatment of a symptomatic Schatzki’s ring in patients who do not have reflux consists of esophageal dilation for relief of the obstructive symptoms. In patients with a ring who have proven reflux and a mechanically defective sphincter, an antireflux procedure is necessary to obtain relief and avoid repeated dilation.SCLERODERMAScleroderma is a systemic disease accompanied by esophageal abnormalities in approximately 80% of patients. In most, the disease follows a prolonged course. Renal involvement occurs in a small percentage of patients and signals a poor prognosis. The onset of the disease is usually in the third or fourth decade of life, occurring twice as frequently in women as in men.Small vessel

1	a small percentage of patients and signals a poor prognosis. The onset of the disease is usually in the third or fourth decade of life, occurring twice as frequently in women as in men.Small vessel inflammation appears to be an initiating event, with subsequent perivascular deposition of normal col-lagen, which may lead to vascular compromise. In the GI tract, the predominant feature is smooth muscle atrophy. Whether the atrophy in the esophageal musculature is a primary effect or occurs secondary to a neurogenic disorder is unknown. The results of pharmacologic and hormonal manipulation, with agents that act either indirectly via neural mechanisms or directly on the muscle, suggest that scleroderma is a pri-mary neurogenic disorder. Methacholine, which acts directly on smooth muscle receptors, causes a similar increase in LES pressure in normal controls and in patients with scleroderma. Edrophonium, a cholinesterase inhibitor that enhances the effect of acetylcholine when given to

1	causes a similar increase in LES pressure in normal controls and in patients with scleroderma. Edrophonium, a cholinesterase inhibitor that enhances the effect of acetylcholine when given to patients with sclero-derma, causes an increase in LES pressure that is less marked in these patients than in normal controls, suggesting a neurogenic rather than myogenic etiology. Muscle ischemia due to peri-vascular compression has been suggested as a possible mecha-nism for the motility abnormality in scleroderma. Others have observed that in the early stage of the disease, the manomet-ric abnormalities may be reversed by reserpine, an agent that depletes catecholamines from the adrenergic system. This sug-gests that, in early scleroderma, an adrenergic overactivity may be present that causes a parasympathetic inhibition, supporting SclerodermammHg35 –0Esophagus25 cmEsophagus30 cmEsophagus35 cmSSSS35 –0035 –Figure 25-42. Esophageal motility record in a patient with sclero-derma showing

1	a parasympathetic inhibition, supporting SclerodermammHg35 –0Esophagus25 cmEsophagus30 cmEsophagus35 cmSSSS35 –0035 –Figure 25-42. Esophageal motility record in a patient with sclero-derma showing aperistalsis in the distal two-thirds of the esopha-geal body with peristalsis in the proximal portion. (Reproduced with permission from Waters PF, DeMeester TR: Foregut motor disorders and their surgical management, Med Clin North Am. 1981 Nov;65(6):1235-1268.)a neurogenic mechanism for the disease. In advanced disease manifested by smooth muscle atrophy and collagen deposition, reserpine no longer produces this reversal. Consequently, from a clinical perspective, the patient can be described as having a poor esophageal pump and a poor valve.The diagnosis of scleroderma can be made manometrically by the observation of normal peristalsis in the proximal striated esophagus, with absent peristalsis in the distal smooth muscle por-tion (Fig. 25-42). The LES pressure is progressively weakened as

1	by the observation of normal peristalsis in the proximal striated esophagus, with absent peristalsis in the distal smooth muscle por-tion (Fig. 25-42). The LES pressure is progressively weakened as the disease advances. Because many of the systemic sequelae of the disease may be nondiagnostic, the motility pattern is fre-quently used as a specific diagnostic indicator. Gastroesophageal reflux commonly occurs in patients with scleroderma because they have both hypotensive sphincters and poor esophageal clearance. This combined defect can lead to severe esophagitis and stricture formation. The typical barium swallow shows a dilated, barium-filled esophagus, stomach, and duodenum, or a hiatal hernia with distal esophageal stricture and proximal dilatation (Fig. 25-43).Traditionally, esophageal symptoms have been treated with PPIs, antacids, elevation of the head of the bed, and multiple dilations for strictures, with generally unsatisfac-tory results. The degree of esophagitis is usually

1	symptoms have been treated with PPIs, antacids, elevation of the head of the bed, and multiple dilations for strictures, with generally unsatisfac-tory results. The degree of esophagitis is usually severe and may lead to marked esophageal shortening as well as stric-ture. Scleroderma patients have frequently had numerous dilations before they are referred to the surgeon. The surgi-cal management is somewhat controversial, but the major-ity of opinion suggests that a partial fundoplication (anterior or posterior) performed laparoscopically is the procedure of choice. The need for a partial fundoplication is dictated by the likelihood of severe dysphagia if a total fundoplication is performed in the presence of aperistalsis. Esophageal short-ening may require a Collis gastroplasty in combination with a partial fundoplication. Surgery reduces esophageal acid exposure but does not return it to normal because of the poor Brunicardi_Ch25_p1009-p1098.indd 105001/03/19 6:04 PM

1	in combination with a partial fundoplication. Surgery reduces esophageal acid exposure but does not return it to normal because of the poor Brunicardi_Ch25_p1009-p1098.indd 105001/03/19 6:04 PM 1051ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Figure 25-43. Barium esophagogram of a patient with sclero-derma and stricture. Note the markedly dilated esophagus and retained food material. (Reproduced with permission from Waters PF, DeMeester TR: Foregut motor disorders and their surgical management, Med Clin North Am. 1981 Nov;65(6):1235-1268.)Figure 25-44. The esophagus on the left shows a stacking of rings, demonstrating eosinophilic esophagus. The esophagus on the right is a normal barium swallow.EOSINOPHILIC ESOPHAGITISEosinophilic esophagitis (EE) was first described in 1977, but it has become well known only in the last two decades. The condi-tion is characterized by a constellation of symptoms, endoscopic and radiologic findings, and distinctive pathology. The etiology of

1	it has become well known only in the last two decades. The condi-tion is characterized by a constellation of symptoms, endoscopic and radiologic findings, and distinctive pathology. The etiology of eosinophilic esophagitis is not entirely known but its simi-larities, immunologically, to asthma suggest that it is a form of “allergic esophagitis.”SymptomsThe presentation of eosinophilic esophagitis is chest pain (often postprandial) and dysphagia. Dysphagia may occur with liquids or solids, but solid food dysphagia is most common. Because dysphagia and chest pain are characteristic of GERD, EE is often confused with GERD; however, EE does not respond to proton pump inhibitors. The evaluation of the patient with EE and dysphagia and chest pain with esophagram and endoscopy usually reveals the diagnosis.SignsA barium swallow should be the first test obtained in the patient with dysphagia. EE has a characteristic finding often called the “ringed esophagus” or the “feline esophagus,” as the

1	diagnosis.SignsA barium swallow should be the first test obtained in the patient with dysphagia. EE has a characteristic finding often called the “ringed esophagus” or the “feline esophagus,” as the esophageal rings are felt to look like the stripes on a housecat (Fig. 25-44). The endoscopic appearance of EE is also characteristic, and also appears as a series of rings (Fig. 25-45).PathologyEndoscopic biopsy specimens should be taken when eosin-ophilic esophagus is suspected. To make the diagnosis of EE, the pathologist should see a minimum of 15 eosinophils per high powered field, usually at the base of the epithelium (Fig. 25-46).TreatmentThe treatment of EE is largely symptomatic and includes test-ing for food allergies and elimination of identified items from the diet. Second-line therapy includes inhaled or ingested cor-ticosteroids, as would be used to treat asthma. If dysphagia is not relieved with steroids, it may be necessary to dilate the clearance function of the body of

1	includes inhaled or ingested cor-ticosteroids, as would be used to treat asthma. If dysphagia is not relieved with steroids, it may be necessary to dilate the clearance function of the body of the esophagus. Only 50% of the patients have a good-to-excellent result. If the esopha-gitis is severe, or there has been a previous failed antireflux procedure and the disease is associated with delayed gastric emptying, a gastric resection with Roux-en-Y gastrojejunos-tomy has proved the best option.Brunicardi_Ch25_p1009-p1098.indd 105101/03/19 6:04 PM 1052SPECIFIC CONSIDERATIONSPART IIFigure 25-46. A cluster of eosinophils are visualized in the esophageal epithelium in a patient with EE.Figure 25-45. The endoscopic appearance of eosinophilic esopha-gitis is characteristically a series of stacked mucosal rings.esophagus. Because of the length of esophageal involvement, rigid dilators (Maloney or Savary) are often used. Great care must be exercised, as the inflamed EE is quite friable. The

1	mucosal rings.esophagus. Because of the length of esophageal involvement, rigid dilators (Maloney or Savary) are often used. Great care must be exercised, as the inflamed EE is quite friable. The mucosal tears easily, and esophageal perforation (full thickness laceration) has been reported with EE dilation.MOTILITY DISORDERS OF THE PHARYNX AND ESOPHAGUSClinical ManifestationsDysphagia (i.e., difficulty in swallowing) is the primary symp-tom of esophageal motor disorders. Its perception by the patient is a balance between the severity of the underlying abnormality causing the dysphagia and the adjustment made by the patient in altering eating habits. Consequently, any complaint of dyspha-gia must include an assessment of the patient’s dietary history. It must be known whether the patient experiences pain, chokes, or vomits with eating; whether the patient requires liquids with the meal, is the last to finish, or is forced to interrupt or avoid a social meal; and whether he or she has

1	experiences pain, chokes, or vomits with eating; whether the patient requires liquids with the meal, is the last to finish, or is forced to interrupt or avoid a social meal; and whether he or she has been admitted to the hos-pital for food impaction. These assessments, plus an evaluation of the patient’s nutritional status, help to determine how severe the dysphagia is and judge the need for surgical intervention, rather than more conservative methods of treating dysphagia.Motility Disorders of the Pharynx and Upper Esophagus—Transit DysphagiaDisorders of the pharyngeal phase of swallowing result from a discoordination of the neuromuscular events involved in chew-ing, initiation of swallowing, and propulsion of the material from the oropharynx into the cervical esophagus. They can be categorized into one or a combination of the following abnor-malities: (a) inadequate oropharyngeal bolus transport; (b) inability to pressurize the pharynx; (c) inability to elevate the larynx; (d)

1	categorized into one or a combination of the following abnor-malities: (a) inadequate oropharyngeal bolus transport; (b) inability to pressurize the pharynx; (c) inability to elevate the larynx; (d) discoordination of pharyngeal contraction and cri-copharyngeal relaxation; and (e) decreased compliance of the pharyngoesophageal segment secondary to neuromuscular dis-ease. The latter may result in incomplete relaxation of the crico-pharyngeus and cervical esophagus during swallowing. Taken together, these disorders are termed transit dysphagia by many.Transit dysphagia is usually congenital or results from acquired disease involving the central and peripheral nervous system. This includes cerebrovascular accidents, brain stem tumors, poliomyelitis, multiple sclerosis, Parkinson’s disease, pseudobulbar palsy, peripheral neuropathy, and operative dam-age to the cranial nerves involved in swallowing. Pure muscular diseases such as radiation-induced myopathy, dermatomyositis, myotonic

1	pseudobulbar palsy, peripheral neuropathy, and operative dam-age to the cranial nerves involved in swallowing. Pure muscular diseases such as radiation-induced myopathy, dermatomyositis, myotonic dystrophy, and myasthenia gravis are less common causes. Rarely, extrinsic compression of the cervical esophagus by thyromegaly, lymphadenopathy, or hyperostosis of the cervi-cal spine can cause transit dysphagia.Diagnostic Assessment of the Cricopharyngeal SegmentTransit dysphagia difficult to assess with standard manometric techniques because of the rapidity of the oropharyngeal phase of swallowing, the elevation of the larynx, and the asymmetry of the cricopharyngeus. Videoor cineradiography is currently the Brunicardi_Ch25_p1009-p1098.indd 105201/03/19 6:04 PM 1053ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25ABFigure 25-47. A. Zenker’s diverticulum, initially discovered 15 years ago and left untreated. B. Note its marked enlargement and evidence of laryngeal inlet aspiration on recent

1	HERNIACHAPTER 25ABFigure 25-47. A. Zenker’s diverticulum, initially discovered 15 years ago and left untreated. B. Note its marked enlargement and evidence of laryngeal inlet aspiration on recent esophagogram. (Reproduced with permission from Waters PF, DeMeester TR: Foregut motor disorders and their surgical management, Med Clin North Am. 1981 Nov;65(6):1235-1268.)Time 0Peak pharyngealpressureAtmosphericpressureABBolus pressureinitialMaximum residual(MaxR)contractionB0finalMinimum Residual(MinR)Subatomic pressureFigure 25-48. A. Schematic drawing of a pharyngeal pressure wave indicating the presence of the bolus pressure. B. Schematic drawing of the manometric recording typically seen during crico-pharyngeal sphincter relaxation.most objective test to evaluate oropharyngeal bolus transport, pharyngeal compression, relaxation of the pharyngoesophageal segment, and the dynamics of airway protection during swal-lowing. It readily identifies a diverticulum (Fig. 25-47), stasis of the

1	pharyngeal compression, relaxation of the pharyngoesophageal segment, and the dynamics of airway protection during swal-lowing. It readily identifies a diverticulum (Fig. 25-47), stasis of the contrast medium in the valleculae, a cricopharyngeal bar, and/or narrowing of the pharyngoesophageal segment. These are anatomic manifestations of neuromuscular disease, and they result from the loss of muscle compliance in portions of the pharynx and esophagus composed of skeletal muscle.Careful analysis of videoor cineradiographic studies com-bined with manometry using specially designed catheters can identify the cause of a pharyngoesophageal dysfunction in most sit-uations (Fig. 25-48). Motility studies may demonstrate inadequate pharyngeal pressurization, insufficient or lack of cricopharyngeal relaxation, marked discoordination of pharyngeal pressurization, cricopharyngeal relaxation and cervical esophageal contraction, or a hypopharyngeal bolus pressure suggesting decreased compli-ance of

1	relaxation, marked discoordination of pharyngeal pressurization, cricopharyngeal relaxation and cervical esophageal contraction, or a hypopharyngeal bolus pressure suggesting decreased compli-ance of the skeletal portion of the cervical esophagus.In many patients with cricopharyngeal dysfunction, including those with Zenker’s diverticulum, it has been difficult to consistently demonstrate a motility abnormality or discoor-dination of pharyngoesophageal events. The abnormality most apt to be present is a loss of compliance in the pharyngoesopha-geal segment manifested by an increased bolus pressure. Cook and colleagues have demonstrated an increased resistance to the movement of a bolus through what appears on manometry to be a completely relaxed cricopharyngeal sphincter. Using simulta-neous manometry and videofluoroscopy, they showed that, in these patients, the cricopharyngeus is only partially relaxed; that is, the sphincter is relaxed enough to allow a drop of its pressure to

1	manometry and videofluoroscopy, they showed that, in these patients, the cricopharyngeus is only partially relaxed; that is, the sphincter is relaxed enough to allow a drop of its pressure to esophageal baseline on manometry, but insufficiently relaxed to allow unimpaired passage of the bolus into the esophagus. This incomplete relaxation is due to a loss of compliance of the muscle in the pharyngoesophageal segment, and may be associ-ated with a cricopharyngeal bar or Zenker’s diverticulum. This decreased compliance of the cricopharyngeal sphincter can be recognized on esophageal manometry by a “shoulder” on the pharyngeal pressure wave, the amplitude of which correlates directly with the degree of outflow obstruction (Fig. 25-49). Increasing the diameter of this noncompliant segment reduces the resistance imposed on the passage of a bolus. Consequently, patients with low pharyngeal pressure (i.e., poor piston function of the pharynx), or patients with increased resistance of the

1	reduces the resistance imposed on the passage of a bolus. Consequently, patients with low pharyngeal pressure (i.e., poor piston function of the pharynx), or patients with increased resistance of the pha-ryngocervical esophageal segment from loss of skeletal muscle compliance, are improved by a cricopharyngeal myotomy. This enlarges the pharyngoesophageal segment and reduces outflow resistance. Esophageal muscle biopsy specimens from patients with Zenker’s diverticulum have shown histologic evidence of the restrictive myopathy in the cricophayngeous muscle. These findings correlate well with the observation of a decreased com-pliance of the upper esophagus demonstrated by videoradiog-raphy and the findings on detailed manometric studies of the pharynx and cervical esophagus. They suggest that the diver-ticulum develops as a consequence of the outflow resistance to bolus transport through the noncompliant muscle of the pharyn-goesophageal segment.The requirements for a successful

1	that the diver-ticulum develops as a consequence of the outflow resistance to bolus transport through the noncompliant muscle of the pharyn-goesophageal segment.The requirements for a successful pharyngoesophageal myotomy are (a) adequate oropharyngeal bolus transport; (b) the presence of an intact swallowing reflex; (c) reasonable coordi-nation of pharyngeal pressurization with cricopharyngeal relax-ation; and (d) a cricopharyngeal bar, Zenker’s diverticulum, or a narrowed pharyngoesophageal segment on videoesophagogram and/or the presence of excessive pharyngoesophageal shoulder pressure on motility study.Zenker’s Diverticulum. In the past, the most common recog-nized sign of cricopharyngeal dysfunction was the presence of a Brunicardi_Ch25_p1009-p1098.indd 105301/03/19 6:04 PM 1054SPECIFIC CONSIDERATIONSPART IIZenker’s diverticulum, originally described by Ludlow in 1769. The eponym resulted from Zenker’s classic clinicopathologic descriptions of 34 cases published in 1878.

1	CONSIDERATIONSPART IIZenker’s diverticulum, originally described by Ludlow in 1769. The eponym resulted from Zenker’s classic clinicopathologic descriptions of 34 cases published in 1878. Pharyngoesophageal diverticula have been reported to occur in 1 of 1000 routine barium examinations, and classically occur in elderly, white males. Zenker’s diverticula tend to enlarge progressively with time due to the decreased compliance of the skeletal portion of the cervical esophagus that occurs with aging.Presenting symptoms include dysphagia associated with the spontaneous regurgitation of undigested, bland material, often interrupting eating or drinking. On occasion, the dyspha-gia can be severe enough to cause debilitation and significant weight loss. Chronic aspiration and repetitive respiratory infec-tion are common associated complaints. Once suspected, the diagnosis is established by a barium swallow. Endoscopy is usually difficult in the presence of a cricopharyngeal diverticu-lum, and

1	infec-tion are common associated complaints. Once suspected, the diagnosis is established by a barium swallow. Endoscopy is usually difficult in the presence of a cricopharyngeal diverticu-lum, and potentially dangerous, owing to obstruction of the true esophageal lumen by the diverticulum and the attendant risk of diverticular perforation.Cricopharyngeal Myotomy. The low morbidity and mor-tality associated with cricopharyngeal and upper esophageal myotomy have encouraged a liberal approach toward its use for almost any problem in the oropharyngeal phase of swallowing. This attitude has resulted in an overall success rate in the relief of symptoms of only 64%. When patients are selected for sur-gery using radiographic or motility markers of disease, a much higher proportion will benefit. Two methods of cricopharyngo-esophageal myotomy are in common use, one using traditional surgical approaches, and one using rigid laryngoscopy and a linear cutting stapler.Open Cricopharyngeal

1	Two methods of cricopharyngo-esophageal myotomy are in common use, one using traditional surgical approaches, and one using rigid laryngoscopy and a linear cutting stapler.Open Cricopharyngeal Myotomy, Diverticulopexy, and Diverticulectomy. The myotomy can be performed under local or general anesthesia through an incision along the anterior border of the left sternocleidomastoid muscle. The pharynx and cervi-cal esophagus are exposed by retracting the sternocleidomastoid muscle and carotid sheath laterally and the thyroid, trachea, and larynx medially (Fig. 25-50). When a pharyngoesophageal diverticulum is present, localization of the pharyngoesophageal segment is easy. The diverticulum is carefully freed from the overlying areolar tissue to expose its neck, just below the inferior pharyngeal constrictor and above the cricopharyngeus muscle. It can be difficult to identify the cricopharyngeus muscle in the absence of a diverticulum. A benefit of local anesthesia is that the patient

1	constrictor and above the cricopharyngeus muscle. It can be difficult to identify the cricopharyngeus muscle in the absence of a diverticulum. A benefit of local anesthesia is that the patient can swallow and demonstrate an area of persistent nar-rowing at the pharyngoesophageal junction. Furthermore, before closing the incision, gelatin can be fed to the patient to ascertain whether the symptoms have been relieved, and to inspect the opening of the previously narrowed pharyngoesophageal seg-ment. Under general anesthesia, and in the absence of a diver-ticulum, the placement of a nasogastric tube to the level of the manometrically determined cricopharyngeal sphincter helps in localization of the structures. The myotomy is extended cephalad by dividing 1 to 2 cm of inferior constrictor muscle of the phar-ynx, and caudad by dividing the cricopharyngeal muscle and the cervical esophagus for a length of 4 to 5 cm. The cervical wound is closed only when all oozing of blood has ceased

1	muscle of the phar-ynx, and caudad by dividing the cricopharyngeal muscle and the cervical esophagus for a length of 4 to 5 cm. The cervical wound is closed only when all oozing of blood has ceased because a hematoma after this procedure is common and is often associated with temporary dysphagia while the hematoma absorbs. Oral ali-mentation is started the day after surgery. The patient is usually discharged on the first or second postoperative day.mm Hg40–0102030400HypopharynxCricopharyngeusFigure 25-50. Cross-section of the neck at the level of the thyroid isthmus that shows the sur-gical approach to the hypopharynx and cervical esophagus. (Reproduced with permission from Waters PF, DeMeester TR: Foregut motor dis-orders and their surgical management, Med Clin North Am. 1981 Nov;65(6):1235-1268.)Swallow volume010Pharyngeal shoulderpressure mmHgControlsZenker’s2030405101520200150100UES area mm25005101520Zenker’sControlsFigure 25-49. Pharyngeal shoulder pressures and diameter of the

1	volume010Pharyngeal shoulderpressure mmHgControlsZenker’s2030405101520200150100UES area mm25005101520Zenker’sControlsFigure 25-49. Pharyngeal shoulder pressures and diameter of the pharyngoesophageal segment in controls and patients with Zenker’s diverticulum. UES = upper esophageal sphincter. (Data from Cook IJ, et al. Zenker’s diverticu-lum: evidence for a restrictive cricopharyngeal myopathy. Gastroenterology. 1989;96:A98.)Brunicardi_Ch25_p1009-p1098.indd 105401/03/19 6:04 PM 1055ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Prevertebral fascia MyotomyZenker’sdiverticulumFigure 25-51. Posterior of the anatomy of the pharynx and cervical esophagus showing pharyngoesophageal myotomy and pexing of the diverticulum to the prevertebral fascia.If a diverticulum is present and is large enough to persist after a myotomy, it may be sutured in the inverted position to the prevertebral fascia using a permanent suture (i.e., diverticu-lopexy) (Fig. 25-51). If the diverticulum is

1	large enough to persist after a myotomy, it may be sutured in the inverted position to the prevertebral fascia using a permanent suture (i.e., diverticu-lopexy) (Fig. 25-51). If the diverticulum is excessively large so that it would be redundant if suspended, or if its walls are thick-ened, a diverticulectomy should be performed. This is best per-formed under general anesthesia by placing a Maloney dilator (48F) in the esophagus, after controlling the neck of the diver-ticulum and after myotomy. A linear stapler is placed across the neck of the diverticulum, and the diverticulum is excised distal to the staple line. The security of this staple line and effective-ness of the myotomy may be tested before hospital discharge with a water-soluble contrast esophagogram. Postoperative complications include fistula formation, abscess, hematoma, recurrent nerve paralysis, difficulties in phonation, and Horner’s syndrome. The incidence of the first two can be reduced by per-forming a

1	complications include fistula formation, abscess, hematoma, recurrent nerve paralysis, difficulties in phonation, and Horner’s syndrome. The incidence of the first two can be reduced by per-forming a diverticulopexy rather than diverticulectomy.Endoscopic Cricopharyngotomy. Endoscopic stapled crico-pharyngotomy and diverticulotomy recently has been described. This procedure is most effective for larger diverticula (>2 cm) and may be impossible to perform for the small diverticulum. The procedure uses a specialized “diverticuloscope” with two retractable valves passed into the hypopharynx. The lips of the diverticuloscope are positioned so that one lip lies in the esopha-geal lumen and the other in the diverticular lumen. The valves of the diverticuloscope are retracted appropriately so as to visu-alize the septum interposed between the diverticulum and the esophagus. An endoscopic linear stapler is introduced into the diverticuloscope and positioned against the common septum with the

1	to visu-alize the septum interposed between the diverticulum and the esophagus. An endoscopic linear stapler is introduced into the diverticuloscope and positioned against the common septum with the anvil in the diverticulum and the cartridge in the esoph-ageal lumen. Firing of the stapler divides the common septum between the posterior esophageal and the diverticular wall over a length of 30 mm, placing three rows of staples on each side. More than one stapler application may be needed, depending on the size of the diverticulum (Fig. 25-52). The patient is allowed to resume liquid feeds immediately and is usually discharged the day after surgery. Complications are rare and may include perforation at the apex of the diverticulum and failure to relieve dysphagia resulting from incomplete myotomy. The former complication can usually be treated with antibiotics, but it may, rarely, require neck drainage.Recurrence of a Zenker’s diverticulum may occur with long follow-up and is more

1	myotomy. The former complication can usually be treated with antibiotics, but it may, rarely, require neck drainage.Recurrence of a Zenker’s diverticulum may occur with long follow-up and is more common after diverticulectomy without myotomy, presumably due to persistence of the under-lying loss of compliance of the cervical esophagus when a myot-omy is not performed. After endoscopic cricopharyngotomy Figure 25-52. The technique for transoral cricopharyngotomy and Zenker’s diverticulotomy.lateral residual “pouches” may be seen on radiographs, but they are rarely responsible for residual or recurrent symptoms if the myotomy has been complete.Postoperative motility studies have shown that the peak pharyngeal pressure generated on swallowing is not affected, the resting cricopharyngeal pressure is reduced but not elimi-nated, and the cricopharyngeal sphincter length is shortened. Consequently, after myotomy, there is protection against esoph-agopharyngeal regurgitation.Motility

1	pressure is reduced but not elimi-nated, and the cricopharyngeal sphincter length is shortened. Consequently, after myotomy, there is protection against esoph-agopharyngeal regurgitation.Motility Disorders of the Esophageal Body and Lower Esophageal SphincterDisorders of the esophageal phase of swallowing result from abnormalities in the propulsive pump action of the esophageal body or the relaxation of the LES. These disorders result from either primary esophageal abnormalities, or from generalized neural, muscular, or collagen vascular disease (Table 25-8). The use of standard and high-resolution esophageal manometry techniques has allowed specific primary esophageal motility disorders to be identified out of a pool of nonspecific motil-ity abnormalities. Primary esophageal motor disorders include achalasia, DES, nutcracker esophagus, and the hypertensive LES. The manometric characteristics of these disorders are shown in Table 25-9.The boundaries between the primary esophageal

1	include achalasia, DES, nutcracker esophagus, and the hypertensive LES. The manometric characteristics of these disorders are shown in Table 25-9.The boundaries between the primary esophageal motor disorders are vague, and intermediate types exist, some of which may combine more than one type of motility pattern. These findings indicate that esophageal motility disorders should be looked at as a spectrum of abnormalities that reflects various stages of destruction of esophageal motor function.Achalasia. The best known and best understood primary motil-ity disorder of the esophagus is achalasia, with an incidence of six Brunicardi_Ch25_p1009-p1098.indd 105501/03/19 6:04 PM 1056SPECIFIC CONSIDERATIONSPART IITable 25-9Manometric characteristics of the primary esophageal motility disordersAchalasiaIncomplete lower esophageal sphincter (LES) relaxation (<75% relaxation)Aperistalsis in the esophageal bodyElevated LES pressure ≤26 mmHgIncreased intraesophageal baseline pressures relative

1	lower esophageal sphincter (LES) relaxation (<75% relaxation)Aperistalsis in the esophageal bodyElevated LES pressure ≤26 mmHgIncreased intraesophageal baseline pressures relative to gastric baselineDiffuse esophageal spasm (DES)Simultaneous (nonperistaltic contractions) (>20% of wet swallows)Repetitive and multipeaked contractionsSpontaneous contractionsIntermittent normal peristalsisContractions may be of increased amplitude and durationNutcracker esophagusMean peristaltic amplitude (10 wet swallows) in distal esophagus ≥180 mmHgIncreased mean duration of contractions (>7.0 s)Normal peristaltic sequenceHypertensive lower esophageal sphincterElevated LES pressure (≥26 mmHg)Normal LES relaxationNormal peristalsis in the esophageal bodyIneffective esophageal motility disordersDecreased or absent amplitude of esophageal peristalsis (<30 mmHg)Increased number of nontransmitted contractionsReproduced with permission from Zuidema GD, Orringer MB: Shackelford’s Surgery of the Alimentary

1	absent amplitude of esophageal peristalsis (<30 mmHg)Increased number of nontransmitted contractionsReproduced with permission from Zuidema GD, Orringer MB: Shackelford’s Surgery of the Alimentary Tract, 3rd ed. Vol 1. Philadelphia, PA: Elsevier/Saunders; 1991.Simultaneous esophageal waves develop as a result of the increased resistance to esophageal emptying caused by the nonre-laxing LES. This conclusion is supported by experimental studies in which a band placed loosely around the GEJ in experimental models did not change sphincter pressures but resulted in impaired relaxation of the LES and outflow resistance. This led to a mark-edly increased frequency of simultaneous waveforms and a decrease in contraction amplitude. The changes were associated with radiographic dilation of the esophagus and were reversible after removal of the band. Observations in patients with pseudo-achalasia due to tumor infiltration, a tight stricture in the distal esophagus, or an antireflux procedure

1	and were reversible after removal of the band. Observations in patients with pseudo-achalasia due to tumor infiltration, a tight stricture in the distal esophagus, or an antireflux procedure that is too tight also provide evidence that dysfunction of the esophageal body can be caused by the increased outflow obstruction of a nonrelaxing LES. The observation that esophageal peristalsis can return in patients with classic achalasia following dilation or myotomy provides further support that achalasia is a primary disease of the LES.The pathogenesis of achalasia is presumed to be a neuro-genic degeneration, which is either idiopathic or due to infec-tion. In experimental animals, the disease has been reproduced by destruction of the nucleus ambiguus and the dorsal motor nucleus of the vagus nerve. In patients with the disease, degenerative changes have been shown in the vagus nerve and in the ganglia in the myenteric plexus of the esophagus itself. This degeneration results in

1	the vagus nerve. In patients with the disease, degenerative changes have been shown in the vagus nerve and in the ganglia in the myenteric plexus of the esophagus itself. This degeneration results in hypertension of the LES, a failure of the sphincter to relax on swallowing, elevation of intraluminal esophageal pres-sure, esophageal dilatation, and a subsequent loss of progressive peristalsis in the body of the esophagus. The esophageal dilatation results from the combination of a nonrelaxing sphincter, which causes a functional retention of ingested material in the esopha-gus, and elevation of intraluminal pressure from repetitive pha-ryngeal air swallowing (Fig. 25-53). With time, the functional disorder results in anatomic alterations seen on radiographic stud-ies, such as a dilated esophagus with a tapering, “bird’s beak”-like narrowing of the distal end (Fig. 25-54). There is usually an air-fluid level in the esophagus from the retained food and saliva, the height of which

1	esophagus with a tapering, “bird’s beak”-like narrowing of the distal end (Fig. 25-54). There is usually an air-fluid level in the esophagus from the retained food and saliva, the height of which reflects the degree of resistance imposed by the nonrelaxing sphincter. As the disease progresses, the esophagus becomes massively dilated and tortuous.A subgroup of patients with otherwise typical features of classic achalasia has simultaneous contractions of their esopha-geal body that can be of high amplitude. This manometric pattern has been termed vigorous achalasia, and chest pain episodes are a common finding in these patients. Since the development of high resolution esophageal manometry technology, the term vigorous achalasia has been replaced with Chicago type 3 achalasia. Dif-ferentiation of type 3 achalasia from DES can be difficult. In both diseases, videoradiographic examination may show a cork-screw deformity of the esophagus and diverticulum formation.Diffuse and Segmental

1	of type 3 achalasia from DES can be difficult. In both diseases, videoradiographic examination may show a cork-screw deformity of the esophagus and diverticulum formation.Diffuse and Segmental Esophageal Spasm. DES is charac-terized by substernal chest pain and/or dysphagia. DES differs from classic achalasia in that it is primarily a disease of the esophageal body, produces a lesser degree of dysphagia, causes more chest pain, and has less effect on the patient’s general con-dition. Nonetheless, it is impossible to differentiate achalasia from DES on the basis of symptoms alone. Esophagogram and esophageal manometry are required to distinguish these two entities. True symptomatic DES is a rare condition, occurring about five times less frequently than achalasia.The causation and neuromuscular pathophysiology of DES are unclear. The basic motor abnormality is rapid wave progression down the esophagus secondary to an abnormality in Table 25-8Esophageal motility disordersPrimary

1	pathophysiology of DES are unclear. The basic motor abnormality is rapid wave progression down the esophagus secondary to an abnormality in Table 25-8Esophageal motility disordersPrimary esophageal motility disordersAchalasia, “vigorous” achalasiaDiffuse and segmental esophageal spasmNutcracker esophagusHypertensive lower esophageal sphincterNonspecific esophageal motility disordersSecondary esophageal motility disordersCollagen vascular diseases: progressive systemic sclerosis, polymyositis and dermatomyositis, mixed connective tissue disease, systemic lupus erythematosus, etc.Chronic idiopathic intestinal pseudoobstructionNeuromuscular diseasesEndocrine and metastatic disordersper 100,000 population per year. Although complete absence of peristalsis in the esophageal body has been proposed as the major abnormality, present evidence indicates achalasia is a primary disorder of the LES. This is based on 24-hour outpatient esophageal motility monitoring, which shows that, even in

1	proposed as the major abnormality, present evidence indicates achalasia is a primary disorder of the LES. This is based on 24-hour outpatient esophageal motility monitoring, which shows that, even in advanced disease, up to 5% of contractions can be peristaltic. 5Brunicardi_Ch25_p1009-p1098.indd 105601/03/19 6:04 PM 1057ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25A34140120100806050403020100–10–2056*60453525159–5–15–25–3550403020100–10–206040200–20100 mmHg10 mins10 secs100 mmHgB3*4*1501401201008060402001501401201008060402005*1501401201008060402006*1451251051008565455–15MealFigure 25-53. Pressurization of esophagus: ambulatory motility tracing of a patient with achalasia. A. Before esophageal myotomy. B. After esophageal myotomy. The tracings have been compressed to exaggerate the motility spikes and baseline elevations. Note the rise in esophageal baseline pressure during a meal represented by the rise off the baseline to the left of panel A. No such rise occurs postmyotomy

1	motility spikes and baseline elevations. Note the rise in esophageal baseline pressure during a meal represented by the rise off the baseline to the left of panel A. No such rise occurs postmyotomy (B).Figure 25-54. Barium esophagogram showing a markedly dilated esophagus and characteristic “bird’s beak” in achalasia. (Repro-duced with permission from Waters PF, DeMeester TR: Foregut motor disorders and their surgical management, Med Clin North Am. 1981 Nov;65(6):1235-1268.)the latency gradient. Hypertrophy of the muscular layer of the esophageal wall and degeneration of the esophageal branches of the vagus nerve have been observed in this disease, although these are not constant findings. Manometric abnormalities in DES may be present over the total length of the esophageal body but usually are confined to the distal two-thirds. In segmental esophageal spasm, the manometric abnormalities are confined to a short segment of the esophagus.The classic manometric findings in these

1	usually are confined to the distal two-thirds. In segmental esophageal spasm, the manometric abnormalities are confined to a short segment of the esophagus.The classic manometric findings in these patients are characterized by the frequent occurrence of simultaneous wave-forms and multipeaked esophageal contractions, which may be of abnormally high amplitude or long duration. Key to the diag-nosis of DES is that there remain some peristaltic waveforms in excess of those seen in achalasia. A criterion of 30% or more peristaltic waveforms out of 10 wet swallows has been used to differentiate DES from vigorous achalasia. However, this figure is arbitrary and often debated.The LES in patients with DES usually shows a normal resting pressure and relaxation on swallowing. A hypertensive sphincter with poor relaxation may also be present. In patients with advanced disease, the radiographic appearance of tertiary contractions appears helical and has been termed corkscrew esophagus or

1	sphincter with poor relaxation may also be present. In patients with advanced disease, the radiographic appearance of tertiary contractions appears helical and has been termed corkscrew esophagus or pseudodiverticulosis (Fig. 25-55). Patients with segmental or diffuse esophageal spasm can compartmentalize the esophagus and develop an epiphrenic or midesophageal diverticulum between two areas of high pressure occurring simultaneously (Fig. 25-56).Nutcracker Esophagus. The disorder, termed nutcracker or supersqueezeresophagus, was recognized in the late 1970s. Other terms used to describe this entity are hypertensive peri-stalsis or high-amplitude peristaltic contractions. It is the most common of the primary esophageal motility disorders. By definition the so-called nutcracker esophagus is a manomet-ric abnormality in patients who are characterized by peristal-tic esophageal contractions with peak amplitudes greater than two SDs above the normal values in individual laboratories.

1	is a manomet-ric abnormality in patients who are characterized by peristal-tic esophageal contractions with peak amplitudes greater than two SDs above the normal values in individual laboratories. Contraction amplitudes in these patients can easily be above 400 mmHg. At the lower end of peak pressure, it is unclear whether nutcracker esophagus causes any symptoms. In fact, chest pain symptoms in nutcracker esophagus patients may be related to GERD rather than intraluminal hypertension. Treatment in these patients should be aimed at the treatment of GERD. At the high end (peak pressures >300 mmHg) chest pain may be the result of the nutcracker physiology, as treatment directed at reducing intraluminal pressure is more effective than when used for those with lower peak pressures.Hypertensive Lower Esophageal Sphincter. Hyperten-sive lower esophageal sphincter (LES) in patients with chest pain or dysphagia was first described as a separate entity by Code and associates. This disorder is

1	Lower Esophageal Sphincter. Hyperten-sive lower esophageal sphincter (LES) in patients with chest pain or dysphagia was first described as a separate entity by Code and associates. This disorder is characterized by an ele-vated basal pressure of the LES with normal relaxation and Brunicardi_Ch25_p1009-p1098.indd 105701/03/19 6:04 PM 1058SPECIFIC CONSIDERATIONSPART IIFigure 25-56. Barium esophagogram showing a high epiphrenic diverticulum in a patient with diffuse esophageal spasm. (Repro-duced with permission from Castell DO: The Esophagus. Boston, MA: Little, Brown; 1992.)normal propulsion in the esophageal body. About one-half of these patients, however, have associated motility disorders of the esophageal body, particularly hypertensive peristalsis and simultaneous waveforms. In the remainder, the disorder exists as an isolated abnormality. Dysphagia in these patients may be caused by a lack of compliance of the sphincter, even in its relaxed state. Myotomy of the LES may be

1	the remainder, the disorder exists as an isolated abnormality. Dysphagia in these patients may be caused by a lack of compliance of the sphincter, even in its relaxed state. Myotomy of the LES may be indicated in patients not responding to medical therapy or dilation. When the symp-tom contribution of the hypertensive sphincter is in doubt, it is possible to inject the LES with botulinum toxin, endoscopically. If symptoms are relieved (temporarily) with this technique, then it is likely that myotomy will provide more permanent benefit.Secondary Esophageal Motility Disorders. Connective tissue disease, particularly scleroderma and the CREST syn-drome, exhibits severe esophageal motility disorders. Addi-tionally, patients treated as infants for esophageal atresia will often develop secondary motility disorders manifest later in life. Symptoms of these disorders are heartburn and dysphagia. The latter may be a result of a peptic stricture rather than the esophageal dysmotility. An

1	motility disorders manifest later in life. Symptoms of these disorders are heartburn and dysphagia. The latter may be a result of a peptic stricture rather than the esophageal dysmotility. An esophageal motility study will usu-ally show severely reduced or absent peristalsis with severely reduced or absent LES pressure. The role of antireflux surgery under these conditions is controversial but, if performed, should be limited to partial fundoplication, as full (Nissen) fundoplica-tion may result in severe dysphagia.Nonspecific Esophageal Motor Disorders and Ineffective Esophageal Motility. Many patients complaining of dys-phagia or chest pain of noncardiac origin demonstrate a vari-ety of wave patterns and contraction amplitudes on esophageal manometry that are clearly out of the normal range, but do not meet the criteria of a primary esophageal motility disor-der. Esophageal motility in these patients frequently shows an increased number of multipeaked or repetitive contractions,

1	range, but do not meet the criteria of a primary esophageal motility disor-der. Esophageal motility in these patients frequently shows an increased number of multipeaked or repetitive contractions, contractions of prolonged duration, nontransmitted contrac-tions, an interruption of a peristaltic wave at various levels of the esophagus, or contractions of low amplitude. These motility abnormalities have been termed nonspecific esophageal motility disorders. Their significance in the causation of chest pain or dysphagia is still unclear. Surgery plays no role in the treatment of these disorders unless there is an associated diverticulum.A clear distinction between primary esophageal motility disorders and nonspecific esophageal motility disorders is often not possible. Patients diagnosed as having nonspecific esophageal motility abnormalities on repeated studies will occasionally show abnormalities consistent with nutcracker esophagus. Similarly, progression from a nonspecific

1	as having nonspecific esophageal motility abnormalities on repeated studies will occasionally show abnormalities consistent with nutcracker esophagus. Similarly, progression from a nonspecific esophageal motility disorder to classic DES has been demonstrated. Therefore, the finding of a nonspecific esophageal motility disorder may represent only a manometric marker of an intermittent, more severe esophageal motor abnormality. Combined ambulatory 24-hour esophageal pH and motility monitoring has shown that an increased esopha-geal exposure to gastric juice is common in patients diagnosed as having a nonspecific esophageal motility disorder. In some situ-ations, the motor abnormalities may be induced by the irritation of refluxed gastric juice; in other situations, it may be a primary event unrelated to the presence of reflux. High-amplitude peristal-sis (nutcracker esophagus) and low-amplitude peristalsis (ineffec-tive esophageal motility) are frequently associated with

1	a primary event unrelated to the presence of reflux. High-amplitude peristal-sis (nutcracker esophagus) and low-amplitude peristalsis (ineffec-tive esophageal motility) are frequently associated with GERD.Diverticula of the Esophageal Body. Diverticula of the esophagus may be characterized by their location in the esoph-agus (proximal, mid-, or distal esophagus), or by the nature of Figure 25-55. Barium esophagogram of patient with diffuse spasm showing the corkscrew deformity.Brunicardi_Ch25_p1009-p1098.indd 105801/03/19 6:04 PM 1059ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Figure 25-57. Barium esophagogram showing a midesophageal diverticulum. Despite the anatomic distortion, the patient was asymptomatic. (Reproduced with permission from Waters PF, DeMeester TR: Foregut motor disorders and their surgical man-agement, Med Clin North Am. 1981 Nov;65(6):1235-1268.)InflamednodesTraction diverticulumFigure 25-58. Illustration of the pathophysiology of midesopha-geal diverticulum

1	and their surgical man-agement, Med Clin North Am. 1981 Nov;65(6):1235-1268.)InflamednodesTraction diverticulumFigure 25-58. Illustration of the pathophysiology of midesopha-geal diverticulum showing traction on the esophageal wall from adhesions to inflamed subcarinal lymph nodes.concomitant pathology. Diverticula associated with motor dis-orders are termed pulsion diverticula and those associated with inflammatory conditions are termed traction diverticula. Pulsion diverticula occur most commonly with nonspecific motility disor-ders, but they can occur with all of the primary motility disorders. In the latter situation, the motility disorder is usually diagnosed before the development of the diverticulum. When associated with achalasia, the development of a diverticulum may temporar-ily alleviate the symptom of dysphagia by becoming a receptacle for ingested food and substitute the symptom of dysphagia for postprandial pain and regurgitation of undigested food. If a motil-ity

1	alleviate the symptom of dysphagia by becoming a receptacle for ingested food and substitute the symptom of dysphagia for postprandial pain and regurgitation of undigested food. If a motil-ity abnormality of the esophageal body or LES cannot be identi-fied, a traction or congenital cause for the diverticulum should be considered.Because development in radiology preceded develop-ment in motility monitoring, diverticula of the esophagus were considered historically to be a primary abnormality, the cause, rather than the consequence, of motility disorders. Conse-quently, earlier texts focused on them as specific entities based upon their location.Epiphrenic diverticula arise from the terminal third of the thoracic esophagus and are usually found adjacent to the diaphragm. They have been associated with distal esophageal muscular hypertrophy, esophageal motility abnormalities, and increased luminal pressure. They are “pulsion” diverticula, and they are associated with diffuse spasm,

1	associated with distal esophageal muscular hypertrophy, esophageal motility abnormalities, and increased luminal pressure. They are “pulsion” diverticula, and they are associated with diffuse spasm, achalasia, or nonspecific motor abnormalities in the body of the esophagus.Whether the diverticulum should be surgically resected or suspended depends on its size and proximity to the vertebral body. When diverticula are associated with esophageal motility disorders, esophageal myotomy from the proximal extent of the diverticulum to the stomach should be combined with diverticu-lectomy. If diverticulectomy alone is performed, one can expect a high incidence of suture line rupture due to the same intralu-minal pressure that initially gave rise to the diverticulum. If the diverticulum is suspended to the prevertebral fascia of the tho-racic vertebra, a myotomy is begun at the neck of the diverticu-lum and extended across the LES. If the diverticulum is excised by dividing the neck, the

1	to the prevertebral fascia of the tho-racic vertebra, a myotomy is begun at the neck of the diverticu-lum and extended across the LES. If the diverticulum is excised by dividing the neck, the muscle is closed over the excision site, and a myotomy is performed on the opposite esophageal wall, starting just above the level of the diverticulum or at the proximal extent of the spastic segment of the esophagus if high resolution motility is used. If complete, the myotomy will cross the LES, reducing distal esophageal peak pressure, and it will increase the likelihood that dysphagia will be replaced with GERD symp-toms. Increasingly, partial fundoplication (anterior or posterior) is performed after LES myotomy to decrease the frequency of disabling GERD developing after myotomy and diverticulec-tomy. When a large diverticulum is associated with a hiatal her-nia, then hiatal hernia repair is added. All these procedures may be performed with traditional or minimally invasive

1	and diverticulec-tomy. When a large diverticulum is associated with a hiatal her-nia, then hiatal hernia repair is added. All these procedures may be performed with traditional or minimally invasive techniques.Midesophageal or traction diverticula were first described in the 19th century (Fig. 25-57). At that time, they were fre-quently noted in patients who had mediastinal LN involve-ment with tuberculosis. It was theorized that adhesions formed between the inflamed mediastinal nodes and the esophagus. By contraction, the adhesions exerted traction on the esophageal wall and led to a localized diverticulum (Fig. 25-58). This theory was based on the findings of early dissections, where adhesions between diverticula and LNs were commonly found. Other con-ditions associated with mediastinal lymphadenopathy, such as pulmonary fungal infections (e.g., aspergillosis), lymphoma, or sarcoid, may create traction esophageal diverticula after success-ful treatment. Rarely, when no underlying

1	lymphadenopathy, such as pulmonary fungal infections (e.g., aspergillosis), lymphoma, or sarcoid, may create traction esophageal diverticula after success-ful treatment. Rarely, when no underlying inflammatory pathol-ogy is identified, a motility disorder may be identified.Most midesophageal diverticula are asymptomatic and incidentally discovered during investigation for nonesophageal complaints. In such patients, the radiologic abnormality may Brunicardi_Ch25_p1009-p1098.indd 105901/03/19 6:04 PM 1060SPECIFIC CONSIDERATIONSPART II100%80%60%40%20%Normal volunteersPat, no dysphagiaPat, dysphagia0%Figure 25-59. Prevalence of effective contractions (i.e., peristaltic contractions with an amplitude >30 mmHg) during meal periods in individual normal volunteers, patients (Pat) without dysphagia, and patients with nonobstructive dysphagia.100%% Symptomatic10 cm5 cm0 cm80%60%40%20%0%Pre Rx17NEso. diameter% Retention0–24mo1725–48mo1649–72mo1473–120mo12Figure 25-60. Esophageal (Eso.)

1	dysphagia, and patients with nonobstructive dysphagia.100%% Symptomatic10 cm5 cm0 cm80%60%40%20%0%Pre Rx17NEso. diameter% Retention0–24mo1725–48mo1649–72mo1473–120mo12Figure 25-60. Esophageal (Eso.) diameter, dysphagia, and esoph-ageal retention in patients with achalasia treated with myotomy and Nissen fundoplication, 10 years after treatment (Rx). (Data from Topart P, Deschamps C, Taillefer R, et al: Long-term effect of total fundoplication on the myotomized esophagus, Ann Thorac Surg. 1992 Dec;54(6):1046-1051.)be ignored. Patients with symptoms of dysphagia, regurgita-tion, chest pain, or aspiration, in whom a diverticulum is dis-covered, should be thoroughly investigated for an esophageal motor abnormality. Occasionally, a patient will present with a bronchoesophageal fistula manifested by a chronic cough on ingestion of meals. The diverticulum in such patients is most likely to have an inflammatory etiology.The indication for surgical intervention is dictated by the degree of

1	by a chronic cough on ingestion of meals. The diverticulum in such patients is most likely to have an inflammatory etiology.The indication for surgical intervention is dictated by the degree of symptomatic disability. Usually, midesophageal diverticula can be suspended due to their proximity to the spine. If a motor abnormality is documented, a myotomy should be performed as described for an epiphrenic diverticulum.OPERATIONS FOR ESOPHAGEAL MOTOR DISORDERS AND DIVERTICULALong Esophageal Myotomy for Motor Disorders of the Esophageal BodyA long esophageal myotomy is indicated for dysphagia caused by any motor disorder characterized by segmental or general-ized simultaneous waveforms in a patient whose symptoms are not relieved by medical therapy. Such disorders include diffuse and segmental esophageal spasm, vigorous or type 3 achalasia, and nonspecific motility disorders associated with a midor epiphrenic esophageal diverticulum. However, the decision to operate must be made by a

1	esophageal spasm, vigorous or type 3 achalasia, and nonspecific motility disorders associated with a midor epiphrenic esophageal diverticulum. However, the decision to operate must be made by a balanced evaluation of the patient’s symptoms, diet, lifestyle adjustments, and nutritional status, with the most important factor being the possibility of improv-ing the patient’s swallowing disability. The symptom of chest pain alone is not an indication for a surgical procedure.The identification of patients with symptoms of dyspha-gia and chest pain who might benefit from a surgical myotomy is difficult. Ambulatory motility studies have shown that when the prevalence of “effective contractions” (i.e., peristaltic waveforms consisting of contractions with an amplitude above 30 mmHg) drops below 50% during meals, the patient is likely to experience dysphagia (Fig. 25-59). This would suggest that relief from the symptom can be expected with an improvement of esophageal contraction amplitude

1	50% during meals, the patient is likely to experience dysphagia (Fig. 25-59). This would suggest that relief from the symptom can be expected with an improvement of esophageal contraction amplitude or amelioration of non-peristaltic waveforms. Prokinetic agents may increase esopha-geal contraction amplitude, but they do not alter the prevalence of simultaneous waveforms. Patients in whom the efficacy of esophageal propulsion is severely compromised because of a high prevalence of simultaneous waveforms usually receive little benefit from medical therapy. In these patients, a surgi-cal myotomy of the esophageal body can improve the patients’ dysphagia, provided the loss of contraction amplitude in the remaining peristaltic waveforms, caused by the myotomy, has less effect on swallowing function than the presence of the excessive simultaneous contractions. This situation is reached when the prevalence of effective waveforms during meals drops below 30% (i.e., 70% of esophageal waveforms

1	than the presence of the excessive simultaneous contractions. This situation is reached when the prevalence of effective waveforms during meals drops below 30% (i.e., 70% of esophageal waveforms are ineffective).In patients selected for surgery, preoperative high-resolution manometry is essential to determine the proximal extent of the esophageal myotomy. Most surgeons extend the myotomy distally across the LES to reduce outflow resistance. Consequently, some form of antireflux protection is needed to avoid gastroesophageal reflux if there has been extensive dissection of the cardia. In this situation, most authors prefer a partial, rather than a full, fundoplication, in order not to add back-resistance that will further interfere with the ability of the myotomized esophagus to empty (Fig. 25-60). If the symptoms of reflux are present preoperatively, 24-hour pH monitoring is required to confirm its presence.The procedure may be performed either open or via thoracoscopy. The open

1	(Fig. 25-60). If the symptoms of reflux are present preoperatively, 24-hour pH monitoring is required to confirm its presence.The procedure may be performed either open or via thoracoscopy. The open technique is performed through a left thoracotomy in the sixth intercostal space (Fig. 25-61). An incision is made in the posterior mediastinal pleura over the esophagus, and the left lateral wall of the esophagus is exposed. The esophagus is not circumferentially dissected unless necessary. A 2-cm incision is made into the abdomen through the parietal peritoneum at the midportion of the left crus. A tongue of gastric fundus is pulled into the chest. This exposes the GEJ and its associated fat pad. The latter is excised to give a clear view of the junction. A myotomy is performed through all muscle layers, extending distally over the stomach 1 to 2 cm below the GEJ, and proximally on the esophagus over the distance of the manometric abnormality. The muscle layer is dissected from the

1	all muscle layers, extending distally over the stomach 1 to 2 cm below the GEJ, and proximally on the esophagus over the distance of the manometric abnormality. The muscle layer is dissected from the mucosa laterally for a distance of 1 cm. Care is taken to divide all minute muscle bands, particularly in the area of the GEJ. The gastric fundic tongue is sutured to the margins of the myotomy over a distance of 3 to 4 cm and replaced into the abdomen. This maintains separation of the muscle and acts as a partial fundoplication to prevent reflux.Brunicardi_Ch25_p1009-p1098.indd 106001/03/19 6:04 PM 1061ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Figure 25-61. Technique of long myotomy: A. Exposure of the lower esophagus through the left sixth intercostal space and incision of the mediastinal pleura in preparation for surgical myotomy. B. Location of a 2-cm incision made through the phrenoesophageal mem-brane into the abdomen along the midlateral border of the left crus. C. Retraction

1	pleura in preparation for surgical myotomy. B. Location of a 2-cm incision made through the phrenoesophageal mem-brane into the abdomen along the midlateral border of the left crus. C. Retraction of tongue of gastric fundus into the chest through the previously made incision. D. Removal of the gastroesophageal fat pad to expose the gastroesophageal junction. E. A myotomy down to the mucosa is started on the esophageal body. F. Completed myotomy extending over the stomach for 1 cm. G. Reconstruction of the cardia after a myotomy, illustrating the position of the sutures used to stitch the gastric fundic flap to the margins of the myotomy. H. Reconstruction of the cardia after a myotomy, illustrating the intra-abdominal position of the gastric tongue covering the distal 4 cm of the myotomy.Brunicardi_Ch25_p1009-p1098.indd 106101/03/19 6:04 PM 1062SPECIFIC CONSIDERATIONSPART IIIf an epiphrenic diverticulum is present, it is excised by dividing the neck with a stapler sized for the

1	106101/03/19 6:04 PM 1062SPECIFIC CONSIDERATIONSPART IIIf an epiphrenic diverticulum is present, it is excised by dividing the neck with a stapler sized for the thickness of the diverticulum (2.0to 4.8-mm staple leg length) followed by a closure of the muscle over the staple line, when possible. The myotomy is then performed on the opposite esophageal wall. If a midesophageal diverticulum is present, the myotomy is made so that it includes the muscle around the neck, and the diver-ticulum is suspended by attaching it to the paravertebral fascia of the thoracic vertebra above the level of the diverticular neck. Before performing any operation for an esophageal diverticu-lum, it is wise to endoscope the patient to wash all food and other debris from the diverticulum.The results of myotomy for motor disorders of the esopha-geal body have improved in parallel with the improved preop-erative diagnosis afforded by manometry. Previous published series report between 40% and 92%

1	of myotomy for motor disorders of the esopha-geal body have improved in parallel with the improved preop-erative diagnosis afforded by manometry. Previous published series report between 40% and 92% improvement of symptoms, but interpretation is difficult due to the small number of patients involved and the varying criteria for diagnosis of the primary motor abnormality. When myotomy is accurately done, 93% of the patients have effective palliation of dysphagia after a mean follow-up of 5 years, and 89% would have the procedure again, if it was necessary. Most patients gain or maintain rather than lose weight after the operation. Postoperative motility studies show that the myotomy reduces the amplitude of esophageal contractions to near zero and eliminates simultaneous peristaltic waves. If the benefit of obliterating the simultaneous waves exceeds the adverse effect on bolus propulsion caused by the loss of peristaltic waveforms, the patient’s dysphagia is likely to be improved by

1	If the benefit of obliterating the simultaneous waves exceeds the adverse effect on bolus propulsion caused by the loss of peristaltic waveforms, the patient’s dysphagia is likely to be improved by the procedure. If not, the patient is likely to continue to complain of dysphagia and to have little improvement as a result of the operation.The thoracoscopic technique may be performed through the left or right chest. There has been little experience gained with doing adequate operations (as described previously with the open exposure) through left thoracoscopy, so most surgeons will combine a right thoracoscopic long myotomy with an abdominal approach for Heller myotomy and partial fundopli-cation. These two procedures may be done at the same setting, by double positioning the patient, or they may be done at two operations. If this is the case, it is best to do the abdominal com-ponent first, as the esophageal outflow obstruction is the source of most of the symptoms. Performing

1	or they may be done at two operations. If this is the case, it is best to do the abdominal com-ponent first, as the esophageal outflow obstruction is the source of most of the symptoms. Performing abdominal myotomy (and diverticulectomy, if present) may be all that is required.Figure 25-61. (Continued )A new procedure, peroral endoscopic myotomy (POEM) allows a long myotomy to be performed from the lumen of the esophagus with an endoscope. This procedure is attractive for, at a minimum, those with type 3 achalasia (vigorous achalasia), where it is necessary to divide esopha-gogastric circular muscle on both sides of the diaphragm to the extent that might not be possible with laparoscopy or thoracoscopy alone. The POEM procedure is started by open-ing the esophageal mucosa several centimeters above the spastic segment with a needle–knife electrosurgery device passed through an endoscope. A long submucosal plane is developed with the endoscope, down to and below the LES. The circular

1	above the spastic segment with a needle–knife electrosurgery device passed through an endoscope. A long submucosal plane is developed with the endoscope, down to and below the LES. The circular muscle of the LES and the esophagus is divided with endoscopic electrosurgery all the way back until normal (nonspastic) esophagus is reached. The submucosal entry site in the esophagus is then closed with endoscopic clips. While the results of POEM are still accumulating, the procedure is attractive because it is extremely minimally invasive and can be done on an outpatient basis.Epiphrenic diverticula cannot be treated with POEM and are most frequently addressed with laparoscopic access, in combination with a laparoscopic division of the LES (Heller myotomy) (Fig. 25-62). If the diverticulum can be completely mobilized through the hiatus, it may be safely excised from below. The neck of the diverticulum is transected with a GIA stapler after passage of a 48F dilator. Not infrequently, the

1	be completely mobilized through the hiatus, it may be safely excised from below. The neck of the diverticulum is transected with a GIA stapler after passage of a 48F dilator. Not infrequently, the diverticulum is sufficiently large that access to the neck of the diverticulum across the hiatus is quite difficult. Addi-tionally, the inflammatory reaction to the diverticulum may further make the transhiatal dissection difficult. Under these circumstances, it is safer to perform the diverticulectomy through a right thoracoscopic approach either at the time of the initial procedure or at a later date, depending upon the frailty of the patient. Following diverticulectomy, it is critical that the esophageal staple line be treated with a great deal of care. Closure of the muscle over the staple line is preferable. Additionally, the patient is kept NPO or on clear liquids for 5 to 7 days, and a contrast study is obtained before advancing to a full liquid or “mushy food” diet. Solid foods are

1	is preferable. Additionally, the patient is kept NPO or on clear liquids for 5 to 7 days, and a contrast study is obtained before advancing to a full liquid or “mushy food” diet. Solid foods are withheld for 2 weeks to decrease the likelihood of staple line leak. But-tressing or sealing the staple line with fibrin glue is also an attractive option.Brunicardi_Ch25_p1009-p1098.indd 106201/03/19 6:04 PM 1063ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Figure 25-62. A. Epiphrenic diverticula are situated above the lower esophageal sphincter on right side of esophagus. B. Stapler amputates neck of diverticulum. C. Muscle reapproximated over staple line, and Heller myotomy is performed.Myotomy of the Lower Esophageal Sphincter (Heller Myotomy)Second only to reflux disease, achalasia is the most common functional disorder of the esophagus to require surgical intervention. The goal of treatment is to relieve the functional outflow obstruction secondary to the loss of relaxation and

1	the most common functional disorder of the esophagus to require surgical intervention. The goal of treatment is to relieve the functional outflow obstruction secondary to the loss of relaxation and compliance of the LES. This requires disrupting the LES muscle. When performed adequately (i.e., reducing sphincter pressure to <10 mmHg), and done early in the course of disease, LES myotomy results in symptomatic improvement with the occasional return of esophageal peristalsis. Reduction in LES resistance can be accomplished intraluminally by hydrostatic balloon dilation, which ruptures the sphincter muscle, by botulinum toxin injection, or by a surgical myotomy that cuts the sphincter. The difference between these three methods appears to be the greater likelihood of reducing sphincter pressure to <10 mmHg by surgical myotomy compared with hydrostatic balloon dilation. However, patients whose sphincter pressure has been reduced by hydrostatic balloon dilation to <10 mmHg have an outcome

1	to <10 mmHg by surgical myotomy compared with hydrostatic balloon dilation. However, patients whose sphincter pressure has been reduced by hydrostatic balloon dilation to <10 mmHg have an outcome similar to those after surgical myotomy (Fig. 25-63). Botulinum toxin injection may achieve similar results, but it has a longer duration of action that may be measured in weeks or months, rather than years. Botulinum toxin injection may best be used as a diagnostic tool, when it is not clear whether a hypertensive LES is the primary cause of dysphagia. Responsiveness to botulinum toxin injection may predict a good response to Heller myotomy.The therapeutic decisions regarding the treatment of patients with achalasia center on four issues. The first issue is the question of whether newly diagnosed patients should be treated with pneumatic dilation or a surgical myotomy. Long-term follow-up studies have shown that pneumatic dilation Brunicardi_Ch25_p1009-p1098.indd 106301/03/19 6:05 PM

1	patients should be treated with pneumatic dilation or a surgical myotomy. Long-term follow-up studies have shown that pneumatic dilation Brunicardi_Ch25_p1009-p1098.indd 106301/03/19 6:05 PM 1064SPECIFIC CONSIDERATIONSPART II10.80.60.40.200122426LES < 10 mmHg0.530.23LES > 10 mmHg48Months% in remission60728496Figure 25-63. Prevalence of clinical remission in 122 patients stratified according to postdilatation lower esophageal sphincter (LES) pressures greater than or <10 mmHg. (Reproduced with per-mission from Ponce J, Garrigues V, Pertejo V, et al: Individual pre-diction of response to pneumatic dilation in patients with achalasia, Dig Dis Sci. 1996 Nov;41(11):2135-2141.)achieves adequate relief of dysphagia and pharyngeal regurgi-tation in 50% to 60% of patients (Fig. 25-64). Close follow-up is required, and if dilation fails, myotomy is indicated. For those patients who have a dilated and tortuous esophagus or an associ-ated hiatal hernia, balloon dilation is dangerous and

1	follow-up is required, and if dilation fails, myotomy is indicated. For those patients who have a dilated and tortuous esophagus or an associ-ated hiatal hernia, balloon dilation is dangerous and surgery is the better option. The outcome of the one controlled random-ized study (38 patients) comparing the two modes of therapy suggests that surgical myotomy as a primary treatment gives better long-term results. Several randomized trials comparing laparoscopic cardiomyotomy with balloon dilation or botuli-num toxin injection have favored the surgical approach as well. 100908070605040%302010001234567Years89101112131415Pneumatic dilatation n = 122Pneumatic dilatation n = 54Myotomy + antireflux n = 22Myotomy n = 65Myotomy n = 81Figure 25-64. Summary of long-term studies reporting the proportion of patients with complete relief or minimal dysphagia (Stage 0–1) stratified according to type of treatment. (Data from: Ellis FH, Jr. Oesophagomyotomy for achalasia: a 22-year experience. Br J Surg.

1	of patients with complete relief or minimal dysphagia (Stage 0–1) stratified according to type of treatment. (Data from: Ellis FH, Jr. Oesophagomyotomy for achalasia: a 22-year experience. Br J Surg. 1993;80:882; Goulbourne IA, Walbaum PR. Long-term results of Heller’s operation for achalasia. J Royal Coll Surg. 1985;30:101; Malthaner RA, Todd TR, Miller L, et al. Long-term results in surgically managed esophageal achalasia. Ann Thorac Surg. 1994;58:1343; Ponce J, Garrigues V, Pertejo V, et al. Individual prediction of response to pneumatic dilation in patients with achalasia. Dig Dis Sci. 1996;41:2135; Eckardt V, Aignherr C, Bernhard G. Predictors of outcome in patients with achalasia treated by pneumatic dilation. Gastroenterology. 1992;103:1732.)Although it has been reported that a myotomy after previous balloon dilation is more difficult, this has not been the experi-ence of these authors unless the cardia has been ruptured in a sawtooth manner. In this situation, operative

1	a myotomy after previous balloon dilation is more difficult, this has not been the experi-ence of these authors unless the cardia has been ruptured in a sawtooth manner. In this situation, operative intervention, either immediately or after healing has occurred, can be difficult. Sim-ilarly, myotomy after botulinum toxin injection has reported to be more difficult, but this is largely a function of the submucosal inflammatory response, which may be a bit unpredictable, and is most intense in the first 6 to 12 weeks after injection. It is impor-tant to wait at least 3 months after botulinum toxin injection to perform cardiomyotomy to minimize the risk of encountering dense inflammation.The second issue is the question of whether a surgical myotomy should be performed through the abdomen or the chest. Myotomy of the LES can be accomplished via either an abdominal or thoracic approach. In the absence of a previous upper abdominal surgery, most surgeons prefer the abdominal approach to

1	or the chest. Myotomy of the LES can be accomplished via either an abdominal or thoracic approach. In the absence of a previous upper abdominal surgery, most surgeons prefer the abdominal approach to LES myotomy as laparoscopy results in less pain and a shorter length of stay than thoracoscopy. In addition, it is a bit easier to ensure a long gastric myotomy when the approach is transabdominal.The third issue—and one that has been long debated—is the question of whether an antireflux procedure should be added to a surgical myotomy. Excellent results have been reported fol-lowing meticulously performed myotomy without an antireflux component. Retrospective studies, with long-term follow-up of large cohorts of patients undergoing Heller myotomy demon-strated that, after 10 years, more than 50% of patients had reflux symptoms without a fundoplication. In a recent randomized clin-ical trial, 7% of patients undergoing Dor fundoplication follow-ing LES myotomy had abnormal 24-hour pH

1	than 50% of patients had reflux symptoms without a fundoplication. In a recent randomized clin-ical trial, 7% of patients undergoing Dor fundoplication follow-ing LES myotomy had abnormal 24-hour pH probes, and 42% of patients with a myotomy only had abnormal reflux profiles. If an antireflux procedure is used as an adjunct to esophageal myotomy, a complete 360° fundoplication should be avoided. Rather, a 270° Belsey fundoplication, a Toupet posterior 180° fundoplication, or a Dor anterior 180° fundoplication should be used to avoid the long-term esophageal dysfunction secondary to the outflow obstruction afforded by the fundoplication itself.The fourth issue centers on whether or not a cure of this disease is achievable. Long-term follow-up studies after surgical myotomy have shown that late deterioration in results occurs after this procedure, regardless of whether an antireflux pro-cedure is done, and also after balloon dilation, even when the sphincter pressure is reduced to below

1	deterioration in results occurs after this procedure, regardless of whether an antireflux pro-cedure is done, and also after balloon dilation, even when the sphincter pressure is reduced to below 10 mmHg. It may be that, even though a myotomy or balloon rupture of the LES muscle reduces the outflow obstruction at the cardia, the underlying motor disorder in the body of the esophagus persists and dete-riorates further with the passage of time, leading to increased impairment of esophageal emptying. The earlier an effective reduction in outflow resistance can be accomplished, the better the outcome will be, and the more likely some esophageal body function can be restored.In performing a surgical myotomy of the LES, there are four important principles: (a) complete division of all circular and collar-sling muscle fibers, (b) adequate distal myotomy to reduce outflow resistance, (c) “undermining” of the muscularis to allow wide separation of the esophageal muscle, and (d) pre-vention of

1	and collar-sling muscle fibers, (b) adequate distal myotomy to reduce outflow resistance, (c) “undermining” of the muscularis to allow wide separation of the esophageal muscle, and (d) pre-vention of postoperative reflux. In the past, the drawback of a surgical myotomy was the need for an open procedure, which often deterred patients from choosing the best treatment option for achalasia. With the advent of minimally invasive surgi-cal techniques two decades ago, laparoscopic cardiomyotomy Brunicardi_Ch25_p1009-p1098.indd 106401/03/19 6:05 PM 1065ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25(Heller myotomy) has become the treatment of choice for most patients with achalasia.Open Esophageal MyotomyOpen techniques of distal esophageal myotomy are rarely used outside reoperations. In fact, primary procedures can almost always be successfully completed via laparoscopy. A modified Heller myotomy can be performed through a left thoracotomy incision in the sixth intercostal space along the

1	primary procedures can almost always be successfully completed via laparoscopy. A modified Heller myotomy can be performed through a left thoracotomy incision in the sixth intercostal space along the upper border of the seventh rib. The esophagus and a tongue of gastric fun-dus are exposed as described for a long myotomy. A myotomy through all muscle layers is performed, extending distally over the stomach to 1 to 2 cm below the junction, and proximally on the esophagus for 4 to 5 cm. The cardia is reconstructed by suturing the tongue of gastric fundus to the margins of the myotomy to prevent rehealing of the myotomy site and to pro-vide reflux protection in the area of the divided sphincter. If an extensive dissection of the cardia has been done, a more for-mal Belsey repair is performed. The tongue of gastric fundus is allowed to retract into the abdomen. Traditionally, nasogastric drainage is maintained for 6 days to prevent distention of the stomach during healing. An oral diet is

1	The tongue of gastric fundus is allowed to retract into the abdomen. Traditionally, nasogastric drainage is maintained for 6 days to prevent distention of the stomach during healing. An oral diet is resumed on the seventh day, after a barium swallow study shows unobstructed passage of the bolus into the stomach without extravasation.In a randomized, long-term follow-up by Csendes and colleagues of 81 patients treated for achalasia, either by forceful dilation or by surgical myotomy, myotomy was associated with a significant increase in the diameter at the GEJ and a decrease in the diameter at the middle third of the esophagus on follow-up radiographic studies. There was a greater reduction in sphincter pressure and improvement in the amplitude of esophageal contractions after myotomy. After dilation, 13% of patients regained some peristalsis, compared with 28% after surgery. These findings were shown to persist over a 5-year follow-up period, at which time 95% of those treated with

1	dilation, 13% of patients regained some peristalsis, compared with 28% after surgery. These findings were shown to persist over a 5-year follow-up period, at which time 95% of those treated with surgical myotomy were doing well. Of those who were treated with dilation, only 54% were doing well, while 16% required redilation, and 22% eventually required surgical myotomy to obtain relief.If simultaneous esophageal contractions are associated with the sphincter abnormality, the so-called vigorous achala-sia, then the myotomy should extend over the distance of the abnormal motility as mapped by the preoperative motility study. Failure to do this will result in continuing dysphagia and a dis-satisfied patient. The best objective evaluation of improvement in the patient following either balloon dilation or myotomy is a scintigraphic measurement of esophageal emptying time. A good therapeutic response improves esophageal emptying toward normal. However, some degree of dysphagia may per-sist

1	dilation or myotomy is a scintigraphic measurement of esophageal emptying time. A good therapeutic response improves esophageal emptying toward normal. However, some degree of dysphagia may per-sist despite improved esophageal emptying, due to disturbances in esophageal body function. When an antireflux procedure is added to the myotomy, it should be a partial fundoplication. A 360° fundoplication is associated with progressive retention of swallowed food, regurgitation, and aspiration to a degree that exceeds the patient’s preoperative symptoms.Laparoscopic CardiomyotomyMore commonly known as a laparoscopic Heller myotomy, after Ernst Heller, a German surgeon who described a “dou-ble myotomy” in 1913, the laparoscopic approach is similar to the Nissen fundoplication in terms of the trocar placement and exposure and dissection of the esophageal hiatus (Fig. 25-65). The procedure begins by division of the short gastric vessels in preparation for fundoplication. Exposure of the GEJ via

1	placement and exposure and dissection of the esophageal hiatus (Fig. 25-65). The procedure begins by division of the short gastric vessels in preparation for fundoplication. Exposure of the GEJ via removal of the gastroesophageal fat pad follows. The anterior vagus nerve is swept right laterally along with the fat pad. Once completed, the GEJ and distal 4 to 5 cm of esophagus should be bared of any overlying tissue, and generally follows dissection of the GEJ. A distal esophageal myotomy is performed. It is generally easiest to begin the myotomy 1 to 2 cm above the GEJ, in an area above that of previous botulinum toxin injections or balloon dilation. Either scissors or a hook-type electrocautery can be used to initiate the incision in the longitudinal and circu-lar muscle. Distally, the myotomy is carried across the GEJ and onto the proximal stomach for approximately 2 to 3 cm. After completion, the muscle edges are separated bluntly from the esophageal mucosa for approximately 50% of

1	is carried across the GEJ and onto the proximal stomach for approximately 2 to 3 cm. After completion, the muscle edges are separated bluntly from the esophageal mucosa for approximately 50% of the esophageal circumference. An antireflux procedure follows completion of the myotomy. Either an anterior hemifundoplication augment-ing the angle of His (Dor) or posterior partial fundoplication (Toupet) can be performed. The Dor type fundoplication is slightly easier to perform, and it does not require disruption of the normal posterior gastroesophageal attachments (a theoretical advantage in preventing postoperative reflux).Per Oral Endoscopic Myotomy (POEM)The POEM procedure was developed in Japan. It is the ultimate minimally invasive myotomy as it requires no incisions through the skin. With the POEM procedure, a very effective myotomy is performed entirely from the lumen of the esophagus. The POEM procedure is started by opening the esophageal mucosa 10 cm above the lower esophageal

1	the POEM procedure, a very effective myotomy is performed entirely from the lumen of the esophagus. The POEM procedure is started by opening the esophageal mucosa 10 cm above the lower esophageal sphincter with a needle–knife electrosurgery device passed through an endoscope. A long submucosal plane is developed with the endoscope, down to and below the LES. The circular muscle of the LES, above and below the gastroesophageal junction, is divided with endoscopic electrosurgery. The submucosal entry site in the esophagus is then closed with endoscopic clips. While the results of POEM are still accumulating, the procedure is attractive because it is extremely minimally invasive, and can be done on an outpatient basis. The major downside of POEM is that an effective antire-flux valve cannot be created, exposing the patient to a 40% to 50% risk of GERD post procedure.Outcome Assessment of the Therapy for AchalasiaCritical analysis of the results of therapy for motor disor-ders of the

1	be created, exposing the patient to a 40% to 50% risk of GERD post procedure.Outcome Assessment of the Therapy for AchalasiaCritical analysis of the results of therapy for motor disor-ders of the esophagus requires objective measurement. The use of symptoms alone as an endpoint to evaluate therapy for achalasia may be misleading. The propensity for patients to unconsciously modify their diet to avoid difficulty swallowing is underestimated, making an assessment of results based on symptoms unreliable. Insufficient reduction in outflow resis-tance may allow progressive esophageal dilation to develop slowly, giving the impression of improvement because the volume of food able to be ingested with comfort increases. A variety of objective measurements may be used to assess success, including LES pressure, esophageal baseline pressure, and scintigraphic assessment of esophageal emptying time. Esophageal baseline pressure is usually negative compared to gastric pressure. Given that the

1	LES pressure, esophageal baseline pressure, and scintigraphic assessment of esophageal emptying time. Esophageal baseline pressure is usually negative compared to gastric pressure. Given that the goal of therapy is to eliminate the outflow resistance of a nonrelaxing sphincter, measure-ments of improvements in esophageal baseline pressure and scintigraphic transit time may be better indicators of success, but these are rarely reported.Brunicardi_Ch25_p1009-p1098.indd 106501/03/19 6:05 PM 1066SPECIFIC CONSIDERATIONSPART IIFigure 25-65. A. Longitudinal muscle is divided. B. Mechanical disruption of lower esophageal sphincter muscle fibers. C. Myotomy must be carried across gastroesophageal junction. D. Gastric extension should equal 2 to 3 cm. E. Anterior (Dor) fundoplication is sutured to the diaphragmatic arch. F. Posterior (Toupet) fundoplication is sutured to cut edges of myotomy. EG jct = esophagogastric junction.Eckardt and associates investigated whether the outcome of

1	to the diaphragmatic arch. F. Posterior (Toupet) fundoplication is sutured to cut edges of myotomy. EG jct = esophagogastric junction.Eckardt and associates investigated whether the outcome of pneumatic dilation in patients with achalasia could be pre-dicted on the basis of objective measurements. Postdilation LES pressure was the most valuable measurement for predict-ing long-term clinical response. A postdilatation sphincter pres-sure <10 mmHg predicted a good response. Approximately 50% of the patients studied had postdilatation sphincter pressures between 10 and 20 mmHg, with a 2-year remission rate of 71%. More important, 16 of 46 patients were left with a postdilatation sphincter pressure of >20 mmHg and had an unacceptable out-come. Overall, only 30% of patients dilated remained in symp-tomatic remission at 5 years.Bonavina and colleagues reported good to excellent results with transabdominal myotomy and Dor fundoplication in 94% of patients after a mean follow-up of 5.4 years.

1	symp-tomatic remission at 5 years.Bonavina and colleagues reported good to excellent results with transabdominal myotomy and Dor fundoplication in 94% of patients after a mean follow-up of 5.4 years. No operative mortality occurred in either of these series, attesting to the safety of the procedure. Malthaner and Pearson reported the long-term clinical results in 35 patients with achalasia, having a minimum follow-up of 10 years (Table 25-10). Twenty-two of these patients underwent primary esophageal myotomy and Belsey hemifundoplication at the Toronto General Hospital. Excellent to good results were noted in 95% of patients at 1 year, declining to 68%, 69%, and 67% at 10, 15, and 20 years, respectively. Two patients underwent early reoperation for an incomplete myotomy, and three underwent an esophagectomy for progressive disease. They concluded that there was a deterioration of the initially good results after surgical myotomy and hiatal repair for achalasia, which is due to late

1	an esophagectomy for progressive disease. They concluded that there was a deterioration of the initially good results after surgical myotomy and hiatal repair for achalasia, which is due to late complications of gastroesophageal reflux.Ellis reported his lifetime experience with transthoracic short esophageal myotomy without an antireflux procedure. One hundred seventy-nine patients were analyzed at a mean follow-up of 9 years, ranging from 6 months to 20 years. Overall, 89% of patients were improved at the 9-year mark. He also observed that the level of improvement deteriorated with time, with excel-lent results (patients continuing to be symptom free) decreasing from 54% at 10 years to 32% at 20 years. He concluded that a short transthoracic myotomy without an antireflux procedure provides excellent long-term relief of dysphagia, and, contrary to Malthaner and Pearson’s experience, does not result in com-plications of gastroesophageal reflux. Both studies document nearly identical

1	excellent long-term relief of dysphagia, and, contrary to Malthaner and Pearson’s experience, does not result in com-plications of gastroesophageal reflux. Both studies document nearly identical results 10 to 15 years following the procedure, and both report deterioration over time, probably due to progres-sion of the underlying disease. The addition of an antireflux procedure if the operation is performed transthoracically has no significant effect on the outcome.Brunicardi_Ch25_p1009-p1098.indd 106601/03/19 6:05 PM 1067ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Figure 25-65. (Continued )Table 25-10Reasons for failure of esophageal myotomyREASONAUTHOR, PROCEDURE (N)ELLIS, MYOTOMY ONLY (N = 81)GOULBOURNE, MYOTOMY ONLY (N = 65)MALTHANER, MYOTOMY + ANTIREFLUX (N = 22)Reflux4%5%18%Inadequate myotomy2%—9%Megaesophagus2%——Poor emptying4%3%—Persistent chest pain1%——Data from Malthaner RA, et al. Long-term results in surgically managed esophageal achalasia. Ann Thorac Surg.

1	myotomy2%—9%Megaesophagus2%——Poor emptying4%3%—Persistent chest pain1%——Data from Malthaner RA, et al. Long-term results in surgically managed esophageal achalasia. Ann Thorac Surg. 1994;58:1343; Ellis FH, Jr. Oesophagomyotomy for achalasia: a 22-year experience. Br J Surg. 1993;80:882; and Goulbourne IA, et al. Long-term results of Heller’s operation for achalasia. J R Coll Surg Edinb. 1985;30:101.Brunicardi_Ch25_p1009-p1098.indd 106701/03/19 6:05 PM 1068SPECIFIC CONSIDERATIONSPART IIThe outcome of laparoscopic myotomy and hemifun-doplication has been well documented. Two reports of over 100 patients have documented relief of dysphagia in 93% of patients. Richter and coworkers reviewed published reports to date, including 254 patients with an average success rate of 93% at 2.5 years. Conversion to an open procedure occurs in 0% to 5% of patients. Complications are uncommon, occurring in <5% of patients. Intraoperative complications consist largely of mucosal perforation, and have

1	Conversion to an open procedure occurs in 0% to 5% of patients. Complications are uncommon, occurring in <5% of patients. Intraoperative complications consist largely of mucosal perforation, and have been more likely to occur after botulinum toxin injection. The incidence of objective reflux dis-ease as evidenced by abnormal acid exposure is <10%.A number of randomized clinical trials in the past decade have compared the outcomes of laparoscopic Heller myotomy to pneumatic dilation and to botulinum toxin injection. In each of these trials, laparoscopic Heller myotomy and partial fun-doplication was superior to the alternative treatment. Lastly, a randomized clinical trial examining the need for fundoplica-tion following Heller myotomy demonstrated a great deal more reflux in patients without fundoplication, and no better swallow-ing in the Heller-only group. The best treatment for achalasia is a laparoscopic Heller myotomy and partial fundoplication. The role of POEM in the management

1	fundoplication, and no better swallow-ing in the Heller-only group. The best treatment for achalasia is a laparoscopic Heller myotomy and partial fundoplication. The role of POEM in the management of classic (nonspastic) achalasia is yet to be established.Esophageal Resection for End-Stage Motor Disorders of the EsophagusPatients with dysphagia and long-standing benign disease, whose esophageal function has been destroyed by the disease process or multiple previous surgical procedures, are best man-aged by esophagectomy. Fibrosis of the esophagus and cardia can result in weak contractions and failure of the distal esopha-geal sphincter to relax. The loss of esophageal contractions can result in the stasis of food, esophageal dilatation, regurgitation, and aspiration. The presence of these abnormalities signals end-stage motor disease. In these situations, esophageal replace-ment is usually required to establish normal alimentation. Before proceeding with esophageal resection for

1	these abnormalities signals end-stage motor disease. In these situations, esophageal replace-ment is usually required to establish normal alimentation. Before proceeding with esophageal resection for patients with end-stage benign disease, the choice of the organ to substitute for the esophagus (i.e., stomach, jejunum, or colon) should be considered. The choice of replacement is affected by a num-ber of factors, as described later in “Techniques of Esophageal Reconstruction.” If minimally invasive esophagectomy is to be performed, thoracoscopic dissection should be combined with abdominal dissection. Attempts at MIS transhiatal esophagec-tomy for the massively dilated esophagus may result in large volume bleeding from mediastinal vessels that become enlarged with esophageal dilation, and such bleeding must be directly controlled for hemostasis to be adequate and the operation to be safe.CARCINOMA OF THE ESOPHAGUSSquamous carcinoma accounts for the majority of esophageal carcinomas

1	such bleeding must be directly controlled for hemostasis to be adequate and the operation to be safe.CARCINOMA OF THE ESOPHAGUSSquamous carcinoma accounts for the majority of esophageal carcinomas worldwide. Its incidence is highly variable, ranging from approximately 20 per 100,000 in the United States and Britain, to 160 per 100,000 in certain parts of South Africa and the Henan Province of China, and even 540 per 100,000 in the Guriev district of Kazakhstan. The environmental factors responsible for these localized high-incidence areas have not been conclusively identified, though additives to local foodstuffs (nitroso compounds in pickled vegetables and smoked meats) and mineral deficiencies (zinc and molybdenum) have been suggested. In Western societies, smoking and alcohol consumption are strongly linked with squamous carcinoma. Other definite associations link squamous carcinoma with long-standing achalasia, lye strictures, tylosis (an autosomal dominant disorder characterized

1	are strongly linked with squamous carcinoma. Other definite associations link squamous carcinoma with long-standing achalasia, lye strictures, tylosis (an autosomal dominant disorder characterized by hyperkeratosis of the palms and soles), and human papillomavirus.Adenocarcinoma of the esophagus, once an unusual malig-nancy, is diagnosed with increasing frequency (Fig. 25-66) and now accounts for more than 50% of esophageal cancer in most Western countries. The shift in the epidemiology of esophageal cancer from predominantly squamous carcinoma seen in associ-ation with smoking and alcohol to adenocarcinoma in the setting of BE is one of the most dramatic changes that has occurred in the history of human neoplasia. Although esophageal carcinoma is a relatively uncommon malignancy, its prevalence is explod-ing, largely secondary to the well-established association among gastroesophageal reflux, BE, and esophageal adenocarcinoma. Although BE was once a nearly uniformly lethal disease,

1	is explod-ing, largely secondary to the well-established association among gastroesophageal reflux, BE, and esophageal adenocarcinoma. Although BE was once a nearly uniformly lethal disease, sur-vival has improved slightly because of advances in the under-standing of its molecular biology, screening and surveillance practices, improved staging, minimally invasive surgical tech-niques, and neoadjuvant therapy.Furthermore, the clinical picture of esophageal adenocar-cinoma is changing. It now occurs not only considerably more frequently but also in younger patients, and it is often detected at an earlier stage. These facts support rethinking the traditional approach of assuming palliation is appropriate in all patients. The historical focus on palliation of dysphagia in an elderly patient with comorbidities should change when dealing with a young patient with dependent children and a productive life ahead. The potential for cure becomes of paramount importance.The gross appearance

1	with comorbidities should change when dealing with a young patient with dependent children and a productive life ahead. The potential for cure becomes of paramount importance.The gross appearance resembles that of squamous cell car-cinoma. Microscopically, adenocarcinoma almost always origi-nates in Barrett’s mucosa and resembles gastric cancer. Rarely, it arises in the submucosal glands and forms intramural growths that resemble the mucoepidermal and adenoid cystic carcinomas of the salivary glands.The most important etiologic factor in the development of primary adenocarcinoma of the esophagus is a metaplastic columnar-lined or Barrett’s esophagus, which occurs in approxi-mately 10% to 15% of patients with GERD. When studied pro-spectively, the incidence of adenocarcinoma in a patient with BE is one in 100 to 200 patient-years of follow-up (i.e., for every 100 patients with BE followed for 1 year, one will develop adenocarcinoma). Although this risk appears to be small, it is at

1	with BE is one in 100 to 200 patient-years of follow-up (i.e., for every 100 patients with BE followed for 1 year, one will develop adenocarcinoma). Although this risk appears to be small, it is at least 40 to 60 times that expected for a similar population without BE. This risk is similar to the risk for developing lung cancer in a person with a 20-pack-per-year history of smoking. Endoscopic surveillance for patients with BE is recommended for two reasons: (a) at present there is no reliable evidence that medical therapy removes the risk of neoplastic transformation, and (b) malignancy in BE is curable if detected at an early stage.Clinical ManifestationsEsophageal cancer generally presents with dysphagia, although increasing numbers of relatively asymptomatic patients are now identified on surveillance endoscopy, or present with nonspecific upper GI symptoms and undergo screening endoscopy. Extension of the primary tumor into the tracheobronchial tree can occur primarily with

1	on surveillance endoscopy, or present with nonspecific upper GI symptoms and undergo screening endoscopy. Extension of the primary tumor into the tracheobronchial tree can occur primarily with squamous cell carcinoma and can cause stridor, tracheoesophageal fistula, and resultant coughing, choking, and aspiration 6Brunicardi_Ch25_p1009-p1098.indd 106801/03/19 6:05 PM 1069ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25U.S. esophageal cancer incidence19851993199720012005Incidence per 100,00002520151051989NCI esophageal cancer research investment$21.8M$21.7M$21.6M srallod fo snoilliMilliBo snolod fsralFiscal year020032004200520062007252015105054321Esophageal cancer funding Total NCI budget $22.3M$4.8B$4.7B$4.7B$4.6B$4.8B$22.7MU.S. esophageal cancer mortalityMortality per 100,000198519931997200120050252015105White malesOverall rateAfrican American malesWhite femalesAfrican American females1989Figure 25-66. Incidence and mortality rate trends for esophageal cancer. NCI = National Cancer

1	malesOverall rateAfrican American malesWhite femalesAfrican American females1989Figure 25-66. Incidence and mortality rate trends for esophageal cancer. NCI = National Cancer Institute. (Reproduced with permis-sion from the National Cancer Institute. Last updated September, 2008.)pneumonia. Rarely, severe bleeding from the primary tumor or from erosion into the aorta or pulmonary vessels occurs. Either vocal cord may be invaded, causing paralysis, but most commonly, paralysis is caused by invasion of the left recurrent laryngeal nerve by the primary tumor or LN metastasis. Systemic organ metastases are usually manifested by jaundice or bone pain. The situation is different in high-incidence areas where screening is practiced. In these communities, the most prominent early symptom is pain on swallowing rough or dry food. In patients that present with back pain at the time of esophageal cancer diagnosis, there is usually distant metastasis or celiac encasement.Dysphagia usually presents

1	swallowing rough or dry food. In patients that present with back pain at the time of esophageal cancer diagnosis, there is usually distant metastasis or celiac encasement.Dysphagia usually presents late in the natural history of the disease because the lack of a serosal layer on the esopha-gus allows the smooth muscle to dilate with ease. As a result, the dysphagia becomes severe enough for the patient to seek medical advice only when more than 60% of the esophageal circumference is infiltrated with cancer. Consequently, the dis-ease is usually advanced if symptoms herald its presence. Tra-cheoesophageal fistula may be present in some patients on their first visit to the hospital, and more than 40% will have evidence of distant metastases. With tumors of the cardia, anorexia and weight loss usually precede the onset of dysphagia. The physical signs of esophageal tumors are those associated with the pres-ence of distant metastases.General Approach to Esophageal CancerTherapy of

1	loss usually precede the onset of dysphagia. The physical signs of esophageal tumors are those associated with the pres-ence of distant metastases.General Approach to Esophageal CancerTherapy of esophageal cancer is dictated by the stage of the can-cer at the time of diagnosis. Put simply, one needs to determine if the disease is confined to the esophagus, (T1–T2, N0), locally advanced (T1–3, N1), or disseminated (any T, any N, M1). If cancer is confined to the esophagus, removal of the tumor with adjacent lymph nodes may be curative. Very early tumors con-fined to the mucosa (T in situ, T1a, intramucosal cancer) may be addressed with endoscopic treatment. When the tumor is locally aggressive, modern therapy dictates a multimodality approach in a surgically fit patient. Multimodality therapy is either che-motherapy followed by surgery or radiation and chemotherapy followed by surgery. When given before surgery, these treat-ments are referred to as neoadjuvant or induction therapy. For

1	is either che-motherapy followed by surgery or radiation and chemotherapy followed by surgery. When given before surgery, these treat-ments are referred to as neoadjuvant or induction therapy. For disseminated cancer, treatment is aimed at palliation of symp-toms. If the patient has dysphagia, as many do, the most rapid form of palliation is the endoscopic placement of an expandable esophageal stent. For palliation of GEJ cancer, radiation may be the first choice, as stents placed across the GEJ create a great deal of gastroesophageal reflux.Staging of Esophageal CancerChoosing the best therapy for an individual patient requires accurate staging. Staging starts with the history and physical. LN disease remote from the tumor, particularly in the cervi-cal region, may be palpable on neck examination and generally indicates cancer dissemination. This is often referred to as M1a disease, indicating that these patients should not be treated with therapy directed toward locally advanced

1	examination and generally indicates cancer dissemination. This is often referred to as M1a disease, indicating that these patients should not be treated with therapy directed toward locally advanced cancer. Other meta-static LNs are rarely palpable but are equally ominous, espe-cially the umbilical LN in GEJ cancer.Computed tomographic (CT) scanning of the chest, abdo-men, and pelvis provides information on local invasion of the primary cancer, LN involvement, or disseminated disease. The most common sites of esophageal cancer metastases are lung, liver, and peritoneal surfaces, including the omentum and small bowel mesentery. If masses are identified that are Brunicardi_Ch25_p1009-p1098.indd 106901/03/19 6:05 PM 1070SPECIFIC CONSIDERATIONSPART IInot characteristic for cancer or are in a location that precludes resection with the cancer specimen, positron emission tomogra-phy (PET) scanning may be able to tell whether the masses are metabolically active (likely to be cancer) or

1	in a location that precludes resection with the cancer specimen, positron emission tomogra-phy (PET) scanning may be able to tell whether the masses are metabolically active (likely to be cancer) or not. A PET active focus corresponding to a mass on CT scan outside of the field of esophageal resection should be biopsied before resection is performed.The introduction of endoscopic ultrasound (EUS) has made it possible to identify patients who are potentially curable before surgical therapy. Using an endoscope, the depth of the wall penetration by the tumor and the presence of LN metasta-ses can be determined with 80% accuracy. A curative resection should be encouraged if EUS indicates that the tumor has not invaded adjacent organs (T4b), and/or fewer than six enlarged LNs are imaged. Thoracoscopic and laparoscopic staging of esophageal cancer may add benefit when the nature of enlarged LNs remote from the cancer cannot be determined or when advanced imaging systems (PET and

1	Thoracoscopic and laparoscopic staging of esophageal cancer may add benefit when the nature of enlarged LNs remote from the cancer cannot be determined or when advanced imaging systems (PET and high-resolution spiral CT) are not available.Occasionally, diagnostic laparoscopy and jejunostomy tube placement may precede induction chemoradiation in the patient with severe dysphagia and weight loss from a locally advanced cancer. In summary, esophageal cancer is diagnosed with endoscopic biopsy and is staged with CT scanning of the chest and abdomen, EUS, and PET scan for all patients with CT or EUS evidence of advanced disease (T2 or greater, N1-2 or NX). Experience with esophageal resection in patients with early stage disease has identified characteristics of esophageal cancer that are associated with improved survival. A number of studies suggest that only metastasis to LNs and tumor penetration of the esophageal wall have a significant and independent influence on prognosis. Factors

1	with improved survival. A number of studies suggest that only metastasis to LNs and tumor penetration of the esophageal wall have a significant and independent influence on prognosis. Factors known to be important in the survival of patients with advanced disease, such as cell type, degree of cellular differentiation, or location of tumor in the esophagus, have no effect on survival of patients who have undergone resection for early disease. Studies also showed that patients having five or fewer LN metastases have a better outcome. Using these data, Skinner developed the wall penetration, LN, and distant organ metastases system for staging.The wall penetration, LN, and distant organ metastases system differed somewhat from the previous efforts to develop a satisfactory staging criteria for carcinoma of the esophagus. Most surgeons agreed that the 1983 tumor, nodes, and metastasis system left much to be desired. In the third edition of the manual for Staging of Cancer of the American

1	carcinoma of the esophagus. Most surgeons agreed that the 1983 tumor, nodes, and metastasis system left much to be desired. In the third edition of the manual for Staging of Cancer of the American Joint Committee on Cancer (AJCC) in 1988, an effort was made to provide a finer discrimination between stages than had been contained in the previous edition in 1983. In 2016, further refinements of the staging system of esophageal cancer were approved by the AJCC, recognizing the difference in survival afforded by resection of limited LN disease adjacent to the tumor, compared to multilevel LN disease and positive LNs remote from the primary. Table 25-11 shows the AJCC definitions for the primary tumor, lymph nodes, distant metastasis, and overall staging schema for both squamous cell carcinoma and adenocarcinoma.Clinical Approach to Carcinoma of the Esophagus and CardiaThe selection of a curative vs. a palliative operation for cancer of the esophagus is based on the location of the tumor,

1	and adenocarcinoma.Clinical Approach to Carcinoma of the Esophagus and CardiaThe selection of a curative vs. a palliative operation for cancer of the esophagus is based on the location of the tumor, the patient’s age and health, the extent of the disease, and preoperative stag-ing. Figure 25-67 shows an algorithm of the clinical decisions important in the selection of curative or palliative therapy.Tumor Location. The selection of surgical therapy for patients with carcinoma of the esophagus depends not only on the ana-tomic stage of the disease and an assessment of the swallowing capacity of the patient but also on the location of the primary tumor.It is estimated that 8% of the primary malignant tumors of the esophagus occur in the cervical portion (Fig. 25-68). They are almost always squamous cell cancer, with a rare adenocar-cinoma arising from a congenital inlet patch of columnar lining. These tumors, particularly those in the postcricoid area, repre-sent a separate pathologic

1	squamous cell cancer, with a rare adenocar-cinoma arising from a congenital inlet patch of columnar lining. These tumors, particularly those in the postcricoid area, repre-sent a separate pathologic entity for two reasons: (a) they are more common in females and appear to be a unique entity in this regard; and (b) the efferent lymphatics from the cervical esophagus drain completely differently from those of the tho-racic esophagus. The latter drain directly into the paratracheal and deep cervical or internal jugular LNs with minimal flow in a longitudinal direction. Except in advanced disease, it is unusual for intrathoracic LNs to be involved.Cervical esophageal cancer is frequently unresectable because of early invasion of the larynx, great vessels, or trachea. Radical surgery, including esophagolaryngectomy may occa-sionally be performed for these lesions, but the ensuing mor-bidity makes this a less than desirable approach in the face of uncertain cure. Thus, for most patients

1	esophagolaryngectomy may occa-sionally be performed for these lesions, but the ensuing mor-bidity makes this a less than desirable approach in the face of uncertain cure. Thus, for most patients with cervical esophageal cancer, stereotactic radiation with concomitant chemotherapy is the most desirable treatment.Tumors that arise within the middle third of the esopha-gus are squamous carcinomas most commonly and are fre-quently associated with LN metastasis, which are usually in the thorax but may be in the neck or abdomen, and may skip areas in between. Although it is generally felt that individu-als with midthoracic cancer and abdominal LN metastases are incurable with surgery, there are some emerging data that suggest that cervical LN metastases, if isolated, can be resected with benefit. Generally, T1 and T2 cancers with-out LN metastases are treated with resection only, but there is more and more data to suggest that LN involvement or transmural cancer (T3) warrants treatment with

1	Generally, T1 and T2 cancers with-out LN metastases are treated with resection only, but there is more and more data to suggest that LN involvement or transmural cancer (T3) warrants treatment with neoadjuvant chemoradiation therapy followed by resection. Although some surgeons prefer a transhiatal esophagectomy for all tumor locations, most surgeons believe that resection of mid-esophageal cancer should be performed under direct vision with either thoracoscopy (video-assisted thoracic surgery [VATS]) or with thoracotomy.Tumors of the lower esophagus and cardia are usually adenocarcinomas. Unless preoperative and intraoperative stag-ing clearly demonstrate an incurable lesion, resection in con-tinuity with a LN dissection should be performed. Because of the propensity of GI tumors to spread for long distances sub-mucosally, long lengths of grossly normal GI tract should be resected. The longitudinal lymph flow in the esophagus can result in skip areas, with small foci of tumor above

1	for long distances sub-mucosally, long lengths of grossly normal GI tract should be resected. The longitudinal lymph flow in the esophagus can result in skip areas, with small foci of tumor above the primary lesion, which underscores the importance of a wide resection of esophageal tumors. Wong has shown that local recurrence at the anastomosis can be prevented by obtaining a 10-cm margin of normal esophagus above the tumor. Anatomic studies have also shown that there is no submucosal lymphatic barrier between the esophagus and the stomach at the cardia, and Wong has Brunicardi_Ch25_p1009-p1098.indd 107001/03/19 6:05 PM 1071ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Table 25-11American Joint Committee on Cancer (AJCC) Staging Schema for Esophageal CancerTXT0TisT1T1aT1bT2T3T4T4aT4bNXN0N1N2N3M0M1Primary tumor cannot be assessed.No evidence of primary tumor.High-grade dysplasia.Tumor invades lamina propria, muscularis mucosae, or submucosa.Tumor invades lamina propria or muscularis

1	tumor cannot be assessed.No evidence of primary tumor.High-grade dysplasia.Tumor invades lamina propria, muscularis mucosae, or submucosa.Tumor invades lamina propria or muscularis mucosae.Tumor invades submucosa.Tumor invades muscularis propria.Tumor invades adventitia.Tumor invades adjacent structures.Resectable tumor invading pleura, pericardium, or diaphragm.Unresectable tumor invading other adjacent structures, such as aorta, vertebral body, trachea, etc.Regional lymph nodes cannot be assessed.No regional lymph node metastasis.Metastases in 1–2 regional lymph nodes.Metastases in 3–6 regional lymph nodes.Metastases in ≥7 regional lymph nodes.No distant metastasis.Distant metastasis.SQUAMOUS CELL CARCINOMA Pathological (pTNM)When And And And And Then the stagepT is... pN is... M is... G is... location is... group is...Tis N0 M0 N/A Any 0T1a N0 M0 G1 Any IAT1a N0 M0 G2–3 Any IBT1a N0 M0 GX Any IAT1b N0 M0 G1–3 Any IBT1b N0 M0 GX Any IBT2 N0 M0 G1 Any IBT2 N0 M0 G2–3 Any

1	is... G is... location is... group is...Tis N0 M0 N/A Any 0T1a N0 M0 G1 Any IAT1a N0 M0 G2–3 Any IBT1a N0 M0 GX Any IAT1b N0 M0 G1–3 Any IBT1b N0 M0 GX Any IBT2 N0 M0 G1 Any IBT2 N0 M0 G2–3 Any IIAT2 N0 M0 GX Any IIAT3 N0 M0 G1–3 Lower IIAT3 N0 M0 G1 Upper/middle IIAT3 N0 M0 G2–3 Upper/middle IIBClinical (cTNM)When And And Then the cT is... cN is... M is... stage group is...Tis N0 M0 0T1 N0–1 M0 IT2 N0–1 M0 IIT3 N0 M0 IIT3 N1 M0 IIIT1–3 N2 M0 IIIT4 N0–2 M0 IVAAny T N3 M0 IVAAny T Any N M1 IVBPostneoadjuvant Therapy (ypTNM)When yp And yp And Then the stageT is... N is... M is... group is...T0–2 N0 M0 IT3 N0 M0 IIT0–2 N1 M0 IIIAT3 N1 M0 IIIBT0–3 N2 M0 IIIBT4a N0 M0 IIIBT4a N1–2 M0 IVAT4a NX M0 IVAT4b N0–2 M0 IVAAny T N3 M0 IVAAny T Any N M1 IVBClinical (cTNM)When And And Then the cT is... cN is... M is... stage group is...Tis N0 M0 0T1 N0 M0 IT1 N1 M0 IIAT2 N0 M0 IIBT3 N0 M0 GX Lower/upper/middle IIBT3 N0 M0 Any Location

1	T Any N M1 IVBClinical (cTNM)When And And Then the cT is... cN is... M is... stage group is...Tis N0 M0 0T1 N0 M0 IT1 N1 M0 IIAT2 N0 M0 IIBT3 N0 M0 GX Lower/upper/middle IIBT3 N0 M0 Any Location X IIBT1 N1 M0 Any Any IIBT1 N2 M0 Any Any IIIAT2 N1 M0 Any Any IIIAT2 N2 M0 Any Any IIIBT3 N1–2 M0 Any Any IIIBT4a N0–1 M0 Any Any IIIBT4a N2 M0 Any Any IVAT4b N0–2 M0 Any Any IVAAny T N3 M0 Any Any IVAAny T Any N M1 Any Any IVB(Continued)ADENOCARCINOMAT2 N1 M0 IIIT3 N0–1 M0 IIIT4a N0–1 M0 IIIT1–4a N2 M0 IVAT4b N0–2 M0 IVAAny T N3 M0 IVAAny T Any N M1 IVBBrunicardi_Ch25_p1009-p1098.indd 107101/03/19 6:05 PM 1072SPECIFIC CONSIDERATIONSPART IITable 25-11American Joint Committee on Cancer (AJCC) Staging Schema for Esophageal CancerPostneoadjuvant Therapy (ypTNM)When yp And yp And Then the stage T is... N is... M is... group is...T0–2 N0 M0 IT3 N0 M0 IIT0–2 N1 M0 IIIAT3 N1 M0 IIIBT0–3 N2 M0 IIIBT4a N0 M0 IIIBT4a N1–2 M0 IVAT4a NX M0 IVAT4b N0–2 M0 IVAAny T N3 M0 IVAAny T Any

1	Then the stage T is... N is... M is... group is...T0–2 N0 M0 IT3 N0 M0 IIT0–2 N1 M0 IIIAT3 N1 M0 IIIBT0–3 N2 M0 IIIBT4a N0 M0 IIIBT4a N1–2 M0 IVAT4a NX M0 IVAT4b N0–2 M0 IVAAny T N3 M0 IVAAny T Any N M1 IVBUsed with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Pathological (pTNM)When And And And Then the stage pT is... pN is... M is... G is... group is...Tis N0 M0 N/A 0T1a N0 M0 G1 IAT1a N0 M0 GX IAT1a N0 M0 G2 IBT1b N0 M0 G1–2 IBT1b N0 M0 GX IBT1 N0 M0 G3 ICT2 N0 M0 G1–2 ICT2 N0 M0 G3 IIAT2 N0 M0 GX IIAT1 N1 M0 Any IIBT3 N0 M0 Any IIBT1 N2 M0 Any IIIAT2 N1 M0 Any IIIAT2 N2 M0 Any IIIBT3 N1–2 M0 Any IIIBT4a N0–1 M0 Any IIIBT4a N2 M0 Any IVAT4b N0–2 M0 Any IVAAny T N3 M0 Any IVAAny T Any N M1 Any IVB*Could include combined Rx and chemo neoadjuvant therapyprior to resection to increase resectability and potentialsurvival in patients 75 or under.Curative enbloc

1	T N3 M0 Any IVAAny T Any N M1 Any IVB*Could include combined Rx and chemo neoadjuvant therapyprior to resection to increase resectability and potentialsurvival in patients 75 or under.Curative enbloc resectionIntraoperativestagingAgePhysiologicfitnessClinical stagingEndoscopicultrasoundPalliation75 yearsPalliation FEV1 1.25 Ejection fraction 40%PalliationRecurrent nerve paralysisHorner's syndromePersistent spinal painParalysis of diaphragmFistula formationMalignant pleural effusionEndoscopic tumor length 9 cmAbnormal esophageal axisMultiple enlarged nodes or distantorgan metastasis on CTMore than 20% weight lossLoss of appetite (relative)PalliationTransmural tumors with 4enlarged nodesPalliationUnresectable primaryCavitary spreadDistant metastasisExtension through mediastinal wallMultiple gross lymph node metastasesMicroscopic nodal metastasis at margins ofthe en bloc dissectionPalliative symptomsDysphagiaObstructionPain of ulcerationBleedingInfectionAnxietyRequirements for

1	gross lymph node metastasesMicroscopic nodal metastasis at margins ofthe en bloc dissectionPalliative symptomsDysphagiaObstructionPain of ulcerationBleedingInfectionAnxietyRequirements for palliative transhiatal resection* Free of distant organ metastases Complete excision of primary tumor possibleNonsurgicalpalliationFigure 25-67. Algorithm for the evaluation of esophageal cancer patients to select the proper therapy: curative en bloc resection, palliative transhiatal resection, or nonsurgical palliation. CT = computed tomography; FEV1 = forced expiratory volume in 1 second. (Reproduced with permission from DeMeester TR: Esophageal carcinoma: current controversies, Semin Surg Oncol. 1997 Jul-Aug;13(4):217-233.)shown that 50% of the local recurrences in patients with esopha-geal cancer who are resected for cure occur in the intrathoracic stomach along the line of the gastric resection. Considering that the length of the esophagus ranges from 17 to 25 cm, and

1	with esopha-geal cancer who are resected for cure occur in the intrathoracic stomach along the line of the gastric resection. Considering that the length of the esophagus ranges from 17 to 25 cm, and the length of the lesser curvature of the stomach is approximately 12 cm, a curative resection requires a cervical division of the esophagus and a >50% proximal gastrectomy in most patients with carcinoma of the distal esophagus or cardia.Age. Resection for cure of carcinoma of the esophagus in a patient older than 80 years is rarely indicated because of the additional operative risk and the shorter life expectancy. Despite this general guideline, octogenarians with a high-performance status and excellent cardiopulmonary reserve may be consid-ered candidates for esophagectomy, and recent case series have established its success in highly selected patients. It is in this group of patients that the lesser physiologic impact of minimally (Continued)Brunicardi_Ch25_p1009-p1098.indd

1	case series have established its success in highly selected patients. It is in this group of patients that the lesser physiologic impact of minimally (Continued)Brunicardi_Ch25_p1009-p1098.indd 107201/03/19 6:05 PM 1073ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25LocationIncidenceCervicalUpperthoracicMiddlethoracicLowerthoracicCardia8%3%32%25%32%Figure 25-68. Incidence of carcinoma of the esophagus and cardia based on tumor location.induction chemoradiation therapy, more pronounced dysphagia and associated malnutrition should be addressed before the initiation of chemoradiation. A laparoscopic jejunostomy tube can be placed prior to induction therapy or at the time of esophagectomy. There are emerging data that 5 days’ pretreatment with immune-enhancing nutrition, rich in fish oils, decreases cardiac and other complications, following esophagectomy.Clinical Staging. Clinical factors that indicate an advanced stage of carcinoma and exclude surgery with curative intent are recurrent

1	cardiac and other complications, following esophagectomy.Clinical Staging. Clinical factors that indicate an advanced stage of carcinoma and exclude surgery with curative intent are recurrent nerve paralysis, Horner’s syndrome, persistent spinal pain, paralysis of the diaphragm, fistula formation, and malig-nant pleural effusion. Factors that make surgical cure unlikely include a tumor >8 cm in length, abnormal axis of the esopha-gus on a barium radiogram, more than four enlarged LNs on CT, a weight loss more than 20%, and loss of appetite. Stud-ies indicate that there are several favorable parameters associ-ated with tumors <4 cm in length, there are fewer with tumors between 4 and 8 cm, and there are no favorable criteria for tumors >8 cm in length. Consequently, the finding of a tumor >8 cm in length should exclude curative resection; the finding of a smaller tumor should encourage an aggressive approach.Preoperative Staging With Advanced Imaging. For years, clinical staging,

1	>8 cm in length should exclude curative resection; the finding of a smaller tumor should encourage an aggressive approach.Preoperative Staging With Advanced Imaging. For years, clinical staging, contrast radiography, endoscopy, and CT scan-ning formed the backbone of esophageal cancer staging. More recently, preoperative decision making is guided by endoscopic ultrasonography and PET scanning.EUS provides the most reliable method of determining depth of cancer invasion. In the absence of enlarged LNs, the degree of wall invasion dictates surgical therapy. If a small focus of esophageal cancer is confined to the mucosa, endoscopic mucosal resection (EMR) is a preferable option. If the tumor invades into the submucosa, without visible lymph node involvement, most individuals would suggest esophagectomy with LN dissection, as positive nodes can be found in 20% to 25% of those with cancer limited to the mucosa and submucosa. If EUS demonstrates spread through the wall of the esophagus,

1	esophagectomy with LN dissection, as positive nodes can be found in 20% to 25% of those with cancer limited to the mucosa and submucosa. If EUS demonstrates spread through the wall of the esophagus, especially if LNs are enlarged, then induction chemoradiation therapy (neoadjuvant therapy) should be strongly considered. Lastly, when the EUS demonstrates invasion of the trachea, bronchus, aorta, or spine, then surgical resection is rarely indicated. If there is invasion into the pleura (T4a), then surgical resection can be considered in the absence of a malignant effusion. Thus, it can be seen that the therapy of esophageal cancer is largely driven by the findings of an endoscopic ultrasonography. It is difficult to provide modern treatment of esophageal cancer without access to this modality.PET scanning, usually combined with an axial CT scan (CTPET), usually is performed on patients with locally advanced cancer or questionable lesions on CT scan to deter-mine whether metastases are

1	scanning, usually combined with an axial CT scan (CTPET), usually is performed on patients with locally advanced cancer or questionable lesions on CT scan to deter-mine whether metastases are present. The PET scan uses the injection of radiolabeled deoxyglucose, which is taken up in metabolically active tissues such as cancer. PET-positive areas must be correlated with the CT scan findings to assess the sig-nificance of “hot spots.” CTPET scanning has been especially useful before the initiation of chemoradiation therapy. An early response to chemoradiotherapy, by PET scan, improves the prognosis whether or not resection is ultimately performed. Conversely, if a PET-avid tumor shows no change in metabolic activity after 2 weeks of induction chemoradiation therapy, it is unlikely that further chemoor radiation therapy will be of invasive surgery may reduce the morbidity and mortality associ-ated with open twoor three-field esophagectomy.Cardiopulmonary Reserve. Patients undergoing

1	further chemoor radiation therapy will be of invasive surgery may reduce the morbidity and mortality associ-ated with open twoor three-field esophagectomy.Cardiopulmonary Reserve. Patients undergoing esophageal resection should have sufficient cardiopulmonary reserve to tol-erate the proposed procedure. The respiratory function is best assessed with the forced expiratory volume in 1 second, which ideally should be 2 L or more. Any patient with a forced expi-ratory volume in 1 second of <1.25 L is a poor candidate for thoracotomy because he or she has a 40% risk of dying from respiratory insufficiency within 4 years. In patients with poor pulmonary reserve, the transhiatal esophagectomy should be considered, as the pulmonary morbidity of this operation is less than is seen following thoracotomy. Clinical evaluation and electrocardiogram are not sufficient indicators of cardiac reserve. Echocardiography and dipyridamole thallium imaging provide accurate information on wall motion,

1	Clinical evaluation and electrocardiogram are not sufficient indicators of cardiac reserve. Echocardiography and dipyridamole thallium imaging provide accurate information on wall motion, ejection fraction, and myocardial blood flow. A defect on thallium imaging may require further evaluation with preoperative coronary angiogra-phy. A resting ejection fraction of <40%, particularly if there is no increase with exercise, is an ominous sign. In the absence of invasive testing, observed stair-climbing is an economical (albeit not quantitative) method of assessing cardiopulmonary reserve. Most individuals who can climb three flights of stairs without stopping will do well with two-field open esophagectomy, espe-cially if an epidural catheter is used for postoperative pain relief.Nutritional Status. The factor most predictive of postoperative complication is the nutritional status of the patient. Profound weight loss, more than 20 lb, associated with hypoalbuminemia (albumin <3.5 g/dL) is

1	factor most predictive of postoperative complication is the nutritional status of the patient. Profound weight loss, more than 20 lb, associated with hypoalbuminemia (albumin <3.5 g/dL) is associated with a much higher rate of complications and mortality than patients who enter curative surgery in better nutritional condition. Because malnourished patients generally have locally advanced esophageal cancer, if not metastatic disease, one should consider the placement of a feeding tube before the beginning of induction chemoradiation therapy. Although mild amounts of dysphagia are improved by Brunicardi_Ch25_p1009-p1098.indd 107301/03/19 6:05 PM 1074SPECIFIC CONSIDERATIONSPART IIany benefit. These patients have a worse prognosis and may be referred for resection or palliation without incurring the morbid-ity or expense of a full course of chemoand radiation therapy.Palliation of Esophageal CancerPalliation of esophageal cancer is indicated for individuals with metastatic esophageal

1	the morbid-ity or expense of a full course of chemoand radiation therapy.Palliation of Esophageal CancerPalliation of esophageal cancer is indicated for individuals with metastatic esophageal cancer or cancer invading adjacent organs (T4b) who are unable to swallow, or individuals with fistulae into the tracheobronchial tree. Aortic esophageal fistulas are extremely rare and nearly 100% lethal. Dysphagia as a result of esophageal cancer can be graded from grade I, eating normally, to grade VI, unable to swallow saliva (Table 25-12). Grades I to III often can be managed with radiation therapy, usually in combination with chemotherapy. When surgical resection is not anticipated in the future, this is termed definitive chemoradia-tion therapy and usually is palliative. Radiation dose is increased from 45 Gy to 60 Gy administered over 8 weeks, rather than the 4 weeks given for chemoradiation induction therapy. In 20% of patients, a complete response to chemoradiation therapy will not only

1	from 45 Gy to 60 Gy administered over 8 weeks, rather than the 4 weeks given for chemoradiation induction therapy. In 20% of patients, a complete response to chemoradiation therapy will not only palliate the symptoms but will also leave the patient with undetectable cancer of the esophagus. Although some of these patients are truly cured, cancer will recur in many either locally or systemically 1 to 5 years following definitive chemo-radiation. In a few patients, definitive chemoradiation will be successful in all sites but the esophagus. After a 12-month wait from initial treatment and no other sites of tumor detectable except the esophagus, some of these patients may be candidates for salvage esophagectomy.For individuals with dysphagia grades IV and higher, addi-tional treatment generally is necessary. The mainstay of therapy is in-dwelling esophageal stents. Covered removable stents may be used to seal fistulae or when stent removal becomes desir-able in the future. When large,

1	is necessary. The mainstay of therapy is in-dwelling esophageal stents. Covered removable stents may be used to seal fistulae or when stent removal becomes desir-able in the future. When large, locally invasive tumors or meta-static esophageal cancer precludes any future hope of resection, uncovered expandable metal stents are the treatment of choice. The major limitations to stenting exist in cancers at the GEJ. A stent placed across the GEJ will result in severe gastroesopha-geal reflux and heartburn that can be quite disabling. In cancers at this level, radiation therapy alone may be preferable. If feed-ing access is desirable, a laparoscopic jejunostomy is usually the procedure of choice.Surgical TreatmentThe surgical treatment of esophageal cancer is dependent upon the location of the cancer, the depth of invasion, LN metastases, the fitness of the patient for operation, and the culture and beliefs of the individuals and institutions in which the treatment is performed. In an

1	the cancer, the depth of invasion, LN metastases, the fitness of the patient for operation, and the culture and beliefs of the individuals and institutions in which the treatment is performed. In an ideal world, there would be a single, stage-specific method of treating esophageal cancer because the evidence would be unassailable and noncontroversial. Randomized clinical trials and meta-analyses would prove beyond a shadow of a doubt the value of surgery vs. nonoperative therapy and would dictate the type and extent of surgery that would optimally balance immediate morbidity and mortality with duration and quality of life conferred by the procedure and the perioperative management of the esophagectomy patient. Despite many noble attempts to establish this high level of evidence, many questions relating to the appropriate therapy of esophageal cancer remain. About the only area of complete agreement is that esophagectomy should not be performed if an R0 resection is not possible. In

1	relating to the appropriate therapy of esophageal cancer remain. About the only area of complete agreement is that esophagectomy should not be performed if an R0 resection is not possible. In other words, if the surgeon does not believe he or she can remove all LNs invaded by cancer and provide a tumor-free radial margin and esophagus and stomach margins that are tumor free, then a resection should not be performed.Mucosally Based Cancer. In patients with BE, and especially those with high-grade dysplasia, subcentimeter nodules are frequently discovered. Nodules should be resected in entirety, as they often harbor adenocarcinoma. Five years ago, such resection was performed with a transhiatal esophagectomy, but more recently EMR offers another method for removing intramucosal cancer. In this clinical situation, EMR is typi-cally combined with EUS to rule out more invasive disease. EUS, however, is unable to differentiate between cancer that is confined to the mucosa (T1a) and that

1	this clinical situation, EMR is typi-cally combined with EUS to rule out more invasive disease. EUS, however, is unable to differentiate between cancer that is confined to the mucosa (T1a) and that which invades the submu-cosa (T1b). Tumors invading the submucosa are not amenable to endoscopic mucosal resection because of the high-frequency (20–25%) concurrent finding of positive LNs, which cannot be removed without esophagectomy. On the other hand, intramu-cosal cancers have little risk of spreading to regional LNs. The current approach used involves performing EMR on all nodules identified in a field of Barrett’s esophagus, and then T staging is performed by histologic analysis. This approach dictates the need for future therapy such as esophagectomy.For this reason, small intramucosal carcinomas may be removed with EMR in the following manner. The area beneath the nodule is infiltrated with saline through a sclerotherapy needle. A specialized suction cap is mounted on the end of

1	carcinomas may be removed with EMR in the following manner. The area beneath the nodule is infiltrated with saline through a sclerotherapy needle. A specialized suction cap is mounted on the end of the endoscope, and the nodule is drawn up into the cap; a snare is then applied to resect the tissue. Alternatively, a rubber band can be delivered, and the snare can be used to resect above the level of the rubber band. This specimen is then removed and sent to pathology. As long as the tumor is found to be confined to the mucosa and all margins are negative, the resection is complete. A positive margin or involvement of the submucosa warrants esophagectomy. Most importantly, these patients are at high risk for developing small nodular carcinomas elsewhere in their Barrett’s segment, and routine surveillance on a 3to 6-month basis must be continued indefinitely. Alternatively, one can consider radiofrequency ablation of the remainder of the high-grade dysplasia after careful surveillance

1	surveillance on a 3to 6-month basis must be continued indefinitely. Alternatively, one can consider radiofrequency ablation of the remainder of the high-grade dysplasia after careful surveillance biopsy specimens demonstrate no further sign of cancer. This approach to the early esophageal cancer Table 25-12Functional grades of dysphagiaGRADEDEFINITIONINCIDENCE AT DIAGNOSIS (%)IEating normally11IIRequires liquids with meals21IIIAble to take semisolids but unable to take any solid food30IVAble to take liquids only40VUnable to take liquids, but able to swallow saliva7VIUnable to swallow saliva12Data from Takita H, Vincent RG, Caicedo V, et al. Squamous cell carcinoma of the esophagus: a study of 153 cases, J Surg Oncol. 1977;9(6):547-554.Brunicardi_Ch25_p1009-p1098.indd 107401/03/19 6:05 PM 1075ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25should not be used when there is any suspicion of mediastinal or abdominal lymphadenopathy. Although it is currently rare that EMR provides

1	6:05 PM 1075ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25should not be used when there is any suspicion of mediastinal or abdominal lymphadenopathy. Although it is currently rare that EMR provides definitive therapy of small nodular esophageal cancers, this may become more of the norm as greater surveillance reveals earlier cancers and proficiency of the technique by surgeons and gastroenterologists increases.Minimally Invasive Transhiatal Esophagectomy. Minimally invasive transhiatal esophagectomy is an increasingly popular procedure; however, the number of these operations performed around the world remains small. Mini-invasive surgery (MIS) transhiatal esophagectomy was first performed by Aureo DePaula in Brazil and has been modified and adopted by many individuals around the world. This operation combines the advantages of transhiatal esophagectomy at minimizing pulmonary complications with the advantages of laparoscopy (less pain, quicker rehabilitation). Several variations of

1	This operation combines the advantages of transhiatal esophagectomy at minimizing pulmonary complications with the advantages of laparoscopy (less pain, quicker rehabilitation). Several variations of MIS transhiatal esophagectomy have been developed. For the earliest lesions, such as high-grade dysplasia or intramucosal carcinoma, a vagal sparing procedure can be entertained. In such a procedure, the vagal trunks are separated from the esophagus at the level of the diaphragm and the lesser curvature dissection of the stomach allows the vagus and left gastric pedicle to remain intact. Clearly, this dissection, which hugs the stomach and esophagus, provides no LN staging and is thus inadequate for all high-grade dysplasia and intramucosal cancer.MIS transhiatal esophagectomy is usually performed through five or six small incisions in the upper abdomen and a transverse cervical incision for removing the specimen and performing the cervical esophagogastrostomy. To remove the esophagus

1	through five or six small incisions in the upper abdomen and a transverse cervical incision for removing the specimen and performing the cervical esophagogastrostomy. To remove the esophagus from the posterior mediastinum, especially the area behind the pulmonary vessels and the tracheal bifurcation, which cannot be visualized even with a long laparoscope placed in the posterior mediastinum, it is preferred to use a vein stripping “inversion” technique (Fig. 25-69A). The details of this operation are too lengthy to include in this text, but include the laparoscopic creation of a neo-esophagus (gastric conduit) along the greater curvature of the stomach using the right gastroepiploic artery as the primary vascular pedicle. The conduit can be created through a mini-laparotomy or laparoscopically. A Kocher maneuver releases the duodenum, and a pyloroplasty may be performed (optional). Retrograde esophageal stripping is performed by dividing the esophagus below the GEJ and sliding a vein

1	A Kocher maneuver releases the duodenum, and a pyloroplasty may be performed (optional). Retrograde esophageal stripping is performed by dividing the esophagus below the GEJ and sliding a vein stripper from the neck down into the abdomen followed by an inversion of the esophagus in the posterior mediastinum and removal through the neck (Fig. 25-69B). This technique is reserved for patients with high-grade dysplasia. For small cancers at the GEJ, the esophagus can be stripped in an antegrade fashion by sliding the vein stripper down from the cervical incision and out the tail of the lesser curvature (Fig. 25-69C). The tail of the lesser curvature is pulled out a port site high in the epigastrium while the esophagus is inverted into itself. For GEJ cancers, a wide celiac access LN dissection, splenic artery, hepatic artery, and posterior mediastinal LN dissection can be performed as well or better than through a laparotomy. The gastric conduit is pulled up to the neck with a chest tube

1	splenic artery, hepatic artery, and posterior mediastinal LN dissection can be performed as well or better than through a laparotomy. The gastric conduit is pulled up to the neck with a chest tube and anastomosed to the cervical esophagus in an end-to-side fashion using a surgical stapler or with a handsewn anastomosis. Complications of this technique are primarily limited to leak from the esophagogastric anastomosis, which is self-limited and usually heals within 1 to 3 weeks, spontaneously.Figure 25-69. A. Laparoscopic retrograde inversion. B. Laparo-scopic antegrade inversion. A silk suture holds the tunnel after the esophagus is removed. C. The esophageal conduit is returned to the neck after passing a chest tube down the tunnel and suturing the conduit to the chest tube.Brunicardi_Ch25_p1009-p1098.indd 107501/03/19 6:05 PM 1076SPECIFIC CONSIDERATIONSPART IIOpen Transhiatal Esophagectomy. Transhiatal esophagec-tomy, also known as blunt esophagectomy or esophagectomy without a

1	107501/03/19 6:05 PM 1076SPECIFIC CONSIDERATIONSPART IIOpen Transhiatal Esophagectomy. Transhiatal esophagec-tomy, also known as blunt esophagectomy or esophagectomy without a thoracotomy, was first performed in 1933 by a British surgeon, but was popularized in the last quarter of the 20th century by Mark Orringer from the University of Michigan. Although this operation may violate many of the principles of cancer resec-tion, including extended radical LN dissection, this operation has performed as well as any of the more radical procedures in randomized trials, and in large database analyses. With transhia-tal esophagectomy, the elements of dissection are similar to that described in the section entitled Minimally Invasive Transhiatal Esophagectomy, including the creation of the gastric tube and the posterior mediastinal dissection through the hiatus. Because this dissection is performed with the fingertips rather than under direct vision with surgical instruments, it requires an

1	tube and the posterior mediastinal dissection through the hiatus. Because this dissection is performed with the fingertips rather than under direct vision with surgical instruments, it requires an enlargement of the diaphragmatic hiatus. The lower mediastinal LN basins can be resected as can the upper abdominal LNs, making this an attrac-tive option for GEJ cancers. The mediastinal LNs above the infe-rior pulmonary vein are not removed with this technique, but they rarely result in a point of isolated cancer recurrence.Of all procedures for esophageal cancer, this operation is the quickest to perform in experienced hands and lies in an intermedi-ate position between minimally invasive esophagectomy and the Ivor Lewis procedure with respect to complications and recovery.Minimally Invasive Twoand Three-Field Esophagectomy. After a rocky start, minimally invasive esophagectomy using a thoracic dissection through VATS has become reasonably popular. In general, this operation is performed

1	Three-Field Esophagectomy. After a rocky start, minimally invasive esophagectomy using a thoracic dissection through VATS has become reasonably popular. In general, this operation is performed with an anastomosis created in the neck (three-field), but it may be performed with the anastomosis stapled in the high thorax (two-field). Both procedures will be described.With a minimally invasive three-field esophagectomy, the patient is placed in the left lateral decubitus position. Double lumen intubation is required. Videoscopic access to the thorax is obtained in the midaxillary line in the ninth intercostal space and an angled telescope illuminates the chest superiorly. A mini-thoracotomy at about the sixth intercostal space anteriorly allows introduction of conventional surgical instruments, and a high trocar allows retraction of the lung away from the esophagus. In a three-field approach, the esophagus is dissected along its length to include division of the azygos vein and

1	instruments, and a high trocar allows retraction of the lung away from the esophagus. In a three-field approach, the esophagus is dissected along its length to include division of the azygos vein and harvesting of the LNs in the upper, middle, and lower posterior mediastinum. Hilar, and posterior mediastinal nodes are all removed and sent with the specimen or individually. The thoracic duct is divided at the level of the diaphragm and removed with the specimen.Following complete intrathoracic dissection, the patient is placed in the supine position and five laparoscopic ports are placed as with the MIS transhiatal esophagectomy. The abdominal portions of the operation are identical to those described previously in the section entitled “Minimally Invasive Transhiatal Esophagectomy,” and the gastric conduit is then sewn to the tip of the fully mobilized GEJ and lesser curvature sleeve. A feeding tube is placed, and the pyloroplasty may be performed laparoscopically. A transverse

1	and the gastric conduit is then sewn to the tip of the fully mobilized GEJ and lesser curvature sleeve. A feeding tube is placed, and the pyloroplasty may be performed laparoscopically. A transverse cervical incision and dissection between the sternocleidomastoid and the anterior strap muscles allows access to the cervical esophagus. Great care is made to avoid stretching the recurrent laryngeal nerve. The esophagus and proximal stomach is then pulled up into the neck with the gastric conduit following. Cervical anastomosis is then performed.The MIS transthoracic two-field esophagectomy is slightly different. In this operation, the abdominal portions of the operation are done first, including placement of the feeding tube, the creation of the conduit, and the sewing of the tip of the conduit to the fully dissected GEJ. The patient is then rolled into the left lateral decubitus position and, through right thoracoscopy, the esophagus is dissected and divided 10 cm above the tumor. Once

1	to the fully dissected GEJ. The patient is then rolled into the left lateral decubitus position and, through right thoracoscopy, the esophagus is dissected and divided 10 cm above the tumor. Once freed, the specimen is pulled out through the mini-thoracotomy, and an end-to-end anastomosis stapler is introduced through the high corner of the gastric conduit and out a stab wound along the greater curvature. The anvil of the stapler is placed in the proximal esophagus and held with a purse-string, the stapler is docked, the anastomosis is created, and a gastrotomy is then closed with another firing of the GIA stapler. The three-field esophagectomy has the advantage of placing the anastomosis in the neck where leakage is unlikely to create a severe systemic consequence. On the other hand, placement of the anastomosis in the high chest minimizes the risks of injury to structures in the neck, particularly the recurrent laryngeal nerve. Although the leak of the intrathoracic anastomosis may

1	of the anastomosis in the high chest minimizes the risks of injury to structures in the neck, particularly the recurrent laryngeal nerve. Although the leak of the intrathoracic anastomosis may be more likely to bear septic consequences, the incidence of leak is diminished. Other complications of this approach relate to pulmonary and cardiac status. In many series, the most common complication is pneumonia, the second is atrial fibrillation, and the third is anastomotic leak.Ivor Lewis (En Bloc) Esophagectomy. The theory behind radical transthoracic esophagectomy is that greater removal of LNs and periesophageal tissues diminishes the chance of a posi-tive radial margin and LN recurrence. Although there are no ran-domized data demonstrating this to be superior to other forms of esophagectomy, there are many retrospective data demonstrat-ing improved survival with greater numbers of LNs harvested. A recent study from Sloan-Kettering demonstrates a direct rela-tionship between the number

1	there are many retrospective data demonstrat-ing improved survival with greater numbers of LNs harvested. A recent study from Sloan-Kettering demonstrates a direct rela-tionship between the number of negative nodes harvested and long-term survival. Although such a survival advantage may be related to the completeness of resection, extended radical resec-tions may also be a surrogate for experienced surgeons working in great institutions. As a time-honored operation, there is no doubt that en bloc esophagectomy is the standard to which less radical techniques must be compared.Generally, this operation is started in the abdomen with an upper midline laparotomy and extensive LN dissection in and about the celiac access and its branches, extending into the porta hepatis and along the splenic artery to the tail of the pan-creas. All LNs are removed en bloc with the lesser curvature of the stomach. Unless the tumor extends into the stomach, recon-struction is performed with a greater

1	artery to the tail of the pan-creas. All LNs are removed en bloc with the lesser curvature of the stomach. Unless the tumor extends into the stomach, recon-struction is performed with a greater curvature gastric tube. For GEJ cancers extending significantly into the gastric cardia or fundus, the proximal stomach is removed, and reconstruction is performed with an isoperistaltic section of left colon between the upper esophagus and the remnant stomach, or the colon is connected to a Roux-en-Y limb of jejunum, if total gastrectomy is necessary. In the majority of cases, colon interposition is unnecessary, and a gastric conduit is used.Following closure of the abdominal incision, the patient is placed in the left lateral decubitus position and an anterolateral thoracotomy is performed through the sixth intercostal space. The azygos vein is divided and the posterior mediastinum is entirely cleaned out to include the thoracic duct, all periaor-tic tissues, and all tissue in the upper

1	the sixth intercostal space. The azygos vein is divided and the posterior mediastinum is entirely cleaned out to include the thoracic duct, all periaor-tic tissues, and all tissue in the upper mediastinum along the course of the current laryngeal nerves and in the peribronchial, Brunicardi_Ch25_p1009-p1098.indd 107601/03/19 6:05 PM 1077ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25hilar, and tracheal LN stations. The proximal stomach is pulled up into the thorax where a conduit is created (if not performed previously) and a handsewn or stapled anastomosis is made between the upper thoracic esophagus and the gastric conduit or transverse colon. Chest tubes are placed, and the patient is taken to the intensive care unit.Because this is the most radical of dissections, com-plications are most common, including pneumonia, respira-tory failure, atrial fibrillation, chylothorax, anastomotic leak, conduit necrosis, gastrocutaneous fistula, and, if dissection is too near the recurrent

1	are most common, including pneumonia, respira-tory failure, atrial fibrillation, chylothorax, anastomotic leak, conduit necrosis, gastrocutaneous fistula, and, if dissection is too near the recurrent laryngeal nerves, hoarseness will occur with an increased risk of aspiration. Tracheobronchial injury resulting in fistulas between the bronchus and conduit may also occur, however rarely. Although this procedure and three-field esophagectomy are fraught with the highest complica-tion rate, the long-term outcome of this procedure provides the greatest survival in many single-center series and retrospective reviews.Three-Field Open Esophagectomy. Three-field open esoph-agectomy is very similar to a minimally invasive three-field except that all access is through open incisions. This proce-dure is preferred by certain Japanese surgeons and LN counts achieved through this kind of operation may run from 45 to 60 LNs. Most Western surgeons question the benefit of such radical surgery when it

1	is preferred by certain Japanese surgeons and LN counts achieved through this kind of operation may run from 45 to 60 LNs. Most Western surgeons question the benefit of such radical surgery when it is hard to define a survival advantage. Nonetheless, high intrathoracic cancers probably deserve such an aggressive approach if cure is the goal.Salvage Esophagectomy. Salvage esophagectomy is the nomenclature applied to esophagectomy performed after failure of definitive radiation and chemotherapy. The most frequent scenario is one in which distant disease (bone, lung, brain, or wide LN metastases) renders the patient nonoperable at initial presentation. Then, systemic chemotherapy, usually with radiation of the primary tumor, destroys all foci of metastasis, as demonstrated by CT and CT-PET, but the primary remains present and symptomatic. Following a period of observation, to make sure no new disease will become evident, salvage esophagectomy is performed, usually with an open two-field

1	but the primary remains present and symptomatic. Following a period of observation, to make sure no new disease will become evident, salvage esophagectomy is performed, usually with an open two-field approach. Surprisingly, the cure rate of salvage esophagectomy is not inconsequential. One in four patients undergoing this operation will be disease free 5 years later, despite the presence of residual cancer in the operative specimen. Because of the dense scarring created by radiation treatment, this procedure is the most technically challenging of all esophagectomy techniques.Comparative Studies of Esophagectomy TechniqueTransthoracic vs. Transhiatal Esophagectomy. There has been a great debate as to whether en bloc esophagectomy will provide a greater long-term benefit and cure rate in esophageal cancer than transhiatal esophagectomy. In a recent 7-year fol-low-up of a Dutch study addressing GEJ and lower esophageal cancers, there does not appear to be any benefit to the more

1	in esophageal cancer than transhiatal esophagectomy. In a recent 7-year fol-low-up of a Dutch study addressing GEJ and lower esophageal cancers, there does not appear to be any benefit to the more extensive dissection despite higher morbidity and mortality. In a subgroup analysis of those with one to eight positive LNs, it did appear that the en bloc transthoracic resection may add to longevity. In another large database analysis of the Surveil-lance, Epidemiology, and End Results database, transthoracic and transhiatal esophagectomy were compared. In this study, the transhiatal esophagectomy had a greater long-term survival, but when adjusted by cancer stage, this survival benefit disap-peared. The mortality and morbidity after transhiatal esopha-gectomy appeared to be less. Suffice it to say that this debate over the best procedure for esophagectomy remains an open question.The role of the minimally invasive surgical procedures for a cancer cure will require further study and longer

1	say that this debate over the best procedure for esophagectomy remains an open question.The role of the minimally invasive surgical procedures for a cancer cure will require further study and longer follow-up. It would appear from preliminary analysis that the transhiatal esophagectomy, like its open cousin, may be performed with less morbidity and mortality than the VATS procedure. Long-term survival analyses will require careful follow-up for at least 5 to 10 years after cancer treatment. A recent European multi-center randomized trial comparing open and minimally invasive approaches revealed a highly significant reduction in pulmo-nary complications in the patients who underwent the minimally invasive approach. There was no difference in procedure-related mortality between the approaches.Alternative TherapiesRadiation Therapy. Primary treatment with radiation ther-apy does not produce results comparable with those obtained with surgery. Currently, the use of radiotherapy is

1	TherapiesRadiation Therapy. Primary treatment with radiation ther-apy does not produce results comparable with those obtained with surgery. Currently, the use of radiotherapy is restricted to patients who are not candidates for surgery, and it is usually combined with chemotherapy. Radiation alone is used for pal-liation of dysphagia, but the benefit is short lived, lasting only 2 to 3 months. Furthermore, the length and course of treatment are difficult to justify in patients with a limited life expectancy. Radiation is effective in patients who have hemorrhage from the primary tumor.Adjuvant Chemotherapy. The proposal to use adjuvant che-motherapy in the treatment of esophageal cancer began when it became evident that most patients develop postoperative sys-temic metastasis without local recurrence. This observation led to the hypothesis that undetected systemic micrometasta-sis had been present at the time of diagnosis, and if effective systemic therapy was added to local regional

1	recurrence. This observation led to the hypothesis that undetected systemic micrometasta-sis had been present at the time of diagnosis, and if effective systemic therapy was added to local regional therapy, survival should improve.Recently, this hypothesis has been supported by the obser-vation of epithelial tumor cells in the bone marrow in 37% of patients with esophageal cancer who were resected for cure. These patients had a greater prevalence of relapse at 9 months after surgery compared to those patients without such cells. Such studies emphasize that hematogenous dissemination of viable malignant cells occurs early in the disease, and that sys-temic chemotherapy may be helpful if the cells are sensitive to the agent. On the other hand, systemic chemotherapy may be a hindrance, because of its immunosuppressive properties, if the cells are resistant. Unfortunately, current technology is not able to test tumor cell sensitivity to chemotherapeutic drugs. This requires that the

1	because of its immunosuppressive properties, if the cells are resistant. Unfortunately, current technology is not able to test tumor cell sensitivity to chemotherapeutic drugs. This requires that the choice of drugs be made solely on the basis of their clinical effectiveness against grossly similar tumors.The decision to use preoperative rather than postopera-tive chemotherapy was based on the ineffectiveness of chemo-therapeutic agents when used after surgery, and animal studies suggesting that agents given before surgery were more effec-tive. The claim that patients who receive chemotherapy before resection are less likely to develop resistance to the drugs is unsupported by hard evidence. The claim that drug delivery is enhanced because blood flow is more robust before patients undergo surgical dissection is similarly flawed, due to the fact that if enough blood reaches the operative site to heal the wound or anastomosis, then the flow should be sufficient to

1	patients undergo surgical dissection is similarly flawed, due to the fact that if enough blood reaches the operative site to heal the wound or anastomosis, then the flow should be sufficient to Brunicardi_Ch25_p1009-p1098.indd 107701/03/19 6:05 PM 1078SPECIFIC CONSIDERATIONSPART IIdeliver chemotherapeutic drugs. There are, however, data sup-porting the claim that preoperative chemotherapy in patients with esophageal carcinoma can, if effective, facilitate surgical resection by reducing the size of the tumor. This is particularly beneficial in the case of squamous cell tumors above the level of the carina. Reducing the size of the tumor may provide a safer margin between the tumor and the trachea and allow an anastomosis to a tumor-free cervical esophagus just below the cricopharyngeus. Involved margin at this level usually requires a laryngectomy to prevent subsequent local recurrence.Preoperative Chemotherapy. Eight randomized prospec-tive studies of neoadjuvant chemotherapy vs.

1	Involved margin at this level usually requires a laryngectomy to prevent subsequent local recurrence.Preoperative Chemotherapy. Eight randomized prospec-tive studies of neoadjuvant chemotherapy vs. surgery alone have demonstrated mixed results. For adenocarcinomas of the distal esophagus and proximal stomach, preoperative neoadju-vant 5-fluorouracil (5-FU) and cisplatin chemotherapy has been shown to provide a survival advantage over surgery alone in a well-powered study from the United Kingdom (MRC trial). This trial is one of the few to include enough patients (800) to detect small differences. The trial had a 10% absolute survival benefit at 2 years for the neoadjuvant chemotherapy group. In a second trial from the United Kingdom (MAGIC trial) of distal esopha-geal and proximal gastric adenocarcinomas, the use of epirubi-cin in combination with cisplatin and 5-FU also demonstrated a survival advantage for the induction chemotherapy arm with 4 years median follow-up. As a result of

1	adenocarcinomas, the use of epirubi-cin in combination with cisplatin and 5-FU also demonstrated a survival advantage for the induction chemotherapy arm with 4 years median follow-up. As a result of these two trials, stan-dard treatment of locally advanced adenocarcinoma in Europe calls for neoadjuvant chemotherapy with one of these two regi-mens. Most failures are due to distant metastatic disease, under-scoring the need for improved systemic therapy. Postoperative septic and respiratory complications may be more common in patients receiving chemotherapy.Preoperative Combination Chemoand Radiotherapy. Preoperative chemoradiotherapy using cisplatin and 5-FU in combination with radiotherapy has been reported by several investigators to be beneficial in both adenocarcinoma and squa-mous cell carcinoma of the esophagus. There have been 10 randomized prospective studies (Table 25-13). A recent meta-analysis of these trials demonstrates a 13% survival advantage for neoadjuvant

1	cell carcinoma of the esophagus. There have been 10 randomized prospective studies (Table 25-13). A recent meta-analysis of these trials demonstrates a 13% survival advantage for neoadjuvant chemoradiation therapy, which is more pro-nounced for patients with adenocarcinoma than for those with squamous carcinoma (Table 25-14). It was also observed that the benefit for chemotherapy alone (7%) was not as dramatic as for chemoradiotherapy used in the neoadjuvant setting. Addi-tionally, other work has demonstrated the importance of obtain-ing an R0 (tumor-free) resection as the most important variable determining long-term survival. Although there are no direct, randomized comparisons between chemotherapy and chemora-diation therapy, it appears that the addition of radiation may improve local response of the tumor and may allow a greater opportunity for the surgeon to obtain an R0 resection.The timing of surgery after chemoradiation induction is generally felt to be optimal between 6 and 8

1	of the tumor and may allow a greater opportunity for the surgeon to obtain an R0 resection.The timing of surgery after chemoradiation induction is generally felt to be optimal between 6 and 8 weeks following the completion of induction therapy. Earlier than this time, active inflammation may make the resection hazardous, and the patients have not had time to recover fully from the chemoradia-tion. After 8 weeks, edema in the periesophageal tissue starts to turn to scar tissue, making dissection more difficult.With chemoradiation, the complete response rates for ade-nocarcinoma range from 17% to 24% (Table 25-15). No tumor is detected in the specimen after esophagectomy. Patients dem-onstrating a complete response to chemoradiation have a better survival rate than those without complete response, but distant failure remains common.At present, the strongest predictors of outcome of patients with esophageal cancer are the anatomic extent of the tumor at diagnosis and the completeness of

1	but distant failure remains common.At present, the strongest predictors of outcome of patients with esophageal cancer are the anatomic extent of the tumor at diagnosis and the completeness of tumor removal by surgical resection. After incomplete resection of an esophageal cancer, the 5-year survival rates are 0% to 5%. In contrast, after com-plete resection, independent of stage of disease, 5-year sur-vival ranges from 15% to 40%, according to selection criteria and stage distribution. The importance of early recognition and adequate surgical resection cannot be overemphasized. Figure 25-70 is a global algorithm for the management of esophageal carcinoma.SARCOMA OF THE ESOPHAGUSSarcomas and carcinosarcomas are rare neoplasms, account-ing for approximately 0.1% to 1.5% of all esophageal tumors. They present with the symptom of dysphagia, which does not differ from the dysphagia associated with the more common epithelial carcinoma. Tumors located within the cervical or high thoracic

1	tumors. They present with the symptom of dysphagia, which does not differ from the dysphagia associated with the more common epithelial carcinoma. Tumors located within the cervical or high thoracic esophagus can cause symptoms of pulmonary aspiration secondary to esophageal obstruction. Large tumors originating at the level of the tracheal bifurcation can produce symptoms of airway obstruction and syncope by direct com-pression of the tracheobronchial tree and heart (Fig. 25-71). The duration of dysphagia and age of the patients affected with these tumors are similar to those with carcinoma of the esophagus.A barium swallow usually shows a large polypoid intralu-minal esophageal mass, causing partial obstruction and dilata-tion of the esophagus proximal to the tumor (Fig. 25-72). The smooth polypoid nature of the lesion, although not diagnostic, is distinctive enough to suggest the presence of a sarcoma rather than the more common ulcerating, stenosing carcinoma.Esophagoscopy

1	The smooth polypoid nature of the lesion, although not diagnostic, is distinctive enough to suggest the presence of a sarcoma rather than the more common ulcerating, stenosing carcinoma.Esophagoscopy commonly shows an intraluminal necrotic mass. When biopsy is attempted, it is important to remove the necrotic tissue until bleeding is seen on the tumor’s surface. When this is not done, the biopsy specimen will show only tis-sue necrosis. Even when viable tumor is obtained on biopsy, it has been these authors’ experience that it cannot be defini-tively identified as carcinoma, sarcoma, or carcinosarcoma on the basis of the histology of the portion biopsied. Biopsy results cannot be totally relied on to identify the presence of sarcoma, and it is often the polypoid nature of the lesion that arouses sus-picion that it may be something other than carcinoma.Polypoid sarcomas of the esophagus, in contrast to infil-trating carcinomas, remain superficial to the muscularis propria and are less

1	arouses sus-picion that it may be something other than carcinoma.Polypoid sarcomas of the esophagus, in contrast to infil-trating carcinomas, remain superficial to the muscularis propria and are less likely to metastasize to regional LNs. In one series of 14 patients, local extension or tumor metastasis would have prevented a potentially curative resection in only five. Thus, the presence of a large polypoid tumor should not deter the surgeon from resecting the lesion.Sarcomatous lesions of the esophagus can be divided into epidermoid carcinomas with spindle cell features, such as car-cinosarcoma, and true sarcomas that arise from mesenchymal tissue, such as leiomyosarcoma, fibrosarcoma, and rhabdo-myosarcoma. Based on current histologic criteria for diagno-sis, fibrosarcoma and rhabdomyosarcoma of the esophagus are extremely rare lesions.Surgical resection of polypoid sarcoma of the esophagus is the treatment of choice because radiation therapy has little

1	fibrosarcoma and rhabdomyosarcoma of the esophagus are extremely rare lesions.Surgical resection of polypoid sarcoma of the esophagus is the treatment of choice because radiation therapy has little Brunicardi_Ch25_p1009-p1098.indd 107801/03/19 6:05 PM 1079ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Table 25-13Randomized trials of neoadjuvant chemoradiotherapy vs. surgery, or neoadjuvant chemotherapy vs. surgeryYEAR ACTIVATEDTREATMENT SCHEDULE (RADIOTHERAPY)TREATMENT SCHEDULE (CHEMOTHERAPY)CONCURRENT OR SEQUENTIALTUMOR TYPESAMPLE SIZEMEDIAN FOLLOWUP (MO)Chemoradiotherapy198335 Gy, 1.75 Gy/fraction over 4 wkTwo cycles: cisplatin 20 mg/m2 d 1–5; bleomycin 5 mg/m2 d 1–5SequentialSCC7818a198640 Gy, 2 Gy/fraction over 4 wkTwo cycles: cisplatin 100 mg/m2 d 1; 5-fluorouracil 1000 mg/m2 d 1–4ConcurrentSCC6912a198820 Gy, 2 Gy/fraction over 12 dTwo cycles: cisplatin 100 mg/m2 d 1; 5-fluorouracil 600 mg/m2 d 2–5, 22–25SequentialSCC8612a198945 Gy, 1.5 Gy/fraction over 3 wkTwo cycles:

1	d 1–4ConcurrentSCC6912a198820 Gy, 2 Gy/fraction over 12 dTwo cycles: cisplatin 100 mg/m2 d 1; 5-fluorouracil 600 mg/m2 d 2–5, 22–25SequentialSCC8612a198945 Gy, 1.5 Gy/fraction over 3 wkTwo cycles: cisplatin 20 mg/m2 d 1–5; 5-fluorouracil 300 mg/m2 d 1–21; vinblastine 1 mg/m2 d 1–4ConcurrentSCC and adenocarcinoma10098198937 Gy, 3.7 Gy/fraction over 2 wkTwo cycles: cisplatin 80 mg/m2 d 0–2SequentialSCC29355199040 Gy, 2.7 Gy/fraction over 3 wkTwo cycles: cisplatin 75 mg/m2 d 7; 5-fluorouracil 15 mg/kg d 1–5ConcurrentAdenocarcinoma11324199040 Gy, 2.7 Gy/fraction over 3 wkTwo cycles: cisplatin 75 mg/m2 d 7; 5-fluorouracil 15 mg/kg d 1–5ConcurrentSCC6110199435 Gy, 2.3 Gy/fraction over 3 wkOne cycle: cisplatin 80 mg/m2 d 1; 5-fluorouracil 800 mg/m2 d 2–5ConcurrentSCC and adenocarcinoma25665200650.4 Gy, 1.8 Gy/fraction over 5.6 wkTwo cycles: cisplatin 60 mg/m2 d 1; 5-fluorouracil 1000 mg/m2 d 3–5ConcurrentSCC and adenocarcinoma5660199945.6 Gy, 1.2 Gy/fraction over 28 dTwo cycles: cisplatin 60

1	Gy, 1.8 Gy/fraction over 5.6 wkTwo cycles: cisplatin 60 mg/m2 d 1; 5-fluorouracil 1000 mg/m2 d 3–5ConcurrentSCC and adenocarcinoma5660199945.6 Gy, 1.2 Gy/fraction over 28 dTwo cycles: cisplatin 60 mg/m2 d 1; 5-fluorouracil 1000 mg/m2 d 3–5ConcurrentSCC10125Chemotherapy1982—Two cycles: cisplatin 120 mg/m2 d 1; vindesine 3 mg/m2 d 1, 8; bleomycin 10 U/m2 d 3–6—SCC39201983—Two cycles: cisplatin 20 mg/m2 d 1–5; bleomycin 5 mg/m2 d 1–5—SCC10618a1988c—Three cycles: cisplatin 20 mg/m2 d 1–5; 5-fluorouracil 1000 mg/m2 d 1–5—SCC46751988—Two cycles: cisplatin 100 mg/m2 d 1; bleomycin 10 mg/m2 d 3–8; vinblastine 3 mg/m2 d 1, 8—SCC4617a1989—Two cycles: cisplatin 100 mg/m2 d 1; 5-fluorouracil 1000 mg/m2 d 1–5—SCC147171990—Two cycles: cisplatin 80 mg/m2 d 1; etoposide 200 mg/m2 d 1–5—SCC16019a1990—Three cycles: cisplatin 100 mg/m2 1; 5-fluorouracil 1000 mg/m2 days 1–5—SCC and adeno-carcinoma467561992—Two cycles: cisplatin 100 mg/m2 d 1; 5-fluorouracil 1000 mg/m2 d 1–5—SCC96241992—Two cycles:

1	cycles: cisplatin 100 mg/m2 1; 5-fluorouracil 1000 mg/m2 days 1–5—SCC and adeno-carcinoma467561992—Two cycles: cisplatin 100 mg/m2 d 1; 5-fluorouracil 1000 mg/m2 d 1–5—SCC96241992—Two cycles: cisplatin 80 mg/m2 d 1; 5-fluorouracil 1000 mg/m2 d 1–4—SCC and adeno-carcinoma80237aEstimated as median survival.bUnpublished thesis.cYear of activation not reported, but imputed.dOnly available as an abstract.SCC = squamous cell carcinoma.Reproduced with permission from Gebski V, Burmeister B, Smithers BM, et al: Survival benefits from neoadjuvant chemoradiotherapy or chemotherapy in oesophageal carcinoma: a meta-analysis, Lancet Oncol. 2007 Mar;8(3):226-234.Brunicardi_Ch25_p1009-p1098.indd 107901/03/19 6:05 PM 1080SPECIFIC CONSIDERATIONSPART IITable 25-14Results of the meta-analysis applied to effects of preoperative chemoradiotherapy and chemotherapy on 2-y survival for patients with various levels of riskRISK GROUP2-Y SURVIVAL RATE (%)EXPECTED 2-Y MORTALITYCONTROL (%)TREATEDa (%)ARR

1	effects of preoperative chemoradiotherapy and chemotherapy on 2-y survival for patients with various levels of riskRISK GROUP2-Y SURVIVAL RATE (%)EXPECTED 2-Y MORTALITYCONTROL (%)TREATEDa (%)ARR (%)NNTChemoradiotherapyHigh208064.815.27Medium356552.712.38Low505040.59.510ChemotherapyHigh208072.012.08Medium356558.56.515Low505045.05.020aBased on a 19% relative mortality reduction for those receiving concurrent chemoradiotherapy and a 10% relative mortality reduction for those receiving chemotherapy.ARR = absolute risk reduction; NNT = number needed to treat to prevent one death.Reproduced with permission from Gebski V, Burmeister B, Smithers BM, et al: Survival benefits from neoadjuvant chemoradiotherapy or chemotherapy in oesophageal carcinoma: a meta-analysis, Lancet Oncol. 2007 Mar;8(3):226-234.success and the tumors remain superficial, with local invasion or distant metastases occurring late in the course of the disease. As with carcinoma, the absence of both wall penetration and

1	and the tumors remain superficial, with local invasion or distant metastases occurring late in the course of the disease. As with carcinoma, the absence of both wall penetration and LN metastases is necessary for curative treatment, and surgi-cal resection is consequently responsible for the majority of the reported 5-year survivals. Resection also provides an excellent means of palliating the patient’s symptoms. The surgical tech-nique for resection and the subsequent restoration of the GI con-tinuity is similar to that described for carcinoma.In these authors’ experience, four of the eight patients with carcinosarcoma survived for 5 years or longer. Even though this number is small, it suggests that resection produces better Table 25-15Results of neoadjuvant therapy in adenocarcinoma of the esophagusINSTITUTIONYEARNO. OF PATIENTSREGIMENCOMPLETE PATHOLOGIC RESPONSE (%)SURVIVALMD Anderson199035P, E, 5-FU342% at 3 ySLMC199218P, 5-FU, RT1740% at 3 yVanderbilt199339P, E, 5-FU, RT1947%

1	the esophagusINSTITUTIONYEARNO. OF PATIENTSREGIMENCOMPLETE PATHOLOGIC RESPONSE (%)SURVIVALMD Anderson199035P, E, 5-FU342% at 3 ySLMC199218P, 5-FU, RT1740% at 3 yVanderbilt199339P, E, 5-FU, RT1947% at 4 yMichigan199321P, VBL, 5-FU, RT2434% at 5 yMGH199416P, 5-FU042% at 4 yMGH199422E, A, P558% at 2 yA = doxorubicin; E = etoposide; 5-FU = 5-fluorouracil; MGH = Massachusetts General Hospital; P = cisplatin; RT = radiation therapy; SLMC = St. Louis University Medical Center; VBL = vinblastine.Reproduced with permission from Wright CD, Mathisen DJ, Wain JC, et al: Evolution of treatment strategies for adenocarcinoma of the esophagus and gastroesophageal junction, Ann Thorac Surg. 1994 Dec;58(6):1574-1578.results in epithelial carcinoma with spindle cell features than in squamous cell carcinoma of the esophagus. Similarly, with leiomyosarcoma of the esophagus, the same scattered reports exist with little information on survival. Of seven patients with leiomyosarcoma, two died from their

1	of the esophagus. Similarly, with leiomyosarcoma of the esophagus, the same scattered reports exist with little information on survival. Of seven patients with leiomyosarcoma, two died from their disease—one in 3 months and the other 4 years and 7 months after resection. The other five patients were reported to have survived more than 5 years.It is difficult to evaluate the benefits of resection for leio-myoblastoma of the esophagus because of the small number of reported patients with tumors in this location. Most leiomyo-blastomas occur in the stomach, and 38% of these patients suc-cumb to the cancer in 3 years. Fifty-five percent of patients with extragastric leiomyoblastoma also die from the disease, within an average of 3 years. Consequently, leiomyoblastoma should be considered a malignant lesion and apt to behave like a leiomyosarcoma. The presence of nuclear hyperchromatism, increased mitotic figures (more than one per high-power field), tumor size larger than 10 cm, and

1	a malignant lesion and apt to behave like a leiomyosarcoma. The presence of nuclear hyperchromatism, increased mitotic figures (more than one per high-power field), tumor size larger than 10 cm, and clinical symptoms of longer than 6 months’ duration are associated with a poor prognosis.BENIGN TUMORS AND CYSTSBenign tumors and cysts of the esophagus are relatively uncom-mon. From the perspectives of both the clinician and the patholo-gist, benign tumors may be divided into those that are within the muscular wall and those that are within the lumen of the esophagus.Intramural lesions are either solid tumors or cysts, and the vast majority are leiomyomas. They are made up of varying por-tions of smooth muscle and fibrous tissue. Fibromas, myomas, fibromyomas, and lipomyomas are closely related and occur rarely. Other histologic types of solid intramural tumors have been described, such as lipomas, neurofibromas, hemangiomas, osteochondromas, granular cell myoblastomas, and glomus

1	related and occur rarely. Other histologic types of solid intramural tumors have been described, such as lipomas, neurofibromas, hemangiomas, osteochondromas, granular cell myoblastomas, and glomus tumors, but they are medical curiosities.Intraluminal lesions are polypoid or pedunculated growths that usually originate in the submucosa, develop mainly into the lumen, and are covered with normal stratified squamous epi-thelium. The majority of these tumors are composed of fibrous tissue of varying degrees of compactness with a rich vascular supply. Some are loose and myxoid (e.g., myxoma and myxo-fibroma), some are more collagenous (e.g., fibroma), and some contain adipose tissue (e.g., fibrolipoma). These different types of tumor are frequently collectively designated fibrovascular Brunicardi_Ch25_p1009-p1098.indd 108001/03/19 6:05 PM 1081ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Barium swallow, endoscopyTumor staging(CT chest and abdomen,endoscopic ultrasonography)Late disease

1	108001/03/19 6:05 PM 1081ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Barium swallow, endoscopyTumor staging(CT chest and abdomen,endoscopic ultrasonography)Late disease orsignificant comorbidityDistant organ metastasisImminent cardiac pulmonary or hepatic failureSevere debilityAdvanced diseaseSupportive careCurativeen bloc resectionPalliative surgeryLocal recurrenceNo metastasesComplete excisionpossibleUnresectable proximalor bleeding tumorLaser ablative therapyStentAirway fistula orunresectable primarytumor or localrecurrenceChemotherapyEarly diseaseTumor suspected notto be through the wall and/or less than8 lymph nodes involvedThrough the wall and multiplelymph node metastasisAdvanced diseaseChemoradiationPreoperative chemoradiation followed by en bloc resectionClinical evaluationTreatment failure orrecurrenceDistant metastasisNo local recurrenceFigure 25-70. Suggested global algorithm for the management of carcinoma of the esophagus. CT = computed tomography.polyps, or simply

1	failure orrecurrenceDistant metastasisNo local recurrenceFigure 25-70. Suggested global algorithm for the management of carcinoma of the esophagus. CT = computed tomography.polyps, or simply as polyps. Pedunculated intraluminal tumors should be removed. If the lesion is not too large, endoscopic removal with a snare is feasible.LeiomyomaLeiomyomas constitute more than 50% of benign esophageal tumors. The average age at presentation is 38, which is in sharp contrast to that seen with esophageal carcinoma. Leiomyomas are twice as common in males. Because they originate in smooth muscle, 90% are located in the lower two-thirds of the esophagus. They are usually solitary, but multiple tumors have been found on occasion. They vary greatly in size and shape. Actually, tumors as small as 1 cm in diameter and as large as 10 lb have been removed.Typically, leiomyomas are oval. During their growth, they remain intramural, having the bulk of their mass protruding toward the outer wall of the

1	in diameter and as large as 10 lb have been removed.Typically, leiomyomas are oval. During their growth, they remain intramural, having the bulk of their mass protruding toward the outer wall of the esophagus. The overlying mucosa is freely movable and normal in appearance. Dysphagia and pain are the most common complaints, the two symptoms occurring more frequently together than separately. Bleeding directly related to the tumor is rare, and when hematemesis or melena occur in a patient with an esophageal leiomyoma, other causes should be investigated.A barium swallow is the most useful method to demon-strate a leiomyoma of the esophagus (Fig. 25-73). In profile, the tumor appears as a smooth, semilunar, or crescent-shaped filling defect that moves with swallowing, is sharply demarcated, and is covered and surrounded by normal mucosa. Esophagoscopy should be performed to exclude the reported observation of a coexistence with carcinoma. The freely movable mass, which bulges into the

1	and is covered and surrounded by normal mucosa. Esophagoscopy should be performed to exclude the reported observation of a coexistence with carcinoma. The freely movable mass, which bulges into the lumen, should not be biopsied because of an increased chance of mucosal perforation at the time of surgical enucleation. Endoscopic ultrasound is also a useful adjunct in the workup of leiomyoma and provides detail related to the ana-tomic extent and relationship to surrounding structures.Despite their slow growth and limited potential for malig-nant degeneration, leiomyomas should be removed unless there are specific contraindications. The majority can be removed by simple enucleation. If, during removal, the mucosa is inadver-tently entered, the defect can be repaired primarily. After tumor removal, the outer esophageal wall should be reconstructed by closure of the muscle layer. The location of the lesion and the Brunicardi_Ch25_p1009-p1098.indd 108101/03/19 6:05 PM 1082SPECIFIC

1	removal, the outer esophageal wall should be reconstructed by closure of the muscle layer. The location of the lesion and the Brunicardi_Ch25_p1009-p1098.indd 108101/03/19 6:05 PM 1082SPECIFIC CONSIDERATIONSPART IIABFigure 25-71. A. Computed tomographic scan of a leiomyosarcoma (black arrow) that caused compression of the heart and symptoms of syncope. B. Surgical specimen of leiomyosarcoma shown in A with a pedunculated luminal lesion (white arrow) and a large extraesophageal component (black arrow). There was no evidence of lymph node metastasis at the time of operation.ABFigure 25-72. A. Barium swallow showing a large polypoid intraluminal esophageal mass causing partial obstruction and dilation of the proximal esophagus. B. Operative specimen showing 9-cm polypoid leiomyoblastoma.Brunicardi_Ch25_p1009-p1098.indd 108201/03/19 6:05 PM 1083ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25extent of surgery required will dictate the approach. Lesions of the proximal and middle

1	108201/03/19 6:05 PM 1083ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25extent of surgery required will dictate the approach. Lesions of the proximal and middle esophagus require a right thoracotomy, whereas distal esophageal lesions require a left thoracotomy. Vid-eothoracoscopic and laparoscopic approaches are now frequently used. The mortality rate associated with enucleation is low, and success in relieving the dysphagia is near 100%. Large lesions or those involving the GEJ may require esophageal resection.Esophageal CystCysts may be congenital or acquired. Congenital cysts are lined wholly or partly by columnar ciliated epithelium of the respiratory type, by glandular epithelium of the gastric type, by squamous epithelium, or by transitional epithelium. In some, epithelial lining cells may be absent. Confusion over the embry-ologic origin of congenital cysts has led to a variety of names, such as enteric, bronchogenic, duplication, and mediastinal cysts. Acquired retention

1	cells may be absent. Confusion over the embry-ologic origin of congenital cysts has led to a variety of names, such as enteric, bronchogenic, duplication, and mediastinal cysts. Acquired retention cysts also occur, probably as a result of obstruction of the excretory ducts of the esophageal glands.Enteric and bronchogenic cysts are the most common, and they arise as a result of developmental abnormalities dur-ing the formation and differentiation of the lower respiratory tract, esophagus, and stomach from the foregut. During its embryologic development, the esophagus is lined successively with simple columnar, pseudostratified ciliated columnar, and, finally, stratified squamous epithelium. This sequence probably accounts for the fact that the lining epithelium may be any or a combination of these; the presence of cilia does not necessarily indicate a respiratory origin.Cysts vary in size from small to very large, and they are usually located intramurally in the middleto lower-third

1	of these; the presence of cilia does not necessarily indicate a respiratory origin.Cysts vary in size from small to very large, and they are usually located intramurally in the middleto lower-third of the esophagus. Their symptoms are similar to those of a leio-myoma. The diagnosis similarly depends on radiographic, endoscopic, and endosonographic findings. Surgical excision by enucleation is the preferred treatment. During removal, a fistulous tract connecting the cysts to the airways should be sought, particularly in patients who have had repetitive bron-chopulmonary infections.ESOPHAGEAL PERFORATIONPerforation of the esophagus constitutes a true emergency. It most commonly occurs following diagnostic or therapeutic pro-cedures. Spontaneous perforation, referred to as Boerhaave’s syndrome, accounts for only 15% of cases of esophageal per-foration, foreign bodies for 14%, and trauma for 10%. Pain is a striking and consistent symptom and strongly suggests that an esophageal rupture

1	accounts for only 15% of cases of esophageal per-foration, foreign bodies for 14%, and trauma for 10%. Pain is a striking and consistent symptom and strongly suggests that an esophageal rupture has occurred, particularly if located in the cervical area following instrumentation of the esophagus, or sub-sternally in a patient with a history of resisting vomiting. If sub-cutaneous emphysema is present, the diagnosis is almost certain.Spontaneous rupture of the esophagus is associated with a high mortality rate because of the delay in recognition and treat-ment. Although there usually is a history of resisting vomiting, in a small number of patients, the injury occurs silently, without any antecedent history. When the chest radiogram of a patient with an esophageal perforation shows air or an effusion in the pleural space, the condition is often misdiagnosed as a pneumo-thorax or pancreatitis. An elevated pleural amylase caused by the extrusion of saliva through the perforation may fix

1	an effusion in the pleural space, the condition is often misdiagnosed as a pneumo-thorax or pancreatitis. An elevated pleural amylase caused by the extrusion of saliva through the perforation may fix the diag-nosis of pancreatitis in the mind of an unwary physician. If the chest radiogram is normal, a mistaken diagnosis of myocardial infarction or dissecting aneurysm is often made.Spontaneous rupture usually occurs into the left pleural cavity or just above the GEJ. About 50% of patients have concomitant GERD, suggesting that minimal resistance to the transmission of abdominal pressure into the thoracic esophagus is a factor in the pathophysiology of the lesion. During vomiting, high peaks of intragastric pressure can be recorded, frequently exceeding 200 mmHg, but because extragastric pressure remains almost equal to intragastric pressure, stretching of the gastric wall is minimal. The amount of pressure transmitted to the esophagus varies considerably, depending on the position of

1	remains almost equal to intragastric pressure, stretching of the gastric wall is minimal. The amount of pressure transmitted to the esophagus varies considerably, depending on the position of the GEJ. When it is in the abdomen and exposed to intra-abdominal pressure, the pressure transmitted to the esophagus is much less than when it is exposed to the negative thoracic pressure. In the latter situation, the pressure in the lower esophagus will frequently equal intragastric pressure if the glottis remains closed. Cadaver studies have shown that when this pressure exceeds 150 mmHg, rupture of the esophagus is apt to occur. When a hiatal hernia is present and the sphincter remains exposed to abdominal pressure, the lesion produced is usually a Mallory-Weiss mucosal tear, and bleeding rather than perforation is the problem. This is due to the stretching of the supradiaphragmatic portion of the gastric wall. In this situation, the hernia sac represents an extension of the abdominal cavity,

1	perforation is the problem. This is due to the stretching of the supradiaphragmatic portion of the gastric wall. In this situation, the hernia sac represents an extension of the abdominal cavity, and the GEJ remains exposed to abdominal pressure.DiagnosisAbnormalities on the chest radiogram can be variable and should not be depended upon to make the diagnosis. This is because the abnormalities are dependent on three factors: (a) the time interval between the perforation and the radiographic examination, (b) the site of perforation, and (c) the integrity of the mediastinal pleura. Mediastinal emphysema, a strong indica-tor of perforation, takes at least 1 hour to be demonstrated and is present in only 40% of patients. Mediastinal widening second-ary to edema may not occur for several hours. The site of perfo-ration also can influence the radiographic findings. In cervical perforation, cervical emphysema is common and mediastinal emphysema rare; the converse is true for thoracic

1	hours. The site of perfo-ration also can influence the radiographic findings. In cervical perforation, cervical emphysema is common and mediastinal emphysema rare; the converse is true for thoracic perforations. Figure 25-73. Barium esophagogram showing a classical, smooth, contoured, punched-out defect of a leiomyoma.Brunicardi_Ch25_p1009-p1098.indd 108301/03/19 6:05 PM 1084SPECIFIC CONSIDERATIONSPART IIFrequently, air will be visible in the erector spinae muscles on a neck radiogram before it can be palpated or seen on a chest radiogram (Fig. 25-74). The integrity of the mediastinal pleura influences the radiographic abnormality in that rupture of the pleura results in a pneumothorax, a finding that is seen in 77% of patients. In two-thirds of patients, the perforation is on the left side; in one-fifth, it is on the right side; and in one-tenth, it is bilateral. If pleural integrity is maintained, mediastinal emphy-sema (rather than a pneumothorax) appears rapidly. A pleural

1	left side; in one-fifth, it is on the right side; and in one-tenth, it is bilateral. If pleural integrity is maintained, mediastinal emphy-sema (rather than a pneumothorax) appears rapidly. A pleural effusion secondary to inflammation of the mediastinum occurs late. In 9% of patients, the chest radiogram is normal.The diagnosis is confirmed with a contrast esophagram, which will demonstrate extravasation in 90% of patients. The use of a water-soluble medium such as Gastrografin is preferred. Of concern is that there is a 10% false-negative rate. This may be due to obtaining the radiographic study with the patient in the upright position. When the patient is upright, the passage of water-soluble contrast material can be too rapid to demonstrate a small perforation. The studies should be done with the patient in the right lateral decubitus position (Fig. 25-75). In this, the contrast material fills the entire length of the esophagus, allow-ing the actual site of perforation and its

1	done with the patient in the right lateral decubitus position (Fig. 25-75). In this, the contrast material fills the entire length of the esophagus, allow-ing the actual site of perforation and its interconnecting cavities to be visualized in almost all patients.ManagementThe key to optimum management is early diagnosis. The most favorable outcome is obtained following primary closure of the perforation within 24 hours, resulting in 80% to 90% survival. Figure 25-76 is an operative photograph taken through a left thoracotomy of an esophageal rupture following a pneumatic dilation for achalasia. The most common location for the injury is the left lateral wall of the esophagus, just above the GEJ. Figure 25-74. Chest radiogram showing air in the deep muscles of the neck following perforation of the esophagus (arrow). This is often the earliest sign of perforation and can be present without evidence of air in the mediastinum.Figure 25-75. Radiographic study of a patient with a

1	perforation of the esophagus (arrow). This is often the earliest sign of perforation and can be present without evidence of air in the mediastinum.Figure 25-75. Radiographic study of a patient with a perforation of the esophagus using water-soluble contrast material. The patient is placed in the lateral decubitus position with the left side up to allow complete filling of the esophagus and demonstration of the defect.Figure 25-76. Left thoracotomy in a patient with an esophageal rupture at the gastroesophageal junction following forceful dila-tion of the lower esophagus for achalasia (the surgical clamp is on the stomach, and the Penrose drain encircles the esophagus). The injury consists of a mucosal perforation and extensive splitting of the esophageal muscle from just below the Penrose drain to the stomach.To get adequate exposure of the injury, a dissection similar to that described for esophageal myotomy is performed. A flap of stomach is pulled up and the soiled fat pad at the

1	drain to the stomach.To get adequate exposure of the injury, a dissection similar to that described for esophageal myotomy is performed. A flap of stomach is pulled up and the soiled fat pad at the GEJ is removed. The edges of the injury are trimmed and closed pri-marily (Fig. 25-77). The closure is reinforced with the use of a pleural patch or construction of a Nissen fundoplication.Mortality associated with immediate closure varies between 8% and 20%. After 24 hours, survival decreases to <50%, and is not influenced by the type of operative therapy (i.e., drainage alone or drainage plus closure of the perforation). If the time delay before closing a perforation approaches 24 hours and the tissues are inflamed, division of the cardia and resection of the diseased portion of the esophagus are recommended. The remainder of the esophagus is mobilized, and as much normal esophagus as pos-sible is saved and brought out as an end cervical esophagostomy. In some situations, the retained

1	are recommended. The remainder of the esophagus is mobilized, and as much normal esophagus as pos-sible is saved and brought out as an end cervical esophagostomy. In some situations, the retained esophagus may be so long that Brunicardi_Ch25_p1009-p1098.indd 108401/03/19 6:05 PM 1085ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25it loops down into the chest. The contaminated mediastinum is drained and a feeding jejunostomy tube is inserted. The recov-ery from sepsis is often immediate, dramatic, and reflected by a marked improvement in the patient’s condition over a 24-hour period. On recovery from the sepsis, the patient is discharged and returns on a subsequent date for reconstruction with a substernal colon interposition. Failure to apply this aggressive therapy can result in a mortality rate in excess of 50% in patients in whom the diagnosis has been delayed.Nonoperative management of esophageal perforation has been advocated in select situations. The choice of conserva-tive

1	rate in excess of 50% in patients in whom the diagnosis has been delayed.Nonoperative management of esophageal perforation has been advocated in select situations. The choice of conserva-tive therapy requires skillful judgment and necessitates care-ful radiographic examination of the esophagus. This course of management usually follows an injury occurring during dila-tion of esophageal strictures or pneumatic dilations of achalasia. Conservative management should not be used in patients who have free perforations into the pleural space. Cameron proposed three criteria for the nonoperative management of esophageal perforation: (a) the esophagram must show the perforation to be contained within the mediastinum and drain well back into the esophagus (Fig. 25-78), (b) symptoms should be mild, and (c) there should be minimal evidence of clinical sepsis. If these Figure 25-77. The technique of closure of an esophageal perfora-tion through a left thoracotomy. A. A tongue of stomach is pulled

1	and (c) there should be minimal evidence of clinical sepsis. If these Figure 25-77. The technique of closure of an esophageal perfora-tion through a left thoracotomy. A. A tongue of stomach is pulled up through the esophageal hiatus, and the gastroesophageal fat pad is removed; the edges of the mucosal injury are trimmed and closed using interrupted modified Gambee stitches. B. Reinforcement of the closure with a parietal pleural patch.conditions are met, it is reasonable to treat the patient with hyper-alimentation, antibiotics, and cimetidine to decrease acid secre-tion and diminish pepsin activity. Oral intake is resumed in 7 to 14 days, dependent on subsequent radiographic examinations.MALLORY-WEISS SYNDROMEIn 1929, Mallory and Weiss described four patients with acute upper GI bleeding who were found at autopsy to have mucosal tears at the GEJ. This syndrome, characterized by acute upper GI bleeding following vomiting, is considered to be the cause of up to 15% of all severe

1	who were found at autopsy to have mucosal tears at the GEJ. This syndrome, characterized by acute upper GI bleeding following vomiting, is considered to be the cause of up to 15% of all severe upper GI bleeds. The mechanism is similar to spontaneous esophageal perforation: an acute increase in intra-abdominal pressure against a closed glottis in a patient with a hiatal hernia.Mallory-Weiss tears are characterized by arterial bleeding, which may be massive. Vomiting is not an obligatory factor, as there may be other causes of an acute increase in intra-abdominal pressure, such as paroxysmal coughing, seizures, and retching. The diagnosis requires a high index of suspicion, par-ticularly in the patient who develops upper GI bleeding follow-ing prolonged vomiting or retching. Upper endoscopy confirms the suspicion by identifying one or more longitudinal fissures in the mucosa of the herniated stomach as the source of bleeding.In the majority of patients, the bleeding will stop

1	endoscopy confirms the suspicion by identifying one or more longitudinal fissures in the mucosa of the herniated stomach as the source of bleeding.In the majority of patients, the bleeding will stop sponta-neously with nonoperative management. In addition to blood replacement, the stomach should be decompressed and anti-emetics administered, as a distended stomach and continued vomiting aggravate further bleeding. A Sengstaken-Blakemore tube will not stop the bleeding, as the pressure in the balloon is not sufficient to overcome arterial pressure. Endoscopic injec-tion of epinephrine may be therapeutic if bleeding does not stop spontaneously. Only occasionally will surgery be required to stop blood loss. The procedure consists of laparotomy and high gastrotomy with oversewing of the linear tear. Mortality is uncommon, and recurrence is rare.Figure 25-78. Barium esophagogram showing a stricture and a contained perforation following dilation. The injury meets Cameron criteria: It is

1	tear. Mortality is uncommon, and recurrence is rare.Figure 25-78. Barium esophagogram showing a stricture and a contained perforation following dilation. The injury meets Cameron criteria: It is contained within the mediastinum and drawn back into the esophagus, the patient had mild symptoms, and there was no evidence of clinical sepsis. Nonoperative management was successful.Brunicardi_Ch25_p1009-p1098.indd 108501/03/19 6:05 PM 1086SPECIFIC CONSIDERATIONSPART IITable 25-16Endoscopic grading of corrosive esophageal and gastric burnsFirst degree: Mucosal hyperemia and edemaSecond degree: Limited hemorrhage, exudate ulceration, and pseudomembrane formationThird degree: Sloughing of mucosa, deep ulcers, massive hemorrhage, complete obstruction of lumen by edema, charring, and perforationTable 25-17Location of caustic injury (n = 62)Pharynx10%Esophagus70% Upper15% Middle65% Lower2% Whole18%Stomach20% Antral91% Whole9%Both stomach and esophagus14%CAUSTIC INJURYAccidental caustic

1	25-17Location of caustic injury (n = 62)Pharynx10%Esophagus70% Upper15% Middle65% Lower2% Whole18%Stomach20% Antral91% Whole9%Both stomach and esophagus14%CAUSTIC INJURYAccidental caustic lesions occur mainly in children, and, in general, rather small quantities of caustics are taken. In adults or teenagers, the swallowing of caustic liquids is usually deliberate, during a suicide attempt, and greater quantities are swallowed. Alkalis are more frequently swallowed accidentally than acids, because strong acids cause an immediate burning pain in the mouth.PathologyThe swallowing of caustic substances causes an acute and a chronic injury. During the acute phase, care focuses on con-trolling the immediate tissue injury and the potential for per-foration. During the chronic phase, the focus is on treatment of strictures and disturbances in pharyngeal swallowing. In the acute phase, the degree and extent of the lesion are dependent on several factors: the nature of the caustic substance,

1	is on treatment of strictures and disturbances in pharyngeal swallowing. In the acute phase, the degree and extent of the lesion are dependent on several factors: the nature of the caustic substance, its con-centration, the quantity swallowed, and the time the substance is in contact with the tissues.Acids and alkalis affect tissue in different ways. Alkalis dissolve tissue, and therefore penetrate more deeply, while acids cause a coagulative necrosis that limits their penetration. Animal experiments have shown that there is a correlation between the depth of the lesion and the concentration of sodium hydroxide solution. When a solution of 3.8% comes into contact with the esophagus for 10 seconds, it causes necrosis of the mucosa and the submucosa but spares the muscular layer. A concentration of 22.5% penetrates the whole esophageal wall and into the periesophageal tissues. Cleansing products can contain up to 90% sodium hydroxide. The strength of esophageal contractions varies

1	of 22.5% penetrates the whole esophageal wall and into the periesophageal tissues. Cleansing products can contain up to 90% sodium hydroxide. The strength of esophageal contractions varies according to the level of the esophagus, being weakest at the striated muscle–smooth muscle interface. Consequently, clearance from this area may be somewhat slower, allowing caustic substances to remain in contact with the mucosa longer. This explains why the esophagus is preferentially and more severely affected at this level than in the lower portions.The lesions caused by lye injury occur in three phases. First is the acute necrotic phase, lasting 1 to 4 days after injury. During this period, coagulation of intracellular proteins results in cell necrosis, and the living tissue surrounding the area of necrosis develops an intense inflammatory reaction. Second is the ulcer-ation and granulation phase, starting 3 to 5 days after injury. During this period, the superficial necrotic tissue sloughs,

1	of necrosis develops an intense inflammatory reaction. Second is the ulcer-ation and granulation phase, starting 3 to 5 days after injury. During this period, the superficial necrotic tissue sloughs, leav-ing an ulcerated, acutely inflamed base, and granulation tissue fills the defect left by the sloughed mucosa. This phase lasts 10 to 12 days, and it is during this period that the esophagus is the weakest. Third is the phase of cicatrization and scarring, which begins the third week following injury. During this period, the previously formed connective tissue begins to contract, result-ing in narrowing of the esophagus. Adhesions between granulat-ing areas occur, resulting in pockets and bands. It is during this period that efforts must be made to reduce stricture formation.Clinical ManifestationsThe clinical picture of an esophageal burn is determined by the degree and extent of the lesion. In the initial phase, complaints consist of pain in the mouth and substernal region,

1	ManifestationsThe clinical picture of an esophageal burn is determined by the degree and extent of the lesion. In the initial phase, complaints consist of pain in the mouth and substernal region, hypersali-vation, pain on swallowing, and dysphagia. The presence of fever is strongly correlated with the presence of an esopha-geal lesion. Bleeding can occur, and, frequently, the patient vomits. These initial complaints disappear during the quiescent period of ulceration and granulation. During the cicatrization and scarring phase, the complaint of dysphagia reappears and is due to fibrosis and retraction, resulting in narrowing of the esophagus. Of the patients who develop strictures, 60% do so within 1 month, and 80% within 2 months. If dysphagia does not develop within 8 months, it is unlikely that a stricture will occur. Serious systemic reactions such as hypovolemia and acidosis resulting in renal damage can occur in cases in which the burns have been caused by strong acids.

1	is unlikely that a stricture will occur. Serious systemic reactions such as hypovolemia and acidosis resulting in renal damage can occur in cases in which the burns have been caused by strong acids. Respiratory com-plications such as laryngospasm, laryngoedema, and occasion-ally pulmonary edema can occur, especially when strong acids are aspirated.Inspection of the oral cavity and pharynx can indicate that caustic substances were swallowed, but does not reveal that the esophagus has been burned. Conversely, esophageal burns can be present without apparent oral injuries. Because of this poor correlation, early esophagoscopy is advocated to establish the presence of an esophageal injury. To lessen the chance of perfo-ration, the scope should not be introduced beyond the proximal esophageal lesion. The degree of injury can be graded according to the criteria listed in Table 25-16. Even if the esophagoscopy is normal, strictures may appear later. Radiographic examina-tion is not a

1	lesion. The degree of injury can be graded according to the criteria listed in Table 25-16. Even if the esophagoscopy is normal, strictures may appear later. Radiographic examina-tion is not a reliable means to identify the presence of early esophageal injury, but it is important in later follow-up to iden-tify strictures. The most common locations of caustic injuries are shown in Table 25-17.TreatmentTreatment of a caustic lesion of the esophagus is directed toward management of both the immediate and late consequences of the injury. The immediate treatment consists of limiting the burn by administering neutralizing agents. To be effective, this must be done within the first hour. Lye or other alkali can be neutralized with half-strength vinegar, lemon juice, or orange juice. Acid can be neutralized with milk, egg white, or antacids. Sodium bicarbonate is not used because it generates carbon dioxide, Brunicardi_Ch25_p1009-p1098.indd 108601/03/19 6:05 PM 1087ESOPHAGUS AND

1	can be neutralized with milk, egg white, or antacids. Sodium bicarbonate is not used because it generates carbon dioxide, Brunicardi_Ch25_p1009-p1098.indd 108601/03/19 6:05 PM 1087ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25which might increase the danger of perforation. Emetics are contraindicated because vomiting renews the contact of the caustic substance with the esophagus and can contribute to perforation if too forceful. Hypovolemia is corrected, and broad-spectrum antibiotics are administered to lessen the inflammatory reaction and prevent infectious complications. If necessary, a feeding jejunostomy tube is inserted to provide nutrition. Oral feeding can be started when the dysphagia of the initial phase has regressed.In the past, surgeons waited until the appearance of a stric-ture before starting treatment. Currently, dilations are started the first day after the injury, with the aim of preserving the esophageal lumen by removing the adhesions that occurred in the

1	a stric-ture before starting treatment. Currently, dilations are started the first day after the injury, with the aim of preserving the esophageal lumen by removing the adhesions that occurred in the injured segments. However, this approach is controversial in that dilations can traumatize the esophagus, causing bleed-ing, and perforation, and there are data indicating that exces-sive dilations cause increased fibrosis secondary to the added trauma. The use of steroids to limit fibrosis has been shown to be effective in animals, but their effectiveness in human beings has not been established.Extensive necrosis of the esophagus frequently leads to perforation, and it is best managed by resection. When there is extensive gastric involvement, the esophagus is nearly always necrotic or severely burned, and total gastrectomy and near-total esophagectomy are necessary. The presence of air in the esopha-geal wall is a sign of muscle necrosis and impending perforation and is a strong

1	severely burned, and total gastrectomy and near-total esophagectomy are necessary. The presence of air in the esopha-geal wall is a sign of muscle necrosis and impending perforation and is a strong indication for esophagectomy.Management of acute injury is summarized in the algo-rithm in Fig. 25-79. Some authors have advocated the use of an intraluminal esophageal stent (Fig. 25-80) in patients who are operated on and found to have no evidence of extensive esophagogastric necrosis. In these patients, a biopsy of the posterior gastric wall should be performed to exclude occult injury. If, histologically, there is a question of viability, a second-look operation should be done within 36 hours. If a stent is inserted, it should be kept in position for 21 days, and removed after a satisfactory barium esophagogram. Esopha-goscopy should be done, and if strictures are present, dilations initiated.Once the acute phase has passed, attention is turned to the prevention and management of

1	barium esophagogram. Esopha-goscopy should be done, and if strictures are present, dilations initiated.Once the acute phase has passed, attention is turned to the prevention and management of strictures. Both antegrade dilation with a Hurst or Maloney bougie and retrograde dila-tion with a Tucker bougie have been satisfactory. In a series of 1079 patients, early dilations started during the acute phase gave excellent results in 78%, good results in 13%, and poor results in 2%. During the treatment, 55 patients died. In contrast, of 333 patients whose strictures were dilated when they became symptomatic, only 21% had excellent results, 46% good, and 6% poor, with three dying during the process. The length of time the surgeon should persist with dilation before consideration of esophageal resection is problematic. An adequate lumen should be re-established within 6 months to 1 year, with progressively longer intervals between dilations. If, during the course of treat-ment, an adequate

1	resection is problematic. An adequate lumen should be re-established within 6 months to 1 year, with progressively longer intervals between dilations. If, during the course of treat-ment, an adequate lumen cannot be established or maintained (i.e., smaller bougies must be used), operative intervention should be considered. Surgical intervention is indicated when there is (a) complete stenosis in which all attempts from above and below have failed to establish a lumen, (b) marked irregu-larity and pocketing on barium swallow, (c) the development of a severe periesophageal reaction or mediastinitis with dilatation, (d) a fistula, (e) the inability to dilate or maintain the lumen above a 40F bougie, or (f) a patient who is unwilling or unable to undergo prolonged periods of dilation.Ingestion of caustic agentObservation24–48 hoursExploratorylaparotomySecond lookat 36 hoursIntraluminal esophageal stentPosterior gastric wall biopsyJejunostomy1° burn2° & 3° burnEsophagogastric

1	of caustic agentObservation24–48 hoursExploratorylaparotomySecond lookat 36 hoursIntraluminal esophageal stentPosterior gastric wall biopsyJejunostomy1° burn2° & 3° burnEsophagogastric resectionCervical esophagostomyJejunostomyResection of adjacent involved organsFull thicknessnecrosisof esophagusand stomachViableesophagusandstomachQuestionableesophagusandstomach Esophagoscopy(Within 12 hours)Figure 25-79. Algorithm summarizing the management of acute caustic injury.Figure 25-80. The use of an esophageal stent to prevent stricture. The stent is constructed from a chest tube and placed in the esopha-gus at the time of an exploratory laparotomy. A Penrose drain is placed over the distal end as a flap valve to prevent reflux. The stent is supported at its upper end by attaching it to a suction catheter that is secured to the nares. Continuous suction removes saliva and mucus trapped in the pharynx and upper esophagus.Brunicardi_Ch25_p1009-p1098.indd 108701/03/19 6:05 PM 1088SPECIFIC

1	catheter that is secured to the nares. Continuous suction removes saliva and mucus trapped in the pharynx and upper esophagus.Brunicardi_Ch25_p1009-p1098.indd 108701/03/19 6:05 PM 1088SPECIFIC CONSIDERATIONSPART IIThe variety of abnormalities seen requires that creativity be used when considering esophageal reconstruction. Skin tube esophagoplasties are now used much less frequently than they were in the past, and are mainly of historical interest. Currently, the stomach, jejunum, and colon are the organs used to replace the esophagus, through either the posterior mediastinum or the retrosternal route. A retrosternal route is chosen when there has been a previous esophagectomy or there is extensive fibrosis in the posterior mediastinum. When all factors are considered, the order of preference for an esophageal substitute is (a) colon, (b) stomach, and (c) jejunum. Free jejunal grafts based on the supe-rior thyroid artery have provided excellent results. Whatever method is

1	of preference for an esophageal substitute is (a) colon, (b) stomach, and (c) jejunum. Free jejunal grafts based on the supe-rior thyroid artery have provided excellent results. Whatever method is selected, it must be emphasized that these procedures cannot be taken lightly; minor errors of judgment or technique may lead to serious or even fatal complications.Critical in the planning of the operation is the selection of cervical esophagus, pyriform sinus, or posterior pharynx as the site for proximal anastomosis. The site of the upper anastomosis depends on the extent of the pharyngeal and cervical esophageal damage encountered. When the cervical esophagus is destroyed and a pyriform sinus remains open the anastomosis can be made to the hypopharynx (Fig. 25-81). When the pyriform sinuses are completely stenosed, a transglottic approach is used to perform an anastomosis to the posterior oropharyngeal wall (Fig. 25-82). This allows excision of supraglottic strictures and elevation and

1	are completely stenosed, a transglottic approach is used to perform an anastomosis to the posterior oropharyngeal wall (Fig. 25-82). This allows excision of supraglottic strictures and elevation and anterior tilting of the larynx. In both of these situations, the patient must relearn to swallow. Recovery is long and difficult and may require several endoscopic dilations—and often reop-erations. Sleeve resections of short strictures are not successful because the extent of damage to the wall of the esophagus can be greater than realized, and almost invariably the anastomosis is carried out in a diseased area.The management of a bypassed damaged esophagus after injury is problematic. If the esophagus is left in place, ulcer-ation from gastroesophageal reflux or the development of carcinoma must be considered. The extensive dissection neces-sary to remove the esophagus, particularly in the presence of marked periesophagitis, is associated with significant morbidity. Leaving the esophagus

1	be considered. The extensive dissection neces-sary to remove the esophagus, particularly in the presence of marked periesophagitis, is associated with significant morbidity. Leaving the esophagus in place preserves the function of the Figure 25-82. Anastomosis of the bowel to the posterior orophar-ynx. The anastomosis is done through an inverted trapezoid incision above the thyroid cartilage (dotted line). A triangle-shaped piece of the upper half of the cartilage is resected. Closure of the oropharynx is done so that the larynx is pulled up (sagittal section).Figure 25-81. Anastomosis of the bowel to a preserved pyriform sinus. To identify the site, a finger is inserted into the free pyriform sinus through a suprahyoid incision (dotted line). This requires removing the lateral inferior portion of the thyroid cartilage as shown in cross-section.vagus nerves, and, in turn, the function of the stomach. On the other hand, leaving a damaged esophagus in place can result in multiple blind

1	of the thyroid cartilage as shown in cross-section.vagus nerves, and, in turn, the function of the stomach. On the other hand, leaving a damaged esophagus in place can result in multiple blind sacs and subsequent development of medias-tinal abscesses years later. Most experienced surgeons recom-mend that the esophagus be removed unless the operative risk is unduly high.ACQUIRED FISTULAThe esophagus lies in close contact with the membranous por-tion of the trachea and left bronchus, predisposing to the for-mation of fistula to these structures. Most acquired esophageal fistulas are to the tracheobronchial tree and secondary to either esophageal or pulmonary malignancy. Traumatic fistulas and those associated with esophageal diverticula account for the remainder. Fistulas associated with traction diverticula are usu-ally due to mediastinal inflammatory disease, and traumatic fistulas usually occur secondary to penetrating wounds, lye ingestion, or iatrogenic injury.These fistulas are

1	traction diverticula are usu-ally due to mediastinal inflammatory disease, and traumatic fistulas usually occur secondary to penetrating wounds, lye ingestion, or iatrogenic injury.These fistulas are characterized by paroxysmal cough-ing following the ingestion of liquids, and by recurrent or chronic pulmonary infections. The onset of cough immediately after swallowing suggests aspiration, whereas a brief delay (30–60 seconds) suggests a fistula.Spontaneous closure is rare, owing to the presence of malignancy or a recurrent infectious process. Surgical treat-ment of benign fistulas consists of division of the fistulous tract, resection of irreversibly damaged lung tissue, and closure of the esophageal defect. To prevent recurrence, a pleural flap should be interposed. Treatment of malignant fistulas is difficult, par-ticularly in the presence of prior irradiation. Generally, only palliative treatment is indicated. This can best be done by using a specially designed esophageal

1	malignant fistulas is difficult, par-ticularly in the presence of prior irradiation. Generally, only palliative treatment is indicated. This can best be done by using a specially designed esophageal endoprosthesis that bridges and occludes the fistula, allowing the patient to eat. A salivary tube is also a good option for proximal esophageal fistulas. This tube has a proximal “lip” that rests on the cricopharyngeal muscle and thereby directs the saliva into the tube and past the fis-tula. Rarely, esophageal diversion, coupled with placement of a feeding jejunostomy, can be used as a last resort.Brunicardi_Ch25_p1009-p1098.indd 108801/03/19 6:05 PM 1089ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25of the internal mammary artery and the internal mammary or innominate vein. Removal of the sternoclavicular joint aids in performing the vascular and distal esophageal anastomosis (Fig. 25-83).Reconstruction After Total EsophagectomyNeither the intrathoracic stomach nor the intrathoracic

1	sternoclavicular joint aids in performing the vascular and distal esophageal anastomosis (Fig. 25-83).Reconstruction After Total EsophagectomyNeither the intrathoracic stomach nor the intrathoracic colon functions as well as the native esophagus after an esophagogas-trectomy. The choice between these organs will be influenced by several factors, such as the adequacy of their blood supply and the length of resected esophagus that they are capable of bridging. If the stomach shows evidence of disease, or has been contracted or reduced by previous gastric surgery, the length available for esophageal replacement may not be adequate. The presence of diverticular disease, unrecognized carcinoma, or colitis prohibits the use of the colon. The blood supply of the colon is more affected by vascular disease than the blood supply of the stomach, which may prevent its use. Of the two, the colon provides the longest graft. The stomach can usually reach to the neck if the amount of lesser curvature

1	disease than the blood supply of the stomach, which may prevent its use. Of the two, the colon provides the longest graft. The stomach can usually reach to the neck if the amount of lesser curvature resected does not interfere with the blood supply to the fundus. Gastric interposition has the advantage that only one anastomosis is required. On the other hand, there is greater potential for aspiration of gastric juice or stricture of the cervical anastomosis from chronic reflux when stomach is used for replacement.Following an esophagogastrectomy, patients may have discomfort during or shortly after eating. The most common symptom is a postprandial pressure sensation or a feeling of being full, which probably results from the loss of the gastric reservoir. This symptom is less common when the colon is used as an esophageal substitute, probably because the distal third of the stomach is retained in the abdomen and the interposed colon provides an additional reservoir function.King and

1	the colon is used as an esophageal substitute, probably because the distal third of the stomach is retained in the abdomen and the interposed colon provides an additional reservoir function.King and Hölscher have reported a 40% and 50% inci-dence of dysphagia after reestablishing GI continuity with the stomach following esophagogastrectomy. This incidence is similar to Orringer’s results after using the stomach to replace the esophagus in patients with benign disease. More than one-half of the patients experienced dysphagia postoperatively; TECHNIQUES OF ESOPHAGEAL RECONSTRUCTIONOptions for esophageal substitution include gastric advance-ment, colonic interposition, and either jejunal free transfer or advancement into the chest. Rarely, combinations of these grafts will be the only possible option. The indications for esopha-geal resection and substitution include malignant and end-stage benign disease. The latter includes refluxor drug-induced stricture formation that cannot be

1	option. The indications for esopha-geal resection and substitution include malignant and end-stage benign disease. The latter includes refluxor drug-induced stricture formation that cannot be dilated without damage to the esophagus, a dilated and tortuous esophagus secondary to severe motility disorders, lye-induced strictures, and multiple previous antireflux procedures. The choice of esophageal substitution has significant impact upon the technical difficulty of the procedure and influences the long-term outcome.Partial Esophageal ResectionDistal benign lesions, with preserved proximal esophageal func-tion, are best treated with the interposition of a segment of prox-imal jejunum into the chest and primary anastomosis. A jejunal interposition can reach to the inferior border of the pulmonary hilum with ease, but the architecture of its blood supply rarely allows the use of the jejunum proximal to this point. Because the anastomosis is within the chest, a thoracotomy is necessary.The

1	hilum with ease, but the architecture of its blood supply rarely allows the use of the jejunum proximal to this point. Because the anastomosis is within the chest, a thoracotomy is necessary.The jejunum is a dynamic graft and contributes to bolus transport, whereas the stomach and colon function more as a conduit. The stomach is a poor choice in this circumstance because of the propensity for the reflux of gastric contents into the proximal remaining esophagus following an intratho-racic esophagogastrostomy. It is now well recognized that this occurs and can lead to incapacitating symptoms and esophageal destruction in some patients. Short segments of colon, on the other hand, lack significant motility and have a propensity for the development of esophagitis proximal to the anastomosis.Replacement of the cervical portion of the esophagus, while preserving the distal portion, is occasionally indicated in cervical esophageal or head and neck malignancy, and follow-ing the ingestion of

1	of the cervical portion of the esophagus, while preserving the distal portion, is occasionally indicated in cervical esophageal or head and neck malignancy, and follow-ing the ingestion of lye. Free transfer of a portion of jejunum to the neck has become a viable option and is successful in the majority of cases. Revascularization is achieved via use Figure 25-83. A. The portion of the thoracic inlet to be resected to provide space for a free jejunal graft and access to the internal mammary artery (shaded area). B. Cross-section showing the space available after resection of the sternoclavicular joint and one-half of the manubrium. (Reproduced with permission from Shields TW: General Thoracic Surgery, 3rd ed. Philadelphia, PA: Lea & Febiger; 1989.)Brunicardi_Ch25_p1009-p1098.indd 108901/03/19 6:06 PM 1090SPECIFIC CONSIDERATIONSPART IItwo-thirds of this group required postoperative dilation, and one-fourth had persistent dysphagia and required home dilation. In contrast, dysphagia

1	6:06 PM 1090SPECIFIC CONSIDERATIONSPART IItwo-thirds of this group required postoperative dilation, and one-fourth had persistent dysphagia and required home dilation. In contrast, dysphagia is uncommon, and the need for dilation is rare following a colonic interposition. Isolauri reported on 248 patients with colonic interpositions and noted a 24% incidence of dysphagia 12 months after the operation. When it occurred, the most common cause was recurrent mediastinal tumor. The high incidence of dysphagia with the use of the stomach is prob-ably related to the esophagogastric anastomosis in the neck and the resulting difficulty of passing a swallowed bolus.Another consequence of the transposition of the stomach into the chest is the development of postoperative duodenogastric reflux, probably due to pyloric denervation, and adding a pyloroplasty may worsen this problem. Following gastric advancement, the pylorus lies at the level of the esophageal hiatus, and a distinct pressure

1	probably due to pyloric denervation, and adding a pyloroplasty may worsen this problem. Following gastric advancement, the pylorus lies at the level of the esophageal hiatus, and a distinct pressure differential develops between the intrathoracic gastric and intra-abdominal duodenal lumina. Unless the pyloric valve is extremely efficient, the pressure differential will encourage reflux of duodenal contents into the stomach. Duodenogastric reflux is less likely to occur following colonic interposition because there is sufficient intra-abdominal colon to be compressed by the abdominal pressure and the pylorus and duodenum remain in their normal intra-abdominal position.Although there is general acceptance of the concept that an esophagogastric anastomosis in the neck results in less post-operative esophagitis and stricture than one at a lower level, reflux esophagitis following a cervical anastomosis does occur, albeit at a lower rate than when the anastomosis is at a lower level. Most

1	esophagitis and stricture than one at a lower level, reflux esophagitis following a cervical anastomosis does occur, albeit at a lower rate than when the anastomosis is at a lower level. Most patients undergo cervical esophagogastrostomy for malignancy; thus, the long-term sequelae of an esophagogastric anastomosis in the neck are not of concern. However, patients who have had a cervical esophagogastrostomy for benign dis-ease may develop problems associated with the anastomosis in the fourth or fifth postoperative year that are severe enough to require anastomotic revision. This is less likely in patients who have had a colonic interposition for esophageal replace-ment. Consequently, in patients who have a benign process or a potentially curable carcinoma of the esophagus or cardia, a colonic interposition is used to obviate the late problems associ-ated with a cervical esophagogastrostomy. Colonic interposition for esophageal substitution is a more complex procedure than gastric

1	colonic interposition is used to obviate the late problems associ-ated with a cervical esophagogastrostomy. Colonic interposition for esophageal substitution is a more complex procedure than gastric advancement, with the potential for greater perioperative morbidity, particularly in inexperienced hands.Composite ReconstructionOccasionally, a combination of colon, jejunum, and stomach is the only reconstructive option available. This situation may arise when there has been previous gastric or colonic resection, when dysphagia has recurred after a previous esophageal resec-tion, or following postoperative complications such as ischemia of an esophageal substitute. Although not ideal, combinations of colon, jejunum, and stomach used to restore GI continuity function surprisingly well and allow alimentary reconstruction in an otherwise impossible situation.Vagal Sparing Esophagectomy With Colon InterpositionTraditional esophagectomy typically results in bilateral vagot-omy and its

1	and allow alimentary reconstruction in an otherwise impossible situation.Vagal Sparing Esophagectomy With Colon InterpositionTraditional esophagectomy typically results in bilateral vagot-omy and its attendant consequences. It is likely that symptoms such as dumping, diarrhea, early satiety, and weight loss seen in 15% to 20% of patients postesophagectomy are at least in part, if not completely, due to vagal interruption. The technique of vagal sparing esophagectomy with colon interposition has been described in an effort to avoid the morbidities associated with standard esophagectomy.Through an upper midline abdominal incision, the right and left vagal nerves are identified, circled with a tape, and retracted to the right. A limited, highly selective proximal gas-tric vagotomy is performed along the cephalad 4 cm of the lesser curvature. The stomach is divided with an Endo-GIA stapler just below the GEJ. The colon is prepared to provide an interposed segment as previously described.

1	along the cephalad 4 cm of the lesser curvature. The stomach is divided with an Endo-GIA stapler just below the GEJ. The colon is prepared to provide an interposed segment as previously described. A neck incision is made along the anterior border of the left sternocleidomastoid muscle, and the strap muscles are exposed. The omohyoid muscle is divided at its pulley, and the sternohyoid and sternothyroid muscles are divided at their manubrial insertion. The left carotid sheath is retracted laterally and the thyroid and trachea medially. The left inferior thyroid artery is ligated laterally as it passes under the left common carotid artery. The left recurrent laryngeal nerve is identified and protected. The esophagus is dissected circumfer-entially in an inferior direction, from the left neck to the apex of the right chest, to avoid injury to the right recurrent laryngeal nerve. The esophagus is divided at the level of the thoracic inlet, leaving about 3 to 4 cm of cervical esophagus.

1	to the apex of the right chest, to avoid injury to the right recurrent laryngeal nerve. The esophagus is divided at the level of the thoracic inlet, leaving about 3 to 4 cm of cervical esophagus. The proximal esophagus is retracted anteriorly and to the right with the use of two sutures to keep saliva and oral contents from contaminating the neck wound.Returning to the abdomen, the proximal staple line of the gastric division is opened, and the esophagus is flushed with povidone-iodine solution. A vein stripper is passed up the esophagus into the neck wound. The distal portion of the esophagus in the neck is secured tightly around the stripping cable with “endoloops” and an umbilical tape for a trailer. The tip of the stripper is exchanged for a mushroom head, and the stripper is pulled back into the abdomen, inverting the esopha-gus as it transverses the posterior mediastinum. This maneuver strips the branches of the esophageal plexus off the longitudi-nal muscle of the esophagus,

1	back into the abdomen, inverting the esopha-gus as it transverses the posterior mediastinum. This maneuver strips the branches of the esophageal plexus off the longitudi-nal muscle of the esophagus, preserving the esophageal plexus along with the proximal vagal nerves and the distal vagal nerve trunks. In patients with end-stage achalasia, only the mucosa is secured around the stripping cable, so that it alone is stripped and the dilated muscular wall of the esophagus, with its enriched blood supply, remains. The resulting medi-astinal tunnel, or in the case of achalasia the muscular tube, is dilated with a Foley catheter containing 90 mL of fluid in the balloon. The previously prepared interposed portion of the transverse colon is passed behind the stomach and up through the mediastinal tunnel into the neck. An end-to-end anastomo-sis is performed to the cervical esophagus using a single layer technique. The colon is pulled taut and secured to the left crus with four or five

1	tunnel into the neck. An end-to-end anastomo-sis is performed to the cervical esophagus using a single layer technique. The colon is pulled taut and secured to the left crus with four or five interrupted sutures. Five centimeters below the crura an opening is made in the mesentery adjacent to the colon along its mesenteric border, through which an Endo-GIA stapler is passed and the colon is divided. The proximal end, which is the distal end of the interposed colon, is anasto-mosed high on the posterior fundic wall of the stomach, using a triangular stapling anastomotic technique. This is done by stapling longitudinally the stomach and colon together with a 75-mm Endo-GIA stapler, spreading the base of the incision apart, and closing it with a T-55 stapler. Colonic continuity is reestablished by bringing the proximal right colon to the dis-tal staple line in the left colon and performing an end-to-end anastomosis using a double-layer technique.Brunicardi_Ch25_p1009-p1098.indd

1	by bringing the proximal right colon to the dis-tal staple line in the left colon and performing an end-to-end anastomosis using a double-layer technique.Brunicardi_Ch25_p1009-p1098.indd 109001/03/19 6:06 PM 1091ESOPHAGUS AND DIAPHRAGMATIC HERNIACHAPTER 25Although conceptually appealing, preservation of vagal nerve integrity or the gastric reservoir function after vagal spar-ing esophagectomy only recently has been validated. Banki and associates compared patients undergoing vagal sparing esopha-gectomy to those with conventional esophagectomy and colon or gastric interposition. This study showed that vagal sparing esophagectomy preserved gastric secretion, gastric emptying, meal capacity, and body mass index, compared to esophagogas-trectomy with colon interposition or standard esophagectomy with gastric pull-up. Vagal sparing esophagectomy patients functioned, for the most part, similarly to normal subjects, allowing them to eat a normal meal, free of dumping or diarrhea. These

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1	StomachRobert E. Roses and Daniel T. Dempsey 26chapterThe stomach stores and facilitates the digestion and absorption of ingested food and helps regulate appetite. Treatable dis-eases of the stomach are common, and it is accessible and rela-tively forgiving of surgical manipulation. To provide accurate diagnosis and rational treatment, the physician must under-stand gastric anatomy, physiology, and pathophysiology; this includes a sound understanding of the mechanical, secretory, and endocrine processes by which the stomach accomplishes its important functions and a familiarity with the common benign and malignant gastric disorders. Important historical milestones1-6 that influenced the contemporary understand-ing of gastric disease and surgical therapy are summarized in Table 26-1.ANATOMYAnatomic Relationships and Gross MorphologyThe stomach is the most proximal abdominal organ of the diges-tive tract (Fig. 26-1).7 The part of the stomach attached to the esophagus is called the

1	Relationships and Gross MorphologyThe stomach is the most proximal abdominal organ of the diges-tive tract (Fig. 26-1).7 The part of the stomach attached to the esophagus is called the cardia. Just proximal to the cardia at the gastroesophageal (GE) junction is the anatomically indistinct but physiologically demonstrable lower esophageal sphincter. At the distal end, the readily apparent pyloric sphincter con-nects the stomach to the proximal duodenum. The stomach is relatively fixed at these points, but the majority of the stomach is quite mobile with the shorter lesser curvature on the right and the longer greater curvature on the left.The superior-most part of the stomach is the distensible fundus, bounded superiorly by the diaphragm and laterally by the spleen. The angle of His is where the fundus meets the left side of the GE junction. Generally, the inferior extent of the fun-dus is considered to be the horizontal plane of the GE junction, where the body (corpus) of the stomach

1	the fundus meets the left side of the GE junction. Generally, the inferior extent of the fun-dus is considered to be the horizontal plane of the GE junction, where the body (corpus) of the stomach begins. The body of the stomach contains most of the parietal (oxyntic) cells, some of which are also present in the cardia and fundus. At the angularis incisura, the lesser curvature turns rather abruptly to the right, marking the anatomic beginning of the antrum, which comprises the distal 25% to 30% of the stomach.Anatomy 1099Anatomic Relationships and Gross Morphology / 1099Arterial and Venous Blood Supply / 1100Lymphatic Drainage / 1100Innervation / 1101Histology / 1102Physiology 1105Acid Secretion / 1105Pepsinogen Secretion / 1108Intrinsic Factor / 1108Gastric Mucosal Barrier / 1109Gastric Hormones / 1109Gastric Motility and Emptying / 1110Diagnosis of Gastric Disease 1115Signs and Symptoms / 1115Diagnostic Tests / 1115Helicobacter Pylori Infection 1117Peptic Ulcer

1	/ 1109Gastric Hormones / 1109Gastric Motility and Emptying / 1110Diagnosis of Gastric Disease 1115Signs and Symptoms / 1115Diagnostic Tests / 1115Helicobacter Pylori Infection 1117Peptic Ulcer Disease 1120Pathophysiology and Etiology / 1121Clinical Manifestations / 1123Diagnosis / 1123Complications / 1124Medical Treatment of Peptic Ulcer Disease / 1125Surgical Treatment of Peptic Ulcer Disease / 1127Bleeding Peptic Ulcer / 1131Operation for Bleeding Peptic Ulcer / 1133Perforated Peptic Ulcer / 1134Obstructing Peptic Ulcer / 1136Intractable or Nonhealing Peptic Ulcer / 1136Zollinger-Ellison Syndrome / 1136Stress Gastritis and Stress Ulcer 1137Atrophic Gastritis 1138Malignant Neoplasms of the Stomach 1139Adenocarcinoma / 1139Pathology / 1143Gastric Lymphoma / 1149Gastrointestinal Stromal Tumor / 1149Gastric Neuroendocrine Tumors / 1151Benign Gastric Neoplasms 1152Leiomyoma / 1152Lipoma / 1152Gastroparesis / 1152Massive Upper Gastrointestinal Bleeding / 1152Isolated Gastric

1	Stromal Tumor / 1149Gastric Neuroendocrine Tumors / 1151Benign Gastric Neoplasms 1152Leiomyoma / 1152Lipoma / 1152Gastroparesis / 1152Massive Upper Gastrointestinal Bleeding / 1152Isolated Gastric Varices / 1153Hypertrophic Gastropathy (Ménétrier’s Disease) / 1153Watermelon Stomach (Gastric Antral Vascular Ectasia) / 1154Dieulafoy’s Lesion / 1154Bezoars/Diverticula / 1154Foreign Bodies / 1154Mallory-Weiss Syndrome / 1155Volvulus / 1155Gastrostomy 1155Postgastrectomy Problems 1156Dumping Syndrome / 1156Diarrhea / 1157Gastric Stasis / 1157Bile Reflux Gastritis and Esophagitis / 1158Roux Syndrome / 1158Gallstones / 1159Weight Loss / 1159Anemia / 1159Bone Disease / 1159Laparoscopic Gastric Operations 1159Brunicardi_Ch26_p1099-p1166.indd 109901/03/19 7:11 PM 1100The liver, colon, spleen, pancreas, and occasionally the kidney, abut the stomach (Fig. 26-2). The left lateral segment of the liver usually obscures part of the anterior stomach. Infe-riorly, the stomach is attached to

1	pancreas, and occasionally the kidney, abut the stomach (Fig. 26-2). The left lateral segment of the liver usually obscures part of the anterior stomach. Infe-riorly, the stomach is attached to the transverse colon by the gastrocolic omentum. The lesser curvature is tethered to the liver by the hepatogastric ligament, also referred to as the lesser omentum or pars flaccida. Posterior to the stomach is the lesser omental bursa and the pancreas.Arterial and Venous Blood SupplyThe stomach is the most richly vascularized portion of the ali-mentary tube with ample blood flow and a dense intramural vascular anastomotic network. The large majority of the gastric blood supply is from the celiac axis via four named arteries (Fig. 26-3). The left and right gastric arteries form an anasto-motic arcade along the lesser gastric curvature, and the right and left gastroepiploic arteries form an arcade along the greater gastric curvature. The left gastric artery is consistently the larg-est artery to

1	along the lesser gastric curvature, and the right and left gastroepiploic arteries form an arcade along the greater gastric curvature. The left gastric artery is consistently the larg-est artery to the stomach and usually arises directly from the celiac trunk and divides into an ascending and descending branch along the lesser gastric curvature. Approximately 20% of the time, the left gastric artery supplies an aberrant vessel that travels in the gastrohepatic ligament (lesser omentum) to the left side of the liver. Rarely, this is the only arterial blood supply to this part of the liver (replaced left hepatic artery), and inadvertent ligation may lead to clinically significant hepatic ischemia. The more common smaller accessory left hepatic artery may be ligated without significant consequences.The second largest artery to the stomach is the right gas-troepiploic artery, which consistently arises from the gastro-duodenal artery behind the first portion of the duodenum. The left

1	consequences.The second largest artery to the stomach is the right gas-troepiploic artery, which consistently arises from the gastro-duodenal artery behind the first portion of the duodenum. The left gastroepiploic artery arises from the splenic artery, and, together with the right gastroepiploic artery, forms the rich gastroepiploic arcade along the greater curvature. The right gastric artery usually arises from the hepatic artery near the pylorus and hepatoduodenal ligament and runs proximally along the distal stomach. In the fundus along the proximal greater curvature, the short gastric arteries and veins arise from the splenic circulation. There also may be additional vascular branches to the proximal stomach from the phrenic and splenic circulation.The veins draining the stomach generally parallel the arteries. The left gastric (coronary vein) and right gastric veins usually drain into the portal vein, though occasionally the coro-nary vein drains into the splenic vein. The right

1	parallel the arteries. The left gastric (coronary vein) and right gastric veins usually drain into the portal vein, though occasionally the coro-nary vein drains into the splenic vein. The right gastroepiploic vein drains into the superior mesenteric vein near the inferior border of the pancreatic neck, and the left gastroepiploic vein drains into the splenic vein.The richness of the gastric blood supply and its many anastomotic connections have important clinical implications. At least two of the four named gastric arteries may be occluded or ligated without inducing gastric ischemia. This is done rou-tinely when the stomach is mobilized and pedicled on the right gastric and right gastroepiploic vessels to reach into the neck as an esophageal replacement (see Chapter 25) or during sleeve gastrectomy for weight loss when the gastroepiploic arcade is interrupted proximally and distally prior to gastric resection (see Chapter 27). Following radical subtotal gastrectomy dur-ing which the

1	gastrectomy for weight loss when the gastroepiploic arcade is interrupted proximally and distally prior to gastric resection (see Chapter 27). Following radical subtotal gastrectomy dur-ing which the right and left gastric arteries and both gastroepi-ploic arteries are all ligated, the gastric remnant is adequately supplied by short gastric arteries as long as the splenic artery is patent and intact. Angiographic control of gastric bleeding from a deep ulcer or tumor often requires embolization of more than one feeding artery. Because of the rich venous interconnections in the stomach, a transjugular intrahepatic portosystemic shunt (TIPSS) can effectively decompress esophagogastric varices in patients with portal hypertension.8,9Lymphatic DrainageGenerally speaking, the gastric lymphatics parallel the blood vessels (Fig. 26-4).10 The cardia and medial half of the corpus commonly drain to nodes along the left gastric and celiac axis. The lesser curvature side of the antrum usually

1	parallel the blood vessels (Fig. 26-4).10 The cardia and medial half of the corpus commonly drain to nodes along the left gastric and celiac axis. The lesser curvature side of the antrum usually drains to the right gastric and pyloric nodes, while the greater curvature half of the distal stomach drains to the nodes along the right gas-troepiploic chain. The proximal greater curvature side of the stomach usually drains into nodes along the left gastroepiploic or splenic hilum. The nodes along both the greater and lesser Key Points1 Whenever testing suggests Helicobacter pylori infec-tion, treatment should be initiated and eradication confirmed.2 Lifelong acid suppression should be considered in any patient admitted to a hospital because of peptic ulcer dis-ease. Acid suppressive medication may be equivalent to sur-gical vagotomy in preventing recurrent peptic ulcer or ulcer complications.3 If possible, gastric resection for peptic ulcer is avoided in the asthenic or high-risk

1	may be equivalent to sur-gical vagotomy in preventing recurrent peptic ulcer or ulcer complications.3 If possible, gastric resection for peptic ulcer is avoided in the asthenic or high-risk patient.4 Though less common in the United States, gastric cancer is a major cause of cancer-related morbidity and mortality worldwide.5 Diagnostic laparoscopy with peritoneal lavage should be considered in the evaluation of clinical stage 2 and 3 patients with gastric cancer.6 Multimodality therapy for gastric cancer, including resec-tion in combination with perioperative chemotherapy or adjuvant chemoradiotherapy is associated with a survival advantage compared to surgery alone.7 Most patients with primary gastric lymphoma can be treated without gastric resection.8 Localized gastrointestinal stromal tumors of the stomach are treated with full thickness excision. Adjuvant (or neo-adjuvant) imatinib is indicated for higher-risk lesions.9 Gastric neuroendocrine tumors may arise in the presence

1	tumors of the stomach are treated with full thickness excision. Adjuvant (or neo-adjuvant) imatinib is indicated for higher-risk lesions.9 Gastric neuroendocrine tumors may arise in the presence (types 1 and 2) or absence (type 3) of hypergastrinemia. Type 3 gastric neuroendocrine tumors should usually be treated with subtotal gastrectomy and regional lymphadenectomy.10 Roux-en-Y gastrojejunostomy with a large (>50%) proxi-mal gastric remnant should be avoided because marginal ulceration and/or gastric stasis (Roux syndrome) may become problematic.Brunicardi_Ch26_p1099-p1166.indd 110001/03/19 7:11 PM 1101STOMACHCHAPTER 26curvature commonly drain into the celiac nodal basin. There is a rich anastomotic network of lymphatics that drain the stomach, often in a somewhat unpredictable fashion. Thus, a tumor aris-ing in the distal stomach may give rise to positive lymph nodes in the splenic hilum. The rich intramural plexus of lymphatics and veins accounts for the fact that there can be

1	Thus, a tumor aris-ing in the distal stomach may give rise to positive lymph nodes in the splenic hilum. The rich intramural plexus of lymphatics and veins accounts for the fact that there can be microscopic evi-dence of malignant cells in the gastric wall at a resection margin that is several centimeters away from palpable malignant tumor. It also helps explain the not infrequent finding of positive lymph nodes, which may be many centimeters away from the primary tumor, with closer nodes that are uninvolved.Not surprisingly, extensive and meticulous lymphadenec-tomy is considered by many surgeons to be an important part of an operation for gastric cancer. Surgeons and pathologists have numbered the primary and secondary lymph node groups to which the stomach drains (see Fig. 26-4).11,12Innervation13The vagus nerves provide the extrinsic parasympathetic innerva-tion to the stomach, and acetylcholine is the most important neu-rotransmitter. From the vagal nucleus in the floor of the

1	vagus nerves provide the extrinsic parasympathetic innerva-tion to the stomach, and acetylcholine is the most important neu-rotransmitter. From the vagal nucleus in the floor of the fourth cerebral ventricle, the vagus traverses the neck in the carotid sheath and enters the mediastinum, where it gives off the recur-rent laryngeal nerve and divides into several branches around the esophagus. These branches come together again above the esophageal hiatus and form the left (anterior) and right Table 26-1Historic milestones in gastric surgeryDATEEVENTDATEEVENT350 b.c.– 201 a.d. 1363 1586 1600–1700 16881737 1833 1869 1875 1879 1880 1880 1881 1884 1885Existence of gastric ulceration was acknowledged by Diocles of Carystos (350 b.c.), Celsus, and Galen (131–201 a.d.).Guy de Chauliac describes closure of gastric wound.Marcellus Donatus of Mantua describes gastric ulcer at autopsy.Reports of surgeons cutting stomach to remove foreign bodies.Muralto describes duodenal ulcer at

1	describes closure of gastric wound.Marcellus Donatus of Mantua describes gastric ulcer at autopsy.Reports of surgeons cutting stomach to remove foreign bodies.Muralto describes duodenal ulcer at autopsy.Morgagni describes both gastric and duodenal ulcer at autopsy.William Beaumont reports data recorded during his care of Alexis St. Martin who developed a gastric fistula from a left upper quadrant musket wound.Maury reportedly performs feeding gastrostomy to palliate esophageal stricture following consultation with Samuel D. Gross.Sidney Jones in London publishes the first successful gastrostomy for feeding.Paen performed distal gastrectomy and gastroduodenostomy. The patient died 5 d later.Rydygier resected a distal gastric cancer, and the patient died 12 h later.Billroth resects distal gastric cancer and performs gastroduodenostomy (Billroth I). Patient Therese Heller recovers and survives 4 mo.Anton Wolfler performs loop gastrojejunostomy to palliate an obstructing distal gastric

1	gastric cancer and performs gastroduodenostomy (Billroth I). Patient Therese Heller recovers and survives 4 mo.Anton Wolfler performs loop gastrojejunostomy to palliate an obstructing distal gastric cancer.Rydygier reports an unsuccessful gastrojejunostomy for benign gastric outlet obstruction.Billroth performs a successful distal gastrectomy and gastrojejunostomy (Billroth II) for gastric cancer.1886188818921902 1891–1913 1920–1950 1943 1952 1953 1955 1957 1980–2000 1990–current 1995–current2000–currentHeineke performs pyloroplasty.Mikulicz performs similar operation.Jaboulay describes bypassing the intact pylorus with gastroduodenostomy.Finney from Baltimore describes pyloroplasty technique.Different techniques of gastrostomy are described by Witzel (1891), Stamm (1894), and Janeway (1913).Subtotal gastrectomy grows popular as an operation for peptic ulcer. Von Haberer and Finsterer proponents.Dragstedt and Owen describe transthoracic truncal vagotomy to treat peptic

1	and Janeway (1913).Subtotal gastrectomy grows popular as an operation for peptic ulcer. Von Haberer and Finsterer proponents.Dragstedt and Owen describe transthoracic truncal vagotomy to treat peptic ulcer disease. By the early 1950s, it is well recognized that some patients developed gastric stasis after this procedure, and transabdominal truncal vagotomy and drainage (pyloroplasty or gastrojejunostomy) become a standard ulcer operation.Farmer and Smithwick describe good results with truncal vagotomy and hemigastrectomy for peptic ulcer.Edwards and Herrington (Nashville) describe truncal vagotomy and antrectomy for peptic ulcer.Zollinger and Ellison describe the eponymous syndrome.Griffith and Harkins (Seattle) describe parietal cell vagotomy (highly selective vagotomy) for the elective treatment of peptic ulcer disease.Japanese surgeons and other surgical groups from East Asia demonstrate that more aggressive lymphadenectomy may improve survival in patients with gastric

1	elective treatment of peptic ulcer disease.Japanese surgeons and other surgical groups from East Asia demonstrate that more aggressive lymphadenectomy may improve survival in patients with gastric cancer.Evolving role of laparoscopic techniques in the treatment of surgical gastric disease.Dramatic increase in bariatric operations.Development of natural orifice translumenal endoscopic surgery, such as transgastric appendectomy and peroral pyloromyotomy.Development of robotic gastrectomy.Brunicardi_Ch26_p1099-p1166.indd 110101/03/19 7:11 PM 1102SPECIFIC CONSIDERATIONS PART II(posterior) vagal trunks (mnemonic LARP). Near the GE junc-tion the anterior vagus sends a branch (or branches) to the liver in the gastrohepatic ligament, and continues along the lesser curvature as the anterior nerve of Latarjet (Fig. 26-5). Simi-larly, the posterior vagus sends branches to the celiac plexus and continues along the posterior lesser curvature. The nerves of Latarjet send segmental branches

1	nerve of Latarjet (Fig. 26-5). Simi-larly, the posterior vagus sends branches to the celiac plexus and continues along the posterior lesser curvature. The nerves of Latarjet send segmental branches to the body of the stomach before they terminate near the angularis incisura as the “crow’s foot,” sending branches to the antropyloric region. There may be additional branches to the distal stomach and pylorus that travel near the right gastric and/or gastroepiploic arteries. In 50% of patients, there are more than two vagal nerves at the esophageal hiatus. The branch that the posterior vagus sends to the posterior fundus is termed the criminal nerve of Grassi. This branch typically arises above the esophageal hiatus and is easily missed during truncal or highly selective vagotomy (HSV). Vagal fibers originating in the brain synapse with neu-rons in Auerbach’s myenteric plexus and Meissner’s submu-cosal plexus. In the stomach, the vagus nerves affect secretion (including acid), motor

1	Vagal fibers originating in the brain synapse with neu-rons in Auerbach’s myenteric plexus and Meissner’s submu-cosal plexus. In the stomach, the vagus nerves affect secretion (including acid), motor function, and mucosal bloodflow and cytoprotection. They also play a role in appetite control and per-haps even mucosal immunity and inflammation.14,15 Most of the axons contained in the vagal trunks are afferent (i.e., carrying stimuli from the viscera to the brain).The extrinsic sympathetic nerve supply to the stomach originates at spinal levels T5 through T10 and travels in the splanchnic nerves to the celiac ganglion. Postganglionic sympa-thetic nerves then travel from the celiac ganglion to the stomach along the blood vessels.Neurons in the myenteric and submucosal plexuses consti-tute the intrinsic nervous system of the stomach. There may be more intrinsic gastric neurons than extrinsic neurons, but their function is poorly understood.The characterization of the vagus as the

1	the intrinsic nervous system of the stomach. There may be more intrinsic gastric neurons than extrinsic neurons, but their function is poorly understood.The characterization of the vagus as the cholinergic system and the sympathetic system as the adrenergic system of innerva-tion is a misleading oversimplification. Although acetylcholine is an important neurotransmitter mediating vagal function, and epinephrine is important in the sympathetic nerves, both sys-tems (as well as the intrinsic neurons) have various and diverse neurotransmitters, including cholinergic, adrenergic, and pepti-dergic (e.g., substance P and somatostatin).HistologyThere are four distinct layers of the gastric wall: mucosa, sub-mucosa, muscularis propria, and serosa (Fig. 26-6).7 The inner layer of the stomach is the mucosa, which is lined with colum-nar epithelial cells of various types. Beneath the basement mem-brane of the epithelial cells is the lamina propria, which contains connective tissue, blood

1	is the mucosa, which is lined with colum-nar epithelial cells of various types. Beneath the basement mem-brane of the epithelial cells is the lamina propria, which contains connective tissue, blood vessels, nerve fibers, and inflammatory cells. Beneath the lamina propria is a thin muscle layer called the muscularis mucosa, the deep boundary of the mucosal layer of the gut. The epithelium, lamina propria, and muscularis mucosa constitute the mucosa (Fig. 26-7).16 The epithelium of the gastric mucosa is columnar glandular. Scanning electron micrographs show a smooth mucosal carpet punctuated by the openings of the gastric glands or units. The gastric glands are lined with different types of epithelial cells, depending upon their loca-tion in the stomach (Fig. 26-8 and Table 26-2).17,18 There are also endocrine cells present in the gastric glands. Progenitor or stem cells in the isthmus and base of the glands differentiate and BodyFundusEsophagusCardiaPylorusDuodenumPyloric antrum

1	There are also endocrine cells present in the gastric glands. Progenitor or stem cells in the isthmus and base of the glands differentiate and BodyFundusEsophagusCardiaPylorusDuodenumPyloric antrum Lessercurvature GreatercurvatureFigure 26-1. Anatomic regions of the stomach. (Reproduced with permission from Zuidema GD, Yeo CJ: Shackelford’s Surgery of the Alimentary Tract, 5th ed. Vol. II. Philadelphia, PA: Elsevier/Saunders; 2002.)Figure 26-2. Anatomic relationships of the stomach. (Reproduced with permission from Zuidema GD, Yeo CJ: Shackelford’s Surgery of the Alimentary Tract, 5th ed. Vol. II. Philadelphia, PA: Elsevier/Saunders; 2002.)Liver (left lobe) Celiac trunk(splenic, gastric, hepatic)Gastrohepatic omentum Stomach(posterior wall)SpleenPancreasTransverse colonLesser omental spaceGastrocolic ligamentRight kidneyHepatoduodenal ligamentGallbladderT lictBrunicardi_Ch26_p1099-p1166.indd 110201/03/19 7:11 PM 1103STOMACHCHAPTER 26replenish

1	colonLesser omental spaceGastrocolic ligamentRight kidneyHepatoduodenal ligamentGallbladderT lictBrunicardi_Ch26_p1099-p1166.indd 110201/03/19 7:11 PM 1103STOMACHCHAPTER 26replenish sloughed cells on a regular basis. Genetic studies show that there are several different subpopulations of stem cells in the gastric glands and that during conditions of stress even chief cells exhibit the plasticity required to regenerate other types of gastric epithelial cells.19 Throughout the stomach, the luminal carpet consists primarily of mucus-secreting surface epithelial cells (SECs) that extend down into the gland pits for variable distances. These cells also secrete bicarbonate and play an important role in protecting the stomach from injury due to acid, pepsin, and/or ingested irritants. In fact, all epithelial cells of the stomach (except the endocrine cells) contain carbonic anhydrase and are capable of producing bicarbonate.In the cardia, the gastric glands are branched and

1	In fact, all epithelial cells of the stomach (except the endocrine cells) contain carbonic anhydrase and are capable of producing bicarbonate.In the cardia, the gastric glands are branched and secrete primarily mucus and bicarbonate, and little acid. In the fundus and body, the glands are more tubular, and the pits are deep. Parietal and chief cells are common in these glands (Fig. 26-9). Histamine-secreting enterochromaffin-like (ECL) cells and somatostatin-secreting D cells are also found. Parietal cells Figure 26-3. Arterial blood supply to the stomach. a. = artery; v. = vein. (Reproduced with permission from Zuidema GD, Yeo CJ: Shackelford’s Surgery of the Alimentary Tract, 5th ed. Vol. II. Philadelphia, PA: Elsevier/Saunders; 2002.)StomachCeliac a.Short gastric arteries(vasa brevia)SpleenDescending colonPancreasDuodenumTransverse colonPylorusSup. pancreatic duodenal a.Abdominal AortaInferior pancreatic duodenal a.Hepatic a.Left gastric a.Branchesto

1	arteries(vasa brevia)SpleenDescending colonPancreasDuodenumTransverse colonPylorusSup. pancreatic duodenal a.Abdominal AortaInferior pancreatic duodenal a.Hepatic a.Left gastric a.Branchesto greateromentumSplenic a. & v.L. gastroepiploic a. Sup. mesenteric a. & v. Inferior mesenteric a.Ileocolic a.Jejunum21534a4b6128971011Figure 26-4. Lymph node stations draining the stomach according to the Japanese Research Society for Gastric Cancer. Stations 3 to 6 are commonly removed with D1 gastrectomy. Stations 1, 2, and 7 to 12 are commonly removed with D2 gastrectomy. (Reproduced with permis-sion from Hermanek P, Hutter RVP, Sobin LH, et al: TNM Atlas: Illustrated Guide to the TNM/pTNM Classification of Malignant Tumours, 4th ed. Berlin: Springer-Verlag; 1997.)Brunicardi_Ch26_p1099-p1166.indd 110301/03/19 7:11 PM 1104SPECIFIC CONSIDERATIONS PART IIRight vagus n.Hepatic br. left vagusCeliac br. rt. vagusNerve of LaterjetPyloric br.Left vagus n.Figure 26-5. Vagal

1	110301/03/19 7:11 PM 1104SPECIFIC CONSIDERATIONS PART IIRight vagus n.Hepatic br. left vagusCeliac br. rt. vagusNerve of LaterjetPyloric br.Left vagus n.Figure 26-5. Vagal innervation of the stomach. br. = branch; n. = nerve; rt. = right. (Reproduced with permission from Menguy R: Surgery of Peptic Ulcer. Philadelphia, PA: Elsevier/Saunders; 1976.)Surface epitheliumGastric pitsLymphoid noduleLamina propria mucosaSubmucosaSmooth muscle layersSerosaMuscularis mucosaeFigure 26-6. Layers of the gastric wall. (Reproduced with permis-sion from Fawcett DW: Bloom and Fawcett’s Textbook of Histology, 11th ed. Philadelphia, PA: Elsevier/Saunders; 1986.)Figure 26-7. Gastric mucosa. (Used with permission from Emma Furth, MD.)Brunicardi_Ch26_p1099-p1166.indd 110401/03/19 7:11 PM 1105STOMACHCHAPTER 26secrete acid and intrinsic factor into the gastric lumen, and bicarbonate into the intercellular space. They have a character-istic ultrastructural appearance with secretory canaliculi

1	26secrete acid and intrinsic factor into the gastric lumen, and bicarbonate into the intercellular space. They have a character-istic ultrastructural appearance with secretory canaliculi (deep invaginations of the surface membrane) and cytoplasmic tubulo-vesicles containing the acid-producing apparatus H+/K+-ATPase (proton pump) (see Fig. 26-9). There are numerous mitochon-dria; in fact, the parietal cell is the most mitochondria-rich cell in the body. When the parietal cell is stimulated, the cytoplasmic tubulovesicles fuse with the membrane of the secretory cana-liculus; when acid production ceases, the process is reversed. Arguably, parietal cells produce the only truly essential sub-stance made by the stomach (i.e., intrinsic factor). Parietal cells tend to occupy the midportion of the gastric glands found in the corpus of the stomach.Chief cells (also called zymogenic cells) secrete pep-sinogen I, which is maximally activated at a pH of 2.5. They tend to be clustered toward the

1	gastric glands found in the corpus of the stomach.Chief cells (also called zymogenic cells) secrete pep-sinogen I, which is maximally activated at a pH of 2.5. They tend to be clustered toward the base of the gastric glands and have a low columnar shape. Ultrastructurally, chief cells have the characteristics of protein-synthesizing cells: basal granular endoplasmic reticulum, supranuclear Golgi apparatus, and api-cal zymogen granules (Fig. 26-10). When stimulated, the chief cells produce two immunologically distinct proenzyme forms of pepsinogen: predominantly pepsinogen I and some pepsinogen II, most of which is produced by SECs. These proenzymes are activated in an acidic luminal environment.In the antrum, the gastric glands are again more branched and shallow, parietal cells are rare, and gastrin-secreting G cells and somatostatin-secreting D cells are present. A variety of hormone-secreting cells are present in various proportions throughout the gastric mucosa (Fig. 26-11).20

1	and gastrin-secreting G cells and somatostatin-secreting D cells are present. A variety of hormone-secreting cells are present in various proportions throughout the gastric mucosa (Fig. 26-11).20 Histologic analy-sis suggests that in the normal stomach, 13% of the epithelial cells are oxyntic (parietal) cells, 44% are chief (zymogenic) cells, 40% are mucous cells, and 3% are endocrine cells. In gen-eral, the antrum produces gastrin but not acid, and the proximal stomach produces acid but not gastrin. The border between the corpus and antrum migrates proximally with age (especially on the lesser curvature side of the stomach).Deep to the muscularis mucosa is the submucosa, which is rich in branching blood vessels, lymphatics, collagen, vari-ous inflammatory cells, and nerve fibers and ganglion cells of Meissner’s autonomic submucosal plexus. The collagen-rich submucosa gives strength to GI anastomoses. The mucosa and submucosa are folded into the grossly visible gastric rugae, which

1	cells of Meissner’s autonomic submucosal plexus. The collagen-rich submucosa gives strength to GI anastomoses. The mucosa and submucosa are folded into the grossly visible gastric rugae, which tend to flatten out as the stomach becomes distended.Below the submucosa is the thick muscularis propria (also referred to as the muscularis externa), which consists of an incomplete inner oblique layer, a complete middle circular layer (continuous with the esophageal circular muscle and the circular muscle of the pylorus), and a complete outer longitudinal layer (continuous with the longitudinal layer of the esophagus and duodenum). Within the muscularis propria is the rich network of autonomic ganglia and nerves that make up Auerbach’s myen-teric plexus. Specialized pacemaker cells, the interstitial cells of Cajal (ICC), also are present.The outer layer of the stomach is the serosa, also known as the visceral peritoneum. This layer provides significant tensile strength to gastric anastomoses.

1	cells of Cajal (ICC), also are present.The outer layer of the stomach is the serosa, also known as the visceral peritoneum. This layer provides significant tensile strength to gastric anastomoses. When tumors originating in the mucosa penetrate and breach the serosa, microscopic or gross peritoneal metastases are common, presumably from shedding of tumor cells that would not have occurred if the serosa had not been penetrated. In this way, the serosa may be thought of as an outer envelope of the stomach.PHYSIOLOGYThe stomach stores food and facilitates digestion through a variety of secretory and motor functions. Important secretory functions include the production of acid, pepsin, intrinsic factor, mucus, and a variety of GI hormones. Important motor func-tions include food storage (receptive relaxation and accommo-dation), grinding and mixing, controlled emptying of ingested food, and periodic interprandial “housekeeping.”Acid SecretionHydrochloric acid in the stomach hastens both

1	relaxation and accommo-dation), grinding and mixing, controlled emptying of ingested food, and periodic interprandial “housekeeping.”Acid SecretionHydrochloric acid in the stomach hastens both the physical and (with pepsin) the biochemical breakdown of ingested food. In an acidic environment, pepsin and acid facilitate proteolysis. Gastric acid also inhibits the proliferation of ingested patho-gens, which protects against both infectious gastroenteritis and intestinal bacterial overgrowth and helps to maintain a healthy gastrointestinal microbiome.21 Long-term acid suppression with proton pump inhibitors (PPIs) has been associated with an Surface mucous cellsParietal cellsMucous neck cellsArgentaffin cellChief cellsGastric pitIsthmusNeckBaseFigure 26-8. Mammalian gastric gland from the body of the stom-ach. (Reproduced with permission from Ito S, Winchester RJ: The fine structure of the gastric mucosa in the bat, J Cell Biol. 1963 Mar;16(3):541-577.)Brunicardi_Ch26_p1099-p1166.indd

1	of the stom-ach. (Reproduced with permission from Ito S, Winchester RJ: The fine structure of the gastric mucosa in the bat, J Cell Biol. 1963 Mar;16(3):541-577.)Brunicardi_Ch26_p1099-p1166.indd 110501/03/19 7:11 PM 1106SPECIFIC CONSIDERATIONS PART IIincreased risk of community-acquired Clostridium difficile coli-tis and other gastroenteritis, presumably because of the absence of this protective germicidal barrier.22,23Parietal Cell. The parietal cell is stimulated to secrete acid (Fig. 26-12) when one or more of three membrane receptor types is stimulated by acetylcholine (from vagally stimulated enteric neurons), gastrin (from G cells), or histamine (from ECL cells).7,24,25 The enzyme H+/K+-ATPase is the parietal cell proton pump. It is stored within the intracellular tubulovesicles and is the final common pathway for gastric acid secretion. When the parietal cell is stimulated, there is a cytoskeletal rearrangement and fusion of the tubulovesicles with the apical membrane

1	and is the final common pathway for gastric acid secretion. When the parietal cell is stimulated, there is a cytoskeletal rearrangement and fusion of the tubulovesicles with the apical membrane of the secretory canaliculus. The heterodimer assembly of the enzyme subunits into the microvilli of the secretory canalicu-lus results in acid secretion, with extracellular potassium being exchanged for cytosolic hydrogen. Although electroneutral, this is an energy-requiring process because the hydrogen is secreted against a gradient of at least 1 million-fold, which explains why the parietal cell is packed with energy producing mito-chondria. During acid production, potassium and chloride are also secreted into the apical secretory canaliculus through sepa-rate channels, providing potassium to exchange for H+ via the H+/K+-ATPase, and chloride to accompany the secreted hydrogen. At the basolateral membrane, the combined activity of various SCMTVFigure 26-9. Ultrastructural features of the

1	exchange for H+ via the H+/K+-ATPase, and chloride to accompany the secreted hydrogen. At the basolateral membrane, the combined activity of various SCMTVFigure 26-9. Ultrastructural features of the parietal (oxyntic) cell. SC = secretory canaliculus; M = mitochondria; TV = tubulovesicle. (Reproduced with permission from Ming S-C, Goldman H: Pathol-ogy of the Gastrointestinal Tract, 2nd ed. Baltimore, MD: Williams & Wilkins; 1998.)ZGGAGERFigure 26-10. Ultrastructural features of the chief (zymogenic) cell. GA = Golgi apparatus; GER = granular endoplasmic reticu-lum; ZG = zymogen granule. (Reproduced with permission from Ming S-C, Goldman H: Pathology of the Gastrointestinal Tract, 2nd ed. Baltimore, MD: Williams & Wilkins; 1998.)Table 26-2Epithelial cells of the stomachCELL TYPEDISTINCTIVE ULTRASTRUCTURAL FEATURESMAJOR FUNCTIONSSurface-foveolar mucous cellsApical stippled granules up to 1 μm in diameterProduction of neutral glycoprotein and bicarbonate to form a gel on the gastric

1	ULTRASTRUCTURAL FEATURESMAJOR FUNCTIONSSurface-foveolar mucous cellsApical stippled granules up to 1 μm in diameterProduction of neutral glycoprotein and bicarbonate to form a gel on the gastric luminal surface; neutralization of hydrochloric acidaMucous neck cellHeterogeneous granules 1–2 μm in diameter dispersed throughout the cytoplasmProgenitor cell for all other gastric epithelial cells; glycoprotein production; production of pepsinogens I and IIOxyntic (parietal) cellSurface membrane invaginations (canaliculi); tubulovesicle structures; numerous mitochondriaProduction of hydrochloric acid; production of intrinsic factor; production of bicarbonateChief cellModerately dense apical granules up to 2 μm in diameter; prominent supranuclear Golgi apparatus; extensive basolateral granular endoplasmic reticulumProduction of pepsinogens I and II, and of lipaseCardiopyloric mucous cellMixture of granules like those in mucous neck and chief cells; extensive basolateral granular endoplasmic

1	reticulumProduction of pepsinogens I and II, and of lipaseCardiopyloric mucous cellMixture of granules like those in mucous neck and chief cells; extensive basolateral granular endoplasmic reticulumProduction of glycoprotein; production of pepsinogen IIEndocrine cellsSee Figure 26-11 aBicarbonate is probably produced by other gastric epithelial cells in addition to surface-foveolar mucous cells.Reproduced with permission from Ming S-C, Goldman H: Pathology of the Gastrointestinal Tract, 2nd ed. Baltimore, MD: Williams & Wilkins; 1998.Brunicardi_Ch26_p1099-p1166.indd 110601/03/19 7:11 PM 1107STOMACHCHAPTER 26cotransporters and ion exchangers accomplishes intracellular pH regulation and electrolyte homeostasis.24The normal human stomach contains approximately 1 billion parietal cells, and total gastric acid production is pro-portional to parietal cell mass. Almost all of the parietal cells are in the proximal 2/3 stomach, though there are some parietal cells found in gastric antral

1	total gastric acid production is pro-portional to parietal cell mass. Almost all of the parietal cells are in the proximal 2/3 stomach, though there are some parietal cells found in gastric antral glands. The potent acid-suppressing PPI drugs irreversibly interfere with the function of the H+/K+-ATPase molecule. These agents must be incorporated into the activated enzyme to be effective and thus work best when taken before or during a meal (when the parietal cell is stimulated). When PPI therapy is stopped, acid secretory capability gradu-ally returns (within days) as new H+/K+-ATPase is synthesized.Gastrin, acetylcholine, and histamine stimulate the parietal cell to secrete hydrochloric acid (see Fig. 26-12). Gastrin binds to type B cholecystokinin (CCK2) receptors on ECL cells and stimulates ECL cell histamine release, which binds to H2 recep-tors on the parietal cell. This stimulates adenylatecyclase (via a G-protein–linked mechanism) and increases cAMP which acti-vates protein

1	ECL cell histamine release, which binds to H2 recep-tors on the parietal cell. This stimulates adenylatecyclase (via a G-protein–linked mechanism) and increases cAMP which acti-vates protein kinases, leading to increased levels of phospho-proteins and activation of the proton pump. Gastrin also binds to CCK2 receptors on the parietal cell, but this is less important for acid secretion than the gastrin effect on ECL cells. Acetyl-choline from intrinsic neurons binds to M3 muscarinic recep-tors on the parietal cell, which (like gastrin binding to CCK2 receptors) stimulates phospholipase C via a G-protein–linked mechanism leading to increased production of inositol trispho-sphate from membrane bound phospholipids. Inositol trisphos-phate stimulates the release of calcium from intracellular stores, which leads to activation of protein kinases and activation of H+/K+-ATPase. Somatostatin released from mucosal D cells in the antral and oxcyntic mucosa in response to luminal acid binds to

1	stores, which leads to activation of protein kinases and activation of H+/K+-ATPase. Somatostatin released from mucosal D cells in the antral and oxcyntic mucosa in response to luminal acid binds to SSTR2 receptors on parietal cells and inhibits acid release directly. Somatostatin also inhibits acid secretion in a paracrine fashion, binding to nearby ECL cells in the oxcyntic mucosa and decreasing histamine release, and binding to nearby antral G cells to inhibit gastrin release.26Physiologic Acid Secretion.27 Food ingestion is the physi-ologic stimulus for acid secretion (Fig. 26-13). The acid secre-tory response that occurs after a meal is traditionally described Oxyntic mucosaOther3%D19%G49%EC29%Other14%D26%EC25%ECL35%Pyloric mucosaFigure 26-11. Endocrine cells of the stomach—proportion by site. D = d cell (somatostatin); EC = enterochromaffin cell; ECL = enterochromaffin-like cell (histamine); G = g cell (gastrin). (Repro-duced with permission from Feldman M, Friedman LS,

1	by site. D = d cell (somatostatin); EC = enterochromaffin cell; ECL = enterochromaffin-like cell (histamine); G = g cell (gastrin). (Repro-duced with permission from Feldman M, Friedman LS, Sleisenger MH, et al: Sleisenger and Fordtran’s Gastrointestinal and Liver Disease, 7th ed. Philadelphia, PA: Elsevier/Saunders; 2002.)Adenylate cyclaseATPHistamineInterstitiumLumenSomatostatinGi proteinGs proteinIP3PIP2Ca++K+ channelK+ClchannelCl-H+K+CCK8 receptorM3 receptorH+/K+-ATPaseGs proteinActivation ofother kinasescAMPActivatesprotein kinasesGastrinAcetylcholinePLCSomatostatinreceptorH2 receptorGs proteinFigure 26-12. Control of acid secretion in the parietal cell. ATP = adenosine triphosphate; cAMP = cyclic adenosine monophosphate; CCK = cholecystokinin; H2 = histamine 2; IP3 = inositol trisphosphate; PIP2 = phosphatidylinositol 4,5-bisphosphate; PLC = phospholipase C. (Reproduced with permission from Zuidema GD, Yeo CJ: Shackelford’s Surgery of the Alimentary Tract, 5th ed. Vol. II.

1	PIP2 = phosphatidylinositol 4,5-bisphosphate; PLC = phospholipase C. (Reproduced with permission from Zuidema GD, Yeo CJ: Shackelford’s Surgery of the Alimentary Tract, 5th ed. Vol. II. Philadelphia, PA: Elsevier/Saunders; 2002.)Brunicardi_Ch26_p1099-p1166.indd 110701/03/19 7:11 PM 1108SPECIFIC CONSIDERATIONS PART IIin three phases: cephalic, gastric, and intestinal.28,29 The cephalic or vagal phase begins with the thought, sight, smell, and/or taste of food. These stimuli activate several cortical and hypotha-lamic sites (e.g., tractus solitarius, dorsal motor nucleus, and dorsal vagal complex), and signals are transmitted to the stom-ach by the vagal nerves which stimulate enteric submucosal neurons. Acetylcholine is released, leading to stimulation acid secretion from parietal cells. Vagal stimulation also leads to gas-trin release from antral G cells via CGRP, and sensitizes ECL cells to gastrin.30,31 Although the acid secreted per unit of time in the cephalic phase is

1	cells. Vagal stimulation also leads to gas-trin release from antral G cells via CGRP, and sensitizes ECL cells to gastrin.30,31 Although the acid secreted per unit of time in the cephalic phase is greater than in the other two phases, the cephalic phase is shorter. Thus, the cephalic phase accounts for no more than 30% of total acid secretion in response to a meal. Sham feeding (chewing and spitting) stimulates gastric acid secretion only via the cephalic phase, and it results in acid secretion that is about half of that seen in response to IV penta-gastrin or histamine.When food reaches the stomach, the gastric phase of acid secretion begins. This phase lasts until the stomach is empty and accounts for about 60% of the total acid secretion in response to a meal. The gastric phase of acid secretion has several compo-nents. Amino acids and small peptides directly stimulate antral G cells to secrete gastrin, which is carried in the bloodstream to the ECL and parietal cells, stimulating

1	secretion has several compo-nents. Amino acids and small peptides directly stimulate antral G cells to secrete gastrin, which is carried in the bloodstream to the ECL and parietal cells, stimulating acid secretion in an endo-crine fashion. In addition, proximal gastric distention stimulates acid secretion via a vagovagal reflex arc, which is mitigated by truncal or highly selective vagotomy (HSV). Antral disten-tion also stimulates antral gastrin secretion. Finally, ongoing cephalic vagal input stimulates gastrin release, which in turn stimulates histamine release from ECL cells and acid secretion.The intestinal phase of gastric secretion is poorly under-stood. It is thought to be mediated by a hormone released from the proximal small bowel mucosa in response to luminal chyme. This phase starts when gastric emptying of ingested food begins, and it continues as long as nutrients remain in the proximal small intestine. It accounts for about 10% of meal-induced acid

1	chyme. This phase starts when gastric emptying of ingested food begins, and it continues as long as nutrients remain in the proximal small intestine. It accounts for about 10% of meal-induced acid secretion.Interprandial basal acid secretion is 2 to 5 mEq hydrochlo-ric acid per hour, about 10% of maximal acid output (MAO), and it is greater at night. Basal acid secretion probably contrib-utes to the relatively low bacterial counts found in the stomach. Basal acid secretion is reduced 75% to 90% by vagotomy or continuous H2-receptor blockade.The pivotal role that ECL cells play in the regulation of gastric acid secretion is emphasized in Fig. 26-13. The acid stimulatory effect of gastrin is largely mediated by histamine released from mucosal ECL cells. H2-receptor knockout mice do not secrete acid in response to gastrin.24 This explains why the H2-receptor antagonists (H2RAs) are effective inhibitors of acid secretion, even though histamine is only one of three parietal cell

1	not secrete acid in response to gastrin.24 This explains why the H2-receptor antagonists (H2RAs) are effective inhibitors of acid secretion, even though histamine is only one of three parietal cell stimulants. The mucosal D cell, which releases somatosta-tin, is also an important regulator of acid secretion. Somatostatin inhibits histamine release from ECL cells and gastrin release from antral G cells. The function of D cells can be inhibited by Helicobacter pylori infection, resulting in an exaggerated acid secretory response (see “Helicobacter pylori Infection”).Proton pump inhibitors are potent suppressors of gastric acid secretion. This results in hypergastrinemia and conse-quent ECL stimulation. In patients on long-term PPI (median 5.5 years), the degree of hypergastrinemia does not appear to correlate with the length of treatment.32 Chronic PPI use has been associated with ECL hyperplasia and type 1 gastric neuro-endocrine tumor, but so far there has been no evidence linking

1	appear to correlate with the length of treatment.32 Chronic PPI use has been associated with ECL hyperplasia and type 1 gastric neuro-endocrine tumor, but so far there has been no evidence linking these agents to malignant gastric epithelial or neuroendocrine tumors. Gastrin levels return to normal within a few days of PPI cessation, but during this time, some patients may experience gastric hyperacidity and dyspeptic symptoms, which may lead to difficulty in getting patients off the medication.33,34 This is less likely to occur with short-term PPI use and may be amelio-rated by PPI dose tapering and/or initiation of H2 blockers prior to PPI cessation.Pepsinogen SecretionThe most potent physiologic stimulus for pepsinogen secre-tion from chief cells is food ingestion; acetylcholine is the most important mediator. Somatostatin inhibits pepsinogen secre-tion. Pepsinogen I is produced by chief cells in acid producing glands, whereas pepsinogen II is produced by chief cells and by SECs in

1	most important mediator. Somatostatin inhibits pepsinogen secre-tion. Pepsinogen I is produced by chief cells in acid producing glands, whereas pepsinogen II is produced by chief cells and by SECs in both acid producing and gastrin producing (i.e., antral) glands. Pepsinogen is cleaved to the active pepsin enzyme in an acidic environment and is maximally active at pH 2.5, and inactive at pH >5, although pepsinogen II may be activated over a wider pH range than pepsinogen I. Pepsin catalyzes the hydro-lysis of proteins and is denatured at alkaline pH. Serum levels of pepsinogen I and II are increased in helicobacter gastritis, so elevated pepsinogen I and II levels and positive helicobacter serology are presumptive evidence of active helicobacter infec-tion. Longstanding helicobacter infection may lead to atrophic gastritis, suggested by decreased pepsinogen I/II ratio (from chief cell loss) and hypergastriemia (from parietal cell loss and hypochlorhydria).35Intrinsic FactorActivated

1	may lead to atrophic gastritis, suggested by decreased pepsinogen I/II ratio (from chief cell loss) and hypergastriemia (from parietal cell loss and hypochlorhydria).35Intrinsic FactorActivated parietal cells secrete intrinsic factor in addition to hydrochloric acid. Presumably the stimulants are similar, but MealVagusD-cellECLcellParietal cellGastrinSomatostatinHistamine++++–––++++AcetylcholineG-cellFigure 26-13. Physiologic control of acid secretion. ECL = enterochromaffin-like. (Reproduced with permission from Zuidema GD, Yeo CJ: Shackelford’s Surgery of the Alimentary Tract, 5th ed. Vol. II. Philadelphia, PA: Elsevier/Saunders; 2002.)Brunicardi_Ch26_p1099-p1166.indd 110801/03/19 7:11 PM 1109STOMACHCHAPTER 26acid secretion and intrinsic factor secretion may not be linked. Intrinsic factor binds to luminal vitamin B12, and the complex is absorbed in the terminal ileum via mucosal receptors. Vitamin B12 deficiency can be life threatening, and patients with total gastrectomy or

1	factor binds to luminal vitamin B12, and the complex is absorbed in the terminal ileum via mucosal receptors. Vitamin B12 deficiency can be life threatening, and patients with total gastrectomy or pernicious anemia (i.e., patients with no pari-etal cells) require B12 supplementation by a nonenteric route. Some patients develop vitamin B12 deficiency following gastric bypass, presumably because there is insufficient intrinsic fac-tor present in the small proximal gastric pouch and oral B12 intake may be decreased. Under normal conditions, a signifi-cant excess of intrinsic factor is secreted, and acid-suppressive medication does not appear to inhibit intrinsic factor production and release.Gastric Mucosal BarrierThe stomach’s durable resistance to autodigestion by caustic hydrochloric acid and active pepsin is intriguing. Some of the important elements of gastric barrier function and cytoprotection are listed in Table 26-3.36,37 When these defenses break down, ulceration occurs. A

1	acid and active pepsin is intriguing. Some of the important elements of gastric barrier function and cytoprotection are listed in Table 26-3.36,37 When these defenses break down, ulceration occurs. A variety of factors are important in maintain-ing an intact gastric mucosal layer.38 The mucus and bicarbonate secreted by SECs form an unstirred mucous gel with a favorable pH gradient. Cell membranes and tight junctions prevent hydro-gen ions from gaining access to the interstitial space. Hydrogen ions that do break through are buffered by the alkaline tide cre-ated by basolateral bicarbonate secretion from stimulated pari-etal cells. Any sloughed or denuded SECs are rapidly replaced by migration of adjacent cells, a process known as restitution. Mucosal blood flow plays a crucial role in maintaining a healthy mucosa, providing nutrients and oxygen for the cellular func-tions involved in cytoprotection. During acid secretion, there is a tremendous gradient favoring the movement of

1	in maintaining a healthy mucosa, providing nutrients and oxygen for the cellular func-tions involved in cytoprotection. During acid secretion, there is a tremendous gradient favoring the movement of hydrogen ions from the lumen to the interstitium. This “back-diffused” hydrogen is buffered and rapidly removed by the rich blood supply. When “barrier breakers” such as bile or aspirin lead to increased back-diffusion of hydrogen ions from the lumen into the lamina propria and submucosa, there is a protective increase in mucosal blood flow. If this protective response is blocked, gross ulceration can occur. Important mediators of these protec-tive mechanisms include prostaglandins, nitric oxide, intrinsic nerves, and peptides (e.g., calcitonin gene-related peptide, gas-trin-releasing peptide [GRP], gastrin, and heat shock proteins). Sucralfate acts locally to enhance mucosal defenses. Protec-tive reflexes involve afferent sensory neurons, and they can be blocked by the application of

1	[GRP], gastrin, and heat shock proteins). Sucralfate acts locally to enhance mucosal defenses. Protec-tive reflexes involve afferent sensory neurons, and they can be blocked by the application of topical anesthetics to the gastric mucosa, or the experimental destruction of the afferent sensory nerves. In addition to these local defenses, there are important protective factors in saliva, duodenal secretions, and pancreatic or biliary secretions.Gastric Hormones13,39The stomach is quite an elegant endocrine organ. It is the source of important peptides which work in an autocrine (EGF and surface epithelial cells, TGF and parietal cells), paracrine (somatostatin), endocrine (gastrin), and/or neurocrine (ghrelin) fashion.Gastrin. Gastrin is produced by antral G cells and is the major hormonal stimulant of acid secretion during the gastric phase predominantly via an endocrine effect on histamine generating ECL cells and to a lesser extent via a direct effect on parietal cells. A variety of

1	stimulant of acid secretion during the gastric phase predominantly via an endocrine effect on histamine generating ECL cells and to a lesser extent via a direct effect on parietal cells. A variety of molecular forms exist: big gastrin (34 amino acids; G34), little gastrin (17 amino acids; G17), and mini-gastrin (14 amino acids; G14). The large majority of gastrin released by the human antrum is G17. The biologically active pentapeptide sequence at the C-terminal end of gastrin is identical to that of CCK. Luminal peptides and amino acids are the most potent stimulants of gastrin release, and luminal acid is the most potent inhibitor of gastrin secretion. The latter effect is predominantly mediated in a paracrine fashion by somatostatin released from antral D cells. Gastrin-stimulated acid secretion is significantly blocked by H2 antagonists, suggesting that the principal media-tor of gastrin-stimulated acid production is histamine from mucosal ECL cells and not direct stimulation of

1	secretion is significantly blocked by H2 antagonists, suggesting that the principal media-tor of gastrin-stimulated acid production is histamine from mucosal ECL cells and not direct stimulation of parietal cells by gastrin (see Fig. 26-13). In fact, chronic hypergastrinemia is associated with hyperplasia of gastric ECL cells and, rarely, gastric type I gastric neuroendocrine tumors (type I gastric car-cinoid). Gastrin is trophic to gastric parietal cells and to other GI mucosal cells including gastric stem cells. It also is a regulator of gastric cellular proliferation, migration, invasion, apoptosis and angiogenesis.40 Mucosal biopsies of the gastric body from patients with gastrinoma show a thick mucosa with excess pari-etal cells, while similar biopsies in patients years after antrec-tomy (i.e., low gastrin state) show thin mucosa and decreased parietal cells. In animal studies, gastrin administration has been shown to stimulate the growth of established colon cancers and to cause

1	(i.e., low gastrin state) show thin mucosa and decreased parietal cells. In animal studies, gastrin administration has been shown to stimulate the growth of established colon cancers and to cause pancreatic acinar cell hyperplasia.41 Important causes of hypergastrinemia include pernicious anemia, acid-suppressive medication, gastrinoma, retained antrum following distal gas-trectomy and Billroth II surgery, and vagotomy.42,43Ghrelin. Ghrelin, first described in 1999, is a small peptide that is produced mainly in the stomach.44,45 It is produced by specialized P/D1 endocrine cells in gastric oxyntic glands. Ninety percent of the body’s ghrelin stores are in the stomach and duodenum. Ghrelin is a potent secretagogue of pituitary growth hormone and a weak secretogogue for ACTH and pro-lactin. It appears to be a major orexigenic regulator of appetite. Ghrelin crosses the blood brain barrier and stimulates appetite via hypothalamic receptors. It also stimulates appetite periph-erally by

1	It appears to be a major orexigenic regulator of appetite. Ghrelin crosses the blood brain barrier and stimulates appetite via hypothalamic receptors. It also stimulates appetite periph-erally by stimulating vagal afferent fibers in the gastric wall. Table 26-3Important components and mediators of mucosal defenses in the stomachComponentsMucous barrierBicarbonate secretionEpithelial barrier Hydrophobic phospholipids Tight junctions RestitutionMicrocirculation (reactive hyperemia)Afferent sensory neuronsMediatorsProstaglandinsNitric oxideEpidermal growth factorCalcitonin gene-related peptideHepatocyte growth factorHistamineGastrin-releasing peptideBrunicardi_Ch26_p1099-p1166.indd 110901/03/19 7:11 PM 1110SPECIFIC CONSIDERATIONS PART IIWhen ghrelin is elevated, appetite is stimulated, and when it is suppressed, appetite is decreased. Typically, ghrelin levels are elevated before a meal and decreased postprandially. Levels are high during starvation and decreased during

1	stimulated, and when it is suppressed, appetite is decreased. Typically, ghrelin levels are elevated before a meal and decreased postprandially. Levels are high during starvation and decreased during hyperglycemia. Obesity and insulin resistance is associated with low ghrelin levels, but resection of the primary source of this hormone (i.e., the stomach) may partly account for the anorexia and weight loss seen in some patients following gastric resection including sleeve gastrectomy (Fig. 26-14).46-48 The effect of RYGBP on ghrelin physiology is controversial. This very effective weight loss procedure has been shown by some investigators to be asso-ciated with suppression of plasma ghrelin levels (and appetite) in humans (Fig. 26-15A).49,50Other groups have failed to show a significant decrease in ghrelin levels following gastric bypass but have found such decreases following sleeve gastrectomy, another effective weight loss operation (Fig. 26-15B).50 Possibly, subtle differ-ences in

1	in ghrelin levels following gastric bypass but have found such decreases following sleeve gastrectomy, another effective weight loss operation (Fig. 26-15B).50 Possibly, subtle differ-ences in operative technique, patient selection, or experimental (including assay) conditions account for the disparate results of studies on the effect of bariatric surgery on ghrelin levels in obese patients. Interestingly the two common metabolites of ghrelin have different physiologic effects: acyl-ghrelin increases gastric emptying and appetite while deacyl ghrelin decreases gastric emptying and induces satiety.51 Obviously appetite con-trol is complex with redundant and overlapping orexigenic and anorexigenic pathways and signals.39,52Somatostatin. Somatostatin is produced by D cells located throughout the gastric mucosa. The predominant form in humans is somatostatin 14, though somatostatin 28 is present as well. The major stimulus for somatostatin release is antral acidification; acetylcholine

1	the gastric mucosa. The predominant form in humans is somatostatin 14, though somatostatin 28 is present as well. The major stimulus for somatostatin release is antral acidification; acetylcholine from vagal nerve fibers inhibits its release. Somatostatin inhibits acid secretion from parietal cells and gastrin release from G cells. It also decreases histamine release from ECL cells. The proximity of the D cells to these target cells suggests that the primary effect of somatostatin is mediated in a paracrine fashion, but an endocrine (i.e., blood-stream) effect also is possible.Gastrin-Releasing Peptide. GRP is the mammalian equiva-lent of bombesin, a hormone discovered more than two decades ago in an extract of skin from a frog. In the antrum, GRP stimu-lates both gastrin and somatostatin release by binding to recep-tors on the G and D cells. There are nerve terminals ending near the mucosa in the gastric body and antrum, which are rich in GRP immunoreactivity. When GRP is given

1	release by binding to recep-tors on the G and D cells. There are nerve terminals ending near the mucosa in the gastric body and antrum, which are rich in GRP immunoreactivity. When GRP is given peripherally, it stimulates acid secretion, but when it is given centrally into the cerebral ventricles of animals, it inhibits acid secretion, appar-ently via a pathway involving the sympathetic nervous system.Leptin. Leptin is a protein primarily synthesized in adipocytes. It is also made by chief cells in the stomach, the main source of leptin in the GI tract.39,53 Leptin works at least in part via vagally mediated pathways to decrease food intake in animals. Not surprisingly, leptin, a satiety signal hormone, and ghrelin, a hunger signal hormone, are both synthesized in the stomach, an organ increasingly recognized as central to the mechanisms of appetite control.39,52Autocrine Proteins. Gastric surface epithelial cells secrete a variety of proteins that are important regulators of SEC

1	increasingly recognized as central to the mechanisms of appetite control.39,52Autocrine Proteins. Gastric surface epithelial cells secrete a variety of proteins that are important regulators of SEC health, including trefoil factor family proteins and heat shock pro-teins.38 Parietal cells may also be influenced by molecules they secrete including transforming growth factor-α.Gastric Motility and EmptyingGastric motor function has several purposes.43,54-56 Interprandial motor activity clears the stomach of undigested debris, sloughed cells, and mucus. When feeding begins, the stomach relaxes to accommodate the meal. Regulated motor activity then breaks down the food into small particles and controls the output into the duodenum. The stomach accomplishes these functions by coordinated smooth muscle relaxation and contraction of the various gastric segments (proximal, distal, and pyloric). Smooth muscle myoelectric potentials are translated into muscular activity, which is modulated by

1	muscle relaxation and contraction of the various gastric segments (proximal, distal, and pyloric). Smooth muscle myoelectric potentials are translated into muscular activity, which is modulated by extrinsic and intrinsic innerva-tion and hormones. The mechanisms by which gastric distention is translated into a neurohormonal satiety signal have only been partially elucidated.39,52Intrinsic Gastric Innervation. The extrinsic parasympa-thetic and sympathetic gastric innervation was discussed pre-viously in “Innervation.” The intrinsic innervation consists of ganglia and nerves that constitute the enteric nervous system (Fig. 26-16).57 There are a variety of neurotransmitters that effect gastric smooth muscle; these are generally grouped as excitatory (augment muscular activity) and inhibitory (decrease muscular activity). Important excitatory neurotransmitters include acetylcholine, the tachykinins, substance P, and neu-rokinin A. Important inhibitory neurotransmitters include nitric

1	(decrease muscular activity). Important excitatory neurotransmitters include acetylcholine, the tachykinins, substance P, and neu-rokinin A. Important inhibitory neurotransmitters include nitric oxide (NO) and vasoactive intestinal peptide (VIP). Serotonin has been shown to modulate both contraction and relaxation. A variety of other molecules affect motility, including GRP, hista-mine, neuropeptide Y, norepinephrine, and endogenous opioids.Specialized cells in the muscularis propria also are impor-tant modulators of GI motility. These cells, called interstitial cells of Cajal, are distinguishable histologically from neurons and myocytes and appear to amplify both cholinergic excitatory 300200Plasma ghrelin (fmol/mL)100NormalcontrolsGastrectomizedpatients0*Figure 26-14. Ghrelin levels are decreased after gastrectomy. (Reproduced with permission from Ariyasu H, Takaya K, Tagami T, et al: Stomach is a major source of circulating ghrelin, and feed-ing state determines plasma ghrelin-like

1	decreased after gastrectomy. (Reproduced with permission from Ariyasu H, Takaya K, Tagami T, et al: Stomach is a major source of circulating ghrelin, and feed-ing state determines plasma ghrelin-like immunoreactivity levels in humans, J Clin Endocrinol Metab. 2001 Oct;86(10):4753-4758.)Brunicardi_Ch26_p1099-p1166.indd 111001/03/19 7:11 PM 1111STOMACHCHAPTER 26Figure 26-15. A and B. Ghrelin secretion after bariatric surgery. Some investigators have suggested that ghrelin secretion is dramatically decreased after gastric bypass. Other groups have shown statistically insig-nificant changes in ghrelin levels after gastric bypass, (RYGBP) but significant decreases after sleeve gastrectomy (SG). A. green = gastric bypass; blue = obese controls; red = normal weight controls; B. blue = fasting; pink = postprandial. B. Top = RYGBP; B bottom = SG. (A, Repro-duced with permission from Cummings DE, Weigle DS, Frayo RS, et al: Plasma ghre-lin levels after diet-induced weight loss or gastric

1	pink = postprandial. B. Top = RYGBP; B bottom = SG. (A, Repro-duced with permission from Cummings DE, Weigle DS, Frayo RS, et al: Plasma ghre-lin levels after diet-induced weight loss or gastric bypass surgery, N Engl J Med. 2002 May 23;346(21):1623-1630. B, Reproduced with permission from Karamanakos SN, Vagenas K, Kalfarentzos F, et al: Weight loss, appetite suppression, and changes in fasting and postprandial ghrelin and peptide-YY levels after Roux-en-Y gastric bypass and sleeve gastrectomy: A prospec-tive, double blind study, Ann Surg. 2008 Mar;247(3):401-407.)Plasma ghrelin level (pg/mL)Detection limitTime6 a.m.8 a.m.10 a.m.noon2 p.m.4 p.m.6 p.m.8 p.m.10 p.m.080100200300400500600700800BreakfastLunchDinnerA1200P = 0.06P = 0.15P = 0.16P = 0.13P = 0.01P = 0.03P = 0.01P = 0.0310008006004002000PreGhrelin (pg/mL)Ghrelin (pg/mL)3Time (months)Time (months)612Pre36129008007006005004003002001000Band nitrergic inhibitory input to the smooth muscle of the stom-ach and intestine.58 They are

1	(pg/mL)Ghrelin (pg/mL)3Time (months)Time (months)612Pre36129008007006005004003002001000Band nitrergic inhibitory input to the smooth muscle of the stom-ach and intestine.58 They are thought to be the cell of origin for gastrointestinal stromal tumors (GISTs), which are the most common mesenchymal neoplasms in the GI tract.Segmental Gastric Motility.13,58,59 In general, the proximal stomach serves a short-term food storage function and helps regulate basal intragastric tone, and the distal stomach mixes and grinds the food. The pylorus helps the latter process when Brunicardi_Ch26_p1099-p1166.indd 111101/03/19 7:11 PM 1112SPECIFIC CONSIDERATIONS PART IIclosed, facilitating retropulsion of the solid food bolus back into the body of the stomach for additional breakdown. The pylorus opens intermittently to allow metered emptying of liquids and small solid particles into the duodenum.Most of the motor activity of the proximal stomach con-sists of slow tonic contractions and

1	opens intermittently to allow metered emptying of liquids and small solid particles into the duodenum.Most of the motor activity of the proximal stomach con-sists of slow tonic contractions and relaxations, lasting up to 5 minutes. This activity is the main determinant of basal intra-gastric pressure, an important determinant of liquid empty-ing. Rapid phasic contractions may be superimposed on the slower tonic motor activity. When food is ingested, intragastric pressure falls as the proximal stomach relaxes. This proximal relaxation is mediated by two important vagovagal reflexes: receptive relaxation and gastric accommodation. Receptive relaxation refers to the reduction in gastric tone associated with the act of swallowing. This occurs before the food reaches the stomach and can be reproduced by mechanical stimulation of the pharynx or esophagus. Gastric accommodation refers to the proximal gastric relaxation associated with distention of the stomach.60 Accommodation is mediated

1	by mechanical stimulation of the pharynx or esophagus. Gastric accommodation refers to the proximal gastric relaxation associated with distention of the stomach.60 Accommodation is mediated through stretch recep-tors in the gastric wall and does not require esophageal or pha-ryngeal stimulation. Initially, as the meal enters the stomach, there is a drop in intragastric pressure mediated by nitric oxide. As the meal progresses, the intragastric pressure rises, parallel with the onset of satiety. Interestingly, satiety does not seem to be associated with any specific level of intragastric pressure. Obese patients have a delayed onset of satiety, so an obvious hypothesis to be tested is that a pharmacologic-induced increase in gastric tone leads to increased satiety and decreased food intake in this patient group.60 Because receptive relaxation and accommodation are mediated by afferent and efferent vagal fibers, they are significantly altered by truncal and highly selec-tive vagotomy.

1	in this patient group.60 Because receptive relaxation and accommodation are mediated by afferent and efferent vagal fibers, they are significantly altered by truncal and highly selec-tive vagotomy. Both these operations result in decreased gastric compliance, shifting the volume/pressure curve to the left. Pre-sumably for any given amount of food ingested, the intragastric pressure is higher, and perhaps in some patients the onset of satiety is sooner. This may be one explanation for weight loss associated with vagotomy,61 and it also helps explain acceler-ated liquid gastric emptying postvagotomy, which likely con-tributes to dumping symptoms in some patients.NO and VIP are the principal mediators of proximal gas-tric relaxation. But a variety of other agents increase proximal gastric relaxation and compliance, including dopamine, gastrin, CCK, secretin, GRP, and glucagon. Proximal gastric tone also is decreased by duodenal distention, colonic distention, and ileal perfusion with

1	relaxation and compliance, including dopamine, gastrin, CCK, secretin, GRP, and glucagon. Proximal gastric tone also is decreased by duodenal distention, colonic distention, and ileal perfusion with glucose (ileal brake).The distal stomach breaks up solid food and is the main determinant of gastric emptying of solids. Slow waves of myo-electric depolarization sweep down the distal stomach at a rate of about three per minute. These waves originate from the proxi-mal gastric pacemaker, high on the greater curvature.62 The pac-ing cells appear to be the interstitial cells of Cajal, which have been shown to have a similar function in the small intestine and colon. Most of these myoelectric waves are below the thresh-old for smooth muscle contraction in the quiescent state and thus are associated with negligible changes in pressure. Neural and/or hormonal input, which increases the plateau phase of the action potential, can trigger muscle contraction, resulting in a peristaltic wave

1	with negligible changes in pressure. Neural and/or hormonal input, which increases the plateau phase of the action potential, can trigger muscle contraction, resulting in a peristaltic wave associated with the electrical slow wave and of the same frequency (three per minute) (Fig. 26-17). There are measurable abnormalities in gastric slow wave activity in disor-ders of gastric motility such as gastroparesis, but reliable elec-trogastrogaphy to aid in the diagnosis and management of these problems is not yet a clinical reality. It is likely that implantable gastric pacemakers benefit some patients with gastroparesis by favorably impacting this myoelectric coupling, normalizing gas-tric slow wave patterns.During fasting, distal gastric motor activity is controlled by the migrating motor complex (MMC), the “gastrointesti-nal housekeeper” (Fig. 26-18). The purported function of the MMC is to sweep along any undigested food, debris, sloughed cells, and mucus after the fed phase of

1	complex (MMC), the “gastrointesti-nal housekeeper” (Fig. 26-18). The purported function of the MMC is to sweep along any undigested food, debris, sloughed cells, and mucus after the fed phase of digestion is complete. The MMC lasts approximately 100 minutes (longer at night, shorter during daytime) and is divided into four phases. Phase I (about half the length of the entire cycle) is a period of relative motor inactivity. High-amplitude muscular contractions do not occur in phase I of the MMC. Phase II (about 25% of the entire MMC cycle) consists of some irregular, high-amplitude, gener-ally nonpropulsive contractions. Phase III, a period of intense, Figure 26-16. Enteric nervous sys-tem. (Reproduced with permission from Friedman SL, McQuaid KR, Grendell JH: Current Diagnosis and Treatment in Gastroenterology, 2nd ed. New York, NY: McGraw-Hill Education; 2003.)Parasympathetic (vagal) nerveMesenteryPerivascular sympathic nerveMuscularis externa (longitudinal)Muscularis externa

1	in Gastroenterology, 2nd ed. New York, NY: McGraw-Hill Education; 2003.)Parasympathetic (vagal) nerveMesenteryPerivascular sympathic nerveMuscularis externa (longitudinal)Muscularis externa (circular)Subepethelial plexusMuscularis mucosaSubmucosaVillusIntestinal lumenDeep muscularis plexusSerosaTertiary branchSecondary branchGanglionSubmucosal plexusMyenteric plexusBrunicardi_Ch26_p1099-p1166.indd 111201/03/19 7:11 PM 1113STOMACHCHAPTER 26regular (about three per minute), propulsive contractions, only lasts about 5 to 10 minutes. Most phase III complexes of the GI MMC begin in the stomach, and the frequency approximates that of the myoelectric gastric slow wave. Phase IV is a transi-tion period.Neurohormonal control of the MMC is poorly understood, but it appears that different phases are regulated by different mechanisms. For example, vagotomy abolishes phase II of the gastric MMC but has little influence on phase III that persists even in the

1	but it appears that different phases are regulated by different mechanisms. For example, vagotomy abolishes phase II of the gastric MMC but has little influence on phase III that persists even in the autotransplanted stomach, totally devoid of extrinsic neural input. This suggests that phase III is regulated by intrin-sic nerves and/or hormones. Indeed, the initiation of phase III of the MMC in the distal stomach corresponds temporally to elevation in serum levels of motilin, a hormone produced in the duodenal mucosa. Resection of the duodenum abolishes dis-tal gastric phase III in dogs, and resection of the duodenum in humans (e.g., with pancreaticoduodenectomy, the Whipple pro-cedure) commonly results in early postoperative delayed gastric emptying. There are clearly motilin receptors on gastric smooth muscle and nerves. Other modulators of gastric MMC activ-ity include NO, endogenous opioids, intrinsic cholinergic and adrenergic nerves, and duodenal pH. The onset of MMC phase III

1	gastric smooth muscle and nerves. Other modulators of gastric MMC activ-ity include NO, endogenous opioids, intrinsic cholinergic and adrenergic nerves, and duodenal pH. The onset of MMC phase III signals the return of hunger in humans, but oddly ghrelin, a major orexigenic hormone, appears to have little to do with phase III.63Feeding abolishes the MMC and leads to the fed motor pattern. The fed motor pattern of gastric activity starts within 10 minutes of food ingestion and persists until all the food has left the stomach. The neurohormonal initiator of this change is unknown, but CCK and the vagus appear to play some role since sham feeding transiently induces antral motor activity resembling the fed motor pattern which is blocked by the CCK receptor antagonist loxiglumide. Gastric motility during the fed pattern resembles phase II of the MMC, with irregular but con-tinuous phasic contractions of the distal stomach. During the fed state, about half of the myoelectric slow waves

1	motility during the fed pattern resembles phase II of the MMC, with irregular but con-tinuous phasic contractions of the distal stomach. During the fed state, about half of the myoelectric slow waves are associated with strong higher frequency distal gastric contractions. Some are prograde and some are retrograde, serving to mix and grind the solid components of the meal. The magnitude of gastric con-tractions and the duration of the pattern are influenced by the consistency and composition of the meal.The pylorus functions as an effective regulator of gastric emptying and an effective barrier to duodenogastric reflux. Bypass, transection, or resection of the pylorus may lead to uncontrolled gastric emptying of food and the dumping syn-drome (see “Postgastrectomy Problems”). Pyloric dysfunction IntracellularrecordingTensionQuiescentStimulated0 mv0 5 g -70 1122Figure 26-17. The relationship between intracellular electrical activity and muscle cell contraction. Note that contractile

1	IntracellularrecordingTensionQuiescentStimulated0 mv0 5 g -70 1122Figure 26-17. The relationship between intracellular electrical activity and muscle cell contraction. Note that contractile activity is always associated with electrical activity, but the converse is not so. During mechanical quiescence, there are regular depolarizations that do not reach threshold. In the stimulated state, the threshold for contraction is reached, and motor activity is demonstrable. (Reproduced with permission from Kim CH, Malagelada JR: Electrical activity of the stomach: clinical implications, Mayo Clin Proc. 1986 Mar;61(3):205-210.)Figure 26-18. Migrating motor complex, the fasting pattern of GI activity. During phase III of the migrating motor complex, effective peri-staltic waves progress from the stomach to the distal small intestine. (Reproduced with permis-sion from Rees WD, Malagelada JR, Miller LJ, et al: Human interdigestive and postprandial gas-trointestinal motor and gastrointestinal

1	stomach to the distal small intestine. (Reproduced with permis-sion from Rees WD, Malagelada JR, Miller LJ, et al: Human interdigestive and postprandial gas-trointestinal motor and gastrointestinal hormone patterns, Dig Dis Sci. 1982 Apr;27(4):321-329.)AntrumProximal duodenumDistal duodenumJejunum1 min100 mm Hg100 mm Hg100 mm Hg100 mm HgPhase IIPhase IIIPhase IVPhase IBrunicardi_Ch26_p1099-p1166.indd 111301/03/19 7:11 PM 1114SPECIFIC CONSIDERATIONS PART IIor disruption may also result in uncontrolled entry of duode-nal contents into the stomach. Perfusion of the duodenum with lipids, glucose, amino acids, hypertonic saline, or hydrochloric acid results in closure of the pylorus and decreased transpylo-ric flow. Ileal perfusion with fat has the same effect. A variety of neurohumoral pathways are involved with these physiologic responses, and there is evidence that different pathways may be involved for different stimuli.The pylorus is readily apparent grossly as a

1	neurohumoral pathways are involved with these physiologic responses, and there is evidence that different pathways may be involved for different stimuli.The pylorus is readily apparent grossly as a thick ring of muscle and connective tissue. The density of nerve tissue in the pyloric smooth muscle is several folds higher than in the antrum, with increased numbers of neurons staining positive for substance P, neuropeptide Y, VIP, and galanin. Interstitial cells of Cajal are closely associated with pyloric myocytes, and the myoelectric slow wave of the pylorus has the same frequency as that seen in the distal stomach. The motor activity of the pylo-rus is both tonic and phasic. During phase III of the MMC, the pylorus is open as gastric contents are swept into the duodenum. During the fed phase, the pylorus is closed most of the time. It relaxes intermittently, usually in synchronization with lower-amplitude, minor antral contractions. The higher-amplitude, more major antral

1	the fed phase, the pylorus is closed most of the time. It relaxes intermittently, usually in synchronization with lower-amplitude, minor antral contractions. The higher-amplitude, more major antral contractions are usually met with a closed pylorus, facilitating retropulsion and further grinding of food.Modulation of pyloric motor activity is complex. There is evidence for both inhibitory and excitatory vagal pathways. Some contractile vagal effects are mediated by opioid pathways because they are blocked by naloxone. Electrical stimulation of the duodenum causes the pylorus to contract, whereas electrical stimulation of the antrum causes pyloric relaxation. Nitric oxide is an important mediator of pyloric relaxation. Other molecules that may play a physiologic role in controlling pyloric smooth muscle include serotonin, VIP, prostaglandin E1, and galanin (pyloric relaxation); and histamine, CCK, and secretin (pyloric contraction).Gastric Emptying.13 The control of gastric emptying is

1	smooth muscle include serotonin, VIP, prostaglandin E1, and galanin (pyloric relaxation); and histamine, CCK, and secretin (pyloric contraction).Gastric Emptying.13 The control of gastric emptying is com-plex. In general, liquid emptying is faster than solid emptying. Osmolarity, acidity, caloric content, nutrient composition, and particle size are important modulators of gastric emptying. Stimulation of duodenal osmoreceptors, glucoreceptors, and pH receptors clearly inhibits gastric emptying by a variety of neurohumoral mechanisms. CCK has been consistently shown to inhibit gastric emptying at physiologic doses (Fig. 26-19). Recently, it has been noted that the anorexigenic hormone leptin, secreted largely by fat but also by gastric mucosa, inhib-its gastric emptying, perhaps through the same pathway as CCK (which also has properties of a satiety hormone). The orexigenic hormone ghrelin has the opposite effect.Liquid Emptying. The gastric emptying of water or isotonic saline follows

1	same pathway as CCK (which also has properties of a satiety hormone). The orexigenic hormone ghrelin has the opposite effect.Liquid Emptying. The gastric emptying of water or isotonic saline follows first-order kinetics, with a half emptying time around 12 minutes. Thus, if one drinks 200 mL of water, about 100 mL enters the duodenum by 12 minutes, whereas if one drinks 400 mL of water, about 200 mL enters the duodenum by 12 minutes. This emptying pattern of liquids is modified consid-erably as the caloric density, osmolarity, and nutrient composi-tion of the liquid changes (Fig. 26-20). Up to an osmolarity of about 1 M, liquid emptying occurs at a rate of about 200 kcal per hour. Duodenal osmoreceptors and hormones (e.g., secretin and VIP) are important modulators of liquid gastric emptying. Generally, liquid emptying is delayed in the supine position.Traditionally, liquid emptying has been attributed to the activity of the proximal stomach, but it is probably more complicated than

1	Generally, liquid emptying is delayed in the supine position.Traditionally, liquid emptying has been attributed to the activity of the proximal stomach, but it is probably more complicated than previously thought. Clearly, receptive relax-ation and gastric accommodation play a role in gastric empty-ing of liquids. Patients with a denervated (e.g., vagotomized), resected, or plicated (e.g., fundoplication) proximal stomach have decreased gastric compliance and may show accelerated gastric emptying of liquids.Some observations suggest an active role for the distal stomach in liquid emptying. For instance, even if the proximal intragastric pressure is lower than duodenal pressure, normal gastric emptying of liquids can occur. Also, diabetic patients CCK (µg/kg)0.25Low-fat dietHight-fat diet0102030405060700.51.02.0% Suppression of gastric emptyingFigure 26-19. Cholecystokinin (CCK) inhibits gastric emptying. (Reproduced with permission from Covasa M, Ritter RC: Adapta-tion to high-fat

1	Suppression of gastric emptyingFigure 26-19. Cholecystokinin (CCK) inhibits gastric emptying. (Reproduced with permission from Covasa M, Ritter RC: Adapta-tion to high-fat diet reduces inhibition of gastric emptying by CCK and intestinal oleate, Am J Physiol Regul Integr Comp Physiol. 2000 Jan;278(1):R166-R170.)7006005004003001002000Residual gastric volume (mL)Time (min)806040200Figure 26-20. Nutrient composition and caloric density affect liquid gastric emptying. Glucose solution (purple circles), the least calorically dense, emptied the fastest. Other more calorically dense solutions, such as milk protein (green triangles) and pep-tide hydrolysates (red circles and blue triangles), emptied slower. (Reproduced with permission from Calbet JA, MacLean DA. Role of caloric content on gastric emptying in humans, J Physiol. 1997 Jan 15;498 (Pt 2):553-559.)Brunicardi_Ch26_p1099-p1166.indd 111401/03/19 7:11 PM 1115STOMACHCHAPTER 26may have normal proximal gastric motor function and

1	emptying in humans, J Physiol. 1997 Jan 15;498 (Pt 2):553-559.)Brunicardi_Ch26_p1099-p1166.indd 111401/03/19 7:11 PM 1115STOMACHCHAPTER 26may have normal proximal gastric motor function and pro-foundly delayed gastric emptying of liquids. Indeed, antral contractile activity does correlate with liquid gastric emptying, and this distal gastric activity appears to vary with the nutrient composition and caloric content of the liquid meal. Depend-ing on the circumstances, distal gastric motor activity can pro-mote or inhibit gastric emptying of liquids. Distal gastrectomy and pyloric stenting both obviously interfere with distal gastric motor activity, and both accelerate the initial rapid phase of liq-uid gastric emptying.Solid Emptying. Normally, the half-time of solid gastric emp-tying is less than 2 hours. Unlike liquids, which display an initial rapid phase followed by a slower linear phase of emptying, sol-ids have an initial lag phase during which little emptying of sol-ids

1	is less than 2 hours. Unlike liquids, which display an initial rapid phase followed by a slower linear phase of emptying, sol-ids have an initial lag phase during which little emptying of sol-ids occurs. It is during this phase that much of the grinding and mixing occurs. A linear emptying phase follows, during which the smaller particles are metered out to the duodenum. Solid gastric emptying is a function of meal particle size, caloric con-tent, and composition (especially fat). When liquids and solids are ingested together, the liquids empty first. Solids are stored in the fundus and delivered to the distal stomach at constant rates for grinding. Liquids also are sequestered in the fundus, but they appear to be readily delivered to the distal stomach for early emptying. The larger the solid component of the meal, the slower the liquid emptying. Patients bothered by dumping syn-drome are advised to limit the amount of liquid consumed with the solid meal, taking advantage of this

1	solid component of the meal, the slower the liquid emptying. Patients bothered by dumping syn-drome are advised to limit the amount of liquid consumed with the solid meal, taking advantage of this effect. Three prokinetic (metoclopramide, erythromycin, domperidone) may be used to treat delayed gastric emptying. Typical doses and mechanism of action are shown in Table 26-4.DIAGNOSIS OF GASTRIC DISEASESigns and SymptomsThe most common symptoms of gastric disease are pain, weight loss, early satiety, and anorexia. Nausea, vomiting, bloating, and anemia also are frequent complaints. Several of these symptoms (pain, bloating, nausea, and early satiety) are often described by physicians as dyspepsia, synonymous with the common nonmedical term indigestion. Common causes of dyspepsia include gastroesophageal reflux disease (GERD), helicobacter gastritis, and other disorders of the stomach, gallbladder, and pancreas. Although none of the aforementioned symptoms alone is specific for gastric

1	reflux disease (GERD), helicobacter gastritis, and other disorders of the stomach, gallbladder, and pancreas. Although none of the aforementioned symptoms alone is specific for gastric disease, when elicited in the con-text of a careful history and physical examination, they point to a differential diagnosis, which can be refined with certain tests. Early endoscopy should be considered in patients present-ing with recent onset of alarm symptoms (weight loss, anemia, dysphagia, vomiting) particularly those over 55 years of age (Table 26-5).Diagnostic TestsEsophagogastroduodenoscopy. Esophagogastroduodenos-copy (EGD) is a safe and accurate outpatient procedure per-formed under conscious sedation.64 Smaller flexible scopes with excellent optics and a working channel are easily passed trans-nasally in the unsedated patient. Following an 8-hour fast, the flexible scope is advanced under direct vision into the esopha-gus, stomach, and duodenum. The fundus and GE junction are inspected by

1	in the unsedated patient. Following an 8-hour fast, the flexible scope is advanced under direct vision into the esopha-gus, stomach, and duodenum. The fundus and GE junction are inspected by retroflexing the scope. To rule out cancer with a high degree of accuracy, all patients with gastric ulcer diag-nosed on upper GI series or found at EGD should have multiple biopsy specimens of the base and rim of the lesion. Brush cytol-ogy also should be considered. Gastritis should be biopsied both for histologic examination and assessment (see discussion on gastritis in “Helicobacter Pylori Infection”) and for a tissue ure-ase test and histologic evaluation to rule out the presence of H pylori. If Helicobacter infection is detected, it should be treated because of the etiologic association with peptic ulcers, mucosa-associated lymphoid tissue (MALT), and gastric cancer; in addition, eradication may ameliorate symp-toms. The most serious complications of EGD are perforation (which is rare, but

1	ulcers, mucosa-associated lymphoid tissue (MALT), and gastric cancer; in addition, eradication may ameliorate symp-toms. The most serious complications of EGD are perforation (which is rare, but can occur anywhere from the cervical esoph-agus to the duodenum), aspiration, and respiratory depression from excessive sedation. Although EGD is a more sensitive test than double-contrast upper GI series, these modalities should be considered complementary rather than mutually exclusive.Barium Upper GI Study. Plain abdominal X-rays may be helpful in the diagnosis of gastric perforation (pneumoperi-toneum) or delayed gastric emptying (large air-fluid level). Double-contrast upper GI series may be better than EGD at elucidating gastric diverticula, fistula, tortuosity, stricture loca-tion, and size or morphology of hiatal hernia.65 Although there are radiologic characteristics of ulcers that suggest the pres-ence or absence of malignancy, gastric ulcers always require adequate biopsy.Computed

1	or morphology of hiatal hernia.65 Although there are radiologic characteristics of ulcers that suggest the pres-ence or absence of malignancy, gastric ulcers always require adequate biopsy.Computed Tomographic Scanning and Magnetic Resonance Imaging. Usually, significant gastric disease can be diag-nosed without these sophisticated imaging studies. However, one or the other should be part of the routine staging work-up for patients with a malignant gastric tumor. Magnetic resonance imaging (MRI) may prove clinically useful as a quantitative test for gastric emptying, and it may even hold some promise for the 1Table 26-4Drugs that accelerate gastric emptyingAGENTTYPICAL ADULT DOSEMECHANISM OF ACTIONMetoclopramide10 mg PO four times a dayDopamine antagonistErythromycin250 mg PO four times a dayMotilin agonistDomperidone10 mg PO four times a dayDopamine antagonistTable 26-5Alarm symptoms that indicate the need for upper endoscopyAge >55 years with new onset dyspepsiaUnintentional weight

1	dayMotilin agonistDomperidone10 mg PO four times a dayDopamine antagonistTable 26-5Alarm symptoms that indicate the need for upper endoscopyAge >55 years with new onset dyspepsiaUnintentional weight lossPersistent or recurrent vomitingProgressive dysphagiaRecent onset odynophagiaUnexplained iron deficiency anemia or GI bleedingPalpable abdominal mass or lymphadenopathyFamily history of upper gastrointestinal cancerBrunicardi_Ch26_p1099-p1166.indd 111501/03/19 7:11 PM 1116SPECIFIC CONSIDERATIONS PART IIanalysis of myoelectric derangements in patients with gastropa-resis. Virtual gastroscopy using multi detector CT scan or MRI is not yet widely used, but these techniques may prove useful for screening and staging of gastric disease66-68 (Fig. 26-21). CTA or MRA is useful in evaluating the blood supply to the stomach after endovascular treatment of aortic and/or visceral arterial disease or in patients with previous upper abdominal operation in whom gastric conduit construction

1	the blood supply to the stomach after endovascular treatment of aortic and/or visceral arterial disease or in patients with previous upper abdominal operation in whom gastric conduit construction is contemplated, e.g., with esophagectomy.Arteriography can be helpful in the occasional poor-risk patient with exsanguinating gastric hemorrhage, in the patient with occult gastric bleeding, or when CTA or MRA is inconclu-sive in delineating vascular anatomy.Endoscopic Ultrasound. Endoscopic ultrasound (EUS) is useful in the evaluation and management of gastric mass lesions.69-71 Local staging of gastric adenocarcinoma with EUS is quite accurate, and this modality can be used to plan therapy. At many centers, patients with transmural and/or node positive adenocarcinoma of the stomach are considered for preoperative (neoadjuvant) chemoradiation therapy. EUS is the best way to clinically stage these patients locoregion-ally. Suspicious nodes can be sampled with EUS-guided endoscopic needle

1	for preoperative (neoadjuvant) chemoradiation therapy. EUS is the best way to clinically stage these patients locoregion-ally. Suspicious nodes can be sampled with EUS-guided endoscopic needle biopsy. Malignant tumors that are confined to the mucosa on EUS may be amenable to endoscopic muco-sal resection (EMR). EUS also can be used to assess tumor response to chemotherapy. Submucosal masses are commonly discovered during routine EGD. Large submucosal masses should be resected unless benign pathology is a certainty, but observation may be appropriate for some small submucosal masses (e.g., lipoma or leiomyoma). There are endoscopic characteristics of benign and malignant mesenchymal tumors, and thus, EUS can provide reassurance, but no guarantee, that small lesions under observation are probably benign. Thus, EUS-guided needle biopsy should be considered. Submucosal varices also can be assessed by EUS.Gastric Secretory Analysis. Analysis of gastric acid output requires gastric

1	are probably benign. Thus, EUS-guided needle biopsy should be considered. Submucosal varices also can be assessed by EUS.Gastric Secretory Analysis. Analysis of gastric acid output requires gastric intubation, and it is performed infrequently nowadays. This test may be useful in the evaluation of patients with hypergastrinemia, including the Zollinger-Ellison syn-drome (ZES), patients with refractory ulcer or GERD, and patients with recurrent ulcer after operation. Historically, gas-tric analysis was performed most commonly to test for the ade-quacy of vagotomy in postoperative patients with recurrent or persistent ulcer. Now this can be done by assessing peripheral ABCDFigure 26-21. Conventional double-contrast barium study (A) shows a focal protruding mass (arrow) on the gastric fundus. Axial com-puted tomographic scan (B) also shows a protruding polyp (arrow). The three-dimensional computed tomographic gastrographic images in the transparent (C) mode shows an elevated lesion on the

1	Axial com-puted tomographic scan (B) also shows a protruding polyp (arrow). The three-dimensional computed tomographic gastrographic images in the transparent (C) mode shows an elevated lesion on the gastric fundus (arrow). Photograph of the total gastrectomy specimen (D) shows a well-demarcated polypoid mass (arrow); this lesion was confirmed as early gastric carcinoma type I on microscopic examination (not shown). (Reproduced with permission from Shin KS, Kim SH, Han JK, et al: Three-dimensional MDCT gastrography compared with axial CT for the detection of early gastric cancer, J Comput Assist Tomogr. 2007 Sep-Oct;31(5):741-749.)Brunicardi_Ch26_p1099-p1166.indd 111601/03/19 7:11 PM 1117STOMACHCHAPTER 26pancreatic polypeptide levels in response to sham feeding.72 A 50% increase in pancreatic polypeptide within 30 minutes of sham feeding suggests intact vagal function.Normal basal acid output (BAO) is greater than 5 mEq/h. MAO is the average of the two final stimulated 15-minute

1	polypeptide within 30 minutes of sham feeding suggests intact vagal function.Normal basal acid output (BAO) is greater than 5 mEq/h. MAO is the average of the two final stimulated 15-minute periods and is usually 10 to 15 mEq/h. Peak acid output is defined as the highest of the four stimulated periods. Patients with a gastrinoma commonly have a high BAO, often above 30 mEq/h, but consistently above 15 mEq/h unless there has been previous vagotomy or gastric resection. In patients with gastrinoma, the ratio of BAO to MAO exceeds 0.6. Normal acid output in the patient prescribed acid-suppressive medica-tion usually means that the patient is noncompliant. To assess acid-secretory capacity in the absence of medication effect, H2 blockers and PPIs should be withheld for several days before gastric analysis.Scintigraphy. The standard scintigraphic evaluation of gastric emptying involves the ingestion of a test meal with one or two isotopes and scanning the patient under a gamma camera. A

1	analysis.Scintigraphy. The standard scintigraphic evaluation of gastric emptying involves the ingestion of a test meal with one or two isotopes and scanning the patient under a gamma camera. A curve for gastric emptying is plotted, and the half-time is calcu-lated. Normal standards exist at each facility. Duodenogastric reflux can be quantitated by the IV administration of hepato-biliary iminodiacetic acid (HIDA scan), which is concentrated and excreted by the liver into the duodenum. Software allows a semiquantitative assessment of how much of the isotope refluxes into the stomach. Positron emission tomography (PET) scan or CT/PET scan is useful in staging certain patients with gastric malignancy.Tests for Helicobacter pylori. A variety of tests can help the clinician to determine whether the patient has active H pylori infection.73 The predictive value (positive and negative) of any of these tests when used as a screening tool depends on the prevalence of H pylori infection in the

1	the patient has active H pylori infection.73 The predictive value (positive and negative) of any of these tests when used as a screening tool depends on the prevalence of H pylori infection in the screened population. A positive test is quite accurate in predicting H pylori infection, but a negative test can be unreliable. Thus, in the appropriate clinical setting, treatment for H pylori should be initiated on the basis of a positive test, but not necessarily withheld if the test is negative. Helicobacter infection should be treated when the diagnosis is made and eradication is confirmed.A positive serologic test is presumptive evidence of active infection if the patient has never been treated for H pylori. Histologic examination of gastric mucosal biopsy using special stains is the gold standard test for helicobacter infection. Other sensitive tests include commercially available rapid urease tests, which assay for the presence of urease in mucosal biopsy speci-mens (strong

1	the gold standard test for helicobacter infection. Other sensitive tests include commercially available rapid urease tests, which assay for the presence of urease in mucosal biopsy speci-mens (strong presumptive evidence of infection). Urease is an omnipresent enzyme in H pylori strains that colonize the gas-tric mucosa. The carbon labeled urea breath test has become the standard test to confirm eradication of H pylori following appropriate treatment.74 In this test, the patient ingests urea labeled with nonradioactive 13C or 14C. The labeled urea is acted upon by the urease present in the H pylori and converted into ammonia and carbon dioxide. The radiolabeled carbon dioxide is excreted from the lungs and can be detected in the expired air (Fig. 26-22). It also can be detected in a blood sample. The fecal antigen test also is quite sensitive and specific for active H pylori infection and may also be used to confirm cure after treatment. Helicobacter culture may be useful to assess

1	sample. The fecal antigen test also is quite sensitive and specific for active H pylori infection and may also be used to confirm cure after treatment. Helicobacter culture may be useful to assess antimi-crobial resistance in persistently recalcitrant cases.Antroduodenal Motility Testing and Electrogastrography. Antroduodenal motility testing and electrogastrography (EGG) are performed in specialized centers and may be useful in the evaluation of the occasional patient with dyspeptic symptoms. EGG consists of the transcutaneous recording of gastric myo-electric activity. Antroduodenal motility testing is done with a tube placed transnasally or transorally into the distal duodenum. There are pressure-recording sensors extending from the stom-ach to the distal duodenum. The combination of these two tests together with scintigraphy provides a thorough assessment of gastric motility.HELICOBACTER PYLORI INFECTIONOver 50% of people worldwide are infected with Helicobacter pylori.75,76

1	of these two tests together with scintigraphy provides a thorough assessment of gastric motility.HELICOBACTER PYLORI INFECTIONOver 50% of people worldwide are infected with Helicobacter pylori.75,76 Infection with H pylori is a chronic disease and does not resolve spontaneously without specific treatment. World-wide, H pylori–induced gastritis accounts for 80% to 90% of all gastritis. Chronic gastritis associated with H pylori is the most important risk factor for peptic ulcer and gastric adeno-carcinoma. Successful H pylori treatment largely eliminates recurrent peptic ulcer in infected patients, and eradication of H pylori worldwide would eliminate most cases of gastric Figure 26-22. Labeled urea breath test to detect Helicobacter infection. (Repro-duced with permission from Walsh JH, Peterson WL. The treatment of Helico-bacter pylori infection in the management of peptic ulcer disease, N Engl J Med. 1995 Oct 12;333(15):984-991.)[13C] urea13CO2 (µmol)Positive breathurea

1	Walsh JH, Peterson WL. The treatment of Helico-bacter pylori infection in the management of peptic ulcer disease, N Engl J Med. 1995 Oct 12;333(15):984-991.)[13C] urea13CO2 (µmol)Positive breathurea testUreaseBloodHours1213CO2 in breathNH2NH22NH3+CO2H2O + 13C = 0Negative breathurea testBrunicardi_Ch26_p1099-p1166.indd 111701/03/19 7:11 PM 1118SPECIFIC CONSIDERATIONS PART IIadenocarcinoma, a major cause of cancer death worldwide.77 Helicobacter pylori infection is also associated with MALT lymphoma, dyspepsia, hyperplastic gastric polyps, and even immune thrombocytopenic purpura.Human beings are the only reservoir for H pylori. Infection is presumed to occur by oral ingestion of the bacterium, which dramatically alters the gastric microbiome.78 In helicobacter-infected individuals, 90% of gastric bacteria are helicobacter, whereas in helicobacter-negative patients 90% of gastric bac-teria are a combination of firmicutes, actinobacteria, bacte-roidetes, proteobacteria, and

1	90% of gastric bacteria are helicobacter, whereas in helicobacter-negative patients 90% of gastric bac-teria are a combination of firmicutes, actinobacteria, bacte-roidetes, proteobacteria, and fusobacteria. The prevalence of H pylori infection varies among populations and is strongly cor-related with socioeconomic conditions. In developing countries, H pylori infection usually occurs in childhood, and over 80% of adults are infected. Reinfection after curative treatment is common. Infection rates are lower in industrialized countries, and the prevalence of infection in the United States has been declining since the second half of the 19th century as hygiene and sanitation have improved. Nonetheless, H pylori infection is predicted to remain endemic in the United States for the next century. Family members of infected individuals and healthcare workers are at increased risk of infection.With specialized flagella and a rich supply of urease, H pylori is uniquely equipped for survival

1	Family members of infected individuals and healthcare workers are at increased risk of infection.With specialized flagella and a rich supply of urease, H pylori is uniquely equipped for survival in the hostile envi-ronment of the stomach.[79-81] Helicobacter strains that lack either flagella or urease are nonpathogenic. The pathogenesis of helicobacter infection involves survival in the acidic gastric lumen, flagellated movement from the lumen across the mucus layer to the surface epithelial cell, adhesion to the surface epi-thelial cell, and toxin production. Up to 15% of the protein in a Helicobacter organism is composed of cytoplasmic urease that converts periplasmic urea into CO2 and ammonia. This buffers the surrounding acid, allowing the bacteria to survive the inimical luminal environment until it can burrow deeply into the surface mucus, propelled by its flagella (Fig. 26-23). H pylori typically does not invade the surface epithelial cell layer. Rather, it triggers a host

1	environment until it can burrow deeply into the surface mucus, propelled by its flagella (Fig. 26-23). H pylori typically does not invade the surface epithelial cell layer. Rather, it triggers a host immune response by attach-ing to gastric epithelial cells. Important Helicobacter adhesins mediating surface cell injury include neutrophil activating pro-tein A, heat shock protein 60, and sialic acid–binding adhesin. Helicobacter-produced toxins include vacuolating cytotoxin A and cag A (cytotoxin-associated gene A). The initial inflammatory response to Helicobacter infection is characterized by recruit-ment of neutrophils, followed sequentially by T and B lympho-cytes, plasma cells, and macrophages (Fig. 26-24). The resultant chronic gastric inflammation in affected individuals is charac-terized by enhanced mucosal expression of multiple cytokines and the presence of reactive oxygen and nitrogen species, and long-term infection is associated with mucosal cell DNA damage and

1	charac-terized by enhanced mucosal expression of multiple cytokines and the presence of reactive oxygen and nitrogen species, and long-term infection is associated with mucosal cell DNA damage and chromosomal instability and increased apopto-sis (Fig. 26-25).80,81 The net effect is a weakening of mucosal defenses. The mechanism by which the helicobacter organism avoids recognition and destruction by the mucosal immune sys-tem is a topic of interest and active research.82Acute H pylori infection causes a nonerosive pangastritis that is invariably followed by the development of chronic gastritis. Chronic antral gastritis with sparing of the proximal stomach occurs in about 10% of infected patients, and this pre-disposes to peptic ulcer disease (PUD). The other 90% of Heli-cobacter-infected patients develop chronic inflammation of the proximal stomach (corpus dominant gastritis), which can lead to gastric cancer in about 1% to 3% of this group.Figure 26-23. Helicobacter pylori closely

1	patients develop chronic inflammation of the proximal stomach (corpus dominant gastritis), which can lead to gastric cancer in about 1% to 3% of this group.Figure 26-23. Helicobacter pylori closely adherent to the cell membrane (top), and spiral-shaped H pylori attached to epithelial surface and surrounding microvilli (bottom). In the image on the bottom, the bacterial flagella can be seen arising from the upper pole of the bacterium. (Reproduced with permission from Mertz HR, Walsh JH: Peptic ulcer pathophysiology, Med Clin North Am. 1991 Jul;75(4):799-814.)H pylori infection is the major cause of peptic ulceration. Patients with H pylori infection and antral gastritis are three and one-half times more likely to develop PUD than patients without H pylori infection. Up to 90% of patients with duode-nal ulcers, and at least 70% of patients with gastric ulcers, have H pylori infection. It is clear from multiple randomized prospec-tive studies that curing H pylori infection dramatically

1	with duode-nal ulcers, and at least 70% of patients with gastric ulcers, have H pylori infection. It is clear from multiple randomized prospec-tive studies that curing H pylori infection dramatically alters the natural history of PUD, decreasing the recurrent ulcer rate from more than 75% in patients treated with a course of acid-suppressive therapy alone (in whom H pylori is not eradicated) to less than 20% in patients treated with a course of antibacterial therapy (Fig. 26-26).83In patients with duodenal ulcer caused by helicobacter, the associated antral gastritis leads to relative hypergastrinemia by depleting antral somatostatin, the primary inhibitor of antral gastrin release. H pylori infection is associated with decreased Brunicardi_Ch26_p1099-p1166.indd 111801/03/19 7:11 PM 1119STOMACHCHAPTER 26levels of somatostatin, decreased somatostatin messenger RNA production, and fewer somatostatin-producing D cells. The mechanism of decreased antral somatostatin synthesis and

1	PM 1119STOMACHCHAPTER 26levels of somatostatin, decreased somatostatin messenger RNA production, and fewer somatostatin-producing D cells. The mechanism of decreased antral somatostatin synthesis and release may be related to (a) antral alkalinization due to heli-cobacter urease (acid in the antrum releases somatostatin); (b) toxic cytokine effect on antral D cells; and/or (c) Helicobacter production of N-α-methylhistamine, an H3 receptor agonist, which binds H3 receptors on the antral D cell and decreases somatostatin release.84 Since the gastritis does not involve the oxcyntic mucosa, hypergastrinemia leads to hyperacidity and parietal cell hyperplasia. The acid hypersecretion and the antral gastritis are thought to lead to antral epithelial metaplasia in the postpyloric duodenum. This duodenal metaplasia allows H pylori to colonize the duodenal mucosa, and this is where the duodenal ulcer occurs. In fact, in patients with gastric metapla-sia of the duodenum, the risk of developing

1	duodenal metaplasia allows H pylori to colonize the duodenal mucosa, and this is where the duodenal ulcer occurs. In fact, in patients with gastric metapla-sia of the duodenum, the risk of developing a duodenal ulcer increases 50-fold. When H pylori colonizes the duodenum, there is a significant decrease in acid-stimulated duodenal bicarbonate release. When H pylori infection is successfully treated, acid secretory physiology tends to normalize. Relapse of duodenal ulcer after eradication of H pylori may signal reinfection of the gastric mucosa by the organism.Many patients with antral dominant helicobacter gastritis never develop duodenal ulcer, and some patients with peptic ulcer do not have Helicobacter. This obviously suggests that there are other important pathogenetic factors involved in peptic ulcer. And even in the presence of active H pylori infection, strong acid suppression usually heals peptic ulcer, an observa-tion consistent with the old dictum “no acid, no ulcer.” But

1	in peptic ulcer. And even in the presence of active H pylori infection, strong acid suppression usually heals peptic ulcer, an observa-tion consistent with the old dictum “no acid, no ulcer.” But suc-cessful helicobacter treatment eliminates ulcer recurrence and the need for long-term PPI. And long-term PPI in patients with active Helicobacter infection may lead to corpus predominant gastritis, which leads to atrophic gastritis and increases the risk of gastric cancer. Thus, Helicobacter infection should be treated and eradication confirmed.Testing for H pylori infection should be performed in patients with peptic ulcer, gastritis, significant dyspepsia, MALT lymphoma, and early gastric cancer.85 Noninvasive methods for diagnosis of H pylori infection include the urea breath test, serology, and detection of stool antigen. The urea breath test has a sensitivity and specificity of greater than 90% and is useful for initial diagnosis of infection and for follow-up after eradication

1	and detection of stool antigen. The urea breath test has a sensitivity and specificity of greater than 90% and is useful for initial diagnosis of infection and for follow-up after eradication therapy since it is positive only in the presence of active infection. The stool antigen test is another noninvasive test to detect active H pylori infection, but it is recommended that only locally validated tests be used.86 Because H pylori induces a strong immunologic response, serological testing is useful but may not be as accurate as the urea breath test or the stool antigen test, and a positive serology persists after eradica-tion of H pylori infection, so serology is not useful to confirm successful treatment of Helicobacter infection. H pylori infec-tion can also be diagnosed by histologic evaluation of gastric biopsies and/or the rapid urease test on fresh biopsies. Culture of H pylori is not routine and is usually reserved for recurrent infection and for antibiotic sensitivity testing

1	of gastric biopsies and/or the rapid urease test on fresh biopsies. Culture of H pylori is not routine and is usually reserved for recurrent infection and for antibiotic sensitivity testing when second-line therapy has failed. All tests for H pylori have a false negative rate. Empiric Helicobacter treatment can be considered despite negative tests if clinical likelihood of infection is high, e.g., a compliant nonsmoking, non–NSAID-consuming patient facing operation for nonhealing peptic ulcer or a patient with unex-plained gastritis.Patients with a positive test should be treated and eradi-cation confirmed. Spontaneous cure without treatment is very rare. It is important to note that none of the therapeutic regimens reported to date cure H pylori infection in 100% of patients. To be effective, antimicrobial drugs must be combined with gas-tric acid secretion inhibitors or bismuth salts. The Maastricht V/Florence Consensus Report87 provides current recommenda-tions for diagnosis and

1	antimicrobial drugs must be combined with gas-tric acid secretion inhibitors or bismuth salts. The Maastricht V/Florence Consensus Report87 provides current recommenda-tions for diagnosis and treatment of H pylori infection in various clinical scenarios, including recommendations for areas with high metronidazole and clarithromycin resistance.87,88 Ideally, a treatment regimen is chosen with 90% effectiveness. Treat-ment failure requires an alternative course of therapy. Failure to eradicate infection after two tries should prompt Helicobacter culture and sensitivity testing and referral to a specialist. With assiduous treatment, Helicobacter eradication can be achieved in nearly every patient. Patients with atrophic gastritis require endoscopic surveillance (see discussion of gastritis later in this Figure 26-24. Model of Helico-bacter effects on duodenal ulcer pathogenesis. (Reproduced with per-mission from Feldman M, Friedman LS, Sleisenger MH, et al: Sleisenger and Fordtran’s

1	later in this Figure 26-24. Model of Helico-bacter effects on duodenal ulcer pathogenesis. (Reproduced with per-mission from Feldman M, Friedman LS, Sleisenger MH, et al: Sleisenger and Fordtran’s Gastrointestinal and Liver Disease, 7th ed. Philadelphia, PA: Elsevier/Saunders; 2002.)Multiple factors (smoking, ageat acquisition of infection)Somatostatin/gastrindysregulationAcquisition of H pyloriDuodenum ulcerationIncreased acidsecretionGastric metaplasiain duodenumDuodenum bicarbonate secretionInflammation (Duodenitis)Chronic H pyloriinfection in stomachH pylori colonization in duodenumBrunicardi_Ch26_p1099-p1166.indd 111901/03/19 7:11 PM 1120SPECIFIC CONSIDERATIONS PART IIchapter) because the same sequence of inflammation to meta-plasia to dysplasia to carcinoma, that is well known to occur in the esophagus from reflux-induced inflammation (and in the colon from inflammatory bowel disease), is now increasingly well recognized to occur in the stomach with Helicobacter-induced

1	to occur in the esophagus from reflux-induced inflammation (and in the colon from inflammatory bowel disease), is now increasingly well recognized to occur in the stomach with Helicobacter-induced gastritis. Helicobacter also clearly has an etiologic role in the development of gastric lymphoma.PEPTIC ULCER DISEASEPeptic ulcers are focal defects in the gastric or duodenal mucosa that extend into the submucosa or deeper. They may be acute or chronic and, ultimately, are caused by an imbalance between mucosal defenses and acid/peptic injury (Fig. 26-27).89,90 Peptic ulcer remains a common outpatient diagnosis, but the number of Figure 26-25. Pathogen-host interactions in the pathogenesis of Helicobacter pylori infection. ICAM = intercellular adhesion molecule-1; IFN-γ = interferon-γ; LPS = lipopolysaccharide; NF-κB = nuclear factor κB; PAI = pathogenicity island; PMN = polymorphonuclear neutrophil; TNF-α = tumor necrosis factor-α; VCAM = vascular cell adhesion molecule. (Used with

1	= lipopolysaccharide; NF-κB = nuclear factor κB; PAI = pathogenicity island; PMN = polymorphonuclear neutrophil; TNF-α = tumor necrosis factor-α; VCAM = vascular cell adhesion molecule. (Used with permission from Manuel Amieva, Stanford University.)cag+H pyloristraincag-PAI-encodedsecretoryapparatusMultiple adhesions:BabA (binds tolewis B), AIpA,AlpB, HopZPhospholipaseA2Alterationsin mucousglycoproteinsGastricepithelialcellFlagellaMucusActinpolymerizationCagAphosphorylationVacAApoptosisDisruption of epithelialbarrierFasexpressionUreaseLPSPorinsInterleukin-8CagAMacrophageInterleukin-12Th 1 cellTh 2 cellB cellCytokine-inducedchanges in gastric physiologyTh 0 cellICAM and VCAMexpressionPMN recruitmentInterleukin-8Chemotacticinterleukin-8 gradienton proteoglycanscaffoldingNeutrophilT cellB cellBlood vesselNF-˜ B AP-1 GRO-°ENA-78T-cell and B-cellextravasationINF-˛ TNF-°Interleukin-1˙MHC II, B7-1,and

1	gradienton proteoglycanscaffoldingNeutrophilT cellB cellBlood vesselNF-˜ B AP-1 GRO-°ENA-78T-cell and B-cellextravasationINF-˛ TNF-°Interleukin-1˙MHC II, B7-1,and B7-2expressionAnti-H+/K+-ATPaseantibodiesBrunicardi_Ch26_p1099-p1166.indd 112001/03/19 7:11 PM 1121STOMACHCHAPTER 26physician visits, hospital admissions, and elective operations for PUD has decreased steadily and dramatically over the past four decades. Interestingly, the start of these trends all predated the widespread use of acid suppression, or highly selective vagot-omy. The incidence of emergency surgery and the death rate associated with peptic ulcers has not decreased nearly so dra-matically. These epidemiologic changes probably represent the net effect of several factors, including (beneficially) decreased prevalence of H pylori infection, better medical therapy, and increased outpatient management and (detrimentally) the use of NSAIDs and aspirin (with and without ulcer prophylaxis)

1	decreased prevalence of H pylori infection, better medical therapy, and increased outpatient management and (detrimentally) the use of NSAIDs and aspirin (with and without ulcer prophylaxis) in an aging population with multiple risk factors.Figure 26-26. Helicobacter treatment dramatically decreases the recurrence rate of duodenal and gastric ulcer. (Reproduced with permission from Peek RM, Blaser MJL: Pathophysiology of Helico-bacter pylori-induced gastritis and peptic ulcer disease, Am J Med. 1997 Feb;102(2):200-207.)Figure 26-27. Balance of aggressive and defensive factors in the gastric mucosa. (Reproduced with permission from Suerbaum S, Michetti P: Helicobacter pylori infection, N Engl J Med. 2002 Oct 10;347(15):1175-1186.)PUD is one of the most common GI disorders in the United States with a prevalence of about 2%, and a lifetime cumulative prevalence of about 10%, peaking around age 70 years.91 The costs of PUD, including lost work time and productivity, are estimated to be

1	with a prevalence of about 2%, and a lifetime cumulative prevalence of about 10%, peaking around age 70 years.91 The costs of PUD, including lost work time and productivity, are estimated to be above $8 billion per year in the United States. In 1998, approximately 1.5% of all Medicare hospital costs were spent treating PUD, and the crude mortal-ity rate for peptic ulcer was 1.7 per 100,000 individuals. Using the National Inpatient Sample, it can be estimated that the mortality rate in patients hospitalized in 2006 with duodenal ulcer was 3.7% compared to 2.1% for gastric ulcer,92 and the age adjusted hospitalization rate was 56.5 per 100,000, down 21% from the previous decade. Recent studies have shown an increase in the rates of hospitalization and mortality in elderly patients for the peptic ulcer complications of bleeding and perforation.93 This may be due in part to the increasingly com-mon use of NSAIDs and aspirin in this elderly cohort, many of whom also have H pylori

1	for the peptic ulcer complications of bleeding and perforation.93 This may be due in part to the increasingly com-mon use of NSAIDs and aspirin in this elderly cohort, many of whom also have H pylori infection.Pathophysiology and EtiologyA variety of factors may contribute to the development of PUD. Although it is now recognized that the large majority of duode-nal and gastric ulcers are caused by H pylori infection (see previ-ous discussion on H pylori) and/or NSAID use20,94 (Fig. 26-28), the final common pathway to ulcer formation is acid-peptic injury of the gastroduodenal mucosal barrier. Acid suppression heals both duodenal and gastric ulcers and prevents recurrence if continued. In general, H pylori predisposes to ulceration, both by acid hypersecretion and by compromise of mucosal defense mechanisms. NSAID use causes ulcers predominantly by com-promise of mucosal defenses. Duodenal ulcer was traditionally viewed as a disease of increased acid-peptic action on the duo-denal

1	defense mechanisms. NSAID use causes ulcers predominantly by com-promise of mucosal defenses. Duodenal ulcer was traditionally viewed as a disease of increased acid-peptic action on the duo-denal mucosa, whereas gastric ulcer was viewed as a disease of weakened mucosal defenses. An increased understanding of peptic ulcer pathophysiology has blurred this overly simplistic distinction. Clearly, weakened mucosal defenses play a role in both duodenal and gastric ulcers, and acid hypersecretion may result in a duodenal or gastric ulcer in the setting of normal mucosal defenses.Elimination of H pylori infection or NSAID use is important for optimal ulcer healing, and perhaps is even more important in preventing ulcer recurrence and/or complications. A variety of other diseases are known to cause peptic ulcer, including ZES (gastrinoma), antral G-cell hyperfunction and/or hyperplasia, systemic mastocytosis, trauma, burns, and major physiologic stress. Other causative agents include drugs

1	peptic ulcer, including ZES (gastrinoma), antral G-cell hyperfunction and/or hyperplasia, systemic mastocytosis, trauma, burns, and major physiologic stress. Other causative agents include drugs (all NSAIDs, aspirin, and cocaine), smoking, and psychologic stress. In the United States, probably more than 90% of serious peptic ulcer complications can be attributed to H pylori infec-tion, NSAID use, and/or cigarette smoking.Acid Secretion and Peptic Ulcer. A variety of abnormalities related to mucosal acid exposure have been described in patients with duodenal ulcer (Fig. 26-29).95 Although duodenal ulcer patients as a group have a higher mean BAO and mean MAO compared to normal controls, many duodenal ulcer patients have basal and peak acid outputs in the normal range, and there is no correlation between acid secretion and the severity of the ulcer disease. As a group, duodenal ulcer patients produce more acid than normal controls in response to any known acid secre-tory stimulus.

1	correlation between acid secretion and the severity of the ulcer disease. As a group, duodenal ulcer patients produce more acid than normal controls in response to any known acid secre-tory stimulus. Although they usually have normal fasting serum gastrin levels, DU patients often produce more gastric acid at AcidPepsinNSAIDsH pyloriAggressionDefenseRepairBicarbonateBlood flowMucusCell junctionsApical resistanceRestitutionMucoid capProliferationGrowth factors0%10%20%30%40%50%60%70%80%90%100%Duodenal ulcerrecurrenceGastric ulcerrecurrenceRanitidine +antibioticsRanitidine aloneBrunicardi_Ch26_p1099-p1166.indd 112101/03/19 7:11 PM 1122SPECIFIC CONSIDERATIONS PART IIany given dose of gastrin than controls. Considering that many duodenal ulcer patients do produce excessive gastric acid, it has been argued that a “normal” fasting gastrin level in these patients is inappropriately high, and that there is an impaired feedback mechanism, especially in light of the apparently increased

1	it has been argued that a “normal” fasting gastrin level in these patients is inappropriately high, and that there is an impaired feedback mechanism, especially in light of the apparently increased sensitivity of the parietal cell mass to gastrin. Many of these long-standing observations now seem reasonable in light of recently gained understanding of the perturbations in acid and gastrin secretion associated with H pylori infection. Some patients with duodenal ulcer also have increased rates of gastric emptying that deliver an increased acid load per unit of time to the duodenum. Finally, the buffering capacity of the duodenum in many patients with duodenal ulcer is compromised due to decreased duodenal bicarbonate secretion and duodenal gastric metaplasia.In patients with gastric ulcer, acid secretion is vari-able. Currently, five types of gastric ulcer are described, although the original Johnson classification contained three types (Fig. 26-30).96 The most common, Johnson type I

1	acid secretion is vari-able. Currently, five types of gastric ulcer are described, although the original Johnson classification contained three types (Fig. 26-30).96 The most common, Johnson type I gas-tric ulcer, is typically located near the angularis incisura on the lesser curvature, close to the border between antral and corpus mucosa. Patients with type I gastric ulcer usually have normal or decreased acid secretion. Type II gastric ulcer is associ-ated with active or quiescent duodenal ulcer disease, and type III gastric ulcer is prepyloric ulcer disease. Both type II and type III gastric ulcers are associated with normal or increased gastric acid secretion and surgically are treated similar to duode-nal ulcer. Type IV gastric ulcers occur near the GE junction, and acid secretion is normal or below normal. Type V gastric ulcers are medication induced and may occur anywhere in the stomach. Patients with gastric ulcers may have weak mucosal defenses that permit an abnormal amount

1	normal or below normal. Type V gastric ulcers are medication induced and may occur anywhere in the stomach. Patients with gastric ulcers may have weak mucosal defenses that permit an abnormal amount of injurious acid back-diffusion into the mucosa. Duodenogastric reflux may play a role in weakening the gastric mucosal defenses, and a variety of components in duodenal juice, including bile, lysolecithin, and pancreatic juice, have been shown to cause injury and inflam-mation in the gastric mucosa. NSAIDs and aspirin have similar effects. Although chronic gastric ulcer usually is associated with surrounding gastritis, it is unproven that the latter leads to the former.Nonsteroidal Anti-Inflammatory Drugs in Peptic Ulcer Disease. Chronic use of NSAIDs (including aspirin) increases the risk of peptic ulcer disease about fivefold and upper GI bleeding at least twofold.97-100 Complications of PUD (specifi-cally hemorrhage and perforation) are much more common in patients taking NSAIDs. More

1	peptic ulcer disease about fivefold and upper GI bleeding at least twofold.97-100 Complications of PUD (specifi-cally hemorrhage and perforation) are much more common in patients taking NSAIDs. More than half of patients who present with peptic ulcer hemorrhage or perforation report the recent use of NSAIDs, including aspirin. Many of these patients remain asymptomatic until they develop these life-threatening complications.Figure 26-29. Frequency of physiologic abnormali-ties in patients with duodenal ulcer (DU). HCO3 = bicarbonate; MAO = maximal acid output. (Reproduced with permission from Yamada T, Alpers DH, Laine L, et al: Textbook of Gastroenterology, 4th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2003.)2550751000Approximate % of DU patientsPathophysiologic abnormalities in DUvary in frequencyNocturnal acid secretionpH inhibition of gastrin releaseGastric emptyingPentagastrin-stimulated MAODaytime acid secretionDuodenal acid loadDuodenal HCO3 secretionFigure

1	in DUvary in frequencyNocturnal acid secretionpH inhibition of gastrin releaseGastric emptyingPentagastrin-stimulated MAODaytime acid secretionDuodenal acid loadDuodenal HCO3 secretionFigure 26-28. “Causes” of peptic ulcer disease. Z.E. = Zollinger-Ellison syndrome. (Data from Graham DY, Lew GM, Klein PD, et al. Effect of treatment of Helicobacter pylori infec-tion on the long-term recurrence of gas-tric or duodenal ulcer. A randomized, controlled study, Ann Intern Med. 1992 May 1;116(9):705-708.)Conditions associated with peptic ulcerNSAIDuseNone knownZ.E., otherH. pyloriinfectionNSAIDuseNone knownZ.E., otherH. pyloriinfectionDuodenalGastricBrunicardi_Ch26_p1099-p1166.indd 112201/03/19 7:12 PM 1123STOMACHCHAPTER 26The overall risk of significant serious adverse GI events in patients taking NSAIDs is more than three times that of con-trols (Table 26-6). This risk increases to five times in patients more than age 60 years old. In elderly patients taking NSAIDs, the likelihood that

1	taking NSAIDs is more than three times that of con-trols (Table 26-6). This risk increases to five times in patients more than age 60 years old. In elderly patients taking NSAIDs, the likelihood that they will require an operation related to a GI complication is 10 times that of the control group, and the risk that they will die from a GI cause is about four and one-half times higher. This problem is put into perspective when one realizes that approximately 20 million patients in the United States take NSAIDs on a regular basis; perhaps even more regularly take aspirin. Persons who take NSAIDs also have a higher hospitalization rate for serious GI events than those who do not.Factors that clearly put patients at increased risk for NSAID-induced GI complications include age >60, prior GI event, high NSAID dose, concurrent steroid intake, and con-current anticoagulant intake. Proton pump inhibitors have been shown to significantly decrease upper GI bleeding risk in patients on chronic

1	event, high NSAID dose, concurrent steroid intake, and con-current anticoagulant intake. Proton pump inhibitors have been shown to significantly decrease upper GI bleeding risk in patients on chronic warfarin, low dose aspirin, and/or antiplate-let agents.101-103 ANY patient taking NSAIDs or aspirin who has one or more of these risk factors should receive concomitant acid suppressive medication,104 preferably PPI (Table 26-7). High-dose H2 blockers have been shown to be somewhat less effective than PPIs in preventing GI complications in these high-risk patients on antiplatelet therapy, but clearly, they are better than no acid suppression.105Smoking, Stress, and Other Factors. Epidemiologic studies suggest that smokers are about twice as likely to develop PUD as nonsmokers. Smoking increases gastric acid secretion and duodenogastric reflux. Smoking decreases both gastroduodenal prostaglandin production and pancreaticoduodenal bicarbonate production. These observations may be related,

1	gastric acid secretion and duodenogastric reflux. Smoking decreases both gastroduodenal prostaglandin production and pancreaticoduodenal bicarbonate production. These observations may be related, and any or all could explain the observed association between smoking and PUD.Although difficult to measure, both physiologic and psy-chologic stress undoubtedly play a role in the development of peptic ulcer in some patients.106 In 1842, Curling described duodenal ulcer and/or duodenitis in burn patients. Decades later, Cushing described the appearance of acute peptic ulcer-ation in patients with head trauma (Cushing’s ulcer). Even the ancients recognized the undeniable links between PUD and stress. Patients still present with ulcer complications (bleeding, perforation, and obstruction) that are seemingly exacerbated by stressful life events. The use of crack cocaine has been linked to juxtapyloric peptic ulcers with a propensity to perforate. Alco-hol is commonly mentioned as a risk factor

1	seemingly exacerbated by stressful life events. The use of crack cocaine has been linked to juxtapyloric peptic ulcers with a propensity to perforate. Alco-hol is commonly mentioned as a risk factor for PUD, but con-firmatory data are lacking.Clinical ManifestationsMore than 90% of patients with PUD complain of abdominal pain. The pain is typically nonradiating, burning in quality, and located in the epigastrium. The mechanism of the pain is unclear. Patients with duodenal ulcer often experience pain 2 to 3 hours after a meal and at night. Two-thirds of patients with duodenal ulcers will complain of pain that awakens them from sleep. The pain of gastric ulcer more commonly occurs with eating and is less likely to awaken the patient at night. A history of PUD, use of NSAIDs, over-the-counter antacids, or antisecretory drugs is suggestive of the diagnosis. Other signs and symptoms include nausea, bloating, weight loss, stool positive for occult blood, and anemia. Duodenal ulcer is about

1	antacids, or antisecretory drugs is suggestive of the diagnosis. Other signs and symptoms include nausea, bloating, weight loss, stool positive for occult blood, and anemia. Duodenal ulcer is about twice as common in men compared to women, but the incidence of gastric ulcer is similar in men and women. On average, gastric ulcer patients are older than duodenal ulcer patients, and the incidence is increasing in the elderly, perhaps because of increasing NSAID and aspirin.DiagnosisIn the young patient with dyspepsia and without alarm symp-toms, it may be appropriate to initiate empirical PPI therapy for PUD without upper endoscopy or upper GI series. NSAIDs and aspirin should be stopped if the patient is taking these drugs, and Helicobacter should be ruled out with testing and treated if pres-ent. It is prudent to discuss with the patient the small possibility of an alternative diagnosis, including malignancy, even if symp-toms improve with the initiation of empiric therapy. Patients

1	pres-ent. It is prudent to discuss with the patient the small possibility of an alternative diagnosis, including malignancy, even if symp-toms improve with the initiation of empiric therapy. Patients with persistent dyspepsia, and those who cannot stop NSAIDs or aspirin for health reasons should have an upper endoscopy, and all patients, regardless of age, should have this study if any alarm symptoms (see Table 26-5) are present. A double-contrast upper GI X-ray study may be useful. Once an ulcer has been confirmed endoscopically or radiologically, obvious possible causes (Helicobacter, NSAIDs, gastrinoma, cancer) should always be considered. All gastric ulcers should be adequately biopsied, and any sites of gastritis should be biopsied to rule out H pylori, and for histologic evaluation. Additional testing for H pylori may be indicated. It is reasonable to test all peptic IIIIIIIVVNSAID-inducedAcid hypersecretionFigure 26-30. Modified Johnson classification for gastric ulcer. I.

1	Additional testing for H pylori may be indicated. It is reasonable to test all peptic IIIIIIIVVNSAID-inducedAcid hypersecretionFigure 26-30. Modified Johnson classification for gastric ulcer. I. Lesser curve, incisura. II. Body of stomach, incisura + duodenal ulcer (active or healed). III. Prepyloric. IV. High on lesser curve, near gastroesophageal junction. V. Medication-induced (NSAID/acetylsalicylic acid), anywhere in stomach. (Reproduced with permission from Cameron JL: Current Surgical Therapy, 9th ed. Philadelphia, PA: Elsevier/Mosby; 2008.)Brunicardi_Ch26_p1099-p1166.indd 112301/03/19 7:12 PM 1124SPECIFIC CONSIDERATIONS PART IIulcer patients and those with nonulcer dyspepsia for H pylori (Table 26-8). A baseline serum gastrin level to rule out gastri-noma should be considered if the peptic ulcer is unusual (distal duodenal or jejunal) or if the patient is Helicobacter and NSAID negative.ComplicationsThe three most common complications of PUD, in decreas-ing order of

1	if the peptic ulcer is unusual (distal duodenal or jejunal) or if the patient is Helicobacter and NSAID negative.ComplicationsThe three most common complications of PUD, in decreas-ing order of frequency, are bleeding, perforation, and obstruction.92,94,107 Most peptic ulcer–related deaths in the United States are due to bleeding. Inhospital mortality and length of stay can be predicted by the AIMS65 score,108 with a score of 0 predicting negligible mortality and a score of 5 predicting a 30% inhospital mortality. Bleeding peptic ulcers are by far the most common cause of upper GI bleeding in patients admitted to a hospital (Fig. 26-31).109,110 Patients with a bleeding peptic ulcer typically present with melena and/or hematemesis. Naso-gastric aspiration is usually confirmatory of upper GI bleeding. Abdominal pain is quite uncommon. Shock may be present, necessitating aggressive resuscitation and blood transfusion. Table 26-7Patients taking NSAIDs or aspirin need concomitant acid

1	GI bleeding. Abdominal pain is quite uncommon. Shock may be present, necessitating aggressive resuscitation and blood transfusion. Table 26-7Patients taking NSAIDs or aspirin need concomitant acid suppressing medication if any of the following risk factors are present• Age over 60 years• History of acid/peptic disease• Concurrent steroid intake• Concurrent anticoagulant intake• High-dose or chronic NSAID use• High-dose or chronic aspirin use >325 mg/dayTable 26-8Indications for diagnosis and treatment of Helicobacter pyloriEstablished• Active peptic ulcer disease (gastric or duodenal ulcer)• Confirmed history of peptic ulcer disease (not previously treated for H pylori)• Gastric mucosa-associated lymphoid tissue lymphoma (low grade)• After endoscopic resection of early gastric cancer• Uninvestigated dyspepsia (depending on H pylori prevalence)Controversial• Nonulcer dyspepsia• Gastroesophageal reflux disease• Persons using NSAIDs• Unexplained iron deficiency anemia• Populations at

1	dyspepsia (depending on H pylori prevalence)Controversial• Nonulcer dyspepsia• Gastroesophageal reflux disease• Persons using NSAIDs• Unexplained iron deficiency anemia• Populations at higher risk for gastric cancerReproduced with permission from Chey WD, Wong BC; Practice Parameters Committee of the American College of Gastroenterology: American College of Gastroenterology guideline on the management of Helicobacter pylori infection, Am J Gastroenterol. 2007 Aug;102(8):1808-1825.Table 26-6Hospitalization rates for GI events with and without NSAID use in selected large populationsANNUALIZED INCIDENCEb THERAPIES USEDCLINICAL UPPER GI EVENTScCOMPLICATED UPPER GI EVENTSdSTUDYaNSAID CONTROLSTUDY DRUGSCONTROLSTUDY DRUGCONTROLSTUDY DRUGMUCOSANSAIDs (n = 4439)Misoprostol 200 μg four times a day + NSAID (n = 4404)3.1%1.6%1.5%0.7%CLASSIbuprofen 800 mg three times a day, diclofenac 75 mg twice a day (n = 3987)Celecoxib 400 mg twice a day (n = 3995)3.5%2.1%1.5%0.8% (No aspirine:

1	times a day + NSAID (n = 4404)3.1%1.6%1.5%0.7%CLASSIbuprofen 800 mg three times a day, diclofenac 75 mg twice a day (n = 3987)Celecoxib 400 mg twice a day (n = 3995)3.5%2.1%1.5%0.8% (No aspirine: 2.9%)1.4%1.3%0.4%VIGORNaproxen 500 mg twice a day (n = 4047)Rofecoxib 50 mg four times a day (n = 4029)4.5%2.1%1.4%0.6%aMUCOSA and VIGOR trials included only rheumatoid arthritis patients; CLASS trial included osteoarthritis (73%) and rheumatoid arthritis (27%).bIncidence for MUCOSA trial represents doubling of results provided at 6 months (although median follow-up was <6 months). Incidences for VIGOR and CLASS trials represent rates per 100 patient-years, although VIGOR median follow-up was 9 months, and CLASS data include only the first 6 months of the study.cIncludes perforations, obstructions, bleeding, and uncomplicated ulcers discovered on clinically indicated work-up.dIncludes perforation, obstruction, bleeding (documented due to ulcer or erosions in MUCOSA and CLASS; major

1	bleeding, and uncomplicated ulcers discovered on clinically indicated work-up.dIncludes perforation, obstruction, bleeding (documented due to ulcer or erosions in MUCOSA and CLASS; major bleeding in VIGOR).e21% of patients in CLASS study were taking low-dose aspirin.Note: All differences between controls and study drugs were significant except clinical upper GI events in overall CLASS study (P = .09).Reproduced with permission from Laine L: Approaches to nonsteroidal anti-inflammatory drug use in the high-risk patient, Gastroenterology 2001 Feb;120(3):594-606.Brunicardi_Ch26_p1099-p1166.indd 112401/03/19 7:12 PM 1125STOMACHCHAPTER 26Early endoscopy is important to diagnose the cause of the bleed-ing and to assess the need for hemostatic therapy.Three-fourths of the patients who come to the hospital with bleeding peptic ulcer will stop bleeding if given acid sup-pression and nothing by mouth. However, one fourth will con-tinue to bleed or will rebleed after an initial quiescent

1	to the hospital with bleeding peptic ulcer will stop bleeding if given acid sup-pression and nothing by mouth. However, one fourth will con-tinue to bleed or will rebleed after an initial quiescent period, and virtually all the mortalities (and all the operations for bleeding) occur in this group. This group can be fairly well delineated based on clinical factors related to the magnitude of the hemorrhage, comorbidities, age, and endoscopic find-ings. Shock, hematemesis, transfusion requirement exceeding four units in 24 hours, and certain endoscopic stigmata (active bleeding or visible vessel) define this high-risk group. Risk stratification tools have proven useful in predicting rebleed-ing and death, and in identifying a low risk cohort. As can be seen in Table 26-9, the maximal Blatchford score is 23, and the maximal Rockall score is 11. The former does not use endoscopic criteria and may be better in identifying the low-risk cohort. Studies have shown that a Blatchford score of 1

1	score is 23, and the maximal Rockall score is 11. The former does not use endoscopic criteria and may be better in identifying the low-risk cohort. Studies have shown that a Blatchford score of 1 or less, or a Rockall score of 2 or less, identifies patients who are very unlikely to be suffering from life-threatening upper GI bleeding. The shorter modified Blatchford score may be just as useful (BUN, Hgb, pulse, BP; maximal score 16).111 High-risk patients benefit from endoscopic therapy to stop the bleeding, while low-risk patients with low-risk lesions can be promptly discharged and treated as outpatients. The most common endoscopic hemostatic modalities used are injection with epinephrine and electrocautery. In a case with exposed vessel, mechanical hemostasis using clips is useful to control the bleeding.112 Biopsy should be performed to evaluate for H pylori infection. Persistent bleeding or rebleeding after endo-scopic therapy is an indication for repeat endoscopic treatment.

1	control the bleeding.112 Biopsy should be performed to evaluate for H pylori infection. Persistent bleeding or rebleeding after endo-scopic therapy is an indication for repeat endoscopic treatment. Surgery should be considered after two endoscopic failures. Elderly patients and patients with multiple comorbidities do not tolerate repeated episodes of hemodynamically significant hemorrhage, and they may benefit from early elective opera-tion after initially successful endoscopic treatment, especially if they have a high-risk ulcer.Planned surgery under controlled circumstances often yields better outcomes than emergent surgery. Deep bleeding ulcers on the posterior duodenal bulb or lesser gastric curvature are high-risk lesions because they often erode large arteries less amenable to nonoperative treatment, and early operation should be considered.Perforated peptic ulcer usually presents as an acute abdo-men. The patient can often give the exact time of onset of the excruciating

1	nonoperative treatment, and early operation should be considered.Perforated peptic ulcer usually presents as an acute abdo-men. The patient can often give the exact time of onset of the excruciating abdominal pain. Initially, a chemical peritonitis develops from the gastric and/or duodenal secretions, but within hours a bacterial peritonitis supervenes. The patient is in obvi-ous distress, and the abdominal examination shows peritoneal signs. Usually, marked involuntary guarding and rebound ten-derness is evoked by a gentle examination. Upright chest X-ray shows free air in about 80% of patients (Fig. 26-32). Once the diagnosis has been made, the patient is given analgesia and antibiotics, resuscitated with isotonic fluid, and taken to the operating room. Fluid sequestration into the third space of the inflamed peritoneum can be impressive, so preoperative fluid resuscitation is mandatory. Sometimes, the perforation has sealed spontaneously by the time of presentation, and surgery can

1	space of the inflamed peritoneum can be impressive, so preoperative fluid resuscitation is mandatory. Sometimes, the perforation has sealed spontaneously by the time of presentation, and surgery can be avoided if the patient is doing well. Nonoperative man-agement is appropriate only if there is objective evidence that the leak has sealed (i.e., radiologic contrast study), and in the absence of clinical peritonitis.Gastric outlet obstruction occurs in no more than 5% of patients with PUD. It is usually due to duodenal or prepyloric ulcer disease, and it may be acute (from inflammatory swelling and peristaltic dysfunction) or chronic (from cicatrix). Patients typically present with nonbilious vomiting and may have pro-found hypokalemic hypochloremic metabolic alkalosis and dehydration. Pain or discomfort is common. Weight loss may be prominent, depending on the duration of symptoms. A succus-sion splash may be audible with stethoscope placed in the epi-gastrium. Initial treatment is

1	or discomfort is common. Weight loss may be prominent, depending on the duration of symptoms. A succus-sion splash may be audible with stethoscope placed in the epi-gastrium. Initial treatment is nasogastric suction, IV hydration and electrolyte repletion, and acid suppression. The diagnosis is confirmed by endoscopy. Most patients admitted to the hospital nowadays with obstructing ulcer disease require intervention, either balloon dilation or operation. Cancer must be ruled out because most patients who present with the symptoms of gastric outlet obstruction will have a pancreatic, gastric, or duodenal malignancy.Medical Treatment of Peptic Ulcer DiseasePPIs are the mainstay of medical therapy for PUD, but high-dose H2RAs and sucralfate are also quite effective. Patients hospitalized for ulcer complications should receive high-dose intravenous PPI and, when discharged, should be considered for lifelong PPIs unless the definitive cause is eliminated or a definitive operation

1	for ulcer complications should receive high-dose intravenous PPI and, when discharged, should be considered for lifelong PPIs unless the definitive cause is eliminated or a definitive operation performed. Peptic ulcer patients should stop smoking and avoid alcohol and NSAIDs (including aspirin). Patients who require NSAIDs or aspirin to treat other medical conditions should always take concomi-tant PPIs or high dose H2 receptor blockers. Testing for H pylori infection is performed, and if it is found, it should be treated with one of several acceptable regimens (Table 26-10).113 If initial H pylori testing is negative and ulcer symptoms per-sist, an empirical trial of anti–H pylori therapy is reasonable since false-negative H pylori tests are not uncommon. Gener-ally, acid suppression can be stopped after 3 months if the ulcerogenic stimulus (e.g., H pylori, NSAIDs, or aspirin) has been removed. However, long-term maintenance PPI therapy should be considered in all patients admitted

1	be stopped after 3 months if the ulcerogenic stimulus (e.g., H pylori, NSAIDs, or aspirin) has been removed. However, long-term maintenance PPI therapy should be considered in all patients admitted to hospital with ulcer complications, all high-risk patients on NSAIDs or aspi-rin (the elderly or debilitated), and all patients requiring anti-coagulation or antiplatelet agents or those with a history of recurrent ulcer or bleeding. Consideration should also be given to maintenance PPI therapy in refractory smokers with a history of peptic ulcer. Sucralfate acts locally on mucosal defects and is well tolerated, and occasionally it is useful as a supplement to acid suppression.2Peptic ulcer40%24%6%4%6%5%10%5%No obvious causeOtherNeoplasmErosive diseaseMallory-Weiss syndromeOesophagitisVaricesFigure 26-31. Causes of upper GI bleeding. (Reproduced with permission from Dallal HJ, Palmer KR: ABC of the upper gastro-intestinal tract: Upper gastrointestinal haemorrhage, BMJ. 2001 Nov

1	26-31. Causes of upper GI bleeding. (Reproduced with permission from Dallal HJ, Palmer KR: ABC of the upper gastro-intestinal tract: Upper gastrointestinal haemorrhage, BMJ. 2001 Nov 10;323(7321):1115-1117.)Brunicardi_Ch26_p1099-p1166.indd 112501/03/19 7:12 PM 1126SPECIFIC CONSIDERATIONS PART IITable 26-9Risk-stratification tools for upper gastrointestinal hemorrhageaA. BLATCHFORD SCORE AT PRESENTATIONPOINTS Systolic blood pressure 100–109 mmHg1 90–99 mmHg2 <90 mmHg3 Blood urea nitrogen 6.5–7.9 mmol/L2 8.0–9.9 mmol/L3 10.0–24.9 mmol/L4 ≥25 mmol/L6 Hemoglobin for men 12.0–12.9 g/dL1 10.0–11.9 g/dL3 <10.0 g/dL6 Hemoglobin for women 10.0–11.9 g/dL1 <10.0 g/dL6 Other variables at presentation Pulse ≥100 beats/min1 Melena1 Syncope2 Hepatic disease2 Cardiac failure2 B. ROCKALL SCOREVARIABLEPOINTSAge <60 y 60–79 y ≥80 y012Shock Heart rate >100 beats/min Systolic blood pressure <100 mmHg12Coexisting illness Ischemic heart disease, congestive heart failure, other

1	ROCKALL SCOREVARIABLEPOINTSAge <60 y 60–79 y ≥80 y012Shock Heart rate >100 beats/min Systolic blood pressure <100 mmHg12Coexisting illness Ischemic heart disease, congestive heart failure, other major illness Renal failure, hepatic failure, metastatic cancer23Endoscopic diagnosis No lesions observed, Mallory-Weiss syndrome Peptic ulcer, erosive disease, esophagitis Cancer of the upper GI tract012Endoscopic stigmata of recent hemorrhage Clean base ulcer, flat pigmented spot Blood in upper GI tract, active bleeding, visible vessel, clot02aPanel A shows the values used in the Blatchford risk-stratification score, which ranges from 0 to 23, with higher scores indicating higher risk. Panel B shows the Rockall score, with point values assigned for each of three clinical variables (age and the presence of shock and coexisting illnesses) and two endoscopic variables (diagnosis and stigmata of recent hemorrhage). The complete Rockall score ranges from 0 to 11, with higher scores indicating

1	presence of shock and coexisting illnesses) and two endoscopic variables (diagnosis and stigmata of recent hemorrhage). The complete Rockall score ranges from 0 to 11, with higher scores indicating higher risk. Patients with a clinical Rockall score (Age + Shock + Coexisting illness) of 0 or a complete Rockall score of 2 or less are considered to be at low risk for rebleeding or death.Reproduced with permission from Gralnek IM, Barkun AN, Bardou M: Management of acute bleeding from a peptic ulcer, N Engl J Med. 2008 Aug 28;359(9):928-937.COMPLETE ROCKALL SCORECLINICAL ROCKALL SCOREBrunicardi_Ch26_p1099-p1166.indd 112601/03/19 7:12 PM 1127STOMACHCHAPTER 26Surgical Treatment of Peptic Ulcer DiseaseThe indications for surgery in PUD are (in order of decreasing frequency) perforation, obstruction, bleeding, and intractabil-ity or nonhealing.114,115 Gastric cancer must always be consid-ered in patients with gastric ulcer or gastric outlet obstruction. Today, most patients undergoing

1	bleeding, and intractabil-ity or nonhealing.114,115 Gastric cancer must always be consid-ered in patients with gastric ulcer or gastric outlet obstruction. Today, most patients undergoing emergent operation have simple patch of a perforated ulcer or oversewing of a bleed-ing ulcer.[92] Simultaneous performance of vagotomy either truncal or highly selective is increasingly uncommon, probably due to surgeon unfamiliarity with the procedure and reliance on postoperative PPIs to decrease acid secretion. But even in the current era, vagotomy may improve outcomes in emergency ulcer surgery.116-118 Before denying the stable low-risk patient a highly selective vagotomy or truncal vagotomy and drainage as an adjunct to simple patch or oversew, the surgeon should con-sider that many patients having emergency operation for peptic ulcer will not take long-term PPI, do not have Helicobacter, or will continue to smoke or take NSAIDs.Unfortunately, the data from many excellent randomized clinical

1	emergency operation for peptic ulcer will not take long-term PPI, do not have Helicobacter, or will continue to smoke or take NSAIDs.Unfortunately, the data from many excellent randomized clinical trials evaluating elective operation for peptic ulcer over the last several decades may be irrelevant to most patients pre-senting for ulcer surgery today.114 The large majority of these excellent studies were done in the pre-PPI, pre-Helicobacter, pre-NSAID era, and focused on elective operation for intracta-ble disease, an unusual indication for operation nowadays. Thus, today’s surgeon should take great care in applying this literature to inform surgical decision making.Traditionally, the vast majority of peptic ulcers were treated by a variant of one of the three basic operations: parietal cell vagotomy, also called highly selective vagotomy (HSV) or proximal gastric vagotomy, vagotomy and drainage (V+D), and vagotomy and distal gastrectomy. Recurrence rates are lowest but morbidity

1	cell vagotomy, also called highly selective vagotomy (HSV) or proximal gastric vagotomy, vagotomy and drainage (V+D), and vagotomy and distal gastrectomy. Recurrence rates are lowest but morbidity highest with the latter procedure, while the oppo-site is true for HSV (Table 26-11).114,115HSV severs the vagal nerve supply to the proximal two-thirds of the stomach, where essentially all the parietal cells are located, and preserves the vagal innervation to the antrum and pylorus and the remaining abdominal viscera (Fig. 26-33). Thus, the operation decreases total gastric acid secretion by about 75%, and GI side effects are rare. Elective HSV has largely been supplanted by long-term PPI treatment, but the operation, which has a learning curve, may still be useful in the patient (elective or emergent) who is noncompliant with, intolerant of, or cannot afford medical treatment. Historically, HSV has not performed particularly well for type II (gastric and duodenal) and type III

1	or emergent) who is noncompliant with, intolerant of, or cannot afford medical treatment. Historically, HSV has not performed particularly well for type II (gastric and duodenal) and type III (prepyloric) gastric ulcer, perhaps because of hypergastrinemia caused by gastric outlet obstruction and persistent antral stasis. Figure 26-32. Pneumoperitoneum on upright chest X-ray in patient with perforated ulcer.Table 26-10Helicobacter pylori therapies (10–14 days)Clarithromycin triple therapy standard or double dose PPI twice a day clarithromycin 500 mg twice a day amoxicillin 1 g twice a day; or metronidazole 500 mg three times a dayMetronidazole triple therapy standard or double dose PPI twice a day metronidazole 500 mg twice a day amoxicillin 1 g twice a dayLevofloxacin triple therapy standard dose PPI twice a day amoxicillin 1 g twice a day levofloxacin 500 mg dailySequential therapy standard or double dose PPI (10–14 days) amoxicillin 1 g twice a day (5–7 days); then clarithromycin

1	dose PPI twice a day amoxicillin 1 g twice a day levofloxacin 500 mg dailySequential therapy standard or double dose PPI (10–14 days) amoxicillin 1 g twice a day (5–7 days); then clarithromycin 500 mg twice a day and metronidazole 500 mg twice a day (5–7 days)Bismuth quadruple therapy (commonly used when above regimens fail to eradicate H pylori) standard dose PPI twice a day bismuth subsalicylate 300 mg four times a day tetracycline 500 mg four times a day metronidazole 250 mg four times a dayPPI = proton pump inhibitor.Table 26-11Clinical results of surgery for duodenal ulcer PARIETAL CELL VAGOTOMYTRUNCAL VAGOTOMY AND PYLOROPLASTYTRUNCAL VAGOTOMY AND ANTRECTOMYOperative mortality rate (%)0<11Ulcer recurrence rate (%)5–155–15<2Dumping (%) Mild<51010–15 Severe011–2Diarrhea (%) Mild<52520 Severe021–2Reproduced with permission from Mulholland MW, Debas HT: Chronic duodenal and gastric ulcer, Surg Clin North Am. 1987 Jun;67(3):489-507.Brunicardi_Ch26_p1099-p1166.indd 112701/03/19

1	with permission from Mulholland MW, Debas HT: Chronic duodenal and gastric ulcer, Surg Clin North Am. 1987 Jun;67(3):489-507.Brunicardi_Ch26_p1099-p1166.indd 112701/03/19 7:12 PM 1128SPECIFIC CONSIDERATIONS PART IIThe Taylor procedure, a straightforward laparoscopic operation, consists of a posterior truncal vagotomy and anterior seromy-otomy (but anterior HSV is probably equivalent), and it is an attractive and simple alternative to HSV with similar results.Truncal vagotomy and pyloroplasty, and truncal vagot-omy and gastrojejunostomy are the paradigmatic vagotomy and drainage procedures. HSV may be substituted for truncal vagotomy. The advantage of V + D is that it can be performed safely and quickly by the experienced surgeon. The main dis-advantages are the side effect profile (10% of patients have significant dumping and/or diarrhea). During truncal vagotomy (Fig. 26-34), care must be taken not to perforate the esophagus, a potentially lethal complication. Intraoperative

1	(10% of patients have significant dumping and/or diarrhea). During truncal vagotomy (Fig. 26-34), care must be taken not to perforate the esophagus, a potentially lethal complication. Intraoperative frozen sec-tion confirmation of at least two vagal trunks is prudent; addi-tional vagal trunks are common. Unlike HSV, V + D is widely accepted as a successful definitive operation for complicated PUD. It has been described as a useful part of the operative treatment for bleeding duodenal and gastric ulcer, perforated duodenal and gastric ulcer, and obstructing duodenal and gastric (types II and III) ulcer. When applied to gastric ulcer, the ulcer should be excised or biopsied.Truncal vagotomy denervates the antropyloric mechanism, and therefore, some sort of procedure is necessary to ablate or bypass the pylorus. Gastrojejunostomy is a good choice in patients with gastric outlet obstruction or a severely diseased proximal duodenum. The anastomosis is done between the proximal jejunum and

1	bypass the pylorus. Gastrojejunostomy is a good choice in patients with gastric outlet obstruction or a severely diseased proximal duodenum. The anastomosis is done between the proximal jejunum and the most dependent portion of the greater gastric curvature, in either an antecolic or retrocolic fashion (Fig. 26-35). Marginal ulceration is a potential complication. 6–8cm 7cmFigure 26-33. Highly selective vagotomy. (Reproduced with per-mission from Zinner MJ Schwartz SI, Ellis H: Maingot’s Abdominal Operations, 10th ed. Vol. I. Stamford, CT: Appleton & Lange; 1997.)Resected segmentResected segmentCeliac branchHepatic branchFigure 26-34. Truncal vagotomy. (Reproduced with permission from Zollinger RM Jr, Zollinger RM Sr: Zollinger’s Atlas of Surgi-cal Operations, 8th ed. New York, NY: McGraw-Hill Education; 2003.)Figure 26-35. Retrocolic gastrojejunostomy. Note meso-colon sutured to stomach (b, c, d). (Reproduced with per-mission from Zuidema GD, Yeo CJ: Shackelford’s Surgery of the

1	Education; 2003.)Figure 26-35. Retrocolic gastrojejunostomy. Note meso-colon sutured to stomach (b, c, d). (Reproduced with per-mission from Zuidema GD, Yeo CJ: Shackelford’s Surgery of the Alimentary Tract, 5th ed. Vol. II. Philadelphia, PA: Elsevier/Saunders; 2002.)Suture reinforcing the angleMesocolonbcdStomachJejunumBrunicardi_Ch26_p1099-p1166.indd 112801/03/19 7:12 PM 1129STOMACHCHAPTER 26Mechanical complications are also possible such as afferent or efferent loop obstruction, internal hernia, and intussusception. Pyloroplasty is useful in patients who require a pyloroduo-denotomy to deal with the ulcer complication (e.g., posterior bleeding duodenal ulcer), in those with limited or focal scarring in the pyloric region, or when gastrojejunostomy is technically difficult. The most commonly performed pyloroplasty is the Heineke-Mikulicz type (Fig. 26-36). Other occasionally use-ful techniques include the Finney (Fig. 26-37) and the Jaboulay pyloroplasties (Fig. 26-38). These

1	commonly performed pyloroplasty is the Heineke-Mikulicz type (Fig. 26-36). Other occasionally use-ful techniques include the Finney (Fig. 26-37) and the Jaboulay pyloroplasties (Fig. 26-38). These more extensive pyloroplasty techniques may make subsequent distal gastric resection more difficult and/or hazardous.Although vagotomy and antrectomy (V + A) is associated with a very low ulcer recurrence rate and is applicable to many patients with complicated PUD (e.g., bleeding duodenal and gastric ulcer, obstructing peptic ulcer, nonhealing gastric ulcer, and recurrent ulcer), V + A has a higher operative mortality risk (compared with HSV or V + D), and is irreversible. Fol-lowing antrectomy, GI continuity may be reestablished with a Billroth I gastroduodenostomy (Fig. 26-39) or a Billroth II loop gastrojejunostomy (Fig. 26-40). Since antrectomy routinely leaves a 60% to 70% gastric remnant, routine reconstruction as a Roux-en-Y gastrojejunostomy should be avoided (Fig. 26-41). Although

1	loop gastrojejunostomy (Fig. 26-40). Since antrectomy routinely leaves a 60% to 70% gastric remnant, routine reconstruction as a Roux-en-Y gastrojejunostomy should be avoided (Fig. 26-41). Although the Roux-en-Y operation is an excellent procedure for keeping duodenal contents out of the stomach and esophagus, in the presence of a large gastric remnant, this reconstruction will predispose to marginal ulceration and/or gastric stasis.V + A should be avoided in hemodynamically unsta-ble patients, and in patients with extensive inflammation and/or scarring of the proximal duodenum, because secure MucosaPylorusAntrumGambee stitchCBDAFigure 26-36. A through D. Heineke-Mikulicz pyloroplasty. (Reproduced with permission from Zinner MJ: Atlas of Gastric Surgery. New York, NY: Elsevier/Churchill Livingstone; 1992.)Brunicardi_Ch26_p1099-p1166.indd 112901/03/19 7:12 PM 1130SPECIFIC CONSIDERATIONS PART IIanastomosis (Billroth I) or duodenal closure (Billroth II) may be difficult.Distal

1	Livingstone; 1992.)Brunicardi_Ch26_p1099-p1166.indd 112901/03/19 7:12 PM 1130SPECIFIC CONSIDERATIONS PART IIanastomosis (Billroth I) or duodenal closure (Billroth II) may be difficult.Distal gastrectomy without vagotomy (usually about a 50% gastrectomy to include the ulcer) has traditionally been the procedure of choice for type I gastric ulcer. The addition of vagotomy should be considered for type II and III gastric ulcers (because the pathophysiology is more analogous to duodenal ulcer), or if the patient is believed to be at increased risk for recurrent ulcer, or perhaps even if Billroth II reconstruction is contemplated (to decrease the chance of marginal ulcer). Subto-tal gastrectomy (75% distal gastrectomy) without vagotomy is rarely used to treat PUD today, although it was the most popular ulcer operation at the middle of the last century.Pylorus preserving gastrectomy (PPG) was first reported as a surgical option for gastric ulcer that could minimize both dumping and

1	the most popular ulcer operation at the middle of the last century.Pylorus preserving gastrectomy (PPG) was first reported as a surgical option for gastric ulcer that could minimize both dumping and duodenogastric reflux. Though not widely adopted for this indication, in some centers PPG is considered a good minimally invasive surgical option for early gastric cancer.119,120Choice of Operation for Peptic Ulcer. The choice of opera-tion for the individual patient with PUD depends on a variety of factors, including the type of ulcer (duodenal, gastric, recurrent, or marginal), the indication for operation, and the condition of the patient. Other important considerations are intra-abdominal factors (duodenal scarring/inflammation, adhesions, or difficult exposure), the ulcer diathesis status of the patient, the surgeon’s experience and personal preference, whether H pylori infection is present, the need for NSAID therapy, previous treatment, and the likelihood of future compliance with

1	of the patient, the surgeon’s experience and personal preference, whether H pylori infection is present, the need for NSAID therapy, previous treatment, and the likelihood of future compliance with treatment. Table 26-12 shows the surgical options for managing various aspects of PUD. Stomach Stom.AB DC Approximation suture PylorusInvertedincisionGallbladderDuodenumConnellthrough & throughsuture (1st ant. tier)Duod.Cushingseromuscularsuture (2nd ant. tier)Posteriorthrough &through sutureFigure 26-37. A through D. Finney pyloroplasty. ant. = anterior; Duod. = duodenum; Stom. = stomach. (Reproduced with permission from Zuidema GD: Shackelford’s Surgery of the Alimentary Tract, 4th ed. Philadelphia, PA: Elsevier/Saunders; 1996.)Brunicardi_Ch26_p1099-p1166.indd 113001/03/19 7:12 PM 1131STOMACHCHAPTER 26In general, resective procedures have a lower ulcer recurrence rate, but a higher morbidity and mortality rate (see Table 26-11) compared to nonresective ulcer operations. Because

1	26In general, resective procedures have a lower ulcer recurrence rate, but a higher morbidity and mortality rate (see Table 26-11) compared to nonresective ulcer operations. Because ulcer recur-rence often is related to H pylori and/or NSAIDs, it is usually managed adequately without reoperation. Thus, gastric resection to minimize recurrence in duodenal ulcer disease is usually not justified; resection for gastric ulcer remains the standard because of the risk of cancer. Clearly, the modern trend in peptic ulcer operation could be described as “less is more.”121,122Bleeding Peptic UlcerBleeding is the most common cause of ulcer-related death, but only rarely do patients with bleeding gastric or duodenal ulcer require operation today. The success of endoscopic treatment and medical therapy for bleeding PUD has resulted in the selec-tion of a small subgroup of high-risk patients for today’s sur-geon. It is likely that patients currently coming to operation for bleeding PUD are at

1	for bleeding PUD has resulted in the selec-tion of a small subgroup of high-risk patients for today’s sur-geon. It is likely that patients currently coming to operation for bleeding PUD are at higher risk for a poor outcome than ever before. The surgical options for treating bleeding PUD include suture ligation of the bleeder; suture ligation and definitive non-resective ulcer operation (HSV or V + D); and gastric resection (usually, including vagotomy and ulcer excision). Gastric ulcer requires biopsy if not resected.The management of bleeding peptic ulcer is summarized in the algorithm provided in Fig. 26-42. All patients admitted to the hospital with bleeding peptic ulcer should be adequately Stomach Stom.AB DCPylorusInvertedincisionGallbladderDuodenumConnellthrough & throughsuture (1st ant. tier)Duod.Cushingseromuscularsuture (2nd ant. tier)Posteriorthrough &through sutureFigure 26-38. A through D. Jaboulay pyloroplasty. ant. = anterior; Duod. = duodenum; Stom. = stomach.

1	(1st ant. tier)Duod.Cushingseromuscularsuture (2nd ant. tier)Posteriorthrough &through sutureFigure 26-38. A through D. Jaboulay pyloroplasty. ant. = anterior; Duod. = duodenum; Stom. = stomach. (Reproduced with permission from Zuidema GD: Shackelford’s Surgery of the Alimentary Tract, 4th ed. Philadelphia, PA: Elsevier/Saunders; 1996.)Brunicardi_Ch26_p1099-p1166.indd 113101/03/19 7:12 PM 1132SPECIFIC CONSIDERATIONS PART IIresuscitated and started on IV PPI.125 Most patients will stop bleeding with these measures alone, but about 25% will continue to bleed or will rebleed in hospital. It is important to identify this high-risk group early with clinical and endoscopic parameters because, essentially, all the deaths from bleeding ulcer occur in this group. Surgical consultation is mandatory, and endoscopic hemostatic therapy (cautery, epinephrine injection, clipping) is indicated and usually successful in these high-risk patients.126 Indications for operation include massive

1	mandatory, and endoscopic hemostatic therapy (cautery, epinephrine injection, clipping) is indicated and usually successful in these high-risk patients.126 Indications for operation include massive hemorrhage unre-sponsive to initial endoscopic control, recurrent hemorrhage ABCFigure 26-40. A through C. Billroth II antecolic gastrojejunostomy. (Reproduced with permission from Zinner MJ Schwartz SI, Ellis H: Maingot’s Abdominal Operations, 10th ed. Vol. I. Stamford, CT: Appleton & Lange; 1997.)Figure 26-39. A and B. Billroth I gastroduo-denostomy. (Reproduced with permission from Zinner MJ: Atlas of Gastric Surgery. New York, NY: Elsevier/Churchill Livingstone; 1992.)ABBrunicardi_Ch26_p1099-p1166.indd 113201/03/19 7:12 PM 1133STOMACHCHAPTER 26requiring multiple transfusions after two attempts at endoscopic control, ongoing hemorrhage and transfusion with limited availability of blood for transfusion or lack of availability of a therapeutic endoscopist, early rehospitalization for

1	attempts at endoscopic control, ongoing hemorrhage and transfusion with limited availability of blood for transfusion or lack of availability of a therapeutic endoscopist, early rehospitalization for bleeding ulcer, and concurrent indications for surgery such as perforation or obstruction. Patients with massive bleeding from high-risk lesions (e.g., posterior duodenal ulcer with erosion of gastroduo-denalartery, or lesser curvature gastric ulcer with erosion of left gastric artery or branch) should be considered for operation as should those presenting in shock, those requiring more than four units of blood in 24 hours or eight units of blood in 48 hours, and those with ulcers >2 cm in diameter. The mortality rate for surgery for bleeding peptic ulcer is around 20%. Angiography and embolization may be useful in some patients.Operation for Bleeding Peptic Ulcer (Fig. 26-43)The two operations most commonly used for bleeding duode-nal ulcer are oversewing of the ulcer with or without

1	may be useful in some patients.Operation for Bleeding Peptic Ulcer (Fig. 26-43)The two operations most commonly used for bleeding duode-nal ulcer are oversewing of the ulcer with or without vagotomy and drainage,121 or V + A. Oversewing alone results in a higher rebleeding rate but a lower operative mortality rate than defini-tive operation. When the mortality for reoperation for rebleed-ing is considered, the overall mortality is probably comparable for the two approaches. Patients who are in shock or medically unstable should not have gastric resection.An initial pyloromyotomy incision allows access to the bleeding posterior duodenal ulcer, and an expeditious Kocher maneuver allows the surgeon to control the hemorrhage with the left hand if necessary. Heavy suture material on a stout needle is used to place figure-of-eight sutures or a U-stitch to secure the bleeding vessel at the base of the posterior duodenal ulcer. Multiple sutures are usually necessary. Once the surgeon is

1	needle is used to place figure-of-eight sutures or a U-stitch to secure the bleeding vessel at the base of the posterior duodenal ulcer. Multiple sutures are usually necessary. Once the surgeon is unequivocally convinced that hemostasis is secure, a pyloro-plasty can be performed. If the patient is stable, vagotomy may be considered if the surgeon is experienced and the vagotomy straightforward. If the patient is not a high operative risk and V + A is selected, smaller duodenal ulcers are resected with the specimen; larger bleeding duodenal ulcers must often be left behind in the duodenal stump. In this situation, suture hemo-stasis must be attained and a secure duodenal closure accom-plished. The anterior wall of the open duodenum can be sutured to either the proximal or distal lip of the posterior ulcer once the bleeding vessel has been sutured. The duodenal closure can be buttressed with omentum and the duodenum should be decompressed, either with a lateral duodenostomy or

1	of the posterior ulcer once the bleeding vessel has been sutured. The duodenal closure can be buttressed with omentum and the duodenum should be decompressed, either with a lateral duodenostomy or retrograde tube via the proximal jejunum or well secured nasogastric tube secured with tip well into afferent limb. Right upper quadrant closed suction peritoneal drainage is important. Use of a feed-ing jejunostomy is also considered. A Billroth II anastomosis reestablishes gastrointestinal continuity.The initial management of bleeding gastric ulcers and the indications for operation are similar to those for bleeding duodenal ulcer. These lesions tend to occur in older and/or medically complicated patients, and this fact may increase the operative risk. However, experience shows that planned surgery in a resuscitated patient results in a better operative survival rate than emergent operation in a patient who has rebled and is in shock. Distal gastric resection to include the bleeding ulcer

1	in a resuscitated patient results in a better operative survival rate than emergent operation in a patient who has rebled and is in shock. Distal gastric resection to include the bleeding ulcer is the procedure of choice for bleeding gastric ulcer. Second best is V + D with oversewing and biopsy of the ulcer to rule out cancer. Oversewing of the bleeder and biopsy followed by long-term acid suppression is a reasonable alternative in high-risk or unstable patients.50 to 60 cm< 50% gastricremnantFigure 26-41. Roux-en-Y gastrojejunostomy. (Reproduced with permission from Ritchie WP, Steele G, Dean RH: General Surgery. Philadelphia, PA: Lippincott Williams & Wilkins; 1995.)Table 26-12Surgical options in the treatment of duodenal and gastric ulcerINDICATIONDUODENALGASTRICBleeding1. Oversewa2. Oversew, V + D3. V + A1. Oversew and biopsya2. Oversew, biopsy, V + D3. Distal gastrectomybPerforation1. Patcha2. Patch, HSV3. Patch, V + D1. Biopsy and patcha2. Wedge excision, V + D3. Distal

1	V + D3. V + A1. Oversew and biopsya2. Oversew, biopsy, V + D3. Distal gastrectomybPerforation1. Patcha2. Patch, HSV3. Patch, V + D1. Biopsy and patcha2. Wedge excision, V + D3. Distal gastrectomybObstruction1. HSV + GJ2. V + A1. Biopsy; HSV + GJ2. Distal gastrectomybIntractability/nonhealing1. HSVb2. V + D3. V + A1. HSV and wedge excision2. Distal gastrectomyaUnless the patient is in shock or moribund, a definitive procedure should be considered.bOperation of choice in low-risk patient.GJ = gastrojejunostomy; HSV = highly selective vagotomy; V + A = vagotomy and antrectomy; V + D = vagotomy and drainage.Brunicardi_Ch26_p1099-p1166.indd 113301/03/19 7:12 PM 1134SPECIFIC CONSIDERATIONS PART IIPerforated Peptic Ulcer (Fig. 26-44)Perforation is the second most common complication of peptic ulcer, but nowadays it is a much more common indication for operation than bleeding. As with bleeding ulcer, NSAID and/or aspirin use have been inextricably linked with perforated PUD,

1	of peptic ulcer, but nowadays it is a much more common indication for operation than bleeding. As with bleeding ulcer, NSAID and/or aspirin use have been inextricably linked with perforated PUD, especially in the elderly population.61 Surgery is almost always indicated for ulcer perforation, although occasionally nonsurgi-cal treatment can be used in the stable patient without peritoni-tis in whom radiologic studies document a sealed perforation. Patients with acute perforation and GI blood loss (either chronic or acute) should be suspected of having a second ulcer or a GI cancer.The options for surgical treatment of perforated duode-nal ulcer are simple patch closure, patch closure and HSV, or patch closure and V + D. Simple patch closure, currently the most commonly performed operation for perforated peptic ulcer, is the procedure of choice in patients with hemodynamic instability and/or exudative peritonitis signifying a perforation >24 hours old. In stable patients without

1	for perforated peptic ulcer, is the procedure of choice in patients with hemodynamic instability and/or exudative peritonitis signifying a perforation >24 hours old. In stable patients without longstanding perfo-ration, particularly those with chronic symptoms or failure of medical treatment, the addition of HSV should be considered. Vagotomy and drainage is also an acceptable definitive opera-tion for perforated duodenal ulcer, but occasionally side effects are disabling, and if gastrojejunostomy has been performed, Hospital admissionBleeding peptic ulcerBleeding recursin hospitalO.R.20% high risk80% Low riskBleeding stopsDischargeBleeding recursBleeding persists>4 PRBC transfused/24hDeep ulcer eroding big vesselHemodynamic instabilityHemostatic Rx unavailableEndoscopic hemostatic RxConsult surgeonResuscitateContinuous IV PPI dripEGDLifelong acid suppressionTest + Rx H. pyloriAvoid NSAIDs/ASA if possibleYesShock?NoYesTransfusion?NoYesActive bleeding on EGD?NoYesVisible vessel on

1	surgeonResuscitateContinuous IV PPI dripEGDLifelong acid suppressionTest + Rx H. pyloriAvoid NSAIDs/ASA if possibleYesShock?NoYesTransfusion?NoYesActive bleeding on EGD?NoYesVisible vessel on EGD?NoYesAbnormal PT, PTT, or platelets?NoFigure 26-42. Algorithm for the treatment of bleeding peptic ulcer. ASA = acetylsalicylic acid; EGD = esophagogastroduodenoscopy; O.R. = operating room; PPI = proton pump inhibitor; PRBC = unit of packed red blood cells; PT = prothrombin time; PTT = partial throm-boplastin time; Rx = treatment.Brunicardi_Ch26_p1099-p1166.indd 113401/03/19 7:12 PM 1135STOMACHCHAPTER 26Operation for bleeding peptic ulcerYesYesNoGastric ulcerDuodenal ulcerType 1,2,3Distal gastrectomy**Hemodynamically unstable?OrHigh operative risk?Csendes procPauchet procKelling-Madlener proc(see text)*Add lifelong PPI**Add TV for type 2 + 3Oversew + TV/DOversew + TV/ABMI < 21? ordifficult duodenum?Type 4RebleedRebleedDuodenal ulcer*Gastric ulcer*1) Bx and oversew2) Wedge

1	proc(see text)*Add lifelong PPI**Add TV for type 2 + 3Oversew + TV/DOversew + TV/ABMI < 21? ordifficult duodenum?Type 4RebleedRebleedDuodenal ulcer*Gastric ulcer*1) Bx and oversew2) Wedge resectionOversewNoFigure 26-43. Algorithm for operation for bleeding peptic ulcer. BMI = body mass index; Bx = biopsy; PPI = proton pump inhibitor; proc = procedure; TV = truncal vagotomy; TV/A = truncal vagotomy and antrectomy; TV/D = truncal vagotomy and drainage.Operation for Perforated Peptic UlcerYesPatch + HSV,orPatch + TV/DWedge resection and TV/D or HSVOrDistal gastrectomy including perforation*** Duodenal ulcer* Gastric ulcer* PatchBx + PatchOrWedge resectionChronic ulcer HX?OrPerforation on RX?OrNSAIDs/ASA necessary? Gastric ulcer* Duodenal ulcer* Hemodynamically unstable?OrHigh operative risk?OrPerforation >24 hours * In all patients, test and treat for H pylori, and if vagotomy not performed (most patients today) consider lifelong PPI. ** Avoid truncal vagotomy and avoid gastrectomy

1	>24 hours * In all patients, test and treat for H pylori, and if vagotomy not performed (most patients today) consider lifelong PPI. ** Avoid truncal vagotomy and avoid gastrectomy if BMI<21 *** Consider adding vagotomy for type II and type III gastric ulcerNoNoYes**Figure 26-44. Algorithm for operation for perforated peptic ulcer. ASA = acetylsalicylic acid; BMI = body mass index; Bx = biopsy; HSV = highly selective vagotomy; Hx = history; PPI = proton pump inhibitor; Rx = treatment; TV/D = truncal vagotomy and drainage.Brunicardi_Ch26_p1099-p1166.indd 113501/03/19 7:12 PM 1136SPECIFIC CONSIDERATIONS PART IImarginal ulcer can be life-threatening. In the United States and Western Europe, there is clearly a trend away from definitive operation for perforated duodenal ulcer.92,121In the stable patient without multiple operative risk fac-tors, perforated gastric ulcers are best treated by distal gastric resection. Vagotomy is usually added for type II and III gastric ulcers.

1	the stable patient without multiple operative risk fac-tors, perforated gastric ulcers are best treated by distal gastric resection. Vagotomy is usually added for type II and III gastric ulcers. Patch closure with biopsy; or local excision and closure; or biopsy, closure, truncal vagotomy, and drainage are alter-native operations in the unstable or high-risk patient, or in the patient with a perforation in an inopportune location. All perfo-rated gastric ulcers, even those in the prepyloric position, should be biopsied if they are not removed at surgery.Obstructing Peptic UlcerAcute ulcers associated with obstruction due to edema and/or motor dysfunction may respond to intensive antisecretory ther-apy and nasogastric suction. But most patients with significant obstruction from chronic ulceration will require some sort of more substantial intervention. Endoscopic balloon dilation can often transiently improve obstructive symptoms, but many of these patients ultimately fail and come to

1	will require some sort of more substantial intervention. Endoscopic balloon dilation can often transiently improve obstructive symptoms, but many of these patients ultimately fail and come to operation.123The standard operation for obstructing PUD is vagotomy and antrectomy. Alternatively, vagotomy and gastrojejunos-tomy should be considered if a difficult duodenal stump is anticipated with resection. HSV and gastrojejunostomy may be comparable to V + A for obstructing ulcer disease,124 and this procedure is appealing because it can be done laparoscopically and does not complicate future resection, if needed. However, potentially curable gastric or duodenal cancers can be missed with this approach.Intractable or Nonhealing Peptic UlcerIntractability should be an unusual indication for peptic ulcer operation nowadays. The patient referred for surgical evaluation because of intractable PUD should raise red flags for the sur-geon: Maybe the patient has a missed cancer; maybe the patient

1	ulcer operation nowadays. The patient referred for surgical evaluation because of intractable PUD should raise red flags for the sur-geon: Maybe the patient has a missed cancer; maybe the patient is noncompliant (not taking prescribed PPI, still taking NSAIDs, still smoking); maybe the patient has Helicobacter despite the presence of a negative test or previous treatment. Because acid secretion can be totally blocked and H pylori eradicated with modern medication, the question remains: “Why does the patient have a persistent ulcer diathesis?” The surgeon should review the differential diagnosis of nonhealing ulcer before any consideration of operative treatment (Table 26-13).Surgical treatment should be considered in patients with nonhealing or intractable PUD who have multiple recurrences, large ulcers (>2 cm), complications (obstruction, perforation, or hemorrhage), or suspected malignancy. Definitive operation, particularly gastric resection, should be considered most cau-tiously

1	large ulcers (>2 cm), complications (obstruction, perforation, or hemorrhage), or suspected malignancy. Definitive operation, particularly gastric resection, should be considered most cau-tiously in the thin or marginally nourished individual.It is important that the surgeon not fall into the trap of per-forming a large, irreversible operation on these patients, based on the unproven theory that if all other methods have failed to heal the ulcer, a large operation is required. Although there are good data in the surgical literature suggesting that the major-ity of patients do well after the larger elective ulcer operations, most of these data are several decades old and may not be par-ticularly relevant to the modern patient.114 Candidates for ulcer operation today are different than those of 30 to 50 years ago. One might argue that current medical care has healed the typical peptic ulcers, and that patients presenting with true intractabil-ity or nonhealing will be more difficult to

1	of 30 to 50 years ago. One might argue that current medical care has healed the typical peptic ulcers, and that patients presenting with true intractabil-ity or nonhealing will be more difficult to treat and are likely to have chronic problems after a major ulcer operation.If surgery is necessary, a lesser operation may be preferable. It is prudent to avoid truncal vagotomy and/or distal gastrectomy as the initial elective operation for intractable peptic ulcer in the thin or asthenic patient. Alternatives for intractable duo-denal ulcer include HSV with or without gastrojejunostomy (reversible drainage operation). In patients with nonhealing gas-tric ulcer, wedge resection with HSV should be considered in thin or frail patients. Otherwise, distal gastrectomy (to include the ulcer) is recommended. It is unnecessary to add a vagotomy in patients with type I or type IV (juxta-esophageal) gastric ulcers because they are usually associated with acid hyposecretion. Type IV gastric ulcers

1	It is unnecessary to add a vagotomy in patients with type I or type IV (juxta-esophageal) gastric ulcers because they are usually associated with acid hyposecretion. Type IV gastric ulcers may be difficult to resect as part of a distal gas-trectomy, and a variety of surgical techniques have been described to treat these more proximal lesions (Fig. 26-45).Zollinger-Ellison Syndrome127-129ZES is caused by the hypersecretion of gastrin, typically by a duodenal or pancreatic neuroendocrine tumor (i.e., gastrinoma). Most cases (80%) are sporadic, but 20% are inherited. The inherited or familial form of gastrinoma is associated with mul-tiple endocrine neoplasia type I (MEN I), which is characterized by parathyroid, pituitary, and pancreatic (or duodenal) tumors. Gastrinoma is the most common islet cell tumor in patients with MEN I. Patients with MEN I usually have multiple gastrinomas, and surgical cure is usually not achievable; sporadic gastrino-mas are more often solitary and are more

1	islet cell tumor in patients with MEN I. Patients with MEN I usually have multiple gastrinomas, and surgical cure is usually not achievable; sporadic gastrino-mas are more often solitary and are more often amenable to surgical cure. About 50% to 60% of gastrinomas are malignant, with lymph node, liver, or other distant metastases at operation. Five-year survival in patients presenting with metastatic disease is approximately 40%. More than 90% of patients with sporadic, completely resected gastrinoma will be cured.The most common symptoms of ZES are epigastric pain, GERD, and diarrhea. More than 90% of patients with gastri-noma have peptic ulcers. Most ulcers are in a typical location (proximal duodenum), but atypical ulcer location (distal duo-denum, jejunum, or multiple ulcers) should prompt an evalu-ation for gastrinoma. Gastrinoma also should be considered in the differential diagnosis of recurrent or refractory peptic ulcer, secretory diarrhea, gastric rugal hypertrophy,

1	should prompt an evalu-ation for gastrinoma. Gastrinoma also should be considered in the differential diagnosis of recurrent or refractory peptic ulcer, secretory diarrhea, gastric rugal hypertrophy, esophagi-tis with stricture, bleeding or perforated ulcer, familial ulcer, peptic ulcer with hypercalcemia, and gastric neuroendocrine tumor (carcinoid). The majority of patients with ZES have been symptomatic for several years before definitive diagnosis and, 3Table 26-13Differential diagnosis of intractability or nonhealing peptic ulcer diseaseCancer Gastric Pancreatic DuodenalPersistent Helicobacter pylori infection Tests may be false-negative Consider empiric treatmentNoncompliant patient Failure to take prescribed medication Surreptitious use of NSAIDsMotility disorderZollinger-Ellison syndromeBrunicardi_Ch26_p1099-p1166.indd 113601/03/19 7:12 PM 1137STOMACHCHAPTER 26in general, patients with ZES and MEN1 are diagnosed in their 20s and 30s, while those with sporadic ZES more

1	syndromeBrunicardi_Ch26_p1099-p1166.indd 113601/03/19 7:12 PM 1137STOMACHCHAPTER 26in general, patients with ZES and MEN1 are diagnosed in their 20s and 30s, while those with sporadic ZES more typically are diagnosed in their 40s and 50s.ZES is an important element in the differential diagnosis of hypergastrinemia (Fig. 26-46). All patients with gastrinoma have an elevated gastrin level, and hypergastrinemia in the pres-ence of elevated BAO strongly suggests gastrinoma. Patients with gastrinoma usually have a BAO >15 mEq/h or >5 mEq/h if they have had a previous procedure for peptic ulcer. Acid secre-tory medications should be held for several days before gastrin measurement, because acid suppression may falsely elevate gas-trin levels. Causes of hypergastrinemia can be divided into those associated with hyperacidity and those associated with hypo-acidity (see Fig. 26-46). The diagnosis of ZES is confirmed by the secretin stimulation test. An IV bolus of secretin (2 U/kg) is

1	those associated with hyperacidity and those associated with hypo-acidity (see Fig. 26-46). The diagnosis of ZES is confirmed by the secretin stimulation test. An IV bolus of secretin (2 U/kg) is given, and gastrin levels are checked before and after injection. An increase in serum gastrin of 200 pg/mL or greater suggests the presence of gastrinoma. Patients with gastrinoma should have serum calcium and parathyroid hormone levels determined to rule out MEN1 and, if present, parathyroidectomy should be considered before resection of gastrinoma.About 80% of primary tumors are found in the gastrinoma triangle (Fig. 26-47), and many tumors are small (<1 cm), mak-ing preoperative localization difficult. Transabdominal ultra-sound is quite specific, but not very sensitive. CT will detect most lesions >2 cm in size, and MRI is comparable. EUS is more sensitive than noninvasive imaging tests, but it still misses many smaller lesions or lesions in inaccessible locations (e.g., the pan-creatic

1	>2 cm in size, and MRI is comparable. EUS is more sensitive than noninvasive imaging tests, but it still misses many smaller lesions or lesions in inaccessible locations (e.g., the pan-creatic tail). Somatostatin receptor scintigraphy (the octreotide scan) or Gallium-68 dotatate PET/CT are sensitive and specific when the pretest probability of gastrinoma is high and may iden-tify sites of regional or distant metastatic disease (Fig. 26-48). Angiographic localization studies are infrequently performed for gastrinoma. Both diagnostic angiography and transhepatic selective venous sampling of the portal system have been sup-planted by selective arterial secretin infusion, which helps to localize the tumor as inside or outside the gastrinoma triangle. This study too, is rarely performed given increasing availability of endoscopic ultrasonography and accurate nuclear medicine imaging.All patients with sporadic (nonfamilial) gastrinoma should be considered for surgical exploration. The

1	increasing availability of endoscopic ultrasonography and accurate nuclear medicine imaging.All patients with sporadic (nonfamilial) gastrinoma should be considered for surgical exploration. The lesions can be located in over 90% of patients, and a majority are cured by extirpation of the gastrinoma.130,131 A thorough intraoperative exploration of the gastrinoma triangle and pancreas is essential, but other sites (i.e., liver, stomach, small bowel, mesentery, and pelvis) should be evaluated as part of a thorough intra-abdom-inal evaluation to find the primary tumor, which is most often solitary and often in the duodenal wall. The duodenum and pan-creatic head should be extensively mobilized and intraoperative ultrasound should be utilized. Intraoperative EGD with transil-lumination may be considered. If the tumor cannot be located, longitudinal duodenotomy with inspection and palpation of the duodenal wall is performed. Lymph nodes from the portal, peri-pancreatic, and celiac drainage

1	considered. If the tumor cannot be located, longitudinal duodenotomy with inspection and palpation of the duodenal wall is performed. Lymph nodes from the portal, peri-pancreatic, and celiac drainage basins should be removed. Abla-tion or resection of hepatic metastases when identified should be considered.The management of gastrinoma in patients with MEN I is controversial because patients are infrequently cured by opera-tion. Acid hypersecretion in patients with gastrinoma can always be managed with high-dose PPIs. Highly selective vagotomy may make management easier in some patients and should be considered in those with surgically untreatable or unresectable gastrinoma. Gastrectomy for ZES is not indicated.STRESS GASTRITIS AND STRESS ULCERStress gastritis is a peculiar entity that has all but disappeared from the clinical (if not endoscopic) lexicon, largely due to bet-ter critical care and acid suppression or cytoprotective agents (e.g., sucralfate) in the intensive care unit

1	has all but disappeared from the clinical (if not endoscopic) lexicon, largely due to bet-ter critical care and acid suppression or cytoprotective agents (e.g., sucralfate) in the intensive care unit (ICU). Stress gastritis and stress ulcer are probably due to inadequate gastric mucosal Figure 26-45. Operations for gastric ulcer. (Reproduced with permission from Feldman M, Sharschmidt BF, Sleisenger MH: Slei-senger and Fordtran’s Gastrointestinal and Liver Disease: Pathophysiology/Diagnosis/ Management 6th ed. Philadelphia, PA: Elsevier/Saunders; 1998.)Ulcer excisionAntrectomyPauchet procedureKelling-MadlenerprocedureCsendes procedure(Roux reconstruction)Csendes procedureBrunicardi_Ch26_p1099-p1166.indd 113701/03/19 7:12 PM 1138SPECIFIC CONSIDERATIONS PART IIblood flow during periods of intense physiologic stress. Ade-quate mucosal blood flow is important to maintain the mucosal barrier and to buffer any back-diffused hydrogen ions. When blood flow is inadequate, these

1	periods of intense physiologic stress. Ade-quate mucosal blood flow is important to maintain the mucosal barrier and to buffer any back-diffused hydrogen ions. When blood flow is inadequate, these processes fail and mucosal breakdown occurs. Modern intensive care, with emphasis on adequate tissue perfusion and oxygenation, has undoubtedly decreased the severity of gastric mucosal injury seen in the ICU today. Although it is still common to see small mucosal ero-sions when performing upper endoscopy in the ICU, it is rare for these lesions to coalesce into the larger bleeding erosions that plagued the ICU patient 30 to 50 years ago. The rationale for routine acid suppression in the ICU, supported by excellent data from clinical trials and the laboratory, is that less mucosal injury will be caused in the potentially weakened gastric mucosa if there is less luminal acid.132 There are some studies suggest-ing that routine acid suppression leads to overgrowth of gastric bacteria, which

1	be caused in the potentially weakened gastric mucosa if there is less luminal acid.132 There are some studies suggest-ing that routine acid suppression leads to overgrowth of gastric bacteria, which increases the incidence and/or severity of aspira-tion pneumonia in the ICU.133,134 Nevertheless, acid suppression, particularly in the severely ill patient, remains an important part of clinical pathways in most ICUs.135 In the extraordinarily rare patient requiring operation today for hemorrhagic stress gastri-tis, the surgical options include V + D with oversewing of the major bleeding lesions, or near total gastrectomy. Angiographic embolization and endoscopic hemostatic treatment should be considered as well.ATROPHIC GASTRITISAtrophic gastritis is characterized by atrophy or disappearance of gastric glands and loss of parietal and chief cells. The most common cause is chronic H pylori infection, particularly in the corporal distribution (as opposed to the antral distribution which is

1	of gastric glands and loss of parietal and chief cells. The most common cause is chronic H pylori infection, particularly in the corporal distribution (as opposed to the antral distribution which is more typically associated with peptic ulcer disease). Auto-immune destruction of cells (pernicious anemia) and chemical irritation (e.g., bile reflux) can also result in atrophic gastritis. Some patients with atrophic gastritis develop intestinal meta-plasia in the gastric mucosa that may progress to dysplasia and then to gastric cancer. Numerous cofactors have been impli-cated, including diet, altered gastric microbiome, genetics, and hypergastrinemia. Patients with atrophic gastritis are at risk for Elevated serum gastrin(off PPI + H2RA)Measure BAO and gastric pHSecretin stimulation testBAO > 10 mEq/hpH <2BAO lowpH > 2Previous GI surgery• Vagotomy?• Massive SB resectionAtrophicgastritisPerniciousanemiaRenal insuffNoYesConfirm with EGD/Bxtest/treat H pylorigive B12Significant elevation

1	> 10 mEq/hpH <2BAO lowpH > 2Previous GI surgery• Vagotomy?• Massive SB resectionAtrophicgastritisPerniciousanemiaRenal insuffNoYesConfirm with EGD/Bxtest/treat H pylorigive B12Significant elevation in serumgastrin in response to IV secretin?G-cell +/or ECLcell hyperplasiaLoop GJ with antralalkalinizationTakedown GJOrTV and AConsider TV+ antrectomyRetained antrum on“duodenal stump”Resect retained antrumOrConvert B2 to B1AntralstasisS/P gastrectomy B2Confirm with EGD + BxConsider resectionZollinger-EllisonsyndromeOctreotide scanR/O MEN-1Figure 26-46. Algorithm for diagnosis and management of hypergastrinemia. BAO = basal acid output; B1 = Billroth 1; B2 = Billroth 2; Bx = biopsy; ECL = enterochromaffin-like; EGD = esophagogastroduodenoscopy; GJ = gastrojejunostomy; H2RA = histamine 2 receptor antagonist; insuff = insufficiency; MEN1 = multiple endocrine neoplasia type I; PPI = proton pump inhibitor; R/O = rule out; SB = small bowel; S/P = status post; TV = truncal vagotomy; TV and A

1	antagonist; insuff = insufficiency; MEN1 = multiple endocrine neoplasia type I; PPI = proton pump inhibitor; R/O = rule out; SB = small bowel; S/P = status post; TV = truncal vagotomy; TV and A = truncal vagotomy and antrectomy.Brunicardi_Ch26_p1099-p1166.indd 113801/03/19 7:12 PM 1139STOMACHCHAPTER 26gastric cancer and should undergo periodic endoscopic surveil-lance. Metaplastic atrophic gastritis and dysplastic atrophic gastritis in particular, are markers of increased risk for gastric cancer. Patients with high grade dysplasia may benefit from gas-trectomy. Cancer risk is related to the extent of the atrophic gas-tritis and intestinal metaplasia, and grading systems have been developed to stratify cancer risk based on endoscopic findings. Two such systems are the operative link on gastritis assessment (OLGA) and the operative link on gastric intestinal metaplasia (OLGIM) assessment.136,137 These systems define the severity (“stage”) of atrophic gastritis based on the

1	link on gastritis assessment (OLGA) and the operative link on gastric intestinal metaplasia (OLGIM) assessment.136,137 These systems define the severity (“stage”) of atrophic gastritis based on the histologic grading of at least five gastric biopsies (lesser and greater curve antrum; Figure 26-47. Gastrinoma triangle. (Reproduced with permission from Ritchie WP, Steele G, Dean RH: General Surgery. Philadelphia, PA: Lippincott Williams & Wilkins; 1995.)Figure 26-48. Positive gallium-68 dotatate scan in patient with gastrinoma.Table 26-14Types of gastric tumorsMalignant tumors Carcinoma Lymphoma Gastrointestinal stromal tumor (GIST) Neuroendocrine tumorBenign tumors Hyperplastic polyp Adenomatous polyp Leiomyoma Lipoma Schwannoma Hetrotopic pancreaslesser and greater curve corpus; angularis incisura). Since pathol-ogists are more likely to agree on the histological diagnosis of intestinal metaplasia than they are on atrophic gastritis, the latter tool (OLGIM) may be more useful in

1	incisura). Since pathol-ogists are more likely to agree on the histological diagnosis of intestinal metaplasia than they are on atrophic gastritis, the latter tool (OLGIM) may be more useful in stratifying gastric cancer risk. Patients stratified as stage 3 or 4 gastritis and those with pernicious anemia may benefit from surveillance endoscopy every 3 years. Serum markers are also useful in helping to iden-tify patients with atrophic gastritis who usually have increased serum gastrin and iron deficiency due to parietal cell loss and hypochlorhydria or achlorhydria; decreased pepsinogen I levels due to chief cell loss; and B12 deficiency due to parietal cell loss and concomitant loss of intrinsic factor.MALIGNANT NEOPLASMS OF THE STOMACHThe most common primary malignant gastric neoplasm is adeno-carcinoma (95%); lymphoma and GIST account for most of the remaining cases (Table 26-14). Other rare primary malignancies include neuroendocrine tumor, angiosarcoma, carcinosarcoma, and

1	is adeno-carcinoma (95%); lymphoma and GIST account for most of the remaining cases (Table 26-14). Other rare primary malignancies include neuroendocrine tumor, angiosarcoma, carcinosarcoma, and squamous cell carcinoma. Occasionally the stomach is a site of hematogenous metastasis from other sites (e.g., melanoma or breast cancer). Malignant tumors from adjacent organs may also invade the stomach by direct extension (e.g., colon or pancreas) or by peritoneal dissemination (e.g., ovary or appendiceal).AdenocarcinomaEpidemiology. Gastric cancer is the fourth most common can-cer type and the second leading cause of cancer death world-wide. Over the past century, there has been a dramatic decrease in the incidence of gastric cancer in most Western industrialized countries (Fig. 26-49). This decrease has been largely in the so-called intestinal form rather than in the diffuse form of gastric cancer. In Asia and Eastern Europe, gastric cancer remains a leading cause of cancer death. In 2017

1	has been largely in the so-called intestinal form rather than in the diffuse form of gastric cancer. In Asia and Eastern Europe, gastric cancer remains a leading cause of cancer death. In 2017 in the United States, approximately 28,000 new cases of stomach cancer were diagnosed (17,750 in men and 10,250 in women), and 10,960 deaths will be attributed to this disease (6720 in men and 4240 in women).138 The estimated 5-year survival rate is 27%, up from about 15% in 1975.In general, gastric cancer is a disease of the elderly, and it is twice as common in blacks as in whites. In younger patients, tumors are more often of the diffuse variety and tend to be large, aggressive, and poorly differentiated, sometimes involving the entire stomach (linitis plastic). Gastric cancer has a higher inci-dence in groups of lower socioeconomic status.4Brunicardi_Ch26_p1099-p1166.indd 113901/03/19 7:12 PM 1140SPECIFIC CONSIDERATIONS PART IIEtiology. Gastric cancer is more common in patients with

1	in groups of lower socioeconomic status.4Brunicardi_Ch26_p1099-p1166.indd 113901/03/19 7:12 PM 1140SPECIFIC CONSIDERATIONS PART IIEtiology. Gastric cancer is more common in patients with per-nicious anemia, blood group A, or a family history of gastric cancer. When patients migrate from a high-incidence region to a low-incidence region, the risk of gastric cancer decreases in the subsequent generations born in the new region. This strongly suggests an environmental influence on the development of gas-tric cancer. Environmental factors appear to be more important in the pathogenesis of the intestinal form of gastric cancer com-pared to the diffuse form. The commonly accepted risk factors for gastric cancer are listed in Table 26-15.Diet and Drugs A diet high in pickled, salted, or smoked food is found in many regions of high gastric cancer risk. Dietary nitrates have been implicated as a possible cause of gastric cancer. Gastric bacteria (more abundant in the achlorhydric

1	or smoked food is found in many regions of high gastric cancer risk. Dietary nitrates have been implicated as a possible cause of gastric cancer. Gastric bacteria (more abundant in the achlorhydric stomach of patients with atrophic gastritis, a risk factor for gastric cancer) convert nitrate into nitrite, a known carcinogen. A diet high in fresh fruits and vegetables and rich in vitamin C and E has been shown to decrease the risk of gastric cancer. The reduced consumption of nitrate-rich preserved foods seen with the widespread availability of refrigeration has been sug-gested as a cause of the dramatic decrease in gastric cancer seen in North America and Western Europe over the last century. Eastern AsiaCentral and Eastern EuropeLess developed regionsWorldMore developed regionsSouth AmericaWestern AsiaCentral AmericaSouthern EuropePolynesiaSouth-Central AsiaCaribbeanWestern EuropeMelanesiaSouth-Eastern AsiaNorthern EuropeSouthern AfricaAustralia/New ZealandEastern AfricaNorthern

1	AmericaWestern AsiaCentral AmericaSouthern EuropePolynesiaSouth-Central AsiaCaribbeanWestern EuropeMelanesiaSouth-Eastern AsiaNorthern EuropeSouthern AfricaAustralia/New ZealandEastern AfricaNorthern AmericaMiddle AfricaNorthern AfricaWestern AfricaMicronesia40302010010203040FemaleMaleIncidenceMortalityFigure 26-49. Gastric cancer incidence and death rates per 100,000 population in men and women, in different regions and countries. (Reproduced with permission from The Global Cancer Observatory All Rights Reserved, September, 2018.)Table 26-15Factors increasing or decreasing the risk of gastric cancerIncrease risk Family history Diet (high in nitrates, salt, fat) Familial polyposis Gastric adenomas Hereditary nonpolyposis colorectal cancer Helicobacter pylori infection Atrophic gastritis, intestinal metaplasia, dysplasia Previous gastrectomy or gastrojejunostomy (>10 y ago) Tobacco use Ménétrier’s diseaseDecrease risk Aspirin Diet (high fresh fruit and vegetable intake) Vitamin

1	intestinal metaplasia, dysplasia Previous gastrectomy or gastrojejunostomy (>10 y ago) Tobacco use Ménétrier’s diseaseDecrease risk Aspirin Diet (high fresh fruit and vegetable intake) Vitamin CBrunicardi_Ch26_p1099-p1166.indd 114001/03/19 7:12 PM 1141STOMACHCHAPTER 26Tobacco use probably increases the risk of stomach cancer, and alcohol use probably has no effect. Regular aspirin use may be protective.Helicobacter pylori80,139 The risk of gastric cancer in patients with chronic H pylori infection is increased about threefold. Compared to uninfected patients, patients with a history of gas-tric ulcers are more likely to develop gastric cancer (incidence ratio 1.8, 95% confidence interval 1.6–2.0), and patients with a history of duodenal ulcers are at decreased risk for gastric can-cer (incidence ratio 0.6, 95% confidence interval 0.4–0.7). This may be due to the fact that some patients develop antral-pre-dominant disease (predisposing to duodenal ulcer and somehow protecting

1	(incidence ratio 0.6, 95% confidence interval 0.4–0.7). This may be due to the fact that some patients develop antral-pre-dominant disease (predisposing to duodenal ulcer and somehow protecting against gastric cancer), while other patients develop corpus-predominant gastritis, resulting in hypochlorhydria and somehow predisposing to gastric ulcer and gastric cancer (Fig. 26-50).10 The theoretical sequence for development of gas-tric adenocarcinoma is diagrammed in Fig. 26-51.10,80 Recently, it has been demonstrated that bone marrow-derived stem cells play a key role in the pathogenesis of gastric adenocarcinoma in patients with chronic H pylori infection.80 However, it must be recognized that gastric adenocarcinoma is a multifactorial dis-ease. Not all patients with gastric cancer have H pylori, and there are some geographic areas with a high prevalence of chronic H pylori infection and a low prevalence of gastric cancer (the “African enigma”). Finally, H pylori–infected patients seem

1	and there are some geographic areas with a high prevalence of chronic H pylori infection and a low prevalence of gastric cancer (the “African enigma”). Finally, H pylori–infected patients seem to be at decreased risk for the development of adenocarcinoma of the distal esophagus and cardia region.140 Perhaps corporeal gas-tritis decreases acid secretion, creating a less damaging refluxate and thus reducing the risk for Barrett’s esophagus, the precursor lesion for these tumors.Epstein-Barr Virus About 10% of gastric adenocarcinomas carry the EBV virus. Recently it has been suggested that EBV infection is a late step in gastric carcinogenesis, since EBV tran-scripts are present in cancer cells but not in the metaplastic cells of precursor epithelium.141Genetic Factors A variety of genetic abnormalities have been described in gastric cancer (Table 26-16). Most gastric can-cers are aneuploid. The most common genetic abnormalities in sporadic gastric cancer affect the p53 and COX-2 genes.

1	have been described in gastric cancer (Table 26-16). Most gastric can-cers are aneuploid. The most common genetic abnormalities in sporadic gastric cancer affect the p53 and COX-2 genes. Over two-thirds of gastric cancers have deletion or suppression of the important tumor-suppressor gene p53. Additionally, approxi-mately the same proportion have overexpression of COX-2. In the colon, tumors with upregulation of this gene have suppressed apoptosis, more angiogenesis, and higher metastatic potential. Gastric tumors that overexpress COX-2 are more aggressive. Recently, a germline mutation in the CDH1 gene encoding E-cadherin was shown to be associated with hereditary diffuse gastric cancer. Prophylactic total gastrectomy should be consid-ered in patients with these mutations.142Premalignant Conditions of the Stomach Figure 26-52 shows the prevalence of some premalignant conditions associated with the development of early gastric cancer in a series of 1900 cases from Tokyo. By far the

1	Conditions of the Stomach Figure 26-52 shows the prevalence of some premalignant conditions associated with the development of early gastric cancer in a series of 1900 cases from Tokyo. By far the most common precancerous lesion is atrophic gastritis. There is a growing appreciation of the impor-tant influence of the chronic inflammatory milieu on the genome of mucosal cells. Chronic inflammation leads to both genetic Acute pangastritisChronic pangastritisDecreased acidGastric cancerChronic antral gastritisDecreased SSTIncreased gastrinIncreased acidDUFigure 26-50. Helicobacter, gastritis, and the pathogenesis of duo-denal ulcer (DU) or gastric cancer.Figure 26-51. Gastric carcinogenesis. (Reproduced with permission from Yamada T, Alpers DH, Laine L, et al: Textbook of Gastroenterology, 4th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2003.)NormalDysplasiaCancerIntestinal metaplasiaAtrophic gastritisChronic superficial gastritisH pyloriDiet low in vitamin C, EHigh-salt

1	4th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2003.)NormalDysplasiaCancerIntestinal metaplasiaAtrophic gastritisChronic superficial gastritisH pyloriDiet low in vitamin C, EHigh-salt dietBrunicardi_Ch26_p1099-p1166.indd 114101/03/19 7:12 PM 1142SPECIFIC CONSIDERATIONS PART IIand epigenetic changes in mucosal cells, which in the stomach leads to the development of gastritis-associated cancer.143,144Polyps Benign gastric polyps are classified as neoplastic (ade-noma and fundic gland polyps) or nonneoplastic (hyperplastic polyp, inflammatory polyp, hamartomatous polyp).145 In general, inflammatory and hamartomatous polyps have little or no malig-nant potential. Fundic gland polyps, commonly seen in patients on long-term PPI therapy, are not premalignant, but in patients with familial adenomatous polyposis (FAP), dysplasia in these lesions is not uncommon, and there are numerous reports of gas-tric cancer arising in a background of fundic gland polyposis in this

1	with familial adenomatous polyposis (FAP), dysplasia in these lesions is not uncommon, and there are numerous reports of gas-tric cancer arising in a background of fundic gland polyposis in this setting. Hyperplastic polyps usually occur in the setting of chronic inflammation. Large hyperplastic polyps (>2 cm) may harbor dysplasia or carcinoma in situ, and gastric cancer may develop remote from the hyperplastic polyp in an area of associ-ated chronic inflammation. Gastric adenomas are premalignant. Patients with familial adenomatous polyposis (FAP) have a high prevalence of gastric adenomatous polyps (about 50%), and are 10 times more likely to develop adenocarcinoma of the stomach than the general population.146 Screening EGD is indicated in these families. Patients with hereditary nonpolyposis colorectal cancer may also be at risk for gastric cancer.147Atrophic Gastritis Chronic atrophic gastritis (Fig. 26-53) is by far the most common precursor for gastric cancer, particularly the

1	colorectal cancer may also be at risk for gastric cancer.147Atrophic Gastritis Chronic atrophic gastritis (Fig. 26-53) is by far the most common precursor for gastric cancer, particularly the intestinal subtype (see Fig. 26-52). The prevalence of atro-phic gastritis is higher in older age groups, but it is also com-mon in younger people in areas with a high incidence of gastric cancer. In many patients, H pylori is critical in the pathogenesis of atrophic gastritis. Correa described three distinct patterns of chronic atrophic gastritis: autoimmune (involves the acid-secreting proximal stomach), hypersecretory (involving the distal stomach), and environmental (involving multiple random areas at the junction of the oxyntic and antral mucosa).139Intestinal Metaplasia Gastric carcinoma often occurs in an area of intestinal metaplasia, and the risk of gastric cancer is proportional to the extent of intestinal metaplasia of the gastric mucosa. These observations suggest that intestinal

1	occurs in an area of intestinal metaplasia, and the risk of gastric cancer is proportional to the extent of intestinal metaplasia of the gastric mucosa. These observations suggest that intestinal metaplasia is a precursor lesion to gastric cancer. There are different pathologic subtypes of intestinal metaplasia in the stomach, based upon the histologic and biochemical characteristics of the changed muco-sal glands. In the complete type of intestinal metaplasia, the glands are lined with goblet cells and intestinal absorptive cells Table 26-16Genetic abnormalities in gastric cancerABNORMALITIESGENEAPPROXIMATE FREQUENCY %Deletion/suppressionp53FHITAPCDCCE-cadherin60–70605050<5Amplification/overexpressionCOX-2HGF/SFVEGFc-metAIB-1β-catenink-samrasc-erb B-27060504540252010–155–7Microsatellite instability 25–40DNA aneuploidy 60–75Reproduced with permission from Feldman M, Friedman LS, Sleisenger MH, et al: Sleisenger and Fordtran’s Gastrointestinal and Liver Disease, 7th ed. Philadelphia,

1	instability 25–40DNA aneuploidy 60–75Reproduced with permission from Feldman M, Friedman LS, Sleisenger MH, et al: Sleisenger and Fordtran’s Gastrointestinal and Liver Disease, 7th ed. Philadelphia, PA: Elsevier/Saunders; 2002.1900 CasesPrecancerouslesionNumber of casesHyperplasticpolypAdenomaChronic ulcerAtrophicgastritisVerrucousgastritisStomachremnantAberrantpancreas10471318022620Total 19000.532.470.6894.841.370.110100%Figure 26-52. Precancerous lesions of the stomach.Figure 26-53. Chronic atrophic gastritis. (Used with permission from Kristen Stashek, MD.)Brunicardi_Ch26_p1099-p1166.indd 114201/03/19 7:12 PM 1143STOMACHCHAPTER 26(Fig. 26-54). These cells are indistinguishable histologically and biochemically from their small bowel counterparts. There is evi-dence that eradication of H pylori infection leads to significant regression of intestinal metaplasia and improvement in atrophic gastritis. Treatment of H pylori is mandatory for patients with these pathologic findings and

1	H pylori infection leads to significant regression of intestinal metaplasia and improvement in atrophic gastritis. Treatment of H pylori is mandatory for patients with these pathologic findings and H pylori infection.Benign Gastric Ulcer Although once considered a premalig-nant condition, it is likely that the older literature addressing gastric ulcers was confounded by the inclusion of inadequately biopsied ulcers s as “benign,” when, in fact, they were malig-nant. Regardless, all gastric ulcers should be viewed as malig-nant until proven otherwise with adequate biopsy and follow-up.Gastric Remnant Cancer It has long been recognized that stomach cancer can develop in the gastric remnant following subtotal gastrectomy. The extent of risk is controversial, but the phenomenon is real. Most tumors develop >10 years follow-ing the initial operation, and they usually arise in an area of chronic gastritis, metaplasia, and dysplasia. This is often near the anastomosis, but many of these

1	tumors develop >10 years follow-ing the initial operation, and they usually arise in an area of chronic gastritis, metaplasia, and dysplasia. This is often near the anastomosis, but many of these tumors are quite large at pre-sentation. Bile or alkali reflux gastritis has been implicated as a precursor, and the greatest number of cases have been reported following Billroth II gastroenterostomy where some transit of pancreatic and biliary secretions through the stomach is obli-gate. Although Roux-en-Y anastomosis has been suggested to be protective, this hypothesis remains unproven. Stage for stage, the prognosis for gastric stump cancer is similar to that of other proximal gastric cancers.148Other Premalignant States Mutations in the E-cadherin gene (CDH1) are associated with hereditary diffuse gastric cancer (HDGC). HDGC is an autosomal dominant trait with a high degree of penetrance. Indeed, the lifetime risk of gastric cancer in individuals with pathogenic germline mutations is 70%

1	gastric cancer (HDGC). HDGC is an autosomal dominant trait with a high degree of penetrance. Indeed, the lifetime risk of gastric cancer in individuals with pathogenic germline mutations is 70% (in men) and 56% (in women).149 CDH1 is a tumor-suppressor gene and a second somatic hit is required for tumorigenesis. The median age at diagnosis of gastric cancer is 38 years. Presentation with clini-cally significant gastric cancer in this setting is associated with a grave prognosis. Increasing recognition of HDGC has afforded the opportunity for early recognition of pathogenic mutations in relatives of individuals with index cases and utilization of prophy-lactic or early total gastrectomy.142 Multifocal intramucosal carci-noma is a frequent finding on putative prophylactic gastrectomy specimens, even in patients without a preoperative diagnosis of carcinoma, affirming the role of early intervention. Surgery should be conducted with the aim of complete extirpation of the stomach to

1	specimens, even in patients without a preoperative diagnosis of carcinoma, affirming the role of early intervention. Surgery should be conducted with the aim of complete extirpation of the stomach to squamous lined esophagus proximally and normal duodenal mucosa distally. Mutation-carrying females are also at increased risk of breast cancer, most often lobular carcinomas, and should be carefully monitored.Up to 10% of gastric cancer cases appear to be familial without a clear-cut genetic diagnosis. First-degree relatives of patients with gastric cancer have a twoto threefold increased risk of developing the disease. Patients with hereditary nonpol-yposis colorectal cancer have a 10% risk of developing gastric cancer, predominantly the intestinal subtype. The mucous cell hyperplasia of Ménétrier’s disease is generally considered to carry a 5% to 10% risk of adenocarcinoma. Periodic surveil-lance EGD is prudent in all the aforementioned conditions. The glandular hyperplasia associated

1	disease is generally considered to carry a 5% to 10% risk of adenocarcinoma. Periodic surveil-lance EGD is prudent in all the aforementioned conditions. The glandular hyperplasia associated with gastrinoma is not prema-lignant, but ECL hyperplasia and/or carcinoid tumors can occur.PathologyDysplasia It is generally accepted that gastric dysplasia is the universal precursor to gastric adenocarcinoma. Patients with severe dysplasia should be considered for gastric resection if the abnormality is widespread or multifocal, or EMR if the severe dysplasia is localized. Patients with mild dysplasia should be followed with endoscopic biopsy surveillance, and Helicobacter eradication.Early Gastric Cancer Early gastric cancer is defined as adeno-carcinoma limited to the mucosa (T1a) and submucosa (T1b) of the stomach. The entity is common in Asia, where the higher frequency of gastric cancer justified adoption of aggressive surveillance programs. Approximately 10% of patients clini-cally staged

1	of the stomach. The entity is common in Asia, where the higher frequency of gastric cancer justified adoption of aggressive surveillance programs. Approximately 10% of patients clini-cally staged with early gastric cancer will have lymph node metastases. There are several subtypes of early gastric cancer (Table 26-17 and Fig. 26-55). Approximately 70% of early gas-tric cancers are well differentiated, and 30% are poorly differen-tiated. The overall cure rate with adequate gastric resection and lymphadenectomy is 95%. In some Japanese centers, 50% of the gastric cancers treated are early stage (compared to less than 20% of resected gastric adenocarcinomas in the United States). Selected patients with early gastric cancer can be treated with endoscopic resection.150Table 26-17Macroscopic types of superficial gastric cancerType 0-I (protruding)aPolypoid tumorsType 0-II (superficial)Tumors with or without minimal elevation or depression relative to the surrounding mucosa Type

1	types of superficial gastric cancerType 0-I (protruding)aPolypoid tumorsType 0-II (superficial)Tumors with or without minimal elevation or depression relative to the surrounding mucosa Type 0-IIaSlightly elevated tumors (superficial elevated) Type 0-IIbTumors without elevation or depression (superficial flat) Type 0-IIcSlightly depressed tumors (superficial depressed)Type 0-III (excavated)Tumors with deep depressionaTumors with less than 3-mm elevation are usually classified as–IIa, with more elevated tumors being classified as 0-I.Figure 26-54. Complete intestinal metaplasia of the stomach. Note intestinal type crypts lined with goblet cells and intestinal absorptive cells. (Used with permission from Kristen Stashek, MD.)Brunicardi_Ch26_p1099-p1166.indd 114301/03/19 7:12 PM 1144SPECIFIC CONSIDERATIONS PART IIGross Morphology and Histologic Subtypes Gastric cancer has been subdivided into four morphologic subtypes: polypoid, fungating, ulcerative, and scirrhous. The first two

1	CONSIDERATIONS PART IIGross Morphology and Histologic Subtypes Gastric cancer has been subdivided into four morphologic subtypes: polypoid, fungating, ulcerative, and scirrhous. The first two are character-ized by a largely intraluminal mass. Polypoid tumors are not ulcerated; fungating tumors are predominantly intraluminal with ulceration. In the latter two gross subtypes, the bulk of the tumor mass is confined to the wall of the stomach. Ulcerative tumors are self-descriptive. Scirrhous tumors infiltrate the entire thickness of the stomach and cover a very large surface area, commonly involve the entire stomach and have a particularly poor prognosis. Although these latter lesions may be technically resectable with total gastrectomy, it is common for both the esophageal and duodenal margins of resection to show micro-scopic evidence of tumor infiltration; distant metastasis, overt or occult, is frequent and death from recurrent disease within 6 months is common. Palliative

1	margins of resection to show micro-scopic evidence of tumor infiltration; distant metastasis, overt or occult, is frequent and death from recurrent disease within 6 months is common. Palliative chemotherapy may prolong median survival.151The location of the primary tumor in the stomach is essen-tial in planning an operation. Several decades ago, the large majority of gastric cancers were in the distal stomach. Recently, there has been a proximal migration of tumors, so currently, the distribution is closer to 40% distal, 30% middle, and 30% proximal.Histology The most important prognostic indicators in gastric cancer are both histologic: lymph node involvement and depth of tumor invasion. Tumor grade (degree of differentiation: well, moderately, or poorly) is also important prognostically.There are several histologic classifications of gastric cancer. The World Health Organization recognizes several histologic types (Table 26-18). The Japanese classification is similar but more

1	are several histologic classifications of gastric cancer. The World Health Organization recognizes several histologic types (Table 26-18). The Japanese classification is similar but more detailed. The commonly used Lauren classifi-cation separates gastric cancers into intestinal type (53%), dif-fuse type (33%), and unclassified (14%). The intestinal type is associated with chronic atrophic gastritis, severe intestinal meta-plasia, and dysplasia, and tends to be less aggressive than the diffuse type. The diffuse type of gastric cancer is more likely to be poorly differentiated and is associated with younger patients and proximal tumors. The Ming classification also is useful and easy to remember, with only two types—expanding (67%) and infiltrative (33%).Recently, the significance of human epidermal growth factor receptor-2 (HER2) was reported in patients with gastric cancer. In breast cancer, overexpression of HER2 has been reported in 15% to 25% of cases, and it is well recognized as

1	epidermal growth factor receptor-2 (HER2) was reported in patients with gastric cancer. In breast cancer, overexpression of HER2 has been reported in 15% to 25% of cases, and it is well recognized as an unfavorable prognostic factor. The development of molecular targeted agents such as trastuzumab has improved the survival of HER2-positive patients. Likewise, in gastric cancer, HER2 overexpression has been reported in 13% to 30% of patients. HER2 targeting with trastuzumab resulted in improved survival in patients with stage IV gastric cancer, and immunohistochem-istry (IHC) staining for HER2 should be performed in recurrent or metastatic cases.152 Expression of other growth receptors in gastric cancer have been characterized as well, including HER1 (epidermal growth factor rector) and HER3. The latter is associ-ated with poor prognosis, but efforts to target these receptors for therapeutic benefit are still exploratory.153Pathologic Staging Ultimately, prognosis is related to

1	and HER3. The latter is associ-ated with poor prognosis, but efforts to target these receptors for therapeutic benefit are still exploratory.153Pathologic Staging Ultimately, prognosis is related to patho-logic stage. The most widespread system for staging of gastric cancer is the tumor-node-metastasis (TNM) staging system based on depth of tumor invasion, extent of lymph node metastases, and presence of distant metastases. This system was developed by the American Joint Committee on Cancer and the International Union Against Cancer, and it has under gone several modifications since it was originally conceived (Table 26-19).Clinical Manifestations. Most patients who are diagnosed with gastric cancer in the United States have advanced stage III or IV disease at the time of diagnosis. The most common symp-toms are weight loss and decreased food intake due to anorexia and early satiety. Abdominal pain (usually not severe and often ignored) is also common. Other symptoms include nausea,

1	most common symp-toms are weight loss and decreased food intake due to anorexia and early satiety. Abdominal pain (usually not severe and often ignored) is also common. Other symptoms include nausea, vomit-ing, and bloating. Acute GI bleeding is somewhat unusual (5%), but chronic occult blood loss is common and manifests as iron Type IIaType IType IIBType IIcType IIIExcavatedDepressedFlatElevatedProtrudedFigure 26-55. Pathologic types of early gastric cancer. (Repro-duced with permission from Fenoglio-Preiser CM, Noffsinger AE, Belli J, et al: Pathologic and phenotypic features of gastric cancer, Semin Oncol. 1996 Jun;23(3):292-306.)Table 26-18World Health Organization histologic typing of gastric cancerAdenocarcinoma Papillary adenocarcinoma Tubular adenocarcinoma Mucinous adenocarcinoma Signet-ring cell carcinomaAdenosquamous carcinomaSquamous cell carcinomaSmall cell carcinomaUndifferentiated carcinomaOthersReproduced with permission from Ming S-C, Goldman H: Pathology of the

1	cell carcinomaAdenosquamous carcinomaSquamous cell carcinomaSmall cell carcinomaUndifferentiated carcinomaOthersReproduced with permission from Ming S-C, Goldman H: Pathology of the Gastrointestinal Tract, 2nd ed. Baltimore, MD: Williams & Wilkins; 1998.Brunicardi_Ch26_p1099-p1166.indd 114401/03/19 7:12 PM 1145STOMACHCHAPTER 26deficiency anemia and heme-positive stool. Dysphagia is com-mon if the tumor involves the cardia of the stomach. Paraneoplas-tic syndromes such as Trousseau’s syndrome (thrombophlebitis), acanthosis nigricans (hyperpigmentation of the axilla and groin), or peripheral neuropathy are rarely present.Physical examination typically is normal. Other than signs of weight loss, specific positive physical findings usually indi-cate incurability. A focused examination in a patient in whom gastric cancer is a likely part of the differential diagnosis should include an examination of the neck, chest, abdomen, and rectum. Cervical, supraclavicular (on the left referred

1	a patient in whom gastric cancer is a likely part of the differential diagnosis should include an examination of the neck, chest, abdomen, and rectum. Cervical, supraclavicular (on the left referred to as Virchow’s node), and axillary lymph nodes may be enlarged, and can be sampled with fine-needle aspiration cytology. Malignant pleural effusions or ascites, or aspiration pneumonitis may be present. An abdominal mass may indicate a large (usually T4) primary tumor, liver metastases, or carcinomatosis (including Kruken-berg’s tumor of the ovary). A palpable umbilical nodule (Sister Joseph’s nodule) is pathognomonic of advanced disease. Rec-tal exam may reveal heme-positive stool and hard nodularity extraluminally and anteriorly, indicating so-called drop metas-tases, or rectal shelf of Blumer in the pouch of Douglas.Diagnostic Evaluation. Distinguishing between peptic ulcer and gastric cancer on clinical grounds alone can be difficult. Patients over the age of 55 years who have

1	of Blumer in the pouch of Douglas.Diagnostic Evaluation. Distinguishing between peptic ulcer and gastric cancer on clinical grounds alone can be difficult. Patients over the age of 55 years who have new-onset dyspep-sia as well as all patients with dyspepsia and alarm symptoms (weight loss, recurrent vomiting, dysphagia, evidence of GI bleeding, or anemia) or with a family history of gastric cancer should undergo prompt upper endoscopy and biopsy if a muco-sal lesion is noted. Essentially, all patients in whom gastric can-cer is part of the differential diagnosis should have endoscopy and biopsy. If suspicion for cancer is high and the biopsy is negative, the patient should be reendoscoped and more aggres-sively biopsied. In some patients with gastric tumors, upper GI series can be helpful in planning treatment. Although a good double-contrast barium upper GI examination is sensitive for gastric tumors (up to 75% sensitive), in most centers, endos-copy has become the gold standard for

1	in planning treatment. Although a good double-contrast barium upper GI examination is sensitive for gastric tumors (up to 75% sensitive), in most centers, endos-copy has become the gold standard for the diagnosis of gastric malignancy. In addition, recent advances in endoscopy have contributed to the earlier diagnosis of gastric cancer. Magnify-ing endoscopy with narrow-band imaging (NBI) has undergone technological improvements and can observe the microvascu-lar architecture of the mucosa and microsurface pattern of the lesion. Magnifying endoscopy with NBI has been reported to be accurate and reliable in the diagnosis of early gastric cancer.154Preoperative staging of gastric cancer is best accomplished with abdominal/pelvic CT scanning with IV and oral contrast. MRI is probably comparable. The best way to stage the tumor locally is via EUS, which gives fairly accurate (80%) informa-tion about the depth of tumor penetration into the gastric wall, and can usually show enlarged (>5

1	The best way to stage the tumor locally is via EUS, which gives fairly accurate (80%) informa-tion about the depth of tumor penetration into the gastric wall, and can usually show enlarged (>5 mm) perigastric and celiac lymph nodes. However, there are limitations to tumor staging with EUS. It is highly operator dependent and may underes-timate lymph node involvement because normal-sized nodes (<5 mm) can harbor metastases. EUS is most accurate in distin-guishing early gastric cancer (T1) from more advanced tumors.Positron Emission Tomography Scanning Whole-body PET scanning derives its power from the preferential accumulation of positron-emitting 18F-fluorodeoxy glucose in tumor com-pared to nontumor cells. It is most useful in the evaluation of distant metastasis in gastric cancer, but it can also be useful in locoregional staging. PET scan is accurate when combined with spiral CT (PET-CT)155 and should be considered before major surgery in patients with particularly high-risk or

1	it can also be useful in locoregional staging. PET scan is accurate when combined with spiral CT (PET-CT)155 and should be considered before major surgery in patients with particularly high-risk or locally advanced tumors.Staging Laparoscopy and Peritoneal Cytology Laparoscopy has emerged as a valuable adjunct to gastric cancer staging, par-ticularly in patients with more substantial tumors. This modality allows for rapid identification of macrosopic peritoneal metas-tases. Peritoneal lavage identifies an additional subset of patients with microscopic dissemination. The prognostic 5Table 26-19AJCC Staging Classification for 8th EditionsAJCC 8TH EDITIONSTAGETNMIAT1N0M0IBT2N0M0T1N1M0IIAT3N0M0T2N1M0T1N2M0IIBT4aN0M0T3N1M0T2N2M0T1N3aM0IIIAT4bN1M0T4aN1M0T4aN2M0T3N2M0T2N3aM0IIIBT4bN1M0T4bN2M0T4aN3aM0T3N3aM0T2N3bM0T1N3bM0IIICT4bN3aM0T4bN3bM0T4aN3bM0T3N3bM0IVAny TAny NM1T1 Tumor invades lamina propria, muscularis mucosa, or submucosa, T2 Tumor invades muscularis propria, T3 Tumor penetrates

1	TAny NM1T1 Tumor invades lamina propria, muscularis mucosa, or submucosa, T2 Tumor invades muscularis propria, T3 Tumor penetrates subserosal connective tissue without invasion of visceral peritoneum or adjacent structures, T4a Tumor invades serosa (visceral peritoneum), T4b: Tumor invades adjacent structures, N0 No regional lymph node metastasis, N1 Metastasis in 1-2 regional lymph nodes, N2: Metastasis in 3-6 regional lymph nodes, N3: Metastasis in 7 or more regional lymph nodes, N3a: Metastasis in 7-15 regional lymph nodes N3b: Metastasis in 16 or more regional lymph nodes, M0 No distant metastasis, M1 Distant metastasis.Used with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Brunicardi_Ch26_p1099-p1166.indd 114501/03/19 7:12 PM 1146SPECIFIC CONSIDERATIONS PART IIsignificance of the latter has been established by several investigators.156,157 Gastrectomy should be

1	114501/03/19 7:12 PM 1146SPECIFIC CONSIDERATIONS PART IIsignificance of the latter has been established by several investigators.156,157 Gastrectomy should be deferred in patients with positive peritoneal cytology without obvious peritoneal metastases. Patients with gastric cancer who undergo R0 resec-tion (i.e., no gross residual disease) and are found to have posi-tive peritoneal cytology (no gross carcinomatosis) have a much poorer prognosis than those with negative cytology (median survival 14.8 months vs. 98.5 months).156 Stand-alone laparos-copy may influence management in up to 36% of cases and is increasingly advocated to allow appropriate initial treatment selection. The yield is likely highest in patients with T3 or T4 tumors, proximal tumors, or evidence of regional nodal involve-ment158; such patients may benefit from neoadjuvant therapy, and laparoscopy should be offered prior to initiation of treat-ment. Systemic therapy is the cornerstone of therapy for patients

1	involve-ment158; such patients may benefit from neoadjuvant therapy, and laparoscopy should be offered prior to initiation of treat-ment. Systemic therapy is the cornerstone of therapy for patients with Stage IV disease and surgery is generally reserved for pal-liation of symptoms (e.g., an obstructing distal tumor) in patients with metastases identified during laparoscopy.Treatment. Surgical resection is the only potentially curative treatment for gastric cancer,159 and most patients with clini-cally resectable locoregional disease should undergo gastrec-tomy. The goals of curative surgical treatment are resection of all tumor (i.e., R0 resection and adequate lymphadenectomy to afford accurate staging and provide locoregional control. Gener-ally, the surgeon strives for a grossly negative margin of at least 5 cm, although an evidence base for this is lacking and recent retrospective analyses have suggested that more conservative resections may be adequate.160 Conversely, complete

1	margin of at least 5 cm, although an evidence base for this is lacking and recent retrospective analyses have suggested that more conservative resections may be adequate.160 Conversely, complete resection of diffuse tumors sometimes proves challenging, and wider gross margins guided by frozen section are sometimes appro-priate. Prior to extending the resection on the basis of a positive frozen section margin, the surgeon should determine whether the microscopic tumor cells are within the wall or on the serosal. The latter may indicate incurable disseminated disease, render-ing additional resection proximally or distally moot, particularly when it makes the anastomosis or stump closure more difficult or hazardous.More than 15 resected lymph nodes are required for ade-quate staging, a relevant marker of quality of care.161,162 Thera-peutic nihilism should be avoided, and in the low-risk patient, an aggressive attempt to resect all tumor should be made. The primary tumor may be resected

1	marker of quality of care.161,162 Thera-peutic nihilism should be avoided, and in the low-risk patient, an aggressive attempt to resect all tumor should be made. The primary tumor may be resected en bloc with adjacent involved organs (e.g., distal pancreas, transverse colon, or spleen) during the course of curative gastrectomy. Palliative gastrectomy may be indicated in the rare patient with incurable disease, but most patients presenting with stage IV gastric cancer can be managed without major operation.163Extent of Gastrectomy The standard operation for gastric cancer is radical subtotal gastrectomy. Unless required for R0 resection, total gastrectomy confers no additional survival ben-efit and may have adverse nutritional or quality-of-life conse-quences and higher perioperative morbidity and mortality.164 Subtotal gastric resection typically entails ligation of the left and right gastric and gastroepiploic arteries at their origins, as well as the en bloc removal of the distal

1	and mortality.164 Subtotal gastric resection typically entails ligation of the left and right gastric and gastroepiploic arteries at their origins, as well as the en bloc removal of the distal 2/3 of the stomach, includ-ing the pylorus and 2 cm of duodenum, the greater and lesser omentum, and all associated lymphatic tissue (Fig. 26-56). In the absence of involvement by direct extension, the spleen and pancreatic tail are not removed.Reconstruction is usually by Billroth II gastrojejunostomy or Roux-en-Y gastrojejunostomy. The former is associated with shorter operative time and precludes roux limb stasis. The lat-ter mitigates bile reflux and, therefore, may be associated with better quality of life long term. In East Asia, especially Japan, Billroth-I gastroduodenostomy is frequently performed after distal gastrectomy. Billroth-I gastroduodenostomy consists of one anastomosis (which is usually straightforward and keeps the duodenum in the food stream). In the United States,

1	performed after distal gastrectomy. Billroth-I gastroduodenostomy consists of one anastomosis (which is usually straightforward and keeps the duodenum in the food stream). In the United States, traditional surgical teaching eschews gastroduodenostomy following gas-tric cancer resection because of the possibility of anastomotic recurrence and obstruction. A risk of remnant carcinoma attrib-uted to bile reflux has been invoked in support of Roux-en-Y reconstruction over Billroth II or I. Strong evidence linking reconstruction approach to long-term oncologic outcome is lacking.Total gastrectomy with Roux-en-Y esophagojejunostomy may be required for R0 resection (Fig. 26-57) and is frequently the optimal operation for patients with proximal gastric adeno-carcinoma. The construction of a jejunal pouch is associated with superior nutritional recovery in some but not all reports, and is a consideration.102 Proximal subtotal gastric resection, Gastroepiploicvs.ABDuodenumTransverse

1	of a jejunal pouch is associated with superior nutritional recovery in some but not all reports, and is a consideration.102 Proximal subtotal gastric resection, Gastroepiploicvs.ABDuodenumTransverse colonTumorLiverStomachStomachJejunumTransverse colonPancreasFigure 26-56. A and B. Radical subtotal gastrectomy. vs. = vessels. (Reproduced with permission from Daly JM, Cady B, Low DW: Atlas of Surgical Oncology. St. Louis, MO: Elsevier/Mosby-Year Book; 1993.)Brunicardi_Ch26_p1099-p1166.indd 114601/03/19 7:13 PM 1147STOMACHCHAPTER 26a technically feasible alternative to total gastrectomy for some proximal gastric tumors, requires esophagogastrostomy to a denervated distal gastric remnant, and functional outcomes are generally poor.165 Pyloroplasty in this setting virtually guar-antees bile esophagitis, and if the pylorus is left intact, gastric emptying may be problematic. An isoperistaltic jejunal intero-sition (Henley loop) between the esophagus and antrum may mitigate some of the

1	esophagitis, and if the pylorus is left intact, gastric emptying may be problematic. An isoperistaltic jejunal intero-sition (Henley loop) between the esophagus and antrum may mitigate some of the adverse symptoms associated with this operation but adds additional complexity.Extent of Lymphadenectomy The 3rd edition of Japanese classification of gastric carcinoma defines lymph node sta-tions on the basis of anatomic landmarks. Lymph node stations 1 to 12 and 14V are classified as regional and metastasis to any other lymph nodal stations constitute distant disease (M1) (see Fig. 26-4). So-called D1 lymphadenectomy in distal gastrec-tomy requires the dissection of stations 1, 3, 4sb, 4d, 5, 6, and 7. Additional resection of stations 8a, 9, 11p, and 12a constitute D2 lymphadenectomy. D1 lymphadenectomy in total gastrec-tomy requires dissection of stations 1 through 7; D2 lymph-adenectomy includes stations 8a to 12a as well. The operation most commonly performed in the United States for

1	in total gastrec-tomy requires dissection of stations 1 through 7; D2 lymph-adenectomy includes stations 8a to 12a as well. The operation most commonly performed in the United States for gastric cancer is a D1 resection and involves removal of the primary tumor with perigastric nodes. The standard operation for gastric cancer in Asia and specialized U.S. centers is D2 gastrectomy, which involves a more extensive lymphadenectomy (removal of the D1 and D2 nodes). In addition to the tissue removed in a D1 resection, D2 gastrectomy includes the superior peritoneum overlying the mesocolon and, selectively, the pancreas, as well as nodes along the common hepatic and splenic arteries, and the celiac axis. Splenectomy and distal pancreatectomy are not routinely performed because this has been shown to increase the morbidity and mortality of the operation.166,167The purported survival advantage of D2 gastrectomy in gastric cancer is illustrated in Table 26-20, which shows the 5-year survival

1	to increase the morbidity and mortality of the operation.166,167The purported survival advantage of D2 gastrectomy in gastric cancer is illustrated in Table 26-20, which shows the 5-year survival rates for gastric cancer stratified by pathologic stage for the United States and Japan. Randomized prospective trials have not confirmed this survival advantage. Two stud-ies showed increased operative mortality with D2 gastrectomy, but the most recent study did not (Table 26-21).166,168,169 With extended lymphadenectomy, much of the morbidity and mor-tality is attributable to performance of splenectomy and distal pancreatectomy, which are no longer routinely included as part of the D2 gastrectomy. Because D2 lymphadenectomy in total gastrectomy requires the dissection of station 10 (i.e., splenic hilar lymph nodes) splenectomy is still selectively performed, particularly for locally advanced fundic tumors.Longer-term follow-up from the Dutch lymphdenectomy trial demonstrating a

1	(i.e., splenic hilar lymph nodes) splenectomy is still selectively performed, particularly for locally advanced fundic tumors.Longer-term follow-up from the Dutch lymphdenectomy trial demonstrating a disease-specific survival advantage with D2 dissection170 as well as recognition that pancreas and spleen preserving dissection can be performed with low morbidity169 have provided momentum for increased utilization of D2 gas-trectomy at high-volume centers in the United States and Europe. Some experts have argued that the D2 operation affords better staging and informs more rational decision-making regarding multimodality therapy. Without question, D2 provides a bet-ter yield of evaluable nodes. Given the frequent inadequacy of lymph node evaluation with gastrectomy in the United States and the association with poorer outcomes, greater attention to the conduct of the operation and pathologic specimen evaluation is clearly desirable. Whether better outcomes after more exten-sive

1	and the association with poorer outcomes, greater attention to the conduct of the operation and pathologic specimen evaluation is clearly desirable. Whether better outcomes after more exten-sive dissection are an epiphenomenon of improved pathologic staging, or a function of therapeutic benefit, remains unclear.Chemotherapy and Radiation for Gastric Cancer The actu-arial 5-year survival rates for resected gastric adenocarcinoma stages I, II, and III in the United States are approximately 75%, 50%, and 25%, respectively. Because most surgical patients have stage II disease or greater, adjuvant therapy is indicated in the majority of patients who undergo initial resection. Adjuvant chemotherapy alone has not proven effective, at least in studies from Europe and the United States.171 Several studies from Table 26-20Gastric cancer 5-year survival and operative mortality in the United States and Japan MARUYAMA (JAPAN), 1971–1985AMERICAN COLLEGE OF SURGEONS, 1982–1987MEMORIAL SLOAN

1	Several studies from Table 26-20Gastric cancer 5-year survival and operative mortality in the United States and Japan MARUYAMA (JAPAN), 1971–1985AMERICAN COLLEGE OF SURGEONS, 1982–1987MEMORIAL SLOAN KETTERING, 1985–1994No. patients317618,365675Stage I91%50%84%Stage II72%29%61%Stage III44%13%29%Stage IV9%3%25%Operative mortality1%7%3%60 cmFigure 26-57. Reconstruction after total gastrectomy. Jejunal pouch (not shown here) should be considered. (Reproduced with permission from Zinner MJ: Atlas of Gastric Surgery. New York, NY: Elsevier/Churchill Livingstone; 1992.)Brunicardi_Ch26_p1099-p1166.indd 114701/03/19 7:13 PM 1148SPECIFIC CONSIDERATIONS PART IIJapan and Korea have indicated a survival advantage with adju-vant chemotherapy after D2 gastrectomy.172-174 The discordance between outcomes in Asia and those from the United States and Europe have been attributed to differences in disease biology or treatment approaches. Proponents of the latter suggest that D2 lymphadenectomy

1	outcomes in Asia and those from the United States and Europe have been attributed to differences in disease biology or treatment approaches. Proponents of the latter suggest that D2 lymphadenectomy provides sufficient locoregional control and that chemotherapy alone has efficacy after optimal surgery. A preferred adjuvant approach in the United States incorpo-rates chemotherapy and radiation based on the results of the Intergroup trial. This prospective randomized study of adju-vant treatment with chemotherapy (5-fluorouracil and leucovo-rin) and radiation (4500 cGy) demonstrated a survival benefit in resected patients with stage II and III adenocarcinoma of the stomach.175 Only 10% of patients entered in the study actually had D2 gastrectomy, and most (54%) had less than an adequate D1 gastrectomy. Because adequacy of lymphadenectomy has been correlated with survival, particularly in patients with stage III gastric cancer, it has been suggested that the benefits of adjuvant

1	D1 gastrectomy. Because adequacy of lymphadenectomy has been correlated with survival, particularly in patients with stage III gastric cancer, it has been suggested that the benefits of adjuvant chemoradiation shown in this study would be vitiated by a more extensive operation.Neoadjuvant chemotherapy has emerged as a viable alter-native to adjuvant chemoradiotherapy in Europe and the United States. Theoretical advantages of this approach include more consistent completion of multimodality therapy, downstaging, earlier treatment of micrometastatic disease, and the ability to gauge response at the in situ tumor. The MAGIC trial, a random-ized controlled trial comparing perioperative epirubicin, cispla-tin, and 5-flourouracil to surgery alone demonstrated a survival advantage and supported this approach in patients with at least stage II disease.176 A subset of patients with very symptomatic tumors may not be eligible for this approach, and the perception that systemic therapy is an

1	this approach in patients with at least stage II disease.176 A subset of patients with very symptomatic tumors may not be eligible for this approach, and the perception that systemic therapy is an ineffective detour for patients who require locoregional control with surgery is sometimes hard to overcome. Regardless, neoadjuvant approaches are increasingly utilized, and even more rigorous regimens incorporating radio-therapy or targeted agents have been explored, sometimes with promising outcomes.177Recent clinical trials from Asia suggest the potential ben-efit of adjuvant chemotherapy after D2 lymphadenectomy in patients with advanced gastric cancer. These trials compared surgery alone and surgery plus adjuvant chemotherapy including oral fluoropyrimidines in resected advanced gastric cancer.172,173 A study from the Japan Clinical Oncology Group showed a 69% overall 5-year survival rate in patients with clinically curable T2b, T3, and T4 gastric cancer, treated with D2 gastrectomy

1	cancer.172,173 A study from the Japan Clinical Oncology Group showed a 69% overall 5-year survival rate in patients with clinically curable T2b, T3, and T4 gastric cancer, treated with D2 gastrectomy alone.178 A subsequent trial from Korea demonstrated a survival advantage with adjuvant capecitabine and oxaliplatin after D2 gastrectomy compared to D2 gastrectomy alone.173 It is uncer-tain whether this approach can be translated to patients in the United States.Although the prognosis of metastatic or recurrent gastric cancer is poor, systemic chemotherapy provides a significant survival benefit over the best supportive care.179,180 Agents that have shown activity against gastric cancer include 5-fluorouracil (5-FU), cisplatin, doxorubicin, methotrexate, taxanes, and camptothecin. Until recently, 5-FU–based chemotherapy, especially in combination with platinums, played a key role in the treatment for the unresectable gastric cancer as well as several types of cancer, such as colon and

1	5-FU–based chemotherapy, especially in combination with platinums, played a key role in the treatment for the unresectable gastric cancer as well as several types of cancer, such as colon and lung. In the 1990s, the introduction of novel anticancer agents such as camptoth-ecin, taxanes, third-generation platinums, and new oral fluo-ropyrimidines, improved the prognosis of unresectable gastric cancer.179,180It is likely that targeted molecular agents will have an increasing role in treating gastric cancer. Recently, Trastu-zumab, a humanized molecular antibody reactive against the extracellular domain of HER2, increased the effectiveness of cytotoxic chemotherapy in patients with HER2 over-expressing advanced gastric cancer.152 Other large trials are ongoing. Deter-mination of HER2 gene amplification status may have prognos-tic significance.181Endoscopic Resection182 The shortand long-term morbidity associated with gastrectomy and the relatively infrequent dis-semination of superficial

1	status may have prognos-tic significance.181Endoscopic Resection182 The shortand long-term morbidity associated with gastrectomy and the relatively infrequent dis-semination of superficial (i.e., T1) tumors to regional nodes have compelled exploration of endoscopic resection for selected lesions. Numerous East Asian centers have demonstrated that some patients with early gastric cancer are adequately treated with endoscopic mucosal resection (EMR). EMR is most appro-priate for patients in whom the probability of lymph node metas-tasis is low. According to the Japanese treatment guidelines for gastric cancer, EMR is a standard treatment for well differenti-ated gastric cancer confined to the mucosa (T1a), measuring less than 2 cm and without signs of ulceration. Such lesions are associated with a negligible risk of lymph node metastasis. 6Table 26-21Randomized trials comparing D1 and D2 gastrectomy for gastric cancerAUTHORSNUMBER OF PATIENTSTYPE OF SURGERYPOSTOPERATIVE

1	are associated with a negligible risk of lymph node metastasis. 6Table 26-21Randomized trials comparing D1 and D2 gastrectomy for gastric cancerAUTHORSNUMBER OF PATIENTSTYPE OF SURGERYPOSTOPERATIVE COMPLICATIONSPOSTOPERATIVE MORTALITY5 YEAR SURVIVALBonenkamp et al.711D125445 D2431047Cuschieri et al.400D1286.535 D2461333Degiuli et al.267D1123 D217.92.2 Data from Bonenkamp JJ, Hermans J, Sasako M, et al. Extended lymph node dissection for gastric cancer. N Engl J Med. 1999;340:908; Cuschieri A, Fayers P, Fielding J, et al. Postoperative morbidity and mortality after D1 and D2 resections for gastric cancer: preliminary results of the MRC randomized controlled surgical trial. The Surgical Cooperative Group. Lancet. 1996;347:995; and Degiuli M, Sasako M, Ponti A, et al. Morbidity and mortality in the Italian Gastric Cancer Study Group randomized clinical trial of D1 versus D2 resection for gastric cancer. Br J Surg. 2010;97:5.Brunicardi_Ch26_p1099-p1166.indd 114801/03/19 7:13 PM

1	in the Italian Gastric Cancer Study Group randomized clinical trial of D1 versus D2 resection for gastric cancer. Br J Surg. 2010;97:5.Brunicardi_Ch26_p1099-p1166.indd 114801/03/19 7:13 PM 1149STOMACHCHAPTER 26En bloc resection is required to evaluate margins for confirma-tion of complete resection. The development of endoscopic submucosal dissection (ESD) allows en bloc resection of larger tumors. This has increased the feasibility of endoscopic resec-tion of larger lesions (<3 cm) at experienced centers. If patho-logic evaluation of the resected specimen does not demonstrate ulceration, penetration of the muscularis mucosae, or lymphatic invasion, the risk of lymph node metastases is less than 1%. Even the occasional patient with higher risk stigmata may be managed endoscopically, particularly in the presence of comor-bidities that preclude safe operation.Screening for Gastric Cancer. In Japan, it clearly has been shown that patients participating in gastric cancer screening

1	particularly in the presence of comor-bidities that preclude safe operation.Screening for Gastric Cancer. In Japan, it clearly has been shown that patients participating in gastric cancer screening pro-grams have a significantly decreased risk of dying from gastric cancer. Thus, screening is effective in a high-risk population. Screening the general population in the United States (a low-risk country) is probably not justified, but patients clearly at risk for gastric cancer probably should have periodic endoscopy and biopsy. This includes patients with familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer, gastric adenomas, Ménétrier’s disease, intestinal metaplasia or dyspla-sia, and remote gastrectomy or gastrojejunostomy.Gastric LymphomaGastric lymphomas generally account for about 4% of gastric malignancies. Over half of patients with non-Hodgkin’s lym-phoma have involvement of the GI tract. The stomach is the most common site of primary GI lymphoma, and over

1	account for about 4% of gastric malignancies. Over half of patients with non-Hodgkin’s lym-phoma have involvement of the GI tract. The stomach is the most common site of primary GI lymphoma, and over 95% are non-Hodgkin’s type. Most are B-cell type, thought to arise in mucosa associated lymphoid tissue (MALT), although most high-grade gastric lymphomas are without any characteristics of the low-grade MALT neoplasm.183 About half of gastric lym-phomas are histologically low grade, and about half are high grade. Interestingly, the normal stomach is relatively devoid of lymphoid tissue. However, in the setting of chronic gastritis, the stomach acquires MALT, which can undergo malignant degen-eration. Again, H pylori is thought to be the culprit. In popula-tions with a high incidence of gastric lymphoma, there is a high incidence of H pylori infection; patients with gastric lymphoma also usually have H pylori infection.184Low-grade MALT lymphoma, essentially a monoclonal proliferation of

1	lymphoma, there is a high incidence of H pylori infection; patients with gastric lymphoma also usually have H pylori infection.184Low-grade MALT lymphoma, essentially a monoclonal proliferation of B cells, presumably arises from a background of chronic gastritis associated with H pylori. These relatively innocuous tumors then undergo degeneration to high-grade lymphoma, which is the usual variety seen by the surgeon. Remarkably, when the H pylori is eradicated and the gastritis improves, the low-grade MALT lymphoma often disappears. Thus, low-grade MALT lymphoma is not a surgical lesion. Careful follow-up is necessary particularly in those lesions with a t(11:18) translocation, thought to be a risk factor for a more aggressive MALT lesion. If low-grade lymphoma per-sists after H pylori eradication, radiation should be consid-ered for disease clinically confined to the stomach (stage I), while chemotherapy with or without radiation is used for more advanced lesions (Fig.

1	H pylori eradication, radiation should be consid-ered for disease clinically confined to the stomach (stage I), while chemotherapy with or without radiation is used for more advanced lesions (Fig. 26-58).Patients with high-grade gastric lymphoma require aggres-sive oncologic treatment for cure and present with many of the same symptoms as gastric cancer patients. However, systemic symptoms such as fever, weight loss, and night sweats occur in about 50% of patients with gastric lymphoma. The tumors may bleed and/or obstruct. Lymphadenopathy and/or organomegaly suggest systemic disease. Diagnosis is by endoscopy and biopsy. Much of the tumor may be submucosal, and an assiduous attempt at biopsy is necessary. Primary lymphoma is usually nodular with enlarged gastric folds. A diffusely infiltrative pro-cess akin to linitis plastica is more suggestive of secondary gas-tric involvement by lymphoma. A diligent search for extragastric disease is necessary before the diagnosis of localized

1	pro-cess akin to linitis plastica is more suggestive of secondary gas-tric involvement by lymphoma. A diligent search for extragastric disease is necessary before the diagnosis of localized primary gastric lymphoma is made. This includes EUS; CT scanning of the chest, abdomen, and pelvis; and bone marrow biopsy. Most patients with high-grade gastric lymphoma are currently treated with chemotherapy and radiation, without surgical resection. Treatment-related perforation or bleeding is an unusual but recognized complication. For disease limited to the stomach and regional nodes, radical subtotal D2 gastrectomy may be performed, especially for bulky tumors with bleeding and/or obstruction. Palliative gastrectomy for tumor complica-tions also has a role. Certainly, a multidisciplinary team should be involved in managing patients with primary gastric lymphoma.Gastrointestinal Stromal TumorGISTs arise from interstitial cells of Cajal (ICC) and are dis-tinct from leiomyoma and

1	team should be involved in managing patients with primary gastric lymphoma.Gastrointestinal Stromal TumorGISTs arise from interstitial cells of Cajal (ICC) and are dis-tinct from leiomyoma and leiomyosarcoma, which arise from smooth muscle.185,186 Prognosis in patients with GIST tumors depends on tumor size, location, and mitotic count. Metasta-sis, when it occurs, is typically by the hematogenous route. Virtually all GISTs should be resected along with a margin of normal tissue. Most GISTs (and almost no smooth muscle tumors) express c-KIT (CD117) or the related PDGF receptor A, as well as CD34; almost all smooth muscle tumors (and almost no GISTs) express actin and desmin. These markers can often be detected on specimens obtained by fine-needle aspiration187 and are useful in differentiating between GIST and smooth muscle tumor histopathologically. Lesions that are definitively leiomyoma by histopathologic criteria can be observed if small and asymptomatic. Larger or symptomatic

1	between GIST and smooth muscle tumor histopathologically. Lesions that are definitively leiomyoma by histopathologic criteria can be observed if small and asymptomatic. Larger or symptomatic gastric leiomyomas are adequately treated by enucleation or wedge resection. Lesions that are definitively GIST or leio-myosarcoma are best treated by resection with negative mar-gins. Most equivocal lesions should be resected provided that the patient has a reasonable operative risk.Two-thirds of all GISTs occur in the stomach and have a more favorable prognosis than do GISTs occurring in other locations. Epithelial cell stromal GIST is the most common cell type arising in the stomach, and cellular spindle type is the next most common. The glomus tumor type is seen only in the stom-ach. Smaller lesions are usually found incidentally, although they occasionally may ulcerate and cause bleeding. Larger lesions may produce symptoms of weight loss, abdominal pain, fullness, early satiety, and

1	lesions are usually found incidentally, although they occasionally may ulcerate and cause bleeding. Larger lesions may produce symptoms of weight loss, abdominal pain, fullness, early satiety, and bleeding. An abdominal mass may be palpable. Metastasis is by the hematogenous route, most often to liver.Diagnosis is by endoscopy and biopsy, although the inter-pretation of the latter may be problematic. When performed, a transluminal (i.e., endoscopic) approach to biopsy is preferred to a percutaneous one, to avoid to the potential for fragmenta-tion and peritoneal seeding. A nondiagnostic biopsy does not preclude resection of a suspicious appearing lesion. Metastatic workup entails CT of the abdomen, and pelvis (chest X-ray suf-fices in lieu of CT of the chest for most patients). Most GISTs are solitary. Local resection with clear margins is adequate sur-gical treatment but is sometimes impractical for larger prepy-loric or pyloric channel tumors, or those near the GE junction.

1	GISTs are solitary. Local resection with clear margins is adequate sur-gical treatment but is sometimes impractical for larger prepy-loric or pyloric channel tumors, or those near the GE junction. 7Brunicardi_Ch26_p1099-p1166.indd 114901/03/19 7:13 PM 1150SPECIFIC CONSIDERATIONS PART IITrue invasion of adjacent structures by the primary tumor is occasionally seen with larger more aggressive lesions. If safe, en bloc resection of involved surrounding organs is appropriate to remove all tumor.The risk of tumor recurrence or metastasis behavior has been stratified into four groups according to the tumor size and mitotic count.110 Very low risk is defined by size <2 cm and <5 mitoses/50 HPF (high-power field). Low risk is defined by size 2 to 5 cm and <5 mitoses/50 cm. Intermediate risk is defined by size <5 cm and 6 to 10 mitoses/50 HPF or size 5 to 10 cm and >5 mitoses/50 HPF. High risk is defined by size >5 cm and >5 mitoses/50HPF, size >10 cm regardless of mitotic rate or >10

1	is defined by size <5 cm and 6 to 10 mitoses/50 HPF or size 5 to 10 cm and >5 mitoses/50 HPF. High risk is defined by size >5 cm and >5 mitoses/50HPF, size >10 cm regardless of mitotic rate or >10 mitoses/50 HPF regardless of size. As mentioned, stomach lesions are associated with lower risk than are tumors in other locations. Classification based on tumor location, size, and mitotic rate have been proposed to evaluate the risk of recurrence and metastasis and role for adjuvant therapy.188 In an effort to further refine risk stratification, a number of nomo-grams have been introduced incorporating tumor features. These tools draw upon and are subject to the limitations of the institu-tional data from which they are derived, but they are sometimes helpful in counseling patients.Mutations in the oncodriver c-kit and PDGFRA are pres-ent in a majority of GISTs. This has been exploited through the use of imatinib (Gleevec), a tyrosine kinase inhibitor. Sev-eral clinical trials in a

1	in the oncodriver c-kit and PDGFRA are pres-ent in a majority of GISTs. This has been exploited through the use of imatinib (Gleevec), a tyrosine kinase inhibitor. Sev-eral clinical trials in a metastatic disease setting demonstrated marked improvements in median survival from 9 months to greater than 5 years.189 These striking results not only estab-lished imatinib as the primary therapy for metastatic GIST, but they also compelled broader efforts to target solid tumors with small molecule inhibitors. Notably, up to 50% of treated patients develop resistance to imatinib by 2 years, and several Low-grade (indolent) MALTLymphoma persistsStage IIChemo** + XRT*Stage IXRT**XRT: external beam radiation therapy, approximately 30 Gy with 10 Gy boost**Chemo: chemotherapy regimens include chlorambucil, fludarabinel,and cyclosphosphamide, vincristine, prednisone (COP) +/– rituximabClose follow-upNo residual diseaseResidual diseaseFurther chemotherapy*Chemo: chemotherapy regimen usually

1	chlorambucil, fludarabinel,and cyclosphosphamide, vincristine, prednisone (COP) +/– rituximabClose follow-upNo residual diseaseResidual diseaseFurther chemotherapy*Chemo: chemotherapy regimen usually cyclophosphamide,doxorobicin, vincristine, prednisone (CHOP) +/– rituximab**XRT: external beam radiation, approximately 30 Gy with 10 Gy boostFollow-upLymphoma regressionHigh-grade (aggressive)SurgeryStage IVChemo* +/– XRT**Stage I, II, IIIChemo* + XRT**Stage III or IVLymph node involvementt(11:18) translocationConfined to gastric wallNo t(11:18) translocationH pylori eradication therapyand chemo** +/– XRT*H pylori eradication therapyRe-evaluate at 3–6 monthsH pylori eradication therapyRe-evaluate at 12 monthsFigure 26-58. Algorithm for the treatment of gastric lymphoma. MALT = mucosa-associated lymphoid tissue. (Reproduced with permission from Yoon SS, Coit DG, Portlock CS, et al. The diminishing role of surgery in the treatment of gastric lymphoma, Ann Surg. 2004

1	MALT = mucosa-associated lymphoid tissue. (Reproduced with permission from Yoon SS, Coit DG, Portlock CS, et al. The diminishing role of surgery in the treatment of gastric lymphoma, Ann Surg. 2004 Jul;240(1):28-37.)Brunicardi_Ch26_p1099-p1166.indd 115001/03/19 7:13 PM 1151STOMACHCHAPTER 26second-line agents have been utilized for patients with refrac-tory disease, most notable sunitinib.The efficacy of imatinib as adjuvant therapy for high risk GIST has been demonstrated in two randomized clinical trials, ACOSOG Z9001 and SSG XVIII.190,191 The former trial randomized patients to 1 year of adjuvant imatinib or pla-cebo and showed an improvement in recurrence-free survival with imatinib. The latter trial demonstrated an overall survival advantage with 3 years compared to 1 year of therapy. Imatinib is now recommended in high risk groups as an adjuvant therapy, for three years or longer. Preoperative therapy with imatinib may be indicated in selected patients with larger lesions

1	Imatinib is now recommended in high risk groups as an adjuvant therapy, for three years or longer. Preoperative therapy with imatinib may be indicated in selected patients with larger lesions that may be more difficult to completely resect or require multivis-ceral resection.Molecular profiling has been embraced with growing rec-ognition that specific tumor subtypes are insensitive to imatinib. Patients with PDGFRA D842V mutations, for example, do not respond to imatinib.192 Management of metastatic GIST is prin-cipally medical, but surgery has a selected role. An algorithm for the treatment of patients with metastatic GIST is shown in Fig. 26-59.Gastric Neuroendocrine Tumors193,194Compared to neuroendocrine tumors of the midgut and hind-gut, neuroendcorine tumors of the stomach are rare. Gastric neuroendocrine tumors comprise about 1% of all neuroen-docrine tumors and less than 2% of gastric neoplasms. They arise from gastric enterochromaffin-like (ECL) cells and may have malignant

1	Gastric neuroendocrine tumors comprise about 1% of all neuroen-docrine tumors and less than 2% of gastric neoplasms. They arise from gastric enterochromaffin-like (ECL) cells and may have malignant potential. The apparent incidence of gastric neuroendocrine tumors is increasing, perhaps related to increased detection or the increasing use of acid suppres-sive medication. The latter may cause hypergastrinemia, and gastrin has a recognized trophic effect on gastric ECL cells. Nomenclature remains a point of confusion; carcinoid and well-differentiated neuroendocrine tumor (NET) are synony-mous according to WHO classification.Gastric neuroendocrine tumors are classified into one of three different types. Type I is the most common, account-ing for about 75% of cases. Type I lesions occur in patients with chronic hypergastrinemia secondary to pernicious anemia or atrophic gastritis. These lesions occur more frequently in women, are often multiple and small, and have low malignant potential

1	with chronic hypergastrinemia secondary to pernicious anemia or atrophic gastritis. These lesions occur more frequently in women, are often multiple and small, and have low malignant potential (<5% metastasize). The role of long-term acid sup-pression with resultant hypergastrinemia in the pathogenesis of type I gastric carcinoids is unclear. Type II gastric neuroendo-crine tumors are associated with MEN1 and ZES. These lesions also tend to be small and multiple, but they have a somewhat higher malignant potential than type I lesions (10% metasta-size). Type II lesions are more common in the setting of MEN1; they are quite uncommon in patients with sporadic ZES. The constellation of gastric acidity, hypergastrinemia, and gastric neuroendocrine tumors suggests gastrinoma until proven other-wise. Type III gastric neuroendocrine tumors are sporadic. They are most often solitary (usually >2 cm) and occur more com-monly in men. They are not associated with hypergastrinemia. Most patients

1	Type III gastric neuroendocrine tumors are sporadic. They are most often solitary (usually >2 cm) and occur more com-monly in men. They are not associated with hypergastrinemia. Most patients have regional nodal or distant metastases at the time of diagnosis, and some present with symptoms of carcinoid syndrome.Gastric neuroendocrine tumors are usually diagnosed with endoscopy and biopsy. The type can be determined based upon clinical context, patient history, the presence or absence of atro-phic gastric mucosa, gastric pH and gastrin level. Some tumors are submucosal and may be quite small. They are often confused with heterotopic pancreas or small leiomyomas. Biopsy may be difficult because of the submucosal location, and EUS can be helpful in defining the size and depth of the lesion. Plasma 8Metastatic or recurrent GISTRepeat imaging in 1 to 3 monthsResponsive/Stable diseaseResectableConsidersurgeryContinueimatinibUnresectableConsidersurgeryDose escalation toimatinib 400 mg

1	Plasma 8Metastatic or recurrent GISTRepeat imaging in 1 to 3 monthsResponsive/Stable diseaseResectableConsidersurgeryContinueimatinibUnresectableConsidersurgeryDose escalation toimatinib 400 mg bidSunitinibRegorafenibClinical trialUnifocalMultifocalProgressive diseaseImatinib 400 mg four times a day in all KIT, non-D842V PDGFRA mutations,and WT GIST Consider 400 mg twice a day in exon 9 KIT mutationsConsider clinical trial in PDGFRA D842V mutationsFigure 26-59. Algorithm for the treatment of malignant gastrointestinal stromal tumor. (Reproduced with permission from Balachandran VP, DeMatteo RP: Gastrointestinal stromal tumors: who should get imatinib and for how long? Adv Surg. 2014;48:165-183.)Brunicardi_Ch26_p1099-p1166.indd 115101/03/19 7:13 PM 1152SPECIFIC CONSIDERATIONS PART IIchromogranin A levels are frequently elevated. CT scan and octreotide or gallium dotatate scans are useful for staging.Type I and II patients with numerous diminutive lesions can be followed with

1	A levels are frequently elevated. CT scan and octreotide or gallium dotatate scans are useful for staging.Type I and II patients with numerous diminutive lesions can be followed with serial endoscopy. Small lesions con-fined to the mucosa (typically type I or type II lesions) less than 1 cm may be treated endoscopically with EMR if there are only a few lesions (<5). Occasionally a slightly larger lesion (1–2 cm) necessitate local surgical excision. Larger lesions and type III lesions should be removed by D1 or D2 gastrectomy. Antrectomy to mitigate gastric secretion in type I patients with refractory growing lesions was invoked as a viable treatment strategy in the past, but it is rarely indicated.Survival is excellent for node-negative patients (>90% 5-year survival); node-positive patients have a 50% 5-year sur-vival. Gastrinoma should be resected if located in patients with type II carcinoid. The 5-year survival for patients with type I gastric carcinoid is close to 100%; for

1	patients have a 50% 5-year sur-vival. Gastrinoma should be resected if located in patients with type II carcinoid. The 5-year survival for patients with type I gastric carcinoid is close to 100%; for patients with type III lesions, the 5-year survival is less than 50%. Somatostatin ana-logue therapy may delay progression of metastatic disease. Sur-gical debulking may have a role in selected patients with limited metastatic disease.BENIGN GASTRIC NEOPLASMSLeiomyomaThe typical leiomyoma is submucosal and firm. If ulcerated, it has an umbilicated appearance and may bleed. Histologically, these lesions appear to be of smooth muscle origin. Lesions <2 cm are usually asymptomatic and benign. Larger lesions may cause symptoms such as bleeding, obstruction, or pain. Asymp-tomatic lesions <2 cm may be carefully observed or enucleated if fine-needle aspiration and immune markers confirm smooth muscle tumor; larger lesions and symptomatic lesions should be removed by wedge resection (often

1	cm may be carefully observed or enucleated if fine-needle aspiration and immune markers confirm smooth muscle tumor; larger lesions and symptomatic lesions should be removed by wedge resection (often possible laparoscopically). When lesions thought to be leiomyoma are observed rather than resected, the patient should be made aware of their presence and the small possibility for malignancy.LipomaLipomas are benign submucosal fatty tumors that are usually asymptomatic, found incidentally on upper GI series or EGD. Endoscopically, they have a characteristic appearance; there also is a characteristic appearance on EUS. Excision is unnec-essary unless the patient is symptomatic.GastroparesisGastric motility disorders include delayed gastric emptying (gastroparesis), rapid gastric emptying, and motor and sensory abnormalities (e.g., functional dyspepsia). Surgically relevant secondary disorders of gastric motility (e.g., dumping, gastric stasis, and Roux syndrome) are discussed under

1	and motor and sensory abnormalities (e.g., functional dyspepsia). Surgically relevant secondary disorders of gastric motility (e.g., dumping, gastric stasis, and Roux syndrome) are discussed under Postgastrec-tomy Problems. Gastroparesis is the most surgically relevant primary disorder of gastric motility.195,196Most patients with primary gastroparesis present with nausea, vomiting, bloating, early satiety, and/or abdominal pain. Eighty percent of these patients are women; some are diabetic. Postprandial vomiting significantly complicates the management of blood glucose in the latter group, predispos-ing to hypoglycemia following preprandial insulin. In patients with gastroparesis, it is important to rule out mechanical gastric outlet obstruction, and small-bowel obstruction. An upper GI series may suggest slow gastric emptying and relative atony, 9or it may be normal. EGD may show bezoars or retained food but is frequently normal. Gastric emptying scintigraphy shows delayed solid

1	GI series may suggest slow gastric emptying and relative atony, 9or it may be normal. EGD may show bezoars or retained food but is frequently normal. Gastric emptying scintigraphy shows delayed solid emptying, and often delayed liquid emptying. Gastroparesis can be a manifestation of a variety of problems (Table 26-22). Medical treatment includes promotility agents, antiemetics, and, perhaps, botulinum injection into the pylorus.If the diabetic gastroparetic patient is not a candidate for pancreas transplant, both gastrostomy (for decompression) and jejunostomy tubes (for feeding and prevention of hypo-glycemia) can be helpful in managing these patients. Other surgical options include implantation of a gastric pacemaker, pyloroplasty or peroral endoscopic pyloromyotmy (particularly in patients responsive to pyloric Botox injection), and gastric resection.197 Generally, gastric resection should be done only after other therapeutic options have been exhausted.Massive Upper

1	in patients responsive to pyloric Botox injection), and gastric resection.197 Generally, gastric resection should be done only after other therapeutic options have been exhausted.Massive Upper Gastrointestinal BleedingAlthough there are arbitrary definitions of “massive” upper GI bleeding put forth, perhaps the most practical definition in the current era would be acute GI bleeding proximal to the ligament of Treitz, which requires blood transfusion. In multiple series, the stomach and proximal duodenum are by far the most com-mon sources of pathology associated with this diagnosis.109,198 Table 26-22Etiology of gastroparesisIdiopathicEndocrine or metabolic Diabetes mellitus Thyroid disease Renal insufficiencyAfter gastric surgery After resection After vagotomyCentral nervous system disorders Brain stem lesions Parkinson’s diseasePeripheral neuromuscular disorders Myotonia dystrophica Duchenne muscular dystrophyConnective tissue

1	resection After vagotomyCentral nervous system disorders Brain stem lesions Parkinson’s diseasePeripheral neuromuscular disorders Myotonia dystrophica Duchenne muscular dystrophyConnective tissue disorders Scleroderma Polymyositis/dermatomyositisInfiltrative disorders Lymphoma AmyloidosisDiffuse gastrointestinal motility disorder Chronic intestinal pseudo-obstructionMedication-inducedElectrolyte imbalance Potassium, calcium, magnesiumMiscellaneous conditions Infections (especially viral) Paraneoplastic syndrome Ischemic conditions Gastric ulcerReproduced with permission from Parkman HP, Harris AD, Krevsky B, et al: Gastroduodenal motility and dysmotility: an update on techniques available for evaluation, Am J Gastroenterol. 1995 Jun;90(6):869-892.Brunicardi_Ch26_p1099-p1166.indd 115201/03/19 7:13 PM 1153STOMACHCHAPTER 26The most common causes of acute upper GI bleeding in emer-gency department or hospitalized patients are peptic ulcer, gastri-tis, Mallory-Weiss syndrome, and

1	7:13 PM 1153STOMACHCHAPTER 26The most common causes of acute upper GI bleeding in emer-gency department or hospitalized patients are peptic ulcer, gastri-tis, Mallory-Weiss syndrome, and esophagogastric varices. Less common causes include benign or malignant neoplasm, angio-dysplasia, Dieulafoy’s lesion, portal gastropathy, Ménétrier’s disease, and watermelon stomach. Arterioenteric fistula should always be considered in the patient who has an aortic graft or who has undergone repair of a visceral artery aneurysm.The most important issues in the early hospital manage-ment of patients with acute upper GI bleeding are resuscita-tion and risk stratification. Large-bore IV access and Foley catheterization is accomplished, and nasogastric intubation is considered. Risk stratification is essentially accomplished by answering the following questions:a. What is the magnitude and acuity of the hemorrhage? Hy-potension, tachycardia, oliguria, low hematocrit, pallor, al-tered mentation, and/or

1	accomplished by answering the following questions:a. What is the magnitude and acuity of the hemorrhage? Hy-potension, tachycardia, oliguria, low hematocrit, pallor, al-tered mentation, and/or hematemesis suggest a large blood loss that has occurred over a short period of time. This is a high-risk situation.b. Does the patient have significant chronic disease, particu-larly lung, liver, kidney, and/or heart disease, which com-promises physiologic reserve? If yes, this is a high-risk situation.c. Is the patient anticoagulated, or immunosuppressed? If yes, this is a high-risk situation.d. On endoscopy, is the patient bleeding from varices, or is there active bleeding, or is there a visible vessel, or is there a deep ulcer overlying a large vessel (e.g., posterior duode-nal ulcer overlying the gastroduodenal artery)? Could the patient be bleeding from an arterio-enteric fistula? If yes, this is a high-risk situation.When judged to be low risk, most patients will stop bleed-ing with

1	the gastroduodenal artery)? Could the patient be bleeding from an arterio-enteric fistula? If yes, this is a high-risk situation.When judged to be low risk, most patients will stop bleed-ing with supportive treatment and IV PPI. Selected patients may be discharged from the emergency department and managed on an outpatient basis.If the patient is deemed to be high risk based on one or more of the aforementioned questions, then the following should be done immediately:1. Type and cross-match for transfusion of blood products.2. Admit to ICU or monitored bed in specialized unit.3. Consult surgeon.4. Consult gastroenterologist.5. Start intravenous PPI.6. Perform upper endoscopy within 12 hours, after resuscita-tion and correction of coagulopathy. Endoscopic hemosta-sis should be considered in most high-risk patients with acute upper GI bleeding.Although the surgeon should be involved early in the hos-pital course of all high-risk patients with acute upper GI bleed-ing, most of these

1	in most high-risk patients with acute upper GI bleeding.Although the surgeon should be involved early in the hos-pital course of all high-risk patients with acute upper GI bleed-ing, most of these patients will be adequately managed without operation. Mucosal lesions can usually be controlled with endo-scopic hemotherapy and medical management. Occasionally, arteriography can be helpful.199 Operation for bleeding ulcer is discussed previously (see “Operation for Bleeding Peptic Ulcer” and Fig. 26-43).Isolated Gastric VaricesIsolated gastric varices are those that occur in the absence of esophageal varices and are classified as type I (fundic) or type II (distal to fundus including proximal duodenum).200 The presence of isolated gastric varices is usually associated with portal hyper-tension or splenic vein thrombosis. Although there is a significant bleeding risk from isolated gastric varices on long-term follow-up, there is no indication for the routine application of prophylactic

1	or splenic vein thrombosis. Although there is a significant bleeding risk from isolated gastric varices on long-term follow-up, there is no indication for the routine application of prophylactic measures.Patients with acute upper GI bleeding from isolated gas-tric varices should be considered high risk. Although data are limited, octreotide and/or vasopressin infusion may decease bleeding, if tolerated. Balloon tamponade with a Sengstaken-Blakemore tube may provide temporary control of exsanguinat-ing hemorrhage from type isolated gastric varices, but if this is used, endotracheal intubation for airway protection is prudent. Endoscopic treatment with sclerotherapy or varix ligation is less successful than in esophageal varices but should be con-sidered. Interventional radiology should be consulted and bal-loon-occluded retrograde transvenous obliteration considered. A transjugular intrahepatic portosystemic shunt (TIPSS) may be useful if there is nonsegmental portal hypertension. If

1	consulted and bal-loon-occluded retrograde transvenous obliteration considered. A transjugular intrahepatic portosystemic shunt (TIPSS) may be useful if there is nonsegmental portal hypertension. If the patient has splenic vein thrombosis and left-sided (sinistral) or segmental portal hypertension, splenectomy is quite effective in controlling bleeding from isolated gastric varices. The operative mortality is 5%. Liver transplantation should always be consid-ered in the cirrhotic patient.Hypertrophic Gastropathy (Ménétrier’s Disease)There are two clinical syndromes characterized by epithelial hyperplasia and giant gastric folds: ZES and Ménétrier’s dis-ease. The latter is characteristically associated with protein-losing gastropathy and hypochlorhydria. There are large rugal folds in the proximal stomach, and the antrum is usu-ally spared. Mucosal biopsy shows diffuse hyperplasia of the surface mucus-secreting cells and usually decreased parietal cells (Fig. 26-60). It has recently

1	proximal stomach, and the antrum is usu-ally spared. Mucosal biopsy shows diffuse hyperplasia of the surface mucus-secreting cells and usually decreased parietal cells (Fig. 26-60). It has recently been suggested that Méné-trier’s disease is caused by local overexpression of transform-ing growth factor-α in the gastric mucosa, which stimulates the epidermal growth factor receptor, a receptor tyrosine kinase, on gastric SECs. This results in the selective expan-sion of surface mucous cells in the gastric body and fundus. A few patients with this unusual disease have been successfully Figure 26-60. Mucosal biopsy in Ménétrier’s disease. (Repro-duced with permission from Ming S-C, Goldman H: Pathology of the Gastrointestinal Tract, 2nd ed. Baltimore, MD: Williams & Wilkins; 1998.)Brunicardi_Ch26_p1099-p1166.indd 115301/03/19 7:13 PM 1154SPECIFIC CONSIDERATIONS PART IItreated with the epidermal growth factor receptor blocking monoclonal antibody cetuximab.201Most patients with

1	115301/03/19 7:13 PM 1154SPECIFIC CONSIDERATIONS PART IItreated with the epidermal growth factor receptor blocking monoclonal antibody cetuximab.201Most patients with Ménétrier’s disease are middle-aged men who present with epigastric pain, weight loss, diarrhea, and hypoproteinemia. There may be an increased risk of gastric cancer. Sometimes, the disease regresses spontaneously. Occa-sionally it is associated with H pylori infection, and the disease improves with helicobacter eradication. Total gastrectomy may be indicated for bleeding, severe hypoproteinemia, or cancer.Watermelon Stomach (Gastric Antral Vascular Ectasia)The parallel red stripes atop the mucosal folds of the distal stomach give this rare entity its sobriquet. Histologically, gas-tric antral vascular ectasia (GAVE) is characterized by dilated mucosal blood vessels that often contain thrombi, in the lamina propria. Mucosal fibromuscular hyperplasia and hyalinization often are present (Fig. 26-61). The

1	(GAVE) is characterized by dilated mucosal blood vessels that often contain thrombi, in the lamina propria. Mucosal fibromuscular hyperplasia and hyalinization often are present (Fig. 26-61). The histologic appearance can resemble portal hypertensive gastropathy, but the latter usually affects the proximal stomach, whereas watermelon stomach pre-dominantly affects the distal stomach. β-Blockers and nitrates, useful in the treatment of portal hypertensive gastropathy, are ineffective in patients with gastric antral vascular ectasia. Patients with GAVE are usually elderly women with chronic GI blood loss requiring transfusion. Most have an associated autoimmune connective tissue disorder, and at least 25% have chronic liver disease. Nonsurgical treatment options include estrogen and progesterone, and endoscopic treatment with the neodymium yttrium-aluminum garnet (Nd:YAG) laser or argon plasma coagulator.202 Antrectomy may be required to control blood loss, and this operation is quite

1	and endoscopic treatment with the neodymium yttrium-aluminum garnet (Nd:YAG) laser or argon plasma coagulator.202 Antrectomy may be required to control blood loss, and this operation is quite effective but carries increased morbidity in this elderly patient group. Patients with portal hypertension and antral vascular ectasia should be consid-ered for transjugular intrahepatic portosystemic shunt (TIPSS).Dieulafoy’s LesionDieulafoy’s lesion is a congenital arteriovenous malformation characterized by an unusually large tortuous submucosal artery. If this artery is eroded, impressive pulsatile bleeding may occur. To the endoscopist or surgeon, this appears as a stream of arte-rial blood emanating from what appears grossly to be a normal gastric mucosa. The lesion typically occurs in middle-aged or elderly men and may be more common in patients with liver disease.203 Patients typically present with upper GI bleeding, which may be intermittent, and endoscopy can miss the lesion if it is

1	or elderly men and may be more common in patients with liver disease.203 Patients typically present with upper GI bleeding, which may be intermittent, and endoscopy can miss the lesion if it is not actively bleeding. Treatment options include endoscopic hemostatic therapy, angiographic embolization, or operation. At surgery, the lesion may be oversewn or resected.Bezoars/DiverticulaBezoars are concretions of indigestible matter that accumulate in the stomach. Trichobezoars are composed of swallowed hair (Fig. 26-62). Phytobezoars are composed of vegetable matter and, in the United States, are usually seen in association with gastroparesis or gastric outlet obstruction. They also are asso-ciated with persimmon ingestion. Most commonly, bezoars produce obstructive symptoms, but they may cause ulceration and bleeding. Diagnosis is suggested by upper GI series and confirmed by endoscopy. Treatment options include enzyme therapy (papain, cellulase, or acetylcysteine), endoscopic

1	may cause ulceration and bleeding. Diagnosis is suggested by upper GI series and confirmed by endoscopy. Treatment options include enzyme therapy (papain, cellulase, or acetylcysteine), endoscopic dis-ruption and removal, or surgical removal.Gastric diverticula are usually solitary and may be con-genital or acquired. Congenital diverticula are true diverticula and contain a full coat of muscularis propria, whereas acquired diverticula (perhaps caused by pulsion) usually have a negli-gible outer muscle layer. Most gastric diverticula occur in the posterior cardia or fundus (Fig. 26-63) and are usually asymp-tomatic. However, they can become inflamed and may produce pain or bleeding. Perforation is rare. Asymptomatic diverticula do not require treatment, but symptomatic lesions should be removed. This can often be done laparoscopically.Foreign BodiesIngested foreign bodies are usually asymptomatic. Small coins usually pass through the GI tract without difficulty. Sharp or Figure

1	be removed. This can often be done laparoscopically.Foreign BodiesIngested foreign bodies are usually asymptomatic. Small coins usually pass through the GI tract without difficulty. Sharp or Figure 26-62. Trichobezoar forming cast of stomach and duode-num; removed from 15-year-old girl. (Reproduced with permission from DeBakey M, Ochsner A: Bezoars and concretions, Surgery. 1938;Dec;4:934.)Figure 26-61. Gastric antral vascular ectasia (watermelon stom-ach). (Reproduced with permission from Godlman H, Hayek J, Federman M: Gastrointestinal Mucosal Biopsy. New York, NY: Churchill Livingstone; 1996.)Brunicardi_Ch26_p1099-p1166.indd 115401/03/19 7:13 PM 1155STOMACHCHAPTER 26large objects in the stomach should be removed. This can usu-ally be done endoscopically, with an overtube technique. Rec-ognized dangers include aspiration of the foreign body during removal and rupture of drug-containing bags in “body packers.” Both complications can be fatal. Surgical removal is recom-mended in

1	Rec-ognized dangers include aspiration of the foreign body during removal and rupture of drug-containing bags in “body packers.” Both complications can be fatal. Surgical removal is recom-mended in body packers who ingest drug parcels for smuggling and in patients with large jagged objects that cannot be safely removed endoscopically. Corrosive objects (i.e., batteries) should be removed promptly usually endoscopically. Ingested magnets should be removed unless they are small and singular and without other ingested metal objects.Mallory-Weiss SyndromeThe Mallory-Weiss lesion is a longitudinal tear in the mucosa of the GE junction.204 It is presumably caused by forceful vom-iting and/or retching, and it is commonly seen in alcoholics. It presents with upper GI bleeding, often with hematemesis. Endoscopy confirms the diagnosis and may be useful in con-trolling the bleeding, but 90% of patients stop bleeding sponta-neously. Other options to control the bleeding include balloon tamponade,

1	Endoscopy confirms the diagnosis and may be useful in con-trolling the bleeding, but 90% of patients stop bleeding sponta-neously. Other options to control the bleeding include balloon tamponade, angiographic embolization, or selective infusion of vasopressin, systemic vasopressin, and operation. Surgical treat-ment consists of oversewing the bleeding lesion through a long gastrotomy.VolvulusGastric volvulus is a twist of the stomach that usually occurs in association with a large hiatal hernia. It also can occur in patients with an unusually mobile stomach without hiatal hernia. Typically, the stomach twists along its long axis (organoaxial volvulus), and the greater curvature flips up (Fig. 26-64C). If the stomach twists around the transverse axis, it is called mesentero-axial rotation (Fig. 26-64A and Fig. 26-64B). Often, volvulus is a chronic condition that can be surprisingly asymptomatic. In these instances, expectant nonoperative management is typi-cally advised, especially in

1	(Fig. 26-64A and Fig. 26-64B). Often, volvulus is a chronic condition that can be surprisingly asymptomatic. In these instances, expectant nonoperative management is typi-cally advised, especially in the elderly. The risk of strangulation and infarction has been overestimated in asymptomatic patients. Symptomatic patients should be considered for operation, espe-cially if the symptoms are severe and/or progressive. Patients may present with symptoms of pain and pressure related to the intermittently distending and poorly emptying twisted stomach. Pressure on the lung may produce dyspnea, pressure on the peri-cardium may produce palpitations, and pressure on the esopha-gus may produce dysphagia. Symptoms are often relieved with vomiting or passage of a nasogastric tube. Gastric infarction is a surgical emergency, and the patient can be moribund. Gastric necrosis may be extensive or focal. Elective operation for gas-tric volvulus usually involves reduction of the stomach and gas-tropexy

1	a surgical emergency, and the patient can be moribund. Gastric necrosis may be extensive or focal. Elective operation for gas-tric volvulus usually involves reduction of the stomach and gas-tropexy with or without repair of hiatal hernia. Gastropexy alone should be considered for high-risk patients since it can nearly always be performed laparoscopically and may be surprisingly effective in relieving mechanical symptoms.GASTROSTOMYA gastrostomy is performed either for alimentation or for gastric drainage/decompression. Gastrostomy may be done percutane-ously, laparoscopically, or via open technique.205,206 Currently, percutaneous endoscopic gastrostomy is the most common method used. The open techniques include the Stamm method (Fig. 26-65), the Witzel method (Fig. 26-66), and the Janeway method. The Janeway gastrostomy, designed to create a per-manent nondraining gastric stoma that can be intermittently intubated, is more complicated than the other open techniques, and is rarely

1	method. The Janeway gastrostomy, designed to create a per-manent nondraining gastric stoma that can be intermittently intubated, is more complicated than the other open techniques, and is rarely necessary. By far the most common surgical tech-nique is the Stamm gastrostomy, which can be performed open or laparoscopically.Complications of gastrostomy include infection, dis-lodgment, leakage with peritonitis, and aspiration pneumonia. Although gastrostomy tubes usually do prevent tense gastric dil-atation, they may not adequately drain the stomach, especially when the patient is bedridden, and they cannot always be relied upon to prevent pulmonary aspiration of gastric contents.ABCFigure 26-64. A through C. Gastric volvulus. (Reproduced with permission from Buchanan J: Volvulus of the stomach, Br J Surg. 1930;July;18(69):99-112.)Figure 26-63. Upper GI contrast study showing a diverticulum of the stomach. (Used with permission from Marc Levine, MD.)Brunicardi_Ch26_p1099-p1166.indd

1	Br J Surg. 1930;July;18(69):99-112.)Figure 26-63. Upper GI contrast study showing a diverticulum of the stomach. (Used with permission from Marc Levine, MD.)Brunicardi_Ch26_p1099-p1166.indd 115501/03/19 7:13 PM 1156SPECIFIC CONSIDERATIONS PART IIPOSTGASTRECTOMY PROBLEMS207Dumping SyndromeDumping is a phenomenon caused by the destruction or bypass of the pyloric sphincter.208 However, other factors undoubtedly play a role because dumping can occur after operations that pre-serve the pylorus, such as parietal cell vagotomy. Also, an appro-priate stimulus may provoke dumping symptoms, even in some patients who have not undergone surgery. Clinically significant dumping occurs in 5% to 10% of patients after pyloroplasty, pyloromyotomy, or gastrectomy, and consists of a constellation of postprandial symptoms ranging in severity from annoying to disabling. The symptoms are thought to be the result of the abrupt delivery of a hyperosmolar load into the small bowel due to ablation of

1	postprandial symptoms ranging in severity from annoying to disabling. The symptoms are thought to be the result of the abrupt delivery of a hyperosmolar load into the small bowel due to ablation of the pylorus or decreased gastric compliance. Typi-cally, 15 to 30 minutes after a meal, the patient becomes dia-phoretic, weak, light-headed, and tachycardic. These symptoms may be ameliorated by recumbence or saline infusion. Crampy abdominal pain is not uncommon, and diarrhea often follows. This is referred to as early dumping and should be distinguished from postprandial (reactive) hypoglycemia, also called late dumping, which usually occurs later (2–3 hours following a meal) and is relieved by the administration of sugar. A variety Figure 26-65. Stamm gastrostomy. (Reproduced with permission from Zuidema GD, Yeo CJ: Shackelford’s Surgery of the Alimentary Tract, 5th ed. Vol. II. Philadelphia, PA: Elsevier/Saunders; 2002.)Figure 26-66. A through F. Witzel gastros-tomy. (Reproduced with

1	from Zuidema GD, Yeo CJ: Shackelford’s Surgery of the Alimentary Tract, 5th ed. Vol. II. Philadelphia, PA: Elsevier/Saunders; 2002.)Figure 26-66. A through F. Witzel gastros-tomy. (Reproduced with permission from Zuidema GD, Yeo CJ: Shackelford’s Sur-gery of the Alimentary Tract, 5th ed. Vol. II. Philadelphia, PA: Elsevier/Saunders; 2002.)Brunicardi_Ch26_p1099-p1166.indd 115601/03/19 7:13 PM 1157STOMACHCHAPTER 26of hormonal aberrations have been observed in early dumping, including increased serum levels of VIP, CCK, neurotensin, peripheral hormone peptide YY, renin-angiotensin-aldosterone, and decreased atrial natriuretic peptide. Late dumping is associ-ated with hypoglycemia and hyperinsulinemia.Medical therapy for the dumping syndrome consists of dietary modification and somatostatin analogue (octreotide). Often, symptoms improve if the patient avoids liquids during meals. Hyperosmolar liquids (e.g., milk shakes) may be particu-larly troublesome. There is some evidence that

1	analogue (octreotide). Often, symptoms improve if the patient avoids liquids during meals. Hyperosmolar liquids (e.g., milk shakes) may be particu-larly troublesome. There is some evidence that adding dietary fiber compounds at mealtime may improve the syndrome. If dietary manipulation fails, the patient is started on octreotide, 100 μg subcutaneously twice daily. This can be increased up to 500 μg twice daily if necessary. The long-acting depot octreo-tide preparation is useful. Octreotide not only ameliorates the abnormal hormonal pattern seen in patients with dumping symptoms, but it also promotes restoration of a fasting motility pattern in the small intestine (i.e., restoration of the MMC). The α-glucosidase inhibitor acarbose may be particularly helpful in ameliorating the symptoms of late dumping.Only a very small percentage of patients with dumping symptoms ultimately require surgery. Most patients improve with time (months and even years), dietary management, and medication.

1	of late dumping.Only a very small percentage of patients with dumping symptoms ultimately require surgery. Most patients improve with time (months and even years), dietary management, and medication. Therefore, the surgeon should not rush to reoper-ate on the patient with dumping symptoms. Multidisciplinary nonsurgical management must be optimized first. Before reop-eration, a period of inhospital observation is useful to define the severity of the patient’s symptoms and patient compliance with prescribed dietary and medical therapy.The results of remedial operation for dumping are variable and unpredictable. There are a variety of surgical approaches, none of which work consistently well. Addition-ally, there is not a great deal of experience reported in the literature with any of these methods and long-term follow-up is rare. Patients with disabling refractory dumping after gas-trojejunostomy can be considered for simple takedown of this anastomosis provided that the pyloric channel

1	and long-term follow-up is rare. Patients with disabling refractory dumping after gas-trojejunostomy can be considered for simple takedown of this anastomosis provided that the pyloric channel is patent. The reversed intestinal segment is rarely used today—and rightly so. This operation interposes a 10-cm reversed segment of intestine between the stomach and the proximal small bowel. This slows gastric emptying, but often leads to obstruction, requiring reoperation. Isoperistaltic interposition (Henley loop) has not been successful in ameliorating severe dumping over the long term. The Roux-en-Y gastrojejunostomy is asso-ciated with delayed gastric emptying, probably on the basis of disordered motility in the Roux limb. Taking advantage of this disordered physiology, surgeons have used this operation successfully in the management of the dumping syndrome. Although this is probably the procedure of choice in the small group of patients requiring operation for severe dumping fol-lowing

1	operation successfully in the management of the dumping syndrome. Although this is probably the procedure of choice in the small group of patients requiring operation for severe dumping fol-lowing gastric resection, gastric stasis may result, particularly if a large gastric remnant is left. In the presence of significant gastric acid secretion, marginal ulceration is common after both jejunal interposition and Roux-en-Y procedures; thus, concomitant vagotomy and hemigastrectomy should be con-sidered. The theoretical possibility of treating postpyloroplasty dumping with a Roux-en-Y to the proximal duodenum (the duodenal switch, a potentially reversible operation) has not yet been reported (Fig. 26-67). Because pyloric ablation seems to be the dominant factor in the etiology of dumping, it is not sur-prising that conversion of Billroth II to Billroth I anastomosis has not been successful in the treatment of dumping.DiarrheaDiarrhea following gastric surgery may be the result of trun-cal

1	not sur-prising that conversion of Billroth II to Billroth I anastomosis has not been successful in the treatment of dumping.DiarrheaDiarrhea following gastric surgery may be the result of trun-cal vagotomy, dumping, or malabsorption. Truncal vagotomy is associated with clinically significant diarrhea in 5% to 10% of patients. It occurs soon after surgery and usually is not associ-ated with other symptoms, a fact that helps to distinguish it from dumping. The diarrhea may be a daily occurrence, or there may be significant periods of relatively normal bowel function. The symptoms tend to improve over the months and years after the index operation. The cause of postvagotomy diarrhea is unclear. Possible mechanisms include intestinal dysmotility and accel-erated transit, bile acid malabsorption, rapid gastric emptying, and bacterial overgrowth. The latter problem is facilitated by decreased gastric acid secretion and (even small) blind loops. Although bacterial overgrowth can be

1	rapid gastric emptying, and bacterial overgrowth. The latter problem is facilitated by decreased gastric acid secretion and (even small) blind loops. Although bacterial overgrowth can be confirmed with the hydrogen breath test, a simpler test is an empirical trial of oral antibiotics. Some patients with postvagotomy diarrhea respond to cholestyramine, while in others codeine or loperamide may be useful. Octreotide should also be tried. Another theoretical cause of diarrhea following gastric surgery is fat malabsorption due to acid inactivation of pancreatic enzymes or poorly coor-dinated mixing of food and digestive juices. This can be con-firmed with a qualitative test for fecal fat and treated with acid suppression. Postvagotomy diarrhea usually does not respond to treatment with pancreatic enzymes. In the rare patient who is debilitated by postvagotomy diarrhea unresponsive to medical management, operation might be considered, but outcomes can be problematic. The operation of

1	enzymes. In the rare patient who is debilitated by postvagotomy diarrhea unresponsive to medical management, operation might be considered, but outcomes can be problematic. The operation of choice is probably a 10-cm reversed jejunal interposition placed in continuity 100 cm distal to the ligament of Treitz. Another option is the onlay antiperi-staltic distal ileal graft. Both operations can cause obstructive symptoms and/or bacterial overgrowth.209Gastric Stasis210,211Gastric stasis following surgery on the stomach may be due to a problem with gastric motor function or caused by an obstruc-tion. The gastric motility abnormality could have been preexist-ing and unrecognized by the operating surgeon. Alternatively, Figure 26-67. Duodenal switch operation. (Reproduced with per-mission from Hinder RA: Duodenal switch: a new form of pancreati-cobiliary diversion, Surg Clin North Am. 1992 Apr;72(2):487-499.)Brunicardi_Ch26_p1099-p1166.indd 115701/03/19 7:13 PM 1158SPECIFIC

1	from Hinder RA: Duodenal switch: a new form of pancreati-cobiliary diversion, Surg Clin North Am. 1992 Apr;72(2):487-499.)Brunicardi_Ch26_p1099-p1166.indd 115701/03/19 7:13 PM 1158SPECIFIC CONSIDERATIONS PART IIit may be secondary to deliberate or unintentional vagotomy, or resection of the dominant gastric pacemaker. An obstruction may be mechanical (e.g., anastomotic stricture, efferent limb kink from adhesions or constricting mesocolon, or a proximal small-bowel obstruction) or functional (e.g., retrograde peristal-sis in a Roux limb). Gastric stasis presents with vomiting (often of undigested food), bloating, epigastric pain, and weight loss.The evaluation of a patient with suspected postoperative gastric stasis includes EGD, upper GI and small bowel series, gastric emptying scan, and gastric motor testing. Endoscopy shows gastritis and retained food or bezoar. The anastomosis and efferent limb should be evaluated for stricture or narrow-ing. A dilated efferent limb

1	scan, and gastric motor testing. Endoscopy shows gastritis and retained food or bezoar. The anastomosis and efferent limb should be evaluated for stricture or narrow-ing. A dilated efferent limb suggests chronic stasis, either from a motor abnormality (e.g., Roux syndrome) or mechanical small bowel obstruction (e.g., chronic adhesion). If the problem is thought to be primarily a disorder of intrinsic motor func-tion, newer techniques such as EGG and GI manometry should be considered, but chronic distal mechanical obstruction may result in disordered motility in the proximal organ confounding interpretation.Once mechanical obstruction has been ruled out, medi-cal treatment is successful in most cases of motor dysfunction following previous gastric surgery. This consists of dietary modification and promotility agents. Intermittent oral antibiotic therapy may be helpful in treating bacterial overgrowth, with its attendant symptoms of bloating, flatulence, and diarrhea.Gastroparesis

1	and promotility agents. Intermittent oral antibiotic therapy may be helpful in treating bacterial overgrowth, with its attendant symptoms of bloating, flatulence, and diarrhea.Gastroparesis following V + D may be treated with subto-tal gastrectomy but simple loop gastrojejunoctomy (GJ) should be tried if previous drainage was pyloroplasty. Billroth II anas-tomosis with Braun enteroenterostomy may be preferable to Roux-en-Y reconstruction after subtotal gastrectomy for gas-tric stasis, but bile reflux can still occur. Initial operation for gastric stasis is often associated with persistent gastric empty-ing problems that may subsequently require near-total or total gastrectomy, a nutritionally unattractive option. Delayed gastric emptying following ulcer surgery (V + D or V + A) may rep-resent an anastomotic stricture (often due to recurrent ulcer) or proximal small bowel obstruction. Recurrent ulcer may respond to medical therapy with PPI and abstinence from NSAIDs, aspi-rin, and

1	an anastomotic stricture (often due to recurrent ulcer) or proximal small bowel obstruction. Recurrent ulcer may respond to medical therapy with PPI and abstinence from NSAIDs, aspi-rin, and smoking. And if necessary, endoscopic dilation is occa-sionally helpful. However, when associated with symptomatic gastric stasis, reoperation is often necessary. Gastroparesis fol-lowing subtotal gastric resection is best treated with near-total (95%) or total gastric resection and Roux-en-Y reconstruction. If total gastrectomy is performed, a jejunal reservoir should be considered. Gastric pacing is promising, but it has not achieved widespread clinical usefulness in the treatment of postoperative gastric atony.Bile Reflux Gastritis and EsophagitisMost patients who have undergone ablation or resection of the pylorus have bile in the stomach on endoscopic examination, along with some degree of gross or microscopic gastric inflam-mation. Therefore, attributing postoperative symptoms to bile reflux

1	of the pylorus have bile in the stomach on endoscopic examination, along with some degree of gross or microscopic gastric inflam-mation. Therefore, attributing postoperative symptoms to bile reflux is problematic because most asymptomatic patients have bile reflux too. However, it is generally accepted that a small subset of patients have bile reflux gastritis syndrome. These patients present with nausea, bilious vomiting, and epigastric pain, and quantitative evidence of excess enterogastric reflux. Curiously, symptoms often develop months or years after the index operation. The differential diagnosis includes afferent or efferent loop obstruction, gastric stasis, and small-bowel obstruction. Plain abdominal X-rays, upper endoscopy, upper GI series, abdominal CT scan, and gastric emptying scans are helpful in evaluating these possibilities.Bile reflux may be quantified with gastric analysis or esophageal impedance testing or with scintigraphy (bile reflux scan). Typically,

1	emptying scans are helpful in evaluating these possibilities.Bile reflux may be quantified with gastric analysis or esophageal impedance testing or with scintigraphy (bile reflux scan). Typically, enterogastric reflux is greatest after Billroth II gastrectomy or gastrojejunostomy, and least after vagotomy and pyloroplasty, with Billroth I gastrectomy giving intermediate values. Patients who are well into the abnormal range of bile reflux may be considered for remedial surgery if symptoms are severe. Remedial surgery will eliminate the bile from the vomi-tus and may improve the patient’s pain, but it is quite unusual to render these patients completely asymptomatic, especially if they are narcotic dependent.Bile reflux gastritis after distal gastric resection may be treated by one of the following options: Roux-en-Y gastroje-junostomy; interposition of a 40-cm isoperistaltic jejunal loop between the gastric remnant and the duodenum (Henley loop); Billroth II gastro jejunostomy with

1	following options: Roux-en-Y gastroje-junostomy; interposition of a 40-cm isoperistaltic jejunal loop between the gastric remnant and the duodenum (Henley loop); Billroth II gastro jejunostomy with Braun enteroenterostomy; total gastrectomy with Roux esophagojejunostomy. To mini-mize reflux of bile into the stomach or the esophagus, the Roux limb should be at least 45 cm long (preferably 60 cm). The Braun enteroenterostomy should be placed at a similar distance from the stomach. Excessively long limbs may be associated with obstruction or malabsorption. All of these operations can result in marginal ulceration on the jejunal side of the gastrojeju-nostomy and thus are combined with a generous distal gastrec-tomy. If this has already been done at a previous operation, the Roux or Braun operations may be attractively simple. Whether truncal vagotomy should be considered to decrease the risk of marginal ulceration is controversial because acid-suppressing medications may be equally

1	operations may be attractively simple. Whether truncal vagotomy should be considered to decrease the risk of marginal ulceration is controversial because acid-suppressing medications may be equally effective. In addition, the benefits of decreased acid secretion following vagotomy may be out-weighed by problems with vagotomy-associated dysmotility in the gastric remnant. The Roux operation may be associated with an increased risk of emptying problems compared to the other two options, but controlled data are lacking. Patients with debilitating bile reflux after gastrojejunostomy can be consid-ered for simple takedown of this anastomosis provided that the pyloric channel is open.Primary bile reflux gastritis (i.e., no previous operation) is rare, and may be treated with the duodenal switch operation, essentially an end-to-end Roux-en-Y to the proximal duodenum (see Fig. 26-68). The Achilles’ heel of this operation is, not sur-prisingly, marginal ulceration. Thus, it should be combined

1	operation, essentially an end-to-end Roux-en-Y to the proximal duodenum (see Fig. 26-68). The Achilles’ heel of this operation is, not sur-prisingly, marginal ulceration. Thus, it should be combined with highly selective vagotomy, and/or long-term acid suppressive medication.Bile gastritis or esophagitis is a recognized complication after esophagogastrectomy with or without pyloroplasty. This can be effectively treated by division of the duodenum immedi-ately distal to the pylorus with drainage of the prepyloric antrum into a Roux limb. Preservation of the right gastroepiploic pedi-cal is important. Proximal subtotal gastrectomy with esophago-antral anastomosis should be avoided, but when performed, the pylorus should be left intact.Roux SyndromeA subset of patients who have had distal gastrectomy and Roux-en-Y gastrojejunostomy will have great difficulty with gastric emptying in the absence of mechanical obstruc-tion. These patients present with vomiting, epigastric pain, and weight

1	and Roux-en-Y gastrojejunostomy will have great difficulty with gastric emptying in the absence of mechanical obstruc-tion. These patients present with vomiting, epigastric pain, and weight loss. This clinical scenario has been labeled the Brunicardi_Ch26_p1099-p1166.indd 115801/03/19 7:13 PM 1159STOMACHCHAPTER 26Roux syndrome. Endoscopy may show retained food or bezoars, dilation of the gastric remnant, and/or dilation of the Roux limb. Anastomotic inflammation and stricture from mar-ginal ulceration is a confounding finding. An upper GI series confirms these findings and may show delayed gastric empty-ing. This is better quantified by a gastric emptying scan, which always shows delayed solid emptying and may show delayed liquid emptying as well.GI motility testing shows abnormal motility in the Roux limb, with much of the propulsive activity toward, rather than away from, the stomach.212 Gastric motility also may be abnor-mal. Presumably, the disordered motility in the Roux

1	motility in the Roux limb, with much of the propulsive activity toward, rather than away from, the stomach.212 Gastric motility also may be abnor-mal. Presumably, the disordered motility in the Roux limb occurs in all patients with this operation. Why only a subset develops the Roux syndrome is unclear. Perhaps patients with disordered gastric motility are at most risk. The disorder seems to be more common in patients with a generous gastric remnant. Truncal vagotomy also has been implicated.Medical treatment consists of promotility agents. Surgi-cal treatment consists of paring down the gastric remnant. Care should be taken to preserve adequate blood supply to the new gastric pouch. If the left gastric artery is intact, a vertically ori-ented lesser curvature based pouch (similar to gastric bypass) with excision of the fundus can be considered. If gastric motility is severely disordered, 95% gastrectomy or total gastrectomy should be considered. The Roux limb should be resected if it

1	bypass) with excision of the fundus can be considered. If gastric motility is severely disordered, 95% gastrectomy or total gastrectomy should be considered. The Roux limb should be resected if it is dilated and flaccid, unless doing so puts the patient at risk for short bowel.GallstonesGallstone formation following gastric surgery generally is thought to be secondary to vagal denervation of the gallblad-der with attendant gallbladder dysmotility and stasis. Although prophylactic cholecystectomy is not justified with most gas-tric surgery, it should be considered if the gallbladder appears abnormal, especially if subsequent cholecystectomy is likely to be difficult. If preoperative evaluation reveals sludge or gall-stones, or if intraoperative evaluation reveals stones, incidental cholecystectomy should be considered.Weight LossWeight loss is common in patients who have had a vagotomy and/or gastric resection. The degree of weight loss tends to par-allel the magnitude of the

1	cholecystectomy should be considered.Weight LossWeight loss is common in patients who have had a vagotomy and/or gastric resection. The degree of weight loss tends to par-allel the magnitude of the operation. It may be insignificant in the obese but devastating in the asthenic patient. The surgeon should always consider the possible nutritional consequences before performing a gastric resection for benign disease in a thin patient. The causes of weight loss after gastric surgery generally fall into one of two categories: altered dietary intake or malabsorption. If a stool stain for fecal fat is negative, it is likely that decreased caloric intake is the cause. This is the most common cause of weight loss after gastric surgery, and it may be due to small stomach syndrome, postoperative gastroparesis, anorexia due to loss of ghrelin, or self-imposed dietary modifi-cation because of dumping and/or diarrhea. Consultation with an experienced dietitian may prove invaluable.AnemiaIron

1	gastroparesis, anorexia due to loss of ghrelin, or self-imposed dietary modifi-cation because of dumping and/or diarrhea. Consultation with an experienced dietitian may prove invaluable.AnemiaIron absorption takes place primarily in the proximal GI tract, and it is facilitated by an acidic environment. Intrinsic factor, essential for the enteric absorption of vitamin B12, is made by the parietal cells of the stomach. Vitamin B12 bioavailability also is facilitated by an acidic environment. Folate-rich vegetables may be poorly tolerated if gastric emptying is delayed or if 10gastric capacity is limited. Since iron, B12, and folate play vital roles in hematopoiesis, it is easy to understand why patients who have had a gastric operation are at risk for anemia. Ane-mia is the most common metabolic side effect in patients who have had a gastric bypass for morbid obesity. It also occurs in up to one-third of patients who have had a vagotomy and/or gastric resection. Iron deficiency is the

1	side effect in patients who have had a gastric bypass for morbid obesity. It also occurs in up to one-third of patients who have had a vagotomy and/or gastric resection. Iron deficiency is the most common cause, but vitamin B12 or folate deficiency also occurs, even in patients who have not had total gastrectomy. Of course, patients who have had a total gastrectomy will all develop B12 deficiency with-out some type of regular nonenteral vitamin B12 administration. Gastric bypass patients should be given oral iron supplements and monitored for iron, B12, and folate deficiency. Patients who have had a vagotomy and/or gastrectomy should be similarly monitored with periodic determination of hematocrit, red blood cell indices, iron and transferrin levels, B12, and folate levels. Marginal nutrient status should be corrected with oral and/or parenteral supplementation.Bone DiseaseGastric surgery sometimes disturbs calcium and vitamin D metabolism. Calcium absorption occurs primarily in the

1	status should be corrected with oral and/or parenteral supplementation.Bone DiseaseGastric surgery sometimes disturbs calcium and vitamin D metabolism. Calcium absorption occurs primarily in the duo-denum, which is bypassed with gastrojejunostomy. Fat mal-absorption may occur because of blind loop syndrome and bacterial overgrowth or because of inefficient mixing of food and digestive enzymes. This can significantly affect the absorp-tion of vitamin D, a fat-soluble vitamin. Both abnormalities of calcium and vitamin D metabolism can contribute to metabolic bone disease in patients following gastric surgery. The prob-lems usually manifest as pain and/or fractures many years after the index operation. Musculoskeletal symptoms should prompt a study of bone density. Dietary supplementation of calcium and vitamin D may be useful in preventing these complications. Routine skeletal monitoring of patients at high-risk (e.g., elderly males and females and postmenopausal females) may prove

1	of calcium and vitamin D may be useful in preventing these complications. Routine skeletal monitoring of patients at high-risk (e.g., elderly males and females and postmenopausal females) may prove useful in identifying skeletal deterioration that may be slowed or stopped with appropriate treatment after gastric surgery.LAPAROSCOPIC GASTRIC OPERATIONSThe most common laparoscopic gastric operations performed today are for GERD and obesity. However, all conventional gastric operations can be performed with minimal access techniques.213 Some are more technically challenging (e.g., partial or total gastric resection) are of debatable advantage over conventional open approaches. Certainly, highly selective vagotomy, vagotomy and gastrojejunostomy, and gastrostomy lend themselves to a minimal access approach. Laparoscopic local excision is often feasible for GI stromal tumors, leiomyo-mas, or gastric diverticula. Difficult to access lesions near the GE junction or pylorus may be removed

1	access approach. Laparoscopic local excision is often feasible for GI stromal tumors, leiomyo-mas, or gastric diverticula. Difficult to access lesions near the GE junction or pylorus may be removed through an anterior gastrotomy; more recent approaches utilizing transgastric ports or combined laparoscopic and endoscopic approaches show promise in allowing removal of practically any small gastric lesion with limited incisions.In Japan and Korea, laparoscopic and robotic assisted approaches have been applied increasingly in the manage-ment of gastric cancer.214,215 Indeed, laparoscopic subtotal gas-trectomy has supplanted the traditional open operation as the preferred operation for patients with earlier stage tumors, and laparocopic total gastrectomy for proximal tumors is performed with regularity and excellent outcomes. The Asian experience Brunicardi_Ch26_p1099-p1166.indd 115901/03/19 7:13 PM 1160SPECIFIC CONSIDERATIONS PART IIhas firmly established the feasibility of safe

1	regularity and excellent outcomes. The Asian experience Brunicardi_Ch26_p1099-p1166.indd 115901/03/19 7:13 PM 1160SPECIFIC CONSIDERATIONS PART IIhas firmly established the feasibility of safe laparoscopic D2 gastrectomy. Translation of this experience to the United States, however, is not easily accomplished. Studies from Asia suggest that expertise in the laparoscopic approach require upwards of 40 cases, a challenging baseline given the much lower incidence of gastric cancer in the United States.216-219 A more advanced spectrum of disease and higher mean BMI in Western coun-tries are additional barriers to widespread implementation of laparoscopic resection for gastric cancer. Notwithstanding, several high volume centers in the United States have reported excellent outcomes after laparoscopic gastrectomy. As robotic technology that facilitates dissection and anastomosis with articulated instrumentation and enhanced visualization becomes increasingly ubiquitous, the pendulum

1	laparoscopic gastrectomy. As robotic technology that facilitates dissection and anastomosis with articulated instrumentation and enhanced visualization becomes increasingly ubiquitous, the pendulum will likely swing toward increasing utilization of minimal access approaches for all gas-tric operations.220REFERENCESEntries highlighted in bright blue are key references. 1. Beaumont W. Experiments and Observations on the Gastric Juice and the Physiology of Digestion. Plattsburgh: PP Allen; 1833. 2. Wangensteen OH, Wangensteen SD. Gastric surgery. In: The Rise of Surgery. Minneapolis: University of Minnesota Press; 1978. 3. Herrington JL. Historical aspects of gastric surgery. In: Scott HW, Jr, Sawyers JL, eds. Surgery of the Stomach, Duodenum, and Small Intestine. 2nd ed. Boston: Blackwell; 1992. 4. Dragstedt LR. Vagotomy for the gastroduodenal ulcer. Ann Surg. 1945;122:973-989. 5. Zollinger RM, Ellison EH. Primary peptic ulcerations of the jejunum associated with islet cell tumors of

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1	Crit Care Med. 2010;38:2222-2228. 136. Isajevs S, et al. Gastritis staging: interobserver agreement by applying OLGA and OLGIM systems. Virchows Arch. 2014;464(4):403-407. 137. Nam JH, et al. OLGA and OLGIM stage distribution accord-ing to age and Helicobacter pylori status in the Korean popula-tion. Helicobacter. 2014;19(2):81-89. 138. The American Cancer Society. Cancer Facts and Figures 2017; pg 4, Table 1. Cancer.org. https://www.cancer.org/content/dam/cancer-org/research/cancer-facts-and-statistics/annual-cancer-facts-and-figures/2017/cancer-facts-and-figures-2017 .pdf Accessed 1-1-2018. 139. Correa P, Houghton J. Carcinogenesis of Helicobacter pylori. Gastroenterology. 2007;133(2):659-672. 140. McColl KE, Watabe H, Derakhshan MH. Role of gastric atrophy in mediating negative association between Heli-cobacter pylori infection and reflux oesophagitis, Barrett’s oesophagus and oesophageal adenocarcinoma. Gut. 2008;57: 721-723. 141. ZurHausen A, van Rees BP, van Beek J, et al.

1	between Heli-cobacter pylori infection and reflux oesophagitis, Barrett’s oesophagus and oesophageal adenocarcinoma. Gut. 2008;57: 721-723. 141. ZurHausen A, van Rees BP, van Beek J, et al. Epstein-Barr virus in gastric carcinomas and gastric stump carci-nomas: alate event in gastric carcinogenesis. J Clin Pathol. 2004;57:487-491. 142. Norton JA, Ham CM, Dam JV, et al. CDH1 truncating muta-tions in the E-cadherin gene: an indication for total gastrec-tomy to treat hereditary diffuse gastric cancer. Ann Surg. 2007;245:873-879. 143. Chiba T, et al. Chronic inflammation and gastric cancer devel-opment (in Japanese). Nihon Rinsho. 2012;70(10):1694-1698. 144. Chiba T, Marusawa H, Ushijima T. Inflammation-associated cancer development in digestive organs: mechanisms and roles for genetic and epigenetic modulation. Gastroenterol-ogy. 2012;143(3):550-563. 145. Park DY, Lauwers GY. Gastric polyps: classification and man-agement. Arch Pathol Lab Med. 2008;132(4):633-640. 146. Shimoyama S, Aoki

1	modulation. Gastroenterol-ogy. 2012;143(3):550-563. 145. Park DY, Lauwers GY. Gastric polyps: classification and man-agement. Arch Pathol Lab Med. 2008;132(4):633-640. 146. Shimoyama S, Aoki F, Kawahara M, et al. Early gastric cancer development in a familial adenomatous polyposis patient. Dig Dis Sci. 2004;49(2):260-265. 147. Gylling A, Abdel-Rahman WM, Juhola M, et al. Is gastric cancer part of the tumour spectrum of hereditary non-pol-yposis colorectal cancer? A molecular genetic study. Gut. 2007;56:926-933. 148. Schaefer N, Sinning C, Standop J, et al. Treatment and prog-nosis of gastric stump carcinoma in comparison with primary proximal gastric cancer. Am J Surg. 2007;194:63-67. 149. van der Post RS, et al. Hereditary diffuse gastric cancer: updated clinical guidelines with an emphasis on germline CDH1 mutation carriers. J Med Genet. 2015;52(6):361-374. 150. Ono H. Early gastric cancer: diagnosis, pathology, treatment techniques, and treatment outcomes. Eur J Gastroenterol

1	emphasis on germline CDH1 mutation carriers. J Med Genet. 2015;52(6):361-374. 150. Ono H. Early gastric cancer: diagnosis, pathology, treatment techniques, and treatment outcomes. Eur J Gastroenterol Hep-atol. 2006;18:863-866. 151. Pyrhonen S, Kuitunen T, Nyandoto P, Kouri M. Randomised comparison of fluorouracil, epidoxorubicin, and methotrex-ate (FEMTX) plus supportive care with supportive care alone in patients with non-resectable gastric cancer. Br J Cancer. 1995;71:587-591. 152. Bang YJ, Van Cutsem E, Feyereislova A, et al. Transtuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gas-troesophageal junction cancer (TOGA): a phase 3 open label randomized controlled trial. Lancet. 2010;376:687-697. 153. Begnami MD, Fuduka E, Fregnani JH, et al. Prognostic impli-cations of altered human epidermal growth factor receptors (HERs) in gastric carcinomas: HER2 and HER3 are predictors of poor outcome. J Clin Oncol.

1	MD, Fuduka E, Fregnani JH, et al. Prognostic impli-cations of altered human epidermal growth factor receptors (HERs) in gastric carcinomas: HER2 and HER3 are predictors of poor outcome. J Clin Oncol. 2011;29(22):3030-3036. 154. Li HY, Dai J, Xue HB, et al. Application of magnifying endoscopy with narrow band imaging in diagnosing gas-tric lesions: a prospective study. Gastrointestinal Endosc. 2012;76:1124-1132. 155. Chen J, Cheong JH, Yun MJ, et al. Improvement in preopera-tive staging of gastric adenocarcinoma with positron emission tomography. Cancer. 2005;103:2383-2390,2005. 156. Wong J, Coit D. Detection of gastric cancer peritoneal metas-tases by peritoneal lavage: current limitations and future per-spectives. Surgery. 2012;152:1-4. 157. Ikoma N, Blum M, Chiang YJ, et al. Yield of staging lapa-roscopy and lavage cytology for radiologically occult peri-toneal carcinomatosis of gastric cancer. Ann Surg Oncol. 2016;23(13):4332-4337. 158. Sarela AI, Lefkowitz R, Brennan MF, et al.

1	staging lapa-roscopy and lavage cytology for radiologically occult peri-toneal carcinomatosis of gastric cancer. Ann Surg Oncol. 2016;23(13):4332-4337. 158. Sarela AI, Lefkowitz R, Brennan MF, et al. Selection of patients with gastric adenocarcinoma for laparoscopic stag-ing. Am J Surg. 2006;191:134-138. 159. Dicken BJ, Bigam DL, Cass C, Mackey JR, Joy AA, Hamilton SM. Gastric adenocarcinoma: review and considerations for future directions. Ann Surg. 2005;241(1):27-39. 160. Squires MH 3rd, Kooby DA, Poultsides GA, et al. Is it time to abandon the 5-cm margin rule during resection of distal gastric adenocarcinoma? A multi-institution study of the U.S. Gastric Cancer Collaborative. Ann Surg Oncol. 2015;22(4):1243-1251. 161. Coburn NG, Swallow CJ Kiss A, Law C. Significant regional variation in adequacy of lymph node assessment and survival in gastric cancer. Cancer. 2006;107(9):2143-2151. 162. Datta J, Lewis RS, Jr, Mamtni R, et al. Implications of inad-equate lymph node staging in

1	in adequacy of lymph node assessment and survival in gastric cancer. Cancer. 2006;107(9):2143-2151. 162. Datta J, Lewis RS, Jr, Mamtni R, et al. Implications of inad-equate lymph node staging in resectable gastric cancer: a contemporary analysis using the National Cancer Data Base. Cancer. 2014;120(18):2855-2865. 163. Saidi RF, ReMine SG, Dudrick PS, et al. Is there a role for palliative gastrectomy in patients with stage IV gastric cancer? World J Surg. 2006;30:21-27. 164. Bozzetti F, Marubini E, Bonfantai G, Gennari L. Subtotal versus total gastrectomy for gastric cancer: five-year survival rates in a multicenter randomized Italian trial. Italian Gastroin-testinal Tumor Study Group. Ann Surg. 1999;230(2):170-178. 165. Karanicolas PJ, Graham D, Gönen DM, Strong VE, Ben-nan MF, Coit DG. Quality of life after gastrectomy for adenocarcinoma: a prospective cohort study. Ann Surg. 2013;257(6):1039-1046. 166. Bonenkamp JJ, Hermans J, Sasako M, et al. Extended lymph node dissection for

1	Quality of life after gastrectomy for adenocarcinoma: a prospective cohort study. Ann Surg. 2013;257(6):1039-1046. 166. Bonenkamp JJ, Hermans J, Sasako M, et al. Extended lymph node dissection for gastric cancer. N Engl J Med. 1999;340:908-914. 167. Bartlett EK, Roses RE, Kelz RR, Drebin JA, Fraker DL, Karakousis GC. Morbidity and mortality after total gastrec-tomy for gastric malignancy using the American College of Surgeons National Surgical Quality Improvement Program database. Surgery. 2014;156(2):298-304. 168. Cuschieri A, Fayers P, Fielding J, et al. Postoperative morbid-ity and mortality after D1 and D2 resections for gastric can-cer: preliminary results of the MRC randomised controlled Brunicardi_Ch26_p1099-p1166.indd 116301/03/19 7:13 PM 1164SPECIFIC CONSIDERATIONS PART IIsurgical trial. The Surgical Cooperative Group. Lancet. 1996;347:995-999. 169. Degiuli M, Sasako M, Ponti A, et al. Randomized clinical trial comparing survival after D1 or D2 gastrectomy for gastric

1	trial. The Surgical Cooperative Group. Lancet. 1996;347:995-999. 169. Degiuli M, Sasako M, Ponti A, et al. Randomized clinical trial comparing survival after D1 or D2 gastrectomy for gastric can-cer. Br J Surg. 2014;101(2):23-31. 170. Songun I, Putter H, Kranenbarg EM, Sasako M, van de Velde CJ. Surgical treatment of gastric cancer: 15-year follow-up results of the randomised nationwide Dutch D1D2 trial. Lan-cet Oncol. 2010;11(5):439-449. 171. GASTRIC (Global Advanced/Adjuvant Stomach Tumor Research International Collaboration) Group; Paoletti X, Oba K, Burzykowski T, et al. Benefit of adjuvant chemo-therapy for resectable gastric cancer: a meta-analysis. JAMA. 2010;303(17):1729-1737. 172. Sasako M, et al. Five-year outcomes of a randomized phase III trial comparing adjuvant chemotherapy with S-1 versus surgery alone in stage II or III gastric cancer. J Clin Oncol. 2011;29:4387-4393. 173. Noh SH, Adjuvant capecitabine plus oxaliplatin for gastric cancer after D2 gastrectomy (CLASSIC):

1	S-1 versus surgery alone in stage II or III gastric cancer. J Clin Oncol. 2011;29:4387-4393. 173. Noh SH, Adjuvant capecitabine plus oxaliplatin for gastric cancer after D2 gastrectomy (CLASSIC): 5-year follow-up of an open-label, randomised phase 3 trial. Lancet Oncol. 2014;15(12):1389-1396. 174. Park SH, Sohn TS, Lee J, et al. Phase III trial to compare adjuvant chemotherapy with capecitabine and cisplatin ver-sus concurrent chemoradiotherapy in gastric cancer: final report of the adjuvant chemoradiotherapy in stomach tumors trial, including survival and subset analyses. J Clin Oncol. 2015;33(28):3130-3136. 175. Macdonald JS, Smalley SR, Benedetti J, et al. Chemoradio-therapy after surgery compared with surgery alone for adeno-carcinoma of the stomach or gastroesophageal junction. N Engl J Med. 2001;345:725-730. 176. Cunningham D, Allum WH, Stenning SP, et al. Perioperative chemotherapy versus surgery alone for resectable gastroesoph-ageal cancer. N Engl J Med.

1	junction. N Engl J Med. 2001;345:725-730. 176. Cunningham D, Allum WH, Stenning SP, et al. Perioperative chemotherapy versus surgery alone for resectable gastroesoph-ageal cancer. N Engl J Med. 2006;355(1):11-20. 177. Ajani JA, Winter K, Okawara GS, et al. Phase II trial of preoperative chemoradiation in patients with localized gas-tric adenocarcinoma (RTOG 9904): quality of combined modality therapy and pathologic response. J Clin Oncol. 2006;24(24):3953-3958. 178. Sasako M, Sano T, Yamamoto S, et al. D2 lymphadenectomy alone or with para-aortic nodal dissection for gastric cancer. N Engl J Med. 2008;359:453-462. 179. VanCutsem E, Moiseyenko VM, Tjulandin S, et al. Phase III study of docetaxel and cisplatin plus fluorouracil compared with cisplatin and fluorouracil as first-line chemotherapy for advanced gastric cancer: areport of the V325 study group. J Clin Oncol. 2006;24:4991-4997. 180. Cunningham D, Starling N, Rao S, et al. Capecitabine and oxaliplatin for advanced

1	chemotherapy for advanced gastric cancer: areport of the V325 study group. J Clin Oncol. 2006;24:4991-4997. 180. Cunningham D, Starling N, Rao S, et al. Capecitabine and oxaliplatin for advanced esophagogastric cancer. N Engl J Med. 2008;358(1):36-46. 181. Gomez-Martin C, Garralda E, Echerri MJ. HER2/neu testing for anti-HER2 based therapies in patients with unresectable and/or metastatic gastric cancer. J Clin Path. 2012;65:751-757. 182. Baptista V, Singh A, Wassef W. Early gastric cancer: an update on endoscopic management. Curr Opin Gastroenterol. 2012;28(6):629-635. 183. Yoon SS, Coit DG, Portlock CS, et al. The diminishing role of surgery in the treatment of gastric lymphoma. Ann Surg. 2004;240:28-37. 184. Farinha P, Gascoyne RD. Helicobacter pylori and MALT lym-phoma. Gastroenterology. 2005;128:1579-1605. 185. Gold JS, DeMatteo RP. Combined surgical and molecular therapy: the gastrointestinal stromal tumor model. Ann Surg. 2006;244(2):176-184. 186. Rubin BP, Heinrich MC,

1	2005;128:1579-1605. 185. Gold JS, DeMatteo RP. Combined surgical and molecular therapy: the gastrointestinal stromal tumor model. Ann Surg. 2006;244(2):176-184. 186. Rubin BP, Heinrich MC, Corless CL. Gastrointestinal stromal tumour. Lancet. 2007;369:1731-1741. 187. Stelow EB, Murad FM, Debol SM, et al. A limited immuno-cytochemical panel for the distinction of subepithelial gastro-intestinal mesenchymal neoplasms sampled by endoscopic ultrasound-guided fine-needle aspiration. Am J Clin Pathol. 2008;129:219-225. 188. Miettinen M, Lasota J. Gastrointestinal stromal tumors: Pathology and prognosis at different sites. Semin Diagn Pathol. 2006;23:70-83. 189. Heinrich MC. Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J Clin Oncol. 2003;21(23):4342-4349. 190. Dematteo RP, Ballman KV, Antonescu CR, et al. Adjuvant imatinib mesylate after resection of localised, primary gas-trointestinal stromal tumour: a randomised, double-blind,

1	RP, Ballman KV, Antonescu CR, et al. Adjuvant imatinib mesylate after resection of localised, primary gas-trointestinal stromal tumour: a randomised, double-blind, placebo-controlled trial. Lancet. 2009;373(9669):1097-1104. 191. Joensuu H, Eriksson M, Sunby Hall K, et al. One vs three years of adjuvant imatinib for operable gastrointestinal stromal tumor: a randomized trial. JAMA. 2012;307(12):1265-1272. 192. Balachandran VP, DeMatteo RP. Adjuvant imatinib for GIST: the pie is shrinking. Ann Surg Oncol. 2014;21(11):3365-3366. 193. Raut CP, Kulke MH, Glickman JN, et al. Carcinoid tumors. Curr Probl Surg. 2006;43:383-450. 194. Perren A, Couvelard A, Scoazec JY, et al. ENETS consensus guidelines for the standards of care in neuroendocrine tumors: pathology: diagnosis and prognostic stratification. Neuroendo-crinology. 2017;105(3):196-200. 195. Parkman HP, Hasler WL, Fisher RS. American Gastroen-terological Association technical review on the diagnosis and treatment of gastroparesis.

1	Neuroendo-crinology. 2017;105(3):196-200. 195. Parkman HP, Hasler WL, Fisher RS. American Gastroen-terological Association technical review on the diagnosis and treatment of gastroparesis. Gastroenterology. 2004;127: 1592-1622. 196. Yin J, Chen JD. Implantable gastric electrical stimulation: ready for prime time? Gastroenterology. 2008;134:665-667. 197. Zehetner J, Ravari F, Ayazi S, et al. Minimally invasive surgi-cal approach for the treatment of gastroparesis. Surg Endosc. 2013;27(1):61-66. 198. Cappell MS, Friedel D. Initial management of acute upper gas-trointestinal bleeding-from initial evaluation to gastrointestinal endoscopy. Med Clin North Am. 2008;92:491-509. 199. Dempsey DT, Burke DR, Reilly RS, McLean GK, Rosato EF. Angiography in poor-risk patients with massive nonvariceal upper gastrointestinal bleeding. Am J Surg. 1990;159:282-286. 200. Zaman A. Portal hypertension related bleeding-management of difficult cases. Clin Liver Dis. 2006;10:353-370. 201. Coffey RJ,

1	upper gastrointestinal bleeding. Am J Surg. 1990;159:282-286. 200. Zaman A. Portal hypertension related bleeding-management of difficult cases. Clin Liver Dis. 2006;10:353-370. 201. Coffey RJ, Washington MK, Corless CL, et al. Ménétrier dis-ease and gastrointestinal stromal tumors: hyperproliferative disorders of the stomach. J Clin Invest. 2007;117:70. 202. Sebastian S, O’Morain CA, Buckley MJ. Current therapeutic options for gastric antral vascular ectasia. Aliment Pharmacol-Ther. 2003;18:157-165. 203. Akhras J, Patel P, Tobi M. Dieulafoy’s lesion-like bleeding: an under-recognized cause of upper gastrointestinal hemor-rhage in patients with advanced liver disease. Dig Dis Sci. 2007;52:722-726. 204. Harbison SP, Dempsey DT. Mallory-Weiss syndrome, in Cam-eron JL, ed. Current Surgical Therapy. 9nd ed. Philadelphia: Mosby; 2008. 205. Schrag SP, Sharma R, Jaik NP, et al. Complications related to percutaneous endoscopic gastrostomy (PEG) tubes. A comprehensive clinical review. J

1	Therapy. 9nd ed. Philadelphia: Mosby; 2008. 205. Schrag SP, Sharma R, Jaik NP, et al. Complications related to percutaneous endoscopic gastrostomy (PEG) tubes. A comprehensive clinical review. J Gastrointestin Liver Dis. 2007;16:407-418. 206. McClave SA. Critical care nutrition: getting involved as a gastrointestinal endoscopist. J Clin Gastroenterol. 2006;40:870-890. 207. Dempsey DT. Reoperative gastric surgery and postgastrec-tomy syndromes. In: Zuidema GD, Yeo CJ, eds. Shackelford’s Surgery of the Alimentary Tract. 5nd ed. Vol 2. Philadelphia: Saunders; 2002:161.Brunicardi_Ch26_p1099-p1166.indd 116401/03/19 7:13 PM 1165STOMACHCHAPTER 26 208. Ukleja A. Dumping syndrome: pathophysiology and treat-ment. Nutr Clin Pract. 2005;20:517-525. 209. Cuschieri A. Postvagotomy diarrhea: is there a place for surgi-cal management? Gut. 1990;31:245-246. 210. Forster-Barthell AW, Murr MM, Nitecki S, et al. Near-total completion gastrectomy for severe postvagotomy gastric stasis: analysis of

1	a place for surgi-cal management? Gut. 1990;31:245-246. 210. Forster-Barthell AW, Murr MM, Nitecki S, et al. Near-total completion gastrectomy for severe postvagotomy gastric stasis: analysis of early and long-term results in 62 patients. J Gastrointest Surg. 1999;3:15-21. 211. Jones MP, Maganti K. A systematic review of surgical therapy for gastroparesis. Am J Gastroenterol. 2003;98: 2122-2129. 212. Van der Milje HC, Kleibeuker JH, Limburg AJ, et al. Mano-metric and scintigraphic studies of the relation between motility disturbances in the Roux limb and the Roux-en-Y syndrome. Am J Surg. 1993;166:11-17. 213. Farrell TM, Hunter JG. Laparoscopic surgery of the stomach and duodenum. In: Zuidema GD, Yeo CJ, eds. Shackelford’s Surgery of the Alimentary Tract. 5nd ed. Vol 2. Philadelphia: Saunders; 2002:202. 214. Lee J, Kim W. Clinical experience of 528 laparoscopic gas-trectomies on gastric cancer in a single institution. Surgery. 2013;153(5):611-618. 215. Zeng YK, Yang ZL, Peng JS, et

1	2002:202. 214. Lee J, Kim W. Clinical experience of 528 laparoscopic gas-trectomies on gastric cancer in a single institution. Surgery. 2013;153(5):611-618. 215. Zeng YK, Yang ZL, Peng JS, et al. Laparoscopy-assisted ver-sus open distal gastrectomy for early gastric cancer. Ann Surg. 2012;256:39-52. 216. Kim HG, Park JH, Jeong SH, et al. Totally laparoscopic distal gastrectomy after learning curve completion: comparison with laparoscopy-assisted distal gastrectomy. J Gastric Cancer. 2013;13(1):26-33. 217. Moon JS, et al. Lessons learned from a comparative analy-sis of surgical outcomes of and learning curves for lap-aroscopy-assisted distal gastrectomy. J Gastric Cancer. 2015;15(1):29-38. 218. Kim HH, Han SU, Kim MC, et al. Long-term results of laparo-scopic gastrectomy for gastric cancer: a large-scale case-con-trol and case-matched Korean multicenter study. J Clin Oncol. 2014;32(7):627-633. 219. Jung DH, Son SY, Park YS, et al. The learning curve asso-ciated with laparoscopic total

1	a large-scale case-con-trol and case-matched Korean multicenter study. J Clin Oncol. 2014;32(7):627-633. 219. Jung DH, Son SY, Park YS, et al. The learning curve asso-ciated with laparoscopic total gastrectomy. Gastric Cancer. 2016;19(1):264-272. 220. Suda K, Uyama I, Kitagawa Y. Technology beats the cur-rent standard: is robotic gastrectomy becoming the standard treatment option for gastric cancer? : “Surgical outcomes after open, laparoscopic, and robotic gastrectomy for gas-tric cancer,” by Kim, Hyuong-Il, et al. Ann Surg Oncol. 2017;24(7):1755-1757.Brunicardi_Ch26_p1099-p1166.indd 116501/03/19 7:13 PM

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1	The Surgical Management of ObesityAnita P. Courcoulas and Philip R. Schauer 27chapterINTRODUCTIONDespite the global pandemic of obesity, there has been little progress in nonsurgical treatment approaches, especially among patients with severe obesity. In addition, the evidence base for bariatric procedures has grown rapidly over the last 10 years, yielding important shortand long-term data on the safety and efficacy of the surgical treatment for obesity and related metabolic disorders. Therefore, the approach for patients con-sidering bariatric and metabolic surgery has now shifted to a well-informed and shared decision-making process as there are significant tradeoffs between the potential risks and benefits of these procedures.1HistoryDuring the 1950s, operations were first performed to treat severe hyperlipidemia with associated obesity.2 These were ileocolic bypass operations to limit absorption and were associated with severe nutritional complications and liver failure

1	to treat severe hyperlipidemia with associated obesity.2 These were ileocolic bypass operations to limit absorption and were associated with severe nutritional complications and liver failure postopera-tively. A more modest jejunoileal bypass was performed next, also a malabsorptive operation, but it bypassed only a portion of the small intestine. Complications after this procedure included severe diarrhea, electrolyte disturbances, protein-calorie malnu-trition, renal stones, and liver failure.In 1969, Mason and Ito performed the first gastric bypass, describing a loop of jejunum connected to a transverse proximal gastric pouch.3 Bile reflux esophagitis was severe postopera-tively, causing Griffin and colleagues to describe the Roux-en-Y modification of the gastric bypass in 1977.4 The gastric pouch was also altered from transverse to vertical using the upper lesser curvature at this time (Fig. 27-1).In 1980, Mason5 first performed the vertical banded gastroplasty (VBG), which was a

1	pouch was also altered from transverse to vertical using the upper lesser curvature at this time (Fig. 27-1).In 1980, Mason5 first performed the vertical banded gastroplasty (VBG), which was a restrictive procedure using a stapled proximal gastric pouch of the upper lesser curvature of the stomach with a restrictive band for its outlet to the rest of the stomach. This operation produced excellent initial weight loss (50% of excess weight or more) with low mor-bidity and mortality. It rapidly became the most commonly performed bariatric operation in the United States during the 1980s. However, by the early 1990s, it became clear that patients who underwent VBG modified their diets to high-calorie soft foods and liquids and some regained weight.6 A significant incidence of stenosis at the cuff and staple line Introduction1167History / 1167State of the Field / 1169The Disease of Obesity1169Overview / 1169U.S. Prevalence of Obesity / 1170Causes of Obesity / 1170Concurrent Medical and

1	cuff and staple line Introduction1167History / 1167State of the Field / 1169The Disease of Obesity1169Overview / 1169U.S. Prevalence of Obesity / 1170Causes of Obesity / 1170Concurrent Medical and Social Problems / 1170Medical Management of Obesity1171Lifestyle Intervention / 1171Pharmacotherapy / 1172Barriers to Treatment / 1173Candidates for Bariatric Surgery1173Indications / 1173Contraindications / 1173Mechanism of Action of Bariatric and Metabolic Surgery1175Overview / 1175Mechanisms of Bariatric Surgery (Weight Loss) / 1175Mechanisms of Metabolic Surgery (Diabetes Improvement) / 1177Preoperative Issues1177Preoperative Preparation / 1177Anesthesiology Issues / 1179Enhanced Recovery After Surgery / 1179Special Equipment and Infrastructure / 1180Bariatric Surgical Procedures1180Laparoscopic Roux-en-Y Gastric Bypass / 1180Laparoscopic Sleeve Gastrectomy / 1185Laparoscopic Adjustable Gastric Banding / 1187Biliopancreatic Diversion and Duodenal Switch / 1189Investigational

1	Roux-en-Y Gastric Bypass / 1180Laparoscopic Sleeve Gastrectomy / 1185Laparoscopic Adjustable Gastric Banding / 1187Biliopancreatic Diversion and Duodenal Switch / 1189Investigational Bariatric Procedures / 1190Follow-Up and Postoperative Care1191Results of Bariatric Surgery1192Short-Term Outcomes / 1192Effectiveness of Bariatric Surgery Compared to Nonsurgical Treatment / 1193Longer-Term Studies / 1193Other Studies / 1198Comparisons Between Procedures / 1199Resolution of Specific Comorbid Conditions / 1199Results of Surgery for Diabetes (Metabolic Surgery) / 1200Complications of Bariatric Surgery1203Surgical Complications / 1203Nonsurgical Complications / 1204Reoperative (Revision and Conversion) Bariatric Surgery1205Introduction / 1205Principles and Preoperative Evaluation / 1205Treatment for Insufficient Weight Loss or Weight Regain / 1206Treatment of Surgical Complications / 1206Special Issues in Bariatric Surgery1207Bariatric Procedures in Adolescents / 1207Cost

1	/ 1205Treatment for Insufficient Weight Loss or Weight Regain / 1206Treatment of Surgical Complications / 1206Special Issues in Bariatric Surgery1207Bariatric Procedures in Adolescents / 1207Cost Effectiveness / 1207Quality Assurance / 1207Plastic Surgery After Weight Loss / 1208Future Important Questions1209Brunicardi_Ch27_p1167-p1218.indd 116723/02/19 2:20 PM 1168JejunoilealBypassGastricBypassHorizontalGastroplastyAdjustable GastricBandingVertical BandedGastroplastyRoux-en-Y gastricBypassBPD with DuodenalSwitchBiliopancreaticDiversionSleeveGastrectomyMalabsorptiveRestrictiveBoth50s60s70s80s90sdisruptions was also problematic.7 Long-term weight loss was poor,8 and by the 1990s in the United States, Roux-en-Y gastric bypass (RYGB) became the procedure of choice for bariatric surgery.In the meantime, in Italy Scopinaro had developed and pop-ularized the biliopancreatic diversion (BPD) in the early 1980s.9 This procedure was also modified to include duodenal switch (DS),10 the only

1	the meantime, in Italy Scopinaro had developed and pop-ularized the biliopancreatic diversion (BPD) in the early 1980s.9 This procedure was also modified to include duodenal switch (DS),10 the only major malabsorptive operation currently in use.The laparoscopic approach to bariatric surgery became available in the 1990s, and Belachew performed the first laparo-scopic adjustable gastric banding (LAGB) operation in 1994.11 Wittgrove and Clark performed the first laparoscopic RYGB the same year.12 LAGB was commonly performed in Europe and Australia during the late 1990s, and in 2001 it was approved for use in the United States. Sleeve gastrectomy (SG) as a primary bariatric operation has grown rapidly in use since 2008.Figure 27-1. History of bariatric surgery. (Reproduced with permission from Arterburn DE, Courcoulas AP: Bariatric surgery for obesity and metabolic condi-tions in adults, BMJ. 2014 Aug 27;349:g3961.)Key Points1 Sixty-five percent of the world’s population live in

1	from Arterburn DE, Courcoulas AP: Bariatric surgery for obesity and metabolic condi-tions in adults, BMJ. 2014 Aug 27;349:g3961.)Key Points1 Sixty-five percent of the world’s population live in countries where overweight and obesity are linked to more deaths than underweight and malnutrition. Obesity is the second leading cause of preventable death in adults in the United States.2 There is an ongoing major shift in procedure utilization with the sleeve gastrectomy and Roux-en-Y gastric bypass being the two most common procedures, worldwide.3 The former classification of bariatric operations as either “restrictive” or “malabsorptive” is being replaced by knowl-edge from investigation into the more basic physiologic and metabolic mechanisms responsible for the effects of bariatric surgery.4 Patients who develop a bowel obstruction after laparoscopic gastric bypass require surgical and not conservative therapy due to the high incidence of internal hernias and the potential for bowel

1	who develop a bowel obstruction after laparoscopic gastric bypass require surgical and not conservative therapy due to the high incidence of internal hernias and the potential for bowel infarction.5 Malabsorptive operations are highly effective in producing durable weight loss and metabolic improvements but have higher surgical complication rates and considerable nutri-tional side effects. Patients undergoing such procedures require complete follow-up and appropriate nutritional supplements.6 Large, longer-term observational studies of bariatric surgery have shown durable weight loss, diabetes remission, lipid improvements, and improved survival with bariatric surgery. Still unclear are specific preand postoperative predictors of those outcomes, long-term complications, microvascular and macrovascular events, mental health outcomes, and costs.7 High quality data have clearly established that bariatric pro-cedures are more effective than medical or lifestyle interven-tions for inducing

1	events, mental health outcomes, and costs.7 High quality data have clearly established that bariatric pro-cedures are more effective than medical or lifestyle interven-tions for inducing weight loss and initial remission of type 2 diabetes, even in less obese patients. Randomized clinical trials showed greater weight loss and type 2 diabetes mellitus remission following bariatric surgery compared with nonsur-gical treatments.8 The incidence of complications after bariatric surgery varies from 4% to over 25% and depends on the duration of follow-up, the definition of complication used, the type of bariatric procedure performed, and individual patient characteristics.9 Emerging areas in bariatric surgery include the use of inter-mediate weight loss devices, adolescent bariatric surgery, and the increase in the need for revision and conversion bar-iatric procedures.Brunicardi_Ch27_p1167-p1218.indd 116823/02/19 2:20 PM 1169THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 2780p <

1	surgery, and the increase in the need for revision and conversion bar-iatric procedures.Brunicardi_Ch27_p1167-p1218.indd 116823/02/19 2:20 PM 1169THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 2780p < 0.0017060Relative Procedure Utilization (%)504030201002006200720082009Year of Procedure2010201120122013Adjustable Gastric BandingRoux-en-Y Gastric BypassSleeve GastrectomyDuodenal SwitchFigure 27-2. Changes in bariatric procedure utilization. (Reproduced with permission from Reames BN, Finks JF, Bacal D, et al: Changes in bariatric surgery procedure use in Michigan, 2006-2013, JAMA. 2014 Sep 3;312(9):959-961.)State of the FieldThere has been an ongoing major shift in bariatric procedures both in the United States and worldwide13 with the rapid adop-tion of the laparoscopic sleeve gastrectomy and the simultane-ous decreasing utilization of the laparoscopic adjustable gastric banding procedure (Fig. 27-2). International trends in the utiliza-tion of bariatric surgical procedures have also

1	and the simultane-ous decreasing utilization of the laparoscopic adjustable gastric banding procedure (Fig. 27-2). International trends in the utiliza-tion of bariatric surgical procedures have also been published. These show that the total number of bariatric surgical procedures performed in 2014 was 579,517. The three most commonly per-formed procedures in the world were SG at 46%, followed by RYGB (40%), and LAGB (7%). The annual percentage changes from 2013 show an increased utilization of SG and a decreased use of RYGB in the United States, Canada, Europe, and Asia and Pacific countries. In Central and South America, however, the use of SG decreased, and RYGB was most commonly used.14Extension of the indication from bariatric surgery for weight loss to metabolic surgery to treat type 2 diabetes (T2DM) even in patients with less than severe obesity has been another more recent development, driven by the availability of more level 1 data.15,16Also, considerable effort is now being

1	2 diabetes (T2DM) even in patients with less than severe obesity has been another more recent development, driven by the availability of more level 1 data.15,16Also, considerable effort is now being devoted to the study of the basic physiologic mechanisms underlying weight loss and, perhaps more importantly, the resolution of comorbid medical problems associated with obesity. Despite the classic “restric-tive” and “malabsorptive” anatomic conceptualizations of bar-iatric surgical procedures (see Fig. 27-1), there is much ongoing research in animal and human models towards understanding the specific underlying mechanisms of action, which may be more physiologic in nature.17 Some of the potential candidates for the mechanisms of action of bariatric procedures include alterations in ghrelin, leptin, glucagon-like peptide-1 (GLP-1), cholecystokinin, peptide YY (PYY), gut microbiota, and bile acids. In the future, bariatric procedures will not be described by anatomic surgical similarities

1	leptin, glucagon-like peptide-1 (GLP-1), cholecystokinin, peptide YY (PYY), gut microbiota, and bile acids. In the future, bariatric procedures will not be described by anatomic surgical similarities but by how they affect key physiological variables, which will provide greater mechanistic insight into how the procedures actually work.THE DISEASE OF OBESITYOverviewWorldwide obesity has more than doubled since 1980. In 2014, 39% of adults age 18 years and over (38% of men and 40% of women) were overweight, and 13% of the world’s adult population (11% of men and 15% of women) were obese. In 2014, an estimated 41 million children under the age of 5 years were overweight or obese.18 Overweight and obesity are now on the rise in lowand middle-income countries, particularly in urban settings. Currently, 65% of the world’s population live in countries where overweight and obesity are linked to more deaths than underweight and malnutrition. Obesity is the second leading cause of preventable

1	Currently, 65% of the world’s population live in countries where overweight and obesity are linked to more deaths than underweight and malnutrition. Obesity is the second leading cause of preventable death in adults in the United States, after tobacco use.19The degrees of obesity are defined by body mass index (BMI = weight [kg]/height [m]2), which correlates body weight with height. The World Health Organization international clas-sification of overweight and obesity is shown in Table 27-1. It should be noted that for Asian populations, classifications remain the same as the international classification, but the pub-lic health action points for interventions are set at a lower BMI threshold. For children, age needs to be considered when defin-ing overweight and obesity, so for children age 5 to 19 years, 1Table 27-1The international classification of adult overweight and obesity according to body mass index (BMI)CLASSIFICATIONBMI (kg/m2)PRINCIPAL CUTOFF POINTSADDITIONAL CUTOFF

1	age 5 to 19 years, 1Table 27-1The international classification of adult overweight and obesity according to body mass index (BMI)CLASSIFICATIONBMI (kg/m2)PRINCIPAL CUTOFF POINTSADDITIONAL CUTOFF POINTSaNormal range 18.50–24.99 18.50–22.9923.00–24.99Overweight≥25.00≥25.00 Preobese 25.00–29.99 25.00–27.4927.50–29.99 Obese≥30.00≥30.00 Obese class I 30.00–34.99 30.00–32.4932.50–34.99 Obese class II 35.00–39.99 35.00–37.4937.50–39.99 Obese class III≥40.00≥40.00aFor Asian populations, classifications remain the same as the international classification, but public health action points for interventions are set at 23, 27.5, 32.5, and 37.5 kg/m.2Data from WHO, 1995, WHO, 2000 and WHO 2004.Brunicardi_Ch27_p1167-p1218.indd 116923/02/19 2:20 PM 1170SPECIFIC CONSIDERATIONSPART IIoverweight is BMI-for-age greater than 1 standard deviation above the World Health Organization (WHO) growth reference median, and obesity is greater than 2 standard deviations above the WHO growth reference

1	is BMI-for-age greater than 1 standard deviation above the World Health Organization (WHO) growth reference median, and obesity is greater than 2 standard deviations above the WHO growth reference median.The fundamental cause of obesity and overweight is an energy imbalance between calories consumed and calories expended. Globally, there has been an increased intake of energy-dense foods that are high in fat and a decrease in physi-cal inactivity due to the increasingly sedentary nature of many forms of work, changing modes of transportation, and increas-ing urbanization. These changes in dietary and physical activ-ity patterns are the result of both societal and environmental changes associated with development. There is also a dearth of supportive policies to counteract these forces in sectors such as health, agriculture, transport, urban planning, environment, food processing, marketing, and education. Obesity as a disease was recognized by the American Medical Association in 2013.

1	such as health, agriculture, transport, urban planning, environment, food processing, marketing, and education. Obesity as a disease was recognized by the American Medical Association in 2013. It is multifactorial in its etiology, and the components of the disease likely include a combination of both environmental and genetic factors.U.S. Prevalence of ObesityAccording to the 2013–2014 National Health and Nutrition Examination Survey (NHANES) data, in the United States the overall age-adjusted prevalence of obesity was 37.7% (95% confidence interval [CI], 35.8–39.7%); among men, it was 35.0% (95% CI, 32.8–37.3%); and among women, it was 40.4% (95% CI, 37.6–43.3%). The corresponding prevalence of class III obesity overall was 7.7% (95% CI, 6.2–9.3%); among men, it was 5.5% (95% CI, 4.0–7.2%); and among women, it was 9.9% (95% CI, 7.5–12.3%). Changes over the decade from 2005 through 2014, adjusted for age, race, smoking status, and education, showed significantly increas-ing trends

1	and among women, it was 9.9% (95% CI, 7.5–12.3%). Changes over the decade from 2005 through 2014, adjusted for age, race, smoking status, and education, showed significantly increas-ing trends among women for overall obesity and for class III obesity, but not among men.20For children in the United States, obesity is defined as a BMI at or above the sex-specific 95th percentile, and extreme obesity is defined as a BMI at or above 120% of the sexspecific 95th percentile on the U.S. Centers for Disease Control and Prevention (CDC) BMI-for-age growth charts. In the most recent NHANES study of U.S. children and adolescents age 2 to 19 years, the prevalence of obesity from 2011 to 2014 was 17.0%, and extreme obesity was 5.8%.21Causes of ObesityBoth genetic and environmental factors contribute to the devel-opment of obesity. Not everyone exposed to the prevailing envi-ronment becomes obese, suggesting that genetic mechanisms are operating at the individual level. Estimates vary, but twin,

1	to the devel-opment of obesity. Not everyone exposed to the prevailing envi-ronment becomes obese, suggesting that genetic mechanisms are operating at the individual level. Estimates vary, but twin, family, and adoption studies show that the rate of heritability of BMI is high, ranging from 40% to 70%.22,23 Eleven rare and monogenic forms of obesity are now recognized, including a deficiency of the leptin and melanocortin-4 receptors, which are expressed in the hypothalamus and are involved in regulating energy homeostasis.24 Heterozygous mutations in the melano-cortin-4 receptor gene are currently the most common cause of monogenic obesity, causative in 2% to 5% of children with severe obesity.Genes and environment interact in a complex process that regulates energy balance and weight. Reducing food intake or increasing physical activity leads to a negative energy bal-ance and a cascade of compensatory adaptive mechanisms that preserve vital functions and are associated with

1	Reducing food intake or increasing physical activity leads to a negative energy bal-ance and a cascade of compensatory adaptive mechanisms that preserve vital functions and are associated with reductions in resting energy expenditure, food preoccupation, and many other changes that depend on the amount and duration of caloric restriction. There is also a counterregulatory increase in appetite and food intake that limits the degree of expected weight loss that is associated with interventions such as exercise programs.22Individuals with obesity have excessive adipose cells, both in size and number. The number of such cells often is deter-mined early in life; adult-onset obesity is largely a product of increase in adipose cell size. Weight gain results from increase in both adipose cell size and number. Adipose tissue may be deposited in large quantities in the subcutaneous layer of the abdominal wall or the viscera. Generally, males tend to have central visceral fat distribution,

1	size and number. Adipose tissue may be deposited in large quantities in the subcutaneous layer of the abdominal wall or the viscera. Generally, males tend to have central visceral fat distribution, whereas females more often have a peripheral fat distribution. Central or visceral fat distri-bution is associated with metabolic diseases such as diabetes, hypertension, and the metabolic syndrome.Concurrent Medical and Social ProblemsRaised BMI is a major risk factor for diseases such as cardio-vascular disease (mainly heart disease and stroke), which were the leading cause of death in 2012, diabetes, osteoarthritis, some cancers (including endometrial, breast, ovarian, prostate, liver, gallbladder, kidney, and colon).18 The risk for these conditions increases with increases in BMI. Childhood obesity is asso-ciated with a higher chance of obesity, premature death, and disability in adulthood. In addition to increased future risks, children with obesity experience sleep apnea, increased

1	obesity is asso-ciated with a higher chance of obesity, premature death, and disability in adulthood. In addition to increased future risks, children with obesity experience sleep apnea, increased risk of fractures, hypertension, early markers of cardiovascular disease, insulin resistance, and psychological effects.The severely obese patient typically presents with multiple chronic and weight-related problems or comorbidities/comorbid conditions. These include degenerative joint disease, low back pain, hypertension, obstructive sleep apnea, gastroesophageal reflux disease (GERD), cholelithiasis, T2DM, dyslipidemia, asthma, hypoventilation syndrome of obesity, right-sided heart failure, migraine headaches, pseudotumor cerebri, venous stasis ulcers, deep venous thrombosis (DVT), fungal skin rashes, skin abscesses, stress urinary incontinence, infertility, dysmenorrhea, depression, and large abdominal wall hernias.There are anatomical, metabolic, and physiological effects of obesity

1	skin rashes, skin abscesses, stress urinary incontinence, infertility, dysmenorrhea, depression, and large abdominal wall hernias.There are anatomical, metabolic, and physiological effects of obesity through which this excess adiposity leads to disease risk factors and chronic diseases themselves (Fig. 27-3).22 Subcu-taneous adipose tissue holds most of the stored lipid at a variety of anatomical sites while visceral adipose tissue is a smaller storage compartment with omental and mesenteric fat mechanistically linked to some of the metabolic disturbances and adverse con-sequences outcomes associated with obesity.25,26 Adipose tissue surrounds and compresses the renal parenchyma and may contrib-ute to the hypertension frequently observed in patients who are obese.27 Obesity is frequently accompanied by an increase in pha-ryngeal soft tissues, which can lead to obstructive sleep apnea.28 Excess adiposity also imposes a significant mechanical load on joints, making it a risk factor for

1	accompanied by an increase in pha-ryngeal soft tissues, which can lead to obstructive sleep apnea.28 Excess adiposity also imposes a significant mechanical load on joints, making it a risk factor for the development of osteoarthri-tis.29 An increase in intraabdominal pressure likely accounts for the elevated risks of gastroesophageal reflux disease (GERD), Barrett’s esophagus, and esophageal adenocarcinoma among those with obesity.30 Chronic overactivity of the sympathetic ner-vous system is present in some patients with obesity and may contribute to pathophysiological processes, including high blood pressure.27 Obesity is also associated with an increased preva-lence of mood, anxiety, and other psychiatric disorders, especially Brunicardi_Ch27_p1167-p1218.indd 117023/02/19 2:20 PM 1171THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27ä Adiposity˜ Lipid production˜ Activity ofthe sympatheticnervous system˜ Activity of therenin–angiotensin–aldosterone systemMechanical stress˜ Adipokine

1	SURGICAL MANAGEMENT OF OBESITYCHAPTER 27ä Adiposity˜ Lipid production˜ Activity ofthe sympatheticnervous system˜ Activity of therenin–angiotensin–aldosterone systemMechanical stress˜ Adipokine synthesis˜ Adipose tissuemacrophages and otherinflammatory cells˜ Proinflammatorycytokines˜ Pharyngealsoft tissue˜ Mechanicalload on joints˜ Intraabdominalpressure˜ InsulinType 2 diabetesNonalcoholicfatty liver diseaseSteatohepatitisCirrhosisCoronaryartery diseaseObstructivesleep apneaOsteoarthritisGastroesophagealreflux diseaseBarrett’s esophagusEsophagealadenocarcinomaCongestive heart failureStrokeChronic kidney diseaseImpaired insulinsignaling and˜ insulin resistanceLipotoxicityDyslipidemiaSystemic andpulmonaryhypertensionRenalcompressionHydrolysis oftriglyceridesRelease offree fatty acidsFigure 27-3. Pathways through which obesity leads to major risk factors and common chronic diseases. Common chronic diseases are shown in red boxes. The dashed arrows indicate an indirect association.

1	27-3. Pathways through which obesity leads to major risk factors and common chronic diseases. Common chronic diseases are shown in red boxes. The dashed arrows indicate an indirect association. (Reproduced with permission from Heymsfield SB, Wadden TA: Mechanisms, Pathophysiology, and Management of Obesity, N Engl J Med. 2017 Jan 19;376(3):254-266.)among persons with severe obesity and in those seeking bariatric surgery.31-33 Decreased quality of life also results due to severe obesity. Most patients seeking surgical treatment of severe obe-sity do so because of the medical issues they face from comorbid conditions or the decreased quality of life they are experiencing as a result of severe obesity.MEDICAL MANAGEMENT OF OBESITYTreatments should be aligned with the severity of obesity, asso-ciated comorbid conditions, and the individual’s functional limitations. There are guidelines available to evaluate an indi-vidual’s health risks and potential treatment options.34,35 Three main

1	asso-ciated comorbid conditions, and the individual’s functional limitations. There are guidelines available to evaluate an indi-vidual’s health risks and potential treatment options.34,35 Three main treatment options exist with sufficient evidence-based support: lifestyle intervention, pharmacotherapy, and bariatric surgery.Lifestyle InterventionLifestyle interventions designed to modify eating behaviors and physical activity are the first option for weight manage-ment, given their low cost and low risk.35 Behavioral therapy, the core of any lifestyle intervention, provides patients with techniques for adopting dietary and activity recommendations. Among these recommendations are regular recording of food intake, physical activity, and weight. Patients review their progress approximately weekly with a trained interventionist Brunicardi_Ch27_p1167-p1218.indd 117123/02/19 2:20 PM 1172SPECIFIC CONSIDERATIONSPART II01020304050Percentage of

1	Patients review their progress approximately weekly with a trained interventionist Brunicardi_Ch27_p1167-p1218.indd 117123/02/19 2:20 PM 1172SPECIFIC CONSIDERATIONSPART II01020304050Percentage of Participants6070809010010%5%High-lntensityLifestyleInterventionPharmacotherapyInterventionLook AHEADDPPTeixeira, et aI.PlaceboOrlistatLorcaserinLiraglutidePhentermine-topiramateNaltrexone-bupropionFigure 27-4. 22Percent weight loss at 1 year with intensive lifestyle interventions or pharmacotherapy combined with lower level lifestyle. Shown are the percentages of participants in randomized, controlled trials who had weight loss of at least 5% or at least 10% of their initial weight at 1 year after intensive lifestyle intervention or pharmacotherapy that typically was combined with lower-intensity lifestyle. Percentages shown are cumulative; the percentage of participants who lost at least 5% of their initial weight includes the percentage who lost at least 10%. Additional data on the

1	lifestyle. Percentages shown are cumulative; the percentage of participants who lost at least 5% of their initial weight includes the percentage who lost at least 10%. Additional data on the percentage of participants with weight loss at 1 year of at least 15% of their initial weight were available for the Look AHEAD36 study (16%), the DPP39 trial (11%), liraglutide40 (14%), phentermine–topiramate (32%), and naltrexone–bupropion (14%). (Reproduced with permis-sion from Heymsfield SB, Wadden TA: Mechanisms, Pathophysiology, and Management of Obesity, N Engl J Med. 2017 Jan 19;376(3):254-266.)who provides support and encouragement, help setting goals, and problem-solving instructions.35 This type of comprehen-sive program results in a mean weight loss of 5% to 8%, and approximately 60% to 65% of patients lose 5% or more of initial weight. The Look AHEAD study randomized 5145 adults with obesity to either an intensive lifestyle intervention (ILI) or to a diabetes support group and

1	to 65% of patients lose 5% or more of initial weight. The Look AHEAD study randomized 5145 adults with obesity to either an intensive lifestyle intervention (ILI) or to a diabetes support group and education group (DSE) to assess the impact on weight loss, T2DM, and cardiovascular outcomes. At 1 year, the intensive intervention group lost an average of 8.6% initial weight compared to 0.7% in the support and educa-tion group. As shown in Fig. 27-4, 68% of participants in the Look AHEAD study lost at least 5% of their initial weight, and 37% of these participants lost at least 10%. Also at 1 year, par-ticipants undergoing the more intensive program experienced improved cardiovascular risk factors and glycemic control.36At 4 years, participants in the intensive intervention group experienced more weight loss (−6.15% ILI compared to −0.88% DSE), better glycemic control, fitness, and an improvement in cardiovascular risk factors.37 Nevertheless, the beneficial clinical effects of the

1	more weight loss (−6.15% ILI compared to −0.88% DSE), better glycemic control, fitness, and an improvement in cardiovascular risk factors.37 Nevertheless, the beneficial clinical effects of the improved weight loss achieved with intensive lifestyle intervention did not reduce morbidity and mortality associated with cardiovascular disease after 9.6 years when the Look AHEAD study was stopped due to futility for that primary endpoint.38 Figure 27-4 shows a comparison of several lifestyle intervention trials (Look AHEAD, the Diabetes Prevention Program [DPP] trial,39 and the trial reported by Teixeira et al40) for >5% and >10% weight loss outcomes. These trials, specifically, were selected because they were judged to be good quality by the Guidelines (2013) for the Management of Overweight and Obesity in Adults and because the trial data were reported as categorical weight loss. Categorical weight loss data from the DPP trial were provided by the DPP Research Group to the authors of the

1	Obesity in Adults and because the trial data were reported as categorical weight loss. Categorical weight loss data from the DPP trial were provided by the DPP Research Group to the authors of the review.22In summary, multidisciplinary lifestyle intervention and weight-management programs are viable and potentially cost-effective treatment options in overweight or obese patients with or without T2DM. Such approaches, however, often fail to achieve durable weight loss of more than 5% to 10%, so they are not effective enough for the severely obese. Importantly, lifestyle and medical approaches do not appear to improve car-diovascular outcomes in studies so far. Thus, further research is needed to evaluate the role for current medical and lifestyle therapeutic regimens for obesity and T2DM, including compari-sons to surgical interventions. Certainly, and at the very least, these approaches are important adjuncts to bariatric surgery.PharmacotherapyMedications may be considered as an

1	including compari-sons to surgical interventions. Certainly, and at the very least, these approaches are important adjuncts to bariatric surgery.PharmacotherapyMedications may be considered as an adjunct to lifestyle modi-fication in adults who have a BMI of 30 or higher or a BMI of 27 to 29 with at least one obesity-related condition.41 Phar-macotherapy and lifestyle intervention together lead to addi-tive weight losses and should be used together and may also be helpful in facilitating the maintenance of reduced weight.34,41,42 Phentermine, the most widely prescribed weight-management medication in the United States, is a sympathomimetic amine that was approved by the FDA in 1959 for short-term use of fewer than 3 months long.41 There are now five newer FDA-approved medications for long-term weight management that include three single drugs and two combination drugs. In 1-year pivotal trials, total weight losses for the three single therapies (orlistat, lorcaserin, and liraglutide),

1	weight management that include three single drugs and two combination drugs. In 1-year pivotal trials, total weight losses for the three single therapies (orlistat, lorcaserin, and liraglutide), the effects of which are mediated by different mechanisms, ranged from 5.8% to 8.8% of initial body weight.41,43-45 The two combination medications (phentermine–topiramate and naltrexone–bupropion) include drugs that act on neural weight-loss mechanisms.46,47 In 1-year pivotal trials, total weight loss for these combination drugs ranged from 6.4% to 9.8% of initial body weight.These medications, when prescribed with lifestyle inter-ventions, produce additional weight loss relative to placebo ranging from approximately 3% of initial weight for orlistat and lorcaserin to 9% for the higher-dose phentermine plus topi-ramate–extended release at 1 year. The proportion of patients achieving clinically meaningful (at least 5%) weight loss ranges from 37% to 47% for lorcaserin, 35% to 73% for orlistat,

1	plus topi-ramate–extended release at 1 year. The proportion of patients achieving clinically meaningful (at least 5%) weight loss ranges from 37% to 47% for lorcaserin, 35% to 73% for orlistat, and 67% to 70% for higher-dose phentermine plus topiramate–extended release. All three of these medications produce greater improvements in cardiometabolic risk factors than placebo, Brunicardi_Ch27_p1167-p1218.indd 117223/02/19 2:20 PM 1173THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27but none has been shown to reduce cardiovascular morbid-ity or mortality. There is limited data for the long-term safety and efficacy of these medications, and some of these drugs may increase heart rate43 or attenuate expected blood pressure reductions.47 In addition, completed trials of hard cardiovascular disease outcomes (heart attack and stroke) in patients treated with these medications have yet to be published, except in the case of liraglutide.43 Figure 27-4 shows a comparison of weight loss outcomes

1	outcomes (heart attack and stroke) in patients treated with these medications have yet to be published, except in the case of liraglutide.43 Figure 27-4 shows a comparison of weight loss outcomes for these medications. The median percentages of participants who had a weight loss of at least 5% or 10% with each of five medications approved for long-term weight man-agement are from a meta-analysis by Khera et al.48In summary, medications approved for long-term obesity treatment, when used as an adjunct to lifestyle intervention, lead to greater mean weight loss and an increased likelihood of achieving clinically meaningful 1-year weight loss compared to placebo.42 Yet weight loss medications are underutilized, likely due to several factors. First, patients are often disappointed by moderate weight loss. Second, there are requirements to pay a substantial portion of costs, which may lead to short-term rather than longer-term use. Third, there remain concerns about medi-cation safety.

1	weight loss. Second, there are requirements to pay a substantial portion of costs, which may lead to short-term rather than longer-term use. Third, there remain concerns about medi-cation safety. Finally, weight regain is common after termina-tion of drug treatment, which is discouraging to both patients and their providers.22,42Barriers to TreatmentOnly a small fraction of patients for whom these medical treat-ments or bariatric surgery are indicated actually pursue and receive them. Past studies have estimated that 1% or fewer of those people with severe obesity who could consider bariatric surgery ever do so. Barriers to general obesity care include the slow recognition among providers that obesity requires long-term management, inadequate physician training in nutrition and obesity, limited reimbursement for the full range of treat-ments, lack of more effective and accessible lifestyle programs, and limited referrals of patients with severe obesity to expe-rienced surgeons.22 Lack

1	reimbursement for the full range of treat-ments, lack of more effective and accessible lifestyle programs, and limited referrals of patients with severe obesity to expe-rienced surgeons.22 Lack of knowledge about the more recent outcomes of bariatric surgery may also play a contributing role.CANDIDATES FOR BARIATRIC SURGERYIndicationsThere has been significant procedure evolution over the last several years indicating an ongoing major shift in bariatric procedures both in the United States and worldwide13 (Fig. 27-5). According to a 2016 report from the American Society of Metabolic and Bariatric Surgery (ASMBS), the two most common procedures in the United States are RYGB and SG, accounting for approximately 25% to 30% and 50% to 60%, respectively, of annual cases.49,50 The utilization of LAGB has declined dramatically to under 10% of cases, and the malabsorptive procedure BPD with or without DS is utilized in less than 1% to 2% of cases (see Fig. 27-2). All of these procedures were

1	LAGB has declined dramatically to under 10% of cases, and the malabsorptive procedure BPD with or without DS is utilized in less than 1% to 2% of cases (see Fig. 27-2). All of these procedures were defined by the Centers for Medicare & Medicaid Services (CMS) as standard approved procedures, noting that SG coverage is based on the discretion of regional carriers throughout the United States.The indications for performing bariatric surgery in class II and class III obesity still remain as described in the National Institutes of Health (NIH) Consensus Conference of 1991, and a summary of the broad selection criteria are shown in Table 27-2.51 In 2016, the second Diabetes Surgery Summit (DSS-II) published guidelines indicating that metabolic surgery 2should also be considered for patients with T2DM and BMI of 30 to 34.9 kg/m2 (class I obesity) if blood sugar is inadequately controlled despite optimal medication treatment.16 In addition, these guidelines recommended that the BMI

1	with T2DM and BMI of 30 to 34.9 kg/m2 (class I obesity) if blood sugar is inadequately controlled despite optimal medication treatment.16 In addition, these guidelines recommended that the BMI threshold for metabolic surgery (surgery for diabetes as the indication) should be reduced by 2.5 kg/m2 for Asian populations at risk.The NIH criteria for bariatric surgery do not set guidelines or limits for age, and surgical practice varies widely. The pediatric obesity epidemic is both increasing and also driving the adult epidemic, and a growing proportion of younger patients are potentially eligible for bariatric surgery. For young patients, there are concerns about assent to surgery and compliance with and adherence to postoperative lifestyle changes, but there are also some emerging data that suggest intervening earlier in the disease process may lead to improved reversal of comorbid conditions compared to adults.52 In addition, there is a longer period of postoperative benefit in terms

1	suggest intervening earlier in the disease process may lead to improved reversal of comorbid conditions compared to adults.52 In addition, there is a longer period of postoperative benefit in terms of improved quality of life and prevention of or reduction in the emotional, social, and physical consequences of obesity (see “Bariatric Procedures in Adolescents”). Alternatively, older patients are more likely to have more numerous and debilitating comorbid conditions and thus have an immediate benefit in quality of life but not necessarily enhanced longevity. There is also some concern that recovery from potential complications is impaired in patients over the age of 65. Most studies in older patients have focused on RYGB and older restrictive procedures with limited follow-up. The results of more recent studies in older patients are generally equivocal in terms of any increased risk of morbidity and mortality or any difference in weight outcomes compared to younger adults.53-55 One

1	of more recent studies in older patients are generally equivocal in terms of any increased risk of morbidity and mortality or any difference in weight outcomes compared to younger adults.53-55 One study has shown that the older patient population, especially those few patients older than age 70 undergoing bariatric surgery, did have an increased risk of mortality and morbidity after RYGB.56 Also, a 2016 study from the Utah Obesity group found that RYGB is protective against mortality even for older patients and also reduces the age-related increase in mortality observed in severely obese individuals not undergoing surgery.57ContraindicationsMedical issues that preclude patients from being good surgi-cal candidates include American Society of Anesthesiologists (ASA) class IV disease of a nature that makes surgical therapy extraordinarily high risk. Psychological instability or the inabil-ity to understand the implications of the proposed operation and what changes will result from it

1	nature that makes surgical therapy extraordinarily high risk. Psychological instability or the inabil-ity to understand the implications of the proposed operation and what changes will result from it in terms of the patient’s life-style are also contraindications. Known and documented active drug or alcohol addiction is a contraindication to surgery (see Table 27-2). Tobacco use should be completely avoided by bar-iatric patients at all times, and smoking cessation should occur 6 weeks prior to surgery. After surgery smoking increases risks of poor wound healing, anastomotic ulcers, and impaired health.58 A poorly controlled eating disorder, especially bulimia, is also a contraindication to surgery. Nonambulatory status is a relative contraindication to surgery and is associated with increased sur-gical risk,59 especially if the obesity is so severe that the patient cannot normally do self-care or would not likely be able to do so after surgery. In addition to excessive morbidity,

1	sur-gical risk,59 especially if the obesity is so severe that the patient cannot normally do self-care or would not likely be able to do so after surgery. In addition to excessive morbidity, the placement of these individuals in care facilities postoperatively for recovery is often impossible due to their size and limitations of physical ability. Finally, lack of sufficient social support or an extremely poor or unsupportive home environment can be contraindications to surgical care, since such environmental factors are important to optimize outcomes once discharged from the hospital.Brunicardi_Ch27_p1167-p1218.indd 117323/02/19 2:20 PM 1174SPECIFIC CONSIDERATIONSPART IIABCDEFGHFigure 27-5. Bariatric surgery procedure evolution. A. Horizontal gastroplasty; B. vertical banded gastroplasty; C. Roux-en-Y gastric bypass; D. transected Roux-en-Y gastric bypass; E. laparoscopic adjustable gastric band; F. biliopancreatic diversion; G. biliopancreatic diversion with duodenal switch; H.

1	C. Roux-en-Y gastric bypass; D. transected Roux-en-Y gastric bypass; E. laparoscopic adjustable gastric band; F. biliopancreatic diversion; G. biliopancreatic diversion with duodenal switch; H. vertical sleeve gastrectomy. (Modified with permission from Arterburn DE, Courcoulas AP: Bariatric surgery for obesity and metabolic conditions in adults, BMJ. 2014 Aug 27;349:g3961.)Brunicardi_Ch27_p1167-p1218.indd 117423/02/19 2:20 PM 1175THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27Table 27-2Patient selection criteria for bariatric surgeryFACTORCRITERIAWeight (adults)BMI ≥40 kg/m2 with no comorbid conditionsBMI ≥35 kg/m2 with obesity-associated comorbidityWeight loss historyFailure of previous nonsurgical attempts at weight reduction, including nonprofessional programsCommitmentExpectation that patient will adhere to postoperative careFollow-up visits with physician(s) and team membersRecommended medical management, including use of dietary supplementsInstructions regarding any

1	that patient will adhere to postoperative careFollow-up visits with physician(s) and team membersRecommended medical management, including use of dietary supplementsInstructions regarding any recommended procedures or testsContraindications/exclusionsProhibitive surgical risk, ASA IVReversible endocrine or other disorders that can cause obesityCurrent drug or alcohol misuseUncontrolled, severe psychiatric illnessUncontrolled, severe bulimiaLack of comprehension of risks, benefits, expected outcomes, alternatives, and lifestyle changesData from Mechanick JI, Youdim A, Jones DB, et al: Clinical practice guidelines for the perioperative nutritional, metabolic, and nonsurgical support of the bariatric surgery patient—2013 update: cosponsored by American Association of Clinical Endocrinologists, The Obesity Society, and American Society for Metabolic & Bariatric Surgery, Obesity (Silver Spring). 2013 Mar;21 Suppl 1:S1-S27.MECHANISM OF ACTION OF BARIATRIC AND METABOLIC SURGERYOverviewThere

1	The Obesity Society, and American Society for Metabolic & Bariatric Surgery, Obesity (Silver Spring). 2013 Mar;21 Suppl 1:S1-S27.MECHANISM OF ACTION OF BARIATRIC AND METABOLIC SURGERYOverviewThere is not yet a clear understanding as to how various bariatric procedures exert their effects on weight loss, metabolism, and glycemic control. Much effort is currently being devoted to gaining a better understanding of these specific mechanisms. A review of what is known from published animal and human studies about mechanisms related the three most common surgical proce-dures is shown in Fig. 27-6.17 A few interim, summary statements can be drawn from this available data. First, neither LRYGB nor SG can be thought of as primarily “restrictive procedures,” and there are changes in behavior and physiology that likely help to maintain the new reduced body weight that are not observed after nonsurgically induced weight loss. LAGB appears to be more dependent on gastric restriction as both the

1	physiology that likely help to maintain the new reduced body weight that are not observed after nonsurgically induced weight loss. LAGB appears to be more dependent on gastric restriction as both the behavioral changes and changes in gut hormone secretion are much less dramatic. Second, both LRYGB and SG are associated with metabolic improvements that are different from those that are caused by weight loss alone, and these mechanisms remain under current active study. For LAGB, the metabolic effects are mostly due to the impact of the resulting weight loss. This growing understanding of the physiol-ogy of these procedures points away from the older, classic 3anatomic classifications of “restrictive” versus “malabsorptive” procedures. This new conceptual approach has important implica-tions for future studies of how bariatric surgery exerts its effects. Earlier hypotheses for the mechanism of action of LRYGB have been classified into either the “foregut hypothesis” or “hindgut

1	for future studies of how bariatric surgery exerts its effects. Earlier hypotheses for the mechanism of action of LRYGB have been classified into either the “foregut hypothesis” or “hindgut hypothesis.”60 The foregut hypothesis states that improvements after LRYGB come from the bypassing of the upper small intestine that results in the reduction of nutrient-dependent hormonal actions that would normally impair glucose tolerance.61 The hindgut hypothesis states instead that the key events are the result of more rapid delivery of nutrients to the distal small intestine causing effects such as increased GLP-1/PYY secretion and the ileal brake.62 Now, the more recently recognized and common metabolic effects of SG and LRYGB may indicate directions for study away from this foregut/hindgut distinction, as the SG does not bypass the foregut or induce nutrients further down in the intestine.Mechanisms of Bariatric Surgery (Weight Loss)Certainly, one component mechanism by which RYGB pro-duces

1	as the SG does not bypass the foregut or induce nutrients further down in the intestine.Mechanisms of Bariatric Surgery (Weight Loss)Certainly, one component mechanism by which RYGB pro-duces weight loss is related to reduced caloric intake and malab-sorption brought about by a smaller gastric volume and bypass of the proximal small bowel, so weight loss following LAGB and SG may be explained, at least in part and early on, by gas-tric restriction63 and resulting reduced food intake. Aside from anatomic changes induced by surgery, there are also changes in physiology that may affect food preferences and energy expen-diture. Gastric emptying and insulin sensitivity increases follow-ing RYGB and SG.63 Functional magnetic resonance imaging has demonstrated a decreased neuronal activation of the food reward-related centers in response to high-calorie foods follow-ing RYGB.64 Neural signaling may be altered and stretch sensi-tive vagal endings in the new pouch and Roux limb, resulting in a

1	food reward-related centers in response to high-calorie foods follow-ing RYGB.64 Neural signaling may be altered and stretch sensi-tive vagal endings in the new pouch and Roux limb, resulting in a feeling of early satiety.65 Hormonal changes are also evi-dent, and in a number of studies looking at the effect of RYGB on ghrelin levels, results are conflicting. Changes in intestinal microbiota is another area of active study.66 Individuals with obesity have different gut flora compared to nonobese subjects. The Firmicutes (mainly Lactobacillus and Clostridium species) to Bacteroidetes ratio (Bacteroides or Prevotella species) is elevated in obese subjects. Following gastric bypass, the Fir-micutes group decrease while Bacteroides/Prevotella increase at 3 and 6 months intervals.67 Bacteria transplant provides some of the benefits of gastric bypass surgery without the surgery.68 It has also been shown that administering oral lactobacillus post-RYGB leads to increased weight loss; this

1	transplant provides some of the benefits of gastric bypass surgery without the surgery.68 It has also been shown that administering oral lactobacillus post-RYGB leads to increased weight loss; this indicates benefits of changing gut microbiota to induce weight loss.69Serum bile acid levels also increase following gastric bypass.70 Gastric bypass diverts undiluted bile acids to the distal bowel. Bile acids activate protein-coupled receptor TGR5 pres-ent in L cells responsible for GLP-1 secretion. They also activate FXR (farsenoid-X receptor) in the jejunum, which regulates lipid and glucose metabolism.71 Furthermore, bile acids lead to rapid clearance of triglycerides.72 Bariatric surgery alters bile acid enterohepatic circulation in favor of weight loss and resolution of nonalcoholic steatohepatitis (NASH).73 Ryan et al demonstrated in a study on mice that the therapeutic value of SG is not limited to mechanical restriction but to an increase in circulating bile acids and associated

1	(NASH).73 Ryan et al demonstrated in a study on mice that the therapeutic value of SG is not limited to mechanical restriction but to an increase in circulating bile acids and associated changes to gut microbiota.74 Hollanda et al studied two cohorts of patients: those who lost more than 50% of their excess weight compared to those who did not. This group suggested that ghrelin and GLP-1 may be mediators of success-ful weight loss as those levels increased, while PYY and GLP-2, Brunicardi_Ch27_p1167-p1218.indd 117523/02/19 2:20 PM 1176SPECIFIC CONSIDERATIONSPART IILRYGBLAGBSGLipidElevated HDLReduced triglyceridesReduced total cholesterol, LDLElevated HDLReduction in triglycerides not as dramatic as LRYGB or SGElevated HDLReduced triglyceridesGlucose homeostasisImproved fasting blood glucose and insulin sensitivity, prior to weight lossImprovements are slower and not as dramatic as after SG or LRYGBImproved fasting blood glucose and insulin sensitivity, prior to weight lossRole of

1	and insulin sensitivity, prior to weight lossImprovements are slower and not as dramatic as after SG or LRYGBImproved fasting blood glucose and insulin sensitivity, prior to weight lossRole of gastric restrictionHas not yet been directly testedFailure of band leads to less gastric restriction and less weight lossGastric restriction is not the critical factor preventing hyperphagiaGastric emptyingFew published studiesNo overall change in gastric emptying rate; Emptying rate of proximal pouch created by band is enhancedMost papers show increaseEnergy expenditureControversialNot reportedUnchanged, but only reported in one studyLeptinCirculating leptin levels lower than expected for body weightChanges to leptin sensitivity not testedPlasma leptin reduced, as expected for body weight; Changes to leptin sensitivity not testedCirculating leptin levels lower than expected for body weight; Body weight changes not driven by changes to leptin sensitivityGhrelinReduced total ghrelin;

1	Changes to leptin sensitivity not testedCirculating leptin levels lower than expected for body weight; Body weight changes not driven by changes to leptin sensitivityGhrelinReduced total ghrelin; Controversial, but no change in acyl-ghrelin levelsIncreased circulating ghrelinReduced total ghrelin; Contro-versial, but no change in acyl-ghrelin levelsCCKNo changeNo changeNot measuredGLP=1 (postprandial)Weight loss-independent postprandial increaseIncreased circulating GLP-1 but much less than RYGB or SGWeight loss-independent increase comparable to LRYGBPYY (postprandial)Increased postprandial PYY levels; Reduced body weight loss in PYY knockout miceNo changeIncreased postprandial PYY levels, comparable to levels after LRYGBBile acidsIncreased plasma bile acidsNot reportedIncreased plasma bile acidsDiet changeDecreased fat intake, more fruits and vegetablesDecrease bread intake and increase in caloric liquids; Greater fat intake and fewer fruits/vegetables than RYGBDecreased fat intake,

1	acidsDiet changeDecreased fat intake, more fruits and vegetablesDecrease bread intake and increase in caloric liquids; Greater fat intake and fewer fruits/vegetables than RYGBDecreased fat intake, similar to LRYGBFood IntoleranceSome dumping syndrome, usually well-tolerated More persistent and problematic than LRYGB; Mainly vomitingLittle or noneFigure 27-6. Mechanisms of effect: comparison of LRYGB, LAGB, and SG. (Reproduced with permission from Stefater MA, Wilson-Pérez HE, Chambers AP, et al: All bariatric surgeries are not created equal: insights from mechanistic comparisons, Endocr Rev. 2012 Aug;33(4):595-622.)Brunicardi_Ch27_p1167-p1218.indd 117623/02/19 2:20 PM 1177THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27gut mass and hypertrophy (citrulline), and the bile acid effect on fibroblast growth factor-19 (FGF-19) appear to have no effect on weight loss.75 In summary, there exists a complex relationship between dietary changes, bile flow changes, altered hormonal milieu and the

1	growth factor-19 (FGF-19) appear to have no effect on weight loss.75 In summary, there exists a complex relationship between dietary changes, bile flow changes, altered hormonal milieu and the gut microbiota that is not yet completely well characterized as it relates to weight loss after bariatric surgery.Mechanisms of Metabolic Surgery (Diabetes Improvement)Understanding the basic mechanism(s) of diabetes improvement following bariatric surgery is an important area of intensive study. Recently published data on worldwide trends in diabetes indicate that the number of adults with diabetes has increased from 108 million in 1980 to 422 million in 2014,76 the major-ity with T2DM. In the United States, diabetes is the number two cause of hospitalizations in adults age 18 years or older, accounting for approximately 11% of all hospital admissions. With no medical cure, the natural course of diabetes is charac-terized by progressive β-cell failure and development of micro-vascular and

1	accounting for approximately 11% of all hospital admissions. With no medical cure, the natural course of diabetes is charac-terized by progressive β-cell failure and development of micro-vascular and macrovascular complications, leading to renal failure, blindness, amputation, and death due to cardiovascular disease (CVD). Bariatric surgery has been renamed metabolic surgery for T2DM treatment and has emerged as an effective tool for control of hyperglycemia.77-86More than 20 years ago Pories et al found that bariatric surgery rapidly normalized blood glucose levels in people with obesity and T2DM, and 10 years later the majority remained dis-ease free.87 He suggested that caloric restriction played a role but that there were likely other factors such as proximal intestinal nutrient exclusion, rapid distal gut nutrient delivery, and the role of gut hormones that would require further investigation. The findings of T2DM improvement and remission after bariatric surgery have now been

1	rapid distal gut nutrient delivery, and the role of gut hormones that would require further investigation. The findings of T2DM improvement and remission after bariatric surgery have now been widely replicated by others, and there is evidence that bariatric surgery prevents or delays incident cases of T2DM. Much work has been done to investigate these spe-cific physiological mechanisms underlying the beneficial gly-cemic effects of bariatric surgery, but they remain incompletely understood. Candidate hypotheses include changes in bile acid metabolism, nutrient sensing and glucose utilization, intestinal adaptation, incretins, possible anti-incretin(s), and the intestinal microbiome. These physiologic and molecular changes lead to reduced hepatic glucose production, increased glucose uptake in tissues, improved insulin sensitivity, and enhanced β-cell func-tion. A schematic of these potential mechanisms of improved glycemic control is shown in Fig. 27-7.It is likely that several of

1	in tissues, improved insulin sensitivity, and enhanced β-cell func-tion. A schematic of these potential mechanisms of improved glycemic control is shown in Fig. 27-7.It is likely that several of these individual factors, acting together and with different impact based on the specific surgical pro-cedure, are responsible for postoperative glycemic improvement. Work in this area is actively ongoing, and genomic, metabolomic, and gut microbiome studies will likely enhance the understanding of these changes. This may potentially lead to identifying novel pathways and potential therapeutic targets to replace bariatric pro-cedures by equally effective, but less invasive, new treatments for obesity-related T2DM. In other words, understanding mechanisms of glycemic improvement after bariatric surgery may allow for the development of treatments to “bypass the bypass.”88PREOPERATIVE ISSUESPreoperative PreparationPatient selection for surgery should be based on a multidisci-plinary team

1	surgery may allow for the development of treatments to “bypass the bypass.”88PREOPERATIVE ISSUESPreoperative PreparationPatient selection for surgery should be based on a multidisci-plinary team approach. All patients should undergo preoperative evaluation for obesity-related comorbidities and causes of obe-sity, with special attention directed to factors that could affect candidacy for bariatric surgery89 (Table 27-3).The preoperative assessment of the patient for bariatric surgery must include input from the nutritionist as an important independent evaluation. Careful assessment of the patient’s eat-ing habits, knowledge, self-awareness, and insight are important. An estimation of the patient’s motivation to change eating habits is important. The nutritionist should have at least one assessment session with the patient and an educational session preoperatively once the decision to proceed with surgery has been determined. The operation to be performed requires specific nutritional

1	session with the patient and an educational session preoperatively once the decision to proceed with surgery has been determined. The operation to be performed requires specific nutritional counseling and education. Psychological assessment is required by most programs and many insurance carriers with a goal of identifying potential contraindications to surgical intervention, such as poorly controlled psychiatric illness or active substance abuse, and identifying strategies to help with long-term weight management.90 There are published recommendations regarding the content of a mental health evaluation for bariatric surgery,91-93 but no consensus guidelines have been published. These evalua-tions are carried out by interview and questionnaires, which rely on clinical interviews including tests of personality or psycho-pathological conditions.94 More comprehensive evaluations also assess bariatric surgery knowledge, weight history, lifestyle hab-its, and potential barriers.91,92

1	tests of personality or psycho-pathological conditions.94 More comprehensive evaluations also assess bariatric surgery knowledge, weight history, lifestyle hab-its, and potential barriers.91,92 Psychological assessment in clinical practice may be inaccurate compared to independent evaluations for research purposes, as patients present themselves in the most favorable light in order to gain access to surgery.95Obstructive sleep apnea (OSA) is prevalent in over 90% of bariatric surgery candidates with approximately one-third undiagnosed.96-101 The Epworth Sleepiness Scale, a standard set of questions evaluating daytime sleepiness, is often used as a screening tool for OSA.38 As OSA is associated with increased risk of mortality102 (and in bariatric surgery patients, with adverse outcomes),59 routine preoperative screening with polysomnog-raphy should be considered.103 In addition, standard preopera-tive management of obese patients with OSA using continuous positive airway pressure

1	routine preoperative screening with polysomnog-raphy should be considered.103 In addition, standard preopera-tive management of obese patients with OSA using continuous positive airway pressure (CPAP) is recommended.104 Asthma and hypoventilation syndrome of obesity are other significant pulmonary diseases often requiring preoperative management. Hypoventilation syndrome of obesity is defined as resting arte-rial partial pressure of oxygen less than 55 mmHg and partial pressure of carbon dioxide greater than 47 mmHg, with accom-panying pulmonary hypertension and polycythemia. Pulmonary consultation is indicated for patients with hypoventilation syn-drome. Postoperative intensive care unit hospitalization, rarely used after bariatric surgery, may be indicated for these patients.Preoperative weight loss can reduce liver volume/size and may help improve the technical aspects of surgery in those people with extreme central obesity and an enlarged liver, and it is sometimes utilized as a

1	weight loss can reduce liver volume/size and may help improve the technical aspects of surgery in those people with extreme central obesity and an enlarged liver, and it is sometimes utilized as a practice-specific recommendation or requirement. Ten percent total body weight loss (TBWL) with energy-restricted diets has been associated with a reduction in hepatic volume,105 variable perceived and measured improved facility in operative technique,106,107 variable effects on short-term108-112 complication rates, and weight loss. Cirrhosis113 has been associated with poor outcomes following bariatric surgery, including progression to liver transplantation.114Preoperative glycemic control should be optimized using diet, physical activity, and medications, as needed. Reasonable targets for preoperative glycemic control include a hemoglo-bin A1c value of 6.5% to 7.0% or less, a fasting blood glucose Brunicardi_Ch27_p1167-p1218.indd 117723/02/19 2:20 PM 1178SPECIFIC

1	targets for preoperative glycemic control include a hemoglo-bin A1c value of 6.5% to 7.0% or less, a fasting blood glucose Brunicardi_Ch27_p1167-p1218.indd 117723/02/19 2:20 PM 1178SPECIFIC CONSIDERATIONSPART IIlevel of ≤110 mg/dL, and a 2-hour postprandial blood glucose concentration of ≤140 mg/dL.115 More liberal preoperative targets with higher A1c should be considered in patients with advanced comorbid conditions or long-standing diabetes where lower targets are not attainable. For patients with active GERD on medication, a preoperative screening upper endoscopy to rule out Barrett’s esophagus and to rule out intrinsic lesions of the stomach or duodenum is recommended. This is especially true for patients planning LRYGB, where the distal stomach and duodenum will be precluded from easy inspection postop-eratively. In addition, the presence of Barrett’s esophagus is a contraindication to SG, which is a reflux-inducing operation. The presence of a hiatal hernia detected on

1	from easy inspection postop-eratively. In addition, the presence of Barrett’s esophagus is a contraindication to SG, which is a reflux-inducing operation. The presence of a hiatal hernia detected on preoperative esoph-agogastroduodenoscopy will alert the surgeon for the need to perform intraoperative repair.Patients with a history of DVT or cor pulmonale should undergo a diagnostic evaluation for DVT. A prophylactic vena caval filter may present a greater risk than benefit in patients A Immediate impact of surgeryB Potential mediators/mechanismsC Effect on glucose homeostasisCaloric restritctionImproved ˜-cell function/functional ˜-cell massAltered bile acid/ FGF-19 signalingReduced hepatic and pancreatic triglyceridesRYGBRYGBSGReduced hepatic glucose productionIncreased glucose utilizationIncreased glucose effectivenessImproved insulin sensitivityAltered microbiomeReduced glucotoxicityWeight lossAltered GI nutrient-sensingAltered gut hormonesAltered neural signalingRapid emptying of

1	glucose effectivenessImproved insulin sensitivityAltered microbiomeReduced glucotoxicityWeight lossAltered GI nutrient-sensingAltered gut hormonesAltered neural signalingRapid emptying of nutrients into the small intestineIntestinal adaptation/Reprogramming of intestinal glucoseRYGBSGEnhanced nutrient/bile delivery to the mid/distal jejunum and ileumRYGBSGSGExclusion of the duodenum and proximal jejunum and ileumRYGBRemoval of the stomach fundusSGFigure 27-7. Schematic of potential mechanisms of improved glycemic control after LRYGB and SG. A. Immediate effects of RYGB and SG due to anatomical changes. B. Potential mediators/mechanisms involved. Cross talk occurs among these factors. C. Effects on glucose homeostasis. (Reproduced with permission from Batterham RL, Cummings DE: Mechanisms of Diabetes Improvement Following Bariatric/Metabolic Surgery, Diabetes Care. 2016 Jun;39(6):893-901.)Brunicardi_Ch27_p1167-p1218.indd 117823/02/19 2:20 PM 1179THE SURGICAL MANAGEMENT OF

1	of Diabetes Improvement Following Bariatric/Metabolic Surgery, Diabetes Care. 2016 Jun;39(6):893-901.)Brunicardi_Ch27_p1167-p1218.indd 117823/02/19 2:20 PM 1179THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27with a history of prior pulmonary embolism (PE) or DVT given the risks of filter-related complications including thrombosis. The overall risk of venous thromboembolism (VTE) after sur-gery is 0.42%, and over 70% of these events occur after hospital discharge, most within 30 days after surgery.116 The risk of VTE is greater in patients undergoing RYGB than in those undergoing LAGB and is more frequent following open surgery. Patients with a VTE event tend to be male, older, and have higher BMIs; they are also more likely to have a history of VTE.116 The risk of VTE is greater in patients with an inferior vena cava filter (hazard ratio [HR] 7.66, 95% CI 4.55–12.91),116 and there is evidence suggest-ing that prophylactic inferior vena caval (IVC) filter placement before bariatric

1	with an inferior vena cava filter (hazard ratio [HR] 7.66, 95% CI 4.55–12.91),116 and there is evidence suggest-ing that prophylactic inferior vena caval (IVC) filter placement before bariatric surgery does not prevent PE and may lead to addi-tional morbidity, which may outweigh its use.117,118Candidates for bariatric surgery should avoid pregnancy preoperatively and for 12 to 18 months postoperatively and Table 27-3Preoperative checklist for bariatric surgery• Complete for History & Physical (H&P) (obesity-related comorbidities, causes of obesity, weight/BMI, weight loss history, commitment, and exclusions related to surgical risk)• Routine labs (including fasting blood glucose and lipid panel, kidney function, liver profile, lipid profile, urine analysis, prothrombin time/INR, blood type, CBC)• Nutrient screening with iron studies, B12 and folic acid (RBC folate, homocysteine, methylmalonic acid optional), and 25-vitamin D (vitamins A and E optional); consider more extensive testing

1	CBC)• Nutrient screening with iron studies, B12 and folic acid (RBC folate, homocysteine, methylmalonic acid optional), and 25-vitamin D (vitamins A and E optional); consider more extensive testing in patients undergoing malabsorptive procedures based on symptoms and risks• Cardiopulmonary evaluation with sleep apnea screening (ECG, CXR, echocardiography if cardiac disease or pulmonary hypertension suspected; DVT evaluation if clinically indicated)• GI evaluation (H pylori screening in high-prevalence areas; gallbladder evaluation and upper endoscopy if clinically indicated)• Endocrine evaluation (A1c with suspected or diagnosed prediabetes or diabetes); TSH with symptoms or increased risk of thyroid disease; androgens with PCOS suspicion (total/bioavailable testosterone, DHEAS, D4-androstenedione); screening for Cushing’s syndrome if clinically suspected (1 mg overnight dexamethasone test, 24-hour urinary free cortisol, 11 PM salivary cortisol)• Clinical nutrition evaluation by

1	screening for Cushing’s syndrome if clinically suspected (1 mg overnight dexamethasone test, 24-hour urinary free cortisol, 11 PM salivary cortisol)• Clinical nutrition evaluation by registered dietician• Psychosocial-behavioral evaluation• Document medical necessity for bariatric surgery• Informed consent• Provide relevant financial information• Continue efforts for preoperative weight loss• Optimize glycemic control• Pregnancy counseling• Smoking cessation counseling• Verify cancer screening by primary care physicianReproduced with permission from Mechanick JI, Youdim A, Jones DB, et al: Clinical practice guidelines for the perioperative nutritional, metabolic, and nonsurgical support of the bariatric surgery patient—2013 update: cosponsored by American Association of Clinical Endocrinologists, The Obesity Society, and American Society for Metabolic & Bariatric Surgery, Obesity (Silver Spring). 2013 Mar;21 Suppl 1:S1-S27.women who become pregnant after bariatric surgery should be

1	The Obesity Society, and American Society for Metabolic & Bariatric Surgery, Obesity (Silver Spring). 2013 Mar;21 Suppl 1:S1-S27.women who become pregnant after bariatric surgery should be counseled and monitored for appropriate weight gain, nutri-tional supplementation, and for fetal health.119 All women of reproductive age should be counseled on contraceptive choices following bariatric surgery as utilization, absorption, and effec-tiveness are inconsistent.120,121 Patients should be provided with educational materials and access to preoperative educational sessions. Multimedia tools for patient education and consent show promise for improving understanding.122-124 There should be a thorough and dynamic consent discussion regarding the risks and benefits, procedural options, and the need for long-term follow-up and vitamin supplementation (including costs required to maintain appropriate follow-up). Consent should include the experience of the surgeon with the specific proce-dure

1	for long-term follow-up and vitamin supplementation (including costs required to maintain appropriate follow-up). Consent should include the experience of the surgeon with the specific proce-dure and whether the program participates in national quality improvement initiatives and certification.Anesthesiology IssuesTwo major challenges that face the anesthesiologist when per-forming a general anesthetic for the severely obese patient are vascular access and airway management. Fiberoptic laryngos-copy is often used for the most difficult class IV or even class III airways should standard laryngoscopy be determined to provide an inadequate view. Videotelescopic intubation systems are suc-cessfully used as well. Significant preoxygenation for 3 minutes or longer prior to intubation is used for the severely obese patient to provide a longer safe duration for intubation should difficul-ties be encountered. However, desaturation must be immediately addressed with reestablishment of

1	used for the severely obese patient to provide a longer safe duration for intubation should difficul-ties be encountered. However, desaturation must be immediately addressed with reestablishment of oxygenated ventilation because this patient group does not tolerate any prolonged desaturation without potential adverse cardiopulmonary consequences.The anesthesiologist must also manage alterations in car-diopulmonary function from the use of a pneumoperitoneum during laparoscopic bariatric procedures. These include the effects of carbon dioxide absorption on required minute venti-lation, the potential for bradyarrhythmias, and the potential for decreased systemic pH with longer procedures in patients with preexisting cardiopulmonary disease. Arterial monitoring of the latter group of patients may be necessary by the anesthesiology team, and a radial arterial line is standard for such patients.125 Drug pharmacokinetics differ in severely obese patients as well. Changes in volume of

1	may be necessary by the anesthesiology team, and a radial arterial line is standard for such patients.125 Drug pharmacokinetics differ in severely obese patients as well. Changes in volume of distribution include smaller-than-normal fraction of total body water, greater adipose tissue content, altered protein binding, and increased blood volume. Possible changes in renal function and hepatic function must be consid-ered when administering drugs. Specific anesthetic drug meta-bolic alterations in the severely obese include a larger volume distribution of thiopentone, resulting in a prolonged effect of the drug. Calculation of the dosage should be done by lean body weight. Benzodiazepines also exhibit a prolonged elimination phase, causing prolongation of their effects. Increased pseudo-cholinesterase activity is present in the severely obese patient, requiring increased dosages of pancuronium. Enflurane metab-olism is increased over the average-sized person, requiring a lower dosage of

1	activity is present in the severely obese patient, requiring increased dosages of pancuronium. Enflurane metab-olism is increased over the average-sized person, requiring a lower dosage of this agent.Enhanced Recovery After SurgeryEnhanced recovery after surgery (ERAS) protocols have been initiated in bariatric surgery and have demonstrated promise to decrease surgical morbidity. Additionally, a recent meta-analy-sis has identified a significant decrease in length of stay (stan-dard mean difference = −2.40 [−33.89, −0.89], P = 0.002).126 In 2016, the ERAS Society published evidence-based guidelines Brunicardi_Ch27_p1167-p1218.indd 117923/02/19 2:20 PM 1180SPECIFIC CONSIDERATIONSPART IIfor perioperative care in bariatric surgery.127 The guidelines include recommendations in preoperative, intraoperative, and postoperative care. These include shorter acting and lower absorption anesthetic agents and opioid minimization as impor-tant intraoperative recommendations.Special Equipment

1	intraoperative, and postoperative care. These include shorter acting and lower absorption anesthetic agents and opioid minimization as impor-tant intraoperative recommendations.Special Equipment and InfrastructureThe special needs of the bariatric patient and program extend from the entry to the hospital and clinic, to the operating room, and throughout the inpatient and outpatient experience. The program needs infrastructure and support at all levels including support staff, physicians and surgeons, administrators, program directors, psychologists, and nutritionists. The physical plant needs to include extra-wide doorways, special seating, a scale that weighs up to 800 lb (363 kg), larger patient gowns, large blood pressure cuffs, and floor-mounted toilets. In the operat-ing room, the table must accommodate 600 to 800 lb (272 to 363 kg) and must position in steep reverse Trendelenburg posi-tion. Larger lower extremity compression devices, extra padding, safety belts, and a footboard

1	must accommodate 600 to 800 lb (272 to 363 kg) and must position in steep reverse Trendelenburg posi-tion. Larger lower extremity compression devices, extra padding, safety belts, and a footboard are required. An angled (30° or 45°) telescope, extra-long graspers and staplers, and a liver retractor system are all standard equipment. Staff sensitivity training for the care of the obese as well as regular education about the com-plications of bariatric surgery are program requirements.BARIATRIC SURGICAL PROCEDURESVBG shown in Figs. 27-1 and 27-5, although still listed as one of the approved operations for the surgical treatment of severe obesity based on the NIH Consensus Conference of 1991,51 is not currently performed due to poor long-term weight loss and technical complications, so it is of historic interest only, and the surgical technique will not be described here.8 The other procedures described in this section will be articulated using a laparoscopic approach as that is the

1	is of historic interest only, and the surgical technique will not be described here.8 The other procedures described in this section will be articulated using a laparoscopic approach as that is the dominant method. RYGB, BPD, and DS may still be performed by some surgeons using an open approach, but this has now become the exception. In this text LRYGB will refer specifically to RYGB performed by the laparoscopic approach, while RYGB will indicate proce-dures performed by the open approach or by both approaches as is the case for many studies of outcomes. Minimization of the morbidity of the open incision, especially incisional hernias and wound complications, as well as earlier hospital discharge and lower 30-day complication rates have all been clearly shown to favor using a laparoscopic approach when feasible.128-130 Lapa-roscopy begins with the safe creation of a pneumoperitoneum, often a difficult step in the bariatric patient. A tracheostomy hook can be inserted through a

1	approach when feasible.128-130 Lapa-roscopy begins with the safe creation of a pneumoperitoneum, often a difficult step in the bariatric patient. A tracheostomy hook can be inserted through a trocar-sized incision to elevate the fascia in the left subcostal region to facilitate the insertion of a Veress needle into an appropriate location for pneumoperi-toneum creation. The use of a Hasson approach for creating a pneumoperitoneum in the bariatric population may be limited by the thick body wall. In the patient with an extremely thick body wall, extra-long trocar ports can be used for laparoscopic surgery. The pneumoperitoneum pressure that is used when per-forming bariatric surgical procedures is generally in the 15 to 18 mmHg range. A high-flow insufflator is mandatory to main-tain the pneumoperitoneum for adequate and safe visualization.When an open surgical approach is used for any of these procedures, an upper midline incision with table mounted retrac-tors is the most commonly

1	pneumoperitoneum for adequate and safe visualization.When an open surgical approach is used for any of these procedures, an upper midline incision with table mounted retrac-tors is the most commonly used approach. The robotic approach to bariatric procedures is also now utilized with purported Figure 27-8. Configuration of laparoscopic gastric bypass. (Reprinted with permission from Cleveland Clinic Center for Medi-cal Art & Photography © 2005-2009. All Rights Reserved.)advantages of reduction of the use of the open technique, improved surgical, length of stay, cost outcomes, and potentially improving ergonomics and resultant surgeon fatigue and injury. A meta-analysis involving 27 studies and over 25,000 patients concluded that there were no significant differences between robotic bariatric surgery and laparoscopic bariatric surgery with respect to overall complications, length of stay, reoperation, conversion, and mortality.131 Another study utilizing Univer-sity Consortium data

1	surgery and laparoscopic bariatric surgery with respect to overall complications, length of stay, reoperation, conversion, and mortality.131 Another study utilizing Univer-sity Consortium data demonstrated no difference in hospital mortality, major complications, readmissions, or length of stay between the robotic and laparoscopic approach.132 In both stud-ies, robotic surgery did increase significantly operative time and hospital costs (>20%) compared to laparoscopic approaches. As yet, larger prospective cohort studies and/or randomized trials have not yet been published, so the role of this technique is still to be defined and further studies are needed.133,134Laparoscopic Roux-en-Y Gastric BypassBackground and Patient Selection. Figure 27-8 depicts the configuration of the LRYGB. It is an appropriate operation for consideration for most patients eligible for bariatric surgery. Relative contraindications specifically for LRYGB include pre-vious gastric surgery, previous antireflux

1	an appropriate operation for consideration for most patients eligible for bariatric surgery. Relative contraindications specifically for LRYGB include pre-vious gastric surgery, previous antireflux surgery, severe iron deficiency anemia, distal gastric or duodenal lesions that require ongoing future surveillance, and Barrett’s esophagus with severe Brunicardi_Ch27_p1167-p1218.indd 118023/02/19 2:20 PM 1181THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 275mm5mm12mm12mm5mm5mmFigure 27-9. Port scheme for laparoscopic gastric bypass. (Reprinted with permission from Cleveland Clinic Center for Medi-cal Art & Photography © 2005-2009. All Rights Reserved.)dysplasia. The major feature of the operation is a proximal gas-tric pouch of small size (<20 mL) that is totally separated from the distal residual stomach. A Roux limb of proximal jejunum is brought up and anastomosed to the pouch. The pathway of that limb can be anterior to the colon and stomach, posterior to both, or posterior to the

1	residual stomach. A Roux limb of proximal jejunum is brought up and anastomosed to the pouch. The pathway of that limb can be anterior to the colon and stomach, posterior to both, or posterior to the colon and anterior to the stomach. The length of the biliopancreatic limb from the ligament of Treitz to the distal enteroenterostomy is 20 to 50 cm, and the length of the Roux limb is 75 to 150 cm.Creating the proximal gastric pouch by totally dividing it from the distal stomach is superior to simply stapling and par-titioning the stomach, since the latter is associated with a high incidence of staple line breakdown.135 The size of the proximal gastric pouch must be small to create adequate restriction and should be based on the lesser curvature of the stomach to pre-vent dilation over time. Length of the Roux limb was associated with higher short-term weight loss for longer length limbs,136 but this difference becomes less meaningful on long-term follow-up and has not been demonstrated

1	Length of the Roux limb was associated with higher short-term weight loss for longer length limbs,136 but this difference becomes less meaningful on long-term follow-up and has not been demonstrated in more recent studies.137 Gastric pouch size and caliber of the gastrojejunostomy have not, in any studies, been shown to be related to weight loss. The gas-trojejunal anastomosis can be constructed in a variety of ways, including hand sewn techniques and linear and circular staplers. Smaller diameter circular staplers are associated with a higher incidence of postoperative stenosis, and linear stapling is asso-ciated with a lower incidence of stenosis compared to circular stapling.138,139Technique. The operation generally is performed using five ports plus a liver retractor as shown in Fig. 27-9. Both the sur-geon, who stands on the patient’s right, and the first assistant, who stands on the patient’s left, have two ports for instruments. The telescope requires a port, usually in the

1	27-9. Both the sur-geon, who stands on the patient’s right, and the first assistant, who stands on the patient’s left, have two ports for instruments. The telescope requires a port, usually in the supraumbilical region. The assistant’s ports are in the left subcostal and flank areas, while the surgeon may have both ports in the right upper quadrant or one on each side of the camera. Division of the proximal jejunum at 40 to 50 cm distal to the ligament of Tre-itz is performed with the linear stapler, using a vascular stapler cartridge. Further division of the mesentery at that location is performed either with the stapler or harmonic scalpel, such that adequate mobilization of the Roux limb is achieved. A Pen-rose drain or a marking suture is placed on the proximal Roux limb for identification and facilitation of advancement to the Figure 27-10. Creating Roux limb during laparoscopic gastric bypass.gastric pouch (Fig. 27-10). The length of the Roux limb (usually 100–150 cm) to be

1	and facilitation of advancement to the Figure 27-10. Creating Roux limb during laparoscopic gastric bypass.gastric pouch (Fig. 27-10). The length of the Roux limb (usually 100–150 cm) to be created is measured. A jejunojejunostomy is then created to the proximal end of the biliopancreatic limb at the previously determined location along the Roux limb. A side-to-side stapled anastomosis is performed (Fig. 27-11). Either singleor double-fired staple technique (the latter using a stapler Brunicardi_Ch27_p1167-p1218.indd 118123/02/19 2:20 PM 1182SPECIFIC CONSIDERATIONSPART IIFigure 27-12. Passage of Roux limb. (Reprinted with permission from Cleveland Clinic Center for Medical Art & Photography © 2005-2009. All Rights Reserved.)321Figure 27-13. Creation of gastric pouch for laparoscopic Roux-en-Y gastric bypass. (Reprinted with permission from Cleveland Clinic Center for Medical Art & Photography © 2005-2009. All Rights Reserved.)Figure 27-11. Enteroenterostomy of lapa-roscopic

1	Roux-en-Y gastric bypass. (Reprinted with permission from Cleveland Clinic Center for Medical Art & Photography © 2005-2009. All Rights Reserved.)Figure 27-11. Enteroenterostomy of lapa-roscopic Roux-en-Y gastric bypass.fired in each direction) is used. The stapler defect is optimally closed with sutures but can be closed with a stapler if great care is taken not to narrow the lumen of the alimentary tract at this location. Once the stapler defect is closed, the mesenteric defect is then also closed with running permanent suture.Passage of the Roux limb toward the stomach is now per-formed. If an antecolic route is to be used, the end of the Roux limb is brought up so as to confirm its ability to reach the stom-ach (Fig. 27-12). If a retrocolic route is to be used, a defect is made in the transverse colon mesentery just to the left and slightly above the ligament of Treitz. The proximal end of the Roux limb is placed into the retrogastric space. The left lobe of the liver is now

1	the transverse colon mesentery just to the left and slightly above the ligament of Treitz. The proximal end of the Roux limb is placed into the retrogastric space. The left lobe of the liver is now retracted using any one of several retractor types. The patient is moved to a reverse Trendelenburg posi-tion. The harmonic scalpel divides the peritoneum in the area of the angle of His, and then it is used to open an area along the lesser curvature of the stomach approximately 3 cm down from the gastroesophageal junction. Another approach for creating access to the lesser curvature of the stomach is to use a white or gray load (vascular load) of the stapler and divide the lesser curvature vessels up to the surface of the stomach. Then a blue load of the stapler is fired one time transversely from the lesser curvature side partially across the stomach, followed by mul-tiple subsequent firings of the stapler upward in the direction of the angle of His, to completely separate the proximal

1	from the lesser curvature side partially across the stomach, followed by mul-tiple subsequent firings of the stapler upward in the direction of the angle of His, to completely separate the proximal gastric pouch from the remainder of the stomach (Fig. 27-13). Option-ally, use of an Ewald tube passed by the anesthesiologist and maneuvered to lie against the lesser curvature of the proximal stomach can help calibrate the pouch size.Once the pouch is created, the Roux limb is brought up to the proximal gastric pouch. For the linear stapled anastomosis, the proximal end of the Roux limb is aligned with the distal gastric pouch end, and the sides of the organs are sutured together to maintain their side-by-side position. A stapler is introduced through a gastrotomy and an enterotomy for the two legs of the stapler, and the anastomosis is created (Fig. 27-14). The stapler defect is closed with sutures and often reinforced with a second Brunicardi_Ch27_p1167-p1218.indd 118223/02/19

1	two legs of the stapler, and the anastomosis is created (Fig. 27-14). The stapler defect is closed with sutures and often reinforced with a second Brunicardi_Ch27_p1167-p1218.indd 118223/02/19 2:21 PM 1183THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27Figure 27-14. Gastrojejunostomy in laparoscopic Roux-en-Y gastric bypass. (Reprinted with permission from Cleveland Clinic Center for Medical Art & Photography © 2005-2009. All Rights Reserved.)Figure 27-15. Oral passage of EEA circular stapler to create gastrojejunostomy for laparoscopic Roux-en-Y gastric bypass. (Reprinted with permission from Cleveland Clinic Center for Medical Art & Photography © 2005-2009. All Rights Reserved.)step of the operation involves suture closure of all mesenteric defects using permanent suture.Alternatively, a completely hand-sewn gastrojejunostomy can be created using two layers of absorbable suture to anasto-mose an approximately 1-cm gastrotomy and enterotomy. The circular anastomosis technique is

1	a completely hand-sewn gastrojejunostomy can be created using two layers of absorbable suture to anasto-mose an approximately 1-cm gastrotomy and enterotomy. The circular anastomosis technique is another approach to complete the gastrojejunostomy and is also a particularly useful technique for “salvage” anastomosis if the gastric pouch is very small and/or high. This is done through placement of the anvil of the sta-pler through the anterior wall of the proximal gastric pouch. This is accomplished by pulling the anvil transorally via an endoscopically placed guidewire (Fig. 27-15), making a gas-trotomy in the pouch that is later closed, or making a gastrotomy in the lower stomach before completing gastric division to cre-ate the pouch, allowing the anvil to be placed into the lumen of the stomach and then be brought through the anterior stomach in an area that is subsequently included in the proximal gastric pouch (Fig. 27-16).Procedure-Specific Complications. Mortality after LRYGB is

1	stomach and then be brought through the anterior stomach in an area that is subsequently included in the proximal gastric pouch (Fig. 27-16).Procedure-Specific Complications. Mortality after LRYGB is now consistently less than 0.5% in most large reported series. Data from several national data sets/studies find a mortal-ity rate of approximately 0.3%, 0.14%, and 0.2% at 30 days overall.59,140 Overall morbidity after LRYGB has also been low. In the Longitudinal Assessment of Bariatric Surgery (LABS) study, a composite endpoint including death, deep-vein throm-bosis or venous thromboembolism, reintervention, or failure to be discharged by 30 days after surgery occurred in 4.8% of those who had undergone LRYGB.59 In the national database of the ASMBS, morbidity alone was 14.87% for 30,864 gas-tric bypass procedures.141 Complications that do occur after LRYGB include a 0.3% incidence of anastomotic leak,142 0.33% layer of sutures. The gastrojejunostomy may be tested for secu-rity by using

1	bypass procedures.141 Complications that do occur after LRYGB include a 0.3% incidence of anastomotic leak,142 0.33% layer of sutures. The gastrojejunostomy may be tested for secu-rity by using either methylene blue injected under pressure through the Ewald tube or a flexible upper endoscopy intraop-eratively to test for air leakage from the anastomosis. The final Brunicardi_Ch27_p1167-p1218.indd 118323/02/19 2:21 PM 1184SPECIFIC CONSIDERATIONSPART IIFigure 27-16. Transgastric passage of circular EEA stapler to create gastrojejunostomy for laparoscopic Roux-en-Y gastric bypass. (Reproduced with permission from Schauer PR, Schirmer BD, Brethauer S: Minimally Invasive Bariatric Surgery. New York, NY: Springer; 2007.)Figure 27-17. Obstruction of contrast at enteroenterostomy with small bowel obstruction from internal hernia after laparoscopic Roux-en-Y gastric bypass.incidence of venous thromboembolism,143 a 3% to 5% incidence of wound infections or problems,140 a 3% to 15% incidence

1	obstruction from internal hernia after laparoscopic Roux-en-Y gastric bypass.incidence of venous thromboembolism,143 a 3% to 5% incidence of wound infections or problems,140 a 3% to 15% incidence of marginal ulcers,144 an approximately 7% incidence of bowel obstruction,145 a 4% incidence of postoperative transfusion,146 and a 1% to 19% incidence of anastomotic stenosis,139 based on the type of anastomosis created. Postoperative nutritional complications after LRYGB include a 66% incidence of iron deficiency, a 5% incidence of iron deficiency anemia, a 50% incidence of vitamin B12 deficiency,147 and an at least 15% incidence of vitamin D deficiency,148 which usually is pres-ent preoperatively. Both early and late dumping occur in an unspecified number of postoperative cases, as the symptoms are difficult to document and overlap with other problems such as hypoglycemia.Several complications that are specific to LRYGB must be emphasized. One of the most important is small bowel

1	symptoms are difficult to document and overlap with other problems such as hypoglycemia.Several complications that are specific to LRYGB must be emphasized. One of the most important is small bowel obstruction. This complication must be treated differently than in the average general surgery patient, whose complication is usually from adhesions and often will resolve with conserva-tive, nonoperative therapy. Patients who have had LRYGB who present with obstructive symptoms generally require surgical therapy on an emergent basis. This is because the etiology of the bowel obstruction after LRYGB is often an internal hernia from inadequate or nonclosure of the mesenteric defects by the sur-geon at the time of operation. Thus, treatment for these patients differs from most patients with small bowel obstruction. One of the most important points of this chapter is to emphasize to gen-eral surgeons to be aware of the need to emergently operate on patients after LRYGB who present with small

1	bowel obstruction. One of the most important points of this chapter is to emphasize to gen-eral surgeons to be aware of the need to emergently operate on patients after LRYGB who present with small bowel obstruc-tion. Currently, centers that perform small bowel transplantation are seeing patient referral for that procedure after small bowel obstruction after LRYGB, where patients developed infarction of most of the bowel from an internal hernia and have short gut syndrome.149 Other patients, for whom surgery is delayed and the bowel infarcts, do not survive. When the surgeon does encounter bowel obstruction after LRYGB, he or she can expect to see proximally dilated bowel. Cutoff of passage of contrast on CT scan at the enteroenterostomy is particularly suggestive of this diagnosis (Fig. 27-17). The surgical treatment of this particular problem can, if addressed early in the course of the obstruction, be treated laparoscopically. The surgeon must place a trocar for the telescope low

1	27-17). The surgical treatment of this particular problem can, if addressed early in the course of the obstruction, be treated laparoscopically. The surgeon must place a trocar for the telescope low enough in the abdomen to ade-quately survey most of the small intestine. The cecum and ter-minal ileum are identified, and the bowel is followed retrograde from the terminal ileum to determine the anatomy. Often much of the small bowel is herniated through a mesenteric defect, and only this technique allows the surgeon to reliably identify the bowel and decompress it appropriately. If the bowel is viable, 4Brunicardi_Ch27_p1167-p1218.indd 118423/02/19 2:21 PM 1185THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27suturing the mesenteric defect is all that is needed for treatment. It should be emphasized that either an antecolic or retrocolic placement of the Roux limb can result in this complication, as internal hernias can arise from either approach.Marginal ulcers are another complication

1	that either an antecolic or retrocolic placement of the Roux limb can result in this complication, as internal hernias can arise from either approach.Marginal ulcers are another complication relatively specific to LRYGB. The patient presents with pain in the epigastric region that is not altered by eating. Diagnosis is by endoscopy. Treatment is medical with proton pump inhibitors, which are effective in 90% of cases. Only those with a gastrogastric fistula to the distal stomach, severe stenosis of the lumen of the gastrojejunostomy, or acute perforation require surgical therapy. Treatment of a perforated marginal ulcer is a laparoscopic Graham patch. Stenosis of the gastrojejunostomy has been reduced by the use of the linear stapling technique.138 Stenosis symptoms usually appear from 6 to 12 weeks postoperatively, but less commonly can occur later. Diagnosis is by upper endoscopy. Treatment is circumferential balloon dilatation. Resolution normally occurs with one to two treatments.

1	12 weeks postoperatively, but less commonly can occur later. Diagnosis is by upper endoscopy. Treatment is circumferential balloon dilatation. Resolution normally occurs with one to two treatments. Less than 10% of patients require reoperation, and those are almost always associated with concurrent marginal ulcers.150In the immediate postoperative period, anastomotic leak is the single serious complication after RYGB, either open or laparoscopic. Careful vigilance and a high index of suspicion for this problem are important since its presentation may be insidi-ous and the patient’s demise, if untreated, may be sudden and complete. Tachycardia, tachypnea, fever, and oliguria are the most common symptoms that should arouse suspicion for this problem. The treatment is surgical, except in rare circumstances where a drain is already in place, no hemodynamic or clinical deterioration is present, and the leak is contained.151 Usual surgi-cal treatment involves repair as feasible, drainage,

1	circumstances where a drain is already in place, no hemodynamic or clinical deterioration is present, and the leak is contained.151 Usual surgi-cal treatment involves repair as feasible, drainage, and creation of a reliable feeding access through a distal Stamm gastrostomy.In the first few hours or day after surgery, hematemesis indicates bleeding from the gastrojejunostomy unless proven otherwise. The dangers to the patient include aspiration, life-threatening hemorrhage, or more commonly intraluminal hematoma of the Roux limb and enteroenterostomy, which then causes an obstruction of the biliopancreatic limb leading to distal gastric staple line rupture.152 In fact, any obstructive symptoms in the first few weeks after surgery or any signs of obstruction of the biliopancreatic limb on postoperative swallow studies due to stenosis of the enteroenterostomy require imme-diate surgical intervention to prevent rupture of the distal gastric staple line. Some reports show that percutaneous

1	swallow studies due to stenosis of the enteroenterostomy require imme-diate surgical intervention to prevent rupture of the distal gastric staple line. Some reports show that percutaneous decompres-sion of the distal stomach can help to ameliorate the problem,153 but operative therapy to decompress the stomach and treat the obstruction is first-line therapy.Laparoscopic Sleeve GastrectomyBackground and Patient Selection. SG was originally introduced as the first of a two-stage operative treatment for patients with super obesity (BMI >60 kg/m2).154,155 Its currently utilization is as a primary single-stage operation, but the pos-sibility of a second-stage treatment remains, especially for the super obese patients, depending on the effectiveness of it as the primary operation. In addition, patients who have longstanding severe GERD may not be good candidates for SG as GERD is worsened by the anatomical configuration of the SG. Barrett’s esophagus is also a contraindication for

1	patients who have longstanding severe GERD may not be good candidates for SG as GERD is worsened by the anatomical configuration of the SG. Barrett’s esophagus is also a contraindication for performing SG, since the potential for future esophageal dysplasia and the need for an available intact stomach for esophageal reconstruction outweigh the potential advantages of the procedure.Figure 27-18. Port scheme for laparoscopic sleeve gastrectomy.Technique. The patient is positioned supine, with foot sup-port to allow reverse Trendelenburg positioning. The surgeon stands to the patient’s right along with the camera driver, while the assistant stands to the patient’s left. Port placement may vary, but a recommended port placement schema is shown in Fig. 27-18. The 15-mm port, helpful for removal of the stomach, is located in either the camera (just to the patient’s left of the umbilicus) or surgeon’s right hand (right upper quadrant near the midline) location. The other of these ports is a

1	of the stomach, is located in either the camera (just to the patient’s left of the umbilicus) or surgeon’s right hand (right upper quadrant near the midline) location. The other of these ports is a 12-mm port. The assistant has two 5-mm ports available in the left upper quadrant laterally, and the surgeon’s left-hand port is a 5-mm port more lateral and superior in the right upper quadrant. A liver retractor is placed in the epigastric region.The operation begins by devascularizing the greater curva-ture of the stomach, beginning 3 to 5 cm proximal to the pylorus. The division of all vessels adjacent to the greater curvature is continued up to the left crus of the diaphragm. A complete mobilization of the fundus in this area and division of posterior fibrous attachments to the antrum and body of the stomach are then performed such that the stomach is attached solely by the lesser curvature blood supply and the pyloric and esophageal regions. Stapled division of the stomach now

1	and body of the stomach are then performed such that the stomach is attached solely by the lesser curvature blood supply and the pyloric and esophageal regions. Stapled division of the stomach now follows. The first firing of the stapler occurs from the point of devascularization of the greater curvature at an angle pointing toward a point about 2 cm lateral to the incisura. The antrum of the stomach is at its thickest here, and so it is important to be certain the stapler load used is sufficiently large enough to allow good approximation and closure of the divided stomach. Two staple firings are performed, which takes the gastric division to past the incisura. After the first staple firing, some surgeons will engage the anesthesiologist to pass a 32to 40-French bougie and position it along the lesser curvature of the stomach. This bougie then serves as a guide for further gastric division. Alter-natively, some surgeons will insert the endoscope instead of the bougie as a guide for

1	the lesser curvature of the stomach. This bougie then serves as a guide for further gastric division. Alter-natively, some surgeons will insert the endoscope instead of the bougie as a guide for gastric division. It can also be used to test for air leaks, bleeding, or obstruction as it is withdrawn after gastric division. Dividing the stomach adjacent to the bougie or endoscope will produce the desired diameter of the gastric sleeve. It is most important not to narrow the stomach lumen at the incisura. During the second and third firing of the stapler to divide the stomach, it is critical to confirm by visualization of both the anterior and posterior surfaces of the stomach that the incisura area is not narrowed. By the third firing of the stapler, usually the angle of the gastric division is now pointed directly toward the angle of His, parallel to the bougie (Fig. 27-19). Brunicardi_Ch27_p1167-p1218.indd 118523/02/19 2:21 PM 1186SPECIFIC CONSIDERATIONSPART IIFigure

1	division is now pointed directly toward the angle of His, parallel to the bougie (Fig. 27-19). Brunicardi_Ch27_p1167-p1218.indd 118523/02/19 2:21 PM 1186SPECIFIC CONSIDERATIONSPART IIFigure 27-20. Completed sleeve gastrectomy. (Reprinted with permission from Cleveland Clinic Center for Medical Art & Photography © 2005-2009. All Rights Reserved.)Figure 27-19. Performing sleeve gastrectomy. (Reprinted with permission from Cleveland Clinic Center for Medical Art & Photography © 2005-2009. All Rights Reserved.)At this point, changing staple loads to lower staple height is advisable.Once the stomach is completely divided up to the angle of His, the staple line is inspected for hemostasis and integ-rity. Some surgeons will reinforce the staple line with a but-tress material, while others will invaginate the staple line with a running serosa to serosa suture. Some surgeons will exchange the bougie at this point for a 32-French Ewald tube and per-form a methylene blue leak test.

1	others will invaginate the staple line with a running serosa to serosa suture. Some surgeons will exchange the bougie at this point for a 32-French Ewald tube and per-form a methylene blue leak test. Alternatively, if an endoscope is used, it is withdrawn with insufflation, and the staple line is inspected for air leaks while submerged in saline. The speci-men is removed through the 15-mm trocar site, usually with only slight enlargement of the site. Figure 27-20 shows the com-pleted operation. Controversy still exists as to the optimal size of the bougie used during the procedure and the relative utility of methods used to oversew or reinforce the staple line.Procedure-Specific Complications. The major factor unique to SG is that it creates a high-pressure gastric tube. This increased intraluminal pressure places the staple line at risk for leakage and increased risk for GERD. As noted previ-ously, controversy exists about both bougie size and staple line reinforcement/oversew as

1	intraluminal pressure places the staple line at risk for leakage and increased risk for GERD. As noted previ-ously, controversy exists about both bougie size and staple line reinforcement/oversew as they relate to clinical outcomes. One summary of the literature shows that the stenosis rate is lower if a 40-French bougie is used, and the leak rate may also be lower without compromising weight loss.156 However, individual insti-tutional experiences with smaller-sized bougies have shown the potential for good weight loss and no increased incidence of stenosis.157 Another controversial area is that of staple line rein-forcement with staple buttressing material or reinforcement with oversewing.158 The overall bleeding rate for the staple line after SG is generally cited as about 2% in collected series.159 There have been no studies that have shown a definitive decrease in this bleeding rate with the use of buttress materials; however, a panel of experts has voiced support for a decreased

1	series.159 There have been no studies that have shown a definitive decrease in this bleeding rate with the use of buttress materials; however, a panel of experts has voiced support for a decreased incidence of bleeding from the staple line if buttress material is used.158 One meta-analysis did show that there is evidence to suggest buttress materials may decrease the staple line leak rate.160 Other prospective randomized studies have failed to show a benefit of buttress materials for leak prevention.161 A more recent study of over 180,000 SG procedures in the Metabolic and Bariat-ric Surgery Accreditation and Quality Improvement Program (MBSAQIP) national database showed that staple line rein-forcement cases were associated with higher leak rates (0.96% vs. 0.65%, odds ratio [OR] 1.20 95% CI 1.00–1.43) and lower bleeding rates (0.75% vs. 1.00%, OR 0.74 95% CI 0.63–0.86) compared to no reinforcement, at the patient level.162 At this time, there is no preponderance of data to support

1	CI 1.00–1.43) and lower bleeding rates (0.75% vs. 1.00%, OR 0.74 95% CI 0.63–0.86) compared to no reinforcement, at the patient level.162 At this time, there is no preponderance of data to support one approach as being superior to others for both staple line bleeding or leak rates. Given this state of the literature, a surgeon should con-sider the risks, benefits, and costs of these surgical techniques and utilize those that, in their hands, minimize morbidity while maximizing clinical outcomes.If there is a relative obstruction or stenosis of the sleeve, which most often occurs at the incisura because of narrowing there during formation of the sleeve, pressure above the level of the obstruction will be even more elevated and create an increased risk for staple line leak. Leaks of the proximal staple line are the most frequent type seen after SG and often are felt to be related to increased intraluminal pressure distally. They may also be related to stapling too close to the angle of

1	staple line are the most frequent type seen after SG and often are felt to be related to increased intraluminal pressure distally. They may also be related to stapling too close to the angle of His, with resultant instability of the tissue directly adjacent to the esophagus in this area. It is important not to staple too close to the angle of His during the final stapling division portion of the stomach so as to not further weaken the staple line in this area. Proximal staple line leaks may also present as late leaks, 6 weeks to months following the procedure. Late leaks are rare Brunicardi_Ch27_p1167-p1218.indd 118623/02/19 2:21 PM 1187THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27Figure 27-21. Laparoscopic adjustable band overall scheme. (Reprinted with permission from Cleveland Clinic Center for Medi-cal Art & Photography © 2005-2009. All Rights Reserved.)following other bariatric procedures, but are seen with SG, and index of suspicion should remain high.Distal staple line leaks

1	Center for Medi-cal Art & Photography © 2005-2009. All Rights Reserved.)following other bariatric procedures, but are seen with SG, and index of suspicion should remain high.Distal staple line leaks are different from proximal staple line leaks and are usually associated with earlier presentation and related to mechanical failure of the staple line to securely approximate the thicker distal gastric tissue. These leaks are more amenable to successful repair with a reoperation, whereas proximal leaks may not improve with oversewing at a reopera-tion unless the mechanics of the relative distal obstruction and high intraluminal pressure of the sleeve are also treated. Endo-scopic intervention to dilate stenotic areas as well may be bene-ficial in the setting of a stenosis with or without a proximal leak. Care must be taken by the endoscopist to not excessively dilate the tract beyond the original size of the bougie used. Another factor that may influence stenosis at the incisura is that

1	leak. Care must be taken by the endoscopist to not excessively dilate the tract beyond the original size of the bougie used. Another factor that may influence stenosis at the incisura is that there may be a relative twisting of the stomach at this location, with the antrum being partially twisted away from the upper por-tion of the sleeve. Endoscopic treatment can help straighten and markedly alleviate the obstruction in such cases. Thus, relatively early endoscopic intervention is appropriate in the patient with a stenosis at the incisura. One study has shown that endoscopic dilation is usually successful in treating stenosis after SG, with a mean of 1.6 dilatations being done an average of 48 days postoperatively.163The patient with the proximal gastric staple line leak because of mechanical factors may experience persistence of the leak for months. Nonsurgical treatment with drainage and stenting can be used initially. Some now advocate for conver-sion of the patient with a

1	mechanical factors may experience persistence of the leak for months. Nonsurgical treatment with drainage and stenting can be used initially. Some now advocate for conver-sion of the patient with a longstanding leak after SG to a RYGB to provide a low-pressure anastomosis above the site of the ste-nosis.164,165 Similarly, persistent stenosis of the sleeve despite conservative therapy and endoscopic dilatation also is an indica-tion for conversion to RYGB.Laparoscopic Adjustable Gastric BandingBackground and Patient Selection. LAGB involves place-ment of an inflatable silicone ring around the proximal stom-ach. The band is attached to a reservoir system that allows adjustment of the tightness of the band. This reservoir system is accessed through a subcutaneously placed port, similar in concept to ports used for chemotherapy via central venous catheters. Figure 27-21 shows the LAGB apparatus in place. Patients who have had previous upper gastric surgery, such as a Nissen

1	similar in concept to ports used for chemotherapy via central venous catheters. Figure 27-21 shows the LAGB apparatus in place. Patients who have had previous upper gastric surgery, such as a Nissen fundoplication, and those with severe GERD are rela-tively poor candidates for LAGB due to altered proximal gastric anatomy interfering with proper band placement or worsening of GERD symptoms. Two major types of bands have been used for this procedure. The original Lap-Band has been used most frequently. The Swedish Band, remarketed as the Realize Band in the United States, is slightly wider and larger in circumfer-ence than the Lap-Band but is no longer being manufactured. The port systems have differences as to profile and methods of attachment to the fascia.Technique. Port placement for LAGB has varied among sur-geons. Usually some combination of two ports for the surgeon’s hands, one or two for the assistant, a port for the telescope, and a liver retractor site are needed. With the

1	LAGB has varied among sur-geons. Usually some combination of two ports for the surgeon’s hands, one or two for the assistant, a port for the telescope, and a liver retractor site are needed. With the patient placed in reverse Trendelenburg position, the procedure begins with division of the peritoneum at the angle of His and then division of the gas-trohepatic ligament in its avascular area (the pars flaccida) to expose the base of the right crus of the diaphragm. If a hia-tal hernia is present, it must be repaired at this point, using a Figure 27-22. Grasper being passed through under stomach to grasp tubing during placement. (Reprinted with permission from Cleveland Clinic Center for Medical Art & Photography © 2005-2009. All Rights Reserved.)standard posterior esophageal dissection to expose the crura and perform suture repair. A grasper is inserted along the base of the anterior surface of the diaphragmatic crura, from right to left, emerging at the angle of His in the area of the

1	the crura and perform suture repair. A grasper is inserted along the base of the anterior surface of the diaphragmatic crura, from right to left, emerging at the angle of His in the area of the divided peri-toneum (Fig. 27-22). The device is then used to pull the band Brunicardi_Ch27_p1167-p1218.indd 118723/02/19 2:21 PM 1188SPECIFIC CONSIDERATIONSPART IIABunderneath the posterior surface of the gastroesophageal junc-tion. This technique, by passing the band through some fibrous tissue in this plane, serves to anchor the band more securely posteriorly. During the initial years of band placement, a ret-rogastric location of the posterior half of the band in the free space of the lesser sac caused an unacceptably high incidence of slippage and prolapse of the band. The adoption of the pars flaccida technique decreased the incidence of such slippage.166Once the band is passed around the proximal stomach, it is locked into its ring configuration through its own self-locking mechanism.

1	flaccida technique decreased the incidence of such slippage.166Once the band is passed around the proximal stomach, it is locked into its ring configuration through its own self-locking mechanism. This involves the tubing end being passed through the orifice of the buckle for the Lap-Band and the suture on the end of the flanged end of the band site being passed through for the Realize Band. Once the band is securely locked in place, the buckle portion of the band is located on the lesser curvature of the stomach (Fig. 27-23A,B). Now the anterior surface of the fundus and proximal stomach is imbricated over the band using several sutures (Fig. 27-24). The tubing of the band system is brought out through the desired site for placement of the port portion of the system. Usually this is a trocar site near the upper abdomen or xiphoid region to place the port most superficially such that it can be palpated postoperatively. The port is secured Figure 27-23. A. Lap-Band in place around

1	a trocar site near the upper abdomen or xiphoid region to place the port most superficially such that it can be palpated postoperatively. The port is secured Figure 27-23. A. Lap-Band in place around stomach. (Reprinted with permission from Cleveland Clinic Center for Medical Art & Photography © 2005-2009. All Rights Reserved.) B. RealizeT (Swedish) Band around stomach.to the anterior abdominal wall fascia. Access to the port for subsequent addition of fluid to the band system is percutane-ously achieved using a Huber or noncutting type needle. The band is initially placed empty of fluid, except priming, in most circumstances.Procedure-Specific Complications. The complications that may occur after LAGB include gastric prolapse, band slippage, band erosion, and port and tubing complications. In addition, failure to lose clinically significant weight is more common fol-lowing this procedure compared to others. Acute gastric pro-lapse is the most common emergent complication that

1	In addition, failure to lose clinically significant weight is more common fol-lowing this procedure compared to others. Acute gastric pro-lapse is the most common emergent complication that requires reoperation after LAGB. Acute, severe pain with immediate dysphagia, vomiting, and inability to take oral food or liquid is the typical presentation. Vomiting may predispose or exac-erbate this problem. Either anterior or posterior prolapse may occur.167 The initial evaluation for prolapse involves obtaining a plain film radiograph. If the band is in a horizontal position instead of its normal oblique position, prolapse must be strongly suspected. Initial treatment for an acute or chronic prolapse is to remove all the fluid from the system. This often allows reduc-tion of the prolapse and resolution of symptoms. If symptoms do not resolve after this, an upper gastrointestinal (UGI) series Brunicardi_Ch27_p1167-p1218.indd 118823/02/19 2:21 PM 1189THE SURGICAL MANAGEMENT OF

1	and resolution of symptoms. If symptoms do not resolve after this, an upper gastrointestinal (UGI) series Brunicardi_Ch27_p1167-p1218.indd 118823/02/19 2:21 PM 1189THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27Figure 27-24. Stomach imbricated over band.is indicated, and if prolapse persists, then reoperation laparo-scopically to reduce the prolapse and resuture the band in place is indicated. Chronic gastric prolapse is more subtle. The band retains its normal oblique angle, but there is symmetric dilation of the gastric pouch above the band. These are initially managed with fluid removal and monitoring of symptoms. Follow-up evaluation can be performed in 4 to 8 weeks, and if the chronic prolapse resolves on UGI, slow refilling of the band may begin.Band erosion is uncommon, reported in 1% to 2% of most series. The patient usually develops either a port site infection or systemic fever and a low-grade abdominal inflammatory sepsis. Endoscopy can be diagnostic, visualizing the white

1	1% to 2% of most series. The patient usually develops either a port site infection or systemic fever and a low-grade abdominal inflammatory sepsis. Endoscopy can be diagnostic, visualizing the white band mate-rial within the stomach. The presence of otherwise unexplained free air on computed tomography (CT) scan should alert the surgeon to this diagnosis as well. Laparoscopic removal of the band is indicated, with repair of the gastric perforation. Often the perforation is already sealed by an inflammatory process, but if not, appropriate management of a gastric perforation must be followed.167Port and tubing problems occur in at least 5% to 15% of patients undergoing LAGB. These require revision of the port/tubing system due to perforation, leaking, or kinking of the tub-ing or turning of the port such that access to the surface of the port for adding fluid is precluded. Usually a procedure under local anesthesia is all that is required to repair or realign the tub-ing or port. The

1	of the port such that access to the surface of the port for adding fluid is precluded. Usually a procedure under local anesthesia is all that is required to repair or realign the tub-ing or port. The incidence of band removal for patient dissatisfac-tion or lack of weight loss has been difficult to assess completely, but this number is increasing annually. Angrisani et al168 reported a 40.9% incidence of band removal after 10-year follow-up. In the Longitudinal Assessment of Bariatric Surgery (LABS) study, 18 subsequent reoperations occurred for every 100 participants with LAGB who were followed up for 3 years.169 Overall, these numbers are expected to increase as follow-up increases.Biliopancreatic Diversion and Duodenal SwitchBackground and Patient Selection. BPD was first described by, and remains championed by, Scopinaro in Italy.9 The operation, which is shown in Fig. 27-25, involves resection of the distal half to two-thirds of the stomach and creation of an alimentary tract of

1	and remains championed by, Scopinaro in Italy.9 The operation, which is shown in Fig. 27-25, involves resection of the distal half to two-thirds of the stomach and creation of an alimentary tract of the most distal 200 cm of ileum, which is anastomosed to the stomach. The biliopancreatic limb is anastomosed to the alimentary tract at approximately 100 cm proximal to the ileocecal valve. This operation is limited in its utilization due to both technical difficulty and the significant percentage of nutritional complications that arise postoperatively.One early problem with BPD was the development of a high incidence of marginal ulcers postoperatively. Hess and Figure 27-25. Diagram of biliopancreatic diversion. (Reprinted with permission from Cleveland Clinic Center for Medical Art & Photography © 2005-2009. All Rights Reserved.)Brunicardi_Ch27_p1167-p1218.indd 118923/02/19 2:21 PM 1190SPECIFIC CONSIDERATIONSPART IIHess10 and Marceau and colleagues170 separately described the

1	Photography © 2005-2009. All Rights Reserved.)Brunicardi_Ch27_p1167-p1218.indd 118923/02/19 2:21 PM 1190SPECIFIC CONSIDERATIONSPART IIHess10 and Marceau and colleagues170 separately described the adaptation of the DS operation, originally proposed by DeMeester and colleagues171 for treatment of bile reflux gastri-tis, to replace the gastric portion of the BPD. This procedure was originally called BPD with DS. For ease of description, it is now simply called the duodenal switch (DS) (Fig. 27-26). Currently, BPD and DS represent together less than 1% to 2% of bariatric operations performed in the United States. Patients who undergo either BPD or DS must be prepared for the consequences of a malabsorptive operation. Frequent and large-quantity bowel movement after any large amount of oral intake is common. Also, patients must agree to close follow-up and a large number of vitamin and mineral supplements to avoid nutritional prob-lems. Given the increased incidence of postoperative

1	intake is common. Also, patients must agree to close follow-up and a large number of vitamin and mineral supplements to avoid nutritional prob-lems. Given the increased incidence of postoperative nutritional and other complications, BPD and DS usually are recommended only for patients who have higher BMIs or for patients who have failed another operation for weight loss or metabolic control. Contraindications to the procedure include geographic distance from the surgeon, lack of financial means to afford supplements, and preexisting calcium, iron, or other nutrient deficiencies.Technique. The technique for BPD and DS is the same for the open and the laparoscopic approach, and they are very techni-cally challenging operations.172 The BPD operation begins with performance of a distal subtotal gastrectomy with a residual 200-mL gastric pouch. The terminal ileum is identified and divided 250 cm proximal to the ileocecal valve. The distal end of that divided ileum is then anastomosed to

1	gastrectomy with a residual 200-mL gastric pouch. The terminal ileum is identified and divided 250 cm proximal to the ileocecal valve. The distal end of that divided ileum is then anastomosed to the stom-ach, creating a 2to 3-cm stoma. The proximal end of the ileum is then anastomosed side-to-side to the terminal ileum Figure 27-26. Diagram of duodenal switch. (Reprinted with permission from Cleveland Clinic Center for Medical Art & Photography © 2005-2009. All Rights Reserved.)approximately 100 cm proximal to the ileocecal valve. Prophy-lactic cholecystectomy is performed due to the high incidence of gallstone formation with the malabsorption of bile salts.The DS procedure differs from the BPD procedure only in the proximal gut portion of the operation. Instead of a distal gas-trectomy, resection of all the stomach except for a narrow lesser curvature tube of the stomach (SG) is performed. The diam-eter of this tube is calibrated with a bougie of 32to 40-French size. The duodenum

1	resection of all the stomach except for a narrow lesser curvature tube of the stomach (SG) is performed. The diam-eter of this tube is calibrated with a bougie of 32to 40-French size. The duodenum is them divided in its first portion, leaving an approximately 2-cm length of duodenum intact beyond the pylorus. This end of the duodenum is then anastomosed to the distal 250 cm of ileum. This anastomosis is often done in an end-to-end fashion with a circular stapler. This is the most dif-ficult portion of the DS procedure, and leak rates are slightly higher than with other anastomoses. The distal bowel configura-tion and cholecystectomy are similar to BPD.Procedure-Specific Complications. Complications that occur after BPD include those seen after RYGB, where intestinal anastomoses and gastric division create potential problems with bleeding and leakage. Scopinaro and col-leagues173 reported GI obstruction in 1.2%, wound infections in 1.2%, and marginal ulcers in 2.8% of patients.

1	gastric division create potential problems with bleeding and leakage. Scopinaro and col-leagues173 reported GI obstruction in 1.2%, wound infections in 1.2%, and marginal ulcers in 2.8% of patients. However, others found the incidence of marginal ulcer to be higher after BPD, leading to the adoption of the DS. Preservation of the pylorus reduces the incidence of dumping (poorly quantitated in most series) after BPD. The duodenoileostomy of DS also has a very low rate of stomal ulcer, unlike the gastroileostomy of BPD.Nutritional complications are by far the most frequent and concerning after both of these operations, particularly on long-term follow-up. Scopinaro and colleagues173 reported a protein malnutrition rate of 7%, iron deficiency anemia rate of less than 5%, and bone demineralization at 5 years of 53%. Other prob-lems that may arise include alopecia from inadequate protein absorption, night blindness from a lack of vitamin A, and gall-stones if the gallbladder is not

1	at 5 years of 53%. Other prob-lems that may arise include alopecia from inadequate protein absorption, night blindness from a lack of vitamin A, and gall-stones if the gallbladder is not removed. However, of all these nutritional complications, protein-calorie malnutrition is the most severe and life-threatening. When it is diagnosed, the treat-ment is parenteral nutrition. Two episodes of required parenteral nutrition are usually considered adequate indication to lengthen the “common channel” of ileum—the ileum between the ileo-ileostomy of the biliopancreatic limb to the alimentary tract and the ileocecal valve. The amount of length that the surgeon should increase the common channel is poorly documented.Investigational Bariatric ProceduresThere is continuous evolution of the approaches to and proce-dures for bariatric surgery. The goals of this dynamic process are to minimize risk, reduce invasiveness, and maximize clini-cal effectiveness. This same benefit-risk approach/paradigm

1	to and proce-dures for bariatric surgery. The goals of this dynamic process are to minimize risk, reduce invasiveness, and maximize clini-cal effectiveness. This same benefit-risk approach/paradigm has also been adopted by the FDA for the design of clinical trials for obesity devices to help facilitate product development and approval.174 In the past, a variety of medical devices to assist with weight reduction have been studied, but only a few have been commercially available. In 2012, to address the need for more intermediate treatment options with devices, the FDA initiated a new paradigm based on a benefit-risk determina-tion to suggest appropriate levels of benefit for devices with different risk levels. In other words, it became more feasible to trial less invasive obesity treatment devices, as the threshold for weight loss was lower if the risk of the device or procedure was lower as well. Since that time, several new devices have been approved. These intermediate devices are

1	devices, as the threshold for weight loss was lower if the risk of the device or procedure was lower as well. Since that time, several new devices have been approved. These intermediate devices are intended to provide 5Brunicardi_Ch27_p1167-p1218.indd 119023/02/19 2:21 PM 1191THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27tools in the middle of the spectrum of care between lifestyle modification and bariatric surgery and are offered to people with BMIs between 30 and 40 kg/m2.The vagus nerve is known to play a role in satiety, metabo-lism, and autonomic control in the upper gastrointestinal tract. Studies have been conducted to determine the efficacy of vagal nerve block therapy with a treatment device that consistently delivers at least 12 hours of therapy a day and a sham control device that has no possibility of delivering therapy. A laparo-scopic abdominal procedure is performed to attach two elec-trodes to the anterior and posterior vagal trunks at the level of the

1	control device that has no possibility of delivering therapy. A laparo-scopic abdominal procedure is performed to attach two elec-trodes to the anterior and posterior vagal trunks at the level of the gastroesophageal junction. Customized electrodes are placed around the nerves and then secured with sutures. These electrodes are then connected to a transcutaneously recharge-able neuroregulator placed in a subcutaneous pocket on the thoracic side wall. Published results show modest weight loss in the vagal nerve blockade group of 9.2% compared to 6.0% TBWL in the sham group at 12 months and 8.8% and 3.8% TBWL in vagal nerve blockade and sham groups, respectively, at 18 months.175,176 More weight loss was sustained through 18 months in the vagal blockade group, and the device was shown to be safe, as there was a low rate of serious complications.Endoscopically placed intragastric balloons (IGBs) are once again an option for overweight and obese patients with a BMI greater than 27 kg/m2.

1	safe, as there was a low rate of serious complications.Endoscopically placed intragastric balloons (IGBs) are once again an option for overweight and obese patients with a BMI greater than 27 kg/m2. The original Garren-Edwards bub-ble (GEB) from the late 1980s was an endoscopically placed and removed balloon filled with 220 mL of air that was left in the stomach for 3 months. Adverse events related to the GEB reported in the medical literature included small-bowel obstruc-tion secondary to unplanned deflation, gastric ulcers with GI hemorrhage, and gastric perforation, so its use was abandoned. A multidisciplinary conference that followed recommended that future IGBs should (a) be effective at promoting weight loss, (b) be filled with liquid (not air), (c) be capable of adjustment to various sizes, (d) have a smooth surface with low ulcerogenic and obstructive potential, (e) contain a radiopaque marker, and (f) be constructed of durable materials.177Newer IGBs have undergone

1	to various sizes, (d) have a smooth surface with low ulcerogenic and obstructive potential, (e) contain a radiopaque marker, and (f) be constructed of durable materials.177Newer IGBs have undergone evaluation and approval by the FDA. These include both a single and a double lumen bal-loon, both placed endoscopically and filled with saline.178,179 Results from these two pivotal trials show weight losses of 7.6% and 10.2% TBWL at 6 months in the device group that exceeded weight loss in the control or sham groups. There were some early removals in 9% to 18% of subjects for failure to tolerate symptoms, early deflations without migration in 6%, and gastric ulcers in 10%. The precise role for these devices is yet to be determined, and they must be paired with a diet and exercise plan to maximize effectiveness. Repeat or sequential balloon therapy may be effective in enhancing and sustaining weight loss, and it is being studied in Europe. Finally, ensuring proper follow-up is important to

1	effectiveness. Repeat or sequential balloon therapy may be effective in enhancing and sustaining weight loss, and it is being studied in Europe. Finally, ensuring proper follow-up is important to reduce adverse events related to ulcers, spontaneous deflation, or migration of the balloon.An endoscopically placed percutaneous gastrostomy tube is approved for weight loss. It facilitates drainage of approxi-mately 30% of the calories consumed in a meal, in conjunction with lifestyle counseling. In a randomized trial, participants lost 12.1% ± 9.6% TBWL compared to 3.5% ± 6.0% TBWL in the lifestyle-only control group. The most frequent complication was abdominal pain and discomfort in the perioperative period and peristomal granulation tissue and peristomal irritation in the postoperative period. Serious adverse events were reported in 3.6% of participants in the device group.180A duodenal-jejunal bypass liner is an endoscopic device that mimics the duodenal-jejunal exclusion component of

1	Serious adverse events were reported in 3.6% of participants in the device group.180A duodenal-jejunal bypass liner is an endoscopic device that mimics the duodenal-jejunal exclusion component of an RYGB and is undergoing trials in the United States. Prior studies assessing the efficacy of the DJBL have shown modest weight loss and improvements in glycemic control. There are associated adverse events of migration, obstruction, and epigas-tric pain. One study demonstrated a high (29%) early device removal rate due to these events.181 A more recent meta-analy-sis showed that the DJBL was associated with significant mean differences in TBWL for the device (12.6%) compared with lifestyle modification. The mean differences in glycated hemo-globin and fasting plasma glucose among subjects with T2DM in this meta-analysis did not reach statistical significance.182Various endoscopic and endoluminal procedures are also being utilized as less invasive approaches for bariatric surgi-cal

1	with T2DM in this meta-analysis did not reach statistical significance.182Various endoscopic and endoluminal procedures are also being utilized as less invasive approaches for bariatric surgi-cal procedures. These include procedures to decrease gastric pouch size and to limit gastrojejunostomy anastomotic size after “failed” LRYGB.183 Overall, reports have been disappointing for effectiveness. Gastric plication is also being approached both laparoscopically and endoscopically to mimic results of an SG but without requiring stapling or gastric resection. Fur-ther studies with long-term safety and efficacy data are required before these investigational procedures can be considered for routine clinical use.FOLLOW-UP AND POSTOPERATIVE CAREPostoperative follow-up is required following bariatric surgery to detect and treat postoperative shortand longer-term complications. Weight regain, internal hernias, ulcerations, and important nutrient deficiencies can occur years after bariatric

1	surgery to detect and treat postoperative shortand longer-term complications. Weight regain, internal hernias, ulcerations, and important nutrient deficiencies can occur years after bariatric surgery. These specific problems are detailed in the “Procedure-Specific Complications” and overall “Complications” sections. The frequency of follow-up varies by surgical procedure and to some extent by surgical practice, but continues, hopefully, for life. Postoperative follow-up is defined as short-term (0–2 years), medium (2–5 years), and long term (≥5 years). Recommendations are that at least 75% of patients are followed for 5 years for LAGB, SG, and LRYGB operations, and 90% are followed closely for 5 years and longer if they have malabsorptive operations (BPD and DS). Although a clinical follow-up system may be in place, it still requires patient compliance, which is generally low for long-term follow-up. In a systematic review, Puzziferri et al also identified that less than 3% of

1	follow-up system may be in place, it still requires patient compliance, which is generally low for long-term follow-up. In a systematic review, Puzziferri et al also identified that less than 3% of bariatric studies included >80% long-term follow-up.184 Vigorous efforts can help to improve follow-up, but these require significant staffing and funding. In the NIH-funded prospective, longitudinal bariatric study, more complete follow-up data and weight measurements were obtained for 79% of RYGB patients in the longer term with the use of these resources.169The goals of short-term follow-up are to maximize care of the patient in the postoperative period; assist in adjustment to new eating, exercise, and lifestyle patterns; be on the alert for and treat postoperative complications; and recommend measures to limit such complications. The goals of long-term follow-up are similar, but focus more on weight regain, the man-agement of comorbid condition relapse, and the emergence of recurrent

1	measures to limit such complications. The goals of long-term follow-up are similar, but focus more on weight regain, the man-agement of comorbid condition relapse, and the emergence of recurrent depression, substance and alcohol misuse, and nutri-tional complications. Vitamin and mineral supplements must be taken regularly for life, including oral supplements for iron, calcium, and vitamin B12 and a multivitamin. Evidence indicates that vitamin and mineral deficiencies, including deficiencies of Brunicardi_Ch27_p1167-p1218.indd 119123/02/19 2:21 PM 1192SPECIFIC CONSIDERATIONSPART IITable 27-4Recommended postoperative nutritional monitoringRECOMMENDATIONLAGBSGLRYGBBPD/DSBone density (DXA)a at 2 yearsYesYesYesYes24 hour urinary calcium excretion at 6 months and annuallyYesYesYesYesVitamin B12 annually (methylmalonic acid and homocysteine optional) then every 3–6 months if supplementedYesYesYesYesFolic acid (red blood cell folic acid optional), iron studies, vitamin D, intact

1	B12 annually (methylmalonic acid and homocysteine optional) then every 3–6 months if supplementedYesYesYesYesFolic acid (red blood cell folic acid optional), iron studies, vitamin D, intact parathyroid hormoneNoNoYesYesVitamin A initially and every 6–12 months thereafterNoNoOptionalYesCopper, zinc, and selenium evaluation with specific findingsNoNoYesYesThiamine evaluation with specific findingsYesYesYesYesaDXA = dual energy X-ray absorptiometry; LAGB = laparoscopic adjustable gastric banding; SG = sleeve gastrectomy; LRYGB = laparoscopic Roux-en-Y gastric bypass; BPD/DS = biliopancreatic diversion with duodenal switch.Data from Mechanick JI, Youdim A, Jones DB, et al: Clinical practice guidelines for the perioperative nutritional, metabolic, and nonsurgical support of the bariatric surgery patient—2013 update: cosponsored by American Association of Clinical Endocrinologists, The Obesity Society, and American Society for Metabolic & Bariatric Surgery, Obesity (Silver Spring). 2013

1	patient—2013 update: cosponsored by American Association of Clinical Endocrinologists, The Obesity Society, and American Society for Metabolic & Bariatric Surgery, Obesity (Silver Spring). 2013 Mar;21 Suppl 1:S1-S27.calcium, vitamin D, iron, zinc, and copper, are common after bariatric surgery.185 Guidelines suggest screening patients for iron, vitamin B12, folic acid, and vitamin D deficiencies preop-eratively, as well.89 Patients should also be given daily nutri-tional supplementation postoperatively, including two adult multivitamin plus mineral supplements (each containing 18 mg of iron, 400 to 800 µg of folic acid, and 50 mg of thiamine), 1200 to 1500 mg of elemental calcium (1800 to 2400 mg for BPD/DS), at least 3000 IU of vitamin D, and vitamin B12, the dose of which varies by route of administration. In addition, all patients should undergo annual screening for specific deficien-cies (Table 27-4).Objective data that should be obtained after all bariatric operations include

1	of administration. In addition, all patients should undergo annual screening for specific deficien-cies (Table 27-4).Objective data that should be obtained after all bariatric operations include weight loss, change in BMI, resolution or improvement in medical comorbidities, and any complications that occur. Assessment of quality of life can help gauge effi-cacy as well, with the Short Form-36 (SF36) questionnaire being one frequently used example. In a retrospective review based on the bariatric outcomes longitudinal database (BOLD) dataset by Spaniolas et al, the effect of postoperative follow-up on 12-month weight loss was studied in 51,081 patients. Com-plete follow-up was independently associated with excess weight loss ≥50% and total weight loss ≥30%.186 To identify the relationship between regular follow-up and resolution of comorbidities, the same group studied a cohort of 46,381 patients (31% RYGB patients) who had minimum of 12-month follow-up. After adjusting for baseline

1	between regular follow-up and resolution of comorbidities, the same group studied a cohort of 46,381 patients (31% RYGB patients) who had minimum of 12-month follow-up. After adjusting for baseline characteristics, the group determined that complete follow-up in the first year after RYGB was independently associated with a higher rate of improvement or remission of comorbid conditions (T2DM, hypertension, and dyslipidemia).187 Frequent and protocolized band adjustments and postoperative support individual/group sessions were shown to be important for longer-term outcomes following LAGB.188 Finally, the 12-month postoperative visit, which coincides with the plateauing of weight loss for most procedures, presents an opportunity to intervene while bariat-ric surgery patients are still engaged. Engaging patients and the use of technology to maintain contact with a medical pro-vider are important tools to maintain follow-up after bariatric surgery.RESULTS OF BARIATRIC SURGERYShort-Term

1	Engaging patients and the use of technology to maintain contact with a medical pro-vider are important tools to maintain follow-up after bariatric surgery.RESULTS OF BARIATRIC SURGERYShort-Term OutcomesThe short-term (1–2 year) outcomes for bariatric surgical pro-cedures are shown in Table 27-5, which summarizes of the majority of the literature from 2009 to 2017. Average 30-day mortality is low (<1.0%) for all procedures except BPD/DS. Mortality after LRYGB is now consistently less than 0.3% to 0.5% in most large reported series.59,140 Morbidity varies by procedure, but it is the lowest for LAGB, followed by SG and then LRYGB, and highest for the malabsorptive procedure BPD/DS. In the Longitudinal Assessment of Bariatric Sur-gery (LABS) study, a composite endpoint including death, deep-vein thrombosis or venous thromboembolism, reinter-vention, or failure to be discharged by 30 days after surgery occurred in 4.8% of those who had undergone LRYGB.59 Short-term results of the SG have

1	thrombosis or venous thromboembolism, reinter-vention, or failure to be discharged by 30 days after surgery occurred in 4.8% of those who had undergone LRYGB.59 Short-term results of the SG have been reported from large national databases. These data show that SG is positioned between LAGB and LRYGB for efficacy of weight loss and resolution of comorbid medical problems and for morbidity and mortality.140 Few longer-term results with SG have been published.189,190In the past, large institutional series of LAGB results have been published from centers in Europe and Australia, showing better results for weight loss than those that have been observed in the United States (13–22% TBWL) (see Table 27-5). Weight loss results with BPD or DS are both excellent and comparable but come with higher surgical morbidity. The results from malabsorptive procedures are also very durable for the small percentage of people who undergo them. One 18-year follow-up study after BPD showed a mean excess

1	surgical morbidity. The results from malabsorptive procedures are also very durable for the small percentage of people who undergo them. One 18-year follow-up study after BPD showed a mean excess weight loss of 70% persisting for that duration of time.173 Although most of the results of BPD or DS are after open operations, one report of laparoscopic DS at an experienced center showed that for 40 patients with an average BMI of 60 kg/m2 the mean hospital stay was 4 days, average operation room time was 3.5 hours, and mean excess weight loss at 9 months was 58%.191 Buchwald and colleagues showed that the average weight loss after BPD and DS in the literature was over 70%, with a mortality rate of 1.1%, a complication rate of 27% to 33%, and a nutritional complication rate of 40% to 77%192 (see Table 27-5).Brunicardi_Ch27_p1167-p1218.indd 119223/02/19 2:21 PM 1193THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27Table 27-5Short-term bariatric surgical outcomes OUTCOMEa ALLSURGICAL

1	77%192 (see Table 27-5).Brunicardi_Ch27_p1167-p1218.indd 119223/02/19 2:21 PM 1193THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27Table 27-5Short-term bariatric surgical outcomes OUTCOMEa ALLSURGICAL PROCEDURELRYGBSGLAGBBPD/DS% Mortality 30-day<1.0%0.3–0.5%0.11%0.05%1.1%% Morbidity 30-dayNA, depends on procedure12–21%3–6%2–4%27–33%% Total body weight loss (TBWL)NA, depends on procedure31–36%25–30%13–22%36–38%% Excess body weight loss (EBWL)NA, depends on procedure48–77%(mean, 68%)49–81%29–50%>70%% Diabetes remission77%60–80%60%35%75%% Dyslipidemiab remission70%63–91%72–82%78%80%% Hypertension remission62%61–81%60–92%43%60%, few reports% Sleep apnea remissionc84%80%80%68%80%aOutcomes are the averages found in the literature at 1 to 2 years postoperatively, unless otherwise noted.bDenotes any component lipid remission.cDenotes clinical remission as repeat sleep studies are uncommonly performed.Further detail on surgical morbidity for each procedure is addressed in both the

1	any component lipid remission.cDenotes clinical remission as repeat sleep studies are uncommonly performed.Further detail on surgical morbidity for each procedure is addressed in both the “Procedure-Specific Complications” and overall “Complications” sections.Effectiveness of Bariatric Surgery Compared to Nonsurgical TreatmentThe following section summarizes important findings of studies that compare bariatric procedures with nonsurgical management of obesity. The results of these studies concerning remission from T2DM will be discussed in more detail in “Results of Surgery for Diabetes.” A systematic review and meta-analysis by Gloy summarized all randomized controlled trials (RCTs) that compared bariatric surgery with nonsurgical treatments for obesity.193 The review analyzed 11 trials comprising nearly 800 people with a BMI of 30 to 52. These studies generally focused on cohorts with T2DM and 1 to 2 years of follow-up. They pro-vided good evidence of the effectiveness of bariatric

1	comprising nearly 800 people with a BMI of 30 to 52. These studies generally focused on cohorts with T2DM and 1 to 2 years of follow-up. They pro-vided good evidence of the effectiveness of bariatric procedures, including LRYGB,78-80 LAGB,77,194 BPD,79 and SG.78 These pro-cedures resulted in greater short-term (1–2 years) weight loss (mean difference −26 kg; 95% CI −31 to −21; P <0.001) and greater remission of T2DM (complete case analysis relative risk of remission: 22.1, 3.2–154.3; P = 0.002; conservative analysis: 5.3, 1.8–15.8; P = 0.003) compared with various nonsurgical treatments.77-80,194 After this meta-analysis, two additional RCTs were published that show similar short-term results for both weight loss and T2DM.83,84In addition, serum triglycerides and high-density lipoproteins were significantly reduced by bariatric procedures, but blood pressure and other lipoproteins were not (although some studies showed reduced medication use for these conditions).193 The Gloy review

1	were significantly reduced by bariatric procedures, but blood pressure and other lipoproteins were not (although some studies showed reduced medication use for these conditions).193 The Gloy review also noted a lack of evidence from RCTs beyond 2 years for mortality, cardiovascular diseases, and adverse events. Another systematic review by Maggard-Gibbons focused on weight loss and glycemic control in class I obese (BMI 30–34.9) adults with T2DM and identified three RCTs with results similar to those seen in class II (BMI 35–39.9) and severely obese populations. However, the review also noted a lack of longer-term studies in people with class I obesity.195Longer-Term StudiesA summary of studies with long-term outcomes are shown in Table 27-6. The following section describes these studies and other larger studies that have contributed data to the growing body of evidence with respect to some short-term and now much longer-term outcomes.Swedish Obese Subjects Study. Much of what is

1	and other larger studies that have contributed data to the growing body of evidence with respect to some short-term and now much longer-term outcomes.Swedish Obese Subjects Study. Much of what is currently known about the long-term results of bariatric surgery come from the Swedish Obese Subjects (SOS) study, which was initi-ated in 1987 as a prospective trial of 2010 subjects undergo-ing bariatric surgery compared to a usual care control group (n = 2037) that were matched on 18 clinical and demographic variables. The most common bariatric procedure performed in SOS was the VBG (68%), followed by gastric banding (19%), and RYGB (13%). Follow-up rates are high and reported at 99% for some endpoints (including mortality). The SOS investiga-tors have published widely on health outcomes beyond 10 years and up to 20 years, including: weight loss, mortality, T2DM remission and incidence, cardiovascular events, incident cancer, psychosocial outcomes, and health care use and costs. Weight

1	10 years and up to 20 years, including: weight loss, mortality, T2DM remission and incidence, cardiovascular events, incident cancer, psychosocial outcomes, and health care use and costs. Weight loss among surgical subjects in SOS was greater than in con-trol subjects (mean changes in body weight at 2, 10, 15, and 20 years were −23%, −17%, −16%, and −18% in the surgery group and 0%, 1%, −1%, and −1% in the control group). After 15 years, the mean percent weight loss by procedure type was 27 + 12% for RYGB, 18 + 11% for VBG, and 13 + 14% for gastric banding.The SOS study also showed major improvements in obesityrelated comorbidities. In the surgical group, there was a 72% remission of T2DM after 2 years (OR for remission: 8.4) and 36% durable remission after 10 years (OR for remission: 3.5). In spite of the considerable relapse of T2DM over time, bariatric surgery was associated with a lower incidence of myocardial infarction and other T2DM complications. The SOS study dem-onstrated

1	In spite of the considerable relapse of T2DM over time, bariatric surgery was associated with a lower incidence of myocardial infarction and other T2DM complications. The SOS study dem-onstrated that bariatric surgery also reduced the risk of incident T2DM by 96%, 84%, and 78% after 2, 10, and 15 years among subjects without the condition at baseline. The SOS study also 6Brunicardi_Ch27_p1167-p1218.indd 119323/02/19 2:21 PM 1194SPECIFIC CONSIDERATIONSPART IITable 27-6Long-term studies of bariatric surgery outcomesaAUTHORSTUDY DESIGNPOPULATIONS AND PROCEDURESFOLLOW-UP DURATIONPUBLISHED OUTCOMESMORTALITY AND SURVIVALLIMITATIONSSjöström et al,196-201 2004, 2007, 2009, and 2012 (Swedish Obese Subjects study [SOS])Prospective observational with matched controls2010 Surgical cases (13% RYGB; 19% banding; 68% VBG) and 2037 matched controls10–20 years, depending on the reportSurgery was associated with: greater weight loss at 2 years (–23% vs. 0%) and at 20 y (–18% vs. –1%)16;

1	RYGB; 19% banding; 68% VBG) and 2037 matched controls10–20 years, depending on the reportSurgery was associated with: greater weight loss at 2 years (–23% vs. 0%) and at 20 y (–18% vs. –1%)16; greater remission of T2DM after 2 y (OR for remission, 8.4; P <.001) and 10 y (OR, 3.5; P <.001); lower incidence of T2DM (HR, 0.17; P <.001)Bariatric surgery treatment: 16 years, 29% lower risk of death from any cause (hazard ratio 0.71, 0.54 to 0.92; P = 0.01) vs. usual care; common causes of death: myocardial infarction (HR, 0.71; P = .02), stroke (HR, 0.66; P = .008), and cancer (in women only; HR, 0.58; P <.001)Not randomized; includes mostly procedures (87%) that are no longer in use; involves patients from a single country with little racial/ethnic diversityAdams et al,202 2007 (Utah Mortality study)Retrospective observational with matched controls7925 RYGB cases and 7925 weight-matched controlsMean, 7.1 yearsOnly mortality outcomes reportedBariatric surgery treatment:

1	(Utah Mortality study)Retrospective observational with matched controls7925 RYGB cases and 7925 weight-matched controlsMean, 7.1 yearsOnly mortality outcomes reportedBariatric surgery treatment: average 7.1 years post treatment, 40% reduction in all cause mortality (hazard ratio HR 0.60, 0.45 to 0.67; P <0.001), 49% (HR 0.51, 0.36 to 0.73; P <0.001), and 92% (HR 0.08, 0.01 to 0.47; P = 0.005), cardiovascular mortality, and T2DM mortality, respectivelyNot randomized; matching based on self-reported height and weight from driver’s license database; includes only RYGB procedures; patients from a single stateAdams et al,203,204 2012, 2017 (Utah Obesity study)Prospective observational with two matched control groups418 RYGB cases; 417 bariatric-surgery seekers who did not undergo operation (control 1); 321 population-based matched controls (control 2)6 years, 12 years6 years: RYGB group lost 27.7% body weight compared with 0.2% weight gain in control group 1 and 0% change in

1	(control 1); 321 population-based matched controls (control 2)6 years, 12 years6 years: RYGB group lost 27.7% body weight compared with 0.2% weight gain in control group 1 and 0% change in control group 2; T2DM remission in 62% of RYGB patients and 8% and 6% in each of the control groups (P <.001); incident T2DM was observed in 2% of RYGB patients but 17% and 15% of each of the control groups at 6 years (P <.001); surgery associated with greater improvements in blood pressure, cholesterol, and quality of life (P <.01)Deaths at 6 years: 12 (2.8%), 14 (3.3%), and 3 (0.93%) for bariatric surgery, control 1, and control 2, respectivelyNot randomized; includes only RYGB procedures; patients from a single stateBrunicardi_Ch27_p1167-p1218.indd 119423/02/19 2:21 PM 1195THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 2712 years: RYGB 26.9% body weight loss, 2.0% and 0.9% in control groups 1, 2, respectively; T2DM remission in 51% RYGB group; odds ratio for the incidence T2DM 0.08 (95% CI,

1	OF OBESITYCHAPTER 2712 years: RYGB 26.9% body weight loss, 2.0% and 0.9% in control groups 1, 2, respectively; T2DM remission in 51% RYGB group; odds ratio for the incidence T2DM 0.08 (95% CI, 0.03 to 0.24) for RYGB vs. control group 1 and 0.09 (95% CI, 0.03 to 0.29) RYGB vs. control group 2 (P <0.001 for both comparisons); RYGB group had higher remission rates and lower incidence rates of hypertension and dyslipidemia than did control group 1 (P <0.05 for all comparisons).Maciejewski et al,205-208 2011, 2012, 2015, 2016 (Department of Veterans Affairs)Retrospective observational with matched controls847 to 1787 to 2500 surgical cases and their matched controls6.7 yearsPatients undergoing RYGB lost 28.6% (95% CI, 19.5%–37.6%) of their baseline weight at 10 years, whereas nonsurgical matches lost 7.3% (95% CI, 1.4%–13.3%) of their baseline weight at 10 years. Patients undergoing RYGB lost 21% (95% CI, 11%–31%) more of their baseline weight at 10 years than nonsurgical matches. A

1	matches lost 7.3% (95% CI, 1.4%–13.3%) of their baseline weight at 10 years. Patients undergoing RYGB lost 21% (95% CI, 11%–31%) more of their baseline weight at 10 years than nonsurgical matches. A total of 405 of 564 patients undergoing RYGB (71.8%) had more than 20% body weight loss and 224 of 564 (39.7%) had more than 30% estimated weight loss at 10 years.At 4 years, patients undergoing LRYGB lost 27.5% (95% CI, 23.8%–31.2%) of their baseline weight, patients undergoing LAGB lost 10.6% (95% CI, 0.6%–20.6%), and patients undergoing SG lost 17.8% (95% CI, 9.7%–25.9%).Surgery was not significantly associated with lower health expenditures 3 years after the procedure, in first study.First study, in 2011, bariatric surgery not significantly associated with reduced mortality.Later study, at the end of 14 years, 263 deaths in the surgical group (mean follow-up, 6.9 years) and 1277 deaths in control group (mean follow-up, 6.6 years). Mortality rates were 2.4% at 1 year, 6.4% at 5

1	at the end of 14 years, 263 deaths in the surgical group (mean follow-up, 6.9 years) and 1277 deaths in control group (mean follow-up, 6.6 years). Mortality rates were 2.4% at 1 year, 6.4% at 5 years, and 13.8% at 10 years for surgical patients; for matched control patients, 1.7% at 1 year, 10.4% at 5 years, and 23.9% at 10 years. Significantly lower mortality after 1 to 5 years (HR, 0.45 [95% CI, 0.36–0.56]) and 5 to 14 years (HR, 0.47 [95% CI, 0.39–0.58]).Not randomized; includes older (mean age, 55 years), primarily male (74%) veterans; mortality studies mostly RYGB procedures(Continued)Brunicardi_Ch27_p1167-p1218.indd 119523/02/19 2:21 PM 1196SPECIFIC CONSIDERATIONSPART IICourcoulas et al,59,169 2009, 2013, 2017 (Longitudinal Assessment of Bariatric Surgery [LABS])Prospective observational4776 in LABS-1, 30 day safety study and 2458 in LABS-2, effectiveness study (70.7% RYGB; 24.8% LAGB; and 5% other procedures)30 days, 3 years, 7 years3 year: 31.5% for RYGB and 15.9% for

1	in LABS-1, 30 day safety study and 2458 in LABS-2, effectiveness study (70.7% RYGB; 24.8% LAGB; and 5% other procedures)30 days, 3 years, 7 years3 year: 31.5% for RYGB and 15.9% for LAGB; T2DM remission in 67.5% of RYGB cases and 28.6% for LAGB; incidence of T2DM was 0.9% after RYGB and 3.2% after LAGB. Dyslipidemia remission in 61.9% RYGB cases and 27.1% AGB cases; HTN remission in 38.2% RYGB cases and 17.4% AGB cases; other procedures’ results not reported7 year: 28.4% for RYGB and 14.9% for LAGB; T2DM remission in 60.2% of RYGB cases and 20.3% for LAGB30 days: 0.3% overall; 0% LAGB, 0.2% LRYGB, and 2.1% open RYGB.3 years: 0.9 per 300 person-years for TYGB and 0.8 per 300 person-years for LAGB, i.e., number of events if 100 people were followed for 3 years7 years: 3.7 per 700 person-years for RYGB and 2.7 per 700 person years for LAGB, i.e., number of events if 100 people were followed for 7 yearsNot randomized; lacks nonsurgical control population; primarily RYGB and LAGB

1	for RYGB and 2.7 per 700 person years for LAGB, i.e., number of events if 100 people were followed for 7 yearsNot randomized; lacks nonsurgical control population; primarily RYGB and LAGB procedures; high follow-up rates (>80% for weight) but some not in-personArterburn et al,212,213 2013 (HMO Research Network)1. Retrospective observational2. Retrospective observational with matched controls4434 RYGB cases with T2DM1395 cases; 72% RYGB, 2% SG, 4% LAGB, 13% otherMedian, 3.1 years2 years68% of patients (95% CI, 66–70) experienced an initial T2DM remission within 5 years after RYGB; among these, 35.1% (95% CI, 32–38) redeveloped T2DM within 5 years; median duration of T2DM remission, 8.3 yearsBariatric cases higher T2DM remission rates [73.7% (95% CI: 70.6, 76.5)] vs. controls [6.9% (95%CI: 6.9, 7.1)]. Bariatric cases lower relapse rates T2DM than controls (adjusted HR: 0.19; 95% CI: 0.15–0.23)Not reportedNo higher risk of death in control vs. bariatric within 2 years (adjusted HR =

1	6.9, 7.1)]. Bariatric cases lower relapse rates T2DM than controls (adjusted HR: 0.19; 95% CI: 0.15–0.23)Not reportedNo higher risk of death in control vs. bariatric within 2 years (adjusted HR = 0.54; 95% CI: 0.22 to 1.23).1. Not randomized; lacks nonsurgical control population; only RYGB procedures2. Not randomized, electronic medical record data, small number SGCarlin et al,215 2013 (Michigan Bariatric Surgery Collaborative)Prospective observational8847 to 35,477, varies depending on publication30 days to 3 years, varies depending on publicationComplication rates for SG (6.3%) were significantly lower than for RYGB (10.0%; P <.001) but higher than LAGB (2.4%; P <.001). Excess body weight loss at 1 year was 13% lower for SG (60%) than for RYGB (69%; P <.001) but was 77% higher for SG than for LAGB (34%; P <.001).Not reportedNot randomized; lacks nonsurgical control; patients from a single stateaData from Courcoulas AP, Yanovski SZ2, Bonds D, et al: Long-term outcomes of bariatric

1	than for LAGB (34%; P <.001).Not reportedNot randomized; lacks nonsurgical control; patients from a single stateaData from Courcoulas AP, Yanovski SZ2, Bonds D, et al: Long-term outcomes of bariatric surgery: a National Institutes of Health symposium, JAMA Surg. 2014 Dec;149(12):1323-1329.Table 27-6Long-term studies of bariatric surgery outcomesa (Continued)AUTHORSTUDY DESIGNPOPULATIONS AND PROCEDURESFOLLOW-UP DURATIONPUBLISHED OUTCOMESMORTALITY AND SURVIVALLIMITATIONSBrunicardi_Ch27_p1167-p1218.indd 119623/02/19 2:21 PM 1197THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27found that bariatric surgery was associated with a reduced inci-dence of fatal or nonfatal cancer among women but not in men. Finally, at 16 years follow-up, surgery was associated with a 29% lower risk of death (the primary endpoint of the study) from any cause compared to usual care, with the most common causes of death being cancer and myocardial infarction196-201 (see Table 27-6).Utah Obesity Studies. Another

1	primary endpoint of the study) from any cause compared to usual care, with the most common causes of death being cancer and myocardial infarction196-201 (see Table 27-6).Utah Obesity Studies. Another important long-term obser-vational study performed in Utah from 1984 to 2002 included 7925 people who had undergone RYGB and 7925 weight, age, and sex matched controls. This study showed a 40% reduction in all-cause mortality (hazard ratio 0.60, 0.45 to 0.67; P <0.001) and a 49% (0.51, 0.36 to 0.73; P <0.001) and 92% (0.08, 0.01 to 0.47; P = 0.005) reduction in death from cardiovascular disease and death related to T2DM, respectively, at an average of 7.1 years later.202A separate ongoing prospective Utah Obesity Study involving over 400 RYGB cases and two nonrandomized, matched control groups—each with over 400 and 300 severely obese subjects (one group were people seeking surgery that did not undergo operation; the other was a population-based severely obese control group)—has found

1	with over 400 and 300 severely obese subjects (one group were people seeking surgery that did not undergo operation; the other was a population-based severely obese control group)—has found that the surgery group lost 27.7% of their initial body weight compared to 0.2% weight gain in control group 1 (surgery seekers), and 0% change in control group 2 (population-based control) at 6 years. Diabetes was in remission in 62% of RYGB patients and only 8% and 6% in each of the control groups, while incident T2DM was observed in 2% of RYGB patients and in 17% and 15% of the control groups at 6 years.203 The 12-year follow-up results were also recently published and showed long-term durability of weight loss and effective remission and prevention of T2DM, hypertension, and dyslipidemia after RYGB.204 Follow-up rates in this study were high at over 90% at 12 years. The mean per-cent change from baseline in body weight in the RYGB group was −26.9% at 12 years compared to −2.0% and 0.9% in each

1	Follow-up rates in this study were high at over 90% at 12 years. The mean per-cent change from baseline in body weight in the RYGB group was −26.9% at 12 years compared to −2.0% and 0.9% in each of the two control groups. There was remission of T2DM in 43 of 84 patients (51%) at 12 years. The OR for the incidence of T2DM at 12 years was 0.08 (95% CI, 0.03–0.24) for the RYGB group versus control group 1 and 0.09 (95% CI, 0.03–0.29) for the RYGB group versus control group 2 (P <0.001 for both comparisons). The RYGB group had higher remission rates and lower incidence rates of hypertension and dyslipidemia than did control group 1 (P <0.05 for all comparisons) (see Table 27-6).Veteran’s Administration Study. A retrospective obser-vational study involving 847 U.S. veterans who were older and more high risk than in typical bariatric studies, found no significant association between bariatric surgery and survival compared to usual care at mean 6.7 years follow-up.205 When matched to control

1	high risk than in typical bariatric studies, found no significant association between bariatric surgery and survival compared to usual care at mean 6.7 years follow-up.205 When matched to control subjects, the outpatient, inpatient, and total expenditures were higher for bariatric surgical cases in the 3 years leading up to the procedure and then went back to the lower cost levels of nonsurgical controls in 3 years after the procedure. The conclusion from this study was that bariatric surgery did not appear to be associated with reduced health care expenditures 3 years after the procedure.206In a follow-up retrospective cohort study, 2500 U.S. veterans (74% men) who underwent bariatric surgery (74% gastric bypass, 15% sleeve gastrectomy, 10% adjustable gastric banding, and 1% other) were matched to 7462 control patients. The primary outcome was all-cause mortality. In this study, surgical patients (n = 2500) had a mean age of 52 years and a mean BMI of 47. Matched control patients (n

1	matched to 7462 control patients. The primary outcome was all-cause mortality. In this study, surgical patients (n = 2500) had a mean age of 52 years and a mean BMI of 47. Matched control patients (n = 7462) had a mean age of 53 years and a mean BMI of 46. At the end of the 14-year study period, there were a total of 263 deaths in the surgical group (mean follow-up, 6.9 years) and 1277 deaths in the matched control group (mean follow-up, 6.6 years). Mor-tality rates were 2.4% at 1 year, 6.4% at 5 years, and 13.8% at 10 years for surgical patients; for matched control patients, 1.7% at 1 year, 10.4% at 5 years, and 23.9% at 10 years. So, in this later study, there was significantly lower all-cause mortal-ity at longer follow-up. 207Ten-year weight change in 1787 veterans who underwent RYGB compared to controls, and separately, 4-year weight change in veterans who underwent RYGB (n = 1785), SG (n = 379), and AGB (n = 246) were reported. Patients undergo-ing RYGB lost 21% more of their

1	compared to controls, and separately, 4-year weight change in veterans who underwent RYGB (n = 1785), SG (n = 379), and AGB (n = 246) were reported. Patients undergo-ing RYGB lost 21% more of their baseline weight at 10 years than nonsurgical matches. A total of 405 of 564 patients under-going RYGB (71.8%) had more than 20% weight loss, and 224 of 564 (39.7%) had more than 30% weight loss at 10 years com-pared with 134 of 1247 (10.8%) and 48 of 1247 (3.9%), respec-tively, for nonsurgical matches. At 4 years, patients undergoing LRYGB lost 27.5% of their baseline weight, patients undergo-ing LAGB lost 10.6%, and patients undergoing SG lost 17.8%. Patients undergoing RYGB lost 16.9% more of their baseline weight than patients undergoing AGB and 9.7% more than patients undergoing SG208 (see Table 27-6).The Longitudinal Assessment of Bariatric Surgery Study. The Longitudinal Assessment of Bariatric Surgery (LABS-1) study, a multicenter observational surgical cohort, prospectively

1	(see Table 27-6).The Longitudinal Assessment of Bariatric Surgery Study. The Longitudinal Assessment of Bariatric Surgery (LABS-1) study, a multicenter observational surgical cohort, prospectively assessed 30-day safety among 4776 severely obese patients who underwent a first bariatric surgical procedure (25% AGB, 62% laparoscopic RYGB, 9% open RYGB, and 3% another procedure) between 2005 and 2007.59 The 30-day mortality in the LABS study was 0.3% for all procedures with a major adverse outcome rate (a predefined composite endpoint that included death, venous thromboembolism, reintervention [percutaneous, endoscopic, or operative], or failure to be discharged from the hospital in 30 days) of 4.1%. These results did vary by procedure and approach, with no mortality in the 1198 patients who had undergone LAGB, 0.2% of the 2975 patients who had undergone LRYGB, and 2.1% of the 437 patients who had undergone open RYGB. Similarly, the rate of adverse outcomes (morbidity) occurred in 4.1%

1	had undergone LAGB, 0.2% of the 2975 patients who had undergone LRYGB, and 2.1% of the 437 patients who had undergone open RYGB. Similarly, the rate of adverse outcomes (morbidity) occurred in 4.1% of patients overall; 1.0% for LAGB, 4.8% for LRYGB, and 7.8% for open RYGB.59 The Longitudinal Assessment of Bariatric Surgery (LABS-2) study is another large prospective multicenter observational bariatric cohort study that was not randomized and did not include a nonsurgical control group. LABS-2 assessed weight change and comorbid conditions in 2458 participants (1738 RYGB—both open and laparoscopic, 610 LAGB, and 110 other procedures) recruited between 2005 and 2009 who were followed for 7 years.169,209 At baseline, 33% had diabetes, 63% had dyslipidemia, and 68% had hypertension. In the LABS-2 cohort, median weight change was 31.5% for RYGB and 15.9% for adjustable gastric banding after 3 years, with much variability in response to each surgical treatment. Remission of T2DM was noted

1	cohort, median weight change was 31.5% for RYGB and 15.9% for adjustable gastric banding after 3 years, with much variability in response to each surgical treatment. Remission of T2DM was noted in 67% and 28% of those who had undergone RYGB and LAGB, respectively. The incidence T2DM was 0.9% and 3.2%, respectively, over the 3 years169 (see Table 27-6). LABS-2 looked at both preand postoperative predictors of weight change and found that very few of many baseline variables studied (Black race, T2DM) were associated Brunicardi_Ch27_p1167-p1218.indd 119723/02/19 2:21 PM 1198SPECIFIC CONSIDERATIONSPART IIwith 3-year weight change, and the effects were small overall. Postoperatively, for RYGB only, three behaviors explained most of the variability (16%) in 3-year weight change: weekly self-weighing, continuing to eat when feeling full more than once a week, and eating continuously during the day. If a person started weekly self-weighing, stopped eating when feeling full, and stopped

1	continuing to eat when feeling full more than once a week, and eating continuously during the day. If a person started weekly self-weighing, stopped eating when feeling full, and stopped eating continuously during the day, they lost 14% more weight than those who made no positive changes (38.8% vs. 24.6% TBWL).210,211At 7 years of follow-up, in LABS, data completeness for weight was high (83%), and the median weight change was 28.4% for RYGB and 14.9% for LAGB. Weight regain between years 3 and 7 was 3.9% of baseline weight for RYGB and 1.4% for LAGB, but 75% of RYGB participants maintained at least 20% total body weight loss, and 50% of LAGB participants maintained at least 16% through 7 years. Remission of T2DM was reported in 60.2% of RYGB cases and 20.3% for LAGB. Mortality was reported as 3.7 per 700 person-years for RYGB and 2.7 per 700 person years for LAGB, i.e., number of events if 100 people were followed for 7 years. Reoperations were also much more common after LAGB

1	reported as 3.7 per 700 person-years for RYGB and 2.7 per 700 person years for LAGB, i.e., number of events if 100 people were followed for 7 years. Reoperations were also much more common after LAGB compared to RYGB at 7 years (see Table 27-6).HMO Research Network. Arterburn and colleagues have leveraged the integrated health network system to study bariatric outcomes using the electronic health/medical record. They studied clinical predictors of diabetes remission and relapse among patients undergoing gastric bypass. Theirs was a retrospective cohort study of adults with uncontrolled or medication-controlled T2DM who underwent gastric bypass in three integrated health care delivery systems in the United States. Remission and relapse events were defined by diabetes medication use and clinical laboratory measures of glycemic control. Of 4434 adults with T2DM who underwent RYGB, 68.2% (95% CI, 66% and 70%) experienced an initial complete diabetes remission within 5 years after

1	and clinical laboratory measures of glycemic control. Of 4434 adults with T2DM who underwent RYGB, 68.2% (95% CI, 66% and 70%) experienced an initial complete diabetes remission within 5 years after surgery. Among these, 35.1% (95% CI, 32% and 38%) relapsed back to T2DM within 5 years. The median duration of their remission was 8.3 years. Predictors of incomplete remission and relapse were poor preoperative glycemic control, insulin use, and longer diabetes duration212 (see Table 27-6).In a second study, they compared rates of diabetes remission, relapse, and all-cause mortality at 2 years between severely obese adults with T2DM who underwent bariatric surgery or received usual medical care. There were 1395 adults with T2DM who had bariatric surgery and 62,322 who did not. Most procedures were RYGB (72.0% laparoscopic; 8.2% open); 4.4% were gastric banding, 2.4% were sleeve gastrectomy, and 13.2% were other procedures. At 2 years, bariatric subjects experienced significantly higher

1	were RYGB (72.0% laparoscopic; 8.2% open); 4.4% were gastric banding, 2.4% were sleeve gastrectomy, and 13.2% were other procedures. At 2 years, bariatric subjects experienced significantly higher diabetes remission rates (73.7% [95% CI: 70.6, 76.5]) compared to nonsurgical subjects (6.9% [95% CI: 6.9, 7.1]). Age, site, duration of diabetes, hemoglobin A1c level, and intensity of diabetes medication treatment were significantly associated with remission. Bariatric subjects also experienced lower relapse rates than nonsurgical subjects (adjusted HR: 0.19; 95% CI: 0.15–0.23) with no higher risk of death (adjusted HR: 0.54; 95% CI: 0.22–1.30) (see Table 27-6).213 This group also studied short-term comparative effectiveness outcomes between procedures (LRYGB and LAGB) and found that LRYGB resulted in much greater weight loss than LAGB but had a higher risk of short-term complications and long-term subsequent hospitalizations.214Michigan Bariatric Surgery Collaborative. The Michigan

1	resulted in much greater weight loss than LAGB but had a higher risk of short-term complications and long-term subsequent hospitalizations.214Michigan Bariatric Surgery Collaborative. The Michigan Bariatric Surgery Collaborative is a statewide consortium of hospitals and surgeons that maintains an externally audited prospective clinical registry. The comparative effectiveness of SG, LRYGB, and LAGB procedures was studied in this data-set. Nearly 3000 SG patients with equal numbers of RYGB and LAGB patients were matched on 23 baseline characteristics. Outcomes assessed included 30-day complications, weight loss, quality of life, and comorbid remission up to 3 years after bar-iatric surgery. Overall complication rates for SG (6.3%) were significantly lower than for RYGB (10%) but higher than for LAGB (2.4%). Serious complication rates were similar for SG (2.4%) and LRYGB (2.5%) but higher than for LAGB (1.0%). Excess body weight loss at 1 year was 13% lower for SG (60%) than for RYGB

1	for LAGB (2.4%). Serious complication rates were similar for SG (2.4%) and LRYGB (2.5%) but higher than for LAGB (1.0%). Excess body weight loss at 1 year was 13% lower for SG (60%) than for RYGB (69%), but was 77% higher for SG than for LAGB (34%). Remission of comorbid conditions was similar between SG and LRYGB215 (see Table 27-6).This group also developed a risk prediction model for seri-ous 30-day complications after bariatric surgery. Overall, 2.5% of patients experienced a serious complication. Significant risk factors included prior venous thromboembolism (OR 1.90, CI 1.41–2.54); mobility limitations (OR 1.61, CI 1.23–2.13); coro-nary artery disease (OR 1.53, CI 1.17–2.02); age over 50 (OR 1.38, CI 1.18–1.61); pulmonary disease (OR 1.37, CI 1.15–1.64); male gender (OR 1.26, CI 1.06–1.50); smoking history (OR 1.20, CI 1.02–1.40); and procedure type.216 Further, to assess the rela-tionship between IVC filter insertion and complications while controlling for differences in

1	CI 1.06–1.50); smoking history (OR 1.20, CI 1.02–1.40); and procedure type.216 Further, to assess the rela-tionship between IVC filter insertion and complications while controlling for differences in baseline patient characteristics and medical venous thromboembolism prophylaxis, this group pub-lished an additional study, and 35,477 patients from 32 hospitals in Michigan were included. Patients receiving IVC filters had higher rates of pulmonary embolism, deep vein thrombosis, venous thromboembolism, serious complications, and death.217Recently, this group has also been evaluating the effect of surgical skill and operative technique on complications follow-ing bariatric surgery.218,219Other StudiesMetabolic and Bariatric Surgery Quality Improvement Pro-gram (MBASQIP). This is a prospective, multi-institutional, national database that has been used to compare SG to RYGB and LAGB. The study from 2011 was short term and compared 30-day, 6-month, and 1-year outcomes including morbidity

1	multi-institutional, national database that has been used to compare SG to RYGB and LAGB. The study from 2011 was short term and compared 30-day, 6-month, and 1-year outcomes including morbidity and mortality, readmissions, and reoperations as well as reduction in BMI and weight-related comorbid conditions. The findings were that SG has higher risk-adjusted morbidity, readmission and reop-eration/intervention rates compared to LAGB, but lower reopera-tion/intervention rates compared to RYGB either laparoscopic or open. There were no differences in mortality. Reduction in BMI and most of the weight-related comorbidities after SG was also between LAGB and RYGB rates.140A later study addressed the impact of various SG techniques on short-term (30-day) outcomes. Using the MBSAQIP data reg-istry, 189,477 SG operations that were performed at over 700 cen-ters in the United States were analyzed. Cases in which staple line reinforcement was used were associated with higher leak rates and

1	189,477 SG operations that were performed at over 700 cen-ters in the United States were analyzed. Cases in which staple line reinforcement was used were associated with higher leak rates and lower bleeding rates. Bougie size ≥38 French was associated with significantly lower leak rates compared to <38 French.162 Longer-term data will eventually be available from this national dataset, but the completeness of follow-up has not yet been determined.Geisinger Health System. This is an electronic medical record database in a large rural integrated health system. They Brunicardi_Ch27_p1167-p1218.indd 119823/02/19 2:21 PM 1199THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27have published long-term results (7–12 years) of the percent-age of TBWL and preoperative predictors for LRYGB in approximately 700 patients. Over 200 preoperative clinical fac-tors were studied. At a median of 9.3 postoperative years fol-lowing surgery, the mean (SD) percentage TBWL was 22.5% (13.1%). Preoperative

1	700 patients. Over 200 preoperative clinical fac-tors were studied. At a median of 9.3 postoperative years fol-lowing surgery, the mean (SD) percentage TBWL was 22.5% (13.1%). Preoperative insulin use, history of smoking, and use of 12 or more medications before surgery were associated with greater long-term weight loss; 6.8%, 2.8%, and 3.1%, respec-tively. Preoperative hyperlipidemia, older age, and higher body mass index were associated with poorer long-term weight loss (−2.8%, −8.8%, and −4.1%, respectively). Again, there were only a few preoperative clinical factors associated with differ-ences in long-term weight loss after RYGB.220This group also developed a method to predict the probability of T2DM remission after RYGB surgery on the basis of preop-erative clinical criteria in a retrospective cohort study. Over 200 clinical variables were used to identify independent predictors of remission within 5 years and to produce a score (DiaRem) to assess this likelihood. Records were

1	cohort study. Over 200 clinical variables were used to identify independent predictors of remission within 5 years and to produce a score (DiaRem) to assess this likelihood. Records were available for 690 patients in the pri-mary cohort, of whom 463 (63%) had achieved partial or com-plete T2DM remission. Four preoperative clinical variables were included in the final model: insulin use, age, HbA1c concentration, and type of antidiabetic drugs. The DiaRem score was developed from that, and it ranges from 0 to 22 with the proportion of patients achieving remission being highest for the lowest scores.221Comparisons Between ProceduresThere have been many systematic reviews of bariatric surgery attempting to summarize and quantify differences in the efficacy and safety of the different surgical procedures. A major challenge in summarizing this literature from the last 10 years is the fact that no single randomized trial has included all of the most com-mon procedures (RYGB, LAGB, SG, and

1	procedures. A major challenge in summarizing this literature from the last 10 years is the fact that no single randomized trial has included all of the most com-mon procedures (RYGB, LAGB, SG, and BPD/DS), so infer-ence must be made through pooled analysis of data from many disparate randomized and non-randomized studies of bariatric surgery with different lengths and completeness of follow-up. There are also no studies that have examined differences in long-term survival, incident cardiovascular events, and quality of life across bariatric procedures.1 Still one of the most compre-hensive systematic reviews by Buchwald included 136 studies and a total of 22,094 bariatric patients. Only 5 of the included studies were randomized trials (28 non-RCTs and 101 uncon-trolled case series) and the review did not include any data on the SG procedure, so will need to be updated. This review found a strong trend towards different weight loss outcomes across procedures: weighted mean percentage

1	review did not include any data on the SG procedure, so will need to be updated. This review found a strong trend towards different weight loss outcomes across procedures: weighted mean percentage of excess weight loss (%EWL) 50% for LAGB; 68% for RYGB; 69% for VBG; and 72% for BPD/DS. The rate of T2DM remission also appeared to differ across procedures: 48% for LAGB; 84% for RYGB; 72% for VBG; and 99% for BPD/DS. A similar pattern of dis-ease remission was observed for dyslipidemia, hypertension, and obstructive sleep apnea, with the greatest rates of remission observed among BPD/DS patients, followed by RYGB patients, with the least disease remission among LAGB patients.192There is still ongoing debate regarding the comparative effectiveness of the three most common procedures currently in use: LRYGB, SG, and LAGB. Several other systematic reviews have concluded that LRYGB is more effective for weight loss than LAGB; however, there have been only two small RCTs with follow-up at 4

1	in use: LRYGB, SG, and LAGB. Several other systematic reviews have concluded that LRYGB is more effective for weight loss than LAGB; however, there have been only two small RCTs with follow-up at 4 and 5 years addressing this issue specifically.65,222,223 There is evolving data from a number of smaller RCTs to examine differences between LRYGB, LAGB, and SG for comorbidity improvement (addressed in “Results of Surgery for Diabetes”), but systematic reviews of nonrandomized studies indicate greater remission of T2DM, dyslipidemia, hypertension, and sleep apnea with LRYGB compared to LAGB. Two recent systematic reviews have compared the outcomes of the SG with other procedures.224,225 One review identified 15 RCTs involving 1191 patients. The percent excess body weight loss (%EBWL) ranged from 49% to 81% for SG, from 62% to 94% for LRYGB, and from 29% to 48% for LAGB, with a follow-up ranging from 6 months to 3 years. The T2DM remission rate ranged from 27% to 75% for SG vs. 42% to 93%

1	49% to 81% for SG, from 62% to 94% for LRYGB, and from 29% to 48% for LAGB, with a follow-up ranging from 6 months to 3 years. The T2DM remission rate ranged from 27% to 75% for SG vs. 42% to 93% for RYGB. The second review only compared SG to RYGB and identified 6 RCTs and two nonrandomized controlled studies with follow-up ranging from 3 months to 2 years. They found that LRYGB achieved significantly greater improvement in BMI than SG (1.8 kg/m2) and greater improvements in metabolic factors. Longer-term comparative effectiveness data on SG are still needed, but the effectiveness of the SG procedure, again, appears to be positioned between the LRYGB and LAGB procedures.Resolution of Specific Comorbid ConditionsBariatric surgery can improve and induce remission of many obesity-related comorbid conditions. Nevertheless, the remis-sion rates can decline over time due to relapse of disease, and as follow-up lengthens, complete and more longer-term follow-up data is needed in some

1	comorbid conditions. Nevertheless, the remis-sion rates can decline over time due to relapse of disease, and as follow-up lengthens, complete and more longer-term follow-up data is needed in some areas.Cardiovascular Disease. A recent systematic review of long-term cardiovascular risk factor reduction after bariatric surgery involved 73 studies and 19,543 subjects with a mean age of 42 years; 76% of subjects were female, and 44%, 24%, and 44% had baseline hypertension, diabetes, and hyperlipidemia, respectively.226 At a mean follow-up of 57.8 months, the aver-age excess weight loss for all bariatric procedures was 54%, and remission/improvement was 63% for hypertension, 73% for T2DM, and 65% for hyperlipidemia. Echocardiographic results from 713 subjects showed statistically significant improve-ments in hemodynamics. There are no long-term RCTs com-paring bariatric surgery with nonsurgical medical treatment of obesity that specifically evaluate cardiovascular endpoints and

1	improve-ments in hemodynamics. There are no long-term RCTs com-paring bariatric surgery with nonsurgical medical treatment of obesity that specifically evaluate cardiovascular endpoints and cardiovascular mortality. However, 12 cohort-matched studies comparing bariatric surgery with nonsurgical controls have been reviewed.227 Collectively, all but two of these studies support a reduced cardiovascular event rate and all-cause mortality rate conferred by bariatric surgery. Of these studies, the Swedish Obesity Subjects (SOS) study still has the longest outcomes.Gastroesophageal Reflux Disease. Patients with obesity and GERD have a higher chance of failing to obtain symptomatic relief from standard antireflux surgery. The recurrence of symp-toms is higher, likely due to a higher incidence of wrap hernia-tion into the mediastinum and other mechanical failure of the fundoplication, which in turn is likely affected by the increased intra-abdominal pressure of the obese condition. The

1	of wrap hernia-tion into the mediastinum and other mechanical failure of the fundoplication, which in turn is likely affected by the increased intra-abdominal pressure of the obese condition. The patient with a BMI over 35 kg/m2 who has GERD has a better chance of symptom improvement by undergoing LRYGB, which is effective for the treatment of GERD.228,229 LRYGB creates such a small gastric pouch that it has a very limited volume for acid production. LAGB may worsen or may improve GERD but to a considerably lesser extent than RYGB. A prospective analysis of 558 consecutive SG (n = 200) and LRYGB (n = 358) patients demonstrated significantly improved subjective GERD symp-toms in the bypass cohort when compared to the SG patients at Brunicardi_Ch27_p1167-p1218.indd 119923/02/19 2:21 PM 1200SPECIFIC CONSIDERATIONSPART II1 year.230 Studies show that SG can increase GERD symptoms postoperatively.Obstructive Sleep Apnea. A systematic review of 13,900 patients (69 studies) showed

1	PM 1200SPECIFIC CONSIDERATIONSPART II1 year.230 Studies show that SG can increase GERD symptoms postoperatively.Obstructive Sleep Apnea. A systematic review of 13,900 patients (69 studies) showed significant improvement or reso-lution of sleep apnea in more than 75% of bariatric surgery patients.231 Comparison of outcomes between procedures dem-onstrated the most benefit with BPD and RYGB and the least with LAGB. However, a randomized control trial comparing the effect of medical and surgical weight loss (LAGB) on sleep apnea found no significant difference in apnea events despite major differences in weight loss. The findings suggested that much of the improvement achieved was in the mild to moderate weight loss range, with little benefit of further weight loss.232Asthma. Another pulmonary symptom that commonly occurs in severely obese patients is asthma. Dixon and colleagues233 studied 23 asthmatic patients who underwent bariatric surgery and found a significant improvement in

1	symptom that commonly occurs in severely obese patients is asthma. Dixon and colleagues233 studied 23 asthmatic patients who underwent bariatric surgery and found a significant improvement in asthma control (e.g., forced expiratory volume in 1 second, forced vital capacity), asthma-related quality of life, and responsiveness to methacholine. Boulet and colleagues234 found similar results in their cohort of 12 patients with asthma who experienced significant weight loss after bariatric surgery.Nonalcoholic Fatty Liver Disease. Nonalcoholic Fatty Liver Disease (NAFLD) is a metabolically related problem associated with obesity. The disease is a spectrum of liver abnormalities including steatosis, steatohepatitis, fibrosis, and cirrhosis of the liver. It is estimated that 20% of U.S. adults have NAFLD, largely because of the high incidence of obesity. NAFLD is present in an estimated 85% of patients with severe obesity.235 Although further research is needed to accurately assess the role

1	NAFLD, largely because of the high incidence of obesity. NAFLD is present in an estimated 85% of patients with severe obesity.235 Although further research is needed to accurately assess the role of bariatric surgery as a potential treatment for NAFLD, there are some reports that support its use. A systematic review of the available literature found many retrospective and prospective observational cohort studies, but no RCTs or case-control series.236Musculoskeletal Disease. Degenerative joint disease and low back pain are among the most common complaints and asso-ciated comorbid problems in the severely obese population. A prospective cohort of 50 obese females age 20 to 74 years were followed for 1 year after LRYGB using the timed-get-up-and-go (TGUG) and health survey SF-36.237 The results showed a significant improvement in musculoskeletal function and likely enhanced ability to progress in rehabilitation. Patients with osteoarthritis of the neck, shoulder, spine, hip, knee,

1	results showed a significant improvement in musculoskeletal function and likely enhanced ability to progress in rehabilitation. Patients with osteoarthritis of the neck, shoulder, spine, hip, knee, ankle, wrist, and hand have been shown to have improved or resolved joint pain after bariatric surgery. Reduction in BMI values of 6.2 to 14.7 kg/m2 has corresponded with back and knee pain resolution in 5% to 100% of patients, whereas pain severity was reduced in 31% to 94% of patients depending on the joint and study.238The LABS-2 Study published data on pain and physical function in over 2200 participants. At year 1, clinically mean-ingful improvements were shown in 57.6% of participants for bodily pain, 76.5% for physical function, and 59.5% for walk time. Additionally, among participants with severe knee or dis-ability (633), or hip pain or disability (500) at baseline, approxi-mately three-fourths experienced joint-specific improvements in knee pain (77.1%) and in hip function

1	with severe knee or dis-ability (633), or hip pain or disability (500) at baseline, approxi-mately three-fourths experienced joint-specific improvements in knee pain (77.1%) and in hip function (79.2%). But between year 1 and year 3, rates of improvement significantly decreased for both bodily pain and for physical function.239Quality of Life. Few long-term studies have assessed the impact of bariatric surgery on overall quality of life. However, three studies of 6 to 10 years’ duration suggest that bariatric pro-cedures are associated with greater improvements in overall and obesity specific measures of quality of life compared to medical treatment or care.240-242 Physical functioning aspects of quality of life seem to be more responsive to bariatric procedures than mental health domains, although more research is needed, espe-cially in patients with less severe (class 1) obesity.Results of Surgery for Diabetes (Metabolic Surgery)Based on an abundance of recent observational studies

1	although more research is needed, espe-cially in patients with less severe (class 1) obesity.Results of Surgery for Diabetes (Metabolic Surgery)Based on an abundance of recent observational studies and RCTs, bariatric surgery is increasingly used with the primary intent to treat T2DM or metabolic disease, hence the term meta-bolic surgery.243 Observational, nonrandomized studies first demonstrated profound improvements in hyperglycemia and other cardiovascular risk factors following metabolic surgery that were followed by RCTs. Resolution or remission of T2DM is typically defined as becoming “nondiabetic” with normal HbA1c, without medications. One meta-analysis (2009) of 19 mostly observational studies (n = 4,070 patients) reported an overall T2DM remission rate of 78% after bariatric surgery with 1 to 3 years follow-up.244 The patients all had BMI >35 and gen-erally early/mild T2DM that likely increased remission rates. In the Swedish Obese Subjects study, the remission rate

1	surgery with 1 to 3 years follow-up.244 The patients all had BMI >35 and gen-erally early/mild T2DM that likely increased remission rates. In the Swedish Obese Subjects study, the remission rate follow-ing surgery was 72% at 2 years and 36% at 10 years compared with 21% and 13%, respectively, for the nonsurgical controls (P <.001).201 Metabolic surgery was also significantly more effective than nonsurgical treatment in preventing new onset cases of T2DM, with a relative risk reduction of 78%.A more recent systematic review (2012) evaluated long-term cardiovascular risk reduction after bariatric surgery in 73 studies and 19,543 patients.226 At a mean follow-up of 57.8 months, the average excess weight loss for all procedures was 54%, and rates of remission or improvement were 63% for hyper-tension, 73% for T2DM, and 65% for hyperlipidemia. Results from 12 cohort-matched, nonrandomized studies comparing bariatric surgery vs. nonsurgical controls demonstrated reduced cardiovascular

1	hyper-tension, 73% for T2DM, and 65% for hyperlipidemia. Results from 12 cohort-matched, nonrandomized studies comparing bariatric surgery vs. nonsurgical controls demonstrated reduced cardiovascular events and death (30–88% reduction) in patients with and without T2DM.227 One of these studies involving male veterans, who were mostly at high cardiovascular risk, reported a 42% reduction in mortality at 10 years compared with medical therapy.207 Similarly, in the Swedish Obese Subjects study, the mortality rate from cardiovascular disease in the bariatric surgical group was lower than for control patients (adjusted hazard ratio, 0.47; P = .002).201 For patients with T2DM in this study, surgery was associated with a 50% reduction in microvascular complica-tions (41.8 per 1000 person-years for control patients and 20.6 per 1000 person-years in the surgery group; hazard ratio, 0.44; P <.001).245 These observational, nonrandomized studies provide evidence that metabolic surgery is superior

1	control patients and 20.6 per 1000 person-years in the surgery group; hazard ratio, 0.44; P <.001).245 These observational, nonrandomized studies provide evidence that metabolic surgery is superior to medical manage-ment alone in improving glycemic control, reducing cardiovascu-lar risk factors, and lowering long-term morbidity and mortality of T2DM, yet supporting RCTs have been lacking until recently.During the past 10 years, 11 such RCTs have been published including 794 patients in total (Table 27-7).16,77-83,85,86,246-253 These RCTs included obese patients with T2DM (n = 794; range 38–150 patients per study) with follow-up from 6 months to 5 years (Fig. 27-27). All common metabolic surgical procedures were represented including LRYGB (9 studies), LAGB (5 studies), SG (2 studies), and BPD (1 study). T2DM severity varied significantly from mild (mean HbA1c 7.7%, <2-year onset, no insulin)253,77 to severe (mean HbA1c 9.3%, duration 8.3 years, 48% on insulin).78 The BMI ranged from

1	BPD (1 study). T2DM severity varied significantly from mild (mean HbA1c 7.7%, <2-year onset, no insulin)253,77 to severe (mean HbA1c 9.3%, duration 8.3 years, 48% on insulin).78 The BMI ranged from 25 to 53 kg/m2, with 11 of 12 studies including patients with BMI <35 kg/m2, also called class 1 obesity. Age, sex, and ethnic background were similar, 7Brunicardi_Ch27_p1167-p1218.indd 120023/02/19 2:21 PM 1201THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27Table 27-7Metabolic surgery randomized controlled trials for type 2 diabetes (n = 794)aSTUDYBMI (kg/m2), % OF PATIENTSDESIGNNO. OF PATIENTS RANDOMIZEDFOLLOW-UP (MONTHS)REMISSION CRITERIA*OUTCOME (REMISSION OR CHANGE IN HbA1C)Dixon77<35, 22%LAGB vs. control6024HbA1c <6.2%73% vs. 13%, P <0.001Schauer78,246,252<35, 36%RYGB vs. SG vs. control15060HbA1c ≤6.0%22% vs. 15% vs. 0, P<0.05Mingrone79,82>35, 100%RYGB vs. BPD vs. control6060HbA1c ≤6.5%42% vs. 68% vs. 0, P = 0.003Ikramuddin 80,247<35, 59%RYGB vs. control12024HbA1c <6%44% vs. 9%,

1	≤6.0%22% vs. 15% vs. 0, P<0.05Mingrone79,82>35, 100%RYGB vs. BPD vs. control6060HbA1c ≤6.5%42% vs. 68% vs. 0, P = 0.003Ikramuddin 80,247<35, 59%RYGB vs. control12024HbA1c <6%44% vs. 9%, P <0.001Liang248<35, 100%RYGB vs. control10112HbA1c <6.5%**90% vs. 0 vs. 0, P <0.0001Halperin83<35, 34%RYGB vs. control3812HbA1c <6.5%58% vs. 16%, P = 0.03Courcoulas84,86<35, 43%RYGB vs. LAGB vs. control6936HbA1c <6.5%40% vs. 29% vs. 0, P = 0.004Wentworth250≤30, 100%LAGB vs. control5124Fasting blood glucose <7.0 mmol/L52% vs. 8%, P = 0.001Parikh251<35, 100%Bariatric surgery (RYGB, LAGB, SG) vs. control576HbA1c <6.5%65% vs. 0, P = 0.0001Ding85<35, 34%LAGB vs. control4512HbA1c <6.5%***33% vs. 23%, P = 0.46Cummings81<35, 25%RYGB vs. control4312HbA1c <6.0%60% vs. 5.9%, P = 0.002*Remission defined as reaching HbA1c value without medication, unless otherwise specified**Remission not precisely defined, extrapolated***On or off medicationsReproduced with permission from Schauer PR, Mingrone G2,

1	as reaching HbA1c value without medication, unless otherwise specified**Remission not precisely defined, extrapolated***On or off medicationsReproduced with permission from Schauer PR, Mingrone G2, Ikramuddin S, et al: Clinical Outcomes of Metabolic Surgery: Efficacy of Glycemic Control, Weight Loss, and Remission of Diabetes, Diabetes Care. 2016 Jun;39(6):902-911.although three studies80,247,248 included a significant number of Asian patients. For most studies, the primary endpoint was remission, defined as an HbA1c target at or below 6.0% to 6.5% without use of diabetes medications.Overall, these RCTs showed that surgery was signifi-cantly more effective than medical treatment in reaching remission and glycemic control (P <.05) (Fig. 27-27). The one exception showing no superiority of surgery involved gastric banding and resulted in a diabetes remission for LAGB vs. medical treatment of 33% and 23%, respectively (P >0.05).85 Overall, surgery decreased HbA1c by 2% to 3.5%, whereas

1	surgery involved gastric banding and resulted in a diabetes remission for LAGB vs. medical treatment of 33% and 23%, respectively (P >0.05).85 Overall, surgery decreased HbA1c by 2% to 3.5%, whereas medical treatment lowered it by 1% to 1.5%, as seen in Fig. 27-28. Most of these studies also showed superiority of surgery over medical treatment in achieving secondary endpoints such as weight loss, remission of metabolic syn-drome, reduction in diabetes and cardiovascular medications, and improvement in triglycerides, lipids, and quality of life. Results were mixed in terms of improvements in systolic and diastolic blood pressure or low-density lipoproteins after sur-gery vs. medical treatment, but many studies did show a cor-responding reduction in medication usage.Although previous guidelines and payer coverage policies had limited metabolic surgery to severely obese patients (BMI ≥35 kg/m2), nearly all RCTs showed that the surgical procedures, especially LRYGB and SG, were equally

1	and payer coverage policies had limited metabolic surgery to severely obese patients (BMI ≥35 kg/m2), nearly all RCTs showed that the surgical procedures, especially LRYGB and SG, were equally effective in patients with BMI 30 to 35 kg/m2. This is particularly important given that many patients with T2DM have a BMI <35 kg/m2. The effect of surgery in these patients with a lower class of obesity is also durable out to at least 5 years.243,252None of these RCTs were sufficiently powered to detect differences in macrovascular or microvascular complications or death, especially at the relatively short follow-up, and no such differences have been detected thus far. Four of the RCTs from Pittsburgh, Seattle, Boston, and Cleveland have combined their patient populations in a pooled study to assess 10-year out-comes. This study, Alliance of Randomized Trials of Medicine vs. Metabolic Surgery (ARMMS), aims to identify long-term risks and benefits of metabolic surgery.The evidence, as

1	to assess 10-year out-comes. This study, Alliance of Randomized Trials of Medicine vs. Metabolic Surgery (ARMMS), aims to identify long-term risks and benefits of metabolic surgery.The evidence, as previously summarized, was the basis for newly established international guidelines on the role of metabolic surgery in treating T2DM. In 2015, the 2nd Diabetes Surgery Summit (DSS-II) Consensus Conference generated Brunicardi_Ch27_p1167-p1218.indd 120123/02/19 2:21 PM 1202SPECIFIC CONSIDERATIONSPART IIParikh 2014 (RYGB/LAGB/SG) [6 mo; 6.5% off meds] (18)Courcoulas 2014 (RYGB/LAGB) [12 mo; 6.5% off meds] (14)Ding 2015 (LAGB) [12 mo; 6.5%] (22)Halperin 2014 (RYGB) [12 mo; 6.5% off meds] (15)Ikramuddin 2013 (RYGB) [12 mo; 7.0%] (13)Liang 2013 (RYGB) [12 mo; 7.0% off meds] (16)Schauer 2012 (RYGB/SG) [12 mo; 6.0%] (12)Cummings 2016 (RYGB) [12 mo; 6.5% off meds] (23)Dixon 2008 (LAGB) [24 mo; 6.2% off meds] (10)Ikramuddin 2015 (RYGB) [24 mo; 7.0%] (21)Mingrone 2012 (RYGB/BPD) [24 mo; 6.5%

1	(RYGB/SG) [12 mo; 6.0%] (12)Cummings 2016 (RYGB) [12 mo; 6.5% off meds] (23)Dixon 2008 (LAGB) [24 mo; 6.2% off meds] (10)Ikramuddin 2015 (RYGB) [24 mo; 7.0%] (21)Mingrone 2012 (RYGB/BPD) [24 mo; 6.5% off meds] (11)Wentworth 2014 (LAGB) [24 mo; 7.0%] (17)Courcoulas 2015 (RYGB/LAGB) [36 mo; 6.5% off meds] (24)Schauer 2014 (RYGB/SG) [36 mo; 6.0%] (19)Mingrone 2015 (RYGB/BPD) [60 mo; 6.5% off meds] (20)Fixed-Effects ModelStudy (Operation) [Follow-Up; HbA1c end point]WeightPeto, Fixed, 95% CIPolo Odds Ratios131861128283492226341214271920411819573199152960402337973862400531100148020002417221957704117265920251440154664.5%5.1%3.9%4.4%12.5%8.4%10.4%3.4%6.7%11.8%6.4%4.9%4.0%8.7%4.9%100.0%21.15 [5.85, 76.51]7.51 [2.24, 25.21]1.68 [0.42, 6.66]5.82 [1.59, 21.39]3.72 [1.72, 8.04]86.76 [33.89, 222.08]6.39 [2.74, 14.88]11.48 [2.63, 50.13]10.83 [3 .79, 30.96]4.25 [1.92, 9.38]30.08 [10.28, 88.06]8.11 [2.37, 27.84]6.44 [1.65, 25.21]5.73 [2.28, 14.42]8.44 [2.46, 29.01]8.45 [6.44, 11.10]SurgeryGlyc. Endp.

1	[2.74, 14.88]11.48 [2.63, 50.13]10.83 [3 .79, 30.96]4.25 [1.92, 9.38]30.08 [10.28, 88.06]8.11 [2.37, 27.84]6.44 [1.65, 25.21]5.73 [2.28, 14.42]8.44 [2.46, 29.01]8.45 [6.44, 11.10]SurgeryGlyc. Endp. NMedical/LifestyleGlyc. Endp. NHeterogeneity: Chi2 = 45 .43, df = 14 (P < 0.0001); I2 = 69%Test for overall effect: Z = 15.36 (P < 0.00001)FavorsMedical/Lifestyle0.0010.11011000FavorsSurgeryIncreasing Length of Follow-UpFigure 27-27. Glycemic endpoints of RCTs by length of follow-up. Forest plot of Peto odds ratios (ORs) of main glycemic end points, as defined in each trial, from published RCTs of bariatric/metabolic surgery compared with medical/lifestyle treatments for diabetes with data arranged in order of increasing length of follow-up. (Reproduced with permission from Cummings DE, Cohen RV: Bariatric/Metabolic Surgery to Treat Type 2 Diabetes in Patients With a BMI <35 kg/m2, Diabetes Care. 2016 Jun;39(6):924-933.)Brunicardi_Ch27_p1167-p1218.indd 120223/02/19 2:21 PM 1203THE

1	RV: Bariatric/Metabolic Surgery to Treat Type 2 Diabetes in Patients With a BMI <35 kg/m2, Diabetes Care. 2016 Jun;39(6):924-933.)Brunicardi_Ch27_p1167-p1218.indd 120223/02/19 2:21 PM 1203THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27Patients WithType 2 DiabetesObeseBMI ˙30 kg/m2or ˙27.5 for AsiansNonobeseBMI <30 kg/m2or <27.5 for AsiansClass III ObeseBMI ˙40 kg/m2or ˙37.5 for AsiansClass II ObeseBMI 35.0–39.9 kg/m2or 32.5–37.4 for AsiansClass I ObeseBMI 30.0–34.9 kg/m2or 27.5–32.4 for AsiansClass II ObeseWith PoorGlycemic ControlRecommendMetabolic SurgeryConsiderMetabolic SurgeryNonsurgicalTreatmentClass II ObeseWith AdequateGlycemic ControlClass I ObeseWith PoorGlycemic ControlClass I ObeseWith AdequateGlycemic ControlExpedited Assessment for Metabolic SurgeryOptimal Lifestyle and Medical RxOptimal Lifestyle and Medical Rx (including injectable meds and insulin)Figure 27-29. Algorithm for the treatment of type 2 diabetes as recommended by the 2nd Diabetes Surgery Summit.

1	and Medical RxOptimal Lifestyle and Medical Rx (including injectable meds and insulin)Figure 27-29. Algorithm for the treatment of type 2 diabetes as recommended by the 2nd Diabetes Surgery Summit. (Reproduced with permission from Rubino F, Nathan DM, Eckel RH, et al: Metabolic Surgery in the Treatment Algorithm for Type 2 Diabetes: A Joint Statement by International Diabetes Organizations, Diabetes Care. 2016 Jun;39(6):861-877.)20–2–4–6Change in HbA1cMean baselineBMI 35 kg/m2Mean baselineBMI >35 kg/m2Wentworth 2014Liang 2013Parikh 2014Ikramuddin 2013Courcoulas 2014Courcoulas 2014Halperin 2014Ding 2015Dixon 2008Schauer 2012Schauer 2012Cummings 2016Mingrone 2012Mingrone 2012SurgeryMedical/LifestyleFigure 27-28. Change in baseline HbA1c in each of the 11 RCTs. (Reproduced with permission from Rubino F, Nathan DM, Eckel RH, et al: Metabolic Surgery in the Treatment Algorithm for Type 2 Diabetes: A Joint Statement by International Diabetes Organiza-tions, Diabetes Care. 2016

1	permission from Rubino F, Nathan DM, Eckel RH, et al: Metabolic Surgery in the Treatment Algorithm for Type 2 Diabetes: A Joint Statement by International Diabetes Organiza-tions, Diabetes Care. 2016 Jun;39(6):861-877.)guidelines that were endorsed by more than 50 diabetes and medical organizations.16 The recommendations include patient selection, preoperative evaluation, choice of procedure, postop-erative follow-up, and indications for surgery. According to the DSS-II guidelines, metabolic surgery should be recommended to treat T2DM in patients with class III obesity (BMI ≥40 kg/m2) regardless of glycemic control and in those with class II obesity (BMI 35.0–39.9 kg/m2) when hyperglycemia is inad-equately controlled by lifestyle and optimal medical therapy (Fig. 27-29). Surgery should also be considered for patients with T2DM and BMI 30.0 to 34.9 kg/m2 if hyperglycemia is inadequately controlled despite optimal treatment with either oral or injectable medications. These BMI

1	should also be considered for patients with T2DM and BMI 30.0 to 34.9 kg/m2 if hyperglycemia is inadequately controlled despite optimal treatment with either oral or injectable medications. These BMI thresholds should be reduced by 2.5 kg/m2 for Asian patients. The new treatment algorithm from DSS-II incorporates appropriate use of all three treatment modalities: lifestyle intervention, drug therapy, and surgery (see Fig. 27-29).16 The 2017 Standards of Care for Dia-betes from the American Diabetes Association include those key indications in its recommendations for metabolic surgery, as well.COMPLICATIONS OF BARIATRIC SURGERYSurgical ComplicationsNone of the surgical procedures are without risks. The periop-erative mortality for the average patient is low (<0.5%) and declining, but can vary significantly across sub-groups with perioperative mortality rates of 2.0% or higher in some patient populations.1 The incidence of complications after the various surgical procedures varies from

1	significantly across sub-groups with perioperative mortality rates of 2.0% or higher in some patient populations.1 The incidence of complications after the various surgical procedures varies from 4% to over 25% and depends on the definition of complication used, the type of bariatric procedure performed, and patient characteristics216,254,255 (see Table 27-5).8Brunicardi_Ch27_p1167-p1218.indd 120323/02/19 2:21 PM 1204SPECIFIC CONSIDERATIONSPART IIIn the 11 RCTs (794 patients) that have compared bariatric surgery to nonsurgical treatment, rates of adverse events were higher among surgical subjects, with follow-up up to 5 years for two of the studies and up to 2 to 3 years for others.82,252,256 There were very few cardiovascular events or deaths in either the surgical or the nonsurgical groups, and the most common adverse events after surgery were iron deficiency anemia (15% with intestinal bypass operations) and reoperations (8%). These RCTs were not large enough to compare safety

1	groups, and the most common adverse events after surgery were iron deficiency anemia (15% with intestinal bypass operations) and reoperations (8%). These RCTs were not large enough to compare safety between pro-cedure types, and most of the comparative data on procedure-to-procedure complications has and will come from larger observational studies.The 30-day mortality in the LABS Study was 0.3% for all procedures with a major adverse outcome rate (a predefined composite endpoint that included; death, venous thromboembo-lism, reintervention [percutaneous, endoscopic, or operative], or failure to be discharged from the hospital in 30 days) of 4.1%.59 Major predictors of an increased risk of complications in LABS were a history of venous thromboembolism, a diagnosis of obstructive sleep apnea, impaired functional status defined as inability to walk 300 feet (91 m), extreme BMI, and undergo-ing an RYGB by the open technique. Other large observational studies, such as SOS, have shown

1	apnea, impaired functional status defined as inability to walk 300 feet (91 m), extreme BMI, and undergo-ing an RYGB by the open technique. Other large observational studies, such as SOS, have shown higher rates of complications, with 14.5% having at least one nonfatal complication over the first 90 days, including pulmonary complications, vomiting, wound infection, hemorrhage, and anastomotic leak. However, the SOS included mostly open and VBG procedures, which are rarely performed today. Despite these older procedures and tech-niques, the 90-day mortality rate in SOS was low at 0.25%.196In a 2007 meta-analysis of 361 studies, mostly nonran-domized observational studies involving over 85,000 patients, Buchwald and colleagues reported important differences in ≤30-day mortality across different laparoscopic bariatric pro-cedures: 0.06% for LAGB; 0.21% for VBG; 0.16% for RYGB; and 1.11% for BPD/DS.257 This review also found significantly higher mortality for open procedures compared

1	laparoscopic bariatric pro-cedures: 0.06% for LAGB; 0.21% for VBG; 0.16% for RYGB; and 1.11% for BPD/DS.257 This review also found significantly higher mortality for open procedures compared with those per-formed laparoscopically. A U.S. study of over 300,000 patients in 12 states examined in-hospital complications before and after implementation of the CMS national policy restricting insurance coverage for bariatric surgery to “centers of excellence.” The study found no significant differences in complications before and after the policy was implemented, and overall complication rates were 7% to 8% with 3.3% to 3.6% being serious, including a 1% reoperation rate during both time periods.1,258 A clinically useful prognostic risk score was also developed and validated in 9382 patients to predict 90-day mortality after LRYGB surgery using five clinical characteristics: BMI ≥50 kg/m2, male sex, hypertension, known risk factor for pulmonary embolism, and age ≥45 years. Patients with 4 to

1	90-day mortality after LRYGB surgery using five clinical characteristics: BMI ≥50 kg/m2, male sex, hypertension, known risk factor for pulmonary embolism, and age ≥45 years. Patients with 4 to 5 of these characteristics are at much higher risk of death (4.3%) by 90 days than those with 0 to 1 characteristics (0.26%).259-261 A systematic review of 15 RCTs of SG found no deaths in just under 800 patients but a 9.2% mean complication rate (range 0–18%).224 In the American College of Surgeons Bariatric Surgery Network database, mor-tality 30 days after SG was 0.11%, positioning its overall com-plication profile between that for LAGB (0.05%) and RYGB (0.14%). The 30-day complication rate was similarly positioned at 5.6% for SG, 1.4% LAGB, and 5.9% for RYGB.140An established problem now is the frequent rate of reop-eration, particularly among LAGB patients. In O’Brien and col-leagues’ prospective cohort of 3227 LAGB patients, revisional procedures occurred in 1116 (35%) and were performed

1	frequent rate of reop-eration, particularly among LAGB patients. In O’Brien and col-leagues’ prospective cohort of 3227 LAGB patients, revisional procedures occurred in 1116 (35%) and were performed for the following reasons: proximal enlargement (26%), port and tubing problems (21%), and erosion (3.4%). The need for revision due to proximal enlargement decreased dramatically over a 17-year period from 40% to 6.4% as the surgical technique evolved; however, the band was ultimately removed in 5.6% of all indi-viduals.262 Other long-term cohorts suggest that LAGB removal rates may be as high as 50%. The O’Brien systematic review of long-term studies indicates that the rate of revisional surgery for LRYGB may be similar to LAGB (22% revision for LRYGB, range 8% to 38%; 26% revision for LAGB, range of 8% to 60%). However, in the LABS Study, there was a higher rate of revision and reoperation for LAGB as compared to RYGB at both 3 and 7 years of follow-up.169 In general, more long-term

1	range of 8% to 60%). However, in the LABS Study, there was a higher rate of revision and reoperation for LAGB as compared to RYGB at both 3 and 7 years of follow-up.169 In general, more long-term data with more complete follow-up with standardized defini-tions and reporting of complications are needed to compare reoperation and complication rates of all bariatric procedures.Nonsurgical ComplicationsPostgastric bypass hypoglycemia (PGBH) in a relatively uncommon but particularly challenging problem that affects an unknown number of patients in the longer term. Prevalence rates in the literature vary from 1% to 11%, depending on the defini-tion.263,264 Hypoglycemia is characterized by documentation of Whipple’s triad (including both autonomic and neuroglycopenic symptoms or signs), at the time of a plasma glucose concentration <55 mg/dL with resolution of symptoms and signs after glucose administration. During these episodes, plasma insulin levels are inappropriately high, indicating

1	time of a plasma glucose concentration <55 mg/dL with resolution of symptoms and signs after glucose administration. During these episodes, plasma insulin levels are inappropriately high, indicating dysregulation of β-cell function. When it was initially described in patients who had undergone partial pancreatectomy, nesidioblastosis was found in the speci-mens that are characterized by hyperplasia and/or dysplasia of the pancreatic islets.265 It was initially thought to be endogenous hyperinsulinemia from increased β-cell mass hyperfunctioning islet cells; however, the current thinking is that the recalcitrant symptoms of hyperinsulinemic hypoglycemia after RYGB are related to the anatomic and physiologic changes and not from an inherent change in β-cell mass. One candidate mediator of increased insulin secretion in PGBH is GLP-1, a peptide released from intestinal neuroendocrine L-cells in response to meals. Consistent with this hypothesis, postprandial GLP-1 levels are increased

1	increased insulin secretion in PGBH is GLP-1, a peptide released from intestinal neuroendocrine L-cells in response to meals. Consistent with this hypothesis, postprandial GLP-1 levels are increased by over tenfold in post-RYGB patients, are higher in those with hyperinsulinemic hypoglycemia and neuroglyco-penia, and correlate inversely with postprandial glucose lev-els.266 PGBH needs to be distinguished from other forms of hypoglycemia such as other functional β-cell disorders such as noninsulinoma pancreatogenous hypoglycemia, insulinoma, reactive hypoglycemia, or early or late dumping syndrome. It is possible that PGBH is a spectrum of hypoglycemia with late dumping being on the end of the spectrum that is more respon-sive to dietary changes alone while more severe PGBH can be associated with severe symptoms.263 First-line therapeutic approaches to PGBH include medical nutrition therapy aimed at reducing intake of high glycemic index carbohydrates and premeal treatment with

1	with severe symptoms.263 First-line therapeutic approaches to PGBH include medical nutrition therapy aimed at reducing intake of high glycemic index carbohydrates and premeal treatment with acarbose. Additional therapies that may be considered include octreotide, diazoxide, calcium channel blockers, GLP-1 receptor antagonists, and providing nutrition solely through a gastrostomy tube placed into the bypassed duo-denum. Reversal of gastric bypass is not uniformly successful, suggesting the importance of underlying genetics and/or com-pensatory mechanisms that may persist after surgical reversal.266 Finally, although pancreatic resection was initially employed for patients with life-threatening hypoglycemia, this procedure Brunicardi_Ch27_p1167-p1218.indd 120423/02/19 2:21 PM 1205THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27is not uniformly successful in remitting hypoglycemia and should not be considered for the majority of patients, who can experience improvement in their symptoms

1	MANAGEMENT OF OBESITYCHAPTER 27is not uniformly successful in remitting hypoglycemia and should not be considered for the majority of patients, who can experience improvement in their symptoms with a combination of medical approaches. A 2017 American Society of Metabolic and Bariatric Surgery (ASMBS) position statement provides a comprehensive summary of this topic and also recommends this multimodal medical approach.267There is data to suggest that babies born to women follow-ing bariatric surgery are at risk for certain complications. In a Swedish study, bariatric surgery was originally associated with reduced risks of gestational diabetes and excessive fetal growth, shorter gestation, and an increased risk of small-for-gestational-age infants. In a later follow-up report, this same group reported a significant association between a history of bariatric surgery and an increased risk of preterm birth and spontaneous preterm birth, in particular.119,268There is also emerging data from

1	reported a significant association between a history of bariatric surgery and an increased risk of preterm birth and spontaneous preterm birth, in particular.119,268There is also emerging data from observational studies that some bariatric procedures may be associated with a greater long-term risk of substance and alcohol use disorders, suicide, and nutritional deficiencies. Pharmacokinetic studies indicate that after LRYGB and SG, the anatomic changes lead to very rapid absorption of alcohol and marked increases in blood alco-hol concentrations for a single small.269,270 In the SOS study, RYGB was associated with increased alcohol consumption and an increase in alcohol abuse events (HR: 4.9) over 20 years.196 Similarly in the LABS study, alcohol use disorders were found to be more common in the second postoperative year (9.6%) in those undergoing RYGB compared to before surgery (7.6%). Risk factors for alcohol use disorders included male gender, younger age, and preoperative smoking

1	in the second postoperative year (9.6%) in those undergoing RYGB compared to before surgery (7.6%). Risk factors for alcohol use disorders included male gender, younger age, and preoperative smoking or alcohol use.271 At 7-year follow-up in LABS, there was a progressive and sig-nificant increase over time in the prevalence of regular alcohol consumption for both RYGB and LAGB. In addition, alcohol use disorders, illicit drug use, and treatment for substance use disorders, increased over the 7-year period for RYGB only.272 Overall, these rates were high, with 20% of RYGB participants reporting incident alcohol use disorder symptoms within 5 years of surgery.In addition, there may be an increased risk for suicide273-275 following bariatric surgery, although the etiology is unclear and the data is varied and complex to interpret.276 The Utah mortality study showed a 58% increase in all non–disease-related causes of death in the RYGB group compared to the matched control population,

1	the data is varied and complex to interpret.276 The Utah mortality study showed a 58% increase in all non–disease-related causes of death in the RYGB group compared to the matched control population, including a small but significant increase in suicides, accidents, and intentional poisonings.202 Similar findings were observed in the second Utah Obesity Study.203 An observational study using Pennsylvania state data found that suicide rates were 13.7 per 10,000 among men and 5.2 per 10,000 among women among postbariatric surgery patients in Pennsylvania over 10 years, which were both significantly higher than age and sex-matched rates in the United States. In addition, the majority (70%) of these deaths occurred in the first 3 years following surgery when clinical follow-up is incomplete.277Finally, there is evidence that vitamin and micronutrient deficiencies are common following bariatric surgery including calcium, vitamin D, iron, zinc, and copper, and others. Guide-lines suggest

1	there is evidence that vitamin and micronutrient deficiencies are common following bariatric surgery including calcium, vitamin D, iron, zinc, and copper, and others. Guide-lines suggest that all patients should be screened for deficiencies preoperatively as some deficiencies predate the surgical pro-cedure (see “Follow-Up Postoperative Care”). After surgery, patients must be provided daily nutritional supplementation and undergo routine long-term monitoring for deficiencies (see Table 27-4). Data continue to suggest that the prevalence of micronutrient deficiencies is increasing, while monitoring is decreasing. Aside from these recommendations, there is insuf-ficient evidence currently regarding optimal dietary and nutri-tional management following bariatric procedures, including how to treat some of the specific complications of bariatric operations such as chronic nausea and vomiting, hypoglyce-mic episodes, failed weight loss, and anastomotic ulcers and

1	including how to treat some of the specific complications of bariatric operations such as chronic nausea and vomiting, hypoglyce-mic episodes, failed weight loss, and anastomotic ulcers and strictures.1,278-280REOPERATIVE (REVISION AND CONVERSION) BARIATRIC SURGERYIntroductionSurgical treatments for chronic diseases such as obesity often require additional or revisional surgical procedures when the primary procedure did not sufficiently treat the underly-ing disease. This is true in joint replacements when treating osteo-arthritis, coronary-artery bypass graft surgeries when treating coronary artery disease, and bariatric surgery in the treatment of obesity.281 Additionally, we suspect that obesity is a heteroge-neous disorder282 and is therefore being treated with a variety of procedures with different mechanisms of action.63 Given this, it is not surprising that some patients are “treatment failures” with respect to improvements in weight, comorbidities, and quality of life. Also,

1	with different mechanisms of action.63 Given this, it is not surprising that some patients are “treatment failures” with respect to improvements in weight, comorbidities, and quality of life. Also, older bariatric surgical procedures such as the jejunal-ileal bypass, the VBG, and early gastric bypass procedures (which utilized a horizontal and/or partitioned stomach) require revision because of a higher complication rate.283,284 Initial bariatric sur-gery cases have averaged greater than 150,000 cases per year for the last 15 years.285 For these reasons, reoperative bariatric sur-gery has become increasingly prevalent over the last decade. Despite its increasing prevalence, there are challenges in assess-ing the frequency and effectiveness of these procedures. Reopera-tive bariatric surgery has been difficult to categorize meaningfully and to quantify due to the multiple procedure codes, many with little specificity. Reports in the literature range from 5% to 50% depending on the

1	has been difficult to categorize meaningfully and to quantify due to the multiple procedure codes, many with little specificity. Reports in the literature range from 5% to 50% depending on the primary procedure.283Multiple retrospective, as well as case-matched and case-controlled studies of revisional bariatric surgery, demonstrate they are effective with benefits to weight loss and overall health,281,283 although this is not without some controversy. The literature supports reoperative bariatric surgery in two situa-tions: treatments of insufficient weight loss or weight regain and the treatment of acute and chronic complications.281,285 There are many revisional procedures and approaches that are effective. There is no data-driven evidence to guide in the selection of which patient will benefit most from revisional bariatric surgery. Additionally, there is no evidence-based consensus as to which revisional surgical approach is most optimal in any given situa-tion.283 Currently,

1	benefit most from revisional bariatric surgery. Additionally, there is no evidence-based consensus as to which revisional surgical approach is most optimal in any given situa-tion.283 Currently, there is also little evidence as to which bariat-ric surgical procedure will be efficacious for any specific given patient.286,287Principles and Preoperative EvaluationAs with any other decisions for surgery, revisional or addi-tional bariatric surgery requires evaluating the risks and the benefits of the procedure for specific patient situations. It has been observed that the weight loss following revisional pro-cedures is less than with a primary procedure.288 Reoperations in general are associated with morbidity and mortality that is higher than with primary bariatric procedures281,288,289 but are acceptably low if careful selection of patients is coupled with 9Brunicardi_Ch27_p1167-p1218.indd 120523/02/19 2:21 PM 1206SPECIFIC CONSIDERATIONSPART IIadequate surgeon experience.281,290

1	are acceptably low if careful selection of patients is coupled with 9Brunicardi_Ch27_p1167-p1218.indd 120523/02/19 2:21 PM 1206SPECIFIC CONSIDERATIONSPART IIadequate surgeon experience.281,290 Reoperative bariatric surgery should be undertaken by experienced bariatric surgeons in cen-ters with the wide range of medical resources to manage these complex patients.281 In some situations, it is reasonable to con-sider a two-stage or an open vs. laparoscopic approach.285 When evaluating a patient for revisional bariatric surgery, it is most important to establish clearly the reason for revision.283,291 Is the issue a surgical complication, insufficient weight loss, or weight regain? Is there a discernable anatomic cause for the patient’s symptoms (e.g., abdominal pain, nausea, vomiting, heartburn or reflux, nutritional deficiency)? In situations of noninitial weight loss or weight regain, what is the best justification that can be made? Are there behavioral, or other nonsurgical

1	heartburn or reflux, nutritional deficiency)? In situations of noninitial weight loss or weight regain, what is the best justification that can be made? Are there behavioral, or other nonsurgical modifications that might significantly help to attain further weight loss? Will the patient be able to set into place the behavioral modification required of the revisional surgical approach? With consideration for revision, it is reasonable to place less emphasis on absolute weight loss and focus on the comorbidity and quality of life states. A comprehensive preoperative evaluation is required to fully answer these and other questions.The preoperative evaluation for bariatric surgical revision should include• Review of the initial surgical operative note to understand the exact initial procedure• Anatomic evaluation of the GI track utilizing upper gastroin-testinal endoscopy and radiology to identify known bariatric surgical complications• An extensive nutritional evaluation• A behavioral

1	evaluation of the GI track utilizing upper gastroin-testinal endoscopy and radiology to identify known bariatric surgical complications• An extensive nutritional evaluation• A behavioral health evaluation with a focus on the adaption to the initial bariatric procedure and potential adaptation to a revisional procedure• A full medical evaluation to determine the patient’s suitability to undergo anesthesia and a surgical procedure288,291The results from these evaluations are used to formulate a hypothesis to explain the patient’s symptoms and outcome from the initial procedure and to assess their suitability for and the potential benefits from the potential revisional surgical options. It will come down to a risk-benefit discussion between the sur-geon, other members of the multidisciplinary support team, and the patient.281Treatment for Insufficient Weight Loss or Weight RegainVertical Banded Gastroplasty. Reversal of VBG is associ-ated with significant weight gain. Revision of the VBG

1	support team, and the patient.281Treatment for Insufficient Weight Loss or Weight RegainVertical Banded Gastroplasty. Reversal of VBG is associ-ated with significant weight gain. Revision of the VBG to a re-VBG was associated with poor outcomes.292 VBGs have been safely converted with open and laparoscopic approaches to RYGB and SG.293-295 Conversion of VBG to BPD/DS pro-cedures has limited data with higher leak rate and mortality.296Adjustable Gastric Band. There are increasing reports of LAGB failure and disappointing weight loss over the last decade.289,297,298 Removal of the band without an additional bar-iatric procedure has been associated with significant weight regain.289 Repositioning or replacement of the band is techni-cally possible; however, the long-term weight loss outcomes are mixed.299-301 When converting an LAGB to another bariatric pro-cedure, there is some evidence to suggest possibly fewer com-plications with a two-step approach when significant adhesions or a

1	are mixed.299-301 When converting an LAGB to another bariatric pro-cedure, there is some evidence to suggest possibly fewer com-plications with a two-step approach when significant adhesions or a thick gastric capsule are present. This two-step approach would include removal of the band, allowing 3 to 6 months for gastric tissue healing, and then completing the conver-sion.302,303 However, one-stage conversions have been reported with acceptable outcomes.297 Good outcomes have been dem-onstrated with conversion of LAGBs to SG, RYGB, and BPD/DS.285,297 Several investigators advocate that a failed restrictive bariatric surgery due to poor weight loss should include a revi-sion option with a malabsorptive component.285Sleeve Gastrectomy. Approximately 5% to 10% of primary SG procedures have been reported to require revision for poor weight loss outcomes.304,305 The literature supports conversion to RYGB and BPD/DS.306 There is controversy regarding resleeve gastrectomy.307,308Roux-en-Y

1	been reported to require revision for poor weight loss outcomes.304,305 The literature supports conversion to RYGB and BPD/DS.306 There is controversy regarding resleeve gastrectomy.307,308Roux-en-Y Gastric Bypass. Approximately, 10% to 20 % of patients after a primary RYGB will have inadequate weight loss or weight regain at 2 years, and a subset of these will require a revisional surgical procedure.309 Options for revision include banding over the Roux-en-Y bypass, gastric pouch and gas-trojejunal revisions, RYGB limb lengthening, and conversion to a duodenal switch.281,285 Each of these options has strengths and weaknesses in specific situations. Endoscopic revisions to reduce the gastric pouch and/or gastrojejunal stomal size have been shown to arrest weight gain with short-term weight loss, but the studies have been small and are noncontrolled.Treatment of Surgical ComplicationsVertical Banded Gastroplasty. Several complications have been indications for revision/conversion of

1	loss, but the studies have been small and are noncontrolled.Treatment of Surgical ComplicationsVertical Banded Gastroplasty. Several complications have been indications for revision/conversion of VBGs: wide outlet, pouch dilation, staple line erosion, stoma stenosis, band erosion, band dehiscence, and GERD. Most often conversion is under-taken, and VBGs have been safely converted to RYGB.281,285Adjustable Gastric Band. The following LAGB complica-tions may require additional or revisional surgery: early band obstruction, severe or chronic gastric prolapse or symmetrical gastroesophageal dilatation, band erosion, port and tubing prob-lems, severe or persistent esophageal dysmotility, or psycho-logical intolerance to restriction of band. The literature supports repair of tubing and port problems and revision of LAGB to SG, RYGB, BPD/DS in oneor two-stage revisional operations.285,297Sleeve Gastrectomy. Complications that may require a revi-sional procedure after sleeve gastrectomy are

1	and revision of LAGB to SG, RYGB, BPD/DS in oneor two-stage revisional operations.285,297Sleeve Gastrectomy. Complications that may require a revi-sional procedure after sleeve gastrectomy are staple line leaks, sleeve stricture and sleeve dilatation, and gastroesophageal reflux. Obstruction due to stricture usually at the angularis inci-sura will require first-line treatment of endoscopic dilation and may require a revision to a RYGB. Staple line leaks are ini-tially controlled with endoscopic stenting or drainage. Acute and chronic leaks may develop into fistulous disease and require conversion to RYGB. Persistent gastroesophageal reflux may also require conversion to RYGB.281,285Roux-en-Y Gastric Bypass. Several complications after pri-mary RYGB have been demonstrated as indications for revi-sional surgery: gastric pouch dilatation, gastrojejunostomy dilation/stricture, marginal ulcers, bowel loss due to internal hernia or volvulus, roux stasis syndrome, gastrogastric fistu-las,

1	for revi-sional surgery: gastric pouch dilatation, gastrojejunostomy dilation/stricture, marginal ulcers, bowel loss due to internal hernia or volvulus, roux stasis syndrome, gastrogastric fistu-las, anastomotic structures or ulcers, and metabolic/endocrine derangements. These require a revision focused on the mecha-nism of the complication. Reversal of an RYGB is reserved for severe instances of intractable nausea/vomiting, extreme weight loss and malnutrition, metabolic abnormalities, nonhealing ulceration or leaks, and patient choice. It has been performed rarely, and case reports indicate successful resolution of endo-crine, metabolic, and nutritional abnormalities with improved Brunicardi_Ch27_p1167-p1218.indd 120623/02/19 2:21 PM 1207THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27metabolic parameters. However, 50% to 88% of patients have been reported to regain significant weight.281,285Biliopancreatic Diversion With Duodenal Switch. Acute complications are similar to RYGB and

1	parameters. However, 50% to 88% of patients have been reported to regain significant weight.281,285Biliopancreatic Diversion With Duodenal Switch. Acute complications are similar to RYGB and treated the same. The most severe chronic complication is protein-calorie malnutri-tion, and incidence ranges from 1% to 6%. Management is meticulous nutritional evaluation and nutritional and pancreatic enzyme support, with surgery as a fall back if weight and pro-tein stores are not stabilized. Surgery would entail lengthening the common channel and is rarely necessary.281SPECIAL ISSUES IN BARIATRIC SURGERYBariatric Procedures in AdolescentsThe major controversy with regard to adolescents undergoing bariatric surgery includes the general aversion to subjecting an adolescent to surgery as well as the concern for the second-ary side effects of bariatric surgery on remaining growth and development. Clearly the younger the patient, the more relevant the latter concern becomes. Bariatric surgery in

1	concern for the second-ary side effects of bariatric surgery on remaining growth and development. Clearly the younger the patient, the more relevant the latter concern becomes. Bariatric surgery in adolescents has been performed more frequently, with only 800 cases a year in 2003 increasing to 1600 cases in 2009, but overall rates are much lower than in adults.310,311 Much of the clinical outcomes data have been extrapolated from the adult literature, and more evidence is needed to demonstrate whether weight loss is dura-ble over time, the impact on obesity-related conditions such as T2DM, hypertension, and others, and how often patients experi-ence shortand longer-term complications.A meta-analysis involving 131 adolescents undergoing bariatric surgery demonstrated a 17.8 to 22.3 kg/m2 decrease in BMI after RYGB.312 They also observed improvement of hyper-tension in more than half the patients and sleep apnea resolution in all 131 patients. There were four mortalities in this cohort,

1	in BMI after RYGB.312 They also observed improvement of hyper-tension in more than half the patients and sleep apnea resolution in all 131 patients. There were four mortalities in this cohort, but only one of them was potentially associated with the pro-cedure (Clostridium difficile colitis 9 months after operation). Morbidity in the adolescent literature ranges from 0% to 38%. The most common complication in the meta-analysis was nutri-ent deficiencies. The ASMBS pediatric guidelines suggest using BMI criteria similar to the adult population but with some modi-fications to comorbidity thresholds.313 They recommend con-sidering surgery in patients with a BMI of 35 kg/m2 or greater with major comorbidities (e.g., T2DM, severe nonalcoholic fatty liver disease, OSA) or a BMI of ≥40 kg/m2 or greater with minor comorbidities (e.g., hypertension, dyslipidemia, insulin resistance). One more recent multicenter, prospective study of bariatric surgery in adolescents, the Teen-Longitudinal

1	or greater with minor comorbidities (e.g., hypertension, dyslipidemia, insulin resistance). One more recent multicenter, prospective study of bariatric surgery in adolescents, the Teen-Longitudinal Assess-ment of Bariatric Surgery (Teen-LABS) study, is following 242 adolescent patients for at least 10 years, who underwent bariatric surgery at 5 academic centers.314 Fifty-one percent of adolescents had four or more major comorbid conditions before surgery. LRYGB, SG, and LAGB were performed in 66%, 28%, and 6% of patients, respectively. There were no deaths during the initial hospitalization or within 30 days of operation; major complications such as reoperation were reported in 19 patients (8%). Minor complications such as dehydration were reported in 15%. All reoperations and 85% of readmissions in the 30-day period were related to the bariatric surgery.315 Three-year follow-up data on the Teen-LABS cohort reported a mean weight loss of 28% for LRYGB and 26% for SG. Remission of

1	in the 30-day period were related to the bariatric surgery.315 Three-year follow-up data on the Teen-LABS cohort reported a mean weight loss of 28% for LRYGB and 26% for SG. Remission of T2DM occurred in 95% of participants who had had the con-dition before surgery, remission of abnormal kidney function occurred in 86%, remission of prediabetes in 76%, remission of elevated blood pressure in 74%, and remission of dyslipidemia in 66%. Rates of improvements in comorbid conditions, includ-ing T2DM, occurred at higher rates than in adults. Hypofer-ritinemia was found in 57% of the participants, and 13% of the participants had undergone one or more additional intraabdomi-nal procedures at 3 years.52 So despite the clinically significant improvements in weight, diabetes, cardiometabolic health, and weight-related quality of life that were observed at 3 years fol-lowing surgery, the reoperation and micronutrient risks warrant longer observation and further study.Cost EffectivenessIn a

1	and weight-related quality of life that were observed at 3 years fol-lowing surgery, the reoperation and micronutrient risks warrant longer observation and further study.Cost EffectivenessIn a Canadian study, including five systematic reviews, two eco-nomic evaluations, two reviews of guidelines, and six primary evidence-based guidelines, the cost effectiveness for the use of bariatric surgery in adolescents was reviewed.316 The limited available evidence suggested superior weight loss, resolution of comorbidities compared to nonsurgical interventions, and potential superior weight loss with RYGB compared to other procedures. Cost-effectiveness data was lacking, but limited evidence suggested that bariatric surgery was cost effective several years after intervention, but not immediately.316 A U.S. cost-effectiveness analysis of bariatric surgery in adolescents has been published. In addition to the cost of the surgery, peri-operative mortality, complications, and quality of life

1	A U.S. cost-effectiveness analysis of bariatric surgery in adolescents has been published. In addition to the cost of the surgery, peri-operative mortality, complications, and quality of life improve-ment were included in the modelled analysis. By the fifth year of follow-up, bariatric surgery was found to be cost effective in adolescent patients when compared to a cohort of patients with obesity who had not undergone surgery.317For adults, the overall impact of bariatric surgery to reduce expenditures sufficiently to achieve cost savings continues to be debated. In a Canadian matched cohort study prior to the laparoscopic era, it was shown that bariatric surgery decreases long-term direct healthcare costs and the initial costs of surgery can be amortized over 3.5 years.318 In two observational studies, bariatric surgery was shown to be cost saving over a relatively short period of time.319,320 In more recent observational studies, including the large SOS study and another an analysis

1	studies, bariatric surgery was shown to be cost saving over a relatively short period of time.319,320 In more recent observational studies, including the large SOS study and another an analysis of 30,000 single payor enrollees in the United States, show no evidence of overall cost savings.206,321,322In general, review of the evidence to date suggests that outpatient costs, including pharmacy costs, are significantly reduced after bariatric surgery. However, long-term inpatient hospital costs are increased or unchanged in those who have undergone bariatric surgery compared with matched nonsurgi-cal patients, so no long-term net cost benefit is achieved. Other modeled cost effectiveness studies are consistent with these results as well.323,324 So it is likely that bariatric procedures are cost effective, but do not produce cost savings, compared with nonsurgical treatments.Quality AssuranceBetween 1998 and 2003, with emergence of the laparoscopic technique for bariatric surgery, there

1	cost effective, but do not produce cost savings, compared with nonsurgical treatments.Quality AssuranceBetween 1998 and 2003, with emergence of the laparoscopic technique for bariatric surgery, there was rapid increase in the number bariatric surgical procedures performed. With bariatric surgeons becoming accustomed to laparoscopic techniques and laparoscopic surgeons learning bariatric procedures and patient care, there were realistic concerns regarding the safety of bar-iatric surgery.325 In 2004, ASMBS utilized the volume-outcome concept of centers of excellence and developed the first bar-iatric surgical accreditation program, ASMBS-Center of Excel-lence (COE). In 2005, the American College of Surgeons (ACS) initiated the ACS Bariatric Surgery Center Network (BSCN). Brunicardi_Ch27_p1167-p1218.indd 120723/02/19 2:21 PM 1208SPECIFIC CONSIDERATIONSPART IIThese accreditation programs verified that bariatric surgery centers had the infrastructure and equipment to care for the

1	120723/02/19 2:21 PM 1208SPECIFIC CONSIDERATIONSPART IIThese accreditation programs verified that bariatric surgery centers had the infrastructure and equipment to care for the morbidly obese, experienced and qualified surgeons and staff, appropriate preand postoperative processes in place, and reported outcome data on all surgical cases.325In 2012, these two bariatric surgical accreditations merged into a single unified program, the Metabolic and Bar-iatric Surgery Accreditation and Quality Improvement Program (MBSAQIP). The MBSAQIP has continued to grant accredita-tion only after a rigorous review process during which a center proves that it can maintain certain physical resources, human resources, and standards of practice.326 Additionally, prospec-tive outcome data is collected at the clinical center and validated based on standardized definitions and submitted to the quality improvement program. Centers receive back risk-stratified anal-ysis of their data as a means to

1	at the clinical center and validated based on standardized definitions and submitted to the quality improvement program. Centers receive back risk-stratified anal-ysis of their data as a means to compare with the national statis-tics/standards and are then required to utilize the data in quality improvement projects at their center. This newer focus on qual-ity improvement over centers of excellence will likely continue to improve the quality of bariatric surgery in the United States. Recently, in a systematic review, bariatric facility accreditation by this program has been associated with improved outcomes (length of stay, mortality, morbidity).327Plastic Surgery After Weight LossPatients who have undergone bariatric surgery are often left with skin and subcutaneous tissue deformities. Additional prob-lems include skin rashes and maceration under folds in the pan-nus, thighs, and breasts; body odor; and poorly fitting clothes. Excess skin can also be a limiting factor in exercise

1	Additional prob-lems include skin rashes and maceration under folds in the pan-nus, thighs, and breasts; body odor; and poorly fitting clothes. Excess skin can also be a limiting factor in exercise and sexual activity. Plastic and reconstructive surgery is now a part of the continuum of care for bariatric surgery patients. Reconstructive surgery requires careful preoperative planning and is based on the patient’s deformities and priorities. Timing of plastic and reconstructive surgery is typically deferred until weight sta-bility at approximately 1 to 2 years postoperatively to ensure improved healing. Excess tissue of the lower torso is the most common area for which patients undergo surgical intervention and a standard abdominoplasty is typically performed. More radical body contouring can include a circumferential abdomi-noplasty and lower body lift.328,329 This procedure involves excision of tissue from the buttocks and lateral thighs, with skin undermining down the thighs.

1	can include a circumferential abdomi-noplasty and lower body lift.328,329 This procedure involves excision of tissue from the buttocks and lateral thighs, with skin undermining down the thighs. Circumferential abdomino-plasty removes redundant skin of the lower abdomen, flattens the abdomen, and incorporates the lower body lift. It requires central undermining to the xiphoid and minimal lateral under-mining of the superior flap. The central abdominal fascia often requires imbrication. If simultaneous abdominal hernia repair is performed, this performs the function of fascial imbrication by creating a repair with some degree of fascial tension. The closure of the superior flap to the inferior skin edge incorporates lateral tension to narrow the waist and advance the anterolateral thighs. Medial thighplasty also may be needed for patients with significant excess medial thigh skin.Mid-back and epigastric deformity, along with sagging breasts, are corrected with an upper body lift. The

1	Medial thighplasty also may be needed for patients with significant excess medial thigh skin.Mid-back and epigastric deformity, along with sagging breasts, are corrected with an upper body lift. The upper body lift is a reverse abdominoplasty, removal of mid-torso excessive skin, and reshaping of the breasts. For highly selected individu-als, and with a well-organized team, a single-stage total body lift, which includes a circumferential abdominoplasty, lower body lift, medial thighplasty, an upper body lift, and breast reshap-ing, can be performed safely in under 8 hours (Figs. 27-30 and 27-31).330 Increasing numbers of patients are seeking these cor-rective procedures, and data about the results is evolving. There Figure 27-30. Preoperative frontal, right lateral, and left anterior oblique views of a 36-year-old, 150-lb (68-kg) 5'6" woman who lost 120 lb (54 kg) 2 years after laparoscopic Roux-en-Y bypass procedure. She desired a one-stage total body lift and bilateral

1	oblique views of a 36-year-old, 150-lb (68-kg) 5'6" woman who lost 120 lb (54 kg) 2 years after laparoscopic Roux-en-Y bypass procedure. She desired a one-stage total body lift and bilateral brachioplasties, which were performed in the manner described in the text. (Used with permission from Dennis Hurwitz, MD, Clinical Professor of Plastic Surgery, University of Pittsburgh.)Brunicardi_Ch27_p1167-p1218.indd 120823/02/19 2:21 PM 1209THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27Figure 27-31. Frontal, right lateral, and left anterior oblique views 6 weeks after surgery for the woman in Fig. 27-24. The scars indicate the circumferential abdominoplasty, lower body lift, upper body lift, breast reshaping, and autoaugmentation through a keyhole pattern and bilateral brachioplasties. All redundant skin has been removed, leaving well-positioned scars and feminine features. (Used with permission from Dennis Hurwitz, MD, Clinical Professor of Plastic Surgery, University of Pittsburgh.)is a

1	skin has been removed, leaving well-positioned scars and feminine features. (Used with permission from Dennis Hurwitz, MD, Clinical Professor of Plastic Surgery, University of Pittsburgh.)is a hypothesis that if body image is improved with corrective surgery, that weight maintenance in the longer-term may also be positively affected. Several matched controlled studies suggest that plastic surgery after bariatric surgery may improve long-term weight loss results.328,331FUTURE IMPORTANT QUESTIONSThe volume and quality of literature in the field of bariatric sur-gery has grown tremendously in the last 10 years. High-quality evidence now shows that bariatric surgical procedures result in greater weight loss than nonsurgical treatments, improved sur-vival, and are more effective at inducing remission of T2DM in people with obesity. More information is still needed about the long-term durability of comorbid health improvements and long-term complications after each of the different

1	remission of T2DM in people with obesity. More information is still needed about the long-term durability of comorbid health improvements and long-term complications after each of the different bar-iatric surgical procedures. In addition, the underlying specific mechanism(s) of action for both bariatric and metabolic surgery is still incompletely understood. Future knowledge will come from translational human studies, the ongoing longer-term stud-ies and data registries, randomized studies comparing surgical to nonsurgical treatments, integrated health care systems data, and national “big data” networks. The following are some of the high-priority questions that future research will address.• What are the specific mechanisms of action responsible for weight loss and the T2DM response to bariatric surgical procedures?• What patient level factors can predict success with weight loss, health improvements, and cost savings after bariatric surgical procedures? Understanding preand

1	to bariatric surgical procedures?• What patient level factors can predict success with weight loss, health improvements, and cost savings after bariatric surgical procedures? Understanding preand postsurgery predictors will help to tailor an individual’s treatment.• Is bariatric surgery more effective than nonsurgical care for the longer-term treatment of T2DM in people with less severe obesity (class 1 obesity, BMI <35)?• With more standardized reporting of complications across bariatric studies, what are the long-term complication rates after different bariatric procedures?• What is the effect of bariatric surgery on long-term microvas-cular and macrovascular event rates?• What are the reproductive and mental health outcomes includ-ing risk for self-harm and suicide, alcohol use disorders, sub-stance abuse, and other risk-taking behaviors?REFERENCESEntries highlighted in bright blue are key references. 1. Arterburn DE, Courcoulas AP. Bariatric surgery for obesity and metabolic

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1	and lung function in obese subjects with asthma. Respirat Med. 2012;106(5):651-660. 235. Beymer C, Kowdley KV, Larson A, Edmonson P, Dellinger EP, Flum DR. Prevalence and predictors of asymptomatic liver disease in patients undergoing gastric bypass surgery. Arch Surg. 2003;138(11):1240-1244. 236. Rabl C, Campos GM. The impact of bariatric surgery on nonalcoholic steatohepatitis. Semin Liver Dis. 2012;32(1): 80-91. 237. Iossi MF, Konstantakos EK, Teel DD, 2nd, et al. Musculoskeletal function following bariatric surgery. Obesity. 2013;21(6):1104-1110. 238. Vincent HK, Heywood K, Connelly J, Hurley RW. Obesity and weight loss in the treatment and prevention of osteoarthritis. PM R. 2012;4(5 suppl):S59-S67. 239. King WC, Chen JY, Belle SH, et al. Change in pain and physical function following bariatric surgery for severe obesity. JAMA. 2016;315(13):1362-1371. 240. Karlsson J, Taft C, Ryden A, Sjostrom L, Sullivan M. Ten-year trends in health-related quality of life after surgical and

1	bariatric surgery for severe obesity. JAMA. 2016;315(13):1362-1371. 240. Karlsson J, Taft C, Ryden A, Sjostrom L, Sullivan M. Ten-year trends in health-related quality of life after surgical and conventional treatment for severe obesity: the SOS intervention study. Int J Obes. 2007;31(8):1248-1261. 241. Kolotkin RL, Davidson LE, Crosby RD, Hunt SC, Adams TD. Six-year changes in health-related quality of life in gastric bypass patients versus obese comparison groups. Surg Obes Relat Dis. 2012;8(5):625-633. 242. Nickel MK, Loew TH, Bachler E. Change in mental symptoms in extreme obesity patients after gastric banding, part II: six-year follow up. Int J Psychiatry Med. 2007;37(1):69-79. 243. Schauer PR, Nor Hanipah Z, Rubino F. Metabolic surgery for treating type 2 diabetes mellitus: now supported by the world’s leading diabetes organizations. Cleve Clin J Med. 2017;84 (7 suppl 1):S47-S56. 244. Buchwald H, Estok R, Fahrbach K, et al. Weight and type 2 diabetes after bariatric surgery:

1	by the world’s leading diabetes organizations. Cleve Clin J Med. 2017;84 (7 suppl 1):S47-S56. 244. Buchwald H, Estok R, Fahrbach K, et al. Weight and type 2 diabetes after bariatric surgery: systematic review and meta-analysis. Am J Med. 2009;122(3):248-256 e245. 245. Sjostrom L, Peltonen M, Jacobson P, et al. Association of bariatric surgery with long-term remission of type 2 diabetes and with microvascular and macrovascular complications. JAMA. 2014;311(22):2297-2304. 246. Schauer PR, Bhatt DL, Kirwan JP, et al. Bariatric surgery versus intensive medical therapy for diabetes—3-year outcomes. N Engl J Med. 2014;370(21):2002-2013. 247. Ikramuddin S, Billington CJ, Lee WJ, et al. Roux-en-Y gastric bypass for diabetes (the Diabetes Surgery Study): 2-year outcomes of a 5-year, randomised, controlled trial. Lancet Diabet Endocrinol. 2015;3(6):413-422. 248. Liang Z, Wu Q, Chen B, Yu P, Zhao H, Ouyang X. Effect of laparoscopic Roux-en-Y gastric bypass surgery on type 2 diabetes mellitus

1	controlled trial. Lancet Diabet Endocrinol. 2015;3(6):413-422. 248. Liang Z, Wu Q, Chen B, Yu P, Zhao H, Ouyang X. Effect of laparoscopic Roux-en-Y gastric bypass surgery on type 2 diabetes mellitus with hypertension: a randomized controlled trial. Diabet Clin Res Pract. 2013;101(1):50-56. 249. Courcoulas AP, Yanovski SZ, Bonds D, et al. Long-term outcomes of bariatric surgery: a National Institutes of Health symposium. JAMA Surg. 2014;149(12):1323-1329. 250. Wentworth JM, Playfair J, Laurie C, et al. Multidisciplinary diabetes care with and without bariatric surgery in overweight people: a randomised controlled trial. Lancet Diabet Endocrinol. 2014;2(7):545-552. 251. Parikh M, Chung M, Sheth S, et al. Randomized pilot trial of bariatric surgery versus intensive medical weight management on diabetes remission in type 2 diabetic patients who do NOT meet NIH criteria for surgery and the role of soluble RAGE as a novel biomarker of success. Ann Surg. 2014;260(4):617-622; discussion

1	on diabetes remission in type 2 diabetic patients who do NOT meet NIH criteria for surgery and the role of soluble RAGE as a novel biomarker of success. Ann Surg. 2014;260(4):617-622; discussion 622-614. 252. Schauer PR, Bhatt DL, Kirwan JP, et al. Bariatric surgery versus intensive medical therapy for diabetes—5-year outcomes. N Engl J Med. 2017;376(7):641-651. This is a randomized clinical trial comparing gastric bypass, gastric sleeve, and intensive medical management for the treatment of type 2 diabetes in people with obesity. It is only one of 2 randomized studies with 5 year follow up, at this time. It shows that surgical treatments are superior to intensive medical treatment for glycemic control. 253. Shah SS Todkar J, Phadake U, et al. Gastric bypass vs. medical/lifestyle care for type 2 diabetes in South Asians with BMI 25-40 kg/m2: the COSMID randomized trial Brunicardi_Ch27_p1167-p1218.indd 121623/02/19 2:21 PM 1217THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27[261-OR].

1	type 2 diabetes in South Asians with BMI 25-40 kg/m2: the COSMID randomized trial Brunicardi_Ch27_p1167-p1218.indd 121623/02/19 2:21 PM 1217THE SURGICAL MANAGEMENT OF OBESITYCHAPTER 27[261-OR]. Presented at the American Diabetes Association’s 76th Scientific Session; June 10-14, 2016; New Orleans, LA. 254. Encinosa WE, Bernard DM, Du D, Steiner CA. Recent improvements in bariatric surgery outcomes. Med Care. 2009; 47(5):531-535. 255. Birkmeyer NJ, Dimick JB, Share D, et al. Hospital complication rates with bariatric surgery in Michigan. JAMA. 2010;304(4):435-442. 256. Rubino F, Kaplan LM, Schauer PR, Cummings DE. The Diabetes Surgery Summit consensus conference: recommendations for the evaluation and use of gastrointestinal surgery to treat type 2 diabetes mellitus. Ann Surg. 2010;251(3):399-405. 257. Buchwald H, Estok R, Fahrbach K, Banel D, Sledge I. Trends in mortality in bariatric surgery: a systematic review and meta-analysis. Surgery. 2007;142(4):621-632; discussion

1	2010;251(3):399-405. 257. Buchwald H, Estok R, Fahrbach K, Banel D, Sledge I. Trends in mortality in bariatric surgery: a systematic review and meta-analysis. Surgery. 2007;142(4):621-632; discussion 632-625. 258. Dimick JB, Nicholas LH, Ryan AM, Thumma JR, Birkmeyer JD. Bariatric surgery complications before vs after implementation of a national policy restricting coverage to centers of excellence. JAMA. 2013;309(8):792-799. 259. DeMaria EJ, Murr M, Byrne TK, et al. Validation of the obesity surgery mortality risk score in a multicenter study proves it stratifies mortality risk in patients undergoing gastric bypass for morbid obesity. Ann Surg. 2007;246(4):578-582; discussion 583-574. 260. DeMaria EJ, Portenier D, Wolfe L. Obesity surgery mortality risk score: proposal for a clinically useful score to predict mortality risk in patients undergoing gastric bypass. Surg Obes Relat Dis. 2007;3(2):134-140. 261. Thomas H, Agrawal S. Systematic review of obesity surgery mortality risk

1	useful score to predict mortality risk in patients undergoing gastric bypass. Surg Obes Relat Dis. 2007;3(2):134-140. 261. Thomas H, Agrawal S. Systematic review of obesity surgery mortality risk score—preoperative risk stratification in bariatric surgery. Obes Surg. 2012;22(7):1135-1140. 262. O’Brien PE, MacDonald L, Anderson M, Brennan L, Brown WA. Long-term outcomes after bariatric surgery: fifteen-year follow-up of adjustable gastric banding and a systematic review of the bariatric surgical literature. Ann Surg. 2013;257(1):87-94. 263. Rariy CM, Rometo D, Korytkowski M. Post-gastric bypass hypoglycemia. Curr Diabet Rep. 2016;16(2):19. 264. Goldfine AB, Patti ME. How common is hypoglycemia after gastric bypass? Obesity. 2016;24(6):1210-1211. 265. Service GJ, Thompson GB, Service FJ, Andrews JC, Collazo-Clavell ML, Lloyd RV. Hyperinsulinemic hypoglycemia with nesidioblastosis after gastric-bypass surgery. N Engl J Med. 2005;353(3):249-254. 266. Patti ME, Goldfine AB. Hypoglycemia

1	Andrews JC, Collazo-Clavell ML, Lloyd RV. Hyperinsulinemic hypoglycemia with nesidioblastosis after gastric-bypass surgery. N Engl J Med. 2005;353(3):249-254. 266. Patti ME, Goldfine AB. Hypoglycemia after gastric bypass: the dark side of GLP-1. Gastroenterology. 2014;146(3):605-608. 267. Eisenberg D, Azagury DE, Ghiassi S, Grover BT, Kim JJ. ASMBS position statement on postprandial hyperinsulinemic hypoglycemia after bariatric surgery. Surg Obes Relat Dis. 2017;13(3):371-378. 268. Stephansson O, Johansson K, Naslund I, Neovius M. Bariatric Surgery and Preterm Birth. N Engl J Med. 2016;375(8):805-806. 269. Maluenda F, Csendes A, De Aretxabala X, et al. Alcohol absorption modification after a laparoscopic sleeve gastrectomy due to obesity. Obes Surg. 2010;20(6):744-748. 270. Steffen KJ, Engel SG, Pollert GA, Li C, Mitchell JE. Blood alcohol concentrations rise rapidly and dramatically after Roux-en-Y gastric bypass. Surg Obes Relat Dis. 2013;9(3): 470-473. 271. King WC, Chen JY,

1	KJ, Engel SG, Pollert GA, Li C, Mitchell JE. Blood alcohol concentrations rise rapidly and dramatically after Roux-en-Y gastric bypass. Surg Obes Relat Dis. 2013;9(3): 470-473. 271. King WC, Chen JY, Mitchell JE, et al. Prevalence of alcohol use disorders before and after bariatric surgery. JAMA. 2012;307(23):2516-2525. 272. King WC, Chen JY, Courcoulas AP, et al. Alcohol and other substance use after bariatric surgery: prospective evidence from a U.S. multicenter cohort study. Surg Obes Relat Dis. 2017;13(8):1392-1402. 273. Peterhansel C, Petroff D, Klinitzke G, Kersting A, Wagner B. Risk of completed suicide after bariatric surgery: a systematic review. Obes Rev. 2013;14(5):369-382. 274. Gribsholt SB, Thomsen RW, Svensson E, Richelsen B. Overall and cause-specific mortality after Roux-en-Y gastric bypass surgery: a nationwide cohort study. Surg Obes Relat Dis. 2017;13(4):581-587. 275. Bhatti JA, Nathens AB, Thiruchelvam D, Grantcharov T, Goldstein BI, Redelmeier DA. Self-harm

1	Roux-en-Y gastric bypass surgery: a nationwide cohort study. Surg Obes Relat Dis. 2017;13(4):581-587. 275. Bhatti JA, Nathens AB, Thiruchelvam D, Grantcharov T, Goldstein BI, Redelmeier DA. Self-harm emergencies after bariatric surgery: a population-based cohort study. JAMA Surg. 2016;151(3):226-232. 276. Courcoulas A. Who, why, and how? Suicide and harmful behaviors after bariatric surgery. Ann Surg. 2017;265(2): 253-254. 277. Tindle HA, Omalu B, Courcoulas A, Marcus M, Hammers J, Kuller LH. Risk of suicide after long-term follow-up from bariatric surgery. Am J Med. 2010;123(11):1036-1042. 278. Parrott J, Frank L, Rabena R, Craggs-Dino L, Isom KA, Greiman L. American Society for Metabolic and Bariatric Surgery integrated health nutritional guidelines for the surgical weight loss patient 2016 update: micronutrients. Surg Obes Relat Dis. 2017;13(5):727-741. 279. Dogan K, Homan J, Aarts EO, de Boer H, van Laarhoven CJ, Berends FJ. Long-term nutritional status in patients following

1	2016 update: micronutrients. Surg Obes Relat Dis. 2017;13(5):727-741. 279. Dogan K, Homan J, Aarts EO, de Boer H, van Laarhoven CJ, Berends FJ. Long-term nutritional status in patients following Roux-en-Y gastric bypass surgery. Clin Nutr. 2018;37(2):612-617. 280. Pellitero S, Martinez E, Puig R, et al. Evaluation of vitamin and trace element requirements after sleeve gastrectomy at long term. Obes Surg. 2017;27(7):1674-1682. 281. Brethauer SA, Kothari S, Sudan R, et al. Systematic review on reoperative bariatric surgery: American Society for Metabolic and Bariatric Surgery Revision Task Force. Surg Obes Relat Dis. 2014;10(5):952-972. 282. Field AE, Camargo CA Jr, Ogino S. The merits of subtyping obesity: one size does not fit all. JAMA. 2013; 310(20):2147-2148. 283. Kellogg TA. Revisional bariatric surgery. Surg Clin North Am. 2011;91(6):1353-1371, x. 284. Moshiri M, Osman S, Robinson TJ, Khandelwal S, Bhargava P, Rohrmann CA. Evolution of bariatric surgery: a historical perspective.

1	bariatric surgery. Surg Clin North Am. 2011;91(6):1353-1371, x. 284. Moshiri M, Osman S, Robinson TJ, Khandelwal S, Bhargava P, Rohrmann CA. Evolution of bariatric surgery: a historical perspective. AJR Am J Roentgenol. 2013;201(1):W40-W48. 285. Switzer NJ, Karmali S, Gill RS, Sherman V. Revisional bariatric surgery. Surg Clin North Am. 2016;96(4):827-842. 286. Ikramuddin S, Livingston EH. New insights on bariatric surgery outcomes. JAMA. 2013;310(22):2401-2402. 287. Kral JG. Selection of patients for anti-obesity surgery. Int J Obes Relat Metab Disord. 2001;25(suppl 1):S107-S112. 288. Zundel N, Hernandez JD. Revisional surgery after restrictive procedures for morbid obesity. Surg Laparosc Endosc Percutan Tech. 2010;20(5):338-343. 289. Fulton C, Sheppard C, Birch D, Karmali S, de Gara C. A comparison of revisional and primary bariatric surgery. Can J Surg. 2017;60(3):205-211. 290. Hallowell PT, Stellato TA, Yao DA, Robinson A, Schuster MM, Graf KN. Should bariatric revisional

1	C. A comparison of revisional and primary bariatric surgery. Can J Surg. 2017;60(3):205-211. 290. Hallowell PT, Stellato TA, Yao DA, Robinson A, Schuster MM, Graf KN. Should bariatric revisional surgery be avoided secondary to increased morbidity and mortality? Am J Surg. 2009;197(3):391-396. 291. Sarr MG. Reoperative bariatric surgery. Surg Endosc. 2007;21(11):1909-1913. 292. van Gemert WG, van Wersch MM, Greve JW, Soeters PB. Revisional surgery after failed vertical banded gastroplasty: restoration of vertical banded gastroplasty or conversion to gastric bypass. Obes Surg. 1998;8(1):21-28. 293. Gagne DJ, Dovec E, Urbandt JE. Laparoscopic revision of vertical banded gastroplasty to Roux-en-Y gastric bypass: outcomes of 105 patients. Surg Obes Relat Dis. 2011;7(4):493-499.Brunicardi_Ch27_p1167-p1218.indd 121723/02/19 2:21 PM 1218SPECIFIC CONSIDERATIONSPART II 294. Schouten R, Wiryasaputra DC, van Dielen FM, van Gemert WG, Greve JW. Influence of reoperations on long-term quality

1	121723/02/19 2:21 PM 1218SPECIFIC CONSIDERATIONSPART II 294. Schouten R, Wiryasaputra DC, van Dielen FM, van Gemert WG, Greve JW. Influence of reoperations on long-term quality of life after restrictive procedures: a prospective study. Obes Surg. 2011;21(7):871-879. 295. Foletto M, Prevedello L, Bernante P, et al. Sleeve gastrectomy as revisional procedure for failed gastric banding or gastroplasty. Surg Obes Relat Dis. 2010;6(2):146-151. 296. Dapri G, Cadiere GB, Himpens J. Laparoscopic conversion of adjustable gastric banding and vertical banded gastroplasty to duodenal switch. Surg Obes Relat Dis. 2009;5(6):678-683. 297. Coblijn UK, Verveld CJ, van Wagensveld BA, Lagarde SM. Laparoscopic Roux-en-Y gastric bypass or laparoscopic sleeve gastrectomy as revisional procedure after adjustable gastric band--a systematic review. Obes Surg. 2013;23(11):1899-1914. 298. Aarts EO, Dogan K, Koehestanie P, Janssen IM, Berends FJ. What happens after gastric band removal without additional

1	gastric band--a systematic review. Obes Surg. 2013;23(11):1899-1914. 298. Aarts EO, Dogan K, Koehestanie P, Janssen IM, Berends FJ. What happens after gastric band removal without additional bariatric surgery? Surg Obes Relat Dis. 2014;10(6):1092-1096. 299. Ardestani A, Lautz DB, Tavakkolizadeh A. Band revision versus Roux-en-Y gastric bypass conversion as salvage operation after laparoscopic adjustable gastric banding. Surg Obes Relat Dis. 2011;7(1):33-37. 300. Lanthaler M, Mittermair R, Erne B, Weiss H, Aigner F, Nehoda H. Laparoscopic gastric re-banding versus laparoscopic gastric bypass as a rescue operation for patients with pouch dilatation. Obes Surg. 2006;16(4):484-487. 301. Muller MK, Attigah N, Wildi S, et al. High secondary failure rate of rebanding after failed gastric banding. Surg Endosc. 2008;22(2):448-453. 302. Obeid NR, Schwack BF, Kurian MS, Ren-Fielding CJ, Fielding GA. Single-stage versus 2-stage sleeve gastrectomy as a conversion after failed adjustable gastric

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1	in adolescents: recent national trends in use and in-hospital outcome. Arch Pediatr Adolesc Med. 2007;161(3):217-221. 311. Zwintscher NP, Azarow KS, Horton JD, Newton CR, Martin MJ. The increasing incidence of adolescent bariatric surgery. J Pediatr Surg. 2013;48(12):2401-2407. 312. Treadwell JR, Sun F, Schoelles K. Systematic review and meta-analysis of bariatric surgery for pediatric obesity. Ann Surg. 2008;248(5):763-776. 313. Michalsky M, Reichard K, Inge T, et al. ASMBS pediatric committee best practice guidelines. Surg Obes Relat Dis. 2012;8(1):1-7. 314. Inge TH, Zeller M, Harmon C, et al. Teen-Longitudinal Assessment of Bariatric Surgery: methodological features of the first prospective multicenter study of adolescent bariatric surgery. J Pediatr Surg. 2007;42(11):1969-1971. 315. Inge TH, Zeller MH, Jenkins TM, et al. Perioperative outcomes of adolescents undergoing bariatric surgery: the Teen-Longitudinal Assessment of Bariatric Surgery (Teen-LABS) study. JAMA Pediatr.

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1	PY, Buchwald H, Shikora SA, Ghosh A, Yang HE, Buessing M. A study on the economic impact of bariatric surgery. Am J Manag Care. 2008;14(9):589-596. 320. Finkelstein EA, Allaire BT, Burgess SM, Hale BC. Financial implications of coverage for laparoscopic adjustable gastric banding. Surg Obes Relat Dis. 2011;7(3):295-303. 321. Neovius M, Narbro K, Keating C, et al. Health care use during 20 years following bariatric surgery. JAMA. 2012;308(11):1132-1141. 322. Weiner JP, Goodwin SM, Chang HY, et al. Impact of bariatric surgery on health care costs of obese persons: a 6-year follow-up of surgical and comparison cohorts using health plan data. JAMA Surg. 2013;148(6):555-562. 323. Picot J, Jones J, Colquitt JL, et al. The clinical effectiveness and cost-effectiveness of bariatric (weight loss) surgery for obesity: a systematic review and economic evaluation. Health Technol Assess. 2009;13(41):1-190, 215-357, iii-iv. 324. Padwal R, Klarenbach S, Wiebe N, et al. Bariatric surgery: a

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1	Small IntestineAli Tavakkoli, Stanley W. Ashley, and Michael J. Zinner 28chapterINTRODUCTORY COMMENTSThe small intestine is a remarkable and complex organ that is not only the principle site of nutrient digestion and absorption but also contains the body’s largest reservoir of immunologi-cally active and hormone-producing cells. Hence, it can be con-ceptualized as the largest organ of the immune and endocrine systems.1 It achieves this diversity of action through unique anatomical features, which provide it with a massive sur-face area, a diversity of cell types, and a complex neural network to coordinate these functions.Despite the size and importance of the small intestine, diseases of this organ are relatively infrequent and can present diagnostic and therapeutic challenges. Despite introduction of novel imaging techniques such as capsule endoscopy and dou-ble balloon endoscopy, diagnostic tests lack sufficient ability to reliably assess the small bowel. Furthermore, few

1	Despite introduction of novel imaging techniques such as capsule endoscopy and dou-ble balloon endoscopy, diagnostic tests lack sufficient ability to reliably assess the small bowel. Furthermore, few high-quality, controlled data on the efficacy of surgical therapies for small bowel diseases are available.1Therefore, sound clinical judgment and a thorough under-standing of anatomy, physiology, and pathophysiology remain essential to the care of patients with suspected small bowel disorders.GROSS ANATOMYThe small intestine is a tubular structure that extends from the pylorus to the cecum. The estimated length varies depending on whether radiologic, surgical, or autopsy measurements are made. In the living, it is thought to measure 4 to 6 meters.2 The small intestine consists of three segments lying in series: the duodenum, the jejunum, and the ileum. The duodenum, the most proximal segment, lies in the retroperitoneum immediately adja-cent to the head and inferior border of the body of

1	lying in series: the duodenum, the jejunum, and the ileum. The duodenum, the most proximal segment, lies in the retroperitoneum immediately adja-cent to the head and inferior border of the body of the pancreas. The duodenum is demarcated from the stomach by the pylorus and from the jejunum by the ligament of Treitz. The jejunum and ileum lie within the peritoneal cavity and are tethered to the Introductory Comments1219Gross Anatomy1219Histology1220Development1221Physiology1222Digestion and Absorption / 1222Barrier and Immune Function / 1225Motility / 1226Endocrine Function / 1227Intestinal Adaptation / 1228Small Bowel Obstruction1228Epidemiology / 1228Pathophysiology / 1229Clinical Presentation / 1229Diagnosis / 1229Therapy / 1231Outcomes / 1232Prevention / 1233Other Causes of Small Bowel Obstruction / 1233Ileus and Other Disorders of Intestinal Motility1233Pathophysiology / 1233Clinical Presentation / 1234Diagnosis / 1234Therapy / 1234Crohn’s Disease1235Pathophysiology /

1	Small Bowel Obstruction / 1233Ileus and Other Disorders of Intestinal Motility1233Pathophysiology / 1233Clinical Presentation / 1234Diagnosis / 1234Therapy / 1234Crohn’s Disease1235Pathophysiology / 1235Clinical Presentation / 1236Diagnosis / 1237Therapy / 1238Outcomes / 1240Intestinal Fistulas1240Pathophysiology / 1240Clinical Presentation / 1240Diagnosis / 1240Therapy / 1241Outcomes / 1241Small Bowel Neoplasms1241Pathophysiology / 1242Clinical Presentation / 1243Diagnosis / 1243Therapy / 1244Outcomes / 1245Radiation Enteritis1245Pathophysiology / 1245Clinical Presentation / 1245Diagnosis / 1245Therapy / 1246Outcomes / 1246Prevention / 1246Meckel’s Diverticula1246Pathophysiology / 1246Clinical Presentation / 1247Diagnosis / 1247Therapy / 1248Acquired Diverticula1248Pathophysiology / 1249Clinical Presentation / 1250Diagnosis / 1250Therapy / 1250Mesenteric Ischemia1250Miscellaneous Conditions1250Obscure GI Bleeding / 1250Small Bowel Perforation / 1251Chylous Ascites /

1	/ 1249Clinical Presentation / 1250Diagnosis / 1250Therapy / 1250Mesenteric Ischemia1250Miscellaneous Conditions1250Obscure GI Bleeding / 1250Small Bowel Perforation / 1251Chylous Ascites / 1252Intussusception / 1253Pneumatosis Intestinalis / 1253Short Bowel Syndrome1254Pathophysiology / 1254Therapy / 1255Outcomes / 1255Brunicardi_Ch28_p1219-p1258.indd 121923/02/19 2:24 PM 1220JejunumIleumKey Points1 The small intestine performs a diverse set of functions.2 Small bowel obstruction is one of the most common surgical diagnoses.3 Most cases of small bowel obstruction are due to adhe-sions from previous surgery and resolve with conservative management.4 Tumors and malignancies of the small bowel are rare and difficult to diagnose. 5 If following surgical resection less than 200 cm of small bowel remains, patients are at risk of developing short bowel syndrome.retroperitoneum by a broad-based mesentery. No distinct ana-tomical landmark demarcates the jejunum from the ileum; the proximal

1	bowel remains, patients are at risk of developing short bowel syndrome.retroperitoneum by a broad-based mesentery. No distinct ana-tomical landmark demarcates the jejunum from the ileum; the proximal 40% of the jejunoileal segment is arbitrarily defined as the jejunum and the distal 60% as the ileum. The ileum is demarcated from the cecum by the ileocecal valve.The small intestine contains internal mucosal folds known as plicae circulares or valvulae conniventes that are visible upon gross inspection. These folds are also visible radiographically and help in the distinction between small intestine and colon, which does not contain them, on abdominal radiographs. These folds are more prominent in the proximal intestine than in the distal small intestine. Other features evident on gross inspection that are more characteristic of the proximal than distal small intestine include larger circumference, thicker wall, less fatty mesentery, and longer vasa recta (Fig. 28-1). Gross examination

1	that are more characteristic of the proximal than distal small intestine include larger circumference, thicker wall, less fatty mesentery, and longer vasa recta (Fig. 28-1). Gross examination of the small-intestinal mucosa also reveals aggregates of lymphoid follicles. Those follicles, located in the ileum, are the most prominent and are designated Peyer’s patches.Most of the duodenum derives its arterial blood from branches of both the celiac and the superior mesenteric arteries. The distal duodenum, the jejunum, and the ileum derive their arterial blood from the superior mesenteric artery. Their venous drainage occurs via the superior mesenteric vein. Lymph drain-age occurs through lymphatic vessels coursing parallel to corre-sponding arteries. This lymph drains through mesenteric lymph nodes to the cisterna chyli, then through the thoracic duct, and ultimately into the left subclavian vein. The parasympathetic and sympathetic innervation of the small intestine is derived from the

1	nodes to the cisterna chyli, then through the thoracic duct, and ultimately into the left subclavian vein. The parasympathetic and sympathetic innervation of the small intestine is derived from the vagus and splanchnic nerves, respectively.HISTOLOGYThe wall of the small intestine consists of four distinct layers: mucosa, submucosa, muscularis propria, and serosa (Fig. 28-2).The mucosa is the innermost layer and it consists of three layers: epithelium, lamina propria, and muscularis mucosae. The epithelium is exposed to the intestinal lumen and is the surface through which absorption from and secretion into the lumen occurs. The lamina propria is located immediately external to the epithelium and consists of connective tissue and a heterogeneous population of cells. It is demarcated from the more external submucosa by the muscularis mucosae, a thin sheet of smooth muscle cells.The mucosa is organized into villi and crypts (crypts of Lieberkuhn). Villi are finger-like projections of

1	the more external submucosa by the muscularis mucosae, a thin sheet of smooth muscle cells.The mucosa is organized into villi and crypts (crypts of Lieberkuhn). Villi are finger-like projections of epithelium and underlying lamina propria that contain blood and lymphatic (lacteals) vessels that extend into the intestinal lumen. Intes-tinal, epithelial cellular proliferation is confined to the crypts, each of which carries 250 to 300 cells. All epithelial cells in each crypt are derived from an unknown number of multipotent stem cells located at or near the crypt’s base. Our understanding of these crypt cells is rapidly expanding. It appears that there are two subgroups of intestinal stem cells, with specific cell markers. Bmi1-positive cells are usually quiescent, radiation-resistant cells that are induced by injury, while LGR5-positive cells facilitate homeostatic vs. injury-induced regeneration and are radiation sensitive.3The stem cells can differentiate along one of four path-ways

1	that are induced by injury, while LGR5-positive cells facilitate homeostatic vs. injury-induced regeneration and are radiation sensitive.3The stem cells can differentiate along one of four path-ways that ultimately yield enterocytes and goblet, enteroendo-crine, and Paneth cells. Except for Paneth cells, these lineages complete their terminal differentiation during an upward migra-tion from each crypt to adjacent villi. The journey from the crypt to the villus tip is completed in 2 to 5 days and terminates with cells being removed by apoptosis and/or exfoliation. Thus, the small-intestinal epithelium undergoes continuous renewal, mak-ing it one of the body’s most dynamic tissues. The high cellular turnover rate contributes to mucosal resiliency but also makes the intestine uniquely susceptible to certain forms of injury such as that induced by radiation and chemotherapy.Figure 28-1. Gross features of jejunum contrasted with those of ileum. Relative to the ileum, the jejunum has a

1	to certain forms of injury such as that induced by radiation and chemotherapy.Figure 28-1. Gross features of jejunum contrasted with those of ileum. Relative to the ileum, the jejunum has a larger diameter, a thicker wall, more prominent plicae circulares, a less fatty mesentery, and longer vasa recta.Brunicardi_Ch28_p1219-p1258.indd 122023/02/19 2:24 PM 1221SMALL INTESTINECHAPTER 284. MucosaCircular layerLongitudinallayer2. Muscularis propriaSubserous layer1. SerosaVascular network,longisection of villusSimple columnar epitheliumwith mucous cellsLamina propria,smooth muscle cells, blood vesselsCentral lymph capillary (lacteal)Muscularis mucosae3. Submucosa4321Opening of crypts (of Lieberkühn)Figure 28-2. Layers of wall of the small intestine. The individual layers and their prominent features are repre-sented schematically.Enterocytes are the predominant absorptive cell of the intestinal epithelium. Their apical (lumen-facing) cell mem-brane contains specialized digestive

1	features are repre-sented schematically.Enterocytes are the predominant absorptive cell of the intestinal epithelium. Their apical (lumen-facing) cell mem-brane contains specialized digestive enzymes, transporter mechanisms, and microvilli that are estimated to increase the absorptive surface area of the small intestine by up to 40-fold. Goblet cells produce mucin believed to play a role in mucosal defense against pathogens. Enteroendocrine cells are charac-terized by secretory granules containing regulatory agents and are discussed in greater detail in the “Endocrine Function” section. Paneth cells are located at the base of the crypt and contain secretory granules containing growth factors, diges-tive enzymes, and antimicrobial peptides, through which they control the host-microbe interaction and influence the intestinal microbiome. In addition, the intestinal epithelium contains M cells and intraepithelial lymphocytes. These two components of the immune system are discussed in

1	and influence the intestinal microbiome. In addition, the intestinal epithelium contains M cells and intraepithelial lymphocytes. These two components of the immune system are discussed in this chapter.The submucosa consists of dense connective tissue and a heterogeneous population of cells, including leukocytes and fibroblasts. The submucosa also contains an extensive network of vascular and lymphatic vessels, nerve fibers, and ganglion cells of the submucosal (Meissner’s) plexus.The muscularis propria consists of an outer, longitudinally-oriented layer and an inner, circularly-oriented layer of smooth muscle fibers. Located at the interface between these two layers are ganglion cells of the myenteric (Auerbach’s) plexus.The serosa consists of a single layer of mesothelial cells and is a component of the visceral peritoneum.DEVELOPMENTThe first recognizable precursor of the small intestine is the embryonic gut tube, formed from the endoderm during the fourth week of gestation. The

1	a component of the visceral peritoneum.DEVELOPMENTThe first recognizable precursor of the small intestine is the embryonic gut tube, formed from the endoderm during the fourth week of gestation. The gut tube is divided into forgut, midgut, and hindgut. Other than the duodenum, which is a forgut structure, the rest of the small intestine is derived from the midgut. The gut tube initially communicates with the yolk sac; however, the communication between these two struc-tures narrows by the sixth week to form the vitelline duct. The yolk sac and vitelline duct usually undergo obliteration by the end of gestation. Incomplete obliteration of the vitelline duct results in the spectrum of defects associated with Meckel’s diverticuli.Also during the fourth week of gestation, the mesoderm of the embryo splits. The portion of mesoderm that adheres to the endoderm forms the visceral peritoneum, while the portion that adheres to the ectoderm forms the parietal peritoneum. This mesodermal

1	of the embryo splits. The portion of mesoderm that adheres to the endoderm forms the visceral peritoneum, while the portion that adheres to the ectoderm forms the parietal peritoneum. This mesodermal division results in the formation of a coelomic cav-ity that is the precursor of the peritoneal cavity.At approximately the fifth week of gestation, the bowel begins to lengthen to an extent greater than that which can be accommodated by the developing abdominal cavity, resulting in the extracoelomic herniation of the developing bowel. The bowel continues to lengthen during the subsequent weeks and is retracted back into the abdominal cavity during the tenth week of gestation. Subsequently, the duodenum becomes a retroperitoneal structure. Coincident with extrusion and retraction, the bowel undergoes a 270° counterclockwise rotation relative to the posterior abdominal wall. This rotation accounts for the usual locations of the cecum in the right lower quadrant and the duodenojejunal

1	undergoes a 270° counterclockwise rotation relative to the posterior abdominal wall. This rotation accounts for the usual locations of the cecum in the right lower quadrant and the duodenojejunal junction to the left of midline (Fig. 28-3).The celiac and superior mesenteric arteries and veins are derived from the vitelline vascular system, which in turn is derived from blood vessels formed within the splanchnopleuric mesoderm during the third week of gestation. Neurons found in the small intestine are derived from neural crest cells that begin to migrate away from the neural tube during the third week of gestation. These neural crest cells enter the mesenchyme of the primitive foregut and subsequently migrate to the remainder of the bowel.During the sixth week of gestation, the lumen of the developing bowel becomes obliterated as bowel epithelial proliferation accelerates. Vacuoles form within the bowel substance during the subsequent weeks and coalesce to form the intestinal lumen by

1	developing bowel becomes obliterated as bowel epithelial proliferation accelerates. Vacuoles form within the bowel substance during the subsequent weeks and coalesce to form the intestinal lumen by the ninth week of gestation. Errors in this recanalization may account for defects such as intestinal webs and stenoses. Most intestinal atresias, however, are believed to be related to ischemic episodes occurring after organogenesis has been completed rather than to errors in recanalization.During the ninth week of gestation, the intestinal epithe-lium develops intestine-specific features such as crypt-villus architecture. Organogenesis is complete by approximately the twelfth week of gestation.Brunicardi_Ch28_p1219-p1258.indd 122123/02/19 2:24 PM 1222SPECIFIC CONSIDERATIONSPART IIStomachDuodenumProximal limb of prim.intestinal loopVitelline ductDistal limb of prim. intestinal loopSuperiormesentericarteryStomachTransverse colonCecal budVitelline ductAscending colonJejunoileal

1	limb of prim.intestinal loopVitelline ductDistal limb of prim. intestinal loopSuperiormesentericarteryStomachTransverse colonCecal budVitelline ductAscending colonJejunoileal loopsACDuodenumCecal budTransverse colonSmall intestineHepatic flextureAppendixTransversecolonDescending colonSigmoidcolonCecumBDFigure 28-3. Developmental rotation of the intestine. A. During the fifth week of gestation, the developing intestine herniates out of the coelomic cavity and begins to undergo a counterclockwise rotation about the axis of the superior mesenteric artery. B and C. Intestinal rotation continues, as the developing transverse colon passes anterior to the developing duodenum. D. Final positions of the small intestine and colon resulting from a 270° counterclockwise rotation of the developing intestine and its return into the abdominal cavity.• Oral intake 2000 mL• Saliva 1500 mL• Gastric secretions 2500• Bile 500 mL• Pancreatic secretions 1500 mL• Small intestinal secretions 1000 mL•

1	intestine and its return into the abdominal cavity.• Oral intake 2000 mL• Saliva 1500 mL• Gastric secretions 2500• Bile 500 mL• Pancreatic secretions 1500 mL• Small intestinal secretions 1000 mL• Small intestinal absorption 7500 mL•1500 mL to colonFigure 28-4. Small intestinal fluid fluxes. Typical quantities (in volume per day) of fluid entering and leaving the small intestinal lumen in a healthy adult are shown.PHYSIOLOGYDigestion and AbsorptionThe intestinal epithelium is the interface through which absorp-tion and secretion occur. It has features characteristic of absorp-tive epithelia in general, including epithelial cells with cellular membranes possessing distinct apical (luminal) and basolateral (serosal) domains demarcated by intercellular tight junctions and an asymmetric distribution of transmembrane transporter mechanisms that promotes vectorial transport of solutes across the epithelium.Solutes can traverse the epithelium by active or passive transport. Passive

1	distribution of transmembrane transporter mechanisms that promotes vectorial transport of solutes across the epithelium.Solutes can traverse the epithelium by active or passive transport. Passive transport of solutes occurs through diffusion or convection and is driven by existing electrochemical gradi-ents. Active transport is the energy-dependent net transfer of solutes in the absence of or against an electrochemical gradient.Active transport occurs through transcellular pathways (through the cell), whereas passive transport can occur through either transcellular or paracellular pathways (between cells through the tight junctions). Transcellular transport requires solutes to traverse the cell membranes through specialized membrane proteins, such as channels, carriers, and pumps. The molecular characterization of transporter proteins is evolving rapidly, with different transporter families, each containing many individual genes encoding specific transporters, now identified.

1	The molecular characterization of transporter proteins is evolving rapidly, with different transporter families, each containing many individual genes encoding specific transporters, now identified. Similarly, understanding of the paracellular pathway is evolving. In contrast to what was once believed, it is becoming apparent that paracellular permeability is substrate-specific, dynamic, and subject to regulation by specific tight junction proteins.Water and Electrolyte Absorption and Secretion. Eight to 9 L of fluid enter the small intestine daily. Most of this volume consists of salivary, gastric, biliary, pancreatic, and intestinal secretions. Under normal conditions, the small intestine absorbs over 80 percent of this fluid, leaving approximately 1.5 L that enters the colon (Fig. 28-4). Small-intestinal absorption and secretion are tightly regulated; derangements in water and electrolyte homeostasis characteristic of many of the disorders discussed in this chapter play an

1	28-4). Small-intestinal absorption and secretion are tightly regulated; derangements in water and electrolyte homeostasis characteristic of many of the disorders discussed in this chapter play an important role in contributing to their associated clinical features.Gut epithelia have two pathways for water transport: (a) the paracellular route, which involves transport through the spaces between cells, (b) the transcellular route, through apical and the basolateral cell membranes, with most occurring through Brunicardi_Ch28_p1219-p1258.indd 122223/02/19 2:24 PM 1223SMALL INTESTINECHAPTER 28the transcellular pathway.4 The specific transport mechanisms mediating this transcellular transport are not completely char-acterized, and they may involve passive diffusion through the phospholipid bilayer, cotransport with other ions and nutrients, or diffusion through water channels called aquaporins. Many different types of aquaporins have been identified; however, their contribution to

1	bilayer, cotransport with other ions and nutrients, or diffusion through water channels called aquaporins. Many different types of aquaporins have been identified; however, their contribution to overall intestinal water absorption appears to be relatively minor.5The prevailing model for intestinal epithelial Na+ absorp-tion is shown in Fig. 28-5. Activity of the Na+/K+ ATPase enzyme, which is located in the basolateral membrane and exchanges three intracellular Na+ for every two extracellular K+ in an energy-dependent process, generates the electrochemi-cal gradient that drives the transport of Na+ from the intestinal lumen into the cytoplasm of enterocytes. Na+ ions traverse the apical membrane through several distinct transporter mecha-nisms, including nutrient-coupled sodium transport (e.g., sodium glucose cotransporter-1, SGLT1), sodium channels, and sodium-hydrogen exchangers (NHEs). Absorbed Na+ ions are then extruded from enterocytes through the Na+/K+ ATPase located in the

1	(e.g., sodium glucose cotransporter-1, SGLT1), sodium channels, and sodium-hydrogen exchangers (NHEs). Absorbed Na+ ions are then extruded from enterocytes through the Na+/K+ ATPase located in the basolateral membrane. Similar mechanistic mod-els that account for the transport of other common ions such as K+ and HCO3also exist.Substantial heterogeneity, with respect to both crypt-villus and craniocaudal axes, exists for intestinal epithelial transport mechanisms. This spatial distribution pattern is consistent with a model in which absorptive function resides primarily in the villus and secretory function in the crypt.Intestinal absorption and secretion are subject to modu-lation under physiologic and pathophysiologic conditions by a wide array of hormonal, neural, and immune regulatory media-tors (Table 28-1).Carbohydrate Digestion and Absorption. Approximately 45% of energy consumption in the average Western diet con-sists of carbohydrates, approximately one-half of which is in the

1	(Table 28-1).Carbohydrate Digestion and Absorption. Approximately 45% of energy consumption in the average Western diet con-sists of carbohydrates, approximately one-half of which is in the form of starch (linear or branched polymers of glucose) derived from cereals and plants. Other major sources of dietary carbo-hydrates include sugars derived from milk (lactose), fruits and LUMEN –BLOOD +Na+GlucoseNa+K+Na+Na+H+Figure 28-5. Model of transepithelial Na+ absorption. Na+ traverses the apical membrane of enterocytes through a variety mechanisms, including nutrient-coupled Na+ transport, Na+/H+ exchange, and Na+ channels. Activity of the Na+/K+ATPase located on the basolateral membrane generates the electrochemical gradient that provides the driving force for Na+ absorption.Table 28-1Regulation of intestinal absorption and secretionAgents that stimulate absorption or inhibit secretion of

1	the electrochemical gradient that provides the driving force for Na+ absorption.Table 28-1Regulation of intestinal absorption and secretionAgents that stimulate absorption or inhibit secretion of water Aldosterone Glucocorticoids Angiotensin Norepineprhine Epinephrine Dopamine Somatostatin Neuropeptide Y Peptide YY EnkephalinAgents that simulate secretion or inhibit absorption of water Secretin Bradykinin Prostaglandins Acetylcholine Atrial natriuretic factor Vasopressin Vasoactive intestinal peptide Bombesin Substance P Serotonin Neurotensin Histaminevegetables (fructose, glucose, and sucrose), or purified from sugar cane or beets (sucrose). Processed foods contain a vari-ety of sugars including fructose, oligosaccharides, and polysac-charides. Glycogen derived from meat contributes only a small fraction of dietary carbohydrate.Pancreatic amylase is the major enzyme of starch diges-tion, although salivary amylase initiates the process. The ter-minal products of amylase-mediated

1	only a small fraction of dietary carbohydrate.Pancreatic amylase is the major enzyme of starch diges-tion, although salivary amylase initiates the process. The ter-minal products of amylase-mediated starch digestion are oligosaccharides, maltotriose, maltose, and alpha-limit dextrins (Fig. 28-6). These products, as well as the major disaccharides in the diet (sucrose and lactose), are unable to undergo absorp-tion in this form. They must first undergo hydrolytic cleavage into their constituent monosaccharides; these hydrolytic reac-tions are catalyzed by specific brush border membrane hydro-lases that are expressed most abundantly in the villi of the duodenum and jejunum. The three major monosaccharides that represent the terminal products of carbohydrate digestion are glucose, galactose, and fructose.Under physiologic conditions, most of these sugars are absorbed through the epithelium via the transcellular route. Glucose and galactose are transported through the enterocyte brush

1	and fructose.Under physiologic conditions, most of these sugars are absorbed through the epithelium via the transcellular route. Glucose and galactose are transported through the enterocyte brush border membrane via intestinal Na+–glucose cotrans-porter, SGLT1 (Fig. 28-7). Fructose is transported through the brush border membrane by facilitated diffusion via GLUT5 (a member of the facilitative glucose transporter family). All three monosaccharides are extruded through the basolateral membrane by facilitated diffusion using GLUT2 and five trans-porters. Extruded monosaccharides diffuse into venules and ulti-mately enter the portal venous system.There is evidence of overexpression of hexose transport-ers, specifically SGLT1, in disease states such as diabetes.6 Several approaches aimed at downregulation of small intestinal Brunicardi_Ch28_p1219-p1258.indd 122323/02/19 2:24 PM 1224SPECIFIC CONSIDERATIONSPART IIGlucose, galactose, fructoseAbsorptionDietary starchSalivary

1	aimed at downregulation of small intestinal Brunicardi_Ch28_p1219-p1258.indd 122323/02/19 2:24 PM 1224SPECIFIC CONSIDERATIONSPART IIGlucose, galactose, fructoseAbsorptionDietary starchSalivary amylasePancreatic amylaseBrush-border hydrolasesOligosaccharidesMaltotrioseMaltosea-limit dextransSucrose and lactoseFigure 28-6. Carbohydrate digestion. Dietary carbohydrates, including starch and the disaccharides sucrose and lactose, must undergo hydrolysis into constituent monosaccharides glucose, galactose, and fructose before being absorbed by the intestinal epithelium. These hydrolytic reactions are catalyzed by salivary and pancreatic amylase and by enterocyte brush border hydrolases.LUMENTight junctionTight junctionBLOOD SGLT1GLUT5GLUT5GLUT2FructoseFructoseNa+GlucoseGalactoseGlucoseGalactoseNa+Figure 28-7. Hexose transporters. Glucose and galactose enter the enterocyte through secondary active transport via the sodium-glucose cotransporter (SGLT1) located on the apical (brush

1	28-7. Hexose transporters. Glucose and galactose enter the enterocyte through secondary active transport via the sodium-glucose cotransporter (SGLT1) located on the apical (brush border) membrane. Fructose enters through facilitated diffusion via glucose transporter 5 (GLUT5). Glucose and galactose are extruded basolaterally through facilitated diffusion via glucose transporter 2 (GLUT2). Fructose is extruded basolaterally via GLUT5.Dipeptides + Tripeptides + Amino acidsAbsorptionDietary proteinsPolypeptidesAmino acidsTrypsinChymotrypsinElastaseCarboxypeptidase ACarboxypeptidase BOligopeptidesBrush-borderpeptidasesAmino acids++PepsinFigure 28-8. Protein digestion. Dietary proteins must undergo hydrolysis into constituent single amino acids and diand tri-peptides before being absorbed by the intestinal epithelium. These hydrolytic reactions are catalyzed by pancreatic peptidases (e.g., trypsin) and by enterocyte brush border peptidases.glucose transporter are being investigated as a

1	the intestinal epithelium. These hydrolytic reactions are catalyzed by pancreatic peptidases (e.g., trypsin) and by enterocyte brush border peptidases.glucose transporter are being investigated as a novel therapy for disease states such as diabetes and obesity. In fact, recent con-sensus statements have recognized the small bowel as a thera-peutic target for treatment of diabetes.7Protein Digestion and Absorption. Ten percent to 15% of energy consumption in the average Western diet consists of pro-teins. In addition to dietary proteins, approximately one-half of the protein load that enters the small intestine is derived from endogenous sources, including salivary and gastrointestinal secretions and desquamated intestinal epithelial cells. Protein digestion begins in the stomach with action of pepsins. This is not, however, an essential step because surgical patients who are acholorhydric, or have lost part or all their stomach, are still able to successfully digest proteins.

1	action of pepsins. This is not, however, an essential step because surgical patients who are acholorhydric, or have lost part or all their stomach, are still able to successfully digest proteins. Digestion continues in the duodenum with the actions of a variety of pancreatic peptidases. These enzymes are secreted as inactive proenzymes. This con-trasts with pancreatic amylase and lipase, which are secreted in their active forms. In response to the presence of bile acids, enterokinase is liberated from the intestinal brush border mem-brane to catalyze the conversion of trypsinogen to active tryp-sin; trypsin in turn activates itself and other proteases. The final products of intraluminal protein digestion consist of neutral and basic amino acids and peptides two to six amino acids in length (Fig. 28-8). Additional digestion occurs through the actions of peptidases that exist in the enterocyte brush border and cyto-plasm. Epithelial absorption occurs for both single amino acids and dior

1	(Fig. 28-8). Additional digestion occurs through the actions of peptidases that exist in the enterocyte brush border and cyto-plasm. Epithelial absorption occurs for both single amino acids and dior tripeptides via specific membrane-bound transporters. Absorbed amino acids and peptides then enter the portal venous circulation.Of all amino acids, glutamine appears to be a unique, major source of energy for enterocytes. Active glutamine uptake into enterocytes occurs through both apical and basolateral transport mechanisms.Fat Digestion and Absorption. Approximately 40% of the average Western diet consists of fat. Over 95% of dietary fat is in the form of long-chain triglycerides; the remainder includes phospholipids such as lecithin, fatty acids, cholesterol, and Brunicardi_Ch28_p1219-p1258.indd 122423/02/19 2:24 PM 1225SMALL INTESTINECHAPTER 28Dietary long-chaintriglyceridesShort& medium-chaintriglyceridesLong-chain fatty acidsand monoglyceridesTriglyceridesresynthesizedin

1	122423/02/19 2:24 PM 1225SMALL INTESTINECHAPTER 28Dietary long-chaintriglyceridesShort& medium-chaintriglyceridesLong-chain fatty acidsand monoglyceridesTriglyceridesresynthesizedin enterocytesGastric lipasePancreatic lipaseChyle (lymphatics)Portal venous bloodAbsorbedAbsorbedFigure 28-9. Fat digestion. Long-chain triglycerides, which constitute the majority of dietary fats, must undergo lipolysis into constituent log-chain fatty acids and monoglycerides before being absorbed by the intestinal epithelium. These reactions are catalyzed by gastric and pancreatic lipases. The products of lipolysis are transported in the form of mixed micelles to enterocytes, where they are resynthesized into triglycerides, which are then packaged in the form of chylomicrons that are secreted into the intestinal lymph (chyle). Triglycerides composed of shortand medium-chain fatty acids are absorbed by the intestinal epithelium directly, without undergoing lipolysis, and are secreted into the portal

1	lymph (chyle). Triglycerides composed of shortand medium-chain fatty acids are absorbed by the intestinal epithelium directly, without undergoing lipolysis, and are secreted into the portal venous circulation.fat-soluble vitamins. Over 94% of the ingested fats are absorbed in the proximal jejunum.Since fats are normally water insoluble, key to success-ful digestion of ingested fats is solubalization of them into an emulsion by the mechanical actions of mastication and antral peristalsis. Although lipolysis of triglycerides to form fatty acids and monoglyciderides is initiated in the stomach by gastric lipase, its principal site is the proximal intestine, where pancre-atic lipase is the catalyst (Fig. 28-9).Bile acids act as detergents that help in solubalization of the lipolysis by forming mixed micelles. These micelles are polymolecular aggregates with a hydrophobic core of fat and a hydrophillic surface that act as shuttles, delivering the products of lipolysis to the enterocyte

1	mixed micelles. These micelles are polymolecular aggregates with a hydrophobic core of fat and a hydrophillic surface that act as shuttles, delivering the products of lipolysis to the enterocyte brush border membrane, where they are absorbed. The bile salts, however, remain in the bowel lumen and travel to the terminal ileum, where they are actively resorbed. They enter the portal circulation and are resecreted into bile, thus completing the enterohepatic circulation.Dissociation of lipids from the micelles occurs in a thin layer of water (50 to 500 μm thick) with an acidic microenvi-ronment immediately adjacent to the brush border called the unstirred water layer. Most lipids are absorbed in the proxi-mal jejunum, whereas bile salts are absorbed in the distal ileum through an active process. Fatty acid binding proteins (FABP) are a family of proteins located on the brush border membrane, facilitating diffusion of long-chain fatty acids across the brush border membrane. Cholesterol

1	Fatty acid binding proteins (FABP) are a family of proteins located on the brush border membrane, facilitating diffusion of long-chain fatty acids across the brush border membrane. Cholesterol crosses the brush border mem-brane through an active process that is yet to be completely characterized. Within the enterocytes, triglycerides are resyn-thesized and incorporated into chylomicrons that are secreted into the intestinal lymphatics and ultimately enter the thoracic duct. In these chylomicrons, lipoproteins serve a detergent-like role similar to that served by bile salts in the mixed micelles.The aforementioned steps are required for the digestion and absorption of triglycerides containing long-chain fatty acids. However, triglycerides containing shortand medium-chain fatty acids are more hydrophilic and are absorbed without undergoing intralumenal hydrolysis, micellular solubilization, mucosal reesterification, and chylomicron formation. Instead, they are directly absorbed and

1	more hydrophilic and are absorbed without undergoing intralumenal hydrolysis, micellular solubilization, mucosal reesterification, and chylomicron formation. Instead, they are directly absorbed and enter the portal venous circula-tion rather than the lymphatics. This information provides the rationale for administering nutritional supplements containing medium-chain triglycerides to patients with gastrointestinal dis-eases associated with impaired digestion and/or malabsorption of long-chain triglycerides.Vitamin and Mineral Absorption. Vitamin B12 (cobalamin) malabsorption can result from a variety of surgical manipula-tions. The vitamin is initially bound by saliva-derived R protein. In the duodenum, R protein is hydrolyzed by pancreatic enzymes, allowing free cobalamin to bind to gastric parietal cell-derived intrinsic factor. The cobalamin-intrinsic factor complex can escape hydrolysis by pancreatic enzymes, allowing it to reach the terminal ileum, which expresses specific

1	gastric parietal cell-derived intrinsic factor. The cobalamin-intrinsic factor complex can escape hydrolysis by pancreatic enzymes, allowing it to reach the terminal ileum, which expresses specific receptors for intrin-sic factor. Subsequent events in cobalamin absorption are poorly characterized, but the intact complex probably enters enterocytes through translocation. Because each of these steps is necessary for cobalamin assimilation, gastric resection, gastric bypass, and ileal resection can each result in vitamin B12 insufficiency.Other water-soluble vitamins for which specific carrier-mediated transport processes have been characterized include ascorbic acid, folate, thiamine, riboflavin, pantothenic acid, and biotin. Fat-soluble vitamins A, D, and E appear to be absorbed through passive diffusion. Vitamin K appears to be absorbed through both passive diffusion and carrier-mediated uptake.Calcium is absorbed through both transcellular transport and paracellular diffusion. The

1	passive diffusion. Vitamin K appears to be absorbed through both passive diffusion and carrier-mediated uptake.Calcium is absorbed through both transcellular transport and paracellular diffusion. The duodenum is the major site for transcellular transport; paracellular transport occurs throughout the small intestine. A key step in transcellular calcium transport is mediated by calbindin, a calcium-binding protein located in the cytoplasm of enterocytes. Regulation of calbindin synthesis is the principle mechanism by which vitamin D regulates intes-tinal calcium absorption. Abnormal calcium levels are increas-ingly seen in surgical patients who have undergone a gastric bypass. Although usual calcium supplementation is often in the form of calcium carbonate, which is cheap, in such patients with low acid exposure, calcium citrate is a better formulation for supplemental therapy.Iron and magnesium are each absorbed through both tran-scellular and paracellular routes. A divalent metal

1	with low acid exposure, calcium citrate is a better formulation for supplemental therapy.Iron and magnesium are each absorbed through both tran-scellular and paracellular routes. A divalent metal transporter capable of transporting Fe2+, Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Ni2+, and Pb2+ that has been localized to the intestinal brush border may account for at least a portion of the transcellular absorption of these ions.8Barrier and Immune FunctionAlthough the intestinal epithelium allows for the efficient absorption of dietary nutrients, it must discriminate between pathogens and harmless antigens such as food proteins and com-mensal bacteria, and it must resist invasion by pathogens. Fac-tors contributing to epithelial defense include immunoglobulin A (IgA), mucins, and the relative impermeability of the brush border membrane and tight junctions to macromolecules and Brunicardi_Ch28_p1219-p1258.indd 122523/02/19 2:24 PM 1226SPECIFIC CONSIDERATIONSPART IIIntestinal lumenPeyer’s

1	of the brush border membrane and tight junctions to macromolecules and Brunicardi_Ch28_p1219-p1258.indd 122523/02/19 2:24 PM 1226SPECIFIC CONSIDERATIONSPART IIIntestinal lumenPeyer’s patchLamina propriaFAESEDDCGCIgAPlasmacellM cellBVillusTTbacteria. Other factors likely to play important roles in intesti-nal mucosal defense include antimicrobial peptides such as the defensins.9 The intestinal component of the immune system, known as the gut-associated lymphoid tissue (GALT), contains over 70% of the body’s immune cells.The GALT is conceptually divided into inductive and effector sites.10 Inductive sites include Peyer’s patches, mesenteric lymph nodes, and smaller isolated lymphoid follicles scattered throughout the small intestine (Fig. 28-10). Peyer’s patches are macroscopic aggregates of B-cell follicles and intervening T-cell areas found in the lamina propria of the small intestine, primarily the distal ileum. Overlying Peyer’s patches is a specialized epithelium containing

1	of B-cell follicles and intervening T-cell areas found in the lamina propria of the small intestine, primarily the distal ileum. Overlying Peyer’s patches is a specialized epithelium containing microfold (M) cells. These cells possess an apical membrane with microfolds rather than microvilli, which is characteristic of most intestinal epithelial cells. Using transepithelial vesicular transport, M cells transfer microbes to underlying professional antigen presenting cells (APCs), such as dendritic cells. Dendritic cells, in addition, may sample luminal antigens directly through their dendrite-like processes that extend through epithelial tight junctions. APCs interact with and prime naive lymphocytes, which then exit through the draining lymphatics to enter the mesenteric lymph nodes, where they undergo differentiation. These lymphocytes then migrate into the systemic circulation via the thoracic duct and ultimately accumulate in the intestinal mucosa at effector sites. Alternative

1	they undergo differentiation. These lymphocytes then migrate into the systemic circulation via the thoracic duct and ultimately accumulate in the intestinal mucosa at effector sites. Alternative induction mechanisms, such as antigen presentation within mesenteric lymph nodes, are also likely to exist.Effector lymphocytes are distributed into distinct compart-ments. IgA-producing plasma cells are derived from B cells and are located in the lamina propria. CD4+ T cells are also located in the lamina propria. CD8+ T cells migrate preferentially to the epithelium, but they are also found in the lamina propria. These T cells are central to immune regulation; in addition, the CD8+ T cells have potent cytotoxic (CTL) activity. IgA is transported through the intestinal epithelial cells into the lumen, where it exists in the form of a dimer complexed with a secretory component. This configuration renders IgA resistant to proteolysis by diges-tive enzymes. IgA is believed to both help prevent

1	lumen, where it exists in the form of a dimer complexed with a secretory component. This configuration renders IgA resistant to proteolysis by diges-tive enzymes. IgA is believed to both help prevent the entry of microbes through the epithelium and to promote excretion of anti-gens or microbes that have already penetrated the laminal propria.It has been increasingly recognized that the gastrointestinal tract is colonized with many bacteria that are essential for health. Communication between the microbiota and the host defense allows for protective immune responses against pathogens while preventing adverse inflammatory responses to harmless com-mensal microbes, which could lead to chronic inflammatory dis-orders such as celiac disease and Crohn’s disease.11MotilityMyocytes of the intestinal muscle layers are electrically and mechanically coordinated in the form of syncytia. Contractions of the muscularis propria are responsible for small-intestinal peri-stalsis. Contraction of the

1	muscle layers are electrically and mechanically coordinated in the form of syncytia. Contractions of the muscularis propria are responsible for small-intestinal peri-stalsis. Contraction of the outer longitudinal muscle layer results in bowel shortening; contraction of the inner circular layer results in luminal narrowing. Contractions of the muscularis mucosa contribute to mucosal or villus motility, but not to peristalsis.Several distinctive patterns of muscularis propria activity have been observed to occur in the small intestine. These patterns include ascending excitation and descending inhibition in which muscular contraction occurs proximal to a stimulus, such as the presence of a bolus of ingested food, and muscular relaxation occurs distal to the stimulus (Fig. 28-11). These two reflexes are present even in the absence of any extrinsic innervation to the small intestine and contribute to peristalsis when they are propagated in a coordinated fashion along the length of the

1	reflexes are present even in the absence of any extrinsic innervation to the small intestine and contribute to peristalsis when they are propagated in a coordinated fashion along the length of the intestine. The fed or postprandial pattern begins within 10 to 20 minutes of meal ingestion and abates 4 to 6 hours afterwards. Rhythmic segmentations or pressure waves traveling only short distances also are observed. This segmenting pattern is hypothesized to assist in mixing intraluminal contents and in facilitating their contact with the absorptive mucosal surface. The fasting pattern or interdigestive motor cycle (IDMC) consists of three phases. Phase 1 is characterized by motor quiescence, phase 2 by seemingly disorganized pressure waves occurring at submaximal rates, and phase 3 by sustained pressure waves occurring at maximal rates. This pattern is hypothesized to expel residual debris and bacteria from the small intestine. The median duration of the IDMC ranges from 90 to 120

1	sustained pressure waves occurring at maximal rates. This pattern is hypothesized to expel residual debris and bacteria from the small intestine. The median duration of the IDMC ranges from 90 to 120 minutes. At any given time, different portions of the small intestine can be in different phases of the IDMC.Figure 28-10. Gut-associated lymphoid tissue. Select components of the gut-associated lymphoid tissue (GALT) are schematically represented. Peyer’s patches consist of a specialized follicle-associated epithelium (FAE) containing M cells, a subepithelial dome (SED) rich in dendritic cells (DC), and B-cell follicle containing germinal centers (GC). Plasma cells in the lamina propria produce IgA, which is transported to the intestinal lumen where serves as the first line of defense against pathogens. Other components of the GALT include isolated lymphoid follicles, mesenteric lymph nodes, and regulatory and effector lymphocytes.Brunicardi_Ch28_p1219-p1258.indd 122623/02/19 2:24 PM

1	pathogens. Other components of the GALT include isolated lymphoid follicles, mesenteric lymph nodes, and regulatory and effector lymphocytes.Brunicardi_Ch28_p1219-p1258.indd 122623/02/19 2:24 PM 1227SMALL INTESTINECHAPTER 28EMNSNIMNProximalDistalFigure 28-11. Ascending excitation and descending inhibition. The presence of a food bolus within the intestinal lumen is sensed by a sensory neuron (SN) that relays signals to (a) excitatory motor neurons (EMN) that have projections to intestinal muscle cells located proximal to the food bolus and (b) inhibitory motor neu-rons (IMN) that have projections to intestinal muscle cells located distal to the food bolus. This stereotypical motor reflex is controlled by the enteric nervous system and occurs in the absence of extra-intestinal innervations. It contributes to peristalsis.The regulatory mechanisms driving small-intestinal motil-ity consist of both pacemakers intrinsic to the small intestine and external neurohumoral modulatory

1	It contributes to peristalsis.The regulatory mechanisms driving small-intestinal motil-ity consist of both pacemakers intrinsic to the small intestine and external neurohumoral modulatory signals. The interstitial cells of Cajal are pleomorphic mesenchymal cells located within the muscularis propria of the intestine that generate the electrical slow wave (basic electrical rhythm or pacesetter potential) that plays a pacemaker role in setting the fundamental rhythmicity of small-intestinal contractions. The frequency of the slow wave varies along the longitudinal axis of the intestine: it ranges from 12 waves per minute in the duodenum to 7 waves per minute in the distal ileum. Smooth muscle contraction occurs only when an electrical action potential (spike burst) is superimposed on the slow wave. Thus, the slow wave determines the maximum frequency of contractions; however, not every slow wave is associated with a contraction.This intrinsic contractile mechanism is subject to neural

1	wave. Thus, the slow wave determines the maximum frequency of contractions; however, not every slow wave is associated with a contraction.This intrinsic contractile mechanism is subject to neural and hormonal regulation. The enteric motor system (ENS) pro-vides both inhibitory and excitatory stimuli. The predominant excitatory transmitters are acetylcholine and substance P, and the inhibitory transmitters include nitric oxide, vasoactive intes-tinal peptide, and adenosine triphosphate. In general, the sympa-thetic motor supply is inhibitory to the ENS; therefore, increased sympathetic input into the intestine leads to decreased intestinal smooth muscle activity. The parasympathetic motor supply is more complex, with projections to both inhibitory and excitatory ENS motor neurons. Correspondingly, the effects of parasympa-thetic inputs into intestinal motility are more difficult to predict.Endocrine FunctionEndocrinology as a discipline was born with the discovery of secretin, an

1	the effects of parasympa-thetic inputs into intestinal motility are more difficult to predict.Endocrine FunctionEndocrinology as a discipline was born with the discovery of secretin, an intestinal regulatory peptide that was the first hormone to be identified. Our improving understanding of the physiology of the small intestine has led to identification of many additional intestinal-derived hormones, which make this the largest hormone-producing organ in the body. Over 30 peptide hormone genes have been identified as being expressed in the gastrointestinal tract. Because of differential posttranscriptional and posttranslational processing, over 100 distinct regulatory peptides are produced. In addition, monoamines, such as histamine and dopamine, and eicosanoids with hormone-like activities are produced in the intestine.“Gut hormones” were previously conceptualized as pep-tides produced by the enteroendocrine cells of the intestinal mucosa that are released into the systemic

1	activities are produced in the intestine.“Gut hormones” were previously conceptualized as pep-tides produced by the enteroendocrine cells of the intestinal mucosa that are released into the systemic circulation to reach receptors in target sites in the gastrointestinal tract. Now it is clear that “gut hormone” genes are widely expressed through-out the body, not only in endocrine cells but also in central and peripheral neurons. The products of these genes are general intercellular messengers that can act as endocrine, paracrine, autocrine, or neurocrine mediators. Thus, they may act as true blood-borne hormones as well as through local effects.There are notable homology patterns among individual regulatory peptides found in the gastrointestinal tract. Based on these homologies, approximately one-half of the known regulatory peptides can be classified into families.12 For example, the secretin family includes secretin, glucagon, and glucagon-like peptides, glucose-dependent

1	approximately one-half of the known regulatory peptides can be classified into families.12 For example, the secretin family includes secretin, glucagon, and glucagon-like peptides, glucose-dependent insulinotropic peptide, vasoactive intestinal polypeptide, peptide histidine isoleucine, growth hormone releasing hormone, and pituitary adenylyl cyclase-activating peptide. Other peptide families include those named for insulin, epidermal growth factor, gastrin, pancreatic polypeptide, tachykinin, and somatostatin.Receptor subtype multiplicity and cell-specific expres-sion patterns for these receptor subtypes that are characteristic of these regulatory mediators makes definition of their actions complex. Detailed description of these actions is beyond the scope of this chapter; however, examples of regulatory pep-tides produced by enteroendocrine cells of the small-intestinal epithelium and their most commonly ascribed functions are summarized in Table 28-2. Some of these peptides, or

1	of regulatory pep-tides produced by enteroendocrine cells of the small-intestinal epithelium and their most commonly ascribed functions are summarized in Table 28-2. Some of these peptides, or their analogues, are used in routine clinical practice. For example, Table 28-2Representative regulatory peptides produced in the small intestineHORMONESOURCEaACTIONSSomatostatinD cellInhibits gastrointestinal secretion, motility, and splanchnic perfusionSecretinS cellStimulates exocrine pancreatic secretion, stimulates intestinal secretionCholecystokininI cellSimulates pancreatic exocrine secretion, simulates gallbladder emptying, inhibits sphincter of Oddi contractionMotilinM cellSimulates intestinal motilityPeptide YYL cellInhibits intestinal motility and secretionGlucagon-like Peptide 2L cellStimulates intestinal epithelial proliferationNeurotensinN cellStimulates pancreatic and biliary secretion, inhibits small bowel motility, stimulates intestinal mucosal growthaThis table indicates which

1	intestinal epithelial proliferationNeurotensinN cellStimulates pancreatic and biliary secretion, inhibits small bowel motility, stimulates intestinal mucosal growthaThis table indicates which enteroendocrine cell types located in the intestinal epithelium produce these peptides. These peptides are also widely expressed in nonintestinal tissues.Brunicardi_Ch28_p1219-p1258.indd 122723/02/19 2:24 PM 1228SPECIFIC CONSIDERATIONSPART IITable 28-3Small bowel obstruction: common etiologiesAdhesionsNeoplasms Primary small bowel neoplasms Secondary small bowel cancer (e.g., melanomaderived metastasis) Local invasion by intra-abdominal malignancy (e.g., Desmoid tumors) CarcinomatosisHernias External (e.g., inguinal and femoral) Internal (e.g., following Roux-en-Y gastric bypass surgery)Crohn’s diseaseVolvulusIntussusceptionRadiation-induced stricturePostischemic strictureForeign bodyGallstone ileusDiverticulitisMeckel’s diverticulumHematomaCongenital abnormalities (e.g., webs, duplications,

1	stricturePostischemic strictureForeign bodyGallstone ileusDiverticulitisMeckel’s diverticulumHematomaCongenital abnormalities (e.g., webs, duplications, and malrotation)therapeutic applications of octreotide, a long-acting analogue of somatostatin, include the amelioration of symptoms associ-ated with neuroendocrine tumors (e.g., carcinoid syndrome), postgastrectomy dumping syndrome, enterocutaneous fis-tulas, and the initial treatment of acute hemorrhage due to esophageal varices. The gastrin secretory response to secretin administration forms the basis for the standard test used to establish the diagnosis of Zollinger-Ellison syndrome. Chole-cystokinin is used in evaluations of gallbladder ejection frac-tion, a parameter that may have utility in patients who have symptoms of biliary colic but are not found to have gallstones. Of the peptides listed in Table 28-2, glucagon-like peptide 2 (GLP-2) has been identified as a specific and potent intestino-trophic hormone and is currently

1	but are not found to have gallstones. Of the peptides listed in Table 28-2, glucagon-like peptide 2 (GLP-2) has been identified as a specific and potent intestino-trophic hormone and is currently under clinical evaluation as an intestinotrophic agent in patients suffering from the short bowel syndrome, as discussed in the “Short Bowel Syndrome” section.Intestinal AdaptationThe small intestine has the capacity to adapt in response to vary-ing demands imposed by physiologic and pathologic conditions. Of relevance to many of the diseases discussed in this chapter is the adaptation that occurs in the remnant intestine following surgical resection of a large portion of the small intestine (mas-sive small bowel resection). Postresection intestinal adaptation has been studied extensively using animal models. Within a few hours after bowel resection, the remnant small intestine displays evidence of epithelial cellular hyperplasia. With additional time, villi lengthen, intestinal absorptive

1	animal models. Within a few hours after bowel resection, the remnant small intestine displays evidence of epithelial cellular hyperplasia. With additional time, villi lengthen, intestinal absorptive surface area increases, and digestive and absorptive functions improve. Postresection intes-tinal adaptation in human patients is less well studied, but it seems to follow similar steps as that seen in experimental mod-els, and it takes 1 to 2 years to complete.13The mechanisms responsible for inducing postresection intestinal adaptation are under active investigation. Several classes of effectors that stimulate intestinal growth include spe-cific nutrients, peptide hormones and growth factors, pancreatic secretions, and some cytokines. Nutritional components with intestinal growth-stimulating effects include fiber, fatty acids, triglycerides, glutamine, polyamines, and lectins.Postresection adaptation serves to compensate for the function of intestine that has been resected. Jejunal

1	effects include fiber, fatty acids, triglycerides, glutamine, polyamines, and lectins.Postresection adaptation serves to compensate for the function of intestine that has been resected. Jejunal resection is generally better tolerated, as ileum shows better capacity to compensate. However, the magnitude of this response is limited. If enough small intestine is resected, a devastating condition known as the short bowel syndrome results. This condition is discussed in the “Short Bowel Syndrome” section at the end of this chapter.SMALL BOWEL OBSTRUCTIONEpidemiologyMechanical small bowel obstruction is the most frequently encountered surgical disorder of the small intestine. Although a wide range of etiologies for this condition exist, the obstructing lesion can be conceptualized according to its anatomical relationship to the intestinal wall as:1. intraluminal (e.g., foreign bodies, gallstones, or meconium)2. intramural (e.g., tumors, Crohn’s disease–associated inflam-matory

1	according to its anatomical relationship to the intestinal wall as:1. intraluminal (e.g., foreign bodies, gallstones, or meconium)2. intramural (e.g., tumors, Crohn’s disease–associated inflam-matory strictures)3. extrinsic (e.g., adhesions, hernias, or carcinomatosis)2Intra-abdominal adhesions related to prior abdominal sur-gery account for up to 75% of cases of small bowel obstruction. Over 300,000 patients are estimated to undergo surgery to treat adhesion-induced small bowel obstruction in the United States annually. A 20-year trend analysis between 1988 and 2007 has documented no decrease in this rate during this period, highlighting the ongoing problem of this “old” disease.14 In fact, small bowel resection and lysis of adhesions account for two of the seven procedures that were responsible for 80% of the emergency surgeries in the United States between 2008 and 2011.15Less prevalent etiologies for small bowel obstruction include hernias, malignant bowel obstruction, and Crohn’s

1	for 80% of the emergency surgeries in the United States between 2008 and 2011.15Less prevalent etiologies for small bowel obstruction include hernias, malignant bowel obstruction, and Crohn’s dis-ease. The frequency with which obstruction related to these con-ditions is encountered varies according to the patient population and practice setting. Cancer-related small bowel obstructions are commonly due to extrinsic compression or invasion by advanced malignancies arising in organs other than the small bowel; few are due to primary small bowel tumors. The most commonly encountered etiologies of small bowel obstruction are summarized in Table 28-3. Although congenital abnormali-ties capable of causing small bowel obstruction usually become evident during childhood, they sometimes elude detection and are diagnosed for the first time in adult patients presenting with abdominal symptoms. For example, intestinal malrotation and midgut volvulus should not be forgotten when considering the

1	and are diagnosed for the first time in adult patients presenting with abdominal symptoms. For example, intestinal malrotation and midgut volvulus should not be forgotten when considering the differential diagnosis of adult patients with acute or chronic symptoms of small bowel obstruction, espe-cially those without a history of prior abdominal surgery. A rare etiology of obstruction is the superior mesenteric artery syn-drome, characterized by compression of the third portion of the duodenum by the superior mesenteric artery as it crosses over this portion of the duodenum. This condition should be 34Brunicardi_Ch28_p1219-p1258.indd 122823/02/19 2:24 PM 1229SMALL INTESTINECHAPTER 28considered in young asthenic individuals who have chronic symptoms suggestive of proximal small bowel obstruction.PathophysiologyWith onset of obstruction, gas and fluid accumulate within the intestinal lumen proximal to the site of obstruction. The intestinal activity increases to overcome the

1	bowel obstruction.PathophysiologyWith onset of obstruction, gas and fluid accumulate within the intestinal lumen proximal to the site of obstruction. The intestinal activity increases to overcome the obstruction, accounting for the colicky pain and the diarrhea that some experience even in the presence of complete bowel obstruction. Most of the gas that accumulates originates from swallowed air, although some is produced within the intestine. The fluid consists of swallowed liquids and gastrointestinal secretions (obstruction stimulates intestinal epithelial water secretion). With ongoing gas and fluid accumulation, the bowel distends and intraluminal and intramural pressures rise. The intestinal motility is eventually reduced with fewer contractions. With obstruction, the luminal flora of the small bowel, which is usually sterile, changes and a variety of organisms have been cultured from the contents. Translocation of these bacteria to regional lymph nodes has been demonstrated,

1	of the small bowel, which is usually sterile, changes and a variety of organisms have been cultured from the contents. Translocation of these bacteria to regional lymph nodes has been demonstrated, although the significance of this process is not well understood. If the intramural pressure becomes high enough, intestinal microvascular perfusion is impaired leading to intestinal ischemia, and, ultimately, necrosis. This condition is termed strangulated bowel obstruction.With partial small bowel obstruction, only a portion of the intestinal lumen is occluded, allowing passage of some gas and fluid. The progression of pathophysiologic events described previously tends to occur more slowly than with complete small bowel obstruction, and development of strangulation is less likely.A particularly dangerous form of bowel obstruction is closed loop obstruction in which a segment of intestine is obstructed both proximally and distally (e.g., with volvulus). In such cases, the accumulating gas

1	dangerous form of bowel obstruction is closed loop obstruction in which a segment of intestine is obstructed both proximally and distally (e.g., with volvulus). In such cases, the accumulating gas and fluid cannot escape either proximally or distally from the obstructed segment, lead-ing to a rapid rise in luminal pressure and a rapid progression to strangulation.Clinical PresentationThe symptoms of small bowel obstruction are colicky abdomi-nal pain, nausea, vomiting, and obstipation. Vomiting is a more prominent symptom with proximal obstructions than distal. Character of vomitus is important as with bacterial overgrowth, the vomitus is more feculent, suggesting a more established obstruction. Continued passage of flatus and/or stool beyond 6 to 12 hours after onset of symptoms is characteristic of par-tial rather than complete obstruction. The signs of small bowel obstruction include abdominal distention, which is most pro-nounced if the site of obstruction is in the distal ileum

1	of par-tial rather than complete obstruction. The signs of small bowel obstruction include abdominal distention, which is most pro-nounced if the site of obstruction is in the distal ileum and may be absent if the site of obstruction is in the proximal small intestine. Bowel sounds may be hyperactive initially, but in late stages of bowel obstruction, minimal bowel sounds may be heard. Laboratory findings reflect intravascular volume deple-tion and consist of hemoconcentration and electrolyte abnor-malities. Mild leukocytosis is common.Features of strangulated obstruction include abdominal pain often disproportionate to the degree of abdominal findings, suggestive of intestinal ischemia. Patients often have tachycar-dia, localized abdominal tenderness, fever, marked leukocyto-sis, and acidosis. Any of these findings should alert the clinician to the possibility of strangulation and the need for early surgical intervention.DiagnosisThe diagnostic evaluation should focus on the

1	and acidosis. Any of these findings should alert the clinician to the possibility of strangulation and the need for early surgical intervention.DiagnosisThe diagnostic evaluation should focus on the following goals: (a) distinguish mechanical obstruction from ileus, (b) determine the etiology of the obstruction, (c) discriminate partial from complete obstruction, and (d) discriminate simple from stran-gulating obstruction.Important elements to obtain on history include prior abdominal operations (suggesting the presence of adhesions) and the presence of abdominal disorders (e.g., intra-abdominal cancer or inflammatory bowel disease) that may provide insights into the etiology of obstruction. Upon examination, a meticulous search for hernias (particularly in the inguinal and femoral regions) should be conducted.The diagnosis of small bowel obstruction is usually con-firmed with radiographic examination. The abdominal series consists of (a) a radiograph of the abdomen with the patient

1	should be conducted.The diagnosis of small bowel obstruction is usually con-firmed with radiographic examination. The abdominal series consists of (a) a radiograph of the abdomen with the patient in a supine position, (b) a radiograph of the abdomen with the patient in an upright position, and (c) a radiograph of the chest with the patient in an upright position. The finding most specific for small bowel obstruction is the triad of dilated small bowel loops (>3 cm in diameter), air-fluid levels seen on upright films, and a paucity of air in the colon. The sensitivity of abdominal radiographs in the detection of small bowel obstruction ranges from 70% to 80%. Specificity is low because ileus and colonic obstruction can be associated with findings that mimic those observed with small bowel obstruction. False-negative findings on radiographs can result when the site of obstruction is in the proximal small bowel and when the bowel lumen is filled with fluid but no gas, thereby preventing

1	obstruction. False-negative findings on radiographs can result when the site of obstruction is in the proximal small bowel and when the bowel lumen is filled with fluid but no gas, thereby preventing visualization of air-fluid levels or bowel distention. The latter situation is associated with closed-loop obstruction. Despite these limitations, abdominal radiographs remain an important study in patients with sus-pected small bowel obstruction because of their widespread availability and low cost (Fig. 28-12).Computed tomographic (CT) scanning is becoming increasingly the imaging test of choice for patients with small bowel obstruction, and it is ideally done with oral contrast. CT is 80% to 90% sensitive and 70% to 90% specific in the detec-tion of small bowel obstruction. The findings of small bowel obstruction include a discrete transition zone with dilation of bowel proximally, decompression of bowel distally, intralumi-nal contrast that does not pass beyond the transition zone,

1	of small bowel obstruction include a discrete transition zone with dilation of bowel proximally, decompression of bowel distally, intralumi-nal contrast that does not pass beyond the transition zone, and a colon containing little gas or fluid (Figs. 28-13 and 28-14). CT scanning may also provide evidence for the presence of closed-loop obstruction and strangulation. Closed-loop obstruction is suggested by the presence of a U-shaped or C-shaped dilated bowel loop associated with a radial distribution of mesenteric vessels converging toward a torsion point. Strangulation is sug-gested by thickening of the bowel wall, pneumatosis intestinalis (air in the bowel wall), portal venous gas, mesenteric haziness, and poor uptake of intravenous contrast into the wall of the affected bowel (Fig. 28-15). CT scanning also offers a global evaluation of the abdomen and may therefore reveal the etiol-ogy of obstruction. This feature is important in the acute setting when intestinal obstruction

1	28-15). CT scanning also offers a global evaluation of the abdomen and may therefore reveal the etiol-ogy of obstruction. This feature is important in the acute setting when intestinal obstruction represents only one of many diag-noses in patients presenting with acute abdominal conditions.The CT scan is usually performed after administration of oral water-soluble contrast or diluted barium. The water-soluble contrast has been shown to have prognostic and therapeutic val-ues too. Several studies and several subsequent meta-analysis have shown that water-soluble contrast could in fact have ther-apeutic and prognostic value. The appearance of the contrast Brunicardi_Ch28_p1219-p1258.indd 122923/02/19 2:24 PM 1230SPECIFIC CONSIDERATIONSPART IIFigure 28-12. Small bowel obstruction. Plain radiographs (A) supine, which show dilated loops of small bowel in the right upper quadrant; (B) erect, which confirm the presence of airfluid level in the loops of small bowel as well as the stomach,

1	(A) supine, which show dilated loops of small bowel in the right upper quadrant; (B) erect, which confirm the presence of airfluid level in the loops of small bowel as well as the stomach, consistant with small bowel obstruction.Figure 28-13. Small bowel obstruction. A CT scan of a patient presenting with signs and symptoms of bowel obstruction. Image shows grossly dilated loops of small bowel, with decompressed terminal ileum (I) and ascending colon (C), suggesting a complete distal small bowel obstruction. At laparotomy, adhesive bands from a previous surgery were identified and divided.Figure 28-14. Chronic partial small bowel obstruction. This patient presented with a several months history of chronic abdominal pain and intermittent vomiting. The coronal CT image shows grossly dilated loops of proximal small bowel on the left side (wide arrow), with decompressed loops of small bowel on the right side (narrow arrow). The dilated segment shows evidence of feculization of bowel

1	loops of proximal small bowel on the left side (wide arrow), with decompressed loops of small bowel on the right side (narrow arrow). The dilated segment shows evidence of feculization of bowel contents, consistent with the chronic nature of the obstruction. Patient’s vomitus had characteristic feculent smell and quality. At exploratory laparotomy, adhesive bands were identified and divided.in the colon within 24 hours of administration is predictive of nonsurgical resolution of bowel obstruction with a sensitivity of 92% and a specificity of 93%.16A limitation of CT scanning is its low sensitivity (<50%) in the detection of low-grade or partial small bowel obstruction. A subtle transition zone may be difficult to identify in the axial images obtained during CT scanning. In such cases, contrast examinations of the small bowel, either small bowel series (small bowel follow-through) or enteroclysis, can be helpful. For standard small bowel series, contrast is swallowed or instilled into

1	examinations of the small bowel, either small bowel series (small bowel follow-through) or enteroclysis, can be helpful. For standard small bowel series, contrast is swallowed or instilled into the stomach through a nasogastric tube. Abdominal radiographs are then taken serially as the contrast travels distally in the intestine. Although barium can be used, water-soluble contrast agents, such as gastrograffin, should be used if the possibility of intestinal perforation exists. These examinations are more labor-intensive and less rapidly performed than CT scanning but may offer greater sensitivity in the detection of luminal and mural etiologies of obstruction, such as primary intestinal tumors. For enteroclysis, 200 to 250 mL of barium followed by 1 to 2 L of a solution of methylcellulose in water is instilled into the proximal jejunum via a long nasoenteric catheter. The double-contrast technique used in enterocolysis Brunicardi_Ch28_p1219-p1258.indd 123023/02/19 2:24 PM

1	in water is instilled into the proximal jejunum via a long nasoenteric catheter. The double-contrast technique used in enterocolysis Brunicardi_Ch28_p1219-p1258.indd 123023/02/19 2:24 PM 1231SMALL INTESTINECHAPTER 28Figure 28-15. Intestinal pneumatosis. This CT scan shows intestinal pneumatosis (arrow). The cause of this radiological finding was intestinal ischemia. Patient was taken emergently to the operating room and underwent resection of an infarcted segment of small bowel.permits a better assessment of mucosal surface and detection of relatively small lesions, even through overlapping small bowel loops. Enterocolysis is rarely performed in the acute setting but offers greater sensitivity than small bowel series in the detection of lesions that may be causing partial small bowel obstruction. Recently, CT enterocolysis has been used, and it was reported to be superior to plain X-ray small bowel contrast studies.TherapySmall bowel obstruction is usually associated with a marked

1	Recently, CT enterocolysis has been used, and it was reported to be superior to plain X-ray small bowel contrast studies.TherapySmall bowel obstruction is usually associated with a marked depletion of intravascular volume due to decreased oral intake, vomiting, and sequestration of fluid in bowel lumen and wall. Therefore, fluid resuscitation is integral to treatment. Isotonic fluid should be given intravenously, and an indwelling bladder catheter may be placed to monitor urine output. Central-venous or pulmonary-artery catheter monitoring are not generally indi-cated unless the patient has underlying cardiac disease and severe dehydration. Broad-spectrum antibiotics are not indi-cated unless there is concern for bowel ischemia and surgery is planned.The stomach should be continuously evacuated of air and fluid using a nasogastric (NG) tube. Effective gastric decom-pression decreases nausea, distention, and the risk of vomiting and aspiration. Longer nasoenteric tubes, with tips

1	evacuated of air and fluid using a nasogastric (NG) tube. Effective gastric decom-pression decreases nausea, distention, and the risk of vomiting and aspiration. Longer nasoenteric tubes, with tips placed into the jejunum or ileum, were favored in the past but are rarely used today, as they are associated with higher complication rates than NG tubes, with no proven greater efficacy in several studies.While a period of close observation and nonoperative management has been the mainstay of treatment for partial bowel obstruction, the standard therapy for complete small bowel obstruction has generally been expeditious surgery, with the dictum that “the sun should never rise and set on a complete bowel obstruction.” The rationale for favoring early surgical intervention is to minimize the risk for bowel strangu-lation, which is associated with an increased risk for morbidity and mortality. Clinical signs and currently available laboratory tests and imaging studies do not reliably permit

1	for bowel strangu-lation, which is associated with an increased risk for morbidity and mortality. Clinical signs and currently available laboratory tests and imaging studies do not reliably permit the distinction between patients with simple obstruction and those with stran-gulated obstruction prior to the onset of irreversible ischemia. Therefore, the goal is to operate before the onset of irreversible ischemia. This treatment approach has, however, undergone significant reassessment in recent years, with many advocating for nonoperative approaches in management of these patients, providing closed-loop obstruction is ruled out and there is no evidence of intestinal ischemia. In a study of 145 patients with CT-diagnosed high-grade compete small bowel obstruction, 46% of the overall cohort were managed nonoperatively. More specifically, of the 104 patients who did not meet criteria for immediate surgery, 66 patients were successfully managed nonoperatively.17Thus, conservative therapy

1	were managed nonoperatively. More specifically, of the 104 patients who did not meet criteria for immediate surgery, 66 patients were successfully managed nonoperatively.17Thus, conservative therapy in the form of NG decompres-sion and fluid resuscitation is now commonly recommended in the initial management of nonischemic bowel obstruction. Non-operative management has been documented to be successful in 65% to 81% of patients with partial small bowel obstruction. Of those successfully treated nonoperatively, only 5% to 15% have been reported to have symptoms that were not substantially improved within 48 hours after initiation of therapy. Therefore, most patients with partial small obstruction whose symptoms do not improve within 48 hours after initiation of nonopera-tive therapy should be considered for surgery. In a study using the National Inpatient Sample, this principle was further high-lighted. The authors concluded that a 2-day limit of watchful waiting before surgery is not

1	be considered for surgery. In a study using the National Inpatient Sample, this principle was further high-lighted. The authors concluded that a 2-day limit of watchful waiting before surgery is not associated with an increase in mortality or postoperative morbidity, although inpatient costs were higher.18The observation that administration of water-soluble oral contrast has not only diagnostic but also therapeutic and prog-nostic value has led to the creation of several protocols and path-ways for management of patients presenting with small bowel obstruction. An example of such a pathway that is utilized at our institution is outlined in Fig. 28-16. Several studies and subsequent meta-analyses have shown that use of water-soluble contrast not only predicts likelihood of success of nonoperative management but also reduces the need for surgery (odds ratio 0.44), length of stay by about 2 days, and time to resolution by about 28 hours, without an increase in morbidity or

1	of nonoperative management but also reduces the need for surgery (odds ratio 0.44), length of stay by about 2 days, and time to resolution by about 28 hours, without an increase in morbidity or mortality.16The operative procedure performed for small bowel obstruction varies according to the etiology of the obstruc-tion. For example, adhesions are lysed, tumors are resected, and hernias are reduced and repaired. Regardless of the etiol-ogy, the affected intestine should be examined, and nonviable bowel should be resected. Criteria suggesting viability are nor-mal color, peristalsis, and marginal arterial pulsations. Usu-ally, visual inspection alone is adequate in judging viability. In borderline cases, a Doppler probe may be used to check for pulsatile flow to the bowel, and arterial perfusion can be veri-fied by visualizing intravenously administered fluorescein dye in the bowel wall under ultraviolet illumination. Neither tech-nique has, however, been found to be superior to

1	perfusion can be veri-fied by visualizing intravenously administered fluorescein dye in the bowel wall under ultraviolet illumination. Neither tech-nique has, however, been found to be superior to clinical judg-ment. In general, if the patient is hemodynamically stable, short lengths of bowel of questionable viability should be resected, and primary anastomosis of the remaining intestine should be performed. However, if the viability of a large proportion of the intestine is in question, a concerted effort to preserve intestinal tissue should be made. In such situations, the bowel of uncertain viability should be left intact and the patient reexplored in 24 to 48 hours in a “second-look” operation. At that time, definitive resection of nonviable bowel is completed.Brunicardi_Ch28_p1219-p1258.indd 123123/02/19 2:24 PM 1232SPECIFIC CONSIDERATIONSPART IINoNoYesYesAdhesive smallbowel obstructionSigns and symptomsof strangulation andintestinal ischemia?Operating roomfor

1	123123/02/19 2:24 PM 1232SPECIFIC CONSIDERATIONSPART IINoNoYesYesAdhesive smallbowel obstructionSigns and symptomsof strangulation andintestinal ischemia?Operating roomfor explorationNPOIVFNG tubeSerial abdominal exam100 mL of water-solublecontrast through NGKUB after 8 hoursHas contrastreached colon?High likelihood for nonoperativeresolution of bowel obstructionRemove NGStart sipsRepeat KUBafter 24 hoursHas contrastreached colon?Strongly consider surgerywithin 72 hours of admissionYesFigure 28-16. Management of small bowel obstruction.Successful laparoscopic surgery for bowel obstruction is being reported with greater frequency. In a propensity score-matched study of patients who underwent adhesiolysis for small bowel obstruction, the laparoscopic approach was associated with significantly lower rates of overall complications, surgical site infections, and a shorter length of hospital stay (4 vs. 10 days).19 Since distended loops of bowel can interfere with adequate

1	with significantly lower rates of overall complications, surgical site infections, and a shorter length of hospital stay (4 vs. 10 days).19 Since distended loops of bowel can interfere with adequate visualization, early cases of proximal small bowel obstruction that are likely due to a single adhesive band are best suited for this approach. Presence of bowel distention and multiple adhesions can cause these procedures to be difficult, with a reported conversion rate of 17% to 33%. One of the major concerns with the laparoscopic approach has been the risk of iatrogenic bowel injury. A pooled analysis of 11 nonrandomized comparative studies has, however, shown that the risk of bowel injury and reoperation were not different between the two procedures, although the laparoscopic approach was associated with greater surgical time.20OutcomesThe perioperative mortality rate associated with surgery for nonstrangulating small bowel obstruction is less than 5%, with most deaths occurring in

1	associated with greater surgical time.20OutcomesThe perioperative mortality rate associated with surgery for nonstrangulating small bowel obstruction is less than 5%, with most deaths occurring in elderly patients with significant comorbidities. Mortality rates associated with surgery for stran-gulated obstruction is higher, highlighting the need for prompt intervention in this group. Long-term prognosis is related to the etiology of obstruction. Many patients who are treated conser-vatively for adhesive small bowel obstruction do not require future readmissions; less than 20% of such patients will have a readmission over the subsequent 5 years with another episode of bowel obstruction.In a study of 286 patients who had undergone surgical intervention for adhesive small bowel obstruction, the risk of recurrent obstruction was 5.5% at 1 year, 11.3% at 3 years, and 13.5% at 5 years. The risk of reoperation for recurrent obstruction was 3.7% at 1 year, 4.8% at 3 years, and 5.8% at 5

1	the risk of recurrent obstruction was 5.5% at 1 year, 11.3% at 3 years, and 13.5% at 5 years. The risk of reoperation for recurrent obstruction was 3.7% at 1 year, 4.8% at 3 years, and 5.8% at 5 years.21 Considering the frequency of small bowel obstruction and the varied degree of clinical severity and presentation, there is often variation in the care of patients admitted with bowel obstruction. Studies have shown that a standard hospital-wide policy can help improve care of patients with bowel obstruction, reducing their time to surgery and shortening their length of hospital stay.22Brunicardi_Ch28_p1219-p1258.indd 123223/02/19 2:24 PM 1233SMALL INTESTINECHAPTER 28PreventionWith adhesive small bowel obstruction representing a large therapeutic burden, prevention of postoperative adhesions has become an area of great interest. Good surgical technique, careful handling of tissue, and minimal use and exposure of peritoneum to foreign bodies, forms the cornerstone of adhe-sion

1	adhesions has become an area of great interest. Good surgical technique, careful handling of tissue, and minimal use and exposure of peritoneum to foreign bodies, forms the cornerstone of adhe-sion prevention. These measures alone are often inadequate. In patients undergoing colorectal or pelvic surgery, hospital read-mission rates of greater than 30% over the subsequent 10 years have been reported for adhesive small bowel obstruction.23Use of laparoscopic surgery, when possible, has been strongly promoted. A recent study using the Swedish National Inpatient Register has shown that, compared to laparoscopy, open surgery is associated with a fourfold increase in risk of small bowel obstruction within 5 years of the index procedure, even after accounting for other risk factors such as age, comor-bidity, and previous abdominal surgery.24In those undergoing open surgery, several strategies for adhesion prevention have been tried; however, the only therapy that has shown some success has

1	comor-bidity, and previous abdominal surgery.24In those undergoing open surgery, several strategies for adhesion prevention have been tried; however, the only therapy that has shown some success has been the use of hyaluronan-based agents, such as Sperafilm. The use of this barrier has been clearly shown to reduce the incidence of postoperative bowel adhesions; however, their effect in actually reducing the inci-dence of small bowel obstruction remains less well defined.25 The use of these products is often left to the discretion of the surgeon and the clinical context. Wrapping of an intestinal anas-tomosis with the material may be associated with increased leak rates and is generally discouraged.26Other Causes of Small Bowel ObstructionEarly postoperative bowel obstruction, as defined by signs, symptoms, and radiographic signs of SBO occurring within 30 days following surgery, been reported to occur in 0.7% to 9% of patients, with a higher rate in patients undergoing pelvic

1	by signs, symptoms, and radiographic signs of SBO occurring within 30 days following surgery, been reported to occur in 0.7% to 9% of patients, with a higher rate in patients undergoing pelvic sur-gery, especially colorectal procedures.27 CT scanning or small bowel series is often required to make the diagnosis. Obstruc-tion that occurs in the early postoperative period is usually par-tial and only rarely is associated with strangulation. Therefore, a period of extended nonoperative therapy (2–3 weeks) consist-ing of bowel rest, hydration, and TPN administration is usually warranted. However, if complete obstruction is demonstrated or if signs suggestive of peritonitis are detected, expeditious reop-eration should be undertaken without delay. In a series of 180 patients undergoing anterior resection for rectal cancer, 12.8% developed early postoperative bowel obstruction on the median postoperative day 5, with 4 requiring surgical exploration at a median interval of 2 weeks from the

1	resection for rectal cancer, 12.8% developed early postoperative bowel obstruction on the median postoperative day 5, with 4 requiring surgical exploration at a median interval of 2 weeks from the index case.27Crohn’s disease as a cause of small bowel obstruction is discussed in more detail later in this chapter in the “Crohn’s Disease” section.Malignant small bowel obstruction can be a challenging problem. Although it often indicates advanced disease with poor prognosis, 25% to 33% of patients with a history of cancer who present with small bowel obstruction have adhesions as the etiol-ogy of their obstruction and therefore should not be denied appro-priate therapy. Even in cases in which the obstruction is related to recurrent malignancy, palliative resection or bypass can be performed, and in select cases these procedures lead to improved quality of life. In a series of 81 patients with small bowel obstruc-tion, palliation was achieved in over 80% of patients, with over 70% able to

1	and in select cases these procedures lead to improved quality of life. In a series of 81 patients with small bowel obstruc-tion, palliation was achieved in over 80% of patients, with over 70% able to reestablish oral intake. In this series, the surgical mor-bidity was high, with 7% developing an enterocutaneous fistula/Table 28-4Ileus: common etiologiesAbdominal surgeryInfection Sepsis Intra-abdominal abscess Peritonitis PneumoniaElectrolyte abnormalities Hypokalemia Hypomagnesemia Hypermagnesemia HyponatremiaMedications Anticholinergics Opiates Phenothiazines Calcium channel blockers Tricyclic antidepressantsHypothyroidismUreteral colicRetroperitoneal hemorrhageSpinal cord injuryMyocardial infarctionMesenteric ischemiaanastomotic leak and a 30-day mortality rate of 6%.28 Patients with obvious carcinomatosis and multifocal obstruction pose a difficult challenge, given their limited prognosis. Thus, management must be tailored to an individual patient’s prognosis and desires. At the

1	obvious carcinomatosis and multifocal obstruction pose a difficult challenge, given their limited prognosis. Thus, management must be tailored to an individual patient’s prognosis and desires. At the time of surgery, relief of the obstruction may be best achieved by a bypass procedure, avoiding a potentially difficult bowel resection, and even if that is not feasible, a palliative gastrostomy tube can be considered to help resolve nausea and vomiting.ILEUS AND OTHER DISORDERS OF INTESTINAL MOTILITYIleus and intestinal pseudo-obstruction are clinical syndromes caused by impaired intestinal motility and are characterized by symptoms and signs of intestinal obstruction in the absence of a lesion-causing mechanical obstruction. Ileus is a temporary motility disorder that is reversed with time as the inciting factor is corrected. In contrast, chronic intestinal pseudo-obstruction comprises a spectrum of specific disorders associated with irre-versible intestinal dysmotility.Ileus is a

1	time as the inciting factor is corrected. In contrast, chronic intestinal pseudo-obstruction comprises a spectrum of specific disorders associated with irre-versible intestinal dysmotility.Ileus is a major cause of morbidity in hospitalized patients. A degree of intestinal ileus is a normal physiological response to abdominal surgery, which often resolves quickly without any long-term sequela. However, when postoperative ileus is pro-longed, it can cause significant morbidity and cost. Prolonged postoperative ileus is the most frequently implicated cause of delayed discharge following abdominal operations, and its eco-nomic impact has been estimated to be between $750 million and $1 billion annually in the United States.29PathophysiologyNumerous factors capable of impairing intestinal motility, and thus inciting ileus, have been described (Table 28-4). The most Brunicardi_Ch28_p1219-p1258.indd 123323/02/19 2:24 PM 1234SPECIFIC CONSIDERATIONSPART IIfrequently encountered factors

1	and thus inciting ileus, have been described (Table 28-4). The most Brunicardi_Ch28_p1219-p1258.indd 123323/02/19 2:24 PM 1234SPECIFIC CONSIDERATIONSPART IIfrequently encountered factors are abdominal operations, infec-tion and inflammation, electrolyte abnormalities, and drugs.Following most abdominal operations or injuries, the motility of the gastrointestinal tract is transiently impaired. Among the proposed mechanisms responsible for this dysmotility are surgical stress-induced sympathetic reflexes, inflammatory response mediator release, and anesthetic/analgesic side effects; each of which can inhibit intestinal motility. The return of normal motility generally follows a characteristic temporal sequence, with small-intestinal motility returning to normal within the first 24 hours after laparotomy and gastric and colonic motility returning to normal by 48 hours and 2 to 5 days, respectively. Since small bowel motility is returned before colonic and gastric motility, listening

1	after laparotomy and gastric and colonic motility returning to normal by 48 hours and 2 to 5 days, respectively. Since small bowel motility is returned before colonic and gastric motility, listening for bowel sounds is not a reliable indicator that ileus has fully resolved. Functional evidence of coordinated gastrointestinal motility in the form of passing flatus or bowel movement is a more useful indicator. Resolution of ileus may be delayed in the presence of other factors capable of inciting ileus such as the presence of intra-abdominal abscesses or electrolyte abnormalities.Chronic intestinal pseudo-obstruction can be caused by a large number of specific abnormalities affecting intestinal smooth muscle, the myenteric plexus, or the extraintestinal nervous system (Table 28-5). Visceral myopathies constitute a group of diseases characterized by degeneration and fibro-sis of the intestinal muscularis propria. Visceral neuropathies encompass a variety of degenerative disorders of the

1	myopathies constitute a group of diseases characterized by degeneration and fibro-sis of the intestinal muscularis propria. Visceral neuropathies encompass a variety of degenerative disorders of the myenteric and submucosal plexuses. Both sporadic and familial forms of visceral myopathies and neuropathies exist. Systemic disor-ders involving the smooth muscle such as progressive systemic sclerosis and progressive muscular dystrophy, and neurologi-cal diseases such as Parkinson’s disease, can also be compli-cated by chronic intestinal pseudo-obstruction. In addition, viral infections, such as those associated with cytomegalovirus and Epstein-Barr virus, can cause intestinal pseudo-obstruction.Table 28-5Chronic intestinal pseudo-obstruction: etiologiesPrimary CausesFamilial types Familial visceral myopathies (types I, II, and III) Familial visceral neuropathies (types I and II) Childhood visceral myopathies (types I and II)Sporadic types Visceral myopathies Visceral

1	types Familial visceral myopathies (types I, II, and III) Familial visceral neuropathies (types I and II) Childhood visceral myopathies (types I and II)Sporadic types Visceral myopathies Visceral neuropathiesSecondary CausesSmooth muscle disorders Collagen vascular diseases (e.g., scleroderma) Muscular dystrophies (e.g., myotonic dystrophy) AmyloidosisNeurological disorders Chagas disease, Parkinson’s disease, spinal cord injuryEndocrine disorders Diabetes, hypothyroidism, hypoparathyroidismMiscellaneous disorders Radiation enteritisPharmacological causes E.g., phenothiazines and tricyclic antidepressantsViral infectionsClinical PresentationThe clinical presentation of ileus resembles that of small bowel obstruction. Inability to tolerate liquids and solids by mouth, nausea, and lack of flatus or bowel movements are the most common symptoms. Vomiting and abdominal distension may occur. Although bowel sound characteristics are not diagnostic, they are usually diminished or absent, in

1	flatus or bowel movements are the most common symptoms. Vomiting and abdominal distension may occur. Although bowel sound characteristics are not diagnostic, they are usually diminished or absent, in contrast to the hyper-active bowel sounds that usually accompany mechanical small bowel obstruction. The clinical manifestations of chronic intes-tinal pseudo-obstruction include variable degrees of nausea and vomiting and abdominal pain and distention.DiagnosisRoutine postoperative ileus should be expected and requires no diagnostic evaluation. Definition of prolonged postoperative ileus has been varied but generally diagnosed if ileus persists beyond 5 days postoperatively. A recent global survey synthesized the results of the data to define postoperative ileus as “interval from surgery until passage of flatus/stool AND tolerance of an oral diet,” with prolonged postoperative ileus being defined as “two or more of nausea/vomiting, inability to tolerate oral diet over 24 h, absence of

1	passage of flatus/stool AND tolerance of an oral diet,” with prolonged postoperative ileus being defined as “two or more of nausea/vomiting, inability to tolerate oral diet over 24 h, absence of flatus over 24 h, distension, radiologic confirmation occurring on or after day 4 postoperatively without prior resolu-tion of postoperative ileus.”30 Prolonged ileus is reported to occur in 10% to 15% of patients undergoing intestinal surgery.31Once suspected, diagnostic evaluation to detect specific underlying factors capable of inciting ileus and to rule out the presence of mechanical obstruction is warranted.Patient medication lists should be reviewed for the pres-ence of drugs, especially opiates, known to be associated with impaired intestinal motility. Measurement of serum electrolytes may demonstrate electrolyte abnormalities commonly associ-ated with ileus. Abdominal radiographs are often obtained, but the distinction between ileus and mechanical obstruction may be difficult based on

1	demonstrate electrolyte abnormalities commonly associ-ated with ileus. Abdominal radiographs are often obtained, but the distinction between ileus and mechanical obstruction may be difficult based on this test alone. In the postoperative setting, CT scanning is the test of choice as it can demonstrate the presence of an intra-abdominal abscess or other evidence of peritoneal sepsis that may be causing ileus and can exclude the presence of complete mechanical obstruction. Distinction of postoperative ileus from early postoperative obstruction can be difficult but is helpful in developing the appropriate management plan.The diagnosis of chronic pseudo-obstruction is suggested by clinical features and confirmed by radiographic and mano-metric studies. Diagnostic laparotomy or laparoscopy with full-thickness biopsy of the small intestine may be required to establish the specific underlying cause in cases of suspected neural disorder.TherapyThe management of ileus consists of limiting oral

1	full-thickness biopsy of the small intestine may be required to establish the specific underlying cause in cases of suspected neural disorder.TherapyThe management of ileus consists of limiting oral intake and correcting the underlying inciting factor. If vomiting or abdom-inal distention are prominent, the stomach should be decom-pressed using a nasogastric tube. Fluid and electrolytes should be administered intravenously until ileus resolves. If the dura-tion of ileus is prolonged, total parental nutrition (TPN) may be required.Given the frequency of postoperative ileus and its financial impact, many strategies have been tested to reduce its duration. The administration of nonsteroidal anti-inflammatory drugs such as ketorolac and concomitant reductions in opioid dosing have been shown to reduce the duration of ileus in most studies. Similarly, the use of perioperative thoracic epidural anesthesia/analgesia with Brunicardi_Ch28_p1219-p1258.indd 123423/02/19 2:24 PM 1235SMALL

1	to reduce the duration of ileus in most studies. Similarly, the use of perioperative thoracic epidural anesthesia/analgesia with Brunicardi_Ch28_p1219-p1258.indd 123423/02/19 2:24 PM 1235SMALL INTESTINECHAPTER 28regimens containing local anesthetics combined with limitation or elimination of systemically administered opioids has been shown to reduce duration of postoperative ileus, although they have not reduced the overall length of hospital stay.32 Many studies have also suggested that limiting intraand postoperative fluid administration can also result in reduction of postoperative ileus and shortened hospital stay.33 Furthermore, studies have shown that early postoperative feeding after GI surgery is generally well tolerated and can lead to reduced postoperative ileus and a shorter hospital stay. Table 28-6 summarizes some of the measures used to minimize postoperative ileus. Such data have generated significant interest in Early Recovery After Surgery (ERAS) pathways, which

1	hospital stay. Table 28-6 summarizes some of the measures used to minimize postoperative ileus. Such data have generated significant interest in Early Recovery After Surgery (ERAS) pathways, which are a collection of steps taken to expedite postoperative recovery in general. ERAS protocols typically involve 15 to 20 steps that involve the pre-, intraand postoperative phases of care and form a multimodal pathway. Although the contribution of each element to the overall outcome has not been well studied, the bundle of steps leads to reduced length of stay and surgical complications. In cases of GI surgery, many of these steps are targeted towards reducing postoperative ileus, which is often the barrier to early discharge.Although prokinetic agents have been tried to pro-mote return of GI motility, they are associated with efficacytoxicity profiles that are too unfavorable to warrant routine use. Recently, administration of alvimopan, a novel, peripherally active mu-opioid receptor

1	GI motility, they are associated with efficacytoxicity profiles that are too unfavorable to warrant routine use. Recently, administration of alvimopan, a novel, peripherally active mu-opioid receptor antagonist with limited oral absorp-tion, has been shown to reduce duration of postoperative ileus, hospital stay, and rate of readmissions in several prospective, randomized, placebo-controlled trials and the subsequent meta-analysis.34 Any cost savings associated with the use of this drug outside of a clinical trial has, however, been debated.35The therapy of patients with chronic intestinal pseudo-obstruction focuses on palliation of symptoms as well as fluid, electrolyte, and nutritional management. Surgery should be avoided if possible. No standard therapies are curative or delay the natural history of any of the specific disorders causing intes-tinal pseudo-obstruction. Prokinetic agents, such as metoclo-promide and erythromycin, are associated with poor efficacy. Cisapride has been

1	history of any of the specific disorders causing intes-tinal pseudo-obstruction. Prokinetic agents, such as metoclo-promide and erythromycin, are associated with poor efficacy. Cisapride has been associated with palliation of symptoms; however, because of cardiac toxicity and reported deaths, this agent is restricted to compassionate use in the United States.Patients with refractory disease may require strict limitation of oral intake and long-term TPN administration. Despite these measures, some patients will continue to have severe abdomi-nal pain or such copious intestinal secretions that vomiting and fluid and electrolyte losses remain substantial. These patients may require a decompressive gastrostomy or an extended small bowel Table 28-6Measures to reduce postoperative ileusIntraoperative measures Minimalize handling of the bowel Laparoscopic approach, if possible Restricted intraoperative fluid administrationPostoperative measures Avoid nasogastric tubes Early enteral

1	measures Minimalize handling of the bowel Laparoscopic approach, if possible Restricted intraoperative fluid administrationPostoperative measures Avoid nasogastric tubes Early enteral feeding Epidural anesthesia, if indicated Restricted IV fluid administration Correct electrolyte abnormalities Consider mu-opiod antagonistsresection to remove abnormal intestine. Small-intestinal trans-plantation has been applied in these patients with increasing fre-quency; the ultimate role of this modality remains to be defined.CROHN’S DISEASECrohn’s disease is a chronic, idiopathic transmural inflamma-tory disease with skip lesions that may affect any part of the alimentary tract, although there is propensity to affect the dis-tal small bowel. Nearly 80% of patients with Crohn’s disease have small bowel involvement, with 30% having terminal ileitis exclusively. Recent studies suggest a prevalence of about 241 cases per 100,000 in the United States.36 The rates of Crohn’s and ulcerative colitis have

1	involvement, with 30% having terminal ileitis exclusively. Recent studies suggest a prevalence of about 241 cases per 100,000 in the United States.36 The rates of Crohn’s and ulcerative colitis have been increasing globally over the past several decades with substantial regional variations in inci-dence. The highest incidences are reported in western nations and those in northern latitudes, with Canada having the highest reported rates.37 In countries such as China, the prevalence of Crohn’s disease is substantially below that seen in the West, but rates have been rapidly increasing recently.38 The incidence of Crohn’s disease varies among ethnic groups within the same geographic region. For example, members of Eastern European Ashkenazi Jewish population are at a twoto fourfold higher risk of developing Crohn’s disease than members of other popu-lations living in the same location.Most studies suggest that Crohn’s disease is slightly more prevalent in females than in males. The mean

1	of developing Crohn’s disease than members of other popu-lations living in the same location.Most studies suggest that Crohn’s disease is slightly more prevalent in females than in males. The mean age at which patients are diagnosed with Crohn’s disease falls in the third decade of life years, with a second smaller peak in the sixth decade of life, giv-ing it a bimodal distribution. The age at diagnosis can, however, range from early childhood through the entire lifespan.Both genetic and environmental factors appear to influ-ence the risk for developing Crohn’s disease. The relative risk among first-degree relatives of patients with Crohn’s disease is 14 to 15 times higher than that of the general population, with about 20% of patients reporting a family history. The concor-dance rate among monozygotic twins is as high as 67%; how-ever, Crohn’s disease is not associated with simple Mendelian inheritance patterns. Although there is a tendency within fami-lies for either ulcerative

1	among monozygotic twins is as high as 67%; how-ever, Crohn’s disease is not associated with simple Mendelian inheritance patterns. Although there is a tendency within fami-lies for either ulcerative colitis or Crohn’s disease to be present exclusively, mixed kindreds also occur, suggesting the presence of some shared genetic traits as a basis for both diseases.Higher socioeconomic status is associated with an increased risk of Crohn’s disease. Most studies have found breastfeeding to be protective against the development of Crohn’s disease. Crohn’s disease is more prevalent among smokers. Furthermore, smoking is associated with the increased risk for both the need for surgery and the risk of relapse after surgery for Crohn’s disease.PathophysiologyCrohn’s disease is characterized by sustained inflammation. Whether this inflammation represents an appropriate response to a yet unrecognized pathogen or an inappropriate response to a normally innocuous stimulus is unknown. Various

1	sustained inflammation. Whether this inflammation represents an appropriate response to a yet unrecognized pathogen or an inappropriate response to a normally innocuous stimulus is unknown. Various hypotheses on the roles of environmental and genetic factors in the patho-genesis of Crohn’s disease have been proposed. Many infec-tious agents have been suggested to be the causative organism of Crohn’s disease; however, there has been no conclusive evidence to confirm any. Studies using animal models suggest that in a genetically susceptible host, a nonpathogenic gut microbiome is sufficient to induce a chronic inflammatory response resembling Brunicardi_Ch28_p1219-p1258.indd 123523/02/19 2:24 PM 1236SPECIFIC CONSIDERATIONSPART IIthat associated with Crohn’s disease. In these models, the sus-tained intestinal inflammation is the result of either abnormal epithelial barrier function or immune dysregulation. A full dis-cussion of the role of gut immune system and microbiome in the

1	the sus-tained intestinal inflammation is the result of either abnormal epithelial barrier function or immune dysregulation. A full dis-cussion of the role of gut immune system and microbiome in the development of Crohn’s disease is beyond the scope of this work, but it is an area of great interest and under investigation. In general, poor barrier function is hypothesized to permit inap-propriate exposure of lamina propria lymphocytes to antigenic stimuli derived from the intestinal lumen. In addition, a variety of defects in immune regulatory mechanisms, e.g., overrespon-siveness of mucosal T cells to enteric flora-derived antigens, can lead to defective immune tolerance and sustained inflammation.Specific genetic defects associated with Crohn’s disease in human patients are beginning to be defined. For example, the presence of a locus on chromosome 16 (the so-called IBD1 locus) has been linked to Crohn’s disease. The IBD1 locus has been identified as the NOD2 gene. Persons with

1	to be defined. For example, the presence of a locus on chromosome 16 (the so-called IBD1 locus) has been linked to Crohn’s disease. The IBD1 locus has been identified as the NOD2 gene. Persons with allelic variants on both chromosomes have a 40-fold relative risk of Crohn’s disease com-pared to those without variant NOD2 genes. The relevance of this gene to the pathogenesis of Crohn’s disease is biologically plau-sible, as the protein product of the NOD2 gene mediates the innate immune response to microbial pathogens. Other putative IBD loci have been identified on other chromosomes (IBD2 on chromose 12q, and IBD3 on chromose 6), and are under investigation.Although appendectomy has been shown to lower the risk of subsequent development of ulcerative colitis, it was suspected that the surgery may increase the risk of developing Crohn’s disease. A meta-analysis has, however, suggested that the observed increased risk of Crohn’s disease in the first few years after an appendectomy may

1	may increase the risk of developing Crohn’s disease. A meta-analysis has, however, suggested that the observed increased risk of Crohn’s disease in the first few years after an appendectomy may in fact reflect diagnostic dif-ficulty in a group of patients with incipient Crohn’s.39Although the pathological hallmark of Crohn’s disease is focal, transmural inflammation of the intestine, a spectrum of pathological lesions can be present. The earliest lesion characteristic of Crohn’s disease is the aphthous ulcer. These superficial ulcers are up to 3 mm in diameter and are surrounded by a halo of erythema. In the small intestine, aphthous ulcers typically arise over lymphoid aggregates. Granulomas are highly characteristic of Crohn’s disease and are reported to be present in up to 70% of intestinal specimens obtained during surgical resection. These granulomas are noncaseating and can be found in both areas of active disease and apparently normal intestine, in any layer of the bowel wall,

1	specimens obtained during surgical resection. These granulomas are noncaseating and can be found in both areas of active disease and apparently normal intestine, in any layer of the bowel wall, and in mesenteric lymph nodes.As disease progresses, aphthae coalesce into larger, stellateshaped ulcers. Linear or serpiginous ulcers may form when multiple ulcers fuse in a direction parallel to the longitudinal axis of the intestine. With transverse coalescence of ulcers, a cobblestoned appearance of the mucosa may arise.With advanced disease, inflammation can be transmural. Serosal involvement results in adhesion of the inflamed bowel to other loops of bowel or other adjacent organs. Transmural inflammation can also result in fibrosis with stricture formation, intra-abdominal abscesses, fistulas, and, rarely, free perforation. Inflammation in Crohn’s disease can affect discontinuous por-tions of intestine, so-called skip lesions that are separated by intervening normal-appearing

1	fistulas, and, rarely, free perforation. Inflammation in Crohn’s disease can affect discontinuous por-tions of intestine, so-called skip lesions that are separated by intervening normal-appearing intestine.A feature of Crohn’s disease that is grossly evident and helpful in identifying affected segments of intestine during sur-gery is the presence of fat wrapping, which represents encroach-ment of mesenteric fat onto the serosal surface of the bowel (Fig. 28-17). This finding is virtually pathognomonic of Crohn’s Figure 28-17. Crohn’s disease. This intraoperative photograph demonstrates encroachment of mesenteric fat onto the serosal surface of the intestine (“fat wrapping”) that is characteristic of intestinal segments affected by active Crohn’s’ disease.disease. The presence of fat wrapping correlates well with the presence of underlying acute and chronic inflammation.Features that allow for differentiation between Crohn’s dis-ease of the colon and ulcerative colitis include the

1	correlates well with the presence of underlying acute and chronic inflammation.Features that allow for differentiation between Crohn’s dis-ease of the colon and ulcerative colitis include the layers of the bowel wall affected (inflammation in ulcerative colitis is limited to the mucosa and submucosa but may involve the full-thickness of the bowel wall in Crohn’s disease) and the longitudinal extent of inflammation (inflammation is continuous and characteristi-cally affects the rectum in ulcerative colitis but may be discon-tinuous and spare the rectum in Crohn’s disease). In the absence of full expression of features of advanced disease, Crohn’s colitis can sometimes be difficult to distinguish from ulcerative colitis. It is also important to remember that although ulcerative colitis is a disease of the colon, it can be associated with inflammatory changes in the distal ileum (backwash ileitis).Clinical PresentationThe most common symptoms of Crohn’s disease are abdominal pain,

1	is a disease of the colon, it can be associated with inflammatory changes in the distal ileum (backwash ileitis).Clinical PresentationThe most common symptoms of Crohn’s disease are abdominal pain, diarrhea, and weight loss. However, the clinical features are highly variable among individual patients and depend on which segment(s) of the gastrointestinal tract is (are) predomi-nantly affected, the intensity of inflammation, and the presence or absence of specific complications. In fact, some patients with Crohn’s disease may have been initially misdiagnosed as having irritable bowel syndrome or celiac disease.Patients can be classified by their predominant clinical manifestation as having primarily (a) fibrostenotic disease, (b) fistulizing disease, and (c) aggressive inflammatory disease. There is substantial overlap among these disease patterns in individual patients, however. The onset of symptoms is insidious, and once present, their severity follows a waxing and waning course.

1	There is substantial overlap among these disease patterns in individual patients, however. The onset of symptoms is insidious, and once present, their severity follows a waxing and waning course. Constitutional symptoms, particularly weight loss and fever, or growth retardation in children, may also be prominent and are occasionally the sole presenting features of Crohn’s disease.The disease affects the small bowel in 80% of cases and colon alone in 20%. In those with small bowel disease, the major-ity have ileocecal disease. Isolated perineal and anorectal disease occurs in 5% to 10% of affected patients. Uncommon sites of involvement include the esophagus, stomach, and duodenum.An estimated one-fourth of all patients with Crohn’s disease will have an extraintestinal manifestation of their disease. One Brunicardi_Ch28_p1219-p1258.indd 123623/02/19 2:24 PM 1237SMALL INTESTINECHAPTER 28fourth of those affected will have more than one manifestation. Many of these complications can

1	disease. One Brunicardi_Ch28_p1219-p1258.indd 123623/02/19 2:24 PM 1237SMALL INTESTINECHAPTER 28fourth of those affected will have more than one manifestation. Many of these complications can be seen with both Crohn’s disease and ulcerative colitis, although they are more prevalent among patients with Crohn’s disease. The most common extraintestinal manifestations are listed in Table 28-7. The clinical severity of some of these manifestations, such as erythema nodosum and peripheral arthritis, are correlated with the severity of intestinal inflammation. The severity of other manifestations, such as pyoderma gangrenosum and ankylosing spondylitis, bear no apparent relationship to the severity of intestinal inflammation.DiagnosisThe diagnosis is usually established with endoscopic findings in a patient with a compatible clinical history. The diagnosis should be considered in those presenting with acute or chronic abdominal pain, especially when localized to the right lower quadrant,

1	in a patient with a compatible clinical history. The diagnosis should be considered in those presenting with acute or chronic abdominal pain, especially when localized to the right lower quadrant, chronic diarrhea, evidence of intestinal inflammation on radiography or endoscopy, the discovery of a bowel stricture or fistula arising from the bowel, and evidence of inflamma-tion or granulomas on intestinal histology. Disorders associated with clinical presentations that resemble those of Crohn’s dis-ease include ulcerative colitis, functional bowel disorders such as irritable bowel syndrome, mesenteric ischemia, collagen vascular diseases, carcinoma and lymphoma, diverticular dis-ease, and infectious enteritides. Infectious enteritides are most frequently diagnosed in immunocompromised patients, but they can also occur in patients with normal immune function. Acute ileitis caused by Campylobacter and Yersinia species can be difficult to distinguish from that caused by an acute

1	patients, but they can also occur in patients with normal immune function. Acute ileitis caused by Campylobacter and Yersinia species can be difficult to distinguish from that caused by an acute presenta-tion of Crohn’s disease. Typhoid enteritis caused by Salmonella Table 28-7Extraintestinal manifestations of Crohn’s diseaseDermatologic Erythema nodosum Pyoderma gangrenosumRheumatologic Peripheral arthritis Ankylosing spondylitis SacroiliitisOcular Conjunctivitis Uveitis/iritis EpiscleritisHepatobiliary Hepatic steatosis Cholelithiasis Primary sclerosing cholangitis PericholangitisUrologic Nephrolithiasis Ureteral obstructionMiscellaneous Thromboembolic disease Vasculitis Osteoporosis Endocarditis, myocarditis, pleuropericarditis Interstitial lung disease Amyloidosis Pancreatitistyphosa can lead to overt intestinal bleeding and perforation, most often affecting the terminal ileum. The distal ileum and cecum are the most common sites of intestinal involvement by infection due to

1	can lead to overt intestinal bleeding and perforation, most often affecting the terminal ileum. The distal ileum and cecum are the most common sites of intestinal involvement by infection due to Mycobacterium tuberculosis. This condi-tion can result in intestinal inflammation, strictures, and fistula formation, like those seen in Crohn’s disease. Cytomegalovirus (CMV) can cause intestinal ulcers, bleeding, and perforation.No single symptom, sign, or diagnostic test establishes the diagnosis of Crohn’s disease. Instead, the diagnosis is based on a complete assessment of the clinical presentation with confirmatory findings derived from radiographic, endoscopic, and in most cases, pathologic tests. Patients presenting with a history of Crohn’s disease should have their full blood count, electrolytes and renal function, liver function, iron, B12, ESR, and CRP levels checked. The results may be abnormal, show-ing anemia, but these results are nondiagnostic. Colonoscopy with intubation of

1	and renal function, liver function, iron, B12, ESR, and CRP levels checked. The results may be abnormal, show-ing anemia, but these results are nondiagnostic. Colonoscopy with intubation of terminal ileum is the main diagnostic tool and can reveal focal ulcerations adjacent to areas of normal appear-ing mucosa along with polypoid mucosal changes that give a “cobblestone appearance.” Skip areas of involvement are typical with segments of normal-appearing bowel interrupted by large areas of obvious disease; this pattern is different from the con-tinuous involvement in ulcerative colitis. Pseudopolyps, as seen in ulcerative colitis, are also often present. Barium small bowel follow-through, CT enterography, or MR enterography may be used as contrast examinations of the small bowel to reveal strictures or networks of ulcers and fissures. CT scanning may reveal intra-abdominal abscesses and is useful in acute presenta-tions to rule out the presence of other intra-abdominal disorders.

1	strictures or networks of ulcers and fissures. CT scanning may reveal intra-abdominal abscesses and is useful in acute presenta-tions to rule out the presence of other intra-abdominal disorders. Esophagogastroduodenoscopy (EGD) is done for disease of the proximal alimentary tract. Because Crohn’s disease often affects the small bowel, which is difficult to image, capsule endoscopy has been increasing used to make this diagnosis (Fig. 28-18).40Figure 28-18. Crohn’s disease. This image was captured by a wireless capsule endoscope as it was traveling through the small intestine. It demonstrates a superficial ulceration in the small bowel consistent with Crohn’s disease. (Used with permission from Anne T. Wolf, M.D., Department of Medicine, Brigham and Women’s Hospital, Boston, MA.)Brunicardi_Ch28_p1219-p1258.indd 123723/02/19 2:24 PM 1238SPECIFIC CONSIDERATIONSPART IISeveral antibodies have also been identified in patients with inflammatory bowel disease, which may have diagnostic

1	123723/02/19 2:24 PM 1238SPECIFIC CONSIDERATIONSPART IISeveral antibodies have also been identified in patients with inflammatory bowel disease, which may have diagnostic value. The most commonly tested antibodies are antineutrophil cytoplasmic antibody (pANCA) and antisaccharmyces cerevi-siae antibody (ASCA). ASCA+/pANCA–, is associated with a diagnosis of Crohn’s disease, while ASCA–/pANCA+, corre-lates with ulcerative colitis. Although these antibody tests have high specificity, their use has been hampered by low test sensi-tivities. There is ongoing interest in developing other antibody tests to diagnose inflammatory bowel disease and help differ-entiate Crohn’s disease from ulcerative colitis. There have been attempts to develop stool tests to diagnose inflammatory bowel disease, and although fecal calprotectin or lactoferrin can iden-tify patients with intestinal inflammation, they are not routinely done in clinical practice.Because of the insidious, and often nonspecific,

1	and although fecal calprotectin or lactoferrin can iden-tify patients with intestinal inflammation, they are not routinely done in clinical practice.Because of the insidious, and often nonspecific, presenta-tion of Crohn’s disease, a diagnosis of Crohn’s is typically made only after symptoms have been present for several years. How-ever, in acute presentations, the diagnosis is sometimes made intraoperatively or during surgical evaluation. The initial mani-festation of Crohn’s disease can consist of right lower quadrant abdominal mimicking the presentation of acute appendicitis. In patients with this presentation, Crohn’s disease can be discov-ered for the first time during laparotomy or laparoscopy per-formed for presumed appendicitis. In some patients, the initial manifestation of Crohn’s disease is an acute abdomen related to small bowel obstruction, intra-abdominal abscess, or free intestinal perforation. In other patients, perianal abscesses and fistulas requiring surgical

1	Crohn’s disease is an acute abdomen related to small bowel obstruction, intra-abdominal abscess, or free intestinal perforation. In other patients, perianal abscesses and fistulas requiring surgical therapy may be the first manifestation of Crohn’s disease.TherapyBecause no curative therapies are available for Crohn’s dis-ease, the goal of treatment is to palliate symptoms rather than to achieve cure. Medical therapy is used to induce and maintain disease remission. Surgery is reserved for specific indications described later in this chapter. In addition, nutritional support in the form of aggressive enteral regimens or, if necessary, paren-teral nutrition, is used to manage the malnutrition that is com-mon in patients with Crohn’s disease.Medical Therapy. Pharmacologic agents used to treat Crohn’s disease include antibiotics, aminosalicylates, corticosteroids, immunomodulators, and biologic therapies. Antibiotics have an adjunctive role in the treatment of infectious complications

1	Crohn’s disease include antibiotics, aminosalicylates, corticosteroids, immunomodulators, and biologic therapies. Antibiotics have an adjunctive role in the treatment of infectious complications associated with Crohn’s disease. They are also used to treat patients with perianal disease, enterocutaneous fistulas, and active colonic disease.Crohn’s disease activity is assessed using the Crohn’s dis-ease Activity Index or Harvey-Bradshaw Index, and depending on the scores, it can be categorized as asymptomatic, mild, mod-erate, or severe disease to guide therapy. While patients with mild and moderate disease can be managed on an outpatient basis, those with severe or fulminant disease often require hospitaliza-tion for treatment, bowel rest, and possible nutritional support. There are two general approaches to treating Crohn’s disease: top-down (which starts with the most potent agents to achieve remission with a subsequent decrease in medication) or step-up (starts with less potent and

1	approaches to treating Crohn’s disease: top-down (which starts with the most potent agents to achieve remission with a subsequent decrease in medication) or step-up (starts with less potent and often safer drugs, and if symptoms fail to improve advances to the next group of medications).The use of oral 5-aminosalicylic acid (5-ASA) drugs (e.g., mesalamine) is somewhat controversial with mixed results from several randomized studies and meta-analyses. Aminosalicylates are associated with minimal toxicity and are available in a variety of formulations that allow for their delivery to specific regions of the alimentary tract. Thus, many continue to recommend use of mesalamine as an initial step in management of mild symptoms in patients with small bowel Crohn’s disease.Orally administered glucocorticoids are used to treat patients with mild disease that does not respond to aminosalicy-lates, or as initial treatment of patients with moderate disease. Patients with severe active disease

1	glucocorticoids are used to treat patients with mild disease that does not respond to aminosalicy-lates, or as initial treatment of patients with moderate disease. Patients with severe active disease usually require intravenous administration of glucocorticoids. Although glucocorticoids are effective in inducing remission, they are ineffective in prevent-ing relapse, and their adverse side-effect profile makes long-term use hazardous. Therefore, they should be tapered once remis-sion is achieved. Some patients are unable to undergo glucocor-ticoid tapering without suffering recurrence of symptoms. Such patients are said to have steroid dependence. These patients, along with those who do not respond to steroids at all (steroid resistant), should be considered for immune modulator therapies. Controlled ileal-released budesonide is an oral steroid with high first-pass hepatic metabolism and few systemic effects that can be tried in those with ileal and colonic Crohn’s disease.For those

1	Controlled ileal-released budesonide is an oral steroid with high first-pass hepatic metabolism and few systemic effects that can be tried in those with ileal and colonic Crohn’s disease.For those with severe disease, the thiopurine antimetabo-lites azathioprine and its active metabolite, 6-mercaptopurine, have demonstrated efficacy in inducing remission, maintaining remission, and allowing for glucocorticoid tapering in gluco-corticoid-dependent patients. A response to these medications is usually observed in 3 to 6 months, during which patients may need to continue with steroids. There is also some evidence that they decrease the risk of relapse after intestinal resection for Crohn’s disease. These agents are relatively safe but can induce bone marrow suppression and promote infectious complications. For patients who do not respond to the thiopurines, methotrexate is an alternative that is usually initially given intramuscularly before switching to oral form after achieving

1	complications. For patients who do not respond to the thiopurines, methotrexate is an alternative that is usually initially given intramuscularly before switching to oral form after achieving symptomatic con-trol. There is little role for cyclosporine in Crohn’s disease; its efficacy/toxicity profile in this disease is poor.The successful introduction of infliximab (Remicade), an anti-TNFα antibody, heralded the era of biological therapies for inflammatory bowel disease. Infliximab is a chimeric monoclo-nal antitumor necrosis-factor alpha (TNFα) antibody that has been shown to have efficacy in inducing remission and in pro-moting closure of enterocutaneous fistulae. There are two other anti-TNFα antibodies, with no randomized studies comparing efficacy of the drugs head to head. In general, it is thought that there is no significant difference in efficacy between them. While infliximab is a mouse-human chimeric antibody, adalimumab (Humira) is a fully human antibody. Certolizumab

1	it is thought that there is no significant difference in efficacy between them. While infliximab is a mouse-human chimeric antibody, adalimumab (Humira) is a fully human antibody. Certolizumab pegol (Cimzia) is a PEGylated Fab fragment of a humanized TNF inhibitor mono-clonal antibody. These agents are generally used for patients who are resistant to standard therapy, to help taper steroid dosage. They are generally well tolerated, but they should not be used in patients with ongoing septic processes, such as undrained intra-abdomi-nal abscesses. Antibodies against other targets in this inflamma-tory pathway have also been developed, including vedolizumab (Entyvio), a humanized anti–α4β7 integrin monoclonal antibody, with more specific anti-inflammatory effect in the intestine.For patients with perianal disease, antibiotic therapy with metronidazole or ciprofloxacin is the primary step. Two to 4 weeks of therapy is needed before improvements are seen, and often long-term therapy is

1	with perianal disease, antibiotic therapy with metronidazole or ciprofloxacin is the primary step. Two to 4 weeks of therapy is needed before improvements are seen, and often long-term therapy is required to prevent relapse. In cases of relapse, azathioprine can be considered. In patients with fis-tulas, infliximab and azathiprine are drugs of choice.Brunicardi_Ch28_p1219-p1258.indd 123823/02/19 2:24 PM 1239SMALL INTESTINECHAPTER 28Surgical Therapy. With introduction of new treatments, the need for surgery for Crohn’s disease has decreased steadily over the past few decades. Recent meta-analysis estimated the risk of surgery to be 16.3%, 33.3%, and 46.6% at 1, 5, and 10 years respectively.41 Surgery is generally reserved for patients whose disease is unresponsive to aggressive medical therapy or who develop complications of their disease (Table 28-8). Failure of medical management may be the indication for surgery if symp-toms persist despite aggressive therapy for several months

1	therapy or who develop complications of their disease (Table 28-8). Failure of medical management may be the indication for surgery if symp-toms persist despite aggressive therapy for several months or if symptoms recur whenever aggressive therapy is tapered. Sur-gery should be considered if medication-induced complications arise, specifically corticosteroid-related complications, such as cushingoid features, cataracts, glaucoma, systemic hyperten-sion, compression fractures, or aseptic necrosis of the femoral head. Growth retardation constitutes an indication for surgery in 30% of children with Crohn’s disease.One of the most common indications for surgical interven-tion is intestinal obstruction. Abscesses and fistulas are frequently encountered during operations performed for intestinal obstruc-tion in these patients, but they are rarely the only indication for surgery. Most abscesses are amenable to percutaneous drain-age, and fistulas, unless associated with symptoms or metabolic

1	obstruc-tion in these patients, but they are rarely the only indication for surgery. Most abscesses are amenable to percutaneous drain-age, and fistulas, unless associated with symptoms or metabolic derangements, do not require surgical intervention. Less common complications that require surgical intervention are acute gastro-intestinal hemorrhage, perforations, and development of cancer.Although surgery for Crohn’s disease is usually planned, an uncommon, but not rare, scenario is the intraoperative dis-covery of inflammation limited to the terminal ileum during operations performed for presumed appendicitis. This scenario can result from an acute presentation of Crohn’s disease or from acute ileitis caused by bacteria such as Yersinia or Campylo-bacter. Both conditions should be treated medically; ileal resec-tion is not generally indicated. However, the appendix, even if normal appearing, should be removed (unless the cecum is inflamed, increasing the potential morbidity of this

1	medically; ileal resec-tion is not generally indicated. However, the appendix, even if normal appearing, should be removed (unless the cecum is inflamed, increasing the potential morbidity of this procedure) to eliminate appendicitis from the differential diagnosis of abdominal pain in these patients, particularly those with Crohn’s disease who may be destined to have recurring symptoms.When the diagnosis of Crohn’s disease is known and sur-gery is planned, a thorough examination of the entire intestine should be performed. The presence of active disease is suggested by thickening of the bowel wall, narrowing of the lumen, serosal inflammation and coverage by creeping fat, and thickening of Table 28-8Indications for surgical intervention in Crohn’s diseaseAcute onset of severe disease: Crohn’s colitis +/− toxic megacolon (rare)Failure of medical therapy: Persistent symptoms despite long-term steroid use Recurrence of symptoms when high-dose steroids are tapered Drug-induced

1	colitis +/− toxic megacolon (rare)Failure of medical therapy: Persistent symptoms despite long-term steroid use Recurrence of symptoms when high-dose steroids are tapered Drug-induced complications (Cushing’s disease, hypertension)Development of disease complications: Obstruction Perforation Complicated fistulas Hemorrhage Malignancy riskthe mesentery. Skip lesions are present in approximately 20% of cases and should be sought. The length of uninvolved small intestine should be noted.Segmental intestinal resection of grossly evident disease followed by primary anastomosis is the usual procedure of choice. Microscopic evidence of Crohn’s disease at the resection margins does not compromise a safe anastomosis, and frozen section analysis of resection margins is unnecessary. In a ran-domized prospective trial, the effects of achieving 2-cm resec-tion margins beyond grossly evident disease were compared with achieving 12-cm resection margins.42 There were no evident dif-ferences with

1	prospective trial, the effects of achieving 2-cm resec-tion margins beyond grossly evident disease were compared with achieving 12-cm resection margins.42 There were no evident dif-ferences with respect to clinical recurrence rates or anastomotic recurrences. Recurrence rates were similar whether margins were histologically free of or involved with Crohn’s disease. An area of controversy in surgical management of Crohn’s disease has been the ideal anastomotic technique for the bowel after intestinal resection. This issue was addressed in a randomized study of 139 patients undergoing an ileocolic resection for Crohn’s disease, with a mean follow-up of 11.9 months. There were no differ-ences in endoscopic or symptomatic disease recurrence between the groups reconstructed using end-to-end sutured (2-0 PDS) anastomosis versus those with side-to-side staples anastomosis.43An alternative to segmental resection for obstructing lesions is stricturoplasty (Fig. 28-19). This technique allows

1	sutured (2-0 PDS) anastomosis versus those with side-to-side staples anastomosis.43An alternative to segmental resection for obstructing lesions is stricturoplasty (Fig. 28-19). This technique allows for preservation of intestinal surface area and is especially well suited to patients with extensive disease and fibrotic stric-tures who may have undergone previous resection and are at risk for developing short bowel syndrome. In this technique, the bowel is opened longitudinally to expose the lumen. Any ABFigure 28-19. Stricturoplasty. The wall of the strictured bowel is incised longitudinally. Reconstruction is performed by closing the defect transversely in a manner similar to the Heinecke-Mickulicz pyloroplasty for short strictures (A), or the Finney pyloroplasty for longer strictures (B).Brunicardi_Ch28_p1219-p1258.indd 123923/02/19 2:24 PM 1240SPECIFIC CONSIDERATIONSPART IIintraluminal ulcerations should be biopsied to rule out the pres-ence of neoplasia. Depending on the

1	(B).Brunicardi_Ch28_p1219-p1258.indd 123923/02/19 2:24 PM 1240SPECIFIC CONSIDERATIONSPART IIintraluminal ulcerations should be biopsied to rule out the pres-ence of neoplasia. Depending on the length of the stricture, the reconstruction can be fashioned in a manner similar to the Heinecke-Mickulicz pyloroplasty (for strictures less than 12 cm in length) or the Finney pyloroplasty (for longer strictures as much as 25 cm in length). For longer strictures, variations on the standard stricturoplasty, namely the side-to-side isoperistaltic enteroenterostomy, have been advocated and used for strictures with mean lengths of 50 cm.44 Stricturoplasty sites should be marked with metallic clips to facilitate their identification on radiographs and during subsequent operations. Stricturoplasty is associated with recurrence rates that are no different from those associated with segmental resection. Because the affected bowel is left in situ rather than resected, there is the potential for

1	is associated with recurrence rates that are no different from those associated with segmental resection. Because the affected bowel is left in situ rather than resected, there is the potential for cancer developing at the stricturoplasty site. However, as data on this complication are limited to anecdotes, this risk remains a theo-retical one. Stricturoplasty is contraindicated in patients with intra-abdominal abscesses or intestinal fistulas. The presence of a solitary stricture relatively close to a segment for which resection is planned is a relative contraindication. In general, stricturoplasty is performed in cases where single or multiple strictures are identified in diffusely involved segments of bowel, or where previous resections have been performed and mainte-nance of intestinal length is of great importance.Intestinal bypass procedures are sometimes required in the presence of intramesenteric abscesses or if the diseased bowel is coalesced in the form of a dense

1	intestinal length is of great importance.Intestinal bypass procedures are sometimes required in the presence of intramesenteric abscesses or if the diseased bowel is coalesced in the form of a dense inflammatory mass, making its mobilization unsafe. Bypass procedures (gastrojejunostomy) are also used in the presence of duodenal strictures, for which stric-turoplasty and segmental resection can be technically difficult.Since the 1990s, laparoscopic surgical techniques have been applied to patients with Crohn’s disease. The inflamma-tory changes associated with Crohn’s disease such as thickened and for eshortened mesentery, obliterated tissue planes, and fri-able tissues with engorged vasculature can make laparoscopic approach challenging. Randomized studies and a meta-analysis have confirmed that laparoscopic surgery for Crohn’s disease is associated with less postoperative pain, shorter duration of ileus, and a shorter hospital stay. The rates of disease recurrence were similar

1	that laparoscopic surgery for Crohn’s disease is associated with less postoperative pain, shorter duration of ileus, and a shorter hospital stay. The rates of disease recurrence were similar between the two groups.45OutcomesOverall complication rates following surgery for Crohn’s dis-ease range from 15% to 30%. Wound infections, postoperative intra-abdominal abscesses, and anastomotic leaks account for most of these complications.Surgery is not a curative intervention in Crohn’s disease, and many patients develop recurrence. If recurrence is defined endoscopically, 70% recur within 1 year of a bowel resection and 85% by 3 years.46 Clinical recurrence, defined as the return of symptoms confirmed as being due to Crohn’s disease, affects 60% of patients by 5 years and 94% by 15 years after intestinal resection. Reoperation becomes necessary in approximately one-third of patients by 5 years after the initial operation, with a median time to reoperation of 7 to 10 years.47 Of

1	years after intestinal resection. Reoperation becomes necessary in approximately one-third of patients by 5 years after the initial operation, with a median time to reoperation of 7 to 10 years.47 Of patient-modifying factors, smoking is a strong risk factor for disease recurrence.INTESTINAL FISTULASA fistula is defined as an abnormal communication between two epithelialized surfaces. The communication occurs between two parts of the gastrointestinal tract or adjacent organs in an internal fistula (e.g., enterocolonic fistula or colovesicular fistula). An external fistula (e.g., enterocutaneous fistula or rectovaginal fistula) involves the skin or another external surface epithelium. Enterocutaneous fistulas that drain less than 200 mL of fluid per day are known as low-output fistulas, whereas those that drain more than 500 mL of fluid per day are known as high-output fistulas.Over 80% of enterocutaneous fistulas represent iatrogenic complications that occur as the result of

1	whereas those that drain more than 500 mL of fluid per day are known as high-output fistulas.Over 80% of enterocutaneous fistulas represent iatrogenic complications that occur as the result of enterotomies or intes-tinal anastomotic dehiscences. Fistulas that arise spontaneously without antecedent iatrogenic injury are usually manifestations of progression of underlying Crohn’s disease or cancer.PathophysiologyThe manifestations of fistulas depend on which structures are involved. Low-resistance enteroenteric fistulas, which allow luminal contents to bypass a significant proportion of the small intestine, may result in clinically-significant malabsorption. Enterovesicular fistulas often cause recurrent urinary tract infec-tions. The drainage emanating from enterocutaneous fistulas are irritating to the skin and cause excoriation. The loss of enteric luminal contents, particularly from high-output fistulas originat-ing from the proximal small intestine, results in dehydration,

1	are irritating to the skin and cause excoriation. The loss of enteric luminal contents, particularly from high-output fistulas originat-ing from the proximal small intestine, results in dehydration, electrolyte abnormalities, and malnutrition.Fistulas have the potential to close spontaneously. Factors inhibiting spontaneous closure, however, include malnutrition, sepsis, inflammatory bowel disease, cancer, radiation, obstruc-tion of the intestine distal to the origin of the fistula, foreign bodies, high output, short fistulous tract (<2 cm) and epitheli-alization of the fistula tract (Table 28-9).Clinical PresentationIatrogenic enterocutaneous fistulas usually become clinically evident between the fifth and tenth postoperative days. Fever, leukocytosis, prolonged ileus, abdominal tenderness, and wound infection are the initial signs. The diagnosis becomes obvious when drainage of enteric material through the abdominal wound or through existing drains occurs. These fistulas are often

1	and wound infection are the initial signs. The diagnosis becomes obvious when drainage of enteric material through the abdominal wound or through existing drains occurs. These fistulas are often asso-ciated with intra-abdominal abscesses.DiagnosisCT scanning following the administration of enteral contrast is the most useful initial test. Leakage of contrast material from the intestinal lumen can be observed. Intra-abdominal abscesses Table 28-9Factors negatively impacting enteric fistula closurePatient factors Poor nutrition Medications such as steroidsEtiological factors Malignant fistula Fistula related to Crohn’s disease Fistula in radiated fieldsFistula site Gastric DuodenalLocal Factors Persistence of local inflammation and sepsis Presence of a foreign body (e.g., meshes or sutures) Epithelialization of fistula tract Fistula tract <2 cm Distal obstruction to the fistula siteBrunicardi_Ch28_p1219-p1258.indd 124023/02/19 2:24 PM 1241SMALL INTESTINECHAPTER 28should be sought

1	of fistula tract Fistula tract <2 cm Distal obstruction to the fistula siteBrunicardi_Ch28_p1219-p1258.indd 124023/02/19 2:24 PM 1241SMALL INTESTINECHAPTER 28should be sought and drained percutaneously. If the anatomy of the fistula is not clear on CT scanning, a small bowel series or enteroclysis examination can be obtained to demonstrate the fistula’s site of origin in the bowel. This study is also useful to rule out the presence of intestinal obstruction distal to the site of origin. Occasionally, contrast administered into the intestine does not demonstrate the fistula tract. A fistulogram, in which contrast is injected under pressure through a catheter placed per-cutaneously into the fistula tract, may offer greater sensitivity in localizing the fistula origin.TherapyThe treatment of enterocutaneous fistulas should proceed through an orderly sequence of steps48:1. Stabilization. Fluid and electrolyte resuscitation is begun. Nutrition is provided, usually through the

1	treatment of enterocutaneous fistulas should proceed through an orderly sequence of steps48:1. Stabilization. Fluid and electrolyte resuscitation is begun. Nutrition is provided, usually through the parenteral route initially. Sepsis is controlled with antibiotics and drainage of abscesses. The skin is protected from the fistula effluent with ostomy appliances or fistula drains.2. Investigation. The anatomy of the fistula is defined using the aforementioned studies.3. Decision. The available treatment options are considered, and a time line for conservative measures is determined.4. Definitive Management. This entails the surgical procedure and requires appropriate preoperative planning and surgical experience.5. Rehabilitation.The overall objectives are to increase the probability of spontaneous closure. Nutrition and time are the key components of this approach. Most patients will require TPN; however, a trial of oral or enteral nutrition should be attempted in patients with

1	of spontaneous closure. Nutrition and time are the key components of this approach. Most patients will require TPN; however, a trial of oral or enteral nutrition should be attempted in patients with low-output fistulas originating from the distal intestine. The somatostatin analogue octreotide is a useful adjunct, par-ticularly in patients with high-output fistulas. A meta-analysis of several randomized studies confirmed that somatostatin treatment reduced length of hospital stay and time to closure of fistulas; however, its administration did not lead to a significant differ-ence in fistula closure rates.49 Use of negative pressure wound therapy has increased in management of enterocutaneous fistulas. The system can allow better management of the fistula output. In a study of 91 patients with enterocutaneous fistulas, 40% of fis-tulae reached minimal output within a week, and with an average follow-up of 90 days, spontaneous closure rate was 46%.50Timing of Surgical

1	of 91 patients with enterocutaneous fistulas, 40% of fis-tulae reached minimal output within a week, and with an average follow-up of 90 days, spontaneous closure rate was 46%.50Timing of Surgical Intervention. Most surgeons would pur-sue 2 to 3 months of conservative therapy before considering surgical intervention. This approach is based on evidence that 90% of fistulas that are going to close do so within 5 weeks and that surgical intervention after this period is associated with bet-ter outcomes and lower morbidity.51If the fistula fails to resolve during this period, surgery may be required, during which the fistula tract, together with the segment of intestine from which it originates, should be resected. Simple closure of the opening in the intestine from which the fistula originates is associated with high recurrence rates. Patients with intestinal fistulas typically have extensive and dense intra-abdominal adhesions. Thus, operations per-formed for nonhealing fistulas can

1	is associated with high recurrence rates. Patients with intestinal fistulas typically have extensive and dense intra-abdominal adhesions. Thus, operations per-formed for nonhealing fistulas can present formidable chal-lenges. Successful applications of alternative therapies to close intestinal fistulas such as the use of biologic sealants have been reported. The indications for their use remain to be defined.OutcomesOver 50% of intestinal fistulas close spontaneously. A useful mnemonic designates factors that inhibit spontaneous closure of intestinal fistulas: “FRIEND” (Foreign body within the fistula tract, Radiation enteritis, Infection/Inflammation at the fistula origin, Epithelialization of the fistula tract, Neoplasm at the fis-tula origin, Distal obstruction of the intestine).In a 23-year old retrospective review of 153 cases of enterocutaneous fistulas that were treated surgically, most fis-tulas were found to originate from the small bowel and be iat-rogenic in nature, with

1	23-year old retrospective review of 153 cases of enterocutaneous fistulas that were treated surgically, most fis-tulas were found to originate from the small bowel and be iat-rogenic in nature, with patients having undergone five or more previous abdominal surgeries. Operative repair was associated with a 30-day mortality of approximately 4% and a 1-year mor-tality of 15%. Morbidity was over 80%. First attempt at surgical repair was successful in 70% of cases, with an overall closure rate of 84% and some patients requiring up to 3 attempts at surgical repair. The authors identified closure of the abdominal fascia as an important factor in reducing rates of refistulization and postoperative mortality.52 In another similar study, fistula recurrence rates of 30% were documented and were indepen-dently associated with high output fistulas and the type of surgi-cal treatment: operations not involving resection of the fistula had a much higher rate of recurrence.53SMALL BOWEL

1	and were indepen-dently associated with high output fistulas and the type of surgi-cal treatment: operations not involving resection of the fistula had a much higher rate of recurrence.53SMALL BOWEL NEOPLASMSAdenomas are the most common benign neoplasm of the small intestine. Other benign tumors include fibromas, lipomas, hemangiomas, lymphangiomas, and neurofibromas. The prevalence of small bowel tumors identified at autopsy is 0.2% to 0.3%, which is significantly higher than the rate of operation for small bowel tumors. This suggests that majority of small bowel tumors are asymptomatic. These lesions are most frequently encountered in the duodenum as incidental findings during esophagogastroduodenoscopic (EGD) examinations (Fig. 28-20). The reported prevalence of Figure 28-20. Duodenal polyp. This polyp was incidentally encountered during EGD. It was biopsied and found to be an adenoma.Brunicardi_Ch28_p1219-p1258.indd 124123/02/19 2:24 PM 1242SPECIFIC

1	of Figure 28-20. Duodenal polyp. This polyp was incidentally encountered during EGD. It was biopsied and found to be an adenoma.Brunicardi_Ch28_p1219-p1258.indd 124123/02/19 2:24 PM 1242SPECIFIC CONSIDERATIONSPART IIduodenal polyps, as detected during EGD performed for other reasons, range from 0.3% to 4.6%.Benign neoplasms account for 30% to 50% of small bowel tumors and include adenomas, lipomas, hematomas, and hemangiomas. Primary small bowel cancers are rare but have been increasing in incidence, with an estimated incidence of 10,190 cases in 2017 in the United States. Among small bowel cancers, adenocarcinomas comprise 35% to 50% of all cases, carcinoid tumors comprise 20% to 40%, and lymphomas comprise approximately 10% to 15%. In a retrospective review of a large U.S. database (SEER) between 1992 and 2006, of a total number of 10,945 small intestine cancers, 4315 were neuroendocrine in origin, 3412 were carcinomas, 2023 were lymphomas, and 1084 were sarcomas.54

1	database (SEER) between 1992 and 2006, of a total number of 10,945 small intestine cancers, 4315 were neuroendocrine in origin, 3412 were carcinomas, 2023 were lymphomas, and 1084 were sarcomas.54 Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors arising in the small intestine and comprise the vast majority of tumors that were formerly classified as leiomyomas, leiomyosarcomas, and smooth muscle tumors of the intestine. The small intestine is frequently affected by metastases from or local invasion by cancers originating at other sites. Melanoma, in particular, is associated with a propensity for metastasis to the small intestine.Most patients with small-intestinal cancers are in their fifth or sixth decade of life. Reported risk factors for developing small-intestinal cancers include consumption of red meat, inges-tion of smoked or cured foods, Crohn’s disease, celiac sprue, hereditary nonpolyposis colorectal cancer (HNPCC), familial adenomatous polyposis

1	cancers include consumption of red meat, inges-tion of smoked or cured foods, Crohn’s disease, celiac sprue, hereditary nonpolyposis colorectal cancer (HNPCC), familial adenomatous polyposis (FAP), and Peutz-Jeghers syndrome.PathophysiologyThe small intestine contains over 90% of the mucosal surface area of the gastrointestinal tract but only 1.1% to 2.4% of all gastrointestinal malignancies. Proposed explanations for the low frequency of small-intestinal neoplasms include (a) dilu-tion of environmental carcinogens in the liquid chyme present in the small-intestinal lumen; (b) rapid transit of chyme, limiting the contact time between carcinogens and the intestinal mucosa; (c) a relatively low concentration of bacteria in small-intestinal chyme and, therefore, a relatively low concentration of carcino-genic products of bacterial metabolism; (d) mucosal protection by secretory IgA and hydrolases such as benzpyrene hydroxy-lase that may render carcinogens less active; and (e) efficient

1	of carcino-genic products of bacterial metabolism; (d) mucosal protection by secretory IgA and hydrolases such as benzpyrene hydroxy-lase that may render carcinogens less active; and (e) efficient epithelial cellular apoptotic mechanisms that serve to eliminate clones harboring genetic mutations.Recent advances have begun to clarify the molecular pathogenesis of small-intestinal adenocarcinomas and GISTs; there has been less progress with respect to the pathogen-esis of the other small-intestinal malignancies (Table 28-10). Small-intestinal adenocarcinomas are believed to arise from Table 28-10Features of small intestinal malignanciesTUMOR TYPECELL OF ORIGINFREQUENCYaPREDOMINANT SITEAdenocarcinomaEpithelial cell35–50%DuodenumCarcinoidEnterochromaffin cell20–40%IleumLymphomaLymphocyte10–15%IleumGISTInterstitial cell of Cajal10–15%–aFrequencies given as percentages of small intestinal malignancies comprised by each of the tumor types. Gastrointestinal stromal tumors (GISTs) display no

1	cell of Cajal10–15%–aFrequencies given as percentages of small intestinal malignancies comprised by each of the tumor types. Gastrointestinal stromal tumors (GISTs) display no regional variation in prevalence within the small intestine.preexisting adenomas through a sequential accumulation of genetic abnormalities in a model like that described for the pathogenesis of colorectal cancer. Adenomas are histologi-cally classified as tubular, villous, and tubulovillous. Tubular adenomas have the least aggressive features. Villous adenomas have the most aggressive features and tend to be large, sessile, and located in the second portion of the duodenum. Malignant degeneration has been reported to be present in up to 45% of vil-lous adenomas by the time of diagnosis. Patients with FAP have a nearly 100% cumulative lifetime risk of developing duodenal adenomas that have the potential to undergo malignant trans-formation. The risk of duodenal cancer in these patients is over 100-fold greater

1	100% cumulative lifetime risk of developing duodenal adenomas that have the potential to undergo malignant trans-formation. The risk of duodenal cancer in these patients is over 100-fold greater than in the general population. Indeed, duode-nal cancer is the leading cause of cancer-related death among patients with FAP who have undergone colectomy. Patients with Peutz-Jeghers syndrome develop hamartomatous polyps; however, these polyps can contain adenomatous foci that can undergo malignant transformation (Fig. 28-21).A defining feature of GISTs is their gain of function mutation of proto-oncogene KIT, a receptor tyrosine kinase. Figure 28-21. Small bowel polyp in Peutz-Jeghers syndrome. This image was captured by a wireless capsule endoscope as it was traveling through the small intestine. (Used with permission from Anne T. Wolf, M.D., Department of Medicine, Brigham and Women’s Hospital, Boston, MA.)Brunicardi_Ch28_p1219-p1258.indd 124223/02/19 2:24 PM 1243SMALL INTESTINECHAPTER

1	(Used with permission from Anne T. Wolf, M.D., Department of Medicine, Brigham and Women’s Hospital, Boston, MA.)Brunicardi_Ch28_p1219-p1258.indd 124223/02/19 2:24 PM 1243SMALL INTESTINECHAPTER 28Pathological KIT signal transduction is believed to be a cen-tral event in GIST pathogenesis. The majority of GISTs have activating mutations in the c-kit proto-oncogene, which cause KIT to become constitutively activated, presumably leading to persistence of cellular growth or survival signals. Because the interstitial cells of Cajal normally express KIT, these cells have been implicated as the cell of origin for GISTs. KIT expression is assessed by staining the tissues for CD117 antigen, which is part of the KIT receptor and is present in 95% of GISTs.Clinical PresentationMost small-intestinal neoplasms are asymptomatic until they become large. Partial small bowel obstruction, with associ-ated symptoms of crampy abdominal pain and distention, nau-sea, and vomiting, is the most common

1	neoplasms are asymptomatic until they become large. Partial small bowel obstruction, with associ-ated symptoms of crampy abdominal pain and distention, nau-sea, and vomiting, is the most common mode of presentation. Obstruction can be the result of either luminal narrowing by the tumor itself or intussusception, with the tumor serving as the lead point. Hemorrhage, usually indolent, is the second most common mode of presentation.Physical examination may be unrevealing, but it also may reveal a palpable abdominal mass in those with large tumors. Findings of intestinal obstruction may be present in some patients. A fecal occult blood test may be positive. Jaundice secondary to biliary obstruction or hepatic metastasis may be present. Cachexia, hepatomegaly, and ascites may be present with advanced disease.Although the clinical presentation is usually not specific for tumor type, some general comments are appropriate. Adeno-carcinomas, as well as adenomas (from which most are believed to

1	disease.Although the clinical presentation is usually not specific for tumor type, some general comments are appropriate. Adeno-carcinomas, as well as adenomas (from which most are believed to arise), are most commonly found in the duodenum, except in patients with Crohn’s disease, in whom most are found in the ileum. Lesions in the periampullary location can cause obstruc-tive jaundice or pancreatitis. Adenocarcinomas located in the duodenum tend to be diagnosed earlier in their progression than those located in the jejunum or ileum, which are rarely diagnosed prior to the onset of locally advanced or metastatic disease.Carcinoid tumors of the small intestine are also usually diagnosed after the development of metastatic disease. These tumors are associated with a more aggressive behavior than the more common appendiceal carcinoid tumors. Approximately 25% to 50% of patients with carcinoid tumor-derived liver metastases will develop manifestations of the carcinoid syn-drome. These

1	than the more common appendiceal carcinoid tumors. Approximately 25% to 50% of patients with carcinoid tumor-derived liver metastases will develop manifestations of the carcinoid syn-drome. These manifestations include diarrhea, flushing, hypo-tension, tachycardia, and fibrosis of the endocardium and valves the right heart. Candidate tumor-derived mediators of the car-cinoid syndrome such as serotonin, bradykinin, and substance P undergo nearly complete metabolism during the first passage through the liver. As a result, symptoms of carcinoid syndrome are rare in the absence of liver metastases.Lymphoma may involve the small intestine primarily or as a manifestation of disseminated systemic disease. Primary small-intestinal lymphomas are most commonly located in the ileum, which contains the highest concentration of lymphoid tissue in the intestine. Although partial small bowel obstruction is the most common mode of presentation, 10% of patients with small-intestinal lymphoma present

1	the highest concentration of lymphoid tissue in the intestine. Although partial small bowel obstruction is the most common mode of presentation, 10% of patients with small-intestinal lymphoma present with bowel perforation.The small intestine is the second most common site of GIST tumors after the stomach, containing 25% to 35% of GISTs. There appears to be no regional variation in the preva-lence of GISTs within the small intestine. GISTs have a greater propensity to be associated with overt hemorrhage than the other small-intestinal malignancies (Fig. 28-22).Metastatic tumors involving the small intestine can induce intestinal obstruction and bleeding.DiagnosisBecause of the absent or nonspecific symptoms associated with most small-intestinal neoplasms, these lesions are rarely diagnosed preoperatively. Laboratory tests are nonspecific, except for elevated urinary 5-hydroxyindole acetic acid (5-HIAA) and serum chromogranin A (CgA) levels in patients with carcinoid syndrome, both of

1	preoperatively. Laboratory tests are nonspecific, except for elevated urinary 5-hydroxyindole acetic acid (5-HIAA) and serum chromogranin A (CgA) levels in patients with carcinoid syndrome, both of which have low sensitivity. Elevated carcinoembryonic antigen (CEA) levels are associated with small-intestinal adenocarcinomas, but only in the presence of liver metastases.Contrast radiography of the small intestine may demon-strate benign and malignant lesions. Enterocolysis is reported to have a sensitivity of over 90% in the detection of small bowel tumors and is the test of choice, particularly for tumors located in the distal small bowel. Upper GI with small bowel follow-through examinations have reported sensitivities ranging from only 30% to 44% (Fig. 28-23). CT scanning can detect abnor-malities in 70% to 80% of cases with small bowel tumor and assess for metastatic spread. Tumors associated with significant bleeding can be localized with angiography or radioisotope-tagged red

1	abnor-malities in 70% to 80% of cases with small bowel tumor and assess for metastatic spread. Tumors associated with significant bleeding can be localized with angiography or radioisotope-tagged red blood cell (RBC) scans.Tumors located in the duodenum can be visualized and biopsied on EGD. In addition, endoscopic ultrasonography (EUS) can offer additional information such as the layers of the intestinal wall involved by the lesion. Occasionally, the distal ileum can suc-cessfully be visualized during colonoscopy. Intraoperative enter-oscopy can be used to directly visualize small-intestinal tumors beyond the reach of standard endoscopic techniques. For more distal small bowel lesions, capsule endoscopy and double-balloon Figure 28-22. Jejunal gastrointestinal stromal tumor (GIST). This patient presented with overt obscure GI bleeding and was found to have a 7-cm jejunal GIST. The picture represents the laparo-scopic view of the mass (black arrow), arising from the antimesenteric

1	patient presented with overt obscure GI bleeding and was found to have a 7-cm jejunal GIST. The picture represents the laparo-scopic view of the mass (black arrow), arising from the antimesenteric side of the small bowel (*). He underwent a successful laparoscopic resection.Brunicardi_Ch28_p1219-p1258.indd 124323/02/19 2:24 PM 1244SPECIFIC CONSIDERATIONSPART IIFigure 28-23. Small bowel lesion identified during a small bowel follow-through (arrow). This patient had previously had a metastatic renal lesion to his duodenum requiring a Whipple procedure. During follow-up imaging 3 years later, he was found to have this new lesion in his jejunum. He underwent a laparoscopic small bowel resection. Pathology revealed a benign jejunal sessile polyp.endoscopy have been used to evaluate small bowel. CT and MR enterography are also increasing utilized as noninvasive tests to look for small bowel masses. PET scans can also help assess metabolic activity of lesions and risk of

1	evaluate small bowel. CT and MR enterography are also increasing utilized as noninvasive tests to look for small bowel masses. PET scans can also help assess metabolic activity of lesions and risk of malignancy.TherapyBenign neoplasms of the small intestine that are symptomatic should be surgically resected or removed endoscopically, if fea-sible. Tumors located in the duodenum, including asymptomatic lesions incidentally found during EGD, can pose the great-est therapeutic challenges. These lesions should be biopsied; symptomatic tumors and adenomas, because of their malignant potential, should be removed. In general, duodenal tumors less than 1 cm in diameter are amenable to endoscopic polypectomy. Lesions greater than 2 cm in diameter are technically difficult to remove endoscopically and may need to be removed surgi-cally. Surgical options include transduodenal polypectomy and segmental duodenal resection. Tumors located in the second portion of the duodenum near the ampulla of

1	and may need to be removed surgi-cally. Surgical options include transduodenal polypectomy and segmental duodenal resection. Tumors located in the second portion of the duodenum near the ampulla of Vater may require pancreaticoduodenectomy. EUS may offer utility for duodenal tumors ranging in size between 1 and 2 cm in diameter, with those limited to the mucosa being amenable to endoscopic pol-ypectomy. Endoscopic resection of biopsy-proven benign duo-denal periampullary adenomas leads to equivalent efficacy to surgery but with lower morbidity. Adenomas can recur; there-fore, surveillance endoscopy is required after these procedures.55Duodenal adenomas occurring in the setting of FAP require an especially aggressive approach to management. Patients with FAP should undergo screening EGD starting sometime during their second or third decade of life. Adenomas detected should be removed endoscopically, if possible, followed by surveillance endoscopy in 6 months and yearly thereafter, in

1	sometime during their second or third decade of life. Adenomas detected should be removed endoscopically, if possible, followed by surveillance endoscopy in 6 months and yearly thereafter, in the absence of recurrence. If surgery is required, pancreaticoduodenectomy is generally necessary because adenomas in patients with FAP tend to be multiple and sessile, with a predilection for the peri-ampullary region. Further, localized resections are complicated by high recurrence rates. Given the potential for recurrences in the duodenal remnant following pylorus-preserving pancreati-coduodenectomy, there is rationale for recommending the appli-cation of standard pancreaticoduodenectomy in these patients. However, recurrences have been reported even following this procedure; therefore, continuing surveillance is necessary. For most adenocarcinomas of the duodenum, except those in the third or fourth portion of the duodenum where a local resection could be considered, pancreaticoduodenectomy

1	surveillance is necessary. For most adenocarcinomas of the duodenum, except those in the third or fourth portion of the duodenum where a local resection could be considered, pancreaticoduodenectomy is required.The surgical therapy of jejunal and ileal malignancies usually consists of wide-local resection of the intestine harboring the lesion. For adenocarcinomas, a wide excision of corresponding mesentery is done to achieve regional lymphadenectomy, as is done for adenocarcinomas of the colon. In the presence of locally-advanced or metastatic disease, palliative intestinal resection or bypass is performed. Chemotherapy has no proven efficacy in the adjuvant or palliative treatment of small-intestinal adenocarcinomas.The goal of surgical therapy for carcinoids is resection of all visible disease. Localized small-intestinal carcinoid tumors should be treated with segmental intestinal resection and regional lymphadenectomy. Nodal metastases are unusual with tumors less than 1 cm in

1	disease. Localized small-intestinal carcinoid tumors should be treated with segmental intestinal resection and regional lymphadenectomy. Nodal metastases are unusual with tumors less than 1 cm in diameter, but they are present with 75% to 90% of tumors larger than 3 cm in diameter. In approximately 30% of cases, multiple small-intestinal carcinoid tumors are present (Fig. 28-24). Therefore, the entire small intestine should Figure 28-24. Small bowel carcinoid tumor. This patient pre-sented with history of abdominal pain and on CT was found to have a circumferentially thickened loop of distal small bowel with associated mesenteric stranding and lymphadenopathy. An octreotide scan demonstrated abnormal activity in the area, concerning for a carcinoid tumor. He underwent an open small bowel resection. Pathology revealed a multifocal carcinoid tumor with 50 distinct nodules and metastasis to mesenteric lymph nodes.Brunicardi_Ch28_p1219-p1258.indd 124423/02/19 2:24 PM 1245SMALL

1	bowel resection. Pathology revealed a multifocal carcinoid tumor with 50 distinct nodules and metastasis to mesenteric lymph nodes.Brunicardi_Ch28_p1219-p1258.indd 124423/02/19 2:24 PM 1245SMALL INTESTINECHAPTER 28be examined before planning extent of resection. In the presence of metastatic disease, tumor debulking should be conducted as it can be associated with long-term survival and amelioration of symptoms of the carcinoid syndrome. Response rates of 30% to 50% have been reported to chemotherapy regimens based on agents such as doxorubicin, 5-fluorouracil, and streptozocin. However, none of these regimens is associated with a clearly demonstrable impact on the natural history of disease. Octreo-tide is the most effective pharmacologic agent for management of symptoms of carcinoid syndrome.Localized small-intestinal lymphoma should be treated with segmental resection of the involved intestine and adjacent mesentery. If the small intestine is diffusely affected by lym-phoma,

1	syndrome.Localized small-intestinal lymphoma should be treated with segmental resection of the involved intestine and adjacent mesentery. If the small intestine is diffusely affected by lym-phoma, chemotherapy rather than surgical resection should be the primary therapy. The value to adjuvant chemotherapy after resection of localized lymphoma is controversial.Small-intestinal GISTs should be treated with segmental intestinal resection. If the diagnosis is known prior to resec-tion, wide lymphadenectomy can be avoided as GISTs are rarely associated with lymph node metastases. GISTs are resistant to conventional chemotherapy agents. Imatinib (Gleevec) is a tyrosine kinase inhibitor with potent activity against tyrosine kinase KIT, and it is used in those with metastatic disease. Clini-cal trials have shown that 80% of patients with unresectable or metastatic GISTs derive clinical benefit from the administra-tion of Imatinib, with 50% to 60% having objective evidence of reduction in

1	trials have shown that 80% of patients with unresectable or metastatic GISTs derive clinical benefit from the administra-tion of Imatinib, with 50% to 60% having objective evidence of reduction in tumor volume.56 Imatinib has shown great promise as a neoadjuvant and adjuvant therapy for GISTs. Studies have emphasized the potential for development of tumor resistance to this agent. In this setting, an alternative tyrosine kinas inhibitor, sunitinib, has been used with good results.Metastatic cancers affecting the small intestine that are symptomatic should be treated with palliative resection or bypass except in the most advanced cases. Systemic therapy may be offered if effective chemotherapy exists for the primary cancer.OutcomesComplete resection of duodenal adenocarcinomas is associ-ated with postoperative 5-year survival rates ranging from 50% to 60%. Complete resection of adenocarcinomas located in the jejunum or ileum is associated with 5-year survival rates of 20% to 30%.57

1	with postoperative 5-year survival rates ranging from 50% to 60%. Complete resection of adenocarcinomas located in the jejunum or ileum is associated with 5-year survival rates of 20% to 30%.57 Five-year survival rates of 75% to 95% fol-lowing resection of localized small-intestinal carcinoid tumors have been reported. In the presence of carcinoid tumor-derived liver metastases, 5-year survival rates of 19% to 54% have been reported. The overall 5-year survival rate for patients diagnosed with intestinal lymphoma ranges from 20% to 40%. For patients with localized lymphoma amenable to surgical resection, the 5-year survival rate is 60%.The recurrence rate following resection of GISTs aver-ages 35%. The 5-year survival rate following surgical resection has been reported to range from 35% to 60%. Both tumor size and mitotic index are independently correlated with prognosis. Low-grade tumors (mitotic index <10 per high-power field) measuring less than 5 cm in diameter are associated with

1	60%. Both tumor size and mitotic index are independently correlated with prognosis. Low-grade tumors (mitotic index <10 per high-power field) measuring less than 5 cm in diameter are associated with excel-lent prognosis.RADIATION ENTERITISRadiation therapy is a component of multi-modality therapy for many intra-abdominal and pelvic cancers such as those of the cervix, endometrium, ovary, bladder, prostate, and rectum. An undesired side effect of radiation therapy is radiation-induced injury to the small intestine, which can present clinically as two distinct syndromes: acute and chronic radiation enteritis. Acute radiation enteritis is a transient condition that occurs in approximately 75% of patients undergoing radiation therapy for abdominal and pelvic cancers. Chronic radiation is enteritis is inexorable and develops in approximately 5% to 15% of these patients.PathophysiologyRadiation induces cellular injury directly and through the gen-eration of free radicals. The principal

1	enteritis is inexorable and develops in approximately 5% to 15% of these patients.PathophysiologyRadiation induces cellular injury directly and through the gen-eration of free radicals. The principal mechanism of radiation-induced cell death is believed to be apoptosis resulting from free-radical–induced breaks in double-stranded DNA. Because radiation has its greatest impact on rapidly proliferating cells, the small-intestinal epithelium is acutely susceptible to radiationinduced injury. Pathological correlates of this acute injury include villus blunting and a dense infiltrate of leukocytes and plasma cells within the crypts. With severe cases, mucosal sloughing, ulceration, and hemorrhage are observed. The inten-sity of injury is related to the dose of radiation administered, with most cases occurring in patients who have received at least 4500 cGy. Risk factors for acute radiation enteritis include con-ditions that may limit splanchnic perfusion such as hyperten-sion, diabetes

1	cases occurring in patients who have received at least 4500 cGy. Risk factors for acute radiation enteritis include con-ditions that may limit splanchnic perfusion such as hyperten-sion, diabetes mellitus, coronary artery disease, and restricted mobility of the small intestine due to adhesions. Injury is potentiated by concomitant administration of chemotherapeutic agents, such as doxorubicin, 5-fluorouracil, actinomycin D, and methotrexate, that act as radiation-sensitizers. Because of the intestinal epithelium’s capacity for regeneration, the mucosal injury that is characteristic of acute radiation enteritis resolves after the cessation of radiation therapy.In contrast, chronic radiation enteritis is characterized by a progressive occlusive vasculitis that leads to chronic ischemia and fibrosis that affects all layers of the intestinal wall, rather than the mucosa alone. These changes can lead to strictures, abscesses, and fistulas, which are responsible for the clinical

1	and fibrosis that affects all layers of the intestinal wall, rather than the mucosa alone. These changes can lead to strictures, abscesses, and fistulas, which are responsible for the clinical manifestations of chronic radiation enteritis.Clinical PresentationThe most common manifestations of acute radiation enteritis are nausea, vomiting, diarrhea, and crampy abdominal pain. Symptoms are generally transient and subside after the discon-tinuation of radiation therapy. Because the diagnosis is usually obvious, given the clinical context, no specific diagnostic tests are required. However, if patients develop signs suggestive of peritonitis, CT scanning should be performed to rule out the presence of other conditions capable of causing acute abdominal syndromes.The clinical manifestations of chronic radiation enteritis usually become evident within 2 years of radiation administra-tion, although they can begin as early as several months or as late as decades afterwards. The most common

1	of chronic radiation enteritis usually become evident within 2 years of radiation administra-tion, although they can begin as early as several months or as late as decades afterwards. The most common clinical presenta-tions are diarrhea or one of partial small bowel obstruction with nausea, vomiting, intermittent abdominal distention, crampy abdominal pain, and weight loss. The terminal ileum is the most frequently affected segment. Other manifestations of chronic radiation enteritis include complete bowel obstruction, acute or chronic intestinal hemorrhage, and abscess or fistula formation.DiagnosisEvaluation of patients suspected of having chronic radia-tion enteritis should include review of the records of their Brunicardi_Ch28_p1219-p1258.indd 124523/02/19 2:24 PM 1246SPECIFIC CONSIDERATIONSPART IIradiation treatments for information on total radiation dose administered, fractionation, and volume of treatment. Areas that received high doses should be noted, as lesions

1	CONSIDERATIONSPART IIradiation treatments for information on total radiation dose administered, fractionation, and volume of treatment. Areas that received high doses should be noted, as lesions subse-quently found in imaging studies usually localize to areas that had received high radiation doses. Enterocolysis is the most accurate imaging test for diagnosing chronic radiation enteri-tis, with reported sensitivities and specificities of over 90% (Fig. 28-25). CT scan findings are neither very sensitive nor specific for chronic radiation enteritis. However, CT scanning should be obtained to rule out the presence of recurrent can-cer since its clinical manifestations may overlap with those of chronic radiation enteritis.TherapyMost cases of acute radiation enteritis are self-limited. Support-ive therapy, including the administration of antiemetics, is usu-ally sufficient. Patients with diarrhea-induced dehydration may require hospital admission and parenteral fluid administration.

1	therapy, including the administration of antiemetics, is usu-ally sufficient. Patients with diarrhea-induced dehydration may require hospital admission and parenteral fluid administration. Rarely are symptoms severe enough to necessitate reduction in or cessation of radiation therapy.In contrast, the treatment of chronic radiation enteritis represents a formidable challenge. Antidiarrheal agents may have a role in the management of diarrhea while, in those with obstructive symptoms, a low residue diet may be tried. Surgery for this condition is difficult, is associated with high morbidity rates, and should be avoided in the absence of specific indica-tions such as high-grade obstruction, perforation, hemorrhage, intra-abdominal abscesses, and fistulas. The goal of surgery is limited resection of diseased intestine with primary anastomosis Figure 28-25. Radiation enteritis. This contrast radiograph reveals widely separated loops of small bowel with luminal narrowing, loss of mucosal

1	of diseased intestine with primary anastomosis Figure 28-25. Radiation enteritis. This contrast radiograph reveals widely separated loops of small bowel with luminal narrowing, loss of mucosal folds, and ulceration. This patient had received radiation therapy for a pelvic malignancy 8 years before this examination.between healthy bowel segments. However, the characteristi-cally diffuse nature of fibrosis and dense adhesions among bowel segments can make limited resection difficult to achieve. Further, it is difficult to distinguish between normal and irra-diated intestine intraoperatively by either gross inspection or even frozen section analysis. This distinction is important as anastomoses between irradiated segments of intestine have been associated with leak rates as high as 50%.58 If limited resec-tion is not achievable, an intestinal bypass procedure may be an option, except in cases for which hemorrhage is the surgical indication. There remain cases in which resections

1	If limited resec-tion is not achievable, an intestinal bypass procedure may be an option, except in cases for which hemorrhage is the surgical indication. There remain cases in which resections extensive enough to cause short bowel syndrome are unavoidable. This condition is discussed in detail below in the “Short Bowel Syn-drome” section.OutcomesAcute radiation injury to the intestine is self-limited; its sever-ity is not correlated with the probability of chronic radiation enteritis developing. Surgery for chronic radiation enteritis is associated with high morbidity rates and reported mortality rates averaging 10%.PreventionIn view of significant morbidity associated with radiation enteri-tis, groups have studies possible measures to reduce or prevent such side effects. Keeping radiation exposure to below 5000 cGy is associated with minimal long-term side effects and is recom-mended where clinically possible.Uses of multibeam radiation techniques to minimize the area of maximal

1	exposure to below 5000 cGy is associated with minimal long-term side effects and is recom-mended where clinically possible.Uses of multibeam radiation techniques to minimize the area of maximal radiation exposure, as well as tilt tables to move the bowel out of the pelvic during radiation, are increasingly utilized. Few small studies have suggested that oral sulphasala-zine may help reduce the incidence of acute radiation-induced enteritis.59In patients undergoing pelvic surgery that are likely to require postoperative radiation therapy, surgical techniques that keep the small bowel out of the pelvic have been recommended. These measures include use of absorbable mesh sling to sepa-rate the pelvic from the true abdominal cavity and prevent the small bowel from being exposed to pelvic radiation.60MECKEL’S DIVERTICULAMeckel’s diverticulum is the most prevalent congenital anom-aly of the gastrointestinal tract, affecting approximately 2% of the general population. Meckel’s diverticuli

1	DIVERTICULAMeckel’s diverticulum is the most prevalent congenital anom-aly of the gastrointestinal tract, affecting approximately 2% of the general population. Meckel’s diverticuli are designated true diverticuli because their walls contain all the layers found in normal small intestine. Their location varies among individual patients, but they are usually found in the ileum within 100 cm of the ileocecal valve (Fig. 28-26). Approximately 60% of Meckel’s diverticuli contain heterotopic mucosa, of which over 60% consist of gastric mucosa. Pancreatic acini are the next most common; others include Brunner’s glands, pancre-atic islets, colonic mucosa, endometriosis, and hepatobiliary tissues. A useful, although crude, mnemonic describing Meck-el’s diverticuli is the “rule of twos”: 2% prevalence, 2:1 male predominance, location 2 feet proximal to the ileocecal valve in adults, and half of those who are symptomatic are under 2 years of age.PathophysiologyDuring the eighth week of

1	prevalence, 2:1 male predominance, location 2 feet proximal to the ileocecal valve in adults, and half of those who are symptomatic are under 2 years of age.PathophysiologyDuring the eighth week of gestation, the omphalomesenteric (vitelline) duct normally undergoes obliteration. Failure or Brunicardi_Ch28_p1219-p1258.indd 124623/02/19 2:24 PM 1247SMALL INTESTINECHAPTER 28Figure 28-26. Meckel’s diverticulum. This intraoperative photograph shows Meckel’s diverticulum in ileum that has been eviscerated.incomplete vitelline duct obliteration results in a spectrum of abnormalities, the most common of which is Meckel’s divertic-ulum. Other abnormalities include omphalomesenteric fistula, enterocyst, and a fibrous band connecting the intestine to the umbilicus. A remnant of the left vitelline artery can persist to form a mesodiverticular band tethering a Meckel’s diverticulum to the ileal mesentery.Bleeding associated with Meckel’s diverticulum is usu-ally the result of ileal mucosal

1	artery can persist to form a mesodiverticular band tethering a Meckel’s diverticulum to the ileal mesentery.Bleeding associated with Meckel’s diverticulum is usu-ally the result of ileal mucosal ulceration that occurs adjacent to acid-producing, heterotopic gastric mucosa located within the diverticulum. Intestinal obstruction associated with Meckel’s diverticulum can result from several mechanisms:1. Volvulus of the intestine around the fibrous band attaching the diverticulum to the umbilicus2. Entrapment of intestine by a mesodiverticular band (Fig. 28-27)3. Intussusception with the diverticulum acting as a lead point4. Stricture secondary to chronic diverticulitisMeckel’s diverticuli can be found in inguinal or femoral hernia sacs (known as Littre’s hernia). These hernias, when incarcerated, can cause intestinal obstruction.Figure 28-27. Meckel’s diverticulum with mesodiverticular band (A). One mechanism by which Meckel’s diverticuli can cause small bowel obstruction is entrapment

1	can cause intestinal obstruction.Figure 28-27. Meckel’s diverticulum with mesodiverticular band (A). One mechanism by which Meckel’s diverticuli can cause small bowel obstruction is entrapment of the intestine by a mesodiverticular band (B).Clinical PresentationMeckel’s diverticuli are asymptomatic unless associated com-plications arise. The lifetime incidence rate of complications arising in patients with Meckel’s diverticuli has been estimated to be approximately 4% to 6%.61,62 Although initial data had suggested that the risk of developing a complication related to Meckel’s diverticulum decreases with age, this has been ques-tioned. In a population-based reviews at Olmsted County, Cullen and colleagues suggested that the risk of developing Meckel’s diverticulum–related complications does not change with age.62The most common presentations associated with symp-tomatic Meckel’s diverticuli are bleeding, intestinal obstruction, and diverticulitis. Bleeding is the most common

1	does not change with age.62The most common presentations associated with symp-tomatic Meckel’s diverticuli are bleeding, intestinal obstruction, and diverticulitis. Bleeding is the most common presentation in children with Meckel’s diverticuli, representing over 50% of Meckel’s diverticulum-related complications among patients less than 18 years of age. Bleeding associated with Meckel’s diverticuli is rare among patients older than 30 years of age.Intestinal obstruction is the most common presentation in adults with Meckel’s diverticuli. Diverticulitis, present in 20% of patients with symptomatic Meckel’s diverticuli, is associated with a clinical syndrome that is indistinguishable from acute appendicitis. Neoplasms, most commonly carcinoid tumors, are present in 0.5% to 3.2% of symptomatic Meckel’s diverticuli that are resected.DiagnosisMost Meckel’s diverticuli are discovered incidentally on radio-graphic imaging, during endoscopy, or at the time of surgery. In the absence of

1	Meckel’s diverticuli that are resected.DiagnosisMost Meckel’s diverticuli are discovered incidentally on radio-graphic imaging, during endoscopy, or at the time of surgery. In the absence of bleeding, Meckel’s diverticuli rarely are diag-nosed prior to the time of surgical intervention. For those pre-senting with symptoms suggestive of a Meckel’s diverticulum, confirmatory imaging can be challenging. The sensitivity of CT scanning for the detection of Meckel’s diverticuli is too low to be clinically useful. Enterocolysis is associated with an accuracy of 75% but is usually not applicable during acute presentations of complications related to Meckel’s diverticuli. Radionuclide scans (99MTc–pertechnetate) can be helpful in the diagnosis of Meckel’s diverticulum; this test is, however, positive only when the diverticulum contains associated ectopic gastric mucosa that is capable of uptake of the tracer (Fig. 28-28). The accuracy of radionuclide scanning is reported to be 90% in pediatric

1	only when the diverticulum contains associated ectopic gastric mucosa that is capable of uptake of the tracer (Fig. 28-28). The accuracy of radionuclide scanning is reported to be 90% in pediatric patients but less than 50% in adults. Angiography can localize the site of bleeding during acute hemorrhage related to Meckel’s diverticuli.Brunicardi_Ch28_p1219-p1258.indd 124723/02/19 2:24 PM 1248SPECIFIC CONSIDERATIONSPART IIFigure 28-28. Meckel’s diverticulum with ectopic gastric tissue. The diagnosis was made in this patient using 99MTc–pertechnetate scintig-raphy. The study revealed an abnormal focus of radiotracer accumulation in the right lower quadrant (arrow).TherapyThe surgical treatment of symptomatic Meckel’s diverticuli should consist of diverticulectomy with removal of associated bands connecting the diverticulum to the abdominal wall or intestinal mesentery. If the indication for diverticulectomy is bleeding, segmental resection of ileum that includes both the

1	of associated bands connecting the diverticulum to the abdominal wall or intestinal mesentery. If the indication for diverticulectomy is bleeding, segmental resection of ileum that includes both the diverticulum and the adjacent ileal peptic ulcer should be per-formed. Segmental ileal resection may also be necessary if the diverticulum contains a tumor or if the base of the diverticulum is inflamed or perforated.The management of incidentally found (asymptomatic) Meckel’s diverticuli is controversial. Until recently, most authors recommended against prophylactic removal of asymptomatic Meckel’s diverticuli, given the low lifetime incidence of com-plications. Supporting this approach, a meta-analysis has shown that 758 prophylactic diverticulectomies needed to be performed to prevent one Meckel’s-related death.63 Others have had greater enthusiasm for prophylactic diverticulectomy has appeared in the literature.64 Proponents of this approach cite the minimal mor-bidity associated with

1	death.63 Others have had greater enthusiasm for prophylactic diverticulectomy has appeared in the literature.64 Proponents of this approach cite the minimal mor-bidity associated with removing Meckel’s diverticuli and the possibility that previous estimates of the lifetime incidence of complications related to Meckel’s diverticuli may be erroneously low. Many have advocated a selective approach, with a recom-mendation to remove diverticuli in patients younger than 50 years of age, or those with band attachments, those with ectopic tissue, or those >2 cm in length on the assumption that these diverticuli are more likely to develop complications. No controlled data sup-porting or refuting these recommendations exist.ACQUIRED DIVERTICULAAcquired diverticuli are designated false diverticuli because their walls consist of mucosa and submucosa but lack a com-plete muscularis. Acquired diverticuli are more common in the duodenum, and tend to be located near the ampulla; such diverticuli are

1	their walls consist of mucosa and submucosa but lack a com-plete muscularis. Acquired diverticuli are more common in the duodenum, and tend to be located near the ampulla; such diverticuli are known as periampullary, juxtapapillary, and perivaterian diverticuli. Approximately 75% of juxtapapillary diverticuli arise on the medial wall of the duodenum. Acquired diverticuli in the jejunum or ileum are known as jejunoileal diverticuli. Eighty percent of jejunoileal diverticuli are localized to the jejunum, 15% to the ileum, and 5% to both jejunum and ileum. Diverticuli in the jejunum tend to be large and accom-panied by multiple other diverticuli, whereas those in the ileum tend to be small and solitary.The prevalence of duodenal diverticuli, as detected on upper GI examinations (Fig. 28-29), has been reported to range from 0.16% to 6%.65 Their prevalence, as detected during ERCP examinations, has been reported to range from 5% to 27%. A 23% prevalence rate has been reported in an autopsy

1	has been reported to range from 0.16% to 6%.65 Their prevalence, as detected during ERCP examinations, has been reported to range from 5% to 27%. A 23% prevalence rate has been reported in an autopsy series. The prevalence of duodenal diverticuli increases with age; they are Brunicardi_Ch28_p1219-p1258.indd 124823/02/19 2:24 PM 1249SMALL INTESTINECHAPTER 28Figure 28-29. Duodenal diverticulum. This contrast radiograph demonstrates a duodenal diverticulum (arrows) that extends medially into the substance of the head of the pancreas.Figure 28-30. Jejunoileal diverticuli. This picture demonstrates incidental jejunal diverticuli identified during a laparoscopic cholecystectomy. The diverticuli are typically located on the mesenteric aspect of the jejunum. Resection was not indicated as the diverticuli were asymptomatic.rare in patients under the age of 40 years. The mean age of diagnosis ranges from 56 to 76 years.The prevalence of jejunoileal diverticuli (Fig. 28-30) has been

1	as the diverticuli were asymptomatic.rare in patients under the age of 40 years. The mean age of diagnosis ranges from 56 to 76 years.The prevalence of jejunoileal diverticuli (Fig. 28-30) has been estimated to range from 1% to 5%.66 Their prevalence increases with age; most patients diagnosed with these diver-ticuli are in the sixth and seventh decades of life.PathophysiologyThe pathogenesis of acquired diverticuli is hypothesized to be related to acquired abnormalities of intestinal smooth muscle or dysregulated motility, leading to herniation of mucosa and submucosa through weakened areas of muscularis.Acquired diverticuli can be associated with bacterial over-growth, leading to vitamin B12 deficiency, megaloblastic anemia, malabsorption, and steatorrhea. Periampullary duodenal diver-ticuli have been described to become distended with intralumi-nal debris and to compress the common bile duct or pancreatic duct, thus causing obstructive jaundice or pancreatitis, respec-tively.

1	have been described to become distended with intralumi-nal debris and to compress the common bile duct or pancreatic duct, thus causing obstructive jaundice or pancreatitis, respec-tively. Jejunoileal diverticuli can also cause intestinal obstruction through intussusception or compression of adjacent bowel.Brunicardi_Ch28_p1219-p1258.indd 124923/02/19 2:25 PM 1250SPECIFIC CONSIDERATIONSPART IIClinical PresentationAcquired diverticuli are asymptomatic unless associated com-plications arise. Such complications are estimated to occur in 6% to 10% of patients with acquired diverticuli and include intestinal obstruction, diverticulitis, hemorrhage, perforation, and malabsorption. Periampullary duodenal diverticuli may be associated with choledocholithiasis, cholangitis, recurrent pan-creatitis, and sphincter of Oddi dysfunction. However, a clear link between the presence of the diverticuli and the development of these conditions has not been demonstrated. Symptoms such as intermittent

1	and sphincter of Oddi dysfunction. However, a clear link between the presence of the diverticuli and the development of these conditions has not been demonstrated. Symptoms such as intermittent abdominal pain, flatulence, diarrhea, and consti-pation are reported to be present in 10% to 30% of patients with jejunoileal diverticuli. The relationship between these symp-toms and the presence of the diverticuli is similarly unclear.DiagnosisMost acquired diverticuli are discovered incidentally on radiographic imaging, during endoscopy, or at the time of surgery. On ultrasound and CT scanning, duodenal diverticuli may be mistaken for pancreatic pseudocysts and fluid collections, biliary cysts, and periampullary neoplasms. These lesions can be missed on endoscopy, particularly with forward-viewing endoscopes, and are best diagnosed on upper gastrointestinal radiographs. Enterocolysis is the most sensitive test for detecting jejunoileal diverticuli.TherapyAsymptomatic-acquired diverticuli

1	endoscopes, and are best diagnosed on upper gastrointestinal radiographs. Enterocolysis is the most sensitive test for detecting jejunoileal diverticuli.TherapyAsymptomatic-acquired diverticuli should be left alone. Bacte-rial overgrowth associated with acquired diverticuli is treated with antibiotics. Other complications, such as bleeding and diverticulitis, are treated with segmental intestinal resection for diverticuli located in the jejunum or ileum.Bleeding and obstruction related to lateral duodenal diverticuli are generally treated with diverticulectomy alone. Treatment of such complications in medial duodenal diverticuli that penetrate the substance of the pancreas can be very challenging. Complications related to these medial duodenal diverticuli should be managed nonoperatively if possible, using endoscopy. In emergent situations, bleeding related to medial duodenal diverticuli can be controlled using a lateral duodenotomy and oversewing of the bleeding vessel. Similarly,

1	if possible, using endoscopy. In emergent situations, bleeding related to medial duodenal diverticuli can be controlled using a lateral duodenotomy and oversewing of the bleeding vessel. Similarly, perforation can be managed with wide drainage rather than complex surgery. Whether diverticulectomy should be done in patients with biliary or pancreatic symptoms is controversial and is not routinely recommended.MESENTERIC ISCHEMIAMesenteric ischemia can present as one of two distinct clinical syndromes: acute mesenteric ischemia and chronic mesenteric ischemia.Four distinct pathophysiologic mechanisms can lead to acute mesenteric ischemia:1. Arterial embolus2. Arterial thrombosis3. Vasospasm (also known as nonocclusive mesenteric isch-emia or NOMI)4. Venous thrombosisEmbolus is the most common cause of acute mesen-teric ischemia and is responsible for over 50% of cases. The embolic source is usually in the heart, most often the left atrial or ventricular thrombi or valvular lesions.

1	common cause of acute mesen-teric ischemia and is responsible for over 50% of cases. The embolic source is usually in the heart, most often the left atrial or ventricular thrombi or valvular lesions. Indeed, up to 95% of patients with acute mesenteric ischemia due to emboli will have a documented history of cardiac disease. Embolism to the superior mesenteric artery accounts for 50% of cases; most of these emboli become wedged and cause occlusion at branch points in the midto distal superior mesenteric artery, usually distal to the origin of the middle colic artery. In contrast, acute occlusions due to thrombosis tend to occur in the proximal mes-enteric arteries, near their origins. Acute thrombosis is usually superimposed on preexisting atherosclerotic lesions at these sites. NOMI is the result of vasospasm and is usually diagnosed in critically ill patients receiving vasopressor agents.Mesenteric venous thrombosis accounts for 5% to 15% of cases of acute mesenteric ischemia and

1	the result of vasospasm and is usually diagnosed in critically ill patients receiving vasopressor agents.Mesenteric venous thrombosis accounts for 5% to 15% of cases of acute mesenteric ischemia and involves the supe-rior mesenteric vein in 95% of cases.67 The inferior mesenteric vein is only rarely involved. Mesenteric venous thrombosis is classified as primary if no etiologic factor is identifiable, or as secondary if an etiologic factor, such as heritable or acquired coagulation disorders, is identified.Regardless of the pathophysiologic mechanism, acute mes-enteric ischemia can lead to intestinal mucosal sloughing within 3 hours of onset and full-thickness intestinal infarction by 6 hours.In contrast, chronic mesenteric ischemia develops insidi-ously, allowing for development of collateral circulation, and, therefore, rarely leads to intestinal infarction. Chronic mesen-teric arterial ischemia results from atherosclerotic lesions in the main splanchnic arteries (celiac, superior

1	circulation, and, therefore, rarely leads to intestinal infarction. Chronic mesen-teric arterial ischemia results from atherosclerotic lesions in the main splanchnic arteries (celiac, superior mesenteric, and inferior mesenteric arteries). In most patients with symptoms attributable to chronic mesenteric ischemia, at least two of these arteries are either occluded or severely stenosed. A chronic form of mesenteric venous thrombosis can involve the portal or splenic veins and may lead to portal hypertension, with result-ing esophagogastric varices, splenomegaly, and hypersplenism.Severe abdominal pain, out of proportion to the degree of tenderness on examination is the hallmark of acute mesenteric ischemia, regardless of the pathophysiologic mechanism. The pain is typically perceived to be colicky and most severe in the mid-abdomen. Associated symptoms can include nausea, vomiting, and diarrhea. Physical findings are characteristically absent early in the course of ischemia. With the

1	colicky and most severe in the mid-abdomen. Associated symptoms can include nausea, vomiting, and diarrhea. Physical findings are characteristically absent early in the course of ischemia. With the onset of bowel infarction, abdominal distension, peritonitis, and passage of bloody stools occur.Chronic mesenteric ischemia presents insidiously. Post-prandial abdominal pain is the most prevalent symptom, produc-ing a characteristic aversion to food (“food fear”) and weight loss. These patients are often thought to have a malignancy and suffer a prolonged period of symptoms before the correct diag-nosis is made.Most patients with chronic mesenteric venous thrombosis are asymptomatic because of the presence of extensive collat-eral venous drainage routes; this condition is usually discovered as an incidental finding on imaging studies. However, some patients with chronic mesenteric venous thrombosis present with bleeding from esophagogastric varices.The diagnosis and management of these

1	as an incidental finding on imaging studies. However, some patients with chronic mesenteric venous thrombosis present with bleeding from esophagogastric varices.The diagnosis and management of these disorders, which are of primary vascular origin, are discussed in the section on “Mesen-teric Artery Occlusive Disease” in Chapter 23 “Arterial Disease.”MISCELLANEOUS CONDITIONSObscure GI BleedingUp to 90% of lesions responsible for GI bleeding are within the reach of EGD and colonoscopy. Obscure GI bleeding refers to gastrointestinal bleeding for which no source has been identified by routine endoscopic studies (EGD and colonoscopy). Overt Brunicardi_Ch28_p1219-p1258.indd 125023/02/19 2:25 PM 1251SMALL INTESTINECHAPTER 28GI bleeding refers to the presence of hematemesis, melena, or hematochezia. In contrast, occult GI bleeding occurs in the absence of overt bleeding and is identified on laboratory tests (e.g., iron-deficiency anemia) or examination of the stool (e.g., positive guaiac

1	In contrast, occult GI bleeding occurs in the absence of overt bleeding and is identified on laboratory tests (e.g., iron-deficiency anemia) or examination of the stool (e.g., positive guaiac test). Obscure GI bleeding is occult in 20% of cases.68Obscure bleeding can be frustrating for both the patient and the clinician, and this is particularly true for obscure-overt bleeding, which cannot be localized despite aggressive diag-nostic measures. Most of the small bowel is beyond the reach of these examinations and, hence, contains most lesions respon-sible for obscure GI bleeding. Small intestinal angiodysplasias account for approximately 75% of cases in adults, and neo-plasms account for approximately 10%. Meckel’s diverticulum is the most common etiology of obscure GI bleeding in children. Other etiologies of obscure GI bleeding include Crohn’s dis-ease, infectious enteritides, nonsteroidal anti-inflammatory drug (NSAID)-induced ulcers and erosions, vasculitis, ischemia, var-ices,

1	Other etiologies of obscure GI bleeding include Crohn’s dis-ease, infectious enteritides, nonsteroidal anti-inflammatory drug (NSAID)-induced ulcers and erosions, vasculitis, ischemia, var-ices, diverticuli, and intussusception.The diagnostic evaluation of patients with obscure GI bleeding should be tailored to the severity to bleeding and to the availability of technology and expertise. Enteroscopy is playing an increasingly important role. Several endoscopic techniques for visualizing the small intestine are available: push enteros-copy, sonde enteroscopy, intraoperative enteroscopy, double balloon endoscopy, and wireless capsule enteroscopy.Push enteroscopy entails advancing a long endoscope (such as a pediatric or adult colonoscope or a specialized instru-ment) beyond the ligament of Treitz into the proximal jejunum. This procedure can allow for visualization of approximately 60 cm of the proximal jejunum. Diagnostic yield in patients with obscure GI bleeding ranges from 3% to

1	Treitz into the proximal jejunum. This procedure can allow for visualization of approximately 60 cm of the proximal jejunum. Diagnostic yield in patients with obscure GI bleeding ranges from 3% to 65%. In addition to diagnosis, push enteroscopy allows for cauterization of bleed-ing sites.In Sonde enteroscopy, a long, thin fiberoptic instrument is propelled through the intestine by peristalsis following infla-tion of a balloon at the instrument’s tip. Visualization is done during instrument withdrawal; approximately 50% to 75% of the small intestinal mucosa can be examined. However, this instrument lacks biopsy or therapeutic capability. Further, it lacks tip deflection capability, limiting complete mucosal visu-alization, and has therefore been abandoned in favor of capsule endoscopy.Wireless capsule enteroscopy is an excellent tool in the patient who is hemodynamically stable but continues to bleed. This technique has reported success rates as high as 90% in identifying a small

1	capsule enteroscopy is an excellent tool in the patient who is hemodynamically stable but continues to bleed. This technique has reported success rates as high as 90% in identifying a small bowel pathology. In a randomized study of patients with obscure GI bleeding, evaluation with capsule endoscopy vs. small bowel contrast study had a much higher diagnostic yield (30% vs. 7%, respectively); however, this did not translate into an improvement in outcomes. The rates of rebleeding, hospitalization, need for blood transfusion, and therapeutic interventions were similar between the two arms.The inability to perform biopsies or carry out any thera-peutic interventions of capsule endoscope likely prevents the improved diagnostic yield of the test from translating into improved patient outcomes and highlights the continuing chal-lenge with evaluation of the small bowel.69For patients in whom bleeding from an obscure GI source has apparently stopped, push enteroscopy or capsule enteroscopy is

1	highlights the continuing chal-lenge with evaluation of the small bowel.69For patients in whom bleeding from an obscure GI source has apparently stopped, push enteroscopy or capsule enteroscopy is a reasonable initial study. If these examinations do not reveal a potential source of bleeding, then enterocolysis should be performed. Standard small bowel follow-through examinations are associated with a low diagnostic yield in this setting and should be avoided. 99MTc–pertechnetate scintigraphy to diagnose Meckel’s diverticulum should be considered, although its yield in patients older than 40 years of age is extremely low. If still no diagnosis has been made, a “watch-and-wait” approach is reasonable, although angiography should be considered if the episode of bleeding was overt. Angiography can reveal angiodysplasia and vascular tumors in the small intestine even in the absence of ongoing bleeding. In many instances, however, angiography is done in patients with persistent bleeding

1	can reveal angiodysplasia and vascular tumors in the small intestine even in the absence of ongoing bleeding. In many instances, however, angiography is done in patients with persistent bleeding from an obscure GI source, and it is also often performed after a 99MTc-labeled RBC scan, which, if positive, is followed by angiography to localize the source of bleeding. Patients who remain undiagnosed but continue to bleed and those with recurrent episodic bleeding significant enough to require blood transfusions should then undergo exploratory laparoscopy or laparotomy with intraoperative enteroscopy. An endoscope (usually a colonoscope) is inserted into the small bowel through peroral intubation or through an enterotomy made in the small bowel or cecum. The endoscope is advanced by successively telescoping short segments of intestine onto the end to the instrument. In addition to the endoscopic image, the transilluminated bowel should be examined externally with the operating room

1	telescoping short segments of intestine onto the end to the instrument. In addition to the endoscopic image, the transilluminated bowel should be examined externally with the operating room lights dimmed, as this maneuver may facilitate the identification of angiodysplasias. Identified lesions should be marked with a suture placed on the serosal surface of the bowel; these lesions can be resected after completion of endoscopy. Examination should be performed during instrument insertion rather than withdrawal because instrument-induced mucosal trauma can be confused with angiodysplasias.Figure 28-31 provides a diagnostic and management algo-rithm for patients with obscure GI bleeding.Small Bowel PerforationPrior to the 1980s, duodenal perforation due to peptic ulcer dis-ease was the most common form of small bowel perforation. Today, iatrogenic injury incurred during gastrointestinal endos-copy is the most common cause of small bowel perforation. Other etiologies of small bowel

1	most common form of small bowel perforation. Today, iatrogenic injury incurred during gastrointestinal endos-copy is the most common cause of small bowel perforation. Other etiologies of small bowel perforation include infections (especially tuberculosis, typhoid, and CMV), Crohn’s disease, ischemia, drugs (e.g., potassiumand NSAID-induced ulcers), radiation-induced injury, Meckel’s and acquired diverticuli, neoplasms (especially lymphoma, adenocarcinoma, and mela-noma), and foreign bodies.Among iatrogenic injuries, duodenal perforation dur-ing endoscopic retrograde cholangiography (ERCP) with endoscopic sphincterotomy (ES) is the most common. With improved technique and technology, the incidence of this is decreasing but remains at around 0.5%.70 The Stapfer classifi-cation is commonly used to categorize different types of ERCP-related perforations. These are:• Type I: Free bowel wall perforation• Type II: Retroperitoneal duodenal perforation secondary to periampullary injury• Type

1	used to categorize different types of ERCP-related perforations. These are:• Type I: Free bowel wall perforation• Type II: Retroperitoneal duodenal perforation secondary to periampullary injury• Type III: Perforation of the pancreatic or bile duct• Type IV: Retroperitoneal air aloneType II (retroperitoneal duodenal injuries) are the most common and can often be managed nonsurgically. Manifesta-tions of such contained duodenal perforation following ERCP can resemble those of ERCP-induced pancreatitis, including hyperamylasemia.Brunicardi_Ch28_p1219-p1258.indd 125123/02/19 2:25 PM 1252SPECIFIC CONSIDERATIONSPART IICT scanning is the most sensitive test for diagnosing duo-denal perforations; positive findings include pneumoperitoneum for free perforations, but more commonly retroperitoneal air, contrast extravasation, and paraduodenal fluid collections. If all patients undergoing a therapeutic ERCP are imaged with a CT scan following the procedure, up to 30% will have evidence of

1	air, contrast extravasation, and paraduodenal fluid collections. If all patients undergoing a therapeutic ERCP are imaged with a CT scan following the procedure, up to 30% will have evidence of air in the retroperitoneum, but the majority are asymptomatic. These patients do not require any specific therapy.71True cases of retroperitoneal perforations of the duodenum can be managed nonoperatively in the absence of progression and sepsis. However, intraoperitoneal duodenal perforations require surgical repair with pyloric exclusion and gastrojeju-nostomy or tube duodenostomy. Iatrogenic small bowel perfora-tion incurred during endoscopy, if immediately recognized, can sometimes be repaired using endoscopic techniques.Perforation of the jejunum and ileum occurs into the peri-toneal cavity and usually causes overt symptoms and signs, such as abdominal pain, tenderness, and distention accompa-nied by fever and tachycardia. Plain abdominal radiographs may reveal free intraperitoneal air if

1	usually causes overt symptoms and signs, such as abdominal pain, tenderness, and distention accompa-nied by fever and tachycardia. Plain abdominal radiographs may reveal free intraperitoneal air if intraperitoneal perforation has occurred. If perforation is suspected but not clinically obvious, CT scanning should be performed. Jejunal and ileal perforations require surgical repair or segmental resection.Chylous AscitesChylous ascites refers to the accumulation of triglyceride-rich peritoneal fluid with a milky or creamy appearing, caused by the presence of intestinal lymph in the peritoneal cavity. Chy-lomicrons, produced by the intestine and secreted into lymph during the absorption of long-chain fatty acids, account for the characteristic appearance and triglyceride content of chyle.The most common etiologies of chylous ascites in Western countries are abdominal malignancies and cirrhosis. In Eastern and developing countries, infectious etiologies, such as tuber-culosis and

1	most common etiologies of chylous ascites in Western countries are abdominal malignancies and cirrhosis. In Eastern and developing countries, infectious etiologies, such as tuber-culosis and filariasis, account for most cases. Chylous ascites can also develop as a complication of abdominal and thoracic operations and trauma. Operations particularly associated with this complication include abdominal aortic aneurysm repair, retroperitoneal lymph node dissection, inferior vena cava resec-tion, and liver transplantation. Other etiologies of chylous asci-tes include congenital lymphatic abnormalities (e.g., primary lymphatic hypoplasia), radiation, pancreatitis, and right-sided heart failure.Three mechanisms have been postulated to cause chylous ascites: (a) exudation of chyle from dilated lymphatics on the wall of the bowel and in the mesentery caused by obstruction of lymphatic vessels at the base of the mesentery or the cisterna chili (e.g., by malignancies), (b) direct leakage of

1	lymphatics on the wall of the bowel and in the mesentery caused by obstruction of lymphatic vessels at the base of the mesentery or the cisterna chili (e.g., by malignancies), (b) direct leakage of chyle through a lymphoperitoneal fistula (e.g., those that develop as a result of trauma or surgery), and (c) exudation of chyle through the wall of dilated retroperitoneal lymphatic vessels (e.g., in congenital lymphangiectasia or thoracic duct obstruction).Patients with chylous ascites develop abdominal distention over a period of weeks to months. Postoperative chylous ascites can present acutely during the first postoperative week. Delayed presentations following surgery can occur if the mechanism of ascites formation is adhesion-induced lymphatic obstruction rather than lymphatic vessel disruption.Obscure gastrointestinal bleedingRule out upper and lower GIbleeding;EGD and colonoscopyMinor bleeding(intermittent)Major bleeding(persistent)Initiate appropriatetherapyRepeat EGD/Colonoscopy

1	gastrointestinal bleedingRule out upper and lower GIbleeding;EGD and colonoscopyMinor bleeding(intermittent)Major bleeding(persistent)Initiate appropriatetherapyRepeat EGD/Colonoscopy ifrebleedsTreat source, e.g.,small bowelresectionLocalize bleeding:Serial clamping or intraoperativeenteroscopy followed by resectionPositivePositiveNegativeUnstableStableTaggedRBC scanNegativeandpatient stableSource ofbleeding identifiedSourceuncertainAngiographyand treatmentOperatingroomEnteroclysisEnteroscopyCapsuleendoscopyFigure 28-31. Diagnostic and management algorithm for obscure gastrointestinal bleeding.Brunicardi_Ch28_p1219-p1258.indd 125223/02/19 2:25 PM 1253SMALL INTESTINECHAPTER 28Paracentesis is the most important diagnostic test. Chyle typically has a cloudy and turbid appearance; however, it may be clear in fasting patients (such as those in the immediate postoperative period). Fluid triglyceride concentrations above 110 mg/dL are diagnostic. CT scanning may be useful in identifying

1	it may be clear in fasting patients (such as those in the immediate postoperative period). Fluid triglyceride concentrations above 110 mg/dL are diagnostic. CT scanning may be useful in identifying pathological intraabdominal lymph nodes and masses and in identifying extent and localization of fluid. Lymphangiography and lymphoscintigraphy may help localize lymph leaks and obstruction; this information is particularly useful for surgical planning.There is little data on optimal management of patients with chylous ascites. The general approach is to focus on evaluating and treating the underlying causes, especially for patients with infec-tious, inflammatory, or hemodynamic etiologies for this condition.Most patients respond to administration of a high-protein and low-fat diet supplemented with medium-chain triglycerides. This regimen is designed to minimize chyle production and flow. Medium-chain triglycerides are absorbed by the intestinal epithelium and are transported to liver

1	with medium-chain triglycerides. This regimen is designed to minimize chyle production and flow. Medium-chain triglycerides are absorbed by the intestinal epithelium and are transported to liver through the portal vein; they do not contribute to chylomicron formation.Patients who do not respond to this approach should be fasted and placed on TPN. Octreotide can further decrease lymph flow. Paracentesis is indicated for respiratory difficul-ties related to abdominal distention. Overall, two-thirds of patients will respond to conservative therapy. However, one-third of patients will require surgical therapy for chylous ascites. In general, postoperative and trauma-related cases that fail to respond to initial nonoperative therapy are best managed by surgical repair. Lymphatic leaks are localized and repaired with fine nonabsorbable sutures. If extravasation of chyle is localized to the periphery of the small bowel mesentery, then a limited small bowel resection can be performed

1	are localized and repaired with fine nonabsorbable sutures. If extravasation of chyle is localized to the periphery of the small bowel mesentery, then a limited small bowel resection can be performed instead. For patients who are poor surgical candidates and who do not respond to prolonged conservative therapy, peritoneovenous shunting may be an option. However, these shunts are associated with high rates of complications, including sepsis and disseminated intra-vascular coagulation. Because of the viscosity of chyle, these shunts are associated with a high occlusion rate.IntussusceptionIntussusception refers to a condition where one segment of the intestine becomes drawn in to the lumen of the distal segment of the bowel. It is usually seen in the pediatric population, where the ileum intussuscepts into the cecum (ileocolic intussuscep-tion). In children, it is often an idiopathic condition and treated nonsurgically by radiological reduction.Intussusceptions are far less common and

1	into the cecum (ileocolic intussuscep-tion). In children, it is often an idiopathic condition and treated nonsurgically by radiological reduction.Intussusceptions are far less common and usually have a distinct pathologic lead point, which can be malignant in up to one-half of cases.72 They commonly present with a history of intermittent abdominal pain and signs and symptoms of bowel obstruction. CT scan is the investigation of choice, where a “tar-get sign” may be seen (Fig. 28-32). Treatment is surgical resec-tion of the involved segment and the lead point, which needs to undergo pathological evaluation to rule out an underlying malignancy.With increasing use of CT imaging, target signs are some-times seen on CT scans of patients who do not have a clinical presentation indicative of bowel obstruction. In such cases, the finding is of little clinical significance and is probably related to normal peristalsis.In patients who have undergone a Roux-en-Y gastric bypass surgery, an

1	of bowel obstruction. In such cases, the finding is of little clinical significance and is probably related to normal peristalsis.In patients who have undergone a Roux-en-Y gastric bypass surgery, an atypical form of intussusception has been increasingly described. In these cases, the distal bowel is drawn in to the lumen of the proximal bowel (retrograde intussuscep-tion). These intussusceptions are usually not associated with a lead point and may represent a motility disorder of the bowel following the Roux-en-Y reconstruction. Surgical reductions without resection have been successfully reported in these patients.73Pneumatosis IntestinalisPneumatosis intestinalis indicates the presence of gas within the bowel wall. It may affect any region of the GI tract, but it is most commonly seen in the jejunum. Pneumatosis intestinalis is not a disease but merely a sign that can be idiopathic or associated with many intestinal or nonintestinal disorders, such as obstruc-tive pulmonary disease

1	the jejunum. Pneumatosis intestinalis is not a disease but merely a sign that can be idiopathic or associated with many intestinal or nonintestinal disorders, such as obstruc-tive pulmonary disease and asthma. Most cases of pneumatosis intestinalis are secondary to an identifiable cause, and 15% are idiopathic. The pathogenesis of pneumatosis intestinalis is not fully understood.The surgical interest in this finding is the association of it with bowel ischemia and infarction, both of which necessitate emergent surgical intervention (Fig. 28-33). Thus, patients with this radiological finding need to be fully evaluated and moni-tored closely to rule out such intraabdominal catastrophes.ABFigure 28-32. Small bowel intussusception. A. Target sign seen on CT scans in patients with small bowel intussusception (arrow). B. The distal bowel is clearly within the lumen of the proximal bowel (arrow).Brunicardi_Ch28_p1219-p1258.indd 125323/02/19 2:25 PM 1254SPECIFIC

1	with small bowel intussusception (arrow). B. The distal bowel is clearly within the lumen of the proximal bowel (arrow).Brunicardi_Ch28_p1219-p1258.indd 125323/02/19 2:25 PM 1254SPECIFIC CONSIDERATIONSPART IISHORT BOWEL SYNDROMEIntestinal resection is performed for many of the diseases dis-cussed in this chapter and generally is associated with minimal morbidity. However, when extent of resection is great enough, a devastating condition known as short bowel syndrome may result. Although the best definition of short bowel syndrome is likely a functional one, reflecting a state of significant malabsorption of both macronutrients and micronutrients, some have used a more anatomical definition with it being arbi-trarily defined as the presence of less than 200 cm of residual small bowel in adult patients.74In adults, the most common etiologies of short bowel syn-drome are acute mesenteric ischemia, malignancy, and Crohn’s disease. Seventy-five percent of cases result from resection

1	in adult patients.74In adults, the most common etiologies of short bowel syn-drome are acute mesenteric ischemia, malignancy, and Crohn’s disease. Seventy-five percent of cases result from resection of a large amount of small bowel at a single operation. Twenty-five percent of cases result from the cumulative effects of multiple operations during which small intestine is resected. This latter pattern is typical of patients with Crohn’s disease who develop short bowel syndrome; the former is typical of patients with acute mesenteric ischemia who develop intestinal infarction. In pediat-ric patients, intestinal atresias, volvulus, and necrotizing entero-colitis are the most common etiologies of short bowel syndrome.The prevalence of short bowel syndrome is hard to esti-mate due to its multifactorial nature and wide spectrum of presentation and treatment, which may include home total par-enteral nutrition (TPN).PathophysiologyResection of less than 50% of the small intestine is generally

1	nature and wide spectrum of presentation and treatment, which may include home total par-enteral nutrition (TPN).PathophysiologyResection of less than 50% of the small intestine is generally well tolerated. However, clinically significant malabsorption occurs when greater than 50% to 80% of the small intestine has been resected. Among adult patients who lack a functional colon, lifelong TPN dependence is likely to persist if there is less than 100 cm of residual small intestine. Among adult patients who have an intact and functional colon, lifelong TPN dependence is likely to persist if there is less than 60 cm of residual small intestine. Among infants with short bowel syn-drome, weaning from TPN-dependence has been achieved with as little as 10 cm of residual small intestine.Residual bowel length is not the only factor predictive of achieving independence from TPN (enteral autonomy), 5Figure 28-33. STEP procedure. This illustration depicts the serial transverse enteroplasty (STEP)

1	length is not the only factor predictive of achieving independence from TPN (enteral autonomy), 5Figure 28-33. STEP procedure. This illustration depicts the serial transverse enteroplasty (STEP) procedure. Lengthening of dilated small intestine is accomplished by serial applications of an intestinal stapling device, with firings oriented perpendicular to the long axis of the intestine. (Used with permission from Patrick Javid, M.D. and Tom Jaksic, M.D., Department of Surgery, Children’s Hospital, Boston, MA.)Table 28-11Risk factors for development of short bowel syndrome after massive small bowel resectionSmall bowel length <200 cmAbsence of ileocecal valveAbsence of colonDiseased remaining bowel (e.g., Crohn’s disease)Ileal resectionhowever. Other determinants of the severity of malabsorption include the presence or absence of an intact colon, as indicated previously. The colon has the capacity to absorb large fluid and electrolyte loads. In addition, the colon can play an important,

1	include the presence or absence of an intact colon, as indicated previously. The colon has the capacity to absorb large fluid and electrolyte loads. In addition, the colon can play an important, albeit small, role in nutrient assimilation by absorbing short chain fatty acids. Second, an intact ileocecal valve is believed to be associated with decreased malabsorption. The ileocecal valve delays transit of chyme from the small intestine into the colon, thereby prolonging the contact time between nutrients and the small-intestinal absorptive mucosa. Third, healthy, rather than diseased, residual small intestine is associated with decreased severity of malabsorption. Fourth, resection of jejunum is better tolerated than resection of ileum, as the capacity for bile salt and vitamin B12 absorption is specific to the ileum (Table 28-11).During the first 1 to 2 years following massive small bowel resection, the remaining intestine undergoes compensa-tory adaptation, as discussed previously.

1	is specific to the ileum (Table 28-11).During the first 1 to 2 years following massive small bowel resection, the remaining intestine undergoes compensa-tory adaptation, as discussed previously. Clinically, the period of adaptation is associated with reductions in volume and fre-quency of bowel movements, increases in the capacity for enteral nutrient assimilation, and reductions in TPN requirements. As this process completes, some patients are successfully weaned off TPN. Understanding the mechanisms mediating intestinal adaptation may suggest strategies for enhancing adaptation in patients with short bowel syndrome who are unable to achieve independence from TPN. To date, the phenomenon of intestinal adaptation in patients remains poorly understood.13Malabsorption in patients who have undergone mas-sive small bowel resection is exacerbated by a characteristic hypergastrinemia-associated gastric acid hypersecretion that persists for 1 to 2 years postoperatively. The increased acid

1	undergone mas-sive small bowel resection is exacerbated by a characteristic hypergastrinemia-associated gastric acid hypersecretion that persists for 1 to 2 years postoperatively. The increased acid Brunicardi_Ch28_p1219-p1258.indd 125423/02/19 2:25 PM 1255SMALL INTESTINECHAPTER 28load delivered to the duodenum inhibits absorption by a variety of mechanisms, including the inhibition of digestive enzymes, most of which function optimally under alkaline conditions.TherapyMedical Therapy. For patients after massive small bowel resection, the initial treatment priorities include management of the primary condition precipitating the intestinal resection and the repletion of fluid and electrolytes lost in the severe diarrhea that characteristically occurs. Most patients will require TPN, at least initially. Enteral nutrition should be gradually introduced, once ileus has resolved. High-dose histamine-2 receptor antago-nists or proton pump inhibitors should be administered to reduce

1	at least initially. Enteral nutrition should be gradually introduced, once ileus has resolved. High-dose histamine-2 receptor antago-nists or proton pump inhibitors should be administered to reduce gastric acid secretion. Antimotility agents, such as loperamide hydrochloride or diphenoxylate, may be administered to delay small-intestinal transit. Octreotide can be administered to reduce the volume of gastrointestinal secretions, although, in animal models, its use is associated with an inhibition of intestinal adaptation.During the period of adaptation, generally lasting 1 to 2 years postoperatively, TPN and enteral nutrition are titrated to allow for independence from TPN. Patients who remain dependent on TPN face substantial TPN-associated morbidities including catheter sepsis, venous thrombosis, liver and kidney failure, and osteoporosis. Liver failure is a significant source of morbidity and often leads to liver transplantation (always in combination with small bowel

1	venous thrombosis, liver and kidney failure, and osteoporosis. Liver failure is a significant source of morbidity and often leads to liver transplantation (always in combination with small bowel transplantation). Due to these complications, patients with short bowel syndrome on TPN have a reduced life expectancy, with 5-year survival rates of 50% to 75%.Nontransplant Surgical Therapy. Among patients with sto-mas, restoration of intestinal continuity should be performed whenever possible, to capitalize on the absorptive capacity of all residual intestine. Other forms of nontransplant surgery designed to improve intestinal absorption are associated with unclear efficacy and/or substantial morbidities and therefore should not be applied routinely.The goal of these operations is to increase nutrient and fluid absorption by either slowing intestinal transit or increasing intestinal length. Operations designed to slow intestinal transit include segmental reversal of the small bowel,

1	nutrient and fluid absorption by either slowing intestinal transit or increasing intestinal length. Operations designed to slow intestinal transit include segmental reversal of the small bowel, interposition of a segment of colon between segments of small bowel, construc-tion of small-intestinal valves, and electrical pacing of the small intestine. Reported experience with these procedures is limited to case reports or series of a few cases. Objective evidence of increased absorption is lacking; further, these procedures are frequently associated with intestinal obstruction.The intestinal lengthening operation for which has the lon-gest history is the longitudinal intestinal lengthening and tailor-ing (LILT) procedure, first described by Bianchi in 1980.75 The procedure entails separation of the dual vasculature of the small intestine, followed by longitudinal division of the bowel with subsequent isoperistaltic end-to-end anastomosis. This proce-dure has the potential to double the

1	of the dual vasculature of the small intestine, followed by longitudinal division of the bowel with subsequent isoperistaltic end-to-end anastomosis. This proce-dure has the potential to double the length of small intestine to which it is applied. This procedure has generally been used for pediatric patients with dilated residual small bowel.An alternative surgical approach to lengthening the small bowel is the serial transverse enteroplasty procedure (STEP) has been described. This procedure is designed to accom-plish lengthening of dilated small intestine without the need for separating its dual vasculature (Fig. 28-33). A report from an international registry of 111 patients showed that 47% of patients achieved enteral autonomy at a median follow-up of 21 months.76Intestinal Transplantation. This complex procedure is being increasingly performed to treat patients with short bowel syn-drome. The currently accepted indication for intestinal trans-plantation is the presence of

1	complex procedure is being increasingly performed to treat patients with short bowel syn-drome. The currently accepted indication for intestinal trans-plantation is the presence of life-threatening complications attributable to intestinal failure and/or long-term TPN therapy. Specific complications for which intestinal transplantation is indicated include (a) impending or overt liver failure, (b) throm-bosis of major central veins, (c) frequent episodes of catheter-related sepsis, and (d) frequent episodes of severe dehydration.Of the transplants involving the intestine, 37% were intestine-alone transplants, 30% included intestine, liver, and pancreas, and 24% were intestine and liver.77Isolated intestinal transplantation is used for patients with intestinal failure who have no significant liver disease or failure of other organs. Combined intestine/liver transplantation is used for patients with both intestinal and liver failure. Multivisceral transplantation has been used for

1	liver disease or failure of other organs. Combined intestine/liver transplantation is used for patients with both intestinal and liver failure. Multivisceral transplantation has been used for patients with giant desmoid tumors involving the vascular supply of the liver and pancreas as well as that of the intestine, for diffuse gastrointestinal motil-ity disturbances, and for diffuse splanchnic thrombosis.Nearly 80% of survivors have full intestinal graft function with no need for TPN. However, morbidities associated with intestinal transplantation are substantial and include acute and chronic rejection, CMV infection, and posttransplant lymphop-roliferative disease.Alternative Therapies. Pharmacologic and biologic thera-pies designed to expand intestinal mucosal surface area or to enhance the efficiency of intestinal absorption are beginning to undergo clinical evaluation. Promising regimens include GLP-2 and the combination of glutamine and growth hormone with a modified,

1	to enhance the efficiency of intestinal absorption are beginning to undergo clinical evaluation. Promising regimens include GLP-2 and the combination of glutamine and growth hormone with a modified, high-carbohydrate diet.OutcomesApproximately 50% to 70% of patients with short bowel syn-drome who initially require TPN are ultimately able to achieve independence from TPN.73 Prognosis for achieving enteral autonomy is better among pediatric patients than among adults.Information on survival among patients with short bowel syndrome is limited. In a recently reported study of 124 adults with short bowel syndrome due to nonmalignant etiologies, the survival rates at 2 and 5 years of follow up were 86% and 45%, respectively.77 Patients with end-enterostomies and those hav-ing less than 50 cm of residual small intestine had significantly worse survivals than those without these features.No randomized trials comparing intestinal transplanta-tion to chronic TPN administration among patients

1	of residual small intestine had significantly worse survivals than those without these features.No randomized trials comparing intestinal transplanta-tion to chronic TPN administration among patients with short bowel syndrome have been reported. One-, 5-, and 10-year graft survival rates of intestine-alone recipients were 80%, 44%, and 26%; while those for intestine and liver and intestine, liver, and pancreas were 62%, 45%, 36% and 69%, 48%, 33%, respectively.78REFERENCESEntries highlighted in bright blue are key references. 1. Tavakkolizadeh A, Whang EE, Ashley SW, Zinner MJ. Small intestine. In: Brunicardi F, Andersen D, Billiar T, et al, eds. Principles of Surgery. 9th ed. New York: McGraw-Hill; 2004:28-1 to 28-32. 2. McMinn RMH. Last’s Anatomy: Regional and Applied. 9th ed. Singapore: Churchill Livingstone; 1994:337.Brunicardi_Ch28_p1219-p1258.indd 125523/02/19 2:25 PM 1256SPECIFIC CONSIDERATIONSPART II 3. Yan KS, Chia LA, Li X, et al. The intestinal stem cell markers Bmi1

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1	and global survey. J Gastrointest Surg. 2013;17(5):962-972. 31. Wolff BG, Viscusi ER, Delaney CP, Du W, Techner L. Patterns of gastrointestinal recovery after bowel resection and total abdominal hysterectomy: pooled results from the placebo arms of alvimopan phase III North American clinical trials. Am Coll Surg. 2007;205(1):43-51. 32. Gendall KA, Kennedy RR, Watson AJ, Frizelle FA. The effect of epidural analgesia on postoperative outcome after colorectal surgery. Colorectal Dis. 2007;9(7):584-598. 33. Noblett SE, Snowden CP, Shenton BK, Horgan AF. Randomized clinical trial assessing the effect of Doppler-optimized fluid management on outcome after elective colorectal resection. Br J Surg. 2006;93:1069-1076. 34. Tan EK, Cornish J, Darzi AW, Tekkis PP. Meta-analysis: alvimopan vs. placebo in the treatment of post-operative ileus. Aliment Pharmacol Ther. 2007;25(1):47-57. 35. Gaines SL, Giroux K, Thomas S, Gregory JS. Real world efficacy of alvimopan on elective bowel resection

1	in the treatment of post-operative ileus. Aliment Pharmacol Ther. 2007;25(1):47-57. 35. Gaines SL, Giroux K, Thomas S, Gregory JS. Real world efficacy of alvimopan on elective bowel resection patients: an analysis of statistical versus clinical significance. Am J Surg. 2012;203(3):308-311. 36. Kappelman MD, Moore KR, Allen JK, Cook SF. Recent trends in the prevalence of Crohn’s disease and ulcerative colitis in a commercially insured US population. Dig Dis Sci. 2013;58(2):519-525. 37. Molodecky NA, Soon IS, Rabi DM, et al. Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology. 2012;142(1):46-54. 38. Zheng JJ, Zhu XS, Huangfu Z, Gao ZX, Guo ZR, Wang Z. Crohn’s disease in mainland China: a systematic analysis of 50 years of research. Chin J Dig Dis. 2005;6(4):175-181. 39. Kaplan GG, Jackson T, Sands BE, Frisch M, Andersson RE, Korzenik J. The risk of developing Crohn’s disease after an appendectomy: a

1	of 50 years of research. Chin J Dig Dis. 2005;6(4):175-181. 39. Kaplan GG, Jackson T, Sands BE, Frisch M, Andersson RE, Korzenik J. The risk of developing Crohn’s disease after an appendectomy: a meta-analysis. Am J Gastroenterol. 2008;103(11):2925-2931. 40. Nikolaaus S, Schreiber S. Diagnosis of inflammatory bowel disease. Gastroenterology. 2007;133(5):1670-1689. 41. Frolkis AD, Dykeman J, Negrón ME, et al. Risk of surgery for inflammatory bowel diseases has decreased over time: a systematic review and meta-analysis of population-based studies. Gastroenterology. 2013;145(5):996-1006. 42. Fazio VW, Marchetti F, Church JM, et al. Effect of resection margins on the recurrence of Crohn’s disease of the small bowel. Ann Surg. 1996;224(4):563-571. 43. McLeod RS, Wolff BG, Ross S, Parkes R, McKenzie M. Recurrence of Crohn’s disease after ileocolic resection is not affected by anastomotic type: results of a multicenter, randomized, controlled trial. Dis Colon Rectum. 2009;52(5):

1	R, McKenzie M. Recurrence of Crohn’s disease after ileocolic resection is not affected by anastomotic type: results of a multicenter, randomized, controlled trial. Dis Colon Rectum. 2009;52(5): 919-927.Brunicardi_Ch28_p1219-p1258.indd 125623/02/19 2:25 PM 1257SMALL INTESTINECHAPTER 28 44. Michelassi F, Upadhyay GA. Side-to-side isoperistaltic strictureplasty in the treatment of extensive Crohn’s disease. J Surg Res. 2004;117(1):71-78. 45. Tan JJ, Tjandra JJ. Laparoscopic surgery for Crohn’s disease: a meta-analysis. Dis Colon Rectum. 2007;50(5):576-585. 46. Delaney CP, Fazio VW. Crohn’s disease of the small bowel. Surg Clin North Am. 2001;81:137-158. 47. Penner RM, Madsen KL, Fedorak RN. Postoperative Crohn’s disease. Inflamm Bowel Dis. 2005;11:765-777. 48. Evenson AR, Shrikhande G, Fischer JE. Abdominal abscess and enteric fistula. In: Zinner MJ, Ashley SW, eds. Maingot’s Abdominal Operations. 11th ed. New York: McGraw Hill; 2007:184. 49. Coughlin S, Roth L, Lurati G, Faulhaber

1	Fischer JE. Abdominal abscess and enteric fistula. In: Zinner MJ, Ashley SW, eds. Maingot’s Abdominal Operations. 11th ed. New York: McGraw Hill; 2007:184. 49. Coughlin S, Roth L, Lurati G, Faulhaber M. Somatostatin analogues for the treatment of enterocutaneous fistulas: a systematic review and meta-analysis. World J Surg. 2012;36(5):1016-1029. 50. Wainstein DE, Fernandez E, Gonzalez D, Chara O, Berkowski D. Treatment of high-output enterocutaneous fistulas with a vacuum-compaction device. A ten-year experience. World J Surg. 2008;32(3):430-435. 51. Fazio VW, Coutsoftides T, Steiger E. Factors influencing the outcome of treatment of small bowel cutaneous fistula. World J Surg. 1983;7:481-488. 52. Owen RM, Love TP, Perez SD, et al. Definitive surgical treatment of enterocutaneous fistula: outcomes of a 23-year experience. JAMA Surg. 2013;148(2):118-126. 53. Martinez JL, Luque-de-Leon E, Ballinas-Oseguera G, Mendez JD, Juarez-Oropeza MA, Roman-Ramos R. Factors predictive of

1	fistula: outcomes of a 23-year experience. JAMA Surg. 2013;148(2):118-126. 53. Martinez JL, Luque-de-Leon E, Ballinas-Oseguera G, Mendez JD, Juarez-Oropeza MA, Roman-Ramos R. Factors predictive of recurrence and mortality after surgical repair of enterocutaneous fistula. J Gastrointest Surg. 2012;16(1):156-163. 54. Qubaiah O, Devesa SS, Platz CE, Huycke MM, Dores GM. Small intestinal cancer: a population-based study of incidence and survival patterns in the United States, 1992 to 2006. Cancer Epidemiol Biomarkers Prev. 2010;19:1908-1918. 55. Ceppa EP, Burbridge RA, Rialon KL, et al. Endoscopic versus surgical ampullectomy: an algorithm to treat disease of the ampulla of Vater. Ann Surg. 2013;257(2):315-322. 56. Judson I, Demetri G. Advances in the treatment of gastrointestinal stromal tumors. Ann Oncol. 2007;18:S20-S24. 57. Agrawal S, McCarron EC, Gibbs JF, Nava HR, Wilding GE, Rajput A. Surgical management and outcome in primary adenocarcinoma of the small bowel. Ann Surg Onc.

1	tumors. Ann Oncol. 2007;18:S20-S24. 57. Agrawal S, McCarron EC, Gibbs JF, Nava HR, Wilding GE, Rajput A. Surgical management and outcome in primary adenocarcinoma of the small bowel. Ann Surg Onc. 2007;14:2263-2269. 58. Girvent M, Carlson GL, Anderson I, et al. Intestinal failure after surgery for complicated radiation enteritis. Ann R Coll Surg Engl. 2000;82:198-201. 59. Kiliç D, Egehan I, Ozenirler S, Dursun A. Double-blinded, randomized, placebo-controlled study to evaluate the effectiveness of sulphasalazine in preventing acute gastrointestinal complications due to radiotherapy. Radiother Oncol. 2000;57:125-129. 60. Waddell BE, Lee RJ, Rodriguez-Bigas MA, Weber TK, Petrelli NJ. Absorbable mesh sling prevents radiation-induced bowel injury during “sandwich” chemoradiation for rectal cancer. Arch Surg. 2000;135:1212-1217. 61. Yahchouchy EK, Marano AF, Etienne JC, et al. Meckel’s diverticulum. J Am Coll Surg. 2001;192(5):654-662. 62. Cullen JJ, Kelly KA, Moir CR, et al. Surgical

1	cancer. Arch Surg. 2000;135:1212-1217. 61. Yahchouchy EK, Marano AF, Etienne JC, et al. Meckel’s diverticulum. J Am Coll Surg. 2001;192(5):654-662. 62. Cullen JJ, Kelly KA, Moir CR, et al. Surgical management of Meckel’s diverticulum. An epidemiologic, population-based study. Ann Surg. 1994;220(4):564-568. 63. Zani A, Eaton S, Rees CM, et al. Incidentally detected Meckel diverticulum: to resect or not to resect? Ann Surg. 2008;247:276-281. 64. Park JJ, Wolff BG, Tollefson MK, Walsh EE, Larson DR. Meckel diverticulum: the Mayo Clinic experience with 1476 patients (1950-2002). Ann Surg. 2005;241(3):529-533. 65. Lobo DN, Balfour TW, Iftikhar SY, et al. Periampullary diverticula and pancreaticobiliary disease. Br J Surg. 1999; 86:588-597. 66. Chow DC, Babaian M, Taubin HL. Jejunoileal diverticula. Gastroenterologist. 1997;5:78-84. 67. Kumar S, Sarr MG, Kamath PS: Mesenteric venous thrombosis. N Engl J Med. 2001;345:1683-1688. 68. Gralnek IM. Obscure-overt gastrointestinal bleeding.

1	Gastroenterologist. 1997;5:78-84. 67. Kumar S, Sarr MG, Kamath PS: Mesenteric venous thrombosis. N Engl J Med. 2001;345:1683-1688. 68. Gralnek IM. Obscure-overt gastrointestinal bleeding. Gastroenterology. 2005;128:1424-1430. 69. Laine L, Sahota A, Shah A. Does capsule endoscopy improve outcomes in obscure gastrointestinal bleeding? Randomized trial versus dedicated small bowel radiography. Gastroenterology. 2010;138(5):1673-1680. 70. Andriulli A, Loperfido S, Napolitano G, et al. Incidence rates of post-ERCP complications: a systematic survey of prospective studies. Am J Gastroenterol. 2007;102(8):1781-1788. 71. Genzlinger JL, McPhee MS, Fisher JK, et al. Significance of retroperitoneal air after endoscopic retrograde cholangiopancreatography with sphincterotomy. Am J Gastroenterol. 1999;94(5):1267-1270. 72. Varban O, Ardestani A, Azagury D, Kis B, Brooks DC, Tavakkoli A. Contemporary management of adult intussusception: who needs a resection? World J Surg.

1	Am J Gastroenterol. 1999;94(5):1267-1270. 72. Varban O, Ardestani A, Azagury D, Kis B, Brooks DC, Tavakkoli A. Contemporary management of adult intussusception: who needs a resection? World J Surg. 2013;37(8):1872-1877. 73. Varban O, Ardestani A, Azagury D, et al. Resection or reduction? The dilemma of managing retrograde intussusception after Roux-en-Y gastric bypass. Surg Obes Relat Dis. 2013;9(5): 725-730. 74. Buchman AL, Solapio J, Fryer J. AGA technical review on short bowel syndrome and intestinal transplantation. Gastroenterology. 2003;124(4):1111-1134. 75. Bianchi A. Intestinal loop lengthening—a technique for increasing small-intestinal length. J Pediatr Surg. 1980;15(2): 145-151. 76. Jones BA, Hull MA, Potanos KM, et al. Report of 111 consecutive patients enrolled in the International Serial Transverse Enteroplasty (STEP) data registry: a retrospective observational study. J Am Coll Surg. 2013;216(3):438-446. 77. Messing B, Crenn P, Beau P, Boutron-Ruault MC, Rambaud JC,

1	Serial Transverse Enteroplasty (STEP) data registry: a retrospective observational study. J Am Coll Surg. 2013;216(3):438-446. 77. Messing B, Crenn P, Beau P, Boutron-Ruault MC, Rambaud JC, Matuchansky C. Long-term survival and parenteral nutrition dependence in adult patients with short bowel syndrome. Gastroenterology. 1999;117(5):1043-1050. 78. Cai J. Intestine and multivisceral transplantation in the United States: a report of 20-year national registry data (1990-2009). Clin Transpl. 2009;83-101.Brunicardi_Ch28_p1219-p1258.indd 125723/02/19 2:25 PM

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1	Colon, Rectum, and AnusMary R. Kwaan, David B. Stewart Sr, and Kelli Bullard Dunn 29chapterEMBRYOLOGY AND ANATOMYEmbryologyThe embryonic gastrointestinal tract begins developing during the fourth week of gestation. The primitive gut is derived from the endoderm and divided into three segments: foregut, midgut, and hindgut. Both midgut and hindgut contribute to the colon, rec-tum, and anus.The midgut develops into the small intestine, ascending colon, and proximal transverse colon, and receives blood supply from the superior mesenteric artery. During the sixth week of gestation, the midgut herniates out of the abdominal cavity and then rotates 270° counterclockwise around the superior mesen-teric artery to return to its final position inside the abdominal cavity during the tenth week of gestation. The hindgut develops into the distal transverse colon, descending colon, rectum, and Embryology and Anatomy 1259Embryology / 1259Anatomy / 1260Congenital Anomalies / 1263Normal

1	week of gestation. The hindgut develops into the distal transverse colon, descending colon, rectum, and Embryology and Anatomy 1259Embryology / 1259Anatomy / 1260Congenital Anomalies / 1263Normal Physiology 1263Fluid and Electrolyte Exchanges / 1263Short-Chain Fatty Acids / 1263Colonic Microflora and Intestinal Gas / 1263Motility, Defecation, and Continence / 1263Clinical Evaluation 1264Clinical Assessment / 1264Endoscopy / 1264Imaging / 1265Physiologic and Pelvic Floor Investigations / 1266Laboratory Studies / 1266Evaluation of Common Symptoms / 1267General Surgical Considerations 1270Resections / 1270Anastomoses / 1274Ostomies and Preoperative Stoma Planning / 1275Functional Results / 1278Anesthesia Considerations / 1278Operative Preliminaries / 1279Inflammatory Bowel Disease 1279General Considerations / 1279Ulcerative Colitis / 1282Operative Management / 1283Crohn’s Disease / 1283Indeterminate Colitis / 1286Diverticular Disease 1286Inflammatory Complications (Diverticulitis) /

1	Considerations / 1279Ulcerative Colitis / 1282Operative Management / 1283Crohn’s Disease / 1283Indeterminate Colitis / 1286Diverticular Disease 1286Inflammatory Complications (Diverticulitis) / 1286Hemorrhage / 1288Giant Colonic Diverticulum / 1288Right-Sided Diverticula / 1288Adenocarcinoma and Polyps 1288Incidence / 1288Epidemiology (Risk Factors) / 1288Pathogenesis of Colorectal Cancer / 1289Polyps / 1290Inherited Colorectal Carcinoma / 1291Prevention: Screening and Surveillance / 1293Routes of Spread and Natural History / 1295Staging and Preoperative Evaluation / 1295Therapy for Colonic Carcinoma / 1296Therapy for Rectal Carcinoma / 1299Follow-Up and Surveillance / 1302Treatment of Recurrent Colorectal Carcinoma / 1302Minimally Invasive Techniques for Resection / 1302Other Neoplasms 1303Rare Colorectal Tumors / 1303Retrorectal/Presacral Tumors / 1304Anal Canal and Perianal Tumors / 1304Other Benign Colorectal Conditions 1305Rectal Prolapse and Solitary Rectal Ulcer

1	Neoplasms 1303Rare Colorectal Tumors / 1303Retrorectal/Presacral Tumors / 1304Anal Canal and Perianal Tumors / 1304Other Benign Colorectal Conditions 1305Rectal Prolapse and Solitary Rectal Ulcer Syndrome / 1305Volvulus / 1306Megacolon / 1308Colonic Pseudo-Obstruction (Ogilvie’s Syndrome) / 1308Ischemic Colitis / 1309Infectious Colitis / 1309Anorectal Diseases 1310Hemorrhoids / 1310Anal Fissure / 1313Anorectal Sepsis and Cryptoglandular Abscess / 1313Perianal Abscess / 1314Ischiorectal Abscess / 1314Intersphincteric Abscess / 1314Supralevator Abscess / 1316Perianal Sepsis in the Immunocompromised Patient / 1316Necrotizing Soft Tissue Infection of the Perineum / 1316Fistula In Ano / 1317Rectovaginal Fistula / 1317Perianal Dermatitis / 1319Sexually Transmitted Diseases / 1320Pilonidal Disease / 1320Hidradenitis Suppurativa / 1320Trauma 1321Penetrating Colorectal Injury / 1321Blunt Colorectal Injury / 1321Iatrogenic Injury / 1321Anal Sphincter Injury and Incontinence / 1322Foreign

1	Disease / 1320Hidradenitis Suppurativa / 1320Trauma 1321Penetrating Colorectal Injury / 1321Blunt Colorectal Injury / 1321Iatrogenic Injury / 1321Anal Sphincter Injury and Incontinence / 1322Foreign Body / 1323The Immunocompromised Patient 1323Human Immunodeficiency Virus / 1323Immunosuppression for Transplantation 1323The Neutropenic Patient 1323Brunicardi_Ch29_p1259-p1330.indd 125923/02/19 2:28 PM 1260proximal anus, all of which receive their blood supply from the inferior mesenteric artery. During the sixth week of gestation, the distal-most end of the hindgut, the cloaca, is divided by the urorectal septum into the urogenital sinus and the rectum.The distal anal canal is derived from ectoderm and receives its blood supply from the internal pudendal artery. The dentate line divides the endodermal hindgut from the ectodermal distal anal canal.AnatomyThe large intestine extends from the ileocecal valve to the anus. It is divided anatomically and functionally into the colon,

1	the endodermal hindgut from the ectodermal distal anal canal.AnatomyThe large intestine extends from the ileocecal valve to the anus. It is divided anatomically and functionally into the colon, rectum, and anal canal. The wall of the colon and rectum comprise four distinct layers: mucosa, submucosa, muscularis propria (inner circular muscle, outer longitudinal muscle), and serosa. In the colon, the outer longitudinal muscle is separated into three teniae coli, which converge proximally at the appendix and dis-tally at the rectum, where the outer longitudinal muscle layer is circumferential. In the distal rectum, the inner smooth muscle layer coalesces to form the internal anal sphincter. The intraperi-toneal colon and proximal one-third of the rectum are covered by serosa; the mid and lower rectum lack serosa.Colon Landmarks. The colon begins at the junction of the terminal ileum and cecum and extends approximately 150 cm (3 to 5 feet) to the rectum. The rectosigmoid junction is found

1	rectum lack serosa.Colon Landmarks. The colon begins at the junction of the terminal ileum and cecum and extends approximately 150 cm (3 to 5 feet) to the rectum. The rectosigmoid junction is found at approximately the level of the sacral promontory and is arbitrarily described as the point at which the three teniae coli coalesce to form the outer longitudinal smooth muscle layer of the rectum. The cecum is the widest diameter portion of the colon (normally 7.5–8.5 cm) and has the thinnest muscular wall. As a result, the cecum is most vulnerable to perforation and least vulnerable to obstruction. The ascending colon is usu-ally fixed to the retroperitoneum. The hepatic flexure marks the transition to the transverse colon. The transverse colon is relatively mobile, but it is tethered by the gastrocolic ligament and colonic mesentery. The greater omentum is attached to the anterior/superior edge of the transverse colon. These attach-ments explain the characteristic triangular appearance

1	gastrocolic ligament and colonic mesentery. The greater omentum is attached to the anterior/superior edge of the transverse colon. These attach-ments explain the characteristic triangular appearance of the transverse colon observed during colonoscopy. The splenic flexure marks the transition from the transverse colon to the descending colon. The attachments between the splenic flexure and the spleen (the lienocolic ligament) can be short and dense, making mobilization of this flexure during colectomy challeng-ing. The descending colon is relatively fixed to the retroperi-toneum. The sigmoid colon is the narrowest part of the large intestine and is extremely mobile. Although the sigmoid colon is usually located in the left lower quadrant, redundancy and mobility can result in a portion of the sigmoid colon residing in the right lower quadrant. This mobility explains why volvulus is most common in the sigmoid colon and why diseases affect-ing the sigmoid colon, such as diverticulitis,

1	the sigmoid colon residing in the right lower quadrant. This mobility explains why volvulus is most common in the sigmoid colon and why diseases affect-ing the sigmoid colon, such as diverticulitis, may occasionally present as right-sided abdominal pain. The narrow caliber of the sigmoid colon makes this segment of the large intestine the most vulnerable to obstruction.Colon Vascular Supply. The arterial supply to the colon is highly variable (Fig. 29-1). In general, the superior mesenteric artery branches into the ileocolic artery (absent in up to 20% of people), which supplies blood flow to the terminal ileum and proximal ascending colon; the right colic artery, which supplies the ascending colon; and the middle colic artery, which supplies the transverse colon. The inferior mesenteric artery branches into the left colic artery, which supplies the descending colon; several sigmoidal branches, which supply the sigmoid colon; and the superior rectal artery, which supplies the proximal

1	branches into the left colic artery, which supplies the descending colon; several sigmoidal branches, which supply the sigmoid colon; and the superior rectal artery, which supplies the proximal rec-tum. The terminal branches of each artery form anastomoses with the terminal branches of the adjacent artery and communi-cate via the marginal artery of Drummond. This arcade is com-plete in only 15% to 20% of people.Key Points1 Resection principles: The mesenteric clearance technique dictates the extent of resection and is determined by the nature of the primary pathology, the intent of resection, the location of the lesion, and the condition of the mesentery.2 Function after resection: Bowel function is often com-promised after colorectal resection, especially after low anterior resection. For this reason, it is important to obtain a history of prior anorectal trauma and/or incontinence before considering a low anastomosis.3 Ostomies/Stomas: Preoperative marking for a planned stoma is

1	For this reason, it is important to obtain a history of prior anorectal trauma and/or incontinence before considering a low anastomosis.3 Ostomies/Stomas: Preoperative marking for a planned stoma is critical for a patient’s quality of life. Ideally, a stoma should be located within the rectus muscle, in a location where the patient can easily see and manipulate the appliance, and away from previous scars, bony promi-nences, or abdominal creases.4 Inflammatory bowel disease: Both Crohn’s disease and ulcerative colitis are associated with an increased risk of colorectal carcinoma. Risk depends on the amount of colon involved and the duration of disease.5 Pathogenesis of colorectal cancer: A variety of mutations have been identified in colorectal cancer. Mutations may cause activation of oncogenes (K-ras) and/or inactivation of tumor suppressor genes (adenomatous polyposis coli [APC], deleted in colorectal carcinoma [DCC], p53).6 Minimally invasive resection: Laparoscopy and HAL have

1	oncogenes (K-ras) and/or inactivation of tumor suppressor genes (adenomatous polyposis coli [APC], deleted in colorectal carcinoma [DCC], p53).6 Minimally invasive resection: Laparoscopy and HAL have been shown to be both safe and efficacious for colorec-tal resection. Robotic surgery increasingly has been used, especially for rectal/pelvic dissection.7 Anal epidermoid carcinoma: Unlike rectal adenocarci-noma, anal epidermoid carcinoma is treated primarily with chemoradiation. Surgery is reserved for patients with persistent or recurrent disease.8 Rectal prolapse: Rectal prolapse occurs most commonly in elderly women. Transabdominal repair (rectopexy with or without resection) offers more durability than perineal proctosigmoidectomy, but it carries greater operative risk.9 Hemorrhoids: Hemorrhoids are cushions of submucosal tissue containing venules, arterioles, and smooth muscle fibers. They are thought to play a role in maintaining continence. Resection is only indicated for

1	Hemorrhoids are cushions of submucosal tissue containing venules, arterioles, and smooth muscle fibers. They are thought to play a role in maintaining continence. Resection is only indicated for refractory symptoms.10 Fistula in ano: Treatment of fistula in ano depends on the location of the fistula, amount of anal sphincter involved in the fistula, and the underlying disease process.Brunicardi_Ch29_p1259-p1330.indd 126023/02/19 2:28 PM 1261COLON, RECTUM, AND ANUSCHAPTER 29Except for the inferior mesenteric vein, the veins of the colon parallel their corresponding arteries and bear the same terminology (Fig. 29-2). The inferior mesenteric vein ascends in the retroperitoneal plane over the psoas muscle and continues posterior to the pancreas to join the splenic vein. During a col-ectomy, this vein is often mobilized independently and ligated at the inferior edge of the pancreas.Colon Lymphatic Drainage. The lymphatic drainage of the colon originates in a network of lymphatics in

1	this vein is often mobilized independently and ligated at the inferior edge of the pancreas.Colon Lymphatic Drainage. The lymphatic drainage of the colon originates in a network of lymphatics in the muscula-ris mucosa. Lymphatic vessels and lymph nodes follow the regional arteries. Lymph nodes are found on the bowel wall (epicolic), along the inner margin of the bowel adjacent to the arterial arcades (paracolic), around the named mesenteric ves-sels (intermediate), and at the origin of the superior and inferior mesenteric arteries (main).Colon Nerve Supply. The colon is innervated by both sympathetic (inhibitory) and parasympathetic (stimulatory) nerves, which parallel the course of the arteries. Sympathetic nerves arise from T6–T12 and L1–L3. The parasympathetic innervation to the right and transverse colon is from the vagus nerve; the parasympathetic nerves to the left colon arise from sacral nerves S2–S4 to form the nervi erigentes.Anorectal Landmarks. The rectum is approximately

1	and transverse colon is from the vagus nerve; the parasympathetic nerves to the left colon arise from sacral nerves S2–S4 to form the nervi erigentes.Anorectal Landmarks. The rectum is approximately 12 to 15 cm in length. Three distinct submucosal folds, the valves of Houston, extend into the rectal lumen. Posteriorly, the presecral fascia separates the rectum from the presacral venous plexus and the pelvic nerves. At S4, the rectosacral fascia (Waldeyer’s fascia) extends anteriorly and caudally and attaches to the fas-cia propria at the anorectal junction. Anteriorly, Denonvilliers’ fascia separates the rectum from the prostate and seminal ves-icles in men and from the vagina in women. The lateral liga-ments support the lower rectum.The anatomic anal canal extends from the dentate or pectinate line to the anal verge. The dentate or pectinate line marks the transition point between columnar rectal mucosa and squamous anoderm. The anal transition zone includes mucosa proximal to the

1	line to the anal verge. The dentate or pectinate line marks the transition point between columnar rectal mucosa and squamous anoderm. The anal transition zone includes mucosa proximal to the dentate line that shares histologic characteris-tics of columnar, cuboidal, and squamous epithelium. Although the anal transition zone was long thought to extend only 1 to 2 cm proximal to the dentate line, it is known that the proxi-mal extent of this zone is highly variable and can be as far as 15 cm proximal to the dentate line. The dentate line is sur-rounded by longitudinal mucosal folds, known as the columns of Morgagni, into which the anal crypts empty. These crypts are the source of cryptoglandular abscesses (Fig. 29-3). In contrast to the anatomic anal canal, the surgical anal canal begins at the Middlecolic a.Rightcolic a.Leftcolic a.Ileocolic a.Superiormesenteric a.Superiorrectal a.Sigmoidal a.Inferiormesenteric a.Figure 29-1. Arterial blood supply to the colon. a. =

1	canal begins at the Middlecolic a.Rightcolic a.Leftcolic a.Ileocolic a.Superiormesenteric a.Superiorrectal a.Sigmoidal a.Inferiormesenteric a.Figure 29-1. Arterial blood supply to the colon. a. = artery.Inferiormesenteric v.Middlecolic v.Rightcolic v.Superiormesenteric v.Portal v.Leftcolic v.Sigmoidal v.Superiorrectal v.Ileocolic v.Figure 29-2. Venous drainage of the colon. v. = vein. (Reproduced with permission from Bell RH, Rikkers LF, Mulholland M: Diges-tive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)Brunicardi_Ch29_p1259-p1330.indd 126123/02/19 2:28 PM 1262SPECIFIC CONSIDERATIONSPART IIanorectal junction and terminates at the anal verge. The surgical anal canal measures 2 to 4 cm in length and is generally longer in men than in women. It begins at the anorectal junction and terminates at the anal verge.In the distal rectum, the inner smooth muscle is thickened and comprises the internal anal sphincter that is surrounded by the

1	It begins at the anorectal junction and terminates at the anal verge.In the distal rectum, the inner smooth muscle is thickened and comprises the internal anal sphincter that is surrounded by the subcutaneous, superficial, and deep external sphincter. The deep external anal sphincter is an extension of the puborectalis muscle. The puborectalis, iliococcygeus, and pubococcygeus muscles form the levator ani muscle of the pelvic floor (Fig. 29-4).Anorectal Vascular Supply. The superior rectal artery arises from the terminal branch of the inferior mesenteric artery and supplies the upper rectum. The middle rectal artery arises from the internal iliac; the presence and size of these arteries are highly variable. The inferior rectal artery arises from the inter-nal pudendal artery, which is a branch of the internal iliac artery. A rich network of collaterals connects the terminal arterioles of each of these arteries, thus making the rectum relatively resis-tant to ischemia (Fig. 29-5).The

1	branch of the internal iliac artery. A rich network of collaterals connects the terminal arterioles of each of these arteries, thus making the rectum relatively resis-tant to ischemia (Fig. 29-5).The venous drainage of the rectum parallels the arterial supply. The superior rectal vein drains into the portal system via the inferior mesenteric vein. The middle rectal vein drains into the internal iliac vein. The inferior rectal vein drains into the internal pudendal vein, and subsequently into the internal iliac vein. A submucosal plexus deep to the columns of Morgagni forms the hemorrhoidal plexus and drains into all three veins.Anorectal Lymphatic Drainage. Lymphatic drainage of the rectum parallels the vascular supply. Lymphatic channels in the upper and middle rectum drain superiorly into the inferior mes-enteric lymph nodes. Lymphatic channels in the lower rectum drain both superiorly into the inferior mesenteric lymph nodes and laterally into the internal iliac lymph nodes. The

1	into the inferior mes-enteric lymph nodes. Lymphatic channels in the lower rectum drain both superiorly into the inferior mesenteric lymph nodes and laterally into the internal iliac lymph nodes. The anal canal has a more complex pattern of lymphatic drainage. Proximal to the dentate line, lymph drains into both the inferior mesen-teric lymph nodes and the internal iliac lymph nodes. Distal to the dentate line, lymph primarily drains into the inguinal lymph nodes, but can also drain into the inferior mesenteric lymph nodes and internal iliac lymph nodes.AnalcanalColumn of MorgagniDentate lineAnal cryptAnal glandAnodermTransitionzoneFigure 29-3. The lining of the anal canal. (Reproduced with permission from Goldberg SM, Gordon PH, Nivatvongs S: Essentials of Anorectal Surgery. Philadelphia, PA: JB Lippincott Company; 1980.)Deep external sphincterand puborectalis mm.Conjoinedlongitudinal m.Superficial externalsphincter m.Subcutaneous externalsphincter m.Valve of HoustonInternal

1	PA: JB Lippincott Company; 1980.)Deep external sphincterand puborectalis mm.Conjoinedlongitudinal m.Superficial externalsphincter m.Subcutaneous externalsphincter m.Valve of HoustonInternal rectalplexusMuscularis submucosa ani m.Transverse septumof ischiorectal fossaExternal rectalplexusInternalsphincter m.Figure 29-4. The distal rectum and anal canal. m. = muscle.Brunicardi_Ch29_p1259-p1330.indd 126223/02/19 2:28 PM 1263COLON, RECTUM, AND ANUSCHAPTER 29Anorectal Nerve Supply. Both sympathetic and parasym-pathetic nerves innervate the anorectum. Sympathetic nerve fibers are derived from L1–L3 and join the preaortic plexus. The preaortic nerve fibers then extend below the aorta to form the hypogastric plexus, which subsequently joins the para-sympathetic fibers to form the pelvic plexus. Parasympathetic nerve fibers are known as the nervi erigentes and originate from S2–S4. These fibers join the sympathetic fibers to form the pel-vic plexus. Sympathetic and parasympathetic fibers

1	Parasympathetic nerve fibers are known as the nervi erigentes and originate from S2–S4. These fibers join the sympathetic fibers to form the pel-vic plexus. Sympathetic and parasympathetic fibers then supply the anorectum and adjacent urogenital organs.The internal anal sphincter is innervated by sympathetic and parasympathetic nerve fibers; both types of fibers inhibit sphincter contraction. The external anal sphincter and puborec-talis muscles are innervated by the inferior rectal branch of the internal pudendal nerve. The levator ani receives innervation from both the internal pudendal nerve and direct branches of S3 to S5. Sensory innervation to the anal canal is provided by the inferior rectal branch of the pudendal nerve. While the rec-tum is relatively insensate, the anal canal below the dentate line receives somatic innervation.Congenital AnomaliesPerturbation of the embryologic development of the midgut and hindgut may result in anatomic abnormalities of the colon, rec-tum,

1	the dentate line receives somatic innervation.Congenital AnomaliesPerturbation of the embryologic development of the midgut and hindgut may result in anatomic abnormalities of the colon, rec-tum, and anus. Failure of the midgut to rotate and return to the abdominal cavity during the tenth week of gestation results in varying degrees of intestinal malrotation and colonic nonfix-ation. Failure of canalization of the primitive gut can result in colonic duplication. Incomplete descent of the urogenital sep-tum may result in imperforate anus and associated fistulas to the genitourinary tract. Many infants with congenital anomalies of the hindgut have associated abnormalities in the genitourinary tract.NORMAL PHYSIOLOGYFluid and Electrolyte ExchangesWater, Sodium, Potassium, Chloride, Bicarbonate, and Ammonia. The colon is a major site for water absorption and electrolyte exchange. Under normal circumstances, approxi-mately 90% of the water contained in ileal fluid is absorbed in the colon

1	and Ammonia. The colon is a major site for water absorption and electrolyte exchange. Under normal circumstances, approxi-mately 90% of the water contained in ileal fluid is absorbed in the colon (1000–2000 mL/d), but up to 5000 mL of fluid can be absorbed daily. Sodium is absorbed actively via sodiumpotassium (Na+/K+) ATPase. The colon can absorb up to 400 mEq of sodium per day. Water accompanies the transported sodium and is absorbed passively along an osmotic gradient. Potassium is actively secreted into the colonic lumen and absorbed by passive diffusion. Chloride is absorbed actively via a chloride-bicarbonate exchange.Bacterial degradation of protein and urea produces ammo-nia. Ammonia is subsequently absorbed and transported to the liver. Absorption of ammonia depends in part on intraluminal pH. A decrease in colonic bacteria (e.g., due to broad-spectrum antibiotic use) and/or a decrease in intraluminal pH (e.g., due to lactulose administration) will decrease ammonia

1	part on intraluminal pH. A decrease in colonic bacteria (e.g., due to broad-spectrum antibiotic use) and/or a decrease in intraluminal pH (e.g., due to lactulose administration) will decrease ammonia absorption.Short-Chain Fatty AcidsShort-chain fatty acids (acetate, butyrate, and propionate) are produced by bacterial fermentation of dietary carbohydrates. Short-chain fatty acids are an important source of energy for the colonic mucosa, and metabolism by colonocytes provides energy for processes such as active transport of sodium. Lack of a dietary source for production of short-chain fatty acids, or diversion of the fecal stream by an ileostomy or colostomy, may result in mucosal atrophy and inflammation, the latter termed “diversion colitis.”Colonic Microflora and Intestinal GasApproximately 30% of fecal dry weight is composed of bacteria (1011–1012 bacteria/g of feces). Anaerobes are the predominant class of microorganism, and Bacteroides species are the most common (1011–1012

1	30% of fecal dry weight is composed of bacteria (1011–1012 bacteria/g of feces). Anaerobes are the predominant class of microorganism, and Bacteroides species are the most common (1011–1012 organisms/mL). Escherichia coli are the most numerous aerobes (108–1010 organisms/mL). Endogenous microflora are crucial for the breakdown of carbohydrates and proteins in the colon and participate in the metabolism of biliru-bin, bile acids, estrogen, and cholesterol. Colonic bacteria also are necessary for production of vitamin K. Endogenous bacteria also are thought to suppress the emergence of pathogenic micro-organisms, such as Clostridium difficile, a phenomenon termed “colonization resistance.” However, the high bacterial load of the large intestine may contribute to sepsis in critically ill patients and may contribute to intra-abdominal sepsis, abscess, and wound infection following colectomy. Burgeoning research on the human gut microbiome offers new concepts of bacterial community

1	ill patients and may contribute to intra-abdominal sepsis, abscess, and wound infection following colectomy. Burgeoning research on the human gut microbiome offers new concepts of bacterial community structure and its impact on gastrointestinal disease. Use of 16S ribosomal RNA, universally present in bacteria, allows for a more complete understanding of colonic samples, without the use of stool cultures.Intestinal gas arises from swallowed air, diffusion from the blood, and intraluminal production. Nitrogen, oxygen, car-bon dioxide, hydrogen, and methane are the major components of intestinal gas. Nitrogen and oxygen are largely derived from swallowed air. Carbon dioxide is produced by the reaction of bicarbonate and hydrogen ions and by the digestion of triglyc-erides to fatty acids. Hydrogen and methane are produced by colonic bacteria. The production of methane is highly variable. The gastrointestinal tract usually contains between 100 and 200 mL of gas, and 400 to 1200 mL/d are

1	and methane are produced by colonic bacteria. The production of methane is highly variable. The gastrointestinal tract usually contains between 100 and 200 mL of gas, and 400 to 1200 mL/d are released as flatus, depending on the type of food ingested.Motility, Defecation, and ContinenceMotility. Unlike the small intestine, the large intestine does not demonstrate cyclic motor activity characteristic of the migra-tory motor complex. Instead, the colon displays intermittent Internaliliac arterySuperiorrectal arteryMiddlerectal arteryInferiorrectal arteryInferiormesenteric arteryMiddlesacral arteryLevatorani muscleFigure 29-5. Arterial supply to the rectum and anal canal.Brunicardi_Ch29_p1259-p1330.indd 126323/02/19 2:28 PM 1264SPECIFIC CONSIDERATIONSPART IIcontractions of either low or high amplitude. Low-amplitude, short-duration contractions occur in bursts and appear to move the colonic contents both antegrade and retrograde. It is thought that these bursts of motor activity

1	or high amplitude. Low-amplitude, short-duration contractions occur in bursts and appear to move the colonic contents both antegrade and retrograde. It is thought that these bursts of motor activity delay colonic transit and thus increase the time available for absorption of water and exchange of electrolytes. High-amplitude, prolonged duration, propagated contractions (HAPCs) occur in a more coordinated fashion and create “mass movements,” four to ten times per day, mostly after meals and awakening. Bursts of “rectal motor complexes” also have been described. In general, cholinergic activation increases colonic motility.Defecation. Defecation is a complex, coordinated mechanism involving colonic mass movement, increased intra-abdominal and rectal pressure, and relaxation of the pelvic floor. Disten-tion of the rectum causes a reflex relaxation of the internal anal sphincter (the rectoanal inhibitory reflex) that allows the contents to make contact with the anal canal. This “sampling

1	Disten-tion of the rectum causes a reflex relaxation of the internal anal sphincter (the rectoanal inhibitory reflex) that allows the contents to make contact with the anal canal. This “sampling reflex” allows the sensory epithelium to distinguish solid stool from liquid stool and gas. If defecation does not occur, the rec-tum relaxes and the urge to defecate passes (accommodation response). Defecation proceeds by coordinating increasing intra-abdominal pressure via a Valsalva maneuver with rectal contraction, relaxation of the puborectalis muscle, and opening of the anal canal.Continence. The maintenance of fecal continence is at least as complex as the mechanism of defecation. Continence requires adequate rectal wall compliance to accommodate the fecal bolus, appropriate neurogenic control of the pelvic floor and sphincter mechanism, and functional internal and exter-nal sphincter muscles. At rest, the puborectalis muscle creates a “sling” around the distal rectum, forming a

1	control of the pelvic floor and sphincter mechanism, and functional internal and exter-nal sphincter muscles. At rest, the puborectalis muscle creates a “sling” around the distal rectum, forming a relatively acute angle that distributes intra-abdominal forces onto the pelvic floor. With defecation, this angle straightens, allowing down-ward force to be applied along the axis of the rectum and anal canal. The internal and external sphincters are tonically active at rest. The internal sphincter is responsible for most of the rest-ing, involuntary sphincter tone (resting pressure). The external sphincter is responsible for most of the voluntary sphincter tone (squeeze pressure). Branches of the pudendal nerve innervate both the internal and external sphincter. The hemorrhoidal cush-ions may contribute to continence by mechanically blocking the anal canal. Finally, liquid stools exacerbate abnormalities with these anatomic and physiologic mechanisms, so a formed stool contributes to

1	may contribute to continence by mechanically blocking the anal canal. Finally, liquid stools exacerbate abnormalities with these anatomic and physiologic mechanisms, so a formed stool contributes to maintaining continence. Thus, impaired con-tinence may result from poor rectal compliance, injury to the internal and/or external sphincter or puborectalis, or neuropathy.CLINICAL EVALUATIONClinical AssessmentObtaining a complete history and performing a physical exami-nation are the starting points for evaluating any patient with suspected disease of the colon, rectum, or anus. Special atten-tion should be paid to the patient’s past medical and surgical history to detect underlying conditions that might contribute to a gastrointestinal problem. If patients have had prior intes-tinal surgery, it is essential that one understand the resultant gastrointestinal anatomy. A history of anorectal surgery may be critical for patients with either abdominal or anorectal com-plaints. The obstetrical

1	it is essential that one understand the resultant gastrointestinal anatomy. A history of anorectal surgery may be critical for patients with either abdominal or anorectal com-plaints. The obstetrical history in women is essential to detect occult pelvic floor and/or anal sphincter damage. Identifying a family history of colorectal disease, especially inflammatory bowel disease, polyps, and colorectal cancer, is crucial. In addi-tion to a family history of colorectal disease, a history of other malignancies may suggest the presence of a genetic syndrome. Medication use must be detailed as many drugs cause gastroin-testinal symptoms. Before recommending operative interven-tion, the adequacy of medical treatment must be ascertained. In addition to examining the abdomen, visual inspection of the anus and perineum and careful digital rectal exam are essential.EndoscopyAnoscopy. The anoscope is a useful instrument for examina-tion of the anal canal. Anoscopes are made in a variety of sizes

1	the anus and perineum and careful digital rectal exam are essential.EndoscopyAnoscopy. The anoscope is a useful instrument for examina-tion of the anal canal. Anoscopes are made in a variety of sizes and measure approximately 8 cm in length. A larger anoscope provides better exposure for anal procedures such as rubber band ligation or sclerotherapy of hemorrhoids. The anoscope, with obturator in place, should be adequately lubricated and gently inserted into the anal canal. The obturator is withdrawn, inspec-tion of the visualized anal canal is done, and the anoscope should then be withdrawn. It is rotated 90° and reinserted to allow visual-ization of all four quadrants of the canal. If the patient complains of severe perianal pain and cannot tolerate a digital rectal exami-nation, anoscopy should not be attempted without anesthesia.Proctoscopy. The rigid proctoscope is useful for examination of the rectum and distal sigmoid colon and is occasionally used therapeutically. The standard

1	should not be attempted without anesthesia.Proctoscopy. The rigid proctoscope is useful for examination of the rectum and distal sigmoid colon and is occasionally used therapeutically. The standard proctoscope is 25 cm in length and available in various diameters. Most often, a 15or 19-mm diameter proctoscope is used for diagnostic examinations. A smaller “pediatric” proctoscope (11-mm diameter) is better tol-erated by patients with anal stricture. Suction is necessary for an adequate proctoscopic examination. An operating platform for transanal surgery known as transanal endoscopic microsurgery (TEM) has a much wider diameter and can be used for exci-sions of large polyps and tumors. Transanal minimally invasive surgery (TAMIS) can achieve similar resections to TEM, but it does not utilize a proctoscope and instead depends on insuf-flation to create a working space in the rectum while utilizing a circular wound protector to open the anus.Flexible Sigmoidoscopy and Colonoscopy. Video

1	a proctoscope and instead depends on insuf-flation to create a working space in the rectum while utilizing a circular wound protector to open the anus.Flexible Sigmoidoscopy and Colonoscopy. Video or fiber-optic flexible sigmoidoscopy and colonoscopy provide excellent visualization of the colon and rectum. Sigmoidoscopes measure 60 cm in length. Full depth of insertion may allow visualiza-tion as high as the splenic flexure, although the mobility and redundancy of the sigmoid colon often limit the extent of the examination. Partial preparation with enemas is usually adequate for sigmoidoscopy, and most patients can tolerate this proce-dure without sedation. Colonoscopes measure 100 to 160 cm in length and are capable of examining the entire colon and terminal ileum. A complete oral bowel preparation is usually necessary for colonoscopy, and the duration and discomfort of the procedure usually require conscious sedation. Both sigmoid-oscopy and colonoscopy can be used diagnostically

1	preparation is usually necessary for colonoscopy, and the duration and discomfort of the procedure usually require conscious sedation. Both sigmoid-oscopy and colonoscopy can be used diagnostically and thera-peutically. Electrocautery should generally not be used in the absence of a complete bowel preparation because of the risk of explosion of intestinal methane or hydrogen gases. Diagnostic colonoscopes possess a single channel through which instru-ments such as snares, biopsy forceps, or electrocautery can be passed; this channel also provides suction and irrigation capa-bility. Therapeutic colonoscopes possess two channels to allow simultaneous suction/irrigation and the use of snares, biopsy forceps, or electrocautery.Brunicardi_Ch29_p1259-p1330.indd 126423/02/19 2:28 PM 1265COLON, RECTUM, AND ANUSCHAPTER 29Capsule Endoscopy. Capsule endoscopy uses a small ingest-ible camera. After swallowing the camera, images of the mucosa of the gastrointestinal tract are captured,

1	RECTUM, AND ANUSCHAPTER 29Capsule Endoscopy. Capsule endoscopy uses a small ingest-ible camera. After swallowing the camera, images of the mucosa of the gastrointestinal tract are captured, transmitted by radiofre-quency to a belt-held receiver, and then downloaded to a com-puter for viewing and analysis. Capsule endoscopy largely has been used to detect small bowel lesions. Recent advances in the development of maneuverable capsules may improve the sensi-tivity of this procedure.1 Finally, concern over the possibility of an acute obstruction has led to the development of a dissolvable capsule that can detect obstruction lesions. Although this tech-nology is promising, the ultimate utility of capsule endoscopy remains unknown.ImagingPlain X-Rays and Contrast Studies. Despite advanced radio-logic techniques, plain X-rays and contrast studies continue to play an important role in the evaluation of patients with sus-pected colon and rectal diseases. Plain X-rays of the abdomen (supine,

1	techniques, plain X-rays and contrast studies continue to play an important role in the evaluation of patients with sus-pected colon and rectal diseases. Plain X-rays of the abdomen (supine, upright, and diaphragmatic views) are useful for detect-ing free intra-abdominal air, bowel gas patterns suggestive of small or large bowel obstruction, and volvulus. Contrast studies are useful for evaluating obstructive symptoms, delineating fis-tulous tracts, and diagnosing small perforations or anastomotic leaks. Although Gastrografin cannot provide the mucosal detail provided by barium, this water-soluble contrast agent is rec-ommended if perforation or leak is suspected. Double-contrast barium enema (use of barium followed by the insufflation of air into the colon) has been reported to be 70% to 90% sensitive for the detection of mass lesions greater than 1 cm in diameter. Detection of small lesions can be extremely difficult, especially in a patient with extensive diverticulosis. For this

1	90% sensitive for the detection of mass lesions greater than 1 cm in diameter. Detection of small lesions can be extremely difficult, especially in a patient with extensive diverticulosis. For this reason, a colo-noscopy is preferred for evaluating nonobstructing mass lesions in the colon. Double-contrast barium enema has been used as a back-up examination if colonoscopy is incomplete.Computed Tomography. Computed tomography (CT) com-monly is employed in the evaluation of patients with abdominal complaints. Its utility is primarily in the detection of extralu-minal disease, such as intra-abdominal abscesses and pericolic inflammation, and in staging colorectal carcinoma because of its sensitivity in detection of hepatic metastases.Extravasation of oral or rectal contrast may also confirm the diagnosis of perforation or anastomotic leak. Nonspecific findings such as bowel wall thickening or mesenteric strand-ing may suggest inflammatory bowel disease, enteritis/colitis, or ischemia. A

1	the diagnosis of perforation or anastomotic leak. Nonspecific findings such as bowel wall thickening or mesenteric strand-ing may suggest inflammatory bowel disease, enteritis/colitis, or ischemia. A standard CT scan is relatively insensitive for the detection of intraluminal lesions.Computed Tomography Colonography. CT colonography (virtual colonoscopy) is a radiologic technique that is designed to overcome some of the limitations of traditional CT scan-ning. This technology uses helical CT and three-dimensional reconstruction to detect intraluminal colonic lesions. Oral bowel preparation, oral and rectal contrast, and colon insufflation have been used to maximize sensitivity. Experience with this technol-ogy has shown a sensitivity and specificity for detecting 1 cm or larger polyps of 85% to 90% in most studies, making it compa-rable to traditional colonoscopy. Although this technology has yet to be widely adopted, it remains an alternative to traditional colonoscopy for select

1	of 85% to 90% in most studies, making it compa-rable to traditional colonoscopy. Although this technology has yet to be widely adopted, it remains an alternative to traditional colonoscopy for select patients.2Magnetic Resonance Imaging. The main use of magnetic resonance imaging (MRI) in colorectal disorders is in evaluation of pelvic lesions. MRI is more sensitive than CT for detecting bony involvement or pelvic sidewall extension of rectal tumors. MRI accurately determines the extent of spread of rectal cancer into the adjacent mesorectum and can reliably predict diffi-culty achieving radial margin clearance of a rectal cancer by surgery alone. When the radial margin is threatened, neoadju-vant chemoradiation is indicated. MRI also can be helpful in the detection and delineation of complex fistulas in ano. The use of an endorectal coil may increase sensitivity.Positron Emission Tomography. Positron emission tomog-raphy (PET) is used for imaging tissues with high levels of anaerobic

1	fistulas in ano. The use of an endorectal coil may increase sensitivity.Positron Emission Tomography. Positron emission tomog-raphy (PET) is used for imaging tissues with high levels of anaerobic glycolysis, such as malignant tumors. 13F-fluorode-oxyglucose (FDG) is injected as a tracer; metabolism of this molecule then results in positron emission. PET has been used as an adjunct to CT in the staging of colorectal cancer and may prove useful in discriminating recurrent cancer from fibrosis. By combining PET and CT technology (PET/CT), anatomic correlation between regions of high isotope accumulation (“hot spots”) on PET and abnormalities on CT can be determined. PET/CT increasingly is used to diagnose recurrent and/or meta-static colorectal cancer. However, the efficacy and utility of this technology remains unproven.Scintigraphy to Assess Gastrointestinal Bleeding. Scin-tigraphy to assess for gastrointestinal bleeding (technetium-99-tagged red blood cell [RBC] scan; “tagged RBC

1	this technology remains unproven.Scintigraphy to Assess Gastrointestinal Bleeding. Scin-tigraphy to assess for gastrointestinal bleeding (technetium-99-tagged red blood cell [RBC] scan; “tagged RBC scan”) is a nuclear medicine test that uses 99mTc-erythrocytes and dynamic images to localize a bleeding source. Patients must be actively bleeding at the time of imaging, and a normal distribution of 99mTc-erythrocytes in vasculature, liver, spleen, penile circula-tion with mild uptake in kidneys and bladder can interfere with localization in bowel segments near those structures. Patients must be stable enough to tolerate imaging intervals of up to 4 hours, but slow bleeding at a rate of 0.05 to 0.2 mL/minute can be detected.3Single Photon Emission Computed Tomography (SPECT/CT). Radiolabeled erythrocytes are also used for SPECT/CT, but cross-sectional images provide a more specific localization of the bleeding source, which can be very helpful for surgical planning, especially if direct

1	erythrocytes are also used for SPECT/CT, but cross-sectional images provide a more specific localization of the bleeding source, which can be very helpful for surgical planning, especially if direct visualization via endoscopy has not been successful.4Angiography. Angiography is occasionally used for the detec-tion of bleeding within the colon or small bowel. To visual-ize hemorrhage angiographically, bleeding must be relatively brisk (approximately 0.5 to 1.0 mL per minute). If extravasa-tion of contrast is identified, infusion of vasopressin or angio-graphic embolization can be therapeutic. If surgical resection is required, the angiographic catheter can be left in place to assist with identification of the bleeding site intraoperatively.CT and magnetic resonance angiography are also useful for assessing patency of visceral vessels. This technique uses three-dimensional reconstruction to detect vascular lesions. If an abnormality is found, more traditional techniques (angiography,

1	for assessing patency of visceral vessels. This technique uses three-dimensional reconstruction to detect vascular lesions. If an abnormality is found, more traditional techniques (angiography, surgery) may then be used to further define and/or correct the problem.Endorectal and Endoanal Ultrasound. Endorectal ultra-sound is primarily used to evaluate the depth of invasion of neoplastic lesions in the rectum. The normal rectal wall appears as a five-layer structure (Fig. 29-6). Ultrasound can reliably dif-ferentiate most benign polyps from invasive tumors based on the integrity of the submucosal layer. Ultrasound can also dif-ferentiate superficial T1-T2 from deeper T3-T4 tumors. Overall, Brunicardi_Ch29_p1259-p1330.indd 126523/02/19 2:28 PM 1266SPECIFIC CONSIDERATIONSPART IIthe accuracy of ultrasound in detecting depth of mural invasion ranges between 81% and 94%. This modality also can detect enlarged perirectal lymph nodes, which may suggest nodal metastases; accuracy of

1	accuracy of ultrasound in detecting depth of mural invasion ranges between 81% and 94%. This modality also can detect enlarged perirectal lymph nodes, which may suggest nodal metastases; accuracy of detection of pathologically positive lymph nodes is 58% to 83%. Ultrasound may also prove useful for early detection of local recurrence after surgery.5Endoanal ultrasound is used to evaluate the layers of the anal canal. Internal anal sphincter, external anal sphincter, and puborectalis muscle can be differentiated. Endoanal ultrasound is particularly useful for detecting sphincter defects and for out-lining complex anal fistulas. MRI has also proven to be helpful in delineating the anatomy of fistulae, especially in complex cases. This modality increasingly is utilized for the evaluation of anal fistulae.Physiologic and Pelvic Floor InvestigationsAnorectal physiologic testing uses a variety of techniques to investigate the function of the pelvic floor. These techniques are useful in the

1	anal fistulae.Physiologic and Pelvic Floor InvestigationsAnorectal physiologic testing uses a variety of techniques to investigate the function of the pelvic floor. These techniques are useful in the evaluation of patients with incontinence, constipa-tion, rectal prolapse, obstructed defecation, and other functional disorders of the pelvic floor.Manometry. Anorectal manometry is performed by placing a pressure-sensitive catheter in the lower rectum. The catheter is then withdrawn through the anal canal and pressures recorded. A balloon attached to the tip of the catheter also can be used to test anorectal sensation. The resting pressure in the anal canal reflects the function of the internal anal sphincter (nor-mal 40–80 mmHg), whereas the squeeze pressure, defined as the maximum voluntary contraction pressure minus the resting pressure, reflects function of the external anal sphincter (normal 40–80 mmHg above resting pressure). The high-pressure zone estimates the length of the anal

1	contraction pressure minus the resting pressure, reflects function of the external anal sphincter (normal 40–80 mmHg above resting pressure). The high-pressure zone estimates the length of the anal canal (normal 2.0–4.0 cm). The rectoanal inhibitory reflex can be detected by inflating a balloon in the distal rectum; absence of this reflex is characteristic of Hirschsprung’s disease.Neurophysiology. Neurophysiologic testing assesses func-tion of the pudendal nerves and recruitment of puborectalis muscle fibers. Pudendal nerve terminal motor latency mea-sures the speed of transmission of a nerve impulse through the distal pudendal nerve fibers (normal 1.8–2.2 ms); prolonged latency suggests the presence of neuropathy. Electromyographic (EMG) recruitment assesses the contraction and relaxation of the puborectalis muscle during attempted defecation. Normally, recruitment increases when a patient is instructed to “squeeze” and decreases when a patient is instructed to “push.”

1	and relaxation of the puborectalis muscle during attempted defecation. Normally, recruitment increases when a patient is instructed to “squeeze” and decreases when a patient is instructed to “push.” Inappro-priate recruitment is an indication of paradoxical contraction (nonrelaxation of the puborectalis). Needle EMG has been used to map both the pudendal nerves and the anatomy of the internal and external sphincters. However, this examination is painful and poorly tolerated by most patients. Needle EMG has largely been replaced by pudendal nerve motor latency testing to assess pudendal nerve function and endoanal ultrasound to map the sphincters.Rectal Evacuation Studies. Rectal evacuation studies include the balloon expulsion test and video defecography. Balloon expulsion assesses a patient’s ability to expel an intrarectal bal-loon. Video defecography provides a more detailed assessment of defecation. In this test, barium paste is placed in the rectum, and defecation is then

1	a patient’s ability to expel an intrarectal bal-loon. Video defecography provides a more detailed assessment of defecation. In this test, barium paste is placed in the rectum, and defecation is then recorded fluoroscopically. Defecography is used to help diagnose obstructed defecation from nonrelax-ation of the puborectalis muscle or anal sphincter dyssynergy, increased perineal descent, rectal prolapse and intussusception, rectocele, and enterocele. The addition of vaginal contrast and intraperitoneal contrast is useful in delineating complex disor-ders of the pelvic floor.Laboratory StudiesFecal Occult Blood Testing and Fecal Immunohistochemical Testing. Fecal occult blood testing (FOBT) has been used as a screening test for colonic neoplasms in asymptomatic, average-risk individuals. The efficacy of this test is based on serial test-ing because the majority of colorectal malignancies will bleed intermittently. FOBT has been a nonspecific test for peroxidase contained in

1	The efficacy of this test is based on serial test-ing because the majority of colorectal malignancies will bleed intermittently. FOBT has been a nonspecific test for peroxidase contained in hemoglobin; consequently, occult bleeding from any gastrointestinal source will produce a positive result. Simi-larly, many foods (red meat, some fruits and vegetables, and vitamin C) will produce a false-positive result. Increased speci-ficity for cancer detection is possible by using fecal immuno-chemical test (FIT). Reported sensitivity of 79% and specificity Mucosal surfaceMucosa/Muscularis mucosaSubmucosaMuscularis propriaSerosa/Perirectal fatBAFigure 29-6. A. Schematic of the layers of the rectal wall observed on endorectal ultrasonography. B. Normal endorectal ultrasonog-raphy. (A. Used with permission from Charles O. Finne III, MD, Minneapolis, MN.)Brunicardi_Ch29_p1259-p1330.indd 126623/02/19 2:28 PM 1267COLON, RECTUM, AND ANUSCHAPTER 29of 94% has led to widespread use of FIT in

1	permission from Charles O. Finne III, MD, Minneapolis, MN.)Brunicardi_Ch29_p1259-p1330.indd 126623/02/19 2:28 PM 1267COLON, RECTUM, AND ANUSCHAPTER 29of 94% has led to widespread use of FIT in current population-based screening approaches. These tests rely on monoclonal or polyclonal antibodies to react with the intact globin portion of human hemoglobin and are more specific for identifying occult bleeding from the colon or rectum. Any positive FOBT or FIT mandates further investigation, usually by colonoscopy.6 More recently, stool DNA testing has been proposed for early detec-tion of colorectal cancer.7Stool Studies. Stool studies are often helpful in evaluating the etiology of diarrhea. Wet-mount examination reveals the pres-ence of fecal leukocytes, which may suggest colonic inflamma-tion or the presence of an invasive organism such as invasive E coli or Shigella species. Stool cultures can detect pathogenic bacteria, ova, and/or parasites. C difficile colitis is diagnosed by

1	or the presence of an invasive organism such as invasive E coli or Shigella species. Stool cultures can detect pathogenic bacteria, ova, and/or parasites. C difficile colitis is diagnosed by detecting bacterial toxin in the stool. Steatorrhea may be diag-nosed by adding Sudan red stain to a stool sample.Tumor Markers. Carcinoembryonic antigen (CEA) may be elevated in 60% to 90% of patients with colorectal cancer. Preoperative CEA level has recently been suggested to be a prognostic indicator.8 Despite this, CEA is not an effective screening tool for this malignancy. Many practitioners follow serial CEA levels after curative-intent surgery in order to detect early recurrence of colorectal cancer. However, this tumor marker is nonspecific, and no survival benefit associated with its serial measurements has yet been proven. It is also important to note that CEA may be mildly elevated in patients who smoke tobacco. Other biochemical markers (ornithine decarboxylase, urokinase) have been

1	measurements has yet been proven. It is also important to note that CEA may be mildly elevated in patients who smoke tobacco. Other biochemical markers (ornithine decarboxylase, urokinase) have been proposed, but none has yet proven sensi-tive or specific for detection, staging, or predicting prognosis of colorectal carcinoma.Genetic Testing. Although familial colorectal cancer syn-dromes, such as familial adenomatous polyposis (FAP) and hereditary nonpolyposis colon cancer (HNPCC) are rare, infor-mation about the specific genetic abnormalities underlying these disorders has led to significant interest in the role of genetic testing for colorectal cancer.9Tests for mutations in the adenomatous polyposis coli (APC) gene responsible for FAP and in mismatch repair genes responsible for HNPCC are commercially available and extremely accurate in families with known mutations. However, in the absence of an identified mutation, a negative result is uninformative. For individuals from

1	HNPCC are commercially available and extremely accurate in families with known mutations. However, in the absence of an identified mutation, a negative result is uninformative. For individuals from high-risk families without an identified mutation, increased surveillance is recommended.10 Although many of these mutations are also present in sporadic colorectal cancer, the accuracy of genetic testing in average-risk individuals is considerably lower, and these tests are not recommended for screening. Due to the potential psychosocial implications of genetic testing, it is strongly recommended that professional genetic counselors be involved in the care of any patient considering these tests.Evaluation of Common SymptomsPain Abdominal Pain Abdominal pain is a nonspecific symptom with myriad causes. Abdominal pain related to the colon and rectum can result from obstruction (either inflammatory or neo-plastic), inflammation, perforation, or ischemia. Plain X-rays and judicious use of

1	causes. Abdominal pain related to the colon and rectum can result from obstruction (either inflammatory or neo-plastic), inflammation, perforation, or ischemia. Plain X-rays and judicious use of contrast studies and/or a CT scan can often confirm the diagnosis. Gentle retrograde contrast studies (Gastrografin enema) may be useful in delineating the degree of colonic obstruction. Sigmoidoscopy and/or colonoscopy performed by an experienced endoscopist can assist in the diagnosis of ischemic colitis, infectious colitis, and inflamma-tory bowel disease. However, if perforation or near complete obstruction is suspected, colonoscopy and/or sigmoidoscopy are generally contraindicated. Evaluation and treatment of abdomi-nal pain from a colorectal source should follow the usual surgi-cal principles of a thorough history and physical examination, appropriate diagnostic tests, resuscitation, and appropriately timed surgical intervention.Pelvic Pain Pelvic pain can originate from the distal

1	of a thorough history and physical examination, appropriate diagnostic tests, resuscitation, and appropriately timed surgical intervention.Pelvic Pain Pelvic pain can originate from the distal colon and rectum or from adjacent urogenital structures. Tenesmus may result from proctitis or from a rectal or retrorectal mass, or fecal impaction in a constipated patient. Cyclical pain associated with menses, especially when accompanied by rectal bleeding, suggests a diagnosis of endometriosis. Pelvic inflammatory dis-ease also can produce significant abdominal and pelvic pain. The extension of a peridiverticular abscess or periappendiceal abscess into the pelvis may also cause pain. CT scan and/or MRI may be useful in differentiating these diseases. Proctoscopy (if tolerated) also can be helpful. Occasionally, laparoscopy will yield a diagnosis, although with access to high-quality imaging, indications for diagnostic surgery should be rare.Anorectal Pain. Anorectal pain is most often

1	helpful. Occasionally, laparoscopy will yield a diagnosis, although with access to high-quality imaging, indications for diagnostic surgery should be rare.Anorectal Pain. Anorectal pain is most often secondary to an anal fissure, perirectal abscess and/or fistula, or a throm-bosed hemorrhoid. Physical examination can usually differenti-ate these conditions. Other, less common causes of anorectal pain include anal canal neoplasms, perianal skin infection, and dermatologic conditions. Proctalgia fugax results from levator spasm and may present without any other anorectal findings. Physical exam is critical in evaluating patients with anorec-tal pain. If a patient is too tender to examine in the office, an examination under anesthesia is necessary. MRI or other imag-ing studies may be helpful in select cases where the etiology of pain is elusive.Lower Gastrointestinal Bleeding. The first goal in evaluat-ing and treating a patient with gastrointestinal hemorrhage is adequate

1	may be helpful in select cases where the etiology of pain is elusive.Lower Gastrointestinal Bleeding. The first goal in evaluat-ing and treating a patient with gastrointestinal hemorrhage is adequate resuscitation. The second goal is to identify the source of hemorrhage. Because the most common source of gastroin-testinal hemorrhage is esophageal, gastric, or duodenal, naso-gastric aspiration should always be performed; return of bile suggests that the source of bleeding is distal to the ligament of Treitz. If aspiration reveals blood or nonbile secretions, or if symptoms suggest an upper intestinal source, esophagogastro-duodenoscopy should be performed. Anoscopy and/or limited proctoscopy can identify hemorrhoidal bleeding. A techne-tium-99 (99mTc)-tagged red blood cell (tagged RBC scan) scan is extremely sensitive and is able to detect as little as 0.1 mL/h of bleeding; however, localization is imprecise. If the 99mTc-tagged RBC scan is positive, angiography can then be both

1	scan) scan is extremely sensitive and is able to detect as little as 0.1 mL/h of bleeding; however, localization is imprecise. If the 99mTc-tagged RBC scan is positive, angiography can then be both diagnostic and potentially therapeutic. If the patient is hemodynamically stable, a rapid bowel preparation (over 4–6 hours) can be per-formed to allow colonoscopy. Colonoscopy may identify the cause of the bleeding, and cautery or injection/application of epinephrine into the bleeding site may be used to control hem-orrhage. A SPECT/CT may be helpful if other modalities have not achieved localization, particularly in distinguishing between small intestinal and colon sources. Colectomy may be required if bleeding persists despite interventions. Intraoperative colo-noscopy and/or enteroscopy may assist in localizing bleeding. If colectomy is required, a segmental resection is preferred if the bleeding source can be localized. “Blind” subtotal colectomy Brunicardi_Ch29_p1259-p1330.indd

1	may assist in localizing bleeding. If colectomy is required, a segmental resection is preferred if the bleeding source can be localized. “Blind” subtotal colectomy Brunicardi_Ch29_p1259-p1330.indd 126723/02/19 2:28 PM 1268SPECIFIC CONSIDERATIONSPART IIvery rarely may be required in a patient who is hemodynami-cally unstable with ongoing colonic hemorrhage of an unknown source. In this setting, just prior to proceeding with a “blind” subtotal colectomy, it is crucial to irrigate the rectosigmoid and reexamine the mucosa of the anal canal and rectum by anoscopy and proctoscopy to ensure the source of ongoing bleeding is not distal to the planned resection margin (Fig. 29-7).Occult blood loss from the gastrointestinal tract may mani-fest as iron-deficiency anemia or may be detected with FOBT or FIT. Because colon neoplasms bleed intermittently and rarely present with rapid hemorrhage, the presence of occult fecal blood should always prompt a colonoscopy. Unexplained iron-deficiency

1	FOBT or FIT. Because colon neoplasms bleed intermittently and rarely present with rapid hemorrhage, the presence of occult fecal blood should always prompt a colonoscopy. Unexplained iron-deficiency anemia is also an indication for colonoscopy.Hematochezia is commonly caused by hemorrhoids or a fissure. Sharp, knife-like pain and bright red rectal bleeding with bowel movements suggest the diagnosis of fissure. Pain-less, bright red rectal bleeding with bowel movements is often secondary to a friable internal hemorrhoid that is easily detected by anoscopy. In the absence of a painful, obvious fissure, any patient with rectal bleeding should undergo a careful digital rectal examination, anoscopy, and proctosigmoidoscopy. Fail-ure to diagnose a source in the distal anorectum should prompt colonoscopy.Constipation and Obstructed Defecation. Constipation is an extremely common complaint, affecting more than 4 million people in the United States. Despite the prevalence of this prob-lem,

1	colonoscopy.Constipation and Obstructed Defecation. Constipation is an extremely common complaint, affecting more than 4 million people in the United States. Despite the prevalence of this prob-lem, there is lack of agreement about an appropriate definition of constipation. Patients may describe infrequent bowel move-ments, hard stools, or excessive straining. A careful history of these symptoms often clarifies the nature of the problem.Constipation has many causes. Underlying metabolic, pharmacologic, endocrine, psychological, and neurologic causes often contribute to the problem. A stricture or mass lesion should be excluded by colonoscopy, barium enema, or CT colonography. After these causes have been excluded, eval-uation focuses on differentiating slow-transit constipation from outlet obstruction. Transit studies, in which radiopaque markers are swallowed and then followed radiographically, are useful for diagnosing slow-transit constipation. In this study, patients ingest

1	outlet obstruction. Transit studies, in which radiopaque markers are swallowed and then followed radiographically, are useful for diagnosing slow-transit constipation. In this study, patients ingest radiopaque studies and are followed radiographically for 5 days. Retention of 20% or greater of these markers in the colon demonstrated slow transit. If these markers are congregated in the rectosigmoid colon and rectum, obstructed defecation/outlet obstruction is suggested. Anorectal manometry and EMG can detect nonrelaxation of the puborectalis, which contributes to outlet obstruction. The absence of an anorectal inhibitory reflex Massive life-threatening bleedingPositiveRebleedingRebleedingModerate bleedingcontinuedBleeding stopped or slowed downPositivePositivePositivePositivePositivePositivePositivePositiveNegativeNegativeNegativeNegativeNegativeNegativeNegativeNegativeNegativeFailFailorAcute colonic bleedingVolume resuscitationplus blood transfusionNG aspiratenegativeNG

1	colonic bleedingVolume resuscitationplus blood transfusionNG aspiratenegativeNG aspiratepositiveProctoscopyRule out anorectal bleedingGastroduodenoscopy+ endoscopic treatmentElective colonoscopyMesenteric arteriographyMesenteric arteriographyUrgent colonoscopy99MTc RBC scintigraphyColonoscopic treatmentor explore, segmentalresectionEndoscopictreatmentVasopressinor emboliVasopressinor emboliExplore, intraoperativeendoscopyExplore, intraoperativeendoscopyExplore, intraoperativeendoscopyExplore, intraoperativeendoscopyObserveSegmentalresectionTotalcolectomyTotalcolectomyTotalcolectomyTotalcolectomySegmentalresectionSegmentalresectionSegmentalresectionSegmentalresectionSegmentalresectionSegmentalresectionSee moderate bleedingor massive bleedingFigure 29-7. Algorithm for treatment of colorectal hemorrhage. NG = nasogastric; 99mTc = technetium-99; RBC = red blood cell. (Repro-duced with permission of Gordon PH, Nivatvongs S: Principles and Practice of Surgery for the Colon, Rectum, and

1	hemorrhage. NG = nasogastric; 99mTc = technetium-99; RBC = red blood cell. (Repro-duced with permission of Gordon PH, Nivatvongs S: Principles and Practice of Surgery for the Colon, Rectum, and Anus, 2nd ed. New York, NY: Marcel Dekker, Inc; 1999.)Brunicardi_Ch29_p1259-p1330.indd 126823/02/19 2:28 PM 1269COLON, RECTUM, AND ANUSCHAPTER 29suggests Hirschsprung’s disease and may prompt a rectal muco-sal biopsy. Defecography can identify rectal prolapse, intussus-ception, rectocele, or enterocele.Medical management is the mainstay of therapy for consti-pation and includes fiber, increased fluid intake, and laxatives. Outlet obstruction from nonrelaxation of the puborectalis or anal sphincter dyssynergy often responds to biofeedback. Surgery to correct rectocele and rectal prolapse (with or without sigmoid resection) has a variable effect on symptoms of constipation but can be successful in selected patients. Subtotal colectomy is considered only for patients with severe slow-transit

1	or without sigmoid resection) has a variable effect on symptoms of constipation but can be successful in selected patients. Subtotal colectomy is considered only for patients with severe slow-transit consti-pation (colonic inertia) refractory to maximal medical inter-ventions. While this operation almost always increases bowel movement frequency, complaints of diarrhea, incontinence, and abdominal pain are not infrequent, and patients should be care-fully selected and counseled.11Diarrhea and Irritable Bowel Syndrome. Diarrhea is also a common complaint and is usually a self-limited symptom of infectious gastroenteritis. If diarrhea is chronic or is accom-panied by bleeding or abdominal pain, further investigation is warranted. Bloody diarrhea and pain are characteristic of colitis; etiology can be an infection (invasive E coli, Shigella, Salmonella, Campylobacter, Entamoeba histolytica, or C difficile), inflammatory bowel disease (ulcerative colitis or Crohn’s coli-tis), or

1	etiology can be an infection (invasive E coli, Shigella, Salmonella, Campylobacter, Entamoeba histolytica, or C difficile), inflammatory bowel disease (ulcerative colitis or Crohn’s coli-tis), or ischemia. Stool wet-mount and culture can often diag-nose infection. Sigmoidoscopy or colonoscopy can be helpful in diagnosing inflammatory bowel disease or ischemia. However, if the patient has abdominal tenderness, particularly with peri-toneal signs, or any other evidence of perforation, endoscopy is contraindicated.Chronic diarrhea may present a more difficult diagnos-tic dilemma. Chronic ulcerative colitis, Crohn’s colitis, infec-tion, malabsorption, and short gut syndrome can cause chronic diarrhea. Rarely, carcinoid syndrome and islet cell tumors (vasoactive intestinal peptide–secreting tumor [VIPoma], somatostatinoma, gastrinoma) present with this symptom. Large villous lesions may cause secretory diarrhea. Collagenous colitis can cause diarrhea without any obvious mucosal

1	tumor [VIPoma], somatostatinoma, gastrinoma) present with this symptom. Large villous lesions may cause secretory diarrhea. Collagenous colitis can cause diarrhea without any obvious mucosal abnor-mality. Along with stool cultures, tests for malabsorption, and metabolic investigations, colonoscopy can be invaluable in dif-ferentiating these causes. Biopsies should be taken even if the colonic mucosa appears grossly normal.Irritable bowel syndrome is a particularly troubling con-stellation of symptoms consisting of crampy abdominal pain, bloating, constipation, and urgent diarrhea. Workup reveals no underlying anatomic or physiologic abnormality. Once other disorders have been excluded, dietary restrictions and avoid-ance of caffeine, alcohol, and tobacco may help to alleviate symptoms. Antispasmodics and bulking agents may be helpful.Incontinence. The true incidence of fecal incontinence is unknown, but has been estimated to occur in 10 to 13 individu-als per 1000 people older than

1	Antispasmodics and bulking agents may be helpful.Incontinence. The true incidence of fecal incontinence is unknown, but has been estimated to occur in 10 to 13 individu-als per 1000 people older than age 65 years. Incontinence ranges in severity from occasional leakage of gas and/or liquid stool to daily loss of solid stool. The underlying cause of incontinence is often multifactorial, and diarrhea is often contributory. In gen-eral, causes of incontinence can be classified as neurogenic or anatomic. Neurogenic causes include diseases of the central ner-vous system and spinal cord along with pudendal nerve injury. Anatomic causes include congenital abnormalities, procidentia (rectal prolapse), overflow incontinence secondary to impac-tion or an obstructing neoplasm, and trauma. The most common Figure 29-8. A. Endoanal ultrasonography showing the normal layers of the anal canal. B. Endoanal ultrasonography with anterior sphincter defect from birthing injury. EAS = external anal

1	most common Figure 29-8. A. Endoanal ultrasonography showing the normal layers of the anal canal. B. Endoanal ultrasonography with anterior sphincter defect from birthing injury. EAS = external anal sphincter; IAS = internal anal sphincter. (Used with permission from Charles O. Finne III, MD, Minneapolis, MN.)ABtraumatic cause of incontinence is injury to the anal sphincter during vaginal delivery. Other causes include anorectal surgery, impalement, and pelvic fracture.After a thorough medical evaluation to detect underly-ing conditions that might contribute to incontinence, evalua-tion focuses on assessment of the anal sphincter and pudendal nerves. Pudendal nerve terminal motor latency testing may detect neuropathy. Anal manometry can detect low resting and squeeze pressures. Physical examination and defecography can detect rectal prolapse. Endoanal ultrasound is invaluable in diagnosing sphincter defects (Fig. 29-8).Therapy depends on the underlying abnormality. Diarrhea should be

1	and defecography can detect rectal prolapse. Endoanal ultrasound is invaluable in diagnosing sphincter defects (Fig. 29-8).Therapy depends on the underlying abnormality. Diarrhea should be treated medically (fiber, antidiarrheal agents). Even in the absence of frank diarrhea, the addition of dietary fiber may improve continence. Some patients may respond to bio-feedback and this approach may be considered in patients who fail dietary modification. Many patients with a sphincter defect are candidates for an overlapping sphincteroplasty. Sacral nerve Brunicardi_Ch29_p1259-p1330.indd 126923/02/19 2:28 PM 1270SPECIFIC CONSIDERATIONSPART IIstimulation been shown to decrease episodes of fecal incon-tinence and has proven durability in the long term (5 years). The artificial bowel sphincter may be useful in patients who fail other interventions. Other options include radiofrequency energy to the anal canal (SECCA procedure), magnetic anal sphincter, and injectable submucosal bulking

1	may be useful in patients who fail other interventions. Other options include radiofrequency energy to the anal canal (SECCA procedure), magnetic anal sphincter, and injectable submucosal bulking agents, but long-term efficacy has not yet been proven.12-14 Finally, a stoma can provide relief for severely incontinent patients who have failed or are not candidates for other interventions.GENERAL SURGICAL CONSIDERATIONSColorectal resections are performed for a wide variety of condi-tions, including neoplasms (benign and malignant), inflamma-tory bowel diseases, and other benign conditions. Although the indication and urgency for surgery will alter some of the tech-nical details, the operative principles of colorectal resections, anastomoses, and use of ostomies are well established.ResectionsThe mesenteric clearance technique dictates the extent of colonic resection and is determined by the nature of the primary pathology (malignant or benign), the intent of the resection (curative or

1	mesenteric clearance technique dictates the extent of colonic resection and is determined by the nature of the primary pathology (malignant or benign), the intent of the resection (curative or palliative), the precise location(s) of the primary pathology, and the condition of the mesentery (thin and soft or thick and indurated). In general, a proximal mesenteric ligation will eliminate the blood supply to a greater length of colon and require a more extensive “colectomy.” Curative resection of a colorectal cancer is usually best accomplished by performing a proximal mesenteric vessel ligation and radical mesenteric clearance of the lymphatic drainage basin of the tumor site (Fig. 29-9). Resection of a benign process does not require wide mesenteric clearance.Emergency Resection. Emergency resection may be required because of obstruction, perforation, or hemorrhage. In this set-ting, the bowel is almost always unprepared and the patient may be unstable. The surgical principles

1	resection may be required because of obstruction, perforation, or hemorrhage. In this set-ting, the bowel is almost always unprepared and the patient may be unstable. The surgical principles described earlier apply, and an attempt should be made to resect the involved segment along with its lymphovascular supply. If the resection involves the right colon or proximal transverse colon (right or extended right colectomy), a primary ileocolonic anastomosis can usually be performed safely as long as the remaining bowel appears healthy and the patient is stable. For left-sided tumors, the traditional approach has involved resection of the involved bowel and end colostomy, with or without a mucus fistula. However, there is an increasing body of data to suggest that a primary anasto-mosis without a bowel preparation or with an on-table lavage, with or without a diverting ileostomy, may be equally safe in this setting. If the proximal colon appears unhealthy (vascular compromise, serosal

1	a bowel preparation or with an on-table lavage, with or without a diverting ileostomy, may be equally safe in this setting. If the proximal colon appears unhealthy (vascular compromise, serosal tears, perforation), a subtotal colectomy can be performed with a small bowel to rectosigmoid anastomo-sis. Resection and diversion (ileostomy or colostomy) remain safe and appropriate if the bowel appears compromised or if the patient is unstable, malnourished, or immunosuppressed.Minimally Invasive Techniques of Resection. With advances in minimally invasive technology, many proce-dures that previously have required laparotomy can now be performed laparoscopically, with hand-assisted laparoscopy (HAL), or robotically.15,16 Potential advantages of minimally invasive surgery include improved cosmetic result, decreased postoperative pain, and earlier return of bowel function. More-over, some experimental data suggest that minimally invasive 1operations have less immunosuppressive impact on the

1	result, decreased postoperative pain, and earlier return of bowel function. More-over, some experimental data suggest that minimally invasive 1operations have less immunosuppressive impact on the patient and thus might improve postoperative outcome and even long-term survival. To date, most studies have demonstrated equiva-lence between laparoscopic, HAL, and open resection in terms of extent of resection. Return of bowel function and length of hospital stay are highly variable. Long-term outcome has yet to be determined; however, short-term quality of life appears to be improved by laparoscopy.17,18 Laparoscopic total mesorec-tal excision for rectal cancer, however, may not be appropriate. The most recent advances in minimally invasive surgery involve use of robotics and telemanipulation in which the surgeon oper-ates from a console remote from the patient. These procedures have been rapidly gaining in popularity, especially for pelvic and rectal resections. Early studies suggest

1	in which the surgeon oper-ates from a console remote from the patient. These procedures have been rapidly gaining in popularity, especially for pelvic and rectal resections. Early studies suggest equivalence between robotic resections and laparoscopic/HAL resections.19In addition, some proponents have suggested that robotic procedures may be easier to learn (a shorter “learning curve”) and that robotic surgery may be ergonomically better for the operating surgeon. Nevertheless, long-term superiority, or even equivalence, has yet to be demonstrated, and these advanced technologies are likely to be associated with significant cost.Colectomy. A variety of terms are used to describe different types of colectomy (Fig. 29-10).Ileocolic Resection An ileocolic resection describes a limited resection of the terminal ileum, cecum, and appendix. It is used to remove disease involving these segments of the intestine (e.g., ileocecal Crohn’s disease) and benign lesions or incur-able cancers

1	resection of the terminal ileum, cecum, and appendix. It is used to remove disease involving these segments of the intestine (e.g., ileocecal Crohn’s disease) and benign lesions or incur-able cancers arising in the terminal ileum, cecum, and, occa-sionally, the appendix. If curable malignancy is suspected, more radical resections, such as a right hemicolectomy, are gener-ally indicated. The ileocolic vessels are ligated and divided. A variable length of small intestine may be resected depending on the disease process. A primary anastomosis is created between the distal small bowel and the ascending colon. It is technically difficult to perform an anastomosis at or just proximal to the ileocecal valve; therefore, if the most distal ileum needs to be resected, the cecum is generally also removed.Right Colectomy A right colectomy is used to remove lesions or disease in the right colon and is oncologically the most appro-priate operation for curative intent resection of proximal colon

1	removed.Right Colectomy A right colectomy is used to remove lesions or disease in the right colon and is oncologically the most appro-priate operation for curative intent resection of proximal colon carcinoma. The ileocolic vessels, right colic vessels, and right branches of the middle colic vessels are ligated and divided. Approximately 10 cm of terminal ileum are usually included in the resection. A primary ileal-transverse colon anastomosis is almost always possible.Extended Right Colectomy An extended right colectomy may be used for curative intent resection of lesions located at the hepatic flexure or proximal transverse colon. A standard right colectomy is extended to include ligation of the middle colic vessels at their base. The right colon and proximal trans-verse colon are resected, and a primary anastomosis is created between the distal ileum and distal transverse colon. Such an anastomosis relies on the marginal artery of Drummond. If the blood supply to the distal

1	resected, and a primary anastomosis is created between the distal ileum and distal transverse colon. Such an anastomosis relies on the marginal artery of Drummond. If the blood supply to the distal transverse colon is questionable, the resection is extended distally beyond the splenic flexure to well-perfused descending colon where the ileocolic anastomosis can be performed safely.Transverse Colectomy Lesions in the mid and distal trans-verse colon may be resected by ligating the middle colic vessels and resecting the transverse colon, followed by a colocolonic anastomosis. However, an extended right colectomy with an Brunicardi_Ch29_p1259-p1330.indd 127023/02/19 2:28 PM 1271COLON, RECTUM, AND ANUSCHAPTER 29anastomosis between the terminal ileum and descending colon may be a safer anastomosis with an equivalent functional result.Left Colectomy For lesions or disease states confined to the distal transverse colon, splenic flexure, or descending colon, a left colectomy is performed.

1	with an equivalent functional result.Left Colectomy For lesions or disease states confined to the distal transverse colon, splenic flexure, or descending colon, a left colectomy is performed. The left branches of the middle colic vessels, the left colic vessels, and the first branches of the sigmoid vessels are ligated. A colocolonic anastomosis can usually be performed.Extended Left Colectomy An extended left colectomy is an option for removing lesions in the distal transverse colon. In this operation, the left colectomy is extended proximally to include the right branches of the middle colic vessels.Sigmoid Colectomy Lesions in the sigmoid colon require ligation and division of the sigmoid branches of the inferior mesenteric artery. In general, the entire sigmoid colon should be resected to the level of the peritoneal reflection and an BADCFEFigure 29-9. Extent of resection for carcinoma of the colon. A. Cecal cancer. B. Hepatic flexure cancer. C. Transverse colon cancer. D.

1	to the level of the peritoneal reflection and an BADCFEFigure 29-9. Extent of resection for carcinoma of the colon. A. Cecal cancer. B. Hepatic flexure cancer. C. Transverse colon cancer. D. Splenic flexure cancer. E. Descending colon cancer. F. Sigmoid colon cancer.Brunicardi_Ch29_p1259-p1330.indd 127123/02/19 2:28 PM 1272SPECIFIC CONSIDERATIONSPART IIanastomosis created between the descending colon and upper rectum. Full mobilization of the splenic flexure is often required to create a tension-free anastomosis.Total and Subtotal Colectomy Total or subtotal colectomy is occasionally required for patients with fulminant colitis, attenu-ated FAP, or synchronous colon carcinomas. In this procedure, the ileocolic vessels, right colic vessels, middle colic vessels, and left colic vessels are ligated and divided. The superior rectal vessels are preserved. If it is desired to preserve the sigmoid, the distal sigmoid vessels are left intact, and an anastomosis is created between the

1	are ligated and divided. The superior rectal vessels are preserved. If it is desired to preserve the sigmoid, the distal sigmoid vessels are left intact, and an anastomosis is created between the ileum and distal sigmoid colon (subtotal colectomy with ileosigmoid anastomosis). If the sigmoid is to be resected, the sigmoidal vessels are ligated and divided, and the ileum is anastomosed to the upper rectum (total abdominal colectomy with ileorectal anastomosis). If an anastomosis is contraindicated, an end ileostomy is created, and the remain-ing sigmoid or rectum is managed either as a mucus fistula or a Hartmann’s pouch.Proctocolectomy Total Proctocolectomy In this procedure, the entire colon, rectum, and anus are removed, and the ileum is brought to the skin as a Brooke ileostomy.Restorative Proctocolectomy (Ileal Pouch–Anal Anasto-mosis) The entire colon and rectum are resected, but the anal sphincter muscles and a variable portion of the distal anal canal are preserved. Bowel

1	Proctocolectomy (Ileal Pouch–Anal Anasto-mosis) The entire colon and rectum are resected, but the anal sphincter muscles and a variable portion of the distal anal canal are preserved. Bowel continuity is restored by anastomosis of an ileal reservoir to the anal canal. The original technique included a transanal mucosectomy and hand-sewn ileoanal anastomosis. Proponents of this technique argue that mucosectomy guarantees removal of all of the diseased mucosa, including the anal transi-tion zone, and therefore decreases the risk of ongoing disease, dysplasia, and carcinoma. Opponents cite the increased risk of incontinence after mucosectomy and argue that even meticulous technique invariably leaves behind mucosal “islands” that are subsequently hidden under the anastomosis. However, persistent or recurrent dysplasia in the anal transition zone is uncommon (4.5%), and cancers occur even more rarely.19 Moreover, the “double-staple” technique using the circular stapling devices is

1	persistent or recurrent dysplasia in the anal transition zone is uncommon (4.5%), and cancers occur even more rarely.19 Moreover, the “double-staple” technique using the circular stapling devices is considerably simpler than mucosectomy and a hand-sewn anas-tomosis and may be associated with a better functional outcome (Fig. 29-11).20-22 Regardless of the anastomotic technique, many surgeons recommend that patients undergo annual surveillance of the anastomosis and/or anal transition zone by digital rectal exam and anoscopy or proctoscopy.The neorectum is made by anastomosis of the terminal ileum aligned in a “J,” “S,” or “W” configuration. Because func-tional outcomes are similar and because the J-pouch is the sim-plest to construct, it has become the most used configuration. ABCDEFGHIJKLFigure 29-10. Terminology of types of colorectal resections: A→C Ileocecectomy; + A + B→D Ascending colectomy; + A + B→F Right hemicolectomy; + A + B→G Extended right hemi-colectomy; + E + F→G + H

1	29-10. Terminology of types of colorectal resections: A→C Ileocecectomy; + A + B→D Ascending colectomy; + A + B→F Right hemicolectomy; + A + B→G Extended right hemi-colectomy; + E + F→G + H Transverse colectomy; G→I Left hemicolectomy; F→I Extended left hemicolectomy; J + K Sigmoid colectomy; + A + B→J Subtotal colectomy; + A + B→K Total col-ectomy; + A + B→L Total proctocolectomy. (Reproduced with per-mission from Fielding LP, Goldberg SM: Rob & Smith’s Operative: Surgery of the Colon, Rectum, and Anus. London: Elsevier; 1993.)BCAFigure 29-11. After a total colectomy and resection of the rec-tum (A), the anal canal with a short cuff of transitional mucosa and sphincter muscles is preserved (B). An ileal J-pouch has been constructed and is anastomosed to the anal canal using a double-staple technique (C). (Reproduced with permission from Bell RH, Rikkers LF, Mulholland M: Digestive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins;

1	using a double-staple technique (C). (Reproduced with permission from Bell RH, Rikkers LF, Mulholland M: Digestive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)Brunicardi_Ch29_p1259-p1330.indd 127223/02/19 2:29 PM 1273COLON, RECTUM, AND ANUSCHAPTER 29With increasing experience in laparoscopic and robotic colec-tomy, some centers have begun performing total proctocolec-tomy with ileal pouch–anal reconstruction using minimally invasive surgical techniques.15 Most surgeons perform a proxi-mal ileostomy to divert succus from the newly created pouch in an attempt to minimize the consequences of leak and sepsis, especially in patients who are malnourished or immunosup-pressed (Fig. 29-12). The ileostomy is then closed 6 to 12 weeks later, after a contrast study confirms the integrity of the pouch. In low-risk patients, however, there are reports of successful creation of an ileoanal pouch without a diverting stoma.Anterior Resection. Anterior

1	contrast study confirms the integrity of the pouch. In low-risk patients, however, there are reports of successful creation of an ileoanal pouch without a diverting stoma.Anterior Resection. Anterior resection is the general term used to describe resection of the rectum from an abdominal approach to the pelvis with no need for a perineal, sacral, or other incision. Three types of anterior resection have been described.High Anterior Resection A high anterior resection is the term used to describe resection of the distal sigmoid colon and upper rectum and is the appropriate operation for benign lesions and disease at the rectosigmoid junction such as diverticulitis. The upper rectum is mobilized, but the pelvic peritoneum is not divided and the rectum is not mobilized fully from the concavity of the sacrum. The inferior mesenteric artery is ligated at its base, and the inferior mesenteric vein, which follows a different course than the artery, is ligated separately. A primary

1	concavity of the sacrum. The inferior mesenteric artery is ligated at its base, and the inferior mesenteric vein, which follows a different course than the artery, is ligated separately. A primary anasto-mosis (usually end-to-end) between the colon and rectal stump with a short cuff of peritoneum surrounding its anterior two thirds generally can be performed.Low Anterior Resection. A low anterior resection is used to remove lesions in the upper and mid rectum. The rectosigmoid is mobilized, the pelvic peritoneum is opened, and the inferior mesenteric artery is ligated and divided either at its origin from the aorta or just distal to the takeoff of the left colic artery. The rectum is mobilized from the sacrum by sharp dissection under direct view within the endopelvic fascial plane. The dissec-tion may be performed distally to the anorectal ring, extend-ing posteriorly through the rectosacral fascia to the coccyx and anteriorly through Denonvilliers’ fascia to the vagina in women or

1	dissec-tion may be performed distally to the anorectal ring, extend-ing posteriorly through the rectosacral fascia to the coccyx and anteriorly through Denonvilliers’ fascia to the vagina in women or the seminal vesicles and prostate in men. The rectum and accompanying mesorectum are divided at the appropriate level, depending on the nature of the lesion. A low rectal anastomosis usually requires mobilization of the splenic flexure and ligation and division of the inferior mesenteric vein just inferior to the pancreas. Circular stapling devices have greatly facilitated the conduct and improved the safety of the colon to extraperitoneal rectal anastomosis.Extended Low Anterior Resection An extended low anterior resection is necessary to remove lesions located in the distal rectum, but several centimeters above the sphincter. The rectum is fully mobilized to the level of the levator ani muscle just as for a low anterior resection, but the anterior dissection is extended along the

1	several centimeters above the sphincter. The rectum is fully mobilized to the level of the levator ani muscle just as for a low anterior resection, but the anterior dissection is extended along the rectovaginal septum in women and distal to the semi-nal vesicles and prostate in men. After resection at this level, a coloanal anastomosis can be created using one of a variety of techniques. An end-to-end stapled or hand-sewn anastomo-sis has traditionally been the procedure of choice. However, the functional consequences of a “straight” anastomosis have led to consideration for creation of a colon J-pouch to increase the capacity of the neorectal reservoir. A side-to-end anastomosis can be constructed by placing the anvil of an EEA stapler 3 to 4 cm away from the stapled end of the proximal colon, with simi-lar functional outcomes to the colon J-pouch reconstruction.20-22 Because the risk of an anastomotic leak and subsequent sepsis is higher when an anastomosis is created in the distal

1	colon, with simi-lar functional outcomes to the colon J-pouch reconstruction.20-22 Because the risk of an anastomotic leak and subsequent sepsis is higher when an anastomosis is created in the distal rectum or anal canal, creation of a temporary ileostomy should be consid-ered in this setting, especially if the patient has received neoad-juvant radiation.23Although an anastomosis may be technically feasible very low in the rectum or anal canal, it is important to note that post-operative function may be poor. Because the descending colon lacks the distensibility of the rectum, the reservoir function may be compromised. Pelvic radiation, prior anorectal surgery, and obstetrical trauma may cause unsuspected sphincter damage. Finally, a very low anastomosis may involve and compromise the upper sphincter. Creation of a colon J-pouch or transverse coloplasty may improve function, but few long-term studies have addressed this issue.20-22A history of sphincter damage or any degree of

1	the upper sphincter. Creation of a colon J-pouch or transverse coloplasty may improve function, but few long-term studies have addressed this issue.20-22A history of sphincter damage or any degree of inconti-nence is a relative contraindication for a coloanal anastomosis. In such patients, an end colostomy may be a more satisfac-tory option.Hartmann’s Procedure and Mucus Fistula. Hartmann’s procedure refers to a colon or rectal resection without an 2Figure 29-12. Ileal S-pouch anal anastomosis with temporary loop ileostomy. (Reproduced with permission from Bell RH, Rikkers LF, Mulholland M: Digestive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)Brunicardi_Ch29_p1259-p1330.indd 127323/02/19 2:29 PM 1274SPECIFIC CONSIDERATIONSPART IIanastomosis in which a colostomy or ileostomy is created and the distal colon or rectum is left as a blind pouch. The term is typically used when the left or sigmoid colon is resected and the closed off rectum

1	in which a colostomy or ileostomy is created and the distal colon or rectum is left as a blind pouch. The term is typically used when the left or sigmoid colon is resected and the closed off rectum is left in the pelvis. If the distal colon is long enough to reach the abdominal wall, a mucus fistula can be created by opening the defunctioned bowel and suturing the open lumen to the skin.Abdominoperineal Resection. An abdominoperineal resec-tion (APR) involves removal of the entire rectum, anal canal, and anus with construction of a permanent colostomy from the descending or sigmoid colon. The abdominal-pelvic portion of this operation proceeds in the same fashion as described for an extended low anterior resection. The perineal dissection can be performed with the patient in lithotomy position (often by a second surgeon) or in the prone position after closure of the abdomen and creation of the colostomy. For cancer, the perineal dissection is designed to excise the anal canal with a

1	(often by a second surgeon) or in the prone position after closure of the abdomen and creation of the colostomy. For cancer, the perineal dissection is designed to excise the anal canal with a wide cir-cumferential margin including a cylindrical cuff of the levator muscle. A deliberate resection of the levator muscles near their bony attachments, in order to avoid opening the space between the tumor and the levator ani, is known as the extralevator abdomi-noperineal resection (ELAPE). ELAPE is useful for low, locally advanced rectal cancers, but routine use for all rectal cancer has not been shown to improve cancer outcomes.24 Primary wound closure is usually successful, but a large perineal defect, espe-cially if preoperative radiation has been used, may require a vascularized flap closure in some patients.25 For benign disease, proctectomy may be performed using an intersphincteric dissec-tion between the internal and external sphincters. This approach minimizes the perineal wound,

1	in some patients.25 For benign disease, proctectomy may be performed using an intersphincteric dissec-tion between the internal and external sphincters. This approach minimizes the perineal wound, making it easier to close because the levator muscle remains intact.AnastomosesAnastomoses may be created between two segments of bowel in a multitude of ways. The geometry of the anastomosis may be end-to-end, end-to-side, side-to-end, or side-to-side. The anas-tomotic technique may be hand-sewn or stapled (Fig. 29-13). The submucosal layer of the intestine provides the strength of the bowel wall and must be incorporated in the anastomosis to assure healing. The choice of anastomosis depends on the opera-tive anatomy and surgeon preference. Although many surgeons advocate one method over another, none has been proven to be superior. Accurate approximation of two well-vascularized, healthy limbs of bowel without tension in a normotensive, well-nourished patient almost always results in a

1	none has been proven to be superior. Accurate approximation of two well-vascularized, healthy limbs of bowel without tension in a normotensive, well-nourished patient almost always results in a good outcome. Anastomoses at highest risk of leak or stricture are those that are in the distal rectal or anal canal, involve irradiated or dis-eased intestine including perforation with peritoneal soilage, are inadvertently fashioned above a partial distal obstruction, or are performed in malnourished, immunosuppressed, or ill patient.Anastomotic Configuration End-to-End An end-to-end anastomosis can be performed when two segments of bowel are roughly the same caliber. This technique is most often employed in rectal resections, but may be used for colocolostomy or small bowel anastomoses.End-to-Side An end-to-side configuration is useful when one limb of bowel is larger than the other. This most commonly occurs in the setting of chronic obstruction.Side-to-End A side-to-end anastomosis is used

1	end-to-side configuration is useful when one limb of bowel is larger than the other. This most commonly occurs in the setting of chronic obstruction.Side-to-End A side-to-end anastomosis is used when the proxi-mal bowel is of smaller caliber than the distal bowel. Ileorectal anastomoses commonly make use of this configuration. A side-to-end anastomosis may have a less tenuous blood supply than an end-to-end anastomosis.Side-to-Side A side-to-side anastomosis allows a large, well-vascularized connection to be created on the antimesenteric side of two segments of intestine. This technique is commonly used in ileocolic and small bowel anastomoses.Anastomotic Technique Hand-Sutured Technique Any of the configurations described earlier may be created using a hand-sutured or stapled technique. Hand-sutured anastomoses may be single layer, using either running or interrupted stitches, or double layer. A double-layer anastomosis usually consists of a continuous inner layer and an interrupted

1	Hand-sutured anastomoses may be single layer, using either running or interrupted stitches, or double layer. A double-layer anastomosis usually consists of a continuous inner layer and an interrupted outer layer. Suture material may be either perma-nent or absorbable. After distal rectal or anal canal resection, a transanal, hand-sewn coloanal anastomosis may be necessary to restore bowel continuity. This can be done in conjunction with an anal canal mucosectomy to allow the anastomosis to be cre-ated at the dentate line.Stapled Techniques Linear cutting/stapling devices are used to divide the bowel and to create side-to-side anastomoses. The anastomosis may be reinforced with interrupted sutures if desired. Circular cutting/stapling devices can create end-to-end, end-to-side, or side-to-end anastomoses. These instruments are particularly useful for creating low rectal or anal canal anas-tomoses where the anatomy of the pelvis makes a hand-sewn anastomosis technically difficult or

1	anastomoses. These instruments are particularly useful for creating low rectal or anal canal anas-tomoses where the anatomy of the pelvis makes a hand-sewn anastomosis technically difficult or impossible.Following resection of the colorectum, a stapled end-to-end colorectal, coloanal canal, or ileal pouch–anal canal anasto-mosis may be created by one of two techniques. With the open purse-string technique, the distal rectal stump purse-string is placed by hand, and the assembled circular stapler is inserted into the anus and guided up to the rectal purse-string. The sta-pler is opened, and the distal purse-string is tied. A purse-string is placed in the distal end of the proximal colon; the proximal colon is placed over the anvil and the purse-string tightened. The stapler is closed and fired (Fig. 29-14). With the alterna-tive double-staple technique, the distal rectum or anal canal is closed with a transverse staple line. The circular stapler is inserted through the anus without its

1	(Fig. 29-14). With the alterna-tive double-staple technique, the distal rectum or anal canal is closed with a transverse staple line. The circular stapler is inserted through the anus without its anvil until the cartridge effaces the transverse staple line. The stapler is opened, causing the trocar to perforate through the rectal stump adjacent to the transverse staple line. The anastomosis in then completed as described earlier (see Fig. 29-11). If the stapler cannot advance to the end of the rectal stump, further dissection of the stump may be necessary to optimize tissue apposition. After firing and removing the stapler, the resulting anastomotic rings should be inspected to ensure that they are full-thickness and concentric, and in cases of rectal cancer, the distal anastomotic ring should be sent to pathology as a specimen (true distal margin). A gap in an anastomotic ring suggests that the circular staple line is incomplete and the anastomosis should be reinforced with suture

1	should be sent to pathology as a specimen (true distal margin). A gap in an anastomotic ring suggests that the circular staple line is incomplete and the anastomosis should be reinforced with suture circumferentially, if technically feasible. A temporary proximal ileostomy may be indicated as well. Most surgeons will also leak test an anastomosis by instilling water or saline into the pelvis and insufflating the rectum with air via a proc-toscope to looking or alternatively instilling methylene blue or betadine into the rectum to look for extravasation. A leak test strongly suggests a defect and/or disruption of the anastomosis. As such, the suture should line reinforced and sometimes may require reanastomosis.23Brunicardi_Ch29_p1259-p1330.indd 127423/02/19 2:29 PM 1275COLON, RECTUM, AND ANUSCHAPTER 29Ostomies and Preoperative Stoma PlanningDepending on the clinical situation, a stoma may be temporary or permanent. It may be end-on or a loop. However, regardless of the indication

1	AND ANUSCHAPTER 29Ostomies and Preoperative Stoma PlanningDepending on the clinical situation, a stoma may be temporary or permanent. It may be end-on or a loop. However, regardless of the indication for a stoma, placement and construction are crucial for function.The preoperative preparation of a patient who is expected to require a stoma should include a consultation with an enter-ostomal therapy (ET) nurse. ET nurses are specially trained and credentialed by the Wound, Ostomy, and Continence Nurses Society. Preoperative planning includes counseling, education, and stoma siting. Postoperatively, the ET nurse assists with local skin care and pouching. Other considerations in stoma planning include evaluation of other medical conditions that may impact on a patient’s ability to manage a stoma (e.g., eyesight, manual dexterity). Family or other caregivers can prove invaluable in caring for these patients.Preoperative stoma siting is crucial for a patient’s postop-erative function and

1	stoma (e.g., eyesight, manual dexterity). Family or other caregivers can prove invaluable in caring for these patients.Preoperative stoma siting is crucial for a patient’s postop-erative function and quality of life. A poorly placed stoma can result in leakage and skin breakdown. Ideally, a stoma should be placed in a location that the patient can easily see and manipulate, within the rectus muscle, and below the belt line (Fig. 29-15). Because the abdominal landmarks in a supine, anesthetized patient may be dramatically different from those in an awake, standing, or sitting patient, the stoma site should always be marked with a tattoo, skin scratch, or permanent marker preoperatively, if possible. In an emergency operation where the stoma site has not been marked, an attempt should be made to place a stoma within the rectus muscle and away from 3BCAFigure 29-13. A. Sutured end-to-end colocolic anastomosis. B. Sutured end-to-side ileocolic anastomosis. C. Stapled side-to-side,

1	be made to place a stoma within the rectus muscle and away from 3BCAFigure 29-13. A. Sutured end-to-end colocolic anastomosis. B. Sutured end-to-side ileocolic anastomosis. C. Stapled side-to-side, functional end-to-end ileocolic anastomosis. (Reproduced with permission from Bell RH, Rikkers LF, Mulholland M: Digestive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)Brunicardi_Ch29_p1259-p1330.indd 127523/02/19 2:29 PM 1276SPECIFIC CONSIDERATIONSPART IIboth the costal margin and iliac crest. In emergencies, placement high on the abdominal wall is preferred to a more caudal site.For all stomas, a circular skin incision is created, and the subcutaneous tissue is dissected to the level of the anterior rec-tus sheath. The anterior rectus sheath is incised in a cruciate fashion, the muscle fibers separated bluntly, and the posterior sheath identified and incised. Care should be taken to avoid injuring and causing bleeding from the inferior

1	is incised in a cruciate fashion, the muscle fibers separated bluntly, and the posterior sheath identified and incised. Care should be taken to avoid injuring and causing bleeding from the inferior epigastric artery and vein. The size of the defect depends on the size of the bowel used to create the stoma, but it should be as small as possible without compromising the intestinal blood supply (usually the width of two to three fingers). The bowel is then brought through the defect and secured with sutures. The abdominal incision is usually closed and dressed prior to maturing the stoma to avoid contaminating the wound. In order to make appliance use easier, a protruding nipple is fashioned by everting the bowel. Three or four interrupted absorbable sutures are placed through the edge of the bowel, then through the serosa, approximately 2 cm proximal to the edge, and then through the dermis (Brooke tech-nique). After the stoma is everted, the mucocutaneous junction is sutured

1	edge of the bowel, then through the serosa, approximately 2 cm proximal to the edge, and then through the dermis (Brooke tech-nique). After the stoma is everted, the mucocutaneous junction is sutured circumferentially with interrupted absorbable suture (Fig. 29-16).Ileostomy Temporary Ileostomy A temporary ileostomy is often used to “protect” an anastomosis that is at risk for leakage (low in the rectum, in an irradiated field, in an immunocompromised or malnourished patient, and during some emergency operations). In this setting, the stoma is often constructed as a loop ileostomy (see Fig. 29-12). A segment of distal ileum is brought through the defect in the abdominal wall as a loop. An enterotomy is created and the stoma matured as described earlier. The loop may be secured with or without an underlying rod. A divided loop may also be created by firing a linear cutting/stapler across the distal limb of the loop flush with the skin followed by mat-uration of the proximal limb of the

1	an underlying rod. A divided loop may also be created by firing a linear cutting/stapler across the distal limb of the loop flush with the skin followed by mat-uration of the proximal limb of the loop. This technique pre-vents incomplete diversion that occasionally occurs with a loop ileostomy.The advantage of a loop or divided loop ileostomy is that subsequent closure can often be accomplished without a formal laparotomy. An elliptical incision is created around the stoma and the bowel gently dissected free of the subcutaneous tissues and fascia. A hand-sewn or stapled anastomosis can then be created and the intestine returned to the peritoneal cavity. This ABDCEFigure 29-14. Technique of end-to-end colorectal anastomosis using a circular stapler. A. The patient is in modified lithotomy position. B. After resection of the rectosigmoid and placement of purse-string sutures proximally and distally, the stapler is inserted into the anal canal and opened. C. Rectal purse-string suture

1	position. B. After resection of the rectosigmoid and placement of purse-string sutures proximally and distally, the stapler is inserted into the anal canal and opened. C. Rectal purse-string suture is tied to secure the rectal stump to the rod of the stapler, and the colonic purse-string is tied to secure the colon to the anvil of the stapler. D. The stapler is closed and fired. E. The stapler is removed, leaving a circular stapled end-to-end anastomosis.Brunicardi_Ch29_p1259-p1330.indd 127623/02/19 2:29 PM 1277COLON, RECTUM, AND ANUSCHAPTER 29avoids a long laparotomy incision and generally is well toler-ated. The timing of ileostomy closure should take into account anastomotic healing as well as the patient’s overall condition. A flexible endoscopy exam and a contrast enema (Gastrografin) are recommended prior to closure to ensure that the anastomosis has not leaked and is patent. A patient’s nutritional status should be optimized. Because the timing of adjuvant chemotherapy

1	are recommended prior to closure to ensure that the anastomosis has not leaked and is patent. A patient’s nutritional status should be optimized. Because the timing of adjuvant chemotherapy effects survival, cancer patients receiving adjuvant chemother-apy usually should defer ileostomy closure until completion of treatment.Permanent Ileostomy A permanent ileostomy is sometimes required after total proctocolectomy or in patients with obstruc-tion. An end ileostomy is the preferred configuration for a per-manent ileostomy because a symmetric protruding nipple can be fashioned more easily than with a loop ileostomy (see Fig. 29-16). The end of the small intestine is brought through the abdominal wall defect and matured. Stitches are often used to secure the bowel to the posterior fascia.Complications of Ileostomy Stoma necrosis may occur in the early postoperative period and is usually caused by skel-etonizing the distal small bowel and/or creating an overly tight fascial defect.

1	of Ileostomy Stoma necrosis may occur in the early postoperative period and is usually caused by skel-etonizing the distal small bowel and/or creating an overly tight fascial defect. Limited mucosal necrosis above the fascia may be treated expectantly, but necrosis below the level of the fascia requires surgical revision. Stoma retraction may occur early or late and may be exacerbated by obesity. Local revision may be necessary. The creation of an ileostomy bypasses the fluid-absorbing capability of the colon, and dehydration with fluid and electrolyte abnormalities is not uncommon. Ideally, ileos-tomy output should be maintained at less than 1500 mL/d to avoid this problem. Bulk agents and opioids (Lomotil, Imodium, tincture of opium) are useful. The somatostatin analogue, octreotide, has been used with varying success in this setting. Skin irritation can also occur, especially if the stoma appliance fits poorly. Skin-protecting agents and custom pouches can help to solve this

1	has been used with varying success in this setting. Skin irritation can also occur, especially if the stoma appliance fits poorly. Skin-protecting agents and custom pouches can help to solve this problem. Obstruction may occur intra-abdominally or at the site where the stoma exits the fascia. Parastomal hernia is less common after an ileostomy than after a colostomy but can cause poor appliance fitting, pain, obstruction, or strangulation. In general, symptomatic parastomal hernias should be repaired. A variety of techniques to repair these hernias have been described, including local repair (either with or without mesh), laparoscopic repair, and stoma resiting. Prolapse is a rare, late complication and is often associated with a parastomal hernia.Colostomy. Most colostomies are created as end colostomies rather than loop colostomies (Fig. 29-17). The bulkiness of the colon makes a loop colostomy awkward for use of an appliance, and prolapse is more likely with this configuration.

1	as end colostomies rather than loop colostomies (Fig. 29-17). The bulkiness of the colon makes a loop colostomy awkward for use of an appliance, and prolapse is more likely with this configuration. Most colos-tomies are created on the left side of the colon. An abdominal wall defect is created and the end of the colon mobilized through it. Because a protruding stoma is considerably easier to pouch, colostomies should also be matured in a Brooke fashion. The distal bowel may be brought through the abdominal wall as a mucus fistula or left intra-abdominally as a Hartmann’s pouch. Tacking the distal end of the colon to the abdominal wall or tagging it with permanent suture can make identification of the stump easier if the colostomy is closed at a later date. Closure of an end colostomy has traditionally required a laparotomy, but increasingly minimally invasive techniques have been adopted. The stoma is dissected free of the abdominal wall and the distal bowel identified. An end-to-end

1	traditionally required a laparotomy, but increasingly minimally invasive techniques have been adopted. The stoma is dissected free of the abdominal wall and the distal bowel identified. An end-to-end anastomosis is then created.MidlineincisionStoma siteLateral edgeof rectus sheathWaistlineHiplineFigure 29-15. Marking of an ideal site for ileostomy. (Reproduced with permission from Bell RH, Rikkers LF, Mulholland M: Diges-tive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)BACFigure 29-16. Brooke ileostomy. A. Four sutures incorporating the cut end of the ileum, the seromuscular layer at the level of the anterior rectus fascia, and the subcuticular edge of the skin are placed at 90° to each other. B. The sutures are tied to produce sto-mal eversion, and (C) simple sutures from the cut edge of the bowel to the subcuticular tissue complete the maturation of the ileostomy. (Reproduced with permission from Bell RH, Rikkers LF, Mulhol-land M:

1	and (C) simple sutures from the cut edge of the bowel to the subcuticular tissue complete the maturation of the ileostomy. (Reproduced with permission from Bell RH, Rikkers LF, Mulhol-land M: Digestive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)Brunicardi_Ch29_p1259-p1330.indd 127723/02/19 2:29 PM 1278SPECIFIC CONSIDERATIONSPART IIComplications of Colostomy Colostomy necrosis may occur in the early postoperative period and results from an impaired vascular supply (skeletonization of the distal colon or a tight fas-cial defect). Like ileostomy necrosis, limited suprafascial necro-sis may be followed expectantly, but necrosis below the fascia requires surgery. Retraction may also occur but is less problem-atic with a colostomy than with an ileostomy because the stool is less irritating to the skin than succus entericus. Obstruction is unusual, but may also occur. Parastomal hernia is the most common late complication of a colostomy and

1	ileostomy because the stool is less irritating to the skin than succus entericus. Obstruction is unusual, but may also occur. Parastomal hernia is the most common late complication of a colostomy and requires repair if it is symptomatic. Prolapse occurs rarely, but is more com-mon with a loop colostomy. Interestingly, it is almost always the efferent limb of the loop that prolapses. Dehydration is rare after colostomy, and skin irritation is less common than with ileostomy.Functional ResultsFunction following segmental colonic resection and primary anastomosis is generally excellent. A small percentage of patients following subtotal or total colectomy and ileosigmoid or ileorectal anastomosis may experience diarrhea and bowel frequency. This is especially true if the patient is elderly, if sig-nificant length of small bowel has been resected, and if residual proctocolitis is poorly controlled. In general, the more distal the anastomosis, the greater is the risk of troublesome diarrhea

1	sig-nificant length of small bowel has been resected, and if residual proctocolitis is poorly controlled. In general, the more distal the anastomosis, the greater is the risk of troublesome diarrhea and frequency. However, some patients develop significant diarrhea after right colectomy due to malabsorption of bile acids; in these cases, bile acid binding resins (e.g., cholestyramine) sometimes can be helpful.Function following anterior resection is highly dependent on the level of anastomosis, the use of preor postoperative pelvic radiation, and underlying sphincter function. Following low anterior or extended low anterior resection, some surgeons prefer to construct a short (5-cm) colon J-pouch to anastomose to the distal rectum or anal canal in order to increase the capacity of the neorectum. The reservoirs are thought to lessen urgency, frequency, and incontinence, but some patients have difficulty initiating defecation, and long-term superiority over a “straight” anastomosis has

1	The reservoirs are thought to lessen urgency, frequency, and incontinence, but some patients have difficulty initiating defecation, and long-term superiority over a “straight” anastomosis has yet to be proven. In addition, these reservoirs can be technically difficult, especially in an obese male with a narrow pelvis.20-22The physical and psychological problems associated with a permanent Brooke ileostomy led to development of the con-tinent Kock pouch ileostomy. Unfortunately, complications, especially complications related to valve slippage, are common. Despite variations of technique designed to improve the func-tion of the continent ileostomy, most surgeons have abandoned this operation and instead perform restorative proctocolectomy with ileal pouch–anal anastomosis.Although ileal pouch–anal reconstruction is anatomically appealing, functional outcome is far from perfect.26,27 Patients should be counseled to expect 8 to 10 bowel movements per day. Up to 50% have some degree of

1	pouch–anal reconstruction is anatomically appealing, functional outcome is far from perfect.26,27 Patients should be counseled to expect 8 to 10 bowel movements per day. Up to 50% have some degree of nocturnal incontinence. Pouchitis (see the following paragraph) occurs in nearly 50% of patients who undergo the operation for chronic ulcerative colitis, and small bowel obstruction is not uncommon. Other less com-mon complications include difficulties with pouch evacuation, pouch-perineal and/or pouch-vaginal fistula, and anal stricture. Pouch failure rate averages 5% to 10%. Patients who are sub-sequently diagnosed with Crohn’s disease have a considerably higher pouch failure rate (approximately 50%), whereas patients with indeterminate colitis have an intermediate pouch failure rate (15–20%). Despite these drawbacks, the vast majority of patients are satisfied and prefer ileal pouch–anal reconstruction to permanent ileostomy.26,27Pouchitis is an inflammatory condition that affects

1	Despite these drawbacks, the vast majority of patients are satisfied and prefer ileal pouch–anal reconstruction to permanent ileostomy.26,27Pouchitis is an inflammatory condition that affects both ileoanal pouches and continent ileostomy reservoirs. The inci-dence of pouchitis ranges from 30% to 55%. Symptoms include increased diarrhea, hematochezia, abdominal pain, fever, and malaise. Diagnosis is made endoscopically with biopsies. Dif-ferential diagnosis includes infection and undiagnosed Crohn’s disease. The etiology of pouchitis is unknown. Some believe pouchitis results from fecal stasis within the pouch, but empty-ing studies are not confirmatory. Antibiotics (metronidazole ± ciprofloxacin) are the mainstays of therapy, and most patients will respond rapidly to either oral preparations or enemas. Some patients develop chronic pouchitis that necessitates ongoing suppressive antibiotic therapy. Salicylate and corticosteroid enemas have also been used with some success.28-31

1	or enemas. Some patients develop chronic pouchitis that necessitates ongoing suppressive antibiotic therapy. Salicylate and corticosteroid enemas have also been used with some success.28-31 Biologic agents targeting TNFa (infliximab, vedulizumab, ustekinumab) are also being studied.32-34 Reintroduction of normal flora by ingestion of probiotics and/or an elemental diet have been sug-gested as a possible treatment in refractory cases. Occasionally, pouch excision is necessary to control the symptoms of chronic pouchitis.30Anesthesia ConsiderationsLocal Anesthesia. Many anorectal procedures can be per-formed with local anesthetic alone. Intravenous sedation is often provided to calm the patient. Injection of 0.5% lidocaine (short acting) and 0.25% bupivacaine (long acting) into the perianal skin, sphincter, and area around the pudendal nerves usually provides an adequate block. The addition of dilute epinephrine decreases bleeding and prolongs the anesthetic effect.Regional

1	the perianal skin, sphincter, and area around the pudendal nerves usually provides an adequate block. The addition of dilute epinephrine decreases bleeding and prolongs the anesthetic effect.Regional Anesthesia. Epidural, spinal, and caudal anesthetics can be used for anorectal procedures and transanal resections. In patients with severe medical comorbidity, regional anesthesia may occasionally be used for laparotomy and colectomy. Post-operative epidural anesthesia provides excellent pain relief and improves pulmonary function especially after an open operation.General Anesthesia. General anesthesia is required for the vast majority of intra-abdominal procedures. Patients should Figure 29-17. Intraperitoneal end colostomy.Brunicardi_Ch29_p1259-p1330.indd 127823/02/19 2:29 PM 1279COLON, RECTUM, AND ANUSCHAPTER 29undergo a thorough preoperative cardiovascular evaluation. In patients with significant comorbid disease, an anesthesia con-sultation may be appropriate.Positioning. Most

1	RECTUM, AND ANUSCHAPTER 29undergo a thorough preoperative cardiovascular evaluation. In patients with significant comorbid disease, an anesthesia con-sultation may be appropriate.Positioning. Most abdominal colectomies can be performed in the supine position. Anterior resection and APR require lithotomy positioning to facilitate the pelvic dissection and mobilization of the splenic flexure. Adequate padding should be provided for the patient’s sacrum, and care should be taken to avoid stirrup pressure on the peroneal nerves.Anorectal procedures may be performed in lithotomy or in the prone jackknife position. Some surgeons prefer the prone jackknife position because exposure may be better, espe-cially for anterior lesions. Distal posterior lesions can usually be accessed from either position, but more proximal posterior lesions are better accessed in the prone position.Operative PreliminariesBowel Preparation. The rationale for bowel preparation is that decreasing the bacterial load

1	but more proximal posterior lesions are better accessed in the prone position.Operative PreliminariesBowel Preparation. The rationale for bowel preparation is that decreasing the bacterial load in the colon and rectum will decrease the incidence of postoperative infection. Mechanical bowel preparation uses cathartics to rid the colon of solid stool the night before surgery. The most commonly used regimens include polyethylene glycol (PEG) solutions or magnesium citrate. PEG solutions require patients to drink a large volume of fluid and may cause bloating and nausea. Magnesium citrate solutions are generally better tolerated but are more likely to cause fluid and electrolyte abnormalities. Both are equally efficacious in bowel cleansing. Preparatory formulations have been recently introduced in tablet form in an attempt to improve tolerance. However, these methods of bowel cleansing require ingestion of 40 or more tablets with water over several hours. To date, these formulations have

1	in tablet form in an attempt to improve tolerance. However, these methods of bowel cleansing require ingestion of 40 or more tablets with water over several hours. To date, these formulations have not been proven to be superior to the more traditional products. Antibiotic prophylaxis also is recommended. The addition of oral antibiotics to the preopera-tive mechanical bowel preparation has been thought to decrease postoperative infection by further decreasing the bacterial load of the colon. A recent analysis of the Surgical Care Improve-ment Project-1 (SCIP-1) suggests that oral antibiotics reduce postoperative wound infection, especially if a mechanical bowel preparation is not used.35-37Longstanding, convincing data support the efficacy of parenteral antibiotic prophylaxis at the time of surgery. Broad-spectrum parenteral antibiotic(s) with activity against aerobic and anaerobic enteric pathogens should be administered just prior to the skin incision and redosed as needed depending

1	surgery. Broad-spectrum parenteral antibiotic(s) with activity against aerobic and anaerobic enteric pathogens should be administered just prior to the skin incision and redosed as needed depending on the length of the operation. There is no proven benefit to using antibiotics postoperatively after an uncomplicated colectomy.Despite widespread use of mechanical bowel prepara-tion, the necessity of bowel cleansing prior to colectomy has been questioned. European surgeons in particular have advo-cated abandoning this practice. Arguments against mechani-cal bowel preparation include dehydration and electrolyte abnormalities that often result from bowel cleansing, as well as the risk of spillage of liquid stool left over from the “prep.” Arguments in favor of mechanical bowel preparation included easier manipulation of an “empty” colon (especially in mini-mally invasive procedures) and avoidance of a “stool column” above an anastomosis, especially in the pelvis. Interestingly, a recent

1	easier manipulation of an “empty” colon (especially in mini-mally invasive procedures) and avoidance of a “stool column” above an anastomosis, especially in the pelvis. Interestingly, a recent meta-analysis of 14 randomized controlled trials sug-gested that mechanical bowel preparation does not prevent sur-gical site infection and should be abandoned in clinical practice. However, these studies did not include the use of oral antibiotics in the mechanical preparation groups, and further studies will be needed to determine the optimal preparation regimen.35-37Ureteral Stents. Ureteral stents may be useful for identify-ing the ureters intraoperatively and are placed via cystoscopy after the induction of general anesthesia and removed at the end of the operation. Stents can be invaluable during reopera-tive pelvic surgery or when there is significant retroperitoneal inflammation (such as complicated diverticulitis), as well as in obese patients. Lighted stents may be helpful in

1	during reopera-tive pelvic surgery or when there is significant retroperitoneal inflammation (such as complicated diverticulitis), as well as in obese patients. Lighted stents may be helpful in laparoscopic and robotic resections. Patients often have transient hematuria postoperatively, but major complications are rare.Multidisciplinary Teams. Patients with complex colorectal disease often benefit from a multidisciplinary approach to their care. Patients with pelvic floor disorders (especially inconti-nence) often require evaluation by both a colorectal surgeon and a urologist or urogynecologist. Preoperative evaluation of cancer patients by a medical oncologist and/or radiation oncolo-gist is crucial for planning either neoadjuvant or adjuvant ther-apy. Intraoperatively, complex pelvic resections often require the involvement of not only a colorectal surgeon but also a urologist, gynecologic oncologist, neurosurgeon, and/or plastic surgeon. Radiation oncologists should be involved in

1	often require the involvement of not only a colorectal surgeon but also a urologist, gynecologic oncologist, neurosurgeon, and/or plastic surgeon. Radiation oncologists should be involved in the opera-tion if brachytherapy catheters are to be placed for intracavi-tary radiation or if intraoperative radiation therapy is planned. Rarely, psychiatric disorders may manifest as colorectal prob-lems (especially functional disorders and chronic pain), and involvement of a psychiatrist or psychologist may be beneficial.INFLAMMATORY BOWEL DISEASEGeneral ConsiderationsEpidemiology. Inflammatory bowel disease includes ulcer-ative colitis, Crohn’s disease, and indeterminate colitis. Ulcerative colitis occurs in 8 to 15 people per 100,000 in the United States and Northern Europe. The incidence is consid-erably lower in Asia, Africa, and South America, and among the nonwhite population in the United States. Ulcerative coli-tis incidence peaks during the third decade of life and again in the seventh

1	lower in Asia, Africa, and South America, and among the nonwhite population in the United States. Ulcerative coli-tis incidence peaks during the third decade of life and again in the seventh decade of life. The incidence of Crohn’s disease is slightly lower, 1 to 5 people per 100,000. Crohn’s disease also affects Northern European and Caucasian populations dispro-portionately. Crohn’s disease has a similar bimodal incidence, with most cases occurring between ages 15 to 30 years and ages 60 to 70 years.38 In 15% of patients with inflammatory bowel disease, differentiation between ulcerative colitis and Crohn’s colitis is impossible; these patients are classified as having indeterminate colitis.Etiology. Inflammatory bowel disease is a multifactorial con-dition that includes environmental, genetic, and immune causal elements; the variation in disease distributions and severity, as well as the differential responses to medical therapy, reflect a complex pathophysiology that is not

1	genetic, and immune causal elements; the variation in disease distributions and severity, as well as the differential responses to medical therapy, reflect a complex pathophysiology that is not reducible to a single cause. Nonetheless, there are several consistent observations among IBD populations that allow some degree of generalization. The consistent differences in IBD incidence between different geographic regions strongly suggest that environmental factors such as diet and exposure to microorganisms have a causal role. Alcohol and oral contraceptive use have also been implicated, as has tobacco use, in the etiology and exacerbation of Crohn’s disease.Brunicardi_Ch29_p1259-p1330.indd 127923/02/19 2:29 PM 1280SPECIFIC CONSIDERATIONSPART IIIBD is a genetic disease, though one that is polygenic, explaining why IBD frequently affects multiple family mem-bers across more than one generation, while also explaining the large number of genes implicated in the development of IBD. Most

1	polygenic, explaining why IBD frequently affects multiple family mem-bers across more than one generation, while also explaining the large number of genes implicated in the development of IBD. Most of the data on the genetics of IBD focus on Crohn’s disease. Although ulcerative colitis has an association with at least 20 genetic loci based on genome-wide association studies (GWAS), this form of IBD has a weaker genetic link than does Crohn’s disease. Approximately 10% to 30% of IBD patients will have at least one other family member also affected by IBD.39,40 Additionally, there is 50% disease concordance among monozygotic twins and a 10% disease concordance among dizy-gotic twins.41Many of the genetic variants most consistently associated with IBD involve loci involved in innate immune function. These include NOD2 (nucleotide-binding oligomerization domain-containing protein 2), which is located on chromosome 16 and which is responsible for coordinating the function of several genes

1	These include NOD2 (nucleotide-binding oligomerization domain-containing protein 2), which is located on chromosome 16 and which is responsible for coordinating the function of several genes leading to the production of proinflammatory cytokines in response to gut microbes. This genetic variant is arguably the most strongly associated with IBD, being strongly connected with Crohn’s disease, although it is also associated with severe pouchitis in patients with a history of ulcerative colitis.42,43The ATG16L1 gene is located on chromosome 2, and its product is involved in the immune response to muramyl dipep-tide, a component of both gram-positive and gram-negative bac-teria that is recognized by the immune system. A related gene, IRGM, is located on chromosome 5, and its product is pivotal in the interferon-gamma–mediated clearance of intracellular pathogens. Variants in this gene are associated with a higher incidence of ileocolic resections in Crohn’s disease patients.42-44Patients

1	in the interferon-gamma–mediated clearance of intracellular pathogens. Variants in this gene are associated with a higher incidence of ileocolic resections in Crohn’s disease patients.42-44Patients with IBD appear to have a chronic immune dys-regulation, which may lead to an interplay with gut microbes which are also present in non-IBD patients, but which elicit pathologic immune responses in the IBD population leading to chronic, idiopathic inflammation of the alimentary tract. Bacteria such as Mycobacterium paratuberculosis and Liste-ria monocytogenes, as well as viruses such as paramyxovirus and measles virus, have been suggested as having a role in the development of Crohn’s disease. With the decreased cost of sequencing and with the expansion of reference databases for identification of organisms, microbiome studies have been applied to the study of IBD patients, both to learn about the pathogenesis of IBD as well as for disease prognostication. Recent studies have identified an

1	of organisms, microbiome studies have been applied to the study of IBD patients, both to learn about the pathogenesis of IBD as well as for disease prognostication. Recent studies have identified an abundance of Serratia marc-escens, E coli, and Candida tropicalis in the guts of patients with Crohn’s disease. In a study of 543 stool samples, patients with ulcerative colitis and primary sclerosing cholangitis (PSC) demonstrated a distinct bacterial community structure, with an enrichment of bacteria belonging to the Veillonella genus, which is associated with several diseases characterized by inflammation and fibrosis.45-49A defect in the gut mucosal barrier, which increases exposure to intestinal microbes as well as proinflammatory substances, is a potential etiologic factor related to immune dys-regulation. An autoimmune mechanism has also been postulated. Although there is no clear evidence linking an immunologic disorder to inflammatory bowel disease, the similarity of many of the

1	dys-regulation. An autoimmune mechanism has also been postulated. Although there is no clear evidence linking an immunologic disorder to inflammatory bowel disease, the similarity of many of the extraintestinal manifestations to rheumatologic disorders has made this theory attractive. In summary, IBD is primarily characterized by intestinal inflammation, and medical therapy is focused on reducing or preventing that inflammation.45-49Pathology and Differential Diagnosis. Although ulcer-ative colitis and Crohn’s colitis share many pathologic and clinical similarities, these conditions can be differentiated in 85% of patients. Ulcerative colitis is a mucosal process in which the colonic mucosa and submucosa are infiltrated with inflammatory cells. The mucosa may be atrophic, and crypt abscesses are common. Endoscopically, the mucosa is fre-quently friable and may possess multiple inflammatory pseu-dopolyps. In long-standing ulcerative colitis, the colon may be foreshortened and the

1	are common. Endoscopically, the mucosa is fre-quently friable and may possess multiple inflammatory pseu-dopolyps. In long-standing ulcerative colitis, the colon may be foreshortened and the mucosa replaced by scar. In quies-cent ulcerative colitis, the colonic mucosa may appear normal both endoscopically and microscopically. Ulcerative colitis may affect the rectum (proctitis), rectum and sigmoid colon (proctosigmoiditis), rectum and left colon (left-sided colitis), or the rectum and varying lengths of colon extending proxi-mal to the splenic flexure (pancolitis). Ulcerative colitis does not primarily affect the small intestine, but the terminal ileum may demonstrate inflammatory changes (“backwash ileitis”). A key feature of ulcerative colitis is the continuous involve-ment of the rectum and colon; rectal sparing or skip lesions suggest a diagnosis of Crohn’s disease. Symptoms are related to the degree of mucosal inflammation and the extent of coli-tis. Patients typically complain

1	and colon; rectal sparing or skip lesions suggest a diagnosis of Crohn’s disease. Symptoms are related to the degree of mucosal inflammation and the extent of coli-tis. Patients typically complain of bloody diarrhea and crampy abdominal pain. Proctitis may produce tenesmus. Severe abdominal pain and fever raise the concern of fulminant coli-tis or toxic megacolon. Physical findings are nonspecific and range from minimal abdominal tenderness and distention to frank peritonitis. In the nonemergent setting, the diagnosis is typically made by colonoscopy and mucosal biopsy.In contrast to ulcerative colitis, Crohn’s disease is a trans-mural inflammatory process that can affect any part of the gas-trointestinal tract from mouth to anus. Mucosal ulcerations, an inflammatory cell infiltrate, and noncaseating granulomas are characteristic pathologic findings. Chronic inflammation may ultimately result in fibrosis, strictures, and fistulas in either the colon or small intestine. The endoscopic

1	noncaseating granulomas are characteristic pathologic findings. Chronic inflammation may ultimately result in fibrosis, strictures, and fistulas in either the colon or small intestine. The endoscopic appearance of Crohn’s colitis is characterized by deep serpiginous ulcers and a “cob-blestone” appearance. Skip lesions and rectal sparing are com-mon. Symptoms of Crohn’s disease depend on the severity of inflammation and/or fibrosis and the location of inflammation in the gastrointestinal tract. Acute inflammation may produce diarrhea, crampy abdominal pain, and fever. Strictures may pro-duce symptoms of obstruction. Weight loss is common, both because of obstruction and from protein loss. Perianal Crohn’s disease may present with pain, swelling, and drainage from fis-tulas or abscesses. Physical findings are also related to the site and severity of disease.In 15% of patients with colitis from inflammatory bowel disease, differentiation of ulcerative colitis from Crohn’s colitis is

1	Physical findings are also related to the site and severity of disease.In 15% of patients with colitis from inflammatory bowel disease, differentiation of ulcerative colitis from Crohn’s colitis is impossible either grossly or microscopically (indeterminate colitis).50 These patients typically present with symptoms similar to ulcerative colitis. Endoscopic and pathologic findings usually include features common to both diseases. Increasingly, sero-logic markers have been employed to differentiate ulcerative colitis from Crohn’s disease. The anti-Saccharomyces cerevi-siae antibody (ASCA) and perinuclear anticytoplasmic antibody (pANCA) may be useful in differentiating these two processes but require prospective study.50 Further differential diagnoses include infectious colitides, especially cytomegalovirus (CMV), Campylobacter jejuni, Entamoeba histolytica, toxigenic E Coli, C difficile, Neisseria gonorrhoeae, Salmonella, and Shigella species.Brunicardi_Ch29_p1259-p1330.indd

1	cytomegalovirus (CMV), Campylobacter jejuni, Entamoeba histolytica, toxigenic E Coli, C difficile, Neisseria gonorrhoeae, Salmonella, and Shigella species.Brunicardi_Ch29_p1259-p1330.indd 128023/02/19 2:29 PM 1281COLON, RECTUM, AND ANUSCHAPTER 29Extraintestinal Manifestations. The liver is a common site of extracolonic disease in inflammatory bowel disease. Fatty infiltration of the liver is present in 40% to 50% of patients, and cirrhosis is found in 2% to 5%. Fatty infiltration may be reversed by medical or surgical treatment of colonic disease, but cirrhosis is irreversible. Primary sclerosing cholangitis is a progressive disease characterized by intraand extrahepatic bile duct stric-tures. Forty percent to 60% of patients with primary sclerosing cholangitis have ulcerative colitis. Colectomy will not reverse this disease, and the only effective therapy is liver transplanta-tion. Pericholangitis is also associated with inflammatory bowel disease and may be diagnosed with a

1	Colectomy will not reverse this disease, and the only effective therapy is liver transplanta-tion. Pericholangitis is also associated with inflammatory bowel disease and may be diagnosed with a liver biopsy. Bile duct carcinoma is a rare complication of long-standing inflamma-tory bowel disease. Patients who develop bile duct carcinoma in the presence of inflammatory bowel disease are, on average, 20 years younger than other patients with bile duct carcinoma.51Arthritis also is a common extracolonic manifesta-tion of inflammatory bowel disease, and the incidence is 20 times greater than in the general population. Arthritis usually improves with treatment of the colonic disease. Sacroiliitis and ankylosing spondylitis are associated with inflammatory bowel disease, although the relationship is poorly understood. Medical and surgical treatment of the colonic disease does not impact symptoms.51Erythema nodosum is seen in 5% to 15% of patients with inflammatory bowel disease and usually

1	is poorly understood. Medical and surgical treatment of the colonic disease does not impact symptoms.51Erythema nodosum is seen in 5% to 15% of patients with inflammatory bowel disease and usually coincides with clini-cal disease activity. Women are affected three to four times more frequently than men. The characteristic lesions are raised, red, and predominantly on the lower legs. Pyoderma gangreno-sum is an uncommon but serious condition that occurs almost exclusively in patients with inflammatory bowel disease. The lesion begins as an erythematous plaque, papule, or bleb, usu-ally located on the pretibial region of the leg and occasionally near a stoma. The lesions progress and ulcerate, leading to a painful, necrotic wound. Pyoderma gangrenosum may respond to resection of the affected bowel in some patients. In others, this disorder is unaffected by treatment of the underlying bowel disease. One of the challenges in managing pyoderma is that this manifestation of IBD exhibits

1	bowel in some patients. In others, this disorder is unaffected by treatment of the underlying bowel disease. One of the challenges in managing pyoderma is that this manifestation of IBD exhibits pathergy, where the disease will manifest and have its severity exacerbated by the creation of surgical-sites. Depending on the circumstances, a history or the presence of pyoderma should prompt consideration for avoid-ance of a stoma.51Up to 10% of patients with inflammatory bowel dis-ease will develop ocular lesions. These include uveitis, iritis, episcleritis, and conjunctivitis, as well as macular degenerative, hyperpigmented pigmented epithelium (CHRPE). They usu-ally develop during an acute exacerbation of the inflammatory bowel disease. The etiology is unknown.51Principles of Nonoperative Management. Medical therapy for inflammatory bowel disease focuses on decreasing inflam-mation and alleviating symptoms, and many of the agents used are the same for both ulcerative colitis and Crohn’s

1	therapy for inflammatory bowel disease focuses on decreasing inflam-mation and alleviating symptoms, and many of the agents used are the same for both ulcerative colitis and Crohn’s disease. In general, mild to moderate flares may be treated in the outpatient setting. More severe signs and symptoms mandate hospitaliza-tion. Pancolitis generally requires more aggressive therapy than limited disease. Because ulcerative proctitis and proctosigmoid-itis are limited to the distal large intestine, topical therapy with salicylate and/or corticosteroid suppositories and enemas can be extremely effective. Systemic therapy is rarely required in these patients.Salicylates Sulfasalazine (Azulfidine), 5-acetyl salicylic acid (5-ASA), and related compounds are first-line agents in the medical treatment of mild to moderate inflammatory bowel dis-ease. These compounds decrease inflammation by inhibition of cyclooxygenase and 5-lipoxygenase in the gut mucosa. They require direct contact with affected

1	of mild to moderate inflammatory bowel dis-ease. These compounds decrease inflammation by inhibition of cyclooxygenase and 5-lipoxygenase in the gut mucosa. They require direct contact with affected mucosa for efficacy. Mul-tiple preparations are available for administration to different sites in the small intestine and colon (sulfasalazine, mesalamine [Pentasa, Asacol, Rowasa]).Antibiotics Antibiotics are often used to decrease the intra-luminal bacterial load in Crohn’s disease. Metronidazole has been reported to improve Crohn’s colitis and perianal disease, but the evidence is weak. Fluoroquinolones may also be effec-tive in some cases. In the absence of fulminant colitis or toxic megacolon, antibiotics are not used to treat ulcerative colitis.Corticosteroids Corticosteroids (either oral or parenteral) are a key component of treatment for an acute exacerbation of either ulcerative colitis or Crohn’s disease. Corticosteroids are nonspecific inhibitors of the immune system, and 75%

1	or parenteral) are a key component of treatment for an acute exacerbation of either ulcerative colitis or Crohn’s disease. Corticosteroids are nonspecific inhibitors of the immune system, and 75% to 90% of patients will improve with the administration of these drugs. However, corticosteroids have a number of serious side effects, and use of these agents should be limited to the short-est course possible. In addition, corticosteroids should be used judiciously in children because of the potential adverse effect on growth. Failure to wean corticosteroids is a relative indication for surgery.Because of the systemic effects of corticosteroids, an effort has been made to develop drugs that act locally and have limited systemic absorption. Agents such as budesonide, beclomethasone dipropionate, and tixocortol pivalate undergo rapid hepatic degradation that significantly decreases systemic toxicity. Budesonide is available as an oral preparation. Corti-costeroid enemas provide effective

1	and tixocortol pivalate undergo rapid hepatic degradation that significantly decreases systemic toxicity. Budesonide is available as an oral preparation. Corti-costeroid enemas provide effective local therapy for proctitis and proctosigmoiditis and have fewer side effects than systemic corticosteroids.Immunomodulating Agents Azathioprine and 6-mercatopu-rine (6-MP) are antimetabolite drugs that interfere with nucleic acid synthesis and thus decrease proliferation of inflammatory cells. These agents are useful for treating ulcerative colitis and Crohn’s disease in patients who have failed salicylate therapy or who are dependent on, or refractory to, corticosteroids. It is important to note, however, that the onset of action of these drugs takes 6 to 12 weeks, and concomitant use of corticoste-roids almost always is required.Cyclosporine is an immunosuppressive agent that inter-feres with T-lymphocyte function. While cyclosporine is not routinely used to treat inflammatory bowel

1	corticoste-roids almost always is required.Cyclosporine is an immunosuppressive agent that inter-feres with T-lymphocyte function. While cyclosporine is not routinely used to treat inflammatory bowel disease, up to 80% of patients with an acute flare of ulcerative colitis will improve with its use. However, the majority of these patients will ulti-mately require colectomy. Cyclosporine is also occasionally used to treat exacerbations of Crohn’s disease, and approxi-mately two-thirds of patients will note some improvement. Improvement is generally apparent within 2 weeks of beginning cyclosporine therapy. Long-term use of cyclosporine is limited by its significant toxicities (e.g., nephrotoxicity, hirsutism, gum hypertrophy).Methotrexate is a folate antagonist that also has been used to treat inflammatory bowel disease. Although the efficacy of this agent is unproven, there are reports that more than 50% of patients will improve with administration of this

1	that also has been used to treat inflammatory bowel disease. Although the efficacy of this agent is unproven, there are reports that more than 50% of patients will improve with administration of this drug.52Brunicardi_Ch29_p1259-p1330.indd 128123/02/19 2:29 PM 1282SPECIFIC CONSIDERATIONSPART IIBiologic Agents In an effort to improve treatment for steroid-refractory inflammatory bowel disease, a class of agents has been developed based on inhibition of tumor necrosis factor alpha (TNF-α). Intravenous infusion of these agents decreases inflammation systemically. Infliximab is a monoclonal antibody directed against TNF-α and was the first biologic agent used to treat Crohn’s disease. More than 50% of patients with moderate to severe Crohn’s disease will improve with infliximab therapy. Infliximab is a chimeric monoclonal antibody directed against TNF-α and it was the first biologic agent used to treat Crohn’s disease. Based on the ACCENT I and II trials, infliximab was associated

1	Infliximab is a chimeric monoclonal antibody directed against TNF-α and it was the first biologic agent used to treat Crohn’s disease. Based on the ACCENT I and II trials, infliximab was associated with a greater incidence of clinical remission, the ability to discontinue corticosteroids, and a longer length of remission compared to placebo.53,54 There are also multiple studies demonstrating an improvement in fistulizing perianal Crohn’s disease, although studies define “improvement” in a variety of ways, some of which do not require fistula to com-pletely involute.53-56Because infliximab is a chimera partially consisting of mouse antibody, human antibodies directed against infliximab can mitigate the efficacy of this drug. For this reason, adali-mumab and certolizumab, that have no nonhuman component, were developed. These two drugs can be administered by sub-cutaneous injection, allowing patients to self-administer these therapies and to avoid the cost and inconvenience of

1	no nonhuman component, were developed. These two drugs can be administered by sub-cutaneous injection, allowing patients to self-administer these therapies and to avoid the cost and inconvenience of presenting to an infusion center. The ultimate goal of biologic agents, just as with other medical therapies for IBD, is mucosal healing.The use of biologic agents for the treatment of ulcerative colitis is an area where opinions between gastroenterologists and surgeons are more divergent. Unlike in the case of Crohn’s dis-ease, there is a putative surgical cure for ulcerative colitis, which casts the risks of long-term immunosuppression in a different light than in the setting of Crohn’s where there is no curative therapy available. For patients with moderate to severe ulcer-ative colitis not responding to other medical therapies, there is evidence supporting the use of infliximab (UC SUCCESS) and adalimumab (ULTRA I AND II). The use of infliximab as rescue therapy for inpatients with

1	not responding to other medical therapies, there is evidence supporting the use of infliximab (UC SUCCESS) and adalimumab (ULTRA I AND II). The use of infliximab as rescue therapy for inpatients with severe, steroid-dependent ulcerative colitis has more recently been investigated. Though gastroen-terologists may view this intervention as maintenance therapy that begins in an inpatient setting only to be continued following hospital discharge, it is still unclear whether inpatient infliximab is even a reliable bridge to elective surgery. Many of the stud-ies supporting inpatient rescue therapy with infliximab did not focus their analyses on patients with extensive (pan) colitis, the group at highest risk for requiring an unplanned admission due to a disease flare, and the subgroup of ulcerative colitis patients most likely to fail rescue therapy and to require colectomy.57-62Whether the preoperative use of biologics is associated with a higher incidence of postoperative complications

1	colitis patients most likely to fail rescue therapy and to require colectomy.57-62Whether the preoperative use of biologics is associated with a higher incidence of postoperative complications is a matter of contention. There are individual studies demonstrat-ing a higher incidence of postoperative sepsis, intra-abdominal abscess and readmissions for patients undergoing ileocolectomy for Crohn’s disease who also received preoperative infliximab within three months of surgery. Systematic reviews on this topic, primarily focusing on Crohn’s disease, have concluded that perioperative infliximab may or may not be associated with a higher incidence of postoperative complications, with most of these complications being infectious in nature.63Nutrition Patients with inflammatory bowel disease are often malnourished. Abdominal pain and obstructive symptoms may decrease oral intake. Diarrhea can cause significant protein loss. Ongoing inflammation produces a catabolic physiologic state.

1	are often malnourished. Abdominal pain and obstructive symptoms may decrease oral intake. Diarrhea can cause significant protein loss. Ongoing inflammation produces a catabolic physiologic state. Parenteral nutrition should be strongly considered early in the course of therapy for either Crohn’s disease or ulcerative colitis. The nutritional status of the patient also should be considered when planning operative intervention, and nutritional param-eters such as serum albumin, prealbumin, and transferrin should be assessed. In extremely malnourished patients, especially those who are also being treated with corticosteroids, creation of a stoma is often safer than a primary anastomosis.Ulcerative ColitisUlcerative colitis is a dynamic disease characterized by remis-sions and exacerbations. The clinical spectrum ranges from an inactive or quiescent phase to low-grade active disease to ful-minant disease. The onset of ulcerative colitis may be insidi-ous, with minimal bloody stools, or

1	The clinical spectrum ranges from an inactive or quiescent phase to low-grade active disease to ful-minant disease. The onset of ulcerative colitis may be insidi-ous, with minimal bloody stools, or the onset can be abrupt, with severe diarrhea and bleeding, tenesmus, abdominal pain, and fever. The severity of symptoms depends on the degree and extent of inflammation. Although anemia is common, massive hemorrhage is rare. Physical findings are often nonspecific.The diagnosis of ulcerative colitis is almost always made endoscopically. Because the rectum is invariably involved, proctoscopy may be adequate to establish the diagnosis. The earliest manifestation is mucosal edema, which results in a loss of the normal vascular pattern. In more advanced disease, char-acteristic findings include mucosal friability and ulceration. Pus and mucus may also be present. While mucosal biopsy is often diagnostic in the chronic phase of ulcerative colitis, biopsy in the acute phase will often reveal

1	mucosal friability and ulceration. Pus and mucus may also be present. While mucosal biopsy is often diagnostic in the chronic phase of ulcerative colitis, biopsy in the acute phase will often reveal only nonspecific inflammation. Evaluation with colonoscopy or barium enema during an acute flare is contraindicated because of the risk of perforation.Barium enema has been used to diagnose chronic ulcer-ative colitis and to determine the extent of disease. However, this modality is less sensitive than colonoscopy and may not detect early disease. In long-standing ulcerative colitis, the colon is foreshortened and lacks haustral markings (“lead pipe” colon). Because the inflammation in ulcerative colitis is purely mucosal, strictures are highly uncommon. Any stricture diag-nosed in a patient with ulcerative colitis must be presumed to be malignant until proven otherwise.Indications for Surgery. Indications for surgery in ulcer-ative colitis may be emergent or elective. Emergency surgery is

1	ulcerative colitis must be presumed to be malignant until proven otherwise.Indications for Surgery. Indications for surgery in ulcer-ative colitis may be emergent or elective. Emergency surgery is required for patients with massive life-threatening hemorrhage, toxic megacolon, or fulminant colitis who fail to respond rap-idly to medical therapy. Patients with signs and symptoms of fulminant colitis should be treated aggressively with bowel rest, hydration, broad-spectrum antibiotics, and parenteral corticoste-roids. Colonoscopy and barium enema are contraindicated, and antidiarrheal agents should be avoided. Deterioration in clinical condition or failure to improve within 24 to 48 hours mandates surgery.Indications for elective surgery include intractability despite maximal medical therapy and high-risk development of major complications of medical therapy such as aseptic necrosis of joints secondary to chronic steroid use. Elective surgery also is indicated in patients at significant

1	and high-risk development of major complications of medical therapy such as aseptic necrosis of joints secondary to chronic steroid use. Elective surgery also is indicated in patients at significant risk of developing colorec-tal carcinoma. The risk of malignancy increases with pancolonic disease and the duration of symptoms and is approximately 2% after 10 years, 8% after 20 years, and 18% after 30 years. Unlike sporadic colorectal cancers, carcinoma developing in the con-text of ulcerative colitis is more likely to arise from areas of Brunicardi_Ch29_p1259-p1330.indd 128223/02/19 2:29 PM 1283COLON, RECTUM, AND ANUSCHAPTER 29flat dysplasia and may be difficult to diagnose at an early stage. For this reason, it is recommended that patients with longstanding ulcerative colitis undergo colonoscopic surveillance with multiple (40–50), random biopsies to identify dysplasia before invasive malignancy develops. However, the adequacy of this type of screening is controversial. Recently,

1	colonoscopic surveillance with multiple (40–50), random biopsies to identify dysplasia before invasive malignancy develops. However, the adequacy of this type of screening is controversial. Recently, magnifying chro-moendoscopy has been used to improve sensitivity. This tech-nique uses topical dyes that are applied to the colonic mucosa at the time of endoscopy (Lugol’s solution, methylene blue, indigo carmine, and others). These dyes highlight contrast between normal and dysplastic epithelium, allowing more precise biopsy of suspicious areas.64,65 Surveillance is recommended annu-ally after 8 years in patients with pancolitis, and annually after 15 years in patients with left-sided colitis. Although low-grade dysplasia was long thought to represent minimal risk, more recent studies show that invasive cancer may be present in up to 20% of patients with low-grade dysplasia. For this rea-son, any patient with dysplasia should be advised to undergo proctocolectomy. Controversy exists

1	that invasive cancer may be present in up to 20% of patients with low-grade dysplasia. For this rea-son, any patient with dysplasia should be advised to undergo proctocolectomy. Controversy exists over whether prophylac-tic proctocolectomy should be recommended for patients who have had chronic ulcerative colitis for greater than 10 years in the absence of dysplasia. Proponents of this approach note that surveillance colonoscopy with multiple biopsies samples only a small fraction of the colonic mucosa, and dysplasia and carci-noma are often missed. Opponents cite the relatively low risk of progression to carcinoma (approximately 2.4%) if all biopsies lack dysplasia. Neither approach has been shown definitively to decrease mortality from colorectal cancer.Operative ManagementEmergent Operation In a patient with fulminant colitis or toxic megacolon, total abdominal colectomy with end ileostomy (with or without a mucus fistula), rather than total proctocolectomy, is recommended.

1	Operation In a patient with fulminant colitis or toxic megacolon, total abdominal colectomy with end ileostomy (with or without a mucus fistula), rather than total proctocolectomy, is recommended. Although the rectum is invariably diseased, most patients improve dramatically after an abdominal colectomy, and this operation avoids a difficult and time-consuming pelvic dissection in a critically ill patient. Rarely, a loop ileostomy and decompressing colostomy may be necessary if the patient is too unstable to withstand colectomy. Definitive surgery may then be undertaken at a later date once the patient has recovered. Complex techniques, such as an ileal pouch–anal reconstruction, generally are contraindicated in the emergent setting. However, massive hemorrhage that includes bleeding from the rectum may necessitate proctectomy and creation of either a permanent ileostomy or an ileal pouch–anal anastomosis.Elective Operation Elective resection for ulcerative colitis usually is

1	from the rectum may necessitate proctectomy and creation of either a permanent ileostomy or an ileal pouch–anal anastomosis.Elective Operation Elective resection for ulcerative colitis usually is performed for refractory inflammation and/or the risk of malignancy (dysplasia). Because of the risk of ongo-ing inflammation, the risk of malignancy, and the availability of restorative proctocolectomy, most surgeons recommend operations that include resection of the rectum. Total procto-colectomy with end ileostomy has been the “gold standard” for treating patients with chronic ulcerative colitis. This operation removes the entire affected intestine and avoids the functional disturbances associated with ileal pouch–anal reconstruction. Most patients function well physically and psychologically after this operation. Total proctocolectomy with continent ileostomy (Kock’s pouch) was developed to improve function and quality of life after total proctocolectomy, but morbidity is significant, and

1	this operation. Total proctocolectomy with continent ileostomy (Kock’s pouch) was developed to improve function and quality of life after total proctocolectomy, but morbidity is significant, and restorative proctocolectomy is generally preferred today. Since its introduction in 1980, restorative proctocolectomy with ileal pouch–anal anastomosis has become the procedure of choice for most patients who require total proctocolectomy but wish to avoid a permanent ileostomy (see Figs. 29-11 and 29-12).66 Abdominal colectomy with ileorectal anastomosis may be appropriate for a patient with indeterminate colitis and rectal sparing.Crohn’s DiseaseSimilar to ulcerative colitis, Crohn’s disease is characterized by exacerbations and remissions. Crohn’s disease, however, may affect any portion of the intestinal tract, from mouth to anus. Diagnosis may be made by colonoscopy or esophagogastroduo-denoscopy or by barium small bowel study or enema, depending on which part of the intestine is most

1	the intestinal tract, from mouth to anus. Diagnosis may be made by colonoscopy or esophagogastroduo-denoscopy or by barium small bowel study or enema, depending on which part of the intestine is most affected. The presence of skip lesions is key in differentiating Crohn’s colitis from ulcer-ative colitis, and rectal sparing occurs in approximately 40% of patients. The most common site of involvement of Crohn’s dis-ease is the terminal ileum and cecum (ileocolic Crohn’s disease), followed by the small bowel, and then by the colon and rectum. Perianal and anal canal Crohn’s disease manifest by complex anal fistulae and/or abscesses, anal ulcers, and large skin tags may be the initial site of presentation in up to 4% of cases.Indications for Surgery. Because Crohn’s disease is cur-rently incurable and because it can affect any part of the gas-trointestinal tract, the therapeutic rationale is fundamentally different from that of ulcerative colitis. Ulcerative colitis may be cured by

1	incurable and because it can affect any part of the gas-trointestinal tract, the therapeutic rationale is fundamentally different from that of ulcerative colitis. Ulcerative colitis may be cured by removal of the affected intestinal segment (the colon and rectum). In Crohn’s disease, it is impossible to remove all the at-risk intestine; therefore, surgical therapy is reserved for complications of the disease.Crohn’s disease may present as an acute inflammatory process or as a chronic fibrotic process. During the acute inflam-matory phase, patients may present with intestinal inflamma-tion complicated by fistulae and/or intra-abdominal abscesses. Maximal medical therapy should be instituted, including antiinflammatory medications, bowel rest, and antibiotics. Parenteral nutrition should be considered if the patient is malnourished. Most intra-abdominal abscesses can be drained percutaneously with the use of CT scan guidance. Although the majority of these patients will ultimately

1	be considered if the patient is malnourished. Most intra-abdominal abscesses can be drained percutaneously with the use of CT scan guidance. Although the majority of these patients will ultimately require surgery to remove the dis-eased segment of bowel, these interventions allow the patient’s condition to stabilize, nutrition to be optimized, and inflamma-tion to decrease prior to embarking on a surgical resection. Once an operation is undertaken, fistulae generally require resection of the segment of bowel with active Crohn’s disease; the sec-ondary sites of the fistula are often otherwise normal and do not generally require resection after division of the fistula. Simple closure of the secondary fistula site usually suffices.Chronic fibrosis may result in strictures in any part of the gastrointestinal tract. Because the fibrotic process is gradual, free perforation proximal to the obstructing stricture is rare. Chronic strictures almost never improve with medical therapy.

1	of the gastrointestinal tract. Because the fibrotic process is gradual, free perforation proximal to the obstructing stricture is rare. Chronic strictures almost never improve with medical therapy. Strictures may be treated with resection or stricturoplasty. Distal ileal strictures are sometimes amenable to colonoscopic balloon dilatation. Optimal timing for surgery should take into account the patient’s underlying medical and nutritional status.Once an operation is undertaken for Crohn’s disease, sev-eral principles should guide intraoperative decision making. In general, a laparotomy for Crohn’s disease should be performed through a midline incision because of the possible need for a stoma. Laparoscopy is also increasingly used in this setting. Because many patients with Crohn’s disease often will require multiple operations, the length of bowel removed should be Brunicardi_Ch29_p1259-p1330.indd 128323/02/19 2:29 PM 1284SPECIFIC CONSIDERATIONSPART IIminimized. Bowel should be

1	often will require multiple operations, the length of bowel removed should be Brunicardi_Ch29_p1259-p1330.indd 128323/02/19 2:29 PM 1284SPECIFIC CONSIDERATIONSPART IIminimized. Bowel should be resected to an area with grossly nor-mal margins; frozen sections are not necessary. Finally, a primary anastomosis may be created safely if the patient is medically stable, nutritionally replete, and taking few immunosuppressive medications. Creation of a stoma should be strongly considered in any patient who is hemodynamically unstable, septic, malnour-ished, or receiving high-dose immunosuppressive therapy and among patients with extensive intra-abdominal contamination.Ileocolic and Small Bowel Crohn’s Disease. The terminal ileum and cecum are involved in Crohn’s disease in up to 41% of patients; the small intestine is involved in up to 35% of patients. The most common indications for surgery are internal fistula or abscess (30–38% of patients) and obstruction (35–37% of patients). Psoas

1	the small intestine is involved in up to 35% of patients. The most common indications for surgery are internal fistula or abscess (30–38% of patients) and obstruction (35–37% of patients). Psoas abscess may result from ileocolic Crohn’s dis-ease. Sepsis should be controlled with percutaneous drainage of abscess(es) and antibiotics, if possible. Parenteral nutrition may be necessary in patients with chronic obstruction. The extent of resection depends on the amount of involved intestine. Short segments of inflamed small intestine and right colon should be resected and a primary anastomosis created if the patient is sta-ble, nutrition is adequate, and immunosuppression is minimal. Isolated chronic strictures should also be resected. In patients with multiple fibrotic strictures that would require extensive small bowel resection, stricturoplasty is a safe and effective alternative to resection. Short strictures are amenable to a trans-verse stricturoplasty, while longer strictures may be

1	extensive small bowel resection, stricturoplasty is a safe and effective alternative to resection. Short strictures are amenable to a trans-verse stricturoplasty, while longer strictures may be treated with a side-to-side small bowel anastomosis (Fig. 29-18).ABCDEFigure 29-18. Alternative stricturoplasty techniques. A. A short stricture is opened along the antimesenteric surface of the bowel wall. B. The enterotomy is closed transversely. C. A long stricture is opened along the antimesenteric surface of the bowel wall. D. The bowel is folded into an inverted “U.” E. A side-to-side anastomosis is made. (Reproduced with permission from Corman ML. Colon & Rectal Surgery, 2nd ed. Philadelphia, PA: Lippincott Williams & Wilkins; 1989.)Brunicardi_Ch29_p1259-p1330.indd 128423/02/19 2:29 PM 1285COLON, RECTUM, AND ANUSCHAPTER 29Risk of recurrence after resection for ileocolic and small bowel Crohn’s disease is high. More than 50% of patients will experience a recurrence within 10 years,

1	1285COLON, RECTUM, AND ANUSCHAPTER 29Risk of recurrence after resection for ileocolic and small bowel Crohn’s disease is high. More than 50% of patients will experience a recurrence within 10 years, and the majority of these will require a second operation.Crohn’s Colitis. Crohn’s disease of the large intestine may present as fulminant colitis or toxic megacolon. In this set-ting, treatment is identical to treatment of fulminant colitis and toxic megacolon secondary to ulcerative colitis. Resuscitation and medical therapy with bowel rest, broad-spectrum antibiot-ics, and parenteral corticosteroids should be instituted. If the patient’s condition worsens or fails to rapidly improve, total abdominal colectomy with end ileostomy is recommended. An elective proctectomy may be required if the patient has refrac-tory Crohn’s proctitis. Alternatively, if the rectum is spared, an ileorectal anastomosis may be appropriate once the patient has recovered.Other indications for surgery in chronic

1	patient has refrac-tory Crohn’s proctitis. Alternatively, if the rectum is spared, an ileorectal anastomosis may be appropriate once the patient has recovered.Other indications for surgery in chronic Crohn’s colitis are intractability, complications of medical therapy, and risk of or development of malignancy. Unlike ulcerative colitis, Crohn’s colitis may be segmental, and rectal sparing is often observed. A segmental colectomy may be appropriate if the remaining colon and/or rectum appear normal. An isolated colonic stricture may also be treated by segmental colectomy. Although it was long thought that Crohn’s disease did not increase the risk of colorectal carcinoma, it is now recognized that Crohn’s colitis (especially pancolitis) carries nearly the same risk for cancer as ulcerative colitis. Annual surveillance colonoscopy with multiple biopsies is recommended for patients with long-standing Crohn’s colitis (>7 years in duration). As in ulcerative colitis, dysplasia is an

1	colitis. Annual surveillance colonoscopy with multiple biopsies is recommended for patients with long-standing Crohn’s colitis (>7 years in duration). As in ulcerative colitis, dysplasia is an indication for total proctocolectomy. Ileal pouch–anal reconstruction is not recommended in these patients because of the risk for development of Crohn’s disease within the pouch and the high risk of complications, such as fistula, abscess, stricture, pouch dysfunction, and pouch failure.Anal and Perianal Crohn’s Disease. Anal and perianal mani-festations of Crohn’s disease are very common and occur in 35% of all patients with Crohn’s disease. Isolated anal Crohn’s disease is uncommon, affecting only 3% to 4% of patients. Detection of anal Crohn’s disease, therefore, should prompt evaluation of the remainder of the gastrointestinal tract.The most common perianal lesions in Crohn’s disease are skin tags that are minimally symptomatic. Fissures are also common. Typically, a fissure from Crohn’s

1	remainder of the gastrointestinal tract.The most common perianal lesions in Crohn’s disease are skin tags that are minimally symptomatic. Fissures are also common. Typically, a fissure from Crohn’s disease is partic-ularly deep or broad and perhaps better described as an anal ulcer. These fissures are often multiple and located in a lateral position rather than anterior or posterior midline as seen in an idiopathic fissure in ano. A classic-appearing fissure in ano located laterally should raise the suspicion of Crohn’s disease. Perianal abscess and fistulas are common and can be particu-larly challenging. Fistulas tend to be complex and often have multiple tracts (Fig. 29-19). Hemorrhoids are not more common in patients with Crohn’s disease than in the general population, although many patients tend to attribute any anal or perianal symptom to “hemorrhoids.”Treatment of anal and perianal Crohn’s disease focuses on alleviation of symptoms. Perianal skin irritation from diarrhea often

1	tend to attribute any anal or perianal symptom to “hemorrhoids.”Treatment of anal and perianal Crohn’s disease focuses on alleviation of symptoms. Perianal skin irritation from diarrhea often responds to medical therapy directed at small bowel or colonic disease. In general, skin tags and hemorrhoids should not be excised unless they are extremely symptomatic because of the risk of creating chronic, nonhealing wounds. Fissures may respond to local or systemic therapy; sphincterotomy is relatively contraindicated because of the risk of creating a chronic, nonhealing wound and because of the increased risk of incontinence in a patient with diarrhea from underlying colitis or small bowel disease. Anal ulcers associated with Crohn’s dis-ease are usually not very painful unless there is an underlying abscess. Thus, in patients with significant anal pain, an exami-nation under anesthesia is indicated to exclude an underlying abscess or fistula and to assess the rectal mucosa. In the absence

1	abscess. Thus, in patients with significant anal pain, an exami-nation under anesthesia is indicated to exclude an underlying abscess or fistula and to assess the rectal mucosa. In the absence of active Crohn’s proctitis, one can proceed cautiously with a partial internal sphincterotomy if the examination under anes-thesia reveals a classic-appearing posterior or anterior fissure and anal stenosis.Recurrent abscess(es) or complex anal fistulae should raise the possibility of Crohn’s disease. Treatment focuses on control of infection, delineation of complex anatomy, treat-ment of underlying mucosal disease, and sphincter preserva-tion. Abscesses often can be drained locally, and mushroom catheters are useful for maintaining drainage. Endoanal ultra-sound and pelvic MRI are useful for mapping complex fistulous tracts. Liberal use of setons can control many fistulas and avoid division of the sphincter. Many patients with anal Crohn’s dis-ease function well with multiple setons left in

1	complex fistulous tracts. Liberal use of setons can control many fistulas and avoid division of the sphincter. Many patients with anal Crohn’s dis-ease function well with multiple setons left in place for years. Endoanal advancement flaps may be considered for definitive therapy if the rectal mucosa is uninvolved but will not heal due to rectal inflammation. In 10% to 15% of cases, intractable peri-anal sepsis requires proctectomy.Rectovaginal fistula can be a particularly difficult problem in these patients. A rectal or vaginal mucosal advancement flap may be used if the rectal mucosa appears healthy and scarring of the rectovaginal septum is minimal. Occasionally, proctec-tomy is the best option for women with highly symptomatic rectovaginal fistulae. Although proximal diversion is often employed to protect complex perianal reconstruction, there is no evidence that diversion alone increases healing of anal and perianal Crohn’s disease.Medical treatment of underlying proctitis with

1	employed to protect complex perianal reconstruction, there is no evidence that diversion alone increases healing of anal and perianal Crohn’s disease.Medical treatment of underlying proctitis with salicylate and/or corticosteroid enemas may be helpful; however, control of infection is the primary goal of therapy. Metronidazole has been used with some success in this setting. Anti-TNF-α agents (infliximab and adalimumab) have shown some efficacy in heal-ing chronic fistulas secondary to Crohn’s disease. The success of these agents has led to a concerted effort to identify other 4Figure 29-19. Photograph of a patient with multiple perianal fistulas secondary to Crohn’s disease.Brunicardi_Ch29_p1259-p1330.indd 128523/02/19 2:29 PM 1286SPECIFIC CONSIDERATIONSPART IIimmunomodulators that might prove useful. Proinflammatory cytokines such as interleukin-12 and interferon-γ are potential targets. Inhibition of immune cell migration has also been sug-gested as an approach. However, it is

1	prove useful. Proinflammatory cytokines such as interleukin-12 and interferon-γ are potential targets. Inhibition of immune cell migration has also been sug-gested as an approach. However, it is of paramount importance to drain any and all abscesses before initiating immunosuppres-sive therapy such as corticosteroids or anti-TNF-α monoclonal antibodies.54-55Indeterminate ColitisApproximately 15% of patients with inflammatory bowel dis-ease manifest clinical and pathologic characteristics of both ulcerative colitis and Crohn’s disease. Endoscopy, barium enema, and biopsy may be unable to differentiate ulcerative colitis from Crohn’s colitis in this setting. The indications for surgery are the same as those for ulcerative colitis: intracta-bility, complications of medical therapy, and risk of or devel-opment of malignancy. In the setting of indeterminate colitis in a patient who prefers a sphincter-sparing operation, a total abdominal colectomy with end ileostomy may be the best initial

1	or devel-opment of malignancy. In the setting of indeterminate colitis in a patient who prefers a sphincter-sparing operation, a total abdominal colectomy with end ileostomy may be the best initial procedure. Pathologic examination of the entire colon may then allow a more accurate diagnosis. If the diagnosis suggests ulcer-ative colitis, an ileal pouch–anal anastomosis procedure can be performed. If the diagnosis remains in question, the safest surgi-cal option is completion proctectomy with end ileostomy (simi-lar to Crohn’s colitis). Ileal pouch–anal reconstruction may also be considered with the understanding that the pouch failure rate is between 15% and 20%.50DIVERTICULAR DISEASEDiverticular disease is a clinical term used to describe the pres-ence of symptomatic diverticula. Diverticulosis refers to the presence of diverticula without inflammation. Diverticulitis refers to inflammation and infection associated with diverticula. The majority of colonic diverticula are false

1	refers to the presence of diverticula without inflammation. Diverticulitis refers to inflammation and infection associated with diverticula. The majority of colonic diverticula are false diverticula in which the mucosa and muscularis mucosa have herniated through the colonic wall. These diverticula occur between the teniae coli, at points where nutrient arterial blood vessels penetrate the colonic wall (presumably creating an area of relative weakness in the colonic muscle). They are thought to be pulsion diverticula resulting from high intraluminal pressure. Diverticular bleeding can be massive but usually is self-limited. True diverticula, which comprise all layers of the bowel wall, are rare and are usually congenital in origin.Diverticulosis is extremely common in the United States and Europe. It is estimated that half of the population older than age 50 years has colonic diverticula. The sigmoid colon is the most common site of diverticulosis (Fig. 29-20). Diverticulosis is

1	and Europe. It is estimated that half of the population older than age 50 years has colonic diverticula. The sigmoid colon is the most common site of diverticulosis (Fig. 29-20). Diverticulosis is thought to be an acquired disorder, but the etiology is poorly understood. The most accepted theory is that a lack of dietary fiber results in smaller stool volume, requiring high intraluminal pressure and high colonic wall tension for propulsion. Chronic contraction then results in muscular hypertrophy and develop-ment of the process of segmentation in which the colon acts like separate segments instead of functioning as a continuous tube. As segmentation progresses, the high pressures are directed radi-ally toward the colon wall rather than to development of propul-sive waves that move stool distally. The high radial pressures directed against the bowel wall create pulsion diverticula. A loss of tensile strength and a decrease in elasticity of the bowel wall with age have also been

1	stool distally. The high radial pressures directed against the bowel wall create pulsion diverticula. A loss of tensile strength and a decrease in elasticity of the bowel wall with age have also been proposed etiologies. Although none of these theories has been proven, a high-fiber diet does appear to decrease the incidence of diverticulosis. Although diverticulosis is common, most cases are asymptomatic, and complications occur in the minority of people with this condition.Inflammatory Complications (Diverticulitis)Diverticulitis refers to inflammation and infection associated with a diverticulum and is estimated to occur in 10% to 25% of people with diverticulosis. Peridiverticular and pericolic infection results from a perforation (either macroscopic or microscopic) of a diverticulum, which leads to contamination, inflammation, and infection. The spectrum of disease ranges from mild, uncomplicated diverticulitis that can be treated in the outpatient setting, to free perforation and

1	leads to contamination, inflammation, and infection. The spectrum of disease ranges from mild, uncomplicated diverticulitis that can be treated in the outpatient setting, to free perforation and diffuse peritonitis that requires emergency laparotomy. Most patients present with left-sided abdominal pain, with or without fever, and leukocytosis. A mass may be present. Plain radiographs are useful for detect-ing free intra-abdominal air. CT scan is extremely useful for defining pericolic inflammation, phlegmon, or abscess. Contrast enemas and/or endoscopy are relatively contraindicated because of the risk of perforation. The differential diagnosis includes malignancy, ischemic colitis, infectious colitis, and inflamma-tory bowel disease.Uncomplicated Diverticulitis. Uncomplicated diverticulitis is characterized by left lower quadrant pain and tenderness. CT findings include pericolic soft tissue stranding, colonic wall thickening, and/or phlegmon. Most patients with uncomplicated

1	is characterized by left lower quadrant pain and tenderness. CT findings include pericolic soft tissue stranding, colonic wall thickening, and/or phlegmon. Most patients with uncomplicated diverticulitis will respond to outpatient therapy with broadspectrum oral antibiotics and a low-residue diet. Antibiotics should be continued for 7 to 10 days. About 10% to 20% of patients with more severe pain, tenderness, fever, and leuko-cytosis are treated in the hospital with parenteral antibiotics Figure 29-20. Diverticulosis of sigmoid colon on barium enema. (Reproduced with permission from James EC, Corry RJ, Perry JCF: Basic Surgical Practice. Philadelphia, PA: Hanley & Belfus; 1987.)Brunicardi_Ch29_p1259-p1330.indd 128623/02/19 2:29 PM 1287COLON, RECTUM, AND ANUSCHAPTER 29and bowel rest. Most patients improve within 48 to 72 hours. Failure to improve may suggest abscess formation. CT can be extremely useful in this setting, and many pericolic abscesses can be drained percutaneously.

1	Most patients improve within 48 to 72 hours. Failure to improve may suggest abscess formation. CT can be extremely useful in this setting, and many pericolic abscesses can be drained percutaneously. Deterioration in a patient’s clini-cal condition and the development of peritonitis are indications for laparotomy.Most patients with uncomplicated diverticulitis will recover without surgery, and 50% to 70% will have no further episodes. It has long been believed that the risk of complications increases with recurrent disease. For this reason, elective sig-moid colectomy has often been recommended after the second episode of diverticulitis, especially if the patient has required hospitalization. Resection has often been recommended after the first episode in very young patients and is often recommended after the first episode of complicated diverticulitis. These gen-eral guidelines have been questioned in recent years, and more recent studies suggest that the risk of complications and/or

1	after the first episode of complicated diverticulitis. These gen-eral guidelines have been questioned in recent years, and more recent studies suggest that the risk of complications and/or need for emergent resection does not increase with recurrent disease. Moreover, the rate of complications is rare after elective sur-gery and recurrences do not increase the rate of complications. As such, the rate of resection in all patients, including young patients and those with complicated disease, has decreased.67-71Many surgeons now will not advise colectomy even after two documented episodes of diverticulitis assuming the patient is completely asymptomatic and that carcinoma has been excluded by colonoscopy. Immunosuppressed patients are gen-erally still advised to undergo colectomy after a single episode of documented diverticulitis. Medical comorbidities should be considered when evaluating a patient for elective resection, and the risks of recurrent disease should be weighed against the

1	episode of documented diverticulitis. Medical comorbidities should be considered when evaluating a patient for elective resection, and the risks of recurrent disease should be weighed against the risks of the operation.69,70 Because colon carcinoma may present in an identical fashion to diverticulitis (either complicated or uncom-plicated), all patients must be evaluated for malignancy after resolution of the acute episode. Colonoscopy is recommended 4 to 6 weeks after recovery. Inability to exclude malignancy is another indication for resection.In the elective setting, a sigmoid colectomy with a primary anastomosis is the procedure of choice. The resection should always be extended to the rectum distally because the risk of recurrence is high if a segment of sigmoid colon is retained. The proximal extent of the resection should include all thick-ened or inflamed bowel; however, resection of all diverticula is unnecessary. Increasingly, laparoscopy is being used for elec-tive sigmoid

1	proximal extent of the resection should include all thick-ened or inflamed bowel; however, resection of all diverticula is unnecessary. Increasingly, laparoscopy is being used for elec-tive sigmoid colectomy for diverticular disease.Complicated Diverticulitis. Complicated diverticulitis includes diverticulitis with abscess, obstruction, diffuse peri-tonitis (free perforation), or fistulas between the colon and adjacent structures. Colovesical, colovaginal, and coloenteric fistulas are long-term sequelae of complicated diverticulitis. The Hinchey staging system is often used to describe the severity of complicated diverticulitis: Stage I includes colonic inflam-mation with an associated pericolic abscess; stage II includes colonic inflammation with a retroperitoneal or pelvic abscess; stage III is associated with purulent peritonitis; and stage IV is associated with fecal peritonitis. Treatment depends on the patient’s overall clinical condition and the degree of peritoneal

1	stage III is associated with purulent peritonitis; and stage IV is associated with fecal peritonitis. Treatment depends on the patient’s overall clinical condition and the degree of peritoneal contamination and infection. Small abscesses (<2 cm in diame-ter) may be treated with parenteral antibiotics. Larger abscesses are best treated with CT-guided percutaneous drainage (Fig. 29-21) and antibiotics.72 Many of these patients will ultimately require resection, but percutaneous drainage may allow a one-stage, elective procedure and may obviate the need for colec-tomy if full recovery follows the drainage.Urgent or emergent laparotomy may be required if an abscess is inaccessible to percutaneous drainage, if the patient’s condition deteriorates or fails to improve, or if the patient pres-ents with free intra-abdominal air or peritonitis. In almost all cases, an attempt should be made to resect the affected seg-ment of bowel. Patients with small, localized pericolic or pel-vic abscesses

1	with free intra-abdominal air or peritonitis. In almost all cases, an attempt should be made to resect the affected seg-ment of bowel. Patients with small, localized pericolic or pel-vic abscesses (Hinchey stages I and II) may be candidates for a sigmoid colectomy with a primary anastomosis (a one-stage operation). Among patients with larger abscesses, peritoneal soiling, or peritonitis, sigmoid colectomy with end colostomy and Hartmann’s pouch is the most commonly used procedure.71 Success also has been reported after sigmoid colectomy, pri-mary anastomosis, with or without on-table lavage, and proxi-mal diversion (loop ileostomy). This option may be appropriate in stable patients and offers the great advantage that the subse-quent operation to restore bowel continuity is simpler than is BAFigure 29-21. A. Computed tomography scan demonstrating pelvic abscess from perforated diverticular disease. B. Posterolateral computed tomography–guided drainage of abdominal abscess from

1	than is BAFigure 29-21. A. Computed tomography scan demonstrating pelvic abscess from perforated diverticular disease. B. Posterolateral computed tomography–guided drainage of abdominal abscess from perforated diverticular disease. (Used with permission from Charles O. Finne III, MD, Minneapolis, MN.)Brunicardi_Ch29_p1259-p1330.indd 128723/02/19 2:29 PM 1288SPECIFIC CONSIDERATIONSPART IItakedown of a Hartmann’s pouch. The presence of inflammation and phlegmon may increase the risk of ureteral damage during mobilization of the sigmoid colon, and preoperative placement of ureteral catheters can be invaluable. In extremely unstable patients, or in the presence of such severe inflammation that resection would harm adjacent organs, proximal diversion and local drainage have been employed. However, this approach is generally avoided because of high morbidity and mortality rates, along with the requirement for multiple operations. More recently, several studies have suggested that

1	However, this approach is generally avoided because of high morbidity and mortality rates, along with the requirement for multiple operations. More recently, several studies have suggested that laparoscopic lavage and drainage without bowel resection may be safe and effective even in the presence of free perforation. However, a 20% risk of surgical reoperation can be expected.73,74Obstructive symptoms occur in approximately 67% of patients who develop acute diverticulitis, and complete obstruc-tion occurs in 10%. Patients with incomplete obstruction often respond to fluid resuscitation, nasogastric suction, and gentle, low-volume water or Gastrografin enemas. Relief of obstruction allows full bowel preparation and elective resection. A high-volume oral bowel preparation is contraindicated in the pres-ence of obstructive symptoms. Obstruction that does not rapidly respond to medical management mandates laparotomy. Sigmoid colectomy with end colostomy is the safest procedure to per-form

1	in the pres-ence of obstructive symptoms. Obstruction that does not rapidly respond to medical management mandates laparotomy. Sigmoid colectomy with end colostomy is the safest procedure to per-form in this setting. However, colectomy and primary anasto-mosis, with or without on-table lavage (depending on extent of fecal load in the proximal colon), and proximal diversion may be appropriate if the patient is stable and the proximal and distal bowel appear healthy.Approximately 5% of patients with complicated diverticu-litis develop fistulas between the colon and an adjacent organ. Colovesical fistulas are most common, followed by colovaginal and coloenteric fistulas. Colocutaneous fistulas are a rare com-plication of diverticulitis. Two key points in the evaluation of fistulas are to define the anatomy of the fistula and exclude other diagnoses. Contrast enema and/or small bowel stud-ies are extremely useful in defining the course of the fistula. CT scan can identify associated

1	define the anatomy of the fistula and exclude other diagnoses. Contrast enema and/or small bowel stud-ies are extremely useful in defining the course of the fistula. CT scan can identify associated abscesses or masses. The dif-ferential diagnosis includes malignancy, Crohn’s disease, and radiation-induced fistulas. While Crohn’s disease and radiation injury may be suspected based on the patient’s medical history, colonoscopy or sigmoidoscopy usually is required to rule out malignancy. In addition, in a patient who has received radia-tion therapy, a fistula must be considered to be recurrent cancer until proven otherwise. Once the anatomy of the fistula has been defined and other diagnoses excluded, operative management should include resection of the affected segment of the colon involved with diverticulitis (usually with a primary anastomo-sis) and simple repair of the secondarily involved organ. Sus-picion of carcinoma may mandate a wider, en bloc resection.HemorrhageBleeding from

1	with diverticulitis (usually with a primary anastomo-sis) and simple repair of the secondarily involved organ. Sus-picion of carcinoma may mandate a wider, en bloc resection.HemorrhageBleeding from a diverticulum results from erosion of the peri-diverticular arteriole and may result in massive hemorrhage. Most significant lower gastrointestinal hemorrhage occurs in elderly patients in whom both diverticulosis and angiodyspla-sia are common. Consequently, the exact bleeding source may be difficult to identify. Fortunately, in 80% of patients, bleed-ing stops spontaneously. Clinical management should focus on resuscitation and localization of the bleeding site as described for lower gastrointestinal hemorrhage. Colonoscopy may occa-sionally identify a bleeding diverticulum that may then be treated with epinephrine injection or cautery. Angiography may be diagnostic and therapeutic in this setting. In the rare instance in which diverticular hemorrhage persists or recurs, laparotomy and

1	treated with epinephrine injection or cautery. Angiography may be diagnostic and therapeutic in this setting. In the rare instance in which diverticular hemorrhage persists or recurs, laparotomy and segmental colectomy may be required.Giant Colonic DiverticulumGiant colonic diverticula are extremely rare. Most occur on the antimesenteric side of the sigmoid colon. Patients may be asymptomatic or may present with vague abdominal com-plaints such as pain, nausea, or constipation. Plain radiographs may suggest the diagnosis. Barium enema is usually diagnos-tic. Complications of a giant diverticulum include perforation, obstruction, and volvulus. Resection of the involved colon and diverticulum is recommended.Right-Sided DiverticulaThe cecum and ascending colon infrequently are involved in diverticulosis coli. Even more uncommon is a true solitary diverticulum, which contains all layers of the bowel wall and is thought to be congenital in origin. Right-sided diverticula occur more often

1	coli. Even more uncommon is a true solitary diverticulum, which contains all layers of the bowel wall and is thought to be congenital in origin. Right-sided diverticula occur more often in younger patients than do left-sided diver-ticula and are more common in people of Asian descent than in other populations. Most patients with right-sided diverticula are asymptomatic. However, diverticulitis does occur occasionally. Because patients are young and present with right lower quad-rant pain, they are often thought to suffer from acute appendici-tis, and the diagnosis of right-sided diverticulitis is subsequently made in the operating room. If there is a single large diverticu-lum and minimal inflammation, a diverticulectomy may be performed, but an ileocecal resection is usually the preferred operation in this setting. Hemorrhage rarely occurs and should be treated in the same fashion as hemorrhage from a left-sided diverticulum.ADENOCARCINOMA AND POLYPSIncidenceColorectal carcinoma is

1	operation in this setting. Hemorrhage rarely occurs and should be treated in the same fashion as hemorrhage from a left-sided diverticulum.ADENOCARCINOMA AND POLYPSIncidenceColorectal carcinoma is the most common malignancy of the gastrointestinal tract. Over 130,000 new cases are diagnosed annually in the United States, and more than 50,000 patients die of this disease each year, making colorectal cancer the third most lethal cancer in the United States.75The incidence is similar in men and women and has remained fairly constant over the past 20 years; however, the widespread adoption of current national screening programs is gradually decreasing the incidence of this common and lethal disease in people over 50 years of age. However, people younger than 50 have been experiencing and increase incidence and worse mortality.75 Early detection and improvements in medical and surgical care are thought to be responsible for the decreasing mortality of colorectal cancer observed in recent

1	incidence and worse mortality.75 Early detection and improvements in medical and surgical care are thought to be responsible for the decreasing mortality of colorectal cancer observed in recent years.Epidemiology (Risk Factors)Identification of risk factors for development of colorectal can-cer is essential to establish screening and surveillance programs in appropriately targeted populations.Aging. Aging is the dominant risk factor for colorectal cancer, with incidence rising steadily after age 50 years. More than 90% of cases diagnosed are in people older than age 50 years. This is the rationale for initiating screening tests of asymptomatic patients at average risk of developing colorectal cancer at age 50 years. However, individuals of any age can develop colorec-tal cancer, so symptoms such as a significant change in bowel Brunicardi_Ch29_p1259-p1330.indd 128823/02/19 2:29 PM 1289COLON, RECTUM, AND ANUSCHAPTER 29habits, rectal bleeding, melena, unexplained anemia, or weight

1	such as a significant change in bowel Brunicardi_Ch29_p1259-p1330.indd 128823/02/19 2:29 PM 1289COLON, RECTUM, AND ANUSCHAPTER 29habits, rectal bleeding, melena, unexplained anemia, or weight loss require a thorough evaluation.Hereditary Risk Factors. Approximately 80% of colorectal cancers occur sporadically, while 20% arise in patients with a known family history of colorectal cancer. Advances in the understanding of these familial disorders have led to interest in early diagnosis using genetic testing. Because of the medical, legal, and ethical considerations that are involved in this type of testing, all patients should be offered genetic counseling if a familial syndrome is suspected.Environmental and Dietary Factors. The observation that colorectal carcinoma occurs more commonly in populations that consume diets high in animal fat and low in fiber has led to the hypothesis that dietary factors contribute to carcinogenesis. A diet high in saturated or polyunsaturated fats

1	in populations that consume diets high in animal fat and low in fiber has led to the hypothesis that dietary factors contribute to carcinogenesis. A diet high in saturated or polyunsaturated fats increases risk of colorectal cancer, while a diet high in oleic acid (olive oil, coco-nut oil, fish oil) does not increase risk. Animal studies suggest that fats may be directly toxic to the colonic mucosa and thus may induce early malignant changes. In contrast, a diet high in vegetable fiber appears to be protective. A correlation between alcohol intake and incidence of colorectal carcinoma has also been suggested. Ingestion of calcium; selenium; vitamins A, C, and E; carotenoids; and plant phenols may decrease the risk of developing colorectal cancer. Obesity and sedentary lifestyle dramatically increase cancer-related mortality in a number of malignancies, including colorectal carcinoma. This knowl-edge is the basis for primary prevention strategies to eliminate colorectal cancer by

1	increase cancer-related mortality in a number of malignancies, including colorectal carcinoma. This knowl-edge is the basis for primary prevention strategies to eliminate colorectal cancer by altering diet and lifestyle.76Inflammatory Bowel Disease. Patients with long-standing colitis from inflammatory bowel disease are at increased risk for the development of colorectal cancer. It is hypothesized that chronic inflammation predisposes the mucosa to malignant changes, and there is some evidence that degree of inflamma-tion influences risk. In general, the duration and extent of colitis correlate with risk. Other factors thought to increase risk include the presence of primary sclerosing cholangitis and family his-tory of colorectal cancer.Other Risk Factors. Cigarette smoking is associated with an increased risk of colonic adenomas, especially after more than 35 years of use. Patients with ureterosigmoidostomy may also be at increased risk for both adenoma and carcinoma

1	is associated with an increased risk of colonic adenomas, especially after more than 35 years of use. Patients with ureterosigmoidostomy may also be at increased risk for both adenoma and carcinoma formation.77Acromegaly, which is associated with increased levels of circulating human growth hormone and insulin-like growth factor-1, increases risk as well. Pelvic irradiation may increase the risk of developing rectal carcinoma. However, it is unclear whether this represents a direct effect of radiation damage or is instead a correlation between the development of rectal cancer and a history of another pelvic malignancy; for example, among patients who develop prostate cancer and are treated with radia-tion, the risk of rectal cancer increases significantly.78Pathogenesis of Colorectal CancerGenetic Defects. An intense research effort has focused on elucidating the genetic defects and molecular abnormalities asso-ciated with the development and progression of colorectal adeno-mas and

1	Defects. An intense research effort has focused on elucidating the genetic defects and molecular abnormalities asso-ciated with the development and progression of colorectal adeno-mas and carcinoma. Mutations may cause activation of oncogenes (K-ras) and/or inactivation of tumor suppressor genes (APC, deleted in colorectal carcinoma [DCC], p53). Colorectal carcinoma is thought to develop from adenomatous polyps by accumulation of these mutations in what has come to be known as the adenoma-carcinoma sequence (Fig. 29-22).9,79Defects in the APC gene were first described in patients with Familial Adenomatous Polyposis (FAP). By investigating these families, characteristic mutations in the APC gene were identified. They are now known to be present in 80% of spo-radic colorectal cancers as well.The APC gene is a tumor suppressor gene. Mutations in both alleles are necessary to initiate polyp formation. The majority of mutations are premature stop codons, which result in a truncated APC

1	well.The APC gene is a tumor suppressor gene. Mutations in both alleles are necessary to initiate polyp formation. The majority of mutations are premature stop codons, which result in a truncated APC protein. In FAP, the site of mutation correlates with the clinical severity of the disease. For example, mutations in either the 3′ or 5′ end of the gene result in attenuated forms of FAP (AFAP), whereas mutations in the center of the gene result in more virulent disease. Thus, knowledge of the specific mutation in a family may help guide clinical decision-making.10APC inactivation alone does not result in a carcinoma. Instead, this mutation sets the stage for the accumulation of genetic damage that results in malignancy. Additional mutations may include activation or inactivation of a variety of genes.One of the most commonly involved genes in colorectal cancer is K-ras. K-ras, a signaling molecule in the epidermal growth factor receptor (EGFR) pathway, is classified as a proto-oncogene

1	of genes.One of the most commonly involved genes in colorectal cancer is K-ras. K-ras, a signaling molecule in the epidermal growth factor receptor (EGFR) pathway, is classified as a proto-oncogene because mutation of only one allele will perturb the cell cycle. The K-ras gene product is a G-protein involved in intracellular signal transduction. When active, K-ras binds gua-nosine triphosphate (GTP); hydrolysis of GTP to guanosine diphosphate (GDP) then inactivates the G-protein. Mutation of KRAS results in an inability to hydrolyze GTP, thus leaving the G-protein permanently in the active form. It is thought that this then leads to uncontrolled cell division. Because K-ras muta-tion results in uncontrolled downstream signaling, anti-EGFR agents are ineffective in treating K-ras mutant tumors. As such, K-ras mutation status is important in deciding when to utilize anti-EGFR therapies. Other EGFR signaling molecules such as BRAF have also been implicated in colorectal cancer

1	mutant tumors. As such, K-ras mutation status is important in deciding when to utilize anti-EGFR therapies. Other EGFR signaling molecules such as BRAF have also been implicated in colorectal cancer patho-genesis and progression, and ongoing research is focusing on elucidating their roles in this disease.5NormalepitheliumDysplasticepitheliumEarlyadenomaIntermediateadenomaLateadenomaCarcinomaMetastasisAPCK-RASDCC/DPC4/JV18?p53Other changesFigure 29-22. Schematic showing progression from normal colonic epithelium to carcinoma of the colon.Brunicardi_Ch29_p1259-p1330.indd 128923/02/19 2:29 PM 1290SPECIFIC CONSIDERATIONSPART IIAnother common mutation occurs in the MYH gene on chromosome 1p. MYH is a base excision repair gene, and bial-lelic deletion results in changes in other downstream molecules. Since its discovery, MYH mutations have been associated with an AFAP phenotype in addition to sporadic cancers. Unlike APC gene mutations that are expressed in an autosomal domi-nant

1	molecules. Since its discovery, MYH mutations have been associated with an AFAP phenotype in addition to sporadic cancers. Unlike APC gene mutations that are expressed in an autosomal domi-nant pattern, the requirement for biallelic mutation in MYH results in an autosomal recessive pattern of inheritance.80,81The tumor suppressor gene p53 has been well character-ized in a number of malignancies. The p53 protein appears to be crucial for initiating apoptosis in cells with irreparable genetic damage. Mutations in p53 are present in 75% of colorectal cancers.Deletion of the tumor suppressor phosphatase and ten-sin homolog (PTEN) appears to be involved in a number of hamartomatous polyposis syndromes. Deletions in PTEN have been identified in juvenile polyposis, Peutz-Jeghers syndrome, Cowden’s syndrome, and PTEN hamartoma syndrome, in addi-tion to multiple endocrine neoplasia type IIB. Peutz-Jeghers syndrome is also associated with mutation in STK11, a serine-threonin kinase gene. The

1	syndrome, and PTEN hamartoma syndrome, in addi-tion to multiple endocrine neoplasia type IIB. Peutz-Jeghers syndrome is also associated with mutation in STK11, a serine-threonin kinase gene. The genetic changes that underlie serratis polyposis syndrome(s) are currently poorly understood.9Genetic Pathways. The mutations involved in colorectal cancer pathogenesis and progression are now recognized to accumulate via one of three major genetic pathways: the loss of heterozygosity (LOH; chromosomal instability) pathway, the microsatellite instability (MSI) pathway, and the CpG island methylation (CIMP; serrated methylated) pathway.The Loss of Heterozygosity Pathway The LOH pathway is characterized by chromosomal deletions and tumor aneu-ploidy. Eighty percent of colorectal carcinomas appear to arise from mutations in the LOH pathway. This pathway was first described in patients with FAP in whom mutations of the APC gene were found to be inherited.Another example of LOH occurs in the region

1	arise from mutations in the LOH pathway. This pathway was first described in patients with FAP in whom mutations of the APC gene were found to be inherited.Another example of LOH occurs in the region of chromo-some 18q. This region has been found to be deleted in up to 70% of colorectal cancers. Two tumor suppressor genes, DCC and SMAD4, are located in this region, and as such, deletion of 18q may result in the loss of one or both of these genes. DCC is a tumor suppressor gene, and loss of both alleles is required for malignant degeneration. The main role of this molecule appears to be in the central nervous system, where it is involved in neural differentiation and axonal migration. This observa-tion has led to the hypothesis that DCC may be involved in differentiation and cellular adhesion in colorectal cancer, but this theory remains unproven. DCC mutations are present in more than 70% of colorectal carcinomas and may negatively impact prognosis. SMAD4 functions in the signaling

1	in colorectal cancer, but this theory remains unproven. DCC mutations are present in more than 70% of colorectal carcinomas and may negatively impact prognosis. SMAD4 functions in the signaling cascade of transforming growth factor beta and beta-catenin (also a down-stream effector of the APC gene). Loss of either of these genes is thought to promote cancer progression.82The Microsatellite Instability Pathway. Many of the remain-ing colorectal carcinomas are thought to arise from mutations in the MSI pathway, which is characterized by errors in mismatch repair during DNA replication. These errors in mismatch repair were first described in Hereditary Nonpolyposis Colon Cancer (HNPCC; Lynch syndrome) Lynch 9, but are now recognized to be present in many sporadic tumors as well. A number of genes have been identified that appear to be crucial for recognizing and repairing DNA replication errors. These mismatch repair genes include MSH2, MLH1, PMS1, PMS2, and MSH6/GTBP. A mutation in one

1	genes have been identified that appear to be crucial for recognizing and repairing DNA replication errors. These mismatch repair genes include MSH2, MLH1, PMS1, PMS2, and MSH6/GTBP. A mutation in one of these genes predisposes a cell to mutations, which may occur in proto-oncogenes or tumor suppressor genes. Accumulation of these errors then leads to genomic instability and ultimately to carcinogenesis.Microsatellites are regions of the genome in which short base-pair segments are repeated several times, regions that are particularly prone to replication error. Consequently, a mutation in a mismatch repair gene produces variable lengths of these repetitive sequences, a finding that has been described as MSI.Tumors associated with MSI appear to have different bio-logic characteristics than do tumors that result from the LOH pathway. Tumors with MSI are more likely to be in the right colon and possess diploid DNA and are associated with a better prognosis than tumors that arise from the

1	do tumors that result from the LOH pathway. Tumors with MSI are more likely to be in the right colon and possess diploid DNA and are associated with a better prognosis than tumors that arise from the LOH pathway that are microsatellite stable. Tumors arising from the LOH pathway tend to occur in the more distal colon, often have chromosomal aneuploidy, and are associated with a poorer prognosis.CpG Island Methylation Pathway In the CIMP pathway, genes do not accumulate mutations (deletions or insertions of bases), but instead are activated or inactivated by methylation. This process has been called epigenetic alteration to differenti-ate it from the more traditional genetic alterations or true muta-tions. In normal cells, methylation is critical for regulation of gene expression. In cancer, aberrant methylation (either hyperor hypomethylation), usually of a promoter region, results in abnormal activation or inactivation of genes. This gene silenc-ing or, alternatively, activation

1	aberrant methylation (either hyperor hypomethylation), usually of a promoter region, results in abnormal activation or inactivation of genes. This gene silenc-ing or, alternatively, activation results in a phenotype similar to that present with a true gene mutation. This pathway has also been called the serrated methylated pathway because of the observation that serrated polyps often harbor aberrant meth-ylation in contrast to adenomatous polyps that are more often associated with mutations in the APC gene (LOH pathway).83Although these classifications are useful for understanding the mechanisms underlying carcinogenesis, they are not mutu-ally exclusive. For example, a mismatch repair gene may be inactivated by methylation. Errors in mismatch repair may then allow mutations to inactivate a tumor suppressor gene. In addi-tion, there is considerable interest in targeting molecules in each of these pathways in order to design better anticancer agents. Finally, ongoing research is

1	a tumor suppressor gene. In addi-tion, there is considerable interest in targeting molecules in each of these pathways in order to design better anticancer agents. Finally, ongoing research is focusing on the utility of molecular profiling in predicting prognosis and/or response to treatment.10PolypsIt is now well accepted that the majority of colorectal carcino-mas evolve from adenomatous polyps; this sequence of events is the adenoma-carcinoma sequence. Polyp is a nonspecific clini-cal term that describes any projection from the surface of the intestinal mucosa regardless of its histologic nature. Colorec-tal polyps may be classified as neoplastic (tubular adenoma, villous adenoma, tubulovillous adenomas, serrated adenomas/polyps), hyperplastic, hamartomatous (juvenile, Peutz-Jeghers, Cronkite-Canada), or inflammatory (pseudopolyp, benign lymphoid polyp).Neoplastic Polyps. Adenomatous polyps are common, occur-ring in up to 25% of the population older than 50 years of age in the

1	Cronkite-Canada), or inflammatory (pseudopolyp, benign lymphoid polyp).Neoplastic Polyps. Adenomatous polyps are common, occur-ring in up to 25% of the population older than 50 years of age in the United States. By definition, these lesions are dysplas-tic. The risk of malignant degeneration is related to both the size and type of polyp. Tubular adenomas are associated with malignancy in only 5% of cases, whereas villous adenomas may harbor cancer in up to 40%. Tubulovillous adenomas are at intermediate risk (22%). Invasive carcinomas are rare in Brunicardi_Ch29_p1259-p1330.indd 129023/02/19 2:29 PM 1291COLON, RECTUM, AND ANUSCHAPTER 29polyps smaller than 1 cm; the incidence of invasive carcinoma increases with size. The risk of carcinoma in a polyp larger than 2 cm is 35% to 50%. Although most neoplastic polyps do not evolve to cancer, most colorectal cancers originate as a polyp. It is this fact that forms the basis for secondary prevention strate-gies to eliminate colorectal

1	most neoplastic polyps do not evolve to cancer, most colorectal cancers originate as a polyp. It is this fact that forms the basis for secondary prevention strate-gies to eliminate colorectal cancer by targeting the neoplastic polyp for removal before malignancy develops.Polyps may be pedunculated or sessile. Most pedunculated polyps are amenable to colonoscopic snare excision. Removal of sessile polyps is often more challenging. Special colonoscopic techniques, including saline lift, piecemeal snare excision, and endoscopic mucosal resection facilitate successful removal of many sessile polyps. For rectal sessile polyps, transanal opera-tive excision is preferred because it produces an intact, single pathology specimen that can be used to determine the need for further therapy. Interpretation of the precise depth of invasion of a cancer arising in a sessile polyp after piecemeal excision is often impossible. The site of sessile polypectomies should be marked by tattoo marking to

1	of the precise depth of invasion of a cancer arising in a sessile polyp after piecemeal excision is often impossible. The site of sessile polypectomies should be marked by tattoo marking to guide subsequent endoscopic sur-veillance and to facilitate identification of the involved bowel segment should operative resection be necessary.84 Colectomy is reserved for cases in which colonoscopic removal is impos-sible, such as large, flat lesions, or if a focus of invasive cancer is confirmed in the specimen.Complications of polypectomy include perforation and bleeding. A small perforation (microperforation) in a fully pre-pared, stable patient may be managed with bowel rest, broad-spectrum antibiotics, and close observation. Signs of sepsis, peritonitis, or deterioration in clinical condition are indications for laparotomy. Bleeding may occur immediately after polypec-tomy or may be delayed. The bleeding will usually stop sponta-neously, but colonoscopy may be required to apply endoscopic

1	are indications for laparotomy. Bleeding may occur immediately after polypec-tomy or may be delayed. The bleeding will usually stop sponta-neously, but colonoscopy may be required to apply endoscopic clips, resnare a bleeding stalk, cauterize the lesion, or inject/apply epinephrine. Occasionally angiography and infusion of vasopressin may be necessary. Rarely, colectomy is required.Hyperplastic Polyps. Hyperplastic polyps are extremely com-mon in the colon. These polyps are usually small (<5 mm) and show histologic characteristics of hyperplasia without any dys-plasia. They are not considered premalignant, but they cannot be distinguished from adenomatous polyps colonoscopically and are therefore often removed. In contrast, large hyperplas-tic polyps (>2 cm) may have a risk of malignant degeneration. Hyperplastic polyposis is a rare disorder in which multiple large hyperplastic polyps occur in young adults. These patients are at increased risk for the development of colorectal

1	degeneration. Hyperplastic polyposis is a rare disorder in which multiple large hyperplastic polyps occur in young adults. These patients are at increased risk for the development of colorectal cancer.Serrated Polyps. Serrated polyps, including sessile serrated adenomas and traditional serrated adenomas, are a recently recognized, histologically distinct group of neoplastic polyps. Endoscopically they are flat lesions and frequently difficult to visualize. These lesions were long thought to be similar to hyperplastic polyps with minimal malignant potential. How-ever, it has become clear that some of these polyps will develop into invasive cancers. In addition, a familial serrated polyposis syndrome has been described. Serrated polyps should be treated like adenomatous polyps.85Hamartomatous Polyps (Juvenile Polyps). In contrast to adenomatous and serrated polyps, hamartomatous polyps (juve-nile polyps) usually are not premalignant. These lesions are the characteristic polyps of

1	Polyps (Juvenile Polyps). In contrast to adenomatous and serrated polyps, hamartomatous polyps (juve-nile polyps) usually are not premalignant. These lesions are the characteristic polyps of childhood but may occur at any age. Bleeding is a common symptom, and intussusception and/or obstruction may occur. Because the gross appearance of these polyps is identical to adenomatous polyps, these lesions should also be treated by polypectomy. In contrast to adenomatous pol-yposis syndromes, these conditions are often associated with mutation in BMPR1A and SMAD4.Familial juvenile polyposis is an autosomal dominant dis-order in which patients develop hundreds of polyps in the colon and rectum. Unlike solitary juvenile polyps, these lesions may degenerate into adenomas and eventually carcinoma. Annual screening should begin between the ages of 10 and 12 years. Treatment is surgical and depends in part on the degree of rectal involvement. If the rectum is relatively spared, a total abdomi-nal

1	screening should begin between the ages of 10 and 12 years. Treatment is surgical and depends in part on the degree of rectal involvement. If the rectum is relatively spared, a total abdomi-nal colectomy with ileorectal anastomosis may be performed with subsequent close surveillance of the retained rectum. If the rectum is carpeted with polyps, total proctocolectomy is the more appropriate operation. These patients are candidates for ileal pouch–anal reconstruction to avoid a permanent stoma.Peutz-Jeghers syndrome is characterized by polyposis of the small intestine and, to a lesser extent, polyposis of the colon and rectum. Characteristic melanin spots are often noted on the buccal mucosa and lips of these patients. The polyps of Peutz-Jeghers syndrome are generally considered to be hamar-tomas and are not thought to be at significant risk for malignant degeneration. However, carcinoma may occasionally develop. Because the entire length of the gastrointestinal tract may be affected,

1	and are not thought to be at significant risk for malignant degeneration. However, carcinoma may occasionally develop. Because the entire length of the gastrointestinal tract may be affected, surgery is reserved for symptoms such as obstruction or bleeding or for patients in whom polyps develop adenoma-tous features. Screening consists of a baseline colonoscopy and upper endoscopy at age 20 years, followed by annual flexible sigmoidoscopy thereafter. Clinicians should ensure patients are screened for associated extraintestinal malignancies (breast, upper gastrointestinal tract, pancreas, cervix, ovaries, and testicles).Cronkite-Canada syndrome is a disorder in which patients develop gastrointestinal polyposis in association with alopecia, cutaneous pigmentation, and atrophy of the fingernails and toenails. Diarrhea is a prominent symptom, and vomiting, mal-absorption, and protein-losing enteropathy may occur. Most patients die of this disease despite maximal medical therapy, and

1	and toenails. Diarrhea is a prominent symptom, and vomiting, mal-absorption, and protein-losing enteropathy may occur. Most patients die of this disease despite maximal medical therapy, and surgery is reserved for complications of polyposis such as obstruction.Cowden’s syndrome is an autosomal dominant disorder with hamartomas of all three embryonal cell layers. Facial trich-ilemmomas, breast cancer, thyroid disease, and gastrointestinal polyps are typical of the syndrome. Patients should be screened for cancers. Treatment is otherwise based on symptoms.Inflammatory Polyps (Pseudopolyps). Inflammatory pol-yps occur most commonly in the context of inflammatory bowel disease, but they may also occur after amebic colitis, ischemic colitis, and schistosomal colitis. These lesions are not premalig-nant, but they cannot be distinguished from adenomatous polyps based on gross appearance and therefore should be removed. Microscopic examination shows islands of normal, regenerat-ing mucosa

1	but they cannot be distinguished from adenomatous polyps based on gross appearance and therefore should be removed. Microscopic examination shows islands of normal, regenerat-ing mucosa (the polyp) surrounded by areas of mucosal loss. Polyposis may be extensive, especially in patients with severe colitis, and may mimic FAP.Inherited Colorectal CarcinomaMany of the genetic defects originally described in hereditary cancers have subsequently been found in sporadic tumors. Although the majority of colorectal cancer is sporadic, several hereditary syndromes provide paradigms for the study of this disease. Insight gained from studying inherited colorectal cancer Brunicardi_Ch29_p1259-p1330.indd 129123/02/19 2:29 PM 1292SPECIFIC CONSIDERATIONSPART IIsyndromes has led to better understanding of the genetics of colorectal carcinoma.Familial Adenomatous Polyposis. This rare autosomal dominant condition accounts for only about 1% of all colorec-tal adenocarcinomas. Nevertheless, this

1	of the genetics of colorectal carcinoma.Familial Adenomatous Polyposis. This rare autosomal dominant condition accounts for only about 1% of all colorec-tal adenocarcinomas. Nevertheless, this syndrome has provided tremendous insight into the molecular mechanisms underlying colorectal carcinogenesis. The genetic abnormality in FAP is a mutation in the APC gene, located on chromosome 5q. Of patients with FAP, APC mutation testing is positive in 75% of cases. While most patients with FAP will have a known family history of the disease, up to 25% present without other affected family members. Clinically, patients develop hundreds to thou-sands of adenomatous polyps shortly after puberty. The lifetime risk of colorectal cancer in FAP patients approaches 100% by age 50 years.Flexible sigmoidoscopy of first-degree relatives of FAP patients beginning at age 10 to 15 years has been the traditional mainstay of screening. Today, following genetic counseling, APC gene testing may be used to

1	of first-degree relatives of FAP patients beginning at age 10 to 15 years has been the traditional mainstay of screening. Today, following genetic counseling, APC gene testing may be used to screen family members, pro-viding an APC mutation has been identified. If APC testing is positive in a relative of a patient with a known APC mutation, annual flexible sigmoidoscopy beginning at age 10 to 15 years is done until polyps are identified. If APC testing is negative, the relative can be screened starting at age 50 years per average-risk guidelines. If APC testing is refused or unavailable, or if a mutation cannot be identified, annual flexible sigmoidoscopy beginning at age 10 to 15 years is performed until age 24 years. Screening flexible sigmoidoscopy is then done every 2 years until age 34 years, every 3 years until age 44 years, and then every 3 to 5 years.FAP patients are also at risk for the development of adeno-mas anywhere in the gastrointestinal tract, particularly in the

1	age 34 years, every 3 years until age 44 years, and then every 3 to 5 years.FAP patients are also at risk for the development of adeno-mas anywhere in the gastrointestinal tract, particularly in the duodenum. Periampullary carcinoma is a particular concern. Upper endoscopy is therefore recommended for surveillance every 1 to 3 years beginning at age 25 to 30 years.Once the diagnosis of FAP has been made and polyps are developing, treatment is surgical. Four factors affect the choice of operation: age of the patient; presence and severity of symp-toms; extent of rectal polyposis; and presence and location of cancer or desmoid tumors. Three operative procedures can be considered: total proctocolectomy with an end (Brooke) ileos-tomy; total abdominal colectomy with ileorectal anastomosis; and restorative proctocolectomy with ileal pouch–anal anas-tomosis (IPAA) with or without a temporary ileostomy. Most patients elect to have an ileal pouch–anal anastomosis in the absence of a distal

1	and restorative proctocolectomy with ileal pouch–anal anas-tomosis (IPAA) with or without a temporary ileostomy. Most patients elect to have an ileal pouch–anal anastomosis in the absence of a distal rectal cancer, a mesenteric desmoid tumor that prevents the ileum from reaching the anus, or poor sphinc-ter function. Mucosectomy has been advocated in patients with FAP undergoing ileal pouch–anal anastomosis because of the risk of neoplasia in the anal transition zone, but the requirement for this procedure remains controversial.86,87 Although patient satisfaction with IPAA remains high, function may not be ideal, and up to 50% of patients experience some degree of incon-tinence. Total abdominal colectomy with an ileorectal anasto-mosis is also an option in these patients, but requires vigilant surveillance of the retained rectum for development of rectal cancer. There is increasing data suggesting that the administra-tion of cyclooxygenase-2 (COX-2) inhibitors (celecoxib, sulin-dac)

1	surveillance of the retained rectum for development of rectal cancer. There is increasing data suggesting that the administra-tion of cyclooxygenase-2 (COX-2) inhibitors (celecoxib, sulin-dac) may slow or prevent the development of polyps.88,89FAP may be associated with extraintestinal manifesta-tions such as congenital hypertrophy of the retinal pigmented epithelium, desmoid tumors, epidermoid cysts, mandibular osteomas (Gardner’s syndrome), and central nervous system tumors (Turcot’s syndrome). Although they arise in a minority of FAP patients, desmoid tumors in particular can make surgi-cal management difficult and are a source of major morbidity and mortality in these patients. These lesions arise from fibro-blasts, and although technically benign, can be highly locally invasive. These tumors often involve the bowel mesentery, pel-vis, and abdominal wall. In some cases, surgery is thought to be the “trigger.” Local recurrence after attempted resection is common; therefore, surgery

1	tumors often involve the bowel mesentery, pel-vis, and abdominal wall. In some cases, surgery is thought to be the “trigger.” Local recurrence after attempted resection is common; therefore, surgery is to be avoided if at all possible. Desmoid tumors are often hormone responsive, and growth may be inhibited in some patients with tamoxifen. COX-2 inhibitors and nonsteroidal, anti-inflammatory drugs may also be benefi-cial in this setting. A recent report suggests that imatinib may also be effective.90,91Attenuated Familial Adenomatous Polyposis. AFAP is a recognized variant of FAP. Patients present later in life with fewer polyps (usually 10–100) predominantly located in the right colon, when compared to classic FAP. Colorectal carci-noma develops in more than 50% of these patients, but occurs later (average age, 55 years). Patients are also at risk for duo-denal polyposis. However, in contrast to FAP, APC gene muta-tions are present in only about 30% of patients with AFAP. When

1	but occurs later (average age, 55 years). Patients are also at risk for duo-denal polyposis. However, in contrast to FAP, APC gene muta-tions are present in only about 30% of patients with AFAP. When present, these mutations are expressed in an autosomal dominant pattern.Mutations in MYH also result in the AFAP phenotype but are expressed in an autosomal recessive pattern. It has been sug-gested that MYH mutations may be responsible for AFAP in patients who do not have a detectable APC gene mutation.80,81Genetic testing is often offered to patients with suspected AFAP. When positive, genetic counseling and testing may be used to screen at-risk family members. If the family mutation is unknown, screening colonoscopy is recommended beginning at age 13 to 15 years, then every 4 years to age 28 years, and then every 3 years. These patients are often candidates for a total abdominal colectomy with ileorectal anastomosis because the limited polyposis in the rectum can usually be treated by

1	28 years, and then every 3 years. These patients are often candidates for a total abdominal colectomy with ileorectal anastomosis because the limited polyposis in the rectum can usually be treated by colo-noscopic snare excision.92 Prophylaxis with COX-2 inhibitors also may be appropriate. Because of the more subtle phenotype in these patients, it is important to rule out other familial syn-dromes such as HNPCC (Lynch syndrome) and the more com-mon familial colorectal cancer.Lynch Syndrome (Hereditary Nonpolyposis Colon Cancer; HNPCC). Lynch syndrome is more common than FAP, but it is still extremely rare (1–3% of all colon cancers). The genetic defects associated with Lynch syndrome arise from errors in mismatch repair, the phenotypic result being MSI. Lynch syn-drome is inherited in an autosomal dominant pattern and is char-acterized by the development of colorectal carcinoma at an early age (average age, 40–45 years). Approximately 70% of affected individuals will develop

1	in an autosomal dominant pattern and is char-acterized by the development of colorectal carcinoma at an early age (average age, 40–45 years). Approximately 70% of affected individuals will develop colorectal cancer. Cancers appear in the proximal colon more often than in sporadic colorectal cancer and have a better prognosis regardless of stage. The risk of syn-chronous or metachronous colorectal carcinoma is 40%. Lynch syndrome may also be associated with extracolonic malignan-cies, including endometrial carcinoma, which is most com-mon in women, and ovarian, pancreas, stomach, small bowel, biliary, and urinary tract carcinomas. The diagnosis is made based on family history. The Amsterdam I criteria for clini-cal diagnosis of Lynch syndrome are three affected relatives Brunicardi_Ch29_p1259-p1330.indd 129223/02/19 2:29 PM 1293COLON, RECTUM, AND ANUSCHAPTER 29with histologically verified adenocarcinoma of the large bowel (one must be a first-degree relative of one of the others)

1	129223/02/19 2:29 PM 1293COLON, RECTUM, AND ANUSCHAPTER 29with histologically verified adenocarcinoma of the large bowel (one must be a first-degree relative of one of the others) in two successive generations of a family with one patient diagnosed before age 50 years. The presence of other related carcinomas should raise the suspicion of this syndrome. Revised criteria Amsterdam II requires three or more relatives with an HNPCC related malignancy in which at least one is a first degree relative of the others, two generations are affected, at least one cancer occurred before age 50, FAP has been excluded, and pathology of the tumors has been reviewed and confirmed. In a patient with an established diagnosis of colorectal cancer, tumor testing for presence of mismatch repair gene products (immunohisto-chemistry) and/or MSI can sometimes serve as screening for this syndrome.10,93,94Lynch syndrome results from mutations in mismatch repair genes, and like FAP, specific mutations are

1	(immunohisto-chemistry) and/or MSI can sometimes serve as screening for this syndrome.10,93,94Lynch syndrome results from mutations in mismatch repair genes, and like FAP, specific mutations are associated with different phenotypes. For example, mutations in PMS2 or MSH6 result in a more attenuated form of Lynch syndrome when compared to mutations in other genes. MSH6 inactivation also appears to be associated with a higher risk for endometrial cancer. Further significance of these specific mutations remains to be determined.Screening colonoscopy is recommended annually for at-risk patients beginning at either age 20 to 25 years or 10 years younger than the youngest age at diagnosis in the family, which-ever comes first. Because of the high risk of endometrial carci-noma, transvaginal ultrasound or endometrial aspiration biopsy is also recommended annually after age 25 to 35 years. Because there is a 40% risk of developing a second colon cancer, total colectomy with ileorectal

1	ultrasound or endometrial aspiration biopsy is also recommended annually after age 25 to 35 years. Because there is a 40% risk of developing a second colon cancer, total colectomy with ileorectal anastomosis is recommended once adenomas or a colon carcinoma is diagnosed. Annual proctos-copy is necessary because the risk of developing rectal cancer remains high. Similarly, prophylactic hysterectomy and bilateral salpingo-oophorectomy should be considered in women who have completed childbearing.93-95Familial Colorectal Cancer. Nonsyndromic familial colorec-tal cancer accounts for 10% to 15% of patients with colorec-tal cancer. The lifetime risk of developing colorectal cancer increases with a family history of the disease. The lifetime risk of colorectal cancer in a patient with no family history of this disease (average-risk population) is approximately 6%, but rises to 12% if one first-degree relative is affected and to 35% if two first-degree relatives are affected. Age of onset

1	history of this disease (average-risk population) is approximately 6%, but rises to 12% if one first-degree relative is affected and to 35% if two first-degree relatives are affected. Age of onset also impacts risk, and a diagnosis before the age of 50 years is associated with a higher incidence in family members. Screening colonoscopy is recommended every 5 years beginning at age 40 years or begin-ning 10 years before the age of the earliest diagnosed patient in the pedigree. While there are no specific genetic abnormalities that are associated with familial colorectal cancer, any of the defects found in either the LOH pathway or MSI pathway may be present in these patients.Prevention: Screening and SurveillanceBecause the majority of colorectal cancers are thought to arise from adenomatous polyps, preventive measures focus on identi-fication and removal of these premalignant lesions. In addition, many cancers are asymptomatic, and screening may detect these tumors at an early and

1	polyps, preventive measures focus on identi-fication and removal of these premalignant lesions. In addition, many cancers are asymptomatic, and screening may detect these tumors at an early and curable stage (Table 29-1). Although screening for colorectal cancer decreases the incidence of can-cer and cancer-related mortality, the optimal method of screen-ing remains controversial. Screening guidelines are meant for asymptomatic patients.95-98 Any patient with a gastrointestinal complaint (bleeding, change in bowel habits, pain, etc) requires a complete evaluation, usually by colonoscopy.Fecal Occult Blood Testing and Fecal Immunohistochemical Testing. FOBT is known to reduce colorectal cancer mortality by 33% and metastatic disease by 50%. However, FOBT is rela-tively insensitive, missing up to 50% of cancers and the majority of adenomas. Its specificity is low because 90% of patients with positive tests do not have colorectal cancer. FIT is more sensitive and specific for cancer.

1	up to 50% of cancers and the majority of adenomas. Its specificity is low because 90% of patients with positive tests do not have colorectal cancer. FIT is more sensitive and specific for cancer. Mortality benefits for its use are inferred from FOBT literature. Compliance with annual testing is low and costs are significant if one includes the colonoscopy examina-tions done to evaluate patients with positive FOBT/FIT. Nonethe-less, the direct evidence that FOBT screening is efficacious and decreases both the incidence and mortality of colorectal cancer is so strong that national guidelines recommend annual FOBT/FIT screening for asymptomatic, average-risk Americans older than 50 years of age as one of several accepted strategies. A positive FOBT/FIT should be followed by colonoscopy.97-100Stool DNA. Neoplastic lesions of the colon shed cells into the lumen posing an opportunity for detection via DNA testing. A commercially available multitarget stool DNA test evaluates stool samples

1	DNA. Neoplastic lesions of the colon shed cells into the lumen posing an opportunity for detection via DNA testing. A commercially available multitarget stool DNA test evaluates stool samples for mutant KRAS, methylated BMP3 and the pro-moter region of NDRG4. In a large North American prospective study, this test has recently been found to be 92% sensitive for detection of colorectal cancer. Compared to FIT, stool DNA testing has a lower specificity (74%) raising concerns about how to manage stool DNA-positive patients who have a negative colonoscopic evaluation. Sensitivity for advanced precancerous lesions was 42%.100,101 This test, in combination with FIT, is supported as a screening modality every 1 to 3 years by the U.S. Preventative Task Force, and every 3-year utilization is sup-ported by NCCN guidelines.100 Nevertheless, additional studies will be necessary to determine if these tests are comparable or superior to more traditional methods has been techniques.Flexible

1	is sup-ported by NCCN guidelines.100 Nevertheless, additional studies will be necessary to determine if these tests are comparable or superior to more traditional methods has been techniques.Flexible Sigmoidoscopy. Screening by flexible sigmoidos-copy every 5 years may lead to a 60% to 70% reduction in mor-tality from colorectal cancer, chiefly by identifying high-risk individuals with adenomas. However, it is important to recog-nize that lesions in the proximal colon cannot be identified, and for this reason, flexible sigmoidoscopy has often been paired with air-contrast barium enema to detect transverse and right colon lesions. Patients found to have a polyp, cancer, or other lesion on flexible sigmoidoscopy will require colonoscopy.101-102Fecal Occult Blood Testing and Flexible Sigmoidoscopy. Several trials have shown that FOBT screening is least effec-tive at detecting rectosigmoid cancers.97-99 This is precisely the area screened by flexible sigmoidoscopy; thus, the combination

1	Several trials have shown that FOBT screening is least effec-tive at detecting rectosigmoid cancers.97-99 This is precisely the area screened by flexible sigmoidoscopy; thus, the combination of the two tests has been suggested as a reasonable screening strategy. Winawer and colleagues, in a study of 12,479 subjects, showed that the combination of FOBT annually with flexible sigmoidoscopy every 5 years resulted in lower mortality from colorectal cancer and better survival in patients with colorectal cancer.99 A similar benefit was confirmed in long-term (11-year) follow-up from the Norwegian Colorectal Cancer Prevention Trial. Such data led to the American Cancer Society recommen-dations that one of the acceptable screening regimens for aver-age-risk Americans is the combination of FOBT/FIT annually and flexible sigmoidoscopy every 5 years; this combination was preferred over either test alone. Recent NCCN guidelines offer the option of flexible sigmoidoscopy with stool-based testing

1	annually and flexible sigmoidoscopy every 5 years; this combination was preferred over either test alone. Recent NCCN guidelines offer the option of flexible sigmoidoscopy with stool-based testing Brunicardi_Ch29_p1259-p1330.indd 129323/02/19 2:29 PM 1294SPECIFIC CONSIDERATIONSPART IIevery 10 years. The addition of air-contrast barium enema to assess the proximal colon may improve sensitivity as well.96Colonoscopy. Colonoscopy is currently the most accurate and most complete method for examining the large bowel. This procedure is highly sensitive for detecting even small polyps (<1 cm) and allows biopsy, polypectomy, control of hemor-rhage, and dilation of strictures. However, colonoscopy does require mechanical bowel preparation, and the discomfort asso-ciated with the procedure requires conscious sedation in most patients. Colonoscopy is also considerably more expensive than other screening modalities and requires a well-trained endos-copist. The risk of a major complication

1	conscious sedation in most patients. Colonoscopy is also considerably more expensive than other screening modalities and requires a well-trained endos-copist. The risk of a major complication after colonoscopy (perforation and hemorrhage) is extremely low (0.2–0.3%). Nevertheless, deaths have been reported.Air-Contrast Barium Enema. Air-contrast barium enema is also highly sensitive for detecting polyps greater than 1 cm in diameter (90% sensitivity). Unfortunately, there are no studies proving its efficacy for screening large populations. Accuracy is greatest in the proximal colon but may be compromised in the sigmoid colon if there is significant diverticulosis. The major disadvantages of barium enema are the need for mechanical bowel preparation and the requirement for colonoscopy if a lesion is discovered.Computed Tomography Colonography (Virtual Colonos-copy). Advances in imaging technology have created a num-ber of less invasive, but highly accurate tools for screening. CT

1	if a lesion is discovered.Computed Tomography Colonography (Virtual Colonos-copy). Advances in imaging technology have created a num-ber of less invasive, but highly accurate tools for screening. CT colonography makes use of helical CT technology and three-dimensional reconstruction to image the intraluminal colon. At present, patients require a mechanical bowel preparation. The colon is then insufflated with air, a spiral CT is performed, and both two-dimensional and three-dimensional images are gener-ated. In the hands of a qualified radiologist, sensitivity appears to be as good as colonoscopy for colorectal cancers and polyps greater than 1 cm in size.103 Colonoscopy is required if a lesion is identified. CT colonography has also been used for imag-ing the proximal colon in cases of obstruction or if a colonos-copy cannot be completed in selected patients. Limitations of this technique include false-positive results from retained stool, diverticular disease, haustral folds, motion

1	or if a colonos-copy cannot be completed in selected patients. Limitations of this technique include false-positive results from retained stool, diverticular disease, haustral folds, motion artifacts, and an inability to detect flat adenomas.Guidelines for Screening. Current American Cancer Society guidelines advocate screening for the average-risk population (asymptomatic, no family history of colorectal carcinoma, no personal history of polyps or colorectal carcinoma, no familial syndrome) beginning at age 50 years. Recommended proce-dures include yearly FOBT/FIT, flexible sigmoidoscopy every Table 29-1Advantages and disadvantages of screening modalities for asymptomatic individuals ADVANTAGESDISADVANTAGESFecal occult blood testing (FOBT)Ease of use and noninvasiveLow costGood sensitivity with repeat testingMay not detect most polypsLow specificityColonoscopy required for positive resultPoor compliance with serial testingThree successive stools requiredFecal immunohistochemical test

1	with repeat testingMay not detect most polypsLow specificityColonoscopy required for positive resultPoor compliance with serial testingThree successive stools requiredFecal immunohistochemical test (FIT)Ease of use and noninvasiveLow costMore sensitive and specific than FOBTOnly one stool sample requiredMay not detect most polypsColonoscopy required for positive resultMultitarget stool DNAEase of use and noninvasiveMore sensitive than FITMay not detect most polypsColonoscopy required for positive resultLess specific than FITSigmoidoscopyExamines colon most at riskVery sensitive for polyp detection in left colonDoes not require full bowel preparation (enemas only)InvasiveUncomfortableSlight risk of perforation or bleedingMay miss proximal lesionsColonoscopy required if polyp identifiedColonoscopyExamines entire colonHighly sensitive and specificTherapeuticMost invasiveUncomfortable and requires sedativeRequires bowel preparationRisk of perforation or bleedingCostlyDouble-contrast

1	entire colonHighly sensitive and specificTherapeuticMost invasiveUncomfortable and requires sedativeRequires bowel preparationRisk of perforation or bleedingCostlyDouble-contrast barium enemaExamines entire colonGood sensitivity for polyps >1 cmExamines entire colonRequires bowel preparationLess sensitivity for polyps <1 cmMay miss lesions in the sigmoid colonColonoscopy required for positive resultComputed tomography colonography (virtual colonoscopy)NoninvasiveSensitivity may be as good as colonoscopyRequires bowel preparationInsensitive for small polypsMinimal experience and dataColonoscopy required for positive resultBrunicardi_Ch29_p1259-p1330.indd 129423/02/19 2:29 PM 1295COLON, RECTUM, AND ANUSCHAPTER 295 years, FOBT/FIT and flexible sigmoidoscopy in combination, air-contrast barium enema every 5 years, or colonoscopy every 10 years. Patients with other risk factors should be screened earlier and more frequently (Table 29-2).93-96,104,105Routes of Spread and Natural

1	barium enema every 5 years, or colonoscopy every 10 years. Patients with other risk factors should be screened earlier and more frequently (Table 29-2).93-96,104,105Routes of Spread and Natural HistoryCarcinoma of the colon and rectum arises in the mucosa. The tumor subsequently invades the bowel wall and eventually adja-cent tissues and other viscera. Tumors may become bulky and circumferential, leading to colon obstruction. Local invasion (especially in the rectum or sigmoid colon) may occasionally cause obstruction of other organs such as the ureter.Regional lymph node involvement is the most common form of spread of colorectal carcinoma and usually precedes distant metastasis or the development of carcinomatosis. The likelihood of nodal metastasis increases with tumor size, poorly differentiated histology, lymphovascular invasion, and depth of invasion. The T stage (depth of invasion) is the single most significant predictor of lymph node spread. Carcinoma in situ (Tis) in which

1	histology, lymphovascular invasion, and depth of invasion. The T stage (depth of invasion) is the single most significant predictor of lymph node spread. Carcinoma in situ (Tis) in which there is no penetration of the muscularis mucosa (basement membrane) has also been called high-grade dysplasia and should carry no risk of lymph node metastasis. Small lesions confined to the bowel wall (T1 and T2) are associated with lymph node metastasis in 5% to 20% of cases, whereas larger tumors that invade through the bowel wall or into adja-cent organs (T3 and T4) are likely to have lymph node metas-tasis in more than 50% of cases. The number of lymph nodes with metastases correlates with the presence of distant disease and inversely with survival. Four or more involved lymph nodes (N2 disease) predict a poor prognosis. In colon cancer, lym-phatic spread usually follows the major venous outflow from the involved segment of the colon. Lymphatic spread from the rectum follows two routes. In the

1	a poor prognosis. In colon cancer, lym-phatic spread usually follows the major venous outflow from the involved segment of the colon. Lymphatic spread from the rectum follows two routes. In the upper rectum, drainage ascends along the superior rectal vessels to the inferior mesen-teric nodes. In the lower rectum, lymphatic drainage may course along the middle rectal vessels. Nodal spread along the inferior rectal vessels to the internal iliac nodes or groin is rare unless the tumor involves the anal canal or the proximal lymphatics are blocked with tumor (Fig. 29-23).The most common site of distant metastasis from colorec-tal cancer is the liver. These metastases arise from hematog-enous spread via the portal venous system. Like lymph node metastasis, the risk of hepatic metastasis increases with tumor size and tumor grade. However, even small tumors may pro-duce distant metastasis. The lung is also a site of hematogenous spread, but this rarely occurs in isolation. Carcinomatosis

1	with tumor size and tumor grade. However, even small tumors may pro-duce distant metastasis. The lung is also a site of hematogenous spread, but this rarely occurs in isolation. Carcinomatosis (dif-fuse peritoneal metastases) occurs by peritoneal seeding and has a dismal prognosis.Staging and Preoperative EvaluationClinical Presentation. Symptoms of colon and rectal can-cers are nonspecific and generally develop when the cancer is locally advanced. The classic first symptoms are a change in bowel habits and rectal bleeding. Abdominal pain, bloating, and other signs of obstruction typically occur with larger tumors and Table 29-2Screening guidelines for colorectal cancerPOPULATIONINITIAL AGERECOMMENDED SCREENING TESTAverage risk50 yAnnual FOBT orFlexible sigmoidoscopy every 5 y orAnnual FOBT and flexible sigmoidoscopy every 5 y orAir-contrast barium enema every 5 y orColonoscopy every 10 yAdenomatous polyps50 yColonoscopy at first detection; then colonoscopy in 3 yIf no further polyps,

1	and flexible sigmoidoscopy every 5 y orAir-contrast barium enema every 5 y orColonoscopy every 10 yAdenomatous polyps50 yColonoscopy at first detection; then colonoscopy in 3 yIf no further polyps, colonoscopy every 5 yIf polyps, colonoscopy every 3 yAnnual colonoscopy for >5 adenomasColorectal cancerAt diagnosisPretreatment colonoscopy; then at 12 mo after curative resection; then colonoscopy after 3 y; then colonoscopy every 5 y, if no new lesionsUlcerative colitis, Crohn’s colitisAt diagnosis; then after 8 y for pancolitis, after 15 y for left-sided colitisColonoscopy with multiple biopsies every 1–2 yFAP10–12 yAnnual flexible sigmoidoscopyUpper endoscopy every 1–3 y after polyps appearAttenuated FAP20 yAnnual flexible sigmoidoscopyUpper endoscopy every 1–3 y after polyps appearHNPCC20–25 yColonoscopy every 1–2 yEndometrial aspiration biopsy every 1–2 yFamilial colorectal cancer first-degree relative40 y or 10 y before the age of the youngest affected relativeColonoscopy every 5

1	yColonoscopy every 1–2 yEndometrial aspiration biopsy every 1–2 yFamilial colorectal cancer first-degree relative40 y or 10 y before the age of the youngest affected relativeColonoscopy every 5 yIncrease frequency if multiple family members are affected, especially before 50 yFAP = familial adenomatous polyposis; FOBT = fecal occult blood testing; HNPCC = hereditary nonpolyposis colon cancer.Data from Smith et al,79 Pignone et al,97 and Levin et al.67Brunicardi_Ch29_p1259-p1330.indd 129523/02/19 2:29 PM 1296SPECIFIC CONSIDERATIONSPART IIsuggest more advanced disease. Because of the caliber of the bowel and the consistency of the stool, left-sided tumors are more likely to cause obstruction than are right-sided tumors. Rectal tumors may cause bleeding, tenesmus, and pain. How-ever, it is important to note that many patients may be asymp-tomatic and/or present with unexplained anemia, weight loss, or poor appetite.Staging. Colorectal cancer staging is based on tumor depth and the

1	is important to note that many patients may be asymp-tomatic and/or present with unexplained anemia, weight loss, or poor appetite.Staging. Colorectal cancer staging is based on tumor depth and the presence or absence of nodal or distant metastases. Older staging systems, such as the Dukes’ Classification and its Astler-Coller modification, have been replaced by the tumor-node-metastasis (TNM) staging system described by the American Joint Committee on Cancer (AJCC). The AJCC TNM classification has recently been updated to reflect sur-vival outcomes based upon the Surveillance Epidemiology and End Results (SEER) registry (Table 29-3).106-109 Stage I disease includes adenocarcinomas that are invasive through the muscu-laris mucosa but are confined to the submucosa (T1) or the mus-cularis propria (T2) in the absence of nodal metastases. Stage II disease consists of tumors that invade through the bowel wall into the subserosa or nonperitonealized pericolic or perirectal tissues (T3) or

1	propria (T2) in the absence of nodal metastases. Stage II disease consists of tumors that invade through the bowel wall into the subserosa or nonperitonealized pericolic or perirectal tissues (T3) or into other organs or tissues or through the vis-ceral peritoneum (T4) without nodal metastases. Stage III dis-ease includes any T stage with nodal metastases, and stage IV disease denotes distant metastases.The preoperative imaging evaluation usually identifies stage IV disease. In colon cancer, differentiating stages I, II, and III depends on histologic examination of the resected specimen. In rectal cancer, endorectal ultrasound or MRI may predict the stage (ultrasound stage, uTxNx) preoperatively, but the final determination depends on pathologic examination of the resected tumor and adjacent lymph nodes (pathologic stage, pTxNx). Dis-ease stage correlates with 5-year survival. Patients with stages I and II disease can expect excellent survival rates. The pres-ence of nodal metastases

1	lymph nodes (pathologic stage, pTxNx). Dis-ease stage correlates with 5-year survival. Patients with stages I and II disease can expect excellent survival rates. The pres-ence of nodal metastases (stage III) decreases survival. In rectal cancer, staging has been further refined, and outcomes suggest that subgroups of patients within each stage may have very dif-ferent prognoses. If the mesorectum around a rectal cancer is involved or threatened (only 1–2 mm of clearance), there is a very high likelihood of local recurrence and a poor prognosis. This circumferential or radial margin is probably best assessed preoperatively by MRI. Although nodal involvement is the sin-gle most important prognostic factor in colorectal carcinoma, tumor characteristics, such as degree of differentiation, muci-nous or signet-ring cell histology, vascular invasion, and DNA aneuploidy, also adversely affect prognosis. Preoperative CEA also has been suggested to be a prognostic indicator.8 Molecular

1	muci-nous or signet-ring cell histology, vascular invasion, and DNA aneuploidy, also adversely affect prognosis. Preoperative CEA also has been suggested to be a prognostic indicator.8 Molecular profiling is currently being studied in an effort to further improve prognostic indicators.110 The 5-year survival rate with stage IV disease is low. However, in well-selected patients, metastasec-tomy, especially of isolated liver or lung lesions, can result in cure. In these patients, the involvement of a multidisciplinary team and/or tumor board is highly recommended (Fig. 29-24).Preoperative Evaluation. Once a colon or rectal carcinoma has been diagnosed, a staging evaluation should be undertaken. The colon must be evaluated for synchronous tumors, usually by colonoscopy. Synchronous disease will be present in up to 5% of patients. For rectal cancers, digital rectal examination and rigid or flexible proctoscopy with biopsy should be performed to assess tumor size, location, morphology,

1	will be present in up to 5% of patients. For rectal cancers, digital rectal examination and rigid or flexible proctoscopy with biopsy should be performed to assess tumor size, location, morphology, histology, and fixa-tion. Endorectal ultrasound or MRI can be invaluable in staging rectal cancer and is used to classify the ultrasound T and N stage of rectal cancers (see Fig. 29-24). A chest/abdominal/pelvic CT scan should be obtained to evaluate for distant metastases. Pelvic CT scan, and sometimes MRI, can be useful in large rec-tal tumors and in recurrent disease to determine the extent of local invasion. Among patients with obstructive symptoms, a water-soluble contrast study (Gastrografin enema) may be use-ful for delineating the degree of obstruction. It is important to avoid mechanical bowel preparation (for either colonoscopy or surgery) in a patient who appears to be obstructed. PET scan may be useful in evaluating lesions seen on CT scan and in patients in whom a risky or

1	bowel preparation (for either colonoscopy or surgery) in a patient who appears to be obstructed. PET scan may be useful in evaluating lesions seen on CT scan and in patients in whom a risky or highly morbid operation is planned (pelvic exenteration, sacrectomy). Preoperative CEA is often obtained and may be useful for postoperative follow-up.Therapy for Colonic CarcinomaPrinciples of Resection. The objective in treatment of car-cinoma of the colon is to remove the primary tumor along with its lymphovascular supply. Because the lymphatics of the colon accompany the main arterial supply, the length of bowel resected depends on which vessels are supplying the segment involved with the cancer. Any adjacent organ or tissue, such as the omentum, that has been invaded should be resected en bloc with the tumor. If all of the tumor cannot be removed, a palliative procedure should be considered, although it impor-tant to note that “debulking” is rarely effective in colorectal adenocarcinoma.The

1	the tumor. If all of the tumor cannot be removed, a palliative procedure should be considered, although it impor-tant to note that “debulking” is rarely effective in colorectal adenocarcinoma.The presence of synchronous cancers or adenomas or a strong family history of colorectal neoplasms suggests that the entire colon is at risk for carcinoma (often called a field defect), and a subtotal or total colectomy should be considered. Meta-chronous tumors (a second primary colon cancer) identified during follow-up studies should be treated similarly. However, the surgeon must be aware of which mesenteric vessels have been ligated at the initial colectomy because that may influence the viability of the remaining colon and the choice of procedure.The number of lymph nodes recovered in the surgical specimen has long served as a proxy for the oncologic adequacy of resection. A number of studies previously have suggested that Inferiormesenteric a.Superiorrectal a.Middlerectal a.Inferiorrectal

1	specimen has long served as a proxy for the oncologic adequacy of resection. A number of studies previously have suggested that Inferiormesenteric a.Superiorrectal a.Middlerectal a.Inferiorrectal a.Common iliac a.Figure 29-23. Lymphatic drainage of the rectum. a. = artery.Brunicardi_Ch29_p1259-p1330.indd 129623/02/19 2:29 PM 1297COLON, RECTUM, AND ANUSCHAPTER 29STAGETNM0TisN0M0IT1, T2N0M0IIAT3N0M0IIBT4aN0M0IICT4bN0M0IIIAT1–T2N1/N1cM0IIIAT1N2aM0IIIBT3–T4aN1/N1cM0IIIBT2–T3N2aM0IIIBT1–T2N2bM0IIICT4aN2aM0IIICT3–T4aN2bM0IIICT4bN1–N2M0IVAAny TAny NM1aIVBAny TAny NM1bIVCAny TAny NM1cUsed with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Table 29-3TNM Staging of colorectal carcinoma DEFINITIONTumor Stage (T)TXPrimary tumor cannot be assessedT0No evidence of primary tumorTisCarcinoma in situ, intramucosal carcinoma (involvement of lamina propria with no extension through

1	Stage (T)TXPrimary tumor cannot be assessedT0No evidence of primary tumorTisCarcinoma in situ, intramucosal carcinoma (involvement of lamina propria with no extension through muscularis mucosae)T1Tumor invades the submucosa (through the muscularis mucosa but not into the muscularis propria)T2Tumor invades the muscularis propriaT3Tumor invades through the muscularis propria into pericolorectal tissuesT4Tumor invades the visceral peritoneum or invades or adheres to adjacent organ or structure T4aTumor invades through the visceral peritoneum (including gross perforation of the bowel through tumor and continuous invasion of tumor through areas of inflammation to the surface of the visceral peritoneum) T4bTumor directly invades or adheres to adjacent organs or structuresNodal Stage (N)NXRegional lymph nodes cannot be assessedN0No regional lymph node metastasisN1One to three regional lymph nodes are positive (tumor in lymph nodes measuring ≥0.2 mm), or any number of tumor deposits are

1	lymph nodes cannot be assessedN0No regional lymph node metastasisN1One to three regional lymph nodes are positive (tumor in lymph nodes measuring ≥0.2 mm), or any number of tumor deposits are present and all identifiable lymph nodes are negative N1aOne regional lymph node is positive N1bTwo or three regional lymph nodes are positive N1cNo regional lymph nodes are positive, but there are tumor deposits in the• subserosa• mesentery• or nonperitonealized pericolic, or perirectal/mesorectal tissues.N2Four or more regional nodes are positive N2aFour to six regional lymph nodes are positive N2bSeven or more regional lymph nodes are positiveDistant Metastasis (M)M0No distant metastasis by imaging, etc.; no evidence of tumor in distant sites or organs (This category is not assigned by pathologists.)M1Metastasis to one or more distant sites or organs or peritoneal metastasis is identified M1aMetastasis to one site or organ is identified without peritoneal metastasis M1bMetastasis to two or

1	to one or more distant sites or organs or peritoneal metastasis is identified M1aMetastasis to one site or organ is identified without peritoneal metastasis M1bMetastasis to two or more sites or organs is identified without peritoneal metastasis M1cMetastasis to the peritoneal surface is identified alone or with other site or organ metastasesBrunicardi_Ch29_p1259-p1330.indd 129723/02/19 2:29 PM 1298SPECIFIC CONSIDERATIONSPART IIFigure 29-24. A. Endorectal ultrasonography showing a T3 rectal carcinoma. The dotted line is being used to measure the diameter of the lesion. B. Rectal cancer MRI. (A, Used with permission from Charles O. Finne III, MD, Minneapolis, MN.) a minimum of 12 lymph nodes in the resected specimen are nec-essary for adequate staging. In addition, patients in whom more nodes are harvested have better long-term outcome. As such, a 12-node minimum has been suggested as an appropriate bench-mark for assessing quality of care. However, several investi-gators recently

1	nodes are harvested have better long-term outcome. As such, a 12-node minimum has been suggested as an appropriate bench-mark for assessing quality of care. However, several investi-gators recently have called this into question, noting that the number of lymph nodes examined does not correlate with stag-ing, use of adjuvant chemotherapy, or patient survival. Others have suggested that the number of negative lymph nodes and/or the lymph node ratio (positive lymph nodes to total lymph nodes) may further improve staging.106,111-114If unexpected metastatic disease is encountered at the time of a laparotomy, the decision about whether to proceed with resection of the primary tumor depends on the volume of dis-tant disease, location and size of the primary tumor, the opera-tion required to remove the primary tumor, and the operative approach. If the metastatic disease is low volume (isolated or potentially resectable liver lesions) and the resection of the primary tumor is straightforward

1	the primary tumor, and the operative approach. If the metastatic disease is low volume (isolated or potentially resectable liver lesions) and the resection of the primary tumor is straightforward (segmental abdominal colec-tomy), it is probably reasonable to proceed with resection. On the other hand, if the metastatic disease is high volume (carci-nomatosis), especially if the primary tumor is minimally symp-tomatic, the operation should be aborted in order to facilitate early systemic chemotherapy. Some centers favor starting the operation with a diagnostic laparoscopy in cases where risk of discovering metastasis is high in order to minimize the magni-tude of the operation should surgery be aborted. With recent advances in chemotherapy, many of these patients will never develop a complication from the primary tumor requiring surgi-cal intervention. Other palliative approaches include a bypass or proximal stoma for obstructing lesions.115,116Stage-Specific Therapy Stage 0 (Tis, N0,

1	from the primary tumor requiring surgi-cal intervention. Other palliative approaches include a bypass or proximal stoma for obstructing lesions.115,116Stage-Specific Therapy Stage 0 (Tis, N0, M0) Polyps containing carcinoma in situ (high-grade dysplasia) carry no risk of lymph node metastasis. However, the presence of high-grade dysplasia increases the risk of finding an invasive carcinoma within the polyp. For this reason, these polyps should be excised completely, and patho-logic margins should be free of dysplasia. Most pedunculated polyps and many sessile polyps may be completely removed endoscopically. These patients should be followed with fre-quent colonoscopy to ensure that the polyp has not recurred and that an invasive carcinoma has not developed. In cases where the polyp cannot be removed entirely, a segmental resection is recommended.Stage I: The Malignant Polyp (T1, N0, M0) Occasionally a polyp that was thought to be benign will be found to harbor invasive carcinoma after

1	be removed entirely, a segmental resection is recommended.Stage I: The Malignant Polyp (T1, N0, M0) Occasionally a polyp that was thought to be benign will be found to harbor invasive carcinoma after polypectomy. Treatment of a malig-nant polyp is based on the risk of local recurrence and the risk of lymph node metastasis.59 The risk of lymph node metastases depends primarily on the depth of invasion. Invasive carcinoma in the head of a pedunculated polyp with no stalk involvement carries a low risk of metastasis (<1%) and may be completely resected endoscopically. For sessile polyps, the depth of inva-sion predicts risk of lymphovascular spread. A recent classifica-tion stratifies risk by depth of submucosal spread. Superficial lesions (submucosa 1; Sm1) are low risk, whereas Sm2 and Sm3 are intermediate and high risk.117 Lymphovascular inva-sion, poorly differentiated histology, tumor budding, or tumor within 1 mm of the resection margin greatly increases the risk of local

1	and Sm3 are intermediate and high risk.117 Lymphovascular inva-sion, poorly differentiated histology, tumor budding, or tumor within 1 mm of the resection margin greatly increases the risk of local recurrence and metastatic spread. Segmental colectomy is then indicated. Invasive carcinoma arising in a sessile polyp extending into the submucosa and is usually best treated with segmental colectomy (Fig. 29-25).Stages I and II: Localized Colon Carcinoma (T1-3, N0, M0) The majority of patients with stages I and II colon cancer will be cured with surgical resection. Few patients with completely resected stage I disease will develop either local or distant recur-rence, and adjuvant chemotherapy does not improve survival in these patients.117 However, up to 46% of patients with com-pletely resected stage II disease will ultimately die from colon cancer. For this reason, adjuvant chemotherapy has been sug-gested for selected patients with stage II disease (young patients, tumors with

1	resected stage II disease will ultimately die from colon cancer. For this reason, adjuvant chemotherapy has been sug-gested for selected patients with stage II disease (young patients, tumors with “high-risk” histologic findings). It remains contro-versial as to whether chemotherapy improves survival rates in these patients. In some cases, molecular profiling may predict prognosis, although it is important to note that these tools have not been shown to predict response to therapy. At present, Brunicardi_Ch29_p1259-p1330.indd 129823/02/19 2:29 PM 1299COLON, RECTUM, AND ANUSCHAPTER 29molecular profiling for selecting patients to receive chemother-apy remains unproven.Stage III: Lymph Node Metastasis (Tany, N1, M0) Patients with lymph node involvement are at significant risk for both local and distant recurrence, and adjuvant chemotherapy has been recommended routinely in these patients. 5-Fluorouracil–based regimens (with leucovorin) and oxaliplatin (FOLFOX) reduce recurrences and

1	and distant recurrence, and adjuvant chemotherapy has been recommended routinely in these patients. 5-Fluorouracil–based regimens (with leucovorin) and oxaliplatin (FOLFOX) reduce recurrences and improve survival in this patient popula-tion. It is important to note, however, that a subgroup of patients with stage III disease will do well without chemotherapy. MSI status in particular predicts good prognosis. Subset analysis from the CRYSTAL trial has shown that patients with MSI-high stage III disease do not benefit from 5-fluorouracil–based chemotherapy. Molecular profiling, therefore, may be helpful in determining which stage III patients can safely avoid systemic chemotherapy.118Stage IV: Distant Metastasis (Tany, Nany, M1) Survival is extremely limited in stage IV colon carcinoma. Systemic che-motherapy is recommended in almost all cases of distant spread. However, unlike many other malignancies, highly selected patients with isolated, resectable metastases may benefit from

1	Systemic che-motherapy is recommended in almost all cases of distant spread. However, unlike many other malignancies, highly selected patients with isolated, resectable metastases may benefit from resection (metastasectomy). The most common site of metasta-sis is the liver. Of patients with systemic disease, approximately 15% will have metastases limited to the liver. Of these, 20% are potentially resectable for cure. Survival is improved in these patients (20–40% 5-year survival) when compared to patients who do not undergo resection. Hepatic resection of synchronous metastases from colorectal carcinoma may be performed as a combined procedure or in two stages. The second most com-mon site of metastasis is the lung, occurring in approximately 20% of patients with colorectal carcinoma. Although very few of these patients will be potentially resectable, among those who are (about 1–2% of all colorectal cancer patients), long-term survival benefit is approximately 30% to 40%. There are

1	very few of these patients will be potentially resectable, among those who are (about 1–2% of all colorectal cancer patients), long-term survival benefit is approximately 30% to 40%. There are limited reports of successful resection of metastases in other sites (ovary and retroperitoneum are most common). Cytore-ductive surgery and intraperitoneal chemotherapy (HIPEC) has been suggested for patients with carcinomatosis, but remains unproven for colorectal cancer and carries high morbidity.119,120The remainder of patients with stage IV disease cannot be cured surgically, and therefore, the focus of treatment should be palliation. Methods such as colonic stenting for obstruct-ing lesions of the left colon also provide good palliation. More limited surgical intervention such as a diverting stoma or bypass procedure may be appropriate in patients with stage IV disease who develop obstruction. Hemorrhage in an unresectable tumor can sometimes be controlled with angiographic embolization.

1	stoma or bypass procedure may be appropriate in patients with stage IV disease who develop obstruction. Hemorrhage in an unresectable tumor can sometimes be controlled with angiographic embolization. External beam radiation also has been used for palliation. The involvement of a palliative care team in the management of these patients is critical.116Therapy for Rectal CarcinomaPrinciples of Resection. The biology of rectal adenocarci-noma is thought to be similar to the biology of colonic adeno-carcinoma, and the operative principles of complete resection of the primary tumor, its lymphatic bed, and any other involved organ apply to surgical resection of rectal carcinoma. However, the anatomy of the pelvis and proximity of other structures (ureters, bladder, prostate, vagina, iliac vessels, and sacrum) make resection more challenging and often require a different approach than for colonic adenocarcinoma. Moreover, it is more difficult to achieve negative radial margins in rectal

1	and sacrum) make resection more challenging and often require a different approach than for colonic adenocarcinoma. Moreover, it is more difficult to achieve negative radial margins in rectal cancers that extend through the bowel wall because of the anatomic limita-tions of the pelvis. Therefore, local recurrence is higher than with similar stage colon cancers. However, unlike the intraperi-toneal colon, the relative paucity of small bowel and other radi-ation-sensitive structures in the pelvis makes it easier to treat rectal tumors with radiation. Therapeutic decisions, therefore, are based on the location and depth of the tumor and its relation-ship to other structures in the pelvis.Local Therapy. The distal 10 cm of the rectum are accessible transanally. For this reason, several local approaches have been proposed for treating rectal neoplasms. Transanal excision (full thickness or mucosal) is an excellent approach for noncircum-ferential, benign, villous adenomas of the rectum.

1	approaches have been proposed for treating rectal neoplasms. Transanal excision (full thickness or mucosal) is an excellent approach for noncircum-ferential, benign, villous adenomas of the rectum. Transanal endoscopic microsurgery (TEM) and transanal minimally invasive surgery (TAMIS) make use of a specially designed proctoscope, magnifying system, and instruments similar to those used in laparoscopy to allow local excision of lesions higher in the rectum (up to 15 cm). Although this technique has been used for selected T1, and some T2, carcinomas, local excision does not allow pathologic examination of the lymph nodes and might therefore understage patients. Moreover, local recurrence rates are high after transanal excision, and salvage surgery, while often curative, has been reported to be associ-ated with poorer survival than with initial radical surgery. Cur-rent recommendation is to limit local excision of T1 lesions to patients with well to moderately differentiated small

1	to be associ-ated with poorer survival than with initial radical surgery. Cur-rent recommendation is to limit local excision of T1 lesions to patients with well to moderately differentiated small lesions (<3 cm) and/or in patients medically unfit for radical resection. In general, local excision of any rectal neoplasm should be con-sidered an excisional biopsy because final pathologic examina-tion of the specimen may reveal an invasive carcinoma that then mandates more radical therapy.120,121Ablative techniques, such as electrocautery or endocavi-tary radiation, also have been used. The disadvantage of these techniques is that no pathologic specimen is retrieved to confirm the tumor stage. Fulguration is generally reserved for extremely high-risk, symptomatic patients with a limited life span who cannot tolerate more radical surgery.120Radical Resection. Radical resection is preferred to local therapy for most rectal carcinomas. Radical resection involves removal of the involved

1	life span who cannot tolerate more radical surgery.120Radical Resection. Radical resection is preferred to local therapy for most rectal carcinomas. Radical resection involves removal of the involved segment of the rectum along with its lymphovascular supply. Although any microscopically nega-tive margin has been suggested to be adequate, most surgeons Pedunculated polypInvasive caSessile polypStalkNeckMuscularismucosaFigure 29-25. Levels of invasive carcinoma in pedunculated and sessile polyps. ca = carcinoma.Brunicardi_Ch29_p1259-p1330.indd 129923/02/19 2:29 PM 1300SPECIFIC CONSIDERATIONSPART IIstill attempt to obtain a 2-cm distal mural margin for curative resections.Total mesorectal excision (TME) is a technique that uses sharp dissection along anatomic planes to ensure complete resection of the rectal mesentery during low and extended low anterior resections. For upper rectal or rectosigmoid resections, a partial mesorectal excision of at least 5 cm distal to the tumor

1	resection of the rectal mesentery during low and extended low anterior resections. For upper rectal or rectosigmoid resections, a partial mesorectal excision of at least 5 cm distal to the tumor appears adequate. TME both decreases local recurrence rates and improves long-term survival rates. Moreover, this tech-nique is associated with less blood loss and less risk to the pel-vic nerves and presacral plexus than is blunt dissection. The principles of TME should be applied to all radical resections for rectal cancer.Recurrence of rectal cancer generally has a poor prog-nosis. Extensive involvement of other pelvic organs (usually occurring in the setting of tumor recurrence) may require a pelvic exenteration. The rectal and perineal portions of this operation are similar to an APR, but en bloc resection of the ureters, bladder, and prostate or uterus and vagina are also per-formed. A permanent colostomy and an ileal conduit to drain the urinary tract may be necessary. The sacrum may

1	bloc resection of the ureters, bladder, and prostate or uterus and vagina are also per-formed. A permanent colostomy and an ileal conduit to drain the urinary tract may be necessary. The sacrum may also be resected if necessary (sacrectomy) up to the level of the S2-S3 junction. These operations are best performed in tertiary centers with multidisciplinary teams consisting of a colon and rectal surgeon, urologist, neurosurgeon, and plastic surgeon.Stage-Specific Therapy (Fig. 29-26). Pretreatment staging of rectal carcinoma often relies on endorectal ultrasound or MRI to determine the T and N status of a rectal cancer. Ultrasound is highly accurate at assessing tumor depth, but it is less accurate in diagnosing nodal involvement. Ultrasound evaluation can guide choice of therapy in most patients. MRI is useful to assess mesorectal involvement. When the radial margin is threatened or involved, neoadjuvant chemoradiation is recommended.122Stage 0 (Tis, N0, M0) Villous adenomas harboring

1	MRI is useful to assess mesorectal involvement. When the radial margin is threatened or involved, neoadjuvant chemoradiation is recommended.122Stage 0 (Tis, N0, M0) Villous adenomas harboring carci-noma in situ (high-grade dysplasia) are ideally treated with local excision. A 1-cm margin should be obtained. Rarely, radical resection will be necessary if transanal excision is not techni-cally possible (large circumferential lesions).Stage I: Localized Rectal Carcinoma (T1-2, N0, M0) Although local excision has been used for small, favorable ses-sile uT1N0 and uT2N0 rectal cancers, local recurrence rates may be as high as 20% and 40%, respectively. Local excision increasingly is offered to patients with small, low-risk lesions, but it does not allow physicians to assess regional lymph nodes. For this reason, radical resection is recommended in all good-risk patients. Lesions with unfavorable histologic characteristics and those located in the distal third of the rectum, in particular,

1	For this reason, radical resection is recommended in all good-risk patients. Lesions with unfavorable histologic characteristics and those located in the distal third of the rectum, in particular, are prone to recurrence. In high-risk patients and in patients who refuse radical surgery because of the risk of need for a permanent colostomy, local excision may be adequate, but strong consideration should be given to adjuvant or neoadju-vant chemoradiation to improve local control. The efficacy of adjuvant or neoadjuvant chemoradiation followed by transanal excision in patients who can tolerate radical surgery has been High risk orrefuses radicalresectionTransanalexcision +/–neoadjuvant oradjuvantchemoradiationRadicalresectionRadicalresectionRadicalresectionContinuechemotherapyConsiderresectionChemoradiationvs. palliativeprocedurestent, laserablation, stoma,resectionLow risk andaccepts

1	palliativeprocedurestent, laserablation, stoma,resectionLow risk andaccepts radicalresectionNeoadjuvantchemoradiationNeoadjuvantchemoradiationAsymptomaticSymptomaticRestageRestageUnresectable/multiplemetastaticsitesResectable/singlemetastatic siteChemotherapyNo metastasesNo metastasesRectal adenocarcinomaStage II(T3-4, N0, M0)Stage III(Tany, N1-3, M0)Staging evaluationCT chest/abdomen/pelvisEndorectal U/S+/– MRIColonoscopy/CT colonography (if possible)Stage IV(Tany, Nany, M1)Stage I(T1-2, N0, M0)RestageFigure 29-26. Diagnostic algorithm for rectal cancer. CT = computed tomography; MRI = magnetic resonance imaging; U/S = ultrasound.Brunicardi_Ch29_p1259-p1330.indd 130023/02/19 2:29 PM 1301COLON, RECTUM, AND ANUSCHAPTER 29controversial. Early results from ACOSOG Z6041, in which patients with T2 rectal cancers received neoadjuvant chemo-radiation followed by transanal excision, showed a pathologic complete response rate of 44%.123 At 3 years, disease-free sur-vival was 88%, which is

1	with T2 rectal cancers received neoadjuvant chemo-radiation followed by transanal excision, showed a pathologic complete response rate of 44%.123 At 3 years, disease-free sur-vival was 88%, which is comparable to cancer outcomes after a formal resection. However, population-based data suggests that survival after local excision for rectal cancer is suboptimal and should not be offered as a matter of routine.124Locally Advanced Rectal Cancer (Stages II and III) Stage II: Localized Rectal Carcinoma (T3-4, N0, M0). Larger rectal tumors, especially if located in the distal rectum, are more likely to recur locally. There are two schools of thought, each differing in their approach, concerning how to control local recurrences. Advocates of total mesorectal resection sug-gest that optimization of operative technique will obviate the need for any adjuvant chemoradiation to control local recur-rence after resection of stages I, II, and III rectal cancers. The opposing school suggests that

1	of operative technique will obviate the need for any adjuvant chemoradiation to control local recur-rence after resection of stages I, II, and III rectal cancers. The opposing school suggests that stages II and III rectal cancers will benefit from chemoradiation. They argue that such therapy reduces local recurrences and prolongs survival whether given preoperatively or postoperatively. The advantages of preopera-tive chemoradiation include tumor shrinkage, increased likeli-hood of resection and of a sphincter-sparing procedure, tumor downstaging by treating locally involved lymph nodes, and decreased risk to the small intestine. Disadvantages include possible overtreatment of early-stage tumors, impaired wound healing, and pelvic fibrosis increasing the risk of operative com-plications. Postoperative radiation allows accurate pathologic staging of the resected tumor and lymph nodes and avoids the wound healing problems associated with preoperative radiation. However, bulky tumors,

1	Postoperative radiation allows accurate pathologic staging of the resected tumor and lymph nodes and avoids the wound healing problems associated with preoperative radiation. However, bulky tumors, tumors involving adjacent organs, and very low rectal tumors may be much more difficult to resect without preoperative radiation and may require a more exten-sive operation.124-127Stage III: Lymph Node Metastasis (Tany, N1, M0). Many surgeons now recommend chemotherapy and radiation either preor postoperatively for node-positive rectal cancers. The advantages and disadvantages are similar to those listed for stage II disease, except that the likelihood of overtreating an early-stage lesion is considerably less.Over the past two decades, a wide variety of studies have addressed the issue of adjuvant and neoadjuvant therapy for locally advanced rectal cancer. Many of these studies demon-strated both improved local control and prolonged survival and resulted in the 1990 National Institutes of

1	and neoadjuvant therapy for locally advanced rectal cancer. Many of these studies demon-strated both improved local control and prolonged survival and resulted in the 1990 National Institutes of Health (NIH) con-sensus conference recommendation for postoperative chemo-radiation therapy in these patients. There is little controversy regarding chemoradiation therapy for stage III (node-positive) disease. However, advances in surgical technique, such as TME, for locally advanced node-negative cancers (T3-4, N0; stage II) have improved local control with surgery alone, prompting some to abandon adjuvant chemoradiation in these patients, especially for those with cancers in the proximal rectum. Although the data from these studies are intriguing, other reports have shown that chemoradiation improves local control and survival even in patients who undergo TME. Thus, most colorectal surgeons in the United States continue to recommend adjuvant or neo-adjuvant therapy for patients with locally

1	local control and survival even in patients who undergo TME. Thus, most colorectal surgeons in the United States continue to recommend adjuvant or neo-adjuvant therapy for patients with locally advanced disease. Many European surgeons now rely heavily on MRI staging to determine the need for neoadjuvant chemoradiation. They use neoadjuvant chemoradiation if the radial margin is threatened or involved by the cancer or if anal sphincter or other local organ invasion is present. In the United States, chemoradia-tion therapy is still recommended for all patients with stage III disease and the majority of patients with stage II disease. In select patients with T3 tumors, favorable histology, and nega-tive radial margins, chemoradiation may not be necessary, but larger prospective studies are required before this approach can be recommended.125-127Appropriate timing of chemoradiation for locally advanced rectal cancer has been debated. Historically, preopera-tive chemoradiation has been

1	required before this approach can be recommended.125-127Appropriate timing of chemoradiation for locally advanced rectal cancer has been debated. Historically, preopera-tive chemoradiation has been advocated based on tumor shrink-age/downstaging, improved resectability, and the possibility of performing a sphincter-sparing operation in some patients. In addition, the absence of small bowel adhesions in the pelvis may decrease toxicity. However, preoperative radiation therapy may increase operative complications and impairs wound heal-ing. Although preoperative endorectal ultrasound and MRI have improved our ability to stage rectal cancer, clinical “overstag-ing” can be problematic, and neoadjuvant therapy may there-fore overtreat patients with pT1-2, N0 tumors. Advocates of postoperative radiation therapy cite more accurate pathologic staging and fewer operative/postoperative complications. How-ever, large, bulky tumors may be unresectable or require a more extensive operation (APR,

1	radiation therapy cite more accurate pathologic staging and fewer operative/postoperative complications. How-ever, large, bulky tumors may be unresectable or require a more extensive operation (APR, pelvic exenteration) without preop-erative therapy. In addition, postoperative pelvic radiation may compromise function of the neorectum.Comparisons of perioperative toxicity and oncologic out-come have been addressed by the German CAO/ARO/AIO-94 trial. In this study, preand postoperative chemoradiation were associated with equivalent acute toxicity and equivalent post-operative complication rates. Postoperative chemoradiation, however, doubled the risk of postoperative stricture forma-tion. In addition, preoperative chemoradiation halved the risk of local recurrence (6% vs. 12%). Based on these data, most surgeons consider preoperative chemoradiation to be the most appropriate therapy for locally advanced rectal cancer.126 In the United States, this generally consists of 5-FU based

1	these data, most surgeons consider preoperative chemoradiation to be the most appropriate therapy for locally advanced rectal cancer.126 In the United States, this generally consists of 5-FU based chemother-apy and 5 to 6 weeks of external beam radiation (“long course”) followed by surgery 6 to 8 weeks later. It is important to note, however, that many European centers utilize a “short course” preoperative radiation regimen consisting of 5 days of radia-tion followed by surgery within 1 to 2 weeks. At present, these modalities have not been compared in any randomized, prospec-tive trial.125,127With advances in chemoradiation, an increasing number pf patients with locally advanced rectal cancer will have com-plete shrinkage of their tumor (a clinical complete response; cCR). In light of the potential morbidity of proctectomy, it has been suggested that select patients can be managed nonopera-tively (“watch and wait”). However, data from current stud-ies are contradictory and concern

1	the potential morbidity of proctectomy, it has been suggested that select patients can be managed nonopera-tively (“watch and wait”). However, data from current stud-ies are contradictory and concern remains about the ability to predict which patients with clinical complete response actually have a pathologic complete response.128 Patients selected for nonoperative management must be examined by a surgeon at a frequent intervals. Additional adjuvant chemotherapy adminis-tered after the decision for a nonoperative approach is another important consideration.128,129 At present, this approach is not recommended outside of a specialty center and/or clinical trial.Stage IV: Distant Metastasis (Tany, Nany, M1) Like stage IV colon carcinoma, survival is limited in patients with distant metastasis from rectal carcinoma. Isolated hepatic Brunicardi_Ch29_p1259-p1330.indd 130123/02/19 2:29 PM 1302SPECIFIC CONSIDERATIONSPART IIand/or pulmonary metastases are rare, but when present may be

1	from rectal carcinoma. Isolated hepatic Brunicardi_Ch29_p1259-p1330.indd 130123/02/19 2:29 PM 1302SPECIFIC CONSIDERATIONSPART IIand/or pulmonary metastases are rare, but when present may be resected for cure in selected patients.119,120 Some patients will require palliative procedures. Radical resection may be required to control pain, bleeding, or tenesmus, but highly morbid pro-cedures such as pelvic exenteration and sacrectomy should generally be avoided in this setting. Local therapy using cau-tery, endocavitary radiation, or laser ablation may be adequate to control bleeding or prevent obstruction. Intraluminal stents may be useful in the uppermost rectum but often cause pain and tenesmus lower in the rectum. Occasionally, a proximal divert-ing colostomy will be required to alleviate obstruction. A mucus fistula should be created if possible to vent the distal colon. It is critical that the morbidity of any procedure be realistically weighed against potential benefit in

1	alleviate obstruction. A mucus fistula should be created if possible to vent the distal colon. It is critical that the morbidity of any procedure be realistically weighed against potential benefit in these patients with limited life expectancy. The assistance of a palliative care team can be invaluable in this setting.116Follow-Up and SurveillancePatients who have been treated for one colorectal cancer are at risk for the development of recurrent disease (either locally or systemically) or metachronous disease (a second primary tumor). In theory, metachronous cancers should be preventable by using surveillance colonoscopy to detect and remove polyps before they progress to invasive cancer. For most patients, a colonoscopy should be performed within 12 months after the diagnosis of the original cancer (or sooner if the colon was not examined in its entirety prior to the original resection). If that study is normal, colonoscopy should be repeated every 3 to 5 years thereafter.The

1	original cancer (or sooner if the colon was not examined in its entirety prior to the original resection). If that study is normal, colonoscopy should be repeated every 3 to 5 years thereafter.The optimal method of following patients for recurrent cancer remains controversial. The goal of close follow-up observation is to detect resectable recurrence and to improve survival. Re-resection of local recurrence and resection of dis-tant metastasis to liver, lung, or other sites are often technically challenging and highly morbid, with only a limited chance of achieving long-term survival. Thus, only selected patients who would tolerate such an approach should be followed intensively. Because most recurrences occur within 2 years of the original diagnosis, surveillance focuses on this time period. Patients who have undergone local resection of rectal tumors also should be followed with frequent endoscopic examinations (every 3–6 months for 3 years, then every 6 months for 2 years). CEA

1	Patients who have undergone local resection of rectal tumors also should be followed with frequent endoscopic examinations (every 3–6 months for 3 years, then every 6 months for 2 years). CEA is often followed every 3 to 6 months for 2 years. CT scans are often performed annually for 5 years, but there are few data to support this practice. More intensive surveillance is appro-priate in high-risk patients such as those with possible Lynch syndrome or T3, N+ cancers. Although intensive surveillance improves detection of resectable recurrences, it is important to note that a survival benefit has never been proven. Therefore, the risks and benefits of intensive surveillance must be weighed and treatment individualized.Treatment of Recurrent Colorectal CarcinomaBetween 20% and 40% of patients who have undergone cura-tive intent surgery for colorectal carcinoma will eventually develop recurrent disease. Most recurrences occur within the first 2 years after the initial diagnosis, but

1	who have undergone cura-tive intent surgery for colorectal carcinoma will eventually develop recurrent disease. Most recurrences occur within the first 2 years after the initial diagnosis, but preoperative chemo-radiation therapy may delay recurrence. While most of these patients will present with distant metastases, a small propor-tion will have isolated local recurrence and may be considered for salvage surgery. Recurrence after colon cancer resection usually occurs at the local site within the abdomen or in the liver or lungs. Resection of other involved organs may be nec-essary. Recurrence of rectal cancer can be considerably more difficult to manage because of the proximity of other pelvic structures. If the patient has not received chemotherapy and radiation, then adjuvant therapy should be administered prior to salvage surgery. Radical resection may require extensive resection of pelvic organs (pelvic exenteration with or without sacrectomy). Ideally, the aim of a salvage

1	should be administered prior to salvage surgery. Radical resection may require extensive resection of pelvic organs (pelvic exenteration with or without sacrectomy). Ideally, the aim of a salvage operation should be to resect all of the tumor with negative margins. However, if the ability to achieve a negative margin is in question, the addi-tion of intraoperative radiation therapy (usually brachytherapy) can help improve local control. Pelvic MRI is useful for identi-fying tumor extension that would prevent successful resection (extension of tumor into the pelvic sidewall, involvement of the iliac vessels or bilateral sacral nerves, sacral invasion above the S2-S3 junction). Patients should also undergo a thorough pre-operative evaluation to identify distant metastases (CT of chest, abdomen, and pelvis, and PET scan) before undergoing such an extensive procedure. Nevertheless, radical salvage surgery can prolong survival in selected patients.Minimally Invasive Techniques for

1	abdomen, and pelvis, and PET scan) before undergoing such an extensive procedure. Nevertheless, radical salvage surgery can prolong survival in selected patients.Minimally Invasive Techniques for ResectionLaparoscopic colectomy for cancer has been controversial. Early reports of high port site recurrence dampened enthusiasm for this technique.130 The ability to perform an adequate onco-logic resection for cancer has also been questioned. Several tri-als have laid to rest many of these fears. The Clinical Outcomes of Surgical Therapy (COST) Study Group, the Colon Car-cinoma Laparoscopic or Open Resection (COLOR) trial, and the United Kingdom Medical Research Council Conventional versus Laparoscopic-Assisted Surgery in Colorectal Cancer (CLASSICC) trial all have shown oncologic equivalence between open and laparoscopic techniques. In these multi-institutional studies, the rates of cancer recurrence, survival, and quality of life were similar, suggesting that, in the hands of an

1	between open and laparoscopic techniques. In these multi-institutional studies, the rates of cancer recurrence, survival, and quality of life were similar, suggesting that, in the hands of an appropriately trained surgeon, laparoscopic colectomy is appropriate for colon cancer.131-136 The recent introduction of robotic surgery offers an additional minimally invasive approach. Early studies suggest that robotic surgery may be the oncologic equivalent to laparo-scopic surgery for colon cancer.137Laparoscopic surgery for rectal cancer has been more controversial. Multiple studies of laparoscopic total mesorectal excision for rectal cancer have demonstrated decreased blood loss, earlier return of bowel function and shorter length of stay compared with open TME. While a laparoscopic approach to mobilization of the left colon and splenic flexure can be used in rectal resection procedures, laparoscopic TME refers to comple-tion of the pelvic dissection laparoscopically and not through the

1	to mobilization of the left colon and splenic flexure can be used in rectal resection procedures, laparoscopic TME refers to comple-tion of the pelvic dissection laparoscopically and not through the abdominal extraction site. Technical challenges in the retraction of the rectum and surrounding soft tissues and transection of the distal rectum mandate careful evaluation oncologic outcomes. Two recent randomized controlled trials from the United States (ACOSOG Z6051)138 and Australia and New Zealand (ALaC-aRT: Australasian Laparoscopic Cancer of the Rectum Trial)139 have shown that laparoscopic surgery is not superior to open surgery in this regard. When the totality of evidence from nine randomized trials is evaluated, CRM was positive in 7.9% of laparoscopic and 6.1% of open rectal resections, a difference that was not statistically significant.17 In trials that reported the completeness of mesorectal excision (n = 5), inadequate TME was significantly more likely in laparoscopic

1	a difference that was not statistically significant.17 In trials that reported the completeness of mesorectal excision (n = 5), inadequate TME was significantly more likely in laparoscopic (13.2%) com-pared with open (10.4%) resections. Long-term recurrence and 6Brunicardi_Ch29_p1259-p1330.indd 130223/02/19 2:29 PM 1303COLON, RECTUM, AND ANUSCHAPTER 29survival data from ACOSOG and ALaCaRT studies will be needed to determine what role laparoscopy should play in rectal cancer surgery. Two earlier trials, COLOR (COlon cancer Lapa-roscopic or Open Resection) II134 and COREAN (Comparison of Open versus laparoscopic surgery for mid and low Rectal cancer After Neoadjuvant chemoradiotherapy),135 have shown equivalent 3-year recurrence and survival. It is possible that spe-cific subsets of patients are more appropriate for open surgery. For example, ALaCaRT authors thought laparoscopic surgery may be less successful in patients who had neoadjuvant therapy, larger T3 tumors, or higher BMIs,

1	are more appropriate for open surgery. For example, ALaCaRT authors thought laparoscopic surgery may be less successful in patients who had neoadjuvant therapy, larger T3 tumors, or higher BMIs, but their study was under-powered to make definitive conclusions in these patients. A laparoscopic approach should not be considered for T4 tumors.The inferior quality of laparoscopic rectal cancer surgery suggested by recent randomized trials is attributed to techni-cal limitations, leading to the suggestion that robotic surgery, with its wristed instruments, fixed platform, and potentially improved visualization could offer an improvement. The results of the ROLARR (Robotic vs Laparoscopic Resection for Rectal Cancer) randomized trial will offer more data on the quality of TME and margins after robotic surgery; however, a trial com-paring open to robotic dissection will also be important.141OTHER NEOPLASMSRare Colorectal TumorsNeuroendocrine Tumors. Neuroendocrine tumors occur most commonly

1	robotic surgery; however, a trial com-paring open to robotic dissection will also be important.141OTHER NEOPLASMSRare Colorectal TumorsNeuroendocrine Tumors. Neuroendocrine tumors occur most commonly in the gastrointestinal tract, and up to 25% of these tumors are found in the rectum. Well-differentiated neuroendo-crine tumors (also known as carcinoid tumors) are commonly identified on colonoscopy as small (<1 cm) nodules and have a benign clinical course, with overall survival is greater than 80%. However, the risk of malignancy increases with size, and more than 60% of tumors greater than 2 cm in diameter are associated with distant metastases. Rectal neuroendocrine tumors appear to be less likely to secrete vasoactive substances than carcinoids in other locations, and carcinoid syndrome is uncommon in the absence of hepatic metastases. Small carcinoids can be locally resected transanally. Larger tumors, poorly differentiated tumors (such as small cell or large cell neuroendocrine

1	is uncommon in the absence of hepatic metastases. Small carcinoids can be locally resected transanally. Larger tumors, poorly differentiated tumors (such as small cell or large cell neuroendocrine carcinomas), and those with obvious invasion into the muscularis require more radical surgery. Neuroendocrine tumors in the proximal colon are less common and are more likely to be malignant. Size also correlates with risk of malignancy, and tumors less than 2 cm in diameter rarely metastasize. However, the majority of neuroendocrine tumors in the proximal colon present as bulky lesions, and up to two-thirds of patients will have metastatic spread at the time of diagnosis. These tumors should usually be treated with radical resection. Because well-differentiated neuroendocrine tumors are typically slow growing, patients with distant metastases may expect reasonably long survival. Symptoms of carcinoid syndrome can often be alleviated with somatostatin analogues (octreotide) and/or

1	typically slow growing, patients with distant metastases may expect reasonably long survival. Symptoms of carcinoid syndrome can often be alleviated with somatostatin analogues (octreotide) and/or interferon-α. Tumor debulking can offer effective palliation in selected patients.142-144Mixed Adenoneuroendocrine Carcinomas. Mixed adeno-neuroendocrine carcinomas, also known as composite carcinoid carcinomas, adenocarcinoids tumors, amphicrine or collision tumors, have histologic features of both neuroendocrine tumors and adenocarcinomas. The natural history of these tumors more closely parallels that of adenocarcinomas than neuroendocrine tumors, and regional and systemic metastases are common. Carcinoid carcinoma of the colon and rectum should be treated according to the same oncologic principles as followed for man-agement of adenocarcinoma.Lipomas. Lipomas are benign lesions that occur most com-monly in the submucosa of the colon and rectum. The major-ity of lipomas are asymptomatic

1	as followed for man-agement of adenocarcinoma.Lipomas. Lipomas are benign lesions that occur most com-monly in the submucosa of the colon and rectum. The major-ity of lipomas are asymptomatic and discovered incidentally. Small asymptomatic lesions do not require resection. However, larger lesions may occasionally cause bleeding, obstruction, or intussusception, especially when greater than 2 cm in diameter. Larger lipomas should be resected by colonoscopic techniques or by a colotomy and enucleation or limited colectomy.144Lymphoma. Gastrointestinal lymphoma may be primary or generalized/secondary. Primary GI lymphomas occur most fre-quently in the terminal ileum and cecum. Lymphoma involv-ing the colon and rectum is rare, but it accounts for about 10% of all gastrointestinal lymphomas. Presentation, treatment and prognosis differ between patients with lymphoma occurring as a localized entity in the colon and rectum versus those inoccur-ing in patients who have generalized lymphoma

1	Presentation, treatment and prognosis differ between patients with lymphoma occurring as a localized entity in the colon and rectum versus those inoccur-ing in patients who have generalized lymphoma with colorectal involvement. Symptoms in isolated rectal lymphoma include bleeding, obstruction, and pain, and these tumors may be clini-cally indistinguishable from adenocarcinomas. The cecum is most often involved, probably as a result of spread from the terminal ileum. Symptoms include bleeding and obstruction. Bowel resec-tion is the treatment of choice for isolated colorectal lymphoma. Adjuvant therapy may be given based on the stage of disease.144Leiomyoma and Leiomyosarcoma. Leiomyomas are benign tumors of the smooth muscle of the bowel wall and occur most commonly in the upper gastrointestinal tract. Most patients are asymptomatic, and lesions are often diagnosed inciden-tally when a mass is seen on endoscopy or felt on digital rec-tal examination. However, large lesions can cause

1	tract. Most patients are asymptomatic, and lesions are often diagnosed inciden-tally when a mass is seen on endoscopy or felt on digital rec-tal examination. However, large lesions can cause bleeding or obstruction. Because it is difficult to differentiate a benign leio-myoma from a malignant leiomyosarcoma, these lesions should be resected. Recurrence is common after local resection, but most small leiomyomas can be adequately treated with limited resection. Lesions larger than 5 cm should be treated with radi-cal resection because the risk of malignancy is high.Leiomyosarcoma is rare in the gastrointestinal tract. When this malignancy occurs in the large intestine, the rectum is the most common site. Leiomyosarcoma of the rectum is usually low grade, and, as such, can be difficult to differentiate from leiomyoma. Definitive diagnosis is usually made after resection. Symptoms, when they occur, are usually bleeding or obstruc-tion. A radical resection is indicated for most of these

1	differentiate from leiomyoma. Definitive diagnosis is usually made after resection. Symptoms, when they occur, are usually bleeding or obstruc-tion. A radical resection is indicated for most of these tumors. Despite complete resection, recurrence is not uncommon, and prognosis is generally poor.144Gastrointestinal Stromal Tumor (GIST). Gastrointestinal Stromal Tumors (GIST) are most common in the proximal GI tract but do occasionally occur in the colorectum (5–10%) and may be mistaken for leiomyomas. GISTs are mesenchy-mal tumors that arise from the interstitial cells of Cajal. The vast majority (>95%) of GISTs express CD117 (KIT), and as such, are sensitive to tyrosine kinase inhibitors (TKIs), such as imatinib mesylate and sunitinib malate. Risk stratification is based on tumor size and mitotic activity, and 30% to 50% are malignant. Although small GISTs may be asymptomatic and discovered incidentally, larger lesions can cause bleeding, obstruction, or abdominal pain. Treatment of

1	mitotic activity, and 30% to 50% are malignant. Although small GISTs may be asymptomatic and discovered incidentally, larger lesions can cause bleeding, obstruction, or abdominal pain. Treatment of choice is surgi-cal resection (either local excision or radical resection) with Brunicardi_Ch29_p1259-p1330.indd 130323/02/19 2:29 PM 1304SPECIFIC CONSIDERATIONSPART IImicroscopically negative margins, if possible; however, local recurrence is common. For larger marginally resectable tumors, TKIs (imatinib) can be used to shrink the tumor. These agents can also be considered for adjuvant therapy after resection and are useful for treating metastatic disease.144Retrorectal/Presacral TumorsTumors occurring in the retrorectal space are rare. This region lies between the upper two-thirds of the rectum and the sacrum above the rectosacral fascia. It is bound by the rectum anteri-orly, the presacral fascia posteriorly, and the endopelvic fascia laterally (lateral ligaments). The retrorectal

1	rectum and the sacrum above the rectosacral fascia. It is bound by the rectum anteri-orly, the presacral fascia posteriorly, and the endopelvic fascia laterally (lateral ligaments). The retrorectal space contains mul-tiple embryologic remnants derived from a variety of tissues (neuroectoderm, notochord, and hindgut). As such, tumors that develop in this space are often heterogeneous.Congenital lesions are most common, comprising almost two-thirds of retrorectal lesions. The remainder are classified as neurogenic, osseous, inflammatory, or miscellaneous lesions. Malignancy is more common in the pediatric population than in adults, and solid lesions are more likely to be malignant than are cystic lesions. Inflammatory lesions may be solid or cystic (abscess) and usually represent extensions of infection either in the perirectal space or in the abdomen.Developmental cysts constitute the majority of congenital lesions and may arise from all three germ cell layers. Dermoid and epidermoid

1	of infection either in the perirectal space or in the abdomen.Developmental cysts constitute the majority of congenital lesions and may arise from all three germ cell layers. Dermoid and epidermoid cysts are benign lesions that arise from the ecto-derm. Enterogenous cysts arise from the primitive gut. Anterior meningocele and myelomeningocele arise from herniation of the dural sac through a defect in the anterior sacrum. A “scimitar sign” (sacrum with a rounded, concave border without any bony destruction) is the pathognomonic radiographic appearance of this condition.Solid lesions include teratomas, chordomas, neurologic tumors, or osseous lesions. Teratomas are true neoplasms and contain tissue from each germ cell layer. They often contain both cystic and solid components. Teratomas are more com-mon in children than in adults, but when found in adults, 30% are malignant. Chordomas arise from the notochord and are the most common malignant tumor in this region. These are

1	are more com-mon in children than in adults, but when found in adults, 30% are malignant. Chordomas arise from the notochord and are the most common malignant tumor in this region. These are slow-growing, invasive cancers that show characteristic bony destruc-tion. Neurogenic tumors include neurofibromas and sarcomas, neurilemomas, ependymomas, and ganglioneuromas. Osseous lesions include osteomas and bone cysts, as well as neoplasms such as osteogenic sarcoma, Ewing’s tumor, chondromyxosar-coma, and giant cell tumors.Patients may present with pain (lower back, pelvic, or lower extremity), gastrointestinal symptoms, or urinary tract symptoms. Most lesions are palpable on digital rectal examina-tion. While plain X-rays and CT scans often are used to evalu-ate these lesions, pelvic MRI is the most sensitive and specific imaging study. Myelogram is occasionally necessary if there is central nervous system involvement. Treatment is almost always surgical resection. The approach depends in

1	most sensitive and specific imaging study. Myelogram is occasionally necessary if there is central nervous system involvement. Treatment is almost always surgical resection. The approach depends in part on the nature of the lesion and its location. Lesions high in the pelvis may be approached via a transabdominal route, whereas low lesions may be resected transsacrally. Intermediate lesions may require a combined abdominal and sacral operation. Although survival is excellent after resection of benign lesions, local recurrence is not uncommon. Prognosis after resection of malignant lesions is highly variable and reflects the biology of the underlying tumor.The role of biopsy in this setting has been controversial. Historically, the recommendation was to avoid biopsy because of the risk of infection or needle tract seeding. This recommen-dation has recently been challenged, especially for large and/or unusual tumors that would be better treated with multimodality neoadjuvant therapy

1	of infection or needle tract seeding. This recommen-dation has recently been challenged, especially for large and/or unusual tumors that would be better treated with multimodality neoadjuvant therapy (GIST, sarcoma, metastatic adenocarci-noma). A recent study confirmed the utility of needle biopsy of solid lesions and refuted concerns about needle tract seeding. As such, most solid lesions should be biopsied regardless of resectability. Biopsy or aspiration of cystic lesions, especially meningoceles, should still be avoided because of the risk of infection.144-147Anal Canal and Perianal TumorsCancers of the anal canal are uncommon and account for approx-imately 2% of all colorectal malignancies. Neoplasms of the anal canal have traditionally been divided into those affecting the anal margin (distal to the dentate line) and those affecting the anal canal (proximal to the dentate line) based on lymphatic drainage patterns. Lymphatics from the anal canal proximal to the dentate line

1	margin (distal to the dentate line) and those affecting the anal canal (proximal to the dentate line) based on lymphatic drainage patterns. Lymphatics from the anal canal proximal to the dentate line drain cephalad via the superior rectal lymphat-ics to the inferior mesenteric nodes and laterally along both the middle rectal vessels and inferior rectal vessels through the ischiorectal fossa to the internal iliac nodes. Lymph from the anal canal distal to the dentate line usually drains to the inguinal nodes. It can also drain to the superior rectal lymph nodes or along the inferior rectal lymphatics to the ischiorectal fossa if primary drainage routes are blocked with tumor (Fig. 29-27).A more clinically useful classification divides anal lesions into those that are perianal (can be completely visualized with gentle eversion of the buttocks) and those that are intra-anal (cannot be completely visualized with gentle eversion of the buttocks). In many cases, therapy depends on whether

1	visualized with gentle eversion of the buttocks) and those that are intra-anal (cannot be completely visualized with gentle eversion of the buttocks). In many cases, therapy depends on whether the tumor is perianal or intra-anal.Squamous Intraepithelial Lesions. Anal canal and peri-anal dysplasia have long had a potpourri of nomenclature. Anal intraepithelial neoplasia (AIN), Bowen’s disease, and carci-noma in situ all refer to human papillomavirus (HPV)–induced Inferiormesenteric a.Superiorrectal a.Middlerectal a.Inferiorrectal a.Common iliac a.Figure 29-27. Lymphatic drainage of the anal canal. a. = artery.Brunicardi_Ch29_p1259-p1330.indd 130423/02/19 2:29 PM 1305COLON, RECTUM, AND ANUSCHAPTER 29dysplasia. Because these entities are pathologically identical, there has been an effort to standardize nomenclature. High-grade squamous intraepithelial lesions (HSIL) include highand intermediate-grade dysplasia, AINII and AINIII, Bowen’s disease, and carcinoma in situ. Low-grade

1	to standardize nomenclature. High-grade squamous intraepithelial lesions (HSIL) include highand intermediate-grade dysplasia, AINII and AINIII, Bowen’s disease, and carcinoma in situ. Low-grade squamous intraepi-thelial lesions (LSIL) includes low-grade dysplasia and AINI. Recently, the terms High-grade AIN (HGAIN; AINIII) and low-grade AIN (LGAIN; AIN I/II) have been suggested.146-148 Both highand low-grade lesions are associated with infection with HPV, especially types 16 and 18.High-grade lesions are precursors to invasive squamous cell carcinoma (epidermoid carcinoma) and may appear as a plaque or may only be apparent with high-resolution anoscopy and application of acetic acid and/or Lugol’s iodine solution. The incidence of both squamous intraepithelial lesions and epidermoid carcinoma of the anus has increased dramatically among human immunodeficiency virus (HIV)–positive men who have sex with men. This increase is thought to result from increased rates of HPV infection along

1	of the anus has increased dramatically among human immunodeficiency virus (HIV)–positive men who have sex with men. This increase is thought to result from increased rates of HPV infection along with HIV-induced immu-nosuppression. Treatment of high-grade dysplasia is ablation. Because of a high recurrence and/or reinfection rate, these patients require close surveillance. High-risk patients should be followed with frequent anal Papanicolaou (Pap) smears every 3 to 6 months. An abnormal Pap smear should be followed by an examination under anesthesia and anal mapping using high-resolution anoscopy. High-resolution anoscopy shows areas with abnormal telangiectasias that are consistent with high-grade dysplasia. Many centers now consider this technique for repeated ablation of high-grade lesions to be the optimal method for following these patients.148-150 It should be noted, however, that the practice has not been universally adopted, and it is unclear whether close surveillance in

1	lesions to be the optimal method for following these patients.148-150 It should be noted, however, that the practice has not been universally adopted, and it is unclear whether close surveillance in lower-risk (nonimmu-nosuppressed) patients is necessary. Rarely, extensive disease may require resection with flap closure. Medical therapy for HPV has also been proposed. Topical immunomodulators such as imiquimod (Aldara) have been shown to induce regression in some series.151 Topical 5-fluorouracil has also been used in this setting. Finally, the introduction of a vaccine against HPV may help decrease the incidence of this disease in the future.Epidermoid Carcinoma. Epidermoid carcinoma of the anus includes squamous cell carcinoma, cloacogenic carcinoma, tran-sitional carcinoma, and basaloid carcinoma. The clinical behav-ior and natural history of these tumors are similar. Epidermoid carcinoma is a slow-growing tumor and usually presents as an intra-anal or perianal mass. Pain and

1	carcinoma. The clinical behav-ior and natural history of these tumors are similar. Epidermoid carcinoma is a slow-growing tumor and usually presents as an intra-anal or perianal mass. Pain and bleeding may be present. Perianal epidermoid carcinoma may be treated in a similar fash-ion as squamous cell carcinoma of the skin in other locations because wide local excision can usually be achieved without resecting the anal sphincter. Intra-anal epidermoid carcinoma cannot be excised locally, and first-line therapy relies on chemo-therapy and radiation (the Nigro protocol: 5-fluorouracil, mitomycin C, and 30 Gy of external beam radiation). This regimen cures 70% to 80% of these tumors. It is important to note that epidermoid carcinomas continue to respond after com-pletion of chemoradiation. Lesions that persist greater than 3 to 6 months after therapy may represent persistent disease and should be biopsied. Recurrence usually requires radical resec-tion (APR). Metastasis to inguinal lymph

1	that persist greater than 3 to 6 months after therapy may represent persistent disease and should be biopsied. Recurrence usually requires radical resec-tion (APR). Metastasis to inguinal lymph nodes is a poor prog-nostic sign.148Verrucous Carcinoma (Buschke-Lowenstein Tumor, Giant Condyloma Acuminata). Verrucous carcinoma is a locally aggressive form of condyloma acuminata. Although these lesions do not metastasize, they can cause extensive local tissue destruction and may be grossly indistinguishable from epider-moid carcinoma. Wide local excision is the treatment of choice when possible, but radical resection may sometimes be required. Topical immunomodulators such as imiquimod (Aldara) may shrink some tumors, but they are almost never curative.151 Very large lesions may respond to external beam radiation, but resec-tion is almost always required.Basal Cell Carcinoma. Basal cell carcinoma of the anus is rare and resembles basal cell carcinoma elsewhere on the skin (raised, pearly

1	beam radiation, but resec-tion is almost always required.Basal Cell Carcinoma. Basal cell carcinoma of the anus is rare and resembles basal cell carcinoma elsewhere on the skin (raised, pearly edges with central ulceration). This is a slow-growing tumor that rarely metastasizes. Wide local excision is the treatment of choice, but recurrence occurs in up to 30% of patients. Radical resection and/or radiation therapy may be required for large lesions.Adenocarcinoma. Adenocarcinoma of the anus is extremely rare and usually represents downward spread of a low rectal ade-nocarcinoma. Adenocarcinoma may occasionally arise from the anal glands or may develop in a chronic fistula. Radical resection, usually after neoadjuvant chemoradiation, is usually required.Extramammary perianal Paget’s disease is adenocarci-noma in situ arising from the apocrine glands of the perianal area. The lesion is typically plaque-like and may be indistin-guishable from high-grade intraepithelial lesions.

1	disease is adenocarci-noma in situ arising from the apocrine glands of the perianal area. The lesion is typically plaque-like and may be indistin-guishable from high-grade intraepithelial lesions. Characteristic Paget’s cells are seen histologically. These tumors are often associated with a synchronous gastrointestinal adenocarcinoma, so a complete evaluation of the intestinal tract should be per-formed. Wide local excision is usually adequate treatment for perianal Paget’s disease.Melanoma. Anorectal melanoma is rare, comprising less than 1% of all anorectal malignancies and 1% to 2% of melanomas. Diagnosis is often delayed, and symptoms are attributed to hem-orrhoidal disease. Despite many advances in the treatment of cutaneous melanoma, prognosis for patients with anorectal dis-ease remains poor. Overall 5-year survival is less than 10%, and many patients present with systemic metastasis and/or deeply invasive tumors at the time of diagnosis. A few patients with anorectal melanoma,

1	poor. Overall 5-year survival is less than 10%, and many patients present with systemic metastasis and/or deeply invasive tumors at the time of diagnosis. A few patients with anorectal melanoma, however, present with isolated local or locoregional disease that is potentially resectable for cure, and both radical resection (APR) and wide local excision have been advocated. Recurrence is common and usually occurs systemi-cally regardless of the initial surgical procedure. Local resection with free margins does not increase the risk of local or regional recurrence, and APR offers no survival advantage over local excision. Because of the morbidity associated with APR, wide local excision is recommended for initial treatment of localized anal melanoma. In some patients, wide local excision may not be technically feasible, and APR may be required if the tumor involves a significant portion of the anal sphincter or is circum-ferential. The addition of adjuvant chemotherapy, biochemo-therapy,

1	not be technically feasible, and APR may be required if the tumor involves a significant portion of the anal sphincter or is circum-ferential. The addition of adjuvant chemotherapy, biochemo-therapy, vaccines, or radiotherapy may be of benefit in some patients, but efficacy remains unproven.152,153OTHER BENIGN COLORECTAL CONDITIONSRectal Prolapse and Solitary Rectal Ulcer SyndromeRectal Prolapse. Rectal prolapse refers to a circumferential, full-thickness protrusion of the rectum through the anus and has also been called “first-degree” prolapse, “complete” prolapse, or 7Brunicardi_Ch29_p1259-p1330.indd 130523/02/19 2:29 PM 1306SPECIFIC CONSIDERATIONSPART IIprocidentia. Internal prolapse occurs when the rectal wall intus-suscepts but does not protrude and is probably more accurately described as internal intussusception. Mucosal prolapse is a partial-thickness protrusion often associated with hemorrhoidal disease and is usually treated with banding or hemorrhoidectomy.In adults,

1	as internal intussusception. Mucosal prolapse is a partial-thickness protrusion often associated with hemorrhoidal disease and is usually treated with banding or hemorrhoidectomy.In adults, this condition is far more common among women, with a female-to-male ratio of 6:1. Prolapse becomes more prevalent with age in women and peaks in the sev-enth decade of life. In men, prevalence is unrelated to age. Symptoms include tenesmus, a sensation of tissue protruding from the anus that may or may not spontaneously reduce, and a sensation of incomplete evacuation. Mucus discharge and leak-age may accompany the protrusion. Patients also present with a myriad of functional complaints, from incontinence and diar-rhea to constipation and outlet obstruction.A thorough preoperative evaluation, including colonic transit studies, anorectal manometry, tests of pudendal nerve terminal motor latency, EMG, and cinedefecography, may be useful. The colon should be evaluated by colonoscopy, aircontrast

1	colonic transit studies, anorectal manometry, tests of pudendal nerve terminal motor latency, EMG, and cinedefecography, may be useful. The colon should be evaluated by colonoscopy, aircontrast barium enema, or CT colonography to exclude neo-plasms or diverticular disease. Cardiopulmonary condition should be thoroughly evaluated because comorbidities may influence the choice of surgical procedure.The primary therapy for rectal prolapse is surgery, and more than 100 different procedures have been described to treat this condition. Operations can be categorized as either abdominal or perineal. Abdominal operations have taken three major approaches: (a) reduction of the perineal hernia and closure of the cul-de-sac (Moschowitz repair); (b) fixation of the rectum, either with a prosthetic mesh or fascia lata sling (Ripstein and Wells rectopexy; ventral rectopexy) or by suture rectopexy; or (c) resection of redundant sigmoid colon (Fig. 29-28). In some cases, resection is combined with

1	mesh or fascia lata sling (Ripstein and Wells rectopexy; ventral rectopexy) or by suture rectopexy; or (c) resection of redundant sigmoid colon (Fig. 29-28). In some cases, resection is combined with rectal fixation (resection rectopexy). The recently described ventral rectopexy involves dissection of the anterior rectum down to the pelvic floor. Mesh is sutured to the anterior rectum at one end and anchored to the sacral promontory at the other end.154 Abdominal procedures for rectal prolapse are increasingly performed laparoscopically or robotically. Perineal approaches have focused on tightening the anus with a variety of prosthetic materials, reefing the rectal mucosa (Delorme procedure), or resecting the prolapsed bowel from the perineum (perineal rectosigmoidectomy or Altemeier procedure) (Fig. 29-29).Because rectal prolapse occurs most commonly in elderly women, the choice of operation depends in part on the patient’s overall medical condition. Abdominal rectopexy (with or

1	procedure) (Fig. 29-29).Because rectal prolapse occurs most commonly in elderly women, the choice of operation depends in part on the patient’s overall medical condition. Abdominal rectopexy (with or with-out sigmoid resection) offers the most durable repair, with recurrence occurring in less than 10% of patients. Perineal rectosigmoidectomy avoids an abdominal operation and may be preferable in high-risk patients but is associated with a higher recurrence rate. Reefing the rectal mucosa is effective for patients with limited prolapse. Anal encirclement procedures generally have been abandoned.Solitary Rectal Ulcer Syndrome. Solitary rectal ulcer syn-drome and colitis cystica profunda are commonly associated with internal intussusception. Patients may complain of pain, bleeding, mucus discharge, or outlet obstruction. In solitary rec-tal ulcer syndrome, one or more ulcers are present in the distal rectum, usually on the anterior wall. In colitis cystica profunda, nodules or a mass may

1	or outlet obstruction. In solitary rec-tal ulcer syndrome, one or more ulcers are present in the distal rectum, usually on the anterior wall. In colitis cystica profunda, nodules or a mass may be found in a similar location. Evalu-ation should include anorectal manometry, defecography, and either colonoscopy or barium enema to exclude other diagnoses. Biopsy of an ulcer or mass is mandatory to exclude malignancy or infection due to cytomegalovirus (CMV) in an immunosup-pressed patient. Nonoperative therapy (high-fiber diet, def-ecation training to avoid straining, and laxatives or enemas) is effective in the majority of patients. Biofeedback has also been reported to be effective in some patients. Surgery (either abdominal or perineal repair of prolapse as described earlier) is reserved for highly symptomatic patients who have failed all medical interventions.VolvulusVolvulus occurs when an air-filled segment of the colon twists about its mesentery. The sigmoid colon is involved in up

1	highly symptomatic patients who have failed all medical interventions.VolvulusVolvulus occurs when an air-filled segment of the colon twists about its mesentery. The sigmoid colon is involved in up to 90% of cases, but volvulus can involve the cecum (<20%) or trans-verse colon. A volvulus may reduce spontaneously, but more commonly produces bowel obstruction, which can progress to strangulation, gangrene, and perforation. Chronic constipation may produce a large, redundant colon (chronic megacolon) that predisposes to volvulus, especially if the mesenteric base is narrow.The symptoms of volvulus are those of acute bowel obstruction. Patients present with abdominal distention, nau-sea, and vomiting. Symptoms rapidly progress to generalized abdominal pain and tenderness. Fever and leukocytosis are her-alds of gangrene and/or perforation. Occasionally, patients will report a long history of intermittent obstructive symptoms and distention, suggesting intermittent chronic volvulus.Sigmoid

1	are her-alds of gangrene and/or perforation. Occasionally, patients will report a long history of intermittent obstructive symptoms and distention, suggesting intermittent chronic volvulus.Sigmoid Volvulus. Sigmoid volvulus can often be differenti-ated from cecal or transverse colon volvulus by the appearance of plain X-rays of the abdomen. Sigmoid volvulus produces a characteristic bent inner tube or coffee bean appearance, with the convexity of the loop lying in the right upper quadrant (opposite the site of obstruction). Gastrografin enema shows a 8Figure 29-28. Transabdominal proctopexy for rectal prolapse. The fully mobilized rectum is sutured to the presacral fascia. A. Anterior view. B. Lateral view. If desired, a sigmoid colectomy can be performed con-comitantly to resect the redundant colon.BABrunicardi_Ch29_p1259-p1330.indd 130623/02/19 2:29 PM 1307COLON, RECTUM, AND ANUSCHAPTER 29narrowing at the site of the volvulus and a pathognomonic bird’s beak (Fig. 29-30).Unless

1	colon.BABrunicardi_Ch29_p1259-p1330.indd 130623/02/19 2:29 PM 1307COLON, RECTUM, AND ANUSCHAPTER 29narrowing at the site of the volvulus and a pathognomonic bird’s beak (Fig. 29-30).Unless there are obvious signs of gangrene or peritoni-tis, the initial management of sigmoid volvulus is resuscitation followed by endoscopic detorsion. Detorsion is usually most easily accomplished by using a rigid proctoscope, but a flexible sigmoidoscope or colonoscope may also be effective. A rec-tal tube may be inserted to maintain decompression. Although these techniques are successful in reducing sigmoid volvulus in the majority of patients, the risk of recurrence is high (up to 40%). For this reason, an elective sigmoid colectomy should be performed after the patient has been stabilized and undergone an adequate bowel preparation.Clinical evidence of gangrene or perforation mandates immediate surgical exploration without an attempt at endoscopic decompression. Similarly, the presence of

1	undergone an adequate bowel preparation.Clinical evidence of gangrene or perforation mandates immediate surgical exploration without an attempt at endoscopic decompression. Similarly, the presence of necrotic mucosa, ulcer-ation, or dark blood noted on endoscopy examination suggests strangulation and is an indication for operation. If dead bowel is present at laparotomy, a sigmoid colectomy with end colostomy (Hartmann’s procedure) may be the safest operation to perform.Cecal Volvulus. Cecal volvulus results from nonfixation of the right colon. In the majority of cases, rotation occurs around the ileocolic blood vessels and vascular impairment occurs early, although 10% to 30% of the cecum folds upon itself (cecal bascule). Plain X-rays of the abdomen show a characteristic kidney-shaped, air-filled structure in the left upper quadrant (opposite the site of obstruction), and a Gastrografin enema confirms obstruction at the level of the volvulus.Unlike sigmoid volvulus, cecal volvulus

1	air-filled structure in the left upper quadrant (opposite the site of obstruction), and a Gastrografin enema confirms obstruction at the level of the volvulus.Unlike sigmoid volvulus, cecal volvulus can almost never be detorsed endoscopically. Moreover, because vascular com-promise occurs early in the course of cecal volvulus, surgical exploration is necessary when the diagnosis is made. Right hemicolectomy with a primary ileocolic anastomosis can usu-ally be performed safely and prevents recurrence. Simple detor-sion or detorsion and cecopexy are associated with a high rate of recurrence.Transverse Colon Volvulus. Transverse colon volvulus is extremely rare. Nonfixation of the colon and chronic consti-pation with megacolon may predispose to transverse colon AFCEDBFigure 29-29. Perineal rectosigmoidectomy shown in lithotomy position. A. A circular incision is made 2 cm proximal to the dentate line. B. The anterior peritoneal reflection is opened. C. The mesentery is divided and

1	rectosigmoidectomy shown in lithotomy position. A. A circular incision is made 2 cm proximal to the dentate line. B. The anterior peritoneal reflection is opened. C. The mesentery is divided and ligated. D. The peritoneum may be sutured to the bowel wall. E. The bowel is resected. F. A hand-sewn anastomosis is performed.Brunicardi_Ch29_p1259-p1330.indd 130723/02/19 2:29 PM 1308SPECIFIC CONSIDERATIONSPART IIvolvulus. The radiographic appearance of transverse colon volvulus resembles sigmoid volvulus, but Gastrografin enema will reveal a more proximal obstruction. Although colonoscopic detorsion is occasionally successful in this setting, most patients require emergent exploration and resection.MegacolonMegacolon describes a chronically dilated, elongated, hypertro-phied large bowel. Megacolon may be congenital or acquired and is usually related to chronic mechanical or functional obstruction. In general, the degree of megacolon is related to the duration of obstruction. Evaluation

1	may be congenital or acquired and is usually related to chronic mechanical or functional obstruction. In general, the degree of megacolon is related to the duration of obstruction. Evaluation must always include examination of the colon and rectum (either endoscopically or radiographically) to exclude a surgically correctable mechanical obstruction.Congenital megacolon caused by Hirschsprung’s disease results from the failure of migration of neural crest cells to the distal large intestine. The resulting absence of ganglion cells in the distal colon results in a failure of relaxation and causes a functional obstruction. The proximal, healthy bowel becomes progressively dilated. Surgical resection of the aganglionic seg-ment is curative. Although Hirschsprung’s disease primarily is a disease of infants and children, it occasionally presents later in adulthood, especially if an extremely short segment of the bowel is affected (ultrashort-segment Hirschsprung’s disease).Acquired

1	a disease of infants and children, it occasionally presents later in adulthood, especially if an extremely short segment of the bowel is affected (ultrashort-segment Hirschsprung’s disease).Acquired megacolon may result from infection or chronic constipation. Infection with the protozoan Trypanosoma cruzi (Chagas’ disease) destroys ganglion cells and produces both megacolon and megaesophagus. Chronic constipation from slow transit or secondary to medications (especially anticholinergic medications) or neurologic disorders (paraplegia, poliomyelitis, amyotrophic lateral sclerosis, multiple sclerosis) may produce progressive colonic dilatation. Diverting ileostomy or subtotal colectomy with an ileorectal anastomosis is occasionally neces-sary in these patients.Colonic Pseudo-Obstruction (Ogilvie’s Syndrome)Colonic pseudo-obstruction (Ogilvie’s syndrome) is a func-tional disorder in which the colon becomes massively dilated in the absence of mechanical obstruction. Pseudo-obstruction

1	(Ogilvie’s Syndrome)Colonic pseudo-obstruction (Ogilvie’s syndrome) is a func-tional disorder in which the colon becomes massively dilated in the absence of mechanical obstruction. Pseudo-obstruction most commonly occurs in hospitalized patients and is associated with the use of narcotics, bed rest, and comorbid disease. Pseudo-obstruction is thought to result from autonomic dysfunction and severe adynamic ileus. The diagnosis is made based on the pres-ence of massive dilatation of the colon (usually predominantly the right and transverse colon) in the absence of a mechanical obstruction. Initial treatment consists of cessation of narcotics, anticholinergics, or other medications that may contribute to ileus. Strict bowel rest and intravenous hydration are crucial. Most patients will respond to these measures. In patients who fail to improve, colonoscopic decompression often is effective. However, this procedure is technically challenging, and great care must be taken to avoid causing

1	to these measures. In patients who fail to improve, colonoscopic decompression often is effective. However, this procedure is technically challenging, and great care must be taken to avoid causing perforation. Up to 40% of patients recur. Intravenous neostigmine (an acetylcholines-terase inhibitor), administered as a single 2 mg dose, also is extremely effective in decompressing the dilated colon and is associated with a low rate of recurrence (20%).155 However, neostigmine may produce transient but profound bradycardia and may be inappropriate in patients with cardiopulmonary ABFigure 29-30. Sigmoid volvulus: (A) Illustration and (B) Gastrografin enema showing “bird-beak” sign (arrow). (B. Reproduced with per-mission from James EC, Corry RJ, Perry JCF: Basic Surgical Practice. Philadelphia, PA: Hanley & Belfus; 1987.)Brunicardi_Ch29_p1259-p1330.indd 130823/02/19 2:29 PM 1309COLON, RECTUM, AND ANUSCHAPTER 29disease. Because the colonic dilatation is typically greatest in the

1	PA: Hanley & Belfus; 1987.)Brunicardi_Ch29_p1259-p1330.indd 130823/02/19 2:29 PM 1309COLON, RECTUM, AND ANUSCHAPTER 29disease. Because the colonic dilatation is typically greatest in the proximal colon, placement of a rectal tube is rarely effective. It is crucial to exclude mechanical obstruction (usually with a Gastrografin enema) prior to medical or endoscopic treatment.Ischemic ColitisIntestinal ischemia occurs most commonly in the colon. Unlike small bowel ischemia, colonic ischemia rarely is associated with major arterial or venous occlusion. Instead, most colonic isch-emia appears to result from low flow and/or small vessel occlu-sion. Risk factors include vascular disease, diabetes mellitus, vasculitis, hypotension, and tobacco use. In addition, ligation of the inferior mesenteric artery during aortic surgery predisposes to colonic ischemia. Occasionally, thrombosis or embolism may cause ischemia. Although the splenic flexure is the most com-mon site of ischemic colitis,

1	artery during aortic surgery predisposes to colonic ischemia. Occasionally, thrombosis or embolism may cause ischemia. Although the splenic flexure is the most com-mon site of ischemic colitis, any segment of the colon may be affected. The rectum is relatively spared because of its rich col-lateral circulation.Signs and symptoms of ischemic colitis reflect the extent of bowel ischemia. In mild cases, patients may have diarrhea (usually bloody) without abdominal pain. With more severe ischemia, intense abdominal pain (often out of proportion to the clinical examination), tenderness, fever, and leukocytosis are present. Peritonitis and/or systemic toxicity are signs of full-thickness necrosis and perforation.The diagnosis of ischemic colitis is often based on the clinical history and physical examination. Plain films may reveal thumb printing, which results from mucosal edema and submucosal hemorrhage. CT often shows nonspecific colonic wall thickening and pericolic fat stranding.

1	examination. Plain films may reveal thumb printing, which results from mucosal edema and submucosal hemorrhage. CT often shows nonspecific colonic wall thickening and pericolic fat stranding. Angiography is usually not helpful because major arterial occlusion is rare. While sigmoidoscopy may reveal characteristic dark, hemor-rhagic mucosa, the risk of precipitating perforation is high. For this reason, sigmoidoscopy is relatively contraindicated in any patient with significant abdominal tenderness. Contrast studies (Gastrografin or barium enema) are similarly contraindicated during the acute phase of ischemic colitis.Treatment of ischemic colitis depends on clinical severity. Unlike ischemia of the small bowel, the majority of patients with ischemic colitis can be treated medically. Bowel rest and broad-spectrum antibiotics are the mainstay of therapy, and 80% of patients will recover with this regimen. Hemodynamic parameters should be optimized, especially if hypotension and low flow

1	rest and broad-spectrum antibiotics are the mainstay of therapy, and 80% of patients will recover with this regimen. Hemodynamic parameters should be optimized, especially if hypotension and low flow appear to be the inciting cause. Long-term sequelae include stricture (10–15%) and chronic segmental ischemia (15–20%). Colonoscopy should be performed after recovery to evaluate strictures and to rule out other diagnoses such as inflammatory bowel disease or malignancy. Failure to improve after 2 to 3 days of medical management, progression of symp-toms, and deterioration in clinical condition are indications for surgical exploration. In this setting, all necrotic bowel should be resected. Primary anastomosis should be avoided. Occasion-ally, repeated exploration (a second-look operation) may be necessary.Infectious ColitisPseudomembranous Colitis (Clostridium difficile Colitis). Pseudomembranous colitis is caused by C difficile, a gram-positive anaerobic bacillus. Clostridium difficile

1	be necessary.Infectious ColitisPseudomembranous Colitis (Clostridium difficile Colitis). Pseudomembranous colitis is caused by C difficile, a gram-positive anaerobic bacillus. Clostridium difficile colitis is extremely common and is the leading cause of nosocomially acquired diarrhea. The spectrum of disease ranges from watery diarrhea to fulminant, life-threatening colitis. Clostridium dif-ficile is carried in the large intestine of many healthy adults. Colitis is thought to result from overgrowth of this organism after depletion of the normal commensal flora of the gut with the use of antibiotics. Although clindamycin was the first anti-microbial agent associated with C difficile colitis, almost any antibiotic may cause this disease. Moreover, although the risk of C difficile colitis increases with prolonged antibiotic use, even a single dose of an antibiotic may cause the disease. Immunosup-pression, medical comorbidities, prolonged hospitalization or nursing home residence, and

1	increases with prolonged antibiotic use, even a single dose of an antibiotic may cause the disease. Immunosup-pression, medical comorbidities, prolonged hospitalization or nursing home residence, and bowel surgery increase the risk.The pathogenic changes associated with C difficile colitis result from production of two toxins: toxin A (an enterotoxin) and toxin B (a cytotoxin). Diagnosis of this disease was tradition-ally made by culturing the organism from the stool. Detection of one or both toxins (either by cytotoxic assays or by immuno-assays) has proven to be more rapid, sensitive, and specific.156 The diagnosis may also be made endoscopically by detection of characteristic ulcers, plaques, and pseudomembranes.157Management should include immediate cessation of the offending antimicrobial agent. Patients with mild disease (diar-rhea but no fever or abdominal pain) may be treated as outpatients with a 10-day course of oral metronidazole. Oral vancomycin is a second-line agent used

1	agent. Patients with mild disease (diar-rhea but no fever or abdominal pain) may be treated as outpatients with a 10-day course of oral metronidazole. Oral vancomycin is a second-line agent used in patients allergic to metronidazole or in patients with recurrent disease. More severe diarrhea associ-ated with dehydration and/or fever and abdominal pain is best treated with bowel rest, intravenous hydration, and oral met-ronidazole or vancomycin. Proctosigmoiditis may respond to vancomycin enemas. Recurrent colitis occurs in up to 20% of patients and may be treated by a longer course of oral vanco-mycin (up to 1 month) or rifaximin (a rapamycin derivative). Reintroduction of normal flora by ingestion of probiotics or stool transplantation has been suggested as a possible treatment for recurrent or refractory disease. Fulminant colitis, characterized by septicemia and/or evidence of perforation, requires emergent laparotomy. A total abdominal colectomy with end ileostomy may be

1	for recurrent or refractory disease. Fulminant colitis, characterized by septicemia and/or evidence of perforation, requires emergent laparotomy. A total abdominal colectomy with end ileostomy may be lifesaving. Over the past decade, C difficile colitis has increased in prevalence, and new, more virulent strains have appeared, making this disease increasingly challenging to treat.157,158Other Infectious Colitides. A variety of other infections with bacteria, parasites, fungi, or viruses may cause colonic inflammation. Common bacterial infections include enterotoxic E coli, C jejuni, Yersinia enterocolitica, S typhi, Shigella, and N gonorrhoeae. Less commonly, Mycobacterium tuberculosis, M bovis, Actinomycosis israelii, or Treponema pallidum (syphi-lis) may cause colitis or proctitis. Parasitic infections such as amebiasis, cryptosporidiosis, and giardiasis are also relatively common. Fungal infections (Candida species, histoplasmosis) are extremely rare in otherwise healthy

1	Parasitic infections such as amebiasis, cryptosporidiosis, and giardiasis are also relatively common. Fungal infections (Candida species, histoplasmosis) are extremely rare in otherwise healthy individuals. The most common viral infections that produce colitic symptoms are HIV, herpes simplex viruses, and CMV.Most symptoms are nonspecific and consist of diarrhea (with or without bleeding), crampy abdominal pain, and mal-aise. A thorough history may offer clues to the etiology (other medical conditions, especially immunosuppression; recent travel or exposures; and ingestions). Diagnosis is usually made by identification of a pathogen in the stool, either by microscopy or culture. Serum immunoassays may also be useful (amebia-sis, HIV, CMV). Occasionally, endoscopy with biopsy may be required. Treatment is tailored to the infection.Brunicardi_Ch29_p1259-p1330.indd 130923/02/19 2:29 PM 1310SPECIFIC CONSIDERATIONSPART IIANORECTAL DISEASESAny patient with anal/perianal symptoms

1	required. Treatment is tailored to the infection.Brunicardi_Ch29_p1259-p1330.indd 130923/02/19 2:29 PM 1310SPECIFIC CONSIDERATIONSPART IIANORECTAL DISEASESAny patient with anal/perianal symptoms requires a careful his-tory and physical, including a digital rectal examination. Other studies such as defecography, manometry, CT scan, MRI, con-trast enema, endoscopy, endoanal ultrasound, or exam under anesthesia may be required to arrive at an accurate diagnosis.HemorrhoidsHemorrhoids are cushions of submucosal tissue containing venules, arterioles, and smooth muscle fibers that are located in the anal canal (see Fig. 29-4). Three hemorrhoidal cushions are found in the left lateral, right anterior, and right poste-rior positions. Hemorrhoids are thought to function as part of the continence mechanism and aid in complete closure of the anal canal at rest. Because hemorrhoids are a normal part of anorectal anatomy, treatment is only indicated if they become symptom-atic. Excessive

1	mechanism and aid in complete closure of the anal canal at rest. Because hemorrhoids are a normal part of anorectal anatomy, treatment is only indicated if they become symptom-atic. Excessive straining, increased abdominal pressure, and hard stools increase venous engorgement of the hemorrhoidal plexus and cause prolapse of hemorrhoidal tissue. Bleeding, thrombo-sis, and symptomatic hemorrhoidal prolapse may result.External hemorrhoids are located distal to the dentate line and are covered with anoderm. Because the anoderm is richly innervated, thrombosis of an external hemorrhoid may cause significant pain. It is for this reason that external hemorrhoids should not be ligated or excised without adequate local anes-thetic. A skin tag is redundant fibrotic skin at the anal verge, often persisting as the residua of a thrombosed external hemor-rhoid. Skin tags are often confused with symptomatic hemor-rhoids. External hemorrhoids and skin tags may cause itching and difficulty with

1	persisting as the residua of a thrombosed external hemor-rhoid. Skin tags are often confused with symptomatic hemor-rhoids. External hemorrhoids and skin tags may cause itching and difficulty with hygiene if they are large. Treatment of exter-nal hemorrhoids and skin tags is only indicated for symptomatic relief.Internal hemorrhoids are located proximal to the dentate line and covered by insensate anorectal mucosa. Internal hem-orrhoids may prolapse or bleed, but they rarely become painful unless they develop thrombosis and necrosis (usually related to severe prolapse, incarceration, and/or strangulation). Inter-nal hemorrhoids are graded according to the extent of prolapse. First-degree hemorrhoids bulge into the anal canal and may prolapse beyond the dentate line on straining. Second-degree hemorrhoids prolapse through the anus but reduce spontane-ously. Third-degree hemorrhoids prolapse through the anal canal and require manual reduction. Fourth-degree hemorrhoids prolapse but

1	hemorrhoids prolapse through the anus but reduce spontane-ously. Third-degree hemorrhoids prolapse through the anal canal and require manual reduction. Fourth-degree hemorrhoids prolapse but cannot be reduced and are at risk for strangulation.Combined internal and external hemorrhoids straddle the dentate line and have characteristics of both internal and external hemorrhoids. Hemorrhoidectomy is often required for large, symptomatic, combined hemorrhoids. Postpartum hemorrhoids result from straining during labor, which results in edema, thrombosis, and/or strangulation. Hemorrhoidectomy is often the treatment of choice, especially if the patient has had chronic hemorrhoidal symptoms. Portal hypertension was long thought to increase the risk of hemorrhoidal bleeding because of the anastomoses between the portal venous system (middle and upper hemorrhoidal plexuses) and the systemic venous system (inferior rectal plexuses). It is now understood that hemorrhoidal disease is no more

1	between the portal venous system (middle and upper hemorrhoidal plexuses) and the systemic venous system (inferior rectal plexuses). It is now understood that hemorrhoidal disease is no more common in patients with portal hypertension than in the normal population. Rectal varices, however, may occur and may cause hemorrhage in these patients. In general, rectal varices are best treated by lowering portal venous pres-sure. Rarely, suture ligation may be necessary if massive bleed-ing persists. Surgical hemorrhoidectomy should be avoided in these patients because of the risk of massive, difficult-to-control variceal bleeding.Treatment Medical Therapy Bleeding from firstand second-degree hem-orrhoids often improves with the addition of dietary fiber, stool softeners, increased fluid intake, and avoidance of straining. Associated pruritus often may improve with improved hygiene. Many over-the-counter topical medications are desiccants and are relatively ineffective for treating

1	intake, and avoidance of straining. Associated pruritus often may improve with improved hygiene. Many over-the-counter topical medications are desiccants and are relatively ineffective for treating hemorrhoidal symptoms.Rubber Band Ligation Persistent bleeding from first-, sec-ond-, and selected third-degree hemorrhoids may be treated by rubber band ligation.Mucosa located 1 to 2 cm proximal to the dentate line is grasped and pulled into a rubber band applier. After firing the ligator, the rubber band strangulates the underlying tissue, causing scarring and preventing further bleeding or prolapse (Fig. 29-31). In general, only one or two quadrants are banded per visit. Severe pain will occur if the rubber band is placed at or distal to the dentate line where sensory nerves are located. Other complications of rubber band ligation include urinary retention, infection, and bleeding. Urinary retention occurs in approximately 1% of patients and is more likely if the ligation has

1	Other complications of rubber band ligation include urinary retention, infection, and bleeding. Urinary retention occurs in approximately 1% of patients and is more likely if the ligation has inadvertently included a portion of the internal sphincter. Necrotizing infection is an uncommon, but life-threatening complication. Severe pain, fever, and urinary retention are early signs of infection and should prompt immediate evaluation of the patient usually with an exam under anesthesia. Treatment includes debridement of necrotic tissue, drainage of associated abscesses, and broad-spectrum antibiotics. Bleeding may occur approximately 7 to 10 days after rubber band ligation, at the time when the ligated pedicle necroses and sloughs. Bleeding is usually self-limited, but persistent hemorrhage may require exam under anesthesia and suture ligation of the pedicle.Infrared Photocoagulation Infrared photocoagulation is an effective office treatment for small firstand second-degree hemorrhoids.

1	may require exam under anesthesia and suture ligation of the pedicle.Infrared Photocoagulation Infrared photocoagulation is an effective office treatment for small firstand second-degree hemorrhoids. The instrument is applied to the apex of each hem-orrhoid to coagulate the underlying plexus. All three quadrants may be treated during the same visit. Larger hemorrhoids and hemorrhoids with a significant amount of prolapse are not effec-tively treated with this technique.Sclerotherapy The injection of bleeding internal hemorrhoids with sclerosing agents is another effective office technique for treatment of first-, second-, and some third-degree hemorrhoids. One to 3 mL of a sclerosing solution (phenol in olive oil, sodium morrhuate, or quinine urea) is injected into the submucosa of each hemorrhoid. Few complications are associated with sclero-therapy, but infection and fibrosis have been reported.Excision of Thrombosed External Hemorrhoids Acutely thrombosed external hemorrhoids

1	hemorrhoid. Few complications are associated with sclero-therapy, but infection and fibrosis have been reported.Excision of Thrombosed External Hemorrhoids Acutely thrombosed external hemorrhoids generally cause intense pain and a palpable perianal mass during the first 24 to 72 hours after thrombosis. The thrombosis can be effectively treated with an elliptical excision performed in the office under local anesthe-sia. Because the clot is usually loculated, simple incision and drainage is rarely effective. After 72 hours, the clot begins to resorb, and the pain resolves spontaneously. Excision is unnec-essary, but sitz baths and analgesics are often helpful.Operative Hemorrhoidectomy A number of surgical proce-dures have been described for elective resection of symptomatic hemorrhoids. All are based on decreasing blood flow to the hem-orrhoidal plexuses and excising redundant anoderm and mucosa.9Brunicardi_Ch29_p1259-p1330.indd 131023/02/19 2:29 PM 1311COLON, RECTUM, AND

1	All are based on decreasing blood flow to the hem-orrhoidal plexuses and excising redundant anoderm and mucosa.9Brunicardi_Ch29_p1259-p1330.indd 131023/02/19 2:29 PM 1311COLON, RECTUM, AND ANUSCHAPTER 29Closed Submucosal Hemorrhoidectomy The Parks or Fer-guson hemorrhoidectomy involves resection of hemorrhoidal tissue and closure of the wounds with absorbable suture. The procedure may be performed in the prone or lithotomy position under local, regional, or general anesthesia. The anal canal is examined and an anal speculum inserted. The hemorrhoid cush-ions and associated redundant mucosa are identified and excised using an elliptical incision starting just distal to the anal verge and extending proximally to the anorectal ring. It is crucial to identify the fibers of the internal sphincter and carefully brush these away from the dissection in order to avoid injury to the sphincter. The apex of the hemorrhoidal plexus is then ligated and the hemorrhoid excised. The wound is then

1	and carefully brush these away from the dissection in order to avoid injury to the sphincter. The apex of the hemorrhoidal plexus is then ligated and the hemorrhoid excised. The wound is then closed with a running absorbable suture. All three hemorrhoidal cushions may be removed using this technique; however, care should be taken to avoid resecting a large area of perianal skin in order to avoid postoperative anal stenosis (Fig. 29-32).Open Hemorrhoidectomy This technique, often called the Milligan and Morgan hemorrhoidectomy, follows the same principles of excision described earlier, but the wounds are left open and allowed to heal by secondary intention.Whitehead’s Hemorrhoidectomy Whitehead’s hemorrhoid-ectomy involves circumferential excision of the hemorrhoidal cushions just proximal to the dentate line. After excision, the rectal mucosa is then advanced and sutured to the dentate line. While some surgeons still use Whitehead’s hemorrhoidectomy, most have abandoned this approach

1	to the dentate line. After excision, the rectal mucosa is then advanced and sutured to the dentate line. While some surgeons still use Whitehead’s hemorrhoidectomy, most have abandoned this approach because of the risk of ectro-pion (Whitehead’s deformity).Procedure for Prolapse and Hemorrhoids/Stapled Hemor-rhoidectomy Procedure for prolapse and hemorrhoids (PPH) is also referred to as a stapled hemorrhoidopexy. Best suited for patients with secondand third-degree hemorrhoids, this out-patient procedure uses a stapling device similar in appearance and mechanism of action to an end-to-end anastomotic (EEA) stapling device used for rectal surgery. Just as with an EEA sta-pler, proximal and distal tissue donuts, in this case consisting of mucosa and submucosa, are generated by the PPH stapler though the primary means by which this procedure provides relief for internal hemorrhoids is by pexying the redundant hemorrhoidal tissue, ligating the venules feeding the hemorrhoidal plexus and

1	though the primary means by which this procedure provides relief for internal hemorrhoids is by pexying the redundant hemorrhoidal tissue, ligating the venules feeding the hemorrhoidal plexus and fixing redundant mucosa proximal to the dentate line. Several studies suggest that this procedure is safe and effective, that it is associated with less postoperative pain and disability, and that it has an equivalent risk of postoperative complications when compared to excisional hemorrhoidectomy. Complications associated with this procedure include chronic anal pain, bac-teremia, rectovaginal fistula, formation of an obstructing rectal stricture and even rectal perforation. In at least one systematic review comparing outcomes between PPH and excisional hem-orrhoidectomy, overall incidence of complications was similar, ElasticbandsElasticbandsElasticbandsFigure 29-31. Rubber band ligation of internal hemorrhoids. The mucosa just proximal to the internal hemorrhoids is

1	overall incidence of complications was similar, ElasticbandsElasticbandsElasticbandsFigure 29-31. Rubber band ligation of internal hemorrhoids. The mucosa just proximal to the internal hemorrhoids is banded.Brunicardi_Ch29_p1259-p1330.indd 131123/02/19 2:29 PM 1312SPECIFIC CONSIDERATIONSPART IIthough the incidence of recurrent hemorrhoids was lower fol-lowing excisional hemorrhoidectomy.159,160Doppler-Guided Hemorrhoidal Artery Ligation Another recent approach to treating symptomatic hemorrhoids is Doppler-guided hemorrhoidal artery ligation (also called trans-anal hemorrhoidal dearterialization). In this procedure, a Dop-pler probe is used to identify the artery or arteries feeding the hemorrhoidal plexus. These vessels are then ligated. Early reports have shown promise, but long-term durability remains to be determined.161Complications of Hemorrhoidectomy. Postoperative pain following excisional hemorrhoidectomy requires analgesia usually with oral narcotics. Nonsteroidal

1	durability remains to be determined.161Complications of Hemorrhoidectomy. Postoperative pain following excisional hemorrhoidectomy requires analgesia usually with oral narcotics. Nonsteroidal anti-inflammatory drugs, muscle relaxants, topical analgesics, and comfort mea-sures, including sitz baths, are often useful as well. Urinary retention is a common complication following hemorrhoidec-tomy and has been reported to be as high as 10% to 50% in some series. The risk of urinary retention can be minimized by limiting intraoperative and perioperative intravenous flu-ids and by providing adequate analgesia. Pain can also lead to fecal impaction. Risk of impaction may be decreased by pre-operative enemas or a limited mechanical bowel preparation, liberal use of laxatives postoperatively, and adequate pain con-trol. While a small amount of bleeding, especially with bowel movements, is to be expected, massive hemorrhage can occur after hemorrhoidectomy. Bleeding may occur in the immediate

1	adequate pain con-trol. While a small amount of bleeding, especially with bowel movements, is to be expected, massive hemorrhage can occur after hemorrhoidectomy. Bleeding may occur in the immediate postoperative period (often in the recovery room) as a result of inadequate ligation of the vascular pedicle. This type of hemorrhage mandates an urgent return to the operating room where suture ligation of the bleeding vessel will often solve the problem. Bleeding may also occur 7 to 10 days after hemor-rhoidectomy when the necrotic mucosa overlying the vascular pedicle sloughs. While some of these patients may be safely observed, others will require an exam under anesthesia to ligate the bleeding vessel or to oversew the wounds if no specific site of bleeding is identified. Infection is uncommon after hemor-rhoidectomy; however, necrotizing soft tissue infection can occur with devastating consequences. Severe pain, fever, and urinary retention may be early signs of infection. If

1	is uncommon after hemor-rhoidectomy; however, necrotizing soft tissue infection can occur with devastating consequences. Severe pain, fever, and urinary retention may be early signs of infection. If infection is suspected, emergent examination under anesthesia, drainage of abscess, and/or debridement of all necrotic tissue are required.Long-term sequelae of hemorrhoidectomy include incon-tinence, anal stenosis, and ectropion (Whitehead’s deformity). Many patients experience transient incontinence to flatus, but these symptoms are usually short-lived, and few patients have ABCDEFFigure 29-32. Technique of closed submucosal hemorrhoidectomy. A. The patient is in prone jackknife position. B. A Fansler anoscope is used for exposure. C. A narrow ellipse of anoderm is excised. D. A submucosal dissection of the hemorrhoidal plexus from the underlying anal sphincter is performed. E. Redundant mucosa is anchored to the proximal anal canal, and the wound is closed with a running absorbable

1	dissection of the hemorrhoidal plexus from the underlying anal sphincter is performed. E. Redundant mucosa is anchored to the proximal anal canal, and the wound is closed with a running absorbable suture. F. Additional quadrants are excised to complete the procedure.Brunicardi_Ch29_p1259-p1330.indd 131223/02/19 2:29 PM 1313COLON, RECTUM, AND ANUSCHAPTER 29permanent fecal incontinence. Anal stenosis may result from scarring after extensive resection of perianal skin. Ectropion may occur after Whitehead’s hemorrhoidectomy.Anal FissureA fissure in ano is a tear in the anoderm distal to the dentate line. The pathophysiology of anal fissure is thought to be related to trauma from either the passage of hard stool or prolonged diarrhea. A tear in the anoderm causes spasm of the internal anal sphincter, which results in pain, increased tearing, and decreased blood supply to the anoderm. This cycle of pain, spasm, and ischemia contributes to development of a poorly healing wound that

1	anal sphincter, which results in pain, increased tearing, and decreased blood supply to the anoderm. This cycle of pain, spasm, and ischemia contributes to development of a poorly healing wound that becomes a chronic fissure. The vast major-ity of anal fissures occur in the posterior midline. Ten percent to 15% occur in the anterior midline. Less than 1% of fissures occur off midline.Symptoms and Findings. Anal fissure is extremely common. Characteristic symptoms include tearing pain with defecation and hematochezia (usually described as blood on the toilet paper). Patients may also complain of a sensation of intense and painful anal spasm lasting for several hours after a bowel move-ment. On physical examination, the fissure can often be seen in the anoderm by gently separating the buttocks. Patients are often too tender to tolerate digital rectal examination, anoscopy, or proctoscopy. An acute fissure is a superficial tear of the distal anoderm and almost always heals with medical

1	Patients are often too tender to tolerate digital rectal examination, anoscopy, or proctoscopy. An acute fissure is a superficial tear of the distal anoderm and almost always heals with medical management. Chronic fissures develop ulceration and heaped-up edges with the white fibers of the internal anal sphincter visible at the base of the ulcer. There often is an associated external skin tag and/or a hypertrophied anal papilla internally. These fissures are more challenging to treat and may require surgery. A lateral loca-tion of a chronic anal fissure may be evidence of an underlying disease such as Crohn’s disease, HIV, syphilis, tuberculosis, or leukemia. If the diagnosis is in doubt or there is suspicion of another cause for the perianal pain such as abscess or fistula, an examination under anesthesia may be necessary.Treatment. Therapy focuses on breaking the cycle of pain, spasm, and ischemia thought to be responsible for development of fissure in ano. First-line therapy to

1	under anesthesia may be necessary.Treatment. Therapy focuses on breaking the cycle of pain, spasm, and ischemia thought to be responsible for development of fissure in ano. First-line therapy to minimize anal trauma includes bulk agents, stool softeners, and warm sitz baths. The addition of 2% lidocaine jelly or other analgesic creams can provide additional symptomatic relief. Nitroglycerin ointment has been used locally to improve blood flow but often causes severe headaches. Both oral and topical calcium channel block-ers (diltiazem and nifedipine) have also been used to heal fis-sures and may have fewer side effects than topical nitrates.162 Newer agents, such as arginine (a nitric oxide donor) and topical bethanechol (a muscarinic agonist), have also been used to treat fissures. Medical therapy is effective in most acute fissures, but it will heal only approximately 50% of chronic fissures.162Botulinum toxin (Botox) causes temporary muscle paraly-sis by preventing acetylcholine

1	therapy is effective in most acute fissures, but it will heal only approximately 50% of chronic fissures.162Botulinum toxin (Botox) causes temporary muscle paraly-sis by preventing acetylcholine release from presynaptic nerve terminals. Injection of botulinum toxin is used in some centers as an alternative to surgical sphincterotomy for chronic fissure. Although there are few long-term complications from the use of botulinum toxin, healing appears to be equivalent to other medical therapies.163,164Surgical therapy has traditionally been recommended for chronic fissures that have failed medical therapy, and lateral internal sphincterotomy is the procedure of choice. The aim of this procedure is to decrease spasm of the internal sphincter by dividing a portion of the muscle. Approximately 30% of the internal sphincter fibers are divided laterally by using either an open (Fig. 29-33) or closed (Fig. 29-34) technique. Healing is achieved in more than 95% of patients using this technique,

1	of the internal sphincter fibers are divided laterally by using either an open (Fig. 29-33) or closed (Fig. 29-34) technique. Healing is achieved in more than 95% of patients using this technique, and most patients experience immediate pain relief. Recurrence occurs in less than 10% of patients, and the risk of incontinence (usually to flatus) ranges from 5% to 15%. Advancement flaps (VY) with or without sphincterotomy have also been reported to successfully treat chronic fissures.Anorectal Sepsis and Cryptoglandular AbscessRelevant Anatomy. The majority of anorectal suppurative disease results from infections of the anal glands (cryptoglan-dular infection) found in the intersphincteric plane. Their ducts traverse the internal sphincter and empty into the anal crypts at the level of the dentate line. Infection of an anal gland results in the formation of an abscess that enlarges and spreads along one of several planes in the perianal and perirectal spaces. The perianal space

1	of the dentate line. Infection of an anal gland results in the formation of an abscess that enlarges and spreads along one of several planes in the perianal and perirectal spaces. The perianal space surrounds the anus and laterally becomes con-tinuous with the fat of the buttocks. The intersphincteric space separates the internal and external anal sphincters. It is continu-ous with the perianal space distally and extends cephalad into the rectal wall. The ischiorectal space (ischiorectal fossa) is located lateral and posterior to the anus and is bounded medially by the external sphincter, laterally by the ischium, superiorly by the levator ani, and inferiorly by the transverse septum. The ischiorectal space contains the inferior rectal vessels and lym-phatics. The two ischiorectal spaces connect posteriorly above the anococcygeal ligament but below the levator ani muscle, forming the deep postanal space. The supralevator spaces lie above the levator ani on either side of the rectum

1	connect posteriorly above the anococcygeal ligament but below the levator ani muscle, forming the deep postanal space. The supralevator spaces lie above the levator ani on either side of the rectum and commu-nicate posteriorly. The anatomy of these spaces influences the location and spread of cryptoglandular infection (Fig. 29-35).As an abscess enlarges, it spreads in one of several direc-tions. A perianal abscess is the most common manifestation and appears as a painful swelling at the anal verge. Spread through the external sphincter below the level of the puborectalis pro-duces an ischiorectal abscess. These abscesses may become extremely large and may not be visible in the perianal region. ClosureInternal sphincterincisedExternalsphincter m.Internalsphincter m.FissureFissure-in-anoADCBFigure 29-33. A through D. Open lateral internal sphincterotomy for fissure in ano. m = muscle.Brunicardi_Ch29_p1259-p1330.indd 131323/02/19 2:29 PM 1314SPECIFIC CONSIDERATIONSPART IIDigital

1	29-33. A through D. Open lateral internal sphincterotomy for fissure in ano. m = muscle.Brunicardi_Ch29_p1259-p1330.indd 131323/02/19 2:29 PM 1314SPECIFIC CONSIDERATIONSPART IIDigital rectal exam will reveal a painful swelling laterally in the ischiorectal fossa. Intersphincteric abscesses occur in the inter-sphincteric space and are notoriously difficult to diagnose, often requiring an examination under anesthesia. Pelvic and supralevator abscesses are uncommon and may result from extension of an intersphincteric or ischiorectal abscess upward or extension of an intraperitoneal abscess downward (Fig. 29-36).Diagnosis. Severe anal pain is the most common presenting complaint. A palpable mass is often detected by inspection of the perianal area or by digital rectal examination. Occasion-ally, patients will present with fever, urinary retention, or lifethreatening sepsis. The diagnosis of a perianal or ischiorectal abscess can usually be made with physical exam alone (either in the

1	patients will present with fever, urinary retention, or lifethreatening sepsis. The diagnosis of a perianal or ischiorectal abscess can usually be made with physical exam alone (either in the office or in the operating room). However, complex or atypical presentations may require imaging studies such as CT or MRI to fully delineate the anatomy of the abscess.Treatment. Anorectal abscesses should be treated by drain-age as soon as the diagnosis is established. If the diagnosis is in question, an examination and drainage under anesthesia are often the most expeditious ways both to confirm the diagnosis and to treat the problem. Delayed or inadequate treatment may occasionally cause extensive and life-threatening suppuration with massive tissue necrosis and septicemia. Antibiotics are only indicated if there is extensive overlying cellulitis or if the patient is immunocompromised, has diabetes mellitus, or has valvular heart disease. Antibiotics alone are ineffective at treat-ing

1	only indicated if there is extensive overlying cellulitis or if the patient is immunocompromised, has diabetes mellitus, or has valvular heart disease. Antibiotics alone are ineffective at treat-ing perianal or perirectal infection.Perianal AbscessMost perianal abscesses can be drained under local anesthesia in the office, clinic, or emergency department. Larger, more com-plicated abscesses may require drainage in the operating room. A skin incision is created, and a disk of skin excised to prevent premature closure. No packing is necessary, and sitz baths are started the next day (Fig. 29-37).Ischiorectal AbscessAn ischiorectal abscess causes diffuse swelling in the ischiorectal fossa that may involve one or both sides, forming a “horseshoe” abscess. Simple ischiorectal abscesses are drained through an incision in the overlying skin. Horseshoe abscesses require drainage of the deep postanal space and often require counterin-cisions over one or both ischiorectal spaces (Fig.

1	are drained through an incision in the overlying skin. Horseshoe abscesses require drainage of the deep postanal space and often require counterin-cisions over one or both ischiorectal spaces (Fig. 29-38).Intersphincteric AbscessIntersphincteric abscesses are notoriously difficult to diagnose because they produce little swelling and few perianal signs of infection. Pain is typically described as being deep and “up inside” the anal area and is usually exacerbated by coughing or sneezing. The pain is so intense that it usually precludes a digital rectal examination. The diagnosis is made based on a high index of suspicion and usually requires an examination Anal fissureSentinel pileADCBFigure 29-34. A through D. Closed lateral internal sphincterotomy for fissure in ano.Brunicardi_Ch29_p1259-p1330.indd 131423/02/19 2:29 PM 1315COLON, RECTUM, AND ANUSCHAPTER 29Levator ani m.Internal sphincter m.Puborectalis anddeep external sphincter m.Superficial externalsphincter

1	131423/02/19 2:29 PM 1315COLON, RECTUM, AND ANUSCHAPTER 29Levator ani m.Internal sphincter m.Puborectalis anddeep external sphincter m.Superficial externalsphincter m.Subcutaneousexternal sphincter m.PeritoneumUreterSupralevator spaceIschiorectal spacePerianal spacePeritoneumRectorectal spaceRectosacral fasciaSupralevator spaceLevator ani m.Deep postanal spaceSuperficialpostanal spaceABFigure 29-35. Anatomy of perianorectal spaces. (A) Anterior view and (B) lateral view. m = muscle.Internalsphincter m.Perianal spaceabscessLevatorani m.Externalsphincter m.Longitudinal m.Pelvirectal spaceabscessIntersphincteric spaceabscessIschiorectal fossaabscessABFigure 29-36. A and B. Pathways of anorectal infection in perianal spaces. m = muscle.Brunicardi_Ch29_p1259-p1330.indd 131523/02/19 2:29 PM 1316SPECIFIC CONSIDERATIONSPART IIunder anesthesia. Once identified, an intersphincteric abscess can be drained through a limited, usually posterior, internal sphincterotomy.Supralevator

1	2:29 PM 1316SPECIFIC CONSIDERATIONSPART IIunder anesthesia. Once identified, an intersphincteric abscess can be drained through a limited, usually posterior, internal sphincterotomy.Supralevator AbscessThis type of abscess is uncommon and can be difficult to diag-nose. Because of its proximity to the peritoneal cavity, supral-evator abscesses can mimic intra-abdominal conditions. Digital rectal examination may reveal an indurated, bulging mass above the anorectal ring. It is essential to identify the origin of a supralevator abscess prior to treatment. If the abscess is sec-ondary to an upward extension of an intersphincteric abscess, it should be drained through the rectum. If it is drained through the ischiorectal fossa, a complicated, suprasphincteric fistula may result. If a supralevator abscess arises from the upward extension of an ischiorectal abscess, it should be drained through the ischiorectal fossa. Drainage of this type of abscess through the rectum may result in an

1	abscess arises from the upward extension of an ischiorectal abscess, it should be drained through the ischiorectal fossa. Drainage of this type of abscess through the rectum may result in an extrasphincteric fistula. If the abscess is secondary to intra-abdominal disease, the pri-mary process requires treatment and the abscess is drained via the most direct route (transabdominally, rectally, or through the ischiorectal fossa).Perianal Sepsis in the Immunocompromised PatientThe immunocompromised patient with perianal pain presents a diagnostic dilemma. Because of leukopenia, these patients may develop serious perianal infection without any of the cardinal signs of inflammation. While broad-spectrum antibiotics may cure some of these patients, an exam under anesthesia should not be delayed because of neutropenia. An increase in pain or fever and/or clinical deterioration mandates an exam under anesthesia. Any indurated area should be incised and drained, biopsied to exclude a leukemic

1	because of neutropenia. An increase in pain or fever and/or clinical deterioration mandates an exam under anesthesia. Any indurated area should be incised and drained, biopsied to exclude a leukemic infiltrate, and cultured to aid in the selection of antimicrobial agents.165Necrotizing Soft Tissue Infection of the PerineumNecrotizing soft tissue infection of the perineum is a rare, but lethal, condition. Most of these infections are polymicrobial and synergistic. The source of sepsis is commonly an undrained or inadequately drained cryptoglandular abscess or a urogenital infection. Occasionally, these infections may be encountered postoperatively (e.g., after hemorrhoidectomy). Immunocom-promised patients and diabetic patients are at increased risk.Physical examination may reveal necrotic skin, bullae, or crepitus. Patients often have signs of systemic toxicity and may be hemodynamically unstable. A high index of suspicion is necessary because perineal signs of severe infection may be

1	skin, bullae, or crepitus. Patients often have signs of systemic toxicity and may be hemodynamically unstable. A high index of suspicion is necessary because perineal signs of severe infection may be minimal and prompt surgical intervention can be lifesaving.Surgical debridement of all nonviable tissue is required to treat all necrotizing soft tissue infections. Multiple operations may be necessary to ensure that all necrotic tissue has been resected. Broad-spectrum antibiotics are frequently employed, but adequate surgical debridement remains the mainstay of ther-apy. Colostomy may be required if extensive resection of the sphincter is required or if stool contamination of the perineum makes wound management difficult. Despite early recognition and adequate surgical therapy, the mortality of necrotizing peri-neal soft tissue infections remains approximately 50%.ABCFigure 29-37. A through C. Technique of drainage of perianal abscess.Figure 29-38. Drainage of horseshoe abscess. The

1	of necrotizing peri-neal soft tissue infections remains approximately 50%.ABCFigure 29-37. A through C. Technique of drainage of perianal abscess.Figure 29-38. Drainage of horseshoe abscess. The deep postanal space is entered, incising the anococcygeal ligament. Counter drain-age incisions are made for each limb of the ischiorectal space.Brunicardi_Ch29_p1259-p1330.indd 131623/02/19 2:30 PM 1317COLON, RECTUM, AND ANUSCHAPTER 29surgical treatment is dictated by the location of the internal and external openings and the course of the fistula. The exter-nal opening is usually visible as a red elevation of granu-lation tissue with or without concurrent drainage. The internal opening may be more difficult to identify. Injection of hydrogen peroxide or dilute methylene blue may be helpful. Care must be taken to avoid creating an artificial internal opening (thus often converting a simple fistula into a complex fistula).Simple intersphincteric fistulas can often be treated by

1	helpful. Care must be taken to avoid creating an artificial internal opening (thus often converting a simple fistula into a complex fistula).Simple intersphincteric fistulas can often be treated by fistulotomy (opening the fistulous tract), curettage, and healing by secondary intention (see Fig. 29-40A). “Horseshoe” fistulas usu-ally have an internal opening in the posterior midline and extend anteriorly and laterally to one or both ischiorectal spaces by way of the deep postanal space. Treatment of a transsphincteric fistula depends on its location in the sphincter complex. Fistulas that include less than 30% of the sphincter muscles can often be treated by sphincterotomy without significant risk of major incontinence (see Fig. 29-40B). High transsphincteric fistulas, which encircle a greater amount of muscle, are more safely treated by initial place-ment of a seton. Similarly, suprasphincteric fistulas are usually treated with seton placement (see Fig. 29-40C). Extrasphincteric

1	a greater amount of muscle, are more safely treated by initial place-ment of a seton. Similarly, suprasphincteric fistulas are usually treated with seton placement (see Fig. 29-40C). Extrasphincteric fistulas are rare, and treatment depends on both the anatomy of the fistula and its etiology. In general, the portion of the fistula outside the sphincter should be opened and drained. A primary tract at the level of the dentate line may also be opened if pres-ent. Complex fistulas with multiple tracts may require numerous procedures to control sepsis and facilitate healing. Liberal use of drains and setons is helpful. Failure to heal may ultimately require fecal diversion (see Fig. 29-40D). Complex and/or non-healing fistulas may result from Crohn’s disease, malignancy, radiation proctitis, or unusual infection. Proctoscopy should be performed in all cases of complex and/or nonhealing fistulas to assess the health of the rectal mucosa. Biopsies of the fistula tract should be taken to

1	or unusual infection. Proctoscopy should be performed in all cases of complex and/or nonhealing fistulas to assess the health of the rectal mucosa. Biopsies of the fistula tract should be taken to rule out malignancy.A seton is a drain placed through a fistula to maintain drain-age and/or induce fibrosis. Cutting setons consist of a suture or a rubber band that is placed through the fistula and intermittently tightened in the office. Tightening the seton results in fibrosis and gradual division of the sphincter, thus eliminating the fis-tula while maintaining continuity of the sphincter. A noncutting seton is a soft plastic drain (often a vessel loop) placed in the fistula to maintain drainage. The fistula tract may subsequently be laid open with less risk of incontinence because scarring pre-vents retraction of the sphincter. Alternatively, the seton may be left in place for chronic drainage. Higher fistulas may be treated by an endorectal advancement flap. Fibrin glue and a variety

1	pre-vents retraction of the sphincter. Alternatively, the seton may be left in place for chronic drainage. Higher fistulas may be treated by an endorectal advancement flap. Fibrin glue and a variety of collagen-based plugs also have been used to treat persistent fistulas with variable results. A more recent technique, ligation of the intersphincteric fistula tract (LIFT), also shows promise. In this procedure, the fistula is identified in the intersphincteric plane (usually by placement of a lacrimal probe), divided, and the two ends ligated. Early reports have shown success with this technique, but long-term outcome is not yet known.166-168Rectovaginal FistulaA rectovaginal fistula is a connection between the vagina and the rectum or anal canal proximal to the dentate line. Recto-vaginal fistulas are classified as low (rectal opening close to the dentate line and vaginal opening in the fourchette), middle (vaginal opening between the fourchette and cervix), or high (vaginal opening

1	fistulas are classified as low (rectal opening close to the dentate line and vaginal opening in the fourchette), middle (vaginal opening between the fourchette and cervix), or high (vaginal opening near the cervix). Low rectovaginal fistulas are 10Posterior(curved tracts)Transverse Anal LineAnterior(straight tracts)3 cmExceptionFigure 29-39. Goodsall’s rule to identify the internal opening of fistulas in ano.Fistula In AnoDrainage of an anorectal abscess results in cure for about 50% of patients. The remaining 50% develop a persistent fistula in ano. The fistula usually originates in the infected crypt (internal opening) and tracks to the external opening, usually the site of prior drainage. The course of the fistula can often be predicted by the anatomy of the previous abscess.While the majority of fistulas are cryptoglandular in ori-gin, trauma, Crohn’s disease, malignancy, radiation, or unusual infections (tuberculosis, actinomycosis, and chlamydia) may also produce

1	the majority of fistulas are cryptoglandular in ori-gin, trauma, Crohn’s disease, malignancy, radiation, or unusual infections (tuberculosis, actinomycosis, and chlamydia) may also produce fistulas. A complex, recurrent, or nonhealing fis-tula should raise the suspicion of one of these diagnoses.Diagnosis. Patients present with persistent drainage from the internal and/or external openings. An indurated tract is often palpable. Although the external opening is often easily identifi-able, identification of the internal opening may be more chal-lenging. Goodsall’s rule can be used as a guide in determining the location of the internal opening (Fig. 29-39). In general, fis-tulas with an external opening anteriorly connect to the internal opening by a short, radial tract. Fistulas with an external open-ing posteriorly track in a curvilinear fashion to the posterior midline. However, exceptions to this rule often occur if an ante-rior external opening is greater than 3 cm from the anal

1	external open-ing posteriorly track in a curvilinear fashion to the posterior midline. However, exceptions to this rule often occur if an ante-rior external opening is greater than 3 cm from the anal margin. Such fistulas usually track to the posterior midline.Fistulas are categorized based on their relationship to the anal sphincter complex, and treatment options are based on these classifications. An intersphincteric fistula tracks through the distal internal sphincter and intersphincteric space to an external opening near the anal verge (Fig. 29-40A). A trans-sphincteric fistula often results from an ischiorectal abscess and extends through both the internal and external sphincters (Fig. 29-40B). A suprasphincteric fistula originates in the inter-sphincteric plane and tracks up and around the entire external sphincter (Fig. 29-40C). An extrasphincteric fistula originates in the rectal wall and tracks around both sphincters to exit later-ally, usually in the ischiorectal fossa (Fig.

1	the entire external sphincter (Fig. 29-40C). An extrasphincteric fistula originates in the rectal wall and tracks around both sphincters to exit later-ally, usually in the ischiorectal fossa (Fig. 29-40D).Treatment. The goal of treatment of fistula in ano is eradica-tion of sepsis without sacrificing continence. Because fistulous tracks encircle variable amounts of the sphincter complex, Brunicardi_Ch29_p1259-p1330.indd 131723/02/19 2:30 PM 1318SPECIFIC CONSIDERATIONSPART IIcommonly caused by obstetric injuries or trauma from a foreign body. Mid-rectovaginal fistulas may result from more severe obstetric injury, but they also occur after surgical resection of a mid-rectal neoplasm, radiation injury, or extension of an und-rained abscess. High rectovaginal fistulas result from operative or radiation injury. Complicated diverticulitis may cause a colo-vaginal fistula. Crohn’s disease can cause rectovaginal fistulas at all levels, as well as colovaginal and enterovaginal

1	operative or radiation injury. Complicated diverticulitis may cause a colo-vaginal fistula. Crohn’s disease can cause rectovaginal fistulas at all levels, as well as colovaginal and enterovaginal fistulas.Diagnosis. Patients describe symptoms varying from the sen-sation of passing flatus from the vagina to the passage of solid stool from the vagina. Most patients experience some degree of fecal incontinence. Contamination may result in vaginitis. Large fistulas may be obvious on anoscopic and/or vaginal speculum examination, but smaller fistulas may be difficult to locate. Occasionally, a barium enema or vaginogram may identify these fistulas. Endorectal ultrasound may also be useful. With the patient in the prone position, installation of methylene blue into the rectum while a tampon is in the vagina may confirm the presence of a small fistula.Treatment. The treatment of rectovaginal fistula depends on the size, location, etiology, and condition of surrounding tis-sues. Because up to

1	the vagina may confirm the presence of a small fistula.Treatment. The treatment of rectovaginal fistula depends on the size, location, etiology, and condition of surrounding tis-sues. Because up to 50% of fistulas caused by obstetric injury heal spontaneously, it is prudent to wait 3 to 6 months before embarking on surgical repair in these patients. If the fistula was caused by a cryptoglandular abscess, drainage of the abscess may allow spontaneous closure.Low and mid-rectovaginal fistulas are usually best treated with an endorectal advancement flap. The principle of this procedure is based on the advancement of healthy mucosa, submucosa, and circular muscle over the rectal opening (the high-pressure side of the fistula) to promote healing (Fig. 29-41). If a sphincter injury is present, an overlapping sphincteroplasty ABCDFigure 29-40. The four major categories of fistula in ano (left side of drawings) and the usual operative procedure to correct the fistula (right side of drawings).

1	overlapping sphincteroplasty ABCDFigure 29-40. The four major categories of fistula in ano (left side of drawings) and the usual operative procedure to correct the fistula (right side of drawings). A. Intersphincteric fistula with simple low tract. B. Uncomplicated transsphincteric fistula. C. Uncomplicated suprasphinc-teric fistula. D. Extrasphincteric fistula secondary to anal fistula.Brunicardi_Ch29_p1259-p1330.indd 131823/02/19 2:30 PM 1319COLON, RECTUM, AND ANUSCHAPTER 29should be performed concurrently. Fecal diversion is rarely required. High rectovaginal, colovaginal, and enterovaginal fis-tulas are usually best treated via a transabdominal approach. The diseased tissue, which caused the fistula (upper rectum, sigmoid colon, or small bowel), is resected and the hole in the vagina closed. Healthy tissue, such as omentum or muscle, frequently is interposed between the bowel anastomosis and the vagina to prevent recurrence.Rectovaginal fistulas caused by Crohn’s disease,

1	vagina closed. Healthy tissue, such as omentum or muscle, frequently is interposed between the bowel anastomosis and the vagina to prevent recurrence.Rectovaginal fistulas caused by Crohn’s disease, radia-tion injury, or malignancy almost never heal spontaneously. In Crohn’s disease, treatment is based on adequate drainage of perianal sepsis and nutritional support. An endorectal advance-ment flap may be performed if the rectum is spared from active Crohn’s disease. Fistulas resulting from radiation damage are not amenable to local repair with an advancement flap because of damage to the surrounding rectal and vaginal tissues. Such midand high rectovaginal fistulas are occasionally repaired successfully with a transabdominal approach in which healthy tissue (omentum, muscle, or nonradiated bowel) is interposed between the damaged rectum and vagina. Fistulas caused by malignancy should be treated with resection of the tumor. Because differentiating radiation damage from malignancy can

1	bowel) is interposed between the damaged rectum and vagina. Fistulas caused by malignancy should be treated with resection of the tumor. Because differentiating radiation damage from malignancy can be extremely difficult, all fistulas resulting from radiation should be biopsied to rule out the presence of cancer.Perianal DermatitisPruritus Ani. Pruritus ani (severe perianal itching) is a com-mon problem with a multitude of etiologies. Surgically cor-rectable (anatomic) causes include prolapsing hemorrhoids, ectropion, fissure, fistula, and neoplasms. Perianal infection may also present with pruritus ani. Infections may be caused by fungus (Candida species and Epidermophyton organisms), parasites (Enterobius vermicularis [pinworms], Pediculus pubis [a louse], and Sarcoptes scabiei [scabies]), bacteria Probe inrectovaginalfistulaRectal mucosa andinternal sphinctermuscle incisedAttenuatedrectovaginalseptumExcess flap ofmucosa excisedInternal sphinctermuscle mobilizedInternal

1	[scabies]), bacteria Probe inrectovaginalfistulaRectal mucosa andinternal sphinctermuscle incisedAttenuatedrectovaginalseptumExcess flap ofmucosa excisedInternal sphinctermuscle mobilizedInternal sphinctermuscle approximatedFlap of mucosaand internalsphincter muscleFigure 29-41. Endorectal advancement flap for rectovaginal fistula. (Reproduced with permission from Gordon PH, Nivatvongs S: Principles and Practice of Surgery for the Colon, Rectum, and Anus, 2nd ed. New York, NY: Marcel Dekker, Inc; 1999.)Brunicardi_Ch29_p1259-p1330.indd 131923/02/19 2:30 PM 1320SPECIFIC CONSIDERATIONSPART II(Corynebacterium minutissimum [erythrasma] and T pallidum [syphilis]), or viruses (HPV [condyloma acuminata]). Antibi-otic use may also cause itching, usually by precipitating fungal infection. Noninfectious dermatologic causes include seborrhea, psoriasis, and contact dermatitis. Contact dermatitis can be par-ticularly troublesome because many over-the-counter topical agents used by patients to

1	dermatologic causes include seborrhea, psoriasis, and contact dermatitis. Contact dermatitis can be par-ticularly troublesome because many over-the-counter topical agents used by patients to relieve itching may exacerbate the problem. Occasionally, systemic diseases such as jaundice and diabetes may present with pruritus ani.Despite the myriad of causes, the majority of pruritus ani is idiopathic and probably related to local hygiene, neurogenic, or psychogenic causes. Treatment focuses on removal of irritants, improving perianal hygiene, dietary adjustments, and avoiding scratching. Biopsy and/or culture may be required to rule out an infectious or dermatologic cause. Hydrocortisone ointment 0.5% to 1.0% can provide symptomatic relief but should not be used for prolonged periods of time because of the risk of dermal atrophy. Skin barriers such as Calmoseptine can also provide relief. Systemic antihistamines or tricyclic antidepressants have also been used with some

1	periods of time because of the risk of dermal atrophy. Skin barriers such as Calmoseptine can also provide relief. Systemic antihistamines or tricyclic antidepressants have also been used with some success.Nonpruritic Lesions. Several perianal skin conditions may present with perianal skin changes. Leprosy, amebiasis, actino-mycosis, and lymphogranuloma venereum produce characteris-tic perianal lesions. Neoplasms such as squamous intraepithelial lesions, Paget’s disease, and invasive carcinomas may also appear first in the perianal skin. Biopsy can usually distinguish these diagnoses.Sexually Transmitted DiseasesBacterial Infections. Proctitis is a common symptom of ano-rectal bacterial infection. Neisseria gonorrhoeae is the most common bacterial cause of proctitis and causes pain, tenes-mus, rectal bleeding, and mucus discharge. Chlamydia tracho-matis infection may be asymptomatic or may produce similar symptoms. Treponema pallidum, the microbe causing syphilis, causes a chancre at

1	rectal bleeding, and mucus discharge. Chlamydia tracho-matis infection may be asymptomatic or may produce similar symptoms. Treponema pallidum, the microbe causing syphilis, causes a chancre at the site of inoculation, which may be asymp-tomatic or may present as an atypical fissure (primary syphilis). Condyloma lata are characteristic of secondary syphilis. Chan-croid, caused by Haemophilus ducreyi, is a disease manifested by multiple painful, bleeding lesions. Inguinal lymphadenopathy and fluctuant, draining lymph nodes are characteristic. Donova-nia granulomatis infection produces shiny, red masses on the perineum (granuloma inguinale). Diarrheal illnesses caused by organisms such as Campylobacter or Shigella may also be sexually transmitted. Treatment consists of antimicrobial agents directed against the infecting organism.Parasitic Infections. Entamoeba histolytica is an increas-ingly common sexually transmitted disease. Amebas produce ulcerations in the gastrointestinal mucosa

1	directed against the infecting organism.Parasitic Infections. Entamoeba histolytica is an increas-ingly common sexually transmitted disease. Amebas produce ulcerations in the gastrointestinal mucosa and can infect any part of the gut. Symptoms include diarrhea, abdominal pain, and tenesmus. Giardia lamblia is also common and produces diarrhea, abdominal pain, and malaise.Viral Infections Herpes Simplex Virus Herpes proctitis is extremely common. Proctitis is usually caused by type 2 herpes simplex virus and less commonly by type 1 herpes simplex virus. Patients complain of severe, intractable perianal pain and tenesmus. Pain often precedes the development of characteristic vesicles, and these patients may require an examination under anesthesia to exclude another diagnosis such as an intersphincteric abscess. Diagnosis is confirmed by viral culture of tissue or vesicular fluid.Human Papillomavirus HPV causes condyloma acuminata (anogenital warts) and is associated with squamous

1	intersphincteric abscess. Diagnosis is confirmed by viral culture of tissue or vesicular fluid.Human Papillomavirus HPV causes condyloma acuminata (anogenital warts) and is associated with squamous intraepithe-lial lesions and squamous cell carcinoma (see previous section, “Anal Canal and Perianal Tumors”). Condylomas occur in the perianal area or in the squamous epithelium of the anal canal. Occasionally, the mucosa of the lower rectum may be affected. There are approximately 30 serotypes of HPV. As previously mentioned, HPV types 16 and 18, in particular, appear to pre-dispose to malignancy and often cause flat dysplasia in skin unaffected by warts. In contrast, HPV types 6 and 11 commonly cause warts, but do not appear to cause malignant degeneration.Treatment of anal condyloma depends on the location and extent of disease. Small warts on the perianal skin and distal anal canal may be treated in the office with topical application of bichloracetic acid or podophyllin. Although 60%

1	on the location and extent of disease. Small warts on the perianal skin and distal anal canal may be treated in the office with topical application of bichloracetic acid or podophyllin. Although 60% to 80% of patients will respond to these agents, recurrence and reinfection are common. Imiquimod (Aldara) is an immunomodulator that was recently introduced for topical treatment of several viral infections, including anogenital condyloma.100 Initial reports suggest that this agent is highly effective in treating condyloma located on the perianal skin and distal anal canal. Larger and/or more numerous warts require excision and/or fulguration in the operating room. Excised warts should be sent for pathologic examination to rule out dysplasia or malignancy. It is impor-tant to note that prior use of podophyllin may induce histologic changes that mimic dysplasia. The recent introduction of a vaccine against HPV holds promise for preventing anogenital condylomas.169,170Human Immunodeficiency

1	of podophyllin may induce histologic changes that mimic dysplasia. The recent introduction of a vaccine against HPV holds promise for preventing anogenital condylomas.169,170Human Immunodeficiency Virus See later section, “The Immunocompromised Patient.”Pilonidal DiseasePilonidal disease (cyst, infection) consists of a hair-containing sinus or abscess occurring in the intergluteal cleft. Although the etiology is unknown, it is speculated that the cleft creates a suction that draws hair into the midline pits when a patient sits. These ingrown hairs may then become infected and pres-ent acutely as an abscess in the sacrococcygeal region. Once an acute episode has resolved, recurrence is common.An acute abscess should be incised and drained as soon as the diagnosis is made. Because these abscesses are usually very superficial, this procedure can often be performed in the office, clinic, or emergency department under local anesthetic. Because midline wounds in the region heal poorly, some

1	are usually very superficial, this procedure can often be performed in the office, clinic, or emergency department under local anesthetic. Because midline wounds in the region heal poorly, some sur-geons recommend using an incision lateral to the intergluteal cleft. A number of procedures have been proposed to treat a chronic pilonidal sinus. The simplest method involves unroof-ing the tract, curetting the base, and marsupializing the wound. The wound must then be kept clean and free of hair until heal-ing is complete (often requiring weekly office visits for wound care). Alternatively, a small lateral incision can be created and the pit excised. This method is effective for most primary pilo-nidal sinuses. In general, extensive resection should be avoided. Complex and/or recurrent sinus tracts may require more exten-sive resection and closure with a Z-plasty, advancement flap, or rotational flap.Hidradenitis SuppurativaHidradenitis suppurativa is an infection of the cutaneous

1	sinus tracts may require more exten-sive resection and closure with a Z-plasty, advancement flap, or rotational flap.Hidradenitis SuppurativaHidradenitis suppurativa is an infection of the cutaneous apo-crine sweat glands. Infected glands rupture and form subcutane-ous sinus tracts. The infection may mimic complex anal fistula disease, but stops at the anal verge because there are no apocrine Brunicardi_Ch29_p1259-p1330.indd 132023/02/19 2:30 PM 1321COLON, RECTUM, AND ANUSCHAPTER 29glands in the anal canal. Treatment involves incision and drain-age of acute abscesses and unroofing of all chronically inflamed fistulas and debridement of granulation tissue. Radical excision and skin grafting are almost never necessary.TRAUMAPenetrating Colorectal InjuryColorectal injury is common following penetrating trauma to the abdomen and has historically been associated with high mortality. In the first half of the 20th century, the mortality rate from colorectal injury was as high as 90%. The

1	penetrating trauma to the abdomen and has historically been associated with high mortality. In the first half of the 20th century, the mortality rate from colorectal injury was as high as 90%. The introduction of exteriorization of colonic injuries and fecal diversion during World War II dramatically decreased mortality, and this princi-ple has governed the management of large bowel injury for over 50 years. Recently, however, this practice was challenged, and trauma surgeons are increasingly performing primary repairs in selected patients.Management of colonic injury depends on the mechanism of injury, the delay between the injury and surgery, the over-all condition and stability of the patient, the degree of perito-neal contamination, and the condition of the injured colon. A primary repair may be considered in hemodynamically stable patients with few additional injuries and minimal contamination if the colon appears otherwise healthy. Contraindications to pri-mary repair include

1	repair may be considered in hemodynamically stable patients with few additional injuries and minimal contamination if the colon appears otherwise healthy. Contraindications to pri-mary repair include shock, injury to more than two other organs, mesenteric vascular damage, and extensive fecal contamina-tion. A delay of greater than 6 hours between the injury and the operation also is associated with increased morbidity and mor-tality and is a relative contraindication to primary repair. Inju-ries caused by high-velocity gunshot wounds or blast injuries are often associated with multiple intra-abdominal injuries and tissue loss and therefore are usually treated by fecal diversion after debridement of all nonviable tissue. Patient factors, such as medical comorbidities, advanced age, and the presence of tumor or radiation injury, must also be considered (Table 29-4).Like injuries to the intraperitoneal colon, penetrating trauma to the rectum traditionally has been associated with high

1	presence of tumor or radiation injury, must also be considered (Table 29-4).Like injuries to the intraperitoneal colon, penetrating trauma to the rectum traditionally has been associated with high morbidity and mortality. Primary repair of the rectum is more difficult than primary repair of the colon, however, and most rectal injuries are associated with significant contamination. For that reason, the majority of penetrating rectal injuries should be treated with proximal fecal diversion. Distal washout (copious irrigation of the rectum) and presacral drains are not routinely recommended.158 Small, clean rectal injuries may be closed pri-marily without fecal diversion in an otherwise stable patient. Intractable rectal bleeding may require angiographic emboliza-tion. Very rarely, hemorrhage or extensive tissue loss (espe-cially if the anal sphincter is severely damaged) may require an emergent APR. However, this operation should be avoided, if at all possible, because of the morbidity

1	or extensive tissue loss (espe-cially if the anal sphincter is severely damaged) may require an emergent APR. However, this operation should be avoided, if at all possible, because of the morbidity associated with an extensive pelvic dissection in a severely injured patient.171Blunt Colorectal InjuryBlunt injury to the colon and rectum is considerably less com-mon than penetrating injury. Nevertheless, blunt trauma can cause colon perforation, and shear injury to the mesentery can devascularize the intestine. Management of these injuries should follow the same principles outlined for management of penetrat-ing injuries. Small perforations with little contamination in a stable patient may be closed primarily; more extensive injury requires fecal diversion. A serosal hematoma alone does not Table 29-4Criteria for use of an ostomyInjuring agent factors High-velocity bullet wounds Shotgun wounds Explosive blast wounds Crush injuryPatient factors Presence of tumor Radiated tissue Medical

1	Table 29-4Criteria for use of an ostomyInjuring agent factors High-velocity bullet wounds Shotgun wounds Explosive blast wounds Crush injuryPatient factors Presence of tumor Radiated tissue Medical condition Advanced ageInjury factors Inflamed tissue Advanced infection Distal obstruction Local foreign body Impaired blood supply Mesenteric vascular damage Shock with blood pressure <80/60 mmHg Hemorrhage >1000 mL More than two organs (especially kidney) injured Interval to operation >6 h (pancreatic, splenic, hepatic) Extensive injury requiring resection Major abdominal wall loss Thoracoabdominal penetrationReproduced with permission from Gordon PH, Nivatvongs S: Principles and Practice of Surgery for the Colon, Rectum, and Anus, 2nd ed. New York, NY: Marcel Dekker, Inc; 1999.mandate resection, but the bowel should be carefully inspected to ensure that there is not an associated perforation or significant bowel ischemia.Blunt injury to the rectum may result from significant trauma,

1	but the bowel should be carefully inspected to ensure that there is not an associated perforation or significant bowel ischemia.Blunt injury to the rectum may result from significant trauma, such as a pelvic crush injury, or may result from local trauma caused by an enema or foreign body. Crush injuries, especially with an associated pelvic fracture, are often associ-ated with significant rectal damage and contamination. These patients require debridement of all nonviable tissue, proximal fecal diversion, and a distal rectal washout, with or without drain placement. Blunt trauma from an enema or foreign body may produce a mucosal hematoma, which requires no surgical treatment if the mucosa is intact. Small mucosal tears may be closed primarily if the bowel is relatively clean and there is little contamination.Iatrogenic InjuryIntraoperative Injury. The colon and rectum are at risk for inadvertent injury during other procedures, especially during pelvic operations. The key to managing

1	little contamination.Iatrogenic InjuryIntraoperative Injury. The colon and rectum are at risk for inadvertent injury during other procedures, especially during pelvic operations. The key to managing these injuries is early recognition. The vast majority of iatrogenic colorectal injuries may be closed primarily if there is little contamination and if the patient is otherwise stable. Delayed recognition of colorectal injuries may result in significant peritonitis and life-threatening sepsis. In these cases, fecal diversion is almost always required, and the patient may need repeated exploration for drainage of abscesses.Injury From Barium Enema. Colorectal injury from a bar-ium enema is an extremely rare complication associated with a high rate of morbidity and mortality. Perforation with spillage Brunicardi_Ch29_p1259-p1330.indd 132123/02/19 2:30 PM 1322SPECIFIC CONSIDERATIONSPART IIof barium, especially above the peritoneal reflection, may result in profound peritonitis, sepsis,

1	spillage Brunicardi_Ch29_p1259-p1330.indd 132123/02/19 2:30 PM 1322SPECIFIC CONSIDERATIONSPART IIof barium, especially above the peritoneal reflection, may result in profound peritonitis, sepsis, and a systemic inflam-matory response. If the perforation is recognized early, it may be closed primarily and the abdomen irrigated to remove stool and barium. However, if the patient has developed sepsis, fecal diversion (with or without bowel resection) is almost always required. Rarely, a small mucosal injury to the extraperitoneal rectum may be managed with bowel rest, broad-spectrum anti-biotics, and close observation.Colonoscopic Perforation. Perforation is the most common major complication after either diagnostic or therapeutic colo-noscopy. Fortunately, this complication is rare and occurs in less than 1% of procedures. Perforation may result from trauma from the tip of the instrument, from shear forces related to the formation of a “loop” in the colonoscope, or from barotrauma

1	occurs in less than 1% of procedures. Perforation may result from trauma from the tip of the instrument, from shear forces related to the formation of a “loop” in the colonoscope, or from barotrauma from insufflation. Biopsy or fulguration can also cause perfo-ration. Polypectomy using electrocautery may produce a full-thickness burn, resulting in postpolypectomy syndrome in which a patient develops abdominal pain, fever, and leukocytosis with-out evidence of diffuse peritonitis.Management of colonoscopic perforation depends on the size of the perforation, the duration of time since the injury, the overall condition of the patient, and the underlying diagnosis. A large perforation recognized during the procedure requires surgical exploration. Because the bowel has almost always been prepared prior to the colonoscopy, there is usually little contamination associated with these injuries, and most can be repaired primarily. If there is significant contamination, if there has been a delay

1	prior to the colonoscopy, there is usually little contamination associated with these injuries, and most can be repaired primarily. If there is significant contamination, if there has been a delay in diagnosis with resulting peritonitis, or if the patient is hemodynamically unstable, proximal diversion with or without resection is the safest approach. It is also important to know the indication for and findings at the time of colonos-copy. If the patient has an underlying neoplasm and is stable, definitive resection is best. Occasionally, a patient will develop abdominal pain and localized signs of perforation after what was thought to be an uneventful colonoscopy. Many of these patients will have a “microperforation,” which will resolve with bowel rest, broad-spectrum antibiotics, and close observation. Evidence of peritonitis or any deterioration in clinical condition mandates exploration. Similarly, free retroperitoneal or intra-peritoneal air may be discovered incidentally after

1	observation. Evidence of peritonitis or any deterioration in clinical condition mandates exploration. Similarly, free retroperitoneal or intra-peritoneal air may be discovered incidentally after colonoscopy. In a completely asymptomatic patient, this finding is thought to result from barotrauma and dissection of air through tissue planes without a free perforation. Many of these patients can be successfully treated with bowel rest and broad-spectrum antibi-otics. Surgical exploration is indicated if any clinical deteriora-tion occurs.Anal Sphincter Injury and IncontinenceThe most common cause of anal sphincter injury is obstetric trauma during vaginal delivery. The risk of sphincter injury is increased by a laceration that extends into the rectum (fourth-degree tear), infection of an episiotomy or laceration repair, prolonged labor, and possibly by use of a midline episiotomy. Sphincter damage may also result from hemorrhoidectomy, sphincterotomy, abscess drainage, or fistulotomy.

1	or laceration repair, prolonged labor, and possibly by use of a midline episiotomy. Sphincter damage may also result from hemorrhoidectomy, sphincterotomy, abscess drainage, or fistulotomy. Patients with incontinence and a suspected sphincter injury can be evaluated with anal manometry, EMG, pudendal nerve motor latency, and endoanal ultrasound. Mild incontinence, even in the presence of a sphincter defect, may respond to dietary changes and/or biofeedback. More severe incontinence may require surgical repair.The anal sphincter can also be injured by penetrating or blunt mechanisms (impalement, blast injury, crush injuries of the pelvis). Because damage to the anal sphincter is not life-threatening, definitive repair of the sphincter is often deferred until other injuries have been repaired and the patient’s clinical condition is stable. Isolated sphincter injuries that do not involve the rectum may be repaired primarily. Rectal injury accompa-nied by sphincter injury should be

1	repaired and the patient’s clinical condition is stable. Isolated sphincter injuries that do not involve the rectum may be repaired primarily. Rectal injury accompa-nied by sphincter injury should be treated with fecal diversion and distal rectal washout, with or without drain placement. Sig-nificant perineal tissue loss may require extensive debridement and a diverting colostomy.Surgical Repairs. The most common method of repair of the anal sphincter is a wrap-around sphincteroplasty (Fig. 29-42).172 The procedure involves mobilization of the divided sphincter muscle and reapproximation without tension. The internal and external sphincters may be overlapped together or separately. Postanal intersphincteric levatorplasty is less commonly used to repair sphincter defects but may be useful for incontinence caused by prolapse and/or loss of the anorectal angle (see “Continence”). The approach is via the intersphincteric plane posteriorly. It may be performed concomitantly with a

1	useful for incontinence caused by prolapse and/or loss of the anorectal angle (see “Continence”). The approach is via the intersphincteric plane posteriorly. It may be performed concomitantly with a perineal repair of rectal prolapse. The levator ani muscle is approximated to restore the anorectal angle, and the puborectalis and external sphincter muscles are tightened with sutures. These elective pro-cedures usually do not require a diverting colostomy.In cases where there has been significant loss of sphincter muscle or in which prior repairs have failed, more complex tech-niques, such as gracilis muscle transposition with or without Inferior rectal nervePudendal nerveFibrotic portion ofexternal sphincterTransverse superficialperineal muscleABCDFigure 29-42. Overlapping sphincteroplasty for incontinence from sphincter disruption. A. The external sphincter muscle with scar at site of injury is mobilized. B. The muscle edges are aligned in an overlapping fashion. C. Mattress sutures

1	for incontinence from sphincter disruption. A. The external sphincter muscle with scar at site of injury is mobilized. B. The muscle edges are aligned in an overlapping fashion. C. Mattress sutures are used to approximate the muscle. D. The completed operation.Brunicardi_Ch29_p1259-p1330.indd 132223/02/19 2:30 PM 1323COLON, RECTUM, AND ANUSCHAPTER 29chronic, low-frequency electrostimulation, have been used with some success.173 In this procedure, the gracilis muscle is mobi-lized from the thigh, detached from its insertion on the tibial tuberosity, tunneled through the perineum, and wrapped around the anal canal. Another alternative in patients who have failed other repairs is the artificial anal sphincter. This device consists of an inflatable silastic cuff, a pressure-regulating balloon, and a control pump. Patients deflate the cuff manually to open the anal canal; the cuff then reinflates spontaneously to maintain clo-sure of the anal canal. Frequent infections and erosion can

1	and a control pump. Patients deflate the cuff manually to open the anal canal; the cuff then reinflates spontaneously to maintain clo-sure of the anal canal. Frequent infections and erosion can lead to device loss.173,174 Sacral nerve stimulation via an implanted pulse generator is a technique used for neurogenic incontinence when the sphincter is intact.12-14 In some patients, an end stoma provides the best relief for intractable incontinence.176Foreign BodyForeign body entrapment in the rectum is not uncommon. Depending on the level of entrapment, a foreign body may cause damage to the rectum, rectosigmoid, or descending colon. Generalized abdominal pain suggests intraperitoneal perfo-ration. Evaluation of the patient includes inspection of the perineum and a careful abdominal examination to detect any evidence of perforation. Plain films of the abdomen are manda-tory to detect free intra-abdominal air.Foreign bodies lodged low in the rectum may often be removed under conscious

1	to detect any evidence of perforation. Plain films of the abdomen are manda-tory to detect free intra-abdominal air.Foreign bodies lodged low in the rectum may often be removed under conscious sedation with or without a local anes-thetic block. Objects impacted higher in the rectum may require regional or general anesthesia for removal. Only rarely will a laparotomy be required to remove the object, either through manual manipulation of the object to expel from the anus, or via colotomy. After removal of the foreign body, it is crucial to evaluate the rectum and sigmoid colon for injury. Proctos-copy and/or flexible sigmoidoscopy should be performed. A hematoma without evidence of perforation requires no surgical treatment. Perforation of the rectum or sigmoid colon should be managed as described in the preceding sections.THE IMMUNOCOMPROMISED PATIENTHuman Immunodeficiency VirusPatients infected with HIV may present with a myriad of gastro-intestinal symptoms. Diarrhea, in particular,

1	in the preceding sections.THE IMMUNOCOMPROMISED PATIENTHuman Immunodeficiency VirusPatients infected with HIV may present with a myriad of gastro-intestinal symptoms. Diarrhea, in particular, is extremely com-mon. The severity of gastrointestinal disease depends in part on the degree of immunosuppression; however, both ordinary and opportunistic pathogens may affect patients at any stage of the disease. Opportunistic infections with bacteria (Salmonella, Shi-gella, Campylobacter, Chlamydia, and Mycobacterium species), fungi (histoplasmosis, coccidiosis, Cryptococcus), protozoa (toxoplasmosis, cryptosporidiosis, isosporiasis), and viruses (CMV, herpes simplex virus) can cause diarrhea, abdominal pain, and weight loss. CMV in particular may cause severe enterocolitis and is the most common infectious cause of emer-gency laparotomy in acquired immunodeficiency syndrome (AIDS) patients. Clostridium difficile colitis is a major concern in these patients, especially because many patients

1	infectious cause of emer-gency laparotomy in acquired immunodeficiency syndrome (AIDS) patients. Clostridium difficile colitis is a major concern in these patients, especially because many patients are main-tained on suppressive antibiotic therapy. The incidence of gas-trointestinal malignancy is also increased in patients with HIV infection.177 Kaposi’s sarcoma is the most common malignancy in AIDS patients and can affect any part of the gastrointestinal tract. Patients may be asymptomatic or may develop bleeding or obstruction. Gastrointestinal lymphoma (usually non-Hodg-kin’s lymphoma) is also common. The incidence of colorectal carcinoma may also be increased in this population, although definitive data are lacking.Perianal disease is extremely common in patients with HIV infection. Because HIV is sexually transmitted, it is com-mon to find concomitant infection with other sexually trans-mitted diseases such as Chlamydia, herpes simplex virus, and HPV (anal condyloma). Anal

1	Because HIV is sexually transmitted, it is com-mon to find concomitant infection with other sexually trans-mitted diseases such as Chlamydia, herpes simplex virus, and HPV (anal condyloma). Anal condyloma in particular are very common, and the incidence of dysplasia (HGAIN) is high in the HIV-infected population.105 Abscesses and fistulas may be more difficult to diagnose in these patients and may be com-plex. Many patients require an examination under anesthesia with biopsy and cultures to determine the etiology of many of these perianal problems. The introduction of highly active anti-retroviral therapy (HAART) has changed the natural history of HIV infection, but it remains to be seen how these medications will affect the incidence and outcome of colorectal disease in this patient population.177IMMUNOSUPPRESSION FOR TRANSPLANTATIONThe gastrointestinal tract is a common site for posttransplanta-tion complications that are responsible for significant morbid-ity and mortality. In

1	population.177IMMUNOSUPPRESSION FOR TRANSPLANTATIONThe gastrointestinal tract is a common site for posttransplanta-tion complications that are responsible for significant morbid-ity and mortality. In these patients, infection and medication are the most common causes of diarrhea. Immunosuppressant medications, in particular, may cause diarrhea. CMV infection is common and may be severe. Clostridium difficile colitis also occurs commonly. Diverticulitis appears to be more common in some populations of transplant patients and may be more likely to present with abscess or free perforation. Elective resection after recovery from one episode of confirmed diverticulitis may be indicated in the transplant population.178 Graft-versus-host disease is unique to transplant patients and often requires endoscopy and biopsy to diagnose gastrointestinal involvement. Patients are subject to the same opportunistic infections outlined earlier; however, sexually transmitted infections and Kaposi’s

1	endoscopy and biopsy to diagnose gastrointestinal involvement. Patients are subject to the same opportunistic infections outlined earlier; however, sexually transmitted infections and Kaposi’s sarcoma are somewhat less prevalent. Perianal disease is some-what less common in the transplant population than in patients infected with HIV; however, similar infections may occur, and immunosuppression often makes diagnosis and treatment challenging.With increasing long-term survival among transplant recipients, the development of posttransplant malignancy has become a major concern. Posttransplant lymphoprolifera-tive disease is increasingly common and may occur anywhere in the gastrointestinal tract. The risk of colorectal carcinoma is increased in patients with predisposing conditions such as ulcerative colitis. However, immunosuppression alone does not appear to increase the incidence of colorectal cancer, and current screening recommendations are similar to those for the average risk

1	as ulcerative colitis. However, immunosuppression alone does not appear to increase the incidence of colorectal cancer, and current screening recommendations are similar to those for the average risk population. In contrast, the incidence of anal squamous cell carcinoma is dramatically increased in transplant patients, and patients with known HPV infection should undergo more vigor-ous screening.THE NEUTROPENIC PATIENTNeutropenic enterocolitis (typhlitis) is a life-threatening prob-lem with a mortality rate of greater than 50%. This syndrome is characterized by abdominal pain and distention, fever, diarrhea (often bloody), nausea, and vomiting in a patient with fewer than 1000 neutrophils/μL blood from any cause (bone marrow transplantation, solid-organ transplantation, or chemotherapy). Its etiology is poorly understood. Histologic features can be Brunicardi_Ch29_p1259-p1330.indd 132323/02/19 2:30 PM 1324SPECIFIC CONSIDERATIONSPART IIseen on biopsy or surgical resection and

1	Its etiology is poorly understood. Histologic features can be Brunicardi_Ch29_p1259-p1330.indd 132323/02/19 2:30 PM 1324SPECIFIC CONSIDERATIONSPART IIseen on biopsy or surgical resection and include a paucity of inflammatory and leukemic infiltrates but with mucosal and submucosal edema, villous sloughing, stromal hemorrhage, and patchy-to-complete epithelial necrosis. CT scan of the abdomen often shows a dilated cecum with pericolic stranding. However, a normal-appearing CT scan does not exclude the diagnosis. Some patients will respond to bowel rest, broad-spectrum anti-biotics, parenteral nutrition, and granulocyte infusion or col-ony-stimulating factors.179 Evidence of perforation, generalized peritonitis, and deterioration in clinical condition are indications for operation.Neutropenic patients often develop perianal pain, and diagnosis may be difficult because of a lack of inflammatory response to infection. While broad-spectrum antibiotics may cure some of these patients,

1	patients often develop perianal pain, and diagnosis may be difficult because of a lack of inflammatory response to infection. While broad-spectrum antibiotics may cure some of these patients, an examination under anesthesia should not be delayed because of neutropenia. An increase in pain or fever and/or clinical deterioration mandates an exam under anesthesia. Any indurated area should be incised and drained, biopsied to exclude a leukemic infiltrate, and cultured to aid in the selection of antimicrobial agents.REFERENCESEntries highlighted in bright blue are key references. 1. Keller J, Fibbe C, Rosien U, et al. Recent advances in capsule endoscopy: development of maneuverable capsules. Expert Rev Gastroenterol Hepatol. 2012;6:561-566. 2. Mang T, Bogoni L, Salganicoff M, et al. Computer-aided detection of colorectal polyps in CT colonography with and without fecal tagging: a stand-alone evaluation. Invest Radiol. 2012;47:99-108. 3. Grady E. Gastrointestinal bleeding scintigraphy in

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1	for colorectal-cancer screening. N Engl J Med. 2014;370(14):1287-1297. 8. Thirunavukarasu P, Talati C, Munjal S, Attwood K, Edge SB, Francescutti V. Effect of incorporation of pretreatment serum carcinoembryonic antigen levels into AJCC staging for colon cancer on 5-year survival. JAMA Surg. 2015;150(8):747-755. doi: 10.1001/jamasurg.2015.0871. PMID: 26083632. 9. Offit K. Genetic prognostic markers for colorectal cancer. N Engl J Med. 2000;342:124-125. 10. Lynch HT, Lynch JF, Lynch PM, et al. Hereditary colorectal cancer syndromes: molecular genetics, genetic counseling, diagnosis and management. Fam Cancer. 2008;7:27-39. This reference is included in an issue of Familial Cancer that is devoted to discussion of hereditary forms of colorectal can-cer. The paper summarizes the evaluation of patients suspected to have familial colorectal cancer with a special emphasis on HNPCC (Lynch Syndrome) and FAP. Recommendations are provided for genetic testing, surveillance strategies, and

1	of patients suspected to have familial colorectal cancer with a special emphasis on HNPCC (Lynch Syndrome) and FAP. Recommendations are provided for genetic testing, surveillance strategies, and treatment. 11. FitzHarris GP, Garcia-Aguilar J, Parker SC, et al. Quality of life after subtotal colectomy for slow-transit constipa-tion: both quality and quantity count. Dis Colon Rectum. 2003;46:433-440. 12. Paquette IM, Varma MG, Kaiser AM, Steele SR, Raf-ferty JF. The American Society of Colon and Rectal Sur-geons’ clinical practice guideline for the treatment of fecal incontinence. Dis Colon Rectum. 2015;58:623-636. The Standards Task Force of the American Society of Colon and Rectal Surgeons evaluated existing data on treatment of fecal incontinence based upon quality. The guidelines provided in this manuscript represent evidence based management of this condition. 13. Ganio E, Ratto C, Masin A, et al. Neuromodulation for fecal incontinence: outcome in 16 patients with definitive

1	in this manuscript represent evidence based management of this condition. 13. Ganio E, Ratto C, Masin A, et al. Neuromodulation for fecal incontinence: outcome in 16 patients with definitive implant. The initial Italian Sacral Neurostimulation Group (GINS) experience. Dis Colon Rectum, 2001;44:965-970. 14. Forte ML1, Andrade KE, Lowry AC, Butler M, Bliss DZ, Kane RL. Systematic review of surgical treatments for fecal incontinence. Dis Colon Rectum. 2016;59(5):443-469. doi: 10.1097/DCR.0000000000000594 15. Ky AJ, Sonoda T, Milsom JW. One-stage laparoscopic restor-ative proctocolectomy: an alternative to the conventional approach? Dis Colon Rectum. 2002;45:207-210. 16. Lezoche E, Feliciotti F, Paganini AM, et al. Laparoscopic colonic resections versus open surgery: a prospective non-randomized study on 310 unselected cases. Hepatogastroen-terology. 2000;47:697-708. 17. Martinez-Perez A, Cara MC, Brunetti F, de’Angelis N. Patho-logic outcomes of laparoscopic vs. open mesorectal excision

1	study on 310 unselected cases. Hepatogastroen-terology. 2000;47:697-708. 17. Martinez-Perez A, Cara MC, Brunetti F, de’Angelis N. Patho-logic outcomes of laparoscopic vs. open mesorectal excision for rectal cancer: a systematic review and meta-analysis. JAMA Surg. 2017;8:165665. doi:10.1001/jamasurg.20165665 18. Weeks JC, Nelson H, Gelber S, et al. Short-term quality-of-life outcomes following laparoscopic-assisted colectomy vs. open colectomy for colon cancer: a randomized trial. JAMA. 2002;287:321-328. 19. deSouza AL, Prasad LM, Ricci J, et al. A comparison of open and robotic total mesorectal excision for rectal adenocarci-noma. Dis Colon Rectum. 2011;54:275-282. 20. Biondo S, Frago R, Codina Cazador A, et al. Long-term func-tional results from a randomized clinical study of transverse coloplasty compared with colon J-pouch after low anterior resection for rectal cancer. Surgery. 2013;153:383-392. 21. Heah SM, Seow-Choen F, Eu KW, et al. Prospective, random-ized trial comparing

1	coloplasty compared with colon J-pouch after low anterior resection for rectal cancer. Surgery. 2013;153:383-392. 21. Heah SM, Seow-Choen F, Eu KW, et al. Prospective, random-ized trial comparing sigmoid vs. descending colonic J-pouch after total rectal excision. Dis Colon Rectum. 2002;45: 322-328. 22. Machado M, Nygren J, Goldman S, et al. Similar outcome after colonic pouch and side-to-end anastomosis in low ante-rior resection for rectal cancer: a prospective randomized trial. Ann Surg. 2003;238:214-220. 23. Ricciardi R, Roberts PL, Marcello PW, Hall JF, Read TE, Schoetz DJ. Anastomotic leak testing after colo-rectal resec-tion: what are the data? Arch Surg. 2009;144(5):407-411. 24. Prytz M, Angenete E, Bock D, Haglind E. Extralevator abdom-inoperineal excision for low rectal cancer—extensive surgery to be used with discretion based on 3 year local recurrence results. Ann Surg. 2016;263(3):516-521. 25. Holm T, Ljung A, Haggmark T, Jurell G, Lagergren J. Extended abdominoperineal

1	surgery to be used with discretion based on 3 year local recurrence results. Ann Surg. 2016;263(3):516-521. 25. Holm T, Ljung A, Haggmark T, Jurell G, Lagergren J. Extended abdominoperineal resection with gluteus maximus flap reconstruction of the pelvic floor for rectal cancer. Br J Surg. 2007;94:232-238. 26. Farouk R, Pemberton JH, Wolff BG, et al. Functional out-comes after ileal pouch-anal anastomosis for chronic ulcer-ative colitis. Ann Surg. 2000;231:919-926. 27. Bullard KM, Madoff RD, Gemlo BT. Is ileoanal pouch func-tion stable with time? Results of a prospective audit. Dis Colon Rectum. 2002;45:299-304. 28. Segal JP, Ding NS, Worley G, et al. Systematic review with meta-analysis: the management of chronic refractory pouchitis with an evidence-based treatment algorithm. Aliment Pharma-col Ther. 2017;45(5):581-592. doi:10.1111/apt.13905.Brunicardi_Ch29_p1259-p1330.indd 132423/02/19 2:30 PM 1325COLON, RECTUM, AND ANUSCHAPTER 29 29. Sandborn W, McLeod R, Jewell D.

1	Aliment Pharma-col Ther. 2017;45(5):581-592. doi:10.1111/apt.13905.Brunicardi_Ch29_p1259-p1330.indd 132423/02/19 2:30 PM 1325COLON, RECTUM, AND ANUSCHAPTER 29 29. Sandborn W, McLeod R, Jewell D. Pharmacotherapy for inducing and maintaining remission in pouchitis. Cochrane Database Syst Rev. 2000;2:CD001176. 30. Stocchi L, Pemberton JH. Pouch and pouchitis. Gastroenterol Clin North Am. 2001;30:223-241. 31. Sandborn W, McLeod R, Jewell D. Pharmacotherapy for inducing and maintaining remission in pouchitis. Cochrane Database Syst Rev. 2000;2:CD001176. 32. Tran-Minh ML, Allez M, Gornet JM. Successful treatment with ustekinumab for chronic refractory pouchitis. J Crohns Colitis. 2017;11(9):1156. doi:10.1093/ecco-jcc/jjx018. 33. Kelly OB, Rosenberg M, Tyler AD, et al. Infliximab to treat refractory inflammation after pelvic pouch surgery for ulcer-ative colitis. J Crohns Colitis. 2016;10(4):410-417. doi: 10.1093/ecco-jcc/jjv225 34. Schmid M, Frick JS, Malek N, Goetz M. Successful

1	refractory inflammation after pelvic pouch surgery for ulcer-ative colitis. J Crohns Colitis. 2016;10(4):410-417. doi: 10.1093/ecco-jcc/jjv225 34. Schmid M, Frick JS, Malek N, Goetz M. Successful treat-ment of pouchitis with Vedolizumab, but not fecal micro-biota transfer (FMT), after proctocolectomy in ulcerative colitis. Int J Colorectal Dis. 2017;32(4):597-598. doi: 10.1007/s00384-017-2761-4 35. Cannon JA, Altom LK, Deierhoi RJ, et al. Preoperative oral antibiotics reduce surgical site infection following elective colorectal resections. Dis Colon Rectum. 2012;55:1160-1166. 36. Scarborough JE, Mantyh CR, Sun Z, Migaly J. Com-bined mechanical and oral antibiotic bowel preparation reduces incisional surgical site infection and anastomotic leak rates after elective colorectal resection. Ann Surg. 2015;262(2):331-337. This study assessed the effect of com-bined mechanical and oral antibiotic bowel preparation (prep) and 30-day outcomes after colorectal resection using data from the 2012

1	Surg. 2015;262(2):331-337. This study assessed the effect of com-bined mechanical and oral antibiotic bowel preparation (prep) and 30-day outcomes after colorectal resection using data from the 2012 Colectomy-Targeted American College of Sur-geons National Surgical Quality Improvement (ACS NSQIP) data (4999 patients). The combination of oral and mechani-cal bowel preparation decreased the incidence of surgical site infection, anastomotic leak, and hospital readmission when compared to mechanical prep alone, oral antibiotic prep alone, or no prep. 37. Cao F, Li J, Li F. Mechanical bowel preparation for elective colorectal surgery: updated systematic review and meta-anal-ysis. Int J Colorectal Dis. 2012;27:803-810. 38. Hadithi M, Cazemier M, Meijer GA, et al. Retrospective anal-ysis of old-age colitis in the Dutch inflammatory bowel dis-ease population. World J Gastroenterol. 2008;14:3183-3187. 39. Bonen DK, Cho JH. The genetics of inflammatory bowel dis-ease. Gastroenterology.

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1	Dis Colon Rectum. 2010;53(11):1487-1494. 44. Sehgal R, Berg A, Polinski JI, et al. Mutations in IRGM are associated with more frequent need for surgery in patients with ileocolonic Crohn’s disease. Dis Colon Rectum. 2012;55(2):115-121. 45. Hoarau G, Mukherjee PK, Gower-Rousseau C, et al. Bac-teriome and mycobiome interactions underscore microbial dysbiosis in familial Crohn’s disease. mBio. 2016;7(5). pii: e01250-16. 46. Strisciuglio C, Duijvestein M, Verhaar AP, et al. Impaired autophagy leads to abnormal dendritic cell-epithelial cell interactions. J Crohns Colitis. 2013;7(7):534-541. 47. Brinar M, Vermeire S, Cleynen I, et al. Genetic variants in autophagy-related genes and granuloma formation in a cohort of surgically treated Crohn’s disease patients. J Crohns Colitis. 2012;6(1):43-50. 48. Strisciuglio C, Duijvestein M, Verhaar AP, et al. Impaired autophagy leads to abnormal dendritic cell-epithelial cell interactions. J Crohns Colitis. 2013;7(7):534-541. 49. Brinar M, Vermeire S,

1	C, Duijvestein M, Verhaar AP, et al. Impaired autophagy leads to abnormal dendritic cell-epithelial cell interactions. J Crohns Colitis. 2013;7(7):534-541. 49. Brinar M, Vermeire S, Cleynen I, et al. Genetic variants in autophagy-related genes and granuloma formation in a cohort of surgically treated Crohn’s disease patients. J Crohns Colitis. 2012;6(1):43-50. 50. Tremaine WJ. Is indeterminate colitis determinable? Curr Gastroenterol Rep. 2012;14:162-165. 51. Lakatos PL, Lakatos L, Kiss LS, et al. Treatment of extraintes-tinal manifestations in inflammatory bowel disease. Digestion. 2012;86(suppl 1):28-35. 52. Alfadhli AA, McDonald JW, Feagan BG. Methotrexate for induction of remission in refractory Crohn’s disease. Cochrane Database Syst Rev. 2005;1:CD003459. 53. Hanauer SB, Feagan BG, Lichtenstein GR, et al. Main-tenance infliximab for Crohn’s disease: the ACCENT I randomised trial. Lancet. 2002;359:1541-1549. This study reports a randomized, controlled trial to assess main-tenance

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1	of MYH associated with somatic G:C–>T:A mutations in colorectal tumors. Nat Genet. 2002;30:227-232. 81. Lefevre JH, Rodrigue CM, Mourra N, et al. Implication of MYH in colorectal polyposis. Ann Surg. 2006;244: 874-879. MYH is a base excision repair gene that removes adenines from DNA that have been mispaired with a nucle-otide product of oxidative reaction (8oxoG). Without MYH, mispaired adenines will lead to somatic transversions G:C→ T:A in genes such as APC or K-ras. The authors reviewed 433 patients who underwent prophylactic surgery for colorectal polyposis over a 25-year time frame and evaluated 31 patients who did not have APC, finding biallelic MYH mutations in 6 patients. They conclude that MYH is a new gene responsible for approximately 1.4% of all adenomatous polyposis and 20% of polyposis without an APC mutation identified. 82. Martin M, Simon-Assmann P, Kedinger M, et al. DCC regu-lates cell adhesion in human colon cancer derived HT-29 cells and associates with ezrin. Eur

1	polyposis without an APC mutation identified. 82. Martin M, Simon-Assmann P, Kedinger M, et al. DCC regu-lates cell adhesion in human colon cancer derived HT-29 cells and associates with ezrin. Eur J Cell Biol. 2006;85: 769-783. 83. Lao VV, Grady WM. Epigenetics and colorectal cancer. Nat Rev Gastroenterol Hepatol. 2011;8:686-700. 84. Ferlitsch M, Moss A, Hassan C, et al. Colorectal polypectomy and endoscopic mucosal resection (EMR): European Society of Gastrointestinal Endoscopy (ESGE) clinical guideline. Endoscopy. 2017;49(3):270-297. doi:10.1055/s-0043-102569. 85. Rex DK, Ahnen DJ, Baron JA, et al. Serrated lesions of the colorectum: review and recommendations from an expert panel. Am J Gastroenterol. 2012;107:1315-1329. An expert panel provide their recommendation after a thorough literature review and 2-day conference. Hyperplastic polyps, sessile ser-rated adenomas/polyps, and traditional serrated adenomas are all included in the category of serrated lesions of the colon. This

1	review and 2-day conference. Hyperplastic polyps, sessile ser-rated adenomas/polyps, and traditional serrated adenomas are all included in the category of serrated lesions of the colon. This review outlines the histologic features of these lesions. Sessile serrated adenomas are predominantly flat, often with indistinct borders, right sided. They are associated with inter-val colon adenocarcinomas and are thought to progress to cancer, possibly through a promotor CpG island hypermeth-ylation pathway. The panel recommends that these lesions are completely excised and points to the variability in detection rates between endoscopists, hypothesizing that difficulty in endoscopic detection is related to the high rate of subsequent cancer diagnosis in these patients. 86. Regimbeau JM, Panis Y, Pocard M, et al. Handsewn ileal pouch-anal anastomosis on the dentate line after total proc-tectomy: technique to avoid incomplete mucosectomy and the need for long-term follow-up of the anal

1	Y, Pocard M, et al. Handsewn ileal pouch-anal anastomosis on the dentate line after total proc-tectomy: technique to avoid incomplete mucosectomy and the need for long-term follow-up of the anal transition zone. Dis Colon Rectum. 2001;44:43-50. 87. Remzi F, Fazio VW, Delaney CP, et al. Dysplasia of the anal transitional zone after ileal pouch-anal anastomosis: results of prospective evaluation after a minimum of ten years. Dis Colon Rectum. 2003;46:6-13. 88. Janne PA, Mayer RJ. Chemoprevention of colorectal cancer. N Engl J Med. 2000;342:1960-1968. 89. Steinbach G, Lynch PM, Phillips RK, et al. The effect of cele-coxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N Engl J Med. 2000;342:1946-1952. 90. Kasper B, Gruenwald V, Reichardt P, et al. Imatinib induces sustained progression arrest in RECIST progressive desmoid tumours: Final results of a phase II study of the German Inter-disciplinary Sarcoma Group (GISG). Eur J Cancer. 2017;76: 60-67. doi:

1	induces sustained progression arrest in RECIST progressive desmoid tumours: Final results of a phase II study of the German Inter-disciplinary Sarcoma Group (GISG). Eur J Cancer. 2017;76: 60-67. doi: 10.1016/j.ejca.2017.02.001. 91. Church J, Xhaja X, LaGuardia L, O’Mally M, Burke C, Kalady M. Desmoids and genotype in familial adenomatous polyposis. Dis Colon Rectum. 2015;58(4):444-448. 92. Bulow C, Vasen H, Järvinen H, et al. Ileorectal anastomosis is appropriate for a subset of patients with familial adenomatous polyposis. Gastroenterology. 2000;119:1454-1460. 93. Hampel H, Panescu J, Lockman J, et al. Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer). N Engl J Med. 2005;352:1851-1860. 94. Jarvinen HJ, Aarnio M, Mustonen H, et al. Controlled 15-year trial on screening for colorectal cancer in families with hereditary nonpolyposis colorectal cancer. Gastroenterology. 2000;118:829-834. 95. National Quality Forum. Appendix A: Specifications of the National

1	screening for colorectal cancer in families with hereditary nonpolyposis colorectal cancer. Gastroenterology. 2000;118:829-834. 95. National Quality Forum. Appendix A: Specifications of the National Voluntary Consensus Standards for Breast and Colon Cancer. Available at: https://www.qualityforum.org/Publica-tions/2009/05/National_Voluntary_Consensus_Standards_for_Quality_of_Cancer_Care.aspx. Accessed July 23, 2018. 96. Lieberman DA, Weiss DG, Bond JH, et al. Use of colonos-copy to screen asymptomatic adults for colorectal cancer. Brunicardi_Ch29_p1259-p1330.indd 132623/02/19 2:30 PM 1327COLON, RECTUM, AND ANUSCHAPTER 29Veterans Affairs Cooperative Study Group 380. N Engl J Med. 2000;343:162-168. 97. Pignone M, Rich M, Teutsch SM, et al. Screening for colorec-tal cancer in adults at average risk: a summary of the evidence for the U.S. Preventive Services Task Force. Ann Intern Med. 2002;137:132-141. 98. U.S. Preventive Services Task Force, Bibbins-Domingo K, Grossman DC, et al.

1	average risk: a summary of the evidence for the U.S. Preventive Services Task Force. Ann Intern Med. 2002;137:132-141. 98. U.S. Preventive Services Task Force, Bibbins-Domingo K, Grossman DC, et al. Screening for colorectal cancer: US Preventive Services Task Force recommendation statement. JAMA. 2016;315:2564-2575. 99. Mandel JS, Church TR, Bond JH, et al. The effect of fecal occult-blood screening on the incidence of colorectal cancer. N Engl J Med. 2000;343:1603-1607. 100. Hardcastle JD, Armitage NC, Chamberlain J, et al. Fecal occult blood screening for colorectal cancer in the general popula-tion. Results of a controlled trial. Cancer. 1986;58:397-403. 101. Winawer SJ, Flehinger BJ, Schottenfeld D, et al. Screening for colorectal cancer with fecal occult blood testing and sigmoid-oscopy. J Natl Cancer Inst. 1993;85:1311-1318. 102. Levin B, Brooks D, Smith RA, et al. Emerging technologies in screening for colorectal cancer: CT colonography, immu-nochemical fecal occult blood

1	J Natl Cancer Inst. 1993;85:1311-1318. 102. Levin B, Brooks D, Smith RA, et al. Emerging technologies in screening for colorectal cancer: CT colonography, immu-nochemical fecal occult blood tests, and stool screening using molecular markers. CA Cancer J Clin. 2003;53:44-55. 103. Yee J, Akerkar GA, Hung RK, et al. Colorectal neoplasia: per-formance characteristics of CT colonography for detection in 300 patients. Radiology. 2001;219:685-692. 104. Imperiale TF, Wagner DR, Lin CY, et al. Risk of advanced proximal neoplasms in asymptomatic adults according to the distal colorectal findings. N Engl J Med. 2000;343:169-174. 105. Holme O, Løberg M, Kalager M, et al. Effect of flexible sig-moidoscopy screening on colorectal cancer incidence and mor-tality: a randomized clinical trial. JAMA. 2014;312:606-615. 106. Greene FL, Fleming PD, Fleming ID, et al. AJCC Cancer Staging Manual. Springer: New York; 2002. 107. Chang GJ, Kaiser AM, Mills S, Rafferty JE, Buie WD, on behalf of the Standards

1	FL, Fleming PD, Fleming ID, et al. AJCC Cancer Staging Manual. Springer: New York; 2002. 107. Chang GJ, Kaiser AM, Mills S, Rafferty JE, Buie WD, on behalf of the Standards Practice Task Force of the American Society of Colon and Rectal Surgeons. Practice parameters for the management of colon cancer. Dis Colon Rectum. 2012;55:831-843. 108. Gunderson LL, Sargent DJ, Tepper JE, et al. Impact of T and N substage on survival and disease relapse in adjuvant rec-tal cancer: a pooled analysis. Int J Radiat Oncol Biol Phys. 2002;54:386-396. 109. Monson JRT, Weiser MR, Buie WD, Chang GJ, Rafferty JF; Prepared by the Standards practice Task Force of the American Society of Colon and Rectal Surgeons. Practice parameters for the management of rectal cancer (revised). Dis Colon Rectum. 2013;56:535-550. 110. Qiu H, Sirivongs P, Rothenberger M, et al. Molecular prognos-tic factors in rectal cancer treated by radiation and surgery. Dis Colon Rectum. 2000;43:451-459. 111. Johnson PM, Porter GA,

1	H, Sirivongs P, Rothenberger M, et al. Molecular prognos-tic factors in rectal cancer treated by radiation and surgery. Dis Colon Rectum. 2000;43:451-459. 111. Johnson PM, Porter GA, Ricciardi R, et al. Increasing negative lymph node count is independently associated with improved long-term survival in stage IIIB and IIIC colon cancer. J Clin Oncol. 2006;24:3570-3575. 112. Chang GJ, Rodriguez-Bigas MA, Skibber JM, et al. Lymph node evaluation and survival after curative resec-tion of colon cancer: systematic review. J Natl Cancer Inst. 2007;99:433-441. 113. Ricciardi R, Madoff RD, Rothenberger DA, et al. Population-based analyses of lymph node metastases in colorectal cancer. Clin Gastroenterol Hepatol. 2006;4:1522-1527. 114. Ricciardi R, Baxter NN. Association versus causation versus quality improvement: setting benchmarks for lymph node eval-uation in colon cancer. J Natl Cancer Inst. 2007;99:414-415. 115. Puthillath A, Dunn KB, Rajput A, et al. Safety and efficacy of first-line

1	quality improvement: setting benchmarks for lymph node eval-uation in colon cancer. J Natl Cancer Inst. 2007;99:414-415. 115. Puthillath A, Dunn KB, Rajput A, et al. Safety and efficacy of first-line chemotherapy in unresected metastatic colorectal cancer. Clin Colorectal Cancer. 2007;6:710-715. 116. Francescutti V, Miller A, Satchidanand Y, Alvarez-Perez A, Dunn KB. Management of bowel obstruction in patients with stage IV cancer: predictors of outcome after surgery. Ann Surg Oncol. 2013;20(3):707-714. doi: 10.1245/s10434-012-2662-2. 117. Kashida H, Kudo SE. Early colorectal cancer: concept, diag-nosis, and management. Int J Clin Oncol. 2006;11(1):1-8. 118. Sargent DJ, Marsoni S, Monges G, et al. Defective mismatch repair as a predictive marker for lack of efficacy of fluoro-uracil-based adjuvant therapy in colon cancer. J Clin Oncol. 2010;28:3219-3226. 119. Mahmoud N, Bullard Dunn K. Metastasectomy for stage IV colorectal cancer. Dis Colon Rectum. 2010;53:1080-1092. 120. Demmy TL,

1	adjuvant therapy in colon cancer. J Clin Oncol. 2010;28:3219-3226. 119. Mahmoud N, Bullard Dunn K. Metastasectomy for stage IV colorectal cancer. Dis Colon Rectum. 2010;53:1080-1092. 120. Demmy TL, Dunn KB. Surgical and nonsurgical therapy for lung metastasis: indications and outcomes. Surg Oncol Clin N Am. 2007;16:579-605. 121. Stitzenberg KB, Sanoff HK, Penn DC, Meyers MO, Tepper JE. Practice patterns and long-term survival for early-stage rectal cancer. J Clin Oncol. 2013;31(34):4276-4282. 122. Taylor FGM, Quirke P, Heald RJ, et al. Preoperative high-res-olution magnetic resonance imaging can identify good prog-nosis stage I, II and III rectal cancer best managed by surgery alone. Ann Surg. 2011;253:711-719. 123. Garcia-Aguilar J, Shi Q, Thomas CR, Jr, et al. A phase II trial of neoadjuvant chemoradiation and local excision for T2N0 rectal cancer: preliminary results of the ACOSOG Z6041 trial. Ann Surg Oncol. 2012;19:384-391. 124. Garcia-Aguilar J, Renfro LA, Chow OS, et al. Organ

1	chemoradiation and local excision for T2N0 rectal cancer: preliminary results of the ACOSOG Z6041 trial. Ann Surg Oncol. 2012;19:384-391. 124. Garcia-Aguilar J, Renfro LA, Chow OS, et al. Organ preserva-tion for clinical T2N0 distal rectal cancer using neoadjuvant chemoradiotherapy and local excision (ACOSOG Z6041): results of an open-label, single-arm, multi-institutional, phase 2 trial. Lancet Oncol. 2015;16(15):1537-1546. 125. Sun Z, Adam MA, Kim J, et al. Determining the optimal timing for initiation of adjuvant chemotherapy after resec-tion for Stage II and III colon cancer. Dis Colon Rectum. 2016;59(2):87-93. 126. Sauer R, Liersch T, Merkel S, et al. Preoperative versus postoperative chemoradiotherapy for rectal cancer. N Engl J Med. 2004;351:1731-1740. The German Rectal Cancer Study Group randomized patients with clinical stage II and III rectal cancer to preoperative or postoperative long-course chemoradiation. The study occurred between 1994 and 2002 and, of note, the

1	Study Group randomized patients with clinical stage II and III rectal cancer to preoperative or postoperative long-course chemoradiation. The study occurred between 1994 and 2002 and, of note, the approach to staging did not include MRI. Among the 799 patients who were analyzed, fewer local recurrences were seen in patients assigned to preoperative chemoradiation compared with patients assigned to postopera-tive chemoradiation (6% vs. 13%; P = 0.006) at 5 years. The authors also found that in the long term, 72% of local recur-rences were associated with distant metastases. A subgroup analysis showed that the strongest difference in hazard ratios between preoperative and postoperative chemoradiation were in the patients who had intersphincteric or abdominoperineal resections (hazard ratio 2.24; P = 0.03). 127. Cercek A, Garcia-Aguilar J. Rectal cancer: neoadjuvant ther-apy. In: Steele SR, Hull TL, Read TE, Saclarides TJ, Senagore AJ, Whitlow CB, eds. The ASCRS Textbook of Colon and

1	2.24; P = 0.03). 127. Cercek A, Garcia-Aguilar J. Rectal cancer: neoadjuvant ther-apy. In: Steele SR, Hull TL, Read TE, Saclarides TJ, Senagore AJ, Whitlow CB, eds. The ASCRS Textbook of Colon and Rectal Surgery. 3rd ed. New York: Springer; 2016:481-494. 128. Smith FM, Wiland H, Mace A, Pai RK, Kalady MF. Clinical criteria underestimate complete pathological response in rec-tal cancer treated with neoadjuvant chemoradiotherapy. Dis Colon Rectum. 2014;57(3):311-315. 129. Sammour T, Price BA, Krause KJ, Chang GJ. Nonoperative management of “watch and wait” for rectal cancer with com-plete clinical response after neoadjuvant chemoradiotherapy: a critical appraisal. Ann Surg Oncol. 2017;24:1904-1915. doi: 10.1245/s10434-017-5841-3. 130. Berends FJ, Kazemier G, Bonjer HJ, et al. Subcutaneous metastases after laparoscopic colectomy. Lancet. 1994; 344:58.Brunicardi_Ch29_p1259-p1330.indd 132723/02/19 2:30 PM 1328SPECIFIC CONSIDERATIONSPART II 131. Jayne DG, Guillou PJ, Thorpe H, et al.

1	metastases after laparoscopic colectomy. Lancet. 1994; 344:58.Brunicardi_Ch29_p1259-p1330.indd 132723/02/19 2:30 PM 1328SPECIFIC CONSIDERATIONSPART II 131. Jayne DG, Guillou PJ, Thorpe H, et al. Randomized trial of laparoscopic-assisted resection of colorectal carcinoma: 3-year results of the UK MRC CLASICC Trial Group. J Clin Oncol. 2007;25:3061-3068. 132. Jayne DG, Thorpe HC, Copeland J, Quirke P, Brown JM, Guillou PJ. Five-year follow-up of the Medical Research Council CLASICC trial of laparoscopically assisted versus open surgery for colorectal cancer. Br J Surg. 2010;97(11):1638-1645. doi: 10.1002/bjs.7160. This study presents 5-year follow-up from the UK Medical Research Council CLASSIC Trial comparing the safety and efficacy of laparoscopic vs. open surgery for colorectal cancer. Outcomes included overall and disease free survival and local, distant, and port site recurrences. The investigators found no differ-ences between the two groups. They conclude, therefore, that

1	Outcomes included overall and disease free survival and local, distant, and port site recurrences. The investigators found no differ-ences between the two groups. They conclude, therefore, that long-term outcomes are not compromised by a laparoscopic approach. 133. Nelson H, Sargent D, Wieand HS, et al; for the Clinical Out-comes of Surgical Therapy Study Group. Laparoscopically assisted colectomy is as safe and effective as open colectomy in people with colon cancer. Abstracted from: A comparison of laparoscopically assisted and open colectomy for colon cancer. N Engl J Med. 2004;350:2050-2059. Cancer Treat Rev. 2004;30(8):707-709. 134. Hazebroek EJ. COLOR: a randomized clinical trial compar-ing laparoscopic and open resection for colon cancer. Surg Endosc. 2002;16:949-953. 135. Jeong SY, Park JW, Nam BH, et al. Open versus laparoscopic surgery for mid-rectal or low-rectal cancer after neoadjuvant chemoradiotherapy (COREAN trial): survival outcomes of an open-label, non-inferiority,

1	Park JW, Nam BH, et al. Open versus laparoscopic surgery for mid-rectal or low-rectal cancer after neoadjuvant chemoradiotherapy (COREAN trial): survival outcomes of an open-label, non-inferiority, randomised controlled trial. Lancet Oncol. 2014;15(7):767-774. 136. Martínez-Pérez A, Carra MC, Brunetti F, de’Angelis N. Pathologic outcomes of laparoscopic vs open mesorectal excision for rectal cancer: a systematic review and meta-analysis. JAMA Surg. 2017;152(4):e165665. doi: 10.1001/jamasurg.2016.5665. 137. Collinson FJ, Jayne DG, Pigazzi A, et al. An international, multicentre, prospective, randomised, controlled, unblinded, parallel-group trial of robotic-assisted versus standard laparo-scopic surgery for the curative treatment of rectal cancer. Int J Colorectal Dis. 2012; 27:233-241. 138. Fleshman J, Branda M, Sargent DJ, et al. Effect of laparo-scopic-assisted resection vs open resection of stage II or III rectal cancer on pathologic outcomes: the ACOSOG Z6051 randomized clinical

1	J, Branda M, Sargent DJ, et al. Effect of laparo-scopic-assisted resection vs open resection of stage II or III rectal cancer on pathologic outcomes: the ACOSOG Z6051 randomized clinical trial. JAMA. 2015;314(13):1346-1355. 139. Stevenson AR, Solomon MJ, Lumley JW, et al; ALaCaRT Investigators. Effect of laparoscopic-assisted resection vs open resection on pathological outcomes in rectal cancer: the ALaCaRT randomized clinical trial. JAMA. 2015;314(13): 1356-1363. 140. Bonjer HJ, Deijen CL, Abis GA, et al. A randomized trial of laparoscopic versus open surgery for rectal cancer. N Engl J Med. 2015;372(14):1324-1332. 141. Collinson FJ, Jayne DG, Pigazzi A, et al. An international, multicentre, prospective, randomised, controlled, unblinded, parallel-group trial of robotic-assisted versus standard laparo-scopic surgery for the curative treatment of rectal cancer. Int J Colorectal Dis. 2012;27(2):233-241. 142. Rindi G, Arnold R, Bosman FT. Nomenclature and classifica-tion of

1	versus standard laparo-scopic surgery for the curative treatment of rectal cancer. Int J Colorectal Dis. 2012;27(2):233-241. 142. Rindi G, Arnold R, Bosman FT. Nomenclature and classifica-tion of neuroendocrine neoplasms of the digestive system. In Bosman FT, Carneiro F, Hruban RH, Theise ND, eds. WHO Classification of Tumors of Digestive System. Lyon, France: International Agency for Research on Cancer (IARC) Press; 2010:13-14. 143. Volkan Adsay N, Klimstra DS. Neuroendocrine tumors of the gastrointestinal and pancreatobiliary tracts. In: Odze RD, Goldblum JR, eds. Odze and Goldblum Surgical Pathology of the GI Tract, Liver, Biliary Tract and Pancreas. 3rd ed. Philadelphia: Saunders/Elsevier; 2015. 144. Bullard Dunn KM. Other rectal neoplasms. In: Beck DE, Steele SR, Wexner SD, eds. Fundamentals of Anorectal Surgery. 3rd ed. New York: Springer; 2018. 145. Merchea AL, Hubner M, Wenger D, Rose P, Dozois E. The value of preoperative biopsy in the management of solid pre-sacral tumors.

1	of Anorectal Surgery. 3rd ed. New York: Springer; 2018. 145. Merchea AL, Hubner M, Wenger D, Rose P, Dozois E. The value of preoperative biopsy in the management of solid pre-sacral tumors. Dis Colon Rectum. 2013;56:756-760. 146. Merchea A, Dozois EJ. Lesions originating within the retrorec-tal space. J Gastrointest Surg. 2014;18(12):2232-2233. 147. Dozois EJ, Jacofsky DJ, Billings BJ, et al. In: Wards CY, Larson DW. Surgical approach and oncologic outcomes fol-lowing multidisciplinary management of retrorectal sarcomas. Ann Surg Oncol. 2011;18(4):983-988. 148. Steele SR, Varma MG, Melton GB, Ross HM, Rafferty JF, Buie WD. Practice parameters for anal squamous neoplasms. Dis Colon Rectum. 2012;55:735-749. 149. Darragh TM, Colgan TJ, Cox JT, et al. The lower anogenital squamous terminology standardization project for HPV-asso-ciated lesions: background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical

1	standardization project for HPV-asso-ciated lesions: background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology. J Low Genit Tract Dis. 2012;16:205-242. 150. Messick CA, Rodriguez-Bigas. Anal dysplasia. Surg Oncol Clin North Am. 2017;26:33-43. The authors review the litera-ture on anal dysplasia with a focus on the natural history and the impact of ablative techniques with high resolution anos-copy on recurrence rates. High-risk patients are categorized as HIV-positive, immunosuppressed transplant recipients, and men who have sex with men. The authors emphasize the lack of literature in patients who are not non–high risk and overall emphasize close follow-up in all patients with high grade squamous intraepithelial lesion (HSIL)/high grade anal intraepithelial neoplasia (HGAIN). 151. Stanley MA. Imiquimod and the imidazoquinolones: mecha-nism of action and therapeutic potential. Clin Exp Dermatol.

1	lesion (HSIL)/high grade anal intraepithelial neoplasia (HGAIN). 151. Stanley MA. Imiquimod and the imidazoquinolones: mecha-nism of action and therapeutic potential. Clin Exp Dermatol. 2002;27:571-577. 152. Chen H, Cai Y, Liu Y, et al. Incidence, surgical treatment, and prognosis of anorectal melanoma from 1973 to 2011: a population based SEER analysis. Medicine (Baltimore). 2016;95:e2770. 153. Bullard KM, Tuttle TM, Rothenberger DA, et al. Surgi-cal therapy for anorectal melanoma. J Amer Coll Surg. 2003;196:206-211. 154. D’Hoore A, Penninckx F. Laparoscopic ventral recto(colpo)pexy for rectal prolapse: surgical technique and outcome for 109 patients. Surg Endosc. 2006;20:1919-1923. 155. Ponec RJ, Saunders MD, Kimmey MB. Neostigmine for the treatment of acute colonic pseudo-obstruction. N Engl J Med. 1999;341:137-141. 156. Turgeon DK, Novicki TJ, Quick J, et al. Six rapid tests for direct detection of Clostridium difficile and its toxins in fecal samples compared with the fibroblast

1	N Engl J Med. 1999;341:137-141. 156. Turgeon DK, Novicki TJ, Quick J, et al. Six rapid tests for direct detection of Clostridium difficile and its toxins in fecal samples compared with the fibroblast cytotoxicity assay. J Clin Microbiol. 2003;41:667-670. 157. Bagdasarian N, Rao K, Malani PN. Diagnosis and treatment of Clostridium difficile in adults: a systematic review. JAMA. 2015;313(4):398-408. 158. Cecil JA. Clostridium difficile: changing epidemiology, treat-ment and infection prevention measures. Curr Infect Dis Rep. 2012;14:612-619. 159. Manfredelli S, Montalto G, Leonetti G, et al. Conventional (CH) vs. stapled hemorrhoidectomy (SH) in surgical treat-ment of hemorrhoids. Ten years experience. Ann Ital Chir. 2012;83:129-134. 160. Shao WJ, Li GC, Zhang ZH, Yang BL, Sun GD, Chen YQ. Systematic review and meta-analysis of randomized Brunicardi_Ch29_p1259-p1330.indd 132823/02/19 2:30 PM 1329COLON, RECTUM, AND ANUSCHAPTER 29controlled trials comparing stapled haemorrhoidopexy

1	Systematic review and meta-analysis of randomized Brunicardi_Ch29_p1259-p1330.indd 132823/02/19 2:30 PM 1329COLON, RECTUM, AND ANUSCHAPTER 29controlled trials comparing stapled haemorrhoidopexy with conventional haemorrhoidectomy. Br J Surg. 2008;95: 147-160. 161. Schuurman JP, Borel Rinkes IH, Go PM. Hemorrhoidal artery ligation procedure with or without Doppler transducer in grade II and III hemorrhoidal disease: a blinded randomized clinical trial. Ann Surg. 2012;255:840-845. 162. Jonas M, Speake W, Scholefield J. Diltiazem heals glyceryl trinitrate-resistant chronic anal fissures: a prospective study. Dis Colon Rectum. 2002;45:1091-1095. 163. Mentes BB, Irkörücü O, Akin M, et al. Comparison of botu-linum toxin injection and lateral internal sphincterotomy for the treatment of chronic anal fissure. Dis Colon Rectum. 2003;46:232-237. 164. Nelson RL, Thomas K, Morgan J, et al. Nonsurgical therapy for anal fissure. Cochrane Database Syst Rev. 2012;2:CD003431. 165. North JH, Jr,

1	anal fissure. Dis Colon Rectum. 2003;46:232-237. 164. Nelson RL, Thomas K, Morgan J, et al. Nonsurgical therapy for anal fissure. Cochrane Database Syst Rev. 2012;2:CD003431. 165. North JH, Jr, Weber TK, Rodriguez-Bigas MA, et al. The management of infectious and noninfectious anorectal complications in patients with leukemia. J Am Coll Surg. 1996;183:322-328. 166. Jacob TJ, Perakath B, Keighley MR. Surgical interven-tion for anorectal fistula. Cochrane Database Syst Rev. 2010;5:CD006319. This meta-analysis evaluated 10 random-ized trials on outcomes after anorectal fistula procedures, with the ideal procedure stated to be associated with low recurrence rates, minimal incontinence, and good quality of life. Evalu-ated surgical interventions included: fistulotomy, fistulectomy, myo-mucosal advancement flap, fibrin glue, and cutting or loose setons. There were no differences in the procedures compared, except that fibrin glue had more recurrences. Trial sample sizes were noted to be

1	advancement flap, fibrin glue, and cutting or loose setons. There were no differences in the procedures compared, except that fibrin glue had more recurrences. Trial sample sizes were noted to be small, and more recently dis-cussed procedures such as the anal fistula plug and Ligation of Intersphincteric Fistula Tract (LIFT) were not evaluated in this review. 167. De Groof EJ, Cabral VN, Buskens CJ, et al. Systematic review of the evidence and consensus on perianal fistula: an analy-sis of national and international guidelines. Colorectal Dis. 2016;18(4):O119-O134. 168. Shanwani A, Nor AM, Amri N. Ligation of the intersphincteric fistula tract (LIFT): a sphincter-saving technique for fistula-in-ano. Dis Colon Rectum. 2010;53:39-42. 169. Gunter J. Genital and perianal warts: new treatment opportuni-ties for human papillomavirus infection. Am J Obstet Gynecol. 2003;189(3 suppl):S3-S11. 170. Palefsky JM. Anal squamous intraepithelial lesions: relation to HIV and human papillomavirus

1	opportuni-ties for human papillomavirus infection. Am J Obstet Gynecol. 2003;189(3 suppl):S3-S11. 170. Palefsky JM. Anal squamous intraepithelial lesions: relation to HIV and human papillomavirus infection. J Acquir Immune Defic Syndr. 1999;21(suppl 1):S42-S48. 171. Bosarge PL, Como JJ, Fox N, et al. Management of penetrat-ing extraperitoneal rectal injuries: an Eastern Association for the Surgery of Trauma practice management guideline. J Trauma Acute Care Surg. 2016;80(3):546-551. 172. Buie WD, Lowry AC, Rothenberger DA, et al. Clinical rather than laboratory assessment predicts continence after anterior sphincteroplasty. Dis Colon Rectum. 2001;44:1255-1260. 173. Baeten CG, Bailey HR, Bakka A, et al. Safety and efficacy of dynamic graciloplasty for fecal incontinence: report of a prospective, multicenter trial. Dynamic Graciloplasty Therapy Study Group. Dis Colon Rectum. 2000;43:743-751. 174. Hussain ZI, Lim M, Stojkovic SG. Systematic review of peri-anal implants in the treatment

1	multicenter trial. Dynamic Graciloplasty Therapy Study Group. Dis Colon Rectum. 2000;43:743-751. 174. Hussain ZI, Lim M, Stojkovic SG. Systematic review of peri-anal implants in the treatment of faecal incontinence. Br J Surg. 2011;98:1526-1536. 175. Vaizey CJ, Kamm MA, Roy AJ, et al. Double-blind crossover study of sacral nerve stimulation for fecal incontinence. Dis Colon Rectum. 2000;43:298-302. 176. Tan EK, Vaizey C, Cornish J, et al. Surgical strategies for faecal incontinence—a decision analysis between dynamic gracilo-plasty, artificial bowel sphincter, and end stoma. Colorectal Dis. 2008;10:577-586. 177. Cooksley CD, Hwang LY, Waller DK, et al. HIV-related malig-nancies: community-based study using linkage of cancer reg-istry and HIV registry data. Int J STD AIDS. 1999;10:795-802. 178. Goldberg H, Baxter NN, Hertz M, Madoff RD, Ricciardi R, Bullard KM. Colon and rectal complications in heart and lung transplant patients. J Am Coll Surg. 2006;202:55-61. 179. Ullery BW, Pieracci

1	H, Baxter NN, Hertz M, Madoff RD, Ricciardi R, Bullard KM. Colon and rectal complications in heart and lung transplant patients. J Am Coll Surg. 2006;202:55-61. 179. Ullery BW, Pieracci FM, Rodney JRM, Barie PS. Neutropenic enterocolitis. Surg Infect (Larchmt). 2009;10(3):307-314.Brunicardi_Ch29_p1259-p1330.indd 132923/02/19 2:30 PM

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1	The AppendixFadi S. Dahdaleh, David Heidt, and Kiran K. Turaga 30chapterHISTORYAlthough anatomists such as Vesalius and Leonardo Da Vinci had written about the appendix, Claudius Amyand in the early 18th century was the first surgeon to describe a successful appendectomy.1 In subsequent centuries, significant progress was made in the diagnosis and management of appendicitis, especially after Chester McBurney advocated for early appen-dectomy in his 1889 publication.2 Famously, the magician Harry Houdini died of a ruptured appendix after suffering a blow to his abdomen. Following the introduction and widespread use of antibiotics in the 1940s, mortality rates improved further. In 1982, Kurt Semm, a gynecologist, reported on the first laparo-scopic appendectomy, which is now the most widely adopted technique.EMBRYOLOGY, ANATOMY, AND HISTOLOGYPreviously considered a vestigial organ, the appendix is now linked to the development and preservation of gut-associated lymphoid tissue (GALT)

1	adopted technique.EMBRYOLOGY, ANATOMY, AND HISTOLOGYPreviously considered a vestigial organ, the appendix is now linked to the development and preservation of gut-associated lymphoid tissue (GALT) and to the maintenance of intestinal flora. It has been suggested that appendectomy is associated with increased Clostridium difficile infections and increased subse-quent cancer (colon, esophageal) as a result of microbial altera-tion, although this is currently unproven.3 The protective effect of an early appendectomy against development of ulcerative colitis has been proposed to be mechanistically linked to the release of dimeric forms of IgA from plasma B cells and the Th2 response mediated by IL-13–producing natural killer T cells.4The appendix, along with the ileum and the colon, devel-ops from the midgut and first appears at 8 weeks of gestation. As the gut rotates medially, the cecum becomes fixed in the right lower quadrant, thus determining the final position of the appendix. The

1	from the midgut and first appears at 8 weeks of gestation. As the gut rotates medially, the cecum becomes fixed in the right lower quadrant, thus determining the final position of the appendix. The appendix is a true diverticulum of the cecum as it contains all the histological layers of the colon, although cer-tain differences in the irregularity of crypts remain. The average appendix measures 6 to 9 cm and derives its blood supply from the appendicular branch of the ileocolic artery. Visceral innerva-tion occurs along the superior mesenteric plexus (T10-L1) and the vagus nerves. The appendix is intraperitoneal and retrocecal in location, but it can be pelvic (30%) and retroperitoneal (7%).5 Grossly, the appendiceal base can be identified by tracing the convergence of the cecal taeneia.ACUTE APPENDICITISInflammation of the appendix is a significant public health prob-lem with a lifetime incidence of 8.6% in men and 6.7% in women, with the highest incidence occurring in the second and

1	APPENDICITISInflammation of the appendix is a significant public health prob-lem with a lifetime incidence of 8.6% in men and 6.7% in women, with the highest incidence occurring in the second and third decade of life.6 While the rate of appendectomy in developed countries has decreased over the last several decades, it remains one of the most frequent emergent abdominal operations.7The etiology of appendicitis is perhaps due to luminal obstruction that occurs as a result of lymphoid hyperplasia in pediatric populations; in adults, it may be due to fecaliths, fibro-sis, foreign bodies (food, parasites, calculi), or neoplasia.5,8-10 Early obstruction leads to bacterial overgrowth of aerobic organisms in the early period, and subsequently, it leads to mixed flora. Obstruction generally leads to increased intralumi-nal pressure and referred visceral pain to the periumbilical region.10 It is postulated that this leads to impaired venous drain-age, mucosal ischemia leading to bacterial

1	to increased intralumi-nal pressure and referred visceral pain to the periumbilical region.10 It is postulated that this leads to impaired venous drain-age, mucosal ischemia leading to bacterial translocation, and subsequent gangrene and intraperitoneal infection. Escherichia coli and Bacteroides fragilis are the most common aerobic and anaerobic bacteria isolated in perforated appendicitis.11,12 1History 1331Embryology, Anatomy, and Histology 1331Acute Appendicitis 1331Clinical Diagnosis 1332History / 1332Physical Examination / 1332Laboratory Findings / 1332Imaging / 1332Differential Diagnosis / 1335Management of Appendicitis 1335Uncomplicated Appendicitis / 1332Complicated Appendicitis / 1335Operative Intervention 1335Preoperative Preparation / 1335Operative Technique / 1336Novel Techniques / 1336Negative Exploration / 1336Incidental Appendectomy / 1336Special Circumstances 1337Appendicitis in Children / 1337Appendicitis in Older Adults / 1338Appendicitis in Pregnancy /

1	Techniques / 1336Negative Exploration / 1336Incidental Appendectomy / 1336Special Circumstances 1337Appendicitis in Children / 1337Appendicitis in Older Adults / 1338Appendicitis in Pregnancy / 1338Chronic or Recurrent Appendicitis / 1338Outcomes and Postoperative Course 1338Stump Appendicitis / 1338Appendiceal Neoplasms / 1338Gastroenteropancreatic Neuroendocrine Tumors (GEP-NETs or Carcinoid) / 1338Goblet Cell Carcinomas / 1339Lymphomas / 1339Adenocarcinoma / 1339Appendiceal Mucoceles and Mucinous Neoplasms of the Appendix / 1339Pseudomyxoma Peritonei Syndrome / 1340Brunicardi_Ch30_p1331-p1344.indd 133101/03/19 7:05 PM 1332Key Points1 Inflammation of the appendix is a significant public health problem with a lifetime incidence of 8.6% in men and 6.7% in women, with the highest incidence in the second and third decade of life.6 While the rate of appendectomy in devel-oped countries has decreased over the last several decades, it remains one of the most frequent emergent

1	incidence in the second and third decade of life.6 While the rate of appendectomy in devel-oped countries has decreased over the last several decades, it remains one of the most frequent emergent abdominal operations.2 The natural history of appendicitis is unclear, but it appears that progression to perforation is not predictable and that spontaneous resolution is common, suggesting that nonper-forated and perforated appendicitis may, in fact, be different diseases.3 C-reactive protein, bilirubin, Il-6, and procalcitonin have all been suggested to be helpful in the diagnosis of appendicitis, specifically in predicting perforated appendicitis.4 Perforated appendicitis can be managed either operatively or nonoperatively. Immediate surgery is necessary in patients that appear septic, but this is usually associated with higher complications, including abscesses and enterocutaneous fis-tulae, due to dense adhesions and inflammation.5 Single incision appendectomy has not been shown to

1	this is usually associated with higher complications, including abscesses and enterocutaneous fis-tulae, due to dense adhesions and inflammation.5 Single incision appendectomy has not been shown to improve outcomes, including cosmetic outcomes, in prospective randomized studies and has been suggested to have a higher incisional hernia rate.6 While there is no evidence clearly evaluating long-term out-comes of patients undergoing incidental appendectomy with an asymptomatic appendix, the risk of adhesions and future complications after an appendectomy has been suggested to be higher than the risk of future appendicitis and increased economic costs. An incidental appendectomy is currently not advocated.7 Older adult patients are at a higher risk for complications due to their premorbid conditions, and it is prudent to obtain definitive diagnostic imaging prior to taking patients to the operating room.8 Patients with uncomplicated appendicitis do not require fur-ther antibiotics after an

1	and it is prudent to obtain definitive diagnostic imaging prior to taking patients to the operating room.8 Patients with uncomplicated appendicitis do not require fur-ther antibiotics after an appendectomy, while patients with perforated appendicitis are treated with 3 to 7 days of antibiotics.9 The most common mode of presentation for appendiceal car-cinoma is that of acute appendicitis. Patients also may pres-ent with ascites or a palpable mass, or the neoplasm may be discovered during an operative procedure for an unrelated cause.This sequence is not inevitable, however, and some episodes of acute appendicitis may resolve spontaneously. Due to differ-ences in epidemiology, nonperforated and perforated appendicitis are considered different diseases.13 Addition-ally, since not all nonperforated appendicitis progresses to per-forations, it is suggested that the pathogenesis of the two conditions may be different.CLINICAL DIAGNOSISHistoryIt is important to elicit an accurate history

1	appendicitis progresses to per-forations, it is suggested that the pathogenesis of the two conditions may be different.CLINICAL DIAGNOSISHistoryIt is important to elicit an accurate history from the patient and/or family, in the case of pediatric patients. Inflammation of the visceral peritoneum usually progresses to the parietal perito-neum, presenting with migratory pain, which is a classic sign of appendicitis (likelihood ratio+, 2.06 [1.63–2.60]).14 Inflam-mation can often result in anorexia, nausea, vomiting, and fever (Table 30-1). Regional inflammation can also present with an ileus, diarrhea, small bowel obstruction, and hematuria. Perti-nent negative history (including menstrual) must be obtained to rule out other etiologies of abdominal pain.Physical ExaminationMost patients lay quite still due to parietal peritonitis. Patients are generally warm to the touch (with a low-grade fever, ∼38.0°C [100.4°F]) and demonstrate focal tenderness with guarding. McBurney’s point, which

1	quite still due to parietal peritonitis. Patients are generally warm to the touch (with a low-grade fever, ∼38.0°C [100.4°F]) and demonstrate focal tenderness with guarding. McBurney’s point, which is found one-third of the distance between the anterior superior iliac spine and the umbili-cus, is often the point of maximal tenderness in a patient with an anatomically normal appendix. Certain physical signs with their respective eponyms can be helpful in discerning the location of the appendix: Rovsing’s sign, pain in the right lower quad-rant after release of gentle pressure on left lower quadrant (nor-mal position); Dunphy’s sign, pain with coughing (retrocecal 2appendix); obturator sign, pain with internal rotation of the hip (pelvic appendix); iliopsoas sign, pain with flexion of the hip (retrocecal appendix). In addition, pain with rectal or cervical examinations is also suggestive of pelvic appendicitis.Laboratory FindingsPatients with appendicitis usually have leukocytosis of

1	hip (retrocecal appendix). In addition, pain with rectal or cervical examinations is also suggestive of pelvic appendicitis.Laboratory FindingsPatients with appendicitis usually have leukocytosis of 10,000 cells/mm3, with a higher leukocytosis associated with gangrenous and perforated appendicitis (∼17,000 cells/mm3). C-reactive pro-tein, bilirubin, Il-6, and procalcitonin have all been sug-gested to help in the diagnosis of appendicitis, specifically in predicting perforated appendicitis.14,15 The authors believe that a white blood cell (WBC) count and a C-reactive protein are two appropriate lab tests to obtain in the initial work up of appendicitis; a pregnancy test is also essential in women of childbearing age. Lastly, a urinalysis can be valuable in ruling out nephrolithiasis or pyelonephritis.ImagingImaging is often utilized to confirm a diagnosis of appendici-tis because a negative operation rate is acceptable in <10% of male patients and <20% of female patients. Routine use

1	is often utilized to confirm a diagnosis of appendici-tis because a negative operation rate is acceptable in <10% of male patients and <20% of female patients. Routine use of crosssectional imaging somewhat reduces the rate of negative laparot-omies. Imaging studies are most appropriate for patients in whom a diagnosis of appendicitis is unclear or who are at high risk from operative intervention and general anesthesia, such as pregnant patients or patients with multiple comorbidities. Commonly utilized imaging modalities include computerized tomography (CT), ultrasound (US), and magnetic resonance imaging (MRI).CT Scan. A contrast-enhanced CT scan has a sensitivity of 0.96 (95% confidence interval [CI] 0.95–0.97) and specificity of 0.96 (95% CI 0.93–0.97) in diagnosing acute appendicitis.16,17 Features on a CT scan that suggest appendicitis include enlarged lumen and double wall thickness (greater than 6 mm), 3Brunicardi_Ch30_p1331-p1344.indd 133201/03/19 7:05 PM 1333THE

1	Features on a CT scan that suggest appendicitis include enlarged lumen and double wall thickness (greater than 6 mm), 3Brunicardi_Ch30_p1331-p1344.indd 133201/03/19 7:05 PM 1333THE APPENDIXCHAPTER 30Table 30-1Signs and symptoms of appendicitis TRUE POSITIVE LIKELIHOOD RATIO95% CONFIDENCE INTERVALTRUE NEGATIVE LIKELIHOOD RATIO95% CONFIDENCE INTERVALDuration of symptoms (hours) >9 >12 >24 >481.010.960.650.490.97–1.050.90–1.040.47–0.900.36–0.670.941.191.471.200.62–1.420.87–1.631.14–1.901.08–1.34Fever1.640.89–3.010.610.49–0.77Gastrointestinal dysfunction Anorexia Nausea Vomiting1.271.151.631.14–1.411.04–1.361.45–1.840.590.720.750.45–0.770.57–0.910.69–0.80Pain Pain migration Pain progression Direct tenderness Indirect tenderness Psoas sign Rebound Percussion

1	migration Pain progression Direct tenderness Indirect tenderness Psoas sign Rebound Percussion tenderness Guarding Rigidity2.061.391.292.472.311.992.862.482.961.63–2.601.29–1.501.06–1.571.38–4.431.36–3.911.61–2.451.95–4.211.60–3.842.43–3.590.520.460.250.710.850.390.490.570.860.40–0.690.27–0.770.12–0.530.65–0.770.76–0.950.32–0.480.37–0.630.48–0.680.72–1.02Temperature (degrees centigrade) >37.7 >38.51.571.870.90–2.760.66–5.320.650.890.31–1.360.71–1.12White blood cells (109/L) ≥10 ≥154.207.202.11–8.354.31–12.000.200.660.10–0.410.56–0.78C-reactive protein (mg/L) >10 >201.972.391.58–2.451.67–3.410.320.470.20–0.510.28–0.81Conclusions: Individually, disease history, clinical findings, and laboratory tests are weak. But when combined, they yield high discriminatory power.Data from Andersson RE: Meta-analysis of the clinical and laboratory diagnosis of appendicitis, Br J Surg. 2004 Jan;91(1):28-37.wall thickening (greater than 2 mm), periappendiceal fat stranding, appendiceal wall thickening,

1	of the clinical and laboratory diagnosis of appendicitis, Br J Surg. 2004 Jan;91(1):28-37.wall thickening (greater than 2 mm), periappendiceal fat stranding, appendiceal wall thickening, and/or an appendicolith (Fig. 30-1). While there remains a concern of ionizing radia-tion exposure with a CT scan, typical low-dose CT scans result in exposure of 2 to 4 mSv, which is not significantly higher than background radiation (3.1 mSv).18 Recent trials have also suggested that although low-dose CT scans of 2 mSv do not generate high-resolution images, using these lower resolution images does not affect clinical outcomes.19 Intravenous contrast is generally preferred in these studies, but it can be avoided in patients with allergies or low estimated glomerular filtration rate (less than 30 mL/minute for 1.73 m2). Several meta-analyses have suggested that CT scan is more sensitive and specific than ultrasound in diagnosing appendicitis.Ultrasound. Ultrasonography has a sensitivity of 0.85 (95%

1	for 1.73 m2). Several meta-analyses have suggested that CT scan is more sensitive and specific than ultrasound in diagnosing appendicitis.Ultrasound. Ultrasonography has a sensitivity of 0.85 (95% CI 0.79–0.90) and a specificity of 0.90 (95% CI 0.83–0.95).20 Graded compression ultrasonography is used to identify the anteroposterior diameter of the appendix. An easily compressible appendix <5 mm in diameter generally rules out appendicitis. Features on an ultrasound that suggest appendicitis include a diameter of greater than 6 mm, pain with compression, presence of an appendicolith, increased echogenicity of the fat, and periappendiceal fluid.21 Ultrasound is cheaper and more readily available than CT scan, and it does not expose patients to ionizing radiation, but it is user-dependent and has limited util-ity in obese patients. In addition, graded compression is usually Figure 30-1. McBurney’s point. 1 = anterior superior iliac spine; 2 = umbilicus; x = McBurney’s

1	user-dependent and has limited util-ity in obese patients. In addition, graded compression is usually Figure 30-1. McBurney’s point. 1 = anterior superior iliac spine; 2 = umbilicus; x = McBurney’s point.Brunicardi_Ch30_p1331-p1344.indd 133301/03/19 7:05 PM 1334SPECIFIC CONSIDERATIONSPART IITable 30-2Meta-analyses comparing CT scan and US outcomes AUTHORSUMMARYTERASAWAWESTONDORIAAL-KHAYALVAN RANDENYear 20042005200620072008 No. of studies 222157256 No. of patientsCTUSTotal117215162688NRNR5039NRNR13697NRNR13046NRNR671 SensitivityCTUS94% (CI: 91%–95%)86% (CI: 83%–88%)97% (CI: 95%–98%)87% (CI: 85%–89%)94% (CI: 92%–97%)88% (CI: 86%–90%)93% (CI: 92%–95%)84% (CI: 82%–85%)91% (CI: 84%–95%)78% (CI: 67%–86%)CT more sensitive than US in five of five meta-analysesSpecificityCTUS95% (CI: 93%–96%)81% (CI: 78%–84%)95% (CI: 93%–96%)93% (CI: 92%–94%)94% (CI: 94%–96%)93% (CI: 90%–96%)93 (CI: 92%–94%)96 (CI: 95%–96%)90% (CI: 85%–94%)83% (CI: 76%–88%)CT more specific than US in four of five

1	93%–96%)81% (CI: 78%–84%)95% (CI: 93%–96%)93% (CI: 92%–94%)94% (CI: 94%–96%)93% (CI: 90%–96%)93 (CI: 92%–94%)96 (CI: 95%–96%)90% (CI: 85%–94%)83% (CI: 76%–88%)CT more specific than US in four of five meta-analysesPositive predictive valueCTUSNRNR94% (CI: 92%–95%)89% (CI: 87%–90%)NRNR90% (CI: 89%–92%)90% (CI: 89%–91%)NRNRCT has superior positive predictive value in one of two meta-analysesNegative predictive valueCTUSNRNR97% (CI: 96%–98%)92% (CI:91%–93%)NRNR96% (CI: 95%–97%)93% (CI: 92%–94%)NRNRCT has superior negative predictive value in both meta-analysesAccuracyCTUSNRNRNRNRNRNR94% (CI: 93%–94%)92% (CI: 92%–96%)NRNRCT is more accurate in the one study reporting resultsCI = confidence interval; CT = computed tomography; NR = not reported; US = ultrasonography.Brunicardi_Ch30_p1331-p1344.indd 133401/03/19 7:05 PM 1335THE APPENDIXCHAPTER 30painful for patients with peritonitis. A comparison of the effi-cacy of ultrasound v. CT scan is found in Table 30-2.MRI. MRI of the abdomen has

1	133401/03/19 7:05 PM 1335THE APPENDIXCHAPTER 30painful for patients with peritonitis. A comparison of the effi-cacy of ultrasound v. CT scan is found in Table 30-2.MRI. MRI of the abdomen has a sensitivity of 0.95 (95% CI 0.88–0.98) and specificity of 0.92 (95% CI 0.87–0.95) for iden-tification of acute appendicitis.22 MRI is an expensive test that requires significant expertise to perform and interpret and is usually recommended in patients for whom the risk of ionizing radiation outweighs the relative ease of obtaining a contrast CT scan, i.e., pregnant or pediatric patients.Differential DiagnosisCauses of acute abdominal pain that are often confused with acute appendicitis include acute mesenteric adenitis, cecal diverticulitis, Meckel’s diverticulitis, acute ileitis, Crohn’s dis-ease, acute pelvic inflammatory disease, torsion of ovarian cyst or graafian follicle, and acute gastroenteritis. Frequently, no organic pathology is identified. Obtaining an antecedent history of a

1	acute pelvic inflammatory disease, torsion of ovarian cyst or graafian follicle, and acute gastroenteritis. Frequently, no organic pathology is identified. Obtaining an antecedent history of a viral infection (mesenteric adenitis or gastroenteritis) and a cervical exam in women (exquisite tenderness with motion in pelvic inflammatory disease) are essential before planning any intervention. Detailed menstrual history can distinguish mittel-schmerz (no fever or leukocytosis, mid-menstrual cycle pain) and ectopic pregnancies.MANAGEMENT OF APPENDICITISUncomplicated AppendicitisThe preferred approach to manage patients with uncomplicated appendicitis is an appendectomy. Several recent randomized trials and cohort studies have examined the role of nonopera-tive management of adult patients with appendicitis.23,24,25 In each of these well-designed studies with noninferiority as the endpoint, patients were randomized to either receiving antibiot-ics or undergoing an appendectomy, which was

1	appendicitis.23,24,25 In each of these well-designed studies with noninferiority as the endpoint, patients were randomized to either receiving antibiot-ics or undergoing an appendectomy, which was frequently per-formed open. A majority of the patients in the nonoperative arm received intravenous antibiotics for a short course followed by a course of a fluoroquinolone and metronidazole, or oral amoxi-cillin/clavulanic acid.23,26,27 Meta-analysis of the published data found that 26.5% of patients in the nonoperative group required an appendectomy within 1 year. In addition, the rate of adverse events following antibiotics therapy was higher (relative risk [RR] 3.18, 95% CI 1.63–6.21, P = 0.0007), and patients who recurred presented more frequently with complicated appen-dicitis (RR 2.52, 95% CI 1.17–5.43, P = 0.02).28,29 Currently, conservative management can be offered to informed patients using techniques of shared decision-making, but it is not the standard modality of management of

1	CI 1.17–5.43, P = 0.02).28,29 Currently, conservative management can be offered to informed patients using techniques of shared decision-making, but it is not the standard modality of management of appendicitis, except in patients with significant phobia of surgery.30 Societal costs and long-term implications of the conservative strategy have not yet been completely evaluated.Timing of Surgery. Emergent surgery is often performed in patients with appendicitis, but studies have evaluated the perfor-mance of urgent surgery (waiting less than 12 hours) in a semi-elective setting after administering antibiotics upon admission. The studies did not reveal any significant difference in outcomes, except for a slightly longer hospital stay in those undergoing urgent surgery.31-33 Currently, delaying surgery less than 12 hours is acceptable in patients with short duration of symptoms (less than 48 hours) and in nonperforated, nongangrenous appendicitis.Approach of Surgery. Numerous

1	delaying surgery less than 12 hours is acceptable in patients with short duration of symptoms (less than 48 hours) and in nonperforated, nongangrenous appendicitis.Approach of Surgery. Numerous meta-analyses comparing laparoscopic to open appendectomy have demonstrated relative equivalence of the techniques, with laparoscopic appendec-tomy resulting in a shorter length of stay (LOS), faster return to work, and lower superficial wound infection rates, especially in obese patients.34,35 Open appendectomy results in shorter operative times and lower intra-abdominal infection rates.36 Costs of the two techniques are relatively similar because of the offset of costs in laparoscopic techniques by shorter LOS. In the United States, laparoscopic appendectomies are increas-ingly utilized.37Complicated AppendicitisPerforated and gangrenous appendicitis and appendicitis with abscess or phlegmon formation are considered complicated conditions. Patients with perforated appendicitis usually

1	AppendicitisPerforated and gangrenous appendicitis and appendicitis with abscess or phlegmon formation are considered complicated conditions. Patients with perforated appendicitis usually pres-ent after 24 hours of onset, although 20% of patients present within 24 hours. Such patients are often acutely ill and dehy-drated and require resuscitation. Usually, the perforated abscess is walled off in the right lower quadrant, although retroperito-neal abscesses including psoas abscess, liver abscesses, fistu-las, and pylephlebitis (portal vein inflammation) can also occur when left untreated.Perforated appendicitis can be managed either operatively or nonoperatively. Immediate surgery is necessary in patients that appear septic, but this is usually associated with higher complications, including abscesses and enterocuta-neous fistulae due to dense adhesions and inflammation. The management of long-duration, complicated appendici-tis is often staged.38,39 Patients are resuscitated and

1	abscesses and enterocuta-neous fistulae due to dense adhesions and inflammation. The management of long-duration, complicated appendici-tis is often staged.38,39 Patients are resuscitated and treated with IV antibiotics.40,41 Patients with longstanding perforation are better treated with adequate percutaneous image-guided drain-age.42 This strategy is successful in 79% of patients who achieve complete resolution, which occurs more often in lower-grade abscesses, transgluteal drainage, and with CT(vs. ultrasound-) guided drainage43 Operative intervention is performed in patients who fail conservative management and in patients with free intra-peritoneal perforation.Interval Appendectomy. The majority of patients with perfo-rated appendicitis (80%) have resolution of their symptoms with drainage and antibiotics. There remains debate about the value of performing an interval appendectomy 6 to 8 weeks after the original inflammatory episode.44-46 Proponents of this approach cite the

1	drainage and antibiotics. There remains debate about the value of performing an interval appendectomy 6 to 8 weeks after the original inflammatory episode.44-46 Proponents of this approach cite the incidence of recurrent appendicitis (7.4%–8.8%) and the presence of appendiceal neoplasms detected on the appendec-tomy (relevant benign lesions 0.7%, malignant lesions 1.3%).47 Opponents cite the high incidence of no future events after a median follow-up of 34 months in 91% of patients. Currently, shared decision-making is necessary before proceeding with an interval appendectomy.39OPERATIVE INTERVENTIONPreoperative PreparationOnce the decision to proceed with surgical intervention is made, patients can be taken to the operating room rather expeditiously. While resuscitative efforts are important in patients who pres-ent with significant dehydration or in a compromised host, the majority of patients can be taken to the operating room within a short interval. Placement of a Foley catheter

1	in patients who pres-ent with significant dehydration or in a compromised host, the majority of patients can be taken to the operating room within a short interval. Placement of a Foley catheter is optional but not necessary while performing an appendectomy. Preopera-tive antibiotics must be administered at least 30 to 60 minutes prior to skin incision. The choice of antibiotics include cefoxi-tin, ampicillin/sulbactam, and cefazolin plus metronidazole for 4Brunicardi_Ch30_p1331-p1344.indd 133501/03/19 7:05 PM 1336SPECIFIC CONSIDERATIONSPART IIuncomplicated appendicitis. Patients with ß-lactam allergies can be given clindamycin in combination with a fluoroquinolone, gentamicin, or aztreonam. Postoperative antibiotics are usually not necessary.In patients with perforated appendicitis undergoing opera-tive intervention, preoperative antibiotics are necessary to cover gram-negative bacteria and anaerobes. Monotherapy with piper-acillin/tazobactam or combination of cephalosporin with

1	opera-tive intervention, preoperative antibiotics are necessary to cover gram-negative bacteria and anaerobes. Monotherapy with piper-acillin/tazobactam or combination of cephalosporin with metro-nidazole are reasonable choices. The duration of postoperative antibiotics is generally less than 4 days once complete source control has been achieved (STOP-IT trial).48 Patients with incomplete drainage, persistent catheters, complications from surgery, and uncertain resolution of inflammation might need a longer duration of antibiotics.49Operative TechniqueOpen Appendectomy. An open appendectomy is usually per-formed under general anesthesia, although regional anesthesia can be used. After wide prep and drape, an incision is usually made on McBurney’s point either in an oblique fashion (McBur-ney’s incision) or transverse incision (Rocky-Davis incision). A lower midline laparotomy incision is more appropriate for perforated appendicitis with a phlegmon. A muscle-splitting approach can be

1	incision) or transverse incision (Rocky-Davis incision). A lower midline laparotomy incision is more appropriate for perforated appendicitis with a phlegmon. A muscle-splitting approach can be utilized to access the peritoneum in patients that are well paralyzed. The bed is positioned in Trendelen-burg’s with the left side down. The appendix is usually readily identified, but if necessary, it can be found by tracing the ante-rior taenia (taenia Liberia) of the cecum distally. We generally ligate the mesentery early to allow better exposure. If the base of the appendix is viable, ligating the appendix is acceptable. This can be imbricated with a Z-stitch or purse string configura-tion, or alternatively the mucosa can be fulgurated. In the event of retraction of the appendiceal artery or unexpected bleeding, the incision can be extended medially (Fowler extension). Skin closure is usually performed in a layered fashion, but in cases with significant abscess or contamination, closure

1	bleeding, the incision can be extended medially (Fowler extension). Skin closure is usually performed in a layered fashion, but in cases with significant abscess or contamination, closure by sec-ondary intention or delayed primary closure has been consid-ered. Recent trials have suggested no difference in surgical site infection rates between primary and delayed primary closure.50 Placement of surgical drains has not been proven to be benefi-cial in multiple clinical trials for either complicated or uncom-plicated appendicitis.51,52Laparoscopic Appendectomy. Patients undergoing laparo-scopic appendectomy are positioned supine with the left arm tucked for better access. Monitors and assistants are positioned appropriately. Access to the peritoneum can be obtained using either the Hasson technique in a periumbilical fashion or with a Verees or optical trocar in the left upper quadrant 3 cm below the costal margin in the midclavicular line. Five-mm ports are usually placed in the

1	technique in a periumbilical fashion or with a Verees or optical trocar in the left upper quadrant 3 cm below the costal margin in the midclavicular line. Five-mm ports are usually placed in the suprapubic and left lower quadrant areas. It is also technically feasible to place the third port in the right upper quadrant. The bed is positioned in Trendelenburg, with the left side down to sweep the bowel away. The appendix is grasped and elevated upwards to identify the window between the mesoappendix and the cecum (Fig. 30-2). Occasionally, it is essential to release the mesenteric attachments of the cecum to mobilize a retrocecal or pelvic appendix to obtain this view. Using a Maryland grasper, the window is created, and the mesoappendix is divided with cautery, clip, or a bipolar energy source. The base of the appendix is divided either with an endoscopic stapler or after placing an endoloop. In the case of a nonviable appendix base, a staple line through the cecum that avoids the

1	source. The base of the appendix is divided either with an endoscopic stapler or after placing an endoloop. In the case of a nonviable appendix base, a staple line through the cecum that avoids the ileocecal valve might be sufficient, unless sig-nificant inflammation is present. The appendix is retrieved through the midline port in a specimen bag, especially if an appendiceal lesion is suspected. If a periappendiceal phlegmon is encountered or if the operation is being performed for perfo-rated appendicitis, careful sweeping of the bowel with a blunt dissector can release the appendix. It is important to carefully separate adjacent bowel, which can be friable in such settings. Conversion to open surgery should be considered for failure to progress. Typically, once the base of the appendix is identified, it is generally more helpful to divide the stump first. An endo-scopic stapler or endoloop can be used for the base, provided the base is viable. Occasionally, an ileocecectomy is

1	is identified, it is generally more helpful to divide the stump first. An endo-scopic stapler or endoloop can be used for the base, provided the base is viable. Occasionally, an ileocecectomy is necessary when resection of the base of the appendix or cecum is likely to impinge on the ileocecal valve. The mesoappendix is similarly divided with either a stapler with thin leg length staples, a clip, cautery, or energy device.Novel TechniquesThree novel techniques have been investigated in the performance of an appendectomy: single incision appendectomy, natural orifice transluminal endoscopic surgery (NOTES), and robotic appendec-tomy. Single incision appendectomy has not been shown to improve outcomes, including cosmetic outcomes, in pro-spective randomized studies and has been suggested to have a higher incisional hernia rate.53 NOTES surgery has been shown to have better cosmetic outcome and less postoperative pain in a meta-analysis of NOTES procedures including appendectomies,

1	to have a higher incisional hernia rate.53 NOTES surgery has been shown to have better cosmetic outcome and less postoperative pain in a meta-analysis of NOTES procedures including appendectomies, although only 40 patients were included in the analysis.54 The risk of luminal contamination and closure of enteral or vaginal mucosa remain suboptimal; for this reason, there has not been widespread dissemination of this technique.55 Robotic appendectomy allows flexible motions of intraperitoneal instruments and is therefore superior in ergonomics for the surgeon.56 However, it is extremely expensive and requires larger ports based on most of the current platforms; thus, this technique is also not utilized widely.Negative ExplorationUpon performing a laparoscopy or laparotomy for suspected appendicitis, if one finds no evidence of appendicitis, a thor-ough exploration of the peritoneum must be performed to rule out contributing pathology. A normal appendix is often removed to reduce future

1	if one finds no evidence of appendicitis, a thor-ough exploration of the peritoneum must be performed to rule out contributing pathology. A normal appendix is often removed to reduce future diagnostic dilemma.57 Management of incidentally found common conditions is summarized in Table 30-3.Incidental AppendectomyThe practice of prophylactic appendectomy has been considered during other operations to prevent the future risk of appendici-tis.6,58 It is routinely performed in children undergoing chemo-therapy, compromised hosts with an unclear physical exam, patients with Crohn’s disease with a normal cecum, patients traveling to remote places with no urgent care, and in patients undergoing cytoreductive operations for ovarian malignancies.59 While there is no evidence clearly evaluating long-term out-comes of patients undergoing incidental appendectomy with an asymptomatic appendix, the risk of adhesions and future complications after an appendectomy has been suggested to be higher than

1	out-comes of patients undergoing incidental appendectomy with an asymptomatic appendix, the risk of adhesions and future complications after an appendectomy has been suggested to be higher than the risk of future appendicitis and increased economic costs. For these reasons, an incidental appendectomy is currently not advocated. 56Brunicardi_Ch30_p1331-p1344.indd 133601/03/19 7:05 PM 1337THE APPENDIXCHAPTER 30SurgeonAssistantAnesthesiologistFigure 30-2. Operating room setup.SPECIAL CIRCUMSTANCESAppendicitis in ChildrenAlmost 1 in 8 children undergo a workup for the diagnosis of appendicitis.60,61 Of these, infants and young children are most likely to present with perforated disease (51%–100%), while school-age children have lower rates of perforation.62,63,64 While most age groups demonstrate the same symptoms previously described in adults, neonates can also present with abdominal distension and lethargy or irritability. The Pediatric Appendici-tis Score has components similar to

1	the same symptoms previously described in adults, neonates can also present with abdominal distension and lethargy or irritability. The Pediatric Appendici-tis Score has components similar to the Alvarado Score and is scored of 10 points, with maximum weight (2 points each) for right lower quadrant tenderness and pain with cough, percussion or hopping. A score of 7 or greater indicates that the patient has a high chance of having appendicitis (78%–96% percent).65In the pediatric population, special considerations must be made to exclude relevant differential diagnoses such as intus-susception (currant jelly stools, abdominal mass), gastroenteritis (often no luekocytosis), malrotation (pain out of proportion), pregnancy (ectopic), mesenteric adenitis, torsion of the omen-tum, and ovarian or testicular torsion.Table 30-3Management of Intraoperative Findings Mimicking AppendicitisOvarian TorsionConservative management with detorsion and oophoropexyCrohn’s terminal ileitisAppendectomy if

1	testicular torsion.Table 30-3Management of Intraoperative Findings Mimicking AppendicitisOvarian TorsionConservative management with detorsion and oophoropexyCrohn’s terminal ileitisAppendectomy if base uninflamedMeckel’s diverticulitisSegmental small bowel resection and primary anastomosisAppendiceal MassLaparoscopic appendectomy/ileocecectomy without capsular disruption or spillage and retrieval in a bagBrunicardi_Ch30_p1331-p1344.indd 133701/03/19 7:05 PM 1338SPECIFIC CONSIDERATIONSPART IIWith regard to the management of children with appen-dicitis, early appendicitis is treated preferably with a laparo-scopic appendectomy, which has better outcomes than open appendectomies in children.66,67 For patients with complicated appendicitis, urgent appendectomy is advocated in the setting of no abscess or mass. Laparoscopic appendectomy appears to retain its benefits in this setting as well.68,69 In the setting of a perforation, antibiotics are continued after surgery for at least 3

1	of no abscess or mass. Laparoscopic appendectomy appears to retain its benefits in this setting as well.68,69 In the setting of a perforation, antibiotics are continued after surgery for at least 3 days, and preferably 5 days (APSA guidelines).70,71 Manage-ment of perforated appendicitis with abscess is similar to adults, although no adverse effects of an early laparoscopic appendec-tomy have been seen even in this setting.39,72,73,74Nonoperative management of appendicitis has also been studied in children.75-77 It may be safe for children with early presentation (less than 48 hours), limited inflammation (WBC less than 18,000/cu.ml), appendicoliths, and no evidence of rup-ture on imaging.78 Patients are usually administered IV antibi-otics until inflammation reduces and then transitioned to oral antibiotics.79 This is usually effective in reducing inflammation (88%–92%), but has a recurrence rate of 22% at 1 year and increased resource utilization.80Appendicitis in Older AdultsOlder

1	to oral antibiotics.79 This is usually effective in reducing inflammation (88%–92%), but has a recurrence rate of 22% at 1 year and increased resource utilization.80Appendicitis in Older AdultsOlder adult patients can have diminished inflammation and thus present with perforation or abscess more frequently.81,82 Such patients are at a higher risk for complications because of their premorbid conditions, and it is more prudent to obtain definitive diagnostic imaging prior to taking patients to the operating room. Laparoscopic appendectomy is safe and might allow patients to reduce pain and their hospital stay.83 Appendicitis in PregnancyAppendicitis occurs in 1 in 800 to 1 in 1000 pregnancies, mostly in the first and second trimesters. Its incidence is rare in the antepartum state, and it can occur in the postpartum state in geriatric pregnancies (maternal age greater than 35 years).84 While the majority of the clinical features are similar, patients can also present with heartburn,

1	can occur in the postpartum state in geriatric pregnancies (maternal age greater than 35 years).84 While the majority of the clinical features are similar, patients can also present with heartburn, bowel irregularity, flatulence, or a change in bowel habits. The point of maximum tender-ness is usually displaced on physical exam. Ultrasonography is the preferred imaging modality, although nonvisualization can occur. Sensitivity can vary from 67% to 100%, and specificity varies from 93% to 96%.39 An alternative imaging modality is MRI, with a sensitivity of 94% and specificity of 97%.85 While CT can be performed in pregnancy, the risk of fetal irradiation leads many practitioners to avoid it unless other modalities are inconclusive.86 When discussing options with the patient and the patient’s family, it is important to note that the risk of fetal loss is up to 36% if appendiceal perforation occurs.87 Therefore, there remains a lower threshold to operate on such patients, with an

1	patient’s family, it is important to note that the risk of fetal loss is up to 36% if appendiceal perforation occurs.87 Therefore, there remains a lower threshold to operate on such patients, with an acceptable negative exploration rate of as high as 30%. Lapa-roscopic appendectomies can be safely performed in pregnant patients, although studies suggest a variable but reproducible higher rate of fetal loss (around 7% vs. 3%) than open tech-niques. Lower intra-abdominal pressures (10–12 mmHg) during insufflation have been suggested to reduce early labor. Nonoper-ative management has also been proposed for pregnant patients, but treatment failure rates have been reported as high as 25%.Chronic or Recurrent AppendicitisPatients with recurrent right lower quadrant abdominal pain not associated with a febrile illness with imaging findings sugges-tive of an appendicolith or dilated appendix are classified as having chronic appendicitis.88 Patients often report resolution of symptoms with an

1	with a febrile illness with imaging findings sugges-tive of an appendicolith or dilated appendix are classified as having chronic appendicitis.88 Patients often report resolution of symptoms with an appendectomy. In the absence of imaging abnormalities, prophylactic appendectomy is not encouraged.45OUTCOMES AND POSTOPERATIVE COURSEAppendectomy is a relatively safe procedure with an extremely low mortality rate (less than 1%). The commonest adverse events include soft tissue infections, either superficial or deep (including abscesses). Patients with uncomplicated appendicitis do not require further antibiotics after an appendec-tomy, while patients with perforated appendicitis are treated with 3 to 7 days of antibiotics (4 days from the STOP-IT trial).89 Patients with wound infections can be managed with simple wound opening and packing, and delayed primary closure has not been shown to be beneficial.90 In laparoscopic cases, these are usually the periumbilical ports.91 Patients with

1	be managed with simple wound opening and packing, and delayed primary closure has not been shown to be beneficial.90 In laparoscopic cases, these are usually the periumbilical ports.91 Patients with deep space abscesses are managed with percutaneous drainage and antibiot-ics. Fistulas (appendicocutaneous or appendicovesicular) are managed conservatively as the first step. Bowel obstructions and infertility are infrequent but reported.Stump AppendicitisAn uncommon complication after surgery is the development of appendicitis in an incompletely excised appendiceal stump (greater than 0.5 cm stump length). Optimal management requires reexcision of the appendiceal base, but diagnosis can be difficult and requires careful assessment of the patient’s history, physical exam, and imaging studies.92 Use of the “appendiceal critical view” (appendix placed at 10 o’clock, taenia coli/libera at 3 o’clock, and terminal ileum at 6 o’clock) and identification of where the taeniae coli merge and

1	Use of the “appendiceal critical view” (appendix placed at 10 o’clock, taenia coli/libera at 3 o’clock, and terminal ileum at 6 o’clock) and identification of where the taeniae coli merge and disappear is paramount to identifying and ligating the base of the appendix during the ini-tial operation (Fig. 30-3). In patients who have had prior appen-dectomy, a low index of suspicion is important to prevent delay in diagnosis and complications. Prior appendectomy should not be an absolute criterion in ruling out acute appendicitis.Appendiceal NeoplasmsThe incidence of appendiceal neoplasms is estimated at around 1% of all appendectomy specimens, although the true incidence of appendiceal neoplasms is not known.93 Neoplasms that occur in the appendix are predominantly gastroenteropancreatic neu-roendocrine tumors (or GEP-NETs, previously called carci-noids), mucinous neoplasms, or adenocarcinomas.94-96 Almost one-third of the neoplasms of the appendix present with acute appendicitis, while

1	tumors (or GEP-NETs, previously called carci-noids), mucinous neoplasms, or adenocarcinomas.94-96 Almost one-third of the neoplasms of the appendix present with acute appendicitis, while the others are often incidentally detected or are detected after regional spread of disease.97Gastroenteropancreatic Neuroendocrine Tumors (GEP-NETs or Carcinoid)Appendiceal carcinoid tumors are submucosal rubbery masses that are detected incidentally on the appendix.98 Carcinoid tumors of the appendix are relatively indolent but can develop nodal or hepatic metastases.99 Infrequently, these can be associ-ated with a carcinoid syndrome if there are hepatic metastases (2.9%).100 Upon incidental findings of a suspected carcinoid, the surgeon must evaluate the nodal basin along the ileocolic ped-icle and also examine the liver for any signs of metastases. For lesions that are less than 1 cm (95% of all lesions), a negative margin appendectomy is adequate. For tumors 2 cm or larger, a right hemicolectomy

1	examine the liver for any signs of metastases. For lesions that are less than 1 cm (95% of all lesions), a negative margin appendectomy is adequate. For tumors 2 cm or larger, a right hemicolectomy is recommended. For lesions 1 to 2 cm in size, there is no consensus on a completion colectomy. A right colectomy is often performed for mesenteric invasion, enlarged 78Brunicardi_Ch30_p1331-p1344.indd 133801/03/19 7:05 PM 1339THE APPENDIXCHAPTER 30Figure 30-3. A and B. Appendiceal critical view.ABnodes, or positive or unclear margins. Measurement of serum chromogranin A is recommended.Goblet Cell CarcinomasThese lesions were mistakenly called goblet cell carcinoids, implying a rather indolent biology, while goblet cell carcinomas are adenocarcinoid with both adenocarcinoma and neuroendocrine features.101,102 Such lesions carry a worse prognosis than carcinoids but slightly better than adenocarcinomas. There is a high risk of peritoneal recurrence in such cases. For incidentally

1	features.101,102 Such lesions carry a worse prognosis than carcinoids but slightly better than adenocarcinomas. There is a high risk of peritoneal recurrence in such cases. For incidentally detected lesions, a systematic surveillance of the peritoneum must be per-formed, and a peritoneal cancer index score must be documented if disease is present.103 In the absence of metastatic disease, a right hemicolectomy is generally appropriate, although some advocate for a right colectomy only for tumors 2 cm or larger.104LymphomasAppendiceal lymphomas are rare (1%–3% of lymphomas, usu-ally non-Hodgkin’s) and difficult to diagnose preoperatively (appendiceal diameter can be 2.5 cm or larger).105,106 Manage-ment includes an appendectomy in most cases.AdenocarcinomaPrimary adenocarcinoma of the appendix is a rare neoplasm with three major histologic subtypes: mucinous adenocarcinoma, colonic adenocarcinoma, and adenocarcinoid. The most common mode of presentation for appendiceal carcinoma is

1	is a rare neoplasm with three major histologic subtypes: mucinous adenocarcinoma, colonic adenocarcinoma, and adenocarcinoid. The most common mode of presentation for appendiceal carcinoma is acute appendicitis. Patients also may present with ascites or a palpable mass, or the neoplasm may be discovered during an operative procedure for an unrelated cause. The recommended treatment for all patients with adenocarcinoma of the appendix is a formal right hemicolectomy. Appendiceal adenocarcinomas have a propensity for early perforation, although they are not clearly associated with a worsened prognosis. Overall 5-year sur-vival is 55% and varies with stage and grade. Patients with appendiceal adenocarcinoma are at significant risk for both syn-chronous and metachronous neoplasms, approximately half of which will originate from the gastrointestinal tract.Appendiceal Mucoceles and Mucinous Neoplasms of the AppendixThe term appendiceal mucocele broadly describes a mucus-filled appendix that

1	half of which will originate from the gastrointestinal tract.Appendiceal Mucoceles and Mucinous Neoplasms of the AppendixThe term appendiceal mucocele broadly describes a mucus-filled appendix that could be secondary to neoplastic or nonneoplastic 9pathologies (mucosal hyperplasia, simple or retention cysts, mucinous cystadenomas, mucinous cystadenocarcinoma). The most common form of presentation is incidental; however, pre-sentation with appendicitis occurs in a third of cases.107,108 On cross-sectional imaging, a low attenuation, round, well encapsu-lated cystic mass in the right or quadrant is often encountered, and features such as wall irregularity and soft tissue thickening are suggestive of a neoplastic process. It is important to carefully assess for the presence of ascites, peritoneal disease, and scal-loping of the liver surface on imaging upon initial evaluation. A reliable diagnosis cannot be established using imaging alone, and it is recommended that surgical excision

1	disease, and scal-loping of the liver surface on imaging upon initial evaluation. A reliable diagnosis cannot be established using imaging alone, and it is recommended that surgical excision without capsular disruption is undertaken.109 The importance of careful handling of a mucocele and the avoidance of rupture cannot be overem-phasized because the intraperitoneal spread of neoplastic cells at subsequent development of pseudomyxoma peritonei are nearly certain in cases of adenocarcinoma.110,111 When suspecting a mucinous neoplasm of the appendix, it is imperative to systemat-ically examine the peritoneum and document a peritoneal cancer index score if mucin is present. Biopsies to examine the content of epithelial cell, neoplastic cells, and mucin can be helpful.In cases where a homogeneous cyst without nodularity or signs of dissemination is encountered, laparoscopic excision is acceptable, provided that a stapler is fired across the base of the cecum to avoid a positive margin.

1	cyst without nodularity or signs of dissemination is encountered, laparoscopic excision is acceptable, provided that a stapler is fired across the base of the cecum to avoid a positive margin. The specimen should be placed in a plastic bag and carefully removed through a small incision. In the absence of mesenteric or peritoneal involvement, an appendectomy with concurrent appendiceal lymphadenec-tomy is sufficient, as the chances of lymph node involvement are quite low. If peritoneal spread is evident upon exploration, it is important to obtain biopsies and document the peritoneal dis-ease burden. An appendectomy is acceptable if the patient has acute appendicitis, but suboptimal debulking is discouraged. In addition, colorectal, ovarian, and endometrial cancers can coex-ist in the setting of appendiceal mucoceles, and careful examina-tion of intra-abdominal structures is important.When there is discordance between the primary lesion histology and the peritoneum, the peritoneal

1	setting of appendiceal mucoceles, and careful examina-tion of intra-abdominal structures is important.When there is discordance between the primary lesion histology and the peritoneum, the peritoneal histology is usu-ally given priority. For instance, if patients had a neoplasm in the appendix but adenocarcinoma in the peritoneum, the patient would be considered as having adenocarcinoma (AJCC M1b) disease. The recent AJCC 8th edition and the PSOGI 2016 Brunicardi_Ch30_p1331-p1344.indd 133901/03/19 7:05 PM 1340SPECIFIC CONSIDERATIONSPART IITable 30-4AJCC 8th edition and the PSOGI 2016 classification consensus of mucinous neoplasia of the appendixLESIONPERITONEAL DISEASE AT DIAGNOSISPROGNOSISTREATMENTLow-grade appendiceal mucinous neoplasm (LAMN)Confined to the appendixExcellent-curativeNegative margin appendectomy, rarely need ileocecectomyLAMNPeri-appendiceal Acellular mucin dissecting through the wall (t4a) or adjacent organs (t4b)Excellent-low risk of recurrenceNegative margin

1	margin appendectomy, rarely need ileocecectomyLAMNPeri-appendiceal Acellular mucin dissecting through the wall (t4a) or adjacent organs (t4b)Excellent-low risk of recurrenceNegative margin appendectomy, resection of acellular mucinLAMNPeri-appendiceal Epithelial cells dissecting through the wall (t4a) or adjacent organs (t4b)Excellent-high risk of recurrenceNegative margin appendectomy, peritoneal surveillance with second look laparoscopy vs. HIPECLAMNDistant epithelial cells or acellular mucin (M1a)Low grade mucinous carcinoma peritoneiExcellent-high risk of recurrenceNegative margin appendectomy, omentectomy, HIPECHigh-grade appendiceal mucinous neoplasm (HAMN-rare)Management is identical to a LAMN with risk stratification as shown above but slightly worse prognosis.Mucinous adenocarcinomaConfined to the appendixVery GoodRight hemicolectomyMucinous adenocarcinomaPeritoneal DisseminationHigh grade mucinous carcinoma peritonei with or without signet ring cellsWell Differentiated-Very

1	to the appendixVery GoodRight hemicolectomyMucinous adenocarcinomaPeritoneal DisseminationHigh grade mucinous carcinoma peritonei with or without signet ring cellsWell Differentiated-Very goodModerately differentiated –GoodPoorly differentiated/signet ring cell histology:10 year survival of 10-20%Cytoreductive surgery and HIPEC, with systemic chemotherapy for high grade histologiesAdenocarcinoma (non-mucinous, including goblet cell histology)Management identical to the mucinous histologies, with more extensive use of systemic chemotherapySerrated Adenoma (rare)Confined to appendixExcellent-curativeAppendectomyAdenoma (rare)Confined to appendixExcellent-curativeAppendectomyData from American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017 and Carr NJ, Cecil TD, Mohamed F, et al: A Consensus for Classification and Pathologic Reporting of Pseudomyxoma Peritonei and Associated Appendiceal Neoplasia: The Results of

1	Springer New York, 2017 and Carr NJ, Cecil TD, Mohamed F, et al: A Consensus for Classification and Pathologic Reporting of Pseudomyxoma Peritonei and Associated Appendiceal Neoplasia: The Results of the Peritoneal Surface Oncology Group International (PSOGI) Modified Delphi Process, Am J Surg Pathol. 2016 Jan;40(1):14-26.classification consensus has resulted in a therapy-directed clas-sification of mucinous neoplasms of the appendix, summarized in Table 30-4.112Pseudomyxoma Peritonei SyndromePatients with appendiceal mucinous neoplasms develop perito-neal dissemination leading to pseudomyxoma peritonei (PMP) syndrome. This can occur in gastric, ovarian, pancreatic, and colorectal primary tumors as well.111 Patients with this syn-drome can have varied prognosis ranging from curative to pal-liative. Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) are considered the standard of care for patients with PMP syndrome from appendiceal primaries.113-115Early

1	to pal-liative. Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) are considered the standard of care for patients with PMP syndrome from appendiceal primaries.113-115Early detection and management of limited peritoneal dis-ease is favorable and preferred as opposed to extensive intra-peritoneal mucin development. The surgical technique involves parietal and visceral peritonectomies, and intraperitoneal administration of heated (42oC [108oF]) chemotherapy (usually mitomycin) in the abdomen. Previously considered a morbid surgery, high volume centers and standardized practices have made the morbidity and mortality similar to any major open GI procedure. This technique can also be performed laparoscopi-cally when the disease is detected early and is low volume.REFERENCESEntries highlighted in bright blue are key references. 1. Amyand C. Of an inguinal rupture, with a pin in the appendix coeci, incrusted with stone; and some observations on wounds in the guts.

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1	C, Maitre S, et al. Amoxicillin plus clavulanic acid versus appendicectomy for treatment of acute uncom-plicated appendicitis: an open-label, non-inferiority, ran-domised controlled trial. Lancet. 2011;377(9777):1573-1579. 24. Hansson J, Korner U, Khorram-Manesh A, Solberg A, Lundholm K. Randomized clinical trial of antibiotic therapy versus appendicectomy as primary treatment of acute appen-dicitis in unselected patients. Br J Surg. 2009;96(5):473-481. 25. Styrud J, Eriksson S, Nilsson I, et al. Appendectomy versus antibiotic treatment in acute appendicitis. a prospective multicenter randomized controlled trial. World J Surg. 2006;30(6):1033-1037. 26. Di Saverio S, Sibilio A, Giorgini E, et al. The NOTA Study (Non Operative Treatment for Acute Appendicitis): prospec-tive study on the efficacy and safety of antibiotics (amoxi-cillin and clavulanic acid) for treating patients with right lower quadrant abdominal pain and long-term follow-up of conservatively treated suspected

1	on the efficacy and safety of antibiotics (amoxi-cillin and clavulanic acid) for treating patients with right lower quadrant abdominal pain and long-term follow-up of conservatively treated suspected appendicitis. Ann Surg. 2014;260(1):109-117. 27. Salminen P, Paajanen H, Rautio T, et al. Antibiotic therapy vs appendectomy for treatment of uncomplicated acute appendicitis: the APPAC randomized clinical trial. JAMA. 2015;313(23):2340-2348. 28. Harnoss JC, Zelienka I, Probst P, et al. Antibiotics versus surgical therapy for uncomplicated appendicitis: system-atic review and meta-analysis of controlled trials (PROS-PERO 2015: CRD42015016882). Ann Surg. 2017;265(5): 889-900. 29. Varadhan KK, Neal KR, Lobo DN. Safety and efficacy of antibiotics compared with appendicectomy for treatment of uncomplicated acute appendicitis: meta-analysis of ran-domised controlled trials. BMJ. 2012;344:e2156. 30. Sartelli M, Viale P, Catena F, et al. 2013 WSES guidelines for management of intra-abdominal

1	uncomplicated acute appendicitis: meta-analysis of ran-domised controlled trials. BMJ. 2012;344:e2156. 30. Sartelli M, Viale P, Catena F, et al. 2013 WSES guidelines for management of intra-abdominal infections. World J Emerg Surg. 2013;8(1):3. 31. Ingraham AM, Cohen ME, Bilimoria KY, et al. Effect of delay to operation on outcomes in adults with acute appendicitis. Arch Surg. 2010;145(9):886-892. 32. Abou-Nukta F, Bakhos C, Arroyo K, et al. Effects of delaying appendectomy for acute appendicitis for 12 to 24 hours. Arch Surg. 2006;141(5):504-506; discussion 506-507. 33. Stahlfeld K, Hower J, Homitsky S, Madden J. Is acute appen-dicitis a surgical emergency? Am Surg. 2007;73(6):626-629; discussion 629-630. 34. Katkhouda N, Mason RJ, Towfigh S, et al. Laparoscopic versus open appendectomy: a prospective randomized dou-ble-blind study. Ann Surg. 2005;242(3):439-448; discussion 448-450. 35. Enochsson L, Hellberg A, Rudberg C, et al. Laparoscopic vs open appendectomy in overweight

1	a prospective randomized dou-ble-blind study. Ann Surg. 2005;242(3):439-448; discussion 448-450. 35. Enochsson L, Hellberg A, Rudberg C, et al. Laparoscopic vs open appendectomy in overweight patients. Surg Endosc. 2001;15(4):387-392. 36. Wei HB, Huang JL, Zheng ZH, et al. Laparoscopic versus open appendectomy: a prospective randomized comparison. Surg Endosc. 2010;24(2):266-2699. 37. Nguyen NT, Zainabadi K, Mavandadi S, et al. Trends in uti-lization and outcomes of laparoscopic versus open appendec-tomy. Am J Surg. 2004;188(6):813-820. 38. Simillis C, Symeonides P, Shorthouse AJ, Tekkis PP. A meta-analysis comparing conservative treatment versus acute appendectomy for complicated appendicitis (abscess or phleg-mon). Surgery. 2010;147(6):818-829. 39. Andersson RE, Petzold MG. Nonsurgical treatment of appen-diceal abscess or phlegmon: a systematic review and meta-analysis. Ann Surg. 2007;246(5):741-748. 40. Ciftci AO, Tanyel FC, Büyükpamukçu N, Hicsonmez A. Com-parative trial of four

1	treatment of appen-diceal abscess or phlegmon: a systematic review and meta-analysis. Ann Surg. 2007;246(5):741-748. 40. Ciftci AO, Tanyel FC, Büyükpamukçu N, Hicsonmez A. Com-parative trial of four antibiotic combinations for perforated appendicitis in children. Eur J Surg. 1997;163(8):591-596. 41. Schropp KP, Kaplan S, Golladay ES, et al. A randomized clinical trial of ampicillin, gentamicin and clindamycin versus cefotaxime and clindamycin in children with ruptured appen-dicitis. Surg Gynecol Obstet. 1991;172(5):351-356. 42. Andersson RE. The natural history and traditional manage-ment of appendicitis revisited: spontaneous resolution and predominance of prehospital perforations imply that a correct diagnosis is more important than an early diagnosis. World J Surg. 2007;31(1):86-92. 43. St Peter SD, Aguayo P, Fraser JD, et al. Initial laparoscopic appendectomy versus initial nonoperative management Brunicardi_Ch30_p1331-p1344.indd 134101/03/19 7:05 PM 1342SPECIFIC

1	Peter SD, Aguayo P, Fraser JD, et al. Initial laparoscopic appendectomy versus initial nonoperative management Brunicardi_Ch30_p1331-p1344.indd 134101/03/19 7:05 PM 1342SPECIFIC CONSIDERATIONSPART IIand interval appendectomy for perforated appendicitis with abscess: a prospective, randomized trial. J Pediatr Surg. 2010;45(1):236-240. 44. Dixon MR, Haukoos JS, Park IU, et al. An assessment of the severity of recurrent appendicitis. Am J Surg. 2003;186: 718-722; discussion 722. 45. Lai HW, Loong CC, Chiu JH, Chau GY, Wu CW, Lui WY. Interval appendectomy after conservative treatment of an appendiceal mass. World J Surg. 2006;30(3):352-357. 46. Rashid A, Nazir S, Kakroo SM, Chalkoo MA, Razvi SA, Wani AA. Laparoscopic interval appendectomy versus open interval appendectomy: a prospective randomized controlled trial. Surg Laparosc Endosc Percutan Tech. 2013;23(1):93-96. 47. Wright GP, Mater ME, Carroll JT, Choy JS, Chung MH. Is there truly an oncologic indication for interval

1	randomized controlled trial. Surg Laparosc Endosc Percutan Tech. 2013;23(1):93-96. 47. Wright GP, Mater ME, Carroll JT, Choy JS, Chung MH. Is there truly an oncologic indication for interval appendectomy? Am J Surg. 2015;209(3):442-446. 48. Sawyer RG, Claridge JA, Nathens AB, et al. Trial of short-course antimicrobial therapy for intraabdominal infection. N Engl J Med. 2015;372(21):1996-2005. 49. Solomkin JS, Mazuski JE, Bradley JS, et al. Diagnosis and management of complicated intra-abdominal infection in adults and children: guidelines by the Surgical Infection Society and the Infectious Diseases Society of America. Clin Infect Dis. 2010;50(2):133-164. 50. Siribumrungwong B, Chantip A, Noorit P, et al. Comparison of superficial surgical site infection between delayed primary versus primary wound closure in complicated appendicitis: a randomized controlled trial. Ann Surg. 2017;267(4):631-637. 51. Tander B, Pektas O, Bulut M. The utility of peritoneal drains in children with

1	primary wound closure in complicated appendicitis: a randomized controlled trial. Ann Surg. 2017;267(4):631-637. 51. Tander B, Pektas O, Bulut M. The utility of peritoneal drains in children with uncomplicated perforated appendicitis. Pediatr Surg Int. 2003;19:548-550. 52. Toki A, Ogura K, Horimi T, et al. Peritoneal lavage versus drainage for perforated appendicitis in children. Surg Today. 1995;25(3):207-210. 53. Ceci F, Orsini S, Tudisco A, et al. Single-incision laparoscopic appendectomy is comparable to conventional laparoscopic and laparotomic appendectomy: our single center single sur-geon experience. G Chir. 2013;34(7-8):216-219. 54. Coomber RS, Sodergren MH, Clark J, Teare J, Yang GZ, Darzi A. Natural orifice translumenal endoscopic surgery applications in clinical practice. World J Gastrointest Endosc. 2012;4(3):65-74. 55. Strickland AD, Norwood MG, Behnia-Willison F, Olakkengil SA, Hewett PJ. Transvaginal natural orifice translumenal endoscopic surgery (NOTES): a survey of

1	J Gastrointest Endosc. 2012;4(3):65-74. 55. Strickland AD, Norwood MG, Behnia-Willison F, Olakkengil SA, Hewett PJ. Transvaginal natural orifice translumenal endoscopic surgery (NOTES): a survey of women’s views on a new technique. Surg Endosc. 2010;24(10):2424-2431. 56. Akl MN, Magrina JF, Kho RM, Magtibay PM. Robotic appen-dectomy in gynaecological surgery: technique and pathologi-cal findings. Int J Med Robot. 2008;4(3):210-213. 57. Chiarugi M, Buccianti P, Decanini L, et al. “What you see is not what you get.” A plea to remove a ‘normal’ appendix during diagnostic laparoscopy. Acta Chir Belg. 2001;101(5):243-245. 58. Wang HT, Sax HC. Incidental appendectomy in the era of managed care and laparoscopy. J Am Coll Surg. 2001;192(2): 182-188. 59. Fisher KS, Ross DS. Guidelines for therapeutic deci-sion in incidental appendectomy. Surg Gynecol Obstet. 1990;171(1):95-98. 60. Scholer SJ, Pituch K, Orr DP, Dittus RS. Clinical outcomes of children with acute abdominal pain. Pediatrics.

1	deci-sion in incidental appendectomy. Surg Gynecol Obstet. 1990;171(1):95-98. 60. Scholer SJ, Pituch K, Orr DP, Dittus RS. Clinical outcomes of children with acute abdominal pain. Pediatrics. 1996;98(4 pt 1): 680-685. 61. Reynolds SL, Jaffe DM. Diagnosing abdominal pain in a pediatric emergency department. Pediatr Emerg Care. 1992;8(3):126-128. 62. Lee SL, Stark R, Yaghoubian A, Shekherdimian S, Kaji A. Does age affect the outcomes and management of pediatric appendicitis? J Pediatr Surg. 2011;46(12):2342-2345. 63. Rothrock SG, Pagane J. Acute appendicitis in children: emer-gency department diagnosis and management. Ann Emerg Med. 2000;36(1):39-51. 64. Colvin JM, Bachur R, Kharbanda A. The presentation of appendicitis in preadolescent children. Pediatr Emerg Care. 2007;23(12):849-855. 65. Bundy DG, Byerley JS, Liles EA, Perrin EM, Katznel-son J, Rice HE. Does this child have appendicitis? JAMA. 2007;298(4):438-451. 66. Bickell NA, Aufses AH Jr, Rojas M, Bodian C. How time affects the

1	DG, Byerley JS, Liles EA, Perrin EM, Katznel-son J, Rice HE. Does this child have appendicitis? JAMA. 2007;298(4):438-451. 66. Bickell NA, Aufses AH Jr, Rojas M, Bodian C. How time affects the risk of rupture in appendicitis. J Am Coll Surg. 2006;202(3):401-406. 67. Nomura O, Ishiguro A, Maekawa T, Nagai A, Kuroda T, Sakai H. Antibiotic administration can be an independent risk factor for therapeutic delay of pediatric acute appendicitis. Pediatr Emerg Care. 2012;28(8):792-795. 68. Aziz O, Athanasiou T, Tekkis PP, et al. Laparoscopic versus open appendectomy in children: a meta-analysis. Ann Surg. 2006;243(1):17-27. 69. Sauerland S, Jaschinski T, Neugebauer EA. Laparoscopic ver-sus open surgery for suspected appendicitis. Cochrane Data-base Syst Rev. 2010;(10):CD001546. 70. Lee SL, Islam S, Cassidy LD, et al. Antibiotics and appendici-tis in the pediatric population: an American Pediatric Surgical Association Outcomes and Clinical Trials Committee system-atic review. J Pediatr Surg.

1	S, Cassidy LD, et al. Antibiotics and appendici-tis in the pediatric population: an American Pediatric Surgical Association Outcomes and Clinical Trials Committee system-atic review. J Pediatr Surg. 2010;45(11):2181-2185. 71. Chen C, Botelho C, Cooper A, Hibberd P, Parsons SK. Current practice patterns in the treatment of perforated appendicitis in children. J Am Coll Surg. 2003;196(2):212-221. 72. Bufo AJ, Shah RS, Li MH, et al. Interval appendectomy for perforated appendicitis in children. J Laparoendosc Adv Surg Tech A. 1998;8(4):209-214. 73. Weber TR, Keller MA, Bower RJ, Spinner G, Vierling K. Is delayed operative treatment worth the trouble with perforated appendicitis in children? Am J Surg. 2003;186(6):685-658; discussion 688-689. 74. Nadler EP, Reblock KK, Vaughan KG, Meza MP, Ford HR, Gaines BA. Predictors of outcome for children with perfo-rated appendicitis initially treated with non-operative manage-ment. Surg Infect (Larchmt). 2004;5(4):349-356. 75. Svensson JF, Patkova

1	HR, Gaines BA. Predictors of outcome for children with perfo-rated appendicitis initially treated with non-operative manage-ment. Surg Infect (Larchmt). 2004;5(4):349-356. 75. Svensson JF, Patkova B, Almström M, et al. Nonoperative treatment with antibiotics versus surgery for acute nonperfo-rated appendicitis in children: a pilot randomized controlled trial. Ann Surg. 2015;261(1):67-71. 76. Ein SH, Langer JC, Daneman A. Nonoperative management of pediatric ruptured appendix with inflammatory mass or abscess: presence of an appendicolith predicts recurrent appendicitis. J Pediatr Surg. 2005;40(10):1612-1615. 77. Lopez ME, Wesson DE. Medical treatment of pediatric appen-dicitis: are we there yet? JAMA Pediatr. 2017;171(5):419-420. 78. Minneci PC, Mahida JB, Lodwick DL, et al. Effectiveness of patient choice in nonoperative vs surgical management of pediatric uncomplicated acute appendicitis. JAMA Surg. 2016;151(5):408-415. 79. Tanaka Y, Uchida H, Kawashima H, et al. Long-term out-comes

1	of patient choice in nonoperative vs surgical management of pediatric uncomplicated acute appendicitis. JAMA Surg. 2016;151(5):408-415. 79. Tanaka Y, Uchida H, Kawashima H, et al. Long-term out-comes of operative versus nonoperative treatment for uncom-plicated appendicitis. J Pediatr Surg. 2015;50(11):1893-1897. 80. Steiner Z, Buklan G, Stackievicz R, et al. Conservative treat-ment in uncomplicated acute appendicitis: reassessment of practice safety. Eur J Pediatr. 2017;176(4):521-527. 81. Sheu BF, Chiu TF, Chen JC, Tung MS, Chang MW, Young YR. Risk factors associated with perforated appendicitis in elderly patients presenting with signs and symptoms of acute appendicitis. ANZ J Surg. 2007;77(8):662-666. 82. Young YR, Chiu TF, Chen JC, et al. Acute appendicitis in the octogenarians and beyond: a comparison with younger geri-atric patients. Am J Med Sci. 2007;334(4):255-259.Brunicardi_Ch30_p1331-p1344.indd 134201/03/19 7:05 PM 1343THE APPENDIXCHAPTER 30 83. Harrell AG, Lincourt

1	beyond: a comparison with younger geri-atric patients. Am J Med Sci. 2007;334(4):255-259.Brunicardi_Ch30_p1331-p1344.indd 134201/03/19 7:05 PM 1343THE APPENDIXCHAPTER 30 83. Harrell AG, Lincourt AE, Novitsky YW, et al. Advantages of laparoscopic appendectomy in the elderly. Am Surg. 2006;72(6):474-480. 84. Andersen B, Nielsen TF. Appendicitis in pregnancy: diag-nosis, management and complications. Acta Obstet Gynecol Scand. 1999;78(9):758-762. 85. Bree RL, Ralls PW, Balfe DM, et al. Evaluation of patients with acute right upper quadrant pain. American College of Radiology. ACR Appropriateness Criteria. Radiology. 2000;215(suppl):153-157. 86. McGory ML, Zingmond DS, Tillou A, Hiatt JR, Ko CY, Cryer HM. Negative appendectomy in pregnant women is associ-ated with a substantial risk of fetal loss. J Am Coll Surg. 2007;205(4):534-540. 87. Cohen-Kerem R, Railton C, Oren D, Lishner M, Koren G. Pregnancy outcome following non-obstetric surgical interven-tion. Am J Surg.

1	risk of fetal loss. J Am Coll Surg. 2007;205(4):534-540. 87. Cohen-Kerem R, Railton C, Oren D, Lishner M, Koren G. Pregnancy outcome following non-obstetric surgical interven-tion. Am J Surg. 2005;190(3):467-473. 88. Giuliano V, Giuliano C, Pinto F, Scaglione M. Chronic appen-dicitis “syndrome” manifested by an appendicolith and thick-ened appendix presenting as chronic right lower abdominal pain in adults. Emerg Radiol. 2006;12(3):96-98. 89. Andersen BR, Kallehave FL, Andersen HK. Antibiotics versus placebo for prevention of postoperative infection after appen-dicectomy. Cochrane Database Syst Rev. 2003;(2):CD001439. 90. Rucinski J, Fabian T, Panagopoulos G, Schein M, Wise L. Gangrenous and perforated appendicitis: a meta-analytic study of 2532 patients indicates that the incision should be closed primarily. Surgery. 2000;127(2):136-141. 91. Fleming FJ, Kim MJ, Messing S, Gunzler D, Salloum R, Monson JR. Balancing the risk of postoperative surgical infec-tions: a multivariate

1	should be closed primarily. Surgery. 2000;127(2):136-141. 91. Fleming FJ, Kim MJ, Messing S, Gunzler D, Salloum R, Monson JR. Balancing the risk of postoperative surgical infec-tions: a multivariate analysis of factors associated with lapa-roscopic appendectomy from the NSQIP database. Ann Surg. 2010;252(6):895-900. 92. Liang MK, Lo HG, Marks JL. Stump appendicitis: a compre-hensive review of literature. Am Surg. 2006;72(2):162-166. 93. Connor SJ, Hanna GB, Frizelle FA. Appendiceal tumors: retrospective clinicopathologic analysis of appendiceal tumors from 7,970 appendectomies. Dis Colon Rectum. 1998;41(1):75-80. 94. Turaga KK, Pappas SG, Gamblin T. Importance of histologic subtype in the staging of appendiceal tumors. Ann Surg Oncol. 2012;19(5):1379-1385. 95. McGory ML, Maggard MA, Kang H, O’Connell JB, Ko CY. Malignancies of the appendix: beyond case series reports. Dis Colon Rectum. 2005;48(12):2264-2271. 96. Deans GT, Spence RA. Neoplastic lesions of the appendix. Br J Surg.

1	Kang H, O’Connell JB, Ko CY. Malignancies of the appendix: beyond case series reports. Dis Colon Rectum. 2005;48(12):2264-2271. 96. Deans GT, Spence RA. Neoplastic lesions of the appendix. Br J Surg. 1995;82(3):299-306. 97. Rutledge RH, Alexander JW. Primary appendiceal malignan-cies: rare but important. Surgery. 1992;111(3):244-250. 98. Moertel CG, Dockerty MB, Judd ES. Carcinoid tumors of the vermiform appendix. Cancer. 1968;21(2):270-278. 99. Rorstad O. Prognostic indicators for carcinoid neuroen-docrine tumors of the gastrointestinal tract. J Surg Oncol. 2005;89(3):151-160. 100. Carr NJ, Sobin LH. Neuroendocrine tumors of the appendix. Semin Diagn Pathol. 2004;21(2):108-119. 101. Tang LH, Shia J, Soslow RA, et al. Pathologic classifi-cation and clinical behavior of the spectrum of goblet cell carcinoid tumors of the appendix. Am J Surg Pathol. 2008;32(10):1429-1443. 102. Pham TH, Wolff B, Abraham SC, Drelichman E. Surgical and chemotherapy treatment outcomes of goblet cell

1	of goblet cell carcinoid tumors of the appendix. Am J Surg Pathol. 2008;32(10):1429-1443. 102. Pham TH, Wolff B, Abraham SC, Drelichman E. Surgical and chemotherapy treatment outcomes of goblet cell carci-noid: a tertiary cancer center experience. Ann Surg Oncol. 2006;13(3):370-376. 103. Yan TD, Brun EA, Sugarbaker PH. Discordant histology of primary appendiceal adenocarcinoid neoplasms with perito-neal dissemination. Ann Surg Oncol. 2008;15(5):1440-1446. 104. Varisco B, McAlvin B, Dias J, Franga D. Adenocarci-noid of the appendix: is right hemicolectomy necessary? A meta-analysis of retrospective chart reviews. Am Surg. 2004;70(7):593-599. 105. Crump M, Gospodarowicz M, Shepherd FA. Lymphoma of the gastrointestinal tract. Semin Oncol. 1999;26(3):324-337. 106. Pickhardt PJ, Levy AD, Rohrmann CA Jr, et al. Non-Hodg-kin’s lymphoma of the appendix: clinical and CT find-ings with pathologic correlation. AJR Am J Roentgenol. 2002;178(5):1123-1127. 107. Raijman I, Leong S, Hassaram S,

1	Rohrmann CA Jr, et al. Non-Hodg-kin’s lymphoma of the appendix: clinical and CT find-ings with pathologic correlation. AJR Am J Roentgenol. 2002;178(5):1123-1127. 107. Raijman I, Leong S, Hassaram S, Marcon NE. Appen-diceal mucocele: endoscopic appearance. Endoscopy. 1994;26(3):326-328. 108. Hamilton DL, Stormont JM. The volcano sign of appendiceal mucocele. Gastrointest Endosc. 1989;35(5):453-456. 109. Stocchi L, Wolff BG, Larson DR, Harrington JR. Sur-gical treatment of appendiceal mucocele. Arch Surg. 2003;138(6):585-589; discussion 589-590. 110. Smith JW, Kemeny N, Caldwell C, Banner P, Sigurdson E, Huvos A. Pseudomyxoma peritonei of appendiceal origin. The Memorial Sloan-Kettering Cancer Center experience. Cancer. 1992;70(2):396-401. 111. Hinson FL, Ambrose NS. Pseudomyxoma peritonei. Br J Surg. 1998;85(10):1332-1339. 112. Carr NJ, Cecil TD, Mohamed F, et al. A consensus for classi-fication and pathologic reporting of pseudomyxoma peritonei and associated appendiceal neoplasia:

1	Br J Surg. 1998;85(10):1332-1339. 112. Carr NJ, Cecil TD, Mohamed F, et al. A consensus for classi-fication and pathologic reporting of pseudomyxoma peritonei and associated appendiceal neoplasia: the results of the Peri-toneal Surface Oncology Group International (PSOGI) Modi-fied Delphi Process. Am J Surg Pathol. 2016;40(1):14-26. 113. Gough DB, Donohue JH, Schutt AJ, et al. Pseudomyxoma peritonei. Long-term patient survival with an aggressive regional approach. Ann Surg. 1994;219(2):112-119. 114. Stewart JHt, Shen P, Russell GB, et al. Appendiceal neoplasms with peritoneal dissemination: outcomes after cytoreductive surgery and intraperitoneal hyperthermic chemotherapy. Ann Surg Oncol. 2006;13(5):624-634. 115. Sugarbaker PH. New standard of care for appendiceal epi-thelial neoplasms and pseudomyxoma peritonei syndrome? Lancet Oncol. 2006;7(1):69-76.Brunicardi_Ch30_p1331-p1344.indd 134301/03/19 7:05 PM

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1	LiverDavid A. Geller, John A. Goss, Ronald W. Busuttil, and Allan Tsung 31chapterHistory of Liver Surgery 1346Liver Anatomy 1346Segmental Anatomy / 1346Hepatic Artery / 1348Portal Vein / 1348Hepatic Veins and Inferior Vena Cava / 1349Bile Duct and Hepatic Ducts / 1350Neural Innervation and Lymphatic Drainage / 1350Liver Physiology 1351Bilirubin Metabolism / 1352Formation of Bile / 1352Drug Metabolism / 1352Liver Function Tests / 1352Hepatocellular Injury / 1353Abnormal Synthetic Function / 1353Cholestasis / 1353Jaundice / 1353Molecular Signaling Pathways in the Liver 1354Acute Phase Reaction / 1354LPS Signaling / 1355Nitric Oxide / 1355Heme Oxygenase System / 1356Toll-Like Receptors / 1357Radiologic Evaluation of the Liver 1357Ultrasound / 1357Computed Tomography / 1358Magnetic Resonance Imaging / 1359Positron Emission Tomography / 1360Acute Liver Failure 1360Etiology / 1361Clinical Presentation / 1361Diagnosis and Clinical Management / 1361Prognosis / 1362Liver Transplantation

1	Imaging / 1359Positron Emission Tomography / 1360Acute Liver Failure 1360Etiology / 1361Clinical Presentation / 1361Diagnosis and Clinical Management / 1361Prognosis / 1362Liver Transplantation / 1362Emerging Technologies / 1362Cirrhosis and Portal Hypertension 1362Morphologic Classification of Cirrhosis / 1362Etiology of Cirrhosis / 1363Clinical Manifestations of Cirrhosis / 1364Laboratory Findings Associated With Cirrhosis / 1364Liver Biopsy / 1364Hepatic Reserve and Assessment of Surgical Risk in the Cirrhotic Patient / 1365Child-Turcotte-Pugh Score / 1365Model for End-Stage Liver Disease Scoring System / 1365Portal Hypertension / 1365Imaging of the Portal Venous System and Measurement of Portal Venous Pressure / 1366Etiology and Clinical Features of Portal Hypertension / 1366Management of Gastroesophageal Varices / 1367Prevention of Variceal Bleeding / 1367Management of Acute Variceal Hemorrhage / 1367Luminal Tamponade / 1367Transjugular Intrahepatic Portosystemic

1	/ 1366Management of Gastroesophageal Varices / 1367Prevention of Variceal Bleeding / 1367Management of Acute Variceal Hemorrhage / 1367Luminal Tamponade / 1367Transjugular Intrahepatic Portosystemic Shunt / 1367Balloon-Occluded Retrograde Transvenous Obliteration / 1367Surgical Shunting / 1367Nonshunt Surgical Management of Refractory Variceal Bleeding / 1368Hepatic Transplantation / 1368Budd-Chiari Syndrome / 1368Infections of the Liver 1369Pyogenic Liver Abscesses / 1369Amebic Abscesses / 1369Hydatid Disease / 1370Ascariasis / 1371Schistosomiasis / 1371Viral Hepatitis / 1371Evaluation of an Incidental Liver Mass 1372Hepatic Cysts 1373Congenital Cysts / 1373Biliary Cystadenoma / 1373Polycystic Liver Disease / 1373Caroli’s Disease / 1374Benign Liver Lesions 1374Cyst / 1374Hemangioma / 1375Adenoma / 1375Focal Nodular Hyperplasia / 1376Bile Duct Hamartoma / 1376Malignant Liver Tumors 1376Hepatocellular Carcinoma / 1376Cholangiocarcinoma / 1377Gallbladder Cancer / 1378Metastatic

1	/ 1375Adenoma / 1375Focal Nodular Hyperplasia / 1376Bile Duct Hamartoma / 1376Malignant Liver Tumors 1376Hepatocellular Carcinoma / 1376Cholangiocarcinoma / 1377Gallbladder Cancer / 1378Metastatic Colorectal Cancer / 1378Neuroendocrine Tumors / 1379Other Metastatic Tumors / 1379Treatment Options for Liver Cancer 1379Hepatic Resection / 1379Liver Transplantation / 1380Radiofrequency Ablation / 1380Ethanol Ablation, Cryosurgery, and Microwave Ablation / 1380Chemoembolization and Hepatic Artery Pump Chemoperfusion / 1381Yttrium-90 Microspheres / 1381Stereotactic Radiosurgery and Intensity-Modulated Radiation Therapy / 1381Downstaging / 1381Systemic Chemotherapy / 1381Hepatic Resection Surgical Techniques 1381Nomenclature / 1381Techniques and Devices for Dividing the Hepatic Parenchyma / 1382Steps in Commonly Performed Hepatic Resections / 1383Pringle and Ischemic Preconditioning / 1385Preoperative Portal Vein Embolization / 1385Staged Hepatectomy, ALPPS, and Repeat Hepatic

1	/ 1382Steps in Commonly Performed Hepatic Resections / 1383Pringle and Ischemic Preconditioning / 1385Preoperative Portal Vein Embolization / 1385Staged Hepatectomy, ALPPS, and Repeat Hepatic Resection for Recurrent Liver Cancer / 1386Laparoscopic Liver Resection 1386Brunicardi_Ch31_p1345-p1392.indd 134520/02/19 2:36 PM 1346HISTORY OF LIVER SURGERYThe ancient Greek myth of Prometheus reminds us that the liver is the only organ that regenerates. According to Greek mythology, Zeus was furious with the Titan Prometheus because he gave fire to mortals. In return, Zeus chained Prometheus to Mount Caucasus and sent his giant eagle to eat his liver during the day, only to have it regenerate at night. Although this is folk-lore, the principles are correct that after hepatic resection, the remnant liver will hypertrophy over weeks to months to regain most of its original liver mass. It is interesting to note that the ancient Greeks seem to have been aware of this fact, because the

1	the remnant liver will hypertrophy over weeks to months to regain most of its original liver mass. It is interesting to note that the ancient Greeks seem to have been aware of this fact, because the Greek word for the liver, h¯epar, derives from the verb h¯epaomai, which means “mend” or “repair.” Hence h¯epar roughly translates as “repairable.”1 The importance of the liver dates back to even biblical times, for the Babylonians (c. 2000 b.c.) considered the liver to be the seat of the soul. There are scattered reports of liver surgery for battlefield injuries, but the first recorded elec-tive hepatic resection was done in 1888 in Germany by Langen-buch. There followed reports of liver resections in the United States (Tiffany, 1890) and Europe (Lucke, 1891), as well as the first large series of hepatic resections by Keen in 1899.2,3 In 1908, Pringle described in Annals of Surgery the “arrest of hepatic hemorrhage due to trauma” by compression of the porta hepatis, a maneuver that now

1	of hepatic resections by Keen in 1899.2,3 In 1908, Pringle described in Annals of Surgery the “arrest of hepatic hemorrhage due to trauma” by compression of the porta hepatis, a maneuver that now bears his name.4 Possibly due to the potential for massive hemorrhage during liver surgery, very little progress in surgical techniques was recorded for the next half-century. Work by Rex, Cantlie, and others laid the groundwork for experimental and clinical reports in the 1950s by Couinaud, Hjortsjo, Healey, Lortat-Jacob, and Starzl.5,6 These seminal contributions paved the way for the modern era of hepatic resection surgery.LIVER ANATOMYThe liver is the largest organ in the body, weighing approxi-mately 1500 g. It resides in the right upper abdominal cavity beneath the diaphragm and is protected by the rib cage. It is reddish brown and is surrounded by a fibrous sheath known as Glisson’s capsule. The liver is held in place by several ligaments (Fig. 31-1). The round ligament is the remnant

1	by the rib cage. It is reddish brown and is surrounded by a fibrous sheath known as Glisson’s capsule. The liver is held in place by several ligaments (Fig. 31-1). The round ligament is the remnant of the obliterated umbilical vein and enters the left liver hilum at the front edge of the falciform ligament. The falciform ligament separates the left lateral and left medial segments along the umbilical fissure and anchors the liver to the anterior abdominal wall. Deep in the plane between the caudate lobe and the left lateral segment is the fibrous ligamentum venosum (Arantius’ ligament), which is the obliterated ductus venosus and is covered by the plate of Arantius. The left and right triangular ligaments secure the two sides of the liver to the diaphragm. Extending from the trian-gular ligaments anteriorly on the liver are the coronary liga-ments. The right coronary ligament also extends from the right undersurface of the liver to the peritoneum overlying the right kidney, thereby

1	ligaments anteriorly on the liver are the coronary liga-ments. The right coronary ligament also extends from the right undersurface of the liver to the peritoneum overlying the right kidney, thereby anchoring the liver to the right retroperitoneum. These ligaments (round, falciform, triangular, and coronary) can be divided in a bloodless plane to fully mobilize the liver to facilitate hepatic resection. Centrally and just to the left of the gallbladder fossa, the liver attaches via the hepatoduodenal and the gastrohepatic ligaments (Fig. 31-2). The hepatoduodenal ligament is known as the porta hepatis and contains the com-mon bile duct, the hepatic artery, and the portal vein. From the right side and deep (dorsal) to the porta hepatis is the foramen of Winslow, also known as the epiploic foramen (see Fig. 31-2). This passage connects directly to the lesser sac and allows com-plete vascular inflow control to the liver when the hepatoduode-nal ligament is clamped using the Pringle

1	foramen (see Fig. 31-2). This passage connects directly to the lesser sac and allows com-plete vascular inflow control to the liver when the hepatoduode-nal ligament is clamped using the Pringle maneuver.Segmental AnatomyThe liver is grossly separated into the right and left lobes by the plane from the gallbladder fossa to the inferior vena cava (IVC), known as Cantlie’s line.5 The right lobe typically accounts for 60% to 70% of the liver mass, with the left lobe (and caudate lobe) making up the remainder. The caudate lobe lies to the left and anterior of the IVC and contains three subsegments: Key Points1 When operating on the liver, gallbladder, pancreas, or adjacent organs, recognition of the normal or variant vas-cular and biliary anatomy is essential to avoiding surgical complications.2 The liver is the largest gland in the body and performs a diverse spectrum of functions.3 Computed tomography and magnetic resonance imaging with contrast enhancement constitute the mainstays for

1	liver is the largest gland in the body and performs a diverse spectrum of functions.3 Computed tomography and magnetic resonance imaging with contrast enhancement constitute the mainstays for the radiologic evaluation of the liver.4 Acute liver failure rapidly progresses to hepatic coma and death even with maximal medical therapy. The only defin-itive treatment is orthotopic liver transplantation.5 Acute variceal bleeding should be managed with aggres-sive resuscitation and prompt endoscopic diagnosis with hemorrhage control. The transjugular intrahepatic por-tosystemic shunt procedure can be considered for cases refractory to medical treatment.6 Common benign lesions of the liver include cysts, hem-angiomas, focal nodular hyperplasia, and hepatocellular adenomas. In most instances, these lesions can be reliably diagnosed by their characteristic features on imaging.7 Many options exist for the treatment of hepatocellular car-cinomas, and these cases are best managed by a

1	these lesions can be reliably diagnosed by their characteristic features on imaging.7 Many options exist for the treatment of hepatocellular car-cinomas, and these cases are best managed by a multidis-ciplinary liver transplant team.8 Surgical resection is the treatment of choice for hilar chol-angiocarcinoma. Under a protocol with strict eligibility criteria, patients with unresectable tumors can be con-sidered for liver transplantation following neoadjuvant chemoradiation, with survival rates that compare favorably with the rates for resection.9 The resectability of colorectal cancer metastases to the liver is primarily determined by the volume of the future liver remnant and the health of the background liver and not actual tumor number.10 Laparoscopic liver resections can be performed safely by experienced surgeons in selected patients and have been shown to produce short-term patient benefits with com-parable long-term oncologic results compared to open hepatic

1	can be performed safely by experienced surgeons in selected patients and have been shown to produce short-term patient benefits with com-parable long-term oncologic results compared to open hepatic resections.Brunicardi_Ch31_p1345-p1392.indd 134620/02/19 2:36 PM 1347LIVERCHAPTER 31Figure 31-1. Hepatic ligaments suspending the liver to the diaphragm and anterior abdominal wall.Liver in situForamen ofWinslowGastrohepaticligamentOpen hepato-duodenal ligamentFigure 31-2. In situ liver hilar anatomy with hepatoduodenal and gastrohepatic ligaments. Foramen of Winslow is depicted.Right lobeLeft lobeRight lobeLeft lobeVIIIIVaIVbIIIIIVVIIVIIVCCaudate lobeIIIIVVIIVIIIIVbFigure 31-3. Couinaud’s liver segments (I through VIII) num-bered in a clockwise manner. The left lobe includes segments II to IV, the right lobe includes segments V to VIII, and the caudate lobe is segment I. IVC = inferior vena cava.the Spiegel lobe, the paracaval portion, and the caudate process.7 The falciform ligament

1	IV, the right lobe includes segments V to VIII, and the caudate lobe is segment I. IVC = inferior vena cava.the Spiegel lobe, the paracaval portion, and the caudate process.7 The falciform ligament does not separate the right and left lobes, but rather it divides the left lateral segment from the left medial segment. The left lateral and left medial segments also are referred to as sections as defined in the Brisbane 2000 terminology, which is outlined later in the section titled “Hepatic Resection.” A significant advance in our understanding of liver anatomy came from the cast work studies of the French surgeon and anatomist Couinaud in the early 1950s. Couinaud divided the liver into eight segments, numbering them in a clockwise direc-tion beginning with the caudate lobe as segment I.6 Segments II and III comprise the left lateral segment, and segment IV is the left medial segment (Fig. 31-3). Thus, the left lobe is made up of the left lateral segment (Couinaud’s segments II and

1	Segments II and III comprise the left lateral segment, and segment IV is the left medial segment (Fig. 31-3). Thus, the left lobe is made up of the left lateral segment (Couinaud’s segments II and III) and the left medial segment (segment IV). Segment IV can be subdivided into segment IVA and segment IVB. Segment IVA is cepha-lad and just below the diaphragm, spanning from segment VIII to the falciform ligament adjacent to segment II. Segment IVB is caudad and adjacent to the gallbladder fossa. Many anatomy textbooks also refer to segment IV as the quadrate lobe. Quadrate lobe is an outdated term, and the preferred term is segment IV or left medial segment. Most surgeons still refer to segment I as the DiaphragmRight triangularligamentLeft triangularligamentFalciformligamentRoundligamentcaudate lobe, rather than segment I. The right lobe is comprised of segments V, VI, VII, and VIII, with segments V and VIII mak-ing up the right anterior lobe and segments VI and VII making up the

1	lobe, rather than segment I. The right lobe is comprised of segments V, VI, VII, and VIII, with segments V and VIII mak-ing up the right anterior lobe and segments VI and VII making up the right posterior lobe.Brunicardi_Ch31_p1345-p1392.indd 134720/02/19 2:36 PM 1348SPECIFIC CONSIDERATIONSPART IIAdditional functional anatomy was highlighted by Bis-muth based on the distribution of the hepatic veins. The three hepatic veins run in corresponding scissura (fissures) and divide the liver into four sectors.8 The right hepatic vein runs along the right scissura and separates the right posterolateral sector from the right anterolateral sector. The main scissura contains the middle hepatic vein and separates the right and left livers. The left scissura contains the course of the left hepatic vein and separates the left posterior and left anterior sectors.Hepatic ArteryThe liver has a dual blood supply consisting of the hepatic artery and the portal vein. The hepatic artery delivers

1	vein and separates the left posterior and left anterior sectors.Hepatic ArteryThe liver has a dual blood supply consisting of the hepatic artery and the portal vein. The hepatic artery delivers approximately 25% of the blood supply, and the portal vein approximately 75%. The hepatic artery arises from the celiac axis (trunk), which gives off the left gastric, splenic, and common hepatic arteries (Fig. 31-4). The common hepatic artery then divides into the gastroduodenal artery and the hepatic artery proper. The right gastric artery typically originates off of the hepatic artery proper, but this is variable. The hepatic artery proper divides into the right and left hepatic arteries. This “classic” or standard arterial anatomy is present in only approximately 76% of cases, with the remaining 24% having variable anatomy. It is critical to understand the arterial (and biliary) anatomic variants to avoid surgical complications when operating on the liver, gallbladder, pancreas, or adjacent

1	24% having variable anatomy. It is critical to understand the arterial (and biliary) anatomic variants to avoid surgical complications when operating on the liver, gallbladder, pancreas, or adjacent organs.The most common hepatic arterial variants are shown in Fig. 31-5. Approximately 10% to 15% of the time there is a replaced or accessory right hepatic artery arising from the supe-rior mesenteric artery (SMA). When there is a replacement or accessory right hepatic artery, it travels posterior to the por-tal vein and then takes up a right lateral position before diving into the liver parenchyma. This can be recognized visually on a preoperative computed tomography (CT) or magnetic reso-nance imaging (MRI) scan and confirmed by palpation in the hilum where a separate right posterior pulsation is felt distinct from that of the hepatic artery proper that lies anteriorly in the hepatoduodenal ligament to the left of the common bile duct. In approximately 3% to 10% of cases, there exists

1	is felt distinct from that of the hepatic artery proper that lies anteriorly in the hepatoduodenal ligament to the left of the common bile duct. In approximately 3% to 10% of cases, there exists a replace-ment (or accessory) left hepatic artery coming off of the left gastric artery and running obliquely in the gastrohepatic liga-ment anterior to the caudate lobe before entering the hilar plate at the base of the umbilical fissure. Other less common variants (approximately 1–2% each) are the presence of both replaced right and replaced left hepatic arteries, as well as a completely replaced common hepatic artery coming off the SMA (see Fig. 31-5). Although not well demonstrated in the illustration, the clue for a completely replaced common hepatic artery com-ing off the SMA is the presence of a strong arterial pulsation to the right of and posterior to the common bile duct, rather than the left side and anterior, in the porta hepatis. Another important point is that the right hepatic

1	of a strong arterial pulsation to the right of and posterior to the common bile duct, rather than the left side and anterior, in the porta hepatis. Another important point is that the right hepatic artery passes deep and posterior to the common bile duct approximately 88% of the time but crosses anterior to the common bile duct in approximately 12% of cases. The cystic artery feeding the gallbladder usually arises from the right hepatic artery in Calot’s triangle.Portal VeinThe portal vein is formed by the confluence of the splenic vein and the superior mesenteric vein. The inferior mesenteric vein usually drains into the splenic vein upstream from the conflu-ence (Fig. 31-6). The main portal vein traverses the porta hepa-tis before dividing into the left and right portal vein branches. The left portal vein typically branches from the main portal vein outside of the liver with a sharp bend to the left and consists of the transverse portion followed by a 90° turn at the base of the

1	The left portal vein typically branches from the main portal vein outside of the liver with a sharp bend to the left and consists of the transverse portion followed by a 90° turn at the base of the umbilical fissure to become the umbilical portion before enter-ing the liver parenchyma (Fig. 31-7). The left portal vein then divides to give off the segment II and III branches to the left lat-eral segment, as well as the segment IV branches that supply the left medial segment. The left portal vein also provides the domi-nant inflow branch to the caudate lobe (although branches can arise from the main and right portal veins also), usually close to the bend between the transverse and umbilical portions. The division of the right portal vein is usually higher in the hilum and may be close to (or inside) the liver parenchyma at the hilar plate. Twenty percent to 35% of individuals have aberrant por-tal venous anatomy, with portal vein trifurcation or an aberrant branch from the left portal

1	inside) the liver parenchyma at the hilar plate. Twenty percent to 35% of individuals have aberrant por-tal venous anatomy, with portal vein trifurcation or an aberrant branch from the left portal vein supplying the right anterior lobe being the most frequent.The portal vein drains the splanchnic blood from the stom-ach, pancreas, spleen, small intestine, and majority of the colon to the liver before returning to the systemic circulation. The portal vein pressure in an individual with normal physiology is low at 3 to 5 mmHg. The portal vein is valveless, however, and in the setting of portal hypertension, the pressure can be 1Figure 31-4. Arterial anatomy of the upper abdomen and liver, including the celiac trunk and hepatic artery branches. a. = artery; LHA = left hepatic artery; RHA = right hepatic artery.RHALHAHepatic arteryproperRight gastricarteryCommon hepatic arteryLeft gastricarteryCeliac trunkSplenic arteryGastroduodenal arteryBrunicardi_Ch31_p1345-p1392.indd 134820/02/19

1	hepatic artery.RHALHAHepatic arteryproperRight gastricarteryCommon hepatic arteryLeft gastricarteryCeliac trunkSplenic arteryGastroduodenal arteryBrunicardi_Ch31_p1345-p1392.indd 134820/02/19 2:36 PM 1349LIVERCHAPTER 31quite high (20 to 30 mmHg). This results in decompression of the systemic circulation through portocaval anastomoses, most commonly via the coronary (left gastric) vein, which produces esophageal and gastric varices with a propensity for major hem-orrhage. Another branch of the main portal vein is the superior pancreaticoduodenal vein (which comes off low in an anterior lateral position and is divided during pancreaticoduodenec-tomy). Closer to the liver, the main portal vein typically gives off a short branch (posterior lateral) to the caudate process on the right side. It is important to identify this branch and ligate it during hilar dissection for anatomic right hemihepatectomy to avoid avulsion.Hepatic Veins and Inferior Vena CavaThere are three hepatic veins

1	It is important to identify this branch and ligate it during hilar dissection for anatomic right hemihepatectomy to avoid avulsion.Hepatic Veins and Inferior Vena CavaThere are three hepatic veins (right, middle, and left) that pass obliquely through the liver to drain the blood to the suprahe-patic IVC and eventually the right atrium (Fig. 31-8). The right hepatic vein drains segments V through VIII; the middle hepatic vein drains segment IV as well as segments V and VIII; and the left hepatic vein drains segments II and III. The caudate lobe Replaced right hepaticartery from SMA (10%–15%)Replaced left hepatic artery from left gastric artery (3%–10%)Replaced right and replaced left hepatic arteries (1%–2%)Completely replaced commonhepatic artery from SMA (1%–2%)Figure 31-5. Common hepatic artery anatomic variants. SMA = superior mesenteric artery.Coronary v.Portal v.Superior mesenteric v.Inferiormesenteric v.Splenic v.Figure 31-6. Portal vein anatomy. The portal vein is formed by the

1	artery anatomic variants. SMA = superior mesenteric artery.Coronary v.Portal v.Superior mesenteric v.Inferiormesenteric v.Splenic v.Figure 31-6. Portal vein anatomy. The portal vein is formed by the confluence of the splenic and superior mesenteric veins. The inferior mesenteric vein drains into the splenic vein. The coronary (left gastric) vein drains into the portal vein in the vicinity of the confluence. v. = vein.Main portal veinRightportal veinLeftportal veinUmbilicalportion LPVIIIIIIIVAIVBVVIVIIVIIIVIIITransverseportion LPVFigure 31-7. Anatomy of the left portal vein (LPV). Note the transverse and umbilical portions of the LPV.Brunicardi_Ch31_p1345-p1392.indd 134920/02/19 2:36 PM 1350SPECIFIC CONSIDERATIONSPART IIis unique because its venous drainage feeds directly into the IVC. In addition, the liver usually has a few small, variable short hepatic veins that directly enter the IVC from the undersurface of the liver. The left and middle hepatic veins form a common trunk

1	IVC. In addition, the liver usually has a few small, variable short hepatic veins that directly enter the IVC from the undersurface of the liver. The left and middle hepatic veins form a common trunk approximately 95% of the time before entering the IVC, whereas the right hepatic vein inserts separately (in an oblique orientation) into the IVC. There is a large inferior accessory right hepatic vein in 15% to 20% of cases that runs in the hepa-tocaval ligament. This can be a source of torrential bleeding if control of it is lost during right hepatectomy. The hepatic vein branches bisect the portal branches inside the liver parenchyma (i.e., the right hepatic vein runs between the right anterior and posterior portal veins; the middle hepatic vein passes between the right anterior and left portal vein; and the left hepatic vein crosses between the segment II and III branches of the left portal vein).Bile Duct and Hepatic DuctsWithin the hepatoduodenal ligament, the common bile duct lies

1	vein; and the left hepatic vein crosses between the segment II and III branches of the left portal vein).Bile Duct and Hepatic DuctsWithin the hepatoduodenal ligament, the common bile duct lies anteriorly and to the right. It gives off the cystic duct to the gallbladder and becomes the common hepatic duct before divid-ing into the right and left hepatic ducts. In general, the hepatic ducts follow the arterial branching pattern inside the liver. The right anterior hepatic duct usually enters the liver above the hilar plate, whereas the right posterior duct dives behind the right portal vein and can be found on the surface of the caudate pro-cess before entering the liver. The left hepatic duct typically has a longer extrahepatic course before giving off segmental branches behind the left portal vein at the base of the umbilical fissure. Considerable variation exists, and in 30% to 40% of cases, there is a nonstandard hepatic duct confluence with acces-sory or aberrant ducts (Fig.

1	portal vein at the base of the umbilical fissure. Considerable variation exists, and in 30% to 40% of cases, there is a nonstandard hepatic duct confluence with acces-sory or aberrant ducts (Fig. 31-9). The cystic duct itself also has a variable pattern of drainage into the common bile duct. This can lead to potential injury or postoperative bile leakage during cholecystectomy or hepatic resection, and the surgeon needs to expect these variants. The gallbladder sits adherent to hepatic segments IVB (left lobe) and V (right lobe).Neural Innervation and Lymphatic DrainageThe parasympathetic innervation of the liver comes from the left vagus, which gives off the anterior hepatic branch, and the right vagus, which gives off the posterior hepatic branch. The sympathetic innervation involves the greater thoracic splanchnic nerves and the celiac ganglia, although the function of these nerves is poorly understood. The denervated liver after hepatic transplantation seems to function with

1	the greater thoracic splanchnic nerves and the celiac ganglia, although the function of these nerves is poorly understood. The denervated liver after hepatic transplantation seems to function with normal capacity. A common source of referred pain to the right shoulder and scapula as well as the right side or back is the right phrenic Right lobePosteriorsegmentstructuresMiddlehepaticv.Anterior segmentstructuresGall bladderPortal v.Hepatic a.FalciformligamentLeft lobeMedialsegmentstructuresLateralsegmentstructuresMiddle HVRight HVLeft HVIVC and 3 HVsIVC Figure 31-8. Confluence of the three hepatic veins (HVs) and the inferior vena cava (IVC). Note that the middle and left HVs drain into a common trunk before entering the IVC. a. = artery; v. = vein. (Adapted with permission from Cameron JL: Atlas of Surgery. Vol. I, Gallbladder and Biliary Tract, the Liver, Portasystemic Shunts, the Pancreas. Toronto: BC Decker; 1990.)Brunicardi_Ch31_p1345-p1392.indd 135020/02/19 2:36 PM

1	Cameron JL: Atlas of Surgery. Vol. I, Gallbladder and Biliary Tract, the Liver, Portasystemic Shunts, the Pancreas. Toronto: BC Decker; 1990.)Brunicardi_Ch31_p1345-p1392.indd 135020/02/19 2:36 PM 1351LIVERCHAPTER 31nerve, which is stimulated by tumors that stretch Glisson’s cap-sule or by diaphragmatic irritation.Lymph is produced within the liver and drains via the perisinusoidal space of Disse and periportal clefts of Mall to larger lymphatics that drain to the hilar cystic duct lymph node (Calot’s triangle node), as well as the common bile duct, hepatic artery, and retropancreatic and celiac lymph nodes. This is par-ticularly important for resection of hilar cholangiocarcinoma, which has a high incidence of lymph node metastases. The hepatic lymph also drains cephalad to the cardiophrenic lymph nodes, and the latter can be pathologically identified on a stag-ing CT or MRI scan.LIVER PHYSIOLOGYThe liver is the largest gland in the body and has an extraordi-nary spectrum of

1	lymph nodes, and the latter can be pathologically identified on a stag-ing CT or MRI scan.LIVER PHYSIOLOGYThe liver is the largest gland in the body and has an extraordi-nary spectrum of functions. These include processes such as storage, metabolism, production, and secretion. One crucial role is the processing of absorbed nutrients through the metabolism of glucose, lipids, and proteins. The liver maintains glucose con-centrations in a normal range over both short and long periods by performing several important roles in carbohydrate metabo-lism. In the fasting state, the liver ensures a sufficient supply of glucose to the central nervous system. The liver can produce glucose by breaking down glycogen through glycogenolysis and by de novo synthesis of glucose through gluconeogenesis from noncarbohydrate precursors such as lactate, amino acids, and glycerol. In the postprandial state, excess circulating glucose is removed by glycogen synthesis or glycolysis and lipogenesis. The liver

1	noncarbohydrate precursors such as lactate, amino acids, and glycerol. In the postprandial state, excess circulating glucose is removed by glycogen synthesis or glycolysis and lipogenesis. The liver also plays a central role in lipid metabolism through the formation of bile and the production of cholesterol and fatty acids. Protein metabolism occurs in the liver through amino acid deamination, resulting in the production of ammonia as well as the production of a variety of amino acids. In addition to rpA: Normal bifurcation 57%B: Trifurcation of 3 ducts 12%C: R anterior (C1, 16%) or R posterior (C2, 4%) duct draining into CHDD: R posterior (D1, 5%) or R anterior duct (D2, 1%) draining into the left hepatic ductE: Absence of hepatic duct confluence 3%F: Drainage of R posterior duct into cystic duct 2%rprprprprprprprplhlhlhlhlhlhlhrararararararararaIVIVIIIIIIIIIIIIF2%E3%D6%C20%A57%B12%4%1%1%2%5%16%C2C1D2E2E1D1Figure 31-9. Main variations of hepatic duct confluence. As

1	duct into cystic duct 2%rprprprprprprprplhlhlhlhlhlhlhrararararararararaIVIVIIIIIIIIIIIIF2%E3%D6%C20%A57%B12%4%1%1%2%5%16%C2C1D2E2E1D1Figure 31-9. Main variations of hepatic duct confluence. As described by Couinaud in 1957, the bifurcation of the hepatic ducts has a vari-able pattern in approximately 40% of cases. CHD = common hepatic duct; lh = left hepatic; R = right; ra = right anterior; rp = right posterior. (Reproduced with permission from Blumgart LH, Fong Y: Surgery of the Liver and Biliary Tract, 3rd ed, Vol. I. London: Elsevier; 2000.)Brunicardi_Ch31_p1345-p1392.indd 135120/02/19 2:36 PM 1352SPECIFIC CONSIDERATIONSPART IImetabolism, the liver also is responsible for the synthesis of most circulating plasma proteins. Among these proteins are albumin, factors of the coagulation and fibrinolytic systems, and compounds of the complement cascade. Furthermore, the detoxification of many substances through drug metabo-lism occurs in the liver, as do immunologic responses

1	and fibrinolytic systems, and compounds of the complement cascade. Furthermore, the detoxification of many substances through drug metabo-lism occurs in the liver, as do immunologic responses through the many immune cells found in its reticuloendothelial system.9Bilirubin MetabolismBilirubin is the breakdown product of normal heme catabolism. Bilirubin is bound to albumin in the circulation and sent to the liver. In the liver, it is conjugated to glucuronic acid to form bilirubin diglucuronide in a reaction catalyzed by the enzyme glucuronyl transferase, making it water soluble. This glucuro-nide is then excreted into the bile canaliculi. A small amount dissolves in the blood and is then excreted in the urine. The majority of conjugated bilirubin is excreted in the intestine as waste because the intestinal mucosa is relatively impermeable to conjugated bilirubin. However, it is permeable to unconjugated bilirubin and urobilinogens, a series of bilirubin derivatives formed by the

1	because the intestinal mucosa is relatively impermeable to conjugated bilirubin. However, it is permeable to unconjugated bilirubin and urobilinogens, a series of bilirubin derivatives formed by the action of bacteria. Thus, some of the bilirubin and urobilinogens are reabsorbed in the portal circulation; they are again excreted by the liver or enter the circulation and are excreted in the urine.10Formation of BileBile is a complex fluid containing organic and inorganic sub-stances dissolved in an alkaline solution that flows from the liver through the biliary system and into the small intestine. The main components of bile are water, electrolytes, and a variety of organic molecules including bile pigments, bile salts, phospho-lipids (e.g., lecithin), and cholesterol. The two fundamental roles of bile are to aid in the digestion and absorption of lipids and lipid-soluble vitamins and to eliminate waste products (bilirubin and cholesterol) through secretion into bile and elimination in

1	of bile are to aid in the digestion and absorption of lipids and lipid-soluble vitamins and to eliminate waste products (bilirubin and cholesterol) through secretion into bile and elimination in feces. Bile is produced by hepatocytes and secreted through the biliary system. In between meals, bile is stored in the gallblad-der and concentrated through the absorption of water and elec-trolytes. Upon entry of food into the duodenum, bile is released from the gallbladder to aid in digestion. The human liver can produce about 1 L of bile daily.Bile salts, in conjunction with phospholipids, are respon-sible for the digestion and absorption of lipids in the small intestine. Bile salts are sodium and potassium salts of bile acids conjugated to amino acids. The bile acids are derivatives of cho-lesterol synthesized in hepatocytes. Cholesterol, ingested from the diet or derived from hepatic synthesis, is converted into the bile acids cholic acid and chenodeoxycholic acid. These bile acids are

1	synthesized in hepatocytes. Cholesterol, ingested from the diet or derived from hepatic synthesis, is converted into the bile acids cholic acid and chenodeoxycholic acid. These bile acids are conjugated to either glycine or taurine before secre-tion into the biliary system. Bacteria in the intestine can remove glycine and taurine from bile salts. They can also convert some of the primary bile acids into secondary bile acids by removing a hydroxyl group, producing deoxycholic acid from cholic acid and lithocholic acid from chenodeoxycholic acid.Bile salts secreted into the intestine are efficiently reab-sorbed and reused. Approximately 90% to 95% of the bile salts are absorbed from the small intestine at the terminal ileum. The remaining 5% to 10% enter the colon and are converted to the secondary salts, deoxycholic acid and lithocholic acid. The mixture of primary and secondary bile salts and bile acids is absorbed primarily by active transport in the terminal ileum. The absorbed bile

1	salts, deoxycholic acid and lithocholic acid. The mixture of primary and secondary bile salts and bile acids is absorbed primarily by active transport in the terminal ileum. The absorbed bile salts are transported back to the liver in the portal vein and reexcreted in the bile. Those lost in the stool are replaced by synthesis in the liver. The continuous process of secretion of bile salts in the bile, their passage through the intestine, and their subsequent return to the liver is termed the enterohepatic circulation.10Drug MetabolismThe liver plays an important role in providing mechanisms for ridding the body of foreign molecules (xenobiotics) that are absorbed from the environment. In most cases, a drug is relatively lipophilic to ensure good absorption. The liver par-ticipates in the elimination of these lipid-soluble drugs by trans-forming them into more readily excreted hydrophilic products. There are two main reactions important for drug metabolism. Phase 1 reactions include

1	elimination of these lipid-soluble drugs by trans-forming them into more readily excreted hydrophilic products. There are two main reactions important for drug metabolism. Phase 1 reactions include oxidation, reduction, and hydrolysis of molecules. These result in metabolites that are more hydro-philic than the original chemicals. The cytochrome P450 system is a family of hemoproteins important for oxidative reactions involving drugs and toxic substances. Phase 2 reactions, also known as conjugation reactions, are synthetic reactions that involve addition of subgroups to the drug molecule. These sub-groups include glucuronate, acetate, glutathione, glycine, sul-fate, and methyl groups. These drug reactions occur mainly in the smooth endoplasmic reticulum of hepatocytes.Many factors can affect drug metabolism in the liver. When the rate of metabolism of a drug is increased (i.e., enzyme induction), the duration of the drug action will decrease. How-ever, when the metabolism of a drug

1	drug metabolism in the liver. When the rate of metabolism of a drug is increased (i.e., enzyme induction), the duration of the drug action will decrease. How-ever, when the metabolism of a drug is decreased (i.e., enzyme inhibition), then the drug will circulate for a longer period of time. It is important to note that some drugs may be converted to active products by metabolism in the liver. An example is acetaminophen when taken in larger doses. Normally, acet-aminophen is conjugated by the liver to harmless glucuronide and sulfate metabolites that are water soluble and eliminated in the urine. During an overdose, the normal metabolic path-ways are overwhelmed, and some of the drug is converted to a reactive and toxic intermediate by the cytochrome P450 system. Glutathione normally reacts with this intermediate, leading to the production and subsequent excretion of a harmless prod-uct. However, as glutathione stores are diminished, the reactive intermediate cannot be detoxified and

1	with this intermediate, leading to the production and subsequent excretion of a harmless prod-uct. However, as glutathione stores are diminished, the reactive intermediate cannot be detoxified and it combines with lipid membranes of hepatocytes, which results in cellular necrosis. Thus, treatment of acetaminophen overdoses consists of replen-ishing glutathione stores by supplementing with sulfhydryl compounds such as acetylcysteine.Liver Function TestsLiver function tests is a term frequently used to refer to mea-surement of the levels of a group of serum markers for evalu-ation of liver dysfunction. Most commonly, levels of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (AP), γ-glutamyltranspeptidase (GGT), and biliru-bin are included in this panel. This term is a misnomer, how-ever, because most of these tests measure not liver function but rather cell damage. More accurate measurement of the liver’s synthetic function is provided by serum albumin

1	term is a misnomer, how-ever, because most of these tests measure not liver function but rather cell damage. More accurate measurement of the liver’s synthetic function is provided by serum albumin levels and pro-thrombin time (PT). Although measuring liver enzyme levels is important in the assessment of a patient’s liver disease, these test results can be nonspecific. Thus, evaluation of patients with suspected liver disease should always involve careful interpreta-tion of abnormalities in these liver test results in the context of a thorough history and physical examination. The approach to 2Brunicardi_Ch31_p1345-p1392.indd 135220/02/19 2:36 PM 1353LIVERCHAPTER 31evaluating abnormal laboratory values also can be simplified by categorizing the type of abnormality that predominates (hepa-tocellular damage, abnormal synthetic function, or cholestasis).Hepatocellular InjuryHepatocellular injury of the liver is usually indicated by abnor-malities in levels of the liver

1	(hepa-tocellular damage, abnormal synthetic function, or cholestasis).Hepatocellular InjuryHepatocellular injury of the liver is usually indicated by abnor-malities in levels of the liver aminotransferases AST and ALT. These enzymes participate in gluconeogenesis by catalyz-ing the transfer of amino groups from aspartic acid or alanine to ketoglutaric acid to produce oxaloacetic acid and pyruvic acid, respectively (these enzymes are also referred to as serum glutamic-oxaloacetic transaminase [SGOT] and serum glutamic-pyruvic transaminase [SGPT]). AST is found in liver, cardiac muscle, skeletal muscle, kidney, brain, pancreas, lungs, and red blood cells and thus is less specific for disorders of the liver. ALT is predominately found in the liver and thus is more specific for liver disease. Hepatocellular injury is the trigger for release of these enzymes into the circulation. Common causes of elevated aminotransferase levels include viral hepatitis, alcohol abuse, medications, genetic

1	Hepatocellular injury is the trigger for release of these enzymes into the circulation. Common causes of elevated aminotransferase levels include viral hepatitis, alcohol abuse, medications, genetic disorders (Wilson’s disease, hemochroma-tosis, α1-antitrypsin deficiency), and autoimmune diseases.The extent of serum aminotransferase elevations can sug-gest certain etiologies of the liver injury. However, the levels of the enzymes in these tests correlate poorly with the severity of hepatocellular necrosis because they may not be significantly elevated in conditions of hepatic fibrosis or cirrhosis. In alco-holic liver disease, an AST to ALT ratio of >2:1 is common. Mild elevations of transaminase levels can be found in nonal-coholic fatty liver disease, chronic viral infection, or medica-tion-induced injury. Moderate increases in the levels of these enzymes are common in acute viral hepatitis. In conditions of ischemic insults, toxin ingestions (i.e., acetaminophen), and ful-minant

1	injury. Moderate increases in the levels of these enzymes are common in acute viral hepatitis. In conditions of ischemic insults, toxin ingestions (i.e., acetaminophen), and ful-minant hepatitis, AST and ALT levels can be elevated to the thousands.Abnormal Synthetic FunctionAlbumin synthesis is an important function of the liver and thus can be measured to evaluate the liver’s synthetic function. The liver produces approximately 10 g of albumin per day. How-ever, albumin levels are dependent on a number of factors such as nutritional status, renal dysfunction, protein-losing enteropa-thies, and hormonal disturbances. In addition, level of albumin is not a marker of acute hepatic dysfunction due to albumin’s long half-life of 15 to 20 days.Most clotting factors (except factor VIII) are synthesized exclusively in the liver, and thus their levels can also be used as a measure of hepatic synthetic function. Measurements of the pro-thrombin time (PT) and international normalized ratio

1	synthesized exclusively in the liver, and thus their levels can also be used as a measure of hepatic synthetic function. Measurements of the pro-thrombin time (PT) and international normalized ratio (INR) are some of the best tests of hepatic synthetic function. PT measures the rate of conversion of prothrombin to thrombin. To standard-ize the reporting of PT and avoid interlaboratory variability, the INR was developed. The INR is the ratio of the patient’s PT to the mean control PT. Because vitamin K is involved in the γ-carboxylation of factors used to measure PT (factors II, VII, IX, and X), values also may be prolonged in other conditions such as vitamin K deficiency and warfarin therapy.CholestasisCholestasis is a condition in which bile flow from the liver to the duodenum is impaired. Disturbances in bile flow may be due to intrahepatic causes (hepatocellular dysfunction) or extrahepatic causes (biliary tree obstruction). Cholestasis often results in the release of certain

1	Disturbances in bile flow may be due to intrahepatic causes (hepatocellular dysfunction) or extrahepatic causes (biliary tree obstruction). Cholestasis often results in the release of certain enzymes and thus can be detected by measur-ing the serum levels of bilirubin, AP, and GGT. Bilirubin is a breakdown product of hemoglobin metabolism. Unconjugated bilirubin is insoluble and thus is transported to the liver bound to albumin. In the liver, it is conjugated to allow excretion in bile. Measured total bilirubin levels can be normal or high in patients with significant liver disease because of the liver’s abil-ity to conjugate significant amounts of bilirubin. Thus, to help aid in the diagnosis of hyperbilirubinemia, fractionation of total bilirubin is usually performed to distinguish between conjugated (direct) and unconjugated (indirect) bilirubin. Indirect bilirubin is a term frequently used to refer to unconjugated bilirubin in the circulation because the addition of another

1	between conjugated (direct) and unconjugated (indirect) bilirubin. Indirect bilirubin is a term frequently used to refer to unconjugated bilirubin in the circulation because the addition of another chemical is nec-essary to differentiate this fraction from the whole. Normally, >90% of serum bilirubin is unconjugated. The testing process for conjugated bilirubin, in contrast, is direct without the addi-tion of other agents. The direct bilirubin test measures the lev-els of conjugated bilirubin and δ bilirubin (conjugated bilirubin bound to albumin).The patterns of elevation of the different fractions of bili-rubin provide important diagnostic clues as to the cause of cho-lestasis. In general, an elevated indirect bilirubin level suggests intrahepatic cholestasis, and an elevated direct bilirubin level suggests extrahepatic obstruction. Mechanisms that can result in increases in unconjugated bilirubin levels include increased bilirubin production (hemolytic disorders and resorption of

1	level suggests extrahepatic obstruction. Mechanisms that can result in increases in unconjugated bilirubin levels include increased bilirubin production (hemolytic disorders and resorption of hematomas) or defects (inherited or acquired) in hepatic uptake or conjugation. The rate-limiting step in bilirubin metabolism is the excretion of bilirubin from hepatocytes, so conjugated hyperbilirubinemia can be seen in inherited or acquired dis-orders of intrahepatic excretion or extrahepatic obstruction. Conjugated bilirubin that cannot be excreted accumulates in hepatocytes, which results in its secretion into the circulation. Because conjugated bilirubin is water soluble, it can be found in the urine of patients with jaundice.AP is an enzyme with a wide tissue distribution but is found primarily in the liver and bones. In the liver, it is expressed by the bile duct epithelium. In conditions of biliary obstruction, levels rise as a result of increased synthesis and release into the serum.

1	in the liver and bones. In the liver, it is expressed by the bile duct epithelium. In conditions of biliary obstruction, levels rise as a result of increased synthesis and release into the serum. Because the half-life of serum AP is approximately 7 days, it may take several days for levels to normalize even after resolution of the biliary obstruction.GGT is another enzyme found in hepatocytes and released from the bile duct epithelium. Elevation of GGT is an early marker and also a sensitive test for hepatobiliary disease. Like AP elevation, however, it is nonspecific and can be produced by a variety of disorders in the absence of liver disease. Increased levels of GGT can be induced by certain medications, alcohol abuse, pancreatic disease, myocardial infarction, renal failure, and obstructive pulmonary disease. For this reason, elevated GGT levels are often interpreted in conjunction with other enzyme abnormalities. For example, a raised GGT level with increased AP level supports a

1	pulmonary disease. For this reason, elevated GGT levels are often interpreted in conjunction with other enzyme abnormalities. For example, a raised GGT level with increased AP level supports a liver source.JaundiceJaundice refers to the yellowish staining of the skin, sclera, and mucous membranes with the pigment bilirubin. Hyperbilirubi-nemia usually is detectable as jaundice when blood levels rise above 2.5 to 3 mg/dL. Jaundice can be caused by a wide range of benign and malignant disorders. However, when present, it may indicate a serious condition, and thus knowledge of the differential diagnosis of jaundice and a systematic approach to Brunicardi_Ch31_p1345-p1392.indd 135320/02/19 2:36 PM 1354SPECIFIC CONSIDERATIONSPART IIthe workup of the patient is necessary. Workup of a patient with jaundice is simplified by organizing the possible causes of the disorder into groups based on the location of bilirubin metabo-lism. As mentioned previously, bilirubin metabolism can take place

1	with jaundice is simplified by organizing the possible causes of the disorder into groups based on the location of bilirubin metabo-lism. As mentioned previously, bilirubin metabolism can take place in three phases: prehepatic, intrahepatic, and posthepatic. The prehepatic phase includes the production of bilirubin from the breakdown of heme products and its transport to the liver. The majority of the heme results from red blood cell metabolism and the rest from other heme-containing organic compounds such as myoglobin and cytochromes. In the liver, the insoluble unconjugated bilirubin is then conjugated to glucuronic acid to allow for solubility in bile and excretion. The posthepatic phase of bilirubin metabolism consists of excretion of soluble bilirubin through the biliary system into the duodenum. Dysfunction in any of these phases can lead to jaundice.10Prehepatic. Jaundice as a result of elevated levels of uncon-jugated bilirubin occurs from faulty prehepatic metabolism and

1	the duodenum. Dysfunction in any of these phases can lead to jaundice.10Prehepatic. Jaundice as a result of elevated levels of uncon-jugated bilirubin occurs from faulty prehepatic metabolism and usually arises from conditions that interfere with proper conju-gation of bilirubin in the hepatocyte. Insufficient conjugation is often seen in processes that result in excessive heme metabolism. Subsequently, the conjugation system is overwhelmed, which results in unconjugated hyperbilirubinemia. Causes of hemoly-sis include inherited and acquired hemolytic anemias. Inherited hemolytic anemias include genetic disorders of the red blood cell membrane (hereditary spherocytosis and eliptocytosis), enzyme defects (glucose-6-phosphate dehydrogenase deficiency), and defects in hemoglobin structure (sickle cell anemia and thalas-semias). Hemolytic anemias can also be acquired, and these can be further divided into those with immune-mediated and those with non–immune-mediated causes.

1	structure (sickle cell anemia and thalas-semias). Hemolytic anemias can also be acquired, and these can be further divided into those with immune-mediated and those with non–immune-mediated causes. Immune-mediated hemo-lytic anemias result in a positive finding on a direct Coombs test and have a variety of autoimmune and drug-induced causes. In contrast, direct Coombs test results are negative in nonimmune hemolytic anemias. The causes in this latter category are varied and include drugs and toxins that directly damage red blood cells, mechanical trauma (heart valves), microangiopathy, and infections. Prehepatic dysfunction of bilirubin metabolism also can result from failure in the transport of unconjugated bilirubin to the liver by albumin in any condition that leads to plasma protein loss. A poor nutritional state or excess protein loss as seen in burn patients can lead to elevated levels of unconjugated bilirubin in the circulation and jaundice.Intrahepatic. Intrahepatic causes of

1	A poor nutritional state or excess protein loss as seen in burn patients can lead to elevated levels of unconjugated bilirubin in the circulation and jaundice.Intrahepatic. Intrahepatic causes of jaundice involve the intracellular mechanisms for conjugation and excretion of bile from the hepatocyte. The enzymatic processes in hepatocytes can be affected by any condition that impairs hepatic blood flow and subsequent function of the liver (ischemic or hypoxic events). Furthermore, there are multiple inherited disorders of enzyme metabolism that can result in either unconjugated or conjugated hyperbilirubinemia. Gilbert’s syndrome is a genetic variant characterized by diminished activity of the enzyme glucuronyltransferase, which results in decreased conjugation of bilirubin to glucuronide. It is a benign condition that affects approximately 4% to 7% of the population. Typically, the dis-ease results in transient mild increases in unconjugated bilirubin levels and jaundice during

1	It is a benign condition that affects approximately 4% to 7% of the population. Typically, the dis-ease results in transient mild increases in unconjugated bilirubin levels and jaundice during episodes of fasting, stress, or illness. These episodes are self-limited and usually do not require fur-ther treatment. Another inherited disorder of bilirubin conjuga-tion is Crigler-Najjar syndrome. It is a rare disease found in neonates and can result in neurotoxic sequelae from bilirubin encephalopathy.In addition to defects in conjugation, disorders in bilirubin excretion in hepatocytes can also lead to jaundice. Rotor’s syn-drome and Dubin-Johnson syndrome are two uncommon genetic disorders that disrupt secretion of conjugated bilirubin from the hepatocyte into the bile and result in conjugated hyperbilirubi-nemia. There are also multiple acquired conditions that result in inflammation and intrahepatic cholestasis by affecting hepato-cyte mechanisms for conjugation and excretion of bile.

1	hyperbilirubi-nemia. There are also multiple acquired conditions that result in inflammation and intrahepatic cholestasis by affecting hepato-cyte mechanisms for conjugation and excretion of bile. Viruses, alcohol abuse, sepsis, and autoimmune disorders all can result in inflammation in the liver with subsequent disruption of bilirubin transport in the liver. In addition, jaundice can also occur from the cytotoxic effects of many medications, including acetamino-phen, oral contraceptives, and anabolic steroids.Posthepatic. Posthepatic causes of jaundice are usually the result of intrinsic or extrinsic obstruction of the biliary duct sys-tem that prevents the flow of bile into the duodenum. There is a wide spectrum of pathologies that may present with obstructive jaundice. Intrinsic obstruction can occur from biliary diseases, including cholelithiasis, choledocholithiasis, benign and malig-nant biliary strictures, cholangiocarcinoma, cholangitis, and disorders of the papilla of Vater.

1	can occur from biliary diseases, including cholelithiasis, choledocholithiasis, benign and malig-nant biliary strictures, cholangiocarcinoma, cholangitis, and disorders of the papilla of Vater. Extrinsic compression of the biliary tree is commonly due to pancreatic disorders. Patients with pancreatitis, pseudocysts, and malignancies can present with jaundice due to external compression of the biliary system. Finally, with the growing armamentarium of endoscopic tools and minimally invasive surgical approaches, surgical complica-tions are becoming more frequent causes of extrahepatic cho-lestasis. Misadventures with surgical clips, retained stones, and inadvertent ischemic insults to the biliary system can result in obstructive jaundice recognized at any time from immediately postoperatively to many years later.MOLECULAR SIGNALING PATHWAYS IN THE LIVERAcute Phase ReactionThe liver is the site of synthesis of acute phase proteins that consists of a group of plasma proteins that are

1	to many years later.MOLECULAR SIGNALING PATHWAYS IN THE LIVERAcute Phase ReactionThe liver is the site of synthesis of acute phase proteins that consists of a group of plasma proteins that are rapidly released in response to inflammatory conditions elsewhere in the body. The synthesis of these proteins in the liver is mediated by a num-ber of inflammatory mediators. Cytokines such as tumor necro-sis factor alpha (TNF-α), interferon-γ (IFN-γ), interleukin-1 (IL-1), interleukin-6 (IL-6), and interleukin-8 (IL-8) are released by inflammatory cells into the circulation at sites of injury and modulate the acute phase response. In response to these cyto-kines, the liver increases synthesis and release of a wide vari-ety of proteins including ceruloplasmin, complement factors, C-reactive protein (CRP), d-dimer protein, alpha 1-antitrysin, and serum amyloid A. There are proteins, such as serum albu-min and transferrin, that also decrease (negative acute phase proteins) in response to

1	protein (CRP), d-dimer protein, alpha 1-antitrysin, and serum amyloid A. There are proteins, such as serum albu-min and transferrin, that also decrease (negative acute phase proteins) in response to inflammation.The acute phase response of the liver can be initiated in response to infection, trauma, or malignancy. The purpose of the release of these proteins from the liver is to contain infectious processes, prevent further tissue damage and to begin repara-tive and regeneration processes to restore body homeostasis. For example, products of the complement pathways can attach to microbes to allow for phagocytosis and act as chemoattractants to the areas of inflammation. CRP is an important acute phase protein that is also involved in the clearance of microorganisms by binding to their membranes and functioning as an opsonin to Brunicardi_Ch31_p1345-p1392.indd 135420/02/19 2:36 PM 1355LIVERCHAPTER 31facilitate phagocytosis. Other proteins such as α1-antitrypsin are protease

1	membranes and functioning as an opsonin to Brunicardi_Ch31_p1345-p1392.indd 135420/02/19 2:36 PM 1355LIVERCHAPTER 31facilitate phagocytosis. Other proteins such as α1-antitrypsin are protease inhibitors and restrict the protease activity of enzymes of inflammatory cells. Thus, the secretion of acute phase pro-teins from the liver during the acute phase response is an early defense measure against harmful stimuli prior to the full activa-tion of the immune response.11LPS SignalingThe liver is a complex organ with an important function in immune surveillance and clearance of bacteria and their prod-ucts. This function is facilitated by the fact that the liver receives all the drainage of the gastrointestinal tract via the portal blood flow, making it the last barrier preventing bacteria and their toxins from reaching the systemic circulation. The importance of preventing bacteria and their products from reaching the systemic blood stream is evident in patients who are infected with

1	and their toxins from reaching the systemic circulation. The importance of preventing bacteria and their products from reaching the systemic blood stream is evident in patients who are infected with gram-negative bacteria. Gram-negative bacteria infection produces an acute inflammatory reaction that can lead to septic shock and multiple organ failure. The complications of gram-negative sepsis are initiated by endotoxin (lipopolysaccharide [LPS]). LPS is a glycolipid constituent of gram-negative bacte-ria outer membranes composed of a hydrophilic polysaccharide portion and a hydrophobic domain called lipid A. The lipid A structure is the LPS component responsible for the biological effects of LPS. Mere nanogram amounts of LPS injected into humans are needed to result in the manifestations of septic shock. The profound effects of LPS are caused not only by the direct effect of LPS itself but also by the activation of LPS-sensitive cells, resulting in the excessive release of cytokines

1	of septic shock. The profound effects of LPS are caused not only by the direct effect of LPS itself but also by the activation of LPS-sensitive cells, resulting in the excessive release of cytokines and other inflammatory mediators.Since sepsis from gram-negative bacterial infection con-tinues to be a major cause of morbidity and mortality, significant efforts have been made to identify the molecules involved in LPS-binding and signaling. Lipopolysaccharide binding protein (LBP), CD14, MD2, and Toll-like receptors have all been iden-tified as important mediators in the pathway of LPS stimulation. LBP is an acute-phase protein synthesized by hepatocytes that binds the lipid A moiety of LPS and forms a soluble LBP-LPS complex. This LBP-LPS complex then interacts with CD14, a receptor identified as important in LPS recognition, resulting in the release of inflammatory cytokines and mediators. Studies have shown that although LBP is important, it is not required for LPS to interact with

1	as important in LPS recognition, resulting in the release of inflammatory cytokines and mediators. Studies have shown that although LBP is important, it is not required for LPS to interact with CD14; however, its presence markedly decreases the concentration of LPS necessary for cellular acti-vation. This may be important especially at the low concentra-tions of LPS found under physiological conditions. CD14 exists in two forms, membrane (mCD14) and soluble (sCD14) form. The interaction of LPS with membrane CD14 or soluble CD14 is important in host clearance of LPS. This interaction is also responsible for the toxic effects of LPS seen in the liver and systemic circulation after the release of inflammatory cytokines and mediators. While membrane CD14 is a membrane protein found on the surface of myeloid lineage and mediates the activa-tion of these cells by LPS, soluble CD14 is found in the serum and enables responses to LPS by cells that do not express CD14. In addition to its

1	surface of myeloid lineage and mediates the activa-tion of these cells by LPS, soluble CD14 is found in the serum and enables responses to LPS by cells that do not express CD14. In addition to its important role in the release of LBP as an acute phase reactant during LPS-mediated inflammatory insults, the liver is also one of the major sources of soluble CD14 into the circulation.The binding of the LBP-LPS complex to CD14 is not enough to transduce an intracellular LPS signal. Membrane CD14 is a glycosyl phosphatidylinositol-anchored protein without a membrane-spanning domain. Thus, signaling fur-ther downstream of LPS requires additional elements. In stud-ies using chemically modified, radioiodinated LPS capable of cross-linking to nearby proteins, LPS has been shown to cross-link specifically to two other molecules, TLR4 and MD-2. TLR4 is a member of the family of proteins called Toll-like receptors and has been identified as the transmembrane corecep-tor to CD14. TLR4 was

1	specifically to two other molecules, TLR4 and MD-2. TLR4 is a member of the family of proteins called Toll-like receptors and has been identified as the transmembrane corecep-tor to CD14. TLR4 was originally identified as the molecular sensor for bacterial LPS when studies demonstrated that muta-tions in the tlr4 gene were responsible for defective LPS sig-naling in mutant mice. Thus, initiation of LPS signal cascade requires the interaction of LPS directly with the heteromeric receptor complex of CD14, TLR4, and MD-2. Activation of this complex senses the presence of bacterial LPS at the cell sur-face and then transmits a signal into the cytoplasm through two distinct pathways. One pathway is dependent upon an adaptor known as myeloid differentiation factor 88 (MyD88). The other pathway is MyD88-independent and relies on an adaptor known as Toll/IL-1 receptor domain-containing adaptor-inducing IFNβ (TRIF).The liver is the main organ involved in the clearance of LPS from the

1	pathway is MyD88-independent and relies on an adaptor known as Toll/IL-1 receptor domain-containing adaptor-inducing IFNβ (TRIF).The liver is the main organ involved in the clearance of LPS from the bloodstream and so plays a critical role in the identification and processing of LPS. Kupffer cells are the resident macrophages of the liver and have been shown to par-ticipate in LPS clearance. Studies have demonstrated that the majority of radiolabelled LPS injected intravenously is quickly cleared from the circulation and found in the liver, primarily localized to the Kupffer cells. Kupffer cells also contribute to the inflammatory cascade by producing cytokines in response to LPS. Interestingly, hepatocytes, the parenchymal cells of the liver, also have all the components required for LPS recognition and signaling and can participate in the response to LPS and process LPS for clearance.Although the liver is essential in the host response to gram-negative bacteria infection by

1	for LPS recognition and signaling and can participate in the response to LPS and process LPS for clearance.Although the liver is essential in the host response to gram-negative bacteria infection by contributing to LPS clearance and to the LPS-induced inflammatory reaction, evidence reveals that LPS may actually have a reciprocal role in the pathogenesis of liver disorders. The relationship between LPS and liver disease is not a novel concept. Early studies have shown the correlation between the presence or absence of gut-derived LPS and the development of liver injury. Attempts to eliminate gut-derived LPS have had protective effects in various animal models of liver injury, including alcohol-induced liver disease. Other stud-ies have shown the synergism between LPS and hepatotoxins in worsening liver injury.In summary, the liver is essential in the clearance of LPS, but it can also contribute to the negative systemic effects seen in gram-negative bacterial sepsis by excessive

1	in worsening liver injury.In summary, the liver is essential in the clearance of LPS, but it can also contribute to the negative systemic effects seen in gram-negative bacterial sepsis by excessive activation of the LPS signaling pathway. In addition, there is evidence that this signaling pathway may participate in the pathogenesis of a vari-ety of liver disease. An understanding and characterization of the LPS pathway within the liver is an important step to under-standing the molecular basis for the lethal effect of LPS during sepsis and liver disorders.12,13Nitric OxideNitric oxide (NO) is a diffusible, free radical gas that was first identified in 1980 as endothelium-derived relaxing factor. Its physiologic and pathophysiologic importance was first discov-ered in the cardiovascular system with its vital role as a vaso-dilator. However, its mediation in a variety of other diverse biological activities has since been discovered. In the liver, the Brunicardi_Ch31_p1345-p1392.indd

1	with its vital role as a vaso-dilator. However, its mediation in a variety of other diverse biological activities has since been discovered. In the liver, the Brunicardi_Ch31_p1345-p1392.indd 135520/02/19 2:36 PM 1356SPECIFIC CONSIDERATIONSPART IIinfluence of NO on normal physiology as well as in states of disease has been extensively studied. The activation of inflam-matory cascades in the liver almost universally includes the upregulation of the inducible or inflammatory isoform of nitric oxide synthase (iNOS) and subsequent NO production. The functions of iNOS and NO in the liver is complex, and a clear dichotomy of their roles in liver dysfunction, whether being pro-tective or detrimental, has been demonstrated.Nitric oxide can be produced by one of three NO synthases: (NOS)-neuronal NOS (nNOS), inducible or inflammatory NOS (iNOS), and endothelial NOS (eNOS). These enzymes catalyze the conversion of l-arginine to NO and l-citrulline. Neuronal and endothelial NOS are

1	NOS (nNOS), inducible or inflammatory NOS (iNOS), and endothelial NOS (eNOS). These enzymes catalyze the conversion of l-arginine to NO and l-citrulline. Neuronal and endothelial NOS are constitutively expressed in a wide range of tissues. The activity of iNOS and eNOS are primarily controlled by calcium-mediated signaling that results in transient activation of these enzymes to produce small amounts of NO. Inducible NOS, as the name implies, is not normally expressed in rest-ing states in most tissues but is upregulated by gene transcrip-tion under conditions of stress. In contrast to nNOS and eNOS, the amount of NO produced by iNOS is large and sustained. Although first identified in macrophages, the expression of iNOS has been shown in most cell types if appropriately stimulated. Interestingly, studies on the liver with hepatocytes provided the first evidence that parenchymal cells could express iNOS. It is now known that iNOS can be expressed in all cell types of the liver, but

1	studies on the liver with hepatocytes provided the first evidence that parenchymal cells could express iNOS. It is now known that iNOS can be expressed in all cell types of the liver, but hepatocyte expression appears to be the most promi-nent. Studies have shown that many inflammatory mediators, including cytokines, microbial products, and oxidative stress, are all capable of stimulating iNOS expression in the liver.The chemical action of NO in biological systems has been difficult to study due to its short-lived nature. NO is highly reac-tive with other molecules due to its one unpaired electron. These interactions can either result in nitrosation or oxidation with subsequent varied effects on cellular processes. Nitric oxide can also signal through cyclic nucleotides by activating the soluble isoform of guanylyl cyclase, increasing levels of cGMP. The functions of cGMP include acting as second messengers that transmit signals by activating downstream kinases or through the

1	the soluble isoform of guanylyl cyclase, increasing levels of cGMP. The functions of cGMP include acting as second messengers that transmit signals by activating downstream kinases or through the activation of cyclic nucleotide-gated channels. In addition to cGMP signaling, NO has also been found to modulate the expression of many genes.The role of NO in inflammatory states of the liver is complex and is at times conflicting. Under physiologic condi-tions, NO is important in maintaining hepatic perfusion. How-ever, in inflammatory conditions, such as ischemia/reperfusion (I/R), nitric oxide can play either a protective or harmful role depending on the enzymatic source (inducible versus endothe-lial nitric oxide synthase) and the type of ischemia reperfusion (cold vs. warm). It appears that the low level of constitutively expressed eNOS-derived NO is primarily beneficial in models of I/R injury with vasodilation and subsequent improvement in hepatic microcirculation as the proposed

1	that the low level of constitutively expressed eNOS-derived NO is primarily beneficial in models of I/R injury with vasodilation and subsequent improvement in hepatic microcirculation as the proposed mechanism of protec-tion. Interestingly, activation of iNOS in similar models sug-gests a potentially harmful role for iNOS. Nitric oxide, through its reaction with reactive nitrogen and oxygen intermediates generated in the course of reperfusion injury, can contribute to much of the hepatocellular damage depending on the intracellu-lar ratio of these intermediates to nitric oxide. The production of iNOS and NO are also closely tied to multiple other inflamma-tory mediators in the liver, and activation of these downstream signals may explain some of the detrimental effects of NO in I/R injury of the liver. Thus, given its diverse biological effects as a signaling molecule, it is not surprising that NO plays both pro-tective and potentially harmful role in the setting of hepatic I/R

1	injury of the liver. Thus, given its diverse biological effects as a signaling molecule, it is not surprising that NO plays both pro-tective and potentially harmful role in the setting of hepatic I/R injury. The final effect of NO varies in different liver diseases and depends on the overall hepatic environment. The potential use of NO pharmacologic manipulation to treat hepatic disease will require careful balance of the risks and benefits of this sim-ple, yet extremely complicated, molecule.14,15Heme Oxygenase SystemThe heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme to yield biliverdin, carbon monoxide (CO), and free iron. The HO system, which is activated in response to multiple cellular stresses, has been shown to be an endogenous cytoprotectant in a variety of inflammatory conditions. There are currently three heme oxygenase isozymes identified. HO-1 is the inducible form of heme oxygenase, while HO-2 and HO-3 are constitutively expressed. The function

1	of inflammatory conditions. There are currently three heme oxygenase isozymes identified. HO-1 is the inducible form of heme oxygenase, while HO-2 and HO-3 are constitutively expressed. The function of HO in heme deg-radation is essential due to the potentially toxic effects of heme. An excess of heme can cause cellular damage from oxidative stress due to its production of reactive oxygen species. Thus, the HO system is an important defense mechanism against free heme-mediated oxidative stress.HO-1 has been shown to be induced in a variety of organs during diverse conditions such as hypoxia, endotoxemia, ischemia/reperfusion (I/R), hyperthermia, and radiation. It is thought that HO-1 is involved in maintaining redox homeostasis during cellular stress. In the liver, HO-1 is thought to normally modulate hepatic microvasculature tone through its generation of CO and, like nitric oxide, its activation of guanylyl cyclase. This important role is demonstrated in animal models of por-tal

1	modulate hepatic microvasculature tone through its generation of CO and, like nitric oxide, its activation of guanylyl cyclase. This important role is demonstrated in animal models of por-tal hypertension where inhibition of HO-1 exacerbates hyper-tension. Since HO-1 is induced as a protective mechanism in response to various stimuli, targeted induction of HO-1 has been studied as a therapeutic strategy for protection against inflam-matory processes. HO-1 overexpression exerts hepatoprotective effects in models of I/R injury, hemorrhagic shock and resus-citation, acetaminophen-induced hepatonecrosis, and sepsis-mediated liver injury.Although HO-1 has been shown to provide protective effects in a variety of inflammatory states, the specific mecha-nisms by which HO-1 mediates its protective effects are remains to be fully elucidated. Originally thought to be only potentially toxic waste, the byproducts generated during heme catabolism now appear to play important roles against cellular

1	effects are remains to be fully elucidated. Originally thought to be only potentially toxic waste, the byproducts generated during heme catabolism now appear to play important roles against cellular stress. The well-known hazardous effects of high doses of CO are attribut-able to its ability to bind hemoglobin and myoglobin, preventing the release of oxygen to tissues. However, only recently have the physiological and beneficial roles of CO been identified. CO is produced in injured tissues via induction of HO-1 and contrib-utes to the attenuation of proinflammatory processes. Similar to NO, CO plays an important role in maintaining the micro-circulation though its activation of soluble guanylyl cyclase and subsequent elevation of intracellular cyclic 3’5’-guanosine monophosphate (cGMP). The signaling activities of cGMP lead to smooth muscle relaxation and inhibition and platelet aggre-gation. In addition, CO has also been shown to inhibit proin-flammatory cytokines (TNF-α, IL-1) and

1	The signaling activities of cGMP lead to smooth muscle relaxation and inhibition and platelet aggre-gation. In addition, CO has also been shown to inhibit proin-flammatory cytokines (TNF-α, IL-1) and chemokines while simultaneously inducing anti-inflammatory cytokines (IL-10). Exogenous low-dose CO has been shown to protect the liver from I/R injury and endotoxemia.Biliverdin and bilirubin are other metabolites of heme that are also recognized as possible mediators of HO-1’s protective Brunicardi_Ch31_p1345-p1392.indd 135620/02/19 2:36 PM 1357LIVERCHAPTER 31function. The cytosolic enzyme biliverdin reductase catalyzes the reduction of biliverdin to bilirubin. Both biliverdin and bilirubin have important endogenous antioxidant properties. Free iron, the third byproduct of heme oxidation is known to be cytotoxic by catalyzing the production of hydroxyl radicals. However, HO-1 induction is associated with increased levels of ferritin, the free iron sequestering protein. Thus, the

1	is known to be cytotoxic by catalyzing the production of hydroxyl radicals. However, HO-1 induction is associated with increased levels of ferritin, the free iron sequestering protein. Thus, the increase in ferritin with the subsequent decrease in intracellular concentra-tions of free iron results in a net antioxidant effect. Importantly, both bilirubin and ferritin have been shown to protect against liver injury in a variety of I/R models.In summary, HO-1 is upregulated and protective in mul-tiple conditions of hepatic stress. Until recently, the degradation products of the HO system were thought to only be potentially toxic waste. It now appears that CO, biliverdin/bilirubin, ferritin are important in the maintenance of cellular redox homeostasis and may play a role in the mechanism of hepatoprotection in disease. Studies involving induction of HO-1 expression and use of its metabolic products hold therapeutic promises for novel protective agents against disorders of hepatic

1	of hepatoprotection in disease. Studies involving induction of HO-1 expression and use of its metabolic products hold therapeutic promises for novel protective agents against disorders of hepatic inflammation.16,17Toll-Like ReceptorsThe liver is a central regulator of the systemic immune response following acute insults to the body. Not only does it play a cru-cial role in modulating the systemic inflammatory response to infection or injury, it is also subject to injury and dysfunc-tion from these same processes. Recent advances in the study of mechanisms for the activation of the innate immune system have pointed to the Toll-like receptors (TLRs) as a common pathway for immune recognition of microbial invasion and tis-sue injury. By recognizing either microbial products or endog-enous molecules released from damaged sites, the TLR system is capable of alerting the host of danger by activating the innate immune system. Initially, this is manifested by the production of inflammatory

1	molecules released from damaged sites, the TLR system is capable of alerting the host of danger by activating the innate immune system. Initially, this is manifested by the production of inflammatory mediators and the rapid uptake of invading microbes and their products. When excessive, this inflamma-tory response can contribute to organ damage and dysfunction.To date, 13 TLRs have been described in mice, and 11 in humans. TLRs are a family of proteins that are mammalian homologues to the Drosophila Toll, a protein that functions in development and immunity. The cytoplasmic portion of Toll-like receptors is similar to that of the IL-1 receptor (IL-1R) fam-ily and is called the Toll/IL-1 receptor (TIR) domain. Unlike the IL-1 receptor extracellular portion that consists of an immuno-globulin-like domain, the Toll-like receptors have leucine-rich repeats in their extracellular portion. The TLR receptors have many structural similarities both extracellularly and intracel-lularly, but

1	domain, the Toll-like receptors have leucine-rich repeats in their extracellular portion. The TLR receptors have many structural similarities both extracellularly and intracel-lularly, but they differ from each other in ligand specificities, expression patterns, and with some variability in the signaling pathways they activate.The TLR receptors were initially identified as compo-nents of the innate immune system that acted as a front-line defense mechanism against infections. Their recognition of patterns on pathogens, such as microbial peptides, lipopolysac-charide, lipoteichoic acids, bacterial DNA, and single-stranded RNA, resulted in the activation of an inflammatory response meant for controlling the invading organisms. In situations of noninfectious inflammation such as seen in trauma, clinicians have long recognized similar activation of the same inflamma-tory pathways and systemic manifestations. This observation, among others, led to the hypothesis that the immune system is

1	clinicians have long recognized similar activation of the same inflamma-tory pathways and systemic manifestations. This observation, among others, led to the hypothesis that the immune system is designed to recognize any threats, whether from pathogens or tissue damage, that may lead to disruption of homeostasis. In conditions of sterile inflammation, the activation of immune cells is through the release of endogenous danger molecules, normal cell constituents released by damage or dying cells or components of the extracellular matrix, released by the action of proteases at the site of tissue damage. Recent observations show that both microbial products and endogenous danger molecules can be recognized through the TLR system.Perhaps more than any of the other TLR family members, TLR4 sits at the interface of microbial and sterile inflammation. Whereas the role of TLR4 in the recognition of lipopolysac-charide (LPS) is well established, only recently has it become apparent that TLR4

1	at the interface of microbial and sterile inflammation. Whereas the role of TLR4 in the recognition of lipopolysac-charide (LPS) is well established, only recently has it become apparent that TLR4 also participates in the recognition of endogenous danger molecules. In vivo evidence for TLR4-mediated danger signaling comes from studies of acute tissue injury in hemorrhagic shock, trauma, and I/R models. In each case, TLR4-mutant animals exhibited reduced injury or inflam-mation compared to wild-type controls. In efforts to identify the ligands responsible for TLR4-dependent signaling in noninfec-tious insults, multiple molecules have been suggested. These include heat shock proteins, fibrinogen, hyaluronic acid, hepa-ran sulfate, and high mobility group box-1. Although a central role for TLR4 in recognizing tissue injury is building, studies are beginning to suggest that other TLR family members may also participate in the recognition of endogenous molecules released by tissue injury.

1	in recognizing tissue injury is building, studies are beginning to suggest that other TLR family members may also participate in the recognition of endogenous molecules released by tissue injury. The very recent realization that certain TLR family members also respond to endogenous molecules released from stressed or damaged tissues points to a molecular basis for a shared mechanism of innate immune activation by infection and injury.18-20RADIOLOGIC EVALUATION OF THE LIVERUltrasoundAbdominal ultrasound is a commonly applied imaging modal-ity used to evaluate abdominal symptoms. Ultrasound tech-nology is based on the pulse-echo principle. The ultrasound transducer converts electrical energy to high-frequency sound energy that is transmitted into tissue. Although some of the ultrasound waves are transmitted through the tissue, some are reflected back, and the ultrasound image is produced when the ultrasound receiver detects those reflected waves. This real-time gray-scale (B-mode)

1	waves are transmitted through the tissue, some are reflected back, and the ultrasound image is produced when the ultrasound receiver detects those reflected waves. This real-time gray-scale (B-mode) imaging is augmented by Doppler flow imaging. Doppler ultrasound not only can detect the pres-ence of blood vessels but also can determine the direction and velocity of blood flow. Ultrasonography is a useful initial imag-ing test of the liver because it is inexpensive, widely available, involves no radiation exposure, and is well tolerated by patients. It is excellent for diagnosing biliary pathology and focal liver lesions. In addition, liver injury can be evaluated in trauma patients using the focused abdominal sonography for trauma examination. Limitations of ultrasound include incomplete imaging of the liver, most often at the dome or beneath ribs on the surface, and incomplete visualization of lesion boundaries. Moreover, obesity and overlying bowel gas also can interfere with image

1	imaging of the liver, most often at the dome or beneath ribs on the surface, and incomplete visualization of lesion boundaries. Moreover, obesity and overlying bowel gas also can interfere with image quality. Thus, ultrasonographically detected masses usually require further evaluation by other imaging modalities due to the lower sensitivity and specificity of ultrasound com-pared with CT and MRI.The advent of contrast-enhanced ultrasound has improved the ability of this modality to differentiate among benign and malignant lesions. The injection of gas microbubble agents can Brunicardi_Ch31_p1345-p1392.indd 135720/02/19 2:36 PM 1358SPECIFIC CONSIDERATIONSPART IIincrease the sensitivity and specificity of ultrasound in detect-ing and diagnosing liver lesions. Microbubbles are <10 μm and, when given intravenously, allow for more effective echo enhancement. Contrast-enhanced ultrasound imaging of the liver improves delineation of liver lesions through identifica-tion of dynamic

1	μm and, when given intravenously, allow for more effective echo enhancement. Contrast-enhanced ultrasound imaging of the liver improves delineation of liver lesions through identifica-tion of dynamic enhancement patterns and the vascular mor-phology of the lesion. In addition, some agents exhibit a late liver-specific phase in which the bubbles are taken up by cells in the reticuloendothelial system and accumulate in normal liver parenchyma after the vascular enhancement has faded.The use of intraoperative ultrasound of the liver has rap-idly expanded over the years with the increasing number and complexity of hepatic resections being performed.21 It has the ability to provide the surgeon with real-time accurate informa-tion useful for surgical planning. Intraoperative ultrasound is considered the gold standard for detecting liver lesions, and studies have shown that it can identify 20% to 30% more lesions than other preoperative imaging modalities. Impor-tantly, it has been shown to

1	the gold standard for detecting liver lesions, and studies have shown that it can identify 20% to 30% more lesions than other preoperative imaging modalities. Impor-tantly, it has been shown to influence surgical management in almost 50% of planned liver resections for malignancies. Applications for intraoperative ultrasound of the liver include tumor staging, visualization of intrahepatic vascular structures (Fig. 31-10), and guidance of resection plane by assessment of the relationship of a mass to the vessels. In addition, biopsy of lesions and ablation of tumors can be guided by intraoperative ultrasound.Ultrasound elastography, also referred to as transient elastography, can be used to assess the degree of fibrosis or cirrhosis in the liver. Low-frequency vibrations transmitted through the liver induce an elastic shear wave that is detected by pulse-echo ultrasonography as the wave propagates through the liver. The velocity of the wave correlates with the stiffness of the

1	through the liver induce an elastic shear wave that is detected by pulse-echo ultrasonography as the wave propagates through the liver. The velocity of the wave correlates with the stiffness of the organ—the wave travels faster through fibrotic or cirrhotic tissues. Ultrasound elastography has been found in large cohorts of individuals to have a sensitivity of 87% and a specificity of 91% for the diagnosis of cirrhosis when compared with liver biopsy.22 Unlike liver biopsy, ultrasound elastography is non-invasive and can be repeated often without additional risk to the patient. Furthermore, this rapid test can acquire information from a larger area of the tissue relative to needle biopsy, provid-ing a better understanding of the entire hepatic parenchyma and reducing sampling error.Computed TomographyCT produces a digitally processed cross-sectional image of the body from a large series of X-ray images. The introduction of helical (spiral) CT has improved the imaging capabilities of

1	TomographyCT produces a digitally processed cross-sectional image of the body from a large series of X-ray images. The introduction of helical (spiral) CT has improved the imaging capabilities of this technique compared to earlier conventional axial CT by combining a continuous patient-table motion with continuous rotation of the CT gantry and allowing rapid acquisition of a volume of data within a single breath hold. With the recent advent of multidetector row CT scanners, high-resolution images can be obtained in submillimeter section thickness within a short scan time, with virtually no penalty in increased radiation dose. Together, these technologic advances have led to reduced motion artifacts due to variations in inspiration, facili-tated optimal contrast delivery, and allowed for the capability to generate high-resolution reformations in any desired plane. In a single examination, modern-day CT scans provide detailed morphologic information on the number, size, distribution,

1	the capability to generate high-resolution reformations in any desired plane. In a single examination, modern-day CT scans provide detailed morphologic information on the number, size, distribution, and vascularity of liver lesions, all of which are vital in guiding the clinical management and therapeutic plan.Contrast medium is routinely used in CT evaluation of the liver because of the similar densities of most pathologic liver masses and normal hepatic parenchyma. A CT scan with a dualor triple-phase bolus of intravenous contrast agent is performed to achieve the greatest enhancement of contrast between nor-mal and pathologic tissues.23 Ideally, contrast media should be selectively delivered to either the tumor or the liver, but not both. Radiologists use the dual blood supply of the liver and the hemodynamics of hepatic tumors to achieve this goal. The liver is unique in that it has a dual blood supply. As previously noted, the portal vein supplies approximately 75% of the blood

1	liver and the hemodynamics of hepatic tumors to achieve this goal. The liver is unique in that it has a dual blood supply. As previously noted, the portal vein supplies approximately 75% of the blood Figure 31-10. Intraoperative liver ultrasound images of the portal veins, hepatic veins, and inferior vena cava (IVC). Upper panel shows the portal vein bifurcation with echogenic Glissonian sheath. The confluence of the three hepatic veins (right hepatic vein [RHV], middle hepatic vein [MHV], and left hepatic vein [LHV]) and the IVC is shown in the middle panel. An accessory LHV is present in this patient. Lower panel is a color Doppler image showing flow.Brunicardi_Ch31_p1345-p1392.indd 135820/02/19 2:36 PM 1359LIVERCHAPTER 31flow and the hepatic artery the remaining 25%. However, many liver tumors receive the majority of their blood supply from the hepatic artery. After injection of the contrast agent, the rapid scan time of helical CT allows for CT sections through the liver in

1	liver tumors receive the majority of their blood supply from the hepatic artery. After injection of the contrast agent, the rapid scan time of helical CT allows for CT sections through the liver in both the arterial dominant phase (20 to 30 seconds after the beginning of contrast delivery) and venous or por-tal dominant phase (60 to 70 seconds after contrast injection) (Fig. 31-11). Thus, many hepatic tumors that derive the major-ity of their blood supply from the hepatic artery as well as other hypervascular lesions are well delineated in the arterial phase. On the other hand, the portal phase provides optimal enhance-ment of the normal liver parenchyma because the majority of its blood supply is derived from the portal vein. This allows for detection of hypovascular lesions because they will appear hypoattenuated in relation to the brighter normal liver paren-chyma. Furthermore, the arterial and portal phase images allow for noninvasive mapping of the hepatic arterial and venous

1	will appear hypoattenuated in relation to the brighter normal liver paren-chyma. Furthermore, the arterial and portal phase images allow for noninvasive mapping of the hepatic arterial and venous anat-omy, information that is crucial in the preoperative planning for patients undergoing liver surgery.CT cholangiography has emerged as a new imaging modal-ity for biliary disease. This technique usually involves the use of contrast agents, which are excreted by hepatocytes into the bile ducts. CT cholangiography, therefore, provides information on hepatocyte function and bile flow, in addition to high-resolution depiction of the biliary tree. It has compared well to endoscopic retrograde cholangiopancreatography (ERCP) in identifying obstructive biliary disease.24 One of the major advantages of CT cholangiography over other imaging modalities is the abil-ity to depict small nondilated peripheral biliary radicals. This technique may be useful in the context of live liver donation or

1	of CT cholangiography over other imaging modalities is the abil-ity to depict small nondilated peripheral biliary radicals. This technique may be useful in the context of live liver donation or complex biliary surgery to aid in the preoperative depiction of biliary anatomy. It also may be applicable in the postopera-tive setting for the detection of biliary leakage or obstruction. A limitation of CT cholangiography is that the biliary tree may not be well visualized in patients with excessively dilated bile ducts or in those with hyperbilirubinemia, as bilirubin excretion is impaired in these cases.Magnetic Resonance ImagingMRI is a technique that produces images based on magnetic fields and radio waves. The MRI scanner creates a powerful magnetic field that aligns the hydrogen atoms in the body, and radio waves are used to alter the alignment of this magnetiza-tion. Different tissues absorb and release radio wave energy at different rates, and this information is used to construct an

1	body, and radio waves are used to alter the alignment of this magnetiza-tion. Different tissues absorb and release radio wave energy at different rates, and this information is used to construct an image of the body. Most tissues can be differentiated by differ-ences in their characteristic T1 and T2 relaxation times. T1 is a measure of how quickly a tissue can become magnetized, and T2 measures how quickly it loses its magnetization. As with CT technology, advances in MRI now provide the opportunity to perform single-breath T1-weighted imaging and respiration-triggered T2-weighted imaging. The development of breath-hold imaging techniques has eliminated many of the motion artifacts that previously limited the sensitivity and application of MRI for imaging of the liver. Compared with CT scanning, the major advantages of MRI pertain to higher soft tissue contrast resolution and excellent depiction of fluid-containing structures, while obviating the need for ionizing radiation.As with

1	scanning, the major advantages of MRI pertain to higher soft tissue contrast resolution and excellent depiction of fluid-containing structures, while obviating the need for ionizing radiation.As with the iodinated contrast media use in CT scanning, multiple contrast agents have been developed for MRI to increase the difference in signal intensity between normal liver and pathologic lesions. Various gadolinium-based compounds have been used as MRI contrast agents that behave in a manner very similar to iodine in CT. Liver-specific MRI contrast agents also have been developed that rely either on excretion by Kupffer cells, such as ferumoxide (Feridex, Advanced Magnet-ics, Cambridge, MA), or on secretion in bile by hepatocytes, including gadoxetate (Eovist or Primovist, Bayer-Schering, Berlin, Germany). These agents combine the information obtained during a standard MRI with additional functional data, which in turn yields improved detection and characteriza-tion of lesions within the

1	Germany). These agents combine the information obtained during a standard MRI with additional functional data, which in turn yields improved detection and characteriza-tion of lesions within the liver.25Just as ultrasound elastography is useful in the diagno-sis of hepatic fibrosis and cirrhosis, magnetic resonance (MR) elastography appears promising as an imaging modality in 3Figure 31-11. Computed tomographic (CT) images of hepatic veins and Couinaud’s liver segments. The images show the three hepatic veins and inferior vena cava (IVC) (upper panel), as well as Couinaud’s liver segments (lower panels). LHV = left hepatic vein; MHV = middle hepatic vein; RHV = right hepatic vein.Brunicardi_Ch31_p1345-p1392.indd 135920/02/19 2:36 PM 1360SPECIFIC CONSIDERATIONSPART IIreducing the need for liver biopsy. In this technique, a vibration device is used to induce a shear wave in the liver. A modified MRI machine detects the shear wave, then generates a color-coded image that depicts the

1	for liver biopsy. In this technique, a vibration device is used to induce a shear wave in the liver. A modified MRI machine detects the shear wave, then generates a color-coded image that depicts the wave velocity, and hence stiffness, throughout the organ. Although preliminary studies have shown that MR elastography can detect cirrhosis with a high degree of accuracy, the clinical utility of this modality, especially given its relatively high cost, remains to be determined.Magnetic resonance cholangiopancreatography (MRCP) enables rapid, noninvasive depiction of both the biliary tree and the pancreatic duct without the use of ionizing radiation or intravenous contrast media. One of the most common clinical indications for MRCP is biliary obstruction. MRCP provides visualization of dilated bile ducts, and the high spatial and con-trast resolution often enables accurate assessment of the level of occlusion in the biliary tree. MRCP also can be enhanced with liver-specific MRI contrast

1	bile ducts, and the high spatial and con-trast resolution often enables accurate assessment of the level of occlusion in the biliary tree. MRCP also can be enhanced with liver-specific MRI contrast agents that are actively secreted into the bile, but the clinical indications for such studies are still a matter of intensive investigation.26Positron Emission TomographyPositron emission tomography (PET) is a nuclear medicine test that produces images of metabolic activity in tissues by detect-ing gamma rays emitted by a radioisotope incorporated into a metabolically active molecule. Fluorodeoxyglucose (FDG) is the most common metabolic molecule used in PET imaging. Although traditional imaging such as CT, ultrasound, and MRI provide anatomic information, PET offers functional imaging of tissues with high metabolic activity, including most types of metastatic tumors. PET imaging increasingly is used as a tool in the diagnostic evaluation of a patient with potentially resectable hepatic

1	with high metabolic activity, including most types of metastatic tumors. PET imaging increasingly is used as a tool in the diagnostic evaluation of a patient with potentially resectable hepatic disease. In nonrandomized trials, PET demonstrated bet-ter sensitivity and specificity than CT scanning for the detec-tion of both intrahepatic and extrahepatic disease.27 Integrated PET/CT improves diagnostic accuracy over standard PET or CT alone and has been shown to be sensitive in the detection of liver metastases derived from a wide range of cancers, including colorectal (Fig. 31-12), breast, or lung primaries.27More than 20% of patients with colorectal cancer ini-tially present with hepatic metastasis, and a large percentage of patients undergoing resection for their primary colorectal cancer eventually experience disease recurrence in the liver. The role of FDG-PET/CT in colorectal cancers lies predomi-nantly in tumor staging and follow-up, particularly in the detec-tion of occult

1	eventually experience disease recurrence in the liver. The role of FDG-PET/CT in colorectal cancers lies predomi-nantly in tumor staging and follow-up, particularly in the detec-tion of occult intrahepatic metastases or extrahepatic disease. Although hepatic resection of colorectal metastases provides survival rates nearing 50%, the presence of extrahepatic dis-ease is a poor prognosticator and usually precludes aggressive surgical intervention. Thus, accurate information regarding the extent of the disease is necessary for management of patients with colorectal metastases. PET/CT has also been shown to be more accurate than contrast-enhanced CT in tumor surveillance after radiofrequency ablation.28 The sensitivity of FDG-PET, however, is lowered by neoadjuvant chemotherapy, most likely secondary to reduced metabolic activity within the tumor.Although the role of PET/CT in the clinical management of liver metastases has been well-established, its utility in the diagnostic workup of

1	secondary to reduced metabolic activity within the tumor.Although the role of PET/CT in the clinical management of liver metastases has been well-established, its utility in the diagnostic workup of primary liver tumors is still debated. In hepatocellular carcinoma (HCC), FDG uptake correlates with the degree of differentiation—high-grade HCC lesions have increased FDG uptake compared to low-grade HCCs. As a result, the overall sensitivity of FDG-PET/CT in the detection of HCCs is reported to be only 50% to 65%, rendering this modality insufficient when used alone in the diagnosis of primary HCCs. For this reason, dual-tracer PET has been introduced to improve sensitivity in detecting all HCC. This modality combines the use of FDG, which accumulates in poorly differentiated tumors, with 11C-acetate, a tracer preferentially accumulated by well-differentiated HCC lesions. Although the clinical benefits of dual-tracer PET/CT have yet to be fully established, this com-bined modality has

1	11C-acetate, a tracer preferentially accumulated by well-differentiated HCC lesions. Although the clinical benefits of dual-tracer PET/CT have yet to be fully established, this com-bined modality has the potential to become a valuable tool in the diagnosis and staging of HCC. In cholangiocarcinoma tumors, FDG avidity depends on the morphologic characteristics and location of the lesion. Therefore, FDG-PET and FDG-PET/CT have not been shown to be highly beneficial in the diagnosis of primary cholangiocarcinoma, but they may be beneficial in the detection of regional and distal metastases, which can affect clinical decision making and patient management.ACUTE LIVER FAILUREAcute liver failure (ALF) occurs when the rate and extent of hepatocyte death exceeds the liver’s regenerative capabilities. It was initially described as a specific disease entity in the 1950s. Figure 31-12. Computed tomography (CT)–positron emission tomography (PET) scans before and after resection of liver

1	capabilities. It was initially described as a specific disease entity in the 1950s. Figure 31-12. Computed tomography (CT)–positron emission tomography (PET) scans before and after resection of liver metastasis from colorectal cancer in a 54-year-old patient. CT scan shows large 10-cm right lobe liver metastasis (left panel), and PET scan findings are strongly positive (middle panel). Two years after right hepatectomy, the patient has no evidence of recurrence and significant hypertrophy of the left lobe (right panel).Brunicardi_Ch31_p1345-p1392.indd 136020/02/19 2:36 PM 1361LIVERCHAPTER 31It also has been referred to as fulminant hepatic failure. ALF is a rare disorder affecting approximately 2000 patients annually in the United States. ALF is defined by the development of hepatic encephalopathy occurring within 26 weeks of severe liver injury in a patient without a history of previous liver dis-ease or portal hypertension.29 The manifestations of ALF may include cerebral edema,

1	occurring within 26 weeks of severe liver injury in a patient without a history of previous liver dis-ease or portal hypertension.29 The manifestations of ALF may include cerebral edema, hemodynamic instability, increased sus-ceptibility to bacterial and fungal infections, renal failure, coag-ulopathy, and metabolic disturbances. Even with current medical care, ALF can progress rapidly to hepatic coma and death. The most common cause of death is intracranial hypertension due to cerebral edema, followed by sepsis and multisystem organ failure. The causes of ALF, which are the most important variables in determining outcome, are numerous and can include viral infection as well as drug overdose, reaction, and toxicity. It has been determined that the etiologic factor leading to ALF varies according to geographic location.30 Before the introduc-tion of orthotopic liver transplantation (OLT), the chance for survival was <20%. Currently, most series report 5-year survival rates of >70% for

1	to geographic location.30 Before the introduc-tion of orthotopic liver transplantation (OLT), the chance for survival was <20%. Currently, most series report 5-year survival rates of >70% for affected patients.31EtiologyDifferences in etiology, management, and patient outcomes have been described for various regions of the globe. In the East and developing portions of the world, the most common causes of ALF are viral infections, primarily hepatitis B, A, and E.20 In these areas, there are a relatively small number of drug-induced cases. In contrast, 65% of cases of ALF in the West are thought to be due to drugs and toxins, with acetaminophen (paracetamol) being the most common etiologic agent in the United States, Australia, United Kingdom, and most of Europe. In France and Spain, where acetaminophen sales are restricted, the rate of acetaminophen-induced ALF is quite low.32 Acetaminophen-induced ALF is also uncommon in South America. The U.S. Acute Liver Failure Study Group

1	where acetaminophen sales are restricted, the rate of acetaminophen-induced ALF is quite low.32 Acetaminophen-induced ALF is also uncommon in South America. The U.S. Acute Liver Failure Study Group identified several other causes of ALF, including autoimmune hepatitis, hypoperfusion of the liver (in cardiomyopathy or cardiogenic shock), pregnancy-related conditions, and Wilson’s disease.33 Even with exhaustive efforts to identify a cause, approximately 20% of all cases of ALF remain indeterminate in origin.Clinical PresentationIn a multicenter study involving 17 tertiary care centers and 308 patients in the United States, 73% of all patients with ALF were female, with a median age of 38 years.34 The most common ethnic group affected was whites (74%), followed by Hispanics (9%) and African Americans (3%). Patients were ill for a median of 6 days before the onset of encephalopathy and had a median of 2 days between the onset of jaundice and the development of encephalopathy. Hepatic

1	Americans (3%). Patients were ill for a median of 6 days before the onset of encephalopathy and had a median of 2 days between the onset of jaundice and the development of encephalopathy. Hepatic coma grade at presentation was approximately equally distributed across grades I to IV. Eighty-four percent of the patients in the study were referred from outside hospitals, 40% had a serum creatinine level exceeding 2.0 mg/dL, and 14% had an arterial pH of <7.30. In addition, 44% of the patients acquired a culture-proven infection.Diagnosis and Clinical ManagementWhen the medical history is obtained, it is important to address the possibility of exposure to viral infections, medications, and other possible toxins. The possibility of previous liver disease needs to be explored. The physical examination must assess and document the patient’s mental status as well as attempt to identify findings of chronic liver disease. The initial laboratory examination must evaluate the severity of the ALF

1	must assess and document the patient’s mental status as well as attempt to identify findings of chronic liver disease. The initial laboratory examination must evaluate the severity of the ALF as well as attempt to identify the cause (Table 31-1). A liver biopsy should be performed if certain disease entities such as autoimmune hepatitis or lymphoma are a possibility. Because of the asso-ciated coagulopathy, if a liver biopsy is needed, it is usually safest to obtain the tissue via a transjugular approach. Patients with ALF should be admitted to the hospital and monitored fre-quently. Due to the rapidity with which this disease process may progress, a liver transplant center should be contacted, and the affected patient should be transferred to the center early in the evaluation period.If acetaminophen overdose is suspected to have occurred within a few hours of presentation, administration of activated charcoal may be useful to reduce the volume of acetamino-phen present in the

1	acetaminophen overdose is suspected to have occurred within a few hours of presentation, administration of activated charcoal may be useful to reduce the volume of acetamino-phen present in the gastrointestinal (GI) tract. N-acetylcysteine (NAC), the clinically effective antidote for acetaminophen over-dose, should be administered as early as possible to any patient with suspected acetaminophen-associated ALF.35 NAC also should be administered to patients with ALF of unclear etiol-ogy because replenishing glutathione may be beneficial in this patient population as well.36 NAC can be administered either orally (140 mg/kg initial dose, followed by 70 mg/kg every 4 hours × 17 doses) or via the intravenous route (loading dose of 150 mg/kg, followed by a maintenance dose of 50 mg/kg every 4 hours × 12 doses). For patients who are suspected of having drug-induced hepatotoxicity, it is important to obtain details regarding all prescription and nonprescription drugs, herbs, and dietary

1	4 hours × 12 doses). For patients who are suspected of having drug-induced hepatotoxicity, it is important to obtain details regarding all prescription and nonprescription drugs, herbs, and dietary supplements that may have been taken in the previous year. Most instances of drug-induced hepatotoxicity occur in the first 6 months after drug initiation. Any suspected offending agent must be discontinued, and an attempt should be made to administer only essential medications.The majority of patients with ALF need to be monitored in the intensive care unit (ICU) setting, and specific attention needs to be given to fluid management, ulcer prophylaxis, hemody-namic monitoring, electrolyte management, and surveillance for and treatment of infection. Surveillance cultures should be performed to identify bacterial and fungal infections as early as possible. Serum phosphorus levels need to be monitored. 4Table 31-1Acute liver failure laboratory evaluationComplete blood countComplete metabolic

1	identify bacterial and fungal infections as early as possible. Serum phosphorus levels need to be monitored. 4Table 31-1Acute liver failure laboratory evaluationComplete blood countComplete metabolic panelAmylase and lipase levelsLiver function testsProthrombin time/international normalized ratioFactor V levelFactor VII levelArterial blood gas concentrationsArterial serum ammonia levelABO typingAcute hepatitis panelAutoimmune marker levelsCeruloplasmin levelToxicology screeningAcetaminophen levelHIV screeningPregnancy test (females)HIV = human immunodeficiency virus.Brunicardi_Ch31_p1345-p1392.indd 136120/02/19 2:36 PM 1362SPECIFIC CONSIDERATIONSPART IIHypophosphatemia, a sign of hepatic regeneration, may indi-cate a higher likelihood of spontaneous recovery and needs to be corrected via intravenous (IV) administration of phosphate. Sedation should be avoided, and the head of the bed should be elevated at least 30°. Neurologic examinations should be per-formed frequently.

1	via intravenous (IV) administration of phosphate. Sedation should be avoided, and the head of the bed should be elevated at least 30°. Neurologic examinations should be per-formed frequently. Intracranial pressure monitoring is reserved for patients in whom a neurologic examination is no longer reli-able. CT scans of the head should be performed to rule out mass lesion or hemorrhage, but they provide only limited informa-tion regarding increased intracranial pressure. The administra-tion of blood products for thrombocytopenia and prolonged PT is recommended only in the setting of hemorrhage or before invasive procedures. Acute renal failure is a frequent complica-tion in patients with ALF, and efforts should be made to protect renal function by maintaining sufficient perfusion and avoid-ing nephrotoxic medications. Should renal replacement therapy become necessary, continuous venovenous hemodialysis should be used rather than intermittent hemodialysis because continu-ous venovenous

1	nephrotoxic medications. Should renal replacement therapy become necessary, continuous venovenous hemodialysis should be used rather than intermittent hemodialysis because continu-ous venovenous hemodialysis provides better hemodynamic and intracranial pressure stability. The most severely affected patients have a poor prognosis with medical management alone and require liver transplantation. Identifying these patients early in the clinical course is important both to maximize the time available to obtain a donor liver allograft for those in need and to avoid transplant in those who will recover without it.PrognosisAccurate identification of ALF patients who will recover spon-taneously is important because of the severe shortage of donor liver allografts and the potential complications of lifelong non-specific immunosuppression. The most widely applied prognos-tic scoring system is the King’s College Hospital ALF criteria.37 This scoring system has separate criteria predicting a poor

1	lifelong non-specific immunosuppression. The most widely applied prognos-tic scoring system is the King’s College Hospital ALF criteria.37 This scoring system has separate criteria predicting a poor medical management outcome for acetaminophen-related and non–acetaminophen-related forms of ALF (Table 31-2). Many other prognostic models exist such as the Acute Physiology and Chronic Health Evaluation II (APACHE II) score, the Clichy criteria,38 and actin-free Gc-globulin serum concentration.39 Overall, prognostic scoring systems have proven to have acceptable specificity but low sensitivity in determining patient outcome and therefore should not replace the judgment of an experienced clinician.40Liver TransplantationDespite advances in medical management, OLT remains the only definitive therapy for patients unable to regenerate sufficient hepatocyte mass in a timely manner. The advent of OLT has coincided with a rise in overall ALF survival rates from approxi-mately 20% in the

1	therapy for patients unable to regenerate sufficient hepatocyte mass in a timely manner. The advent of OLT has coincided with a rise in overall ALF survival rates from approxi-mately 20% in the pretransplantation era to >70% at the pres-ent time. One-year posttransplantation survival for patients with ALF has been reported to be as high as 80% to 90%.31 Although these improvements in survival rates are impressive, it should be noted that 10% of patients still die while awaiting OLT, which confirms that the potential for improved patient outcome still has not been realized because of the ongoing liver allograft shortage.Emerging TechnologiesAs mentioned earlier, patient survival could be improved if additional time could be gained for the patient while awaiting liver replacement or hepatocyte regeneration. The development of a support device to replace the acutely failing liver has been a highly sought after (and elusive) goal. Several systems have been tested without definitive

1	regeneration. The development of a support device to replace the acutely failing liver has been a highly sought after (and elusive) goal. Several systems have been tested without definitive evidence of efficacy. Transient improvement in hepatic encephalopathy has been observed in several trials, but improvement in hepatocyte function and long-term benefit have not been realized.41 Liver support trials are dif-ficult to perform due to access to liver replacement, the rarity of affected patients, and the heterogeneous causes and varying levels of disease severity. Therefore, additional data are necessary, and liver support systems should be used only as part of an approved clinical trial. Focus has now shifted toward xenotransplantation,42 organ engineering, and cell transplantation.43 In the meantime, living donation and auxiliary liver transplantation can help over-come the organ shortage.44CIRRHOSIS AND PORTAL HYPERTENSIONCirrhosis, the final sequela of chronic hepatic insult, is

1	the meantime, living donation and auxiliary liver transplantation can help over-come the organ shortage.44CIRRHOSIS AND PORTAL HYPERTENSIONCirrhosis, the final sequela of chronic hepatic insult, is char-acterized by the presence of fibrous septa throughout the liver subdividing the parenchyma into hepatocellular nodules (Fig. 31-13).45 Cirrhosis is the consequence of sustained wound healing in response to chronic liver injury. Approximately 40% of cirrhotic patients are asymptomatic, but progressive deteriora-tion leading to the need for liver transplantation or death is typi-cal after the development of end-stage liver disease (ESLD). The complications of ESLD include progressive hyperbilirubinemia, malnutrition, decreased synthetic function of the liver, coagu-lopathy, portal hypertension (i.e., ascites and variceal bleeding), hepatic encephalopathy, and life-limiting fatigue. ESLD car-ries a 5-year mortality of 50%, with 70% of deaths due to liver failure.46 In the United States,

1	(i.e., ascites and variceal bleeding), hepatic encephalopathy, and life-limiting fatigue. ESLD car-ries a 5-year mortality of 50%, with 70% of deaths due to liver failure.46 In the United States, cirrhosis accounts for 30,000 deaths per year and is the most common nonneoplastic cause of death among patients with hepatobiliary and digestive diseases. An additional 10,000 to 12,000 deaths occur annually due to HCC, the most rapidly increasing neoplasm in the United States.46Morphologic Classification of CirrhosisMorphologically, cirrhosis can be described as micronodular, macronodular, or mixed. Micronodular cirrhosis is characterized Table 31-2King’s College selection criteria for liver transplantation in acute liver failureCAUSESELECTION CRITERIAAcetaminophenArterial pH < 7.30 irrespective of hepatic coma gradeOrProthrombin time >100 s + serum creatinine level >3.4 mg/dL + grade III or IV hepatic comaNot acetaminophenProthrombin time >100 s irrespective of hepatic coma gradeOrAny

1	of hepatic coma gradeOrProthrombin time >100 s + serum creatinine level >3.4 mg/dL + grade III or IV hepatic comaNot acetaminophenProthrombin time >100 s irrespective of hepatic coma gradeOrAny three of the following, irrespective of hepatic coma grade: Cryptogenic or drug-induced hepatitis Jaundice to coma interval >7 d Prothrombin time >50 s Serum bilirubin level >17.5 mg/dL Age < 10 y or > 40 yBrunicardi_Ch31_p1345-p1392.indd 136220/02/19 2:36 PM 1363LIVERCHAPTER 31by thick regular septa, small uniform regenerative nodules, and involvement of virtually every hepatic lobule. Macronodular cirrhosis frequently has septa and regenerative nodules of vary-ing sizes. The regenerative nodules consist of irregularly sized hepatocytes with large nuclei and cell plates of varying thick-ness. Mixed cirrhosis is present when regeneration is occurring in a micronodular liver and over time converts to a macronodu-lar pattern. This morphologic categorization is limited, and cir-rhosis is a

1	Mixed cirrhosis is present when regeneration is occurring in a micronodular liver and over time converts to a macronodu-lar pattern. This morphologic categorization is limited, and cir-rhosis is a dynamic process in which nodule size varies over time. The three patterns correlate poorly with etiology, and the same pattern can result from a variety of disease processes. Conversely, a single disease process can demonstrate several morphologic patterns. Irrespective of etiology and morphologic pattern, the cirrhotic liver frequently demonstrates right hepatic lobe atrophy, caudate lobe and left lateral segment hypertrophy, recanalization of the umbilical vein, a nodular surface contour, dilatation of the portal vein, gastroesophageal varices, and sple-nomegaly on radiographic evaluation.Etiology of CirrhosisCirrhosis can result from a wide range of disease processes, including viral, autoimmune, drug-induced, alcohol-induced, nonalcoholic fatty liver disease, and metabolic diseases

1	of CirrhosisCirrhosis can result from a wide range of disease processes, including viral, autoimmune, drug-induced, alcohol-induced, nonalcoholic fatty liver disease, and metabolic diseases (Table 31-3). In the diagnosis of alcoholic liver disease, docu-mentation of chronic alcohol abuse is imperative. Liver biopsy will reveal the typical findings of alcoholic hepatitis, including hepatocyte necrosis, Mallory bodies, neutrophil infiltration, and perivenular inflammation.Nonalcoholic fatty liver disease (NAFLD) covers a wide spectrum of disorders including simple fatty liver, nonalcoholic steatohepatitis (NASH), fibrosis/cirrhosis and NASH-associated hepatocellular carcinoma.47 NAFLD is now the most common chronic liver disease worldwide48 and NASH is a progressive form of NAFLD characterized by steatosis with hepatocellular injury and chronic inflammation.49 NASH affects 3% to 5% of the population, and approximately 1 in 10 NASH patients will progress to cirrhosis, thereby placing

1	by steatosis with hepatocellular injury and chronic inflammation.49 NASH affects 3% to 5% of the population, and approximately 1 in 10 NASH patients will progress to cirrhosis, thereby placing them at risk for the well-described consequences of cirrhosis, including hepatocellular carcinoma.50 Although hepatocellular carcinoma arises less fre-quently in patients with NASH compared to other liver diseases (e.g., hepatitis C viral infection), the overall higher prevalence and more rapidly increasing incidence of NASH relative to other chronic liver diseases mean that the majority of HCC will arise in the setting of NAFLD in the near future.51,52Patients with nonalcoholic steatohepatitis (NASH) often endorse a history of diabetes mellitus or metabolic syndrome. The diagnosis of NASH requires the demonstration of steato-hepatitis on biopsy, the lack of a history of significant alcohol consumption, and exclusion of other causes of hepatic steatosis. Although cryptogenic cirrhosis, or

1	the demonstration of steato-hepatitis on biopsy, the lack of a history of significant alcohol consumption, and exclusion of other causes of hepatic steatosis. Although cryptogenic cirrhosis, or cirrhosis without an apparent cause, accounted for a third of all cases in the past, this propor-tion has declined over time as it becomes increasingly appar-ent that many of such patients may actually have unrecognized NASH.Due to the high prevalence of fatty liver disease, many patients considered for hepatic surgery will have background hepatic steatosis or steatohepatitis. In addition, chemotherapy treatment (e.g., irinotecan) for colorectal cancer liver metasta-ses induces steatosis and steatohepatitis in the nontumor-bearing liver.53 This has important implications as fatty liver disease can increase morbidity after liver resection.54,55 Thus, under-standing the deleterious effects of steatosis and steatohepatitis Figure 31-13. Histology of cirrhotic liver with regenerating mac-ronodules.

1	morbidity after liver resection.54,55 Thus, under-standing the deleterious effects of steatosis and steatohepatitis Figure 31-13. Histology of cirrhotic liver with regenerating mac-ronodules. Upper panel: Grossly cirrhotic liver. Lower panel: Regenerative nodules and bridging fibrosis representative of cir-rhosis seen on standard light microscopy (hematoxylin and eosin stain).Table 31-3Etiology of cirrhosisViral hepatitis (hepatitis B, C, and D)CryptogenicAlcohol abuseMetabolic abnormalities Iron overload (hemochromatosis) Copper overload (Wilson’s disease) α1-Antitrypsin deficiency Glycogen storage disease (types IA, III, and IV) Tyrosinemia GalactosemiaNonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH)Hepatic vein outflow abnormalities Budd-Chiari syndrome Cardiac failureAutoimmune hepatitisToxins and drugsBrunicardi_Ch31_p1345-p1392.indd 136320/02/19 2:36 PM 1364SPECIFIC CONSIDERATIONSPART IIis crucial for the multidisciplinary care of patients

1	failureAutoimmune hepatitisToxins and drugsBrunicardi_Ch31_p1345-p1392.indd 136320/02/19 2:36 PM 1364SPECIFIC CONSIDERATIONSPART IIis crucial for the multidisciplinary care of patients undergoing liver surgery.Chronic hepatitis C infection is the most common cause of chronic liver disease and the most frequent indication for liver transplantation in the United States. The identification of chronic hepatitis C infection is facilitated by serologic assays that detect antibody to hepatitis C and molecular assays that quantify hepatitis C viral RNA. Chronic hepatitis B, on the other hand, can be diagnosed based on the detection of hepatitis B surface antigen (HBsAg) more than 4 to 6 months after initial infection. Additional tests of hepatitis B viral replication, such as the hepatitis B e antigen (HBeAg) and hepatitis B viral DNA, can be used to confirm ongoing infection and to guide appropri-ate antiviral therapy.Autoimmune causes of cirrhosis include primary bili-ary cirrhosis,

1	B e antigen (HBeAg) and hepatitis B viral DNA, can be used to confirm ongoing infection and to guide appropri-ate antiviral therapy.Autoimmune causes of cirrhosis include primary bili-ary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis. Patients with primary biliary cirrhosis may be asymp-tomatic or may present with a history of fatigue, pruritus, and skin hyperpigmentation that is not related to jaundice. Anti-mitochondrial antibodies will test positive in the vast majority of cases. Affected patients also may have marked elevations in serum cholesterol, while hyperbilirubinemia is seen late in the course of the disease. Primary sclerosing cholangitis is a chronic cholestatic disease of the liver associated with ulcer-ative colitis and Crohn’s disease. The clinical presentation can include pruritus, steatorrhea, fat-soluble vitamin deficiencies, and metabolic bone disease. The diagnosis is often established by imaging of the biliary tree, which reveals a

1	presentation can include pruritus, steatorrhea, fat-soluble vitamin deficiencies, and metabolic bone disease. The diagnosis is often established by imaging of the biliary tree, which reveals a characteristic picture of diffuse, multifocal strictures with focal dilation of the bile ducts resulting in a beaded appearance. Complications are common and can include biliary strictures, cholangitis, choleli-thiasis, and cholangiocarcinoma. Autoimmune hepatitis is often accompanied by an elevation in serum globulins, particularly gamma globulins. Liver biopsy will show nonspecific changes such as a portal mononuclear cell infiltrate with the character-istic presence of plasma cells. Many patients with autoimmune hepatitis will respond to treatment with prednisone with or with-out azathioprine.Hereditary hemochromatosis is the most common meta-bolic disorder causing cirrhosis. This entity should be suspected if the patient’s clinical presentation includes skin hyperpigmen-tation, diabetes

1	hemochromatosis is the most common meta-bolic disorder causing cirrhosis. This entity should be suspected if the patient’s clinical presentation includes skin hyperpigmen-tation, diabetes mellitus, pseudogout, cardiomyopathy, or a fam-ily history of cirrhosis. Elevated plasma ferritin and increased iron saturation levels suggest the presence of iron overload, but these findings also can be seen in other diseases of the liver. Confirmatory testing can be achieved by means of genetic test-ing, liver biopsy, or by assessing the response to phlebotomy. Other uncommon metabolic disorders leading to cirrhosis include Wilson’s disease and α1-antitrypsin deficiency.Clinical Manifestations of CirrhosisThe clinical history associated with cirrhosis can include fatigue, anorexia, weight loss, jaundice, abdominal pain, peripheral edema, ascites, GI bleeding, and hepatic encephalopathy. On physical examination, a number of findings have been described in patients with cirrhosis. Spider angiomata

1	abdominal pain, peripheral edema, ascites, GI bleeding, and hepatic encephalopathy. On physical examination, a number of findings have been described in patients with cirrhosis. Spider angiomata and palmar ery-thema are believed to be caused by alterations in sex hormone metabolism. Finger clubbing may be a consequence of hypo-albuminemia, while the pathogenesis of white nail beds and Dupuytren’s contractures are less well understood. Males may develop features of feminization such as gynecomastia, loss of chest and axillary hair, and testicular atrophy. Splenomegaly is common, whereas the cirrhotic liver itself may be enlarged, nor-mal sized, or small. Ascites and pleural effusion can be seen with fluid accumulation. Portal hypertension can manifest as caput medusae and/or the presence of the Cruveilhier-Baumgarten murmur, a venous hum that can be auscultated in the epigas-trium resulting from collaterals between the portal system and the remnant of the umbilical vein. Jaundice

1	of the Cruveilhier-Baumgarten murmur, a venous hum that can be auscultated in the epigas-trium resulting from collaterals between the portal system and the remnant of the umbilical vein. Jaundice usually does not appear until the bilirubin rises above 2 to 3 mg/dL. Asterixis can be detected in patients with hepatic encephalopathy. Other manifestations include fetor hepaticus, as well as features sug-gestive of malnutrition such as weakness, weight loss, and tem-poral muscle wasting.Although fat stores and muscle mass are reduced, rest-ing energy expenditure is increased. Muscle cramps occur frequently in the cirrhotic patient and are felt to correlate with ascites, low mean arterial pressure, and plasma renin activity. Abdominal hernias are common with ascites and should be electively repaired only in patients with well-compensated cir-rhosis; otherwise, the hernia should be repaired at the time of or after hepatic transplantation. HCC can occur in all forms of cirrhosis, and every

1	only in patients with well-compensated cir-rhosis; otherwise, the hernia should be repaired at the time of or after hepatic transplantation. HCC can occur in all forms of cirrhosis, and every cirrhotic patient should undergo screening for the development of HCC every 6 months via imaging and measurement of a serum α-fetoprotein (AFP) level. Cirrhosis is associated with increased cardiac output and heart rate as well as decreased systemic vascular resistance and blood pres-sure. Patients with cirrhosis are more prone to infections due to impaired phagocytic activity of the reticuloendothelial system. Bacterial infections, often of intestinal origin, are common and must be suspected in a patient with unexplained pyrexia or clini-cal deterioration. Spontaneous bacterial peritonitis also is seen in cases of cirrhosis with ascites. Intrinsic drug metabolism is reduced in the cirrhotic liver, and this fact needs to be recog-nized when prescribing medications.Laboratory Findings Associated

1	seen in cases of cirrhosis with ascites. Intrinsic drug metabolism is reduced in the cirrhotic liver, and this fact needs to be recog-nized when prescribing medications.Laboratory Findings Associated With CirrhosisLaboratory findings vary in the cirrhotic patient depending on the degree of compensation; however, in general, a number of trends are seen. The cirrhotic patient usually has a mild normo-cytic normochromic anemia. The white blood cell and platelet counts are reduced, and the bone marrow is macronormoblastic. The PT is prolonged and does not respond to vitamin K therapy, and the serum albumin level is depressed. Urobilinogen is pres-ent and urinary sodium excretion is diminished in the presence of ascites. The serum levels of bilirubin, transaminases, and alkaline phosphatase may all be elevated. However, normal liver function test results do not eliminate the possibility of cirrhosis.Liver BiopsyThe diagnosis of cirrhosis can be made in many cases from a constellation of

1	may all be elevated. However, normal liver function test results do not eliminate the possibility of cirrhosis.Liver BiopsyThe diagnosis of cirrhosis can be made in many cases from a constellation of clinical features, laboratory values, and radio-graphic findings. Histopathologic examination of liver tissue is occasionally needed to confirm the diagnosis of cirrhosis and in determining disease etiology, activity, and progression. Liver biopsy can be performed via a percutaneous, transjugular, or laparoscopic approach. If needed, ultrasound or CT guidance can be helpful in obtaining an adequate sample and avoiding other viscera.Various serologic markers of hepatic fibrosis are currently being investigated to help predict the presence of cirrhosis with-out the need for liver biopsy. However, no currently available marker is sufficiently accurate for clinical use. Ultrasound elas-tography, which measures the stiffness of the liver by inducing Brunicardi_Ch31_p1345-p1392.indd

1	However, no currently available marker is sufficiently accurate for clinical use. Ultrasound elas-tography, which measures the stiffness of the liver by inducing Brunicardi_Ch31_p1345-p1392.indd 136420/02/19 2:36 PM 1365LIVERCHAPTER 31an elastic shear wave that propagates through the tissue, shows promise as a noninvasive test in identifying patients with advanced fibrosis and cirrhosis. (See earlier section, “Radio-logic Evaluation of the Liver.”)Hepatic Reserve and Assessment of Surgical Risk in the Cirrhotic PatientAssessing the hepatic reserve of the cirrhotic patient is impor-tant because cirrhosis and portal hypertension can have a negative impact on the outcome of nontransplant surgical pro-cedures. Patients with liver disease undergoing surgery are at increased risk for surgical and anesthesia-related complications. The actual risk depends on the type of anesthetic used, the spe-cific surgical procedure performed, and the severity of liver disease. Previous studies have

1	and anesthesia-related complications. The actual risk depends on the type of anesthetic used, the spe-cific surgical procedure performed, and the severity of liver disease. Previous studies have demonstrated that emergency operations, cardiac surgery, hepatic resections, and abdominal surgery, particularly cholecystectomy, gastric resection, and colectomy, generate the highest operative risk among cirrhotic patients. Additionally, preoperative patient characteristics such as anemia, ascites, encephalopathy, malnutrition, hypoalbumin-emia, hypoxemia, infection, jaundice, portal hypertension, and prolonged PT have also been associated with inferior outcomes after surgery. Nontransplant surgical procedures are contraindi-cated in patients with acute fulminant hepatitis and those with severe decompensated chronic hepatitis.A number of laboratory tests have been used to assess hepatic reserve in patients with cirrhosis. Tests of indocyanine green, sorbitol, and galactose elimination

1	decompensated chronic hepatitis.A number of laboratory tests have been used to assess hepatic reserve in patients with cirrhosis. Tests of indocyanine green, sorbitol, and galactose elimination capacity as well as the carbon-13 galactose breath test and carbon-13 aminopyrine breath test have all been disappointing clinically due to their dependence on flow to the liver as well as the unavailability and complexity of the tests. The monoethylglycinexylidide (MEGX) test, which measures MEGX formation after the administration of lidocaine, has been shown to be approximately 80% sensitive and specific in diagnosing cirrhosis. However, this test loses both sensitivity and specificity as the serum bilirubin level rises and interferes with the fluorescent readout system.Child-Turcotte-Pugh ScoreThe Child-Turcotte-Pugh (CTP) score was originally developed to evaluate the risk of portocaval shunt procedures performed for portal hypertension and subsequently has been shown to be useful in

1	ScoreThe Child-Turcotte-Pugh (CTP) score was originally developed to evaluate the risk of portocaval shunt procedures performed for portal hypertension and subsequently has been shown to be useful in predicting surgical risks of other intra-abdominal operations on cirrhotic patients (Table 31-4). Numerous studies have demonstrated overall surgical mortality rates of 10% for patients with class A cirrhosis, 30% for those with class B cir-rhosis, and 75% to 80% for those with class C cirrhosis.56 The CTP score is derived from five variables as shown in Table 31-4. The problems with the CTP score are the presence of subjective variables (encephalopathy and ascites), its narrow range (5 to 15 points), and the equal weighting given to each variable. Multiple retrospective studies have demonstrated that perioperative mor-tality and morbidity rates correlate well with the CTP score, and for over 30 years, this measure had been used as the principal predictor of operative risk.Model for

1	that perioperative mor-tality and morbidity rates correlate well with the CTP score, and for over 30 years, this measure had been used as the principal predictor of operative risk.Model for End-Stage Liver Disease Scoring SystemThe Model for End-Stage Liver Disease (MELD) is a linear regression model based on three objective laboratory values (INR, bilirubin level, and creatinine level). It was originally developed as a tool to predict mortality after transjugular intrahepatic portosystemic shunt (TIPS) but has been validated and used as the sole method of liver transplant allocation in the United States since 2002. The MELD formula is as follows:MELD Score = 9.57 Ln(SCr) + 3.78 Ln(Tbil) + 11.2 Ln(INR) + 6.43where Ln represents natural logarithm, SCr is serum creatinine level (in milligrams per deciliter), and Tbil is serum bilirubin level (in milligrams per deciliter).A number of studies have examined the relative values of MELD and CTP scores in predicting postoperative mortality

1	per deciliter), and Tbil is serum bilirubin level (in milligrams per deciliter).A number of studies have examined the relative values of MELD and CTP scores in predicting postoperative mortality in cirrhotic patients undergoing nontransplant surgical procedures. Northup and colleagues demonstrated that MELD score was the only statistically significant predictor of 30-day mortality.57 In this study, mortality increased by approximately 1% for each MELD point up to a score of 20 and by 2% for each MELD point above 20. A comparison of the MELD model with the CTP classification showed good correlation between the two measures in predicting mortality, especially in the setting of emergency surgery.58 In these studies, the relative risk of mortal-ity increased by 14% for each 1-point increase in MELD score. As a result, it has been proposed that patients with a MELD score below 10 can safely undergo elective surgery, those with MELD between 10 and 15 may undergo surgery with caution, while

1	MELD score. As a result, it has been proposed that patients with a MELD score below 10 can safely undergo elective surgery, those with MELD between 10 and 15 may undergo surgery with caution, while those with MELD scores in excess of 15 should not be subjected to elective surgical procedures.59Portal HypertensionThe portal venous system contributes approximately 75% of the blood and 72% of the oxygen supplied to the liver. In the average adult, 1000 to 1500 mL/min of portal venous blood is supplied to the liver. However, this amount can be significantly increased in the cirrhotic patient. The portal venous system is without valves and drains blood from the spleen, pancreas, gallbladder, and abdominal portion of the alimentary tract into the liver. Tributaries of the portal vein communicate with veins draining directly into the systemic circulation. These collateral communications occur at the gastroesophageal junction, anal canal, falciform ligament, splenic venous bed and left renal

1	with veins draining directly into the systemic circulation. These collateral communications occur at the gastroesophageal junction, anal canal, falciform ligament, splenic venous bed and left renal vein, and retroperitoneum (Fig. 31-14). The normal portal venous pressure is 5 to 10 mmHg, and at this pressure, very little blood is shunted from the portal venous system into the systemic circulation. As portal venous pressure increases, however, the Table 31-4Child-Turcotte-Pugh (CTP) scoreVARIABLE1 POINT2 POINTS3 POINTSBilirubin level< 2 mg/dL2–3 mg/dL> 3 mg/dLAlbumin level> 3.5 g/dL2.8– 3.5 g/dL< 2.8 g/dLInternational normalized ratio< 1.71.7–2.2> 2.2EncephalopathyNoneControlledUncontrolledAscitesNoneControlledUncontrolledChild-Turcotte-Pugh class Class A = 5–6 points Class B = 7–9 points Class C = 10–15 pointsBrunicardi_Ch31_p1345-p1392.indd 136520/02/19 2:36 PM 1366SPECIFIC CONSIDERATIONSPART IIcollateral communications with the systemic circulation dilate, and a large amount of

1	C = 10–15 pointsBrunicardi_Ch31_p1345-p1392.indd 136520/02/19 2:36 PM 1366SPECIFIC CONSIDERATIONSPART IIcollateral communications with the systemic circulation dilate, and a large amount of blood may be shunted around the liver and into the systemic circulation.Imaging of the Portal Venous System and Measurement of Portal Venous PressureThe patency of the portal vein and the nature of the collateral circulation should be established. An understanding of portal vein patency and anatomy is crucial before undertaking porto-systemic shunts, hepatic resection, or hepatic transplantation. The simplest initial investigation is abdominal ultrasonography. A large portal vein suggests portal hypertension but is not diag-nostic. Doppler ultrasound is capable of outlining the anatomy of the portal vein, excluding the presence of thrombosis, and identifying the direction of portal venous blood flow. Doppler ultrasound also is useful in evaluating blood flow through sur-gical shunts and TIPS.

1	vein, excluding the presence of thrombosis, and identifying the direction of portal venous blood flow. Doppler ultrasound also is useful in evaluating blood flow through sur-gical shunts and TIPS. Abdominal CT and magnetic resonance angiography both are capable of revealing portal vein anatomy as well as patency. Visceral angiography and portal venography are reserved for cases that cannot be evaluated satisfactorily by noninvasive methods and require further clarification of portal patency or anatomy.The most accurate method of determining portal hyperten-sion is hepatic venography. The most commonly used procedure involves placing a balloon catheter directly into the hepatic vein and measuring the free hepatic venous pressure (FHVP) with the balloon deflated and the wedged hepatic venous pressure (WHVP) with the balloon inflated to occlude the hepatic vein. The hepatic venous pressure gradient (HVPG) is then calcu-lated by subtracting the free from the wedged venous pressure (HVPG =

1	pressure (WHVP) with the balloon inflated to occlude the hepatic vein. The hepatic venous pressure gradient (HVPG) is then calcu-lated by subtracting the free from the wedged venous pressure (HVPG = WHVP – FHVP). The HVPG represents the pressure in the hepatic sinusoids and portal vein and is a measure of por-tal venous pressure. Clinically significant portal hypertension is evident when HVPG exceeds 10 mmHg.Etiology and Clinical Features of Portal HypertensionThe causes of portal hypertension can be divided into three major groups: presinusoidal, sinusoidal, and postsinusoidal.60 Although multiple disease processes can result in portal hyper-tension (Table 31-5), in the United States, the most common cause of portal hypertension is usually an intrahepatic one, namely, cirrhosis. The most significant clinical finding asso-ciated with portal hypertension is the development of gastro-esophageal varices, which are mainly supplied by the anterior branch of the left gastric (coronary)

1	most significant clinical finding asso-ciated with portal hypertension is the development of gastro-esophageal varices, which are mainly supplied by the anterior branch of the left gastric (coronary) vein.a1aFGE1ABbD24cde 3C5Figure 31-14. Intra-abdominal venous flow pathways leading to engorged veins (varices) from portal hypertension. 1, Coro-nary vein; 2, superior hemorrhoidal veins; 3, paraumbilical veins; 4, Retzius’ veins; 5, veins of Sappey; A, portal vein; B, splenic vein; C, superior mesenteric vein; D, inferior mesenteric vein; E, inferior vena cava; F, superior vena cava; G, hepatic veins; a, esophageal veins; a1, azygos system; b, vasa brevia; c, middle and inferior hem-orrhoidal veins; d, intestinal; e, epigastric veins.Table 31-5Etiology of portal hypertensionPresinusoidal Sinistral/extrahepatic Splenic vein thrombosis Splenomegaly Splenic arteriovenous fistula Intrahepatic Schistosomiasis Congenital hepatic fibrosis Nodular regenerative hyperplasia Idiopathic

1	vein thrombosis Splenomegaly Splenic arteriovenous fistula Intrahepatic Schistosomiasis Congenital hepatic fibrosis Nodular regenerative hyperplasia Idiopathic portal fibrosis Myeloproliferative disorder Sarcoid Graft-versus-host diseaseSinusoidal Intrahepatic Cirrhosis Viral infection Alcohol abuse Primary biliary cirrhosis Autoimmune hepatitis Primary sclerosing cholangitis Metabolic abnormalityPostsinusoidal Intrahepatic Vascular occlusive disease Posthepatic Budd-Chiari syndrome Congestive heart failure Inferior vena caval web Constrictive pericarditisBrunicardi_Ch31_p1345-p1392.indd 136620/02/19 2:36 PM 1367LIVERCHAPTER 31Portal hypertension also results in splenomegaly with enlarged, tortuous, and even aneurysmal splenic vessels. Spleno-megaly is frequently associated with functional hypersplenism, causing leukopenia, thrombocytopenia, and anemia. Ascites occurs in the setting of severe portal hypertension in combina-tion with hepatocyte

1	is frequently associated with functional hypersplenism, causing leukopenia, thrombocytopenia, and anemia. Ascites occurs in the setting of severe portal hypertension in combina-tion with hepatocyte dysfunction. The umbilical vein may recan-nulate and dilate, leading to visible collaterals on the abdominal wall. Anorectal varices are present in approximately 45% of cirrhotic patients and must be distinguished from hemorrhoids, which do not communicate with the portal system and are not present at increased incidence in patients with portal hyperten-sion. Large spontaneous venous shunts may form between the portal venous system and the left renal vein or the IVC, but these shunts are ineffective in reducing portal venous pressures and preventing bleeding from gastroesophageal varices.Management of Gastroesophageal VaricesThe most significant manifestation and the leading cause of mor-bidity and mortality related to portal hypertension is variceal bleeding. Approximately 30% of patients

1	of Gastroesophageal VaricesThe most significant manifestation and the leading cause of mor-bidity and mortality related to portal hypertension is variceal bleeding. Approximately 30% of patients with compensated cir-rhosis and 60% of patients with decompensated cirrhosis have esophageal varices. One third of all patients with varices will experience variceal bleeding. Each episode of bleeding is associ-ated with a 20% to 30% risk of mortality. If left untreated, 70% of patients who survive the initial bleed will experience recurrent variceal hemorrhage within 2 years of the index hemorrhage.Prevention of Variceal BleedingCurrent measures aimed at preventing variceal bleeding include the administration of nonselective β-blockers and prophylac-tic endoscopic surveillance with variceal band ligation. Meta-analyses have demonstrated that nonselective β-blockers such as propranolol and nadolol reduce the index variceal bleed by approximately 45% and decrease bleeding mortality by 50%.61

1	ligation. Meta-analyses have demonstrated that nonselective β-blockers such as propranolol and nadolol reduce the index variceal bleed by approximately 45% and decrease bleeding mortality by 50%.61 However, approximately 20% of patients do not respond to β-blockade, and another 20% cannot tolerate β-blockade due to medication side effects. Endoscopic surveillance with prophy-lactic variceal band ligation has been associated with a lower incidence of a first variceal bleed.62 Variceal band ligation is rec-ommended for patients with medium to large varices, performed every 1 to 2 weeks until obliteration, followed by esophagogas-troduodenoscopy (EGD) 1 to 3 months later and surveillance EGD every 6 months to monitor for recurrence of varices.Management of Acute Variceal HemorrhagePatients with acute variceal hemorrhage should be admitted to an ICU for resuscitation and management. Blood resuscitation should be performed carefully to reach a hemoglobin level of approxi-mately 8 g/dL.

1	with acute variceal hemorrhage should be admitted to an ICU for resuscitation and management. Blood resuscitation should be performed carefully to reach a hemoglobin level of approxi-mately 8 g/dL. Overzealous replacement of blood products and administration of saline can lead to both rebleeding and increased mortality. Administration of fresh frozen plasma and platelets can be considered in patients with severe coagulopathy. Use of recom-binant factor VIIa has not been shown to be more beneficial than standard therapy and therefore is not recommended at this time. Cirrhotic patients with variceal bleeding have a high risk of devel-oping bacterial infections, which are associated with increased risks of rebleeding and mortality. Spontaneous bacterial peritoni-tis accounts for approximately half of these infections, with uri-nary tract infections and pneumonias comprising the remainder. The use of short-term prophylactic antibiotics (e.g., ceftriaxone 1 g/d intravenously) has been

1	half of these infections, with uri-nary tract infections and pneumonias comprising the remainder. The use of short-term prophylactic antibiotics (e.g., ceftriaxone 1 g/d intravenously) has been shown both to decrease the rate of bacterial infections and to increase survival.Vasoactive medications decrease blood flow to the gastro-esophageal varices and can be initiated as soon as the diagnosis of variceal bleeding is made. Although vasopressin is the most potent available vasoconstrictor, its use is limited by its systemic vasoconstrictive effects that can produce hypertension, myocar-dial ischemia, arrhythmias, ischemic abdominal pain, and limb gangrene. Octreotide, a somatostatin analog, has the advantage that it can be administered for 5 days or longer, and it is currently the preferred pharmacologic agent for initial management of acute variceal bleeding. In addition to pharmacologic therapy, endos-copy with variceal band ligation should be carried out as soon as possible. This

1	pharmacologic agent for initial management of acute variceal bleeding. In addition to pharmacologic therapy, endos-copy with variceal band ligation should be carried out as soon as possible. This combination of pharmacologic and endoscopic therapy has been shown both to improve the initial control of bleeding and to increase the 5-day hemostasis rate.62Luminal TamponadeWhen medical and endoscopic measures fail to control variceal hemorrhage, balloon tamponade using a Sengstaken-Blakemore tube will control refractory bleeding in up to 90% of patients. However, its application is limited due to the potential for com-plications, which include aspiration, airway obstruction, and esophageal perforation due to overinflation or pressure necro-sis. Therefore, the use of a Sengstaken-Blakemore tube should not exceed 36 hours to avoid tissue necrosis, and this treatment modality should only be considered a temporary bridge to more definitive measures of variceal hemorrhage control.Transjugular

1	should not exceed 36 hours to avoid tissue necrosis, and this treatment modality should only be considered a temporary bridge to more definitive measures of variceal hemorrhage control.Transjugular Intrahepatic Portosystemic ShuntThe TIPS procedure involves implantation of a metallic stent between an intrahepatic branch of the portal vein and a hepatic vein radicle. The needle track is dilated until a portal pressure gradient of ≤12 mmHg is achieved. TIPS can be performed in 95% of patients by an experienced interventional radiolo-gist, controls variceal bleeding in >90% of cases refractory to medical treatment, and should not affect subsequent hepatic transplantation. Possible complications include bleeding either intra-abdominally or via the biliary tree, infections, renal failure, decreased hepatic function, and hepatic encephalopathy, which occur in 25% to 30% of patients after the TIPS procedure. A high rate of thrombosis is seen and can be attributed to inti-mal hyperplasia of

1	hepatic function, and hepatic encephalopathy, which occur in 25% to 30% of patients after the TIPS procedure. A high rate of thrombosis is seen and can be attributed to inti-mal hyperplasia of the metallic stent. Frequent follow-up with repeated interventions such as dilation or restenting often are needed to maintain TIPS patency.Balloon-Occluded Retrograde Transvenous ObliterationThe balloon-occluded retrograde transvenous obliteration (BRTO) procedure has been used for the specific manage-ment of bleeding gastric varices in patients with spontaneous gastrorenal or splenorenal shunts shown on contrast-enhanced cross-sectional imaging. Using a transjugular or transfemoral approach, a balloon-occlusion catheter is directed through the left renal vein into the spontaneous shunt, which is then obliter-ated with the use of a sclerosing agent. BRTO effectively con-trols hemorrhage from gastric varices and preserves portal flow to the liver, thereby reducing the risk of hepatic

1	which is then obliter-ated with the use of a sclerosing agent. BRTO effectively con-trols hemorrhage from gastric varices and preserves portal flow to the liver, thereby reducing the risk of hepatic encephalopathy relative to TIPS. The occlusion of spontaneous shunts, how-ever, can theoretically exacerbate portal hypertension, precipi-tate hemorrhage from esophageal varices, and exacerbate the accumulation of ascites.Surgical ShuntingThe need for surgical shunts has been reduced since the intro-duction of the TIPS procedure and hepatic transplantation. At this time, the recommendation is that surgical shunts be consid-ered only in patients who have MELD scores of <15, who are 5Brunicardi_Ch31_p1345-p1392.indd 136720/02/19 2:36 PM 1368SPECIFIC CONSIDERATIONSPART IInot candidates for hepatic transplantation, or who have limited access to TIPS therapy and the necessary follow-up. The aim of the surgical shunt is to reduce portal venous pressure, maintain total hepatic and portal

1	hepatic transplantation, or who have limited access to TIPS therapy and the necessary follow-up. The aim of the surgical shunt is to reduce portal venous pressure, maintain total hepatic and portal blood flow, and avoid the high inci-dence of complicating hepatic encephalopathy. Patient survival is determined by hepatic reserve.The portacaval shunt, as first described by Eck in 1877, either joins the portal vein to the IVC in an end-to-side fashion and completely disrupts portal vein flow to the liver, or joins it in a side-to-side fashion and thereby maintains partial por-tal venous flow to the liver. Currently this shunt is rarely per-formed due to the high incidence of hepatic encephalopathy and decreased liver function resulting from the reduction of portal perfusion. The Eck fistula also makes subsequent hepatic trans-plantation much more technically difficult.The mesocaval shunt uses an 8or 10-mm polytetrafluo-roethylene (PTFE) graft to connect the superior mesenteric vein to

1	also makes subsequent hepatic trans-plantation much more technically difficult.The mesocaval shunt uses an 8or 10-mm polytetrafluo-roethylene (PTFE) graft to connect the superior mesenteric vein to the IVC. The mesocaval shunt is technically easier to perform and can be easily ligated during subsequent hepatic transplan-tation. The smaller caliber of the shunt avoids the deleterious effects of portal blood flow deprivation on hepatic function. Small-diameter portosystemic shunts have been reported to reduce the incidence of encephalopathy but at the expense of increased risks of shunt thrombosis and rebleeding.The surgical shunt currently used most often is the distal splenorenal or Warren shunt (Fig. 31-15). This shunt is techni-cally the most difficult to perform. It requires division of the gastroesophageal collaterals and allows venous drainage of the stomach and lower esophagus through the short gastrosplenic veins into the spleen, and ultimately decompresses the left upper

1	of the gastroesophageal collaterals and allows venous drainage of the stomach and lower esophagus through the short gastrosplenic veins into the spleen, and ultimately decompresses the left upper quadrant by allowing the splenic vein to drain directly into the left renal vein via an end-to-side splenic to left renal vein anas-tomosis. This shunt has the advantages of being associated with a lower rate of hepatic encephalopathy and decompensation and not interfering with subsequent liver transplantation.Nonshunt Surgical Management of Refractory Variceal BleedingIn the patient with extrahepatic portal vein thrombosis and refractory variceal bleeding, the Sugiura procedure may be considered. The Sugiura procedure consists of extensive devas-cularization of the stomach and distal esophagus along with tran-section of the esophagus, splenectomy, truncal vagotomy, and pyloroplasty. As with performance of surgical shunts, patient survival is dependent on hepatic reserve at the time of the

1	along with tran-section of the esophagus, splenectomy, truncal vagotomy, and pyloroplasty. As with performance of surgical shunts, patient survival is dependent on hepatic reserve at the time of the sur-gical procedure. Experience in Western countries is somewhat limited, and a number of modifications have been made to the original Sugiura procedure over time.Hepatic TransplantationPatients with cirrhosis, portal hypertension, and variceal bleed-ing usually die as a result of hepatic failure and not acute blood loss. Therefore, hepatic transplantation must be considered in the patient with ESLD because it represents the patient’s only chance for definitive therapy and long-term survival. Hepatic transplantation also can be considered for the patient with vari-ceal bleeding refractory to all other forms of management. Sur-vival after hepatic transplantation is not affected adversely by the previous performance of endoscopic variceal band ligation, TIPS, or splenorenal or mesocaval

1	to all other forms of management. Sur-vival after hepatic transplantation is not affected adversely by the previous performance of endoscopic variceal band ligation, TIPS, or splenorenal or mesocaval shunts. Previous creation of an Eck fistula, however, does make hepatic transplantation much more technically difficult, and therefore this procedure should be avoided in the transplant candidate. In addition to sav-ing the patient’s life, hepatic transplantation reverses most of the hemodynamic and humoral changes associated with cirrhosis.Budd-Chiari SyndromeBudd-Chiari syndrome (BCS) is an uncommon congestive hepatopathy characterized by the obstruction of hepatic venous outflow. The incidence of BCS is 1 in 100,000 of the general population worldwide.63 Patients may present with acute signs and symptoms of abdominal pain, ascites, and hepatomegaly or more chronic symptoms related to long-standing portal hyper-tension. BCS is defined as primary when the obstructive process involves an

1	and symptoms of abdominal pain, ascites, and hepatomegaly or more chronic symptoms related to long-standing portal hyper-tension. BCS is defined as primary when the obstructive process involves an endoluminal venous thrombosis. BCS is consid-ered as a secondary process when the veins are compressed or invaded by a neighboring lesion originating outside the vein.A thorough evaluation demonstrates one or more throm-botic risk factors in approximately 75% to 90% of patients with primary BCS.63 Primary myeloproliferative disorders, such as Figure 31-15. Surgical shunts for portal hyper-tension. Types of portacaval anastomoses. A. Nor-mal anatomy. B. Side-to-side portacaval shunt. C. End-to-side portacaval shunt. D. Mesocaval shunt. E. Distal splenorenal (Warren) shunt. (Reproduced with permission from Doherty GM, Way LW: Current Surgical Diagnosis and Treatment, 12th ed. New York, NY: McGraw-Hill Education; 2006.)ABCDEBrunicardi_Ch31_p1345-p1392.indd 136820/02/19 2:36 PM

1	with permission from Doherty GM, Way LW: Current Surgical Diagnosis and Treatment, 12th ed. New York, NY: McGraw-Hill Education; 2006.)ABCDEBrunicardi_Ch31_p1345-p1392.indd 136820/02/19 2:36 PM 1369LIVERCHAPTER 31essential thrombocythemia or polycythemia rubra, account for approximately 35% to 50% of the primary cases of BCS. All known inherited thrombophilias also have been implicated in the development of BCS. Activated protein C resistance, generally related to factor V Leiden mutation, is present in approximately 25% of patients. Anticardiolipin antibodies, hyperhomocystein-emia, and oral contraceptive use all have been shown to be risk factors for BCS.63Clinically significant BCS is usually the result of obstruc-tion of two or more of the major hepatic veins. The obstruction results in hepatomegaly, liver congestion, and right upper quad-rant pain. In addition, liver perfusion via the portal vein may be decreased, and 70% of affected patients have noninflammatory

1	results in hepatomegaly, liver congestion, and right upper quad-rant pain. In addition, liver perfusion via the portal vein may be decreased, and 70% of affected patients have noninflammatory centrilobular necrosis on biopsy. Although ALF is rare, most patients will go on to develop chronic portal hypertension and ascites. Caudate lobe hypertrophy occurs in approximately 50% of cases and is due to the fact that the caudate lobe has direct venous drainage into the IVC. This caudate lobe hypertrophy, in turn, can result in further obstruction of the IVC.Abdominal ultrasonography is the initial investigation of choice and can demonstrate the absence of hepatic vein flow, spider web hepatic veins, and collateral hepatic veins.64 CT or MRI of the abdomen also is capable of demonstrating hepatic vein thrombosis and evaluating the IVC but is limited in that it cannot show direction of blood flow. The definitive radio-graphic study to evaluate BCS is hepatic venography to deter-mine the

1	vein thrombosis and evaluating the IVC but is limited in that it cannot show direction of blood flow. The definitive radio-graphic study to evaluate BCS is hepatic venography to deter-mine the presence and extent of hepatic vein thrombus as well as measure IVC pressures.Initial treatment consists of diagnosing and medically managing the underlying disease process and preventing exten-sion of the hepatic vein thrombosis through systemic anticoagu-lation. The BCS-associated portal hypertension and ascites can be medically managed in a manner similar to that in most cir-rhotic patients. Radiologic and surgical intervention should be reserved for patients whose condition is nonresponsive to medi-cal therapy. Percutaneous angioplasty and TIPS, in combination with thrombolytic therapy, are the preferred strategies to restore the outflow of blood from the liver. Thrombolytic therapy alone may be attempted for acute thrombosis. Surgical shunting, namely with the side-to-side portacaval shunt,

1	strategies to restore the outflow of blood from the liver. Thrombolytic therapy alone may be attempted for acute thrombosis. Surgical shunting, namely with the side-to-side portacaval shunt, essentially turns the portal vein into a hepatic outflow tract. Most patients with a portacaval shunt show improvement in hepatic function and fibrosis at 1 year without significant hepatic encephalopathy.64 However, the enthusiasm for this procedure has been curbed due to the relatively high rate of operative mortality and shunt dysfunction. Patients with progressive BCS and manifestations of ESLD will ultimately require hepatic transplantation.INFECTIONS OF THE LIVERThe liver contains the largest portion of the reticuloendothelial system in the human body and is therefore able to handle the continuous low-level exposure to enteric bacteria that it receives through the portal venous system. Due to the high level of retic-uloendothelial cells in the liver, nonviral infections are unusual.Pyogenic

1	low-level exposure to enteric bacteria that it receives through the portal venous system. Due to the high level of retic-uloendothelial cells in the liver, nonviral infections are unusual.Pyogenic Liver AbscessesPyogenic liver abscesses are the most common liver abscesses seen in the United States. They may be single or multiple and are more frequently found in the right lobe of the liver.65 The abscess cavities are variable in size and, when multiple, may coalesce to give a honeycomb appearance. Approximately 40% of abscesses are monomicrobial, an additional 40% are polymicrobial, and 20% are culture-negative. The most com-mon infecting agents are gram-negative bacteria. Escherichia coli is found in two thirds of cases, and other common organ-isms include Streptococcus faecalis, Klebsiella, and Proteus vulgaris. Anaerobic organisms such as Bacteroides fragilis also are seen frequently. In patients with endocarditis and infected indwelling catheters, Staphylococcus and Streptococcus

1	and Proteus vulgaris. Anaerobic organisms such as Bacteroides fragilis also are seen frequently. In patients with endocarditis and infected indwelling catheters, Staphylococcus and Streptococcus species are more commonly found.In the past, pyogenic liver abscesses often resulted from infections of the intestinal tract such as acute appendicitis and diverticulitis, which then spread to the liver via the portal circu-lation. With improved imaging modalities and earlier diagnosis of these intra-abdominal infections, this particular etiology of pyogenic liver abscesses has become less common. Pyogenic liver abscesses also occur as a result of impaired biliary drain-age, subacute bacterial endocarditis, infected indwelling cath-eters, dental work, or the direct extension of infections such as diverticulitis or Crohn’s disease into the liver. There appears to be an increasing incidence due to infection by opportunis-tic organisms among immunosuppressed patients, including transplant and

1	as diverticulitis or Crohn’s disease into the liver. There appears to be an increasing incidence due to infection by opportunis-tic organisms among immunosuppressed patients, including transplant and chemotherapy recipients as well as patients with acquired immunodeficiency syndrome (AIDS).Patients commonly present with right upper quadrant pain and fever. Jaundice occurs in up to one-third of affected patients. A thorough history and physical examination are usually helpful in identifying the underlying cause of the liver abscess. Leuko-cytosis, an elevated sedimentation rate, and an elevated AP level are the most common laboratory findings. Significant abnor-malities in the results of the remaining liver function tests are unusual. Blood cultures will only reveal the causative organism in approximately 50% of cases. Ultrasound examination of the liver reveals pyogenic abscesses as round or oval hypoechoic lesions with well-defined borders and a variable number of internal echoes. CT

1	approximately 50% of cases. Ultrasound examination of the liver reveals pyogenic abscesses as round or oval hypoechoic lesions with well-defined borders and a variable number of internal echoes. CT scan is highly sensitive in the localization of pyogenic liver abscesses, which appear hypodense with periph-eral enhancement and may contain air-fluid levels indicating a gas-producing infectious organism (Fig. 31-16). MRI of the abdomen can also detect pyogenic abscesses with a high level of sensitivity but plays a limited role because of its inability to be used for image-guided diagnosis and therapy.The current cornerstones of treatment include correction of the underlying cause and IV antibiotic therapy. Empiric antibiotic therapy should cover gram-negative and anaerobic organisms; percutaneous needle aspiration and culture of the aspirate may be useful in guiding subsequent antibiotic therapy. IV antibiotic therapy should be continued for at least 8 weeks and can be expected to be

1	needle aspiration and culture of the aspirate may be useful in guiding subsequent antibiotic therapy. IV antibiotic therapy should be continued for at least 8 weeks and can be expected to be effective in 80% to 90% of patients. Placement of a percutaneous drainage catheter is beneficial only for a minority of patients, as most pyogenic abscesses are quite viscous and catheter drainage is often ineffective.Surgical drainage either via the laparoscopic or open approach may become necessary if initial therapies fail. Ana-tomic surgical resection can be performed in patients with recalcitrant abscesses. It must be kept in mind throughout the evaluation and treatment of the presumed pyogenic abscess that a necrotic hepatic malignancy must not be mistaken for a hepatic abscess. Therefore, early diagnosis and progression to surgical resection should be advocated for patients who do not respond to initial antibiotic therapy.Amebic AbscessesEntamoeba histolytica is a parasite that is endemic

1	diagnosis and progression to surgical resection should be advocated for patients who do not respond to initial antibiotic therapy.Amebic AbscessesEntamoeba histolytica is a parasite that is endemic world-wide, infecting approximately 10% of the world’s population. Brunicardi_Ch31_p1345-p1392.indd 136920/02/19 2:36 PM 1370SPECIFIC CONSIDERATIONSPART IIAmebiasis is most common in subtropical climates, especially in areas with poor sanitation. E histolytica exists as cysts in a vegetative form that are capable of surviving outside the human body. The cystic form passes through the stomach and small bowel unharmed and then transforms into a trophozoite in the colon. Here it invades the colonic mucosa forming typical flask-shaped ulcers, enters the portal venous system, and is carried to the liver. Occasionally, the trophozoite will pass through the hepatic sinusoid and into the systemic circulation, which results in lung and brain abscesses.Amebae multiply and block small intrahepatic

1	liver. Occasionally, the trophozoite will pass through the hepatic sinusoid and into the systemic circulation, which results in lung and brain abscesses.Amebae multiply and block small intrahepatic portal radi-cles with consequent focal infarction of hepatocytes. They con-tain a proteolytic enzyme that also destroys liver parenchyma. The abscesses formed are variable in size and can be single or multiple. The amebic abscess is most commonly located in the superior-anterior aspect of the right lobe of the liver near the diaphragm and has a necrotic central portion that contains a thick, reddish brown, pus-like material. This material has been likened to anchovy paste or chocolate sauce. Amebic abscesses are the most common type of liver abscesses worldwide.Amebiasis should be considered in patients who have trav-eled to an endemic area and present with right upper quadrant pain, fever, hepatomegaly, and hepatic abscess.46 Leukocytosis is common, whereas elevated transaminase levels and

1	patients who have trav-eled to an endemic area and present with right upper quadrant pain, fever, hepatomegaly, and hepatic abscess.46 Leukocytosis is common, whereas elevated transaminase levels and jaundice are unusual. The most common biochemical abnormality is a mildly elevated AP level. Even though this disease process is secondary to a colonic infection, the presence of diarrhea is unusual. Most patients have a positive fluorescent antibody test for E histo-lytica, and test results can remain positive for some time after a clinical cure. This serologic test has a high sensitivity, and therefore amebiasis is unlikely if the test results are negative.Ultrasound and CT scanning of the abdomen are both very sensitive but nonspecific for the detection of amebic abscesses.65 Amebic abscesses usually appear on CT as well-defined low-density round lesions that have enhancement of the wall, some-what ragged in appearance with a peripheral zone of edema. The central cavity may have

1	abscesses usually appear on CT as well-defined low-density round lesions that have enhancement of the wall, some-what ragged in appearance with a peripheral zone of edema. The central cavity may have septations as well as fluid levels. CT scanning is also useful in the detection of extrahepatic involvement.Metronidazole 750 mg three times a day for 7 to 10 days is the treatment of choice and is successful in 95% of cases. Defervescence usually occurs in 3 to 5 days, but the time nec-essary for the abscess to resolve depends on the initial size at presentation and varies from 30 to 300 days.65 Both ultrasound and CT of the liver can be used as follow-up after the initiation of medical therapy. Aspiration of the abscess rarely is needed and should be reserved for patients with large abscesses, those who do not respond to medical therapy, or those who appear to be superinfected. Furthermore, abscesses of the left lobe of the liver at risk for rupture into the pericardium should be

1	those who do not respond to medical therapy, or those who appear to be superinfected. Furthermore, abscesses of the left lobe of the liver at risk for rupture into the pericardium should be treated with aspiration and drainage.Hydatid DiseaseHydatid disease is due to infection by the tapeworm Echinococ-cus granulosus in its larval or cyst stage.66 The tapeworm lives in canids, which are infected by eating the viscera of sheep that contain hydatid cysts. Scolices, contained in the cysts, adhere to the small intestine of dogs and become adult taenia, which attach to the intestinal wall. Each worm sheds approximately 500 ova into the bowel. The infected ova-containing feces of dogs contaminate grass and farmland, and the ova are ingested by intermediate hosts such as sheep, cattle, pigs, and humans. The ova have chitinous envelopes that are dissolved by gastric juice. The liberated ovum then burrows through the intestinal mucosa and is carried by the portal vein to the liver, where it

1	humans. The ova have chitinous envelopes that are dissolved by gastric juice. The liberated ovum then burrows through the intestinal mucosa and is carried by the portal vein to the liver, where it develops into an adult cyst. Most cysts are caught in the hepatic sinusoids, and therefore 70% of hydatid cysts form in the liver. A few ova pass through the liver and are held up in the pul-monary capillary bed or enter the systemic circulation, forming cysts in the lung, spleen, brain, or bones.Hydatid disease is most common in sheep-raising areas, where dogs have access to infected offal. These include South Australia, New Zealand, Africa, Greece, Spain, and the Middle East. Hydatid cysts commonly involve the right lobe of the liver, usually the anterior-inferior or posterior-inferior segments. The uncomplicated cyst may be silent and found only incidentally or at autopsy. Occasionally, the affected patient presents with symptoms such as dull right upper quadrant pain or abdomi-nal

1	The uncomplicated cyst may be silent and found only incidentally or at autopsy. Occasionally, the affected patient presents with symptoms such as dull right upper quadrant pain or abdomi-nal distention. Cysts may become secondarily infected, involve other organs, or even rupture, which leads to an allergic or ana-phylactic reaction.The diagnosis of hydatid disease is based on the findings of an enzyme-linked immunosorbent assay (ELISA) for echino-coccal antigens, and results are positive in approximately 85% of infected patients.66 The ELISA results may be negative in an infected patient if the cyst has not leaked or does not contain scolices or if the parasite is no longer viable. Eosinophilia is seen in approximately 30% of infected patients. Ultrasonography and CT scanning of the abdomen are both quite sensitive for detecting hydatid cysts. The appearance of the cysts on images depends on the stage of cyst development. Typically, hydatid cysts are well-defined hypodense lesions

1	are both quite sensitive for detecting hydatid cysts. The appearance of the cysts on images depends on the stage of cyst development. Typically, hydatid cysts are well-defined hypodense lesions with a distinct wall. Ring-like cal-cifications of the pericysts are present in 20% to 30% of cases. As healing occurs, the entire cyst calcifies densely, and a lesion with this appearance is usually dead or inactive. Daughter cysts generally occur in a peripheral location within the main cyst and are typically slightly hypodense compared with the mother cyst. MRI of the abdomen may be useful to evaluate the pericyst, cyst matrix, and daughter cyst characteristics.Unless the cysts are small or the patient is not a suitable candidate for surgical resection, the treatment of hydatid disease Figure 31-16. Computed tomographic scan of pyogenic liver abscesses. Multiple hepatic abscesses are seen in a patient after an episode of diverticulitis. Note the loculated large central abscess as well as the

1	tomographic scan of pyogenic liver abscesses. Multiple hepatic abscesses are seen in a patient after an episode of diverticulitis. Note the loculated large central abscess as well as the left lateral segment abscess.Brunicardi_Ch31_p1345-p1392.indd 137020/02/19 2:36 PM 1371LIVERCHAPTER 31is surgically based because of the high risk of secondary infec-tion and rupture. Medical treatment with albendazole relies on drug diffusion through the cyst membrane. The concentration of drug achieved in the cyst is uncertain but is better than that of mebendazole, and albendazole can be used as initial treat-ment for small, asymptomatic cysts. For most cysts, surgical resection involving laparoscopic or open complete cyst removal with instillation of a scolicidal agent is preferred and usually is curative. If complete cystectomy is not possible, then formal anatomic liver resection can be undertaken. During surgical resection, caution must be exercised to avoid rupture of the cyst with release

1	If complete cystectomy is not possible, then formal anatomic liver resection can be undertaken. During surgical resection, caution must be exercised to avoid rupture of the cyst with release of protoscolices into the peritoneal cavity. Perito-neal contamination can result in an acute anaphylactic reaction or peritoneal implantation of scolices with daughter cyst forma-tion and inevitable recurrence.Echinococcus multilocularis occurs in the Northern Hemisphere and can infect the liver in a fashion similar to that described earlier, although the cysts are multilocular. Infection of the lung also is common (alveolar echinococcosis). Canine species such as wolves, foxes, and dogs ingest infected vis-cera of an intermediate host (e.g., rodents, moose) and become infected; humans become infected incidentally by ingesting contaminated food or water. Treatment consists of albendazole; however, infection in the lung produces a more generalized granulomatous reaction, can present in a manner

1	incidentally by ingesting contaminated food or water. Treatment consists of albendazole; however, infection in the lung produces a more generalized granulomatous reaction, can present in a manner similar to that of a malignancy, and often requires resection.AscariasisAscaris infection is particularly common in the Far East, India, and South Africa. Ova of the roundworm Ascaris lumbricoides arrive in the liver by retrograde locomotion in the bile ducts from the GI tract. The adult worm is 10 to 20 cm long and may lodge in the common bile duct, causing partial bile duct obstruction and secondary cholangitic abscesses. The Ascaris may serve as a nidus for the development of intrahepatic gallstones. The clini-cal presentation in an affected patient may include biliary colic, acute cholecystitis, acute pancreatitis, or hepatic abscesses.67 Plain abdominal radiographs, abdominal ultrasound, and ERCP can demonstrate the Ascaris as linear filling defects within the bile ducts. Occasionally,

1	acute pancreatitis, or hepatic abscesses.67 Plain abdominal radiographs, abdominal ultrasound, and ERCP can demonstrate the Ascaris as linear filling defects within the bile ducts. Occasionally, worms can be seen moving into and out of the biliary tree from the duodenum. Treatment consists of the administration of piperazine citrate, mebendazole, or alben-dazole in combination with endoscopic extraction of the worms. Surgical intervention may become necessary if the Ascaris can-not be removed via ERCP.SchistosomiasisSchistosomiasis affects >200 million people in 74 countries. Hepatic schistosomiasis occurs when emboli of the ova in the intestines reach the liver via the mesenteric venous system. Eggs excreted in the feces hatch in water to release free-swimming embryos, which enter snails and develop into fork-tailed cercar-iae. They then reenter human skin during contact with infected water. They burrow down to the capillary bed and enter the bloodstream, leading to widespread

1	snails and develop into fork-tailed cercar-iae. They then reenter human skin during contact with infected water. They burrow down to the capillary bed and enter the bloodstream, leading to widespread hematogenous dissemina-tion. Those entering the intrahepatic portal system grow rapidly, resulting in a granulomatous reaction. The degree of consequent portal fibrosis is related to the adult worm load.Schistosomiasis has three stages of clinical symptomatol-ogy: the first includes itching after the entry of cercariae through the skin; the second includes fever, urticaria, and eosinophilia; and the third involves hepatic fibrosis followed by presinusoidal portal hypertension. During this third phase, the liver shrinks, the spleen enlarges, and the patient may develop complications of portal hypertension while hepatic function is maintained. Active infection is detected by stool examination. Serologic tests indicate past exposure but do not provide information regarding the timing of

1	hypertension while hepatic function is maintained. Active infection is detected by stool examination. Serologic tests indicate past exposure but do not provide information regarding the timing of infection. A negative serologic test result excludes the presence of schistosomal infection. Serum levels of transaminases are usually normal, but the AP level may be mildly elevated. A decreased serum albumin level is usually the result of frequent GI bleeds and malnutrition.Medical treatment of schistosomiasis includes education on hygiene and the avoidance of infected water. Treatment with praziquantel 40 to 75 mg/kg as a single dose is the treatment of choice for all forms of schistosomiasis and produces few side effects. GI bleeding usually is controlled by endoscopic variceal ligation. However, in a patient with refractory GI portal hyper-tensive bleeding, distal splenorenal shunt or gastric devascular-ization and splenectomy may be considered.Viral HepatitisThe role of the surgeon in

1	in a patient with refractory GI portal hyper-tensive bleeding, distal splenorenal shunt or gastric devascular-ization and splenectomy may be considered.Viral HepatitisThe role of the surgeon in the management of viral hepatitis is somewhat limited. However, the disease entities of hepatitis A, B, and C need to be kept in mind during any evaluation for liver disease. Hepatitis A usually results in an acute self-limited ill-ness and only rarely leads to fulminant hepatic failure. Patients can present with fatigue, malaise, nausea, vomiting, anorexic fever, and right upper quadrant abdominal pain. The most com-mon physical findings are jaundice and hepatomegaly. Because the disease is self-limited, the treatment is usually supportive. Patients who develop fulminant infection require aggressive therapy and should be transferred to a center capable of per-forming liver transplantation.Hepatitis B and C, on the other hand, can both lead to chronic liver disease, cirrhosis, and HCC. The

1	therapy and should be transferred to a center capable of per-forming liver transplantation.Hepatitis B and C, on the other hand, can both lead to chronic liver disease, cirrhosis, and HCC. The prevalence of chronic hepatitis B infection in the U.S. population is estimated to be 0.27%, but hepatitis B remains a major burden in resource-limited countries, accounting for 30% of cirrhosis and 53% of HCC cases. The ultimate goal of treatment for chronic hepati-tis B is viral suppression, thereby preventing the development of clinical outcomes such as cirrhosis, liver failure, and HCC. The cornerstone of current antiviral therapy includes pegylated interferon and nucleoside analogs such as tenofovir or entecavir.68 These agents have been proven to reduce complications of cirrho-sis and HCC and perhaps reverse previous damage to the liver. Interferon therapy produces various side effects including fatigue, flu-like symptoms, mood changes, bone marrow suppression, and stimulation of

1	HCC and perhaps reverse previous damage to the liver. Interferon therapy produces various side effects including fatigue, flu-like symptoms, mood changes, bone marrow suppression, and stimulation of autoimmunity. On the other hand, the nucleoside analogs are generally well-tolerated by patients. Compared with lamivudine, the nucleoside analogs are less likely to produce resistance and are more likely to be clinically effective. Despite its high rate of viral resistance, lamivudine may be the preferred treatment in some countries because of its relatively low cost.Acute hepatitis C viral (HCV) infection typically devel-ops 2 to 26 weeks after exposure to the virus, and presenting symptoms can include jaundice, nausea, dark urine, and right upper quadrant abdominal pain. Diagnosis is confirmed by testing for the presence of HCV RNA and anti-HCV antibod-ies in the serum; viral RNA is first detectable in the serum by polymerase chain reaction (PCR) within days to weeks follow-ing the

1	by testing for the presence of HCV RNA and anti-HCV antibod-ies in the serum; viral RNA is first detectable in the serum by polymerase chain reaction (PCR) within days to weeks follow-ing the exposure, whereas antibodies will not appear until 2 to 6 months after. If the diagnosis of acute hepatitis is established and the virus is not spontaneously cleared within 12 weeks, patients should generally be treated with pegylated interferon Brunicardi_Ch31_p1345-p1392.indd 137120/02/19 2:36 PM 1372SPECIFIC CONSIDERATIONSPART IImonotherapy. High rates of sustained viral response, in excess of 80%, have been achieved with the early treatment of acute HCV infection.69 Unfortunately, patients with acute HCV infec-tion are typically asymptomatic, and the vast majority of cases go undetected. Untreated, most of these patients will eventually develop chronic infection.Chronic HCV infection often follows a progressive course over many years and can ultimately result in cirrhosis, HCC, and the

1	most of these patients will eventually develop chronic infection.Chronic HCV infection often follows a progressive course over many years and can ultimately result in cirrhosis, HCC, and the need for liver transplantation. Cirrhosis secondary to hepa-titis C remains the leading indication for liver transplantation in the United States, Europe, and Japan. The decision to treat a patient with chronic HCV infection is complex and involves consideration of multiple factors including the natural history of the disease, stage of fibrosis, and the efficacy and adverse effects related to the treatment regimen. Patients with genotype 1 are now treated with triple-agent therapy including pegylated interferon, ribavirin, and a protease inhibitor. The protease inhibitors telaprevir and boceprevir were recently approved for the treatment of chronic HCV genotype 1 infection, and their addition to the treatment regimen has been shown to increase the rate of sustained viral response from 40% to 70%.

1	recently approved for the treatment of chronic HCV genotype 1 infection, and their addition to the treatment regimen has been shown to increase the rate of sustained viral response from 40% to 70%. These prote-ase inhibitors do no exhibit significant antiviral activity against other HCV genotypes, and therefore genotypes 2, 3, and 4 are treated with interferon and ribavirin alone. Genotypes 2 and 3 are generally more responsive to treatment than genotypes 1 and 4, but the therapeutic response also is dependent on other factors such as baseline viral load, ethnicity, and the patient’s genetic background and compliance with the regimen.EVALUATION OF AN INCIDENTAL LIVER MASSA liver mass often is identified incidentally during a radio-logic imaging procedure performed for another indication. For example, a liver mass may be discovered during evaluation for gallbladder disease or kidney stones. In addition, with advances in imaging technology, previously undetected lesions not

1	indication. For example, a liver mass may be discovered during evaluation for gallbladder disease or kidney stones. In addition, with advances in imaging technology, previously undetected lesions not infre-quently are now identified. Although many of these lesions are benign and will require no further treatment, the concern for malignancy requires a thorough evaluation. Thus, an orderly approach should be taken to the workup of an incidental liver lesion to minimize unnecessary testing.70The evaluation of an incidental liver mass begins with a history and physical examination (Fig. 31-17). The patient should be asked about abdominal pain, weight loss, previous Figure 31-17. Algorithm for diagnostic workup of an incidental liver lesion. The evaluation includes history and physical examination, blood work, imaging studies, and liver biopsy (if needed). AFP = α-fetoprotein; BUN = blood urea nitrogen; CA 19-9 = cancer antigen 19-9; CBC = complete blood count; CEA = carcinoembryonic

1	blood work, imaging studies, and liver biopsy (if needed). AFP = α-fetoprotein; BUN = blood urea nitrogen; CA 19-9 = cancer antigen 19-9; CBC = complete blood count; CEA = carcinoembryonic antigen; creat = creatinine; CT = computed tomography; EGD = esophagogas-troduodenoscopy; glu = glucose; Gyn = gynecologic; HTN = hypertension; MRI = magnetic resonance imaging; OCP = oral contraceptive pill; PAP = Papanicolaou; US = ultrasound.DiagnosisAbdominal pain/weight lossLiver disease/cirrhosis/alcohol useHepatitis/blood transfusion/tattoosOCP/hormone use/cancer historyJaundice/scleral icterusPalpable mass/hepatomegalyStigmata of portal HTNCBC, platelet countLytes/BUN/creat/glu/albuminLiver function tests/ammoniaCoagulation studiesHepatitis screenTumor markers (CEA, AFP, CA 19-9)USCT or MRI scanNuclear med.AngiogramOccult primary eval.EGDColonoscopyMammogramGyn/PAP smear(Percutaneous or laparoscopic)Liver biopsy (if needed)Additional imaging studiesLaboratory testsHistory and physical

1	scanNuclear med.AngiogramOccult primary eval.EGDColonoscopyMammogramGyn/PAP smear(Percutaneous or laparoscopic)Liver biopsy (if needed)Additional imaging studiesLaboratory testsHistory and physical examMass identified incidentally by US or CTBrunicardi_Ch31_p1345-p1392.indd 137220/02/19 2:36 PM 1373LIVERCHAPTER 31liver disease, cirrhosis, alcohol use, viral hepatitis, blood trans-fusions, tattoos, oral contraceptive use (in women), and personal or family history of cancer. On physical examination, jaundice, scleral icterus, hepatomegaly, splenomegaly, palpable mass, or stigmata of portal hypertension should be noted. After comple-tion of the history and physical examination, blood work should be performed, including complete blood count; platelet count; measurement of levels of electrolytes, blood urea nitrogen, creatinine, glucose, and albumin; liver function tests; serum ammonia level; coagulation studies; hepatitis screen; and mea-surement of levels of the tumor markers

1	electrolytes, blood urea nitrogen, creatinine, glucose, and albumin; liver function tests; serum ammonia level; coagulation studies; hepatitis screen; and mea-surement of levels of the tumor markers carcinoembryonic anti-gen, AFP, and cancer antigen 19-9.The differential diagnosis for an incidental liver mass includes cysts, benign solid lesions, and primary or metastatic cancers (Table 31-6). Ultrasound or CT is commonly performed to evaluate respiratory or abdominal symptoms, and these imag-ing studies usually lead to the discovery of an incidental liver lesion. Further radiologic evaluation by dualor triple-phase CT scan or MRI is often necessary to fully characterize the extent and nature of the lesion. Different types of liver masses have distinct appearances and patterns of contrast enhancement on these studies, facilitating the clinician in the diagnosis and for-mulation of the treatment plan. The use of liver-specific contrast agents in MRI provides information on hepatocyte

1	enhancement on these studies, facilitating the clinician in the diagnosis and for-mulation of the treatment plan. The use of liver-specific contrast agents in MRI provides information on hepatocyte function in combination with the structural data obtained during a standard MRI, and yields improved detection and characterization of liver lesions. CT cholangiography or MRCP can be obtained, par-ticularly when visualization of the biliary tract is desired. These modalities may be useful in the depiction of benign or malignant strictures resulting in biliary obstruction. In the evaluation of metastases to the liver from a variety of primary cancers, FDG-PET/CT has emerged as an indispensable tool in disease staging and in the follow-up after treatment. The techniques available for imaging of liver lesions are described in detail earlier in the section “Radiologic Evaluation of the Liver.”Liver biopsy is indicated when biochemical analysis and diagnostic imaging fail to lead to a

1	of liver lesions are described in detail earlier in the section “Radiologic Evaluation of the Liver.”Liver biopsy is indicated when biochemical analysis and diagnostic imaging fail to lead to a definitive diagnosis. Percu-taneous liver biopsy with ultrasound or CT guidance is the sim-plest, fastest, and most commonly performed approach to obtain hepatic tissue for histologic examination. Absolute contraindi-cations to percutaneous liver biopsy include significant coagu-lopathy (as in patients with decompensated cirrhosis), biliary Table 31-6Classification of liver lesionsBenign Cyst Hemangioma Focal nodular hyperplasia Adenoma Biliary hamartoma AbscessMalignant Hepatocellular carcinoma Cholangiocarcinoma (bile duct cancer) Gallbladder cancer Metastatic colorectal cancer Metastatic neuroendocrine cancer (carcinoid) Other metastatic cancersdilatation, and suspicion for hemangioma or echinococcal cyst. Obesity and the presence of ascites are relative contraindica-tions that can present a

1	cancer (carcinoid) Other metastatic cancersdilatation, and suspicion for hemangioma or echinococcal cyst. Obesity and the presence of ascites are relative contraindica-tions that can present a challenge to the percutaneous approach. In these patients, laparoscopic liver biopsy can be considered. The laparoscopic technique is likely to have a higher diagnos-tic yield in cirrhotic patients with ascites and/or coagulopathy, in whom bleeding risk is excessive by the percutaneous route. Laparoscopy also offers the opportunity to stage the extent of disease in patients with various intra-abdominal malignancies.HEPATIC CYSTSCongenital CystsThe majority of hepatic cysts are asymptomatic. Hepatic cysts are usually identified incidentally and can occur at any time throughout life. The most common benign lesion found in the liver is the congenital or simple cyst. The exact prevalence of simple hepatic cysts in the U.S. population is not known, but the female to male ratio is approximately 4:1,

1	benign lesion found in the liver is the congenital or simple cyst. The exact prevalence of simple hepatic cysts in the U.S. population is not known, but the female to male ratio is approximately 4:1, and the prevalence is approximately 2.8% to 3.6%.71 Simple cysts are the result of excluded hyperplastic bile duct rests. Simple cysts usually are identified in hepatic imaging studies as thin-walled, homoge-neous, fluid-filled structures with few to no septations. The cyst epithelium is cuboidal and secretes a clear nonbilious serous fluid. With the exception of large cysts, simple cysts are usually asymptomatic. Large simple cysts may cause abdominal pain, epigastric fullness, and early satiety. Occasionally the affected patient presents with an abdominal mass.Asymptomatic simple cysts are best managed conserva-tively. The preferred treatment for symptomatic cysts is ultra-soundor CT-guided percutaneous cyst aspiration followed by sclerotherapy. This approach is approximately 90%

1	are best managed conserva-tively. The preferred treatment for symptomatic cysts is ultra-soundor CT-guided percutaneous cyst aspiration followed by sclerotherapy. This approach is approximately 90% effective in controlling symptoms and ablating the cyst cavity. If percu-taneous treatment is unavailable or ineffective, treatment may include either laparoscopic or open surgical cyst fenestration. The laparoscopic approach is being used more frequently and is 90% effective. The excised cyst wall is sent for pathologic analysis to exclude the presence of carcinoma, and the remain-ing cyst wall must be carefully inspected for evidence of neo-plastic change. If such change is present, complete resection is required, either by enucleation or formal hepatic resection.Biliary CystadenomaBiliary cystadenomas are slow-growing, unusual, benign lesions that most commonly present as large lesions in the right lobe of the liver. Although these lesions are usually benign, they can undergo malignant

1	are slow-growing, unusual, benign lesions that most commonly present as large lesions in the right lobe of the liver. Although these lesions are usually benign, they can undergo malignant transformation. Patients with biliary cystad-enomas commonly present with abdominal pain. An abdominal mass occasionally can be identified on physical examination. In contrast to simple cysts, biliary cystadenomas have walls that appear thicker with soft tissue nodules and septations that usu-ally enhance. The protein content of the fluid can be variable and can affect the radiographic images on CT and MRI. Surgical resection is the preferred mode of treatment.Polycystic Liver DiseaseAdult polycystic liver disease (PCLD) occurs as an autosomal dominant disease and usually presents in the third decade of life. Approximately 44% to 76% of affected families are found to have mutations of PKD1, and approximately 75% have muta-tions of PKD2.72 The prevalence and number of hepatic cysts

1	third decade of life. Approximately 44% to 76% of affected families are found to have mutations of PKD1, and approximately 75% have muta-tions of PKD2.72 The prevalence and number of hepatic cysts Brunicardi_Ch31_p1345-p1392.indd 137320/02/19 2:36 PM 1374SPECIFIC CONSIDERATIONSPART IIare higher in females and increase with advancing age and with increasing severity of renal cystic disease and renal dysfunc-tion. Patients with a small number of cysts or with small cysts (<2 cm) usually remain asymptomatic. In contrast, patients who develop many or large cysts, with a cyst-to-parenchymal vol-ume ratio of >1, usually develop clinical symptoms, including abdominal pain, distension, shortness of breath, and early satiety. Disease progression often results in renal failure and the need for hemodialysis. In most patients, the liver parenchymal vol-ume and synthetic function are preserved despite extensive cystic disease. Hepatic decompensation, variceal hemorrhage, ascites, and

1	for hemodialysis. In most patients, the liver parenchymal vol-ume and synthetic function are preserved despite extensive cystic disease. Hepatic decompensation, variceal hemorrhage, ascites, and encephalopathy develop rarely in patients with PCLD and only in those with massive cystic disease. The most common liver-specific complications associated with PCLD are intracystic hemorrhage, infection, and posttraumatic rup-ture. The most common abnormal biochemical test finding is a modestly elevated γ-glutamyltransferase level, and the most useful imaging tests are CT or MRI of the abdomen, which will demonstrate the characteristic polycystic appearance. Other conditions that may be associated with PCLD include cerebral aneurysm, diverticulosis, mitral valve prolapse, and inguinal hernia.The principal aim of treatment for PCLD is to ameliorate symptoms by decreasing liver volume. Medical therapy options for PCLD remain experimental at this time. Somatostatin ana-logs such as octreotide

1	principal aim of treatment for PCLD is to ameliorate symptoms by decreasing liver volume. Medical therapy options for PCLD remain experimental at this time. Somatostatin ana-logs such as octreotide and lanreotide have been shown to modestly reduce liver volume and are generally well tolerated. Sirolimus and other mammalian target of rapamycin (mTOR) inhibitors possess antiproliferative effects and thus have been postulated to slow disease progression. The effectiveness of these medical measures in relieving symptoms among patients with PCLD, however, remains to be proven.73Cyst aspiration and sclerotherapy entail puncture of a cyst with an aspiration needle followed by injection of a scleros-ing agent that causes destruction of the epithelial lining thereby inhibiting fluid production. Common agents used for sclerosis include ethanol, minocycline, and tetracycline. This technique may be considered if the patient has one or a few dominant cysts, each measuring over 5 cm. Appropriate

1	Common agents used for sclerosis include ethanol, minocycline, and tetracycline. This technique may be considered if the patient has one or a few dominant cysts, each measuring over 5 cm. Appropriate candidates for sclerotherapy can experience a complete resolution of symp-toms, but patients with numerous cysts often do not improve when this technique is used. This procedure is generally well tolerated, with the most common complication being pain from the instillation of ethanol.Cyst fenestration, or surgical unroofing of the cyst, can be performed via an open or laparoscopic approach in symptomatic patients.74 This approach allows multiple cysts to be treated dur-ing a single procedure, but carries the risk of potential surgical complications, including ascites, pleural effusion, hemorrhage, and biliary leakage. Immediate symptom relief can be achieved in up to 92% of cases, but 22% of patients eventually develop recurrence of their symptoms.Hepatic resection can be considered for

1	and biliary leakage. Immediate symptom relief can be achieved in up to 92% of cases, but 22% of patients eventually develop recurrence of their symptoms.Hepatic resection can be considered for PCLD patients with massive hepatomegaly, when fenestration alone is unlikely to significantly reduce liver volume. Appropriate candidates are those with portions of liver that harbor numerous cysts, but have at least one spared segment with predominantly normal liver parenchyma. Because the intrahepatic vascular and biliary anat-omy can be distorted by the cysts, PCLD patients undergoing hepatic resection are at increased risk for hemorrhagic and bili-ary complications. Furthermore, adhesion formation after liver resection can increase the technical complexity of future OLT. Significant symptom relief has been reported in up to 86% of PCLD patients following hepatic resection.OLT represents the only definitive therapy for patients with symptomatic PCLD. This therapeutic option is indicated in

1	has been reported in up to 86% of PCLD patients following hepatic resection.OLT represents the only definitive therapy for patients with symptomatic PCLD. This therapeutic option is indicated in patients with severely disabling symptoms that lead to a poor quality of life or in those who have developed untreatable com-plications such as portal hypertension and nutritional depriva-tion. If the patient has severe renal insufficiency from polycystic kidney disease, consideration should be given to combined liver-kidney transplantation. Because of the genetic basis of PCLD, living-donor transplantation should be considered only if the presence of PCLD in the donor can be ruled out.Caroli’s DiseaseCaroli’s disease is a syndrome of congenital ductal plate mal-formations of the intrahepatic bile ducts and is characterized by segmental cystic dilatation of the intrahepatic biliary radicals.74 Caroli’s disease also is associated with an increased incidence of biliary lithiasis, cholangitis,

1	ducts and is characterized by segmental cystic dilatation of the intrahepatic biliary radicals.74 Caroli’s disease also is associated with an increased incidence of biliary lithiasis, cholangitis, and biliary abscess formation. Caroli’s disease usually occurs in the absence of cirrhosis and is associated with cystic renal disease.75 The most common presenting symptoms include fever, chills, and abdominal pain. Most patients present by the age of 30 years, and males and females are affected equally. Rarely, patients can present later in life with complications secondary to portal hyperten-sion. Approximately 33% of affected patients develop biliary lithiasis, and 7% develop cholangiocarcinoma. The diagnosis of Caroli’s disease is made based on imaging studies. Magnetic resonance cholangiopancreatography, ERCP, and percutaneous transhepatic cholangiography provide more detailed imaging of the biliary tree and confirm communication of the intrahepatic cysts with the biliary tree, which

1	ERCP, and percutaneous transhepatic cholangiography provide more detailed imaging of the biliary tree and confirm communication of the intrahepatic cysts with the biliary tree, which is necessary to solidify the diagnosis. Treatment consists of biliary drainage, with ERCP and percutaneous transhepatic cholangiography serving as first-line therapeutic modalities. If the disease is limited to a single lobe of the liver, hepatic resection can be beneficial. Liver resec-tion can be considered in the patient with hepatic decompensa-tion or unresponsive recurrent cholangitis and possibly in the patient with a small (T1 or T2) cholangiocarcinoma.BENIGN LIVER LESIONSThe liver is an organ that is commonly involved either primarily or secondarily with vascular, metabolic, infectious, and malig-nant processes. Many classification schemes are used to help narrow the differential diagnosis of liver lesions: solid or cystic, single or multiple, cell of origin (hepatocellular, cholangiocel-lular, or

1	processes. Many classification schemes are used to help narrow the differential diagnosis of liver lesions: solid or cystic, single or multiple, cell of origin (hepatocellular, cholangiocel-lular, or mesenchymal), and benign or malignant. Benign liver lesions occur in up to 20% of the general population and are much more common than malignant tumors. The most common benign lesions are cysts, hemangiomas, focal nodular hyperplasia (FNH), and hepatocellular adenomas (see Table 31-6). Many of these lesions have typical features in imaging studies that help confirm the diagnosis.CystHepatic cysts are the most frequently encountered liver lesion overall and are described in detail in the section “Hepatic Cysts.” Cystic lesions of the liver can arise primarily (congeni-tal) or secondarily from trauma (seroma or biloma), infection (pyogenic or parasitic), or neoplastic disease. Congenital cysts are usually simple cysts containing thin serous fluid and are 6Brunicardi_Ch31_p1345-p1392.indd

1	trauma (seroma or biloma), infection (pyogenic or parasitic), or neoplastic disease. Congenital cysts are usually simple cysts containing thin serous fluid and are 6Brunicardi_Ch31_p1345-p1392.indd 137420/02/19 2:36 PM 1375LIVERCHAPTER 31reported to occur in 5% to 14% of the population, with higher prevalence in women. In most cases, congenital cysts are differ-entiated from secondary cysts (infectious or neoplastic origin) in that they have a well-defined thin wall and no solid component and are filled with homogeneous, clear fluid. For benign solid liver lesions, the differential diagnosis includes hemangioma, adenoma, FNH, and bile duct hamartoma.HemangiomaHemangiomas (also referred to as hemangiomata) are the most common solid benign masses that occur in the liver. They con-sist of large endothelial-lined vascular spaces and represent congenital vascular lesions that contain fibrous tissue and small blood vessels that eventually grow. They are predominantly seen in women and

1	of large endothelial-lined vascular spaces and represent congenital vascular lesions that contain fibrous tissue and small blood vessels that eventually grow. They are predominantly seen in women and occur in 2% to 20% of the population. They can range from small (≤1 cm) to giant cavernous hemangiomas (10 to 25 cm). Most hemangiomas are discovered incidentally with little clinical consequence. However, large lesions can cause symptoms as a result of compression of adjacent organs or intermittent thrombosis, which in turn results in further expan-sion of the lesion. Spontaneous rupture (bleeding) is rare, but surgical resection can be considered if the patient is symptom-atic. Resection can be accomplished by enucleation or formal hepatic resection, depending on the location and involvement of intrahepatic vascular structures and hepatic ducts.The majority of hemangiomas can be diagnosed by liver imaging studies. On biphasic contrast CT scan, large hemangio-mas show asymmetrical

1	of intrahepatic vascular structures and hepatic ducts.The majority of hemangiomas can be diagnosed by liver imaging studies. On biphasic contrast CT scan, large hemangio-mas show asymmetrical nodular peripheral enhancement that is isodense with large vessels and exhibit progressive centripetal enhancement fill-in over time (Fig. 31-18). On MRI, hemangio-mas are hypointense on T1-weighted images and hyperintense on T2-weighted images.76 With gadolinium enhancement, hem-angiomas show a pattern of peripheral nodular enhancement similar to that seen on contrast CT scans. Caution should be exercised in ordering a liver biopsy if the suspected diagnosis is hemangioma because of the risk of bleeding from the biopsy site, especially if the lesion is at the edge of the liver.AdenomaHepatic adenomas are benign solid neoplasms of the liver. They are most commonly seen in premenopausal women older than 30 years of age and are typically solitary, although multiple adenomas also can occur. Prior or

1	are benign solid neoplasms of the liver. They are most commonly seen in premenopausal women older than 30 years of age and are typically solitary, although multiple adenomas also can occur. Prior or current use of estrogens (oral contraceptives) is a clear risk factor for development of liver adenomas, although they can occur even in the absence of oral contraceptive use. On gross examination, they appear soft and encapsulated and are tan to light brown. Histologically, adeno-mas lack bile duct glands and Kupffer cells, have no true lobules, and contain hepatocytes that appear congested or vacuolated due to glycogen deposition. On CT scan, adenomas usually have Figure 31-18. Computed tomographic scans showing classic appearance of benign liver lesions. Focal nodular hyperplasia (FNH) is hyper-vascular on arterial phase, isodense to liver on venous phase, and has a central scar (upper panels). Adenoma is hypovascular (lower left panel). Hemangioma shows asymmetrical peripheral

1	is hyper-vascular on arterial phase, isodense to liver on venous phase, and has a central scar (upper panels). Adenoma is hypovascular (lower left panel). Hemangioma shows asymmetrical peripheral enhancement (lower right panel).Brunicardi_Ch31_p1345-p1392.indd 137520/02/19 2:36 PM 1376SPECIFIC CONSIDERATIONSPART IIsharply defined borders and can be confused with metastatic tumors. With venous phase contrast, they can look hypodense or isodense in comparison with background liver, whereas on arterial phase contrast, subtle hypervascular enhancement often is seen (see Fig. 31-18). On MRI scans, adenomas are hyper-intense on T1-weighted images and enhance early after gado-linium injection. With the use of liver-specific MRI contrast agents such as gadoxetate (Eovist or Primovist, Bayer-Schering, Berlin, Germany), hepatic adenomas can be better distinguished from FNH by their enhancement characteristics during the hepa-tobiliary phase of imaging. The new MRI contrast agent,

1	Bayer-Schering, Berlin, Germany), hepatic adenomas can be better distinguished from FNH by their enhancement characteristics during the hepa-tobiliary phase of imaging. The new MRI contrast agent, gado-benate dimeglumine (MultiHance, Bracco Diagnostics, Milan, Italy), is eliminated through both renal and biliary excretion. Therefore, liver lesions that contain hepatocytes with intact bili-ary excretion mechanism will take up this contrast agent and be easily distinguished from lesions that do not. This contrast agent has improved our ability to differentiate hepatic adenoma from FNH with a high degree of accuracy.Hepatic adenomas carry a significant risk of spontaneous rupture with intraperitoneal bleeding. The clinical presentation may be abdominal pain, and in 10% to 25% of cases, hepatic adenomas present with spontaneous intraperitoneal hemorrhage. Hepatic adenomas also have a risk of malignant transformation to a well-differentiated HCC. Therefore, it usually is recom-mended that

1	adenomas present with spontaneous intraperitoneal hemorrhage. Hepatic adenomas also have a risk of malignant transformation to a well-differentiated HCC. Therefore, it usually is recom-mended that large hepatic adenomas (>4–5 cm) be surgically resected.Focal Nodular HyperplasiaFNH is a solid, benign lesion of the liver believed to be a hyper-plastic response to an anomalous artery. Similar to adenomas, they are more common in women of childbearing age, although the link to oral contraceptive use is not as clear as with adeno-mas. A good-quality biphasic CT scan usually is diagnostic of FNH, on which such lesions appear well circumscribed with a typical central scar (see Fig. 31-18). They show intense homo-geneous enhancement on arterial phase contrast images and are often isodense or invisible compared with background liver on the venous phase. On MRI scans, FNH lesions are hypoin-tense on T1-weighted images and isointense to hyperintense on T2-weighted images. After gadolinium

1	invisible compared with background liver on the venous phase. On MRI scans, FNH lesions are hypoin-tense on T1-weighted images and isointense to hyperintense on T2-weighted images. After gadolinium administration, lesions are hyperintense but become isointense on delayed images. The fibrous septa extending from the central scar are also more readily seen with MRI. Unlike adenomas, FNH lesions usually do not rupture spontaneously and have no significant risk of malignant transformation. Therefore, the management of FNH is usually reassurance and prospective observation irrespective of size. Surgical resection can be recommended, however, when patients are symptomatic or when hepatic adenoma or HCC can-not be definitively excluded. Oral contraceptive or estrogen use should be stopped when either FNH or adenoma is diagnosed.Bile Duct HamartomaBile duct hamartomas are typically small liver lesions, 2 to 4 mm in size, visualized on the surface of the liver at laparotomy. They are firm,

1	either FNH or adenoma is diagnosed.Bile Duct HamartomaBile duct hamartomas are typically small liver lesions, 2 to 4 mm in size, visualized on the surface of the liver at laparotomy. They are firm, smooth, and whitish yellow in appearance. They can be difficult to differentiate from small metastatic lesions, and excisional biopsy often is required to establish the diagnosis.MALIGNANT LIVER TUMORSMalignant tumors in the liver can be classified as primary (cancers that originate in the liver) or metastatic (cancers that spread to the liver from an extrahepatic primary site) (see Table 31-6). Primary cancers in the liver that originate from hepato-cytes are known as hepatocellular carcinomas (HCCs or hepa-tomas), whereas cancers arising in the bile ducts are known as cholangiocarcinomas.In the United States, approximately 150,000 new cases of colorectal cancer are diagnosed each year, and the majority of patients (approximately 60%) will develop hepatic metas-tases over their lifetime.

1	the United States, approximately 150,000 new cases of colorectal cancer are diagnosed each year, and the majority of patients (approximately 60%) will develop hepatic metas-tases over their lifetime. Hence, the most common tumor seen in the liver is metastatic colorectal cancer. This compares with approximately 30,000 new cases of HCC diagnosed annually in the United States. Interestingly, in a Western series of 1000 con-secutive new liver cancer patients seen at a university medical center, 47% had HCC, 17% had colorectal cancer metastases, 11% had cholangiocarcinomas, 7% had neuroendocrine metas-tases, and 18% had other tumors.77 Although these figures do not reflect the incidence or prevalence of these liver cancers, they are indicative of referral patterns in a tertiary academic medical center with a large liver transplantation team and active hepatology clinic.Hepatocellular CarcinomaHCC is the fifth most common malignancy worldwide, with an estimated 750,000 new cases diagnosed

1	center with a large liver transplantation team and active hepatology clinic.Hepatocellular CarcinomaHCC is the fifth most common malignancy worldwide, with an estimated 750,000 new cases diagnosed annually. Because of its high fatality, it is the third most common cause of cancer death worldwide.78 Major risk factors are viral hepatitis (B or C), alcoholic cirrhosis, hemochromatosis, and NASH. In Asia, the risk is as high as 35 to 117 per 100,000 persons per year, whereas in the United States, the risk is only 7 per 100,000 per-sons per year.78 Although cirrhosis is not present in all cases, it has been estimated to be present 70% to 90% of the time. In a person with cirrhosis, the annual conversion rate to HCC is 2% to 6%.79 In patients with chronic HCV infection, cirrhosis usually is present before the HCC develops; however, in cases of hepatitis B virus infection, HCC tumors can occur before the onset of cirrhosis. HCCs are typically hypervascular with blood supplied predominantly

1	before the HCC develops; however, in cases of hepatitis B virus infection, HCC tumors can occur before the onset of cirrhosis. HCCs are typically hypervascular with blood supplied predominantly from the hepatic artery. Thus, the lesion often appears hypervascular during the arterial phase of CT studies (Fig. 31-19) and relatively hypodense during the delayed phases due to early washout of the contrast medium by the arterial blood. MRI imaging also is effective in character-izing HCC. HCC is variable on T1-weighted images and usu-ally hyperintense on T2-weighted images. As with contrast CT, HCC enhances in the arterial phase after gadolinium injection because of its hypervascularity and becomes hypointense in the delayed phases due to contrast washout. HCC has a tendency to invade the portal vein, and the presence of an enhancing portal vein thrombus is highly suggestive of HCC.The treatment of HCC is complex and is best managed by a multidisciplinary liver transplant team. A complete

1	vein, and the presence of an enhancing portal vein thrombus is highly suggestive of HCC.The treatment of HCC is complex and is best managed by a multidisciplinary liver transplant team. A complete algorithm for the evaluation and management of HCC is shown in Fig. 31-20. For patients without cirrhosis who develop HCC, resection is the treatment of choice. For patients with Child’s class A cirrhosis with preserved liver function and no portal hypertension, resection also is considered. If resection is not possible because of poor liver function and the HCC meets transplant criteria (discussed later), liver transplantation is the treatment of choice.80,81The Barcelona-Clinic Liver Cancer Group has refined its HCC management strategy and has developed the American Association for the Study of Liver Diseases Practice Guidelines.82 Management guidelines vary slightly in Asia, Europe, the 7Brunicardi_Ch31_p1345-p1392.indd 137620/02/19 2:36 PM 1377LIVERCHAPTER 31United States, and

1	of Liver Diseases Practice Guidelines.82 Management guidelines vary slightly in Asia, Europe, the 7Brunicardi_Ch31_p1345-p1392.indd 137620/02/19 2:36 PM 1377LIVERCHAPTER 31United States, and other countries based in part on availability of organ donors for liver transplantation. Living donor liver trans-plantation also is an alternative for patients with HCC awaiting transplantation to avoid dropout as a candidate for cadaveric donor liver transplantation due to tumor progression.81 Specific treatment options are described in the next section.CholangiocarcinomaCholangiocarcinoma, or bile duct cancer, is the second most common primary malignancy of the liver. Cholangiocarcinoma is an adenocarcinoma of the bile ducts; it forms in the biliary epithelial cells and can be subclassified into peripheral (intra-hepatic) bile duct cancer and central (extrahepatic) bile duct cancer. Extrahepatic bile duct cancer can be located distally or proximally. When proximal, it is referred to as a

1	peripheral (intra-hepatic) bile duct cancer and central (extrahepatic) bile duct cancer. Extrahepatic bile duct cancer can be located distally or proximally. When proximal, it is referred to as a hilar chol-angiocarcinoma (Klatskin’s tumor). Hilar cholangiocarcinoma originates in the wall of the bile duct at the hepatic duct conflu-ence and usually presents with obstructive jaundice rather than an actual liver mass. In contrast, a peripheral (or intrahepatic) cholangiocarcinoma represents a tumor mass within a hepatic lobe or at the periphery of the liver. A biopsy specimen from the cholangiocarcinoma will show adenocarcinoma, but patholo-gists are often unable to differentiate metastatic adenocarcinoma to the liver from primary bile duct adenocarcinoma. Therefore, a search for a primary site should be undertaken in cases in which an incidentally discovered liver lesion is proven to be an adenocarcinoma on biopsy.Hilar cholangiocarcinoma is difficult to diagnose and typi-cally

1	site should be undertaken in cases in which an incidentally discovered liver lesion is proven to be an adenocarcinoma on biopsy.Hilar cholangiocarcinoma is difficult to diagnose and typi-cally presents as a stricture of the proximal hepatic duct causing painless jaundice. It preferentially grows along the length of the bile ducts, often involving the periductal lymphatics with frequent lymph node metastases. Surgical resection offers the only chance for cure of cholangiocarcinoma.83 The location and extent of tumor dictate the operative approach. In one series of 225 patients with hilar cholangiocarcinoma, 65 (29%) were deemed to have unresectable tumors by initial imaging.84 Of the remaining 160 patients who underwent exploratory surgery with curative intent, 80 (50%) were found to have inoperable tumors. Histologically negative margins, concomitant hepatic resection, and well-differentiated tumor histology were associ-ated with improved outcome after resection. In another series of

1	inoperable tumors. Histologically negative margins, concomitant hepatic resection, and well-differentiated tumor histology were associ-ated with improved outcome after resection. In another series of 61 patients undergoing surgical exploration for hilar cholangio-carcinoma, the 5-year actuarial survival rates for an R0 or R1 resection were 45% and 26%, respectively.85 In a large series reported by Nagino and colleagues, 132 patients with hilar chol-angiocarcinoma underwent extended hepatectomy with resec-tion of the caudate lobe and extrahepatic bile duct, and/or portal vein resection (n = 63) after portal vein embolization.86 The 3and 5-year survival rates were 41.7% and 26.8%, respectively.In the absence of associated primary sclerosing cholangi-tis (PSC), surgical resection is the treatment of choice for hilar cholangiocarcinoma. However, approximately 10% of patients with cholangiocarcinoma have PSC.87 Furthermore, cholangio-carcinoma in the setting of PSC is frequently

1	the treatment of choice for hilar cholangiocarcinoma. However, approximately 10% of patients with cholangiocarcinoma have PSC.87 Furthermore, cholangio-carcinoma in the setting of PSC is frequently multicentric and often is associated with underlying liver disease, with eventual cirrhosis and portal hypertension. As a result, experience has shown that resection of cholangiocarcinoma in patients with PSC yields dismal results. This led transplant centers to con-sider OLT for patients with hilar cholangiocarcinoma. The ini-tial results of transplantation were disappointing, however, with high recurrence and overall 3-year survival rates of <30%.88Because the growth of hilar cholangiocarcinoma indicates that this disease spreads in a locoregional manner, a rationale for the use of neoadjuvant chemoradiation was developed by the transplant team at the University of Nebraska in the late 1980s. This was adapted in 1993 by the transplant team at the Mayo Clinic, which led to the current Mayo

1	chemoradiation was developed by the transplant team at the University of Nebraska in the late 1980s. This was adapted in 1993 by the transplant team at the Mayo Clinic, which led to the current Mayo Clinic protocol.89 The pretransplant Mayo protocol consists of external-beam radiation therapy plus a protracted course of intravenous 5-fluorouracil followed by iridium-192 brachytherapy.90 Patients then undergo an abdominal exploration with staging. If findings are negative, patients are given capecitabine for 2 of every 3 weeks until transplantation. Even after restaging with CT/MRI and endo-scopic ultrasonography, approximately 15% to 20% of patients will have positive findings for tumor on abdominal Figure 31-19. Computed tomographic (CT) images of hepato-cellular carcinoma (HCC) and peripheral cholangiocarcinoma. CT scans reveal a large (upper panel) and small (middle panel) hypervascular HCC. A hypovascular left lobe peripheral cholangio-carcinoma (CholangioCA) is also shown (lower

1	cholangiocarcinoma. CT scans reveal a large (upper panel) and small (middle panel) hypervascular HCC. A hypovascular left lobe peripheral cholangio-carcinoma (CholangioCA) is also shown (lower panel).Brunicardi_Ch31_p1345-p1392.indd 137720/02/19 2:36 PM 1378SPECIFIC CONSIDERATIONSPART IIexploration.87,90 The 5-year survival rate for those undergoing transplantation for cholangiocarcinoma at the Mayo Clinic is approximately 70% and compares favorably with the rate for resection.87,90 Current eligibility criteria for this Mayo Clinic protocol include unresectable hilar cholangiocarcinoma or hilar cholangiocarcinoma with PSC. The tumor must have a radial dimension of ≤3 cm with no intrahepatic or extrahepatic metastases, and the patient must not have undergone prior radiation therapy or transperitoneal biopsy.90 Many centers have adopted similar protocols with comparable results.91Peripheral, or intrahepatic, cholangiocarcinoma is less common than hilar cholangiocarcinoma. In a

1	or transperitoneal biopsy.90 Many centers have adopted similar protocols with comparable results.91Peripheral, or intrahepatic, cholangiocarcinoma is less common than hilar cholangiocarcinoma. In a series of 53 patients at Memorial Sloan-Kettering Cancer Center who underwent surgical exploration for a diagnosis of intrahepatic cholangiocarcinoma, 33 (62%) were found to have resectable tumors.92 Actuarial 3-year survival for patients undergoing resection was 55%. Factors predictive of poor survival included vascular invasion, histologically positive margins, and multiple tumors. In a large series in Taiwan, 373 patients with peripheral cholangiocarcinoma underwent surgical treatment from 1977 to 2001. Absence of mucobilia, nonpapillary tumor type, tumor of advanced stage, nonhepatectomy, and lack of postoperative chemotherapy were five independent prognostic factors that adversely affected overall survival.93 Liver transplantation has been performed for peripheral

1	nonhepatectomy, and lack of postoperative chemotherapy were five independent prognostic factors that adversely affected overall survival.93 Liver transplantation has been performed for peripheral cholangiocarcinoma94; however, currently all but one center in the United States have eschewed this approach because of organ shortages and relatively high recurrence rates.Gallbladder CancerGallbladder cancer is a rare aggressive tumor with a very poor prognosis. Over 90% of patients have associated cholelithia-sis. In one study examining the mode of presentation over a 10-year period from 1990 to 2000 in 44 patients diagnosed with gallbladder cancer, the diagnosis was found to be made preoperatively in 57%, intraoperatively in 11%, and inciden-tally after cholecystectomy in 32%.95 Surgical approaches can be classified into (a) reoperation for an incidental finding of gallbladder cancer after cholecystectomy, and (b) radical resec-tion in patients with advanced disease. The results are

1	approaches can be classified into (a) reoperation for an incidental finding of gallbladder cancer after cholecystectomy, and (b) radical resec-tion in patients with advanced disease. The results are dismal for radical resection in patients with advanced disease and positive hilar lymph nodes.96,97 For incidental gallbladder can-cer beyond stage T1, reoperation with central liver resection, hilar lymphadenectomy, and evaluation of cystic duct stump is most commonly performed.98,99 The role of formal lobectomy or extended lobectomy as well as common bile duct resection is more controversial. In a single-center study of 23 patients undergoing attempted curative treatment by surgical resec-tion, survival was 85% at 1 year, 63% at 2 years, and 55% at 3 years.99 In a multicenter study encompassing 115 patients with incidentally discovered gallbladder cancer who underwent re-resection,98 residual disease in the liver was identified in 46% of patients (0% of those with stage T1 disease, 10%

1	115 patients with incidentally discovered gallbladder cancer who underwent re-resection,98 residual disease in the liver was identified in 46% of patients (0% of those with stage T1 disease, 10% of those with T2 tumors, and 36% of those with T3 disease). T stage also was associated with the risk of metastasis to locoregional lymph nodes (lymph node metastasis for T1 of 13%; for T2, 31%; and for T3, 46%). In another study, a German registry of incidental gallbladder cancer identified 439 patients. Patients with tumors staged as T2 or T3 after cholecystectomy had better survival if they underwent reoperation than if they were managed with observation.100 Hence, reoperation should be considered for all patients who have T2 or T3 tumors or for whom the accuracy of staging is in question.Metastatic Colorectal CancerOver 50% to 60% of patients diagnosed with colorectal cancer will develop hepatic metastases during their lifetime. Resection for hepatic metastases has been a routine part of

1	Colorectal CancerOver 50% to 60% of patients diagnosed with colorectal cancer will develop hepatic metastases during their lifetime. Resection for hepatic metastases has been a routine part of treatment for 8ResectionNoncirrhotic/Child’s ASingle lesionNo metastasisOLTx evaluation1 lesion ˜5 cm3 lesions ˜3 cmChild’s A/B/CNo gross vasc. invasionNo metastasisNot Tx candidateComorbid factors°4 lesionsGross vasc. invasionLN (+) or metastasisLDLT ?Suitable donorPalliative careChild’s CBili °3 HCC IdentifiedResection candidate?Transplant candidate?YesYesNoSorafenibYesYesNoOLTxClinicaltrialsNeoadjuvant therapyRFA/TACE/90 YttriumUNOS list (cadaver)MELD score (? >3 mos)Perc/lap. RFASingle lesion<5 cmChild’s A/BTace/90 YttriumMulti-focal >5 cmChild’s A/B/CBili <3NewagentsFigure 31-20. Algorithm for the management of hepatocellular carcinoma (HCC). The treatment algorithm for HCC begins with determining whether the patient is a resection candidate or liver transplant candidate. Bili = bilirubin

1	the management of hepatocellular carcinoma (HCC). The treatment algorithm for HCC begins with determining whether the patient is a resection candidate or liver transplant candidate. Bili = bilirubin level (in milligrams per deciliter); Child’s = Child-Turcotte-Pugh class; lap = laparoscopic; LDLT = living-donor liver transplantation; LN = lymph node; MELD = Model for End-Stage Liver Disease; OLTx = orthotopic liver transplantation; Perc = percutaneous; RFA = radiofrequency ablation; TACE = transarterial chemoemboli-zation; Tx = transplantation; UNOS = United Network for Organ Sharing; vasc. = vascular.Brunicardi_Ch31_p1345-p1392.indd 137820/02/19 2:36 PM 1379LIVERCHAPTER 31colorectal cancer since the publication of a large single-center experience demonstrating its safety and efficacy.101 Predictors of poor outcome in that study included node-positive primary, disease-free interval <12 months, more than one tumor, tumor size >5 cm, and carcinoembryonic antigen level >200 ng/mL.

1	Predictors of poor outcome in that study included node-positive primary, disease-free interval <12 months, more than one tumor, tumor size >5 cm, and carcinoembryonic antigen level >200 ng/mL. Traditional teaching suggested that hepatic resection for meta-static colorectal cancer to the liver, if technically feasible, should be performed only for fewer than four metastases.102 However, later studies challenged this paradigm. In a series of 235 patients who underwent hepatic resection for metastatic colorectal cancer, the 10-year survival rate of patients with four or more nodules was 29%, nearly comparable to the 32% sur-vival rate of patients with only a solitary tumor metastasis.103 In the Memorial Sloan-Kettering Cancer Center series of 98 patients with four or more colorectal hepatic metastases who underwent resection between 1998 and 2002, the 5-year actu-arial survival was 33%.104 Furthermore, improved chemothera-peutic regimens and surgical techniques have produced aggressive

1	metastases who underwent resection between 1998 and 2002, the 5-year actu-arial survival was 33%.104 Furthermore, improved chemothera-peutic regimens and surgical techniques have produced aggressive strategies for the management of this disease. Many groups now consider volume of future liver remnant and the health of the background liver, and not actual tumor number, as the primary determinants in selection for an operative approach.105,106 Hence, resectability is no longer defined by what is actually removed, but indications for hepatic resection now center on what will remain after resection.107 Use of neo-adjuvant chemotherapy, portal vein embolization, two-stage hepatectomy, simultaneous ablation, and resection of extrahepatic tumor in select patients have increased the number of patients eligible for a surgical approach.108,109Neuroendocrine TumorsHepatic metastases from neuroendocrine tumors have a protracted natural history and commonly are associated with debilitating

1	of patients eligible for a surgical approach.108,109Neuroendocrine TumorsHepatic metastases from neuroendocrine tumors have a protracted natural history and commonly are associated with debilitating endocrinopathies. Several groups have advocated an aggressive surgical approach of debulking surgery, both to control symp-toms and to extend survival.110,111 In a series of 170 patients undergoing resection of hepatic metastases from neuroendocrine tumors between 1977 and 1998 at the Mayo Clinic, overall sur-vival was 61% and 35% at 5 and 10 years, respectively.112 There was no difference in survival between patients with carcinoid tumors and those with islet cell tumors. Major hepatectomy was performed in 91 patients (54%), and recurrence rate was 84% at 5 years. Belghiti’s group has described a two-stage strategy used in 41 patients with a primary neuroendocrine tumor and synchronous bilobar liver metastases.113 In the first stage, the primary tumor is resected and limited resection of

1	a two-stage strategy used in 41 patients with a primary neuroendocrine tumor and synchronous bilobar liver metastases.113 In the first stage, the primary tumor is resected and limited resection of metastases in the left hemiliver, combined with right portal vein ligation, is performed. After 8 weeks of hypertrophy, a right hepatec-tomy or extended right hepatectomy (also referred to as a right trisectionectomy; resection of Couinaud’s segments IV, V, VI, VII, and VIII of the liver) is performed.113 In patients treated using this strategy, the 2-, 5-, and 8-year Kaplan-Meier over-all survival rates were 94%, 94%, and 79%, respectively, and disease-free survival rates were 85%, 50%, and 26%, respec-tively. Because systemic therapy has had little success in the treatment of advanced tumors, a broader approach using multi-modal therapy has been used to increase survival and improve hormone-related symptoms. These therapies include radiofre-quency or microwave ablation and intra-arterial

1	a broader approach using multi-modal therapy has been used to increase survival and improve hormone-related symptoms. These therapies include radiofre-quency or microwave ablation and intra-arterial therapy with chemoembolization or radioembolization (yttrium-90). Some centers perform liver transplantation for selected patients (carci-noid histology; primary tumor removed with curative resection; primary tumor drained by portal system; ≤50% hepatic paren-chyma involved; good response or stable disease for at least 6 months during pretransplantation period; and age 55 years or younger), although this is not routine.114Other Metastatic TumorsNearly every cancer has the propensity to metastasize to the liver. Historically, enthusiasm was low for resecting metastases other than those from a colorectal cancer primary. This was due in part to the recognition that many other primary cancers (such as breast cancer) represent a systemic disease when liver metas-tases are present. However, more

1	cancer primary. This was due in part to the recognition that many other primary cancers (such as breast cancer) represent a systemic disease when liver metas-tases are present. However, more recent studies have shown acceptable 5-year survival rates in the 20% to 40% range for resection of hepatic metastases from breast, renal, and other GI tumors.115,116 In a large study of hepatic resection for non-colorectal, nonendocrine liver metastases in 1452 patients, neg-ative prognostic factors were nonbreast origin, age >60 years, disease-free interval of <12 months, need for major hepatec-tomy, performance of R2 resection, and presence of extrahe-patic metastases.115TREATMENT OPTIONS FOR LIVER CANCERIn general, the major treatment options for liver cancer can be categorized as shown in Table 31-7. The decision making for any given patient is complex and is best managed by a multidis-ciplinary liver tumor board. The treatments listed in Table 31-7 are not mutually exclusive; the important

1	31-7. The decision making for any given patient is complex and is best managed by a multidis-ciplinary liver tumor board. The treatments listed in Table 31-7 are not mutually exclusive; the important point is to select the most appropriate initial treatment after a complete evaluation. In general, surveillance imaging (CT or MRI) is performed every 3 to 4 months during the first year after diagnosis to observe for response, progression, or recurrence. The treatment plan is indi-vidualized and modified according to the response of the patient.Hepatic ResectionFor primary liver cancers or hepatic metastases, hepatic resection is the gold standard and treatment of choice. Although there are anecdotal reports of long-term survival after ablation and other regional liver therapies, liver resection remains the only real option for cure. For HCC in the setting of cirrhosis, liver trans-plantation also offers the potential for long-term survival, albeit with the consequences of

1	liver resection remains the only real option for cure. For HCC in the setting of cirrhosis, liver trans-plantation also offers the potential for long-term survival, albeit with the consequences of immunosuppression. Hepatic resection 9Table 31-7Treatment options for liver cancerHepatic resectionLiver transplantationAblation techniquesRadiofrequency ablation• Ethanol ablation• Cryoablation• Microwave ablationRegional liver therapies• Chemoembolization/embolization• Hepatic artery pump chemoperfusion• Internal radiation therapy (yttrium-90 internal radiation)External-beam radiation therapy• Stereotactic radiosurgery (CyberKnife, Trilogy, Synergy)• Intensity-modulated radiation therapySystemic chemotherapyMultimodality approachBrunicardi_Ch31_p1345-p1392.indd 137920/02/19 2:36 PM 1380SPECIFIC CONSIDERATIONSPART IIalso has been advocated for HCC in select patients with cirrho-sis before secondary liver transplantation, although not without some controversy.117 Many large series of

1	CONSIDERATIONSPART IIalso has been advocated for HCC in select patients with cirrho-sis before secondary liver transplantation, although not without some controversy.117 Many large series of patients undergoing major hepatectomy now report mortality rates of <5%.118-121 Pre-viously, a 1-cm tumor margin was considered desirable; how-ever, recent studies have reported comparable survival rates with smaller margins.122-124 Technical innovation in liver surgery and a better understanding of perioperative care have even allowed surgeons to perform resections in cases with IVC involvement with extracorporeal liver surgery.125 The technical aspects of anatomic hepatic lobectomies are described later.Liver TransplantationThe rationale supporting OLT for HCC includes the fact that most HCCs (>80%) arise in the setting of cirrhosis.88,126 The cirrhotic liver often does not have enough reserve to tolerate a formal resection. Also, HCC tumors are commonly multifo-cal and are underestimated by

1	arise in the setting of cirrhosis.88,126 The cirrhotic liver often does not have enough reserve to tolerate a formal resection. Also, HCC tumors are commonly multifo-cal and are underestimated by current CT or MRI imaging.127 Furthermore, recurrence rates are high at 5 years after resec-tion (>50%). Hence, OLT is an appealing treatment because it removes both the cancer and the cirrhotic liver that leads to can-cer. More than 7000 liver transplantations are performed each year in the United States, with 1-year survival rates approaching 90%. In May 2017, approximately 14,463 patients were on the waiting list for liver transplantation.128Initial series of OLT for HCC reported in the 1990s included advanced cases of HCC, and the 5-year survival rates were only 20% to 50%.88 This compared poorly with overall 5-year survival rates of 70% to 75% for OLT in the Organ Pro-curement and Transplantation Network/United Network for Organ Sharing (OPTN/UNOS) database. Mazzaferro and col-leagues at

1	with overall 5-year survival rates of 70% to 75% for OLT in the Organ Pro-curement and Transplantation Network/United Network for Organ Sharing (OPTN/UNOS) database. Mazzaferro and col-leagues at Milan subsequently showed that survival rates were markedly improved when OLT was limited to patients with early-stage HCC (stage I or stage II) with one tumor ≤5 cm, or up to three tumors no larger than 3 cm, along with the absence of gross vascular invasion or extrahepatic spread.129 Multiple studies have validated these findings, and many groups have proposed an expansion of the Milan criteria.81As noted previously, the 6to 40-point MELD score was adopted by OPTN/UNOS in 2002 for allocation of deceased donor liver organs in the United States. In an attempt to pri-oritize patients with preserved liver function and progressive HCC, patients with stage II HCC are allocated exception points (currently 28 MELD points which activates 6 months after liver transplant listing, increasing every 3

1	liver function and progressive HCC, patients with stage II HCC are allocated exception points (currently 28 MELD points which activates 6 months after liver transplant listing, increasing every 3 months and capping at 34 points as long as they continue to meet transplant criteria). This allocation has had a positive effect for HCC liver transplant can-didates, leading to decreased waiting list dropout and increased transplant rates with excellent long-term outcomes.130 The goal is to better equate death rates on the liver transplant waiting list for patients with stage I or stage II HCC with rates for patients with chronic liver disease without HCC. Although indications for liver transplantation have increased, the supply of donor liv-ers has failed to keep pace with the numbers of potential recipi-ents. A partial solution has been the use of living donor grafts. This is especially true in Asia where the incidence of HCC is high and the rate of cadaveric donation is low. Living donor

1	recipi-ents. A partial solution has been the use of living donor grafts. This is especially true in Asia where the incidence of HCC is high and the rate of cadaveric donation is low. Living donor grafts include right and left lobes, as well as dual grafts from separate donors to provide adequate hepatic mass to the recipi-ent. The use of living donor grafts also allows for transplant programs to push the boundaries by accepting patients beyond the Milan criteria with good results.112Radiofrequency AblationIn 1891, d’Arsonval discovered that radiofrequency (RF) waves delivered as an alternating electric current (>10 kHz) could pass through living tissue without causing pain or neuromuscular excitation. The resistance of the tissue to the rapidly alternating current produced heat. This discovery contributed to the devel-opment of the surgical application of electrocautery. In 1908, Beer used RF coagulation to destroy urinary bladder tumors. Cushing and Bovie later applied RF ablation to

1	contributed to the devel-opment of the surgical application of electrocautery. In 1908, Beer used RF coagulation to destroy urinary bladder tumors. Cushing and Bovie later applied RF ablation to intracranial tumors. In 1961, Lounsberry studied the histologic changes of the liver after RF ablation (RFA) in animal models. He found that RF caused local tissue destruction with uniform necrosis. In the early 1990s, two groups proposed that RFA can be an effective method for destroying unresectable malignant liver tumors.132,133 Both groups found that RFA produced lesions with well-demarcated areas of necrosis without viable tumor cells present. Clinical reports after short-term follow-up sug-gested that RFA was safe and effective in the treatment of liver tumors.134-136 However, Abdalla and colleagues exam-ined data for 358 consecutive patients with colorectal liver metastases treated with curative intent over a 10-year period (1992 to 2002).137 Liver-only recurrence after RFA was four

1	colleagues exam-ined data for 358 consecutive patients with colorectal liver metastases treated with curative intent over a 10-year period (1992 to 2002).137 Liver-only recurrence after RFA was four times the rate after resection (44% vs. 11% of patients), and RFA alone or in combination with resection did not provide survival rates comparable to those with resection alone. None-theless, RFA remains a common procedure that can be per-formed by a percutaneous, minimally invasive laparoscopic, or open approach.138,139 It also has been used successfully to ablate small HCCs as a bridge to liver transplantation.140 Results were reported for the first randomized clinical trial involving RFA treatment for HCC in 291 Chinese patients with three or fewer HCC tumors ranging in size from 3 to 7.5 cm.141 Patients were randomly assigned to treatment arms of RFA alone (n = 100), transarterial chemoembolization (TACE) alone (n = 95), or combined TACE plus RFA (n = 96). At a median follow-up of 28.5

1	Patients were randomly assigned to treatment arms of RFA alone (n = 100), transarterial chemoembolization (TACE) alone (n = 95), or combined TACE plus RFA (n = 96). At a median follow-up of 28.5 months, median survival was 22 months in the RFA group, 24 months in the TACE group, and 37 months in the TACE plus RFA group. Patients treated with TACE plus RFA had sig-nificantly better overall survival than those treated with TACE alone (P <.001) or RFA alone (P <.001). Sucandy and col-leagues examined long-term 5and 10-year overall survival in 320 patients that had hepatic RFA for HCC or colorectal cancer liver metastases (CLM).142 The majority of patients (71%) had a single tumor ablation. Minimum 5-year follow-up was available in 89% patients, with a median follow-up of 115.3 months. In the HCC group, the 5and 10-year overall survivals were 38.5% and 23.4%, respectively, while in the CLM group, the 5and 10-year overall survivals were 27.6% and 15%, respectively.Ethanol Ablation,

1	In the HCC group, the 5and 10-year overall survivals were 38.5% and 23.4%, respectively, while in the CLM group, the 5and 10-year overall survivals were 27.6% and 15%, respectively.Ethanol Ablation, Cryosurgery, and Microwave AblationPercutaneous ethanol injection has been shown to be a safe and effective treatment for small HCCs.136 The ethanol usually is delivered by percutaneous injection under ultrasound or CT guidance. Percutaneous ethanol injection also is used to treat small HCC tumors as a bridge to liver transplantation in some centers to avoid patient dropout.80 Although cryosurgery was used in the late 1980s and 1990s for ablation of liver tumors, many have abandoned this approach in favor of RFA because of the latter’s fewer side effects and ease of use. Microwave ablation is a thermal ablative technique used in the management of unresectable liver tumors to produce a coagulation necrosis. Brunicardi_Ch31_p1345-p1392.indd 138020/02/19 2:36 PM 1381LIVERCHAPTER 31In a

1	a thermal ablative technique used in the management of unresectable liver tumors to produce a coagulation necrosis. Brunicardi_Ch31_p1345-p1392.indd 138020/02/19 2:36 PM 1381LIVERCHAPTER 31In a multicenter phase 2 U.S. trial using a 915-MHz micro-wave generator, 87 patients underwent 94 ablation procedures for 224 hepatic tumors.143 Forty-five percent of the procedures were performed using an open approach, 7% laparoscopically, and 48% percutaneously. The average tumor size was 3.6 cm (range, 0.5 to 9.0 cm). At a mean follow-up of 19 months, 47% of the patients were alive with no evidence of disease. Local recurrence at the ablation site occurred in 2.7% of tumors, and regional recurrence occurred in 43% of patients. There were no procedure-related deaths. Further studies are required to define the role of this technology in relation to the other abla-tion options available.Chemoembolization and Hepatic Artery Pump ChemoperfusionChemoembolization is the process of injecting

1	to define the role of this technology in relation to the other abla-tion options available.Chemoembolization and Hepatic Artery Pump ChemoperfusionChemoembolization is the process of injecting chemotherapeu-tic drugs combined with embolization particles into the hepatic artery that supplies the liver tumor using a percutaneous, trans-femoral approach. It is most commonly used for treatment of unresectable HCC. Three randomized trials and a meta-analysis have shown a survival benefit with chemoembolization.144-147 In a study by Lo and colleagues, 80 Asian patients were randomly assigned to receive either chemoembolization with cisplatin in lipiodol or symptomatic treatment only.144 Chemoembolization resulted in a marked tumor response, and the actuarial survival was significantly better in the chemoembolization group (1and 3-year survival of 57% and 26%, respectively) than in the con-trol group (1and 3-year survival of 32% and 3%, respectively). In another randomized trial, a

1	better in the chemoembolization group (1and 3-year survival of 57% and 26%, respectively) than in the con-trol group (1and 3-year survival of 32% and 3%, respectively). In another randomized trial, a Barcelona group compared chemoembolization with doxorubicin versus supportive care and showed that chemoembolization significantly improved survival.145 Finally, in a large prospective cohort study of 8510 patients with unresectable HCC in Japan who received trans-catheter arterial lipiodol chemoembolization, the 5-year survival rate was 26% and median survival time was 34 months.146 The TACE-related mortality rate after the initial therapy was 0.5%. Complications of TACE include liver dysfunction or liver fail-ure, hepatic abscess, and hepatic artery thrombosis. Multiple studies also have shown promising results for chemoemboliza-tion with drug-eluting beads in treatment of HCC.148In the 1990s, hepatic artery pump chemoperfusion with floxuridine for colorectal cancer metastases to the

1	shown promising results for chemoemboliza-tion with drug-eluting beads in treatment of HCC.148In the 1990s, hepatic artery pump chemoperfusion with floxuridine for colorectal cancer metastases to the liver was used both for treatment of inoperable disease and in the adjuvant setting.149 However, in the modern era of improved chemothera-peutic options, this treatment modality is seldom used outside of a clinical trial.Yttrium-90 MicrospheresSelective internal radioembolization or transarterial radioem-bolization (TARE) is a promising new treatment modality for patients with inoperable primary or metastatic liver tumors. The treatment is a minimally invasive transcatheter therapy in which radioactive microspheres are infused into the hepatic arteries via a transfemoral percutaneous approach. The yttrium-90 micro-spheres are directly injected into the hepatic artery branches that supply the tumor. Once infused, the microspheres deliver doses of high-energy, low-penetration radiation

1	The yttrium-90 micro-spheres are directly injected into the hepatic artery branches that supply the tumor. Once infused, the microspheres deliver doses of high-energy, low-penetration radiation selectively to the tumor. The main indications are inoperable HCC150 and colorec-tal cancer hepatic metastases for which systemic chemotherapy has failed.151,152 In a study involving 137 patients with unresect-able chemorefractory liver metastases treated with radioembo-lization, there was a response rate of 42.8% (2.1% complete response, 40.7% partial response) according to World Health Organization criteria.152 One-year survival rate was 47.8%, and 2-year survival rate was 30.9%. Median survival was 457 days for patients with colorectal tumor metastases, 776 days for those with neuroendocrine tumor metastases, and 207 days for those with noncolorectal, nonneuroendocrine tumor metastases. The two products available in the United States are SIR-Spheres (Sirtex, Sydney, Australia) and

1	tumor metastases, and 207 days for those with noncolorectal, nonneuroendocrine tumor metastases. The two products available in the United States are SIR-Spheres (Sirtex, Sydney, Australia) and TheraSphere (Nordian, Ottawa, Canada).Stereotactic Radiosurgery and Intensity-Modulated Radiation TherapyAlthough stereotactic radiosurgery (with CyberKnife and other systems) is in widespread use for brain and spinal tumors, body application to HCC or metastatic liver tumors has only recently occurred. In a phase 1 study, 31 patients with unresectable HCCs and 10 with unresectable cholangiocarcinomas completed a six-fraction course of stereotactic body radiotherapy.153 The treat-ment was well tolerated, and median survival was 11.7 and 15.0 months for the two groups, respectively. A similar safety profile was observed in a study in the Netherlands.154 Further clinical trials are required to define the future role of stereo-tactic radiosurgery in treatment of HCC and metastatic tumors.

1	safety profile was observed in a study in the Netherlands.154 Further clinical trials are required to define the future role of stereo-tactic radiosurgery in treatment of HCC and metastatic tumors. Intensity-modulated radiation therapy (IMRT) is another tech-nologic advancement that facilitates the targeted delivery of external-beam radiation. Early clinical data suggested favorable outcomes with IMRT for the treatment of patients with unre-sectable HCC, and ongoing trials are further examining the role of IMRT for these locally advanced tumors.DownstagingIn more advanced-stage patients not eligible for MELD excep-tion points, hepatic-directed therapy including TACE and tumor ablation with radiofrequency, microwave, and ethanol ablation have been found to be effective in shrinking tumors to meet Milan criteria (downstaging). Multiple centers have used down-staging to allow for OLT in patients whose tumors responded and shrank to meet eligibility criteria.155,156Systemic

1	tumors to meet Milan criteria (downstaging). Multiple centers have used down-staging to allow for OLT in patients whose tumors responded and shrank to meet eligibility criteria.155,156Systemic ChemotherapyChemotherapy has not demonstrated great efficacy in patients with HCC, especially in patients with significant cirrhosis. For treatment of HCC, the multikinase inhibitor sorafenib has shown some efficacy in a phase 3 randomized international multicenter trial. The SHARP trial (Sorafenib HCC Assessment Random-ized Protocol) enrolled 602 patients with Child’s class A cir-rhosis and inoperable HCC. At interim analysis, the trial was discontinued because a survival benefit was found in the treat-ment group. The median overall survival for patients receiv-ing sorafenib was 10.7 months versus 7.9 months for patients in the control arm. Based on these findings, sorafenib received accelerated Food and Drug Administration approval for the treatment of advanced unresectable HCC.157 Future

1	7.9 months for patients in the control arm. Based on these findings, sorafenib received accelerated Food and Drug Administration approval for the treatment of advanced unresectable HCC.157 Future studies will likely examine the role of other molecularly targeted agents and combinations of sorafenib with other treatment modalities.HEPATIC RESECTION SURGICAL TECHNIQUESNomenclatureDue to the confusion in language with regard to anatomic descrip-tions of hepatic resections, a common nomenclature was intro-duced at the International Hepato-Pancreato-Biliary Association Brunicardi_Ch31_p1345-p1392.indd 138120/02/19 2:36 PM 1382SPECIFIC CONSIDERATIONSPART IImeeting in Brisbane, Australia, in 2000 (Table 31-8).158,159 The goal was to provide universal terminology for liver anatomy and hepatic resections because there was much overlap among the designations for hepatic lobes, sections, sectors, and segments used by surgeons worldwide (Fig. 31-21). The most common or prevailing anatomic

1	resections because there was much overlap among the designations for hepatic lobes, sections, sectors, and segments used by surgeons worldwide (Fig. 31-21). The most common or prevailing anatomic pattern was used as the basis for naming liver anatomy, and the surgical procedure nomenclature adopted for hepatic resections was based on the assigned anatomic ter-minology.160 Adoption of a common language should enable hepatic surgeons to better understand and interpret liver surgery publications from different continents and disseminate their knowledge to the next generation of hepatobiliary surgeons. Nonetheless, even today, the literature is full of both old and new liver resection terminology, so the surgeon in training must be familiar with all the various classifications.Techniques and Devices for Dividing the Hepatic ParenchymaHepatic resection surgery has evolved over the past 50 years. A better understanding of liver anatomy and physiology, coupled with improved anesthesia

1	and Devices for Dividing the Hepatic ParenchymaHepatic resection surgery has evolved over the past 50 years. A better understanding of liver anatomy and physiology, coupled with improved anesthesia techniques and widespread use of intraoperative ultrasound, has led to virtually “bloodless” liver surgery in the modern era (the year 2000 to the present). Innova-tions in technology have expanded the list of liver parenchymal transection devices161-163 and hemostatic agents (Table 31-9). Use of each device or agent has a learning curve, and undoubt-edly every experienced hepatic surgeon has his or her personal preferences.One major trend has been the application of vascular sta-pling devices for division of the hepatic and portal veins.164-166 Based on early reports of successful stapling of extrahepatic Table 31-8Brisbane 2000 liver terminologyOLDER HEPATIC RESECTION TERMINOLOGYBRISBANE 2000 HEPATIC RESECTION TERMINOLOGY Right hepatic lobectomy Left hepatic lobectomy Right hepatic

1	of extrahepatic Table 31-8Brisbane 2000 liver terminologyOLDER HEPATIC RESECTION TERMINOLOGYBRISBANE 2000 HEPATIC RESECTION TERMINOLOGY Right hepatic lobectomy Left hepatic lobectomy Right hepatic trisegmentectomy Left hepatic trisegmentectomy Left lateral segmentectomy Right posterior lobectomy Caudate lobectomy Right hepatectomy or right hemihepatectomy (V, VI, VII, VIII) Left hepatectomy or left hemihepatectomy (II, III, IV) Right trisectionectomy or extended right hepatectomy (or hemihepatectomy, IV, V, VI, VII, VIII) Left trisectionectomy or extended left hepatectomy (or hemihepatectomy, II, III, IV, V, VIII) Left lateral sectionectomy or bisegmentectomy (II, III) Right posterior sectionectomy (VI, VII) Caudate lobectomy or segmentectomy (I)ALTERNATIVE “SECTOR” TERMINOLOGY Right anterior sectorectomy Right posterior sectorectomy or right lateral sectorectomy Left medial sectorectomy or left paramedian sectorectomy (bisegmentectomy, III, IV) Left lateral sectorectomy

1	anterior sectorectomy Right posterior sectorectomy or right lateral sectorectomy Left medial sectorectomy or left paramedian sectorectomy (bisegmentectomy, III, IV) Left lateral sectorectomy (segmentectomy, II)IVCRHVMHVVIIVIIIIVaIIVIVIVbIIIFalciform lig.Portal veinIVCLHVIFigure 31-21. Hepatic anatomy. Hepatic segments removed in the formal major hepatic resections are indicated. IVC = inferior vena cava; LHV = left hepatic vein; MHV = middle hepatic vein; RHV = right hepatic vein.Table 31-9Techniques and devices for dividing liver parenchyma and achieving hemostasisBlunt fracture and clipsMonopolar cautery (Bovie)Bipolar cauteryArgon beam coagulatorCUSA ultrasonic dissectorHydro-Jet water-jet dissectorHarmonic Scalpel, AutoSonix ultrasonic transector-coagulatorLigaSure tissue fusion systemSurgRx EnSeal tissue sealing and transection systemGyrus PK cutting forcepsEndovascular staplersTissueLink sealing devicesHabib 4X Laparoscopic sealerInLine bipolar linear coagulatorTopical agents

1	EnSeal tissue sealing and transection systemGyrus PK cutting forcepsEndovascular staplersTissueLink sealing devicesHabib 4X Laparoscopic sealerInLine bipolar linear coagulatorTopical agents (fibrin glues, Surgicel, Gelfoam, Avitene, Tisseel, Floseal, Crosseal)Brunicardi_Ch31_p1345-p1392.indd 138220/02/19 2:36 PM 1383LIVERCHAPTER 31vessels, stapling devices are now being used in the parenchymal transection phase, which remains a source of potential blood loss due to back bleeding from the middle hepatic vein.167,168 One advantage of the stapling technique is the speed with which the transection can be performed, which minimizes surface bleeding and period of ischemia for the remnant liver. How-ever, a major disadvantage of the stapling technique is the cost of multiple stapler cartridges. This is balanced by the decreased expenses reported with avoidance of ICU admission and blood transfusion, as well as shortened operating room time. Another consideration in the use of staplers

1	This is balanced by the decreased expenses reported with avoidance of ICU admission and blood transfusion, as well as shortened operating room time. Another consideration in the use of staplers for parenchymal transec-tion is the potential for bile leaks. However, in a large series of 101 consecutive right hemihepatectomies performed using the stapling technique, there was only one reported bile leak (1%), which sealed after ERCP.168Steps in Commonly Performed Hepatic ResectionsA fundamental understanding of hepatic anatomy is vital for any surgeon with the desire to perform hepatobiliary surgery. Each hepatic resection surgery can be broken down into a series of orderly steps. The key to being a proficient hepatic surgeon is not to operate swiftly but rather to accomplish the operation by completing the steps in an orchestrated fashion. Mastery of the operative steps coupled with knowledge of liver anatomy and the common anatomic variants provides the foundation for safe hepatic

1	by completing the steps in an orchestrated fashion. Mastery of the operative steps coupled with knowledge of liver anatomy and the common anatomic variants provides the foundation for safe hepatic surgery. There are many different techniques and sequences for accomplishing each of the anatomic (and nonana-tomic) hepatic operations. The authors present their preferred approach in a stepwise fashion for right hepatic lobectomy (right hemihepatectomy), left hepatic lobectomy (left hemihepa-tectomy), and left lateral segmentectomy (left lateral sectionec-tomy). Provision of a detailed approach for every type of liver resection is beyond the scope of this chapter, and readers are referred to several excellent descriptions.169Steps Common to All Open Major Hepatic Resections 1. Make the skin incision—right subcostal with or without a partial or complete left subcostal extension across the mid-line, depending on the patient’s habitus and liver/tumor anatomy.2. Open and explore the abdomen,

1	subcostal with or without a partial or complete left subcostal extension across the mid-line, depending on the patient’s habitus and liver/tumor anatomy.2. Open and explore the abdomen, and place a fixed table retractor (e.g., Thompson or Bookwalter).3. Examine the liver with bimanual palpation. Perform liver ultrasound, and confirm the operation to be performed.4. Take down the round and falciform ligaments, and expose the anterior surface of the hepatic veins.5. For a left hepatectomy, divide the left triangular ligament; for a right hepatectomy, mobilize the right lobe from the right coronary and triangular ligaments.6. Open the gastrohepatic ligament, palpate the porta hepatis, and assess for accessory or replaced hepatic arteries.7. Perform a cholecystectomy; leave the gallbladder with the cystic duct intact if the gallbladder is involved by the tumor.Right Hepatic Lobectomy (Right Hepatectomy or Hemihepatectomy) 8. Mobilize the liver from the anterior aspect of the IVC in

1	with the cystic duct intact if the gallbladder is involved by the tumor.Right Hepatic Lobectomy (Right Hepatectomy or Hemihepatectomy) 8. Mobilize the liver from the anterior aspect of the IVC in “piggyback” fashion; ligate the short hepatic veins up to the right hepatic vein (RHV).9. Perform a right hilar dissection—gently lower the hilar plate, then doubly ligate and divide the right hepatic artery (RHA), superior to the right side of the common bile duct.10. Doubly ligate and divide a replaced or accessory RHA if present.11. Expose the portal vein, identifying its right and left branches. There is a small lateral portal vein branch off the right portal vein (RPV) to the caudate lobe that should be controlled and ligated to allow the exposure of additional length on the RPV. Divide the RPV either with a vascular stapler or between vascular clamps.12. Dissect the avascular tissue along the suprahepatic vena cava between the right and middle hepatic veins. Pass a silastic tube of a

1	RPV either with a vascular stapler or between vascular clamps.12. Dissect the avascular tissue along the suprahepatic vena cava between the right and middle hepatic veins. Pass a silastic tube of a Jackson-Pratt drain through this gap.13. Notch or divide the caudate process crossing to the right hepatic lobe, and bring the drain up and through this notch.14. Hang the liver over the drain by pulling up as you divide through the liver parenchyma.15. Repeat ultrasound and confirm the transection plane, staying just to the right of the middle hepatic vein (MHV) unless the tumor extends over it.16. Cauterize approximately 1 cm into the liver parenchyma, then switch to a hydro-jet dissection device in combination with Bovie electrocautery and suture ligation.17. Continue parenchymal division until the RHV is encoun-tered. During this division, identification, control, ligation, and transection of the right hepatic duct (RHD) are obtained late in the parenchymal transection

1	division until the RHV is encoun-tered. During this division, identification, control, ligation, and transection of the right hepatic duct (RHD) are obtained late in the parenchymal transection process.18. Divide the RHV between vascular clamps and suture ligate the RHV.19. Examine the transected liver edge for bleeding; place a figure-of-eight ligating vascular suture if bleeding is encountered.20. Ensure hemostasis of the transected liver edge with an argon beam coagulator and suture ligation.21. Inspect the transection surface for bile leaks. These should be clipped or suture ligated. Applying a dilute solution of hydro-gen peroxide can facilitate the visualization of bile leaks.22. Inspect the IVC and right retroperitoneal space for hemostasis.23. Perform completion ultrasound to confirm left portal vein (LPV) inflow and outflow in the remaining hepatic veins.24. Fix the proximal falciform ligament back to the diaphragm side with figure-of-eight sutures.25. Apply tissue sealant to

1	left portal vein (LPV) inflow and outflow in the remaining hepatic veins.24. Fix the proximal falciform ligament back to the diaphragm side with figure-of-eight sutures.25. Apply tissue sealant to the cut surface of the liver, and place a Jackson-Pratt drain in the right subphrenic space and close the abdomen (Fig. 31-22).Comments Although some liver surgeons advocate a one-step division of the entire intrahepatic Glissonian pedicle as described by Launois and Jamieson,170 it is the authors’ prefer-ence to divide the RHA, RHD, and RPV in an extrahepatic fash-ion. As for the transection plane, the key is to perform accurate ultrasound visualization and mapping of the MHV and to stay just to the right of it. Weaving in and out or bisecting the MHV can leading to torrential back bleeding. Also, for bulky right lobe tumors adherent to the diaphragm or retroperitoneum, an anterior approach with division of the parenchyma can be per-formed before right lobe mobilization.152,153Left Hepatic

1	for bulky right lobe tumors adherent to the diaphragm or retroperitoneum, an anterior approach with division of the parenchyma can be per-formed before right lobe mobilization.152,153Left Hepatic Lobectomy (Left Hepatectomy or Hemihepatectomy) 8. Widely open the gastrohepatic ligament flush with the undersurface of the left lateral section and the caudate lobe. 9. Doubly ligate and divide a replaced or accessory left hepatic artery (LHA) if present.Brunicardi_Ch31_p1345-p1392.indd 138320/02/19 2:36 PM 1384SPECIFIC CONSIDERATIONSPART II10. Clamp the round ligament (ligament teres) and pull it ante-riorly as a handle to expose the left hilum.11. Divide any existing parenchymal bridge between segments III and IVB.12. Dissect the left hilum at the base of the umbilical fissure and lower the hilar plate anterior to the left portal pedicle.13. Incise the peritoneum overlying the hilum from the left side, and doubly ligate the LHA (after test clamping and confirming a palpable pulse in

1	the hilar plate anterior to the left portal pedicle.13. Incise the peritoneum overlying the hilum from the left side, and doubly ligate the LHA (after test clamping and confirming a palpable pulse in the RHA).14. Dissect the portal vein at the base of the umbilical fissure (it will take a nearly 90° bend from the transverse to the umbilical portion).15. Expose the portal vein, identifying the right and left branches. Control the small portal vein branch off the LPV to the caudate lobe to allow the exposure of additional length. Divide the LPV either with a vascular stapler or between vascular clamps.16. Ligate and divide the ligamentum venosum caudally.17. Identify the long extrahepatic course of the left hepatic duct (LHD) behind the portal vein. Ligate and divide the LHD at the umbilical fissure.18. Fold the left lateral segment up and back to the right, expos-ing the window at the base of the left hepatic vein (LHV) as it enters the IVC. This is facilitated by dividing any loose

1	fissure.18. Fold the left lateral segment up and back to the right, expos-ing the window at the base of the left hepatic vein (LHV) as it enters the IVC. This is facilitated by dividing any loose areolar tissue overlying the ligamentum venosum, which is divided proximally.19. Pass a large, blunt right-angle clamp in the window between the RHV and the MHV, and hug the back of the MHV, aim-ing for the deep edge of the LHV. Do not force it or perfo-rate the IVC or MHV.20. Pass the silastic tube of a Jackson-Pratt drain through this window.21. Notch or divide the caudate process crossing to the left hepatic lobe and bring the drain up and through this notch.22. Hang the liver over the drain by pulling up as you divide through the liver parenchyma.23. Repeat ultrasound and confirm the transection plane on the anterior surface, staying close to the demarcated line. Do not bisect the MHV as it passes tangentially from the left to the right lobe.24. Cauterize down approximately 1 cm in the

1	plane on the anterior surface, staying close to the demarcated line. Do not bisect the MHV as it passes tangentially from the left to the right lobe.24. Cauterize down approximately 1 cm in the liver paren-chyma, then switch to a hydro-jet dissection device in com-bination with Bovie electrocautery and suture ligation.25. Continue parenchymal division until the left/middle hepatic veins are encountered.26. Divide the LHV and MHV between vascular clamps and suture the ligate the LHV/MHV.27. Check the transected edge of the liver for surgical bleed-ing; ensure hemostasis of the transected edge with an argon beam coagulator and suture ligation.28. Inspect the transection surface for bile leaks. These should be clipped or suture ligated. Apply dilute solution of hydro-gen peroxide to facilitate the visualization of bile leaks.29. Perform completion ultrasound to confirm RPV inflow and RHV outflow.30. Apply tissue sealant to the transected surface of the liver. Place a Jackson-Pratt drain

1	the visualization of bile leaks.29. Perform completion ultrasound to confirm RPV inflow and RHV outflow.30. Apply tissue sealant to the transected surface of the liver. Place a Jackson-Pratt drain in the left subphrenic space and close the abdomen (Fig. 31-23).Comments Because the right posterior duct arises from the left hepatic duct (LHD) in approximately 20% of cases (see Fig. 31-9) and the right anterior duct comes off the LHD in approximately 5% of cases,6 it is vital to divide the LHD at the base of the umbilical fissure and not more centrally in the hilum as it bifurcates. If the LHD were divided as it appears to bifurcate from the right hepatic duct, then approximately 20% to Figure 31-22. Completed right hepatic lobectomy (right hepa-tectomy) with the right portal vein, right hepatic artery, and right bile duct ligated and divided. The right hepatic vein is ligated and divided. Middle hepatic vein branches inside the liver are divided.Figure 31-23. Completed left hepatic

1	hepatic artery, and right bile duct ligated and divided. The right hepatic vein is ligated and divided. Middle hepatic vein branches inside the liver are divided.Figure 31-23. Completed left hepatic lobectomy (left hepatec-tomy) resecting segments II, III, and IV.Brunicardi_Ch31_p1345-p1392.indd 138420/02/19 2:37 PM 1385LIVERCHAPTER 3125% of the time, either the right posterior or right anterior duct would be transected. After the LHD is divided as described ear-lier (Step 17), the liver parenchyma is scored and divided hori-zontally approximately 1 cm above the left hilum; the surgeon thus assumes that an aberrant right anterior or posterior duct is coming off the LHD in the hilum and preserves it. Then as the parenchymal transection reaches the left side of the gallblad-der fossa, the transection plane turns vertical to run parallel to Cantlie’s line (or the left edge of the gallbladder bed). The left lobe of the liver will be well demarcated at this point (after the vascular

1	the transection plane turns vertical to run parallel to Cantlie’s line (or the left edge of the gallbladder bed). The left lobe of the liver will be well demarcated at this point (after the vascular inflow has been divided), which guides the transection plane on the anterior surface. In general, the transection plane should be close to the demarcation line to minimize the amount of devascularized liver remaining. When dividing the LHV and MHV, the surgeon should keep in mind that they have a com-mon trunk approximately 90% of the time. If it is not easy to open the window deep to the MHV and LHV, then division of the MHV and LHV can be accomplished after the parenchymal transection.Left Lateral Segmentectomy (Left Lateral Sectionectomy) 8. Widely open the gastrohepatic ligament flush with the undersurface of the left lateral section and the caudate lobe. 9. Doubly ligate and divide a replaced or accessory LHA if present.10. Clamp the round ligament and pull it anteriorly as a handle

1	the undersurface of the left lateral section and the caudate lobe. 9. Doubly ligate and divide a replaced or accessory LHA if present.10. Clamp the round ligament and pull it anteriorly as a handle to expose the left hilum.11. Divide any existing parenchymal bridge between segments III and IVB.12. Carry the dissection down from the end of the round liga-ment, and the segment III pedicle will be encountered.13. Incise the peritoneal reflection on the left side of the round ligament as it inserts into the umbilical fissure. This will facilitate encircling the segment III and II pedicles, which can be divided separately. When encircling the segment II pedicle, take care to avoid injury to the caudate inflow ves-sels coming off the LPV.14. Divide the liver parenchyma, staying flush on the left side of the falciform ligament using Bovie electrocautery.15. Divide the LHV as the parenchymal transection is complete.16. A Pringle maneuver usually is not required for a left lateral

1	on the left side of the falciform ligament using Bovie electrocautery.15. Divide the LHV as the parenchymal transection is complete.16. A Pringle maneuver usually is not required for a left lateral sectionectomy because complete devascularization occurs before transection and little back bleeding is encountered.Comments If the segment III and II LHA branches are large, they can be individually ligated in the left hilum before the pedicles (with portal vein and hepatic duct branches) are taken. If the tumor is more peripheral in the left lateral segment, then the segment III and II pedicles can be divided with a vascular stapler inside the liver during the parenchymal transection.Pringle and Ischemic PreconditioningPringle described clamping of the portal triad a century ago in the landmark paper “Notes on the Arrest of Hepatic Hemorrhage Due to Trauma.”4 Although the Pringle maneuver was initially described for controlling bleeding due to traumatic liver injury, it is commonly used

1	paper “Notes on the Arrest of Hepatic Hemorrhage Due to Trauma.”4 Although the Pringle maneuver was initially described for controlling bleeding due to traumatic liver injury, it is commonly used during elective hepatic resections.173,174 The goal is to minimize blood loss and hypotension, which add sig-nificant morbidity to the operation. Furthermore, intraoperative blood transfusion has been shown to be an independent risk fac-tor for increased postoperative infection as well as worse patient survival in some studies. Therefore, all efforts should be made to minimize blood loss during hepatic resection.Although the liver has been shown to tolerate up to 1 hour of warm ischemia, some technical variations of the Pringle maneuver include intermittent vascular occlusion with cycles of approximately 15 minutes on and 5 minutes off. Experimental and clinical studies have demonstrated the efficacy of intermit-tent vascular occlusion in decreasing ischemia/reperfusion injury compared with

1	15 minutes on and 5 minutes off. Experimental and clinical studies have demonstrated the efficacy of intermit-tent vascular occlusion in decreasing ischemia/reperfusion injury compared with continuous vascular occlusion, with less eleva-tion of postoperative liver enzyme levels.175 Another variation is selective hemihepatic vascular occlusion, which can reduce the severity of visceral congestion and total liver ischemia. In one prospective trial of total versus selective portal triad clamping, both techniques of inflow clamping were found to be equally effective for patients with normal livers, but greater liver dam-age was observed with total inflow occlusion in patients with cirrhotic livers.176In an attempt to decrease the ischemic damage associated with inflow occlusion, some hepatic surgeons have advocated the use of ischemic preconditioning.177 Ischemic precondition-ing refers to the brief interruption of blood flow to an organ, fol-lowed by a short reperfusion period, and then

1	surgeons have advocated the use of ischemic preconditioning.177 Ischemic precondition-ing refers to the brief interruption of blood flow to an organ, fol-lowed by a short reperfusion period, and then a more prolonged period of ischemia. In a randomized clinical trial involving 100 patients undergoing major hepatic resection, Clavien and col-leagues reported significantly less liver injury in the group who received ischemic preconditioning with a 10-minute clamp, a 10-minute reperfusion, and then a 30-minute clamp than in those who received a 30-minute clamp alone.178 Patients with steatosis also were especially protected by ischemic preconditioning, and the mechanism was shown to be related in part to preservation of the adenosine triphosphate content of liver tissue.Preoperative Portal Vein EmbolizationThe observation that tumor thrombosis of a major portal vein branch induced ipsilateral lobar atrophy and contralateral lobe hypertrophy led to the concept of intentional preoperative

1	EmbolizationThe observation that tumor thrombosis of a major portal vein branch induced ipsilateral lobar atrophy and contralateral lobe hypertrophy led to the concept of intentional preoperative portal vein embolization (PVE) to induce compensatory hypertrophy of the remnant liver. This procedure was first described in the 1980s and is accomplished via a percutaneous, transhepatic route.160,161 Numerous studies have subsequently confirmed that PVE is effective in inducing hypertrophy of nonembolized hepatic segments.86,181 PVE usually is performed in the setting of a planned right trisectionectomy or extended left hepatec-tomy (also referred to as a left trisectionectomy; resection of Couinaud’s segments II, III, IV, V, and VIII of the liver) or extended hepatic lobectomy when it is thought that the patient’s remnant liver will be too small to support liver function. The future liver remnant volume (e.g., the volume of segments II, III, and I) in a patient undergoing a planned right

1	that the patient’s remnant liver will be too small to support liver function. The future liver remnant volume (e.g., the volume of segments II, III, and I) in a patient undergoing a planned right trisectionec-tomy can be directly measured by helical CT and then divided by the total estimated liver volume to calculate the percentage of the future liver remnant. If the future liver remnant is thought to be too small, then PVE should be considered to increase the size of the future liver remnant.182 In general, surgery is planned approximately 4 weeks after PVE to allow adequate time for hypertrophy.There is no universal agreement on what constitutes a future liver remnant adequate to avoid postoperative liver fail-ure. It is thought that 25% to 30% of the total liver volume is ade-quate in patients with a normal liver.183 Vauthey and associates reported that major postoperative complications were increased when the estimated future liver remnant was <25%.184 Farges and colleagues

1	in patients with a normal liver.183 Vauthey and associates reported that major postoperative complications were increased when the estimated future liver remnant was <25%.184 Farges and colleagues conducted a prospective study to assess the benefits of PVE before right hepatectomy. They demonstrated Brunicardi_Ch31_p1345-p1392.indd 138520/02/19 2:37 PM 1386SPECIFIC CONSIDERATIONSPART IIthat PVE had no beneficial effect on the postoperative course in patients with normal livers but significantly reduced postop-erative complications in patients with chronic liver diseases.185 A larger remnant may be necessary even in patients with nor-mal livers when a complex hepatectomy is planned or when the background liver is steatotic.186 This is especially relevant with the increased incidence of fatty liver disease. A larger remnant may also be needed when patients have received pre-operative chemotherapy. Some have suggested that 40% of the total hepatic volume should remain to minimize

1	of fatty liver disease. A larger remnant may also be needed when patients have received pre-operative chemotherapy. Some have suggested that 40% of the total hepatic volume should remain to minimize postoperative complications in patients who have underlying liver disease or who have received preoperative chemotherapy for colorectal cancer metastases.187,188 In a recent study encompassing 112 patients who underwent PVE, major complications, hepatic insufficiency, length of hospital stay, and 90-day mortality rate were significantly greater in patients with a standardized future liver remnant of ≤20% or a degree of hypertrophy of <5% than in patients with higher values.189 In another study, the authors performed PVE during neoadjuvant chemotherapy for colorec-tal cancer metastases. After a median wait of 30 days after PVE, patients receiving neoadjuvant chemotherapy showed median liver growth of 22% in the contralateral (nonembolized) lobe compared with 26% for those not receiving

1	a median wait of 30 days after PVE, patients receiving neoadjuvant chemotherapy showed median liver growth of 22% in the contralateral (nonembolized) lobe compared with 26% for those not receiving chemotherapy (not a statistically significant difference), which indicated that liver growth occurs after PVE even when cytotoxic chemotherapy is administered.190 PVE-related complications occur at a relatively low rate and include bleeding, hemobilia, liver abscess, incom-plete embolization, and small bowel obstruction. To augment the ability to increase liver function reserve in patients who have undergone PVE, some groups have added ipsilateral hepatic artery embolization and ipsilateral hepatic vein embolization.191Staged Hepatectomy, ALPPS, and Repeat Hepatic Resection for Recurrent Liver CancerA two-stage hepatectomy is a sequential resection strategy to remove all metastatic liver tumors when it is impossible to resect all disease in a single operative procedure. The first-stage

1	CancerA two-stage hepatectomy is a sequential resection strategy to remove all metastatic liver tumors when it is impossible to resect all disease in a single operative procedure. The first-stage hepatectomy usually consists of clearance of the left hemiliver by nonanatomic resection, followed by right portal vein ligation or embolization to induce left lobe hypertrophy.192,193 This is fol-lowed by a second-stage major right hepatectomy or extended right hepatectomy to resect the right liver metastases. This approach is most commonly used in cases of initially unresect-able colorectal hepatic metastases and has yielded very good results.193Another technique to increase future liver remnant to avoid post-hepatectomy liver failure is known as “Associating Liver Partition and Portal Vein Ligation for Staged Hepatec-tomy (ALPPS)” and was described in 2012. This technique consists of operative portal vein ligation with in situ liver tran-section along the future line of resection leaving

1	for Staged Hepatec-tomy (ALPPS)” and was described in 2012. This technique consists of operative portal vein ligation with in situ liver tran-section along the future line of resection leaving the arterial and hepatic vein branches intact.194 There have been several techni-cal modifications to the original description, and this remains a hot topic that is actively debated (ALPPS vs. PVE) at the current liver surgery meetings.The majority of patients undergoing hepatic resection for colorectal cancer metastases experience a recurrence. For those with limited disease recurrence confined to the liver, repeat hepatectomy is a reasonable option and can be performed with low morbidity and mortality in experienced hands.195 In one study, 126 patients who underwent a second liver resection for colorectal cancer metastases had 1-, 3-, and 5-year survival rates of 86%, 51%, and 34%, respectively. By multivariate analysis, the presence of more than one lesion and a tumor size of >5 cm were

1	colorectal cancer metastases had 1-, 3-, and 5-year survival rates of 86%, 51%, and 34%, respectively. By multivariate analysis, the presence of more than one lesion and a tumor size of >5 cm were independent prognostic indicators of reduced survival.196 In another study, 40 patients underwent a second hepatectomy for liver metastases from colorectal cancer and experienced a survival benefit similar to that from the first hepatectomy; how-ever, the results suggested that this approach should be limited to those patients who do not have extrahepatic disease and for whom >1 year has elapsed since the first operation.197 A meta-analysis of 21 studies examining clinical outcomes after first and second liver resections for colorectal cancer metastases showed that repeat hepatectomy was safe and provided a sur-vival benefit equal to that from the first liver resection.198Repeat hepatectomy also has been performed in patients with HCC. Nakajima and colleagues reported on follow-up of 94

1	and provided a sur-vival benefit equal to that from the first liver resection.198Repeat hepatectomy also has been performed in patients with HCC. Nakajima and colleagues reported on follow-up of 94 patients who underwent curative liver resection for HCC from 1991 to 1996.199 Of these, 57 patients had isolated recur-rent disease in the liver. Twelve of these 57 patients underwent repeat hepatic resection, whereas the other 45 patients received ablation therapy. The overall survival rate in those undergoing a second hepatectomy was 90% at 2 years; however, the disease-free survival rate was only 31% at 2 years, significantly lower than the 62% rate after initial hepatectomy. Likewise, in another group of 84 patients who underwent second hepatectomy for recurrent HCC, the overall 5-year survival rate was 50%, but the recurrence-free survival rate was only 10%.200 In a report of 67 patients undergoing a second resection for HCC, over-all 1-, 3-, and 5-year survival rates were 93%, 70%,

1	rate was 50%, but the recurrence-free survival rate was only 10%.200 In a report of 67 patients undergoing a second resection for HCC, over-all 1-, 3-, and 5-year survival rates were 93%, 70%, and 56%, respectively.201 Multivariate analysis showed that absence of portal invasion at the second resection, single HCC at primary hepatectomy, and disease-free interval of ≥1 year after primary hepatectomy were independent prognostic factors after the sec-ond resection.LAPAROSCOPIC LIVER RESECTIONCherqui and colleagues first reported in 2000 that laparoscopic hepatic surgery was feasible.202 Since this initial report, laparo-scopic liver surgery has expanded from the simple unroofing of hepatic cysts to resection of peripheral benign lesions to formal anatomic lobectomies for malignancy and laparoscopic hepa-tectomy for living donor liver transplantation. While minimally invasive approaches have been widely adopted in other areas of abdominal surgery, there was initial apprehension regarding

1	hepa-tectomy for living donor liver transplantation. While minimally invasive approaches have been widely adopted in other areas of abdominal surgery, there was initial apprehension regarding laparoscopic liver resection (LLR), hampering its widespread adoption.203 Great strides have been made in the past decade with techniques of laparoscopic liver resection,204,205 and two International Laparoscopic Liver Resection Consensus Confer-ences have been convened in Louisville (2008) and Morioko (2014).206,207 Indications for liver resection should not be altered by the availability of minimally invasive liver resection tech-niques. Currently, over 9500 cases of laparoscopic liver resec-tion have been reported worldwide, with over 50% of the cases being done for malignancy.208Pure laparoscopic and hand-assisted laparoscopic liver resection are the two most commonly used techniques for mini-mally invasive liver resection surgery,209,210 while robotic liver resection is being used by several

1	and hand-assisted laparoscopic liver resection are the two most commonly used techniques for mini-mally invasive liver resection surgery,209,210 while robotic liver resection is being used by several groups.211 Advantages of the hand-port include tactile feedback, facilitation of liver mobiliza-tion, and ease of ability to control bleeding. Also, when doing a large parenchymal resection, the hand-port can be comparable in size to the extraction port utilized in the purely laparoscopic Brunicardi_Ch31_p1345-p1392.indd 138620/02/19 2:37 PM 1387LIVERCHAPTER 31approach. The hand-assisted approach can be ideal for surgeons beginning the transition to laparoscopic liver resection and for more experienced laparoscopic HPB surgeons doing laparo-scopic major hepatectomies or as an alternative to conversion to open surgery.Benefits of laparoscopic liver resection include less blood loss, decreased morbidity, decreased postoperative pain and narcotic requirements, faster return of bowel

1	to conversion to open surgery.Benefits of laparoscopic liver resection include less blood loss, decreased morbidity, decreased postoperative pain and narcotic requirements, faster return of bowel function, and shorter length of hospital stay compared to open hepatic resection.212 Long-term oncologic outcomes for HCC and CLM have been shown to be comparable for laparoscopic vs. open liver resection using propensity score matching and meta-analysis studies.213-215 The learning curve for laparoscopic liver resection has been reported to be around 60 cases,216 although this may be greater for laparoscopic major hepatectomy.217-219 Cost analy-sis has shown that laparoscopic liver resection is cost effective compared to open liver resection, where the added cost of the operating room disposables are more than offset by the savings associated with ∼50% reduction in the hospital length of stay.221,222Initial experience with laparoscopic living-donor hepatec-tomy for transplantation was with

1	are more than offset by the savings associated with ∼50% reduction in the hospital length of stay.221,222Initial experience with laparoscopic living-donor hepatec-tomy for transplantation was with left lateral segmentectomy during liver allograft procurement for pediatric transplants.222 With continued advances in laparoscopic liver surgery, LLR has been applied to adult-to-adult donor right hepatectomy,223,224 although this approach remains controversial.In summary, laparoscopic liver resection can now be per-formed safely by experienced surgeons in selected patients. Compared to open hepatic resection, the laparoscopic approach has benefits of less blood loss, reduced postoperative pain, and a shorter length of hospital stay, with similar oncologic out-comes for resection of HCC and limited colorectal liver metastases.REFERENCESEntries highlighted in bright blue are key references. 1. Wikipedia. Prometheus. Available at: http://en.wikipedia.org/wiki/Prometheus. Accessed July 26,

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1	to extended liver resection: methodology and clinical associations. Surgery. 2000;127(5):512-519. 185. Farges O, Belghiti J, Kianmanesh R, et al. Portal vein emboli-zation before right hepatectomy: prospective clinical trial. Ann Surg. 2003;237(2):208-217. 186. Hemming AW, Reed AI, Howard RJ, et al. Preoperative por-tal vein embolization for extended hepatectomy. Ann Surg. 2003;237(5):686-691. 187. Azoulay D, Castaing D, Smail A, et al. Resection of nonre-sectable liver metastases from colorectal cancer after percu-taneous portal vein embolization. Ann Surg. 2000;231(4): 480-486. 188. Kubota K, Makuuchi M, Kusaka K, et al. Measurement of liver volume and hepatic functional reserve as a guide to decision-making in resectional surgery for hepatic tumors. Hepatology. 1997;26(5):1176-1181. 189. Ribero D, Abdalla EK, Madoff DC, Donadon M, Loyer EM, Vauthey JN. Portal vein embolization before major hepatec-tomy and its effects on regeneration, resectability and out-come. Br J Surg.

1	D, Abdalla EK, Madoff DC, Donadon M, Loyer EM, Vauthey JN. Portal vein embolization before major hepatec-tomy and its effects on regeneration, resectability and out-come. Br J Surg. 2007;94(11):1386-1394. 190. Covey AM, Brown KT, Jarnagin WR, et al. Combined portal vein embolization and neoadjuvant chemotherapy as a treat-ment strategy for resectable hepatic colorectal metastases. Ann Surg. 2008;247(3):451-455. 191. Hwang S, Lee SG, Ko GY, et al. Sequential preoperative ipsi-lateral hepatic vein embolization after portal vein embolization to induce further liver regeneration in patients with hepatobili-ary malignancy. Ann Surg. 2009;249(4):608-616. 192. Jaeck D, Oussoultzoglou E, Rosso E, Greget M, Weber JC, Bachellier P. A two-stage hepatectomy procedure combined with portal vein embolization to achieve curative resection for initially unresectable multiple and bilobar colorectal liver metastases. Ann Surg. 2004;240(6):1037-1049. 193. Adam R, Miller R, Pitombo M, et al. Two-stage

1	to achieve curative resection for initially unresectable multiple and bilobar colorectal liver metastases. Ann Surg. 2004;240(6):1037-1049. 193. Adam R, Miller R, Pitombo M, et al. Two-stage hepatec-tomy approach for initially unresectable colorectal hepatic metastases. Surg Oncol Clin North Am. 2007;16(3):525-536. Very useful summary of the two-stage hepatectomy approach for resection of hepatic colorectal cancer metastases. 194. Schnitzbauer AA, Lang SA, Goessmann H, et al. Right portal vein ligation combined with in situ splitting induces rapid left lateral liver lobe hypertrophy enabling 2-staged extended right hepatic resection in small-for-size settings. Ann Surg. 2012;255(3):405-414. Initial paper describing the ALPPS technique to induce massive future liver remnant hypertrophy and outcomes.Brunicardi_Ch31_p1345-p1392.indd 139120/02/19 2:37 PM 1392SPECIFIC CONSIDERATIONSPART II 195. Adam R, Bismuth H, Castaing D, et al. Repeat hepatectomy for colorectal liver metastases.

1	outcomes.Brunicardi_Ch31_p1345-p1392.indd 139120/02/19 2:37 PM 1392SPECIFIC CONSIDERATIONSPART II 195. Adam R, Bismuth H, Castaing D, et al. Repeat hepatectomy for colorectal liver metastases. Ann Surg. 1997;225(1):51-60. 196. Petrowsky H, Gonen M, Jarnagin W, et al. Second liver resec-tions are safe and effective treatment for recurrent hepatic metastases from colorectal cancer: a bi-institutional analysis. Ann Surg. 2002;235(6):863-871. 197. Sa Cunha A, Laurent C, Rault A, Couderc P, Rullier E, Saric J. A second liver resection due to recurrent colorectal liver metastases. Arch Surg. 2007;142(12):1144-1149. 198. Antoniou A, Lovegrove RE, Tilney HS, et al. Meta-analysis of clinical outcome after first and second liver resection for colorectal metastases. Surgery. 2007;141(1):9-18. 199. Nakajima Y, Ko S, Kanamura T, et al. Repeat liver resec-tion for hepatocellular carcinoma. J Am Coll Surg. 2001;192(3):339-344. 200. Itamoto T, Nakahara H, Amano H, et al. Repeat hepatec-tomy for

1	Y, Ko S, Kanamura T, et al. Repeat liver resec-tion for hepatocellular carcinoma. J Am Coll Surg. 2001;192(3):339-344. 200. Itamoto T, Nakahara H, Amano H, et al. Repeat hepatec-tomy for recurrent hepatocellular carcinoma. Surgery. 2007;141(5):589-597. 201. Minagawa M, Makuuchi M, Takayama T, Kokudo N. Selec-tion criteria for repeat hepatectomy in patients with recurrent hepatocellular carcinoma. Ann Surg. 2003;238(5):703-710. 202. Cherqui D, Husson E, Hammoud R, et al. Laparoscopic liver resections: a feasibility study in 30 patients. Ann Surg. 2000;232(6):753-762. 203. Reddy SK, Tsung A, Geller DA. Laparoscopic liver resection. World J Surg. 2011;35(7):1478-1486. 204. Nguyen KT, Gamblin TC, Geller DA. World review of laparoscopic liver resection – 2,804 patients. Ann Surg, 2009;250:831-841. Comprehensive review of the indications, techniques, benefits, and outcomes of laparoscopic liver resection. 205. Koffron AJ, Auffenberg G, Kung R, Abecassis M. Evaluation of 300 minimally

1	Comprehensive review of the indications, techniques, benefits, and outcomes of laparoscopic liver resection. 205. Koffron AJ, Auffenberg G, Kung R, Abecassis M. Evaluation of 300 minimally invasive liver resections at a single institu-tion: less is more. Ann Surg. 2007;246(3):385-392. 206. Buell JF, Cherqui D, Geller DA, et al. The international posi-tion on laparoscopic liver surgery: the Louisville statement, 2008. Ann Surg. 2009;250:825-830. 207. Wakabayashi G, Cherqui D, Geller DA, et al. Recommen-dations for laparoscopic liver resection: a report from the second international consensus conference held in Morioka. Ann Surg. 2015;261:619-629. Up-to-date recommendations from the second international consensus conference consist-ing of a 9-member jury and an expert panel that addressed 17 questions for laparoscopic liver resection. 208. Ciria R, Cherqui D, Geller DA, Briceno J, Wakabayashi G. Comparative short term benefits of laparoscopic liver resec-tion: 9,000 cases and climbing.

1	questions for laparoscopic liver resection. 208. Ciria R, Cherqui D, Geller DA, Briceno J, Wakabayashi G. Comparative short term benefits of laparoscopic liver resec-tion: 9,000 cases and climbing. Ann Surg. 2016;263:761-777. 209. Cardinal JS, Reddy SK, Tsung A, Marsh JW, Geller DA. Lap-aroscopic major hepatectomy: pure laparoscopic approach versus hand-assisted technique. J Hepatobiliary Pancreat Sci. 2013;20:114-119. 210. Cheek SM, Sucandy I, Geller DA. Hand-assisted laparoscopic left hepatectomy: how I do it. J Hepatobiliary Pancreat Sci. 2016;23(12):E30-E32. 211. Tsung A, Geller DA, Sukato DC, et al. Robotic versus lapa-roscopic hepatectomy: a matched comparison. Ann Surg. 2014;259:549-555. 212. Nguyen KT, Marsh JW, Tsung A, Steel JL, Gamblin TC, Geller DA. Comparative benefits of laparoscopic versus open hepatic resection: a critical appraisal. Arch Surg. 2011;146:348-356. 213. Takahara T, Wakabayashi G, Beppu T, et al. Long-term and perioperative outcomes of laparoscopic versus

1	versus open hepatic resection: a critical appraisal. Arch Surg. 2011;146:348-356. 213. Takahara T, Wakabayashi G, Beppu T, et al. Long-term and perioperative outcomes of laparoscopic versus open liver resection for hepatocellular carcinoma with propensity score matching: a multi-institutional Japanese study. J Hepatobiliary Pancreat Sci. 2015;22:721-727. 214. Zhou YM, Shao WY, Zhao YF, Xu DH, Li B. Meta-analysis of laparoscopic versus open resection for hepatocellular carci-noma. Dig Dis Sci. 2011;56(7):1937-1943. 215. Schiffman SC, Kim KH, Tsung A, Marsh JW, Geller DA. Laparoscopic versus open liver resection for metastatic colorectal cancer: a meta-analysis of 610 patients. Surgery. 2015;157:211-222. 216. Vigano L, Laurent A, Tayar C, et al. The learning curve in laparoscopic liver resection: improved feasibility and repro-ducibility. Ann Surg 2009;250:772-782. 217. Brown KM, Geller DA. What is the learning curve for laparoscopic major hepatectomy? J Gastrointest Surg.

1	liver resection: improved feasibility and repro-ducibility. Ann Surg 2009;250:772-782. 217. Brown KM, Geller DA. What is the learning curve for laparoscopic major hepatectomy? J Gastrointest Surg. 2016;20:1065-1071. 218. Dagher I, O’Rourke N, Geller DA, et al. Laparoscopic major hepatectomy: an evolution in standard of care. Ann Surg. 2009;250:856-860. 219. Lin NC, Nitta H, Wakabayashi G. Laparoscopic major hepa-tectomy: a systematic literature review and comparison of 3 techniques. Ann Surg. 2013;257:205-213. 220. Vanounou T, Steel JL, Nguyen KT, Tsung A, Marsh JW, Geller DA, Gamblin TC. Comparing the clinical and economic impact of laparoscopic versus open liver resection. Ann Surg Oncol. 2010;17:998-1009. 221. Cleary SP, Han HS, Yamamoto M, Wakabayashi G, Asbun HJ. The comparative costs of laparoscopic and open liver resec-tion: a report for the 2nd International Consensus Con-ference on Laparoscopic Liver Resection. Surg Endosc. 2016;30(11):4691-4696. 222. Cherqui D, Soubrane O,

1	of laparoscopic and open liver resec-tion: a report for the 2nd International Consensus Con-ference on Laparoscopic Liver Resection. Surg Endosc. 2016;30(11):4691-4696. 222. Cherqui D, Soubrane O, Husson E, et al. Laparoscopic living donor hepatectomy for liver transplantation in children. Lancet 2002;359:392-396. 223. Koffron AJ, Kung R, Baker T, Fryer J, Clark L, Abecassis M. Laparoscopic-assisted right lobe donor hepatectomy. Am J Transplant. 2006;6:2522-2525. 224. Soubrane O, Perdigao Cotta F, Scatton O. Pure laparo-scopic right hepatectomy in a living donor. Am J Transplant. 2013;13:2467-2471.Brunicardi_Ch31_p1345-p1392.indd 139220/02/19 2:37 PM

1	Gallbladder and the Extrahepatic Biliary SystemKelly R. Haisley and John G. Hunter 32chapterANATOMYGallbladderThe gallbladder is a pear-shaped sac that measures around 7 to 10 cm long, with an average capacity of 30 to 50 mL. When obstructed, the gallbladder can distend markedly and contain up to 300 mL of fluid. The gallbladder is located in an anatomic fossa on the inferior surface of the liver. Cantle’s line, a vertical plane running from the gallbladder fossa anteriorly to the infe-rior vena cava (IVC) posteriorly divides the liver into right and left lobes. The gallbladder itself is divided into four anatomic areas: the fundus, the body, the infundibulum, and the neck. The fundus is the rounded, blind end that normally extends 1 to 2 cm beyond the liver’s margin and contains most of the smooth muscle of the organ. The body functions as the main storage area and contains most of the elastic tissue allowing for disten-tion. As the body tapers towards the neck of the gallbladder, a

1	the smooth muscle of the organ. The body functions as the main storage area and contains most of the elastic tissue allowing for disten-tion. As the body tapers towards the neck of the gallbladder, a mucosal outpouching is present at the junction of the neck and the cystic duct, known as the infundibulum or Hartmann’s pouch. Beyond this, the neck of the gallbladder lies in the deep-est part of the gallbladder fossa and can extend slightly into the free portion of the hepatoduodenal ligament, where it connects with the cystic duct (Fig. 32-1).1The same peritoneal lining that covers the liver extends to cover the fundus and the inferior surface of the gallblad-der. Occasionally, part or all of the gallbladder is embedded deep inside the liver parenchyma (an intrahepatic gallbladder). Rarely, the gallbladder has a complete peritoneal covering on all sides and is suspended in a mesentery off the inferior surface of the liver.The mucosal lining of the gallbladder is formed by a single,

1	the gallbladder has a complete peritoneal covering on all sides and is suspended in a mesentery off the inferior surface of the liver.The mucosal lining of the gallbladder is formed by a single, highly redundant, simple columnar epithelium that contains cholesterol and fat globules. The mucus secreted into the gall-bladder originates in tubuloalveolar glands that are found in the mucosal lining of the infundibulum and neck of the gallbladder, but are absent from the body and fundus. The epithelial lining of the gallbladder is supported by a lamina propria. The gallblad-der differs histologically from the rest of the gastrointestinal (GI) tract in that it lacks a muscularis mucosa and submucosa. The muscular layer has circular, longitudinal, and oblique fibers, but without well-defined layers. The adventitia contains connective tissue, nerves, vessels, lymphatics, and adipocytes. The gallbladder is covered by serosa except where the gallblad-der is embedded in the liver.The cystic

1	layers. The adventitia contains connective tissue, nerves, vessels, lymphatics, and adipocytes. The gallbladder is covered by serosa except where the gallblad-der is embedded in the liver.The cystic artery that supplies the gallbladder is usually a branch of the right hepatic artery (>90% of the time). The course of the cystic artery may vary, but it nearly always is found within the hepatocystic triangle (triangle of Calot), the area bound by the cystic duct, common hepatic duct, and the inferior edge of the liver. When the cystic artery reaches the neck of the gall-bladder, it divides into anterior and posterior divisions. Venous return is carried either through small veins that enter directly into the liver or, rarely, to a large cystic vein that carries blood back to the portal vein. Gallbladder lymphatics drain into nodes Anatomy 1393Gallbladder / 1393Bile Ducts / 1394Anatomic Variants / 1396Physiology 1396Bile Formation and Composition / 1396Gallbladder Function / 1397Sphincter

1	Gallbladder lymphatics drain into nodes Anatomy 1393Gallbladder / 1393Bile Ducts / 1394Anatomic Variants / 1396Physiology 1396Bile Formation and Composition / 1396Gallbladder Function / 1397Sphincter of Oddi / 1397Diagnostic Studies 1398Blood Tests / 1398Transabdominal Ultrasonography / 1398Computed Tomography / 1398Hepatobiliary Scintigraphy / 1399Magnetic Resonance Imaging / 1399Endoscopic Retrograde Cholangiopancreatography / 1400Endoscopic Choledochoscopy / 1400Endoscopic Ultrasound / 1401Percutaneous Transhepatic Cholangiography / 1401Gallstone Disease 1401Prevalence and Incidence / 1401Natural History / 1401Gallstone Formation / 1402Symptomatic Gallstones / 1404Cholangiohepatitis / 1410Procedural Interventions for Gallstone Disease 1410Percutaneous Transhepatic Cholecystostomy Tubes / 1410Endoscopic Interventions / 1410Cholecystectomy / 1410Common Bile Duct Exploration / 1413Common Bile Duct Drainage Procedures / 1414Other Benign Diseases and Lesions 1414Biliary Dyskinesia

1	/ 1410Endoscopic Interventions / 1410Cholecystectomy / 1410Common Bile Duct Exploration / 1413Common Bile Duct Drainage Procedures / 1414Other Benign Diseases and Lesions 1414Biliary Dyskinesia and Sphincter of Oddi Dysfunction / 1414Acalculous Cholecystitis / 1415Choledochal (Biliary) Cysts / 1417Primary Sclerosing Cholangitis / 1417Bile Duct Strictures / 1418Injury to the Biliary Tract 1419Gallbladder / 1419Extrahepatic Bile Ducts / 1419Tumors 1421Carcinoma of the Gallbladder / 1421Cholangiocarcinoma / 1423Brunicardi_Ch32_p1393-p1428.indd 139311/02/19 2:42 PM 1394at the neck of the gallbladder. Frequently, a visible lymph node (Lund’s or Mascagni’s node, often referred to as Calot’s node) overlies the insertion of the cystic artery into the gallbladder wall. The gallbladder receives parasympathetic, sympathetic and sensory innervation through nerve fibers running largely through the gastro hepatic ligament. Parasympathetic (cholinergic) fibers arise from the hepatic branches

1	parasympathetic, sympathetic and sensory innervation through nerve fibers running largely through the gastro hepatic ligament. Parasympathetic (cholinergic) fibers arise from the hepatic branches of the vagus nerve to stimulate activity in the gallbladder, bile ducts, and liver. These vagal branches also have peptide-containing nerves contain-ing agents such as substance P, somatostatin, enkephalins, and vasoactive intestinal polypeptide (VIP).2 The sympathetic and sensory braches of the gallbladder, liver, and bile ducts pass through the celiac plexus and control gallbladder relaxation and mediate the pain of biliary colic.Bile DuctsThe extrahepatic biliary tree consists of the right and left hepatic ducts, the common hepatic duct, the cystic duct, and the com-mon bile duct. Exiting the liver, the left hepatic duct is longer than the right and has a greater propensity for dilatation as a consequence of distal obstruction. The two ducts join close to their emergence from the liver to

1	liver, the left hepatic duct is longer than the right and has a greater propensity for dilatation as a consequence of distal obstruction. The two ducts join close to their emergence from the liver to form the common hepatic duct. The common hepatic duct typically extends 1 to 4 cm, has a diameter of approximately 4 mm, and lies anterior to the portal vein and to the right of the hepatic artery.The cystic duct exits the gallbladder and joins the common hepatic duct at an acute angle to form the common bile duct. The segment of the cystic duct immediately adjacent to the gall-bladder neck bears a variable number of mucosal folds called the spiral valves of Heister. While they do not have any valvular function, they can make cannulation of the cystic duct difficult. The length and course of the cystic duct can be quite variable. It may be short or absent and have a high union with the hepatic duct, or it may be long and running parallel to, behind, or spiral-ing around to the common

1	of the cystic duct can be quite variable. It may be short or absent and have a high union with the hepatic duct, or it may be long and running parallel to, behind, or spiral-ing around to the common hepatic duct before joining it, some-times as far distally as at the duodenum. Variations of the cystic duct and its point of union with the common hepatic duct are surgically important and misidentification can lead to bile duct injuries (Fig. 32-2).The union of the cystic duct and the common hepatic duct marks the start of the common bile duct. This segment is typi-cally about 7 to 11 cm in length and 5 to 10 mm in diameter, ijoklmabdefgchqrptnsFigure 32-1. Anterior aspect of the biliary anatomy. a = right hepatic duct; b = left hepatic duct; c = common hepatic duct; d = portal vein; e = proper hepatic artery; f = gastroduodenal artery; g = right gastro-epiploic artery; h = common bile duct; i = fundus of the gallbladder; j = body of gallbladder; k = infundibulum of the gallbladder; l =

1	hepatic artery; f = gastroduodenal artery; g = right gastro-epiploic artery; h = common bile duct; i = fundus of the gallbladder; j = body of gallbladder; k = infundibulum of the gallbladder; l = cystic duct; m = cystic artery; n = superior pancreaticoduodenal artery; o = neck of the gallbladder; p = pancreatic duct; q = common hepatic artery; r = right gastric artery; s = ampulla of Vater; t = supraduodenal artery. Note the situation of the hepatic bile duct confluence anterior to the right branch of the portal vein, and the posterior course of the right hepatic artery behind the common hepatic duct.Key Points1 The physiology of the gallbladder, biliary tree, and sphincter of Oddi are regulated by a complex interplay of hormones and neuronal inputs designed to coordinate bile release with food consumption. Dysfunctions related to this activity are linked to the development of gallbladder pathologies as de-scribed in this chapter.2 In Western countries, the most common type of

1	with food consumption. Dysfunctions related to this activity are linked to the development of gallbladder pathologies as de-scribed in this chapter.2 In Western countries, the most common type of gallstones are cholesterol stones. The pathogenesis of these stones relates to supersaturation of bile with cholesterol and sub-sequent precipitation.3 Laparoscopic cholecystectomy has been demonstrated to be safe and effective, and it has become the treatment of choice for symptomatic gallstones. Knowledge of the various ana-tomic anomalies of the cystic duct and artery is critical in guiding the dissection of these structures and avoiding injury to the common bile duct during cholecystectomy.4 Common bile duct injuries, although uncommon, can be devastating to patients. Proper exposure of the hepatocystic (Calot’s) triangle to obtain the critical view of safety and careful identification of the anatomic structures are keys to avoiding these injuries. Once a bile duct injury is diag-nosed,

1	hepatocystic (Calot’s) triangle to obtain the critical view of safety and careful identification of the anatomic structures are keys to avoiding these injuries. Once a bile duct injury is diag-nosed, the best outcomes are seen at large referral centers with experienced biliary surgeons, and patient transfer may be required.5 The main risk factor for gallbladder disease in Western coun-tries is cholelithiasis. The main complications include cho-lecystitis, choledocholithiasis, cholangitis, and biliary pancreatitis. Cholelithiasis is also the major risk factor for the development of gallbladder cancer.6 Carcinomas of the gallbladder or bile ducts generally have a poor prognosis because patients usually present late in the disease process and have poor response to chemotherapy and radiation. Surgery offers the best chance for survival and has good long-term outcomes in patients with early-stage disease.Brunicardi_Ch32_p1393-p1428.indd 139411/02/19 2:42 PM 1395GALLBLADDER AND THE

1	Surgery offers the best chance for survival and has good long-term outcomes in patients with early-stage disease.Brunicardi_Ch32_p1393-p1428.indd 139411/02/19 2:42 PM 1395GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32though its diameter can increase slightly with age and follow-ing cholecystectomy. The upper third (supraduodenal portion) passes downward in the free edge of the hepatoduodenal liga-ment, to the right of the hepatic artery and anterior to the portal vein. The middle third (retroduodenal portion) of the common bile duct curves behind the first portion of the duodenum and diverges laterally from the portal vein and the hepatic arteries. The lower third (pancreatic portion) can curve behind the head of the pancreas in a groove, or traverse through it to enter the wall of the second portion of the duodenum. The duct then runs obliquely downward within the wall of the duodenum for 1 to 2 cm before opening on a papilla of mucous membrane (ampulla of Vater),

1	the wall of the second portion of the duodenum. The duct then runs obliquely downward within the wall of the duodenum for 1 to 2 cm before opening on a papilla of mucous membrane (ampulla of Vater), about 10 cm distal to the pylorus.The union of the common bile duct and the main pan-creatic duct follows one of three configurations. In about 70% of people, these ducts unite outside the duodenal wall and tra-verse the duodenal wall as a single duct. In about 20%, they join within the duodenal wall and have a short or no common duct, but open through the same opening into the duodenum. In about 10%, they exit via separate openings into the duodenum, termed pancreas divisum. The sphincter of Oddi, a thick coat of circular smooth muscle, surrounds the common bile duct at the ampulla of Vater (Fig. 32-3). It controls the flow of bile, and in some cases pancreatic juice, into the duodenum.The extrahepatic bile ducts are lined by a columnar mucosa with numerous mucous glands that are

1	Vater (Fig. 32-3). It controls the flow of bile, and in some cases pancreatic juice, into the duodenum.The extrahepatic bile ducts are lined by a columnar mucosa with numerous mucous glands that are concentrated in the com-mon bile duct. A fibro areolar tissue containing scant smooth muscle cells surrounds the mucosa. A distinct muscle layer is not present in the human common bile duct. The arterial supply to the bile ducts is derived from the gastroduodenal and the right hepatic arteries, with major trunks running along the medial and lateral walls of the common duct (sometimes referred to as 3 o’clock and 9 o’clock). The nerve supply to the common bile duct is the same as for the gallbladder, with the density of nerve fibers and ganglia increasing near the sphincter of Oddi.1,2ABCDEFGHFigure 32-2. Variations of the cystic duct anatomy. A. Low junction between the cystic duct and common hepatic duct. B. Cystic duct adher-ent to the common hepatic duct. C. High junction between the

1	32-2. Variations of the cystic duct anatomy. A. Low junction between the cystic duct and common hepatic duct. B. Cystic duct adher-ent to the common hepatic duct. C. High junction between the cystic and the common hepatic duct. D. Cystic duct drains into right hepatic duct. E. Long cystic duct that joins common hepatic duct behind the duodenum. F. Absence of cystic duct. G. Cystic duct crosses posterior to common hepatic duct and joins it anteriorly. H. Cystic duct courses anterior to common hepatic duct and joins it posteriorly.Pancreatic ductCommonbile duct Sphincterof OddiDuodenumAmpulla ofVaterDuodenal wallFigure 32-3. The sphincter of Oddi.Brunicardi_Ch32_p1393-p1428.indd 139511/02/19 2:43 PM 1396SPECIFIC CONSIDERATIONSPART IIABCDEFFigure 32-5. Variations in the arterial supply to the gallbladder. A. Cystic artery from right hepatic artery, about 80% to 90%. B. Cystic artery off the right hepatic artery arising from the superior mesenteric artery (accessory or replaced),

1	to the gallbladder. A. Cystic artery from right hepatic artery, about 80% to 90%. B. Cystic artery off the right hepatic artery arising from the superior mesenteric artery (accessory or replaced), about 10%. C. Two cystic arteries, one from the right hepatic, the other from the common hepatic artery, rare. D. Two cystic arteries, one from the right hepatic, the other from the left hepatic artery, rare. E. The cystic artery branching from the right hepatic artery and running anterior to the common hepatic duct, rare. F. Two cystic arteries arising from the right hepatic artery, rare.Anatomic VariantsThe classic description of the extrahepatic biliary tree and its arteries applies only in about one-third of patients.3 The gall-bladder may have abnormal positions, be intrahepatic, be rudi-mentary (a small, nonfunctional hypoplastic remnant), or have anomalous forms or duplications. A partially or completely intrahepatic gallbladder is associated with an increased inci-dence of

1	be rudi-mentary (a small, nonfunctional hypoplastic remnant), or have anomalous forms or duplications. A partially or completely intrahepatic gallbladder is associated with an increased inci-dence of cholelithiasis, and may be encountered at the time of cholecystectomy. Isolated congenital absence of the gallblad-der is very rare, with a reported incidence of 0.03%. Before the diagnosis is made, the presence of an intrahepatic gallbladder or anomalous position must first be ruled out. Duplication of the gallbladder with two separate cavities and two separate cystic ducts has an incidence of about one in every 4000 persons. This occurs in two major varieties: the more common form in which each gallbladder has its own cystic duct that empties indepen-dently into the same or different parts of the extrahepatic biliary tree, and the less common variant in which the two cystic ducts merge before they enter the common bile duct. Duplication is only clinically important when some pathologic

1	of the extrahepatic biliary tree, and the less common variant in which the two cystic ducts merge before they enter the common bile duct. Duplication is only clinically important when some pathologic process affects one or both organs. Even rarer variants include a left-sided gallbladder (often with a cystic duct that empties into the left hepatic or common bile duct), retrodisplacement of the gall-bladder at the posterior-inferior surface of the liver, transverse positioning of the gallbladder, or a floating gallbladder in which the gallbladder is hanging by a mesentery (Fig. 32-4).Additional small bile ducts (of Luschka) may drain directly from the liver fossa into the body of the gallbladder. If present, but not recognized at the time of a cholecystectomy, a bile leak and subsequent accumulation of bile (biloma) may occur in the abdomen. An accessory right hepatic duct occurs in about 5% of cases. Variations in how the common bile duct enters the duode-num are described earlier, in

1	of bile (biloma) may occur in the abdomen. An accessory right hepatic duct occurs in about 5% of cases. Variations in how the common bile duct enters the duode-num are described earlier, in the “Bile Ducts” section.Anomalies of the hepatic artery and the cystic artery are quite common, occurring in as many as 50% of cases. While the right hepatic artery usually originates from the proper hepatic branch of the celiac trunk, up to 20% of patients will have a replaced right hepatic artery coming off the superior mesenteric artery. In about 5% of cases, there are two right hepatic arteries, one from the proper hepatic artery and the other from the superior mesenteric artery (accessory right hepatic artery). While the right hepatic artery typically runs posterior to the bile ducts, variations may allow it to course anterior to the common duct, making it vulnerable during surgical procedures, particularly if it runs par-allel to the cystic duct or in the mesentery of the gallbladder. The

1	may allow it to course anterior to the common duct, making it vulnerable during surgical procedures, particularly if it runs par-allel to the cystic duct or in the mesentery of the gallbladder. The cystic artery arises from the right hepatic artery in about 90% of cases, but it may arise from the left hepatic, common hepatic, gastroduodenal, or superior mesenteric arteries (Fig. 32-5).3Figure 32-4. Floating gallbladder suspended on mesenteryPHYSIOLOGYBile Formation and CompositionThe liver produces bile continuously and excretes it into the bile canaliculi. Bile leaves the liver through the right and left hepatic ducts, into the common hepatic duct and then the com-mon bile duct. With an intact sphincter of Oddi, tonic contrac-tion diverts bile flow into the gallbladder for storage, while mealtime stimulation allows for its passage into the duode-num. The normal adult consuming an average diet produces 500 to 1000 mL of bile a day. The secretion of bile is respon-sive to neurogenic,

1	mealtime stimulation allows for its passage into the duode-num. The normal adult consuming an average diet produces 500 to 1000 mL of bile a day. The secretion of bile is respon-sive to neurogenic, hormonal, and chemical stimuli. Parasym-pathetic stimulation from the hepatic branches of the vagus nerve increases secretion of bile, whereas sympathetic nerve stimulation via the celiac plexus results in decreased bile flow. Hydrochloric acid, partly digested proteins, and fatty acids entering the duodenum from the stomach after a meal stimulate the release of secretin from the S-cells of the duodenum, and increases bile production and flow.Bile is mainly composed of water, mixed with bile salts and acids, cholesterol, phospholipids (lecithin), proteins, and bilirubin. It also contains several minor components such as Brunicardi_Ch32_p1393-p1428.indd 139611/02/19 2:43 PM 1397GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32electrolytes and vitamins. Sodium, potassium, calcium,

1	minor components such as Brunicardi_Ch32_p1393-p1428.indd 139611/02/19 2:43 PM 1397GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32electrolytes and vitamins. Sodium, potassium, calcium, and chlorine have the same concentration in bile as in plasma or extracellular fluid. The pH of hepatic bile is usually neutral or slightly alkaline, though a high protein diet will shift the bile to a more acidic ph. The primary bile salts, cholate and che-nodeoxycholate, are synthesized in the liver from cholesterol metabolism. They are conjugated there with taurine and glycine and act within the bile as anions (bile acids) that are balanced by sodium. These bile acids are then excreted into the bile by hepatocytes and aid in the digestion and absorption of fats in the intestines.4 About 80% of the secreted conjugated bile acids are reabsorbed in the terminal ileum. The remainder is dehydroxyl-ated (deconjugated) by gut bacteria, forming the secondary bile acids deoxycholate and

1	80% of the secreted conjugated bile acids are reabsorbed in the terminal ileum. The remainder is dehydroxyl-ated (deconjugated) by gut bacteria, forming the secondary bile acids deoxycholate and lithocholate. These are absorbed in the colon and can then be transported back to the liver. Eventu-ally, about 95% of the bile acid pool is reabsorbed, the so-called enterohepatic circulation. Only a small amount (5%) is excreted in the stool, allowing the relatively small quantity of bile acids produced to have maximal effect.The color of the bile is due to the presence of the pigment bilirubin (orange or yellow) and its oxidized form, biliverdin (green), which are the metabolic products of the breakdown of hemoglobin, and are present in bile in concentrations 100 times greater than in plasma. Bilirubin conjugated in the liver can be excreted through the urine as urobilinogen (yellow). Remaining excess bile pigment passes into the intestines where bacteria con-vert it into stercobilinogen

1	Bilirubin conjugated in the liver can be excreted through the urine as urobilinogen (yellow). Remaining excess bile pigment passes into the intestines where bacteria con-vert it into stercobilinogen (brown), which is excreted through the stool.Gallbladder FunctionThe gallbladder, bile ducts, and the sphincter of Oddi act together to store and regulate the flow of bile. The main function of the gallbladder is to concentrate and store hepatic bile in order to deliver it in a coordinated fashion to the duode-num in response to a meal. Absorption and Secretion. In the fasting state, approxi-mately 80% of the bile secreted by the liver is stored in the gallbladder. This storage is made possible by the fact that the gallbladder mucosa has the greatest absorptive power per unit area of any structure in the body. It rapidly absorbs sodium, chloride, and water against significant gradients, concentrating the bile as much as 10-fold and leading to a marked change in bile composition. This rapid

1	in the body. It rapidly absorbs sodium, chloride, and water against significant gradients, concentrating the bile as much as 10-fold and leading to a marked change in bile composition. This rapid absorptive capacity is one of the protective mechanisms that prevent a potentially dangerous rise in pressure within the biliary system as bile is produced and stored. In addition, gradual relaxation of the gallbladder as well as routine emptying of the gallbladder’s excess bile stores during the fasting period also play a role in maintaining a low resting intraluminal pressure in the biliary tree.The mucosal cells of the gallbladder itself secrete at least two important products into the gallbladder lumen: glycopro-teins and hydrogen ions. The mucosal glands in the infundibu-lum and the neck of the gallbladder secrete mucus glycoproteins that are believed to protect the mucosa from the corrosive action of bile and to facilitate the passage of bile through the cystic duct. This same mucus

1	of the gallbladder secrete mucus glycoproteins that are believed to protect the mucosa from the corrosive action of bile and to facilitate the passage of bile through the cystic duct. This same mucus creates the colorless “white bile” seen in hydrops of the gallbladder as a result of cystic duct obstruction blocking the entry of bile pigments into the gallbladder. The transport of hydrogen ions by the gallbladder epithelium also plays an important role in decreasing the pH of stored bile. This acidification helps prevent the precipitation of calcium salts, which can act as a nidus for stone formation.4Motor Activity. Normal gallbladder filling is facilitated by tonic contraction of the sphincter of Oddi, which creates a small but effective pressure gradient between the bile ducts and the gallbladder. In association with phase II of the interdigestive migrating myenteric motor complex (MMC) in the gut, the gallbladder repeatedly empties small volumes of bile into the duodenum. This

1	gallbladder. In association with phase II of the interdigestive migrating myenteric motor complex (MMC) in the gut, the gallbladder repeatedly empties small volumes of bile into the duodenum. This process is mediated at least in part by the hormone motilin. In response to a meal, the gallbladder delivers larger volumes to the intestine by a combination of gallbladder contraction and synchronized sphincter of Oddi relaxation. One of the main stimuli to this coordinated effort of gallbladder emp-tying is the hormone cholecystokinin (CCK). CCK is released endogenously from the enteroendocrine cells in the duodenum in response to a meal.5 When stimulated by eating, the gallbladder empties 50% to 70% of its contents within 30 to 40 minutes. Over the following 60 to 90 minutes, the gallbladder gradually refills as CCK levels drop. Other minor hormonal and neural pathways also are involved in the coordinated action of the gall-bladder and the sphincter of Oddi. Defects in the motor activity

1	gradually refills as CCK levels drop. Other minor hormonal and neural pathways also are involved in the coordinated action of the gall-bladder and the sphincter of Oddi. Defects in the motor activity of the gallbladder that inhibit correct emptying are thought to play a role in cholesterol nucleation and gallstone formation.Neurohormonal Regulation. Neurally mediated reflexes are very important in maintaining the functions of the gallbladder, sphincter of Oddi, stomach, and duodenum to coordinate the flow of bile into the intestines at the correct times. The vagus nerve stimulates contraction of the gallbladder by parasympa-thetic innervation while splanchnic sympathetic nerves from the celiac plexus are inhibitory to its motor activity. For this reason, parasympathomimetic or cholinergic drugs, including nicotine and caffeine, contract the gallbladder. Conversely, anticholiner-gic drugs such as atropine lead to gallbladder relaxation. Antral distention of the stomach causes both

1	drugs, including nicotine and caffeine, contract the gallbladder. Conversely, anticholiner-gic drugs such as atropine lead to gallbladder relaxation. Antral distention of the stomach causes both gallbladder contraction and relaxation of the sphincter of Oddi.In addition to neural inputs, hormonal receptors are located on the smooth muscles, vessels, nerves, and epithelium of the gallbladder and biliary tree. CCK is a peptide that comes from the enteroendocrine cells of the duodenum and proximal jejunum. CCK is released into the bloodstream in response to the presence of hydrochloric acid, fat, and amino acids in the duodenum.6 CCK has a plasma half-life of 2 to 3 minutes and is metabolized by both the liver and the kidneys. CCK acts directly on smooth muscle receptors of the gallbladder and stimulates gallbladder contraction. It also relaxes the terminal bile duct, the sphincter of Oddi, and the duodenum to allow forward bile flow. CCK stimulation of the gallbladder and biliary tree

1	and stimulates gallbladder contraction. It also relaxes the terminal bile duct, the sphincter of Oddi, and the duodenum to allow forward bile flow. CCK stimulation of the gallbladder and biliary tree is also mediated by cholinergic vagal neurons. For this reason, patients who have had a vagotomy may have a diminished response to CCK stimulation, resulting in an increase in the size and volume of the gallbladder.Hormones such as vasoactive intestinal polypeptide (VIP) and somatostatin are potent inhibitors of gallbladder contrac-tion. Patients treated with somatostatin analogues and those with somatostatinomas have a high incidence of gallstones, presum-ably due to the inhibition of gallbladder contraction and empty-ing. Other hormones such as substance P and enkephalin affect gallbladder motility, but their exact physiologic role is less clear.5Sphincter of OddiThe sphincter of Oddi regulates the flow of bile and pancre-atic juice into the duodenum, prevents the regurgitation of

1	motility, but their exact physiologic role is less clear.5Sphincter of OddiThe sphincter of Oddi regulates the flow of bile and pancre-atic juice into the duodenum, prevents the regurgitation of duodenal contents into the biliary tree, and diverts bile into 1Brunicardi_Ch32_p1393-p1428.indd 139711/02/19 2:43 PM 1398SPECIFIC CONSIDERATIONSPART IIthe gallbladder. It is a complex structure that is functionally independent from the duodenal musculature and creates a high-pressure zone between the bile duct and the duodenum. The sphincter of Oddi spans approximately 4 to 6 mm in length and has a basal resting pressure of about 13 mmHg above the duodenal pressure. On manometry, the sphincter shows pha-sic contractions with a frequency of about four per minute and amplitude of 12 to 140 mmHg. The spontaneous motility of the sphincter of Oddi is regulated by the interstitial cells of Cajal through intrinsic and extrinsic inputs from hormones and neu-rons acting on the smooth muscle

1	mmHg. The spontaneous motility of the sphincter of Oddi is regulated by the interstitial cells of Cajal through intrinsic and extrinsic inputs from hormones and neu-rons acting on the smooth muscle cells.7 Relaxation occurs in response to raising levels of the gastrointestinal hormones CCK, glucagon, and secretin. This leads to diminished amplitude of phasic contractions and reduced basal pressure of the sphinc-ter, allowing increased flow of bile into the duodenum. During fasting, the sphincter of Oddi activity is coordinated with the periodic partial gallbladder emptying that occurs during phase II of the migrating myoelectric motor complexes.8 Pharmacologic administration of certain gastrointestinal hormones, such as glucagon, can temporarily decrease sphincter of Oddi baseline pressure and facilitate diagnostic studies.7DIAGNOSTIC STUDIESA variety of diagnostic modalities are available for the patient with suspected disease of the gallbladder or bile ducts. In 1924, the diagnosis

1	and facilitate diagnostic studies.7DIAGNOSTIC STUDIESA variety of diagnostic modalities are available for the patient with suspected disease of the gallbladder or bile ducts. In 1924, the diagnosis of gallstones was revolutionized by the intro-duction of oral cholecystography by Graham and Cole. For decades, it was the mainstay of investigation for gallstones. It involved oral administration of a radiopaque compound that is absorbed, excreted by the liver, and passed into the gallblad-der. Stones are noted on a film as filling defects in a visualized, pacified gallbladder. In the later half of the 20th century, bili-ary imaging improved dramatically with the development of hepatobiliary scintigraphy (radionucleotide scanning), as well as transhepatic and endoscopic retrograde cholangiography (ERCP), which allowed for more detailed imaging of the bili-ary tree. Later, ultrasonography, computed tomography (CT), and magnetic resonance imaging (MRI) would further improve the ability to

1	(ERCP), which allowed for more detailed imaging of the bili-ary tree. Later, ultrasonography, computed tomography (CT), and magnetic resonance imaging (MRI) would further improve the ability to image the biliary tract.Blood TestsWhen patients with suspected diseases of the gallbladder or the extrahepatic biliary tree are evaluated, a complete blood count and liver function tests are routinely requested. An elevated white blood cell (WBC) count may indicate or raise suspicion of acute cholecystitis (infection within the gallbladder). If asso-ciated with an elevation of bilirubin, alkaline phosphatase, and transaminases, cholangitis (infection within the biliary tree) should be suspected. Cholestasis (an obstruction to bile flow) is generally characterized by an elevation of conjugated bilirubin and a rise in alkaline phosphatase, but it may have no transa-minitis. Such a pattern may suggest choledocholithiasis (stones in the common bile duct) or an obstructing lesion such as a

1	bilirubin and a rise in alkaline phosphatase, but it may have no transa-minitis. Such a pattern may suggest choledocholithiasis (stones in the common bile duct) or an obstructing lesion such as a stricture or cholangiocarcinoma. In patients with simple symp-tomatic cholelithiasis, biliary colic, or chronic cholecystitis (a chronic inflammatory state of the gallbladder without infec-tion), blood tests will often be normal.Transabdominal UltrasonographyTransabdominal ultrasound is the initial investigation of any patient suspected to have disease of the biliary tree.9 It is non-invasive, painless, does not submit the patient to radiation, and can be performed on critically ill patients. Adjacent organs can also frequently be examined at the same time. However, its reliability and interpretation are dependent upon the skills and experience of the operator. In addition, obese patients, patients with ascites, and patients with distended bowel may be difficult to examine with ultrasound as

1	are dependent upon the skills and experience of the operator. In addition, obese patients, patients with ascites, and patients with distended bowel may be difficult to examine with ultrasound as the quality of the images obtained in these situations can be poor.Ultrasound will show stones in the gallbladder with sen-sitivity and specificity of >90%, and can also reliably detect other pathologies of the biliary tree. Stones are acoustically dense and reflect the ultrasound waves back to the ultrasonic transducer. Because stones block the passage of sound waves to the region behind them, they also produce an acoustic shadow (Fig. 32-6). Stones move with changes in position. Polyps, on the other hand, may be calcified and reflect shadows, but they do not move with change in posture. Some stones form a layer in the gallbladder; others a form sediment or sludge. A thickened gallbladder wall, pericholecystic fluid, and local tenderness with direct pressure by the ultrasound probe over the

1	form a layer in the gallbladder; others a form sediment or sludge. A thickened gallbladder wall, pericholecystic fluid, and local tenderness with direct pressure by the ultrasound probe over the fundus of the gallbladder (sonographic Murphy’s sign) may indicate acute cholecystitis. When a stone obstructs the neck of the gallbladder, the gallbladder may become very large, but thin walled. A contracted, thick-walled gallbladder can be indicative of chronic cholecystitis.The extrahepatic bile ducts are also well visualized by transabdominal ultrasound, with the exception of the retro-duodenal portion. Dilation of the biliary tree in a patient with jaundice suggests an extrahepatic obstruction as the cause for the jaundice. Frequently, the site and, sometimes, the cause of the obstruction can be determined by ultrasound. Small stones in the common bile duct frequently get lodged at the distal end of it, behind the duodenum, and are, therefore, difficult to detect. A dilated common bile

1	be determined by ultrasound. Small stones in the common bile duct frequently get lodged at the distal end of it, behind the duodenum, and are, therefore, difficult to detect. A dilated common bile duct on ultrasound, small stones in the gallbladder, and a classic clinical presentation allows one to assume that a stone or stones are causing the obstruction. Peri-ampullary tumors can be difficult to diagnose on ultrasound, but above the retroduodenal portion, the level of obstruction and the cause may be visualized quite well. Ultrasound can also be helpful in evaluating tumor invasion and flow in the portal vein, an important guideline for resectability of periampullary tumors.10Computed TomographyAbdominal CT scans are frequently used in the workup of undif-ferentiated abdominal pain and thus often diagnose gallbladder Figure 32-6. An ultrasonography of the gallbladder. White arrows indicate stones within the gallbladder; black arrowheads show acoustic shadows from

1	abdominal pain and thus often diagnose gallbladder Figure 32-6. An ultrasonography of the gallbladder. White arrows indicate stones within the gallbladder; black arrowheads show acoustic shadows from stones.Brunicardi_Ch32_p1393-p1428.indd 139811/02/19 2:43 PM 1399GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32disease. CT scanning is inferior to ultrasonography in diagnos-ing gallstones but is similar in sensitivity for acute cholecystitis. The major application of CT scan, however, is to define the course and status of the extrahepatic biliary tree and adjacent structures, and to evaluate for alternate causes of a patients clini-cal presentation.11 CT is also the initial test of choice in evaluat-ing patients with suspected malignancy of the gallbladder, the extrahepatic biliary system, or nearby organs such as the head of the pancreas. Use of CT scan is an integral part of the dif-ferential diagnosis of obstructive jaundice of unknown origin (Fig. 32-7).Hepatobiliary

1	system, or nearby organs such as the head of the pancreas. Use of CT scan is an integral part of the dif-ferential diagnosis of obstructive jaundice of unknown origin (Fig. 32-7).Hepatobiliary ScintigraphyHepatobiliary scintigraphy, or hepatobiliary iminodiace-tic acid (HIDA) scanning, is another option for noninvasive evaluation of the liver, gallbladder, bile ducts, and duode-num that provides both anatomic and functional information. 99mTechnetium-labeled derivatives of iminodiacetic acid are injected intravenously, taken up by the Kupffer cells in the liver, and excreted in the bile. Uptake by the liver is usually detected within 10 minutes, and the gallbladder, bile ducts, and duodenum are typically visualized within 60 minutes in fasting subjects. The primary use of biliary scintigraphy is in the diag-nosis of acute cholecystitis, which appears as a nonvisualized gallbladder, with prompt filling of the common bile duct and duodenum. The lack of gallbladder filling is due to

1	is in the diag-nosis of acute cholecystitis, which appears as a nonvisualized gallbladder, with prompt filling of the common bile duct and duodenum. The lack of gallbladder filling is due to inflamma-tory closure of the cystic duct preventing bile backflow into the gallbladder (Fig. 32-8). Evidence of cystic duct obstruction on biliary scintigraphy is highly diagnostic for acute cholecys-titis. The sensitivity and specificity for the diagnosis are about 95% each. False-positive results can occur in patients in the nonfasting state, those receiving parenteral nutrition, or in the setting of gallbladder stasis, recent narcotic use, or alcoholism. Filling of the gallbladder and common bile duct with delayed or absent filling of the duodenum indicates an obstruction at the ampulla. Biliary leaks as a complication of surgery of the gallbladder or the biliary tree can be confirmed and frequently localized by biliary scintigraphy.12 HIDA scanning with evalu-ation of the gallbladder ejection

1	as a complication of surgery of the gallbladder or the biliary tree can be confirmed and frequently localized by biliary scintigraphy.12 HIDA scanning with evalu-ation of the gallbladder ejection fraction with or without CCK provocation may also be helpful in diagnosing chronic bili-ary dyskinesia in patients with atypical symptoms. While an ejection fraction of <35% is considered abnormal, the exact interpretations and clinical implications of this finding remain a matter of some debate.13Magnetic Resonance ImagingAvailable since the mid-1990s, MRI provides anatomic details of the liver, gallbladder, and pancreas similar to those obtained from CT. Many MRI techniques (i.e., heavily T2-weighted sequences, pulse sequences with or without contrast materials) can generate high-resolution anatomic images of the biliary tree and the pancreatic duct. MRI with magnetic resonance chol-angiopancreatography (MRCP) offers a focused, noninvasive test for the diagnosis of biliary tract and

1	anatomic images of the biliary tree and the pancreatic duct. MRI with magnetic resonance chol-angiopancreatography (MRCP) offers a focused, noninvasive test for the diagnosis of biliary tract and pancreatic disease (Fig. 32-9).14 It has a sensitivity and specificity of 95% and 89%, respectively, for detecting choledocholithiasis.15 In many centers, MRCP is the preferred imaging modality for precise evaluation of biliary and pancreatic duct pathology, reserving endoscopic retrograde cholangiopancreatography (ERCP) for therapeutic purposes only.Figure 32-7. Computed tomography scan of the upper abdomen from a patient with cancer of the distal common bile duct. The cancer obstructs the common bile duct as well as the pancreatic duct. 1 = the portal vein; 2 = a dilated intrahepatic bile duct; 3 = dilated cystic duct and the neck of the gallbladder; 4 = dilated common hepatic duct; 5 = the bifurcation of the common hepatic artery into the gastroduodenal artery and the proper hepatic

1	duct; 3 = dilated cystic duct and the neck of the gallbladder; 4 = dilated common hepatic duct; 5 = the bifurcation of the common hepatic artery into the gastroduodenal artery and the proper hepatic artery; 6 = dilated pancreatic duct; 7 = the splenic vein.ABFigure 32-8. HIDA scanning. A. Normal HIDA scan showing filling of the extrahepatic biliary tree and gallbladder (white arrow). B. HIDA san in a patient with acute cholecystitis showing no filling of the gallbladder.Brunicardi_Ch32_p1393-p1428.indd 139911/02/19 2:43 PM 1400SPECIFIC CONSIDERATIONSPART IIEndoscopic Retrograde CholangiopancreatographyWhile the use of endoscopic retrograde cholangiopancrea-tography (ERCP) in biliary disease is particularly valuable for its therapeutic capabilities, its diagnostic role should not be overlooked. Using a side-viewing endoscope, the common bile duct can be cannulated through the ampulla of Vater and a cholangiogram performed using fluoroscopy (Fig. 32-10). The procedure requires at

1	overlooked. Using a side-viewing endoscope, the common bile duct can be cannulated through the ampulla of Vater and a cholangiogram performed using fluoroscopy (Fig. 32-10). The procedure requires at least intravenous (IV) sedation and in some cases general anesthesia. The advantages of ERCP include direct visualization of the ampullary region and direct access to the distal common bile duct for cholangiography or choledochoscopy. The test is rarely needed for uncomplicated gallstone disease. However, for cases of choledocholithiasis, obstructive jaundice, biliary strictures, or cholangitis, ERCP has the advantage of being both diagnostic and therapeutic. If ductal stones are identified on the endoscopic cholangiogram, biliary sphincterotomy and stone extraction can be performed, clearing the common bile duct of stones. If another etiology such as a biliary stricture is found, diagnostic brushings can be obtained at the time of the procedure. In the hands of experts, the success rate

1	the common bile duct of stones. If another etiology such as a biliary stricture is found, diagnostic brushings can be obtained at the time of the procedure. In the hands of experts, the success rate of common bile duct cannulation and cholan-giography is >90%. Notable complications of diagnostic ERCP include pancreatitis, which occurs in approximately 3.5% of patients, as well as rare occurrences of bleeding, perforation, or infection (cholangitis).16,17Endoscopic CholedochoscopyThe development of small fiber-optic cameras that can be threaded through endoscopes used for endoscopic retrograde cholangiopancreatography (ERCP) has facilitated the develop-ment of intraductal endoscopy. By providing direct visualiza-tion of the biliary and pancreatic ducts, this technology has been shown to increase the effectiveness of ERCP in the diag-nosis of certain biliary diseases.18 Intraductal endoscopy has been shown to have therapeutic applications that include bili-ary stone lithotripsy and

1	increase the effectiveness of ERCP in the diag-nosis of certain biliary diseases.18 Intraductal endoscopy has been shown to have therapeutic applications that include bili-ary stone lithotripsy and directed stone extraction in high-risk surgical patients.19 It can also allow for direct visualization and sampling of concerning lesions in order to evaluate for malig-nancy (Fig. 32-11). Studies have thus far shown intraductal endoscopy to be safe and effective, though complications such as bile duct perforation, minor bleeding, and cholangitis have Figure 32-9. Magnetic resonance cholangiopancreatography. This view shows the course of the extrahepatic bile ducts (arrow) and the pancreatic duct (arrowheads).BAFigure 32-10. Endoscopic retrograde cholangiography. A. A schematic picture showing the side-viewing endoscope in the duodenum and a catheter in the common bile duct. B. An endoscopic cholangiogram showing stones in the common bile duct. The catheter has been placed through the

1	the side-viewing endoscope in the duodenum and a catheter in the common bile duct. B. An endoscopic cholangiogram showing stones in the common bile duct. The catheter has been placed through the ampulla of Vater into the distal common bile duct (arrow). Note the duodenal shadow indicated with arrowheads.Brunicardi_Ch32_p1393-p1428.indd 140011/02/19 2:43 PM 1401GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32been described.20 Further refinement of this technology will likely enhance ERCP as a diagnostic and therapeutic tool.Endoscopic UltrasoundEndoscopic ultrasound (EUS) has improved significantly in recent years and offers additional diagnostic utility to the workup of biliary disease. It requires a specialized 30° endo-scope with either a radial or linear ultrasound transducer at its tip. The results are operator dependent and require a skilled endoscopist but offer noninvasive imaging of the bile ducts and adjacent structures. Endoscopic ultrasound can also be used to

1	at its tip. The results are operator dependent and require a skilled endoscopist but offer noninvasive imaging of the bile ducts and adjacent structures. Endoscopic ultrasound can also be used to identify choledocholithiasis. It is useful for evaluation of the retroduodenal potion of the bile duct, which is difficult to visu-alize with transabdominal ultrasonography. Although EUS is less sensitive than ERCP for biliary stones, the technique is less invasive as it does not require cannulation of the sphincter of Oddi. EUS is also of particular value in the evaluation of tumors near or behind the duodenum and their resectability. Using a linear EUS scope that has a biopsy channel, fine-needle aspira-tion (FNA) of tumors or lymph nodes, therapeutic injections, or drainage procedures under direct ultrasonic guidance can be performed.21Percutaneous Transhepatic CholangiographyIn settings in which the biliary tree cannot be accessed endo-scopically, antegrade cholangiography can be

1	under direct ultrasonic guidance can be performed.21Percutaneous Transhepatic CholangiographyIn settings in which the biliary tree cannot be accessed endo-scopically, antegrade cholangiography can be performed by accessing the intrahepatic bile ducts percutaneously with a small needle under fluoroscopic guidance. Once the position in a bile duct has been confirmed, a guidewire is inserted and a catheter is passed over the wire (Fig. 32-12). Through the catheter, an antegrade cholangiogram can be obtained and therapeutic inter-ventions such as tissue sampling, biliary drain insertions, or stent placements performed. Percutaneous transhepatic cholangiog-raphy (PTC) can also be performed through previously placed percutaneous biliary drainage tubes, if present. PTC has little role in the management of patients with uncomplicated gallstone disease but can be useful in patients with bile duct strictures or tumors, as it can define the anatomy of the biliary tree proximal to the affected

1	of patients with uncomplicated gallstone disease but can be useful in patients with bile duct strictures or tumors, as it can define the anatomy of the biliary tree proximal to the affected segment. As with any invasive procedure, there are potential risks. For PTC, these are mainly bleeding, cholan-gitis, bile leak, and other catheter-related problems.12GALLSTONE DISEASEPrevalence and IncidenceGallstone disease (cholelithiasis) is one of the most common afflictions of the digestive tract. Autopsy reports show that gallstones are present in between 10% and 15% of adults.22 The prevalence of gallstones is related to many factors, including diet, age, gender, BMI, and ethnic background with increased prevalence in patients of Native American and Latin American descent. Certain conditions also predispose to the development of gallstones including pregnancy, non-HDL hyperlipidemia, Crohn’s disease, and certain blood disorders such as heredi-tary spherocytosis, sickle cell disease, and

1	also predispose to the development of gallstones including pregnancy, non-HDL hyperlipidemia, Crohn’s disease, and certain blood disorders such as heredi-tary spherocytosis, sickle cell disease, and thalassemia. Sur-geries that alter the normal neural or hormonal regulation of the biliary tree including terminal ileal resection and gastric or duodenal surgery increase the risk of cholelithiasis. Rapid weight loss following bariatric surgery or lifestyle changes can also precipitate gallstone formation by creating an imbal-ance in bile composition. Medications such as somatostatin analogues and estrogen-containing oral contraceptives are also associated with an increased risk of developing gallstones.22 Women are three times more likely to develop gallstones than men, and first-degree relatives of patients with gallstones have a twofold greater prevalence, possibly indicating a genetic predisposition.23Natural HistoryDespite the high prevalence of cholelithiasis, most patients will

1	of patients with gallstones have a twofold greater prevalence, possibly indicating a genetic predisposition.23Natural HistoryDespite the high prevalence of cholelithiasis, most patients will remain asymptomatic from their gallstones throughout life. For unknown reasons, some patients progress to a symptom-atic stage, with typical symptoms of postprandial right upper quadrant pain (biliary colic) caused by a stone obstructing the cystic duct. In addition to pain, gallstones may progress to cause complications such as acute cholecystitis, choledocholithiasis, cholangitis, gallstone pancreatitis, gallstone ileus, and gallblad-der cancer. Rarely, one of these complications of gallstones may be the initial presenting picture.Gallstones in patients without biliary symptoms are com-monly diagnosed incidentally during unrelated abdominal imag-ing or at the time of surgery for an unrelated diagnosis. Several studies have examined the likelihood of developing biliary colic or developing

1	diagnosed incidentally during unrelated abdominal imag-ing or at the time of surgery for an unrelated diagnosis. Several studies have examined the likelihood of developing biliary colic or developing significant complications of gallstone disease after incidental diagnosis in the asymptomatic patient. About 80% of these patients will remain symptom free.24 However, 2% to 3% will become symptomatic per year (i.e., develop biliary colic). Once symptomatic, patients tend to have recurring bouts of biliary colic. Complicated gallstone disease (cholecystitis, choledocholithiasis, gallstone pancreatitis, etc.) develops in 3% to 5% of symptomatic patients per year.25Because few patients develop complications without previ-ous biliary symptoms, prophylactic cholecystectomy in asymp-tomatic persons with gallstones is rarely indicated.24 Exceptions exist for individuals who will be isolated from medical care for extended periods of time, or in populations with increased risk of gallbladder

1	with gallstones is rarely indicated.24 Exceptions exist for individuals who will be isolated from medical care for extended periods of time, or in populations with increased risk of gallbladder cancer, in which case a prophylactic cholecys-tectomy may be advisable. The presence of porcelain gallblad-der, marked by significant calcifications thought to be related to Figure 32-11. A view from the choledochoscope showing cholangiocarcinoma.Brunicardi_Ch32_p1393-p1428.indd 140111/02/19 2:43 PM 1402SPECIFIC CONSIDERATIONSPART IIgallstones, is a rare premalignant condition and is an absolute indication for cholecystectomy, even when asymptomatic.Gallstone FormationGallstones form as a result of solids settling out of solution. The major organic solutes in bile are bilirubin, bile salts, phospho-lipids, and cholesterol. Gallstones are classified by their choles-terol content as either cholesterol stones or pigment stones. Pigment stones can be further classified as either black or brown.

1	and cholesterol. Gallstones are classified by their choles-terol content as either cholesterol stones or pigment stones. Pigment stones can be further classified as either black or brown. In Western countries, about 80% of gallstones are cholesterol stones and about 15% to 20% are black pigment stones.22 Brown pigment stones account for only a small percentage. Both types of pigment stones are more common in Asia.Cholesterol Stones. Pure cholesterol stones are uncommon and account for <10% of all stones. They usually occur as a sin-gle large stone with a smooth surface. The majority of choles-terol stones are mixed but are at least 70% cholesterol by weight in addition to variable amounts of bile pigments and calcium. These stones are usually multiple, of variable size, and may be 2SafetywireinsertedExternaldrainagecatheter21-guageneedleBileducttumorGuidewire insertedthrough introducerDrainagecatheterABCDEFFigure 32-12. Schematic diagram of percutaneous transhepatic cholangiogram and

1	insertedthrough introducerDrainagecatheterABCDEFFigure 32-12. Schematic diagram of percutaneous transhepatic cholangiogram and drainage for obstructing proximal cholangiocarcinoma. A. Dilated intrahepatic bile duct is entered percutaneously with a fine needle. B. Small guidewire is passed through the needle into the duct. C. A plastic catheter has been passed over the wire, and the wire is subsequently removed. A cholangiogram can be performed through the catheter. D. An external drainage catheter in place. E. Long wire placed via the catheter and advanced past the tumor and into the duodenum. F. Internal stent has been placed through the tumor.Brunicardi_Ch32_p1393-p1428.indd 140211/02/19 2:43 PM 1403GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32Figure 32-13. Gallbladder with cholesterol stones. A. Stones of multiple shapes and sizes. B. Solitary large stone. C. Multiple stones of varying composition. (Reproduced with permission from Slesinger MH, Fordtran JS:

1	with cholesterol stones. A. Stones of multiple shapes and sizes. B. Solitary large stone. C. Multiple stones of varying composition. (Reproduced with permission from Slesinger MH, Fordtran JS: Gastrointestinal Diseases. Philadelphia, PA: Elsevier/Saunders; 1989.)100100Moles % Bile salts100808080606060Moles % CholesterolMoles % LecithinMicellarliquidMetastablesupersaturatedzone4040402020200002 or morephasesFigure 32-14. The three major components of bile plotted on triangular coordinates, cholesterol, bile salts and phospholipids (lecithin). A given point represents the relative molar ratios of each. The area labeled “micellar liquid” shows the range of concentrations in which cholesterol is fully solubilized. The shaded area directly above this region corresponds to a metastable zone, supersaturated with cholesterol. Above the shaded area, bile has exceeded the solubilization capacity of cholesterol and precipitation of cholesterol crystals and stones occurs.hard and faceted or

1	supersaturated with cholesterol. Above the shaded area, bile has exceeded the solubilization capacity of cholesterol and precipitation of cholesterol crystals and stones occurs.hard and faceted or irregular, multilobed, and soft (Fig. 32-13). Colors range from whitish yellow to green or black. Most cho-lesterol stones (>90%) are radiolucent, though some have a high calcium carbonate component and become radioopaque.The primary event in the formation of cholesterol stones is supersaturation of bile with cholesterol. Cholesterol is highly nonpolar and its solubility in water and bile depends on the relative concentration of cholesterol, bile salts, and lecithin (the main phospholipid in bile). Cholesterol is secreted into bile and is surrounded by bile salts and phospholipids to form a soluble vesicle complex. When cholesterol hypersecretion is present, either through increased intake or dysfunctional processing, supersaturation occurs. When cholesterol concentrations exceed the ability

1	vesicle complex. When cholesterol hypersecretion is present, either through increased intake or dysfunctional processing, supersaturation occurs. When cholesterol concentrations exceed the ability of the bile salts and phospholipid to maintain solu-bility, the cholesterol precipitates out of solution into a solid, forming a cholesterol stone (Fig. 32-14).26 Cholesterol hyperse-cretion is almost always the cause of supersaturation rather than reduced secretion of phospholipid or bile salts.2Pigmented Stones. Pigmented stones contain <20% choles-terol and are dark because of the presence of calcium bilirubi-nate. Black and brown pigment stones have little in common and should be considered as separate entities.Black pigment stones are usually small, brittle, dark, and sometimes spiculated. They are formed by supersaturation of unconjugated bilirubin within the bile. Deconjugation of bili-rubin occurs normally in bile at a slow rate. Thus, excessive levels of conjugated bilirubin

1	They are formed by supersaturation of unconjugated bilirubin within the bile. Deconjugation of bili-rubin occurs normally in bile at a slow rate. Thus, excessive levels of conjugated bilirubin excretion, as occurs in hemolytic disorders like hereditary spherocytosis and sickle cell disease will lead to an increased rate of production of unconjugated bilirubin. Cirrhosis and hepatic dysfunction may also lead to increased secretion of unconjugated bilirubin directly from the liver. The insoluble unconjugated bilirubin will then precipitate with calcium as insoluble calcium bilirubinate, forming a pig-ment stone. Due to their high calcium content, pigment stones are often radiopaque. Like cholesterol stones, they almost always form in the gallbladder. In Asian countries such as Japan, black stones account for a much higher percentage of gallstones than in the Western hemisphere.Brown stones are usually <1 cm in diameter, brownish-yel-low, soft, and often mushy. They may form either in

1	stones account for a much higher percentage of gallstones than in the Western hemisphere.Brown stones are usually <1 cm in diameter, brownish-yel-low, soft, and often mushy. They may form either in the gallblad-der or in the bile ducts secondary to bacterial infection and bile stasis. Bacteria such as Escherichia coli secrete β-glucuronidase that enzymatically cleaves conjugated bilirubin to produce the insoluble unconjugated bilirubin. This unconjugated bilirubin then precipitates with calcium, and along with dead bacterial cell bodies, forms soft brown stones in the biliary tree. Brown stones are typically found in Asian populations and are associated with stasis secondary to parasite infection with Ascaris lumbricoides BCABrunicardi_Ch32_p1393-p1428.indd 140311/02/19 2:43 PM 1404SPECIFIC CONSIDERATIONSPART II(roundworm) or Clonorchis sinensis (liver fluke). In Western populations, brown stones most often occur as primary bile duct stones in patients with biliary strictures or

1	CONSIDERATIONSPART II(roundworm) or Clonorchis sinensis (liver fluke). In Western populations, brown stones most often occur as primary bile duct stones in patients with biliary strictures or other common bile duct stones that cause stasis and bacterial contamination.2,27Symptomatic GallstonesSymptomatic Cholelithiasis. Patients with symptomatic gall-stone disease typically present with recurrent attacks of pain. The pain develops when a stone obstructs the cystic duct, resulting in a progressive increase of tension in the gallbladder wall as it contracts in response to a meal. This postprandial right upper quadrant or epigastric pain is often referred to as biliary colic. If untreated, about two-thirds of these patients will develop chronic noninfectious inflammation of the gallbladder wall, termed chronic cholecystitis. The pathologic changes, which often do not correlate well with symptoms, vary from an apparently nor-mal gallbladder with minor chronic inflammation in the mucosa,

1	wall, termed chronic cholecystitis. The pathologic changes, which often do not correlate well with symptoms, vary from an apparently nor-mal gallbladder with minor chronic inflammation in the mucosa, to a shrunken, nonfunctioning gallbladder with transmural fibro-sis and adhesions to nearby structures. The mucosa is initially normal or hypertrophied but later becomes atrophied, with the epithelium protruding into the muscle coat, leading to the forma-tion of the so-called Aschoff-Rokitansky sinuses.Clinical Manifestations The chief symptom associated with symptomatic cholelithiasis is pain (biliary colic). The pain is con-stant and increases in severity over the first half hour or so after a meal and can last 1 to 5 hours. It is located in the epigastrium or right upper quadrant and frequently radiates to the right upper back or between the scapulae (Fig. 32-15). The pain is severe and comes on abruptly, typically during the night or after a fatty meal. It often is associated with

1	radiates to the right upper back or between the scapulae (Fig. 32-15). The pain is severe and comes on abruptly, typically during the night or after a fatty meal. It often is associated with nausea and sometimes vomiting. Patients generally suffer discrete, recurrent attacks of pain, between which they feel well. Physical examination may reveal mild right upper quadrant tenderness during an episode of pain. If the patient is pain free, the physical examination is usually unremarkable. Labora-tory values, such as WBC count and liver function tests, are usu-ally normal in patients with uncomplicated gallstones.Atypical presentations of gallstone disease are common and a high index of suspicion for biliary disease must be main-tained when evaluating patients with abdominal complaints. Association with meals is present in only about 50% of patients. Some patients report milder attacks of pain but relate it to meals. The pain may be located primarily in the back or the left upper or right

1	with meals is present in only about 50% of patients. Some patients report milder attacks of pain but relate it to meals. The pain may be located primarily in the back or the left upper or right lower quadrant. Bloating and belching may be present and associated with the attacks of pain. In patients with atypi-cal presentations, other conditions that may be causing upper abdominal pain should be ruled out, even in the presence of gallstones. These include but are not limited to peptic ulcer dis-ease, gastroesophageal reflux disease, herpes zoster, abdominal wall hernias, inflammatory bowel disease, diverticular disease, pancreatitis, liver disease, renal calculi, pleuritic pain, and car-diac pain.When the pain lasts >24 hours without resolving, an impacted stone in the cystic duct or acute cholecystitis (see later “Acute Cholecystitis” section) should be suspected. An impacted stone without cholecystitis will result in what is called hydrops of the gallbladder. Bile will be unable to

1	cholecystitis (see later “Acute Cholecystitis” section) should be suspected. An impacted stone without cholecystitis will result in what is called hydrops of the gallbladder. Bile will be unable to enter the gall-bladder due to the obstructed cystic duct, but the gallbladder epithelium will continue to secrete mucus, and the gallbladder will become distended with clear-white mucinous material. The gallbladder may be palpable but usually is not tender. Hydrops of the gallbladder may result in edema of the gallbladder wall, inflammation, infection, and perforation. Although hydrops may persist with few consequences, early cholecystectomy is generally indicated to avoid complications.Diagnosis The diagnosis of symptomatic cholelithiasis or chronic cholecystitis depends on the presence of typical symp-toms and the demonstration of stones on diagnostic imaging. An abdominal ultrasound is the standard diagnostic test for gallstones as it is noninvasive and highly sensitive (see earlier

1	typical symp-toms and the demonstration of stones on diagnostic imaging. An abdominal ultrasound is the standard diagnostic test for gallstones as it is noninvasive and highly sensitive (see earlier “Ultrasonography” section).28 Gallstones are occasionally iden-tified on abdominal CT scans that were obtained as part of a broader workup of abdominal pain. In these cases, if the patient has typical symptoms, it is reasonable to proceed with interven-tion. Stones diagnosed incidentally on CT or plain radiographs in patients without symptoms should be left in place. Occasion-ally, patients with typical attacks of biliary pain have no evi-dence of stones on ultrasound but have evidence of sludge in the gallbladder. If a patient has attacks of typical biliary pain and sludge is detected, cholecystectomy is warranted.In addition to sludge and stones, cholesterolosis and adeno-myomatosis of the gallbladder may cause typical biliary symp-toms and may be detected on ultrasound or CT.

1	cholecystectomy is warranted.In addition to sludge and stones, cholesterolosis and adeno-myomatosis of the gallbladder may cause typical biliary symp-toms and may be detected on ultrasound or CT. Cholesterolosis is caused by the accumulation of cholesterol in macrophages in the 221514764501352441429614396322151911252311353323ABFigure 32-15. A. Sites of the most severe pain during an episode of biliary colic in 107 patients with gallstones (% values add up to >100% because of multiple responses). The subxiphoid and right subcostal areas were the most common sites; note that the left sub-costal area was not an unusual site of pain. B. Sites of pain radiation (%) during an episode of biliary colic in the same group of patients.Brunicardi_Ch32_p1393-p1428.indd 140411/02/19 2:43 PM 1405GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32gallbladder lamina propria, either locally or as polyps. It produces the classic studded macroscopic appearance of a “strawberry gallbladder.”

1	AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32gallbladder lamina propria, either locally or as polyps. It produces the classic studded macroscopic appearance of a “strawberry gallbladder.” Adenomyomatosis (cholecystitis glandularis prolif-erans) is characterized on microscopy by hypertrophic smooth muscle bundles and by the ingrowths of mucosal glands into the muscle layer (epithelial sinus formation). Granulomatous polyps develop in the lumen at the fundus, and the gallbladder wall is thickened. Septae or strictures may be seen within the gallblad-der. In symptomatic patients, cholecystectomy is the treatment of choice for patients with these conditions.29Treatment Nonsurgical management of gallstone disease using medications or lithotripsy has had disappointing long-term results. These modalities are not considered to be part of the primary treatment algorithm for gallstone disease.30 Surgi-cal cholecystectomy offers the best long-term results for patients with symptomatic

1	These modalities are not considered to be part of the primary treatment algorithm for gallstone disease.30 Surgi-cal cholecystectomy offers the best long-term results for patients with symptomatic gallstones. About 90% of patients with typical biliary symptoms and stones are rendered symp-tom free after cholecystectomy. For patients with atypical symptoms such as dyspepsia, flatulence, belching, bloating, and dietary fat intolerance, the results are not as favorable. The laparoscopic approach has been proven to be safe and effective and has become the standard of care for symptomatic gallstone disease, replacing open cholecystectomy in routine cases.29,31 3Due to the possibility of developing complications related to gallstone disease, patients with symptomatic choleli-thiasis should be offered elective cholecystectomy. While wait-ing for surgery, or if surgery has to be postponed, the patient should be advised to avoid dietary fats and large meals. Dia-betic patients with symptomatic

1	elective cholecystectomy. While wait-ing for surgery, or if surgery has to be postponed, the patient should be advised to avoid dietary fats and large meals. Dia-betic patients with symptomatic gallstones should be encour-aged to have a cholecystectomy promptly, as they are more prone to developing severe acute cholecystitis. Pregnant women with symptomatic gallstones who cannot be managed expectantly with diet modifications can safely undergo laparo-scopic cholecystectomy. The operation should be performed during the second trimester if possible.Acute Cholecystitis. Acute cholecystitis, or infection of the gallbladder, is associated with gallstones in 90% to 95% of cases. Rarely, acalculous cholecystitis can occur, usually in patients with other acute systemic diseases (see later “Acalculous Chole-cystitis” section). Obstruction of the cystic duct by a gall-stone is the initiating event that leads to gallbladder distention, inflammation, and edema of the gallbladder wall. In <1% of

1	Chole-cystitis” section). Obstruction of the cystic duct by a gall-stone is the initiating event that leads to gallbladder distention, inflammation, and edema of the gallbladder wall. In <1% of acute cholecystitis, the cause is a tumor obstructing the cystic duct. Why inflammation develops only occasionally with cystic duct obstruction is unknown, but it is probably related to the duration of obstruction. Initially, acute cholecystitis is an inflammatory process, probably mediated by the mucosal toxin lysolecithin, a product of lecithin, as well as bile salts and platelet-activat-ing factor. An increase in prostaglandin synthesis amplifies the inflammatory response. In acute cholecystitis, the gallbladder wall becomes grossly thickened and reddish with subserosal hemorrhages. Pericholecystic fluid often is present. The mucosa may show hyperemia and patchy necrosis. In severe cases, about 5% to 10%, the inflammatory process progresses and leads to ischemia and necrosis of the

1	fluid often is present. The mucosa may show hyperemia and patchy necrosis. In severe cases, about 5% to 10%, the inflammatory process progresses and leads to ischemia and necrosis of the gallbladder wall. More frequently, the gallstone is dislodged and the inflammation resolves.Not all episodes of uncomplicated acute cholecystitis involve infection. Secondary bacterial contamination is thought to occur in only 15% to 30% of patients. With some severe infections, gangrenous cholecystitis can develop, and an abscess or perforation may occur. When they happen, perforations are usually contained in the subhepatic space by the omentum and adjacent organs. However, free perforation with peritonitis, intrahepatic perforation with intrahepatic abscesses, and per-foration into adjacent organs (duodenum or colon) with cho-lecystoenteric fistula have been described. When gas-forming organisms are part of the secondary bacterial infection, gas may be seen in the gallbladder lumen and in the wall

1	or colon) with cho-lecystoenteric fistula have been described. When gas-forming organisms are part of the secondary bacterial infection, gas may be seen in the gallbladder lumen and in the wall of the gallblad-der on abdominal radiographs and CT scans, an entity called emphysematous cholecystitis.Clinical Manifestations About 80% of patients with acute cholecystitis give a history compatible with chronic cholecysti-tis. Acute cholecystitis often begins as an attack of biliary colic with relapsing and remitting pain in the right upper quadrant or epigastrium that may radiate to the right back or interscapular area. In contrast to biliary colic, the pain of acute cholecystitis does not subside. It is unremitting, may persist for several days, and is usually more severe than the pain associated with uncom-plicated gallstone disease. The patient is often febrile, complains of anorexia, nausea, and vomiting, and may be reluctant to move as the inflammatory process creates focal

1	associated with uncom-plicated gallstone disease. The patient is often febrile, complains of anorexia, nausea, and vomiting, and may be reluctant to move as the inflammatory process creates focal peritonitis. On physi-cal examination, tenderness and guarding are usually present in the right upper quadrant. A mass, the gallbladder and adherent omentum, is occasionally palpable; however, guarding may pre-vent identification of this. Murphy’s sign, an inspiratory arrest with deep palpation in the right subcostal area, is characteristic of acute cholecystitis.Laboratory evaluation commonly reveals a mild to mod-erate leukocytosis (12,000–15,000 cells/mm3). However, a normal WBC does not rule out the diagnosis. An unusually high WBC count (>20,000 cells/mm3) suggests a complicated form of cholecystitis such as gangrenous cholecystitis, perfo-ration, or associated cholangitis. In uncomplicated acute cho-lecystitis, serum liver chemistries are usually normal, but a mild elevation of serum

1	such as gangrenous cholecystitis, perfo-ration, or associated cholangitis. In uncomplicated acute cho-lecystitis, serum liver chemistries are usually normal, but a mild elevation of serum bilirubin (<4 mg/mL) may be present along with mild elevation of alkaline phosphatase, transami-nases, and amylase.28 Severe jaundice is suggestive of obstruc-tion of the bile ducts. This can be a result of common bile duct stones or severe pericholecystic inflammation secondary to impaction of a stone in the infundibulum of the gallblad-der that mechanically obstructs the bile duct, known as Mir-izzi’s syndrome (Fig. 32-16). In elderly patients and in those with diabetes mellitus, acute cholecystitis may have a subtle Figure 32-16. Mirizzi’s syndrome. Impaction of a large stone in the neck of the gallbladder causing obstruction at the level of the confluence of the cystic duct and common hepatic duct.Brunicardi_Ch32_p1393-p1428.indd 140511/02/19 2:43 PM 1406SPECIFIC

1	in the neck of the gallbladder causing obstruction at the level of the confluence of the cystic duct and common hepatic duct.Brunicardi_Ch32_p1393-p1428.indd 140511/02/19 2:43 PM 1406SPECIFIC CONSIDERATIONSPART IIpresentation resulting in a delay in diagnosis. These patients may also have higher rates of treatment related morbidity com-pared to younger and healthier patients.The differential diagnosis for acute cholecystitis includes but is not limited to peptic ulcer disease, pancreatitis, appen-dicitis, hepatitis, perihepatitis (Fitz-Hugh–Curtis syndrome), myocardial ischemia, pneumonia, pleuritis, and herpes zoster involving the intercostal nerve.Diagnosis Ultrasonography is considered the most useful ini-tial radiologic test for diagnosing acute cholecystitis, with a sen-sitivity and specificity of 70% to 90%. Ultrasound is effective at documenting the presence or absence of stones, and it can show gallbladder wall thickening and pericholecystic fluid, both of which are highly

1	specificity of 70% to 90%. Ultrasound is effective at documenting the presence or absence of stones, and it can show gallbladder wall thickening and pericholecystic fluid, both of which are highly suggestive of acute cholecystitis (Fig. 32-17). Focal tenderness over the gallbladder when compressed by the sonographic probe (sonographic Murphy’s sign) also supports the diagnosis of acute cholecystitis. Biliary scintigraphy (HIDA scanning) may be of help in atypical cases if the diagnosis remains in question after initial workup. Lack of filling of the gallbladder after 4 hours indicates an obstructed cystic duct and, in the clinical setting of suspected acute cholecystitis, confirms the diagnosis with a reported sensitivity above 90%.32 Con-versely, a normal HIDA scan with clear filling of the gallblad-der rules out the diagnosis of acute cholecystitis. CT scans are frequently performed on patients with acute abdominal pain of unknown etiology, as they can evaluate for a number of

1	of the gallblad-der rules out the diagnosis of acute cholecystitis. CT scans are frequently performed on patients with acute abdominal pain of unknown etiology, as they can evaluate for a number of poten-tial pathologic processes at once. In patients with acute chole-cystitis, a CT scan can demonstrate thickening of the gallbladder wall, pericholecystic fluid, and the presence of gallstones, but it is somewhat less sensitive than ultrasonography.Treatment Patients who present with acute cholecystitis should receive IV fluids, broad-spectrum antibiotics, and anal-gesia. The antibiotics should cover gram-negative enteric organ-isms as well as anaerobes. Although the inflammation in acute cholecystitis may be sterile in some patients, it is difficult to know who is secondarily infected. Therefore, antibiotics have become a standard part of the initial management of acute cho-lecystitis in most centers.Cholecystectomy is the definitive treatment for acute cho-lecystitis. In the past, the

1	antibiotics have become a standard part of the initial management of acute cho-lecystitis in most centers.Cholecystectomy is the definitive treatment for acute cho-lecystitis. In the past, the timing of cholecystectomy has been a matter of debate. Early cholecystectomy performed within 72 hours of the onset of the illness is preferred over delayed cholecystectomy that is performed 6 to 10 weeks after initial medical treatment and recuperation. Several studies have shown that unless the patient is unfit for surgery, early cholecystectomy should be recommended as soon as possible, as it offers the patient a definitive solution in one hospital admission, quicker recovery times, similar complication rates, and an earlier return to work.33,34Laparoscopic cholecystectomy is the procedure of choice for acute cholecystitis. The conversion rate to an open cholecys-tectomy has fallen in recent years to less than 5% as laparoscopic equipment and experience has improved.35 While laparoscopic

1	choice for acute cholecystitis. The conversion rate to an open cholecys-tectomy has fallen in recent years to less than 5% as laparoscopic equipment and experience has improved.35 While laparoscopic cholecystectomy for acute cholecystitis may be more tedious and take longer than an elective cholecystectomy for symp-tomatic cholelithiasis, the laparoscopic approach remains safe and effective, even in the setting of acute and sometimes severe inflammation. Open cholecystectomy must remain an option in particularly difficult cases, or in patients suspected of having prohibitive intraabdominal adhesions, but it is rarely the primary treatment choice.When patients are medically unfit for surgery due to the severity of their illness or medical comorbidities, they can be treated with antibiotics and biliary decompression with cho-lecystostomy tube placement, which is usually effective in stabilizing the patient.36 For those who do recover after chole-cystostomy, the tube can be removed once

1	and biliary decompression with cho-lecystostomy tube placement, which is usually effective in stabilizing the patient.36 For those who do recover after chole-cystostomy, the tube can be removed once the track is mature (approximately 4 weeks) and cholangiography through it shows a patent cystic duct. Elective laparoscopic cholecystectomy can be scheduled within approximately 6 to 8 weeks, assum-ing their medical fitness recovers.37 Failure to improve after cholecystostomy may be due to gangrene of the gallbladder or perforation, in which case, damage control surgery may be unavoidable.Choledocholithiasis. Common bile duct (CBD) stones may be small or large, single or multiple, and are found in 6% to 12% of patients with stones in the gallbladder. The incidence increases with age. About 20% to 25% of patients above the age of 60 with symptomatic gallstones have stones in the common bile duct as well as in the gallbladder.38 The vast majority of ductal stones in Figure

1	age. About 20% to 25% of patients above the age of 60 with symptomatic gallstones have stones in the common bile duct as well as in the gallbladder.38 The vast majority of ductal stones in Figure 32-17. Ultrasonography from a patient with acute cholecystitis. The white arrowheads indicate the thickened gallbladder wall. There are several stones in the gallbladder (white arrows) throwing acoustic shadows (black arrowheads). Trace pericholecystic fluid can be seen surrounding the gallbladder (black arrows).Brunicardi_Ch32_p1393-p1428.indd 140611/02/19 2:43 PM 1407GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32Western countries are formed within the gallbladder and migrate down the cystic duct into the common bile duct. These are clas-sified as secondary CBD stones, in contrast to the primary CBD stones that form in the bile duct itself. Secondary stones are usu-ally cholesterol stones, whereas primary stones are usually of the brown pigment type. The primary stones are

1	to the primary CBD stones that form in the bile duct itself. Secondary stones are usu-ally cholesterol stones, whereas primary stones are usually of the brown pigment type. The primary stones are associated with biliary stasis and infection, and they are more commonly seen in Asian populations. Biliary stasis leading to the development of primary CBD stones can be caused by biliary strictures, papillary stenosis, tumors, or other (secondary) stones.Clinical Manifestations Choledochal stones may be silent and often are discovered incidentally. They may cause complete or incomplete obstruction, or they may manifest with cholangitis or gallstone pancreatitis. The typical pain caused by a stone in the bile duct is very similar to that of biliary colic caused by impaction of a stone in the cystic duct. Nausea and vomiting are common. Physical examination may be normal, but mild epigas-tric or right upper quadrant tenderness as well as mild icterus are common. The symptoms may also be

1	cystic duct. Nausea and vomiting are common. Physical examination may be normal, but mild epigas-tric or right upper quadrant tenderness as well as mild icterus are common. The symptoms may also be intermittent, such as pain and transient jaundice caused by a stone that temporar-ily impacts the ampulla but subsequently moves away, acting as a ball valve. A small stone may pass through the ampulla spontaneously with resolution of symptoms. Finally, the stones may become completely impacted, causing severe progressive jaundice. Elevation of serum bilirubin, alkaline phosphatase, and transaminases are commonly seen in patients with bile duct stones. However, in about one-third of patients with common bile duct stones, the liver chemistries are normal, particularly if the obstruction is incomplete or intermittent.Diagnosis Ultrasonography is useful for documenting stones in the gallbladder (if still present), as well as determining the size of the common bile duct. As stones in the bile

1	or intermittent.Diagnosis Ultrasonography is useful for documenting stones in the gallbladder (if still present), as well as determining the size of the common bile duct. As stones in the bile ducts tend to move down to the distal part of the common duct behind the duodenum, bowel gas can preclude their detection on ultraso-nography. A dilated common bile duct (>8 mm in diameter) on ultrasonography in a patient with gallstones, jaundice, and bili-ary pain is highly suggestive of common bile duct stones. If the presence of bile duct stones is in question, magnetic resonance cholangiopancreatography (MRCP) provides excellent anatomic detail and has a sensitivity and specificity of 95% and 89%, respectively, for detecting choledocholithiasis.14 Endoscopic retrograde cholangiopancreatography (ERCP) is highly effec-tive at diagnosing choledocholithiasis and in experienced hands, cannulation of the ampulla of Vater and diagnostic cholangiog-raphy are achieved in >90% of cases. However, due

1	(ERCP) is highly effec-tive at diagnosing choledocholithiasis and in experienced hands, cannulation of the ampulla of Vater and diagnostic cholangiog-raphy are achieved in >90% of cases. However, due to the risks associated with the procedure, it is rarely used as a purely diag-nostic modality, rather being reserved for cases in which a thera-peutic intervention such as stone extraction or sphincterotomy is planned. Endoscopic ultrasound has been demonstrated to be as good as ERCP for detecting common bile duct stones (sensitivity of 95% and specificity of 97%). However, EUS has fewer thera-peutic capabilities and requires endoscopic expertise, making it less desirable except in specific clinical senarios.39 Percutaneous transhepatic cholangiography (PTC) is rarely needed in patients with common bile duct stones but can be performed for both diagnostic and therapeutic reasons in patients with contraindica-tions to endoscopic or surgical approaches.Treatment For patients with

1	with common bile duct stones but can be performed for both diagnostic and therapeutic reasons in patients with contraindica-tions to endoscopic or surgical approaches.Treatment For patients with symptomatic gallstones and sus-pected common bile duct stones, bile duct clearance and cho-lecystectomy are indicated. This may be safely achieved either with preoperative ERCP followed by surgery or by going directly to surgery with intraoperative cholangiogram and common bile duct exploration to address retained stones. Both approaches are considered safe and effective, and no formal recommendation exists to definitively support one over the other.40,41If upfront laparoscopic cholecystectomy is pursued, the surgery should include an intraoperative cholangiogram to doc-ument the presence or absence of bile duct stones. If stones are identified, laparoscopic common bile duct exploration via the cystic duct or with formal choledochotomy allows the stones to be retrieved in the same setting (see

1	of bile duct stones. If stones are identified, laparoscopic common bile duct exploration via the cystic duct or with formal choledochotomy allows the stones to be retrieved in the same setting (see “Choledochal Explora-tion” section). If the expertise and/or instrumentation for lapa-roscopic common bile duct exploration are not available, the patient can be awoken and scheduled for ERCP with sphincter-otomy the following day. An open common bile duct explora-tion is an option if the endoscopic and laparoscopic methods are not feasible. If a choledochotomy is performed, primary repair can be considered in large ducts, while smaller ducts should be repaired over a T-tube. To do this, a standard T-tube should be modified by cutting the ends short enough to allow placement within the duct and dividing the T longitudinally to facilitate easy removal from the duct later on (Fig. 32-18). If a common Figure 32-18. T-tube placement. A. A standard T-tube that has been cut and modified for use

1	dividing the T longitudinally to facilitate easy removal from the duct later on (Fig. 32-18). If a common Figure 32-18. T-tube placement. A. A standard T-tube that has been cut and modified for use in the biliary tract. B. The T-tube is placed through a ductotomy in the common bile duct with the defect closed over the tube. The opposite end is brought out through the abdominal wall for decompression of the bile ducts.ABBrunicardi_Ch32_p1393-p1428.indd 140711/02/19 2:43 PM 1408SPECIFIC CONSIDERATIONSPART IIbile duct exploration was performed and a T tube left in place, a T-tube cholangiogram should be obtained before its removal, at least several weeks after its placement.In very severe cases, stones impacted in the ampulla may be unable to be cleared by endoscopic approaches or common bile duct exploration (open or laparoscopic). In these cases, transduodenal sphincterotomy can be considered. If one is entirely unable to disimpact the duct, choledochoduodenostomy or Roux-en-Y

1	bile duct exploration (open or laparoscopic). In these cases, transduodenal sphincterotomy can be considered. If one is entirely unable to disimpact the duct, choledochoduodenostomy or Roux-en-Y choledochojejunostomy may be the only option to restore biliary continuity.42If the stones were left in place at the time of surgery or diagnosed shortly after the cholecystectomy, they are classified as retained. Those diagnosed months or years later are termed recurrent (Fig. 32-19). Retained or recurrent stones following cholecystectomy are best treated endoscopically. A generous sphincterotomy will allow for stone retrieval as well as spon-taneous passage of stones. Alternately, retained stones can be cleared via a mature T-tube tract (4 weeks) if one was placed at the time of surgery. To do this, the T-tube is removed and a catheter passed through the tract into the common bile duct. Under fluoroscopic guidance, the stones can be retrieved with baskets or balloons. A similar approach will

1	the T-tube is removed and a catheter passed through the tract into the common bile duct. Under fluoroscopic guidance, the stones can be retrieved with baskets or balloons. A similar approach will allow for stone clearance by percutaneous transhepatic cholecystostomy (PTC) if there is no other way to reach the duct. Repeat surgery should be a last resort if other interventions have failed.Cholangitis. Cholangitis is one of the main complications of choledochal stones. Acute cholangitis is an ascending bac-terial infection associated with partial or complete obstruc-tion of the bile ducts.43 Hepatic bile is sterile, and bile in the bile ducts is kept sterile by continuous antegrade bile flow and by the presence of antibacterial substances in bile, such as immunoglobulin. Mechanical hindrance to bile flow facilitates ascending bacterial contamination from the bowel. Positive bile cultures are common in the presence of bile duct stones as well as with other causes of obstruction. Biliary

1	to bile flow facilitates ascending bacterial contamination from the bowel. Positive bile cultures are common in the presence of bile duct stones as well as with other causes of obstruction. Biliary bacterial contamination alone does not lead to clinical cholangitis; the combination of both significant bacterial contamination and biliary obstruction is required for its development. Gallstones are the most common cause of obstruction in cholangitis. Other causes include primary sclerosing cholangitis, benign and malignant strictures, parasites, instrumentation of the ducts, and indwelling stents, as well as partially obstructed biliary-enteric anastomoses. The most common organisms cultured from bile in patients with cholangitis include E coli, Klebsiella pneumoniae, Streptococcus faecalis, Enterobacter, and Bacteroides fragilis.43Clinical Manifestations Cholangitis may present as anything from a mild, self-limited episode to a fulminant, potentially life-threatening septicemia.

1	Enterobacter, and Bacteroides fragilis.43Clinical Manifestations Cholangitis may present as anything from a mild, self-limited episode to a fulminant, potentially life-threatening septicemia. Patients with gallstone-induced cholangitis are most commonly older and female. The most common presentation is fever, epigastric or right upper quad-rant pain, and jaundice. These classic symptoms, known as Charcot’s triad, are present in about two-thirds of patients. The illness can progress rapidly with the development of shock and altered mental status, known as Reynolds’ pentad (e.g., fever, jaundice, right upper quadrant pain, septic shock, and mental status changes). However, the presentation may be atypical, with little if any fever, jaundice, or pain. This occurs most commonly in the elderly, who may have unremarkable symptoms until the process is already quite advanced. Patients with indwelling stents are at particularly high risk for cholan-gitis, though rarely become jaundiced as a

1	who may have unremarkable symptoms until the process is already quite advanced. Patients with indwelling stents are at particularly high risk for cholan-gitis, though rarely become jaundiced as a patent stent will prevent the obstruction of bile flow. On abdominal examina-tion, the findings are indistinguishable from those of acute cholecystitis.44Diagnosis Leukocytosis, hyperbilirubinemia, and elevation of alkaline phosphatase and transaminases are common and, when present, support the clinical diagnosis of cholangitis. Ultraso-nography is helpful, as it will document the presence of gall-bladder stones, demonstrate dilated ducts, and possibly pinpoint a site of obstruction. CT scanning and MRI can show pancreatic and periampullary masses, if present, in addition to the ductal dilatation. However, abdominal imaging will rarely elucidate the exact cause of cholangitis, and the initial diagnosis is gener-ally made clinically.Treatment The initial treatment of patients with cholangi-tis

1	However, abdominal imaging will rarely elucidate the exact cause of cholangitis, and the initial diagnosis is gener-ally made clinically.Treatment The initial treatment of patients with cholangi-tis includes broad-spectrum IV antibiotics to cover enteric organisms and anaerobes, fluid resuscitation, and rapid biliary ABFigure 32-19. Retained common bile duct stones. The patient pre-sented 3 weeks after laparoscopic cholecystectomy. A. An ultra-sound shows a normal or mildly dilated common bile duct with a stone. Note the location of the right hepatic artery anterior to the common hepatic duct (an anatomic variation). B. An endoscopic retrograde cholangiography from the same patient shows multiple stones in the common bile duct. Only the top one showed on ultra-sound as the other stones lie in the distal common bile duct behind the duodenum.Brunicardi_Ch32_p1393-p1428.indd 140811/02/19 2:43 PM 1409GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32decompression. This is most

1	in the distal common bile duct behind the duodenum.Brunicardi_Ch32_p1393-p1428.indd 140811/02/19 2:43 PM 1409GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32decompression. This is most often accomplished through ERCP and sphincterotomy. ERCP will show the level and the reason for the obstruction, allow for culture of the bile, permit the removal of stones if present, and accomplish drainage of the bile ducts. Placement of drainage catheters or stents can also be performed if needed. In cases in which ERCP is not available, PTC, EUS, or surgical drainage can be utilized. The selection of the appropriate approach will depend on the type and location of the suspected obstruction as well as the avail-ability of local resources and expertise. Cholecystostomy tubes are not indicated in the acute management of cholangitis as the primary source of the infection is extrinsic to the gallbladder.Patients with cholangitis can deteriorate rapidly and may require intensive care unit

1	in the acute management of cholangitis as the primary source of the infection is extrinsic to the gallbladder.Patients with cholangitis can deteriorate rapidly and may require intensive care unit monitoring and vasopressor support. However, most patients will respond to biliary decompression and supportive measures. In the current era, acute cholangitis is associated with an overall mortality rate of approximately 5%. When associated with renal failure, cardiac impairment, hepatic abscesses, and malignancies, the morbidity and mortal-ity rates are much higher. Patients who have suffered an episode of acute cholangitis related to gallstone disease should be rec-ommended to undergo elective cholecystectomy approximately 6 weeks after the resolution of their cholangitis.45 Those whose cholangitis was related to another cause of biliary obstruction should be followed and treated for the specific etiology of their obstruction but do not necessarily require cholecystectomy if gallstones

1	was related to another cause of biliary obstruction should be followed and treated for the specific etiology of their obstruction but do not necessarily require cholecystectomy if gallstones were not the causative etiology of their cholangitis. Patients with indwelling stents and cholangitis usually require repeated imaging and stent exchange to mitigate the risk of recurrent infections.Gallstone Pancreatitis. Gallstones in the common bile duct can provoke attacks of acute pancreatitis through transient or persistent obstruction of the pancreatic duct by a stone passing through or impacted in the ampulla. The exact mechanism by which obstruction of the pancreatic duct leads to pancreatitis is unclear, but it may be related to increased ductal pressures causing leakage of pancreatic enzymes into the glandular tissue. The initial management of gallstone pancreatitis is supportive, including admission for bowel rest, IV hydration, and pain control. Antibiotics are not indicated in the

1	into the glandular tissue. The initial management of gallstone pancreatitis is supportive, including admission for bowel rest, IV hydration, and pain control. Antibiotics are not indicated in the absence of signs of infected pancreatic necrosis. Imaging of the biliary tree with ultrasound, CT, or MRCP is essential to confirm the diagnosis. When gallstones are present and the pancreatitis is mild and self-limited, the stone has probably passed. For these patients, a cholecystectomy with intraoperative cholangiogram is indi-cated as soon as the pancreatitis has clinically resolved. It is strongly recommended that cholecystectomy be performed dur-ing the same admission whenever possible due to the high rate of recurrence and increased morbidity of subsequent attacks of pancreatitis.46 If gallstones are present obstructing the duct and the pancreatitis is severe, an ERCP with sphincterotomy and stone extraction may be necessary. This must be balanced with the risk of ERCP-induced

1	If gallstones are present obstructing the duct and the pancreatitis is severe, an ERCP with sphincterotomy and stone extraction may be necessary. This must be balanced with the risk of ERCP-induced pancreatitis and thus is usually only employed if supportive measures are failing.Gallstone Ileus. Gallstone ileus can occur when a large gall-stone erodes through the wall of the gallbladder directly into the intestine via a choledochoenteric fistula (Fig. 32-20A). These stones can then pass through the intestinal tract until they reach an area of fixed obstruction. Proximal stones can become impacted in the pylorus or proximal duodenum causing gastric outlet obstruction (Bouveret syndrome). Those that travel dis-tally may become lodged at surgical anastomoses or the ileoce-cal valve, where they can become impacted and cause small bowel obstruction. Gallstone ileus is responsible for less than 1% of all intestinal obstructions.47 These patients present with symptoms of obstipation, nausea,

1	can become impacted and cause small bowel obstruction. Gallstone ileus is responsible for less than 1% of all intestinal obstructions.47 These patients present with symptoms of obstipation, nausea, and abdominal pain. Plain films may show an obstructive bowel gas pattern but may fail to identify a radiolucent stone. Ultrasound evaluation may be lim-ited by extensive bowel gas. CT is highly sensitive and specific for gallstone ileus and will help to determine the location of the obstruction. Management of gallstone ileus focuses on relieving the intestinal obstruction and removing the stone. In cases of very proximal obstructions in the stomach or duodenum, endo-scopic retrieval can be effective. For more distal stones, surgical enterolithotomy can be accomplished either laparoscopically or open. This procedure entails the removal of the stone through ABFigure 32-20. Gallstone Ileus. A. A choledochoenteric fistula has formed between the gallbladder and the duodenum, allowing a

1	or open. This procedure entails the removal of the stone through ABFigure 32-20. Gallstone Ileus. A. A choledochoenteric fistula has formed between the gallbladder and the duodenum, allowing a gallstone to pass into the intestinal tract. B. Intraoperative photo showing a longitudinal enterotomy and extraction of an impacted stone from the distal small bowel.Brunicardi_Ch32_p1393-p1428.indd 140911/02/19 2:43 PM 1410SPECIFIC CONSIDERATIONSPART IIan enterotomy that is then either repaired or resected depend-ing on its size (Fig. 32-20B). Stones that have successfully traversed the ileocecal valve are likely to pass without further intervention. The role of pursuing cholecystectomy and/or cho-ledochoenteric fistula closure at the time of enterolithotomy or addressing it at a later time remains a topic of debate, but it should be considered to reduce the risk of recurrence.47CholangiohepatitisCholangiohepatitis, also known as recurrent pyogenic cholan-gitis, is endemic to the Orient.

1	a topic of debate, but it should be considered to reduce the risk of recurrence.47CholangiohepatitisCholangiohepatitis, also known as recurrent pyogenic cholan-gitis, is endemic to the Orient. It also has been encountered in Asian population in the United States, Europe, and Australia. It affects both sexes equally and occurs most frequently in the third and fourth decades of life. Cholangiohepatitis is caused by bacterial contamination (commonly E coli, Klebsiella spe-cies, Bacteroides species, or Enterococcus faecalis) of the bili-ary tree, and often it is associated with biliary parasites such as Clonorchis sinensis, Opisthorchis viverrini, and A lumbri-coides. Bacterial enzymes cause deconjugation of bilirubin, which precipitates as bile sludge. The sludge and dead bacterial cell bodies form brown pigment stones, the nucleus of which may contain an adult Clonorchis worm, an ovum, or an ascarid. These stones can form throughout the biliary tree and cause par-tial obstructions that

1	form brown pigment stones, the nucleus of which may contain an adult Clonorchis worm, an ovum, or an ascarid. These stones can form throughout the biliary tree and cause par-tial obstructions that contribute to repeated bouts of cholangi-tis, biliary strictures, further stone formation, infection, hepatic abscesses, or liver failure (secondary biliary cirrhosis).48Patients with cholangiohepatitis usually present with pain in the right upper quadrant or epigastrium, fever, and jaundice. Relapsing symptoms are one of the most characteristic features of the disease. The episodes may vary in severity but, without intervention, will gradually lead to malnutrition and hepatic insufficiency. An ultrasound may detect stones in the biliary tree, pneumobilia from infection by gas-forming organisms, liver abscesses, and, occasionally, strictures. The gallbladder may be thickened and inflamed in about 20% of patients but rarely contains gallstones. ERCP or MRCP can be utilized for biliary imaging

1	liver abscesses, and, occasionally, strictures. The gallbladder may be thickened and inflamed in about 20% of patients but rarely contains gallstones. ERCP or MRCP can be utilized for biliary imaging for cholangiohepatitis. They can detect obstruc-tions and define strictures and stones. ERCP (or PTC if nec-essary) has the additional benefit of allowing for emergent decompression of the biliary tree in the septic patient. Hepatic abscesses may be drained percutaneously. The long-term goal of therapy is to extract stones and debris and relieve strictures. It may take several procedures, and in severe, refractory cases in which stones and strictures cannot be relieved, it may require a hepaticojejunostomy to reestablish biliary–enteric continuity. Occasionally, resection of involved areas of the liver may offer the best form of treatment. Recurrences are common, and the prognosis is poor once hepatic insufficiency has developed.49PROCEDURAL INTERVENTIONS FOR GALLSTONE

1	involved areas of the liver may offer the best form of treatment. Recurrences are common, and the prognosis is poor once hepatic insufficiency has developed.49PROCEDURAL INTERVENTIONS FOR GALLSTONE DISEASEPercutaneous Transhepatic Cholecystostomy TubesIn cases in which a patient with cholecystitis is deemed to be too ill to safely undergo cholecystectomy, a cholecystostomy tube may be placed into the gallbladder to decompress and drain a distended, inflamed, hydropic, or purulent gallbladder.36 Surgi-cal cholecystostomy with a large catheter placed under local anesthesia is rarely required today. Rather, percutaneous tran-shepatic cholecystostomy (PTC) tubes are most often pigtail catheters inserted percutaneously under ultrasound guidance.50 The catheter is inserted over a guidewire that has been passed through the abdominal wall, the liver, and into the gallblad-der (Fig. 32-21). By passing the catheter through the liver, the risk of uncontrolled bile leak around the catheter and

1	has been passed through the abdominal wall, the liver, and into the gallblad-der (Fig. 32-21). By passing the catheter through the liver, the risk of uncontrolled bile leak around the catheter and into the peritoneal cavity is minimized. The catheter can be removed when the inflammation has resolved and the patient’s condition has improved. A patent cystic duct should be confirmed by a tube cholangiogram prior to its removal. Interval cholecystec-tomy should be considered if the patient’s fitness has improved, particularly in individuals whose etiology of cholecystitis was gallstones.Endoscopic InterventionsEndoscopic advances in the last few decades have made endos-copy and ERCP a valuable therapeutic tool in the management of gallstone disease, particularly in the setting of common bile duct stones or abnormalities. Using a 90-degree side-viewing endoscope, the duodenum can be entered and the ampulla of Vater on the medial wall of the second portion of the duode-num visualized. This

1	duct stones or abnormalities. Using a 90-degree side-viewing endoscope, the duodenum can be entered and the ampulla of Vater on the medial wall of the second portion of the duode-num visualized. This can then be cannulated to allow wire and catheter access to the biliary tree, facilitating retrograde chol-angiogram, diagnostic brushings, stenting, dilations, or fluoro-scopically guided basket or balloon retrieval of common bile duct stones. When CBD stones are present, endoscopic sphinc-terotomy should be performed, which will allow for passage of larger stones both at the time of bile duct clearance and in the case of any ongoing choledocholithiasis (Fig. 32-22). In the hands of experts, ERCP has high rates of successful cannulation and bile duct clearance, and it is a safe and tolerable procedure. Debate remains when comparing ERCP to surgical common bile duct exploration in terms of timing and outcomes for choledo-cholithiasis, but both are considered acceptable treatments.41 In

1	procedure. Debate remains when comparing ERCP to surgical common bile duct exploration in terms of timing and outcomes for choledo-cholithiasis, but both are considered acceptable treatments.41 In special cases, such as the presence of Roux-en-Y anatomy or a previous hepaticojejunostomy, ERCP can be difficult. How-ever, such anatomy does not preclude the option for endoscopic intervention. Laparoscopic-assisted ERCP (in which the rem-nant stomach is accessed surgically and the endoscope passed into the duodenum) or double-balloon ERCP can be utilized to reach the biliary tree.CholecystectomyCholecystectomy is one of the most common abdominal sur-geries performed in Western countries, with over 750,000 Figure 32-21. Percutaneous cholecystostomy. A pigtail catheter has been placed through the abdominal wall, the right lobe of the liver, and into the gallbladder.Brunicardi_Ch32_p1393-p1428.indd 141011/02/19 2:43 PM 1411GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32being

1	the abdominal wall, the right lobe of the liver, and into the gallbladder.Brunicardi_Ch32_p1393-p1428.indd 141011/02/19 2:43 PM 1411GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32being performed each year in the United States alone.51 Carl Langenbuch performed the first successful open cholecystectomy in 1882, and for >100 years, it was the standard treatment for symptomatic gallbladder stones. In 1987, laparoscopic chole-cystectomy was introduced by Philippe Mouret in France and quickly revolutionized the treatment of gallstone disease. It not only supplanted open cholecystectomy, but it also more or less ended attempts for noninvasive management of gallstones (such as extracorporeal shock wave or cholangioscopic lithotripsy) or medical therapies (such as bile salts). Laparoscopic cholecystec-tomy offers a cure for gallstones with a minimally invasive pro-cedure, minor pain and scarring, and early return to full activity. Today, laparoscopic cholecystectomy is the

1	Laparoscopic cholecystec-tomy offers a cure for gallstones with a minimally invasive pro-cedure, minor pain and scarring, and early return to full activity. Today, laparoscopic cholecystectomy is the treatment of choice for symptomatic gallstones and the complications of gallstone disease.Few absolute contraindications exist to laparoscopic cholecystectomy, but they include hemodynamic instability, uncontrolled coagulopathy, or frank peritonitis. In addition, patients with severe obstructive pulmonary disease (COPD) or congestive heart failure (e.g., cardiac ejection fraction <20%) might not tolerate the increased intraabdominal pressures of pneumoperitoneum with carbon dioxide and may require open cholecystectomy. Conditions formerly believed to be relative contraindications such as acute cholecystitis, gangrene and empyema of the gallbladder, biliary-enteric fistulae, obesity, pregnancy, ventriculoperitoneal shunts, cirrhosis, and previous upper abdominal procedures are now

1	as acute cholecystitis, gangrene and empyema of the gallbladder, biliary-enteric fistulae, obesity, pregnancy, ventriculoperitoneal shunts, cirrhosis, and previous upper abdominal procedures are now considered risk factors for a potentially difficult cholecystectomy, but they do not preclude an attempt at laparoscopy. While laparoscopic outcomes have steadily improved and laparoscopic cholecystectomy has been shown multiple times to be safe and feasible, conversion to an open operation should always remain an option, and it is not a failure. Conversion to open may be necessary if the patient is unable to tolerate pneumoperitoneum, a complication occurs that cannot be fixed laparoscopically, important anatomic struc-tures cannot be clearly identified, or when no progress is made over a set period of time. In the elective setting, conversion to an open procedure is needed in about 5% of patients.51 Emer-gent procedures or patients with complicated gallstone disease can be more

1	a set period of time. In the elective setting, conversion to an open procedure is needed in about 5% of patients.51 Emer-gent procedures or patients with complicated gallstone disease can be more challenging, and the incidence of conversion has been reported to be between 10% and 30%. The possibility of conversion to open should always be discussed with the patient preoperatively.Serious complications of cholecystectomy are rare. The mortality rate for laparoscopic cholecystectomy is about 0.1%. Wound infection and cardiopulmonary complication rates are considerably lower following laparoscopic cholecystectomy than are those for an open procedure.52 While laparoscopic cho-lecystectomy has historically been associated with a higher rate of injury to the bile ducts than the open approach, modern data appears to show this trend disappearing as familiarity with lapa-roscopic techniques and technologies have improved.53Patients undergoing cholecystectomy should have a complete blood count

1	data appears to show this trend disappearing as familiarity with lapa-roscopic techniques and technologies have improved.53Patients undergoing cholecystectomy should have a complete blood count and liver function tests preoperatively. Prophylaxis against deep venous thrombosis with either low molecular weight heparin or compression stockings is indicated. ABCFigure 32-22. An endoscopic sphincterotomy. A. The sphincterotome in place. B. Completed sphincterotomy. C. Endoscopic picture of ampulla before and after sphincterotomy.Brunicardi_Ch32_p1393-p1428.indd 141111/02/19 2:43 PM 1412SPECIFIC CONSIDERATIONSPART IIThe patient should be instructed to empty their bladder before coming to the operating room to avoid the need for urinary cath-eterization. An orogastric tube can be placed if the stomach is distended with gas, but it is generally removed at the end of the operation.Laparoscopic Cholecystectomy. The patient is typically positioned supine with the operating surgeon standing

1	the stomach is distended with gas, but it is generally removed at the end of the operation.Laparoscopic Cholecystectomy. The patient is typically positioned supine with the operating surgeon standing at the patient’s left side. Split-leg positioning with the surgeon stand-ing between the patient’s legs can also provide ergonomic access to the right upper quadrant. Tucking one arm can be helpful if a cholangiogram is planned to allow easier maneu-vering of the fluoroscopy machine around the patient. Pneu-moperitoneum is established with carbon dioxide gas, either with an open technique (Hasson), optical viewing trocar, or closed-needle technique (Veress). Typical access is at the supra-umbilical region, though in the case of previous surgery or scars, alternate access sites should be considered. Once an adequate pneumoperitoneum is established, a 5or 10-mm trocar is inserted through the supraumbilical incision, through which a 5or 10-mm 30° laparoscope is introduced. Traditionally,

1	Once an adequate pneumoperitoneum is established, a 5or 10-mm trocar is inserted through the supraumbilical incision, through which a 5or 10-mm 30° laparoscope is introduced. Traditionally, three additional ports are then placed with a 10or 12-mm port in the epigastrium, a 5-mm port in the right midclavicular line, and a 5-mm port in the right flank (Fig. 32-23). Additional ports may be placed as needed to aid with retraction in difficult cases.Through the lateral-most port, the assistant uses a locking instrument to grasp the gallbladder fundus and retract it over the liver edge and upward towards the patient’s right shoulder. This will help visualize the body of the gallbladder and the hilar area. Exposure may be facilitated by placing the patient ABFCEDFigure 32-23. Laparoscopic cholecystectomy. A. The trocar placement. B. The fundus has been grasped and retracted cephalad to expose the proximal gallbladder and the hepatoduodenal ligament. Another grasper retracts the gallbladder

1	A. The trocar placement. B. The fundus has been grasped and retracted cephalad to expose the proximal gallbladder and the hepatoduodenal ligament. Another grasper retracts the gallbladder infundibulum posterolaterally to better expose the triangle of Calot (hepatocystic triangle bound by the common hepatic duct, cystic duct, and liver margin). C. Intraoperative photo of the critical view of safety. The hepatocystic triangle has been cleared of fat and fibrous tissue, the lower one-third of the gallbladder is separated from the liver to expose the cystic plate, and two and only two structures are seen entering the gallbladder. D. A clip is being placed on the cystic duct–gallbladder junction. E. A small opening has been made in the cystic duct, and a cholangiogram catheter is being inserted. F. Additional clips have been placed, the cystic duct has been divided, and the cystic artery is being divided.Brunicardi_Ch32_p1393-p1428.indd 141211/02/19 2:43 PM 1413GALLBLADDER AND THE

1	F. Additional clips have been placed, the cystic duct has been divided, and the cystic artery is being divided.Brunicardi_Ch32_p1393-p1428.indd 141211/02/19 2:43 PM 1413GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32in reverse Trendelenburg position with slight tilting of the table to bring the right side up. Through the midclavicular port, the surgeon uses a grasper in the left hand to retract the gallbladder infundibulum laterally and expose the neck of the gallbladder and hepatoduodenal ligament. It may be necessary to take down any adhesions between the omentum, duodenum, or colon to the gallbladder in order to reach the infundibulum. The majority of the dissection can then be performed with the right hand through the epigastric port, utilizing a combination of electrocautery and sharp and blunt dissection.Dissection starts at the infundibulum of the gallbladder, just above the takeoff of the cystic duct. The peritoneum, fat, and loose areolar tissue around the

1	and sharp and blunt dissection.Dissection starts at the infundibulum of the gallbladder, just above the takeoff of the cystic duct. The peritoneum, fat, and loose areolar tissue around the gallbladder and the cystic duct–gallbladder junction is dissected off and reflected inferi-orly toward the bile duct. This is continued until the gallbladder neck and the proximal cystic duct are clearly identified. The next step is the identification of the cystic artery, which usually runs parallel to and somewhat behind the cystic duct, and often lies behind a prominent lymph node (Lund’s node, often called Calot’s node). At this point, a critical view of safety should be obtained. This requires that the hepatocystic triangle is cleared of fat and fibrous tissue, the lower third of the gallbladder is separated from the liver to expose the cystic plate, and two and only two structures (cystic duct and cystic artery) are going into the gallbladder (see Fig. 32-19).54 At this point, an

1	is separated from the liver to expose the cystic plate, and two and only two structures (cystic duct and cystic artery) are going into the gallbladder (see Fig. 32-19).54 At this point, an intraopera-tive cholangiogram can be performed if indicated (see “Intraop-erative Cholangiogram” section).With a critical view of safety obtained, the cystic duct and artery are clipped with two clips at the base and one clip on the gallbladder side. They can then be safely divided. Sometimes, a very dilated cystic duct may be too large for clips. Such ducts can be successfully managed by ligation with an endoloop, lapa-roscopic stapler, or suture closure. Finally, the gallbladder is dissected off the liver bed using electrocautery while watching for potential abnormal posterior branches of ducts or arteries. Before the gallbladder is completely removed from the liver edge, it can be used as a retractor for a final evaluation of the operative field. The surgeon should be sure to evaluate for

1	or arteries. Before the gallbladder is completely removed from the liver edge, it can be used as a retractor for a final evaluation of the operative field. The surgeon should be sure to evaluate for bleed-ing points or bile staining, and confirm placement of the clips on the cystic duct and artery. The gallbladder is then divided from its final attachments and removed either through the epigastric or umbilical incision, often with the aid of a retrieval bag. The fascial defect and skin incision may need to be enlarged in order to remove the specimen, particularly if the stones are large or the gallbladder is very inflamed. Any bile or blood that has accumulated during the procedure should be cleaned away, and if stones were spilled, they should be retrieved and removed. If the gallbladder was severely inflamed or gangrenous, or if any bile or blood is expected to accumulate, a closed-suction drain can be placed through one of the 5-mm ports and left underneath the right liver lobe

1	was severely inflamed or gangrenous, or if any bile or blood is expected to accumulate, a closed-suction drain can be placed through one of the 5-mm ports and left underneath the right liver lobe close to the gallbladder fossa, though this is not routinely required.Open Cholecystectomy. The same surgical principles apply for laparoscopic and open cholecystectomies. Open cholecystec-tomy has become an uncommon procedure, usually performed either as a conversion from laparoscopic cholecystectomy or as a second procedure in patients who require laparotomy for another reason. The approach can either be through a midline laparotomy, or more commonly through a right subcostal inci-sion. The gallbladder is dissected free from the liver bed, usu-ally starting at the fundus and working proximally toward the hepatocystic triangle. Once the cystic artery and cystic duct have been dissected and clearly identified, they are ligated and divided, and the gallbladder is removed. In particularly

1	toward the hepatocystic triangle. Once the cystic artery and cystic duct have been dissected and clearly identified, they are ligated and divided, and the gallbladder is removed. In particularly difficult cases, in which the gallbladder is partially obliterated or ductal or arterial anatomy cannot be identified, a partial cholecystec-tomy may be performed. This includes removal of as much gall-bladder mucosa as possible and attempted closure of the cystic duct stump with wide drainage of the area.Intraoperative Cholangiogram. Intraoperative cholangio-gram is an optional but valuable tool for evaluating the extra-hepatic bile ducts, identifying common bile duct stones, or clarifying aberrant ductal anatomy. The use of routine versus selective cholangiography remains a topic of debate with a lack of definitive evidence on either side.55-57 However, routine intra-operative cholangiography will detect stones in approximately 7% of patients, and it assists with outlining anatomy and

1	with a lack of definitive evidence on either side.55-57 However, routine intra-operative cholangiography will detect stones in approximately 7% of patients, and it assists with outlining anatomy and detect-ing injury.58 Selective intraoperative cholangiogram should be performed when the patient has a history of abnormal liver func-tion tests, pancreatitis, jaundice, a large duct and small stones, a dilated duct on preoperative ultrasonography, or if preoperative endoscopic cholangiography for the aforementioned reasons was unsuccessful. Although there is no consensus recommenda-tion on the use of routine versus selective cholangiography, all surgeons performing cholecystectomy should be familiar with the procedure. If a cholangiogram is to be performed, a clip is placed on the proximal cystic duct, and a small incision is made on its anterior surface, just inferior to the clip. A cholangiogram catheter is passed into the cystic duct and secured with a clamp or clip. The fluoroscopy

1	cystic duct, and a small incision is made on its anterior surface, just inferior to the clip. A cholangiogram catheter is passed into the cystic duct and secured with a clamp or clip. The fluoroscopy machine is then positioned over the patient and a cholangiogram performed by injection of contrast through the cholangiocatheter during live fluoroscopic dynamic imaging. An ideal cholangiogram includes filling of the right and left hepatic ducts, emptying into the duodenum, and no visualized filling defects (Fig. 32-24). Care must be taken not to introduce air bubbles into the system during contrast injec-tion as these will appear as filling defects on the cholangiogram images. If no contrast is visualized in the duodenum, a dose of glucagon can be utilized to relax the sphincter of Oddi and facilitate contrast flow. Once the cholangiogram is completed, the catheter is removed. Laparoscopic ultrasonography is as accurate as intraoperative cholangiography in detecting com-mon bile duct

1	facilitate contrast flow. Once the cholangiogram is completed, the catheter is removed. Laparoscopic ultrasonography is as accurate as intraoperative cholangiography in detecting com-mon bile duct stones, and it is less invasive. However, it requires more skill to perform and interpret and is not always readily available.59Common Bile Duct ExplorationCommon bile duct stones that are detected preor intraopera-tively may be managed with common bile duct exploration (CBDE) at the time of the cholecystectomy. While preopera-tive ERCP is also an appropriate option for known bile duct stones, laparoscopic CBDE can be used as a primary approach to choledocholithiasis safely and with good outcomes, even in higher risk populations such as the elderly.60 If stones in the duct are small, they may sometimes be simply flushed into the duo-denum with saline irrigation via the cholangiography catheter. This can be facilitated by the administration of IV glucagon to relax the sphincter of Oddi. If

1	sometimes be simply flushed into the duo-denum with saline irrigation via the cholangiography catheter. This can be facilitated by the administration of IV glucagon to relax the sphincter of Oddi. If irrigation is unsuccessful, several options exist to clear the duct, including fluoroscopic or endo-scopic approaches.With access to the cystic duct by a small ductotomy, a bal-loon catheter is used to dilate the cystic duct, and a wire basket can be passed down the common bile duct under fluoroscopic Brunicardi_Ch32_p1393-p1428.indd 141311/02/19 2:43 PM 1414SPECIFIC CONSIDERATIONSPART IIguidance to catch and remove the stones (Fig. 32-25). Alter-nately, endoscopic evaluation with a flexible choledocho-scope will allow for direct visualization and retrieval of the stones within the common duct. To do this, reliable catheter access must be obtained with an introducer sheath placed either through one of the laparoscopic ports or a new stab incision in the anterior abdominal wall. The

1	duct. To do this, reliable catheter access must be obtained with an introducer sheath placed either through one of the laparoscopic ports or a new stab incision in the anterior abdominal wall. The cystic duct should first be dilated with a small balloon catheter to allow for passage of the introducer and scope and for effective retrieval of larger stones. Once the scope is within the common bile duct, irrigation is used to distend the lumen. Stones may then be caught in a wire basket under direct visualization or simply pushed into the duo-denum. Once the common bile duct has been cleared of stones, the cystic duct is ligated below the level of the ductotomy and divided, and the cholecystectomy is completed.While the cystic duct is the preferred route of access for common bile duct exploration, occasionally an incision into the common bile duct itself (choledochotomy) is necessary. The flexible choledochoscope is then passed into the duct for visu-alization and clearance of stones.

1	occasionally an incision into the common bile duct itself (choledochotomy) is necessary. The flexible choledochoscope is then passed into the duct for visu-alization and clearance of stones. The choledochotomy can be closed primarily of the duct is very large, or over a T-tube. If available, common bile duct exploration can be highly advan-tageous as it provides the opportunity to treat the entirety of the disease in a single event, rather that subjecting patients to multiple procedures. However, the procedure can be techni-cally challenging to perform and requires the availability of the proper equipment and surgical expertise.61Common Bile Duct Drainage ProceduresIn very rare cases in which stones or obstructions cannot be cleared by either ERCP with sphincterotomy or CBDE, and the patient is suffering clinical effects from their common duct stones, an additional choledochal drainage procedure may become necessary. In the case of an open operation, transduodenal sphincterotomy can

1	is suffering clinical effects from their common duct stones, an additional choledochal drainage procedure may become necessary. In the case of an open operation, transduodenal sphincterotomy can be attempted by incising the duodenum transversely and cutting the sphincter of Oddi at the 11 o’clock position, taking care to avoid injury to the pancreatic duct. The impacted stones can then be manually removed or simply allowed to pass through the sphincterotomy.Bypass procedures can also be used to restore continu-ity of bile flow in the setting of irretrievable impacted stones. For short distance bypasses, a Choledochoduodenostomy is performed by mobilizing the second part of the duodenum (a Kocher maneuver) and anastomosing it side to side with the common bile duct (Fig. 32-26A-C). If the distance is too great to safely complete a choledochoduodenostomy without ten-sion, a choledochojejunostomy can be done by bringing up a roughly 45-cm limb of jejunum and anastomosing it end to side to

1	is too great to safely complete a choledochoduodenostomy without ten-sion, a choledochojejunostomy can be done by bringing up a roughly 45-cm limb of jejunum and anastomosing it end to side to the common bile duct (Fig. 32-26D-E). If the entirety of the extrahepatic biliary tree must be bypassed, hepaticojejunostomy allows for drainage of the hepatic ducts directly a loop of jeju-num (Fig. 32-26F-G). These choledochal drainage procedures can also be used to manage common bile duct strictures or as a palliative procedure for malignant obstruction in the periampul-lary region.OTHER BENIGN DISEASES AND LESIONSBiliary Dyskinesia and Sphincter of Oddi DysfunctionBiliary dyskinesia is an umbrella term that refers to disorders affecting the normal motility and function of the gallbladder and sphincter of Oddi. These disorders are becoming increas-ingly recognized as improvements in imaging allow for more detailed evaluations of biliary tract function. Patients with bili-ary dyskinesia may

1	of Oddi. These disorders are becoming increas-ingly recognized as improvements in imaging allow for more detailed evaluations of biliary tract function. Patients with bili-ary dyskinesia may present with typical biliary type symptoms, but without evidence of stones or sludge on abdominal imaging. ABFigure 32-24. A. An intraoperative cholangiogram. The bile ducts are of normal size, with no intraluminal filling defects. The left and the right hepatic ducts are visualized, the distal common bile duct tapers down, and the contrast empties into the duodenum. Cholangiography grasper that holds the catheter and the cystic duct stump partly projects over the common hepatic duct. B. An intraoperative cholangiogram showing a common bile duct stone (arrow) with very little contrast passing into the duodenum.Brunicardi_Ch32_p1393-p1428.indd 141411/02/19 2:43 PM 1415GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32A decreased gallbladder ejection fraction on HIDA scanning (EF <35%) is

1	duodenum.Brunicardi_Ch32_p1393-p1428.indd 141411/02/19 2:43 PM 1415GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32A decreased gallbladder ejection fraction on HIDA scanning (EF <35%) is considered diagnostic of biliary dyskinesia. In these patients, studies suggest that symptoms will be improved or resolved by cholecystectomy in up to 90% of cases.62Sphincter of Oddi dysfunction can occur as a primary pre-sentation of episodic biliary type pain with abnormal liver func-tion tests or as recurrent biliary type pain after cholecystectomy. More severe cases may present with recurrent jaundice or pan-creatitis. If other causes are ruled out, such as retained stones, strictures or periampullary tumors, a stenotic or dyskinetic sphincter of Oddi should be suspected. A benign stenosis of the outlet of the common bile duct is usually associated with inflam-mation, fibrosis, or muscular hypertrophy. The pathogenesis is unclear, but trauma from the passage of stones, sphincter

1	of the outlet of the common bile duct is usually associated with inflam-mation, fibrosis, or muscular hypertrophy. The pathogenesis is unclear, but trauma from the passage of stones, sphincter motil-ity disorders, and congenital anomalies have been suggested. A dilated common bile duct that is difficult to cannulate during ERCP or delayed emptying of contrast from the biliary tree after ERCP are useful diagnostic features. Ampullary manometry and specific provocation tests are available in specialized units to aid in the diagnosis. Once identified, sphincterotomy will typi-cally yield good results.63Acalculous CholecystitisAcalculous cholecystitis is an acute inflammation of the gall-bladder that occurs in the absence of gallstones. It is a rare entity that typically develops in critically ill patients in the intensive care unit.64 Patients on parenteral nutrition, with extensive burns, sepsis, major operations, multiple trauma, or prolonged illness with multiple organ system failure

1	ill patients in the intensive care unit.64 Patients on parenteral nutrition, with extensive burns, sepsis, major operations, multiple trauma, or prolonged illness with multiple organ system failure are at risk for developing acalculous cholecystitis. The cause is unknown, but gallblad-der distention, bile stasis, and ischemia have been implicated as causative factors. After resection, pathologic examination of the gallbladder wall after an episode of acalculous cholecystitis reveals edema of the serosa and muscular layers, with patchy thrombosis of arterioles and venules.65The ability to recognize the symptoms and signs of acalculous cholecystitis can depend on the condition and mental status of the patient, but acalculous cholecystitis can be similar to acute calculous cholecystitis, with right upper quadrant pain and tenderness, fever, and leukocytosis. In the sedated or unconscious patient, the clinical features are often masked, but fever and elevated WBC count, as well as

1	with right upper quadrant pain and tenderness, fever, and leukocytosis. In the sedated or unconscious patient, the clinical features are often masked, but fever and elevated WBC count, as well as eleva-tion of alkaline phosphatase and bilirubin, are indications for AIIIIIIEBFGCDFigure 32-25. Laparoscopic common bile duct exploration. I. Transcystic basket retrieval using fluoroscopy. A. The basket has been advanced past the stone and opened. B. The stone has been entrapped in the basket, and together, they are removed from the cystic duct. II. Transcystic choledochoscopy and stone removal. C. The basket has been passed through the working channel of the scope, and the stone is entrapped under direct vision. D. Entrapped stone. E. A view from the choledochoscope with stone captured in basket. III. Choledochotomy and stone removal. F. A small incision is made in the common bile duct. G. The common bile duct is cleared of stones.Brunicardi_Ch32_p1393-p1428.indd 141511/02/19 2:43 PM

1	III. Choledochotomy and stone removal. F. A small incision is made in the common bile duct. G. The common bile duct is cleared of stones.Brunicardi_Ch32_p1393-p1428.indd 141511/02/19 2:43 PM 1416SPECIFIC CONSIDERATIONSPART IIAIIIIIIBCDEFGFigure 32-26. Biliary enteric anastomoses. There are three types. I. Choledochoduodenostomy. A. The distal common bile duct is opened longitudinally, as is the duodenum. B. Interrupted sutures are placed between the common bile duct and the duodenum. C. Completed cho-ledochoduodenostomy. II. Choledochojejunostomy. D. The common bile duct and small bowel are divided. E. A limb of jejunum is brought up in a Roux-en-Y configuration and anastomosed to the bile duct. III. Hepaticojejunostomy. F. The entire extrahepatic biliary tree has been resected and the reconstruction completed with a Roux-en-Y limb of jejunum. G. Percutaneous transhepatic stents are placed across hepati-cojejunostomy (optional).Brunicardi_Ch32_p1393-p1428.indd 141611/02/19

1	the reconstruction completed with a Roux-en-Y limb of jejunum. G. Percutaneous transhepatic stents are placed across hepati-cojejunostomy (optional).Brunicardi_Ch32_p1393-p1428.indd 141611/02/19 2:43 PM 1417GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32further investigation. Ultrasonography is usually the diagnos-tic test of choice, as it can be done bedside in the intensive care unit. It can demonstrate the distended gallbladder with thickened wall, biliary sludge, pericholecystic fluid, and the presence or absence of abscess formation. CT scanning can aid in the diagnosis of acalculous cholecystitis and addition-ally allows a more general evaluation of the abdomen and chest to rule out other sources of infection. A HIDA scan can also be useful if it shows nonvisualization of the gallblad-der, but it is less sensitive and can have higher false-positive rates in patients who are in a prolonged fasting state, on total parenteral nutrition, or have liver disease. Once the

1	of the gallblad-der, but it is less sensitive and can have higher false-positive rates in patients who are in a prolonged fasting state, on total parenteral nutrition, or have liver disease. Once the diagnosis is confirmed, acalculous cholecystitis requires urgent inter-vention as rapid deterioration can occur. This should include early broad-spectrum antibiotics and fluid resuscitation. If the patient is stable to undergo an abdominal operation, lapa-roscopic cholecystectomy is the most definitive treatment, and it can be safely performed even in the setting of severe acute inflammation.64 However, if patients are critically ill and unfit for surgery, percutaneous cholecystostomy is the best treatment choice (see Fig. 32-18). About 90% of patients will improve with a percutaneous cholecystostomy tube. Interval cholecystectomy can be discussed with the patient after they have recovered from their acute illness, but it is not strictly required in the absence of gallstone or other

1	tube. Interval cholecystectomy can be discussed with the patient after they have recovered from their acute illness, but it is not strictly required in the absence of gallstone or other identi-fied gallbladder pathology.Choledochal (Biliary) CystsCholedochal cysts are congenital cystic dilatations of the extra-hepatic and/or intrahepatic biliary tree. They are rare, with an incidence of between 1:100,000 and 1:150,000 in populations of Western countries, but are more common in populations of Eastern countries occurring in as many as 1:1000 individuals. Choledochal cysts affect females three to eight times more often than males. Although frequently found in infancy or childhood, nearly one-half are diagnosed in adults. The cause is unknown, but it is believed that weakness of the bile duct wall and increased pressure secondary to partial biliary obstruction can contribute to biliary cyst formation. More than 90% of patients have an anomalous pancreaticobiliary duct junction, with the

1	wall and increased pressure secondary to partial biliary obstruction can contribute to biliary cyst formation. More than 90% of patients have an anomalous pancreaticobiliary duct junction, with the pancreatic duct joining the common bile duct outside the duode-nal wall, creating a long common channel (>1.5 cm). This may allow free reflux of pancreatic secretions into the biliary tract, leading to inflammatory changes, increased biliary pressure, and cyst formation. The cysts are lined with cuboidal epithelium and can vary in size from small dilations to giant cystic masses. The typical clinical triad of biliary cysts includes abdominal pain, jaundice, and a palpable mass, though this constellation is seen in less than one-half of patients. Adults may present with chol-angitis. Blood tests will often be normal though elevations of transaminases can be seen in cases of infection or obstruction. Ultrasonography or CT scanning will confirm the diagnosis, but ERCP or MRCP are essential to

1	will often be normal though elevations of transaminases can be seen in cases of infection or obstruction. Ultrasonography or CT scanning will confirm the diagnosis, but ERCP or MRCP are essential to formally assess the biliary anatomy and to plan the appropriate surgical treatment. The risk of cholangiocarcinoma in patients with choledochal cysts is 20to 30-fold higher than in the general population and varies with the patient’s age and type of cyst. For this reason, excision is recommended whenever possible when high-risk choledochal cysts are diagnosed.Choledochal cysts are classified into five types depend-ing on the location and structure of the cysts. The subcatego-ries of choledochal cysts are defined in Fig. 32-27. Type I cysts (fusiform CBD dilations) are the most common form, account-ing for approximately 50% of cases, and have the highest risk of malignancy (>60%). For types I and II (saccular diverticula of the common bile duct), excision of the cystic dilations in the

1	account-ing for approximately 50% of cases, and have the highest risk of malignancy (>60%). For types I and II (saccular diverticula of the common bile duct), excision of the cystic dilations in the extrahepatic biliary tree, including cholecystectomy, with either simple cyst excision or duct resection with Roux-en-Y hepaticojejunostomy is ideal. Type III cysts (intraduodenal) cre-ate a treatment challenge as full resection would require pan-creaticoduodenectomy. Given that type III cyst are associated with the lowest malignancy risk of any choledochal cyst (~2%), sphincterotomy and surveillance is generally recommended over formal excision.66 In Type IV (multiple cysts), excision of all cystic tissue and reconstruction is again recommended. For type IVa, which is characterized by multiple cysts with intrahe-patic involvement, additional segmental resection of the liver may be required if intrahepatic stones, strictures, or abscesses are present. Type V choledochal cysts (Caroli

1	cysts with intrahe-patic involvement, additional segmental resection of the liver may be required if intrahepatic stones, strictures, or abscesses are present. Type V choledochal cysts (Caroli disease) are very rare and account for less than 1% of patients with choledochal cysts. These cysts are multiple and can affect the entire liver. In advanced stages, this may result in cirrhosis and liver failure necessitating liver transplantation.Primary Sclerosing CholangitisPrimary sclerosing cholangitis (PSC) is an uncommon disease characterized by inflammatory strictures involving the intrahe-patic and extrahepatic biliary tree. It is a progressive disease that eventually results in secondary biliary cirrhosis. Sometimes, biliary strictures are clearly secondary to bile duct stones, acute cholangitis, previous biliary surgery, or toxic agents, and are termed secondary sclerosing cholangitis. However, primary sclerosing cholangitis is a disease entity of its own, with no clear attributing

1	previous biliary surgery, or toxic agents, and are termed secondary sclerosing cholangitis. However, primary sclerosing cholangitis is a disease entity of its own, with no clear attributing cause. Autoimmune reaction, chronic low-grade bac-terial or viral infection, toxic reaction, and genetic factors have all been suggested to play a role in its pathogenesis. PSC is commonly associated with other autoimmune diseases includ-ing ulcerative colitis in about two-thirds of patients, Riedel’s thyroiditis, and retroperitoneal fibrosis. The human leukocyte antigen haplotypes HLA-B8, DR3, DQ2, and DRw52A, com-monly found in patients with autoimmune diseases, also are more frequently seen in patients with primary sclerosing chol-angitis than in controls. The mean age of presentation for PSC is 30 to 45 years, and men are affected twice as often as women. Most patients are symptomatic when diagnosed, and may com-plain of intermittent jaundice, fatigue, weight loss, pruritus, or abdominal pain.

1	to 45 years, and men are affected twice as often as women. Most patients are symptomatic when diagnosed, and may com-plain of intermittent jaundice, fatigue, weight loss, pruritus, or abdominal pain. Initial presentation with acute cholangitis is rare without preceding biliary tract intervention or surgery. A minority of patients are diagnosed incidentally by elevated liver function tests, particular when found in a patient with ulcerative colitis. While the clinical presentation and laboratory results may suggest the PSC, ERCP revealing multiple dilatations and strictures (beading) of the intraand extrahepatic biliary tree confirms the diagnosis. The hepatic duct bifurcation is often the most severely affected segment. A liver biopsy may not be diagnostic, but it is important to determine the degree of hepatic fibrosis and the presence of cirrhosis.The clinical course in sclerosing cholangitis is highly vari-able, but cyclic remissions and exacerbations are typical. Some patients

1	the degree of hepatic fibrosis and the presence of cirrhosis.The clinical course in sclerosing cholangitis is highly vari-able, but cyclic remissions and exacerbations are typical. Some patients will remain asymptomatic for years, while others prog-ress rapidly with the obliterative inflammatory changes leading to secondary biliary cirrhosis and liver failure. In patients with associated ulcerative colitis, the course of each disease seems independent of the other and colectomy has no effect on the Brunicardi_Ch32_p1393-p1428.indd 141711/02/19 2:43 PM 1418SPECIFIC CONSIDERATIONSPART IIcourse of primary sclerosing cholangitis. Of the patients with sclerosing cholangitis, 10% to 15% will develop cholangio-carcinoma, which can present at any time during the disease process and does not necessarily correlate with the extent of the sclerosing cholangitis or the development of liver failure.67 Cholangiocarcinoma in the setting of PSC frequently follows an aggressive course. Patients

1	necessarily correlate with the extent of the sclerosing cholangitis or the development of liver failure.67 Cholangiocarcinoma in the setting of PSC frequently follows an aggressive course. Patients need to be followed by serial ERCP and liver biopsies to evaluate for the development of complica-tions such as strictures, cancers, or cirrhosis.There is no known curative treatment for primary scleros-ing cholangitis and medical management is largely supportive. Corticosteroids, immunosuppressants, ursodeoxycholic acid, and antibiotics have been attempted with disappointing results. If biliary strictures occur, they can be dilated and stented either endoscopically or percutaneously. These measures have given short-term improvements in symptoms and serum bilirubin lev-els but provide long-term results in less than half of patients. Surgical management with resection of the extrahepatic biliary tree and hepaticojejunostomy has produced reasonable results in patients with extrahepatic and

1	results in less than half of patients. Surgical management with resection of the extrahepatic biliary tree and hepaticojejunostomy has produced reasonable results in patients with extrahepatic and bifurcation strictures, but without cirrhosis or significant hepatic fibrosis.68 In patients with pri-mary sclerosing cholangitis and advanced liver disease, liver transplantation is the only option. It offers excellent results, with overall 5-year survival as high as 85%. Unfortunately, recur-rence of PSC can occur in 10% to 20% of patients and may require retransplantation.68Bile Duct StricturesBenign bile duct strictures can have numerous causes. However, the vast majority are related to operative injury, most commonly during cholecystectomy. Other causes include fibrosis due to chronic pancreatitis, common bile duct stones, acute cholan-gitis, biliary obstruction due to cholecystolithiasis (Mirizzi’s syndrome), sclerosing cholangitis, cholangiohepatitis, and stric-tures of a

1	pancreatitis, common bile duct stones, acute cholan-gitis, biliary obstruction due to cholecystolithiasis (Mirizzi’s syndrome), sclerosing cholangitis, cholangiohepatitis, and stric-tures of a biliary-enteric anastomosis. Bile duct strictures that go unrecognized or are improperly managed can lead to severe complications such as recurrent cholangitis, secondary biliary cirrhosis, and portal hypertension.69Bile duct strictures most commonly result in recurrent episodes of cholangitis but may present with isolated jaundice without infection. Liver function tests usually show evidence of cholestasis with elevations of bilirubin and alkaline phosphatase. Imaging with ultrasound or CT can show dilated bile ducts proximal to the stricture, as well as provide informa-tion about the level of the stenosis. MRCP gives more detailed anatomic information about the location and the degree of dilatation. If the diagnosis remains in question, cholangiog-raphy (endoscopic or more rarely

1	level of the stenosis. MRCP gives more detailed anatomic information about the location and the degree of dilatation. If the diagnosis remains in question, cholangiog-raphy (endoscopic or more rarely percutaneous) will outline the biliary tree, define the stricture and its location, and allow for therapeutic interventions (Fig. 32-28). The treatment of biliary strictures depends on the location and the cause of the stricture. Percutaneous or endoscopic dilatation and/or stent placement will provide good results in more than one half of Type IIIType IIType IType VType IVbType IVaFigure 32-27. Classification of choledochal cysts. Type I, fusiform or cystic dilations of the extrahepatic biliary tree, is the most common type, making up >50% of the choledochal cysts. Type II, saccular diverticulum of an extrahepatic bile duct. Rare, <5% of choledochal cysts. Type III, bile duct dilatation within the duodenal wall (choledochoceles), makes up about 5% of choledochal cysts. Types IVa and IVb,

1	of an extrahepatic bile duct. Rare, <5% of choledochal cysts. Type III, bile duct dilatation within the duodenal wall (choledochoceles), makes up about 5% of choledochal cysts. Types IVa and IVb, mul-tiple cysts, make up 5% to 10% of choledochal cysts. Type IVa affects both extrahepatic and intrahepatic bile ducts, whereas type IVb cysts affect the extrahepatic bile ducts only. Type V (Caroli disease), intrahepatic biliary cysts, is very rare and makes up 1% of choledochal cysts.Brunicardi_Ch32_p1393-p1428.indd 141811/02/19 2:44 PM 1419GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32patients. For persistent or complex strictures, surgical resec-tion and reconstruction with Roux-en-Y choledochojejunos-tomy or hepaticojejunostomy may be necessary and will result in good or excellent outcomes in 80% to 90% of patients.70 Choledochoduodenostomy may be a choice for strictures in the distal-most part of the common bile duct if a tension free repair can be achieved.INJURY TO THE

1	outcomes in 80% to 90% of patients.70 Choledochoduodenostomy may be a choice for strictures in the distal-most part of the common bile duct if a tension free repair can be achieved.INJURY TO THE BILIARY TRACTGallbladderInjuries to the gallbladder itself are uncommon but can occur in the setting of penetrating trauma (gunshot or stab wounds) or medical procedures (liver biopsy or surgery). Nonpenetrating trauma to the gallbladder is extremely rare but can cause con-tusion, avulsion, laceration, rupture, or traumatic cholecystitis. Regardless of the etiology of gallbladder injury, the treatment of choice is cholecystectomy. The prognosis is typically good but depends on the extent of related injury, as damage to nearby organs is not uncommon.Extrahepatic Bile DuctsRarely, penetrating trauma to the extrahepatic bile ducts does occur, and it is usually associated with trauma to other viscera. The vast majority of injuries to the extrahepatic bili-ary system, however, are iatrogenic,

1	to the extrahepatic bile ducts does occur, and it is usually associated with trauma to other viscera. The vast majority of injuries to the extrahepatic bili-ary system, however, are iatrogenic, usually occurring during cholecystectomy. These injuries are among the most feared and litigated complications in surgery, and can result in sig-nificant morbidity.71,72 Biliary tract injury can also occur during common bile duct exploration, division or mobilization of the duodenum during gastrectomy, or dissection of the hepatic hilum during liver resections.The incidence of bile duct injury during cholecystectomy is estimated to be relatively low (about 0.2%).73 While ini-tial experience with laparoscopic cholecystectomy appeared to show a higher rate of injury to the bile ducts compared to the open approach, these trends appear to be disappearing as lapa-roscopic technology and familiarity with the techniques of the procedure have improved.53 A number of different factors are thought to be

1	open approach, these trends appear to be disappearing as lapa-roscopic technology and familiarity with the techniques of the procedure have improved.53 A number of different factors are thought to be associated with bile duct injury during laparo-scopic cholecystectomy. These include acute or chronic inflam-mation, obesity, anatomic variations, and surgical technique. Inadequate exposure or failure to correctly identify structures before ligating or dividing them are the most common causes of significant biliary injury (see “Anatomic Variants” section). Excessive cephalad retraction of the gallbladder may align the cystic duct with the common bile duct, and the latter may then be mistakenly clipped and divided. Careless use of electrocau-tery can lead to thermal injury. Dissection deep into the liver parenchyma may cause injury to intrahepatic ducts, and poor clip placement close to the hilar area or to structures not well visualized can result in a clip across a bile

1	deep into the liver parenchyma may cause injury to intrahepatic ducts, and poor clip placement close to the hilar area or to structures not well visualized can result in a clip across a bile duct.74,75Techniques to avoid injury to the bile ducts during chole-cystectomy are important to understand. The use of an angled, 30° or 45° laparoscope instead of an end-viewing camera will help visualize the anatomic structures, in particular those around the triangle of Calot. An angled scope also will aid in the proper placement of clips. The routine use of intraoperative cholangi-ography during every cholecystectomy as a method to prevent bile duct injury remains controversial.55 Nonetheless, the fre-quency of bile duct injuries is cut by 50% when an intraop-erative cholangiogram is performed. Critical to the successful use of cholangiography is accurate interpretation of the imag-ing. It is important to check that the whole biliary system fills with contrast, including both major ducts on

1	Critical to the successful use of cholangiography is accurate interpretation of the imag-ing. It is important to check that the whole biliary system fills with contrast, including both major ducts on the right and the left hepatic duct, and that there is no extravasation of contrast. While routine use may reduce or limit the extent of injury, or help identify it early, it does not seem to prevent it entirely.76 No consensus recommendation exists on the use of selective versus routine cholangiography.Perhaps the most universally agreed upon method for mitigating the risk of bile duct injury during laparoscopic cholecystectomy is obtaining the critical view of safety. This requires that the hepatocystic triangle is dissected free of fat and fibrous tissue, the lower third of the gallbladder is sepa-rated from the cystic plate, and there are two and only two structures running into the gallbladder, the cystic duct, and the cystic artery (see Fig. 32-23).54 Newer technologies such as

1	is sepa-rated from the cystic plate, and there are two and only two structures running into the gallbladder, the cystic duct, and the cystic artery (see Fig. 32-23).54 Newer technologies such as fluorescence cholangiography to help identify biliary anatomy intraoperatively have shown promising early results, though large-scale applications remain to be seen.77Diagnosis. Only about 25% of major bile duct injuries (com-mon bile duct or hepatic duct) are recognized at the time of surgery. In these cases, intraoperative bile leakage, recognition of the correct anatomy, or an abnormal cholangiogram led to the diagnosis of a bile duct injury. In those that go unrecognized at the time of surgery, more than half will re-present within the first month postoperatively, though some can present months or years later with strictures, cholangitis, or cirrhosis from a remote bile duct injury.4Figure 32-28. An endoscopic retrograde cholangiography show-ing stricture of the common hepatic duct

1	months or years later with strictures, cholangitis, or cirrhosis from a remote bile duct injury.4Figure 32-28. An endoscopic retrograde cholangiography show-ing stricture of the common hepatic duct (arrow). The patient had recently had a laparoscopic cholecystectomy; clips from the opera-tion can be seen projected over the common bile duct.Brunicardi_Ch32_p1393-p1428.indd 141911/02/19 2:44 PM 1420SPECIFIC CONSIDERATIONSPART IIBile duct injuries typically result in either leaks or obstruc-tions related to strictures. Bile leak, most commonly from the cystic duct stump, a transected aberrant right hepatic duct, or a lateral injury to the main bile duct, usually presents with abdom-inal pain, fever, and a mild elevation of liver function tests. If a drain was placed at the time of surgery, bilious fluid may be seen. A CT scan or ultrasound can show either a fluid collection in the gallbladder fossa (biloma), or free fluid (bile) in the peri-toneum (Fig. 32-29A). ERCP (Fig. 32-29B) or

1	bilious fluid may be seen. A CT scan or ultrasound can show either a fluid collection in the gallbladder fossa (biloma), or free fluid (bile) in the peri-toneum (Fig. 32-29A). ERCP (Fig. 32-29B) or HIDA scan can be utilized to better localize the site of the bile leak.Obstruction or stricture should be suspected in patients with progressive elevations of liver function tests or jaundice after cholecystectomy. CT scan or ultrasound can demonstrate the dilated part of the biliary tree, and may identify the level of the bile duct obstruction. MRI cholangiography, if available, provides an excellent, noninvasive delineation of the biliary anatomy both proximal and distal to the injury. Endoscopic or percutaneous cholangiography may also be helpful to confirm the diagnosis, depending on the location and type of injury.Management. The management of bile duct injuries depends on the type, extent, and level of the injury, as well as the tim-ing of its diagnosis. Initial proper treatment of

1	location and type of injury.Management. The management of bile duct injuries depends on the type, extent, and level of the injury, as well as the tim-ing of its diagnosis. Initial proper treatment of bile duct injury can avoid the development of further complications or bile duct strictures. If an injury is discovered that exceeds the capacity of the available surgical expertise, the patient should be transferred to a tertiary care center. In these situations, drains should be placed in the surgical bed and antibiotics initiated. If a complete obstructive transection has occurred, it may also be necessary to place a percutaneous transhepatic drainage catheter to decom-press the biliary tree prior to transfer.If identified at the time of surgery, bile leaks from small bile ducts (<3 mm) or those draining a single hepatic segment can safely be ligated. If the injured duct is ≥4 mm, however, it is likely to drain multiple segments or an entire lobe and thus needs to be repaired or

1	mm) or those draining a single hepatic segment can safely be ligated. If the injured duct is ≥4 mm, however, it is likely to drain multiple segments or an entire lobe and thus needs to be repaired or reimplanted. Minor injuries to the com-mon bile duct or the common hepatic duct are traditionally managed with placement of a T-tube that has been modified by cutting the ends to allow for its placement in and removal from the bile duct (see Fig. 32-18). If the injury is small, the T-tube may be placed through it as if it were a formal choledochotomy. In more extensive injuries, the T-tube should be placed through a separate choledochotomy and the injury closed over the T-tube end to minimize the risk of subsequent stricture formation.Major bile duct injuries identified intraoperatively such as complete transection of the common hepatic or common bile duct are best managed at the time of injury. In many of these major injuries, the bile duct has not only been transected, but a variable

1	as complete transection of the common hepatic or common bile duct are best managed at the time of injury. In many of these major injuries, the bile duct has not only been transected, but a variable length of the duct may have been removed with the surgical specimen. This injury usually requires reconstruction with a biliary-enteric anastomosis, and is best performed as soon as possible following the injury. If there is no or minimal loss of ductal length, a duct-to-duct repair may be done over a T-tube that is placed through a separate incision. In any repair that is chosen, it is critical to perform a tension-free anastomosis to minimize the high risk of postoperative stricture formation.Bile leaks identified postoperatively can usually be man-aged with percutaneous drainage of intra-abdominal fluid col-lections followed by endoscopic biliary stenting. With most leaks, regardless of the location, stenting of the common bile duct will provide a low resistance route for bile flow into

1	fluid col-lections followed by endoscopic biliary stenting. With most leaks, regardless of the location, stenting of the common bile duct will provide a low resistance route for bile flow into the duodenum, decreasing flow through the leak and allowing it to heal. This is particularly effective for leaks from the cystic duct stump. Rarely, surgery is required to repair a large leak if endoscopic interventions have failed or peritonitis is present.Major bile duct injuries diagnosed in the later postopera-tive period may not be amenable to immediate reconstruction due to acute inflammation. They may need to be managed with transhepatic biliary catheter placement for biliary decompres-sion as well as percutaneous drainage of intra-abdominal bile collections, if any. When the acute inflammation has resolved 6 to 8 weeks later, operative repair is performed.Patients with bile duct stricture from an injury or as a sequela of previous repair usually present with either progres-sive elevation

1	has resolved 6 to 8 weeks later, operative repair is performed.Patients with bile duct stricture from an injury or as a sequela of previous repair usually present with either progres-sive elevation of liver function tests or cholangitis. The initial management usually includes endoscopic attempts at dilation or stenting. Balloon dilatation of a stricture usually requires multiple procedures and rarely provides long-term relief. Self-expanding metal or plastic stents can provide temporary or, in the high-risk patient, permanent drainage of the biliary tree. If the stricture is unable to be addressed endoscopically, ABFigure 32-29. A. Computed tomographic scan of a patient with bile leak after cholecystectomy. The short arrows indicate the intraperitoneal collections. Both air and bile are seen in the gallbladder bed (long arrow) as well as a surgical clip. B. An endoscopic retrograde cholangi-ography from the same patient showing contrast extravasation from the cystic duct stump

1	are seen in the gallbladder bed (long arrow) as well as a surgical clip. B. An endoscopic retrograde cholangi-ography from the same patient showing contrast extravasation from the cystic duct stump (arrow). Note the filling of the pancreatic duct.Brunicardi_Ch32_p1393-p1428.indd 142011/02/19 2:44 PM 1421GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32percutaneous transhepatic biliary drainage catheter placement may be necessary for decompression, and to define the anat-omy, location and extent of the damage. Definitive treatment of refractory biliary strictures entails resection of the affected segment and reconstruction with a biliary-enteric anastomosis.Outcome. Good results can be expected in the majority of patients with bile duct injuries, with the best results coming when the injury is recognized immediately and repaired by an experienced biliary tract surgeon.78 The perioperative mortality rate is reported to be less than 10%, but common morbidities associated with

1	the injury is recognized immediately and repaired by an experienced biliary tract surgeon.78 The perioperative mortality rate is reported to be less than 10%, but common morbidities associated with bile duct repairs include cholangitis, external biliary fistula, bile leak, subhepatic and subphrenic abscesses, and hemobilia. Restenosis of a biliary enteric anastomosis occurs in about 10% of patients, and typically presents within 2 years, but can manifest up to 20 years after the initial procedure. In general, treatment of proximal strictures is associated with a lower success rate than distal ones. The worst results are seen in patients with many operative revisions and in those who have evidence of liver failure or portal hypertension. However, previ-ous repair does not preclude a successful outcome, particularly in patients with good liver function. In the most severe cases, patients with refractory strictures and deteriorating liver func-tion may become candidates for liver

1	successful outcome, particularly in patients with good liver function. In the most severe cases, patients with refractory strictures and deteriorating liver func-tion may become candidates for liver transplant.TUMORSCarcinoma of the GallbladderCancer of the gallbladder is a rare malignancy that occurs pre-dominantly in the elderly. It is an aggressive tumor, with a poor prognosis that is usually not diagnosed until it has become advanced and is causing symptoms. The median survival for gallbladder cancer is around 6 months with a reported 5-year survival rate of 5%.79 In a minority of cases, early cancers are identified incidentally following cholecystectomy for cho-lelithiasis, in which case, 5-year survival is over 80%.80Incidence. Gallbladder cancer is the sixth most common GI malignancy in Western countries.80 It accounts for 2% to 4% of all malignant GI tumors, with about 4000 new cases diagnosed annually in the United States. It is two to six times more com-mon in females than

1	in Western countries.80 It accounts for 2% to 4% of all malignant GI tumors, with about 4000 new cases diagnosed annually in the United States. It is two to six times more com-mon in females than males, and the peak incidence is in the seventh decade of life. Its occurrence in random autopsy series is about 0.4%, but 0.3% to 3% of patients undergoing chole-cystectomy for gallstone disease are found incidentally to have gallbladder cancer.80 There are also significant ethnic variations in the incidence of gallbladder cancer, with rates being particu-larly high in native populations of the United States, Mexico, and Chile. The overall incidence of gallbladder cancer in the United States is approximately 1.5 cases per 100,000 residents. For Native American females with gallstones, the incidence is around 7.1 per 100,000. For women in the native populations of Chile, gallbladder cancer occurs in 27.3 per 100,000 individu-als. Asian populations, particularly those of Korean descent, are

1	is around 7.1 per 100,000. For women in the native populations of Chile, gallbladder cancer occurs in 27.3 per 100,000 individu-als. Asian populations, particularly those of Korean descent, are also at increased risk of developing gallbladder cancer.80Etiology. The pathogenesis of gallbladder cancer has not been fully defined but is likely related to a combination of chronic inflammation, infection, genetics, and environmental exposures such as heavy metals and tobacco. Cholelithiasis is the most important risk factor for gallbladder carcinoma, and up to 85% of patients with carcinoma of the gallbladder have gallstones. However, <3% of patients with gallstones have gallbladder cancer, and the 20-year risk of developing cancer remains low; <0.5% for the overall population and 1.5% for high-risk groups. Larger stones (>3 cm) are associated with a 10-fold increased risk of cancer.81 The risk of developing cancer of the gallblad-der is higher in patients with symptomatic than asymptomatic

1	groups. Larger stones (>3 cm) are associated with a 10-fold increased risk of cancer.81 The risk of developing cancer of the gallblad-der is higher in patients with symptomatic than asymptomatic gallstones, and it is more commonly seen in the setting of cho-lesterol stones.Polypoid lesions of the gallbladder, which are present in as many as 5% of adults, are also associated with increased risk of cancer. This is particularly true for polyps measuring >10 mm, which carry a 25% risk of malignancy.82 Solitary or sessile polys, or those showing rapid growth on serial imaging, particularly if in the presence of gallstones or age >50 are also concerning for malignancy. When such findings are identified, the patient should have their gallbladder removed, even if they are asymptomatic. Polyps that are not removed should be monitored on serial imaging. The finding of a “Porcelain” gallbladder, or dense circumferential calcifications of the gallbladder wall, is associated with an approximately

1	are not removed should be monitored on serial imaging. The finding of a “Porcelain” gallbladder, or dense circumferential calcifications of the gallbladder wall, is associated with an approximately 10% risk of gallbladder car-cinoma. While this condition was previously considered to be an absolute indication for cholecystectomy, more recent studies suggest that given the low rate of malignancy, observation is safe and acceptable. Nevertheless, resection remains a reason-able option, particularly if the patient is symptomatic, and the decision should ultimately be made only after discussing risks and benefits of each approach with the patient.97 Patients with certain types of choledochal cysts also have an increased risk of developing cancer anywhere in the biliary tree, but the inci-dence is highest in the gallbladder and cholecystectomy should be performed with any surgical intervention on the choledochal cyst. Primary sclerosing cholangitis, anomalous pancreatico-biliary duct

1	is highest in the gallbladder and cholecystectomy should be performed with any surgical intervention on the choledochal cyst. Primary sclerosing cholangitis, anomalous pancreatico-biliary duct junction, and exposure to carcinogens (azotoluene, nitrosamines) also are associated with cancer of the gallbladder, and screening with abdominal ultrasound should be considered in these patients.Pathology. Between 80% and 90% of gallbladder cancers are adenocarcinomas. Squamous cell, adenosquamous, oat cell, and other anaplastic lesions rarely occur. The histologic subtypes of gallbladder adenocarcinomas include papillary, nodular, and tubular. Less than 10% are of the papillary type, but these are associated with an overall better outcome, as they are most com-monly diagnosed while localized to the gallbladder. Cancer of the gallbladder can spread through lymphatics, venous drainage, or by direct invasion into the liver parenchyma. Lymphatic flow from the gallbladder drains first to the cystic

1	gallbladder. Cancer of the gallbladder can spread through lymphatics, venous drainage, or by direct invasion into the liver parenchyma. Lymphatic flow from the gallbladder drains first to the cystic duct node (Lund’s node or Calot’s node), then pericholedochal and hilar nodes, and finally to the peripancreatic, duodenal, periportal, celiac, and superior mes-enteric artery nodes. The gallbladder veins drain directly into the adjacent liver, usually segments IVb and V, where tumor inva-sion is common (Fig. 32-30). The gallbladder wall differs histo-logically from the intestines in that it lacks a muscularis mucosa and submucosa. Lymphatics are present in the subserosal layer only. Therefore, cancers that have not grown through the muscu-lar layer have minimal risk of nodal disease. Unfortunately, only a small portion of gallbladder cancers (10–25%) are identified while they are still localized to the gallbladder. The majority will already have nodal involvement, extension into adjacent

1	only a small portion of gallbladder cancers (10–25%) are identified while they are still localized to the gallbladder. The majority will already have nodal involvement, extension into adjacent liver, or distant metastasis at the time of diagnosis.80,83Clinical Manifestations and Diagnosis. Signs and symp-toms of carcinoma of the gallbladder are generally indistinguish-able from those associated with cholecystitis and cholelithiasis, and this can lead to delays in treatment or misdiagnosis. These include abdominal discomfort, right upper quadrant pain, nau-sea, and vomiting. Jaundice, weight loss, anorexia, ascites, 5Brunicardi_Ch32_p1393-p1428.indd 142111/02/19 2:44 PM 1422SPECIFIC CONSIDERATIONSPART IIand abdominal masses are less common presenting symptoms. Common misdiagnoses include chronic cholecystitis, acute cho-lecystitis, choledocholithiasis, hydrops of the gallbladder, and pancreatic cancer. Laboratory findings, if abnormal, are most often consistent with biliary

1	include chronic cholecystitis, acute cho-lecystitis, choledocholithiasis, hydrops of the gallbladder, and pancreatic cancer. Laboratory findings, if abnormal, are most often consistent with biliary obstruction. Ultrasonography often reveals a thickened, irregular gallbladder wall (>3mm) with hypervascularity or a mass replacing the gallbladder. It may also visualize tumor invasion of the liver, lymphadenopathy, or a dilated biliary tree. The sensitivity of ultrasonography in detect-ing gallbladder cancer ranges from 70% to 100%. A CT scan may be helpful in identifying a gallbladder mass and evaluating for nodal spread or local invasion into adjacent organs or vascu-lature. If questions about local invasion remain, MRCP allows for complete assessment of biliary, vascular, nodal, hepatic, and adjacent organ involvement.84 Endoscopic ultrasound (EUS) can be a useful tool in staging and evaluating for local invasion, as well as obtaining tissue diagnosis through fine needle aspiration

1	and adjacent organ involvement.84 Endoscopic ultrasound (EUS) can be a useful tool in staging and evaluating for local invasion, as well as obtaining tissue diagnosis through fine needle aspiration (FNA). Tissue diagnosis can also be obtained by CT or ultra-sound-guided biopsy of the tumor, though this is not required prior to cholecystectomy if the tumor appears resectable on imaging. In jaundiced patients, a percutaneous transhepatic or endoscopic cholangiogram may be helpful to delineate the extent of biliary tree involvement. The role of PET scanning in gallbladder cancer is yet to be fully defined but can be utilized in both staging and surveillance.Treatment. Surgical resection remains the only curative option for gallbladder cancer. While most patients are unresectable at the time of diagnosis, if preoperative staging suggests a potentially resectable tumor, exploration for tissue diagno-sis, formal pathologic staging, and possible curative resection are warranted.Tumors

1	time of diagnosis, if preoperative staging suggests a potentially resectable tumor, exploration for tissue diagno-sis, formal pathologic staging, and possible curative resection are warranted.Tumors limited to the lamina propria or muscular layer of the gallbladder (T1) are usually identified incidentally, after laparoscopic cholecystectomy for gallstone disease. There is near universal agreement that simple laparoscopic cholecys-tectomy is an adequate treatment for T1 lesions and results in a near 100% overall 5-year survival rate. When the tumor invades the perimuscular connective tissue without extension beyond the serosa or into the liver (T2 tumors), an extended cholecystectomy should be performed.85 This includes addi-tional resection of liver segments IVb and V, as well as lymph-adenectomy of the cystic duct and pericholedochal, portal, right celiac, and posterior pancreatoduodenal lymph nodes. Given the extent of this operation, an open approach is stan-dard. One-half of

1	of the cystic duct and pericholedochal, portal, right celiac, and posterior pancreatoduodenal lymph nodes. Given the extent of this operation, an open approach is stan-dard. One-half of patients with T2 tumors are found to have nodal disease on pathologic examination, highlighting the importance of regional lymphadenectomy as part of surgery for T2 cancers.86 For tumors that grow beyond the serosa, or invade the liver or other adjacent organs (T3), there is a higher likelihood of intraperitoneal or distant spread. However, if no peritoneal or nodal involvement is found, complete tumor excision with an extended right hepatectomy and possible cau-date lobectomy with lymphadenectomy must be performed for adequate tumor clearance. In addition, if a T2 or T3 tumor is identified incidentally after laparoscopic cholecystectomy, and the patient is returning to the OR for liver resection and lymph-adenectomy, the previous laparoscopic port sites must also be excised due to the high risk of

1	after laparoscopic cholecystectomy, and the patient is returning to the OR for liver resection and lymph-adenectomy, the previous laparoscopic port sites must also be excised due to the high risk of recurrence in these locations. T4 tumors are those that have grown into major blood vessels or two or more structures outside the liver, and they are typically considered unresectable.Due to the high frequency of late diagnosis, palliative procedures for unresectable cancer, jaundice, or duodenal obstructions remain the most frequently performed surgery for gallbladder cancers. Today, patients with obstructive jaundice can frequently be managed with either endoscopic or percutane-ously placed biliary stents. Various regimens of neoadjuvant, adjuvant, and definitive chemoradiotherapy have been trialed in gallbladder cancer. Overall, benefits have been marginal, but treatment may improve survival time by several months. These therapies can be offered to patients in conjunction with

1	been trialed in gallbladder cancer. Overall, benefits have been marginal, but treatment may improve survival time by several months. These therapies can be offered to patients in conjunction with resec-tion for curative intent or as definitive therapy, but no standard recommendation exists for their use.85-87Prognosis. Most patients with gallbladder cancer have unre-sectable disease at the time of diagnosis. The overall 5-year sur-vival rate of all patients with gallbladder cancer is <5%, with a median survival of 6 months.87 However, patients with T1 dis-ease treated with cholecystectomy have an excellent prognosis (85–100% 5-year survival rate). The 5-year survival rate for T2 lesions treated with an extended cholecystectomy (liver segment IVb/V resection) and lymphadenectomy is >70% compared to 25% to 40% for T2 patients treated with simple cholecystec-tomy. Patients with advanced (T3 or T4) but resectable gallblad-der cancer are reported to have 5-year survival rates of 20% to

1	compared to 25% to 40% for T2 patients treated with simple cholecystec-tomy. Patients with advanced (T3 or T4) but resectable gallblad-der cancer are reported to have 5-year survival rates of 20% to 50%, supporting aggressive resection in those patients who can tolerate surgery. The median survival for patients with distant metastasis at the time of presentation is only 1 to 3 months.Recurrence after resection of gallbladder cancer occurs most commonly in the liver or in the celiac or retropancreatic nodes. The prognosis for recurrent disease is very poor, and the main goal of follow-up is to provide palliative care. The most common problems are pruritus and cholangitis associated with obstructive jaundice, bowel obstruction secondary to carcino-matosis, and pain. Death occurs most commonly secondary to biliary sepsis or liver failure.6Figure 32-30. Computed tomography scan of a patient with gall-bladder cancer. The image shown is at the level of the liver hilum. The portal vein is

1	secondary to biliary sepsis or liver failure.6Figure 32-30. Computed tomography scan of a patient with gall-bladder cancer. The image shown is at the level of the liver hilum. The portal vein is bifurcating into the left and right portal branch. The tumor has invaded segment IV of the liver (arrowheads) and obstructed the common hepatic duct, resulting in intrahepatic ductal dilatation (arrows).Brunicardi_Ch32_p1393-p1428.indd 142211/02/19 2:44 PM 1423GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32CholangiocarcinomaCholangiocarcinoma is a rare tumor arising from the biliary epithelium and may occur anywhere along the biliary tree. About half are located at the hepatic duct bifurcation (Klatskin tumors), with 40% occurring more distally and 10% being intrahepatic.88 Surgical resection offers the only chance for cure, but unfortunately many patients have advanced disease at the time of diagnosis. Therefore, palliative procedures aimed to provide biliary drainage and

1	resection offers the only chance for cure, but unfortunately many patients have advanced disease at the time of diagnosis. Therefore, palliative procedures aimed to provide biliary drainage and prevent liver failure and cholangitis are often the only therapeutic possibilities available.Incidence. The autopsy incidence of bile duct carcinoma is about 0.3%. The overall incidence of cholangiocarcinoma in the United States is about 1 per 100,000 people per year, with approximately 2500 new cases diagnosed annually. The disease has a slight male predominance and an average age of presenta-tion between 50 and 70 years.88Etiology. Most cases of extrahepatic cholangiocarcinoma develop de novo with no identifiable risk factors. However, there is an increased risk of cholangiocarcinoma in patients with choledochal cysts, ulcerative colitis, hepatolithiasis, biliary-enteric anastomoses, hepatitis B and C, cirrhosis, bili-ary tract infections with Clonorchis (liver flukes), and chronic typhoid

1	with choledochal cysts, ulcerative colitis, hepatolithiasis, biliary-enteric anastomoses, hepatitis B and C, cirrhosis, bili-ary tract infections with Clonorchis (liver flukes), and chronic typhoid carriers. Exposure to dietary nitrosamines, Thorotrast, or dioxin also puts patients at increased risk for cholangiocar-cinoma.88 Patients with primary sclerosing cholangitis have a 5% to 10% lifetime risk of developing cholangiocarcinoma with typical disease onset in their 40s. For this reason, these patients require regular screening.67 Features common to most risk factors include biliary stasis, bile duct stones, and infection.Pathology. Over 95% of bile duct cancers are ductal adeno-carcinomas with the vast majority occurring in the extrahepatic biliary tree. Morphologically, they are divided into nodular (the most common type), scirrhous, diffusely infiltrating, or pap-illary. Anatomically, they are divided into distal, perihilar or intrahepatic tumors. Intrahepatic cholangiocarcinomas

1	into nodular (the most common type), scirrhous, diffusely infiltrating, or pap-illary. Anatomically, they are divided into distal, perihilar or intrahepatic tumors. Intrahepatic cholangiocarcinomas make up approximately 10% of cases and are typically treated like hepatocellular carcinoma, with hepatectomy when possible and transplant when unresectable. About half of all cholangiocar-cinomas are located in the perihilar region with the remaining 40% occurring more distally in the common bile duct.Perihilar cholangiocarcinomas, also referred to as Klatskin tumors, are further classified based on anatomic loca-tion by the Bismuth-Corlette classification (Fig. 32-31). Type I tumors are confined to the common hepatic duct, but type II tumors involve the bifurcation without involvement of the secondary intrahepatic ducts. Type IIIa and IIIb tumors extend into the right and left secondary intrahepatic ducts, respec-tively. Type IV tumors involve both the right and left secondary intrahepatic

1	intrahepatic ducts. Type IIIa and IIIb tumors extend into the right and left secondary intrahepatic ducts, respec-tively. Type IV tumors involve both the right and left secondary intrahepatic ducts.Clinical Manifestations and Diagnosis. Painless jaundice is the most common initial presentation in patients with cholangio-carcinoma. Pruritus, mild right upper quadrant pain, anorexia, fatigue, and weight loss may also be present. Cholangitis is the presenting symptom in about 10% of patients. Except for jaundice, physical examination is usually normal in patients with cholangiocarcinoma. Occasionally, asymptomatic patients are found to have cholangiocarcinoma while being evaluated for elevated liver function tests. Tumor markers, such as CA 125 and carcinoembryonic antigen (CEA), can be elevated in cholangiocarcinoma but tend to be nonspecific because they also increase in other GI and gynecologic malignancies. The tumor marker most commonly used to aid the diagnosis of

1	can be elevated in cholangiocarcinoma but tend to be nonspecific because they also increase in other GI and gynecologic malignancies. The tumor marker most commonly used to aid the diagnosis of chol-angiocarcinoma is CA 19-9, which has a sensitivity of 79% and specificity of 98% if the serum value is >129 U/mL.89 How-ever, mild elevations in CA 19-9 can also be seen in cholangitis, biliary obstruction, other GI and gynecologic neoplasms, and patients who lack the Lewis blood type antigen.90The initial workup for suspected cholangiocarcinoma includes abdominal imaging with ultrasound or CT scanning. Perihilar tumors will cause dilatation of the intrahepatic bili-ary tree, but a normal or collapsed gallbladder and extrahepatic bile ducts distal to the tumor. Distal bile duct cancer will lead to dilatation of the extraand intrahepatic bile ducts as well as the gallbladder. Initial imaging is important to determine the level of obstruction and to rule out the presence of bile duct stones

1	to dilatation of the extraand intrahepatic bile ducts as well as the gallbladder. Initial imaging is important to determine the level of obstruction and to rule out the presence of bile duct stones as the cause of the obstructive jaundice (Fig. 32-32). It is usually difficult to visualize the tumor itself on ultrasound, CT, or even MRCP, but any of these modalities can provide an outline of biliary anatomy, an estimate of the level of obstruc-tion, evaluation of portal vein patency, and screening for nearby lymphadenopathy. Detailed evaluation of the biliary anatomy and tumor itself is best completed through cholangiography. ERCP is generally adequate, but in cases where the proximal extent of the tumor remains in question, PTC may be required to determine resectability.Tissue diagnosis may be difficult to obtain. Current diag-nostic techniques including fine-needle aspiration (percutaneous or endoscopic), and biliary brushings have been shown to have a low sensitivity in detecting

1	may be difficult to obtain. Current diag-nostic techniques including fine-needle aspiration (percutaneous or endoscopic), and biliary brushings have been shown to have a low sensitivity in detecting malignancy, anywhere between 15% and 60%. Choledochoscopy with direct visualization and sam-pling of intraluminal masses may be able to improve diagnosis rates but is only available in specialized centers (see Fig. 32-10). Patients with potentially resectable disease should, therefore, be offered surgical exploration based on radiographic findings and clinical suspicion.91Type IIIbType IIType IType IVType IIIaFigure 32-31. Bismuth-Corlette classification of perihilar bile duct tumors (Klatskin tumors).Brunicardi_Ch32_p1393-p1428.indd 142311/02/19 2:44 PM 1424SPECIFIC CONSIDERATIONSPART IITreatment. Surgical excision is the only potentially cura-tive treatment for cholangiocarcinoma. In the past one to two decades, improvements in surgical techniques have resulted in lower mortality and

1	excision is the only potentially cura-tive treatment for cholangiocarcinoma. In the past one to two decades, improvements in surgical techniques have resulted in lower mortality and better outcomes for patients undergoing aggressive surgical excision for cholangiocarcinoma.92Despite improvements in ultrasonography, CT scanning, and MRI, more than one-half of patients who are explored are found to have peritoneal implants, nodal or hepatic metastasis, or locally advanced disease that precludes resection. Patients suspected of having resectable disease should first undergo diagnostic laparoscopy. Those who are found to have previously unidentified metastatic disease should undergo cholecystectomy and surgical bypass for biliary decompression.93For curative resection, the location and local exten-sion of the tumor dictates the extent of the surgery required. Distal bile duct tumors are often resectable but may require pancreaticoduodenectomy (Whipple procedure). For patients with distal

1	exten-sion of the tumor dictates the extent of the surgery required. Distal bile duct tumors are often resectable but may require pancreaticoduodenectomy (Whipple procedure). For patients with distal bile duct cancer found to be unresectable on sur-gical exploration, Roux-en-Y hepaticojejunostomy, chole-cystectomy, and gastrojejunostomy to prevent gastric outlet obstruction should be performed. Perihilar tumors involving the bifurcation or proximal common hepatic duct (Bismuth-Corlette type I or II) with no signs of vascular involvement are candidates for local tumor excision with portal lymphadenec-tomy, cholecystectomy, common bile duct excision, and bilat-eral Roux-en-Y hepaticojejunostomies. If the tumor involves the right or left hepatic duct (Bismuth-Corlette type IIIa or IIIb), right or left hepatic lobectomy, respectively, should also be performed. Frequently, resection of the adjacent caudate lobe is required because of direct extension into caudate bili-ary radicals or

1	right or left hepatic lobectomy, respectively, should also be performed. Frequently, resection of the adjacent caudate lobe is required because of direct extension into caudate bili-ary radicals or parenchyma.91 Type IV Klatskin tumors, those with more extensive involvement of both hepatic ducts and intrahepatic spread, are often considered unresectable or only treatable with liver transplantation.The best outcomes in perihilar cholangiocarcinoma are seen in patients who undergo neoadjuvant chemoradiation followed by liver transplantation. However, there are very strict inclusion criteria for transplantation, and few patients qualify.88 Patients with primary sclerosing cholangitis who develop chol-angiocarcinoma should be treated with liver transplant when-ever possible.Nonoperative biliary decompression can be performed for patients with unresectable disease on initial presentation. Endoscopic placement of expandable metal stents is often the preferred approach. For very proximal or

1	decompression can be performed for patients with unresectable disease on initial presentation. Endoscopic placement of expandable metal stents is often the preferred approach. For very proximal or intrahepatic tumors, percutaneous drainage catheters may be necessary to fully decompress the biliary tree (see Fig. 32-12). There is a sig-nificantly higher risk of cholangitis in patients with drainage catheters or stents compared to those with surgical bypasses. In addition, stent occlusion is not uncommon. Nevertheless, operative intervention is not warranted in patients with meta-static disease.94There is no proven role for adjuvant chemotherapy in the treatment of cholangiocarcinoma. Adjuvant radiation therapy has also not been shown to increase either quality of life or survival in resected patients. Patients with unresect-able disease can be offered palliative chemotherapy, typically with gemcitabine and cisplatin, but the response rates are low (10–20%), and the survival benefit is

1	patients. Patients with unresect-able disease can be offered palliative chemotherapy, typically with gemcitabine and cisplatin, but the response rates are low (10–20%), and the survival benefit is marginal. The combina-tion of radiation and chemotherapy may be more effective than either treatment alone for unresectable disease, but no data from randomized trials are available. Giving chemoradiation to these patients can be difficult because of the high incidence of cholangitis. External-beam radiation has not been shown to be an effective treatment for unresected disease. The use of interstitial (intraoperative) radiation, brachytherapy with iridium-192 via percutaneous or endoscopic stents, and com-bined interstitial and external-beam radiation for unresectable cholangiocarcinoma has been reported with some encouraging results. However, no randomized, prospective trials have been reported.91 Photodynamic therapy has been proposed as a palliative measure for patients with

1	has been reported with some encouraging results. However, no randomized, prospective trials have been reported.91 Photodynamic therapy has been proposed as a palliative measure for patients with unresectable disease and ABFigure 32-32. A. An endoscopic retrograde cholangiogram in a patient with cancer of the common hepatic duct (arrowheads). The common bile duct is of normal size, as is the cystic duct (arrow), but the proximal biliary tree is dilated. The gallbladder is not visualized because of tumor obstructing its neck. B. An ultrasound from the same patient showing dilated ducts and tumor obstructing the common hepatic duct (arrow). The walls of the bile ducts adjacent to the obstruction are thickened by tumor infiltration (arrowheads).Brunicardi_Ch32_p1393-p1428.indd 142411/02/19 2:44 PM 1425GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32has been found to prolong survival and improve quality of life in patients with biliary stents.95,96Prognosis. Most patients

1	2:44 PM 1425GALLBLADDER AND THE EXTRAHEPATIC BILIARY SYSTEMCHAPTER 32has been found to prolong survival and improve quality of life in patients with biliary stents.95,96Prognosis. Most patients with perihilar cholangiocarcinoma present with advanced, unresectable disease. Median survival in this population is between 5 and 8 months. The most common causes of death are hepatic failure and cholangitis. The overall 5-year survival rate for patients with resectable perihilar cholan-giocarcinoma is between 10% and 30%, but for patients with neg-ative margins, it may be as high as 40%. The operative mortality for perihilar cholangiocarcinoma is 6% to 8%. Patients with distal cholangiocarcinoma are more likely to have resectable disease and improved prognosis compared to perihilar cholangiocarci-noma. The overall 5-year survival rate for resectable distal dis-ease is 30% to 50%, and the median survival is 32 to 38 months. Patients who receive liver transplantation for cholangiocarcinoma

1	The overall 5-year survival rate for resectable distal dis-ease is 30% to 50%, and the median survival is 32 to 38 months. Patients who receive liver transplantation for cholangiocarcinoma can experience 5-year disease free survival rates as high as 68%.The greatest risk factors for recurrence after resection are the presence of positive margins and lymph node–positive tumors. Therapy for recurrent disease concentrates on palliation of symptoms and additional surgery is not recommended for patients with recurrent disease.REFERENCESEntries highlighted in bright blue are key references. 1. Clemente CD. Gray’s Anatomy. Philadelphia: Lea & Febiger; 1985:132. 2. Klein AS, Lillemoe KD, Yeo CJ, et al. Liver, biliary tract, and pancreas. In: O’Leary JP, ed. Physiologic Basis of Surgery. Baltimore: Williams & Wilkins; 1996:441. 3. Molmenti EP, Pinto PA, Klein J, et al. Normal and variant arte-rial supply of the liver and gallbladder. Pediatr Transplant. 2003;7:80-82. 4. Boyer J. Bile

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1	T, Shirai Y, Hatakeyama K. Radical second resection provides survival benefit for patients with T2 gallbladder car-cinoma first discovered after laparoscopic cholecystectomy. World J Surg. 2002;26:867-871. 87. Noshiro H, Chijiiwa K, Yamaguchi K, et al. Factors affecting surgical outcome for gallbladder carcinoma. Hepatogastroen-terology. 2003;50:939-944. 88. Razumilava N, Gores GJ. Cholangiocarcinoma. Lancet. 2014;383(9935):2168-2179. 89. Nehls O, Gregor M, Klump B. Serum and bile markers for chol-angiocarcinoma. Semin Liver Dis. 2004;24:139-154. 90. Siqueira E, Schoen RE, Silverman W, et al. Detecting cholan-giocarcinoma in patients with primary sclerosing cholangitis. Gastrointest Endosc. 2005;56:40-47. 91. Lillemoe KD, Cameron JL. Surgery for hilar cholangiocarci-noma: the Johns Hopkins approach. J Hepatobiliary Pancreat Surg. 2000;7:115-121. 92. Mulholland MW, Yahanda A, Yeo CJ. Multidisciplinary management of perihilar bile duct cancer. J Am Coll Surg.

1	the Johns Hopkins approach. J Hepatobiliary Pancreat Surg. 2000;7:115-121. 92. Mulholland MW, Yahanda A, Yeo CJ. Multidisciplinary management of perihilar bile duct cancer. J Am Coll Surg. 2001;193(4):440-447. 93. Vollmer CM, Drebin JA, Middleton WD, et al. Utility of staging laparoscopy in subsets of peripancreatic and biliary malignan-cies [Comment]. Ann Surg. 2002;235:1-7. 94. Strasberg SM. ERCP and surgical intervention in pancre-atic and biliary malignancies. Gastrointest Endosc. 2002;56: S213-S217. 95. Ortner ME, Caca K, Berr F, et al. Successful photodynamic therapy for nonresectable cholangiocarcinoma: a randomized prospective study. Gastroenterology. 2003;125:1355-1363. 96. Cheon YK, Lee TY, Lee SM, Yoon JY, Shim CS. Longterm out-come of photodynamic therapy compared with biliary stenting alone in patients with advanced hilar cholangiocarcinoma. HPB (Oxford). 2012;14(3):185-193.97. DesJardins H, Duy L, Scheirey C, Schnelldorfer T. Porcelain Gallbladder: Is Observation a Safe

1	stenting alone in patients with advanced hilar cholangiocarcinoma. HPB (Oxford). 2012;14(3):185-193.97. DesJardins H, Duy L, Scheirey C, Schnelldorfer T. Porcelain Gallbladder: Is Observation a Safe Option in Select Popula-tions? J Am Coll Surg. 2018 Jun;226(6):1064–1069.Brunicardi_Ch32_p1393-p1428.indd 142711/02/19 2:44 PM

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1	PancreasWilliam E. Fisher, Dana K. Andersen, John A. Windsor, Vikas Dudeja, and F. Charles Brunicardi 33chapterANATOMYThe pancreas is perhaps the most unforgiving organ in the human body, leading most surgeons to avoid even palpating it unless necessary. Situated deep in the center of the abdomen, the pancreas is surrounded by numerous important structures and major blood vessels. Seemingly minor trauma to the pan-creas can result in the release of pancreatic enzymes and cause life-threatening pancreatitis. Therefore, knowledge of the rela-tionships of the pancreas to surrounding structures is critically important for all surgeons to ensure that pancreatic injury is avoided during abdominal surgery.Gross AnatomyThe pancreas is a retroperitoneal organ that lies in an oblique position, sloping upward from the C-loop of the duodenum to the splenic hilum (Fig. 33-1). In an adult, the pancreas weighs 75 to 100 g and is about 15 to 20 cm long. The fact that the pan-creas is situated so

1	sloping upward from the C-loop of the duodenum to the splenic hilum (Fig. 33-1). In an adult, the pancreas weighs 75 to 100 g and is about 15 to 20 cm long. The fact that the pan-creas is situated so deeply in the abdomen and is sealed in the retroperitoneum explains the poorly localized and sometimes ill-defined nature with which pancreatic pathology presents. Patients with pancreatic cancer without bile duct obstruction usually present after months of vague upper abdominal discom-fort, or no antecedent symptoms at all. Due to its retroperitoneal location, pain associated with pancreatitis often is characterized as penetrating through to the back.Regions of the PancreasSurgeons typically describe the location of pathology within the pancreas in relation to four regions: the head, neck, body, and tail. The head of the pancreas is nestled in the C-loop of the duodenum and is posterior to the transverse mesocolon. Just pos-terior to the head of the pancreas lie the vena cava, the right

1	and tail. The head of the pancreas is nestled in the C-loop of the duodenum and is posterior to the transverse mesocolon. Just pos-terior to the head of the pancreas lie the vena cava, the right renal artery, and both renal veins. The neck of the pancreas lies directly anterior to the portal vein. At the inferior border of the neck of the pancreas, the superior mesenteric vein joins the splenic vein and then continues toward the porta hepatis as the portal vein. The inferior mesenteric vein often joins the splenic vein near Anatomy1429Gross Anatomy / 1429Regions of the Pancreas / 1429Pancreatic Duct Anatomy / 1432Vascular and Lymphatic Anatomy / 1433Neuroanatomy / 1435Histology and Physiology1435Exocrine Pancreas / 1435Endocrine Pancreas / 1437Islet Distribution / 1439Acute Pancreatitis1439Definition, Incidence, and Epidemiology / 1439Etiology / 1440Gallstones / 1440Alcohol / 1440Iatrogenic / 1441Hereditary Pancreatitis / 1441Tumors / 1441Hyperlipidemia / 1441Drugs /

1	Pancreatitis1439Definition, Incidence, and Epidemiology / 1439Etiology / 1440Gallstones / 1440Alcohol / 1440Iatrogenic / 1441Hereditary Pancreatitis / 1441Tumors / 1441Hyperlipidemia / 1441Drugs / 1441Pathophysiology / 1441Precipitating Events / 1441Intrapancreatic Events / 1442Systemic Events / 1443Management of Acute Pancreatitis / 1443Diagnosis / 1444Pain Management / 1444Predicting Severity / 1444Classification of Severity / 1445Determining Etiology / 1446Fluid Resuscitation / 1446Nutritional Support / 1446Cross-Sectional Imaging / 1446Therapeutic Endoscopic Retrograde Cholangiopancreatography / 1447Antibiotics / 1447Managing Local Complications / 1447Managing Organ Failure / 1448Cholecystectomy / 1449Diabetes / 1449Chronic Pancreatitis1450Definition, Incidence, and Prevalence / 1450Etiology / 1450Genetic Causes / 1450Alcohol / 1451Hyperparathyroidism / 1452Hyperlipidemia / 1452Classification / 1452Chronic Calcific (Lithogenic) Pancreatitis / 1452Chronic Obstructive

1	/ 1450Etiology / 1450Genetic Causes / 1450Alcohol / 1451Hyperparathyroidism / 1452Hyperlipidemia / 1452Classification / 1452Chronic Calcific (Lithogenic) Pancreatitis / 1452Chronic Obstructive Pancreatitis / 1453Chronic Inflammatory Pancreatitis / 1454Tropical (Nutritional) Pancreatitis / 1455Asymptomatic Pancreatic Fibrosis / 1455Idiopathic Pancreatitis / 1455Pathology / 1456Presentation, Natural History, and Complications / 1460Complications / 1465Treatment / 1468Pancreatic Neoplasms1480Neoplasms of the Endocrine Pancreas / 1480Insulinoma / 1480Noninsulinoma Hyperinsulinemia Hypoglycemia Syndrome / 1481Gastrinoma / 1481Vasoactive Intestinal Peptide-Secreting Tumor / 1482Glucagonoma / 1482Somatostatinoma / 1483Nonfunctioning Pancreatic Endocrine Tumors / 1483Neoplasms of the Exocrine Pancreas / 1484Workup of Asymptomatic Pancreatic Cystic Neoplasms / 1504Brunicardi_Ch33_p1429-p1516.indd 142901/03/19 6:44 PM 1430Key Points1 Incomplete fusion of the dorsal and ventral

1	Exocrine Pancreas / 1484Workup of Asymptomatic Pancreatic Cystic Neoplasms / 1504Brunicardi_Ch33_p1429-p1516.indd 142901/03/19 6:44 PM 1430Key Points1 Incomplete fusion of the dorsal and ventral pancreatic ducts results in pancreas divisum, but a variety of ductal anomalies can be seen. Magnetic resonance cholangiopancreatography as well as endoscopic retrograde cholangiopancreatography can identify these ductal anomalies, and clarification of the ductal pattern of the pancreas is important before attempts at interventions.2 The “replaced right hepatic artery” occurs in 15% of patients and needs to be identified preoperatively to prevent inadvertent injury with resulting hepatic necrosis. Anomalous hepatic arterial anatomy can result in hepatic ischemia during dissection of the porta hepatis as well. “Thin cut” multidetector computed tomographic images are usually able to identify the relevant arterial and venous patterns around the pancreas.3 Regardless of the etiology, the

1	hepatis as well. “Thin cut” multidetector computed tomographic images are usually able to identify the relevant arterial and venous patterns around the pancreas.3 Regardless of the etiology, the management of the early phase of acute pancreatitis is critical to achieve a success-ful outcome. Aggressive fluid resuscitation and early enteral feeding both reduce the risk of complications. It is no longer considered appropriate to “rest the pancreas” if the patient can tolerate enteral nutrients.4 Surgical intervention in acute pancreatitis is reserved for patients with infected collections or infected necrosis only, or to relieve an impacted gallstone in the ampulla if endo-scopic or radiologic treatments are unsuccessful. Infection is usually confirmed by a pattern of air in the retroperitoneum on computed tomographic scan or by documentation of bac-teria on Gram’s stain or culture from fine-needle aspiration of a suspected infected fluid collection. Fine-needle aspira-tion of

1	on computed tomographic scan or by documentation of bac-teria on Gram’s stain or culture from fine-needle aspiration of a suspected infected fluid collection. Fine-needle aspira-tion of suspicious fluid collections should not be converted to percutaneous drainage unless infection is confirmed and the consensus decision has been made that percutaneous drainage is appropriate for the individual patient.5 The appearance of chronic pancreatitis on computed tomo-graphic scan varies dramatically, and multiple diagnostic studies are usually needed to establish the extent of disease. Calcific pancreatitis is not a marker of alcoholic pancreati-tis alone, and it rarely indicates autoimmune pancreatitis. Endoscopic ultrasound provides a better assessment of the disease than computed tomography and is useful to disclose indolent or unsuspected cancer, which can occur in up to 10% of patients.6 The nidus of inflammation in chronic pancreatitis due to any cause is the head of the gland. Therefore,

1	useful to disclose indolent or unsuspected cancer, which can occur in up to 10% of patients.6 The nidus of inflammation in chronic pancreatitis due to any cause is the head of the gland. Therefore, treatment approaches that address the disease in the head have the best long-term results. The Whipple procedure, the Beger procedure, and the Frey procedure, with or without longitu-dinal duct drainage, are the best surgical options, as all three approaches remove all or most of the disease in the head of the gland. Although the limited pancreatic procedures (Beger/Frey) have a lower initial rate of endocrine dysfunc-tion, the long-term risk of diabetes is more related to the progression of the underlying disease than to the effects of operation. Level 1 studies confirm that the duodenum pre-serving options are associated with a lower immediate mor-bidity and mortality and therefore, in the absence of a mass or concerns about cancer, are better options than a Whipple procedure for chronic

1	options are associated with a lower immediate mor-bidity and mortality and therefore, in the absence of a mass or concerns about cancer, are better options than a Whipple procedure for chronic pancreatitis.7 The precursor lesion that probably leads to most cases of ductular adenocarcinoma is the ductal epithelial hyperplasia/dysplasia process described by the pancreatic intraepithelial neoplasia classification system. Pancreatic intraepithelial neoplasia 2 and pancreatic intraepithelial neoplasia 3 lesions may be associated with other, nonspecific changes in pancre-atic morphology seen on imaging studies, or they may only be seen histologically. Resection margins for pancreatic neo-plasms should be examined for advanced pancreatic intraep-ithelial neoplasia stage patterns of ductal hyperplasia to ensure adequate resection status.8 A low threshold for ordering a computed tomography scan with “pancreatic protocol” should be maintained for older adult patients with unexplained,

1	hyperplasia to ensure adequate resection status.8 A low threshold for ordering a computed tomography scan with “pancreatic protocol” should be maintained for older adult patients with unexplained, persistent, although vague, abdominal pain. New-onset diabetes in an older adult patient, especially if combined with vague abdominal pain, should prompt a search for pancreatic cancer.9 Intraductal papillary mucinous neoplasms are small macro-scopic polypoid or plaque-like adenomas that develop in the main pancreatic duct or in side-branch ducts, and secrete mucin. They are often silent symptomatically but cause char-acteristic appearances of small cyst-like collections of mucus or diffuse dilatation of the main pancreatic duct with mucus. These premalignant lesions may be multifocal or single and can evolve into invasive adenocarcinoma in a similar pattern as with other adenomatous polypoid lesions of the gastroin-testinal tract. They have been diagnosed with increasing fre-quency and

1	and can evolve into invasive adenocarcinoma in a similar pattern as with other adenomatous polypoid lesions of the gastroin-testinal tract. They have been diagnosed with increasing fre-quency and account for more than one-third of pancreatic resections at some centers. Main-duct intraductal papillary mucinous neoplasms are an indication for resection; side-branch intraductal papillary mucinous neoplasms have a lower incidence of malignancy and are sometimes followed with serial imaging surveillance.its junction with the portal vein. Sometimes, the inferior mesen-teric vein joins the superior mesenteric vein or merges with the superior mesenteric portal venous junction to form a trifurca-tion (Fig. 33-2). The superior mesenteric artery lies parallel to and just to the left of the superior mesenteric vein. The uncinate process and the head of the pancreas wrap around the right side of the portal vein and end posteriorly near the space between the superior mesenteric vein and superior

1	mesenteric vein. The uncinate process and the head of the pancreas wrap around the right side of the portal vein and end posteriorly near the space between the superior mesenteric vein and superior mesenteric artery. Venous branches draining the pancreatic head and uncinate process enter along the right lateral and posterior sides of the portal vein. There are usually no anterior venous tributaries, and a plane can usually be developed between the neck of the pancreas and the portal and superior mesenteric veins during pancreatic resection, unless the tumor is invading the vein anteriorly. The common bile duct runs in a deep groove on the posterior aspect of the pancreatic head until it passes through the pancreatic parenchyma to join the main pancreatic duct at the ampulla of Vater. The body and tail of the pancreas lie just anterior to the splenic artery and vein. The vein runs in a groove on the back of the pancreas and is fed by multiple fragile venous branches from the pancreatic

1	and tail of the pancreas lie just anterior to the splenic artery and vein. The vein runs in a groove on the back of the pancreas and is fed by multiple fragile venous branches from the pancreatic paren-chyma. These branches must be divided to perform a spleen-sparing distal pancreatectomy. The splenic artery runs parallel and just superior to the vein along the posterior superior edge of the body and tail of the pancreas. The splenic artery often is Brunicardi_Ch33_p1429-p1516.indd 143001/03/19 6:44 PM 1431PANCREASCHAPTER 33Body of pancreasTail of pancreasNeck of pancreasStomachLiverPortal veinSplenic veinGallbladderDuodenumHead of pancreasUncinate processof pancreasLeft kidneyVena cavaDuodenumAortaStomachHead of pancreasLeft kidneySMASMADuodenumJejunum4th portionof duodenumHead of pancreasPortal veinIMVSMVHead of pancreasDuodenumJejunumTail of pancreasStomachLiverPortal veinHead of pancreasDuodenumSMVFigure 33-1. Pancreatic anatomy as seen on computed tomography. Knowledge of the

1	veinIMVSMVHead of pancreasDuodenumJejunumTail of pancreasStomachLiverPortal veinHead of pancreasDuodenumSMVFigure 33-1. Pancreatic anatomy as seen on computed tomography. Knowledge of the relationship of the pancreas with surrounding structures is important to ensure that injury is avoided during abdominal surgery. IMV = inferior mesenteric vein; SMA = superior mesenteric artery; SMV = superior mesenteric vein.PortalveinSplenicveinInferiormesentericveinSuperiormesentericveinSuperiormesentericveinSuperiormesentericveinInferiormesentericveinInferiormesentericveinSplenicveinSplenicveinPortalveinPortalveinFigure 33-2. Variations in portal venous anatomy. The superior mesenteric vein joins the splenic vein and then continues toward the porta hepatis as the portal vein. The inferior mesenteric vein often joins the splenic vein near its junction with the portal vein, but sometimes joins the superior mesenteric vein; or the three veins merge as a trifurcation to form the portal

1	mesenteric vein often joins the splenic vein near its junction with the portal vein, but sometimes joins the superior mesenteric vein; or the three veins merge as a trifurcation to form the portal vein.Brunicardi_Ch33_p1429-p1516.indd 143101/03/19 6:44 PM 1432SPECIFIC CONSIDERATIONSPART IIBlindaccessory duct30%60%Minor papilla10%Dorsal anlageVentral anlageVentral budLiver budLesser oraccessorypapillaGreaterpapillaFusion oftwo ductsBile ductDorsal budDuct ofsantoriniDuct ofwirsungFigure 33-3. Embryology of pancreas and duct variations. The duct of Wirsung from the ventral bud connects to the bile duct, while the duct of Santorini from the larger dorsal bud connects to the duodenum. With gut rotation, the two ducts fuse in most cases such that the majority of the pancreas drains through the duct of Wirsung to the major papilla. The duct of Santorini can persist as a blind accessory duct or drain through the lesser papilla. In a minority of patients, the ducts remain separate, and

1	the duct of Wirsung to the major papilla. The duct of Santorini can persist as a blind accessory duct or drain through the lesser papilla. In a minority of patients, the ducts remain separate, and the majority of the pancreas drains through the duct of Santorini, a condition referred to as pancreas divisum.tortuous. The anterior surface of the body of the pancreas is cov-ered by peritoneum. Once the gastrocolic omentum is divided, the body and tail of the pancreas can be seen along the floor of the lesser sac, just posterior to the stomach.Pancreatic pseudocysts commonly develop in this area, and the posterior aspect of the stomach can form the anterior wall of the pseudocyst, allowing drainage into the stomach. The base of the transverse mesocolon attaches to the inferior margin of the body and tail of the pancreas. The transverse mesoco-lon often forms the inferior wall of pancreatic pseudocysts or inflammatory processes, allowing surgical drainage through the transverse mesocolon.

1	and tail of the pancreas. The transverse mesoco-lon often forms the inferior wall of pancreatic pseudocysts or inflammatory processes, allowing surgical drainage through the transverse mesocolon. The body of the pancreas is anterior to the aorta at the origin of the superior mesenteric artery. The neck of the pancreas is anterior to the vertebral body of L1 and L2, and blunt anteroposterior trauma can compress the neck of the pancreas against the spine, causing parenchymal and, sometimes, ductal injury. The neck divides the pancreas into approximately two equal halves. The small portion of the pan-creas anterior to the left kidney is referred to as the tail and is nestled in the hilum of the spleen near the splenic flexure of the left colon. Awareness of these anatomic relationships is impor-tant to avoid injury to the pancreatic tail during left colectomy or splenectomy.Pancreatic Duct AnatomyAn understanding of embryology is required to appreciate the common variations in pancreatic

1	to avoid injury to the pancreatic tail during left colectomy or splenectomy.Pancreatic Duct AnatomyAn understanding of embryology is required to appreciate the common variations in pancreatic duct anatomy. The pancreas is formed by the fusion of a ventral and dorsal bud (Fig. 33-3). The duct from the smaller ventral bud, which arises from the hepatic diverticulum, connects directly to the common bile duct. The duct from the larger dorsal bud, which arises from the duodenum, drains directly into the duodenum. The duct of the ventral anlage becomes the duct of Wirsung, and the duct from the dorsal anlage becomes the duct of Santorini. With gut rotation, the ventral anlage rotates to the right and around the posterior side of the duodenum to fuse with the dorsal bud. The ventral anlage becomes the inferior portion of the pancreatic head and the unci-nate process, while the dorsal anlage becomes the body and tail of the pancreas. The ducts from each anlage usually fuse together in the

1	the inferior portion of the pancreatic head and the unci-nate process, while the dorsal anlage becomes the body and tail of the pancreas. The ducts from each anlage usually fuse together in the pancreatic head such that most of the pancreas drains through the duct of Wirsung, or main pancreatic duct, into the common channel formed from the bile duct and pancreatic duct. The length of the common channel is variable. In about one-third of patients, the bile duct and pancreatic duct remain distinct to the end of the papilla, the two ducts merge at the end of the papilla in another one-third, and in the remaining one-third, a true common channel is present for a distance of several millimeters. Commonly, the duct from the dorsal anlage, the duct of Santorini, persists as the lesser pancreatic duct, and sometimes drains directly into the duo-denum through the lesser papilla just proximal to the major papilla. In approximately 30% of patients, the duct of Santorini ends as a blind accessory

1	duct, and sometimes drains directly into the duo-denum through the lesser papilla just proximal to the major papilla. In approximately 30% of patients, the duct of Santorini ends as a blind accessory duct and does not empty into the duodenum. In 10% of patients, the ducts of Wirsung and Santorini fail to fuse.1 This results in the majority of the pancreas draining through the duct of Santorini and the lesser papilla, while the inferior portion of the pancreatic head and uncinate process drains through the duct of Wirsung and major papilla. This nor-mal anatomic variant, which occurs in one out of 10 patients, is referred to as pancreas divisum (see Fig. 33-3). In a minority of these patients, the minor papilla can be inadequate to handle the flow of pancreatic juices from the majority of the gland. This rela-tive outflow obstruction can result in pancreatitis and is some-times treated by sphincteroplasty of the minor papilla.The main pancreatic duct is usually only 2 to 3 mm in

1	of the gland. This rela-tive outflow obstruction can result in pancreatitis and is some-times treated by sphincteroplasty of the minor papilla.The main pancreatic duct is usually only 2 to 3 mm in diameter and runs midway between the superior and inferior borders of the pancreas, usually closer to the posterior than to the anterior surface. Pressure inside the pancreatic duct is about twice that in the common bile duct, which is thought to prevent reflux of bile into the pancreatic duct. The main pancreatic duct joins with the common bile duct and empties at the ampulla of Vater or major papilla, which is located on the medial aspect of the second portion of the duodenum. The muscle fibers around the ampulla form the sphincter of Oddi, which controls the flow of pancreatic and biliary secretions into the duodenum. Contrac-tion and relaxation of the sphincter is regulated by complex neu-ral and hormonal factors. When the accessory pancreatic duct or lesser duct drains into the

1	secretions into the duodenum. Contrac-tion and relaxation of the sphincter is regulated by complex neu-ral and hormonal factors. When the accessory pancreatic duct or lesser duct drains into the duodenum, a lesser papilla can be identified approximately 2 cm proximal to the ampulla of Vater.1Brunicardi_Ch33_p1429-p1516.indd 143201/03/19 6:44 PM 1433PANCREASCHAPTER 33BileductPortalveinAbdominal aortaCeliac trunkSplenic arteryDorsalpancreatic arteryGreatpancreaticarteryCaudalarteryInferiorpancreatic arteryAnterior inferiorpancreaticoduodenal arteryAnterior superiorpancreatico-duodenalarteryPosterior inferiorpancreaticoduodenal arteryPosterior superiorpancreatico-duodenalarteryGastroduodenalarteryHepatic arteryproperCommon hepaticarterySuperiormesenteric arterySpleenDuodenumFigure 33-4. Arterial supply to the pancreas. Multiple arcades in the head and body of the pancreas provide a rich blood supply. The head of the pancreas cannot be resected without devascularizing the duodenum

1	supply to the pancreas. Multiple arcades in the head and body of the pancreas provide a rich blood supply. The head of the pancreas cannot be resected without devascularizing the duodenum unless a rim of pancreas containing the pancreaticoduodenal arcade is preserved.Vascular and Lymphatic AnatomyThe blood supply to the pancreas comes from multiple branches from the celiac and superior mesenteric arteries (Fig. 33-4). The common hepatic artery gives rise to the gastroduodenal artery before continuing toward the porta hepatis as the proper hepatic artery. The right gastric artery branches off the gastroduodenal artery just superior to the duodenum. The gastroduodenal artery also supplies the superior pancreaticoduodenal artery which divides into the anterior and superior pancreaticoduodenal arteries. These travel inferiorly within the pancreaticoduodenal groove giving off small branches to the duodenum and head of the pancreas. The superior pancreaticoduodenal arteries join the

1	arteries. These travel inferiorly within the pancreaticoduodenal groove giving off small branches to the duodenum and head of the pancreas. The superior pancreaticoduodenal arteries join the inferior pancreaticoduodenal arteries to complete the arcade. The inferior pancreaticoduodenal artery is a branch off the supe-rior mesenteric artery. Therefore, it is impossible to resect the head of the pancreas without devascularizing the duodenum, unless a rim of pancreas containing the pancreaticoduodenal arcade is preserved. The inferior pancreaticoduodenal artery needs to be controlled when dissecting the head of the pancreas off the SMA during a Whipple procedure. The gastroduodenal artery travels inferiorly anterior to the neck of the pancreas and posterior to the duodenal bulb. A posterior ulcer in the duodenal bulb can erode into the gastroduodenal artery in this location. At the inferior border of the duodenum, the gastroduodenal artery then gives rise to the right gastroepiploic

1	ulcer in the duodenal bulb can erode into the gastroduodenal artery in this location. At the inferior border of the duodenum, the gastroduodenal artery then gives rise to the right gastroepiploic artery then can con-tinue on to join the inferior pancreaticoduodenal artery.Variations in the arterial anatomy occur in one out of five patients. The right hepatic artery, common hepatic artery, or gas-troduodenal arteries can arise from the superior mesenteric artery. In 15% to 20% of patients, the right hepatic artery will arise from the superior mesenteric artery and travel upwards toward the liver along the posterior aspect of the head of the pancreas (referred to as a replaced right hepatic artery). It is important to look for this variation on preoperative com-puted tomographic (CT) scans and in the operating room so the 2replaced hepatic artery is recognized and injury is avoided. The body and tail of the pancreas are supplied by multiple branches of the splenic artery. The splenic

1	and in the operating room so the 2replaced hepatic artery is recognized and injury is avoided. The body and tail of the pancreas are supplied by multiple branches of the splenic artery. The splenic artery arises from the celiac trunk and travels along the posterior-superior border of the body and tail of the pancreas toward the spleen. The inferior pancreatic artery usually arises from the superior mesenteric artery and runs to the left along the inferior border of the body and tail of the pancreas, parallel to the splenic artery. Three vessels run perpen-dicular to the long axis of the pancreatic body and tail and con-nect the splenic artery and inferior pancreatic artery. They are, from medial to lateral, the dorsal, great, and caudal pancreatic arteries. These arteries form arcades within the body and tail of the pancreas and account for the rich blood supply of the organ.The venous drainage of the pancreas follows a pattern similar to that of the arterial supply (Fig. 33-5). The

1	the body and tail of the pancreas and account for the rich blood supply of the organ.The venous drainage of the pancreas follows a pattern similar to that of the arterial supply (Fig. 33-5). The veins are usually superficial to the arteries within the parenchyma of the pancreas. There is an anterior and posterior venous arcade within the head of the pancreas. Typically, the superior vein drains directly into the portal vein just above the neck of the pancreas and is often a larger branch of the portal vein which is divided during the Whipple procedure. The posterior infe-rior arcade drains directly into the inferior mesenteric vein at the inferior border of the neck of the pancreas and this is also divided during a Whipple procedure. The anterior inferior pan-creaticoduodenal vein joins the right gastroepiploic vein and the middle colic vein to form a common venous trunk, which enters into the superior mesenteric vein. Traction on the trans-verse colon during colectomy can tear these

1	right gastroepiploic vein and the middle colic vein to form a common venous trunk, which enters into the superior mesenteric vein. Traction on the trans-verse colon during colectomy can tear these fragile veins, which then retract into the parenchyma of the pancreas, making control tedious. There also are numerous small venous branches com-ing from the pancreatic parenchyma directly into the lateral and posterior aspect of the portal vein. Venous return from the body and tail of the pancreas drains into the splenic vein.The lymphatic drainage from the pancreas is diffuse and widespread (Fig. 33-6). The profuse network of lymphatic Brunicardi_Ch33_p1429-p1516.indd 143301/03/19 6:44 PM 1434SPECIFIC CONSIDERATIONSPART IIBileductPortal veinLeft gastric veinSplenic veinInferior mesenteric veinMiddle colic veinAnteroinferiorpancreatico-duodenalveinAnterosuperiorpancreatico-duodenalveinPosterosuperiorpancreatico-duodenalveinRightgastroepiploicveinSuperior mesenteric

1	mesenteric veinMiddle colic veinAnteroinferiorpancreatico-duodenalveinAnterosuperiorpancreatico-duodenalveinPosterosuperiorpancreatico-duodenalveinRightgastroepiploicveinSuperior mesenteric veinAbdominalaortaSpleenFigure 33-5. Venous drainage from the pancreas. The venous drainage of the pancreas follows a pattern similar to the arterial supply, with the veins usually superficial to the arteries. Anterior traction on the transverse colon can tear fragile branches along the inferior border of the pancreas, which then retract into the parenchyma of the pancreas. Venous branches draining the pancreatic head and uncinate process enter along the right lateral and posterior sides of the portal vein. There are usually no anterior venous tributaries, and a plane can usually be developed between the neck of the pancreas and the portal and superior mesenteric veins.Celiaclymph nodeHepaticlymph nodePyloriclymph nodeAnteriorpancreatico-duodenallymph nodePosteriorpancreaticoduodenallymph

1	between the neck of the pancreas and the portal and superior mesenteric veins.Celiaclymph nodeHepaticlymph nodePyloriclymph nodeAnteriorpancreatico-duodenallymph nodePosteriorpancreaticoduodenallymph nodeSuperior mesentericlymph nodeInferiorbodySuperior body Splenic lymphnodeDuodenumSpleenFigure 33-6. Lymphatic supply to the pancreas. The lymphatic drainage from the pancreas is diffuse and widespread, which explains the high incidence of lymph node metastases and local recurrence of pancreatic cancer. The pancreatic lymphatics also communicate with lymph nodes in the transverse mesocolon and mesentery of the proximal jejunum. Tumors in the body and tail of the pancreas are often unresect-able because they metastasize to these lymph nodes. (Reproduced with permission from Bell RH, Rikkers LF, Mulholland M: Digestive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)Brunicardi_Ch33_p1429-p1516.indd 143401/03/19 6:44 PM 1435PANCREASCHAPTER

1	LF, Mulholland M: Digestive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)Brunicardi_Ch33_p1429-p1516.indd 143401/03/19 6:44 PM 1435PANCREASCHAPTER 33SpleenPancreasEsophagusCeliac ganglionRight vagus nerveLeft vagus nerveSplanchnic nervesDuodenumAbdominal aortaFigure 33-7. Innervation of the pancreas. The pancreas has a rich supply of afferent sensory fibers that travel superiorly to the celiac ganglia. Interruption of these somatic fibers with a celiac plexus block can interfere with transmission of pancreatic pain. (Reproduced with permission from Bell RH, Rikkers LF, Mulholland M: Digestive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)vessels and lymph nodes draining the pancreas provides egress to tumor cells arising from the pancreas. This diffuse lymphatic drainage contributes to the fact that pancreatic cancer often presents with positive lymph nodes and a high incidence of local recurrence

1	tumor cells arising from the pancreas. This diffuse lymphatic drainage contributes to the fact that pancreatic cancer often presents with positive lymph nodes and a high incidence of local recurrence after resection. Lymph nodes can be palpated along the distal bile duct and posterior aspect of the head of the pan-creas in the pancreaticoduodenal groove, where the mesenteric vein passes under the neck of the pancreas, along the inferior border of the body, at the celiac axis and along the hepatic artery ascending into the porta hepatis, and along the splenic artery and vein. The pancreatic lymphatics also communicate with lymph nodes in the transverse mesocolon and mesentery of the proxi-mal jejunum. Tumors in the body and tail of the pancreas often metastasize to these nodes and lymph nodes along the splenic vein and in the hilum of the spleen.NeuroanatomyThe pancreas is innervated by the sympathetic and parasympathetic nervous systems. The acinar cells responsible for exocrine

1	nodes along the splenic vein and in the hilum of the spleen.NeuroanatomyThe pancreas is innervated by the sympathetic and parasympathetic nervous systems. The acinar cells responsible for exocrine secretion, the islet cells responsible for endocrine secretion, and the islet vasculature are innervated by both systems. The parasympathetic system stimulates endocrine and exocrine secretion and the sympathetic system inhibits secretion.2 The pancreas is also innervated by neurons that secrete amines and peptides, such as somatostatin, vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), and galanin. The exact role of these neurons in pancreatic physiology is uncertain, but they do appear to affect both exocrine and endocrine function. The pancreas also has a rich supply of afferent sensory fibers, which are responsible for the intense pain associated with advanced pancreatic cancer, as well as acute and chronic pancreatitis. These somatic fibers travel superiorly

1	of afferent sensory fibers, which are responsible for the intense pain associated with advanced pancreatic cancer, as well as acute and chronic pancreatitis. These somatic fibers travel superiorly to the celiac ganglia (Fig. 33-7). Interruption of these somatic fibers with a celiac plexus block can stop transmission of pain sensation.HISTOLOGY AND PHYSIOLOGYThe exocrine pancreas accounts for about 85% of the pancreatic mass; 10% of the gland is accounted for by extracellular matrix, and 4% by blood vessels and the major ducts, whereas only 2% of the gland is comprised of endocrine tissue. The endocrine and exocrine pancreas are sometimes thought of as function-ally separate, but these different components of the organ are coordinated to allow an elegant regulatory feedback system for digestive enzyme and hormone secretion. This complex system regulates the type of digestion, its rate, and the processing and distribution of absorbed nutrients. This coordination is facili-tated by the

1	enzyme and hormone secretion. This complex system regulates the type of digestion, its rate, and the processing and distribution of absorbed nutrients. This coordination is facili-tated by the physical approximation of the islets and the exo-crine pancreas, the presence of specific islet hormone receptors on the plasma membranes of pancreatic acinar cells, and the existence of an islet-acinar portal blood system.Although patients can live without a pancreas when insu-lin and digestive enzyme replacement are administered, the loss of this islet-acinar coordination leads to impairments in diges-tive function. Although only approximately 20% of the normal pancreas is required to prevent insufficiency, in many patients undergoing pancreatic resection, the remaining pancreas is not normal, and pancreatic endocrine and exocrine insufficiency can develop with removal of smaller portions of the gland.Exocrine PancreasThe pancreas secretes approximately 500 to 800 mL per day of colorless,

1	and pancreatic endocrine and exocrine insufficiency can develop with removal of smaller portions of the gland.Exocrine PancreasThe pancreas secretes approximately 500 to 800 mL per day of colorless, odorless, alkaline, isosmotic pancreatic juice. Pan-creatic juice is a combination of acinar cell and duct cell secre-tions. The acinar cells secrete amylase, proteases, and lipases, enzymes responsible for the digestion of all three food types: carbohydrate, protein, and fat. The acinar cells are pyramid-shaped, with their apices facing the lumen of the acinus. Near the apex of each cell are numerous enzyme-containing zymogen Brunicardi_Ch33_p1429-p1516.indd 143501/03/19 6:44 PM 1436SPECIFIC CONSIDERATIONSPART IIAcinar cellsCentroacinar cellsIntercalated ductFigure 33-8. Acinar cell. Zymogen granules fuse with the apical membrane and release multiple enzymes to digest carbohydrates, proteins, and fat. (Reproduced with permission from Bloom W, Fawcett DW: A Textbook of Histology, 10th

1	granules fuse with the apical membrane and release multiple enzymes to digest carbohydrates, proteins, and fat. (Reproduced with permission from Bloom W, Fawcett DW: A Textbook of Histology, 10th ed. Philadelphia, PA: Saunders; 1975.)granules that fuse with the apical cell membrane (Fig. 33-8). Unlike the endocrine pancreas, where islet cells specialize in the secretion of one hormone type, individual acinar cells secrete all types of enzymes. However, the ratio of the different enzymes released is adjusted to the composition of digested food through nonparallel regulation of secretion.Pancreatic amylase is secreted in its active form and com-pletes the digestive process already begun by salivary amylase. Amylase is the only pancreatic enzyme secreted in its active form, and it hydrolyzes starch and glycogen to glucose, malt-ose, maltotriose, and dextrins. These simple sugars are trans-ported across the brush border of the intestinal epithelial cells by active transport mechanisms.

1	starch and glycogen to glucose, malt-ose, maltotriose, and dextrins. These simple sugars are trans-ported across the brush border of the intestinal epithelial cells by active transport mechanisms. Gastric hydrolysis of protein yields peptides that enter the intestine and stimulate intestinal endocrine cells to release cholecystokinin (CCK)-releasing pep-tide, CCK, and secretin, which then stimulate the pancreas to secrete enzymes and bicarbonate into the intestine.The proteolytic enzymes are secreted as proenzymes that require activation. Trypsinogen is converted to its active form, trypsin, by another enzyme, enterokinase, which is produced by the duodenal mucosal cells. Trypsin, in turn, activates the other proteolytic enzymes. Trypsinogen activation within the pancreas is prevented by the presence of inhibitors that are also secreted by the acinar cells. A failure to express a normal tryp-sinogen inhibitor, pancreatic secretory trypsin inhibitor (PSTI), also known as serine

1	by the presence of inhibitors that are also secreted by the acinar cells. A failure to express a normal tryp-sinogen inhibitor, pancreatic secretory trypsin inhibitor (PSTI), also known as serine protease inhibitor Kazal type 1 (SPINK1), is a cause of familial pancreatitis. Inhibition of trypsinogen activation ensures that the enzymes within the pancreas remain in an inactive precursor state and are activated only within the duodenum. Trypsinogen is expressed in several isoforms, and a missense mutation on the cationic trypsinogen, or PRSS1, results in premature, intrapancreatic activation of trypsinogen. This accounts for about two-thirds of cases of hereditary pan-creatitis. Chymotrypsinogen is activated to form chymotrypsin. Elastase, carboxypeptidase A and B, and phospholipase are also activated by trypsin. Trypsin, chymotrypsin, and elastase cleave bonds between amino acids within a target peptide chain, and carboxypeptidase A and B cleave amino acids at the end of peptide

1	also activated by trypsin. Trypsin, chymotrypsin, and elastase cleave bonds between amino acids within a target peptide chain, and carboxypeptidase A and B cleave amino acids at the end of peptide chains. Individual amino acids and small dipeptides are then actively transported into the intestinal epithelial cells. Pancreatic lipase hydrolyzes triglycerides to 2-monoglyceride and fatty acid. Pancreatic lipase is secreted in an active form. Colipase is also secreted by the pancreas and binds to lipase, changing its molecular configuration and increasing its activity. Phospholipase A2 is secreted by the pancreas as a proenzyme that becomes activated by trypsin. Phospholipase A2 hydrolyzes phospholipids and, as with all lipases, requires bile salts for its action. Carboxylic ester hydrolase and cholesterol esterase hydrolyze neutral lipid substrates like esters of cholesterol, fat-soluble vitamins, and triglycerides. The hydrolyzed fat is then packaged into micelles for transport into

1	and cholesterol esterase hydrolyze neutral lipid substrates like esters of cholesterol, fat-soluble vitamins, and triglycerides. The hydrolyzed fat is then packaged into micelles for transport into the intestinal epithelial cells, where the fatty acids are reassembled and packaged inside chylomicrons for transport through the lymphatic system into the bloodstream (Table 33-1).The centroacinar and intercalated duct cells secrete the water and electrolytes present in the pancreatic juice. About 40 acinar cells are arranged into a spherical unit called an acinus. Centroacinar cells are located near the center of the acinus and are responsible for fluid and electrolyte secretion. These cells contain the enzyme carbonic anhydrase, which is needed for bicarbonate secretion. The amount of bicarbonate secreted varies with the pancreatic secretory rate, with greater concen-trations of bicarbonate being secreted as the pancreatic secre-tory rate increases. Chloride secretion varies inversely

1	secreted varies with the pancreatic secretory rate, with greater concen-trations of bicarbonate being secreted as the pancreatic secre-tory rate increases. Chloride secretion varies inversely with bicarbonate secretion such that the sum of these two remains constant. In contrast, sodium and potassium concentrations are kept constant throughout the spectrum of secretory rates3 (Fig. 33-9). The hormone secretin is released from cells in the duodenal mucosa in response to acidic chyme passing through the pylorus into the duodenum. Secretin is the major stimulant for bicarbonate secretion, which buffers the acidic fluid entering the duodenum from the stomach. CCK also stimulates bicarbon-ate secretion, but to a much lesser extent than secretin. CCK potentiates secretin-stimulated bicarbonate secretion. Gastrin and acetylcholine, both stimulants of gastric acid secretion, are also weak stimulants of pancreatic bicarbonate secretion.4 Truncal vagotomy produces a myriad of complex effects

1	secretion. Gastrin and acetylcholine, both stimulants of gastric acid secretion, are also weak stimulants of pancreatic bicarbonate secretion.4 Truncal vagotomy produces a myriad of complex effects on the downstream digestive tract, but the sum effect on the exocrine pancreas is a reduction in bicarbonate and fluid secretion.5 The endocrine pancreas also influences the adjacent exocrine pan-creatic secretions. Somatostatin, pancreatic polypeptide (PP), and glucagon are all thought to inhibit exocrine secretion.The acinar cells release pancreatic enzymes from their zymogen granules into the lumen of the acinus, and these pro-teins combine with the water and bicarbonate secretions of the centroacinar cells. The pancreatic juice then travels into small intercalated ducts. Several small intercalated ducts join to form an interlobular duct. Cells in the interlobular ducts contribute fluid and electrolytes to adjust the final concentrations of the pancreatic fluid. Interlobular ducts then

1	ducts join to form an interlobular duct. Cells in the interlobular ducts contribute fluid and electrolytes to adjust the final concentrations of the pancreatic fluid. Interlobular ducts then join to form about 20 secondary ducts that empty into the main pancreatic duct. Brunicardi_Ch33_p1429-p1516.indd 143601/03/19 6:44 PM 1437PANCREASCHAPTER 33Table 33-1Pancreatic enzymesENZYMESUBSTRATEPRODUCTCarbohydrate Amylase (active)Starch, glycogenGlucose, maltose, maltotriose, dextrinsProtein Endopeptidases Trypsinogen (inactive) Enterokinase Trypsin (active) Chymotrypsinogen (inactive) EnterokinaseTrypsin Chymotrypsin (active) Proelastase (inactive) EnterokinaseTrypsin Elastase (active) Exopeptidases Procarboxy peptidase A&B (inactive) Enterokinase Carboxypeptidase A&B (active)Cleave bonds between amino acidsCleave amino acids from end of peptide chainsAmino acids, dipeptides—Fat Pancreatic lipase (active) Phospholipase A2 (inactive) Trypsin Phospholipase A2 (active) Cholesterol

1	bonds between amino acidsCleave amino acids from end of peptide chainsAmino acids, dipeptides—Fat Pancreatic lipase (active) Phospholipase A2 (inactive) Trypsin Phospholipase A2 (active) Cholesterol esteraseTriglyceridesPhospholipaseNeutral lipids2-Monoglycerides, fatty acids——Concentration (mEq/L)18016014012010080604020 000.40.81.21.6Secretory rate (mL/min)Pancreatic juice180160140120100806040200PlasmaNaHCO3ClKNa=151.5HCO3=26.0Cl=110.0K=5.38Figure 33-9. Composition of pancreatic exocrine secretions. Greater concentrations of bicarbonate are secreted at higher secretory rates, and chloride secretion varies inversely with bicarbonate secretion. In contrast, sodium and potassium concentrations are independent of the secretory rate. (Reproduced with permission from Bro-Rasmussen F, Killmann SA, Thaysen JH: The composition of pancreatic juice as compared to sweat, parotid saliva and tears, Acta Physiol Scand. 1956 Sep 26;37(2-3):97-113.)Table 33-2Pancreatic Islet Peptide

1	F, Killmann SA, Thaysen JH: The composition of pancreatic juice as compared to sweat, parotid saliva and tears, Acta Physiol Scand. 1956 Sep 26;37(2-3):97-113.)Table 33-2Pancreatic Islet Peptide ProductsHORMONESISLET CELLFUNCTIONSInsulinBetaDecreases gluconeogenesis, glycogenolysis, fatty acid breakdown, and ketogenesisIncreased glycogenesis, protein synthesis and glucose uptakeGlucagonAlphaOpposite effects of insulin; increases hepatic glycogenolysis and gluconeogenesisSomatostatinDeltaInhibits secretion and action of all pancreatic and gut peptides, inhibits cell growthPancreatic PolypeptidePP or FInhibits pancreatic exocrine secretion and facilitates hepatic action of insulinAmylin (IAPP)BetaCounter-regulates insulin secretion and functionPancreastatinBetaDecreases insulin and somatostatin secretion, increases glucagon secretion, decreases exocrine secretionGhrelinEpsilonDecreases insulin secretion and actionPeptide YY (PYY)not knownIncreases insulin secretion and beta cell

1	secretion, increases glucagon secretion, decreases exocrine secretionGhrelinEpsilonDecreases insulin secretion and actionPeptide YY (PYY)not knownIncreases insulin secretion and beta cell growthDestruction of the branching ductal tree from recurrent inflam-mation, scarring, and deposition of stones in chronic pancreatitis eventually contributes to destruction of the exocrine pancreas and exocrine pancreatic insufficiency.Endocrine PancreasThere are nearly 1 million islets of Langerhans in the normal adult pancreas. They vary greatly in size from 40 to 900 μm. Larger islets are located closer to the major arterioles and smaller islets are embedded more deeply in the parenchyma of the pancreas. Most islets contain 3000 to 4000 cells of five major types: alpha cells that secrete glucagon, beta-cells that secrete insulin, delta cells that secrete somatostatin, epsilon cells that secrete ghrelin, and PP cells that secrete PP (Table 33-2).Insulin is the best-studied pancreatic hormone. The

1	that secrete insulin, delta cells that secrete somatostatin, epsilon cells that secrete ghrelin, and PP cells that secrete PP (Table 33-2).Insulin is the best-studied pancreatic hormone. The discovery of insulin in 1920 by Frederick Banting, an orthopedic Brunicardi_Ch33_p1429-p1516.indd 143701/03/19 6:44 PM 1438SPECIFIC CONSIDERATIONSPART IIsurgeon, and Charles Best, a medical student, was recognized with the awarding of the Nobel Prize in Physiology or Medicine. They produced diabetes in dogs by performing total pancreatectomy and then treated them with crude pancreatic extracts from dog and calf pancreata using techniques to prevent the breakdown of insulin by the proteolytic enzymes of the exocrine pancreas. Insulin was subsequently purified and found to be a 56-amino acid peptide with two chains, an alpha and a beta chain, joined by two disulfide bridges and a connecting peptide, or C-peptide. Proinsulin is made in the endoplasmic reticulum and then is transported to the

1	with two chains, an alpha and a beta chain, joined by two disulfide bridges and a connecting peptide, or C-peptide. Proinsulin is made in the endoplasmic reticulum and then is transported to the Golgi complex, where it is packaged into granules and the C-peptide is cleaved off. There are two phases of insulin secretion. In the first phase, stored insulin is released. This phase lasts about 5 minutes after a glucose challenge. The second phase of insulin secretion is a longer, sustained release due to ongoing production of new insulin. beta-cell synthesis of insulin is regulated by plasma glucose levels, neural signals, and the paracrine influence of other islet cells. The diagnosis of diabetes is made by using oral and intravenous (IV) glucose tolerance tests. Oral glucose not only enters the bloodstream but also stimulates the release of enteric hormones such as gastric inhibitory peptide (also known as glucose-dependent insulinotropic polypeptide or GIP), glucagon-like peptide-1

1	the bloodstream but also stimulates the release of enteric hormones such as gastric inhibitory peptide (also known as glucose-dependent insulinotropic polypeptide or GIP), glucagon-like peptide-1 (GLP-1), and CCK that augment the secretion of insulin and are therefore referred to as incretins. As a result, oral glucose is a more vigorous stimulus to insulin secretion than IV glucose. In the oral glucose tolerance test (OGTT), the patient is fasted overnight, and a basal glucose value is determined. Forty g/m2 or 75 g of glucose is given orally over 10 minutes. Blood samples are taken every 30 minutes for 2 hours. Normal values and criteria for diabetes vary by age, but essentially all values should be <200 mg/dL, and the 120-minute value should be <140 mg/dL.Insulin secretion by the beta-cell is also influenced by plasma levels of amino acids such as arginine, lysine, leucine, and free fatty acids. Glucagon, GIP, GLP-1, and CCK stimulate insulin release, while somatostatin, amylin,

1	is also influenced by plasma levels of amino acids such as arginine, lysine, leucine, and free fatty acids. Glucagon, GIP, GLP-1, and CCK stimulate insulin release, while somatostatin, amylin, and pancreastatin inhibit insulin release.6 Cholinergic fibers and alpha-sympa-thetic fibers stimulate insulin release, while beta-sympathetic fibers inhibit insulin secretion.Insulin’s glucoregulatory function is to inhibit endogenous (hepatic) glucose production and to facilitate glucose transport into cells, thus lowering plasma glucose levels. Insulin also inhibits glycogenolysis, fatty acid breakdown, and ketone formation, and stimulates protein synthesis. There is a considerable amount of functional reserve in insulin secretory capacity. If the remaining portion of the pancreas is healthy, about 80% of the pancreas can be resected without the patient becoming diabetic.7 In patients with chronic pancreatitis, or other conditions in which much of the gland is diseased, resection of a smaller

1	80% of the pancreas can be resected without the patient becoming diabetic.7 In patients with chronic pancreatitis, or other conditions in which much of the gland is diseased, resection of a smaller fraction of the pancreas can result in pancreatogenic, or type 3c diabetes (Table 33-3).Insulin receptors are dimeric, tyrosine kinase–containing transmembrane proteins that are located on all cells. Insulin defi-ciency (seen in type 1 and type 3c diabetes) results in an overex-pression or upregulation of insulin receptors, which causes an enhanced sensitivity to insulin in muscle and adipocytes (and therefore increases the risk of insulin-induced hypoglycemia). Type 2 diabetes is associated with a downregulation of insulin receptors and relative hyperinsulinemia, with resulting insulin resistance. Some forms of diabetes are associated with selected impairments of hepatic or peripheral insulin receptors, such as pancreatogenic or type 3c diabetes (T3cDM) or maturity-onset diabetes of the

1	Some forms of diabetes are associated with selected impairments of hepatic or peripheral insulin receptors, such as pancreatogenic or type 3c diabetes (T3cDM) or maturity-onset diabetes of the young (MODY).Glucagon is a 29-amino-acid, single-chain peptide that promotes hepatic glycogenolysis and gluconeogenesis and counteracts the effects of insulin through its hyperglycemic action. Glucose is the primary regulator of glucagon secretion, as it is with insulin, but it has an inhibitory rather than stimulatory effect. Glucagon release is stimulated by hypoglycemia and by the amino acids arginine and alanine. GLP-1 inhibits glucagon secretion in vivo, and insulin and somatostatin inhibit glucagon Table 33-3Clinical and laboratory findings in types of diabetes mellitusPARAMETERTYPE 1TYPE 2TYPE 3C IDDMNIDDMPancreatogenicKetoacidosisCommonRareRareHyperglycemiaSevereUsually mildMildHypoglycemiaCommonRareCommonPeripheral insulin sensitivityNormal or increasedDecreasedIncreasedHepatic insulin

1	3C IDDMNIDDMPancreatogenicKetoacidosisCommonRareRareHyperglycemiaSevereUsually mildMildHypoglycemiaCommonRareCommonPeripheral insulin sensitivityNormal or increasedDecreasedIncreasedHepatic insulin sensitivityNormalNormal or decreasedDecreasedInsulin levelsLowHighLowGlucagon levelsNormal or highNormal or highLowPP levelsNormal or low (late)HighLowGIP levelsNormal or lowNormal or highLowGLP-1 levelsNormalNormal or highNormal or highTypical age of onsetChildhood or adolescenceAdulthoodAnyAbbreviations: IDDM = insulin dependent diabetes mellitus; NIDDM = non–insulin-dependent diabetes mellitus; PP = pancreatic polypeptide; GIP = glucose-dependent insulinotropic polypeptide; GLP-1 = glucagon-like peptide 1.Reproduced with permission from Slezak LA, Andersen DK: Pancreatic resection: effects on glucose metabolism, World J Surg. 2001 Apr;25(4):452-460.Brunicardi_Ch33_p1429-p1516.indd 143801/03/19 6:44 PM 1439PANCREASCHAPTER 33secretion in a paracrine fashion within the islet. The same

1	on glucose metabolism, World J Surg. 2001 Apr;25(4):452-460.Brunicardi_Ch33_p1429-p1516.indd 143801/03/19 6:44 PM 1439PANCREASCHAPTER 33secretion in a paracrine fashion within the islet. The same neural impulses that regulate insulin secretion also regulate glucagon secretion, so that the two hormones work together in a balance of actions to maintain glucose levels. Cholinergic and alpha-sympathetic fibers stimulate glucagon release, while beta-sympathetic fibers inhibit glucagon release.8 In pancreatogenic or type 3c diabetes, glucagon responsiveness to a fall in blood glucose is lost, thereby increasing the risk for hypoglycemia.Although originally isolated from the hypothalamus, somatostatin is a peptide that is now known to have a wide ana-tomic distribution, not only in neurons but also in the pancreas, gut, and other tissues. It is a highly conserved peptide hormone, as it is found in lower vertebrates, and is now realized to be of fundamental importance in regulatory

1	but also in the pancreas, gut, and other tissues. It is a highly conserved peptide hormone, as it is found in lower vertebrates, and is now realized to be of fundamental importance in regulatory processes throughout the body. One gene encodes for a common precursor that is dif-ferentially processed to generate tissue-specific amounts of two bioactive products, somatostatin-14, and somatostatin-28. These peptides inhibit endocrine and exocrine secretion and affect neurotransmission, GI and biliary motility, intestinal absorption, vascular tone, and cell proliferation.Five different somatostatin receptors (SSTRs) have been cloned and the biologic properties of each are different.9 The hexapeptide and octapeptide analogues such as octreotide bind only to SSTR2, SSTR3, and SSTR5. These analogues have a longer serum half-life, and their potent inhibitory effect has been used clinically to treat both endocrine and exocrine disor-ders. For example, octreotide has been shown to decrease

1	have a longer serum half-life, and their potent inhibitory effect has been used clinically to treat both endocrine and exocrine disor-ders. For example, octreotide has been shown to decrease fistula output and speed the time it takes for enteric and pancreatic fistulas to close.10Endocrine release of somatostatin occurs during a meal. The major stimulant is probably intraluminal fat. Acidification of the gastric and duodenal mucosa also releases somatostatin in isolated perfused organ preparations. Acetylcholine from the cholinergic neurons inhibits somatostatin release.Pancreatic polypeptide (PP) is a 36-amino-acid, straight-chain peptide discovered by Kimmel in 1968 during the process of insulin purification. Protein is the most potent enteral stimu-lator of PP release, closely followed by fat, whereas glucose has a weaker effect.11 Hypoglycemia, whether or not it is insulin induced, strongly stimulates PP secretion through cholinergic stimulation.12 Phenylalanine, tryptophan, and

1	by fat, whereas glucose has a weaker effect.11 Hypoglycemia, whether or not it is insulin induced, strongly stimulates PP secretion through cholinergic stimulation.12 Phenylalanine, tryptophan, and fatty acids in the duodenum stimulate PP release, probably by inducing CCK, GIP, and secretin release. Vagal stimulation of the pancreas is the most important regulator of PP secretion. In fact, vagotomy eliminates the rise in PP levels usually seen after a meal. This can be used as a test for the completeness of a surgical vagot-omy or for the presence of diabetic autonomic neuropathy.PP has been shown to inhibit choleresis (bile secretion), gallbladder contraction, and secretion by the exocrine pan-creas. However, PP’s most important role is in glucose regu-lation through its regulation of hepatic insulin receptor gene expression. A deficiency in PP secretion due to proximal pan-createctomy, severe chronic pancreatitis, or cystic fibrosis, is associated with diminished hepatic insulin

1	hepatic insulin receptor gene expression. A deficiency in PP secretion due to proximal pan-createctomy, severe chronic pancreatitis, or cystic fibrosis, is associated with diminished hepatic insulin sensitivity due to reduced hepatic insulin receptor availability.13 This effect is reversed by PP administration.14Recent studies have shown that a fifth islet peptide, ghre-lin, is secreted from a distinct population of islet cells, called epsilon cells.15,16 Ghrelin also is present in the gastric fundus in large amounts and stimulates growth hormone secretion via growth hormone releasing hormone release from the pituitary. It is an orexigenic, or appetite-stimulating, peptide the plasma levels of which are increased in obesity. Ghrelin has also been shown to block insulin effects on the liver, and inhibits the beta-cell response to incretin hormones and glucose.17 Therefore, ghrelin secretion from and within the islet may modulate the responses of other islet cells to nutrient and

1	liver, and inhibits the beta-cell response to incretin hormones and glucose.17 Therefore, ghrelin secretion from and within the islet may modulate the responses of other islet cells to nutrient and hormonal stimuli.In addition to the five main peptides secreted by the pan-creas, there are a number of other peptide products of the islet cells, including amylin, peptide YY (PYY), and pancreastatin, as well as neuropeptides such as VIP, galanin, and serotonin. Amylin or islet amyloid polypeptide (IAPP) is a 37-amino-acid polypeptide that is predominantly expressed by the pancreatic beta-cells, where it is stored along with insulin in secretory granules.18 The function of IAPP seems to be the modulation or counterregulation of insulin secretion and function. Pancre-astatin is a recently discovered pancreatic islet peptide prod-uct that inhibits insulin, and possibly somatostatin release, and augments glucagon release.19,20 In addition to this effect on the endocrine pancreas,

1	discovered pancreatic islet peptide prod-uct that inhibits insulin, and possibly somatostatin release, and augments glucagon release.19,20 In addition to this effect on the endocrine pancreas, pancreastatin inhibits pancreatic exocrine secretion.21 PYY is structurally related to PP and was initially found in hormone-secreting “L” cells of the small intestine, where it colocalizes with GLP-1. Recently, PYY has been local-ized to the islets22 where it appears to regulate insulin secretion through an autocrine mechanism.Islet DistributionThe beta-cells are generally located in the central portion of each islet and make up about 70% of the total islet cell mass. The other cell types are located predominantly in the periphery. The delta cells are least plentiful, making up only 5%; the 〈-cells make up 10%, and the PP cells make up 15%.23 In contrast to the acinar cells that secrete the full gamut of exocrine enzymes, the islet cells seem to specialize in the secretion of predominantly one

1	make up 10%, and the PP cells make up 15%.23 In contrast to the acinar cells that secrete the full gamut of exocrine enzymes, the islet cells seem to specialize in the secretion of predominantly one hormone. However, individual islet cells can secrete mul-tiple hormones. For example, the beta-cells secrete both insulin and amylin, which counter regulates the actions of insulin. In reality, more than 20 different hormones are secreted by the islets, and the exact functions of this milieu are very complex. There is diversity among the islets depending on their location within the pancreas. The beta and delta cells are evenly distrib-uted throughout the pancreas, but islets in the head and uncinate process (ventral anlage) have a higher percentage of PP cells and fewer alpha cells, whereas islets in the body and tail (dorsal anlage) contain the majority of alpha cells and few PP cells. This is clinically significant because pancreatoduodenectomy removes 95% of the PP cells in the

1	islets in the body and tail (dorsal anlage) contain the majority of alpha cells and few PP cells. This is clinically significant because pancreatoduodenectomy removes 95% of the PP cells in the pancreas. This may partially explain the higher incidence of glucose intolerance after the Whipple procedure compared to a distal pancreatectomy with an equivalent amount of tissue resected. In addition, chronic pan-creatitis, which disproportionately affects the pancreatic head, is associated with PP deficiency and pancreatogenic diabetes.24 The relative preponderance of alpha cells in the body and tail of the pancreas explains the typical location of glucagonomas.ACUTE PANCREATITISDefinition, Incidence, and EpidemiologyAcute pancreatitis is an inflammatory disorder of the pancreas that is characterized by edema and, when severe, necrosis. It is a common and challenging disease that can develop local and systemic complications. As such, it ranges from a mild, self-limiting inflammation of the

1	by edema and, when severe, necrosis. It is a common and challenging disease that can develop local and systemic complications. As such, it ranges from a mild, self-limiting inflammation of the pancreas to severe and critical disease characterized by infected pancreatic necrosis, multiple Brunicardi_Ch33_p1429-p1516.indd 143901/03/19 6:44 PM 1440SPECIFIC CONSIDERATIONSPART IITable 33-4Etiologies of acute pancreatitisAlcoholBiliary tract diseaseHyperlipidemiaHereditaryHypercalcemiaTrauma External Surgical Endoscopic retrograde cholangiopancreatographyIschemia Hypoperfusion Atheroembolic VasculitisPancreatic duct obstruction Neoplasms Pancreas divisum Ampullary and duodenal lesionsInfectionsVenomDrugsIdiopathicReproduced with permission from Townsend CM, Sabiston DC: Sabiston’s Textbook of Surgery: the biological basis of modern surgical practice, 16th ed. Philadelphia, PA: Saunders/Elsevier; 2000.organ failure, and a high mortality.25 The traditional view is that acute pancreatitis

1	of Surgery: the biological basis of modern surgical practice, 16th ed. Philadelphia, PA: Saunders/Elsevier; 2000.organ failure, and a high mortality.25 The traditional view is that acute pancreatitis completely resolves with no morphological, functional, or symptomatic sequelae. But necrotizing pancreati-tis can leave significant scarring, strictures, and impairment of exocrine and endocrine pancreatic function. The overall clinical outcome has improved over recent decades, even in the absence of specific treatments that target outcome-determining patho-physiology, probably because of a more standardized approach to diagnosis, monitoring, and management.Acute pancreatitis is the most common inpatient princi-pal gastrointestinal discharge diagnosis in the United States (274,119 in 2009), with an increasing incidence (30% since 2000) and is associated with the highest aggregate inpatient costs at 2.6 billion dollars per year.26 The crude mortality rate of 1 per 100,000 population ranks

1	an increasing incidence (30% since 2000) and is associated with the highest aggregate inpatient costs at 2.6 billion dollars per year.26 The crude mortality rate of 1 per 100,000 population ranks it as the 14th most common overall and the 9th most common noncancer cause of gastrointestinal deaths. Worldwide, the incidence of acute pancreatitis ranges from 5 to 80 per 100,000 population, with the highest incidence recorded in Finland and the United States.27 The incidence of acute pancreatitis also shows significant variation related to the prevalence of etiological factors and ethnicity. The annual inci-dence of acute pancreatitis in Native Americans is 4 per 100,000 population; in whites, it is 5.7; and in blacks it is 20.7.28 Smok-ing is an independent risk factor for acute pancreatitis.29EtiologyMany factors are causally related to the onset of acute pan-creatitis, but the mechanism is often poorly understood. The most common causes are gallstones and alcohol (Table 33-4),

1	factors are causally related to the onset of acute pan-creatitis, but the mechanism is often poorly understood. The most common causes are gallstones and alcohol (Table 33-4), accounting for up to 80% of cases, but it is not uncommon to diagnose acute pancreatitis in the absence of these etiological factors (“idiopathic acute pancreatitis”), and it is important that a systematic approach is taken to the identification of other, less common and potentially modifiable factors. The median age at index presentation of acute pancreatitis varies with etiology: with alcoholand drug-induced pancreatitis presenting in the third or fourth decade compared with gallstone and trauma in the sixth decade. The gender difference is probably more related to etiology: in males alcohol is more often the cause while in females it is gallstones.GallstonesEvidence that passage of a gallstone is related to the onset of acute pancreatitis comes from the characteristic transient derangement of liver function

1	while in females it is gallstones.GallstonesEvidence that passage of a gallstone is related to the onset of acute pancreatitis comes from the characteristic transient derangement of liver function tests and the high retrieval rate of gallstones from feces within 10 days of an attack of acute pancreatitis compared with those without acute pancreatitis (88% vs. 11%).30 The mechanism by which small gallstones cause acute pancreatitis in migrating through to the duodenum is not clear. Opie made the seminal observation of a gallstone impacted in the sphincter of Oddi in two fatal cases of acute pancreatitis, which lead to the “common channel” hypothesis. It was proposed that this allowed bile to reflux into the pancre-atic duct, but this cannot be reliably reproduced in experimen-tal models. Another proposal was that transient incompetence caused by the passage of a stone through the sphincter might allow duodenal fluid and bile to reflux into the pancreatic duct, but this is not supported

1	proposal was that transient incompetence caused by the passage of a stone through the sphincter might allow duodenal fluid and bile to reflux into the pancreatic duct, but this is not supported by the failure of this to commonly occur after endoscopic sphincterotomy. A third possibility is that acute pancreatitis is due to the gallstone obstructing the pancreatic duct and leading to ductal hypertension. It has been postulated that this backpressure might lead to minor ductal dis-ruption, extravasation of pancreatic juice into the less alkaline interstitium of the pancreas, and promotion of enzyme activa-tion. When gallstones and other etiological factors cannot be identified, there is still the possibility of finding microlithiasis, seen as birefringent crystals, on bile microscopy.31 This occult microlithiasis is probably responsible for up to half of those with idiopathic acute pancreatitis.AlcoholAlcohol ingestion is associated with acute pancreatitis, and sus-tained alcohol

1	This occult microlithiasis is probably responsible for up to half of those with idiopathic acute pancreatitis.AlcoholAlcohol ingestion is associated with acute pancreatitis, and sus-tained alcohol ingestion is associated with recurrent acute pan-creatitis and development of chronic pancreatitis in susceptible individuals who have been drinking for more than a decade. The type of alcohol consumed is less important than the amount consumed (typically 100–150 grams per day) and the pattern of drinking. It is common for patients with alcohol-associated acute pancreatitis to have a history of excess alcohol consumption prior to the first attack. There are several mechanisms by which ethanol causes acute pancreatitis by acting on the acinar and stellate cells.32 The acinar cell metabolizes ethanol by oxidative and nonoxidative pathways, and exhibits changes that predis-pose the cell to autodigestive injury, necroinflammation, and cell death. The stellate cells are activated on exposure to

1	by oxidative and nonoxidative pathways, and exhibits changes that predis-pose the cell to autodigestive injury, necroinflammation, and cell death. The stellate cells are activated on exposure to ethanol to a myofibroblast phenotype, stimulating synthesis of proinflam-matory mediators and cytokines. Ethanol causes a brief secretory increase followed by inhibition. The secretory burst coupled with ethanol induced spasm of the sphincter of Oddi probably incites acute pancreatitis. Ethanol also induces ductal permeability, which allows prematurely activated enzymes to cause damage to the pancreatic parenchyma. Ethanol also increases the protein content of pancreatic juice and decreases bicarbonate levels and trypsin inhibitor concentration. The formation of protein plugs may also contribute by causing an obstructive element to pancre-atic outflow, more often seen in chronic pancreatitis.Brunicardi_Ch33_p1429-p1516.indd 144001/03/19 6:44 PM 1441PANCREASCHAPTER 33Figure 33-10. Schema of

1	causing an obstructive element to pancre-atic outflow, more often seen in chronic pancreatitis.Brunicardi_Ch33_p1429-p1516.indd 144001/03/19 6:44 PM 1441PANCREASCHAPTER 33Figure 33-10. Schema of key loco-regional pathophysiological events in the pancreas and intestine and how they interact to drive the severity and outcome of acute pancreatitis. (Adapted with permission from Flint RS, Windsor JA: The role of the intestine in the pathophysiology and management of severe acute pancreatitis, HPB (Oxford). 2003;5(2):69-85.)IatrogenicAcute pancreatitis can occur due to trauma to the ducts or parenchyma after surgical procedures, including biopsy, bile duct exploration, distal gastrectomy, and splenectomy. As the pancreas is susceptible to ischemia, it can also occur second-ary to splanchnic hypoperfusion with cardiopulmonary bypass, cardiac transplant, hemorrhagic shock, and major trauma. The most common iatrogenic cause is ERCP in which acute pancre-atitis occurs after about 5% to 10%

1	hypoperfusion with cardiopulmonary bypass, cardiac transplant, hemorrhagic shock, and major trauma. The most common iatrogenic cause is ERCP in which acute pancre-atitis occurs after about 5% to 10% of procedures, and in many series, it is the third most common identified etiological fac-tor. The risk of post-ERCP acute pancreatitis is increased if the contrast agent is infused repeatedly under high pressure by the endoscopist and in patients with sphincter of Oddi dysfunction. Recent evidence demonstrates that the risk can be decreased with prophylactic rectal nonsteroidal drugs,33 and this may be a better strategy than prophylactic pancreatic duct stenting.34Hereditary PancreatitisHereditary pancreatitis is an autosomal dominant disorder usually related to mutations of the cationic trypsinogen gene (PRSS1). Mutations in this gene cause premature activation of trypsinogen to trypsin and causes abnormalities of ductal secre-tion, both of which promote acute pancreatitis. Mutations in

1	gene (PRSS1). Mutations in this gene cause premature activation of trypsinogen to trypsin and causes abnormalities of ductal secre-tion, both of which promote acute pancreatitis. Mutations in the SPINK1 protein, which blocks the active binding site of trypsin, is likely to also have a role in predisposing to acute pancreatitis. Variations in penetration and phenotype are common, and there are many other mutations that have become implicated. Mutant enzymes activated within acinar cells can overwhelm the first line of defense (pancreatic secretory trypsin inhibitor) and resist backup defenses (e.g., proteolytic degradation, enzyme Y, and trypsin itself) allowing activated mutant cationic trypsin to trig-ger the entire zymogen activation cascade.35TumorsA pancreatic or periampullary tumor should be considered in patients with idiopathic acute pancreatitis, especially in those over 50 years old. Approximately 1% to 2% of patients with acute pancreatitis have a pancreatic tumor, and an

1	should be considered in patients with idiopathic acute pancreatitis, especially in those over 50 years old. Approximately 1% to 2% of patients with acute pancreatitis have a pancreatic tumor, and an episode of acute pancreatitis can be the first clinical indicator. Cross-sectional imaging after the resolution of the acute pancreatitis is required.HyperlipidemiaPatients with types I and V hyperlipoproteinemia can experience episodes of abdominal pain, and these often occur in association with marked hypertriglyceridaemia. Lipase is thought to liberate toxic fatty acids into the pancreatic microcirculation, leading to microcirculatory impairment and ischemia. Dietary modifica-tions and drug treatment are used to lower triglycerides.DrugsIsolated cases of acute pancreatitis have been associated with exposure to certain drugs, such as thiazide diuretics, furosemide, estrogen replacement therapy, and steroid therapy in children. In addition, certain chemotherapy agents and anti-immune

1	with exposure to certain drugs, such as thiazide diuretics, furosemide, estrogen replacement therapy, and steroid therapy in children. In addition, certain chemotherapy agents and anti-immune drugs have been associated with acute pancreatitis, and lipid-based drugs or solutions, such as propofol, have been shown to cause acute pancreatitis.PathophysiologyAcute pancreatitis occurs in various degrees of severity, the determinants of which are multifactorial. The generally prevalent belief today is that pancreatitis begins with the activation of diges-tive zymogens inside acinar cells, which cause acinar cell injury. Studies suggest that the ultimate severity of the resulting pancre-atitis may be determined by the events that occur subsequent to acinar cell injury.36 These include inflammatory cell recruitment and activation, as well as generation and release of cytokines and other chemical mediators that cause systemic inflammation and multiple organ dysfunction/failure (Figure

1	cell recruitment and activation, as well as generation and release of cytokines and other chemical mediators that cause systemic inflammation and multiple organ dysfunction/failure (Figure 33-10).Precipitating EventsIn 1896, Chiari proposed that pancreatitis was due to the premature, intrapancreatic activation of digestive enzymes, resulting in “auto-digestion” of the organ. Since then the Brunicardi_Ch33_p1429-p1516.indd 144101/03/19 6:44 PM 1442SPECIFIC CONSIDERATIONSPART IIFigure 33-11. Schematic representation of the acinar cell events in acute pancreatitis. When acinar cells are pathologically stimulated, their lysosomal (L) and zymogen (Z) contents colocalize, and consequently trypsinogen is activated to trypsin by cathepsin B. Increased cytosolic calcium is required for colocalization. Once trypsin has permeabilized the contents of the cytosol, cathepsin B and other contents of these colocalized organelles are released. Once in the cytosol, cathepsin B activates apoptosis

1	Once trypsin has permeabilized the contents of the cytosol, cathepsin B and other contents of these colocalized organelles are released. Once in the cytosol, cathepsin B activates apoptosis by causing cytochrome c to be released from the mitochondria. Activation of PKC results in a sudden activation of nuclear factor kappa beta (NFκβ), which in turn triggers the release of cytokines that attract inflammatory response cells that mediate local and systemic inflammation cascades.intra-acinar activation of zymogens has been demonstrated consistently in multiple animal models of acute pancreatitis and is considered a key precipitating event.37,38 The key role of trypsin activation in acute pancreatitis has gained additional support from recent studies showing that mice lacking trypsinogen-7 (the isoform of trypsinogen that is activated during acute pancreatitis in mice) have significantly less pancreatic injury during acute pancreatitis39 and that intra-acinar expression of active trypsin

1	isoform of trypsinogen that is activated during acute pancreatitis in mice) have significantly less pancreatic injury during acute pancreatitis39 and that intra-acinar expression of active trypsin causes pancreatitis in mice.40 The role of trypsin activation in the pathophysiology of acute pancreatitis has also been suggested by clinical studies. Hereditary pancreatitis is associated with mutations that lead to elevated intracellular trypsin activation,41 and activation of trypsinogen causes clinical pancreatitis.42Significant progress has been made in understanding the mechanisms by which injurious stimuli lead to intra-acinar activation of trypsinogen and autodigestion of the gland (Figure 33-11). Under physiologic conditions, several protective mechanisms have evolved to prevent autodigestion of the pancreas by these enzymes. This includes synthesis of enzymes as inactive precursors, separation of the site of production, and activation of the enzymes and presence of trypsin

1	autodigestion of the pancreas by these enzymes. This includes synthesis of enzymes as inactive precursors, separation of the site of production, and activation of the enzymes and presence of trypsin inhibitors in the pancreas. It is thought that acute pancreatitis occurs when these protective mechanisms are overwhelmed with erroneously activated enzymes, causing injury. It has been shown that intra-acinar activation of trypsinogen goes hand-in-hand with inhibition of acinar secretion.43,44 Furthermore, with injurious stimuli the zymogens responsible for initiating the disease are not secreted outside, but colocalize with cytoplasmic vacuoles that contain lysosomal enzymes such as cathepsin B45 that activate trypsinogen. Thus, inhibition of cathepsin B by pharmacological inhibitors46 or by genetic deletion of cathepsin B eliminates trypsin activation and decreases the severity of pancreatitis in animal models.47 What leads to the colocalization of zymogens and lysosomal hydrolases is

1	genetic deletion of cathepsin B eliminates trypsin activation and decreases the severity of pancreatitis in animal models.47 What leads to the colocalization of zymogens and lysosomal hydrolases is unclear, but injurious stimuli leading to sustained cytosolic calcium increase have been indicted. Blocking this calcium increase prevents colocalization and activation of trypsin, and it decreases injury due to pancreatitis.48 Based on these data, pre-ERCP supplementation of magnesium, a natural antagonist of calcium, is currently being evaluated as a strategy to decrease post-ERCP pancreatitis.49 Recent work has led to the novel hypothesis that the lysosomal hydrolase cathepsin B activates trypsinogen to trypsin within the colocalization vacuoles. Trypsin then permeabilizes these colocalization vacuoles causing the release of cathepsin B into the cytosol. Once in the cytosol, cathepsin B initiates apoptotic cell death by permeabilizing mitochondrial membranes, which allows cytochrome C to

1	vacuoles causing the release of cathepsin B into the cytosol. Once in the cytosol, cathepsin B initiates apoptotic cell death by permeabilizing mitochondrial membranes, which allows cytochrome C to be released into the cytosol. This initiates the apoptotic cascade and results in the apoptotic death of the acinar cells50 (see Figure 33-11).Intrapancreatic EventsAlthough intra-acinar events initiate acute pancreatitis, events occurring subsequent to acinar cell injury determine the severity of pancreatitis. Activated neutrophils are attracted and activated in the pancreas releasing superoxide (the respiratory burst) and proteolytic enzymes (cathepsins, elastase, and collagenase) that cause further pancreatic injury. In addition, macrophages release cytokines (including tumor necrosis factor-alpha (TNF-α) and interleukins (IL-1, IL-2, IL-6, and IL-8) that mediate local and systemic inflammation.38These inflammatory mediators cause an increased pancre-atic vascular permeability, leading

1	(TNF-α) and interleukins (IL-1, IL-2, IL-6, and IL-8) that mediate local and systemic inflammation.38These inflammatory mediators cause an increased pancre-atic vascular permeability, leading to hemorrhage, edema, and Acinar cell deathTo pancreatic ductTrypsinogenCathepsin BActive trypsinEarly stagesLeaky colocalizedorganellesColocalizationSustainedRise in Ca2+PKC activationInsultSystemic in‘ammationRelease of cytokinesand chemokinesRecruitment ofin‘ammatory cellsLater stagesNF–KBactivationLocal injury: PancreatitisZLBrunicardi_Ch33_p1429-p1516.indd 144201/03/19 6:44 PM 1443PANCREASCHAPTER 33Figure 33-12. Computed tomography scan showing well perfused interstitial edematous acute pancreatitis of the neck and tail of the pancreas with a confluent area of necrosis of the pancreatic body. The pancreas is surrounded by fluid, inflammation, and possible peripancreatic fat necrosis.Table 33-5Local complications of acute pancreatitisCONTENT ACUTE (<4 WEEKS, NO DEFINED WALL)CHRONIC (>4

1	The pancreas is surrounded by fluid, inflammation, and possible peripancreatic fat necrosis.Table 33-5Local complications of acute pancreatitisCONTENT ACUTE (<4 WEEKS, NO DEFINED WALL)CHRONIC (>4 WEEKS, DEFINED WALL)NO INFECTIONINFECTIONNO INFECTIONINFECTIONFluidAcute pancreatic fluid collection (APFC)Infected APFCPseudocystInfected pseudocystSolid ± fluidAcute necrotic collection (ANC)Infected ANCWalled off necrosis (WON)Infected WONReproduced with permission from Windsor JA, Petrov MS: Acute pancreatitis reclassified, Gut. 2013 Jan;62(1):4-5.microthrombi. Fluid may collect in and around the pancreas. The failure of the pancreatic microcirculation, a feature of more severe acute pancreatitis, results in pancreatic hypoperfusion and necrosis. Acute inflammation of the pancreatic parenchyma and peripancreatic tissues, but with no recognizable necrosis, is termed interstitial edematous pancreatitis.25 When necrosis is present, as evidenced by pancreatic hypoperfusion with contrast CT,

1	and peripancreatic tissues, but with no recognizable necrosis, is termed interstitial edematous pancreatitis.25 When necrosis is present, as evidenced by pancreatic hypoperfusion with contrast CT, it is termed necrotizing pancreatitis (Figure 33-12). The updated morphological definitions and the contrast enhanced CT criteria for the diagnosis of the local complications of acute pancreatitis are in the revised Atlanta statement51 and summa-rized in Table 33-5.52Systemic EventsAn important aspect of the pathophysiology of acute pancre-atitis is the mechanism by which events occurring in the pan-creas induce systemic inflammation and multiorgan failure. The NFκB-dependent inflammatory pathway is an important mechanism (see Figure 33-11). Activation of NFκB parallels trypsin activation in acute pancreatitis but appears to be inde-pendent of it, as it occurs in trypsin knockout mice.39 Sustained calcium increase, which leads to trypsinogen activation, is criti-cal for NFκB activation since

1	pancreatitis but appears to be inde-pendent of it, as it occurs in trypsin knockout mice.39 Sustained calcium increase, which leads to trypsinogen activation, is criti-cal for NFκB activation since attenuation of cytosolic calcium abrogates NFκB activation.53 Once activated, NFκB regulates synthesis of multiple cytokines and chemokines, leading to recruitment of various inflammatory cells that then magnify and propagate systemic inflammation.54 The infiltrating neutrophils can also further augment the pancreatic injury.55,56 Inhibition of many of these cytokines have led to reduced local and systemic injury in animal models of acute pancreatitis. But these results did not translate to clinical improvement.57 New strategies are required to reduce the systemic inflammatory response.58-60Organ failure can develop at any stage of acute pancreati-tis, associated with an overwhelming proinflammatory response early, or later secondary to the development of infected local complications. The

1	failure can develop at any stage of acute pancreati-tis, associated with an overwhelming proinflammatory response early, or later secondary to the development of infected local complications. The drivers of the systemic response are poorly understood, although factors include the elaboration of proin-flammatory cytokines, and it appears that mesenteric lymph, bypassing the liver and containing these constituents, may con-tribute to the development of organ failure61 (see Figure 33-10). The development of pancreatic necrosis, the breakdown of the intestinal barrier, and the suppression of the immune response through the compensatory inflammatory response contribute to the development of infected pancreatic necrosis, the incidence of which peaks in the third to fourth week. This is usually asso-ciated with deterioration in the patient’s condition and may be associated with the late development of the systemic inflam-matory response syndrome (SIRS) and multiorgan dysfunction

1	is usually asso-ciated with deterioration in the patient’s condition and may be associated with the late development of the systemic inflam-matory response syndrome (SIRS) and multiorgan dysfunction syndrome/failure (MODS/F).Organ failure is scored using the Marshall or Sequential Organ Failure Assessment (SOFA) systems (Table 33-6). The three organ systems most frequently involved are cardiovas-cular, respiratory, and renal. Multiple organ failure is defined as two or more organs registering two or more points on these scoring systems.51 Monitoring organ failure over time and in response to treatment is important in the care and timing of intervention in these patients.Management of Acute PancreatitisGeneral Considerations. The management of acute pancre-atitis covers a wide spectrum of severity. All patients with sus-pected acute pancreatitis should be admitted to hospital. Those with mild acute pancreatitis usually remain in hospital for less than a week, while those with severe

1	severity. All patients with sus-pected acute pancreatitis should be admitted to hospital. Those with mild acute pancreatitis usually remain in hospital for less than a week, while those with severe and critical acute pan-creatitis may require many weeks or months of intensive treat-ment. The risk of mortality reflects this spectrum of severity. The risk is less than 1% for those with mild disease, increasing to around 10% for those with moderate disease, but for severe (20–40%) and critical (>50%) disease the mortality risk is much higher. The earlier identification of these high-risk categories and their transfer to specialized centers is an important priority of management.62The management of acute pancreatitis is multidisciplinary, and it is important that this is coordinated care plan is carefully supervised.63 The essential requirements for the management Brunicardi_Ch33_p1429-p1516.indd 144301/03/19 6:44 PM 1444SPECIFIC CONSIDERATIONSPART IITable 33-6Sequential organ failure

1	is carefully supervised.63 The essential requirements for the management Brunicardi_Ch33_p1429-p1516.indd 144301/03/19 6:44 PM 1444SPECIFIC CONSIDERATIONSPART IITable 33-6Sequential organ failure assessment (SOFA) score in acute pancreatitis 01234Respiration (PaO2FIO2) (mmHg)>400≤400≤300≤200 with respiratory support≤100 with respiratory supportCoagulation Platelets (xl01 per μL)>150≤150≤100≤50≤20Liver Bilirubin (μmol/L)<2020–3233–101102–204>204Cardiovascular HypotensionNo hypotensionMAP <70 mmHgDopamine ≤5 or dobutamine (any dose)aDopamine >5 or epi ≤0.1a or norepi ≤0.1aDopamine >15 or epi >0.1a or norepi >0.1aCentral nervous system Glasgow coma score1513–1410–126–9<6Kidney Creatinine (μmol/L) or urine output<110110–170171–299300–440 or <500 mL/day>440 or <200 mL/dayaAdrenergic agents administered for at least 1 h (doses given in μg/kg per min). A score of 2 or more in any two systems indicates the presence of multiple organ failure.Abbreviations: MAP =

1	mL/dayaAdrenergic agents administered for at least 1 h (doses given in μg/kg per min). A score of 2 or more in any two systems indicates the presence of multiple organ failure.Abbreviations: MAP = mean arterial pressure; Epi = epineptvine; Norepi = norepinephrine.of acute pancreatitis are accurate diagnosis, appropriate triage, high-quality supportive care, and monitoring for and treatment of complications.64DiagnosisThe diagnosis of acute pancreatitis requires the patient to pres-ent with abdominal pain consistent with acute pancreatitis (acute onset of a severe constant epigastric pain that often radiates through to the mid back) and the elevation of serum amylase or lipase (>3 times upper limit of normal). Imaging (usually by contrast-enhanced CT scanning) is only required for the diagnosis of acute pancreatitis when these diagnostic criteria are not met.51 Because of the many causes of hyperamylasemia, it is important to use either the pancreatic isoenzyme of amylase or

1	the diagnosis of acute pancreatitis when these diagnostic criteria are not met.51 Because of the many causes of hyperamylasemia, it is important to use either the pancreatic isoenzyme of amylase or lipase.63The serum amylase concentration increases almost imme-diately with the onset of disease and peaks within several hours and remains elevated for 3 to 5 days.64 There is no correlation between the extent of serum amylase elevation and severity of pancreatitis; in fact, a milder form of acute pancreatitis is often associated with higher levels of serum amylase compared with that in a more severe form of the disease. It is important to note that hyperamylasemia can also occur in association with other diseases. For example, it can occur in a patient with small bowel obstruction, perforated duodenal ulcer, or other intra-abdominal inflammatory conditions. In contrast, a patient with acute pancreatitis can have a normal serum amylase level, for several different reasons. In patients with

1	duodenal ulcer, or other intra-abdominal inflammatory conditions. In contrast, a patient with acute pancreatitis can have a normal serum amylase level, for several different reasons. In patients with hyperlipidemia, values might appear to be normal because of interference with chemical determination of serum amylase by lipids. In some cases, urinary clearance of pancreatic enzymes from the circu-lation increases during pancreatitis, meaning that urinary levels may be more sensitive than serum levels. For these reasons, it is recommended that amylase concentrations also be measured in the urine. Urinary amylase levels usually remain elevated for several days after serum levels have returned to normal. In patients with severe pancreatitis associated with significant necrosis, the pancreas may not have the capacity to release large amounts of enzymes into the circulation. With more severe dis-ease, there is also hemoconcentration from third space fluid loss, and this can affect the serum

1	not have the capacity to release large amounts of enzymes into the circulation. With more severe dis-ease, there is also hemoconcentration from third space fluid loss, and this can affect the serum concentration of amylase.The clinical signs of acute pancreatitis include abdominal tenderness, often with signs of peritonitis in the upper abdomen. Rarely, pancreatic fluid and bleeding from the pancreas into the retroperitoneum may result in a bruise-like discoloration around the umbilicus (Cullen’s sign) or in the flanks (Grey Turner’s sign). Another rare sign is tetany as a result of hypocalcaemia. In addition to hemoconcentration, patients with acute pancreati-tis often have azotemia with elevated blood urea nitrogen and creatinine levels, hyperglycemia, and hypoalbuminemia.Pain ManagementPain is the cardinal symptom of acute pancreatitis, and its relief is a clinical priority. There is a lack of high-quality evidence to guide the choice of analgesic. Because of unpredictable

1	is the cardinal symptom of acute pancreatitis, and its relief is a clinical priority. There is a lack of high-quality evidence to guide the choice of analgesic. Because of unpredictable absorption, analgesia should be administered intravenously, at least at the outset and before oral intake has been established. Those with mild pain can usually be managed with a nonsteroidal anti-inflammatory drugs (e.g., metamizole 2 g/8 h IV), while those with more severe pain are best managed with opioid analgesia (e.g., buprenorphine 0.3 mg/4 h IV). Administration of buprenorphine, pentazocine, procaine hydrochloride, and meperidine are all of value in controlling abdominal pain. Morphine is to be avoided because of its potential to cause sphincter of Oddi spasm.Predicting SeverityWhereas classification relates to the present or past severity of acute pancreatitis, prediction is about the future and ultimate severity and outcome of the patient. Accurately predicting acute

1	classification relates to the present or past severity of acute pancreatitis, prediction is about the future and ultimate severity and outcome of the patient. Accurately predicting acute Brunicardi_Ch33_p1429-p1516.indd 144401/03/19 6:44 PM 1445PANCREASCHAPTER 33Table 33-7Ranson’s prognostic signs of pancreatitisCriteria for acute pancreatitis not due to gallstonesAt admissionDuring the initial 48 h Age >55 y Hematocrit fall >10 points WBC >16,000/mm3 BUN elevation >5 mg/dL Blood glucose >200 mg/dL Serum calcium <8 mg/dL Serum LDH >350 IU/L Arterial PO2 <60 mmHg Serum AST >250 U/dL Base deficit >4 mEq/L Estimated fluid sequestration >6 LCriteria for acute gallstone pancreatitisAt admissionDuring the initial 48 h Age >70 y Hematocrit fall >10 points WBC >18,000/mm3 BUN elevation >2 mg/dL Blood glucose >220 mg/dL Serum calcium <8 mg/dL Serum LDH >400 IU/L Base deficit >5 mEq/L Serum AST >250 U/dL Estimated fluid sequestration >4 LNote: Fewer than three positive criteria predict

1	mg/dL Blood glucose >220 mg/dL Serum calcium <8 mg/dL Serum LDH >400 IU/L Base deficit >5 mEq/L Serum AST >250 U/dL Estimated fluid sequestration >4 LNote: Fewer than three positive criteria predict mild, uncomplicated disease, whereas more than six positive criteria predict severe disease with a mortality risk of 50%.Abbreviations: AST = aspartate transaminase; BUN = blood urea nitrogen; LDH = lactate dehydrogenase; PO2 = partial pressure of oxygen; WBC = white blood cell count.Data from Ranson JHC. Etiological and prognostic factors in human acute pancreatitis: a review. Am J Gastroenterol. 1982;77:633 and from Ranson JH, Rifkind KM, Roses DF, et al. Prognostic signs and the role of operative management in acute pancreatitis. Surg Gynecol Obstet. 1974;139:69.pancreatitis severity is important in making triage decisions about whether a patient should be transferred to a tertiary hospital or an intensive care unit and in making decisions about fluid therapy and whether an ERCP is

1	important in making triage decisions about whether a patient should be transferred to a tertiary hospital or an intensive care unit and in making decisions about fluid therapy and whether an ERCP is indicated, as well as other issues.65 There is a very long history of attempts to find prognostic or predictive markers that accurately stratify the risk, with the most widely used being the Ranson’s criteria (Table 33-7) or modified Glasgow criteria. Both use clinical and biochemical parameters scored over the first 48 hours of admission. When there are three or more positive criteria, the disease is considered “predicted severe.” There are many other approaches to predicting severity. At 24 hours after admission an APACHE II score of 8 or more or a serum C-reactive protein level of >150 mg/dL has a similar accuracy in predicting severity as Ranson’s criteria.66 The more recently proposed Bedside Index for Severity of Acute Pancreatitis (BISAP) is calculated from blood urea nitrogen (> 25

1	has a similar accuracy in predicting severity as Ranson’s criteria.66 The more recently proposed Bedside Index for Severity of Acute Pancreatitis (BISAP) is calculated from blood urea nitrogen (> 25 mg/dL), impaired mental status (GCS <15), presence of systemic inflammatory response syndrome, age >60 years, and pleural effusion. Although it has the advantage of simplicity and can be performed within the first 24 hours of admission, it performed no better than other predictors.67 The presence of SIRS also has prognostic significance.68 There remains some controversy as to how important obesity is as a risk factor for severe and critical acute pancreatitis.69 Another approach has been taken in seeking to predict those with the “harmless acute pancreatitis score”70 using three factors that can be determined on admission: absence of rebound tenderness or guarding, normal hematocrit, and normal serum creatinine. The accuracy of this approach appears to be over 90% and triages most patients

1	be determined on admission: absence of rebound tenderness or guarding, normal hematocrit, and normal serum creatinine. The accuracy of this approach appears to be over 90% and triages most patients away from intensive care.Unfortunately, these and many other single and combined predictors of severity have an accuracy of around 70%66. This means that there is misclassification error of 30% that limits the value in predicting the severity of acute pancreatitis in indi-vidual patients. In the absence of any new biomarkers of pancre-atitis severity, making better use of existing predictors through sequencing tests, combining tests, or using artificial neural network methodologies has shown some promise.71 Scoring systems should augment clinical judgment, but not replace it.64 Over the first 2 to 3 days the clinician must be alert to patients with an elevated BUN or creatinine and/or persistent SIRS after adequate fluid resuscitation because these patients are at risk of developing severe

1	to 3 days the clinician must be alert to patients with an elevated BUN or creatinine and/or persistent SIRS after adequate fluid resuscitation because these patients are at risk of developing severe acute pancreatitis.64Classification of SeverityAccurately classifying or staging acute pancreatitis severity is important for clinical decision-making, communication, and enrolment into trials. The wide spectrum of pancreatitis severity was not captured in the previous binary classifica-tions (mild or severe). The key determinants of severity are local complications (absent, sterile, or infected) and systemic complications (absent, transient organ failure, persistent organ failure).25,72 Two classification systems have recently been proposed: the three grades (mild, moderately severe, and severe) of the Revised Atlanta Criteria (RAC)51 and the four categories (mild, moderate, severe, critical) of the Determi-nants Based Classification (DBC)73 (Table 33-8). The DBC Table 33-8Definitions for

1	of the Revised Atlanta Criteria (RAC)51 and the four categories (mild, moderate, severe, critical) of the Determi-nants Based Classification (DBC)73 (Table 33-8). The DBC Table 33-8Definitions for the classification of acute pancreatitis severity according to Revised Atlanta Classification11 and the Determinants Based Classification14. (Transient organ failure has a duration of <48 hours, persistent organ failure has duration of >48 hours.)Determinant Based Classification (2012)Revised Atlanta Classification (2013)MildNo local complicationNo systemic complicationNo local complicationNo systemic complicationModerateSterile local complication and/orTransient organ failureLocal or systemic complications without persistent organ failureorexacerbation of preexisting comorbiditySevereinfected local complicationorPersistent organ failurePersistent organ failure (single or multiple)CriticalInfected local complication and Persistent organ failure-Brunicardi_Ch33_p1429-p1516.indd

1	local complicationorPersistent organ failurePersistent organ failure (single or multiple)CriticalInfected local complication and Persistent organ failure-Brunicardi_Ch33_p1429-p1516.indd 144501/03/19 6:44 PM 1446SPECIFIC CONSIDERATIONSPART IITable 33-9The Modified Determinant Based Classification (MDBC) (Acevedo) of acute pancreatitis severity compared with the Revised Atlanta Classification (RAC) and Determinants Based Classification (DBC). Note that the DBC has a narrower definition for local complications than RAC, leading to a slightly broader range of mild acute pancreatitis in this table.RACMild(No OF, No LC)Moderately severe(TOF and/or LC)Severe(POF)DBCMild(No OF, No LC)Moderate(TOF and/or SN)Severe(POF or IN)Critical(POF and IN)MDBC ExcludedGroup 1(TOF and/or SN)Group 2(IN without POF)Group 3(POF without IN)Group 4(POF and IN)Abbreviations: OF = organ failure; LC = local complication; TOF = transient organ failure; POF = persistent organ failure; SN = sterile necrosis; IN

1	POF)Group 3(POF without IN)Group 4(POF and IN)Abbreviations: OF = organ failure; LC = local complication; TOF = transient organ failure; POF = persistent organ failure; SN = sterile necrosis; IN = infected necrosis.was developed on the principle of casual inference, derived by meta-analysis, and refined by an international multidis-ciplinary process,73 and both classifications have been inde-pendently validated, which suggests that they are broadly equivalent. More recently a prospective multicenter study has modified the DBC to address the ongoing issue that patients considered to have severe acute pancreatitis represent several subgroups with different morbidity, mortality, and interven-tion profiles74 (Table 33-9).The classification of patient severity is helpful in tracking the clinical trajectory of a patient, and it can be applied on a daily or more frequent basis. It can also be used in retrospect for audit purposes.Determining EtiologyThe history of alcohol ingestion must be

1	trajectory of a patient, and it can be applied on a daily or more frequent basis. It can also be used in retrospect for audit purposes.Determining EtiologyThe history of alcohol ingestion must be ascertained and preferably confirmed with blood ethanol levels. Gallstones should be investigated by ultrasonography. A gallstone etiology is more likely in females over the age of 50 with an elevation of alkaline phosphatase (>300 iu/L), alanine transferase (>100 iu/L), and amylase (>4000 iu/L). In the absence of gallstones and alcohol, a systematic approach to the identification of another factor will include taking a history of drugs, trauma, ERCP, infection, and measuring serum triglycerides, calcium, and others (see Table 33-4).Fluid ResuscitationFluid therapy to restore and maintain circulating blood volume is the most important intervention in the early management of acute pancreatitis75. However, a recent systematic review has shown that the evidence base for fluid therapy is

1	circulating blood volume is the most important intervention in the early management of acute pancreatitis75. However, a recent systematic review has shown that the evidence base for fluid therapy is scant,76 and most recommendations are based on expert opinion.64 It is not known which fluid to give, how aggressively to adminis-ter it, or what goal to use to guide and monitor the response to it. While there are proponents for vigorous fluid therapy (5–10 mL per kilogram per hour), especially in the first 24 hours, and for specific resuscitation goals,77 it is prob-ably best to resuscitate with a balanced crystalloid and aim to restore normal blood volume, blood pressure, and urine output. In one study, lactated Ringer’s solution was supe-rior to normal saline in reducing the systemic inflammatory response.78 Caution needs to be exercised in those with car-diac and renal disease and in the elderly, where the risks of over-resuscitation are greater.Nutritional SupportIn contrast to

1	inflammatory response.78 Caution needs to be exercised in those with car-diac and renal disease and in the elderly, where the risks of over-resuscitation are greater.Nutritional SupportIn contrast to analgesia and fluid therapy, there is a sound evidence base for nutritional support in acute pancreatitis. It is no longer acceptable to “rest the pancreas” by avoiding enteral nutrition, now the mainstay of nutritional support.79 Parenteral nutrition is now known to be more expensive, riskier, and not more effective than enteral nutrition and should only be offered if the patient’s calculated nutritional requirements cannot be achieved by the enteral route. Early initiation of enteral nutrition (within the first 24 hours of admission) is not superior to delaying an oral diet until 72 hours.80 If this is not tolerated over 48 to 72 hours, then nasogastric tube feedings can be started and increased in step-wise fashion over 2 to 3 days.81 The tube can be advanced to the jejunum, by

1	hours.80 If this is not tolerated over 48 to 72 hours, then nasogastric tube feedings can be started and increased in step-wise fashion over 2 to 3 days.81 The tube can be advanced to the jejunum, by endoscopy or fluoroscopy, if there is evidence of feeding intolerance. A delay in commencing enteral nutrition may contribute to the development of intestinal ileus and feeding intolerance, but aggressive early enteral feeding, particularly before adequate resuscitation, may put the patient at risk of nonocclusive mesenteric ischemia. There is no evidence to support the use of elemental or immune-enhancing formulas over standard polymeric formulas.82 In predicted mild acute pancreatitis the recommencement of oral fluids and then food was delayed until resolution of abdominal pain and normalization of serum levels of amylase, but it appears safe to allow patients to resume intake ad libitum (i.e., patient-controlled nutrition). If after 3 to 5 days there is evidence of feeding

1	and normalization of serum levels of amylase, but it appears safe to allow patients to resume intake ad libitum (i.e., patient-controlled nutrition). If after 3 to 5 days there is evidence of feeding intolerance, tube feeding should be commenced.Cross-Sectional ImagingIt may be necessary to perform a CT scan to diagnose acute pancreatitis in patients who are severely ill or in those presenting with undifferentiated abdominal pain. But there is no advantage in using CT scanning to predict the severity of acute pancreatitis.51,63 The primary purpose of cross-sectional imaging is the diagnosis of local complications; in particular, the development and extent of pancreatic necrosis and the different collections (see Table 33-5). CT scanning is also important to guide the insertion of percutaneous drains, now assuming a greater role in the management of the local complications (discussed later). Magnetic resonance imaging (MRI) is superior to CT scanning in detecting any solid content

1	drains, now assuming a greater role in the management of the local complications (discussed later). Magnetic resonance imaging (MRI) is superior to CT scanning in detecting any solid content within collections (Figure 33-13). And when a bleed is suspected, in association with a local complication, an arterial phase CT scan (CTa) is useful in detecting a pseudoaneurysm, active bleeding, and/or hematoma.3Brunicardi_Ch33_p1429-p1516.indd 144601/03/19 6:44 PM 1447PANCREASCHAPTER 33ABFigure 33-13. Corresponding computed tomography (CT) (A) and MR (B) images of a patient with a symptomatic pseudocyst. CT image reveals a well circumscribed homogenous collection (arrows) exerting mass effect on antrum of stomach. The T2-weighted MR image clearly distinguishes necrotic pancreas (black arrows) from fluid (white arrows). (Reproduced with permission from Bollen TL: Imaging of acute pancreatitis: update of the revised Atlanta classification, Radiol Clin North Am. 2012

1	pancreas (black arrows) from fluid (white arrows). (Reproduced with permission from Bollen TL: Imaging of acute pancreatitis: update of the revised Atlanta classification, Radiol Clin North Am. 2012 May;50(3):429-445.)Therapeutic Endoscopic Retrograde CholangiopancreatographyRandomized trials have demonstrated that early ERCP (within 24 or 48 hours of admission) reduce complications, but not mortality, in patients with predicted severe gallstone associated acute pancreatitis. While the benefits of this inva-sive modality are clear (e.g., treatment of cholangitis and release of impacted stone), this can be offset by the risks of increasing the severity of pancreatitis, bleeding, cholan-gitis, and perforation. More recent evidence has suggested that early ERCP confers no benefit in the absence of con-comitant cholangitis,83 as the offending common duct stone usually passes before ERCP can be performed. This may be evidenced by improvement in the liver function tests over the first 2 to

1	of con-comitant cholangitis,83 as the offending common duct stone usually passes before ERCP can be performed. This may be evidenced by improvement in the liver function tests over the first 2 to 3 days. If there is persistent cholestasis, an MRCP can be used to detect a common duct stone and can be used as a prerequisite for attempting an ERCP.84 Persistent cholesta-sis without cholangitis may require an ERCP but not usually in the acute setting.AntibioticsAlthough the use of broad-spectrum antibiotics to treat estab-lished infection in acute pancreatitis is a well-established prac-tice, there has been considerable controversy surrounding the use of prophylactic antibiotics.63 The overuse of antibiotics has been associated with a documented rise in fungal infec-tions and resistant organisms. Overall, it appears that the most recent and generally better designed studies do not support the use of prophylactic antibiotics to reduce the frequency of pancreatic infectious complications,

1	Overall, it appears that the most recent and generally better designed studies do not support the use of prophylactic antibiotics to reduce the frequency of pancreatic infectious complications, surgical intervention, and death.85Managing Local ComplicationsVigilance is required for the timely and accurate diagnosis of local complications. The decisions regarding how and when to intervene are often difficult. While guided by the information gained by cross-sectional imaging, the decision to intervene is based on the clinical status and trajectory of the patient and the poor response to maximal intensive care support. This means close monitoring of the patient by serial examination, supplemented by regular measurement of inflammatory markers (e.g., C-reactive protein) and a pancreatic protocol CT scan if a local complication is suspected and intervention considered warranted. In practice, intervention is delayed in order to allow demarcation and to reduce the risk of bleeding,

1	protocol CT scan if a local complication is suspected and intervention considered warranted. In practice, intervention is delayed in order to allow demarcation and to reduce the risk of bleeding, disseminated infection, and collateral damage to adjacent organs by an intervention (Figure 33-14). Appreciation of this has resulted in a notable trend toward delayed Figure 33-14. Operative view of infected acute pancreatitis. Peripancreatic infection, characterized by mucopurulent exu-date, extends far beyond the boundaries of the pancreas in the retroperitoneum.Brunicardi_Ch33_p1429-p1516.indd 144701/03/19 6:44 PM 1448SPECIFIC CONSIDERATIONSPART IIStomachPeritoneal spacePeripancreaticfluid collectionwith necrotic tissueEndoscope(with balloon dilatation)PancreasRPOSTERIORLATERAL VIEWNecrotic tissuein snareLStomachSpleenPeripancreaticfluid collectionwith necrotic tissueAccess toretroperitonealspaceCostal marginPancreasPancreasLiverNecrotictissueLong

1	VIEWNecrotic tissuein snareLStomachSpleenPeripancreaticfluid collectionwith necrotic tissueAccess toretroperitonealspaceCostal marginPancreasPancreasLiverNecrotictissueLong graspingforcepsLaparoscopeStomachSpleenPeripancreaticfluid collectionwith necrotic tissueEndoscopeStomach wallFluid collectionAortaPOSTERIORFigure 33-15. Two minimally invasive interventions for local complications of acute pancreatitis: A. video-assisted retroperitoneal debridement and B. endoscopic transgastric necrosectomy. (Reproduced with permission from Bakker OJ, van Santvoort HC, van Brunschot S, et al: Endoscopic transgastric vs surgical necrosectomy for infected necrotizing pancreatitis: a randomized trial, JAMA. 2012 Mar 14; 307(10):1053-1061.)intervention, now uncommon before 3 to 4 weeks from the onset of symptoms. An important emerging approach is the increasing use of percutaneous catheter drainage in patients with suspected infected collections.86 Fine-needle aspiration is now rarely used to confirm

1	An important emerging approach is the increasing use of percutaneous catheter drainage in patients with suspected infected collections.86 Fine-needle aspiration is now rarely used to confirm infection because the insertion of a needle at the time of planned drainage allows confirmation of the suspected infection. Preemptive drainage with one or more catheters often produces improvement or stabilization of the patient’s overall clinical status.87 In this way, drainage “buys time” and allows the lesion to become more walled off and safer to treat. Recent data suggests that primary percutaneous catheter drainage may be the only intervention required in a third to a half of patients and that this proportion might increase further if there were a policy of regular catheter exchange, upsizing, and irrigation.87 A proportion of patients do, however, require further treatment when they fail to respond and there is a wide array of minimally invasive options to choose from88 (Figure 33-15).

1	and irrigation.87 A proportion of patients do, however, require further treatment when they fail to respond and there is a wide array of minimally invasive options to choose from88 (Figure 33-15). These interventions can be classified on the basis of the method of visualization, route taken to the lesion, and the purpose of the intervention.89 In practice, the approach taken will depend on local expertise and equipment as well as the location and type of the specific local complication. A large Dutch randomized trial has shown that open surgical techniques should only be considered in those who fail to respond to the step-up approach, that is, prior percutaneous drainage and minimally invasive intervention.90 The exception is to be found in the rare situation where an abdominal compartment syndrome requires open decompression, but this is usually earlier than the optimal time to intervene for local complications. A recent landmark randomized trial has compared two minimally invasive

1	syndrome requires open decompression, but this is usually earlier than the optimal time to intervene for local complications. A recent landmark randomized trial has compared two minimally invasive techniques, endoscopic transgastric drainage, and the videoscope-assisted retroperitoneal debridement through a flank incision (see Figure 33-15). The data shows that the former approach is superior, although the latter has a role when the walled-off necrosis is remote from the stomach or duodenum, as in the left flank.91The management of an acute noninfected pseudocyst is usually conservative, as about half of these will resolve sponta-neously. When symptoms of pain or the inability to eat persist or infection occurs, intervention is required. The indications for intervention are therefore no longer based on size and duration alone. Pseudocysts persist because of communication with the main pancreatic duct and/or distal ductal stenosis. Percutaneous drainage should be avoided in this

1	no longer based on size and duration alone. Pseudocysts persist because of communication with the main pancreatic duct and/or distal ductal stenosis. Percutaneous drainage should be avoided in this situation because of the risk of external pancreatic fistula.92 EUS-guided internal drainage into stomach or duodenum or transpapillary stenting is the pre-ferred approach.Managing Organ FailureThe specific management of multiple organ failure is beyond the scope of this chapter. The early identification of organ dysfunc-tion and failure is important because it is a key determinant of severity and outcome and to facilitate the timely transfer of the patient to an intensive care unit to optimize management, pro-vide organ support and allow more intensive monitoring. The severity of organ failure can be scored (see Table 33-6). The responsiveness of organ failure to resuscitation over the first 48 hours is an important prognostic clue; those that respond have transient organ failure and have

1	can be scored (see Table 33-6). The responsiveness of organ failure to resuscitation over the first 48 hours is an important prognostic clue; those that respond have transient organ failure and have a better outlook than those who do not respond and have persistent organ failure.93 Organ failure that develops later in the disease course is usually Brunicardi_Ch33_p1429-p1516.indd 144801/03/19 6:44 PM 1449PANCREASCHAPTER 33Table 33-10Algorithm for the evaluation and management of acute pancreatitis 1. Diagnosis • History of abdominal pain consistent with acute pancreatitis • >3x elevation of pancreatic enzymes • CT scan if required to confirm diagnosis 2. Initial assessment/management (first 4 hrs) • Analgesia • Fluid resuscitation • Predict severity of pancreatitis • Ranson’s criteria • HAPS score • Assess systemic response • SIRS score • SOFA (organ failure) 3. Reassessment/management (4 to 6 hrs) • Assess response to fluid resuscitation • mean arterial pressure • heart

1	criteria • HAPS score • Assess systemic response • SIRS score • SOFA (organ failure) 3. Reassessment/management (4 to 6 hrs) • Assess response to fluid resuscitation • mean arterial pressure • heart rate • urine output • hematocrit • Determine etiology • Ultrasound for gallstones/sludge • History of alcohol consumption • Laboratory evaluation of other causes • MRCP and/or Urgent ERCP if concomitant cholangitis is present • not for cholestasis or predicted severe disease per se • Transfer to ICU or specialist center as needed • Deterioration or failure to respond to initial management • Intensive support for persistent organ failure • Commence enteral nutrition • Once normovolemia restored (usually after 6 hours) • Commence via NG tube if no gastric stasis • No prophylactic antibiotics or probiotics 4. Conservative management and monitoring (at least daily) • Clinical evaluation • Assess cardiovascular, respiratory, and renal function • Detect peritonitis and abdominal compartment

1	or probiotics 4. Conservative management and monitoring (at least daily) • Clinical evaluation • Assess cardiovascular, respiratory, and renal function • Detect peritonitis and abdominal compartment syndrome • Daily C-reactive protein • Classify severity (mild, moderate, severe, critical) • Detect intolerance of NG EN • Advance tube for NJ feeding if needed • Consider supplemental parenteral nutrition by day 4 5. Indications for “pancreatic protocol CT scan” (rarely in first week) • For significant clinical deterioration and elevated CRP • For suspicion of local pancreatic complications • For suspected bowel ischemia • For acute bleeding (CTa) (if stable enough and consider embolization) • For abdominal compartment syndrome 6. Invasive intervention • For deteriorating patient with suspected infected local complication • “Step up approach” with initial drain guided by current CT scan (percutaneous or endoscopic drainage) • Delay for 3 to 4 weeks with intensive care support, if

1	suspected infected local complication • “Step up approach” with initial drain guided by current CT scan (percutaneous or endoscopic drainage) • Delay for 3 to 4 weeks with intensive care support, if possible • If failure to respond or secondary deterioration, repeat CT scan, and select appropriate minimally invasive technique based on available expertise and equipment • Video-assisted retroperitoneal debridement or percutaneous nephroscopic debridement • Endoscopic transluminal debridement • Ongoing large bore drainage and irrigation 7. Indication for laparotomy • Failed “step-up approach” for further debridement/drainage • Acute abdomen (perforation or ischemia) • Severe abdominal compartment syndrome (rarely)secondary to infection of a local complication and should be managed accordingly (see Figure 33-9).CholecystectomyWhile it is widely accepted that cholecystectomy is essential to prevent recurrent gallstone associated pancreatitis, the ques-tion relates to the timing of it.

1	(see Figure 33-9).CholecystectomyWhile it is widely accepted that cholecystectomy is essential to prevent recurrent gallstone associated pancreatitis, the ques-tion relates to the timing of it. Index cholecystectomy, done in the same admission and prior to discharge, appears safe and can almost always be accomplished laparoscopically.94 But index cholecystectomy is not suitable for all patients, particu-larly some who have had local pancreatic complications, which includes a large inflammatory mass that extends into the porta hepatis. These patients may require an interval cholecystec-tomy after resolution of the inflammatory process. If surgery is required for the management of local complications, then a cholecystectomy is often performed at that time.DiabetesRecent evidence indicates that prediabetes and diabetes are com-mon after acute pancreatitis and occur in nearly 40% of patients after hospital discharge.95 The prevalence of newly diagnosed diabetes is much higher after acute

1	prediabetes and diabetes are com-mon after acute pancreatitis and occur in nearly 40% of patients after hospital discharge.95 The prevalence of newly diagnosed diabetes is much higher after acute pancreatitis (23%) than the prevalence of diabetes in the general population (4–9%). The risk of diabetes increases by at least twofold after 5 years as compared with 12 months. Interestingly, the severity of acute pancreatitis appears to have minimal effect on risk of diabetes.95 The implica-tion is that patients recovered from an attack of acute pancreatitis may need follow-up and screening for glucose intolerance.The management of acute pancreatitis remains a formidable challenge (Table 33-10) due to the variety and severity of the many associated complications (Table 33-11), and continues to evolve. Although specific treatments for acute pancreatitis remain elusive, progress has been made in the management of pain, fluid resuscitation, antibiotic prophylaxis, enteral nutrition,

1	to evolve. Although specific treatments for acute pancreatitis remain elusive, progress has been made in the management of pain, fluid resuscitation, antibiotic prophylaxis, enteral nutrition, therapeutic ERCP, and cholecystectomy. Progress has also been made in the intensive care management of systemic complications and in the development of less invasive interventions for the treatment of local complications, particularly infected pancreatic necrosis.4Brunicardi_Ch33_p1429-p1516.indd 144901/03/19 6:44 PM 1450SPECIFIC CONSIDERATIONSPART IITable 33-11Complications of acute pancreatitis I. Local A. Pancreatic phlegmon B. Pancreatic abscess C. Pancreatic pseudocyst D. Pancreatic ascites E. Involvement of adjacent organs, with hemorrhage, thrombosis, bowel infarction, obstructive jaundice, fistula formation, or mechanical obstruction II. Systemic A. Pulmonary 1. Pneumonia, atelectasis 2. Acute respiratory distress syndrome 3. Pleural

1	thrombosis, bowel infarction, obstructive jaundice, fistula formation, or mechanical obstruction II. Systemic A. Pulmonary 1. Pneumonia, atelectasis 2. Acute respiratory distress syndrome 3. Pleural effusion B. Cardiovascular 1. Hypotension 2. Hypovolemia 3. Sudden death 4. Nonspecific ST-T wave changes 5. Pericardial effusion C. Hematologic 1. Hemoconcentration 2. Disseminated intravascular coagulopathy D. GI hemorrhage 1. Peptic ulcer 2. Erosive gastritis 3. Portal vein or splenic vein thrombosis with varices E. Renal 1. Oliguria 2. Azotemia 3. Renal artery/vein thrombosis F. Metabolic 1. Hyperglycemia 2. Hypocalcemia 3. Hypertriglyceridemia 4. Encephalopathy 5. Sudden blindness (Purtscher’s retinopathy) G. Central nervous system 1. Psychosis 2. Fat emboli 3. Alcohol withdrawal syndrome H. Fat necrosis 1. Intra-abdominal saponification 2. Subcutaneous tissue necrosisReproduced with permission from Isselbacher KJ: Harrison’s Principles of Internal Medicine, 13th ed. New York, NY:

1	necrosis 1. Intra-abdominal saponification 2. Subcutaneous tissue necrosisReproduced with permission from Isselbacher KJ: Harrison’s Principles of Internal Medicine, 13th ed. New York, NY: McGraw-Hill Education; 1994.CHRONIC PANCREATITISDefinition, Incidence, and PrevalenceChronic pancreatitis is an incurable, chronic inflammatory con-dition that is multifactorial in its etiology, highly variable in its presentation, and a challenge to treat successfully. Autopsy studies indicate that evidence of chronic inflammation, such as fibrosis, duct ectasia, and acinar atrophy is seen in up to 5% of the population,96 although these data are difficult to interpret because many of these changes are also present in asymptomatic elderly patients.97 Population studies suggest a prevalence that ranges from 5 to 40 persons per 100,000 population, with con-siderable geographic variation.98 Differences in diagnostic crite-ria, regional nutrition, alcohol consumption, and medical access account for

1	from 5 to 40 persons per 100,000 population, with con-siderable geographic variation.98 Differences in diagnostic crite-ria, regional nutrition, alcohol consumption, and medical access account for variations in the frequency of the diagnosis, but the overall incidence of the disease has risen progressively over the past 50 years.EtiologyThere are multiple etiologies of chronic pancreatitis, includ-ing genetic mutations, alcohol exposure, duct obstruction due to trauma, gallstones, and tumors, metabolic diseases such as hyperlipidemia and hyperparathyroidism, and auto-immune dis-ease. In addition, nutritional causes include so-called tropical pancreatitis, which has been thought to result from ingestion of certain starches. A significant number of patients have no discernible cause of the disease despite extensive testing, and are said to have “idiopathic” chronic pancreatitis.Genetic CausesIn 1952, Comfort and Steinberg reported a kindred of “heredi-tary chronic relapsing

1	of the disease despite extensive testing, and are said to have “idiopathic” chronic pancreatitis.Genetic CausesIn 1952, Comfort and Steinberg reported a kindred of “heredi-tary chronic relapsing pancreatitis” after treating the proband, a 24-year-old woman, at the Mayo Clinic.99 Subsequently, familial patterns of chronic, nonalcoholic pancreatitis have been described worldwide, and a familiar pattern has emerged. Typically, patients first present in childhood or adolescence with abdominal pain and are found to have chronic calcific pan-creatitis on imaging studies. Progressive pancreatic dysfunction is common, and many patients present with symptoms due to pancreatic duct obstruction. The risk of subsequent carcinoma formation is increased, reaching a prevalence, in some series, of 40%, but the age of onset for carcinoma is typically >50 years old.100 The disorder is characterized by an autosomal dominant pattern of inheritance, with 80% penetrance and variable expres-sion. The

1	40%, but the age of onset for carcinoma is typically >50 years old.100 The disorder is characterized by an autosomal dominant pattern of inheritance, with 80% penetrance and variable expres-sion. The incidence is equal in both sexes.Whitcomb and colleagues,101 and separately LeBodic and associates,102 performed gene-linkage analysis and identified a linkage for hereditary pancreatitis to chromosome 7q35. Subse-quently, the region was sequenced and revealed eight trypsino-gen genes. Mutational analysis revealed a missense mutation resulting in an Arg-to-His substitution at position 117 of the cat-ionic trypsinogen gene, or PRSS1, one of the primary sites for proteolysis of trypsin. This gain-of-function mutation results in an excess production of trypsinogen, which results in persistent and uncontrolled proteolytic activity and autodestruction within the pancreas.103 The position 117 mutation of PRSS1 and an addi-tional mutation, now known collectively as the R122H and N291 mutations

1	uncontrolled proteolytic activity and autodestruction within the pancreas.103 The position 117 mutation of PRSS1 and an addi-tional mutation, now known collectively as the R122H and N291 mutations of PRSS1, account for about two-thirds of cases of hereditary pancreatitis. Masson and associates described a gain-of-function mutation in the anionic trypsinogen gene, PRSS2, that is also present in some cases.104Similarly, SPINK1, an inflammation-induced trypsin inhibitor secreted in acinar cells, has been found to have a role in hereditary pancreatitis. SPINK1 specifically inhibits trypsin action by competitively blocking the active site of the enzyme. Witt and colleagues investigated96 unrelated children with chronic pancreatitis in Germany and found a variety of SPINK1 mutations in 23% of the patients.105 Several studies have now confirmed an association of loss-of-function SPINK1 mutations with familial and idiopathic forms of chronic pancreatitis, as well as so-called tropical

1	of the patients.105 Several studies have now confirmed an association of loss-of-function SPINK1 mutations with familial and idiopathic forms of chronic pancreatitis, as well as so-called tropical pancreatitis.106,107 SPINK1 mutations Brunicardi_Ch33_p1429-p1516.indd 145001/03/19 6:44 PM 1451PANCREASCHAPTER 33BasolateralmembraneAcinar callKeyZymogensClaudin-2CFTRCalcium regulationCAZymogens: Including;trypsinogens (PRSS1, PRSS2)chymotrypsin C (CTRC)(SPINK1 in“ammation-induced trypsin inhibitor)Calcium sensing receptor (CASR)CFTRDuodenumZymogensecretionAbnormalclaudin-2localizationDuct cellBicarbonateSodiumWaterTrypsinogenZymogensEKTrypsinActive digestiveenzymesCASRFigure 33-16. Schematic model of genetic causes of chronic pan-creatitis. A pancreatic acinus is portrayed showing the pathway for digestive enzymes (zymogens) to be secreted by the acinar cell into the ductal system where water, sodium, and bicarbonate are secreted by the duct and centroacinar (CA) cells. Mutations in

1	for digestive enzymes (zymogens) to be secreted by the acinar cell into the ductal system where water, sodium, and bicarbonate are secreted by the duct and centroacinar (CA) cells. Mutations in at least five genes have been identified as risk factors for chronic pancreatitis: gain-of function mutations in the cationic trypsinogen genes (PRSS1, PRSS2) cause hereditary pancreatitis. Cationic and anionic trypsinogen are normally active in the duodenum by entero-kinase (EK). Premature activation of the trypsin in the acinar cell leads to zymogen activation, local cellular injury, and inflamma-tion. Mutations of the trypsin inhibitors SPINK1 or chymotrypsin C (CTRC), or of the calcium sensing receptor CASR, result in pre-mature activation of trypsinogen. Mutations in the cystic fibrosis transmembrane receptor CFTR results in trypsinogen stasis within the ducts due to insufficient secretion by duct cells. A mutation in claudin-2 (CLDN2) results in abnormal expression in acinar cells,

1	transmembrane receptor CFTR results in trypsinogen stasis within the ducts due to insufficient secretion by duct cells. A mutation in claudin-2 (CLDN2) results in abnormal expression in acinar cells, instead of its normal location between duct cells, and is associated with the accelerated development of chronic pancreatitis in alcohol abusers. (Modified with permission from Solomon S, Whitcomb DC: Genetics of pancreatitis: an update for clinicians and genetic coun-selors, Curr Gastroenterol Rep. 2012 Apr;14(2):112-711.)are common in the general population as well, and the frequency of these mutations varies in different cohorts of idiopathic chronic pancreatitis, from 6.4% in France108 to 25.8% in the United States.109 Thus, hereditary pancreatitis results from one or more mutational defects that incapacitate an auto-protective process that normally prevents proteolysis within the pancreas.Cystic fibrosis, originally termed cystic fibrosis of the pancreas, results from a variety of

1	that incapacitate an auto-protective process that normally prevents proteolysis within the pancreas.Cystic fibrosis, originally termed cystic fibrosis of the pancreas, results from a variety of mutations of the cystic fibro-sis transmembrane receptor (CFTR). The CFTR is present in pancreatic duct cells and controls the amount of chloride and bicarbonate secreted into the normally alkaline pancreatic juice. The CFTR gene contains over 4300 nucleotides, divided into 24 exons, which encode a 1480-amino acid protein. Over 1000 polymorphisms have been reported, and many are common. The CFTR mutation associated with the classic pulmonary dis-ease, F508, is rarely observed in chronic pancreatitis. But other CFTR mutations have been noted to be associated with chronic idiopathic pancreatitis, auto-immune pancreatitis, and pancreas divisum, in which the pulmonary, intestinal, and cutaneous manifestations of the disease are silent.110Many studies have been undertaken to determine whether

1	auto-immune pancreatitis, and pancreas divisum, in which the pulmonary, intestinal, and cutaneous manifestations of the disease are silent.110Many studies have been undertaken to determine whether specific genetic abnormalities are associated with alcoholic chronic pancreatitis and which might confer susceptibility to the disease.105 In 2012, a landmark study by Whitcomb and associates demonstrated a likely genetic cause of the predisposition to alcohol-induced chronic pancreatitis in men.111 In a genome-wide association study of more than 2000 patients, these researchers discovered that a common DNA variant on the X chromosome is present in 26% of men without pancreatitis, but jumps to nearly 50% of men diagnosed with alcoholic pancreatitis. The variant involves the claudin 2 (CLDN2) gene, which encodes a tight junction protein normally present in ductal cells. In cases of chronic pancreatitis, the CLDN2 protein is abnormally expressed in acinar cells and may alter the secretory

1	gene, which encodes a tight junction protein normally present in ductal cells. In cases of chronic pancreatitis, the CLDN2 protein is abnormally expressed in acinar cells and may alter the secretory dynamics of enzyme release. The abnormality does not appear to cause pancreatitis, but if pancreatitis occurs for any reason in a person with the CLDN2 variant, it is more likely that the person will develop chronic pancreatitis; the risk is increased even further among alcohol users. Only 10% of women have the X chromosome–linked variant on both X chromosomes, and most women with the CLDN2 variant on one X chromosome appear to be protected from alcoholic chronic pancreatitis by the other X chromosome, if it is normal. Men, with only one X chromosome, have no protection if they inherit a CLDN2 mutation. This helps to explain the high prevalence of alcoholic chronic pancreatitis among men, although the mechanism remains unclear. This study does not demonstrate a genetic cause for all cases

1	mutation. This helps to explain the high prevalence of alcoholic chronic pancreatitis among men, although the mechanism remains unclear. This study does not demonstrate a genetic cause for all cases of alcohol-related chronic pancreatitis, but it shows that a genetic element contributes to many patients with the disease (Fig. 33-16).AlcoholIn 1878, Friedreich proposed that “a general chronic interstitial pancreatitis may result from excessive alcoholism (drunkard’s pancreas).”112 Since that observation, numerous studies have shown that a causal relationship exists between alcohol and chronic pancreatitis, but the prevalence of alcohol as the etiol-ogy of the disease in Western countries ranges widely, from 38% to 94%113 (Fig. 33-17).There is a linear relationship between exposure to alcohol and the development of chronic pancreatitis.114 The risk of dis-ease is present in patients with even a low or occasional expo-sure to alcohol (1 to 20 g/d), perhaps due to the CLDN2 gene mutation

1	and the development of chronic pancreatitis.114 The risk of dis-ease is present in patients with even a low or occasional expo-sure to alcohol (1 to 20 g/d), perhaps due to the CLDN2 gene mutation described previously, so there is no threshold level of alcohol exposure below which there is no risk of develop-ing chronic pancreatitis. Furthermore, although the risk of dis-ease is dose related and highest in heavy (>150 g/d, or about 11 1 oz shots, or 12 beers per day) drinkers, the prevalence of chronic pancreatitis among confirmed alcohol abusers is only 5% to 15%.115 However, the duration of alcohol consumption is definitely associated with the development of pancreatic dis-ease. The onset of disease typically occurs between ages 35 to 40 years, after 16 to 20 years of heavy alcohol consumption. Recurrent episodes of acute pancreatitis are typically followed by chronic symptoms after 4 or 5 years.116In their 1946 classic study, Comfort, Gambrill, and Baggenstoss proposed that chronic

1	Recurrent episodes of acute pancreatitis are typically followed by chronic symptoms after 4 or 5 years.116In their 1946 classic study, Comfort, Gambrill, and Baggenstoss proposed that chronic pancreatitis was the result of multiple episodes of acute inflammation, with residual and Brunicardi_Ch33_p1429-p1516.indd 145101/03/19 6:44 PM 1452SPECIFIC CONSIDERATIONSPART IITimeDegree of pancreatic damage= Episode of acute pancreatitisNecrosis-fibrosis sequenceFigure 33-18. “Multiple hit” theory of the etiology of chronic pancreatitis. Multiple episodes of acute pancreatitis cause progres-sively more organized inflammatory changes that ultimately result in chronic inflammation and scarring. (Reproduced with permission from Apte MV, Wilson JS: Alcohol-induced pancreatic injury, Best Pract Res Clin Gastroenterol. 2003 Aug;17(4):593-612.)Very heavy drinking (15%)Alcohol + genetic (3%)Idiopathic(42%)Genetic(24%)CFTR(14%)CFTR +SPINK1(3%)SPINK1(4%)PRSS1(3%)Hyperlipidemia,Autoimmune,Other

1	Res Clin Gastroenterol. 2003 Aug;17(4):593-612.)Very heavy drinking (15%)Alcohol + genetic (3%)Idiopathic(42%)Genetic(24%)CFTR(14%)CFTR +SPINK1(3%)SPINK1(4%)PRSS1(3%)Hyperlipidemia,Autoimmune,Other (4%)Obstructive(9%)Gallstone /severe AP(3%)Figure 33-17. Etiologies of chronic pancreatitis. (Reproduced with permission from Whitcomb DC: Going MAD: development of a “matrix academic division” to facilitate translating research to personalized medicine, Acad Med. 2011 Nov;86(11):1353-1359.)progressively increasing chronic inflammation.117 Subsequently, Kondo and associates showed that other, additional factors were necessary for repeated exposure to alcohol to cause chronic pancreatitis.118 Regardless of the requirement for other predisposing or facilitative factors, the concept that multiple episodes (or a prolonged course) of pancreatic injury ultimately leads to chronic disease is widely accepted as the pathophysiologic sequence119 (Fig. 33-18).Although direct alcohol exposure to the

1	episodes (or a prolonged course) of pancreatic injury ultimately leads to chronic disease is widely accepted as the pathophysiologic sequence119 (Fig. 33-18).Although direct alcohol exposure to the pancreatic ductal system, or elevated levels of alcohol in the bloodstream, has been shown to alter the integrity and function of pancreatic ducts and acini directly,120 most investigators believe that alco-hol metabolites such as acetaldehyde, combined with oxidant injury, result in local parenchymal injury that is preferentially targeted to the pancreas in predisposed individuals. Repeated or severe episodes of toxin-induced injury activate a cascade of cytokines, which, in turn, induces pancreatic stellate cells (PSCs) to produce collagen and cause fibrosis (Fig. 33-19).A high-protein, low-bicarbonate, low-volume secretory output is seen after chronic alcohol exposure, which may con-tribute to the precipitation of proteins in secondary ducts in the early stages of chronic

1	low-bicarbonate, low-volume secretory output is seen after chronic alcohol exposure, which may con-tribute to the precipitation of proteins in secondary ducts in the early stages of chronic pancreatitis.121 Calcium is complexed to protein plugs in small ductules, secondary ducts, and, eventu-ally, in the main ductal system, which causes ductal cell injury and obstruction of the secretory system, which further promotes an inflammatory response.Cigarette smoking has been strongly associated with chronic pancreatitis,122 but until recently it was unclear whether this was a causative risk factor. Studies have now shown that smoking actually accelerates the development of alcoholic pancreatitis,123 and the risk of cancer in chronic pancreatitis is increased significantly by smoking. In hereditary pancreatitis, smoking has been found to lower the age of onset of carcinoma by about 20 years.124 Smoking therefore appears to be an inde-pendent risk factor for the late complications of

1	hereditary pancreatitis, smoking has been found to lower the age of onset of carcinoma by about 20 years.124 Smoking therefore appears to be an inde-pendent risk factor for the late complications of alcoholic pan-creatitis, if not an early cofactor for the development of fibrosis.HyperparathyroidismHypercalcemia is a known cause of pancreatic hypersecretion,125 and chronic hypercalcemia caused by untreated hyperparathyroidism is associated with chronic calcific pancreatitis.126 Hypercalcemia is also a stimulant for pancreatic calcium secretion, which contributes to calculus formation and obstructive pancreatopathy. The treatment is correction of the hyperparathyroidism and assessment of any additional endocrinopathies.HyperlipidemiaIn addition to the risk of acute pancreatitis, hyperlipidemia and hypertriglyceridemia predispose women to chronic pancreatitis when they receive estrogen replacement therapy.127 Fasting tri-glyceride levels <300 mg/dL are below the threshold for this to

1	and hypertriglyceridemia predispose women to chronic pancreatitis when they receive estrogen replacement therapy.127 Fasting tri-glyceride levels <300 mg/dL are below the threshold for this to occur, and the mechanism of estrogen potentiation of hyperlip-idemia-induced chronic pancreatitis is unknown. It is assumed that chronic changes occur after repeated subclinical episodes of acute inflammation. Aggressive therapy of hyperlipidemia is therefore important in perior postmenopausal patients who are candidates for estrogen therapy.ClassificationA major impediment to a better understanding of the etiology, frequency, and severity of chronic pancreatitis has been the dif-ficulty with which investigators and clinicians have struggled to identify a useful classification system. Multiple classification systems have been proposed. The TIGAR-O scheme catego-rizes chronic pancreatitis according to risk factors and etiolo-gies, such as (a) toxic-metabolic, (b) idiopathic, (c) genetic, (d)

1	systems have been proposed. The TIGAR-O scheme catego-rizes chronic pancreatitis according to risk factors and etiolo-gies, such as (a) toxic-metabolic, (b) idiopathic, (c) genetic, (d) autoimmune, (e) recurrent and severe acute pancreatitis, or (f) obstructive.128 A recent classification system based on his-topathology as well as etiology was delineated by Singer and Chari.129Chronic Calcific (Lithogenic) PancreatitisThis type is the largest subgroup in the classification scheme proposed by Singer and Chari and includes patients with calcific pancreatitis of most etiologies. Although the majority of patients with calcific pancreatitis have a history of alcohol abuse, stone formation and parenchymal calcification can develop in a variety of etiologic subgroups; hereditary pancreatitis and tropical pan-creatitis are particularly noteworthy for the formation of stone disease. The clinician should therefore avoid the assumption that calcific pancreatitis confirms the diagnosis of alcohol

1	tropical pan-creatitis are particularly noteworthy for the formation of stone disease. The clinician should therefore avoid the assumption that calcific pancreatitis confirms the diagnosis of alcohol abuse.Brunicardi_Ch33_p1429-p1516.indd 145201/03/19 6:44 PM 1453PANCREASCHAPTER 33Normal pancreasAlcoholMetabolic / oxidative stressSentinel eventAcinar cell injury(+ / – Necrosis)Inflammatory response:proinflammatory(Early)Anti-inflammatory (Later)Alcoholmetabolic/oxidative stressRecurrent acute pancreatitis(RAP)Anti-inflammatoryFibrosisHealedStressRAPNormal (Recovered):Normal:Stellate cells(Inactive)CytokinesChronicpancreatitis:FibrosisEarly acutepancreatitis: NeutrophilLymphocyteMacrophageStellate cell(Active)Collagen, etc.Late acutepancreatitis:M(Anti-inflam-matory)Necrosis = Stress, ETOHxxxxxxxxxxxxxFigure 33-19. The sentinel acute pancreatitis event (SAPE) hypothesis for the development of chronic pancreatitis. A critical episode of acute pancreatitis activates cytokine-induced

1	ETOHxxxxxxxxxxxxxFigure 33-19. The sentinel acute pancreatitis event (SAPE) hypothesis for the development of chronic pancreatitis. A critical episode of acute pancreatitis activates cytokine-induced transformation of pancreatic stellate cells, which results in collagen production and fibrosis. ETOH = ethyl alcohol. (Adapted with permission from Schneider A, Whitcomb DC: Hereditary pancreatitis: a model for inflammatory diseases of the pancreas, Best Pract Res Clin Gastroenterol. 2002 Jun;16(3):347-363.)Chronic Obstructive PancreatitisThis refers to chronic inflammatory changes that are caused by the compression or occlusion of the proximal ductal system by tumor, gallstone, posttraumatic scar, or inadequate duct caliber (as in pancreas divisum). Obstruction of the main pancreatic duct by inflammatory (posttraumatic) or neoplastic processes can result in diffuse fibrosis, dilated main and secondary pan-creatic ducts, and acinar atrophy. The patient may have little in

1	duct by inflammatory (posttraumatic) or neoplastic processes can result in diffuse fibrosis, dilated main and secondary pan-creatic ducts, and acinar atrophy. The patient may have little in Brunicardi_Ch33_p1429-p1516.indd 145301/03/19 6:44 PM 1454SPECIFIC CONSIDERATIONSPART IINormal pancreasAbsence of duct of SantoriniNormal pancreaswith duct of SantoriniPancreas divisumSmall ductof WirsungPancreas divisumNo duct of WirsungABCDFigure 33-20. Pancreas divisum. Normal pancreatic duct anatomy and the variations of partial or complete pancreas divisum are shown. (Reproduced with permission from Beger HG: The pancreas: an integrated textbook of basic science, medicine, and surgery. London: Blackwell-Science; 1998.)the way of pain symptoms or may present with signs of exo-crine insufficiency. Intraductal stone formation is rare, and both functional and structural abnormalities may improve when the obstructive process is relieved or removed. Trauma to the pan-creas frequently results in

1	Intraductal stone formation is rare, and both functional and structural abnormalities may improve when the obstructive process is relieved or removed. Trauma to the pan-creas frequently results in duct injury and leakage, which may result in pseudocyst formation as well as local scar formation. Inadequately treated pancreatic trauma may result in persistent inflammatory changes in the distal gland.130Pancreas divisum represents a special case of obstruc-tive pancreatitis. It is the most common congenital anomaly involving the pancreas and occurs in up to 10% of children. It is thought to predispose the pancreas to recurrent acute pan-creatitis and chronic pancreatitis, due to functional obstruction of a diminutive duct of Santorini that fails to communicate with Wirsung’s duct (Fig. 33-20). However, the classic pic-ture of obstructive pancreatopathy with a dilated dorsal duct is unusual in pancreas divisum, so a decompressive opera-tion or a lesser papilla sphincteroplasty is

1	33-20). However, the classic pic-ture of obstructive pancreatopathy with a dilated dorsal duct is unusual in pancreas divisum, so a decompressive opera-tion or a lesser papilla sphincteroplasty is frequently not fea-sible or unsuccessful. Endoscopic stenting through the lesser papilla may result in temporary relief of symptoms, and this response would increase the possibility that a permanent sur-gical or endoscopic intervention will be successful. Although some authors emphasize the pathologic implications of pan-creas divisum,131 others express skepticism that it represents a true risk to pancreatic secretory capacity or contributes to the development of chronic pancreatitis.132,133A recent French study reveals that pancreas divisum is equally prevalent among patients with idiopathic chronic pancreatitis and normal con-trols (7%), and is minimally increased in patients with PRSS1 and SPINK1 mutations. It is accompanied by CFTR mutations in 47% of patients, however, suggesting that

1	pancreatitis and normal con-trols (7%), and is minimally increased in patients with PRSS1 and SPINK1 mutations. It is accompanied by CFTR mutations in 47% of patients, however, suggesting that the presence of pancreas divisum together with mutational events may increase the susceptibility to pancreatitis.134Chronic Inflammatory PancreatitisChronic inflammatory pancreatitis is characterized by diffuse fibrosis and a loss of acinar elements with a predominant mono-nuclear cell infiltration throughout the gland.A variant of chronic pancreatitis is a nonobstructive, diffusely infiltrative disease associated with fibrosis, a mono-nuclear cell (lymphocyte, plasma cell, or eosinophil) infiltrate, and an increased titer of one or more autoantibodies.135 This type, referred to as autoimmune pancreatitis (AIP), is associated with a variety of illnesses with suspected or proven autoimmune etiology, such as Sjögren’s syndrome, rheumatoid arthritis, and type 1 diabetes mellitus. AIP has been

1	(AIP), is associated with a variety of illnesses with suspected or proven autoimmune etiology, such as Sjögren’s syndrome, rheumatoid arthritis, and type 1 diabetes mellitus. AIP has been characterized as either type I, with accompanying systemic or multiorgan dysfunction, or type II, which is restricted to the pancreas.Compressive stenosis of the intrapancreatic portion of the common bile duct is frequently seen in both types of AIP, along with symptoms of obstructive jaundice. Increased levels of serum β-globulin or immunoglobulin G4 are also present. Steroid therapy is uniformly successful in ameliorating the dis-ease, including any associated bile duct compression.136 CFTR mutations that result in dislocation of the transmembrane pro-tein have been found in AIP, and steroid therapy results in a nor-malization of the CFTR localization and a resumption of normal chloride and bicarbonate secretion.137 The differential diagnosis includes lymphoma, plasmacytoma (“pseudotumor” of the

1	results in a nor-malization of the CFTR localization and a resumption of normal chloride and bicarbonate secretion.137 The differential diagnosis includes lymphoma, plasmacytoma (“pseudotumor” of the pan-creas) and diffuse infiltrative carcinoma. Although the diagnosis is confirmed on pancreatic biopsy, presumptive treatment with steroids is usually undertaken, especially when clinical and lab-oratory findings, such as an elevation in IgG4 levels, support the diagnosis. Failure to obtain a cytologic specimen may lead to an unnecessary resectional procedure, and an untreated inflam-matory component may cause sclerosis of the extrahepatic or intrahepatic bile ducts, with eventual liver failure.138Brunicardi_Ch33_p1429-p1516.indd 145401/03/19 6:44 PM 1455PANCREASCHAPTER 33Tropical (Nutritional) PancreatitisChronic pancreatitis is highly prevalent among adolescents and young adults in Indonesia, southern India, and tropical Africa. Abdominal pain develops in adolescence, followed by

1	PancreatitisChronic pancreatitis is highly prevalent among adolescents and young adults in Indonesia, southern India, and tropical Africa. Abdominal pain develops in adolescence, followed by the development of a brittle form of pancreatogenic diabetes. Parenchymal and intraductal calcifications are seen, and the pancreatic duct stones may be quite large.139 Many of the patients appear malnourished, some present with extreme emaciation, and a characteristic cyanotic coloration of the lips may be seen.140 In addition to protein-caloric malnutrition, toxic products of some indigenous foodstuffs have also been thought to contribute to the disease. Because of the geographic concentration of this early-onset form of chronic pancreatitis, it has been termed tropical pancreatitis, although the exact etiology remains unclear.Clinically, tropical pancreatitis presents much like heredi-tary pancreatitis, and a familial pattern among cases is not unusual. SPINK1 mutations have been documented in

1	etiology remains unclear.Clinically, tropical pancreatitis presents much like heredi-tary pancreatitis, and a familial pattern among cases is not unusual. SPINK1 mutations have been documented in 20% to 55% of patients with tropical pancreatitis, and CFTR mutations have been reported as well.106,107 The accelerated deteriora-tion of endocrine and exocrine function, the chronic pain due to obstructive disease, and the recurrence of symptoms despite decompressive procedures characterize the course of disease. As immigrants from the tropical regions increasingly find their way to all parts of the world, an awareness of this severe form of chronic pancreatitis is helpful for those who treat patients with pancreatic disease.Asymptomatic Pancreatic FibrosisPancreatic fibrosis is seen in some asymptomatic elderly patients, in tropical populations, or in asymptomatic alcohol users. There is diffuse perilobar fibrosis and a loss of acinar cell mass, but there is not a main ductular component.

1	elderly patients, in tropical populations, or in asymptomatic alcohol users. There is diffuse perilobar fibrosis and a loss of acinar cell mass, but there is not a main ductular component. In addition, the presence of fibrosis and decreased exocrine function in patients with diabetes has raised the question of whether long-standing diabetes is a cause of chronic pancreatitis.141 Patients with this entity are usually asymptomatic in terms of typical pancreatic pain, and a recent histopathologic study of patients with typi-cal chronic pancreatitis and “diabetic exocrine pancreatopathy” reveals significant differences in morphology, including a virtual absence of duct distortion or obstruction (Fig. 33-21).142 It remains unknown whether this form of chronic inflammation precedes or contributes to the roughly twofold increase in the risk of pancreatic cancer in patients with long-standing diabetes.Idiopathic PancreatitisWhen a definable cause for chronic pancreatitis is lacking, the term

1	to the roughly twofold increase in the risk of pancreatic cancer in patients with long-standing diabetes.Idiopathic PancreatitisWhen a definable cause for chronic pancreatitis is lacking, the term idiopathic is used to categorize the illness. Classically, the idiopathic group includes young adults and adolescents who lack a family history of pancreatitis but who may represent indi-viduals with spontaneous gene mutations encoding regulatory proteins in the pancreas. A variable percentage of SPINK1 and CFTR mutations have been described in various studies. In addi-tion, the idiopathic group has included a large number of older patients for whom no obvious cause of recurrent or chronic pan-creatitis can be found.143 However, because the prevalence of biliary calculi increases steadily with age, it is not surprising that, as methods of biliary stone detection have improved, many elderly “idiopathic” pancreatitis patients are found to have bili-ary tract disease.144An increasing number of

1	it is not surprising that, as methods of biliary stone detection have improved, many elderly “idiopathic” pancreatitis patients are found to have bili-ary tract disease.144An increasing number of mutations of the SPINK1 and CFTR genes have been identified in association with various forms of chronic pancreatitis. However, the role of genetic analysis in the management of these patients remains unclear, as guidelines have yet to be developed to allow physicians to use the data consistently. Although the clinical management of patients who harbor a minor CFTR mutation and chronic pan-creatitis, for example, is still dictated by the clinical manifesta-tions of the pancreatitis, recent data suggest that the etiology of chronic pancreatitis, rather than the morphology, may determine the response to surgical treatment.145A shortcoming of these clinical classification systems is the lack of histologic criteria of chronic inflammation due to the usual absence of a biopsy specimen. The

1	response to surgical treatment.145A shortcoming of these clinical classification systems is the lack of histologic criteria of chronic inflammation due to the usual absence of a biopsy specimen. The differentiation of recurrent acute pancreatitis from chronic pancreatitis with exac-erbations of pain can be difficult to establish and is not facili-tated by the current system. Similarly, cystic fibrosis is known to cause fibrosis and acinar dysfunction but is not included in the classification despite increasing evidence for its possible role in idiopathic chronic pancreatitis.146 Therefore, further refinements in the classification system for chronic pancreatitis Interacinarfibrosis0102030405060708090100AcinaratrophyInterlobularfibrosisStromalinflammationDuctaldistortionDuctalepithelialattenuationIslet amyloidCPDEPFigure 33-21. Comparative frequencies of key histolopathologic features of chronic pancreatitis (CP n = 7) and diabetic exocrine pancrea-topathy (DEP n = 9) (Reproduced with

1	amyloidCPDEPFigure 33-21. Comparative frequencies of key histolopathologic features of chronic pancreatitis (CP n = 7) and diabetic exocrine pancrea-topathy (DEP n = 9) (Reproduced with permission from Majumder S, Zhang L, Philip N, et al. Exocrine Pancreatopathy (EP) Associated With Diabetes Mellitus (DM) Is Histologically Distinct From Chronic Pancreatitis (CP): An International Multi-Reader Blinded Study, Gastroenterol 2016 April;150(4) Suppl 1: S191 (abstr).)Brunicardi_Ch33_p1429-p1516.indd 145501/03/19 6:44 PM 1456SPECIFIC CONSIDERATIONSPART IIFigure 33-22. Histology of early chronic pancreatitis. High-power microscopic (40x) histology of chronic pancreatitis shows an infil-tration of mononuclear inflammatory cells throughout the inter-stitium of the pancreas, with little fibrosis. (Used with permission from Rhonda Yantiss, Weill Cornell Medical College.)Figure 33-23. Gross appearance of chronic pancreatitis. Areas of fibrosis and scarring are seen adjacent to other areas

1	(Used with permission from Rhonda Yantiss, Weill Cornell Medical College.)Figure 33-23. Gross appearance of chronic pancreatitis. Areas of fibrosis and scarring are seen adjacent to other areas within the gland in which the lobar architecture is grossly preserved. A dilated pancreatic duct indicates the presence of downstream obstruction in this specimen removed from a patient with chronic pancreatitis. (Used with permis-sion from Rhonda Yantiss, Weill Cornell Medical College.)Figure 33-24. Histology of severe chronic pancreatitis. High-power microscopic (40x) histologic appearance of advanced chronic pancre-atitis shows extensive sheets of fibrosis and loss of acinar tissue, with preservation of islet tissue in scattered areas. (Used with permission from Rhonda Yantiss, Weill Cornell Medical College.)are needed to allow a better prediction of its clinical course and a more accurate diagnosis of a likely etiologic agent.PathologyHistology. In early chronic pancreatitis, the histologic

1	College.)are needed to allow a better prediction of its clinical course and a more accurate diagnosis of a likely etiologic agent.PathologyHistology. In early chronic pancreatitis, the histologic changes are unevenly distributed and are characterized by induration, nodular scarring, and lobular regions of fibrosis (Fig. 33-22). As the disease progresses, there is a loss of normal lobulation, with thicker sheets of fibrosis surrounding a reduced acinar cell mass and dilatation of ductular structures (Fig. 33-23). The ductular epithelium is usually atypical and may display features of dysplasia, as evidenced by cuboidal cells with hyperplastic features, accompanied by areas of mononuclear cell infiltrates or patchy areas of necrosis. Cystic changes may be seen, but areas of relatively intact acinar elements and normal-appearing islets persist. In severe chronic pancreatitis, there is considerable replacement of acinar tissue by broad, coalescing areas of fibrosis, and the islet size and

1	acinar elements and normal-appearing islets persist. In severe chronic pancreatitis, there is considerable replacement of acinar tissue by broad, coalescing areas of fibrosis, and the islet size and number are reduced (Fig. 33-24). Small arteries appear thickened, and neural trunks become prominent.147Tropical pancreatitis and hereditary pancreatitis are histo-logically indistinguishable from chronic alcoholic pancreatitis. In obstructive chronic pancreatitis, calculi are absent, although periacinar fibrosis and dilated ductular structures are prominent. In pancreatic lobular fibrosis seen in elderly subjects, small ducts are dilated, sometimes with small calculi trapped within. Hypertrophy of ductular epithelia is thought to cause this small-duct disease, which is accompanied by perilobular fibrosis.148Fibrosis. A common feature of all forms of chronic pancreatitis is the perilobular fibrosis that forms surrounding individual acini, then propagates to surround small lobules, and

1	fibrosis.148Fibrosis. A common feature of all forms of chronic pancreatitis is the perilobular fibrosis that forms surrounding individual acini, then propagates to surround small lobules, and eventually coalesces to replace larger areas of acinar tissue. The pathogenesis of this process involves the activation of pancreatic stellate cells (PSCs) that are found adjacent to acini and small arteries.149 The extended cytoplasmic processes of PSCs encircle the acini but appear quiescent in the normal gland, where they contain lipid vacuoles and cytoskeletal proteins. In response to pancreatic injury, the PSCs become activated and proliferate (similarly to hepatic stellate cells), lose their lipid vesicles, and transform into myofibroblast-like cells. These cells respond to proliferative factors such as transforming growth factor ®, platelet-derived growth factor, and proinflammatory cytokines that synthesize and secrete type I and III collagen and fibronectin. Studies indicate that vitamin

1	such as transforming growth factor ®, platelet-derived growth factor, and proinflammatory cytokines that synthesize and secrete type I and III collagen and fibronectin. Studies indicate that vitamin A metabolites, similar to those present in quiescent PSCs, can inhibit the collagen production of activated cultured PSCs.150 This raises the possibility that early intervention may be possible to interrupt or prevent the fibrosis resulting from ongoing activation of PSCs.The overall pathogenic sequence proposed by Schneider and Whitcomb151 whereby alcohol induces acute pancreati-tis and, with ongoing exposure, promotes the development of chronic fibrosis, is summarized in Fig. 33-19. PSCs surround-ing the acinus are activated in acute pancreatitis but may be inactivated by anti-inflammatory cytokines and, in the absence of further injury, may revert to a quiescent state. The role of proinflammatory macrophages, cytokines, and PSCs in models of acute and chronic pancreatitis represents an

1	and, in the absence of further injury, may revert to a quiescent state. The role of proinflammatory macrophages, cytokines, and PSCs in models of acute and chronic pancreatitis represents an important area of current research.Stone Formation. Pancreatic stones are composed largely of calcium carbonate crystals trapped in a matrix of fibrillar and other material. The fibrillar center of most stones contains no Brunicardi_Ch33_p1429-p1516.indd 145601/03/19 6:44 PM 1457PANCREASCHAPTER 33calcium but rather a mixture of other metals. This suggests that stones form from an initial noncalcified protein precipitate, which serves as a focus for layered calcium carbonate precipita-tion. The same low molecular weight protein is present in stones and protein plugs and was initially named pancreatic stone pro-tein, or PSP.152 PSP was found to be a potent inhibitor of calcium carbonate crystal growth and has subsequently been renamed lithostathine.153 Independently, a 15-kDa fibrillar protein

1	stone pro-tein, or PSP.152 PSP was found to be a potent inhibitor of calcium carbonate crystal growth and has subsequently been renamed lithostathine.153 Independently, a 15-kDa fibrillar protein iso-lated from the pancreas was named pancreatic thread protein, and it has been shown to be homologous with lithostathine. Finally, a protein product of the reg gene, so named because it is expressed in association with regenerating islets in models of pancreatic injury, was isolated and called reg protein and was subsequently found to be homologous with lithostathine.154 The PSP/pancreatic thread protein/reg/lithostathine gene encodes for a 166-amino acid product that undergoes posttranslational modification to produce isoforms present in pancreatic juice. The protein is expressed in all rodents and mammals, both in the pancreas as well as in brain tissue, where it is found in particu-larly high concentrations in pyramidal neurons in Alzheimer’s disease and Down syndrome. It is also found

1	and mammals, both in the pancreas as well as in brain tissue, where it is found in particu-larly high concentrations in pyramidal neurons in Alzheimer’s disease and Down syndrome. It is also found in the renal tubules, which is consistent with its biologic action of prevent-ing calcium carbonate precipitation.Calcium and bicarbonate ions are normally present in pan-creatic juice in high concentrations, and the solubility product of calcium carbonate is greatly exceeded under normal condi-tions. Microcrystals of calcium carbonate can be seen in normal pancreatic juice but are usually clinically silent. Lithostathine is a potent inhibitor of calcium carbonate crystal formation, at a concentration of only 0.1 μmol/L. However, lithostathine con-centrations in normal pancreatic juice are in the range of 20 to 25 μmol/L, so a constant suppression of calcium carbonate crystal formation is present in the normal pancreas.In alcoholics and in patients with alcoholic chronic pan-creatitis,

1	in the range of 20 to 25 μmol/L, so a constant suppression of calcium carbonate crystal formation is present in the normal pancreas.In alcoholics and in patients with alcoholic chronic pan-creatitis, lithostathine expression and secretion are dramati-cally inhibited155 (Fig. 33-25). In addition, elevated levels of precipitated lithostathine in the duct fluid in chronic pancreati-tis patients suggests that the availability of the protein may be further reduced by the action of increased proteases and other proteins present in the duct fluid of alcoholic patients. Increased pancreatic juice protein levels in alcoholic men are reversible by abstinence from alcohol,156 so the availability and effectiveness of lithostathine may be restored in patients with early-stage dis-ease by timely intervention. Nevertheless, calcific stone forma-tion represents an advanced stage of disease, which can further promote injury or symptoms due to mechanical damage to duct epithelium or obstruction of the

1	Nevertheless, calcific stone forma-tion represents an advanced stage of disease, which can further promote injury or symptoms due to mechanical damage to duct epithelium or obstruction of the ductular network.Duct Distortion. Although calcific stone disease is normally a marker for an advanced stage of disease, parenchymal and ductular calcifications do not always correlate with symptoms. Obstructing main duct stones are commonly observed and are thought to be an indication for endoscopic or surgical removal. The ball-valve effect of a stone in a secreting system produces inevitable episodes of duct obstruction, usually accompanied by pain. But some patients with complete duct obstruction have prolonged periods of painlessness. Ductular hypertension has been documented in patients with proximal stenosis of the main pancreatic duct, and prolonged ductular distention after secre-tin administration is taken as a sign of ductular obstruction.157 Although calculus disease and duct

1	with proximal stenosis of the main pancreatic duct, and prolonged ductular distention after secre-tin administration is taken as a sign of ductular obstruction.157 Although calculus disease and duct enlargement appear together as late stages of chronic pancreatitis, controversy persists over whether they are associated, are independent events, or are caus-ally related.Radiology. Radiologic imaging of chronic pancreatitis assists in four areas: (a) diagnosis, (b) the evaluation of severity of disease, (c) detection of complications, and (d) assistance in determining treatment options.158 With the advent of cross-sectional imaging techniques such as CT and MRI, the contour, content, ductal pattern, calcifications, calculi, and cystic disease of the pancreas are all readily discernible. Transabdominal ultrasonography is frequently used as a screening method for patients with abdominal symptoms or trauma, and the extension of ultrasonic imaging to include endoscopic ultrasound (EUS) and

1	ultrasonography is frequently used as a screening method for patients with abdominal symptoms or trauma, and the extension of ultrasonic imaging to include endoscopic ultrasound (EUS) and laparoscopic US have resulted in the highest-resolution images that are capable of detecting very small (<1 cm) abnormalities in the pancreas. EUS is now frequently used as a preliminary step in the evaluation of patients with pancreatic disease, and magnetic resonance cholangiopancreatography (MRCP) is increasingly being used to select patients who are candidates for the most invasive imaging method, ERCP. The staging of disease is important in the care of patients, and a combination of imaging methods is usually used (Table 33-12).Ultrasonography is frequently used as an initial imaging method in patients with abdominal symptoms, and changes con-sistent with pancreatic duct dilatation, intraductal filling defects, cystic changes, and a heterogeneous texture are seen in chronic pancreatitis (Fig.

1	with abdominal symptoms, and changes con-sistent with pancreatic duct dilatation, intraductal filling defects, cystic changes, and a heterogeneous texture are seen in chronic pancreatitis (Fig. 33-26). The sensitivity of transabdominal ultrasonography ranges from 48% to 96%, and it is operator dependent.159 However, the contour, texture, and ductal pattern are usually quite discernible, and it is a reliable method for peri-odic reexamination to determine the efficacy of treatment.EUS has heavily impacted the evaluation and manage-ment of patients with chronic pancreatitis. Although it is more operator dependent than transabdominal ultrasonography, EUS provides not only imaging capability but also adds the capac-ity to obtain cytologic and chemical samples of tissue and fluid aspirated with linear array monitoring (Fig. 33-27). EUS images obtained through a high-frequency (7.5to 12.5-mHz) transducer are able to evaluate subtle changes in 2to 3-mm 50102030µg lithostathine/mg total

1	with linear array monitoring (Fig. 33-27). EUS images obtained through a high-frequency (7.5to 12.5-mHz) transducer are able to evaluate subtle changes in 2to 3-mm 50102030µg lithostathine/mg total proteinCCPAlc.OPDControlsFigure 33-25. Lithostathine levels in chronic calcific pancreatitis (CCP) patients, patients with alcohol abuse (Alc.), patients with other pancreatic disease (OPD), and controls. (Reproduced with permis-sion from Beger HG: The pancreas: an integrated textbook of basic science, medicine, and surgery. London: Blackwell-Science; 1998.)Brunicardi_Ch33_p1429-p1516.indd 145701/03/19 6:44 PM 1458SPECIFIC CONSIDERATIONSPART IITable 33-12Cambridge classification of pancreatic morphology in chronic pancreatitisCLASSIFICATIONERCP FINDINGSCT AND US FINDINGSNormalNo abnormal SBDsNormal gland size, shape; homogeneous parenchymaEquivocalMPD normalOne of the following: less than three abnormal SBDs; MPD 2–4 mm; gland enlarged more than two times normal size; heterogeneous

1	SBDsNormal gland size, shape; homogeneous parenchymaEquivocalMPD normalOne of the following: less than three abnormal SBDs; MPD 2–4 mm; gland enlarged more than two times normal size; heterogeneous parenchymaMildMPD normalTwo or more of the following: less than three abnormal SBDs; MPD 2–4 mm; slight gland enlargement; heterogeneous parenchymaModerate MPD changesSmall cysts <10 mm; MPD irregularitySBD changesFocal acute pancreatitis; increased echogenicity of MPD walls; gland-contour irregularitySevereAny of the above changes plus one or more of the following: cysts <10 mm; intraductal filling defects; calculi; MPD obstruction or stricture; severe MPD irregularity; contiguous organ invasion—Abbreviations: CT = computed tomography; ERCP = endoscopic retrograde cholangiopancreatography; MPD = main pancreatic duct; SBD = side-branch duct; US = ultrasound.Reproduced with permission from Beger HG: The Pancreas. London: Blackwell-Science; 1998.Figure 33-26. Sonography in chronic

1	MPD = main pancreatic duct; SBD = side-branch duct; US = ultrasound.Reproduced with permission from Beger HG: The Pancreas. London: Blackwell-Science; 1998.Figure 33-26. Sonography in chronic pancreatitis. Transabdomi-nal sonogram of patient with chronic pancreatitis demonstrates het-erogeneity of the pancreatic parenchyma, dilated ductal systems, and cyst formation. (Reproduced with permission from Bolondi L, Li Bassi S, Gaiani S, et al: Sonography of chronic pancreatitis, Radiol Clin North Am. 1989 Jul;27(4):815-833.)Figure 33-27. Endoscopic ultrasound of chronic pancreatitis. The endoscopic ultrasound appearance of the parenchyma is heteroge-neous, and dilated ducts are seen, indicating early obstructive pan-creatopathy. (Used with permission from Mark Topazian, Division of Digestive Diseases, Department of Medicine, Mayo Clinic.)structures within the pancreas and can detect indolent neoplasms in the setting of chronic inflammation. Small intraductal lesions, intraductal mucus,

1	Diseases, Department of Medicine, Mayo Clinic.)structures within the pancreas and can detect indolent neoplasms in the setting of chronic inflammation. Small intraductal lesions, intraductal mucus, cystic lesions, and subtle ductular abnormali-ties are recognizable by EUS (Table 33-13). This allows ERCP to be reserved for these patients who require therapeutic maneu-vers, or for the evaluation of more complex problems. EUS is comparable to ERCP in the detection of advanced changes in chronic pancreatitis and may be more sensitive than ERCP in the detection of mild disease.160CT scanning has affected the diagnosis of pancreatic dis-ease more broadly than any other method. With the advent of faster helical CT scanning and CT angiography, visualization of the nature, extent, location, and relative relationships of pancre-atic structures and lesions is possible with great clarity. Duct dil-atation, calculous disease, cystic changes, inflammatory events, and anomalies are all detectable

1	relative relationships of pancre-atic structures and lesions is possible with great clarity. Duct dil-atation, calculous disease, cystic changes, inflammatory events, and anomalies are all detectable with a resolution of 3 to 4 mm (Fig. 33-28). CT scanning has a false-negative rate of <10% for chronic pancreatitis, but early or mild chronic disease may go undetected by CT imaging. The earliest changes are dilatation of secondary ducts and heterogeneous parenchymal changes, which are detectable by EUS and ERCP. Another drawback of CT scanning is its lower sensitivity for detecting small neo-plasms, which are seen with increased frequency in chronic pancreatitis and may be invisible to all modalities except EUS.An MRI, in both the cross-sectional mode and the coro-nally oriented heavily weighted T2 or high spin ratio imaging Brunicardi_Ch33_p1429-p1516.indd 145801/03/19 6:44 PM 1459PANCREASCHAPTER 33Table 33-13Endoscopic ultrasound features of chronic pancreatitisENDOSCOPIC

1	weighted T2 or high spin ratio imaging Brunicardi_Ch33_p1429-p1516.indd 145801/03/19 6:44 PM 1459PANCREASCHAPTER 33Table 33-13Endoscopic ultrasound features of chronic pancreatitisENDOSCOPIC ULTRASOUND FEATUREIMPLICATIONDuctal changes Duct size >3 mmDuctal dilation Tortuous pancreatic ductDuctal irregularity Intraductal echogenic fociStones or calcification Echogenic duct wallDuctal fibrosis Side-branch ectasiaPeriductal fibrosisParenchymal changes Inhomogeneous echo patternEdema Reduced echogenic foci (1–3 mm)Edema Enhanced echogenic fociCalcifications Prominent interlobular septaeFibrosis Lobular outer gland marginFibrosis, glandular atrophy Large, echo-poor cavities (>5 mm)PseudocystReproduced with permission from Catalano MF, Lahoti S, Geenen JE, et al. Prospective evaluation of endoscopic ultrasonography, endoscopic retrograde pancreatography, and secretin test in the diagnosis of chronic pancreatitis, Gastrointest Endosc. 1998 Jul;48(1):11-17.(MRCP) that can disclose

1	of endoscopic ultrasonography, endoscopic retrograde pancreatography, and secretin test in the diagnosis of chronic pancreatitis, Gastrointest Endosc. 1998 Jul;48(1):11-17.(MRCP) that can disclose fluid-filled ducts and cystic lesions, has added greatly to the imaging options for chronic pancreatitis (Fig. 33-29). The resolution of cross-sectional MRI scanning is now approaching that of CT scanning, although the availability of MRI scanners and the complexity of the images produced have limited their large-scale use for routine imaging of the pancreas. MRCP has been shown to be an effective screening technique for disclosing ductal abnormalities that correlates closely with the contrast-filled ducts imaged by ERCP.161 The advantages of MRCP include its noninvasive methodology and Figure 33-28. Computed tomographic imaging of chronic pancre-atitis. A dilated pancreatic duct is seen, with evidence of intraductal stones and parenchymal calcification. (Reproduced with permission from

1	33-28. Computed tomographic imaging of chronic pancre-atitis. A dilated pancreatic duct is seen, with evidence of intraductal stones and parenchymal calcification. (Reproduced with permission from Forsmark CE: Management of chronic pancreatitis, Gastroen-terology. 2013 Jun;144(6):1282-1291.)Figure 33-29. Coronal T2-weighted magnetic resonance image showing evidence of chronic pancreatitis, including a pancreatic duct dilatation and side-branch clubbing. (Used with permission from Mellena Bridges, MD, Mayo Clinic Jacksonville, Department of Radiology.)its ability to image obstructed ducts that are not opacified by ERCP injection. It is therefore a useful screening study to detect duct abnormalities and to confirm the need for interventional procedures. Oral, IV, and intraductal contrast are unnecessary for MRCP, and its lack of ionizing radiation makes this the saf-est method to image the ductal system in high-risk patients.For the diagnosis and staging of chronic pancreatitis, ERCP is

1	unnecessary for MRCP, and its lack of ionizing radiation makes this the saf-est method to image the ductal system in high-risk patients.For the diagnosis and staging of chronic pancreatitis, ERCP is considered to be the gold standard. It also serves as a vehicle that enables other diagnostic and therapeutic maneu-vers, such as biopsy or brushing for cytology, or the use of stents to relieve obstruction or drain a pseudocyst (Fig. 33-30). Figure 33-30. Pancreatic duct stenting. At endoscopic retrograde cholangiopancreatography, a stent is placed in the proximal pan-creatic duct to relieve obstruction and reduce symptoms of pain. Pancreatic duct stents are left in place for only a limited time to avoid further inflammation.Brunicardi_Ch33_p1429-p1516.indd 145901/03/19 6:44 PM 1460SPECIFIC CONSIDERATIONSPART IIFigure 33-31. Pain location in chronic pancreatitis. (Reproduced with permission from Greenfield LJ, Mulholland MW, Oldham KT, et al: Surgery, Scientific Principles and

1	CONSIDERATIONSPART IIFigure 33-31. Pain location in chronic pancreatitis. (Reproduced with permission from Greenfield LJ, Mulholland MW, Oldham KT, et al: Surgery, Scientific Principles and Practice, 3rd ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2001.)Unfortunately, ERCP also carries a risk of procedure-induced pancreatitis that occurs in approximately 5% of patients.161 Patients at increased risk include those with sphincter of Oddi dysfunction and those with a previous history of post-ERCP pancreatitis. Post-ERCP pancreatitis occurs after uncompli-cated procedures, as well as after those that require prolonged manipulation. Severe pancreatitis and deaths have occurred after ERCP. It should be reserved for patients in whom the diagnosis is unclear despite the use of other imaging methods, or in whom a diagnostic or therapeutic maneuver is specifically indicated.Presentation, Natural History, and ComplicationsPresenting Signs and Symptoms. Pain is the most common symptom of

1	methods, or in whom a diagnostic or therapeutic maneuver is specifically indicated.Presentation, Natural History, and ComplicationsPresenting Signs and Symptoms. Pain is the most common symptom of chronic pancreatitis. It is usually midepigastric in location but may localize or involve either the left or right upper quadrant of the abdomen. Occasionally, it is perceived in the lower midabdomen but is frequently described as penetrating through to the back (Fig. 33-31). The pain is typically steady and boring, but not colicky. It persists for hours or days and may be chronic with exacerbations caused by eating or drink-ing alcohol. Chronic alcoholics also describe a steady, constant pain that is temporarily relieved by alcohol, followed by a more severe recurrence hours later.Patients with chronic pancreatic pain typically flex their abdomen and either sit or lie with their hips flexed, or lie on their side in a fetal position. Unlike ureteral stone pain or biliary colic, the pain

1	with chronic pancreatic pain typically flex their abdomen and either sit or lie with their hips flexed, or lie on their side in a fetal position. Unlike ureteral stone pain or biliary colic, the pain causes the patient to be still. Nausea or vomit-ing may accompany the pain, but anorexia is the most common associated symptom.Pain from chronic pancreatitis has been ascribed to multiple etiologies. Ductal hypertension, due to strictures or stones, may predispose to pain that is initiated or exacerbated by eating. Chronic pain without exacerbation may be related to parenchymal disease or retroperitoneal inflammation with per-sistent neural involvement. Acute exacerbations of pain in the setting of chronic pain may be due to acute increases in duct pressure or recurrent episodes of acute inflammation in the set-ting of chronic parenchymal disease. Nealon and Matin have described these various pain syndromes as being predictive of the response to various surgical procedures.162 Pain that

1	in the set-ting of chronic parenchymal disease. Nealon and Matin have described these various pain syndromes as being predictive of the response to various surgical procedures.162 Pain that is found in association with ductal hypertension is most readily relieved by pancreatic duct decompression, through endoscopic stenting or surgical decompression.The surgical relief of pain due to obstructive pancreatopa-thy may be dependent on the degree of underlying fibrosis and the etiology of the disease rather than the presence of ductal obstruction, per se, according to a recent studies from Johns Hopkins. Cooper et al studied 35 patients with chronic pain associated with evidence of duct obstruction who were treated with local resection of the pancreatic head and longitudinal pan-creaticojejunostomy (LR-LPJ), or Frey procedure.163 The degree of pain resolution after surgery was compared to the degree of underlying parenchymal fibrosis. After a follow-up that averaged 22 months, patients

1	(LR-LPJ), or Frey procedure.163 The degree of pain resolution after surgery was compared to the degree of underlying parenchymal fibrosis. After a follow-up that averaged 22 months, patients with more than 80% fibrosis had 100% pain relief, whereas only 60% patients with less than 10% fibrosis experienced substantial or complete pain relief. Subsequently, this group studied 60 patients who had undergone either the Frey procedure or the Whipple procedure for refractory pain due to chronic pancreatitis.145 In addition to histopathologic findings, they also analyzed the etiology of the disease. Of patients with “toxic” etiologies of acquired disease (i.e., a history of alcohol or tobacco abuse), 89% experienced prolonged pain relief, whereas only 39% of those with hereditary or idiopathic disease achieved this result. Further, these results were independent of the degree of pancreatic fibrosis. These findings suggest that the etiology of the disease may be the most important predictor of

1	achieved this result. Further, these results were independent of the degree of pancreatic fibrosis. These findings suggest that the etiology of the disease may be the most important predictor of the ben-efit of a resectional or hybrid procedure, and that patients with idiopathic or hereditary disease might be considered for an alter-native approach, such as total pancreatectomy with islet auto-transplantation (see following section).The pain of chronic pancreatitis may decrease or disappear completely over a period of years, as symptoms of exocrine and endocrine deficiency become apparent.164 This is referred to as burned out pancreatitis and correlates with the progression of disease from a mild or moderate stage to severe destruction of the pancreas. Although this evolution of painful to nonpainful disease is sometimes used as a justification to avoid intervention in painful chronic pancreatitis, noninterventional approaches to the treatment of chronic pancreatitis are inevitably

1	to nonpainful disease is sometimes used as a justification to avoid intervention in painful chronic pancreatitis, noninterventional approaches to the treatment of chronic pancreatitis are inevitably accompanied by the development of narcotic addiction, inability to work, and the sequelae of chronic illness.Although increased ductal pressure, and therefore paren-chymal pressure, has been thought to be the cause of pain in chronic obstructive pancreatitis, the role of chronic inflamma-tion per se, and the development of actual nerve damage in the diseased gland, are also thought to contribute to pain.165 Chronic inflammation results in the infiltration of tissue by macrophages, which secrete prostaglandins and other nociceptive agents that cause chronic stimulation of afferent neural fibers. Inflamma-tory damage to the perineurial layers surrounding the unmyelin-ated pancreatic nerves and a focal infiltration of inflammatory cells around nerves suggest that neural fibers are a target

1	Inflamma-tory damage to the perineurial layers surrounding the unmyelin-ated pancreatic nerves and a focal infiltration of inflammatory cells around nerves suggest that neural fibers are a target for the cellular response to inflammation in the pancreas.166Strategies to relieve pain are therefore based on three approaches: (a) reducing secretion and/or decompress the secre-tory compartment, (b) resecting the focus of chronic inflamma-tory change, or (c) interrupting the transmission of afferent neural impulses through neural ablative procedures. A trial of antisecre-tory therapy or endoscopic duct drainage may select those patients who will benefit preferentially from a decompressive procedure.Brunicardi_Ch33_p1429-p1516.indd 146001/03/19 6:45 PM 1461PANCREASCHAPTER 33Central Nervous System Abnormalities• Functional reorganization• Central sensitization (cortical)Central NervousSystem Abnormalities• Central sensitization (spinal)Obstructive Abnormalities• Pancreatic duct

1	System Abnormalities• Functional reorganization• Central sensitization (cortical)Central NervousSystem Abnormalities• Central sensitization (spinal)Obstructive Abnormalities• Pancreatic duct hypertension• Pancreatic parenchymal hypertension• Pancreas morphologyPancreatic and extrapancreatic complications• Pseudocysts• Duodenal or/and bile duct obstruction• Peptic ulcer• Splenic vein thrombosisAdditional causes• CCK elevation• Increased norepinephrine levels• Mesenteric ischemia (Type 3c diabetes)• Diabetic neuropathy (Type 3c diabetes)Peripheral NervousSystem Abnormalities• Pancreatic nociception• Pancreatic neuropathy and neuroplasticityAdverse effects to treatment• Opioid induced bowel dysfunction• Complications to surgery and endoscopic therapy• Bacterial overgrowth due to changed bowel motilityCentral Nervous System Abnormalities• Impaired inhibitory pain modulationFigure 33-32. Pain mechanisms in chronic pancreatitis. (Reproduced with permission from Demir IE,

1	to changed bowel motilityCentral Nervous System Abnormalities• Impaired inhibitory pain modulationFigure 33-32. Pain mechanisms in chronic pancreatitis. (Reproduced with permission from Demir IE, Tieftrunk E, Maak M, et al: Pain mechanisms in chronic pancreatitis: of a master and his fire, Langenbecks Arch Surg. 2011 Feb;396(2):151-160.)Patients with chronic pain due to chronic pancreatitis are often treated for years with escalating doses of narcotics, which imposes the additional problem of opioid dependency on this population. Further, it is now apparent that the pain pattern of many patients may be described as being either “visceral pain,” caused by inflammation in and around the pancreas, or “central sensitization of pain,” which is a syndrome of increased pain perception and sensitivity caused by the prolonged presence of pain167 (Fig. 33-32). Recent studies have suggested a method to differentiate these pain processes and further suggest that drugs that decrease or

1	and sensitivity caused by the prolonged presence of pain167 (Fig. 33-32). Recent studies have suggested a method to differentiate these pain processes and further suggest that drugs that decrease or ameliorate the central sensitization of pain, such as pregabalin and tramadol, may be especially useful in chronic pancreatitis patients.168 These methods of differential diagnosis of pain syndromes now appear to offer the means by which it is possible to further identify and predict which patients will ben-efit from a surgical approach to chronic pancreatitis.169Malabsorption and Weight Loss. When pancreatic exocrine capacity falls below 10% of normal, diarrhea and steatorrhea develop170 (Fig. 33-33). Patients describe a bulky, foul-smelling, loose (but not watery) stool that may be pale in color and float on the surface of toilet water. Frequently, patients will describe a greasy or oily appearance to the stool, or may describe an “oil slick” on the water’s surface. In severe

1	pale in color and float on the surface of toilet water. Frequently, patients will describe a greasy or oily appearance to the stool, or may describe an “oil slick” on the water’s surface. In severe steatorrhea, an orange, 6040207010255075Fecal fat excretion (g/day)Normal lipase output (%)Figure 33-33. Relationship of lipase output to fat malabsorption. Excess fecal fat appears when the pancreatic lipase output falls below 10% of normal secretory values. (Data from DiMagno EP, Go VL, Summerskill WH. Relations between pancreatic enzyme out-puts and malabsorption in severe pancreatic insufficiency, N Engl J Med. 1973 Apr 19;288(16):813-815.)Brunicardi_Ch33_p1429-p1516.indd 146101/03/19 6:45 PM 1462SPECIFIC CONSIDERATIONSPART IIoily stool is often reported. As exocrine deficiency increases, symptoms of steatorrhea are often accompanied by weight loss. Patients may describe a good appetite despite weight loss or diminished food intake due to abdominal pain.In severe symptomatic chronic

1	symptoms of steatorrhea are often accompanied by weight loss. Patients may describe a good appetite despite weight loss or diminished food intake due to abdominal pain.In severe symptomatic chronic pancreatitis, anorexia or nausea may occur with or separate from abdominal pain. The combination of decreased food intake and malabsorption of nutrients usually results in chronic weight loss. As a result, many patients with severe chronic pancreatitis are below ideal body weight.Lipase deficiency tends to manifest itself before trypsin deficiency, so the presence of steatorrhea may be the first func-tional sign of pancreatic insufficiency.171 As pancreatic exocrine function deteriorates further, the secretion of bicarbonate into the duodenum is reduced, which causes duodenal acidification and further impairs nutrient absorption.172 Pancreatic exocrine insufficiency is frequently asymptomatic, however, and pancre-atic exocrine function is difficult to measure, so a diagnosis of chronic

1	and further impairs nutrient absorption.172 Pancreatic exocrine insufficiency is frequently asymptomatic, however, and pancre-atic exocrine function is difficult to measure, so a diagnosis of chronic pancreatitis is sufficient to justify a trial of pancreatic enzyme supplements. Each meal should be followed by 90,000 United States Pharmacopeia units of lipase, and the metabolic and symptomatic status of the patients should be followed.173Pancreatogenic Diabetes. The islets comprise only 2% of the mass of the pancreas, but they are preferentially conserved when pancreatic inflammation occurs. In chronic pancreatitis, acinar tissue loss and replacement by fibrosis is greater than the degree of loss of islet tissue. Islets are typically smaller than normal and may be isolated from their surrounding vascular network by the fibrosis. With progressive destruction of the gland, endocrine insufficiency commonly occurs. Frank diabetes is seen initially in about 20% of patients with chronic

1	vascular network by the fibrosis. With progressive destruction of the gland, endocrine insufficiency commonly occurs. Frank diabetes is seen initially in about 20% of patients with chronic pancreatitis, and impaired glucose metabolism can be detected in up to 70% of patients. In a study of 500 patients with predominantly alcoholic chronic pancreatitis, diabetes developed in 83% within 25 years of the clinical onset of chronic pancreatitis, and more than half of the diabetic patients required insulin treatment.174 Ketoacidosis and diabetic nephropathy are relatively uncommon in pancreato-genic diabetes (see Table 33-3), but retinopathy and neuropathy are seen to occur with a similar frequency as in type 1 and type 2 diabetes.175Pancreatogenic diabetes is most common in cases of chronic pancreatitis, and it is often seen after surgical resec-tion for benign or malignant disease176,177 (Fig. 33-34). Distal Hemochromatosis8%Cystic ÿbrosis4%Pancreatic resection3%Chronic

1	of chronic pancreatitis, and it is often seen after surgical resec-tion for benign or malignant disease176,177 (Fig. 33-34). Distal Hemochromatosis8%Cystic ÿbrosis4%Pancreatic resection3%Chronic pancreatitis76%Pancreaticneoplasia9%A Distribution of T1DM, T2DM and T3cDMB Distribution of causes of T3cDMT3cDM8%T1DM12%T2DM80%Figure 33-34. Distribution of types of diabetes (A) and causes of type 3c (pancreatogenic) diabetes (B) based on studies of 1922 diabetic patients referred to an academic medical center as reported by Hardt et al. (Data from Hardt PD, Brendel MD, Kloer HU et al: Is pancreatic diabetes (type 3c diabetes) underdiagnosed and misdiagnosed? Diabetes Care. 2008 Feb;31 Suppl 2:S165-S169.)pancreatectomy and Whipple procedures have a higher inci-dence of diabetes than do drainage procedures, and the severity of diabetes is usually worse after subtotal or total pancreatec-tomy. Pancreatogenic, or type 3c diabetes (T3cDM), is seen in cystic fibrosis, in association with

1	procedures, and the severity of diabetes is usually worse after subtotal or total pancreatec-tomy. Pancreatogenic, or type 3c diabetes (T3cDM), is seen in cystic fibrosis, in association with pancreatic cancer, and in cases of severe hemochromatosis.178The etiology and pathophysiology of pancreatogenic diabetes is distinct from that of either autoimmune (type 1) or obesity-related (type 2) diabetes. In type 3c diabetes, the loss of functioning pancreatic tissue by disease or surgical removal results in a global deficiency of all three glucoregulatory islet cell hormones: insulin, glucagon, and PP. In addition, there is a paradoxical combination of enhanced peripheral sensitivity to insulin and decreased hepatic sensitivity to insulin.178,179 As a result, insulin therapy is frequently difficult; patients are hyper-glycemic when insulin replacement is insufficient (due to unsup-pressed hepatic glucose production) or hypoglycemic when insulin replacement is barely excessive (due to

1	difficult; patients are hyper-glycemic when insulin replacement is insufficient (due to unsup-pressed hepatic glucose production) or hypoglycemic when insulin replacement is barely excessive (due to enhanced periph-eral insulin sensitivity and a deficiency of pancreatic glucagon secretion to counteract the hypoglycemia). This form of diabetes is referred to as brittle diabetes and requires special attention.PP deficiency correlates with the severity of chronic pan-creatitis, and impairments in the hepatic action of insulin are reversed in PP-deficient chronic pancreatitis patients by admin-istration of PP.180 In addition, a study of type 1 and type 3c diabetic patients treated with insulin pump therapy revealed that the addition of a continuous subcutaneous infusion of PP reduced the insulin requirements needed for glycemic control.181 Studies are currently underway to identify a clinically suitable PP analog or PP receptor agonist.Laboratory Studies. The diagnosis of chronic

1	the insulin requirements needed for glycemic control.181 Studies are currently underway to identify a clinically suitable PP analog or PP receptor agonist.Laboratory Studies. The diagnosis of chronic pancreatitis depends on the clinical presentation, a limited number of indirect measurements that correlate with pancreatic function, and selected imaging studies (Table 33-14). The direct measurement of pancreatic enzymes (e.g., lipase and amylase) by blood test is highly sensitive and fairly specific in acute pancreatitis but is seldom helpful in the diagnosis of chronic pancreatitis. The pancreatic endocrine product that correlates most strongly with chronic pancreatitis is the PP response to a test meal (Fig. 33-35). Severe chronic pancreatitis is associated with a blunted or absent PP response to feeding but, as with many other tests, a normal PP response does not rule out the presence of early disease.14Brunicardi_Ch33_p1429-p1516.indd 146201/03/19 6:45 PM 1463PANCREASCHAPTER

1	response to feeding but, as with many other tests, a normal PP response does not rule out the presence of early disease.14Brunicardi_Ch33_p1429-p1516.indd 146201/03/19 6:45 PM 1463PANCREASCHAPTER 33The measurement of pancreatic exocrine secretion requires aspiration of pancreatic juice from the duodenum after nutrient (Lundh test meal) or hormonal (CCK or secretin) stimulation.182,183 Direct aspiration of pancreatic juice by endoscopic cannulation of the duct is performed in some centers, but it is not risk free, comfortable for the patient, or more sensitive than luminal intubation methods.184Indirect tests of pancreatic exocrine function are based on the measurement of metabolites of compounds that are altered (“digested”) by pancreatic exocrine products and can be quantified by serum or urine measurements. A commonly used indirect test is the bentiromide test, in which N-benzoyl-Ltyrosyl-p-aminobenzoic acid is ingested by the subject, and the urinary excretion of the

1	by serum or urine measurements. A commonly used indirect test is the bentiromide test, in which N-benzoyl-Ltyrosyl-p-aminobenzoic acid is ingested by the subject, and the urinary excretion of the proteolytic metabolite p-aminobenzoic Table 33-14Tests for chronic pancreatitis I. Measurement of pancreatic products in blood A. Enzymes B. Pancreatic polypeptide II. Measurement of pancreatic exocrine secretion A. Direct measurements 1. Enzymes 2. Bicarbonate B. Indirect measurement 1. Bentiromide test 2. Schilling test 3. Fecal fat, chymotrypsin, or elastase concentration 4. [14C]-olein absorption III. Imaging techniques A. Plain film radiography of abdomen B. Ultrasonography C. Computed tomography D. Endoscopic retrograde cholangiopancreatography E. Magnetic resonance cholangiopancreatography F. Endoscopic ultrasonography04080120160Time (min)206010014018004080120160200–40IR-pancreatic polypeptide (pmol/L)NLCPFigure 33-35. Pancreatic polypeptide (PP) response to a test

1	ultrasonography04080120160Time (min)206010014018004080120160200–40IR-pancreatic polypeptide (pmol/L)NLCPFigure 33-35. Pancreatic polypeptide (PP) response to a test meal. Immunoreactive PP (IR-PP) responses in control subjects (NL, n = 6) and patients with severe chronic pancreatitis (CP) accom-panied by PP deficiency (CP, n = 5) are shown. A test meal was administered at 0 minutes. Means ± standard error of the mean are shown. (Reproduced with permission from Brunicardi FC, Chaiken RL, Ryan AS, et al. Pancreatic polypeptide administration improves abnormal glucose metabolism in patients with chronic pancreatitis, J Clin Endocrinol Metab. 1996 Oct;81(10):3566-3572.)acid (PABA) is measured.185 Although the sensitivity of the test is as high as 100% in patients with severe chronic pancreati-tis, it identifies only 40% to 50% of patients with mild disease, and reduced PABA excretion is found in patients with a variety of other GI, hepatic, and renal diseases. The quantification of stool

1	it identifies only 40% to 50% of patients with mild disease, and reduced PABA excretion is found in patients with a variety of other GI, hepatic, and renal diseases. The quantification of stool fat has also been used as a measure of pancreatic lipase secretion, either through the direct measurement of total fecal fat levels while the subject consumes a diet of known fat con-tent, or by the measurement of exhaled 14CO2 after ingestion of [14C]-triolein or [14C]-olein. This so-called triolein breath test is less cumbersome than intubation methods and avoids the necessity of stool collections and analysis, but it also has a high false-negative rate.186Fecal levels of chymotrypsin187 and elastase188 have been proposed as simpler, less expensive tests of exocrine function and correlate well with loss of pancreatic function. As with other test methods, however, these tests lose their sensitivity in patients with mild to moderate chronic pancreatitis and may be more sensitive for other

1	with loss of pancreatic function. As with other test methods, however, these tests lose their sensitivity in patients with mild to moderate chronic pancreatitis and may be more sensitive for other causes of pancreatic dysfunction, including cystic fibrosis. Fecal elastase C1 measurements have become widespread in their use, and levels above 200 μg/g are considered normal, whereas levels below 100 μg/g are indica-tive of pancreatic exocrine insufficiency.189 The sensitivity and specificity of fecal elastase C1 measurements fall short of those needed for definitive diagnosis of pancreatic exocrine insuffi-ciency, however.190Radiologic imaging has become the principal method of diagnosis of chronic pancreatitis, with the codification of clas-sification systems that correlate with proven disease. ERCP has been considered the most sensitive radiologic test for the diagnosis of chronic pancreatitis, with specific ERCP findings that are highly correlative with the degree or stage of chronic

1	ERCP has been considered the most sensitive radiologic test for the diagnosis of chronic pancreatitis, with specific ERCP findings that are highly correlative with the degree or stage of chronic disease191 (Table 33-15). CT scanning is sensitive for the diag-nosis of chronic pancreatitis when calcification, duct dilata-tion, or cystic disease is present, but it is not accurate in the absence of these findings. CT is helpful as a screening study to guide interventional therapy or other diagnostic modalities,158 although EUS has become the preferred method for the diagno-sis of pancreatic disease and offers the advantage of very-high-resolution images of the pancreatic parenchyma, the main and secondary ductal systems, cystic lesions, and calcific changes. Table 33-15Cambridge classification of chronic pancreatitis by endoscopic retrograde cholangiopancreatographyGRADEMAIN PANCREATIC DUCTSIDE BRANCHESNormalNormalNormalSuggestiveNormal<3 AbnormalMildNormal≥3 AbnormalModerateAbnormal>3

1	of chronic pancreatitis by endoscopic retrograde cholangiopancreatographyGRADEMAIN PANCREATIC DUCTSIDE BRANCHESNormalNormalNormalSuggestiveNormal<3 AbnormalMildNormal≥3 AbnormalModerateAbnormal>3 AbnormalSevereAbnormal plus at least one of the following: large cavity, duct obstruction, dilation or duct irregularity, intraductal filing defects Reproduced with permission from Axon AT, Classen M, Cotton PB, et al: Pancreatography in chronic pancreatitis: international definitions, Gut. 1984 Oct;25(10):1107-1112.Brunicardi_Ch33_p1429-p1516.indd 146301/03/19 6:45 PM 1464SPECIFIC CONSIDERATIONSPART II246810060NonalcoholicsPancreatico-duodenectomyalcoholicsAlcoholics; 40–80%distal pancreatectomyAlcoholics; 80–95%distal pancreatectomyYears after surgical procedurePercent surviving1214708090100Figure 33-36. Effect of alcohol use on survival after surgical procedures. The cumulative survival of patients with chronic pancreatitis fol-lowing pancreaticoduodenectomy or distal pancreatectomy

1	33-36. Effect of alcohol use on survival after surgical procedures. The cumulative survival of patients with chronic pancreatitis fol-lowing pancreaticoduodenectomy or distal pancreatectomy is shown for nonalcoholic and alcoholic patients. (Reproduced with permission from Frey CF, Child CG, Fry W. Pancreatectomy for chronic pancreatitis, Ann Surg. 1976 Oct;184(4):403-413.)EUS findings may be inconclusive in mild or “minimal change” pancreatitis, however, and improved criteria for an EUS-based diagnosis are still a work in progress.192 Most importantly, EUS is highly reliable in ruling out pancreatic carcinoma when CT findings are normal or equivocal.Prognosis and Natural History. The prognosis for patients with chronic pancreatitis is dependent on the etiology of the disease, the development of complications, and on the age and socioeconomic status of the patient. The influence of treat-ment is less evident in long-term studies, although the general absence of randomized, prospective

1	of complications, and on the age and socioeconomic status of the patient. The influence of treat-ment is less evident in long-term studies, although the general absence of randomized, prospective trials clouds the issue of whether specific forms of therapy alter the long-term outlook for patients with the disease.Several studies have demonstrated that, although symp-toms of pain decrease over time in about half of the patients, this decline is also accompanied by a progression of exocrine and endocrine insufficiency.193 In general, the likelihood of eventual pain relief is dependent upon the stage of disease at diagnosis, and the persistence of alcohol use in patients with alcoholic chronic pancreatitis. Miyake and colleagues found that pain relief was achieved in 60% of alcoholic patients who suc-cessfully discontinued drinking, but in only 26% who did not.194The long-term survival of patients with chronic pancreatitis is less than for patients without pancreatitis. In an

1	patients who suc-cessfully discontinued drinking, but in only 26% who did not.194The long-term survival of patients with chronic pancreatitis is less than for patients without pancreatitis. In an international multicenter study of >2000 patients, Lowenfels and colleagues found that the 10and 20-year survival rates for patients with chronic pancreatitis were 70% and 45%, respectively, compared to 93% and 65% for patients without pancreatitis.195 The mortality risk was found to be 1.6-fold higher in patients who continued to abuse alcohol, compared to those who did not. Continued alcohol abuse has a similar effect on the response to surgical treatment (Fig. 33-36), and results in a twofold increase in mortality over a 10to 14-year follow-up period.195In addition to progressive endocrine and exocrine dysfunc-tion, and the risk of the specific complications outlined here and in Table 33-16, the other significant long-term risk for the patient with chronic pancreatitis is the development

1	exocrine dysfunc-tion, and the risk of the specific complications outlined here and in Table 33-16, the other significant long-term risk for the patient with chronic pancreatitis is the development of pancre-atic carcinoma.196 There is a progressive, cumulative increased risk of carcinoma development in patients with chronic pancre-atitis, which continues throughout the subsequent lifetime of the patient (Fig. 33-37). The incidence of carcinoma in patients with chronic pancreatitis ranges from 1.5% to 6%,196 which is at least 10-fold greater than that of patients of similar age seen in a hospital setting. In patients with chronic pancreatitis accom-panied by diabetes, the risk of carcinoma has been found to be increased 12to 33-fold compared to healthy, comparably aged controls.197,198 In patients with advanced chronic pancreatitis referred for surgical therapy, indolent, undiagnosed carcinoma can be seen in as many as 10% of patients.199The development of carcinoma in the setting of

1	patients with advanced chronic pancreatitis referred for surgical therapy, indolent, undiagnosed carcinoma can be seen in as many as 10% of patients.199The development of carcinoma in the setting of chronic pancreatitis is no doubt related to the dysregulation of cellu-lar proliferation and tissue repair processes in the setting of chronic inflammation, as is seen throughout the alimentary tract and elsewhere. In the setting of chronic pancreatitis, car-cinoma development can be especially cryptic, and the diagno-sis of early-stage tumors is particularly difficult. Awareness of this risk justifies close surveillance for cancer in patients with chronic pancreatitis. Periodic measurement of tumor markers such as CA19-9, and periodic imaging of the pancreas with CT scan and EUS seem logical in order to detect the development Table 33-16Complications of chronic pancreatitisIntrapancreatic complications Pseudocysts Duodenal or gastric obstruction Thrombosis of splenic

1	EUS seem logical in order to detect the development Table 33-16Complications of chronic pancreatitisIntrapancreatic complications Pseudocysts Duodenal or gastric obstruction Thrombosis of splenic vein Abscess Perforation Erosion into visceral artery Inflammatory mass in head of pancreas Bile duct stenosis Portal vein thrombosis Duodenal obstruction Duct strictures and/or stones Ductal hypertension and dilatation Pancreatic carcinomaExtrapancreatic complications Pancreatic duct leak with ascites or fistula Pseudocyst extension beyond lesser sac into mediastinum, retroperitoneum, lateral pericolic spaces, pelvis, or adjacent visceraBrunicardi_Ch33_p1429-p1516.indd 146401/03/19 6:45 PM 1465PANCREASCHAPTER 33Table 33-17Definitions of pancreatic fluid collectionsTERMDEFINITIONPeripancreatic fluid collectionA collection of enzyme-rich pancreatic juice that occurs early in the course of acute pancreatitis, or that forms after a pancreatic duct leak; located in or near the

1	fluid collectionA collection of enzyme-rich pancreatic juice that occurs early in the course of acute pancreatitis, or that forms after a pancreatic duct leak; located in or near the pancreas; it lacks a well-organized wall of granulation or fibrous tissueEarly pancreatic (sterile) necrosisA focal or diffuse area of nonviable pancreatic parenchyma, typically occupying >30% of the gland and containing liquefied debris and fluidLate pancreatic (sterile) necrosisAn organized collection of sterile necrotic debris and fluid with a well-defined margin or wall within the normal domain of the pancreasAcute pseudocystA collection of pancreatic juice enclosed within a perimeter of early granulation tissue, usually as a consequence of acute pancreatitis that has occurred within the preceding 3–4 wkChronic pseudocystA collection of pancreatic fluid surrounded by a wall of normal granulation and fibrous tissue, usually persisting for >6 wkPancreatic abscessAny of the above in which gross

1	3–4 wkChronic pseudocystA collection of pancreatic fluid surrounded by a wall of normal granulation and fibrous tissue, usually persisting for >6 wkPancreatic abscessAny of the above in which gross purulence (pus) is present, with bacterial or fungal organisms documented to be presentModified with permission from Baron TH, Harewood GC, Morgan DE, et al. Outcome differences after endoscopic drainage of pancreatic necrosis, acute pancreatic pseudocysts, and chronic pancreatic pseudocysts, Gastrointest Endosc. 2002 Jul;56(1):7-17.Years after diagnosis of pancreatitisCumulative incidence of pancreatic cancer (%)12345605101520(1160)(599)(244)(64)Figure 33-37. Cumulative risk of pancreatic cancer in patients with chronic pancreatitis. The number of patients evaluated at different time intervals is shown in parentheses. (Reproduced with permis-sion from Lowenfels AB, Maisonneuve P, Cavallini G, et al. Pancre-atitis and the risk of pancreatic cancer. International Pancreatitis Study Group, N

1	is shown in parentheses. (Reproduced with permis-sion from Lowenfels AB, Maisonneuve P, Cavallini G, et al. Pancre-atitis and the risk of pancreatic cancer. International Pancreatitis Study Group, N Engl J Med. 1993 May 20;328(20):1433-1437.)of carcinoma in the patient with chronic pancreatitis, although no evidence exists to indicate that this alters the outcome of patients who develop pancreatic cancer. Surgical procedures, particularly drainage procedures performed for presumed chronic pancreatitis, should always include biopsy of the tissue to exclude the diagnosis of malignancy.ComplicationsPseudocyst. A chronic collection of pancreatic fluid surrounded by a nonepithelialized wall of granulation tissue and fibrosis is referred to as a pseudocyst. Pseudocysts occur in up to 10% of patients with acute pancreatitis, and in 20% to 38% of patients with chronic pancreatitis, and thus, they comprise the most com-mon complication of chronic pancreatitis.200-202 The identification and

1	patients with acute pancreatitis, and in 20% to 38% of patients with chronic pancreatitis, and thus, they comprise the most com-mon complication of chronic pancreatitis.200-202 The identification and treatment of pseudocysts requires definition of the various forms of pancreatic fluid collections that occur (Table 33-17). In chronic pancreatitis, a pancreatic duct leak with extravasa-tion of pancreatic juice results in a peripancreatic fluid collection (PPFC). Over a period of 3 to 4 weeks, the PPFC is sealed by an inflammatory reaction that leads to development of a wall of acute granulation tissue without much fibrosis. This is referred to as an acute pseudocyst. Acute pseudocysts may resolve spontane-ously in up to 50% of cases, over a course of 6 weeks or longer.203 Pseudocysts >6 cm resolve less frequently than smaller ones but may regress over a period of weeks to months. Pseudocysts are multiple in 17% of patients,202 or they may be multilobulated. They may occur

1	>6 cm resolve less frequently than smaller ones but may regress over a period of weeks to months. Pseudocysts are multiple in 17% of patients,202 or they may be multilobulated. They may occur intrapancreatically or extend beyond the region of the pancreas into other cavities or compartments (Fig. 33-38).Pseudocysts may become secondarily infected, in which case they become abscesses. They can compress or obstruct adjacent organs or structures, leading to superior mesenteric-portal vein thrombosis or splenic vein thrombosis.204 They can erode into visceral arteries and cause intracystic hemorrhage or pseudoaneurysms (Fig. 33-39). They also can perforate and cause peritonitis or intraperitoneal bleeding.205Figure 33-38. Extensive pseudocyst disease. A computed tomo-graphic scan in a patient with alcoholic chronic pancreatitis dem-onstrates multiloculated pseudocyst disease.Pseudocysts usually cause symptoms of pain, fullness, or early satiety. Asymptomatic pseudocysts can be managed

1	with alcoholic chronic pancreatitis dem-onstrates multiloculated pseudocyst disease.Pseudocysts usually cause symptoms of pain, fullness, or early satiety. Asymptomatic pseudocysts can be managed expectantly and may resolve spontaneously or persist without complication.201,203 Symptomatic or enlarging pseudocysts require treatment, and any presumed pseudocyst without a documented antecedent episode of acute pancreatitis requires investigation to determine the etiology Brunicardi_Ch33_p1429-p1516.indd 146501/03/19 6:45 PM 1466SPECIFIC CONSIDERATIONSPART IIFigure 33-39. Pseudoaneurysm of the gastroduodenal artery. A pseudocyst can erode into an adjacent artery, which results in contained hemorrhage otherwise known as a pseudoaneurysm. A contrast-injected computed tomographic scan reveals active bleed-ing (area marked B) into a pseudocyst (arrows) as a result of this process. (Reproduced with permission from Balthazar EJ: CT diagnosis and staging of acute pancreatitis, Radiol Clin

1	active bleed-ing (area marked B) into a pseudocyst (arrows) as a result of this process. (Reproduced with permission from Balthazar EJ: CT diagnosis and staging of acute pancreatitis, Radiol Clin North Am. 1989 Jan;27(1):19-37.)of the lesion, including a cystic neoplasm.205 Although pseudocysts comprise roughly two-thirds of all pancreatic cystic lesions, they resemble cystadenomas and cystadenocarcinoma radiographically. An incidentally discovered cystic lesion should be examined by EUS and aspirated to determine whether it is a true pancreatic cystic neoplasm or a pseudocyst.The timing and method of treatment requires careful con-sideration. Pitfalls in the management of pseudocysts result from the incorrect (presumptive) diagnosis of a cystic neoplasm masquerading as a pseudocyst, a failure to appreciate the solid or debris-filled contents of a pseudocyst that appears to be fluid filled on CT scan, and a failure to document true adherence with an adjacent portion of the stomach

1	failure to appreciate the solid or debris-filled contents of a pseudocyst that appears to be fluid filled on CT scan, and a failure to document true adherence with an adjacent portion of the stomach before attempting transgas-tric internal drainage.If the pseudocyst has failed to resolve with conserva-tive therapy and symptoms persist, internal drainage is usu-ally preferred to external drainage to avoid the complication of a pancreaticocutaneous fistula. Pseudocysts communicate with the pancreatic ductal system in up to 80% of cases,206 so external drainage creates a pathway for pancreatic duct leak-age to and through the catheter exit site. Internal drainage may be performed with either endoscopic methods (transgastric or transduodenal puncture and multiple stent placements, with or without a nasocystic irrigation catheter), or surgical methods (a true cystoenterostomy, biopsy of cyst wall, and evacuation of all debris and contents). Surgical options include a cystogastros-tomy

1	without a nasocystic irrigation catheter), or surgical methods (a true cystoenterostomy, biopsy of cyst wall, and evacuation of all debris and contents). Surgical options include a cystogastros-tomy (Fig. 33-40), a Roux-en-Y cystojejunostomy, or a cysto-duodenostomy. Cystojejunostomy is the most versatile method, and it can be applied to pseudocysts that penetrate into the trans-verse mesocolon, the paracolic gutters, or the lesser sac. Cys-togastrostomy can be performed endoscopically207 (Fig. 33-41), laparoscopically,208 or by a combined laparoscopic-endoscopic method.209Because pseudocysts often communicate with the pan-creatic ductal system, two newer approaches to pseudocyst management are based on main duct drainage, rather than pseu-docyst drainage per se. Transpapillary stents inserted at the time Figure 33-40. Cystogastrostomy drainage of a retrogastric pancre-atic pseudocyst. A larger opening is made through the common wall of a retrogastric pseudocyst, and a portion of the

1	at the time Figure 33-40. Cystogastrostomy drainage of a retrogastric pancre-atic pseudocyst. A larger opening is made through the common wall of a retrogastric pseudocyst, and a portion of the pseudocyst wall is submitted for histologic confirmation of the diagnosis. Suture reinforcement of the communication is performed to avoid the com-plication of bleeding. (Reproduced with permission from Bell RH, Rikkers LF, Mulholland M: Digestive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)StomachFNAneedlePseudocystDuodenumPancreasLiverPseudocystDoublepigtailedstentsPancreasStomachEUSscopeABFigure 33-41. Technique of endoluminal cystogastrostomy. A. Endoscopic ultrasound (EUS)-guided transgastric puncture of pancreatic pseudocyst. B. Transgastric stents placed across fused posterior wall of stomach and anterior wall of pseudocyst. (Repro-duced with permission from Chauhan SS, Forsmark CE. Evidence-based treatment of pancreatic pseudocysts,

1	stents placed across fused posterior wall of stomach and anterior wall of pseudocyst. (Repro-duced with permission from Chauhan SS, Forsmark CE. Evidence-based treatment of pancreatic pseudocysts, Gastroenterology. 2013 Sep;145(3):511-5113.)Brunicardi_Ch33_p1429-p1516.indd 146601/03/19 6:45 PM 1467PANCREASCHAPTER 33ABFigure 33-42. Transpapillary drainage of a pancreatic pseudocyst. A. Endoscopic passage of a flexible wire through the major papilla, through the pancreatic duct, and into a communicating pseudocyst. B. Placement of a stent over the wire into the pseudocyst with transpapillary drainage. (Reproduced with permission from Kozarek RA, Brayko CM, Harlan J, et al. Endoscopic drainage of pancreatic pseudocysts, Gastrointest Endosc. 1985 Oct;31(5):322-327.)of ERCP may be directed into a pseudocyst through the ductal communication itself (Fig. 33-42), or they can be left across the area of suspected duct leakage to facilitate decompression and cyst drainage, analogous to the

1	into a pseudocyst through the ductal communication itself (Fig. 33-42), or they can be left across the area of suspected duct leakage to facilitate decompression and cyst drainage, analogous to the use of common bile duct stents in the setting of a cystic duct leak.206 In a surgical series of patients with chronic pancreatitis, ductal dilatation, and a coex-isting pseudocyst, Nealon and Walser showed that duct drainage alone, without a separate cystoenteric anastomosis, was as suc-cessful as a combined drainage procedure.92 Furthermore, the “duct drainage only” group enjoyed a shorter hospital stay and fewer complications than the group who underwent a separate cystoenterostomy. These observations suggest that transductal drainage may be a safe and effective approach to the manage-ment of pseudocystic disease.The complications of endoscopic drainage of pseudocysts often require surgical intervention. Bleeding from the cystoenterostomy and inoculation of a pseudocyst with failure of

1	of pseudocystic disease.The complications of endoscopic drainage of pseudocysts often require surgical intervention. Bleeding from the cystoenterostomy and inoculation of a pseudocyst with failure of resolution and persistence of infection may require surgical treatment. Bleeding risks may be lessened by the routine use of EUS in the selection of the site for transluminal stent placement.210 Endoscopic treatment of pseudocysts requires large-bore catheters or multiple stents and an aggressive approach to management for success to be achieved. Failure of nonsurgical therapy, with subsequent salvage procedures to remove infected debris and establish complete drainage, is associated with increased risks for complications and death.211,212 The most experienced therapeutic endoscopists report a complication rate of 17% to 19% for the treatment of sterile pseudocysts, and deaths as a result of endoscopic therapy have occurred.212 Therefore, the use of endoscopic methods to treat sterile or

1	a complication rate of 17% to 19% for the treatment of sterile pseudocysts, and deaths as a result of endoscopic therapy have occurred.212 Therefore, the use of endoscopic methods to treat sterile or infected pancreatic necrosis has a higher complication rate and is limited to specialized centers.Resection of a pseudocyst is sometimes indicated for cysts located in the pancreatic tail or when a midpancreatic duct disruption has resulted in a distally located pseudocyst. Distal pancreatectomy for removal of a pseudocyst, with or without splenectomy, can be a challenging procedure in the setting of prior pancreatitis. An internal drainage procedure of the com-municating duct or of the pseudocyst itself should be considered when distal resection is being contemplated.Pancreatic Ascites. When a disrupted pancreatic duct leads to pancreatic fluid extravasation that does not become sequestered as a pseudocyst, but drains freely into the peritoneal cavity, pan-creatic ascites occurs.

1	a disrupted pancreatic duct leads to pancreatic fluid extravasation that does not become sequestered as a pseudocyst, but drains freely into the peritoneal cavity, pan-creatic ascites occurs. Occasionally, the pancreatic fluid tracks superiorly into the thorax, and a pancreatic pleural effusion occurs. Referred to as internal pancreatic fistulae, both compli-cations are seen more often in patients with chronic pancreatitis rather than after acute pancreatitis. Pancreatic ascites and pleu-ral effusion occur together in 14% of patients, and 18% have a pancreatic pleural effusion alone.213Patients demonstrate the general demographics of chronic pancreatitis and usually present with a subacute or recent his-tory of progressive abdominal swelling despite weight loss. Pain and nausea are rarely present. The abdominal CT scan discloses ascites and the presence of chronic pancreatitis or a partially collapsed pseudocyst (Fig. 33-43). Paracentesis or thoracente-sis reveals noninfected fluid

1	present. The abdominal CT scan discloses ascites and the presence of chronic pancreatitis or a partially collapsed pseudocyst (Fig. 33-43). Paracentesis or thoracente-sis reveals noninfected fluid with a protein level >25 g/L and a markedly elevated amylase level. Serum amylase may also be elevated, presumably from reabsorption across the parietal membrane. Serum albumin may be low, and patients may have Figure 33-43. Pancreatic ascites. Computed tomographic scan of a patient with a ruptured pancreatic pseudocyst resulting in intra-peritoneal pancreatic fluid. (Reproduced with permission from Cameron JL, Cameron AM: Current Surgical Therapy, 11th ed. Philadelphia, PA: Elsevier; 2014.)Brunicardi_Ch33_p1429-p1516.indd 146701/03/19 6:45 PM 1468SPECIFIC CONSIDERATIONSPART IIFigure 33-44. Internal drainage for leaking pancreatic duct. A Roux-en-Y pancreaticojejunostomy is performed at the site of duct rupture to accomplish internal drainage of the pancreatic duct leak. (Reproduced with

1	drainage for leaking pancreatic duct. A Roux-en-Y pancreaticojejunostomy is performed at the site of duct rupture to accomplish internal drainage of the pancreatic duct leak. (Reproduced with permission from Beger HG: The Pancreas. London: Blackwell-Science; 1998.)coexisting liver disease. Paracentesis is therefore critical to dif-ferentiate pancreatic from hepatic ascites.ERCP is most helpful to delineate the location of the pan-creatic duct leak and to elucidate the underlying pancreatic duc-tal anatomy. Pancreatic duct stenting may be considered at the time of ERCP, but if nonsurgical therapy is undertaken and then abandoned, repeat imaging of the pancreatic duct is appropriate to guide surgical treatment.Antisecretory therapy with the somatostatin analogue octreotide acetate, together with bowel rest and parenteral nutrition, is successful in more than half of patients.214,215 Reap-position of serosal surfaces to facilitate closure of the leak is considered a part of therapy, and

1	with bowel rest and parenteral nutrition, is successful in more than half of patients.214,215 Reap-position of serosal surfaces to facilitate closure of the leak is considered a part of therapy, and this is accomplished by com-plete paracentesis. For pleural effusions, a period of chest tube drainage may facilitate closure of the internal fistula.214 Surgi-cal therapy is reserved for those who fail to respond to medical treatment. If the leak originates from the central region of the pancreas, a Roux-en-Y pancreaticojejunostomy is performed to the site of duct leakage215 (Fig. 33-44). If the leak is in the tail, a distal pancreatectomy may be considered, or an internal drainage procedure can be performed. The results of surgical treatment are usually favorable if the ductal anatomy has been carefully delineated preoperatively.Pancreatic-Enteric Fistula. The erosion of a pancreatic pseu-docyst into an adjacent hollow viscus can result in a pancreatic-enteric fistula. The most common

1	been carefully delineated preoperatively.Pancreatic-Enteric Fistula. The erosion of a pancreatic pseu-docyst into an adjacent hollow viscus can result in a pancreatic-enteric fistula. The most common site of communication is the transverse colon or splenic flexure. The fistula usually presents with evidence of GI or colonic bleeding and sepsis. If the fis-tula communicates with the stomach or duodenum, it may close spontaneously or persist as a pancreatic-enteric fistula. When the fistula involves the colon, operative correction is usually required.215Head-of-Pancreas Mass. In up to 30% of patients with advanced chronic pancreatitis, an inflammatory mass develops in the head of the pancreas.216 The clinical presentation includes severe pain and frequently includes stenosis of the distal common bile duct, duodenal stenosis, compression of the portal vein, and stenosis of the proximal main pancreatic duct (Table 33-18). Mutations and polymorphisms of p53 have been found in these

1	common bile duct, duodenal stenosis, compression of the portal vein, and stenosis of the proximal main pancreatic duct (Table 33-18). Mutations and polymorphisms of p53 have been found in these patients, and a focus of ductular carcinoma was found in 3.7% of patients with pancreatic head enlargement in one series.217 It was concluded that an accelerated transformation from hyperplasia to dysplasia exists in patients with pancreatic head enlargement, although the etiology for this process remains unclear.Splenic and Portal Vein Thrombosis. Vascular complications of chronic pancreatitis are fortunately infrequent because they are difficult to treat successfully. Portal vein compression and occlusion can occur as a consequence of an inflammatory mass in the head of the pancreas, and splenic vein thrombosis occurs in association with chronic pancreatitis in 4% to 8% of cases.218 Variceal formation can occur as a consequence of either portal or splenic venous occlusion, and splenic vein

1	vein thrombosis occurs in association with chronic pancreatitis in 4% to 8% of cases.218 Variceal formation can occur as a consequence of either portal or splenic venous occlusion, and splenic vein thrombosis with gas-tric variceal formation is referred to as left-sided or sinistral por-tal hypertension. Although bleeding complications are infrequent, the mortality risk of bleeding is >20%. When gastroesophageal varices are caused by splenic vein thrombosis, the addition of splenectomy to prevent variceal hemorrhage is prudent when sur-gery is otherwise indicated to correct other problems.TreatmentMedical Therapy. The medical treatment of chronic or recurrent pain in chronic pancreatitis requires the use of analgesics, a cessation of alcohol use, oral enzyme therapy, and the selective use of antisecretory therapy. Interventional procedures to block visceral afferent nerve conduction or to treat obstructions of the main pancreatic duct are also an adjunct to medical

1	the selective use of antisecretory therapy. Interventional procedures to block visceral afferent nerve conduction or to treat obstructions of the main pancreatic duct are also an adjunct to medical treatment.Analgesia. Oral analgesics are prescribed as needed, alone or with analgesia-enhancing agents such as gabapentin.168 Ade-quate pain control usually requires the use of narcotics, but these should be titrated to achieve pain relief with the lowest effective dose. Opioid addiction is common, and the use of long-acting analgesics by transdermal patch together with oral agents for pain exacerbations slightly reduces the sedative effects of high-dose oral narcotics.Table 33-18Signs and symptoms of chronic pancreatitis with and without a pancreatic head massSIGNS AND SYMPTOMSWITH HEAD ENLARGEMENT (n = 138) (%)WITHOUT HEAD ENLARGEMENT (n = 141) (%)Daily severe pain6740Cholestasis4611Duodenal obstruction307Diabetes mellitus1830Vascular involvement158Reproduced with permission from

1	ENLARGEMENT (n = 138) (%)WITHOUT HEAD ENLARGEMENT (n = 141) (%)Daily severe pain6740Cholestasis4611Duodenal obstruction307Diabetes mellitus1830Vascular involvement158Reproduced with permission from Beger HG: The pancreas: an integrated textbook of basic science, medicine, and surgery. London: Blackwell-Science; 1998.Brunicardi_Ch33_p1429-p1516.indd 146801/03/19 6:45 PM 1469PANCREASCHAPTER 33It is essential for patients to abstain from alcohol. In addition to removing the causative agent, alcohol abstention results in pain reduction or relief in 60% to 75% of patients with chronic pancreatitis.219 Despite this benefit, roughly half of alcoholic chronic pancreatitis patients continue to abuse alcohol.Enzyme Therapy. Pancreatic enzyme administration serves to reverse the effects of pancreatic exocrine insufficiency. Adequate pancreatic enzyme replacement reverses the exo-crine insufficiency seen in most patients, and it prevents sec-ondary complications such as metabolic bone

1	pancreatic exocrine insufficiency. Adequate pancreatic enzyme replacement reverses the exo-crine insufficiency seen in most patients, and it prevents sec-ondary complications such as metabolic bone disease due to inadequate absorption of the fat-soluble vitamins A, D, E, and K. In addition, pancreatic enzyme replacement may reduce or alleviate the pain experienced by patients. The choice of enzyme supplement and the dose should be selected based on whether malabsorption or pain (or both) are the indica-tions for therapy220 (Table 33-19). Conventional (nonen-teric-coated) enzyme preparations are partially degraded by gastric acid but are available within the duodenal and jejunal regions to bind to CCK-releasing peptide and downregulate the release of CCK. This theoretically reduces the enteric sig-nal for pancreatic exocrine secretion, which reduces the pres-sure within a partially or completely obstructed pancreatic duct.221 Enteric-coated preparations result in little to no pain

1	enteric sig-nal for pancreatic exocrine secretion, which reduces the pres-sure within a partially or completely obstructed pancreatic duct.221 Enteric-coated preparations result in little to no pain relief, presumably due to their reduced bioavailability in the proximal gut. Due to the loss of pancreatic enzymes by acid hydrolysis and proteolysis, relatively large doses are required to achieve effective levels of enzyme within the proximal small bowel. Enteric-coated preparations are protected from acid degradation but are presumably not released in the criti-cal proximal gut in sufficient quantity to inhibit the stimulus for endogenous pancreatic enzyme secretion. Nonalcoholic patients may experience more effective pain relief than alco-holic patients, but it is recommended that all patients with chronic pancreatitis pain begin a trial of nonenteric-coated enzyme supplements together with an acid-suppressive medi-cation for 1 month. If pain relief is achieved, therapy is con-tinued.

1	with chronic pancreatitis pain begin a trial of nonenteric-coated enzyme supplements together with an acid-suppressive medi-cation for 1 month. If pain relief is achieved, therapy is con-tinued. If enzyme therapy fails, further investigation of the pancreatic ductal system by ERCP guides the therapy based on specific anatomical findings (Fig. 33-45).Antisecretory Therapy. Somatostatin administration has been shown to inhibit pancreatic exocrine secretion and CCK Table 33-19Pancreatic enzyme preparationsPRODUCTFORMULATIONMANUFACTURERLIPASE CONTENT (USP)/PILL OR CAPSULEZenpepEnteric-coated porcineAptalis3000, 5000, 10,000, 15,000, 20,000CreonEnteric-coated porcineAbbott3000, 6000, 12,000, 24,000PancreazeEnteric-coated porcineOrtho-McNeil-Janssen4200, 10,500, 16,800, 21,000PertzyeEnteric-coated porcine mixed with bicarbonate granulesDigestive Care8000, 16,000UltresaEnteric-coated porcineAptalis13,800, 20,700, 23,000ViokaceTablet non-enteric-coated porcineAptalis10,440, 20,880Note: USP =

1	porcine mixed with bicarbonate granulesDigestive Care8000, 16,000UltresaEnteric-coated porcineAptalis13,800, 20,700, 23,000ViokaceTablet non-enteric-coated porcineAptalis10,440, 20,880Note: USP = United States Pharmacopeia, the standard for lipase content in the United States. An average meal requires roughly 90,000 USP units of lipase for fat digestion. Enzyme therapy of exocrine deficiency typically begins with a dose of 50,000 USP units per meal with subsequent adjustment.Reproduced with permission from Forsmark CE: Management of chronic pancreatitis, Gastroenterology. 2013 Jun;144(6):1282-1291.release.222 The somatostatin analogue octreotide acetate has therefore been investigated for pain relief in patients with chronic pancreatitis. In a double-blind, prospective, randomized 4-week trial, 65% of patients who received 200 μg of octreotide acetate subcutaneously three times daily reported pain relief, compared with 35% of placebo-treated subjects.223 Patients who had the best

1	trial, 65% of patients who received 200 μg of octreotide acetate subcutaneously three times daily reported pain relief, compared with 35% of placebo-treated subjects.223 Patients who had the best results were patients with chronic abdominal pain, suggestive of obstructive pancreatopathy. However, in another trial that used a 3-day duration of treatment, no significant pain relief was observed.224 Anecdotal reports suggest that severe pain exacerbations in chronic pancreatitis can benefit from a combination of octreotide therapy and TPN, and a pilot study of the effectiveness of the sustained-release form of octreotide suggested that it was as effective as three-times-per-day admin-istration of the short-acting form of the drug.225Neurolytic Therapy. Celiac plexus neurolysis with alcohol injection has been an effective form of analgesic treatment in patients with pancreatic carcinoma. However, the use of radio-logically or endoscopically guided celiac plexus blockade in chronic

1	injection has been an effective form of analgesic treatment in patients with pancreatic carcinoma. However, the use of radio-logically or endoscopically guided celiac plexus blockade in chronic pancreatitis has been disappointing. Due to the risk of alcohol injury and the need for repeated injections, celiac plexus blockade in chronic pancreatitis has used short-acting analgesics or other drugs rather than 50% alcohol. A trial of EUS-guided celiac plexus blockade revealed successful pain relief in 55% of patients, but the benefit lasted beyond 6 months in only 10% of patients.226 The procedure therefore appears safe, but the effect is short lived in those patients who obtain pain relief.Endoscopic Management. The techniques of endoscopic treat-ment of pancreatic duct obstruction, stone disease, pseudocyst formation, pancreatic duct leak, and for the diagnosis and man-agement of associated pancreatic tumors have expanded greatly over the past 20 years. Newer endoscopes with expanded

1	disease, pseudocyst formation, pancreatic duct leak, and for the diagnosis and man-agement of associated pancreatic tumors have expanded greatly over the past 20 years. Newer endoscopes with expanded thera-peutic capabilities have been introduced, and the role of EUS and EUS-guided needle and catheter insertion has expanded the abil-ity of the therapeutic endoscopist in the diagnosis and treatment of chronic pancreatitis and its complications.227Pancreatic duct stenting is used for treatment of proxi-mal pancreatic duct stenosis, decompression of a pancreatic duct leak, and for drainage of pancreatic pseudocysts that can be catheterized through the main pancreatic duct. Pancreatic duct stents can induce an inflammatory response within the Brunicardi_Ch33_p1429-p1516.indd 146901/03/19 6:45 PM 1470SPECIFIC CONSIDERATIONSPART IIMake a correct diagnosis • Appropriate history • Corroborating imaging tests • MRI/MRCP • EUS • CT • Functional tests if imaging tests equivocal •

1	6:45 PM 1470SPECIFIC CONSIDERATIONSPART IIMake a correct diagnosis • Appropriate history • Corroborating imaging tests • MRI/MRCP • EUS • CT • Functional tests if imaging tests equivocal • Tube-based secretin test • Endoscopic-based secretin test • Assess for alternative diseases and complications and treat if present • Pancreatic cancer or IPMN • Pseudocyst • Bile duct obstruction • Duodenal obstructionMedical therapy • Measure pain severity, character, and impact on QOL • Refer for formal structured smoking and alcohol cessation programs • Counsel on good nutrition and initiate supplementation with vitamin D and calcium • Baseline bone mineral density tasting • Provide information on local and national support groups • Initiate analgesics (starting with Tramadol) • Increase dose and potenay slowly as required • Initiate adjunctive agents in those with persistent pain or requiring higher dosages or potency of narcotics • Pregabalin, Gabapentin • SSRI •

1	• Increase dose and potenay slowly as required • Initiate adjunctive agents in those with persistent pain or requiring higher dosages or potency of narcotics • Pregabalin, Gabapentin • SSRI • SSNRI • Tricyclic antidepressants • Assess for evidence of coexistent exocrine or endocrine insufficiency and treat if present • Fecal elastase or serum trypsin • HgB A1C or GTT • Initiate steroids if autoimmune pancreatitisInflammatory mass in pancreatic head • With or without dilated pancreatic duct • With or without duodenal or biliary obstructionSurgical therapy in ÿt patients • DPPHR • Berger operation • Frey operation • Berne operation • Whipple operationDiscuss options with patient, includingthat data supports superiority of surgery.Surgery remains an option for failure ofendoscopic therapy • Endoscopic therapy • Pancreatic and biliary sphincterotomy • Stricture dilation and stenting • Lithotripsy • Stone extraction • Surgical therapy • Modified Puestow or Frey

1	therapy • Endoscopic therapy • Pancreatic and biliary sphincterotomy • Stricture dilation and stenting • Lithotripsy • Stone extraction • Surgical therapy • Modified Puestow or Frey operation• Continued medical therapy• Surgical therapy • “V-plasty” • Total pancreatectomy, with islet cell autotransplantationDilated pancreatic duct (° 6 mm)Small duct disease (pancreatic duct˛ 6 mm)Treatment effective?Continue treatment with periodicreassessmentYesNoAssess anatomy of pancreas andpancreatic ductFigure 33-45. Management algorithm for chronic pancreatitis. IPMN = intraductal papillary mucinous neoplasm; QOL = quality of life; SSRI = selective serotonin reuptake inhibitor; SSNRI = selective serotonin-norepinephrine reuptake inhibitor; GTT = glucose tolerance test. (Reproduced with permission from Forsmark CE: Management of chronic pancreatitis, Gastroenterology. 2013 Jun;144(6):1282-1291.)Brunicardi_Ch33_p1429-p1516.indd 147001/03/19 6:45 PM 1471PANCREASCHAPTER 33duct, so

1	permission from Forsmark CE: Management of chronic pancreatitis, Gastroenterology. 2013 Jun;144(6):1282-1291.)Brunicardi_Ch33_p1429-p1516.indd 147001/03/19 6:45 PM 1471PANCREASCHAPTER 33duct, so prolonged stenting is usually avoided. Patients with sphincter of Oddi dyskinesia are at high risk for developing post-ERCP pancreatitis after biliary sphincterotomy, and the prophylactic placement of a pancreatic duct stent or the admin-istration of rectal indomethacin reduces the amylase level and development of pancreatitis after biliary sphincterotomy.33,228 Pancreatic duct leaks are seen in 37% of patients with acute pancreatitis, and pancreatic duct stenting appears to facilitate the resolution of the leak.229 Similarly, pancreatic duct stenting has been used to treat postsurgical pancreatic duct leaks and posttraumatic leaks.229-231Pancreas divisum (see Fig. 33-3) is thought to cause pain and chronic pancreatitis due to functional or mechanical obstruc-tion of the dorsal duct

1	pancreatic duct leaks and posttraumatic leaks.229-231Pancreas divisum (see Fig. 33-3) is thought to cause pain and chronic pancreatitis due to functional or mechanical obstruc-tion of the dorsal duct draining exclusively, or predominantly, through the lesser papilla. A study from Marseille reported good long-term results in 24 patients after minor papilla sphinc-terotomy and dorsal duct stenting.232 The number of patients with chronic pain decreased from 83% before stenting to 29% after stenting, but pancreatitis or recurrent papillary stenosis occurred in 38%. Patients that responded best were those with intermittent pain, and this subset may be preferentially treated with endoscopic therapy. Patients with recurrent pain and a dilated dorsal duct may be candidates for internal drainage of the obstructed duct with either an extended Puestow procedure or a Frey procedure (see later in this section).Idiopathic pancreatitis patients have been treated with endoscopic stenting, pancreatic

1	the obstructed duct with either an extended Puestow procedure or a Frey procedure (see later in this section).Idiopathic pancreatitis patients have been treated with endoscopic stenting, pancreatic duct sphincterotomy, and endo-scopic stone removal with good results. In a prospective ran-domized trial, 53% of idiopathic recurrent pancreatitis patients in the control group experienced continued episodes of pancre-atitis, although only 11% of the treated patients had continued symptoms.233Extracorporeal shock wave lithotripsy (ESWL) has been used for pancreatic duct stones, together with endoscopic stent-ing and stone removal.234 A single ESWL session was used in 35 patients with pancreatic duct stones, together with 86 ERCP sessions to complete the stone removal process. After 2.4 years, 80% of patients had significant relief of symptoms (Fig. 33-46). Also, endoscopic intraductal lithotripsy can now be performed in some specialized centers. However, due to the tendency for recurrent

1	80% of patients had significant relief of symptoms (Fig. 33-46). Also, endoscopic intraductal lithotripsy can now be performed in some specialized centers. However, due to the tendency for recurrent stone formation, the use of ESWL or endoscopic litho-tripsy for long-term management of calcific pancreatitis remains uncertain.Surgical Therapy Indications and History The traditional approach to surgi-cal treatment of chronic pancreatitis and its complications has maintained that surgery should be considered only when the medical therapy of symptoms has failed. Nealon and Thomp-son published a landmark study in 1993, however, that showed that the progression of chronic obstructive pancreatitis could be delayed or prevented by pancreatic duct decompression.235 No other therapy has been shown to prevent the progression of chronic pancreatitis, and this study demonstrated the role of surgery in the early management of the disease (Table 33-20). Small-duct disease or “minimal change chronic

1	to prevent the progression of chronic pancreatitis, and this study demonstrated the role of surgery in the early management of the disease (Table 33-20). Small-duct disease or “minimal change chronic pancreatitis” are causes for uncertainty over the choice of operation, however. Major resections have a high complication rate, both early and late, in chronic alcoholic pancreatitis, and lesser procedures often result in symptomatic recurrence. Therefore, the choice of operation and the timing of surgery are based on each patient’s pancreatic anatomy, the likelihood (or lack thereof) that further ABFigure 33-46. Extracorporeal shock wave lithotripsy treatment of pancreatic duct stones. The endoscopic retrograde cholangiopancrea-tography images are shown (A) before and (B) after extracorporeal shock wave lithotripsy therapy of pancreatic duct obstruction due to calculus formation. (Reproduced with permission from Kozarek RA, Brandabur JJ, Ball TJ, et al. Clinical outcomes in patients who

1	shock wave lithotripsy therapy of pancreatic duct obstruction due to calculus formation. (Reproduced with permission from Kozarek RA, Brandabur JJ, Ball TJ, et al. Clinical outcomes in patients who undergo extracorporeal shock wave lithotripsy for chronic calcific pancreatitis, Gastrointest Endosc. 2002 Oct;56(4):496-500.)Table 33-20Effect of surgical drainage on progression of chronic pancreatitisTREATMENT GROUP24-MONTH EVALUATIONOperated (n = 47)Mild to moderate 48 (87%); severe 6 (13%)Nonoperated (n = 36)Mild to moderate 8 (22%); severe 28 (78%)Eighty-three patients with chronic pancreatitis were evaluated by exocrine, endocrine, nutritional, and endoscopic retrograde cholangiopancreatography studies, and all had mild to moderate disease and dilated pancreatic ducts. A Puestow-type duct decompression procedure was performed in 47 patients, and all subjects were restaged by the same methods 24 months later.Reproduced with permission from Nealon WH, Thompson JC. Progressive loss of

1	decompression procedure was performed in 47 patients, and all subjects were restaged by the same methods 24 months later.Reproduced with permission from Nealon WH, Thompson JC. Progressive loss of pancreatic function in chronic pancreatitis is delayed by main pancreatic duct decompression. A longitudinal prospective analysis of the modified puestow procedure, Ann Surg. 1993 May;217(5):458-466.Brunicardi_Ch33_p1429-p1516.indd 147101/03/19 6:45 PM 1472SPECIFIC CONSIDERATIONSPART IIFigure 33-47. Head-of-pancreas mass after Puestow procedure. The computed tomographic appearance of an inflammatory mass occupying the head of the pancreas, which developed 2 years after Puestow decompression of the body and tail of pancreas.medical and endoscopic therapy will halt the symptoms of the disease, and the chance that a good result will be obtained with the lowest risk of morbidity and mortality. Finally, preparation for surgery should include restoration of protein-caloric homeo-stasis,

1	and the chance that a good result will be obtained with the lowest risk of morbidity and mortality. Finally, preparation for surgery should include restoration of protein-caloric homeo-stasis, abstinence from alcohol and tobacco, and a detailed review of the risks and likely outcomes to establish a bond of trust and commitment between the patient and the surgeon.Historically, the surgery for chronic pancreatitis before the second half of the 20th century was a true demonstration of trial and error.236 Obtaining good surgical outcomes before the avail-ability of CT scans and ERCP was either the result of serendip-ity or due to the skill and creativity of the surgeon. In 1911, Link described an operation he devised on the spot, when a laparot-omy in a young woman with abdominal pain revealed a fluctu-ant, obstructed pancreatic duct. After performing a dochotomy and evacuating multiple stones, he inserted a rubber tube, and exteriorized the pancreatostomy just above her navel.237 He

1	a fluctu-ant, obstructed pancreatic duct. After performing a dochotomy and evacuating multiple stones, he inserted a rubber tube, and exteriorized the pancreatostomy just above her navel.237 He later described the operation as having been a success for the next 30 years of the patient’s life, during which the patient managed the care of the drainage tube without apparent problems.238With the demonstration in 1942 by Priestley that total pancreatectomy was technically feasible,239 and the report in 1946 by Whipple that proximal pancreatic resection was ben-eficial in (three) patients with chronic pancreatitis,240 the option of surgical resection as treatment for chronic pancreatitis was established. By the mid 1950s, however, growing disappoint-ment with the high risk of resection and the lack of long-term benefit overshadowed the surgical treatment of chronic pancre-atitis. The choice of resection vs. drainage was largely based on surgeon preference until the 1970s, when the

1	and the lack of long-term benefit overshadowed the surgical treatment of chronic pancre-atitis. The choice of resection vs. drainage was largely based on surgeon preference until the 1970s, when the widespread adoption of ERCP and CT scans provided the ability to pre-operatively diagnose obstructive and sclerotic disease, and this resulted in the rational selection of operative procedures. During this period, the major drawbacks to surgical therapy remained the recurrence of symptoms despite surgery, the correspond-ing development of an inflammatory (or malignant) mass in the undrained pancreatic head (Fig. 33-47), or the high morbidity and mortality of major resectional procedures that predisposed patients to a cascade of metabolic problems.241Sphincteroplasty The sphincter of Oddi and the pancreatic duct sphincter serve as gatekeepers for the passage of pancreatic juice into the duodenum (Fig. 33-48). Stenosis of either sphincter (scle-rosing papillitis), due to scarring from

1	and the pancreatic duct sphincter serve as gatekeepers for the passage of pancreatic juice into the duodenum (Fig. 33-48). Stenosis of either sphincter (scle-rosing papillitis), due to scarring from pancreatitis or from the passage of gallstones, may result in obstruction of the pancreatic duct and chronic pain.242 As gallstone pancreatitis became a popu-lar diagnosis in the 1940s and 1950s, attention was focused on the ampullary region as a possible cause of chronic symptoms, and surgical sphincteroplasty was advocated. Although endoscopic techniques are now used routinely to perform sphincterotomy of either the common bile duct or pancreatic duct, a true (permanent) PancreasCommon bile ductDuodenal wallMajor papillaSphincter of ampullaMucosaSubmucosa Sphincter of pancreatic duct Pancreatic ductSphincter of common bile ductLongitudinal muscleof duodenal wallCircular muscle of duodenal wallFigure 33-48. Schematic diagram of the ampullary, biliary, and pancreatic duct sphincters. The

1	ductSphincter of common bile ductLongitudinal muscleof duodenal wallCircular muscle of duodenal wallFigure 33-48. Schematic diagram of the ampullary, biliary, and pancreatic duct sphincters. The point of merger of the bile duct and pancreatic duct is highly variable, and a true sphincter of the pancreatic duct may be poorly developed. (Reproduced with permission from Yamada T, Alpers DH, Kalloo AN, et al: Textbook of Gastroenterology, 5th ed. Oxford: Wiley-Blackwell; 2009.)Brunicardi_Ch33_p1429-p1516.indd 147201/03/19 6:45 PM 1473PANCREASCHAPTER 33Figure 33-49. Operative sphincteroplasty of the biliary and pan-creatic duct. The ampullary and bile duct sphincters are divided, as is the pancreatic duct sphincter, with suture apposition of the mucosal edges of the incision. sphincteroplasty can only be performed surgically. Transduode-nal sphincteroplasty with incision of the septum between the pan-creatic duct and common bile duct may offer significant relief for the rare patient

1	can only be performed surgically. Transduode-nal sphincteroplasty with incision of the septum between the pan-creatic duct and common bile duct may offer significant relief for the rare patient with a focal obstruction and inflammation isolated to this region (Fig. 33-49).Drainage Procedures After the early reports of success with pancreatostomy for the relief of symptoms of chronic pancreatitis,238 Cattell described pancreaticojejunostomy for relief of pain in unresectable pancreatic carcinoma.243 Shortly thereafter, Duval244 and, separately, Zollinger and associates245 described the caudal Roux-en-Y pancreaticojejunostomy for the treatment of chronic pancreatitis in 1954 (Fig. 33-50). The so-called Duval procedure was used for decades by some surgeons, but it almost invariably failed due to restenosis and segmental obstruction of the pancreas due to progressive scarring. In 1958, Puestow and Gillesby described these segmental narrowings and dilatations of the ductal system as a

1	due to restenosis and segmental obstruction of the pancreas due to progressive scarring. In 1958, Puestow and Gillesby described these segmental narrowings and dilatations of the ductal system as a “chain of lakes,” and proposed a longitudinal decompression of the body and tail of the pancreas into a Roux limb of jejunum246 (Fig. 33-51). Four of Puestow and Gillesby’s 21 initial cases were side-to-side anastomoses, and 2 years after their report, Partington and Rochelle described a much simpler version of the longitudinal, or side-to-side Roux-en-Y pancreaticojejunostomy that became universally known as the Puestow procedure247 (Fig. 33-52).Successful pain relief after the Puestow-type decompres-sion procedure has been reported in 75% to 85% of patients for the first few years after surgery, but pain recurs in >20% of patients after 5 years due to progressive disease even in patients who are abstinent from alcohol.164Figure 33-50. Duval’s caudal pancreaticojejunostomy. (Reproduced

1	but pain recurs in >20% of patients after 5 years due to progressive disease even in patients who are abstinent from alcohol.164Figure 33-50. Duval’s caudal pancreaticojejunostomy. (Reproduced with permission from Greenlee HB: The role of surgery for chronic pancreatitis and its complications, Surg Annu. 1983;15:283-305.)Figure 33-51. Puestow and Gillesby’s longitudinal pancreatico-jejunostomy. Originally described as an invaginating anastomosis that drained the entire body and tail, the anastomosis was created after amputating the tail of the gland and opening the duct along the long axis of the gland. (Reproduced with permission from Greenlee HB: The role of surgery for chronic pancreatitis and its complica-tions, Surg Annu. 1983;15:283-305.)With the advent of therapeutic endoscopy and techniques for transluminal stone removal and lithotripsy, multiple series have reported the successful endoscopic treatment of pancreatic duct calculi, although the long-term outcomes of these

1	and techniques for transluminal stone removal and lithotripsy, multiple series have reported the successful endoscopic treatment of pancreatic duct calculi, although the long-term outcomes of these efforts has been uneven.248-251 Endoscopic removal of pancreatic duct stones is usually coupled to prolonged pancreatic duct stenting, Brunicardi_Ch33_p1429-p1516.indd 147301/03/19 6:45 PM 1474SPECIFIC CONSIDERATIONSPART IIABFigure 33-52. Longitudinal dochotomy in obstructing calcific pancreatitis. A longitudinal pancreatotomy typically discloses seg-mental stenosis of the pancreatic duct and the presence of intra-ductal calculi in a patient with chronic calcific pancreatitis (A). Following mobilization of a Roux limb of jejunum, a longitudinal pancreaticojejunostomy is performed to permit extensive drainage of the pancreatic duct system (B). This technique, described by Partington and Rochelle, is the typical method used for the Puestow procedure. which carries the risk of further

1	extensive drainage of the pancreatic duct system (B). This technique, described by Partington and Rochelle, is the typical method used for the Puestow procedure. which carries the risk of further inflammation.252,253 Despite the risk of perioperative complications, the surgical management of pancreatic duct stones and stenosis has been shown to be superior to endoscopic treatment in randomized clinical trials in which the long, side-to-side technique of pancreaticojejunos-tomy is used.254-256Resectional Procedures Distal Pancreatectomy For patients with focal inflammatory changes localized to the body and tail, or in whom no significant ductal dilatation exists, the technique of partial (40–80%) dis-tal pancreatectomy has been advocated (Fig. 33-53). Although distal pancreatectomy is less morbid than more extensive resec-tional procedures, the operation leaves untreated a major portion of the gland, and is therefore associated with a significant risk of symptomatic recurrence. It has

1	morbid than more extensive resec-tional procedures, the operation leaves untreated a major portion of the gland, and is therefore associated with a significant risk of symptomatic recurrence. It has been a more popular operation in British centers, where its success seems to be greater, perhaps due to the lower incidence of alcoholic chronic pancreatitis.257 However, long-term outcomes reveal good pain relief in only 60% of patients, with completion pancreatectomy required for pain relief in 13% of patients.Laparoscopic distal pancreatectomy has been shown to be feasible for the removal of focal lesions of the distal pancreas,258 but it is more difficult in the setting of chronic pancreatitis.Ninety-Five Percent Distal Pancreatectomy In 1965, Fry and Child proposed the more radical 95% distal pancreatectomy, which was intended for patients with sclerotic (small duct) disease and which attempted to avoid the morbidity of total pancreatectomy by preserving the rim of pancreas in the

1	distal pancreatectomy, which was intended for patients with sclerotic (small duct) disease and which attempted to avoid the morbidity of total pancreatectomy by preserving the rim of pancreas in the pancreaticoduodenal groove, along with its associated blood vessels and distal common bile duct.241 The operation was found to be associated with pain relief in 60% to 77% of patients long term, but it is accompanied by a high risk of brittle diabetes, hypoglycemic coma, and malnutrition. Although the operation was the first attempt to resect the pancreatic head while preserving the duodenum and distal bile duct, the extensive degree of metabolic complications led to its failure as viable treatment for the symptoms of pancreatic sclerosis.Proximal Pancreatectomy In 1946, Whipple reported a series of five patients treated with either pancreaticoduodenectomy or total pancreatectomy for symptomatic chronic pancreatitis, with one operative death.240 Subsequently, proximal pancreatectomy or

1	series of five patients treated with either pancreaticoduodenectomy or total pancreatectomy for symptomatic chronic pancreatitis, with one operative death.240 Subsequently, proximal pancreatectomy or pancreaticoduodenectomy, with or without pylorus preser-vation (Fig. 33-54), has been widely used for the treatment of chronic pancreatitis.259 In the three largest modern (circa 2000) series of the treatment of chronic pancreatitis by the Whipple Splenic arteryFigure 33-53. Distal (spleen-sparing) pancreatectomy. A distal pancreatectomy for chronic pancreatitis is usually performed with en bloc splenectomy, using either an open or laparoscopic tech-nique. In the presence of minimal inflammation, a spleen-sparing version can be performed, as shown here.Brunicardi_Ch33_p1429-p1516.indd 147401/03/19 6:45 PM 1475PANCREASCHAPTER 33procedure, pain relief 4 to 6 years after operation was found in 71% to 89% of patients. However, mortality ranged from 1.5% to 3%, and major complications

1	6:45 PM 1475PANCREASCHAPTER 33procedure, pain relief 4 to 6 years after operation was found in 71% to 89% of patients. However, mortality ranged from 1.5% to 3%, and major complications occurred in 25% to 38% of patients at the Johns Hopkins Hospital,260 the Mayo Clinic,261 and the Massachusetts General Hospital.262 In follow-up, 25% to 48% of patients developed diabetes, and about the same per-centage required exocrine therapy. Advocates of the Whipple procedure as treatment for chronic pancreatitis suggest that the high rate of symptomatic relief outweighs the metabolic conse-quences and the mortality risk of the procedure, but increasingly this approach is being reserved for those patients with suspected occult malignancy.Total Pancreatectomy Priestley and associates first described successful total pancreatectomy in 1944 in a patient with hyperinsulinism,239 and two of Whipple’s original five cases of chronic pancreatitis reported in 1946 were treated with total

1	first described successful total pancreatectomy in 1944 in a patient with hyperinsulinism,239 and two of Whipple’s original five cases of chronic pancreatitis reported in 1946 were treated with total pancreatectomy.240 Subsequently, surgeons who used total pancreatectomy found that the operation produces no better pain relief for their patients than pancreaticoduodenectomy (about 80–85%). Moreover, the metabolic consequences of total pancreatectomy in the absence of islet cell transplantation can be profound and life-threatening. The patients have a “brittle” form of diabetes in which avoidance of hyperand hypoglycemia is problematic.263 In addition, lethal episodes of hypoglycemia are common in severe apancreatic diabetes. These are due to hypoglycemic unresponsiveness, due to the absence of pancreatic glucagon, and to hypoglycemia unawareness, despite an ongoing need to treat with exogenous insulin.177 In a series of >100 patients treated with total pancreatectomy, Gall and

1	absence of pancreatic glucagon, and to hypoglycemia unawareness, despite an ongoing need to treat with exogenous insulin.177 In a series of >100 patients treated with total pancreatectomy, Gall and colleagues showed that half of all the late deaths after this operation were due to (iatrogenic) hypoglycemia.264 Despite newer forms of insulin, insulin delivery systems, and continuous blood glucose monitoring systems, severe pancreatogenic diabetes remains an adverse outcome, as complete prevention of the physiologic consequences of total pancreatectomy remains an unfulfilled goal. Even with the growing acceptance of islet auto-transplantation as an adjunct to the procedure (see later in CholedochojejunostomyGastrojejunostomyPancreaticojejunostomyCholedochojejunostomyDuodenojejunostomyPancreaticojejunostomyABFigure 33-54. The pancreaticoduodenectomy (Whipple procedure) can be performed either with the standard technique, which includes distal gastrectomy (A), or with preservation of the

1	33-54. The pancreaticoduodenectomy (Whipple procedure) can be performed either with the standard technique, which includes distal gastrectomy (A), or with preservation of the pylorus (B). The pylorus-sparing version of the procedure is used most commonly. (Repro-duced from Wu GY, Aziz K, Whalen GF: An Internist’s Illustrated Guide to Gastrointestinal Surgery. Totowa: Humana Press; 2003.)this section), total pancreatectomy itself is now used only rarely for the treatment of refractory chronic pancreatitis.Hybrid Procedures In 1980, Beger and associates described the Duodenum-preserving Pancreatic Head Resection or DPPHR265 (Fig. 33-55), and they published long-term results with DPPHR for the treatment of chronic pancreatitis in 1985266 and again in 1999.267 In 388 patients who were followed for an average of 6 years after DPPHR, pain relief was reportedly maintained in 91%, mortality was <1%, and diabetes developed in 21%, with 11% demonstrating a reversal of their preoperative

1	followed for an average of 6 years after DPPHR, pain relief was reportedly maintained in 91%, mortality was <1%, and diabetes developed in 21%, with 11% demonstrating a reversal of their preoperative diabetic sta-tus. These authors also compared the DPPHR procedure with the pylorus-sparing Whipple procedure in a randomized trial of 40 patients with chronic pancreatitis.268 The mortality was reportedly zero in both groups, and the morbidity was also com-parable. Pain relief (over 6 months) was seen in 94% of DPPHR patients, but in only 67% of Whipple patients. Furthermore, the insulin secretory capacity and glucose tolerance were noted to deteriorate in the Whipple group, but they actually improved in the DPPHR patients.The DPPHR requires the careful dissection of the gas-troduodenal artery and the creation of two anastomoses (Fig. 33-56), and it carries a similar complication risk as the Whipple procedure due to the risk of pancreatic leakage and intra-abdominal fluid collections.In

1	and the creation of two anastomoses (Fig. 33-56), and it carries a similar complication risk as the Whipple procedure due to the risk of pancreatic leakage and intra-abdominal fluid collections.In 1987, Frey and Smith described the local resection of the pancreatic head with longitudinal pancreaticojejunostomy (LR-LPJ), which included excavation of the pancreatic head, including the ductal structures in continuity with a long dochot-omy of the dorsal duct269 (Fig. 33-57). The Frey procedure pro-vides thorough decompression of the pancreatic head as well as the body and tail of the gland, and a long-term follow-up suggested that improved outcomes are associated with this more extensive decompressive procedure. Frey and Amikura reported their results in 50 patients followed for >7 years, and they found complete or substantial pain relief in 87% of patients. There was no operative mortality, but 22% of patients developed postop-erative complications.270Brunicardi_Ch33_p1429-p1516.indd

1	they found complete or substantial pain relief in 87% of patients. There was no operative mortality, but 22% of patients developed postop-erative complications.270Brunicardi_Ch33_p1429-p1516.indd 147501/03/19 6:45 PM 1476SPECIFIC CONSIDERATIONSPART IICommonbile ductPancreaticductPancreaticremnantABFigure 33-55. The duodenum-preserving pancreatic head resection described by Beger and colleagues. A. The completed resection after transection of the pancreatic neck, and subtotal removal of the pan-creatic head, with preservation of the distal common bile duct and duodenum. B. Completion of the reconstruction with anastomosis to the distal pancreas and to the proximal pancreatic rim by the same Roux limb of jejunum. (Reproduced with permission from Bell RH, Rikkers LF, Mulholland M: Digestive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)Figure 33-56. Intraoperative view of the Beger procedure. The gastroduodenal artery is encircled by a vessel

1	Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)Figure 33-56. Intraoperative view of the Beger procedure. The gastroduodenal artery is encircled by a vessel loop. Just below, the intrapancreatic portion of the common bile duct is exposed as it courses toward the ampulla. A rim of well-vascularized pancreatic tissue remains in the duodenal C-loop. Preservation of the posterior branch of the gastroduodenal artery is essential to preserve viability of these structures.Key steps in the performance of the LR-LPJ include pres-ervation of the pancreatic neck as well as the capsule of the posterior pancreatic head. In the pancreaticoduodenectomy and the DPPHR, the pancreatic neck is freed up from the portal and superior mesenteric vein confluence and divided. In the LR-LPJ, the neck of the pancreas is preserved intact as are the body and tail of the pancreas. Not having to divide the pancreatic neck, as in the pancreaticoduodenectomy or DPPHR, reduces

1	In the LR-LPJ, the neck of the pancreas is preserved intact as are the body and tail of the pancreas. Not having to divide the pancreatic neck, as in the pancreaticoduodenectomy or DPPHR, reduces the risk of the operation because it avoids intraoperative prob-lems with the venous structures lying posterior to the gland. To reduce the risk of penetrating the posterior capsule of the head, Frey recommended in his 1994 report that the posterior limit of resection be the back wall of the opened duct of Wirsung and duct to the uncinate (Fig. 33-58).Subsequent to Frey’s own modification of the technique, other surgeons have described modifications of the extent or technique of the LR-LPJ. Andersen and Topazian advocated performing the LR-LPJ as it was originally described, in which the entirety of the ducts are excised from the head (Fig. 33-59), and described the use of the ultrasonic aspirator and dissector for this purpose.271 This device permits precise removal of the ducts and adjacent

1	of the ducts are excised from the head (Fig. 33-59), and described the use of the ultrasonic aspirator and dissector for this purpose.271 This device permits precise removal of the ducts and adjacent tissue with good visualization and without complications. There is little pancreatic tissue behind these ducts, and the pancreatic capsule is continuously palpated as the dissection proceeds to ensure a safe margin of resection. The intrapancreatic portion of the common bile duct is usually exposed, and avoiding injury to it is enhanced by the ultrasonic aspirator. The majority of the parenchyma of the uncinate pro-cess is spared, and the excavation of the pancreatic head is made contiguous with a generous dochotomy of the dorsal duct. Whether merely unroofing as opposed to removal of the proximal ducts contributes to better pain relief is not known and awaits a randomized trial to compare the two versions of the LR-LPJ. Izbicki and colleagues at the University of Hamburg also recommend a

1	ducts contributes to better pain relief is not known and awaits a randomized trial to compare the two versions of the LR-LPJ. Izbicki and colleagues at the University of Hamburg also recommend a more extensive excavation of the pancreatic head, and they use a technique that they refer to as the Hamburg modification of the LR-LPJ272 (Fig. 33-60). This wider excavation of the pancreatic head is created in continu-ity with the dorsal dochotomy, and it is followed by a single, side-to-side pancreaticojejunostomy.In 2001, Ho and Frey subsequently described merely exca-vating the core of the pancreatic head and draining the exca-vation with a Roux-en-Y pancreaticojejunostomy, but without any effort to include the dorsal duct273,274 (Fig. 33-61). In 2003, Brunicardi_Ch33_p1429-p1516.indd 147601/03/19 6:45 PM 1477PANCREASCHAPTER 33Figure 33-57. Frey procedure. The local resection of the pancreatic head with longitudinal pancreaticojejunostomy (LR-LPJ) provides complete decompression of

1	6:45 PM 1477PANCREASCHAPTER 33Figure 33-57. Frey procedure. The local resection of the pancreatic head with longitudinal pancreaticojejunostomy (LR-LPJ) provides complete decompression of the entire pancreatic ductal system. Reconstruction is performed with a side-to-side Roux-en-Y pan-creaticojejunostomy. (Reproduced with permission from Bell RH, Rikkers LF, Mulholland M: Digestive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)Figure 33-58. Operative view of excavated head of the pancreas during the Frey procedure. The main pancreatic duct is opened widely down to the level of the ampulla, and the head of the pan-creas is excavated in a conical fashion so as to allow complete decompression of the chronically obstructed and inflamed pancre-atic ducts. (Reproduced with permission from Aspelund G et al. Improved outcomes for benign disease with limited pancreatic head resection, J Gastrointest Surg. 2005 Mar;9(3):400-409.)Figure

1	pancre-atic ducts. (Reproduced with permission from Aspelund G et al. Improved outcomes for benign disease with limited pancreatic head resection, J Gastrointest Surg. 2005 Mar;9(3):400-409.)Figure 33-59. Complete excavation of the pancreatic head and distal pancreatic dochotomy. A true excavation and removal of the proximal ductal system is combined with a distal pancreatic dochotomy. Reconstruction is performed with a single side-to-side Roux-en-Y pancreaticojejunostomy. (Reproduced with permission from Andersen DK, Topazian MD. Pancreatic head excavation: a variation on the theme of duodenum-preserving pancreatic head resection, Arch Surg. 2004 Apr;139(4):375-379.)Brunicardi_Ch33_p1429-p1516.indd 147701/03/19 6:46 PM 1478SPECIFIC CONSIDERATIONSPART IIFigure 33-60. The Hamburg modification of the local resection of the pancreatic head with longitudinal pancreaticojejunostomy. (Reproduced with permission from Shackelford RT, Yeo CJ, Peters JH: Shackelford’s Surgery of the

1	modification of the local resection of the pancreatic head with longitudinal pancreaticojejunostomy. (Reproduced with permission from Shackelford RT, Yeo CJ, Peters JH: Shackelford’s Surgery of the Alimentary Tract. New York, NY: Saunders/Elsevier; 2007.)Farkas and colleagues described a similar excavation of the cen-tral portion of the pancreatic head without any effort to include the duct of the body in the lateral pancreaticojejunostomy,275 and they reported excellent results with what they termed an organ-preserving pancreatic head resection (OPPHR) in a ran-domized comparison to the pylorus-preserving pancreaticoduo-denectomy (PPPD).276This approach was advocated by Gloor and associates in Bern as an alternative to the DPPHR procedure in patients with portal hypertension277 and was described as the Berne modifica-tion of the DPPHR (Fig. 33-62). Köninger and colleagues in Heidelberg subsequently published a randomized, controlled trial of the “Berne” version of the excavation

1	described as the Berne modifica-tion of the DPPHR (Fig. 33-62). Köninger and colleagues in Heidelberg subsequently published a randomized, controlled trial of the “Berne” version of the excavation method compared to the “classic” Beger procedure.278 Operative times and length of stay were shorter in the group undergoing excavation of the pancreatic head, while long-term outcomes and quality-of-life scores were identical over 2 years postoperatively.The common element of these variations on the theme of LR-LPJ remains the excavation or “coring out” of the central portion of the pancreatic head. It remains uncertain, however, whether and to what degree the dochotomy needs to be extended into the body and tail. The logical conclusion of all of these efforts is that the head of the pancreas is the nidus of the chronic inflammatory process in chronic pancreatitis and that removal of the central portion of the head of the gland is the key to the successful resolution of pain in the long

1	is the nidus of the chronic inflammatory process in chronic pancreatitis and that removal of the central portion of the head of the gland is the key to the successful resolution of pain in the long term.Complications Initial and long-term results of the LR-LPJ demonstrate pain relief that is equivalent to that of pancreati-coduodenectomy and the DPPHR.279,280 The observed mortal-ity rate has been virtually zero, and therefore, less than with the Whipple procedure. Major complications were less with the LR-LPJ (16%) than with pancreaticoduodenectomy (40%) or DPPHR (25%) in one single-site series, and the incidence of new postoperative diabetes after LR-LPJ was 8% with an aver-age follow-up of 3 years.1996Line of enterotomyJejunal limbsecured with 1strow of suturesNormal pancreatic ductPancreatic head cored outABFigure 33-61. Excavation of pancreatic head without longitudinal pancreaticojejunostomy. Comparisons of the Three Operative Procedures: Pancre-aticoduodenectomy (Whipple

1	head cored outABFigure 33-61. Excavation of pancreatic head without longitudinal pancreaticojejunostomy. Comparisons of the Three Operative Procedures: Pancre-aticoduodenectomy (Whipple procedure), DPPHR (Beger procedure), and LR-LPJ (Frey procedure). There has been considerable interest to apply evidence-based methods to the study of the three operations currently advocated for the treat-ment of chronic pancreatitis. The best studies, or level 1 data by the Strength of Recommendation Taxonomy, are prospective, randomized controlled trials comparing two or more operations from a single or multi-institutional study. Retrospective, cohort-based studies are regarded as level 2 data by the Strength of Recommendation Taxonomy criteria.To date, ten published level 1 studies268,276,278-285 and three level 2 studies199,286,287 have examined various comparisons between these three operations. In the level 1 study of Klempa and colleagues281 and that of Buchler et al,288 DPPHR patients had a

1	three level 2 studies199,286,287 have examined various comparisons between these three operations. In the level 1 study of Klempa and colleagues281 and that of Buchler et al,288 DPPHR patients had a shorter hospital stay, greater weight gain, less postopera-tive diabetes, and exocrine dysfunction than standard Whipple patients over a 3to 5-year follow-up. Pain control was similar between the two procedures. Similar results were observed in a Brunicardi_Ch33_p1429-p1516.indd 147801/03/19 6:46 PM 1479PANCREASCHAPTER 33Figure 33-62. The Berne modification of the local resection of the pancreatic head with longitudinal pancreaticojejunostomy.recent level 2 retrospective study of 123 patients287 and in the level 1 study by Keck et al.287In a level 1 study of 61 patients randomized to PPPD or LR-LPJ, Izbicki and colleagues found a lower postoperative complication rate associated with the Frey procedure (19%) compared to the PPPD group (53%), and better global quality-of-life scores (71%

1	LR-LPJ, Izbicki and colleagues found a lower postoperative complication rate associated with the Frey procedure (19%) compared to the PPPD group (53%), and better global quality-of-life scores (71% vs. 43%, respectively).279 Both operations were equally effective in controlling pain over a 2-year follow-up. Similar results were seen in the study by Farkas and associates276 who employed a similar method of excavation of the pancreatic head that their group described as an organ-preserving pancreatic head resection (OPPHR) and found that OPPHR was associated with a shorter operating time, less postoperative morbidity, shorter hospital stay, and better quality of life than PPPD. The superior outcomes of the Frey procedure were found to be durable in a 15-year follow-up study by Bachmann et al285 who found that survival and function were superior after the Frey procedure.Late Morbidity and Mortality In 2005, Izbicki’s group reported on a level 1 study of 74 patients randomized to the

1	et al285 who found that survival and function were superior after the Frey procedure.Late Morbidity and Mortality In 2005, Izbicki’s group reported on a level 1 study of 74 patients randomized to the DPPHR or LR-LPJ who were then followed for an average of 8.5 years.286 There were no significant differences between the groups with regard to global quality of life, pain scores, late mortality, and exocrine or endocrine insufficiency. The level 1 study by Köninger, which compared the classic DPPHR with excavation of the pancreatic head, showed identical outcomes at 2 years after an initial reduction in morbidity associated with the excavation procedure.278 These results were echoed in the level 2 study by Aspelund and associates, which demonstrated fewer complications with both the DPPHR and LR-LPJ proce-dures compared to pancreaticoduodenectomy, a lower incidence of new diabetes (8%) for both DPPHR and LR-LPJ compared to the Whipple procedure (25%), but no significant differences in

1	and LR-LPJ proce-dures compared to pancreaticoduodenectomy, a lower incidence of new diabetes (8%) for both DPPHR and LR-LPJ compared to the Whipple procedure (25%), but no significant differences in outcomes or pain relief between DPPHR and LR-LPJ.199 Finally, level 2 data support the efficacy of both DPPHR and LR-LPJ in patients with dilated as well as nondilated ducts.286,289,290Long-term exocrine and/or endocrine insufficiency in chronic pancreatitis patients treated surgically is a product of the surgical intervention as well as the progression of the underlying disease. Although the short-term (3-year) incidence of new diabe-tes after operation appears less with the LR-LPJ and DPPHR than with the PPPD, the late incidence of diabetes appears similar in all groups. After an average of 7 years of follow-up after LR-LPJ or PPPD, survival, pain relief, and pancreatic function were similar in both groups. The rate of diabetes was slightly lower after LR-LPJ (61%) than after PPPD

1	of 7 years of follow-up after LR-LPJ or PPPD, survival, pain relief, and pancreatic function were similar in both groups. The rate of diabetes was slightly lower after LR-LPJ (61%) than after PPPD (65%), but these had both more than doubled from their preoperative status.275-281 Therefore, although the limited pancreatic procedures of DPPHR and LR-LPJ have a lower initial rate of endocrine dysfunction, the long-term risk of diabetes is more related to the progression of the underlying disease than to the effects of operation.The level 1 studies confirm that the duodenum preserving options are associated with a lower immediate morbidity and mortality and therefore, in the absence of a mass or concerns about cancer, are better options than a Whipple procedure for chronic pancreatitis. The choice of LR-LPJ, DPPHR, or OPPHR depends largely on surgeon experience, and the LR-LPJ is most common in the United States.Total Pancreatectomy With Islet Auto-Transplantation Islet cell

1	The choice of LR-LPJ, DPPHR, or OPPHR depends largely on surgeon experience, and the LR-LPJ is most common in the United States.Total Pancreatectomy With Islet Auto-Transplantation Islet cell transplantation for the treatment of diabetes is an attractive adjunct to pancreatic surgery in the treatment of benign pancre-atic disease. Despite the difficulties in recovering islets from a chronically inflamed gland, Najarian and associates demonstrated the utility of autotransplantation of islets in patients with chronic pancreatitis in 1980.291 Subsequently, through refinements in the methods of harvesting and gland preservation, and through standardization of the methods by which islets are infused into the portal venous circuit for intrahepatic engraftment, the suc-cess of total pancreatectomy combined with islet autotransplan-tation has steadily increased to achieve insulin independence in the majority of patients treated in recent series.292,293 Although 2 to 3 million islets are

1	combined with islet autotransplan-tation has steadily increased to achieve insulin independence in the majority of patients treated in recent series.292,293 Although 2 to 3 million islets are required for successful engraftment in an allogeneic recipient, the auto-transplant recipient can usually achieve long-term, insulin-independent status after engraftment of only 300,000 to 400,000 islets (about one-third to one-half of the number of islets in the normal pancreas).294The ability to recover a sufficient quantity of islets from a sclerotic gland is dependent on the degree of fibrotic dis-ease present, so the selection of patients as candidates for autologous islet transplantation is important. The impressive improvement in quality of life measures and pain relief seen after total pancreatectomy with islet auto-transplantation (TP-IAT) indicate that it is a highly successful form of therapy for some patients (Fig. 33-63).295 The outcomes of TP-IAT are sig-nificantly better in

1	pancreatectomy with islet auto-transplantation (TP-IAT) indicate that it is a highly successful form of therapy for some patients (Fig. 33-63).295 The outcomes of TP-IAT are sig-nificantly better in pediatric patients than in adults, largely due to the prevalence of hereditary and idiopathic causes of chronic pancreatitis in pediatric patients.296 These studies suggest that further definition is needed regarding criteria for considering TP-IAT vs. hybrid or resectional procedures for patients with persistent symptoms. With the emerging evidence that hybrid procedures seem to offer better outcomes in patients with toxic etiologies of chronic pancreatitis, and that TP-IAT appears to Brunicardi_Ch33_p1429-p1516.indd 147901/03/19 6:46 PM 1480SPECIFIC CONSIDERATIONSPART II001224364860MonthsEffectNum DF1131252842994299Den DF0.26268.294.840.608<0.001<0.001F statisticP valueAgeTimeAge x TimePrevalence of narcotic use (%)72849610812020406080100Pediatric patientAdultFigure 33-63. Prevalence

1	DF1131252842994299Den DF0.26268.294.840.608<0.001<0.001F statisticP valueAgeTimeAge x TimePrevalence of narcotic use (%)72849610812020406080100Pediatric patientAdultFigure 33-63. Prevalence of narcotic use after total pancreatectomy with islet auto-transplantation (TP-IAT) in adult and pediatric patients. (Reproduced with permission from Chinnakotla S, Beilman GJ, Dunn TB, et al. Factors Predicting Outcomes After a Total Pancreatectomy and Islet Autotransplantation Lessons Learned From Over 500 Cases, Ann Surg. 2015 Oct;262(4):610-622.)preferentially benefit patients with hereditary and idiopathic forms of the disease, the importance of the careful definition of the etiology of chronic pancreatitis, including genetic testing, is steadily increasing.PANCREATIC NEOPLASMSNeoplasms of the Endocrine PancreasNeoplasms of the endocrine pancreas are relatively uncommon but do occur with enough frequency (five cases per million population) that most surgeons will encounter them in an urban

1	Endocrine PancreasNeoplasms of the endocrine pancreas are relatively uncommon but do occur with enough frequency (five cases per million population) that most surgeons will encounter them in an urban practice. The cells of the endocrine pancreas, or islet cells, origi-nate from neural crest cells, also referred to as amine precursor uptake and decarboxylation cells. Multiple endocrine neopla-sia (MEN) syndromes occur when these cells cause tumors in multiple sites. The MEN1 syndrome involves pituitary tumors, parathyroid hyperplasia, and pancreatic neoplasms. Although most pancreatic endocrine tumors are nonfunctional, some are functional, secreting peptide products that produce interesting clinical presentations. Neoplasms of the endocrine pancreas that are not associated with excess hormone levels and a recogniz-able clinical syndrome are considered nonfunctional. Special immunohistochemical stains allow pathologists to confirm the peptide products being produced within the cells of

1	levels and a recogniz-able clinical syndrome are considered nonfunctional. Special immunohistochemical stains allow pathologists to confirm the peptide products being produced within the cells of a pancre-atic endocrine tumor. However, the histologic characteristics of these neoplasms do not predict their clinical behavior, and malignancy is usually determined by the presence of local inva-sion and lymph node or hepatic metastases. Unfortunately, most pancreatic endocrine tumors are malignant, but the course of the disease is far more favorable than that seen with pancreatic exo-crine cancer. The key to diagnosing these rare tumors is recogni-tion of the classic clinical syndrome; confirmation is achieved by measuring serum levels of the elevated hormone. Localiza-tion of the tumor can be a challenging step, but once accom-plished, the surgery is relatively straightforward. The goals of surgery range from complete resection, often accomplished with insulinomas, to controlling symptoms

1	a challenging step, but once accom-plished, the surgery is relatively straightforward. The goals of surgery range from complete resection, often accomplished with insulinomas, to controlling symptoms with debulking proce-dures. Unresectable disease in the liver is often addressed with chemoembolization.As with pancreatic exocrine tumors, the initial diagnos-tic imaging test of choice for pancreatic endocrine tumors is a multidetector CT scan with four phases of contrast and fine cuts through the pancreas and liver. Neuroendocrine tumors of the pancreas often enhance with contrast. EUS can be superior to CT in localizing these tumors, which can produce dramatic symptoms despite their small (<1 cm) size. In contrast to pan-creatic exocrine tumors, many of the endocrine tumors have somatostatin receptors (SSTRs) that allow them to be detected by a radiolabeled octreotide scan. A radioactive somatostatin analogue is injected intravenously, followed by whole-body radionuclide scanning

1	receptors (SSTRs) that allow them to be detected by a radiolabeled octreotide scan. A radioactive somatostatin analogue is injected intravenously, followed by whole-body radionuclide scanning (Fig. 33-64). The success of this modal-ity in localizing tumors and detecting metastases has decreased the use of older techniques such as angiography and selective venous sampling.InsulinomaInsulinomas are the most common functional pancreatic endocrine neoplasms and present with a typical clinical Brunicardi_Ch33_p1429-p1516.indd 148001/03/19 6:46 PM 1481PANCREASCHAPTER 33syndrome known as Whipple’s triad. The triad consists of symptomatic fasting hypoglycemia, a documented serum glucose level <50 mg/dL, and relief of symptoms with the administration of glucose. Patients can present with a profound syncopal episode or less severe symptoms that are averted by frequent eating. Common symptoms include palpitations, trembling, diaphoresis, confusion or obtundation, and seizure, and family

1	profound syncopal episode or less severe symptoms that are averted by frequent eating. Common symptoms include palpitations, trembling, diaphoresis, confusion or obtundation, and seizure, and family members may report that the patient has undergone a personality change.Routine laboratory studies will uncover a low blood sugar, the cause of all of these symptoms. Serum insulin levels are elevated. C-peptide levels should also be elevated and rule out the unusual case of surreptitious administration of insulin or oral hypoglycemic agents because excess endogenous insulin pro-duction leads to excess C-peptide. The diagnosis can be clinched with a monitored fast in which blood is sampled every 4 to 6 hours for glucose and insulin levels until the patient becomes symptomatic. However, this can be dangerous and must be done with close supervision.Insulinomas are usually localized with CT scanning and EUS. Technical advances in EUS have led to preoperative identification of >90% of

1	this can be dangerous and must be done with close supervision.Insulinomas are usually localized with CT scanning and EUS. Technical advances in EUS have led to preoperative identification of >90% of insulinomas.297 Visceral angiography with venous sampling is rarely required to accurately localize the tumor. Insulinomas are evenly distributed throughout the head, body, and tail of the pancreas.298 Unlike most endocrine pancreatic tumors, the majority (90%) of insulinomas are benign Figure 33-64. Radioactive octreotide scan demonstrating pancre-atic endocrine tumor in the body of the pancreas (arrow).and solitary, and only 10% are malignant. They are typically cured by simple enucleation. However, tumors located close to the main pancreatic duct and large (>2 cm) tumors may require a distal pancreatectomy or pancreaticoduodenectomy. Intraop-erative US is useful to determine the tumor’s relation to the main pancreatic duct and guides intraoperative decision making. Approximately 90% of

1	pancreatectomy or pancreaticoduodenectomy. Intraop-erative US is useful to determine the tumor’s relation to the main pancreatic duct and guides intraoperative decision making. Approximately 90% of insulinomas are sporadic, and 10% are associated with the MEN1 syndrome. Insulinomas associated with the MEN1 syndrome are more likely to be multifocal and have a higher rate of recurrence.Noninsulinoma Hyperinsulinemia Hypoglycemia SyndromeA syndrome of noninsulinoma pancreatogenous hypoglycemia was described by Service et al in 1999.299 The syndrome is asso-ciated with beta-cell hypertrophy, islet hyperplasia and increased beta-cell mass. When these findings are accompanied by ectopic islet tissue, multilobulated islets, and ductuloinsular complexes, the definition of nesideoblastosis is met. Nesideoblastosis accompanied by hyperinsulinism was previously considered a disease of neonates, where subtotal or total pancreatectomy was required to correct potentially fatal neonatal

1	is met. Nesideoblastosis accompanied by hyperinsulinism was previously considered a disease of neonates, where subtotal or total pancreatectomy was required to correct potentially fatal neonatal hyperinsulinism. However, dozens of cases of nesideoblastosis associated with hyperinsulinism have now been reported in patients 2 to 5 years after Roux-en-Y gastric bypass for obesity.300 Many of these patients have undergone partial or total pancreatectomy to prevent potentially fatal hypoglycemia. The illness in former bariatric surgery patients appears to result from an idiosyncraticallyprolonged hypersecretion of the incretin hormones GIP and GLP-1 after the gastric bypass. GLP-1 is a potent stimulant of the expression of the transcription factor PDX-1, which nor-mally regulates beta-cell development and growth. The correct treatment of this condition to prevent episodes of hypoglycemia is conversion of the gastric bypass to a form of bariatric proce-dure that restores normal intestinal

1	and growth. The correct treatment of this condition to prevent episodes of hypoglycemia is conversion of the gastric bypass to a form of bariatric proce-dure that restores normal intestinal flow of nutrients, such as the gastric sleeve, or the addition of a restriction element such as an adjustable gastric band. Pancreatic resection without conversion of the Roux-en-Y gastric bypass is not appropriate because this allows the abnormal enteroinsular relationship to continue and hyperinsulinemia persists or recurs after partial pancreatectomy.GastrinomaZollinger-Ellison syndrome (ZES) is caused by a gastrinoma, an endocrine tumor that secretes gastrin, leading to acid hyper-secretion and peptic ulceration. Many patients with ZES present with abdominal pain, peptic ulcer disease, and severe esopha-gitis. However, in the era of effective antacid therapy, the pre-sentation can be less dramatic. Although most of the ulcers are solitary, multiple ulcers in atypical locations that fail to

1	esopha-gitis. However, in the era of effective antacid therapy, the pre-sentation can be less dramatic. Although most of the ulcers are solitary, multiple ulcers in atypical locations that fail to respond to antacids should raise suspicion for ZES and prompt a work-up. At the time of diagnosis, 21% of patients with gastrinoma have diarrhea.The diagnosis of ZES is made by measuring the serum gastrin level. It is important that patients stop taking proton pump inhibitors for this test. In most patients with gastrinomas, the level is >1000 pg/mL. Gastrin levels can be elevated under conditions other than ZES. Common causes of hypergastrin-emia include pernicious anemia, treatment with proton pump inhibitors, renal failure, G-cell hyperplasia, atrophic gastritis, retained or excluded antrum, and gastric outlet obstruction. In equivocal cases, when the gastrin level is not markedly elevated, a secretin stimulation test is helpful.Brunicardi_Ch33_p1429-p1516.indd 148101/03/19 6:46 PM

1	and gastric outlet obstruction. In equivocal cases, when the gastrin level is not markedly elevated, a secretin stimulation test is helpful.Brunicardi_Ch33_p1429-p1516.indd 148101/03/19 6:46 PM 1482SPECIFIC CONSIDERATIONSPART IIIn 70% to 90% of patients, the primary gastrinoma is found in Passaro’s triangle, an area defined by a triangle with points located at the junction of the cystic duct and common bile duct, the second and third portion of the duodenum, and the neck and body of the pancreas (Fig. 33-65). However, because gastri-nomas can be found almost anywhere, whole-body imaging is required. The test of choice is SSTR (octreotide) scintigraphy in combination with CT. The octreotide scan is more sensitive than CT, locating about 85% of gastrinomas and detecting tumors <1 cm. With the octreotide scan, the need for tedious and techni-cally demanding selective angiography and measurement of gas-trin gradients has declined. EUS is another modality that assists in the

1	<1 cm. With the octreotide scan, the need for tedious and techni-cally demanding selective angiography and measurement of gas-trin gradients has declined. EUS is another modality that assists in the preoperative localization of gastrinomas. It is particularly helpful in localizing tumors in the pancreatic head or duodenal wall, where gastrinomas are usually <1 cm in size. A combina-tion of octreotide scan and EUS detects >90% of gastrinomas.It is important to rule out MEN1 syndrome by checking serum calcium levels before surgery because resection of the gastrinoma(s) in these patients rarely results in normalization of serum gastrin concentrations or a prolongation of survival. Only one-fourth of gastrinomas occur in association with the MEN1 syndrome. One-half of patients with gastrinomas will have soli-tary tumors while the remainder will have multiple gastrino-mas. Multiple tumors are more common in patients with MEN1 syndrome. Aggressive surgical treatment is justified in

1	will have soli-tary tumors while the remainder will have multiple gastrino-mas. Multiple tumors are more common in patients with MEN1 syndrome. Aggressive surgical treatment is justified in patients with sporadic gastrinomas. If patients have MEN1 syndrome, the parathyroid hyperplasia is addressed with total parathyroid-ectomy and implantation of parathyroid tissue in the forearm.Approximately 50% of gastrinomas metastasize to lymph nodes or the liver and are therefore considered malignant. Patients who meet criteria for operability should undergo exploration for possible removal of the tumor. Although the tumors are submu-cosal, a full-thickness excision of the duodenal wall is performed if a duodenal gastrinoma is found. All lymph nodes in Passaro’s triangle are excised for pathologic analysis. If the gastrinoma is found in the pancreas and does not involve the main pancreatic duct, it is enucleated. Pancreatic resection is justified for solitary Figure 33-65. Passaro’s triangle.

1	analysis. If the gastrinoma is found in the pancreas and does not involve the main pancreatic duct, it is enucleated. Pancreatic resection is justified for solitary Figure 33-65. Passaro’s triangle. The typical location of a gastrinoma is described by this anatomic region, including the head of the pancreas, duodenum, and the lymphatic bed posterior and superior to the duodenum, as originally described by E. Passaro. (Reproduced with permission from Stable BE, Morrow DJ, Passaro E: The gastrinoma triangle: operative implications, Am J Surg. 1984 Jan;147(1):25-31.)gastrinomas with no metastases. A highly selective vagotomy can be performed if unresectable disease is identified or if the gastrinoma cannot be localized. This may reduce the amount of expensive proton pump inhibitors required. In cases in which hepatic metastases are identified, resection is justified if the pri-mary gastrinoma is controlled and the metastases can be safely and completely removed. Debulking or incomplete

1	In cases in which hepatic metastases are identified, resection is justified if the pri-mary gastrinoma is controlled and the metastases can be safely and completely removed. Debulking or incomplete removal of multiple hepatic metastases is probably not helpful, especially in the setting of MEN1. The application of new modalities such as radiofrequency ablation seems reasonable, but data to sup-port this approach are limited.301 Postoperatively, patients are followed with fasting serum gastrin levels, secretin stimulation tests, octreotide scans, and CT scans. In patients found to have inoperable disease, chemotherapy with streptozocin, doxorubi-cin, and 5-fluorouracil (5-FU) is used. Other approaches such as somatostatin analogues, interferon, and chemoembolization also have been used in gastrinoma with some success.Unfortunately, a biochemical cure is achieved in only about one-third of the patients operated on for ZES. Despite the lack of success, long-term survival rates are good,

1	gastrinoma with some success.Unfortunately, a biochemical cure is achieved in only about one-third of the patients operated on for ZES. Despite the lack of success, long-term survival rates are good, even in patients with liver metastases. The 15-year survival rate for patients without liver metastases is about 80%, while the 5-year survival rate for patients with liver metastases is 20% to 50%. Pancreatic tumors are usually larger than tumors arising in the duodenum, and more often they have lymph node metastases. In gastrinomas, liver metastases decrease survival rates, but lymph node metastases do not. The best results are seen after complete excision of small sporadic tumors originating in the duodenum. Large tumors associated with liver metastases, located outside of Passaro’s triangle, have the worst prognosis.Vasoactive Intestinal Peptide-Secreting TumorIn 1958, Verner and Morrison first described the syndrome associated with a pancreatic neoplasm secreting VIP. The clas-sic

1	have the worst prognosis.Vasoactive Intestinal Peptide-Secreting TumorIn 1958, Verner and Morrison first described the syndrome associated with a pancreatic neoplasm secreting VIP. The clas-sic clinical syndrome associated with this pancreatic endocrine neoplasm consists of severe intermittent watery diarrhea leading to dehydration, and weakness from fluid and electrolyte losses. Large amounts of potassium are lost in the stool. The vasoac-tive intestinal peptide-secreting tumor (VIPoma) syndrome is also called WDHA syndrome due to the presence of watery diarrhea, hypokalemia, and achlorhydria. The massive (5 L/d) and episodic nature of the diarrhea associated with the appropriate electrolyte abnormalities should raise suspicion of the diagnosis. Serum VIP levels must be measured on multiple occasions because the excess secretion of VIP is episodic and single measurements might be normal and misleading. A CT scan localizes most VIPomas, although as with all islet cell tumors, EUS is

1	occasions because the excess secretion of VIP is episodic and single measurements might be normal and misleading. A CT scan localizes most VIPomas, although as with all islet cell tumors, EUS is the most sensitive imaging method. Electrolyte and fluid balance is sometimes dif-ficult to correct preoperatively and must be pursued aggressively. Somatostatin analogues are helpful in controlling the diarrhea and allowing replacement of fluid and electrolytes. VIPomas are more commonly located in the distal pancreas and most have spread outside the pancreas. Palliative debulking operations can some-times improve symptoms for a period, along with somatostatin analogues. Hepatic artery embolization also has been reported as a potentially beneficial treatment.302GlucagonomaDiabetes in association with dermatitis should raise the suspi-cion of a glucagonoma. The diabetes usually is mild. The classic necrolytic migratory erythema manifests as cyclic migrations of lesions with spreading margins

1	with dermatitis should raise the suspi-cion of a glucagonoma. The diabetes usually is mild. The classic necrolytic migratory erythema manifests as cyclic migrations of lesions with spreading margins and healing centers typically on Brunicardi_Ch33_p1429-p1516.indd 148201/03/19 6:46 PM 1483PANCREASCHAPTER 33the lower abdomen, perineum, perioral area, and feet. Patients also complain of an enlarged, sensitive tongue. The diagnosis is confirmed by measuring serum glucagon levels, which are usu-ally >500 pg/mL. Glucagon is a catabolic hormone, and most patients present with malnutrition. The rash associated with glucagonoma is thought to be caused by low levels of amino acids. Preoperative treatment usually includes control of the dia-betes, parenteral nutrition, and octreotide. Like VIPomas, glu-cagonomas are more often in the body and tail of the pancreas and tend to be large tumors with metastases. Again, debulking operations are recommended in good operative candidates to relieve

1	glu-cagonomas are more often in the body and tail of the pancreas and tend to be large tumors with metastases. Again, debulking operations are recommended in good operative candidates to relieve symptoms.SomatostatinomaBecause somatostatin inhibits pancreatic and biliary secretions, patients with a somatostatinoma present with gallstones due to bile stasis, diabetes due to inhibition of insulin secretion, and steatorrhea due to inhibition of pancreatic exocrine secretion and bile secretion. Most somatostatinomas originate in the proximal pancreas or the pancreatoduodenal groove, with the ampulla and periampullary area as the most common site (60%). The most common presentations are abdominal pain (25%), jaundice (25%), and cholelithiasis (19%).303 This rare type of pancreatic endocrine tumor is diagnosed by confirming elevated serum somatostatin levels, which are usually >10 ng/mL. Although most reported cases of somatostatinoma involve metastatic disease, an attempt at complete

1	tumor is diagnosed by confirming elevated serum somatostatin levels, which are usually >10 ng/mL. Although most reported cases of somatostatinoma involve metastatic disease, an attempt at complete excision of the tumor and cholecystectomy is warranted in fit patients.Nonfunctioning Pancreatic Endocrine TumorsAlthough some pancreatic endocrine neoplasms secrete one or more hormones and are associated with interesting characteristic clinical syndromes, most are not associated with elevated serum hormone levels that cause symptoms. Pancreatic endocrine tumors are considered functional if they are associated with a clinical syndrome and nonfunctioning if not associated with clinical symptoms. The majority of pancreatic endocrine tumors (PET), also called pancreatic neuroendocrine tumors (pNET), are malignant because they have the potential for uncontrolled growth and metastasis. Immunohistochemical markers such as synaptophysin, chromogranin A (CgA), and neuron-specific enolase can be

1	are malignant because they have the potential for uncontrolled growth and metastasis. Immunohistochemical markers such as synaptophysin, chromogranin A (CgA), and neuron-specific enolase can be helpful in the diagnosis, but the gross histology is not a reliable predictor of biologic behavior. CgA is used by some as a serum marker to monitor patients for disease recur-rence or response to treatment, but the test performs poorly for this purpose. Patients often present similar to patients with pancreatic adenocarcinoma with vague pain or weight loss, but pNETs are increasingly discovered incidentally when imaging is performed for another reason. The tumor frequently enhances with arterial contrast (Fig. 33-66). Sometimes a cystic compo-nent is seen due to central necrosis. Octreoscan (somatostatin receptor scintigraphy) can be helpful to stage the disease. Sur-gical resection is typically recommended in fit patients in the absence of metastatic disease. For patients with tumors in the

1	receptor scintigraphy) can be helpful to stage the disease. Sur-gical resection is typically recommended in fit patients in the absence of metastatic disease. For patients with tumors in the body and tail of the pancreas, this typically includes splenec-tomy. Enucleation and splenic preservation, although tempting in small tumors, fails to remove regional lymph nodes.With advances in imaging, small indolent pNETs are being discovered with increasing frequency, and some surgeons are considering observation in these cases.304 There are several argu-ments in favor of observation of small (<2 cm) nonfunctional pNETs, particularly in MEN-1 patients. Pancreatic resection has significant morbidity and mortality. In the setting of MEN-1, resection is rarely curative, and most patients require reopera-tion. Also, the survival of these patients, even with metastatic dis-ease, is generally excellent at least compared to pancreatic ductal adenocarcinoma. However, the 5-year survival with

1	reopera-tion. Also, the survival of these patients, even with metastatic dis-ease, is generally excellent at least compared to pancreatic ductal adenocarcinoma. However, the 5-year survival with metastatic pNET is only 16% and radical surgery with curative intent is the standard of care, particularly in sporadic cases that are fit surgical candidates where the primary tumor can be completely excised.Adjuvant treatment after resection is withheld in the absence of radiographically demonstrable metastatic disease even if CgA levels remain elevated. Although these tumors have a slow growth pattern compared to pancreatic ductal adenocar-cinoma, many patients with pNETs will die of their disease even after an apparent complete resection, making surveillance after complete resection important. CT scan and or octreotide scan is recommended annually for 5 years after resection.ABCFigure 33-66. Pancreatic neuroendocrine tumor (PNET) demonstrating enhancement during arterial phase of computed

1	CT scan and or octreotide scan is recommended annually for 5 years after resection.ABCFigure 33-66. Pancreatic neuroendocrine tumor (PNET) demonstrating enhancement during arterial phase of computed tomography scan. Pancreatic head PNET seen in (left) sagital, (middle) coronal, and (right) lateral views of the abdomen.Brunicardi_Ch33_p1429-p1516.indd 148301/03/19 6:46 PM 1484SPECIFIC CONSIDERATIONSPART IIIncomplete resection (debulking) for locally advanced or metastatic pNETs of the pancreas is controversial because of the favorable survival duration of patients without surgery. How-ever, in carefully selected fit patients with a pNET in the head of the pancreas and minimal disease in the liver, a pancreaticoduo-denectomy with wedge resection of the liver metastasis might be appropriate because this avoids the morbidity of gastroin-testinal hemorrhage and biliary and gastric outlet obstruction before death from the metastatic disease. The role of cytoreduc-tive surgery in

1	because this avoids the morbidity of gastroin-testinal hemorrhage and biliary and gastric outlet obstruction before death from the metastatic disease. The role of cytoreduc-tive surgery in metastatic pNET is controversial, but consensus guidelines agree that aggressive resection of the primary tumor, regional lymph nodes, and liver/distant metastases should be pursued if greater than 90% of the tumor burden can be resected, which is the case in only about 10% of patients with metastatic disease.Treatment of metastatic pancreatic neuroendocrine cancer requires a multidisciplinary approach often including a com-bination of cytoreductive surgery when appropriate, directed therapy for the treatment of liver metastases when possible, and systemic medical therapy. Local ablative therapies include radiofrequency ablation (RFA), cryotherapy, microwave coagu-lation, and ethanol injection, although RFA is the most popular and widely studied. These therapies can be performed percuta-neously or

1	radiofrequency ablation (RFA), cryotherapy, microwave coagu-lation, and ethanol injection, although RFA is the most popular and widely studied. These therapies can be performed percuta-neously or during surgery via laparotomy or laparoscopy, can be repeated, and have been shown to complement resection of the primary tumor and amenable liver metastases; making pallia-tive surgery possible for patients that would otherwise not meet criteria. This approach often improves symptoms and 5-year survival is improved to 48%.305Transarterial chemoembolization (TACE) can be employed as palliative therapy in patients with liver metastases not amenable to surgical resection and/or ablation. It relies on the principle that metastatic tumor cells derive the majority of their oxygen supply from the hepatic artery as opposed to hepa-tocytes, which receive oxygen primarily from the portal vein. Performed via angiography, embolization may be performed alone (bland embolization) or in combination with

1	artery as opposed to hepa-tocytes, which receive oxygen primarily from the portal vein. Performed via angiography, embolization may be performed alone (bland embolization) or in combination with chemothera-peutic agents (chemoembolization). Radioembolization, the selective distribution of radioactive yttrium-90 microspheres into the peritumoral vasculature via branches of the hepatic artery, is another alternative.Somatostatin analogs can inhibit release of hormones from functional pNETS and reduce diarrhea in patients with VIPo-mas, glucagonomas, and somatostatinomas, and it can also help the rash of glucagonomas. Somatostatin analogs such as octreotide (Sandostatin LAR Depot), lanreotide (Somatuline Depot), and a new analog pasireotide (Sifnifor LAR) are also used to slow the growth of some functional and nonfunctional pNETS. Disease seen on an octreotide scan has somatostatin receptors and would be expected to respond. Patients with unre-sectable disease are often treated with

1	of some functional and nonfunctional pNETS. Disease seen on an octreotide scan has somatostatin receptors and would be expected to respond. Patients with unre-sectable disease are often treated with somatostatin analogs first before targeted therapy and cytotoxic chemotherapy are utilized because the side effects are minimal.Some targeted drugs can be helpful in treating advanced pNETs that progress despite somatostatin analogs. Sunitinib (Sutent) attacks new blood vessel growth and other targets that help cancer cells grow. Everolimus (Afinitor) works by block-ing a cell protein known as mammalian target of rapamycin (mTOR) and the VEGF pathway, which normally helps cells grow and divide. These targeted agents induce stabilization much more frequently than response, but they tend to carry fewer side effects than traditional cytotoxic chemotherapy. Cytotoxic chemotherapy for pNETS is usually reserved for large tumors or quickly growing tumors that are causing symptoms, or tumors that

1	fewer side effects than traditional cytotoxic chemotherapy. Cytotoxic chemotherapy for pNETS is usually reserved for large tumors or quickly growing tumors that are causing symptoms, or tumors that progressed despite somatostatin analogs and targeted ther-apy. Platinum-based chemotherapy does, however, remain the standard of care for high-grade (poorly differentiated) pNET, yielding high response rates but typically short-lived benefit. The newer cytotoxic combination of capecitabine and temozolo-mide has shown activity in well-differentiated pNET.306Neoplasms of the Exocrine PancreasEpidemiology and Risk Factors. It is estimated that in 2017, 53,670 Americans will be diagnosed with pancreatic cancer and 43,090 will die from the disease. Overall, pancreatic cancer has the worst prognosis of all malignancies with a 5-year survival rate of only 7.2%.307 The incidence of pancreatic cancer continues to increase, perhaps related to the increased incidence of risk factors such as obesity

1	of all malignancies with a 5-year survival rate of only 7.2%.307 The incidence of pancreatic cancer continues to increase, perhaps related to the increased incidence of risk factors such as obesity and diabetes, and as a result, it is predicted that pancreatic cancer will become the leading cause of cancer deaths in the United States by 2050. Pancreatic cancer recently surpassed breast cancer and is now the third leading cause of cancer death behind lung and colorectal cancer. Despite its ubiquity, this dis-ease is extremely difficult to treat, and its exact cause is unknown. However, epidemiologic studies linking various environmental and host factors provide some clues. Recent discoveries using modern molecular biologic techniques have also improved our understanding of the causes of pancreatic cancer. The etiology of pancreatic cancer likely involves a complex interaction of genetic and environmental factors. These factors will become more fully understood as DNA sequencing is used

1	cancer. The etiology of pancreatic cancer likely involves a complex interaction of genetic and environmental factors. These factors will become more fully understood as DNA sequencing is used to screen populations at risk for developing pancreatic cancer.Pancreatic cancer is more common in older adults with most patients being 75 to 84 years old. Pancreatic cancer is more common in African Americans and slightly more com-mon in men than women. The risk of developing pancreatic cancer is two to three times higher if a parent or sibling had the disease. Another risk factor that is consistently linked to pan-creatic cancer is cigarette smoking. Smoking increases the risk of developing pancreatic cancer by at least twofold due to the carcinogens in cigarette smoke.308 Coffee and alcohol consump-tion have been investigated as possible risk factors, but the data are inconsistent. As in other GI cancers, diets high in fat and low in fiber, fruits, and vegetables are thought to be associated

1	have been investigated as possible risk factors, but the data are inconsistent. As in other GI cancers, diets high in fat and low in fiber, fruits, and vegetables are thought to be associated with an increased risk of pancreatic cancer.Diabetes has been known to be associated with pancreatic cancer for many years. In fact, glucose intolerance is present in 80% of patients with pancreatic cancer, and approximately 20% have overt diabetes, a much greater incidence than would be expected to occur by chance. Preexisting type 2 diabetes increases the risk for development of pancreatic cancer by about twofold.309 The new onset of diabetes also can be an early mani-festation of otherwise occult pancreatic cancer. Thus, the new onset of diabetes, or a sudden increase in insulin requirement in an older adult patient with preexisting diabetes, should provoke concern for the presence of pancreatic cancer.Recent epidemiologic studies have confirmed the fact that patients with chronic

1	in an older adult patient with preexisting diabetes, should provoke concern for the presence of pancreatic cancer.Recent epidemiologic studies have confirmed the fact that patients with chronic pancreatitis, especially familial pan-creatitis, have an increased risk of developing pancreatic can-cer.124,196-198 Large, retrospective cohort studies of patients with pancreatitis have revealed up to a 20-fold increase in risk for pancreatic cancer. This increased risk seems to be independent of the type of pancreatitis, a finding consistent with the fact that most studies have shown little effect of alcohol ingestion per se Brunicardi_Ch33_p1429-p1516.indd 148401/03/19 6:46 PM 1485PANCREASCHAPTER 33on the risk of pancreatic carcinoma. The mechanisms involved in carcinogenesis in patients with preexisting pancreatitis are unknown. However, the mutated K-ras oncogene, which is pres-ent in most cases of pancreatic cancer, has been detected in the ductal epithelium of some patients with

1	with preexisting pancreatitis are unknown. However, the mutated K-ras oncogene, which is pres-ent in most cases of pancreatic cancer, has been detected in the ductal epithelium of some patients with chronic pancreatitis.Genetics of Pancreatic Cancer. Pancreatic carcinogenesis probably involves multiple mutations that are inherited and acquired throughout aging. The K-ras oncogene is currently thought to be the most commonly mutated gene in pancreatic cancer, with approximately 90% of tumors having a mutation.310 This prevalent mutation is present in precursor lesions and is therefore thought to occur early and be essential to pancre-atic cancer development. K-ras mutations can be detected in DNA from serum, stool, pancreatic juice, and tissue aspirates of patients with pancreatic cancer, suggesting that the presence of this mutation or others may provide the basis for diagnostic testing in select individuals. The HER2/neu oncogene, homolo-gous to the epidermal growth factor receptor

1	suggesting that the presence of this mutation or others may provide the basis for diagnostic testing in select individuals. The HER2/neu oncogene, homolo-gous to the epidermal growth factor receptor (EGFr), is over-expressed in pancreatic cancers.310 This receptor is involved in signal transduction pathways that lead to cellular proliferation. Multiple tumor-suppressor genes are deleted and/or mutated in pancreatic cancer, including p53, p16, and DPC4 (Smad 4), and in a minority of cases, BRCA2.311 Most pancreatic cancers have three or more of the aforementioned mutations.The genetic landscape of pancreatic adenocarcinoma has recently been investigated using exome capture technol-ogy combined with the SOLiD or Illumina next generation sequencing platforms and copy number analysis. Detailed analysis of 99 tumors reaffirmed the importance of the already known mutations such as KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A, and SF3B1 in pancreatic cancer and identified eight novel

1	analysis of 99 tumors reaffirmed the importance of the already known mutations such as KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A, and SF3B1 in pancreatic cancer and identified eight novel significantly mutated genes involved in chromatin modification (EPC1 and ARID2), DNA dam-age repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4, and MAGEA6). Pathway-based analysis of recurrently altered genes also revealed the involvement of axon-guidance genes, particularly SLIT/ROBO signaling, in pancreatic carcinogenesis.312 Rapid and sensitive sequencing techniques will hopefully lead to better diagnostic and thera-peutic approaches for pancreatic cancer.It is estimated that up to 10% of pancreatic cancers occur as a result of an inherited genetic predisposition. A family his-tory of pancreatic cancer in a first-degree relative increases the risk of pancreatic cancer by about twofold. Rare familial cancer syndromes that are associated with an increased risk of pancreatic

1	of pancreatic cancer in a first-degree relative increases the risk of pancreatic cancer by about twofold. Rare familial cancer syndromes that are associated with an increased risk of pancreatic cancer include BRCA2, the familial atypical multiple mole–melanoma syndrome, hereditary pancreati-tis, familial adenomatous polyposis (FAP), hereditary non-polyposis colorectal cancer, Peutz-Jeghers syndrome, and ataxia-telangiectasia.311Pathology. Pancreatic cancer probably arises through a stepwise progression of cellular changes, just as colon cancer progresses by stages from hyperplastic polyp to invasive cancer. Systematic histologic evaluation of areas surrounding pancreatic cancers has revealed the presence of precursor lesions that have been named pancreatic intraepithelial neoplasia (Fig. 33-67). Three stages of pancreatic intraepithelial neoplasia have been defined. These lesions demonstrate the same oncogene mutations and loss of tumor-suppressor genes found in invasive cancers, the

1	33-67). Three stages of pancreatic intraepithelial neoplasia have been defined. These lesions demonstrate the same oncogene mutations and loss of tumor-suppressor genes found in invasive cancers, the frequency of these abnormalities increasing with progressive cellular atypia and architectural disarray.313 The ability to detect these precursor lesions in humans at a stage where the cancer can still be prevented or cured is an important goal of current pancreatic cancer research.About two-thirds of pancreatic adenocarcinomas arise within the head or uncinate process of the pancreas; 15% are in the body, and 10% are in the tail, with the remaining tumors demonstrating diffuse involvement of the gland. Tumors in the pancreatic body and tail are generally larger at the time of diagnosis, and therefore, less commonly resectable. Tumors in the head of the pancreas are typically diagnosed earlier because they cause obstructive jaundice. Ampullary carci-nomas, carcinomas of the distal bile

1	therefore, less commonly resectable. Tumors in the head of the pancreas are typically diagnosed earlier because they cause obstructive jaundice. Ampullary carci-nomas, carcinomas of the distal bile duct, and periampullary duodenal adenocarcinomas present in a similar fashion to pan-creatic head cancer but have a slightly better prognosis, prob-ably because early obstruction of the bile duct and jaundice leads to the diagnosis.In addition to ductal adenocarcinoma, which makes up about 75% of nonendocrine cancers of the pancreas, there are a variety of less common types of pancreatic cancer. Adenosquamous carcinoma is a variant that has both glandular and squamous differentiation. The biologic behavior of this lesion is unfortunately no better than the typical ductal adenocarcinoma.314 Acinar cell carcinoma is an uncommon type of pancreatic cancer that usually presents as a large tumor, often 10 cm in diameter or more, but the prognosis of patients with these tumors may be better than

1	cell carcinoma is an uncommon type of pancreatic cancer that usually presents as a large tumor, often 10 cm in diameter or more, but the prognosis of patients with these tumors may be better than with ductal cancer.Diagnosis and Staging. Exact pathologic staging of pancre-atic cancer is important because it allows accurate quantitative assessment of results and comparisons between institutions. The tumor-node-metastasis (TNM) staging of pancreatic cancer was updated by the American Joint Committee on Cancer in 2017 (AJCC) (Table 33-21).The important changes in the staging include more stratification for tumor size and stratification for number of lymph nodes involved. In the new system, tumors ≤2 cm remain as T1 lesions but are subcategorized as T1a (≤0.5 cm), T1b (>0.5–>1 cm), and T1c (1–2 cm). Tumors that are >2–≤4 cm are categorized as T2, and tumors >4 cm are categorized as T3. Tumors that involve the celiac axis, superior mesenteric artery, and/or common hepatic artery are still

1	cm). Tumors that are >2–≤4 cm are categorized as T2, and tumors >4 cm are categorized as T3. Tumors that involve the celiac axis, superior mesenteric artery, and/or common hepatic artery are still categorized as T4 regardless of size. Metastasis in one to three regional lymph nodes is considered N1 disease, and involvement of ≥4 regional lymph nodes is N2. Patients without nodal involvement (N0) are stages IA, IB, and IIA based on tumor size (T1–T3, respectively). Patients with one to three regional lymph nodes involved (N2) are stage IIB regardless of tumor size. All patients with N2 disease without distant metastases are considered stage III regardless of tumor size. Patients with vascular involvement (T4) are stage III regardless of nodal involvement, and patients with distant metastases are, of course, stage IV.Ten percent of pancreas cancer cases are diagnosed while the cancer is still confined to the primary site (localized stage); 29% are diagnosed after the cancer has spread

1	are, of course, stage IV.Ten percent of pancreas cancer cases are diagnosed while the cancer is still confined to the primary site (localized stage); 29% are diagnosed after the cancer has spread to regional lymph nodes or directly beyond the primary site; 52% are diagnosed after the cancer has already metastasized (distant stage); and for the remaining 9%, the staging information was unknown. The corresponding 5-year relative survival rates were 31.5% for localized, 11.5% for regional, 2.7% for distant, and 5.1% for unstaged. The overall 5-year relative survival rate for patients with pancreatic cancer for 2007 to 2013 from Surveillance, Epidemiology, and End Results (SEER) was 8.2%.3077Brunicardi_Ch33_p1429-p1516.indd 148501/03/19 6:46 PM 1486SPECIFIC CONSIDERATIONSPART IINormalPanIN-1APanIN-1BPanIN-2KI-67, Topo˜˜°, 14-3-3˛p53, DPC4, BRCA2Cyclin D1MesothelinMucin 1Mucin 1p16PSCA, Mucin5, FascinTelomere K-rasshorteningPanIN-3InvasionABCDFigure 33-67. Pancreatic

1	Topo˜˜°, 14-3-3˛p53, DPC4, BRCA2Cyclin D1MesothelinMucin 1Mucin 1p16PSCA, Mucin5, FascinTelomere K-rasshorteningPanIN-3InvasionABCDFigure 33-67. Pancreatic intraepithelial neoplasia (PanIN). Histology (top panel) showing grades PanIN-1A (A), PanIN-1B (B), PanIN-2 (C), and PanIN-3 (D), and schema of correlation of histology with mutational events (bottom panel) showing cumulative abnormalities of tumor-promoter and tumor suppressor factors such as kRAS, p53, etc, and their corresponding cellular phenotype. (Reproduced with permis-sion from Hruban RH, Takaori K, Klimstra DS, et al: An illustrated consensus on the classification of pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasms, Am J Surg Pathol. 2004 Aug;28(8):977-987.)The most critical deficit in the ability to treat pancreatic cancer effectively is the lack of tools for early diagnosis. The pancreas is situated deep within the abdomen, and the early symptoms of pancreatic cancer often are too

1	ability to treat pancreatic cancer effectively is the lack of tools for early diagnosis. The pancreas is situated deep within the abdomen, and the early symptoms of pancreatic cancer often are too vague to raise sus-picion of the disease. Ultimately, the majority of patients present with pain and jaundice. On physical examination, weight loss is evident and the skin is icteric; a distended gallbladder is palpa-ble in about one-fourth of patients. More fortunate patients have tumors situated such that biliary obstruction and jaundice occurs early and prompts diagnostic tests. Unfortunately, however, the vast majority of patients are not diagnosed until weight loss has occurred—a sign of advanced disease.Although it is often taught that carcinoma of the pancreas presents with painless jaundice (to help distinguish it from cho-ledocholithiasis), this aphorism is not accurate. Most patients do experience pain as part of the symptom complex of pancreatic cancer, and it is often the first

1	(to help distinguish it from cho-ledocholithiasis), this aphorism is not accurate. Most patients do experience pain as part of the symptom complex of pancreatic cancer, and it is often the first symptom. Therefore, awareness of the way pancreatic pain is perceived may help clinicians sus-pect pancreatic cancer. The pain associated with pancreatic can-cer is usually perceived in the epigastrium but can occur in any Brunicardi_Ch33_p1429-p1516.indd 148601/03/19 6:46 PM 1487PANCREASCHAPTER 33the serum of patients with pancreatic cancer. Serum levels are elevated in about 75% of patients with pancreatic cancer. How-ever, CA19-9 is also elevated in about 10% of patients with benign diseases of the pancreas, liver, and bile ducts.15 CA19-9 is thus neither sufficiently sensitive nor specific to allow an early diagnosis of pancreatic cancer. Despite the fact that many tumor markers such as CA19-9 have been studied, there are still no effective screening tests for pancreatic cancer.

1	to allow an early diagnosis of pancreatic cancer. Despite the fact that many tumor markers such as CA19-9 have been studied, there are still no effective screening tests for pancreatic cancer. Research tak-ing advantage of recent advances in genomics, gene expression analysis, and proteomics has demonstrated thousands of genes and corresponding proteins that are differentially expressed in pancreatic tumors that have potential for early detection of pan-creatic cancer.316 Some of these proteins would be expected to be expressed at the cell surface or in pancreatic juice and may become useful as biomarkers for pancreatic cancer in the future.In patients presenting with jaundice, a reasonable first diagnostic imaging study is abdominal ultrasound. If bile duct dilation is not seen, hepatocellular disease is likely. Demonstra-tion of cholelithiasis and bile duct dilation suggests a diagnosis of choledocholithiasis, and the next logical step would be ERCP to clear the bile duct. In the

1	disease is likely. Demonstra-tion of cholelithiasis and bile duct dilation suggests a diagnosis of choledocholithiasis, and the next logical step would be ERCP to clear the bile duct. In the absence of gallstones, malignant obstruction of the bile duct is likely, and a CT scan rather than ERCP would be the next logical step. For patients suspected of having pancreatic cancer who present without jaundice, a CT scan should be the first test.The current diagnostic and staging test of choice for pan-creatic cancer is a multidetector, dynamic, contrast-enhanced CT scan, and the techniques for obtaining high-quality images are constantly improving (Fig. 33-68). The accuracy of CT scan-ning for predicting unresectable disease is about 90% to 95%.317 In contrast, CT scanning is less accurate in predicting resect-able disease. CT scanning will miss small liver metastases, and predicting arterial involvement is sometimes difficult. CT find-ings that indicate a tumor is unresectable include

1	in predicting resect-able disease. CT scanning will miss small liver metastases, and predicting arterial involvement is sometimes difficult. CT find-ings that indicate a tumor is unresectable include involvement of ≥180° of the celiac axis, hepatic or superior mesenteric artery, enlarged lymph nodes outside the boundaries of resection, asci-tes, and distant metastases (e.g., liver). Invasion of the superior mesenteric vein or portal vein is not in itself a contraindication to resection as long as the veins are patent. Tumors are consid-ered “borderline resectable” if there is abutment of ≤180 degrees of the circumference of the SMA, celiac axis, or hepatic artery or if there is a short segment of vein occlusion. Also, patients with CT findings suspicious for metastatic disease, like 1 mm liver lesions too small to characterize or biopsy, are considered “borderline resectable” as are patients with multiple comorbidi-ties or marginal performance status. There is growing consensus that

1	liver lesions too small to characterize or biopsy, are considered “borderline resectable” as are patients with multiple comorbidi-ties or marginal performance status. There is growing consensus that neoadjuvant treatment should be considered in all patients with any radiographic evidence of extension to adjacent vascu-lar structures.Currently, multidetector CT is probably the single most versatile and cost-effective tool for the diagnosis and staging of pancreatic cancer. Abdominal MRI provides essentially the same information as CT scanning. Positron emission tomog-raphy scanning in locally advanced lesions may help rule out distant metastases. EUS can be used to detect small pancreatic masses that could be missed by CT scanning and is commonly used when there is a high suspicion for pancreatic cancer but no mass is identified by the CT scan. EUS has the added advan-tage of providing the opportunity for transluminal biopsy of pancreatic masses, although a tissue diagnosis before

1	pancreatic cancer but no mass is identified by the CT scan. EUS has the added advan-tage of providing the opportunity for transluminal biopsy of pancreatic masses, although a tissue diagnosis before pancreati-coduodenectomy is not required. However, in specific patients a histologic diagnosis may be necessary such as for those in a part of the abdomen, and often, but not always, penetrates to the back. When questioned in retrospect, patients often recall mild and vague pain for many months before diagnosis. A low thresh-old for ordering a CT scan with “pancreatic protocol” should be maintained for elderly patients with unex-plained, persistent, although vague, abdominal pain. As men-tioned previously, new-onset diabetes in an elderly patient, especially if combined with vague abdominal pain, should prompt a search for pancreatic cancer.Unfortunately, at this time there is no sensitive and spe-cific serum marker to assist in the timely diagnosis of pan-creatic cancer. With jaundice,

1	pain, should prompt a search for pancreatic cancer.Unfortunately, at this time there is no sensitive and spe-cific serum marker to assist in the timely diagnosis of pan-creatic cancer. With jaundice, direct hyperbilirubinemia and elevated alkaline phosphatase are expected but do not serve much of a diagnostic role other than to confirm the obvious. With long-standing biliary obstruction, the prothrombin time will be prolonged due to a depletion of vitamin K, a fat-soluble vitamin dependent on bile flow for absorption. CA19-9 is a mucin-associated carbohydrate antigen that can be detected in 8Table 33-21Staging of pancreatic cancerTumor (T)TXTumor cannot be assessedT1Tumor limited to the pancreas,* <2 cmT2Tumor limited to the pancreas,* 2–4 cmT3Tumor limited to the pancreas,* >4 cm; or tumor invading the duodenum or common bile ductT4Tumor invading adjacent organs (stomach, spleen, colon, adrenal gland) or the wall of large vessels (celiac axis or the superior mesenteric

1	>4 cm; or tumor invading the duodenum or common bile ductT4Tumor invading adjacent organs (stomach, spleen, colon, adrenal gland) or the wall of large vessels (celiac axis or the superior mesenteric artery)Regional Lymph Node (N)NXRegional lymph nodes cannot be assessedN0No regional lymph node involvementN1Regional lymph node involvementDistant Metastasis (M)M0No distant metastasisM1Distant metastasis M1aMetastasis confined to liver M1bMetastases in at least one extrahepatic site (e.g., lung, ovary, nonregional lymph node, peritoneum, bone) M1cBoth hepatic and extrahepatic metastasesTUMORNODEMETASTASISSTAGET1N0M0IT2N0M0IIT3N0M0IIT4N0M0IIIAny TN1M0IIIAny TAny NM1IVUsed with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Brunicardi_Ch33_p1429-p1516.indd 148701/03/19 6:46 PM 1488SPECIFIC CONSIDERATIONSPART IIPortal veinStomachHepaticarteryCeliac axisSplenic veinDilated

1	Manual, 8th Ed. Springer New York, 2017.Brunicardi_Ch33_p1429-p1516.indd 148701/03/19 6:46 PM 1488SPECIFIC CONSIDERATIONSPART IIPortal veinStomachHepaticarteryCeliac axisSplenic veinDilated pancreatic ductin body of pancreasGallbladderPortal veinPortal veinVena cavaDilated bile ductwith stentBody of pancreasGallbladderSplenoportalconfluenceDuodenumMass in headof pancreasMass in head,uncinate processof pancreasSMASMASMADuodenumBile duct stentFat plane between massin head of pancreasand portal veinFigure 33-68. Computed tomography scan demonstrating resectable pancreatic cancer. SMA = superior mesenteric artery.neoadjuvant clinical trial or before chemotherapy in advanced tumors. EUS is a sensitive test for portal/superior mesenteric vein invasion, although it is somewhat less effective at detect-ing superior mesenteric artery invasion. When all of the current staging modalities are used, their accuracy in predicting resect-ability has improved.As imaging continuously improves and

1	at detect-ing superior mesenteric artery invasion. When all of the current staging modalities are used, their accuracy in predicting resect-ability has improved.As imaging continuously improves and high-quality imag-ing is always obtained before surgery, the chance of bringing a patient to the operating room with the intent of a curative resec-tion and finding upon exploration that the patient has unresect-able disease is becoming increasingly uncommon.In an attempt to avoid such futile laparotomies, prelimi-nary laparoscopy has been advocated for patients with disease felt to be resectable by CT imaging (Fig. 33-69). Diagnostic laparoscopy with the use of US is reported to improve the accu-racy of predicting resectability to about 98%.318 The technique involves more than simple visualization with the scope and requires the placement of multiple ports and manipulation of the tissues. A general exploration of the peritoneal surfaces is carried out. The ligament of Treitz and the base

1	with the scope and requires the placement of multiple ports and manipulation of the tissues. A general exploration of the peritoneal surfaces is carried out. The ligament of Treitz and the base of the transverse mesocolon are examined for tumor. The gastrocolic ligament is incised, and the lesser sac is examined. The ultrasound probe is used to examine the liver, porta hepatis and the portal vein, the celiac axis, and the superior mesenteric artery.The percentage of patients in whom a positive laparoscopy helps avoid a nontherapeutic laparotomy varies from 10% to 30% in carcinoma of the head of the pancreas, but it may be as high as 50% in patients with tumors in the body and tail of the gland. Resection for pancreatic cancer is being approached laparoscopically, particularly for tumors in the body and tail of the pancreas thus eliminating the need for any separate stag-ing laparoscopy procedure. Also, as the quality of CT scanning has improved, the value of routine diagnostic

1	in the body and tail of the pancreas thus eliminating the need for any separate stag-ing laparoscopy procedure. Also, as the quality of CT scanning has improved, the value of routine diagnostic laparoscopy has decreased. The morbidity of diagnostic laparoscopy is less than that of laparotomy, and the procedure can be performed on an outpatient basis. Patients who are found to have unresectable disease recover more rapidly from a laparoscopy than a lapa-rotomy and can receive palliative chemotherapy and radiation sooner. The potential immunosuppressive effects of a major surgical procedure also are avoided, as well as the negative psy-chologic impact of a major painful operation with little benefit.Biliary obstruction can be relieved with an endoscopic approach in almost all cases. When large (10F) plastic stents are used, most patients do not require replacement for about 3 months. Metallic wall stents last about 5 months on average and usually fail only with tumor ingrowth.319

1	large (10F) plastic stents are used, most patients do not require replacement for about 3 months. Metallic wall stents last about 5 months on average and usually fail only with tumor ingrowth.319 Keeping in mind that patients with unresectable pancreatic cancer usually live <1 year, the requirement for numerous stent changes is unlikely.Diagnostic laparoscopy is possibly best applied to patients with pancreatic cancer on a selective basis. Diagnostic laparos-copy will have a higher yield in patients with large tumors (T3, Figure 33-69. Liver metastases identified at diagnostic laparoscopy.Brunicardi_Ch33_p1429-p1516.indd 148801/03/19 6:46 PM 1489PANCREASCHAPTER 33>4 cm), tumors located in the body or tail, patients with equivo-cal findings of metastasis or ascites on CT scan, and patients with other indications of advanced disease such as marked weight loss or markedly elevated CA19-9 (>1000 U/mL). An algorithm for the diagnosis, staging, and treatment of pan-creatic cancer is

1	with other indications of advanced disease such as marked weight loss or markedly elevated CA19-9 (>1000 U/mL). An algorithm for the diagnosis, staging, and treatment of pan-creatic cancer is shown in Fig. 33-70. In practice, many of these patients are selected for neoadjuvant chemotherapy and then undergo restaging CT and staging laparoscopy prior to surgery.Palliative Surgery and Endoscopy. Most patients with pan-creatic cancer (85–90%) have disease that clearly precludes surgical resection. For these patients, appropriate and effective palliative treatment is critical to the quality of their remaining life. Because of the poor prognosis of the disease, it is not appro-priate to use invasive, toxic, and expensive regimens in patients with extremely advanced disease and poor performance status. When patients do desire antineoplastic therapy, it is important to encourage them to enroll in clinical trials so that therapeutic advances can be made. In general, there are three clinical

1	status. When patients do desire antineoplastic therapy, it is important to encourage them to enroll in clinical trials so that therapeutic advances can be made. In general, there are three clinical prob-lems in advanced pancreatic cancer that require palliation: pain, jaundice, and duodenal obstruction. The mainstay of pain con-trol is oral narcotics. Sustained-release preparations of morphine sulfate are frequently used. Invasion of retroperitoneal nerve trunks accounts for the severe pain experienced by patients with advanced pancreatic cancer. A celiac plexus nerve block can control pain effectively for a period of months, although the procedure sometimes needs to be repeated.320Jaundice is present in the majority of patients with pan-creatic cancer, and the most troublesome aspect for the patient is the accompanying pruritus. Biliary obstruction may also lead to cholangitis, coagulopathy, digestive symptoms, and hepato-cellular failure. In the past, surgeons traditionally

1	for the patient is the accompanying pruritus. Biliary obstruction may also lead to cholangitis, coagulopathy, digestive symptoms, and hepato-cellular failure. In the past, surgeons traditionally performed a biliary and enteric bypass when unresectable disease was found at laparotomy. This is an increasingly uncommon situation for the surgeon because locally advanced unresectable disease is now detected by high-quality preoperative imaging. Metastatic disease is also more reliably predicted by preoperative imaging and, in select cases, staging laparoscopy.In current practice, jaundice is usually palliated by an endoscopic biliary stent, often prior to surgical referral. Duo-denal obstruction is usually a late event in pancreatic cancer and occurs in only about 20% of patients.321 In cases of biliary and duodenal obstruction, several options need to be consid-ered. Endoscopic metallic duodenal stents are an option, but the patient’s poor prognosis, the cost, and the fact that duodenal

1	of biliary and duodenal obstruction, several options need to be consid-ered. Endoscopic metallic duodenal stents are an option, but the patient’s poor prognosis, the cost, and the fact that duodenal stents often do not result in ideal palliation has to be considered. The results of three RCTs examining endoscopic metallic stent-ing for malignant gastric outlet obstruction demonstrated that major and minor complications were comparable to gastrojeju-nostomy but time to tolerating oral intake and hospital stay was shorter.322 Robot-assisted laparoscopic biliary-enteric bypass is now available at many centers. Although this should result in similar palliation as an open bypass and may be associated with more rapid recovery, the potential complications, the patient’s life expectancy, and cost of this procedure must be considered. As many patients today already have a bile duct stent in place by the time of referral to a surgeon, it is not clear that operative biliary bypass is required.

1	of this procedure must be considered. As many patients today already have a bile duct stent in place by the time of referral to a surgeon, it is not clear that operative biliary bypass is required. In patients with extensive metastases, an alternative short-term palliative option to consider in patients with gastric outlet obstruction is a percutaneous endoscopic gas-trostomy tube or gastrojejunal feeding tube that allows decom-pression of the stomach and feeding into the jejunum.If an operative bypass is performed, choledochojejunos-tomy is the preferred approach. Although an easy procedure to perform, choledochoduodenostomy is felt to be unwise because of the proximity of the duodenum to tumor. Some have discour-aged the use of the gallbladder for biliary bypass; however, it CT scanQuad phaseMultidetectorFine cutsUnresectablemassCT or EUSguided biopsyClinicaltrialResectable massNo mass butsuspicion remainsClinical trialDiagnosticlaparoscopy(selective)EUS

1	however, it CT scanQuad phaseMultidetectorFine cutsUnresectablemassCT or EUSguided biopsyClinicaltrialResectable massNo mass butsuspicion remainsClinical trialDiagnosticlaparoscopy(selective)EUS biopsydiagnosticlaparoscopyNeoadjuvantclinicaltrialERCP / EUSObserve/reimageWhippleprocedureFigure 33-70. Diagnostic and treatment algorithm for pancreatic cancer. If computed tomography (CT) scan demonstrates a potentially resectable tumor, patients are offered participation in a clinical trial after histologic confirmation by CT or endoscopic ultrasound (EUS)-guided biopsy. If CT scan demonstrates resectable disease, diagnostic laparoscopy is used selectively in patients with tumors in the body/tail, equivocal findings of metastasis or CT scan, ascites, high CA19-9, or marked weight loss. Patients also have diagnostic laparoscopy if they elect to participate in a neoadjuvant clinical trial. In cases where no mass is demonstrated on CT scan, but suspicion of cancer remains, EUS or endoscopic

1	also have diagnostic laparoscopy if they elect to participate in a neoadjuvant clinical trial. In cases where no mass is demonstrated on CT scan, but suspicion of cancer remains, EUS or endoscopic retrograde cholangiopancreatography (ERCP) with brushings are performed, and the CT may be repeated after an interval of observation.Brunicardi_Ch33_p1429-p1516.indd 148901/03/19 6:46 PM 1490SPECIFIC CONSIDERATIONSPART IIis suitable as long as the cystic duct clearly enters the common duct well above the tumor. The jejunum is brought anterior to the colon, if possible, rather than retrocolic, where the tumor potentially would invade the bowel sooner. Some surgeons use a loop of jejunum with a jejunojejunostomy to divert the enteric stream away from the biliary-enteric anastomosis. Others use a Roux-en-Y limb with the gastrojejunostomy located 50 cm downstream from the hepaticojejunostomy (Fig. 33-71). Poten-tial advantages of the defunctionalized Roux-en-Y limb include the ease with

1	use a Roux-en-Y limb with the gastrojejunostomy located 50 cm downstream from the hepaticojejunostomy (Fig. 33-71). Poten-tial advantages of the defunctionalized Roux-en-Y limb include the ease with which it will reach up to the hepatic hilum, prob-able decreased risk of cholangitis, and easier management of biliary anastomotic leaks. If a gastrojejunostomy is performed, it should be placed dependently and posterior along the greater Figure 33-71. Biliary-enteric bypass to palliate unresectable pan-creatic cancer. (Reproduced with permission from Bell RH, Rikkers LF, Mulholland M: Digestive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)Figure 33-72. Expandable metallic biliary stent. After ERCP cannulation of the distal bile duct (left) the stent is advanced over the cannula and placed across the obstruction in the distal bile duct (right).curvature to improve gastric emptying, and a vagotomy should not be performed. Endoscopic stents are

1	is advanced over the cannula and placed across the obstruction in the distal bile duct (right).curvature to improve gastric emptying, and a vagotomy should not be performed. Endoscopic stents are definitely not as dura-ble as a surgical bypass. Recurrent obstruction and cholangitis is more common with stents and results in inferior palliation. However, the endoscopic approach is associated with consider-ably less initial morbidity and mortality than surgical bypass. Expandable metallic wall stents have superior patency and pro-vide better palliation than plastic stents (Fig. 33-72).If an initial diagnostic laparoscopy reveals a contraindica-tion to the Whipple procedure, such as liver metastases, it is not appropriate to perform a laparotomy simply to create a biliary bypass. In such a patient, it is better to place an endoscopic stent. In contrast, in the uncommon scenario where a laparotomy has already been performed as part of the assessment of resectability and the Whipple

1	a patient, it is better to place an endoscopic stent. In contrast, in the uncommon scenario where a laparotomy has already been performed as part of the assessment of resectability and the Whipple procedure is not possible, a surgical bypass is usually performed. However, if the patient has a functioning endoscopic stent already in place, it may be reasonable to forego surgical bypass.Chemotherapy and Radiation for Locally Advanced/ Metastatic Disease. Patients with locally advanced unresectable disease are treated with chemotherapy and possibly radiation, and patients with stage IV metastatic disease are treated with systemic chemotherapy. The role of RT in unresectable, locoregionally advanced pancreas cancer remains controversial. RT may slow the progression of local disease and possibly alleviate or prevent symptoms including pain, biliary obstruction, bleeding, and bowel obstruction. However, the likelihood of micrometastatic distant disease is high, treatment is not expected to

1	alleviate or prevent symptoms including pain, biliary obstruction, bleeding, and bowel obstruction. However, the likelihood of micrometastatic distant disease is high, treatment is not expected to be curative, and radiation can result in toxicity. Stereotactic body radiotherapy (SBRT) has been used to limit toxicity by targeting high-dose short-course radiation to enhance local response prior to surgery. In a phase 2 multi-institutional trial evaluating gemcitabine and SBRT in patients with locally advanced unresectable pancreatic cancer, 10% of patients with locally advanced disease who would not have been candidates for initial surgery were deemed to have resectable tumors following therapy, and 8% ultimately underwent R0 and node-negative resection.323 A number of chemotherapy regimens are available for pancreatic cancer, but the results are not impressive. Gemcitabine (Gemzar) was approved by the U.S. Food and Drug Administration (FDA) for use in pancreatic cancer in 1996. In

1	are available for pancreatic cancer, but the results are not impressive. Gemcitabine (Gemzar) was approved by the U.S. Food and Drug Administration (FDA) for use in pancreatic cancer in 1996. In patients with unresectable pancreatic cancer, gemcitabine results in symptomatic Brunicardi_Ch33_p1429-p1516.indd 149001/03/19 6:46 PM 1491PANCREASCHAPTER 33improvement, improved pain control and performance status, and weight gain.324 However, survival is improved by only 1 to 2 months. Prior to gemcitabine, 5-fluorouracil (5-FU) was used as the standard treatment for unresectable pancreatic cancer. Both of these drugs are still used today. 5-FU or capecitabine (Xeloda), a similar but orally administered drug, are frequently used as a radiosensitizer during radiation therapy. Single-agent gemcitabine is still commonly used in patients with a poor performance status.Erlotinib (Tarceva) was approved in 2005 based on very minimal improvement in overall survival in combination with

1	gemcitabine is still commonly used in patients with a poor performance status.Erlotinib (Tarceva) was approved in 2005 based on very minimal improvement in overall survival in combination with gemcitabine.325 The study showed that erlotinib in combination with gemcitabine results in a statistically significant improve-ment in overall survival in patients with advanced pancreatic cancer in the first-line setting. Although the absolute benefit in overall survival was modest with a median survival difference between the two arms of only 2 weeks.FOLFIRINOX, a combination of three chemotherapy drugs (5-FU/leucovorin, irinotecan, and oxaliplatin) is now commonly used as first-line treatment for metastatic pancreatic adenocarcinoma in patients with a relatively good performance status. In 2010, a Phase 3 clinical trial showed positive results for patients treated with FOLFIRINOX.326 The objective response rate was improved from 9% to 32%, and median overall survival of patients with

1	2010, a Phase 3 clinical trial showed positive results for patients treated with FOLFIRINOX.326 The objective response rate was improved from 9% to 32%, and median overall survival of patients with metastatic pancreatic cancer improved from 7 to 11 months, but the improvement was associated with increased toxicity, so patient selection is important. Patients treated with FOLFIRINOX may experience more severe side effects than those treated with gemcitabine alone, so this combination is usu-ally reserved for patients with a good performance status.In 2013, another combination therapy was approved as first-line treatment for metastatic pancreatic adenocarcinoma.327 Albumin-bound paclitaxel (Abraxane) was approved to be used in combination with gemcitabine (Gemzar). The median overall survival was improved to 8.5 months in the nabpaclitaxel–gemcitabine group as compared with 6.7 months in the gemcitabine group. Progression-free survival and the response rate were also improved. Rates of

1	was improved to 8.5 months in the nabpaclitaxel–gemcitabine group as compared with 6.7 months in the gemcitabine group. Progression-free survival and the response rate were also improved. Rates of peripheral neuropathy and myelosuppression were increased, but this regimen is less toxic than FOLFIRINOX.In 2015, Irinotecan (ONIVYDE), in combination with 5-FU (fluorouracil) and leucovorin, was approved as treatment for met-astatic pancreatic adenocarcinoma that has progressed following treatment with a gemcitabine based therapy.328 The median over-all survival in patients assigned to nanoliposomal irinotecan plus fluorouracil and folinic acid was 6.1 months vs. 4.2 months with fluorouracil and folinic acid. Common side effects for this second-line therapy were neutropenia, diarrhea, vomiting, and fatigue.These results may warrant treatment in patients who understand the benefits and risks. However, the lack of signifi-cant survival advantage should encourage physicians to refer motivated

1	and fatigue.These results may warrant treatment in patients who understand the benefits and risks. However, the lack of signifi-cant survival advantage should encourage physicians to refer motivated patients for experimental protocols because it is only through continued clinical research that more meaningful treat-ments for pancreatic cancer will be developed.Ablation for Locally Advanced Unresectable Disease. Persistent arterial vascular encasement after neoadjuvant therapy contraindicates resection. Irreversible electroporation utilizes delivery of high-voltage millisecond electrical pulses resulting in permanent disruption of the cellular membranes and subsequent apoptosis. This process leads to cell death, but does not injure the extracellular matrix, thus allowing cellular tumor ablation while preserving structural components of tissues. Collagen-based structures such as vessels or the pancreatic duct are not disrupted. Furthermore, because IRE is not based on thermal damage of

1	while preserving structural components of tissues. Collagen-based structures such as vessels or the pancreatic duct are not disrupted. Furthermore, because IRE is not based on thermal damage of cancer cells, the heat-sink phenomenon is not a concern, and even lesions abutting large vessels can be ablated with radical intent. Irreversible electroporation using the Nanoknife is reported to enable treatment of pancreatic tumors abutting vascular structures without compromise of the vessels or concern for the heat sink effect of nearby blood flow.329 Martin has created a registry and accumulated multi-institutional data on 200 patients with locally advanced pancreatic cancer showing OS of 28.3 months for patients with borderline resectable pancreatic cancer and 23.2 months in patients with unresectable pancreatic cancer. Those numbers compare favorably with the survival of patients treated with chemoradiation alone, which is 13 months in historical controls.330 This modality is new, but

1	pancreatic cancer. Those numbers compare favorably with the survival of patients treated with chemoradiation alone, which is 13 months in historical controls.330 This modality is new, but these early reports indicate it may be safe in combination with chemotherapy and are of particular interest because of the potential to down stage and offer surgery to patients initially diagnosed with locally advanced unresectable disease. It is also important to understand that a significant learning curve exists to achieve safety and optimization of the technique. Proper patient selection, technical ability with intraoperative ultrasound to allow precise IRE electrode bracketing, and standardization of the IRE energy delivery is important. Randomized prospective trials are needed before adoption of this technique can be expanded.Surgical Resection: Pancreaticoduodenectomy. In a patient with appropriate clinical and/or imaging indications of pancreatic cancer, a tissue diagnosis before performing a

1	technique can be expanded.Surgical Resection: Pancreaticoduodenectomy. In a patient with appropriate clinical and/or imaging indications of pancreatic cancer, a tissue diagnosis before performing a pancreaticoduo-denectomy is not essential. Although percutaneous CT-guided biopsy is usually safe, complications such as hemorrhage, pan-creatitis, fistula, and abscess can occur. Tumor seeding along the subcutaneous tract of the needle is uncommon. Likewise, FNA under EUS guidance is safe and well tolerated. The prob-lem with preoperative or even intraoperative biopsy is that many pancreatic cancers are not very cellular and contain a significant amount of fibrous tissue, so a biopsy may be misinterpreted as showing chronic pancreatitis if it does not contain malignant glandular cells. In the face of clinical and radiologic preoperative indications of pancreatic cancer, a negative biopsy should not preclude resection. In patients who are not candidates for resec-tion because of metastatic

1	face of clinical and radiologic preoperative indications of pancreatic cancer, a negative biopsy should not preclude resection. In patients who are not candidates for resec-tion because of metastatic disease, biopsy for a tissue diagnosis becomes important because these patients may be candidates for palliative chemotherapy trials. It is especially important to make an aggressive attempt at tissue diagnosis before surgery in patients whose clinical presentation and imaging studies are more suggestive of alternative diagnoses such as pancreatic lym-phoma or pancreatic islet cell tumors. These patients might avoid surgery altogether in the case of lymphoma or warrant an aggres-sive approach in the case of islet cell carcinoma.Pancreaticoduodenectomy can be performed through a midline incision from xiphoid to umbilicus or through a bilat-eral subcostal incision. The initial portion of the procedure is an assessment of resectability. The liver and visceral and parietal peritoneal surfaces

1	from xiphoid to umbilicus or through a bilat-eral subcostal incision. The initial portion of the procedure is an assessment of resectability. The liver and visceral and parietal peritoneal surfaces are thoroughly assessed. The gastrohepatic omentum is opened, and the celiac axis area is examined for enlarged lymph nodes. The base of the transverse mesocolon is examined for tumor involvement.The ascending and hepatic flexure of the colon are mobi-lized off the duodenum and head of the pancreas and reflected medially. A Kocher maneuver is performed by dissecting behind Brunicardi_Ch33_p1429-p1516.indd 149101/03/19 6:46 PM 1492SPECIFIC CONSIDERATIONSPART IIthe head of the pancreas. The superior mesenteric vein is identi-fied early in the case and dissected up toward the inferior border of the neck of the pancreas. The gastroepiploic vein and artery are ligated to prevent any traction injury. Often, the middle colic vein and right gastroepiploic vein share a common trunk before

1	of the neck of the pancreas. The gastroepiploic vein and artery are ligated to prevent any traction injury. Often, the middle colic vein and right gastroepiploic vein share a common trunk before entering into the superior mesenteric vein. Knowledge of this anatomy helps reduce injury to the veins and unnecessary blood loss. The relation of the tumor to the superior mesenteric vein and artery cannot be accurately assessed by palpation at this point and is not completely determined until later in the opera-tion when the neck of the pancreas is divided and the surgeon is committed to resection. Mesenteric vascular involvement is best determined by a high quality preoperative CT scan.It is important to assess for an aberrant right hepatic artery, which is present in 20% of patients. The aberrant artery com-monly arises from the superior mesenteric artery posterior to the pancreas and ascends parallel and adjacent to the superior mesenteric and portal veins. The presence of an aberrant

1	artery com-monly arises from the superior mesenteric artery posterior to the pancreas and ascends parallel and adjacent to the superior mesenteric and portal veins. The presence of an aberrant right hepatic artery should be apparent on the preoperative CT scan and can be identified intraoperatively by palpation on the back side of the hepatoduodenal ligament, where a prominent pulse will be felt posterior and to the right of the portal vein.The porta hepatis is examined. Enlarged or firm lymph nodes that can be swept down toward the head of the pancreas with the specimen do not preclude resection. If the assessment phase reveals no contraindications to the Whipple procedure (Table 33-22), the resection phase commences.If the pylorus is to be preserved, the stomach and proximal duodenum are mobilized off the pancreas, preserving the gastroepiploic vessels down to the pylorus. The proximal hepatic artery is identified usually by removing a lymph node that commonly lies just anterior to

1	mobilized off the pancreas, preserving the gastroepiploic vessels down to the pylorus. The proximal hepatic artery is identified usually by removing a lymph node that commonly lies just anterior to the artery. The hepatic artery is dissected and traced toward the porta hepatis. The gastroduodenal branch of the hepatic artery is identified. A test clamping is performed to ensure that a strong pulse remains in the proper hepatic artery before division of the gastroduodenal artery. In cases of celiac occlusion, flow comes from the superior mesenteric artery and retrograde through the gastroduodenal artery to the proper hepatic artery. Ligation of the gastroduodenal in this case would be equivalent to hepatic artery ligation. A bypass to the hepatic artery would be required. Once the test clamping is negative and the gastroduodenal artery is divided, the hepatic artery is retracted medially, and the common bile duct is retracted laterally to reveal the anterior surface of the portal vein

1	is negative and the gastroduodenal artery is divided, the hepatic artery is retracted medially, and the common bile duct is retracted laterally to reveal the anterior surface of the portal vein behind them. Dissection is performed only on the anterior surface of the vein. If there is no tumor involvement, the neck of the Table 33-22Findings at explorationFindings contraindicating resection Liver metastases (any size) Celiac lymph node involvement Peritoneal implants Hepatic hilar lymph node involvementFindings not contraindicating resection Invasion at duodenum or distal stomach Involved peripancreatic lymph nodes Involved lymph nodes along the porta hepatis that can be swept down with the specimenpancreas will separate from the vein easily. A large, blunt-tipped clamp is a safe instrument to use for this dissection. The tunnel under the neck of the pancreas can then be completed mostly under direct vision from inferior and superior.The gallbladder is then mobilized from the liver,

1	to use for this dissection. The tunnel under the neck of the pancreas can then be completed mostly under direct vision from inferior and superior.The gallbladder is then mobilized from the liver, the cystic duct and artery are ligated, and the gallbladder is removed. The common hepatic duct is circumferentially dissected. Either the duodenum is divided 2 cm distal to the pylorus (which defines the procedure as a pylorus-preserving pancreaticoduodenectomy, or PPPD) or the antrum is divided, as classically described by Whipple. The jejunum is divided beyond the ligament of Treitz, and the mesentery is ligated until the jejunum can be delivered posterior to the superior mesenteric vessels from left to right.The common hepatic duct is then divided usually just above the entrance of the cystic duct, and the bile duct is dis-sected down to the superior margin of the duodenum. Inferior traction on the distal bile duct opens the plane to make visible the anterior portion of the portal vein.

1	duct, and the bile duct is dis-sected down to the superior margin of the duodenum. Inferior traction on the distal bile duct opens the plane to make visible the anterior portion of the portal vein. The pancreatic neck is divided anterior to the portal vein (Fig. 33-73). The use of cau-tery is avoided in the area of the pancreatic duct. The pancreatic head and uncinate process then are dissected off of the right lateral aspect of the superior mesenteric vein, ligating the fragile branches draining the head and uncinate process into the portal vein (Fig. 33-74). The uncinate process is then dissected off of the posterior and lateral aspect of the superior mesenteric artery. This can be the most tedious portion of the operation, Figure 33-73. Division of the pancreatic neck. The pancreatic neck is separated from the anterior surface of the portal vein and then divided. If there is no tumor involvement, the neck of the pan-creas will separate from the vein easily. A large, blunt-tipped

1	neck is separated from the anterior surface of the portal vein and then divided. If there is no tumor involvement, the neck of the pan-creas will separate from the vein easily. A large, blunt-tipped clamp is a safe instrument to use for this dissection. (Reproduced with per-mission from Bell RH, Rikkers LF, Mulholland M: Digestive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)Brunicardi_Ch33_p1429-p1516.indd 149201/03/19 6:46 PM 1493PANCREASCHAPTER 33Figure 33-74. Dissection of the pancreatic head and uncinate pro-cess. The pancreatic head and uncinate process are dissected off of the right lateral aspect of the superior mesenteric vein and portal vein by ligating the fragile venous branches. (Reproduced with per-mission from Bell RH, Rikkers LF, Mulholland M: Digestive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)but thoroughly clearing all tissue from the mesenteric vessels helps avoid

1	Rikkers LF, Mulholland M: Digestive Tract Surgery: A Text and Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 1996.)but thoroughly clearing all tissue from the mesenteric vessels helps avoid incomplete resection. The wound is irrigated and meticulous hemostasis is assured at this point because the view of the portal vein area and retroperitoneum is more difficult after the reconstruction phase is completed.The reconstruction involves anastomoses of the pancreas first, then the bile duct, and, finally, the duodenum or stomach. There are various techniques for the pancreatic anastomoses. After the pancreatic anastomosis is completed, the choledocho-jejunostomy is performed about 10 cm down the jejunal limb from the pancreatic anastomosis. This is usually performed in an end-to-side fashion with one layer of interrupted sutures. The duodenojejunostomy or gastrojejunostomy is performed another 10 to 15 cm downstream from the biliary anastomosis, using a two-layer

1	an end-to-side fashion with one layer of interrupted sutures. The duodenojejunostomy or gastrojejunostomy is performed another 10 to 15 cm downstream from the biliary anastomosis, using a two-layer technique.Pancreaticoduodenectomy With Vascular Resection. Accurate staging of pancreatic cancer with a high-quality pre-operative CT scan is extremely important. Involvement of the superior mesenteric artery, the celiac axis, or hepatic artery indicate a T4/stage 3 tumor that is locally advanced and unre-sectable. However, involvement of the portal vein or superior mesenteric vein does not necessarily preclude resection as long as there is a patent superior mesenteric vein-portal vein conflu-ence. Reconstruction can often be accomplished with a primary anastomosis of the vein. When resection of more than 2 cm of vein is required, an interposition graft such as the internal jugu-lar vein can be used for a tension-free reconstruction.In a systematic review of 28 retrospective studies from

1	of more than 2 cm of vein is required, an interposition graft such as the internal jugu-lar vein can be used for a tension-free reconstruction.In a systematic review of 28 retrospective studies from high-volume pancreas centers, the morbidity, mortality, and survival outcome after undertaking extended pancreaticoduodenectomy with vascular resection for pancreatic cancer with venous involvement and/or limited arterial involvement was acceptable.331 Another more recent report from five teaching hospitals demonstrated that the Whipple procedure combined with vascular resection and reconstruction could achieve complete removal of tumors without significantly increasing the mortality rate, and the median survival time was higher than that of patients who underwent palliative treatment.332 However, contrary to the findings of analyses from expert pancreas centers, a retrospective cohort analysis using the National Surgical Quality Improvement Program data from 2005 to 2009 demonstrated

1	However, contrary to the findings of analyses from expert pancreas centers, a retrospective cohort analysis using the National Surgical Quality Improvement Program data from 2005 to 2009 demonstrated increased 30-day postoperative morbidity and mortality in pancreaticoduodenectomy with vein resection when compared with pancreaticoduodenectomy alone.333 These data indicate that pancreaticoduodenectomy with vascular resection is only a reasonable option when it is performed after neoadjuvant therapy and at very-high-volume centers where the morbidity of the operation can be kept to a minimum.Minimally Invasive Pancreatectomy. Laparoscopic distal pancreatectomy has been proven to be safe and is appropriate for essentially all indications for pancreatectomy. This approach is associated with decreased blood loss and quicker recovery. Centrally located lesions near the splenoportal confluence must be approached laparoscopically with caution. Robotic technology may make dissection and

1	with decreased blood loss and quicker recovery. Centrally located lesions near the splenoportal confluence must be approached laparoscopically with caution. Robotic technology may make dissection and control of major vessels in this area easier and perhaps safer. Interest in laparoscopic pancreaticoduodenectomy is increasing throughout the United States with some early adopters reporting excellent outcomes comparable to the open procedure.334 Early results indicate this technique is feasible, but considerable expertise is required in both open pancreatic resection as well as advanced laparoscopic techniques to achieve these outcomes. Whether the advantages seen in other areas of minimally invasive surgery apply to the Whipple procedure is an area of current investigation.Variations and Controversies. The preservation of the pylorus has several theoretical advantages, including preven-tion of reflux of pancreaticobiliary secretions into the stomach, decreased incidence of marginal

1	Controversies. The preservation of the pylorus has several theoretical advantages, including preven-tion of reflux of pancreaticobiliary secretions into the stomach, decreased incidence of marginal ulceration, normal gastric acid secretion and hormone release, and improved gastric function. Patients with pylorus-preserving resections have appeared to regain weight better than historic controls in some studies. Return of gastric emptying in the immediate postoperative period may take longer after the pylorus-preserving operation, and it is controversial whether there is any significant improve-ment in long-term quality of life with pyloric preservation.335-336Techniques for the pancreaticojejunostomy include end-to-side or end-to-end and duct-to-mucosa sutures or invagination (Fig. 33-75). Pancreaticogastrostomy has also been investigated.Some surgeons use stents, glue to seal the anastomosis, or octreotide to decrease pancreatic secretions. No matter what com-bination of these

1	33-75). Pancreaticogastrostomy has also been investigated.Some surgeons use stents, glue to seal the anastomosis, or octreotide to decrease pancreatic secretions. No matter what com-bination of these techniques is used, the clinically significant pan-creatic leakage rate is always about 10%. Therefore, the choice of techniques depends more on the surgeon’s personal experience.Traditionally, most surgeons place drains around the pan-creatic and biliary anastomoses because disruption of the pan-creaticojejunostomy cannot be avoided in one out of 10 patients. Brunicardi_Ch33_p1429-p1516.indd 149301/03/19 6:46 PM 1494SPECIFIC CONSIDERATIONSPART IIThis complication can lead to the development of an upper abdominal abscess or can present as an external pancreatic fis-tula. Usually, a pure pancreatic leak is controlled by the drains and will eventually seal spontaneously. Combined pancreatic and biliary leaks are cause for concern because bile will activate the pancreatic enzymes. In its

1	leak is controlled by the drains and will eventually seal spontaneously. Combined pancreatic and biliary leaks are cause for concern because bile will activate the pancreatic enzymes. In its most virulent form, disruption leads to necrotizing retroperitoneal infection, which can erode major arteries and veins of the upper abdomen, including the exposed portal vein and its branches or the stump of the gastro-duodenal artery. Impending catastrophe is often preceded by a small herald bleed from the drain site. Depending on the clinical situation, such an event is an indication to perform an angio-gram or return the patient to the operating room to widely drain the pancreaticojejunostomy and to repair the involved blood vessel. Open packing may be necessary to control diffuse necro-sis and infection. Some studies have questioned the practice of routine drain placement after pancreatectomy with reliance on postoperative percutaneous drainage when leaks occur.337 How-ever, a randomized,

1	infection. Some studies have questioned the practice of routine drain placement after pancreatectomy with reliance on postoperative percutaneous drainage when leaks occur.337 How-ever, a randomized, controlled, multicenter trial showed that patients who develop a leak after pancreaticoduodenectomy are ABCDEFigure 33-75. Techniques for pancreaticojejunostomy. A to D. Duct-to-mucosa, end-to-side E. Intraoperative photographs of end-to-side pancreaticojejunostomy. F to J. End-to-end invagination. K to O. End-to-side invagination.Brunicardi_Ch33_p1429-p1516.indd 149401/03/19 6:46 PM 1495PANCREASCHAPTER 33FGHIJFigure 33-75. (Continued)at a substantially increased risk of mortality if a drain was not placed at the time of resection.337 In contrast, the outcome does not seem to be as dramatically affected by drain placement in the setting of distal pancreatectomy.338 In the absence of a fis-tula, drains should be removed early in the postoperative period, preferably by postoperative day

1	affected by drain placement in the setting of distal pancreatectomy.338 In the absence of a fis-tula, drains should be removed early in the postoperative period, preferably by postoperative day 5.339Many patients with pancreatic cancer are malnourished preoperatively and suffer from gastroparesis in the immedi-ate postoperative period. Routine placement of a feeding jeju-nostomy tube and gastrostomy tube has become less common, and most surgeons use these tubes selectively. Gastrostomy tubes may decrease the length of stay in patients who might Brunicardi_Ch33_p1429-p1516.indd 149501/03/19 6:46 PM 1496SPECIFIC CONSIDERATIONSPART IIKLNOMFigure 33-75. (Continued)be predicted to have severe gastroparesis. Jejunostomy tubes are certainly not benign and can result in leaks and intestinal obstruction. However, parenteral nutrition is also associated with serious complications such as line sepsis, loss of gut muco-sal integrity, and hepatic dysfunction. Enteric tubes should be considered

1	However, parenteral nutrition is also associated with serious complications such as line sepsis, loss of gut muco-sal integrity, and hepatic dysfunction. Enteric tubes should be considered in patients at risk such as malnourished patients who have received neoadjuvant chemotherapy.Because of the high incidence of direct retroperitoneal invasion and regional lymph node metastasis at the time of surgery, trails of more extended resections including extension of the pancreatic resection to the middle body of the pancreas, segmental resection of the portal vein, if necessary, resection of retroperitoneal tissue along the right perinephric area, and lymphadenectomy to the region of the celiac plexus were exam-ined. In the hands of experienced surgeons, these techniques are associated with greater blood loss but no increase in mortality. However, improved survival has not been demonstrated. Total pancreatectomy has also been considered in the past. Although pancreatic leaks are eliminated,

1	blood loss but no increase in mortality. However, improved survival has not been demonstrated. Total pancreatectomy has also been considered in the past. Although pancreatic leaks are eliminated, major morbidity from brittle diabetes and exocrine insufficiency outweigh any theoretical benefit.Pancreatic cancer can recur locally after pancreaticoduo-denectomy. Intraoperative radiotherapy (IORT) delivers radia-tion to the operative bed at the time of resection. Radiation to Brunicardi_Ch33_p1429-p1516.indd 149601/03/19 6:46 PM 1497PANCREASCHAPTER 33surrounding normal areas is minimized, but the radiation is delivered all in one setting, rather than in fractionated doses over time. Favorable results were recently reported among a series of patients with locally advanced unresectable or border-line-resectable PDAC who received intensive neoadjuvant treat-ment followed by exploratory laparotomy and IORT.340Complications of Pancreaticoduodenectomy. The operative mortality rate for

1	or border-line-resectable PDAC who received intensive neoadjuvant treat-ment followed by exploratory laparotomy and IORT.340Complications of Pancreaticoduodenectomy. The operative mortality rate for pancreaticoduodenectomy has decreased to <5% in high-volume centers (where individual surgeons perform more than 15 cases per year), suggesting that patients in rural areas would benefit from referral to large urban centers.341-342 The most common causes of death are sepsis, hemorrhage, and cardiovascular events. Postoperative complications are unfortunately still very common and include delayed gastric emptying, pancreatic fistula, and hemorrhage.Delayed gastric emptying is common after pancreatico-duodenectomy and is treated conservatively as long as complete gastric outlet obstruction is ruled out by a contrast study. In the acute phase, intravenous erythromycin may help, but the prob-lem usually improves with time.Considerable attention has been focused on the preven-tion of

1	is ruled out by a contrast study. In the acute phase, intravenous erythromycin may help, but the prob-lem usually improves with time.Considerable attention has been focused on the preven-tion of pancreatic leak after pancreas resection. Modifications of the anastomotic technique (end-to-side or end-to-end, duct-to-mucosa, or invaginated), the use of jejunum or the stomach for drainage, the use of pancreatic duct stents, the use of octreotide, and various sealants have all been evaluated.Long-acting synthetic analogues of somatostatin have been evaluated as a pharmacologic therapy to reduce pancreatic secretion and the rate of pancreatic fistula after pancreatic resec-tion. Some European studies supported benefit particularly in selected higher risk patients, while previous North American tri-als concluded there was no benefit.343-349 A recent single-center, randomized trial with pasireotide (a newer analog) suggested potential benefit.350Many technical modifications to the classic

1	tri-als concluded there was no benefit.343-349 A recent single-center, randomized trial with pasireotide (a newer analog) suggested potential benefit.350Many technical modifications to the classic pancreaticodu-odenectomy have been described. However, numerous technical variations to the pancreaticoenteric anastomosis have not clearly demonstrated an objective method to consistently decrease the rate of clinically significant postoperative pancreatic fistula, which in most series is about 10%. Yeo compared the incidence of pancreatic fistula in patients who had a pancreaticoduode-nectomy with reconstruction via a pancreaticogastrostomy or pancreaticojejunostomy.351 There was no significant difference between the two techniques in the incidence of pancreatic fis-tula. A recent meta-analysis summarized the results of 16 trials comparing pancreaticogastrostomy to pancreaticojejunostomy. All of the observational clinical studies reported superiority of pancreaticogastrostomy over

1	summarized the results of 16 trials comparing pancreaticogastrostomy to pancreaticojejunostomy. All of the observational clinical studies reported superiority of pancreaticogastrostomy over pancreaticojejunostomy, most likely influenced by publication bias. In contrast, all random-ized prospective trials failed to show advantage of a particular technique, suggesting both techniques provide equally good results.352Other options to consider when performing the pancre-atic anastomosis are the duct-to-mucosa vs. the invagination techniques. Some surgeons choose the technique at the time of operation, depending on the size of the pancreatic duct and the texture of pancreas favoring invagination when the duct is small and the pancreatic texture is soft.353 Other surgeons use the same technique every time. The duct-to-mucosa anastomosis results in a low pancreatic fistula rate, particularly in patients with a large pancreatic duct and a fibrotic pancreas.354Use of a pancreatic duct stent

1	every time. The duct-to-mucosa anastomosis results in a low pancreatic fistula rate, particularly in patients with a large pancreatic duct and a fibrotic pancreas.354Use of a pancreatic duct stent across the anastomosis has been suggested as a means of preventing a pancreatic leak and as an aid in technical precision. Both internal stenting as well as external stenting have been practiced. A recent Cochrane analysis of eight randomized, controlled trials failed to iden-tify any convincing evidence of benefit with internal or external pancreatic duct stents.355 Some previous studies indicated that stents might be harmful. A recent multicenter randomized trial comparing external to internal pancreatic duct stents during pan-creaticoduodenectomy showed a lower rate of pancreatic fistula with internal stents, so this controversy is likely to continue.356Reconstruction with an isolated Roux-en-Y pancreatico-enteric anastomosis has been suggested as a method do decrease postoperative

1	with internal stents, so this controversy is likely to continue.356Reconstruction with an isolated Roux-en-Y pancreatico-enteric anastomosis has been suggested as a method do decrease postoperative pancreatic leak.357-358 The logic behind this tech-nical modification is that the use of separate Roux-en-Y limbs for biliary and pancreatic secretions may protect the pancreatic anastomosis from activated pancreatic enzymes. However, data is limited, and this is not a common practice.Avoiding the pancreatic anastomosis altogether by ductal ligation or occlusion has also been evaluated as a potential technique to reduce the rate of postoperative pancreatic fistula.359-360 Ductal occlusion with neoprene or prolamine, which are nonresorbable glues, has been abandoned due to pancreatic atrophy and loss of exocrine function. Duct occlusion in pancreaticojejunostomy significantly increases the risk of endocrine insufficiency without a decrease in the postoperative complication rate. To avoid

1	and loss of exocrine function. Duct occlusion in pancreaticojejunostomy significantly increases the risk of endocrine insufficiency without a decrease in the postoperative complication rate. To avoid long-term loss of function, absorbable glues, such as fibrin glue, have been evaluated to limit the action of pancreatic enzymes until the anastomosis is healed. Fibrin glue has been used for both duct occlusion and has also been applied to the surface of the pancreatic stump and anastomotic site without clear improvement in pancreatic fistula rate. The effect of BioGlue applied to the anastomotic surface after the Whipple procedure and pancreatic stump after distal pancreatectomy was evaluated in a retrospective cohort study. There were no statistically significant differences in the incidence or severity grades of postoperative pancreatic fistulas.361 A randomized prospective trial of application of fibrin glue to the surface of the pancreaticojejunostomy in high-risk patients did not

1	or severity grades of postoperative pancreatic fistulas.361 A randomized prospective trial of application of fibrin glue to the surface of the pancreaticojejunostomy in high-risk patients did not reduce the incidence of pancreatic fistula or total complications after pancreaticoduodenectomy.362If not combined with a biliary leak, pancreatic fistula, although serious, can usually be managed conservatively. In about 95% of cases, reoperation is not indicated, and prolonged drainage, using drains placed in the original operation or percu-taneously after resection, results in spontaneous closure of the fistula.363Hemorrhage can occur either intraoperatively or postop-eratively. Intraoperative hemorrhage typically occurs during the dissection of the portal vein. A major laceration of the portal vein can occur at a point in the operation at which the portal vein is not yet exposed. Temporary control of hemorrhage is gener-ally possible in this situation by compressing the portal vein and

1	vein can occur at a point in the operation at which the portal vein is not yet exposed. Temporary control of hemorrhage is gener-ally possible in this situation by compressing the portal vein and superior mesenteric vein against the tumor with the surgeon’s left hand behind the head of the pancreas. An experienced assis-tant is needed to divide the neck of the pancreas to the left of the portal vein and achieve proximal and distal control. Sometimes, the vein can be sutured closed with minimal narrowing. Other times, a segmental resection and interposition graft (internal jugular vein) may be needed.Postoperative hemorrhage can occur from inadequate liga-ture of any one of numerous blood vessels during the procedure. Brunicardi_Ch33_p1429-p1516.indd 149701/03/19 6:46 PM 1498SPECIFIC CONSIDERATIONSPART IIHemorrhage can also occur due to digestion of retroperitoneal blood vessels due to a combined biliary-pancreatic leak. Uncom-monly, a stress ulcer, or later, a marginal ulcer, can

1	can also occur due to digestion of retroperitoneal blood vessels due to a combined biliary-pancreatic leak. Uncom-monly, a stress ulcer, or later, a marginal ulcer, can result in GI hemorrhage. Typically, a vagotomy is not performed when pan-creaticoduodenectomy is performed for pancreatic cancer, but patients are placed on proton pump inhibitors.Outcome and Value of Pancreaticoduodenectomy for Cancer. Survival figures indicate that perhaps few patients are cured indefinitely of pancreatic cancer with pancreaticoduode-nectomy. This has led to a nihilistic view toward patients with this disease which has further contributed to poor outcomes. Using the National Cancer Data Base (1995–2004), Bilimoria reported on 9559 patients with early stage potentially resect-able tumors (pretreatment clinical Stage I: T1N0M0 and T2N0M0).364 Multivariate models were employed to identify factors predicting failure to undergo surgery and assess the impact of pancreatectomy on survival. This study

1	clinical Stage I: T1N0M0 and T2N0M0).364 Multivariate models were employed to identify factors predicting failure to undergo surgery and assess the impact of pancreatectomy on survival. This study identified a striking underuse of pancreatectomy in the United States. Of clinical stage I patients, 71.4% (6823/9559) did not undergo surgery; 6.4% (616/9559) were excluded due to comorbidi-ties; 4.2% (403/9559) refused surgery; 9.1% (869/9559) were excluded due to age; and 38.2% (3644/9559) with potentially resectable cancers were not offered surgery. Patients were less likely to undergo surgery if they were older than 65 years, were black, were on Medicare or Medicaid, had pancreatic head lesions, earned lower annual incomes, or had less education. Patients were less likely to receive surgery at low-volume and community centers. Patients who were not offered surgery had worse survival than patients who underwent resection. Over-all survival from PDAC would significantly increase if more

1	at low-volume and community centers. Patients who were not offered surgery had worse survival than patients who underwent resection. Over-all survival from PDAC would significantly increase if more patients with stage I tumors were identified and offered surgery at high-volume centers (Fig. 33-76).Although pancreaticoduodenectomy may be performed with the hope of the rare cure in mind, the operation more importantly provides better palliation than any other treatment, and it is the only modality that offers any meaningful improve-ment in survival. If the procedure is performed without major complications, many months of palliation are usually achieved. However, it is the surgeon’s duty to make sure patients and their families have a realistic understanding of the true goals of pan-creaticoduodenectomy in the setting of pancreatic cancer.Adjuvant Chemotherapy and Radiation. Small studies in the 1980s suggested that adjuvant chemotherapy with 5-FU combined with radiation improves

1	in the setting of pancreatic cancer.Adjuvant Chemotherapy and Radiation. Small studies in the 1980s suggested that adjuvant chemotherapy with 5-FU combined with radiation improves survival by about 9 months after pancreatic resection for pancreatic adenocarcinoma.365 Subsequent, noncontrolled studies have reinforced that concept; however, the data have been criticized due to the low number of patients and low dose of radiation therapy that was given. In addition, gemcitabine has replaced 5-FU as standard therapy in pancreatic cancer but is thought to be too toxic when given with radiotherapy without dose reduction. A recent large European multicenter trial concluded that there was no value to chemo-radiotherapy, although the study suggested the possibility that chemotherapy alone might have survival benefit.366 Random-ized trials have failed to resolve the debate regarding the role of adjuvant radiation therapy in resectable pancreas cancer. A reasonable consideration in a disease

1	have survival benefit.366 Random-ized trials have failed to resolve the debate regarding the role of adjuvant radiation therapy in resectable pancreas cancer. A reasonable consideration in a disease with high rates of distant metastases is to begin with adjuvant chemotherapy, followed by radiation therapy in patients who do not progress, particularly in patients where there may be increased concern about local recurrence such as in patients with close margins.Remarkable results in adjuvant therapy were reported by the Virginia Mason Clinic with combination 5-FU, cisplatinum, interferon-〈, and external beam radiation.367 Although the toxic-ity was high (42% hospitalized for GI toxicity), the promising results prompted larger confirmatory studies. Unfortunately, one such study was stopped due to toxicity, and this protocol has not been widely adopted. More recent results with FOLFIRINOX in the setting of metastatic disease have encouraged clinical tri-als using this regimen in the

1	due to toxicity, and this protocol has not been widely adopted. More recent results with FOLFIRINOX in the setting of metastatic disease have encouraged clinical tri-als using this regimen in the adjuvant setting, which are cur-rently underway (ClinicalTrials.gov identifier: NCT02172976). Nevertheless, pending further study, it is typical in the United States for patients with acceptable functional status to receive some form of adjuvant chemotherapy and sometimes chemora-diotherapy after surgery.Neoadjuvant Treatment. There are several potential advan-tages to the use of chemotherapy or chemoradiation before an attempt at surgical resection. For example, it avoids the risk that adjuvant treatment is delayed by complications of surgery. Neoadjuvant treatment also may decrease the tumor burden at operation, increasing the rate of resectability and killing some tumor cells before they can be spread intraoperatively. Another potential advantage is that it allows patients with occult

1	burden at operation, increasing the rate of resectability and killing some tumor cells before they can be spread intraoperatively. Another potential advantage is that it allows patients with occult meta-static disease to avoid the morbidity of pancreatic resection. As many as 20% of patients treated with neoadjuvant chemoradia-tion develop metastatic disease detected by restaging CT and do not go on to surgery. This approach may separate patients into a subset likely to benefit from resection and a subset in whom sur-gery would be unlikely to provide clinical benefit. Preoperative chemoradiation has been shown not to increase the periopera-tive morbidity or mortality of pancreaticoduodenectomy. It may even decrease the incidence of pancreatic fistula. Prospective randomized trials investigating this concept are ongoing but are difficult to complete due to the high number of patients who fail to complete or receive a full course of either therapy. Studies have shown that neoadjuvant

1	this concept are ongoing but are difficult to complete due to the high number of patients who fail to complete or receive a full course of either therapy. Studies have shown that neoadjuvant therapy is associated with a lower rate of lymph node positivity and improved survival is achieved among the patients who do not develop disease progression dur-ing neoadjuvant therapy and go on to resection. Neoadjuvant therapy should be considered an acceptable alternative to sur-gery first followed by adjuvant therapy for resectable pancre-atic cancer. Patients should be encouraged to consider available 000.20.40.6Cause-specific survival0.81.0Performed, med. survival = 36 months (N = 724)Refused, med. survival = 7 months (N = 82)1020304050Months60708090Figure 33-76. Effect of surgery on survival in early stage pan-creas cancer. Survival comparison for cases (combined stages IA and IB) for which resection was recommended, and resection was either performed or refused. (Reproduced with

1	in early stage pan-creas cancer. Survival comparison for cases (combined stages IA and IB) for which resection was recommended, and resection was either performed or refused. (Reproduced with permission from Chiari ST, Kelly K, Hollingsworth MA, et al. Early detection of sporadic pancreatic cancer: summative review, Pancreas. 2015 Jul;44(5):693-712.)Brunicardi_Ch33_p1429-p1516.indd 149801/03/19 6:46 PM 1499PANCREASCHAPTER 33clinical trials of neoadjuvant therapy for resectable pancreatic cancer.368 Unfortunately, a recent trial attempting to random-ize patients with resectable pancreatic cancer to neoadjuvant versus adjuvant therapy failed to demonstrate an advantage for the neoadjuvant approach. However, the study was inconclu-sive because it was stopped after 73 of a planned 254 patients due to slow accrual, with only 66 eligible for analysis. Other trials are ongoing. The NEOPA trial (ClinicalTrials.gov iden-tifier: NCT01900327) is a prospectively randomized phase 3 trial of

1	due to slow accrual, with only 66 eligible for analysis. Other trials are ongoing. The NEOPA trial (ClinicalTrials.gov iden-tifier: NCT01900327) is a prospectively randomized phase 3 trial of patients receiving neoadjuvant chemoradiation followed by curative surgery vs. primary surgery followed by adjuvant therapy with a primary endpoint of 3-year overall survival.369Most pancreatic surgeons agree that neoadjuvant chemo-therapy, and perhaps chemoradiotherapy, should be offered to patients with locally advanced but resectable disease (vein involvement) and to patients with “borderline resectable dis-ease” (abutment of ≤180 degrees of the circumference of the SMA, celiac axis, or hepatic artery or if there is a short segment of vein occlusion, CT findings suspicious for metastatic disease, like 1 mm liver lesions too small to characterize or biopsy, and patients with multiple comorbidities or marginal performance status). This strategy acknowledges the fact that these patients are at

1	like 1 mm liver lesions too small to characterize or biopsy, and patients with multiple comorbidities or marginal performance status). This strategy acknowledges the fact that these patients are at high risk of early distant recurrence and/or R1 resection with early local recurrence. Neoadjuvant treatment helps select the right patients for surgery and may reduce the incidence of a margin positive resection.Postoperative Surveillance. Recurrence after successful resection usually manifests as hepatic metastases. Adjuvant che-motherapy with or without radiation is usually administered for 6 months. During this time period, patients are monitored with frequent physical examinations and laboratory tests, including CA19-9. CT scans are typically ordered every 3 months in the first 2 years after resection or when a rising CA19-9 or new symptoms suggest recurrence. Surgical therapy for recurrent disease is usually reserved only for select patients with lim-ited disease who remain reasonable

1	resection or when a rising CA19-9 or new symptoms suggest recurrence. Surgical therapy for recurrent disease is usually reserved only for select patients with lim-ited disease who remain reasonable operative candidates who develop symptomatic gastric outlet or bowel obstruction.Ampullary and Periampullary Cancer. Ampullary cancers need to be distinguished from periampullary cancers. The ampulla is the junction of the biliary and pancreatic ducts within the duodenum. Periampullary cancer includes tumors arising from the distal bile duct, duodenal mucosa, or pancreas just adjacent to the ampulla, and the ampulla can be overgrown by cancers that arise from these adjacent areas, making it impos-sible to determine the true site of origin. Clinically, the term periampullary cancer is, therefore, a nonspecific term used to refer to a variety of tumors arising at the intersection of these four sites. The term ampullary cancer is more specific and is reserved for tumors that arise at the

1	a nonspecific term used to refer to a variety of tumors arising at the intersection of these four sites. The term ampullary cancer is more specific and is reserved for tumors that arise at the ampulla. Based on their location, ampullary cancers are usually detected relatively early due to the appearance of jaundice and have a more favorable prognosis. The ampulla of Vater is lined by an epithelial layer that transitions from pancreatic and biliary ductal epithelium to duodenal mucosal epithelium. Ampullary adenocarcinomas can therefore have an intestinal and/or pancreaticobiliary his-tologic morphology, with the former having a better progno-sis. Patients with ampullary cancer have a 10-year survival of about 35%, which is a much better prognosis than patients with pancreatic adenocarcinoma. The difference in survival is not entirely explained by an earlier presentation and lower inci-dence of lymph node metastases. There are biologic, particularly molecular, differences between

1	The difference in survival is not entirely explained by an earlier presentation and lower inci-dence of lymph node metastases. There are biologic, particularly molecular, differences between ampullary and pancreatic adenocarcinoma of the pancreas.370 Intestinal type ampullary cancers have a lower incidence of EGFr and mutant p53 over-expression, and fewer activating K-ras mutations. These tumors are more likely to have genetic changes similar to colon cancer such as microsatellite instability and adenomatous polyposis coli mutations.Management of Periampullary Adenomas. Benign tumors such as ampullary adenomas can also originate at the ampulla. The accuracy of endoscopic biopsy in distinguishing ampullary cancer from benign adenoma is poor, with false-negative rates from 25% to 56% even if sphincterotomy precedes the biopsy. However, benign villous adenomas of the ampullary region can be excised locally. This technique is applicable only for small tumors (approximately 2 cm or less)

1	if sphincterotomy precedes the biopsy. However, benign villous adenomas of the ampullary region can be excised locally. This technique is applicable only for small tumors (approximately 2 cm or less) with no evidence of malig-nancy upon biopsy. EUS may help to accurately determine if there is invasion into the duodenal wall. In the absence of inva-sion, adenomas may be amenable to an endoscopic or trans-duodenal excision. A longitudinal duodenotomy is made and the tumor is excised with a 2to 3-mm margin of normal duo-denal mucosa. In some centers, small periampullary adenomas can also be removed endoscopically. A preoperative diagnosis of cancer is a contraindication to transduodenal excision, and pancreaticoduodenectomy should be performed. Likewise, if final pathologic examination of a locally excised tumor reveals invasive cancer, the patient should be returned to the operat-ing room for a pancreaticoduodenectomy. An important sub-set of patients are those with FAP who develop

1	a locally excised tumor reveals invasive cancer, the patient should be returned to the operat-ing room for a pancreaticoduodenectomy. An important sub-set of patients are those with FAP who develop periampullary or duodenal adenomas. These lesions have a high incidence of harboring carcinoma and frequently recur unless the mucosa at risk is resected. A standard (not pylorus-sparing) Whipple is the procedure of choice in FAP patients with periampullary lesions.Cystic Neoplasms of the Pancreas. A cystic neoplasm needs to be considered when a patient presents with a fluid-containing pancreatic lesion. Asymptomatic cystic neoplasms of the pancreas may be more frequent than previously recognized and are being identified with increasing frequency as the use of abdominal CT scanning and MRI has increased. Pancreatic cysts are now thought to be present in about 9% of the popula-tion age 80 and older.371 When symptoms are clearly attributable to a pancreatic cyst, resection is indicated in

1	increased. Pancreatic cysts are now thought to be present in about 9% of the popula-tion age 80 and older.371 When symptoms are clearly attributable to a pancreatic cyst, resection is indicated in patients who are fit candidates for surgery. However, management of asymp-tomatic pancreatic cysts is nuanced and can trigger significant anxiety for patients and their surgeons. Invasive surveillance and aggressive surgical intervention can cause harm, decrease quality of life, and increase costs. While the overall risk that an incidental pancreatic cyst is malignant is very low (about 1 in 10,000), the risks of surgery are very significant with a 2% to 5% mortality and 30% to 40% morbidity. However, some of these neoplasms slowly undergo malignant transformation and thus represent an opportunity for surgical cure, which is uncom-mon after transformation to invasive pancreatic adenocarci-noma. The dilemma for the surgeon is to identify the minority of cysts that pose a significant risk and

1	for surgical cure, which is uncom-mon after transformation to invasive pancreatic adenocarci-noma. The dilemma for the surgeon is to identify the minority of cysts that pose a significant risk and provide individual patients with an accurate assessment of their unique risk-benefit ratio of resection vs. surveillance.Surveillance programs are of questionable value in patients who are not candidates for surgery due to age and or multiple comorbidities and limited life expectancy. Surgeons also need to clearly explain to the patient the risks and benefits of surveil-lance itself. MRI is the preferred surveillance imaging modality Brunicardi_Ch33_p1429-p1516.indd 149901/03/19 6:46 PM 1500SPECIFIC CONSIDERATIONSPART IIover computed tomography because MRI does not expose the patient to radiation and better demonstrates the structural rela-tionship between the pancreatic duct and associated cyst. Also, MRI is less invasive than EUS. EUS is therefore reserved for further evaluation of

1	and better demonstrates the structural rela-tionship between the pancreatic duct and associated cyst. Also, MRI is less invasive than EUS. EUS is therefore reserved for further evaluation of higher risk cysts. Another problem to con-front is when to stop surveillance in a cyst that has been stable during observation. Some clinicians stop following after 5 years but data is lacking to guide this decision. In addition, patients who have undergone resection of a pancreatic cystic neoplasm with high grade dysplasia may warrant continued surveillance after surgery particularly when there is a possible field effect such as in IPMN or concern that all of the disease has not been resected. Individualized decision-making and multidisciplinary input is ideal.372Pseudocysts. The most common cystic lesion of the pancreas is the pseudocyst, which, of course, has no epithelial lining and is a nonneoplastic complication of pancreatitis or pancreatic duct injury. As discussed in “Complications of

1	lesion of the pancreas is the pseudocyst, which, of course, has no epithelial lining and is a nonneoplastic complication of pancreatitis or pancreatic duct injury. As discussed in “Complications of Chronic Pancreatitis,” the diagnosis is usually straightforward from the clinical history. Although not usually necessary, analysis of pseudocyst fluid would reveal a high amylase content. The danger comes in mistaking a cystic pancreatic neoplasm for a pseudocyst and incorrectly draining a cystic neoplasm into the GI tract rather than resecting the neoplasm. For this reason, biopsy of the pseudocyst wall is a requirement in the management of pancreatic pseudocysts.Cystadenoma. Serous cystadenomas are essentially considered benign tumors without malignant potential. Serous cystadenocarcinoma has been reported very rarely (<1%). Therefore, malignant potential should not be used as an argument for surgical resection, and the majority of these lesions can be safely observed in the absence of

1	been reported very rarely (<1%). Therefore, malignant potential should not be used as an argument for surgical resection, and the majority of these lesions can be safely observed in the absence of symptoms due to mass effect or rapid growth. The average rate of growth is about 0.5 cm per year. About 50% of cystadenomas are asymptomatic and detected as an incidental finding. Most symptomatic patients have mild upper abdominal pain, epigastric fullness, or moderate weight loss. Occasionally, cystadenomas can grow to a size capable of producing jaundice or GI obstruction due to mass effect (Fig. 33-77). For symptomatic patients with serous cystadenoma, surgical resection is indicated. For lesions in the tail, splenectomy is not necessary, given the benign nature of the tumor. In appropriate candidates, a laparoscopic approach to distal pancreatectomy with or without splenic preservation can be considered. These cysts are frequently found in older women in which pancreatic resection for

1	candidates, a laparoscopic approach to distal pancreatectomy with or without splenic preservation can be considered. These cysts are frequently found in older women in which pancreatic resection for a benign neoplasm should be avoided in the absence of significant symptoms. All regions of the pancreas are affected, with half in the head/uncinate process, and half in the neck, body, or tail of the pancreas. They have a spongy appearance, and multiple small cysts (microcystic) are more common than larger cysts (macrocystic or oligocystic). These lesions contain thin serous fluid that does not stain positive for mucin and is low in CEA (<200 ng/mL). Typical imaging characteristics include a well-circumscribed cystic mass, small septations, fluid close to water density, and sometimes, a central scar with calcification. If a conservative management is adopted, it is important to be sure of the diagnosis. EUS-FNA should yield nonviscous fluid with low CEA and amylase levels, and if cells

1	scar with calcification. If a conservative management is adopted, it is important to be sure of the diagnosis. EUS-FNA should yield nonviscous fluid with low CEA and amylase levels, and if cells are obtained, which is rare, they are cuboidal and have a clear cytoplasm.Mucinous Cystadenoma and Cystadenocarcinoma. Muci-nous cystic neoplasms (MCNs) encompass a spectrum ranging from benign but potentially malignant to carcinoma with a very aggressive behavior (Table 33-23). There is often heterogene-ity within the lesions with benign and malignant-appearing regions, making it impossible to exclude malignancy with biopsy. MCNs are commonly seen in perimenopausal women, and about two-thirds are located in the body or tail of the pan-creas. Like cystadenomas, most MCNs are now incidental find-ings identified during imaging performed for other reasons. When symptoms are present, they are usually nonspecific and include upper abdominal discomfort or pain, early satiety, and weight loss. On

1	identified during imaging performed for other reasons. When symptoms are present, they are usually nonspecific and include upper abdominal discomfort or pain, early satiety, and weight loss. On imaging studies, the cysts have thick walls and do not communicate with the main pancreatic duct (Fig. 33-78). There may be nodules or calcifications within the wall of the cyst. The cysts are lined by tall columnar epithelium that fills the cyst with viscous mucin. The submucosal layer consists of a highly cellular stroma of spindle cells with elongated nuclei similar to the “ovarian stroma,” which is a key pathologic feature distinguishing these lesions. Elevated CEA levels in the fluid (≥200 ng/mL) are consistent with mucinous lesions and may suggest malignant transformation.373 Solid areas may con-tain atypical cells or invasive cancer, and extensive sampling of the specimen is necessary to accurately predict prognosis. Figure 33-77. Mucinous cystic neoplasm in tail of pancreas.Table

1	may con-tain atypical cells or invasive cancer, and extensive sampling of the specimen is necessary to accurately predict prognosis. Figure 33-77. Mucinous cystic neoplasm in tail of pancreas.Table 33-23World Health Organization classification of primary tumors of the exocrine pancreas A. Benign 1. Serous cystadenoma (16%) 2. Mucinous cystadenoma (45%) 3. Intraductal papillary-mucinous adenoma (32%) 4. Mature cystic teratoma B. Borderline 1. Mucinous cystic tumor with moderate dysplasia 2. Intraductal papillary mucinous tumor with moderate dysplasia 3. Solid pseudopapillary tumor C. Malignant 1. Ductal adenocarcinoma 2. Serous/mucinous cystadenocarcinoma (29%) 3. Intraductal mucinous papillary tumorBrunicardi_Ch33_p1429-p1516.indd 150001/03/19 6:46 PM 1501PANCREASCHAPTER 33NoYes<1 cm1–2 cm2–3 cm>3 cmNoNoa. Pancreatitis may be an indication for surgery for relief of symptoms.b. Differential diagnosis includes mucin. Mucin can move with change in patient position, may be dislodged

1	cm2–3 cm>3 cmNoNoa. Pancreatitis may be an indication for surgery for relief of symptoms.b. Differential diagnosis includes mucin. Mucin can move with change in patient position, may be dislodged oncyst lavage and does not have Doppler ’ow. Features of true tumor nodule include lack of mobility, presence ofDoppler ’ow and FNA of nodule showing tumor tissuec. Presence of any one of thickened walls, intraductal mucin or mural nodules is suggestive of main ductinvolvement. In their absence main duct involvement is inconclusive.d. Studies from Japan suggest that on follow-up of subjects with suspected BD-IPMN there is increased incidenceof pancreatic ductal adenocarcinoma unrelated to malignant transformation of the BD-IPMN(s) being followed.However, it is unclear if imaging surveillance can detect early ductal adenocarcinoma, and, if so, at what intervalsurveillance imaging should be performed.Are any of the following high-risk stigmata of malignancy present?i) obstructive jaundice in a

1	detect early ductal adenocarcinoma, and, if so, at what intervalsurveillance imaging should be performed.Are any of the following high-risk stigmata of malignancy present?i) obstructive jaundice in a patient with cystic lesion of the head of the pancreas,ii) enhancing solid component within cyst, iii) main pancreatic duct ‰10 mm in sizeAre any of the following worrisome features present?Clinical: PancreatitisaImaging: i) cyst ‰3 cm, ii) thickened/enhancing cyst walls, iii) main duct size 5–9 mm, iii) non-enhancingmural nodule iv) abrupt change in caliber of pancreatic duct with distal pancreatic atrophy.What is the size of largest cyst?InconclusiveIf yes, perform endoscopic ultrasoundConsidersurgery,if clinicallyappropriateAre any of these features present?i) Deÿnite mural nodule(s)bii) Main duct features suspicious for involvementciii) Cytology: suspicious or positive for malignancyCT/MRIin 2–3 yearsdCT/MRIyearly × 2 years,then lengthenintervalif no changedEUS in 3–6 months,

1	Main duct features suspicious for involvementciii) Cytology: suspicious or positive for malignancyCT/MRIin 2–3 yearsdCT/MRIyearly × 2 years,then lengthenintervalif no changedEUS in 3–6 months, thenlengthen interval alternating MRIwith EUS as appropriate.dConsider surgery in young,ÿt patients with need forprolonged surveillanceClose surveillance alternatingMRI with EUS every 3–6 months.Strongly consider surgery in young,ÿt patientsYesFigure 33-78. Algorithm for management of pancreatic cystic neoplasms. CEA = carcinoembryonic antigen; CT = computed tomography; ERCP = endoscopic retrograde cholangiopancreatography; EUS = endoscopic ultrasound; FNA = fine-needle aspiration; Hx = history; IPMN = intraductal papillary mucinous neoplasm of the pancreas; MCN = mucinous cystic neoplasm; MRCP = magnetic resonance cholangiopancreatography. (Reproduced with permission from Tanaka M, Adsay V, Chari S, et al. International consensus guidelines 2012 for the management of IPMN and MCN of the

1	= magnetic resonance cholangiopancreatography. (Reproduced with permission from Tanaka M, Adsay V, Chari S, et al. International consensus guidelines 2012 for the management of IPMN and MCN of the pancreas, Pancreatology. 2012 May-Jun;12(3):183-197.)Resection is the treatment of choice for most mucin-producing cystic tumors. Malignancy cannot be ruled out without removal and extensive sampling of the entire tumor. Malignancy has been reported in 6% to 36% of MCNs. Current thinking is that all of these tumors will eventually evolve into cancer if left untreated. However, for individual patients, the surgeon is often faced with a difficult decision. The risk of pancreas surgery in an older patient with multiple comorbidities and a relatively short life expectancy regardless of their pancreatic cyst is frequently weighed against the potential future risk that the cyst, particularly a small cyst, will transform into an incurable invasive cancer.The utility of detailed DNA analysis of

1	cyst is frequently weighed against the potential future risk that the cyst, particularly a small cyst, will transform into an incurable invasive cancer.The utility of detailed DNA analysis of pancreatic cyst fluid to diagnose mucinous and malignant cysts has been evalu-ated in the PANDA study.374 The study concluded that cyst fluid K-ras mutation was helpful in the diagnosis of mucinous cysts with a 96% specificity. Components of DNA analysis detect-ing malignant cysts included allelic loss amplitude over 82% and high DNA amount. The criteria of high amplitude K-ras mutation followed by allelic loss showed maximum specificity (96%) for malignancy. However, this test lacks sensitivity. In clinical practice, the surgeon must take all of these complemen-tary factors into consideration when determining the malignant potential of a pancreatic cystic neoplasm.Because most MCNs are located in the body and tail of the pancreas, distal pancreatectomy is the most common treatment. For small

1	determining the malignant potential of a pancreatic cystic neoplasm.Because most MCNs are located in the body and tail of the pancreas, distal pancreatectomy is the most common treatment. For small lesions, it may be appropriate to preserve the spleen, but splenectomy ensures removal of the lymph node basin that can potentially be involved. It is very important not to rupture the cyst during resection, and the tumor should be removed intact, not morselized. Therefore, a laparoscopic approach may not be appropriate for larger lesions. Completely resected MCNs without atypia are usually cured especially if small (<3 cm). Even patients with moderate dysplasia or carcinoma in situ are usually cured by complete resection. Noninvasive MCNs require no surveillance after resection. For MCNs with an associated invasive carcinoma, prognosis depends on the extent of the invasive component, tumor stage, and resectability. The 2-year survival rate and 5-year survival rate of patients with

1	with an associated invasive carcinoma, prognosis depends on the extent of the invasive component, tumor stage, and resectability. The 2-year survival rate and 5-year survival rate of patients with Brunicardi_Ch33_p1429-p1516.indd 150101/03/19 6:46 PM 1502SPECIFIC CONSIDERATIONSPART IIresected MCN with an associated invasive carcinoma are about 67% and 50%, respectively.375Intraductal Papillary Mucinous Neoplasm. Intraductal papillary mucinous neoplasms (IPMNs) usually occur within the head of the pancreas and arise within the pancreatic ducts. The ductal epithelium forms a papillary projection into the duct, and mucin production causes intraluminal cystic dilation of the pancreatic ducts (Fig. 33-79). Imaging studies demonstrate diffuse dilation of the pancreatic duct, and the pancreatic parenchyma is often atrophic due to chronic duct obstruction. However, classic features of chronic pancreatitis, such as calcification and a beaded appearance of the duct, are not present. At

1	parenchyma is often atrophic due to chronic duct obstruction. However, classic features of chronic pancreatitis, such as calcification and a beaded appearance of the duct, are not present. At ERCP, mucin can be seen extruding from the ampulla of Vater, a so-called fish-eye lesion that is virtually diagnostic of IPMN (Fig. 33-80). Initial reports suggested a male predominance, but more recent series indicate an equal distribution. Patients are usually in their seventh to eighth decade of life and present with abdominal pain or recurrent pancreatitis, thought to be caused by obstruction of the pancreatic duct by thick mucin. Some patients 9Figure 33-79. Computed tomography appearance of massive mul-tiseptated serous cystadenoma in head of pancreas with central stel-late scar (left) and resected specimen (right).Figure 33-80. Intraductal papillary mucinous neoplasm histology. Papillary projections of ductal epithelium resemble villous morphology and contain mucin-filled vesicles.

1	resected specimen (right).Figure 33-80. Intraductal papillary mucinous neoplasm histology. Papillary projections of ductal epithelium resemble villous morphology and contain mucin-filled vesicles. (Reproduced with permission from Asiyanbola B, Andersen DK. IPMN. Editorial Update. accesssurgery.com McGraw-Hill Education; 2008.)(5–10%) have steatorrhea, diabetes, and weight loss secondary to pancreatic insufficiency.Some IPMNs primarily involve the main pancreatic duct, while others involve the branch ducts. The mean fre-quency of malignancy in main duct IPMN (MD-IPMN) is 62% (Fig. 33-81). Considering this high incidence of malignant lesions and the low 5-year survival rates (31–54%), interna-tional consensus guidelines recommend resection for all surgi-cally fit patients with MD-IPMN.372 If the margin is positive for high-grade dysplasia, additional resection should be attempted to obtain at least moderate-grade dysplasia at the surgical margin.The surgical management of IPMNs is

1	If the margin is positive for high-grade dysplasia, additional resection should be attempted to obtain at least moderate-grade dysplasia at the surgical margin.The surgical management of IPMNs is complicated by the fact that the lesion itself is small and preoperative imaging stud-ies show a dilated pancreatic duct but not necessarily the mass. Mucus can dilate the duct proximal and distal to the lesion. Fur-thermore, these lesions can spread microscopically along the duct, and there can be skip areas of normal duct between the diseased portions. Therefore, thorough preoperative imaging including EUS, MRCP, or ERCP, and sometimes pancreatic ductoscopy, which can also be repeated intraoperatively, is use-ful (see Fig. 33-80). The surgeon needs to be prepared to extend the resection, if necessary, based on intraoperative findings and frozen section of the margin. Extending the resection to the point of total pancreatectomy is controversial due to the mor-bidity of this operation. Like

1	based on intraoperative findings and frozen section of the margin. Extending the resection to the point of total pancreatectomy is controversial due to the mor-bidity of this operation. Like MCNs, the IPMNs require careful histologic examination of the entire specimen for an invasive cancer (Fig. 33-82).Survival of patients with IPMN, even when malignant and invasive, can be quite good. As with MCN, patients with bor-derline tumors or carcinoma in situ are usually cured. For this reason, if recurrence occurs in the remaining pancreas, further resection is warranted because several series have shown that some of these cases are salvageable. Patients with IPMN are also at risk for other malignancies and should undergo colonos-copy and close surveillance.Branch-duct type IPMNs (BD-IPMN) are often found in the uncinate process, are sometimes asymptomatic, and are less frequently associated with malignant transformation (6–46%). Asymptomatic, small suspected BD-IPMNs are frequently

1	are often found in the uncinate process, are sometimes asymptomatic, and are less frequently associated with malignant transformation (6–46%). Asymptomatic, small suspected BD-IPMNs are frequently Brunicardi_Ch33_p1429-p1516.indd 150201/03/19 6:47 PM 1503PANCREASCHAPTER 33ABCFigure 33-81. Intraductal papillary mucinous neoplasm (IPMN). A. Examples of “fish-eye deformity” of IPMN. Mucin is seen extruding from the ampulla. B. Mucin coming from pancreatic duct when neck of pancreas is transected during Whipple procedure (left). Intraoperative pancreatic ductoscopy to assess the pancreatic tail (right). C. Views of pancreatic duct during ductoscopy; normal (left) and IPMN (right).Brunicardi_Ch33_p1429-p1516.indd 150301/03/19 6:47 PM 1504SPECIFIC CONSIDERATIONSPART IIobserved with serial imaging. High-risk features such as mural nodules, a dilated main duct, positive cytology or cyst fluid CEA >200 need to be ruled out. In the absence of these fea-tures, continued observation with

1	imaging. High-risk features such as mural nodules, a dilated main duct, positive cytology or cyst fluid CEA >200 need to be ruled out. In the absence of these fea-tures, continued observation with serial imaging is appropriate, especially in patients who are not ideal operative candidates. The mean frequency of invasive cancer in resected BD-IPMN is 18%. BD-IPMN mostly occurs in elderly patients, and the annual malignancy rate is only 2% to 3%. These factors support conservative management with follow-up in patients who do not have any symptoms or risk factors predicting malignancy such as mural nodule, rapidly increasing cyst size, and high-grade atypia in cytology. There is insufficient data to support immediate resection for all BD-IPMNs <3 cm without “high-risk stigmata” and “worrisome features.” Branch-duct IPMNs ≥3 cm should be resected.372Four histologic subtypes of IPMNs have been character-ized: gastric, intestinal, pancreatobiliary, and oncocytic. Most of BD-IPMNs are

1	features.” Branch-duct IPMNs ≥3 cm should be resected.372Four histologic subtypes of IPMNs have been character-ized: gastric, intestinal, pancreatobiliary, and oncocytic. Most of BD-IPMNs are composed of gastric-type epithelium. However, intestinal type is more common in MD-IPMN. In a recent report, the four subtypes of IPMNs were associated with significant dif-ferences in survival.376 Patients with gastric-type IPMN had the best prognosis, whereas those with intestinal and pancreatobili-ary type had a bad prognosis.Workup of Asymptomatic Pancreatic Cystic NeoplasmsIncidentally discovered asymptomatic pancreatic cystic neo-plasms are evaluated by MRI with MRCP to check for “high-risk stigmata or worrisome features.” An enhancing solid component or main pancreatic duct (MPD) dilation ≥10 mm are considered “high-risk stigmata.” Cysts ≥3 cm, thickened enhancing cyst walls, nonenhancing mural nodules, MPD size of 5 to 9 mm, abrupt change in the MPD caliber with distal pancreatic atrophy,

1	are considered “high-risk stigmata.” Cysts ≥3 cm, thickened enhancing cyst walls, nonenhancing mural nodules, MPD size of 5 to 9 mm, abrupt change in the MPD caliber with distal pancreatic atrophy, and lymphadenopathy are considered “worrisome features.” All cysts with high risk stigmata are resected. All cysts with wor-risome features and cysts > 3 cm without worrisome features are further evaluated with EUS. If EUS shows a definite mural nodule, main duct features suspicious for involvement or the cytology is positive or suspicious for malignancy, then resection is recommended. In the absence of worrisome features, no fur-ther initial work-up is recommended, but surveillance is still required.The interval between surveillance imaging is based on the size of the cyst with a lengthening of the interval once sta-bility is established. Patients with noninvasive MCNs require no surveillance after resection, but patients with IPMNs need surveillance after resection. In the absence of

1	of the interval once sta-bility is established. Patients with noninvasive MCNs require no surveillance after resection, but patients with IPMNs need surveillance after resection. In the absence of residual lesions, repeat MRI at 2 and 5 years may be reasonable. If there is low or moderate-grade dysplasia at the margin, MRI every 6 months is recommended.Resection is indicated in all surgically fit patients with MD-IPMN, and additional resection is indicated if there is high-grade dysplasia at the margin. Resection is also indicated in all surgically fit patients with MCN. BD-IPMN <3 cm without wor-risome features or high-risk stigmata can undergo surveillance.When patients are deemed unacceptable for resection due to comorbidities, ablation of the cyst can be considered. How-ever, at this time, cyst ablation is considered experimental and should be done as part of a clinical trial. Gastroenterologists have limited experience with injection of a cytotoxic agent into the cyst in an

1	this time, cyst ablation is considered experimental and should be done as part of a clinical trial. Gastroenterologists have limited experience with injection of a cytotoxic agent into the cyst in an attempt to ablate the cyst epithelium. Ethanol has been used and more recently this has been combined with paclitaxel. The combination of ethanol and paclitaxel injection resulted in elimination of the cysts, as determined by CT scan-ning, in 29 out of 47 (62%) of patients in a median follow-up period of 21.7 months.377Recently, the American Gastroenterological Association (AGA) published guidelines for asymptomatic mucinous cysts (http://www.gastro.org/guidelines/pancreatic-cysts) that are different from all previously published guidelines in the following areas: 2-year interval for cyst of any size undergoing surveillance, stopping surveillance after 5 years if no change, surgery only if more than one concerning feature on MRI confirmed on EUS and only in centers with high volumes of

1	any size undergoing surveillance, stopping surveillance after 5 years if no change, surgery only if more than one concerning feature on MRI confirmed on EUS and only in centers with high volumes of pancreatic surgery, and no surveillance after surgery if no invasive cancer or dysplasia. Although based on extensive literature review and synthesis, these recommendations have resulted in significant controversy because, in an effort to reduce the costs of health care delivery and perhaps decrease inadvertent harm to patients, they advocate less frequent follow-up and a higher threshold before offering EUS and/or surgery.Solid-Pseudopapillary Tumor. Solid-pseudopapillary tumors are rare and typically occur in young women. Previous names for this entity include, solid and cystic, solid and papillary, cystic and papillary, and papillary-cystic tumor. They are typically well circumscribed on CT (Fig. 33-83). The cysts are not true epithelial-lined cysts but rather represent a

1	solid and papillary, cystic and papillary, and papillary-cystic tumor. They are typically well circumscribed on CT (Fig. 33-83). The cysts are not true epithelial-lined cysts but rather represent a necrotic/degenerative process. Histology may be similar to neuroendocrine tumors, but they do not stain positive for neuroendocrine markers such as chromogranin. Most are cured by resection, but liver and peritoneal metastases have been reported.Other Cystic Neoplasms. Rarely, typical ductal adenocarci-noma of the pancreas may undergo cystic degeneration due to central necrosis. Occasionally, this will create difficulty in the proper preoperative diagnosis and should be kept in mind when deciding to conservatively follow a cystic pancreatic neoplasm. It is more common, 5% to 10%, for neuroendocrine tumors of the pancreas to contain cysts. These cysts are filled with sero-sanguineous fluid rather than necrotic debris. Lymphoepithelial Figure 33-82. Operative specimen of pancreas with

1	tumors of the pancreas to contain cysts. These cysts are filled with sero-sanguineous fluid rather than necrotic debris. Lymphoepithelial Figure 33-82. Operative specimen of pancreas with multifocal intraductal papillary mucinous neoplasms (black arrow) and a focus of invasive adenocarcinoma (white arrow). (Reproduced with permission from Asiyanbola B, Andersen DK. IPMN. Editorial Update. accesssurgery.com McGraw-Hill Education; 2008.)Brunicardi_Ch33_p1429-p1516.indd 150401/03/19 6:47 PM 1505PANCREASCHAPTER 33Figure 33-83. Abdominal computed tomographic scan of a 25-year-old woman demonstrating a well-circumscribed cystic lesion with septation in body/tail of pancreas. At surgery, the tumor was adherent to the splenic artery. Pathologic diagnosis was solid-pseudopapillary carcinoma.cysts of the pancreas usually occur in men in their fifth to sixth decade. These benign lesions may be unilocular or multilocular and vary widely in size. The contents of the cyst are also vari-able and

1	of the pancreas usually occur in men in their fifth to sixth decade. These benign lesions may be unilocular or multilocular and vary widely in size. The contents of the cyst are also vari-able and may be thin serous fluid or cheesy/caseous material if there is increased keratin formation. A substantial number of patients with von Hippel-Lindau syndrome develop pancreatic cysts that resemble serous cystadenomas. There may be mul-tiple lesions scattered throughout the pancreas. Patients with polycystic kidney and hepatic disease may also develop benign pancreatic cysts (cystadenomas). With all of these rare cystic neoplasms, careful clinical history, high-quality pancreatic imaging, and sampling of the cyst fluid for analysis will guide proper treatment.Pancreatic Lymphoma. Lymphoma can affect the pancreas. Primary involvement of the pancreas with no disease outside the pancreas also occurs. The clinical presentation often is similar to pancreatic adenocarcinoma, with vague abdominal

1	the pancreas. Primary involvement of the pancreas with no disease outside the pancreas also occurs. The clinical presentation often is similar to pancreatic adenocarcinoma, with vague abdominal pain and weight loss. Identification of a large mass often involving the head and body of the pancreas should raise suspicion. Percuta-neous or EUS-guided biopsy will confirm the diagnosis in most cases. If the diagnosis cannot be confirmed preoperatively, lapa-roscopic exploration and biopsy are indicated.378 There is no role for resection in the management of pancreatic lymphoma. Endoscopic stenting to relieve jaundice followed by chemo-therapy is the standard treatment, and long-term remission is often achieved.REFERENCESEntries highlighted in bright blue are key references. 1. Silen W. Surgical anatomy of the pancreas. Surg Clin North Am. 1964;44:1253. 2. Havel PJ, Taborsky GJ, Jr. The contribution of the autonomic nervous system to changes of glucagon and insulin secretion during

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1	2005;11:2456-2461. 359. Di Carlo V, Chiesa R, Pontiroli AE, et al. Pancreatoduodenectomy with occlusion of the residual stump by Neoprene injection. World J Surg. 1989;13:105-110; discussion 110-111. 360. Tran K, Van Eijck C, Di Carlo V, et al. Occlusion of the pancreatic duct versus pancreaticojejunostomy: a prospective randomized trial. Ann Surg. 2002;236:422-428, discussion 428. 361. Fisher WE, Chai C, Hodges SE, Wu MF, Hilsenbeck SG, Brunicardi FC. Effect of BioGlue on the incidence of pancreatic fistula following pancreas resection. J Gastrointest Surg. 2008;12:882-890. 362. Lillemoe KD, Cameron JL, Kim MP, et al. Does fibrin glue sealant decrease the rate of pancreatic fistula after pancreaticoduodenectomy? Results of a prospective randomized trial. J Gastrointest Surg. 2004;8:766-772, discussion 772-774. 363. Kazanjian KK, Hines OJ, Eibl G, Reber HA. Management of pancreatic fistulas after pancreaticoduodenectomy: results in 437 consecutive patients. Arch Surg.

1	2004;8:766-772, discussion 772-774. 363. Kazanjian KK, Hines OJ, Eibl G, Reber HA. Management of pancreatic fistulas after pancreaticoduodenectomy: results in 437 consecutive patients. Arch Surg. 2005;140:849-854, discussion 854-856. 364. Bilimoria KY, Bentrem DJ, Ko CY, et al. National failure to operate on early stage pancreatic cancer. Ann Surg. 2007;246(2):173-180. doi: 10.1097/SLA.0b013e3180691579 365. Group GTS. Further evidence of effective adjuvant combined radiation and chemotherapy following curative resection of pancreatic cancer. Cancer. 1997;59:2006-2010. 366. Neoptolemos JP, Dunn JA, Stocken DD, et al. Adjuvant chemoradiotherapy and chemotherapy in resectable pancreatic cancer: a randomised controlled trial. Lancet. 2001;358:1576-1585. 367. Rocha FG1, Hashimoto Y, Traverso LW, et al. Interferon-based adjuvant chemoradiation for resected pancreatic head cancer: long-term follow-up of the Virginia Mason Protocol. Ann Surg. 2016;263(2):376-384. doi:

1	Hashimoto Y, Traverso LW, et al. Interferon-based adjuvant chemoradiation for resected pancreatic head cancer: long-term follow-up of the Virginia Mason Protocol. Ann Surg. 2016;263(2):376-384. doi: 10.1097/SLA.0000000000001190 368. Russo S, Saif MW. Neoadjuvant therapy for pancreatic cancer: an ongoing debate. Therap Adv Gastroenterol. 2016;9(4):429-436. 369. Tachezy M, Gebauer F, Petersen C, et al. Sequential neoadjuvant chemoradiotherapy (CRT) followed by curative surgery vs. primary surgery alone for resectable, non-metastasized pancreatic adenocarcinoma: NEOPA—a randomized multicenter phase III study (NCT01900327, DRKS00003893, ISRCTN82191749). BMC Cancer. 2014;14:411. 370. Gingras MC, Covington KR, Chang DK, et al. Ampullary cancers harbor ELF3 tumor suppressor gene mutations and exhibit frequent WNT dysregulation. Cell Rep. 2016;14(4):907-919. 371. Laffan TA, Horton KM, Klein AP, et al. Prevalence of unsuspected pancreatic cysts on MDCT. AJR Am J Roentgenol.

1	mutations and exhibit frequent WNT dysregulation. Cell Rep. 2016;14(4):907-919. 371. Laffan TA, Horton KM, Klein AP, et al. Prevalence of unsuspected pancreatic cysts on MDCT. AJR Am J Roentgenol. 2008;191(3):802-807. doi: 10.2214/AJR.07.3340 372. Tanaka M, Adsay V, Chari S, et al. International consensus guidelines 2012 for the management of IPMN and MCN of the pancreas. Pacreatology. 2012(12):183-197.Brunicardi_Ch33_p1429-p1516.indd 151401/03/19 6:47 PM 1515PANCREASCHAPTER 33 373. Brugge WR, Lewandrowski K, Lee-Lewandrowski E, et al. Diagnosis of pancreatic cystic neoplasms: a report of the cooperative pancreatic cyst study. Gastroenterology. 2004;126:1330-1336. 374. Khalid A, Zahid M, Finkelstein SD, et al. Pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study. Gastrointest Endosc. 2009;69:1095-1102. 375. Yoon WJ, Brugge WR. Pancreatic cystic neoplasms: diagnosis and management. Gastroenterol Clin North Am.

1	pancreatic cysts: a report of the PANDA study. Gastrointest Endosc. 2009;69:1095-1102. 375. Yoon WJ, Brugge WR. Pancreatic cystic neoplasms: diagnosis and management. Gastroenterol Clin North Am. 2012;41(1):103-118. 376. Furukawa T, Hatori T, Fujita I, et al. Prognostic relevance of morphological types of intraductal papillary mucinous neoplasms of the pancreas. Gut. 2011;60(4):509-516. 377. Brugge WR. Management and outcomes of pancreatic cystic lesions. Dig Liver Dis. 2008;40(11):854-859. 378. Boni L, Benevento A, Dionigi G, Cabrini L, Dionigi R. Primary pancreatic lymphoma. Surg Endosc. 2002;16:1107-1108.Brunicardi_Ch33_p1429-p1516.indd 151501/03/19 6:47 PM

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1	The SpleenAdrian E. Park, Eduardo M. Targarona, Adam S. Weltz, and Carlos Rodriguez-Otero Luppi 34chapterHISTORICAL BACKGROUNDThe spleen has been the subject of man’s musings since almost the establishment of the written word. It has been largely mis-understood, often maligned, and certainly underappreciated as a major organ for more than two millennia. The ancients, pre-sumably through patient observation and occasional anatomic exploration, often consigned the spleen to a vestigial role.Our current understanding of the central role played by the spleen in regulating the immune system and influencing meta-bolic and endocrine functions has been built upon knowledge gleaned only over the past few decades. Our early notions of the spleen as a dispensable filter of blood or seat of emotion have been dispelled as our understanding of its structure and function has evolved, informing our surgical approach to this worthy and fascinating organ. Many of the “founding fathers of medicine”

1	have been dispelled as our understanding of its structure and function has evolved, informing our surgical approach to this worthy and fascinating organ. Many of the “founding fathers of medicine” have weighed in on the anatomy and function of the spleen over the centuries. Hippocrates in the fourth century BC was one of the first to write on the spleen.1-3 He taught broadly on the need for balance and equilibrium between the patient and his environment. Illness arose from disharmony in nature, particularly among the patient’s four humors: blood, phlegm, black bile (melancholia), and yellow bile. Hippocrates wrote of a direct connection between the brain and spleen and its particu-lar association with the black bile. These ideas would influence thinking about the role of the spleen for more than 1000 years.2Aristotle, later in the same era, famously stated that, “Nature makes nothing in vain,” yet held the spleen to be an organ of minor importance whose main role was to

1	spleen for more than 1000 years.2Aristotle, later in the same era, famously stated that, “Nature makes nothing in vain,” yet held the spleen to be an organ of minor importance whose main role was to counterbal-ance the liver.4 He also described how the “hot character” of the spleen aided in digestion.Galen, in the second century AD, engaged in more seri-ous anatomic investigation espousing the early belief that func-tion followed structure. His investigations, though pioneering, lacked sufficient rigor, evidenced by his contention that black bile or melancholia flowed from the liver to the spleen and then through the short gastric vessels into the stomach to be excreted. The influence of Galen’s teaching endured for more than 1200 years. All the more remarkable considering the cur-rent hallowed measure of influence of surgical publication, the impact factor, which is most commonly calculated using a 2-year frame of reference.5,6In the early 17th century, several physician scientists,

1	measure of influence of surgical publication, the impact factor, which is most commonly calculated using a 2-year frame of reference.5,6In the early 17th century, several physician scientists, Mal-pighi being the most prominent, began testing hypotheses on splenic function by splenectomizing dogs. He reportedly fol-lowed several dogs 5 years postoperatively, noting their healthy survival though apparent ravenous hunger and enhanced sexual appetites. The spleen, still in the era of “balanced humors,” was thus felt to play a role in balancing various appetites as well. In addition to melancholy, the spleen became associated with anger and, paradoxically, was also seen as the “seat of laughter.”7The claim for the first human splenectomy may have pre-dated that of canine splenectomy. Andriano Zaccavello was credited in 1549 with having performed a splenectomy on a middle-aged woman. This claim remains shrouded in contro-versy, and the indication for the surgery and whether in fact

1	Zaccavello was credited in 1549 with having performed a splenectomy on a middle-aged woman. This claim remains shrouded in contro-versy, and the indication for the surgery and whether in fact splenectomy was performed have been called into question. The patient apparently survived, but may in fact have under-gone resection of an ovarian cyst rather than her spleen!8 Most patients who underwent splenectomy in the three centuries that followed fared badly. The vast majority of splenectomies per-formed were partial. Most of these patients required surgery for Historical Background 1517Embryology and Anatomy 1518Physiology and Pathophysiology 1520Indications for Splenectomy 1522Benign Disorders / 1524Malignant Conditions / 1527Miscellaneous Disorders and Lesions / 1529Imaging for Evaluation of Size and Pathology 1532Preoperative Considerations 1532Vaccination and Patient Education / 1532Deep Vein Thrombosis Prophylaxis / 1533Splenectomy Techniques 1533Patient Preparation / 1533Open

1	of Size and Pathology 1532Preoperative Considerations 1532Vaccination and Patient Education / 1532Deep Vein Thrombosis Prophylaxis / 1533Splenectomy Techniques 1533Patient Preparation / 1533Open Splenectomy / 1533Laparoscopic Splenectomy / 1534Hand-Assisted Splenectomy / 1534Single-Incision Laparoscopic Surgery Splenectomy / 1535Robotic Splenectomy / 1536Partial Splenectomy / 1536Inadvertent Intraoperative Splenic Injury / 1538Preoperative Grading Score to Predict Technical Difficulty in Laparoscopic Splenectomy / 1536Splenectomy Outcomes 1538Overwhelming Postsplenectomy Infection / 1538Complications / 1539Hematologic Outcomes / 1539Cancer / 1539Ultrasound / 1540Computed Tomography / 1540Plain Radiography / 1540Magnetic Resonance Imaging / 1540Angiography / 1541Nuclear Imaging / 1541Brunicardi_Ch34_p1517-p1548.indd 151723/02/19 2:36 PM 1518left upper quadrant stab wounds sustained in battles or duels resulting in partial or complete splenic prolapse.9It was in the early 18th

1	151723/02/19 2:36 PM 1518left upper quadrant stab wounds sustained in battles or duels resulting in partial or complete splenic prolapse.9It was in the early 18th century that the growing body of anatomic microstructural knowledge began to turn the tide on the long-held theory of health and disease deriving from a balance of the four humors. William Henson believed the spleen to be a ductless vascular gland similar to the thyroid and adrenals. In 1777, he wrote of the lymphatic nature of the spleen and its filtering function and even suggested its role in hematopoiesis.10Rudolf Virchow, one of the first to discover leukemia, implicated the spleen as figuring prominently in all leukemia patients. He suspected that the spleen was responsible for gen-erating the leukocytes in large quantities in these patients. There soon followed an enthusiastic effort by surgeons to cure leu-kemia by splenectomy. Dr. Thomas Bryant performed the first splenectomy in 1866 in a patient with leukemia.

1	in these patients. There soon followed an enthusiastic effort by surgeons to cure leu-kemia by splenectomy. Dr. Thomas Bryant performed the first splenectomy in 1866 in a patient with leukemia. The patient died, as did all 14 patients who underwent splenectomy for leukemia over the next 15 years. After his second consecutive mortality in this setting, Bryant declared that “the operation is physiologically unsound & surgically unsafe for leukemia and should not be performed.”11 In 1908, Johnson reported a series of 99 splenectomies for leukemia with an 85% mortality rate. Unfortunately, it took several decades for his words to be heeded.In 1916, a medical student from Prague named Paul Kaznelson wrote on the key role played by the spleen in the destruction of platelets leading to the first reported (and success-ful) splenectomy for a patient with idiopathic thrombocytopenia purpura.2As surgeons’ experience with the procedure grew, the associated morbidity and mortality decreased. By

1	reported (and success-ful) splenectomy for a patient with idiopathic thrombocytopenia purpura.2As surgeons’ experience with the procedure grew, the associated morbidity and mortality decreased. By 1920, the Mayo Clinic reported that splenectomy had a reduced mortality rate of 11%.1O’Donnell in 1929 was the first to describe fatal post-splenectomy sepsis in a child who had undergone the sur-gery for hemolytic anemia.3 It took Springer’s 1973 review of almost 2800 postsplenectomy patients and the 2.5% inci-dence of sepsis-induced mortality (vs. 0.01% in the general population) to reorient surgeons to more conservative splenic procedures.2,3The advent of minimally invasive surgery and laparo-scopic splenectomy in the early 1990s represented a clear advance, benefitting the patient through this evolution of sur-gical technique. Most large series of laparoscopic splenectomy for benign and malignant indication now report a mortality rate of <1%.12,13 As even more contemporary research

1	this evolution of sur-gical technique. Most large series of laparoscopic splenectomy for benign and malignant indication now report a mortality rate of <1%.12,13 As even more contemporary research reveals the spleen to play a central role in immune, metabolic, and endocrine function, it follows that the surgeon’s role going forward will be to preserve this organ and its functions when-ever possible.EMBRYOLOGY AND ANATOMYConsisting of an encapsulated mass of vascular and lymphoid tissue, the spleen is the largest reticuloendothelial organ in the body. Arising from the primitive mesoderm as an outgrowth of the left side of the dorsal mesogastrium, by the fifth week of gestation, the spleen is evident in an embryo 8 mm long.Development begins through the formation of the splanch-nic mesodermal plate, derived from the mesoderm, at embryonic day 12. The embryonic spleen is first colonized by erythroid and myeloid progenitor cells at 2 weeks of gestation. Follow-ing soon thereafter, the

1	plate, derived from the mesoderm, at embryonic day 12. The embryonic spleen is first colonized by erythroid and myeloid progenitor cells at 2 weeks of gestation. Follow-ing soon thereafter, the hematopoietic stem cells take up resi-dence in the forming spleen.14 The spleen assumes an important hematopoietic role until the fifth month of gestation. After birth, splenic erythropoietic function may persist in some hematologic disorders.15Key Points1 The human spleen plays a key immunologic role in defense against a number of organisms, particularly encapsulated bacteria.2 The spleen can cause significant morbidity and/or hema-tologic disturbance if it becomes hyperfunctioning (hyper-splenism) or hypertrophied (splenomegaly).3 There is a broad spectrum of nontraumatic diseases for which elective splenectomy can be curative or palliative. They can be broadly categorized as red blood cell disor-ders and hemoglobinopathies, white blood cell disorders, platelet disorders, bone marrow

1	elective splenectomy can be curative or palliative. They can be broadly categorized as red blood cell disor-ders and hemoglobinopathies, white blood cell disorders, platelet disorders, bone marrow disorders, infections and abscesses, cysts and tumors, storage diseases and infiltra-tive disorders, and miscellaneous conditions.4 Inadvertent intraoperative splenic injury is a scenario for which every abdominal surgeon should be prepared. Avail-ability of a predetermined algorithm, with emphasis on the patient’s condition, facilitates intraoperative decision making.5 Partial splenectomy may be a suitable alternative to total splenectomy for certain conditions of hypersplen-ism or splenomegaly, particularly in children in whom preservation of splenic immunologic function is especially important.6 Preoperative splenic artery embolization for elective sple-nectomy has benefits and disadvantages. It may be most suitable in cases of enlarged spleen. Conclusive evidence is lacking.7 Vaccination

1	splenic artery embolization for elective sple-nectomy has benefits and disadvantages. It may be most suitable in cases of enlarged spleen. Conclusive evidence is lacking.7 Vaccination of the splenectomized patient remains the most effective prevention strategy against OPSI. Preopera-tive vaccination before elective splenectomy is most prudent.8 Laparoscopic splenectomy provides equal hematologic outcomes with decreased morbidity compared with the open operation. The laparoscopic approach has emerged as the standard for elective, nontraumatic splenectomy.9 Overwhelming postsplenectomy infection (OPSI) is an uncommon but potentially grave disease. Children and those undergoing splenectomy for hematologic malig-nancy are at elevated risk.10 Antibiotic prophylactic strategies against OPSI vary widely. Data regarding their use are lacking.Brunicardi_Ch34_p1517-p1548.indd 151823/02/19 2:36 PM 1519THE SPLEENCHAPTER 34The organ continues its differentiation and migration to the left upper

1	widely. Data regarding their use are lacking.Brunicardi_Ch34_p1517-p1548.indd 151823/02/19 2:36 PM 1519THE SPLEENCHAPTER 34The organ continues its differentiation and migration to the left upper quadrant, where it comes to rest with its smooth, diaphragmatic surface facing posterosuperiorly.16The most common anomaly of splenic embryology is the accessory spleen. Present in up to 20% of the population, one or more accessory spleens may also occur in up to 30% of patients with hematologic disease. Over 80% of accessory spleens are found in the region of the splenic hilum and vascu-lar pedicle. Other locations for accessory spleens in descending order of frequency are the gastrocolic ligament, the pancreas tail, the greater omentum, the stomach’s greater curve, the splenocolic ligament, the small and large bowel mesentery, the left broad ligament in women, and the left spermatic cord in men (Fig. 34-1).10,16The abdominal surface of the diaphragm separates the spleen from the lower

1	the small and large bowel mesentery, the left broad ligament in women, and the left spermatic cord in men (Fig. 34-1).10,16The abdominal surface of the diaphragm separates the spleen from the lower left lung and pleura and the ninth to eleventh ribs. The visceral surface faces the abdominal cavity and contains gastric, colic, renal, and pancreatic impressions. Spleen size and weight vary with age, with both diminishing in the elderly and in those with underlying pathologic conditions. The average adult spleen is 7 to 11 cm in length and weighs 150 g (range, 70–250 g).The spleen’s superior border separates the diaphragmatic surface from the gastric impression of the visceral surface and often contains one or two notches, which are particularly pro-nounced when the spleen is greatly enlarged.Of particular clinical relevance, the spleen is suspended in position by several ligaments and peritoneal folds to the colon (splenocolic ligament), the stomach (gastrosplenic liga-ment), the

1	particular clinical relevance, the spleen is suspended in position by several ligaments and peritoneal folds to the colon (splenocolic ligament), the stomach (gastrosplenic liga-ment), the diaphragm (phrenosplenic ligament), and the kidney, ABCDEFGFigure 34-1. Sites where accessory spleens are found in order of importance. A. Hilar region, 54%; B. pedicle, 25%; C. tail of pan-creas, 6%; D. splenocolic ligament, 2%; E. greater omentum, 12%; F. mesentery, 0.5%; G. left ovary, 0.5%.Gastrosplenic ligamentLesser sacGreater omentumSplenocolic ligamentSustentaculum lienisPhrenicocolic ligamentFigure 34-2. Suspensory ligaments of the spleen.adrenal gland, and tail of the pancreas (splenorenal ligament) (Fig. 34-2). In a related historical footnote it was widely held less than 200 years ago that a “wandering spleen” led women to experience hypochondria. Dietl in 1863 finally clarified that “it was not a patient’s temperament but rather relaxation, exten-sion or the hypoplasia of splenic

1	that a “wandering spleen” led women to experience hypochondria. Dietl in 1863 finally clarified that “it was not a patient’s temperament but rather relaxation, exten-sion or the hypoplasia of splenic ligaments that made a spleen wander.”17 The gastrosplenic ligament contains the short gas-tric vessels; the remaining ligaments are avascular, with rare exceptions, such as in patients with portal hypertension. The relationship of the pancreas to the spleen also has important clinical implications. In cadaveric anatomic series, the tail of the pancreas has been demonstrated to lie within 1 cm of the splenic hilum 75% of the time and to actually abut the spleen in 30% of patients.2The spleen derives most of its blood from the splenic artery, the longest and most tortuous of the three main branches of the celiac artery. The splenic artery can be characterized by the pattern of its terminal branches. The distributed type of splenic artery is the most common (70%) and is distinguished by a

1	of the celiac artery. The splenic artery can be characterized by the pattern of its terminal branches. The distributed type of splenic artery is the most common (70%) and is distinguished by a short trunk with many long branches entering over three-fourths of the spleen’s medial surface. The less common magis-tral type of splenic artery (30%) has a long main trunk dividing near the hilum into short terminal branches, and these enter over 25% to 30% of the spleen’s medial surface. The spleen also receives some of its blood supply from the short gastric vessels that branch from the left gastroepiploic artery running within the gastrosplenic ligament. The splenic vein joins the superior mesenteric vein to form the portal vein and accommodates the major venous drainage of the spleen.When a normal, freshly excised spleen is sectioned, the cut surface is finely granular and predominantly dark red with whit-ish nodules distributed liberally across its expanse. This gross observation reflects

1	freshly excised spleen is sectioned, the cut surface is finely granular and predominantly dark red with whit-ish nodules distributed liberally across its expanse. This gross observation reflects the spleen’s microstructure. The splenic parenchyma is composed of two main elements: the red pulp, constituting approximately 75% of total splenic volume, and the white pulp (Fig. 34-3). At the interface between the red and white pulp is the narrow marginal zone.Brunicardi_Ch34_p1517-p1548.indd 151923/02/19 2:36 PM 1520SPECIFIC CONSIDERATIONSPART IIBlood enters the red pulp through cords comprised of fibro-blasts and reticular fibers, which contain many macrophages and lack an endothelial lining. The blood then passes from these “open” cords to venous sinuses, which are surrounded and sepa-rated by the same reticulum, and ultimately drains into tributar-ies of the splenic vein. An understanding of the microanatomy of these sinuses has elucidated the mechanical filtration function of the

1	by the same reticulum, and ultimately drains into tributar-ies of the splenic vein. An understanding of the microanatomy of these sinuses has elucidated the mechanical filtration function of the spleen. Unlike the cords of the red pulp, the sinuses of the red pulp are lined by endothelial cells. These cells contain unique stress fibers that connect the endothelial cells and that contain actin and myosin–like filaments capable of producing a sliding action. When activated, these filaments can create slits or gaps between the endothelial cells through which blood can then pass from the cords.14 Aging erythrocytes with stiffer mem-branes get stuck trying to pass into the sinus and are phagocy-tized by macrophages within the red pulp.15The red pulp thus serves as a dynamic filtration system, enabling macrophages to remove microorganisms, cellular debris, antigen-antibody complexes, and senescent erythrocytes from the circulation.Around the terminal millimeters of splenic arterioles, a

1	enabling macrophages to remove microorganisms, cellular debris, antigen-antibody complexes, and senescent erythrocytes from the circulation.Around the terminal millimeters of splenic arterioles, a periarticular lymphatic sheath replaces the native adventitia of the vessel. The sheath is comprised of T lymphocytes and intermittent aggregations of B lymphocytes or lymphoid fol-licles. When antigenically stimulated, the follicles, serving as centers of lymphocyte proliferation, develop germinal centers, which regress as the stimulus or infection subsides. This white pulp consists of nodules that normally are ≤1 mm in size but can increase to several centimeters when nodules coalesce, as occurs in certain lymphoproliferative disorders. At the junction between the white and red pulp is the marginal zone, where lymphocytes are more loosely aggregated.As well as serving as a transit area, the marginal zone is home to its own unique population of cells. Notably two spe-cific types of

1	the marginal zone, where lymphocytes are more loosely aggregated.As well as serving as a transit area, the marginal zone is home to its own unique population of cells. Notably two spe-cific types of macrophages reside there, marginal zone macro-phages and marginal zone metallophilic macrophages. The former play an important role in the targeting and clearance of certain bacterial pathogens. The latter have been shown to be the main producers of interferons A and B in response to a viral challenge.14PHYSIOLOGY AND PATHOPHYSIOLOGYThe spleen is contained by a 1to 2-mm thick capsule. In humans, the capsule is rich in collagen and contains some elas-tin fibers. Many mammals have splenic capsules and trabecu-lae with abundant smooth muscle cells, which upon autonomic stimulation contract to expel large volumes of stored blood into the general circulation. The human splenic capsule and trabecu-lae, by contrast, contain few or no smooth muscle cells.Total splenic inflow of blood is

1	to expel large volumes of stored blood into the general circulation. The human splenic capsule and trabecu-lae, by contrast, contain few or no smooth muscle cells.Total splenic inflow of blood is approximately 250 to 300 mL/min. Blood flows through successively tapering arteries to arterioles, traverses the white pulp, crosses the marginal zone, and enters the red pulp. From that entry, the flow rate through the spleen may vary greatly. Animal studies measuring the tran-sit times of isotopically labeled blood through the spleen have revealed three distinct velocities of flow. Humans have long been recognized to have both a fast or closed circulation—with blood passing directly from arterioles into venous sinuses—and a slower or open circulation. Most of the spleen’s filtration func-tion occurs via the slower circulation. During open circulation, blood percolates through the reticular space and splenic cords, thus gaining access through gaps or slits in the endothelial cell lining to

1	occurs via the slower circulation. During open circulation, blood percolates through the reticular space and splenic cords, thus gaining access through gaps or slits in the endothelial cell lining to the sinuses as previously described. Flowing into and out of the venous sinuses through these gaps, the blood is exposed to extensive contact with splenic macrophages. These are responsible for the innate immune response of the spleen, which occurs largely within the marginal zone. The white pulp, by contrast, is involved only in adaptive immunity. In addition, because the passage of plasma through these spaces does not slow in a similar manner, a temporary and unique adhesive con-tact between blood cells and components of the splenic cord may occur. That there is a selective slowing of blood cell flow versus plasma flow is further evidenced by the fact that within the spleen, the erythrocyte concentration (hematocrit) is twice that of the general circulation. During this contact with

1	cell flow versus plasma flow is further evidenced by the fact that within the spleen, the erythrocyte concentration (hematocrit) is twice that of the general circulation. During this contact with splenic macrophages, it is likely that the removal of both cellular debris and senescent blood cells occurs.18The process by which the spleen removes erythrocyte inclusions, such as Heinz bodies (intracellular altered hemo-globin), without cell lysis while red blood cells travel through the spleen is not well understood. The spleen acts as the major site for clearance from the blood of damaged or aged red blood cells and, in addition, has a part in the removal of abnormal white blood cells and platelets. A minimum of 2 days of the erythrocyte’s 120-day life cycle is spent sequestered in the spleen. Daily, approximately 20 mL of aged red blood cells are removed. Evidence suggests that, as erythrocytes age, previ-ously undetected antigens on their surfaces may attach to auto-antibodies in the

1	Daily, approximately 20 mL of aged red blood cells are removed. Evidence suggests that, as erythrocytes age, previ-ously undetected antigens on their surfaces may attach to auto-antibodies in the circulation; then macrophages may bind to the antibodies and initiate phagocytosis. It is probable that the erythrocyte is damaged over time by multiple passages through the spleen as well as delayed transit through the congested and relatively hypoxic and acidotic environment of the splenic cords.The spleen can also serve as an extra medullary site for hematopoiesis, if required. Another role played by the spleen is in recycling iron. Erythrocytes in large numbers are destroyed intravascularly throughout the body. The released hemoglobin is then bound to haptoglobin, which is ultimately scav-enged from the circulation in the spleen.191Trabeculae (depicted as both capsular and white lined material)Splenic capsuleRed pulpWhite pulpFigure 34-3. Splenic architecture. (Used with permission from

1	from the circulation in the spleen.191Trabeculae (depicted as both capsular and white lined material)Splenic capsuleRed pulpWhite pulpFigure 34-3. Splenic architecture. (Used with permission from Ivan George, University of Maryland School of Medicine.)Brunicardi_Ch34_p1517-p1548.indd 152023/02/19 2:36 PM 1521THE SPLEENCHAPTER 34The spleen plays a vital, although not indispensable, role in host defense evidenced by the healthy survival of splenec-tomized patients. Both innate and adaptive immune responses (historically categorized as cell-mediated and humoral immu-nity) occur within the spleen.In addition to the previously noted activities of the mar-ginal zone macrophages, marginal zone B cells serve to detect circulating pathogens and respond quickly to either differenti-ate into immunoglobulin M (IgM)–producing plasma cells or to function as antigen-presenting cells (APCs), which facilitate pathogen removal and destruction.It is APC entry in the white pulp in particular that is

1	M (IgM)–producing plasma cells or to function as antigen-presenting cells (APCs), which facilitate pathogen removal and destruction.It is APC entry in the white pulp in particular that is key to the initiation of the adaptive immune response. Antigens are thus presented to immunocompetent centers within the lymphoid follicles. This gives rise to the elaboration of immunoglobulins (predominantly IgM). After an antigen challenge, such an acute IgM response results in the release of opsonic antibodies from the white pulp of the spleen. Antigen clearance is then facilitated by the splenic and hepatic reticuloendothelial systems.The structure and immunophysiology of the white pulp is very similar to that of lymph nodes, with the notable difference being that material enters the lymph node in the lymph whereas it is delivered to the white pulp in the blood.20The spleen also produces opsonins, tuftsin, and proper-din. Circulating monocytes are converted within the red pulp into fixed

1	in the lymph whereas it is delivered to the white pulp in the blood.20The spleen also produces opsonins, tuftsin, and proper-din. Circulating monocytes are converted within the red pulp into fixed macrophages that account for the spleen’s remarkable phagocytic activity.The spleen also appears to be a major source of the protein properdin, important in the initiation of the alternate pathway of complement activation. The splenic reticuloendothelial system is better able to clear bacteria that are poorly or inadequately opsonized from the circulation than is the hepatic reticuloendo-thelial system. Encapsulated bacteria generally fit such a pro-file, hence the risk posed by pneumococcus and Haemophilus influenzae to an asplenic patient. There appears to be sufficient physiologic capacity within the complement cascade to with-stand the loss of tuftsin and properdin production without an increase in patient vulnerability after splenectomy.21-23In patients with chronic hemolytic disorders,

1	the complement cascade to with-stand the loss of tuftsin and properdin production without an increase in patient vulnerability after splenectomy.21-23In patients with chronic hemolytic disorders, splenic tissue may become permanently hypertrophied. The reticular spaces of the red pulp become distended with macrophages engorged with the products of erythrocyte breakdown, and splenomegaly can result. It is important to distinguish between splenomegaly and hypersplenism, two similar but distinct terms that are critical to understand when discussing splenic pathology. Splenomegaly refers simply to abnormal enlargement of the spleen. Splenomeg-aly is described variably within the surgical literature as moderate, massive, and hyper, which reflects a lack of con-sensus. Most would agree, however, that splenomegaly applies to organs weighing ≥500 g and/or averaging ≥15 cm in length.Massive splenomegaly similarly lacks a consensus defini-tion but has been described variably as spleens >1 kg in

1	that splenomegaly applies to organs weighing ≥500 g and/or averaging ≥15 cm in length.Massive splenomegaly similarly lacks a consensus defini-tion but has been described variably as spleens >1 kg in mass or >22 cm in length (Fig. 34-4).9 Spleens palpable below the left costal margin are thought to be at least double normal size, with an estimated weight of ≥750 g.24There is not a single, universally accepted standard, but most would agree that an ex vivo mass of >1 kg or a pole-to-pole length of >15 cm generally qualifies as splenomegaly. Hypersplenism often is found in association with splenomegaly but is not synonymous with it. Hypersplenism is defined as the presence of one or more cytopenias in the context of a normally functioning bone marrow.2ABCFigure 34-4. Splenomegaly. A. Computed tomography (CT) scan. B. Three-dimensional reconstruction of CT scan. C. Postoperative specimen.Brunicardi_Ch34_p1517-p1548.indd 152123/02/19 2:36 PM 1522SPECIFIC CONSIDERATIONSPART IIDisorders

1	tomography (CT) scan. B. Three-dimensional reconstruction of CT scan. C. Postoperative specimen.Brunicardi_Ch34_p1517-p1548.indd 152123/02/19 2:36 PM 1522SPECIFIC CONSIDERATIONSPART IIDisorders causing hypersplenism can be categorized as either (a) those in which increased destruction of abnormal blood cells occurs in an intrinsically normal spleen (e.g., hemolytic anemias) or (b) primary disorders of the spleen resulting in increased sequestration and destruction of normal blood cells (e.g., infiltrative disorders).The life cycles of cellular elements vary widely in human blood. A neutrophil in circulation has a normal half-life of approximately 6 hours. The spleen’s role in the normal clearance of neutrophils is not well established. It is clear that hypersplen-ism may result in neutropenia through sequestration of normal white blood cells or the removal of abnormal ones. Platelets, on the other hand, generally survive in the circulation for 10 days. Under normal circumstances,

1	through sequestration of normal white blood cells or the removal of abnormal ones. Platelets, on the other hand, generally survive in the circulation for 10 days. Under normal circumstances, a third of the total platelet pool is sequestered in the spleen. Thrombocytopenia may result from excessive sequestration of platelets as well as accelerated platelet destruction in the spleen. Splenomegaly may result in sequestration of up to 80% of the platelet pool. The spleen may also contribute to the immunologic alteration of platelets, which leads to thrombocytopenia in the absence of splenomegaly (e.g., idiopathic thrombocytopenic purpura [ITP]).25The immunologic functions of the spleen are consistent with those of other lymphoid organs. It is a site of bloodborne antigen presentation and the initiation of Tand B-lymphocyte activities involved in humoral and cellular immune responses. Alteration of splenic immune function often gives rise to anti-body production, which results in blood

1	initiation of Tand B-lymphocyte activities involved in humoral and cellular immune responses. Alteration of splenic immune function often gives rise to anti-body production, which results in blood cell destruction.Although the spleen contributes to the process of eryth-rocyte maturation, in adult humans there is little evidence of normal hematopoietic function. The spleen does have a minor role in hematopoiesis in the fourth month in the human fetus, and reactivation can occur in childhood if the bone marrow fails to meet the hematologic needs. Splenic hematopoiesis giving rise to abnormal red blood cells is seen in adults with myelopro-liferative disorders. In addition, in response to some anemias, elements of the red pulp may revert to hematopoiesis.INDICATIONS FOR SPLENECTOMYGenerally speaking, splenectomy is performed for the purposes of cure or palliation of hematological disease including condi-tions of hypersplenism, to relieve the mass effect and symptoms associated with

1	speaking, splenectomy is performed for the purposes of cure or palliation of hematological disease including condi-tions of hypersplenism, to relieve the mass effect and symptoms associated with splenomegaly, to control infection or hemorrhage and finally to diagnose splenic pathology. For the purposes of this chapter, we will divide these indications into the following categories: (a) benign conditions, including red blood cell disor-ders, hemoglobinopathies, and platelet disorders (Table 34-1a); (b) malignant conditions, including white blood cell disorders, bone marrow disorders (myeloproliferative disorders) and tumors of the spleen (Table 34-1b); and (c) miscellaneous conditions and lesions of the spleen including infections and abscesses, cysts, vascular anomalies, and more (Table 34-1c). We will conclude this chapter with a brief discussion on splenic salvage.Overall, the most common indication for splenectomy is trauma to the spleen, whether external trauma (blunt or

1	(Table 34-1c). We will conclude this chapter with a brief discussion on splenic salvage.Overall, the most common indication for splenectomy is trauma to the spleen, whether external trauma (blunt or penetrat-ing) or iatrogenic injury (e.g., at the time of other operations). Inadvertent intraoperative injury to the spleen, necessitating removal, also called “incidental splenectomy” is discussed in a 3Table 34-1aIndications for and expected response to splenectomy in various benign diseases, including red blood cell disorders, hemoglobinopathies, and platelet disordersDISEASE/CONDITIONINDICATIONS FOR SPLENECTOMYRESPONSE TO SPLENECTOMYEssential thrombocythemiaOnly for advanced disease (i.e., transformation to myeloid metaplasia or AML) with severe symptomatic splenomegalyRelief of abdominal pain and early satietyGlucose-6-phosphate dehydrogenase deficiency (G6PD)Excessive transfusion requirements; failure of medical therapy (controversial)May be curativeHereditary spherocytosisHemolytic

1	pain and early satietyGlucose-6-phosphate dehydrogenase deficiency (G6PD)Excessive transfusion requirements; failure of medical therapy (controversial)May be curativeHereditary spherocytosisHemolytic anemia, recurrent transfusions, intractable leg ulcersImproves or eliminates anemiaIdiopathic thrombocytopenic purpura (ITP)Failure of medical therapy, recurrent disease75%–85% rate of long-term responsePolycythemia veraOnly for advanced disease (i.e., transformation to myeloid metaplasia or AML) with severe symptomatic splenomegalyRelief of abdominal pain and early satietyPyruvate kinase deficiencyOnly in severe cases, recurrent transfusionsDecreased transfusion requirement, palliative onlySickle cell diseaseHistory of acute sequestration crisis, splenic symptoms, or infarction (consider concomitant cholecystectomy)Palliative, variable responseThalassemiaExcessive transfusion requirements, symptomatic splenomegaly, infarction, or hypersplenismDiminished transfusion requirements, relief

1	cholecystectomy)Palliative, variable responseThalassemiaExcessive transfusion requirements, symptomatic splenomegaly, infarction, or hypersplenismDiminished transfusion requirements, relief of symptomsThrombotic thrombocytopenic purpura (TTP)Excessive plasma exchange requirementTypically curativeWarm-antibody autoimmune hemolytic anemiaFailure of medical (steroid) therapy60%–80% response rate, recurrences commonBrunicardi_Ch34_p1517-p1548.indd 152223/02/19 2:36 PM 1523THE SPLEENCHAPTER 34Table 34-1bIndications for and expected response to splenectomy in various malignant diseases, including white blood cell disorders, myeloproliferative disorders, and nonhematologic tumors of the spleenDISEASE/CONDITIONINDICATIONS FOR SPLENECTOMYRESPONSE TO SPLENECTOMYAcute myeloid leukemia (AML)Intolerable symptomatic splenomegalyRelief of abdominal pain and early satietyChronic lymphocytic leukemia (CLL)Cytopenias and anemia75% response rateChronic myelogenous leukemia (CML)Symptomatic

1	symptomatic splenomegalyRelief of abdominal pain and early satietyChronic lymphocytic leukemia (CLL)Cytopenias and anemia75% response rateChronic myelogenous leukemia (CML)Symptomatic splenomegalyRelief of abdominal pain and early satietyChronic myelomonocytic leukemia (CMML)Symptomatic splenomegalyRelief of abdominal pain and early satietyMyelofibrosis (agnogenic myeloid metaplasia)Severe symptomatic splenomegaly76% clinical response at 1 y, high risk of hemorrhagic, thrombotic, and infectious complications (26%)Hairy cell leukemiaSevere symptomatic splenomegaly, severe transfusion requirements; failure of medical therapyCurativeHodgkin’s lymphomaSurgical staging in selected casesVariedNon-Hodgkin’s lymphomaCytopenias, symptomatic splenomegalyImproved complete blood count values, relief of symptomsMetastatic tumor of the spleen (most commonly breast, lung, and melanoma)If symptomatic, or as part of cancer treatmentVariedPrimary tumor of the spleenFor diagnosis and treatment of

1	of symptomsMetastatic tumor of the spleen (most commonly breast, lung, and melanoma)If symptomatic, or as part of cancer treatmentVariedPrimary tumor of the spleenFor diagnosis and treatment of cancerVariedTable 34-1cIndications for and expected response to splenectomy in various miscellaneous conditionsDISEASE/CONDITIONINDICATIONS FOR SPLENECTOMYRESPONSE TO SPLENECTOMYAbscess of the spleenMultiloculated, or failure of conservative measures for unilocularCurativeAmyloidosisSymptomatic splenomegalyImproves symptoms; does not correct underlying diseaseFelty’s syndromeNeutropenia80% durable response rateGaucher’s diseaseHypersplenismImproves cytopenias; does not correct underlying diseaseNiemann-Pick diseaseSymptomatic splenomegalyImproves symptoms; does not correct underlying diseasePortal/sinistral hypertensionSplenic vein thrombosis, symptomatic splenomegalyPalliativeSarcoidosisHypersplenism or symptomatic splenomegalyImproves symptoms and cytopenias; does not correct underlying

1	hypertensionSplenic vein thrombosis, symptomatic splenomegalyPalliativeSarcoidosisHypersplenism or symptomatic splenomegalyImproves symptoms and cytopenias; does not correct underlying diseaseSplenic artery aneurysmBest for distal lesions near splenic hilumCurativeSymptomatic nonparasitic cystsPartial splenectomy for small cysts; unroofing for large cystsCurativeSymptomatic parasitic cystsTherapy of choiceCurative; exercise caution not to spill cyst contentsWandering spleenAbdominal pain or splenomegaly (venous congestion)CurativeTraumatic ruptureGrades 4/5, or failure of conservative management of lower gradesCurativeBrunicardi_Ch34_p1517-p1548.indd 152323/02/19 2:36 PM 1524SPECIFIC CONSIDERATIONSPART IIlater section. Management of splenic injury in the trauma patient is also beyond the scope of this chapter and discussed else-where. The most common indications for elective splenec-tomy are malignancy and hematologic autoimmune disorders, principally, idiopathic thrombocytopenic

1	the scope of this chapter and discussed else-where. The most common indications for elective splenec-tomy are malignancy and hematologic autoimmune disorders, principally, idiopathic thrombocytopenic purpura (ITP) and autoimmune hemolytic anemia (AIHA).Benign DisordersRed Blood Cell Disorders Congenital Hereditary Spherocytosis. Hereditary spherocytosis (HS) is the most common type of hemolytic anemia for which splenectomy is indicated and the third most common type of congenital hemo-lytic anemia overall.26 HS results from an inherited dysfunction or deficiency in one of the erythrocyte membrane proteins (alpha or beta spectrin, ankyrin, band 3 protein, or protein 4.2). The result-ing destabilization of the membrane lipid bilayer allows a patho-logic release of membrane lipids. The red blood cell assumes a more spherical, less deformable shape, and the spherocytic eryth-rocytes are sequestered and destroyed in the spleen and hemolytic anemia ensues. HS is inherited primarily (70–80%

1	cell assumes a more spherical, less deformable shape, and the spherocytic eryth-rocytes are sequestered and destroyed in the spleen and hemolytic anemia ensues. HS is inherited primarily (70–80% of the time) in an autosomal dominant fashion; the estimated prevalence in Western populations is roughly 1 in 2000.26Patients with typical HS forms may have mild jaundice. Splenomegaly usually is palpable on physical examination. Laboratory examination reveals varying degrees of anemia: patients with mild forms of the disease may not have anemia; patients with moderate to severe forms may have hemoglobin levels as low as 4 to 6 g/dL. The mean corpuscular volume is typically low to normal or slightly decreased. For screening, a combined elevated mean corpuscular hemoglobin concentra-tion and an elevated erythrocyte distribution width are an excel-lent predictor. Other laboratory indicators of HS include those providing evidence of rapid red blood cell destruction, includ-ing elevated

1	and an elevated erythrocyte distribution width are an excel-lent predictor. Other laboratory indicators of HS include those providing evidence of rapid red blood cell destruction, includ-ing elevated reticulocyte count, elevated lactate dehydrogenase level, and increased level of unconjugated bilirubin. Sphero-cytes are readily apparent on peripheral blood film.Risks and benefits should be assessed carefully before splenectomy and cholecystectomy are performed for HS.27 The main indications are moderate to severe symptomatic hemo-lytic anemia, growth retardation, skeletal changes, leg ulcers, and extramedullary hemopoietic tumors in young patients.26,28 If gallstones coexist with spherocytosis, the gallbladder should be removed, but prophylactic cholecystectomy without gall-stones is controversial. Near total splenectomy is advocated in children. Dramatic clinical improvement—despite persistent hemolysis—usually occurs after splenectomy in patients with moderate to severe disease.

1	Near total splenectomy is advocated in children. Dramatic clinical improvement—despite persistent hemolysis—usually occurs after splenectomy in patients with moderate to severe disease. Because children can be affected with HS, the timing of splenectomy is important and is aimed at reducing the diminutive possibility of overwhelming postsple-nectomy sepsis. Delaying such an operation until the patient is between the ages of 4 and 6—unless the anemia and hemolysis accelerate—is recommended by most experts.29Red Blood Cell Enzyme Deficiencies. Red blood cell enzyme deficiencies associated with hemolytic anemia may be classified into two groups: deficiencies of enzymes involved in glycolytic pathways, such as pyruvate kinase deficiency, and deficien-cies of enzymes needed to maintain a high ratio of reduced to oxidized glutathione in the red blood cell, protecting it from oxidative damage, such as glucose-6-phosphate dehydrogenase (G6PD) deficiency.304Pyruvate Kinase Deficiency Pyruvate

1	ratio of reduced to oxidized glutathione in the red blood cell, protecting it from oxidative damage, such as glucose-6-phosphate dehydrogenase (G6PD) deficiency.304Pyruvate Kinase Deficiency Pyruvate kinase (PK) deficiency is the most common glycolytic defect causing congenital nons-pherocytic hemolytic anemia.31 Since its first description in the early 1960s, vast amounts of information have been elucidated about the genetic diversity of the disease, red blood cell clear-ance, long-term complications and treatment options includ-ing transfusion and splenectomy. PK deficiency affects people worldwide, with a slight preponderance among those of Northern European or Chinese descent. Its estimated prevalence in the Caucasian population is 51 per million.32,33 Clinical manifesta-tions of the disease vary widely, from transfusion-dependent severe anemia in early childhood to well-compensated mild ane-mia in adolescents or adults. Prenatal hydrops fetalis has also been reported.31 Pyruvate

1	disease vary widely, from transfusion-dependent severe anemia in early childhood to well-compensated mild ane-mia in adolescents or adults. Prenatal hydrops fetalis has also been reported.31 Pyruvate kinase enzyme activity is the gold standard for initial diagnostic testing or by detection of spe-cific mutations at the complementary DNA or genomic level. Splenomegaly is common, and in severe cases, splenectomy can alleviate transfusion requirements.31 As with other disorders that cause hemolytic anemia in children, splenectomy should be delayed if possible to at least 4 years of age to reduce the risk of postsplenectomy infection.Glucose-6-Phosphate Dehydrogenase Deficiency The most common red blood cell enzyme deficiency overall is G6PD deficiency. It is far more prevalent than PK deficiency with more than 400 million people affected worldwide, although most experience only moderate health risks and no longevity reduction.34 Clinical manifestations—chronic hemolytic ane-mia, acute

1	with more than 400 million people affected worldwide, although most experience only moderate health risks and no longevity reduction.34 Clinical manifestations—chronic hemolytic ane-mia, acute intermittent hemolytic episodes, or no hemolysis—depend on the variant of G6PD deficiency. The mainstay of therapy is avoidance of drugs known to precipitate hemolysis in patients with G6PD deficiency. Transfusions are given in cases of symptomatic anemia. Conventional wisdom is that splenectomy is not indicated in this disease, and certainly the overwhelming majority of patients with G6PD deficiency will neither require nor benefit from splenectomy. However, one report described a small case series of six symptomatic G6PD deficiency patients who had severe hemolytic anemia and required transfusion, all of whom were identified to share a common mutation at exon 10. All underwent splenectomy. A complete response occurred in four patients (transfusion requirement eliminated), and a partial

1	all of whom were identified to share a common mutation at exon 10. All underwent splenectomy. A complete response occurred in four patients (transfusion requirement eliminated), and a partial response occurred in one patient (transfusion requirement reduced); no follow-up data were provided for the remaining patient. This study indi-cates that for a carefully select group of patients with severe hemolytic anemia attributable to G6PD deficiency, splenec-tomy may be of benefit, although more data is needed before strong recommendation can be made.34Acquired Warm-Antibody Autoimmune Hemolytic Anemia. Autoim-mune hemolytic anemias (AIHAs) are characterized by the destruction of red blood cells, whose erythrocyte life span is diminished by autoantibodies leveled against antigens. AIHA is classified as either primary or secondary, depending on whether an underlying cause, such as a disease or toxin, is iden-tified. AIHA is also divided into “warm” and “cold” catego-ries, based on the

1	classified as either primary or secondary, depending on whether an underlying cause, such as a disease or toxin, is iden-tified. AIHA is also divided into “warm” and “cold” catego-ries, based on the temperature at which the autoantibodies exert their effect.35 In cold-agglutinin disease, severe symptoms are uncommon and splenectomy is almost never indicated; there-fore, this entity is not discussed further in this section. However, warm-antibody AIHA has clinical consequences with which the surgeon should be familiar.Brunicardi_Ch34_p1517-p1548.indd 152423/02/19 2:36 PM 1525THE SPLEENCHAPTER 34Warm-antibody AIHA, although occurring primarily in midlife, can affect individuals at all ages. The disorder is more common among women, and fully half of warm-antibody AIHA cases are idiopathic. Clinical presentation may be acute or gradual. Findings include mild jaundice and symptoms and signs of anemia. One-third to one-half of patients present with splenomegaly. Sometimes in such cases

1	Clinical presentation may be acute or gradual. Findings include mild jaundice and symptoms and signs of anemia. One-third to one-half of patients present with splenomegaly. Sometimes in such cases the spleen is palpable on physical examination. The diagnosis relies on demonstrat-ing hemolysis as indicated by anemia, reticulocytosis, and/or products of red blood cell destruction, including bilirubin, in the blood, urine, and stool. A positive result on direct Coombs’ test confirms the AIHA diagnosis by distinguishing autoimmune from other forms of hemolytic anemia.Treatment of AIHA depends on the severity of the disease and whether it is primary or secondary. Severe symptomatic anemia demands prompt attention, often requiring red blood cell transfusion. The mainstay treatment for both primary and secondary forms of symptomatic, unstable AIHA remains longterm corticosteroid administration.36 Therapy should continue until a response is noted by a rise in hematocrit and fall in

1	both primary and secondary forms of symptomatic, unstable AIHA remains longterm corticosteroid administration.36 Therapy should continue until a response is noted by a rise in hematocrit and fall in reticu-locyte count, which generally occurs within 3 weeks.Clinical response to splenectomy for AIHA varies, and the evidence from a number of small case series is conflicting. For example, one 2004 series reported a favorable response to splenectomy in patients with AIHA secondary to chronic lym-phocytic leukemia, whereas more recent series found no benefit from splenectomy in patients with AIHA secondary to systemic lupus erythematosus or inflammatory bowel disease.37,38Favorable responses to splenectomy have been reported in patients with warm-antibody AIHA, with a recent series show-ing complete remission of refractory AIHA following lapa-roscopic splenectomy at 35-month follow-up in patients over 60 years old.36 Transient responses are more common, how-ever, and many patients

1	complete remission of refractory AIHA following lapa-roscopic splenectomy at 35-month follow-up in patients over 60 years old.36 Transient responses are more common, how-ever, and many patients eventually experience hemolysis again despite splenectomy.37,38 The decision regarding splenectomy in the case of AIHA should be individualized based on careful consideration of the clinical history and frank discussion with the patient. It is considered as a third-line therapy after failure of steroids or anti-CD20 antibody administration.39Hemoglobinopathies. Sickle cell disease is an inherited chronic hemolytic anemia that results from the mutant sickle cell hemoglobin (HbS) within the red blood cell and is inher-ited in an autosomal dominant fashion. Persons who inherit an HbS gene from one parent (heterozygous) are carriers; those who inherit an HbS gene from both parents (homozygous) have sickle cell anemia.40In sickle cell disease, the underlying abnormality is the mutation of adenine to

1	(heterozygous) are carriers; those who inherit an HbS gene from both parents (homozygous) have sickle cell anemia.40In sickle cell disease, the underlying abnormality is the mutation of adenine to thymine in the sixth codon of the β-globin gene, which results in the substitution of valine for glutamic acid as the sixth amino acid of the β-globin chain. Mutant β chains included in the hemoglobin tetramer create HbS. Deoxy-genated HbS is insoluble and becomes polymerized and sickled. The subsequent lack of deformability of the red blood cell, in addition to other processes, results in microvascular congestion, which may lead to thrombosis, ischemia, and tissue necrosis. The disorder is characterized by painful intermittent episodes.Sequestration occurs in the spleen, with splenomegaly resulting early in the disease course. In most patients, subse-quent infarction of the spleen and autosplenectomy occur at some later time. The most frequent indications for splenectomy in sickle cell

1	early in the disease course. In most patients, subse-quent infarction of the spleen and autosplenectomy occur at some later time. The most frequent indications for splenectomy in sickle cell disease are recurrent acute sequestration crises, hypersplenism, and splenic abscess. The occurrence of one major acute sequestration crisis, characterized by rapid painful enlargement of the spleen and circulatory collapse, generally is considered sufficient grounds for splenectomy. Both partial and total splenectomy have been shown to control clinical symp-toms in children but may not change hematologic parameters.41 Preoperative preparation should include special attention to adequate hydration and avoidance of hypothermia.Transfusions are often indicated for anemia, for mod-erately severe episodes of acute chest syndrome (i.e., a new infiltrate on chest radiograph associated with new symptoms, such as fever, cough, sputum production, or hypoxia), and pre-operatively before splenectomy.

1	of acute chest syndrome (i.e., a new infiltrate on chest radiograph associated with new symptoms, such as fever, cough, sputum production, or hypoxia), and pre-operatively before splenectomy. Patients experiencing stroke or a severe crisis may require hydration and an exchange trans-fusion, which may be performed manually or with automated apheresis equipment. Hydroxyurea is an oral chemotherapeutic agent that upregulates fetal hemoglobin, which interferes with polymerization of HbS and thus reduces the sickling process.42Thalassemia. Thalassemia is the term for a group of inherited disorders of hemoglobin synthesis prevalent among people of Mediterranean extraction and classified according to the hemo-globin chain (α, β, or γ) affected. As a group, the thalassemias are the most common genetic diseases known to arise from a single gene defect.43,44 Most forms of this disorder are inherited in Mendelian recessive fashion from asymptomatic carrier par-ents. In the so-called thalassemia

1	diseases known to arise from a single gene defect.43,44 Most forms of this disorder are inherited in Mendelian recessive fashion from asymptomatic carrier par-ents. In the so-called thalassemia belt that extends throughout the shores of the Mediterranean as well as through the Arabian Peninsula, Turkey, Iran, India, and southeastern Asia, the inci-dence of thalassemia is between 2.5% and 15%. However, thal-assemias have been found in people of all ethnic origins.44In all forms of thalassemia, the primary defect is absent or reduced production of hemoglobin chains. From this abnormal-ity, two significant consequences arise: (a) reduced functioning of hemoglobin tetramers, yielding hypochromia and microcy-tosis; and (b) unbalanced biosynthesis of individual α and β subunits, which results in insoluble red blood cells that cannot release oxygen normally and may precipitate with cell aging. Both underproduction of hemoglobin and excess production of unpaired hemoglobin subunits contribute

1	insoluble red blood cells that cannot release oxygen normally and may precipitate with cell aging. Both underproduction of hemoglobin and excess production of unpaired hemoglobin subunits contribute to thalassemia-associated morbidity and mortality.A diagnosis of thalassemia major (homozygous form) is made by demonstrating hypochromic microcytic anemia asso-ciated with randomly distorted red blood cells and nucleated erythrocytes (target cells) on peripheral blood smear.7 Elevated reticulocyte count and white blood cell count are among the associated findings. Because α chains are needed to form both fetal hemoglobin and adult hemoglobin, α-thalassemia becomes symptomatic in utero or at birth. By contrast, β-thalassemia becomes symptomatic at 4 to 6 months because β chains are involved only in adult hemoglobin synthesis.The clinical spectrum of the thalassemias is wide. Heterozygous carriers of the disease are usually asymptomatic. Homozygous individuals, on the other hand, typically

1	in adult hemoglobin synthesis.The clinical spectrum of the thalassemias is wide. Heterozygous carriers of the disease are usually asymptomatic. Homozygous individuals, on the other hand, typically present before 2 years of age with pallor, growth retardation, jaundice, and abdominal swelling due to liver and spleen enlargement. Among other characteristics of thalassemia major are intractable leg ulcers, head enlargement, frequent infections, and the need for periodic blood transfusions. Untreated individuals usually die in late infancy or early childhood from severe anemia.7Treatment for thalassemia involves red blood cell transfu-sions to maintain a hemoglobin level of >9 mg/dL, along with Brunicardi_Ch34_p1517-p1548.indd 152523/02/19 2:36 PM 1526SPECIFIC CONSIDERATIONSPART IIintensive parenteral chelation therapy with deferoxamine. Sple-nectomy is indicated for patients with excessive transfusion requirements (>200 mL/kg per year), discomfort due to spleno-megaly, or painful

1	parenteral chelation therapy with deferoxamine. Sple-nectomy is indicated for patients with excessive transfusion requirements (>200 mL/kg per year), discomfort due to spleno-megaly, or painful splenic infarction. Careful assessment of the risk-benefit ratio is essential. Thalassemia patients are at high risk for pulmonary hypertension after splenectomy; however, the precise pathophysiology of this sequela remains unclear.45,46 The increase in infectious complications is likely to be due to a coexisting immune deficiency, in large part brought about by iron overload, which may be associated both with the thalas-semia itself and with transfusions. The disproportionately high rate of overwhelming postsplenectomy infection in thalassemia patients has led some investigators to consider partial splenec-tomy in children; some success in reducing mortality has been reported.47 However, splenectomy should be delayed until after the age of 4 years unless it is absolutely necessary.Platelet

1	splenec-tomy in children; some success in reducing mortality has been reported.47 However, splenectomy should be delayed until after the age of 4 years unless it is absolutely necessary.Platelet Disorders Idiopathic Thrombocytopenic Purpura Idiopathic thrombo-cytopenic purpura (ITP), also called immune thrombocytopenic purpura, is an autoimmune disorder characterized by a low platelet count and mucocutaneous and petechial bleeding. The low platelet count stems from premature removal of platelets opsonized by antiplatelet immunoglobulin G autoantibodies produced in the spleen. This clearance occurs through the inter-action of platelet autoantibodies with Fc receptors expressed on tissue macrophages, predominantly in the spleen and liver. The estimated incidence of ITP is 100 persons per million annually, about one-half of whom are children.48 Adult-onset and child-hood-onset ITP are strikingly different in their clinical course and management.Patients with ITP typically present with

1	million annually, about one-half of whom are children.48 Adult-onset and child-hood-onset ITP are strikingly different in their clinical course and management.Patients with ITP typically present with petechiae or ecchymoses, although some experience major bleeding from the outset. Bleeding may occur from mucosal surfaces in the form of gingival bleeding, epistaxis, menorrhagia, hematuria, or even melena. The severity of bleeding frequently corresponds to the deficiency in platelets: Patients with counts greater than 50,000/mm3 usually present with incidental findings; those with counts between 30,000 and 50,000/mm3 often have easy bruis-ing; those with platelet counts between 10,000 and 30,000/mm3 may develop spontaneous petechiae or ecchymoses; and those with counts less than 10,000/mm3 are at risk for internal bleeding.48 The incidence of major intracranial hemorrhage is approximately 1%, and it usually occurs early in the disease course. The duration of the bleeding helps to

1	are at risk for internal bleeding.48 The incidence of major intracranial hemorrhage is approximately 1%, and it usually occurs early in the disease course. The duration of the bleeding helps to distinguish acute from chronic forms of ITP. Children often present at a young age (peak age of approximately 5 years) with sudden onset of petechiae or purpura several days to weeks after an infectious illness. In contrast, adults experience a more chronic form of disease with an insidious onset. Splenomegaly is uncommon with ITP in both adults and children, and its occurrence should prompt a search for a separate cause of thrombocytopenia. Up to 10% of children, however, have a palpable spleen tip.Diagnosis of ITP is based on exclusion of other possibili-ties in the presence of a low platelet count and mucocutane-ous bleeding. Other diseases resulting in secondary forms of ITP, such as systemic lupus erythematosus, antiphospholipid syndrome, lymphoproliferative disorders, human

1	platelet count and mucocutane-ous bleeding. Other diseases resulting in secondary forms of ITP, such as systemic lupus erythematosus, antiphospholipid syndrome, lymphoproliferative disorders, human immunodefi-ciency virus (HIV) infection, and hepatitis C, should be identi-fied and treated when present. In addition, any history of use of a drug known to cause thrombocytopenia, such as certain antimicrobials, anti-inflammatories, antihypertensives, and 5Table 34-2Second-line treatment options for immune thrombocytopeniaSplenectomyAdvantages• Effective, time-honored treatment• Most effective response (overall 88%, partial 22%, complete 66%)Disadvantages• Risk of overwhelming postsplenectomy infection• Increased risk of thrombotic events• Morbidity and mortality associated with an operationRituximabAdvantages• Nonsurgical• Good experience since 1999• Initial immediate response 63%• 31% response at 2 yearsDisadvantages• Severe toxicity in 2%–6%Thrombopoietin Receptor

1	with an operationRituximabAdvantages• Nonsurgical• Good experience since 1999• Initial immediate response 63%• 31% response at 2 yearsDisadvantages• Severe toxicity in 2%–6%Thrombopoietin Receptor AgonistAdvantages• Nonsurgical• Oral agent self-administered weekly• 80% response rateDisadvantages• Long-term treatment required• Potential toxicity• Long-term data lacking at this timeantidepressants, should be sought. In addition to low platelet count, another laboratory finding characteristic of ITP is the presence of large, immature platelets (megakaryocytes) on peripheral blood smear.The usual first line of therapy is oral prednisone at a dos-age of 1.0 to 1.5 mg/kg per day.49,50 No consensus exists as to the optimal duration of steroid therapy, but most responses occur within the first 3 weeks. Response rates range from 50% to 75%, but relapses are common. IV immunoglobulin, given at 1.0 g/kg per day for 2 to 3 days, is indicated for internal bleeding when platelet counts remain less

1	weeks. Response rates range from 50% to 75%, but relapses are common. IV immunoglobulin, given at 1.0 g/kg per day for 2 to 3 days, is indicated for internal bleeding when platelet counts remain less than 5000/mm3, when extensive pur-pura exists, or to preoperatively boost platelets. IV immunoglob-ulin is thought to impair clearance of immunoglobulin G–coated platelets by competing for binding to tissue macrophage recep-tors. An immediate response is common, but a sustained remis-sion is not. The medical approach to ITP has been modified with the advent of rituximab and thrombopoietin receptor agonists.51 Therapeutic recommendations are summarized in Table 34-2. Splenectomy is selectively indicated for failure of medical ther-apy, for prolonged use of steroids with undesirable effects, and in selected cases after first relapse.7 Prolonged use of steroids can be defined in various ways, but a persistent need for more than 10 to 20 mg/d for 3 to 6 months to maintain a platelet count of

1	in selected cases after first relapse.7 Prolonged use of steroids can be defined in various ways, but a persistent need for more than 10 to 20 mg/d for 3 to 6 months to maintain a platelet count of greater than 30,000/mm3 generally prompts referral for sple-nectomy. Splenectomy is an effective option for refractory ITP and provides a permanent response without subsequent need for steroids in 75% to 85% of patients. Two recent reviews and meta-analyses have assessed the global results of splenectomy for ITP, specifically after the use of laparoscopic techniques. Brunicardi_Ch34_p1517-p1548.indd 152623/02/19 2:36 PM 1527THE SPLEENCHAPTER 34In 2004, Kojouri and colleagues reviewed 135 case series from 1966 to 2004, pooling 4955 patients.51 The long-term plate-let count response was assessed, as was the ability to predict response and the incidence of complications. Complete response was achieved in 66% of cases with a follow-up ranging from 1 to 153 months, and complete and partial

1	as was the ability to predict response and the incidence of complications. Complete response was achieved in 66% of cases with a follow-up ranging from 1 to 153 months, and complete and partial responses occurred in as many as 88% of patients, regardless of the duration of follow-up. They also analyzed 12 preoperative demographic, clinical, and laboratory parameters and found no predictive capability of platelet response in any of them. Mortality was very low (1%), and morbidity was 10%. Limitations of this review included old case series and a low percentage of laparoscopic splenectomies. In a 2009 systemic review involving 1223 patients, Mikhael and colleagues evaluated the shortand long-term outcomes after laparoscopic splenectomy.52 The conversion rate to open sur-gery was 5.6%, and the immediate nonresponder rate was 8.2%; however, eventually a clinical response was achieved in 72% of the patients on long-term follow-up (5 years). The initial con-cerns regarding the potential for

1	immediate nonresponder rate was 8.2%; however, eventually a clinical response was achieved in 72% of the patients on long-term follow-up (5 years). The initial con-cerns regarding the potential for missing accessory spleens, lon-ger operative times, and increased cost related to laparoscopic versus open splenectomy have been resolved. Laparoscopy is now the approach of choice for elective splenectomy for ITP, with recent studies showing improved short-term results and comparable long-term results to conventional open splenectomy in this condition.53-56In children with ITP, the course is typically self-limited, with durable and complete remission in greater than 70% of patients regardless of therapy. Because of the good prognosis without treatment, the decision to intervene surgically is contro-versial and is largely to obviate intracranial hemorrhage as dis-cussed earlier. Thus, children with typical ITP—and certainly those without hemorrhage—are managed principally by obser-vation,

1	contro-versial and is largely to obviate intracranial hemorrhage as dis-cussed earlier. Thus, children with typical ITP—and certainly those without hemorrhage—are managed principally by obser-vation, with short-term therapy in select cases.57 Urgent sple-nectomy, in conjunction with aggressive medical therapy, may play a role in the rare circumstance of severe, life-threatening bleeding in both children and adults.Thrombotic Thrombocytopenic Purpura Thrombotic throm-bocytopenic purpura (TTP) is a serious disorder characterized by thrombocytopenia, microangiopathic hemolytic anemia, and neurologic complications. Abnormal platelet clumping occurs in arterioles and capillaries, reducing the lumen of these vessels and predisposing the patient to microvascular thrombotic epi-sodes. The reduced lumen size also causes shearing stresses on erythrocytes, which leads to deformed red blood cells subject to hemolysis. Hemolysis may also be due in part to sequestration and destruction of

1	reduced lumen size also causes shearing stresses on erythrocytes, which leads to deformed red blood cells subject to hemolysis. Hemolysis may also be due in part to sequestration and destruction of erythrocytes in the spleen. Research has dem-onstrated that the underlying abnormality is likely related to the persistence of unusually large multimers of von Willebrand fac-tor associated with platelet clumping in the patient’s blood.58,59TTP occurs in approximately 3.7 individuals per million, but this rare disorder’s dramatic clinical sequelae and favorable response to early therapy demand an understanding of its clini-cal presentation to ensure an early diagnosis. Clinical features of the disorder include petechiae, fever, neurologic symptoms, renal failure, and, infrequently, cardiac symptoms such as heart failure or arrhythmias. Petechial hemorrhages in the lower extremities are the most common presenting sign. Along with fever, patients may experience flu-like symptoms, malaise, or

1	such as heart failure or arrhythmias. Petechial hemorrhages in the lower extremities are the most common presenting sign. Along with fever, patients may experience flu-like symptoms, malaise, or fatigue. Neurologic changes range from generalized headaches to altered mental status, seizures, and even coma. Generally, however, the mere presence of petechiae and thrombocytopenia is sufficient to lead to the diagnosis of TTP and consideration of treatment.The diagnosis is confirmed by the peripheral blood smear, which shows schistocytes, nucleated red blood cells, and baso-philic stippling. Although other conditions such as tight aortic stenosis or prosthetic valves may lead to the presence of schis-tocytes, these conditions generally are not accompanied by thrombocytopenia. TTP may be distinguished from autoimmune causes of thrombocytopenia, such as Evans’ syndrome (ITP and autoimmune hemolytic anemia) or systemic lupus erythemato-sus, by a negative result on Coombs’ test.Plasma exchange

1	from autoimmune causes of thrombocytopenia, such as Evans’ syndrome (ITP and autoimmune hemolytic anemia) or systemic lupus erythemato-sus, by a negative result on Coombs’ test.Plasma exchange is the first-line therapy for TTP. This treatment consists of the daily removal of a single volume of the patient’s plasma and its replacement with fresh-frozen plasma until the thrombocytopenia, anemia, and associated symptoms are corrected. Therapy is then tapered over 1 to 2 weeks.40 Splenectomy should be considered in patients who experience relapse or who require multiple plasma exchanges to control severe symptoms, and generally is well tolerated without sig-nificant morbidity.59,60Malignant ConditionsWhite Blood Cell Disorders. The role of splenectomy in patients with white blood cell disorders varies. As for the myelogenous diseases mentioned previously, splenectomy for white blood cell disorders can be effective therapy for symp-tomatic splenomegaly and hypersplenism, improving some

1	varies. As for the myelogenous diseases mentioned previously, splenectomy for white blood cell disorders can be effective therapy for symp-tomatic splenomegaly and hypersplenism, improving some clinical parameters but generally not altering the course of the underlying disease or long-term prognosis. Historically, sple-nectomy has played a role during surgical staging for Hodg-kin’s disease, although this practice has become largely obsolete with the advent of imaging technologies (Computed tomogra-phy [CT] scan and 18F-fluorodeoxyglucose positron emission tomography [18F-FDG PET]).61-63 Careful consideration of the intended benefits of splenectomy must be weighed against the significant perioperative and postsplenectomy risks in this often complex patient population.64Hairy Cell Leukemia Hairy cell leukemia (HCL) is an uncom-mon blood disorder, representing only 2% of all adult leukemias. HCL is characterized by splenomegaly, pancytopenia, and large numbers of abnormal lymphocytes in

1	cell leukemia (HCL) is an uncom-mon blood disorder, representing only 2% of all adult leukemias. HCL is characterized by splenomegaly, pancytopenia, and large numbers of abnormal lymphocytes in the bone marrow. These lymphocytes contain irregular hair-like cytoplasmic projections identifiable on the peripheral smear. Many HCL patients have few symptoms and require no specific therapy. Splenectomy for HCL was historically performed as a palliative procedure, alleviating the symptoms associated with splenomegaly and normalizing peripheral blood counts in the majority of patients, but not leading to morphological bone marrow remissions. However, with the advent of diverse new drugs (e.g., ritux-imab, pentostatin, cladribine), splenectomy has become rarely performed.65 Splenectomy should be considered after exhaustive systemic therapy or for those with nontraumatic rupture, and it has also been used to treat pregnant women with HCL to delay onset of chemotherapy.66Hodgkin’s

1	should be considered after exhaustive systemic therapy or for those with nontraumatic rupture, and it has also been used to treat pregnant women with HCL to delay onset of chemotherapy.66Hodgkin’s Lymphoma Hodgkin’s Lymphoma (HL) is a dis-order of the lymphoid system characterized by the presence of Reed-Sternberg cells (which actually form the minority of the Hodgkin’s tumor). More than 90% of patients with HL pres-ent with lymphadenopathy above the diaphragm. Lymph nodes can become particularly bulky in the mediastinum, which may result in shortness of breath, cough, or obstructive pneumonia. Brunicardi_Ch34_p1517-p1548.indd 152723/02/19 2:36 PM 1528SPECIFIC CONSIDERATIONSPART IILymphadenopathy below the diaphragm is rare on presen-tation but can arise with disease progression. The spleen is often an occult site of spread, but massive splenomegaly is not common. In addition, large spleens do not necessarily signify involvement.62Four major histologic types exist: lymphocyte

1	The spleen is often an occult site of spread, but massive splenomegaly is not common. In addition, large spleens do not necessarily signify involvement.62Four major histologic types exist: lymphocyte predomi-nance type, nodular sclerosis type, mixed cellularity type, and lymphocyte depletion type. The histologic type, along with location of disease and symptomatology, influence survival for patients with HD. Stage I disease is limited to one anatomic region; stage II disease is defined by the presence of two or more contiguous or noncontiguous regions on the same side of the diaphragm; stage III disease involves disease on both sides of the diaphragm, but limited to lymph nodes, spleen, and Waldeyer’s ring (the ring of lymphoid tissue formed by the lin-gual, palatine, and nasopharyngeal tonsils); and stage IV disease includes involvement of the bone marrow, lung, liver, skin, gas-trointestinal tract, or any organ or tissue other than the lymph nodes or Waldeyer’s ring.62Staging

1	tonsils); and stage IV disease includes involvement of the bone marrow, lung, liver, skin, gas-trointestinal tract, or any organ or tissue other than the lymph nodes or Waldeyer’s ring.62Staging laparotomy for HL is less commonly performed in the current era of minimally invasive surgery and advanced imaging techniques. More liberal use of diagnostic tools (CT scan and 18F-FDG PET) and chemotherapy for patients with HL has drastically reduced the indications for surgical staging. Current indications for surgical staging include clini-cal suspicion of lymphoma without evidence of peripheral disease or patients requiring restaging for suspicion of failure after chemotherapy.62,67Non-Hodgkin’s Lymphoma Non-Hodgkin’s lymphoma (NHL) encompasses all malignancies derived from the lymphoid sys-tem except classic HL.63 A proliferation of any one of the three predominant lymph cell types—natural killer cells, T cells, or B cells—may be included in the category of NHL. Because of the wide net

1	except classic HL.63 A proliferation of any one of the three predominant lymph cell types—natural killer cells, T cells, or B cells—may be included in the category of NHL. Because of the wide net cast by NHL, the clinical presentations of the dis-orders under its umbrella vary. The subentities of NHL may be clinically classified into nodal or extranodal, as well as indolent, aggressive, and very aggressive groups. Patients with indolent lymphomas may present with mild or no symptoms and seek medical attention for a swollen lymph node, whereas the aggres-sive and very aggressive lymphomas create easily noticeable symptoms, such as pain, swelling due to obstruction of vessels, fever, and night sweats. Surgical staging is no longer indicated for NHL because the combination of history and physical exam-ination, chest radiograph and abdominal/pelvic CT scan, biopsy of involved lymph nodes (including laparoscopically directed nodal and liver biopsies), and bone marrow biopsy is

1	and physical exam-ination, chest radiograph and abdominal/pelvic CT scan, biopsy of involved lymph nodes (including laparoscopically directed nodal and liver biopsies), and bone marrow biopsy is suffi-cient.63 Splenomegaly exists in some, but not all, forms of NHL. Splenectomy does not alter the natural history of the disease, but it is indicated in cases where a diagnosis cannot be established by obtaining peripheral tissue and clinical suspicion remains or for management of symptoms related to an enlarged spleen as well as for improvement of cytopenias.68-72Chronic Lymphocytic Leukemia Chronic lymphocytic leuke-mia (CCL) is currently considered a subtype of NHL. The main characteristic of CLL is a progressive accumulation of old and nonfunctional lymphocytes.63,73 Symptoms of CLL are nonspe-cific and include weakness, fatigue, fever without illness, night sweats, and frequent bacterial and viral infections. The most fre-quent finding is lymphadenopathy. When the spleen is enlarged,

1	and include weakness, fatigue, fever without illness, night sweats, and frequent bacterial and viral infections. The most fre-quent finding is lymphadenopathy. When the spleen is enlarged, it may be massive or barely palpable below the costal margin. Splenectomy is indicated to improve cytopenias and was shown to be 75% effective in a combined group of patients who had either CLL or nonmalignant HD.29 Splenectomy may facilitate chemotherapy in patients whose cell counts were prohibitively low before spleen removal. Palliative splenectomy also is indi-cated for symptomatic splenomegaly.Bone Marrow Disorders (Myeloproliferative Disorders). The myeloproliferative disorders are characterized by an abnor-mal growth of cell lines in the bone marrow. They include chronic myeloid leukemia, acute myeloid leukemia, chronic myelomono-cytic leukemia, essential thrombocythemia, polycythemia vera, and myelofibrosis, also known as agnogenic myeloid metaplasia (see “Myelofibrosis [Agnogenic Myeloid

1	myeloid leukemia, chronic myelomono-cytic leukemia, essential thrombocythemia, polycythemia vera, and myelofibrosis, also known as agnogenic myeloid metaplasia (see “Myelofibrosis [Agnogenic Myeloid Metaplasia]” later in this chapter). The common underlying problem leading to sple-nectomy in these disorders is symptomatic splenomegaly. Symp-toms due to splenomegaly are due to mass effect and consist of early satiety, poor gastric emptying, heaviness or pain in the left upper quadrant, and even diarrhea. Hypersplenism, when it occurs in these conditions, usually is associated with splenomeg-aly. Splenectomy performed in the setting of the myeloprolifera-tive disorders is generally for treatment of the pain, early satiety, and other symptoms of splenomegaly. Radiation has been used since 1903 to treat symptomatic splenomegaly in myeloprolif-erative disorders, but today it is principally used in situations in which splenectomy is not an option.Chronic Myelogenous Leukemia Chronic

1	1903 to treat symptomatic splenomegaly in myeloprolif-erative disorders, but today it is principally used in situations in which splenectomy is not an option.Chronic Myelogenous Leukemia Chronic myelogenous leu-kemia (CML) is a disorder of the primitive pluripotent stem cells in the bone marrow that results in a significant increase in erythroid, megakaryotic, and pluripotent progenitors in the peripheral blood smear. The genetic hallmark is a transposition between the bcr gene on chromosome 9 and the abl gene on chromosome 22. CML accounts for 7% to 15% of all leukemias, with an incidence of 1.5 in 100,000 in the United States.44 It is often asymptomatic, but CML can cause fatigue, anorexia, sweating, and left upper quadrant pain and early satiety sec-ondary to splenomegaly. Enlargement of the spleen is found in roughly one-half of patients with CML. Current therapy includes imatinib or allogeneic stem cell transplantation. Splenectomy is indicated to relieve symptoms of massive

1	of the spleen is found in roughly one-half of patients with CML. Current therapy includes imatinib or allogeneic stem cell transplantation. Splenectomy is indicated to relieve symptoms of massive splenomegaly, but it does not prevent blast crisis or alter the disease process.45,70Acute Myeloid Leukemia Like CML, acute myeloid leu-kemia (AML) involves the abnormal growth of stem cells in the bone marrow. Unlike CML, AML has a presentation that is more rapid and dramatic. The proliferation and accumula-tion of hematopoietic stem cells in the bone marrow and blood inhibit the growth and maturation of normal red blood cells, white blood cells, and platelets. Death usually results within weeks to months if AML goes untreated. The incidence of AML is approximately 9200 new cases each year in the United States, and it accounts for 1.2% of all cancer deaths.74 Patients with other myeloproliferative disorders, such as polycythemia vera, primary thrombocytosis, or myeloid metaplasia, are at

1	the United States, and it accounts for 1.2% of all cancer deaths.74 Patients with other myeloproliferative disorders, such as polycythemia vera, primary thrombocytosis, or myeloid metaplasia, are at increased risk for leukemic transformation to AML. Presenting signs and symptoms of AML include a viral-like illness with fever, malaise, and frequently bone pain due to the expansion of the medullary space. Standard treatment is combined induction therapy with daunorubicin, cytarabine, and stem cell transplan-tation. Splenectomy is indicated in AML only in the uncom-mon circumstance that left upper quadrant pain and early satiety become unbearable. The benefit must be weighed against the heightened risk of postsplenectomy infection in AML patients immunocompromised due to neutropenia and chemotherapy.74Brunicardi_Ch34_p1517-p1548.indd 152823/02/19 2:36 PM 1529THE SPLEENCHAPTER 34Chronic Myelomonocytic Leukemia Like CML and AML, chronic myelomonocytic leukemia (CMML) is characterized

1	152823/02/19 2:36 PM 1529THE SPLEENCHAPTER 34Chronic Myelomonocytic Leukemia Like CML and AML, chronic myelomonocytic leukemia (CMML) is characterized by a proliferation of hematopoietic elements in the bone marrow and blood. CMML differs from CML in that it is associated with monocytosis in the peripheral smear (>1 × 103 monocytes/mm3) and in the bone marrow. Splenomegaly occurs in one-half of these patients, and splenectomy can result in symptomatic relief.75Essential Thrombocythemia Essential thrombocythemia (ET) represents abnormal growth of the megakaryocyte cell line, resulting in increased levels of platelets in the bloodstream. The diagnosis is made after the exclusion of other chronic myeloid disorders such as CML, polycythemia vera, and myelofibrosis that may also present with thrombocytosis.76 Clinical manifesta-tions of ET include vasomotor symptoms, thrombohemorrhagic events, recurrent fetal loss, and the transformation to myelofi-brosis with myeloid metaplasia or

1	with thrombocytosis.76 Clinical manifesta-tions of ET include vasomotor symptoms, thrombohemorrhagic events, recurrent fetal loss, and the transformation to myelofi-brosis with myeloid metaplasia or AML. Hydroxyurea is used to reduce thrombotic events in ET but does not alter transforma-tion to myelofibrosis or leukemia. Splenomegaly occurs in one-third to one-half of patients with ET. Splenectomy is not felt to be helpful in the early stages of ET and is best reserved for the later stages of disease, when myeloid metaplasia has devel-oped.69 Even in these circumstances, candidates should be cho-sen selectively because significant bleeding has been reported to complicate splenectomy in these patients.Polycythemia Vera Polycythemia vera (PV) is a clonal, chronic, progressive myeloproliferative disorder characterized by an increase in red blood cell mass, frequently accompanied by leukocytosis, thrombocytosis, and splenomegaly. Patients with PV typically enjoy longer survival than those

1	disorder characterized by an increase in red blood cell mass, frequently accompanied by leukocytosis, thrombocytosis, and splenomegaly. Patients with PV typically enjoy longer survival than those affected by hematologic malignancies but remain at risk for transformation to myelofibrosis or AML. The disease is rare, with an annual incidence of 5 to 17 cases per million population.76,77 Physical findings include ruddy cyanosis, conjunctival plethora, hepato-megaly, splenomegaly, and hypertension. Treatment should be tailored to the risk status of the patient and ranges from phle-botomy and aspirin administration to the use of chemothera-peutic agents. As in ET, splenectomy is not helpful in the early stages of disease and is best reserved for patients with late-stage disease in whom myeloid metaplasia has developed and spleno-megaly-related symptoms are severe.76,77Myelofibrosis (Agnogenic Myeloid Metaplasia) The term myelofibrosis may be used to describe either the generic condi-tion

1	metaplasia has developed and spleno-megaly-related symptoms are severe.76,77Myelofibrosis (Agnogenic Myeloid Metaplasia) The term myelofibrosis may be used to describe either the generic condi-tion of fibrosis of the bone marrow (which may be associated with a number of benign and malignant disorders) or a specific, chronic, malignant hematologic disease associated with sple-nomegaly, the presence of red blood cell and white blood cell progenitors in the bloodstream, marrow fibrosis, and extramed-ullary hematopoiesis, otherwise known as agnogenic myeloid metaplasia (AMM). AMM also can be referred to as myeloscle-rosis, idiopathic myeloid metaplasia, and osteosclerosis. In this chapter, the term myelofibrosis is synonymous with AMM.In AMM, fibrosis of the bone marrow is believed to be a response to a clonal proliferation of hematopoietic stem cells. Marrow failure is common. The true incidence of AMM is unknown due to the scarcity of epidemiologic data, but one study estimated its U.S.

1	to a clonal proliferation of hematopoietic stem cells. Marrow failure is common. The true incidence of AMM is unknown due to the scarcity of epidemiologic data, but one study estimated its U.S. incidence at 1.46 per 100,000 popula-tion.78-80 The diagnosis is made by a careful examination of the peripheral blood smear and bone marrow. Nucleated red blood cells and immature myeloid elements in the blood are present in 96% of cases and strongly suggest the diagnosis. Teardrop poikilocytosis is another frequent finding. Care must be taken, however, to exclude a history of a primary neoplasm (such as lymphoma or adenocarcinoma of the stomach, lung, prostate, or breast) or tuberculosis because patients with these conditions may develop secondary myelofibrosis.Treatment depends on symptoms: Asymptomatic patients are closely followed, whereas symptomatic patients undergo therapeutic intervention targeted to their symptoms. The only curative therapy is allogeneic bone marrow transplantation in

1	patients are closely followed, whereas symptomatic patients undergo therapeutic intervention targeted to their symptoms. The only curative therapy is allogeneic bone marrow transplantation in younger, high-risk patients. Supportive therapy for clinically symptomatic anemia includes steroids, danazol, erythropoietin, or blood transfusion.78,80 Splenomegaly-related symptoms are best treated with splenectomy. Although some chemotherapeu-tic agents (busulfan, hydroxyurea, interferon-α) and low-dose radiation can reduce splenic size, their discontinuation usually results in rapid splenic regrowth.A thorough preoperative workup must precede sple-nectomy in patients with AMM. The candidate must possess acceptable cardiac, pulmonary, hepatic, and renal reserve for the operation. The coagulation system should be examined; testing should include measurement of coagulation factors V and VIII and fibrin split products, platelet count, and bleeding time. Low platelet counts may require

1	system should be examined; testing should include measurement of coagulation factors V and VIII and fibrin split products, platelet count, and bleeding time. Low platelet counts may require administration of ste-roids and/or platelet transfusion at the time of surgery. Sple-nectomy provides durable, effective palliation for nearly all patients with AMM, although postoperative complications are more common in patients with AMM than in those with other hematologic indications. The Mayo Clinic recently published its 30-year experience with 314 myelofibrosis patients who underwent splenectomy. Nearly half of the operations (49%) were performed to alleviate the mechanical symptoms of sple-nomegaly; the remainder were undertaken to manage anemia, thrombocytopenia, or portal hypertension. Response to splenec-tomy was 76% overall at 1 year; overall complication rate was 28%, including 21 perioperative deaths.80 Thrombosis, hemor-rhage, and infection complications were common, with

1	Response to splenec-tomy was 76% overall at 1 year; overall complication rate was 28%, including 21 perioperative deaths.80 Thrombosis, hemor-rhage, and infection complications were common, with preop-erative thrombocytopenia an independent predictor of mortality risk. These data underscore the severity of this malignancy and emphasize the need for careful patient selection when consider-ing splenectomy in AMM.79Tumors and Metastasis. Primary tumors of the spleen are typ-ically benign or malignant variants of vascular neoplasms. The most common primary tumors of the spleen are sarcomas, many of which have been linked to various environmental and occu-pational exposures such as vinyl chloride or thorium dioxide. Isolated splenic metastases are extremely unusual but may occur in the setting of a concomitant carcinoma.81 Lung carcinoma is the tumor that most commonly spreads to the spleen, although, colorectal, ovarian, and melanoma may also metastasize to the spleen.82,83 If after a

1	of a concomitant carcinoma.81 Lung carcinoma is the tumor that most commonly spreads to the spleen, although, colorectal, ovarian, and melanoma may also metastasize to the spleen.82,83 If after a thorough examination, an isolated splenic metastasis is confirmed, a laparoscopic splenectomy with intact spleen retrieval may be considered.81-83Miscellaneous Disorders and LesionsInfections and Abscesses. Primary infections of the spleen although uncommon in immunocompetent adults, are particu-larly reported in the immunocompromised population or those with a history of intravenous recreational drug abuse.84,85 The potential effects of certain systemic infections on the spleen merit close attention, mostly because of the dreaded risk of spontaneous splenic rupture. Infectious mononucleosis due to Brunicardi_Ch34_p1517-p1548.indd 152923/02/19 2:36 PM 1530SPECIFIC CONSIDERATIONSPART IIeither Epstein-Barr virus or cytomegalovirus infection imparts a small but often-discussed risk of

1	due to Brunicardi_Ch34_p1517-p1548.indd 152923/02/19 2:36 PM 1530SPECIFIC CONSIDERATIONSPART IIeither Epstein-Barr virus or cytomegalovirus infection imparts a small but often-discussed risk of spontaneous splenic rup-ture in both adults and children. The true incidence may be underreported, however. Recent case reports abound in the lit-erature regarding spontaneous splenic rupture due to a variety of infectious causes (malaria, Listeria infection, fungal infec-tions, dengue, and Q fever, to name a few) as well as a vari-ety of neoplastic and other noninfectious causes (lymphoma, angiosarcoma, amyloidosis, pregnancy). The presumed patho-physiologic mechanism is infiltration of the splenic paren-chyma with inflammatory cells, which distorts the architecture and fibrous support system of the spleen and thins the splenic capsule.86 In this setting, splenic rupture can occur spontane-ously or after a seemingly minor external trauma or even a Valsalva maneuver.Abscesses of the spleen

1	of the spleen and thins the splenic capsule.86 In this setting, splenic rupture can occur spontane-ously or after a seemingly minor external trauma or even a Valsalva maneuver.Abscesses of the spleen are uncommon, with an incidence reported to be 0.14% to 0.7%.87-89 They occur more frequently in tropical locations, where they are associated with throm-bosed splenic vessels and infarction in patients with sickle cell anemia. Five distinct mechanisms of splenic abscess formation have been described: (a) hematogenous infection; (b) contigu-ous infection; (c) hemoglobinopathy; (d) immunosuppression, including HIV infection and chemotherapy; and (e) trauma. The presentation of splenic abscess frequently is delayed, with most patients enduring symptoms for 16 to 22 days before diagno-sis. Clinical manifestations include fever, left upper quadrant pain, leukocytosis, and splenomegaly in about one-third of patients. The diagnosis is confirmed by ultrasound or CT scan, which has a 95%

1	Clinical manifestations include fever, left upper quadrant pain, leukocytosis, and splenomegaly in about one-third of patients. The diagnosis is confirmed by ultrasound or CT scan, which has a 95% sensitivity and specificity. Common organ-isms are aerobic microbes (streptococci and Escherichia coli), but other microorganisms have also been isolated (Mycobac-terium tuberculosis and Salmonella typhi). Upon discovery of a splenic abscess, broad-spectrum antibiotics should be initi-ated, with adjustment to more specific therapy based on culture results and continuation of treatment for a minimum of 14 days. Splenectomy is the operation of choice, but percutaneous and open drainage are options for patients who either cannot toler-ate splenectomy or where the clinical scenario warrants splenic preservation, particularly in children.90 Percutaneous drainage is often successful for patients with unilocular disease and may result in avoidance of splenectomy. Patients with multilocular disease

1	preservation, particularly in children.90 Percutaneous drainage is often successful for patients with unilocular disease and may result in avoidance of splenectomy. Patients with multilocular disease will often require multiple drains and therefore often benefit from total splenectomy.Cysts. Splenic cysts (Fig. 34-5) can be categorized according to a number of criteria; one clinically relevant scheme is to char-acterize true splenic cysts as either parasitic or nonparasitic.86Parasitic Infections. Parasitic infection is the most common cause of splenic cysts worldwide, and the majority are due to Echinococcus species. These cysts are more commonly found in areas where the pathogen is endemic. Symptoms, when present, generally are related to the presence of a mass lesion in the left upper quadrant or a lesion that impinges on the stomach. Ultra-sound can establish the presence of a cystic lesion and occasion-ally incidentally detect asymptomatic lesions as well. Serologic testing for

1	quadrant or a lesion that impinges on the stomach. Ultra-sound can establish the presence of a cystic lesion and occasion-ally incidentally detect asymptomatic lesions as well. Serologic testing for echinococcal antibodies can confirm or exclude the cystic lesion as parasitic, an important piece of informa-tion when planning operative therapy. Symptomatic parasitic cysts are best treated with splenectomy. Avoidance of spillage of parasitic cyst contents into the peritoneal cavity to avoid the possibility of anaphylactic shock is an important principle in surgical management despite its rare occurrence.91Cysts resulting from trauma are termed pseudocysts due to their lack of an epithelial cell lining. Less common examples of nonparasitic cysts are dermoid, epidermoid, and epithelial cysts.92 The treatment of nonparasitic cysts depends on whether or not they produce symptoms. Asymptomatic nonparasitic cysts may be observed with close follow-up by ultrasound to exclude significant growth

1	The treatment of nonparasitic cysts depends on whether or not they produce symptoms. Asymptomatic nonparasitic cysts may be observed with close follow-up by ultrasound to exclude significant growth or expansion. Patients should be advised of the risk of cyst rupture with even minor abdominal trauma if they elect nonoperative management for large cysts. Small symptomatic nonparasitic cysts may be excised with splenic preservation, and large symptomatic nonparasitic cysts may be unroofed. Both of these operations may be performed laparoscopically.86Storage Diseases and Infiltrative Disorders Gaucher’s Disease Gaucher’s disease is an inherited lipid stor-age disorder characterized by the deposition of glucocerebroside in cells of the macrophage-monocyte system. The underlying abnormality is a deficiency in the activity of a lysosomal hydro-lase. Abnormal glycolipid storage results in organomegaly, particularly hepatomegaly and splenomegaly.93,94 Patients with Gaucher’s disease frequently

1	deficiency in the activity of a lysosomal hydro-lase. Abnormal glycolipid storage results in organomegaly, particularly hepatomegaly and splenomegaly.93,94 Patients with Gaucher’s disease frequently experience symptoms related to splenomegaly, including early satiety and abdominal discom-fort, and to hypersplenism, including thrombocytopenia, normo-cytic anemia, and mild leukopenia. These latter findings occur as a result of excessive sequestration of formed blood elements in the spleen. Other symptoms in patients with Gaucher’s dis-ease include bone pain, pathologic fractures, and jaundice. Splenectomy may alleviate hematologic abnormalities in these patients with hypersplenism, but it does not correct the underly-ing disease process.95Niemann-Pick Disease Niemann-Pick disease is an inherited disorder of abnormal lysosomal storage of sphingomyelin and cholesterol in cells of the macrophage-monocyte system. Four types of the disease (A, B, C, and D) exist, with unique clinical

1	an inherited disorder of abnormal lysosomal storage of sphingomyelin and cholesterol in cells of the macrophage-monocyte system. Four types of the disease (A, B, C, and D) exist, with unique clinical presentations. Types A and B result from a deficiency in lyso-somal hydrolase and are the forms most likely to demonstrate splenomegaly with its concomitant symptoms. Symptoms of splenomegaly are relieved by splenectomy.Amyloidosis Amyloidosis is a disorder of abnormal extra-cellular protein deposition and protein conformation disorder associated with a clonal plasma cell dyscrasia. There are mul-tiple forms of amyloidosis, each with its own individual clini-cal presentation, and the severity of disease may range from asymptomatic to multiorgan failure. Patients with primary amy-loidosis, associated with plasma cell dyscrasia, have splenic involvement in approximately 5% of cases. Secondary amy-loidosis, associated with chronic inflammatory conditions, also may present with an enlarged

1	with plasma cell dyscrasia, have splenic involvement in approximately 5% of cases. Secondary amy-loidosis, associated with chronic inflammatory conditions, also may present with an enlarged spleen and even spontaneous rup-ture.96 Symptoms of splenomegaly are relieved by splenectomy.Sarcoidosis Sarcoidosis is an inflammatory disease of young adults characterized by noncaseating granulomas in affected tissues. Signs and symptoms of the disease range in severity and typically are nonspecific, such as fatigue and malaise. Any organ system may be involved. The most commonly involved organ is the lung, followed by the spleen. Splenomegaly occurs in approximately 25% of patients. Massive splenomegaly (>1 kg) is rare with a reported incidence of 3%.97,98 Other affected tissues include the lymph nodes, eyes, joints, liver, spleen, and heart. When splenomegaly occurs and causes symp-toms related to size or hypersplenism, splenectomy effectively relieves symptoms and corrects hematologic

1	lymph nodes, eyes, joints, liver, spleen, and heart. When splenomegaly occurs and causes symp-toms related to size or hypersplenism, splenectomy effectively relieves symptoms and corrects hematologic abnormalities such Brunicardi_Ch34_p1517-p1548.indd 153023/02/19 2:36 PM 1531THE SPLEENCHAPTER 34as anemia and thrombocytopenia. Spontaneous splenic rupture has been reported in sarcoidosis.94Splenic Artery Aneurysm Although splenic artery aneurysm is rare, it is the most common visceral artery aneurysm. Women are four times more likely to be affected than men. The aneu-rysm usually arises in the middle to distal portion of the splenic artery.99,100 Indications for treatment include presence of symp-toms, pregnancy, intention to become pregnant, and presence of pseudoaneurysms associated with inflammatory processes. Aneurysm resection or ligation alone is acceptable for amenable lesions in the mid-splenic artery, but distal lesions in close prox-imity to the splenic hilum should be

1	with inflammatory processes. Aneurysm resection or ligation alone is acceptable for amenable lesions in the mid-splenic artery, but distal lesions in close prox-imity to the splenic hilum should be treated with concomitant splenectomy. An excellent prognosis follows elective treatment. Splenic artery embolization has been used to treat splenic artery aneurysm; however, this may result in painful splenic infarction or abscess.Portal Hypertension Portal hypertension can result from numerous causes but is usually due to liver cirrhosis. Sple-nomegaly and splenic congestion often accompany portal hypertension, which leads to sequestration and destruction of circulating cells in the spleen. Splenectomy is not indicated for hypersplenism per se in patients with portal hypertension as there is no correlation between the degree of pancytopenia and long-term survival in these patients.7 In rare circumstances in which splenectomy is required to reduce bleeding from esopha-geal varices

1	is no correlation between the degree of pancytopenia and long-term survival in these patients.7 In rare circumstances in which splenectomy is required to reduce bleeding from esopha-geal varices exacerbated by thrombocytopenia, a concomitant splenorenal shunt procedure may be performed to decompress the portal system.Sinistral hypertension secondary to splenic vein thrombo-sis, on the other hand, is potentially curable with splenectomy. Patients that are bleeding from isolated gastric varices who have normal liver function test results, especially those with a pre-vious history of pancreatitis, should be examined for splenic vein thrombosis and treated with splenectomy if findings are positive.Idiopathic portal hypertension (IPH) is extremely rare, and it is characterized by the absence of cirrhosis or other clear etiologies such as hepatic or portal vein thrombosis, cardiac failure, or hematologic disorders.101-104 Hypersplen-ism often develops in these patients due to hyperactivity

1	cirrhosis or other clear etiologies such as hepatic or portal vein thrombosis, cardiac failure, or hematologic disorders.101-104 Hypersplen-ism often develops in these patients due to hyperactivity of the spleen; defined as a triad of splenomegaly, pancytopenia, 6ACBDFigure 34-5. A. Computed tomography (CT) scan of giant splenic cyst. B. Three-dimensional CT reconstruction of splenic cyst. C and D. Macroscopic aspect of a multicystic spleen lesion.Brunicardi_Ch34_p1517-p1548.indd 153123/02/19 2:37 PM 1532SPECIFIC CONSIDERATIONSPART IIand normocellularity of bone marrow warranting intervention by way of splenic artery embolization or splenectomy. Par-tial splenic embolization (PSE) has recently been utilized with good success in prohibitively high operative risk patients with severe hypersplenism.105Felty’s Syndrome The triad of rheumatoid arthritis, spleno-megaly, and neutropenia is called Felty’s syndrome. It exists in approximately 3% of all patients with rheumatoid arthritis,

1	Syndrome The triad of rheumatoid arthritis, spleno-megaly, and neutropenia is called Felty’s syndrome. It exists in approximately 3% of all patients with rheumatoid arthritis, two-thirds of whom are women. Immune complexes coat the surface of white blood cells, which leads to their sequestration and clear-ance in the spleen with subsequent neutropenia. This neutro-penia (<2000 neutrophils/mm3) increases the risk for recurrent infections and often drives the decision for splenectomy. The size of the spleen is variable, from nonpalpable in 5% to 10% of patients to massively enlarged upwards of 4 times heavier than normal in others. Corticosteroids, hematopoietic growth factors, methotrexate, and splenectomy have all been used to treat the neutropenia of Felty’s syndrome. More recently, ritux-imab has been tried as a second line agent in refractory Felty’s syndrome.106,107Overall response to splenectomy is excellent, with >80% of patients showing a durable increase in white blood cell

1	has been tried as a second line agent in refractory Felty’s syndrome.106,107Overall response to splenectomy is excellent, with >80% of patients showing a durable increase in white blood cell count. More than one-half of patients who had infections before surgery may clear their infections after surgery.108 Besides symptomatic neutropenia, other indications for sple-nectomy include transfusion-dependent anemia and profound thrombocytopenia.Wandering Spleen A very uncommon anatomic abnormality is the “wandering spleen.” In this condition, the spleen “floats” inside the abdominal cavity due to an anomaly during embryo-genesis and may present itself in a variety of ways includ-ing acute abdomen.109 The wandering spleen is not normally attached to adjacent viscera in the splenic fossa. This may lead to splenic torsion and infarction. Splenopexy or splenectomy may be required.110Partial Splenectomy and Splenic Salvage. The increased awareness of asplenia-related life-threatening

1	This may lead to splenic torsion and infarction. Splenopexy or splenectomy may be required.110Partial Splenectomy and Splenic Salvage. The increased awareness of asplenia-related life-threatening complications such as overwhelming postsplenectomy infection (OPSI) has led to the development of parenchyma sparing splenic resec-tions for select patients and disorders. The first successful partial splenectomy was reported in 1979 by Shapiro and was followed by Uranus who would perform the first laparoscopic partial splenectomy in 1995.111,112 Previously, many surgeons had been reluctant to perform partial splenectomy because of the technical difficulties and bleeding risk. However, with a better understanding of the segmental vascular anatomy of the spleen and the development of improved laparoscopic skills and technologies, laparoscopic partial splenectomy has been used successfully in patients with hematologic diseases such as hereditary spherocytosis in children, who may benefit the

1	skills and technologies, laparoscopic partial splenectomy has been used successfully in patients with hematologic diseases such as hereditary spherocytosis in children, who may benefit the most from splenic preservation, as well as for benign splenic cysts.113-119 The technique of partial splenectomy will be dis-cussed later in this chapter.IMAGING FOR EVALUATION OF SIZE AND PATHOLOGYThorough assessment of anatomic detail and functional status of the spleen are essential for proper surgical planning. Spe-cial preoperative consideration needs to be given to patients with splenomegaly because minimally invasive methods of resection may be fraught with additional difficulty in patients with very large spleens, even in skilled hands. Other indica-tions for splenic imaging include trauma, investigations of left upper quadrant pain, characterization of splenic lesions such as tumors, cysts, and abscesses, and guidance for percutaneous procedures.165,166Preoperative imaging of the spleen is

1	of left upper quadrant pain, characterization of splenic lesions such as tumors, cysts, and abscesses, and guidance for percutaneous procedures.165,166Preoperative imaging of the spleen is primarily performed to obtain an accurate assessment of the splenic volume in order to confirm and document splenomegaly as well as to exclude any large splenic lesion that could affect the surgical resection plane. Identification of the presence of accessory spleens in the preoperative setting is also important, although lack of acces-sory tissue on the imaging should not preclude a thorough intra-operative search.The guidelines of the European Association for Endo-scopic Surgery suggest that for all patients in whom splenec-tomy is indicated, preoperative imaging should be obtained.24 Two of the most commonly used imaging modalities include ultrasound and CT, both enabling measurement of splenic size and volume. When desired, the splenic volume may be calcu-lated using a formula for the volume of

1	commonly used imaging modalities include ultrasound and CT, both enabling measurement of splenic size and volume. When desired, the splenic volume may be calcu-lated using a formula for the volume of a prolate ellipsoid:Volume (cc) = length (cm) × width (cm) × height (cm) × 0.52.64Other imaging modalities, although not as commonly used, include nuclear medicine studies and magnetic resonance imaging (MRI).PREOPERATIVE CONSIDERATIONSAs part of preoperative discussion prior to splenectomy, patients should be consulted on potential complications associated with this procedure, including overwhelming postsplenectomy sepsis, splenic vein thrombosis, bleeding, arterial thrombosis (myocardial infarction, stroke), deep vein thrombosis, and pul-monary hypertension.Vaccination and Patient EducationConsidering that infection is the most common complication, patient education and vaccinations against encapsulated pathogens are the mainstay of preventive therapy.52,120 Although rare, the most

1	that infection is the most common complication, patient education and vaccinations against encapsulated pathogens are the mainstay of preventive therapy.52,120 Although rare, the most feared and extreme infectious complica-tion is overwhelming postsplenectomy sepsis (OPSI). (See later section, “Overwhelming Postsplenectomy Infection,” for detailed discussion.) Patients undergoing splenectomy for hematologic or malignant indications have the greatest risk, whereas patients who undergo splenectomy for trauma or iatro-genic injury have the lowest risk. OPSI is more common in the pediatric population, with 4.4% of children less than 16 years of age versus 0.9% of adults developing this life-threatening condi-tion. The risk has been observed to be the greatest in the first 2 years after splenectomy; however, asplenic patients remain at lifelong risk.121-123 Considering that the spleen is the site for spe-cial adaptation of macrophages that target encapsulated organ-isms, asplenic patients

1	however, asplenic patients remain at lifelong risk.121-123 Considering that the spleen is the site for spe-cial adaptation of macrophages that target encapsulated organ-isms, asplenic patients are at higher risk of infection caused by Streptococcus pneumoniae (responsible for >50% of OPSI), H influenzae type b, Neisseria meningitidis, and Capnocytoph-aga canimorsus (transmitted by dog bites).124In the setting of elective splenectomy, patients should be vaccinated two weeks prior to surgery to optimize anti-gen recognition and processing. If splenectomy is performed emergently, vaccinations can be administered postoperatively and consideration should be given to delaying administration 7Brunicardi_Ch34_p1517-p1548.indd 153223/02/19 2:37 PM 1533THE SPLEENCHAPTER 34for 2 weeks to avoid the transient immunosuppression asso-ciated with surgery. International guidelines also recommend annual influenza vaccine for asplenic patients. The influenza vaccination provides protection from

1	the transient immunosuppression asso-ciated with surgery. International guidelines also recommend annual influenza vaccine for asplenic patients. The influenza vaccination provides protection from influenza syndrome and secondary bacterial infection. This immunization is associated with a 54% reduced risk of death compared with unimmunized asplenic persons.125Preoperative and postoperative patient education regard-ing OPSI is paramount because patients with OPSI may rap-idly progress from a febrile illness to circulatory collapse and death within a matter of hours. In one study, 28% of asplenic patients were unaware of the potential infection risks, and the main reasons for this lack of awareness were that correct advice was not given or that that advice was forgotten.121,126 The use of currently available vaccines against pneumococcus and other encapsulated organisms has led to a drop in the overall inci-dence of OPSI to <1%. The mechanism by which vaccination protects asplenic

1	of currently available vaccines against pneumococcus and other encapsulated organisms has led to a drop in the overall inci-dence of OPSI to <1%. The mechanism by which vaccination protects asplenic patients is not entirely understood. Serum anti-body titers do not necessarily correspond to clinical immunity. Moreover, antibody levels after pneumococcus vaccination decline steadily within 5 to 10 years. Revaccination is reason-ably recommended for these patients, although the efficacy of this measure is not proven.121-124,126Deep Vein Thrombosis ProphylaxisDeep vein thrombosis (DVT) after splenectomy is not infre-quent, especially in cases involving splenomegaly and myelo-proliferative disorders.76 Risk of portal vein thrombosis (PVT) may reach 50% for patients presenting with both splenomegaly and myeloproliferative disorders.127,128Postsplenectomy PVT typically presents with anorexia, abdominal pain, leukocytosis, and thrombocytosis. Effective PVT treatment is possible by

1	splenomegaly and myeloproliferative disorders.127,128Postsplenectomy PVT typically presents with anorexia, abdominal pain, leukocytosis, and thrombocytosis. Effective PVT treatment is possible by maintaining a high index of sus-picion, achieving early diagnosis with contrast enhanced CT, and starting anticoagulation immediately. DVT prophylaxis, including use of sequential compression devices and subcu-taneous administration of heparin (5000 U), should be initi-ated for patients undergoing splenectomy.16,77 Each patient’s risk factors for DVT should be evaluated, and when elevated risk exists (obesity, history of prior venous thromboembolism, known hypercoagulable state, older age), a postoperative anti-thrombotic regimen of up to two weeks of low molecular weight heparin should be maintained.SPLENECTOMY TECHNIQUESPatient PreparationAssessment of the potential need for transfusion of blood products and optimization of preoperative coagulation status are necessary. It is the authors’

1	TECHNIQUESPatient PreparationAssessment of the potential need for transfusion of blood products and optimization of preoperative coagulation status are necessary. It is the authors’ practice to order blood typing and antibody screening tests for normosplenic patients under-going elective splenectomy. Anemic patients should be trans-fused before surgery to a hemoglobin level of 10 g/dL. In more complex cases, including patients with splenomegaly, at least 2 to 4 units of cross-matched blood should be available at the time of surgery. Thrombocytopenia may be transiently cor-rected with platelet transfusions. Thrombocytopenic patients preferably should not undergo transfusion before the day of surgery and ideally not before the intraoperative ligation of the splenic artery. Several authors recommended a platelet count of 30 × 109 / L before the surgery; this may require treatment with IV immunoglobulin or oral corticosteroids if the platelets are below this number. Pooled normal human

1	recommended a platelet count of 30 × 109 / L before the surgery; this may require treatment with IV immunoglobulin or oral corticosteroids if the platelets are below this number. Pooled normal human immunoglobulin is effective in elevating the platelet count in approximately 75% of patients.129-131Patients who have been maintained on corticosteroid therapy preoperatively should receive parenteral corticosteroid therapy perioperatively. Bowel preparation is not routinely performed for patients undergoing elective splenectomy. All splenectomy patients do receive DVT prophylaxis, as discussed previously. After endotracheal intubation, a nasogastric (NG) tube is inserted for stomach decompression.Open SplenectomyAlthough laparoscopic surgery increasingly has achieved acceptance as the standard approach for normosplenic patients requiring splenectomy, open splenectomy (OS) is still widely practiced. The largest published series is a report of the Nationwide Inpatient Sample (NIS), where of

1	approach for normosplenic patients requiring splenectomy, open splenectomy (OS) is still widely practiced. The largest published series is a report of the Nationwide Inpatient Sample (NIS), where of 37,006 nontrau-matic splenectomies identified during a 6-year study period (2005–2010), 81.4% of those cases were approached by open surgery.132Traumatic rupture of the spleen continues as the most common indication for OS. Several other clinical scenarios favor an OS approach, including massive splenomegaly, ascites, portal hypertension, multiple prior operations, extensive splenic irradiation, and possible splenic abscess.During OS, the patient is placed in the supine position with the surgeon situated at the patient’s right. A left subcostal incision paralleling the left costal margin and lying two finger-breadths below it is preferred for most elective splenectomies. A midline incision is optimal for exposure when the spleen is ruptured or massively enlarged. The spleen is mobilized by

1	two finger-breadths below it is preferred for most elective splenectomies. A midline incision is optimal for exposure when the spleen is ruptured or massively enlarged. The spleen is mobilized by dividing ligamentous attachments, usually beginning with the splenocolic ligament (Fig. 34-6). In patients with signifi-cant splenomegaly, once lesser sac access has been achieved through either the gastrosplenic or gastrohepatic attachments, ligating the splenic artery in continuity along the superior bor-der of the pancreas may be preferable. This maneuver may serve several purposes: allowing safer manipulation of the Figure 34-6. Splenocolic ligament is divided at the beginning of open splenectomy.Brunicardi_Ch34_p1517-p1548.indd 153323/02/19 2:37 PM 1534SPECIFIC CONSIDERATIONSPART II5-mm cephalad working port5-mm camera port with 30’ scope2-mm accessory port (flank)10/12-mm caudad working portFigure 34-7. Patient positioning and trocar placement for laparoscopic splenectomy.spleen and

1	working port5-mm camera port with 30’ scope2-mm accessory port (flank)10/12-mm caudad working portFigure 34-7. Patient positioning and trocar placement for laparoscopic splenectomy.spleen and dissection of the splenic hilum, facilitating some shrinkage of the spleen, and providing an autotransfusion of erythrocytes and platelets. Further medial mobilization of the spleen is achieved by incising its lateral peritoneal attachments, most notably the splenophrenic ligament. Then follows indi-vidual ligation and sequential division of the short gastric ves-sels, steps that if carefully executed reduce the risk of these vessels’ retracting and bleeding. Splenic hilar dissection then takes place. Whenever possible, care should be taken to dis-sect and individually ligate the splenic artery and vein (in that order) before dividing them. As noted in the discussion of splenic anatomy, the tail of the pancreas lies within 1 cm of the splenic hilum in 75% of patients; therefore, during hilar

1	vein (in that order) before dividing them. As noted in the discussion of splenic anatomy, the tail of the pancreas lies within 1 cm of the splenic hilum in 75% of patients; therefore, during hilar dissec-tion, great care must be taken to avoid injuring the pancreas. Once the spleen is excised, hemostasis is secured by irrigating, suctioning, and scrupulously inspecting the bed of dissection. The splenic bed is not routinely drained. A thorough search for accessory spleens must be undertaken when a hematologic disorder has occasioned splenectomy. At the completion of sur-gery, the nasogastric tube is removed.Laparoscopic SplenectomyLaparoscopic splenectomy (LS) has become the procedure of choice over the last two decades, since it has been first described Delaitre and Maignien in 1991.133 In fact, LS is now the gold standard for elective splenectomy in patients with normal-sized spleens. In experienced hands, LS is associated with decreased intraoperative blood loss, shorter hospital

1	In fact, LS is now the gold standard for elective splenectomy in patients with normal-sized spleens. In experienced hands, LS is associated with decreased intraoperative blood loss, shorter hospital length of stay, and lower morbidity rates when compared to OS.134,135 Since the introduction of the lateral approach, most LS proce-dures are now performed with the patient in the right lateral decubitus position (Fig. 34-7).136 A midway “double-access” technique in which the patient is in a 45° right lateral decubitus position has also been advocated. This positioning permits con-comitant surgery, such as laparoscopic cholecystectomy, more easily than does the lateral approach. The double-access 8technique requires the placement of five or six trocars. The lateral approach routinely involves the use of three or four trocars posi-tioned as shown in Fig. 34-7. Use of an angled (30° or 45°) lapa-roscope (2, 5, or 10 mm) greatly facilitates the procedure. Exposure of the vital anatomy in a

1	the use of three or four trocars posi-tioned as shown in Fig. 34-7. Use of an angled (30° or 45°) lapa-roscope (2, 5, or 10 mm) greatly facilitates the procedure. Exposure of the vital anatomy in a manner that allows for a more intuitive sequence of dissection, paralleling that of OS, may be considered an additional advantage of the lateral approach.Placement of trocars in the left upper quadrant should be performed under laparoscopic visualization, particularly if any degree of splenomegaly exists, because the latter can sig-nificantly reduce the available operating space. As with OS, the splenocolic ligament and the lateral peritoneal attachments are divided with resultant medial mobilization of the spleen. The short gastric vessels may be divided usually with hemo-static energy sources such as ultrasonic dissection, diathermy, or radiofrequency ablation. With the lower pole of the spleen gently retracted, the splenic hilum is accessible to further appli-cations of clips or an

1	such as ultrasonic dissection, diathermy, or radiofrequency ablation. With the lower pole of the spleen gently retracted, the splenic hilum is accessible to further appli-cations of clips or an endovascular stapling device. The splenic artery and vein are divided separately when possible. Good long-term outcomes, however, are increasingly being achieved with mass hilar stapling (Fig. 34-8). Using the lateral approach with the spleen thus elevated, the surgeon can easily visualize the tail of the pancreas and avoid injury when placing the endo-vascular stapler within the sack and allows piecemeal extrac-tion; a blunt instrument should be used to disrupt and remove the spleen to avoid the risk of sack rupture, spillage of contents, and subsequent splenosis (Fig. 34-9 and Fig. 34-10).Hand-Assisted SplenectomyWhen LS is performed in patients with splenomegaly, there have been reports of high rates of both complications and con-version to open splenectomy.137 Hand-assisted laparoscopic

1	SplenectomyWhen LS is performed in patients with splenomegaly, there have been reports of high rates of both complications and con-version to open splenectomy.137 Hand-assisted laparoscopic surgery (HALS) has been described as an alternative to the LS approach.24,138,139 Spleens greater than 22 cm in craniocau-dal length or 19 cm in width may benefit from HALS over a purely laparoscopic approach.139 It has been reported that the Brunicardi_Ch34_p1517-p1548.indd 153423/02/19 2:37 PM 1535THE SPLEENCHAPTER 34Figure 34-8. Splenic hilum can be divided laparoscopically en masse once the spleen has been rotated medially having been mobi-lized from its lateral attachments.ABFigure 34-9. Spleen extraction. A. Spleen is placed into a ripstop nylon bag before morcellation. B. Splenic morcellation.use of this technique has resulted in a marked reduction in aver-age operative time for patients with massive splenomegaly (146 vs. 295 minutes). Although HALS does require a small incision (7–8 cm)

1	of this technique has resulted in a marked reduction in aver-age operative time for patients with massive splenomegaly (146 vs. 295 minutes). Although HALS does require a small incision (7–8 cm) for hand insertion and specimen extraction, no differences in length of stay were observed when comparing these patients to those managed purely laparoscopically.139When performing HALS splenectomy, patient position-ing is similar to that of LS (Fig. 34-11). For patients with mas-sive spleens, lateral positioning is altered slightly, such that the patient is placed supine with the left side elevated at 45°. Depending on the hand dominance of the surgeon, the hand-assist device can be placed in either a midline position for right-hand dominant or a subcostal position for left-hand dominant surgeons. A 7to 8-cm incision should be made 2 to 4 cm caudal to the inferior pole of the enlarged spleen. The hand-assisted technique allows for a tactile feedback and atraumatic manipu-lation of the

1	A 7to 8-cm incision should be made 2 to 4 cm caudal to the inferior pole of the enlarged spleen. The hand-assisted technique allows for a tactile feedback and atraumatic manipu-lation of the enlarged spleen.The nondominant hand provides medial retraction and rotation of the spleen through a hand-assist port, while the domi-nant hand carries out the dissection using laparoscopic instru-ments. The anterior and posterior attachments of the spleen are taken down as in the laparoscopic approach, and the hilar pedicle is ligated using an endoscopic vascular stapler.Single-Incision Laparoscopic Surgery SplenectomySingle-incision laparoscopic surgery (SILS) splenectomy emphasizes the concept of surgery through one small transab-dominal incision rather than the standard multiple trocar sites, with theoretical benefits of less pain and better cosmetics. The incision can be hidden periumbilically and is used as the specimen extraction site at the end of the case. This approach for solid organs

1	theoretical benefits of less pain and better cosmetics. The incision can be hidden periumbilically and is used as the specimen extraction site at the end of the case. This approach for solid organs poses several technical challenges. Since all instruments are closely aligned together, “fencing” of instru-ments and the laparoscope and limited degrees of movement are commonly encountered. The spleen, being a solid organ, cannot be grasped, and thus retraction may be more challeng-ing in these cases. Furthermore, it has been reported that peri-umbilical port position may result in technical challenges when dealing with high body mass index or tall patients, precluding Brunicardi_Ch34_p1517-p1548.indd 153523/02/19 2:37 PM 1536SPECIFIC CONSIDERATIONSPART IIFigure 34-10. Morcellation and extraction of the spleen within nylon sac extending through the 10-mm trocar site. (Reproduced with per-mission from Park A, Marcaccio M, Sternbach M, et al: Laparoscopic vs open splenectomy, Arch Surg.

1	of the spleen within nylon sac extending through the 10-mm trocar site. (Reproduced with per-mission from Park A, Marcaccio M, Sternbach M, et al: Laparoscopic vs open splenectomy, Arch Surg. 1999 Nov;134(11):1263-1269.)the surgeon from adequately reaching the spleen. Other alter-natives to single port placements have been reported, although to date, no proven benefits of SILS splenectomy have been demonstrated.140-142Robotic SplenectomyThe da Vinci surgical robot (Intuitive Surgical, Sunnyvale, CA) was cleared by the U.S. Food and Drug Administration (FDA) for use in humans in the year 2000 and has been applied to clinical practice in a variety of abdominal procedures includ-ing splenectomy since 2002.143,144 The term robotic surgery, referencing the da Vinci device should more accurately be described as computer assisted surgery (CAS), as it requires a surgeon sitting at a console controlling distant end effectors. A truly robotic, automated system has not yet been devised nor

1	be described as computer assisted surgery (CAS), as it requires a surgeon sitting at a console controlling distant end effectors. A truly robotic, automated system has not yet been devised nor deployed to perform splenectomy. The reported advantages of CAS have inspired many surgeons to investigate its potential and to broaden its application in the minimally invasive surgery armamentarium. Some of these advantages include increased degrees of freedom as compared to standard “straight-stick” lap-aroscopy, improved optics including three-dimensional imag-ing of the operative field, improved instrument stabilization and reduction in hand tremor, and finally purported enhanced ergonomic and comfort factors for the operating surgeon.145-147 However, detractors of this emerging technology cite high capi-tal expenses as well as on-going disposable costs and the loss of haptic feedback as major downsides of CAS such that the benefits of the robotic platform are still not fully clear in the

1	high capi-tal expenses as well as on-going disposable costs and the loss of haptic feedback as major downsides of CAS such that the benefits of the robotic platform are still not fully clear in the extant literature. Recent studies suggest that ergonomic ben-efits of robotic surgery may not be as pronounced as previously thought, as “robotic surgeons” still manifested chronic pain related to poor ergonomics.148There have been few published works comparing conven-tional laparoscopic splenectomy to CAS splenectomy. In one recent retrospective case-matched analysis, the authors com-pared hospital length of stay, operating room times and cost between these two groups. Although they were able to conclude that the application of CAS was feasible and safe for splenec-tomy, they cited a nearly 30-minute increase in operative time and over $4000 increase in cost of the procedures.149 This is consistent with a prior study supporting the notion that robotic-assisted splenectomy takes longer and

1	increase in operative time and over $4000 increase in cost of the procedures.149 This is consistent with a prior study supporting the notion that robotic-assisted splenectomy takes longer and is more costly than con-ventional laparoscopic surgery.150Splenectomy has also been reported using a single-incision CAS approach.151 However, this procedure is likely associated with an even steeper learning curve than traditional multiport robotic-assisted splenectomy, which calls into question the util-ity of this technique.Robotic splenectomy has been performed in the pediatric population, also proving feasibility and safety, but much like the adult population, larger series are needed to determine the true benefits of this procedure as compared to conventional laparo-scopic splenectomy in children.152The role of CAS in the patient requiring more complex surgery, for example, partial splenectomy or resection of a mas-sive spleen, is yet to be determined. At present there appears to be no

1	role of CAS in the patient requiring more complex surgery, for example, partial splenectomy or resection of a mas-sive spleen, is yet to be determined. At present there appears to be no argument supporting the application of CAS over standard laparoscopy for routine splenectomy in terms of clinical or cost advantage. The entry into the market of competing devices is likely to favorably alter these considerations.Partial SplenectomyThe past few decades have witnessed ever-widening endorse-ment for and practice of partial splenectomy. This technique, initially reported in the early 18th century, is particularly indi-cated to minimize the risk of postsplenectomy sepsis in children. Certain lipid storage disorders leading to splenomegaly (e.g., Gaucher’s disease), some forms of traumatic splenic injury (blunt and penetrating), spherocytosis in children, and focal benign splenic lesions are amenable to treatment with partial splenectomy.153 Both the laparoscopic and open approaches for

1	injury (blunt and penetrating), spherocytosis in children, and focal benign splenic lesions are amenable to treatment with partial splenectomy.153 Both the laparoscopic and open approaches for partial splenectomy have been well described.153 The spleen must be adequately mobilized, and the splenic hilar vessels attached to the targeted segment, ligated, and divided. The devascularized segment of spleen is transected along an obvi-ous line of demarcation.A useful technical tip is to transect the parenchyma 1 cm inside the ischemic demarcation line to minimize blood loss.154 Bleeding from the cut surface of the spleen usually is limited and can be controlled by various methods, including cauter-ization, argon coagulation, or application of direct hemostatic agents such as cellulose gauze and fibrin glue.Inadvertent Intraoperative Splenic InjuryInadvertent intraoperative injury to the spleen is a noted occur-rence in the surgical literature, familiar to and dreaded by the abdominal

1	and fibrin glue.Inadvertent Intraoperative Splenic InjuryInadvertent intraoperative injury to the spleen is a noted occur-rence in the surgical literature, familiar to and dreaded by the abdominal surgeon. The true incidence is unknown. The gravity of such injury is not to be underestimated. Significant short-term morbidity is associated with injury to the spleen, including increased blood loss, need for transfusion, and pro-longed hospital stay.155Intraoperative injury to the spleen has been linked with numerous operations, such as gastric fundoplication, colectomy, Brunicardi_Ch34_p1517-p1548.indd 153623/02/19 2:37 PM 1537THE SPLEENCHAPTER 34BCAFigure 34-11. A. Patient table placement for hand-assisted laparoscopic splenectomy (HALS) in case of splenomegaly. B and C. Intraopera-tive images of HALS.Brunicardi_Ch34_p1517-p1548.indd 153723/02/19 2:37 PM 1538SPECIFIC CONSIDERATIONSPART IIparaesophageal hernia repair, nephrectomy, and abdominal and pelvic vascular surgery. There

1	images of HALS.Brunicardi_Ch34_p1517-p1548.indd 153723/02/19 2:37 PM 1538SPECIFIC CONSIDERATIONSPART IIparaesophageal hernia repair, nephrectomy, and abdominal and pelvic vascular surgery. There are also reports of splenic injuries after endoscopic procedures, such as colonoscopy.Improper traction on the spleen against its peritoneal attachments is the most common mechanism of intraopera-tive injury. Capsular tears are the most common type of injury, but parenchymal lacerations and subcapsular hematomas also occur. The lower pole is more commonly injured, owing to its orientation and the greater concentration of peritoneal attach-ments found here.As with all hemorrhage, prompt temporary control of bleeding should be obtained by direct compression of the spleen itself, packing of the left upper quadrant, compression of the vessels at the splenic hilum, or pressure on the splenic artery at the superior pancreatic margin. The spleen should then be mobi-lized from its peritoneal

1	the left upper quadrant, compression of the vessels at the splenic hilum, or pressure on the splenic artery at the superior pancreatic margin. The spleen should then be mobi-lized from its peritoneal attachments and the nature of the injury assessed. Overall, the patient’s condition is the primary deter-minant of whether splenic salvage can be attempted, although hilar injury is best managed by splenectomy. When dealing with capsular tears (most common injury), strong consideration should be given to splenorrhaphy techniques: application of topical hemostatics, suture plication of disrupted parenchyma with or without omental buttress, and the use of bioabsorbable mesh sheets.The time-honored surgical tenets of liberal exposure and visualization are particularly germane to the avoidance of splenic injury. Incisions and approaches must be tailored to both patient circumstances and surgeon experience. There is some evidence to support the assertion that use of the lapa-roscopic approach

1	splenic injury. Incisions and approaches must be tailored to both patient circumstances and surgeon experience. There is some evidence to support the assertion that use of the lapa-roscopic approach may reduce the incidence of splenic injury for certain operations. As with all hemorrhage, prompt tempo-rary control of bleeding is required. Direct compression of the spleen itself, packing of the left upper quadrant, compression of the vessels at the splenic hilum, or pressure on the splenic artery at the superior pancreatic margin can slow or stop hem-orrhage and allow more deliberate consideration of manage-ment options.The type of injury plays a role as well; it has been sug-gested that hilar injury is best managed by splenectomy.80 Bar-ring these unfavorable circumstances, however, and recalling that the majority of intraoperative splenic injuries are capsular tears, it is reasonable to expect that splenic preservation can be achieved in many appropriately selected situations.

1	and recalling that the majority of intraoperative splenic injuries are capsular tears, it is reasonable to expect that splenic preservation can be achieved in many appropriately selected situations. Presented with one of these situations, the surgeon has at his or her dis-posal a number of useful and well-described splenorrhaphy techniques: application of topical hemostatics, suture plication of disrupted parenchyma with or without omental buttress, and the use of bioabsorbable mesh sheets.Currently, incidental splenectomies during laparoscopic procedures such as colorectal resections are rare events, but they are associated with worse short-term outcomes.156Preoperative Grading Score to Predict Technical Difficulty in Laparoscopic SplenectomyA splenectomy grading system based on preoperative parame-ters was developed to predict the surgical difficulty and morbid-ity for elective laparoscopic splenectomies.157,158 Preoperative data concerning demographic, clinical, pathological,

1	parame-ters was developed to predict the surgical difficulty and morbid-ity for elective laparoscopic splenectomies.157,158 Preoperative data concerning demographic, clinical, pathological, anatomi-cal, laboratory and radiological factors were compared with three surgical outcomes: operative time, intraoperative bleeding, and surgical conversion. Four preoperative parameters (male Table 34-3Difficulty ScoreAge≤40 years040–60 years1≥60 years2GenderFemale0.5Male1Pathology groupITP0.5Other benign1Malignant2Spleen weighta<400 gr1400–1000 gr3>1000 gr5Difficulty gradeLow≤4Medium4–6High≥6Note: Minimum possible score: 2 points; maximum possible score: 10 points.aSpleen weight formula: width (cm) × length (cm) × height (cm) × 0.6 = splenic weight in grams.gender, age, type of pathology, and spleen weight) were found to be associated with a difficult splenectomy (Table 34-3). This grading score is simple to calculate from the physical examina-tion, laboratory tests, and US or CT images and

1	weight) were found to be associated with a difficult splenectomy (Table 34-3). This grading score is simple to calculate from the physical examina-tion, laboratory tests, and US or CT images and could be highly practical in a daily clinical setting. It could facilitate training and development of skills while simultaneously fostering dissemina-tion of laparoscopic procedures.SPLENECTOMY OUTCOMESChanges in blood composition resulting from splenectomy include the appearance of Howell-Jolly bodies and siderocytes. After splenectomy, leukocytosis and increased platelet counts are common as well. Although platelet counts most often rise within 2 days, they may not peak for several weeks in patients with preoperative thrombocytopenia (see “Hematologic Out-comes” later). Similarly, within 1 day after splenectomy, the white blood cell count typically rises, and such elevation may continue for several months.Overwhelming Postsplenectomy InfectionThe prevalence of asplenia in the United States

1	after splenectomy, the white blood cell count typically rises, and such elevation may continue for several months.Overwhelming Postsplenectomy InfectionThe prevalence of asplenia in the United States is estimated to be 1 million; which is comparable to the number of patients carrying the human immunodeficiency virus (HIV).185,186 As with other forms of immunodeficiency, asplenic patients bear an increased susceptibility to specific types of infections for the remainder of their lives. Asplenic patients are at highest risk for infection with encapsulated organisms, most commonly Streptococcus pneumoniae, but also Haemophilus influenza Brunicardi_Ch34_p1517-p1548.indd 153823/02/19 2:37 PM 1539THE SPLEENCHAPTER 34(in particular subtype B) and Neisseria meningitides.21,185-192 Although the overwhelming majority of splenectomized patients experience no ill consequence from the absence of their spleen, the potentially catastrophic consequences of overwhelming postsplenectomy infection

1	overwhelming majority of splenectomized patients experience no ill consequence from the absence of their spleen, the potentially catastrophic consequences of overwhelming postsplenectomy infection (OPSI) demand lifelong vigilance and intimate knowledge of appropriate precautions and preven-tative measures.ComplicationsComplications of splenectomy may be classified as pulmonary, hemorrhagic, infectious, pancreatic, and thromboembolic.12,159 Left lower lobe atelectasis is the most common complication after OS; pleural effusion and pneumonia also can occur. Hem-orrhage can occur intraoperatively or postoperatively, present-ing as subphrenic hematoma. Transfusions have become less common since the advent of LS, although the indication for operation influences the likelihood of transfusion as well. Sub-phrenic abscess and wound infection are among the periopera-tive infectious complications. The placement of a drain in the left upper quadrant may be associated with postoperative

1	as well. Sub-phrenic abscess and wound infection are among the periopera-tive infectious complications. The placement of a drain in the left upper quadrant may be associated with postoperative sub-phrenic abscess and is not routinely recommended. Pancreati-tis, pseudocyst, and pancreatic fistula are among the pancreatic complications that may result from intraoperative trauma to the pancreas during dissection of the splenic hilum.Hematologic OutcomesThe results of splenectomy may be appraised according to the level of hematologic response (e.g., rise in platelet and hemo-globin levels) in those disorders in which the spleen contributes to the hematologic problem. Hematologic responses may be divided into initial and long-term responses. For thrombocyto-penia, an initial response typically is defined as a rise in plate-let count within several days of splenectomy. Reported series demonstrate the effectiveness of LS in providing a long-term platelet response in approximately 80% of

1	is defined as a rise in plate-let count within several days of splenectomy. Reported series demonstrate the effectiveness of LS in providing a long-term platelet response in approximately 80% of individuals with ITP (Table 34-4). These results are consistent with the long-term success rate associated with OS.For chronic hemolytic anemias, a rise in hemoglobin levels to >10 g/dL without the need for transfusion signifies a successful response to splenectomy. By this criterion, splenec-tomy has been reported to be successful for the vast majority of patients with chronic hemolytic anemia. For hemolytic anemia due to spherocytosis, the success rate is usually higher, ranging from 90% to 100%.Splenectomy results also may be examined in terms of surgical and postsurgical characteristics, including operative time, recovery time, and morbidity and mortality rates, all of which tend to vary according to hematologic indication (see Tables 34-4 and 34-5).12Results of few prospective, randomized

1	operative time, recovery time, and morbidity and mortality rates, all of which tend to vary according to hematologic indication (see Tables 34-4 and 34-5).12Results of few prospective, randomized trials comparing LS and OS have been published. However, several recent meta-analyses of published comparative series including 38 papers with more than 2914 patients indicate that the laparoscopic approach typically results in longer operative times, shorter hos-pital stays, lower morbidity rates, similar blood loss, and similar mortality rates compared with OS.12,120,136 Questions of the cost effectiveness of LS persist, although analysis of this issue is hindered by a lack of universally accepted metrics as well as a paucity of recent objective data. Proponents of LS argue that the generally higher operating room charges are offset by the reduced hospital stay and presumably shorter time of lost productivity.160-162 For those institutions with experienced per-sonnel and technical

1	higher operating room charges are offset by the reduced hospital stay and presumably shorter time of lost productivity.160-162 For those institutions with experienced per-sonnel and technical capability, the laparoscopic approach has emerged as the standard for elective, nontraumatic splenectomy.CancerA Taiwanese population-based study found that individuals who had splenectomy have higher risks of developing certain types of cancer (adjusted hazard ratios were 2.64 and 1.29 for nontraumatic and traumatic reasons, respectively). Splenectomy patients were found to have significantly higher risks in esopha-gus, stomach, liver, other head and neck, non-Hodgkin’s lym-phoma, and leukemia cancers. Although the exact mechanism for the possible association between splenectomy and cancer remains unclear, a plausible explanation is that the spleen is thought to be involved in immunological defenses and provides active response through humoral and cell-mediated pathways and that splenectomy may

1	a plausible explanation is that the spleen is thought to be involved in immunological defenses and provides active response through humoral and cell-mediated pathways and that splenectomy may impair immune surveillance in the host.163,164Table 34-4Outcome after splenectomyOUTCOME VARIABLESTUDIES (N)PATIENTS (N)POOLED RESULTS AND CIPMortality382914–0.01 (–0–02, 0)1Complications Pooled382914–0.11 (–0.16, –0.05)<.00001 Minor362914–0.03 (–0.05, –0.01).13 Severe362745–0.07 (–0.11, –0.03)<.00001 Thrombosis352695–0.01 (–0.02, 0.01)1 Organ injury3426390.01 (–0, 0.02)1Acces. spleen2921350.02 (–0.01, 0.05).87Operative time18137057.4 min (43.3, 71.4)<.00001Blood loss10759–41 mL (–87, 4.71)<.00001Length of stay201566–2.48 d (–2.89, –2.07)<.00001Reproduced with permission from Bai YN, Jiang H, Prasoon P. A meta-analysis of perioperative outcomes of laparoscopic splenectomy for hematological diseases, World J Surg. 2012 Oct;36(10):2349-2345.Brunicardi_Ch34_p1517-p1548.indd 153923/02/19 2:37 PM

1	P. A meta-analysis of perioperative outcomes of laparoscopic splenectomy for hematological diseases, World J Surg. 2012 Oct;36(10):2349-2345.Brunicardi_Ch34_p1517-p1548.indd 153923/02/19 2:37 PM 1540SPECIFIC CONSIDERATIONSPART IIUltrasoundUltrasound is the least invasive mode of splenic imaging. It is rapid, relatively easy to perform, and does not expose the patient to ionizing radiation. It is often the first imaging modality used to evaluate the spleen in a trauma patient, although questions of sensitivity and specificity remain.133,167 In the elective setting, such as for routine diagnostic purposes or for preoperative plan-ning, it is the least costly modality available, and the sensitivity of ultrasound for detecting textural lesions of the spleen can be quite good in experienced hands.168When examining a normal spleen, differentiation between red and white pulp is not possible, and a homogeneous acous-tic echotexture should be visualized. Splenic artery and vein patency may

1	hands.168When examining a normal spleen, differentiation between red and white pulp is not possible, and a homogeneous acous-tic echotexture should be visualized. Splenic artery and vein patency may be assessed using Doppler imaging. Splenic artery anatomy has been classified commonly into two patterns: dis-tributed and magistral. This variability in vascular distribution visualized by Doppler imaging becomes important when there is consideration of performing a partial splenectomy due to the possibility of segmental blood flow and its effect on area of resection.Percutaneous ultrasound-guided procedures for splenic disease (e.g., cyst aspiration, biopsy), historically avoided due to the risk of hemorrhage and other complications, are becom-ing more common as the safety of these procedures has become increasingly demonstrated.165,166Computed TomographyCT affords a high degree of resolution and detail of the splenic parenchyma, vasculature, and its relationship to neighboring

1	procedures has become increasingly demonstrated.165,166Computed TomographyCT affords a high degree of resolution and detail of the splenic parenchyma, vasculature, and its relationship to neighboring structures, making it the preferred imaging modality for many surgeons. CT has become an invaluable tool in the evaluation and management of the blunt trauma patient, and standardized scoring systems for splenic trauma based on CT images now aid in management decisions.169 In the nontrauma setting, CT is extremely useful for assessment of splenomegaly, identification of solid and cystic lesions, and guidance of percutaneous pro-cedures.3 The use of iodinated contrast material adds diagnostic clarity to CT imaging of the spleen, although at the cost of, the small but real risks, possible renal impairment or allergic reac-tions. Three-dimensional reconstruction after CT scan may help to predict the difficulty of the procedure and to choose the best surgical approach.170,171The appearance of

1	impairment or allergic reac-tions. Three-dimensional reconstruction after CT scan may help to predict the difficulty of the procedure and to choose the best surgical approach.170,171The appearance of normal splenic tissue on a noncon-trast CT is uniform parenchymal attenuation with values ranging between 40 and 60 Hounsfield units (HU).172 On a contrast-enhanced CT, the appearance of the spleen depends largely on the timing of the intravenous bolus administration of contrast material. Due to the different rates of flow through the red and white pulp, the spleen appears heterogeneously enhanc-ing during the first minute after initiation of intravenous admin-istration of contrast material during the arterial and early portal venous phases.23 The frequency of these artifacts increases with advancing patient age. When evaluating for splenic abscess, a contrast-enhanced CT should be utilized.64,173Three-dimensional CT volumetry is a novel modality that measures the volume of the spleen.

1	advancing patient age. When evaluating for splenic abscess, a contrast-enhanced CT should be utilized.64,173Three-dimensional CT volumetry is a novel modality that measures the volume of the spleen. This tool may be of some benefit in planning technically challenging cases involving sple-nomegaly. A recent retrospective review of laparoscopic sple-nectomy patients who underwent CT volumetry preoperatively demonstrated a higher conversion rate when the spleen was measured to be greater than 2700 cc.170 This information may aid in proper informed consent as well as preoperative planning to assist in determination of which patients may benefit from a minimally invasive or open surgical approach.139Plain RadiographyRarely is plain radiography used for primary splenic imaging. Plain films can indirectly provide an outline of the spleen in the left upper quadrant or suggest splenomegaly by revealing dis-placement of adjacent air-filled structures (e.g., the stomach or splenic flexure of the

1	provide an outline of the spleen in the left upper quadrant or suggest splenomegaly by revealing dis-placement of adjacent air-filled structures (e.g., the stomach or splenic flexure of the colon). Plain films may also demonstrate splenic calcifications. Splenic calcifications often are found in association with splenomegaly but are otherwise a nonspe-cific finding. Splenic calcifications can indicate a number of benign, neoplastic, or infectious processes, including phlebo-lith, splenic artery aneurysm, sickle cell changes, tumors (e.g., hemangioma, hemangiosarcoma, lymphoma), echinococcosis, or tuberculosis.168Magnetic Resonance ImagingAlthough MRI offers excellent detail and versatility in abdomi-nal imaging, it is more expensive than CT scan or ultrasound and offers no obvious advantage for primary imaging of the spleen. MRI can be a valuable adjunct to the more commonly used imaging techniques when splenic disease is suspected but not definitively diagnosed.168,173Magnetic

1	for primary imaging of the spleen. MRI can be a valuable adjunct to the more commonly used imaging techniques when splenic disease is suspected but not definitively diagnosed.168,173Magnetic resonance (MR) signal characteristics of the spleen are related to the relative ratio of red and white pulp and the relationship of the timing of the intravenous (IV) contrast bolus and the time of image acquisition. The spleen will gener-ally have a homogeneous MR signal on noncontrast images. Table 34-5Laparoscopic splenectomy results by hematologic indicationITP (N = 151)TTP (N = 7)ANEMIA (N = 40)MALIGNANCY (N = 28)OR time (min)128146149165EBL (mL)13796116238LOS (days)2.23.02.22.6Conversions from LS to OS3 (2%)1 (14%)1 (3%)1 (4%)Complications14 (9%)01 (3%)3 (11%)EBL = estimated blood loss; ITP = idiopathic thrombocytopenic purpura; LOS = length of hospital stay; LS = laparoscopic splenectomy; OR = operating room; OS = open splenectomy; TTP = thrombotic thrombocytopenic purpura.Data from

1	= idiopathic thrombocytopenic purpura; LOS = length of hospital stay; LS = laparoscopic splenectomy; OR = operating room; OS = open splenectomy; TTP = thrombotic thrombocytopenic purpura.Data from Brunicardi FC, Andersen DK, Billiar TR, et al: Schwartz’s Principles of Surgery, 8th ed. New York, NY: McGraw-Hill; 2005.Brunicardi_Ch34_p1517-p1548.indd 154023/02/19 2:37 PM 1541THE SPLEENCHAPTER 34On contrast MRI, the spleen appears to have heterogeneous enhancement during the arterial phase of contrast enhancement.AngiographyAngiography of the spleen most commonly refers to inva-sive arterial imaging, and when it is combined with thera-peutic splenic arterial embolization (SAE), there are multiple applications for this procedure: localization and treatment of hemorrhage in select trauma patients; delivery of a variety of therapies in patients with cirrhosis or portal and sinistral hyper-tension and in transplant patients; and adjunct (or, more con-troversially, as an alternative) to

1	patients; delivery of a variety of therapies in patients with cirrhosis or portal and sinistral hyper-tension and in transplant patients; and adjunct (or, more con-troversially, as an alternative) to splenectomy for treatment of hematologic disorders such as ITP or hypersplenism.174-176 Preoperative or intraoperative SAE for elective splenectomy is also a common, although not universal, practice. Few pro-spective data have been published in the last 5 years on pre-operative SAE.177 Preoperative SAE is purported not only to facilitate less intraoperative blood loss but also possibly to allow a laparoscopic approach in patients whose spleens had previ-ously been considered too large for, or otherwise not amenable to, safe laparoscopic resection. Limited success in using partial SAE as an alternative to therapeutic splenectomy in chronic ITP has been previously reported.178 Its detractors argue that the need for increased analgesics and occasional extended hospital stay preoperatively,

1	to therapeutic splenectomy in chronic ITP has been previously reported.178 Its detractors argue that the need for increased analgesics and occasional extended hospital stay preoperatively, the possibility of pancreatitis, and the well-described risks of invasive arteriography associated with the passage of wires and catheters through the vasculature, may negate any presumed benefits of preoperative SAE.Nuclear ImagingRadioscintigraphy with technetium-99m sulfur colloid demon-strates splenic location and size. It may be especially helpful in locating accessory spleens after unsuccessful splenectomy for ITP and has recently proven useful in diagnosing splenosis.179,180 Unfortunately, no conclusive outcome benefit has been shown for preoperative technetium scanning before splenectomy.52,181 When dealing with diseases of platelet sequestration, indium-labeled autologous platelet scanning (ILAPS) demonstrates whether platelet sequestration is predominantly in the spleen, liver, or both.

1	When dealing with diseases of platelet sequestration, indium-labeled autologous platelet scanning (ILAPS) demonstrates whether platelet sequestration is predominantly in the spleen, liver, or both. This becomes important in deciding whether or not a patient will benefit from a splenectomy. ILAPS is a nuclear imaging modality in which autologous platelets are reinfused into the patient after ex vivo labeling. Subsequent scintigraphy demonstrates the site(s) of platelet sequestration and clearance. It has been proposed that patients with purely or predominantly splenic sequestration determined by ILAPS may be more likely to respond to splenectomy than those exhibiting hepatic, mixed, or diffuse patterns.176An emerging and novel application for spleen scintigraphy may be as a noninvasive method to diagnose nonalcoholic ste-atohepatitis (NASH). Conventional imaging methods are reli-able for the detection of moderate to severe fatty changes in the liver, though they are not reliable for

1	to diagnose nonalcoholic ste-atohepatitis (NASH). Conventional imaging methods are reli-able for the detection of moderate to severe fatty changes in the liver, though they are not reliable for detecting NASH or hepatic fibrosis. NASH, which may lead to cirrhosis, can result from nonalcoholic fatty liver disease (NAFLD), the most common cause of steatosis. With the alarming rise of obesity worldwide, NAFLD is also increasingly common with prevalence rang-ing from 6.3% to 33%.15,182 The diagnosis of progression from NAFLD to NASH has been dependent on histologic assessment of tissue obtained from liver biopsy. Characteristics unique to NASH have been reported, among them the association of splenic enlargement, not seen to a similar degree in NAFLD.13In addition, the ratio of liver-to-spleen uptake determined by scintigraphy has been found to be predictably altered in NASH patients. The liver-to-spleen uptake ratio is significantly decreased in NASH patients, but not NAFLD patients,

1	uptake determined by scintigraphy has been found to be predictably altered in NASH patients. The liver-to-spleen uptake ratio is significantly decreased in NASH patients, but not NAFLD patients, leading some to conclude that technetium-99m-phytate scintigraphy is a reliable tool to differentiate NASH from NAFLD.183 Although additional studies are needed to identify the role of these nuclear medicine studies in prognostication and monitoring of those patients at high risk for the development of NASH, recent stud-ies show promise in this regard.184Microbiology and Pathogenesis. Life-threatening infec-tion in the asplenic patient is attributable to four main factors: loss of splenic macrophages, diminished tuftsin production, loss of the spleen’s reticuloendothelial screening function, and dysregulated coagulation.187 In the normal host, these factors work in concert to eliminate opsonized bacteria from the blood-stream. This system is particularly suited for the removal of encapsulated

1	coagulation.187 In the normal host, these factors work in concert to eliminate opsonized bacteria from the blood-stream. This system is particularly suited for the removal of encapsulated bacteria, whose polysaccharide coating is a natu-ral defense against opsonization (S pneumoniae, H influenzae, and N meningitidis are the classic examples). Infections with protozoa that invade the red blood cell, such as Babesia microti (transmitted by tick bites), Ehrlichia, and Plasmodium, occur more frequently in splenectomized individuals than in normal hosts. Other potential infectious bacterial sources include group A streptococci, C canimorsus (transmitted by dog bites), group B streptococci, Enterococcus species, Bacteroides species, Salmonella species, and Bartonella species.124 In the absence of the spleen, elimination of these pathogens from the bloodstream falls solely to the liver, a process that has been demonstrated to be less effective.12,126 Further, the pathophysiology of infection

1	the spleen, elimination of these pathogens from the bloodstream falls solely to the liver, a process that has been demonstrated to be less effective.12,126 Further, the pathophysiology of infection in asplenic patients has also been implicated in their increased risk of thrombosis and pulmonary hypertension.187More recently, the bacterial patterns of splenectomy sepsis have been changing. After the introduction of vaccinations and new oral antibiotics, postsplenectomy patients can suffer from diverse strains of bacterial infection, which are not strictly cor-related with the splenic function. In recent cohort series, gram negative bacteria are prevalent, representing 45% to 50% of infections in asplenic patients.193,194 In vaccinated patients, the rate of sepsis by pneumococcus is very low. In fact, encapsulated bacteria, such as S pneumoniae, N meningitidis, and H influenzae, were rarely encountered in those series in whom vaccination was routinely adopted.193-196Sepsis by uncommon

1	In fact, encapsulated bacteria, such as S pneumoniae, N meningitidis, and H influenzae, were rarely encountered in those series in whom vaccination was routinely adopted.193-196Sepsis by uncommon bacteria as well by protozoa infec-tions such as malaria and babesiosis are also known to affect asplenic patients.196-200Clinical Features. OPSI is uniformly fatal without treat-ment, and thus sepsis in a splenectomized patient is a medical emergency.21,187,192 Therefore, any clinical suggestion of infec-tion, including seemingly isolated fevers, must be viewed with a high index of suspicion and treated empirically as thorough investigation proceeds. OPSI may begin with a relatively mild-appearing prodrome of symptoms. In addition to fever, nonspe-cific symptoms such as malaise, myalgias, headache, vomiting, diarrhea, abdominal pain, and others should be viewed with alarm in the asplenic patient. This process can progress rapidly to fulminant bacteremic septic shock, with hypotension,

1	headache, vomiting, diarrhea, abdominal pain, and others should be viewed with alarm in the asplenic patient. This process can progress rapidly to fulminant bacteremic septic shock, with hypotension, anuria, and disseminated intravascular coagulation.The true incidence of OPSI is not precisely known because defining criteria vary among published series. Overall lifetime risk remains low, ranging from <1% to 5%.12,21,126,201 Among 9Brunicardi_Ch34_p1517-p1548.indd 154123/02/19 2:37 PM 1542SPECIFIC CONSIDERATIONSPART IIthose who develop OPSI, some characteristics can be identified that impart greater risk. Reason for splenectomy is the single most influential determinant of OPSI risk. Case series demon-strate that those who undergo splenectomy for hematologic dis-ease (malignancy, myelodysplasia, or hemoglobinopathy) are far more susceptible to OPSI than patients who undergo sple-nectomy for trauma or iatrogenic reasons. Age is also an impor-tant consideration, with children 5 years

1	or hemoglobinopathy) are far more susceptible to OPSI than patients who undergo sple-nectomy for trauma or iatrogenic reasons. Age is also an impor-tant consideration, with children 5 years of age or less and adults 50 years or older being at elevated risk. Finally, time interval from spleen removal must be considered. A large number of OPSI cases occur many years to decades after splenectomy.186,189 This observation underscores both the threat of this lethal dis-ease and the need for lifelong vigilance.Antibiotics and the Asplenic Patient. Antibiotic therapy for the asplenic patient can be considered in three contexts: therapy for established or presumed infections, prophylaxis in anticipation of invasive procedures (e.g., dental procedures), and general prophylaxis. The most critical action in the treat-ment of established or presumed OPSI is the immediate use of broad-spectrum intravenous antibiotics, ideally after the collec-tion of blood cultures. Vancomycin provides

1	critical action in the treat-ment of established or presumed OPSI is the immediate use of broad-spectrum intravenous antibiotics, ideally after the collec-tion of blood cultures. Vancomycin provides broad-spectrum Gram-positive coverage, including coverage against penicillin-resistant S pneumonia.190 Ceftriaxone should be added to include Gram-negative coverage for N meningitidis and H influenza.190 Early implementation of antibiotics and goal-directed therapy for sepsis can significant reduce mortality rates.21,191 For the lat-ter two indications, unfortunately, evidence supporting efficacy is scant, and guidelines for antibiotic prophylaxis are not uni-form. Optimal duration of chemoprophylaxis in children also remains unclear; however, a daily dose of antibiotics until 5 years of age or at least 5 years after splenectomy are commonly recommended, although some advocate continuation into at least early adulthood.121,122,192 Concerns regarding compliance and bacterial resistance have

1	at least 5 years after splenectomy are commonly recommended, although some advocate continuation into at least early adulthood.121,122,192 Concerns regarding compliance and bacterial resistance have been raised, which have led some authors to suggest that lifelong daily antibiotic prophylaxis be recommended only for those patients whose antibody titers fail to respond appropriately to vaccination or, alternately, that asplenic patients be advised to carry at all times a reserve sup-ply of antibiotic to be self-administered at the earliest sign of infection.122 Considering the grave consequences of OPSI and its relatively low incidence, controlled trials resulting in mean-ingful data on this issue seem unlikely to be performed.Education. Several risk management strategies are com-monly recommended to patients following splenectomy, including wearing a medical bracelet, carrying a laminated medical alert card, possessing a medical letter with specific empiric therapy instructions

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1	ST, Scott DJ, Burdick JS, Rege RV, Jones DB. Laparoscopic marsupialization and hemisplenectomy for splenic cysts. J Laparoendosc Adv Surg Tech A. 2001;11(4): 243-249. 114. Fan H, Zhang D, Zhao X, Pan F, Jin ZK. Laparoscopic partial splenectomy for large splenic epidermoid cyst. Chin Med J (Engl). 2011;124(11):1751-1753. 115. Mouttalib S, Rice HE, Snyder D, et al. Evaluation of par-tial and total splenectomy in children with sickle cell dis-ease using an Internet-based registry. Pediatr Blood Cancer. 2012;59(1):100-104. 116. Buesing KL, Tracy ET, Kiernan C, et al. Partial splenectomy for hereditary spherocytosis: a multi-institutional review. J Pediatr Surg. 2011;46(1):178-183. 117. Hery G, Becmeur F, Mefat L, et al. Laparoscopic partial sple-nectomy: indications and results of a multicenter retrospective study. Surg Endosc. 2008;22(1):45-49. 118. Dutta S, Price VE, Blanchette V, Langer JC. A laparoscopic approach to partial splenectomy for children with hereditary spherocytosis. Surg

1	retrospective study. Surg Endosc. 2008;22(1):45-49. 118. Dutta S, Price VE, Blanchette V, Langer JC. A laparoscopic approach to partial splenectomy for children with hereditary spherocytosis. Surg Endosc. 2006;20(11):1719-1724. 119. Szczepanik AB, Meissner AJ. Partial splenectomy in the management of nonparasitic splenic cysts. World J Surg. 2009;33(4):852-856. 120. Bai YN, Jiang H, Prasoon P. A meta-analysis of perioperative outcomes of laparoscopic splenectomy for hematological dis-orders. World J Surg. 2012;36(10):2349-2358. 121. Jones P, Leder K, Woolley I, Cameron P, Cheng A, Spelman D. Postsplenectomy infection strategies for prevention in gen-eral practice. Aust Fam Physician. 2010;39(6):383-386. 122. Denholm JT, Jones PA, Spelman DW, Cameron PU, Woolley IJ. Spleen registry may help reduce the incidence of overwhelming postsplenectomy infection in Victoria. Med J Aust. 2010;192(1):49-50. 123. Holdsworth RJ, Irving AD, Cuschieri A. Postsplenectomy sep-sis and its mortality

1	help reduce the incidence of overwhelming postsplenectomy infection in Victoria. Med J Aust. 2010;192(1):49-50. 123. Holdsworth RJ, Irving AD, Cuschieri A. Postsplenectomy sep-sis and its mortality rate: actual versus perceived risks. Br J Surg. 1991;78(9):1031-1038. 124. Waghorn DJ. Overwhelming infection in asplenic patients: current best practice preventive measures are not being fol-lowed. J Clin Pathol. 2001;54(3):214-218. 125. Bonanni P, Grazzini M, Niccolai G, et al. Recommended vac-cinations for asplenic and hyposplenic adult patients. Hum Vaccin Immunother. 2017;13(2):359-368. 126. Taylor MD, Genuit T, Napolitano LM. Overwhelming post-splenectomy sepsis and trauma: time to consider revaccina-tion? J Trauma. 2005;59(6):1482-1485. 127. Targarona EM. Portal vein thrombosis after laparoscopic splenectomy: the size of the risk. Surg Innov. 2008;15(4): 266-270. 128. Ruiz-Tovar J, Priego P. Portal vein thrombosis after splenic and pancreatic surgery. Adv Exp Med Biol. 2017;906:

1	laparoscopic splenectomy: the size of the risk. Surg Innov. 2008;15(4): 266-270. 128. Ruiz-Tovar J, Priego P. Portal vein thrombosis after splenic and pancreatic surgery. Adv Exp Med Biol. 2017;906: 241-251. 129. British Committee for Standards in Haematology Gen-eral Haematology Task F. Guidelines for the investigation and management of idiopathic thrombocytopenic pur-pura in adults, children and in pregnancy. Br J Haematol. 2003;120(4):574-596. 130. Martin Arnau B, Turrado Rodriguez V, Tartaglia E, Bollo Rodriguez J, Targarona EM, Trias Folch M. Impact of preop-erative platelet count on perioperative outcome after laparo-scopic splenectomy for idiopathic thrombocytopenic purpura. Cir Esp. 2016;94(7):399-403. 131. Rijcken E, Mees ST, Bisping G, et al. Laparoscopic sple-nectomy for medically refractory immune thrombocytope-nia (ITP): a retrospective cohort study on longtime response predicting factors based on consensus criteria. Int J Surg. 2014;12(12):1428-1433. 132. Matharoo GS,

1	refractory immune thrombocytope-nia (ITP): a retrospective cohort study on longtime response predicting factors based on consensus criteria. Int J Surg. 2014;12(12):1428-1433. 132. Matharoo GS, Afthinos JN, Gibbs KE. Trends in splenectomy: where does laparoscopy stand? JSLS. 2014;18(4). 133. Myers J. Focused assessment with sonography for trauma (FAST): the truth about ultrasound in blunt trauma. J Trauma. 2007;62(6 suppl):S28. 134. Park A, Marcaccio M, Sternbach M, Witzke D, Fitzgerald P. Laparoscopic vs open splenectomy. Arch Surg. 1999; 134(11):1263-1269. 135. Winslow ER, Brunt LM. Perioperative outcomes of lapa-roscopic versus open splenectomy: a meta-analysis with an emphasis on complications. Surgery. 2003;134(4):647-653; discussion 654-645. 136. Park A, Gagner M, Pomp A. The lateral approach to laparo-scopic splenectomy. Am J Surg. 1997;173(2):126-130. 137. Pietrabissa A, Morelli L, Peri A, et al. Laparoscopic treatment of splenomegaly: a case for hand-assisted laparoscopic

1	approach to laparo-scopic splenectomy. Am J Surg. 1997;173(2):126-130. 137. Pietrabissa A, Morelli L, Peri A, et al. Laparoscopic treatment of splenomegaly: a case for hand-assisted laparoscopic surgery. Arch Surg. 2011;146(7):818-823. 138. Patel AG, Parker JE, Wallwork B, et al. Massive splenomeg-aly is associated with significant morbidity after laparoscopic splenectomy. Ann Surg. 2003;238(2):235-240. 139. Kercher KW, Matthews BD, Walsh RM, Sing RF, Backus CL, Heniford BT. Laparoscopic splenectomy for massive spleno-megaly. Am J Surg. 2002;183(2):192-196. 140. Monclova JL, Targarona EM, Vidal P, et al. Single incision versus reduced port splenectomy—searching for the best alternative to conventional laparoscopic splenectomy. Surg Endosc. 2013;27(3):895-902. 141. Vatansev C, Ece I, Jr. Single incision laparoscopic splenec-tomy with double port. Surg Laparosc Endosc Percutan Tech. 2009;19(6):e225-e227. 142. Barbaros U, Dinccag A. Single incision laparoscopic splenectomy: the first two

1	incision laparoscopic splenec-tomy with double port. Surg Laparosc Endosc Percutan Tech. 2009;19(6):e225-e227. 142. Barbaros U, Dinccag A. Single incision laparoscopic splenectomy: the first two cases. J Gastrointest Surg. 2009;13(8):1520-1523. 143. Maeso S, Reza M, Mayol JA, et al. Efficacy of the Da Vinci surgical system in abdominal surgery compared with that of laparoscopy: a systematic review and meta-analysis. Ann Surg. 2010;252(2):254-262. 144. Chapman WH, 3rd, Albrecht RJ, Kim VB, Young JA, Chitwood WR, Jr. Computer-assisted laparoscopic splenec-tomy with the da Vinci surgical robot. J Laparoendosc Adv Surg Tech A. 2002;12(3):155-159. 145. Moore LJ, Wilson MR, Waine E, Masters RS, McGrath JS, Vine SJ. Robotic technology results in faster and more robust surgical skill acquisition than traditional laparoscopy. J Robot Surg. 2015;9(1):67-73. 146. Park BS, Ryu DY, Son GM, Cho YH. Factors influenc-ing on difficulty with laparoscopic total extraperitoneal repair according to

1	than traditional laparoscopy. J Robot Surg. 2015;9(1):67-73. 146. Park BS, Ryu DY, Son GM, Cho YH. Factors influenc-ing on difficulty with laparoscopic total extraperitoneal repair according to learning period. Ann Surg Treat Res. 2014;87(4):203-208. 147. Smith CD, Farrell TM, McNatt SS, Metreveli RE. Assessing laparoscopic manipulative skills. Am J Surg. 2001;181(6):547-550. 148. Lee GI, Lee MR, Green I, Allaf M, Marohn MR. Surgeons’ physical discomfort and symptoms during robotic surgery: a comprehensive ergonomic survey study. Surgical endoscopy. 2017;31(4):1697-1706. 149. Gelmini R, Franzoni C, Spaziani A, Patriti A, Casciola L, Saviano M. Laparoscopic splenectomy: conventional versus robotic approach—a comparative study. J Laparoendosc Adv Surg Tech A. 2011;21(5):393-398. 150. Bodner J, Kafka-Ritsch R, Lucciarini P, Fish JH, 3rd, Schmid T. A critical comparison of robotic versus conventional laparo-scopic splenectomies. World J Surg. 2005;29(8):982-985; discussion

1	J, Kafka-Ritsch R, Lucciarini P, Fish JH, 3rd, Schmid T. A critical comparison of robotic versus conventional laparo-scopic splenectomies. World J Surg. 2005;29(8):982-985; discussion 985-986. 151. Corcione F, Bracale U, Pirozzi F, Cuccurullo D, Angelini PL. Robotic single-access splenectomy using the Da Vinci Single-Site(R) platform: a case report. Int J Med Robot. 2014;10(1):103-106. 152. Klein MD, Langenburg SE, Kabeer M, Lorincz A, Knight CG. Pediatric robotic surgery: lessons from a clinical experience. J Laparoendosc Adv Surg Tech A. 2007;17(2):265-271.Brunicardi_Ch34_p1517-p1548.indd 154523/02/19 2:37 PM 1546SPECIFIC CONSIDERATIONSPART II 153. Wang X, Wang M, Zhang H, Peng B. Laparoscopic partial splenectomy is safe and effective in patients with focal benign splenic lesion. Surg Endosc. 2014;28(12):3273-3278. 154. de la Villeon B, Zarzavadjian Le Bian A, Vuarnesson H, et al. Laparoscopic partial splenectomy: a technical tip. Surg Endosc. 2015;29(1):94-99. 155. Cadili A,

1	Surg Endosc. 2014;28(12):3273-3278. 154. de la Villeon B, Zarzavadjian Le Bian A, Vuarnesson H, et al. Laparoscopic partial splenectomy: a technical tip. Surg Endosc. 2015;29(1):94-99. 155. Cadili A, de Gara C. Complications of splenectomy. Am J Med. 2008;121(5):371-375. 156. Isik O, Aytac E, Ashburn J, et al. Does laparoscopy reduce splenic injuries during colorectal resections? An assess-ment from the ACS-NSQIP database. Surg Endosc. 2015;29(5):1039-1044. 157. Rodriguez-Otero Luppi C, Targarona Soler EM, Balague Ponz C, et al. Clinical, anatomical, and pathological grading score to predict technical difficulty in laparoscopic splenectomy for non-traumatic diseases. World J Surg. 2017;41(2):439-448. 158. Gonçalves D, Morais M, Costa-Pinho A, Bessa-Melo R, Graca L, Costa-Maia J. Validation of a difficulty grading score in laparoscopic splenectomy. J Laparoendosc Adv Surg Tech A. 2018;28(3):242-247. 159. James AW, Rabl C, Westphalen AC, Fogarty PF, Posselt AM, Campos GM.

1	J. Validation of a difficulty grading score in laparoscopic splenectomy. J Laparoendosc Adv Surg Tech A. 2018;28(3):242-247. 159. James AW, Rabl C, Westphalen AC, Fogarty PF, Posselt AM, Campos GM. Portomesenteric venous thrombosis after lapa-roscopic surgery: a systematic literature review. Arch Surg. 2009;144(6):520-526. 160. Li B, Liu J, Shangguan Y, Liu B, Qi Y. Laparoscopy-assisted small incision splenectomy and open splenectomy in the treatment of hematologic diseases: a single-institution com-parative experience. Surg Laparosc Endosc Percutan Tech. 2013;23(3):309-311. 161. Zhou J, Liu P, Yin Z, Zhao Y, Wang X. Safety and cost-effectiveness analysis of laparoscopic splenectomy by secondary pedicle division using monopolar electrocautery. Hepatogastroenterology. 2013;60(126):1302-1306. 162. Cordera F, Long KH, Nagorney DM, et al. Open versus laparoscopic splenectomy for idiopathic thrombocytope-nic purpura: clinical and economic analysis. Surgery. 2003; 134(1):45-52. 163. Sun LM,

1	F, Long KH, Nagorney DM, et al. Open versus laparoscopic splenectomy for idiopathic thrombocytope-nic purpura: clinical and economic analysis. Surgery. 2003; 134(1):45-52. 163. Sun LM, Chen HJ, Jeng LB, Li TC, Wu SC, Kao CH. Sple-nectomy and increased subsequent cancer risk: a nationwide population-based cohort study. Am J Surg. 2015;210(2): 243-251. 164. Kristinsson SY, Gridley G, Hoover RN, Check D, Landgren O. Long-term risks after splenectomy among 8,149 cancer-free American veterans: a cohort study with up to 27 years follow-up. Haematologica. 2014;99(2):392-398. 165. McInnes MD, Kielar AZ, Macdonald DB. Percutaneous image-guided biopsy of the spleen: systematic review and meta-analysis of the complication rate and diagnostic accu-racy. Radiology. 2011;260(3):699-708. 166. Singh AK, Shankar S, Gervais DA, Hahn PF, Mueller PR. Image-guided percutaneous splenic interventions. Radio-graphics. 2012;32(2):523-534. 167. Zarzaur BL, Kozar RA, Fabian TC, Coimbra R. A survey of American

1	Shankar S, Gervais DA, Hahn PF, Mueller PR. Image-guided percutaneous splenic interventions. Radio-graphics. 2012;32(2):523-534. 167. Zarzaur BL, Kozar RA, Fabian TC, Coimbra R. A survey of American Association for the Surgery of Trauma member practices in the management of blunt splenic injury. J Trauma. 2011;70(5):1026-1031. 168. Kamaya A, Weinstein S, Desser TS. Multiple lesions of the spleen: differential diagnosis of cystic and solid lesions. Semin Ultrasound CT MR. 2006;27(5):389-403. 169. Thompson BE, Munera F, Cohn SM, et al. Novel computed tomography scan scoring system predicts the need for inter-vention after splenic injury. J Trauma. 2006;60(5):1083-1086. 170. Filicori F, Stock C, Schweitzer AD, et al. Three-dimensional CT volumetry predicts outcome of laparoscopic splenectomy for splenomegaly: retrospective clinical study. World J Surg. 2013;37(1):52-58. 171. Berindoague R, Targarona EM, Balague C, et al. Can we pre-dict immediate outcome after laparoscopic splenectomy

1	for splenomegaly: retrospective clinical study. World J Surg. 2013;37(1):52-58. 171. Berindoague R, Targarona EM, Balague C, et al. Can we pre-dict immediate outcome after laparoscopic splenectomy for splenomegaly? Multivariate analysis of clinical, anatomic, and pathologic features after 3D reconstruction of the spleen. Surgical innovation. 2007;14(4):243-251. 172. Vancauwenberghe T, Snoeckx A, Vanbeckevoort D, Dymarkowski S, Vanhoenacker FM. Imaging of the spleen: what the clinician needs to know. Singapore Med J. 2015;56(3):133-144. 173. Karakas HM, Tuncbilek N, Okten OO. Splenic abnormali-ties: an overview on sectional images. Diagn Interv Radiol. 2005;11(3):152-158. 174. Dent D, Alsabrook G, Erickson BA, et al. Blunt splenic injuries: high nonoperative management rate can be achieved with selective embolization. J Trauma. 2004;56(5): 1063-1067. 175. Koconis KG, Singh H, Soares G. Partial splenic emboliza-tion in the treatment of patients with portal hypertension: a review of the

1	with selective embolization. J Trauma. 2004;56(5): 1063-1067. 175. Koconis KG, Singh H, Soares G. Partial splenic emboliza-tion in the treatment of patients with portal hypertension: a review of the english language literature. J Vasc Interv Radiol. 2007;18(4):463-481. 176. Cuker A, Cines DB. Evidence-based mini-review: is indium-labeled autologous platelet scanning predictive of response to splenectomy in patients with chronic immune thrombo-cytopenia? Hematology Am Soc Hematol Educ Program. 2010;2010:385-386. 177. Wu Z, Zhou J, Pankaj P, Peng B. Comparative treatment and literature review for laparoscopic splenectomy alone versus preoperative splenic artery embolization splenectomy. Surg Endosc. 2012;26(10):2758-2766. 178. Mousa A, Armbruster J, Adongay J, AbuRahma AF. Splenic artery embolization as a treatment option for chronic pancyto-penia secondary to hypersplenism: a case report and review of literature. Vasc Endovascular Surg. 2012;46(6):501-503. 179. Williams G, Rosen MP,

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1	liver dis-ease. Prim Care. 2017;44(4):599-607. 183. Bolton-Maggs PH, Langer JC, Iolascon A, Tittensor P, King MJ; General Haematology Task Force of the British Committee for Standards in Haematology. Guidelines for the diagnosis and management of hereditary spherocytosis—2011 update. Br J Haematol. 2012;156(1):37-49. 184. Tovo CV, de Mattos AZ, Coral GP, Branco FS, Suwa E, de Mattos AA. Noninvasive imaging assessment of non-alcoholic fatty liver disease: focus on liver scintigraphy. World J Gastro-enterol. 2015;21(15):4432-4439. 185. Rubin LG, Schaffner W. Clinical practice. Care of the asplenic patient. N Engl J Med. 2014;371(4):349-356. 186. Theilacker C, Ludewig K, Serr A, et al. Overwhelming post-splenectomy infection: a prospective multicenter cohort study. Clin Infect Dis. 2016;62(7):871-878. 187. O’Neal HR, Jr, Niven AS, Karam GH. Critical illness in patients with asplenia. Chest. 2016;150(6):1394-1402. 188. Chong J, Jones P, Spelman D, Leder K, Cheng AC. Over-whelming

1	HR, Jr, Niven AS, Karam GH. Critical illness in patients with asplenia. Chest. 2016;150(6):1394-1402. 188. Chong J, Jones P, Spelman D, Leder K, Cheng AC. Over-whelming post-splenectomy sepsis in patients with asplenia and hyposplenia: a retrospective cohort study. Epidemiol Infect. 2017;145(2):397-400. 189. Sinwar PD. Overwhelming post splenectomy infection syndrome—review study. Int J Surg. 2014;12(12):1314-1316. 190. Lynch AM, Kapila R. Overwhelming postsplenectomy infec-tion. Infect Dis Clin North Am. 1996;10(4):693-707.Brunicardi_Ch34_p1517-p1548.indd 154623/02/19 2:37 PM 1547THE SPLEENCHAPTER 34 191. Brigden ML. Overwhelming postsplenectomy infection still a problem. West J Med. 1992;157(4):440-443. 192. Dionne B, Dehority W, Brett M, Howdieshell TR. The asplenic patient: post-insult immunocompetence, infection, and vacci-nation. Surg Infect (Larchmt). 2017;18(5):536-544. 193. Dendle C, Sundararajan V, Spelman T, Jolley D, Woolley I. Splenectomy sequelae: an analysis of

1	immunocompetence, infection, and vacci-nation. Surg Infect (Larchmt). 2017;18(5):536-544. 193. Dendle C, Sundararajan V, Spelman T, Jolley D, Woolley I. Splenectomy sequelae: an analysis of infectious outcomes among adults in Victoria. Med J Aust. 2012;196(9):582-586. 194. Ejstrud P, Kristensen B, Hansen JB, Madsen KM, Schonhey-der HC, Sorensen HT. Risk and patterns of bacteraemia after splenectomy: a population-based study. Scand J Infect Dis. 2000;32(5):521-525. 195. Edgren G, Almqvist R, Hartman M, Utter GH. Splenectomy and the risk of sepsis: a population-based cohort study. Ann Surg. 2014;260(6):1081-1087. 196. Leone G, Pizzigallo E. Bacterial infections following splenec-tomy for malignant and nonmalignant hematologic diseases. Mediterr J Hematol Infect Dis. 2015;7(1):e2015057. 197. Yu RK, Shepherd LE, Rapson DA. Capnocytophaga canimor-sus, a potential emerging microorganism in splenectomized patients. Br J Haematol. 2000;109(4):679. 198. Sica S, Di Mario A, Salutari P, et al.

1	RK, Shepherd LE, Rapson DA. Capnocytophaga canimor-sus, a potential emerging microorganism in splenectomized patients. Br J Haematol. 2000;109(4):679. 198. Sica S, Di Mario A, Salutari P, et al. Morganella morganii pericarditis after resolvent splenectomy for immune pan-cytopenia following allogeneic bone marrow transplanta-tion for acute lymphoblastic leukemia. Clin Infect Dis. 1995;21(4):1052-1053. 199. Demar M, Legrand E, Hommel D, Esterre P, Carme B. Plas-modium falciparum malaria in splenectomized patients: two case reports in French Guiana and a literature review. Am J Trop Med Hyg. 2004;71(3):290-293. 200. Rosner F, Zarrabi MH, Benach JL, Habicht GS. Babesiosis in splenectomized adults. Review of 22 reported cases. Am J Med. 1984;76(4):696-701. 201. Okabayashi T, Hanazaki K. Overwhelming postsplenectomy infection syndrome in adults—a clinically preventable disease. World J Gastroenterol. 2008;14(2):176-179.Brunicardi_Ch34_p1517-p1548.indd 154723/02/19 2:37 PM

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1	Abdominal Wall, Omentum, Mesentery, and RetroperitoneumScott Kizy and Sayeed Ikramuddin 35chapterABDOMINAL WALLGeneral ConsiderationsIncision and closure of the abdominal wall is among the most common surgical procedures performed. Knowledge of its lay-ered anatomy is critical in the management of surgical patients. Cranially defined by the costal margin and xiphoid process and caudally ending over the pubic bones of the pelvis, the abdomi-nal wall provides support and protection to the peritoneal and retroperitoneal organs.Surgical AnatomyEmbryologically, the abdominal wall is derived from the meso-derm and envelops the future abdominal contents as bilateral migrating layers originating from the paravertebral area. The leading bars of these sheets fuse in the midline as the linea alba at 7 weeks after gestation and reach the umbilicus at 8 weeks.The abdominal wall consists of nine distinct layers: skin, subcutaneous tissue, superficial fascia, external oblique muscle, internal

1	at 7 weeks after gestation and reach the umbilicus at 8 weeks.The abdominal wall consists of nine distinct layers: skin, subcutaneous tissue, superficial fascia, external oblique muscle, internal oblique muscles, transversus abdominus muscle, trans-versalis fascia, preperitoneal adipose tissue, and peritoneum. The subcutaneous tissue is composed of superficial adipose tis-sue that is contiguous with Camper’s fascia anteriorly. Deep to this, Scarpa’s fascia consists of a fibrous matrix of tissue that fuses with the anterior layer of fascia of the flank and back.The muscles of the abdominal wall consist of the rectus abdominus medially and the external oblique, internal oblique, and transversus abdominis laterally. The rectus abdominus is a paired longitudinal muscle spanning the length of the abdomen. Divided by the linea alba, both rectus muscles originate at the pubic symphysis and crest and insert on the xiphoid process, the fifth and sixth ribs, and the seventh costal cartilage.

1	the abdomen. Divided by the linea alba, both rectus muscles originate at the pubic symphysis and crest and insert on the xiphoid process, the fifth and sixth ribs, and the seventh costal cartilage. Three ten-dinous insertions cross the rectus muscle along its length. The muscle is contained within an aponeurotic sheath formed from the fusion of differing components of the lateral fascial layers.Laterally, the three muscular layers (external oblique, internal oblique, and transversus abdominis) have obliquely oriented fibers relative to one another. The external oblique arises from the eighth rib and inserts medially into the linea alba and anterior iliac crests. The fibers of this muscular layer travel medial and caudal from its insertion. The internal oblique originates from the thoracolumbar fascia. Its fibers travels cra-nially and anteriorly, inserting on the lower costal margin and the xiphoid process. The deep muscular layer, the transversus abdominis muscle, begins at the

1	fascia. Its fibers travels cra-nially and anteriorly, inserting on the lower costal margin and the xiphoid process. The deep muscular layer, the transversus abdominis muscle, begins at the costal margin and lumbar fas-cia, runs horizontally and anteriorly, and inserts on the linea alba, xiphoid process, and pubis symphysis (Fig. 35-1).Altogether, these muscular layers provide fascial aponeu-rotic sheaths anteriorly. These fascial layers provide variable contributions to the separate layers of the rectus sheath and ultimately fuse in the midline as the linea alba. The rectus sheath that encloses the rectus muscles has differing composi-tions above and below the arcuate line. The anterior rectus sheath is composed of the external oblique aponeurosis through-out its length. The internal oblique aponeurosis is bilaminar and contributes to the anterior and posterior rectus sheaths above the arcuate line. Below this line, the internal aponeurosis contrib-utes only to the anterior rectus

1	aponeurosis is bilaminar and contributes to the anterior and posterior rectus sheaths above the arcuate line. Below this line, the internal aponeurosis contrib-utes only to the anterior rectus sheath. The transversus abdomi-nis aponeurosis contributes the posterior rectus sheath above the arcuate line and the anterior rectus sheath below the arcuate line. Therefore, below the arcuate line, all the aponeurotic layers of the lateral musculature form the anterior sheath, leaving the transversalis fascia as the only posterior fascial covering. This layer is a weak fibrous layer separated from the peritoneum by preperitoneal fat (Fig. 35-2).Along the posterior abdominal wall are folds correspond-ing to underlying vasculature and embryologic remnants. The median umbilical fold is formed by the obliterated urachus trav-eling from the dome of the bladder to the umbilicus in the mid-line. The bilateral medial folds are formed by remnants of the umbilical arteries. Lastly, the lateral folds are

1	urachus trav-eling from the dome of the bladder to the umbilicus in the mid-line. The bilateral medial folds are formed by remnants of the umbilical arteries. Lastly, the lateral folds are associated with the inferior epigastric vessels.The deep blood supply of the abdominal wall is supplied mostly from the inferior and superior epigastric arteries. The superior epigastric artery is the final branch of the internal tho-racic artery. It enters the rectus sheath below the costal margin 1Abdominal Wall 1549General Considerations / 1549Surgical Anatomy / 1549Physiology / 1550Abdominal Surgical Incisions and Closure / 1550Congenital Abnormalities / 1553Acquired Abnormalities / 1554Omentum 1557Surgical Anatomy / 1557Physiology / 1558Acquired Disorders / 1558Mesentery 1559Surgical Anatomy / 1559Sclerosing Mesenteritis / 1559Mesenteric Cysts / 1560Mesenteric Tumors / 1561Retroperitoneum 1561Surgical Anatomy / 1561Retroperitoneal Infections / 1561Retroperitoneal Fibrosis /

1	Anatomy / 1559Sclerosing Mesenteritis / 1559Mesenteric Cysts / 1560Mesenteric Tumors / 1561Retroperitoneum 1561Surgical Anatomy / 1561Retroperitoneal Infections / 1561Retroperitoneal Fibrosis / 1562Brunicardi_Ch35_p1549-p1566.indd 154912/02/19 9:58 AM 1550Key Points1 There are differences in the anatomic structure in the rectus sheath above and below the arcuate line. Below the arcuate line, all the lateral fascial layers combine and travel anteriorly forming the anterior rectus sheath and leaving the posterior aspect of the lower portion of the rectus muscles without an aponeurotic covering. Above the arcuate line, the posterior rectus sheath is formed by a portion of the internal oblique aponeurosis and the transversus abdominus sheath, and the anterior rectus sheath is formed by the remaining fibers of the internal oblique and the external oblique aponeuroses.2 Two randomized trials have found that closure of midline incisions with small fascial stitches of five to eight mm

1	the remaining fibers of the internal oblique and the external oblique aponeuroses.2 Two randomized trials have found that closure of midline incisions with small fascial stitches of five to eight mm length and five mm width is beneficial in preventing inci-sional hernias of the abdominal wall.3 Primary repair of ventral incisional hernias results in high recurrence rates, and repair utilizing other methods, includ-ing prosthetic mesh and component separation, are preferred techniques of repair.4 Laparoscopic incisional hernia repair results in similar recur-rence rates and wound infections, compared to open repair. Laparoscopic repair may result in a faster recovery and shorter hospitalization; however, there may be an increase in bowel injury when compared to an open repair.5 Desmoid tumors should be monitored for asymptomatic patients as there is a possibility of spontaneous regression. For patients with rapidly growing or symptomatic tumors resection is recommended. If complete

1	tumors should be monitored for asymptomatic patients as there is a possibility of spontaneous regression. For patients with rapidly growing or symptomatic tumors resection is recommended. If complete pathologic resection is not achievable without significant morbidity, more modest resection is recommended along with treatment with adju-vant therapies.6 The omentum provides an immunogenic and fibrotic response to foreign stimuli, allowing the abdomen to wall off infections preventing diffuse peritonitis.7 The role of surgery in the treatment of sclerosing mesenteri-tis is minimal and is most often undertaken to obtain tissue for diagnosis. Most cases should be treated medically with surgical interventions reserved for cases of bowel obstruc-tion and ischemia.8 Surgical interventions for retroperitoneal fibrosis include obtaining tissue for pathologic diagnosis, relieving ureteral obstructions via ureterolysis or ureteral stenting, and reliev-ing vascular obstructions via endovascular

1	fibrosis include obtaining tissue for pathologic diagnosis, relieving ureteral obstructions via ureterolysis or ureteral stenting, and reliev-ing vascular obstructions via endovascular stenting of affected vessels. Most cases are treated successfully with the use of steroids.and travels along the posterior surface of the anterior sheath and forms an anastomosis with the inferior epigastric artery at the umbilicus. The inferior epigastric artery arises from the external iliac artery. These arteries provide a collateral circula-tion between the vasculature of the upper and lower extremities (Fig. 35-3). The abdominal wall is also supplied by branches of the subcostal and lumbar arteries. Superficially, the abdominal wall subcutaneous and skin tissue is supplied by branches of the superficial epigastric arteries, femoral arteries, superficial exter-nal pudendal, and superficial circumflex arteries. Venous drain-age of the abdominal wall is variable but typically follows the

1	superficial epigastric arteries, femoral arteries, superficial exter-nal pudendal, and superficial circumflex arteries. Venous drain-age of the abdominal wall is variable but typically follows the aforementioned arteries. Above the umbilicus, the lymphatics of the abdominal wall drain into the superficial axillary nodes. Below the umbilicus, these drain into the inguinal nodes. Lym-phatics near the umbilicus can drain along the falciform liga-ment toward the hepatic nodes.Innervation of the abdominal wall is segmental, leading to a dermatomal sensory pattern. Afferent branches of the T4 to L1 nerve roots provide sensation of the abdominal wall (see Fig. 35-3). The muscles of the abdominal wall are inner-vated by the efferent branches of spinal nerves T6 to T12.PhysiologyAside from providing protection of the intraabdominal and retroperitoneal organs, the abdominal wall muscles assist with flexion, extension, and rotation of the torso along with the muscles of the back and trunk.

1	protection of the intraabdominal and retroperitoneal organs, the abdominal wall muscles assist with flexion, extension, and rotation of the torso along with the muscles of the back and trunk. Working as a unit, the rectus muscles and external/internal obliques activate to flex the trunk anteriorly and laterally. Rotation of the torso is performed by simultaneous stimulation of the contralateral external oblique and ipsilateral internal oblique. To rotate the torso to the right requires simultaneous contraction of the right internal oblique muscle and left external oblique muscle, and vice versa for leftward rotation. Altogether, the muscles of the abdominal wall can act to raise intraabdominal pressure providing assistance with respiration, coughs, defecation, uri-nation, and parturition.Abdominal Surgical Incisions and ClosureKnowledge of the abdominal wall anatomy is an important aspect of safe entry into the abdomen. The goal of an efficacious incision is to provide adequate

1	Surgical Incisions and ClosureKnowledge of the abdominal wall anatomy is an important aspect of safe entry into the abdomen. The goal of an efficacious incision is to provide adequate exposure to perform the proce-dure with minimal perturbation of the abdominal wall function.Incisions for open abdominal surgery are generally located in proximity to operative targets. There are two general types of incisions: longitudinal or transverse/oblique (Fig. 35-4). There does not appear to be differences in early or late postoperative complications or recovery time between these two types of inci-sions. However, transverse incisions may be associated with lower incisional hernia rates but higher rates of wound infec-tions. Overall, without clear evidence of superiority the choice of incision remains a surgeon-dependent decision. Several retractor systems can be used to provide exposure using these open incisions. Examples include the Bookwalter, Omni-Tract, and Thompson retractors (Fig.

1	remains a surgeon-dependent decision. Several retractor systems can be used to provide exposure using these open incisions. Examples include the Bookwalter, Omni-Tract, and Thompson retractors (Fig. 35-5).The most common longitudinal incision is the midline incision. Providing access to most intraabdominal organs and some retroperitoneal structures, the midline incision is carried down to the linea alba to allow access to the abdomen with mini-mal injury to skeletal muscles, nerves, and vessels. Paramedian longitudinal incisions are made lateral to the midline through the rectus sheath or in the pararectus location. These incisions restrict access to the contralateral abdomen and pelvis and risk damage to the musculature, vessels, and nerves.Closure of the midline incision requires reapproximation without undue tension or strangulation of the tissue to prevent Brunicardi_Ch35_p1549-p1566.indd 155012/02/19 9:58 AM 1551ABDOMINAL WALL, OMENTUM, MESENTERY, AND RETROPERITONEUMCHAPTER

1	without undue tension or strangulation of the tissue to prevent Brunicardi_Ch35_p1549-p1566.indd 155012/02/19 9:58 AM 1551ABDOMINAL WALL, OMENTUM, MESENTERY, AND RETROPERITONEUMCHAPTER 35perioperative dehiscence or incisional hernia formation. Clas-sically, the midline fascia of the incision is closed using sutures placed 1 cm from the edge and a width of 1 cm apart. Two European randomized controlled trials demonstrated reduced rates of incisional hernia with shorter stitch width (5–8 mm) compared to the standard 1 cm stitches. Studies are ongoing in the United States at this time. Several studies have evaluated the use of prophylactic mesh implanta-tion in the closure of the midline incision. While these studies demonstrate reduced incisional hernia formation in the short-term, more long-term data is needed to determine the inci-dence of mesh-related complications. Furthermore, the optimal location of mesh implantation and type of mesh used are still being evaluated.When

1	more long-term data is needed to determine the inci-dence of mesh-related complications. Furthermore, the optimal location of mesh implantation and type of mesh used are still being evaluated.When performing a transverse or oblique incision, a sur-geon can either divide or separate the muscle fibers of the abdom-inal wall. The classic McBurney’s incision for appendectomy, an oblique incision performed one-third of the way from the iliac spine to the umbilicus, is an example of a muscle-splitting approach. A subcostal incision can be used to access the upper abdomen, liver, gallbladder, spleen, pancreas, or adrenals. On closure of these incisions, two layers must be approximated. The deep layer includes the internal oblique, transversus abdominus muscle, and the transversalis fascia. The superficial layer includes the anterior aponeurotic tissue of the rectus sheath medially and the external muscle/aponeurosis laterally. A bilateral subcostal incision, or a chevron incision, can be

1	superficial layer includes the anterior aponeurotic tissue of the rectus sheath medially and the external muscle/aponeurosis laterally. A bilateral subcostal incision, or a chevron incision, can be used to access organs of the upper abdomen and diaphragm. A Mercedes-Benz modification includes a midline incision superiorly, providing further access to the upper abdomen or the lower mediastinum.Pelvic procedures are commonly performed through a Pfannenstiel incision. This incision is performed via a trans-verse skin incision carried down to the anterior rectus sheath. The sheath is also transversely incised and dissected off the underlying rectus muscle. The rectus muscles are separated, and access through the transversalis fascia is performed lon-gitudinally. Closure of this incision requires approximation of the peritoneum and rectus muscles and closure of the anterior rectus sheath.Laparoscopic port site incision placement must be care-fully planned based on approach angles and

1	requires approximation of the peritoneum and rectus muscles and closure of the anterior rectus sheath.Laparoscopic port site incision placement must be care-fully planned based on approach angles and working distances both to the operative site and between ports. Placement of a 278910789106511L1L2L31010(A) Anterior view(B) Rectus abdominis (RA)(C) External oblique (EO) (layer 1)(D) Internal oblique (IO) (layer 2)(E) Transversus abdominis (layer 3)Lateral viewsEOIliac crestIntertendinousband (IT)RAIOTARSRSRSEOEOEORAITRAITRAITRALinea albaPyramidalisITITFigure 35-1. Muscles of the anterior abdominal wall. A. The anterior abdominal wall musculature is shown with the rectus sheath reflected on the left side. B. Rectus abdominis. C. External oblique. D. Internal oblique. E. Transversus abdominus. (Reproduced with permission from Moore KL, Agur AM: Essential Clinical Anatomy, 5th edition. Philadelphia, PA: Lippincott Williams & Wilkins; 2014.)Brunicardi_Ch35_p1549-p1566.indd

1	abdominus. (Reproduced with permission from Moore KL, Agur AM: Essential Clinical Anatomy, 5th edition. Philadelphia, PA: Lippincott Williams & Wilkins; 2014.)Brunicardi_Ch35_p1549-p1566.indd 155112/02/19 9:58 AM 1552SPECIFIC CONSIDERATIONSPART IInasogastric tube and Foley catheter may help to decompress the stomach and bladder reducing injury to these structures on entry. Initial entry into the abdomen may be completed using the open Hasson or closed Veress needle techniques. The Hasson tech-nique involves direct visualization and systematic opening of each fascial layer encountered on entry. The closed technique utilizes the Veress needle to access the abdomen in a controlled fashion. Access to the peritoneum is confirmed using one or a combination of several techniques (saline drop method or measurement of intraabdominal pressure), and gas insuffla-tion is begun. The abdomen can then be accessed using either a visual entry port or a nonvisualized entry port at the site of the

1	method or measurement of intraabdominal pressure), and gas insuffla-tion is begun. The abdomen can then be accessed using either a visual entry port or a nonvisualized entry port at the site of the needle track or an alternative site. Choice of entry method is still controversial and based on surgeon preference. Retrospec-tive reviews suggest that complications may be lower using the Hasson technique, but randomized trials (although small) have not found significant differences in techniques.Abdominal incisions can lead to significant patient mor-bidity. Complications include hematomas/seromas, surgical site infections, fascial dehiscence, incisional hernias, and nerve injuries among others. In general, minimizing opera-tive incision lengths to only that which is necessary for a safe operation is prudent to reduce these complications and morbidity.Right externaloblique muscleFibers of rightexternal obliqueaponeurosisFibers of leftinternal obliqueaponeurosisFibers passing from

1	operation is prudent to reduce these complications and morbidity.Right externaloblique muscleFibers of rightexternal obliqueaponeurosisFibers of leftinternal obliqueaponeurosisFibers passing from superficial to deep(and vice versa) at linea albaLeft internaloblique muscleIntermuscular exchange of fibers between aponeuroses ofcontralateral external and internal oblique muscles.Fibers of left externaloblique aponeurosis,which run deep on theright side and runningsuperficially on the leftsideDeep fibers of left external oblique aponeurosisUmbilical ringDeep fibers of right external oblique aponeurosisIntramuscular exchange of superficial and deepfibers within aponeuroses of contralateralexternal oblique muscles. Transverse sections(B) Anterior view(A) Anterior viewsLineaalbaRectus abdominisAponeurosisof externalobliqueAponeurosisof internalobliqueAponeurosisof transversusabdominisParietal peritoneumSkinTransversus abdominisInternal obliqueExternal obliqueDeep membranous layerof

1	externalobliqueAponeurosisof internalobliqueAponeurosisof transversusabdominisParietal peritoneumSkinTransversus abdominisInternal obliqueExternal obliqueDeep membranous layerof subcutaneoustissueTransversalis fasciaExtraperitoneal fatRectussheathLinea alba(note fibers passingfrom superficial to deep,and vice versa, as inright side of figure A)Superficial fatty layer ofsubcutaneoustissueInvesting (deep) fascia:SuperficialIntermediateDeepFigure 35-2. Fiber direction and cross-sectional anatomy of the abdominal wall. A. Muscular and aponeurosis fiber direction of external and internal obliques. B. Cross-sectional anatomy of the anterior abdominal wall above and below the arcuate line. The posterior leaf of the rectus sheath exists above the arcuate line. Below this line, all aponeurotic sheaths converge and travel anterior to the rectus muscles, leaving the posterior rectus uncovered by a fascial layer. (Reproduced with permission from Moore KL, Agur AM: Essential Clinical Anatomy,

1	converge and travel anterior to the rectus muscles, leaving the posterior rectus uncovered by a fascial layer. (Reproduced with permission from Moore KL, Agur AM: Essential Clinical Anatomy, 5th edition. Philadelphia, PA: Lippincott Williams & Wilkins; 2014.)Brunicardi_Ch35_p1549-p1566.indd 155212/02/19 9:58 AM 1553ABDOMINAL WALL, OMENTUM, MESENTERY, AND RETROPERITONEUMCHAPTER 35Congenital AbnormalitiesThe folds of mesodermal cells that form the abdominal wall lay-ers begin to form in the early weeks after gestation. The folds develop in the cephalic, caudal, and right/left lateral directions and converge at the umbilicus around 8 weeks. At around 6 weeks of development the contents of the abdominal cavity outgrow the space allowed by the surrounding layers of the Thoraco-abdominalnerves (T7-T11)Lateral cutaneous branchof subcostal nerve (T12)Anterior cutaneous branchof subcostal nerve (T12)Iliohypogastric nerve (L1)Ilio-inguinal nerve (L1)Xiphoid processAnterior superior iliac

1	(T7-T11)Lateral cutaneous branchof subcostal nerve (T12)Anterior cutaneous branchof subcostal nerve (T12)Iliohypogastric nerve (L1)Ilio-inguinal nerve (L1)Xiphoid processAnterior superior iliac spine (ASIS)AnteriorcutaneousbranchesLateralcutaneousbranchesInguinal ligamentSuperiorepigastric a.External obliqueInternal oblique TransversusabdominisInferior epigastric a.Deep circumflexiliac a.Superficialepigastric a.Superficialcircumflex iliac a.External iliac a.Femoral a.Transversalis fasciaMusculophrenic a.11th posteriorintercostal a.10th posteriorintercostal a.Subcostal a.Internal thoracic a.Anterior viewFigure 35-3. Neurovascular anatomy of the anterior abdominal wall. The right image demonstrates the arteries of the deep abdominal wall. The superior epigastric and inferior epigastric form an anastomosis along the posterior aspect of the rectus muscle. The image on the left demonstrates the dermatomal distribution of cutaneous nerves of the abdominal wall. (Reproduced with permission

1	an anastomosis along the posterior aspect of the rectus muscle. The image on the left demonstrates the dermatomal distribution of cutaneous nerves of the abdominal wall. (Reproduced with permission from Moore KL, Agur AM: Essential Clinical Anatomy, 5th edition. Philadelphia, PA: Lippincott Williams & Wilkins; 2014.)CDEFHGABFigure 35-4. Various open abdominal wall incisions. A. Mid-line incision. B. Paramedian incision. C. Right subcostal incision and “saber slash” extension (dashed line). D. Bilateral subcostal incision (also chevron incision) with “Mercedes Benz” extension (dashed line). E. Rocky-Davis incision and Weir extension (dashed line). F. McBurney incision. G. Transverse incision. H. Pfannen-stiel incision.Figure 35-5. Thompson retractor for exposure of intra-abdominal structures utilizing an upper midline incision.Brunicardi_Ch35_p1549-p1566.indd 155312/02/19 9:58 AM 1554SPECIFIC CONSIDERATIONSPART IIabdominal wall, resulting in temporary herniation of the abdom-inal

1	utilizing an upper midline incision.Brunicardi_Ch35_p1549-p1566.indd 155312/02/19 9:58 AM 1554SPECIFIC CONSIDERATIONSPART IIabdominal wall, resulting in temporary herniation of the abdom-inal contents through a central defect. At this time, the vitelline duct (omphalomesenteric duct) and allantois also pass through the central defect. The vitelline duct serves as a conduit to the embryologic midgut and the yolk sac. As the midgut develops outside of the abdomen, it undergoes a 270° counterclockwise rotation and reenters the abdomen at around 12 weeks. Failure of the midgut to reenter the abdomen leads to the congenital abdominal wall defect known as an omphalocele, in which the contents of the defect protrude through an open umbilicus and are covered by an amniotic/peritoneal membrane. Gastroschisis, on the other hand, results from either malformation or disrup-tion of the abdominal wall, either from genetic defects or vas-cular compromise. Gastroschisis presents as protruding

1	Gastroschisis, on the other hand, results from either malformation or disrup-tion of the abdominal wall, either from genetic defects or vas-cular compromise. Gastroschisis presents as protruding viscera through a defect lateral to the umbilicus (usually along the right side) without the amniotic sac covering.The vitelline duct usually involutes at around the eighth to ninth week after gestation. Failure of vitelline duct regression can lead to several abnormalities depending on the spectrum of involution. This spectrum includes total persistence of the vitel-line duct leading to omphalomesenteric fistula causing drainage of intestinal contents at the umbilicus, to partial closure leading to omphalomesenteric cyst. If the vitelline remnant persists at the ileal border, a Meckel’s diverticulum forms. The vitelline duct may also persist as a fibrous attachment of the intestine to the abdominal wall predisposing the patient to bowel obstruc-tions. Fistulas, cysts, and fibrous attachments

1	forms. The vitelline duct may also persist as a fibrous attachment of the intestine to the abdominal wall predisposing the patient to bowel obstruc-tions. Fistulas, cysts, and fibrous attachments should be resected when diagnosed.The urachus is the proximal portion of the allantois form-ing as the bladder descends into its pelvic position. The urachus closes and forms the median umbilical ligament of the abdomi-nal wall as previously described. Failure of the urachus to close results in urinary fistula or cyst. These are treated by urachal excision and closure of the bladder defect.Acquired AbnormalitiesAbdominal Wall Hernias. A protrusion or bulge of abdomi-nal contents through the abdominal wall muscle/fascia repre-sents an abdominal wall hernia. This may be present at birth or acquired from weakening or disruption of the overlying fascia, or from failed healing of a surgical incision. Hernias may present as asymptomatic bulges that increase with Val-salva maneuvers, or with

1	from weakening or disruption of the overlying fascia, or from failed healing of a surgical incision. Hernias may present as asymptomatic bulges that increase with Val-salva maneuvers, or with significant discomfort. On physical exam, the patient’s abdominal wall should be evaluated with the patient both standing and in the recumbent position. Her-nias may reduce spontaneously or with manual pressure. If a hernia is incarcerated, it cannot be reduced and generally requires surgical correction. If intestine is incarcerated in the hernia defect, bowel obstruction may ensue, which represents a surgical emergency. Incarcerated hernias present with sig-nificant pain, nausea, and vomiting. A hernia is considered strangulated if blood supply to its contents is compromised. Localized ischemia may lead to infarction and eventual perfo-ration if left untreated.Nonincisional hernias are named based on their location on the abdominal wall. Epigastric hernias are defects in the abdominal wall

1	may lead to infarction and eventual perfo-ration if left untreated.Nonincisional hernias are named based on their location on the abdominal wall. Epigastric hernias are defects in the abdominal wall located between the umbilicus and the xiphoid process. These hernias are usually small but may be associ-ated with multiple defects. They result from multiple factors, including muscle weakness, congenitally weakened epigastric fascia, or increases in intra-abdominal pressure. Epigastric hernias rarely contain bowel and usually contain portions of the omentum or falciform ligament. Given the rarity of incarcera-tion, repair of an epigastric hernia is indicated for symptomatic patients only. Laparoscopic repair can be attempted, but this type of hernia usually can be managed with a small incision where the defect is closed with interrupted sutures.Umbilical hernias may be congenital or acquired. Umbili-cal hernias are common in newborns, especially in premature infants. Closure of an

1	incision where the defect is closed with interrupted sutures.Umbilical hernias may be congenital or acquired. Umbili-cal hernias are common in newborns, especially in premature infants. Closure of an umbilical defect occurs after birth as the muscles of the rectus abdominis grow toward one another. Most umbilical hernias close spontaneously by 5 years of age and can be monitored as they will spontaneously resolve. Indications for repair include incarceration, symptomatic hernia, failure to decrease in size or if the defect fails to close by the age of 5 years.In adults, umbilical hernias form because of increased abdominal pressure due to pregnancy, obesity, or ascites. Females are at higher risk for this type of hernia than men. Small, asymptomatic hernias may be followed clinically. How-ever, if an umbilical hernia enlarges in size, causes symptoms, or incarcerates surgical treatment should be offered. Hernias can be repaired laparoscopically or with an open procedure. Mesh should

1	if an umbilical hernia enlarges in size, causes symptoms, or incarcerates surgical treatment should be offered. Hernias can be repaired laparoscopically or with an open procedure. Mesh should be employed for large defects where the fascial edges cannot be approximated without tension. In this case, mesh should be placed as a sublay technique (below the fascia) and sutured in place to prevent migration.Patients with cirrhosis and associated ascites with an umbilical hernia pose a significant clinical dilemma. Umbilical defects enlarge in these patients because of high intra-abdominal pressure associated with uncontrolled ascites. With severe liver disease, these patients are at high risk of operative complica-tions. Most hernias contain ascites; however, omentum and bowel may also enter the defect. Given the high pressure, skin breakdown may ensue leading to hernia rupture or weeping as well as risk of spontaneous bacterial peritonitis. All attempts should be made to control the

1	the defect. Given the high pressure, skin breakdown may ensue leading to hernia rupture or weeping as well as risk of spontaneous bacterial peritonitis. All attempts should be made to control the patient’s ascites prior to repair. Therefore, asymptomatic patients should be managed conserva-tively with aggressive management of ascites. Liver transplant candidates should undergo repair at the time of transplanta-tion as pretransplant repair has high morbidity and mortality. Patients with incarcerated hernias or with thinning or ruptured skin overlying the hernia should be treated emergently.Hernias that occur along the arcuate line are known as Spigelian hernias. While rare, these hernias form due to the anatomic weakness of lack of a posterior rectus sheath below the arcuate line. As the hernia develops, peritoneum that passes through the arcuate line will pass laterally toward the external oblique muscle given the overlying aponeurosis (Fig. 35-6). Most patients present with pain and

1	develops, peritoneum that passes through the arcuate line will pass laterally toward the external oblique muscle given the overlying aponeurosis (Fig. 35-6). Most patients present with pain and swelling in the mid to lower abdomen. Incarceration is common as up to 20% of patients present with a nonreducible hernia. Given the high rate of incar-ceration, surgical repair is usually recommended. Either open or laparoscopic repair can be performed. The defect is closed by approximating the medial and lateral edges of the transversalis fascia to the rectus sheath.Incisional Hernias. Hernias that develop at sites of pre-vious abdominal incisions are known as incisional hernias. Hernias can develop at the site of any previous abdominal incision. Up to 20% of midline incisions will develop her-nias eventually. Vertical incisions may have a higher risk of hernia formation than transverse or oblique incisions. Upper Brunicardi_Ch35_p1549-p1566.indd 155412/02/19 9:58 AM 1555ABDOMINAL WALL,

1	eventually. Vertical incisions may have a higher risk of hernia formation than transverse or oblique incisions. Upper Brunicardi_Ch35_p1549-p1566.indd 155412/02/19 9:58 AM 1555ABDOMINAL WALL, OMENTUM, MESENTERY, AND RETROPERITONEUMCHAPTER 35abdominal incisions are also at higher risk than lower inci-sions. Laparoscopic port sites may also develop hernias. The etiology of incisional hernias is complex. Several patient-derived factors increase the risk of hernia, including diabe-tes, immunosuppressant use, obesity, smoking, malnutrition, and connective tissue disorders. Local operative factors may also be implicated, including technique, wound infection, or high tension at the time of closure. Hernias can develop up to 10 years after surgery but normally occur in the early postop-erative period. Incisional hernias can present as asymptom-atic bulges or with severe discomfort. Multiple hernias can be present along the length of the incision. Elective surgery should be recommended in

1	period. Incisional hernias can present as asymptom-atic bulges or with severe discomfort. Multiple hernias can be present along the length of the incision. Elective surgery should be recommended in patients who are symptomatic. Small defects pose a higher risk of incarceration and should be repaired. To improve operative outcome, patient associated factors, including smoking and obesity, should be remedied prior to surgical repair.Surgical management of incisional hernias include either primary tissue or mesh repairs. Hernias can also be repaired via a laparoscopic or open approach. Simple suture repair is associ-ated with recurrence rates as high as 54%. A Cochrane review of several randomized controlled trials found that open mesh repair improved hernia recurrence rates when compared to simple closure (33% with simple repair vs. 16% with mesh repair). Mesh repairs are, however, associated with a higher rate of infections.To reduce tension at the suture line associated with pri-mary

1	simple closure (33% with simple repair vs. 16% with mesh repair). Mesh repairs are, however, associated with a higher rate of infections.To reduce tension at the suture line associated with pri-mary suture repair Ramirez described the components separa-tion technique in 1990. This procedure entails dividing portions of the bilateral external oblique aponeuroses forming musculo-fascial advancement flaps. The posterior rectus sheath can also be incised, allowing up to 10 cm of medial mobilization and ten-sionless approximation of the midline fascia. This technique can cause large skin flaps and initially had high rates of infection. Overtime, techniques have been developed to decrease flap for-mation and decreased rates of surgical site infection. Endoscopic component separation can also be used to mobilize flaps with minimal ischemia to overlying subcutaneous and skin tissue, theoretically decreasing infection rates. Mobilization of midline fascia is reduced with endoscopic methods.

1	be used to mobilize flaps with minimal ischemia to overlying subcutaneous and skin tissue, theoretically decreasing infection rates. Mobilization of midline fascia is reduced with endoscopic methods. Mesh can also be utilized to reinforce the repair. Overall, component separation without mesh compares to standard mesh repair with respect to hernia recurrence, but precludes the risk of mesh implantation. When mesh is added to component separation, recurrence rates may be as low as 4% to 10% depending on follow up period.Mesh repair has become the standard for elective manage-ment of most incisional hernias. Position of mesh placement is controversial. Mesh can be placed above the midline fascia (overlay), bridged across fascial defects (interlay), underneath fascia (sublay), or within the abdominal cavity (underlay). A systematic review found that sublay placement of mesh may reduce hernia recurrence and prevent wound related compli-cations. The sublay technique is performed by

1	the abdominal cavity (underlay). A systematic review found that sublay placement of mesh may reduce hernia recurrence and prevent wound related compli-cations. The sublay technique is performed by developing the plane between the rectus muscle and the posterior sheath, and affixing mesh in this space. The anterior sheath can be approxi-mated if there is no tension.Material used for mesh manufacturing can be classified into two classes: synthetic and biologic. Synthetic meshes can be either permanent or degradable, while all biologic meshes are degradable. Permanent mesh is currently made of either poly-propylene, polyethylene terephthalate polyester, or expanded polytetrafluoroethylene. Permanent mesh is durable and of rel-atively low cost. Degradable synthetic mesh, including Vicryl mesh, is eventually eliminated and loses structural support, but it does offer the advantage of lower mesh infection rates. Degrad-able mesh is associated with high recurrence rates, but it can be used

1	is eventually eliminated and loses structural support, but it does offer the advantage of lower mesh infection rates. Degrad-able mesh is associated with high recurrence rates, but it can be used for temporary abdominal wall closure in contaminated or infected fields. Newer synthetic biomaterial meshes, includ-ing Gore BioA or Phasix, degrade over a longer period of time and may reduce recurrence rates, but long-term effectiveness is unknown. Biologic meshes are decellularized, collagen-rich porcine, bovine, or human tissue. These meshes are designed to allow host cellular ingrowth, promoting incorporation and eventual replacement of the mesh with host tissue. Biologic meshes are a high cost alternative to synthetic degradable mesh and can be used in infected fields. However, their efficacy in preventing recurrence is unclear. Composite products contain two components and are used during intraperitoneal repair. One side of composite mesh, which is placed on the abdominal wall side of

1	in preventing recurrence is unclear. Composite products contain two components and are used during intraperitoneal repair. One side of composite mesh, which is placed on the abdominal wall side of implantation, is made of typical nondegradable synthetic material and promotes integration of host tissue. The other side is covered in a synthetic or biologic material, allowing contact with viscera and preventing adhesion formation. These materi-als include polyglactin, collagen, cellulose, titanium, omega-3, and hyaluronate. This allays concerns of direct mesh contact 3Figure 35-6. Computed tomography scan imaging of Spigelian hernia. Hernia contents traverse through the arcuate line but remain covered by the external oblique aponeurosis. (Reproduced with permission from Martin M, Paquette B, Badet N, et al: Spigelian hernia: CT findings and clinical relevance, Abdom Imaging. 2013 Apr;38(2):260-264.)Brunicardi_Ch35_p1549-p1566.indd 155512/02/19 9:58 AM 1556SPECIFIC

1	M, Paquette B, Badet N, et al: Spigelian hernia: CT findings and clinical relevance, Abdom Imaging. 2013 Apr;38(2):260-264.)Brunicardi_Ch35_p1549-p1566.indd 155512/02/19 9:58 AM 1556SPECIFIC CONSIDERATIONSPART IIwith viscera that may cause adhesions, erosion, and eventual fistula formation. Pore size and mesh weight are also important aspects of mesh design. Recently, large-pore, lightweight mesh has been developed. This was initially thought to delay incorpo-ration, but that has not been seen in practice. They do allow the theoretical advantage of increased incorporation of host tissue and potentially better elasticity and improved postoperative pain when compared to microporous heavier meshes. There is also initial data which suggests lower rates of mesh infection with the use of large pore mesh.Initially described by LeBlanc and Booth in 1993, laparo-scopic repair is now an accepted modality for treatment of inci-sional hernias. Several studies have found that laparoscopic

1	large pore mesh.Initially described by LeBlanc and Booth in 1993, laparo-scopic repair is now an accepted modality for treatment of inci-sional hernias. Several studies have found that laparoscopic repair has a lower incidence of surgical site and mesh infections compared to open repair. It also seems that lapa-roscopic repair allows faster recovery with less postoperative pain. A meta-analysis of 11 studies comparing laparoscopic and open ventral hernia repair found no difference in hernia recur-rence and lower rates of wound infection and wound drainage. There was however, a higher risk of bowel injury in the laparo-scopic group. Another meta-analysis of six randomized con-trolled trials had similar findings. Follow-up in these studies is relatively short, and more long-term data is needed at this time to compare these two modalities of repair.Laparoscopic hernia repair is performed by initially plac-ing lateral ports for midline defects and contralaterally placed ports for lateral

1	at this time to compare these two modalities of repair.Laparoscopic hernia repair is performed by initially plac-ing lateral ports for midline defects and contralaterally placed ports for lateral defects. Adhesions between the abdominal wall and intestine are carefully taken down, and the hernia contents are completely reduced. The sac is normally left in situ. Once the fascial defect is defined, a mesh is properly shaped and fash-ioned over the hernia. Transfascial sutures are placed circum-ferentially to position the mesh with sufficient overlap (4–5 cm) with healthy abdominal wall. Spiral tacks may be placed accord-ing to surgeon preference. Even more recently, robotic surgery has been established as another surgical modality in the treat-ment of ventral hernias. The theoretical advantage of improved visualization and articulating instruments may improve out-comes, but the cost-effectiveness of robotic repair is unclear. Overall, more studies are needed to evaluate the role of

1	of improved visualization and articulating instruments may improve out-comes, but the cost-effectiveness of robotic repair is unclear. Overall, more studies are needed to evaluate the role of robotics in ventral hernia surgery.Although still rare, given the increase in laparoscopic procedures, the incidence of laparoscopic port site hernias is becoming more common. Given the size of the hernias, there is a substantial risk of bowel strangulation and ischemia. These hernias commonly present as Richter’s hernia, or a hernia con-taining only a portion of bowel wall. A recent meta-analysis found that the incidence of port site hernia after laparoscopic procedure was less than 1%. Patients can present either early or several years after surgery. Risk factors are similar to other incisional hernias. The most common site of herniation is at an umbilical incision, but it may be found elsewhere. In adults, hernias usually occur in ports that are greater than 5 mm in size, but they can occur in

1	The most common site of herniation is at an umbilical incision, but it may be found elsewhere. In adults, hernias usually occur in ports that are greater than 5 mm in size, but they can occur in any size ports in children. Depending on the timing of presentation, these are usually repaired via an open approach by increasing the size of the skin incision, reducing the hernia, and approximating all layers of fascia.Rectus Abdominis Diastasis. Rectus abdominis diastasis (diastasis recti) is an anatomic term referring to an abnormal separation of rectus muscles and a laxity at the linea alba. Although there is controversy regarding normal separation dis-tances between rectus muscles, a distance of two centimeters is usually considered abnormal in the midline abdomen above the umbilicus. This can either be a congenital or acquired abnor-mality. This is not a true hernia as the midline fascia is intact, and as such incarceration and strangulation do not occur. Risk factors for acquired

1	can either be a congenital or acquired abnor-mality. This is not a true hernia as the midline fascia is intact, and as such incarceration and strangulation do not occur. Risk factors for acquired rectus abdominis diastasis include condi-tions that elevate intraabdominal pressure, including obesity and pregnancy, as well as conditions which weaken the abdominal wall, including connective tissue disorders or prior abdominal surgery. Risk factors of developing a diastasis recti after preg-nancy include older age at the time of pregnancy, multiple preg-nancies, and recurrent ceasarean sections. Postpartum exercise reduces the risk of developing diasthesis recti. Most patients with diastasis recti can be diagnosed based on physical exam where a fusiform bulge is usually apparent. This bulge worsens with contraction of the rectus muscles or Valsalva maneuver. If imaging is needed, ultrasonography can be used to confirm dia-thesis and rule out hernia. CT scan can also be used to confirm

1	worsens with contraction of the rectus muscles or Valsalva maneuver. If imaging is needed, ultrasonography can be used to confirm dia-thesis and rule out hernia. CT scan can also be used to confirm diagnosis and measure distance between muscle pillars. Rectus diastasis does not require surgical repair and may be improved via weight loss and exercise. Indications for repair include dis-ability of abdominal wall muscular function or cosmesis. Sur-gical repair includes both open and laparoscopic plication of the rectus sheath. Mesh can also be used to bridge the muscle; however, complication rates increase with mesh usage. These procedures do, unfortunately, have a high risk of recurrence long term and introduce a new risk of incisional hernia.Rectus Sheath Hematoma. Disruption of one of the branches of the inferior epigastric artery as well as an inability to tampon-ade the hemorrhage results in a rectus sheath hematoma. This occurs commonly around the arcuate line where the artery and

1	branches of the inferior epigastric artery as well as an inability to tampon-ade the hemorrhage results in a rectus sheath hematoma. This occurs commonly around the arcuate line where the artery and its branches are relatively fixed causing vulnerability to shearing forces. Several risk factors are associated with rectus hematoma formation via either proclivity to disruption of blood vessels or by inability to cease bleeding. Trauma to the abdominal wall, including iatrogenic trauma with laparoscopic trocar placement, can lead to disruption of blood vessels. Vigorous contraction of the rectus muscle, either with coughing, sneezing, or exercise, can also induce hemorrhage formation. Chronic pulmonary dis-ease can lead to hemorrhage because of coughing fits. Patients on anticoagulation also present with higher risk of hematoma formation. This condition presents with acute abdominal pain and a palpable abdominal wall mass. Rectus sheath hematoma may be mistaken with intraperitoneal

1	also present with higher risk of hematoma formation. This condition presents with acute abdominal pain and a palpable abdominal wall mass. Rectus sheath hematoma may be mistaken with intraperitoneal pathology, including appendicitis if on the right side. However, in patients with rec-tus sheath hematomas, pain usually increases with contraction of the rectus muscles as opposed to intraperitoneal conditions. In addition, palpation of a mass that does not change during contraction of the rectus muscle, known as Fothergill’s sign, is also associated with rectus sheath pathology. The diagnosis should be confirmed via ultrasound or CT scan with intravenous contrast. Obtaining a type and screen, hemoglobin/hematocrit, and coagulation factors are critical in the management of these patients.Treatment of patients with rectus sheath hematoma depends on the hemodynamic stability of the patient as well as the size of the hematoma. Hemodynamically stable patients with small hematomas, stable

1	of patients with rectus sheath hematoma depends on the hemodynamic stability of the patient as well as the size of the hematoma. Hemodynamically stable patients with small hematomas, stable serial hemoglobin/hematocrits, and normal coagulation factors may be observed without hospital-ization. Hemodynamically stable patients with larger or bilateral hematomas and decreases in hemoglobin should be monitored in the hospital setting, with serial hemoglobin levels, compres-sion of the hematoma, and bedrest. If anticoagulated, reversal is necessary and transfusions of packed red blood cells may 4Brunicardi_Ch35_p1549-p1566.indd 155612/02/19 9:58 AM 1557ABDOMINAL WALL, OMENTUM, MESENTERY, AND RETROPERITONEUMCHAPTER 35be required in some situations. Patients in hypovolemic shock should be aggressively resuscitated with the use of blood prod-ucts and treated via angiographic embolization. Angiographic intervention may also be required if the hematoma increases in size or if clinical

1	be aggressively resuscitated with the use of blood prod-ucts and treated via angiographic embolization. Angiographic intervention may also be required if the hematoma increases in size or if clinical deterioration occurs. Surgical therapy can be performed if angiographic intervention has failed. Surgical treatment includes operative evacuation of hematoma and liga-tion of bleeding vessels.Desmoid Tumors. Also known as aggressive fibromatosis, desmoid tumors are fibroblastic neoplasms with aggressive infiltrative behavior but no metastatic potential. These tumors can occur anywhere in the body but commonly occur in the abdomen or abdominal wall. Desmoid tumors are rare and usu-ally occur sporadically. They are, however, also associated with familial adenomatous polyposis (FAP), with an even greater risk in patients with Gardner’s syndrome. Of patients with FAP, 10% to 15% develop desmoid tumors. After prophylactic col-ectomy, desmoid tumors becomes the primary cause of death in

1	an even greater risk in patients with Gardner’s syndrome. Of patients with FAP, 10% to 15% develop desmoid tumors. After prophylactic col-ectomy, desmoid tumors becomes the primary cause of death in patients with FAP. Risk factors for sporadic development of desmoid tumors include previous surgical incision, pregnancy, hormonal exposure, and trauma. Females have a higher pre-dilection for formation of desmoid tumors. Diagnosis can be performed via core-needle or incisional biopsy. Larger tumor size, young patient age, and extra-abdominal tumor location all predict poor recurrence free survival.The gold standard of treatment of abdominal wall desmoid tumors, historically, is margin-negative resection with immedi-ate mesh reconstruction. However, more recently there has been controversy as to whether complete microscopic resection is necessary. It is now commonly agreed that while complete microscopic resection is ideal, with the emergence of adjuvant therapy a positive margin may not

1	to whether complete microscopic resection is necessary. It is now commonly agreed that while complete microscopic resection is ideal, with the emergence of adjuvant therapy a positive margin may not require additional surgery, especially if re-resection would cause high morbidity. There is also some evidence to suggest a period of close watchful wait-ing, as some tumors appear to remain stable or even regress over time. In one cohort of 106 abdominal wall desmoids managed initially without surgery, 16% of patients went on to require surgery in a follow-up period of three years. Interestingly, 29 patients had spontaneous regression of their tumors over that time. The National Comprehensive Cancer Network (NCCN) now suggests initial close observation for patients with asymptomatic, non–life-threatening tumors. Surgery is indicated in patients with symptomatic disease, risk of invasion of surrounding structures, or enlarging tumors. There may be a role for adjuvant or neoadjuvant

1	tumors. Surgery is indicated in patients with symptomatic disease, risk of invasion of surrounding structures, or enlarging tumors. There may be a role for adjuvant or neoadjuvant radiation therapy, although the data are unclear at this time. Primary radiation therapy may be an option for patients who are not surgical candidates. There may also be a role for systemic therapy, especially if tumors are unre-sectable. Options include hormonal therapy, nonsteroidal anti-inflammatory agents, cytotoxic chemotherapies (doxorubicin or carboplatin), or imatinib.Other Abdominal Wall Tumors. Various tumors may also be found within the abdominal wall including lipomas and neu-rofibromas (Fig. 35-7). Surgical resection is recommended for symptomatic or enlarging lesions. Abdominal wall malignancies are exceedingly rare and include several histologic subtypes of sarcomas, dermatofibrosarcoma protuberans, schwannomas, and melanomas. Workup of abdominal wall tumors should include core needle biopsy

1	exceedingly rare and include several histologic subtypes of sarcomas, dermatofibrosarcoma protuberans, schwannomas, and melanomas. Workup of abdominal wall tumors should include core needle biopsy or excisional biopsy if the tumor is small enough. Magnetic resonance imaging (MRI) or CT scan with IV contrast should be utilized to define local extent of disease. MRI is preferred as this modality provides more detail on extent of disease. Chest CT should also be obtained to rule out pulmo-nary metastasis in high-grade tumors. Chest X-ray may be suf-ficient to stage tumors that are low grade, given the lower risk of distant disease. Surgical resection is the mainstay of treatment for nonmetastatic disease. For most soft tissue sarcomas, 1 cm margins are usually sufficient, but 2 cm margins may be needed for dermatofibrosarcoma protuberans. Tumors of the superficial abdominal wall should be resected with the underlying fascia, which may require use of mesh to prevent abdominal wall laxity

1	be needed for dermatofibrosarcoma protuberans. Tumors of the superficial abdominal wall should be resected with the underlying fascia, which may require use of mesh to prevent abdominal wall laxity or hernia.Given the rarity of these tumors, the efficacy of adjuvant or neoadjuvant chemoradiation is unclear. Adjuvant or neo-adjuvant therapy may be recommended in patients with large (>5 cm) or high-grade tumors. Some centers administer neoad-juvant radiation therapy to patients with high-risk tumors to monitor response. Chemotherapy may also be used in certain situations. If tumors involve underlying viscera, en bloc resec-tion may be required. Primary closure may be feasible, but pros-thetic mesh use (even in the setting of bowel resection) may be required. Options for abdominal wall closure after resection include absorbable or biologic mesh reinforcement, and myocu-taneous flap reconstruction.OMENTUMSurgical AnatomyThe omentum is a fibrous adipose apron providing support and

1	closure after resection include absorbable or biologic mesh reinforcement, and myocu-taneous flap reconstruction.OMENTUMSurgical AnatomyThe omentum is a fibrous adipose apron providing support and protection of the intraabdominal viscera. Embryologically, the omentum originated from the dorsal mesogastrium. Anatomi-cally, the omentum is divided into the greater and lesser omen-tums. The greater omentum begins to form during the fourth week of gestation. Initially, the omentum forms as a double-layered structure, with the spleen developing between the two layers. As development proceeds, the layers of mesentery fuse, the spleen assumes its position in the peritoneum, and the gas-trosplenic ligament forms. The greater omentum, therefore, is a double-layered sheet of visceral fibroadipose tissue descending from the greater curvature of the stomach, covering the small intestines. The omentum folds back on itself and attaches onto the anterior peritoneum of the transverse colon (Fig.

1	tissue descending from the greater curvature of the stomach, covering the small intestines. The omentum folds back on itself and attaches onto the anterior peritoneum of the transverse colon (Fig. 35-8). 5Figure 35-7. Abdominal wall lipoma.Brunicardi_Ch35_p1549-p1566.indd 155712/02/19 9:59 AM 1558SPECIFIC CONSIDERATIONSPART IIIn an adult, the greater omentum lies between the abdominal wall and the hollow viscera, usually extending into the pelvis. The greater omental tissue connecting the stomach to transverse colon, as well as the stomach to the spleen, are known as the gastrocolic and gastrosplenic ligaments, respectively. The blood supply to the greater omentum is derived from the right and left gastroepiploic arteries. The venous system parallels the arterial supply and ultimately drains into the portal system. Lymphatic drainage of the greater omentum occurs via the subpyloric or splenic nodes depending on laterality ultimately culminating in the celiac nodes and subsequently

1	drains into the portal system. Lymphatic drainage of the greater omentum occurs via the subpyloric or splenic nodes depending on laterality ultimately culminating in the celiac nodes and subsequently the thoracic duct.Forming the anterior boundary of the lesser sac, the lesser omentum extends between the liver and lesser curvature of the stomach. Also known as the hepatoduodenal and hepatogastric ligaments, the portal triad (including the common bile duct, por-tal vein, and hepatic artery) is located within the inferolateral margin of the lesser omentum. This free edge of lesser omentum forms the foramen of Winslow, which is used to encircle the portal triad during a pringle maneuver (see Fig. 35-8).PhysiologyInitially described by the British surgeon Rutherford Morison as the “policeman of the abdomen,” the greater omentum is under-stood as a peritoneal defense organ. The omentum forms fibrin adhesions at sites of inflammation, effectively attempting to wall off peritoneal

1	of the abdomen,” the greater omentum is under-stood as a peritoneal defense organ. The omentum forms fibrin adhesions at sites of inflammation, effectively attempting to wall off peritoneal infections. The greater omentum is known to respond to a foreign stimulus by expanding stro-mal tissue that expresses chemotactic, inflammatory, and hemo-static factors, which promote tissue inflammation and subsequent repair. Forming a fibrin bridge between omental tis-sue and the injured site allows passage of inflammatory factors and immune cells. The Graham Patch repair of a perforated ulcer utilizes the complex inflammatory interactions of the omentum to improve leak rates after repair.The visceral fat of the greater omentum is also involved in metabolic functions of the body. Increased visceral fat, including the greater omentum, is an independent risk factor for insulin resistance and high triglyceride levels. This may be, in part, mediated by the increase in visceral inflammation seen in

1	fat, including the greater omentum, is an independent risk factor for insulin resistance and high triglyceride levels. This may be, in part, mediated by the increase in visceral inflammation seen in intra-abdominal obesity. Several adipokines, or cell-signaling molecules secreted by adipose tissue, are secreted by the greater omentum and act on peripheral tissues, affecting metabolic health throughout the body.Acquired DisordersOmental Infarction. Omental infarction is a rare cause of acute abdominal pain. Omental infarction may be primary or 6DiaphragmGastrohepaticligamentRight colic (hepatic)flexureAscending colonGreater omentum(gastrocolic ligament)Falciform ligamentRound ligamentof liverHepatoduodenalligament (containingportal triad)Arrow passingthrough omentalforamen intoomental bursaGastrophrenicligamentCoronaryligament (cut)StomachSpleenDescending colonPhrenicocolicligamentInferior recess ofomental bursa(between layers ofgreater omentum)GastrocolicligamentTransverse

1	bursaGastrophrenicligamentCoronaryligament (cut)StomachSpleenDescending colonPhrenicocolicligamentInferior recess ofomental bursa(between layers ofgreater omentum)GastrocolicligamentTransverse colon(sectioned)GastrosplenicligamentLeft colic (splenic)flexureTransversemesocolonLiver: Diaphragmatic surface Visceral surfaceAnterior view** Parts of greater omentum* Parts of lesser omentum********Figure 35-8. Greater and lesser omentum. The greater omentum begins along the greater curvature of the stomach, drapes over the transverse colon into the pelvis, and folds back on itself inserting along the posterior wall of the transverse colon. The greater omentum includes the gastrophrenic and gastro splenic ligament. The lesser omentum includes the gastrohepatic and hepatoduodenal ligaments, covering the lesser sac of the abdomen. The free lateral edge of the lesser omentum includes the portal triad, forming the foramen of Winslow below. (Reproduced with permission from Moore KL, Agur AM:

1	the lesser sac of the abdomen. The free lateral edge of the lesser omentum includes the portal triad, forming the foramen of Winslow below. (Reproduced with permission from Moore KL, Agur AM: Essential Clinical Anatomy, 5th edition. Philadelphia, PA: Lippincott Williams & Wilkins; 2014.)Brunicardi_Ch35_p1549-p1566.indd 155812/02/19 9:59 AM 1559ABDOMINAL WALL, OMENTUM, MESENTERY, AND RETROPERITONEUMCHAPTER 35secondary depending on the etiology. Primary infarction may be caused by torsion of the omentum from sudden or force-ful movements, thrombosis or vasculitis of omental vessels, or omental venous outflow obstruction. Secondary causes of torsion are most often due to hernias, tumors, or adhesions. Only about 250 cases of primary omental infarction have been reported in the literature. This occurs most often in male and obese patients. Omental infarction may mimic other intra-abdominal pathologies such as appendicitis, cholecystitis, or diverticulitis. Abdominal exam usually

1	This occurs most often in male and obese patients. Omental infarction may mimic other intra-abdominal pathologies such as appendicitis, cholecystitis, or diverticulitis. Abdominal exam usually demonstrates perito-neal tenderness, with a possible palpable mass. Ultrasonogra-phy (US) and abdominal CT scan are used to assist diagnosis. US may demonstrate a hyperechoic, noncompressible intra-abdominal mass attached to the abdominal wall. CT scan demonstrates a streaking whirling pattern of fatty tissue in the anterior abdomen (Fig. 35-9). Regardless, only a small percent-age of patients are preoperatively diagnosed. Omental infarction can be treated conservatively, but this leads to several possible complications, including abscess or adhesion formation. Lapa-roscopic exploration and resection of infarcted tissue is the usual treatment of choice and leads to rapid resolution of symptoms.Omental Cysts. Cystic lesions of the omentum are rare dis-orders, related in pathogenesis to mesenteric

1	of infarcted tissue is the usual treatment of choice and leads to rapid resolution of symptoms.Omental Cysts. Cystic lesions of the omentum are rare dis-orders, related in pathogenesis to mesenteric cysts. Most are thought to form through degeneration or inclusion of lymphatic structures. Case reports described these lesions presenting as vague abdominal pain or nausea or vomiting. Cysts may also present as a lead point for omental torsion and infarction. Physi-cal exam may or may not reveal evidence of an intra-abdominal mass. Cysts may also be diagnosed incidentally on imaging exams performed for other reasons. CT scan and US are largely diagnostic and reveal a well-circumscribed cystic appearing lesion arising from the greater omentum. A very small percent-age may transform to malignancy. Most are resected, especially if symptomatic, via open or laparoscopic approaches. Cysts may relapse if treated conservatively via laparoscopic unroofing or percutaneous drainage.Omental

1	malignancy. Most are resected, especially if symptomatic, via open or laparoscopic approaches. Cysts may relapse if treated conservatively via laparoscopic unroofing or percutaneous drainage.Omental Neoplasms. Most omental neoplasms are meta-static disease. Ovarian cancer is the most common cancer to have omental involvement. Other metastatic cancers include gastrointestinal tract tumors, melanoma, endometrial cancer, and kidney cancer.Primary tumors of the omentum are exceedingly rare. Benign masses may include lipomas, myxomas, and desmoid tumors. Extra gastrointestinal stromal tumors are a rare malig-nant tumor of the omentum that have been described in several case series. These tumors share many genetic and immuno-phenotypic similarities to classical GISTS, including c-kit and PDGFRA mutations. In one review of case reports, the median age of diagnosis was noted to be 65 years and had equal predi-lection for male and female patients.MESENTERYSurgical AnatomyThe mesentery is a

1	mutations. In one review of case reports, the median age of diagnosis was noted to be 65 years and had equal predi-lection for male and female patients.MESENTERYSurgical AnatomyThe mesentery is a contiguous structure suspending and fix-ing bowel to the abdominal wall providing housing for arterial, venous, nervous, and lymphatic structures connecting hollow viscera with the body. The mesentery is derived from the meso-dermal germ layer becoming the dorsal mesentery. Previous theories of development included sliding and regression models of mesenteric development. However, with a new contiguous model of development of the mesentery, several key simple steps are proposed to take place. Initially, the mesentery provides a point of suspension from vascular connections. As the intestine and mesentery elongate, they undergo a 270° counterclockwise rotation leaving the primordial abdominal cavity. Eventually, the duodenum and ascending/descending colon flatten against the posterior abdominal

1	elongate, they undergo a 270° counterclockwise rotation leaving the primordial abdominal cavity. Eventually, the duodenum and ascending/descending colon flatten against the posterior abdominal wall returning the bowel back into the abdomen and taking its normal shape. The resultant develop-ment of the white line of Toldt along the lateral border of the ascending and descending large bowel provides an avascular fascial plan between the colon, its mesentery, and the underlying retroperitoneal space. The small intestine mesentery, transverse colon mesentery, and sigmoid colon mesentery remain mobile.Defects in the proper rotation and fixation of the bowel results in the spectrum of congenital disorders known as intesti-nal malrotation. In this scenario, the intestine and mesentery are simply suspended from vascular connection, making intestinal volvulus common. Defects that form in anatomical positions may act as routes for intestinal herniation. Common sites of her-niation include the

1	suspended from vascular connection, making intestinal volvulus common. Defects that form in anatomical positions may act as routes for intestinal herniation. Common sites of her-niation include the paraduodenal or mesocolic areas, leading to either acute or chronic intestinal obstruction in pediatric or adult populations. Areas where mesenteric attachment is incomplete may predispose patients to volvulus. Attachments of the cecum may degrade overtime leading to an area of laxity and possible twisting. The sigmoid mesentery can also increase in size over time also leading to possible sigmoid volvulus. Rarely, other areas of the colon can develop volvulus if differential mesen-teric lengths form.Sclerosing MesenteritisSclerosing mesenteritis is a rare disorder characterized by idio-pathic fibrosis of the mesentery, affecting hollow viscera as well as mesenteric vessels. The disease is part of a spectrum of inflammation and fibrosis, which when localized is known as mesenteric

1	fibrosis of the mesentery, affecting hollow viscera as well as mesenteric vessels. The disease is part of a spectrum of inflammation and fibrosis, which when localized is known as mesenteric lipodystrophy and when diffuse is known as mes-enteric panniculitis. The cause of this disease is unknown, but it may be instigated by antecedent abdominal surgery, an auto-immune disease, a paraneoplastic syndrome, a previous infec-tion (including typhoid, tuberculosis, influenza, and rheumatic fever), or vascular insult. This disease most commonly effects Figure 35-9. Computed tomography scan findings indicative of omental infarction. The area demonstrated by the bold arrow appears congested with a streaking whirling pattern of fatty tissue in the anterior abdomen. (Reproduced with permission from Barai KP, Knight BC: Diagnosis and management of idiopathic omental infarc-tion: A case report, Int J Surg Case Rep. 2011;2(6):138-140.)Brunicardi_Ch35_p1549-p1566.indd 155912/02/19 9:59 AM

1	from Barai KP, Knight BC: Diagnosis and management of idiopathic omental infarc-tion: A case report, Int J Surg Case Rep. 2011;2(6):138-140.)Brunicardi_Ch35_p1549-p1566.indd 155912/02/19 9:59 AM 1560SPECIFIC CONSIDERATIONSPART IIwhite patients between 50 and 70 years of age, although it has been rarely described in children. Most studies report a predi-lection for male patients. Most patients with this disease pres-ent with abdominal pain. Other symptoms include nausea and vomiting, weight loss, anorexia, and altered bowel habits. This may appear as a chronic or acute disorder. On physical exam, patients may be found to have tenderness and distension. Up to 50% of patients are found to have an abdominal mass that often transmits aortic pulsations.Abdominal CT with IV contrast is used to assist in diag-nosis (Fig. 35-10). The most common finding is that of a soft tissue mass with a higher density than normal mesenteric tis-sue. Although it is sometimes difficult to distinguish

1	to assist in diag-nosis (Fig. 35-10). The most common finding is that of a soft tissue mass with a higher density than normal mesenteric tis-sue. Although it is sometimes difficult to distinguish mesen-teric fibrosis from a mesenteric tumor, two CT findings may add specificity. The “tumor pseudocapsule” refers to a hypodense zone around the associated fibrotic mass, and the “fat ring sign” refers to an area of preserved fat near mesenteric vessels cours-ing through areas of fibrosis. These lesions may also be calcified on CT scan.Pathologic confirmation is required to confirm the diagno-sis. This usually requires laparoscopic or open biopsy to provide adequate tissue for confirmation. Treatment of sclerosing mesenteritis is complex, with surgery having a mini-mal role. Patients who present with bowel ischemia may require bowel and mesenteric resection; however, the extent and loca-tion of mesenteric involvement may preclude complete resec-tion. If obstructive symptoms are dominant,

1	with bowel ischemia may require bowel and mesenteric resection; however, the extent and loca-tion of mesenteric involvement may preclude complete resec-tion. If obstructive symptoms are dominant, intestinal bypass may be indicated. Aggressive surgical treatment is not indicated because in many cases symptoms may improve with medical treatment or even without intervention. Steroids, hormonal ther-apy, colchicine, thalidomide, and cyclophosphamide have all been reported to be beneficial.Mesenteric CystsMesenteric cysts are a rare benign disorder with an incidence ranging from 1 in 27,000 to 1 in 250,000 admissions. Cysts are thought to be caused by disruption of the lymphatics in the mes-entery either by traumatic disruption, mechanical obstruction, or congenital lymphatic malformations. Most cysts are unilocular, but they also may have multiple loculations. They are usually lined with a single layer of columnar epithelial cells. Presenta-tion of mesenteric cyst is varied, with some

1	Most cysts are unilocular, but they also may have multiple loculations. They are usually lined with a single layer of columnar epithelial cells. Presenta-tion of mesenteric cyst is varied, with some being found inciden-tally on imaging exams and others causing acute abdominal pain because of cyst rupture or bowel torsion. Chronic symptoms are usually nonspecific, including abdominal pain or discomfort, anorexia, distension, nausea, vomiting, or changes in bowel habits. Symptoms are due to local compression of abdominal structures. Up to 45% of cysts are found incidentally.Physical exam revels an abdominal mass in up to 60% of patients. The classic Tillaux’s sign is an abdominal mass lesion that is only mobile laterally, contrasting omental cysts which are usually freely mobile in all directions. CT scan and ultra-sound can be used to make an accurate diagnosis. Cystic lesions usually appear as a fluid filled mass without solid components (Fig. 35-11). It can sometimes be difficult to

1	CT scan and ultra-sound can be used to make an accurate diagnosis. Cystic lesions usually appear as a fluid filled mass without solid components (Fig. 35-11). It can sometimes be difficult to differentiate cystic masses from solid tumors based on imaging. Mesenteric cys-tic lymphangioma may present as numerous cysts on imaging. Up to 3% of mesenteric cysts contain malignancy, mostly as a sarcomatous lesion. In one recently published series, 19% of patients harbored malignancy. Solid components within the cystic structure are associated with higher rates of malignancy.Most mesenteric cysts are treated surgically. Marsupial-ization and simple aspiration have high rates of recurrence and 7Figure 35-11. Computed tomography scan demonstrating a mes-enteric cyst. White arrow points to a homogenous cystic structure located in the mesentery of the small bowel. (Reproduced with per-mission from Jain V, DeMuro JP, Geller M, et al. A case of laparo-scopic mesenteric cyst excision. Case Rep Surg.

1	cystic structure located in the mesentery of the small bowel. (Reproduced with per-mission from Jain V, DeMuro JP, Geller M, et al. A case of laparo-scopic mesenteric cyst excision. Case Rep Surg. 2012;2012:594095.)ABFigure 35-10. Computed tomography scan findings of sclerosing mesenteritis in (A) coronal view and (B) cross-sectional view. The area demonstrated with arrows depicts an area of higher density and edema representing inflamed mesentery. (Reproduced with per-mission from Daumas A, Agostini S, Villeret J, et al. Spontaneous resolution of severe, symptomatic mesocolic panniculitis: a case report, BMC Gastroenterol. 2012 Jun 6;12:59.)Brunicardi_Ch35_p1549-p1566.indd 156012/02/19 9:59 AM 1561ABDOMINAL WALL, OMENTUM, MESENTERY, AND RETROPERITONEUMCHAPTER 35are generally discouraged. Benign lesions should be enuclu-ated, and malignant cysts should be resected with clear margins. Intestines or surrounding viscera may require resection if the associated vasculature is excised.

1	Benign lesions should be enuclu-ated, and malignant cysts should be resected with clear margins. Intestines or surrounding viscera may require resection if the associated vasculature is excised. Resection can be performed either via a laparoscopic or open procedure.Mesenteric TumorsPrimary mesenteric tumors are rare but represent several histo-logic patterns. Benign tumors include desmoid tumors, lipomas, and cystic lymphangiomas. The most common malignant neo-plasm of the mesentery is lymphoma. On imaging, lymphoma may appear as bulky adenopathy, usually surrounding and not obstructing nearby structures. Lymphomas should not be resected, but they may require operative biopsy for diagnosis. Other malignant tumors of the mesentery include gastrointesti-nal stromal tumors, carcinoids, liposarcoma, leiomyosarcoma, malignant fibrous histiocytomoas, lipoblastomas, or lymphan-giosarcoma. Treatment of malignant mesenteric masses usually involves wide resection; however, given the proximity

1	leiomyosarcoma, malignant fibrous histiocytomoas, lipoblastomas, or lymphan-giosarcoma. Treatment of malignant mesenteric masses usually involves wide resection; however, given the proximity to mes-enteric vessels, resection may not be feasible or require removal of large portions of bowel.RETROPERITONEUMSurgical AnatomyThe retroperitoneum is bound by the peritoneum anteriorly, the iliopsoas and lumbar muscles posteriorly, the diaphragm superiorly, and the levator ani muscles inferiorly. It is divided into the three spaces: the anterior pararenal space, the perirenal space, and the posterior pararenal space (Fig. 35-12). The ante-rior pararenal space refers to the area anterior to the renal fascia but posterior to the peritoneum. This area contains the ascend-ing and descending colon, the duodenum, and the pancreas. Posterior to this space is the perirenal space, which houses the inferior vena cava, the aorta, kidneys, and adrenal glands. The posterior pararenal space is in continuity

1	duodenum, and the pancreas. Posterior to this space is the perirenal space, which houses the inferior vena cava, the aorta, kidneys, and adrenal glands. The posterior pararenal space is in continuity with preperitoneal fat of the anterior abdomen. Given the compliance of the anterior boundary of the retroperitoneum and the rigidity of other mar-gins, tumors, hematomas, and abscesses tend to expand anteri-orly toward the peritoneal cavity.With the expansion of minimally invasive techniques in surgery, the retroperitoneoscopic approach has emerged as a potential modality for access to retroperitoneal organs. Patients are positioned in the prone or lateral decubitus positions. The retroperitoneoscopic approach allows access to the kidneys, adrenal glands, and retroperitoneal lymph nodes. One system-atic review found no difference when comparing laparoscopic to retroperitoneoscopic adrenalectomy in terms of operative outcomes, complications, or postoperative recovery.

1	lymph nodes. One system-atic review found no difference when comparing laparoscopic to retroperitoneoscopic adrenalectomy in terms of operative outcomes, complications, or postoperative recovery. Retroperi-toneoscopic approach did, however, lead to shorter hospital stay likely because of reduced postoperative pain. The majority of the studies evaluating the retroperitoneoscopic approach are retrospective, and more randomized trials are needed to provide further guidance.Retroperitoneal InfectionsInfections of the retroperitoneum can be due to primary hema-togenous spread of microbes or due to secondary infection from retroperitoneal or nearby organs. Examples include abscesses due to a perforated retrocecal appendix, diverticulitis, a con-tained perforated duodenal ulcer, iatrogenic perforation of the gastrointestinal tract, or pancreatitis. Patients may develop back, Inferiorvena cavaDiaphragmEsophagusAbdominal aortaSplenorenal ligamentPancreasRoot of

1	duodenal ulcer, iatrogenic perforation of the gastrointestinal tract, or pancreatitis. Patients may develop back, Inferiorvena cavaDiaphragmEsophagusAbdominal aortaSplenorenal ligamentPancreasRoot of transversemesocolonDuodenojejunaljunctionLeft kidneyPsoas majorInferior mesenteric arterySite of descending mesocolonRoot of sigmoidmesocolonRectumSite of barearea of liverHepatic portalveinDuodenumSuperiormesenteric vein and arterySite of ascending colonRoot of mesenteryTesticular (or ovarian) vesselsUreterAnterior viewRight kidneyRightparacolic gutterFigure 35-12. Anatomy of the retroperitoneum. (Reproduced with permission from Moore KL, Agur AM: Essential Clinical Anatomy, 5th edition. Philadelphia, PA: Lippincott Williams & Wilkins; 2014.)Brunicardi_Ch35_p1549-p1566.indd 156112/02/19 9:59 AM 1562SPECIFIC CONSIDERATIONSPART IIflank, or groin pain and suffer from fevers or chills. Depend-ing on severity, patients may present with fulminant sepsis. Abscesses may become quite large

1	9:59 AM 1562SPECIFIC CONSIDERATIONSPART IIflank, or groin pain and suffer from fevers or chills. Depend-ing on severity, patients may present with fulminant sepsis. Abscesses may become quite large given the substantial retro-peritoneal space. On physical exam, patients may present with erythema of the umbilicus or flank. Abscesses are usually found easily on CT scan of the abdomen with IV contrast, which can also show if the collection is loculated. Treatment of retroperi-toneal abscesses includes source control via treatment of the underlying condition, drainage of well-defined collections, and IV antibiotics. Image guided drainage is preferred, but it may be unsuccessful if the abscess is multiloculated or in an inacces-sible area. For these cases, operative drainage may be required. Given the insidious nature of this disease and a lack of abdomi-nal findings on physical exam, recognition of a retroperitoneal abscess may be delayed. Delays in diagnosis and insufficient drainage

1	Given the insidious nature of this disease and a lack of abdomi-nal findings on physical exam, recognition of a retroperitoneal abscess may be delayed. Delays in diagnosis and insufficient drainage may lead to high morbidity and mortality. Depending on severity, mortality of retroperitoneal abscess can be as high as 25%. Rarely, patients may develop necrotizing fasciitis of the retroperitoneum, a condition with high mortality.Retroperitoneal FibrosisRetroperitoneal fibrosis is a rare disease characterized by inflammation and fibrosis of the tissue of the retroperitoneum. It exists as a spectrum of disease with chronic periaortitis, which affects the retroperitoneal tissue near large arteries of the retro-peritoneum. Fibrosis gradually expands, encasing the ureters, inferior vena cava, aorta, mesenteric vessels, or sympathetic nerves. Bilateral involvement is noted in up to 70% of cases. The condition may either be idiopathic or due to a secondary cause, including aortic aneurysms,

1	aorta, mesenteric vessels, or sympathetic nerves. Bilateral involvement is noted in up to 70% of cases. The condition may either be idiopathic or due to a secondary cause, including aortic aneurysms, pancreatitis, certain drugs (Ergot-derivatives, β-blockers, hydralazine, methyldopa, among others), malignancies (including lymphoma, carcinoids, sarco-mas, colorectal, breast, and others), infections such as tuber-culosis, radiation, retroperitoneal hematoma, surgery, asbestos, or tobacco use. Retroperitoneal fibrosis has been described in association with several autoimmune disorders including anky-losing spondylitis, systemic lupus erythematosus, Wegener’s granulomatosis, and polyarteritis nodosa.Idiopathic retroperitoneal fibrosis accounts for 70% of cases and is thought to be an immune-mediated disorder. Theo-ries regarding the pathogenesis of idiopathic fibrosis of the retro-peritoneum include exaggerated local reactions to aortic or iliac atherosclerosis or autoimmune deposition of

1	disorder. Theo-ries regarding the pathogenesis of idiopathic fibrosis of the retro-peritoneum include exaggerated local reactions to aortic or iliac atherosclerosis or autoimmune deposition of fibroinflammatory cytokines. Retroperitoneal fibrosis is also a manifestation of IgG4-related disease, a multisystem disease characterized by lymphocytic infiltrate and variable degrees of fibrosis of sev-eral affected organs. One study has associated retroperitoneal fibrosis with the HLA-DRB1*03 allelle, which has been linked to autoimmune diseases such as systemic lupus erythematosus, type 1 diabetes mellitus, and myasthenia gravis.Idiopathic retroperitoneal fibrosis is rare, with an incidence of 1.3 per 100,000 people per year. It most commonly affects individuals in the fourth to sixth decades of life. Some studies suggest a 2:1 male-to-female predominance, but others have not found a gender predilection. Patients with this condition present with nonspecific findings. Most patients are

1	of life. Some studies suggest a 2:1 male-to-female predominance, but others have not found a gender predilection. Patients with this condition present with nonspecific findings. Most patients are diagnosed after ure-teral obstruction. Patients may complain of dull or acute back or flank pain. Systemic complaints include anorexia, weight loss, nausea, vomiting, fever, and malaise. Decreased urinary out-put may ensue if the ureters become fibrotic. Because of renal artery impingement, patients are commonly hypertensive on exam. Other physical exam findings include lower extremity edema and diminished lower extremity pulses if the vessels of the lower extremities are compressed. New hydrocele or varico-cele can also be associated with fibrosis of the retroperitoneum. Laboratory analysis is also nonspecific. If there is renal obstruc-tion, patients may have elevated serum blood urea nitrogen and creatinine. Erythrocyte sedimentation rate and C-reactive pro-tein is elevated in most

1	is also nonspecific. If there is renal obstruc-tion, patients may have elevated serum blood urea nitrogen and creatinine. Erythrocyte sedimentation rate and C-reactive pro-tein is elevated in most patients with retroperitoneal fibrosis. Antinuclear antibodies may also be elevated, highlighting the autoimmune nature of this disease.Contrast-enhanced CT scan is the modality of choice to visualize the extent of disease. On CT scan, fibrotic ret-roperitoneum appears to have similar attenuation to muscle tissue (Fig. 35-13). The fibrotic mass can appear to encase the aorta and often compresses the inferior vena cava. If renal insufficiency precludes the use of IV contrast, MRI can be used to clarify the extent of fibrosis. Renal US can also be used to document ureteral compression and hydronephrosis. In some cases, a compressing mass lesion can also be found on abdominal US. Lower-extremity US may show deep venous thrombosis.Pathologic examination of tissue is necessary to confirm the

1	In some cases, a compressing mass lesion can also be found on abdominal US. Lower-extremity US may show deep venous thrombosis.Pathologic examination of tissue is necessary to confirm the diagnosis and to rule out malignancy. Differential of ret-roperitoneal masses include lymphomas or sarcomas, desmoid Figure 35-13. Computed tomography findings of retroperitoneal fibrosis. Arrows point to a soft tissue mass surrounding the aorta and common iliac arteries. Also present is hydronephrosis of the right kidney because of ureteral compression. (Reproduced with permission from Vagilo A. Salvarani C, Buzio C. Retroperitoneal Fibrosis, Lancet. 2006 Jan 21;367(9506):241-251.)Brunicardi_Ch35_p1549-p1566.indd 156212/02/19 9:59 AM 1563ABDOMINAL WALL, OMENTUM, MESENTERY, AND RETROPERITONEUMCHAPTER 35tumors, and infections such as tuberculosis. Biopsy can be obtained either via image-guided or surgical techniques, which may be performed laparoscopically or during an open procedure.Once the

1	35tumors, and infections such as tuberculosis. Biopsy can be obtained either via image-guided or surgical techniques, which may be performed laparoscopically or during an open procedure.Once the diagnosis of retroperitoneal fibrosis is estab-lished, treatment is initiated. Surgical treatment is aimed at relieving ureteral obstruction either by ureterolysis or stenting. Surgery is reserved for patients who develop renal insufficiency as any surgical intervention in these patients car-ries several associated risks. In patients with secondary fibro-sis, treatment is aimed at the underlying etiology or discontinuation of the causative medication. For patients with idiopathic retroperitoneal fibrosis, corticosteroids are the mainstay of treatment. Patients are usually started on one month of high-dose prednisone, followed by maintenance therapy and subsequent taper. Alternatively, tamoxifen can be used if patients have contraindications to steroids. Within days of instituting therapy,

1	of high-dose prednisone, followed by maintenance therapy and subsequent taper. Alternatively, tamoxifen can be used if patients have contraindications to steroids. Within days of instituting therapy, symptoms and inflammatory markers should improve. Over the course of a few weeks, renal func-tion should improve. Interval imaging studies are usually obtained after 1 month to evaluate for efficacy. If steroids fail to cause regression of disease, patients can be started on immunosuppressant medications including methotrexate, aza-thioprine, cyclophosphamide, or mycophenolate mofetil. Recurrence of retroperitoneal fibrosis varies between studies from 10% to 30%. In patients who respond to steroid therapy, 5-year survival is as high as 90%.BIBLIOGRAPHYEntries highlighted in bright blue are key references.Aguirre DA, Santosa AC, Casola G, Sirlin CB. Abdominal wall her-nias: imaging features, complications, and diagnostic pitfalls at multi-detector row CT. RadioGraphics.

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1	M, Petrakis G, et al. Rectus sheath hematoma in a single secondary care institution: a retrospective study. Hernia. 2015;19(3):509-512.Collins D, Hogan AM, O’Shea D, Winter DC. The omentum: ana-tomical, metabolic, and surgical aspects. J Gastrointest Surg. 2009;13:1138-1146. doi: 10.1007/s11605-009-0855-1Dedemadi G, Georgoulis G, Kontopanos D, et al. Extragastroin-testinal stromal tumors of the omentum: review apropos of a case with a novel gain-of-function KIT mutation. J Gastrointest Cancer. 2009;40:73-78. doi: 10.1007/s12029-009-9089-4Giovanni C, Riccardo V, Grazia EM, et al. Laparoscopic treatment of an omental cyst: a case report and review of the literature. Surg Laparosc Endosc Percutan Tech. 2005;15(1):33-35.Katagiri H, Honjo K, Nasu M, et al. Omental infarction due to omental torsion. Case Rep Surg. 2013;2013:373810. doi: 10.1155/2013/373810Kranendonk MEG, van Herwaarden JA, Stupkova T, et al. Inflam-matory characteristics of distinct abdominal adipose tissue depots relate

1	Case Rep Surg. 2013;2013:373810. doi: 10.1155/2013/373810Kranendonk MEG, van Herwaarden JA, Stupkova T, et al. Inflam-matory characteristics of distinct abdominal adipose tissue depots relate differently to metabolic risk factors for cardiovascular disease. Atherosclerosis. 2017;239:419-427. doi: 10.1016/j.atherosclerosis.2015.01.035Morison R. Remarks on some functions of the Omentum. Br Med J. 1906;1(2350):76-78.Motie MR, Asadi M. Large omental cyst: a case report and review of the literature. Acta Medica Iranica. 2011;49(10):690-693.Mouaqit O, Jahid A, Ifrine L, et al. Primary omental gastrointestinal stromal tumors. Clin Res Hepatol Gastroenterol. 2011;35: 590-593. doi: http://dx.doi.org/10.1016/j.clinre.2010.11.012Occhionorelli S, Zese M, Cappellari L, et al. Acute abdomen due to primary omental torsion and infarction. Case Rep Surg. 2014;2014:208382. doi: 10.1155/2014/208382Park TU, Oh JH, Chang IT, et al. Omental infarction: case series and review of the literature. J Emerg

1	primary omental torsion and infarction. Case Rep Surg. 2014;2014:208382. doi: 10.1155/2014/208382Park TU, Oh JH, Chang IT, et al. Omental infarction: case series and review of the literature. J Emerg Med. 2017;42:149-154. doi: 10.1016/j.jemermed.2008.07.023Platell C, Cooper D, Papadimitriou JM, Hall JC. The omentum. World J Gastroenterol. 2000;6:169-176. doi: 10.3748/wjg.v6.i2.169Rangel-Moreno J, Moyron-Quiroz JE, Carragher DM, et al. Omental milky spots develop in the absence of lymphoid tissue-inducer cells and support B and T cell responses to peritoneal antigens. Immunity. 2017;30:731-743. doi: 10.1016/j.immuni.2009.03.014Rao TN, Parvathi T, Suvarchala A. Omental lymphangioma in adults—rare presentation report of a case. Case Rep Surg. 2012;2012:629482. doi: 10.1155/2012/629482Schwartz R, Reames M, McGrath P, et al. Primary solid neoplasms of the greater omentum. Surgery. 1991;109:543-539.Seow-En I, Seow-Choen F, Lim TKH, Leow WQ. Primary omen-tal gastrointestinal stromal tumour

1	R, Reames M, McGrath P, et al. Primary solid neoplasms of the greater omentum. Surgery. 1991;109:543-539.Seow-En I, Seow-Choen F, Lim TKH, Leow WQ. Primary omen-tal gastrointestinal stromal tumour (GIST) presenting with a large abdominal mass and spontaneous haemoperitoneum. BMJ Case Rep. 2014;2014:bcr2014205528. doi: 10.1136/bcr-2014-205528Tarantino G, Lobello R, Scopacasa F, et al. The contribution of omen-tal adipose tissue to adipokine concentrations in patients with the metabolic syndrome. Clin Invest Med. 2007;30:E192–E199.Uramatsu M, Saida Y, Nagao J, et al. Omental cyst: report of a case. Surg Today. 2001;31:1104-1106. doi: 10.1007/s595-001-8068-0Yoo E, Kim JH, Kim M-J, et al. Greater and lesser omenta: nor-mal anatomy and pathologic processes. RadioGraphics. 2007;27:707-720. doi: 10.1148/rg.273065085MesenteryAdams SD, Stanton MP. Malrotation and intestinal atre-sias. Early Hum Dev. 2017;90:921-925. doi: 10.1016/j.earlhumdev.2014.09.017Akram S, Pardi DS, Schaffner JA, Smyrk

1	10.1148/rg.273065085MesenteryAdams SD, Stanton MP. Malrotation and intestinal atre-sias. Early Hum Dev. 2017;90:921-925. doi: 10.1016/j.earlhumdev.2014.09.017Akram S, Pardi DS, Schaffner JA, Smyrk TC. Sclerosing mesenteritis: clinical features, treatment, and outcome in ninety-two patients. Clin Gastroenterol Hepatol. 2017;5: 589-596. doi: 10.1016/j.cgh.2007.02.032Avincsal MO, Otani K, Kanzawa M, et al. Sclerosing mes-enteritis: a real manifestation or histological mimic of Brunicardi_Ch35_p1549-p1566.indd 156412/02/19 9:59 AM 1565ABDOMINAL WALL, OMENTUM, MESENTERY, AND RETROPERITONEUMCHAPTER 35IgG4-related disease? Pathol Int. 2016;66(3):158-163. doi: 10.1111/pin.12386Coffey JC, O’Leary DP. The mesentery: structure, function, and role in disease. Lancet Gastroenterol Hepatol. 2017;1(3): 238-247. doi: 10.1016/S2468-1253(16)30026-7Daskalogiannaki M, Voloudaki A, Prassopoulos P, et al. CT evalu-ation of mesenteric panniculitis. Am J Roentgenol. 2000;174: 427-431. doi:

1	2017;1(3): 238-247. doi: 10.1016/S2468-1253(16)30026-7Daskalogiannaki M, Voloudaki A, Prassopoulos P, et al. CT evalu-ation of mesenteric panniculitis. Am J Roentgenol. 2000;174: 427-431. doi: 10.2214/ajr.174.2.1740427Del Gobbo A, Bimbatti M, Ferrero S. A case report of mesenteric mucinous cystoadenoma with review of the literature. BMC Gastroenterol. 2010;10:105. doi: 10.1186/1471-230X-10-105Dequanter D, Lefebvre J, Belva P, Takieddine M, Vaneukem P. Mes-enteric cyst. A case treated by laparoscopy and a review of the literature. Surg Endosc. 2002;16(10):1493.Ezhapilli SR, Moreno CC, Small WC, et al. Mesenteric masses: approach to differential diagnosis at MRI with histopathologic correlation. J Magn Reson Imaging. 2014;40(4):753-769. doi: 10.1002/jmri.24690Halabi WJ, Jafari MD, Kang CY, et al. Colonic volvulus in the United States: trends, outcomes, and predictors of mortality. Ann Surg. 2014;259(2):293-301.Horton KM, Lawler LP, Fishman EK. CT findings in sclerosing mesenteritis

1	CY, et al. Colonic volvulus in the United States: trends, outcomes, and predictors of mortality. Ann Surg. 2014;259(2):293-301.Horton KM, Lawler LP, Fishman EK. CT findings in sclerosing mesenteritis (panniculitis): spectrum of disease. RadioGraph-ics. 2003;23:1561-1567. doi: 10.1148/rg.1103035010Hussein MRA, Abdelwahed SR. Mesenteric panniculitis: an update. Exp Rev Gastroenterol Hepatol. 2015;9:67-78. doi: 10.1586/17474124.2014.939632Kikiros CS, Edis AJ. Mesenteric panniculitis resulting in bowel obstruction: response to steroids. N Z J Surg. 1989;59(3): 287-290. doi: 10.1111/j.1445-2197.1989.tb01565.xKirby R, Rajasagaram N, Ghusn M. Primary mesenteric gastroin-testinal stromal tumour. J Surg Case Rep. 2014;2014:rju050. doi: 10.1093/jscr/rju050Park I-S, Kye B-H, Kim H-S, et al. Primary mesenteric carcinoid tumor. J Korean Surg Soc. 2013;84:114-117. doi: 10.4174/jkss.2013.84.2.114Parra-Davila E, McKenney M, Sleeman D, et al. Mesenteric panniculitis: case report and literature

1	mesenteric carcinoid tumor. J Korean Surg Soc. 2013;84:114-117. doi: 10.4174/jkss.2013.84.2.114Parra-Davila E, McKenney M, Sleeman D, et al. Mesenteric panniculitis: case report and literature review. Am Surg. 1998;64:768-771.Peng L, Yang L, Wu N, Wu B. Primary primitive neuroectoder-mal tumor arising in the mesentery and ileocecum: a report of three cases and review of the literature. Exp Ther Med. 2015;9(4):1299-1303. doi: 10.3892/etm.2015.2242Rajendran S, Khan A, Murphy M, O’Hanlon D. The diagnosis and treatment of a symptomatic mesenteric cyst. BMJ Case Reports. 2014;2014:bcr2013202410. doi: 10.1136/bcr-2013-202410Salar O, El-Sharkawy AM, Singh R, Speake W. Internal her-nias: a brief review. Hernia. 2013;17:373-377. doi: 10.1007/s10029-012-1023-1Tan JJ-Y, Tan K-K, Chew S-P. Mesenteric cysts: an institution experience over 14 years and review of literature. World J Surg. 2009;33:1961-1965. doi: 10.1007/s00268-009-0133-0van Putte-Katier N, van Bommel EFH, Elgersma OE, Hendriksz TR.

1	cysts: an institution experience over 14 years and review of literature. World J Surg. 2009;33:1961-1965. doi: 10.1007/s00268-009-0133-0van Putte-Katier N, van Bommel EFH, Elgersma OE, Hendriksz TR. Mesenteric panniculitis: prevalence, clinicoradiological presentation and 5-year follow-up. Br J Radiol. 2014;87:20140451. doi: 10.1259/bjr.20140451RetroperitoneumConstantinides VA, Christakis I, Touska P, Palazzo FF. System-atic review and meta-analysis of retroperitoneoscopic versus laparoscopic adrenalectomy. Br J Surg. 2012;99:1639-1648. doi: 10.1002/bjs.8921Goenka AH, Shah SN, Remer EM. Imaging of the retroperitoneum. Radiol Clin North Am. 2017;50(2):333-355. doi: 10.1016/j.rcl.2012.02.004Huang S-H, Lo W-O, Lin C-M, et al. Retroperitoneal abscess: 7-year experience of 29 cases in a tertiary care center in Taiwan. Urol Sci. 2015;26:218-221. doi: http://dx.doi.org/10.1016/j.urols.2015.01.009Kermani TA, Crowson CS, Achenbach SJ, Luthra HS. Idio-pathic retroperitoneal fibrosis: a

1	a tertiary care center in Taiwan. Urol Sci. 2015;26:218-221. doi: http://dx.doi.org/10.1016/j.urols.2015.01.009Kermani TA, Crowson CS, Achenbach SJ, Luthra HS. Idio-pathic retroperitoneal fibrosis: a retrospective review of clini-cal presentation, treatment, and outcomes. Mayo Clin Proc. 2011;86(4):297-303. doi: 10.4065/mcp.2010.0663Knezevic S, Ignjatovic I, Lukic S, et al. Primary retroperitoneal mucinous cystadenoma: a case report. World J Gastroenterol. 2015;21(17):5427-5431. doi: 10.3748/wjg.v21.i17.5427Martorana D, Vaglio A, Greco P, et al. Chronic periaortitis and HLA–DRB1*03: another clue to an autoimmune origin. Arthri-tis Care Res. 2006;55:126-130. doi: 10.1002/art.21698Messiou C, Moskovic E, Vanel D, et al. Primary retroperitoneal soft tissue sarcoma: imaging appearances, pitfalls and diag-nostic algorithm. Eur J Surg Oncol. 2017;43(7):1191-1198. doi: 10.1016/j.ejso.2016.10.032Miralas P, Skandalakis J. Surgical Anatomy of the retroperitoneal spaces part II: the architecture

1	and diag-nostic algorithm. Eur J Surg Oncol. 2017;43(7):1191-1198. doi: 10.1016/j.ejso.2016.10.032Miralas P, Skandalakis J. Surgical Anatomy of the retroperitoneal spaces part II: the architecture of the retroperitoneal space. Am Surg. 2010;76(1):33-42.Osman S, Lehnert B, Eloseimy S, et al. A comprehensive review of the retroperitoneal anatomy, neoplasms, and pattern of disease spread. Curr Probl Diagn Radiol. 2013;42(5):191-208.Scali EP, Chandler TM, Heffernan EJ, Coyle J, Harris AC, Chang SD. Primary retroperitoneal masses: what is the differential diagnosis? Abdom Imaging. 2015;40:1887-1903. doi: 10.1007/s00261-014-0311-xScheel P, Feeley N. Retroperitoneal fibrosis: the clinical, labora-tory, and radiographic presentation. Medicine (Baltimore). 2009;88(4):202-207.Stone JR. Aortitis, periaortitis, and retroperitoneal fibrosis, as mani-festations of IgG4-related systemic disease. Curr Opin Rheu-matol. 2011;23(1):88-94.Vaglio A, Salvarani C, Buzio C. Retroperitoneal fibrosis.

1	periaortitis, and retroperitoneal fibrosis, as mani-festations of IgG4-related systemic disease. Curr Opin Rheu-matol. 2011;23(1):88-94.Vaglio A, Salvarani C, Buzio C. Retroperitoneal fibrosis. Lancet. 2017;367(9506):241-251. doi: 10.1016/S0140-6736(06)68035-5van Bommel EFH, Siemes C, Hak LE, et al. Long-term renal and patient outcome in idiopathic retroperitoneal fibrosis treated with prednisone. Am J Kidney Dis. 2017;49:615-625. doi: 10.1053/j.ajkd.2007.02.268Brunicardi_Ch35_p1549-p1566.indd 156512/02/19 9:59 AM

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1	INTRODUCTIONSarcomas are a heterogeneous group of neoplasms that arise predominantly from cells of the embryonic mesoderm. While the majority of sarcomas are soft tissue sarcomas, other types of sarcoma include bone sarcomas (osteosarcoma, chondrosarcoma, and rare bone tumors like chordoma, angiosar-coma, and leiomyosarcoma of bone) and Ewing’s sarcoma/peripheral primitive neuroectodermal tumor, which can occur either in the bone or in the soft tissues. The primary focus of this chapter is soft tissue sarcomas. Most primary soft tissue sarco-mas originate in an extremity (50–60%); the next most common sites are the trunk (19%), retroperitoneum (15%), and head and neck (9%). The anatomic site of a primary sarcoma influ-ences treatment and outcome.1Soft tissue sarcomas include more than 70 histologic sub-types (Table 36-1). Historically, the most common subtypes in adults (excluding Kaposi’s sarcoma) were malignant fibrous histiocytoma (28%), liposarcoma (15%), leiomyosarcoma (12%),

1	70 histologic sub-types (Table 36-1). Historically, the most common subtypes in adults (excluding Kaposi’s sarcoma) were malignant fibrous histiocytoma (28%), liposarcoma (15%), leiomyosarcoma (12%), synovial sarcoma (10%), and malignant peripheral nerve sheath tumor (6%).2 Today, malignant fibrous histiocytoma is classified as either leiomyosarcoma, pleomorphic undifferenti-ated sarcoma, myxofibrosarcoma, or dedifferentiated liposar-coma based on cellular differentiation and genetics. Embryonal/alveolar rhabdomyosarcomas are the most common soft tissue sarcomas of childhood, whereas pleomorphic rhabdomyosar-coma occurs predominantly in adults, and although it shares part 12of the name, it has a different biology and should not be treated as a pediatric sarcoma.During the past 25 years, patients with extremity sarcomas have been treated with a multimodality approach, which has led to some improvements in survival, local control, and quality of life.3 However, patients with abdominal

1	patients with extremity sarcomas have been treated with a multimodality approach, which has led to some improvements in survival, local control, and quality of life.3 However, patients with abdominal sarcomas con-tinue to have high rates of recurrence and poor overall survival.4 The overall 5-year survival rate for patients with all stages of soft tissue sarcoma is 50% to 60%. Of the patients who die of sarcoma, most succumb to lung metastasis, which 80% of the time occurs within 2 to 3 years after initial diagnosis.INCIDENCEIn the United States in 2012, approximately 11,280 new cases of soft tissue sarcoma were diagnosed, and 3900 deaths were attributable to this disease.5 The incidence of soft tissue sarcomas increased 1.3% per year from 1995 to 2009, and subsequently leveled off while mortality rates increased slightly (0.5% per year) from 2001 to 2014.5 The true incidence of sarcoma is thought to be higher than reported, and gastrointestinal stromal tumors (GISTs) likely account

1	mortality rates increased slightly (0.5% per year) from 2001 to 2014.5 The true incidence of sarcoma is thought to be higher than reported, and gastrointestinal stromal tumors (GISTs) likely account for an additional 5000 new sarcoma cases per year.1 Overall, sarcomas affect 5 to 6 individuals per 100,000 inhabitants per year,6 accounting for less than 1% of all malignancies in adults and 15% of malignancies in children.7345Soft Tissue SarcomasRicardo J. Gonzalez, Alessandro Gronchi, and Raphael E. Pollock 36chapterIntroduction1567Incidence1567Epidemiology1568Radiation Exposure / 1568Occupational Chemical Exposure / 1568Trauma / 1568Chronic Lymphedema / 1568Molecular Pathogenesis1569Translocation-Associated Sarcomas / 1569Amplification-Associated Sarcomas / 1569Oncogenic Mutations / 1569Complex Genomic Rearrangements / 1570Initial Assessment1570Clinical Presentation / 1570Diagnostic Imaging / 1570Biopsy Techniques / 1571Pathologic Assessment and Classification / 1572Staging and

1	1569Complex Genomic Rearrangements / 1570Initial Assessment1570Clinical Presentation / 1570Diagnostic Imaging / 1570Biopsy Techniques / 1571Pathologic Assessment and Classification / 1572Staging and Prognostic Factors / 1573Treatment of Extremity and Trunk Wall Sarcoma1574Surgery / 1575Radiation Therapy / 1577Systemic Therapy / 1578Concurrent Chemoradiation Therapy / 1580Posttreatment Surveillance / 1580Management of Recurrent Sarcoma / 1581Special Clinical Situations1582Myxoid Liposarcoma / 1582Retroperitoneal Sarcoma / 1582Gastrointestinal Sarcoma / 1583Breast Sarcoma / 1584Uterine Sarcoma / 1584Gastrointestinal Stromal Tumors1585Radiologic Assessment / 1585Management of Localized Disease / 1585Management of Locally Advanced or Metastatic Disease / 1585Multidisciplinary Treatment / 1586Postoperative Imatinib / 1586Preoperative Imatinib / 1587Desmoids1588Dermatofibrosarcoma Protuberans1588Pediatric Sarcomas1589Rhabdomyosarcoma / 1589Nonrhabdomyosarcoma Soft Tissue Sarcomas /

1	/ 1586Postoperative Imatinib / 1586Preoperative Imatinib / 1587Desmoids1588Dermatofibrosarcoma Protuberans1588Pediatric Sarcomas1589Rhabdomyosarcoma / 1589Nonrhabdomyosarcoma Soft Tissue Sarcomas / 1589Research Perspectives1590Conclusions1590Brunicardi_Ch36_p1567-p1598.indd 156701/03/19 6:38 PM 1568Table 36-1Relative frequency of histologic subtypes of soft tissue sarcomaHISTOLOGIC SUBTYPESNO.%Liposarcoma18815Leiomyosarcoma14812Unclassified sarcoma14011Synovial sarcoma12510Malignant peripheral nerve sheath tumor726Rhabdomyosarcoma605Fibrosarcoma383Ewing sarcoma252Angiosarcoma252Osteosarcoma141Epithelioid sarcoma141Chondrosarcoma131Clear cell sarcoma121Alveolar soft part sarcoma71Malignant hemangiopericytoma50.4Data from Coindre JM, Terrier P, Guillou L, et al. Predictive value of grade for metastasis development in the main histologic types of adult soft tissue sarcomas: a study of 1240 patients from the French Federation of Cancer Centers Sarcoma Group, Cancer. 2001 May

1	of grade for metastasis development in the main histologic types of adult soft tissue sarcomas: a study of 1240 patients from the French Federation of Cancer Centers Sarcoma Group, Cancer. 2001 May 15;91(10):1914-1926.EPIDEMIOLOGYExcept for malignant peripheral nerve sheath tumors in patients with neurofibromatosis, sarcomas do not seem to result from progression or dedifferentiation of a benign soft tissue tumor. While most sarcomas are of unknown cause, a few sarcoma sub-types have been observed in settings suggesting etiology.Radiation ExposureExternal radiation therapy is a rare but well-established risk factor for soft tissue sarcoma that may be associated with radiation-induced mutations of the p53 gene.8 The incidence of sarcoma among patients who are often treated with radiation for cancer of the breast, cervix, ovary, testes, or lymphatic system is 8 to 50 times the general-population risk.9,10 In a review of 160 patients with postirradiation sarcomas, the most common

1	for cancer of the breast, cervix, ovary, testes, or lymphatic system is 8 to 50 times the general-population risk.9,10 In a review of 160 patients with postirradiation sarcomas, the most common histologic types were osteogenic sarcoma, pleomorphic undifferentiated sarcoma, angiosarcoma, and lymphangiosarcoma.9 The risk of developing a sarcoma increased with radiation dose, and the median time between radiation therapy and diagnosis of sarcoma was 10 years.9 A review of 44 patients with radiation-associated sarcomas identified between 1989 and 2009 noted that the average period from initial radiation treatment to diagnosis was 16 years and that radiation-associated sarcomas occurred most commonly in patients treated for breast cancer (36% of the patients in the series) and lymphoma (34% of the patients in the series).11 The 5-year overall survival rate for patients presenting without metastasis was 44%. A recent review of undifferentiated pleomorphic sarcoma characterizing outcomes

1	of the patients in the series).11 The 5-year overall survival rate for patients presenting without metastasis was 44%. A recent review of undifferentiated pleomorphic sarcoma characterizing outcomes between sporadic and radiation-associated presentation identified a higher incidence of local recurrence and worse overall and disease specific survival among patients who presented with radiation-associated undifferentiated pleomorphic sarcoma.12Occupational Chemical ExposureExposure to herbicides such as phenoxyacetic acids and to wood preservatives containing chlorophenols has been linked to an increased risk of soft tissue sarcoma.13 Several chemi-cal carcinogens, including thorium oxide (Thorotrast), vinyl chloride, and arsenic, have been associated with hepatic angiosarcomas.14TraumaAlthough patients with sarcoma often report a history of trauma, no causal relationship has been established. More often, a minor injury calls attention to a preexisting tumor.Chronic LymphedemaIn 1948,

1	patients with sarcoma often report a history of trauma, no causal relationship has been established. More often, a minor injury calls attention to a preexisting tumor.Chronic LymphedemaIn 1948, Stewart and Treves first described the association between chronic lymphedema after axillary dissection and sub-sequent lymphangiosarcoma (Fig. 36-1).15 Lymphangiosarcoma has been estimated to occur in 0.07% of patients who undergo axillary node dissection.16 It also has been reported to occur after filarial infections and in the lower extremities of patients with congenital lymphedema.17,18 Lymphangiosarcoma is gen-erally an aggressive tumor; average survival of patients with lymphangiosarcoma is 19 months.19Key Points1 Sarcomas are a heterogeneous group of tumors that can occur throughout the body and encompass more than 50 subtypes with distinct histologic lines of differentiation.2 Approximately two-thirds of soft tissue sarcomas arise in the extremities; the remaining one-third is

1	the body and encompass more than 50 subtypes with distinct histologic lines of differentiation.2 Approximately two-thirds of soft tissue sarcomas arise in the extremities; the remaining one-third is distributed between the retroperitoneum, trunk, abdomen, head, and neck.3 Multimodality treatment, including surgical resection, radia-tion therapy, and, in selected cases, systemic chemotherapy, has been applied to patients with locally advanced, high-grade, extremity sarcomas.4 Overall 5-year survival rate for patients with all stages of soft tissue sarcoma is 50% to 60%.5 These rare tumors account for less than 1% of cancer in adults (estimated 10,000 cases per year in the United States) and represent 15% of cancers in children.6 The treatment algorithm for soft tissue sarcomas depends on tumor stage, site, and histology.7 Of the patients who die of sarcoma, most will succumb to metastatic disease in the lungs, which 80% of the time occurs within 2 to 3 years of the initial

1	depends on tumor stage, site, and histology.7 Of the patients who die of sarcoma, most will succumb to metastatic disease in the lungs, which 80% of the time occurs within 2 to 3 years of the initial diagnosis.8 Progress in the understanding of soft tissue sarcoma biology is crucial for the development of new treatments.Brunicardi_Ch36_p1567-p1598.indd 156801/03/19 6:38 PM 1569SOFT TISSUE SARCOMASCHAPTER 36Figure 36-1. A 57-year-old with a chronic, progressive lymph-edema of the left upper extremity developed lymphangiosarcoma 10 years after breast cancer treatment.Table 36-2Fusion transcripts in soft tissue sarcomaDIAGNOSISCHROMOSOMAL ABNORMALITYGENES INVOLVEDAlveolar rhabdomyosarcomat(2;13)(q35;q14)t(1;13)(p36;q14)PAX3-FKHRPAX7-FKHRAlveolar soft part sarcomat(X;17)(p11.2;q25)TFE3-ASPLAngiomatoid fibrous histiocytomat(12;16)(q13;p11)FUS-ATF1Clear cell sarcomat(12;22)(q13;q12)EWS-ATF1Congenital fibrosarcoma/congenital mesoblastic

1	soft part sarcomat(X;17)(p11.2;q25)TFE3-ASPLAngiomatoid fibrous histiocytomat(12;16)(q13;p11)FUS-ATF1Clear cell sarcomat(12;22)(q13;q12)EWS-ATF1Congenital fibrosarcoma/congenital mesoblastic nephromat(12;15)(p13;q25)ETV6-NTRK3Dermatofibrosarcoma protuberanst(17;22)(q22;q13)PDFGB-COL1A1Desmoplastic small round cell tumort(11;22)(p13;q12)EWS-WT1Endometrial stromal sarcomat(7;17)(p15;q21)JAZF1-JJAZ1Ewing’s sarcoma/peripheral primitive neuroectodermal tumort(11;22)(q24;q12)t(21;22)(q22;q12)t(7;22)(p22;q12)t(17;22)(q12;q12)t(2;22)(q33;q12)t(16;21)(p11;q22)EWS-FLI1EWS-ERGEWS-ETV1EWS-FEVEWS-E1AFFUS-ERGLow-grade fibromyxoid sarcomat(7;16)(q33;p11)FUS-CREB3I2Inflammatory myofibroblastic tumort(1;2)(q22;p23)t(2;19)(p23;p13)t(2;17)(p23;q23)TPM3-ALKTPM4-ALKCLTC-ALKMyxoid liposarcomat(12;16)(q13;p11)t(12;22)(q13;q12)TLS-CHOPEWS-CHOPMyxoid chondrosarcomat(9;22)(q22;q12)t(9;15)(q22;q21)t(9;17)(q22;q11)EWS-CHNTFC12-CHNTAF2N-CHNSynovial sarcomat(x;18)(p11;q11)SSX1-SYTSSX2-SYTSSX4-SYTMOLECULAR

1	chondrosarcomat(9;22)(q22;q12)t(9;15)(q22;q21)t(9;17)(q22;q11)EWS-CHNTFC12-CHNTAF2N-CHNSynovial sarcomat(x;18)(p11;q11)SSX1-SYTSSX2-SYTSSX4-SYTMOLECULAR PATHOGENESISSarcomas can be broadly classified into three groups accord-ing to the genetic events underlying their development: specific translocations or gene amplification, defining oncogenic muta-tions, and complex genomic rearrangements.20 In general, sar-comas resulting from identifiable molecular events tend to occur in younger patients with histology suggesting a clear line of differentiation. The identifiable molecular events include point mutations, translocations causing overexpression of an autocrine grow factor, and oncogenic fusion transcription factor produc-ing a cellular environment prone to malignant transformation. In contrast, sarcomas without identifiable genetic changes or expression profile signatures tend to occur in older patients and exhibit pleomorphic cytology and p53 dysfunction.21 Improved understanding of

1	sarcomas without identifiable genetic changes or expression profile signatures tend to occur in older patients and exhibit pleomorphic cytology and p53 dysfunction.21 Improved understanding of the molecular pathogenesis of sarcomas has revealed several potential targets against which investigators are working to develop subtype-specific targeted therapy.Translocation-Associated SarcomasTo date, translocations have been identified in 14 subtypes of soft tissue sarcoma, accounting for 20% to 30% of all sarcomas22 (Table 36-2). Translocations result in in-frame gene fusion, which in turn results in fused products encoding oncoproteins that function as transcriptional activators or repressors.23,24 The best characterized gene fusions are in Ewing’s sarcoma (EWS-FLI1), clear cell sarcoma (EWS-ATF1), myxoid/round cell liposarcoma (TLS-CHOP), alveolar rhabdomyosarcoma (PAX3-FHKR), desmoplastic small round cell tumor (EWS-WT1), and synovial sarcoma (SS18-SSX). Fusion gene–related sarcomas

1	myxoid/round cell liposarcoma (TLS-CHOP), alveolar rhabdomyosarcoma (PAX3-FHKR), desmoplastic small round cell tumor (EWS-WT1), and synovial sarcoma (SS18-SSX). Fusion gene–related sarcomas have been estimated to account for 30% or more of all sarcomas.25Direct or indirect interactions between fusion transcripts and cell cycle regulators have been elucidated by several inves-tigators and identify these transcripts as potentially promising molecular therapeutic targets.26 However, fusion genes in sar-coma have been successfully targeted in only a few cases, in which fusion resulted in overexpression of a growth factor or growth factor receptor. Several growth factors and their recep-tors (e.g., epidermal growth factor receptor) previously reported to play an important role in autocrine or paracrine stimulation of carcinoma growth have been associated with high histologic grade and poor prognosis in soft tissue sarcomas.Amplification-Associated SarcomasOncogenes are genes that can

1	or paracrine stimulation of carcinoma growth have been associated with high histologic grade and poor prognosis in soft tissue sarcomas.Amplification-Associated SarcomasOncogenes are genes that can induce malignant transformation and tend to drive cell proliferation. Several oncogenes have been associated with soft tissue sarcomas, including MDM2, N-myc, c-erbB2, and members of the ras family. These onco-genes produce specific oncoproteins that either play a role in nuclear function and cellular signal transduction or function as growth factors or growth factor receptors. This typically occurs in dedifferentiated liposarcoma, where the amplification of MDM2 drives the neoplastic process. Amplification of these genes has been shown to correlate with adverse outcome in sev-eral types of soft tissue sarcoma.23Oncogenic MutationsGISTs are the classic example of sarcomas in which tumorigen-esis is primarily driven by a single activating mutation, in the Brunicardi_Ch36_p1567-p1598.indd

1	tissue sarcoma.23Oncogenic MutationsGISTs are the classic example of sarcomas in which tumorigen-esis is primarily driven by a single activating mutation, in the Brunicardi_Ch36_p1567-p1598.indd 156901/03/19 6:38 PM 1570SPECIFIC CONSIDERATIONSPART IIgene encoding KIT receptor tyrosine kinase or platelet-derived growth factor receptor-α (PDGFRA).20 The majority of GISTs have mutations in either exon 11 or exon 9 of KIT and respond dramatically to the tyrosine kinase inhibitor imatinib mesylate, although this treatment rarely produces cure.Complex Genomic RearrangementsThe largest group of sarcomas is the group with complex cytoge-netic alterations, which includes high-grade spindle cell sarco-mas and pleomorphic sarcomas.20 Many sarcomas in this group exhibit inactivation of tumor suppressor genes. The two genes most relevant to soft tissue sarcoma are retinoblastoma (Rb) and p53. Mutations or deletions in Rb can lead to retinoblastoma, the most common malignant ocular neoplasm of

1	genes. The two genes most relevant to soft tissue sarcoma are retinoblastoma (Rb) and p53. Mutations or deletions in Rb can lead to retinoblastoma, the most common malignant ocular neoplasm of childhood. Survi-vors of retinoblastoma are at risk for developing soft tissue and bone sarcomas later in life. Patients with germline mutations in p53 (Li-Fraumeni syndrome) have a high incidence of soft tissue sarcomas Mutant p53 expression is thought to correlate with poor overall survival.23 Strategies to target p53 mutation are being investigated for treatment of some sarcomas.Neurofibromatosis type 1 (von Recklinghausen’s disease) occurs in approximately 1 of every 3000 people and is due to various mutations in the NF-1 tumor suppressor gene, located on chromosome 17. Patients with neurofibromatosis type 1 have an estimated 3% to 15% additional risk of malignant disease compared with the general population lifetime risk, including malignant peripheral nerve sheath tumors (MPNST) and GIST.

1	type 1 have an estimated 3% to 15% additional risk of malignant disease compared with the general population lifetime risk, including malignant peripheral nerve sheath tumors (MPNST) and GIST. In turn, 25% to 50% of patients with MPNST have a mutation in NF-1.27INITIAL ASSESSMENTThe clinical behavior of most soft tissue sarcomas is determined by anatomic location (depth in relation to the investing fascia), histologic subtype and grade of aggressiveness, and size. The dominant pattern of metastasis is hematogenous, primarily to the lungs. Lymph node metastasis is rare (affecting <5% of patients) except in a few histologic subtypes, including epi-thelioid sarcoma, pediatric rhabdomyosarcoma, clear cell sar-coma, angiosarcoma, and, more rarely, synovial sarcoma and myxofibrosarcoma.28Clinical PresentationSoft tissue sarcomas most commonly present as an asymptom-atic mass. Extremity sarcomas may present as a deep venous thrombosis, particularly in patients without significant risk

1	PresentationSoft tissue sarcomas most commonly present as an asymptom-atic mass. Extremity sarcomas may present as a deep venous thrombosis, particularly in patients without significant risk factors for thrombosis.29 Tumors in the distal extremities are generally smaller, whereas tumors in the proximal extremities and retroperitoneum can grow quite large before becoming apparent. Tumors often grow centrifugally and can compress surrounding normal structures. Infrequently, tumor impinge-ment on bone or neurovascular bundles produces pain, edema, and swelling. Less frequently, tumors cause obstructive gastro-intestinal symptoms or neurologic symptoms related to com-pression of lumbar or pelvic nerves. Often an extremity mass is discovered after a traumatic event that draws attention to a preexisting lesion.The differential diagnosis of a soft tissue mass should include consideration of lipoma (which is 100 times more com-mon than sarcoma), lymphangioma, leiomyoma, neurinoma, primary or

1	lesion.The differential diagnosis of a soft tissue mass should include consideration of lipoma (which is 100 times more com-mon than sarcoma), lymphangioma, leiomyoma, neurinoma, primary or metastatic carcinoma, melanoma, and lymphoma. Superficial small lesions (<5 cm) that are new or that are not enlarging as indicated by clinical history can be observed. Figure 36-2. A 55-year-old man with a leiomyosarcoma involving the inferior vena cava. Note the displacement of the inferior vena cava to the right hemiabdomen adjacent to the liver (arrow).Enlarging masses and masses larger than 5 cm or deep to the fascia should be evaluated with a history, imaging, and biopsy.30Diagnostic ImagingDiagnostic imaging of the primary should be performed before any invasive procedure to avoid the possibility of soft tissue swelling or hemorrhage complicating the image interpretation. Pretreatment diagnostic imaging is helpful for defining the size and anatomic location of a tumor and its proximity to

1	of soft tissue swelling or hemorrhage complicating the image interpretation. Pretreatment diagnostic imaging is helpful for defining the size and anatomic location of a tumor and its proximity to adjacent structures; staging disease with respect to regional or metastatic spread; guiding percutaneous biopsy; and establishing whether a tumor is benign or malignant and low grade or high grade.Radiographs are useful in the evaluation of primary bone tumors but not in the evaluation of soft tissue sarcomas of the extremities unless there is underlying bone involvement from an adjacent soft tissue tumor or mineralization patterns sugges-tive of histologic subtype. Magnetic resonance imaging (MRI) is the preferred imaging technique for soft tissue sarcomas of the extremities, whereas computed tomography (CT) is most use-ful for evaluating retroperitoneal, intra-abdominal, and truncal sarcomas.31 CT of the chest should be performed to assess for lung metastases in patients with high-grade

1	tomography (CT) is most use-ful for evaluating retroperitoneal, intra-abdominal, and truncal sarcomas.31 CT of the chest should be performed to assess for lung metastases in patients with high-grade tumors larger than 5 cm; while chest X-ray is sufficient for smaller or low-grade lesions. Abdominal/pelvic CT should be performed in patients with myxoid round cell liposarcomas, leiomyosarcomas, epi-thelioid sarcomas, or angiosarcomas because of their propensity to metastasize to the abdomen and/or pelvis.1 Total Spine MRI has been advocated for myxoid round cell liposarcoma.1 MRI of the brain should be considered for patients with alveolar soft part sarcomas and angiosarcomas because of their propensity to metastasize to the brain.Ultrasonography. Ultrasonography may have a diagnostic role in patients with soft tissue sarcoma who cannot undergo MRI. Ultrasonography can also be a useful adjunct to MRI when findings on MRI are indeterminate and for delineating adjacent vascular

1	role in patients with soft tissue sarcoma who cannot undergo MRI. Ultrasonography can also be a useful adjunct to MRI when findings on MRI are indeterminate and for delineating adjacent vascular structures. Finally, ultrasonography can be used for postoperative surveillance and to guide biopsies.Computed Tomography. Chest CT should be performed to evaluate for lung metastasis at presentation and before any radi-cal treatment. CT is also the preferred imaging technique for evaluating retroperitoneal sarcomas (Fig. 36-2).31 Current CT Brunicardi_Ch36_p1567-p1598.indd 157001/03/19 6:38 PM 1571SOFT TISSUE SARCOMASCHAPTER 36techniques can provide a detailed image of the abdomen and pelvis and can delineate adjacent organs and vascular struc-tures. For extremity sarcomas, CT may be useful if MRI is not available or cannot be used. When histologic assessment of an extremity sarcoma reveals a myxoid liposarcoma, CT of the abdomen and pelvis should be done because this subtype is known to

1	MRI is not available or cannot be used. When histologic assessment of an extremity sarcoma reveals a myxoid liposarcoma, CT of the abdomen and pelvis should be done because this subtype is known to metastasize to the abdomen.32Magnetic Resonance Imaging. MRI is the most useful imag-ing modality for extremity sarcomas because of its superior soft tissue contrast resolution and multiplanar capabilities. MRI accurately delineates muscle groups and distinguishes among bone, vascular structures, and tumor. Sagittal and coronal views allow evaluation of anatomic compartments in three dimensions (Fig. 36-3). Soft tissue sarcomas of the extremities usually present on MRI as a heterogeneous mass. Their signal inten-sity tends to be equal to or slightly higher than that of adjacent skeletal muscle on T1-weighted images and heterogeneous and high on T2-weighted images. Hemorrhagic, cystic, or necrotic changes may also be observed in the tumor. If adjacent vas-cular structures must be delineated,

1	T1-weighted images and heterogeneous and high on T2-weighted images. Hemorrhagic, cystic, or necrotic changes may also be observed in the tumor. If adjacent vas-cular structures must be delineated, special MRI techniques may be performed, including magnetic resonance angiography. MRI may also be an important adjunct to cytologic analysis in distinguishing benign lesions such as lipomas, hemangiomas, schwannomas, neurofibromas, and intramuscular myxomas from their malignant counterparts. In patients undergoing pre-operative chemotherapy, contrast-enhanced T1-weighted MRI can be useful in evaluating intratumoral necrosis.MRI is also valuable for assessing tumor recurrence after surgery. A baseline image is usually obtained 3 months after surgery. Some clinicians forego routine postoperative imaging of the primary extremity tumor site in asymptomatic patients, citing the difficulties in detecting early recurrence in scarred, irradiated tissue.31 Others advocate routine imaging every 3 to

1	imaging of the primary extremity tumor site in asymptomatic patients, citing the difficulties in detecting early recurrence in scarred, irradiated tissue.31 Others advocate routine imaging every 3 to 4 months for the first 2 years, every 6 months in years 3 through 5, and then annually.Positron Emission Tomography. Positron emission tomogra-phy (PET) is a functional imaging modality that measures tumor uptake of the glucose analog [18F] fluorodeoxyglucose (FDG). Figure 36-3. A 62-year-old man presented with right thigh mass. Magnetic resonance imaging demonstrated an 18 × 15 cm2 dedifferentiated liposarcoma within the posterior compartment. Note the atypical fatty mass (left) with a large necrotic and peripherally enhancing nodule (left).PET imaging allows evaluation of the entire body. Although PET/CT may be useful in specific circumstances, FDG-PET is not currently recommended for the initial staging of patients with soft tissue sarcoma.Roberge and colleagues compared FDG-PET/CT

1	Although PET/CT may be useful in specific circumstances, FDG-PET is not currently recommended for the initial staging of patients with soft tissue sarcoma.Roberge and colleagues compared FDG-PET/CT versus chest CT alone in the initial staging of 75 patients with soft tissue sarcoma and found that only one patient had disease upstaged as a result of PET, whereas two had false-positive find-ings and three had indeterminate findings with no subsequent development of metastasis.33 Previous studies that reported a marginal benefit of PET/CT for detecting metastasis at the time of sarcoma staging included patients with more heterogeneous tumors, such as osseous tumors, soft tissue osteosarcomas, Ewing’s sarcoma, and rhabdomyosarcoma.34-36In patients with sarcoma, PET has primarily been used to assist with tumor grading and to assess response to chemotherapy.37-40 In 50 patients with resectable high-grade soft tissue tumors scheduled for preoperative chemotherapy and tumor resection, a 35%

1	with tumor grading and to assess response to chemotherapy.37-40 In 50 patients with resectable high-grade soft tissue tumors scheduled for preoperative chemotherapy and tumor resection, a 35% or greater reduction in tumor FDG uptake following an initial cycle of chemotherapy was associated with histopathologic tumor response defined as pathologic necrosis in 95% or more of the resected specimen.41 While this is helpful in determining response controversy exists as to whether this translates into a predictor of overall survival.Biopsy TechniquesFine-Needle Aspiration. At centers where cytopathologists have experience with evaluation of mesenchymal tumors, fine-needle aspiration is an acceptable method of diagnosing most soft tissue sarcomas, particularly when the results correlate closely with clinical and radiologic findings.42 Fine-needle aspiration of primary tumors has a lower diagnostic accuracy rate (60–90%) than core needle biopsy and is often not sufficient for establish-ing

1	with clinical and radiologic findings.42 Fine-needle aspiration of primary tumors has a lower diagnostic accuracy rate (60–90%) than core needle biopsy and is often not sufficient for establish-ing a specific histologic diagnosis and grade.43 However, fine-needle aspiration is the procedure of choice to confirm or rule out the presence of a metastatic focus or local recurrence.44Although fine-needle aspiration of superficial lesions can often be done in the clinic, fine-needle aspiration of deeper tumors may need to be done by an interventional radiologist Brunicardi_Ch36_p1567-p1598.indd 157101/03/19 6:38 PM 1572SPECIFIC CONSIDERATIONSPART IIunder sonographic or CT guidance. Generally, a 21to 23-gauge needle is introduced into the mass after appropriate cleansing of the skin and injection of local anesthetic. Negative pressure is applied, and the needle is moved back and forth several times in various directions. After the negative pressure is released, the needle is withdrawn,

1	of local anesthetic. Negative pressure is applied, and the needle is moved back and forth several times in various directions. After the negative pressure is released, the needle is withdrawn, and the contents of the needle are used to prepare smears.45 A cytopathologist then examines the slides to determine whether sufficient diagnostic material is present.Core Needle Biopsy. Core needle biopsy is safe, accurate,46,47 and economical48 and has become the preferred technique for diagnosing soft tissue lesions. Dupuy and colleagues found that core needle biopsy had an accuracy of 93% in 221 patients with musculoskeletal neoplasms.46Image guidance (ultrasound or CT) can prevent sam-pling of nondiagnostic necrotic or cystic areas of the tumor and thus increase the positive yield rate. Image guidance also per-mits biopsy of tumors in otherwise inaccessible locations and tumors located near vital structures with less risk of injury or complication.The tissue sample obtained from core needle

1	also per-mits biopsy of tumors in otherwise inaccessible locations and tumors located near vital structures with less risk of injury or complication.The tissue sample obtained from core needle biopsy is usually sufficient for several diagnostic tests, such as electron microscopy, cytogenetic analysis, and flow cytometry. The risk for needle track seeding is negligible, and the reported compli-cation rate for core needle biopsy is less than 1%.46,47Incisional Biopsy. Historically, an open surgical biopsy was the gold standard for achieving adequate tissue for definitive and specific histologic diagnosis of bone or soft tissue sarcomas. Contemporary guidelines recommend incisional biopsy when core needle biopsy cannot produce adequate tissue for diagnosis or when findings on core needle biopsy are nondiagnostic.The disadvantages of incisional biopsy include the need to schedule the procedure, the need for general anesthesia, and high costs. In addition, an inappropriately placed

1	biopsy are nondiagnostic.The disadvantages of incisional biopsy include the need to schedule the procedure, the need for general anesthesia, and high costs. In addition, an inappropriately placed incision can necessitate more extensive definitive resection to incorporate the biopsy incision. In a series of 107 patients with soft tissue sarcoma, planned surgical treatments had to be changed because of poorly oriented biopsies in 25% of cases.49 Complication rates up to 17% have been reported after incisional biopsies.45 Potential complications include hematoma, infection, wound dehiscence, and tumor fungation, any of which can delay defini-tive treatment.45Incisional biopsies should be performed only by surgeons experienced in the management of soft tissue sarcoma, ideally in a center specializing in the treatment of sarcoma and by the surgeon who will perform the definitive surgery. The biopsy incision should be oriented longitudinally along the extremity to allow a subsequent wide

1	in the treatment of sarcoma and by the surgeon who will perform the definitive surgery. The biopsy incision should be oriented longitudinally along the extremity to allow a subsequent wide local excision that encompasses the biopsy site, scar, and tumor en bloc minimizing the risk for increasing the complexity of the subsequent curative procedure. A poorly oriented biopsy incision often necessitates an exces-sively large surgical defect for a wide local excision, which in turn can result in a larger postoperative radiation therapy field to encompass all tissues at risk. Adequate hemostasis must be achieved at the time of biopsy to prevent dissemination of tumor cells into adjacent tissue planes by hematoma.Excisional Biopsy. Excisional biopsy can be performed for easily accessible (superficial) extremity or truncal lesions smaller than 3 cm. However, excisional biopsy rarely provides benefits over other biopsy techniques. Excisional biopsies should not be performed for lesions

1	(superficial) extremity or truncal lesions smaller than 3 cm. However, excisional biopsy rarely provides benefits over other biopsy techniques. Excisional biopsies should not be performed for lesions involving the hands and feet because such biopsies may complicate definitive reexcision. For sarcomas with initial diagnosis confirmed with excisional biopsy, microscopic residual disease has been reported in up to 69% of reexcision specimens50,51; without reexcision, the reported rate of local recurrence is 30% to 40% when margins are positive or uncertain.Wide en bloc excision is seldom performed as a diagnostic procedure. When en bloc excision is done for diagnosis, the mar-gin status is often not adequately evaluated during pathologic assessment of the specimen. Unless detailed descriptions of the surgical procedure and the pathology specimen are provided, the margins should be classified as uncertain or unknown, a classifi-cation associated with the same prognosis as resection

1	of the surgical procedure and the pathology specimen are provided, the margins should be classified as uncertain or unknown, a classifi-cation associated with the same prognosis as resection margins that are positive for tumor cells. In patients with uncertain or unknown margins, reexcision should be performed if possible to ensure negative margins. The biopsy site or tract (if applicable) should be included en bloc with the re-resected specimen.Pathologic Assessment and ClassificationSarcoma is generally diagnosed by morphologic assessment based on microscopic examination of histologic sections by an experienced sarcoma pathologist. However, even expert sar-coma pathologists disagree on the specific histologic diagnosis and the tumor grade in 25% to 40% of cases.52Morphologic assessment can be supported by ancillary techniques, including conventional cytogenetics; immunohis-tochemistry; and molecular genetic testing, which is useful for classifying soft tissue sarcoma subtypes with

1	can be supported by ancillary techniques, including conventional cytogenetics; immunohis-tochemistry; and molecular genetic testing, which is useful for classifying soft tissue sarcoma subtypes with multiple genetic aberrations. Other molecular diagnostic techniques include cytogenetic analysis, fluorescence in situ hybridization, and polymerase chain reaction–based methods.53 However, molecu-lar genetic techniques are associated with significant technical limitations and should be interpreted in the context of the sar-coma’s morphologic features.Some experts have suggested that pathologic classification of soft tissue sarcomas has more prognostic significance than does tumor grade when other pretreatment variables are taken into account. Tumors with limited metastatic potential include des-moid, atypical lipomatous tumor (also called well-differentiated liposarcoma), dermatofibrosarcoma protuberans, and solitary fibrous tumor. Tumors with an intermediate risk of metastatic spread

1	des-moid, atypical lipomatous tumor (also called well-differentiated liposarcoma), dermatofibrosarcoma protuberans, and solitary fibrous tumor. Tumors with an intermediate risk of metastatic spread usually have a large myxoid component and include myx-oid liposarcoma, myxofibrosarcoma, and extraskeletal myxoid chondrosarcoma. Among the highly aggressive tumors with sub-stantial metastatic potential are angiosarcoma, clear cell sarcoma, pleomorphic and dedifferentiated liposarcoma, leiomyosarcoma, MPNST, rhabdomyosarcoma, and synovial sarcoma.It has recently been noted that malignant fibrous histio-cytoma is not associated with a distinct gene cluster, suggest-ing that malignant fibrous histiocytoma is not a separate tumor entity but rather a common morphologic appearance of various sarcoma subtypes.54,55 For example, most tumors initially diag-nosed as malignant fibrous histiocytoma in the retroperitoneum have been reclassified using genomic profiling as dedifferenti-ated

1	sarcoma subtypes.54,55 For example, most tumors initially diag-nosed as malignant fibrous histiocytoma in the retroperitoneum have been reclassified using genomic profiling as dedifferenti-ated liposarcomas,56 whereas those in the extremities have been reclassified as leiomyosarcoma, myxofibrosarcoma, or pleo-morphic undifferentiated sarcoma.Guidelines for the pathologic reporting of sarcoma have been established.1 Included in the report should be the primary diagnosis, anatomic site, depth, size, and histologic grade, pres-ence or absence of necrosis, status of excision margins and lymph nodes, TNM stage, and additional features of the tumor (i.e., mitotic rate and presence or absence of vascular invasion).Brunicardi_Ch36_p1567-p1598.indd 157201/03/19 6:38 PM 1573SOFT TISSUE SARCOMASCHAPTER 36Staging and Prognostic FactorsSoft tissue sarcoma is most commonly staged using either the American Joint Committee on Cancer (AJCC) system (generally used in the United States) or the World

1	36Staging and Prognostic FactorsSoft tissue sarcoma is most commonly staged using either the American Joint Committee on Cancer (AJCC) system (generally used in the United States) or the World Health Organization sys-tem. A unique aspect of sarcoma staging is the inclusion of tumor grade, which is one of the most important prognostic factors.57The seventh edition of the AJCC staging system for soft tissue sarcomas is based on histologic grade of aggressive-ness, tumor size and depth, and the presence of nodal or distant metastases.58 This system does not apply to GIST, fibromatosis (desmoid tumor), Kaposi’s sarcoma, or infantile fibrosarcoma.Histologic Grade of Aggressiveness. Histologic grade is the most important prognostic factor for patients with soft tissue sarcoma. For accurate determination of grade, an adequate tis-sue sample must be appropriately fixed, stained, and reviewed by an experienced sarcoma pathologist. The features that define grade are cellularity, differentiation

1	of grade, an adequate tis-sue sample must be appropriately fixed, stained, and reviewed by an experienced sarcoma pathologist. The features that define grade are cellularity, differentiation (good, moderate, or poor/anaplastic), pleomorphism, necrosis (absent, <50%, or ≥50%), and number of mitoses per high-power field (<10, 10–19, or ≥20). Tumor grade has been shown to predict metastasis and overall survival.59 Metastasis has been estimated to occur in 5% to 10% of low-grade lesions, 25% to 30% of intermediate-grade lesions, and 50% to 60% of high-grade lesions.The number of grades varies according to the classifica-tion system used. The most common classification systems, those of the National Cancer Institute and the French Federation of Cancer Centers, use three-tier tumor grades.60 The National Cancer Institute system is based primarily on histologic sub-type, location, and amount of necrosis. The French Federation of Cancer Centers system is based on tumor differentiation (good,

1	National Cancer Institute system is based primarily on histologic sub-type, location, and amount of necrosis. The French Federation of Cancer Centers system is based on tumor differentiation (good, moderate, or poor/anaplastic), number of mitoses per high-power field (<10, 10–19, or ≥20), and amount of tumor necrosis (absent, <50%, or ≥50%). A comparative analysis of the two systems suggested that the French Federation of Can-cer Centers system has better prognostic capability, predicting 5-year survival rates of 90%, 70%, and 40% for grade 1, 2, and 3 tumors, respectively.60Following the recommendation of the College of American Pathologists, the committee that developed the 2008 AJCC staging system changed the system from a four-grade to a three-grade system in which the grades are well differentiated (grade 1), moderately differentiated (grade 2), and poorly differentiated (grade 3).61 Grade 1 is considered low grade, and grades 2 and 3 are considered high grade.Tumor Size and

1	differentiated (grade 1), moderately differentiated (grade 2), and poorly differentiated (grade 3).61 Grade 1 is considered low grade, and grades 2 and 3 are considered high grade.Tumor Size and Location. Tumor size is an important prog-nostic variable in soft tissue sarcomas. Sarcomas have classi-cally been stratified into two size groups; T1 lesions are 5 cm or smaller, and T2 lesions are larger than 5 cm. The staging of soft tissue sarcomas has changed considerably in the eighth edition of the AJCC Cancer Staging Handbook.62 Previously, all soft tissue sarcoma sites were staged as one. In the new edi-tion, subsites have been created, and they include head and neck; extremity and trunk; gastrointestinal tract; genitourinary tract; viscera and peritoneum; gynecological sites; breast lung, pleura and mediastinum; and other histologies. Size criteria for head and neck tumors have been reduced from the classic cri-teria because of a worse prognosis at smaller sizes. Trunk and extremity

1	pleura and mediastinum; and other histologies. Size criteria for head and neck tumors have been reduced from the classic cri-teria because of a worse prognosis at smaller sizes. Trunk and extremity sarcoma has been reclassified relative to size criteria for tumor stage, changing the four categories: T1 tumors are smaller than 5 cm; T2 tumors are 5 to 10 cm; T3 tumors are 10 to 15 cm; and T4 tumors are larger than 15 cm.62Figure 36-4. A 74-year-old man with a history of an extraskeletal myxoid chondrosarcoma of the gluteal region developed multiple lung metastases.Anatomic tumor location was incorporated into the AJCC staging system in 1998. Soft tissue sarcomas above the superfi-cial investing fascia of the extremity or trunk are designated “a” lesions within the T category, whereas tumors invading or deep to the fascia and all retroperitoneal, mediastinal, and visceral tumors are designated “b” lesions. However, in this most recent AJCC staging system, the superficial versus deep

1	invading or deep to the fascia and all retroperitoneal, mediastinal, and visceral tumors are designated “b” lesions. However, in this most recent AJCC staging system, the superficial versus deep distinction is less important and has been eliminated.62Nodal Metastasis. Overall, lymph node metastases arising from soft tissue sarcomas are rare,28 but the incidence of nodal involvement is higher for epithelioid sarcoma, pediatric rhab-domyosarcoma, clear cell sarcoma, synovial sarcoma, myxo-fibrosarcoma, and angiosarcoma. In the seventh edition of the AJCC staging system, sarcoma associated with nodal metas-tases was reclassified as stage III rather than stage IV because several studies reported better survival for patients with isolated regional lymph node metastases treated with radical lymphad-enectomy than for patients with distant metastases.28,63-65 How-ever, in the eighth edition of the AJCC, nodal disease has been revisited: N1 disease behaves similarly between stages III and IV

1	than for patients with distant metastases.28,63-65 How-ever, in the eighth edition of the AJCC, nodal disease has been revisited: N1 disease behaves similarly between stages III and IV and is now captured as stage IV.62 Patients with clinically or radiologically suspicious regional nodes should have metastases confirmed or ruled out by either fine-needle aspiration or core biopsy before radical lymphadenectomy.Distant Metastasis. Distant metastases occur most often in the lungs (Fig. 36-4). Selected patients with pulmonary metas-tases may survive for long periods after surgical resection and chemotherapy. Other potential sites of metastasis include bone (Fig. 36-5), brain (Fig. 36-6), and liver (Fig. 36-7). Visceral and retroperitoneal sarcomas have a higher incidence of liver and peritoneal metastases.Prognostic Factors. Prognostic variables in soft tissue sar-coma include primary tumor size, grade, and depth, all of which are incorporated into the staging system, as well as

1	peritoneal metastases.Prognostic Factors. Prognostic variables in soft tissue sar-coma include primary tumor size, grade, and depth, all of which are incorporated into the staging system, as well as histology, Brunicardi_Ch36_p1567-p1598.indd 157301/03/19 6:38 PM 1574SPECIFIC CONSIDERATIONSPART IIFigure 36-5. A 36-year-old woman with history of multifocal sclerosing osteosarcoma of the humerus developed diffuse bony metastases 2 years after diagnosis.Figure 36-6. A 64-year-old man with a history of a T2 high-grade pleomorphic sarcoma of the thigh who developed brain metastases 14 months after diagnosis.Figure 36-7. A 33-year-old woman with osteosarcoma of the sternum who presented with a liver metastasis 3 years after diagnosis.tumor site, and presentation (local recurrence or initial diagno-sis). Patient factors such as older age and gender have also been associated with recurrence and mortality in several studies.66 A positive microscopic margin and early recurrence after

1	diagno-sis). Patient factors such as older age and gender have also been associated with recurrence and mortality in several studies.66 A positive microscopic margin and early recurrence after resection of an extremity sarcoma have been shown to be associated with decreased survival.67Several groups have reported that Ki-67, a proliferation marker, is correlated with a poor clinical outcome in high-grade extremity sarcomas.68,69 E-cadherin and catenins, proteins essen-tial for intercellular junctions, have been associated with poor outcome in patients with soft tissue sarcoma.68 Similarly, higher CD100 expression has been shown to correlate with higher pro-liferative potential and poorer outcome.69Prognostic Nomograms. Prognostic nomograms for soft tis-sue sarcoma have been introduced for use in patient counsel-ing, selecting appropriate surveillance strategies, and selecting patients for clinical trials.70 One such nomogram, developed by Kattan and colleagues at Memorial

1	for use in patient counsel-ing, selecting appropriate surveillance strategies, and selecting patients for clinical trials.70 One such nomogram, developed by Kattan and colleagues at Memorial Sloan-Kettering Cancer Center, considers age, histology, grade, location, depth, and size to determine the likelihood of 12-year sarcoma-specific survival.70 Two validation studies using the nomogram demon-strated good predictive value.71 More recently, the same group of investigators developed histology subtype-specific nomo-grams for patients with liposarcoma, synovial sarcoma, and GIST72 and demonstrated that they were accurate in predicting disease-specific survival. Other investigators have just devel-oped a site-specific nomogram for patients with retroperitoneal sarcoma, demonstrating an accurate prediction of survival and disease recurrence.73TREATMENT OF EXTREMITY AND TRUNK WALL SARCOMAThe goals of treatment of soft tissue sarcoma are to maxi-mize the likelihood of long-term

1	an accurate prediction of survival and disease recurrence.73TREATMENT OF EXTREMITY AND TRUNK WALL SARCOMAThe goals of treatment of soft tissue sarcoma are to maxi-mize the likelihood of long-term recurrence-free survival while minimizing morbidity and maximizing function. In the past two decades, a multimodality treatment approach with optimal sequencing of treatments for individual patients has been shown to improve survival.74 Furthermore, patients with soft tissue sarcoma treated at high-volume centers have been shown to have improved survival and functional outcomes.75 Care at such centers is particularly important for patients with high-risk and advanced disease.The overall 5-year survival rate for patients with all stages of soft tissue sarcoma is 50% to 60%. For patients with extremity sarcomas, a multidisciplinary treatment approach has resulted in local control rates exceeding 90% and 5-year survival rates exceeding 70%. Most patients who die of soft tissue sarcoma die of

1	sarcomas, a multidisciplinary treatment approach has resulted in local control rates exceeding 90% and 5-year survival rates exceeding 70%. Most patients who die of soft tissue sarcoma die of metastatic disease, which becomes evident within 2 to 3 years of initial diagnosis in 80% of cases.Recommendations for evaluation and treatment of patients presenting with soft tissue masses are summarized in Table 36-3.Brunicardi_Ch36_p1567-p1598.indd 157401/03/19 6:38 PM 1575SOFT TISSUE SARCOMASCHAPTER 36Table 36-3Recommendations for the management of soft tissue masses1. Soft tissue tumors that are enlarging or greater than 3 cm should be evaluated with radiologic imaging (ultrasonography or computed tomography [CT]), and a tissue diagnosis should be made using core needle biopsy.2. Once a sarcoma diagnosis is established, obtain imaging (magnetic resonance imaging for extremity lesions and CT for other anatomic locations) and evaluate for metastatic disease with chest CT for

1	a sarcoma diagnosis is established, obtain imaging (magnetic resonance imaging for extremity lesions and CT for other anatomic locations) and evaluate for metastatic disease with chest CT for intermediateor high-grade (grade 2 or 3) or large (T2) tumors.3. A wide local excision with 1to 2-cm margins is adequate therapy for low-grade lesions and T1 tumors.4. Radiation therapy plays a critical role in the management of large (T2), intermediateor high-grade tumors.5. Patients with locally advanced high-grade sarcomas or distant metastases should be evaluated for chemotherapy.6. An aggressive surgical approach should be taken in the treatment of patients with an isolated local recurrence or resectable distant metastases.SurgeryPrimary tumors with no evidence of distant metastasis are managed with surgery alone or, when wide pathologic margins cannot be achieved because of anatomic constraints and/or the grade is high, surgery plus radiation therapy. The type of surgical resection is

1	with surgery alone or, when wide pathologic margins cannot be achieved because of anatomic constraints and/or the grade is high, surgery plus radiation therapy. The type of surgical resection is determined by several factors, including tumor location, tumor size, depth of invasion, involvement of nearby structures, need for skin grafting or autogenous tissue reconstruction, and the patient’s performance status. In 1985, the National Institutes of Health developed a consensus statement recommending limb-sparing surgery for most patients with high-grade extremity sarcomas.76 However, for patients with primary or recurrent tumors that cannot be grossly resected with a limb-sparing procedure and preservation of function (<5% of patients), amputation remains the treatment of choice.Margin status after surgical resection has been shown to be an independent prognostic factor.77,78 The goal of surgical resec-tion is to achieve a complete resection because microscopically positive or grossly

1	after surgical resection has been shown to be an independent prognostic factor.77,78 The goal of surgical resec-tion is to achieve a complete resection because microscopically positive or grossly positive resection margins are associated with increased risk of local recurrence and death.79 If an unex-pected positive margin is found on pathologic examination of the resection specimen, reexcision should be performed if fea-sible. In patients with a positive margin, particularly in patients with macroscopic residual disease, local control is unlikely even with the addition of postoperative radiation therapy, emphasiz-ing the importance of a well-planned initial operation.80Wide Local Excision. The preferred treatment for extrem-ity sarcomas is wide local excision that includes resection of the biopsy site. The goal of wide local excision is to remove the tumor with approximately 1 to 2 cm of surrounding nor-mal soft tissue,77 but narrower margins may be necessary to preserve uninvolved

1	the biopsy site. The goal of wide local excision is to remove the tumor with approximately 1 to 2 cm of surrounding nor-mal soft tissue,77 but narrower margins may be necessary to preserve uninvolved critical neurovascular structures and may be adequate for patients undergoing radiation therapy.81 Dis-section should proceed through grossly normal tissue planes not abutting the tumor. Soft tissue sarcomas are generally sur-rounded by a zone of compressed reactive tissue that forms a pseudocapsule; this pseudocapsule should not be used to guide resection (enucleation) as microscopic disease exists within this reactive zone. If the tumor is adjacent to or displacing major neurovascular structures, these do not need to be resected, but the adventitia or perineurium should be removed.1 For some massive tumors of the extremities, wide local excision entails a radical or complete anatomic compartment resection. Surgical clips should be placed to delineate the extent of the resection bed for

1	massive tumors of the extremities, wide local excision entails a radical or complete anatomic compartment resection. Surgical clips should be placed to delineate the extent of the resection bed for patients likely to require postoperative radiation therapy.Recent reports demonstrate encouraging results follow-ing radical en bloc resection with vascular reconstruction in the lower extremities.82,83 While en bloc resection with vascular reconstruction has been associated with increased rates of post-operative complications, reported local recurrence and 5-year survival rates are similar to those for patients not requiring ves-sel resection.84,85 Similarly, studies have shown acceptable func-tional outcomes with resection of the sciatic, tibial, and peroneal nerves with appropriate reconstruction and rehabilitation.86Bone invasion from extremity soft tissue sarcoma, which can generally be identified using high-quality cross-sectional imaging such as MRI, has been estimated to occur in

1	and rehabilitation.86Bone invasion from extremity soft tissue sarcoma, which can generally be identified using high-quality cross-sectional imaging such as MRI, has been estimated to occur in about 5% of patients and is associated with reduced overall survival.87 In cases of bone invasion, bone resection is required to obtain an adequate surgical margin and to achieve local control. Although tumor resection and repair of skeletal defects are possible, the likelihood of postoperative complications may be increased, and functional outcomes may be less favorable. Lin and colleagues88 recently analyzed 55 patients with soft tissue sarcomas abutting bone and reported that in the absence of frank cortical bone pen-etration, periosteum was an adequate surgical margin in patients treated with wide local excision and radiation.Soft tissue sarcomas arising in the distal extremities, particularly the hands and feet, present unique technical chal-lenges. While distal-extremity tumors are often

1	local excision and radiation.Soft tissue sarcomas arising in the distal extremities, particularly the hands and feet, present unique technical chal-lenges. While distal-extremity tumors are often detected at a smaller size (<5 cm) than proximal-extremity tumors, resec-tion and reconstruction techniques are often more complex for distal-extremity tumors, and preoperative planning is criti-cal to obtain favorable functional outcomes. Identifying the proximity of the tumor to underlying critical structures (e.g., bone, tendon, or neurovascular structures) using MRI is essen-tial for surgical planning. In a reported series of patients with sarcomas of the hands or feet treated with limited surgery only, 32% of patients had local recurrences.89 Preservation of function and acceptable recurrence rates with limited surgery and adjuvant radiation therapy for soft tissue sarcomas of the distal extremities have been reported.90 For locally advanced tumors, repair of bone defects, vascular

1	rates with limited surgery and adjuvant radiation therapy for soft tissue sarcomas of the distal extremities have been reported.90 For locally advanced tumors, repair of bone defects, vascular reconstruction, tendon transfers, and soft tissue reconstruction using regional or free flaps have resulted in good functional outcomes.91 Amputation remains a reasonable option for patients with soft tissue sar-comas of the distal extremities when acceptable oncologic or functional outcomes cannot be achieved using available limb salvage techniques.In an interesting study conducted in Ontario and Quebec, investigators found patients expecting a difficult recovery and patients with uncertain expectations had worse functional out-comes than patients anticipating an easy recovery, indicating that preoperative education including consultation with reha-bilitation services may optimize outcomes.92 Furthermore, all patients undergoing resection of extremity sarcomas should undergo physical therapy

1	education including consultation with reha-bilitation services may optimize outcomes.92 Furthermore, all patients undergoing resection of extremity sarcomas should undergo physical therapy beginning immediately after surgery and continuing until maximum function is achieved.1Brunicardi_Ch36_p1567-p1598.indd 157501/03/19 6:38 PM 1576SPECIFIC CONSIDERATIONSPART IILocoregional Lymphadenectomy. Several studies have reported improved survival for patients with isolated regional lymph node metastases treated with radical lymphadenectomy.28,63-65 Patients with clinically or radiologically suspicious regional nodes should have metastases confirmed by biopsy before radi-cal lymphadenectomy. An ultrasound-guided fine-needle aspi-ration or core biopsy of lymph nodes in selected patients with suspicious clinical or radiologic findings. The utility of sentinel lymph node biopsy has remained controversial despite the rec-ognition that several histologic subtypes of high-grade sarcoma are known

1	clinical or radiologic findings. The utility of sentinel lymph node biopsy has remained controversial despite the rec-ognition that several histologic subtypes of high-grade sarcoma are known to have a propensity for lymph node metastasis. However, there have been no prospective studies of the sen-sitivity and specificity of sentinel lymph node biopsy for such tumors, and as such, sentinel node biopsy for sarcoma should be performed in either highly selected patients or in the setting of a clinical trial.Amputation. Amputation is the treatment of choice for the 5% of patients with primary or recurrent extremity tumors whose tumors cannot be grossly resected with limb-sparing proce-dures and preservation of function. Historically, local excision of large, high-grade soft tissue sarcomas resulted in local fail-ure rates of 50% to 70%, even when a margin of normal tissue around the tumor was excised; consequently, radical resection or amputation was recommended. Today, however, the

1	resulted in local fail-ure rates of 50% to 70%, even when a margin of normal tissue around the tumor was excised; consequently, radical resection or amputation was recommended. Today, however, the addition of radiation therapy to less radical surgical resection has made limb salvage possible in most cases.A comparison of amputation versus limb-sparing sur-gery followed by adjuvant radiation therapy performed by the National Cancer Institute between 1975 and 1981 demonstrated no significant difference between the two groups in local recur-rence or overall survival rate.93 Potter and colleagues50 later reviewed the entire National Cancer Institute experience with 123 patients treated with conservative surgery plus radiation therapy and 83 treated with amputation. The local recurrence rate was significantly higher in the surgery and adjuvant radia-tion therapy group: 8% versus 0% in the amputation group. However, survival rates did not differ between the groups. Several large

1	rate was significantly higher in the surgery and adjuvant radia-tion therapy group: 8% versus 0% in the amputation group. However, survival rates did not differ between the groups. Several large single-institution studies have since also reported favorable local control rates with conservative resection plus radiation therapy.94-96Isolated Regional Perfusion. Isolated regional perfusion is a limb-sparing technique in which a soft tissue sarcoma is perfused with high concentrations of tumor necrosis factor-alpha (TNF-α) and melphalan under hyperthermic conditions. The use of TNF-α is not approved by the U.S. Food and Drug Administration (FDA) and is used only in European countries. The technique is generally used for locally advanced, multifo-cal, or locally recurrent disease; it has also served as a pallia-tive treatment to achieve local control for patients with distant metastases.Limb perfusion requires isolating the main artery and vein of the perfused limb from the systemic

1	also served as a pallia-tive treatment to achieve local control for patients with distant metastases.Limb perfusion requires isolating the main artery and vein of the perfused limb from the systemic circulation. The ana-tomic approach is determined by tumor site: external iliac ves-sels are used for thigh tumors, femoral or popliteal vessels for calf tumors, and axillary vessels for upper extremity tumors. The vessels are dissected, and all collateral vessels are ligated. The main artery and vein are then cannulated and connected to a pump oxygenator similar to that used in cardiopulmonary bypass. Either a tourniquet or an Esmarch band is applied to the limb to achieve complete vascular isolation. Chemotherapeutic agents are then added to the perfusion circuit and circulated for 90 minutes. Systemic leakage from the perfused limb is moni-tored continuously with 99Tc-radiolabeled human serum albumin injected into the perfusate, and radioactivity above the precor-dial area is recorded

1	Systemic leakage from the perfused limb is moni-tored continuously with 99Tc-radiolabeled human serum albumin injected into the perfusate, and radioactivity above the precor-dial area is recorded with a Geiger counter. During the entire procedure, hyperthermia of the perfused limb is maintained by external heating and by warming the perfusate to 40°C. At the end of the procedure, the limb is washed out, the cannulas are extracted, and the blood vessels are repaired.Despite the 40-year history of using isolated limb per-fusion to treat extremity sarcomas, many questions about this technique remain to be answered. The optimal chemotherapeu-tic agent in the perfusion circuit, the benefits of hyperthermia, and the effectiveness of hyperthermic perfusion as neoadjuvant or adjuvant treatment remain to be elucidated. Studies published to date have involved heterogeneous patient groups and various chemotherapeutic agents. Despite these limitations, response rates from 18% to 80% and overall

1	to be elucidated. Studies published to date have involved heterogeneous patient groups and various chemotherapeutic agents. Despite these limitations, response rates from 18% to 80% and overall 5-year survival rates from 50% to 70% have been reported.97-101 However, survival out-comes following isolated limb perfusion have not yet been directly compared with survival outcomes after more conven-tional treatment approaches.In the initial report of isolated regional perfusion for extremity sarcomas, published in 1974, McBride reported results in 79 patients with extremity sarcomas who had been treated with isolated limb perfusion during the previous 14 years.97 All patients received melphalan and dactinomycin. The overall 5-year survival rate was 57%, and only 13 patients had subsequent amputation for recurrent disease. Over the next 20 years, isolated perfusion for treatment of extremity sarcoma fell out of favor for several reasons. Most notably, improved survival and decreased local

1	for recurrent disease. Over the next 20 years, isolated perfusion for treatment of extremity sarcoma fell out of favor for several reasons. Most notably, improved survival and decreased local recurrence rates could be obtained with less radical therapy, including conservative surgical exci-sion combined with radiation to allow limb sparing in patients who were previously thought to require amputation.A 1992 report by Lienard and colleagues101 renewed interest in isolated limb perfusion for extremity tumors. Those investigators reported a 100% response rate among patients with extremity melanomas and sarcomas treated with high-dose recombinant TNF-α plus interferon-γ and melphalan in an iso-lated perfusion circuit. This report led to larger studies geared specifically to patients with sarcoma. The largest of these stud-ies, the European Multicenter Study, was reported by Egger-mont and colleagues in 1996.99 In that study of 186 patients, the overall tumor response rate was 82%, and the

1	The largest of these stud-ies, the European Multicenter Study, was reported by Egger-mont and colleagues in 1996.99 In that study of 186 patients, the overall tumor response rate was 82%, and the clinical and pathologic complete response rate was 29%. Although all of the study participants were reported to initially be candidates for amputation, the rate of limb salvage following isolated limb perfusion was 82%.99 Subsequent studies have shown high local response and limb salvage rates and acceptable local and sys-temic toxic effects.102However, results in the United States have been inferior to those reported in Europe. In a study by Fraker and colleagues, the complete response rate was 26%, and an additional 30% of patients had a partial response. Fourteen patients (32%) under-went amputation for progressive tumors, while the remaining 30 patients (68%) were able to undergo limb-sparing surgery after isolated limb perfusion.100 The inferior results in the U.S.-based studies are

1	amputation for progressive tumors, while the remaining 30 patients (68%) were able to undergo limb-sparing surgery after isolated limb perfusion.100 The inferior results in the U.S.-based studies are thought to be due to patient selection biases and the degree of treatment before limb perfusion.While isolated limb perfusion for extremity sarcoma has fallen out of favor, recent reports of isolated limb infusion from the H. Lee Moffitt Cancer Center have shown promising Brunicardi_Ch36_p1567-p1598.indd 157601/03/19 6:38 PM 1577SOFT TISSUE SARCOMASCHAPTER 36results.103 Patients with extremity sarcoma and who are con-sidered for amputation are offered the option of isolated limb infusion with high-dose melphalan and actinomycin-D on pro-tocol. Isolated limb infusion is a less invasive technique that can be repeated. Percutaneous cannulas are placed prior to infu-sion, the extremity is isolated similar to limb perfusion with an Esmarch band or tourniquet, and the perfusion is

1	technique that can be repeated. Percutaneous cannulas are placed prior to infu-sion, the extremity is isolated similar to limb perfusion with an Esmarch band or tourniquet, and the perfusion is normothermic and acidotic. Results from a multicenter retrospective study have demonstrated an overall response rate of 58%, and after a median follow-up of 21 months, there was an overall limb sal-vage rate of 78%. The benefits over limb perfusion remain the ability to repeat the technique in patients with disease response and less operative morbidity and risk of vascular injury from open surgery and cannulation of the iliac vessels. Although to date the technique has been well established for patients with locally advanced extremity disease for melanoma, its applica-tion for advanced, locally recurrent extremity sarcoma deserves further study.Radiation TherapyRadiation therapy is part of the standard treatment for high-grade extremity and trunk wall soft tissue sarcomas either in the preor

1	extremity sarcoma deserves further study.Radiation TherapyRadiation therapy is part of the standard treatment for high-grade extremity and trunk wall soft tissue sarcomas either in the preor postoperative setting. Patients with low-grade tumors or small, superficial high-grade tumors that have been resected with adequate margins may safely avoid radiation therapy.The evidence supporting adjuvant radiation therapy for patients eligible for conservative surgical resection comes from two randomized trials104,105 and three large single-institution reports.106-108 In a randomized trial by the National Cancer Institute, 91 patients with high-grade extremity tumors were treated with limb-sparing surgery followed by chemotherapy alone or radiation therapy plus chemotherapy. The 10-year local control rate was 98% for patients receiving radiation therapy compared with 70% for those not receiving radiation therapy (P = .0001).104 Similarly, in a randomized trial from Memorial Sloan-Kettering

1	rate was 98% for patients receiving radiation therapy compared with 70% for those not receiving radiation therapy (P = .0001).104 Similarly, in a randomized trial from Memorial Sloan-Kettering Cancer Center, 164 patients underwent conservative surgery followed by observation or brachytherapy. For patients with high-grade tumors, the 5-year local control rate was 66% in the observation group and 89% in the brachytherapy group (P = .003).105 For patients with low-grade tumors, no significant difference was observed between treatment groups.109Until recently, the standard treatment guidelines required radiation therapy after surgery for all patients with intermediate or high-grade tumors of any size. However, small tumors (≤5 cm) have not generally been associated with local recurrence, and radiation therapy for such tumors may not be necessary.105 In a series of 174 patients reported by Geer and colleagues, post-operative radiation therapy did not improve 5-year local recur-rence or

1	radiation therapy for such tumors may not be necessary.105 In a series of 174 patients reported by Geer and colleagues, post-operative radiation therapy did not improve 5-year local recur-rence or overall survival rates for patients with small soft tissue sarcomas.110 Karakousis and colleagues reported a 5-year local recurrence rate of 6% for 80 patients with extremity sarcomas treated with wide local excision and observation, a rate similar to that for the 64 patients who underwent resection with nar-rower surgical margins and postoperative radiation therapy.111The optimal mode of radiation therapy (external-beam radiation therapy, brachytherapy, or intensity-modulated radia-tion therapy [IMRT]) and timing of radiation therapy (preopera-tive, intraoperative, or postoperative) have yet to be defined. External-beam radiation therapy can be delivered using pho-tons or particle beams (electrons, protons, pions, or neutrons). Conventional fractionation is usually 1.8 to 2 Gy per day. CT

1	be defined. External-beam radiation therapy can be delivered using pho-tons or particle beams (electrons, protons, pions, or neutrons). Conventional fractionation is usually 1.8 to 2 Gy per day. CT is an integral part of radiation therapy, used to define the gross tumor volume and to estimate the margin of tissue at risk for microscopic tumor involvement. The optimal radiation margin is not well defined: a margin of 5 to 7 cm is standard, but some centers advocate wider margins for tumors larger than 15 cm. At most institutions, the typical preoperative dose is 50 Gy given in 25 fractions, and resection is performed 4 to 8 weeks after completion of radiation therapy to allow acute radiation changes to subside. Postoperative radiation therapy planning is based on tumor site, tumor grade, surgical margins, and institutional preferences. The entire surgical scar and drain sites should be included in the field so that a near-full dose can be administered to the superficial skin. Metallic

1	margins, and institutional preferences. The entire surgical scar and drain sites should be included in the field so that a near-full dose can be administered to the superficial skin. Metallic clips placed in the tumor bed during surgery can help define the limits of the resection and aid in radiation therapy planning. Doses of 60 to 70 Gy are usually necessary for postoperative treatment.No consensus exists on the optimal sequence of radia-tion therapy and surgery. The available data come largely from single-institution, nonrandomized studies. Proponents of pre-operative radiation therapy note that multidisciplinary planning with radiation oncologists, medical oncologists, and surgeons is easier early in the course of therapy. In addition, for some radiosensitive histologic subtypes, such as myxoid liposarcoma, preoperative radiation therapy may shrink the tumor, facilitat-ing resection with negative margins. Furthermore, a tissue bed undisturbed by resection has better tissue

1	such as myxoid liposarcoma, preoperative radiation therapy may shrink the tumor, facilitat-ing resection with negative margins. Furthermore, a tissue bed undisturbed by resection has better tissue oxygenation and can be successfully treated with lower doses of radiation. In addi-tion, Nielsen and colleagues112 demonstrated that preoperative radiation fields are smaller than postoperative radiation fields and that the average number of joints included in the field is lower with preoperative than postoperative radiation therapy, which may result in improved functional outcome. Critics of preoperative radiation therapy cite the difficulty of pathologic assessment of margins and the increased rate of postoperative wound complications.113 However, reconstructive surgical tech-niques with advanced tissue transfer procedures are being used more often in these high-risk wounds and reportedly result in better outcomes. The higher doses generally required for post-operative radiation therapy

1	tissue transfer procedures are being used more often in these high-risk wounds and reportedly result in better outcomes. The higher doses generally required for post-operative radiation therapy have also been shown to be associ-ated with greater long-term functional impairment.The only randomized comparison of preoperative and postoperative radiation therapy to date was performed by the National Cancer Institute of Canada Clinical Trials–Canadian Sarcoma Group.114 This trial was designed to examine compli-cations and functional outcome. The 190 patients enrolled from October 1994 to December 1997 were randomized to preopera-tive radiation therapy (50 Gy) or postoperative radiation therapy (66 Gy). With a median follow-up time of 3.3 years, the recur-rence and progression-free survival rates were similar in the two groups. The incidence of wound complications was higher in the preoperative group versus the postoperative group (35% vs. 17%), and the incidence of wound complications was

1	were similar in the two groups. The incidence of wound complications was higher in the preoperative group versus the postoperative group (35% vs. 17%), and the incidence of wound complications was signifi-cantly higher for tumors of the lower extremity (43%) than for those of the upper extremity (5%).114 Late radiation toxic effects (e.g., fibrosis, joint stiffness, and edema) were more common with postoperative than preoperative radiation therapy (48% vs. 32%) because of higher postoperative radiation doses and larger treatment field sizes.115Brachytherapy involves the placement of multiple radioactive seeds through catheters inserted in the tumor resection bed. The primary benefit of brachytherapy is the shorter overall treatment time of 4 to 6 days, compared to the 4 to 6 weeks generally required for preoperative or postoperative Brunicardi_Ch36_p1567-p1598.indd 157701/03/19 6:38 PM 1578SPECIFIC CONSIDERATIONSPART IIradiation therapy regimens. A cost-analysis comparison of

1	required for preoperative or postoperative Brunicardi_Ch36_p1567-p1598.indd 157701/03/19 6:38 PM 1578SPECIFIC CONSIDERATIONSPART IIradiation therapy regimens. A cost-analysis comparison of adjuvant brachytherapy versus adjuvant external-beam irradiation for soft tissue sarcomas showed that costs were lower with brachytherapy.116 The implications of cost have been studied after radical resections and immediate adjuvant brachytherapy with either staged or immediate reconstruction noting a lower cost with staged reconstruction and tissue transfer techniques.117 Brachytherapy can also be used for recurrent disease previously treated with external-beam radiation. Guidelines established at Memorial Sloan-Kettering Cancer Center recommend spacing the afterloading catheters in 1-cm increments while leaving a 2-cm margin around the surgical bed.105 After adequate wound healing is confirmed, usually after the fifth postoperative day, the catheters are loaded with seeds containing

1	increments while leaving a 2-cm margin around the surgical bed.105 After adequate wound healing is confirmed, usually after the fifth postoperative day, the catheters are loaded with seeds containing iridium-192 that deliver 42 to 45 Gy of radiation to the tumor bed over 4 to 6 days. Subsequent studies at the H. Lee Moffitt Cancer Center that sought to determine outcomes between staged immediate and delayed reconstruction noted advantages in improved local control, wound healing, and less radiation-associated toxicity with staged reconstruction.118 The primary disadvantage of brachytherapy is that it requires significant expertise, extended inpatient hospital stays, and bed rest.IMRT delivers radiation more precisely to the tumor than external-beam irradiation while minimizing the volume of sur-rounding tissues exposed to high radiation doses. The proposed benefits of preoperative IMRT include reduced risk of postop-erative wound infections because of minimization of the dose to the

1	of sur-rounding tissues exposed to high radiation doses. The proposed benefits of preoperative IMRT include reduced risk of postop-erative wound infections because of minimization of the dose to the skin119 and protection of underlying bone (e.g., femur) as a result of concave dose distributions.120 There have been no prospective randomized trials comparing the long-term out-comes following IMRT versus other types of radiation therapy. In a retrospective analysis of IMRT, patients with negative and positive/close (within 1 mm) margins were found to have 5-year local control rates of 94%.121 In addition, the rates of posttreat-ment edema and joint stiffness with IMRT were lower than the expected rates with conventional radiation therapy.Local toxic effects of radiation therapy vary according to radiation dose, field size, and timing (preoperative or postop-erative). With preoperative radiation therapy, the most frequent wound complications are wound dehiscence, wound necrosis,

1	according to radiation dose, field size, and timing (preoperative or postop-erative). With preoperative radiation therapy, the most frequent wound complications are wound dehiscence, wound necrosis, persistent drainage, infection, seroma formation, ulceration, and cellulitis.114 Postoperative irradiation of free flaps is often asso-ciated with wound complications, and patients should be advised that secondary surgical repair may be necessary; therefore, con-sideration for preoperative radiation rather than postoperative radiation for larger tumors requiring flap reconstruction is a logical and sound approach. Wound complication rates of 13% to 37% have been reported for preoperative radiation therapy, compared to 5% to 20% for postoperative radiation therapy.121 If catheters are loaded after the fifth postoperative day and/or if staged reconstruction is used, rates of wound complications after brachytherapy are improved if not similar to those after postoperative radiation

1	loaded after the fifth postoperative day and/or if staged reconstruction is used, rates of wound complications after brachytherapy are improved if not similar to those after postoperative radiation therapy.118Long-term (chronic) effects of radiation therapy (those occurring >1 year after completion of therapy) are generally related to fibrosis/contractures, lymphedema, neurologic injury, osteitis, and fractures, all of which can cause substantial func-tional impairment.122 Variables associated with poorer func-tional outcome after radiation therapy include larger tumors, higher doses of radiation (>63 Gy), longer radiation fields (>35 cm), poor radiation technique, neural sacrifice, postop-erative fractures, and wound complications.115,123 Additionally, complications of any kind are less likely after treatment for upper extremity sarcoma than after treatment for lower extremity sarcoma.113,114Definitive radiation therapy that delivers maximal-tissue-tolerance doses of radiation may

1	likely after treatment for upper extremity sarcoma than after treatment for lower extremity sarcoma.113,114Definitive radiation therapy that delivers maximal-tissue-tolerance doses of radiation may be appropriate for selected patients with unresectable soft tissue sarcomas. In a study of 112 patients with unresectable soft tissue sarcomas, tumor size and radiation dose were found to influence local control and survival.124 The local control rate was 51% for tumors smaller than 5 cm and 9% for tumors larger than 10 cm, and patients who received at least 64 Gy had better local control and survival.Systemic TherapyDespite improvements in local control rates, metastasis and death remain significant problems for patients with high-risk soft tissue sarcomas. Patients considered at high risk of death from sarcoma include those presenting with metastatic disease, localized sarcomas at nonextremity sites, or sarcomas of intermediateor high-grade histology larger than 5 cm.59,105Standard

1	of death from sarcoma include those presenting with metastatic disease, localized sarcomas at nonextremity sites, or sarcomas of intermediateor high-grade histology larger than 5 cm.59,105Standard Chemotherapy. For most patients with sarcoma, results of conventional chemotherapy regimens have been poor. The chemosensitivity of soft tissue sarcoma varies by histologic subtype.30 Synovial sarcoma, myxoid/round cell liposarcoma, and uterine leiomyosarcoma are sensitive to chemotherapy,125 whereas pleomorphic liposarcoma, myxofibrosarcoma, epithe-lioid sarcoma, leiomyosarcoma, MPNSTs, angiosarcoma, and desmoplastic round cell tumors have intermediate sensitivity to chemotherapy. Relatively chemoresistant histologic subtypes include clear cell sarcoma, endometrial stromal sarcoma, alveo-lar soft part sarcoma, and extraskeletal myxoid chondrosarcoma. Considering the variability of responses by histologic subtype, it is not surprising that clinical trials of standard chemotherapy, which

1	soft part sarcoma, and extraskeletal myxoid chondrosarcoma. Considering the variability of responses by histologic subtype, it is not surprising that clinical trials of standard chemotherapy, which often include heterogeneous populations with respect to tumor grade and histology, have demonstrated no overall sur-vival benefit.Doxorubicin and ifosfamide are the two most active agents against soft tissue sarcoma, with consistently reported response rates of 20% or greater and positive dose-response curves.126,127 The European guidelines recommend doxorubicin 75 mg/m2 every 3 weeks as first-line treatment for advanced disease.30 Treatment duration is based on response, but a maximum of six cycles is generally recommended because of the risk of cumula-tive cardiotoxicity. Ifosfamide is the recommended second-line treatment and is recommended for first-line treatment in patients with cardiac morbidity. The standard dose of ifosfamide is 9 to 10 g/m2; however, single-institution series

1	recommended second-line treatment and is recommended for first-line treatment in patients with cardiac morbidity. The standard dose of ifosfamide is 9 to 10 g/m2; however, single-institution series using higher-dose regimens (>10 g/m2) or standard-dose ifosfamide combined with doxorubicin have shown response rates of 20% to 60%.127 Synovial sarcomas have been shown to be particularly sensi-tive to ifosfamide. Ifosfamide-associated toxic effects include hemorrhagic cystitis, neurotoxicity, and renal tubular acidosis. Historically, combination therapy with doxorubicin plus ifos-famide, dacarbazine, or both has resulted in increased response rates but no improvement in overall survival.128 Dacarbazine as a single agent has also demonstrated activity in clinical trials.Over the past decade, several additional chemotherapeutic agents, including gemcitabine, taxanes, and trabectedin, have been noted to be active against soft tissue sarcomas. Gemcitabine as a single agent was reported to

1	several additional chemotherapeutic agents, including gemcitabine, taxanes, and trabectedin, have been noted to be active against soft tissue sarcomas. Gemcitabine as a single agent was reported to produce responses in 18% of patients with advanced sarcoma.129 Gemcitabine combined with docetaxel has been reported to produce response rates as high as 53% in patients with uterine leiomyosarcoma.129,130 Gemcitabine Brunicardi_Ch36_p1567-p1598.indd 157801/03/19 6:38 PM 1579SOFT TISSUE SARCOMASCHAPTER 36combined with vinorelbine has also been associated with clini-cal benefit in patients with advanced sarcomas.131 The taxanes (docetaxel and paclitaxel) have been found to be active against angiosarcomas, particularly of the face and scalp, likely because of their potent antiangiogenic effects.132,133Novel Chemotherapeutic Agents. Aldoxorubicin is a doxo-rubicin derivative that serves as a prodrug of doxorubicin that covalently binds to albumin in the blood until reaching the acidic

1	Chemotherapeutic Agents. Aldoxorubicin is a doxo-rubicin derivative that serves as a prodrug of doxorubicin that covalently binds to albumin in the blood until reaching the acidic tumor environment releasing doxorubicin into the tis-sue. A recent international, multicenter, phase 2b, open-label, randomized study enrolled 126 patients from 2012 to 2013. Single-agent aldoxorubicin therapy showed superior efficacy over doxorubicin by prolonging progression-free survival and improving rates of 6-month progression-free survival and tumor response, warranting further study.134Trabectedin, a marine-derived alkaloid that binds DNA, affecting transcription and inducing the formation of DNA double-strand breaks, has shown benefit in the treatment of advanced soft tissue sarcomas, particularly leiomyosarcoma, myxoid liposarcoma, and other translocation-related sarcomas.135 Trabectedin is generally well tolerated but can be associated with prolonged and severe neutropenia, thrombocytopenia, and

1	myxoid liposarcoma, and other translocation-related sarcomas.135 Trabectedin is generally well tolerated but can be associated with prolonged and severe neutropenia, thrombocytopenia, and hepatic toxic effects.Palifosfamide is a stabilized formulation of the active metabolite of ifosfamide that has been reported to be better tolerated than ifosfamide.136 Early trials have suggested anti-tumor activity comparable or superior to that of ifosfamide without nephrotoxicity; however, recent negative results of the PICASSO III Trial, a phase 3, placebo-controlled study of doxorubicin with or without palifosfamide in patients with metastatic soft tissue sarcoma, have neatly secured the fate of palifosfamide in the treatment of soft tissue sarcoma.137Targeted Therapies. Several targeted agents are being investigated for the treatment of soft tissue sarcomas. Among these are tyrosine kinase inhibitors (e.g., imatinib, suni-tinib, sorafenib, and dasatinib) that have been developed and approved

1	being investigated for the treatment of soft tissue sarcomas. Among these are tyrosine kinase inhibitors (e.g., imatinib, suni-tinib, sorafenib, and dasatinib) that have been developed and approved for treatment of GIST. Clinical data accumulated in phase 2 trials also support the use of tyrosine kinase inhibitors (e.g., imatinib, sorafenib, and sunitinib) in the management of other advanced sarcomas.128 Anti–vascular endothelial growth factor antibodies such as bevacizumab have demonstrated activity in patients with metastatic or unresectable angiosar-coma, solitary fibrous tumor, and epithelioid hemangioendo-thelioma.138 Pazopanib is an oral angiogenesis inhibitor that targets vascular endothelial growth factor receptors, platelet-derived growth factor receptor (PDGFR), and c-kit. In a recent phase 3 study, pazopanib showed efficacy against placebo in second or further line of therapy in patients with advanced soft tissue sarcoma.139 Inhibitors of the mammalian target of rapamycin

1	recent phase 3 study, pazopanib showed efficacy against placebo in second or further line of therapy in patients with advanced soft tissue sarcoma.139 Inhibitors of the mammalian target of rapamycin pathway, including temsirolimus, everolimus, and ridaforolimus, have also shown activity against some soft tis-sue sarcomas (i.e., PEComas).140Olaratumab is a human antiplatelet-derived growth factor receptor α monoclonal antibody that has antitumor activity in human sarcoma xenografts. Recently, a phase 1b and random-ized phase 2 study of olaratumab and doxorubicin versus doxo-rubicin alone has demonstrated improvements in both objective response rates (18.2% vs. 11.9%, P = 0.34) and median over-all survival (26.5 vs. 14.7 months [stratified hazard ratio 0.46, 0.30–0.71, P = 0.0003]). Additional studies are warranted for this promising combination of agents.141Benefits of Systemic Therapy. The use of adjuvant and neoad-juvant chemotherapy for soft tissue sarcomas remains controver-sial.

1	studies are warranted for this promising combination of agents.141Benefits of Systemic Therapy. The use of adjuvant and neoad-juvant chemotherapy for soft tissue sarcomas remains controver-sial. More than a dozen individual randomized trials of adjuvant chemotherapy have failed to demonstrate improvement in disease-free or overall survival for patients with soft tissue sarcoma. How-ever, several limitations of these individual trials may explain the lack of observed improvement. First, the chemotherapy regimens used were suboptimal, consisting of single-agent therapy (most commonly with doxorubicin) and insufficiently intensive dosing schedules. Second, the patient groups were not large enough to reveal clinically significant differences in survival rates. Finally, most studies included patients at low risk of metastasis and death, namely those with small (<5 cm) and low-grade tumors.The Sarcoma Meta-Analysis Collaboration analyzed 1568 patients from 14 trials of doxorubicin-based

1	patients at low risk of metastasis and death, namely those with small (<5 cm) and low-grade tumors.The Sarcoma Meta-Analysis Collaboration analyzed 1568 patients from 14 trials of doxorubicin-based adjuvant chemotherapy to evaluate the effect of adjuvant chemotherapy on localized, resectable soft tissue sarcomas.142 At a median follow-up time of 9.4 years, doxorubicin-based chemotherapy significantly improved the time to local and distant recurrence and recurrence-free survival rates. However, the absolute benefit in overall survival was only 4%, which was not significant (P = .12). In a subset analysis, patients with extremity tumors had a 7% benefit in terms of overall survival (P = .029).142After this meta-analysis, randomized controlled trials of more contemporary anthracycline/ifosfamide dosing combinations with relatively small numbers of patients have yielded conflicting results. In an Italian cooperative trial, adjuvant chemotherapy improved median disease-free and overall

1	dosing combinations with relatively small numbers of patients have yielded conflicting results. In an Italian cooperative trial, adjuvant chemotherapy improved median disease-free and overall survival times in patients with high-risk extremity soft tissue sarcomas.143 In that study, 104 patients with high-grade tumors 5 cm or larger were randomized to definitive surgery or surgery plus adjuvant chemotherapy consisting of epirubicin (60 mg/m2 per day on days 1 and 2) and ifosfamide (1.8 g/m2 per day on days 1 through 5) for five cycles. With a median follow-up time of almost 5 years, disease-free survival times were 16 months in the surgery-alone group and 48 months in the combined-treatment group (P = .04), and median overall survival times were 46 months in the surgery-alone group and 75 months in the combined-treatment group (P = .03).143 However, several years later, the surgery-alone and combined-treatment groups had equivalent relapse rates and deaths, which resulted in

1	and 75 months in the combined-treatment group (P = .03).143 However, several years later, the surgery-alone and combined-treatment groups had equivalent relapse rates and deaths, which resulted in statistically similar overall survival.144In an effort to further assess the role of chemotherapy in patients with stage III extremity sarcoma, a cohort analy-sis of the combined databases of The University of Texas MD Anderson Cancer Center and Memorial Sloan-Kettering Can-cer Center was performed. Data on 674 patients with stage III extremity sarcoma who received either preoperative or postop-erative doxorubicin-based chemotherapy were reviewed. The 5-year disease-specific survival rate was 61%.145 Cox regres-sion analysis showed a time-varying effect of chemotherapy with an associated benefit during the first year while receiving chemotherapy. However, the clinical benefits of chemotherapy in patients with stage III sarcomas were not sustained beyond 1 year. Grobmyer and colleagues

1	during the first year while receiving chemotherapy. However, the clinical benefits of chemotherapy in patients with stage III sarcomas were not sustained beyond 1 year. Grobmyer and colleagues compared the outcomes of patients treated at two institutions (1990–2001) with surgery only or surgery plus preoperative chemotherapy containing doxorubicin and ifosfamide. In this analysis, chemotherapy was associated with an improvement in the 3-year disease-specific survival rate that was most pronounced in patients with tumors larger than 10 cm (62% for surgery alone vs. 83% for neoadju-vant chemotherapy and surgery).146Brunicardi_Ch36_p1567-p1598.indd 157901/03/19 6:38 PM 1580SPECIFIC CONSIDERATIONSPART IIMore recently, the European Organization for Research and Treatment of Cancer (EORTC) completed a phase 3 ran-domized study (trial EORTC-62931; conducted from 1995 through 2003) comparing surgery alone versus surgery plus adjuvant ifosfamide (5 g/m2) plus doxorubicin (75 mg/m2) with

1	completed a phase 3 ran-domized study (trial EORTC-62931; conducted from 1995 through 2003) comparing surgery alone versus surgery plus adjuvant ifosfamide (5 g/m2) plus doxorubicin (75 mg/m2) with growth factor support (lenograstim) every 21 days for five cycles in 351 patients with resected grade II or III soft tissue sarcoma at any site. The estimated relapse-free survival rate was 52% in both arms, and the overall survival rate was better in the control arm (69% vs. 64%).147 Although most individual studies are underpowered, data from all of these studies suggest that chemotherapy regimens that incorporate ifosfamide may provide some disease-free survival benefit but do not improve long-term overall survival for the majority of patients with soft tissue sarcoma.In 2008, two updates to the 1997 Sarcoma Meta-Anal-ysis Collaboration were published.148,149 O’Connor and col-leagues included all of the trials in the original meta-analysis and added data from four additional trials, for

1	the 1997 Sarcoma Meta-Anal-ysis Collaboration were published.148,149 O’Connor and col-leagues included all of the trials in the original meta-analysis and added data from four additional trials, for a total of 18 trials with 2170 patients.148 The results showed a benefit of chemotherapy in terms of disease-free survival at 5 years and recurrence-free survival at 10 years but again failed to demon-strate a benefit in terms of long-term overall survival. The sec-ond update, by Pervaiz and colleagues, which did not include the EORTC-62931 trial, showed that adjuvant chemotherapy was associated with a significant decrease in the risk of death (hazard ratio, 0.77; P = .01).149Because the evidence regarding adjuvant systemic therapy for stage III soft tissue sarcoma is inconclusive, considerable variation still exists in treatment recommendations even though patients with large, stage II or stage III soft tissue sarcomas are at high risk for recurrence and metastasis. Chemotherapy may be

1	variation still exists in treatment recommendations even though patients with large, stage II or stage III soft tissue sarcomas are at high risk for recurrence and metastasis. Chemotherapy may be considered to downstage large tumors to enable limb-sparing procedures, particularly for tumors known to be chemosensi-tive. It is likely that subsets of high-risk patients with extremity soft tissue sarcoma defined on the basis of tumor size or histol-ogy derive significant benefit from systemic chemotherapy. For example, retrospective cohort analyses have noted a disease-specific survival benefit in patients with large, high-grade lipo-sarcomas and synovial sarcomas of the extremity treated with ifosfamide plus doxorubicin versus no chemotherapy.150Neoadjuvant (Preoperative) Chemotherapy. The use of neoadjuvant (preoperative) chemotherapy for soft tissue sarco-mas is based on the belief that only 30% to 50% of patients respond to standard adjuvant (postoperative) chemotherapy. The rationale

1	of neoadjuvant (preoperative) chemotherapy for soft tissue sarco-mas is based on the belief that only 30% to 50% of patients respond to standard adjuvant (postoperative) chemotherapy. The rationale for using neoadjuvant chemotherapy is that it enables oncologists to identify patients whose disease is sensi-tive to a particular chemotherapy regimen by assessing response while the primary tumor is in situ. Patients whose tumors do not respond to short courses of neoadjuvant chemotherapy can thus be spared the toxic effects of prolonged adjuvant chemotherapy. Another advantage of neoadjuvant chemotherapy is that it may shrink tumors, enabling less morbid operations. The theoreti-cal disadvantages of neoadjuvant chemotherapy are related to myelosuppression and potential postoperative wound healing complications.A recent randomized study comparing three preoperative cycles of full-dose anthracycline-ifosfamide–based chemother-apy with three preoperative plus two postoperative cycles of the

1	complications.A recent randomized study comparing three preoperative cycles of full-dose anthracycline-ifosfamide–based chemother-apy with three preoperative plus two postoperative cycles of the same regimen in high-risk extremity and trunk wall soft tissue sarcomas showed equivalence between the two approaches, sug-gesting the possibility of limiting chemotherapy administration to the three preoperative courses, improving the ratio between toxicity and expected benefit.151A subanalysis on response showed how tumor attenuation on CT scan and MRI obtained by the administration of such preoperative treatment was associated with a higher percentage of pathologic necrosis152 and better outcome.153Eilber and colleagues examined treatment-induced patho-logic necrosis in patients who received neoadjuvant therapy for high-grade extremity sarcomas.154 The addition of ifosfamide to other agents (doxorubicin alone or doxorubicin and cisplatin) increased the rate of pathologic necrosis to 48%

1	neoadjuvant therapy for high-grade extremity sarcomas.154 The addition of ifosfamide to other agents (doxorubicin alone or doxorubicin and cisplatin) increased the rate of pathologic necrosis to 48% compared to 13% with other combinations. The 5and 10-year local recur-rence rates were significantly lower for patients with 95% or greater pathologic necrosis (6% and 11%, respectively) than for patients with less than 95% pathologic necrosis (17% and 23%, respectively).Concurrent Chemoradiation TherapyTreatment approaches that combine systemic chemotherapy with radiosensitizers and concurrent external-beam radiation therapy may improve disease-free survival by treating micro-scopic disease and enhancing the treatment of macroscopic disease. Concurrent chemoradiation therapy with doxorubicin-based regimens reportedly produces favorable local control rates for patients with sarcoma.150 Since those findings were published, several groups have evaluated routes of administra-tion, alternative

1	regimens reportedly produces favorable local control rates for patients with sarcoma.150 Since those findings were published, several groups have evaluated routes of administra-tion, alternative chemotherapeutic agents, and the toxicity of combined therapies.Theoretical advantages notwithstanding, concurrent chemoradiation therapy decreases the total treatment time for patients with high-risk sarcoma. This decrease represents a sub-stantial advantage over current sequential combined-modality treatment approaches, for which the total duration of radiation therapy, chemotherapy, surgery, and rehabilitation frequently exceeds 6 to 9 months.Posttreatment SurveillanceCurrently, posttreatment surveillance is recommended for all patients with soft tissue sarcomas based on a few reports involving small numbers of patients, indicating that local recur-rence can be successfully treated with radical reexcision with or without radiation therapy.155,156 Similarly, several groups have reported that

1	small numbers of patients, indicating that local recur-rence can be successfully treated with radical reexcision with or without radiation therapy.155,156 Similarly, several groups have reported that survival can be prolonged by resection of pulmo-nary metastases.157-159The National Comprehensive Cancer Network (NCCN) recommends a history and physical and chest CT or radiog-raphy every 3 to 6 months for 2 to 3 years after completion of treatment. Because most cases of distant metastasis occur within 2 to 3 years of initial diagnosis, the NCCN guidelines indicate that follow-up intervals can be lengthened to every 6 months, and imaging can be done annually during years 2 through 5.1 Consideration should also be given to imaging the primary tumor site; most experts recommend that the tumor site be evaluated every 6 months with MRI for extremity tumors or CT for intra-abdominal or retroperitoneal tumors. Guidelines have been established for using MRI to distinguish recurrences from

1	site be evaluated every 6 months with MRI for extremity tumors or CT for intra-abdominal or retroperitoneal tumors. Guidelines have been established for using MRI to distinguish recurrences from typical postsurgical changes: a discrete nodule with low signal intensity on T1-weighted images and higher signal intensity on T2-weighted images that enhances after adminis-tration of intravenous contrast material is strongly suggestive of recurrence and should be biopsied. Ultrasonography may be an alternative to MRI or CT for assessing for recurrence in the extremities.Brunicardi_Ch36_p1567-p1598.indd 158001/03/19 6:38 PM 1581SOFT TISSUE SARCOMASCHAPTER 36Recurrence is common after surgery for abdominal soft tissue sarcomas. CT is useful for detecting recurrences at primary and distant anatomic sites in the abdomen and pelvis. After surgery, CT every 3 to 6 months during the first 2 years and every 6 months for 3 years thereafter has been recommended. However, today many experienced

1	sites in the abdomen and pelvis. After surgery, CT every 3 to 6 months during the first 2 years and every 6 months for 3 years thereafter has been recommended. However, today many experienced surgeons are advocating less aggressive imaging for asymptomatic patients, particularly after a second recurrence of retroperitoneal sarcoma, arguing that there is insufficient evidence to suggest that survival is improved by earlier detection.Whooley and colleagues reviewed the efficacy of the sur-veillance strategy used at Roswell Park Cancer Institute for 174 patients with soft tissue sarcomas of the extremities.160 Patients were evaluated every 3 months for the first 2 years, every 4 to 6 months during year 3, and every 6 months during years 4 and 5. Local recurrence occurred in 18% of patients at a median time after completion of treatment of 14 months, and all but one of the recurrences were detected with physical examination alone. Fifty-seven patients had distant recurrences (at a

1	at a median time after completion of treatment of 14 months, and all but one of the recurrences were detected with physical examination alone. Fifty-seven patients had distant recurrences (at a median of 18 months after treatment), of which 36 were asymptomatic and diagnosed by surveillance imaging. The investigators determined that the positive predictive value of chest radiography during follow-up was 92%.160 However, evaluation of the primary tumor site by CT or MRI was ineffective in detecting recurrences. The authors recommended that patient characteristics, location of the primary tumor, previous treatment, and physician familiarity with changes after surgery and radiation therapy should all be considered in determining the need for radiographic imaging.Management of Recurrent SarcomaUp to 20% of patients with extremity sarcoma develop locally recurrent disease, which is often accompanied by distant metastases; thus, all patients with recurrent extremity sarcoma should undergo

1	to 20% of patients with extremity sarcoma develop locally recurrent disease, which is often accompanied by distant metastases; thus, all patients with recurrent extremity sarcoma should undergo a full staging assessment. Patients with microscopically positive surgical margins are at increased risk of local recurrence. In a series of 179 patients with locally recurrent extremity soft tissue sarcoma at Memorial Sloan-Kettering Cancer Center, the median interval to local recurrence was 16 months; 65% of patients developed a local recurrence by 2 years, and 90% by 4 years.155 The majority of patients (89%) were treated with additional limb-sparing surgery, and 73% received additional adjuvant therapy; the disease-specific survival after treatment of first local recurrence was 55% at 4 years. Independent prognostic factors for disease-specific survival after local recurrence included tumor grade, local recurrence size, and local recurrence-free interval. These data indicate that an

1	years. Independent prognostic factors for disease-specific survival after local recurrence included tumor grade, local recurrence size, and local recurrence-free interval. These data indicate that an isolated local recurrence should be treated aggressively with resection with negative margins.For patients with extremity sarcomas, achieving negative margins on resection of recurrent disease frequently requires amputation. However, in some patients with recurrent extrem-ity sarcoma, function-preserving resection combined with addi-tional radiation therapy, with or without chemotherapy, can produce acceptable rates of local control.161-163 Nori and col-leagues reported a local control rate of 69% among 40 patients with recurrent tumors treated with reexcision and brachytherapy to a median dose of 45 Gy.163 In a similar series, Midis and col-leagues reported that limb-sparing surgery was possible in 66% of patients, and the 5-year local recurrence-free survival rate was 72% in those

1	dose of 45 Gy.163 In a similar series, Midis and col-leagues reported that limb-sparing surgery was possible in 66% of patients, and the 5-year local recurrence-free survival rate was 72% in those patients.161The primary determinant of survival in patients with soft tissue sarcoma is the development of distant metastases. Patients with extremity sarcomas generally develop pulmonary metastases.160 Less common sites of metastasis for soft tissue sarcomas include bone (7%), liver (4%),49 and lymph nodes (5–7%).28 Myxoid liposarcoma of the extremity is known to metastasize to the abdomen and pelvis; therefore, staging CT of these regions must be performed before definitive local therapy is administered.32Management of Recurrent and Distant Metastatic Sarcoma. In selected individuals with distant metastatic disease, surgical resection of a primary soft tissue sarcoma may be appropriate as a palliative procedure. The decision should be based on the patient’s symptoms, which often include

1	metastatic disease, surgical resection of a primary soft tissue sarcoma may be appropriate as a palliative procedure. The decision should be based on the patient’s symptoms, which often include pain; ability to achieve local tumor control; comorbidities; anticipated morbidity of the surgical procedure; and the extent of metastases.The most common initial site of distant metastasis of soft tissue sarcomas is the lung. Selected patients with a limited number of pulmonary nodules (less than four nodules), long disease-free intervals, and no endobronchial invasion may become long-term survivors after pulmonary resection (Fig. 36-8); 15% to 40% of patients with complete resection of metastatic disease confined to the lung are long-term survivors.159,160,164 In a retrospective multi-institutional study of 255 patients with lung metastases, the 5-year overall survival rate after metastasectomy was 38%.157 Favorable prognostic factors in that study included microscopically tumor-free

1	study of 255 patients with lung metastases, the 5-year overall survival rate after metastasectomy was 38%.157 Favorable prognostic factors in that study included microscopically tumor-free margins, age younger than 40 years, and grade 1 or 2 tumor.157 For patients who are surgical candidates, pulmonary resection alone can be more cost-effective than watchful waiting, chemotherapy, or chemotherapy plus surgery.158Chemotherapy for Distant Metastatic Sarcoma. Doxorubicin, either alone or combined with other agents, has been the primary treatment modality for patients with advanced or distant metastatic sarcomas for several decades.128 Although most patients with metastatic disease are not curable, some Figure 36-8. A 69-year-old patient with a history of a dedifferenti-ated liposarcoma of the retroperitoneum developed a solitary lung metastasis 6 years after surgical resection.Brunicardi_Ch36_p1567-p1598.indd 158101/03/19 6:38 PM 1582SPECIFIC CONSIDERATIONSPART IIpatients with

1	of the retroperitoneum developed a solitary lung metastasis 6 years after surgical resection.Brunicardi_Ch36_p1567-p1598.indd 158101/03/19 6:38 PM 1582SPECIFIC CONSIDERATIONSPART IIpatients with limited disease experience stabilization of disease with multidisciplinary treatment, which often includes surgery and radiation therapy in addition to chemotherapy. Several factors predict better outcome for patients with recurrent metastatic sarcoma undergoing chemotherapy, including good performance status, previous response to chemotherapy, younger age, absence of hepatic metastases, low-grade tumor, and long disease-free interval.165 Isolated liver metastases, if stable over several months, may be amenable to resection,166 radiofrequency ablation,167 or chemoembolization.168As data accumulate regarding the sensitivity of sarcoma subtypes to particular chemotherapies, it is critical that histologydriven treatment approaches be used. New therapies are also being identified based on the

1	regarding the sensitivity of sarcoma subtypes to particular chemotherapies, it is critical that histologydriven treatment approaches be used. New therapies are also being identified based on the unique molecular signatures of sarcomas.128Palliative Radiation Therapy. Definitive radiation therapy can be considered when no acceptable surgical option is avail-able (e.g., in patients with significant medical comorbidities). In this setting, radiation doses greater than 63 Gy yielded superior tumor control, but doses greater than 68 Gy resulted in increased rates of major complications.169SPECIAL CLINICAL SITUATIONSMyxoid LiposarcomaMyxoid liposarcomas belong to the group of soft tissue sarco-mas with lipomatous differentiation. However, myxoid liposar-comas differ from the other liposarcoma subtypes with respect to morphology (i.e., myxoid stroma and lipomatous differen-tiation) and clinical behavior. Myxoid liposarcomas frequently present as slow-growing, deep tumors in the lower

1	subtypes with respect to morphology (i.e., myxoid stroma and lipomatous differen-tiation) and clinical behavior. Myxoid liposarcomas frequently present as slow-growing, deep tumors in the lower extremity and can metastasize to other soft tissue locations, including the retroperitoneum and extremities.170,171 For this reason, CT of the chest, abdomen, and pelvis is recommended for adequate stag-ing and surveillance of myxoid liposarcoma.Retroperitoneal SarcomaMost retroperitoneal tumors are malignant, and about one-third are soft tissue sarcomas. Also to be considered in the differen-tial diagnosis of a retroperitoneal tumor are primary germ cell tumors, lymphoma, and metastatic testicular cancer. Approxi-mately 1000 new cases of retroperitoneal sarcoma are diagnosed annually in the United States, and these tumors account for 10% to 15% of all adult tissue sarcomas. Approximately two-thirds of retroperitoneal sarcomas are high grade (either grade 2 or 3), and liposarcoma and

1	the United States, and these tumors account for 10% to 15% of all adult tissue sarcomas. Approximately two-thirds of retroperitoneal sarcomas are high grade (either grade 2 or 3), and liposarcoma and leiomyosarcoma are the most common histologies.Retroperitoneal sarcomas generally present as large masses: 70% are larger than 10 cm at diagnosis.172 They typi-cally do not produce symptoms until they grow large enough to compress or invade contiguous structures, although pain, early satiety, and obstructive gastrointestinal symptoms may occur early in the disease course in some patients. Evaluation of a patient with a retroperitoneal mass begins with an accurate his-tory that should exclude signs and symptoms associated with lymphoma (e.g., fever and night sweats). A complete physical examination, with particular attention to all nodal basins and with a testicular examination in men, is critically important. Laboratory assessment can be helpful; elevated lactate dehy-drogenase levels may

1	with particular attention to all nodal basins and with a testicular examination in men, is critically important. Laboratory assessment can be helpful; elevated lactate dehy-drogenase levels may suggest lymphoma, and elevated β-human chorionic gonadotropin levels or α-fetoprotein levels may indi-cate a germ cell tumor.Figure 36-9. A 50-year-old man with a large right dedifferentiated liposarcoma. Note the atypical fat surrounding the right kidney and displacing the viscera to the left hemiabdomen and the large dedifferentiated mineralized solid nodule lateral to the right kidney.Although the general principles of evaluation and man-agement for retroperitoneal sarcomas are similar to those for extremity sarcomas, there are some differences. Contrast-enhanced CT of the abdomen and pelvis is used to define the extent of the tumor and its relationship to surrounding struc-tures, particularly vascular structures, for surgical planning; contrast-enhanced CT can also often distinguish between

1	to define the extent of the tumor and its relationship to surrounding struc-tures, particularly vascular structures, for surgical planning; contrast-enhanced CT can also often distinguish between well-differentiated and dedifferentiated liposarcoma. CT imaging is also done to evaluate the liver for the evidence of metastases, the peritoneal cavity for evidence of discontiguous disease, and the kidneys for assessment of function. Angiography or mag-netic resonance arteriography/venography can also be used to delineate vascular anatomy when involvement of critical vascu-lar structures is suspected. Thoracic CT should be performed to evaluate for potential lung metastases because 11% of patients with retroperitoneal sarcoma present with synchronous meta-static disease. CT-guided core needle biopsy is appropriate to provide a tissue diagnosis; however, well-differentiated liposar-coma may be diagnosed with CT imaging alone, and negative biopsy findings should not delay operative

1	needle biopsy is appropriate to provide a tissue diagnosis; however, well-differentiated liposar-coma may be diagnosed with CT imaging alone, and negative biopsy findings should not delay operative intervention.Complete surgical resection is the most effective treatment for primary or recurrent retroperitoneal sarcoma (Fig. 36-9). En bloc resection often necessitates sacrificing contiguous struc-tures such as the colon, kidney, spleen, pancreas, psoas muscle, small bowel, inferior vena cava, and aorta.173 In a review of 25 patients who underwent resection of retroperitoneal sarcoma with major blood vessel involvement in a 16-year time span, postoperative morbidity and mortality rates were 36% and 4%, respectively. Vessel patency rates were greater than 88% with a median follow-up time of 19.3 months.83,174 Local control and survival rates were favorable in patients with tumor-free resec-tion margins. The authors concluded that vascular resection is the treatment of choice in sarcomas

1	19.3 months.83,174 Local control and survival rates were favorable in patients with tumor-free resec-tion margins. The authors concluded that vascular resection is the treatment of choice in sarcomas that involve major blood vessels in the retroperitoneum.83 Similar considerations were made by other groups reporting specifically on inferior vena cava resection in the context of multivisceral resection for ret-roperitoneal sarcoma and on surgical morbidity after extended surgical resection of retroperitoneal sarcoma. Extended proce-dures, including also vessels, are feasible and safe if carried out in experienced centers. While the goal of sarcoma resection is Brunicardi_Ch36_p1567-p1598.indd 158201/03/19 6:38 PM 1583SOFT TISSUE SARCOMASCHAPTER 36wide excision, this is unlikely to be achievable in most patients with retroperitoneal sarcomas. Surgery is considered marginal in most cases, even when macroscopically complete, but every attempt should be made to minimize this

1	to be achievable in most patients with retroperitoneal sarcomas. Surgery is considered marginal in most cases, even when macroscopically complete, but every attempt should be made to minimize this marginality by liberally resecting surrounding organs when involved. The extension of surgery should then take into consideration a trade-off between expected morbidity and benefit and should be best carried out at high-volume centers, where technical skills and knowledge of the natural history of this very rare disease can be found.In an analysis of 500 patients with retroperitoneal soft tissue sarcoma treated at Memorial Sloan-Kettering Cancer Center, the median survival time was 103 months for those who underwent complete resection versus 18 months for those who underwent incomplete resection or observation without resection.172 In general, surgical resection should not be offered unless radiographic evidence indicates the potential for complete resection; however, palliative surgical

1	or observation without resection.172 In general, surgical resection should not be offered unless radiographic evidence indicates the potential for complete resection; however, palliative surgical resection may be considered to reduce symptoms of intestinal obstruction, pain, or bleeding.175 In particular, in patients with atypical lipomatous tumors, an aggressive surgical approach including incomplete resection or debulking is justified to palliate symptoms and may provide a potential survival benefit.176 Such an approach is not justified for dedifferentiated liposarcomas or other high-grade retroperitoneal sarcomas because these tumors have high rates of distant metastasis and local recurrence.Adjuvant Therapy. Most studies have failed to show a sur-vival benefit from adjuvant chemotherapy for retroperitoneal sarcoma.177-179 Because of the high rates of local recurrence, radiation therapy has been proposed for treating microscopic residual disease as an adjunct to surgical resection.

1	for retroperitoneal sarcoma.177-179 Because of the high rates of local recurrence, radiation therapy has been proposed for treating microscopic residual disease as an adjunct to surgical resection. However, the optimal technique and timing of radiation therapy have not been established, and the potential benefits of radiation therapy must be weighed against the increased risk of treatment-related toxic effects.Radiation treatment of retroperitoneal sarcomas is com-plex because tumors are usually large, which necessitates large treatment fields close to radiosensitive structures (e.g., bowel). Several techniques have been used, including preoperative and postoperative external-beam radiation therapy, intraoperative radiation therapy, and brachytherapy.180 Preoperative radiation therapy is feasible and well tolerated. Toxic effects may be less severe with preoperative radiation therapy given that the tumor borders are definable, the tumor displaces radiosensitive viscera away from the

1	feasible and well tolerated. Toxic effects may be less severe with preoperative radiation therapy given that the tumor borders are definable, the tumor displaces radiosensitive viscera away from the treatment field, and effective doses of radiation may be lower preoperatively.181Several studies have shown favorable local control rates for intermediateand high-grade retroperitoneal sarcoma treated with preoperative radiation therapy and complete resection.180 However, most studies have failed to show a survival benefit.182 This situation prompted the initiation of a multicenter, ran-domized trial sponsored by the American College of Surgeons Oncology Group (ACOSOG) comparing surgery to surgery with preoperative radiation (ACOSOG Z9031). Unfortunately, the study was closed prematurely in 2006 because of low patient accrual. A similar study is now ongoing in Europe, sponsored by the Soft Tissue and Bone Sarcoma Group (STBSG) of the EORTC.Current recommendations for radiation therapy for

1	because of low patient accrual. A similar study is now ongoing in Europe, sponsored by the Soft Tissue and Bone Sarcoma Group (STBSG) of the EORTC.Current recommendations for radiation therapy for patients with retroperitoneal sarcoma at high volumes centers are based on disease characteristics at presentation.183 For high-risk patients, defined as those with large, high-grade tumors or recurrent low-grade tumors, preoperative radiation therapy to a total dose of 50 Gy followed by surgical resection is considered. Postoperative radiation is discouraged unless the resected tumor bed is clearly away from dose-limiting structures.Treatment of Recurrence. Retroperitoneal sarcomas recur more often than extremity and trunk wall ones. Retroperito-neal leiomyosarcomas, in addition to recurring locally in the tumor bed and metastasizing to the lungs, frequently spread to the liver. Retroperitoneal sarcomas can also recur diffusely throughout the peritoneal cavity (sarcomatosis). Resection of

1	in the tumor bed and metastasizing to the lungs, frequently spread to the liver. Retroperitoneal sarcomas can also recur diffusely throughout the peritoneal cavity (sarcomatosis). Resection of recurrent retroperitoneal sarcoma is similar to resection of recurrent extremity sarcoma. However, the likelihood that a recurrent retroperitoneal sarcoma will be resectable declines precipitously with each recurrence. In a large series of patients treated at Memorial Sloan-Kettering Cancer Center, the authors were able to resect recurrent tumors in 57% of patients with a first recurrence but only 20% of patients with a second recur-rence and 10% of patients with a third recurrence.68 In up to 25% of patients, well-differentiated retroperitoneal liposarcoma recurs in a poorly differentiated form or recurs with areas of dedifferentiation. Dedifferentiated retroperitoneal liposarcoma is more aggressive than its well-differentiated precursor and has a greater propensity for distant

1	form or recurs with areas of dedifferentiation. Dedifferentiated retroperitoneal liposarcoma is more aggressive than its well-differentiated precursor and has a greater propensity for distant metastasis.Gastrointestinal SarcomaPatients with gastrointestinal sarcoma most often present with nonspecific gastrointestinal symptoms that are determined by the site of the primary tumor. In a series from Memorial Sloan-Kettering Cancer Center, early satiety and dyspepsia were noted in patients with tumors of the upper gastrointestinal tract, whereas tenesmus and changes in bowel habits were common in patients with tumors of the lower gastrointestinal tract.184 In a series of 80 patients with various smooth-muscle tumors of the gastrointestinal tract, Chou and colleagues185 identified the most common presenting symptoms and signs as gastrointes-tinal bleeding (44%), abdominal mass (38%), and abdominal pain (21%).Establishing the diagnosis of a gastrointestinal sarcoma preoperatively is often

1	presenting symptoms and signs as gastrointes-tinal bleeding (44%), abdominal mass (38%), and abdominal pain (21%).Establishing the diagnosis of a gastrointestinal sarcoma preoperatively is often difficult. Radiologic assessment, includ-ing CT of the abdomen or pelvis, is sometimes useful to deter-mine the anatomic location, size, and extent of disease. Patients with localized disease frequently present with a large intra-abdominal mass. However, there is no radiographic evidence of regional lymph node metastases, which would be typical of an adenocarcinoma of similar size and anatomic location. In patients with advanced gastrointestinal sarcoma, CT may dem-onstrate disseminated intra-abdominal masses with or without concomitant ascites and invasion of tissue planes.Endoscopy (esophagoduodenoscopy or colonoscopy) has become the mainstay for evaluating symptoms related to the gastrointestinal tract. For tumors involving the stomach, upper endoscopy with endoscopic ultrasonography and

1	or colonoscopy) has become the mainstay for evaluating symptoms related to the gastrointestinal tract. For tumors involving the stomach, upper endoscopy with endoscopic ultrasonography and biopsy are important diagnostic tests used to distinguish gastrointestinal sarcoma from adenocarcinoma of the stomach. Endoscopic biopsy of these tumors is preferred over CT-guided biopsy if feasible. This distinction is clinically significant because the extent of resection (local excision versus gastrectomy) and the role of regional lymphadenectomy differ for these two condi-tions. For gastrointestinal sarcomas, lymphatic spread is not the primary route of metastasis; consequently, lymphadenectomy is not routinely performed as part of resection. The general rec-ommendation for gastrointestinal sarcoma, based on published data and the primary pattern of distant (vs. local) failure, is to Brunicardi_Ch36_p1567-p1598.indd 158301/03/19 6:38 PM 1584SPECIFIC CONSIDERATIONSPART IIresect the tumor

1	based on published data and the primary pattern of distant (vs. local) failure, is to Brunicardi_Ch36_p1567-p1598.indd 158301/03/19 6:38 PM 1584SPECIFIC CONSIDERATIONSPART IIresect the tumor with a 2to 4-cm margin of normal tissue. However, some cases may be technically challenging because of the tumor’s anatomic location or size. For example, for gastric tumors located near the gastroesophageal junction, achieving adequate surgical margins may not be possible without a total or proximal subtotal gastrectomy. This recommendation is much different when considering resection of gastrointestinal stromal tumors (GIST) where a gross margin negative resection is rec-ommended are rarely is a total gastrectomy required. Similarly, large leiomyosarcomas arising from the stomach with invasion of adjacent organs should be resected together with the adjacent involved viscera en bloc.For sarcomas of the small or large intestine, segmental bowel resection is the standard treatment. For sarcomas

1	adjacent organs should be resected together with the adjacent involved viscera en bloc.For sarcomas of the small or large intestine, segmental bowel resection is the standard treatment. For sarcomas of the jejunum, ileum, and colon, the tumor is excised en bloc with the involved segment of intestine and its mesentery; radical mesenteric lymphadenectomy is not attempted. For sarcomas originating in the rectum, the tumor resection technique is based on the anatomic location and size of the tumor. For small, low rectal lesions, clear margins may be achievable with a transanal excision. Large or locally invasive lesions may require more extensive operations for complete tumor extirpation.186,187Breast SarcomaSarcomas of the breast are rare tumors, accounting for less than 1% of all breast malignancies and less than 5% of all soft tissue sarcomas. A variety of histologic subtypes have been reported within the breast, including angiosarcoma, stromal sarcoma, fibrosarcoma, and malignant

1	malignancies and less than 5% of all soft tissue sarcomas. A variety of histologic subtypes have been reported within the breast, including angiosarcoma, stromal sarcoma, fibrosarcoma, and malignant fibrous histiocytoma.Angiosarcoma of the breast accounts for about 50% of all sarcomas of the breast and has increasingly been associated with radiation therapy for treatment of primary breast cancer.10 The period between radiation therapy and diagnosis of radiation-associated breast sarcoma has been reported to range from 3 to 20 years, with an incidence of 0.3% at 10 years and 0.5% at 15 years.188 In a retrospective study of 55 patients with angiosarcoma of the breast, patients with radiation-associated angiosarcoma were on average 30 years older and were less likely to present with distant metastases than radiation-naive patients. Clinically, radiation-associated angiosarcoma of the breast may occur in the irradiated chest wall after mastectomy or in the irradiated breast following

1	metastases than radiation-naive patients. Clinically, radiation-associated angiosarcoma of the breast may occur in the irradiated chest wall after mastectomy or in the irradiated breast following segmental resection. The findings at presentation of a patient with cutaneous angiosarcoma often include an expanding erythematous patch, red papular eruptions, bluish-black lesions, or bruise-like discoloration overlying an area of induration. Mammography is often nonspecific, and diagnosis requires punch or incisional biopsy.Cystosarcoma phyllodes are generally not considered to be sarcomas because these tumors are thought to originate from hormonally responsive stromal cells of the breast and are usu-ally benign. In patients with these tumors, infiltrating tumor margins, severe stromal overgrowth, atypia, and cellularity have all been identified as risk factors for metastases.189As with sarcomas at other anatomic sites, histopathologic grade and tumor size are important prognostic factors

1	atypia, and cellularity have all been identified as risk factors for metastases.189As with sarcomas at other anatomic sites, histopathologic grade and tumor size are important prognostic factors for sarco-mas of the breast. The likelihood of local recurrence increases as tumor size increases; tumors smaller than 5 cm are associated with better overall survival. Local and distant recurrences are more common in patients with high-grade lesions. Complete excision with negative margins is the primary therapy. Simple mastectomy confers no additional benefit if complete excision can be accomplished by segmental mastectomy. Because of low rates of regional lymphatic spread, axillary dissection is not rou-tinely indicated. Neoadjuvant chemotherapy or radiation ther-apy may be considered for patients with large, high-risk tumors.Uterine SarcomaSarcomas account for less than 5% of uterine malignancies. Uterine sarcomas have been classified into four histologic sub-groups: uterine

1	for patients with large, high-risk tumors.Uterine SarcomaSarcomas account for less than 5% of uterine malignancies. Uterine sarcomas have been classified into four histologic sub-groups: uterine leiomyosarcoma, endometrial stromal sarcoma, malignant mixed Müllerian tumor (carcinosarcoma), and undif-ferentiated endometrial sarcoma. Five-year overall survival rates for patients with uterine sarcoma are 30% to 50%.190 Total abdominal hysterectomy (TAH) is recommended for localized disease. Bilateral salpingo-oophorectomy is mandatory only in endometrial stromal sarcoma. Because uterine sarcomas are rare, the benefits of adjuvant therapy (e.g., chemotherapy, hor-monal therapy) have not been adequately evaluated. Pelvic post-operative irradiation has been studied instead in a randomized fashion. The results of such study have been reported, showing no benefit in survival in favor of radiation therapy.191Uterine leiomyosarcomas are smooth-muscle tumors and account for 35% to 40% of uterine

1	The results of such study have been reported, showing no benefit in survival in favor of radiation therapy.191Uterine leiomyosarcomas are smooth-muscle tumors and account for 35% to 40% of uterine sarcomas. Leiomyosarcoma can affect women in their twenties, although it is more com-monly diagnosed between 50 and 60 years of age. Standard treatment is TAH with or without ovarian preservation depend-ing on the patient’s wishes and menopausal status. Lymph node metastasis is present in less than 5% of patients at diagnosis, and lymphadenectomy is not recommended. Adjuvant pelvic radia-tion therapy can be considered for selected high-risk patients. Adjuvant chemotherapy is controversial. Gemcitabine plus docetaxel has been noted to be well tolerated and highly active, with a response rate of 53% in patients with unresectable uter-ine leiomyosarcoma.130 Doxorubicin and trabectedin have also demonstrated activity when used as firstor second-line therapy.Endometrial stromal sarcomas account

1	53% in patients with unresectable uter-ine leiomyosarcoma.130 Doxorubicin and trabectedin have also demonstrated activity when used as firstor second-line therapy.Endometrial stromal sarcomas account for approximately 7% to 10% of uterine sarcomas. Mitotic count is used to clas-sify endometrial stromal sarcomas as low grade (<10 mito-ses per 10 high-power fields) or high-grade (>10 mitoses per 10 high-power fields). In general, low-grade tumors demon-strate an indolent clinical course, while high-grade tumors are more aggressive with a poorer prognosis. Unlike other uterine sarcomas subtypes, endometrial stromal sarcomas express pro-gesterone receptors and have been found to be responsive to hormonal manipulation as an adjuvant therapy or for treatment of recurrent disease.192,193 Surgical treatment for these tumors includes TAH and bilateral salpingo-oophorectomy in premeno-pausal women; postoperative hormone replacement therapy is contraindicated.194 Recurrent or advanced disease

1	treatment for these tumors includes TAH and bilateral salpingo-oophorectomy in premeno-pausal women; postoperative hormone replacement therapy is contraindicated.194 Recurrent or advanced disease may respond to antiestrogen therapy. Tamoxifen is not recommended because it may be proestrogenic in this setting.Malignant mixed müllerian tumor accounts for 50% of uterine sarcomas and arises predominantly in postmenopausal women. This tumor is regarded as epithelial and is treated not with agents typically used to treat sarcoma but with agents used to treat ovarian and endometrial cancers.Undifferentiated endometrial sarcoma is an aggressive malignancy that does not express estrogen or progesterone receptors. It is associated with a poor prognosis even in patients presenting with localized disease. TAH with or without pres-ervation of the ovaries is recommended; postoperative pelvic radiation therapy may also be administered. Systemic agents for other soft tissue sarcomas are used for

1	disease. TAH with or without pres-ervation of the ovaries is recommended; postoperative pelvic radiation therapy may also be administered. Systemic agents for other soft tissue sarcomas are used for recurrent and/or meta-static disease.Brunicardi_Ch36_p1567-p1598.indd 158401/03/19 6:38 PM 1585SOFT TISSUE SARCOMASCHAPTER 36GASTROINTESTINAL STROMAL TUMORSGISTs, which account for the majority of gastrointestinal sar-comas, have distinctive molecular features that have been char-acterized over the last decade. These tumors share phenotypic similarities with the intestinal pacemaker cells known as the interstitial cells of Cajal195; interstitial cells of Cajal and GIST cells express the hematopoietic progenitor cell marker CD34 and the growth factor receptor c-Kit.196 Expression of the c-Kit gene protein product, CD117, has emerged as an important defining feature of GISTs. Using these diagnostic criteria, the incidence of GIST has been estimated to be 6 to 15 cases per million

1	the c-Kit gene protein product, CD117, has emerged as an important defining feature of GISTs. Using these diagnostic criteria, the incidence of GIST has been estimated to be 6 to 15 cases per million individuals per year.197-199 Until recently, systemic treat-ment for patients with unresectable or metastatic GIST was of little benefit because these tumors were resistant to conventional chemotherapy. Since the recognition that KIT activation occurs in most GISTs, KIT inhibition has emerged as an adjunct to sur-gery in select patients with resectable disease and as a primary treatment modality for patients with stage IV disease.Approximately 80% of GISTs have a mutation in the gene encoding the KIT receptor tyrosine kinase, and 5% to 10% have a mutation in the gene encoding the related PDGFRA recep-tor tyrosine kinase; such mutations result in the expression of mutant proteins with constitutive tyrosine kinase activity.1 The remaining GISTs do not have a detectable mutation, but lack of

1	recep-tor tyrosine kinase; such mutations result in the expression of mutant proteins with constitutive tyrosine kinase activity.1 The remaining GISTs do not have a detectable mutation, but lack of a mutation does not preclude a diagnosis of GIST if the tumor is morphologically typical of GIST. The presence and type of KIT (exon 11 or exon 9) or PDGFRA (exon 18) muta-tion has been found to predict tumor response to imatinib. In a phase 2 trial, patients with KIT exon 11 mutations had bet-ter response rates (83.5% vs. 47.8%) and survival than those with KIT exon 9 mutations or those without KIT or PDGFRA mutation.200 These findings have subsequently been confirmed in two additional phase 3 trials conducted by the EORTC– Italian Sarcoma Group–Australasian Gastrointestinal Trials Group (EORTC-62005).201,202The most common locations for GISTs are the stomach (60%) and small intestine (30%), but GISTs can arise anywhere along the gastrointestinal tract.203 Gastric GISTs have been shown to

1	most common locations for GISTs are the stomach (60%) and small intestine (30%), but GISTs can arise anywhere along the gastrointestinal tract.203 Gastric GISTs have been shown to be associated with a more favorable prognosis than GISTs at other sites.204 GISTs are most commonly diagnosed by upper endoscopy and/or CT of the abdomen as an incidental finding in an asymptomatic patient or in a patient being evalu-ated for symptoms of early satiety, abdominal pain, or gastroin-testinal bleeding. GIST most frequently metastasizes to the liver and/or abdominal cavity.Radiologic AssessmentStandard imaging techniques apply for GIST as for other intraabdominal sarcomas. In general, oral and IV contrast enhanced spiral CT is the staging modality of choice for GIST. CT scan of the abdomen and pelvis allows for assessment of the primary lesion and the presence or absence of intraabdomi-nal disseminated disease or metastatic disease to the liver (the two most common locations for distant

1	pelvis allows for assessment of the primary lesion and the presence or absence of intraabdomi-nal disseminated disease or metastatic disease to the liver (the two most common locations for distant metastasis of GIST). FDG-PET has been reported to be useful for preoperative stag-ing of GISTs because it may reveal early metastases and estab-lish baseline metabolic activity and may be considered in select patients where equivocal findings are identified on CT or in the setting of following metabolic response to therapy. PET has been shown to be highly sensitive in detecting early response to imatinib treatment and in predicting long-term response in patients with metastatic GIST. If PET is to be used for moni-toring response to therapy, baseline PET should be performed before initiation of treatment. Useful and effective CT-based criteria by Choi et al for detection of GIST and for predicting prognosis of GIST have also been proposed and may be used readily without incurring the cost and

1	Useful and effective CT-based criteria by Choi et al for detection of GIST and for predicting prognosis of GIST have also been proposed and may be used readily without incurring the cost and radiation exposure of PET/CT.205Management of Localized DiseaseComplete surgical resection with negative margins is the rec-ommended treatment for localized GISTs. Extended anatomic resection, wide margins, and lymphadenectomy are not required; therefore, total gastrectomy for gastric primaries is rarely required even with the largest of lesions. Resection of even locally advanced tumors is associated with improved survival.206 The 5-year survival rate for all patients with GISTs ranges from 20% to 44%, and the 5-year survival rate for patients with com-pletely excised early-stage tumors is up to 75%.206 An analysis of 200 patients by DeMatteo and colleagues found a disease-specific survival rate of 54% for patients with grossly complete resection of primary GIST, and the median survival duration

1	An analysis of 200 patients by DeMatteo and colleagues found a disease-specific survival rate of 54% for patients with grossly complete resection of primary GIST, and the median survival duration for patients with metastatic disease was only 20 months.58As for other soft tissue sarcomas, tumor size has con-sistently been identified as an important prognostic factor for GIST. Mitotic activity has also been identified as an important prognostic factor and is generally categorized as fewer than 5, 5 to 10, or more than 10 mitoses per high-power field. The National Institutes of Health207 and the Armed Forces Insti-tute of Pathology203 have proposed prognostic criteria for risk stratification of surgically treated, localized primary GIST. Both groups take into account tumor size and mitotic count; the Armed Forces Institute of Pathology also includes tumor site as a prognostic variable. Accurate risk stratification is essential for selecting patients most likely to benefit from adjuvant

1	count; the Armed Forces Institute of Pathology also includes tumor site as a prognostic variable. Accurate risk stratification is essential for selecting patients most likely to benefit from adjuvant treatment.Management of Locally Advanced or Metastatic DiseaseTreatment with imatinib mesylate (Gleevec, ST1571), a selec-tive inhibitor of the KIT protein tyrosine kinase, has resulted in impressive clinical responses in a large percentage of patients with unresectable or metastatic GISTs. On the basis of the initial results in a single patient with metastatic GIST, the EORTC Soft Tissue and Bone Sarcoma Group initiated a phase 1 study to test the safety and efficacy of imatinib.208 In that study, 53% of patients with GISTs had confirmed partial responses; investiga-tors concluded that imatinib is safe and effective against this disease.208 A multicenter, international trial of imatinib for GIST was begun in July 2000 at four treatment centers: Dana-Farber Cancer Institute, Oregon Health

1	is safe and effective against this disease.208 A multicenter, international trial of imatinib for GIST was begun in July 2000 at four treatment centers: Dana-Farber Cancer Institute, Oregon Health Sciences University, Fox Chase Cancer Center, and University Hospital of Helsinki, Finland.209 A total of 147 patients with unresectable or metastatic GISTs were randomized to 400 or 600 mg of imatinib daily for up to 24 months. Objective response was demonstrated in 79 patients (54%); all had partial responses, and there was no significant difference in response rate between imatinib doses.210 Fourteen percent of patients experienced disease progression. The toxic-ity profile was acceptable; the predominant effects were gastro-intestinal effects (diarrhea, nausea), periorbital edema, muscle cramps, and fatigue. However, 21% of patients experienced serious (grade 3 or 4) adverse events, including gastrointestinal bleeding in 5% of patients, most likely related to the rapid tumor response of

1	and fatigue. However, 21% of patients experienced serious (grade 3 or 4) adverse events, including gastrointestinal bleeding in 5% of patients, most likely related to the rapid tumor response of mural lesions.Brunicardi_Ch36_p1567-p1598.indd 158501/03/19 6:38 PM 1586SPECIFIC CONSIDERATIONSPART IIA phase 3 randomized Intergroup trial was simultaneously performed to assess the clinical activity of imatinib at two dose levels for patients with unresectable or metastatic GIST express-ing the c-Kit tyrosine kinase.211 From December 15, 2000, to September 1, 2001, 746 patients were accrued and randomized to low-dose (400 mg/d) or high-dose (800 mg/d) imatinib. The primary endpoint of the trial was survival. Preliminary toxicity data from 325 patients revealed a 23% incidence of grade 3 or 4 adverse events, including nausea and vomiting, gastrointestinal bleeding, abdominal pain, edema, fatigue, and rash.In February 2002, the FDA approved imatinib for treat-ment of GIST based on the

1	or 4 adverse events, including nausea and vomiting, gastrointestinal bleeding, abdominal pain, edema, fatigue, and rash.In February 2002, the FDA approved imatinib for treat-ment of GIST based on the results of these promising clinical trials. Both the Intergroup trial mentioned in the preceding paragraph and a separate phase 3 trial compared the efficacy of low-dose (400 mg/d) and high-dose (800 mg/d) imatinib in patients with metastatic or unresectable GISTs.212,213 Both stud-ies showed equivalent response rates and overall survival for the two doses but increased toxicity for the 800-mg/d dose. Current recommendations include consideration of dose escalation to 800 mg/d for patients who experience disease progression at a dose of 400 mg/d and for patients with advanced GIST and KIT exon 9 mutations.1,214The optimal duration of imatinib treatment, the duration of benefit from imatinib, and the long-term toxicity of imatinib have not been established. When feasible, imatinib should

1	9 mutations.1,214The optimal duration of imatinib treatment, the duration of benefit from imatinib, and the long-term toxicity of imatinib have not been established. When feasible, imatinib should be continued in the absence of disease progression. A random-ized trial reported worse median progression-free survival in patients who stopped imatinib after 1 year than in patients who continued beyond 1 year (progression-free survival of 6 months vs. 18 months).215 Less than 4% of patients with GISTs have experienced serious adverse events with imatinib. Mild gastrointestinal toxicity is the most frequently reported adverse event, but gastrointestinal tract hemorrhage, presum-ably from rapid tumor necrosis, has also been reported. Thus, all patients with GISTs treated on clinical protocols should be evaluated and followed by a team of medical professionals that includes a surgeon.Many patients with GIST develop resistance to imatinib. Primary resistance is defined as clinical progression

1	be evaluated and followed by a team of medical professionals that includes a surgeon.Many patients with GIST develop resistance to imatinib. Primary resistance is defined as clinical progression that devel-ops during the first 6 months of treatment and is most commonly seen in patients with KIT exon 9 mutation, PDGFRA exon 18 mutation, or no mutations.216 Secondary resistance is defined as progression that develops more than 6 months after the start of treatment in a patient with an initial response.217 Imatinib resis-tance should be managed by either dose escalation or transition to treatment with sunitinib.1In 2006, sunitinib malate (SU11248, Sutent, Pfizer) emerged as an alternative systemic treatment for patients unable to tolerate imatinib and patients with imatinib-refractory GIST. Sunitinib is a tyrosine kinase inhibitor that targets multiple kinases, including the vascular endothelial growth factor receptors, PDGFRA, KIT, and FLT3. Sunitinib has both antiangiogenic and

1	GIST. Sunitinib is a tyrosine kinase inhibitor that targets multiple kinases, including the vascular endothelial growth factor receptors, PDGFRA, KIT, and FLT3. Sunitinib has both antiangiogenic and antiproliferative activity. In a phase 3, randomized, placebo-controlled trial, sunitinib was associated with a significant improvement in median time to progression (27.3 weeks vs. 6.4 weeks with placebo) in patients with imatinib-resistant GIST.135 In addition, sunitinib therapy was well tolerated; diarrhea, fatigue, and nausea were the most common adverse effects. Sunitinib has also been associated with hand-foot skin reaction, hypertension, cardiotoxicity, and hypothyroidism.218-220 In 2006, sunitinib was approved by the FDA for treatment of patients with resistance or intolerance to imatinib.The third FDA-approved drug recently made available for treatment of patients with imatiniband sunitinib-resistant GIST is regorafenib. Regorafenib is a structurally unique inhibitor of multiple

1	third FDA-approved drug recently made available for treatment of patients with imatiniband sunitinib-resistant GIST is regorafenib. Regorafenib is a structurally unique inhibitor of multiple cancer-associated kinases, including KIT and PDGFR, with broad-spectrum anticancer activity in preclinical and early-phase trials. Because KIT and PDGFR-α remain drivers of GIST after resistance to imatinib and sunitinib, a multicenter, single-stage phase 2 trial examined regorafenib in patients with advanced GIST after failure of at least imatinib and sunitinib.221 Thirty-four patients were enrolled from February to December 2010 and given regorafenib orally, 160 mg daily, on days 1 to 21 of a 28-day cycle. Clinical benefit was noted in 79% of patients with a median progression-free survival of 10 months. This trial was then followed by a phase 3, international, placebo-controlled, randomized trial of regorafenib for metastatic GIST after failure of imatinib and sunitinib.222 Patients with

1	10 months. This trial was then followed by a phase 3, international, placebo-controlled, randomized trial of regorafenib for metastatic GIST after failure of imatinib and sunitinib.222 Patients with progres-sion of disease on the placebo arm were crossed to the treatment arm of the study. Patients treated with regorafenib had a median progression-free survival of 4.8 months compared to 0.9 months on the placebo arm. Other tyrosine kinase inhibitors have dem-onstrated modest activity against GIST and target more than one family of protein kinases.223 Among these are sorafenib, dasatinib, and nilotinib for the treatment of imatinib-resistant GIST, and these are now generally reserved for therapy after progression with regorafenib.222Multidisciplinary TreatmentAlthough imatinib has improved survival of patients with advanced GIST, most patients with advanced GIST are not cured with imatinib. Some patients develop secondary resistance to imatinib with one or more sites of disease

1	survival of patients with advanced GIST, most patients with advanced GIST are not cured with imatinib. Some patients develop secondary resistance to imatinib with one or more sites of disease progression after 6 months of clinical response (Fig. 36-10A [before imatinib], Fig. 36-10B [after imatinib]). The mechanisms of imatinib resistance are currently being investigated. Surgery has been shown to be beneficial for selected patients with isolated disease progression during imatinib therapy.224-227 Surgical resection of residual metastatic disease responding to imatinib-sensitive GIST has also been shown to result in progression-free survival in 70% to 96% of patients with imatinibor sunitinib-sensitive GISTs.226-228 The optimal timing of surgery in relation to ima-tinib therapy for patients with metastatic disease remains to be determined. It is not possible to compare outcomes for patients treated with kinase inhibitors alone and patients treated with kinase inhibitors plus surgical

1	with metastatic disease remains to be determined. It is not possible to compare outcomes for patients treated with kinase inhibitors alone and patients treated with kinase inhibitors plus surgical resection outside the context of randomized trials given the heterogeneity of patients and biases associated with selection of patients for surgical resection.Postoperative ImatinibGiven the promising results of imatinib therapy for metastatic and locally advanced GIST, the next step was to study the efficacy of imatinib as adjuvant (postoperative) treatment in patients with surgically resectable disease, particularly those at high risk for recurrence because of large tumor size or high mitotic count. The ACOSOG first evaluated the efficacy of 1 year of postoperative imatinib in a single-arm phase 2 trial with 106 patients with high-risk GIST and compared the results with historical controls. Adjuvant treatment with imatinib for GIST patients was then examined in two key trials. In the

1	phase 2 trial with 106 patients with high-risk GIST and compared the results with historical controls. Adjuvant treatment with imatinib for GIST patients was then examined in two key trials. In the ACOSOG randomized, double-blind, phase 3 Z9001 study,230 treatment with 12 months of imatinib was compared with placebo, fol-lowing complete resection of a primary GIST smaller than 3 cm. Primary and secondary endpoints were recurrence-free survival Brunicardi_Ch36_p1567-p1598.indd 158601/03/19 6:38 PM 1587SOFT TISSUE SARCOMASCHAPTER 36ABFigure 36-10. A 57-year-old man with a history of a pelvic gastrointestinal stromal tumor involving the small bowel mesen-tery treated preoperatively with 6 months of imatinib (Gleevec). A. Before imatinib. B. After imatinib.(RFS) and overall survival (OS), respectively. Results showed a significant benefit in RFS, but not OS, with 12 months of imatinib. Based on these results, imatinib for the adjuvant treat-ment of adult patients following resection

1	respectively. Results showed a significant benefit in RFS, but not OS, with 12 months of imatinib. Based on these results, imatinib for the adjuvant treat-ment of adult patients following resection of KIT-positive GIST was approved by the FDA in 2008 and by the European Medical Agency (EMA) in 2009.A more recent study conducted by the Scandinavian Sarcoma Group (SSG) and the Sarcoma Group of the Arbe-itsgemeinschaft Internistische Onkologie (AIO; SSGXVIII/AIO trial) compared 12 versus 36 months of adjuvant imatinib 400 mg/d in patients with GIST at high risk of recurrence (defined as a GIST tumor diameter >10 cm, or mitotic count >10 per 50 high-powered fields, or tumor diameter >5 cm and mitotic count >5 per 50 high-power fields, or tumor rupture).231 Results showed that both RFS and OS significantly improved with 36 months of imatinib: the 5-year RFS rates for patients receiving 36 versus 12 months of imatinib were 65.6% versus 47.9%, respectively, and the 5-year OS rates were 92%

1	significantly improved with 36 months of imatinib: the 5-year RFS rates for patients receiving 36 versus 12 months of imatinib were 65.6% versus 47.9%, respectively, and the 5-year OS rates were 92% versus 81.7%, respectively.The NCCN and the European Society of Medical Oncol-ogy now recommend that imatinib be considered for patients at intermediate or high risk of recurrence after resection and that at least 36 months of adjuvant imatinib be considered for patients at high risk of recurrence.232 Further, both the FDA and EMA updated the label, extending the duration of adjuvant therapy to at least 36 months in patients at high risk of recurrence.Whether longer treatment durations may be of further benefit is still an open question that will be addressed by future studies. However, paralleling what is commonly done in the metastatic setting, many investigators believe that even adju-vant imatinib should become a chronic therapy. While moving toward more prolonged adjuvant treatment

1	what is commonly done in the metastatic setting, many investigators believe that even adju-vant imatinib should become a chronic therapy. While moving toward more prolonged adjuvant treatment durations, it is all the more essential to identify the appropriate patients to treat to avoid the burden of adverse events or increased financial liabil-ity for patients who will not derive therapeutic benefit from imatinib. Risk stratification based on patients’ risk of recur-rence is a key component to optimizing adjuvant treatment. The most practical stratification scheme to use for making a decision for adjuvant therapy is the modified National Insti-tutes of Health consensus criteria.233 High-risk GIST patients, whose tumor harbors a sensitive genotype, should be treated by adjuvant imatinib because they have a poor prognosis. On the contrary, neither low-risk nor intermediate-risk GIST patients need adjuvant therapy, even if their tumor carries a sensitive genotype. In fact, evidence has

1	they have a poor prognosis. On the contrary, neither low-risk nor intermediate-risk GIST patients need adjuvant therapy, even if their tumor carries a sensitive genotype. In fact, evidence has been provided that the out-come of these patients with intermediateor low-risk GIST is good. When using other risk stratification schemes, such as the Armed Forces Institute of Pathology table, Memorial Sloan-Kettering Cancer Center nomogram, or the heat map,204,234,235 there is a consensus to treat all patients having 30% or higher risk of recurrence, if their tumor carries a sensitive genotype. There is also a consensus not to treat patients having 10% or less risk of recurrence, even if their tumor carries a sensitive genotype. All patients having a risk between 10% and 30% should be evaluated on a case-by-case basis, and advantages/disadvantages of treatment should be made clear and discussed with the patient. Whenever a decision for adjuvant therapy is made, treatment duration of at least

1	a case-by-case basis, and advantages/disadvantages of treatment should be made clear and discussed with the patient. Whenever a decision for adjuvant therapy is made, treatment duration of at least 36 months should be con-sidered independently from the risk.Beside the risk of recurrence, the other important factor to consider is the tumor genotype. In other words, as found in both the metastatic and adjuvant settings, GIST tumors with KIT exon 11 and PDGFRA non-D842V mutations are sensitive to imatinib. Patients with these mutations are suitable for adjuvant imatinib if the risk determined by stratification tools is signifi-cant. Patients with KIT exon 9 mutations should also be treated; a higher dose (800 mg/d) may be more appropriate but remains to be studied clinically. Patients with PDGFRA D842V–mutated tumors should not be treated with adjuvant imatinib, nor should patients with KIT and PDGFRA wild-type tumors associated with neurofibromatosis type 1 or the pediatric

1	with PDGFRA D842V–mutated tumors should not be treated with adjuvant imatinib, nor should patients with KIT and PDGFRA wild-type tumors associated with neurofibromatosis type 1 or the pediatric GIST/Carney-Stratakis syndromes, whatever the risk. Patients with sporadic wild-type GIST could be treated on an individual basis.Preoperative ImatinibPatients with marginally resectable GIST or at significant risk for operative morbidity should be considered for preoperative imatinib with close monitoring. Since the optimal duration of preoperative therapy is unknown, imatinib should be continued until maximal response is achieved or until there is evidence of progression.236 Preoperative imatinib can be stopped imme-diately before surgery and resumed when oral medications are restarted.Brunicardi_Ch36_p1567-p1598.indd 158701/03/19 6:38 PM 1588SPECIFIC CONSIDERATIONSPART IIPreoperative imatinib in patients with primary GIST or resectable metastatic GIST has been evaluated in the context of

1	158701/03/19 6:38 PM 1588SPECIFIC CONSIDERATIONSPART IIPreoperative imatinib in patients with primary GIST or resectable metastatic GIST has been evaluated in the context of two randomized phase 2 studies. The Radiation Treatment Oncology Group (RTOG 0132/ACRIN 6665) evaluated the efficacy of preoperative imatinib (600 mg/d) for 8 to 10 weeks before surgery and 24 months after surgery in patients with primary (n = 30) or potentially resectable recurrent or metastatic (n = 22) tumors.237 Primarily stable disease was noted during imatinib treatment, and the 2-year progression-free survival rates were 83% for primary GIST and 77% for recurrent or metastatic GIST. In another study, 19 patients undergoing surgical resection at a single institution were randomized to preoperative imatinib (600 mg/d) for 3, 5, or 7 days followed by surgical resection and postoperative imatinib for 24 months. The response rate assessed using FDG-PET was 69%, and the median disease-free survival time

1	(600 mg/d) for 3, 5, or 7 days followed by surgical resection and postoperative imatinib for 24 months. The response rate assessed using FDG-PET was 69%, and the median disease-free survival time following treatment with surgery and imatinib was 46 months.238 Similar results were observed in a prospective series of patients treated at a major institution.239All of these studies show that neoadjuvant treatment is feasible, but to maximize the benefit of preoperative therapy, the following factors need to be considered. First, the patient should in principle have a favorable mutational status; other-wise, the treatment would be in vain, allowing the tumor to con-tinue growing. Fortunately, the majority of GISTs will respond, but it should not be forgotten that, especially in gastric loca-tion, the amount of insensitive mutations in the localized setting is less uncommon than what has been previously reported.240 However, we do not absolutely need to know the mutational status in

1	loca-tion, the amount of insensitive mutations in the localized setting is less uncommon than what has been previously reported.240 However, we do not absolutely need to know the mutational status in advance, but we should be aware that mutation is an issue. Alternatively, if mutation status is not determined prior to treatment, we may also check response very early, either by CT, PET, or contrast-enhanced ultrasound. If a radiographic response is detected within a month, then the mutation status is likely favorable, and the treatment could be continued with-out necessarily pursuing mutation testing. If not, then mutation status should be investigated before continuing the treatment.Second, the resectability of the tumor and the extent of resection necessary should be considered. Other than presenta-tions of clearly inoperable tumors (which are usually treated with imatinib upfront) or symptomatic tumors requiring urgent intervention (hemorrhage, perforation, etc), there are few

1	than presenta-tions of clearly inoperable tumors (which are usually treated with imatinib upfront) or symptomatic tumors requiring urgent intervention (hemorrhage, perforation, etc), there are few rea-sons today to perform extended procedures (i.e., multivisceral resections or formal organ resections) without first attempting preoperative therapy. For instance, patients with large GISTs who may require a long midline incision for resection may benefit from neoadjuvant imatinib to downstage the operation, potentially converting an open laparotomy approach to a laparo-scopic one. Another typical circumstance in which neoadjuvant therapy may be beneficial is in patients with GISTs arising in the esophagus, gastroesophageal junction, duodenum, or distal rectum. Preoperative treatment may shrink the tumor and allow a more conservative local excision. In general, such patients would normally undergo postoperative adjuvant treatment as well because of the expected recurrence risk. The chance

1	the tumor and allow a more conservative local excision. In general, such patients would normally undergo postoperative adjuvant treatment as well because of the expected recurrence risk. The chance of obtaining a response is high, and the benefit for tumor shrink-age obvious, so this approach should always be discussed with patients affected by bulky and/or poorly located disease (esoph-agus, gastroesophageal junction, duodenum, distal rectum) as well as for those who would be candidates for an adjuvant treat-ment anyway.Third, in the studies mentioned earlier, patients underwent surgery after a limited treatment duration (3 months at best). It is now well known that the preoperative treatment may result in sustained tumor shrinkage if given for a longer duration. Sur-gical resection may then be performed between 6 and 12 months or sooner if treatment effect plateaus. This allows for optimal tumor shrinkage, or at least shrinkage to the point where there is no further benefit to be

1	then be performed between 6 and 12 months or sooner if treatment effect plateaus. This allows for optimal tumor shrinkage, or at least shrinkage to the point where there is no further benefit to be gained by further neoadjuvant therapy without the risk of developing secondary resistance.DESMOIDSDesmoid tumors are not low-grade sarcomas but can be locally aggressive, although they do not metastasize. Approximately half of these tumors arise in the extremities; the remaining lesions are located on the trunk or in the retroperitoneum. Abdominal wall desmoids are associated with pregnancy and are thought to be the result of hormonal influence. Although usually sporadic, desmoids may occur in association with famil-ial adenomatous polyposis, a presentation that is referred to as Gardner’s syndrome and is linked to germline mutations in the APC gene. Sporadic cases of desmoid fibromatosis are com-monly linked to mutations in CTNNB1, the gene for β-catenin.The primary therapy for desmoid

1	and is linked to germline mutations in the APC gene. Sporadic cases of desmoid fibromatosis are com-monly linked to mutations in CTNNB1, the gene for β-catenin.The primary therapy for desmoid tumors has long been considered surgical resection with wide local excision to achieve negative margins. However, local recurrence occurs in up to one-third of patients independently of the quality of surgical margins.241-243 Up to two-thirds of the patients operated on with positive margins do not recur. This is why there is growing evidence that the primary approach could be more conservative. Function-sparing operations should be the goal, even if a positive margin is left on a critical structure. Moreover, some authors advocate the possibility to observe patients at presentation, limiting surgery to those who progress or fail medical therapies. It has in fact been reported that by this approach, up to 50% of patients skip surgical resection.244,245 Radiation therapy may be effective in

1	to those who progress or fail medical therapies. It has in fact been reported that by this approach, up to 50% of patients skip surgical resection.244,245 Radiation therapy may be effective in patients with unresectable tumors or as adjuvant therapy following surgery for recurrent disease, although long-term side effects and the risk of radiation-induced sarcoma should always be considered. When used, a dose of 50 to 54 Gy is usually recommended. Systemic treatment is another option when surgery is not indicated. Hormonal therapies such as tamoxifen have been reported to be beneficial, as have nonsteroidal anti-inflammatory drugs, which are known to affect the β-catenin signaling pathways. Chemotherapy is also effective, although usually reserved for patients with tumor-associated symptoms who have not responded to other interventions. Combinations of methotrexate and vinblastine have been shown to have activity, as have single-agent pegylated liposomal doxorubicin246 and

1	symptoms who have not responded to other interventions. Combinations of methotrexate and vinblastine have been shown to have activity, as have single-agent pegylated liposomal doxorubicin246 and sorafenib.247 Imatinib has also been studied with unconvincing results.248-250DERMATOFIBROSARCOMA PROTUBERANSDermatofibrosarcoma protuberans is a rare low-grade sarcoma arising in the dermis that rarely metastasizes but is locally aggressive. The overall annual incidence has been estimated at 4.2 cases per million individuals,251 and the incidence is higher among blacks than whites (6.5 vs. 3.9 per million per year). Approximately 40% of cases arise on the trunk, and most of the remaining tumors are distributed between the head and neck and the extremities. Dermatofibrosarcoma protuberans presents as a nodular, cutaneous mass that grows slowly and persistently. Satellite lesions may be found in patients with larger tumors. Brunicardi_Ch36_p1567-p1598.indd 158801/03/19 6:38 PM 1589SOFT

1	as a nodular, cutaneous mass that grows slowly and persistently. Satellite lesions may be found in patients with larger tumors. Brunicardi_Ch36_p1567-p1598.indd 158801/03/19 6:38 PM 1589SOFT TISSUE SARCOMASCHAPTER 36Standard treatment is wide local excision, which generally results in local recurrence rates of less than 10%.252 Although local recurrence rates as high as 30% to 50% have been reported in population-based series, the associated 5-year survival rate is greater than 99%.251Dermatofibrosarcoma protuberans arises from a specific chromosomal translocation involving chromosomes 17 and 22, in which the collagen 1 α 1 gene is fused to the gene for PDGF β-chain (PDGFB).253 The resultant deregulated expression of PDGFB leads to continuous activation of the PDGFR protein tyrosine kinase, which promotes tumor cell growth. The identification of this chromosomal translocation in more than 90% of cases of dermatofibrosarcoma protuberans has led to the development of targeted

1	kinase, which promotes tumor cell growth. The identification of this chromosomal translocation in more than 90% of cases of dermatofibrosarcoma protuberans has led to the development of targeted therapy. Inhibiting PDGFR with imatinib has been shown to induce clinical and radiologic improvement in patients with unresectable dermatofibrosarcoma protuberans.21 These data have resulted in the approval by the FDA of imatinib for treatment of patients with locally advanced dermatofibrosarcoma protuberans.PEDIATRIC SARCOMASSoft tissue sarcomas in children are relatively rare, accounting for 7% to 8% of all pediatric cancers and totaling approximately 600 new cases per year.254 Pediatric sarcomas have traditionally been divided into two groups: rhabdomyosarcoma and nonrhab-domyosarcoma soft tissue sarcomas.RhabdomyosarcomaAssociated with skeletal muscle, rhabdomyosarcomas are the most common soft tissue tumors among children younger than 15 years and can occur at any site comprised of

1	tissue sarcomas.RhabdomyosarcomaAssociated with skeletal muscle, rhabdomyosarcomas are the most common soft tissue tumors among children younger than 15 years and can occur at any site comprised of striated muscle. Patients with these tumors generally present with a painless enlarging mass; about 24% of tumors are located in the geni-tourinary system, 20% in the extremities, 20% in the head and neck, 16% in the parameningeal region, and 22% in other sites.255Rhabdomyosarcoma is a small round cell tumor that dem-onstrates muscle differentiation upon light microscopy and immunohistochemical analysis. Two primary histologic sub-types account for 90% of cases: embryonal (70%) and alveolar (20%). Alveolar rhabdomyosarcoma is associated with cytoge-netic translocation [t(2:13)(q35:q14)] in 85% to 90% of cases and [t(1:13)(p36:q14)] in 10% of cases.256 These translocations affect biologic activity at the levels of protein function and gene expression, thereby affecting the control of cell

1	to 90% of cases and [t(1:13)(p36:q14)] in 10% of cases.256 These translocations affect biologic activity at the levels of protein function and gene expression, thereby affecting the control of cell growth, apopto-sis, differentiation, and motility and ultimately contributing to tumorigenic behavior.256 Whereas alveolar rhabdomyosarcomas often have translocations, most embryonal rhabdomyosarcomas have an allelic loss at chromosome 11p15.5 that is thought to inactivate a tumor suppressor gene.256,257 Both of these dis-tinct molecular subtypes of rhabdomyosarcoma are thought to have similar alterations in downstream targets such as the p53 and Rb pathways.256 Further insight into these genetic altera-tions may lead to a better understanding of the pathogenesis of rhabdomyosarcoma and provide novel targets for therapeutic approaches.Extent of disease is the strongest predictor of long-term outcome. Several staging systems for rhabdomyosarcoma are available. The Intergroup Rhabdomyosarcoma

1	novel targets for therapeutic approaches.Extent of disease is the strongest predictor of long-term outcome. Several staging systems for rhabdomyosarcoma are available. The Intergroup Rhabdomyosarcoma Study Group system is based on surgical-pathologic groupings. Multidis-ciplinary evaluation including pediatric oncologists, surgical subspecialists, and radiation oncologists is critical to plan the best treatment approach to maximize local tumor control while minimizing long-term treatment effects.Complete surgical resection is the treatment of choice for rhabdomyosarcoma when function and cosmesis can be preserved. Patients who are able to undergo a complete tumor resection with negative (group I) or microscopic surgical mar-gins (group II) are able to undergo less intensive systemic ther-apy and still have overall survival rates approaching 90%.258 At some anatomic sites, in particular the head and neck and genito-urinary system, surgery is often avoided because the associated

1	ther-apy and still have overall survival rates approaching 90%.258 At some anatomic sites, in particular the head and neck and genito-urinary system, surgery is often avoided because the associated morbidity would be substantial. Recent findings suggest that chemotherapy alone can adequately control many such tumors. In the second International Society of Paediatric Oncology study of rhabdomyosarcoma (MMT84), the choice of local treatment was based on response to initial chemotherapy such that radical surgery and radiation therapy were avoided in 66% of patients. Among the patients who subsequently developed local relapse, the 5-year overall survival rate after salvage therapy was 46%.258Unlike other soft tissue sarcomas, rhabdomyosarcomas have a high propensity for lymph node metastasis, with rates up to 20% to 30% for sites such as the extremities, parates-ticular nodes, and prostate. Lymph node sampling and, more recently, sentinel lymph node mapping have been used to evalu-ate

1	with rates up to 20% to 30% for sites such as the extremities, parates-ticular nodes, and prostate. Lymph node sampling and, more recently, sentinel lymph node mapping have been used to evalu-ate regional node status in children with rhabdomyosarcoma.About 15% to 20% of patients with rhabdomyosarcoma have distant metastasis at presentation, most commonly (40–50% of cases) to the lungs, followed by bone marrow and bone. However, all patients with rhabdomyosarcoma are assumed to have micrometastatic disease at presentation. Therefore, multia-gent chemotherapy is recommended for all patients with rhab-domyosarcoma. Combination regimens including vincristine, dactinomycin, and cyclophosphamide continue to be the basis of effective curative therapy.254 Although various combinations including doxorubicin, ifosfamide, cisplatin, and etoposide have been shown to be active against rhabdomyosarcoma, they have not improved outcomes.258,259 Radiation therapy is given to most patients with

1	doxorubicin, ifosfamide, cisplatin, and etoposide have been shown to be active against rhabdomyosarcoma, they have not improved outcomes.258,259 Radiation therapy is given to most patients with microscopic residual disease (group II) after resection.The prognosis for children with rhabdomyosarcomas is related to tumor site, surgical-pathologic grouping, and tumor histology. The 5-year disease-free survival rate for all patients has been reported to be 65%. Five-year disease-free survival rates for patients in groups I, II, III, and IV have been reported to be 84%, 74%, 62%, and 23%, respectively (see Table 36-3).260Nonrhabdomyosarcoma Soft Tissue SarcomasApproximately 60% of soft tissue sarcomas in children are nonrhabdomyosarcomas. These include numerous histologic subtypes, which are generally categorized into four groups: (a) fibrosarcoma, (b) Kaposi’s sarcoma, (c) other “specified” soft tissue sarcomas (e.g., synovial, angiosarcoma, hemangiopericy-toma, leiomyosarcoma,

1	are generally categorized into four groups: (a) fibrosarcoma, (b) Kaposi’s sarcoma, (c) other “specified” soft tissue sarcomas (e.g., synovial, angiosarcoma, hemangiopericy-toma, leiomyosarcoma, liposarcoma, and extraosseous Ewing’s sarcoma), and (d) “unspecified” soft tissue sarcoma.261 The most common subtypes are synovial sarcoma, MPNST, and fibro-sarcoma. No single histology accounts for more than 15% of all cases.As with adult tumors, the evaluation of the soft tissue mass begins with a history and physical examination followed by imaging, which usually includes MRI. A CT scan of the chest is important for evaluation of metastatic disease. A core needle biopsy is generally required to establish a diagnosis. Surgery remains the primary treatment of nonrhabdomyosarcoma, and Brunicardi_Ch36_p1567-p1598.indd 158901/03/19 6:38 PM 1590SPECIFIC CONSIDERATIONSPART IIlocal control of large, high-grade tumors is improved with radia-tion therapy. The prognostic factors for children with

1	158901/03/19 6:38 PM 1590SPECIFIC CONSIDERATIONSPART IIlocal control of large, high-grade tumors is improved with radia-tion therapy. The prognostic factors for children with nonrhab-domyosarcoma are similar to those for adults, and the role of chemotherapy for high-risk tumors is unclear, as for adults.RESEARCH PERSPECTIVESAs the molecular alterations associated with various sar-coma subtypes are elucidated, many new potential targets for therapeutic intervention will be identified. A wide variety of DNA alterations have been observed in sarcomas that result in mutated genes encoding proteins ranging from transcription fac-tors to tyrosine kinases to cytokines. The challenge in identify-ing therapeutic targets in sarcoma is to identify those that are specifically important to cellular function. The ideal therapeutic target has been described as a single molecule that is critical for pathogenesis, is expressed and active, is involved in a single pathway amenable to blockade (i.e.,

1	function. The ideal therapeutic target has been described as a single molecule that is critical for pathogenesis, is expressed and active, is involved in a single pathway amenable to blockade (i.e., no alternative bypass path-ways exist), and is critical for sarcoma cell survival.25CONCLUSIONSSoft tissue sarcomas are a heterogeneous family of rare tumors, accounting for approximately 1% of malignancies in adults. The etiology in the vast majority of patients is sporadic, and the man-agement of such diverse tumors is complex. Diagnosis by light microscopy is inexact, but molecular diagnosis, although still in its infancy, holds great promise. The natural history of soft tissue sarcomas is well established. Approximately two-thirds of cases arise in the extremities, and the remaining one-third are distrib-uted between the retroperitoneum, trunk, abdomen, and head and neck. The management algorithm for soft tissue sarcomas is complex and depends on tumor stage, site, and histology. The

1	are distrib-uted between the retroperitoneum, trunk, abdomen, and head and neck. The management algorithm for soft tissue sarcomas is complex and depends on tumor stage, site, and histology. The most common site of metastasis is the lungs, and metastasis generally occurs within 3 years of diagnosis.Soft tissue sarcomas have unique molecular profiles that contribute to varying responses to systemic therapy. Doxorubicinbased regimens have been the mainstay of treatment for the past two decades; however, it is now clear that specific histologic subtypes have increased sensitivity to specific agents. For exam-ple, angiosarcomas are more sensitive to paclitaxel, while leio-myosarcoma is sensitive to gemcitabine and docetaxel. Progress in understanding of soft tissue sarcoma biology is crucial for the development of additional therapeutic targets. Drug engineering will enable molecular-based therapies to become increasingly incorporated into clinical trials and, with success, into standard

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1	a wait-and-see policy according to tumor presentation. J Clin Oncol. 2011;29: 3553-3558. 243. Bonvalot S, Eldweny H, Haddad V, et al. Extra-abdominal primary fibromatosis: aggressive management could be avoided in a subgroup of patients. Eur J Surg Oncol. 2008;34: 462-468. 244. Fiore M, Rimareix F, Mariani L, et al. Desmoid-type fibromatosis: a front-line conservative approach to select patients for surgical treatment. Ann Surg Oncol. 2009;16: 2587-2593. 245. Constantinidou A, Scurr M, Jones R, et al. Treatment of aggressive fibromatosis with pegylated liposomal doxorubi-cin: The Royal Marsden Hospital experience. J Clin Oncol. (Meeting Abstracts), Abstract 10519, 2009. 246. Gounder MM, Lefkowitz RA, Keohan ML, et al. Activity of sorafenib against desmoid tumor/deep fibromatosis. Clin Cancer Res. 2011;17:4082-4090. 247. Dufresne A, Penel N, Salas S, et al. Updated outcome with long term follow-up of imatinib for the treatment of progres-sive or recurrent aggressive fibromatosis

1	Cancer Res. 2011;17:4082-4090. 247. Dufresne A, Penel N, Salas S, et al. Updated outcome with long term follow-up of imatinib for the treatment of progres-sive or recurrent aggressive fibromatosis (desmoid tumor): a FNCLCC/French Sarcoma Group phase II trial. Age. 2009;40:59. 248. Chugh R, Wathen JK, Patel SR, et al. Efficacy of imatinib in aggressive fibromatosis: results of a phase II multicenter Sarcoma Alliance for Research through Collaboration (SARC) trial. Clin Cancer Res. 2010;16:4884-4891. 249. Penel N, Le Cesne A, Bui B, et al. Imatinib for progressive and recurrent aggressive fibromatosis (desmoid tumors): an FNCLCC/French Sarcoma Group phase II trial with a long-term follow-up. Ann Oncol. 2011;22:452-457. 250. Criscione VD, Weinstock MA. Descriptive epidemiology of dermatofibrosarcoma protuberans in the United States, 1973 to 2002. J Am Acad Dermatol. 2007;56:968-973. 251. Fiore M, Miceli R, Mussi C, et al. Dermatofibrosarcoma protuberans treated at a single

1	dermatofibrosarcoma protuberans in the United States, 1973 to 2002. J Am Acad Dermatol. 2007;56:968-973. 251. Fiore M, Miceli R, Mussi C, et al. Dermatofibrosarcoma protuberans treated at a single institution: a surgical disease with a high cure rate. J Clin Oncol. 2005;23:7669-7675. 252. McArthur GA. Molecular targeting of dermatofibrosarcoma protuberans: a new approach to a surgical disease. J Natl Compr Canc Netw. 2007;5:557-562.Brunicardi_Ch36_p1567-p1598.indd 159601/03/19 6:38 PM 1597SOFT TISSUE SARCOMASCHAPTER 36 253. Grovas A, Fremgen A, Rauck A, et al. The National Cancer Data Base report on patterns of childhood cancers in the United States. Cancer. 1997;80:2321-2332. 254. Meyer WH, Spunt SL. Soft tissue sarcomas of childhood. Cancer Treat Rev. 2004;30:269-280. 255. Barr FG, Chatten J, D’Cruz CM, et al. Molecular assays for chromosomal translocations in the diagnosis of pediatric soft tissue sarcomas. JAMA. 1995;273:553-557. 256. Xia SJ, Pressey JG, Barr FG. Molecular

1	Chatten J, D’Cruz CM, et al. Molecular assays for chromosomal translocations in the diagnosis of pediatric soft tissue sarcomas. JAMA. 1995;273:553-557. 256. Xia SJ, Pressey JG, Barr FG. Molecular pathogenesis of rhabdomyosarcoma. Cancer Biol Ther. 2002;1:97-104. 257. Scrable H, Witte D, Shimada H, et al. Molecular differential pathology of rhabdomyosarcoma. Genes Chromosomes Cancer. 1989;1:23-35. 258. Flamant F, Rodary C, Rey A, et al. Treatment of non-metastatic rhabdomyosarcomas in childhood and adolescence. Results of the second study of the International Society of Paediatric Oncology: MMT84. Eur J Cancer. 1998;34:1050-1062. 259. Crist WM, Anderson JR, Meza JL, et al. Intergroup rhabdomyosarcoma study-IV: results for patients with nonmetastatic disease. J Clin Oncol. 2001;19:3091-3102. 260. Crist WM, Garnsey L, Beltangady MS, et al. Prognosis in children with rhabdomyosarcoma: a report of the intergroup rhabdomyosarcoma studies I and II. Intergroup Rhabdomyo-sarcoma Committee.

1	WM, Garnsey L, Beltangady MS, et al. Prognosis in children with rhabdomyosarcoma: a report of the intergroup rhabdomyosarcoma studies I and II. Intergroup Rhabdomyo-sarcoma Committee. J Clin Oncol. 1990;8:443-452. 261. Loeb DM, Thornton K, Shokek O. Pediatric soft tissue sarcomas. Surg Clin North Am. 2008;88:615-627, vii.Brunicardi_Ch36_p1567-p1598.indd 159701/03/19 6:38 PM

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1	Inguinal HerniasChandan Das, Tahir Jamil, Stephen Stanek, Ziya Baghmanli, James R. Macho, Joseph Sferra, and F. Charles Brunicardi 37chapterINTRODUCTIONInguinal herniorrhaphy is one of the most commonly performed operations in the United States.1 Based on estimates made by the National Center for Health Statistics, in 2010 nearly 515,000 inguinal hernia operations were performed in hospitals, and an additional 450,000 were performed in ambulatory surgery centers.1Approximately 75% of abdominal wall hernias occur in the groin. Of inguinal hernia repairs, 90% are performed in men, and 10% are performed in women. This is thought to be because the lifetime risk of inguinal hernia is 27% in men and 3% in women.2 The incidence of inguinal hernia in men has a bimodal distribution, with peaks before the first year of age and after age 40. Abramson demonstrated the age-dependence of inguinal hernias in 1978. Those age 25 to 34 years had a life-time prevalence rate of 15%, whereas those age

1	before the first year of age and after age 40. Abramson demonstrated the age-dependence of inguinal hernias in 1978. Those age 25 to 34 years had a life-time prevalence rate of 15%, whereas those age 75 years and over had a rate of 47% (Table 37-1).3 Approximately 70% of femoral hernia repairs are performed in women; however, the most common subtype of groin hernia in men and women is still the indirect inguinal hernia. Inguinal hernias are five times more common than femoral hernias.4Globally, the inguinal hernia repair has become one of the most important procedures in improving quality of life and preventing disability. In one study, an international coopera-tive organization performed over 1033 hernia repairs on 926 patients, and their impact was measured in disability adjusted life years (DALYs). They were able to avoid 5014 DALYs or 5.41 DALYs per patient.5HistorySurgical repair of hernias has been documented as far back as in ancient Egyptian and Greek civilizations.4 In the

1	years (DALYs). They were able to avoid 5014 DALYs or 5.41 DALYs per patient.5HistorySurgical repair of hernias has been documented as far back as in ancient Egyptian and Greek civilizations.4 In the past, early management of inguinal hernias often involved a conservative approach with operative management reserved only for compli-cations. Surgery often involved routine excision of the testicle, and wounds were closed with cauterization or allowed to close by secondary intention. These procedures were 1performed without aseptic technique, and infection and recur-rence rates were high.From the late 1700s to the early 1800s, physicians including Hesselbach, Cooper, Camper, Scarpa, Richter, and Gimbernat identified vital components of the inguinal region from cadaveric dissection. This improved understanding of the anatomy and pathophysiology of inguinal hernias. These findings, coupled with the development of aseptic technique, led surgeons such as Marcy, Kocher, and Lucas-Championnière

1	understanding of the anatomy and pathophysiology of inguinal hernias. These findings, coupled with the development of aseptic technique, led surgeons such as Marcy, Kocher, and Lucas-Championnière to perform sac dissection, high ligation, and closure of the internal ring. This led to improved outcomes, but recurrence rates remained unacceptably high.At around this time, Bassini (1844–1924) pioneered a new method that transformed inguinal hernia repair into a success-ful venture with minimal morbidity. The success of the Bassini repair over its predecessors ushered in an era of tissue-based repairs. The Bassini repair was then modified into the McVay and Shouldice repairs. All three of these techniques, as well as modern variations such as the Desarda operation, are currently practiced.6The next major advancement in inguinal hernia repair was in the 1980s. At this time, Lichtenstein applied a piece of mesh to the floor of the inguinal canal, allowing for a truly tension-free repair.

1	next major advancement in inguinal hernia repair was in the 1980s. At this time, Lichtenstein applied a piece of mesh to the floor of the inguinal canal, allowing for a truly tension-free repair. This technique demonstrated superior outcomes compared to previous tissue-based repairs. There were several other advantages of this process. In addition to being truly tension-free, the mesh could restore the strength of the transversalis fascia, and importantly, the technique had a very short learning curve. The superior outcomes have been widely reproduced regardless of hernia size and type, and they were achievable among both expert and nonexpert hernia surgeons.7With the advent of minimally invasive surgery, inguinal hernia repair underwent its most recent transformation. Laparo-scopic inguinal hernia repair offers an alternative approach, minimizes postoperative pain,8 and improves recovery. Since the initial description by Ger, the laparoscopic method has become more sophisticated.

1	hernia repair offers an alternative approach, minimizes postoperative pain,8 and improves recovery. Since the initial description by Ger, the laparoscopic method has become more sophisticated. Refinements in approach and technique have Introduction1599History / 1599Anatomy / 1600Pathophysiology / 1604Diagnosis1606History / 1606Physical Examination / 1607Imaging / 1607Treatment1608Prophylactic Antibiotics / 1609Open Approach / 1609Laparoscopic Approach / 1613Robot-Assisted Inguinal Hernia Repair / 1616Prosthesis Considerations / 1616Complications1618Hernia Recurrence / 1618Pain / 1619Cord and Testes Injury / 1620Laparoscopic Complications / 1620Hematomas and Seromas / 1621Outcomes1621Brunicardi_Ch37_p1599-p1624.indd 159929/01/19 2:02 PM 1600Key Points1 Conservative management of asymptomatic inguinal hernias is recommended.2 A thorough understanding of groin anatomy is essential to successful surgical treatment of inguinal hernias.3 Elective repair of inguinal hernias can be

1	asymptomatic inguinal hernias is recommended.2 A thorough understanding of groin anatomy is essential to successful surgical treatment of inguinal hernias.3 Elective repair of inguinal hernias can be undertaken using a laparoscopic, robotic, or open approach.4 Robotic-assisted hernia surgery is quickly becoming adopted by general surgeons because of its better ergonomics and visualization.5 The use of prosthetic mesh as a reinforcement significantly improves recurrence rates, whether the repair is open or laparoscopic.6 Recurrence, pain, and quality of life are the metrics by which hernia repair outcomes are measured.7 Laparoscopic inguinal hernia repair results in less pain; how-ever, mastery of this technique has a longer learning curve.led to the development of the intraperitoneal onlay mesh,9,10 the transabdominal preperitoneal (TAPP) repair,11 and the totally extraperitoneal (TEP) repair.12 Irrespective of the approach, suc-cessful surgical treatment of inguinal hernias depends

1	mesh,9,10 the transabdominal preperitoneal (TAPP) repair,11 and the totally extraperitoneal (TEP) repair.12 Irrespective of the approach, suc-cessful surgical treatment of inguinal hernias depends on a sound grasp of inguinal anatomy.AnatomyThe inguinal canal is an approximately 4to 6-cm long cone-shaped region situated in the anterior portion of the pelvic basin (Fig. 37-1). The canal begins on the posterior abdominal wall, where the spermatic cord passes through a hiatus in the transver-salis fascia also known as the deep (internal) inguinal ring. The canal concludes medially at the superficial (external) inguinal ring, the point at which the spermatic cord crosses a defect in the external oblique aponeurosis. The boundaries of the ingui-nal canal are the external oblique aponeurosis anteriorly, the internal oblique muscle laterally, the transversalis fascia and transversus abdominis muscle posteriorly, the internal oblique and transversus abdominis muscle superiorly, and the

1	anteriorly, the internal oblique muscle laterally, the transversalis fascia and transversus abdominis muscle posteriorly, the internal oblique and transversus abdominis muscle superiorly, and the inguinal (Poupart’s) ligament inferiorly. The spermatic cord traverses the inguinal canal, and it contains three arteries, three veins, two nerves, the pampiniform venous plexus, and the vas deferens. It is enveloped in three layers of spermatic fascia.Additional important structures surrounding the inguinal canal include the iliopubic tract, the lacunar ligament, Cooper’s ligament, and the conjoined tendon (Fig. 37-2). The iliopubic tract is an aponeurotic band that begins at the anterior superior iliac spine and inserts into Cooper’s ligament from above. It forms on the deep inferior margin of the transversus abdominis and transversalis fascia. The shelving edge of the inguinal ligament is a structure that connects the iliopubic tract to the inguinal ligament. The iliopubic tract helps form

1	transversus abdominis and transversalis fascia. The shelving edge of the inguinal ligament is a structure that connects the iliopubic tract to the inguinal ligament. The iliopubic tract helps form the inferior margin of the internal inguinal ring as it courses medially, where it continues as the anteromedial border of the femoral canal. The lacunar ligament, or ligament of Gimbernat, is the triangular fanning of the inguinal ligament as it joins the pubic 2tubercle. Cooper’s (pectineal) ligament is the lateral portion of the lacunar ligament that is fused to the periosteum of the pubic tubercle. The conjoined tendon is commonly described as the fusion of the inferior fibers of the internal oblique and transversus abdominis aponeurosis at the point where they insert on the pubic tubercle.Inguinal hernias are generally classified as direct, indirect, or femoral based upon the site of herniation relative to surround-ing structures. Indirect hernias protrude lateral to the inferior

1	hernias are generally classified as direct, indirect, or femoral based upon the site of herniation relative to surround-ing structures. Indirect hernias protrude lateral to the inferior epigastric vessels, through the deep inguinal ring. Direct hernias protrude medial to the inferior epigastric vessels, within Hesselbach’s triangle. The borders of the triangle are the ingui-nal ligament inferiorly, the lateral edge of rectus sheath medi-ally, and the inferior epigastric vessels superolaterally. Femoral hernias protrude through the small and inflexible femoral ring. They traverse the empty space between the femoral vein and the lymphatic channels. The borders of the femoral ring include the iliopubic tract and inguinal ligament anteriorly, Cooper’s liga-ment posteriorly, the lacunar ligament medially, and the femoral vein laterally. The Nyhus classification categorizes hernia defects by location, size, and type (Table 37-2).Laparoscopic inguinal hernia repair requires a thorough

1	medially, and the femoral vein laterally. The Nyhus classification categorizes hernia defects by location, size, and type (Table 37-2).Laparoscopic inguinal hernia repair requires a thorough knowledge of inguinal anatomy from a posterior perspective (Fig. 37-3). Intraperitoneal points of reference are the five peritoneal folds, bladder, inferior epigastric vessels, and psoas muscle (Fig. 37-4). Two potential spaces exist within the pre-peritoneum. Between the peritoneum and the posterior lamina of the transversalis fascia is Bogros’s (preperitoneal) space. This area contains preperitoneal fat and areolar tissue. The most medial aspect of the preperitoneal space, that which lies superior to the bladder, is known as the space of Retzius. The posterior perspective also allows visualization of the myopectineal orifice of Fruchaud, a relatively weak portion of the abdominal wall that is divided by the inguinal ligament (Fig. 37-5).The vascular space is situated between the posterior and

1	of the myopectineal orifice of Fruchaud, a relatively weak portion of the abdominal wall that is divided by the inguinal ligament (Fig. 37-5).The vascular space is situated between the posterior and anterior laminae of the transversalis fascia, and it houses the Table 37-1Inguinal hernia prevalence by ageAGE (Y)25–3435–4445–5455–6465–7475+Current prevalence (%)121520262934Lifetime prevalence (%)151928344047Current = repaired hernias excluded; lifetime = repaired hernias included.Brunicardi_Ch37_p1599-p1624.indd 160029/01/19 2:02 PM 1601INGUINAL HERNIASCHAPTER 37Abdominal ringLateral leafPubic tubercleSpermatic cordInterrupted sutures taken to suture the medial leaf to the inguinal ligament Interrupted sutures between the upper border of the strip and conjoined muscle and internal oblique muscleInternal oblique muscle seen through the splitting incision made in the medial leafReflected medial leaf after a strip has been separatedFigure 37-2. Ligaments that contribute to the

1	oblique muscleInternal oblique muscle seen through the splitting incision made in the medial leafReflected medial leaf after a strip has been separatedFigure 37-2. Ligaments that contribute to the inguinal canal include the inguinal ligament, Cooper’s ligament, and the lacunar ligament. The iliopubic tract originates and inserts in a similar fashion to the inguinal ligament, but in a deeper position. m. = muscle.Abdominal ringLateral leafMedial leafPubic tubercleSpermatic cordInterrupted sutures taken to suture the medial leaf to the inguinal ligament Figure 37-1. Location and orientation of the inguinal canal within the pelvic basin. Boundaries of the canal include: transversus abdominus and transversalis fascia posterior; internal oblique muscle superior; external oblique aponeurosis anterior; inguinal ligament inferior. m. = muscle.inferior epigastric vessels. The inferior epigastric artery supplies the rectus abdominis. It is derived from the external iliac artery, and it

1	anterior; inguinal ligament inferior. m. = muscle.inferior epigastric vessels. The inferior epigastric artery supplies the rectus abdominis. It is derived from the external iliac artery, and it anastomoses with the superior epigastric, a continuation of the internal thoracic artery. The epigastric veins course paral-lel to the arteries within the rectus sheath, posterior to the rectus muscles. Inspection of the internal inguinal ring will reveal the deep location of the inferior epigastric vessels.Nerves of interest in the inguinal region are the ilioin-guinal, iliohypogastric, genitofemoral, and lateral femoral cutaneous nerves (Figs. 37-6 and 37-7). The ilioinguinal and iliohypogastric nerves arise together from the first lumbar nerve (L1). The ilioinguinal nerve emerges from the lateral border of the psoas major and passes obliquely across the quadratus lumborum. At a point just medial to the anterior Brunicardi_Ch37_p1599-p1624.indd 160129/01/19 2:02 PM 1602SPECIFIC

1	the lateral border of the psoas major and passes obliquely across the quadratus lumborum. At a point just medial to the anterior Brunicardi_Ch37_p1599-p1624.indd 160129/01/19 2:02 PM 1602SPECIFIC CONSIDERATIONSPART IIInferior epigastric vesselsCooper’s ligamentPubic tubercleArcuate lineUmbilicusLinea albaRectus muscleDeepinguinalringIliopubictractExternal iliacvesselsIndirecthernia siteDirecthernia siteObturatorvesselsTransversusabdominismusclearchSuperioranteriorcrusSpermatic cordSpermatic vesselsFemoral canalFigure 37-3. Anatomy of the groin region from the posterior perspective.AA UmbilicusB Median umbilical ligament (urachus)C Medial umbilical ligament (obliterated umbilical vein)D Lateral umbilical ligament (inferior epigastric vessels)E Lateral fossa (indirect hernia)F Medial fossa (direct hernia)G Supravesical fossaBEFBladderGCDFigure 37-4. Posterior view of intraperitoneal folds and associated fossa: A. Umbilicus. B. Median umbilical ligament. C. Medial

1	hernia)F Medial fossa (direct hernia)G Supravesical fossaBEFBladderGCDFigure 37-4. Posterior view of intraperitoneal folds and associated fossa: A. Umbilicus. B. Median umbilical ligament. C. Medial umbilical ligament (obliterated umbilical vein). D. Lateral umbilical ligament (inferior epigastric vessels). E. Lateral fossa (indirect hernia). F. Medial fossa (direct hernia). G. Supravesical fossa. (Modified with permission from Rowe JS, Skandalakis JE, Gray SW: Multiple bilateral inguinal hernias, Am Surg. 1973 May;39(5):269-270.)Table 37-2Nyhus classification systemType IIndirect hernia; internal abdominal ring normal; typically in infants, children, small adultsType IIIndirect hernia; internal ring enlarged without impingement on the floor of the inguinal canal; does not extend to the scrotumType IIIADirect hernia; size is not taken into accountType IIIBIndirect hernia that has enlarged enough to encroach upon the posterior inguinal wall; indirect sliding or scrotal hernias are

1	scrotumType IIIADirect hernia; size is not taken into accountType IIIBIndirect hernia that has enlarged enough to encroach upon the posterior inguinal wall; indirect sliding or scrotal hernias are usually placed in this category because they are commonly associated with extension to the direct space; also includes pantaloon herniasType IIICFemoral herniaType IVRecurrent hernia; modifiers A–D are sometimes added, which correspond to indirect, direct, femoral, and mixed, respectivelysuperior iliac spine, it pierces the transversus and internal oblique muscles to enter the inguinal canal and exits through the superficial inguinal ring. It supplies somatic sensation to the skin of the upper and medial thigh. In males, it also inner-vates the base of the penis and upper scrotum. In females, it innervates the mons pubis and labium majus. The iliohypo-gastric nerve arises from T12–L1. After it pierces the deep abdominal wall, it courses between the internal oblique and transversus abdominis,

1	innervates the mons pubis and labium majus. The iliohypo-gastric nerve arises from T12–L1. After it pierces the deep abdominal wall, it courses between the internal oblique and transversus abdominis, supplying both. It then divides into lat-eral and anterior cutaneous branches. A common variant is for the iliohypogastric and ilioinguinal nerves to exit around the superficial inguinal ring as a single entity. The genitofemoral nerve arises from L1 to L2, courses along the retroperitoneum, and emerges on the anterior aspect of the psoas. It then divides into genital and femoral branches. The genital branch enters the inguinal canal lateral to the inferior epigastric vessels, and it courses ventral to the iliac vessels and iliopubic tract. In males, it travels through the superficial inguinal ring and sup-plies the ipsilateral scrotum and cremaster muscle. In females, it supplies the ipsilateral mons pubis and labium majus. The Brunicardi_Ch37_p1599-p1624.indd 160229/01/19 2:02 PM

1	ring and sup-plies the ipsilateral scrotum and cremaster muscle. In females, it supplies the ipsilateral mons pubis and labium majus. The Brunicardi_Ch37_p1599-p1624.indd 160229/01/19 2:02 PM 1603INGUINAL HERNIASCHAPTER 37Epigastric vesselsInternal spermaticvesselsIliopsoas muscleIliopubictractMyopectinealorificeExternal iliac a. and v.Vas deferensObturator n.Obturator vesselsLacunar ligamentCooper's ligamentFigure 37-5. Posterior view of the myopectineal orifice of Fruchaud. a. = artery; n. = nerve; v. = vein.Ilioinguinal n.Iliohypogastric n.Iliac m.Lateral femoralcutaneous n.Femoral n.Inguinal ligamentGenitofemoral n.(femoral branch)(genital branch)Iliopectinal archPectineal ligamentIliopubic tractLacunar ligamentFigure 37-6. Retroperitoneal view of major inguinal nerves and their courses. m. = muscle; n. = nerve.femoral branch courses along the femoral sheath, supply-ing the skin of the upper anterior thigh. The lateral femoral cutaneous nerve arises from L2 to L3, emerges

1	courses. m. = muscle; n. = nerve.femoral branch courses along the femoral sheath, supply-ing the skin of the upper anterior thigh. The lateral femoral cutaneous nerve arises from L2 to L3, emerges lateral to the psoas muscle at the level of L4, and crosses the iliacus mus-cle obliquely toward the anterior superior iliac spine. It then passes inferiorly to the inguinal ligament where it divides to supply the lateral thigh (Fig. 37-8).The preperitoneal anatomy seen in laparoscopic hernia repair led to characterization of important anatomic areas of interest, known as the triangle of doom, the triangle of pain, and the circle of death (Fig. 37-9).7 The triangle of doom is bordered medially by the vas deferens and laterally by the vessels of the spermatic cord. The contents of the space include the external iliac vessels, deep circumflex iliac vein, femoral nerve, and gen-ital branch of the genitofemoral nerve. The triangle of pain is a region bordered by the iliopubic tract and gonadal

1	the external iliac vessels, deep circumflex iliac vein, femoral nerve, and gen-ital branch of the genitofemoral nerve. The triangle of pain is a region bordered by the iliopubic tract and gonadal vessels, and it encompasses the lateral femoral cutaneous, femoral branch of the genitofemoral and femoral nerves. The circle of death is a vascular continuation formed by the common iliac, internal iliac, obturator, inferior epigastric, and external iliac vessels.Brunicardi_Ch37_p1599-p1624.indd 160329/01/19 2:03 PM 1604SPECIFIC CONSIDERATIONSPART IIFigure 37-7. Anterior view of the five major nerves of the inguinal region.Femoral branch ofgenitofemoral n.Ilioinguinal n.Lateral femoralcutaneous n.Medial and intermediatefemoral cutaneous nn.Saphenous n.Iliohypogastric n.Genital branch ofgenitofemoral n.Figure 37-8. Sensory dermatomes of the major nerves in the groin area. n. = nerve.PathophysiologyInguinal hernias may be congenital or acquired. Most adult inguinal hernias are considered

1	n.Figure 37-8. Sensory dermatomes of the major nerves in the groin area. n. = nerve.PathophysiologyInguinal hernias may be congenital or acquired. Most adult inguinal hernias are considered acquired defects in the abdomi-nal wall. There is however, a known hereditary association that is not well understood.13 The most likely risk factor for inguinal hernia is weakness in the abdominal wall musculature; how-ever, there are several other risk-factors that have been studied (Table 37-3). Congenital hernias, which make up the majority of pediatric hernias, can be considered a developmental defect rather than an acquired weakness. During the normal course of development, the testes descend from the intra-abdominal space into the scrotum in the third trimester. Their descent is guided by the gubernaculum through an evagination of the peritoneum, which protrudes through the inguinal canal and becomes the processus vaginalis. Between 36 and 40 weeks’ gestation, the processus vaginalis closes

1	through an evagination of the peritoneum, which protrudes through the inguinal canal and becomes the processus vaginalis. Between 36 and 40 weeks’ gestation, the processus vaginalis closes and eliminates the peritoneal open-ing at the internal inguinal ring.14 Failure of the peritoneum to close results in a patent processus vaginalis (PPV). In preterm babies, indirect inguinal hernias as a result of PPV is very high (Fig. 37-10). However, overall, the risk of developing a symp-tomatic hernia during childhood in the presence of a known PPV is relatively low.15Overall, there is limited data regarding the etiology of inguinal hernia development. Several studies have documented strenuous physical activity as a risk factor for acquired inguinal hernia.16 A case-controlled study of over 1400 male patients with inguinal hernia revealed that a positive family history was associated with an eightfold lifetime incidence of Brunicardi_Ch37_p1599-p1624.indd 160429/01/19 2:03 PM 1605INGUINAL

1	patients with inguinal hernia revealed that a positive family history was associated with an eightfold lifetime incidence of Brunicardi_Ch37_p1599-p1624.indd 160429/01/19 2:03 PM 1605INGUINAL HERNIASCHAPTER 37Inferolateral border:iliopubic tractDeep circumflexiliac a. & v.Lateral border:reflected peritoneumLat. femoral cutaneous n.Ant. femoral cutaneous n. or other variable branchesFemoral br. of genitofemoral n.Femoral n.Superomedial border:gonadal vesselsLateral border:gonadal vesselsGenital branch ofgenitofemoral nerveIliac veinIliac arteryMedial border:ductus deferensPosterior border:peritoneal edgeDeep ringABFigure 37-9. Borders and contents of the (A) triangle of doom and (B) triangle of pain. a. = artery; Ant. = anterior; br. = branch; Lat. = lateral; n. = nerve; v. = vein. (Modified with permission from Colborn GL, Skandalakis JE: Laparoscopic cadaveric anatomy of the inguinal area, Probl Gen Surg. 1995;12(1):13-20.)inguinal hernia.17 Chronic obstructive pulmonary disease

1	with permission from Colborn GL, Skandalakis JE: Laparoscopic cadaveric anatomy of the inguinal area, Probl Gen Surg. 1995;12(1):13-20.)inguinal hernia.17 Chronic obstructive pulmonary disease also significantly increases the risk of direct inguinal hernias, thought to be due to repeated instances of intra-abdominal pressure during coughing.18 Several studies have suggested a protective effect of obesity. In a large, population-based prospective study of American individuals (First National Health and Nutrition Examination Survey), the risk of inguinal hernia development in obese men was only 50% that of normal-weight men, while the risk in overweight men was 80% that of nonobese men. A possible explanation is the increased difficulty in detecting inguinal hernias in obese individuals.18Epidemiologic studies have identified risk factors that may predispose to a hernia. Microscopic examination of skin of inguinal hernia patients demonstrated significantly decreased ratios of type I to

1	studies have identified risk factors that may predispose to a hernia. Microscopic examination of skin of inguinal hernia patients demonstrated significantly decreased ratios of type I to type III collagen. Type III collagen does not contribute to wound tensile strength as significantly as type I collagen. Additional analyses of similar skin revealed disaggregated collagen tracts with decreased collagen fiber density.19 Collagen disorders such as Ehlers-Danlos syndrome are also associated with an increased incidence of hernia formation (Table 37-4). Recent studies have found an association between concentrations of extracellular matrix Brunicardi_Ch37_p1599-p1624.indd 160529/01/19 2:03 PM 1606SPECIFIC CONSIDERATIONSPART IITable 37-3Presumed causes of groin herniationCoughingChronic obstructive pulmonary diseaseObesityStraining Constipation ProstatismPregnancyBirthweight <1500 gFamily history of a herniaValsalva’s maneuverAscitesUpright positionCongenital connective tissue

1	obstructive pulmonary diseaseObesityStraining Constipation ProstatismPregnancyBirthweight <1500 gFamily history of a herniaValsalva’s maneuverAscitesUpright positionCongenital connective tissue disordersDefective collagen synthesisPrevious right lower quadrant incisionArterial aneurysmsCigarette smokingHeavy liftingPhysical exertionFigure 37-10. Varying degrees of closure of the processus vagi-nalis (PV). A. Closed PV. B. Minimally patent PV. C. Moderately patent PV. D. Scrotal hernia.Table 37-4Connective tissue disorders associated with groin herniationOsteogenesis imperfectaCutis laxa (congenital elastolysis)Ehlers-Danlos syndromeHurler-Hunter syndromeMarfan’s syndromeCongenital hip dislocation in childrenPolycystic kidney diseaseα1-Antitrypsin deficiencyWilliams syndromeAndrogen insensitivity syndromeRobinow’s syndromeSerpentine fibula syndromeAlport’s syndromeTel Hashomer camptodactyly syndromeLeriche’s syndromeTesticular feminization syndromeRokitansky-Mayer-Küster

1	insensitivity syndromeRobinow’s syndromeSerpentine fibula syndromeAlport’s syndromeTel Hashomer camptodactyly syndromeLeriche’s syndromeTesticular feminization syndromeRokitansky-Mayer-Küster syndromeGoldenhar’s syndromeMorris syndromeGerhardt’s syndromeMenkes’ syndromeKawasaki diseasePfannenstiel syndromeBeckwith-Wiedemann syndromeRubinstein-Taybi syndromeAlopecia-photophobia syndromeDIAGNOSISHistoryWorkup for inguinal hernia begins with a detailed history. The most common symptom of inguinal hernia is a groin mass that protrudes while standing, coughing, or straining. It is sometimes described as reducible while lying down. Symptoms that are extrainguinal such as a change in bowel habits or urinary symptoms are far less common but should be recognized as having the potential to be ominous. The pain is thought to be due to compression of the nerves by the sac, causing generalized pressure, localized sharp pain, or referred pain. Referred pain may involve the scrotum, tes-ticle, or

1	The pain is thought to be due to compression of the nerves by the sac, causing generalized pressure, localized sharp pain, or referred pain. Referred pain may involve the scrotum, tes-ticle, or inner thigh.Important considerations of the patient’s history include the duration and timing of symptoms. Sudden onset symp-toms are more concerning. Questions should also be directed to characterize whether the hernia is reducible. Patients will often reduce the hernia by pushing the contents back into the abdomen, thereby providing temporary relief. As the defect size increases and more intra-abdominal contents fill the hernia sac, the hernia may become harder to reduce and incarcerate, prompting urgent surgical intervention.Certain elements of the review of systems such as chronic constipation, cough, or urinary retention should prompt the sur-geon to perform a thorough workup to rule out any underlying malignancy.elements and hernia formation.20 Although a significant amount of work

1	cough, or urinary retention should prompt the sur-geon to perform a thorough workup to rule out any underlying malignancy.elements and hernia formation.20 Although a significant amount of work remains to elucidate the biologic nature of hernias, current evidence suggests they have a multifactorial etiology with both environmental and hereditary influences.Brunicardi_Ch37_p1599-p1624.indd 160629/01/19 2:03 PM 1607INGUINAL HERNIASCHAPTER 37Figure 37-11. Digital examination of the inguinal canal.Table 37-5Differential diagnosis of groin herniaMalignancy Lymphoma Retroperitoneal sarcoma Metastasis Testicular tumorPrimary testicular Varicocele Epididymitis Testicular torsion Hydrocele Ectopic testicle Undescended testicleFemoral artery aneurysm or pseudoaneurysmLymph nodeSebaceous cystHidradenitisCyst of the canal of Nuck (female)Saphenous varixPsoas abscessHematomaAscitesPhysical ExaminationPhysical examination is essential to the diagnosis of inguinal hernia. The patient should be

1	of the canal of Nuck (female)Saphenous varixPsoas abscessHematomaAscitesPhysical ExaminationPhysical examination is essential to the diagnosis of inguinal hernia. The patient should be examined in a standing position to increase intra-abdominal pressure, with the groin and scro-tum fully exposed. Inspection is performed first, with the goal of identifying an abnormal bulge along the groin or within the scrotum. If an obvious bulge is not detected, palpation is per-formed to confirm the presence of the hernia.Palpation is performed by advancing the index fin-ger through the scrotum towards the external inguinal ring (Fig. 37-11). This allows the inguinal canal to be explored. The patient is then asked to perform a Valsalva maneuver to increase intraabdominal pressure. These maneuvers will reveal an abnor-mal bulge and allow the clinician to determine whether the her-nia is reducible or not. Examination of the contralateral side affords the clinician the opportunity to compare the

1	will reveal an abnor-mal bulge and allow the clinician to determine whether the her-nia is reducible or not. Examination of the contralateral side affords the clinician the opportunity to compare the presence and extent of herniation between sides. This is especially useful in the case of a small hernia. In addition to inguinal hernia, a number of other diagnoses may be considered in the differential of a groin bulge (Table 37-5).While very difficult to ascertain, there are certain physi-cal examination maneuvers that can be performed to help distinguish direct vs. indirect inguinal hernias. The inguinal occlusion test entails the examiner blocking the internal ingui-nal ring with a finger as the patient is instructed to cough. A controlled impulse suggests an indirect hernia, while persistent herniation suggests a direct hernia. Transmission of the cough impulse to the tip of the finger implies an indirect hernia, while an impulse palpated on the dorsum of the finger implies a direct

1	herniation suggests a direct hernia. Transmission of the cough impulse to the tip of the finger implies an indirect hernia, while an impulse palpated on the dorsum of the finger implies a direct hernia. When results of physical examination are com-pared against operative findings, there is a probability some-what higher than chance (i.e., 50%) of correctly diagnosing the type of hernia.21,22External groin anatomy is difficult to assess in obese patients, making the physical diagnosis of inguinal hernia chal-lenging. A further challenge to the physical examination is the identification of a femoral hernia. Femoral hernias should be palpable below the inguinal ligament, lateral to the pubic tubercle. In obese patients, a femoral hernia may be missed or misdiagnosed as a hernia of the inguinal canal. In contrast, a prominent inguinal fat pad in a thin patient, otherwise known as a femoral pseudohernia, may prompt an erroneous diagnosis of femoral hernia.ImagingIn the case of an ambiguous

1	canal. In contrast, a prominent inguinal fat pad in a thin patient, otherwise known as a femoral pseudohernia, may prompt an erroneous diagnosis of femoral hernia.ImagingIn the case of an ambiguous diagnosis, radiologic investigations may be used as an adjunct to history and physical examination. Imaging in obvious cases is unnecessary. The most common radiologic modalities include ultrasonography (US), computed tomography (CT), and magnetic resonance imaging (MRI). Each technique has certain advantages over physical examination alone; however, each modality is associated with potential limitations.US is the least invasive technique and does not impart any radiation to the patient. Anatomic structures can be more easily identified by the presence of bony landmarks; however, because there are few bones in the inguinal canal, other structures such as the inferior epigastric vessels are used to define groin anatomy. Positive intra-abdominal pressure is used to elicit the herniation of

1	are few bones in the inguinal canal, other structures such as the inferior epigastric vessels are used to define groin anatomy. Positive intra-abdominal pressure is used to elicit the herniation of abdominal contents. Movement of these contents through the canal is essential to making the diagnosis with US, Brunicardi_Ch37_p1599-p1624.indd 160729/01/19 2:03 PM 1608SPECIFIC CONSIDERATIONSPART IIFigure 37-12. Computed tomography scan depicting a large right inguinal hernia (arrow). A smaller left inguinal hernia is also visualized.and lack of this movement may lead to a false negative. A recent meta-analysis demonstrated that ultrasound detects inguinal hernia with a sensitivity of 86%, specificity of 77%.23 In thin patients, normal movement of the spermatic cord and posterior abdominal wall against the anterior abdominal wall may lead to false-positive diagnoses of hernia.24CT and MRI provide static images that are able to delin-eate groin anatomy, to detect groin hernias, and to

1	wall against the anterior abdominal wall may lead to false-positive diagnoses of hernia.24CT and MRI provide static images that are able to delin-eate groin anatomy, to detect groin hernias, and to exclude potentially confounding diagnoses (Fig. 37-12). Meta-analysis determined standard CT detects inguinal hernia with a sensitiv-ity of 80%, specificity of 65%. Although direct herniography has a higher sensitivity and specificity than CT, its invasiveness and limited availability restrict its routine use.23 As CT imaging increases in resolution, its sensitivity in detecting inguinal her-nia is expected to expand; however, this has yet to be clinically confirmed by surgical correlation.25MRI is most commonly utilized in cases where physical examination detects a groin bulge, but where ultrasonography is inconclusive. In a 1999 study of 41 patients with clinical find-ings of inguinal hernia, laparoscopy revealed that MRI was an effective diagnostic test with a sensitivity of 95%,

1	ultrasonography is inconclusive. In a 1999 study of 41 patients with clinical find-ings of inguinal hernia, laparoscopy revealed that MRI was an effective diagnostic test with a sensitivity of 95%, specificity of 96%.26 The expense of MRI precludes its routine use to diag-nose inguinal hernias.TREATMENTSurgical repair of hernias can be performed open, laparoscopic, or with robotic assistance. Surgical repair is the definitive treat-ment of inguinal hernias. The most common reason for elective repair is pain. Incarceration and strangulation are the primary indications for urgent repair. Symptomatic hernias should be operated on electively, and minimally symptomatic or asymptomatic hernias should undergo watchful waiting.27 Repair of minimally symptomatic inguinal hernia in patients with significant medical comorbidities surgery should be deferred and the patient medically optimized. If despite optimal management of comorbidities, the patient remains high-risk, open repair under local

1	medical comorbidities surgery should be deferred and the patient medically optimized. If despite optimal management of comorbidities, the patient remains high-risk, open repair under local anesthesia can be safely performed.28 Although the natural history of untreated inguinal hernias is 3poorly defined, the rates of incarceration and strangulation are low in the asymptomatic population. As a result, nonoperative management is an appropriate consideration in minimally symp-tomatic patients. Prospective studies and meta-analyses have demonstrated no difference in intention-to-treat outcomes, qual-ity of life, or cost-effectiveness between watchful waiting and elective repair among healthy inguinal hernia patients.27,29 A 2012 systematic review found that 72% of asymptomatic ingui-nal hernia patients developed symptoms and had surgical repair within 7.5 years of diagnosis.30 Nevertheless, the complication rates of immediate and delayed elective repair are equivalent.29,31 A nonoperative

1	patients developed symptoms and had surgical repair within 7.5 years of diagnosis.30 Nevertheless, the complication rates of immediate and delayed elective repair are equivalent.29,31 A nonoperative strategy is safe for minimally symptomatic inguinal hernia patients.32Nonoperative inguinal hernia treatment targets pain, pres-sure, and protrusion of abdominal contents in the symptomatic patient population. The recumbent position aids in hernia reduc-tion via the effects of gravity and a relaxed abdominal wall. Trusses externally confine hernias to a reduced state and inter-mittently relieve symptoms in up to 65% of patients; however, they do not prevent complications, and they may be associated with an increased rate of incarceration.33 The risks of incarcera-tion and strangulation appear to decrease over the first year, likely because gradual enlargement of the abdominal wall defect facilitates spontaneous reduction of hernia contents. The sheer volume of protruding tissue in an

1	to decrease over the first year, likely because gradual enlargement of the abdominal wall defect facilitates spontaneous reduction of hernia contents. The sheer volume of protruding tissue in an inguinal hernia does not nec-essarily signify severe morbidity.Femoral and symptomatic inguinal hernias carry higher complication risks, and so surgical repair is performed earlier for these patients. Irrespective of symptoms, one study found the 3-month and 2-year cumulative incidences of strangulation were 2.8% and 4.5%, respectively, for inguinal hernias and 22% and 45%, respectively, for femoral hernias.34 Data from the Swedish Hernia Registry demonstrates that emergent operation is associ-ated with a sevenfold increase in all-cause mortality over that of elective surgery among 107,838 groin hernia repairs.35 For this reason, it is recommended that femoral hernias and symptomatic inguinal hernias be electively repaired, when possible.Incarceration occurs when hernia contents fail to

1	hernia repairs.35 For this reason, it is recommended that femoral hernias and symptomatic inguinal hernias be electively repaired, when possible.Incarceration occurs when hernia contents fail to reduce; however, a minimally symptomatic, chronically incarcerated hernia may also be treated nonoperatively. Taxis should be attempted for incarcerated hernias without sequelae of strangulation, and the option of surgical repair should be discussed prior to the maneuver. To perform taxis, analgesics and light sedatives are administered, and the patient is placed in the Trendelenburg position. The hernia sac is elongated with both hands, and while slight countertraction is maintained, reduction of the contents is attempted circumferentially in a small stepwise fashion to ease their reduction into the abdomen.The indication for emergent inguinal hernia repair is impending compromise of intestinal contents. As such, strangu-lation of hernia contents is a surgical emergency. Clinical signs that

1	the abdomen.The indication for emergent inguinal hernia repair is impending compromise of intestinal contents. As such, strangu-lation of hernia contents is a surgical emergency. Clinical signs that indicate strangulation include tenderness, fever, leukocy-tosis, and hemodynamic instability. The hernia bulge is usu-ally warm, tender, and the overlying skin is often erythematous or discolored. Symptoms of bowel obstruction in patients with sliding or incarcerated inguinal hernias may also indicate stran-gulation. Taxis should not be performed when strangulation is suspected, as reduction of potentially gangrenous tissue into the abdomen may result in an intra-abdominal catastrophe. Preop-eratively, the patient should receive fluid resuscitation, naso-gastric decompression, and prophylactic intravenous antibiotics.Brunicardi_Ch37_p1599-p1624.indd 160829/01/19 2:03 PM 1609INGUINAL HERNIASCHAPTER 37Prophylactic AntibioticsThe debate as to whether or not to administer preoperative

1	intravenous antibiotics.Brunicardi_Ch37_p1599-p1624.indd 160829/01/19 2:03 PM 1609INGUINAL HERNIASCHAPTER 37Prophylactic AntibioticsThe debate as to whether or not to administer preoperative prophylactic antibiotics in elective inguinal hernia repair still remains controversial as elective hernia repair is considered a clean procedure and as such are exempt from SCIP surgical prophylaxis guidelines. A Cochrane review of 17 randomized controlled trials in 2012 revealed an overall decrease in infec-tion rates (3.1% vs. 4.5%, odds ratio [OR] 0.64, 95% confi-dence interval [CI] 0.50–0.82) when prophylactic antibiotics are administered in patients. In subgroup analyses, the differ-ence was smaller in patients without mesh placement (3.5% vs. 4.9%, OR 0.71, 95% CI 0.51–1.00) than in those with mesh placement (2.4% vs. 4.2%, OR 0.56, 95% CI 0.38–0.81). However, with inguinal hernia repair, overall wound infection rates were higher than those expected for clean operations, as a result,

1	with mesh placement (2.4% vs. 4.2%, OR 0.56, 95% CI 0.38–0.81). However, with inguinal hernia repair, overall wound infection rates were higher than those expected for clean operations, as a result, they were unable to definitively recommend for or against antimicrobial prophylaxis.36,37 Although there is no uni-versal guideline regarding the administration of prophylactic antibiotics for open elective hernia repair, the routine indexing of cases for quality improvement databases have resulted in the routine administration of prophylactic perioperative antibiotics in inguinal hernia repairs.Open ApproachThe most commonly performed type of hernia operation still remains the open inguinal hernia repair. These repairs can be performed tension-free with mesh or by reconstruction of the floor with tissue. Tissue repairs are less common and are pri-marily indicated in infected fields.Exposure of the anterior inguinal region is common to the open approaches. An oblique or horizontal incision

1	tissue. Tissue repairs are less common and are pri-marily indicated in infected fields.Exposure of the anterior inguinal region is common to the open approaches. An oblique or horizontal incision is performed over the groin (Fig. 37-13). The incision begins two fingerbreadths inferior and medial to the anterior superior iliac spine. It is then extended medially for approximately 6 to 8 cm. The subcutaneous tissue is dissected using electrocautery. Scarpa’s fascia is divided to expose the external oblique aponeurosis. A small incision is made in the external oblique aponeurosis parallel to the direction of the muscle fibers. Standard groin incisionEx. obliqueSpermatic cordCamper’sfasciaScarpa’sfasciaHemostatExternal ringInguinalfloorSQ fatARepaireddefect intransversalisfasciaDirect herniasacIndirect herniasacIllioinguinal nerveBFigure 37-13. A. Layers of the abdominal wall in the anterior open approach to hernia repair. B. Identification of indirect and direct hernia sacs with

1	herniasacIndirect herniasacIllioinguinal nerveBFigure 37-13. A. Layers of the abdominal wall in the anterior open approach to hernia repair. B. Identification of indirect and direct hernia sacs with retraction of the spermatic cord and ilioinguinal nerve. Ex. = external; SQ = subcutaneous.Brunicardi_Ch37_p1599-p1624.indd 160929/01/19 2:03 PM 1610SPECIFIC CONSIDERATIONSPART IIFigure 37-14. Anterior open exposure of the inguinal canal. m. = muscle; n. = nerve; v. = vein.Metzenbaum scissors are introduced and spread beneath the fibers to sweep away the underlying ilioinguinal nerve. The scissors are then used to incise the aponeurosis superior to the inguinal ligament, splitting the external inguinal ring.The flaps of the external oblique aponeurosis are elevated with Hemostat clamps. The internal oblique fibers are dissected bluntly from the overlying external oblique flaps. Dissection of the inferior flap reveals the shelving edge of the inguinal ligament. The iliohypogastric and

1	internal oblique fibers are dissected bluntly from the overlying external oblique flaps. Dissection of the inferior flap reveals the shelving edge of the inguinal ligament. The iliohypogastric and ilioinguinal nerves are iden-tified and preserved. Effort should be made to avoid remov-ing nerves from their natural bed and disrupting the protective investing fascia. The pubic tubercle is identified, and the cord structures are dissected off of the pubis, encircled, and elevated with a Penrose drain. The cord is elevated 2 cm over the pubic symphysis in an avascular plane, and cremasteric fibers are pre-served to avoid injuring cord structures (Fig. 37-14).An indirect hernia sac will generally be found on the antero-medial surface of the spermatic cord after division of the crem-asteric muscle in the direction of its fibers. The genital nerve is visualized along the inferolateral surface of the cord adjacent to the external spermatic vein. The floor of the inguinal canal is fully

1	muscle in the direction of its fibers. The genital nerve is visualized along the inferolateral surface of the cord adjacent to the external spermatic vein. The floor of the inguinal canal is fully assessed for direct hernias. If a hernia is not visualized upon entry into the inguinal canal, the preperitoneal space should be explored for a femoral hernia. In addition to sac identification, the vas deferens and vessels of the spermatic cord must be identi-fied to allow dissection of the sac from the cord. Blunt dissection facilitates dissection of the sac from the cord. The dissection is carried proximally toward the deep inguinal ring.In cases where the viability of sac contents is in question, the sac should be incised, and hernia contents should be evalu-ated for signs of ischemia. The defect should be enlarged to augment blood flow to the sac contents. Viable contents may be reduced into the peritoneal cavity, while nonviable contents resected. In elective cases, the sac may be

1	defect should be enlarged to augment blood flow to the sac contents. Viable contents may be reduced into the peritoneal cavity, while nonviable contents resected. In elective cases, the sac may be amputated at the internal inguinal ring or inverted into the preperitoneum. Both methods are effective; however, patients undergoing sac exci-sion had significantly increased postoperative pain in a prospec-tive trial.38 Dissection of a densely adherent sac may result in injury to cord structures and should be avoided; however, sac ligation at the internal inguinal ring is necessary in these cases. A hernia sac that extends into the scrotum may require division within the inguinal canal as extensive dissection and reduction risks injury to the testicular blood supply, resulting in testicular swelling, orchitis, and atrophy.At this point, the inguinal canal is reconstructed, either with native tissue or with prostheses. The following sections describe the most commonly performed types of

1	swelling, orchitis, and atrophy.At this point, the inguinal canal is reconstructed, either with native tissue or with prostheses. The following sections describe the most commonly performed types of tissue-based and prosthetic-based reconstructions.Tissue Repairs. Tissue-based herniorrhaphy is a suitable alter-native when prosthetic materials cannot be used safely. Indica-tions for tissue repairs include operative field contamination, emergency surgery, and when the viability of hernia contents is uncertain.Bassini Repair The Bassini repair was a historic advancement in operative technique. Its current use is limited as modern tech-niques reduce recurrence. The original repair includes dissection of the spermatic cord, dissection of the hernia sac with high liga-tion, and extensive reconstruction of the floor of the inguinal canal (Fig. 37-15). After exposing the inguinal floor, the transversalis fascia is incised from the pubic tubercle to the internal inguinal ring. Preperitoneal

1	of the floor of the inguinal canal (Fig. 37-15). After exposing the inguinal floor, the transversalis fascia is incised from the pubic tubercle to the internal inguinal ring. Preperitoneal fat is bluntly dissected from the upper margin of the posterior side of the transversalis fascia to permit adequate tissue mobilization. A triple-layer repair is then performed. The internal oblique, transversus abdominis, and transversalis fascia are fixed to the shelving edge of the inguinal ligament and pubic periosteum with interrupted sutures. The lateral aspect of the repair reinforces the medial border of the internal inguinal ring.Shouldice Repair The Shouldice repair recapitulates principles of the Bassini repair, and its distribution of tension over several tissue layers results in lower recurrence rates (Fig. 37-16). Dur-ing dissection of the cord, the genital branch of the genitofemoral nerve is routinely divided, resulting in ipsilateral loss of sensation to the scrotum in men or the

1	rates (Fig. 37-16). Dur-ing dissection of the cord, the genital branch of the genitofemoral nerve is routinely divided, resulting in ipsilateral loss of sensation to the scrotum in men or the mons pubis and labium majus in women. With the posterior inguinal floor exposed, an incision in the transversalis fascia is made between the pubic tubercle and internal ring. Care is taken to avoid injury to preperitoneal struc-tures, which are bluntly dissected to mobilize the upper and lower fascial flaps. At the pubic tubercle, the iliopubic tract is sutured to the lateral edge of the rectus sheath using a synthetic, nonab-sorbable, monofilament suture. This continuous suture progresses laterally, approximating the edge of the inferior transversalis flap to the posterior aspect of the superior flap. At the internal inguinal ring, the suture continues back in the medial direction, approxi-mating the edge of the superior transversalis fascia flap to the shelving edge of the inguinal ligament. At

1	At the internal inguinal ring, the suture continues back in the medial direction, approxi-mating the edge of the superior transversalis fascia flap to the shelving edge of the inguinal ligament. At the pubic tubercle, this suture is tied to the tail of the original stitch. The next suture begins at the internal inguinal ring, and it continues medially, apposing the aponeuroses of the internal oblique and transversus abdominis to the external oblique aponeurotic fibers. At the pubic tubercle, the suture doubles back through the same structures lat-erally towards the tightened internal ring.McVay Repair The McVay repair addresses both inguinal and femoral ring defects. This technique is indicated for femoral hernias and in cases where the use of prosthetic material is contraindicated (Fig. 37-17). Once the spermatic cord has been isolated, an incision in the transversalis fascia permits entry into the preperitoneal space. The upper flap is mobilized by gentle blunt dissection of

1	(Fig. 37-17). Once the spermatic cord has been isolated, an incision in the transversalis fascia permits entry into the preperitoneal space. The upper flap is mobilized by gentle blunt dissection of underlying tissue. Cooper’s ligament is bluntly dissected to expose its surface. A 2 to 4 cm relaxing incision is made in the anterior rectus sheath vertically from the Brunicardi_Ch37_p1599-p1624.indd 161029/01/19 2:03 PM 1611INGUINAL HERNIASCHAPTER 37SpermaticcordPoupart'sligament Internal abdominaloblique muscleExternal abdominaloblique aponeurosisPreperitoneal fatTransversalis fasciaABEOTATFILIOFigure 37-15. Bassini repair. A. The transversalis fascia is opened. B. Reconstruction of the posterior wall by suturing the transver-salis fascia (TF), the transversus abdominis muscle (TA), and the internal oblique muscle (IO) medially to the inguinal ligament (IL) laterally. EO = external oblique aponeurosis.ABFigure 37-16. Shouldice repair. A. The iliopubic tract is sutured to the

1	and the internal oblique muscle (IO) medially to the inguinal ligament (IL) laterally. EO = external oblique aponeurosis.ABFigure 37-16. Shouldice repair. A. The iliopubic tract is sutured to the medial flap of the transversalis fascia and the internal oblique and transverse abdominis muscles. B. The second of the four suture lines, reversing toward the pubic tubercle approximating the inter-nal oblique and transversus muscles to the inguinal ligament. Two more suture lines affix the internal oblique and transversus muscles medially.Cooper’s ligamentFigure 37-17. McVay Cooper’s ligament repair.pubic tubercle. This incision is essential to reduce tension on the repair; however, it may result in increased postoperative pain and higher risk of ventral abdominal herniation. Using either interrupted or continuous suture, the superior transversalis flap is then fastened to Cooper’s ligament, and the repair is contin-ued laterally along Cooper’s ligament to occlude the femoral ring. Lateral

1	or continuous suture, the superior transversalis flap is then fastened to Cooper’s ligament, and the repair is contin-ued laterally along Cooper’s ligament to occlude the femoral ring. Lateral to the femoral ring, a transition stitch is placed, affixing the transversalis fascia to the inguinal ligament. The transversalis is then sutured to the inguinal ligament laterally to the internal ring.Desarda Repair The Desarda hernia repair was recently described in 2001, and it consists of a mesh-free repair utilizing a strip of external oblique aponeurosis.An oblique skin incision is made, and dissection is carried down to the external oblique fascia. The integrity of the fascia is preserved as much as possible. The cremasteric muscle is then incised, and the spermatic cord along with the cremasteric muscle is separated from the inguinal floor. Excision of the sac is done in all cases except in small direct hernias, where it is inverted.The medial leaf of the external oblique aponeurosis is

1	muscle is separated from the inguinal floor. Excision of the sac is done in all cases except in small direct hernias, where it is inverted.The medial leaf of the external oblique aponeurosis is sutured to the inguinal ligament from the pubic tubercle to the abdominal ring using 1–0 Ethilon or Prolene interrupted sutures. The first two sutures are taken at the junction of the anterior rec-tus sheath and EOA. The last suture is taken so as to sufficiently Brunicardi_Ch37_p1599-p1624.indd 161129/01/19 2:03 PM 1612SPECIFIC CONSIDERATIONSPART IInarrow the abdominal ring without constricting the spermatic cord (Fig. 37-18). Each suture is passed first through the ingui-nal ligament, then the transversalis fascia, and then the EOA. The index finger of the left hand is used to protect the femo-ral vessels and retract the cord structures laterally while taking lateral sutures. A splitting incision is then taken in the EOA, partially separating a strip. This splitting incision is extended

1	vessels and retract the cord structures laterally while taking lateral sutures. A splitting incision is then taken in the EOA, partially separating a strip. This splitting incision is extended medially up to the pubic symphysis and laterally 1 to 2 cm beyond the reconstructed abdominal ring.The free border of the strip of the EOA is now sutured to the internal oblique or conjoined tendon lying close to it with 1–0 Ethilon or Prolene interrupted sutures. This is followed by closure of the superficial fascia and the skin as usual.39-41Prosthetic Repairs. The popularization of tension-free pros-thetic mesh repairs signified a paradigm shift in the surgical concept of inguinal hernia pathophysiology. Mesh-based her-nioplasty is the most commonly performed general surgical procedure, owing to the technique’s efficacy and improved out-comes. The techniques of the most commonly performed pros-thetic repairs are presented in this section.Lichtenstein Tension-Free Repair The Lichtenstein

1	to the technique’s efficacy and improved out-comes. The techniques of the most commonly performed pros-thetic repairs are presented in this section.Lichtenstein Tension-Free Repair The Lichtenstein technique allows for a tension-free repair of the inguinal floor by buttressing the floor with a prosthetic mesh (Fig. 37-18). Initial exposure and mobilization of cord structures is identical to other open approaches. The inguinal canal is dissected to expose the shelving edge of the inguinal ligament, the pubic tubercle, and sufficient area for mesh. The most commonly used mesh is “flat iron” shaped with a keyhole for cord egress, it is available in several sizes. It should be noted that when selecting the size, it must be large enough to extend 2 to 3 cm superior to Figure 37-18. The Desarda repair. A. The medial leaf of the external oblique aponeurosis is sutured to the inguinal ligament. 1 Medial leaf, 2 interrupted sutures taken to suture the medial leaf to the inguinal ligament, 3

1	repair. A. The medial leaf of the external oblique aponeurosis is sutured to the inguinal ligament. 1 Medial leaf, 2 interrupted sutures taken to suture the medial leaf to the inguinal ligament, 3 pubic tubercle, 4 abdominal ring, 5 spermatic cord, 6 lateral leaf B. Undetached strip of the external oblique aponeurosis forming the posterior wall. 1 Reflected medial leaf after a strip has been separated, 2 internal oblique muscle seen through the splitting incision made in the medial leaf, 3 interrupted sutures between the upper border of the strip and conjoined muscle and internal oblique muscle, 4 interrupted sutures between the lower border of the strip and the inguinal ligament, 5 pubic tubercle, 6 abdominal ring, 7 spermatic cord, 8 lateral leaf.64123568751234Hesselbach’s Triangle. The medial edge of the mesh is affixed to the anterior rectus sheath such that it overlaps the pubic tubercle by 1.5 to 2 cm. This refinement to the original Lichtenstein technique minimizes medial

1	The medial edge of the mesh is affixed to the anterior rectus sheath such that it overlaps the pubic tubercle by 1.5 to 2 cm. This refinement to the original Lichtenstein technique minimizes medial recurrence.42For fixation of the inferior margin of the mesh, a per-manent, synthetic, monofilament suture is used taking care to avoid placing sutures directly into the periosteum of the pubic tubercle. Fixation is continued along the shelving edge of the inguinal ligament from medial to lateral, ending at the internal ring. The upper tail of the mesh is then fixed to the internal oblique aponeurosis and the medial edge to the rectus sheath using a synthetic, absorbable suture.In the case of a femoral hernia, a triangular extension of the inferior aspect of the mesh is sutured to Cooper’s ligament medially and to the inguinal ligament laterally. The lateral tails of the mesh are tailored to fit snugly around the cord at the internal ring, but not too tight to strangulate it. The tails are

1	medially and to the inguinal ligament laterally. The lateral tails of the mesh are tailored to fit snugly around the cord at the internal ring, but not too tight to strangulate it. The tails are then sutured to the inguinal ligament with an interrupted stitch and placed beneath the external oblique aponeurosis.Plug and Patch Technique. A modification of the Lichtenstein repair, the Plug and Patch technique was developed by Gilbert and later popularized by Rutkow and Robbins.43 Prior to placing the prosthetic mesh patch over the inguinal floor, a three-dimensional prosthetic plug is placed in the space previously occupied by the hernia sac (Fig. 37-19). In the case of an indirect hernia, the plug is placed alongside the spermatic cord through the internal ring. Prosthetic plugs of various sizes are available, and one of appropriate size is fixed to the margins of the internal ring with interrupted sutures.44 For direct hernias, the sac is reduced, and the plug is sutured to Cooper’s

1	sizes are available, and one of appropriate size is fixed to the margins of the internal ring with interrupted sutures.44 For direct hernias, the sac is reduced, and the plug is sutured to Cooper’s ligament, the inguinal ligament, and Brunicardi_Ch37_p1599-p1624.indd 161229/01/19 2:03 PM 1613INGUINAL HERNIASCHAPTER 37Figure 37-19. Lichtenstein tension-free hernioplasty. m. = muscle; n. = nerve; v. = vein.the internal oblique aponeurosis. While the technique has good overall outcomes, there have been some isolated case report series of complications involving the presence of the plug, including bowel obstruction and chronic pain.Wound Closure Once the reconstruction of the inguinal canal is complete, the cord contents are returned to their anatomic posi-tion. The external oblique aponeurosis is then reapproximated continuously from medial to lateral using an absorbable suture. The external ring should be reconstructed in close apposition to the spermatic cord to avoid the

1	aponeurosis is then reapproximated continuously from medial to lateral using an absorbable suture. The external ring should be reconstructed in close apposition to the spermatic cord to avoid the appearance of recurrence on future examination. Scarpa’s fascia and skin are appropriately closed.Laparoscopic ApproachLaparoscopic inguinal hernia repairs have become increas-ingly popular given the noninferiority studies, improved aes-thetics, and increased surgeon experience with the procedure. Principal endoscopic methods include the transabdominal preperitoneal (TAPP) repair, the totally extraperitoneal (TEP) repair, and the less-commonly performed intraperitoneal onlay mesh (IPOM) repair.Of note, awake patients do not tolerate abdominal insuffla-tion well; therefore, laparoscopic repair necessitates the admin-istration of general anesthesia and its inherent risks. Any patient with a contraindication to the use of general anesthesia should not undergo laparoscopic hernia repair.

1	necessitates the admin-istration of general anesthesia and its inherent risks. Any patient with a contraindication to the use of general anesthesia should not undergo laparoscopic hernia repair. Occasionally, induction of general anesthesia may result in reduction of an incarcer-ated or strangulated inguinal hernia. If the surgeon suspects this might have occurred, the abdomen should be explored for non-viable tissue either via laparoscopy or upon conversion to an open laparotomy.The indications for laparoscopic inguinal hernia repair are similar to those for open repair. Most surgeons would agree that the endoscopic approach to bilateral or recurrent inguinal hernias is superior to the open approach.45 Concurrent inguinal hernia repair can be considered if a hernia patient is scheduled to undergo another laparoscopic procedure without gross contamination, such as prostatectomy.46,47 International Endohernia Society (IEHS) guidelines offer a grade A recommendation that TEP and TAPP

1	to undergo another laparoscopic procedure without gross contamination, such as prostatectomy.46,47 International Endohernia Society (IEHS) guidelines offer a grade A recommendation that TEP and TAPP are preferred alternatives to Lichtenstein repair for recurrent hernias after open anterior repair.48,49 The possibility of bilateral repair should be discussed with all patients undergoing endoscopic inguinal hernia surgery.The operating room configuration is identical for TAPP, TEP, and IPOM procedures. The patient is placed in the Trendelenburg position, and video screens are placed at the foot of the bed. The surgeon stands contralateral to the hernia, and the assistant stands opposite the surgeon. The patient’s arms are tucked to the sides. Figure 37-20 demonstrates a typical operating room setup for endoscopic inguinal hernia repair. The following sections outline the most commonly performed endoscopic inguinal hernia repair techniques.Transabdominal Preperitoneal Procedure. The

1	room setup for endoscopic inguinal hernia repair. The following sections outline the most commonly performed endoscopic inguinal hernia repair techniques.Transabdominal Preperitoneal Procedure. The transab-dominal approach confers the advantage of an intraperitoneal perspective, which is useful for bilateral hernias, large hernia defects, and scarring from previous lower abdominal surgery. The abdominal cavity is accessed using a dissecting trocar or open Hasson technique. Pneumoperitoneum to a level of 15 mmHg is achieved. Two 5-mm trocars are placed lateral and slightly inferior to the umbilical trocar, avoiding injury to the inferior epigastric vessels (Fig. 37-21). The patient is then placed in the Trendelenburg position, and the pelvis is inspected.The bladder, median and medial umbilical ligaments, external iliac, and inferior epigastric vessels are visualized. An incision is made in the peritoneum at the medial umbilical liga-ment, 3 to 4 cm superior to the hernia defect, and

1	ligaments, external iliac, and inferior epigastric vessels are visualized. An incision is made in the peritoneum at the medial umbilical liga-ment, 3 to 4 cm superior to the hernia defect, and it is carried lat-erally to the anterior superior iliac spine. For bilateral inguinal hernia repair, bilateral peritoneal incisions are advisable, leav-ing a midline bridge of tissue to avoid injuring a potential patent urachus. The inferior edge of incised peritoneum is retracted, and the preperitoneum is dissected to expose the spermatic cord. If a direct hernia is encountered, the sac is inverted and fixed to Cooper’s ligament to prevent development of hematoma or seroma. An indirect hernia sac will usually protrude anterior to the spermatic cord. In this case, the sac is grasped and elevated superiorly from the cord and the space below is developed bluntly to allow for mesh placement. The sac is dissected from its adhesions, and the cord is skeletonized.The mesh usually measures 10 × 15 cm

1	from the cord and the space below is developed bluntly to allow for mesh placement. The sac is dissected from its adhesions, and the cord is skeletonized.The mesh usually measures 10 × 15 cm to completely cover the myopectineal orifice (Fig. 37-22). It is rolled length-wise and placed through the 12-mm trocar. It is unrolled in the preperitoneal space and secured medially to Cooper’s ligament using an endoscopic tacker. During this fixation, the surgeon palpates the end of the tacker from the abdominal surface to ensure its proper angle and to stabilize the pelvis. The mesh is then pulled taut and fixed laterally to the anterior superior iliac spine. Tacks are placed above the iliopubic tract to avoid injury to the lateral cutaneous nerve of the thigh and the femoral branch of the genitofemoral nerve. The peritoneal edges are reapproxi-mated using tacks or intracorporeal sutures as the mesh is sta-bilized. The peritoneum should be closed completely to avoid contact between the mesh

1	nerve. The peritoneal edges are reapproxi-mated using tacks or intracorporeal sutures as the mesh is sta-bilized. The peritoneum should be closed completely to avoid contact between the mesh and the intestine. The abdomen is desufflated, and the trocars are removed. The fascial defect of the 12-mm port and the skin incisions are appropriately closed.Totally Extraperitoneal Procedure. The advantage of the TEP repair is the access to the preperitoneal space without intra-peritoneal infiltration. Consequently, this approach minimizes the risk of injury to intra-abdominal organs and port site hernia-tion through an iatrogenic defect in the abdominal wall. As with TAPP, TEP is indicated for repair of bilateral inguinal hernias or Brunicardi_Ch37_p1599-p1624.indd 161329/01/19 2:03 PM 1614SPECIFIC CONSIDERATIONSPART IIFigure 37-20. Operating room setup for laparoscopic inguinal hernia repair.for unilateral hernias when scarring makes the anterior approach challenging.A small horizontal

1	CONSIDERATIONSPART IIFigure 37-20. Operating room setup for laparoscopic inguinal hernia repair.for unilateral hernias when scarring makes the anterior approach challenging.A small horizontal incision is made inferior to the umbi-licus. Subcutaneous tissue is dissected to the level of the ante-rior rectus sheath, which is then incised lateral to the linea alba. The rectus muscle is retracted superolaterally, and a dissecting balloon is advanced through the incision toward the pubic sym-physis. Under direct visualization with a 30° laparoscope, the balloon is inflated slowly to bluntly dissect the preperitoneal space (Fig. 37-23). The dissecting balloon is replaced with a 12-mm balloon trocar, and pneumopreperitoneum is achieved by insufflation to 15 mmHg. A 5-mm trocar is placed suprapubically in the midline, and another is placed inferior to the insufflation port (see Fig. 37-21). The patient is placed in the Trendelenburg position, and the operation proceeds in an identical fashion

1	in the midline, and another is placed inferior to the insufflation port (see Fig. 37-21). The patient is placed in the Trendelenburg position, and the operation proceeds in an identical fashion to TAPP. No modifications are necessary to repair bilateral ingui-nal hernias with the TEP approach. Any peritoneal rents should be repaired prior to desufflation to prevent mesh from contact-ing intraperitoneal structures. Following mesh placement, the preperitoneal space is desufflated slowly under direct vision to ensure proper mesh positioning. Trocars are removed, and the anterior rectus sheath is closed with an interrupted suture. If there Brunicardi_Ch37_p1599-p1624.indd 161429/01/19 2:03 PM 1615INGUINAL HERNIASCHAPTER 37BA Figure 37-21. Trocar placement for (A) transabdominal preperitoneal repair and (B) totally extraperitoneal repair.Figure 37-22. View of mesh placement in posterior repairs. A large mesh overlaps the myopectineal orifice.Figure 37-23. Balloon dissection of the

1	repair and (B) totally extraperitoneal repair.Figure 37-22. View of mesh placement in posterior repairs. A large mesh overlaps the myopectineal orifice.Figure 37-23. Balloon dissection of the preperitoneal space in a totally extraperitoneal inguinal hernia repair.Brunicardi_Ch37_p1599-p1624.indd 161529/01/19 2:04 PM 1616SPECIFIC CONSIDERATIONSPART IIis violation of the peritoneum during insufflation of the dissec-tion balloon and subsequent pneumoperitoneum, visualization can be compromised. To address this, a Veress needle or angio-catheter can be placed in the LUQ, which will allow desufflation of the peritoneum and restore visualization.Intraperitoneal Onlay Mesh Procedure. In contrast to TAPP and TEP, the IPOM procedure permits the posterior approach without preperitoneal dissection. It is an attractive procedure in cases where the anterior approach is unfeasible, in recurrent her-nias that are refractory to other approaches, or where extensive preperitoneal scarring would

1	It is an attractive procedure in cases where the anterior approach is unfeasible, in recurrent her-nias that are refractory to other approaches, or where extensive preperitoneal scarring would make TEP or TAPP challenging. Port placement and inguinal hernia identification are identical to TAPP. Hernia sac contents are reduced; however, the sac itself is not inverted from the preperitoneal space. Instead, mesh is placed directly over the defect and fixed in place with sutures or spiral tacks. Because these anchors are placed through the peritoneum without preperitoneal inspection, the lateral cutane-ous nerve of the thigh and the genitofemoral nerve are especially prone to injury. Furthermore, intraperitoneal mesh migration is a documented phenomenon that can lead to postoperative mor-bidity, recurrence, and reoperation.Robot-Assisted Inguinal Hernia RepairApplication of a robotic platform to hernia repair has been adapted by general surgeons across the country. The endowrist

1	mor-bidity, recurrence, and reoperation.Robot-Assisted Inguinal Hernia RepairApplication of a robotic platform to hernia repair has been adapted by general surgeons across the country. The endowrist capabilities provides greatly improved manual dexterity and a relatively short learning curve. Though both total extraperito-neal repair and transabdominal preperitoneal repair can be adapted to a robotic platform, the latter has gained more traction among surgeons.Many papers have explored the efficacy and cost-effectiveness of robot-assisted herniorraphy. Retrospective data have had mixed results when comparing robot-assisted surgery vs. laparoscopy. One recent study has shown longer operative time,50 another analysis has shown increased cost.51 It should be noted, however, that there is a decrease in cost with robotic surgery as the volume of procedures increases at each center, though it is still unlikely that the costs will ever converge to that of laparoscopic surgery. A

1	there is a decrease in cost with robotic surgery as the volume of procedures increases at each center, though it is still unlikely that the costs will ever converge to that of laparoscopic surgery. A retrospective, single-institution study has shown greatly reduced complication rates with robotic assisted surgery in obese patients; however, this was compared against open inguinal hernia repair (10.8% vs. 3.2%, P = 0.047), the two groups were covariate matched for preoperative risk.52 Studies have also shown excellent long-term (36-month) quality of life indicators in robot-assisted TAPP, though this was a single surgeon survey.53 Further randomized trials will shed more light into cost issues as surgeons gain more experience with robotic application that would lead to shorter operative time and minimize additional instrument use.Similar to laparoscopy, robot-assisted repair is ideal for recurrent inguinal hernia patients who had previous anterior repair and bilateral hernias.

1	time and minimize additional instrument use.Similar to laparoscopy, robot-assisted repair is ideal for recurrent inguinal hernia patients who had previous anterior repair and bilateral hernias. Contraindications to robotic her-nia repair are the same as for laparoscopic repair and include coagulopathy and/or severe cardiopulmonary disease precluding induction of general anesthesia and pneumoperitoneum. Previ-ous preperitoneal repair is a relative contraindication along with the presence of a large incarcerated inguinal hernia.Patient evaluation should proceed similarly to workup for laparoscopic inguinal herniorraphy.Technique. Patients are instructed to void in preoperative area to avoid Foley catheter placement, though some surgeons advocate 4routine Foley catheter placement. Ideally, the operating table should have capability of synchronization with robotic arms to prevent injury to the patient during repositioning during the pro-cedure. The patient is placed in supine position

1	the operating table should have capability of synchronization with robotic arms to prevent injury to the patient during repositioning during the pro-cedure. The patient is placed in supine position with arms tucked at both sides. Appropriate padding of extremities is important to avoid neuropraxia and trauma from robotic arm movements. Three trocars are typically used for TAPP repair. Open Hasson technique is employed for initial trocar placement at umbilicus; this can be an 8-mm trocar or alternatively a 12-mm with a tele-scoped 8-mm trochar. Additionally, two 8-mm trocars are placed in each side of the mid-abdomen, slightly above the level of umbi-licus. After trocar placement, the robot is docked and targeted, and the patient is placed in Trendelenburg position. Typically, the surgeon will use robotic shears attached to electrocautery, Cadiere forceps, and a needle holder as the primary instruments. This combination provides optimal cost-effectiveness because the majority of the

1	will use robotic shears attached to electrocautery, Cadiere forceps, and a needle holder as the primary instruments. This combination provides optimal cost-effectiveness because the majority of the cost associated with robotic application is due to disposable instruments. Exposure starts with incising the parietal peritoneum from the medial umbilical ligament to the anterior superior iliac spine. A peritoneal flap is developed by blunt and sharp dissection with robotic shears in the prepreitoneal space. Special care is taken to leave the preperitoneal fat pad contain-ing nerves and vessels with the anterior abdominal wall. Small vessels can be coagulated with application of electrocautery with scissors. With the aid of pneumoperitoneum, the preference is first to perform a lateral dissection in the space of Bogros. Dissection continues in this plane laterally towards the anterior superior iliac spine. The generous development of a peritoneal flap will ensure successful mesh placement

1	in the space of Bogros. Dissection continues in this plane laterally towards the anterior superior iliac spine. The generous development of a peritoneal flap will ensure successful mesh placement at the end. Then the space of Retzius is entered medially exposing the pubic symphysis. In the absence of haptic feedback, visual recognition of the pubic symphysis is crucial as this serves as an important landmark for further dis-section. Inferior epigastric vessels are readily identified. Next, an inferior peritoneal flap is developed to avoid rolling of mesh during closure. Direct, indirect, and femoral spaces are carefully examined. Cadiere forceps are then used to grasp the hernia sac to provide traction. Any cord lipoma is carefully dissected free from the cord structures, and the testicular vessels, pampiniform plexus, and ductus deferens are separated from hernia sac. These structures can usually be identified at the neck of sac. Reduction is successful when the sac stays reduced

1	vessels, pampiniform plexus, and ductus deferens are separated from hernia sac. These structures can usually be identified at the neck of sac. Reduction is successful when the sac stays reduced after traction is released. The next step is placement of the mesh. Lightweight barbed mesh and anatomically preshaped mesh are routinely used. Mesh should be an appropriate size to cover the myopectineal orifice entirely, and the peritoneal dissection will need to be large enough to accommodate this size mesh. It is rolled and placed through one of the ports by the bedside assistant. Then it is unrolled and placed in the pelvis overlapping the pubic symphysis by several centimeters medially; this is essential as the majority of recur-rences occur in this area. Utilization of tacking devices are not necessary, which helps to reduce procedural cost; however, this is surgeon preference. Finally, the peritoneal flap is placed back over the mesh layer and sutured back into place with a running

1	not necessary, which helps to reduce procedural cost; however, this is surgeon preference. Finally, the peritoneal flap is placed back over the mesh layer and sutured back into place with a running locking suture that is facilitated by the increased intracorporeal dexterity of the robotic instruments. Then the fascia of the umbili-cal trochar site is closed with 0-absorbable suture (Fig 37-24), and the skin is closed with absorbable monofilament suture.Prosthesis ConsiderationsThe success of prosthetic repairs has generated considerable debate about the desirable physical attributes of mesh and their fixation. An ideal mesh should be easy to handle, flexible, Brunicardi_Ch37_p1599-p1624.indd 161629/01/19 2:04 PM 1617INGUINAL HERNIASCHAPTER 37Figure 37-24. Steps in robotic TAPP repair. A. Image of a direct inguinal hernia. B. There is no visible hernia on the contralateral side. C. Hernia contents and sac are dissected and cleared for mesh placement. D. Unrolling and placement of

1	A. Image of a direct inguinal hernia. B. There is no visible hernia on the contralateral side. C. Hernia contents and sac are dissected and cleared for mesh placement. D. Unrolling and placement of mesh E. Satisfactory placement of mesh. F. Closure of peritoneum. G. Completed repair of hernia with comparison to contralateral side.ABCDEFGBrunicardi_Ch37_p1599-p1624.indd 161729/01/19 2:04 PM 1618SPECIFIC CONSIDERATIONSPART IIstrong, immunologically inert, contraction-resistant, infection-resistant, and inexpensive to manufacture.54 The following section reviews the most common types of mesh and fixatives currently available.Synthetic Mesh Material. Polypropylene and polyester are the most common synthetic prosthetic materials used in her-nia repair. These materials are permanent and hydrophobic, and they promote a local inflammatory response that results in cellular infiltration and scarring with slight contraction in size. Other synthetic mesh materials are under investigation with

1	and they promote a local inflammatory response that results in cellular infiltration and scarring with slight contraction in size. Other synthetic mesh materials are under investigation with the goals of minimizing postoperative pain and preventing infection or recurrence. In selecting mesh material, considerations include mesh absorbability, thickness, weight, porosity, and strength.Variations in the fiber diameter and fiber count of mesh materials categorize them as heavyweight or lightweight in den-sity, though there does not seem to be a universally agreed upon set of criteria for either. Commonly used lightweight mesh materi-als include β-d-glucan, titanium-coated polypropylene, and poly-propylene–poliglecaprone. These materials have greater elasticity and less theoretical surface area contact with surrounding tissues than their heavyweight counterparts.55 They are hypothesized to reduce scarring and chronic pain without compromising the strength of the repair. The use of

1	area contact with surrounding tissues than their heavyweight counterparts.55 They are hypothesized to reduce scarring and chronic pain without compromising the strength of the repair. The use of lightweight mesh use in TEP and TAPP repairs is associated with fewer 3-month cumulative mesh-related complications.54 A 2012 meta-analysis of 2310 patients undergoing open or laparoscopic hernia repairs found a lower incidence of chronic pain (relative risk [RR] 0.61, CI 0.50–0.74) following use of lightweight mesh versus heavyweight mesh and no significant difference in rates of recurrence.55When available, lightweight mesh should be considered for all prosthetic repairs to minimize postoperative chronic pain. A disadvantage of currently available commercial pros-theses is their high cost. In settings where resources are limited, prosthetic repairs are performed using alternative materials. Polypropylene and polyethylene mosquito nets are inexpensive and ubiquitous in sub-Saharan Africa and

1	where resources are limited, prosthetic repairs are performed using alternative materials. Polypropylene and polyethylene mosquito nets are inexpensive and ubiquitous in sub-Saharan Africa and India, and they have similar mechanical properties to commercially available hernio-plasty meshes. Meta-analysis of 577 hernioplasties performed using sterilized mosquito nets demonstrated similar rates of short-term mesh-related complications (6.1%) and recurrence (0.17%) to those using commercial meshes.56 Furthermore, the disability-adjusted life years (DALYs) prevented by inguinal hernia repair signify a comparable impact to that of vaccination in sub-Saharan Africa.57,58 Expensive prostheses are not neces-sarily needed for hernia surgery, whether in resource-limited or in resource-abundant settings, and the anticipated benefits should be evaluated with consideration of increased costs.Biologic Mesh. Although indications for the use of biologic prostheses have not been absolutely defined,

1	and the anticipated benefits should be evaluated with consideration of increased costs.Biologic Mesh. Although indications for the use of biologic prostheses have not been absolutely defined, they are commonly reserved for contaminated cases or when domain expansion is necessary in the face of high infection risk. This is partially on account of their high cost and high recurrence rates. There are numerous biologic materials available with differing properties, but in general, they have a lower tensile strength and subse-quently higher rates of rupture than synthetic prostheses.59 They also have varying degrees of tensile strength and tissue biocom-patibility between them. In ventral hernia repairs, xenograft material was associated with a lower rate of recurrence than allograft material.60 A review of biologic materials concludes cross-linked graft materials are more durable and less prone to failure than non–cross-linked grafts.61 Nevertheless, their 5diminished ability to remodel

1	A review of biologic materials concludes cross-linked graft materials are more durable and less prone to failure than non–cross-linked grafts.61 Nevertheless, their 5diminished ability to remodel adversely affects rates of infec-tion and incorporation. While new prosthetic materials continue to be developed, no single biologic warrants routine use. These materials will continue to evolve, and they remain an important tool for challenging cases when used judiciously.Fixation Technique. Independent of prosthesis material, the method of its fixation remains disputed. Suturing, stapling, and tacking prostheses entail tissue perforation, which may cause inflammation, neurovascular injury, and chronic pain devel-opment. Conversely, improper prosthesis fixation may result in mesh migration, repair failure, meshoma pain, and hernia recurrence. Mesh may be fixed with fibrin-derived glue, and self-gripping mesh has been developed to minimize trauma to surrounding tissues and to reduce the risk

1	failure, meshoma pain, and hernia recurrence. Mesh may be fixed with fibrin-derived glue, and self-gripping mesh has been developed to minimize trauma to surrounding tissues and to reduce the risk for entrapment neuropathy. For hernias repaired via a strictly preperitoneal approach, prosthesis fixation may not be necessary at all.Fibrin glue fixation is a successful alternative to tack fixation in hernia repair with a synthetic prosthesis. Recent studies comparing fibrin glue fixation and suture fixation in open hernia repair show superior rates of chronic pain with both Lichtenstein and Plug and Patch techniques.62,63 Meta-analyses of endoscopic hernia repair determined the incidence of chronic postoperative pain after tacker fixation was significantly higher than after fibrin glue fixation, with one showing a relative risk of 4.64 (CI 1.9–11.7). Rates of other postoperative complications and recurrence were similar between both fixation methods.62,63 Glue fixation is a promising

1	with one showing a relative risk of 4.64 (CI 1.9–11.7). Rates of other postoperative complications and recurrence were similar between both fixation methods.62,63 Glue fixation is a promising technical refinement, and several studies have shown long-term benefit; however, its questionable efficacy in larger hernias and cost remain considerations.In TEP repairs, fixation of mesh may not be compulsory. A prospective randomized trial comparing fixation and no fixation in TEP repairs found a significant increase in new pain and equiv-alent recurrence rates in the fixation group several months after repair.64 A 2012 meta-analysis comparing laparoscopic tacker mesh fixation to no mesh fixation found no statistically signifi-cant differences in operative duration, pain, mesh-related compli-cations, recurrence, or length of stay between the two methods.65 Studies of three-dimensional, ergonomically contoured mesh without fixation, as well as self-gripping meshes, have yielded similar

1	recurrence, or length of stay between the two methods.65 Studies of three-dimensional, ergonomically contoured mesh without fixation, as well as self-gripping meshes, have yielded similar results.66 In the preperitoneal approach, the reapproxi-mation of surrounding tissues and physiologic intra-abdominal pressure hypothetically prevent mesh migration. Due to higher theoretical risk of mesh migration, repair without fixation is not recommended for anterior or transperitoneal approaches.COMPLICATIONSAs with other clean operations, the most common complications of inguinal hernia repair include bleeding, infection, seroma, urinary retention, ileus, and injury to adjacent structures (Table 37-6). Complications specific to herniorrhaphy include hernia recurrence, chronic inguinal and pubic pain, and injury to the spermatic cord or testis. The incidence, prevention, and treatment of these complications are discussed in the ensuing section.Hernia RecurrenceWhen a patient develops pain,

1	pain, and injury to the spermatic cord or testis. The incidence, prevention, and treatment of these complications are discussed in the ensuing section.Hernia RecurrenceWhen a patient develops pain, bulging, or a mass at the site of an inguinal hernia repair, clinical entities such as seroma, persistent cord lipoma, and hernia recurrence should be considered. Com-mon medical issues associated with recurrence include malnu-trition, immunosuppression, diabetes, steroid use, and smoking. Technical causes of recurrence include improper mesh size, tissue Brunicardi_Ch37_p1599-p1624.indd 161829/01/19 2:04 PM 1619INGUINAL HERNIASCHAPTER 37Table 37-6Complications of groin hernia repairsRecurrenceChronic groin pain Nociceptive Somatic Visceral Neuropathic Iliohypogastric Ilioinguinal Genitofemoral Lateral cutaneous FemoralCord and testicular Hematoma Ischemic orchitis Testicular atrophy Dysejaculation Division of vas deferens Hydrocele Testicular descentBladder injuryWound

1	cutaneous FemoralCord and testicular Hematoma Ischemic orchitis Testicular atrophy Dysejaculation Division of vas deferens Hydrocele Testicular descentBladder injuryWound infectionSeromaHematoma Wound Scrotal RetroperitonealOsteitis pubisProsthetic complications Contraction Erosion Infection Rejection FractureLaparoscopic Vascular injury Intra-abdominal Retroperitoneal Abdominal wall Gas embolism Visceral injury Bowel perforation Bladder perforation Trocar site complications Hematoma Hernia Wound infection Keloid Bowel obstruction Trocar or peritoneal closure site hernia Adhesions Miscellaneous Diaphragmatic dysfunction HypercapniaGeneral Urinary Paralytic ileus Nausea and vomiting Aspiration pneumonia Cardiovascular and respiratory insufficiencyischemia, infection, and tension in the reconstruction. A focused physical examination should be performed. As with primary her-nias, US, CT, or MRI can elucidate ambiguous physical findings. When a recurrent hernia is

1	and tension in the reconstruction. A focused physical examination should be performed. As with primary her-nias, US, CT, or MRI can elucidate ambiguous physical findings. When a recurrent hernia is discovered and warrants reoperation, an approach through a virgin plane facilitates its dissection and exposure. Extensive dissection of the scarred field and mesh may result in injury to cord structures, viscera, large blood ves-sels, and nerves. After an initial anterior approach, the posterior endoscopic approach will usually be easier and more effective than another anterior dissection. Conversely, failed preperitoneal repairs should be approached using an open anterior repair.PainPain after inguinal hernia repair is classified into acute or chronic manifestations of three mechanisms: nociceptive (somatic), neuropathic, and visceral pain. Nociceptive pain is the most common of the three. Because it is usually a result of ligamentous or muscular trauma and inflammation, nociceptive pain

1	(somatic), neuropathic, and visceral pain. Nociceptive pain is the most common of the three. Because it is usually a result of ligamentous or muscular trauma and inflammation, nociceptive pain is reproduced with abdominal muscle contraction. Treat-ment consists of rest, nonsteroidal anti-inflammatory drugs (NSAIDs), and reassurance as it resolves spontaneously in most cases. Neuropathic pain occurs as a result of direct nerve dam-age or entrapment. It may present early or late, and it mani-fests as a localized, sharp, burning, or tearing sensation. It may respond to pharmacologic therapy and to local steroid or anes-thetic injections when indicated. Visceral pain refers to pain conveyed through afferent autonomic pain fibers. It is usually poorly localized and may occur during ejaculation as a result of sympathetic plexus injury.Chronic postoperative pain remains an important measure of clinical outcome that has been reported in as many as 63% of inguinal hernia repair cases.67-69

1	as a result of sympathetic plexus injury.Chronic postoperative pain remains an important measure of clinical outcome that has been reported in as many as 63% of inguinal hernia repair cases.67-69 Despite the significant anatomic variation in the three inguinal nerves, literature reviews suggest identification of all three nerves is possible in 70% to 90% of cases.70 Meticulous nerve identification may prevent injury that results in debilitating chronic postoperative pain syndromes. Notwithstanding, moderate-to-severe pain adversely affects physical activity, social interactions, health care utilization, employment, and productivity in 6% to 8% of patients.67,68,71-74 Pain in this subset of patients comprises a tremendous individ-ual and societal burden.Postherniorrhaphy inguinodynia is a debilitating chronic complication. Its incidence is independent of the method of her-nia repair.73 Selective ilioinguinal, iliohypogastric, and genito-femoral neurolysis/neurectomy, removal of mesh

1	a debilitating chronic complication. Its incidence is independent of the method of her-nia repair.73 Selective ilioinguinal, iliohypogastric, and genito-femoral neurolysis/neurectomy, removal of mesh and fixation material, and revision of the repair are the three most common options for treatment. Nevertheless, anatomic variation and cross-innervation of the inguinal nerves in the retroperitoneum and inguinal canal make selective neurectomy less reliable.75-78 When inguinodynia is refractory to pharmacologic and interven-tional measures, triple neurectomy with removal of meshoma is routinely performed with acceptable outcomes in the majority of patients.74,76,77,79-84 Refractory inguinodynia with concurrent orchialgia also requires resection of the paravasal nerves.84A relatively newly described technique that has cited good outcomes is the laparoscopic triple neurectomy. This involves laparoscopic approach to and division of the main trunks of the ilioinguinal and iliohypogastric

1	described technique that has cited good outcomes is the laparoscopic triple neurectomy. This involves laparoscopic approach to and division of the main trunks of the ilioinguinal and iliohypogastric nerves and additional division of the genitofemoral nerve in the lumbar plexus.84,85 Several studies with moderate numbers of patients treated showed dura-ble reduction in pain scores.Other chronic pain syndromes include local nerve entrapment, meralgia paresthesia, and osteitis pubis. At greatest Brunicardi_Ch37_p1599-p1624.indd 161929/01/19 2:04 PM 1620SPECIFIC CONSIDERATIONSPART IIrisk of entrapment are the ilioinguinal and iliohypogastric nerves in anterior repairs and the genitofemoral and lateral femoral cutaneous nerves in endoscopic repairs. Clinical manifestations of nerve entrapment mimic acute neuropathic pain, and they occur with a dermatomal distribution. Injury to the lateral femoral cutaneous nerve results in meralgia paresthesia, a condition characterized by persistent

1	mimic acute neuropathic pain, and they occur with a dermatomal distribution. Injury to the lateral femoral cutaneous nerve results in meralgia paresthesia, a condition characterized by persistent paresthesia of the lateral thigh. Initial treatment of nerve entrapment consists of rest, ice, NSAIDs, physical therapy, and possible local corticosteroid and anesthetic injection. This can be followed by a trial of gabapentin86 or its analogues. Osteitis pubis is characterized by inflammation of the pubic symphysis and usually presents as medial groin or symphyseal pain that is reproduced by thigh adduction. Avoiding the pubic periosteum when placing sutures and tacks reduces the risk of developing osteitis pubis. CT scan or MRI excludes hernia recurrence, and bone scan is confirmatory for the diagnosis. Initial treatment is identical to that of nerve entrapment; however, if pain remains intractable, orthopedic surgery consultation should be sought for possible bone resection and curettage.

1	Initial treatment is identical to that of nerve entrapment; however, if pain remains intractable, orthopedic surgery consultation should be sought for possible bone resection and curettage. Irrespective of treatment, the condition often takes six months to resolve.87Cord and Testes InjuryInjury to spermatic cord structures may result in ischemic orchitis or testicular atrophy. Ischemic orchitis is most com-monly caused by injury to the pampiniform plexus and not to the testicular artery. It usually manifests within 1 week of ingui-nal hernia repair as an enlarged, indurated, and painful testis, and it is almost certainly self-limited. It occurs in <1% of pri-mary hernia repairs; however, this figure is larger for recurrent inguinal hernia repairs.88 US will demonstrate testicular blood flow to differentiate between ischemia and necrosis. Emergent orchiectomy is only necessary in the case of necrosis. Injury to the testicular artery itself may lead to testicular atrophy, which is

1	flow to differentiate between ischemia and necrosis. Emergent orchiectomy is only necessary in the case of necrosis. Injury to the testicular artery itself may lead to testicular atrophy, which is manifest over a protracted period but does not always lead to testicular necrosis. This is because despite compromise of the artery, there is collateral flow from the inferior epigastric, vesi-cal, prostatic, and scrotal arteries that supply the testes, and in the case of insufficiency, there is atrophy. Treatment for isch-emic orchitis most frequently consists of reassurance, NSAIDs, and comfort measures. Intraoperatively, proximal ligation of large hernia sacs to avoid cord manipulation minimizes the risk of injury.Injury to the vas deferens within the cord may lead to infertility. In open inguinal hernia repairs, isolating the vas deferens along with the cord structures using digital manipulation may cause injury or disruption. In endoscopic approach, grasping the vas may result in a

1	inguinal hernia repairs, isolating the vas deferens along with the cord structures using digital manipulation may cause injury or disruption. In endoscopic approach, grasping the vas may result in a crush injury. Transections of the vas deferens should be addressed with a urologic consult and early anastomosis, if possible. Historically, surgeons and their patients speculated that synthetic material would increase the risks of mesh rejection, carcinogenesis, and inflammation; however, as mesh became used more frequently, these concerns did not manifest. Nevertheless, one study found prosthetic mesh may exert long-term deleterious effects upon the vas deferens, causing azoospermia.89 Similar studies report varied results, though. A recent prospective study from the Swedish Hernia Registry discovered no difference in rates of patient-reported infertility between the general population and patients who underwent either mesh or tissue-based inguinal hernia repair.90 Chronic scarring may

1	discovered no difference in rates of patient-reported infertility between the general population and patients who underwent either mesh or tissue-based inguinal hernia repair.90 Chronic scarring may lead to vas deferens obstruction, resulting in decreased fertility rates and a dysejaculation syndrome. Pain and burning during ejaculation are usually self-limited, and more common causes, such as sexually transmitted diseases, should be excluded.In females, the round ligament is the analog to the sper-matic cord, and it maintains uterine anteversion. Injury to the artery of the round ligament does not result in clinically signifi-cant morbidity.Laparoscopic ComplicationsIn general, the risks of the TEP technique mirror those of open anterior repairs, as the peritoneal space is not violated. Com-plications of transabdominal laparoscopy include urinary reten-tion, paralytic ileus, visceral injuries, vascular injuries, and less commonly, bowel obstruction, hypercapnia, gas embolism, and

1	Com-plications of transabdominal laparoscopy include urinary reten-tion, paralytic ileus, visceral injuries, vascular injuries, and less commonly, bowel obstruction, hypercapnia, gas embolism, and pneumothorax. The most common complications of endoscopic inguinal hernia repair are presented in this section.Urinary Retention. The most common cause of urinary reten-tion after hernia repair is general anesthesia, which is routine in endoscopic hernia repairs. Among 880 patients undergoing inguinal hernia repair with local anesthesia only, 0.2% devel-oped urinary retention, while the rate of urinary retention was 13% among 200 patients undergoing repair with general or spinal anesthesia.91 Overall, the risk of development of postop-erative urinary retention is 2% to 3%.92-95 Other risk factors for postoperative urinary retention include pain, narcotic analgesia, and perioperative bladder distention. Initial treatment of urinary retention requires decompression of the bladder with

1	factors for postoperative urinary retention include pain, narcotic analgesia, and perioperative bladder distention. Initial treatment of urinary retention requires decompression of the bladder with short-term catheterization. Patients will generally require an overnight admission and trial of normal voiding before discharge. Failure to void normally requires reinsertion of the catheter for up to a week. Chronic requirement of a urinary catheter is rare, though older patients may require prolonged catheterization. Risk of urinary retention can be minimized by ensuring voiding prior to surgery and minimization of perioperative fluid administration.96Ileus and Bowel Obstruction. The laparoscopic transab-dominal approach is associated with a higher incidence of ileus than other modes of repair. This complication is self-limited; however, it necessitates sustained inpatient observation, intra-venous fluid maintenance, and possibly nasogastric decom-pression. Abdominal imaging may be

1	repair. This complication is self-limited; however, it necessitates sustained inpatient observation, intra-venous fluid maintenance, and possibly nasogastric decom-pression. Abdominal imaging may be helpful to confirm the diagnosis and to exclude bowel obstruction. Prolonged absence of bowel function, in conjunction with a suspicious abdominal series, should raise concern for obstruction. In this case, CT of the abdomen is helpful to distinguish anatomic sites of obstruc-tion, inflammation, and ischemia. In TAPP repairs, obstruction occurs most commonly secondary to herniation of bowel loops through peritoneal defects or large trocar insertion sites; how-ever, the use of smaller trocars and the preponderance of TEP repairs have reduced the frequency of this complication. True obstruction warrants reoperation.Visceral Injury. Small bowel, colon, and bladder are at risk for injury in laparoscopic hernia repair. The presence of intra-abdominal adhesions from previous surgeries may

1	warrants reoperation.Visceral Injury. Small bowel, colon, and bladder are at risk for injury in laparoscopic hernia repair. The presence of intra-abdominal adhesions from previous surgeries may predispose to visceral injuries. Direct bowel injuries may also result from tro-car placement. In reoperative abdominal surgery, open Hasson technique and direct visualization of trocars are recommended to reduce the likelihood of visceral injury. Bowel injury may also occur secondary to electrocautery and instrument trauma outside of the camera field. Missed bowel injuries are associated with increased mortality. If injury to the bowel is suspected, its entire length should be examined, and conversion to open repair may be necessary.Brunicardi_Ch37_p1599-p1624.indd 162029/01/19 2:04 PM 1621INGUINAL HERNIASCHAPTER 37Bladder injuries are less common than visceral injuries, and they are usually associated with perioperative bladder dis-tention or extensive dissection of perivesical adhesions.

1	HERNIASCHAPTER 37Bladder injuries are less common than visceral injuries, and they are usually associated with perioperative bladder dis-tention or extensive dissection of perivesical adhesions. As with bladder injuries encountered in open surgery, cystotomies must be repaired in several layers with 1 to 2 weeks of Foley catheter decompression. A confirmatory cystogram may be performed before catheter removal to confirm healing of the injury.Vascular Injury. The most severe vascular injuries usually occur in iliac or femoral vessels, either by misplaced sutures in anterior repairs, endoscopic tacker use, or by trocar injury or direct dissection in laparoscopic repairs. In these cases, exsan-guination may be swift. Conversion to an open approach may be necessary, and bleeding should be temporarily controlled with mechanical compression until vascular control is obtained.The most commonly injured vessels in laparoscopic hernia repair include the inferior epigastrics and external iliac

1	controlled with mechanical compression until vascular control is obtained.The most commonly injured vessels in laparoscopic hernia repair include the inferior epigastrics and external iliac arter-ies. Although apparent upon initial approach, these vessels may be obscured during mesh positioning, and tacks or staples may injure them. Oftentimes, due to tamponade effect, injury to the inferior epigastric vessels is not apparent until the adjacent tro-car is removed. If injured, the inferior epigastrics may be ligated with a percutaneous suture passer or endoscopic vessel clips.If the tissue pressure exerted by pneumoperitoneum is greater than an injured vessel’s hydrostatic intraluminal pres-sure, bleeding will not manifest until pneumoperitoneum is released. The presentation of an inferior epigastric vein injury is often delayed because of this effect, and it may result in a significant rectus sheath hematoma. Accordingly, the surgeon should be aware of this intraoperative

1	an inferior epigastric vein injury is often delayed because of this effect, and it may result in a significant rectus sheath hematoma. Accordingly, the surgeon should be aware of this intraoperative consideration.Hematomas and SeromasHematomas may present as localized collections or as dif-fuse bruising over the operative site. Injury to spermatic cord vessels may result in a scrotal hematoma. Although they are self-limited, characteristic dark blue discoloration of the entire scrotum may alarm patients. Intermittent warm and cold com-pression aids in resolution. Hematomas may also develop in the incision, retroperitoneum, rectus sheath, and peritoneal cavity. The latter three sites are more frequently associated with lapa-roscopic repair. Bleeding within the peritoneum or preperitoneal space may not be readily apparent on physical examination. For this reason, close monitoring of subjective complaints, vital signs, urine output, and physical parameters is necessary.Seromas are fluid

1	may not be readily apparent on physical examination. For this reason, close monitoring of subjective complaints, vital signs, urine output, and physical parameters is necessary.Seromas are fluid collections that most commonly develop within one week of synthetic mesh repairs. Large hernia sac remnants may fill with physiologic fluid and mimic seromas. Patients often mistake seromas for early recurrence. Treatment consists of reassurance and warm compression to accelerate resolution. To avoid secondary infection, seromas should not be aspirated unless they cause discomfort or they restrict activ-ity for a prolonged time.OUTCOMESThe incidence of recurrence is the most-cited measure of post-operative outcome following inguinal hernia repair. In evaluat-ing the various available techniques, other salient signifiers of outcome include complication rates, operative duration, hospital stay, and quality of life. The following section summarizes the evidence-based outcomes of the various

1	other salient signifiers of outcome include complication rates, operative duration, hospital stay, and quality of life. The following section summarizes the evidence-based outcomes of the various approaches to inguinal hernia repair.Among tissue repairs, the Shouldice operation is the most commonly performed technique, and it is most frequently executed at specialized centers. A 2012 meta-analysis from the Cochrane database demonstrated significantly lower rates of hernia recurrence (OR 0.62, CI 0.45–0.85) in patients undergoing Shouldice operations when compared with other open tissue-based methods.97 In experienced hands, the overall recurrence rate for the Shouldice repair is about 1%.98 Although it is an elegant procedure, its meticulous nature requires significant technical expertise to achieve favorable outcomes, and it is associated with longer operative duration and longer hospital stay. One study found the recurrence rate for Shouldice repairs decreased from 9.4% to 2.5%

1	to achieve favorable outcomes, and it is associated with longer operative duration and longer hospital stay. One study found the recurrence rate for Shouldice repairs decreased from 9.4% to 2.5% after surgeons performed the repair six times.99 Compared with mesh repairs, the Shouldice technique resulted in significantly higher rates of recurrence (OR 3.65, CI 1.79–7.47); however, it is the most effective tissue-based repair when mesh is unavailable or contraindicated.97Hernia recurrence is drastically reduced as a result of the Lichtenstein tension-free repair.100 Compared with open elective tissue-based repairs, mesh repair is associated with fewer recur-rences (OR 0.37, CI 0.26–0.51) and with shorter hospital stay and faster return to usual activities.101,102 In a multi-institutional series, 3019 inguinal hernias were repaired using the Lichten-stein technique, with an overall recurrence rate of 0.2%.103 Among other tension-free repairs, the Lichtenstein technique remains the most

1	3019 inguinal hernias were repaired using the Lichten-stein technique, with an overall recurrence rate of 0.2%.103 Among other tension-free repairs, the Lichtenstein technique remains the most commonly performed procedure worldwide. Meta-analysis demonstrates no significant differences in out-comes between the Lichtenstein and the Plug and Patch tech-niques; however, intra-abdominal plug migration and erosion into contiguous structures occurs in approximately 6% of cases.101,104,105 The Stoppa technique results in longer operative duration than the Lichtenstein technique. Nevertheless, postop-erative acute pain, chronic pain, and recurrence rates are similar between the two methods.106 Perhaps the most compelling advan-tage of the Lichtenstein technique is that nonexpert surgeons rapidly achieve similar outcomes to their expert counterparts. Guidelines issued by the European Hernia Society recommend the Lichtenstein repair for adults with either unilateral or bilat-eral inguinal

1	achieve similar outcomes to their expert counterparts. Guidelines issued by the European Hernia Society recommend the Lichtenstein repair for adults with either unilateral or bilat-eral inguinal hernias as the preferred open technique.102 Com-pared to open approaches, endoscopic primary inguinal hernia repair produces equivalent recurrence rates and improved recov-ery time, pain prevention, and return to normal activities.107 In a study of 168 patients randomized to either TEP or Lichtenstein repair, the 5-year recurrence rates were extremely low in both groups.108,109 Similarly, a study of 200 male patients randomized to either ambulatory TEP or Lichtenstein repair demonstrated no recurrences in either group after one year.110 Because endoscopic surgery requires specialized instruments and longer operative times, its cost is higher than conventional open repair; however, the potential financial benefit of shorter recovery and decreased pain may offset these costs in the

1	instruments and longer operative times, its cost is higher than conventional open repair; however, the potential financial benefit of shorter recovery and decreased pain may offset these costs in the long-term.Perhaps the most salient difference between open and endo-scopic techniques is the number of cases needed to develop techni-cal proficiency. In a randomized controlled trial performed by the VA Cooperative Study, two-year recurrence rates were 10.1% in patients undergoing endoscopic repair and 4.9% in those undergo-ing open repair, and the outcomes of endoscopic repairs improved after each surgeon performed at least 250 cases.111 More recently, Lal and colleagues found that surgeons sustained a decrease from 9% to 2.9% in postoperative recurrences after performing 100 TEP operations.112 Other studies also suggest surgeons develop proficiency in these endoscopic techniques after performing 30 67Brunicardi_Ch37_p1599-p1624.indd 162129/01/19 2:04 PM 1622SPECIFIC

1	TEP operations.112 Other studies also suggest surgeons develop proficiency in these endoscopic techniques after performing 30 67Brunicardi_Ch37_p1599-p1624.indd 162129/01/19 2:04 PM 1622SPECIFIC CONSIDERATIONSPART IIto 100 cases; however, this estimate has decreased precipitously since laparoscopic technique was first introduced.111,113,114Although controversy persists regarding the utility of TEP versus TAPP, reviews to date find no significant differences in operative duration, length of stay, time to recovery, or short-term recurrence rate between the two approaches. In TAPP repair, the risk of intra-abdominal injury is higher than in TEP repair. This finding prompted the IEHS to recommend TAPP should only be attempted by surgeons with sufficient experience.49 A Cochrane systematic review found rates of port-site hernias and visceral injuries were higher for the TAPP technique, while TEP may be associated with a higher rate of conversion to an alternative approach; however,

1	review found rates of port-site hernias and visceral injuries were higher for the TAPP technique, while TEP may be associated with a higher rate of conversion to an alternative approach; however, neither finding was sufficiently compelling to recommend one technique over the other.114The frequency with which the aforementioned ingui-nal hernia repair techniques are performed reinforces the importance of broad experience. The authors recommend that surgeons become proficient in several techniques to address dif-ferent manifestations of inguinal hernias. Surgeons should tailor this experience to optimize outcomes for each patient.REFERENCESEntries highlighted in bright blue are key references. 1. National Center for Health Statistics. National Hospital Discharge Survey and National Survey of Ambulatory Surgery, 2010 . Available at: https://www.cdc.gov/nchs/index .htm. Accessed August 4, 2018. 2. Abramson JH, Gofin J, Hopp C, et al. The epidemiology of inguinal hernia. A survey in

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1	JS, Gibbs JO, Reda DJ, et al. Does delaying repair of an asymptomatic hernia have a penalty? Am J Surg. 2008;195(1):89-93. 32. Miserez M, Peeters E, Aufenacker T, et al. Update with level 1 studies of the European Hernia Society guidelines on the treatment of inguinal hernia in adult patients. Hernia. 2014;18(2):151-163. 33. Law NW, Trapnell JE. Does a truss benefit a patient with inguinal hernia? BMJ. 1992;304(6834):1092. 34. Gallegos NC, Dawson J, Jarvis M, Hobsley M. Risk of strangulation in groin hernias. Br J Surg. 1991;78(10): 1171-1173. 35. Nilsson H, Stylianidis G, Haapamäki M, Nilsson E, Nordin P. Mortality after groin hernia surgery. Ann Surg. 2007;245(4):656-660.Brunicardi_Ch37_p1599-p1624.indd 162229/01/19 2:04 PM 1623INGUINAL HERNIASCHAPTER 37 36. Sanchez-Manuel FJ, Lozano-García J, Seco-Gil JL. Antibi-otic prophylaxis for hernia repair. Cochrane Database Syst Rev. 2012;(2):CD003769. 37. Yin Y, Song T, Liao B, Luo Q, Zhou Z. Antibiotic prophylaxis in patients

1	Lozano-García J, Seco-Gil JL. Antibi-otic prophylaxis for hernia repair. Cochrane Database Syst Rev. 2012;(2):CD003769. 37. Yin Y, Song T, Liao B, Luo Q, Zhou Z. Antibiotic prophylaxis in patients undergoing open mesh repair of inguinal hernia: a meta-analysis. Am Surg. 2012;78(3):359-365. 38. Delikoukos S, Lavant L, Hlias G, Palogos K, Gikas D. The role of hernia sac ligation in postoperative pain in patients with elective tension-free indirect inguinal hernia repair: a prospective randomized study. Hernia. 2007;11(5):425-428. 39. Desarda MP. Inguinal herniorrhaphy with an undetached strip of external oblique aponeurosis: a new approach used in 400 patients. Eur J Surg. 2001;167(6):443-448. 40. Desarda MP. New method of inguinal hernia repair: a new solution. ANZ J Surg. 2001;71(4):241-244. 41. Desarda MP. Physiological repair of inguinal hernia: a new technique (study of 860 patients). Hernia. 2006;10(2):143-146. 42. Amid PK, Shulman AG, Lichtenstein IL. Critical scrutiny of the

1	MP. Physiological repair of inguinal hernia: a new technique (study of 860 patients). Hernia. 2006;10(2):143-146. 42. Amid PK, Shulman AG, Lichtenstein IL. Critical scrutiny of the open “tension-free” hernioplasty. Am J Surg. 1993;165(3):369-371. 43. Gilbert AI. Sutureless repair of inguinal hernia. Am J Surg. 1992;163(3):331-335. 44. Millikan KW, Cummings B, Doolas A. The Millikan modified mesh-plug hernioplasty. Arch Surg. 2003;138(5):525-529; discussion 529-530. 45. Voyles CR, Hamilton BJ, Johnson WD, Kano N. Meta-analysis of laparoscopic inguinal hernia trials favors open hernia repair with preperitoneal mesh prosthesis. Am J Surg. 2002;184(1):6-10. 46. Antunes AA, Dall’oglio M, Crippa A, Srougi M. Inguinal hernia repair with polypropylene mesh during radical retropubic prostatectomy: an easy and practical approach. BJU Int. 2005;96(3):330-333. 47. Lee BC, Rodin DM, Shah KK, Dahl DM. Laparoscopic inguinal hernia repair during laparoscopic radical prostatectomy. BJU Int.

1	an easy and practical approach. BJU Int. 2005;96(3):330-333. 47. Lee BC, Rodin DM, Shah KK, Dahl DM. Laparoscopic inguinal hernia repair during laparoscopic radical prostatectomy. BJU Int. 2007;99(3):637-639. 48. Bittner R, Arregui ME, Bisgaard T, et al. Guidelines for laparoscopic (TAPP) and endoscopic (TEP) treatment of inguinal hernia (International Endohernia Society [IEHS]). Surg Endosc. 2011;25(9):2773-2843. 49. Bittner R, Montgomery MA, Arregui E, et al. Update of guidelines on laparoscopic (TAPP) and endoscopic (TEP) treatment of inguinal hernia (International Endohernia Society). Surg Endosc. 2015;29(2):289-321. 50. Waite KE, Herman MA, Doyle PJ. Comparison of robotic versus laparoscopic transabdominal preperitoneal (TAPP) inguinal hernia repair. J Robot Surg. 2016;10(3):239-244. 51. Higgins RM, Frelich MJ, Bosler ME, Gould JC. Cost analysis of robotic versus laparoscopic general surgery procedures. Surg Endosc. 2017;31(1):185-192. 52. Kolachalam R, Dickens E, D’Amico L, et

1	RM, Frelich MJ, Bosler ME, Gould JC. Cost analysis of robotic versus laparoscopic general surgery procedures. Surg Endosc. 2017;31(1):185-192. 52. Kolachalam R, Dickens E, D’Amico L, et al. Early outcomes of robotic-assisted inguinal hernia repair in obese patients: a multi-institutional, retrospective study. Surg Endosc. 2018;32(1):229-235. 53. Iraniha A, Peloquin J. Long-term quality of life and outcomes following robotic assisted TAPP inguinal hernia repair. J Robot Surg. 2018;12(2):261-269. 54. Pickett LC. Prosthetic choice in open inguinal hernia repair. In: Jacob BP, Ramshaw B, eds. The SAGES Manual of Hernia Repair. New York: Springer; 2013:19-26. 55. Sajid MS, Leaver C, Baig MK, Sains P. Systematic review and meta-analysis of the use of lightweight versus heavy-weight mesh in open inguinal hernia repair. Br J Surg. 2012;99(1):29-37. 56. Sorensen CG, Rosenberg J. The use of sterilized mosquito nets for hernioplasty: a systematic review. Hernia. 2012;16(6): 621-625. 57. Luboga

1	inguinal hernia repair. Br J Surg. 2012;99(1):29-37. 56. Sorensen CG, Rosenberg J. The use of sterilized mosquito nets for hernioplasty: a systematic review. Hernia. 2012;16(6): 621-625. 57. Luboga S, Macfarlane SB, von Schreeb J, et al. Increasing access to surgical services in sub-saharan Africa: priorities for national and international agencies recommended by the Bellagio Essential Surgery Group. PLoS Med. 2009;6(12):e1000200. 58. Jacobs DO. Improving surgical services in developing nations: getting to go. World J Surg. 2010;34(11):2509-2510. 59. Earle DB, Mark LA. Prosthetic material in inguinal hernia repair: how do I choose? Surg Clin North Am. 2008;88(1):179-201. 60. Beale EW, Hoxworth RE, Livingston EH, Trussler AP. The role of biologic mesh in abdominal wall reconstruction: a systematic review of the current literature. Am J Surg. 2012;204(4):510-517. 61. Smart NJ, Bloor S. Durability of biologic implants for use in hernia repair: a review. Surg Innov.

1	reconstruction: a systematic review of the current literature. Am J Surg. 2012;204(4):510-517. 61. Smart NJ, Bloor S. Durability of biologic implants for use in hernia repair: a review. Surg Innov. 2012;19(3):221-229. 62. Campanelli G, Sfeclan C, Cavalli M, Biondi A. Reducing postoperative pain: the use of Tisseel for mesh fixation in inguinal hernia repair. Surg Technol Int. 2012;22:134-139. 63. Fortelny RH, Petter-Puchner AH, Glaser KS, Redl H. Use of fibrin sealant (Tisseel/Tissucol) in hernia repair: a systematic review. Surg Endosc. 2012;26(7):1803-1812. 64. Taylor C, Layani L, Liew V, Ghusn M, Crampton N, White S. Laparoscopic inguinal hernia repair without mesh fixation, early results of a large randomised clinical trial. Surg Endosc. 2008;22(3):757-762. 65. Sajid MS, Ladwa N, Kalra L, McFall M, Baig MK, Sains P. A meta-analysis examining the use of tacker mesh fixation versus glue mesh fixation in laparoscopic inguinal hernia repair. Am J Surg.

1	MS, Ladwa N, Kalra L, McFall M, Baig MK, Sains P. A meta-analysis examining the use of tacker mesh fixation versus glue mesh fixation in laparoscopic inguinal hernia repair. Am J Surg. 2013;206(1):103-111. 66. Morrison JE, Jr, Jacobs VR. Laparoscopic preperitoneal inguinal hernia repair using preformed polyester mesh without fixation: prospective study with 1-year follow-up results in a rural setting. Surg Laparosc Endosc Percutan Tech. 2008;18(1):33-39. 67. Aasvang E, Kehlet H. Surgical management of chronic pain after inguinal hernia repair. Br J Surg. 2005;92(7):795-801. 68. Kehlet H. Chronic pain after groin hernia repair. Br J Surg. 2008;95(2):135-136. 69. Reinpold WM, Nehls J, Eggert A. Nerve management and chronic pain after open inguinal hernia repair: a prospective two phase study. Ann Surg. 2011;254(1):163-168. 70. Alfieri S, Amid PK, Campanelli G, et al. International guide-lines for prevention and management of post-operative chronic pain following inguinal hernia surgery.

1	Ann Surg. 2011;254(1):163-168. 70. Alfieri S, Amid PK, Campanelli G, et al. International guide-lines for prevention and management of post-operative chronic pain following inguinal hernia surgery. Hernia. 2011; 15(3):239-249. 71. Callesen T, Beck K, Kehlet H. Prospective study of chronic pain after groin hernia repair. Br J Surg. 1999;86(12):1528-1531. 72. Bay-Nielsen M, Perkins FM, Kehlet H; Danish Hernia Database. Pain and functional impairment 1 year after inguinal herniorrhaphy: a nationwide questionnaire study. Ann Surg. 2001;233(1):1-7. 73. Aasvang EK, Bay-Nielsen M, Kehlet H. Pain and functional impairment 6 years after inguinal herniorrhaphy. Hernia. 2006;10(4):316-321. 74. Aasvang EK, Kehlet H. The effect of mesh removal and selective neurectomy on persistent postherniotomy pain. Ann Surg. 2009;249(2):327-334. 75. Rab M, Ebmer J, Dellon AL. Anatomic variability of the ilioinguinal and genitofemoral nerve: implications for the treatment of groin pain. Plast Reconstr Surg.

1	Ann Surg. 2009;249(2):327-334. 75. Rab M, Ebmer J, Dellon AL. Anatomic variability of the ilioinguinal and genitofemoral nerve: implications for the treatment of groin pain. Plast Reconstr Surg. 2001; 108(6):1618-1623. 76. Loos MJ, Scheltinga MR, Roumen RM. Tailored neurectomy for treatment of postherniorrhaphy inguinal neuralgia. Surgery. 2010;147(2):275-281. 77. Zacest AC, Magill ST, Anderson VC, Burchiel KJ. Long-term outcome following ilioinguinal neurectomy for chronic pain. J Neurosurg. 2010;112(4):784-789. 78. Klaassen Z, Marshall E, Tubbs RS, Louis RG, Jr, Wartmann CT, Loukas M. Anatomy of the ilioinguinal and iliohypogastric Brunicardi_Ch37_p1599-p1624.indd 162329/01/19 2:04 PM 1624SPECIFIC CONSIDERATIONSPART IInerves with observations of their spinal nerve contributions. Clin Anat. 2011;24(4):454-461. 79. Starling JR, Harms BA, Schroeder ME, Eichman PL. Diagnosis and treatment of genitofemoral and ilioinguinal entrapment neuralgia. Surgery.

1	nerve contributions. Clin Anat. 2011;24(4):454-461. 79. Starling JR, Harms BA, Schroeder ME, Eichman PL. Diagnosis and treatment of genitofemoral and ilioinguinal entrapment neuralgia. Surgery. 1987;102(4):581-586. 80. Starling JR, Harms BA. Ilioinguinal, iliohypogastric, and genitofemoral neuralgia. In: Bendavid R, ed. Prostheses and Abdominal Wall Hernia. Austin, TX: RG Landes Co; 1994: 351-356. 81. Amid PK. A 1-stage surgical treatment for postherniorrhaphy neuropathic pain: triple neurectomy and proximal end implantation without mobilization of the cord. Arch Surg. 2002;137(1):100-104. 82. Kim DH, Murovic JA, Tiel RL, Kline DG. Surgical management of 33 ilioinguinal and iliohypogastric neuralgias at Louisiana State University Health Sciences Center. Neurosurgery. 2005;56(5):1013-1020; discussion 1013-1020. 83. Madura JA, Madura JA 2nd, Copper CM, Worth RM. Inguinal neurectomy for inguinal nerve entrapment: an experience with 100 patients. Am J Surg. 2005;189(3):283-287. 84. Amid

1	discussion 1013-1020. 83. Madura JA, Madura JA 2nd, Copper CM, Worth RM. Inguinal neurectomy for inguinal nerve entrapment: an experience with 100 patients. Am J Surg. 2005;189(3):283-287. 84. Amid PK, Chen DC. Surgical treatment of chronic groin and testicular pain after laparoscopic and open preperitoneal inguinal hernia repair. J Am Coll Surg. 2011;213(4):531-536. 85. Chen DC, Hiatt JR, Amid PK. Operative management of refractory neuropathic inguinodynia by a laparoscopic ret-roperitoneal approach. JAMA Surg. 2013;148(10):962-967. 86. Benito-Leon J, Picardo A, Garrido A, Cuberes R. Gabapentin therapy for genitofemoral and ilioinguinal neuralgia. J Neurol. 2001;248(10):907-908. 87. LeBlanc KE, LeBlanc KA. Groin pain in athletes. Hernia. 2003;7(2):68-71. 88. Fong Y, Wantz GE. Prevention of ischemic orchitis during inguinal hernioplasty. Surg Gynecol Obstet. 1992;174(5):399-402. 89. Shin D, Lipshultz LI, Goldstein M, et al. Herniorrhaphy with polypropylene mesh causing inguinal vasal

1	ischemic orchitis during inguinal hernioplasty. Surg Gynecol Obstet. 1992;174(5):399-402. 89. Shin D, Lipshultz LI, Goldstein M, et al. Herniorrhaphy with polypropylene mesh causing inguinal vasal obstruction: a preventable cause of obstructive azoospermia. Ann Surg. 2005;241(4):553-558. 90. Hallén M, Sandblom G, Nordin P, et al. Male infertility after mesh hernia repair: a prospective study. Surgery. 2011; 149(2):179-184. 91. Finley RK, Jr, Miller SF, Jones LM. Elimination of urinary retention following inguinal herniorrhaphy. Am Surg. 1991;57(8):486-488; discussion 488-489. 92. Aeberhard P, Klaiber C, Meyenberg A, Osterwalder A, Tschudi J. Prospective audit of laparoscopic totally extraperitoneal inguinal hernia repair: a multicenter study of the Swiss Association for Laparoscopic and Thoracoscopic Surgery (SALTC). Surg Endosc. 1999;13(11):1115-1120. 93. Dulucq JL, Wintringer P, Mahajna A. Laparoscopic totally extraperitoneal inguinal hernia repair: lessons learned from 3,100

1	and Thoracoscopic Surgery (SALTC). Surg Endosc. 1999;13(11):1115-1120. 93. Dulucq JL, Wintringer P, Mahajna A. Laparoscopic totally extraperitoneal inguinal hernia repair: lessons learned from 3,100 hernia repairs over 15 years. Surg Endosc. 2009;23(3):482-486. 94. Kapiris S, Mavromatis T, Andrikopoulos S, Georgiades C, Floros D, Diamantopoulos G. Laparoscopic transabdominal preperitoneal hernia repair (TAPP): stapling the mesh is not mandatory. J Laparoendosc Adv Surg Tech A. 2009;19(3):419-422. 95. Swadia ND. Laparoscopic totally extra-peritoneal inguinal hernia repair: 9 year’s experience. Hernia. 2011;15(3):273-279. 96. Petros JG, Rimm EB, Robillard RJ, Argy O. Factors influencing postoperative urinary retention in patients undergoing elective inguinal herniorrhaphy. Am J Surg. 1991;161(4):431-433; discussion 434. 97. Amato B, Moja L, Panico S, et al. Shouldice technique versus other open techniques for inguinal hernia repair. Cochrane Database Syst Rev.

1	Am J Surg. 1991;161(4):431-433; discussion 434. 97. Amato B, Moja L, Panico S, et al. Shouldice technique versus other open techniques for inguinal hernia repair. Cochrane Database Syst Rev. 2012;(4):CD001543. 98. Glassow F. The Shouldice Hospital technique. Int Surg. 1986;71(3):148-153. 99. Kingsnorth AN, Britton BJ, Morris PJ. Recurrent inguinal hernia after local anaesthetic repair. Br J Surg. 1991;68(4):273-275. 100. Lichtenstein IL, Shulman AG, Amid PK. Use of mesh to prevent recurrence of hernias. Postgrad Med. 1990;87(1):155-158, 160. 101. Scott NW, McCormack K, Graham P, Go PM, Ross SJ, Grant AM. Open mesh versus non-mesh for repair of femoral and inguinal hernia. Cochrane Database Syst Rev. 2002;(4):CD002197. 102. Simons MP, Aufenacker T, Bay-Nielsen M, et al. European Hernia Society guidelines on the treatment of inguinal her-nia in adult patients. Hernia. 2009;13(4):343-403. 103. Shulman AG, Amid PK, Lichtenstein IL. The safety of mesh repair for primary inguinal hernias:

1	guidelines on the treatment of inguinal her-nia in adult patients. Hernia. 2009;13(4):343-403. 103. Shulman AG, Amid PK, Lichtenstein IL. The safety of mesh repair for primary inguinal hernias: results of 3,019 operations from five diverse surgical sources. Am Surg. 1992;58(4):255-257. 104. Kingsnorth AN, Porter CS, Bennett DH, Walker AJ, Hyland ME, Sodergren S. Lichtenstein patch or Perfix plug-and-patch in inguinal hernia: a prospective double-blind randomized controlled trial of short-term outcome. Surgery. 2000;127(3):276-283. 105. Li J, Ji Z, Li Y. Comparison of mesh-plug and Lichtenstein for inguinal hernia repair: a meta-analysis of randomized controlled trials. Hernia. 2012;16(5):541-548. 106. Willaert W, De Bacquer D, Rogiers X, Troisi R, Berrevoet F. Open preperitoneal techniques versus lichtenstein repair for elective inguinal hernias. Cochrane Database Syst Rev. 2012;(7):CD008034. 107. McCormack K, Scott NW, Go PM, Ross S, Grant AM; EU Hernia Trialists Collaboration.

1	versus lichtenstein repair for elective inguinal hernias. Cochrane Database Syst Rev. 2012;(7):CD008034. 107. McCormack K, Scott NW, Go PM, Ross S, Grant AM; EU Hernia Trialists Collaboration. Laparoscopic tech-niques versus open techniques for inguinal hernia repair. Cochrane Database Syst Rev. 2003;(1):CD001785. 108. Andersson B, Hallén M, Leveau P, Bergenfelz A, Westerdahl J. Laparoscopic extraperitoneal inguinal hernia repair versus open mesh repair: a prospective randomized controlled trial. Surgery. 2003;133(5):464-472. 109. Hallén M, Bergenfelz A, Westerdahl J. Laparoscopic extraperitoneal inguinal hernia repair versus open mesh repair: long-term follow-up of a randomized controlled trial. Surgery. 2008;143(3):313-317. 110. Lau H, Patil NG, Yuen WK. Day-case endoscopic totally extraperitoneal inguinal hernioplasty versus open Lichtenstein hernioplasty for unilateral primary inguinal hernia in males: a randomized trial. Surg Endosc. 2006;20(1):76-81. 111. Neumayer L,

1	extraperitoneal inguinal hernioplasty versus open Lichtenstein hernioplasty for unilateral primary inguinal hernia in males: a randomized trial. Surg Endosc. 2006;20(1):76-81. 111. Neumayer L, Giobbie-Hurder A, Jonasson O, et al. Open mesh versus laparoscopic mesh repair of inguinal hernia. N Engl J Med. 2004;350(18):1819-1827. 112. Lal P, Kajla RK, Chander J, Ramteke VK. Laparoscopic total extraperitoneal (TEP) inguinal hernia repair: overcoming the learning curve. Surg Endosc. 2004;18(4):642-645. 113. Katkhouda N, Campos GM, Mavor E, Trussler A, Khalil M, Stoppa R. Laparoscopic extraperitoneal inguinal hernia repair. A safe approach based on the understanding of rectus sheath anatomy. Surg Endosc. 1999;13(12):1243-1246. 114. Wake BL, McCormack K, Fraser C, Vale L, Perez J, Grant AM. Transabdominal pre-peritoneal (TAPP) vs totally extraperitoneal (TEP) laparoscopic techniques for ingui-nal hernia repair. Cochrane Database Syst Rev. 2005;(1): CD004703.Brunicardi_Ch37_p1599-p1624.indd

1	pre-peritoneal (TAPP) vs totally extraperitoneal (TEP) laparoscopic techniques for ingui-nal hernia repair. Cochrane Database Syst Rev. 2005;(1): CD004703.Brunicardi_Ch37_p1599-p1624.indd 162429/01/19 2:04 PM

1	Thyroid, Parathyroid, and AdrenalGeeta Lal and Orlo H. Clark 38chapterTHYROIDHistorical BackgroundGoiters (from the Latin guttur, throat), defined as an enlarge-ment of the thyroid, have been recognized since 2700 b.c. even though the thyroid gland was not documented as such until the Renaissance period. In 1619, Hieronymus Fabricius ab Aqua-pendente recognized that goiters arose from the thyroid gland. The term thyroid gland (Greek thyreoeides, shield-shaped) is, however, attributed to Thomas Wharton in his Adenographia (1656). In 1776, the thyroid was classified as a ductless gland by Albrecht von Haller and was thought to have numerous func-tions ranging from lubrication of the larynx to acting as a res-ervoir for blood to provide continuous flow to the brain, and to beautifying women’s necks. Burnt seaweed was considered to be the most effective treatment for goiters.The first accounts of thyroid surgery for the treatment of goiters were given by Roger Frugardi in 1170. In

1	women’s necks. Burnt seaweed was considered to be the most effective treatment for goiters.The first accounts of thyroid surgery for the treatment of goiters were given by Roger Frugardi in 1170. In response to failure of medical treatment, two setons were inserted at right angles into the goiter and tightened twice daily until the goiter separated. The open wound was treated with caustic powder and left to heal. However, thyroid surgery continued to be hazardous with prohibitive mortality rates (>40%) until the latter half of the 19th century, when advances in general anesthesia, antisep-sis, and hemostasis enabled surgeons to perform thyroid sur-gery with significantly reduced mortality and morbidity rates. The most notable thyroid surgeons were Emil Theodor Kocher (1841–1917) and C.A. Theodor Billroth (1829–1894), who per-formed thousands of operations with increasingly successful results. However, as more patients survived thyroid operations, new problems and issues became

1	C.A. Theodor Billroth (1829–1894), who per-formed thousands of operations with increasingly successful results. However, as more patients survived thyroid operations, new problems and issues became apparent. After total thyroid-ectomy, patients (particularly children) became myxedematous with cretinous features. Myxedema was first effectively treated in 1891 by George Murray using a subcutaneous injection of an extract of sheep’s thyroid, and later, Edward Fox demonstrated that oral therapy was equally effective. In 1909, Kocher was awarded the Nobel Prize for medicine in recognition “for his works on the physiology, pathology, and surgery of the thyroid gland.”EmbryologyThe thyroid gland arises as an outpouching of the primitive foregut around the third week of gestation. It originates at the base of the tongue at the foramen cecum. Endoderm cells in the floor of the pharyngeal anlage thicken to form the medial thyroid anlage (Fig. 38-1) that descends in the neck anterior to

1	at the base of the tongue at the foramen cecum. Endoderm cells in the floor of the pharyngeal anlage thicken to form the medial thyroid anlage (Fig. 38-1) that descends in the neck anterior to structures that form the hyoid bone and larynx. During its descent, the anlage remains connected to the foramen cecum via an epithelial-lined tube known as the thyroglossal duct. The epithelial cells making up the anlage give rise to the thyroid fol-licular cells. The paired lateral anlages originate from the fourth branchial pouch and fuse with the median anlage at approxi-mately the fifth week of gestation. The lateral anlages are neu-roectodermal in origin (ultimobranchial bodies) and provide the calcitonin producing parafollicular or C cells, which thus come to lie in the superoposterior region of the gland. Thyroid fol-licles are initially apparent by 8 weeks, and colloid formation begins by the 11th week of gestation.Developmental AbnormalitiesThyroglossal Duct Cyst and Sinus. Thyroglossal

1	the gland. Thyroid fol-licles are initially apparent by 8 weeks, and colloid formation begins by the 11th week of gestation.Developmental AbnormalitiesThyroglossal Duct Cyst and Sinus. Thyroglossal duct cysts are the most commonly encountered congenital cervical anoma-lies. During the fifth week of gestation, the thyroglossal duct lumen starts to obliterate, and the duct disappears by the eighth week of gestation. Rarely, the thyroglossal duct may persist in whole or in part. Thyroglossal duct cysts may occur anywhere along the migratory path of the thyroid, although 80% are found in juxtaposition to the hyoid bone. They are usually asymptom-atic but occasionally become infected by oral bacteria, prompt-ing the patient to seek medical advice. Thyroglossal duct sinuses Thyroid 1625Historical Background / 1625Embryology / 1625Developmental Abnormalities / 1625Thyroid Anatomy / 1627Thyroid Histology / 1629Thyroid Physiology / 1629Evaluation of Patients With Thyroid Disease / 1633Benign

1	Background / 1625Embryology / 1625Developmental Abnormalities / 1625Thyroid Anatomy / 1627Thyroid Histology / 1629Thyroid Physiology / 1629Evaluation of Patients With Thyroid Disease / 1633Benign Thyroid Disorders / 1634Solitary Thyroid Nodule / 1641Malignant Thyroid Disease / 1645Parathyroid 1663Historical Background / 1663Embryology / 1663Anatomy and Histology / 1664Parathyroid Physiology and Calcium Homeostasis / 1664Hyperparathyroidism / 1665Hypoparathyroidism / 1681Adrenal 1681Historical Background / 1681Embryology / 1681Anatomy / 1682Adrenal Physiology / 1682Disorders of the Adrenal Cortex / 1685Disorders of the Adrenal Medulla / 1693The Adrenal Incidentaloma / 1695Adrenal Insufficiency / 1697Adrenal Surgery / 1698Brunicardi_Ch38_p1625-p1704.indd 162501/03/19 11:20 AM 1626result from infection of the cyst secondary to spontaneous or surgical drainage of the cyst and are accompanied by minor inflammation of the surrounding skin. Histologically, thyroglos-sal duct cysts are

1	from infection of the cyst secondary to spontaneous or surgical drainage of the cyst and are accompanied by minor inflammation of the surrounding skin. Histologically, thyroglos-sal duct cysts are lined by pseudostratified ciliated columnar epithelium and squamous epithelium, with heterotopic thyroid tissue present in 20% of cases.The diagnosis usually is established by observing a 1to 2-cm, smooth, well-defined midline neck mass that moves upward with protrusion of the tongue. Routine thyroid imaging is not necessary, although thyroid scintigraphy and ultrasound have been performed to document the presence of normal thy-roid tissue in the neck. Treatment involves the “Sistrunk opera-tion,” which consists of en bloc cystectomy and excision of the central hyoid bone to minimize recurrence. Approximately 1% of thyroglossal duct cysts are found to contain cancer, which is usually papillary (85%). The role of total thyroidectomy in this setting is debated, but it is advised in patients

1	Approximately 1% of thyroglossal duct cysts are found to contain cancer, which is usually papillary (85%). The role of total thyroidectomy in this setting is debated, but it is advised in patients with large tumors, particularly if there are additional thyroid nodules and evidence of cyst wall invasion or lymph node metastases.1 Squamous, Hürthle cell, and anaplastic cancers also have been reported but are rare. Medullary thyroid cancers (MTCs) are, however, not found in thyroglossal duct cysts.Lingual Thyroid. A lingual thyroid represents a failure of the median thyroid anlage to descend normally and may be the only thyroid tissue present. Intervention becomes necessary for obstructive symptoms such as choking, dysphagia, airway obstruction, or hemorrhage. Many of these patients develop hypothyroidism. Medical treatment options include administra-tion of exogenous thyroid hormone to suppress thyroid-stim-ulating hormone (TSH) and radioactive iodine (RAI) ablation followed by hormone

1	hypothyroidism. Medical treatment options include administra-tion of exogenous thyroid hormone to suppress thyroid-stim-ulating hormone (TSH) and radioactive iodine (RAI) ablation followed by hormone replacement. Surgical excision is rarely needed but, if required, should be preceded by an evaluation of normal thyroid tissue in the neck to avoid inadvertently render-ing the patient hypothyroid.Ectopic Thyroid. Normal thyroid tissue may be found any-where in the central neck compartment, including the esopha-gus, trachea, and anterior mediastinum. Thyroid tissue has been observed adjacent to the aortic arch, in the aortopulmonary win-dow, within the upper pericardium, or in the interventricular septum. Often, “tongues” of thyroid tissue are seen to extend off the inferior poles of the gland and are particularly appar-ent in large goiters. Thyroid tissue situated lateral to the carotid sheath and jugular vein, previously termed lateral aberrant thyroid, almost always represents

1	the gland and are particularly appar-ent in large goiters. Thyroid tissue situated lateral to the carotid sheath and jugular vein, previously termed lateral aberrant thyroid, almost always represents metastatic thyroid cancer in lymph nodes, and not remnants of the lateral anlage that had Buccalcavity1234EndodermMedian thyroiddiverticulumTracheo-esophageal tube1stpharyngealpouch2nd pouch 3rd pouch 4th pouch Figure 38-1. Thyroid embryology—early development of the median thyroid anlage as a pharyngeal pouch. (Reproduced with permission from Cady B, Rossi R: Surgery of the Thyroid and Para-thyroid Glands. Philadelphia, PA: WB Saunders; 1991.)Key Points1 There has been a paradigm shift in the surgical manage-ment of Graves’ disease with increased use of total or near-total thyroidectomy, rather than subtotal thyroidectomy.2 Familial nonmedullary thyroid cancer is increasingly being recognized as a separate entity. Surgeons must be aware of the potential for false-negative fine-needle

1	rather than subtotal thyroidectomy.2 Familial nonmedullary thyroid cancer is increasingly being recognized as a separate entity. Surgeons must be aware of the potential for false-negative fine-needle aspi-ration biopsy in this setting.3 Fine-needle aspiration biopsies are now classified into six groups based on the risk of malignancy associated with each group (Bethesda criteria).4 Encapsulated follicular variants of papillary thyroid can-cers are now designated noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP).5 Lobectomy or total/near-total thyroidectomy are consid-ered appropriate treatments for low-risk thyroid cancers. Some small papillary thyroid cancers (<1 cm) can be fol-lowed with active surveillance.6 Focused mini-incision parathyroidectomy, after appropri-ate localization, has become the procedure of choice for the treatment of sporadic primary hyperparathyroidism.7 Parathyroidectomy has been shown to improve the clas-sic and the so-called

1	appropri-ate localization, has become the procedure of choice for the treatment of sporadic primary hyperparathyroidism.7 Parathyroidectomy has been shown to improve the clas-sic and the so-called nonspecific symptoms and metabolic complications of primary hyperparathyroidism.8 Normocalcemic hyperparathyroidism is being increasingly recognized; however, there are no definitive guidelines for management.9 Very high calcium and parathyroid hormone levels in a patient with primary hyperparathyroidism should alert the surgeon to the presence of a possible parathyroid carcinoma.10 Subclinical Cushing’s syndrome is characterized by subtle abnormalities in corticosteroid synthesis, and many of its manifestations appear to be treated by adrenalectomy.11 Fine-needle aspiration biopsy has a very limited role in the evaluation of adrenal incidentalomas unless the patient has previously had a cancer and should only be performed after appropriate biochemical studies have been performed to rule out

1	role in the evaluation of adrenal incidentalomas unless the patient has previously had a cancer and should only be performed after appropriate biochemical studies have been performed to rule out pheochromocytoma.12 Laparoscopic adrenalectomy has become the procedure of choice for excision of most adrenal lesions, except known or suspected cancers.Brunicardi_Ch38_p1625-p1704.indd 162601/03/19 11:20 AM 1627THYROID, PARATHYROID, AND ADRENALCHAPTER 38failed to fuse with the main thyroid, as previously suggested by Crile. Even if not readily apparent on physical examination or ultrasound imaging, the ipsilateral thyroid lobe contains a focus of papillary thyroid cancer (PTC), which may be microscopic.Pyramidal Lobe. Normally the thyroglossal duct atrophies, although it may remain as a fibrous band. In about 50% of indi-viduals, the distal end that connects to the thyroid persists as a pyramidal lobe projecting up from the isthmus, lying just to the left or right of the midline. In the

1	band. In about 50% of indi-viduals, the distal end that connects to the thyroid persists as a pyramidal lobe projecting up from the isthmus, lying just to the left or right of the midline. In the normal individual, the pyra-midal lobe is not palpable, but in disorders resulting in thyroid hypertrophy (e.g., Graves’ disease, diffuse nodular goiter, or lymphocytic thyroiditis), the pyramidal lobe usually is enlarged and palpable.Thyroid AnatomyThe anatomic relations of the thyroid gland and surrounding structures are depicted in Fig. 38-2. The adult thyroid gland is brown in color and firm in consistency and is located posterior to the strap muscles. The normal thyroid gland weighs approx-imately 20 g, but gland weight varies with body weight and iodine intake. The thyroid lobes are located adjacent to the thy-roid cartilage and connected in the midline by an isthmus that is located just inferior to the cricoid cartilage. A pyramidal lobe is present in about 50% of patients. The thyroid

1	adjacent to the thy-roid cartilage and connected in the midline by an isthmus that is located just inferior to the cricoid cartilage. A pyramidal lobe is present in about 50% of patients. The thyroid lobes extend to the midthyroid cartilage superiorly and lie adjacent to the carotid sheaths and sternocleidomastoid muscles laterally. The strap muscles (sternohyoid, sternothyroid, and superior belly of the omohyoid) are located anteriorly and are innervated by the ansa cervicalis (ansa hypoglossi). The thyroid gland is enveloped by a loosely connecting fascia that is formed from the parti-tion of the deep cervical fascia into anterior and posterior divi-sions. The true capsule of the thyroid is a thin, densely adherent fibrous layer that sends out septa that invaginate into the gland, forming pseudolobules. The thyroid capsule is condensed into the posterior suspensory or Berry’s ligament near the cricoid cartilage and upper tracheal rings.Sup. thyroid a. and v.PyramidallobeCommon

1	forming pseudolobules. The thyroid capsule is condensed into the posterior suspensory or Berry’s ligament near the cricoid cartilage and upper tracheal rings.Sup. thyroid a. and v.PyramidallobeCommon carotid a.Common carotid a.Thyroid cartilageInt. jugular v.Int. jugular v.Recurrent laryngeal n.Recurrentlaryngeal n.Vagus n.Vagus n.Vagus n.Arch of aortaThyrocervical trunkInf. thyroid v.Middle thyroid v.Ext. carotid a.Thyroidea ima a.(variable)TracheaSternocleido-mastoid m.Inf. thyroid a.Vertebralv. and a. Thyroid glandStrap musclesABFigure 38-2. Anatomy of the thyroid gland and surrounding structures, viewed anteriorly (A) and in cross-section (B). a. = artery; m. = muscle; n. = nerve; v. = vein.Brunicardi_Ch38_p1625-p1704.indd 162701/03/19 11:20 AM 1628SPECIFIC CONSIDERATIONSPART IIBlood Supply. The superior thyroid arteries arise from the ipsilateral external carotid arteries and divide into anterior and posterior branches at the apices of the thyroid lobes. The infe-rior

1	Supply. The superior thyroid arteries arise from the ipsilateral external carotid arteries and divide into anterior and posterior branches at the apices of the thyroid lobes. The infe-rior thyroid arteries arise from the thyrocervical trunk shortly after their origin from the subclavian arteries. The inferior thy-roid arteries travel upward in the neck posterior to the carotid sheath to enter the thyroid lobes at their midpoint. A thyroidea ima artery arises directly from the aorta or innominate in 1% to 4% of individuals to enter the isthmus or replace a missing inferior thyroid artery. The inferior thyroid artery crosses the recurrent laryngeal nerve (RLN), necessitating identification of the RLN before the arterial branches can be ligated. The venous drainage of the thyroid gland occurs via multiple small surface veins, which coalesce to form three sets of veins—the supe-rior, middle, and inferior thyroid veins. The superior thyroid veins run with the superior thyroid arteries

1	via multiple small surface veins, which coalesce to form three sets of veins—the supe-rior, middle, and inferior thyroid veins. The superior thyroid veins run with the superior thyroid arteries bilaterally. The middle vein or veins are the least consistent. The superior and middle veins drain directly into the internal jugular veins. The inferior veins often form a plexus, which drains into the bra-chiocephalic veins.Nerves. The left RLN arises from the vagus nerve where it crosses the aortic arch, loops around the ligamentum arteriosum, and ascends medially in the neck within the tracheoesophageal groove. The right RLN arises from the vagus at its crossing with the right subclavian artery. The nerve usually passes posterior to the artery before ascending in the neck, its course being more oblique than the left RLN. Along their course in the neck, the RLNs may branch, and pass anterior, posterior, or interdigitate with branches of the inferior thyroid artery (Fig. 38-3). The right

1	more oblique than the left RLN. Along their course in the neck, the RLNs may branch, and pass anterior, posterior, or interdigitate with branches of the inferior thyroid artery (Fig. 38-3). The right RLN may be nonrecurrent in 0.5% to 1% of individuals and often is associated with a vascular anomaly. Nonrecurrent left RLNs are rare but have been reported in patients with situs inversus and a right-sided aortic arch. The RLN may branch in its course in the neck, and identification of a small nerve should alert the surgeon to this possibility. Identification of the nerves or their branches often necessitates mobilization of the most lateral and posterior extent of the thyroid gland, the tubercle of Zucker-kandl, at the level of the cricoid cartilage. The last segments of the nerves often course below the tubercle and are closely approximated to the ligament of Berry. Branches of the nerve may traverse the ligament in 25% of individuals and are particu-larly vulnerable to injury at this

1	below the tubercle and are closely approximated to the ligament of Berry. Branches of the nerve may traverse the ligament in 25% of individuals and are particu-larly vulnerable to injury at this junction. The RLNs terminate by entering the larynx posterior to the cricothyroid muscle.The RLNs innervate all the intrinsic muscles of the larynx, except the cricothyroid muscles, which are innervated by the external laryngeal nerves. Injury to one RLN leads to paralysis of the ipsilateral vocal cord, which comes to lie in the parame-dian or the abducted position. The paramedian position results in a normal but weak voice, whereas the abducted position leads to a hoarse voice and an ineffective cough. Bilateral RLN injury may lead to airway obstruction, necessitating emergency trache-ostomy, or loss of voice. If both cords come to lie in an abducted position, air movement can occur, but the patient has an ineffec-tive cough and is at increased risk of repeated respiratory tract infections

1	loss of voice. If both cords come to lie in an abducted position, air movement can occur, but the patient has an ineffec-tive cough and is at increased risk of repeated respiratory tract infections from aspiration.The superior laryngeal nerves also arise from the vagus nerves. After their origin at the base of the skull, these nerves 1) Nerve in tracheoesophageal groove R: 64% L: 77% 4) Nerve between branches of inferior thyroid artery R: 7% L: 67% 2) Nerve lateral to trachea R: 28% L: 17% R: 8% L: 6%5) Nerve posterior to artery R: 53% L: 69% R: 37% L: 24%3) Nerve far anterior6) Nerve anterior to artery7) Artery absent R: 3% L: 1%Figure 38-3. Relationship of recurrent laryngeal nerve to the inferior thyroid artery—the superior parathyroid is characteristically dorsal to the plane of the nerve, whereas the inferior gland is ventral to the nerve.Brunicardi_Ch38_p1625-p1704.indd 162801/03/19 11:20 AM 1629THYROID, PARATHYROID, AND ADRENALCHAPTER

1	dorsal to the plane of the nerve, whereas the inferior gland is ventral to the nerve.Brunicardi_Ch38_p1625-p1704.indd 162801/03/19 11:20 AM 1629THYROID, PARATHYROID, AND ADRENALCHAPTER 38travel along the internal carotid artery and divide into two branches at the level of the hyoid bone. The internal branch of the superior laryngeal nerve is sensory to the supraglottic larynx. Injury to this nerve is rare in thyroid surgery, but its occurrence may result in aspiration. The external branch of the superior laryngeal nerve lies on the inferior pharyngeal constric-tor muscle and descends alongside the superior thyroid vessels before innervating the cricothyroid muscle. Cernea and col-leagues2 proposed a classification system to describe the rela-tionship of this nerve to the superior thyroid vessels (Fig. 38-4). The type 2a variant, in which the nerve crosses below the tip of the thyroid superior pole, occurs in up to 20% of individuals and places the nerve at a greater risk of

1	thyroid vessels (Fig. 38-4). The type 2a variant, in which the nerve crosses below the tip of the thyroid superior pole, occurs in up to 20% of individuals and places the nerve at a greater risk of injury. Therefore, the superior pole vessels should not be ligated en masse, but should be individually divided, low on the thyroid gland and dissected lateral to the cricothyroid muscle. Injury to this nerve leads to inability to tense the ipsilateral vocal cord and hence difficulty “hitting high notes,” difficulty projecting the voice, and voice fatigue during prolonged speech.Sympathetic innervation of the thyroid gland is provided by fibers from the superior and middle cervical sympathetic ganglia. The fibers enter the gland with the blood vessels and are vasomotor in action. Parasympathetic fibers are derived from the vagus nerve and reach the gland via branches of the laryngeal nerves.Parathyroid Glands. The embryology and anatomy of the parathyroid glands are discussed in detail in

1	fibers are derived from the vagus nerve and reach the gland via branches of the laryngeal nerves.Parathyroid Glands. The embryology and anatomy of the parathyroid glands are discussed in detail in the “Parathyroid Gland” section of this chapter. About 85% of individuals have four parathyroid glands that can be found within 1 cm of the junction of the inferior thyroid artery and the RLN. The supe-rior glands are usually located dorsal to the RLN, whereas the inferior glands are usually found ventral to the RLN (Fig. 38-5).Lymphatic System. The thyroid gland is endowed with an extensive network of lymphatics. Intraglandular lymphatic ves-sels connect both thyroid lobes through the isthmus and also drain to perithyroidal structures and lymph nodes. Regional lymph nodes include pretracheal, paratracheal, perithyroidal, RLN, superior mediastinal, retropharyngeal, esophageal, and upper, middle, and lower jugular chain nodes. These lymph nodes can be classified into seven levels as depicted

1	perithyroidal, RLN, superior mediastinal, retropharyngeal, esophageal, and upper, middle, and lower jugular chain nodes. These lymph nodes can be classified into seven levels as depicted in Fig. 38-6. The central compartment includes nodes located in the area between the two carotid sheaths, whereas nodes lateral to the vessels are present in the lateral compartment. Thyroid can-cers may metastasize to any of these regions, although metas-tases to submaxillary nodes (level I) are rare (<1%). There also can be “skip” metastases to nodes in the lateral ipsilateral neck without central neck nodes.Thyroid HistologyMicroscopically, the thyroid is divided into lobules that contain 20 to 40 follicles (Fig. 38-7). There are about 3 × 106 follicles in the adult male thyroid gland. The follicles are spherical and average 30 μm in diameter. Each follicle is lined by cuboidal epithelial cells and contains a central store of colloid secreted from the epithelial cells under the influence of the

1	are spherical and average 30 μm in diameter. Each follicle is lined by cuboidal epithelial cells and contains a central store of colloid secreted from the epithelial cells under the influence of the pituitary hor-mone TSH. The second group of thyroid secretory cells is the C cells or parafollicular cells, which contain and secrete the hor-mone calcitonin. They are found as individual cells or clumped in small groups in the interfollicular stroma and located in the upper poles of the thyroid lobes.Thyroid PhysiologyIodine Metabolism. The average daily iodine requirement is 0.1 mg, which can be derived from foods such as fish, milk, and eggs or as additives in bread or salt. In the stomach and jeju-num, iodine is rapidly converted to iodide and absorbed into the bloodstream, and from there it is distributed uniformly through-out the extracellular space. Iodide is actively transported into the thyroid follicular cells by an adenosine triphosphate (ATP)–dependent process. The thyroid is

1	it is distributed uniformly through-out the extracellular space. Iodide is actively transported into the thyroid follicular cells by an adenosine triphosphate (ATP)–dependent process. The thyroid is the storage site of >90% of the body’s iodine content and accounts for one-third of the plasma iodine loss. The remaining plasma iodine is cleared via renal excretion.Thyroid Hormone Synthesis, Secretion, and Transport. The synthesis of thyroid hormone consists of several steps 1cmType 1Type 2aType 2bFigure 38-4. Relationship of the external branch of the superior laryngeal nerve and superior thyroid artery originally described by Cernea and colleagues.2 In type 1 anatomy, the nerve crosses the artery ≥1 cm above the superior aspect of the thyroid lobe. In type 2 anatomy, the nerve crosses the artery <1 cm above the thyroid pole (2a) or below (2b) it. (Reproduced with permission from Bliss RD, Gauger PG, Delbridge LW:Surgeon’s approach to the thyroid gland: surgical anatomy and the

1	the artery <1 cm above the thyroid pole (2a) or below (2b) it. (Reproduced with permission from Bliss RD, Gauger PG, Delbridge LW:Surgeon’s approach to the thyroid gland: surgical anatomy and the importance of technique, World J Surg. 2000 Aug;24(8):891-897.)Brunicardi_Ch38_p1625-p1704.indd 162901/03/19 11:20 AM 1630SPECIFIC CONSIDERATIONSPART II(Fig. 38-8). The first, iodide trapping, involves active (ATP-dependent) transport of iodide across the basement mem-brane of the thyrocyte via an intrinsic membrane protein, the sodium/iodine (Na+/I–) symporter. Thyroglobulin (Tg) is a large (660 kDa) glycoprotein, which is present in thyroid follicles and has four tyrosyl residues. The second step in thyroid hormone synthesis involves oxidation of iodide to iodine and iodination of tyrosine residues on Tg, to form monoiodotyrosines (MIT) and diiodotyrosines (DIT). Both processes are catalyzed by thyroid peroxidase (TPO). A recently identified protein, pen-drin, is thought to mediate

1	residues on Tg, to form monoiodotyrosines (MIT) and diiodotyrosines (DIT). Both processes are catalyzed by thyroid peroxidase (TPO). A recently identified protein, pen-drin, is thought to mediate iodine efflux at the apical membrane. The third step leads to coupling of two DIT molecules to form tetra-iodothyronine or thyroxine (T4), and one DIT molecule with one MIT molecule to form 3,5,3′-triiodothyronine (T3) or 3,3′,5′-triiodothyronine reverse (rT3). When stimulated by TSH, thyrocytes form pseudopodia, which encircle portions of cell membrane containing Tg, which in turn, fuse with enzymecontaining lysosomes. In the fourth step, Tg is hydrolyzed to release free iodothyronines (T3 and T4) and monoand diiodo-tyrosines. The latter are deiodinated in the fifth step to yield iodide, which is reused in the thyrocyte. In the euthyroid state, T4 is produced and released entirely by the thyroid gland, whereas only 20% of the total T3 is produced by the thyroid. Most of the T3 is produced by

1	reused in the thyrocyte. In the euthyroid state, T4 is produced and released entirely by the thyroid gland, whereas only 20% of the total T3 is produced by the thyroid. Most of the T3 is produced by peripheral deiodination (removal of 5′-iodine from the outer ring) of T4 in the liver, muscles, kidney, and anterior pituitary, a reaction that is catalyzed by 5′-mono-deiodinase. Some T4 is converted to rT3, the metaboli-cally inactive compound, by deiodination of the inner ring of T4. In conditions such as Graves’ disease, toxic multinodular goi-ter, or a stimulated thyroid gland, the proportion of T3 released from the thyroid may be dramatically elevated. Thyroid hor-mones are transported in serum bound to carrier proteins such as T4-binding globulin, T4-binding prealbumin, and albumin. Only a small fraction (0.02%) of thyroid hormone (T3 and T4) is free (unbound) and is the physiologically active component. T3 is the more potent of the two thyroid hormones, although its circulating

1	Only a small fraction (0.02%) of thyroid hormone (T3 and T4) is free (unbound) and is the physiologically active component. T3 is the more potent of the two thyroid hormones, although its circulating plasma level is much lower than that of T4. T3 is less tightly bound to protein in the plasma than T4, and so it enters tissues more readily. T3 is three to four times more active than T4 per unit weight, with a half-life of about 1 day, compared to approximately 7 days for T4.The secretion of thyroid hormone is controlled by the hypothalamic-pituitary-thyroid axis (Fig. 38-9). The hypo-thalamus produces a peptide, the thyrotropin-releasing hor-mone (TRH), which stimulates the pituitary to release TSH or thyrotropin. TRH reaches the pituitary via the portovenous circulation. TSH, a 28-kDa glycopeptide, mediates iodide trap-ping, secretion, and release of thyroid hormones, in addition to increasing the cellularity and vascularity of the thyroid gland. The TSH receptor (TSH-R) belongs to a

1	mediates iodide trap-ping, secretion, and release of thyroid hormones, in addition to increasing the cellularity and vascularity of the thyroid gland. The TSH receptor (TSH-R) belongs to a family of G-protein–coupled receptors that have seven transmembrane-spanning domains and use cyclic adenosine monophosphate in the signal-transduction pathway. TSH secretion by the anterior pituitary is also regulated via a negative feedback loop by T4 and T3. Because the pituitary has the ability to convert T4 to T3, the latter is thought to be more important in this feedback control. T3 also inhibits the release of TRH.The thyroid gland also is capable of autoregulation, which allows it to modify its function independent of TSH. As an adap-tation to low iodide intake, the gland preferentially synthesizes Lower parathyroidUpper parathyroidInt. jugular v.Recurrent laryngeal n.ThyroidInferior thyroid a.Common carotid a. Figure 38-5. Relationship of the parathyroids to the recurrent laryngeal nerve.

1	Lower parathyroidUpper parathyroidInt. jugular v.Recurrent laryngeal n.ThyroidInferior thyroid a.Common carotid a. Figure 38-5. Relationship of the parathyroids to the recurrent laryngeal nerve. a. = artery; v. = vein.Brunicardi_Ch38_p1625-p1704.indd 163001/03/19 11:20 AM 1631THYROID, PARATHYROID, AND ADRENALCHAPTER 38T3 rather than T4, thereby increasing the efficiency of secreted hormone. In situations of iodine excess, iodide transport, per-oxide generation, and synthesis and secretion of thyroid hor-mones are inhibited. Excessively large doses of iodide may lead to initial increased organification, followed by suppression, a phenomenon called the Wolff-Chaikoff effect. Epinephrine and human chorionic gonadotropin hormones stimulate thyroid hormone production. Thus, elevated thyroid hormone levels are found in pregnancy and gynecologic malignancies such as hydatidiform mole. In contrast, glucocorticoids inhibit thyroid hormone production. In severely ill patients, peripheral

1	hormone levels are found in pregnancy and gynecologic malignancies such as hydatidiform mole. In contrast, glucocorticoids inhibit thyroid hormone production. In severely ill patients, peripheral thyroid hormones may be reduced, without a compensatory increase in TSH levels, giving rise to the euthyroid sick syndrome.Thyroid Hormone Function. Free thyroid hormone enters the cell membrane by diffusion or by specific carriers and is car-ried to the nuclear membrane by binding to specific proteins. T4 is deiodinated to T3 and enters the nucleus via active trans-port, where it binds to the thyroid hormone receptor. The T3 receptor is similar to the nuclear receptors for glucocorticoids, mineralocorticoids, estrogens, vitamin D, and retinoic acid. In humans, two types of T3 receptor genes (α and β) are located on chromosomes 3 and 17. Thyroid receptor expression depends on peripheral concentrations of thyroid hormones and is tissue specific—the α form is abundant in the central nervous

1	and β) are located on chromosomes 3 and 17. Thyroid receptor expression depends on peripheral concentrations of thyroid hormones and is tissue specific—the α form is abundant in the central nervous sys-tem, whereas the β form predominates in the liver. Each gene product has a ligand-independent, amino-terminal domain; IIVVIIIIIIIVVITrapezius m.Sternocleidomastoid m.Central neck nodesUpper jugular nodesSubmaxillary nodesParotidMiddle jugular nodesAnterior mediastinalnodesPosteriortriangleLower jugular nodesExternaljugularnodeSternocleido-mastoid m.Spinal accessory n.Jugulodigastric nodeDeep lateral nodesIntercalatednodeTransversecervical chainof nodesDigastric m.Mandibular &submandibularnodesSubmental nodeHyoidInternal jugularchain of nodesStrap muscleSuperior thyroid nodesAnterior superficialcervical nodesSupra-clavicular nodes ABFigure 38-6. A and B. Lymph nodes in the neck can be divided into six regions. Upper mediastinal nodes constitute level VII. m. = muscle; n. =

1	superficialcervical nodesSupra-clavicular nodes ABFigure 38-6. A and B. Lymph nodes in the neck can be divided into six regions. Upper mediastinal nodes constitute level VII. m. = muscle; n. = nerve.Brunicardi_Ch38_p1625-p1704.indd 163101/03/19 11:20 AM 1632SPECIFIC CONSIDERATIONSPART IIa ligand-binding, carboxy-terminal domain; and centrally located DNA-binding regions. Binding of thyroid hormone leads to the transcription and translation of specific hormone-responsive genes.Thyroid hormones affect almost every system in the body. They are important for fetal brain development and skeletal mat-uration. T3 increases oxygen consumption, basal metabolic rate, and heat production by stimulation of Na+/K+ ATPase in various tissues. It also has positive inotropic and chronotropic effects on the heart by increasing transcription of the Ca2+ ATPase in the sarcoplasmic reticulum and increasing levels of β-adrenergic receptors and concentration of G proteins. Myocardial α recep-tors are

1	on the heart by increasing transcription of the Ca2+ ATPase in the sarcoplasmic reticulum and increasing levels of β-adrenergic receptors and concentration of G proteins. Myocardial α recep-tors are decreased, and actions of catecholamines are amplified. Thyroid hormones are responsible for maintaining the normal hypoxic and hypercapnic drive in the respiratory center of the brain. They also increase gastrointestinal (GI) motility, leading to diarrhea in hyperthyroidism and constipation in hypothyroid-ism. Thyroid hormones also increase bone and protein turnover H2O2 GenerationIodinationDifferentiationGrowthHormone synthesisCREBCREMPAX-8TTF-1TTF-2XXXXXIP3PKCDAGPKAPIP2ATPcAMPPLCGqGSGIACTSHRTSHIGF-1 RIGF-1 NISIodide uptakeMITDITT3T4T3T4HydrolysisNADP+NADPHH2O2H2O22O2TPOTgTgTPOI or HOICouplingOrganificationMITDITMITDITT3T4DehalogenaseI–I–Figure 38-8. Thyroid follicular cell showing the major signaling pathways involved in thyroid cell growth and function and key steps in thy-roid hormone

1	38-8. Thyroid follicular cell showing the major signaling pathways involved in thyroid cell growth and function and key steps in thy-roid hormone synthesis. The basal membrane of the cell in contact with the circulation and its apical surface contact the thyroid follicle. Thy-roid hormone synthesis is initiated by the binding of thyroid-stimulating hormone (TSH) to the TSH receptor (TSHR), a G-protein–coupled transmembrane receptor, on the basal membrane. Activation leads to an increase in cyclic adenosine monophosphate (cAMP), phosphorylation of protein kinase A (PKA), and activation of target cytosolic and nuclear proteins. The protein kinase C (PKC) pathway is stimulated at higher doses of TSH. Iodide is actively transported into the cell via the Na/I symporter (NIS) and flows down an electrical gradient to the apical membrane. There, thyroid peroxidase (TPO) oxidizes iodide and iodinated tyrosyl residues on thyroglobulin (Tg) in the presence of peroxide (H2O2). Monoand

1	down an electrical gradient to the apical membrane. There, thyroid peroxidase (TPO) oxidizes iodide and iodinated tyrosyl residues on thyroglobulin (Tg) in the presence of peroxide (H2O2). Monoand diiodotyrosyl (MIT, DIT) residues are also coupled to form T4 and T3 by TPO. Thyroglobulin carrying T4 and T3 is then internalized by pinocytosis and digested in lysosomes. Thyroid hormone is released into the circulation, while MIT and DIT are deiodinated and recycled. ATP = adenosine triphosphate; CREB = cAMP response element binding protein; CREM = cAMP response element modulator; DAG = diacylglycerol; IGF-1 = insulin-like growth factor 1; IP3 = inositol-3-phosphate; NADP+ = nicotinamide adenine dinucleotide phosphate, oxidized form; NADPH = nicotinamide adenine dinucleotide phosphate; PIP2 = phosphatidylinositol; PLC = phospholipase C; T3 = 3,5′,3-triiodothyronine; T4 = thyroxine. (Reproduced with permission from Kopp P: Pendred’s syndrome and genetic defects in thyroid hormone

1	PIP2 = phosphatidylinositol; PLC = phospholipase C; T3 = 3,5′,3-triiodothyronine; T4 = thyroxine. (Reproduced with permission from Kopp P: Pendred’s syndrome and genetic defects in thyroid hormone synthesis, Rev Endocr Metab Disord. 2000 Jan;1(1-2):109-121.)Figure 38-7. Normal thyroid histology—follicular cells surround colloid.Brunicardi_Ch38_p1625-p1704.indd 163201/03/19 11:20 AM 1633THYROID, PARATHYROID, AND ADRENALCHAPTER 38and the speed of muscle contraction and relaxation. They also increase glycogenolysis, hepatic gluconeogenesis, intestinal glucose absorption, and cholesterol synthesis and degradation.Evaluation of Patients With Thyroid DiseaseTests of Thyroid Function. A multitude of different tests are available to evaluate thyroid function. No single test is sufficient to assess thyroid function in all situations, and the results must be interpreted in the context of the patient’s clinical condition. TSH is the only test necessary in most patients with thyroid nodules

1	assess thyroid function in all situations, and the results must be interpreted in the context of the patient’s clinical condition. TSH is the only test necessary in most patients with thyroid nodules that clinically appear to be euthyroid.Serum Thyroid-Stimulating Hormone (Normal 0.5–5 μU/mL) The tests for serum TSH are based on the following principle: monoclonal TSH antibodies are bound to a solid matrix and bind serum TSH. A second monoclonal antibody binds to a separate epitope on TSH and is labeled with radioisotope, enzyme, or fluorescent tag. Therefore, the amount of serum TSH is propor-tional to the amount of bound secondary antibody (immunomet-ric assay). Serum TSH levels reflect the ability of the anterior pituitary to detect free T4 levels. There is an inverse relation-ship between the free T4 level and the logarithm of the TSH concentration—small changes in free T4 lead to a large shift in TSH levels. The ultrasensitive TSH assay has become the most sensitive and specific

1	the free T4 level and the logarithm of the TSH concentration—small changes in free T4 lead to a large shift in TSH levels. The ultrasensitive TSH assay has become the most sensitive and specific test for the diagnosis of hyperand hypo-thyroidism and for optimizing T4 therapy.Total T4 (Reference Range 55–150 nmol/L) and T3 (Reference Range 1.5–3.5 nmol/L) Total T4 and T3 levels are measured by radioimmunoassay and measure both the free and bound components of the hormones. Total T4 levels reflect the output from the thyroid gland, whereas T3 levels in the nonstimulated thyroid gland are more indicative of peripheral thyroid hor-mone metabolism, and are, therefore, not generally suitable as a general screening test. Total T4 levels are increased not only in hyperthyroid patients, but also in those with elevated Tg levels secondary to pregnancy, estrogen/progesterone use, or congeni-tal diseases. Similarly, total T4 levels decrease in hypothyroidism and in patients with decreased Tg

1	in those with elevated Tg levels secondary to pregnancy, estrogen/progesterone use, or congeni-tal diseases. Similarly, total T4 levels decrease in hypothyroidism and in patients with decreased Tg levels due to anabolic steroid use and protein-losing disorders like nephrotic syndrome. Indi-viduals with these latter disorders may be euthyroid if their free T4 levels are normal. Measurement of total T3 levels is impor-tant in clinically hyperthyroid patients with normal T4 levels, who may have T3 thyrotoxicosis. As discussed previously in “Thyroid Hormone Synthesis, Secretion, and Transport,” total T3 levels often are increased in early hypothyroidism.Free T4 (Reference Range 12–28 pmol/L) and Free T3 (3–9 pmol/L) These radioimmunoassay-based tests are a sensitive and accurate measurement of biologically active thy-roid hormone. Free T4 estimates are not performed as a routine screening tool in thyroid disease. Use of this test is confined to cases of early hyperthyroidism in which

1	of biologically active thy-roid hormone. Free T4 estimates are not performed as a routine screening tool in thyroid disease. Use of this test is confined to cases of early hyperthyroidism in which total T4 levels may be normal but free T4 levels are raised. In patients with end-organ resistance to T4 (Refetoff’s syndrome), T4 levels are increased, but TSH levels usually are normal. Free T3 is most useful in con-firming the diagnosis of early hyperthyroidism, in which levels of free T4 and free T3 rise before total T4 and T3. Free T4 levels may also be measured indirectly using the T3-resin uptake test. If free T4 levels are increased, fewer hormone binding sites are available for binding radiolabeled T3 that has been added to the patient’s serum. Therefore, more T3 binds with an ion-exchange resin, and the T3-resin uptake is increased.Thyrotropin-Releasing Hormone This test is useful to evalu-ate pituitary TSH secretory function and is performed by admin-istering 500 μg of TRH

1	resin, and the T3-resin uptake is increased.Thyrotropin-Releasing Hormone This test is useful to evalu-ate pituitary TSH secretory function and is performed by admin-istering 500 μg of TRH intravenously and measuring TSH levels after 30 and 60 minutes. In a normal individual, TSH levels should increase at least 6 μIU/mL from the baseline. This test also was previously used to assess patients with borderline hyperthyroidism but has largely been replaced by sensitive TSH assays for this purpose.Thyroid Antibodies Thyroid antibodies include anti-Tg, anti-microsomal, or anti-TPO and thyroid-stimulating immuno-globulin (TSI). Anti-Tg and anti-TPO antibody levels do not determine thyroid function, but rather indicate the underlying disorder, usually an autoimmune thyroiditis. About 80% of patients with Hashimoto’s thyroiditis have elevated thyroid anti-body levels; however, levels may also be increased in patients with Graves’ disease, multinodular goiter, and occasionally, thyroid

1	of patients with Hashimoto’s thyroiditis have elevated thyroid anti-body levels; however, levels may also be increased in patients with Graves’ disease, multinodular goiter, and occasionally, thyroid neoplasms.Serum Thyroglobulin Tg is only made by normal or abnormal thyroid tissue. It normally is not released into the circulation in large amounts but increases dramatically in destructive pro-cesses of the thyroid gland, such as thyroiditis, or overactive states such as Graves’ disease and toxic multinodular goiter. The most important use for serum Tg levels is in monitoring patients with differentiated thyroid cancer for recurrence, partic-ularly after total thyroidectomy and RAI ablation. Elevated anti-Tg antibodies can interfere with the accuracy of serum Tg levels and should always be measured when interpreting Tg levels.ThyroidPortalsystemHypothalamusTRHTissueTSH++T4T4T4T3T3T3––II“Free”Figure 38-9. Hypothalamic-pituitary-thyroid hormone axis. In both the hypothalamus and

1	be measured when interpreting Tg levels.ThyroidPortalsystemHypothalamusTRHTissueTSH++T4T4T4T3T3T3––II“Free”Figure 38-9. Hypothalamic-pituitary-thyroid hormone axis. In both the hypothalamus and pituitary, 3,5′,3-triiodothyronine (T3) is primarily responsible for inhibition of thyrotropin-releasing hor-mone (TRH) and thyroid-stimulating hormone (TSH) secretion. T4 = thyroxine. (Reproduced with permission from Greenspan FS, Gardner D: Basic and Clinical Endocrinology, 6th ed. New York, NY: McGraw-Hill Education; 2001.)Brunicardi_Ch38_p1625-p1704.indd 163301/03/19 11:20 AM 1634SPECIFIC CONSIDERATIONSPART IISerum Calcitonin (0–4 pg/mL Basal) This 32-amino-acid polypeptide is secreted by the C cells and functions to lower serum calcium levels, although in humans, it has only minimal physiologic effects. It is also a sensitive marker of MTC.Thyroid Imaging Radionuclide Imaging Both iodine-123 (123I) and iodine-131 (131I) are used to image the thyroid gland. The former emits low-dose

1	effects. It is also a sensitive marker of MTC.Thyroid Imaging Radionuclide Imaging Both iodine-123 (123I) and iodine-131 (131I) are used to image the thyroid gland. The former emits low-dose radiation, has a half-life of 12 to 14 hours, and is used to image lingual thyroids or goiters. In contrast, 131I has a half-life of 8 to 10 days and leads to higher-dose radiation expo-sure. Therefore, this isotope is used to screen and treat patients with differentiated thyroid cancers for metastatic disease. The images obtained by these studies provide information not only about the size and shape of the gland, but also the distribution of functional activity. Areas that trap less radioactivity than the surrounding gland are termed cold (Fig. 38-10), whereas areas that demonstrate increased activity are termed hot. The risk of malignancy is higher in “cold” lesions (20%) compared to “hot” or “warm” lesions (<5%). Technetium Tc 99m pertech-netate (99mTc) is taken up by the thyroid gland and is

1	are termed hot. The risk of malignancy is higher in “cold” lesions (20%) compared to “hot” or “warm” lesions (<5%). Technetium Tc 99m pertech-netate (99mTc) is taken up by the thyroid gland and is increas-ingly being used for thyroid evaluation. This isotope is taken up by the mitochondria, but is not organified. It also has the advantage of having a shorter half-life and minimizes radiation exposure. It is particularly sensitive for nodal metastases. More recently, 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) combined with computed tomography (CT) is being increasingly used to screen for metastases in patients with thyroid cancer in whom other imaging studies are nega-tive. PET scans are not routinely used in the evaluation of thy-roid nodules; however, they may show clinically occult thyroid lesions. There are several recent reports of rates of malignancy in these lesions ranging from 14% to 63%. These incidentally Figure 38-10. Radioactive iodine scan of the

1	clinically occult thyroid lesions. There are several recent reports of rates of malignancy in these lesions ranging from 14% to 63%. These incidentally Figure 38-10. Radioactive iodine scan of the thyroid, with the arrow showing an area of decreased uptake, a cold nodule.discovered nodules should be worked up by ultrasound and fine-needle aspiration biopsy (FNAB).Ultrasound Ultrasound is an excellent noninvasive and por-table imaging study of the thyroid gland with the added advan-tage of no radiation exposure. It is helpful in the evaluation of thyroid nodules, distinguishing solid from cystic ones, and pro-viding information about size and multicentricity. In addition, characteristics such as echotexture, shape, borders and presence of calcifications, and vascularity can provide useful information regarding risk of malignancy. Ultrasound is also especially help-ful for assessing cervical lymphadenopathy (Fig. 38-11) and to guide FNAB. An experienced ultrasonographer is necessary for

1	regarding risk of malignancy. Ultrasound is also especially help-ful for assessing cervical lymphadenopathy (Fig. 38-11) and to guide FNAB. An experienced ultrasonographer is necessary for the best results.Computed Tomography/Magnetic Resonance Imaging Scan CT and magnetic resonance imaging (MRI) studies provide excellent imaging of the thyroid gland and adjacent nodes and are particularly useful in evaluating the extent of large, fixed, or substernal goiters (which cannot be evaluated by ultrasound) and their relationship to the airway and vascular structures. Noncontrast CT scans should be obtained for patients who are likely to require subsequent RAI therapy. If contrast is necessary, therapy needs to be delayed by several months. Combined PET-CT scans are increasingly being used for Tg-positive, RAI-negative tumors.Benign Thyroid DisordersHyperthyroidism. The clinical manifestations of hyperthy-roidism result from an excess of circulating thyroid hormone. Hyperthyroidism may arise

1	RAI-negative tumors.Benign Thyroid DisordersHyperthyroidism. The clinical manifestations of hyperthy-roidism result from an excess of circulating thyroid hormone. Hyperthyroidism may arise from a number of conditions that are listed in Table 38-1. It is important to distinguish disorders such as Graves’ disease and toxic nodular goiters that result from increased production of thyroid hormone from those dis-orders that lead to a release of stored hormone from injury to the thyroid gland (thyroiditis) or from other nonthyroid gland–related conditions. The former disorders lead to an increase in RAI uptake (RAIU), whereas the latter group is characterized by low RAIU. Of these disorders, Graves’ disease, toxic mul-tinodular goiter, and solitary toxic nodule are most relevant to the surgeon.Diffuse Toxic Goiter (Graves’ Disease) Although originally described by the Welsh physician Caleb Parry in a posthumous article in 1825, this disorder is known as Graves’ disease after Robert Graves,

1	Toxic Goiter (Graves’ Disease) Although originally described by the Welsh physician Caleb Parry in a posthumous article in 1825, this disorder is known as Graves’ disease after Robert Graves, an Irish physician who described three patients in 1835. Graves’ disease is by far the most common cause of hyperthyroidism in North America, accounting for 60% to 80% of cases. It is an autoimmune disease with a strong famil-ial predisposition, female preponderance (5:1), and peak inci-dence between the ages of 40 and 60 years. Graves’ disease is characterized by thyrotoxicosis, diffuse goiter, and extrathy-roidal conditions including ophthalmopathy, dermopathy (pre-tibial myxedema), thyroid acropachy, gynecomastia, and other manifestations.Etiology, Pathogenesis, and Pathology. The exact etiology of the initiation of the autoimmune process in Graves’ disease is not known. However, conditions such as the postpartum state, iodine excess, lithium therapy, and bacterial and viral infections have

1	of the initiation of the autoimmune process in Graves’ disease is not known. However, conditions such as the postpartum state, iodine excess, lithium therapy, and bacterial and viral infections have been suggested as possible triggers. Genetic factors also play a role, as haplotyping studies indicate that Graves’ disease is associated with certain human leukocyte antigen (HLA) hap-lotypes, including HLA-B8, HLA-DR3, and HLADQA1*0501 in Caucasian patients, whereas HLA-DRB1*0701 is protective Brunicardi_Ch38_p1625-p1704.indd 163401/03/19 11:20 AM 1635THYROID, PARATHYROID, AND ADRENALCHAPTER 38against it. Polymorphisms of the cytotoxic T-lymphocyte anti-gen 4 (CTLA-4) gene also have been associated with Graves’ disease development. CD40 has also been recognized as a Graves’ susceptibility gene. It has an important role in B-cell function and its upregulation leads to a lower threshold for B-cell activation. It can also lead to enhanced IL-6 secretion and activation of T-cells in

1	gene. It has an important role in B-cell function and its upregulation leads to a lower threshold for B-cell activation. It can also lead to enhanced IL-6 secretion and activation of T-cells in thyrocytes leading to a local inflam-matory response. Other susceptibility genes include PTPN22 (encodes the lymphoid tyrosine phosphatase) and CD25, which encodes for the interleukin-2 receptor α-chain (IL-2Rα).3 Once initiated, the inflammatory process causes sensitized T-helper lymphocytes to stimulate B lymphocytes, which produce anti-bodies directed against the thyroid hormone receptor. TSIs or antibodies that stimulate the TSH-R, as well as TSH-binding inhibiting immunoglobulins or antibodies, have been described. The thyroid-stimulating antibodies stimulate the thyrocytes to grow and synthesize excess thyroid hormone, which is a hall-mark of Graves’ disease. Graves’ disease also is associated with other autoimmune conditions such as type 1 diabetes mellitus, Addison’s disease, pernicious

1	excess thyroid hormone, which is a hall-mark of Graves’ disease. Graves’ disease also is associated with other autoimmune conditions such as type 1 diabetes mellitus, Addison’s disease, pernicious anemia, and myasthenia gravis.Macroscopically, the thyroid gland in patients with Graves’ disease is diffusely and smoothly enlarged, with a concomitant increase in vascularity. Microscopically, the gland is hyperplastic, and the epithelium is columnar with minimal colloid present. The nuclei exhibit mitosis, and papillary pro-jections of hyperplastic epithelium are common. There may be aggregates of lymphoid tissue, and vascularity is markedly increased.Clinical Features. The clinical manifestations of Graves’ disease can be divided into those related to hyperthyroidism and those specific to Graves’ disease. Hyperthyroid symp-toms include heat intolerance, increased sweating and thirst, and weight loss despite adequate caloric intake. Symptoms of increased adrenergic stimulation include

1	Graves’ disease. Hyperthyroid symp-toms include heat intolerance, increased sweating and thirst, and weight loss despite adequate caloric intake. Symptoms of increased adrenergic stimulation include palpitations, nervous-ness, fatigue, emotional lability, hyperkinesis, and tremors. The most common GI symptoms include increased frequency of bowel movements and diarrhea. Female patients often develop amenorrhea, decreased fertility, and an increased incidence of miscarriages. Children experience rapid growth with early bone maturation, whereas older patients may present with cardiovas-cular complications such as atrial fibrillation and congestive heart failure.On physical examination, weight loss and facial flush-ing may be evident. The skin is warm and moist, and African American patients often note darkening of their skin. Tachycar-dia or atrial fibrillation is present, with cutaneous vasodilation leading to a widening of the pulse pressure and a rapid falloff in the transmitted pulse

1	note darkening of their skin. Tachycar-dia or atrial fibrillation is present, with cutaneous vasodilation leading to a widening of the pulse pressure and a rapid falloff in the transmitted pulse wave (collapsing pulse). A fine tremor, muscle wasting, and proximal muscle group weakness with hyperactive tendon reflexes often are present.Approximately 50% of patients with Graves’ disease also develop clinically evident ophthalmopathy, and dermopathy occurs in 1% to 2% of patients. It is characterized by deposi-tion of glycosaminoglycans, leading to thickened skin in the pretibial region and dorsum of the foot (Fig. 38-12). Eye symp-toms include lid lag (von Graefe’s sign), spasm of the upper eyelid revealing the sclera above the corneoscleral limbus (Dal-rymple’s sign), and a prominent stare, due to catecholamine excess. True infiltrative eye disease results in periorbital edema, conjunctival swelling and congestion (chemosis), proptosis, limitation of upward and lateral gaze (from

1	stare, due to catecholamine excess. True infiltrative eye disease results in periorbital edema, conjunctival swelling and congestion (chemosis), proptosis, limitation of upward and lateral gaze (from involvement of the inferior and medial rectus muscles, respectively), keratitis, and even blindness due to optic nerve involvement. The etiology of Graves’ ophthalmopathy is not completely known; however, Figure 38-11. Thyroid ultrasound showing a lymph node (arrow) along the carotid artery.Table 38-1Differential diagnosis of hyperthyroidismINCREASED HORMONE SYNTHESIS (INCREASED RAIU)RELEASE OF PREFORMED HORMONE (DECREASED RAIU)Graves’ disease (diffuse toxic goiter)Toxic multinodular goiterToxic adenomaDrug induced—amiodarone, iodineThyroid cancerStruma ovariiHydatidiform moleTSH-secreting pituitary adenomaThyroiditis—acute phase of Hashimoto’s thyroiditis, subacute thyroiditisFactitious (iatrogenic) thyrotoxicosis“Hamburger thyrotoxicosis”RAIU = radioactive iodine uptake; TSH =

1	pituitary adenomaThyroiditis—acute phase of Hashimoto’s thyroiditis, subacute thyroiditisFactitious (iatrogenic) thyrotoxicosis“Hamburger thyrotoxicosis”RAIU = radioactive iodine uptake; TSH = thyroid-stimulating hormone.Brunicardi_Ch38_p1625-p1704.indd 163501/03/19 11:20 AM 1636SPECIFIC CONSIDERATIONSPART IIorbital fibroblasts and muscles are thought to share a common antigen, the TSH-R. Ophthalmopathy is thought to result from inflammation caused by cytokines released from sensitized killer T lymphocytes and cytotoxic antibodies. Gynecomas-tia is common in young men. Rare bony involvement leads to subperiosteal bone formation and swelling in the metacarpals (thyroid acropachy). Onycholysis, or separation of fingernails from their beds, is a commonly observed finding. On physical examination, the thyroid usually is diffusely and symmetrically enlarged, as evidenced by an enlarged pyramidal lobe. There may be an overlying bruit or thrill over the thyroid gland and a loud venous

1	the thyroid usually is diffusely and symmetrically enlarged, as evidenced by an enlarged pyramidal lobe. There may be an overlying bruit or thrill over the thyroid gland and a loud venous hum in the supraclavicular space.Diagnostic Tests. The diagnosis of hyperthyroidism is made by a suppressed TSH with or without an elevated free T4 or T3 level. If eye signs are present, other tests are generally not needed. However, in the absence of eye findings, an 123I uptake and scan should be performed. An elevated uptake, with a dif-fusely enlarged gland, confirms the diagnosis of Graves’ disease and helps to differentiate it from other causes of hyperthyroid-ism. Technetium scintigraphy (using pertechnetate, which is trapped by the thyroid, but not organified) can also be used to determine etiology. While technetium scans result in low range of normal uptake and high background activity, total-body radi-ation exposure is less than that of 123I scans. If free T4 levels are normal, free T3

1	While technetium scans result in low range of normal uptake and high background activity, total-body radi-ation exposure is less than that of 123I scans. If free T4 levels are normal, free T3 levels should be determined, as they often are elevated in early Graves’ disease or toxic nodules (T3 toxi-cosis). Anti-Tg and anti-TPO antibodies are elevated in up to 75% of patients but are not specific. Elevated TSH-R or thyroidstimulating antibodies (TSAb) are diagnostic of Graves’ disease and are increased in about 90% of patients. CT or MRI scans of the orbits are useful in evaluating Graves’ ophthalmopathy.Treatment. Graves’ disease may be treated by any of three treatment modalities: antithyroid drugs, thyroid ablation with radioactive 131I, and thyroidectomy. The choice of treatment depends on several factors, as discussed in the following sections.Antithyroid Drugs Antithyroid medications generally are administered in preparation for RAI ablation or surgery. The drugs commonly used are

1	on several factors, as discussed in the following sections.Antithyroid Drugs Antithyroid medications generally are administered in preparation for RAI ablation or surgery. The drugs commonly used are propylthiouracil (PTU, 100 to 300 mg three times daily) and methimazole (10 to 30 mg three times daily, then once daily). Methimazole has a longer half-life and can be dosed once daily. Both drugs reduce thyroid hormone production by inhibiting the organic binding of iodine and the coupling of iodotyrosines (mediated by TPO). In addition, PTU also inhibits the peripheral conversion of T4 to T3, making it useful for the treatment of thyroid storm. Both drugs can cross the placenta, inhibiting fetal thyroid function, and are excreted in breast milk, although PTU has a lower risk of transplacental transfer. Methimazole also has been associated with congeni-tal aplasia; therefore, PTU is preferred in pregnant and breast-feeding women. Side effects of treatment include reversible

1	transplacental transfer. Methimazole also has been associated with congeni-tal aplasia; therefore, PTU is preferred in pregnant and breast-feeding women. Side effects of treatment include reversible granulocytopenia, skin rashes, fever, peripheral neuritis, poly-arteritis, vasculitis, hepatitis, and, rarely, agranulocytosis and aplastic anemia. Patients should be monitored for these pos-sible complications and should always be warned to stop PTU or methimazole immediately and seek medical advice should they develop a sore throat or fever. Treatment of agranulocy-tosis involves admission to the hospital, discontinuation of the drug, and broad-spectrum antibiotic therapy. Surgery should be postponed until the granulocyte count reaches 1000 cells/mm3.The dose of antithyroid medication is titrated as needed in accordance with TSH and T4 levels. Most patients have improved symptoms in 2 weeks and become euthyroid in about 6 weeks. Some physicians use the block-replace regimen, by adding T4

1	as needed in accordance with TSH and T4 levels. Most patients have improved symptoms in 2 weeks and become euthyroid in about 6 weeks. Some physicians use the block-replace regimen, by adding T4 (0.05 to 0.10 mg) to prevent hypothyroidism and suppress TSH secretion, because some, but not all, studies sug-gest that this reduces recurrence rates. The length of therapy is debated. Treatment with antithyroid medications is associated ABFigure 38-12. A. Graves’ ophthalmopathy and (B) pretibial myx-edema. This patient demonstrates exophthalmos, proptosis, perior-bital swelling, congestion, and edema of the conjunctiva.Brunicardi_Ch38_p1625-p1704.indd 163601/03/19 11:20 AM 1637THYROID, PARATHYROID, AND ADRENALCHAPTER 38with a high relapse rate when these drugs are discontinued, with 40% to 80% of patients developing recurrent disease after a 1to 2-year course. Patients with small glands are less likely to recur, so that treatment for curative intent is reserved for patients with (a)

1	to 80% of patients developing recurrent disease after a 1to 2-year course. Patients with small glands are less likely to recur, so that treatment for curative intent is reserved for patients with (a) small, nontoxic goiters less than 40 g; (b) mildly elevated thyroid hormone levels; (c) negative or low or titers of thyroid hormone receptor antibodies; and (d) rapid decrease in gland size with antithyroid medications. The catecholamine response of thyrotoxicosis can be alleviated by administering β-blocking agents. β-Blockade should be considered in all patients with symptomatic thyrotoxicosis and is recommended for elderly patients, those with coexistent cardiac disease, and patients with resting heart rates >90 bpm. These drugs have the added effect of decreasing the peripheral conversion of T4 to T3. Pro-pranolol is the most commonly prescribed medication in doses of about 20 to 40 mg four times daily. Higher doses are some-times required due to increased clearance of the

1	conversion of T4 to T3. Pro-pranolol is the most commonly prescribed medication in doses of about 20 to 40 mg four times daily. Higher doses are some-times required due to increased clearance of the medication. Caution should be exercised in patients with asthma. Calcium channel blockers are useful for rate control in patients in whom β-blockers are contraindicated.Radioactive Iodine Therapy (131I) RAI forms the mainstay of Graves’ disease treatment in North America. The major advan-tages of this treatment are the avoidance of a surgical procedure and its concomitant risks, reduced overall treatment costs, and ease of treatment. Antithyroid drugs are given until the patient is euthyroid and then discontinued to maximize drug uptake. The 131I dose is calculated after a preliminary scan and usu-ally consists of 8 to 12 mCi administered orally. After standard treatment with RAI, most patients become euthyroid within 2 months. However, only about 50% of patients treated with RAI are

1	and usu-ally consists of 8 to 12 mCi administered orally. After standard treatment with RAI, most patients become euthyroid within 2 months. However, only about 50% of patients treated with RAI are euthyroid 6 months after treatment, and the remain-ing are still hyperthyroid or already hypothyroid.4 After 1 year, about 2.5% of patients develop hypothyroidism each year. RAI also has been documented to lead to progression of Graves’ oph-thalmopathy (33% after RAI compared to 16% after surgery), and ophthalmopathy is more common in smokers. Although there is no evidence of long-term problems with infertility, and overall cancer incidence rates are unchanged, there is a small increased risk of nodular goiter, thyroid cancer,5 and hyperpara-thyroidism (HPT)6 in patients who have been treated with RAI. Patients treated with RAI have an unexplained increase in their overall and cardiovascular mortality rates when compared to the general population.RAI therapy is therefore most often used in

1	RAI. Patients treated with RAI have an unexplained increase in their overall and cardiovascular mortality rates when compared to the general population.RAI therapy is therefore most often used in older patients with small or moderate-sized goiters, those who have relapsed after medical or surgical therapy, and those in whom antithyroid drugs or surgery are contraindicated. Absolute contraindications to RAI include women who are pregnant (or planning pregnancy within 6 months of treatment) or breastfeeding. Relative contra-indications include young patients (i.e., especially children and adolescents), those with thyroid nodules, and those with oph-thalmopathy. Lack of access to a high-volume thyroid surgeon is also a consideration. The higher the initial dose of 131I, the earlier the onset and the higher the incidence of hypothyroidism.Surgical Treatment In North America, surgery is recom-mended when RAI is contraindicated as in patients who (a) have confirmed cancer or suspicious

1	and the higher the incidence of hypothyroidism.Surgical Treatment In North America, surgery is recom-mended when RAI is contraindicated as in patients who (a) have confirmed cancer or suspicious thyroid nodules, (b) are young, (c) desire to conceive soon (<6 months) after treatment, (d) have had severe reactions to antithyroid medications, (e) have large goiters (>80 g) causing compressive symptoms, and (f) are reluctant to undergo RAI therapy. Relative indications for thyroidectomy include patients, particularly smokers, with mod-erate to severe Graves’ ophthalmopathy, those desiring rapid control of hyperthyroidism with a chance of being euthyroid, and those demonstrating poor compliance to antithyroid medica-tions. Pregnancy is also a relative contraindication, and surgery should be used only when rapid control is needed and antithy-roid medications cannot be used. Surgery is best performed in the second trimester. The goal of thyroidectomy for Graves’ disease should be the

1	be used only when rapid control is needed and antithy-roid medications cannot be used. Surgery is best performed in the second trimester. The goal of thyroidectomy for Graves’ disease should be the complete and permanent control of the disease with minimal morbidity. Patients should be rendered euthyroid before operation with antithyroid drugs that should be continued up to the day of surgery. Lugol’s iodide solution or saturated potassium iodide generally is administered beginning 7 to 10 days preoperatively (three drops twice daily) to reduce vascularity of the gland and decrease the risk of precipitating thyroid storm. The major action of iodine in this situation is to inhibit release of thyroid hormone. If it is not possible to render the patient euthyroid prior to surgery (if the surgery is urgent or the patient is allergic to antithyroid medications), the patient can be prepared with β-blockade and potassium iodide alone. Steroids can be a useful adjunct in this situation.The

1	is urgent or the patient is allergic to antithyroid medications), the patient can be prepared with β-blockade and potassium iodide alone. Steroids can be a useful adjunct in this situation.The extent of thyroidectomy to be performed used to be determined by the desired outcome (risk of recurrence vs. euthy-roidism) and surgeon experience. In patients with coexistent thyroid cancer and those who refused RAI therapy or had severe ophthalmopathy or life-threatening reactions to antithyroid medications (vasculitis, agranulocytosis, or liver failure), total or near-total thyroidectomy was recommended. Ophthalmopa-thy has been demonstrated to stabilize or improve in most patients after total thyroidectomy, presumably from removal of the antigenic stimulus. A subtotal thyroidectomy, leaving a 4to 7-g remnant, was recommended for all remaining patients. Dur-ing subtotal thyroidectomy, remnant tissue may be left on each side (bilateral subtotal thyroidectomy), or a total lobectomy can be

1	a 4to 7-g remnant, was recommended for all remaining patients. Dur-ing subtotal thyroidectomy, remnant tissue may be left on each side (bilateral subtotal thyroidectomy), or a total lobectomy can be performed on one side with a subtotal thyroidectomy on the other side (Hartley-Dunhill procedure). Results were similar with either procedure, but the latter procedure was theoretically associated with fewer complications and requires reentering only one side of the neck should recurrence require reoperation. Most studies, however, show no difference in the rates of com-plications with either approach, although patients undergoing a total resection had higher rates of temporary hypoparathyroid-ism. However, patients treated with subtotal thyroidectomy are prone to recurrence, the rates of which are dependent on rem-nant size. Based on the current evidence, recently revised guide-lines from the American Thyroid Association (ATA) recommend total or near-total thyroidectomy as the procedure

1	are dependent on rem-nant size. Based on the current evidence, recently revised guide-lines from the American Thyroid Association (ATA) recommend total or near-total thyroidectomy as the procedure of choice for the surgical management of Graves’ disease.7 Recurrent thyrotoxicosis usually is managed by radioiodine treatment.Toxic Multinodular Goiter Toxic multinodular goiters usu-ally occur in older individuals, who often have a prior history of a nontoxic multinodular goiter. Over several years, enough thyroid nodules become autonomous to cause hyperthyroidism. The presentation often is insidious in that hyperthyroidism may only become apparent when patients are placed on low doses of thyroid hormone suppression for the goiter. Some patients have T3 toxicosis, whereas others may present only with atrial fibrillation or congestive heart failure. Hyperthyroidism also can be precipitated by iodide-containing drugs such as contrast media and the antiarrhythmic agent amiodarone

1	present only with atrial fibrillation or congestive heart failure. Hyperthyroidism also can be precipitated by iodide-containing drugs such as contrast media and the antiarrhythmic agent amiodarone (Jod-Basedow 1Brunicardi_Ch38_p1625-p1704.indd 163701/03/19 11:20 AM 1638SPECIFIC CONSIDERATIONSPART IIhyperthyroidism). Symptoms and signs of hyperthyroidism are similar to Graves’ disease, but extrathyroidal manifestations are absent.Diagnostic Studies. Blood tests are similar to Graves’ disease with a suppressed TSH level and elevated free T4 or T3 levels. RAI uptake also is increased, showing multiple nodules with increased uptake and suppression of the remaining gland.Treatment. Hyperthyroidism must be adequately controlled. Both RAI and surgical resection may be used for treatment. When surgery is performed, near-total or total thyroidectomy is recommended to avoid recurrence and the consequent increased complication rates with repeat surgery. Care must be taken in identifying the

1	surgery is performed, near-total or total thyroidectomy is recommended to avoid recurrence and the consequent increased complication rates with repeat surgery. Care must be taken in identifying the RLN, which may be found laterally on the thy-roid (rather than posterior) or stretched anteriorly over a nodule. RAI therapy is reserved for elderly patients who represent very poor operative risks, provided there is no airway compression from the goiter and thyroid cancer is not a concern. However, because uptake is less than in Graves’ disease, larger doses of RAI often are needed to treat the hyperthyroidism. Furthermore, RAI-induced thyroiditis has the potential to cause swelling and acute airway compromise and leaves the goiter intact, with the possibility of recurrent hyperthyroidism.Toxic Adenoma Hyperthyroidism from a single hyperfunction-ing nodule typically occurs in younger patients who note recent growth of a long-standing nodule along with the symptoms of hyperthyroidism. Toxic

1	Adenoma Hyperthyroidism from a single hyperfunction-ing nodule typically occurs in younger patients who note recent growth of a long-standing nodule along with the symptoms of hyperthyroidism. Toxic adenomas are characterized by somatic mutations in the TSH-R gene, although G-protein–stimulating gene (gsp) mutations may occur also.8 Most hyperfunctioning or autonomous thyroid nodules have attained a size of at least 3 cm before hyperthyroidism occurs. Physical examination usu-ally reveals a solitary thyroid nodule without palpable thyroid tissue on the contralateral side. RAI scanning shows a “hot” nodule with suppression of the rest of the thyroid gland. These nodules are rarely malignant. Smaller nodules may be man-aged with antithyroid medications and RAI. Larger nodules can require higher doses, which can lead to hypothyroidism. Sur-gery (lobectomy and isthmusectomy) is preferred to treat young patients and those with larger nodules. Percutaneous ethanol injection (PEI) has been

1	doses, which can lead to hypothyroidism. Sur-gery (lobectomy and isthmusectomy) is preferred to treat young patients and those with larger nodules. Percutaneous ethanol injection (PEI) has been reported to have reasonable success rates but has not been directly compared with surgery.Thyroid Storm Thyroid storm is a condition of hyperthyroid-ism accompanied by fever, central nervous system agitation or depression, and cardiovascular and GI dysfunction, including hepatic failure. The condition may be precipitated by abrupt cessation of antithyroid medications, infection, thyroid or non-thyroid surgery, and trauma in patients with untreated thyrotoxi-cosis. Occasionally, thyroid storm may result from amiodarone administration or exposure to iodinated contrast agents or fol-lowing RAI therapy. This condition was previously associated with high mortality rates but can be appropriately managed in an intensive care unit setting. β-Blockers are given to reduce peripheral T4 to T3 conversion

1	This condition was previously associated with high mortality rates but can be appropriately managed in an intensive care unit setting. β-Blockers are given to reduce peripheral T4 to T3 conversion and decrease the hyperthyroid symptoms. Oxygen supplementation and hemodynamic support should be instituted. Nonaspirin compounds can be used to treat pyrexia, and Lugol’s iodine or sodium ipodate (intravenously) should be administered to decrease iodine uptake and thyroid hormone secretion. PTU therapy blocks the formation of new thyroid hormone and reduces peripheral conversion of T4 to T3. Corticosteroids often are helpful to prevent adrenal exhaustion and block hepatic thyroid hormone conversion.Hypothyroidism. Deficiency in circulating levels of thyroid hormone leads to hypothyroidism and, in neonates, to cretin-ism, which is characterized by neurologic impairment and men-tal retardation. Hypothyroidism also may occur in Pendred’s syndrome (associated with deafness) and Turner’s

1	and, in neonates, to cretin-ism, which is characterized by neurologic impairment and men-tal retardation. Hypothyroidism also may occur in Pendred’s syndrome (associated with deafness) and Turner’s syndrome. Conditions that cause hypothyroidism are listed in Table 38-2.Clinical Features Failure of thyroid gland development or function in utero leads to cretinism and characteristic facies similar to those of children with Down syndrome and dwarfism. Failure to thrive and severe mental retardation often are present. Immediate testing and treatment with thyroid hormone at birth can lessen the neurologic and intellectual deficits. Hypothyroid-ism developing in childhood or adolescence results in delayed development and may also lead to abdominal distention, umbili-cal hernia, and rectal prolapse. In adults, symptoms in general are nonspecific, including tiredness, weight gain, cold intoler-ance, constipation, and menorrhagia. Patients with severe hypo-thyroidism or myxedema develop

1	prolapse. In adults, symptoms in general are nonspecific, including tiredness, weight gain, cold intoler-ance, constipation, and menorrhagia. Patients with severe hypo-thyroidism or myxedema develop characteristic facial features due to the deposition of glycosaminoglycans in the subcutane-ous tissues, leading to facial and periorbital puffiness. The skin becomes rough and dry and often develops a yellowish hue from reduced conversion of carotene to vitamin A. Hair becomes dry and brittle, and severe hair loss may occur. There is also a characteristic loss of the outer two thirds of the eyebrows. An enlarged tongue may impair speech, which is already slowed, in keeping with the impairment of mental processes. Patients may also have nonspecific abdominal pain accompanied by disten-tion and constipation. Libido and fertility are impaired in both sexes. Cardiovascular changes in hypothyroidism include bra-dycardia, cardiomegaly, pericardial effusion, reduced cardiac output, and pulmonary

1	constipation. Libido and fertility are impaired in both sexes. Cardiovascular changes in hypothyroidism include bra-dycardia, cardiomegaly, pericardial effusion, reduced cardiac output, and pulmonary effusions. When hypothyroidism occurs as a result of pituitary failure, other features of hypopituitarism, such as pale, waxy skin; loss of body hair; and atrophic genita-lia, may be present.Table 38-2Causes of hypothyroidismPRIMARY (INCREASED TSH LEVELS)SECONDARY (DECREASED TSH LEVELS)TERTIARYHashimoto’s thyroiditisPituitary tumorHypothalamic insufficiencyRAI therapy for Graves’ diseasePituitary resection or ablationResistance to thyroid hormonePostthyroidectomy Excessive iodine intake Subacute thyroiditis Medications: antithyroid drugs, lithium Rare: iodine deficiency, dyshormogenesis RAI = radioactive iodine; TSH = thyroid-stimulating hormone.Brunicardi_Ch38_p1625-p1704.indd 163801/03/19 11:20 AM 1639THYROID, PARATHYROID, AND ADRENALCHAPTER 38Laboratory Findings Hypothyroidism

1	= radioactive iodine; TSH = thyroid-stimulating hormone.Brunicardi_Ch38_p1625-p1704.indd 163801/03/19 11:20 AM 1639THYROID, PARATHYROID, AND ADRENALCHAPTER 38Laboratory Findings Hypothyroidism is characterized by low circulating levels of T4 and T3. Raised TSH levels are found in primary thyroid failure, whereas secondary hypothyroidism is characterized by low TSH levels that do not increase following TRH stimulation. Thyroid autoantibodies are highest in patients with autoimmune disease (Hashimoto’s thyroiditis, Graves’ dis-ease) and may also be elevated in patients with nodular goiter and thyroid neoplasms. An electrocardiogram demonstrates decreased voltage with flattening or inversion of T waves.Treatment T4 is the treatment of choice and is administered in dosages varying from 50 to 200 μg per day, depending on the patient’s size and condition. Starting doses of 100 μg of T4 daily are well tolerated; however, elderly patients and those with coexisting heart disease and

1	50 to 200 μg per day, depending on the patient’s size and condition. Starting doses of 100 μg of T4 daily are well tolerated; however, elderly patients and those with coexisting heart disease and profound hypothyroidism should be started on a considerably lower dose such as 25 to 50 μg daily because of associated hypercholesterolemia and atherosclerosis. The dose can be slowly increased over weeks to months to attain a euthyroid state. A baseline electrocardiogram should always be obtained in patients with severe hypothyroidism before treat-ment. T4 dosage is titrated against clinical response and TSH levels, which should return to normal. The management of patients with subclinical hypothyroidism (normal T4, slightly raised TSH) is controversial. Some evidence suggests that patients with subclinical hypothyroidism and increased antithy-roid antibody levels should be treated because they will sub-sequently develop hypothyroidism. Patients who present with myxedema coma may require

1	subclinical hypothyroidism and increased antithy-roid antibody levels should be treated because they will sub-sequently develop hypothyroidism. Patients who present with myxedema coma may require initial emergency treatment with large doses of IV T4 (300 to 400 μg), with careful monitoring in an intensive care unit setting.9Thyroiditis. Thyroiditis usually is classified into acute, sub-acute, and chronic forms, each associated with a distinct clinical presentation and histology.Acute (Suppurative) Thyroiditis The thyroid gland is inherently resistant to infection due to its extensive blood and lymphatic supply, high iodide content, and fibrous capsule. However, infectious agents can seed it (a) via the hematoge-nous or lymphatic route, (b) via direct spread from persistent pyriform sinus fistulae or thyroglossal duct cysts, (c) as a result of penetrating trauma to the thyroid gland, or (d) due to immu-nosuppression. Streptococcus and anaerobes account for about 70% of cases; however,

1	fistulae or thyroglossal duct cysts, (c) as a result of penetrating trauma to the thyroid gland, or (d) due to immu-nosuppression. Streptococcus and anaerobes account for about 70% of cases; however, other species also have been cultured.10 Acute suppurative thyroiditis is more common in children and often is preceded by an upper respiratory tract infection or otitis media. It is characterized by severe neck pain radiating to the jaws or ear, fever, chills, odynophagia, and dysphonia. Compli-cations such as systemic sepsis, tracheal or esophageal rupture, jugular vein thrombosis, laryngeal chondritis, and perichondritis or sympathetic trunk paralysis may also occur.The diagnosis is established by leukocytosis on blood tests and FNAB for Gram’s stain, culture, and cytology. CT scans may help to delineate the extent of infection and identify abscesses. A persistent pyriform sinus fistula should always be suspected in children with recurrent acute thyroiditis. The sensitivity of

1	may help to delineate the extent of infection and identify abscesses. A persistent pyriform sinus fistula should always be suspected in children with recurrent acute thyroiditis. The sensitivity of identification of fistulae in the acute setting is lowest for barium esophagography (50%) and best for direct endoscopy (100%), with CT scans being intermediate (80%). Both barium esopha-gogram and CT scans have improved sensitivity once the acute inflammation has resolved (100% and 83%, respectively), with CT being better at defining the accurate anatomic pathway and its relationship to the thyroid gland.11 Treatment consists of parenteral antibiotics and drainage of abscesses. Thyroidec-tomy may be needed for persistent abscesses or failure of open drainage. Patients with pyriform sinus fistulae require complete resection of the sinus tract, including the area of the thyroid where the tract terminates, to prevent recurrence. Transnasal flexible fiberoptic laryngoscopy is being

1	sinus fistulae require complete resection of the sinus tract, including the area of the thyroid where the tract terminates, to prevent recurrence. Transnasal flexible fiberoptic laryngoscopy is being increasingly used to identify the internal opening of the pyriform sinus tract and may also allow electrocauterization of the tract, and success rates similar to open surgery have been reported.Subacute Thyroiditis Subacute thyroiditis can occur in the painful or painless forms. Although the exact etiology is not known, painful thyroiditis is thought to be viral in origin or result from a postviral inflammatory response. Genetic pre-disposition may also play a role, as manifested by its strong association with the HLA-B35 haplotype. One model of patho-genesis suggests that viral or thyroid antigens, when presented by macrophages in the context of HLA-B35, stimulate cytotoxic T lymphocytes and damage thyroid follicular cells.Painful thyroiditis most commonly occurs in 30to 40-year-old

1	antigens, when presented by macrophages in the context of HLA-B35, stimulate cytotoxic T lymphocytes and damage thyroid follicular cells.Painful thyroiditis most commonly occurs in 30to 40-year-old women and is characterized by the sudden or grad-ual onset of neck pain, which may radiate toward the mandible or ear. History of a preceding upper respiratory tract infection often can be elicited. The gland is enlarged, exquisitely ten-der, and firm. The disorder classically progresses through four stages. An initial hyperthyroid phase, due to release of thyroid hormone, is followed by a second, euthyroid phase. The third phase, hypothyroidism, occurs in about 20% to 30% of patients and is followed by resolution and return to the euthyroid state in >90% of patients. A few patients develop recurrent disease.In the early stages of the disease, TSH is decreased, and Tg, T4, and T3 levels are elevated due to the release of pre-formed thyroid hormone from destroyed follicles. The erythro-cyte

1	disease.In the early stages of the disease, TSH is decreased, and Tg, T4, and T3 levels are elevated due to the release of pre-formed thyroid hormone from destroyed follicles. The erythro-cyte sedimentation rate is typically >100 mm/h. RAIU also is decreased (<2% at 24 hours), even in euthyroid patients, due to the release of thyroid hormones from destruction of the thyroid parenchyma. Painful thyroiditis is self-limited, and therefore, treatment is primarily symptomatic. Aspirin and other nonste-roidal anti-inflammatory drugs are used for pain relief, but ste-roids may be indicated in more severe cases. Short-term thyroid replacement may be needed and may shorten the duration of symptoms. Thyroidectomy is reserved for the rare patient who has a prolonged course not responsive to medical measures or for recurrent disease.Painless thyroiditis is considered to be autoimmune in ori-gin and may occur sporadically or in the postpartum period; the latter typically occurs at about 6 weeks

1	measures or for recurrent disease.Painless thyroiditis is considered to be autoimmune in ori-gin and may occur sporadically or in the postpartum period; the latter typically occurs at about 6 weeks after delivery in women with high TPO antibody titers in early pregnancy. This timing is thought to coincide with a decrease in the normal immune tolerance of pregnancy and consequent rebound elevation of antibody titers.Painless thyroiditis also is more common in women and usually occurs between 30 and 60 years of age. Physical exami-nation demonstrates a normal sized or minimally enlarged, slightly firm, nontender gland. Laboratory tests and RAIU are similar to those in painful thyroiditis, except for a normal erythrocyte sedimentation rate. The clinical course also paral-lels painful thyroiditis. Patients with symptoms may require β-blockers and thyroid hormone replacement. Thyroidectomy or RAI ablation is only indicated for the rare patient with recur-rent, disabling episodes of

1	thyroiditis. Patients with symptoms may require β-blockers and thyroid hormone replacement. Thyroidectomy or RAI ablation is only indicated for the rare patient with recur-rent, disabling episodes of thyroiditis.Brunicardi_Ch38_p1625-p1704.indd 163901/03/19 11:20 AM 1640SPECIFIC CONSIDERATIONSPART IIChronic Thyroiditis Lymphocytic (Hashimoto’s) Thyroiditis. Lymphocytic thy-roiditis was first described by Hashimoto in 1912 as struma lymphomatosa—a transformation of thyroid tissue to lymphoid tissue. It is the most common inflammatory disorder of the thy-roid and the leading cause of hypothyroidism today.Etiology, Pathogenesis, and Pathology Hashimoto’s thyroid-itis is an autoimmune process that is thought to be initiated by the activation of CD4+ T (helper) lymphocytes with specificity for thyroid antigens. Once activated, T cells can recruit cyto-toxic CD8+ T cells to the thyroid. Hypothyroidism results not only from the destruction of thyrocytes by cytotoxic T cells but also by

1	for thyroid antigens. Once activated, T cells can recruit cyto-toxic CD8+ T cells to the thyroid. Hypothyroidism results not only from the destruction of thyrocytes by cytotoxic T cells but also by autoantibodies, which lead to complement fixation and killing by natural killer cells or block the TSH-R. Antibod-ies are directed against three main antigens—Tg (60%), TPO (95%), and TSH-R (60%)—and, less commonly, the sodium/iodine symporter (25%). Apoptosis (programmed cell death) also has been implicated in the pathogenesis of Hashimoto’s thyroiditis. Chronic thyroiditis also has been associated with increased intake of iodine and administration of medications such as interferon-α, lithium, and amiodarone. Support for an inherited predisposition includes an increased incidence of thyroid autoantibodies in first-degree relatives of patients with Hashimoto’s thyroiditis compared to controls and the occur-rence of the autoantibodies and hypothyroidism in patients with specific chromosomal

1	in first-degree relatives of patients with Hashimoto’s thyroiditis compared to controls and the occur-rence of the autoantibodies and hypothyroidism in patients with specific chromosomal abnormalities such as Turner’s syn-drome and Down syndrome. Associations with HLA-B8, DR3, and DR5 haplotypes of the major histocompatibility complex also have been described. Alterations in CTLA4 have also been shown to increase the risk of developing Hashimoto’s thyroid-itis. Other associated genes include various cytokine genes, GITR (glucocorticoid-induced tumor necrosis factor-receptor) and STAT 3; however, these need further confirmatory studies.11On gross examination, the thyroid gland is usually mildly enlarged throughout and has a pale, gray-tan cut surface that is granular, nodular, and firm. On microscopic examination, the gland is diffusely infiltrated by small lymphocytes and plasma cells and occasionally shows well-developed germinal centers. Thyroid follicles are smaller than normal

1	On microscopic examination, the gland is diffusely infiltrated by small lymphocytes and plasma cells and occasionally shows well-developed germinal centers. Thyroid follicles are smaller than normal with reduced amounts of colloid and increased interstitial connective tissue. The fol-licles are lined by Hürthle or Askanazy cells, which are charac-terized by abundant eosinophilic, granular cytoplasm.Clinical Presentation Hashimoto’s thyroiditis is also more common in women (male-to-female ratio is 1:10 to 20) between the ages of 30 and 50 years old. The most common presenta-tion is that of a minimally or moderately enlarged firm granular gland discovered on routine physical examination or the aware-ness of a painless anterior neck mass, although 20% of patients present with hypothyroidism, and 5% present with hyperthy-roidism (Hashitoxicosis). In classic goitrous Hashimoto’s thy-roiditis, physical examination reveals a diffusely enlarged, firm gland, which also is lobulated. An

1	and 5% present with hyperthy-roidism (Hashitoxicosis). In classic goitrous Hashimoto’s thy-roiditis, physical examination reveals a diffusely enlarged, firm gland, which also is lobulated. An enlarged pyramidal lobe often is palpable.Diagnostic Studies When Hashimoto’s thyroiditis is suspected clinically, an elevated TSH and the presence of thyroid autoan-tibodies usually confirm the diagnosis. FNAB with ultrasound guidance is indicated in patients who present with a solitary suspicious nodule or a rapidly enlarging goiter. Thyroid lym-phoma is a rare but well-recognized, ominous complication of chronic autoimmune thyroiditis and has a prevalence 80 times higher than expected frequency in this population than in a control population without thyroiditis. Studies of clonal simi-larity indicate that lymphoma may, in fact, evolve from Hashi-moto’s thyroiditis.12Treatment Thyroid hormone replacement therapy is indicated in overtly hypothyroid patients, with a goal of maintaining nor-mal

1	that lymphoma may, in fact, evolve from Hashi-moto’s thyroiditis.12Treatment Thyroid hormone replacement therapy is indicated in overtly hypothyroid patients, with a goal of maintaining nor-mal TSH levels. The management of patients with subclinical hypothyroidism (normal T4 and elevated TSH) is controversial. A systematic review of cohort studies showed that in ageand sex-adjusted analyses, subclinical hypothyroidism is associated with a hazard ratio (HR) for coronary heart disease events of 1.89 (95% confidence interval [CI], 1.28 to 2.80; P <.001) and coronary heart disease mortality of 1.58 (95% CI, 1.10 to 2.27; P = .005) for a TSH level of 10 to 19.9 mIU/L.13 The data for TSH levels of 5 to 10 mIU/L were less convincing. An evalu-ation of the 12 randomized controlled trials in this area only showed a trend toward improvement of some lipid parameters, and none of the included trials evaluated overall mortality or car-diac morbidity. For this reason, levothyroxine is recommended

1	area only showed a trend toward improvement of some lipid parameters, and none of the included trials evaluated overall mortality or car-diac morbidity. For this reason, levothyroxine is recommended for all patients with TSH levels >10 μIU/mL and patients with levels of 5 to 10 μIU/mL in the presence of a goiter or anti-TPO antibodies. Treatment is also advised especially for middle-aged patients with cardiovascular risk factors such as hyperlipidemia or hypertension and in pregnant patients. Surgery may occa-sionally be indicated for suspicion of malignancy or for goiters causing compressive symptoms or cosmetic deformity.Riedel’s Thyroiditis Riedel’s thyroiditis is a rare variant of thyroiditis also known as Riedel’s struma or invasive fibrous thyroiditis that is characterized by the replacement of all or part of the thyroid parenchyma by fibrous tissue, which also invades into adjacent tissues. The etiology of this disorder is contro-versial, and it has been reported to occur in

1	replacement of all or part of the thyroid parenchyma by fibrous tissue, which also invades into adjacent tissues. The etiology of this disorder is contro-versial, and it has been reported to occur in patients with other autoimmune diseases. This association, coupled with the pres-ence of lymphoid infiltration and response to steroid therapy, suggests a primary autoimmune etiology. Riedel’s thyroiditis also is associated with other focal sclerosing syndromes includ-ing mediastinal, retroperitoneal, periorbital, and retro-orbital fibrosis and sclerosing cholangitis, suggesting that it may, in fact, be a primary fibrotic disorder. It is now considered a manifestation of IgG4-related systemic disease characterized by elevated serum IgG4 levels and a lymphoplasmacytic infiltrate with an abundance of IgG4 bearing plasma cells.14 The disease occurs predominantly in women between the ages of 30 and 60 years old. It typically presents as a painless, hard anterior neck mass, which progresses

1	of IgG4 bearing plasma cells.14 The disease occurs predominantly in women between the ages of 30 and 60 years old. It typically presents as a painless, hard anterior neck mass, which progresses over weeks to years to produce symptoms of compression, including dysphagia, dyspnea, chok-ing, and hoarseness. Patients may present with symptoms of hypothyroidism and hypoparathyroidism as the gland is replaced by fibrous tissue. Physical examination reveals a hard, “woody” thyroid gland with fixation to surrounding tissues. The diagnosis needs to be confirmed by open thyroid biopsy because the firm and fibrous nature of the gland renders FNAB inadequate.Surgery is the mainstay of the treatment. The chief goal of operation is to decompress the trachea by wedge excision of the thyroid isthmus and to make a tissue diagnosis. More extensive resections are not advised due to the infiltrative nature of the fibrotic process that obscures usual landmarks and struc-tures. Hypothyroid patients are

1	to make a tissue diagnosis. More extensive resections are not advised due to the infiltrative nature of the fibrotic process that obscures usual landmarks and struc-tures. Hypothyroid patients are treated with thyroid hormone replacement. Some patients who remain symptomatic have been reported to experience dramatic improvement after treatment with corticosteroids and tamoxifen. Mycophenolate mofetil and more recently rituximab has also been used to attenuate the Brunicardi_Ch38_p1625-p1704.indd 164001/03/19 11:20 AM 1641THYROID, PARATHYROID, AND ADRENALCHAPTER 38inflammatory process and led to dramatic symptom improve-ments in some patients.15Goiter. Any enlargement of the thyroid gland is referred to as a goiter. The causes of nontoxic goiters are listed in Table 38-3. Goiters may be diffuse, uninodular, or multinodular. Most non-toxic goiters are thought to result from TSH stimulation second-ary to inadequate thyroid hormone synthesis and other paracrine growth factors.16

1	may be diffuse, uninodular, or multinodular. Most non-toxic goiters are thought to result from TSH stimulation second-ary to inadequate thyroid hormone synthesis and other paracrine growth factors.16 Elevated TSH levels induce diffuse thyroid hyperplasia, followed by focal hyperplasia, resulting in nod-ules that may or may not concentrate iodine, colloid nodules, or microfollicular nodules. The TSH-dependent nodules progress to become autonomous. Familial goiters resulting from inher-ited deficiencies in enzymes necessary for thyroid hormone synthesis may be complete or partial. The term endemic goiter refers to the occurrence of a goiter in a significant proportion of individuals in a particular geographic region. In the past, dietary iodine deficiency was the most common cause of endemic goi-ter. This condition has largely disappeared in North America due to routine use of iodized salt and iodination of fertilizers, animal feeds, and preservatives. However, in areas of iodine

1	endemic goi-ter. This condition has largely disappeared in North America due to routine use of iodized salt and iodination of fertilizers, animal feeds, and preservatives. However, in areas of iodine deficiency, such as Central Asia, South America, and Indonesia, up to 90% of the population have goiters. Other dietary goitro-gens that may participate in endemic goiter formation include kelp, cassava, and cabbage. In many sporadic goiters, no obvi-ous cause can be identified.Clinical Features Most patients with nontoxic goiters are asymptomatic, although patients often complain of a pres-sure sensation in the neck. As the goiters become very large, compressive symptoms such as dyspnea and dysphagia ensue. Patients also describe having to clear their throats frequently (catarrh). Dysphonia from RLN injury is rare, except when malignancy is present. Obstruction of venous return at the tho-racic inlet from a substernal goiter results in a positive Pember-ton’s sign—facial flushing and

1	from RLN injury is rare, except when malignancy is present. Obstruction of venous return at the tho-racic inlet from a substernal goiter results in a positive Pember-ton’s sign—facial flushing and dilatation of cervical veins upon raising the arms above the head (Fig. 38-13A). Sudden enlarge-ment of nodules or cysts due to hemorrhage may cause acute pain. Physical examination may reveal a soft, diffusely enlarged gland (simple goiter) or nodules of various size and consistency in case of a multinodular goiter. Deviation or compression of the trachea may be apparent.Diagnostic Tests Patients usually are euthyroid with normal TSH and low-normal or normal free T4 levels. If some nod-ules develop autonomy, patients have suppressed TSH levels or become hyperthyroid. RAI uptake often shows patchy uptake with areas of hot and cold nodules. FNAB is recommended in patients who have a dominant nodule or one that is painful or enlarging, as carcinomas have been reported in 5% to 10% of

1	patchy uptake with areas of hot and cold nodules. FNAB is recommended in patients who have a dominant nodule or one that is painful or enlarging, as carcinomas have been reported in 5% to 10% of multinodular goiters. CT scans are helpful to evaluate the extent of retrosternal extension and airway compression (Fig. 38-13B).Treatment Most euthyroid patients with small, diffuse goi-ters do not require treatment. Some physicians give patients with large goiters exogenous thyroid hormone to reduce the TSH stimulation of gland growth; this treatment may result in decrease and/or stabilization of goiter size and is most effective for small diffuse goiters. Endemic goiters are treated by iodine administration. Surgical resection is reserved for goiters that (a) continue to increase despite T4 suppression, (b) cause obstruc-tive symptoms, (c) have substernal extension (considered a rela-tive indication by some groups), (d) have malignancy suspected or proven by FNAB, and (e) are cosmetically

1	(b) cause obstruc-tive symptoms, (c) have substernal extension (considered a rela-tive indication by some groups), (d) have malignancy suspected or proven by FNAB, and (e) are cosmetically unacceptable. Near-total or total thyroidectomy is the treatment of choice, and patients require lifelong T4 therapy.Solitary Thyroid NoduleSolitary thyroid nodules are present in approximately 4% of individuals in the United States, whereas thyroid cancer has a much lower incidence of 40 new cases per 1 million. Therefore, it is of utmost importance to determine which patients with soli-tary thyroid nodule would benefit from surgery.History. Details regarding the nodule, such as time of onset, change in size, and associated symptoms such as pain, dyspha-gia, dyspnea, or choking, should be elicited. Pain is an unusual symptom and, when present, should raise suspicion for intra-thyroidal hemorrhage in a benign nodule, thyroiditis, or malig-nancy. Patients with MTC may complain of a dull, aching

1	Pain is an unusual symptom and, when present, should raise suspicion for intra-thyroidal hemorrhage in a benign nodule, thyroiditis, or malig-nancy. Patients with MTC may complain of a dull, aching sensation. A history of hoarseness is worrisome, as it may be secondary to malignant involvement of the RLNs. Most impor-tantly, patients should be questioned regarding risk factors for malignancy, such as exposure to ionizing radiation and family history of thyroid and other malignancies associated with thy-roid cancer.External-Beam Radiation Low-dose therapeutic radiation has been used to treat conditions such as tinea capitis (6.5 cGy), thymic enlargement (100 to 400 cGy), enlarged tonsils and adenoids (750 cGy), acne vulgaris (200 to 1500 cGy), and other conditions such as hemangioma and scrofula. Radiation (approximately 4000 cGy) is also an integral part of the manage-ment of patients with Hodgkin’s disease. It is now known that a history of exposure to low-dose ionizing radiation to

1	Radiation (approximately 4000 cGy) is also an integral part of the manage-ment of patients with Hodgkin’s disease. It is now known that a history of exposure to low-dose ionizing radiation to the thyroid gland places the patient at increased risk for developing thyroid cancer. The risk increases linearly from 6.5 to 2000 cGy, beyond which the incidence declines as the radiation causes destruc-tion of the thyroid tissue. The risk is maximum 20 to 30 years after exposure, but these patients require lifelong monitoring. During the nuclear fallout from Chernobyl in 1986, 131I release was accompanied by a marked increase in the incidence of both benign and malignant thyroid lesions noted within 4 years of exposure, particularly in children.17 Most thyroid carcinomas following radiation exposure are papillary, and some of these cancers with a solid type of histology and presence of RET/PTC translocations appear to be more aggressive. In general, there is a 40% chance that patients

1	exposure are papillary, and some of these cancers with a solid type of histology and presence of RET/PTC translocations appear to be more aggressive. In general, there is a 40% chance that patients presenting with a thyroid nodule and a history of radiation have thyroid cancer. Of those patients who have thyroid cancer, the cancer is located in the dominant nodule in 60% of patients, but in the remaining 40% of patients, the cancer is in another nodule in the thyroid gland.Table 38-3Etiology of nontoxic goiterCLASSIFICATIONSPECIFIC ETIOLOGYEndemicIodine deficiency, dietary goitrogens (cassava, cabbage)MedicationsIodide, amiodarone, lithiumThyroiditisSubacute, chronic (Hashimoto’s)FamilialImpaired hormone synthesis from enzyme defectsNeoplasmAdenoma, carcinomaResistance to thyroid hormone—Brunicardi_Ch38_p1625-p1704.indd 164101/03/19 11:20 AM 1642SPECIFIC CONSIDERATIONSPART IIABFigure 38-13. A. Retrosternal extension of a large goiter may result in impeded flow in the superior vena

1	164101/03/19 11:20 AM 1642SPECIFIC CONSIDERATIONSPART IIABFigure 38-13. A. Retrosternal extension of a large goiter may result in impeded flow in the superior vena cava, leading to dilated veins over the chest wall. This may become more prominent when patients raise their arms above the head—Pemberton’s sign. B. Computed tomography scan demonstrating retrosternal extension and consequent tracheal deviation and compression from a large goiter.Brunicardi_Ch38_p1625-p1704.indd 164201/03/19 11:20 AM 1643THYROID, PARATHYROID, AND ADRENALCHAPTER 38Family History A family history of thyroid cancer is a risk factor for the development of both medullary and nonmedullary thyroid cancer. Familial MTCs occur in isolation or in associa-tion with other tumors as part of multiple endocrine neoplasia type 2 (MEN2) syndromes. Nonmedullary thyroid cancers can occur in association with other known familial cancer syn-dromes such as Cowden’s syndrome, Werner’s syndrome (adult progeroid syndrome),

1	type 2 (MEN2) syndromes. Nonmedullary thyroid cancers can occur in association with other known familial cancer syn-dromes such as Cowden’s syndrome, Werner’s syndrome (adult progeroid syndrome), familial adenomatous polyposis, and DICER 1 (Table 38-4). Nonmedullary thyroid cancers can also occur independently of these syndromes as the predominant tumors in the families, and in fact nonsyndromic FNMTC accounts for 95% of cases. The definition of familial nonmedul-lary thyroid cancer (FNMTC) is variable across the literature; however, in most studies, it is defined by the presence of two or more first-degree relatives with follicular cell–derived cancers. FNMTC is now recognized as a distinct clinical entity associated with a high incidence of multifocal tumors and benign thyroid nodules. Some studies report that these patients have higher locoregional recurrence rates and consequently shorter disease-free survival. Several candidate chromosomal loci that predispose to these tumors

1	Some studies report that these patients have higher locoregional recurrence rates and consequently shorter disease-free survival. Several candidate chromosomal loci that predispose to these tumors have been identified, includ-ing MNG1 (14q32), thyroid carcinoma with oxphilia (TCO, on 19p13.2), fPTC/papillary renal neoplasia (PRN, on 1q21), NMTC1 (2q21), and FTEN (8p23.1-p22). Susceptibility genes include SRGAP1 (12q14), TITF-1/NKX2.1 (14q13), FOXE1 (9q22), and the telomere-telomerase complex.18Physical Examination. The thyroid gland is best palpated from behind the patient and with the neck in mild extension. The cricoid cartilage is an important landmark, as the isthmus is situated just below it. Nodules that are hard, gritty, or fixed to surrounding structures such as the trachea or strap muscles are more likely to be malignant. The cervical chain of lymph nodes should be assessed as well as the nodes in the posterior triangle.Diagnostic Investigations. An algorithm for the workup

1	muscles are more likely to be malignant. The cervical chain of lymph nodes should be assessed as well as the nodes in the posterior triangle.Diagnostic Investigations. An algorithm for the workup of a solitary thyroid nodule is shown in Fig. 38-14.Fine-Needle Aspiration Biopsy FNAB has become the single most important test in the evaluation of thyroid masses and can be performed with or without ultrasound guidance. Ultrasound guidance is recommended for nodules that are difficult to pal-pate, for cystic or solid-cystic nodules that recur after the initial aspiration, and for multinodular goiters. A 23-gauge needle is inserted into the thyroid mass, and several passes are made while aspirating the syringe. After releasing the suction on the syringe, the needle is withdrawn and the cells are immediately placed on prelabeled dry glass slides; some are immersed in a 70% alcohol solution while others are air dried. A sample of the aspirate is also placed in a 90% alcohol solution for

1	are immediately placed on prelabeled dry glass slides; some are immersed in a 70% alcohol solution while others are air dried. A sample of the aspirate is also placed in a 90% alcohol solution for cytospin or cell pellet. The slides are stained by Papanicolaou’s or Wright’s stains and examined under the microscope. If a bloody aspirate is obtained, the patient should be repositioned in a more upright position and the biopsy repeated with a finer (25to 30-gauge) needle.After FNAB, the majority of nodules can be classified into several categories that determine further management. To address the issue of variability in the terminology of fine-needle aspiration (FNA), the National Cancer Institute (NCI) hosted the “NCI Thyroid Fine Needle Aspiration State of the Science Conference,” which then defined the Bethesda criteria for thyroid FNA.19 Accordingly, optimum cytology specimens should have at least six follicles each containing at least 10 to 15 cells from at least two aspirates.The

1	defined the Bethesda criteria for thyroid FNA.19 Accordingly, optimum cytology specimens should have at least six follicles each containing at least 10 to 15 cells from at least two aspirates.The FNA is classified as “nondiagnostic or unsatisfac-tory” in 2% to 20% of cases and typically results from a virtu-ally acellular specimen, cyst fluid, or the presence of blood or clotting artifact. The risk of malignancy in this setting ranges from 1% to 4%, and reaspiration under ultrasound guidance is recommended. A “benign” result is obtained in 60% to 70% of thyroid FNAs. The most common lesion in this setting is a follicular nodule (includes adenomatoid nodule, colloid nodule, and follicular adenoma). Other diagnoses include lymphocytic (Hashimoto’s) thyroiditis and granulomatous thyroiditis. False-negative results are reported in up to 3% of cases, and follow-up is recommended. A result of “atypia of unknown significance (AUS) or follicular lesion of unknown significance (FLUS)” is

1	False-negative results are reported in up to 3% of cases, and follow-up is recommended. A result of “atypia of unknown significance (AUS) or follicular lesion of unknown significance (FLUS)” is obtained in 3% to 6% of biopsies. The risk of malignancy in this scenario is difficult to determine; however, it is thought to be in the range of 5% to 15%. Clinical correlation and a repeat FNA are recommended for AUS lesions (which often results in 23Table 38-4Familial cancer syndromes involving nonmedullary thyroid cancerSYNDROMEGENEMANIFESTATIONTHYROID TUMORCowden’s syndromePTENIntestinal hamartomas, benign and malignant breast tumorsFTC, rarely PTC and Hürthle cell tumorsFAPAPCColon polyps and cancer, duodenal neoplasms, desmoidsPTC cribriform growth patternWerner’s syndromeWRNAdult progeroid syndromePTC, FTC, anaplastic cancerCarney complex type 1PRKAR1αCutaneous and cardiac myxomas, breast and adrenal tumorsPTC, FTCMcCune-Albright syndromeGNAS1Polyostotic fibrous dysplasia, endocrine

1	syndromePTC, FTC, anaplastic cancerCarney complex type 1PRKAR1αCutaneous and cardiac myxomas, breast and adrenal tumorsPTC, FTCMcCune-Albright syndromeGNAS1Polyostotic fibrous dysplasia, endocrine abnormalities, café-au-lait spotsPTC clear cellDICER 1 syndrome Pleuropulmonary blastoma, cystic nephroma, ovarian sex cord-stromal tumorsMultinodular goiter, thyroid cancerFAP = familial adenomatous polyposis; FTC = follicular thyroid cancer; PTC = papillary thyroid cancer.Brunicardi_Ch38_p1625-p1704.indd 164301/03/19 11:20 AM 1644SPECIFIC CONSIDERATIONSPART IIa more definitive interpretation), although clinical observation or surgery may be appropriate because of worrisome clinical or ultrasound findings. The category of “follicular neoplasm” is intended to identify nodules that might be follicular carcinomas. The term suspicious for a follicular neoplasm is preferred by some laboratories for this category because up to 35% of cases turn out not to be neoplasms but hyperplastic

1	be follicular carcinomas. The term suspicious for a follicular neoplasm is preferred by some laboratories for this category because up to 35% of cases turn out not to be neoplasms but hyperplastic proliferations of follicular cells, most commonly those of multinodular goiter. Lobectomy is the preferred treatment for this result, and approx-imately 15% to 35% of lesions placed in this category prove to be malignant. Hürthle cell neoplasms are also included in this category. Most papillary and other carcinomas can be diagnosed by FNA, but the features are subtle at times, such as in follicular variant of papillary carcinomas. If the diagnosis is uncertain, the lesions are classified as “suspicious for malignancy.” Lobec-tomy or near-total thyroidectomy is recommended because 60% to 75% turn out to be malignant. This category also includes lesions suspicious for medullary carcinoma and lymphoma, and ancillary testing such as immunohistochemical analysis and flow cytometry may be helpful.

1	out to be malignant. This category also includes lesions suspicious for medullary carcinoma and lymphoma, and ancillary testing such as immunohistochemical analysis and flow cytometry may be helpful. The risk of malignancy in lesions classified as “malignant” by FNA is 97% to 99%, and near-total/total thyroidectomy is recommended.Laboratory Studies Most patients with thyroid nodules are euthyroid. Determining the blood TSH level is helpful. If a patient with a nodule is found to be hyperthyroid, the risk of malignancy is approximately 1%. Serum Tg levels cannot dif-ferentiate benign from malignant thyroid nodules unless the lev-els are extremely high, in which case metastatic thyroid cancer should be suspected. Tg levels are, however, useful in following patients who have undergone total thyroidectomy for thyroid cancer and also for serial evaluation of patients undergoing nonoperative management of thyroid nodules. Serum calcitonin levels should be obtained in patients with MTC or a

1	thyroidectomy for thyroid cancer and also for serial evaluation of patients undergoing nonoperative management of thyroid nodules. Serum calcitonin levels should be obtained in patients with MTC or a family his-tory of MTC or MEN2. There is insufficient evidence to recom-mend routine calcitonin testing for all nodules. All patients with MTC should be tested for RET oncogene mutations and have a 24-hour urine collection with measurement of levels of vanil-lylmandelic acid (VMA), metanephrine, and catecholamine levels to rule out a coexisting pheochromocytoma. About 10% of patients with familial MTC and MEN2A have de novo RET mutations, so that their children are at risk for thyroid cancer.Imaging Ultrasound is helpful for detecting nonpalpable thy-roid nodules, differentiating solid from cystic nodules, and identifying adjacent lymphadenopathy. Ultrasound evaluation can identify features of a nodule that increase the a priori risk of malignancy, such as fine stippled calcification and

1	nodules, and identifying adjacent lymphadenopathy. Ultrasound evaluation can identify features of a nodule that increase the a priori risk of malignancy, such as fine stippled calcification and enlarged regional nodes; however, a tissue diagnosis is strongly recom-mended before thyroidectomy.20 Ultrasound also provides a noninvasive and inexpensive method of following the size of suspected benign nodules diagnosed by FNAB and for identi-fying enlarged lymph nodes. Ultrasound elastography is used to evaluate tissue stiffness noninvasively. This technique takes advantage of the fact that malignant nodules tend to be harder than benign nodules and thus deform less compared with the surrounding normal thyroid parenchyma. Larger studies are warranted before elastography and newer techniques such as contrast-enhanced ultrasound can be routinely included in the evaluation of thyroid nodules.21 CT and MRI are unnecessary in the routine evaluation of thyroid tumors except for large, fixed, or

1	as contrast-enhanced ultrasound can be routinely included in the evaluation of thyroid nodules.21 CT and MRI are unnecessary in the routine evaluation of thyroid tumors except for large, fixed, or substernal lesions. Scanning the thyroid with 123I or 99mTc is rarely necessary, and thyroid scanning currently is recom-mended in the assessment of thyroid nodules only in patients Solitary thyroid noduleFNABBenignMalignantNondiagnosticRepeatFNAB withu/sguidanceCystColloidnoduleAspirateReaccumulates x 3ThyroidectomyObserveContinuedgrowth,compressivesymptoms+/–FNABAUS/FLUSFN orSuspiciousfor FNSuspiciousformalignancyNear-total/ totalThyroidectomyRepeatFNABLobectomyLobectomy ornear-total/totalthyroidectomyFigure 38-14. Management of a solitary thyroid nodule based on Bethesda criteria. a = except in patients with a history of external radia-tion exposure or a family history of thyroid cancer; FNAB = fine-needle aspiration biopsy; AUS = atypia of unknown significance; FLUS = follicular lesion

1	in patients with a history of external radia-tion exposure or a family history of thyroid cancer; FNAB = fine-needle aspiration biopsy; AUS = atypia of unknown significance; FLUS = follicular lesion of unknown significance; FN = follicular neoplasm.Brunicardi_Ch38_p1625-p1704.indd 164401/03/19 11:20 AM 1645THYROID, PARATHYROID, AND ADRENALCHAPTER 38who have follicular thyroid nodules on FNAB and a suppressed TSH. PET scanning does not play a major role in the primary evaluation of thyroid nodules.Management. Malignant tumors are treated by thyroidectomy, as discussed earlier and later in this chapter in “Surgical Treat-ment under Malignant Thyroid Disease.” Simple thyroid cysts resolve with aspiration in about 75% of cases, although some require a second or third aspiration. If the cyst persists after three attempts at aspiration, unilateral thyroid lobectomy is rec-ommended. Lobectomy also is recommended for cysts >4 cm in diameter or complex cysts with solid and cystic

1	If the cyst persists after three attempts at aspiration, unilateral thyroid lobectomy is rec-ommended. Lobectomy also is recommended for cysts >4 cm in diameter or complex cysts with solid and cystic components, as the latter have a higher incidence of malignancy (15%). When FNAB is used in complex nodules, the solid portion should be sampled. If a colloid nodule is diagnosed by FNAB, patients should still be observed with serial ultrasound and Tg measure-ments. If the nodule enlarges, repeat FNAB often is indicated. Although controversial, levothyroxine in doses sufficient to maintain a serum TSH level between 0.1 and 1.0 μU/mL may also be administered. In areas with a high prevalence of iodine deficiency, this can decrease nodule size and potentially prevent the growth of new nodules. In iodine-sufficient populations, the data are less impressive. Randomized controlled trial analyses have shown that less than 25% of benign nodules shrink more than 50% with TSH suppression in

1	In iodine-sufficient populations, the data are less impressive. Randomized controlled trial analyses have shown that less than 25% of benign nodules shrink more than 50% with TSH suppression in iodine-replete populations. Thyroidectomy should be performed if a nodule enlarges on TSH suppression, causes compressive symptoms, or for cos-metic reasons. An exception to this general rule is the patient who has had previous irradiation of the thyroid gland or has a family history of thyroid cancer. In these patients, total or near-total thyroidectomy is recommended because of the high incidence of thyroid cancer and decreased reliability of FNAB in this setting.Malignant Thyroid DiseaseIn the United States, thyroid cancer accounts for <1% of all malignancies (2% of women and 0.5% of men) and is the most rapidly increasing cancer in women. Thyroid cancer is respon-sible for six deaths per million persons annually. Most patients present with a palpable swelling in the neck, which initiates

1	is the most rapidly increasing cancer in women. Thyroid cancer is respon-sible for six deaths per million persons annually. Most patients present with a palpable swelling in the neck, which initiates assessment through a combination of history, physical exami-nation, and FNAB.Molecular Genetics of Thyroid Tumorigenesis. Several oncogenes and tumor suppressor genes are involved in thy-roid tumorigenesis,22 as depicted in Table 38-5. The RET proto-oncogene (Fig. 38-15) plays a significant role in the pathogenesis of thyroid cancers. It is located on chromosome 10 and encodes a receptor tyrosine kinase, which binds sev-eral growth factors such as glial-derived neurotrophic factor and neurturin. The RET protein is expressed in tissues derived from the embryonic nervous and excretory systems. Therefore, Table 38-5Oncogenes, tumor suppressor genes, and other genetic alterations implicated in thyroid tumorigenesisGENEFUNCTIONTUMOROncogenes RETMembrane receptor with tyrosine kinase

1	Therefore, Table 38-5Oncogenes, tumor suppressor genes, and other genetic alterations implicated in thyroid tumorigenesisGENEFUNCTIONTUMOROncogenes RETMembrane receptor with tyrosine kinase activitySporadic and familial MTC, PTC (RET/PTC rearrangements)METSameOverexpressed in PTCTRK1SameActivated in some PTCTSH-RLinked to heterotrimeric G proteinHyperfunctioning adenomaGsα (gsp)Signal transduction molecule (GTP binding)Hyperfunctioning adenoma, follicular adenomaRasSignal transduction proteinFollicular adenoma and carcinoma, PTCPAX8/PPARγ1OncoproteinFollicular adenoma, follicular carcinomaB-Raf (BRAF)Signal transductionPTC, tall cell and poorly differentiated, anaplasticCTNNB1 (β-catenin)Signal transductionUpregulated in poorly differentiated and anaplastic cancersTERT promoterChromosome integrityMutated in thyroid cancers including PTC, FTC and anaplastic cancersTumor suppressors p53Cell cycle regulator, arrests cells in G1, induces apoptosisDedifferentiated PTC, FTC, anaplastic

1	integrityMutated in thyroid cancers including PTC, FTC and anaplastic cancersTumor suppressors p53Cell cycle regulator, arrests cells in G1, induces apoptosisDedifferentiated PTC, FTC, anaplastic cancersp16Cell cycle regulator, inhibits cyclin-dependent kinaseThyroid cancer cell linesPTENProtein tyrosine phosphataseFollicular adenoma and carcinomaOther genetic alterations microRNASmall, noncoding RNASpecific types upregulated in papillary and some follicular carcinomasFTC = follicular thyroid cancer; GTP = guanosine triphosphate; MTC = medullary thyroid cancer; PTC = papillary thyroid cancer.Brunicardi_Ch38_p1625-p1704.indd 164501/03/19 11:20 AM 1646SPECIFIC CONSIDERATIONSPART IIReceptorGDNFTyrosinekinaseIntracellularExtracellularSubstrateATP620618811609634830804768= MEN2A & FMTC mutation= MEN2B mutation= FMTC mutation= Hirschsprung's disease mutation918Figure 38-15. Structure of the RET tyrosine kinase receptor. Multiple endocrine neoplasia type 2A (MEN2A) and type 2B

1	mutation= MEN2B mutation= FMTC mutation= Hirschsprung's disease mutation918Figure 38-15. Structure of the RET tyrosine kinase receptor. Multiple endocrine neoplasia type 2A (MEN2A) and type 2B (MEN2B), famil-ial medullary thyroid cancer (FMTC), and Hirschsprung’s disease result from germline mutations in the RET proto-oncogene. The extracel-lular domain binds the ligand glial-derived neurotrophic factor (GDNF) and contains 28 cysteine residues. Mutations in cysteine residues at codons 609, 611, 618, 620, and 634, which are in the juxtamembrane region of the receptor, are associated with MEN2A and FMTC. The ATP-binding site is located intracellularly near the site, which binds the substrate for the tyrosine kinase catalytic domain. Mutations at codon 918 (Met to Thr) alter the substrate binding pocket located in the intracellular region and cause MEN2B. FMTC is associated with mutations at codons 768 and 804. ATP = adenosine triphosphate. (Reproduced with permission from Wells S,

1	binding pocket located in the intracellular region and cause MEN2B. FMTC is associated with mutations at codons 768 and 804. ATP = adenosine triphosphate. (Reproduced with permission from Wells S, Franz C. Medullary carcinoma of the thyroid gland, World J Surg. 2000 Aug;24(8):952-956.)RET disruption can lead to developmental abnormalities in organs derived from these systems, such as the enteric nervous system (Hirschsprung’s disease) and kidney. Germline muta-tions in the RET proto-oncogene are known to predispose to MEN2A, MEN2B, and familial MTCs, and somatic mutations have been demonstrated in tumors derived from the neural crest, such as MTCs (30%) and pheochromocytomas. The tyrosine kinase domain of RET can fuse with other genes by rearrange-ment. These fusion products also function as oncogenes and have been implicated in the pathogenesis of PTCs. At least 15 RET/PTC rearrangements have been described and appear to be early events in tumorigenesis. Young age and radiation

1	as oncogenes and have been implicated in the pathogenesis of PTCs. At least 15 RET/PTC rearrangements have been described and appear to be early events in tumorigenesis. Young age and radiation expo-sure seem to be independent risk factors for the development of RET/PTC rearrangements. Up to 70% of papillary cancers in children exposed to the radiation fallout from the 1986 Cher-nobyl disaster carry RET/PTC rearrangements, the most com-mon being RET/PTC1 and RET/PTC3. These rearrangements confer constitutive activation of the receptor tyrosine kinases. RET/PTC3 is associated with a solid type of PTC that appears to present at a higher stage and to be more aggressive. It has now been established that RET/PTC signaling involves the mitogen-activated protein kinase (MAPK) pathway via other signaling molecules such as Ras, Raf, and MEK. In normal cells, physi-ologic activation of Raf kinases occurs via direct interaction with guanosine triphosphate (GTP)–bound Ras, a membrane-bound small

1	molecules such as Ras, Raf, and MEK. In normal cells, physi-ologic activation of Raf kinases occurs via direct interaction with guanosine triphosphate (GTP)–bound Ras, a membrane-bound small G protein. Activated Raf, a serine-threonine kinase, in turn phosphorylates MEK, another serine-threonine kinase. This leads to phosphorylation of ERK/MAPK, which phosphor-ylates regulatory molecules in the nucleus, thereby altering gene expression. Aberrant activation of the MAPK pathway leads to tumorigenesis. Aside from RET/PTC alterations, mutations in the Ras genes can also activate the MAPK pathway. Mutated RAS oncogenes have been identified in up to 20% to 40% of thyroid follicular adenomas and carcinomas, multinodular goi-ters, and papillary and anaplastic carcinomas. There are three Raf kinases, A-Raf, B-Raf (BRAF), and C-Raf. Mutations in BRAF also have been implicated in aberrant MAPK pathway activation and tumorigenesis. Of the various identified BRAF mutations, T1799A (V600E amino

1	A-Raf, B-Raf (BRAF), and C-Raf. Mutations in BRAF also have been implicated in aberrant MAPK pathway activation and tumorigenesis. Of the various identified BRAF mutations, T1799A (V600E amino acid substitution) is the most common and occurs frequently in thyroid cancers. Interestingly, BRAF mutations occur in papillary and anaplastic tumors (aver-age prevalence of 44% and 22%, respectively),23 but not in fol-licular thyroid cancers, suggesting a role in the pathogenesis of these malignancies. Studies also show that BRAF mutations are associated with more aggressive clinicopathologic features, including larger tumor size, invasion, and lymphadenopathy, and may have a role as prognostic markers.The p53 gene is a tumor suppressor gene encoding a transcriptional regulator, which causes cell cycle arrest allow-ing repair of damaged DNA, thus helping to maintain genomic integrity. Mutations of p53 are rare in PTCs but common in undifferentiated thyroid cancers and thyroid cancer cell

1	cell cycle arrest allow-ing repair of damaged DNA, thus helping to maintain genomic integrity. Mutations of p53 are rare in PTCs but common in undifferentiated thyroid cancers and thyroid cancer cell lines. Other cell cycle regulators and tumor suppressors such as p15 and p16 are mutated more commonly in thyroid cancer cell lines than in primary tumors. An oncogene resulting from the fusion of the DNA binding domain of the thyroid-transcription fac-tor PAX8 gene to the peroxisome proliferator-activated receptor gamma 1 (PPARγ1) has been noted to play an important role Brunicardi_Ch38_p1625-p1704.indd 164601/03/19 11:21 AM 1647THYROID, PARATHYROID, AND ADRENALCHAPTER 38in the development of follicular neoplasms, including follicular cancers. Mutations in the telomerase reverse transcriptase cata-lytic subunit (TERT) promoter unit have also been recently been identified in well-differentiated thyroid cancers and appear to be related to poor prognosis. Thyroid cancer stem cells have

1	cata-lytic subunit (TERT) promoter unit have also been recently been identified in well-differentiated thyroid cancers and appear to be related to poor prognosis. Thyroid cancer stem cells have also been identified; however, their role in thyroid carcinogenesis remains to be determined.24 Mutations in the kinases PIK3CA and AKT1 are rare in thyroid cancers and tend to occur as late events in tumorigenesis.Specific Tumor Types Papillary Carcinoma Papillary carcinoma accounts for 80% of all thyroid malignancies in iodine-sufficient areas and is the predominant thyroid cancer in children and individuals exposed to external radiation. Papillary carcinoma occurs more often in women, with a 2:1 female-to-male ratio, and the mean age at presentation is 30 to 40 years. Most patients are euthyroid and present with a slow-growing painless mass in the neck. Dys-phagia, dyspnea, and dysphonia usually are associated with locally advanced invasive disease. Lymph node metastases are common,

1	and present with a slow-growing painless mass in the neck. Dys-phagia, dyspnea, and dysphonia usually are associated with locally advanced invasive disease. Lymph node metastases are common, especially in children and young adults, and may be the presenting complaint. “Lateral aberrant thyroid” almost always denotes a cervical lymph node that has been invaded by metastatic cancer. Suspicion of thyroid cancer often originates through physical examination of patients and a review of their history. Diagnosis is established by FNAB of the thyroid mass or lymph node. Once thyroid cancer is diagnosed on FNAB, a complete neck ultrasound is strongly recommended to evalu-ate the contralateral lobe and for lymph node metastases in the central and lateral neck compartments. Distant metastases are uncommon at initial presentation, but may ultimately develop in up to 20% of patients. The most common sites are lungs, fol-lowed by bone, liver, and brain.Pathology. On gross examination, PTCs

1	are uncommon at initial presentation, but may ultimately develop in up to 20% of patients. The most common sites are lungs, fol-lowed by bone, liver, and brain.Pathology. On gross examination, PTCs generally are hard and whitish and remain flat on sectioning with a blade, in contrast to normal tissue or benign nodular lesions that tend to bulge. Mac-roscopic calcification, necrosis, or cystic change may be appar-ent. Histologically, papillary carcinomas may exhibit papillary projections (Fig. 38-16A), a mixed pattern of papillary and fol-licular structures, or a pure follicular pattern (follicular variant). The diagnosis is established by characteristic nuclear cellular features. Cells are cuboidal with pale, abundant cytoplasm, crowded nuclei that may demonstrate “grooving,” and intranu-clear cytoplasmic inclusions (leading to the designation of Orphan Annie nuclei [Fig. 38-16B]), which allow diagnosis by FNAB. Psammoma bodies, which are microscopic, calcified deposits representing

1	cytoplasmic inclusions (leading to the designation of Orphan Annie nuclei [Fig. 38-16B]), which allow diagnosis by FNAB. Psammoma bodies, which are microscopic, calcified deposits representing clumps of sloughed cells, also may be present. Mixed papillary-follicular tumors and follicular variant of papillary thyroid carcinoma (FVPTC) are classified as papil-lary carcinomas because they behave biologically as papillary carcinomas. Two main subtypes of FVPTC are recognized: encapsulated and nonencapsulated (infiltrative). The for-mer is challenging to diagnose. Since the tumors have no invasion, the diagnosis relies on the finding of characteristic nuclei, which can be subjective. In addition, several studies have shown that the encapsulated tumors have an indolent behavior and are genetically distinct from their infiltrative counterparts. As such, these tumors are now designated noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP).25 Multifocality is

1	distinct from their infiltrative counterparts. As such, these tumors are now designated noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP).25 Multifocality is common in papillary carcinoma and may be present in up to 85% of cases on microscopic examination. Mul-tifocality is associated with an increased risk of cervical nodal metastases, and these tumors may rarely invade adjacent struc-tures such as the trachea, esophagus, and RLNs. Other variants of papillary carcinoma include tall cell, insular, columnar, dif-fuse sclerosing, clear cell, trabecular, and poorly differentiated types. These variants account for about 1% of all papillary car-cinomas and are generally associated with a worse prognosis.Minimal or occult/microcarcinoma refers to tumors of 1 cm or less in size with no evidence of local invasiveness through the thyroid capsule or angioinvasion, and that are not associated with lymph node metastases. They are nonpalpable and usually are

1	1 cm or less in size with no evidence of local invasiveness through the thyroid capsule or angioinvasion, and that are not associated with lymph node metastases. They are nonpalpable and usually are incidental findings at operative, histologic, or autopsy examination. Studies have demonstrated occult PTC to be present in 2% to 36% of thyroid glands removed at autopsy. These tumors are also being identified more frequently due to the widespread use of ultrasound. These occult tumors are generally associated with a better prognosis than larger tumors, but they may be more aggressive than previously appreciated. About 25% of patients with these tumors have associated occult lymph node metastases.Prognostic Indicators. In general, patients with PTC have an excellent prognosis with a >95% 10-year survival rate. Sev-eral prognostic indicators have been incorporated into various 4Figure 38-16. A. Histomicrograph of a papillary thyroid cancer (hematoxylin-eosin stain). B. Fine-needle

1	10-year survival rate. Sev-eral prognostic indicators have been incorporated into various 4Figure 38-16. A. Histomicrograph of a papillary thyroid cancer (hematoxylin-eosin stain). B. Fine-needle aspiration biopsy specimen from a papillary thyroid cancer showing typical intranuclear cytoplasmic inclusions in the center of the slide.Brunicardi_Ch38_p1625-p1704.indd 164701/03/19 11:21 AM 1648SPECIFIC CONSIDERATIONSPART IIstaging systems, which enable patients to be stratified into low-risk and high-risk groups. Unfortunately, all of these classifica-tion systems rely on data that are not available preoperatively.In 1987, Hay and colleagues26 at the Mayo Clinic proposed the AGES scoring system, which incorporates Age, histologic Grade, Extrathyroidal invasion, and metastases and tumor Size to predict the risk of dying from papillary cancer. Low-risk patients are young, with well-differentiated tumors, no metas-tases, and small primary lesions, whereas high-risk patients are older,

1	Size to predict the risk of dying from papillary cancer. Low-risk patients are young, with well-differentiated tumors, no metas-tases, and small primary lesions, whereas high-risk patients are older, with poorly differentiated tumors, local invasion, dis-tant metastases, and large primary lesions. The MACIS scale is a postoperative system modified from the AGES scale. This scale incorporates distant Metastases, Age at presentation (<40 or >40 years old), Completeness of original surgical resection, extrathyroidal Invasion, and Size of original lesion (in centime-ters) and classifies patients into four risk groups based on their scores. Cady proposed the AMES system27 to classify differen-tiated thyroid tumors into lowand high-risk groups using Age (men <40 years old, women <50 years old), Metastases, Extra-thyroidal spread, and Size of tumors (< or >5 cm). A simplified system by DeGroot and associates28 uses four groups—class I (intrathyroidal), class II (cervical nodal metastases),

1	Metastases, Extra-thyroidal spread, and Size of tumors (< or >5 cm). A simplified system by DeGroot and associates28 uses four groups—class I (intrathyroidal), class II (cervical nodal metastases), class III (extrathyroidal invasion), and class IV (distant metastases)—to determine prognosis. Another classification system is the TNM system (Tumor, Nodal status, Metastases; Table 38-6), which used by most medical centers in North America and has been recently updated.29 In this new version, minimal extrathyroidal extension is no longer considered T3a disease. Thyroglobulin doubling time (using levels obtained when TSH is <0.1 mIU/L) has also been demonstrated to be an independent prognostic marker for metastatic disease and recurrence.30Several molecular and genetic markers such as tumor DNA aneuploidy, decreased cyclic adenosine monophosphate response to TSH, increased epidermal growth factor binding, presence of N-ras and gsp mutations, overexpression of c-myc, and presence of p53

1	DNA aneuploidy, decreased cyclic adenosine monophosphate response to TSH, increased epidermal growth factor binding, presence of N-ras and gsp mutations, overexpression of c-myc, and presence of p53 mutations also have been associated with a worse prognosis. The presence of BRAF V600E mutation, as previously mentioned, is associated with aggressive tumor characteristics, including extrathyroidal extension, older age at presentation, and lymph node and distant metastases. This muta-tion also appears to be an independent predictor of both tumor recurrence (even for early-stage disease) and tumor-related mortality. Some studies propose that BRAF mutation status on FNAB can be used to tailor initial management including more extensive initial surgical excision, high-dose postoperative RAI therapy, increased TSH suppression, and closer follow-up.31 The correlation of RET/PTC rearrangements and Ras mutations with prognosis is less clear. TERT promoter mutations have been associated with

1	therapy, increased TSH suppression, and closer follow-up.31 The correlation of RET/PTC rearrangements and Ras mutations with prognosis is less clear. TERT promoter mutations have been associated with poor disease-specific and disease-free survival.Surgical Treatment. Most authors agree that patients with high-risk tumors (judged by any of the classification systems discussed earlier in “Prognostic Indicators”) or bilateral tumors should undergo total or near-total thyroidectomy. The optimal surgical strategy in the majority of patients with low-risk (small, unilateral) cancers was controversial for many years, with the focus of the debate centering around outcome data and risks associated with extent of thyroidectomy in this group of patients. Proponents of total thyroidectomy indicate that it enables the use of RAI to effectively detect and treat residual thyroid tissue or metastatic disease and makes serum Tg level a more sensitive marker of recurrent or persistent disease. It is

1	that it enables the use of RAI to effectively detect and treat residual thyroid tissue or metastatic disease and makes serum Tg level a more sensitive marker of recurrent or persistent disease. It is also known that a significant proportion (33% to 50%) of patients who develop a recurrence die from their disease,32 and even though the data are retrospective, long-term, follow-up studies suggest that recur-rence rates are lowered and that survival is improved in patients undergoing near-total or total thyroidectomy28,32-36 (Fig. 38-17). In addition, diminished survival is noted in patients with low-risk disease (mortality rates of 5% at 10 to 20 years), and it is not possible to accurately risk stratify patients preoperatively. In the last 10 years, a large study of >50,000 patients with papillary cancer demonstrated that, in multivariate analyses, total thyroid-ectomy led to a significantly improved recurrence and survival for tumors >1 cm. Furthermore, the authors also showed that

1	papillary cancer demonstrated that, in multivariate analyses, total thyroid-ectomy led to a significantly improved recurrence and survival for tumors >1 cm. Furthermore, the authors also showed that patients with tumors 1 to 2 cm in diameter who were treated with lobectomy had a 24% higher risk of recurrence and a 49% higher risk of thyroid cancer mortality.37 Based on this informa-tion, the American Thyroid Association 2009 guidelines for the evidence-based management of thyroid cancers recommended a near-total or total thyroidectomy for primary cancers >1 cm unless there are contraindications to the surgery.38 However, additional studies since then have demonstrated no survival dif-ferences based on initial surgical procedure when adjusting for complexity/risk and comorbid diseases.39 This finding, coupled with a trend for increased use of ultrasound and Tg measure-ments to assess for recurrences and the declining use of RAI ablation, led to revised guidelines in 2015. Accordingly,

1	finding, coupled with a trend for increased use of ultrasound and Tg measure-ments to assess for recurrences and the declining use of RAI ablation, led to revised guidelines in 2015. Accordingly, either near-total/total thyroidectomy or lobectomy constitute appropriate initial treatment for tumors >1 cm and <4 cm without extrathyroidal extension or lymph node involvement (cN0). Of note, the guidelines do state that the treatment team may elect near-total/total thyroidectomy to facilitate RAI ther-apy, enhance follow-up based on disease features, or if the patient expresses a preference for complete thyroid excision.40There has also been in change in the management of pap-illary microcarcinomas (<1 cm) since at least two trials from Japan have shown that active surveillance (defined as observa-tion without immediate surgery) can be a viable and safe first line of treatment for these very-low-risk tumors without extra-thyroidal extension or lymph node metastases.41 Tumors that progress

1	without immediate surgery) can be a viable and safe first line of treatment for these very-low-risk tumors without extra-thyroidal extension or lymph node metastases.41 Tumors that progress during monitoring are treated by surgery. If surgery is chosen as initial treatment for these patients, a thyroid lobec-tomy is considered sufficient.When PTC is diagnosed by FNAB, the definitive operation can be done without confirming the diagnosis by frozen section during the operation. Patients with a nodule that is suspicious for papillary cancer should be treated by thyroid lobectomy, isthmu-sectomy, and removal of any pyramidal lobe or adjacent lymph nodes. If intraoperative frozen-section examination of a lymph node or primary tumor confirms carcinoma, completion total or near-total thyroidectomy is performed. If a definitive diagnosis cannot be made or the surgeon is concerned about the viability of the parathyroid glands or the status of the RLN, the opera-tion is terminated. When final

1	is performed. If a definitive diagnosis cannot be made or the surgeon is concerned about the viability of the parathyroid glands or the status of the RLN, the opera-tion is terminated. When final histology confirms carcinoma, completion thyroidectomy is performed if deemed necessary based on risk-stratification. During thyroidectomy, enlarged or obviously involved central neck nodes should be removed (ther-apeutic central-compartment, level VI), along with nodes with known lateral neck metastases. Some investigators recommend routine bilateral central neck dissection due to the high inci-dence of microscopic metastases and data showing improved rates of recurrence and survival (compared to historic controls). 5Brunicardi_Ch38_p1625-p1704.indd 164801/03/19 11:21 AM 1649THYROID, PARATHYROID, AND ADRENALCHAPTER 38Table 38-6TNM classification of thyroid tumors PAPILLAEY or FOLLICULAR TUMORSDIAGNOSIS AGETUMORNODEMETASTASISSTAGE<55 yearsAny TAny NM0I<55 yearsAny TAny NM1II≥55

1	PARATHYROID, AND ADRENALCHAPTER 38Table 38-6TNM classification of thyroid tumors PAPILLAEY or FOLLICULAR TUMORSDIAGNOSIS AGETUMORNODEMETASTASISSTAGE<55 yearsAny TAny NM0I<55 yearsAny TAny NM1II≥55 yearsT1N0/NXM0I≥55 yearsT1N1M0II≥55 yearsT2N0/NXM0I≥55 yearsT2N1M0II≥55 yearsT3a/T3bAny NM0II≥55 yearsT4aAny NM0III≥55 yearsT4bAny NM0IVA≥55 yearsAny TAny NM1IVBMEDULLARY THYROID CANCERTUMORNODEMETASTASISSTAGET1N0M0IT2N0M0IIT3N0M0IIT1–3N1aM0IIIT4aAny NM0IVAT1–3N1bM0IVAT4bAny NM0IVBAny TAny NM1IVCANAPLASTIC CANCERTUMORNODEMETASTASISSTAGET1–T3aN0/NXM0IVAT1–T3aN1M0IVBT3bAny NM0IVBT4Any NM0IVBAny TAny NM1IVCDEFINITIONSPrimary tumor (T)TX = Primary tumor cannot be assessedT0 = No evidence of primary tumorT1 = Tumor ≤2 cm in greatest dimension limited to the thyroid T1a = Tumor ≤1 cm in greatest dimension limited to the thyroid T1b = Tumor >1 cm but ≤2 cm in greatest dimension limited to the thyroidT2 = Tumor >2 cm but ≤4 cm in greatest dimension limited to the thyroidT3 = Tumor >4 cm limited to

1	limited to the thyroid T1b = Tumor >1 cm but ≤2 cm in greatest dimension limited to the thyroidT2 = Tumor >2 cm but ≤4 cm in greatest dimension limited to the thyroidT3 = Tumor >4 cm limited to the thyroid, or gross extrathyroidal extension invading only strap muscles T3a = Tumor >4 cm limited to the thyroid T3b = Gross extrathyroidal extension invading only strap muscles (sternohyoid, sternothyroid, thyrohyoid, or omohyoid muscles) from a tumor of any sizeT4 = Includes gross extrathyroidal extension beyond the strap muscles T4a = Gross extrathyroidal extension invading subcutaneous soft tissues, larynx, trachea, esophagus, or recurrent laryngeal nerve from a tumor of any size T4b = Gross extrathyroidal extension invading prevertebral fascia or encasing the carotid artery or mediastinal vessels from a tumor of any sizeRegional lymph nodes (N)NX = Regional lymph nodes cannot be assessedN0 = No evidence of locoregional lymph node metastasis N0a = One or more cytologically or

1	vessels from a tumor of any sizeRegional lymph nodes (N)NX = Regional lymph nodes cannot be assessedN0 = No evidence of locoregional lymph node metastasis N0a = One or more cytologically or histologically confirmed benign lymph nodes N0b = No radiologic or clinical evidence of locoregional lymph node metastasisN1 = Metastasis to regional nodes N1a = Metastasis to level VI or VII (pretracheal, paratracheal, or prelaryngeal/Delphian, or upper mediastinal) lymph nodes. This can be unilateral or bilateral disease. N1b = Metastasis to unilateral, bilateral, or contralateral lateral neck lymph nodes (levels I, II, III, IV, or V) or retropharyngeal lymph nodesDistant Metastasis (M)M0 = No distant metastasisM1 = Distant metastasisUsed with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Brunicardi_Ch38_p1625-p1704.indd 164901/03/19 11:21 AM 1650SPECIFIC CONSIDERATIONSPART IIHowever,

1	MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Brunicardi_Ch38_p1625-p1704.indd 164901/03/19 11:21 AM 1650SPECIFIC CONSIDERATIONSPART IIHowever, these risks need to be balanced with the increased risk of hypoparathyroidism with routine central neck dissection and the fact that some studies do not show any difference in recur-rence rates or rates of low or undetectable Tg levels. The updated 2015 ATA guidelines for thyroid cancer management suggest that prophylactic (ipsilateral or bilateral) dissection may be per-formed in patients with advanced (T3 or T4) papillary thyroid carcinoma, or if the lateral neck nodes are involved with tumor (N1b), or if the information will help in treatment planning.40 The American Head and Neck Society urges the involvement of multidisciplinary teams in the decision-making process for pro-phylactic CND and indicates that it can be considered in patients with high risk of recurrence (to include older

1	the involvement of multidisciplinary teams in the decision-making process for pro-phylactic CND and indicates that it can be considered in patients with high risk of recurrence (to include older or young age, mul-tifocal disease and extrathyroidal extension in addition to the aforementioned factors).42 Further prospective studies are needed before definitive recommendations can be made in this regard.Biopsy-proven lymph node metastases detected clini-cally or by imaging in the lateral neck in patients with papil-lary carcinoma are managed with modified radical or functional neck dissection,40 as described later in this chapter in “Thyroid Surgery.” Dissection of the posterior triangle and suprahyoid dissection usually are not necessary unless there is extensive metastatic disease in levels 2, 3, and 4, but should be performed when appropriate. Prophylactic lateral neck node dissection is not necessary in patients with PTC because these cancers do not appear to metastasize systemically

1	2, 3, and 4, but should be performed when appropriate. Prophylactic lateral neck node dissection is not necessary in patients with PTC because these cancers do not appear to metastasize systemically from lymph nodes, and micrometastases often can be ablated with RAI therapy.Follicular Carcinoma Follicular carcinomas account for 10% of thyroid cancers and occur more commonly in iodinedeficient areas. The overall incidence of this tumor is declining in the United States, probably due to iodine supplementation and improved histologic classification. Women have a higher incidence of follicular cancer, with a female-to-male ratio of 3:1, and a mean age at presentation of 50 years old. Follicular cancers usually present as solitary thyroid nodules, occasion-ally with a history of rapid size increase, and long-standing goi-ter. Pain is uncommon, unless hemorrhage into the nodule has occurred. Unlike papillary cancers, cervical lymphadenopathy is uncommon at initial presentation (about 5%),

1	and long-standing goi-ter. Pain is uncommon, unless hemorrhage into the nodule has occurred. Unlike papillary cancers, cervical lymphadenopathy is uncommon at initial presentation (about 5%), although distant metastases may be present. In <1% of cases, follicular cancers may be hyperfunctioning, leading patients to present with signs and symptoms of thyrotoxicosis. FNAB is unable to distinguish benign follicular lesions from follicular carcinomas. Therefore, preoperative clinical diagnosis of cancer is difficult unless dis-tant metastases are present. Large follicular tumors (>4 cm) in older men are more likely to be malignant.Due to the limitations inherent in the FNAB diagnosis, a number of studies have focused on identifying molecular mark-ers to distinguish benign from malignant follicular lesions. Many of these genetic changes can be identified using tissue obtained during FNAB. While no single marker has met the ideal charac-teristics of being simple to use, reproducible, and

1	lesions. Many of these genetic changes can be identified using tissue obtained during FNAB. While no single marker has met the ideal charac-teristics of being simple to use, reproducible, and cost-effective, several combinations of markers appear to be useful in differen-tiating benign from malignant lesions. A commonly used panel of seven genes used to “rule in” malignancy detects mutations in BRAF, Ras, RET/PTC, and PAX/PPARg and has been associated with a sensitivity of 57% to 75%, specificity of 97% to 100%, PPV of 87% to 100%, and NPV of 79% to 86% in the case of nodules consistent with Follicular/Hürthle cell neoplasm or sus-picious of the same.43 In contrast, the Afirma Gene Expression Classifier (GEC) uses a “rule out” strategy to identify benign nodules. This method uses material from additional FNA passes (in an RNA-preserving solution) to analyze a 167 gene panel, and the results obtained are reported as benign or suspicious.44 It is reported to have a lower PPV of 37% but

1	from additional FNA passes (in an RNA-preserving solution) to analyze a 167 gene panel, and the results obtained are reported as benign or suspicious.44 It is reported to have a lower PPV of 37% but a better NPV of 94% for Follicular/Hürthle cell neoplasms. Next generation sequenc-ing techniques have been used to enhance malignancy detection Less than near-totalthyroidectomyP = 0.02Less thannear-totalthyroidectomy00510152025303524681012Cumulative death (%)Near-total or totalthyroidectomyNear-totalor totalthyroidectomyYears after initial therapy04361135262907227218111200640290698555464416365131432092100032Figure 38-17. Improved survival in patients with papillary or follicular thyroid cancer following total or near-total thyroidectomy compared to those who underwent less than near-total thyroidectomy. (Reproduced with permission from Mazzaferri E, Jhiang S: Long-term impact of initial surgical and medical therapy on papillary and follicular thyroid cancer, Am J Med. 1994

1	near-total thyroidectomy. (Reproduced with permission from Mazzaferri E, Jhiang S: Long-term impact of initial surgical and medical therapy on papillary and follicular thyroid cancer, Am J Med. 1994 Nov;97(5):418-428.)Brunicardi_Ch38_p1625-p1704.indd 165001/03/19 11:21 AM 1651THYROID, PARATHYROID, AND ADRENALCHAPTER 38by including additional mutations and gene arrangements. The advanced version of this assay (ThyroSeqV2) had a sensitivity of 90%, specificity of 93%, PPV of 83%, and NPV of 96% in a study of Follicular/Hüurthle cell neoplasm/suspicious of the same nodules, making it useful as both a “rule in” and “rule out” test.45 Of note, these assays have also been evaluated in “AUS/FLUS” and “suspicious for malignancy” nodules with varying results, i.e., the performance characteristics of these tests have been noted to change depending upon the prevalence of malig-nancy in the tested population (pretest probability). At this time, the ATA guidelines do not advise molecular

1	of these tests have been noted to change depending upon the prevalence of malig-nancy in the tested population (pretest probability). At this time, the ATA guidelines do not advise molecular testing in the work-up of “suspicious for malignancy” nodules. Molecular testing may be used to supplement cytology results for malignancy risk assessment in “AUS/FLUS” or “follicular/Hürthle cell neo-plasm/suspicious of the same” nodules depending on feasibility and informed patient preference.40 Expression arrays also have been used to investigate the role of microRNAs, which are a new class of small, noncoding RNAs that have been implicated in carcinogenesis. The specific microRNAs miR-197 and miR-346 are upregulated in follicular thyroid cancers46 and have the potential to be used as diagnostic markers. Additional studies have also demonstrated the feasibility of studying a panel of microRNAs in a small number of FNA samples. ThyGenX/ThyraMIR uses a mutation panel supplemented with 10 miRNA

1	markers. Additional studies have also demonstrated the feasibility of studying a panel of microRNAs in a small number of FNA samples. ThyGenX/ThyraMIR uses a mutation panel supplemented with 10 miRNA markers,47 whereas Rosetta GX Reveal is exclusively based on miRNA markers; however, both require further validation.48Pathology. Follicular carcinomas usually are solitary lesions, and the majority are surrounded by a capsule. Histologically, follicles are present, but the lumen may be devoid of colloid. Architectural patterns depend on the degree of differentiation demonstrated by the tumor. Malignancy is defined by the pres-ence of capsular and vascular invasion (Fig. 38-18). In general, minimally invasive tumors appear grossly encapsulated and have microscopic invasion through the tumor capsule without extension into the parenchyma and/or invasion into smallto medium-sized vessels (venous caliber) in or immediately out-side the capsule, but not within the tumor. On the other hand,

1	capsule without extension into the parenchyma and/or invasion into smallto medium-sized vessels (venous caliber) in or immediately out-side the capsule, but not within the tumor. On the other hand, widely invasive tumors demonstrate evidence of large vessel invasion and/or broad areas of tumor invasion through the cap-sule. They may, in fact, be unencapsulated. It is important to note that there is a wide variation of opinion among clinicians and pathologists with respect to the above definitions. Tumor infiltration and invasion, as well as tumor thrombus within the middle thyroid or jugular veins, may be apparent at operation.Surgical Treatment and Prognosis. Patients diagnosed by FNAB as having a follicular lesion should undergo thyroid lobectomy because at least 70% to 80% of these patients will have benign adenomas. Total thyroidectomy is recommended by some surgeons in older patients with follicular lesions >4 cm because of the higher risk of cancer in this setting (50%) and

1	patients will have benign adenomas. Total thyroidectomy is recommended by some surgeons in older patients with follicular lesions >4 cm because of the higher risk of cancer in this setting (50%) and certainly should be performed in patients with atypia on FNA, a family history of thyroid cancer, or a history of radiation exposure. Intraoperative frozen-section examination usually is not helpful, but it should be performed when there is evidence of capsular or vascular invasion or when adjacent lymphadenopa-thy is present. Total thyroidectomy should be performed when thyroid cancer is diagnosed. There is debate among experts about whether patients with minimally invasive follicular can-cers should undergo completion thyroidectomy because the prognosis is so good in these patients. A diagnosis of frankly invasive carcinoma or follicular carcinoma with angioinvasion, with or without capsular invasion, necessitates completion of total thyroidectomy primarily so that 131I can be used to

1	of frankly invasive carcinoma or follicular carcinoma with angioinvasion, with or without capsular invasion, necessitates completion of total thyroidectomy primarily so that 131I can be used to detect and ablate metastatic disease. Prophylactic nodal dissection is not needed because nodal involvement is infrequent, but in the unusual patient with nodal metastases, therapeutic neck dissec-tion is recommended. Prophylactic central neck dissection may be considered in patients with large tumors. The cumulative mortality from follicular thyroid cancer is approximately 15% at 10 years and 30% at 20 years. Poor long-term prognosis is predicted by age over 50 years old at presentation, tumor size >4 cm, higher tumor grade, marked vascular invasion, extrathy-roidal invasion, and distant metastases at the time of diagnosis.Hürthle Cell Carcinoma Hürthle cell carcinomas account for approximately 3% of all thyroid malignancies and, under the World Health Organization classification, are

1	at the time of diagnosis.Hürthle Cell Carcinoma Hürthle cell carcinomas account for approximately 3% of all thyroid malignancies and, under the World Health Organization classification, are considered to be a subtype of follicular thyroid cancer. Hürthle cell cancers also are characterized by vascular or capsular invasion and, there-fore, cannot be diagnosed by FNAB. Tumors contain sheets of eosinophilic cells packed with mitochondria, which are derived from the oxyphilic cells of the thyroid gland. Hürthle cell tumors differ from follicular carcinomas in that they are more often multifocal and bilateral (about 30%), usually do not take up RAI (about 5%), are more likely to metastasize to local nodes (25%) and distant sites, and are associated with a higher mortal-ity rate (about 20% at 10 years). Hence, they are considered to be a separate class of tumors by some groups.Management is similar to that of follicular neoplasms, with lobectomy and isthmusectomy being sufficient surgical

1	10 years). Hence, they are considered to be a separate class of tumors by some groups.Management is similar to that of follicular neoplasms, with lobectomy and isthmusectomy being sufficient surgical treatment for unilateral Hürthle cell adenomas. When Hürthle cell neoplasms are found to be invasive on definitive paraffin-section histology, then total thyroidectomy should be performed. These patients should also undergo routine central neck node removal, similar to patients with MTC, and modified radical neck dissection when lateral neck nodes are palpable or identi-fied by ultrasonography. Although RAI scanning and ablation usually are ineffective, they probably should be considered to ablate any residual normal thyroid tissue and occasionally ablate tumors because there is no other good therapy.Postoperative Management of Differentiated Thyroid Cancer Radioiodine Therapy The issue of whether RAI therapy offers any benefit to patients with differentiated thyroid cancer Figure

1	good therapy.Postoperative Management of Differentiated Thyroid Cancer Radioiodine Therapy The issue of whether RAI therapy offers any benefit to patients with differentiated thyroid cancer Figure 38-18. Hematoxylin-eosin–stained section from follicular thyroid carcinoma showing capsular invasion.Brunicardi_Ch38_p1625-p1704.indd 165101/03/19 11:21 AM 1652SPECIFIC CONSIDERATIONSPART IIremains controversial in the absence of prospective, random-ized controlled trials. Long-term cohort studies by Mazzaferri and associates and DeGroot and colleagues demonstrate that postoperative RAI therapy reduces recurrence (Fig. 38-19) and provides a small improvement in survival, even in low-risk patients.28,34 Screening with RAI is more sensitive than chest X-ray or CT scanning for detecting metastases; however, it is less sensitive than Tg measurements for detecting metastatic disease in most differentiated thyroid cancers except Hürthle cell tumors. Screening and treatment are facilitated by

1	however, it is less sensitive than Tg measurements for detecting metastatic disease in most differentiated thyroid cancers except Hürthle cell tumors. Screening and treatment are facilitated by the removal of all normal thyroid tissue, which effectively competes for iodine uptake. Metastatic differentiated thyroid carcinoma can be detected and treated by 131I in about 75% of patients. Multiple studies show that RAI effectively treats >70% of lung micrometastases that are detected by RAI scan in the presence of a normal chest X-ray, whereas the success rates drop to <10% with pulmonary macrometastases. Early detection therefore is very important to improve prognosis.Several features place patients at increased risk for local recurrences or metastases. The 2015 ATA guidelines use various features to risk-stratify tumors.40 Low-risk papillary thyroid cancer includes those without local tumor invasion, all macroscopic tumor resected, absence of aggressive histol-ogy (e.g., tall cell,

1	features to risk-stratify tumors.40 Low-risk papillary thyroid cancer includes those without local tumor invasion, all macroscopic tumor resected, absence of aggressive histol-ogy (e.g., tall cell, columnar cell carcinoma), no known dis-tant metastases (clinical or on RAI scan if done), no vascular invasion, clinical N0 or ≤5 pathologic N1 micrometastases (<0.2 cm in largest dimension), intrathyroidal, encapsulated fol-licular variant of papillary thyroid cancer, intrathyroidal, well differentiated follicular thyroid cancer with capsular invasion and no or minimal (<4 foci) vascular invasion and intrathyroi-dal papillary microcarcinoma (unifocal or multifocal, including BRAFV600E mutated). Intermediate-risk tumors include those showing microscopic invasion of tumor into the perithyroidal soft tissues or RAI-avid metastatic foci in the neck on the first posttreatment whole-body RAI scan. This group also includes tumors with aggressive histology (e.g., tall cell, columnar cell

1	soft tissues or RAI-avid metastatic foci in the neck on the first posttreatment whole-body RAI scan. This group also includes tumors with aggressive histology (e.g., tall cell, columnar cell carcinoma), papillary thyroid cancer with vascular invasion, clinical N1 or >5 pathologic N1 with all involved lymph nodes <3 cm in largest dimension and multifocal papillary microcar-cinoma with extra-thyroidal extension (ETE) and BRAFV600E mutated (if known). High-risk tumors include those demon-strating macroscopic invasion of tumor into the perithyroidal soft tissues (gross ETE), incomplete tumor resection, and pres-ence of distant metastases (or postoperative serum thyroglobu-lin suggestive of distant metastase) or pathologic N1 with any metastatic lymph node ≥3 cm in largest dimension. Follicular thyroid cancers with extensive vascular invasion (>4 foci of vascular invasion) also fall into this category. It is important to note that this risk assessment represents a continuum with recurrence

1	thyroid cancers with extensive vascular invasion (>4 foci of vascular invasion) also fall into this category. It is important to note that this risk assessment represents a continuum with recurrence rates from 1% to 2% for low-risk cancers to >50% for high-risk cancers.The current ATA guidelines recommend RAI therapy after surgical treatment for all patients with high-risk disease, i.e., those with gross ETE and M1 disease. RAI therapy is not rec-ommended for patients with papillary microcarcinomas, either uni -or multifocal. RAI remnant ablation is not routinely recom-mended after thyroidectomy for ATA low-risk DTC patients. However, it may be considered in patients with aggressive histology or vascular invasion. Consideration of RAI is recom-mended for patients with intermediate-risk disease and “gen-erally favored” for patients with microscopic ETE due to the risk of recurrent disease, large (>2–3 cm) or clinically evident lymph nodes (central, mediastinal, and lateral neck) or

1	disease and “gen-erally favored” for patients with microscopic ETE due to the risk of recurrent disease, large (>2–3 cm) or clinically evident lymph nodes (central, mediastinal, and lateral neck) or presence of extranodal extension. Advancing age may also favor RAI use. However, RAI is not needed for patients with a few (<5) microscopic nodal metastases in the central compartment in the absence of other adverse features as there is insufficient evi-dence for its utility in this setting. It is generally favored for patients with lateral neck disease. There is currently no estab-lished role for molecular testing in determining RAI therapy.Remnant ablation can be performed with either thyroid hormone withdrawal or recombinant TSH (rTSH) stimula-tion. This is based on randomized studies showing that both techniques are equally effective in preparing patients for abla-tion, with the latter being associated with an improved quality of life.49,50 In patients with ATA high-risk disease

1	showing that both techniques are equally effective in preparing patients for abla-tion, with the latter being associated with an improved quality of life.49,50 In patients with ATA high-risk disease (including distant metastases), there is insufficient data to recommend thy-rogen-mediated ablation, and hormone withdrawal is preferred. Furthermore, if patients have comorbidities that can be exac-erbated by severe hypothyroidism (cardiac or psychiatric con-ditions), consideration should be given to thyrogen-mediated RAI. If hormone withdrawal is used, T4 therapy should be dis-continued for approximately 6 weeks before scanning with 131I. Patients should receive T3 during this time period to decrease the period of hypothyroidism. T3 has a shorter half-life than T4 (1 day vs. 1 week) and needs to be discontinued for 2 weeks to allow TSH levels to rise before treatment. Levels >30 mU/L are considered optimal, based on noncontrolled studies. A low-iodine diet also is recommended during this

1	to be discontinued for 2 weeks to allow TSH levels to rise before treatment. Levels >30 mU/L are considered optimal, based on noncontrolled studies. A low-iodine diet also is recommended during this 2-week period. The usual protocol involved administering a screening dose of 1 to 3 mCi and measuring uptake 24 hours later. After a total thy-roidectomy, this value should be <1%. A “hot” spot in the neck after initial screening usually represents residual normal tissue in the thyroid bed. Some investigators recommend omitting the scanning dose altogether to minimize thyrocyte “stunning” and subsequent requirement for higher treatment doses. Others recommend scanning only if the size of the remnant cannot be determined by the operative report or ultrasound, or if the results would alter the decision to treat or the dose to be administered. Current guidelines recommend using either 123I or low-activity 131I (1to 3-mCi dose) and delivering a therapeutic dose within 72 hours.The recommended

1	to treat or the dose to be administered. Current guidelines recommend using either 123I or low-activity 131I (1to 3-mCi dose) and delivering a therapeutic dose within 72 hours.The recommended dose of RAI is 30 mCi if remnant abla-tion is performed after total thyroidectomy for ATA low-risk thyroid or intermediate-risk cancer with lower risk features (i.e., low-volume central neck nodal metastases with no other known gross residual disease or any other adverse features). If RAI is given for adjuvant treatment to treat suspected microscopic dis-ease (in the absence of metastatic disease), doses ranging from 30 to 150 mCi are recommended, and there is no solid evidence to show that higher doses reduce the recurrence rates for T3 and N1 disease in this setting.If patients have an elevated Tg level, but negative RAI scan on follow-up, some physicians recommend treating once with 100 mCi of 131I and repeating the imaging 1 to 2 weeks later. Approximately one-third of these patients

1	Tg level, but negative RAI scan on follow-up, some physicians recommend treating once with 100 mCi of 131I and repeating the imaging 1 to 2 weeks later. Approximately one-third of these patients demonstrate uptake on posttreatment imaging, and Tg levels usually decrease in these patients, documenting therapeutic benefit. The maxi-mum dose of radioiodine that can be administered at one time without performing dosimetry is approximately 200 mCi with a cumulative dose of 1000 to 1500 mCi. Up to 500 mCi can be given with proper pretreatment dosimetry. Recent studies also Brunicardi_Ch38_p1625-p1704.indd 165201/03/19 11:21 AM 1653THYROID, PARATHYROID, AND ADRENALCHAPTER 38Initial medical therapyall recurrencesNoneT4 aloneP< .05403530252015105P< .0001Years after initial therapyPercent cancer recurrences605040302010004035302520151050NoneT4 + RAI T4 alone T4 + RAI remnant

1	therapyall recurrencesNoneT4 aloneP< .05403530252015105P< .0001Years after initial therapyPercent cancer recurrences605040302010004035302520151050NoneT4 + RAI T4 alone T4 + RAI remnant ablation34/16315/230101/78922/1120/13551/6035/823/10217/4621/661/847/3782/541/6710/3264/390/386/2290/250/2610/1350/130/181/51Initial medical therapydistant recurrencesNoneT4 aloneP<.0002P<.02Years after initial therapyPercent distant recurrence35302520151050None 8/163 9/112 3/82 0/66 1/54 4/39 0/25 0/13T4 alone 19/789 15/603 7/462 0/378 2/326 4/229 8/135 1/51T4 + RAI 2/230 0/135 1/102 0/84 1/67 0/38 0/26 0/11T4 + RAI remnant ablationABFigure 38-19. Tumor recurrence at a median of 16.7 years after thyroid surgery. The numerator is the number of patients with recurrence, and the denominator is the number of patients in each time interval. The P values

1	recurrence at a median of 16.7 years after thyroid surgery. The numerator is the number of patients with recurrence, and the denominator is the number of patients in each time interval. The P values are derived from log-rank statistical analysis of 40-year life-table data. Figure shows that all recurrences (A) and distant metastases (B) were reduced in patients who received radioactive iodine (RAI) in addition to thyroxine (T4) therapy. (Reproduced with permission from Mazzaferri E, Kloos R: Current approaches to primary therapy for papillary and follicular thyroid cancer, Clin Endocrinol Metab. 2001 Apr;86(4):1447-1363.)Brunicardi_Ch38_p1625-p1704.indd 165301/03/19 11:21 AM 1654SPECIFIC CONSIDERATIONSPART IIshow an increase in the number of second cancers in patients treated with RAI.51 The early and delayed complications of RAI therapy are listed in Table 38-7.Thyroid Hormone T4 is necessary as replacement therapy in patients after total or near-total thyroidectomy, and also has

1	The early and delayed complications of RAI therapy are listed in Table 38-7.Thyroid Hormone T4 is necessary as replacement therapy in patients after total or near-total thyroidectomy, and also has the additional effect of suppressing TSH and reducing the growth stimulus for any possible residual thyroid cancer cells. TSH suppression reduces tumor recurrence rates. Current guidelines advise maintaining initial TSH levels <0.1 mU/mL in patients with high-risk thyroid cancer and in the range of 0.1 to 0.5 mU/mL in patients with intermediate-risk disease. For low-risk patients (with or without remnant ablation) with undetectable serum Tg levels, TSH levels can be maintained at the lower end of the reference range (0.5–2 mU/L). If these patients have low measureable Tg levels, it is recommended that TSH be maintained at or slightly below lower limit of normal (0.1 to 0.5 mU/L) while continuing surveillance for recurrence. In low-risk patients treated with lobectomy alone, it is advised

1	that TSH be maintained at or slightly below lower limit of normal (0.1 to 0.5 mU/L) while continuing surveillance for recurrence. In low-risk patients treated with lobectomy alone, it is advised to keep TSH in the mid to lower reference range (0.5–2 mU/L), and hor-mone therapy may be needed to maintain these levels. Further TSH suppression levels are determined by response to therapy. The risk of tumor recurrence must be balanced with the side effects associated with prolonged TSH suppression, including osteopenia and cardiac problems, particularly in older patients.Follow-Up of Patients With Differentiated Thyroid Cancer Thyroglobulin Measurement Tg and anti-Tg antibody levels should be measured initially at 6 to 12 month intervals and more frequently in patients with high-risk tumors. Further measure-ments are guided by response to therapy. Patients are considered to have an excellent response to treatment if suppressed Tg is <0.2 ng/mL and stimulated Tg is <1 ng/mL with negative

1	Further measure-ments are guided by response to therapy. Patients are considered to have an excellent response to treatment if suppressed Tg is <0.2 ng/mL and stimulated Tg is <1 ng/mL with negative imag-ing. In these patients, Tg levels can be followed every 12 to 24 months while on thyroid hormone as their risk of recurrence is low (1–4%). Patients with structurally or biochemically incomplete (negative imaging but suppressed Tg ≥1 ng/mL or stimulated Tg ≥10 ng/mL or rising anti-Tg levels) or indeter-minate responses (nonspecific imaging findings, suppressed Tg detectable but <1 ng/mL, and stimulated Tg detectable but <10 ng/mL or stable or declining anti-Tg levels) require addi-tional investigations.40 Tg measurements in FNAB aspirates have also been shown to be useful in the detection of nodal metastatic disease.52Imaging After the first posttreatment scan, lowand some intermediate-risk patients with negative TSH-stimulated Tg and cervical ultrasound do not require routine

1	of nodal metastatic disease.52Imaging After the first posttreatment scan, lowand some intermediate-risk patients with negative TSH-stimulated Tg and cervical ultrasound do not require routine diagnostic whole-body radioiodine scans. However, diagnostic whole-body scans 6 to 12 months after remnant ablation may be of value in the follow-up of patients with highor intermediate-risk patients with higher risk features. Other scenarios for follow-up scans include patients with abnormal uptake outside the thyroid bed on posttherapy scan, those with poorly informative postablation scans (e.g., due to high thyroid bed uptake), and patients with Tg antibodies.Cervical ultrasound be performed to evaluate the thyroid bed and central and lateral cervical nodal compartments at 6 and 12 months after thyroidectomy and then annually for at least 3 to 5 years, depending on the patient’s risk for recurrent disease and Tg status. Sonographically suspicious nodes ≥8 to 10 mm on the smallest diameter

1	thyroidectomy and then annually for at least 3 to 5 years, depending on the patient’s risk for recurrent disease and Tg status. Sonographically suspicious nodes ≥8 to 10 mm on the smallest diameter measurement should be biopsied for cytology as well as Tg measurement in the aspirate washout. Smaller nodes can be followed and biopsied if there is contin-ued growth. FDG-PET and PET-CT scans have been shown to be useful to localize recurrent or persistent thyroid cancer in patients who have Tg-positive, RAI scan–negative disease. FDG-PET can also be useful for the initial staging of patients with poorly differentiated thyroid carcinomas or Hürthle cell tumors, particularly in patients with other evidence of disease on imaging or Tg levels. In addition, they may be used as a prog-nostic tool in patients with metastatic disease and to evaluate the response to treatment in patients with metastatic or locally advanced disease.Additional Treatment Modalities Radiotherapy, Thermal Ablation,

1	in patients with metastatic disease and to evaluate the response to treatment in patients with metastatic or locally advanced disease.Additional Treatment Modalities Radiotherapy, Thermal Ablation, and Chemotherapy External-beam radiotherapy is occasionally required to control unresectable, locally invasive, or recurrent disease and to treat metastases in support bones to decrease the risk of fractures. It also is of value for the treatment and control of pain from bony metastases when there is minimal or no RAIU. Stereotac-tic brain radiotherapy and intensity-modulated radiation therapy have both been used successfully for metastatic lesions. Percuta-neous thermal ablation by increasing (radiofrequency ablation) or decreasing temperature (cryoablation) in the lesion to induce irreversible cellular damage has shown promise for lung, bone, and liver lesions.53,54 Single-drug and multidrug chemotherapy has been used with little success in disseminated thyroid can-cer, and there is no

1	cellular damage has shown promise for lung, bone, and liver lesions.53,54 Single-drug and multidrug chemotherapy has been used with little success in disseminated thyroid can-cer, and there is no role for routine chemotherapy. Doxorubicin (Adriamycin) and paclitaxel (Taxol) were previously the most frequently used agents. The former acts as a radiation sensitizer and should be considered in patients undergoing external-beam radiation.Novel Therapies These therapies are directed at the molecular pathways known to be involved in thyroid cancers. Sorafenib Table 38-7Complications of radioactive iodine therapy (131I) and doses at which they are observedACUTELONG-TERMNeck pain, swelling, and tendernessThyroiditis (if remnant present)Sialadenitis (50–450 mCi), taste dysfunctionHemorrhage (brain metastases)Cerebral edema (brain metastases, 200 mCi)Vocal cord paralysisNausea and vomiting (50–450 mCi)Bone marrow suppression (200 mCi)HematologicBone marrow suppression (>500 mCi)Leukemia (>1000

1	metastases)Cerebral edema (brain metastases, 200 mCi)Vocal cord paralysisNausea and vomiting (50–450 mCi)Bone marrow suppression (200 mCi)HematologicBone marrow suppression (>500 mCi)Leukemia (>1000 mCi)FertilityOvarian/testicular damage, infertilityIncreased spontaneous abortion ratePulmonary fibrosisChronic sialadenitis, nodules, taste dysfunctionIncreased risk of cancerAnaplastic thyroid cancerGastric cancerHepatocellular cancerLung cancerBreast cancer (>1000 mCi)Bladder cancerHypoparathyroidismBrunicardi_Ch38_p1625-p1704.indd 165401/03/19 11:21 AM 1655THYROID, PARATHYROID, AND ADRENALCHAPTER 38and lenvatinib are U.S. Food and Drug Administration (FDA) and European Medical Agency (EMA)-approved for use in patients with advanced differentiated thyroid cancer that is non-responsive to RAI after evaluation in phase 3 placebo-controlled double blinded trials (the DECISION study and the SELECT study, respectively).55,56 Both drugs are multikinase inhibitors and target RET kinase

1	to RAI after evaluation in phase 3 placebo-controlled double blinded trials (the DECISION study and the SELECT study, respectively).55,56 Both drugs are multikinase inhibitors and target RET kinase and the vascular endothelial growth factor (VEGF)-receptor; however, lenvatinib also inhib-its the fibroblast growth factor and the platelet-derived growth factor receptor. Sorafenib demonstrated progression-free sur-vival (PFS) improvement by 5 months with about 12% partial response rates, whereas lenvatinib prolonged median PFS by 15.7 months compared with placebo with response rates of 65%, including some complete responses. Vandetanib is mainly a RET-kinase inhibitor, but it also affects the VEGF-receptor and epidermal growth factor receptor. It has been evaluated in a phase 2 trial and also demonstrated an improved PFS.57 However, none of the agents show improvements in overall survival. Moreover, they are associated with significant side effects (diarrhea, fatigue hypertension,

1	also demonstrated an improved PFS.57 However, none of the agents show improvements in overall survival. Moreover, they are associated with significant side effects (diarrhea, fatigue hypertension, hepatotoxicity, bleed-ing, and thrombosis) that affect patient quality of life. As such, they are considered only in patients with metastatic, rapidly progressive, symptomatic disease that is unable to respond to other local treatment approaches and generally in the context of clinical trials. Oncogenic kinase inhibitors that selectively inhibit the mutant V600E BRAF kinase (dabrafenib) has also shown promise in treating a subset of patients with advanced differentiated thyroid cancer.58Medullary Carcinoma MTC accounts for about 5% of thyroid malignancies and arises from the parafollicular or C cells of the thyroid, which, in turn, are derived from the ultimobranchial bod-ies. These cells are concentrated superolaterally in the thyroid lobes, and this is where MTC usually develops. C cells

1	cells of the thyroid, which, in turn, are derived from the ultimobranchial bod-ies. These cells are concentrated superolaterally in the thyroid lobes, and this is where MTC usually develops. C cells secrete calcitonin, a 32-amino-acid polypeptide that functions to lower serum calcium levels, although its effects in humans are mini-mal. Most MTCs occur sporadically. However, approximately 25% occur within the spectrum of several inherited syndromes such as familial MTC, MEN2A, and MEN2B. All these variants are known to result secondary to germline mutations in the RET proto-oncogene. The syndromes also are characterized by geno-type-phenotype correlations, with specific mutations leading to particular clinical manifestations. The salient clinical and genetic features of these syndromes are outlined in Table 38-8. Some clinical features of MEN2B patients are shown in Fig. 38-20.Table 38-8Clinical and genetic features of medullary thyroid cancer syndromesSYNDROMEMANIFESTATIONSRET

1	are outlined in Table 38-8. Some clinical features of MEN2B patients are shown in Fig. 38-20.Table 38-8Clinical and genetic features of medullary thyroid cancer syndromesSYNDROMEMANIFESTATIONSRET MUTATIONSMEN2A MTC, pheochromocytoma, primary hyperparathyroidism, lichen planus amyloidosis Exon 10—codons 609, 611, 618, 620Exon 11—codon 634 (more commonly associated with pheochromocytoma and primary hyperparathyroidism)MEN2BMTC, pheochromocytoma, Marfanoid habitus, mucocutaneous ganglioneuromatosisExon 16—codon 918Familial MTCMTCCodons 609, 611, 618, 620, and 634 Codons 768, 790, 791, or 804 (rare)MEN2A and Hirschsprung’s diseaseMTC, pheochromocytoma, primary hyperparathyroidism, Hirschsprung’s diseaseCodons 609, 618, 620MEN2 = multiple endocrine neoplasia type 2; MTC = medullary thyroid cancer.BAFigure 38-20. Features of MEN2B: thickened lips (A) and mucosal neuromas (A and B).Brunicardi_Ch38_p1625-p1704.indd 165501/03/19 11:21 AM 1656SPECIFIC CONSIDERATIONSPART IIPatients with MTC

1	38-20. Features of MEN2B: thickened lips (A) and mucosal neuromas (A and B).Brunicardi_Ch38_p1625-p1704.indd 165501/03/19 11:21 AM 1656SPECIFIC CONSIDERATIONSPART IIPatients with MTC often present with a neck mass that may be associated with palpable cervical lymphadenopathy (15% to 20%). Pain or aching is more common in patients with these tumors, and local invasion may produce symptoms of dys-phagia, dyspnea, or dysphonia. Distant blood-borne metastases to the liver, bone (frequently osteoblastic), and lung occur later in the disease. The female-to-male ratio is 1.5:1. Most patients present between 50 and 60 years old, although patients with familial disease present at a younger age. Medullary thyroid tumors secrete not only calcitonin and carcinoembryonic anti-gen (CEA), but also other peptides such as calcitonin gene–related peptide, histaminadases, prostaglandins E2 and F2α, and serotonin. Patients with extensive metastatic disease frequently develop diarrhea, which may

1	other peptides such as calcitonin gene–related peptide, histaminadases, prostaglandins E2 and F2α, and serotonin. Patients with extensive metastatic disease frequently develop diarrhea, which may result from increased intestinal motility and impaired intestinal water and electrolyte flux. About 2% to 4% of patients develop Cushing’s syndrome as a result of ectopic production of adrenocorticotropic hormone (ACTH).Pathology. MTCs typically are unilateral (80%) in patients with sporadic disease and multicentric in familial cases, with bilateral tumors occurring in up to 90% of familial patients. Familial cases also are associated with C-cell hyperplasia, which is considered a premalignant lesion. Microscopically, tumors are composed of sheets of infiltrating neoplastic cells separated by collagen and amyloid. Marked heterogeneity is present; cells may be polygonal or spindle shaped. The presence of amyloid is a diagnostic finding, but immunohistochemistry for calcitonin is more commonly

1	and amyloid. Marked heterogeneity is present; cells may be polygonal or spindle shaped. The presence of amyloid is a diagnostic finding, but immunohistochemistry for calcitonin is more commonly used as a diagnostic tumor marker. These tumors also stain positively for CEA and calcito-nin gene–related peptide.Diagnosis. The diagnosis of MTC is established by history, physical examination, raised serum calcitonin, or CEA levels, and FNAB cytology of the thyroid mass. Attention to family history is important because about 25% of patients with MTC have familial disease. Because it is not possible to distinguish sporadic from familial disease at initial presentation, all new patients with MTC should be screened for RET point mutations, pheochromocytoma, and HPT. Screening of patients with famil-ial MTC for RET point mutations has largely replaced using provocative testing with pentagastrin or calcium-stimulated calcitonin levels to make the diagnosis. Calcitonin and CEA are used to identify

1	MTC for RET point mutations has largely replaced using provocative testing with pentagastrin or calcium-stimulated calcitonin levels to make the diagnosis. Calcitonin and CEA are used to identify patients with persistent or recurrent MTC. Calcitonin is a more sensitive tumor marker, but CEA is a better predictor of prognosis.Treatment. The ATA published revised guidelines for the man-agement of medullary cancers in 2015.59 A neck ultrasound is recommended to evaluate the central and lateral neck compart-ments and the superior mediastinum. Serum calcitonin, CEA, calcium levels should also be measured, and RET proto-onco-gene mutation testing should be performed. Pheochromocy-tomas need to be excluded. If patients are found to have a pheochromocytoma, this must be operated on first. Primary hyperparathyroidism, if present, is treated at the time of thyroidectomy. These tumors are generally (>50%) bilateral. Total thyroidectomy is the treatment of choice for patients with MTC because of

1	hyperparathyroidism, if present, is treated at the time of thyroidectomy. These tumors are generally (>50%) bilateral. Total thyroidectomy is the treatment of choice for patients with MTC because of the high incidence of multicentricity, the more aggressive course, and the fact that 131I therapy usually is not effective.Central compartment nodes frequently are involved early in the disease process, so that a bilateral prophylactic central neck node dissection should be routinely performed. In patients with palpable or imaging-detected cervical nodes, symptoms and signs of distant disease or calcitonin levels >500 pg/mL, additional imaging to include a neck and chest CT and a triple-phase liver CT or contrast-enhanced MRI and an axial MRI/bone scan is recommended to assess for metastatic disease. In patients with no distant disease but nodal involvement, an ipsilateral or bilateral lateral neck dissection (levels IIA, III, IV, and V) is performed. Less aggressive neck surgery should

1	disease. In patients with no distant disease but nodal involvement, an ipsilateral or bilateral lateral neck dissection (levels IIA, III, IV, and V) is performed. Less aggressive neck surgery should be consid-ered to preserve speech and swallowing while maintaining locoregional control in patients with limited metastatic disease. The role of prophylactic lateral neck dissection is controversial and may be considered based on calcitonin levels. Some groups favor this procedure if central neck lymph nodes are involved or if the primary tumor is ≥1.5 cm.In the case of locally recurrent or widely metastatic dis-ease, tumor debulking is advised not only to ameliorate symp-toms of pain, flushing, and diarrhea, but also to decrease risk of death from recurrent central neck or mediastinal disease. External-beam radiotherapy is controversial but can be consid-ered for patients with resected T4 disease and for patients with unresectable residual or recurrent tumor and symptomatic bony

1	disease. External-beam radiotherapy is controversial but can be consid-ered for patients with resected T4 disease and for patients with unresectable residual or recurrent tumor and symptomatic bony metastases. Liver metastases tend to be multiple and are typi-cally not amenable to resection, percutaneous ethanol ablation, or radiofrequency ablation. However, chemoembolization may be helpful in this setting. There is no effective chemotherapy regimen.Various targeted therapies directed against the RET kinase have been investigated for the treatment of MTC.43 Many of these also inhibit VEGF receptor due to their close structural similarities. Sorafenib, sunitinib, lenvatinib, and cabozantinib are some such multikinase inhibitors, whereas axitinib and pazopanib act only on VEGFR. Vandetanib inhibits both targets and is also an EGF receptor inhibitor, and cabozantinib targets c-MET in addition to RET and VEGF receptor. Both drugs are currently approved by the FDA and EMA for the treatment

1	both targets and is also an EGF receptor inhibitor, and cabozantinib targets c-MET in addition to RET and VEGF receptor. Both drugs are currently approved by the FDA and EMA for the treatment of advanced and progressive MTC based on data that they prolong progression-free survival, in addition to reducing secretion of calcitonin and CEA.60,61 They are recommended as first-line sys-temic therapy in symptomatic patients with advanced MTC. An anti-CEA monoclonal antibody (labetuzumab) also has shown antitumor response in a small group of patients. Patients with recurrent/metastatic disease should be enrolled in well-designed clinical trials.In patients who have hypercalcemia and an increased PTH at the time of thyroidectomy, only obviously enlarged parathy-roid glands should be removed. The other parathyroid glands should be preserved and marked in patients with normocalce-mia, as only about 20% of patients with MEN2A develop HPT. When a normal parathyroid cannot be maintained on a

1	The other parathyroid glands should be preserved and marked in patients with normocalce-mia, as only about 20% of patients with MEN2A develop HPT. When a normal parathyroid cannot be maintained on a vascu-lar pedicle, it should be removed, biopsied to confirm that it is a parathyroid, and then autotransplanted to the forearm of the nondominant arm, particularly in patients with MEN2A. Reim-plantation into the sternocleidomastoid muscle is also accept-able for patients with known MEN2B and familial MTC.Prophylactic total thyroidectomy is indicated in RET mutation carriers once the mutation is confirmed. The ATA guidelines stratify mutation into various risk levels to offer recommendations regarding age at which a prophylactic Brunicardi_Ch38_p1625-p1704.indd 165601/03/19 11:21 AM 1657THYROID, PARATHYROID, AND ADRENALCHAPTER 38Figure 38-21. Magnetic resonance imaging scan of a patient with anaplastic thyroid cancer. Note heterogeneity consistent with necrosis.thyroidectomy should be

1	PARATHYROID, AND ADRENALCHAPTER 38Figure 38-21. Magnetic resonance imaging scan of a patient with anaplastic thyroid cancer. Note heterogeneity consistent with necrosis.thyroidectomy should be performed and to predict phenotypes, including pheochromocytomas.59 In general, in patients with less aggressive mutations (designated ATA moderate-risk), thy-roidectomy may be delayed >5 years, especially if there is a nor-mal annual serum calcitonin, neck ultrasound, less aggressive family history, or family preference. Children with MEN2A and mutations at codon 634 (designated high-risk) are advised to undergo thyroidectomy at <5 years of age, and those with MEN2B-related mutations (designated highest-risk) should undergo the procedure before age 1. Central neck dissection can be avoided in children who are RET-positive and calcitonin-negative with a normal ultrasound examination. When the cal-citonin is increased or the ultrasound suggests a thyroid cancer, a prophylactic central neck

1	children who are RET-positive and calcitonin-negative with a normal ultrasound examination. When the cal-citonin is increased or the ultrasound suggests a thyroid cancer, a prophylactic central neck dissection is indicated.Postoperative Follow-Up and Prognosis. Patients are fol-lowed by annual measurements of calcitonin and CEA levels, in addition to history and physical examination. Other modalities used to localize recurrent disease include ultrasound, CT, MRI, and more recently, FDG-PET/CT scans. Prognosis is related to disease stage. The 10-year survival rate is approximately 80% but decreases to 45% in patients with lymph node involvement. Survival also is significantly influenced by disease type. It is best in patients with non-MEN familial MTC, followed by those with MEN2A, and then those with sporadic disease. Progno-sis is the worst (survival of 35% at 10 years) in patients with MEN2B. Performing prophylactic surgery in RET oncogene mutation carriers not only improves

1	and then those with sporadic disease. Progno-sis is the worst (survival of 35% at 10 years) in patients with MEN2B. Performing prophylactic surgery in RET oncogene mutation carriers not only improves survival rates but also ren-ders most patients calcitonin free.Anaplastic Carcinoma Anaplastic carcinoma accounts for approximately 1% of all thyroid malignancies in the United States. Women are more commonly affected, and the majority of tumors present in the seventh and eighth decade of life. The typical patient has a long-standing neck mass, which rapidly enlarges and may be painful. Associated symptoms such as dys-phonia, dysphagia, and dyspnea are common. The tumor is large and may be fixed to surrounding structures or may be ulcerated with areas of necrosis (Fig. 38-21). Lymph nodes usually are palpable at presentation. Evidence of metastatic spread also may be present. Diagnosis is confirmed by FNAB revealing char-acteristic giant and multinucleated cells. Differential diagnoses on

1	are palpable at presentation. Evidence of metastatic spread also may be present. Diagnosis is confirmed by FNAB revealing char-acteristic giant and multinucleated cells. Differential diagnoses on FNA can include lymphomas, medullary carcinomas, direct extension from a laryngeal carcinoma, or other metastatic car-cinomas or melanoma. When spindle cell elements are present, primary and metastatic sarcomas need to be considered as well. Immunohistochemical markers can aid with excluding other diagnoses. Core or incisional biopsy occasionally is needed to confirm the diagnosis, especially when there is necrotic material on the FNA.Pathology. On gross inspection, anaplastic tumors are firm and whitish in appearance. Microscopically, sheets of cells with marked heterogeneity are seen. The three main histologic growth patterns are spindle cell, squamoid, and pleomorphic giant cell. Tumors may show a predominance of one pattern or a mixture of various patterns. Foci of more differentiated

1	main histologic growth patterns are spindle cell, squamoid, and pleomorphic giant cell. Tumors may show a predominance of one pattern or a mixture of various patterns. Foci of more differentiated thy-roid tumors, either follicular or papillary, may be seen, suggest-ing that anaplastic tumors arise from more well-differentiated tumors.Treatment and Prognosis. This tumor is one of the most aggressive thyroid malignancies, with few patients surviving 6 months beyond diagnosis. All forms of treatment have been disappointing. The ATA has published guidelines for the man-agement of patients with anaplastic cancer.62 Imaging (ultra-sound, CT, MRI, or PET-CT) should be obtained to assess resectability. All patients should have preoperative laryngos-copy to assess the status of the vocal cords. A total or near-total thyroidectomy with therapeutic lymph node dissection is rec-ommended for patients with an intrathyroidal mass (although lobectomy may also be appropriate, particularly if there is

1	total or near-total thyroidectomy with therapeutic lymph node dissection is rec-ommended for patients with an intrathyroidal mass (although lobectomy may also be appropriate, particularly if there is concern for vocal cord paralysis). If extrathyroidal extension is present, an en bloc resection should be considered if all gross disease can be removed (R1). Tracheostomy should be avoided as long as possible unless there is impending airway loss. Adjuvant radiation which should be offered to patients with a good performance status and no metastatic disease who desire aggressive management. Cytotoxic chemotherapy (with some combination of a taxane, anthracycline, and platinum) is typi-cally given concurrently and has been associated with prolonged survival, although these agents are also being used in a neoadju-vant fashion, particularly in patients with unresectable disease.Lymphoma Lymphomas account for <1% of thyroid malignan-cies, and most are of the non-Hodgkin’s B-cell type.

1	being used in a neoadju-vant fashion, particularly in patients with unresectable disease.Lymphoma Lymphomas account for <1% of thyroid malignan-cies, and most are of the non-Hodgkin’s B-cell type. Although the disease can arise as part of a generalized lymphomatous con-dition, most thyroid lymphomas develop in patients with chronic lymphocytic thyroiditis. Chronic antigenic lymphocyte stimu-lation has been suggested to result in lymphocyte transforma-tion. Patients usually present with symptoms similar to those of patients with anaplastic carcinoma, although the rapidly enlarg-ing neck mass often is painless. Patients may present with acute respiratory distress. Ultrasound can be useful for early diagno-sis, and lymphoma appears as a well-defined hypoechoic mass. The diagnosis usually is suggested by FNAB, but FNAB can be nondiagnostic, particularly in the setting of low-grade lym-phomas. Therefore, needle core or open biopsy may be neces-sary for definitive diagnosis. Staging studies

1	by FNAB, but FNAB can be nondiagnostic, particularly in the setting of low-grade lym-phomas. Therefore, needle core or open biopsy may be neces-sary for definitive diagnosis. Staging studies should be obtained expeditiously to assess the extent of extrathyroidal spread.Brunicardi_Ch38_p1625-p1704.indd 165701/03/19 11:21 AM 1658SPECIFIC CONSIDERATIONSPART IITreatment and Prognosis. Patients with thyroid lymphoma respond rapidly to chemotherapy (CHOP—cyclophosphamide, doxorubicin, vincristine, and prednisone), which also has been associated with improved survival. Combined treatment with radiotherapy and chemotherapy often is recommended. Thy-roidectomy and nodal resection are used to alleviate symptoms of airway obstruction in patients who do not respond quickly to the above regimens or who have completed the regimen before diagnosis. Prognosis depends on the histologic grade of the tumor and whether the lymphoma is confined to the thy-roid gland or is disseminated. The overall

1	or who have completed the regimen before diagnosis. Prognosis depends on the histologic grade of the tumor and whether the lymphoma is confined to the thy-roid gland or is disseminated. The overall 5-year survival rate is about 50%; patients with extrathyroidal disease have markedly lower survival rates.Metastatic Carcinoma The thyroid gland is a rare site of metastases from other cancers, including kidney, breast, lung, and melanoma. Clinical examination and a review of the patient’s history often suggest the source of the metastatic dis-ease, and FNAB usually provides definitive diagnosis. Resec-tion of the thyroid, usually lobectomy, may be helpful in many patients, depending on the status of their primary tumor.Thyroid Surgery Conduct of Thyroidectomy Patients with any recent or remote history of altered phonation or prior neck or upper chest surgery that places the recurrent laryngeal or vagus nerves at risk should undergo vocal cord assessment by direct or indirect laryngos-copy

1	history of altered phonation or prior neck or upper chest surgery that places the recurrent laryngeal or vagus nerves at risk should undergo vocal cord assessment by direct or indirect laryngos-copy before thyroidectomy. Laryngeal examination is also advised in patients with known posterior extension of thyroid cancer and extensive central nodal metastases.40 The patient is positioned supine, with a sandbag between the scapulae. The head is placed on a donut cushion, and the neck is extended to provide maximal exposure. A Kocher transverse collar incision, typically 3 to 5 cm in length, is placed in or parallel to a natu-ral skin crease 1 cm below the cricoid cartilage (Fig. 38-22A), although longer incisions may be needed. The subcutaneous tis-sues and platysma are incised sharply, and subplatysmal flaps are raised superiorly to the level of the thyroid cartilage and inferiorly to the suprasternal notch (Fig. 38-22B). The strap muscles are divided in the midline along the entire

1	subplatysmal flaps are raised superiorly to the level of the thyroid cartilage and inferiorly to the suprasternal notch (Fig. 38-22B). The strap muscles are divided in the midline along the entire length of the mobilized flaps, and the thyroid gland is exposed. On the side to be approached first, the sternohyoid muscles are sepa-rated from the underlying sternothyroid muscle by blunt dissec-tion until the internal jugular vein and ansa cervicalis nerve are identified. The strap muscles rarely need to be divided to gain exposure to the thyroid gland. If this maneuver is necessary, the muscles should be divided high to preserve their innervation by branches of the ansa cervicalis. If there is evidence of direct tumor invasion into the strap muscles, the portion of involved muscle should be resected en bloc with the thyroid gland. The sternothyroid muscle is then dissected off the underlying thyroid by a combination of sharp and blunt dissection, thus exposing the middle thyroid veins.

1	resected en bloc with the thyroid gland. The sternothyroid muscle is then dissected off the underlying thyroid by a combination of sharp and blunt dissection, thus exposing the middle thyroid veins. The thyroid lobe is retracted medially and anteriorly, and the lateral tissues are swept posterolaterally using a peanut sponge. The middle thyroid veins are ligated and divided (Fig. 38-22C). Attention is then turned to the midline where Delphian nodes and the pyramidal lobe are identified. The fascia just cephalad and caudad to the isthmus is divided. The superior thyroid pole is identified by retracting the thyroid first inferiorly and medially, and then the upper pole of the thy-roid is mobilized caudally and laterally. The dissection plane is kept as close to the thyroid as possible, and the superior pole vessels are individually identified, skeletonized, ligated, and divided low on the thyroid gland to avoid injury to the exter-nal branch of the superior laryngeal nerve (Fig.

1	and the superior pole vessels are individually identified, skeletonized, ligated, and divided low on the thyroid gland to avoid injury to the exter-nal branch of the superior laryngeal nerve (Fig. 38-22D). Once these vessels are divided, the tissues posterior and lateral to the superior pole can be swept from the gland in a posteromedial direction, to reduce the risk of damaging vessels supplying the upper parathyroid.The RLNs should then be identified, and the ATA 2015 guidelines strongly recommend visual identification in all cases.40 The course of the right RLN is more oblique than the left RLN. The nerves can be most consistently identified at the level of the cricoid cartilage. The parathyroids usually can be identified within 1 cm of the crossing of the inferior thyroid artery and the RLN, although they also may be ectopic in loca-tion. The lower pole of the thyroid gland should be mobilized by gently sweeping all tissues dorsally. The inferior thyroid vessels are dissected,

1	the RLN, although they also may be ectopic in loca-tion. The lower pole of the thyroid gland should be mobilized by gently sweeping all tissues dorsally. The inferior thyroid vessels are dissected, skeletonized, ligated, and divided as close to the surface of the thyroid gland as possible to minimize devascular-ization of the parathyroids (extracapsular dissection) or injury to the RLN. The RLN is most vulnerable to injury in the vicinity of the ligament of Berry. The nerve often passes through this structure along with small crossing arterial and venous branches (Fig. 38-22E). Any bleeding in this area should be controlled with gentle pressure before carefully identifying the vessel and ligating it. Use of the electrocautery should be avoided in prox-imity to the RLN. Once the ligament is divided, the thyroid can be separated from the underlying trachea by sharp dissection. The pyramidal lobe, if present, must be dissected in a cephalad direction to above the level of the notch in

1	is divided, the thyroid can be separated from the underlying trachea by sharp dissection. The pyramidal lobe, if present, must be dissected in a cephalad direction to above the level of the notch in the thyroid cartilage or higher in continuity with the thyroid gland. If a lobectomy is to be performed, the isthmus is divided flush with the trachea on the contralateral side and suture ligated. The procedure is repeated on the opposite side for a total thyroidectomy.Parathyroid glands located anteriorly on the surface of the thyroid that cannot be dissected from the thyroid with a good blood supply or that have been inadvertently removed during the thyroidectomy should be resected, confirmed as parathy-roid tissue by frozen section, divided into 1-mm fragments, and reimplanted into individual pockets in the sternocleido-mastoid muscle. The sites should be marked with silk sutures and a clip. Various novel techniques using indocyanine fluo-rescence angiography and near-infrared

1	individual pockets in the sternocleido-mastoid muscle. The sites should be marked with silk sutures and a clip. Various novel techniques using indocyanine fluo-rescence angiography and near-infrared autofluorescence have shown utility in the identification and viability assessment of parathyroid glands; however, they are not routinely used at the present time.63,64 If a subtotal thyroidectomy is to be performed, once the superior pole vessels are divided and the thyroid lobe mobilized anteriorly, the thyroid lobe is cross-clamped with a Mayo clamp, leaving approximately 4 g of the posterior por-tion of the thyroid. The thyroid remnant is suture ligated, taking care to avoid injury to the RLN. Routine drain placement rarely is necessary. After adequate hemostasis is obtained, the strap muscles are reapproximated in the midline. The platysma is approximated in a similar fashion. The skin can be closed with subcuticular sutures or clips.Nerve Monitoring Intraoperative RLN and external

1	are reapproximated in the midline. The platysma is approximated in a similar fashion. The skin can be closed with subcuticular sutures or clips.Nerve Monitoring Intraoperative RLN and external laryngeal nerve monitoring techniques are being increasingly used during thyroid and parathyroid surgery. Both continuous monitoring using endotracheal tube electrodes and intermittent monitor-ing by periodic stimulation and laryngeal palpation are used. Many published studies have established the feasibility of nerve monitoring; however, none were able to show that the technique equivocally reduces nerve injury (particularly by experienced Brunicardi_Ch38_p1625-p1704.indd 165801/03/19 11:21 AM 1659THYROID, PARATHYROID, AND ADRENALCHAPTER 38Figure 38-22. Conduct of thyroidectomy. A. Correct placement of thyroidectomy incision. B. Raising subplatysmal flaps. C. Dissection of middle thyroid vein. D. Dissection of the superior pole vessels, which should be individually ligated. E. Dissection at

1	of thyroidectomy incision. B. Raising subplatysmal flaps. C. Dissection of middle thyroid vein. D. Dissection of the superior pole vessels, which should be individually ligated. E. Dissection at the ligament of Berry. Note small artery and vein within the ligament and the recurrent laryngeal nerve coursing laterally. F. Endoscopic thyroidectomy via axillary incisions. m. = muscle; n. = nerve; v. = vein.IncorrectCricoid cartilageCorrectAPlatysmaCauteryFive straight clamps on dermisBMiddle thyroid v.CCricothyroid m.External branch ofsuperior laryngeal n.Superior thyroid vesselsDBrunicardi_Ch38_p1625-p1704.indd 165901/03/19 11:21 AM 1660SPECIFIC CONSIDERATIONSPART IISmall artery & veinin ligament of BerryRecurrent laryngeal n.ThyroidEFFigure 38-22. (Continued)surgeons and especially when the RLN is routinely visually identified) until recently. In 2009, Barczynski and colleagues65 were the first to demonstrate in their prospective randomized trial with 2000 nerves at risk that

1	when the RLN is routinely visually identified) until recently. In 2009, Barczynski and colleagues65 were the first to demonstrate in their prospective randomized trial with 2000 nerves at risk that neuromonitoring was asso-ciated with a statistically significant improvement in transient RLN injury rates when compared to the practice of visualiza-tion of the nerve alone and particularly in patients at higher risk of nerve injury. Of note, there was no difference in permanent RLN injury rates, and a later meta-analysis of all the published studies also failed to show a protective effect of neuromonitor-ing. Despite conflicting data, the technology has become widely adopted. The current ATA guidelines recommend that intraop-erative neural stimulation may be used to facilitate nerve iden-tification of the RLN and confirm its function, especially prior to proceeding with contralateral thyroidectomy.Minimally Invasive Approaches Several approaches to minimally invasive thyroidectomy have

1	of the RLN and confirm its function, especially prior to proceeding with contralateral thyroidectomy.Minimally Invasive Approaches Several approaches to minimally invasive thyroidectomy have been described. Miniincision procedures use a small, 3-cm incision with no flap creation and minimal dissection to deliver the thyroid into the wound and then perform the pretracheal and paratracheal dissec-tion. Video assistance can be used to improve the visualization Brunicardi_Ch38_p1625-p1704.indd 166001/03/19 11:21 AM 1661THYROID, PARATHYROID, AND ADRENALCHAPTER 38via the small incision. Totally endoscopic approaches also have been described, via the supraclavicular, anterior chest, axillary, and breast approach. The axillary, anterior chest, and breast approaches eliminate the skin incision in the neck but are more invasive. The endoscopic approaches can also be performed with the assistance of robotic techniques. More recently, there have been studies of transoral robotic-assisted

1	in the neck but are more invasive. The endoscopic approaches can also be performed with the assistance of robotic techniques. More recently, there have been studies of transoral robotic-assisted thyroidectomy in which the thyroid is approached through the oral cavity.66 These methods are feasible, but clear benefits over the “tradi-tional” open approach via small neck incisions have not been established.4Typically, endoscopic thyroidectomies are performed under general anesthesia. For the axillary approach, a 30-mm skin incision is made in the axilla, and 12-mm and 5-mm trocars are inserted through this incision (Fig. 38-22F). An additional 5-mm trocar is inserted adjacent to the incision. For the anterior chest approach, a 12-mm skin incision is made in the skin of the anterior chest approximately 3 to 5 cm below the border of the ipsilateral clavicle. Two additional 5-mm trocars are inserted by endoscopic guidance below the ipsilateral clavicle, and carbon dioxide (CO2) is then

1	approximately 3 to 5 cm below the border of the ipsilateral clavicle. Two additional 5-mm trocars are inserted by endoscopic guidance below the ipsilateral clavicle, and carbon dioxide (CO2) is then insufflated up to a pressure of 4 mmHg to facilitate creation of a working space. The anterior border of the sternocleidomastoid muscle is then separated from the ster-nohyoid muscle to expose the sternothyroid muscle. The thy-roid gland is exposed by splitting the sternothyroid muscle. The lower pole is retracted upward and dissected from the adipose tissue to identify the RLN. As the RLN is exposed, Berry’s liga-ment is exposed and incised with a 5-mm clip or laparoscopic coagulating shears. The upper pole of the thyroid gland is sepa-rated from the cricothyroid muscle, and the external branch of the superior laryngeal nerve can be identified during this maneu-ver. The upper pole of the thyroid gland then is dissected free.Surgical Removal of Intrathoracic Goiter A goiter is consid-ered

1	of the superior laryngeal nerve can be identified during this maneu-ver. The upper pole of the thyroid gland then is dissected free.Surgical Removal of Intrathoracic Goiter A goiter is consid-ered mediastinal if at least 50% of the thyroid tissue is located intrathoracically. Mediastinal goiters can be primary or second-ary. Primary mediastinal goiters constitute approximately 1% of all mediastinal goiters and arise from accessory (ectopic) thy-roid tissue located in the chest. These goiters are supplied by intrathoracic blood vessels and do not have any connection to thyroid tissue in the neck. The vast majority of mediastinal goi-ters are, however, secondary mediastinal goiters that arise from downward extension of cervical thyroid tissue along the fascial planes of the neck and derive their blood supply from the supe-rior and inferior thyroid arteries. Virtually all intrathoracic goi-ters can be removed via a cervical incision. Patients who have (a) invasive thyroid cancers, (b)

1	their blood supply from the supe-rior and inferior thyroid arteries. Virtually all intrathoracic goi-ters can be removed via a cervical incision. Patients who have (a) invasive thyroid cancers, (b) had previous thyroid operations and may have developed parasitic mediastinal vessels, or (c) pri-mary mediastinal goiters with no thyroid tissue in the neck may require a median sternotomy for removal.49 The chest, however, should be prepared in most cases in the event it is necessary to perform a median sternotomy to control mediastinal bleeding or completely remove an unsuspected invasive cancer. The goiter is approached via a neck incision. The superior pole vessels and the middle thyroid veins are identified and ligated first. Early division of the isthmus helps with subsequent mobilization of the substernal goiter from beneath the sternum. Placement of large 1-0 or 2-0 sutures deep into the goiter, when necessary, helps deliver it. For patients in whom thyroid cancer is suspected or

1	of the substernal goiter from beneath the sternum. Placement of large 1-0 or 2-0 sutures deep into the goiter, when necessary, helps deliver it. For patients in whom thyroid cancer is suspected or demonstrated in an intrathoracic gland, attempts should be made to avoid rupture of the thyroid capsule. When sternotomy is indicated, the sternum usually should be divided to the level of the third intercostal space and then laterally on one side at the space between the third and fourth ribs (Fig. 38-23).Central and Lateral Neck Dissection for Nodal Metastases Central compartment (medial to the carotid sheath) lymph nodes frequently are involved in patients with papillary, medullary, and Hürthle cell carcinomas and should be removed at the time of thyroidectomy, preserving the RLNs and parathyroid glands. Central neck dissection is particularly important in patients with medullary and Hürthle cell carcinoma because of the high fre-quency of microscopic tumor spread and because these

1	glands. Central neck dissection is particularly important in patients with medullary and Hürthle cell carcinoma because of the high fre-quency of microscopic tumor spread and because these tumors cannot be ablated with 131I. An ipsilateral modified radical neck dissection is indicated in the presence of palpable cervical lymph nodes or prophylactically in some patients with medul-lary carcinoma.A modified radical (functional) neck dissection can be per-formed via the cervical incision used for thyroidectomy, which can be extended laterally (Fig. 38-24A) to the anterior margin of the trapezius muscle. The procedure involves removal of all fibro-fatty tissue along the internal jugular vein (levels II, III, and IV) and the posterior triangle (level V). In contrast to a radical neck dissection, the internal jugular vein, the spinal accessory nerve, the cervical sensory nerves, and the sternoclei-domastoid muscle are preserved unless they are adherent to or invaded by tumor. The procedure

1	the internal jugular vein, the spinal accessory nerve, the cervical sensory nerves, and the sternoclei-domastoid muscle are preserved unless they are adherent to or invaded by tumor. The procedure begins by opening the plane between the strap muscles medially and the sternocleidomas-toid muscle laterally. The anterior belly of the omohyoid muscle is retracted laterally, and the dissection is carried posteriorly until the carotid sheath is reached. The internal jugular vein is retracted medially with a vein retractor and the fibro-fatty tissue and lymph nodes are dissected away from it by a combination of sharp and blunt dissection. The lateral dissection is carried along the posterior border of the sternocleidomastoid muscle, remov-ing the tissue from the posterior triangle. The deep dissection plane is the anterior scalenus muscle, the phrenic nerve, the bra-chial plexus, and the medial scalenus muscle. The phrenic nerve is preserved on the scalenus anterior muscle, as are the

1	dissection plane is the anterior scalenus muscle, the phrenic nerve, the bra-chial plexus, and the medial scalenus muscle. The phrenic nerve is preserved on the scalenus anterior muscle, as are the cervical sensory nerves in most patients (Fig. 38-24B). Dissection along the spinal accessory nerve superiorly is most important because this is a frequent site of metastatic disease.123Figure 38-23. Conduct of thyroidectomy. Incisions for a partial sternotomy.Brunicardi_Ch38_p1625-p1704.indd 166101/03/19 11:21 AM 1662SPECIFIC CONSIDERATIONSPART IIComplications of Thyroid Surgery Nerves, parathyroids, and surrounding structures are all at risk of injury during thyroidec-tomy. Injury to the RLN may occur by severance, ligation, or traction, but should occur in <1% of patients undergoing thy-roidectomy by experienced surgeons. The RLN is most vulner-able to injury during the last 2 to 3 cm of its course, but also can be damaged if the surgeon is not alert to the possibility of nerve

1	thy-roidectomy by experienced surgeons. The RLN is most vulner-able to injury during the last 2 to 3 cm of its course, but also can be damaged if the surgeon is not alert to the possibility of nerve branches and the presence of a nonrecurrent nerve, par-ticularly on the right side. If the injury is recognized intraopera-tively, most surgeons advocate primary reapproximation of the perineurium using nonabsorbable sutures. Approximately 20% of patients are at risk of injury to the external branches of the 123McFee incisionABSpinal accessory n.Phrenic n.Vagus n.Scalenusanticus m.LymphnodesCarotid a. Internaljugular v.Figure 38-24. Conduct of neck dissection. A. Incisions for modified radical neck dissection. B. Anatomic relations of structures identified during a modified radical neck dissection. a. = artery; m. = muscle; n. = nerve.Brunicardi_Ch38_p1625-p1704.indd 166201/03/19 11:21 AM 1663THYROID, PARATHYROID, AND ADRENALCHAPTER 38superior laryngeal nerve, especially if superior

1	a. = artery; m. = muscle; n. = nerve.Brunicardi_Ch38_p1625-p1704.indd 166201/03/19 11:21 AM 1663THYROID, PARATHYROID, AND ADRENALCHAPTER 38superior laryngeal nerve, especially if superior pole vessels are ligated en masse. The cervical sympathetic trunk is at risk of injury in invasive thyroid cancers and retroesophageal goiters and may result in Horner’s syndrome. Transient hypocalcemia (from surgical injury or inadvertent removal of parathyroid tis-sue) has been reported in up to 50% of cases, but permanent hypoparathyroidism occurs <2% of the time. Postoperative hypocalcemia is more likely in patients who undergo concomi-tant thyroidectomy and central and lateral neck dissection and in patients with Graves’ disease. Postoperative hematomas or bleeding may also complicate thyroidectomies and rarely neces-sitate emergency reoperation to evacuate the hematoma. Bilat-eral vocal cord dysfunction with airway compromises requires immediate reintubation and tracheostomy. Seromas may

1	and rarely neces-sitate emergency reoperation to evacuate the hematoma. Bilat-eral vocal cord dysfunction with airway compromises requires immediate reintubation and tracheostomy. Seromas may need aspiration to relieve patient discomfort. Wound cellulitis and infection and injury to surrounding structures, such as the carotid artery, jugular vein, and esophagus, are infrequent.PARATHYROIDHistorical BackgroundIn 1849, the curator of the London Zoological Gardens, Sir Richard Owen, provided the first accurate description of the normal parathyroid gland after autopsy examination of an Indian rhinoceros. However, human parathyroids were not grossly and microscopically described until 1879 by Ivar Sandström, a med-ical student in Uppsala, Sweden. He suggested that these glands be named the glandulae parathyroideae, although their function was not known.The association of HPT and the bone disease osteitis fibrosa cystica (described by von Recklinghausen) was recog-nized in 1903. Calcium

1	glandulae parathyroideae, although their function was not known.The association of HPT and the bone disease osteitis fibrosa cystica (described by von Recklinghausen) was recog-nized in 1903. Calcium measurement became possible in 1909, and the association between serum calcium levels and the para-thyroid glands was established. The first successful parathyroid-ectomy was performed in 1925 by Felix Mandl on a 38-year-old man who had severe bone pain secondary to advanced osteitis fibrosa cystica. The patient’s condition dramatically improved after the operation, and he lived for another 7 years before dying of recurrent HPT or renal failure. In 1926, the first parathyroid operation was performed at Massachusetts General Hospital. Edward Churchill, assisted by an intern named Oliver Cope, operated on the famous sea captain Charles Martell for severe primary HPT (PHPT). It was not until his seventh operation, which included total thyroidectomy, that an ectopic adenoma was found

1	Cope, operated on the famous sea captain Charles Martell for severe primary HPT (PHPT). It was not until his seventh operation, which included total thyroidectomy, that an ectopic adenoma was found substernally. Unfortunately, Captain Martell died 6 weeks later, likely due to laryngeal spasm and complications of renal stones and ureteral obstruction. The first successful para-thyroidectomy for HPT in the United States was performed on a 56-year-old woman in 1928 by Isaac Y. Olch at the Barnes Hospital in St. Louis, Missouri. At operation, a parathyroid ade-noma was found attached to the left lower lobe of the thyroid gland. Postoperatively, the patient developed tetany, requiring lifelong supplemental calcium.EmbryologyIn humans, the superior parathyroid glands are derived from the fourth branchial pouch, which also gives rise to the thyroid gland. The third branchial pouches give rise to the inferior para-thyroid glands and the thymus (Fig. 38-25). The parathyroids remain closely

1	branchial pouch, which also gives rise to the thyroid gland. The third branchial pouches give rise to the inferior para-thyroid glands and the thymus (Fig. 38-25). The parathyroids remain closely associated with their respective branchial pouch derivatives. The position of normal superior parathyroid glands is more consistent, with 80% of these glands being found near the posterior aspect of the upper and middle thyroid lobes, at the level of the cricoid cartilage. Approximately 1% of normal upper glands may be found in the paraesophageal or retro-esophageal space. Enlarged superior glands may descend in the tracheoesophageal groove and come to lie caudal to the inferior glands. Truly ectopic superior parathyroid glands are rare, but they may be found in the middle or posterior mediastinum or in the aortopulmonary window.51 As the embryo matures, the thy-mus and inferior parathyroids migrate together caudally in the neck. The most common location for inferior glands is within a

1	or in the aortopulmonary window.51 As the embryo matures, the thy-mus and inferior parathyroids migrate together caudally in the neck. The most common location for inferior glands is within a distance of 1 cm from a point centered where the inferior thyroid artery and RLN cross. Approximately 15% of inferior glands Parathyroid IIIForamen caecumThyroglossal ductMedian thyroidThymusTracheaEsophagusFifth branchialpouchFourthbranchialpouchThirdbranchialpouchParathyroid IVUltimobranchialbodyMedian thyroidLateralthyroid or ultimobranchial bodyEsophagusParathyroid IVParathyroid IIIThymusABFigure 38-25. Parathyroid embryology. Figure demonstrates a schematic view of the pharynx of an 8to 10-mm embryo (A) and locations of the thyroid, parathyroid, and thymic tissues in a 13to 14-mm embryo (B). The lower parathyroids are derived from the third branchial pouch and migrate with the thymus, whereas the upper parathyroids are derived from the fourth branchial pouch and lie in close proximity to the

1	lower parathyroids are derived from the third branchial pouch and migrate with the thymus, whereas the upper parathyroids are derived from the fourth branchial pouch and lie in close proximity to the ultimobranchial bodies. (Reproduced with permission from Randolph G: Surgery of the Thyroid and Parathyroid Glands. Philadelphia, PA: Elsevier/Saunders; 2003.)Brunicardi_Ch38_p1625-p1704.indd 166301/03/19 11:21 AM 1664SPECIFIC CONSIDERATIONSPART IIare found in the thymus. The position of the inferior glands, however, tends to be more variable due to their longer migratory path. Undescended inferior glands may be found near the skull base, angle of the mandible, or superior to the upper parathyroid glands along with an undescended thymus. The frequency of intrathyroidal glands is about 2%.Anatomy and HistologyMost patients have four parathyroid glands. The superior glands usually are dorsal to the RLN at the level of the cricoid cartilage, whereas the inferior parathyroid glands are

1	and HistologyMost patients have four parathyroid glands. The superior glands usually are dorsal to the RLN at the level of the cricoid cartilage, whereas the inferior parathyroid glands are located ventral to the nerve. Normal parathyroid glands are gray and semitransparent in newborns but appear golden yellow to light brown in adults. Parathyroid color depends on cellularity, fat content, and vas-cularity. Moreover, they often are embedded in and sometimes difficult to discern from surrounding fat. Normal parathyroid glands are located in loose tissue or fat and are ovoid. They measure up to 7 mm in size and weigh approximately 40 to 50 mg each. Parathyroid glands usually derive their blood supply from branches of the inferior thyroid artery, although branches from the superior thyroid artery supply at least 20% of upper glands. Branches from the thyroidea ima, and vessels to the trachea, esophagus, larynx, and mediastinum may also be found. The parathyroid glands drain

1	thyroid artery supply at least 20% of upper glands. Branches from the thyroidea ima, and vessels to the trachea, esophagus, larynx, and mediastinum may also be found. The parathyroid glands drain ipsilaterally by the superior, middle, and inferior thyroid veins.Akerström and colleagues,67 in an autopsy series of 503 cadavers, found four parathyroid glands in 84% of cases. Super-numerary glands were present in 13% of patients, most com-monly in the thymus. Only 3% of patients had less than four glands. Similar results were obtained in other dissection studies of 428 human subjects by Gilmour who reported a 6.7% inci-dence of supernumerary glands.68Histologically, parathyroid glands are composed of chief cells and oxyphil cells arranged in trabeculae, within a stroma composed primarily of adipose cells (Fig. 38-26). The parathy-roid glands of infants and children are composed mainly of chief cells, which produce parathyroid hormone (PTH). Acidophilic, mitochondria-rich oxyphil cells are

1	cells (Fig. 38-26). The parathy-roid glands of infants and children are composed mainly of chief cells, which produce parathyroid hormone (PTH). Acidophilic, mitochondria-rich oxyphil cells are derived from chief cells, can be seen around puberty, and increase in numbers in adult-hood. A third group of cells, known as water-clear cells, also are derived from chief cells, are present in small numbers, and are rich in glycogen. Although most oxyphil and water-clear Figure 38-26. Normal parathyroid histology showing chief cells interspersed with adipose cells.cells retain the ability to secrete PTH, their functional signifi-cance is not known.Parathyroid Physiology and Calcium HomeostasisCalcium is the most abundant cation in human beings and has several crucial functions. Extracellular calcium levels are 10,000-fold higher than intracellular levels, and both are tightly controlled. Extracellular calcium is important for excitationcontraction coupling in muscle tissues, synaptic

1	calcium levels are 10,000-fold higher than intracellular levels, and both are tightly controlled. Extracellular calcium is important for excitationcontraction coupling in muscle tissues, synaptic transmission in the nervous system, coagulation, and secretion of other hormones. Intracellular calcium is an important second messenger regulat-ing cell division, motility, membrane trafficking, and secretion. Calcium is absorbed from the small intestine in its inorganic form. Calcium fluxes in the steady state are depicted in Fig. 38-27.Extracellular calcium (900 mg) accounts for only 1% of the body’s calcium stores, the majority of which is sequestered in the skeletal system. Approximately 50% of the serum cal-cium is in the ionized form, which is the active component. The remainder is bound to albumin (40%) and organic anions such as phosphate and citrate (10%). The total serum calcium levels range from 8.5 to 10.5 mg/dL (2.1 to 2.6 mmol/L), and ionized calcium levels range from 4.4 to

1	to albumin (40%) and organic anions such as phosphate and citrate (10%). The total serum calcium levels range from 8.5 to 10.5 mg/dL (2.1 to 2.6 mmol/L), and ionized calcium levels range from 4.4 to 5.2 mg/dL (1.1 to 1.3 mmol/L). Both concentrations are tightly regulated. The total serum calcium level must always be considered in its rela-tionship to plasma protein levels, especially serum albumin. For each gram per deciliter of alteration of serum albumin above or below 4.0 mg/dL, there is a 0.8 mg/dL increase or decrease in protein-bound calcium and, thus, in total serum calcium levels. Total and, particularly, ionized calcium levels are influenced by various hormone systems.Parathyroid Hormone. The parathyroid cells rely on a G-protein–coupled membrane receptor, designated the calcium-sensing receptor (CASR), to regulate PTH secretion by sensing extracellular calcium levels69 (Fig. 38-28). PTH secretion also is stimulated by low levels of 1,25-dihydroxy vitamin D, cat-echolamines,

1	receptor (CASR), to regulate PTH secretion by sensing extracellular calcium levels69 (Fig. 38-28). PTH secretion also is stimulated by low levels of 1,25-dihydroxy vitamin D, cat-echolamines, and hypomagnesemia. The PTH gene is located on chromosome 11. PTH is synthesized in the parathyroid gland as a precursor hormone preproPTH, which is cleaved first to pro-PTH and then to the final 84-amino-acid PTH. Secreted PTH has a half-life of 2 to 4 minutes. In the liver, PTH is metabolized into the active N-terminal component and the relatively inactive C-terminal fraction. The C-terminal component is excreted by the kidneys and accumulates in chronic renal failure.PTH functions to regulate calcium levels via its actions on three target organs, the bone, kidney, and gut. PTH increases the resorption of bone by stimulating osteoclasts and promotes the release of calcium and phosphate into the circulation. At the kidney, calcium is primarily absorbed in concert with sodium in the proximal

1	of bone by stimulating osteoclasts and promotes the release of calcium and phosphate into the circulation. At the kidney, calcium is primarily absorbed in concert with sodium in the proximal convoluted tubule, but fine adjustments occur more distally. PTH acts to limit calcium excretion at the dis-tal convoluted tubule via an active transport mechanism. PTH also inhibits phosphate reabsorption (at the proximal convoluted tubule) and bicarbonate reabsorption. It also inhibits the Na+/H+ antiporter, which results in a mild metabolic acidosis in hyper-parathyroid states. PTH and hypophosphatemia also enhance 1-hydroxylation of 25-hydroxyvitamin D, which is responsible for its indirect effect of increasing intestinal calcium absorption.Calcitonin. Calcitonin is produced by thyroid C cells and func-tions as an antihypercalcemic hormone by inhibiting osteoclast-mediated bone resorption. Calcitonin production is stimulated Brunicardi_Ch38_p1625-p1704.indd 166401/03/19 11:21 AM

1	cells and func-tions as an antihypercalcemic hormone by inhibiting osteoclast-mediated bone resorption. Calcitonin production is stimulated Brunicardi_Ch38_p1625-p1704.indd 166401/03/19 11:21 AM 1665THYROID, PARATHYROID, AND ADRENALCHAPTER 38Figure 38-27. Calcium balance and fluxes in a normal human. Solid arrows depict a direct effect, whereas dashed arrows depict an indirect effect. The thickness of the arrows is representative of the magnitude of the flux. ECF = extracellular fluid; PTH = parathyroid hormone; VIT. = vitamin. (Reproduced with permission from Felig P, Frohman L: Endocrinology and Metabolism. New York, NY: McGraw-Hill Education; 2001.)PTHPTHVIT. DECFCALCIUM900 mgKidneyUrine0.15 0.3 g900 1400 gBone0.25 0.5 g0.25 0.5 g0.25 0.5 g0.1 0.2 g0.4 1.5 g0.35 1.0 gVIT. DPTHby calcium and pentagastrin and also by catecholamines, chole-cystokinin, and glucagon. When administered intravenously to experimental animals, it produces hypocalcemia. At the kidney, calcitonin

1	calcium and pentagastrin and also by catecholamines, chole-cystokinin, and glucagon. When administered intravenously to experimental animals, it produces hypocalcemia. At the kidney, calcitonin increases phosphate excretion by inhibiting its reab-sorption. Calcitonin plays a minimal, if any, role in the regula-tion of calcium levels in humans. However, it is very useful as a marker of MTC and in treating acute hypercalcemic crisis.Vitamin D. Vitamin D refers to vitamin D2 and vitamin D3, both of which are produced by photolysis of naturally occurring ste-rol precursors. Vitamin D2 is available commercially in pharma-ceutical preparations, whereas vitamin D3 is the most important physiologic compound and is produced from 7-dehydrocholes-terol, which is found in the skin. Vitamin D is metabolized in the liver to its primary circulating form, 25-hydroxyvitamin D. Further hydroxylation in the kidney results in 1,25-dihydroxy vitamin D, which is the most metabolically active form of

1	in the liver to its primary circulating form, 25-hydroxyvitamin D. Further hydroxylation in the kidney results in 1,25-dihydroxy vitamin D, which is the most metabolically active form of vitamin D. Vitamin D stimulates the absorption of calcium and phosphate from the gut and the resorption of calcium from the bone.HyperparathyroidismHyperfunction of the parathyroid glands may be classified as primary, secondary, or tertiary. PHPT arises from increased PTH production from abnormal parathyroid glands and results from a disturbance of normal feedback control exerted by serum calcium. Elevated PTH levels may also occur as a compensa-tory response to hypocalcemic states resulting from chronic renal failure or GI malabsorption of calcium. This secondary HPT can be reversed by correction of the underlying problem (e.g., kidney transplantation for chronic renal failure). However, chronically stimulated glands may occasionally become autono-mous, resulting in persistence or recurrence of

1	the underlying problem (e.g., kidney transplantation for chronic renal failure). However, chronically stimulated glands may occasionally become autono-mous, resulting in persistence or recurrence of hypercalcemia after successful renal transplantation, resulting in tertiary HPT.Primary Hyperparathyroidism. PHPT is a common dis-order, affecting 100,000 individuals annually in the United States. PHPT occurs in 0.1% to 0.3% of the general population and is more common in women (1:500) than in men (1:2000). Increased PTH production leads to hypercalcemia via increased GI absorption of calcium, increased production of vitamin D3, and reduced renal calcium clearance. PHPT is characterized by increased parathyroid cell proliferation and PTH secretion that is independent of calcium levels.Etiology The exact cause of PHPT is unknown, although exposure to low-dose therapeutic ionizing radiation and familial predisposition account for some cases. Various diets and inter-mittent exposure to

1	exact cause of PHPT is unknown, although exposure to low-dose therapeutic ionizing radiation and familial predisposition account for some cases. Various diets and inter-mittent exposure to sunshine may also be related. Other causes include renal leak of calcium and declining renal function with age as well as alteration in the sensitivity of parathyroid glands to suppression by calcium. The latency period for development of PHPT after radiation exposure is longer than that for the development of thyroid tumors, with most cases occurring 30 to 40 years after exposure. Patients who have been exposed to radiation have similar clinical presentations and calcium levels when compared to patients without a history of radiation expo-sure. However, the former tends to have higher PTH levels and a higher incidence of concomitant thyroid neoplasms. Lithium therapy has been known to shift the set point for PTH secre-tion in parathyroid cells, thereby resulting in elevated PTH lev-els and mild

1	a higher incidence of concomitant thyroid neoplasms. Lithium therapy has been known to shift the set point for PTH secre-tion in parathyroid cells, thereby resulting in elevated PTH lev-els and mild hypercalcemia. Lithium stimulates the growth of abnormal parathyroid glands in vitro and also in susceptible patients in vivo. PHPT results from the enlargement of a single gland or parathyroid adenoma in approximately 80% of cases, multiple adenomas or hyperplasia in 15% to 20% of patients, and parathyroid carcinoma in 1% of patients. Existence of two enlarged glands or double adenomas is supported by bio-chemical (calcium and PTH), intraoperative PTH (IOPTH), molecular, and histologic data. This entity is less common in younger patients but accounts for up to 10% of older patients with PHPT. It should be emphasized that when more than one abnormal parathyroid gland is identified preoperatively or intra-operatively, the patient has hyperplasia (all glands abnormal) until proven

1	with PHPT. It should be emphasized that when more than one abnormal parathyroid gland is identified preoperatively or intra-operatively, the patient has hyperplasia (all glands abnormal) until proven otherwise.Genetics Most cases of PHPT are sporadic. However, PHPT also occurs within the spectrum of a number of inherited Brunicardi_Ch38_p1625-p1704.indd 166501/03/19 11:21 AM 1666SPECIFIC CONSIDERATIONSPART IIdisorders such as MEN1, MEN2A, isolated familial HPT, and familial HPT with jaw-tumor syndrome. All of these syndromes are inherited in an autosomal dominant fashion. PHPT is the earliest and most common manifestation of MEN1 and develops in 80% to 100% of patients by age 40 years old. These patients also are prone to pancreatic neuroendocrine tumors and pitu-itary adenomas and, less commonly, to adrenocortical tumors, lipomas, skin angiomas, and carcinoid tumors of the bronchus, thymus, or stomach. About 50% of patients develop gastrino-mas, which often are multiple and

1	less commonly, to adrenocortical tumors, lipomas, skin angiomas, and carcinoid tumors of the bronchus, thymus, or stomach. About 50% of patients develop gastrino-mas, which often are multiple and metastatic at diagnosis. Insu-linomas develop in 10% to 15% of cases, whereas many patients have nonfunctional pancreatic endocrine tumors. Prolactinomas occur in 10% to 50% of MEN1 patients and constitute the most common pituitary lesion. MEN1 has been shown to result from germline mutations in the MEN1 gene, a tumor suppressor gene located on chromosome 11q12-13 that encodes menin, a protein that is postulated to interact with the transcription factors JunD and nuclear factor-κB in the nucleus, in addition to replication protein A and other proteins.70 Most MEN1 mutations result in a nonfunctional protein and are scattered throughout the translated nine exons of the gene. This makes presymptomatic screening for mutation carriers difficult. MEN1 mutations also have been found in kindreds

1	protein and are scattered throughout the translated nine exons of the gene. This makes presymptomatic screening for mutation carriers difficult. MEN1 mutations also have been found in kindreds initially suspected to represent isolated famil-ial HPT. HPT develops in about 20% of patients with MEN2A and generally is less severe. MEN2A is caused by germline mutations of the RET proto-oncogene located on chromosome 10. In contrast to MEN1, genotype-phenotype correlations have been noted in this syndrome in that individuals with mutations at codon 634 are more likely to develop HPT. Patients with the familial HPT with jaw-tumor syndrome have an increased predisposition to parathyroid carcinoma. This syndrome maps to a tumor suppressor locus HRPT2 (CDC73 or parafibromin) on chromosome 1. Patients belonging to isolated HPT kindreds also appear to demonstrate linkage to HRPT2. More recently, a subset of patients with MEN-1 phenotype in the absence of MENIN mutations were found to harbor

1	belonging to isolated HPT kindreds also appear to demonstrate linkage to HRPT2. More recently, a subset of patients with MEN-1 phenotype in the absence of MENIN mutations were found to harbor inactivating mutation NH2Ca2+Ca2+Ca2+[Ca2+]iCASRPKCCOOHG proteins PLCIns (1,4,5) P3Regulation of PTHParathyroid cellSystemic effectsKidney25(OH)D3LiverVitamin D1,25(OH)2D3BoneSmall intestineERCa2+Ca2+Ca2+Ca2+Ca2+Ca2+Figure 38-28. Regulation of calcium homeostasis. The calcium-sensing receptor (CASR) is expressed on the surface of the parathyroid cell and senses fluctuations in the concentration of extracellular calcium. Activation of the receptor is thought to increase intracellular cal-cium levels, which, in turn, inhibit parathyroid hormone (PTH) secretion via posttranslational mechanisms. Increased PTH secretion leads to an increase in serum calcium levels by increasing bone resorption and enhancing renal calcium reabsorption. PTH also stimulates renal 1-α-hydroxylase activity, leading to an

1	PTH secretion leads to an increase in serum calcium levels by increasing bone resorption and enhancing renal calcium reabsorption. PTH also stimulates renal 1-α-hydroxylase activity, leading to an increase in 1,25-dihydroxy vitamin D, which also exerts a negative feedback on PTH secretion. PKC = protein kinase C; PLC = phospholipase C. (Reproduced with permission from Carling T: Molecular pathology of parathyroid tumors, Trends Endocrinol Metab. 2001 Mar;12(2):53-58.)Brunicardi_Ch38_p1625-p1704.indd 166601/03/19 11:21 AM 1667THYROID, PARATHYROID, AND ADRENALCHAPTER 38in the tumor suppressor gene CDKN1B on chromosome 12p13 and given the diagnosis of MEN4.71,72 CDKN1B encodes p27kip1, which is involved in cyclin D1 signaling.Approximately 25% to 40% of sporadic parathyroid ade-nomas and some hyperplastic parathyroid glands have loss of heterozygosity (LOH) at 11q13, the site of the MEN1 gene. The parathyroid adenoma 1 oncogene (PRAD1 or CCND1), which encodes cyclin D1, a cell cycle

1	some hyperplastic parathyroid glands have loss of heterozygosity (LOH) at 11q13, the site of the MEN1 gene. The parathyroid adenoma 1 oncogene (PRAD1 or CCND1), which encodes cyclin D1, a cell cycle control protein, is overexpressed in about 18% of parathyroid adenomas. This was demonstrated to result from a rearrangement on chromosome 11 that places the PRAD1 gene under the control of the PTH promoter. Sporadic parathyroid tumors also appear to carry alterations in cyclin dependent kinase inhibitor encoding genes, in particular somatic inactivating mutations of CDKN1B. Other chromosomal regions deleted in parathyroid adenomas and possibly reflecting loss of tumor suppressor genes include 1p, 6q, and 15q, whereas ampli-fied regions suggesting oncogenes have been identified at 16p and 19p. RET mutations are rare in sporadic parathyroid tumors. Sporadic parathyroid cancers are characterized by uniform loss of the tumor suppressor gene RB, which is involved in cell cycle regulation, and

1	RET mutations are rare in sporadic parathyroid tumors. Sporadic parathyroid cancers are characterized by uniform loss of the tumor suppressor gene RB, which is involved in cell cycle regulation, and 60% have HRPT2 (CDC73) mutations. These alterations are rare in benign parathyroid tumors and may have implications for diagnosis. The p53 tumor suppressor gene is also inactivated in a subset (30%) of parathyroid carcinomas.73Clinical Manifestations Patients with PHPT formerly pre-sented with the “classic” pentad of symptoms (i.e., kidney stones, painful bones, abdominal groans, psychic moans, and fatigue overtones). With the advent and widespread use of auto-mated blood analyzers in the early 1970s, there has been an alteration in the “typical” patient with PHPT. They are more likely to be minimally symptomatic or asymptomatic. Currently, most patients present with weakness, fatigue, polydipsia, poly-uria, nocturia, bone and joint pain, constipation, decreased appe-tite, nausea,

1	to be minimally symptomatic or asymptomatic. Currently, most patients present with weakness, fatigue, polydipsia, poly-uria, nocturia, bone and joint pain, constipation, decreased appe-tite, nausea, heartburn, pruritus, depression, and memory loss. Patients with PHPT also tend to score lower than healthy controls when assessed by general multidimensional health assessment tools such as the Medical Outcomes Study Short-Form Health Survey (SF-36) and other specific questionnaires. Furthermore, these symptoms and signs improve in most, but certainly not all, patients after parathyroidectomy. Truly “asymptomatic” PHPT appears to be rare, occurring in <5% of patients, as determined by prospectively administered questionnaires. Complications of PHPT are described in the following section.Renal Disease. Approximately 80% of patients with PHPT have some degree of renal dysfunction or symptoms. Kidney stones were previously reported in up to 80% of patients but now occur in about 20% to 25%.

1	Disease. Approximately 80% of patients with PHPT have some degree of renal dysfunction or symptoms. Kidney stones were previously reported in up to 80% of patients but now occur in about 20% to 25%. The calculi are typically com-posed of calcium phosphate or oxalate. In contrast, PHPT is found to be the underlying disorder in only 3% of patients pre-senting with nephrolithiasis. Nephrocalcinosis, which refers to renal parenchymal calcification, is found in <5% of patients and is more likely to lead to renal dysfunction. Chronic hypercalce-mia also can impair concentrating ability, thereby resulting in polyuria, polydipsia, and nocturia. The incidence of hyperten-sion is variable but has been reported to occur in up to 50% of patients with PHPT. Hypertension appears to be more common in older patients and correlates with the magnitude of renal dys-function and, in contrast to other symptoms, is least likely to improve after parathyroidectomy.Bone Disease. Bone disease, including

1	in older patients and correlates with the magnitude of renal dys-function and, in contrast to other symptoms, is least likely to improve after parathyroidectomy.Bone Disease. Bone disease, including osteopenia, osteoporo-sis, and osteitis fibrosa cystica, is found in about 15% of patients with PHPT. Increased bone turnover, as found in patients with osteitis fibrosa cystica, can be determined by documenting an elevated blood alkaline phosphatase level. Advanced PHPT with osteitis fibrosa cystica now occurs in <5% of patients. It has pathognomonic radiologic findings, which are best seen on X-rays of the hands and are characterized by subperiosteal resorption (most apparent on the radial aspect of the middle pha-lanx of the second and third fingers), bone cysts, and tufting of the distal phalanges (Fig. 38-29). The skull also may be affected and appears mottled with a loss of definition of the inner and outer cortices. Brown or osteoclastic tumors and bone cysts also may be present.

1	phalanges (Fig. 38-29). The skull also may be affected and appears mottled with a loss of definition of the inner and outer cortices. Brown or osteoclastic tumors and bone cysts also may be present. Severe bone disease, resulting in bone pain and tenderness and/or pathologic fractures, is rarely observed nowa-days. However, reductions of bone mineral density (BMD) with osteopenia and osteoporosis are more common. Patients with normal serum alkaline phosphatase levels almost never have clinically apparent osteitis fibrosa cystica. HPT typically results in a loss of bone mass at sites of cortical bone such as the radius and relative preservation of cancellous bone such as that located at the vertebral bodies. Patients with PHPT, however, also may Figure 38-29. X-ray of the hand showing subperiosteal bone resorption most apparent along the radial aspect of the middle pha-lanx, characteristic of osteitis fibrosa cystica.Brunicardi_Ch38_p1625-p1704.indd 166701/03/19 11:21 AM

1	subperiosteal bone resorption most apparent along the radial aspect of the middle pha-lanx, characteristic of osteitis fibrosa cystica.Brunicardi_Ch38_p1625-p1704.indd 166701/03/19 11:21 AM 1668SPECIFIC CONSIDERATIONSPART IIhave osteoporosis of the lumbar spine that improves dramati-cally following parathyroidectomy. Fractures also occur more frequently in patients with PHPT, and the incidence of fractures also decreases after parathyroidectomy. Bone disease correlates with serum PTH and vitamin D levels.Gastrointestinal Complications. PHPT has been associated with peptic ulcer disease. In experimental animals, hypergastrin-emia has been shown to result from PTH infusion into blood ves-sels supplying the stomach, independent of its effects on serum calcium. An increased incidence of pancreatitis also has been reported in patients with PHPT, although this appears to occur only in patients with profound hypercalcemia (Ca2+ ≥12.5 mg/dL). Patients with PHPT also have an increased

1	pancreatitis also has been reported in patients with PHPT, although this appears to occur only in patients with profound hypercalcemia (Ca2+ ≥12.5 mg/dL). Patients with PHPT also have an increased incidence of choleli-thiasis, presumably due to an increase in biliary calcium, which leads to the formation of calcium bilirubinate stones.Neuropsychiatric Complications. Severe hypercalcemia may lead to various neuropsychiatric manifestations such as florid psy-chosis, obtundation, or coma. Other findings such as depression, anxiety, and fatigue are more commonly observed in patients with only mild hypercalcemia. The etiology of these symptoms is not known. Studies demonstrate that levels of certain neu-rotransmitters (monoamine metabolites 5-hydroxyindoleacetic acid and homovanillic acid) are reduced in the cerebrospi-nal fluid of patients with PHPT when compared to controls. Electroencephalogram abnormalities also occur in patients with primary and secondary HPT and normalize following

1	reduced in the cerebrospi-nal fluid of patients with PHPT when compared to controls. Electroencephalogram abnormalities also occur in patients with primary and secondary HPT and normalize following parathyroidectomy.Other Features. PHPT also can lead to fatigue and muscle weakness, which is prominent in the proximal muscle groups. Although the exact etiology of this finding is not known, muscle biopsy studies show that weakness results from a neuropathy, rather than a primary myopathic abnormality. Patients with HPT also have an increased incidence of chondrocalcinosis, gout, and pseudogout, with deposition of uric acid and calcium pyro-phosphate crystals in the joints. Calcification at ectopic sites such as blood vessels, cardiac valves, and skin also has been reported, as has hypertrophy of the left ventricle independent of the presence of hypertension. There is also evidence for subtle cardiovascular manifestations in mild disease, such as changes in endothelial function, increased

1	of the left ventricle independent of the presence of hypertension. There is also evidence for subtle cardiovascular manifestations in mild disease, such as changes in endothelial function, increased vascular stiffness, and perhaps subtle diastolic dysfunction. Several large studies from Europe also suggest that PHPT is associated with increased death rates from cardiovascular disease and cancer even in patients with mild HPT, although this finding was not substantiated in North American studies.Physical Findings Parathyroid tumors are seldom palpable, except in patients with profound hypercalcemia or parathyroid cancer. A palpable neck mass in a patient with PHPT is more likely to be thyroid in origin or a parathyroid cancer. Patients also may demonstrate evidence of band keratopathy, a depo-sition of calcium in Bowman’s membrane just inside the iris of the eye. This nonspecific condition generally is caused by chronic eye diseases such as uveitis, glaucoma, and trauma but also may

1	depo-sition of calcium in Bowman’s membrane just inside the iris of the eye. This nonspecific condition generally is caused by chronic eye diseases such as uveitis, glaucoma, and trauma but also may occur in the presence of conditions associated with high calcium or phosphate levels. Fibro-osseous jaw tumors, and/or the presence of familial disease in patients with PHPT and jaw tumors, if present, should alert the physician to the pos-sibility of parathyroid carcinoma.Differential Diagnosis Hypercalcemia may be caused by a multitude of conditions, as listed in Table 38-9. PHPT and malignancy account for >90% of all cases of hypercalcemia. PHPT is more common in the outpatient setting, whereas malig-nancy is the leading cause of hypercalcemia in hospitalized patients. PHPT can virtually always be distinguished from other diseases causing hypercalcemia by a combination of history, physical examination, and appropriate laboratory investigations.Hypercalcemia associated with malignancy

1	always be distinguished from other diseases causing hypercalcemia by a combination of history, physical examination, and appropriate laboratory investigations.Hypercalcemia associated with malignancy includes three distinct syndromes. Although bone metastases may cause hypercalcemia, patients with solid tumors of the lung, breast, kidney, head and neck, and ovary often have humoral hyper-calcemia of malignancy, without any associated bony metas-tases. In addition, hypercalcemia also may be associated with hematologic malignancies such as multiple myeloma. Humoral hypercalcemia of malignancy is known to be mediated primar-ily by PTH-related peptide (PTHrP), which also plays a role in the hypercalcemia associated with bone metastases and multiple myeloma.Thiazide diuretics cause hypercalcemia by decreasing renal clearance of calcium. This corrects in normal patients within days to weeks after discontinuing the diuretic, but patients with PHPT continue to be hypercalcemic. Thiazide

1	by decreasing renal clearance of calcium. This corrects in normal patients within days to weeks after discontinuing the diuretic, but patients with PHPT continue to be hypercalcemic. Thiazide diuretics can, therefore, exacerbate underlying PHPT and can be used to unmask PHPT in patients with borderline hyper-calcemia. Familial hypocalciuric hypercalcemia 1 (FHH1) is a rare autosomal dominant condition with nearly 100% pen-etrance and results from inherited heterozygous mutations in the CASR gene located on chromosome 3.54 Homozygous germline mutations at this locus result in neonatal severe pri-mary hyperparathyroidism and calcemia, a condition that can rapidly prove fatal. Patients with FHH1 generally have lifelong hypercalcemia, which is not corrected by parathyroidectomy. A milder form of the disease known as familial hypercalciu-ric hypercalcemia results from germline inactivating mutations in the intracytoplasmic tail domain of the CaSR gene. These patients have an appropriate

1	of the disease known as familial hypercalciu-ric hypercalcemia results from germline inactivating mutations in the intracytoplasmic tail domain of the CaSR gene. These patients have an appropriate hypercalciuric response to elevated calcium and PTH in addition to hypermagnesemia and hyper-phosphaturia. Some cases benefit from parathyroidectomy. Recently two new types of FHH (2 and 3) have been described. These are associated with germline inactivating mutations of GNA11(19p13.3) and AP2S1(19q12.2) genes. Both mutations cause hypocalciuric hypercalcemia through aberrant inactiva-tion of CaSR signaling. Although clinical presentation in FHH2 is similar to FHH1, those with FHH3 tend to have higher PTH levels and osteomalacia.71Table 38-9Differential diagnosis of hypercalcemiaHyperparathyroidismMalignancy—hematologic (multiple myeloma), solid tumors (due to PTHrP)Endocrine diseases—hyperthyroidism, Addisonian crisis, VIPomaGranulomatous diseases—sarcoidosis, tuberculosis, berylliosis,

1	(multiple myeloma), solid tumors (due to PTHrP)Endocrine diseases—hyperthyroidism, Addisonian crisis, VIPomaGranulomatous diseases—sarcoidosis, tuberculosis, berylliosis, histoplasmosisMilk-alkali syndromeDrugs—thiazide diuretics, lithium, vitamin A or D intoxicationFamilial hypocalciuric hypercalcemiaPaget’s diseaseImmobilizationPTHrP = parathyroid hormone-related protein; VIP = vasoactive intestinal peptide.Brunicardi_Ch38_p1625-p1704.indd 166801/03/19 11:21 AM 1669THYROID, PARATHYROID, AND ADRENALCHAPTER 38Coexisting primaryhyperparathyroidismand malignancyIntact PTHNormalrangeHypo-parathyroidismPrimaryhyper-parathyroidismLimit ofdetectionHypercalcemiaassociatedwith malignancy968634Intact PTH (pg/mL of hPTH [1-84])Normal50020015010050Figure 38-30. Intact parathyroid hormone (PTH) measurement allows differentiation between the various causes of hypercalcemia. (Reproduced with permission from Endres DB, Villanueva R, Sharp CF, et al. Measurement of parathyroid hormone,

1	(PTH) measurement allows differentiation between the various causes of hypercalcemia. (Reproduced with permission from Endres DB, Villanueva R, Sharp CF, et al. Measurement of parathyroid hormone, Endocrinol Metab Clin North Am. 1989 Sep;18(3):611-629.)Hypercalcemia also is found in approximately 10% of patients with sarcoidosis secondary to increased 25-hydroxy vitamin D 1-hydroxylase activity in lymphoid tissue and pul-monary macrophages, which is not subject to inhibitory feed-back control by serum calcium. Thyroid hormone also has bone-resorption properties, thus causing hypercalcemia in thy-rotoxic states, especially in immobilized patients. Hemoconcen-tration appears to be an important factor in the hypercalcemia associated with adrenal insufficiency and pheochromocytoma, although the latter patients may have associated parathyroid tumors (MEN2A), and some pheochromocytomas are known to secrete PTHrP. Other endocrine lesions such as vasoactive intestinal peptide–secreting tumors

1	latter patients may have associated parathyroid tumors (MEN2A), and some pheochromocytomas are known to secrete PTHrP. Other endocrine lesions such as vasoactive intestinal peptide–secreting tumors may be associated with hypercalcemia due to increased secretion of PTHrP. Milk-alkali syndrome requires the ingestion of large quantities of calcium with an absorbable alkali such as that used in the treatment of peptic ulcer disease with antacids. Ingestions of large quantities of vitamins D and A are infrequent causes of hypercalcemia, as is immobilization.Diagnostic Investigations Biochemical Studies. The presence of an elevated serum cal-cium and intact PTH or two-site PTH levels, without hypocal-ciuria, establishes the diagnosis of PHPT with virtual certainty. These sensitive PTH assays use immunoradiometric or immu-nochemiluminescent techniques and can reliably distinguish PHPT from other causes of hypercalcemia. Furthermore, they do not cross-react with PTHrP (Fig. 38-30). In

1	use immunoradiometric or immu-nochemiluminescent techniques and can reliably distinguish PHPT from other causes of hypercalcemia. Furthermore, they do not cross-react with PTHrP (Fig. 38-30). In patients with Table 38-10Biochemical features of primary hyperparathyroidismSERUM TESTSALTERATIONCalciumIncreased, except in normocalcemic primary hyperparathyroidismIntact PTHIncreased or inappropriately highChlorideIncreased or high normalPhosphateDecreased or low normalChloride-to-phosphate ratioIncreased (usually >33)MagnesiumUnchanged or decreased (in patients with osteitis fibrosa cystica)Uric acidNormal or increasedAlkaline phosphataseNormal or increased (in the presence of high turnover bone disease)Acid-base statusMild hyperchloremic metabolic acidosisCalcium-to-creatinine clearance ratioGenerally >0.02 (vs. <0.01 in FHH) but there are exceptions1,25-dihydroxy vitamin DNormal or increasedUrine tests 24-h urinary calciumNormal or increasedBFHH = benign familial hypocalciuric

1	ratioGenerally >0.02 (vs. <0.01 in FHH) but there are exceptions1,25-dihydroxy vitamin DNormal or increasedUrine tests 24-h urinary calciumNormal or increasedBFHH = benign familial hypocalciuric hypercalcemia; PTH = parathyroid hormone.metastatic cancer and hypercalcemia, intact PTH levels help to determine whether the patient also has concurrent PHPT. Although extremely rare, a patient with hypercalcemia may have a tumor that secretes PTH. FNAB of such a tumor for PTH levels or selective venous catheterization of the veins draining such tumors can help clarify the diagnosis.Patients with PHPT also typically have decreased serum phosphate (∼50%) and elevated 24-hour urinary calcium con-centrations (∼60%). A mild hyperchloremic metabolic acidosis also is present (80%), thereby leading to an elevated chloride-to-phosphate ratio (>33). Urinary calcium levels need not be measured routinely, except in patients who have not had previ-ously documented normocalcemia or have a family history

1	an elevated chloride-to-phosphate ratio (>33). Urinary calcium levels need not be measured routinely, except in patients who have not had previ-ously documented normocalcemia or have a family history of hypercalcemia to rule out FHH. In patients with FHH, 24-hour urinary calcium excretion is characteristically low (<100 mg/d). Furthermore, the serum calcium-to-creatinine clearance ratio (24-hour urine calcium/plasma total calcium/24-hour urine creatinine/plasma creatinine) usually is <0.01 in patients with FHH, whereas it is typically >0.02 in patients with PHPT, although there are exceptions to this. Other biochemical features of PHPT are listed in Table 38-10. Elevated levels of alkaline phosphatase may be found in approximately 10% of patients with PHPT and are indicative of high-turnover bone disease. These patients are prone to developing postoperative hypocal-cemia due to bone hunger. Serum and urine protein electropho-resis may be necessary to exclude multiple

1	of high-turnover bone disease. These patients are prone to developing postoperative hypocal-cemia due to bone hunger. Serum and urine protein electropho-resis may be necessary to exclude multiple myeloma.Brunicardi_Ch38_p1625-p1704.indd 166901/03/19 11:21 AM 1670SPECIFIC CONSIDERATIONSPART IIOccasionally, patients present with normocalcemic PHPT due to vitamin D deficiency, a low serum albumin, excessive hydration, a high-phosphate diet, or a low normal blood calcium set point. These patients have increased total PTH levels with or without increased blood ionized calcium levels and must be distinguished from patients with renal leak hypercalciuria who also have increased PTH levels due to excessive calcium loss in the urine. This can be accomplished by administering thiazide diuretics. In patients with idiopathic hypercalciuria, the urinary calcium level falls, and the secondary increase in the blood PTH level also decreases to normal, whereas patients with normocal-cemic HPT

1	In patients with idiopathic hypercalciuria, the urinary calcium level falls, and the secondary increase in the blood PTH level also decreases to normal, whereas patients with normocal-cemic HPT continue to have elevated urine calcium and blood PTH levels and may, in fact, become hypercalcemic.Radiologic Tests. In patients with profound hypercalcemia or PHPT associated with vitamin D deficiency, hand and skull X-rays may demonstrate osteitis fibrosa cystica, but this is rare in current clinical practice. BMD studies using dual-energy absorptiometry are being increasingly used to assess the effects of PHPT on bone. PHPT primarily leads to one loss at cortical sites such as the distal radius while bone density is preserved at sites such as the lumbar spine. Current evaluation of patients with PHPT includes vertebral imaging by X-ray or vertebral fracture assessment (VFA) or CT scan in addition to BMD stud-ies. Measurement of trabecular bone score (TBS) is optional. In addition, renal

1	with PHPT includes vertebral imaging by X-ray or vertebral fracture assessment (VFA) or CT scan in addition to BMD stud-ies. Measurement of trabecular bone score (TBS) is optional. In addition, renal imaging by ultrasound, X-ray, or CT scan is also recommended. Parathyroid localization studies are not used to confirm the diagnosis of PHPT, but rather to aid in identify-ing the location of the offending gland(s), as discussed later in “Preoperative Localization Tests.”Treatment Indications for Parathyroidectomy and Role of Medical Management. Most authorities agree that patients who have developed complications and have “classic” symptoms of PHPT should undergo parathyroidectomy. However, the treatment of patients with asymptomatic PHPT has been the subject of con-troversy, due, in part, to the fact that there is little agreement on what constitutes an asymptomatic patient.At the National Institutes of Health consensus conference in 1990, “asymptomatic” PHPT was defined as “the

1	part, to the fact that there is little agreement on what constitutes an asymptomatic patient.At the National Institutes of Health consensus conference in 1990, “asymptomatic” PHPT was defined as “the absence of common symptoms and signs of PHPT, including no bone, renal, gastrointestinal, or neuromuscular disorders.” To deter-mine the best course of action for these patients, it is important to consider the natural history of untreated PHPT and the out-comes of treatment options, both medical and surgical.With respect to the natural history, the panel advocated nonoperative management of these patients with mild PHPT based on observational studies, which suggested relative stabil-ity of biochemical parameters over time. However, the consen-sus panel considered certain patients to be candidates for surgery based on testing or other information indicating end-organ effects or a higher likelihood of disease progression, and this led to the establishment of initial guidelines for

1	be candidates for surgery based on testing or other information indicating end-organ effects or a higher likelihood of disease progression, and this led to the establishment of initial guidelines for parathyroidectomy.74 Subsequently, another observational study on the natural history of treated versus untreated HPT was published by Silverberg and colleagues.75 In their cohort of 52 patients with asymptomatic HPT followed without surgery, levels of serum and urinary calcium, PTH, alkaline phosphatase, and vitamin D metabolites remained relatively stable over a 10-year period in most patients. Average bone mass also remained relatively stable. However, the study also reported development of a new indication for surgery in 14 (27%) of 52 of their asymptom-atic patients and, because approximately 50% of their patients were initially treated surgically, overall, about 75% of patients were underwent parathyroidectomy. Age <50 years was predic-tive of progression, and patients undergoing

1	50% of their patients were initially treated surgically, overall, about 75% of patients were underwent parathyroidectomy. Age <50 years was predic-tive of progression, and patients undergoing parathyroidectomy showed not only normalization of calcium and PTH levels but also improved BMD at the spine and hip. Based on these and other studies, the guidelines were reassessed at a second work-shop on asymptomatic PHPT held at the National Institutes of Health in 2002.76Since that time, additional studies have provided further insights into the natural history of treated and untreated HPT. Three of these were randomized, controlled, prospective studies ranging in duration from 1 to 3.5 years. One was an observa-tional study (a continuation of the Columbia University PHPT Project) but was notable for its long duration of follow-up of 15 years.77 These studies confirmed the relative stability of vari-ous biochemical indices, thus validating the need for guidelines. However, the long-term

1	for its long duration of follow-up of 15 years.77 These studies confirmed the relative stability of vari-ous biochemical indices, thus validating the need for guidelines. However, the long-term study suggested that the stability was not indefinite as calcium levels tended to rise in years 13 to 15. In addition, the study also demonstrated that bone density mea-surements remained stable for 8 to 10 years, but cortical bone density worsened after year 10. More concerning was the fact that 60% of patients lost >10% of their BMD over the 15-year observation period. Furthermore, whether patients met the 2002 guidelines for surgery did not appear to predict the risk of pro-gressive disease, with 40% of patients undergoing follow-up eventually needing surgery. Although there are no randomized trials, registry data also suggest that fracture risk is increased for PHPT up to 10 years prior to diagnosis and treatment.Medical options for treating PHPT and its complications include

1	randomized trials, registry data also suggest that fracture risk is increased for PHPT up to 10 years prior to diagnosis and treatment.Medical options for treating PHPT and its complications include antiresorptive treatments such as bisphosphonates, hor-mone replacement therapy (HRT), and selective estrogen recep-tor modulators such as raloxifene.78 Bisphosphonates and HRT are reasonable options in patients for whom skeletal protection is needed, as evidence from randomized, placebo-controlled trials indicates that these medications are very effective at decreasing bone turnover and increasing BMD in PHPT, with the effects being comparable to patients undergoing parathyroidectomy. Caution needs to be exercised due to the nonskeletal effects of HRT, and hence, bisphosphonates are preferred. There are no clinical studies regarding the effects of raloxifene on BMD in HPT, and none of these agents affects calcium or PTH levels. More recently, calcimimetics (modifiers of the sensitivity of

1	There are no clinical studies regarding the effects of raloxifene on BMD in HPT, and none of these agents affects calcium or PTH levels. More recently, calcimimetics (modifiers of the sensitivity of the CASR) have been used in randomized, multicenter controlled trials and have been shown to decrease both serum calcium and PTH levels in both symptomatic and asymptomatic PHPT patients. Unfortunately, bone density failed to improve in medi-cally treated patients. Although this therapy shows promise, long-term outcome data are lacking, and their routine use is not advocated at this time, except in patients who are very poor operative risks or refuse surgery.Successful parathyroidectomy results in resolution of oste-itis fibrosa cystica and decreased formation of renal stones in symptomatic (classic) patients. In addition, it results in improved BMD (6% to 8% in the first year and up to 12% to 15% at 15 years) and fracture risk (by 50% at hip and upper arm and 30% overall) after

1	(classic) patients. In addition, it results in improved BMD (6% to 8% in the first year and up to 12% to 15% at 15 years) and fracture risk (by 50% at hip and upper arm and 30% overall) after adjustment for age, sex, and previous frac-tures over a 20-year observation period.75 There are also data to show that it improves a number of the nonspecific manifesta-tions of PHPT such as fatigue, polydipsia, polyuria and nocturia, Brunicardi_Ch38_p1625-p1704.indd 167001/03/19 11:21 AM 1671THYROID, PARATHYROID, AND ADRENALCHAPTER 38Table 38-11Indications for parathyroidectomy in patients with asymptomatic primary HPT (2014 NIH consensus conference guidelines)• Serum calcium >1 mg/dL above the upper limits of normal• GFR <60 mL/min; 24-h urine for calcium >400 mg/d (>10 mmol/d) and increased stone risk by biochemical stone risk analysis; presence of nephrolithiasis or nephrocalcinosis by X-ray, ultrasound, or CT• Substantially decreased bone mineral density at the lumbar spine, total hip,

1	risk by biochemical stone risk analysis; presence of nephrolithiasis or nephrocalcinosis by X-ray, ultrasound, or CT• Substantially decreased bone mineral density at the lumbar spine, total hip, femoral neck, or distal radius (>2.5 SD below peak bone mass, T score <−2.5; vertebral fracture by X-ray, CT, MRI, or VFA)• Age <50 y• Long-term medical surveillance not desired or possibleGFR = glomerular filtration rate; HPT = hyperparathyroidism; NIH = National Institutes of Health; SD = standard deviation; VFA = vertebral fracture assessmentbone and joint pain, constipation, nausea, and depression in many patients. This also has been demonstrated using symptom questionnaires and various standardized general quality-of-life assessments such as the SF-36 and a specific parathyroidectomy assessment of symptoms scale.79 The increased death rate in patients with PHPT appears to be revers-ible by successful parathyroidectomy, at least in some studies. Lastly, parathyroidectomy can be

1	assessment of symptoms scale.79 The increased death rate in patients with PHPT appears to be revers-ible by successful parathyroidectomy, at least in some studies. Lastly, parathyroidectomy can be accomplished with >95% suc-cess rates with minimal morbidity, even in elderly patients and is the only curative treatment option for PHPT. Previous inves-tigations have also documented that parathyroidectomy is more cost-effective than medical management or follow-up.80Given these findings, it is recommended that parathyroid-ectomy should be offered to virtually all patients except those in whom the operative risks are prohibitive. This is also acknowl-edged by the panel of the latest workshop, which stated that “even though patients may not meet the guidelines for surgi-cal intervention, it is always a reasonable option in those who do not have medical contraindications.” This was first stated in the 2008 guidelines and reiterated in the most recent revi-sion in 2014, which advised

1	it is always a reasonable option in those who do not have medical contraindications.” This was first stated in the 2008 guidelines and reiterated in the most recent revi-sion in 2014, which advised parathyroidectomy for patients with smaller elevations in serum calcium levels (>1 mg/dL above the upper limit of normal) and if BMD measured at any of three sites (radius, spine, or hip) is greater than 2.5 standard deviations below those of genderand race-matched, not age-matched, controls (i.e., peak bone density or T score [rather than Z score] <2.5). In addition, patients <50 years of age were advised to undergo parathyroidectomy. Parathyroidectomy is also indicated for creatinine clearance <60 cc/minute and urine calcium >400 mg/day in the presence of increased stone risk by biochemical stone risk analysis. The significant changes from the previous guidelines pertain to the fact that (a) patients with nephrolithiasis or nephrocalcinosis by X-ray, ultrasound, or CT scan and (b) those

1	stone risk analysis. The significant changes from the previous guidelines pertain to the fact that (a) patients with nephrolithiasis or nephrocalcinosis by X-ray, ultrasound, or CT scan and (b) those with vertebral fracture by X-ray, CT, MRI, or VFA are also candidates for parathyroidectomy.81,82 The current guidelines are summarized in Table 38-11.It is important to point out that the neurocognitive and neuropsychological aspects of PHPT remain a topic of con-troversy with respect to the guidelines for parathyroidectomy. Although there were more studies since the previous iteration 6of the guidelines, there were concerns that while some lacked adequate controls and were plagued by problems related to the instruments used to quantify these nonspecific symptoms, others showed variability in improvement of neurocognitive symptoms following parathyroidectomy. Similarly, uncertainty is also present concerning the cardiovascular consequences of mild HPT. Therefore, the workshop panel

1	in improvement of neurocognitive symptoms following parathyroidectomy. Similarly, uncertainty is also present concerning the cardiovascular consequences of mild HPT. Therefore, the workshop panel emphasizes that these criteria alone should not be used as guidelines for surgical intervention.Since there are no definitive criteria to indicate which patients with mild PHPT will develop progressive disease, more clinical studies are required. Patients who do not undergo sur-gery should undergo routine follow-up as outlined in the recent workshop summary statement, consisting of annual calcium and serum creatinine measurements, and measurements of BMD at three sites every 1 to 2 years.82Preoperative Localization Tests. Localization studies may be classified into noninvasive or invasive modalities. These stud-ies have variable performance characteristics, which, in turn, vary with operator and institutional experience, as outlined in Table 38-12. Localization studies have permitted

1	modalities. These stud-ies have variable performance characteristics, which, in turn, vary with operator and institutional experience, as outlined in Table 38-12. Localization studies have permitted surgeons to perform more limited operations, some of them under local anesthesia. These “minimally invasive” procedures include uni-lateral and focused neck exploration, radio-guided parathyroid-ectomy, and several endoscopic or video-assisted approaches. The use of localization studies has been shown in some studies to be associated with lower morbidity rates (hypoparathyroidism and RLN injury) and decreased operative times, reduced dura-tion of hospital stay, and improved cosmetic outcomes, while maintaining success rates similar to those obtained with tradi-tional bilateral neck explorations. Some studies also show that use of localization studies may be more cost-effective. Overall, it has become routine to localize hyperfunctioning parathyroid glands before parathyroidectomy. It is

1	Some studies also show that use of localization studies may be more cost-effective. Overall, it has become routine to localize hyperfunctioning parathyroid glands before parathyroidectomy. It is important to point out that imaging is not a diagnostic approach, and the decision for exploration should be made before any imaging is performed.99mTc-labeled sestamibi (Fig. 38-31A) is the most widely used and accurate modality with a sensitivity >80% for detec-tion of parathyroid adenomas. Sestamibi (Cardiolite) initially was introduced for cardiac imaging and is concentrated in mitochondria-rich tissue. It was subsequently noted to be useful for parathyroid localization due to the delayed washout of the radionuclide from hypercellular parathyroid tissue compared to thyroid tissue. Sestamibi scans generally are complemented by neck ultrasound (Fig. 38-31B), which can identify adenomas with >75% sensitivity in experienced centers and is most useful in identifying intrathyroidal parathyroids.

1	generally are complemented by neck ultrasound (Fig. 38-31B), which can identify adenomas with >75% sensitivity in experienced centers and is most useful in identifying intrathyroidal parathyroids. Single-photon emis-sion CT, particularly when used with CT, has been shown to be superior to other nuclear medicine–based imaging. Specifically, single-photon emission CT can indicate whether an adenoma is located in the anterior or posterior mediastinum (aortopulmo-nary window), thus enabling the surgeon to modify the opera-tive approach accordingly. CT and MRI scans are less sensitive than sestamibi scans, but they are helpful in localizing large paraesophageal and mediastinal glands. More recently, four-dimensional CT (4D-CT) has shown utility in parathyroid local-ization. This technique incorporates the perfusion of contrast in hyperfunctioning parathyroid tissue over time, thus providing functional information in addition to the anatomic information provided by conventional

1	incorporates the perfusion of contrast in hyperfunctioning parathyroid tissue over time, thus providing functional information in addition to the anatomic information provided by conventional three-dimensional CT imaging. In one study, 4D-CT showed improved sensitivity of 88% compared to Brunicardi_Ch38_p1625-p1704.indd 167101/03/19 11:21 AM 1672SPECIFIC CONSIDERATIONSPART IIthat of sestamibi (65%) and ultrasound (57%) for lateralization of the enlarged gland and also showed superiority when local-ization to the correct quadrant was examined.83 A combination of 4D-CT and ultrasound has been reported to have a positive predictive value of 92% for single-gland disease and 75% for multiple-gland disease.IOPTH was initially introduced in 1993 and is used to determine the adequacy of parathyroid resection (Fig. 38-32).84 According to one commonly used criterion, when the PTH falls by 50% or greater 10 minutes after removal of a parathyroid tumor, as compared to the highest preremoval

1	resection (Fig. 38-32).84 According to one commonly used criterion, when the PTH falls by 50% or greater 10 minutes after removal of a parathyroid tumor, as compared to the highest preremoval value, the test is considered positive, and the operation is terminated. IOPTH measurements, like localization studies, are less reliable in mul-tiglandular disease. Bilateral internal jugular vein sampling has also been used to lateralize tumors intraoperatively but is less accurate.Operative Approaches Unilateral parathyroid exploration was first carried out using intraoperative staining of a biopsy from the normal parathyroid gland with Sudan black dye to rule out a double adenoma. Initially, the choice of side to be explored was random, but the introduction of preoperative localization studies has enabled a more directed approach. In contrast, the focused approach identifies only the enlarged parathyroid gland, and no attempts are made to locate other parathyroid glands. Unilateral neck

1	has enabled a more directed approach. In contrast, the focused approach identifies only the enlarged parathyroid gland, and no attempts are made to locate other parathyroid glands. Unilateral neck explorations have several advantages over bilateral neck exploration, including reduced operative times and complications, such as injury to the RLN and hypoparathyroidism. However, Table 38-12Commonly used parathyroid localization studiesSTUDYADVANTAGESDISADVANTAGESPreoperative, noninvasive Sestamibi-technetium-99m scanAllows planar and SPECT imagingFalse-positive tests due to thyroid neoplasms, lymphadenopathyUltrasoundIdentification of juxtaand intrathyroidal tumorsFalse-positive results due to thyroid nodules, cysts, lymph nodes, esophageal lesions Relatively inexpensiveFalse-negatives result from substernal, ectopic, and undescended tumorsCT scanLocalization of ectopic (mediastinal) glandsNot useful for juxtaor intrathyroidal glands False-positive results from lymph nodes Relatively

1	from substernal, ectopic, and undescended tumorsCT scanLocalization of ectopic (mediastinal) glandsNot useful for juxtaor intrathyroidal glands False-positive results from lymph nodes Relatively high cost Radiation exposure Requires IV contrast Interference from shoulders and metallic clipsMRI scanLocalization of ectopic tumorsExpensive No radiation exposureFalse-positive results from lymph nodes and thyroid nodules No IV contrastCannot be used in claustrophobic patientsFour-dimensional CT scanStructural and functional informationSimilar to CT scanPreoperative, invasiveFNABCan distinguish parathyroid tumor from lymphadenopathy using PTH assayExperienced cytologist neededAngiogramProvides a road map for selective venous samplingExpensive Treatment of mediastinal tumors by embolizationExperienced radiologist needed Neurologic complicationsVenous samplingUseful to lateralize tumor in equivocal cases or negative localization studiesExpensive, experienced radiologist

1	by embolizationExperienced radiologist needed Neurologic complicationsVenous samplingUseful to lateralize tumor in equivocal cases or negative localization studiesExpensive, experienced radiologist neededIntraoperativePTH assayImmediate confirmation of tumor removalExpensive Increased operative time, decreased accuracy in multiple-gland diseaseCT = computed tomography; FNAB = fine-needle aspiration biopsy; IV = intravenous; MRI = magnetic resonance imaging; PTH = parathyroid hormone; SPECT = single-photon emission computed tomography.Brunicardi_Ch38_p1625-p1704.indd 167201/03/19 11:21 AM 1673THYROID, PARATHYROID, AND ADRENALCHAPTER 38ABFigure 38-31. A. Sestamibi scan in a patient with primary hyperparathyroidism showing persistent uptake suggesting a left lower hypercel-lular parathyroid gland. B. Neck ultrasound in a patient with primary hyperparathyroidism showing a left lower parathyroid adenoma.most existing studies comparing the two approaches are retro-spective and do not

1	gland. B. Neck ultrasound in a patient with primary hyperparathyroidism showing a left lower parathyroid adenoma.most existing studies comparing the two approaches are retro-spective and do not analyze the results on an intent-to-treat basis. Another argument against a unilateral exploration is the risk of missing another adenoma on the opposite side of the neck. The incidence of double adenomas has been reported to range from 0% to 10%, with an increased incidence in elderly patients. The risk of missing a second adenoma is higher in populations with a higher incidence of multiple adenomas, such as those with famil-ial HPT, MEN syndromes, and the elderly. Another difficulty inherent with unilateral exploration is the inability to discern whether the combination of an abnormal gland and a normal gland on the initial side constitutes a single adenoma or asym-metric hyperplasia. A recently published update on the 5-year results of a randomized trial comparing unilateral versus bilateral

1	normal gland on the initial side constitutes a single adenoma or asym-metric hyperplasia. A recently published update on the 5-year results of a randomized trial comparing unilateral versus bilateral neck exploration did not note any difference in the rates of recur-rent or persistent disease in the two groups of patients.85 These issues will only be resolved by a large, prospective, multicenter study or improved molecular analytic techniques.Brunicardi_Ch38_p1625-p1704.indd 167301/03/19 11:21 AM 1674SPECIFIC CONSIDERATIONSPART IIRadio-guided parathyroidectomy takes advantage of the ability of parathyroid tumors to retain 99mTc-sestamibi. Before sur-gery, 1 to 2 mCi of the isotope is injected, and a hand-held gamma probe is used to guide the identification of the enlarged gland, taking care to ensure the equilibration of radioactivity counts in all quadrants. Reported advantages include easier localization, par-ticularly in reoperative cases, and the ability to perform the

1	taking care to ensure the equilibration of radioactivity counts in all quadrants. Reported advantages include easier localization, par-ticularly in reoperative cases, and the ability to perform the pro-cedure under local anesthetic or sedation using smaller incisions. Many studies demonstrated the feasibility of this technique; how-ever, it is rarely used now, largely because it offers little advantage over preoperative sestamibi scans and is associated with increased operative times. Like preoperative scanning, it also has reduced accuracy in the presence of multiglandular disease.Endoscopic approaches include both video-assisted and total endoscopic techniques. Total endoscopic parathyroidec-tomy was first described by Gagner in 1996,86 and several other investigators have since reported on this technique. Although port placements are variable, as is the case with endoscopic thy-roidectomy, they all involve creation of a working space in the neck using CO2 insufflation, with the

1	on this technique. Although port placements are variable, as is the case with endoscopic thy-roidectomy, they all involve creation of a working space in the neck using CO2 insufflation, with the reported advantages being superior cosmesis and excellent visualization. Although feasi-ble, these techniques also have been associated with increased operating times, more personnel, and greater expense, and have, in general, not been useful for patients with multiglandu-lar disease, a large thyroid mass, or previous neck surgery and irradiation. Their greatest use has been in patients with tumors at ectopic sites such as the mediastinum where thoracoscopic parathyroidectomy is an excellent alternative to sternotomy. Robotic approaches using a gasless, transaxillary technique are also being used for parathyroidectomy. Reported advantages include improved three-dimensional magnified visualization, refined ergonomic control, more freedom of motion with multi-articulated instruments, and

1	used for parathyroidectomy. Reported advantages include improved three-dimensional magnified visualization, refined ergonomic control, more freedom of motion with multi-articulated instruments, and improved cosmetic result as a result of incision placement in the axilla.Studies have shown that if both sestamibi scan and neck ultrasound studies independently identify the same, enlarged parathyroid gland, and no other gland, it is indeed the abnormal gland in approximately 95% of cases. These patients with spo-radic PHPT are candidates for a focused neck exploration, an approach that is most commonly referred to as minimally inva-sive parathyroidectomy. A standard bilateral neck exploration is planned if parathyroid localization studies or IOPTH are not available; if the localizing studies fail to identify any abnormal parathyroid gland or identify multiple abnormal glands in patients with a family history of PHPT, MEN1, or MEN2A; or if a concomitant thyroid disorder requires bilateral

1	fail to identify any abnormal parathyroid gland or identify multiple abnormal glands in patients with a family history of PHPT, MEN1, or MEN2A; or if a concomitant thyroid disorder requires bilateral exploration. In addition, finding a minimally abnormal parathy-roid gland on the side indicated by localization studies during focal exploration should prompt a bilateral exploration or at least the identification of a normal parathyroid gland on the same side. In patients with MEN1, HPT should be corrected before treatment of gastrinomas because gastrin levels decline after parathyroidectomy.Conduct of Parathyroidectomy (Standard Bilateral Explo-ration) An experienced parathyroid surgeon with a thor-ough knowledge of parathyroid anatomy and embryology and meticulous technique is crucial for the best surgical results. The procedure usually is performed under general anesthesia. The patient is positioned supine on the operating table with the neck extended. For a bilateral exploration, the

1	best surgical results. The procedure usually is performed under general anesthesia. The patient is positioned supine on the operating table with the neck extended. For a bilateral exploration, the neck is explored via a 3to 4-cm incision just caudal to the cricoid cartilage. The initial dissection and exposure is similar to that used for thy-roidectomy. After the strap muscles are separated in the midline, one side of the neck is chosen for exploration. In contrast to a thyroidectomy, the dissection during a parathyroidectomy is maintained lateral to the thyroid, making it easier to identify the parathyroid glands and not disturb their blood supply.Identification of Parathyroids. A bloodless field is important to allow identification of parathyroid glands. The middle thyroid veins are ligated and divided, thus enabling medial and anterior retraction of the thyroid lobe, with the aid of a peanut sponge or placement of 2-0 silk sutures into the thyroid. The space between the carotid

1	ligated and divided, thus enabling medial and anterior retraction of the thyroid lobe, with the aid of a peanut sponge or placement of 2-0 silk sutures into the thyroid. The space between the carotid sheath and thyroid is then opened by gentle sharp and blunt dissection, from the cricoid cartilage superiorly to the thy-mus inferiorly and the RLN is identified. Approximately 85% of the parathyroid glands are found within 1 cm of the junction of the inferior thyroid artery and RLNs. The upper parathyroid glands usually are superior to this junction and dorsal (posterior) to the nerve, whereas the lower glands are located inferior to the junction and ventral (anterior) to the recurrent nerve. Because parathyroid glands are partly surrounded by fat, any fat lobule at typical parathyroid locations should be explored because the normal or abnormal parathyroid gland may be concealed in the fatty tissue. The thin fascia overlying a “suspicious” fat lobule should be incised using a sharp

1	locations should be explored because the normal or abnormal parathyroid gland may be concealed in the fatty tissue. The thin fascia overlying a “suspicious” fat lobule should be incised using a sharp curved hemostat and scalpel. This maneuver often causes the parathyroid gland to “pop” out. Alternatively, gentle, blunt peanut sponge dissection between the carotid sheath and the thyroid gland often reveals a “float” sign, suggesting the site of the abnormal parathyroid gland. Normal parathyroids are light beige and only slightly darker or brown compared to adjacent fat.Parathyroid tissue needs to be distinguished from normal or brown fat tissue, thyroid nodules, lymph nodes, and ectopic thymus. Lymph nodes generally are light beige to whitish gray in color, glassy, and multiple in number, whereas thyroid nod-ules generally are more vascular, firm, dark or reddish brown in color, and have a more variegated appearance. Intraoperatively, 7Standard 24 hr irma

1	and multiple in number, whereas thyroid nod-ules generally are more vascular, firm, dark or reddish brown in color, and have a more variegated appearance. Intraoperatively, 7Standard 24 hr irma pg/ml2000160012008004000correlation r = 0.9161r2 = 0.8393p value <0.00010400800120016002000Figure 38-32. Correlation of the 10-minute incubation time quick parathyroid hormone assay with the 24-hour immunoradiometric (irma) parathyroid hormone assay from 138 paired intraoperative samples from 38 patients undergoing parathyroidectomy. (Repro-duced with permission from Irvin G, Dembrow VD, Prudhomme DL: Clinical usefulness of an intraoperative “quick parathyroid hormone” assay, Surgery. 1993 Dec;114(6):1019-1022.)Brunicardi_Ch38_p1625-p1704.indd 167401/03/19 11:21 AM 1675THYROID, PARATHYROID, AND ADRENALCHAPTER 38a suspicious nodule may be aspirated using a fine needle attached to a syringe containing 1 cc of saline. Very high PTH levels in the aspirate have been shown to be diagnostic

1	AND ADRENALCHAPTER 38a suspicious nodule may be aspirated using a fine needle attached to a syringe containing 1 cc of saline. Very high PTH levels in the aspirate have been shown to be diagnostic in the intraopera-tive identification of parathyroid glands. Several characteristics such as size (>7 mm), weight, and color are used to distinguish normal from hypercellular parathyroid glands. Hypercellular glands generally are darker, more firm, and more vascular than normocellular glands. No single method is 100% reliable, and therefore, the parathyroid surgeon must rely on experience and, at times, advice from a pathologist to help distinguish normal from hypercellular glands. Although several molecular studies have shown use in distinguishing parathyroid adenomas from hyperplasia, this determination also must be made by the sur-geon intraoperatively by documenting the presence of a normal parathyroid gland.Location of Parathyroid Glands. The majority of lower para-thyroid glands are

1	also must be made by the sur-geon intraoperatively by documenting the presence of a normal parathyroid gland.Location of Parathyroid Glands. The majority of lower para-thyroid glands are found in proximity to the lower thyroid pole (Fig. 38-33A). If not found at this location, the thyrothymic liga-ment and thymus should be mobilized. The upper end of the cervical thymus is gently grasped with a right-angle clamp, and the distal portion is bluntly dissected from perithymic fat with a peanut sponge. One can then “walk down” the thymus with suc-cessive right-angle clamps (Fig. 38-33B). Applying light tension along with a “twisting” motion helps to free the upper thymus. The carotid sheath also should be opened from the bifurcation to the base of the neck if the parathyroid tumor cannot be found. If these maneuvers are unsuccessful, an intrathyroidal gland should be sought by using intraoperative ultrasound, incising the thyroid capsule on its posterolateral surface, or by perform-ing an

1	If these maneuvers are unsuccessful, an intrathyroidal gland should be sought by using intraoperative ultrasound, incising the thyroid capsule on its posterolateral surface, or by perform-ing an ipsilateral thyroid lobectomy and “bread-loafing” the thy-roid lobe. Preoperative or intraoperative ultrasonography can be useful for identifying intrathyroidal parathyroid glands. Rarely, the third branchial pouch may maldescend and be found high in the neck (undescended parathymus), anterior to the carotid bulb, along with the missing parathyroid gland. Upper para-thyroid glands are more consistent in position and usually are found near the junction of the upper and middle thirds of the gland, at the level of the cricoid cartilage (Fig. 38-33C). Ectopic upper glands may be found in carotid sheath, tracheoesopha-geal groove, retroesophageal, or in the posterior mediastinum. The locations of ectopic upper and lower parathyroid glands are shown in Fig. 38-34. Every attempt must be made to

1	sheath, tracheoesopha-geal groove, retroesophageal, or in the posterior mediastinum. The locations of ectopic upper and lower parathyroid glands are shown in Fig. 38-34. Every attempt must be made to identify all four glands. Treatment depends on the number of abnormal glands.1. A single adenoma is presumed to be the cause of a patient’s PHPT if only one parathyroid tumor is identified and the other parathyroid glands are normal, a situation present in about 80% of patients with PHPT. Adenomas typically have an atrophic rim of normal parathyroid tissue, but this char-acteristic may be absent. The adenoma is dissected free of surrounding tissue, taking care to stay immediately adjacent to the tumor, without fracturing it. The vascular pedicle is clamped, divided, and ligated. Care should be taken to not rupture the parathyroid gland to decrease the risk of parathy-romatosis. If there is any question about the presumed nor-mal glands, one of them should be biopsied and examined by

1	be taken to not rupture the parathyroid gland to decrease the risk of parathy-romatosis. If there is any question about the presumed nor-mal glands, one of them should be biopsied and examined by frozen section.2. If two abnormal and two normal glands are identified, the patient has double adenomas. Triple adenomas are pres-ent if three glands are abnormal and one is normal. Mul-tiple adenomas are more common in older patients with an incidence of up to 10% in patients >60 years old. The abnormal glands should be excised, provided the re-maining glands are confirmed as such, thus excluding asymmetric hyperplasia after biopsy and frozen section.3. If all parathyroid glands are enlarged or hypercellular, pa-tients have parathyroid hyperplasia that has been shown to occur in about 15% of patients in various series. These glands are often lobulated, usually lack the rim of normal Upper parathyroid glandRecurrentlaryngeal n.Inf. thyroid a. ThymusLowerparathyroidglandThyroidABCFigure

1	of patients in various series. These glands are often lobulated, usually lack the rim of normal Upper parathyroid glandRecurrentlaryngeal n.Inf. thyroid a. ThymusLowerparathyroidglandThyroidABCFigure 38-33. Conduct of parathyroidectomy. A. Exposure of the lower parathyroid gland near the inferior pole of the thyroid gland and anterior to the recurrent laryngeal nerve. B. A thymectomy may be necessary if the lower parathyroid cannot be found in its usual location, or if the patient has familial primary hyperparathyroid-ism or secondary hyperparathyroidism. C. Exposure of the upper parathyroid gland near the insertion of the recurrent laryngeal nerve at the level of the cricothyroid muscle. a. = artery; Inf. = inferior; n. = nerve.Brunicardi_Ch38_p1625-p1704.indd 167501/03/19 11:21 AM 1676SPECIFIC CONSIDERATIONSPART IIparathyroid gland seen in adenomas, and may be variable in size. It often is difficult to distinguish multiple adenomas from hyperplasia with variable gland size.

1	AM 1676SPECIFIC CONSIDERATIONSPART IIparathyroid gland seen in adenomas, and may be variable in size. It often is difficult to distinguish multiple adenomas from hyperplasia with variable gland size. Hyperplasia may be of the chief cell (more common), mixed, or clear cell type. Patients with hyperplasia may be treated by subtotal para-thyroidectomy or by total parathyroidectomy and autotrans-plantation, with the choice of procedure being determined by rates of recurrence, postoperative hypocalcemia, and failure rates of autotransplanted tissue. Initial studies demonstrated equivalent cure rates and postoperative hypocalcemia for the two techniques, with the latter having the added advantage of avoiding recurrence in the neck. However, autotrans-planted tissue may fail to function in about 5% of cases.All four parathyroid glands are identified and carefully mobilized. For patients with hyperplasia, a titanium clip is placed across the most normal gland, leaving a 50-mg rem-nant and

1	5% of cases.All four parathyroid glands are identified and carefully mobilized. For patients with hyperplasia, a titanium clip is placed across the most normal gland, leaving a 50-mg rem-nant and taking care to avoid disturbing the vascular pedicle and that the gland is resected with a sharp scalpel. If possible, it is preferable to subtotally resect an inferior gland, which is more easily accessible in case of recurrence due to its anterior location with respect to the RLN. The resected parathyroid tis-sue is confirmed by frozen section or PTH assay. If the rem-nant appears to be viable, the remaining glands are resected. If there is any question as to the viability of the initially subtotally resected gland, another gland is chosen for subtotal resection, and the initial remnant is removed. Whenever multiple para-thyroids are resected, it is preferable to cryopreserve tissue, so that it may be autotransplanted should the patient become hypo-parathyroid. Parathyroid tissue usually is

1	Whenever multiple para-thyroids are resected, it is preferable to cryopreserve tissue, so that it may be autotransplanted should the patient become hypo-parathyroid. Parathyroid tissue usually is transplanted into the nondominant forearm. A horizontal skin incision is made over-lying the brachioradialis muscle a few centimeters below the antecubital fossa. Pockets are made in the belly of the muscle, and one to two pieces of parathyroid tissue measuring 1 mm each are placed into each pocket. A total of 12 to 14 pieces are transplanted. Autotransplanted tissue also has been reported to function when transplanted into fat.Indications for Sternotomy A sternotomy is usually not rec-ommended at the initial operation, unless the calcium level is >13 mg/dL. Rather, it is preferred to biopsy the normal glands and subsequently close the patient’s neck and obtain localiz-ing studies, if they were not obtained previously. Intraoperative PTH assay during the operation from large veins may be

1	the normal glands and subsequently close the patient’s neck and obtain localiz-ing studies, if they were not obtained previously. Intraoperative PTH assay during the operation from large veins may be helpful. Using highly selective venous catheterization postoperatively also may be needed when noninvasive localization studies are negative, equivocal, or conflicting. Lower parathyroid glands tend to migrate into the anterior mediastinum in the thymus or perithymic fat and usually can be approached via a cervical inci-sion. A sternotomy is needed to deliver these tumors in approxi-mately 5% of cases. Generally, the gland can be approached by a partial sternotomy to the third intercostal space. The midline sternotomy can be extended to the left or right side as required. Upper glands tend to migrate to the posterior mediastinum in the tracheoesophageal groove. Mediastinal glands also may be found in the aortopulmonary window or pericardium, or attached to the ascending aorta, aortic

1	to migrate to the posterior mediastinum in the tracheoesophageal groove. Mediastinal glands also may be found in the aortopulmonary window or pericardium, or attached to the ascending aorta, aortic arch, or its branches.Special Situations Normocalcemic Hyperparathyroidism. This disorder is becoming increasingly recognized in clinical practice (preva-lence from 0.5% to 16%) and is defined by the presence of an elevated PTH level with repeatedly normal calcium (including ionized calcium) levels. In addition, other secondary causes of elevated PTH should be ruled out, namely, vitamin D deficiency, osteomalacia, hypercalciuria (renal leak), and renal insufficiency. Data regarding the natural history of this disorder are limited. In a series of 37 patients, Lowe and colleagues87 showed that 19% of patients became frankly hyper-calcemic within 3 years. In addition, 57% developed osteoporo-sis, 11% developed fragility fractures, and 14% developed nephrolithiasis. Although the study had some

1	of patients became frankly hyper-calcemic within 3 years. In addition, 57% developed osteoporo-sis, 11% developed fragility fractures, and 14% developed nephrolithiasis. Although the study had some limitations, it led the authors to suggest that normocalcemic HPT may represent a variant of “symptomatic” PHPT and may not be an early form of “asymptomatic” disease. Limited studies show that 800.811217440.222128040.20.8626Figure 38-34. Location of ectopic upper and lower parathyroid glands. (Reproduced with permission from Akerström G, Malmaeus J, Bergström R: Surgical anatomy of human parathyroid glands, Surgery. 1984 Jan;95(1):14-21.)Brunicardi_Ch38_p1625-p1704.indd 167601/03/19 11:22 AM 1677THYROID, PARATHYROID, AND ADRENALCHAPTER 38parathyroidectomy is more likely to be unsuccessful in these patients. In the absence of strong data, no guidelines are avail-able for this entity.88 As such, most clinicians follow a conserva-tive course unless patients progress to the classic

1	in these patients. In the absence of strong data, no guidelines are avail-able for this entity.88 As such, most clinicians follow a conserva-tive course unless patients progress to the classic hypercalcemic form or develop nephrolithiasis, reduced bone mineral density, or fragility fractures.Parathyroid Carcinoma. Parathyroid cancer accounts for approximately 1% of PHPT cases. It may be suspected preop-eratively by the presence of severe symptoms, serum calcium levels >14 mg/dL, significantly elevated PTH levels (five times normal), and a palpable parathyroid gland. Local invasion is quite common; approximately 15% of patients have lymph node metastases, and 33% have distant metastases at pre-sentation. Intraoperatively, parathyroid cancer is suggested by the presence of a large, gray-white to gray-brown parathyroid tumor that is adherent to or invasive into surrounding tissues like muscle, thyroid, RLN, trachea, or esophagus. Enlarged lymph nodes also may be present. Frozen sections

1	to gray-brown parathyroid tumor that is adherent to or invasive into surrounding tissues like muscle, thyroid, RLN, trachea, or esophagus. Enlarged lymph nodes also may be present. Frozen sections are generally unreliable. Accurate diagnosis necessitates histologic examina-tion. The major diagnostic criteria include vascular or capsular invasion, trabecular or fibrous stroma, and frequent mitoses. It is, however, important to emphasize that these classic findings are not as frequently noted as previously reported, and some may be found in benign adenomas as well.Treatment of parathyroid cancer consists of neck explora-tion, with en bloc excision of the tumor and the ipsilateral thy-roid lobe, in addition to the removal of contiguous lymph nodes (tracheoesophageal, paratracheal, and upper mediastinal). The recurrent nerve is not sacrificed unless it is directly involved with tumor. Adherent soft tissue structures (strap muscles or other soft tissues) should also be resected.89 Modified

1	The recurrent nerve is not sacrificed unless it is directly involved with tumor. Adherent soft tissue structures (strap muscles or other soft tissues) should also be resected.89 Modified radical neck dissection is recommended in the presence of lateral lymph node metastases. Prophylactic neck dissection is not advised. If the diagnosis is made postoperatively, a decision must be made regarding the adequacy of initial surgery based on a review of operative notes, pathology reports, localization studies, and cal-cium and PTH levels. If any question exists, histologic review by another experienced pathologist can be helpful. Additional procedures can include ipsilateral thyroid lobectomy with resec-tion of contiguous structures and lymph nodes if the features are typical or the patient remains hypercalcemic. Patients with equivocal pathologic findings and normocalcemia may be monitored closely. Reoperation is indicated for locally recur-rent or metastatic disease to control

1	patient remains hypercalcemic. Patients with equivocal pathologic findings and normocalcemia may be monitored closely. Reoperation is indicated for locally recur-rent or metastatic disease to control hypercalcemia. Adjuvant radiation therapy should be considered in patients at high risk of local recurrence such as those with close or positive margins, invasion of surrounding structures, or tumor rupture. Radiation may also be used as primary therapy in unresectable disease or for palliation of bone metastases. Chemotherapy is not very effective. Bisphosphonates have shown some effectiveness in treating hypercalcemia associated with parathyroid carcinoma. Cinacalcet hydrochloride, a calcimimetic, can reduce PTH lev-els by directly binding to the CASR cells on the parathyroid gland and has been shown to be useful in controlling hyper-calcemia in patients with refractory parathyroid carcinoma.90 Other promising approaches include antiparathyroid hormone immunotherapy, octreotide, and the

1	shown to be useful in controlling hyper-calcemia in patients with refractory parathyroid carcinoma.90 Other promising approaches include antiparathyroid hormone immunotherapy, octreotide, and the telomerase inhibitor azido-thymidine, but additional investigations are needed in this area.Familial Hyperparathyroidism. PHPT may occur as a com-ponent of various inherited syndromes such as MEN1 and MEN2A. Inherited PHPT also can occur as isolated familial HPT (non-MEN) or familial HPT with jaw tumors. The diag-nosis of familial HPT is known or suspected in approximately 85% of patients preoperatively. Furthermore, patients with hereditary HPT generally have a higher incidence of multiglan-dular disease, supernumerary glands, and recurrent or persistent disease. Therefore, these patients warrant a more aggressive approach and are not candidates for various focused surgical approaches.91 Although not absolutely necessary, preoperative sestamibi scan and ultrasound can be obtained in patients

1	a more aggressive approach and are not candidates for various focused surgical approaches.91 Although not absolutely necessary, preoperative sestamibi scan and ultrasound can be obtained in patients with inherited HPT to identify potential ectopic glands. A standard bilateral neck exploration is performed, along with a bilateral cervical thymectomy, regardless of the results of localization studies. Both subtotal parathyroidectomy and total parathyroid-ectomy with autotransplantation are appropriate, and parathy-roid tissue also should be cryopreserved. If an adenoma is found in patients with familial HPT, the adenoma and the ipsilateral normal parathyroid glands are resected. The normal-appearing glands on the contralateral side are biopsied and marked, so that only one side of the neck will need to be explored in the event of recurrence. Patients with MEN2A require total thyroidectomy and central neck dissection for prevention/treatment of MTC, a procedure that places the

1	the neck will need to be explored in the event of recurrence. Patients with MEN2A require total thyroidectomy and central neck dissection for prevention/treatment of MTC, a procedure that places the parathyroids at risk. Moreover, HPT is less aggressive in these patients. Hence, only abnormal para-thyroid glands need to be resected at neck exploration. The other normal parathyroid glands should be marked with a clip.Neonatal Hyperparathyroidism. Infants with neonatal HPT present with severe hypercalcemia, lethargy, hypotonia, and mental retardation. This disorder is associated with homozygous mutations in the CASR gene. As indicated earlier, urgent total parathyroidectomy (with autotransplantation and cryopreserva-tion) and thymectomy are indicated. Subtotal resection is associ-ated with high recurrence rates.Parathyromatosis. Parathyromatosis is a rare condition char-acterized by the finding of multiple nodules of hyperfunctioning parathyroid tissue throughout the neck and

1	with high recurrence rates.Parathyromatosis. Parathyromatosis is a rare condition char-acterized by the finding of multiple nodules of hyperfunctioning parathyroid tissue throughout the neck and mediastinum, usu-ally following a previous parathyroidectomy. The true etiology of parathyromatosis is not known. It is postulated to arise either from overgrowth of congenital parathyroid rests (ontogenous parathyromatosis) or seeding at surgery from rupture of parathy-roid tumors or subtotal resection of hyperplastic glands. Para-thyromatosis represents a rare cause of persistent or recurrent HPT92 and can be identified intraoperatively. Aggressive local resection of these deposits can result in normocalcemia but is rarely curative. Some studies suggest that these patients have low-grade carcinoma because of invasion into muscle and other structures distant from the resected parathyroid tumor.Postoperative Care and Follow-Up Patients who have under-gone parathyroidectomy are advised to

1	because of invasion into muscle and other structures distant from the resected parathyroid tumor.Postoperative Care and Follow-Up Patients who have under-gone parathyroidectomy are advised to undergo calcium level checks 2 weeks postoperatively, at 6 months, and then annu-ally. Recurrences are rare (<1%), except in patients with famil-ial HPT. Recurrence rates of 15% at 2 years and 67% at 8 years have been reported for MEN1 patients.Persistent and Recurrent Hyperparathyroidism. Per-sistence is defined as hypercalcemia that fails to resolve after parathyroidectomy and is more common than recurrence, which refers to HPT occurring after an intervening period of at least 6 months of biochemically documented normocalcemia.93 Recurrent disease is far less common than persistent HPT; however, both occur more frequently in the setting of familial HPT and MEN1, in particular. The most common causes for both these states include ectopic parathyroids, unrecognized

1	persistent HPT; however, both occur more frequently in the setting of familial HPT and MEN1, in particular. The most common causes for both these states include ectopic parathyroids, unrecognized 9Brunicardi_Ch38_p1625-p1704.indd 167701/03/19 11:22 AM 1678SPECIFIC CONSIDERATIONSPART IIhyperplasia, or supernumerary glands. More rare causes include parathyroid carcinoma, missed adenoma in a normal position, incomplete resection of an abnormal gland, parathyromatosis, or an inexperienced surgeon. The most common sites of ecto-pic parathyroid glands in patients with persistent or recurrent HPT are paraesophageal (28%), mediastinal (26%), intrathymic (24%), intrathyroidal (11%), carotid sheath (9%), and high cer-vical or undescended (2%) (Fig. 38-35).Once the diagnosis of persistent or recurrent HPT is sus-pected, it should be confirmed by the necessary biochemical tests. Other causes of an elevated serum PTH such as renal insufficiency, renal calcium leak, and GI tract abnormalities

1	HPT is sus-pected, it should be confirmed by the necessary biochemical tests. Other causes of an elevated serum PTH such as renal insufficiency, renal calcium leak, and GI tract abnormalities should be considered. A detailed family history should be per-formed to screen for familial disease, as this will influence the operative approach. In particular, a 24-hour urine collec-tion should be performed to rule out FHH. In redo-parathyroid surgery, the glands are more likely to be in ectopic locations, and postoperative scarring tends to make the procedure more technically demanding. Cure rates are generally lower (80–90% compared with 95–99% for initial operation), and risk of injury to RLNs and permanent hypocalcemia are higher. Therefore, an evaluation of severity of HPT and the patient’s anesthetic risk (using the American Society of Anesthesiology classifica-tion of physical status or the Goldman cardiac index) is impor-tant. There are no published guidelines directly applicable to

1	anesthetic risk (using the American Society of Anesthesiology classifica-tion of physical status or the Goldman cardiac index) is impor-tant. There are no published guidelines directly applicable to this group of patients. In general, patients with significant and ongoing problems such as recurrent kidney stones, a markedly elevated calcium level, or ongoing bone loss will need reex-ploration. Patients in whom the diagnosis remains in question or those with equivocal or minimal symptoms may be considered for conservative management. Preoperative localization studies are routinely performed. Noninvasive studies such as a sesta-mibi scan and ultrasound are obtained, supplemented by 4D-CT scans. If these studies are negative, discordant, or equivocal, obtaining an ultrasound-guided aspirate of a suspicious cervi-cal lesion or a highly selective venous catheterization for PTH levels (by an experienced angiographer) is recommended. Previ-ous operative notes and pathology reports should be

1	of a suspicious cervi-cal lesion or a highly selective venous catheterization for PTH levels (by an experienced angiographer) is recommended. Previ-ous operative notes and pathology reports should be carefully reviewed and reconciled with the information obtained from localization studies before any neck reexploration. An algorithm for the treatment of patients with recurrent and persistent HPT is shown in Fig. 38-36.Generally, these patients are approached with a focused exploration. The lateral approach is frequently used and can be achieved via the previous incision. The plane between the ster-nocleidomastoid and strap muscle is opened and allows for early identification of the RLN. Parathyroid tissue is cryopreserved routinely. Use of adjuncts, such as measuring intraoperative PTH levels, is critical to ensure adequate resection and avoid potentially harmful additional explorations. In case of difficult reexplorations, additional techniques such as bilateral internal jugular vein

1	is critical to ensure adequate resection and avoid potentially harmful additional explorations. In case of difficult reexplorations, additional techniques such as bilateral internal jugular vein sampling for PTH, thyroid lobectomy on the side of the missing gland, cervical thymectomy, and ligation of the ipsi-lateral inferior thyroid artery (after lobectomy, to cause infarc-tion of the missing gland) may be needed. Blind mediastinal exploration is not recommended. In patients who are denied, refuse, or fail exploration, medical options such as cinacalcet may be considered.Figure 38-35. Anatomic location of ectopic parathyroid glands. Numbers represent number of glands found in each location, with a total of 54. (Reproduced with permission from Shen W, Düren M, Morita E, et al: Reoperation for persistent or recurrent primary hyperparathyroidism, Arch Surg. 1996 Aug;131(8):861-867.)High

1	with a total of 54. (Reproduced with permission from Shen W, Düren M, Morita E, et al: Reoperation for persistent or recurrent primary hyperparathyroidism, Arch Surg. 1996 Aug;131(8):861-867.)High cervicalpositionCarotidsheathIntrathymicIntrathyroidalAnteriormediastinum(nonthymic)AortopulmonarywindowPosteriormediastinumParaesophageal5169213315C3C4C5C6C7T1T2T3T4Brunicardi_Ch38_p1625-p1704.indd 167801/03/19 11:22 AM 1679THYROID, PARATHYROID, AND ADRENALCHAPTER 38Recurrent or persistent HPT1) Confirm diagnosis2) Rule out FHH3) Review operative notes and pathologyParathyroidectomyPositiveNegativeNoninvasivelocalization studiesNegativePositiveSelective venous catheterization for PTHIs tumorlocalized?Follow-upif mildhypercalcemia ParathyroidectomyMedical therapyNoYesFigure 38-36. Management of recurrent and persistent hyperpara-thyroidism (HPT). FHH = familial hypocalciuric hypercalcemia; PTH = parathyroid hormone.Hypercalcemic Crisis. Patients with PHPT may occasionally present

1	of recurrent and persistent hyperpara-thyroidism (HPT). FHH = familial hypocalciuric hypercalcemia; PTH = parathyroid hormone.Hypercalcemic Crisis. Patients with PHPT may occasionally present acutely with nausea, vomiting, fatigue, muscle weak-ness, confusion, and a decreased level of consciousness—a complex referred to as hypercalcemic crisis. These symptoms result from severe hypercalcemia from uncontrolled PTH secre-tion, worsened by polyuria, dehydration, and reduced kidney function and may occur with other conditions causing hyper-calcemia. Calcium levels are markedly elevated and may be as high as 16 to 20 mg/dL. Parathyroid tumors tend to be large or multiple and may be palpable. Patients with parathyroid cancer or familial HPT are more likely to present with hypercalcemic crisis.Treatment consists of therapies to lower serum calcium levels followed by surgery to correct HPT. The mainstay of therapy involves rehydration with a 0.9% saline solution to keep urine output >100

1	consists of therapies to lower serum calcium levels followed by surgery to correct HPT. The mainstay of therapy involves rehydration with a 0.9% saline solution to keep urine output >100 cc/h. Once urine output is established, diure-sis with furosemide (which increases renal calcium clearance) is begun. If these methods are unsuccessful, other drugs may be used to lower serum calcium levels as outlined in Table 38-13. Occasionally, in life-threatening cases, hemodialysis may be of benefit.Secondary Hyperparathyroidism. Secondary HPT com-monly occurs in patients with chronic renal failure but also may occur in those with hypocalcemia secondary to inadequate cal-cium or vitamin D intake or malabsorption. The pathophysiol-ogy of HPT in chronic renal failure is complex and appears to be related to hyperphosphatemia (and resultant hypocalcemia), deficiency of 1,25-dihydroxy vitamin D due to loss of renal tissue, low calcium intake, decreased calcium absorption, and abnormal parathyroid

1	to hyperphosphatemia (and resultant hypocalcemia), deficiency of 1,25-dihydroxy vitamin D due to loss of renal tissue, low calcium intake, decreased calcium absorption, and abnormal parathyroid cell response to extracellular calcium or vitamin D in vitro and in vivo. Patients generally are hypocalce-mic or normocalcemic. Aluminum hydroxide, which often was used as a phosphate binder, has been shown to contribute to the osteomalacia observed in this disease. These patients gen-erally are treated medically with a low-phosphate diet, phos-phate binders, adequate intake of calcium and 1, 25-dihydroxy vitamin D, and a high-calcium, low-aluminum dialysis bath. Calcimimetics have been shown to control parathyroid hyper-plasia and osteitis fibrosa cystica associated with secondary HPT in animal studies and to decrease plasma PTH and total and ionized calcium levels in humans.As the indications for parathyroidectomy were not well established, surgical treatment was traditionally recommended

1	studies and to decrease plasma PTH and total and ionized calcium levels in humans.As the indications for parathyroidectomy were not well established, surgical treatment was traditionally recommended for patients with bone pain, pruritus, and (a) a calciumphosphate product ≥70, (b) calcium >11 mg/dL with markedly elevated PTH, (c) calciphylaxis, (d) progressive renal osteodys-trophy, and (e) soft tissue calcification and tumoral calcinosis, despite maximal medical therapy. Following the introduction of calcimimetics, there appears to have been a reduction in para-thyroidectomy rates. Parathyroidectomy has been reported to maintain biochemical targets for up to 5 years and improve bone density, fracture risk, calcinosis, hemoglobin levels, and even long-term survival. Studies also report that in a large series of patients on hemodialysis, calcimimetics increased the likelihood of achieving goal PTH (≤300 pg/mL), calcium, phosphate, and Ca × PO4 product, in addition to reducing the risk

1	in a large series of patients on hemodialysis, calcimimetics increased the likelihood of achieving goal PTH (≤300 pg/mL), calcium, phosphate, and Ca × PO4 product, in addition to reducing the risk of fractures and cardiovascular complications.In the absence of randomized trials comparing medical ther-apy with parathyroidectomy, current recommendations from the National Kidney Foundation’s Kidney Disease Quality Outcomes Initiative (KDOQI) advise parathyroidectomy for patients on maximal medical therapy with (a) severe HPT (defined as PTH >800 pg/mL, (b) hypercalcemia, (c) osteoporosis or pathologic bone fracture, (d) Symptoms and signs such as pruritis, bone pain, severe vascular calcifications, myopathy, and (e) calci-phylaxis.94 Calciphylaxis is a rare, limband life-threatening complication of secondary HPT characterized by painful (some-times throbbing), violaceous, and mottled lesions usually on the extremities, which often become necrotic and progress to non-healing ulcers,

1	of secondary HPT characterized by painful (some-times throbbing), violaceous, and mottled lesions usually on the extremities, which often become necrotic and progress to non-healing ulcers, gangrene, sepsis, and death. Skin biopsy can be helpful to make the diagnosis. These are critically ill, high-risk patients, but successful parathyroidectomy sometimes relieves symptoms. Not all patients with calciphylaxis will have high PTH levels, and parathyroidectomy should not be undertaken in the absence of documented hyperparathyroidism. Assessment of parathyroid mass is thought to be an important factor for pre-dicting the response to medical management. Therefore, some groups recommend parathyroidectomy if the glands are >1 cm (or >500 mm3) on ultrasound. These glands are more likely to have developed nodular hyperplasia and hence might be refrac-tory to medical management.Patients should undergo routine dialysis the day before surgery to correct electrolyte abnormalities. Localization

1	developed nodular hyperplasia and hence might be refrac-tory to medical management.Patients should undergo routine dialysis the day before surgery to correct electrolyte abnormalities. Localization stud-ies are not necessary but can identify ectopic parathyroid glands. A bilateral neck exploration is indicated. The parathyroid glands in secondary HPT are characterized by asymmetric enlargement and nodular hyperplasia. These patients may be treated by sub-total resection, leaving about 50 mg of the most normal parathy-roid gland or total parathyroidectomy and autotransplantation Brunicardi_Ch38_p1625-p1704.indd 167901/03/19 11:22 AM 1680SPECIFIC CONSIDERATIONSPART IIof parathyroid tissue into the brachioradialis muscle of the non-dominant forearm, with parathyroid cryopreservation. Upper thymectomy usually is performed because 15% to 20% of patients have one or more parathyroid glands situated in the thy-mus or perithymic fat. Some groups recommend total parathy-roidectomy without

1	thymectomy usually is performed because 15% to 20% of patients have one or more parathyroid glands situated in the thy-mus or perithymic fat. Some groups recommend total parathy-roidectomy without autotransplantation because it is associated with a lower rate of recurrence.95 While it may be preferable in patients with calciphylaxis, this procedure is contraindicated in patients eligible for renal transplant. Since the evidence needed to determine the superiority of one approach over another is lacking, the choice of procedure is influenced by surgeon pref-erence and experience and various patient factors, as indicated earlier.Tertiary Hyperparathyroidism. Generally, renal transplan-tation is an excellent method of treating secondary HPT, but some patients develop autonomous parathyroid gland function and tertiary HPT. Tertiary HPT can cause problems similar to PHPT, such as pathologic fractures, bone pain and worsened bone disease, renal stones, peptic ulcer disease, pancreatitis,

1	gland function and tertiary HPT. Tertiary HPT can cause problems similar to PHPT, such as pathologic fractures, bone pain and worsened bone disease, renal stones, peptic ulcer disease, pancreatitis, and mental status changes. The transplanted kidney is also at risk from tubulointerstitial calcification and volume depletion. Similar to patients with secondary HPT, many patients with ter-tiary HPT are being treated with cinacalcet. Although the drug is effective and well-tolerated in these patients, the long-term effects on kidney allograft function are not known, and many of these patients have persistence of their hypercalcemic symp-toms. On the other hand, parathyroidectomy has been shown to lead to a more immediate and dramatic reduction in hypercal-cemic symptoms. As such, operative intervention is indicated if autonomous PTH secretion persists for >1 year after a successful transplant in patients with hypophosphatemia, low BMD/severe osteopenia, symptoms, and signs such as

1	intervention is indicated if autonomous PTH secretion persists for >1 year after a successful transplant in patients with hypophosphatemia, low BMD/severe osteopenia, symptoms, and signs such as fatigue, pruritis, bone pain, peptic ulcer disease or nephrocalcinosis, provided they are deemed operative candidates.96 All parathyroid glands should be identified. The traditional surgical management of these patients consisted of subtotal or total parathyroid-ectomy with autotransplantation and an upper thymectomy. Some authors suggest that these patients derive similar benefit from excision of only obviously enlarged glands, while avoid-ing the higher risks of hypocalcemia associated with the for-mer approach. Others recommend that all parathyroid glands be identified and subtotal parathyroidectomy be performed as long-term follow-up studies show that limited excisions in these patients are associated with an up to fivefold increased risk of recurrent or persistent disease. Further

1	be performed as long-term follow-up studies show that limited excisions in these patients are associated with an up to fivefold increased risk of recurrent or persistent disease. Further studies are needed to define the best operative approach for these patients.Complications of Parathyroid Surgery. Parathyroidectomy can be accomplished successfully in >95% of patients with minimal mortality and morbidity, provided the procedure is performed by a surgeon experienced in parathyroid surgery. Table 38-13Medications commonly used to treat hypercalcemiaMEDICATIONDOSAGE AND ADMINISTRATIONMECHANISM, ONSET OF ACTION, AND DURATIONSIDE EFFECTSBisphosphonates (pamidronate, zolendronic acid preferred in patients with malignancy due to rapid action)60–90 mg IV over 4–24 hInhibits osteoclastic bone resorption; rapid onset, 2–3 dMay cause local pain and swelling, low-grade fever, lymphopenia, electrolyte abnormalities, osteonecrosis of the jaw in some patients (iv use)Calcitonin4 IU/kg

1	bone resorption; rapid onset, 2–3 dMay cause local pain and swelling, low-grade fever, lymphopenia, electrolyte abnormalities, osteonecrosis of the jaw in some patients (iv use)Calcitonin4 IU/kg SC/IMInhibits osteoclast function, augments renal calcium excretion; onset of action in hours; but short lived, therefore not useful as sole therapyTransient nausea and vomiting, abdominal cramps, flushing, and local skin reactionMithramycin (plicamycin)25 μg/kg/d IV for 3–4 dInhibits osteoclasts RNA secretion; rapid onset of action (12 h); peaks at 48–72 h and lasts days to several weeksMay cause renal, hepatic, and hematologic complications, nausea and vomitingGallium nitrate200 mg/m2 BSA/d IV for 5 dReduces urinary calcium excretion; onset of action delayed (5–7 d)Nephrotoxicity, nausea, vomiting, hypotension, anemia, hypophosphatemiaGlucocorticoidsHydrocortisone 100 mg IV q8hDelayed onset of action (7–10 d); useful for hematologic malignancies, sarcoidosis, vitamin D intoxication,

1	vomiting, hypotension, anemia, hypophosphatemiaGlucocorticoidsHydrocortisone 100 mg IV q8hDelayed onset of action (7–10 d); useful for hematologic malignancies, sarcoidosis, vitamin D intoxication, hyperthyroidismHypertension, hyperglycemiaCalcimimetics (cinacalcet)Up to 90 mg 3 o 4 times per dayUseful in patients with parathyroid carcinoma and patients with chronic renal failureGastrointestinal complaints, hypotension, hypocalcemiaBSA = body surface area; IM = intramuscular; IV = intravenous; SC = subcutaneous.Brunicardi_Ch38_p1625-p1704.indd 168001/03/19 11:22 AM 1681THYROID, PARATHYROID, AND ADRENALCHAPTER 38Table 38-14Conditions causing hypocalcemiaHypoparathyroidism • Surgical • Neonatal • Familial • Heavy metal deposition • Magnesium depletionResistance to the action of parathyroid hormone • Pseudohypoparathyroidism • Renal failure • Medications—calcitonin, bisphosphonates, mithramycinFailure of normal 1,25-dihydroxy vitamin D productionResistance to the action of

1	parathyroid hormone • Pseudohypoparathyroidism • Renal failure • Medications—calcitonin, bisphosphonates, mithramycinFailure of normal 1,25-dihydroxy vitamin D productionResistance to the action of 1,25-dihydroxy vitamin DAcute complex formation or deposition of calcium • Acute hyperphosphatemia • Acute pancreatitis • Massive blood transfusion (citrate overload) • “Hungry bones”Specific complications include transient and permanent vocal cord palsy and hypoparathyroidism. The latter is more likely to occur in patients who undergo four-gland exploration with biopsies, subtotal resection with an inadequate remnant, or total parathyroidectomy with a failure of autotransplanted tissue. Furthermore, hypocalcemia is more likely to occur in patients with high-turnover bone disease as evidenced by elevated pre-operative alkaline phosphatase levels. Vocal cord paralysis and hypoparathyroidism are considered permanent if they persist for >6 months. Fortunately, these complications are rare,

1	by elevated pre-operative alkaline phosphatase levels. Vocal cord paralysis and hypoparathyroidism are considered permanent if they persist for >6 months. Fortunately, these complications are rare, occurring in approximately 1% of patients undergoing surgery by experi-enced parathyroid surgeons.Patients with symptomatic hypocalcemia or those with calcium levels <8 mg/dL are treated with oral calcium supple-mentation (up to 1–2 g every 4 hours). 1,25-Dihydroxy vitamin D (calcitriol [Rocaltrol] 0.25–0.5 μg twice a day) may also be required, particularly in patients with severe hypercalcemia and elevated serum alkaline phosphatase levels, preoperatively and with osteitis fibrosa cystica. Intravenous calcium supplementa-tion rarely is needed, except in cases of severe, symptomatic hypocalcemia.HypoparathyroidismHypocalcemia can be the result of a multitude of conditions, which are listed in Table 38-14. The parathyroid glands may be congenitally absent in DiGeorge syndrome, which also is

1	can be the result of a multitude of conditions, which are listed in Table 38-14. The parathyroid glands may be congenitally absent in DiGeorge syndrome, which also is characterized by lack of thymic development and, therefore, a thymus-dependent lymphoid system. By far, the most common cause of hypoparathyroidism is thyroid surgery, particularly total thyroidectomy with a concomitant central neck dissection. Patients often develop transient hypocalcemia due to ischemia of the parathyroid glands; permanent hypoparathyroidism is rare. Hypoparathyroidism also may occur after parathyroid surgery, which is more likely if patients undergo a subtotal resection or total parathyroidectomy with parathyroid autotransplantation.Acute hypocalcemia results in decreased ionized calcium and increased neuromuscular excitability. Patients initially develop circumoral and fingertip numbness and tingling. Mental symptoms include anxiety, confusion, and depression. Physical examination reveals positive

1	excitability. Patients initially develop circumoral and fingertip numbness and tingling. Mental symptoms include anxiety, confusion, and depression. Physical examination reveals positive Chvostek’s sign (contraction of facial muscles elicited by tapping on the facial nerve anterior to the ear) and Trousseau’s sign (carpopedal spasm that is elic-ited by occluding blood flow to the forearm with a blood pres-sure cuff for 2–3 minutes). Tetany, which is characterized by tonic-clonic seizures, carpopedal spasm, and laryngeal stridor, may prove fatal and should be avoided. Most patients with post-operative hypocalcemia can be treated with oral calcium and vitamin D supplements; IV calcium infusion is rarely required except in patients with preoperative osteitis fibrosa cystica.ADRENALHistorical BackgroundEustachius provided the first accurate anatomic account of the adrenals in 1563. The anatomic division of the adrenals into the cortex and medulla was described much later, by Cuvier in

1	BackgroundEustachius provided the first accurate anatomic account of the adrenals in 1563. The anatomic division of the adrenals into the cortex and medulla was described much later, by Cuvier in 1805. Subsequently, Thomas Addison in 1855 described the features of adrenal insufficiency, which still bear his name. DeCreccio provided the first description of congenital adrenal hyperplasia (CAH) occurring in a female pseudohermaphrodite in 1865. Pheochromocytomas were first identified by Frankel in 1885, but were not named as such until 1912 by Pick, who noted the characteristic chromaffin reaction of the tumor cells. Adren-aline was identified as an agent from the adrenal medulla that elevated blood pressure in dogs and was subsequently named epinephrine in 1897. The first successful adrenalectomies for pheochromocytoma were performed by Roux in Switzerland and Charles Mayo in the United States.In 1932, Harvey Cushing described 11 patients who had moon facies, truncal obesity,

1	adrenalectomies for pheochromocytoma were performed by Roux in Switzerland and Charles Mayo in the United States.In 1932, Harvey Cushing described 11 patients who had moon facies, truncal obesity, hypertension, and other features of the syndrome that now bears his name. Although several individuals prepared adrenocortical extracts to treat adrenalec-tomized animals, cortisone was first synthesized by Kendall. Aldosterone was identified in 1952, and the syndrome result-ing from excessive secretion of this mineralocorticoid was first described in 1955 by Conn.EmbryologyThe adrenal or suprarenal glands are two endocrine organs in one; an outer cortex and an inner medulla, each with distinct embryologic, anatomic, histologic, and secretory features. The cortex originates around the fifth week of gestation from mesodermal tissue near the gonads on the adrenogenital ridge (Fig. 38-37). Therefore, ectopic adrenocortical tissue may be found in the ovaries, spermatic cord, and testes. The

1	of gestation from mesodermal tissue near the gonads on the adrenogenital ridge (Fig. 38-37). Therefore, ectopic adrenocortical tissue may be found in the ovaries, spermatic cord, and testes. The cortex dif-ferentiates further into a thin, definitive cortex and a thicker, inner fetal cortex. The latter is functional and produces fetal adrenal steroids by the eighth week of gestation, but undergoes involution after birth, resulting in a decrease in adrenal weight during the first three postpartum months. The definitive cor-tex persists after birth to form the adult cortex over the first 3 years of life. In contrast, the adrenal medulla is ectodermal in origin and arises from the neural crest. At around the same time as cortical development, neural crest cells migrate to the para-aortic and paravertebral areas and toward the medial aspect of the developing cortex to form the medulla. Most extra-adrenal neural tissue regresses but may persist at several sites. The larg-est of these is

1	paravertebral areas and toward the medial aspect of the developing cortex to form the medulla. Most extra-adrenal neural tissue regresses but may persist at several sites. The larg-est of these is located to the left of the aortic bifurcation near the inferior mesenteric artery origin and is designated as the organ of Zuckerkandl. Adrenal medullary tissue also may be found in neck, urinary bladder, and para-aortic regions. Several Brunicardi_Ch38_p1625-p1704.indd 168101/03/19 11:22 AM 1682SPECIFIC CONSIDERATIONSPART IIfactors are involved in adrenal development and include insulin-like growth factor 2; gastric inhibitory peptide; and the dosagesensitive, sex-reversal adrenal hypoplasia (DAX1) gene.AnatomyThe adrenal glands are paired, retroperitoneal organs located superior and medial to the kidneys at the level of the elev-enth ribs. The normal adrenal gland measures 5 × 3 × 1 cm and weighs 4 to 5 g. The right gland is pyramidal shaped and lies in close proximity to the right

1	to the kidneys at the level of the elev-enth ribs. The normal adrenal gland measures 5 × 3 × 1 cm and weighs 4 to 5 g. The right gland is pyramidal shaped and lies in close proximity to the right hemidiaphragm, liver, and inferior vena cava (IVC). The left adrenal is closely associated with the aorta, spleen, and tail of the pancreas. Each gland is supplied by three groups of vessels—the superior adrenal arter-ies derived from the inferior phrenic artery, the middle adrenal arteries derived from the aorta, and the inferior adrenal arteries derived from the renal artery. Other vessels originating from the intercostal and gonadal vessels may also supply the adrenals. These arteries branch into about 50 arterioles to form a rich plexus beneath the glandular capsule and require careful dissec-tion, ligation, and division during adrenalectomy. In contrast to the arterial supply, each adrenal usually is drained by a single, major adrenal vein. The right adrenal vein is usually short and

1	ligation, and division during adrenalectomy. In contrast to the arterial supply, each adrenal usually is drained by a single, major adrenal vein. The right adrenal vein is usually short and drains into the IVC, whereas the left adrenal vein is longer and empties into the left renal vein after joining the inferior phrenic vein. Accessory veins occur in 5% to 10% of patients—on the right, these vessels may drain into the right hepatic vein or the right renal vein; on the left, accessory veins may drain directly into the left renal vein. The anatomic relationships of the adre-nals and surrounding structures are depicted in Fig. 38-38.The adrenal cortex appears yellow due to its high lipid content and accounts for about 80% to 90% of the gland’s vol-ume. Histologically, the cortex is divided into three zones—the zona glomerulosa, zona fasciculata, and zona reticularis. The outer area of the zona glomerulosa consists of small cells and is the site of production of the mineralocorticoid

1	into three zones—the zona glomerulosa, zona fasciculata, and zona reticularis. The outer area of the zona glomerulosa consists of small cells and is the site of production of the mineralocorticoid hormone, aldo-sterone. The zona fasciculata is made up of larger cells, which often appear foamy due to multiple lipid inclusions, whereas the zona reticularis cells are smaller. These latter zones are the site of production of glucocorticoids and adrenal androgens. The adrenal medulla constitutes up to 10% to 20% of the gland’s volume and is reddish-brown in color. It produces the catechol-amine hormones epinephrine and norepinephrine. The cells of the adrenal medulla are arranged in cords and are polyhedral in shape. They often are referred to as chromaffin cells because they stain specifically with chromium salts.Adrenal PhysiologyCholesterol, derived from the plasma or synthesized in the adre-nal, is the common precursor of all steroid hormones derived from the adrenal cortex.

1	with chromium salts.Adrenal PhysiologyCholesterol, derived from the plasma or synthesized in the adre-nal, is the common precursor of all steroid hormones derived from the adrenal cortex. Cholesterol initially is cleaved within mitochondria to 5-δ-pregnolone, which in turn is transported to the smooth endoplasmic reticulum where it forms the substrate for various biosynthetic pathways leading to steroidogenesis (Fig. 38-39).Mineralocorticoids. The major adrenal mineralocorticoid hormones are aldosterone, 11-deoxycorticosterone (DOC), and cortisol. Cortisol has minimal effects on the kidney due to hor-mone degradation. Aldosterone secretion is regulated primarily by the renin-angiotensin system. Decreased renal blood flow, decreased plasma sodium, and increased sympathetic tone all stimulate the release of renin from juxtaglomerular cells. Renin, in turn, leads to the production of angiotensin I from its pre-cursor angiotensinogen. Angiotensin I is cleaved by pulmo-nary

1	all stimulate the release of renin from juxtaglomerular cells. Renin, in turn, leads to the production of angiotensin I from its pre-cursor angiotensinogen. Angiotensin I is cleaved by pulmo-nary angiotensin-converting enzyme (ACE) to angiotensin II; the latter is not only a potent vasoconstrictor, but it also leads to increased aldosterone synthesis and release. Hyperkalemia is another potent stimulator of aldosterone synthesis, whereas ACTH, pituitary pro-opiomelanocortin, and antidiuretic hor-mone are weak stimulators.Aldosterone is secreted at a rate of 50 to 250 μg/d (depend-ing on sodium intake) and circulates in plasma chiefly as a complex with albumin. Small amounts of the hormone bind to corticosteroid-binding globulin, and approximately 30% to 50% of secreted aldosterone circulates in a free form. The hormone has a half-life of only 15 to 20 minutes and is rapidly cleared via the liver and kidney. A small quantity of free aldosterone also is excreted in the urine.

1	circulates in a free form. The hormone has a half-life of only 15 to 20 minutes and is rapidly cleared via the liver and kidney. A small quantity of free aldosterone also is excreted in the urine. Mineralocorticoids cross the cell membrane and bind to cytosolic receptors. The receptor-ligand complex subsequently is transported into the nucleus where it induces the transcription and translation of specific genes. Aldo-sterone functions mainly to increase sodium reabsorption and potassium and hydrogen ion excretion at the level of the renal distal convoluted tubule. Less commonly, aldosterone increases sodium absorption in salivary glands and GI mucosal surfaces.9123678CA45BFigure 38-37. Cross-section of the embryo depicting adrenal development: (1) neural tube, (2) chorda, (3) aorta, (4) base of the mesentery, (5) digestive tube, (6) adrenal cortex, (7) undifferenti-ated gonad, (8) mesonephros, and (9) neural crest. Cells migrate from the neural crest to form the ganglia of the

1	(4) base of the mesentery, (5) digestive tube, (6) adrenal cortex, (7) undifferenti-ated gonad, (8) mesonephros, and (9) neural crest. Cells migrate from the neural crest to form the ganglia of the sympathetic trunk (A), sympathetic plexi (B), and the adrenal medulla and paraganglia (C). (Reproduced with permission from Avisse C, Marcus C, Patey M, et al: Surgical anatomy and embryology of the adrenal glands, Surg Clin North Am. 2000 Feb;80(1):403-415.)Brunicardi_Ch38_p1625-p1704.indd 168201/03/19 11:22 AM 1683THYROID, PARATHYROID, AND ADRENALCHAPTER 38AbdominalaortaInferiorvena cavaLeft inferiorphrenica. and v.Right phrenic a.Celiac trunkSuperiormesenteric a.Left adrenal gland Right adrenal gland Left superior adrenal aa.Right superior adrenal aa.Inferior adrenal a.Renal a. and v.Left adrenal v.Inferior adrenal a.Rightadrenalv.Middleadrenala.Figure 38-38. Anatomy of the adrenals and surrounding structures. a. = artery; v. =

1	adrenal aa.Inferior adrenal a.Renal a. and v.Left adrenal v.Inferior adrenal a.Rightadrenalv.Middleadrenala.Figure 38-38. Anatomy of the adrenals and surrounding structures. a. = artery; v. = vein.Pregnenolone17˜-hydroxypregnenoloneAndrostenedioneDHEADHEASCortisolAldosteroneCholesterol12217˜-hydroxyprogesterone311-deoxycortisol42Progesterone311-deoxy-corticosterone4.5Corticosterone518-hydroxy-corticosterone567678Figure 38-39. Synthesis of adrenal steroids. The enzymes involved are (1) p450scc (cholesterol side chain cleavage), (2) 3β-hydroxysteroid dehydrogenase, (3) p450c21 (21β-hydroxylase), (4) p450c11 (11β-hydroxylase), (5) p450c11AS (aldosterone synthase), (6) p450c17 (17α-hydroxylase activity), (7) p450c17 (17,20-lyase/desmolase activity), and (8) sulfokinase. DHEAS = dehydroepiandrosterone sulfate.Glucocorticoids. The secretion of cortisol, the major adrenal glucocorticoid, is regulated by ACTH secreted by the anterior pituitary, which, in turn, is under the control of

1	sulfate.Glucocorticoids. The secretion of cortisol, the major adrenal glucocorticoid, is regulated by ACTH secreted by the anterior pituitary, which, in turn, is under the control of corticotrophin-releasing hormone (CRH) secreted by the hypothalamus. ACTH is a 39-amino-acid protein, which is derived by cleavage from a larger precursor, pro-opiomelanocortin. ACTH is further cleaved into α-melanocyte-stimulating hormone and corticotrophin-like intermediate peptide. ACTH not only stimulates the secretion of glucocorticoids, mineralocorticoids, and adrenal androgens, but is also trophic for the adrenal glands. ACTH secretion may be Brunicardi_Ch38_p1625-p1704.indd 168301/03/19 11:22 AM 1684SPECIFIC CONSIDERATIONSPART IIstimulated by pain, stress, hypoxia, hypothermia, trauma, and hypoglycemia. ACTH secretion fluctuates, peaking in the morn-ing and reaching nadir levels in the late afternoon. Thus, there is a diurnal variation in the secretion of cortisol, with peak cortisol excretion

1	ACTH secretion fluctuates, peaking in the morn-ing and reaching nadir levels in the late afternoon. Thus, there is a diurnal variation in the secretion of cortisol, with peak cortisol excretion also occurring in the early morning and declining dur-ing the day to its lowest levels in the evening (Fig. 38-40). Corti-sol controls the secretion of both CRH and ACTH via a negative feedback loop. A similar mechanism leads to the inhibition of CRH secretion by ACTH.Cortisol is transported in plasma bound primarily to corti-costeroid-binding globulin (75%) and albumin (15%). Approxi-mately 10% of circulating cortisol is free and is the biologically active component. The plasma half-life of cortisol is 60 to 90 minutes and is determined by the extent of binding and rate of inactivation. Cortisol is converted to diand tetrahydrocor-tisol and cortisone metabolites in the liver and the kidney. The majority (95%) of cortisol and cortisone metabolites are con-jugated with glucuronic acid in the

1	is converted to diand tetrahydrocor-tisol and cortisone metabolites in the liver and the kidney. The majority (95%) of cortisol and cortisone metabolites are con-jugated with glucuronic acid in the liver, thus facilitating their renal excretion. A small amount of unmetabolized cortisol is excreted unchanged in the urine.Glucocorticoid hormones enter the cell and bind cytosolic steroid receptors. The activated receptor-ligand complex is then transported to the nucleus where it stimulates the transcription of specific target genes via a “zinc finger” DNA binding ele-ment. Cortisol also binds the mineralocorticoid receptor with an affinity similar to aldosterone. However, the specificity of mineralocorticoid action is maintained by the production of 11β-hydroxysteroid dehydrogenase, an enzyme that inactivates cortisol to cortisone in the kidney. Glucocorticoids have impor-tant functions in intermediary metabolism but also affect con-nective tissue, bone, immune, cardiovascular, renal,

1	that inactivates cortisol to cortisone in the kidney. Glucocorticoids have impor-tant functions in intermediary metabolism but also affect con-nective tissue, bone, immune, cardiovascular, renal, and central nervous systems, as outlined in Table 38-15.Sex Steroids. Adrenal androgens are produced in the zona fasciculata and reticularis from 17-hydroxypregnenolone in response to ACTH stimulation. They include dehydroepiandros-terone (DHEA) and its sulfated counterpart (DHEAS), andro-stenedione, and small amounts of testosterone and estrogen. Adrenal androgens are weakly bound to plasma albumin. They exert their major effects by peripheral conversion to the more potent testosterone and dihydrotestosterone but also have weak intrinsic androgen activity. Androgen metabolites are conju-gated as glucuronides or sulfates and excreted in the urine. Dur-ing fetal development, adrenal androgens promote the formation of male genitalia. In normal adult males, the contribution of adrenal androgens

1	glucuronides or sulfates and excreted in the urine. Dur-ing fetal development, adrenal androgens promote the formation of male genitalia. In normal adult males, the contribution of adrenal androgens is minimal; however, they are responsible for the development of secondary sexual characteristics at puberty. Adrenal androgen excess leads to precocious puberty in boys and virilization, acne, and hirsutism in girls and women.Catecholamines. Catecholamine hormones (epinephrine, norepinephrine, and dopamine) are produced not only in the central and sympathetic nervous system but also the adrenal medulla. The substrate, tyrosine, is converted to catechol-amines via a series of steps shown in Fig. 38-41A. Phenyletha-nolamine N-methyltransferase, which converts norepinephrine to epinephrine, is only present in the adrenal medulla and the organ of Zuckerkandl. Therefore, the primary catecholamine produced may be used to distinguish adrenal medullary tumors from those situated at extra-adrenal

1	present in the adrenal medulla and the organ of Zuckerkandl. Therefore, the primary catecholamine produced may be used to distinguish adrenal medullary tumors from those situated at extra-adrenal sites. Catecholamines are stored in granules in combination with other neuropeptides, ATP, calcium, magnesium, and water-soluble proteins called chromogranins. Hormonal secretion is stimulated by various stress stimuli and mediated by the release of acetylcholine at the preganglionic nerve terminals. In the circulation, these pro-teins are bound to albumin and other proteins. Catecholamines are cleared by several mechanisms including reuptake by sym-pathetic nerve endings, peripheral inactivation by catechol O-methyltransferase and monoamine oxidase, and direct excre-tion by the kidneys. Metabolism of catecholamines takes place 2520151050Noon4 PM8 PMMidnight4 AM8 AMNoonLunchSnackSnackDinnerSleepB’fast200180160140120100806040200Plasma ACTH (pg/mL)Plasma 11-ohcs (µg/dL) Figure 38-40. Diurnal

1	of catecholamines takes place 2520151050Noon4 PM8 PMMidnight4 AM8 AMNoonLunchSnackSnackDinnerSleepB’fast200180160140120100806040200Plasma ACTH (pg/mL)Plasma 11-ohcs (µg/dL) Figure 38-40. Diurnal variation in cortisol levels as determined by half-hourly sampling in a 16-year-old girl. (Reproduced with permission from Krieger DT, Allen W, Rizzo F, et al: Characterization of the normal temporal pattern of plasma corticosteroid levels, J Clin Endocrinol Metab. 1971 Feb;32(2):266-284.)Brunicardi_Ch38_p1625-p1704.indd 168401/03/19 11:22 AM 1685THYROID, PARATHYROID, AND ADRENALCHAPTER 38Table 38-15Functions of glucocorticoid hormonesFUNCTION/SYSTEMEFFECTSGlucose metabolismIncreased hepatic glycogen deposition, gluconeogenesis, decreased muscle glucose uptake and metabolismProtein metabolismDecreased muscle protein synthesis, increased catabolismFat metabolismIncreased lipolysis in adipose tissueConnective tissueInhibition of fibroblasts, loss of collagen, thinning of skin, striae

1	muscle protein synthesis, increased catabolismFat metabolismIncreased lipolysis in adipose tissueConnective tissueInhibition of fibroblasts, loss of collagen, thinning of skin, striae formationSkeletal systemInhibition of bone formation, increased osteoclast activity, potentiate the action of PTHImmune systemIncreases circulation of polymorphonuclear cells; decreases numbers of lymphocytes, monocytes, and eosinophils; reduces migration of inflammatory cells to sites of injuryCardiovascular systemIncreases cardiac output and peripheral vascular toneRenal systemSodium retention, hypokalemia, hypertension via mineralocorticoid effect, increased glomerular filtration via glucocorticoid effectsEndocrine systemInhibits TSH synthesis and release, decreased TBG levels, decreased conversion of T4 to T3PTH = parathyroid hormone; T3 = 3,5’,3-triiodothyronine; T4 = thyroxine; TBG = thyroxine-binding globulin; TSH = thyroid-stimulating hormone.primarily in the liver and kidneys and leads to the

1	to T3PTH = parathyroid hormone; T3 = 3,5’,3-triiodothyronine; T4 = thyroxine; TBG = thyroxine-binding globulin; TSH = thyroid-stimulating hormone.primarily in the liver and kidneys and leads to the formation of metabolites such as metanephrines, normetanephrines, and VMA, which may undergo further glucuronidation or sulfation before being excreted in the urine (Fig. 38-41B).Adrenergic receptors are transmembrane-spanning mol-ecules that are coupled to G proteins. They may be subdivided into α and β subtypes, which are localized in different tissues, have varying affinity to various catecholamines, and mediate distinct biologic effects (Table 38-16). The receptor affinities for α receptors are—epinephrine > norepinephrine >> isopro-terenol; β1 receptors—isoproterenol > epinephrine = norepi-nephrine; and β2 receptors—isoproterenol > epinephrine >> norepinephrine.Disorders of the Adrenal CortexHyperaldosteronism. Hyperaldosteronism may be secondary to stimulation of the renin-angiotensin

1	and β2 receptors—isoproterenol > epinephrine >> norepinephrine.Disorders of the Adrenal CortexHyperaldosteronism. Hyperaldosteronism may be secondary to stimulation of the renin-angiotensin system from renal artery stenosis and to low-flow states such as congestive heart failure and cirrhosis. Hyperaldosteronism resulting from these condi-tions is reversible by treatment of the underlying cause. Primary hyperaldosteronism results from autonomous aldosterone secre-tion, which, in turn, leads to suppression of renin secretion. Pri-mary aldosteronism usually occurs in individuals between the ages of 30 to 50 years old and accounts for 1% of hypertension cases. It is associated with hypokalemia; however, more patients with Conn’s syndrome are being diagnosed with normal potas-sium levels. Most cases result from a solitary functioning adre-nal adenoma (∼70%) and idiopathic bilateral hyperplasia (30%). Adrenocortical carcinoma and glucocorticoid-suppressible hyperaldosteronism are rare,

1	cases result from a solitary functioning adre-nal adenoma (∼70%) and idiopathic bilateral hyperplasia (30%). Adrenocortical carcinoma and glucocorticoid-suppressible hyperaldosteronism are rare, each accounting for <1% of cases. Glucocorticoid-suppressible hyperaldosteronism is an auto-somal dominant form of hypertension in which aldosterone secretion is abnormally regulated by ACTH. This condition is caused by recombinations between linked genes encoding closely related isozymes, 11b-hydroxylase (CYP11B1), and aldosterone synthase (CYP11B2) generating a dysregulated chimeric gene with aldosterone synthase activity. This entity is now designated familial hyperaldosteronism type I (FH-I). Initially, FH-III referred to patients with massive adrenal hyper-plasia refractory to glucocorticoid administration. However, the term is now more commonly used to describe patients with pri-mary hyperaldosteronism due to germline KCNJ5 mutations. This gene encodes an inward rectifier potassium

1	administration. However, the term is now more commonly used to describe patients with pri-mary hyperaldosteronism due to germline KCNJ5 mutations. This gene encodes an inward rectifier potassium channel, and the mutations affected amino acids in or close to the chan-nel’s selectivity filter. Somatic gain of function mutations are found in up to 40% of aldosterone-producing adenomas (APA). Familial hyperaldosteronism type II refers to families in which two first-degree relatives have been diagnosed with primary hyperaldosteronism (adenoma or hyperplasia) and in whom types I and III have been excluded. Other genes mutated in (APA) include CACNA1D (encodes a voltage-gated L-type calcium channel), ATP1A1 (encodes part of the Na+-K+-ATPase), ATP2B3 (encodes the plasma membrane Ca2+ ATPase), CACNA1H (encodes the α1 subunit of the T-type voltage calcium channel), and CTNNB1 (β-catenin).97Symptoms and Signs Patients typically present with hyperten-sion, which is long-standing, moderate to

1	CACNA1H (encodes the α1 subunit of the T-type voltage calcium channel), and CTNNB1 (β-catenin).97Symptoms and Signs Patients typically present with hyperten-sion, which is long-standing, moderate to severe, and may be difficult to control despite multiple-drug therapy. Other symp-toms include muscle weakness, polydipsia, polyuria, nocturia, headaches, and fatigue. Weakness and fatigue are related to the presence of hypokalemia.Diagnostic Studies Laboratory Studies. Hypokalemia is a common finding, and hyperaldosteronism must be suspected in any hypertensive patient who presents with coexisting spontaneous hypokale-mia (K <3.2 mmol/L) or hypokalemia (<3 mmol/L) while on diuretic therapy, despite potassium replacements. However, it is important to note that up to 40% of patients with a confirmed aldosteronoma were normokalemic preoperatively. Once the diagnosis is suspected, further tests are necessary to confirm the diagnosis. Before testing, patients must receive adequate sodium and

1	aldosteronoma were normokalemic preoperatively. Once the diagnosis is suspected, further tests are necessary to confirm the diagnosis. Before testing, patients must receive adequate sodium and potassium. Antihypertensive medications should be held, if possible, and spironolactone, β-blockers, ACE inhibitors, and angiotensin II receptor blockers should be avoided. Patients with primary hyperaldosteronism have an ele-vated plasma aldosterone concentration level with a suppressed plasma renin activity; a plasma aldosterone concentrationto-plasma renin activity ratio of 1:25 to 30 is strongly sugges-tive of the diagnosis.98 False-positive results can occur, particularly in patients with chronic renal failure. Patients with primary hyperaldosteronism also fail to suppress aldosterone levels with sodium loading. This test can be performed by performing a 24-hour urine collection for cortisol, sodium, and aldosterone after 5 days of a high-sodium diet or alternatively giving the

1	levels with sodium loading. This test can be performed by performing a 24-hour urine collection for cortisol, sodium, and aldosterone after 5 days of a high-sodium diet or alternatively giving the Brunicardi_Ch38_p1625-p1704.indd 168501/03/19 11:22 AM 1686SPECIFIC CONSIDERATIONSPART IIFigure 38-41. A. Synthesis of catecholamines. B. Metabolism of catecholamine hormones.Tyrosine hydroxylaseL-amino acid decarboxylase Dopamine-beta-hydroxylasePhenylethanolamine-N-methyltransferaseATyrosineDopa(L-dihydroxyphenylalanine)DopamineNorepinephrineEpinephrineMAOMAOMAOMAODihydroxymandelicacid3-methoxy-4-hydroxymandelic acidCOMTCOMTCOMTEpinephrineNorepinephrineMetanephrineNormetanephrineMAO Monoamine oxidase COMT Catechol O-methyltransferaseBBrunicardi_Ch38_p1625-p1704.indd 168601/03/19 11:22 AM 1687THYROID, PARATHYROID, AND ADRENALCHAPTER 38Table 38-16Catecholamine hormone receptors and effects they mediateRECEPTORTISSUEFUNCTIONα1Blood vesselsContraction GutDecreased motility, increased

1	AM 1687THYROID, PARATHYROID, AND ADRENALCHAPTER 38Table 38-16Catecholamine hormone receptors and effects they mediateRECEPTORTISSUEFUNCTIONα1Blood vesselsContraction GutDecreased motility, increased sphincter tone PancreasDecreased insulin and glucagon release LiverGlycogenolysis, gluconeogenesis EyesPupil dilation UterusContraction SkinSweatingα2Synapse (sympathetic)Inhibits norepinephrine release PlateletAggregationβ1HeartChronotropic, inotropic Adipose tissueLipolysis GutDecreased motility, increased sphincter tone PancreasIncreased insulin and glucagon releaseβ2Blood vesselsVasodilation BronchiolesDilation UterusRelaxationpatient 2 L of saline while in the supine position, 2 to 3 days after being on a low-sodium diet. Plasma aldosterone level <5 ng/dL or a 24-hour urine aldosterone <14 μg after saline loading essentially rules out primary hyperaldosteronism. Once the biochemical diagnosis is confirmed, further evaluation should be directed at determining which patients have a

1	<14 μg after saline loading essentially rules out primary hyperaldosteronism. Once the biochemical diagnosis is confirmed, further evaluation should be directed at determining which patients have a unilat-eral aldosteronoma vs. bilateral hyperplasia because surgery is almost always curative for the former, but usually not the latter. No biochemical studies can make this distinction with 100% sensitivity; thus, imaging studies are necessary.Radiologic Studies. CT scans with 0.5-cm cuts in the adrenal area can localize aldosteronomas with a sensitivity of 90%. A unilateral 0.5to 2-cm adrenal tumor with a normal-appearing contralateral gland confirms an aldosteronoma in the presence of appropriate biochemical parameters. MRI scans are less sen-sitive but more specific, particularly if opposed phase chemi-cal shift images are obtained. MRI scans also have increased use in pregnant patients or those unable to tolerate intravenous contrast. If adrenal hyperplasia is suspected, the algorithm

1	phase chemi-cal shift images are obtained. MRI scans also have increased use in pregnant patients or those unable to tolerate intravenous contrast. If adrenal hyperplasia is suspected, the algorithm depicted in Fig. 38-42 is useful. Selective venous catheteriza-tion and adrenal vein sampling (AVS) for aldosterone have been demonstrated to be 95% sensitive and 90% specific in localiz-ing the aldosteronoma. In this procedure, the adrenal veins are cannulated, and blood samples for aldosterone and cortisol are obtained from both adrenal veins and the vena cava after ACTH administration.99 Measurement of cortisol levels is necessary to confirm proper placement of the catheters in the adrenal veins. A greater than fourfold difference in the aldosterone-to-cortisol ratios between the adrenal veins indicates the presence of a uni-lateral tumor. Some investigators use this study routinely, but it is invasive, requires an experienced interventional radiolo-gist, and can lead to adrenal vein

1	veins indicates the presence of a uni-lateral tumor. Some investigators use this study routinely, but it is invasive, requires an experienced interventional radiolo-gist, and can lead to adrenal vein rupture in approximately 1% of cases. Therefore, most groups advocate use of this modal-ity selectively in ambiguous cases, when the tumor cannot be localized and in patients with bilateral adrenal enlargement to determine whether there is unilateral or bilateral increased secretion of aldosterone.100 Additional indications for forgoing AVS include patients who are suspected of having adrenocor-tical carcinoma, those with comorbid conditions precluding surgery and those with proven familial hyperaldosteronism type I or III. Scintigraphy with 131I-6β-iodomethyl noriodocho-lesterol (NP-59) also may be used for the same purpose. Like cholesterol, this compound is taken up by the adrenal cortex, but unlike cholesterol, it remains in the gland without undergo-ing further metabolism. Adrenal

1	may be used for the same purpose. Like cholesterol, this compound is taken up by the adrenal cortex, but unlike cholesterol, it remains in the gland without undergo-ing further metabolism. Adrenal adenomas appear as “hot” nod-ules with suppressed contralateral uptake, whereas hyperplastic glands show bilaterally increased uptake. This test, however, is not widely available. Newer isotopes such as 11C-metomidate in conjunction with PET-CT have also shown promise in the localization of aldosteronomas.101AdrenalectomyBilateral hyperfunctionor failure to localizeMedical managementAdrenalectomyUnilateralincreased aldosteroneCT scan or MRIUnilateral adrenal tumorusually 0.5 2 cmin diameterBilaterallyabnormal or normaladrenals1) Selective venous catheterization for aldosterone and cortisol or2) NP-59 scanFigure 38-42. Management of an adrenal aldosteronoma. CT = computed tomography; MRI = magnetic resonance imaging.Brunicardi_Ch38_p1625-p1704.indd 168701/03/19 11:22 AM

1	cortisol or2) NP-59 scanFigure 38-42. Management of an adrenal aldosteronoma. CT = computed tomography; MRI = magnetic resonance imaging.Brunicardi_Ch38_p1625-p1704.indd 168701/03/19 11:22 AM 1688SPECIFIC CONSIDERATIONSPART IIFigure 38-43. Some characteristic features of Cushing’s syndrome—moon facies, hirsutism, and acne.Table 38-17Etiology of Cushing’s syndromeACTH-dependent (70%) • Pituitary adenoma or Cushing’s disease (∼70%) • Ectopic ACTH productiona (∼10%) • Ectopic CRH production (<1%)ACTH-independent (20–30%) • Adrenal adenoma (10–15%) • Adrenal carcinoma (5–10%) • Adrenal hyperplasia—pigmented micronodular cortical hyperplasia or gastric inhibitory peptide-sensitive macronodular hyperplasia (5%)Other • Pseudo-Cushing’s syndrome • Iatrogenic—exogenous administration of steroidsaFrom small cell lung tumors, pancreatic islet cell tumors, medullary thyroid cancers, pheochromocytomas, and carcinoid tumors of the lung, thymus, gut, pancreas, and ovary.ACTH =

1	of steroidsaFrom small cell lung tumors, pancreatic islet cell tumors, medullary thyroid cancers, pheochromocytomas, and carcinoid tumors of the lung, thymus, gut, pancreas, and ovary.ACTH = adrenocorticotropic hormone; CRH = corticotrophin-releasing hormone.Treatment Preoperatively, control of hypertension and ade-quate potassium supplementation (to keep K >3.5 mmol/L) are important. Patients generally are treated with spironolactone (an aldosterone antagonist), amiloride (a potassium-sparing diuretic that blocks sodium channels in the distal nephron), nifedipine (a calcium channel blocker), or captopril (an ACE inhibitor). Unilateral tumors producing aldosterone are best managed by adrenalectomy, either by a laparoscopic approach (preferred) or via a posterior open approach. If a carcinoma is suspected because of the large size of the adrenal lesion or mixed hormone secretion, an anterior transabdominal approach is preferred to permit adequate determination of local invasion and

1	is suspected because of the large size of the adrenal lesion or mixed hormone secretion, an anterior transabdominal approach is preferred to permit adequate determination of local invasion and distal metastases. Only 20% to 30% of patients with hyperaldosteron-ism secondary to bilateral adrenal hyperplasia benefit from sur-gery, and as described, selective venous catheterization is useful to predict which patients will respond. For the other patients, medical therapy with spironolactone, amiloride, or triamterene is the mainstay of management. Glucocorticoid-suppressible hyperaldosteronism is treated by administering exogenous dexa-methasone at doses of 0.5 to 1 mg daily. Treatment with spirono-lactone may help decrease glucocorticoid requirements in this condition and avoid symptoms of Cushing’s syndrome. Postop-eratively, some patients experience transient hypoaldosteronism requiring mineralocorticoids for up to 3 months. Recent studies suggest that postresection hyperkalemia may be

1	syndrome. Postop-eratively, some patients experience transient hypoaldosteronism requiring mineralocorticoids for up to 3 months. Recent studies suggest that postresection hyperkalemia may be more common and last longer than previously appreciated; therefore, it should be screened for in patients who are older and who have had a longer duration of hypertension, impaired kidney function, and higher preoperative aldosterone levels, making them a high-risk group.102 Rarely, acute Addison’s disease may occur 2 to 3 days after adrenalectomy. Adrenalectomy is >90% successful in improving hypokalemia and about 70% successful in cor-recting hypertension. Patients who respond to spironolactone therapy and those with a shorter duration of hypertension with minimal renal damage are more likely to achieve improvement in hypertension, whereas male patients, those >50 years old, and those with multiple adrenal nodules, are least likely to benefit from adrenalectomy.Cushing’s Syndrome. Cushing

1	to achieve improvement in hypertension, whereas male patients, those >50 years old, and those with multiple adrenal nodules, are least likely to benefit from adrenalectomy.Cushing’s Syndrome. Cushing described patients with a pecu-liar fat deposition, amenorrhea, impotence (in men), hirsutism, purple striae, hypertension, diabetes, and other features that con-stitute the syndrome (Fig. 38-43). He also recognized that sev-eral of these patients had basophilic tumors of the pituitary gland and concluded that these tumors produced hormones that caused adrenocortical hyperplasia, thus resulting in the manifestations of the syndrome. Today, the term Cushing’s syndrome refers to a complex of symptoms and signs resulting from hypersecretion of cortisol regardless of etiology. In contrast, Cushing’s disease refers to a pituitary tumor, usually an adenoma, which leads to bilateral adrenal hyperplasia and hypercortisolism. Cushing’s syndrome (endogenous) is a rare disease, affecting 10 in 1

1	disease refers to a pituitary tumor, usually an adenoma, which leads to bilateral adrenal hyperplasia and hypercortisolism. Cushing’s syndrome (endogenous) is a rare disease, affecting 10 in 1 mil-lion individuals. It is more common in adults but may occur in children. Women are more commonly affected (male-to-female ratio is 1:8). Although most individuals have sporadic disease, Cushing’s syndrome may be found in MEN1 families and can result from ACTH-secreting pituitary tumors, primary adre-nal neoplasms, or an ectopic ACTH-secreting carcinoid tumor (more common in men) or bronchial adenoma (more common in women).Cushing’s syndrome may be classified as ACTH-dependent or ACTH-independent (Table 38-17). The most common cause of hypercortisolism is exogenous administration of steroids. However, approximately 70% of cases of endogenous Cush-ing’s syndrome are caused by an ACTH-producing pituitary tumor. Primary adrenal sources (adenoma, hyperplasia, and Brunicardi_Ch38_p1625-p1704.indd

1	approximately 70% of cases of endogenous Cush-ing’s syndrome are caused by an ACTH-producing pituitary tumor. Primary adrenal sources (adenoma, hyperplasia, and Brunicardi_Ch38_p1625-p1704.indd 168801/03/19 11:22 AM 1689THYROID, PARATHYROID, AND ADRENALCHAPTER 38Table 38-18Features of Cushing’s syndromeSYSTEMMANIFESTATIONGeneralWeight gain—central obesity, buffalo hump, supraclavicular fat padsIntegumentaryHirsutism, plethora, purple striae, acne, ecchymosisCardiovascularHypertensionMusculoskeletalGeneralized weakness, osteopeniaNeuropsychiatricEmotional lability, psychosis, depressionMetabolicDiabetes or glucose intolerance, hyperlipidemiaRenalPolyuria, renal stonesGonadalImpotence, decreased libido, menstrual irregularitiescarcinoma) account for about 20% of cases, and ectopic ACTH-secreting tumors account for <10% of cases. CRH also may be secreted ectopically in bronchial carcinoid tumors, pheochro-mocytomas, and other tumors. These patients are difficult to distinguish from

1	tumors account for <10% of cases. CRH also may be secreted ectopically in bronchial carcinoid tumors, pheochro-mocytomas, and other tumors. These patients are difficult to distinguish from those with ectopic ACTH production, but can be diagnosed by determining CRH levels. Patients with major depression, alcoholism, pregnancy, chronic renal failure, or stress also may have elevated cortisol levels and symptoms of hypercortisolism. However, these manifestations resolve with treatment of the underlying disorder, and these patients are deemed to have pseudo-Cushing’s syndrome.Primary adrenal hyperplasia may be micronodular, mac-ronodular, or massively macronodular. Adrenal hyperplasia resulting from ACTH stimulation usually is macronodular (3-cm nodules). Primary pigmented nodular adrenocortical disease is a rare cause of ACTH-independent Cushing’s syndrome, which is characterized by the presence of small (<5 mm), black adrenal nodules. Primary pigmented nodular adrenocortical disease may

1	disease is a rare cause of ACTH-independent Cushing’s syndrome, which is characterized by the presence of small (<5 mm), black adrenal nodules. Primary pigmented nodular adrenocortical disease may be associated with Carney complex (atrial myxomas, schwanno-mas, and pigmented nevi) and is thought to be immune related.Symptoms and Signs The classical features of Cushing’s syn-drome are listed in Table 38-18. Early diagnosis of this disease requires a thorough knowledge of these manifestations, coupled with a high clinical suspicion. In some patients, symptoms are less pronounced and may be more difficult to recognize, par-ticularly given their diversity and the absence of a single defin-ing symptom or sign. Progressive truncal obesity is the most common symptom, occurring in up to 95% of patients. This pattern results from the lipogenic action of excessive corti-costeroids centrally and catabolic effects peripherally, along with peripheral muscle wasting. Fat deposition also occurs in

1	This pattern results from the lipogenic action of excessive corti-costeroids centrally and catabolic effects peripherally, along with peripheral muscle wasting. Fat deposition also occurs in unusual sites, such as the supraclavicular space and posterior neck region, leading to the so-called buffalo hump. Purple striae are often visible on the protuberant abdomen. Rounding of the face leads to moon facies, and thinning of subcutaneous tis-sues leads to plethora. There is an increase in fine hair growth on the face, upper back, and arms, although true virilization is more commonly seen with adrenocortical cancers. Endocrine abnormalities include glucose intolerance, amenorrhea, and decreased libido or impotence. In children, Cushing’s syndrome is characterized by obesity and stunted growth. Patients with Cushing’s disease also may present with headaches, visual field defects, and panhypopituitarism. Hyperpigmentation of the skin, if present, suggests an ectopic ACTH-producing tumor with

1	Patients with Cushing’s disease also may present with headaches, visual field defects, and panhypopituitarism. Hyperpigmentation of the skin, if present, suggests an ectopic ACTH-producing tumor with high levels of circulating ACTH.Diagnostic Tests The aims of diagnostic tests in the evalua-tion of patients suspected of having Cushing’s syndrome are twofold: to confirm the presence of Cushing’s syndrome and to determine its etiology (Fig. 38-44).Laboratory Studies. Cushing’s syndrome is characterized by elevated glucocorticoid levels that are not suppressible by exog-enous hormone administration and loss of diurnal variation. This phenomenon is used to screen patients using the overnight low-dose dexamethasone suppression test. In this test, 1 mg of a synthetic glucocorticoid (dexamethasone) is given at 11 p.m. and plasma cortisol levels are measured at 8 a.m. the following morning. Physiologically normal adults suppress cortisol levels to <3 μg/dL, whereas most patients with

1	is given at 11 p.m. and plasma cortisol levels are measured at 8 a.m. the following morning. Physiologically normal adults suppress cortisol levels to <3 μg/dL, whereas most patients with Cushing’s syndrome do not. False-negative results may be obtained in patients with mild disease; therefore, some authors consider the test positive only if cortisol levels are suppressed to <1.8 μg/dL. False-positive results can occur in up to 3% of patients with chronic renal fail-ure, depression, or those taking medications such as phenytoin, which enhance dexamethasone metabolism. In patients with a negative test but a high clinical suspicion, the classic low-dose dexamethasone (0.5 mg every 6 hours for eight doses, or 2 mg over 48 hours) suppression test or urinary cortisol measurement should be performed. Measurement of elevated 24-hour urinary cortisol levels is a very sensitive (95–100%) and specific (98%) modality of diagnosing Cushing’s syndrome and is particularly useful for identifying

1	Measurement of elevated 24-hour urinary cortisol levels is a very sensitive (95–100%) and specific (98%) modality of diagnosing Cushing’s syndrome and is particularly useful for identifying patients with pseudo-Cushing’s syn-drome. A urinary cortisol-free excretion of less than 100 μg/dL (in most laboratories) rules out hypercortisolism. Recently, salivary cortisol measurements using commercially available kits also have demonstrated superior sensitivity in diagnosing Cushing’s syndrome and are being increasingly used. Overall, 24-hour urinary tests for free cortisol and the overnight dexa-methasone suppression test at the 5 μg/dL cutoff have the high-est specificity for the diagnosis of Cushing’s syndrome.103Once a diagnosis of hypercortisolism is established, further testing is aimed at determining whether it is ACTH-dependent or ACTH-independent Cushing’s syndrome. This is best accom-plished by measurement of plasma ACTH levels (normal 10–100 pg/mL). Elevated ACTH levels are found

1	determining whether it is ACTH-dependent or ACTH-independent Cushing’s syndrome. This is best accom-plished by measurement of plasma ACTH levels (normal 10–100 pg/mL). Elevated ACTH levels are found in patients with adrenal hyperplasia due to Cushing’s disease (15–500 pg/mL) and those with CRH-secreting tumors, but the highest levels are found in patients with ectopic sources of ACTH (>1000 pg/mL). In con-trast, ACTH levels are characteristically suppressed (<5 pg/mL) in patients with primary cortisol-secreting adrenal tumors. The high-dose dexamethasone suppression test is used to distinguish between the causes of ACTH-dependent Cushing’s syndrome (pituitary vs. ectopic). The standard test (2 mg dexamethasone every 6 hours for 2 days) or the overnight test (8 mg) may be used, with 24-hour urine collections for cortisol and 17-hydroxy steroids performed over the second day. Failure to suppress urinary cortisol by 50% confirms the diagnosis of an ectopic ACTH-producing tumor. Patients

1	urine collections for cortisol and 17-hydroxy steroids performed over the second day. Failure to suppress urinary cortisol by 50% confirms the diagnosis of an ectopic ACTH-producing tumor. Patients suspected of having ectopic tumors should also undergo testing for MTC and pheochro-mocytoma. Bilateral petrosal vein sampling also is helpful for determining whether the patient has Cushing’s disease or ecto-pic Cushing’s syndrome.Brunicardi_Ch38_p1625-p1704.indd 168901/03/19 11:22 AM 1690SPECIFIC CONSIDERATIONSPART IIThe CRH test also is helpful in determining the etiology of Cushing’s syndrome. Ovine CRH (1 μg/kg) is administered intravenously, followed by serial measurements of ACTH and cortisol at 15-minute intervals for 1 hour. Patients with a primary adrenal hypercortisolism exhibit a blunted response (ACTH peak <10 pg/mL), whereas those with ACTH-dependent Cushing’s syndrome demonstrate a higher elevation of ACTH (>30 pg/mL). CRH stimulation also can enhance the useful-ness of

1	a blunted response (ACTH peak <10 pg/mL), whereas those with ACTH-dependent Cushing’s syndrome demonstrate a higher elevation of ACTH (>30 pg/mL). CRH stimulation also can enhance the useful-ness of petrosal vein sampling. Patients with pituitary tumors also have a higher peak ACTH than those with ectopic ACTHproducing tumors.Radiologic Studies. CT and MRI scans of the abdomen can identify adrenal tumors with 95% sensitivity. They also are helpful in distinguishing adrenal adenomas from carcinomas, as discussed in the subsequent section “Adrenocortical Cancer.” MRI scans have the added advantage of allowing assessment of vascular anatomy. Adrenal adenomas appear darker than the liver on T2-weighted imaging. Radioscintigraphic imag-ing of the adrenals using NP-59 also can be used to distinguish adenoma from hyperplasia. Reports suggest that “cold” adrenal nodules are more likely to be cancerous, although this distinc-tion is not absolute. NP-59 scanning is most useful in identi-fying

1	adenoma from hyperplasia. Reports suggest that “cold” adrenal nodules are more likely to be cancerous, although this distinc-tion is not absolute. NP-59 scanning is most useful in identi-fying patients with an adrenal source of hypercortisolism and primary pigmented micronodular hyperplasia.Thin-section head CT scans are 22% sensitive, and contrast-enhanced brain MRI scans are 33% to 67% sensitive at identifying pituitary tumors. Inferior petrosal sinus sampling for ACTH before and after CRH injection has been helpful in this regard and has a sensitivity approaching 100%. In this study, catheters are placed in both internal jugular veins and a periph-eral vein. A ratio of petrosal to peripheral vein ACTH level of >2 in the basal state and >3 after CRH stimulation is diagnostic 1) Overnight DST2) 24-hour urinary free cortisol3) 11:00 pm salivary cortisol1) Plasma ACTH2) High-dose DST and urinary cortisolConfirm the diagnosisACTH gradient?Determine source of

1	is diagnostic 1) Overnight DST2) 24-hour urinary free cortisol3) 11:00 pm salivary cortisol1) Plasma ACTH2) High-dose DST and urinary cortisolConfirm the diagnosisACTH gradient?Determine source of hypercortisolismDecreased ACTHLack of suppressionCT scan adrenalsIncreased ACTHPositive Increased ACTHLack of suppressionEquivocalresultsFurther testingBilateral petrosalvein samplingAdrenalsourcePituitarysourceEctopic ACTHsourceSTEPS IN DIAGNOSISDIAGNOSTIC STUDIESYesNoFigure 38-44. Diagnosis of Cushing’s syndrome. ACTH = adrenocorticotropic hormone; CT = computed tomography; DST = dexamethasone suppression test.Brunicardi_Ch38_p1625-p1704.indd 169001/03/19 11:22 AM 1691THYROID, PARATHYROID, AND ADRENALCHAPTER 38of a pituitary tumor. In patients suspected of having ectopic ACTH production, CT or MRI scans of the chest and anterior mediastinum are performed first, followed by imaging of the neck, abdomen, and pelvis if the initial studies are negative.Treatment Laparoscopic

1	production, CT or MRI scans of the chest and anterior mediastinum are performed first, followed by imaging of the neck, abdomen, and pelvis if the initial studies are negative.Treatment Laparoscopic adrenalectomy is the treatment of choice for patients with adrenal adenomas. Open adrenalectomy is reserved for large tumors (≥6 cm) or those suspected to be adrenocortical cancers. Bilateral adrenalectomy is curative for primary adrenal hyperplasia.The treatment of choice in Cushing’s disease is transsphe-noidal excision of the pituitary adenoma, which is successful in 80% of patients. Pituitary irradiation has been used for patients with persistent or recurrent disease after surgery. However, it is associated with a high rate of panhypopituitarism, and some patients develop visual deficits. This has led to increased use of stereotactic radiosurgery, which uses CT guidance to deliver high doses of radiotherapy to the tumor (photon or gamma knife) and also bilateral laparoscopic

1	deficits. This has led to increased use of stereotactic radiosurgery, which uses CT guidance to deliver high doses of radiotherapy to the tumor (photon or gamma knife) and also bilateral laparoscopic adrenalectomy. Patients who fail to respond to either treatment are candidates for pharmacologic therapy with adrenal inhibitors (medical adrenalectomy) such as ketoconazole, metyrapone, or aminoglutethimide.Patients with ectopic ACTH production are best managed by treating the primary tumor, including recurrences, if possible. Medical or bilateral laparoscopic adrenalectomy has been used to palliate patients with unresectable disease and those whose ectopic ACTH-secreting tumor cannot be localized.Patients undergoing surgery for a primary adrenal ade-noma secreting glucocorticoids require preoperative and post-operative steroids due to suppression of the contralateral adrenal gland. These patients are also at increased predisposition for infectious and thromboembolic complications, the

1	and post-operative steroids due to suppression of the contralateral adrenal gland. These patients are also at increased predisposition for infectious and thromboembolic complications, the latter due to a hypercoagulable state resulting from an increase in clotting factors including factor VIII and von Willebrand’s factor com-plex, and by impaired fibrinolysis. Duration of steroid therapy is determined by the ACTH stimulation test. Exogenous ste-roids may be needed for up to 2 years but are needed indefi-nitely in patients who have undergone bilateral adrenalectomy. This latter group of patients also may require mineralocorticoid replacement therapy. Typical replacement doses include hydro-cortisone (10–20 mg every morning and 5–10 mg every eve-ning) and fludrocortisone (0.05–0.1 mg/d every morning).Adrenocortical Cancer. Adrenal carcinomas are rare neo-plasms with a worldwide incidence of two per 1 million. These tumors have a bimodal age distribution, with an increased inci-dence in

1	morning).Adrenocortical Cancer. Adrenal carcinomas are rare neo-plasms with a worldwide incidence of two per 1 million. These tumors have a bimodal age distribution, with an increased inci-dence in children and adults in the fourth and fifth decades of life. The majority are sporadic, but adrenocortical carcinomas also occur in association with germline mutations of p53 (Li-Fraumeni syndrome) and MENIN (multiple endocrine neoplasia type 1) genes. Loci on 11p (Beckwith-Wiedemann syndrome), 2p (Carney complex), and 9q also have been implicated. Somatic p53 mutations are present in up to 33% of tumors, and LOH at the p53 locus has been reported in >85% of adreno-cortical carcinomas. In addition, insulin-like growth factor II is overexpressed in 90% of tumors, and approximately 30% harbor somatic activating mutations in the b-catenin gene. Recently identified genes mutated in adrenal cancers include ZNRF3 (an E3 ubiquitin ligase) and others involved in chromatin modeling and several

1	somatic activating mutations in the b-catenin gene. Recently identified genes mutated in adrenal cancers include ZNRF3 (an E3 ubiquitin ligase) and others involved in chromatin modeling and several microRNAs. Patterns of mutations may also help better define prognosis in adrenocortical cancers.104Symptoms and Signs Approximately 50% of adrenocorti-cal cancers are nonfunctioning. The remaining secrete cortisol (30%), androgens (20%), estrogens (10%), aldosterone (2%), or multiple hormones (35%). Patients with functioning tumors often present with the rapid onset of Cushing’s syndrome accompanied by virilizing features. Nonfunctioning tumors more commonly present with an enlarging abdominal mass and abdominal or back pain. Rarely, weight loss, anorexia, and nau-sea may be present.Diagnostic Tests Diagnostic evaluation of these patients begins with measurement of serum electrolyte levels to rule out hypokalemia, urinary catecholamines to rule out pheochromo-cytomas, an overnight 1-mg

1	Tests Diagnostic evaluation of these patients begins with measurement of serum electrolyte levels to rule out hypokalemia, urinary catecholamines to rule out pheochromo-cytomas, an overnight 1-mg dexamethasone suppression test, and a 24-hour urine collection for cortisol, and 17-ketosteroids to rule out Cushing’s syndrome.CT and MRI scans are useful to image these tumors (Fig. 38-45). The size of the adrenal mass on imaging studies is the single most important criterion to help diagnose malig-nancy. In the series reported by Copeland, 92% of adrenal can-cers were >6 cm in diameter.105 The sensitivity, specificity, and likelihood ratio of tumor size in predicting malignancy (based on Surveillance, Epidemiology, and End Results program data) were reported as 96%, 51%, and 2 for tumors ≥4 cm, and 90%, 78%, and 4.1 for tumors ≥6 cm.106 Other CT imaging charac-teristics suggesting malignancy include tumor heterogeneity, irregular margins, and the presence of hemorrhage and adjacent

1	≥4 cm, and 90%, 78%, and 4.1 for tumors ≥6 cm.106 Other CT imaging charac-teristics suggesting malignancy include tumor heterogeneity, irregular margins, and the presence of hemorrhage and adjacent lymphadenopathy or liver metastases. Moderately bright signal intensity on T2-weighted images (adrenal mass–to–liver ratio 1.2:2.8), significant lesion enhancement, and slow washout after injection of gadolinium contrast also indicate malignancy, as does evidence of local invasion into adjacent structures such as the liver, blood vessels (IVC), and distant metastases. FDG-PET or PET-CT scans may have some utility in distinguishing benign from malignant lesions, as discussed in the section on incidentalomas. Once adrenal cancer is diagnosed, CT scans of the chest and pelvis or FDG-PET or PET-CT scans are per-formed for staging. The tumor-node-metastasis (TNM) staging system for adrenocortical carcinoma is depicted in Table 38-19. Up to 70% of patients present with stage III or IV

1	or PET-CT scans are per-formed for staging. The tumor-node-metastasis (TNM) staging system for adrenocortical carcinoma is depicted in Table 38-19. Up to 70% of patients present with stage III or IV disease.Pathology Most adrenocortical cancers are large, weigh-ing between 100 and 1000 g. On gross examination, areas of Figure 38-45. Computed tomography scan of the abdomen show-ing a left adrenocortical cancer with synchronous liver metastasis.Brunicardi_Ch38_p1625-p1704.indd 169101/03/19 11:22 AM 1692SPECIFIC CONSIDERATIONSPART IIhemorrhage and necrosis often are evident. Microscopically, cells are hyperchromatic and typically have large nuclei and prominent nucleoli. It is very difficult to distinguish benign adrenal adenomas from carcinomas by histologic examination alone. Capsular or vascular invasion is the most reliable sign of cancer. Weiss and associates studied a combination of nine criteria for their usefulness in distinguishing malignant from benign adrenal tumors:

1	or vascular invasion is the most reliable sign of cancer. Weiss and associates studied a combination of nine criteria for their usefulness in distinguishing malignant from benign adrenal tumors: nuclear grade III or IV; mitotic rate greater than 5 per 50 high-power fields; atypical mitoses; clear cells comprising 25% or less of the tumor; a diffuse architecture; microscopic necrosis; and invasion of venous, sinusoidal, and capsular structure. Tumors with four or more of these criteria were likely to metastasize and/or recur.107 Rarely, the diagnosis of malignancy of a completely resected adrenal tumor is often only made in retrospect by the finding of metastatic disease many years later. Molecular markers such as Ki67 (indicating proliferative activity) can also be useful in this regard.Treatment The most important predictor of survival in patients with adrenal cancer is the adequacy of resection. Patients who undergo complete resection have 5-year actuarial survival rates ranging

1	most important predictor of survival in patients with adrenal cancer is the adequacy of resection. Patients who undergo complete resection have 5-year actuarial survival rates ranging from 32% to 48%, whereas median survival is <1 year in those undergoing incomplete excision. Therefore, adrenocor-tical carcinomas are treated by excision of the tumor en bloc with any contiguously involved lymph nodes or organs such as the diaphragm, kidney, pancreas, liver, or IVC. This is best accomplished by open adrenalectomy via a generous subcostal incision or a thoracoabdominal incision (on the right side). The incisions should permit wide exposure, minimize chances of capsule rupture and tumor spillage, and allow vascular control of the aorta, IVC, and renal vessels, as needed.Mitotane or o,p-DDD or 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl) ethane, which is a derivative of the insec-ticide DDT, has adrenolytic activity and has been used in the adjuvant setting and for the treatment of

1	1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl) ethane, which is a derivative of the insec-ticide DDT, has adrenolytic activity and has been used in the adjuvant setting and for the treatment of unresectable or metastatic disease. However, the therapeutic effectiveness is conflicting, and consistent improvement in survival rates is lacking. Moreover, the drug is associated with significant GI and neurologic side effects, particularly at the effective doses of 2 to 6 g/d. Terzolo and associates retrospectively evaluated the use of mitotane in the adjuvant setting and reported signifi-cantly increased recurrence-free survival in the treatment group, even in long-term follow-up.108,109 However, a study of several centers in the United States failed to show similar results.110 The routine use of this medication awaits evaluation in random-ized, controlled trials. Determination of blood mitotane levels is helpful to ascertain whether therapeutic and nontoxic levels are present.

1	use of this medication awaits evaluation in random-ized, controlled trials. Determination of blood mitotane levels is helpful to ascertain whether therapeutic and nontoxic levels are present. Adrenocortical tumors commonly metastasize to the liver, lung, and bone.Surgical debulking is recommended for isolated, recurrent disease and has been demonstrated to prolong survival. Sys-temic chemotherapeutic agents used in this tumor include etopo-side, cisplatin, doxorubicin, and, more recently, paclitaxel, but consistent responses are rare, possibly due to the expression of the multidrug resistance gene (MDR-1) in tumor cells. In vitro data indicate that mitotane may be able to reverse this resistance when combined with various chemotherapeutic agents. Results from the First International Randomized Trial in Advanced or Metastatic Adrenocortical Carcinoma Treatment (FIRM-ACT) showed that patients receiving etoposide, doxorubicin, cispla-tin, and mitotane had better response rates and

1	Randomized Trial in Advanced or Metastatic Adrenocortical Carcinoma Treatment (FIRM-ACT) showed that patients receiving etoposide, doxorubicin, cispla-tin, and mitotane had better response rates and progression-free survival rates than patients receiving streptozotocin and mitotane.111 There has been recent interest in the use of sura-min, a growth factor inhibitor, as therapy for adrenocortical carcinoma; however, this requires further study, particularly because this drug may be associated with significant neurotoxic-ity. Gossypol, a naturally occurring insecticide (from the cotton plant Gossypium species), also appears to inhibit the growth of adrenocortical cancer cell lines and tumors in vivo. However, poor response rates combined with high death rates in limited clinical studies have reduced enthusiasm for this agent. Adre-nocortical cancers also are relatively insensitive to conventional external-beam radiation therapy. However, this modality is used in the adjuvant setting in

1	reduced enthusiasm for this agent. Adre-nocortical cancers also are relatively insensitive to conventional external-beam radiation therapy. However, this modality is used in the adjuvant setting in patients with incomplete resections and palliation of bony metastases. Ketoconazole, metyrapone, or aminoglutethimide may also be useful in controlling steroid hypersecretion. Targeted molecular therapies such as VEGF/EGF-receptor inhibitors, tyrosine-kinase inhibitors, and IGF-2 inhibitors have had disappointing results in patients with ACC.Sex Steroid Excess. Adrenal adenomas or carcinomas that secrete adrenal androgens lead to virilizing syndromes. Although women with virilizing tumors develop hirsutism, amenorrhea, infertility, and other signs of masculinization, such as increased muscle mass, deepened voice, and temporal bald-ing, men with these tumors are more difficult to diagnose and, hence, usually present with disease in advanced stages. Chil-dren with virilizing tumors have

1	mass, deepened voice, and temporal bald-ing, men with these tumors are more difficult to diagnose and, hence, usually present with disease in advanced stages. Chil-dren with virilizing tumors have accelerated growth, premature development of facial and pubic hair, acne, genital enlargement, and deepening of their voice. Feminizing adrenal tumors are less common and occur in men in the third to fifth decades of life. These tumors lead to gynecomastia, impotence, and testicular atrophy. Women with these tumors develop irregular menses or dysfunctional uterine bleeding. Vaginal bleeding may occur in postmenopausal women. Girls with these tumors experience precocious puberty with breast enlargement and early menarche.Diagnostic Tests Virilizing tumors produce excessive amounts of the androgen precursor, DHEA, which can be measured Table 38-19TNM Staging for adrenocortical cancerTUMORNODEMETASTASISSTAGET1N0M0IT2N0M0IIT1N1M0IIIT2N1M0IIIT3Any NM0IIIT4Any NM0IIIAny TAny NM1IVPrimary tumor

1	precursor, DHEA, which can be measured Table 38-19TNM Staging for adrenocortical cancerTUMORNODEMETASTASISSTAGET1N0M0IT2N0M0IIT1N1M0IIIT2N1M0IIIT3Any NM0IIIT4Any NM0IIIAny TAny NM1IVPrimary tumor (T): T1, size ≤5 cm without local invasion; T2, size >5 cm without local invasion; T3, any size with local invasion but no involvement of adjacent organs; T4, any size with involvement of adjacent organs.Nodes (N): N0, no involvement of regional nodes; N1, positive regional lymph nodes.Metastasis (M): M0, no known distal metastases: M1, distant metastases present.Used with the permission of the American College of Surgeons. Amin MB, Edge SB, Greene FL, et al. (Eds.) AJCC Cancer Staging Manual, 8th Ed. Springer New York, 2017.Brunicardi_Ch38_p1625-p1704.indd 169201/03/19 11:22 AM 1693THYROID, PARATHYROID, AND ADRENALCHAPTER 38in plasma or urine as 17-ketosteroids. Patients with feminiz-ing tumors also have elevated urinary 17-ketosteroids in addi-tion to increased estrogen levels.

1	PARATHYROID, AND ADRENALCHAPTER 38in plasma or urine as 17-ketosteroids. Patients with feminiz-ing tumors also have elevated urinary 17-ketosteroids in addi-tion to increased estrogen levels. Androgen-producing tumors often are associated with production of other hormones such as glucocorticoids.Treatment Virilizing and feminizing tumors are treated by adrenalectomy. Malignancy is difficult to diagnose histologi-cally but is suggested by the presence of local invasion, recur-rence, or distal metastases. Adrenolytic drugs such as mitotane, aminoglutethimide, and ketoconazole may be useful in control-ling symptoms in patients with metastatic disease.Congenital Adrenal Hyperplasia. CAH refers to a group of disorders that result from deficiencies or complete absence of enzymes involved in adrenal steroidogenesis. 21-Hydroxylase (CYP21A2) deficiency is the most common enzymatic defect, accounting for >90% of cases of CAH. This deficiency pre-vents the production of 11-deoxycortisol and

1	adrenal steroidogenesis. 21-Hydroxylase (CYP21A2) deficiency is the most common enzymatic defect, accounting for >90% of cases of CAH. This deficiency pre-vents the production of 11-deoxycortisol and 11-DOC from progesterone precursors. Deficiency of glucocorticoids and aldosterone leads to elevated ACTH levels and overproduction of adrenal androgens and corticosteroid precursors such as 17hydroxyprogesterone and Δ4-androstenedione. These com-pounds are converted to testosterone in the peripheral tis-sues, thereby leading to virilization. Complete deficiency of 21-hydroxylase presents at birth with virilization, diarrhea, hypovolemia, hyponatremia, hyperkalemia, and hyperpig-mentation. Partial enzyme deficiency may present at birth or later with virilizing features. These patients are less prone to the salt wasting that characterizes complete enzyme deficiency. 11β-Hydroxylase deficiency is the second most common form of CAH and leads to hypertension (from 11-DOC accumula-tion),

1	less prone to the salt wasting that characterizes complete enzyme deficiency. 11β-Hydroxylase deficiency is the second most common form of CAH and leads to hypertension (from 11-DOC accumula-tion), virilization, and hyperpigmentation. Other enzyme defi-ciencies include 3β-hydroxydehydrogenase and 17-hydroxylase deficiency. Congenital adrenal lipoid hyperplasia is the most severe form of CAH, which is caused by cholesterol desmolase deficiency. It leads to the disruption of all steroid biosynthetic pathways, thus resulting in a fatal salt-wasting syndrome in phe-notypic female patients.Diagnostic Tests The particular enzyme deficiency can be diagnosed by karyotype analysis and measurement of plasma and urinary steroids. The most common enzyme defi-ciency, absence of 21-hydroxylase, leads to increased plasma 17-hydroxyprogesterone and progesterone levels because these compounds cannot be converted to 11-deoxycortisol and 11-DOC, respectively. 11β-Hydroxylase deficiency is the next most

1	increased plasma 17-hydroxyprogesterone and progesterone levels because these compounds cannot be converted to 11-deoxycortisol and 11-DOC, respectively. 11β-Hydroxylase deficiency is the next most common disorder and results in elevated plasma 11-DOC and 11-deoxycortisol. Urinary 17-hydroxyprogesterone, andro-gens, and 17-ketosteroids also are elevated. The dexamethasone suppression test (2–4 mg divided four times a day for 7 days) can be used to distinguish adrenal hyperplasia from neoplasia. CT, MRI, and iodocholesterol scans generally are used to local-ize the tumors.Treatment Patients with CAH traditionally have been man-aged medically, with cortisol and mineralocorticoid replacement to suppress the hypothalamic-pituitary-adrenal axis. However, the doses of steroids required often are supraphysiologic and lead to iatrogenic hypercortisolism. More recently, bilateral laparoscopic adrenalectomy has been proposed as an alternative treatment for this disease and has been successfully

1	supraphysiologic and lead to iatrogenic hypercortisolism. More recently, bilateral laparoscopic adrenalectomy has been proposed as an alternative treatment for this disease and has been successfully performed in a limited number of patients for various forms of CAH.Disorders of the Adrenal MedullaPheochromocytomas. Pheochromocytomas are rare tumors with prevalence rates ranging from 0.3% to 0.95% in autopsy series and approximately 1.9% in series using biochemical screening. They can occur at any age, with a peak incidence in the fourth and fifth decades of life, and have no gender predilec-tion. Extra-adrenal tumors, also called functional paraganglio-mas, may be found at sites of sympathetic ganglia in the organ of Zuckerkandl, neck, mediastinum, abdomen, and pelvis. Pheo-chromocytomas often are called the 10 percent tumor because 10% are bilateral, 10% are malignant, 10% occur in pediatric patients, 10% are extra-adrenal, and 10% are familial.Pheochromocytomas occur in families

1	often are called the 10 percent tumor because 10% are bilateral, 10% are malignant, 10% occur in pediatric patients, 10% are extra-adrenal, and 10% are familial.Pheochromocytomas occur in families with MEN2A and MEN2B in approximately 50% of patients. Both syndromes are inherited in an autosomal dominant fashion and are caused by germline mutations in the RET proto-oncogene. Another syndrome with an increased risk of pheochromocytomas is von Hippel-Lindau (VHL) disease, which also is inherited in an autosomal dominant manner. This syndrome also includes retinal angioma, hemangioblastomas of the central nervous system, renal cysts and carcinomas, pancreatic cysts, and epi-didymal cystadenomas. The incidence of pheochromocytomas in the syndrome is approximately 14%. The gene causing VHL has been mapped to chromosome 3p and is a tumor suppres-sor gene. Pheochromocytomas also are included within the tumor spectrum of neurofibromatosis type 1 (NF1 gene) and other neuroectodermal disorders

1	been mapped to chromosome 3p and is a tumor suppres-sor gene. Pheochromocytomas also are included within the tumor spectrum of neurofibromatosis type 1 (NF1 gene) and other neuroectodermal disorders (Sturge-Weber syndrome and tuberous sclerosis), Carney’s syndrome (gastric epithelioid leio-myosarcoma, pulmonary chondroma, and extra-adrenal para-ganglioma), MEN1 syndrome, and the familial paraganglioma and pheochromocytoma syndrome are caused by mutations in the succinyl dehydrogenase family of genes (SDHB, SDHC, and SDHD), which comprise portions of the mitochondrial complex II.112 More recently, mutations in SDHA and SDH5 have also been identified. Additional susceptibility loci include TMEM127 (involved in the mTORC1-signaling pathway) and MAX (myc-associated factor X).Symptoms and Signs Headache, palpitations, and diaphoresis constitute the “classic triad” of pheochromocytomas. Symp-toms such as anxiety, tremulousness, paresthesias, flushing, chest pain, shortness of breath,

1	Signs Headache, palpitations, and diaphoresis constitute the “classic triad” of pheochromocytomas. Symp-toms such as anxiety, tremulousness, paresthesias, flushing, chest pain, shortness of breath, abdominal pain, nausea, vomit-ing, and others are nonspecific and may be episodic in nature. Cardiovascular complications such as myocardial infarction and cerebrovascular accidents may ensue. These symptoms can be incited by a range of stimuli including exercise, micturition, and defecation. The most common clinical sign is hyperten-sion. Pheochromocytomas are one of the few curable causes of hypertension and are found in 0.1% to 0.2% of hypertensive patients. Hypertension related to this tumor may be paroxys-mal with intervening normotension, sustained with paroxysms or sustained hypertension alone. Sudden death may occur in patients with undiagnosed tumors who undergo other surgeries or biopsy.Diagnostic Tests Biochemical Studies. Pheochromocytomas are diagnosed by testing 24-hour urine

1	alone. Sudden death may occur in patients with undiagnosed tumors who undergo other surgeries or biopsy.Diagnostic Tests Biochemical Studies. Pheochromocytomas are diagnosed by testing 24-hour urine samples for catecholamines and their metab-olites as well as by determining plasma metanephrine levels. Urinary metanephrines are 98% sensitive and also about 98% specific for pheochromocytomas, whereas VMA measure-ments are slightly less sensitive and specific. False-positive VMA tests may result from ingestion of caffeine, raw fruits, Brunicardi_Ch38_p1625-p1704.indd 169301/03/19 11:22 AM 1694SPECIFIC CONSIDERATIONSPART IIBAFigure 38-46. A left-sided pheochromocytoma (arrows) imaged by a computed tomography scan of the abdomen (A) and a metaiodoben-zylguanidine scan viewed posteriorly (B).or medications (α-methyldopa). Fractionated urinary catechol-amines (norepinephrine, epinephrine, and dopamine) also are very sensitive but less specific for pheochromocytomas. Because extraadrenal

1	medications (α-methyldopa). Fractionated urinary catechol-amines (norepinephrine, epinephrine, and dopamine) also are very sensitive but less specific for pheochromocytomas. Because extraadrenal sites lack phenylethanolamine N-methyltransferase, these tumors secrete norepinephrine, whereas epinephrine is the main hormone secreted from adrenal pheochromocytomas. Many physiologic and pathologic states can alter the levels of plasma catecholamines. Hence, they often are thought to be less accurate than urinary tests. Both epinephrine and norepi-nephrine should be measured, as tumors often secrete one or the other hormone. Sensitivities of 85% and specificities of 95% have been reported using cutoff values of 2000 pg/mL for norepinephrine and 200 pg/mL for epinephrine. Clonidine is an agent that suppresses neurogenically mediated catecholamine excess but not secretion from pheochromocytomas. A normal clonidine suppression test is defined by a decrease of basal cate-cholamine levels to

1	that suppresses neurogenically mediated catecholamine excess but not secretion from pheochromocytomas. A normal clonidine suppression test is defined by a decrease of basal cate-cholamine levels to <500 pg/mL within 2 to 3 hours after an oral dose of 0.3 mg of clonidine. Chromogranin A is a monomeric, acidic protein, which is stored in the adrenal medulla and other neuroendocrine tumors and released along with catecholamine hormones. It has been reported to have a sensitivity of 83% and a specificity of 96% and is useful in conjunction with catechol-amine measurement for diagnosing pheochromocytomas. Some studies have shown that plasma metanephrines should be the first-line test to identify pheochromocytomas, as the predictive value of a negative test is very high and normal levels exclude pheochromocytoma in patients with preclinical disease or dopa-mine secreting tumors. Although sensitivities of 96% to 100% have been reported, specificity is lower at 85% to 89% and may be much

1	pheochromocytoma in patients with preclinical disease or dopa-mine secreting tumors. Although sensitivities of 96% to 100% have been reported, specificity is lower at 85% to 89% and may be much lower at 77% in elderly patients. Although attractive because of the simplicity of a blood test, measurement of plasma metanephrines is generally reserved for cases for which there is a high index of suspicion.Radiologic Studies. Radiologic studies are useful to localize tumors and to assess the extent of spread once the diagnosis has been made with biochemical tests. CT scans are 85% to 95% sensitive and 70% to 100% specific for pheochromocytomas (Fig. 38-46A). The scans should be performed without con-trast to minimize the risk of precipitating a hypertensive crisis, although some recent studies suggest that intravenous contrast may be used. Images should include the region from the dia-phragm to the aortic bifurcation so as to include the organ of Zuckerkandl. CT scans do not provide

1	suggest that intravenous contrast may be used. Images should include the region from the dia-phragm to the aortic bifurcation so as to include the organ of Zuckerkandl. CT scans do not provide functional information and cannot definitively diagnose pheochromocytomas. MRI scans are 95% sensitive and almost 100% specific for pheo-chromocytomas because these tumors have a characteristic appearance on T2-weighted images or after gadolinium. MRI is also the study of choice in pregnant women as there is no risk of radiation exposure. Metaiodobenzylguanidine (MIBG) is taken up and concentrated by vesicles in the adrenal medullar cells because its structure is similar to norepinephrine. Normal adrenal medullary tissue does not take up appreciable MIBG. 131I-radiolabeled MIBG is, therefore, useful for localizing pheo-chromocytomas (Fig. 38-46B), especially those in ectopic posi-tions. This test has a reported sensitivity of 77% to 89% and a specificity ranging from 88% to 100%.Treatment The

1	for localizing pheo-chromocytomas (Fig. 38-46B), especially those in ectopic posi-tions. This test has a reported sensitivity of 77% to 89% and a specificity ranging from 88% to 100%.Treatment The medical management of pheochromocytomas is aimed chiefly at blood pressure control and volume repletion. Irreversible, long-acting α-blockers such as phenoxybenzamine are started 1 to 3 weeks before surgery at doses of 10 mg twice daily, which may be increased to 300 to 400 mg/d with rehydra-tion. Patients should be warned about orthostatic hypotension. β-Blockers such as propranolol at doses of 10 to 40 mg every 6 to 8 hours often need to be added preoperatively in patients who have persistent tachycardia and arrhythmias. β-Blockers should only be instituted after adequate α-blockade and hydration to avoid the effects of unopposed α stimulation (i.e., hypertensive crisis and congestive heart failure) and are typically initiated 3 to 4 days preoperatively. Patients also should be volume

1	to avoid the effects of unopposed α stimulation (i.e., hypertensive crisis and congestive heart failure) and are typically initiated 3 to 4 days preoperatively. Patients also should be volume repleted preoperatively to avoid postoperative hypotension, which ensues with the loss of vasoconstriction after tumor removal. Other α-blockers such as prazosin, terazosin, and doxazosin, which are selective α1-adrenergic blockers, have a better side effect profile and are preferable to phenoxybenzamine when long-term Brunicardi_Ch38_p1625-p1704.indd 169401/03/19 11:22 AM 1695THYROID, PARATHYROID, AND ADRENALCHAPTER 38pharmacologic therapy is needed, as in patients with metastatic pheochromocytoma. Nicardipine is the most commonly used calcium channel blocker and inhibits norepinephrine-mediated calcium transport into vascular smooth muscle. When used as the primary mode of treatment, it appears to be just as effec-tive as αand β-blockade preoperatively and for intraoperative

1	calcium transport into vascular smooth muscle. When used as the primary mode of treatment, it appears to be just as effec-tive as αand β-blockade preoperatively and for intraoperative hemodynamics.113 In some patients, catecholamine-synthesis inhibitors such as α-methyl-p-l-tyrosine (metyrosine) may need to be added if standard αand β-blockade is poorly tolerated or is ineffective in reaching target blood pressure and when moder-ate intraoperative tumor manipulation is anticipated.Adrenalectomy is the treatment of choice for patients with pheochromocytoma. The chief goal of surgery is to resect the tumor completely with minimal tumor manipulation or rupture of the tumor capsule. Surgery should be performed with both noninvasive and invasive monitors, including an arterial line and central venous lines. In patients with congestive heart failure or underlying coronary artery disease, Swan-Ganz catheters may be necessary. Stress must be avoided during anesthesia induc-tion, and use of

1	venous lines. In patients with congestive heart failure or underlying coronary artery disease, Swan-Ganz catheters may be necessary. Stress must be avoided during anesthesia induc-tion, and use of inhaled agents like isoflurane and enflurane are preferred because they have minimal cardiac depressant effects. Fentanyl, ketamine, and morphine should be avoided as they can potentially stimulate catecholamine release from the tumor. The common medications used for intraoperative blood pres-sure control include nitroprusside, nitroglycerin, phentolamine, and nicardipine. Intraoperative arrhythmias are best managed by short-acting β-blockers such as esmolol. Adrenalectomy usu-ally was performed via an open anterior approach to facilitate detection of bilateral tumors, extra-adrenal lesions, or metastatic lesions. However, most pheochromocytomas <5 cm in diameter can be safely resected laparoscopically. Postoperatively, these patients are prone to hypotension due to loss of adrenergic

1	or metastatic lesions. However, most pheochromocytomas <5 cm in diameter can be safely resected laparoscopically. Postoperatively, these patients are prone to hypotension due to loss of adrenergic stim-ulation and consequent vasodilatation and therefore need large volume resuscitation.Hereditary Pheochromocytomas. Inherited pheochromo-cytomas tend to be multiple and bilateral. Generally, unilat-eral adrenalectomy is recommended in the absence of obvious lesions in the contralateral adrenal gland because of Addison’s disease, requiring lifelong steroid replacement in patients under-going bilateral adrenalectomy. For patients with tumors in both adrenal glands, cortical-sparing subtotal adrenalectomy may preserve adrenocortical function and avoid the morbidity of bilateral total adrenalectomy. Laparoscopic subtotal adrenal-ectomy has been shown to provide short-term clinical results comparable to total adrenalectomy, with reduced surgical morbidity and may be done as a unilateral or

1	Laparoscopic subtotal adrenal-ectomy has been shown to provide short-term clinical results comparable to total adrenalectomy, with reduced surgical morbidity and may be done as a unilateral or bilateral subtotal procedure.114 However, these patients remain at risk for recur-rent pheochromocytoma, which has been reported in 20% of patients with VHL disease a median of 40 months after partial adrenalectomy, and in 33% of MEN2 patients followed for 54 to 88 months after surgery. Autotransplantation of adrenocortical tissue after total adrenalectomy may be another option for these patients. However, the transplanted cortical tissue rarely pro-vides full function, and steroid replacement usually is required.Malignant Pheochromocytomas. Approximately 12% to 29% of pheochromocytomas are malignant, and these tumors are associated with decreased survival. There are no definitive histologic criteria defining malignant pheochromocytomas. In fact, pleomorphism, nuclear atypia, and abundant

1	and these tumors are associated with decreased survival. There are no definitive histologic criteria defining malignant pheochromocytomas. In fact, pleomorphism, nuclear atypia, and abundant mitotic fig-ures are seen in benign tumors. Capsular and vascular invasion may be seen in benign lesions as well. Malignancy usually is diagnosed when there is evidence of invasion into surrounding structures or distant metastases. The most common sites for met-astatic disease are bone, liver, regional lymph nodes, lung, and peritoneum, although the brain, pleura, skin, and muscles may also occasionally be involved. Some studies also suggest that older patient age and larger tumors are associated with a higher risk of malignancy. Although risk of malignancy increases with size for all pheochromocytomas, size does not seem to reliably predict malignancy in pheochromocytomas with local disease only.115 Given this difficulty of defining malignancy clinically (in the absence of metastatic disease), a

1	size does not seem to reliably predict malignancy in pheochromocytomas with local disease only.115 Given this difficulty of defining malignancy clinically (in the absence of metastatic disease), a number of other features such as DNA ploidy, tumor size, and necrosis, neuropeptide Y mRNA expression, and serum neuron-specific enolase expres-sion have been studied. Malignant pheochromocytomas are more likely to express p53 and bcl-2 and have activated telom-erase. Recent data suggest that flow cytometry and molecular markers such as expression of Ki-67, tissue inhibitor of metal-loproteinase, and COX-2 also have shown some use in deter-mining malignancy. When pheochromocytomas develop in the MEN syndromes, they rarely are malignant. In contrast, patients with germline SDHB mutations appear to have a higher propen-sity for extra-adrenal and malignant tumors. In general, soft tis-sue lesions are treated with resection if feasible. External-beam radiation can be used for unresectable

1	to have a higher propen-sity for extra-adrenal and malignant tumors. In general, soft tis-sue lesions are treated with resection if feasible. External-beam radiation can be used for unresectable lesions or symptomatic skeletal metastases. Therapeutic 131I-MIBG irradiation may be useful in patients with diffuse disease showing 123-I-MIBG uptake on a diagnostic scan. Chemotherapy regimens typically use cyclophosphamide, vincristine, and dacarbazine with vari-able response rates. However, molecular targeted therapies such as sunitinib have shown some promising results.The Adrenal IncidentalomaAdrenal lesions discovered during imaging performed for unre-lated reasons are referred to as incidentalomas. This definition excludes tumors discovered on imaging studies performed for evaluating symptoms of hormone hypersecretion or staging patients with known cancer. The incidence of these lesions iden-tified by CT scans ranges from 0.4% to 4.4%.Differential Diagnosis. The differential diagnosis

1	of hormone hypersecretion or staging patients with known cancer. The incidence of these lesions iden-tified by CT scans ranges from 0.4% to 4.4%.Differential Diagnosis. The differential diagnosis of adre-nal incidentalomas is shown in Table 38-20. Nonfunctional cortical adenomas account for the majority (36–94%) of adre-nal incidentalomas in patients without a history of cancer. In a series of patients from the Mayo Clinic, no nonfunctional Table 38-20Differential diagnosis of adrenal incidentalomaFUNCTIONING LESIONSNONFUNCTIONING LESIONSBenignBenign Aldosteronoma Cortisol-producing adenoma Sex steroid–producing adenoma Pheochromocytoma Cortical adenoma Myelolipoma Cyst Ganglioneuroma HemorrhageMalignant Adrenocortical cancer Metastasis Malignant pheochromocytoma Adrenocortical cancerBrunicardi_Ch38_p1625-p1704.indd 169501/03/19 11:22 AM 1696SPECIFIC CONSIDERATIONSPART IIlesion progressed to cause clinical or biochemical abnormali-ties. However, other studies indicate that 5%

1	169501/03/19 11:22 AM 1696SPECIFIC CONSIDERATIONSPART IIlesion progressed to cause clinical or biochemical abnormali-ties. However, other studies indicate that 5% to 20% of patients with apparently nonfunctioning cortical adenomas have under-lying, subtle abnormalities of glucocorticoid secretion, and a rare benign-appearing incidentaloma is a cancer.By definition, patients with incidentalomas do not have clinically overt Cushing’s syndrome, but subclinical Cushing’s syndrome is estimated to occur in approximately 8% of patients. This disorder is characterized by subtle fea-tures of cortisol excess, such as weight gain, skin atrophy, facial fullness, diabetes, and hypertension, accompanied by loss of normal diurnal variation in cortisol secretion, autonomous cor-tisol secretion, and resistance to suppression by dexamethasone. Total cortisol produced and 24-hour urinary cortisol levels may be normal. Examination of the natural history of subclinical Cushing’s syndrome indicates

1	resistance to suppression by dexamethasone. Total cortisol produced and 24-hour urinary cortisol levels may be normal. Examination of the natural history of subclinical Cushing’s syndrome indicates that, although most patients remain asymptomatic, some do progress to clinically evident Cushing’s syndrome. Furthermore, cases of postoperative adre-nal crisis from unrecognized suppression of the contralateral adrenal have been reported, making preoperative identification of this condition imperative, particularly in the era of early dis-charge following laparoscopic adrenalectomy.The adrenal is a common site of metastases of lung and breast tumors, melanoma, renal cell cancer, and lymphoma. In patients with a history of nonadrenal cancer and a unilat-eral adrenal mass, the incidence of metastatic disease has been reported to range from 32% to 73%. Myelolipomas are benign, biochemically nonfunctioning lesions composed of ele-ments of hematopoietic and mature adipose tissue, which are rare

1	disease has been reported to range from 32% to 73%. Myelolipomas are benign, biochemically nonfunctioning lesions composed of ele-ments of hematopoietic and mature adipose tissue, which are rare causes of adrenal incidentaloma. Other less commonly encountered lesions include adrenal cysts, ganglioneuromas, and hemorrhage.Diagnostic Investigations. The diagnostic workup of an adrenal incidentaloma is aimed at identifying patients who would benefit from adrenalectomy (i.e., patients with function-ing tumors and tumors at increased risk of being malignant). It is not necessary for asymptomatic patients whose imaging studies are consistent with obvious cysts, hemorrhage, myelolipomas, or diffuse metastatic disease to undergo additional investigations. All other patients should be tested for underlying hormonally active tumors using (a) a low-dose (1 mg) overnight dexametha-sone suppression test to rule out subclinical Cushing’s syndrome and 17-ketosteroids (if sex steroid excess is

1	for underlying hormonally active tumors using (a) a low-dose (1 mg) overnight dexametha-sone suppression test to rule out subclinical Cushing’s syndrome and 17-ketosteroids (if sex steroid excess is suspected); (b) a 24-hour urine collection for catecholamines, metanephrines, VMA, or plasma metanephrine to rule out pheochromocytoma; and (c) in hypertensive patients, serum electrolytes, plasma aldosterone, and plasma renin to rule out an aldosteronoma. In patients with a high index of suspicion for subclinical Cushing’s (those with hypertension, obesity, or diabetes), three tests (i.e., dexamethasone suppression test, salivary cortisol, and 24-hour urine free cortisol) may be used. Confirmatory tests can be per-formed based on the results of the initial screening studies.Determination of the malignant potential of an inciden-taloma is more difficult. The risk of malignancy in an adrenal lesion is related to its size. Lesions >6 cm in diameter have an approximate risk of malignancy of

1	the malignant potential of an inciden-taloma is more difficult. The risk of malignancy in an adrenal lesion is related to its size. Lesions >6 cm in diameter have an approximate risk of malignancy of about 25%.82 However, this size cutoff is not absolute because adrenal carcinomas also have been reported in lesions <6 cm. Carcinomas account for 2% of lesions <4 cm and 6% of lesions 4.1 to 6 cm in size. This has led to increased use of the imaging characteristics of incidentalomas to predict malignancy. Benign adrenal adenomas tend to be homogeneous, well encapsulated, and have smooth and regular margins. They also tend to be hypoattenuating lesions (<10 Hounsfield units) on CT scanning. In contrast, adrenal cancers tend to be hyperattenuating (>18 Hounsfield units) and inhomo-geneous, have irregular borders, and may show evidence of local invasion or adjacent lymphadenopathy. On MRI T2-weighted imaging, adenomas demonstrate low signal inten-sity when compared to the liver (adrenal

1	have irregular borders, and may show evidence of local invasion or adjacent lymphadenopathy. On MRI T2-weighted imaging, adenomas demonstrate low signal inten-sity when compared to the liver (adrenal mass-to-liver ratio <1.4), whereas carcinomas and metastases have moderate inten-sity (mass-to-liver ratio 1.2:2.8). Pheochromocytomas are extremely bright, with mass-to-liver ratios >3. Unfortunately, the ranges overlap, and signal intensity is not 100% reliable for determining the nature of the lesion. Radionuclide imaging with NP-59 also has been used to distinguish between various adre-nal lesions, with some investigators suggesting that uptake of NP-59 was 100% predictive of a benign lesion (adenoma), whereas absence of imaging was 100% predictive of a nonade-nomatous lesion. However, the technique has not gained wide-spread acceptance because patients need to be given cold iodine 1 week before the study to prevent thyroid uptake, imaging needs to be delayed by 5 to 7 days after

1	the technique has not gained wide-spread acceptance because patients need to be given cold iodine 1 week before the study to prevent thyroid uptake, imaging needs to be delayed by 5 to 7 days after administration of the contrast, and false-positive and false-negative results occur. FDG-PET or PET-CT scans may have some utility in distin-guishing potentially malignant from benign lesions in cases of inconclusive CT densitometry. However, caution must be exer-cised for false-positive (some adenomas and pheochromocyto-mas) and false-negative results (small lesions or those with hemorrhage or necrosis).116 FNAB cannot be used to distinguish adrenal adenomas from carcinomas. This being said, FNAB is useful in the setting of a patient with a history of cancer and a solitary adrenal mass. The positive predictive value of FNAB in this situation has been shown to be almost 100%, although false-negative rates of up to 33% have been reported. Biopsies usually are performed under CT guidance, and

1	predictive value of FNAB in this situation has been shown to be almost 100%, although false-negative rates of up to 33% have been reported. Biopsies usually are performed under CT guidance, and appropriate testing to rule out pheochromocytomas should be undertaken before the procedure to avoid precipitating a hypertensive crisis.Management. An algorithm for the management of patients with incidentalomas is shown in Fig. 38-47. The AACE and American Association of Endocrine Surgeons (AAES) have published management guidelines for patients with adrenal incidentalomas.117 Patients with functional tumors or obviously malignant lesions should undergo adrenalectomy. The optimal management of patients with subclinical Cushing’s syndrome is controversial, especially due to the paucity of data from high-quality prospective trials. In general, operative intervention is advised in patients with subclinical Cushing’s syndrome with suppressed plasma ACTH levels and elevated urinary cortisol levels

1	high-quality prospective trials. In general, operative intervention is advised in patients with subclinical Cushing’s syndrome with suppressed plasma ACTH levels and elevated urinary cortisol levels because these patients are at high risk for progression to overt Cushing’s syndrome. The adrenal incidentaloma guide-lines also recommend adrenalectomy in patients with worsening hypertension, abnormal glucose tolerance, or osteoporosis.For nonfunctional lesions, the risk of malignancy needs to be balanced with operative morbidity and mortality. The AACE/AAES guidelines recommend that lesions with suspicious fea-tures on imaging studies such as heterogeneity, irregular cap-sule, or adjacent nodes should be treated by adrenalectomy. Nonoperative therapy, with close periodic follow-up, is advised for lesions <4 cm in diameter with benign imaging characteris-tics, whereas adrenalectomy is recommended for lesions ≥4 cm in size due to the increased risk of

1	close periodic follow-up, is advised for lesions <4 cm in diameter with benign imaging characteris-tics, whereas adrenalectomy is recommended for lesions ≥4 cm in size due to the increased risk of cancer.1011Brunicardi_Ch38_p1625-p1704.indd 169601/03/19 11:22 AM 1697THYROID, PARATHYROID, AND ADRENALCHAPTER 38Questions1) Low dose DST2) Plasma metanephrines or 24 h urine catecholamines, VMA, metanephrines3) Plasma aldosterone, renin activity, electrolytesAdrenalectomyYesNoPast historyof cancer?YesSolitarymetastasisyesNoConsideradrenalectomySystemic therapySee question 3<4 cm,Benign imaging featuresNoRepeat imaging in 3–6 months, biochemicalevaluation annually˜4 cm, Indeterminate orsuspicious imagingfeaturesAdrenalectomy1Is the tumorfunctioning?2Is the tumormetastatic?3Is it at highrisk of beingmalignant?Figure 38-47. Management algorithm for an adrenal incidentaloma. CT = computed tomography; DST = dexamethasone suppression test; VMA = vanillylmandelic acid.However, several

1	of beingmalignant?Figure 38-47. Management algorithm for an adrenal incidentaloma. CT = computed tomography; DST = dexamethasone suppression test; VMA = vanillylmandelic acid.However, several important points must be considered in the management of these patients. First, size criteria for malig-nancy are not definitive and are derived from a selected series of patients. Second, the actual size of adrenal tumors can be underestimated by at least 1 cm by modalities such as CT and MRI scans because tumors are larger in a cephalocaudal axis. Third, the natural history of incidentalomas is variable and depends on the underlying diagnosis, age of the study popula-tion, and the size of the mass. Older patients are more likely to have nonfunctioning adenomas. Existing data in terms of the long-term behavior of these nonfunctional lesions, although limited, indicate that malignant transformation is uncommon. Furthermore, tumors that increase in size by at least 1 cm over a 2-year follow-up

1	behavior of these nonfunctional lesions, although limited, indicate that malignant transformation is uncommon. Furthermore, tumors that increase in size by at least 1 cm over a 2-year follow-up period and those with subtle hormonal abnor-malities appear to be more likely to enlarge. Overt hormone overproduction is more likely in tumors >3 cm and those with increased NP-59 uptake. Surgeons are more likely to operate on a 40-year-old patient with a 4-cm lesion, while electing to follow an 80-year-old patient with a similar lesion but multiple concurrent comorbidities. Based on the above considerations, some surgeons use a size threshold for adrenalectomy with a nonfunctioning homogeneous tumor of 3 to 4 cm in young patients with no comorbidities and 5 cm in older patients with significant comorbidity.Lesions that grow during follow-up also are treated by adrenalectomy. Myelolipomas generally do not warrant adrenal-ectomy unless there is concern regarding malignancy, which is rare, or

1	comorbidity.Lesions that grow during follow-up also are treated by adrenalectomy. Myelolipomas generally do not warrant adrenal-ectomy unless there is concern regarding malignancy, which is rare, or bleeding into the lesion, which is more likely in myelo-lipomas >4 cm in size. These tumors, even when large, can be removed laparoscopically. Resection of solitary adrenal metas-tases in patients with a history of nonadrenal cancer has been demonstrated to lead to prolonged patient survival. Suspected adrenal metastases also may be resected for diagnosis.There is no consensus regarding the follow-up of patients with adrenal incidentaloma. The AACE/AAES guidelines recommend repeating hormonal screening with a 1-mg dexa-methasone suppression test and urinary catecholamines and metabolites yearly for 5 years as the risk of hypersecretion appears to plateau after this time period. It also recommends repeat imaging at 3 to 6 months and then annually for 1 to 2 years. Less frequent imaging is

1	for 5 years as the risk of hypersecretion appears to plateau after this time period. It also recommends repeat imaging at 3 to 6 months and then annually for 1 to 2 years. Less frequent imaging is reasonable or small (<2 cm), uniform, hypodense cortical nodules in patients without a his-tory of malignant disease. Adrenalectomy is recommended for lesions that grow ≥1 cm or if autonomous hormone secretion develops during follow-up.Adrenal InsufficiencyAdrenal insufficiency may be primary, resulting from adrenal dis-ease, or secondary, due to a deficiency of ACTH (Table 38-21). The most commonly encountered causes of primary adrenal insufficiency are autoimmune disease, infections, and meta-static deposits. Spontaneous adrenal hemorrhage can occur in patients with fulminant meningococcal septicemia (Water-house-Friderichsen syndrome). Bilateral adrenal hemorrhage also can occur secondary to trauma, severe stress, infection, and coagulopathies and, if unrecognized, is lethal. Exogenous

1	(Water-house-Friderichsen syndrome). Bilateral adrenal hemorrhage also can occur secondary to trauma, severe stress, infection, and coagulopathies and, if unrecognized, is lethal. Exogenous glucocorticoid therapy with suppression of the adrenal glands is the most common cause of secondary adrenal insufficiency.Symptoms and Signs. Acute adrenal insufficiency should be suspected in stressed patients with any of the relevant risk factors. It may mimic sepsis, myocardial infarction, or pulmo-nary embolus and presents with fever, weakness, confusion, nausea, vomiting, lethargy, abdominal pain, or severe hypoten-sion. Chronic adrenal insufficiency, such as that occurring in patients with metastatic tumors, may be more subtle. Symptoms include fatigue, salt craving, weight loss, nausea, vomiting, and Brunicardi_Ch38_p1625-p1704.indd 169701/03/19 11:22 AM 1698SPECIFIC CONSIDERATIONSPART IIabdominal pain. These patients may appear hyperpigmented from increased secretion of CRH and ACTH,

1	and Brunicardi_Ch38_p1625-p1704.indd 169701/03/19 11:22 AM 1698SPECIFIC CONSIDERATIONSPART IIabdominal pain. These patients may appear hyperpigmented from increased secretion of CRH and ACTH, with increased α-melanocyte-stimulating hormone side-products.Diagnostic Studies. Characteristic laboratory findings include hyponatremia, hyperkalemia, eosinophilia, mild azote-mia, and fasting or reactive hypoglycemia. The peripheral blood smear may demonstrate eosinophilia in approximately 20% of patients. Adrenal insufficiency is diagnosed by the ACTH stimulation test. ACTH (250 μg) is infused intravenously, and cortisol levels are measured at 0, 30, and 60 minutes. Peak cor-tisol levels <20 μg/dL suggest adrenal insufficiency. ACTH levels also allow primary insufficiency to be distinguished from secondary causes. High ACTH levels with low plasma cortisol levels are diagnostic of primary adrenal insufficiency.Treatment. Treatment must be initiated based on clinical sus-picion alone, even

1	from secondary causes. High ACTH levels with low plasma cortisol levels are diagnostic of primary adrenal insufficiency.Treatment. Treatment must be initiated based on clinical sus-picion alone, even before test results are obtained, or the patient is unlikely to survive. Management includes volume resuscita-tion with at least 2 to 3 L of a 0.9% saline solution or 5% dex-trose in saline solution. Blood should be obtained for electrolyte (decreased Na+ and increased K+), glucose (low), and cortisol (low) levels; ACTH (increased in primary and decreased in sec-ondary); and quantitative eosinophilic count. Dexamethasone (4 mg) should be administered intravenously. Hydrocortisone (100 mg intravenously every 8 hours) also may be used, but it interferes with testing of cortisol levels. Once the patient has been stabilized, underlying conditions such as infection should be sought, identified, and treated. The ACTH stimulation test should be performed to confirm the diagnosis. Glucocorticoids

1	patient has been stabilized, underlying conditions such as infection should be sought, identified, and treated. The ACTH stimulation test should be performed to confirm the diagnosis. Glucocorticoids can then be tapered to maintenance doses (oral hydrocortisone 15–20 mg in the morning and 10 mg in the evening). Mineralo-corticoids (fludrocortisone 0.05–0.1 mg daily) may be required once the saline infusions are discontinued.Adrenal SurgeryChoice of Procedure. Adrenalectomy may be performed via a laparoscopic or open approach. In either approach, the gland may be approached anteriorly, laterally, or posteriorly via the retroperitoneum. The choice of approach depends on the size and nature of the lesion and expertise of the surgeon. Laparo-scopic adrenalectomy has rapidly become the standard proce-dure of choice for the excision of most benign-appearing adrenal lesions <6 cm in diameter. The role of laparoscopic adrenalec-tomy in the management of adrenocortical cancers is

1	the standard proce-dure of choice for the excision of most benign-appearing adrenal lesions <6 cm in diameter. The role of laparoscopic adrenalec-tomy in the management of adrenocortical cancers is controver-sial. The data with respect to local tumor recurrence and intra-abdominal carcinomatosis from laparoscopic adrenalec-tomy for malignant adrenal tumors that were not appreciated as such, preoperatively or intraoperatively, are conflicting. Although laparoscopic adrenalectomy appears to be feasible and safe for solitary adrenal metastasis118 (provided there is no local invasion and the tumor can be resected intact), open adrenalec-tomy or laparoscopic-assisted open adrenalectomy is the safest option for suspected or known adrenocortical cancers and malig-nant pheochromocytomas. Technical considerations and surgeon experience, rather than absolute tumor size, usually determine the size threshold for laparoscopic resection. Hand-assisted lapa-roscopic adrenalectomy may provide a

1	considerations and surgeon experience, rather than absolute tumor size, usually determine the size threshold for laparoscopic resection. Hand-assisted lapa-roscopic adrenalectomy may provide a bridge between laparo-scopic adrenalectomy and conversion to an open procedure. There have been no randomized trials directly comparing open vs. laparoscopic adrenalectomy. However, studies have shown that laparoscopic adrenalectomy is associated with decreased blood loss, postoperative pain, and narcotic use; reduced length of hospital stay; and faster return to work.Laparoscopic Adrenalectomy. The procedure is performed under general anesthesia. Arterial lines are used routinely, and central lines are necessary for patients in whom massive fluid shifts are anticipated (e.g., those with large, active pheochro-mocytomas). A nasogastric tube and Foley catheter are rec-ommended. Routine preoperative antibiotics are not needed, except in patients with Cushing’s syndrome. The adrenals can be removed

1	pheochro-mocytomas). A nasogastric tube and Foley catheter are rec-ommended. Routine preoperative antibiotics are not needed, except in patients with Cushing’s syndrome. The adrenals can be removed laparoscopically via a transabdominal (anterior or lateral) or retroperitoneal (lateral or posterior) approach. The lateral approach is preferred by most laparoscopic surgeons and uses gravity to aid retraction of surrounding organs. Patients, however, need to be repositioned for a bilateral procedure. The anterior transabdominal approach offers the advantage of a con-ventional view of the abdominal cavity and allows a bilateral adrenalectomy to be performed without the necessity of repo-sitioning the patient. The posterior retroperitoneal approach has also been gaining popularity in recent years, particularly in patients with previous anterior abdominal surgery and peri-toneal adhesions. In addition, several centers have successfully utilized robotic approaches for both lateral

1	recent years, particularly in patients with previous anterior abdominal surgery and peri-toneal adhesions. In addition, several centers have successfully utilized robotic approaches for both lateral transabdominal and retroperitoneal laparoscopic adrenal surgery. Single incision laparoscopic adrenalectomy is another option. While these latter approaches are feasible,119 their widespread use awaits analysis of long-term outcomes data and cost analyses. The lateral trans-abdominal approach is widely used and described in detail in the following section.12Table 38-21Etiology of adrenal insufficiencyPRIMARYSECONDARYAutoimmune (autoimmune polyglandular disease types I and II)Exogenous glucocorticoid therapyInfectious—TB, fungi, CMV, HIVBilateral adrenalectomyHemorrhage—spontaneous (Waterhouse-Friderichsen syndrome) and secondary to stress, trauma, infections, coagulopathy, or anticoagulantsPituitary or hypothalamic tumorsMetastasesPituitary hemorrhage (postpartum Sheehan’s

1	(Waterhouse-Friderichsen syndrome) and secondary to stress, trauma, infections, coagulopathy, or anticoagulantsPituitary or hypothalamic tumorsMetastasesPituitary hemorrhage (postpartum Sheehan’s syndrome)Infiltrative disorders—amyloidosis, hemochromatosisTrans-sphenoidal resection of pituitary tumorAdrenoleukodystrophy Congenital adrenal hyperplasia Drugs—ketoconazole, metyrapone, aminoglutethimide, mitotane CMV = cytomegalovirus; HIV = human immunodeficiency virus; TB = tuberculosis.Brunicardi_Ch38_p1625-p1704.indd 169801/03/19 11:22 AM 1699THYROID, PARATHYROID, AND ADRENALCHAPTER 38Umbilicus1234Figure 38-48. Positioning of the patient and placement of trocars for a laparoscopic adrenalectomy. Four trocars are placed from the mid-clavicular to the anterior axillary line.Triangular ligamentInferiorvena cavaPancreasSpleenABLiverRight adrenalLeft adrenalFigure 38-49. Technique of laparoscopic adrenalectomy. Expo-sure of the right adrenal is facilitated by division of the

1	ligamentInferiorvena cavaPancreasSpleenABLiverRight adrenalLeft adrenalFigure 38-49. Technique of laparoscopic adrenalectomy. Expo-sure of the right adrenal is facilitated by division of the triangular ligament (A) and dissection and reflection of the spleen and tail of the pancreas aids in identifying the left adrenal (B).Lateral Transabdominal Approach The patient is placed in the lateral decubitus position, and the operating table is flexed at the waist to open the space between the lower rib cage and the iliac crest (Fig. 38-48). The surgeon and assistant both stand on the same side, facing the front of the patient. Pneumoperi-toneum is created using a Veress needle or insufflation via a Hasson port. In general, four 10-mm trocars are placed between the midclavicular line medially and anterior axillary line later-ally, one to two fingerbreadths below the costal margin (see Fig. 38-48), although additional ports may be placed, if needed. A 30° laparoscope is inserted through the

1	anterior axillary line later-ally, one to two fingerbreadths below the costal margin (see Fig. 38-48), although additional ports may be placed, if needed. A 30° laparoscope is inserted through the second or midcla-vicular port. Most of the dissection is carried out via the two most lateral ports. However, the instruments and ports may be changed to provide optimum exposure, as needed.For a right adrenalectomy, a fan retractor is inserted through the most medial port to retract the liver. An atrau-matic grasper and an L-hook cautery are inserted via the two lateral ports for the dissection. The right triangular ligament is divided, and the liver is rotated medially (Fig. 38-49A). Rarely, the hepatic flexure of the colon may need mobilization during a right adrenalectomy. The right kidney is identified visually and by palpation with an atraumatic grasper. The adrenal gland is identified on the superomedial aspect of the kidney. Gerota’s fascia is incised with the hook cautery.

1	kidney is identified visually and by palpation with an atraumatic grasper. The adrenal gland is identified on the superomedial aspect of the kidney. Gerota’s fascia is incised with the hook cautery. Dissection of the adrenal is started superomedially and then proceeds inferiorly, dissect-ing around the adrenal in a clockwise manner. The periadrenal tissues are grasped or moved with a blunt grasper to facilitate circumferential dissection. The right adrenal vein is identified at its junction with the IVC, ligated with clips, and divided using endoscopic scissors. Alternatively, a vascular stapler may be used to divide the vein endoscopically. There may be a second adrenal vein on the right. Generally, two clips are left on the vena cava side. Although early identification of the adrenal vein is helpful to facilitate mobilization and prevent injury, it can be dissected whenever it is safe to do so. Early ligation of the adrenal vein makes it easier to mobilize the gland but may make

1	vein is helpful to facilitate mobilization and prevent injury, it can be dissected whenever it is safe to do so. Early ligation of the adrenal vein makes it easier to mobilize the gland but may make subsequent dissection more difficult due to venous congestion. The arterial branches to the adrenal gland can be electrocoagu-lated if small or clipped and divided.For a left adrenalectomy, the fan retractor is used to retract the spleen. The splenic flexure is mobilized early, and the lat-eral attachments to the spleen and the tail of the pancreas are divided using the electrocautery (Fig. 38-49B). Gravity allows the spleen and the pancreatic tail to fall medially. The remain-der of the dissection proceeds similarly to that described for the right adrenal. In addition to the adrenal vein, the inferior phrenic vein, which joins the left adrenal vein medially, also needs to be Brunicardi_Ch38_p1625-p1704.indd 169901/03/19 11:22 AM 1700SPECIFIC CONSIDERATIONSPART IIABAnterior

1	vein, the inferior phrenic vein, which joins the left adrenal vein medially, also needs to be Brunicardi_Ch38_p1625-p1704.indd 169901/03/19 11:22 AM 1700SPECIFIC CONSIDERATIONSPART IIABAnterior approachCThoracoabdominal approachPosterior approachFigure 38-50. Incisions for open adrenalectomy. Anterior approach (A), posterior approach (B), and thoracoabdominal approach (C).Anterior Approach The adrenals may be removed via a mid-line incision or bilateral subcostal incision (Fig. 38-50). The for-mer allows adequate infraumbilical exposure for examination of extra-adrenal tumors, whereas the latter provides better superior and lateral exposure. For the right side, the hepatic flexure of the colon is mobilized inferiorly, and the triangular ligament is incised to retract the liver medially and superiorly. A generous Kocher maneuver is used to mobilize the duodenum anteriorly and expose the retroperitoneal fat and the IVC (Fig. 38-51A). Gerota’s fascia is incised, and the gland is

1	and superiorly. A generous Kocher maneuver is used to mobilize the duodenum anteriorly and expose the retroperitoneal fat and the IVC (Fig. 38-51A). Gerota’s fascia is incised, and the gland is freed of surround-ing fibro-fatty tissue and the kidney inferiorly. The lateral and superior surfaces usually are mobilized first. Then, the short, right adrenal vein is dissected, ligated, and divided, taking care not to injure the hepatic veins and IVC. On the left side, the adrenal is located cephalad to the pancreatic tail and just lateral to the aorta. For large tumors, the adrenal is best approached by medial visceral rotation to mobilize the spleen, colon, and pan-creas toward the midline (Fig. 38-51B). An alternative approach is to enter the lesser sac by division of the gastrocolic ligament. The pancreas is mobilized superiorly by incision of its inferior dissected, doubly clipped, and divided. As with the right adrenal vein, the left-sided veins also can be divided with a vascular

1	The pancreas is mobilized superiorly by incision of its inferior dissected, doubly clipped, and divided. As with the right adrenal vein, the left-sided veins also can be divided with a vascular stapler. Once the dissection is complete, the area of the adrenal bed can be irrigated and suctioned. A drain is rarely necessary. The gland is placed in a nylon specimen bag, which is brought out via one of the ports after morcellation, if necessary.Posterior Retroperitoneal Approach The retroperitoneal approach provides a more direct access to the adrenal gland and avoids abdominal adhesions in patients who have had previous abdominal surgery. Furthermore, bilateral adrenalectomy can be performed without repositioning the patient. Intraoperative ultrasound is helpful for identifying the adrenal, but the dis-section and exposure are more difficult because the working space is limited. This makes vascular control difficult and also renders it unsuitable for large (>5 cm) lesions. This

1	adrenal, but the dis-section and exposure are more difficult because the working space is limited. This makes vascular control difficult and also renders it unsuitable for large (>5 cm) lesions. This tech-nique is being increasingly used for small adenomas causing hyperaldosteronism.The patient is placed in the prone-jackknife position, and the operating table is flexed at the waist to open the space between the posterior costal margin and the pelvis. Palpation is used to identify the position of the twelfth rib. Percutane-ous ultrasound is performed to determine the outline of the underlying kidney and adrenal. When done laparoscopically, the surgeon stands on the side of the adrenal to be removed, and the assistant stands on the opposite side. A 1.5-cm incision is placed 2 cm inferior and parallel to the twelfth rib, laterally at the level of the inferior pole of the kidney. Gerota’s space is entered under direct vision using a 12-mm direct viewing trocar with a 0° laparoscope

1	and parallel to the twelfth rib, laterally at the level of the inferior pole of the kidney. Gerota’s space is entered under direct vision using a 12-mm direct viewing trocar with a 0° laparoscope through the muscle layers of the posterior abdominal wall. Alternatively, blunt dissection with the surgeon’s finger also can identify the space behind Gerota’s fascia. The trocar is then replaced by a dissecting balloon, which is manually inflated using a hand pump under direct vision through the laparoscope. A 12-mm trocar is then reinserted into this space, and CO2 is insufflated to 12 to 15 mmHg pressure. The 0° laparoscope is replaced by a 45° laparoscope. Two addi-tional 5or 10-mm trocars are placed, one each on either side of the first port. Laparoscopic ultrasound then is used to help locate the adrenal gland and vessels. The adrenal dissection is begun at the superior pole and then proceeds to the lateral and inferior aspect. The medial dissection usually is performed last, and the

1	the adrenal gland and vessels. The adrenal dissection is begun at the superior pole and then proceeds to the lateral and inferior aspect. The medial dissection usually is performed last, and the vessels are identified and divided as described in the earlier “Lateral Transabdominal Approach” section.Open Adrenalectomy. Open adrenalectomy may be per-formed via four approaches, each with specific advantages and disadvantages. The anterior approach allows examination of the abdominal cavity and resection of bilateral tumors via a single incision. The posterior approach avoids the morbidity of a lapa-rotomy incision, especially in patients with cardiopulmonary disease and those prone to wound complications (Cushing’s syndrome) and avoids abdominal adhesions in patients who have undergone previous abdominal surgery. Recovery time is also quicker and hospitalization shorter. However, the retroperi-toneal exposure is difficult, particularly in obese patients, and the small working space makes

1	abdominal surgery. Recovery time is also quicker and hospitalization shorter. However, the retroperi-toneal exposure is difficult, particularly in obese patients, and the small working space makes it unsuitable for tumors >6 cm in diameter. The lateral approach is best for obese patients and for large tumors because it provides a bigger working space. The thoracoabdominal approach is most useful for en bloc resection of large (>10 cm), malignant lesions. However, it is associated with significant morbidity and should be used selectively.Brunicardi_Ch38_p1625-p1704.indd 170001/03/19 11:22 AM 1701THYROID, PARATHYROID, AND ADRENALCHAPTER 38ABRightadrenalSpleenPancreasPancreasColonLeftadrenal v.LeftkidneyFigure 38-51. Technique of open adrenalectomy. Exposure of the right adrenal is facilitated by a Kocher maneuver to mobilize the duodenum and upward retraction of the liver (A). The left adrenal can be exposed by medial visceral rotation of the spleen and pan-creas (B). v. =

1	is facilitated by a Kocher maneuver to mobilize the duodenum and upward retraction of the liver (A). The left adrenal can be exposed by medial visceral rotation of the spleen and pan-creas (B). v. = vein.peritoneal attachments, thus exposing the left kidney and adre-nal. The gland is then mobilized as on the right side.Posterior Approach The patient is placed prone on the operat-ing table, similar to the laparoscopic approach. A hockey stick or curvilinear incision may be used, and extended through the latissimus dorsi and sacrospinous fascia. The twelfth rib gener-ally is excised at its base, and the eleventh rib is retracted supe-riorly to reveal the pleura and the lateral arcuate ligament of the liver on the right side. The pleura also is mobilized cephalad, and the adrenal and kidney are identified. The superior aspect of the gland is dissected first, and the superior vessels are identi-fied and ligated. This prevents superior retraction of the adrenal gland. The remainder of the

1	are identified. The superior aspect of the gland is dissected first, and the superior vessels are identi-fied and ligated. This prevents superior retraction of the adrenal gland. The remainder of the gland is then dissected and the adre-nal gland and tumor removed. The resulting space generally is filled with perinephric fat and closed in layers. A chest X-ray is obtained postoperatively to rule out a pneumothorax.Lateral Approach The patient is placed in a lateral position with the table flexed, and an incision is made between the elev-enth and twelfth ribs or subcostally. The dissection then is per-formed as indicated previously in “Anterior Approach.”Complications of Adrenal Surgery. Patients with Cushing’s syndrome are more prone to infectious (incisional and intra-abdominal abscess) and thrombotic complications. Creation of pneumoperitoneum may result in injury to various organs from Veress needle and trocar insertion, subcutaneous emphysema, pneumothorax, and hemodynamic

1	and thrombotic complications. Creation of pneumoperitoneum may result in injury to various organs from Veress needle and trocar insertion, subcutaneous emphysema, pneumothorax, and hemodynamic compromise. Excessive retraction and dissection may lead to bleeding from injury to the IVC and renal vessels, or from injury to surrounding organs such as the liver, pancreas, spleen, and stomach. Postopera-tive hemodynamic instability may be evident in patients with pheochromocytomas, and patients are at risk of adrenal insuffi-ciency after bilateral adrenalectomy and sometimes after unilat-eral adrenalectomy (unrecognized Cushing’s syndrome or, very rarely, Conn’s syndrome). Long-term morbidity results mainly from injury to nerve roots during trocar insertion, which can lead to chronic pain syndromes or muscle weakness, although this is more of an issue in case of open procedures.Approximately 30% of patients who undergo bilateral adrenalectomy for Cushing’s disease are at risk of developing

1	or muscle weakness, although this is more of an issue in case of open procedures.Approximately 30% of patients who undergo bilateral adrenalectomy for Cushing’s disease are at risk of developing Nelson’s syndrome from progressive growth of the preexisting pituitary tumor. This leads to increased ACTH levels, hyper-pigmentation, visual field defects, headaches, and extraocular muscle palsies. Transsphenoidal pituitary resection is the ini-tial mode of therapy, and external-beam radiotherapy is used in patients with residual tumor or extrasellar invasion.REFERENCESEntries highlighted in bright blue are key references. 1. Rayess HM, Monk I, Svider PF, Gupta A, Raza SN, Lin HS. Thyroglossal duct cyst carcinoma: a systematic review of clinical features and outcomes. Otolaryngol Head Neck Surg. 2017;156:794-802. 2. Cernea CR, Ferraz AR, Nishio S, Dutra A, Jr, Hojaij FC, dos Santos LR. Surgical anatomy of the external branch of the superior laryngeal nerve. Head Neck.

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1	INTRODUCTIONIn his 1953 classic textbook entitled The Surgery of Infancy and Childhood, Dr. Robert E. Gross summarized the essential challenge of pediatric surgery: “Those who daily operate upon adults, even with the greatest of skill, are sometimes appalled—or certainly are not at their best —when called upon to operate upon and care for a tiny patient. Something more than diminu-tive instruments or scaled-down operative manipulations are necessary to do the job in a suitable manner.” To this day, surgi-cal residents and other trainees often approach the pediatric sur-gical patient with the same mix of fear, trepidation, and anxiety. These same trainees often complete their pediatric surgical rotations with a profound respect for the resilience of young children to undergo complex operations and an appreciation for the precision required from their caregivers, both in the operat-ing room and during the perioperative period. Over the decades, the specialty of pediatric surgery has

1	and an appreciation for the precision required from their caregivers, both in the operat-ing room and during the perioperative period. Over the decades, the specialty of pediatric surgery has evolved considerably in its care for the smallest of surgical patients, such that in utero sur-gery is now an option in an increasing number of circumstances. Similarly, our understanding of the pathophysiology of the dis-eases that pediatric surgeons face has increased to the point that some pediatric surgical diseases are now understood at the level of molecular or cellular signaling pathways. Pediatric surgery provides the opportunity to intervene in a wide array of diseases and to exert a long-lasting impact on the lives of children and their grateful parents. The scope of diseases encountered in the standard practice of pediatric surgery is immense, with patients Pediatric SurgeryDavid J. Hackam, Jeffrey Upperman, Tracy Grikscheit, Kasper Wang, and Henri R. Ford

1	of diseases encountered in the standard practice of pediatric surgery is immense, with patients Pediatric SurgeryDavid J. Hackam, Jeffrey Upperman, Tracy Grikscheit, Kasper Wang, and Henri R. Ford 39chapterIntroduction1705Pediatric Surgical Themes: Pitfalls and Pearls1706General Considerations1707Fluid and Electrolyte Balance / 1707Acid-Base Equilibrium / 1707Blood Volume and Blood Replacement / 1707Parenteral Alimentation and Nutrition / 1708Venous Access / 1709Thermoregulation / 1709Pain Control / 1710Neck Masses1710Lymphadenopathy / 1710Thyroglossal Duct Remnants / 1710Branchial Cleft Anomalies / 1711Lymphatic Malformation / 1711Torticollis / 1712Respiratory System1712Congenital Diaphragmatic Hernia (Bochdalek) / 1712Congenital Lobar Emphysema / 1714Bronchopulmonary Foregut Malformations / 1715Bronchiectasis / 1716Foreign Bodies / 1716Esophagus1717Esophageal Atresia and Tracheoesophageal Fistula / 1717Corrosive Injury of the Esophagus / 1721Gastroesophageal Reflux /

1	Malformations / 1715Bronchiectasis / 1716Foreign Bodies / 1716Esophagus1717Esophageal Atresia and Tracheoesophageal Fistula / 1717Corrosive Injury of the Esophagus / 1721Gastroesophageal Reflux / 1721Gastrointestinal Tract1722An Approach to the Vomiting Infant / 1722Hypertrophic Pyloric Stenosis / 1722Intestinal Obstruction in the Newborn / 1723Duodenal Obstruction / 1724Intestinal Atresia / 1724Malrotation and Midgut Volvulus / 1725Meconium Ileus / 1726Necrotizing Enterocolitis / 1727Short Bowel Syndrome / 1730Intussusception / 1731Appendicitis / 1731Intestinal Duplications / 1733Meckel’s Diverticulum / 1733Mesenteric Cysts / 1733Hirschsprung’s Disease / 1734Anorectal Malformations / 1735Jaundice1737The Approach to the Jaundiced Infant / 1737Biliary Atresia / 1737Choledochal Cyst / 1739Deformities of the Abdominal Wall1740Embryology of the Abdominal Wall / 1740Umbilical Hernia / 1740Patent Urachus / 1740Omphalocele / 1740Gastroschisis / 1741Prune-Belly Syndrome / 1743Inguinal

1	1739Deformities of the Abdominal Wall1740Embryology of the Abdominal Wall / 1740Umbilical Hernia / 1740Patent Urachus / 1740Omphalocele / 1740Gastroschisis / 1741Prune-Belly Syndrome / 1743Inguinal Hernia / 1743Genitalia1744Undescended testis / 1744Vaginal Anomalies / 1745Ovarian Cysts and Tumors / 1745Ambiguous Genitalia / 1746Pediatric Malignancy1747Wilms’ Tumor / 1747Neuroblastoma / 1748Rhabdomyosarcoma / 1749Teratoma / 1750Liver Tumors / 1751Trauma in Children1751Mechanisms of Injury / 1751Initial Management / 1752Evaluation of Injury / 1752Injuries to the Central Nervous System / 1752Thoracic Injuries / 1752Abdominal Injuries / 1752Fetal Intervention1753Fetal Surgery for Lower Urinary Tract Obstruction / 1754Fetal Surgery for Myelomeningocele / 1754The EXIT Procedure / 1754Brunicardi_Ch39_p1705-p1758.indd 170512/02/19 11:26 AM 1706Key Points1 In infants with Bochdalek-type congenital diaphragmatic hernia, the severity of pulmonary hypoplasia and the resul-tant pulmonary

1	170512/02/19 11:26 AM 1706Key Points1 In infants with Bochdalek-type congenital diaphragmatic hernia, the severity of pulmonary hypoplasia and the resul-tant pulmonary hypertension are key determinants of sur-vival. Barotrauma and hypoxia should be avoided.2 During initial management of an infant with esophageal atresia and distal tracheoesophageal fistula, every effort should be made to avoid distending the gastrointestinal tract, especially when using mechanical ventilation. The patient should be evaluated for components of the VAC-TERRL (vertebral, anorectal, cardiac, tracheoesophageal, renal, radial limb) anomalies. Timing and extent of surgery are dictated by the stability of the patient.3 Although malrotation with midgut volvulus occurs most commonly within the first few weeks of life, it should always be considered in the differential diagnosis in a child with bilious emesis. Volvulus is a surgical emergency; therefore, in a critically ill child, prompt surgical

1	few weeks of life, it should always be considered in the differential diagnosis in a child with bilious emesis. Volvulus is a surgical emergency; therefore, in a critically ill child, prompt surgical interven-tion should not be delayed for any reason.4 When evaluating a newborn infant for vomiting, it is criti-cal to distinguish between proximal and distal causes of intestinal obstruction using both prenatal and postnatal history, physical examination, and abdominal radiographs.5 Risk factors for necrotizing enterocolitis (NEC) include prematurity, formula feeding, bacterial infection, and intestinal ischemia. Critical to the management of infants with advanced (Bell stage III) or perforated NEC is timely and adequate source control of peritoneal contamination. Early sequelae of NEC include perforation, sepsis, and death. Later sequelae include short bowel syndrome and stricture.6 In patients with intestinal obstruction secondary to Hirschsprung’s disease, a leveling ostomy or

1	include perforation, sepsis, and death. Later sequelae include short bowel syndrome and stricture.6 In patients with intestinal obstruction secondary to Hirschsprung’s disease, a leveling ostomy or endorectal pull-through should be performed using ganglionated bowel, proximal to the transition zone between ganglionic and aganglionic intestine.7 Prognosis of infants with biliary atresia is directly related to age at diagnosis and timing of portoenterostomy. Infants with advanced age at the time of diagnosis or infants who fail to demonstrate evidence of bile drainage after porto-enterostomy usually require liver transplantation.8 Infants with omphaloceles have greater associated morbid-ity and mortality than infants with gastroschisis due to a higher incidence of congenital anomalies and pulmonary hypoplasia. Gastroschisis can be associated with intestinal atresia, but not with other congenital anomalies. An intact omphalocele can be repaired electively, whereas gastros-chisis requires

1	hypoplasia. Gastroschisis can be associated with intestinal atresia, but not with other congenital anomalies. An intact omphalocele can be repaired electively, whereas gastros-chisis requires urgent intervention to protect the exposed intestine.9 Prognosis for children with Wilms’ tumor is defined by the stage of disease at the time of diagnosis and the histo-logic type (favorable vs. unfavorable). Preoperative che-motherapy is indicated for bilateral involvement, a solitary kidney, or tumor in the inferior vena cava above the hepatic veins. Gross tumor rupture during surgery auto-matically changes the stage to 3 (at a minimum).10 Injury is the leading cause of death in children older than 1 year of age. Blunt mechanisms account for the majority of pediatric injuries. The central nervous system is the most commonly injured organ system and the leading cause of death in injured children.ranging in age from the fetus to 18 years old, and it includes pathologies in the head and neck,

1	system is the most commonly injured organ system and the leading cause of death in injured children.ranging in age from the fetus to 18 years old, and it includes pathologies in the head and neck, thoracic, gastrointestinal, and genitourinary regions. This chapter is not designed to cover the entire spectrum of diseases a pediatric surgeon is expected to master; rather, it presents a synopsis of the most commonly encountered pediatric surgical conditions that a practicing gen-eral surgeon is likely to treat over the course of her or his career.PEDIATRIC SURGICAL THEMES: PITFALLS AND PEARLSThis chapter focuses on the unique considerations regarding the diagnosis and management of surgical diseases in the pediatric population. Many surgical trainees approach the surgical care of children with some degree of fear and trepidation. As any pediatric caregiver will attest to, the surgical management of infants and children requires delicate, careful, and professional interactions with their

1	some degree of fear and trepidation. As any pediatric caregiver will attest to, the surgical management of infants and children requires delicate, careful, and professional interactions with their parents. The stress that the parents of sick children experience in the hospital setting can, at times, be over-whelming. It is due, in part, to the uncertainty regarding a par-ticular prognosis, the feeling of helplessness that evolves when one is unable to care for one’s own child, and in certain cases, the guilt or remorse that one feels for not seeking medical care earlier, or for consenting to a particular procedure. Management of the sick child and his or her family requires not only a cer-tain set of skills but also a unique knowledge base. This section is included to summarize some important general principles in accomplishing this task.1. Children are not little adults, but they are little people. In practical terms, this often-heard refrain implies that children have unique fluid,

1	general principles in accomplishing this task.1. Children are not little adults, but they are little people. In practical terms, this often-heard refrain implies that children have unique fluid, electrolyte, and medication needs. Thus, the dosage of medications and the administration of IV fluids should at all times be based on their weight. The corollary of this point is that infants and young children are extremely sensitive to perturbations in their normal physiology and may be easily tipped into fluid overload or dehydration.2. Sick children whisper before they shout. Children with surgi-cal diseases can deteriorate very quickly. But before they dete-riorate, they often manifest subtle physical findings. These findings—referred to as “whispers”—may include signs such as tachycardia, bradycardia, hypothermia, fever, recurrent emesis, or feeding intolerance. Meticulous attention to these subtle findings may unmask the development of potentially serious, life-threatening

1	bradycardia, hypothermia, fever, recurrent emesis, or feeding intolerance. Meticulous attention to these subtle findings may unmask the development of potentially serious, life-threatening physiological disturbances.3. Always listen to the mother and the father. Surgical diseases in children can be very difficult to diagnose because children are often minimally communicative, and information that they communicate may be confusing, conflicting, or both. In all cases, it is wise to listen to the child’s parents, who have closely observed their child and know him or her best. Most importantly, the child’s parents know with certainty Brunicardi_Ch39_p1705-p1758.indd 170612/02/19 11:26 AM 1707PEDIATRIC SURGERYCHAPTER 39whether or not the child is sick or not, despite not always knowing the precise diagnosis.4. Pediatric tissue must be handled delicately and with pro-found respect.5. Children suffer pain after surgery. Timely and adequate pain management must accompany surgical

1	the precise diagnosis.4. Pediatric tissue must be handled delicately and with pro-found respect.5. Children suffer pain after surgery. Timely and adequate pain management must accompany surgical interventions.6. Pay particular attention to the postoperative pediatric patient whose pain cannot be soothed by the administration of stan-dard amounts of analgesic agents. Ask yourself whether a sig-nificant yet unrecognized postoperative complication exists.GENERAL CONSIDERATIONSFluid and Electrolyte BalanceIn managing the pediatric surgical patient, an understanding of fluid and electrolyte balance is critical as the margin between dehydration and fluid overload is small. This is particularly true in infants, who have little reserve at baseline and even less when ill. Failure to pay meticulous attention to their hydration status can result in significant fluid overload or dehydration. Several surgical diagnoses such as gastroschisis or short-gut syndrome are characterized by a

1	meticulous attention to their hydration status can result in significant fluid overload or dehydration. Several surgical diagnoses such as gastroschisis or short-gut syndrome are characterized by a predisposition to fluid loss. Others require judicious restoration of intravascular volume in order to pre-vent cardiac failure as is the case in patients with congenital diaphragmatic hernia and associated pulmonary hypertension.The infant’s physiologic day is approximately eight hours in duration. Accordingly, careful assessment of the individual patient’s fluid balance, including fluid intake and output for the previous eight hours, is essential to prevent dehydration or fluid overload. Clinical signs of dehydration include tachycardia, decreased urine output, reduced skin turgor, depressed fonta-nelle, absent tears, lethargy, and poor feeding. Fluid overload is often manifested by the onset of a new oxygen requirement, respiratory distress, tachypnea, and tachycardia. The physi-cal

1	fonta-nelle, absent tears, lethargy, and poor feeding. Fluid overload is often manifested by the onset of a new oxygen requirement, respiratory distress, tachypnea, and tachycardia. The physi-cal assessment of the fluid status of each child must include a complete head-to-toe evaluation, with emphasis on determining whether perturbations in normal physiology are present.At 12 weeks’ gestation, the total body water of a fetus is approximately 94 cc/kg. By the time the fetus reaches full term, the total body water has decreased to approximately 80 cc/kg. Total body water drops an additional 5% within the first week of life, and by 1 year of life, total body water approaches adult levels, around 60 to 65 cc/kg. Parallel to the drop in total body water is the reduction in extracellular fluid. These changes are accelerated in the preterm infant who may face additional fluid losses due to coexisting congenital anomalies or surgery. Nor-mal daily maintenance fluids for most children can be

1	These changes are accelerated in the preterm infant who may face additional fluid losses due to coexisting congenital anomalies or surgery. Nor-mal daily maintenance fluids for most children can be estimated using the following formula:100 mL/kg for the first 10 kg, plus 50 mL/kg for 11 to 20 kg, plus 25 mL/kg for each additional kilogram of body weight thereafter.Because IV (I.V.) fluid orders are written as milliliters per hour, this can be conveniently converted to:4 mL/kg/h up to 10 kg, add 2 mL/kg/h for 11 to 20 kg, and add 1 mL/kg/h for each additional kilogram body weight thereafter.For example, a 26-kg child has an estimated maintenance fluid requirement of (10 × 4) + (10 × 2) + (6 × 1) = 66 mL/h in the absence of massive fluid losses or shock. A newborn infant with gastroschisis will manifest significant evaporative losses from the exposed bowel such that fluid requirements can be on the order of 150 to 180 cc/kg/day.Precise management of a neonate’s fluid status requires

1	will manifest significant evaporative losses from the exposed bowel such that fluid requirements can be on the order of 150 to 180 cc/kg/day.Precise management of a neonate’s fluid status requires an understanding of changes in the glomerular filtration rate (GFR) and tubular function of the kidney. The term newborn’s GFR is approximately 21 mL/min/1.73 m2 compared to 70 mL/min/1.73 m2 in an adult. Within the first 2 weeks of life GFR increases to approximately 60, and by 2 years of age it is essentially at adult levels. The capacity to concentrate urine is very limited in preterm and term infants. In comparison to an adult who can concentrate urine to 1200 mOsm/kg, infants can concentrate urine at best to 600 mOsm/kg. While infants are capable of secreting antidiuretic hormone, ADH, the aquaporin water channel–mediated osmotic water permeability of the infant’s collecting tubules is severely limited compared to that of adults, leading to an insensitivity to ADH.Sodium requirements

1	aquaporin water channel–mediated osmotic water permeability of the infant’s collecting tubules is severely limited compared to that of adults, leading to an insensitivity to ADH.Sodium requirements range from 2 mEq/kg per day in term infants up to 5 mEq/kg per day in critically ill preterm infants as a consequence of salt wasting. Potassium require-ments are on the order of 1 to 2 mEq/kg per day. Calcium and magnesium supplementation of IV fluids is essential to prevent laryngospasm, dysrhythmias, and tetany.Acid-Base EquilibriumAcute metabolic acidosis usually implies inadequate tissue perfusion and is a serious disorder in children. Potentially life-threatening causes that are specific for the pediatric population must be sought; they include intestinal ischemia from necro-tizing enterocolitis (in the neonate), midgut volvulus, or incar-cerated hernia. Other causes include chronic bicarbonate loss from the gastrointestinal tract or acid accumulation as in chronic renal failure.

1	(in the neonate), midgut volvulus, or incar-cerated hernia. Other causes include chronic bicarbonate loss from the gastrointestinal tract or acid accumulation as in chronic renal failure. Respiratory acidosis implies hypoventilation, the cause of which should be apparent. Treatment of acute meta-bolic acidosis should be aimed at restoring tissue perfusion by addressing the underlying abnormality first. For severe meta-bolic acidemia where the serum pH is less than 7.25, sodium bicarbonate should be administered using the following guide-line: base deficit × weight in kilograms × 0.5 (in newborns). The last factor in the equation should be 0.4 for smaller children and 0.3 for older children. The dose should be diluted to a concentra-tion of 0.5 mEq/mL because full-strength sodium bicarbonate is hyperosmolar. One-half the corrective dose is given, and the serum pH is measured again. During cardiopulmonary resusci-tation (CPR), one-half the corrective dose can be given as an intravenous

1	is hyperosmolar. One-half the corrective dose is given, and the serum pH is measured again. During cardiopulmonary resusci-tation (CPR), one-half the corrective dose can be given as an intravenous bolus and the other half given slowly intravenously.Respiratory alkalosis is usually caused by hyperventila-tion, which is readily correctable. Metabolic alkalosis most commonly implies gastric acid loss, as in the child with pyloric stenosis, or aggressive diuretic therapy. In the child with gastric fluid loss, IV fluids of 5% dextrose, 0.5% normal saline, and 20 mEq KCl/L usually correct the alkalosis.Blood Volume and Blood ReplacementCriteria for blood transfusion in infants and children remain poorly defined. The decision to transfuse a critically ill pediatric patient may depend on a number of clinical features that include the patient’s age, primary diagnosis, the presence of ongoing bleeding, coagulopathy, hypoxia, hemodynamic compromise, lactic acidosis, cyanotic heart disease, and

1	of clinical features that include the patient’s age, primary diagnosis, the presence of ongoing bleeding, coagulopathy, hypoxia, hemodynamic compromise, lactic acidosis, cyanotic heart disease, and overall severity of illness. A recent survey of transfusion practices among pediatric intensivists showed that the baseline hemoglobin levels that would prompt them to recommend RBC transfusion ranged from 7 to 13 g/dL. Patients with cyanotic heart disease are often transfused to Brunicardi_Ch39_p1705-p1758.indd 170712/02/19 11:26 AM 1708SPECIFIC CONSIDERATIONSPART IIhigher hemoglobin values, although the threshold for transfusion in this population remains to be defined. In general terms, there is a trend towards an avoidance of the use of RBC products whenever possible as current studies suggest that lower hemoglobin concentrations are well tolerated by many groups of patients and that administration of RBCs may have unintended negative consequences, including perhaps an increase in

1	suggest that lower hemoglobin concentrations are well tolerated by many groups of patients and that administration of RBCs may have unintended negative consequences, including perhaps an increase in predisposition to the development of necrotizing enterocolitis, although this finding is controversial. In addition, there is increasing evidence that PRBC transfusion may have adverse effects on the host immune in both children and adults. These effects are poorly understood but may include effects due to RBC storage and due to factors that are particular to the individual RBC donor. The TRIPICU randomized controlled trial by Lacroix et al in 2007, which was performed in stable critically ill children, determined that a restrictive Hb transfusion trigger (70 g/L) was as safe as a liberal Hb trigger (95 g/L) and was associated with reduced blood use. It remains uncertain whether this can be extrapolated to unstable patients. Expert opinion now generally favors an Hb transfusion trigger of

1	(95 g/L) and was associated with reduced blood use. It remains uncertain whether this can be extrapolated to unstable patients. Expert opinion now generally favors an Hb transfusion trigger of 70 g/L in stable critically ill children, which is the same as the recommendation for adult patients (see Chapter 7). A higher threshold should be considered if the child has symptomatic anemia or impaired cardiorespiratory function.A useful guideline for estimating blood volume for the newborn infant is approximately 80 mL/kg of body weight. When packed red blood cells are required, the transfusion requirement is usually administered in 10 mL/kg increments, which is roughly equivalent to a 500-mL transfusion for a 70-kg adult. The following formula may be used to determine the vol-ume (ml) of PRBC to be transfused:(Target hematocrit—Current Hematocrit) × weight (kg) × 80/65 (65 represents the estimated hematocrit of a unit of PRBC)As a general rule, blood is recommended for replacement of

1	to be transfused:(Target hematocrit—Current Hematocrit) × weight (kg) × 80/65 (65 represents the estimated hematocrit of a unit of PRBC)As a general rule, blood is recommended for replacement of volume loss if the child’s perfusion is inadequate despite administration of 2 to 3 boluses of 20 mL/kg of isotonic crystalloid. Consideration should be given for the administration of 10 mL/kg of packed red blood cells as soon as possible. Type O blood can be administered without a cross-match and is relatively safe; type-specific blood can be obtained quite quickly; however, unlike fully cross-matched blood, incompatibilities other than ABO and Rh may exist.In the child, coagulation deficiencies may rapidly assume clinical significance after extensive blood transfusion. It is advisable to have fresh frozen plasma and platelets available if more than 30 mL/kg have been transfused. Plasma is given in a dose of 10 to 20 mL/kg, and platelets are given in a dose of 1 unit/5 kg. Each unit of

1	fresh frozen plasma and platelets available if more than 30 mL/kg have been transfused. Plasma is given in a dose of 10 to 20 mL/kg, and platelets are given in a dose of 1 unit/5 kg. Each unit of platelets consists of 40 to 60 mL of fluid (plasma plus platelets). Following transfusion of PRBCs to neonates with tenuous fluid balance, a single dose of a diuretic (such as furosemide 1 mg/kg) may help to facilitate excretion of the extra fluid load. Many clinicians prefer to administer fresh products to minimize the deleterious effects of red cell storage.In pediatric patients who have lost greater than 30 mL/kg with ongoing bleeding, consideration should be given to initia-tion of a massive transfusion protocol. Such a protocol involves transfusion, based on weight, of 1:1:1 transfusion of RBCs, plasma, and platelets.Parenteral Alimentation and NutritionThe nutritional requirements of the surgical neonate must be met in order for the child to grow and to heal surgical wounds. Table

1	of RBCs, plasma, and platelets.Parenteral Alimentation and NutritionThe nutritional requirements of the surgical neonate must be met in order for the child to grow and to heal surgical wounds. Table 39-1Nutritional requirements for the pediatric surgical patientAGECALORIESPROTEIN(kcal/kg/d)(gram/kg/d)0–6 months100–12026 months–1 year1001.51–3 years1001.24–6 years9017–10 years70111–14 years55115–18 years451If inadequate protein and carbohydrate calories are given, the child may not only fail to recover from surgery but may also exhibit growth failure and impaired development of the central nervous system. In general terms, the adequacy of growth must be assessed frequently by determining both total body weight as well as head circumference. Neonates that are particularly predisposed to protein-calorie malnutrition include those with gastroschisis, intestinal atresia, or intestinal insufficiency from other causes, such as necrotizing enterocolitis. The protein and caloric requirements

1	malnutrition include those with gastroschisis, intestinal atresia, or intestinal insufficiency from other causes, such as necrotizing enterocolitis. The protein and caloric requirements for the surgical neonate are shown in Table 39-1.Nutrition can be provided via either the enteral or parenteral routes. Whenever possible, the enteral route is preferred because it not only promotes the growth and function of the gastrointestinal system, it also ensures that the infant learns how to feed. There are various enteral feeding preparations available; these are outlined in Table 39-2. The choice of formula is based upon the individual clinical state of the child. Pediatric surgeons are often faced with situations where oral feeding is not possible. This problem can be seen in the extremely premature infant who has not yet developed the feeding skills, or in the infant with concomitant craniofacial anomalies that impair sucking, for example. In these instances, enteral feeds can be

1	premature infant who has not yet developed the feeding skills, or in the infant with concomitant craniofacial anomalies that impair sucking, for example. In these instances, enteral feeds can be administered either a nasojejunal or a gastrostomy tube.When the gastrointestinal tract cannot be used because of mechanical, ischemic, inflammatory, or functional disorders, parenteral alimentation must be given. Prolonged parenteral nutrition is delivered via a central venous catheter. Peripheral IV alimentation can be given, utilizing less concentrated but greater volumes of solutions. Long-term parenteral nutrition should include supplemental copper, zinc, and iron to prevent the development of trace metal deficiencies. A major complica-tion of long-term total parenteral nutrition (TPN) is the devel-opment of parenteral nutrition–associated cholestasis, which can eventually progress to liver failure. To prevent this major complication, concomitant enteral feedings should be instituted, and

1	devel-opment of parenteral nutrition–associated cholestasis, which can eventually progress to liver failure. To prevent this major complication, concomitant enteral feedings should be instituted, and the gastrointestinal tract should be used as soon as pos-sible. When proximal stomas are in place, gastrointestinal con-tinuity should be restored as soon as possible. Where intestinal insufficiency is associated with dilation of the small intestine, tapering or intestinal lengthening procedures may be beneficial. Brunicardi_Ch39_p1705-p1758.indd 170812/02/19 11:26 AM 1709PEDIATRIC SURGERYCHAPTER 39Table 39-2Formulas for pediatric surgical neonatesFORMULAkcal/mLPROTEIN (g/mL)FAT (g/mL)CARBOHYDRATE (g/mL)Human milk0.670.0110.040.07Milk-based formula Enfamil 200.670.0150.0380.069Similac 200.670.0150.0360.072Soy-based formula Prosobee0.670.020.0360.07Isomil0.670.0180.0370.068Special formula Pregestimil.67.019.028.091Alimentum.67.019.038.068Preterm Enfamil

1	200.670.0150.0360.072Soy-based formula Prosobee0.670.020.0360.07Isomil0.670.0180.0370.068Special formula Pregestimil.67.019.028.091Alimentum.67.019.038.068Preterm Enfamil Premature.80.024.041.089Other strategies to minimize the development of TPN-related liver disease include meticulous catheter care to avoid infec-tion, which increases cholestatic symptoms, aggressive treat-ment of any infection, and early cycling of parenteral nutrition in older children who can tolerate not receiving continuous dextrose solution for a limited period. Evidence suggests that cholestasis eventually resolves in most cases after parenteral nutrition is discontinued, as measured by levels of total bili-rubin. Preliminary evidence suggests that substituting omega-3 fish oil lipid emulsion in parenteral nutrition for the standard soybean-based emulsions may prevent the development of TPN-related cholestasis and reverse the effects of established liver disease. A phase 2 trial to determine whether

1	nutrition for the standard soybean-based emulsions may prevent the development of TPN-related cholestasis and reverse the effects of established liver disease. A phase 2 trial to determine whether parenteral nutrition–associated liver disease can be reversed or its progres-sion halted by using a parenteral fat emulsion prepared from fish oil as measured by normalization of serum levels of hepatic enzymes and bilirubin is ongoing (ClinicalTrials.gov, identifier NCT00826020).Venous AccessObtaining reliable vascular access in an infant or child is an important task that often becomes the responsibility of the pedi-atric surgeon. The goal should always be to place the catheter in the least invasive, least risky, and least painful manner, and in a location that is most accessible and allows for use of the catheter without complications for as long as it is needed. In infants, cen-tral venous access may be established using a cutdown approach, either in the antecubital fossa, external

1	for use of the catheter without complications for as long as it is needed. In infants, cen-tral venous access may be established using a cutdown approach, either in the antecubital fossa, external jugular vein, facial vein, or proximal saphenous vein. If the internal jugular vein is used, care is taken to prevent venous occlusion. In infants over 3 kg and in older children, percutaneous access of the subclavian, internal jugular, or femoral veins is possible in most cases, and central access is achieved using the Seldinger technique. The use of ultrasound (US) is considered standard of care for placement of central lines in this population for the internal jugular vein and femoral veins, and it significantly improves the safety of the insertion procedure. The catheters are tunneled to an exit site separate from the venotomy site. Where available, PICC lines (peripherally inserted central catheters) may be placed, typically via the antecubital fossa. Regardless of whether the catheter

1	exit site separate from the venotomy site. Where available, PICC lines (peripherally inserted central catheters) may be placed, typically via the antecubital fossa. Regardless of whether the catheter is placed by a cutdown approach or percutaneously, a chest X-ray to confirm central location of the catheter tip and to exclude the presence of a pneumothorax or hemothorax is mandatory. When discussing the placement of central venous catheters with par-ents, it is important to note that the complication rate for central venous lines in children can be high. The incidence of catheter-related sepsis or infection remains a problem, yet should be less than 1% with meticulous attention to catheter insertion care and exit site management. Superior or inferior vena caval occlusion is a significant risk after the placement of multiple lines, particu-larly in the smallest premature patients.ThermoregulationCareful regulation of the ambient environment of infants and children is crucial as these

1	risk after the placement of multiple lines, particu-larly in the smallest premature patients.ThermoregulationCareful regulation of the ambient environment of infants and children is crucial as these patients are extremely thermolabile. Premature infants are particularly susceptible to changes in envi-ronmental temperature. Because they are unable to shiver and lack stores of fat, their potential for thermogenesis is impaired. The innate inability to regulate temperature is compounded by the administration of anesthetic and paralyzing agents. Since these patients lack adaptive mechanisms to cope with the envi-ronment, the environment must be carefully regulated. Attention to heat conservation during transport of the infant to and from the operating room is essential. Transport systems incorporating heating units are necessary for premature infants. In the operat-ing room, the infant is kept warm by the use of overhead heat-ing lamps, a heating blanket, warming of inspired gases, and

1	heating units are necessary for premature infants. In the operat-ing room, the infant is kept warm by the use of overhead heat-ing lamps, a heating blanket, warming of inspired gases, and coverage of the extremities and head with occlusive materials. During abdominal surgery, extreme care is taken to avoid wet and cold drapes. All fluids used to irrigate the chest or abdomen must be warmed to body temperature. Laparoscopic approaches for abdominal operations may result in more stable thermoregu-lation due to decreased heat loss from the smaller wound size. Constant monitoring of the child’s temperature is critical in a lengthy procedure, and the surgeon should continuously com-municate with the anesthesiologist regarding the temperature of the patient. The development of hypothermia in infants and chil-dren can result in cardiac arrhythmias or coagulopathy. These potentially life-threatening complications can be avoided by careful attention to

1	The development of hypothermia in infants and chil-dren can result in cardiac arrhythmias or coagulopathy. These potentially life-threatening complications can be avoided by careful attention to thermoregulation.Brunicardi_Ch39_p1705-p1758.indd 170912/02/19 11:26 AM 1710SPECIFIC CONSIDERATIONSPART IIPain ControlAll children including neonates experience pain; the careful recognition and management of pediatric pain represents an important component of the perioperative management of all pediatric surgical patients. There is a range of pain manage-ment options that can improve the child’s well-being, as well as the parents’ sense of comfort. Given that morphine and fentanyl have an acceptable safety margin, they should be administered to neonates and children when indicated, bear-ing in mind that withholding analgesia poses a significant risk, as does administration of excessive analgesic agents. A recent randomized trial of neonates on ventilators showed that the use of a morphine

1	mind that withholding analgesia poses a significant risk, as does administration of excessive analgesic agents. A recent randomized trial of neonates on ventilators showed that the use of a morphine infusion decreased the incidence of intraventricular hemorrhage by 50%. Additional analge-sic modalities include the use of topical anesthetic ointment (EMLA cream) and the use of regional anesthesia, such as caudal blocks for hernias and epidural or incisional catheter infusions (On-Q) for large abdominal or thoracic incisions. In surgical neonates that have been administered large con-centrations of narcotics over a prolonged period, transient physical dependence should not only be expected but also anticipated. When narcotics are discontinued, symptoms of narcotic withdrawal may develop, including irritability, rest-lessness, and episodes of hypertension and tachycardia. Early recognition of these signs is essential, as is timely treatment using nalaxone and other agents. It is

1	including irritability, rest-lessness, and episodes of hypertension and tachycardia. Early recognition of these signs is essential, as is timely treatment using nalaxone and other agents. It is important to admin-ister pain control in concert with a well-qualified and col-laborative pediatric pain-management team, which typically includes anesthesiologists with expertise in pain management, as well as advance practice nurses who can respond rapidly when the pain control is inadequate or excessive. By ensuring that the pediatric surgical patient has adequate analgesia, the surgeon ensures that the patient receives the most humane and thorough treatment and provides important reassurance to all other members of the healthcare team and to the family that pain control is a very high priority.NECK MASSESThe management of neck masses in children is determined by their location and the length of time that they have been pres-ent. Neck lesions are found either in the midline or lateral

1	MASSESThe management of neck masses in children is determined by their location and the length of time that they have been pres-ent. Neck lesions are found either in the midline or lateral com-partments. Midline masses include thyroglossal duct remnants, thyroid masses, thymic cysts, or dermoid cysts. Lateral lesions include branchial cleft remnants, cystic hygromas, vascular mal-formations, salivary gland tumors, torticollis, and lipoblastoma (a rare benign mesenchymal tumor of embryonal fat occurring in infants and young children). Enlarged lymph nodes and rare malignancies such as rhabdomyosarcoma can occur either in the midline or laterally.LymphadenopathyThe most common cause of a neck mass in a child is an enlarged lymph node, which typically can be found laterally or in the midline. The patient is usually referred to the pedi-atric surgeon for evaluation after the mass has been present for several weeks. A detailed history and physical examination often helps determine the

1	The patient is usually referred to the pedi-atric surgeon for evaluation after the mass has been present for several weeks. A detailed history and physical examination often helps determine the likely etiology of the lymph node and the need for excisional biopsy. Enlarged tender lymph nodes are usually the result of a bacterial infection (Staphy-lococcus or Streptococcus). Treatment of the primary cause (e.g., otitis media or pharyngitis) with antibiotics often is all that is necessary. However, when the involved nodes become fluctuant, incision and drainage are indicated. In many North American institutions, there has been an increasing prevalence of methicillin-resistant Staphylococcus aureus infection of the skin and soft tissues, leading to increased staphylococcal lymphadenitis in children. More chronic forms of lymphadeni-tis, including infections with atypical mycobacteria, as well as cat-scratch fever, are diagnosed based on serologic findings or excisional biopsy. The

1	in children. More chronic forms of lymphadeni-tis, including infections with atypical mycobacteria, as well as cat-scratch fever, are diagnosed based on serologic findings or excisional biopsy. The lymphadenopathy associated with infectious mononucleosis can be diagnosed based on serology. When the neck nodes are firm, fixed, and others are also pres-ent in the axillae or groin, or the history suggests lymphoma, excisional biopsy is indicated. In these cases, it is essential to obtain a chest radiograph to look for the presence of a medias-tinal mass. Significant mediastinal load portends cardiorespira-tory collapse due to loss of venous return and compression of the tracheobronchial tree with general anesthesia.Thyroglossal Duct RemnantsPathology and Clinical Manifestations. The thyroid gland buds off the foregut diverticulum at the base of the tongue in the region of the future foramen cecum at 3 weeks of embryonic life. As the fetal neck develops, the thyroid tissue becomes more

1	gland buds off the foregut diverticulum at the base of the tongue in the region of the future foramen cecum at 3 weeks of embryonic life. As the fetal neck develops, the thyroid tissue becomes more anterior and caudad until it rests in its normal position. The “descent” of the thyroid is intimately connected with the development of the hyoid bone. Residual thyroid tis-sue left behind during the migration may persist and subse-quently present in the midline of the neck as a thyroglossal duct cyst. The mass is most commonly appreciated in the 2to 4-year-old child when the baby fat disappears and irregulari-ties in the neck become more readily apparent. Usually the cyst is encountered in the midline at or below the level of the hyoid bone and moves up and down with swallowing or with protrusion of the tongue. Occasionally it presents as an intrathyroidal mass. Most thyroglossal duct cysts are asymp-tomatic. If the duct retains its connection with the pharynx, infection may occur, and the

1	of the tongue. Occasionally it presents as an intrathyroidal mass. Most thyroglossal duct cysts are asymp-tomatic. If the duct retains its connection with the pharynx, infection may occur, and the resulting abscess will necessitate incision and drainage, occasionally resulting in a salivary fis-tula. Submental lymphadenopathy and midline dermoid cysts can be confused with a thyroglossal duct cyst. Rarely, midline ectopic thyroid tissue masquerades as a thyroglossal duct cyst and may represent the patient’s only thyroid tissue. Therefore, if there is any question regarding the diagnosis or if the thyroid gland cannot be palpated in its normal anatomic position, it is advisable to obtain a nuclear scan to confirm the presence of a normal thyroid gland. Although rarely the case in children, in adults the thyroglossal duct may contain thyroid tissue that can undergo malignant degeneration. The presence of malignancy in a thyroglossal cyst should be suspected when the cyst grows rapidly or

1	adults the thyroglossal duct may contain thyroid tissue that can undergo malignant degeneration. The presence of malignancy in a thyroglossal cyst should be suspected when the cyst grows rapidly or when US demonstrates a complex anechoic pattern or the presence of calcification.Treatment. If the thyroglossal duct cyst presents with an abscess, treatment should first consist of drainage and antibiot-ics. Following resolution of the inflammation, resection of the cyst in continuity with the central portion of the hyoid bone and the tract connecting to the pharynx in addition to ligation at the foramen cecum (the Sistrunk operation), is curative in over 90% of patients. Lesser operations result in unacceptably high recur-rence rates, and recurrence is more frequent following infection. According to a recent review, factors predictive of recurrence included more than two infections prior to surgery, age under 2 years, and inadequate initial operation.Brunicardi_Ch39_p1705-p1758.indd

1	to a recent review, factors predictive of recurrence included more than two infections prior to surgery, age under 2 years, and inadequate initial operation.Brunicardi_Ch39_p1705-p1758.indd 171012/02/19 11:26 AM 1711PEDIATRIC SURGERYCHAPTER 39Branchial Cleft AnomaliesPaired branchial clefts and arches develop early in the fourth gestational week. The first cleft and the first, second, third, and fourth pouches give rise to adult organs. The embryologic com-munication between the pharynx and the external surface may persist as a fistula. A fistula is seen most commonly with the second branchial cleft, which normally disappears, and extends from the anterior border of the sternocleidomastoid muscle superiorly, inward through the bifurcation of the carotid artery, and enters the posterolateral pharynx just below the tonsillar fossa. In contrast, a third branchial cleft fistula passes posterior to the carotid bifurcation. The branchial cleft remnants may con-tain small pieces of

1	pharynx just below the tonsillar fossa. In contrast, a third branchial cleft fistula passes posterior to the carotid bifurcation. The branchial cleft remnants may con-tain small pieces of cartilage and cysts, but internal fistulas are rare. A second branchial cleft sinus is suspected when clear fluid is noted draining from the external opening of the tract at the anterior border of the lower third of the sternomastoid muscle. Rarely, branchial cleft anomalies occur in association with bili-ary atresia and congenital cardiac anomalies, an association that is referred to as Goldenhar’s complex.Treatment. Complete excision of the cyst and sinus tract is necessary for cure. Dissection of the sinus tract is facilitated with passage of a fine lacrimal duct probe through the external opening into the tract and utilizing it as a guide for dissection. Injection of a small amount of methylene blue dye into the tract also may be useful. A series of two or sometimes three small transverse

1	into the tract and utilizing it as a guide for dissection. Injection of a small amount of methylene blue dye into the tract also may be useful. A series of two or sometimes three small transverse incisions in a “stepladder” fashion is preferred to a long oblique incision in the neck, which is cosmetically unde-sirable. Branchial cleft cysts can present as abscesses. In these cases, initial treatment includes incision and drainage with a course of antibiotics to cover Staphylococcus and Streptococ-cus species, followed by excision of the cyst after the infection resolves.Lymphatic MalformationEtiology and Pathology. Lymphatic malformation (cystic hygroma or lymphangioma) occurs as a result of sequestration or obstruction of developing lymph vessels in approximately 1 in 12,000 births. Although the lesion can occur anywhere, the most common sites are in the posterior triangle of the neck, axilla, groin, and mediastinum. The cysts are lined by endo-thelium and filled with lymph.

1	Although the lesion can occur anywhere, the most common sites are in the posterior triangle of the neck, axilla, groin, and mediastinum. The cysts are lined by endo-thelium and filled with lymph. Occasionally unilocular cysts occur, but more often there are multiple cysts “infiltrating” the surrounding structures and distorting the local anatomy. A particularly troublesome variant of lymphatic malformation is that which involves the tongue, floor of the mouth, and struc-tures deep in the neck. Adjacent connective tissue may show extensive lymphocytic infiltration. The mass may be apparent at birth or may appear and enlarge rapidly in the early weeks or months of life as lymph accumulates; most present by age 2 years (Fig. 39-1A). Extension of the lesion into the axilla or mediastinum occurs about 10% of the time and can be demon-strated preoperatively by chest X-ray, US, or computed tomo-graphic (CT) scan, although magnetic resonance imaging (MRI) is preferable. Occasionally lymphatic

1	about 10% of the time and can be demon-strated preoperatively by chest X-ray, US, or computed tomo-graphic (CT) scan, although magnetic resonance imaging (MRI) is preferable. Occasionally lymphatic malformations contain nests of vascular tissue. These poorly supported vessels may bleed and produce rapid enlargement and discoloration of the lesion. Infection within the lymphatic malformations, usually caused by Streptococcus or Staphylococcus, may occur. In the neck, this can cause rapid enlargement, which may result in airway compromise. Rarely, it may be necessary to carry out percutaneous aspiration of a cyst to relieve respiratory distress.The diagnosis of lymphatic malformation by prenatal US, before 30 weeks’ gestation, has detected a “hidden mortality” as well as a high incidence of associated anomalies, including abnormal karyotypes and hydrops fetalis. Occasionally, very large lesions can cause obstruction of the fetal airway. Such obstruction can result in the development of

1	associated anomalies, including abnormal karyotypes and hydrops fetalis. Occasionally, very large lesions can cause obstruction of the fetal airway. Such obstruction can result in the development of polyhydramnios by impairing the ability of the fetus to swallow amniotic fluid. In these circumstances, the airway is usually markedly distorted, which can result in immediate airway obstruction unless the air-way is secured at the time of delivery. Orotracheal intubation or emergency tracheostomy while the infant remains attached to the placenta, the so-called EXIT procedure (ex utero intrapar-tum technique) may be necessary to secure the airway.Treatment. The modern management of most lymphatic malformations includes image-guided sclerotherapy as first-line therapy, which often involves multiple injections. Cyst excision may be used in cases where injection is inadequate. BAFigure 39-1. A. Left cervical cystic hygroma in a 2-day old baby. B. Intraoperative photograph showing a vessel

1	injections. Cyst excision may be used in cases where injection is inadequate. BAFigure 39-1. A. Left cervical cystic hygroma in a 2-day old baby. B. Intraoperative photograph showing a vessel loop around the spinal accessory nerve.Brunicardi_Ch39_p1705-p1758.indd 171112/02/19 11:26 AM 1712SPECIFIC CONSIDERATIONSPART IIFigure 39-2. Prenatal ultrasound of a fetus with a congenital dia-phragmatic hernia. Arrows point to the location of the diaphragm. Arrowhead points to the stomach, which is in the thoracic cavity.Total removal of all gross disease is often not possible because of the extent of the lymphatic malformation and its proximity to, and intimate relationship with, adjacent nerves, muscles, and blood vessels (Fig. 39-1B). Radical ablative surgery is not indicated for these lesions, which are always benign. Conservative excision and unroofing of remaining cysts is advised, with repeated partial excision of residual cysts and sclerotherapy if necessary, preserving all

1	lesions, which are always benign. Conservative excision and unroofing of remaining cysts is advised, with repeated partial excision of residual cysts and sclerotherapy if necessary, preserving all adjacent crucial structures. In cases in which surgical excision is performed, closed-suction drainage is recommended. Nevertheless, fluid may accumulate beneath the surgically created flaps in the area from which the lymphatic malformation was excised, requiring multiple needle aspirations. A combined sclerotherapy/resectional approach is particularly useful for masses that extend to the base of the tongue or the floor of the mouth.TorticollisThe presence of a lateral neck mass in infancy in association with rotation of the head towards the opposite side of the mass indicates the presence of congenital torticollis. This lesion results from fibrosis of the sternocleidomastoid muscle. The mass may be palpated in the affected muscle in approximately two-thirds of cases, or it may be diagnosed

1	torticollis. This lesion results from fibrosis of the sternocleidomastoid muscle. The mass may be palpated in the affected muscle in approximately two-thirds of cases, or it may be diagnosed by US. Histologi-cally, the lesion is characterized by the deposition of collagen and fibroblasts around atrophied muscle cells. In the vast major-ity of cases, physical therapy based on passive stretching of the affected muscle is of benefit. Rarely, surgical transection of the sternocleidomastoid may be indicated.RESPIRATORY SYSTEMCongenital Diaphragmatic Hernia (Bochdalek)Pathology. The septum transversum extends to divide the pleural and coelomic cavities during fetal development. This precursor of the diaphragm normally completes separation of these two cavities at the posterolateral aspects of this mesen-chymally derived structure. The most common variant of a congenital diaphragmatic hernia is a posterolateral defect, also known as a Bochdalek hernia. Diaphragmatic defects allow abdominal

1	this mesen-chymally derived structure. The most common variant of a congenital diaphragmatic hernia is a posterolateral defect, also known as a Bochdalek hernia. Diaphragmatic defects allow abdominal viscera to fill the chest cavity. The abdominal cav-ity is small and underdeveloped and remains scaphoid after birth. Both lungs are hypoplastic, with decreased bronchial and pulmonary artery branching. Lung weight, lung volume, and DNA content are also decreased, and these findings are more striking on the ipsilateral side. This anomaly is encountered more commonly on the left (80–90%). Linkage analyses have recently implicated genetic mutations in syndromic variants of congenital diaphragmatic hernias. In many instances, there is a surfactant deficiency, which compounds the degree of respira-tory insufficiency. Amniocentesis with karyotype may identify chromosomal defects, especially trisomy 18 and 21. Associated anomalies, once thought to be uncommon, were identified in 65 of 166

1	insufficiency. Amniocentesis with karyotype may identify chromosomal defects, especially trisomy 18 and 21. Associated anomalies, once thought to be uncommon, were identified in 65 of 166 patients in one study, predominately of the heart, fol-lowed by abdominal wall defects, chromosomal changes, and other defects.Prenatal ultrasonography is successful in making the diag-nosis of congenital diaphragmatic hernia (CDH) as early as 15 weeks’ gestation, and early antenatal diagnosis is associated with worse outcomes. US findings include herniated abdominal viscera in the chest that may also look like a mass or lung anom-aly, changes in liver position, and mediastinal shift away from the herniated viscera (Fig. 39-2). Accurate prenatal prediction of outcome for fetuses who have CDH remains a challenge. One index of severity for patients with left CDH is the lung-to-head ratio (LHR), which is the product of the length and the width of the right lung at the level of the cardiac atria divided

1	One index of severity for patients with left CDH is the lung-to-head ratio (LHR), which is the product of the length and the width of the right lung at the level of the cardiac atria divided by the head circumference (all measurements in millimeters). An LHR value of less than 1.0 is associated with a very poor prognosis, whereas an LHR greater than 1.4 predicts a more favorable outcome. The utility of the LHR in predicting outcome in patients with CDH has recently been questioned because of the tremendous interobserver variability in calculating this ratio for a par-ticular patient, as well as the lack of reliable measures to deter-mine postnatal disease severity. Because the LHR is not gestational age independent, Jani and colleagues proposed the introduction of a new measurement: the observed to expected (o/e) LHR, to correct for gestational age. The observed LHR may be expressed as a percentage of the expected mean for ges-tational age of the observed/expected lung-to-head ratio

1	observed to expected (o/e) LHR, to correct for gestational age. The observed LHR may be expressed as a percentage of the expected mean for ges-tational age of the observed/expected lung-to-head ratio (o/e LHR), which is considered extreme if <15%, severe at 15% to 25%, moderate at 26% to 35%, and mild at 36% to 45%. The most reliable prenatal predictor of postnatal survival is absence of liver herniation, where in 710 fetuses, there was significantly higher survival rate in fetuses without herniation (74% without herniation vs. 45% with herniation).Following delivery, the diagnosis of CDH is made by CXR (Fig. 39-3). The differential diagnosis includes broncho-pulmonary foregut malformations, in which the intrathoracic loops of bowel may be confused for lung or foregut pathol-ogy. The vast majority of infants with CDH develop immedi-ate respiratory distress, which is due to the combined effects of three factors. First, the air-filled bowel in the chest compresses the mobile

1	The vast majority of infants with CDH develop immedi-ate respiratory distress, which is due to the combined effects of three factors. First, the air-filled bowel in the chest compresses the mobile mediastinum, which shifts to the opposite side of the chest, compromising air exchange in the contralateral lung. Second, pulmonary hypertension develops. This phenomenon results in persistent fetal circulation with resultant decreased pulmonary perfusion and impaired gas exchange. Finally, the lung on the affected side is often hypoplastic, such that it is essentially nonfunctional. Varying degrees of pulmonary hypo-plasia on the opposite side may compound these effects. The second and third factors are thought to be the most important. Neonates with CDH are usually in respiratory distress requiring 1Brunicardi_Ch39_p1705-p1758.indd 171212/02/19 11:26 AM 1713PEDIATRIC SURGERYCHAPTER 39Figure 39-3. Chest X-ray showing a left congenital diaphragmatic hernia.ventilation and intensive care,

1	1Brunicardi_Ch39_p1705-p1758.indd 171212/02/19 11:26 AM 1713PEDIATRIC SURGERYCHAPTER 39Figure 39-3. Chest X-ray showing a left congenital diaphragmatic hernia.ventilation and intensive care, and the overall mortality in most series is around 50%.Treatment. CDH care has been improved through effective use of improved methods of ventilation and timely cannula-tion for extracorporeal membrane oxygenation (ECMO). Many infants are symptomatic at birth due to hypoxia, hypercarbia, and metabolic acidosis. Prompt cardiorespiratory stabilization is mandatory. It is noteworthy that the first 24 to 48 hours after birth are often characterized by a period of relative stability with high levels of PaO2 and relatively good perfusion. This has been termed the “honeymoon period” and is often followed by progressive cardiorespiratory deterioration. In the past, cor-rection of the hernia was believed to be a surgical emergency, and patients underwent surgery shortly after birth. It is now accepted

1	by progressive cardiorespiratory deterioration. In the past, cor-rection of the hernia was believed to be a surgical emergency, and patients underwent surgery shortly after birth. It is now accepted that the presence of persistent pulmonary hyperten-sion that results in right-to-left shunting across the open fora-men ovale or the ductus arteriosus, and the degree of pulmonary hypoplasia, are the leading causes of cardiorespiratory insuffi-ciency. Current management therefore is directed toward man-aging the pulmonary hypertension, and minimizing barotrauma while optimizing oxygen delivery. To achieve this goal, infants are placed on mechanical ventilation using relatively low or “gentle” settings that prevent overinflation of the noninvolved lung. Levels of PaCO2 in the range of 50 to 60 mmHg or higher are accepted as long as the pH remains ≥7.25. If these objec-tives cannot be achieved using conventional ventilation, high frequency oscillatory ventilation (HFOV) may be employed to

1	mmHg or higher are accepted as long as the pH remains ≥7.25. If these objec-tives cannot be achieved using conventional ventilation, high frequency oscillatory ventilation (HFOV) may be employed to avoid the injurious effects of conventional tidal volume venti-lation. Echocardiography will assess the degree of pulmonary hypertension and identify the presence of any coexisting cardiac anomaly. ICU goals include minimal sedation, meticulous atten-tion to endotracheal tube secretions, and gradual changes to ven-tilator settings to avoid inducing pulmonary hypertension via hypoxia. To minimize the degree of pulmonary hypertension, inhaled nitric oxide may be administered, and in some patients, this improves pulmonary perfusion. Nitric oxide is administered into the ventilation circuit and is used in concentrations up to 40 parts per million. Correction of acidosis using bicarbonate solution may minimize the degree of pulmonary hypertension. As the degree of pulmonary hypertension becomes

1	used in concentrations up to 40 parts per million. Correction of acidosis using bicarbonate solution may minimize the degree of pulmonary hypertension. As the degree of pulmonary hypertension becomes hemody-namically significant, right-sided heart failure develops, and systemic perfusion is impaired. Administration of excess IV fluid will compound the degree of cardiac failure and lead to marked peripheral edema. Inotropic support using epinephrine, dopamine, and milrinone alone or in combination may be useful in optimizing cardiac contractility and maintaining mean arterial pressure.Infants with CDH who remain severely hypoxic despite maximal ventilatory care may be candidates for treatment of their respiratory failure ECMO, with access via venovenous (VV) or venoarterial (VA) routes. VV bypass is established with a single cannula through the right internal jugular vein, with blood removed from and infused into the right atrium by separate ports. VA bypass provides additional cardiac

1	bypass is established with a single cannula through the right internal jugular vein, with blood removed from and infused into the right atrium by separate ports. VA bypass provides additional cardiac support, whereas VV bypass requires a well-functioning heart and relies on the lungs for some oxygenation as well. In VA ECMO, the right atrium is cannulated by means of the internal jugular vein and the aortic arch through the right common carotid artery. As much of the cardiac output is directed through the membrane oxygenator as is necessary to provide oxygenated blood to the infant and remove carbon dioxide. The infant is maintained on bypass until the pulmonary hypertension is resolved and lung function, as measured by compliance and the ability to oxy-genate and ventilate, is improved. This is usually seen within 7 to 10 days, but in some infants, it may take up several weeks to occur. Complications associated with ECMO increase after 14 days and include cannula malposition,

1	This is usually seen within 7 to 10 days, but in some infants, it may take up several weeks to occur. Complications associated with ECMO increase after 14 days and include cannula malposition, bleeding in multiple locations, and infection. The use of ECMO is associated with significant risk. Because patients require systemic anticoagu-lation, bleeding complications are the most significant. They may occur intracranially or at the site of cannula insertion, and they can be life-threatening. Systemic sepsis is a significant problem and may necessitate decannulation. Criteria for plac-ing infants on ECMO include the presence of normal cardiac anatomy by echocardiography, the absence of fatal chromosome anomalies, and the expectation that the infant would die with-out ECMO. Traditionally, a threshold of weight greater than 2 kg and gestational age greater than 34 weeks has been applied, although success has been achieved at weights as low as 1.8 kg. Upon decannulation, some centers

1	a threshold of weight greater than 2 kg and gestational age greater than 34 weeks has been applied, although success has been achieved at weights as low as 1.8 kg. Upon decannulation, some centers repair the carotid artery. In instances in which the child is cannulated for a brief period (5 days or less) this may be feasible. A recent study failed to show any benefit from repairing the carotid artery, although this finding remains to be studied further.A strategy that does not involve the use of ECMO but instead emphasizes the use of permissive hypercapnia and the avoidance of barotrauma may provide equal overall outcome in patients with CDH. This likely reflects the fact that mortality is related to the degree of pulmonary hypoplasia and the pres-ence of congenital anomalies, neither of which are correctable by ECMO.Brunicardi_Ch39_p1705-p1758.indd 171312/02/19 11:26 AM 1714SPECIFIC CONSIDERATIONSPART IIFigure 39-4. Congenital lobar emphysema of the left upper lobe in a

1	of which are correctable by ECMO.Brunicardi_Ch39_p1705-p1758.indd 171312/02/19 11:26 AM 1714SPECIFIC CONSIDERATIONSPART IIFigure 39-4. Congenital lobar emphysema of the left upper lobe in a 2-week-old boy. Mediastinal shift is present.The timing of diaphragmatic hernia repair still varies from center to center, particularly when the infant is on ECMO. In patients that are not on ECMO, repair should be performed once the hemodynamic status has been optimized. In neonates that are on ECMO, some surgeons perform early repair on bypass; oth-ers wait until the infant’s lungs are improved and the pulmonary hypertension has subsided and then repair the diaphragm and discontinue bypass within hours of surgery. Still others repair the diaphragm only after the infant is off bypass. Operative repair of the diaphragmatic hernia may be accomplished either by an abdominal or transthoracic approach and can be performed either via open or minimally invasive techniques. Through a subcostal

1	repair of the diaphragmatic hernia may be accomplished either by an abdominal or transthoracic approach and can be performed either via open or minimally invasive techniques. Through a subcostal incision the abdominal viscera are withdrawn from the chest, exposing the defect in the diaphragm. Care must be taken when reducing the spleen and liver, as bleeding from these structures can be fatal. The anterior margin is often apparent, while the posterior muscular rim is attenuated. If the infant is heparinized on bypass, minimal dissection of the muscular margins is per-formed. Electrocautery is used liberally to minimize postopera-tive bleeding. Most infants who require ECMO support prior to hernia repair have large defects, often lacking the medial and posterior margins. About three-fourths of infants repaired on bypass require prosthetic material to patch the defect, suturing it to the diaphragmatic remnant or around ribs or costal cartilages for the large defects. If there is

1	of infants repaired on bypass require prosthetic material to patch the defect, suturing it to the diaphragmatic remnant or around ribs or costal cartilages for the large defects. If there is adequate muscle for closure, a single layer of nonabsorbable horizontal mattress suture, pled-geted or not, closes the defect. Just before the repair is complete, a chest tube may be positioned in the thoracic cavity but is not mandatory. Patients repaired on ECMO are at risk for develop-ing a hemothorax, which can significantly impair ventilation. Anatomic closure of the abdominal wall may be impossible after reduction of the viscera. Occasionally, a prosthetic patch or acellular material may be sutured to the fascia to facilitate closure. The patch can be removed at a later time, and the ventral hernia can be closed at that time or subsequently. In patients who are deemed to be candidates for a minimally invasive approach (stable patients, >2 kg, no pulmonary hypertension), a thoraco-scopic

1	hernia can be closed at that time or subsequently. In patients who are deemed to be candidates for a minimally invasive approach (stable patients, >2 kg, no pulmonary hypertension), a thoraco-scopic repair may be safely performed although concerns have been raised about possible effects of the longer operative time for thoracoscopic repair and higher recurrence rates. If the dia-phragm has been repaired on ECMO, weaning and decannulation are accomplished as soon as possible. All infants are ventilated postoperatively to maintain preductal arterial oxygenation of 80 to 100 torr. Very slow weaning from the ventilator is necessary to avoid recurrent pulmonary hypertension.Fetal tracheal occlusion is an experimental prenatal ther-apy for the treatment of severe congenital diaphragmatic hernia that reverses lung hypoplasia. The rationale for this approach is that the occlusion of the fetal trachea leads to net accumula-tion of lung liquid under pressure, which results in the develop-ment

1	that reverses lung hypoplasia. The rationale for this approach is that the occlusion of the fetal trachea leads to net accumula-tion of lung liquid under pressure, which results in the develop-ment of large fluid-filled lungs. The balloon may be placed into the trachea under laparoscopic guidance, then removed prior to delivery when maximal lung growth has been achieved. The use of fetal tracheal occlusion remains investigational, although early reports are promising.Congenital Lobar EmphysemaCongenital lobar emphysema (CLE) is a condition manifested during the first few months of life as a progressive hyperexpan-sion of one or more lobes of the lung. It can be life-threatening in the newborn period if extensive lung tissue is involved, but in the older infant and in cases in which the lesion is less severely distended it causes less respiratory distress. Air entering during inspiration is trapped in the lobe; on expiration, the lobe can-not deflate and progressively overexpands,

1	lesion is less severely distended it causes less respiratory distress. Air entering during inspiration is trapped in the lobe; on expiration, the lobe can-not deflate and progressively overexpands, causing atelectasis of the adjacent lobe or lobes. This hyperexpansion eventually shifts the mediastinum to the opposite side and compromises the other lung. CLE usually occurs in the upper lobes of the lung (left greater than right), followed next in frequency by the right middle lobe, but it also can occur in the lower lobes. It is caused by intrinsic bronchial obstruction from poor bronchial cartilage development or extrinsic compression. Approximately 14% of children with this condition have cardiac defects, with an enlarged left atrium or a major vessel causing compression of the ipsilateral bronchus.Symptoms range from mild respiratory distress to full-fledged respiratory failure with tachypnea, dyspnea, cough, and late cyanosis. These symptoms may be stationary or they may progress

1	bronchus.Symptoms range from mild respiratory distress to full-fledged respiratory failure with tachypnea, dyspnea, cough, and late cyanosis. These symptoms may be stationary or they may progress rapidly or result in recurrent pneumonia. Occasionally, infants with CLE present with failure to thrive, which likely reflects the increased work associated with the overexpanded lung. A hyperexpanded hemithorax on the ipsilateral side is pathogneumonic for CLE. Diagnosis is typically confirmed by chest X-ray that shows a hyperlucent affected lobe with adja-cent lobar compression and atelectasis. The mediastinum may be shifted as a consequence of mass effect to the contralateral side causing compression and atelectasis of the contralateral lung (Fig. 39-4). Although chest radiograph is usually sufficient, it is sometimes important to obtain at CT scan of the chest to clearly establish the diagnosis of CLE. This should be done only in the stable patient. Unless foreign body or mucous plugging

1	it is sometimes important to obtain at CT scan of the chest to clearly establish the diagnosis of CLE. This should be done only in the stable patient. Unless foreign body or mucous plugging is suspected as a cause of hyperinflation, bronchoscopy is not advisable because it can lead to more air trapping and cause life-threatening respiratory distress in a stable infant. Treatment is resection of the affected lobe, which can be safely performed using either an open or thoracoscopic approach. Unless symp-toms necessitate earlier surgery, resection can usually be per-formed after the infant is several months of age. The prognosis is excellent.Brunicardi_Ch39_p1705-p1758.indd 171412/02/19 11:26 AM 1715PEDIATRIC SURGERYCHAPTER 39Figure 39-5. Computed tomography scan of the chest showing a congenital cystic adenomatoid malformation of the left lower lobe.Figure 39-6. Intraoperative photograph showing left lower lobe congenital cystic adenomatoid malformation seen in Fig.

1	showing a congenital cystic adenomatoid malformation of the left lower lobe.Figure 39-6. Intraoperative photograph showing left lower lobe congenital cystic adenomatoid malformation seen in Fig. 39-5.Bronchopulmonary Foregut MalformationsBronchopulmonary foregut malformations include foregut duplication cysts, congenital pulmonary airway malformations, and pulmonary sequestrations as discussed in the following sections.Congenital Pulmonary Airway Malformations. Previ-ously denoted as congenital cystic adenomatous malformation, (CCAM), congenital pulmonary airway malformations (CPAM) exhibits cystic proliferation of the terminal airway, producing cysts lined by mucus-producing respiratory epithelium, and elastic tissue in the cyst walls without cartilage formation. There may be a single cyst with a wall of connective tissue contain-ing smooth muscle. Cysts may be large and multiple (type I), smaller and more numerous (type II), or they may resemble fetal lung without macroscopic cysts

1	with a wall of connective tissue contain-ing smooth muscle. Cysts may be large and multiple (type I), smaller and more numerous (type II), or they may resemble fetal lung without macroscopic cysts (type III). CPAMs frequently occur in the left lower lobe. However, this lesion can occur in any location and may occur in more than one lobe on more than one side, although this is rare. Clinical symptoms range from none to severe respiratory failure at birth. Over time, these mal-formations can be subject to repeated infections and produce fever and cough in older infants and children. The diagnosis is usually confirmed by CT for surgical planning and charac-teristic features that might delineate other bronchopulmonary foregut malformations (Fig. 39-5). Prenatal US may suggest the diagnosis. Resection is curative and may need to be performed urgently in the infant with severe respiratory distress. Long term, there is a risk of malignant degeneration in unresected CPAMs, but this risk

1	Resection is curative and may need to be performed urgently in the infant with severe respiratory distress. Long term, there is a risk of malignant degeneration in unresected CPAMs, but this risk occurs over decades and has not been fully defined. As a result, resection of the affected lobe is usually per-formed (Fig. 39-6). Antenatal resection may be rarely indicated in those instances in which fetal development is complicated by hydrops as a result of the mechanical and vascular effects of the lung lesion.Pulmonary Sequestration. Pulmonary sequestration is uncommon and consists of a mass of lung tissue, usually in the left lower chest, occurring without the usual connections to the pulmonary artery or tracheobronchial tree, yet with a systemic blood supply from the aorta. There are two kinds of sequestra-tion. Extralobar sequestration is usually a small area of nonaer-ated lung separated from the main lung mass, with a systemic blood supply, located immediately above the left

1	two kinds of sequestra-tion. Extralobar sequestration is usually a small area of nonaer-ated lung separated from the main lung mass, with a systemic blood supply, located immediately above the left diaphragm. It is commonly found in cases of CDH. Intralobar sequestration more commonly occurs within the parenchyma of the left lower lobe but can occur on the right. There is no major connection to the tracheobronchial tree, but a secondary connection may be established, perhaps through infection or via adjacent intra-pulmonary shunts. The blood supply frequently originates from the aorta below the diaphragm; multiple vessels may be present (Fig. 39-7). Venous drainage of both types can be systemic or pulmonary. The cause of sequestration is unknown but most probably involves an abnormal budding of the developing lung that picks up a systemic blood supply and never becomes con-nected with the bronchus or pulmonary vessels. Sequestrations may, in some cases, exhibit mixed pathology with

1	of the developing lung that picks up a systemic blood supply and never becomes con-nected with the bronchus or pulmonary vessels. Sequestrations may, in some cases, exhibit mixed pathology with components consistent with CCAMs. Extralobar sequestration is asymptom-atic and is usually discovered incidentally on chest X-ray. If the diagnosis can be confirmed, e.g., by CT scan, resection is not necessary. Diagnosis of intralobar sequestration may be made prenatally and confirmed on postnatal CT scan. Alternatively, the diagnosis of intralobar sequestration may be established after repeated infections manifested by cough, fever, and con-solidation in the posterior basal segment of the left lower lobe. Increasingly the diagnosis is being made in the early months of life by US, and color Doppler often can be helpful in delin-eating the systemic arterial supply. Removal of the entire left lower lobe is usually necessary since the diagnosis often is made late after multiple infections.

1	Doppler often can be helpful in delin-eating the systemic arterial supply. Removal of the entire left lower lobe is usually necessary since the diagnosis often is made late after multiple infections. Occasionally segmental resection Figure 39-7. Arteriogram showing large systemic artery supply to intralobar sequestration of the left lower lobe.Brunicardi_Ch39_p1705-p1758.indd 171512/02/19 11:26 AM 1716SPECIFIC CONSIDERATIONSPART IIof the sequestered part of the lung can be performed using an open, or ideally, a thoracoscopic approach. If an open approach is used, it is important to open the chest through a low inter-costal space (sixth or seventh) to gain access to the vascular attachments to the aorta. These attachments may insert into the aorta below the diaphragm; in these cases, division of the ves-sels as they traverse the thoracic cavity is essential. Prognosis is generally excellent. However, failure to obtain adequate control of these vessels may result in their retraction

1	of the ves-sels as they traverse the thoracic cavity is essential. Prognosis is generally excellent. However, failure to obtain adequate control of these vessels may result in their retraction into the abdomen and result in uncontrollable hemorrhage. It is also possible to perform a combined thoracoscopic and open approach, wherein the vessels are clipped and divided thoracoscopically and then the lesion safely removed through a limited thoracotomy.Bronchogenic Cyst. Bronchogenic cysts are duplication cysts originating from the airway, regardless of the identity of the lining epithelial identity. They can occur anywhere along the respiratory tract and can present at any age, although typically they present after accumulation of intraluminal contents and not within the newborn period. Histologically, they are hamartoma-tous and usually consist of a single cyst lined with an epithe-lium; the mesenchyme contains cartilage and smooth muscle. They are probably embryonic rests of foregut

1	they are hamartoma-tous and usually consist of a single cyst lined with an epithe-lium; the mesenchyme contains cartilage and smooth muscle. They are probably embryonic rests of foregut origin that have been pinched off from the main portion of the developing tra-cheobronchial tree and are closely associated in causation with other foregut duplication cysts such as those arising from the esophagus. Bronchogenic cysts may be seen on prenatal US but are discovered most often incidentally on postnatal chest X-ray. Although they may be completely asymptomatic, bronchogenic cysts may produce symptoms, usually compressive, depending on the anatomic location and size, which increases over time if there is no egress for building luminal contents. In the para-tracheal region of the neck they can produce airway compres-sion and respiratory distress. In the lung parenchyma, they may become infected and present with fever and cough. In addition, they may cause obstruction of the bronchial lumen

1	produce airway compres-sion and respiratory distress. In the lung parenchyma, they may become infected and present with fever and cough. In addition, they may cause obstruction of the bronchial lumen with distal atelectasis and infection, or they may cause mediastinal com-pression. Rarely, rupture of the cyst can occur. Chest X-ray usu-ally shows a dense mass, and CT scan or MRI delineates the precise anatomic location of the lesion. Treatment consists of resection of the cyst, which may need to be undertaken in emer-gency circumstances for airway or cardiac compression. Resec-tion can be performed either as an open procedure, or more commonly using a thoracoscopic approach. If resection of a common wall will result in injury to the airway, resection of the inner epithelial cyst lining after marsupialization is acceptable.BronchiectasisBronchiectasis is an abnormal and irreversible dilatation of the bronchi and bronchioles associated with chronic suppura-tive disease of the airways.

1	marsupialization is acceptable.BronchiectasisBronchiectasis is an abnormal and irreversible dilatation of the bronchi and bronchioles associated with chronic suppura-tive disease of the airways. Usually patients have an underlying congenital pulmonary anomaly, cystic fibrosis, or immunologic deficiency. Bronchiectasis can also result from chronic infection secondary to a neglected bronchial foreign body. The symptoms include a chronic cough, often productive of purulent secretions, recurrent pulmonary infection, and hemoptysis. The diagnosis is suggested by a chest X-ray that shows increased bronchovas-cular markings in the affected lobe. Chest CT delineates bron-chiectasis with excellent resolution. The preferred treatment for bronchiectasis is medical, consisting of antibiotics, postural drainage, and bronchodilator therapy because many children with the disease show signs of airflow obstruction and bron-chial hyperresponsiveness. Lobectomy or segmental resection is indicated for

1	drainage, and bronchodilator therapy because many children with the disease show signs of airflow obstruction and bron-chial hyperresponsiveness. Lobectomy or segmental resection is indicated for localized disease that has not responded appro-priately to medical therapy. In severe cases, lung transplantation may be required to replace the terminally damaged, septic lung.Foreign BodiesThe inherent curiosity of children and their innate propensity to place new objects into their mouths to fully explore them place them at great risk for aspiration. Aspirated objects can be found either in the airway or in the esophagus; in both cases the results can be life-threatening.Airway Ingestion. Aspiration of foreign bodies most com-monly occurs in the toddler age group. Peanuts are the most common object that is aspirated, although other materials (pop-corn, for instance) may also be involved. A solid foreign body often will cause air trapping, with hyperlucency of the affected lobe or lung seen

1	that is aspirated, although other materials (pop-corn, for instance) may also be involved. A solid foreign body often will cause air trapping, with hyperlucency of the affected lobe or lung seen especially on expiration. Oil from the peanut is very irritating and may cause pneumonia. Delay in diagnosis can lead to atelectasis and infection. The most common ana-tomic location for a foreign body is the right main stem bronchus or the right lower lobe. The child usually will cough or choke while eating but may then become asymptomatic. Total respira-tory obstruction with tracheal foreign body may occur; however, respiratory distress is usually mild if present at all. A unilateral wheeze is often heard on auscultation. This wheeze often leads to an inappropriate diagnosis of “asthma” and may delay the correct diagnosis for some time. Chest X-ray will show a radi-opaque foreign body, but in the case of nuts, seeds, or plastic toy parts, the only clue may be hyperexpansion of the affected

1	delay the correct diagnosis for some time. Chest X-ray will show a radi-opaque foreign body, but in the case of nuts, seeds, or plastic toy parts, the only clue may be hyperexpansion of the affected lobe on an expiratory film or fluoroscopy. Bronchoscopy confirms the diagnosis and allows removal of the foreign body. It can be a very simple procedure or it may be extremely difficult, espe-cially with a smooth foreign body that cannot be grasped easily or one that has been retained for some time. The rigid broncho-scope should be used in all cases, and utilization of the optical forceps facilitates grasping the inhaled object. Epinephrine may be injected into the mucosa when the object has been present for a long period of time, which minimizes bleeding. Bronchiectasis may be seen as an extremely late phenomenon after repeated infections of the poorly aerated lung and may require partial or total resection of the affected lobe. The differential diagnosis of a bronchial foreign body

1	extremely late phenomenon after repeated infections of the poorly aerated lung and may require partial or total resection of the affected lobe. The differential diagnosis of a bronchial foreign body includes an intraluminal tumor (i.e., carcinoid, hemangioma, or neurofibroma).Foreign Bodies and Esophageal Injury. The most common foreign body in the esophagus is a coin, followed by small toy parts. Toddlers are most commonly affected. The coin is retained in the esophagus at one of three locations: the cricopharyngeus, the area of the aortic arch, or the gastroesophageal junction, all of which are areas of normal anatomic narrowing. Symptoms are variable depending on the anatomic position of the foreign body and the degree of obstruction. There is often a relatively asymptomatic period after ingestion. The initial symptoms are gastrointestinal, and include dysphagia, drooling, and dehydra-tion. The longer the foreign body remains in the esophagus with oral secretions unable to transit

1	ingestion. The initial symptoms are gastrointestinal, and include dysphagia, drooling, and dehydra-tion. The longer the foreign body remains in the esophagus with oral secretions unable to transit the esophagus, the greater the incidence of respiratory symptoms including cough, stridor, and wheezing. These findings may be interpreted as signs of upper respiratory infections. Objects that are present for a long period of time—particularly in children who have underlying neurological impairment—may manifest as chronic dysphagia. The chest X-ray is diagnostic in the case of a coin. A contrast swallow, or preferably an esophagoscopy, may be required for nonradiopaque foreign bodies. Coins lodged within the upper Brunicardi_Ch39_p1705-p1758.indd 171612/02/19 11:26 AM 1717PEDIATRIC SURGERYCHAPTER 39Figure 39-8. The five varieties of esophageal atresia and tracheoesophageal fistula. A. Isolated esophageal atresia. B. Esophageal atresia with tracheoesophageal fistula between proximal

1	39Figure 39-8. The five varieties of esophageal atresia and tracheoesophageal fistula. A. Isolated esophageal atresia. B. Esophageal atresia with tracheoesophageal fistula between proximal segment of esophagus and trachea. C. Esophageal atresia with tracheoesophageal fistula between distal esophagus and trachea. D. Esophageal atresia with fistula between both proximal and distal ends of esophagus and trachea. E. Tracheoesophageal fistula without esophageal atresia (H-type fistula).esophagus for less than 24 hours may be removed using Magill forceps during direct laryngoscopy. For all other situations, the treatment is by esophagoscopy, rigid or flexible, and removal of the foreign body. In the case of sharp foreign bodies such as open safety pins, extreme care is required on extraction to avoid injury to the esophagus. Rarely, esophagotomy is required for removal, particularly of sharp objects. Diligent follow-up is required after removal of foreign bodies, especially batteries,

1	to avoid injury to the esophagus. Rarely, esophagotomy is required for removal, particularly of sharp objects. Diligent follow-up is required after removal of foreign bodies, especially batteries, which can cause strictures, and sharp objects, which can injure the underlying esophagus. In the case of a retained battery, this case should be handled as a surgical emergency, as the negative pole of the battery directly damages the surrounding tissue, and tracheoesophageal fistula, aortic exsanguination, and mediasti-nitis have all been described after local tissue necrosis at the site where the battery has lodged.ESOPHAGUSEsophageal Atresia and Tracheoesophageal FistulaThe management of esophageal atresia (EA) and tracheoesopha-geal fistula (TEF) is one of the most gratifying pediatric sur-gical conditions to treat. In the not so distant past, nearly all infants born with EA and TEF died. In 1939 Ladd and Leven achieved the first success repair by ligating the fistula, placing a

1	sur-gical conditions to treat. In the not so distant past, nearly all infants born with EA and TEF died. In 1939 Ladd and Leven achieved the first success repair by ligating the fistula, placing a gastrostomy, and reconstructing the esophagus at a later time. Subsequently, Dr. Cameron Haight, in Ann Arbor, Michigan, performed the first successful primary anastomosis for esopha-geal atresia, which remains the current approach for treatment of this condition. Despite the fact that there are several com-mon varieties of this anomaly and the underlying cause remains obscure, a careful approach consisting of meticulous periopera-tive care and attention to the technical detail of the operation can result in an excellent prognosis in most cases.Anatomic Varieties. The five major varieties of EA and TEF are shown in Fig. 39-8. The most commonly seen variety is esophageal atresia with distal tracheoesophageal fistula (type C), which occurs in approximately 85% of the cases in most series. The

1	TEF are shown in Fig. 39-8. The most commonly seen variety is esophageal atresia with distal tracheoesophageal fistula (type C), which occurs in approximately 85% of the cases in most series. The next most frequent is pure esophageal atresia (type A), occurring in 8% to 10% of patients, followed by tracheoesophageal fistula without esophageal atresia (type E). This occurs in 8% of cases and is also referred to as an H-type fistula, based upon the anatomic similarity to that letter Figure 39-9. Barium esophagram showing H-type tracheoesophageal fistula (arrow).(Fig. 39-9). Esophageal atresia with fistula between both proximal and distal ends of the esophagus and trachea (type D) is seen in approximately 2% of cases, and type B, esophageal atresia with tracheoesophageal fistula between distal esophagus and trachea, is seen in approximately 1% of all cases.Etiology and Pathologic Presentation. The esophagus and trachea share a common embryologic origin. At approximately 4 weeks’

1	distal esophagus and trachea, is seen in approximately 1% of all cases.Etiology and Pathologic Presentation. The esophagus and trachea share a common embryologic origin. At approximately 4 weeks’ gestation, a diverticulum forms off the anterior aspect of the proximal foregut in the region of the primitive pharynx. This diverticulum extends caudally with progressive formation of the laryngo-tracheal groove, thus, creating a separate trachea and esophagus. Successful development of these structures is the consequence of extremely intricate interplay of growth and transcription factors necessary for rostral-caudal and anterior-posterior specification. The variations in clinically observed EA and TEF that must result in failure of successful formation of these structures are depicted in Fig. 39-8. While definitive genetic mutations have been difficult to identify in isolated EA-TEF, mutations in N-myc, Sox2, and CHD7 have been character-ized in syndromic EA-TEF with associated

1	in Fig. 39-8. While definitive genetic mutations have been difficult to identify in isolated EA-TEF, mutations in N-myc, Sox2, and CHD7 have been character-ized in syndromic EA-TEF with associated anomalies.Other congenital anomalies commonly occur in asso-ciation with EA-TEF. For instance, VACTERRL syndrome is associated with vertebral anomalies (absent vertebrae or hemi-vertebrae) and anorectal anomalies (imperforate anus), cardiac Brunicardi_Ch39_p1705-p1758.indd 171712/02/19 11:26 AM 1718SPECIFIC CONSIDERATIONSPART IIFigure 39-10. Type C esophageal atresia with tracheoesophageal fistula. Note the catheter that is coiled in the upper pouch and the presence of gas below the diaphragm, which confirms the presence of the tracheoesophageal fistula.defects, tracheoesophageal fistula, renal anomalies (renal agen-esis, renal anomalies), and radial limb hyperplasia. In nearly 20% of the infants born with esophageal atresia, some variant of congenital heart disease occurs.Clinical

1	renal anomalies (renal agen-esis, renal anomalies), and radial limb hyperplasia. In nearly 20% of the infants born with esophageal atresia, some variant of congenital heart disease occurs.Clinical Presentation of Infants With Esophageal Atresia and Tracheoesophageal Fistula. The anatomic variant of infants with EA-TEF predicts the clinical presentation. When the esophagus ends either as a blind pouch or as a fistula into the trachea (as in types A, B, C, or D), infants present with exces-sive drooling, followed by choking or coughing immediately after feeding is initiated as a result of aspiration through the fistula tract. As the neonate coughs and cries, air is transmitted through the fistula into the stomach, resulting in abdominal dis-tention. As the abdomen distends, it becomes increasingly more difficult for the infant to breathe. This leads to further atelecta-sis, which compounds the pulmonary dysfunction. In patients with type C and D varieties, the regurgitated gastric juice

1	more difficult for the infant to breathe. This leads to further atelecta-sis, which compounds the pulmonary dysfunction. In patients with type C and D varieties, the regurgitated gastric juice passes through the fistula where it collects in the trachea and lungs and leads to a chemical pneumonitis, which further exacerbates the pulmonary status. In many instances, the diagnosis is actually made by the nursing staff who attempt to feed the baby and notice the accumulation of oral secretions.The diagnosis of esophageal atresia is confirmed by the inability to pass an orogastric tube into the stomach (Fig. 39-10). The dilated upper pouch may be occasionally seen on a plain chest radiograph. If a soft feeding tube is used, the tube will coil in the upper pouch, which provides further diagnostic cer-tainty. An important alternative diagnosis that must be consid-ered when an orogastric tube does not enter the stomach is that of an esophageal perforation. This problem can occur in infants

1	cer-tainty. An important alternative diagnosis that must be consid-ered when an orogastric tube does not enter the stomach is that of an esophageal perforation. This problem can occur in infants after traumatic insertion of a nasogastric or orogastric tube. In this instance, the perforation classically occurs at the level of the piriform sinus, and a false passage is created, which prevents the tube from entering the stomach. Whenever there is any diag-nostic uncertainty, a contrast study will confirm the diagnosis of EA and occasionally document the TEF. The presence of a tracheoesophageal fistula can be demonstrated clinically by finding air in the gastrointestinal tract. This can be proven at the bedside by percussion of the abdomen and confirmed by obtain-ing a plain abdominal radiograph. Occasionally, a diagnosis of EA-TEF can be suspected prenatally on US evaluation. Typical features include failure to visualize the stomach and the pres-ence of polyhydramnios. These findings

1	Occasionally, a diagnosis of EA-TEF can be suspected prenatally on US evaluation. Typical features include failure to visualize the stomach and the pres-ence of polyhydramnios. These findings reflect the absence of efficient swallowing by the fetus.In a child with esophageal atresia, it is important to iden-tify whether coexisting anomalies are present. These include cardiac defects in 38%, skeletal defects in 19%, neurologi-cal defects in 15%, renal defects in 15%, anorectal defects in 8%, and other abnormalities in 13%. Examination of the heart and great vessels with echocardiography is important to exclude cardiac defects, as these are often the most important predictors of survival in these infants. The echocardiogram also demonstrates whether the aortic arch is left sided or right sided, which may influence the approach to surgical repair. Vertebral anomalies are assessed by plain radiography, and a spinal US is obtained if any are detected. A patent anus should be confirmed

1	sided, which may influence the approach to surgical repair. Vertebral anomalies are assessed by plain radiography, and a spinal US is obtained if any are detected. A patent anus should be confirmed clinically. The kidneys in a newborn may be assessed clinically by palpation. A US of the abdomen will demonstrate the presence of renal anomalies, which should be suspected in the child who fails to make urine. The presence of extremity anomalies is suspected when there are missing digits and confirmed by plain radiographs of the hands, feet, forearms, and legs. Rib anomalies may also be present. These may include the presence of a 13th rib.Initial Management. The initial treatment of infants with EA-TEF includes attention to the respiratory status, decompression of the upper pouch, and appropriate timing of surgery. Because the major determinant of poor survival is the presence of other severe anomalies, a search for other defects including congeni-tal cardiac disease is undertaken in a

1	timing of surgery. Because the major determinant of poor survival is the presence of other severe anomalies, a search for other defects including congeni-tal cardiac disease is undertaken in a timely fashion. The initial strategy after the diagnosis is confirmed is to place the neonate in an infant warmer with the head elevated at least 30°. A sump catheter is placed in the upper pouch on continuous suction. Both of these strategies are designed to minimize the degree of aspiration from the esophageal pouch. When saliva accumulates in the upper pouch and is aspirated into the lungs, coughing, bronchospasm, and desaturation episodes can occur, which may be minimized by ensuring the patency of the sump catheter. IV antibiotic therapy is initiated, and warmed electrolyte solu-tion is administered. Where possible, the right upper extremity is avoided as a site to start an IV line, as this location may interfere with positioning of the patient during the surgical repair. Some surgeons

1	Where possible, the right upper extremity is avoided as a site to start an IV line, as this location may interfere with positioning of the patient during the surgical repair. Some surgeons place a central line in all patients to facilitate the admin-istration of antibiotics and total parenteral nutrition as needed.The timing of repair is influenced by the stability of the patient. Definitive repair of the EA-TEF is rarely a surgical emergency. If the child is hemodynamically stable and is oxy-genating well, definitive repair may be performed within 1 to 2 days after birth. This allows for a careful determination of the presence of coexisting anomalies and for selection of an expe-rienced anesthetic team.Management of Esophageal Atresia and Tracheoesopha-geal Fistula in the Preterm Infant. The ventilated, prema-ture neonate with EA-TEF and associated hyaline membrane disease represents a patient who may develop severe, progres-sive, cardiopulmonary dysfunction. The tracheoesophageal

1	ventilated, prema-ture neonate with EA-TEF and associated hyaline membrane disease represents a patient who may develop severe, progres-sive, cardiopulmonary dysfunction. The tracheoesophageal fis-tula can worsen the fragile pulmonary status as a result of recurrent aspiration through the fistula, and as a result of increased abdominal distention, which impairs lung expansion. Moreover, the elevated airway pressure that is required to ven-tilate these patients can worsen the clinical course by forcing air through the fistula into the stomach, thereby exacerbating the Brunicardi_Ch39_p1705-p1758.indd 171812/02/19 11:26 AM 1719PEDIATRIC SURGERYCHAPTER 39ABCEDAzygos VeinEsophagusEsophagusAzygos VeinFigure 39-11. Primary repair of type C tracheosophageal fistula. A. Right thoracotomy incision. B. Azygous vein transected, proximal and distal esophagus demonstrated, and fistula identified. C. Tracheoesophageal fistula transected and defect in trachea closed. D. End-to-end anastomosis

1	B. Azygous vein transected, proximal and distal esophagus demonstrated, and fistula identified. C. Tracheoesophageal fistula transected and defect in trachea closed. D. End-to-end anastomosis between proximal and distal esophagus (posterior row). E. Completed anastomosis.degree of abdominal distention and compromising lung expan-sion. In this situation, the first priority is to minimize the degree of positive pressure needed to adequately ventilate the child. This can be accomplished using high frequency oscil-latory ventilation (HFOV). If the gastric distention becomes severe, a gastrostomy tube should be placed. This procedure can be performed at the bedside under local anesthetic, if necessary. The dilated, air-filled stomach can easily be accessed through an incision in the left-upper quadrant of the abdomen. Once the gastrostomy tube is placed and the abdominal pressure is relieved, the pulmonary status can paradoxically worsen. This is because the ventilated gas may pass

1	quadrant of the abdomen. Once the gastrostomy tube is placed and the abdominal pressure is relieved, the pulmonary status can paradoxically worsen. This is because the ventilated gas may pass preferentially through the fistula, which is the path of least resistance, and bypass the lungs thereby worsening the hypoxemia. To correct this problem, the gastrostomy tube may be placed under water seal, elevated, or intermittently clamped. If these maneuvers are to no avail, liga-tion of the fistula may be required. This procedure can be per-formed in the neonatal intensive care unit if the infant is too unstable to be transported to the operating room. These inter-ventions allow for the infant’s underlying hyaline membrane disease to improve, for the pulmonary secretions to clear, and for the infant to reach a period of stability so that definitive repair can be performed.Primary Surgical Correction. In a stable infant, definitive repair is achieved through performance of a primary

1	the infant to reach a period of stability so that definitive repair can be performed.Primary Surgical Correction. In a stable infant, definitive repair is achieved through performance of a primary esopha-goesophagostomy. There are two approaches to this operation: 2open thoracotomy or thoracoscopy. In the open approach, the infant is brought to the operating room, intubated, and placed in the lateral decubitus position with the right side up in prepara-tion for right posterolateral thoracotomy. If a right-sided arch was determined previously by echocardiography, consideration is given to performing the repair through the left chest, although most surgeons believe that the repair can be performed safely from the right side as well. Bronchoscopy may be performed to exclude the presence of additional, upper-pouch fistulae in cases of esophageal atresia (i.e., differentiation of types B, C, and D variants) and identification of a laryngeotracheoesopha-geal cleft.The operative technique

1	additional, upper-pouch fistulae in cases of esophageal atresia (i.e., differentiation of types B, C, and D variants) and identification of a laryngeotracheoesopha-geal cleft.The operative technique for primary repair is as follows (Fig. 39-11). A retropleural approach is generally used as this technique prevents widespread contamination of the thorax if a postoperative anastomotic leak occurs. The sequence of steps is as follows: (a) mobilization of the pleura to expose the struc-tures in the posterior mediastinum; (b) division of the fistula and closure of the tracheal opening; (c) mobilization of the upper esophagus sufficiently to permit an anastomosis without tension and to determine whether a fistula is present between the upper esophagus and the trachea (forward pressure by the anesthesia staff on the sump drain in the pouch can greatly facilitate dissection at this stage of the operation; care must be taken when dissecting posteriorly to avoid violation of either the lumen of

1	staff on the sump drain in the pouch can greatly facilitate dissection at this stage of the operation; care must be taken when dissecting posteriorly to avoid violation of either the lumen of trachea and esophagus); (d) mobilization of the dis-tal esophagus (this needs to be performed judiciously to avoid Brunicardi_Ch39_p1705-p1758.indd 171912/02/19 11:26 AM 1720SPECIFIC CONSIDERATIONSPART IIdevascularization since the blood supply to the distal esopha-gus is segmental from the aorta; most of the esophageal length is obtained from mobilizing the upper pouch since the blood supply travels via the submucosa from above); (e) performing a primary esophagoesophageal anastomosis (most surgeons perform this procedure in a single layer using 5-0 sutures; if there is excess tension, the muscle of the upper pouch can be circumferentially incised without compromising blood supply to increase its length; many surgeons place a transanastomotic feeding tube in order to institute feeds in the

1	of the upper pouch can be circumferentially incised without compromising blood supply to increase its length; many surgeons place a transanastomotic feeding tube in order to institute feeds in the early postoperative period); and (f) placement of a retropleural drain and closure of the incision in layers.When a minimally invasive approach is selected, the patient is prepared for right-sided, transthoracic thoracoscopic repair. The same steps as described earlier for the open repair are undertaken, and the magnification and superb optics that are provided by the thoracoscopic approach provide for superb visualization. Identification of the fistula is performed as a first step; this can be readily ligated and divided between tho-racoscopically placed sutures. The anastomosis is performed in a single layer. The thoracoscopically performed TEF repair requires clear and ongoing communication between the oper-ating surgeons and the anesthesiologist; visualization can be significantly

1	in a single layer. The thoracoscopically performed TEF repair requires clear and ongoing communication between the oper-ating surgeons and the anesthesiologist; visualization can be significantly reduced with sudden changes in lung inflation, potentially leading to the need to convert to an open repair. Although clear guidelines for patient selection for a thoraco-scopic repair as opposed to an open repair remain lacking, rea-sonable selection criteria include patients over 2.5 kg who are hemodynamically stable and without comorbidities.Postoperative Course. The postoperative management strat-egy of patients with EA-TEF is influenced to a great degree by the preference of the individual surgeon and the institutional culture. Many surgeons prefer not to leave the infants intubated postoperatively to avoid the effects of positive pressure on the site of tracheal closure. However, early extubation may not be possible in babies with preoperative lung disease either from pre-maturity or

1	to avoid the effects of positive pressure on the site of tracheal closure. However, early extubation may not be possible in babies with preoperative lung disease either from pre-maturity or pneumonia or when there is any vocal cord edema. When a transanastomotic tube is placed, feeds are begun slowly in the postoperative period. Some surgeons institute parenteral nutrition for several days, using a central line. The retropleural drain is assessed daily for the presence of saliva, indicating an anastomotic leak. Many surgeons obtain a contrast swallow 1 week after repair to assess the caliber of the anastomosis and to determine whether a leak is present. If there is no leak, feedings are started. The principal benefit of the thoracoscopic approach is that postoperative pain is significantly reduced, as is the requirement for postoperative narcotic analgesia.Complications of Surgery. Anastomotic leak occurs in 10% to 15% of patients and may be seen either in the immediate post-operative

1	reduced, as is the requirement for postoperative narcotic analgesia.Complications of Surgery. Anastomotic leak occurs in 10% to 15% of patients and may be seen either in the immediate post-operative period or after several days. Early leakage (i.e., within the first 24 to 48 hours) is manifested by a new pleural effusion, pneumothorax, and sepsis and requires immediate exploration. In these circumstances, the anastomosis may be completely dis-rupted, possibly due to excessive tension. Revision of the anas-tomosis may be possible. If not, cervical esophagostomy and gastrostomy placement is required, with a subsequent procedure to reestablish esophageal continuity. Anastomotic leakage that is detected after several days usually heals without intervention, particularly if a retropleural approach is used. Under these cir-cumstances, broad spectrum antibiotics, pulmonary toilet, and optimization of nutrition are important. After approximately a week or so, a repeat esophagram should be

1	is used. Under these cir-cumstances, broad spectrum antibiotics, pulmonary toilet, and optimization of nutrition are important. After approximately a week or so, a repeat esophagram should be performed, at which time the leakage may have resolved.Strictures at the anastomosis are not infrequent (10–20%), particularly if a leak has occurred. A stricture may become apparent at any time, from the early postoperative period to months or years later. It may present as choking, gagging, or failure to thrive, but it often becomes clinically apparent with the transition to eating solid food. A contrast swallow or esoph-agoscopy is confirmatory, and simple dilatation is usually cor-rective. Occasionally, repeated dilatations are required. These may be performed in a retrograde fashion, during which a silk suture is placed into the oropharynx and delivered from the esophagus through a gastrostomy tube. Tucker dilators are then tied to the suture and passed in a retrograde fashion from the

1	which a silk suture is placed into the oropharynx and delivered from the esophagus through a gastrostomy tube. Tucker dilators are then tied to the suture and passed in a retrograde fashion from the gastrostomy tube and delivered out of the oropharynx. Increas-ing sizes are used, and the silk is replaced at the end of the pro-cedure where it is taped to the side of the face at one end, and to the gastrostomy tube at the other. Alternatively, image-guided balloon dilation over a guide wire may be performed, using intraoperative contrast radiography to determine the precise location of the stricture and to assess the immediate response to the dilation.“Recurrent” tracheoesophageal fistula may represent a missed upper pouch fistula or a true recurrence. This may occur after an anastomotic disruption, during which the recurrent fis-tula may heal spontaneously. Otherwise, reoperation may be required. Recently, the use of fibrin glue has been successful in treating recurrent fistulas,

1	disruption, during which the recurrent fis-tula may heal spontaneously. Otherwise, reoperation may be required. Recently, the use of fibrin glue has been successful in treating recurrent fistulas, although long-term follow-up is lacking.Gastroesophageal reflux commonly occurs after repair of EA-TEF, potentially due to alterations in esophageal motility and the anatomy of the gastroesophageal junction. The clinical manifestations of such reflux are similar to those seen in other infants with primary gastroesophageal reflux disease (GERD). A loose antireflux procedure, such as a Nissen fundoplication, is used to prevent further reflux, but the child may have feed-ing problems after antireflux surgery as a result of the intrinsic dysmotility of the distal esophagus. The fundoplication may be safely performed laparoscopically in experienced hands, although care should be taken to ensure that the wrap is not excessively tight.Special Circumstances. Patients with type E tracheoesoph-ageal

1	safely performed laparoscopically in experienced hands, although care should be taken to ensure that the wrap is not excessively tight.Special Circumstances. Patients with type E tracheoesoph-ageal fistulas (also called H-type) most commonly present beyond the newborn period. Presenting symptoms include recurrent chest infections, bronchospasm, and failure to thrive. The diagnosis is suspected using barium esophagography and confirmed by endoscopic visualization of the fistula. Surgical correction is generally possible through a cervical approach with concurrent placement of a balloon catheter across the fis-tula and requires mobilization and division of the fistula. Out-come is usually excellent.Patients with duodenal atresia and EA-TEF may require urgent treatment due to the presence of a closed obstruction of the stomach and proximal duodenum. In stable patients, treat-ment consists of repair of the esophageal anomaly and correc-tion of the duodenal atresia if the infant is stable

1	a closed obstruction of the stomach and proximal duodenum. In stable patients, treat-ment consists of repair of the esophageal anomaly and correc-tion of the duodenal atresia if the infant is stable during surgery. If not, a staged approach should be utilized consisting of ligation of the fistula and placement of a gastrostomy tube. Definitive repair can then be performed at a later point in time.Primary esophageal atresia (type A) represents a chal-lenging problem, particularly if the upper and lower ends are too far apart for an anastomosis to be created. Under these Brunicardi_Ch39_p1705-p1758.indd 172012/02/19 11:26 AM 1721PEDIATRIC SURGERYCHAPTER 39circumstances, treatment strategies include placement of a gas-trostomy tube and performing serial bougienage to increase the length of the upper pouch. This occasionally allows for primary anastomosis to be performed. Occasionally, when the two ends cannot be brought safely together, esophageal replacement is required using either

1	the upper pouch. This occasionally allows for primary anastomosis to be performed. Occasionally, when the two ends cannot be brought safely together, esophageal replacement is required using either a gastric pull-up or colon interposition (see the following section).Outcome. Various classification systems have been utilized to predict survival in patients with EA-TEF and to stratify treat-ment. A system devised by Waterston in 1962 was used to strat-ify neonates based on birth weight, the presence of pneumonia, and the identification of other congenital anomalies. In response to advances in neonatal care, the surgeons from the Montreal Children’s Hospital proposed a new classification system in 1993. In the Montreal experience only two characteristics inde-pendently affected survival: preoperative ventilator dependence and associated major anomalies. Pulmonary disease as defined by ventilator dependence appeared to be more accurate than pneumonia. When the two systems were compared,

1	ventilator dependence and associated major anomalies. Pulmonary disease as defined by ventilator dependence appeared to be more accurate than pneumonia. When the two systems were compared, the Montreal system more accurately identified children at highest risk. Spitz and colleagues analyzed risk factors in infants who died with EA-TEF. Two criteria were found to be important predictors of outcome: birth weight less than 1500 g and the presence of major congenital cardiac disease. A new classification for predicting outcome in esophageal atresia was therefore proposed: group I: birth weight ≥1500 g, without major cardiac disease, survival 97% (283 of 293); group II: birth weight <1500 g, or major car-diac disease, survival 59% (41 of 70); and group III: birth weight <1500 g, and major cardiac disease, survival 22% (2 of 9).In general, surgical correction of EA-TEF leads to a sat-isfactory outcome with nearly normal esophageal function in most patients. Overall survival rates of greater

1	disease, survival 22% (2 of 9).In general, surgical correction of EA-TEF leads to a sat-isfactory outcome with nearly normal esophageal function in most patients. Overall survival rates of greater than 90% have been achieved in patients classified as stable, in all the various staging systems. Unstable infants have an increased mortality (40–60% survival) because of potentially fatal associated cardiac and chromosomal anomalies or prematurity. However, the use of a staged procedure also has increased survival in even these high-risk infants.Corrosive Injury of the EsophagusInjury to the esophagus after ingestion of corrosive substances most commonly occurs in the toddler age group. Both strong alkali and strong acids produce injury by liquefaction or coag-ulation necrosis, and since all corrosive agents are extremely hygroscopic, the caustic substance will cling to the esophageal epithelium. Subsequent strictures occur at the anatomic nar-rowed areas of the esophagus, cricopharyngeus,

1	agents are extremely hygroscopic, the caustic substance will cling to the esophageal epithelium. Subsequent strictures occur at the anatomic nar-rowed areas of the esophagus, cricopharyngeus, midesophagus, and gastroesophageal junction. A child who has swallowed an injurious substance may be symptom-free but usually will be drooling and unable to swallow saliva. The injury may be restricted to the oropharynx and esophagus, or it may extend to include the stomach. There is no effective immediate anti-dote. Diagnosis is by careful physical examination of the mouth and endoscopy with a flexible or a rigid esophagoscope. It is important to endoscope only to the first level of the burn in order to avoid perforation. Early barium swallow may delineate the extent of the mucosal injury. It is important to realize that the esophagus may be burned without evidence of injury to the mouth. Although previously used routinely, steroids have not been shown to alter stricture development or modify

1	to realize that the esophagus may be burned without evidence of injury to the mouth. Although previously used routinely, steroids have not been shown to alter stricture development or modify the extent of injury and are no longer part of the management of caustic injuries. Antibiotics are administered during the acute period.The extent of injury is graded endoscopically as either mild, moderate, or severe (grade I, II, or III). Circumferential esophageal injuries with necrosis have an extremely high like-lihood of stricture formation. These patients should undergo placement of a gastrostomy tube once clinically stable. A string should be inserted through the esophagus either immediately or during repeat esophagoscopy several weeks later. When estab-lished strictures are present (usually 3 to 4 weeks), dilatation is performed. Fluoroscopically guided balloon dilation of the stric-ture is effective, which should be performed in association with esophagoscopy, and allows for a precise

1	3 to 4 weeks), dilatation is performed. Fluoroscopically guided balloon dilation of the stric-ture is effective, which should be performed in association with esophagoscopy, and allows for a precise evaluation of the nature and extent of the stenosis. The procedure should be performed under general anesthesia, and care must be taken to ensure there is no airway injury. Dislodgment of the endotracheal tube can occur during this procedure, and careful communication with the anesthesiologist is critical during the procedure.In certain circumstances, especially if a gastrostomy tube has been placed, retrograde dilatation may be performed, using graduated dilators brought through the gastrostomy and advanced into the esophagus via the transesophageal string. Management of esophageal perforation during dilation should include antibiotics, irrigation, and closed drainage of the tho-racic cavity to prevent systemic sepsis. When recognition is delayed or if the patient is systemically ill,

1	during dilation should include antibiotics, irrigation, and closed drainage of the tho-racic cavity to prevent systemic sepsis. When recognition is delayed or if the patient is systemically ill, esophageal diver-sion may be required with staged reconstruction at a later time.Although the native esophagus can be preserved in most cases, severe stricture formation that does not respond to dila-tion is best managed by esophageal replacement. The most com-monly used options for esophageal substitution are the colon (right colon or transverse/left colon) and the stomach (gastric tubes or gastric pull-up). Pedicled or free grafts of the jejunum are rarely used. The right colon is based on a pedicle of the middle colic artery, and the left colon is based on a pedicle of the middle colic or left colic artery. Gastric tubes are fashioned from the greater curvature of the stomach based on the pedi-cle of the left gastroepiploic artery. When the entire stomach is used, as in gastric pull-up, the

1	artery. Gastric tubes are fashioned from the greater curvature of the stomach based on the pedi-cle of the left gastroepiploic artery. When the entire stomach is used, as in gastric pull-up, the blood supply is provided by the right gastric artery. The neoesophagus may traverse (a) sub-sternally; (b) through a transthoracic route; or (c) through the posterior mediastinum to reach the neck. A feeding jejunostomy is placed at the time of surgery and tube feedings are instituted once the postoperative ileus has resolved. Long-term follow-up has shown that all methods of esophageal substitution can sup-port normal growth and development, and the children enjoy reasonably normal eating habits. Because of the potential for late complications such as ulceration and stricture, follow-up into adulthood is mandatory, but complications appear to dimin-ish with time.Gastroesophageal RefluxGastroesophageal reflux (GER) occurs to some degree in all children and refers to the passage of gastric

1	adulthood is mandatory, but complications appear to dimin-ish with time.Gastroesophageal RefluxGastroesophageal reflux (GER) occurs to some degree in all children and refers to the passage of gastric contents into the esophagus. By contrast, gastroesophageal reflux disease (GERD) describes the situation where reflux is symptomatic. Typical symptoms include failure to thrive, bleeding, stricture formation, reactive airway disease, aspiration pneumonia, or apnea. Failure to thrive and pulmonary problems are particularly common in infants with GERD, whereas strictures and esopha-gitis are more common in older children and adolescents. GERD is particularly problematic in neurologically impaired children.Clinical Manifestations. Because all infants experience occasional episodes of GER to some degree, care must be taken Brunicardi_Ch39_p1705-p1758.indd 172112/02/19 11:26 AM 1722SPECIFIC CONSIDERATIONSPART IIbefore a child is labeled as having pathologic reflux. A history of repeated

1	degree, care must be taken Brunicardi_Ch39_p1705-p1758.indd 172112/02/19 11:26 AM 1722SPECIFIC CONSIDERATIONSPART IIbefore a child is labeled as having pathologic reflux. A history of repeated episodes of vomiting that interferes with growth and development, or the presence of apparent life-threatening events, are required for the diagnosis of GERD. In older chil-dren, esophageal bleeding, stricture formation, severe heartburn, or the development of Barrett’s esophagus unequivocally con-note pathologic reflux or GERD. In neurologically impaired children, vomiting due to GER must be distinguished from chronic retching.The workup of patients suspected of having GERD includes documentation of the episodes of reflux and evalua-tion of the anatomy. A barium swallow should be performed as an initial test. This will determine whether there is obstruction of the stomach or duodenum (due to duodenal webs or pyloric stenosis) and will determine whether malrotation is present. The frequency

1	initial test. This will determine whether there is obstruction of the stomach or duodenum (due to duodenal webs or pyloric stenosis) and will determine whether malrotation is present. The frequency and severity of reflux should be assessed using a 24-hour pH probe study. Although this test is poorly tolerated, it provides the most accurate determination that GERD is present. Esophageal endoscopy with biopsies may identify the presence of esophagitis, and it is useful to determine the length of intra-abdominal esophagus and the presence of Barrett’s esophagus. Some surgeons obtain a radioisotope “milk scan” to evaluate gastric emptying, although there is little evidence to show that this test changes management when a diagnosis of GERD has been confirmed using the aforementioned modalities.Treatment. Most patients with GERD are treated initially by conservative means. In the infant, propping and thickening the formula with rice cereal are generally recommended. Some authors prefer a

1	patients with GERD are treated initially by conservative means. In the infant, propping and thickening the formula with rice cereal are generally recommended. Some authors prefer a prone, head-up position. In the infant unrespon-sive to position and formula changes and the older child with severe GERD, medical therapy is based on gastric acid reduc-tion with an H2-blocking agent and/or a proton pump inhibitor. Medical therapy is successful in most neurologically normal infants and younger children, many of whom will outgrow their need for medications. In certain patients, however, medical treatment does not provide symptomatic relief and surgery is therefore indicated. The least invasive surgical option includes the placement of a nasojejunal or gastrojejunal feeding tube. Because the stomach is bypassed, food contents do not enter the esophagus, and symptoms are often improved. However, as a long-term remedy, this therapy is associated with several problems. The tubes often become

1	is bypassed, food contents do not enter the esophagus, and symptoms are often improved. However, as a long-term remedy, this therapy is associated with several problems. The tubes often become dislodged, acid reflux still occurs, and bolus feeding is generally not possible. Fundoplica-tion provides definitive treatment for gastroesophageal reflux and is highly effective in most circumstances. The fundus may be wrapped around the distal esophagus either 360o (i.e., Nissen) or to lesser degrees (i.e., Thal or Toupet). At present, the stan-dard approach in most children is to perform these procedures laparoscopically whenever possible. In children with feeding difficulties and in infants under 1 year of age, a gastrostomy tube should be placed at the time of surgery. Early postoperative complications include pneumonia and atelectasis, often due to inadequate pulmonary toilet and pain control with abdominal splinting. Late postoperative complications include wrap break-down with

1	complications include pneumonia and atelectasis, often due to inadequate pulmonary toilet and pain control with abdominal splinting. Late postoperative complications include wrap break-down with recurrent reflux, which may require repeat fundo-plication, and dysphagia due to a wrap performed too tightly, which generally responds to dilation. These complications are more common in children with neurologic impairment. The keys to successful surgical management of patients with GERD include careful patient selection and meticulous operative tech-nique. There are emerging concerns regarding the long-term use of acid reducing agents, which may increase the frequency with which antireflux procedures are performed in children, espe-cially those with neurological impairment.GASTROINTESTINAL TRACTAn Approach to the Vomiting InfantAll infants vomit. Because infant vomiting is so common, it is important to differentiate between normal and abnormal vomit-ing, which may be indicative of a

1	TRACTAn Approach to the Vomiting InfantAll infants vomit. Because infant vomiting is so common, it is important to differentiate between normal and abnormal vomit-ing, which may be indicative of a potentially serious underlying disorder. In order to determine the seriousness of a particular infant’s bouts of emesis, one needs to characterize what the vomit looks like and how sick the baby is. Vomit that looks like feeds and comes up immediately after a feeding is almost always gastroesophageal reflux. This may or may not be of concern, as described earlier. Vomiting that occurs a short while after feed-ing, or vomiting that projects out of the baby’s mouth may be indicative of pyloric stenosis. By contrast, vomit that has any green color in it is always worrisome. This may be reflective of intestinal volvulus, an underlying infection, or some other cause of intestinal obstruction. A more detailed description of the management of these conditions is provided in the follow-ing

1	of intestinal volvulus, an underlying infection, or some other cause of intestinal obstruction. A more detailed description of the management of these conditions is provided in the follow-ing sections.Hypertrophic Pyloric StenosisClinical Presentation. Infants with hypertrophic pyloric stenosis (HPS) typically present with nonbilious vomiting that becomes increasingly projectile, over the course of several days to weeks due to progressive thickening of the pylorus muscle. HPS occurs in approximately 1 in 300 live births and commonly in infants between 3 and 6 weeks of age. Male-to-female ratio is nearly 5:1.Eventually as the pyloric muscle thickening progresses, the infant develops a complete gastric outlet obstruction and is no longer able to tolerate any feeds. Over time, the infant becomes increasingly hungry, unsuccessfully feeds repeatedly, and becomes increasingly dehydrated. Wet diapers become less frequent, and there may even be a perception of less passage of flatus. HPS may

1	increasingly hungry, unsuccessfully feeds repeatedly, and becomes increasingly dehydrated. Wet diapers become less frequent, and there may even be a perception of less passage of flatus. HPS may be associated with jaundice due to an indi-rect hyperbilirubinemia, although the nature of this relation is unclear.The cause of HPS has not been determined. Studies have shown that HPS is found in several generations of the same family, suggesting a familial link. Recently, a genome-wide sig-nificant locus for pyloric stenosis at chromosome 11q23.3 was identified, and the single-nucleotide polymorphism (SNP) with the greatest significance was associated with part of the genome that regulates cholesterol. It is not clear how this links to the development of pyloric stenosis, but it does suggest a potential dietary link.Infants with HPS develop a hypochloremic, hypokale-mic metabolic alkalosis. The urine pH level is high initially, but eventually drops because hydrogen ions are preferentially

1	potential dietary link.Infants with HPS develop a hypochloremic, hypokale-mic metabolic alkalosis. The urine pH level is high initially, but eventually drops because hydrogen ions are preferentially exchanged for sodium ions in the distal tubule of the kidney as the hypochloremia becomes severe (paradoxical aciduria). While in the past the diagnosis of pyloric stenosis was most often made on physical examination by palpation of the typical “olive” in the right upper quadrant and the presence of visible gastric waves on the abdomen, current standard of care is to perform an US, which can diagnose the condition accurately in 95% of patients. Criteria for US diagnosis include a channel length of over 16 mm and pyloric thickness over 4 mm. It is important to note that younger babies may have lower values Brunicardi_Ch39_p1705-p1758.indd 172212/02/19 11:26 AM 1723PEDIATRIC SURGERYCHAPTER 39Pyloric “tumor”MucosaABCFigure 39-12. Fredet-Ramstedt pyloromyotomy. A. Pylorus deliv-ered into

1	lower values Brunicardi_Ch39_p1705-p1758.indd 172212/02/19 11:26 AM 1723PEDIATRIC SURGERYCHAPTER 39Pyloric “tumor”MucosaABCFigure 39-12. Fredet-Ramstedt pyloromyotomy. A. Pylorus deliv-ered into wound and seromuscular layer incised. B. Seromuscular layer separated down to submucosal base to permit herniation of mucosa through pyloric incision. C. Cross-section demonstrating hypertrophied pylorus, depth of incision, and spreading of muscle to permit mucosa to herniate through incision.for pyloric thickness and still be abnormal, and a close clinical correlation with the US result is mandatory. In cases in which the diagnosis remains unclear, upper gastrointestinal evaluation by contrast radiography will reveal delayed passage of contents from the stomach through the pyloric channel and a typical thickened appearance to the pylorus.Treatment. Given frequent fluid and electrolyte abnormali-ties at time of presentation, pyloric stenosis is never a surgical emergency. Fluid

1	channel and a typical thickened appearance to the pylorus.Treatment. Given frequent fluid and electrolyte abnormali-ties at time of presentation, pyloric stenosis is never a surgical emergency. Fluid resuscitation with correction of electrolyte abnormalities and metabolic alkalosis is essential prior to induc-tion of general anesthesia for operation. For most infants, fluid containing 5% dextrose and 0.45% saline with added potassium of 2 to 4 mEq/kg over 24 hours at a rate of approximately 150 to 175 mL/kg for 24 hours will correct the underlying deficit. It is important to ensure that the child has an adequate urine output (>2 cc/kg per hour) as further evidence that rehydration has occurred.After resuscitation, a Fredet-Ramstedt pyloromyotomy is performed (Fig. 39-12). It may be performed using an open or laparoscopic approach. The open pyloromyotomy is per-formed through either an umbilical or a right upper quadrant transverse abdominal incision. The former route is cosmetically

1	using an open or laparoscopic approach. The open pyloromyotomy is per-formed through either an umbilical or a right upper quadrant transverse abdominal incision. The former route is cosmetically more appealing, although the transverse incision provides easier access to the antrum and pylorus. In recent years, the laparo-scopic approach has gained great popularity. Two randomized trials have demonstrated that both the open and laparoscopic approaches may be performed safely with equal incidence of postoperative complications, although the cosmetic result is clearly superior with the laparoscopic approach. Whether done through an open or laparoscopic approach, surgical treatment of pyloric stenosis involves splitting the pyloric muscle while leav-ing the underlying submucosa intact. The incision extends from just proximal to the pyloric vein of Mayo to the gastric antrum; it typically measures between 1 and 2 cm in length. Postop-eratively, IV fluids are continued for several hours,

1	incision extends from just proximal to the pyloric vein of Mayo to the gastric antrum; it typically measures between 1 and 2 cm in length. Postop-eratively, IV fluids are continued for several hours, after which Pedialyte is offered, followed by formula or breast milk, which is gradually increased to 60 cc every 3 hours. Most infants can be discharged home within 24 to 48 hours following surgery. Recently, several authors have shown that ad lib feeds are safely tolerated by the neonate and result in a shorter hospital stay.The complications of pyloromyotomy include perforation of the mucosa (1–3%), bleeding, wound infection, and recur-rent symptoms due to inadequate myotomy. When perforation occurs, the mucosa is repaired with a stitch that is placed to tack the mucosa down and reapproximate the serosa in the region of the tear. A nasogastric tube is left in place for 24 hours. The outcome is generally very good.Intestinal Obstruction in the NewbornThe cardinal symptom of intestinal

1	the serosa in the region of the tear. A nasogastric tube is left in place for 24 hours. The outcome is generally very good.Intestinal Obstruction in the NewbornThe cardinal symptom of intestinal obstruction in the newborn is bilious emesis. Prompt recognition and treatment of neonatal intestinal obstruction can truly be lifesaving.The incidence of neonatal intestinal obstruction is 1 in 2000 live births. The approach to intestinal obstruction in the newborn infant is critical for timely and appropriate interven-tion. When a neonate develops bilious vomiting, one must con-sider a surgical etiology. Indeed, the majority of newborns with bilious emesis have a surgical condition. In evaluating a poten-tial intestinal obstruction, it is helpful to determine whether the intestinal obstruction is either proximal or distal to the ligament of Treitz. One must conduct a detailed prenatal and immediate postnatal history and a thorough physical examination. In all cases of intestinal

1	is either proximal or distal to the ligament of Treitz. One must conduct a detailed prenatal and immediate postnatal history and a thorough physical examination. In all cases of intestinal obstruction, it is vital to obtain abdominal films in the supine and upright (or lateral decubitus) views to assess the presence of air-fluid levels or free air as well as how far downstream air has managed to travel. Importantly, one should recognize that it is difficult to determine whether a loop of bowel is part of either the small or large intestine, as neonatal bowel lacks clear features, such as haustra or plica circulares, normally present in older children or adults. As such, contrast imaging may be necessary for diagnosis in some instances.Proximal intestinal obstructions typically present with bil-ious emesis and minimal abdominal distention. The normal neo-nate should have a rounded, soft abdomen; in contrast, a neonate with a proximal intestinal obstruction typically exhibits a flat or

1	bil-ious emesis and minimal abdominal distention. The normal neo-nate should have a rounded, soft abdomen; in contrast, a neonate with a proximal intestinal obstruction typically exhibits a flat or scaphoid abdomen. On a series of upright and supine abdominal radiographs, one may see a paucity or absence of bowel gas, which normally should be present throughout the gastrointesti-nal tract within 24 hours. Of utmost importance is the exclusion of a malrotation with midgut volvulus from all other intestinal obstructions as this is a surgical emergency.Distal obstructions typically present with bilious emesis and abdominal distention. Passage of black-green meconium should have occurred within the first 24 to 38 hours. Of great 34Brunicardi_Ch39_p1705-p1758.indd 172312/02/19 11:26 AM 1724SPECIFIC CONSIDERATIONSPART IIFigure 39-13. Abdominal X-ray showing “double bubble” sign in a newborn infant with duodenal atresia. The two “bubbles” are numbered.importance, one should determine

1	1724SPECIFIC CONSIDERATIONSPART IIFigure 39-13. Abdominal X-ray showing “double bubble” sign in a newborn infant with duodenal atresia. The two “bubbles” are numbered.importance, one should determine whether there is tenderness or discoloration of the abdomen, visible or palpable loops of intestine, presence or absence of a mass, and whether the anus is patent and in appropriate location. Abdominal radiographs may demonstrate calcifications may indicate complicated meconium ileus; pneumatosis and/or pneumoperitoneum may indicate necrotizing enterocolitis. A contrast enema may show whether there is a microcolon indicative of jejunoileal atresia or meconium ileus. If a microcolon is not present, then the diag-noses of Hirschsprung’s disease, small left colon syndrome, or meconium plug syndrome should be considered.Duodenal ObstructionWhenever the diagnosis of duodenal obstruction is entertained, malrotation and midgut volvulus must be excluded. This topic is covered in further detail

1	should be considered.Duodenal ObstructionWhenever the diagnosis of duodenal obstruction is entertained, malrotation and midgut volvulus must be excluded. This topic is covered in further detail later in this chapter. Other causes of duodenal obstruction include duodenal atresia, duodenal web, stenosis, annular pancreas, or duodenal duplication cyst. Duode-nal obstruction is easily diagnosed on prenatal US, which dem-onstrates the fluid-filled stomach and proximal duodenum as two discrete cystic structures in the upper abdomen. Associated polyhydramnios is common and presents in the third trimester. In 85% of infants with duodenal obstruction, the entry of the bile duct is proximal to the level of obstruction, such that vom-iting is bilious. Abdominal distention is typically not present because of the proximal level of obstruction. In those infants with obstruction proximal to the bile duct entry, the vomiting is nonbilious. The classic finding on abdominal radiography is the “double

1	of the proximal level of obstruction. In those infants with obstruction proximal to the bile duct entry, the vomiting is nonbilious. The classic finding on abdominal radiography is the “double bubble” sign, which represents the dilated stomach and duodenum (Fig. 39-13). In association with the appropriate clin-ical picture, this finding is sufficient to confirm the diagnosis of duodenal obstruction. However, if there is any uncertainty, particularly when a partial obstruction is suspected, a contrast upper gastrointestinal series is diagnostic.Treatment. An orogastric tube is inserted to decompress the stomach and duodenum and the infant is given IV fluids to maintain adequate urine output. If the infant appears ill, or if abdominal tenderness is present, a diagnosis of malrotation and midgut volvulus should be considered, and surgery should not be delayed. Typically, the abdomen is soft, and the infant is very stable. Under these circumstances, the infant should be evaluated

1	and midgut volvulus should be considered, and surgery should not be delayed. Typically, the abdomen is soft, and the infant is very stable. Under these circumstances, the infant should be evaluated thoroughly for other associated anomalies. Approxi-mately one-third of newborns with duodenal atresia have asso-ciated Down syndrome (trisomy 21). These patients should be evaluated for associated cardiac anomalies. Once the workup is complete and the infant is stable, he or she is taken to the operat-ing room, and repair is performed either via an open approach or laparoscopically.Regardless of the surgical approach, the principles are the same. If open, the abdomen is entered through a transverse right upper quadrant supraumbilical incision under general endotra-cheal anesthesia. Associated anomalies should be searched for at the time of the operation. These include malrotation, ante-rior portal vein, a second distal web, and biliary atresia. The surgical treatment of choice for duodenal

1	should be searched for at the time of the operation. These include malrotation, ante-rior portal vein, a second distal web, and biliary atresia. The surgical treatment of choice for duodenal obstruction due to duodenal stenosis or atresia or annular pancreas is a duodeno-duodenostomy. This procedure can be most easily performed using a proximal transverse-to-distal longitudinal (diamond-shaped) anastomosis. In cases where the duodenum is extremely dilated, the lumen may be tapered using a linear stapler with a large Foley catheter (24F or greater) in the duodenal lumen. It is important to emphasize that an annular pancreas is never divided but rather is bypassed to avoid injury to the pancreatic ducts. Treatment of duodenal web includes vertical duodenot-omy, excision of the web, oversewing of the mucosa, and clos-ing the duodenotomy horizontally. Care must be taken to avoid injury to the bile duct, which opens up near the web in all cases. For this reason, some surgeons favor

1	of the mucosa, and clos-ing the duodenotomy horizontally. Care must be taken to avoid injury to the bile duct, which opens up near the web in all cases. For this reason, some surgeons favor performing a duodeno-duodenostomy for children with duodenal web, although such an approach may lead to long-term complications associated with the creation of a blind section of duodenum between the web and the bypass, which can expand over time. Gastrostomy tube placement is not routinely performed. Recently reported survival rates exceed 90%. Late complications from repair of duodenal atresia occur in approximately 12% to 15% of patients and include megaduodenum, intestinal motility disorders, and gastroesophageal reflux.Specific consideration may be given to premature infants with duodenal obstruction. Whereas in the past pediatric sur-geons may have favored delayed repair until the child reached either term or a weight closer to 3 kg, there is no reason to wait, and once the child is stable

1	Whereas in the past pediatric sur-geons may have favored delayed repair until the child reached either term or a weight closer to 3 kg, there is no reason to wait, and once the child is stable from a pulmonary perspective, duo-denal repair can be performed in children as small as 1 kg quite safely, as long as there is meticulous attention to detail and a thorough knowledge of the anatomy.Intestinal AtresiaObstruction due to intestinal atresia can occur at any point along the intestinal tract. Intestinal atresias were previously thought to be the result of in utero mesenteric vascular accidents leading to segmental loss of the intestinal lumen, although more likely they are the result of developmental defects in normal intestinal organogenesis due to disruption of various signaling pathways such as fibroblast growth factor, bone morphogenic protein, and β-catenin pathways. The incidence of intestinal atresia has been estimated to be between 1 in 2000 to 1 in 5000 live births, with

1	such as fibroblast growth factor, bone morphogenic protein, and β-catenin pathways. The incidence of intestinal atresia has been estimated to be between 1 in 2000 to 1 in 5000 live births, with equal representation of the sexes. Infants with jejunal or ileal atresia present with bilious vomiting and progressive abdominal distention. The more distal the obstruction, the more distended the abdomen becomes, and the greater the number of obstructed loops on upright abdominal films (Fig. 39-14).In cases where the diagnosis of complete intestinal obstruction is ascertained by the clinical picture and the pres-ence of staggered air-fluid levels on plain abdominal films, the child can be brought to the operating room after appropriate resuscitation. In these circumstances, there is little extra infor-mation to be gained by performing a barium enema. By contrast, Brunicardi_Ch39_p1705-p1758.indd 172412/02/19 11:26 AM 1725PEDIATRIC SURGERYCHAPTER 39Figure 39-14. Intestinal obstruction in

1	infor-mation to be gained by performing a barium enema. By contrast, Brunicardi_Ch39_p1705-p1758.indd 172412/02/19 11:26 AM 1725PEDIATRIC SURGERYCHAPTER 39Figure 39-14. Intestinal obstruction in the newborn showing sev-eral loops of distended bowel with air fluid levels. This child has jejunal atresia.Figure 39-15. Operative photograph of newborn with “Christmas tree” type of ileal atresia.when there is diagnostic uncertainty, or when distal intestinal obstruction is apparent, a barium enema is useful to establish whether a microcolon is present and to diagnose the presence of meconium plugs, small left colon syndrome, Hirschsprung’s disease, or meconium ileus. Judicious use of barium enema is therefore required in order to safely manage neonatal intestinal obstruction, based on an understanding of the expected level of obstruction.Surgical correction of the small intestinal atresia should be performed relatively urgently, especially when there is a possibility of volvulus. At

1	understanding of the expected level of obstruction.Surgical correction of the small intestinal atresia should be performed relatively urgently, especially when there is a possibility of volvulus. At laparotomy, one of several types of atresia will be encountered. In type 1 there is a mucosal atre-sia with intact muscularis. In type 2, the atretric ends are con-nected by a fibrous band. In type 3A, the two ends of the atresia are separated by a V-shaped defect in the mesentery. Type 3B is an “apple-peel” deformity or “Christmas tree” deformity in which the bowel distal to the atresia receives its blood supply in a retrograde fashion from the ileocolic or right colic artery (Fig. 39-15). In type 4 atresia, there are multiple atresias with a “string of sausage” or “string of beads” appearance. Disparity in lumen size between the proximal distended bowel and the small diameter of collapsed bowel distal to the atresia has led to a num-ber of innovative techniques of anastomosis. However,

1	Disparity in lumen size between the proximal distended bowel and the small diameter of collapsed bowel distal to the atresia has led to a num-ber of innovative techniques of anastomosis. However, under most circumstances, an anastomosis can be performed using the end-to-back technique in which the distal, compressed loop is “fish-mouthed” along its antimesenteric border. The proximal distended loop can be tapered as previously described. Because the distended proximal bowel rarely has normal motility, the extremely dilated portion should be resected prior to per-forming the anastomosis.Occasionally the infant with intestinal atresia will develop ischemia or necrosis of the proximal segment secondary to volvulus of the dilated, bulbous, blind-ending proximal bowel. Under these conditions, primary anastomosis may be performed as described earlier. Alternatively, an end ileostomy and mucus fistula should be created, and the anastomosis should be deferred to another time after the infant

1	primary anastomosis may be performed as described earlier. Alternatively, an end ileostomy and mucus fistula should be created, and the anastomosis should be deferred to another time after the infant stabilizes.Malrotation and Midgut VolvulusEmbryology. During the sixth week of fetal development, the midgut grows too rapidly to be accommodated in the abdominal cavity and therefore herniates into the umbilical cord. Between the 10th and 12th week, the midgut returns to the abdominal cavity, undergoing a 270° counterclockwise rotation around the superior mesenteric artery. Because the duodenum also rotates caudal to the artery, it acquires a C-loop, which traces this path. The cecum rotates cephalad to the artery, which determines the location of the transverse and ascending colon. Subsequently, the duodenum becomes fixed retroperitoneally in its third por-tion and at the ligament of Treitz, while the cecum becomes fixed to the lateral abdominal wall by peritoneal bands. The takeoff of

1	the duodenum becomes fixed retroperitoneally in its third por-tion and at the ligament of Treitz, while the cecum becomes fixed to the lateral abdominal wall by peritoneal bands. The takeoff of the branches of the superior mesenteric artery elon-gates and becomes fixed along a line extending from its emer-gence from the aorta to the cecum in the right lower quadrant. Genetic mutations likely disrupt the signaling critical for normal intestinal rotation. For instance, mutations in the gene BCL6 resulting in absence of left-sided expression of its transcript lead to reversed cardiac orientation, defective ocular development, and malrotation. The essential role of the dorsal gut mesentery in mediating normal intestinal rotation and the role of the fork-head box transcription factor FOXF1 in formation of the dorsal mesentery in mice are consistent with the noted association of intestinal malrotation with alveolar capillary dysplasia, caused by mutations in FOXF1. If rotation is

1	FOXF1 in formation of the dorsal mesentery in mice are consistent with the noted association of intestinal malrotation with alveolar capillary dysplasia, caused by mutations in FOXF1. If rotation is incomplete, the cecum remains in the epigastrium, but the bands fixing the duode-num to the retroperitoneum and cecum continue to form. This results in (Ladd’s) bands extending from the cecum to the lat-eral abdominal wall and crossing the duodenum, which creates the potential for obstruction. The mesenteric takeoff remains confined to the epigastrium, resulting in a narrow pedicle sus-pending all the branches of the superior mesenteric artery and the entire midgut. A volvulus may therefore occur around the mesentery. This twist not only obstructs the proximal jejunum but also cuts off the blood supply to the midgut. Intestinal obstruction and complete infarction of the midgut occur unless the problem is promptly corrected surgically.Presentation and Management. Midgut volvulus can occur

1	supply to the midgut. Intestinal obstruction and complete infarction of the midgut occur unless the problem is promptly corrected surgically.Presentation and Management. Midgut volvulus can occur at any age, though it is seen most often in the first few weeks of life. Bilious vomiting is usually the first sign of volvulus and all infants with bilious vomiting must be evaluated rapidly to ensure that they do not have intestinal malrotation with volvu-lus. The child with irritability and bilious emesis should raise particular suspicions for this diagnosis. If left untreated, vascular Brunicardi_Ch39_p1705-p1758.indd 172512/02/19 11:26 AM 1726SPECIFIC CONSIDERATIONSPART IIFigure 39-16. Abdominal X-ray of a 10-day-old infant with bil-ious emesis. Note the dilated proximal bowel and the paucity of distal bowel gas, characteristic of a volvulus.compromise of the midgut initially causes bloody stools, but it eventually results in circulatory collapse. Additional clues to the presence of

1	of distal bowel gas, characteristic of a volvulus.compromise of the midgut initially causes bloody stools, but it eventually results in circulatory collapse. Additional clues to the presence of advanced ischemia of the intestine include ery-thema and edema of the abdominal wall, which progresses to shock and death. It must be reemphasized that the index of sus-picion for this condition must be high, since abdominal signs are minimal in the early stages. Abdominal films show a paucity of gas throughout the intestine with a few scattered air-fluid levels (Fig. 39-16). When these findings are present, the patient should undergo immediate fluid resuscitation to ensure adequate per-fusion and urine output followed by prompt exploratory lapa-rotomy. In cases where the child is stable, laparoscopy may be considered.Often the patient will not appear ill, and the plain films may suggest partial duodenal obstruction. Under these condi-tions, the patient may have malrotation without volvulus.

1	may be considered.Often the patient will not appear ill, and the plain films may suggest partial duodenal obstruction. Under these condi-tions, the patient may have malrotation without volvulus. This is best diagnosed by an upper gastrointestinal series that shows incomplete rotation with the duodenojejunal junction displaced to the right. The duodenum may show a corkscrew effect diag-nosing volvulus, or complete duodenal obstruction, with the small bowel loops entirely in the right side of the abdomen. Barium enema may show a displaced cecum, but this sign is unreliable, especially in the small infant in whom the cecum is normally in a somewhat higher position than in the older child.When volvulus is suspected, early surgical intervention is mandatory if the ischemic process is to be avoided or reversed. Volvulus occurs clockwise, and it is therefore untwisted coun-terclockwise. This can be remembered using the memory aid “turn back the hands of time.” Subsequently, a Ladd’s

1	be avoided or reversed. Volvulus occurs clockwise, and it is therefore untwisted coun-terclockwise. This can be remembered using the memory aid “turn back the hands of time.” Subsequently, a Ladd’s proce-dure is performed. This operation does not correct the malro-tation, but it does broaden the narrow mesenteric pedicle to prevent volvulus from recurring. This procedure is performed as follows (Fig. 39-17). The bands between the cecum and the abdominal wall and between the duodenum and terminal ileum are divided sharply to splay out the superior mesenteric artery and its branches. This maneuver brings the straightened duodenum into the right lower quadrant and the cecum into the left lower quadrant. The appendix is usually removed to avoid diagnostic errors in later life. No attempt is made to suture the cecum or duodenum in place. With advanced ischemia, reduc-tion of the volvulus without the Ladd’s procedure is accom-plished, and a “second look” 24 to 36 hours later often may show

1	to suture the cecum or duodenum in place. With advanced ischemia, reduc-tion of the volvulus without the Ladd’s procedure is accom-plished, and a “second look” 24 to 36 hours later often may show some vascular recovery. A plastic transparent silo may be placed to facilitate constant evaluation of the intestine and to plan for the timing of reexploration. Clearly necrotic bowel can then be resected conservatively. With early diagnosis and cor-rection, the prognosis is excellent. However, diagnostic delay can lead to mortality or to short-gut syndrome requiring intes-tinal transplantation.A subset of patients with malrotation will demonstrate chronic obstructive symptoms. These symptoms may result from Ladd’s bands across the duodenum, or occasionally, from intermittent volvulus. Symptoms include intermittent abdominal pain and intermittent vomiting that may occasionally be bilious. Infants with malrotation may demonstrate failure to thrive, and they may be diagnosed initially as having

1	intermittent abdominal pain and intermittent vomiting that may occasionally be bilious. Infants with malrotation may demonstrate failure to thrive, and they may be diagnosed initially as having gastroesophageal reflux disease. Surgical correction using Ladd’s procedure as described earlier can prevent volvulus from occurring and improve symp-toms in many instances. In these cases, a laparoscopic approach may be taken, where diagnosis of Ladd’s bands and direct visu-alization of the relevant anatomy may be achieved.Meconium IleusPathogenesis and Clinical Presentation. Infants with cystic fibrosis have characteristic pancreatic enzyme deficiencies and abnormal chloride secretion in the intestine that result in the production of viscous, water-poor meconium. This phenotype is explained by the presence of mutations in the CFTR gene. Meconium ileus occurs when this thick, highly viscous meco-nium becomes impacted in the ileum and leads to high-grade intestinal obstruction. Recently,

1	by the presence of mutations in the CFTR gene. Meconium ileus occurs when this thick, highly viscous meco-nium becomes impacted in the ileum and leads to high-grade intestinal obstruction. Recently, additional mutations were identified in genes encoding multiple apical plasma membrane proteins of infants with meconium ileus. Meconium ileus can be either uncomplicated, in which there is no intestinal perforation, or complicated, in which prenatal perforation of the intestine has occurred or vascular compromise of the distended ileum devel-ops. Antenatal US may reveal the presence of intra-abdominal or scrotal calcifications, or distended bowel loops. These infants present shortly after birth with progressive abdominal disten-tion and failure to pass meconium with intermittent bilious emesis. Abdominal radiographs show dilated loops of intestine. Because the enteric contents are so viscous, air-fluid levels do not form, even when obstruction is complete. Small bubbles of gas become

1	Abdominal radiographs show dilated loops of intestine. Because the enteric contents are so viscous, air-fluid levels do not form, even when obstruction is complete. Small bubbles of gas become entrapped in the inspissated meconium in the dis-tal ileum, where they produce a characteristic “ground glass” appearance.The diagnosis of meconium ileus is confirmed by a con-trast enema that typically demonstrates a microcolon. In patients with uncomplicated meconium ileus, the terminal ileum is filled with pellets of meconium. In patients with complicated meco-nium ileus, intraperitoneal calcifications form, producing an eggshell pattern on plain abdominal X-ray.Management. The treatment strategy depends on whether the patient has complicated or uncomplicated meconium ileus. Patients with uncomplicated meconium ileus can be Brunicardi_Ch39_p1705-p1758.indd 172612/02/19 11:26 AM 1727PEDIATRIC SURGERYCHAPTER 39Figure 39-17. Ladd procedure for malrotation. A. Lysis of cecal and duodenal

1	meconium ileus can be Brunicardi_Ch39_p1705-p1758.indd 172612/02/19 11:26 AM 1727PEDIATRIC SURGERYCHAPTER 39Figure 39-17. Ladd procedure for malrotation. A. Lysis of cecal and duodenal bands. B. Broadening the mesentery. C. Appendectomy.treated nonoperatively. Either dilute water-soluble contrast or N-acetylcysteine (Mucomyst) is infused transanally via catheter under fluoroscopic control into the dilated portion of the ileum. Because these agents act by absorbing fluid from the bowel wall into the intestinal lumen, infants undergoing treatment are at risk of fluid and electrolyte abnormalities so that appropriate resuscitation of the infant during this maneuver is extremely important. The enema may be repeated at 12-hour intervals over several days until all the meconium is evacuated. Inability to reflux the contrast into the dilated portion of the ileum signi-fies the presence of an associated atresia or complicated meco-nium ilus, and thus warrants exploratory laparotomy. If

1	Inability to reflux the contrast into the dilated portion of the ileum signi-fies the presence of an associated atresia or complicated meco-nium ilus, and thus warrants exploratory laparotomy. If surgical intervention is required because of failure of contrast enemas to relieve obstruction, operative irrigation with dilute contrast agent, N-acetylcysteine, or saline through a purse-string suture may be successful. Alternatively, resection of the distended ter-minal ileum is performed, and the meconium pellets are flushed from the distal small bowel. At this point, an end ileostomy may be created. The distal bowel may be brought up as a mucus fistula or sewn to the side of the ileum as a classic Bishop-Koop anastomosis. An end-to-end anastomosis may also be consid-ered in the appropriate setting (Fig. 39-18).Necrotizing EnterocolitisClinical Features. Necrotizing enterocolitis (NEC) is the most frequent and lethal gastrointestinal disorder affecting the intestine of the stressed,

1	setting (Fig. 39-18).Necrotizing EnterocolitisClinical Features. Necrotizing enterocolitis (NEC) is the most frequent and lethal gastrointestinal disorder affecting the intestine of the stressed, preterm neonate. The overall mortal-ity ranges between 10% and 50%. Advances in neonatal care such as surfactant therapy as well as improved methods of mechanical ventilation have resulted in increasing numbers of Brunicardi_Ch39_p1705-p1758.indd 172712/02/19 11:26 AM 1728SPECIFIC CONSIDERATIONSPART IIProximalDistalABCDProximalDistalProximalDistalProximalDistalDistalProximalTypical operative findingEnd to backThomas taperBishop-Koop with distal ventMikulicz enterostomyFigure 39-18. Techniques of intestinal anastomosis for infants with small bowel obstruction. A. End-to-back distal limb has been incised, creating “fishmouth” to enlarge the lumen. B. Bishop-Koop; proximal distended limb joined to side of distal small bowel, which is vented by “chimney” to the abdominal wall. C. Tapering;

1	been incised, creating “fishmouth” to enlarge the lumen. B. Bishop-Koop; proximal distended limb joined to side of distal small bowel, which is vented by “chimney” to the abdominal wall. C. Tapering; portion of antimesenteric wall of proximal bowel excised, with longitudinal closure to minimize disparity in the limbs. D. Mikulicz double-barreled enterostomy is constructed by suturing the two limbs together and then exte-riorizing the double stoma. The common wall can be crushed with a special clamp to create a large stoma. The stoma can be closed in an extraperitoneal manner.low-birth-weight infants surviving neonatal hyaline membrane disease. An increasing proportion of survivors of neonatal respi-ratory distress syndrome will therefore be at risk for developing NEC. Consequently, it is estimated that NEC may eventually surpass respiratory distress syndrome as the principal cause of death in the preterm infant. This is especially relevant, as NEC is a significant risk factor for more

1	that NEC may eventually surpass respiratory distress syndrome as the principal cause of death in the preterm infant. This is especially relevant, as NEC is a significant risk factor for more severe respiratory distress in premature infants.Multiple risk factors have been associated with the devel-opment of NEC. These include prematurity, initiation of enteral feeding, bacterial infection, intestinal ischemia resulting from birth asphyxia, umbilical artery cannulation, persistence of a patent ductus arteriosus, cyanotic heart disease, and maternal cocaine abuse. Nonetheless, the mechanisms by which these complex interacting etiologies lead to the development of the disease remain undefined. The only consistent epidemio-logic precursors for NEC are prematurity and enteral ali-mentation, representing the commonly encountered clinical situation of a stressed infant who is fed enterally. Of note, there is some debate regarding the type and strategy of enteral alimen-tation in the

1	representing the commonly encountered clinical situation of a stressed infant who is fed enterally. Of note, there is some debate regarding the type and strategy of enteral alimen-tation in the pathogenesis of NEC. A prospective randomized 5study showed no increase in the incidence of NEC despite an aggressive feeding strategy.The indigenous intestinal microbial flora has been shown to play a central role in the pathogenesis of NEC. The importance of bacteria in the pathogenesis of NEC is further supported by the finding that NEC occurs in episodic waves that can be abrogated by infection control measures, and the fact that NEC usually develops at least 10 days postnatally, when the GI tract is colonized by coliforms. More recently, outbreaks of NEC have been reported in infants fed formula contaminated with Enterobacter sakazakii. Common bacterial isolates from the blood, peritoneal fluid, and stool of infants with advanced NEC include Escherichia coli, Enterobacter, Klebsiella, and

1	contaminated with Enterobacter sakazakii. Common bacterial isolates from the blood, peritoneal fluid, and stool of infants with advanced NEC include Escherichia coli, Enterobacter, Klebsiella, and occasionally, coagulase-negative Staphylococ-cus species.NEC may involve single or multiple segments of the intes-tine, most commonly the terminal ileum, followed by the colon. The gross findings in NEC include bowel distention with patchy areas of thinning, pneumatosis, gangrene, or frank perforation. The microscopic features include the appearance of a “bland infarct” characterized by full thickness necrosis.Brunicardi_Ch39_p1705-p1758.indd 172812/02/19 11:26 AM 1729PEDIATRIC SURGERYCHAPTER 39Figure 39-19. Abdominal radiograph of infant with necrotizing enterocolitis. Arrows point to area of pneumatosis intestinalis.Clinical Manifestations. Infants with NEC present with a spectrum of disease. In general, the infants are premature and may have sustained one or more episodes of stress,

1	of pneumatosis intestinalis.Clinical Manifestations. Infants with NEC present with a spectrum of disease. In general, the infants are premature and may have sustained one or more episodes of stress, such as birth asphyxia, or they may have congenital cardiac disease. The clin-ical picture of NEC has been characterized as progressing from a period of mild illness to that of severe, life-threatening sepsis by Bell and colleagues. Although not all infants progress through the various “Bell stages,” this classification scheme provides a useful format to describe the clinical picture associated with the development of NEC. In the earliest stage (Bell stage I), infants present with feeding intolerance. This is suggested by vomiting or by the presence of a large residual volume from a previous feeding in the stomach at the time of the next feed-ing. Following appropriate treatment, which consists of bowel rest and IV antibiotics, many of these infants will not progress to more advanced

1	feeding in the stomach at the time of the next feed-ing. Following appropriate treatment, which consists of bowel rest and IV antibiotics, many of these infants will not progress to more advanced stages of NEC. These infants are colloqui-ally described as suffering from an “NEC scare” and represent a population of neonates who are at risk of developing more severe NEC if a more prolonged period of stress supervenes.Infants with Bell stage II have established NEC that is not immediately life-threatening. Clinical findings include abdomi-nal distention and tenderness, bilious nasogastric aspirate, and bloody stools. These findings indicate the development of intestinal ileus and mucosal ischemia, respectively. Abdominal examination may reveal a palpable mass indicating the pres-ence of an inflamed loop of bowel, diffuse abdominal tender-ness, cellulitis, and edema of the anterior abdominal wall. The infant may appear systemically ill, with decreased urine output, hypotension,

1	of an inflamed loop of bowel, diffuse abdominal tender-ness, cellulitis, and edema of the anterior abdominal wall. The infant may appear systemically ill, with decreased urine output, hypotension, tachycardia, and noncardiac pulmonary edema. Hematologic evaluation reveals either leukocytosis or leukope-nia, an increase in the number of bands, and thrombocytopenia. An increase in the blood urea nitrogen and plasma creatinine level may be found, which signify the development of renal dys-function. The diagnosis of NEC may be confirmed by abdomi-nal radiography. The pathognomonic radiographic finding in NEC is pneumatosis intestinalis, which represents invasion of the ischemic mucosa by gas producing microbes (Fig. 39-19). Other findings include the presence of ileus or portal venous gas. The latter is a transient finding that indicates the presence of severe NEC with intestinal necrosis. A fixed loop of bowel may be seen on serial abdominal radiographs, which suggests the possibility

1	The latter is a transient finding that indicates the presence of severe NEC with intestinal necrosis. A fixed loop of bowel may be seen on serial abdominal radiographs, which suggests the possibility that a diseased loop of bowel, potentially with a localized perforation, is present. Although these infants are at risk of progressing to more severe disease, with timely and appropriate treatment, they often recover.Infants with Bell stage III have the most advanced form of NEC. Abdominal radiographs often demonstrate the presence of pneumoperitoneum, indicating that intestinal perforation has occurred. These patients may develop a fulminant course with progressive peritonitis, acidosis, sepsis, disseminated intravas-cular coagulopathy, and death.Pathogenesis of Necrotizing Enterocolitis. Several theories have been proposed to explain the development of NEC. In gen-eral terms, the development of diffuse pneumatosis intestinalis—which is associated with the development of stage II NEC—is

1	theories have been proposed to explain the development of NEC. In gen-eral terms, the development of diffuse pneumatosis intestinalis—which is associated with the development of stage II NEC—is thought to be due to the presence of gas within the wall of the intestine from enteric bacteria, suggesting the causative role of bacteria in the pathogenesis of NEC. Furthermore, the develop-ment of pneumoperitoneum indicates disease progression with severe disruption of the intestinal barrier (intestinal perforation). Finally, systemic sepsis with diffuse multisystem organ dysfunc-tion suggests the role for circulating proinflammatory cytokines in the pathogenesis of NEC. It has also been demonstrated that the premature intestine responds in an exaggerated fashion to bacterial products, rendering the host susceptible to barrier dys-function and the development of NEC. Various groups have shown that NEC pathogenesis requires activation of the bacterial receptor—Toll-like receptor 4 (TLR4)—in

1	the host susceptible to barrier dys-function and the development of NEC. Various groups have shown that NEC pathogenesis requires activation of the bacterial receptor—Toll-like receptor 4 (TLR4)—in the intestinal epithe-lium. The expression of TLR4 is significantly elevated in the premature infant intestine as compared with the full-term infant intestine, a consequence of the role that TLR4 plays in normal intestinal development. When the infant is born prematurely and TLR4 expression levels are elevated, subsequent activation of TLR4 by colonizing bacteria in the neonatal intensive care unit leads to the induction of a severe proinflammatory response and the development of NEC. It is noteworthy that breast milk—long known to be protective against NEC—is able to suppress TLR4 signaling and that synthetic TLR4 antagonists are known to prevent NEC in preclinical models, suggesting the possibility of preventive approaches for this disease.Treatment. In all infants suspected of having

1	and that synthetic TLR4 antagonists are known to prevent NEC in preclinical models, suggesting the possibility of preventive approaches for this disease.Treatment. In all infants suspected of having NEC, feedings are discontinued, a nasogastric tube is placed, and broad-spec-trum parenteral antibiotics are given. The infant is resuscitated, and inotropes are administered to maintain perfusion as needed. Intubation and mechanical ventilation may be required to main-tain oxygenation. Total parenteral nutrition is started. Subse-quent treatment may be influenced by the particular stage of NEC that is present. Patients with Bell stage I are closely moni-tored and generally remain NPO and on IV antibiotics for 7 to 10 days, prior to reinitiating enteral nutrition. If the infant fully recovers, feedings may be reinitiated.Patients with Bell stage II disease merit close observa-tion. Serial physical examinations are performed looking for the development of diffuse peritonitis, a fixed mass,

1	feedings may be reinitiated.Patients with Bell stage II disease merit close observa-tion. Serial physical examinations are performed looking for the development of diffuse peritonitis, a fixed mass, progres-sive abdominal wall cellulitis or systemic sepsis. If infants fail to improve after several days of treatment, consideration should be given to exploratory laparotomy. Paracentesis may be per-formed, and if the Gram stain demonstrates multiple organisms and leukocytes, perforation of the bowel should be suspected, and patients should undergo laparotomy.Brunicardi_Ch39_p1705-p1758.indd 172912/02/19 11:26 AM 1730SPECIFIC CONSIDERATIONSPART IIIn the most severe form of NEC (Bell stage III), patients have definite intestinal perforation or have not responded to nonoperative therapy. Two schools of thought direct fur-ther management. One group favors exploratory laparotomy. At laparotomy, frankly gangrenous or perforated bowel is resected, and the intestinal ends are brought out as

1	schools of thought direct fur-ther management. One group favors exploratory laparotomy. At laparotomy, frankly gangrenous or perforated bowel is resected, and the intestinal ends are brought out as stomas. When there is massive intestinal involvement, marginally viable bowel is retained and a “second-look” procedure is carried out after the infant stabilizes (24–48 hours). Patients with extensive necrosis at the second look may be managed by placing a proximal diverting stoma, resecting bowel that is definitely not viable, and leaving questionably viable bowel behind, distal to the diverted segment. When the intestine is viable except for a localized perforation without diffuse peri-tonitis and if the infant’s clinical condition permits, intestinal anastomosis may be performed. In cases where the diseased, perforated segment cannot be safely resected, drainage cath-eters may be left in the region of the diseased bowel, and the infant is allowed to stabilize.An alternative approach to

1	the diseased, perforated segment cannot be safely resected, drainage cath-eters may be left in the region of the diseased bowel, and the infant is allowed to stabilize.An alternative approach to the management of infants with perforated NEC involves drainage of the peritoneal cavity. This may be performed under local anesthesia at the bedside, and it can be an effective means of stabilizing the des-perately ill infant by relieving increased intra-abdominal pres-sure and allowing ventilation. When successful, this method also allows for drainage of perforated bowel by establishing a controlled fistula. Approximately one-third of infants treated with drainage alone survive without requiring additional oper-ations. Infants that do not respond to peritoneal drainage alone after 48 to 72 hours should undergo laparotomy. This proce-dure allows for the resection of frankly necrotic bowel diver-sion of the fecal stream and facilitates more effective drainage. It is noteworthy that a recent

1	should undergo laparotomy. This proce-dure allows for the resection of frankly necrotic bowel diver-sion of the fecal stream and facilitates more effective drainage. It is noteworthy that a recent randomized controlled trial dem-onstrated that outcomes were similar in infants with NEC that were treated either with primary peritoneal drainage or lapa-rotomy, although this study was criticized for the large number of patients who were excluded from randomization. There was also concern that a number of patients who were thought to have NEC may actually have had spontaneous intestinal per-foration, given their lack of pneumatosis and relatively early onset of presentation; these patients would be anticipated to improve after peritoneal drainage due to the more local nature of their disease process.Necrotizing Enterocolitis in Older Infants. Although NEC is typically a disease that affects preterm infants, several inde-pendent groups have reported a tendency for early onset of NEC in term

1	Enterocolitis in Older Infants. Although NEC is typically a disease that affects preterm infants, several inde-pendent groups have reported a tendency for early onset of NEC in term and near-term infants. In these patients, the pattern of disease was found to be different from that found in premature infants. Specifically, NEC in older infants typically is localized to the end of the small intestine and beginning of the colon, sug-gestive of an ischemic pathophysiology. There are four pertinent associations that are observed in term infants that develop NEC: congenital heart disease, in utero growth restriction, polycythe-mia, and perinatal hypoxic-ischemic events. As with NEC in preterm infants, NEC in older patients is also associated with formula consumption and is very rare in exclusively breastfed infants. Patients with NEC at full term typically present with bloody stools and may be characterized by rapid onset of symp-toms and a fulminant course. Thus, although it is true that

1	breastfed infants. Patients with NEC at full term typically present with bloody stools and may be characterized by rapid onset of symp-toms and a fulminant course. Thus, although it is true that NEC is typically a disease of premature babies, in the appropriate setting, NEC can develop at any age.Spontaneous Intestinal Perforation Versus Necrotizing Enterocolitis. In addition to NEC, preterm infants with intes-tinal pathology may develop spontaneous intestinal perforation (SIP). SIP is a distinct clinical entity from NEC, and it is essen-tially a perforation in the terminal ileum. The histopathology of SIP is different from NEC. Specifically, the mucosa is intact and not necrotic, there is no sign of ischemia, and the submucosa is thinned at the site of perforation. In contrast to NEC, pneuma-tosis intestinalis is absent in SIP. Moreover, the demographics of NEC and SIP are slightly different, in that patients with SIP tend to be slightly more premature, smaller, and more likely to

1	pneuma-tosis intestinalis is absent in SIP. Moreover, the demographics of NEC and SIP are slightly different, in that patients with SIP tend to be slightly more premature, smaller, and more likely to have been on inotropic support. SIP occurs in two separate time points, both within a few days after birth and approximately 10 days later, and in all cases, free air will be present, but pneu-matosis will be absent. Because patients with SIP have isolated disease without necrosis or systemic inflammation, they tend to have a better outcome and are likely to respond better to peri-toneal drainage. In short, the diagnosis of SIP versus NEC has important prognostic significance. Treatment for SIP should pri-marily be surgical, with intestinal resection and stoma creation, followed by stoma reversal once the child is stable.In both SIP and NEC, the timing of stoma closure is a mat-ter of ongoing debate. Whereas in the past, pediatric surgeons typically waited until the child reached 5 kg or

1	once the child is stable.In both SIP and NEC, the timing of stoma closure is a mat-ter of ongoing debate. Whereas in the past, pediatric surgeons typically waited until the child reached 5 kg or so, experience indicates that there is no benefit in waiting this long, and chil-dren tolerate stoma closure very well when they are at much lower weights. One approach is to close the stoma when the cal-culated gestational age is approximately 38 to 40 weeks, which will, on average, be at approximately 6 weeks after the initial surgery. This time point is selected based on the observation that proinflammatory gene expression has normalized by then, and NEC recurrence is very unlikely.Outcome. Survival in patients with NEC is dependent on the stage of disease, the extent of prematurity, and the presence of associated comorbidities. Survival by stage has recently been shown to be approximately 85%, 65%, and 35% for stages I, II, and III, respectively. Strictures develop in 20% of medically or

1	of associated comorbidities. Survival by stage has recently been shown to be approximately 85%, 65%, and 35% for stages I, II, and III, respectively. Strictures develop in 20% of medically or surgically treated patients, and a contrast enema is mandatory before reestablishing intestinal continuity. If all other factors are favorable, the ileostomy is closed when the child is between 2 and 2.5 kg. At the time of stoma closure, the entire intestine should be examined to search for areas of NEC. Patients who develop massive intestinal necrosis are at risk of developing short bowel syndrome, particularly when the total length of the viable intes-tinal segment is less than 40 cm. These patients require TPN to provide adequate calories for growth and development, and may develop parenteral nutrition associated cholestasis and hepatic fibrosis. In a significant number of these patients, transplantation of the liver and small bowel may be required.Short Bowel SyndromeShort bowel syndrome

1	nutrition associated cholestasis and hepatic fibrosis. In a significant number of these patients, transplantation of the liver and small bowel may be required.Short Bowel SyndromeShort bowel syndrome (SBS) is an extremely morbid condition with an increasing incidence. Various congenital and perinatal acquired conditions such as gastroschisis, malrotation, atresia, and NEC may lead to SBS. Medical and surgical treatment options carry high dollar and human costs and morbidities including multiple infections and hospitalizations for vascular access, liver failure in conjunction with parenteral nutrition–associated cholestasis, and death. Medical centers that have developed multidisciplinary clinics focused on treating children with short bowel syndrome have achieved significant success in Brunicardi_Ch39_p1705-p1758.indd 173012/02/19 11:26 AM 1731PEDIATRIC SURGERYCHAPTER 39preventing line infections, reducing cholestasis, and improving nutrition and feeding independence

1	success in Brunicardi_Ch39_p1705-p1758.indd 173012/02/19 11:26 AM 1731PEDIATRIC SURGERYCHAPTER 39preventing line infections, reducing cholestasis, and improving nutrition and feeding independence overall.IntussusceptionIntussusception is the leading cause of intestinal obstruction in the young child. It refers to the condition whereby a segment of intestine becomes drawn into the lumen of the more proximal bowel. The process usually begins in the region of the termi-nal ileum, and extends distally into the ascending, transverse, or descending colon. Rarely, an intussusception may prolapse through the rectum.The cause of intussusception is not clear, although one hypothesis suggests that hypertrophy of the Peyer’s patches in the terminal ileum from an antecedent viral infection acts as a lead point. Peristaltic action of the intestine then causes the bowel distal to the lead point to invaginate into itself. Idio-pathic intussusception occurs in children between the ages of

1	acts as a lead point. Peristaltic action of the intestine then causes the bowel distal to the lead point to invaginate into itself. Idio-pathic intussusception occurs in children between the ages of approximately 6 and 24 months of age. Beyond this age group, one should consider the possibility that a pathologic lead point maybe present. These include polyps, malignant tumors such as lymphoma, enteric duplication cysts or Meckel’s diverticu-lum. Such intussusceptions are rarely reduced by air or con-trast enema, and thus the lead point is identified when operative reduction of the intussusception is performed.Clinical Manifestations. Since intussusception is frequently preceded by a gastrointestinal viral illness, the onset may not be easily determined. Typically, the infant develops paroxysms of crampy abdominal pain and intermittent vomiting. Between attacks, the infant may act normally, but as symptoms progress, increasing lethargy develops. Bloody mucus (“currant-jelly” stool) may

1	of crampy abdominal pain and intermittent vomiting. Between attacks, the infant may act normally, but as symptoms progress, increasing lethargy develops. Bloody mucus (“currant-jelly” stool) may be passed per rectum. Ultimately, if reduction is not accomplished, gangrene of the intussusceptum occurs, and perforation may ensue. On physical examination, an elongated mass is detected in the right upper quadrant or epigastrium with an absence of bowel in the right lower quadrant (Dance’s sign). The mass may be seen on plain abdominal X-ray but is more easily demonstrated on air or contrast enema.Treatment. Patients with intussusception should be assessed for the presence of peritonitis and for the severity of systemic illness. Following resuscitation and administration of IV antibi-otics, the child is assessed for suitability to proceed with radio-graphic versus surgical reduction. In the absence of peritonitis, the child should undergo radiographic reduction. If peritonitis is present,

1	child is assessed for suitability to proceed with radio-graphic versus surgical reduction. In the absence of peritonitis, the child should undergo radiographic reduction. If peritonitis is present, or if the child appears systemically ill, urgent lapa-rotomy is indicated.In the stable patient, the air enema is both diagnostic and may be curative, and it is the preferred method of diagnosis and treatment of intussusception. Air is introduced with a manom-eter, and the pressure that is administered is carefully monitored. Under most instances, this should not exceed 120 mmHg. Suc-cessful reduction is marked by free reflux of air into multiple loops of small bowel and symptomatic improvement as the infant suddenly becomes pain free. Unless both of these signs are observed, it cannot be assumed that the intussusception is reduced. If reduction is unsuccessful, and the infant remains stable, the infant should be brought back to the radiology suite for a repeat attempt at reduction after a

1	that the intussusception is reduced. If reduction is unsuccessful, and the infant remains stable, the infant should be brought back to the radiology suite for a repeat attempt at reduction after a few hours. This strategy has improved the success rate of nonoperative reduction in many centers. In addition, hydrostatic reduction with barium may be useful if pneumatic reduction is unsuccessful. The overall suc-cess rate of radiographic reduction varies based on the experi-ence of the center, and it is typically between 60% and 90%.If nonoperative reduction is successful, the infant may be given oral fluids after a period of observation. Failure to reduce the intussusception mandates surgery. which can be approached through an open or laparoscopic technique. In an open procedure, exploration is carried out through a right lower quadrant incision, delivering the intussuscepted mass into the wound. Reduction usually can be accomplished by gentle distal pressure, where the intussusceptum is

1	is carried out through a right lower quadrant incision, delivering the intussuscepted mass into the wound. Reduction usually can be accomplished by gentle distal pressure, where the intussusceptum is gently milked out of the intussuscipiens (Fig. 39-20). Care should be taken not to pull the bowel out, as this can cause damage to the bowel wall. The blood supply to the appendix is often compromised, and appen-dectomy is therefore often performed. If the bowel is frankly gangrenous, resection and primary anastomosis is performed. In experienced hands, laparoscopic reduction may be performed, even in very young infants. This is performed using a 5-mm lap-aroscope placed in the umbilicus, and two additional 5 mm ports in the left and right lower quadrants. The bowel is inspected, and if it appears to be viable, reduction is performed by milking the bowel or using gentle traction, although this approach is nor-mally discouraged during manual reduction. Atraumatic bowel graspers allow the

1	to be viable, reduction is performed by milking the bowel or using gentle traction, although this approach is nor-mally discouraged during manual reduction. Atraumatic bowel graspers allow the bowel to be handled without injuring it.IV fluids are continued until the postoperative ileus sub-sides. Patients are started on clear liquids, and their diet is advanced as tolerated. Of note, recurrent intussusception occurs in 5% to 10% of patients, independent of whether the bowel is reduced radiographically or surgically. Patients present with recurrent symptoms in the immediate postoperative period. Treatment involves repeat air enema, which is successful in most cases. In patients who experience three or more episodes of intussusception, the presence of a pathologic lead point should be suspected and carefully evaluated using contrast stud-ies. After the third episode of intussusception, many pediatric surgeons will perform an exploratory laparotomy to reduce the bowel and to resect a

1	and carefully evaluated using contrast stud-ies. After the third episode of intussusception, many pediatric surgeons will perform an exploratory laparotomy to reduce the bowel and to resect a pathologic lead point if identified.AppendicitisPresentation. Correct diagnosis of appendicitis in children can be one of the most humbling and challenging tasks facing the pediatric surgeon. The classical presentation is known to all students and practitioners of surgery: generalized abdomi-nal pain that localizes to the right lower quadrant followed by nausea, vomiting, fever, and localized peritoneal irritation in the region of McBurney’s point. When children present in this Figure 39-20. Open reduction of intussusception showing how the bowel is milked backwards to relieve the obstruction.Brunicardi_Ch39_p1705-p1758.indd 173112/02/19 11:26 AM 1732SPECIFIC CONSIDERATIONSPART IImanner, there should be little diagnostic delay. The child should be made NPO, administered IV fluids and

1	173112/02/19 11:26 AM 1732SPECIFIC CONSIDERATIONSPART IImanner, there should be little diagnostic delay. The child should be made NPO, administered IV fluids and broad-spectrum anti-biotics, and brought to the operating room for an appendec-tomy. However, children often do not present in this manner. The coexistence of nonspecific viral syndromes and the inability of young children to describe the location and quality of their pain often result in diagnostic delay. As a result, children with appendicitis often present with perforation, particularly those who are under 5 years of age. Perforation increases the length of hospital stay and makes the overall course of the illness sig-nificantly more complex.Diagnosis of Appendicitis in Children. There have been significant improvements in the role of radiographic studies in the diagnosis of acute appendicitis. While CT is quite reliable in making the diagnosis, US is very useful when performed in experienced centers and good

1	in the role of radiographic studies in the diagnosis of acute appendicitis. While CT is quite reliable in making the diagnosis, US is very useful when performed in experienced centers and good visualization of the appendix is achieved. MRI may be performed where available with high specificity and sensitivity—and avoidance of radiation. US is very useful for excluding ovarian causes of abdominal pain. Despite these radiographic measures, the diagnosis of appendi-citis remains largely clinical, and each clinician should develop his or her own threshold to operate or to observe the patient. A reasonable practice guideline is as follows. When the diagno-sis is clinically apparent, appendectomy should obviously be performed with minimal delay. Localized right lower quadrant tenderness associated with low-grade fever and leukocytosis in boys should prompt surgical exploration. In girls, ovarian or uterine pathology must also be considered. When there is diag-nostic uncertainty, the child

1	with low-grade fever and leukocytosis in boys should prompt surgical exploration. In girls, ovarian or uterine pathology must also be considered. When there is diag-nostic uncertainty, the child may be observed, rehydrated, and reassessed. In girls of menstruating age, an US may be obtained to exclude ovarian pathology (cysts, torsion, or tumor). If all studies are negative, yet the pain persists, and the abdominal findings remain equivocal, diagnostic laparoscopy may be employed to determine the etiology of the abdominal pain. The appendix should be removed even if it appears to be normal, unless another pathologic cause of the abdominal pain is defini-tively identified and the appendectomy would substantially increase morbidity.Surgical Treatment of Appendicitis. The definitive treat-ment for acute appendicitis is appendectomy. Prior to surgery, it is important that patients receive adequate IV fluids in order to correct dehydration that commonly develops as a result of fever and

1	for acute appendicitis is appendectomy. Prior to surgery, it is important that patients receive adequate IV fluids in order to correct dehydration that commonly develops as a result of fever and vomiting in patients with appendicitis. Patients should also be started on antibiotics (such as a second-generation cepha-losporin). Most surgeons will perform a laparoscopic appen-dectomy, which may have some advantage over removing the appendix through a single, larger incision. During the laparo-scopic appendectomy, a small incision is made at the umbilicus, and two additional incisions are made in the lower abdomen. The appendix is typically delivered through the umbilicus, and all incisions are then closed, with dissolvable sutures. If the appendix is not ruptured, the patient may start drinking liq-uids shortly after waking up from the operation, and may be advanced to a solid diet the next day. In general, the same steps are taken when appendectomy is performed through an open approach.

1	liq-uids shortly after waking up from the operation, and may be advanced to a solid diet the next day. In general, the same steps are taken when appendectomy is performed through an open approach. The most common complication after appendectomy is a surgical site infection. Other risks—including bleeding or damage to other structures inside the abdomen—are extremely rare. Recovery from surgery is dependent upon the individual patient. Most children are back to school approximately 1 week from surgery and usually are allowed to return to full physical Figure 39-21. Computed tomography scan of the abdomen showing the presence of a ruptured appendix with pelvic fluid and a fecalith (arrow).activity after 2 to 3 weeks. During the recovery period, over-the-counter pain medication may be required. Older patients tend to require a longer time for full recovery.Management of the Child With Perforated Appendicitis. The signs and symptoms of perforated appendicitis can closely mimic those of

1	Older patients tend to require a longer time for full recovery.Management of the Child With Perforated Appendicitis. The signs and symptoms of perforated appendicitis can closely mimic those of gastroenteritis and include abdominal pain, vom-iting, and diarrhea. Alternatively, the child may present with symptoms of intestinal obstruction. An abdominal mass may be present in the lower abdomen. When the symptoms have been present for more than 4 or 5 days, and an abscess is suspected, it is reasonable to obtain a computerized tomogram of the abdo-men and pelvis with IV, oral, and rectal contrast in order to visu-alize the appendix and the presence of an associated abscess, phlegmon, or fecalith (Fig. 39-21).An individualized approach is necessary for the child who presents with perforated appendicitis. When there is evidence of generalized peritonitis, intestinal obstruction or evidence of systemic toxicity, the child should undergo appendectomy. This should be delayed only for as long

1	When there is evidence of generalized peritonitis, intestinal obstruction or evidence of systemic toxicity, the child should undergo appendectomy. This should be delayed only for as long as is required to ensure ade-quate fluid resuscitation and administration of broad-spectrum antibiotics. The operation can be performed through an open or through a laparoscopic approach. One distinct advantage of the laparoscopic approach is that it provides excellent visualiza-tion of the pelvis and all four quadrants of the abdomen. At the time of surgery, adhesions are gently lysed, abscess cavities are drained and the appendix is removed. Drains are seldom used, and the skin incisions can be closed primarily. If a fecalith is identified outside the appendix on computerized tomography, every effort should be made to retrieve it and to remove it along with the appendix, if at all possible. Often, the child in whom symptoms have been present for more than 4 or 5 days will pres-ent with an abscess

1	should be made to retrieve it and to remove it along with the appendix, if at all possible. Often, the child in whom symptoms have been present for more than 4 or 5 days will pres-ent with an abscess without evidence of generalized peritonitis. Under these circumstances, it is appropriate to perform image-guided percutaneous drainage of the abscess followed by broad-spectrum antibiotic therapy. The inflammation will generally subside within several days, and the appendix can be safely removed as an outpatient 6 to 8 weeks later. If the child’s symp-toms do not improve, or if the abscess is not amenable to per-cutaneous drainage, then laparoscopic or open appendectomy and abscess drainage is required. Patients who present with a phlegmon in the region of a perforated appendix may be man-aged in a similar manner. In general, children who are younger Brunicardi_Ch39_p1705-p1758.indd 173212/02/19 11:26 AM 1733PEDIATRIC SURGERYCHAPTER 39than 4 or 5 years of age do not respond as well

1	in a similar manner. In general, children who are younger Brunicardi_Ch39_p1705-p1758.indd 173212/02/19 11:26 AM 1733PEDIATRIC SURGERYCHAPTER 39than 4 or 5 years of age do not respond as well to an initial nonoperative approach because their bodies do not localize or isolate the inflammatory process. Thus, these patients are more likely to require early surgical intervention. Patients who have had symptoms of appendicitis for no more than 4 days should probably undergo “early” appendectomy because the inflamma-tory response is not as excessive during that initial period and the procedure can be performed safely.Nonoperative Management of Acute Appendicitis. Despite the fact that surgical removal of the acutely inflammation appendix is effective in all cases, there has been a growing rec-ognition that certain children will respond to antibiotics alone and thus avoid surgery. Several trials have shown that acute appendicitis may be treated with antibiotics alone effectively in

1	rec-ognition that certain children will respond to antibiotics alone and thus avoid surgery. Several trials have shown that acute appendicitis may be treated with antibiotics alone effectively in nearly 80% of patients. However, the failure rate is considered unacceptably high for many patients, who effectively will have suffered a delay from definitive care. Furthermore, the hetero-geneity of disease presentation, and varying degree of illness severity, make it quite difficult to predict who will respond to antibiotics alone. This question is currently being answered in the United States in the form of a randomized controlled trial that is recruiting over 1500 patients in eight states, which will be divided into antibiotic therapy versus surgery (ClinicalTrials.gov, identifier NCT02800785).Other Causes of Abdominal Pain That Mimic Appendi-citis in Children. As mentioned earlier, appendicitis can be one of the most difficult diagnoses to establish in children with abdominal pain, in

1	Causes of Abdominal Pain That Mimic Appendi-citis in Children. As mentioned earlier, appendicitis can be one of the most difficult diagnoses to establish in children with abdominal pain, in part because of the large number of diseases that present in a similar fashion. Patients with urinary tract infection can present very similarly to those with appen-dicitis. However, patients with urinary tract infection are less likely to present with vomiting and are likely to also experience difficulty with urination, characterized by pressure, burning, and frequency. Constipation may be commonly confused with appendicitis in its earliest stages. However, patients with consti-pation rarely have fever and will not have abnormalities in their blood work. Ovarian torsion can mimic appendicitis, given the severe abdominal pain that accompanies this condition. How-ever, patients with ovarian torsion are generally asymptomatic until the acute onset of severe pain. By contrast, patients with

1	given the severe abdominal pain that accompanies this condition. How-ever, patients with ovarian torsion are generally asymptomatic until the acute onset of severe pain. By contrast, patients with appendicitis generally experience gradual onset of pain asso-ciated with nausea and vomiting. Finally, children and young adults are always at risk for the development of gastroenteritis. However, unlike appendicitis, patients with gastroenteritis gen-erally present with persistent vomiting and occasionally diar-rhea, which precedes the onset of the abdominal pain.Intestinal DuplicationsDuplications represent mucosa-lined structures that are in con-tinuity with the gastrointestinal tract. Although they can occur at any level in the gastrointestinal tract, duplications are found most commonly in the ileum within the leaves of the mesen-tery. Duplications may be long and tubular but usually are cystic masses. In all cases, they share a common wall with the intes-tine. Symptoms associated with

1	the ileum within the leaves of the mesen-tery. Duplications may be long and tubular but usually are cystic masses. In all cases, they share a common wall with the intes-tine. Symptoms associated with enteric duplication cysts include recurrent abdominal pain, emesis from intestinal obstruction, or hematochezia. Such bleeding typically results from ulceration in the duplication or in the adjacent intestine if the duplication contains ectopic gastric mucosa. On examination, a palpable mass is often identified. Children may also develop intestinal obstruction. Torsion may produce gangrene and perforation.The ability to make a preoperative diagnosis of enteric duplication cyst usually depends on the presentation. CT, US, and technetium pertechnetate scanning can be very helpful. Occasionally, a duplication can be seen on small bowel follow-through or barium enema. In the case of short duplications, resection of the cyst and adjacent intestine with end-to-end anastomosis can be performed.

1	a duplication can be seen on small bowel follow-through or barium enema. In the case of short duplications, resection of the cyst and adjacent intestine with end-to-end anastomosis can be performed. If resection of long duplications would compromise intestinal length, multiple enterotomies and mucosal stripping in the duplicated segment will allow the walls to collapse and become adherent. An alternative method is to divide the common wall using the GIA stapler, forming a com-mon lumen. Patients with duplications who undergo complete excision without compromise of the length of remaining intes-tine have an excellent prognosis.Meckel’s DiverticulumA Meckel’s diverticulum is a remnant of a portion of the embryonic omphalomesenteric (vitelline) duct. It is located on the antimesenteric border of the ileum, usually within 2 ft of the ileocecal valve (Fig. 39-22). It may be found incidentally at surgery or may present with inflammation masquerading as appendicitis. Perforation of a

1	border of the ileum, usually within 2 ft of the ileocecal valve (Fig. 39-22). It may be found incidentally at surgery or may present with inflammation masquerading as appendicitis. Perforation of a Meckel’s diverticulum may occur if the outpouching becomes impacted with food, leading to dis-tention and necrosis. Occasionally, bands of tissue extend from the Meckel’s diverticulum to the anterior abdominal wall, and these may represent lead points around which internal hernias may develop. This is an important cause of intestinal obstruction in the older child who has a scarless abdomen. Similar to dupli-cations, ectopic gastric mucosa may produce ileal ulcerations that bleed and lead to the passage of maroon-colored stools. Pancreatic mucosa may also be present. Diagnosis may be made by technetium pertechnetate scans when the patient presents with bleeding. Treatment is surgical. If the base is narrow and there is no mass present in the lumen of the diverticulum, a wedge resection of

1	pertechnetate scans when the patient presents with bleeding. Treatment is surgical. If the base is narrow and there is no mass present in the lumen of the diverticulum, a wedge resection of the diverticulum with transverse closure of the ileum can be performed. A linear stapler is especially useful in this circumstance. When a mass of ectopic tissue is palpable, if the base is wide, or when there is inflammation, it is prefer-able to perform a resection of the involved bowel and end-to-end ileoileostomy.Mesenteric CystsMesenteric cysts are similar to duplications in their location within the mesentery. However, they do not contain any mucosa or muscular wall. Chylous cysts may result from congenital Figure 39-22. Operative photograph showing the presence of a Meckel’s diverticulum (arrow).Brunicardi_Ch39_p1705-p1758.indd 173312/02/19 11:26 AM 1734SPECIFIC CONSIDERATIONSPART IIlymphatic obstruction. Mesenteric cysts can cause intestinal obstruction or may present as an abdominal

1	173312/02/19 11:26 AM 1734SPECIFIC CONSIDERATIONSPART IIlymphatic obstruction. Mesenteric cysts can cause intestinal obstruction or may present as an abdominal mass. The diagno-sis may be made by abdominal US or CT. Treatment involves surgical excision. This may require resection of the adjacent intestine, particularly for extensive, multicystic lesions. In cases where complete excision is not possible due to the close proxim-ity to vital structures, partial excision or marsupialization should be performed.Hirschsprung’s DiseasePathogenesis. In his classic textbook entitled Pediatric Sur-gery, Dr. Orvar Swenson, who is eponymously associated with one of the classic surgical treatments for Hirschsprung’s dis-ease, described this condition as follows: “Congenital megaco-lon is caused by a malformation in the pelvic parasympathetic system which results in the absence of ganglion cells in Auer-bach’s plexus of a segment of distal colon. Not only is there an absence of ganglion cells,

1	a malformation in the pelvic parasympathetic system which results in the absence of ganglion cells in Auer-bach’s plexus of a segment of distal colon. Not only is there an absence of ganglion cells, but the nerve fibers are large and excessive in number, indicating that the anomaly may be more extensive than the absence of ganglion cells.” This narrative of Hirschsprung’s disease is as accurate today as it was more than 50 years ago and summarizes the essential pathologic fea-tures of this disease: absence of ganglion cells in Auerbach’s plexus and hypertrophy of associated nerve trunks. The cause of Hirschsprung’s disease remains incompletely understood, although current thinking suggests that the disease results from a defect in the migration of neural crest cells, which are the embryonic precursors of the intestinal ganglion cell. Under normal conditions, the neural crest cells migrate into the intes-tine from cephalad to caudad. The process is completed by the 12th week of

1	precursors of the intestinal ganglion cell. Under normal conditions, the neural crest cells migrate into the intes-tine from cephalad to caudad. The process is completed by the 12th week of gestation, but the migration from midtransverse colon to anus takes 4 weeks. During this latter period, the fetus is most vulnerable to defects in migration of neural crest cells. This may explain why most cases of aganglionosis involve the rectum and rectosigmoid. The length of the aganglionic segment of bowel is therefore determined by the most distal region that the migrating neural crest cells reach. In rare instances, total colonic aganglionosis may occur.Recent studies have shed light on the molecular basis for Hirschsprung’s disease. Patients with Hirschsprung’s disease have an increased frequency of mutations in several genes, including GDNF, its receptor Ret, or its coreceptor Gfra-1. Moreover, mutations in these genes also lead to aganglionic megacolon in mice, which provides the

1	frequency of mutations in several genes, including GDNF, its receptor Ret, or its coreceptor Gfra-1. Moreover, mutations in these genes also lead to aganglionic megacolon in mice, which provides the opportunity to study the function of the encoded proteins. Initial investigations indicate that GDNF promotes the survival, proliferation, and migration of mixed populations of neural crest cells in culture. Other studies have revealed that GDNF is expressed in the gut in advance of migrating neural crest cells and is chemoattrac-tive for neural crest cells in culture. These findings raise the possibility that mutations in the GDNF or Ret genes could lead to impaired neural crest migration in utero and the development of Hirschsprung’s disease.Clinical Presentation. The incidence of sporadic Hirschsprung’s disease is 1 in 5000 live births. There are reports of increased frequency of Hirschsprung’s disease in multiple generations of the same family. Occasionally, such families have

1	Hirschsprung’s disease is 1 in 5000 live births. There are reports of increased frequency of Hirschsprung’s disease in multiple generations of the same family. Occasionally, such families have mutations in the genes described earlier, includ-ing the Ret gene. Because the aganglionic colon does not permit normal peristalsis to occur, the presentation of children with Hirschsprung’s disease is characterized by a functional distal intestinal obstruction. In the newborn period, the most common symptoms are abdominal distention, failure to pass meconium, and bilious emesis. Any infant who does not pass meconium beyond 48 hours of life must be investigated for the presence of Hirschsprung’s disease. Occasionally, infants present with a dra-matic complication of Hirschsprung’s disease called enteroco-litis. This pattern of presentation is characterized by abdominal distention and tenderness, and it is associated with manifesta-tions of systemic toxicity that include fever, failure to thrive,

1	This pattern of presentation is characterized by abdominal distention and tenderness, and it is associated with manifesta-tions of systemic toxicity that include fever, failure to thrive, and lethargy. Infants are often dehydrated and demonstrate a leukocytosis or increase in circulating band forms on hemato-logic evaluation. On rectal examination, forceful expulsion of foul-smelling liquid feces is typically observed and represents the accumulation of stool under pressure in an obstructed dis-tal colon. Treatment includes rehydration, systemic antibiotics, nasogastric decompression, and rectal irrigations while the diag-nosis of Hirschsprung’s disease is being confirmed. In children that do not respond to nonoperative management, a decompres-sive stoma is required. It is important to ensure that this stoma is placed in ganglion-containing bowel, which must be confirmed by frozen section at the time of stoma creation.In approximately 20% of cases, the diagnosis of Hirschsprung’s

1	ensure that this stoma is placed in ganglion-containing bowel, which must be confirmed by frozen section at the time of stoma creation.In approximately 20% of cases, the diagnosis of Hirschsprung’s disease is made beyond the newborn period. These children have severe constipation, which has usually been treated with laxatives and enemas. Abdominal distention and failure to thrive may also be present at diagnosis.Diagnosis. The definitive diagnosis of Hirschsprung’s disease is made by rectal biopsy. Samples of mucosa and submucosa are obtained at 1 cm, 2 cm, and 3 cm from the dentate line. This can be performed at the bedside in the neonatal period without anes-thesia, as samples are taken in bowel that does not have somatic innervation and is thus not painful to the child. In older children, the procedure should be performed using IV sedation. The histo-pathology of Hirschsprung’s disease is the absence of ganglion cells in the myenteric plexuses, increased acetylcholinesterase

1	children, the procedure should be performed using IV sedation. The histo-pathology of Hirschsprung’s disease is the absence of ganglion cells in the myenteric plexuses, increased acetylcholinesterase staining, and the presence of hypertrophied nerve bundles.It is important to obtain a barium enema in children in whom the diagnosis of Hirschsprung’s disease is suspected. This test may demonstrate the location of the transition zone between the dilated ganglionic colon and the distal constricted aganglionic rectal segment. Our practice is to obtain this test before instituting rectal irrigations if possible so that the differ-ence in size between the proximal and distal bowel is preserved. Although the barium enema can only suggest, but not reliably establish, the diagnosis of Hirschsprung’s disease, it is very useful in excluding other causes of distal intestinal obstruction. These include small left colon syndrome (as occurs in infants of diabetic mothers), colonic atresia, meconium

1	disease, it is very useful in excluding other causes of distal intestinal obstruction. These include small left colon syndrome (as occurs in infants of diabetic mothers), colonic atresia, meconium plug syndrome, or the unused colon observed in infants after the administration of magnesium or tocolytic agents. The barium enema in total colonic aganglionosis may show a markedly shortened colon. Some surgeons have found the use of rectal manometry helpful, particularly in older children, although it is relatively inaccurate.Treatment. The diagnosis of Hirschsprung’s disease requires surgery in all cases. The classic surgical approach consisted of a multiple stage procedure. This included a colostomy in the newborn period, followed by a definitive pull-through operation after the child was over 10 kg. There are three viable options for the definitive pull through procedure that are currently used. Although individual surgeons may advocate one procedure over another, studies have

1	was over 10 kg. There are three viable options for the definitive pull through procedure that are currently used. Although individual surgeons may advocate one procedure over another, studies have demonstrated that the outcome after each type of operation is similar. For each of 6Brunicardi_Ch39_p1705-p1758.indd 173412/02/19 11:26 AM 1735PEDIATRIC SURGERYCHAPTER 39the operations that is performed, the principles of treatment include confirming the location in the bowel where the transition zone between ganglionic and aganglionic bowel exists, resecting the aganglionic segment of bowel, and performing an anastomosis of ganglionated bowel to either the anus or a cuff of rectal mucosa (Fig. 39-23).It is now well established that a primary pull-through pro-cedure can be performed safely, even in the newborn period. This approach follows the same treatment principles as a staged procedure and saves the patient from an additional surgical Figure 39-23. The three operations for surgical

1	even in the newborn period. This approach follows the same treatment principles as a staged procedure and saves the patient from an additional surgical Figure 39-23. The three operations for surgical correction of Hirschsprung’s disease. A. The Duhamel procedure leaves the rec-tum in place and brings ganglionic bowel into the retrorectal space. B. The Swenson procedure is a resection with end-to-end anastomo-sis performed by exteriorizing bowel ends through the anus. C. The Soave operation is performed by endorectal dissection and removal of mucosa from the aganglionic distal segment and bringing the ganglionic bowel down to the anus within the seromuscular tunnel.procedure. Many surgeons perform the intra-abdominal dissec-tion using the laparoscope. This approach is especially useful in the newborn period as this provides excellent visualization of the pelvis. In children with significant colonic distention, it is important to allow for a period of decompression using a rectal tube

1	the newborn period as this provides excellent visualization of the pelvis. In children with significant colonic distention, it is important to allow for a period of decompression using a rectal tube if a single-staged pull-through is to be performed. In older children with very distended, hypertrophied colon, it may be prudent to perform a colostomy to allow the bowel to decom-press prior to performing a pull-through procedure. However, it should be emphasized that there is no upper age limit for per-forming a primary pull-through.Of the three pull-through procedures performed for Hirschsprung’s disease, the first is the original Swenson pro-cedure. In this operation, the aganglionic rectum is dissected in the pelvis and removed down to the anus. The ganglionic colon is then anastomosed to the anus via a perineal approach. In the Duhamel procedure, dissection outside the rectum is confined to the retrorectal space, and the ganglionic colon is anastomosed posteriorly just above the

1	to the anus via a perineal approach. In the Duhamel procedure, dissection outside the rectum is confined to the retrorectal space, and the ganglionic colon is anastomosed posteriorly just above the anus. The anterior wall of the gangli-onic colon and the posterior wall of the aganglionic rectum are anastomosed, using a stapler. Although both of these procedures are extremely effective, they are limited by the possibility of damage to the parasympathetic nerves that are adjacent to the rectum. To circumvent this potential problem, Soave’s proce-dure involves dissection entirely within the rectum. The rectal mucosa is stripped from the muscular sleeve, and the gangli-onic colon is brought through this sleeve and anastomosed to the anus. This operation may be performed completely from below. In all cases, it is critical that the level at which ganglion-ated bowel exists be determined. Most surgeons believe that the anastomosis should be performed at least 5 cm from the point at which

1	In all cases, it is critical that the level at which ganglion-ated bowel exists be determined. Most surgeons believe that the anastomosis should be performed at least 5 cm from the point at which ganglion cells are found. This avoids performing a pull-through in the transition zone, which is associated with a high incidence of complications due to inadequate emptying of the pull-through segment. Up to one-third of patients who undergo a transition zone pull through will require a reoperation.The main complications of all procedures include post-operative enterocolitis, constipation, and anastomotic stricture. There is also a reported incidence of recurrent Hirschsprung’s disease, which may reflect either residual aganglionic bowel left behind after the pull-through, or the presence of ischemia in the pulled-through segment leading to ganglion cell loss. Long-term results with the three procedures are comparable and generally excellent in experienced hands. These three procedures also

1	in the pulled-through segment leading to ganglion cell loss. Long-term results with the three procedures are comparable and generally excellent in experienced hands. These three procedures also can be adapted for total colonic aganglionosis in which the ileum is used for the pull-through segment.Anorectal MalformationsAnatomic Description. Anorectal malformations describe a spectrum of congenital anomalies that include imperforate anus and persistent cloaca. Anorectal malformations occur in approximately 1 in 5000 live births and affect males and females almost equally. The embryologic basis includes failure of descent of the urorectal septum. The level to which this septum descends determines the type of anomaly that is present, which subsequently influences the surgical approach.In patients with imperforate anus, the rectum fails to descend through the external sphincter complex. Instead, the rectal pouch ends “blindly” in the pelvis, above or below the levator ani muscle. In most

1	with imperforate anus, the rectum fails to descend through the external sphincter complex. Instead, the rectal pouch ends “blindly” in the pelvis, above or below the levator ani muscle. In most cases, the blind rectal pouch com-municates more distally with the genitourinary system or with the perineum through a fistulous tract. Traditionally, anatomic Brunicardi_Ch39_p1705-p1758.indd 173512/02/19 11:26 AM 1736SPECIFIC CONSIDERATIONSPART IIFigure 39-24. Low imperforate anus in a male. Note the well-developed buttocks. The perineal fistula was found at the midline raphe.Figure 39-25. Imperforate anus in a female. A catheter has been placed into the fistula, which is in the vestibule of the vagina.description of imperforate anus has been characterized as either “high” or “low” depending on whether the rectum ends above the levator ani muscle complex or partially descends through this muscle (Fig. 39-24). Based upon this classification system, in male patients with high imperforate

1	on whether the rectum ends above the levator ani muscle complex or partially descends through this muscle (Fig. 39-24). Based upon this classification system, in male patients with high imperforate anus the rectum usually ends as a fistula into the membranous urethra. In females, high imperforate anus often occurs in the context of a persistent clo-aca. In both males and females, low lesions are associated with a fistula to the perineum. In males, the fistula connects with the median raphe of the scrotum or penis. In females, the fistula may end within the vestibule of the vagina, which is located immediately outside the hymen or at the perineum.Because this classification system is somewhat arbitrary, Peña proposed a classification system that specifically and unambiguously describes the location of the fistulous opening. In men, the fistula may communicate with: (a) the perineum (cutaneous perineal fistula); (b) the lowest portion of the poste-rior urethra (rectourethral bulbar

1	the location of the fistulous opening. In men, the fistula may communicate with: (a) the perineum (cutaneous perineal fistula); (b) the lowest portion of the poste-rior urethra (rectourethral bulbar fistula); (c) the upper portion of the posterior urethra (rectourethral prostatic fistula); or (d) the bladder neck (rectovesicular fistula). In females, the ure-thra may open to the perineum between the female genitalia and the center of the sphincter (cutaneous perineal fistula) or into the vestibule of the vagina (vestibular fistula) (Fig. 39-25). In both sexes, the rectum may end in a completely blind fashion (imperforate anus without fistula). In rare cases, patients may have a normal anal canal, yet there may be total atresia or severe stenosis of the rectum.The most frequent defect in males is imperforate anus with rectourethral fistula, followed by rectoperineal fistula, then rectovesical fistula or rectobladder neck. In females, the most frequent defect is the rectovestibular

1	in males is imperforate anus with rectourethral fistula, followed by rectoperineal fistula, then rectovesical fistula or rectobladder neck. In females, the most frequent defect is the rectovestibular defect, followed by the cutaneous perineal fistula. The third most common defect in females is the persistent cloaca. This lesion represents a wide spectrum of malformations in which the rectum, vagina, and urinary tract meet and fuse into a single common channel. On physical examination, a single perineal orifice is observed, and it is located at the place where the urethra normally opens. Typi-cally, the external genitalia are hypoplastic.Associated Malformations. Approximately 60% of patients have an associated malformation. The most common is a urinary tract defect, which occurs in approximately 50% of patients. Skeletal defects are also seen, and the sacrum is most commonly involved. Spinal cord anomalies especially tethered cored are common, particularly in children with high

1	approximately 50% of patients. Skeletal defects are also seen, and the sacrum is most commonly involved. Spinal cord anomalies especially tethered cored are common, particularly in children with high lesions. Gastroin-testinal anomalies occur, most commonly esophageal atresia. Cardiac anomalies may be noted, and occasionally patients pres-ent with a constellation of defects as part of the VACTERLL syndrome (described earlier).Management of Patients With Imperforate Anus. Patients with imperforate anus are usually stable, and the diagnosis is readily apparent. Despite the obstruction, the abdomen is initially not distended, and there is rarely any urgency to intervene. The principles of management center around diagnosing the type of defect that is present (high vs. low), and evaluating the presence of associated anomalies. It may take up to 24 hours before the presence of a fistula on the skin is noted, and thus it is important to observe the neonate for some period of time before

1	the presence of associated anomalies. It may take up to 24 hours before the presence of a fistula on the skin is noted, and thus it is important to observe the neonate for some period of time before defini-tive surgery is undertaken. All patients should therefore have an orogastric tube placed and be monitored for the appearance of meconium in or around the perineum or in the urine. Investiga-tion for associated defects should include an US of the abdomen to assess for the presence of urinary tract anomaly. Other tests should include an echocardiogram and spinal radiographs. An US of the spine should be performed to look for the presence of a tethered cord. To further classify the location of the fistula as either “high” versus “low,” a lateral abdominal radiograph can be obtained with a radiopaque marker on the perineum. By placing the infant in the inverted position, the distance between the most distal extent of air in the rectum and the perineal surface can be measured. This study

1	marker on the perineum. By placing the infant in the inverted position, the distance between the most distal extent of air in the rectum and the perineal surface can be measured. This study is imprecise, however, and may add little to the overall management of these patients.The surgical management of infants with imperforate anus is determined by the anatomic defect. In general, when a low lesion is present, only a perineal operation is required without a colostomy. Infants with a high lesion require a colostomy in the newborn period, followed by a pull-through procedure at approximately 2 months of age. When a persistent cloaca is present, the urinary tract needs to be carefully evaluated at the time of colostomy formation to ensure that normal emptying can occur and to determine whether the bladder needs to be drained by means of a vesicostomy. If there is any doubt about the type of lesion, it is safer to perform a colostomy rather than jeopardize the infant’s long-term chances

1	the bladder needs to be drained by means of a vesicostomy. If there is any doubt about the type of lesion, it is safer to perform a colostomy rather than jeopardize the infant’s long-term chances for continence by an injudicious perineal operation.Brunicardi_Ch39_p1705-p1758.indd 173612/02/19 11:26 AM 1737PEDIATRIC SURGERYCHAPTER 39The type of pull-through procedure favored by most pedi-atric surgeons today is the posterior sagittal anorectoplasty (PSARP procedure), as described by Peña and DeVries. This involves placing the patient in the prone jack-knife position, dividing the levator ani and external sphincter complex in the midline posteriorly, dividing the communication between the gastrointestinal tract and the urinary tract, and bringing down the rectum after sufficient length is achieved. The muscles are then reconstructed and sutured to the rectum. The outcome of 1192 patients who had undergone this procedure has been reviewed by Peña and Hong. Seventy-five percent of

1	is achieved. The muscles are then reconstructed and sutured to the rectum. The outcome of 1192 patients who had undergone this procedure has been reviewed by Peña and Hong. Seventy-five percent of patients were found to have voluntary bowel movements, and nearly 40% were considered totally continent. As a rule, patients with high lesions demonstrate an increase incidence of incontinence, whereas those with low lesions are more likely to be consti-pated. Management of patients with high imperforate anus can be greatly facilitated using a laparoscopic assisted approach, in which the patient is operated on in the supine position, and the rectum is mobilized down to the fistulous connection to the bladder neck. This fistulous connection is then divided, and the rectum is completely mobilized down to below the peritoneal reflection. The operation then proceeds at the perineum, and the location of the muscle complex is determined using the nerve stimulator. A Veress needle is then advanced

1	down to below the peritoneal reflection. The operation then proceeds at the perineum, and the location of the muscle complex is determined using the nerve stimulator. A Veress needle is then advanced through the skin at the indicated site, with the laparoscope providing guidance to the exact intrapelvic orientation. Dilators are then placed over the Veress needle, the rectum is then pulled through this perito-neal opening, and an anoplasty is performed.JAUNDICEThe Approach to the Jaundiced InfantJaundice is present during the first week of life in 60% of term infants and 80% of preterm infants. There is usually accumula-tion of unconjugated bilirubin, but there may also be deposition of direct bilirubin. During fetal life, the placenta is the principal route of elimination of unconjugated bilirubin. In the newborn infant, bilirubin is conjugated through the activity of glucoronyl transferase. In the conjugated form, bilirubin is water soluble, which results in its excretion into the

1	bilirubin. In the newborn infant, bilirubin is conjugated through the activity of glucoronyl transferase. In the conjugated form, bilirubin is water soluble, which results in its excretion into the biliary system and then into the gastrointestinal tract. Newborns have a relatively high level of circulating hemoglobin and relative immaturity of the conjugating machinery. This results in a transient accumulation of bilirubin in the tissues, which is manifested as jaundice. Physi-ologic jaundice is evident by the second or third day of life and usually resolves within approximately 5 to 7 days. By definition, jaundice that persists beyond 2 weeks is considered pathologic.Pathologic jaundice may be due to biliary obstruction, increased hemoglobin load, or to liver dysfunction. The workup of the jaundiced infant therefore should include a search for the following possibilities: (a) obstructive disorders, including biliary atresia, choledochal cyst, and inspissated bile syndrome; (b)

1	of the jaundiced infant therefore should include a search for the following possibilities: (a) obstructive disorders, including biliary atresia, choledochal cyst, and inspissated bile syndrome; (b) hematologic disorders, including ABO incompatibility, Rh incompatibility, spherocytosis; (c) metabolic disorders, includ-ing α-1 antitrypsin deficiency, galactosemia; pyruvate kinase deficiency; and (d) congenital infection, including syphilis and rubella.Biliary AtresiaPathogenesis. Biliary atresia is a rare disease associated with significant morbidity and mortality. This disease is character-ized by a fibroproliferative obliteration of the biliary tree which progresses toward hepatic fibrosis, cirrhosis, and end-stage liver failure. The incidence of this disease is approximately 1 in 8000 to 1 in 18,000. The etiology of biliary atresia is likely multifac-torial. In the classic textbook, Abdominal Surgery of Infancy and Childhood, Ladd and Gross described the cause of biliary atresia as

1	1 in 18,000. The etiology of biliary atresia is likely multifac-torial. In the classic textbook, Abdominal Surgery of Infancy and Childhood, Ladd and Gross described the cause of biliary atresia as an “arrest of development during the solid stage of bile duct formation.” Previously proposed theories on the eti-ology of biliary atresia have focused on defects in hepatogen-esis, prenatal vasculogenesis, immune dysregulation, infectious agents, and exposure to toxins. More recently, genetic mutations in the cfc1 gene, implicated in left-right axis determinations, were identified in patients with biliary atresia-splenic malforma-tion syndrome. Additionally, the detection of higher incidence of maternal microchimerism in the livers of males with biliary atresia has led to the suggestion that consequent expression of maternal antigens may lead to an autoimmune process leading to inflammation and obliteration of the biliary tree. Recent ani-mal studies strongly implicate perinatal exposure

1	consequent expression of maternal antigens may lead to an autoimmune process leading to inflammation and obliteration of the biliary tree. Recent ani-mal studies strongly implicate perinatal exposure to reovirus or rotavirus. Such viral exposure may lead to periportal inflamma-tion mediated by interferon-γ and other cytokines.Clinical Presentation. Infants with biliary atresia present with jaundice at birth or shortly thereafter. The diagnosis of biliary atresia is frequently not entertained by pediatricians in part because physiologic jaundice of the newborn is so common and biliary atresia is so uncommon. As such, it is not unusual for there to be a delay in diagnosis. However, infants with bili-ary atresia characteristically have acholic, pale gray appearing stools, secondary to obstructed bile flow. With further passage of time, these infants manifest progressive failure to thrive, and if untreated, develop stigmata of liver failure and portal hyper-tension, particularly

1	to obstructed bile flow. With further passage of time, these infants manifest progressive failure to thrive, and if untreated, develop stigmata of liver failure and portal hyper-tension, particularly splenomegaly and esophageal varices.The obliterative process of biliary atresia involves the common duct, cystic duct, one or both hepatic ducts, and the gallbladder, in a variety of combinations. The histopathology of patients with biliary atresia includes inflammatory changes within the parenchyma of the liver, as well as fibrous deposi-tion at the portal plates that is observed on trichrome staining of frozen tissue sections. In certain cases, bile duct prolifera-tion may be seen, a relatively nonspecific marker of liver injury. Approximately 25% of patients with biliary atresia have coin-cidental malformations, often associated with polysplenia, and may include intestinal malrotation, preduodenal portal vein, and intrahepatic vena cava.Diagnosis. In general, the diagnosis of biliary

1	malformations, often associated with polysplenia, and may include intestinal malrotation, preduodenal portal vein, and intrahepatic vena cava.Diagnosis. In general, the diagnosis of biliary atresia is made utilizing a combination of studies, as no single test is suffi-ciently sensitive or specific. Fractionation of the serum bilirubin is performed to determine if the associated hyperbilirubinemia is conjugated or unconjugated. Workup commonly includes the analysis of TORCH infection titers as well as viral hepatitis. Typically, a US is performed to assess the presence of other causes of biliary tract obstruction, including choledochal cyst. The absence of a gallbladder is highly suggestive of the diagno-sis of biliary atresia. However, the presence of a gallbladder does not exclude the diagnosis of biliary atresia because in approxi-mately 10% of biliary atresia patients, the distal biliary tract is patent and a gall bladder may be visualized, even though the proximal ducts are

1	the diagnosis of biliary atresia because in approxi-mately 10% of biliary atresia patients, the distal biliary tract is patent and a gall bladder may be visualized, even though the proximal ducts are atretic. It is important to note that the intrahe-patic bile ducts are never dilated in patients with biliary atresia. In many centers, a nuclear medicine scan using technetium 99m IDA (DISIDA), performed after pretreatment of the patient with phenobarbital, has proven to be an accurate and reliable study. Brunicardi_Ch39_p1705-p1758.indd 173712/02/19 11:26 AM 1738SPECIFIC CONSIDERATIONSPART IIIf radionuclide appears in the intestine, there is patency of the biliary tree, and the diagnosis of biliary atresia is excluded. If radionuclide is concentrated by the liver but not excreted despite treatment with phenobarbital, and the metabolic screen, particu-larly α1-antitrypsin determination, is normal, the presumptive diagnosis is biliary atresia. A percutaneous liver biopsy might

1	despite treatment with phenobarbital, and the metabolic screen, particu-larly α1-antitrypsin determination, is normal, the presumptive diagnosis is biliary atresia. A percutaneous liver biopsy might potentially distinguish between biliary atresia and other sources of jaundice such as neonatal hepatitis. When these tests point to or cannot exclude the diagnosis of biliary atresia, surgical exploration is warranted. At surgery, a cholangiogram may be performed if possible, using the gallbladder as a point of access. This may be performed using a laparoscope. The cholangio-gram demonstrates the anatomy of the biliary tree, determines whether extrahepatic bile duct atresia is present, and evaluates whether there is distal bile flow into the duodenum. The cholan-giogram may demonstrate hypoplasia of the extrahepatic biliary system. This condition is associated with hepatic parenchymal disorders that cause severe intrahepatic cholestasis, including α1-antitrypsin deficiency and biliary

1	of the extrahepatic biliary system. This condition is associated with hepatic parenchymal disorders that cause severe intrahepatic cholestasis, including α1-antitrypsin deficiency and biliary hypoplasia (Alagille’s syn-drome). Alternatively, a cursory assessment of the extrahepatic biliary tree may clearly delineate the atresia.Inspissated Bile Syndrome. This term is applied to patients with normal biliary tracts who have persistent obstructive jaun-dice. Increased viscosity of bile and obstruction of the canaliculi are implicated as causes. The condition has been seen in infants receiving parenteral nutrition, but it is also encountered in con-ditions associated with hemolysis, or in cystic fibrosis. In some instances, no etiologic factors can be defined. Neonatal hepatitis may present in a similar fashion to biliary atresia. This disease is characterized by persistent jaundice due to acquired biliary inflammation without obliteration of the bile ducts. There may be a viral etiology,

1	a similar fashion to biliary atresia. This disease is characterized by persistent jaundice due to acquired biliary inflammation without obliteration of the bile ducts. There may be a viral etiology, and the disease is usually self-limited. In this case, cholangiography is both diagnostic and therapeutic.Treatment. If the diagnosis of biliary atresia is confirmed intraoperatively, then surgical treatment is undertaken at the same setting. Currently, first-line therapy consists of creation of a hepatoportoenterostomy, as described by Kasai. The purpose of this procedure is to promote bile flow into the intestine. The procedure is based on Kasai’s observation that the fibrous tissue at the porta hepatis invests microscopically patent biliary duct-ules that, in turn, communicate with the intrahepatic ductal sys-tem (Fig. 39-26). Transecting this fibrous tissue at the portal Figure 39-26. Operative photograph showing Kasai portoenteros-tomy. Arrows denote the site of the anastomosis. Note

1	ductal sys-tem (Fig. 39-26). Transecting this fibrous tissue at the portal Figure 39-26. Operative photograph showing Kasai portoenteros-tomy. Arrows denote the site of the anastomosis. Note the engorged liver.Figure 39-27. Schematic illustration of the Kasai portoenteros-tomy for biliary atresia. An isolated limb of jejunum is brought to the porta hepatis and anastomosed to the transected ducts at the liver plate.plate, invariably encountered cephalad to the bifurcating portal vein, opens these channels and establishes bile flow into a surgi-cally constructed intestinal conduit, usually a Roux-en-Y limb of jejunum (Fig. 39-27). Some authors believe that an intussus-cepted antireflux valve is useful in preventing retrograde bile reflux, although the data suggest that it does not impact out-come. A liver biopsy is performed at the time of surgery to determine the degree of hepatic fibrosis that is present. The diameter of bile ducts at the portal plate is predictive of likeli-hood of

1	A liver biopsy is performed at the time of surgery to determine the degree of hepatic fibrosis that is present. The diameter of bile ducts at the portal plate is predictive of likeli-hood of long-term success of biliary drainage through the por-toenterostomy. Numerous studies also suggest that the likelihood of surgical success is inversely related to the age at the time of portoenterostomy. Infants treated prior to 60 days of life are more likely to achieve successful and long-term biliary drainage than older infants. Although the outlook is less favor-able for patients after the 12th week, it is reasonable to proceed with surgery even beyond this time point, as the alternative is certain liver failure. It is noteworthy that a significant number of patients have had favorable outcomes after undergoing portoen-terostomy despite advanced age at time of diagnosis.Bile drainage is anticipated when the operation is carried out early; however, bile flow does not necessarily imply cure.

1	after undergoing portoen-terostomy despite advanced age at time of diagnosis.Bile drainage is anticipated when the operation is carried out early; however, bile flow does not necessarily imply cure. Approximately one-third of patients remain symptom free after portoenterostomy, the remainder require liver transplantation due to progressive liver failure. Independent risk factors that predict failure of the procedure include bridging liver fibrosis at the time of surgery and postoperative cholangitic episodes. A review of the data of the Japanese Biliary Atresia Registry (JBAR), which 7Brunicardi_Ch39_p1705-p1758.indd 173812/02/19 11:26 AM 1739PEDIATRIC SURGERYCHAPTER 39includes the results of 1381 patients, showed that the 10-year survival rate was 53% without transplantation, and 66.7% with transplantation. A common postoperative complication is cholangitis. There is no effective strategy to completely eliminate this complication, and the effectiveness of long-term prophylactic

1	with transplantation. A common postoperative complication is cholangitis. There is no effective strategy to completely eliminate this complication, and the effectiveness of long-term prophylactic antibiotics has not been fully resolved. The Childhood Liver Research and Education Network (ChiLDREN, formerly the Biliary Atresia Research Consortium) is an active consortium of 15 children’s hospitals in the United States, funded by the National Institutes of Health (NIH) that studies rare cholestatic liver diseases of infants and children (http://childrennetwork.org). An NIH-funded, randomized, double-blinded, placebo-controlled trial designed to determine if adjuvant steroids improve outcome of infants undergoing Kasai portoenterostomy has been completed. This trial showed that among infants with biliary atresia who have undergone hepatoportoenterostomy, high-dose steroid therapy following surgery did not result in statistically significant treatment differences in bile drainage at 6

1	with biliary atresia who have undergone hepatoportoenterostomy, high-dose steroid therapy following surgery did not result in statistically significant treatment differences in bile drainage at 6 months, although a small clinical benefit could not be excluded. Steroid treatment was associated with earlier onset of serious adverse events in children with biliary atresia.Previous authors have published merits of revising the portoenterostomy in select patients if drainage of bile stops. Recently, Bondoc et al reported on their experience with revision of portoenterostomies. Specifically, the authors reported on 183 patients who underwent Kasai portoenterostomy for biliary atresia, of which 24 underwent revision for recurrence of nondrainage after successful bypass. Of the patients who underwent revision for nondrainage, 75% ultimately achieved drainage after the second procedure, of which nearly 50% survived long term with their native livers. The authors conclude that in selected

1	underwent revision for nondrainage, 75% ultimately achieved drainage after the second procedure, of which nearly 50% survived long term with their native livers. The authors conclude that in selected patients in which bile flow was established following the Kasai procedure and then lost, revision of the portoenterostomy is a reasonable treatment option with good success.Choledochal CystClassification. The term choledochal cyst refers to a spec-trum of congenital biliary tract disorders that were previously grouped under the name idiopathic dilation of the common bile duct. After the classification system proposed by Alonso-Lej, five types of choledochal cyst are described. Type I cyst is char-acterized by fusiform dilatation of the bile duct. This is the most common type and is found in 80% to 90% of cases. Type II choledochal cysts appear as an isolated diverticulum protruding from the wall of the common bile duct. The cyst may be joined to the common bile duct by a narrow stalk.

1	80% to 90% of cases. Type II choledochal cysts appear as an isolated diverticulum protruding from the wall of the common bile duct. The cyst may be joined to the common bile duct by a narrow stalk. Type III choledochal cysts arise from the intraduodenal portion of the common bile duct and are also known as choledochoceles. Type IVA cysts consist of multiple dilatations of the intrahepatic and extra-hepatic bile ducts. Type IVB choledochal cysts are multiple dilatations involving only the extrahepatic bile ducts. Type V (Caroli’s disease) consists of multiple dilatations limited to the intrahepatic bile ducts.Choledochal cyst is most appropriately considered the pre-dominant feature in a constellation of pathologic abnormalities that can occur within the pancreato-biliary system. Frequently associated with choledochal cyst is an anomalous junction of the pancreatic and common bile ducts. The etiology of choledochal cyst is controversial. Babbit proposed an abnormal pancreatic and

1	associated with choledochal cyst is an anomalous junction of the pancreatic and common bile ducts. The etiology of choledochal cyst is controversial. Babbit proposed an abnormal pancreatic and biliary duct junction, with the formation of a “common channel” into which pancreatic enzymes are secreted. This process results in weakening of the bile duct wall by gradual enzymatic destruction, leading to dilatation, inflammation, and finally cyst formation. Not all patients with choledochal cyst demonstrate an anatomic common channel, which raises ques-tions regarding the accuracy of this model.Clinical Presentation. Choledochal cyst is more common in females than in males (4:1). Typically, these present in children beyond the toddler age group. The classic symptom triad consists of abdominal pain, mass, and jaundice. However, this complex is actually encountered in fewer than half of the patients. The more usual presentation is that of episodic abdominal pain, often recurring over the

1	pain, mass, and jaundice. However, this complex is actually encountered in fewer than half of the patients. The more usual presentation is that of episodic abdominal pain, often recurring over the course of months or years, and generally asso-ciated with only minimal jaundice that may escape detection. If left undiagnosed, patients may develop cholangitis or pancreatitis. Cholangitis may lead to the development of cirrhosis and portal hypertension. Choledochal cyst can present in the newborn period, where the symptoms are very similar to those of biliary atresia. Often neonates will have an abdominal mass at presentation.Diagnosis. Choledochal cyst is frequently diagnosed in the fetus at a screening prenatal US. In the older child or adoles-cent, abdominal US may reveal a cystic structure arising from the biliary tree. CT will confirm the diagnosis. These studies will demonstrate the dimensions of the cyst and define its rela-tionship to the vascular structures in the porta hepatis,

1	arising from the biliary tree. CT will confirm the diagnosis. These studies will demonstrate the dimensions of the cyst and define its rela-tionship to the vascular structures in the porta hepatis, as well as the intrahepatic ductal configuration. Endoscopic retrograde cholangiopancreatography (ERCP) is reserved for patients in whom confusion remains after evaluation by less invasive imag-ing modalities. Magnetic resonance cholangiopancreatography may provide a more detailed depiction of the anatomy of the cyst and its relationship to the bifurcation of the hepatic ducts and into the pancreas.Treatment. The cyst wall is composed of fibrous tissue and is devoid of mucosal lining. As a result, the treatment of cho-ledochal cyst is surgical excision followed by biliary-enteric reconstruction. There is no role for internal drainage by cys-tenterostomy, which leaves the cyst wall intact and leads to the inevitable development of cholangitis. Rarely, choledochal cyst can lead to the

1	There is no role for internal drainage by cys-tenterostomy, which leaves the cyst wall intact and leads to the inevitable development of cholangitis. Rarely, choledochal cyst can lead to the development of a biliary tract malignancy. This provides a further rationale for complete cyst excision.Resection of the cyst may be performed via open or laparo-scopic approach, and where possible, requires circumferential dis-section. The posterior plane between the cyst and portal vein must be carefully dissected to accomplish removal. The pancreatic duct, which may enter the distal cyst, is vulnerable to injury dur-ing distal cyst excision but can be avoided by avoiding entry into the pancreatic parenchyma. In cases were the degree of pericystic inflammation is dense, it may be unsafe to attempt complete cyst removal. In this instance, it is reasonable to dissect within the posterior wall of the cyst, which allows the inner lining of the back wall to be dissected free from the outer layer that

1	cyst removal. In this instance, it is reasonable to dissect within the posterior wall of the cyst, which allows the inner lining of the back wall to be dissected free from the outer layer that directly overlies the portal vascular structures. The lateral and anterior cyst, as well as the internal aspect of the back wall, is removed, yet the outer posterior wall remains behind. Cyst excision is accomplished, and the proximal bile duct is anastomosed to the intestinal tract typically via a Roux-en Y limb of jejunum. More recently, laparoscopic-assisted resections of choledochal cysts have been described. In these cases, the end-to-side jejunojeju-nostomy is performed extracorporeally, but the remainder of the procedure is completed utilizing minimally invasive techniques.The prognosis for children who have undergone com-plete excision of choledochal cyst is excellent. Complications include anastomotic stricture, cholangitis, and intrahepatic stone Brunicardi_Ch39_p1705-p1758.indd

1	children who have undergone com-plete excision of choledochal cyst is excellent. Complications include anastomotic stricture, cholangitis, and intrahepatic stone Brunicardi_Ch39_p1705-p1758.indd 173912/02/19 11:26 AM 1740SPECIFIC CONSIDERATIONSPART IIformation. These complications may develop a long time after surgery has been completed.DEFORMITIES OF THE ABDOMINAL WALLEmbryology of the Abdominal WallThe abdominal wall is formed by four separate embryologic folds: cephalic, caudal, right, and left lateral folds. Each of these is com-posed of somatic and splanchnic layers and develops toward the anterior center portion of the coelomic cavity, joining to form a large umbilical ring that surrounds the two umbilical arteries, the vein, and the yolk sac or omphalomesenteric duct. These struc-tures are covered by an outer layer of amnion, and the entire unit composes the umbilical cord. Between the 5th and tenth weeks of fetal development, the intestinal tract undergoes rapid growth

1	struc-tures are covered by an outer layer of amnion, and the entire unit composes the umbilical cord. Between the 5th and tenth weeks of fetal development, the intestinal tract undergoes rapid growth outside the abdominal cavity within the proximal portion of the umbilical cord. As development is completed, the intestine gradu-ally returns to the abdominal cavity. Contraction of the umbilical ring completes the process of abdominal wall formation.Failure of the cephalic fold to close results in sternal defects such as congenital absence of the sternum. Failure of the caudal fold to close results in exstrophy of the bladder and, in more extreme cases, exstrophy of the cloaca. Interruption of central migration of the lateral folds results in omphalocele. Gastroschisis, originally thought to be a variant of omphalocele, possibly results from a fetal accident in the form of intrauterine rupture of a hernia of the umbilical cord, although other hypoth-eses have been advanced.Umbilical

1	to be a variant of omphalocele, possibly results from a fetal accident in the form of intrauterine rupture of a hernia of the umbilical cord, although other hypoth-eses have been advanced.Umbilical HerniaFailure of the umbilical ring to close results in a central defect in the linea alba. The resulting umbilical hernia is covered by nor-mal umbilical skin and subcutaneous tissue, but the fascial defect allows protrusion of abdominal contents. Hernias less than a cen-timeter in size at the time of birth usually will close spontaneously by 4 to 5 years of life and in most cases should not undergo early repair. Sometimes the hernia is large enough that the protrusion is disfiguring and disturbing to both the child and the family. In such circumstances, early repair may be advisable (Fig. 39-28).Figure 39-28. Umbilical hernia in a 1-year-old female.Umbilical hernias are generally asymptomatic protrusions of the abdominal wall. They are generally noted by parents or physicians shortly

1	39-28).Figure 39-28. Umbilical hernia in a 1-year-old female.Umbilical hernias are generally asymptomatic protrusions of the abdominal wall. They are generally noted by parents or physicians shortly after birth. All families of patients with umbilical hernia should be counseled about signs of incarcera-tion, which is rare in umbilical hernias and more common in smaller (1 cm or less) rather than larger defects. Incarceration presents with abdominal pain, bilious emesis, and a tender, hard mass protruding from the umbilicus. This constellation of symp-toms mandates immediate exploration and repair of the hernia to avoid strangulation. More commonly, the child is asymptomatic and treatment is governed by the size of the defect, the age of the patient, and the concern that the child and family have regard-ing the cosmetic appearance of the abdomen. When the defect is small and spontaneous closure is likely, most surgeons will delay surgical correction until 5 years of age. If closure

1	have regard-ing the cosmetic appearance of the abdomen. When the defect is small and spontaneous closure is likely, most surgeons will delay surgical correction until 5 years of age. If closure does not occur by this time or a younger child has a very large or symp-tomatic hernia, it is reasonable to proceed to repair.Repair of uncomplicated umbilical hernia is performed under general anesthesia as an outpatient procedure. A small curving incision that fits into the skin crease of the umbilicus is made, and the sac is dissected free from the overlying skin. The fascial defect is repaired with permanent or long-lasting absorb-able, interrupted sutures that are placed in a transverse plane. The skin is closed using subcuticular sutures. The postoperative recovery is typically uneventful and recurrence is rare, but it is more common in children with elevated intraabdominal pres-sures, such as those with a VP shunt.Patent UrachusDuring the development of the coelomic cavity, there is free

1	recurrence is rare, but it is more common in children with elevated intraabdominal pres-sures, such as those with a VP shunt.Patent UrachusDuring the development of the coelomic cavity, there is free communication between the urinary bladder and the abdominal wall through the urachus, which exits adjacent to the omphalo-mesenteric duct. Persistence of this tract results in a communi-cation between the bladder and the umbilicus. The first sign of a patent urachus is moisture or urine flow from the umbilicus. Recurrent urinary tract infection can result. The urachus may be partially obliterated, with a remnant beneath the umbilicus in the extraperitoneal position as an isolated cyst that may be identi-fied by US. A urachal cyst usually presents as an inflammatory mass inferior to the umbilicus. Initial treatment is drainage of the infected cyst followed by cyst excision as a separate proce-dure once the inflammation has resolved.In the child with a persistently draining umbilicus, a

1	Initial treatment is drainage of the infected cyst followed by cyst excision as a separate proce-dure once the inflammation has resolved.In the child with a persistently draining umbilicus, a diag-nosis of patent urachus should be considered. The differential diagnosis includes an umbilical granuloma, which generally responds to local application of silver nitrate. The diagnosis of patent urachus is confirmed by umbilical exploration. The ura-chal tract is excised and the bladder is closed with an absorbable suture. A patent vitelline duct may also present with umbilical drainage. In this circumstance, there is a communication with the small intestine, often at the site of a Meckel’s diverticulum. Treatment includes umbilical exploration with resection of the duct remnant (Fig. 39-29).OmphalocelePresentation. Omphalocele refers to a congenital defect of the abdominal wall in which the bowel and solid viscera are covered by peritoneum and amniotic membrane (Fig. 39-30). The umbil-ical

1	refers to a congenital defect of the abdominal wall in which the bowel and solid viscera are covered by peritoneum and amniotic membrane (Fig. 39-30). The umbil-ical cord inserts into the sac. Omphalocele can vary from a small defect with intestinal contents to giant omphalocele in which the abdominal wall defect measures 4 cm or more in diameter and contains liver. The overall incidence is approximately 1 in 5000 Brunicardi_Ch39_p1705-p1758.indd 174012/02/19 11:26 AM 1741PEDIATRIC SURGERYCHAPTER 39Figure 39-29. Patent vitelline duct. Note the communication between the umbilicus and the small bowel at the site of a Meckel’s diverticulum.Figure 39-30. Giant omphalocele in a newborn male.live births, with 1 in 10,000 that are giant omphaloceles. Omphalocele occurs in association with special syndromes such as exstrophy of the cloaca (vesicointestinal fissure), the Beckwith-Wiedemann constellation of anomalies (macroglos-sia, macrosomia, hypoglycemia, and visceromegaly and

1	with special syndromes such as exstrophy of the cloaca (vesicointestinal fissure), the Beckwith-Wiedemann constellation of anomalies (macroglos-sia, macrosomia, hypoglycemia, and visceromegaly and omphalocele) and Cantrell’s Pentalogy (lower thoracic wall malformations [cleft sternum], ectopia cordis, epigastric omphalocele, anterior midline diaphragmatic hernia and cardiac anomalies). There is a 60% to 70% incidence of associated anomalies, especially cardiac (20–40% of cases) and chromo-somal abnormalities. Chromosomal anomalies are more common in children with smaller defects. Omphalocele is associated with prematurity (10–50% of cases) and intrauterine growth restriction (20% of cases).Treatment. Immediate treatment of an infant with omphalocele consists of attending to the vital signs and maintaining the body 8temperature. A blood glucose should be evaluated because of the association with Beckwith-Wiedemann. The omphalocele should be covered to reduce fluid loss, but moist

1	signs and maintaining the body 8temperature. A blood glucose should be evaluated because of the association with Beckwith-Wiedemann. The omphalocele should be covered to reduce fluid loss, but moist dressings may result in heat loss and are not indicated. No pressure should be placed on the omphalocele sac in an effort to reduce its contents because this maneuver may increase the risk of rupture of the sac or may interfere with abdominal venous return. Prophylac-tic broad-spectrum antibiotics should be administered in case of rupture. The subsequent treatment and outcome is determined by the size of the omphalocele. In general terms, small to medium-sized defects have a significantly better prognosis than extremely large defects in which the liver is present. In these cases, not only is the management of the abdominal wall defect a significant challenge, but these patients often have concomitant pulmonary insufficiency that can lead to significant morbidity and mortality. If possible,

1	management of the abdominal wall defect a significant challenge, but these patients often have concomitant pulmonary insufficiency that can lead to significant morbidity and mortality. If possible, and if the pulmonary status will permit it, a primary repair of the omphalocele should be undertaken. This involves resection of the omphalocele membrane and closure of the fas-cia. A layer of prosthetic material may be required to achieve closure. In infants with a giant omphalocele, the defect cannot be closed primarily because there is not adequate intraperitoneal domain to reduce the viscera (see Fig. 39-30). Some infants may have associated congenital anomalies that complicate surgical repair, and because cardiac anomalies are common, an echocar-diogram should be obtained prior to any procedure. If repair is contraindicated, such as with a very large defect, a nonopera-tive approach can be used. The omphalocele sac can be treated with topical treatments, which serve to harden the sac

1	If repair is contraindicated, such as with a very large defect, a nonopera-tive approach can be used. The omphalocele sac can be treated with topical treatments, which serve to harden the sac to allow for more protective coverage where muscle and skin cannot be used given the large defect. Various authors describe success with iodine-containing solutions, silver sulfadiazine, or saline, and some surgeons rotate these solutions because of the impact of iodine on the thyroid and the difficulty of cleaning off all of the silver sulfadiazine and its association with leukopenia. It typically takes 2 to 3 months before reepithelialization occurs. In the past, mercury compounds were used, but they have been discontinued because of associated systemic toxicity. After epi-thelialization has occurred, attempts should be made to achieve closure of the anterior abdominal wall but may be delayed by associated pulmonary insufficiency. Such procedures typically require complex measures to achieve

1	attempts should be made to achieve closure of the anterior abdominal wall but may be delayed by associated pulmonary insufficiency. Such procedures typically require complex measures to achieve skin closure, including the use of biosynthetic materials or component separation. In cases of giant omphalocele, prolonged hospitalization is typical. If the base is very narrow—which can occur even for babies with very large omphaloceles—it may be wise to open the base in order to allow the abdominal contents and the liver to reenter the abdominal cavity, and thereby achieve abdominal domain. This approach will, by necessity, require sewing in some synthetic material in order to achieve fascial closure, and prolonged hos-pitalization will be required to allow for skin coverage to occur. These patients require high amounts of caloric support, given the major demands for healing.GastroschisisPresentation. Gastroschisis represents a congenital anom-aly characterized by a defect in the anterior

1	require high amounts of caloric support, given the major demands for healing.GastroschisisPresentation. Gastroschisis represents a congenital anom-aly characterized by a defect in the anterior abdominal wall through which the intestinal contents freely protrude. Unlike omphalocele, there is no overlying sac, and the size of the defect is usually <4 cm. The abdominal wall defect is located at the junction of the umbilicus and normal skin, and is almost always to the right of the umbilicus (Fig. 39-31). The umbilicus becomes partly detached, allowing free communication with the Brunicardi_Ch39_p1705-p1758.indd 174112/02/19 11:26 AM 1742SPECIFIC CONSIDERATIONSPART IIFigure 39-31. Gastroschisis in a newborn. Note the location of the umbilical cord and the edematous, thickened bowel.Figure 39-32. Prenatal ultrasound of a 30-week gestation age fetus with a gastroschisis. Arrows point to the bowel outside within the amniotic fluid.Figure 39-33. Use of a silo in a patient with a

1	39-32. Prenatal ultrasound of a 30-week gestation age fetus with a gastroschisis. Arrows point to the bowel outside within the amniotic fluid.Figure 39-33. Use of a silo in a patient with a gastroschisis to allow for the bowel wall edema to resolve so as to facilitate closure of the abdominal wall.abdominal cavity. The appearance of the bowel provides some information with respect to the in-utero timing of the defect. The intestine may be normal in appearance, suggesting that the rupture occurred relatively late during the pregnancy. More commonly, however, the intestine is thick, edematous, discol-ored, and covered with exudate, implying a more longstanding process. Progression to full enteral feeding is usually delayed, with diminished motility that may be related to these changes.Unlike infants born with omphalocele, associated anoma-lies are not usually seen with gastroschisis except for a 10% rate of intestinal atresia. This defect can readily be diagnosed on prenatal US (Fig.

1	infants born with omphalocele, associated anoma-lies are not usually seen with gastroschisis except for a 10% rate of intestinal atresia. This defect can readily be diagnosed on prenatal US (Fig. 39-32). There is no advantage to perform-ing a cesarean section instead of a vaginal delivery. In a decade long retrospective review, early deliver did not affect the thick-ness of bowel peel, yet patients delivered before 36 weeks had significantly longer length of stay in the hospital and time to enteral feeds. Based upon these findings, it is thought that fetal well-being should be the primary determinant of delivery for gastroschisis.Treatment. All infants born with gastroschisis require urgent surgical treatment. Of equal importance, these infants require vigorous fluid resuscitation in the range of 160 to 190 cc/kg per day to replace significant evaporative fluid losses. In many instances, the intestine can be returned to the abdominal cavity, and a primary surgical closure of the

1	the range of 160 to 190 cc/kg per day to replace significant evaporative fluid losses. In many instances, the intestine can be returned to the abdominal cavity, and a primary surgical closure of the abdominal wall is per-formed. Some surgeons believe that they facilitate primary closure with mechanical stretching of the abdominal wall, thor-ough orogastric suctioning with foregut decompression, rectal irrigation, and evacuation of meconium. Care must be taken to prevent markedly increased abdominal pressure during the reduction, which will lead to compression of the inferior vena cava, respiratory embarrassment, and abdominal compartment syndrome. To avoid this complication, it is helpful to moni-tor the bladder or airway pressures during reduction. In infants whose intestine has become thickened and edematous, it may be impossible to reduce the bowel into the peritoneal cavity in the immediate postnatal period. Under such circumstances, a plastic spring-loaded silo can be placed

1	thickened and edematous, it may be impossible to reduce the bowel into the peritoneal cavity in the immediate postnatal period. Under such circumstances, a plastic spring-loaded silo can be placed onto the bowel and secured beneath the fascia or a sutured silastic silo constructed. The silo covers the bowel and allows for graduated reduc-tion on a daily basis as the edema in the bowel wall decreases (Fig. 39-33). It is important to ensure that the silo-fascia junc-tion does not become a constricting point or “funnel,” in which case the intestine will be injured upon return to the peritoneum. In this case, the fascial opening must be enlarged. Surgical clo-sure can usually be accomplished within approximately 1 to 2 weeks. A prosthetic piece of material may be required to bring the edges of the fascia together. If an atresia is noted at the time of closure, it is prudent to reduce the bowel at the first operation and return after several weeks once the edema has resolved to correct

1	of the fascia together. If an atresia is noted at the time of closure, it is prudent to reduce the bowel at the first operation and return after several weeks once the edema has resolved to correct the atresia. Intestinal function does not typically return for several weeks in patients with gastroschisis. This is especially true if the bowel is thickened and edematous. As a result, these patients will require central line placement and institution of total parenteral nutrition in order to grow. Feeding advancement should be slow and typically requires weeks to arrive at full enteral nutrition.Brunicardi_Ch39_p1705-p1758.indd 174212/02/19 11:27 AM 1743PEDIATRIC SURGERYCHAPTER 39There has been recent success with the utilization of non-surgical closure of gastroschisis. In this technique, the umbili-cal cord is placed over the defect, which is then covered with a transparent occlusive dressing. Over the ensuing days, the cord provides a tissue barrier, and the defect spontaneously

1	the umbili-cal cord is placed over the defect, which is then covered with a transparent occlusive dressing. Over the ensuing days, the cord provides a tissue barrier, and the defect spontaneously closes. This approach allows for nonsurgical coverage in a majority of cases of gastroschisis, even in the setting of very large openings. Questions remain regarding the long-term presence of umbilical hernias in these children and the total hospitalization.Prune-Belly SyndromeClinical Presentation. Prune-belly syndrome refers to a dis-order that is characterized by extremely lax lower abdominal musculature, dilated urinary tract including the bladder, and bilateral undescended testes (Fig. 39-34). The term prune-belly syndrome appropriately describes the wrinkled appearance of the anterior abdominal wall that characterizes these patients. Prune-belly syndrome is also known as Eagle-Barrett syn-drome as well as the triad syndrome because of the three major manifestations. The incidence is

1	wall that characterizes these patients. Prune-belly syndrome is also known as Eagle-Barrett syn-drome as well as the triad syndrome because of the three major manifestations. The incidence is significantly higher in males. Patients manifest a variety of comorbidities. The most signifi-cant is pulmonary hypoplasia, which can be unsurvivable in the most severe cases. Skeletal abnormalities include dislocation or dysplasia of the hip and pectus excavatum.The major genitourinary manifestation in prune-belly syn-drome is ureteral dilation. The ureters are typically long and tortuous and become more dilated distally. Ureteric obstruction is rarely present, and the dilation may be caused by decreased smooth muscle and increased collagen in the ureters. Approxi-mately eighty percent of these patients will have some degree of vesicureteral reflux, which can predispose to urinary tract infection. Despite the marked dilatation of the urinary tract, most children with prune-belly syndrome have

1	will have some degree of vesicureteral reflux, which can predispose to urinary tract infection. Despite the marked dilatation of the urinary tract, most children with prune-belly syndrome have adequate renal parenchyma for growth and development. Factors associated with the development of long-term renal failure include the presence of abnormal kidneys on US or renal scan and persis-tent pyelonephritis.Treatment. Despite the ureteric dilation, there is currently no role for ureteric surgery unless an area of obstruction develops. The testes are invariably intraabdominal, and bilateral orchido-pexy can be performed in conjunction with abdominal wall recon-struction at 6 to 12 months of age. Despite orchiopexy, fertility in Figure 39-34. Eagle-Barrett (prune-belly) syndrome. Notice the lax, flaccid abdomen.a boy with prune-belly syndrome is unlikely as spermatogenesis over time is insufficient. Deficiencies in the production of pros-tatic fluid and a predisposition to retrograde

1	lax, flaccid abdomen.a boy with prune-belly syndrome is unlikely as spermatogenesis over time is insufficient. Deficiencies in the production of pros-tatic fluid and a predisposition to retrograde ejaculation contrib-ute to infertility. Abdominal wall repair is accomplished through an abdominoplasty, which typically requires a transverse inci-sion in the lower abdomen extending into the flanks.Inguinal HerniaAn understanding of the management of pediatric inguinal her-nias is a central component of modern pediatric surgical prac-tice. Inguinal hernia repair represents one of the most common operations performed in children. The presence of an inguinal hernia in a child is an indication for surgical repair. The opera-tion is termed a herniorrhaphy because it involves closing off the patent processus vaginalis. This is to be contrasted with the hernioplasty that is performed in adults, which requires a recon-struction of the inguinal floor.Embryology. In order to understand how to

1	processus vaginalis. This is to be contrasted with the hernioplasty that is performed in adults, which requires a recon-struction of the inguinal floor.Embryology. In order to understand how to diagnose and treat inguinal hernias in children, it is critical to understand their embryologic origin. It is very useful to describe these events to the parents, who often are under the misconception that the her-nia was somehow caused by their inability to console their crying child, or the child’s high activity level. Inguinal hernia results from a failure of closure of the processus vaginalis; a finger-like projection of the peritoneum that accompanies the testicle as it descends into the scrotum. Closure of the processus vaginalis normally occurs a few months prior to birth. This explains the high incidence of inguinal hernias in premature infants. When the processes vaginalis remains completely patent, a commu-nication persists between the peritoneal cavity and the groin, resulting in a

1	high incidence of inguinal hernias in premature infants. When the processes vaginalis remains completely patent, a commu-nication persists between the peritoneal cavity and the groin, resulting in a hernia. Partial closure can result in entrapped fluid, which results in the presence of a hydrocele. A communicating hydrocele refers to a hydrocele that is in communication with the peritoneal cavity and can therefore be thought of as a hernia. Using the classification system that is typically applied to adult hernias, all congenital hernias in children are by definition indi-rect inguinal hernias. Children also present with direct inguinal and femoral hernias, although these are much less common.Clinical Manifestation. Inguinal hernias occur more com-monly in males than females (10:1) and are more common on the right side than the left. Infants are at high risk for incar-ceration of an inguinal hernia because of the narrow inguinal ring. Patients most commonly present with a groin bulge

1	more common on the right side than the left. Infants are at high risk for incar-ceration of an inguinal hernia because of the narrow inguinal ring. Patients most commonly present with a groin bulge that is noticed by the parents as they change the diaper (Fig. 39-35). Figure 39-35. Right inguinal hernia in a 4-month-old male. The arrows point to the bulge in the right groin.Brunicardi_Ch39_p1705-p1758.indd 174312/02/19 11:27 AM 1744SPECIFIC CONSIDERATIONSPART IIOlder children may notice the bulge themselves. On examina-tion, the cord on the affected side will be thicker, and pressure on the lower abdomen usually will display the hernia on the affected side. The presence of an incarcerated hernia is mani-fested by a firm bulge that does not spontaneously resolve and may be associated with fussiness and irritability in the child. The infant that has a strangulated inguinal hernia will manifest an edematous, tender bulge in the groin, occasionally with over-lying skin changes. The

1	with fussiness and irritability in the child. The infant that has a strangulated inguinal hernia will manifest an edematous, tender bulge in the groin, occasionally with over-lying skin changes. The child will eventually develop intestinal obstruction, peritonitis, and systemic toxicity.Usually an incarcerated hernia can be reduced. Occasion-ally this may require light sedation. Gentle pressure is applied on the sac from below in the direction of the internal inguinal ring. Following reduction of the incarcerated hernia, the child may be admitted for observation, and herniorrhaphy is per-formed within the next 24 hours to prevent recurrent incarcera-tion. Alternatively, the child may be scheduled for surgery at the next available time slot. If the hernia cannot be reduced, or if evidence of strangulation is present, emergency operation is necessary. This may require a laparotomy and bowel resection.When the diagnosis of inguinal hernia is made in an oth-erwise normal child, operative

1	of strangulation is present, emergency operation is necessary. This may require a laparotomy and bowel resection.When the diagnosis of inguinal hernia is made in an oth-erwise normal child, operative repair should be planned. Spon-taneous resolution does not occur, and therefore a nonoperative approach cannot ever be justified. An inguinal hernia in a female infant or child frequently contains an ovary rather than intestine. Although the gonad usually can be reduced into the abdomen by gentle pressure, it often prolapses in and out until surgical repair is carried out. In some patients, the ovary and fallopian tube constitute one wall of the hernial sac (sliding hernia), and in these patients, the ovary can be reduced effectively only at the time of operation. If the ovary is irreducible, prompt hernia repair is indicated to prevent ovarian torsion or strangulation.When a hydrocele is diagnosed in infancy and there is no evidence of a hernia, observation is proper therapy until the

1	prompt hernia repair is indicated to prevent ovarian torsion or strangulation.When a hydrocele is diagnosed in infancy and there is no evidence of a hernia, observation is proper therapy until the child is older than 12 months. If the hydrocele has not disappeared by 12 months, invariably there is a patent processus vaginalis, and operative hydrocelectomy with excision of the processus vaginalis is indicated. When the first signs of a hydrocele are seen after 12 months of age, the patient should undergo elective hydrocelectomy, which in a child is always performed through a groin incision. Aspiration of hydroceles is discouraged because almost all without a patent processus vaginalis will resorb spon-taneously and those with a communication to the peritoneum will recur and require operative repair eventually. Transillumi-nation as a method to distinguish between hydrocele and hernia is nonspecific. A noncommunicating hydrocele is better identi-fied by palpation of a nonreducible oval

1	repair eventually. Transillumi-nation as a method to distinguish between hydrocele and hernia is nonspecific. A noncommunicating hydrocele is better identi-fied by palpation of a nonreducible oval structure that appears to have a blunt end below the external ring, indicating an isolated fluid collection without a patent connection to the peritoneum.Surgical Repair. The repair of a pediatric inguinal hernia can be extremely challenging, particularly in the premature child with incarceration. A small incision is made in a skin crease in the groin directly over the internal inguinal ring. Scarpa’s fascia is seen and divided. The external oblique muscle is dis-sected free from overlying tissue, and the location of the exter-nal ring is confirmed. The external oblique aponeurosis is then opened along the direction of the external oblique fibers over the inguinal canal. The undersurface of the external oblique is then cleared from surrounding tissue. The cremasteric fibers are separated

1	along the direction of the external oblique fibers over the inguinal canal. The undersurface of the external oblique is then cleared from surrounding tissue. The cremasteric fibers are separated from the cord structures and hernia sac, and these are then elevated into the wound. Care is taken not to grasp the vas deferens. The hernia sac is then dissected up to the internal ring and doubly suture ligated. The distal part of the hernia sac is opened widely to drain any hydrocele fluid. When the hernia is very large and the patient very small, tightening of the internal inguinal ring or even formal repair of the inguinal floor may be necessary, although the vast majority of children do not require any treatment beyond high ligation of the hernia sac.Controversy exists regarding the role for exploration of an asymptomatic opposite side in a child with an inguinal hernia. Several reports indicate that frequency of a patent processus vaginalis on the side opposite the obvious hernia is

1	for exploration of an asymptomatic opposite side in a child with an inguinal hernia. Several reports indicate that frequency of a patent processus vaginalis on the side opposite the obvious hernia is approxi-mately 30%, although this figure decreases with increasing age of the child. Management options include never exploring the opposite side, to exploring only under certain conditions such as in premature infants or in patients in whom incarceration is pres-ent. The opposite side may readily be explored laparoscopically. To do so, a blunt 3-mm trochar is placed into the hernia sac of the affected side. The abdominal cavity is insufflated, and the 2.7-mm 70° camera is placed through the trochar such that the opposite side is visualized. The status of the processes vaginalis on the opposite side can be visualized. However, the presence of a patent processus vaginalis by laparoscopy does not always imply the presence of a hernia.There has been quite widespread adoption of laparoscopic

1	side can be visualized. However, the presence of a patent processus vaginalis by laparoscopy does not always imply the presence of a hernia.There has been quite widespread adoption of laparoscopic approach in the management of inguinal hernias in children, especially those under the age of 2 years. This technique requires insufflation through the umbilicus and the placement of an extra-peritoneal suture to ligate the hernia sac. Proponents of this pro-cedure emphasize the fact that no groin incision is used, so there is a decreased chance of injuring cord structures, and that visu-alization of the contralateral side is achieved immediately. The long-term results of this technique have been quite excellent.Inguinal hernias in children recur in less than 1% of patients, and recurrences usually result from missed hernia sacs at the first procedure, a direct hernia, or a missed femoral hernia. All children should have local anesthetic administered either by caudal injection or by direct

1	result from missed hernia sacs at the first procedure, a direct hernia, or a missed femoral hernia. All children should have local anesthetic administered either by caudal injection or by direct injection into the wound. Spinal anesthesia in preterm infant decreases the risk of postoperative apnea when compared with general anesthesia.GENITALIAUndescended testisEmbryology. The term undescended testicle (cryptorchidism) refers to the interruption of the normal descent of the testis into the scrotum. The testicle may reside in the retroperineum, in the internal inguinal ring, in the inguinal canal, or even at the external ring. The testicle begins as a thickening on the uro-genital ridge in the fifth to sixth week of embryologic life. In the seventh and eighth months, the testicle descends along the inguinal canal into the upper scrotum, and with its progress the processus vaginalis is formed and pulled along with the migrat-ing testicle. At birth, approximately 95% of infants have the

1	along the inguinal canal into the upper scrotum, and with its progress the processus vaginalis is formed and pulled along with the migrat-ing testicle. At birth, approximately 95% of infants have the testicle normally positioned in the scrotum.A distinction should be made between an undescended testicle and an ectopic testicle. An ectopic testis, by definition, is one that has passed through the external ring in the normal pathway and then has come to rest in an abnormal location over-lying either the rectus abdominis or external oblique muscle, or the soft tissue of the medial thigh, or behind the scrotum in the perineum. A congenitally absent testicle results from failure of normal development or an intrauterine accident leading to loss of blood supply to the developing testicle.Brunicardi_Ch39_p1705-p1758.indd 174412/02/19 11:27 AM 1745PEDIATRIC SURGERYCHAPTER 39Clinical Presentation. The incidence of undescended testes is approximately 30% in preterm infants, and 1% to 3% at

1	174412/02/19 11:27 AM 1745PEDIATRIC SURGERYCHAPTER 39Clinical Presentation. The incidence of undescended testes is approximately 30% in preterm infants, and 1% to 3% at term. For diagnosis, the child should be examined in the supine posi-tion, where visual inspection may reveal a hypoplastic or poorly rugated scrotum. Usually a unilateral undescended testicle can be palpated in the inguinal canal or in the upper scrotum. Occa-sionally, the testicle will be difficult or impossible to palpate, indicating either an abdominal testicle or congenital absence of the gonad. If the testicle is not palpable in the supine position, the child should be examined with his legs crossed while seated. This maneuver diminishes the cremasteric reflex and facilitates identification of the location of the testicle. If there is uncer-tainty regarding location of a testis, repeated evaluations over time may be helpful.It is now established that cryptorchid testes demonstrate an increased predisposition

1	testicle. If there is uncer-tainty regarding location of a testis, repeated evaluations over time may be helpful.It is now established that cryptorchid testes demonstrate an increased predisposition to malignant degeneration. In addition, fertility is decreased when the testicle is not in the scrotum. For these reasons, surgical placement of the testicle in the scrotum (orchidopexy) is indicated. It should be emphasized that this procedure does improve the fertility potential, although it is never normal. Similarly, the testicle is still at risk of malignant change, although its location in the scrotum facilitates poten-tially earlier detection of a testicular malignancy. Other reasons to consider orchidopexy include the risk of trauma to the testicle located at the pubic tubercle and incidence of torsion, as well as the psychological impact of an empty scrotum in a developing male. The reason for malignant degeneration is not established, but the evidence points to an inherent

1	incidence of torsion, as well as the psychological impact of an empty scrotum in a developing male. The reason for malignant degeneration is not established, but the evidence points to an inherent abnormality of the testicle that predisposes it to incomplete descent and malignancy rather than malignancy as a result of an abnormal environment.Treatment. Males with bilateral undescended testicles are often infertile. When the testicle is not present within the scrotum, it is subjected to a higher temperature, resulting in decreased spermatogenesis. Mengel and coworkers studied 515 undescended testicles by histology and demonstrated reduced spermatogonia after 2 years of age. It is now recommended that the undescended testicle be surgically repositioned by 1 year of age. Despite orchidopexy, the incidence of infertility is approx-imately two times higher in men with unilateral orchidopexy compared to men with normal testicular descent.The use of chorionic gonadotropin occasionally may be

1	the incidence of infertility is approx-imately two times higher in men with unilateral orchidopexy compared to men with normal testicular descent.The use of chorionic gonadotropin occasionally may be effective in patients with bilateral undescended testes, suggest-ing that these patients are more apt to have a hormone insuf-ficiency than children with unilateral undescended testicle. The combination of micro-penis and bilateral undescended testes is an indication for hormonal evaluation and testoster-one replacement if indicated. If there is no testicular descent after a month of endocrine therapy, operative correction should be undertaken. A child with unilateral cryptorchidism should have surgical correction of the problem. The operation is typi-cally performed through a combined groin and scrotal incision. The cord vessels are fully mobilized, and the testicle is placed in a dartos pouch within the scrotum. An inguinal hernia often accompanies a cryptorchid testis. This should be

1	and scrotal incision. The cord vessels are fully mobilized, and the testicle is placed in a dartos pouch within the scrotum. An inguinal hernia often accompanies a cryptorchid testis. This should be repaired at the time of orchidopexy.Patients with a nonpalpable testicle present a challenge in management. The current approach involves laparoscopy to identify the location of the testicle. If the spermatic cord is found to traverse the internal ring or the testis is found at the ring and can be delivered into the scrotum, a groin incision is made and an orchidopexy is performed. If an abdominal testis is identified that is too far to reach the scrotum, a two-staged Fowler-Stephens approach is used. In the first stage, the testicular vessels are clipped laparoscopically, which promotes the development of new blood vessels along the vas deferens. Several months later, the second stage is performed during which the testis is mobilized laparoscopically along with a swath of peritoneum with

1	of new blood vessels along the vas deferens. Several months later, the second stage is performed during which the testis is mobilized laparoscopically along with a swath of peritoneum with collateralized blood supply along the vas. Preservation of the gubernacular attachments with its collaterals to the testicle may confer improved testicular survival following orchidopex in over 90%. It is, nonetheless, preferable to preserve the testicular vessels whenever possible and complete mobilization of the testicle with its vessels intact.Vaginal AnomaliesSurgical diseases of the vagina in children are either congenital or acquired. Congenital anomalies include a spectrum of dis-eases that range from simple defects (imperforate hymen) to more complex forms of vaginal atresia, including distal, proxi-mal, and, most severe, complete. These defects are produced by abnormal development of müllerian ducts and/or urogenital sinus. The diagnosis is made most often by physical examina-tion.

1	proxi-mal, and, most severe, complete. These defects are produced by abnormal development of müllerian ducts and/or urogenital sinus. The diagnosis is made most often by physical examina-tion. Secretions into the obstructed vagina produce hydrocol-pos, which may present as a large, painful abdominal mass. The anatomy may be defined using US. Pelvic magnetic resonance imaging provides the most thorough and accurate assessment of the pelvic structures. Treatment is dependent on the extent of the defect. For an imperforate hymen, division of the hymen is curative. More complex forms of vaginal atresia require mobi-lization of the vaginal remnants and creation of an anastomosis at the perineum. Laparoscopy can be extremely useful, both in mobilizing the vagina, in draining hydrocolpos, and in evaluat-ing the internal genitalia. Complete vaginal atresia requires the construction of skin flaps or the creation of a neovagina using a segment of colon.The most common acquired disorder of the

1	evaluat-ing the internal genitalia. Complete vaginal atresia requires the construction of skin flaps or the creation of a neovagina using a segment of colon.The most common acquired disorder of the vagina is the straddle injury. This often occurs as young girls fall on blunt objects which cause a direct injury to the perineum. Typical manifestations include vaginal bleeding and inability to void. Unless the injury is extremely superficial, patients should be examined in the operating room where the lighting is optimal and sedation can be administered. Examination under anesthe-sia is particularly important in girls who are unable to void, suggesting a possible urethral injury. Vaginal lacerations are repaired using absorbable sutures, and the proximity to the ure-thra should be carefully assessed. Prior to hospital discharge, it is important that girls are able to void spontaneously. In all cases of vaginal trauma, it is essential that the patient be assessed for the presence of

1	assessed. Prior to hospital discharge, it is important that girls are able to void spontaneously. In all cases of vaginal trauma, it is essential that the patient be assessed for the presence of sexual abuse. In these cases, early contact with the sexual abuse service is necessary so that the appropriate microbiologic and photographic evidence can be obtained.Ovarian Cysts and TumorsPathologic Classification. Ovarian cysts and tumors may be classified as nonneoplastic or neoplastic. Nonneoplastic lesions include cysts (simple, follicular, inclusion, paraovarian, or cor-pus luteum), endometriosis, and inflammatory lesions. Neo-plastic lesions are classified based on the three primordia that contribute to the ovary: mesenchymal components of the uro-genital ridge, germinal epithelium overlying the urogenital ridge, and germ cells migrating from the yolk sac. The most common variety is germ cell tumors. Germ cell tumors are classified based on the degree of differentiation and the

1	overlying the urogenital ridge, and germ cells migrating from the yolk sac. The most common variety is germ cell tumors. Germ cell tumors are classified based on the degree of differentiation and the cellular components Brunicardi_Ch39_p1705-p1758.indd 174512/02/19 11:27 AM 1746SPECIFIC CONSIDERATIONSPART IIinvolved. The least differentiated tumors are the dysgermino-mas, which share features similar to the seminoma in males. Although these are malignant tumors, they are extremely sensi-tive to radiation and chemotherapy. The most common germ cell tumors are the teratomas, which may be mature, immature, or malignant. The degree of differentiation of the neural elements of the tumor determines the degree of immaturity. The sex cord stromal tumors arise from the mesenchymal components of the urogenital ridge. These include the granulosa-theca cell tumors and the Sertoli-Leydig cell tumors. These tumors often produce hormones that result in precocious puberty or hirsutism,

1	of the urogenital ridge. These include the granulosa-theca cell tumors and the Sertoli-Leydig cell tumors. These tumors often produce hormones that result in precocious puberty or hirsutism, respec-tively. Although rare, epithelial tumors do occur in children. These include serous and mucinous cystadenomas.Clinical Presentation. Children with ovarian lesions usually present with abdominal pain. Other signs and symptoms include a palpable abdominal mass, evidence of urinary obstruction, symp-toms of bowel obstruction, and endocrine imbalance. The surgical approach depends on the appearance of the mass at operation (i.e., whether it is benign-appearing or is suspicious for malignancy). In the case of a simple ovarian cyst, surgery depends on the size of the cyst and the degree of symptoms it causes. In general, large cysts (over 4–5 cm) in size should be resected, as they are unlikely to resolve, may be at risk of torsion, and may mask an underlying malignancy. Resection may be

1	it causes. In general, large cysts (over 4–5 cm) in size should be resected, as they are unlikely to resolve, may be at risk of torsion, and may mask an underlying malignancy. Resection may be performed laparoscopically, and ovarian tissue should be spared in all cases.Surgical Management. For ovarian lesions that appear malignant, it is important to obtain tumor markers including α-fetoprotein (teratomas), LDH (dysgerminoma), β-human cho-rionic gonadotropin (choriocarcinoma), and CA-125 (epithelial tumors). Although the diagnostic sensitivity of these markers is not always reliable, they provide material for postoperative follow-up and indicate the response to therapy. When a malig-nancy is suspected, the patient should undergo a formal cancer operation. This procedure is performed through either a mid-line incision or a Pfannenstie approach. Ascites and peritoneal washings should be collected for cytologic study. The liver and diaphragm are inspected carefully for metastatic

1	through either a mid-line incision or a Pfannenstie approach. Ascites and peritoneal washings should be collected for cytologic study. The liver and diaphragm are inspected carefully for metastatic disease. An omentectomy is performed if there is any evidence of tumor present. Pelvic and para-aortic lymph nodes are biopsied, and the primary tumor is resected completely. Finally, the contra-lateral ovary is carefully inspected, and if a lesion is seen, it should be biopsied. Dysgerminomas and epithelial tumors may be bilateral in up to 15% of cases. The surgical approach for a benign lesion of the ovary should include preservation of the ipsi-lateral fallopian tube and preservation of the noninvolved ovary.Ovarian Cysts in the Newborn. Ovarian cysts may be detected by prenatal US. The approach to lesions less than 4 cm should include serial US evaluation every 2 months or so as many of these lesions will resolve spontaneously. Consid-eration should be given to laparoscopic excision of

1	to lesions less than 4 cm should include serial US evaluation every 2 months or so as many of these lesions will resolve spontaneously. Consid-eration should be given to laparoscopic excision of cysts larger than 4 cm to avoid the risks of ovarian torsion or development of abdominal symptoms. For smaller lesions, resolution occurs by approximately 6 months of age. A laparoscopic approach is preferable in these cases. By contrast, complex cysts of any size require surgical intervention at presentation to exclude the pos-sibility of malignancy.Ambiguous GenitaliaEmbryology. Normal sexual differentiation occurs in the sixth fetal week. In every fetus, wolffian (male) and müllerian (female) ducts are present until the onset of sexual differentiation. Normal sexual differentiation is directed by the sex determining region of the Y chromosome (SRY). This is located on the distal end of the short arm of the Y chromosome. SRY provides a genetic switch that initiates gonadal differentiation in

1	the sex determining region of the Y chromosome (SRY). This is located on the distal end of the short arm of the Y chromosome. SRY provides a genetic switch that initiates gonadal differentiation in the mammalian urogenital ridge. Secretion of Müllerian-inhibiting substance (MIS) by the Sertoli cells of the seminiferous tubules results in regression of the müllerian duct, the anlage of the uterus, Fal-lopian tubes, and the upper vagina. The result of MIS secretion therefore is a phenotypic male. In the absence of SRY in the Y chromosome, MIS is not produced, and the müllerian duct derivatives are preserved. Thus, the female phenotype prevails.In order for the male phenotype to develop, the embryo must have a Y chromosome, the SRY must be normal with-out point mutations or deletions, testosterone and MIS must be produced by the differentiated gonad, and the tissues must respond to these hormones. Any disruption of the orderly steps in sexual differentiation may be reflected clinically

1	and MIS must be produced by the differentiated gonad, and the tissues must respond to these hormones. Any disruption of the orderly steps in sexual differentiation may be reflected clinically as variants of the intersex syndromes.These may be classified as (a) true hermaphroditism (with ovarian and testicular gonadal tissue), (b) male pseudohermaph-roditism (testicles only), (c) female pseudohermaphroditism (ovarian tissue only), and (d) mixed gonadal dysgenesis (usually underdeveloped or imperfectly formed gonads).True Hermaphroditism This represents the rarest form of ambiguous genitalia. Patients have both normal male and female gonads, with an ovary on one side and a testis on the other. Occasionally, an ovotestis is present on one or both sides. The majority of these patients have a 46,XX karyotype. Both the tes-tis and the testicular portion of the ovotestis should be removed.Male Pseudohermaphroditism This condition occurs in infants with an XY karyotype but deficient

1	have a 46,XX karyotype. Both the tes-tis and the testicular portion of the ovotestis should be removed.Male Pseudohermaphroditism This condition occurs in infants with an XY karyotype but deficient masculinization of the external genitalia. Bilateral testes are present, but the duct structures differentiate partly as phenotypic females. The causes include inadequate testosterone production due to biosynthetic error, inability to convert testosterone to dihy-drotestosterone due to 5α-reductase deficiency or deficiencies in androgen receptors. The latter disorder is termed testicular feminization syndrome. Occasionally, the diagnosis in these children is made during routine inguinal herniorrhaphy in a phenotypic female at which time testes are found. The testes should be resected due to the risk of malignant degeneration, although this should be performed only after a full discussion with the family has occurred.Female Pseudohermaphroditism The most common cause of female

1	due to the risk of malignant degeneration, although this should be performed only after a full discussion with the family has occurred.Female Pseudohermaphroditism The most common cause of female pseudohermaphroditism is congenital adrenal hyper-plasia. These children have a 46,XX karyotype but have been exposed to excessive androgens in utero. Common enzyme deficiencies include 21-hydroxylase, 11-hydroxylase, and 3β-hydroxysteroid dehydrogenase. These deficiencies result in overproduction of intermediary steroid hormones, which results in masculinization of the external genitalia of the XX fetus. These patients are unable to synthesize cortisol. In 90% of cases, deficiency of 21-hydroxylase causes adrenocorticotropic hor-mone (ACTH) to stimulate the secretion of excessive quantities of adrenal androgen, which masculinizes the developing female (Fig. 39-36). These infants are prone to salt loss, and require cortisol replacement. Those with mineralocorticoid deficiency also require

1	adrenal androgen, which masculinizes the developing female (Fig. 39-36). These infants are prone to salt loss, and require cortisol replacement. Those with mineralocorticoid deficiency also require fluorocortisone replacement.Mixed Gonadal Dysgenesis This syndrome is associated with dysgenetic gonads and retained mullerian structures. The typical karyotype is mosaic, usually 45XO,46XY. A high incidence of Brunicardi_Ch39_p1705-p1758.indd 174612/02/19 11:27 AM 1747PEDIATRIC SURGERYCHAPTER 39Figure 39-36. Ambiguous genitalia manifest as enlarged clitoris and labioscrotal folds in a baby with the adrenogenital syndrome.malignant tumors occur in the dysgenetic gonads, most com-monly gonadoblastoma. Therefore, they should be removed.Management. In the differential diagnosis of patients with intersex anomalies, the following diagnostic steps are necessary: (a) evaluation of the genetic background and family history; (b) assessment of the anatomic structures by physical exami-nation,

1	intersex anomalies, the following diagnostic steps are necessary: (a) evaluation of the genetic background and family history; (b) assessment of the anatomic structures by physical exami-nation, US, and/or chromosome studies; (c) determination of biochemical factors in serum and urine to evaluate the presence of an enzyme defect; and (d) laparoscopy for gonadal biopsy. Treatment should include correction of electrolyte and volume losses, in cases of congenital adrenal hyperplasia, and replace-ment of hormone deficiency. Surgical assignment of gender should never be determined at the first operation. Although his-torically female gender had been assigned, there is abundant and convincing evidence that raising a genotypic male as a female has devastating consequences, not only anatomically but also psychosocially. This is particularly relevant given the role of preand postnatal hormones on gender imprinting and identity. In general terms, surgical reconstruction should be performed

1	but also psychosocially. This is particularly relevant given the role of preand postnatal hormones on gender imprinting and identity. In general terms, surgical reconstruction should be performed after a full genetic workup and with the involvement of pediatric endocrinologists, pediatric plastic surgeons, and ethicists with expertise in gender issues. Discussion with the family also plays an important role. This approach will serve to reduce the anxi-ety associated with these disorders and will help to ensure the normal physical and emotional development of these patients.PEDIATRIC MALIGNANCYCancer is the second leading cause of death in children after trauma and accounts for approximately 11% of all pediatric deaths in the United States. The following description will be restricted to the most commonly encountered tumors in children.Wilms’ TumorClinical Presentation. Wilms’ tumor is the most common primary malignant tumor of the kidney in children. There are approximately 500 new

1	the most commonly encountered tumors in children.Wilms’ TumorClinical Presentation. Wilms’ tumor is the most common primary malignant tumor of the kidney in children. There are approximately 500 new cases annually in the United States, and most are diagnosed between 1 and 5 years with the peak inci-dence at age 3. Advances in the care of patients with Wilms’ tumor has resulted in an overall cure rate of roughly 90%, even in the presence of metastatic spread. The tumor usually develops in otherwise healthy children as an asymptomatic mass in the flank or upper abdomen. Frequently, the mass is discovered by a parent while bathing or dressing the child. Other symptoms include hypertension, hematuria, obstipation, and weight loss. Occasionally the mass is discovered following blunt abdominal trauma.Genetics of Wilms’ Tumor. Wilms’ tumor can arise from both germline and somatic mutations and can occur in the presence or absence of a family history. Nearly 97% of Wilms’ tumors are sporadic

1	trauma.Genetics of Wilms’ Tumor. Wilms’ tumor can arise from both germline and somatic mutations and can occur in the presence or absence of a family history. Nearly 97% of Wilms’ tumors are sporadic in that they occur in the absence of a heritable or congenital cause or risk factor. When a heritable risk factor is identified, the affected children often present at an earlier age, and the tumors are frequently bilateral. Most of these tumors are associated with germline mutations. It is well established that there is a genetic predisposition to Wilms’ tumor in WAGR syndrome, which consists of Wilms’ tumor, aniridia, genitouri-nary abnormalities, and mental retardation. In addition, there is an increased incidence of Wilms’ tumor in certain overgrowth conditions, particularly Beckwith–Wiedemann syndrome and hemihypertrophy. WAGR syndrome has been shown to result from the deletion of one copy each of the Wilms’ tumor gene, WT1, and the adjacent aniridia gene, PAX6, on chromosome 11p13.

1	syndrome and hemihypertrophy. WAGR syndrome has been shown to result from the deletion of one copy each of the Wilms’ tumor gene, WT1, and the adjacent aniridia gene, PAX6, on chromosome 11p13. Beckwith–Wiedemann syndrome is an overgrowth syn-drome that is characterized by visceromegaly, macroglossia, and hyperinsulinemic hypoglycemia. It arises from mutations at the 11p15.5 locus. There is evidence to suggest that analysis of the methylation status of several genes in the 11p15 locus could predict the individual risk to the development of Wilms’ tumor. Importantly, most patients with Wilms’ tumor do not have mutations at these genetic loci.Surgical Treatment. Before operation, all patients suspected of having Wilms’ tumor should undergo abdominal and chest computerized tomography. These studies characterize the mass, identify the presence of metastases, and provide information on the opposite kidney (Fig. 39-37). CT scanning also indicates the presence of nephrogenic rests, which are

1	characterize the mass, identify the presence of metastases, and provide information on the opposite kidney (Fig. 39-37). CT scanning also indicates the presence of nephrogenic rests, which are precursor lesions to Wilms’ tumor. An abdominal US should be performed to evalu-ate the presence of renal vein or vena caval extension.The management of patients with Wilms’ tumor has been carefully analyzed within the context of large studies involving thousands of patients. These studies have been coordinated by the National Wilms’ Tumor Study Group (NWTSG) in North America and the International Society of Paediatric Oncology Figure 39-37. Wilms’ tumor of the right kidney (arrow) in a 3-year-old girl.Brunicardi_Ch39_p1705-p1758.indd 174712/02/19 11:27 AM 1748SPECIFIC CONSIDERATIONSPART IITable 39-3Staging of Wilms’ tumorStage I: Tumor limited to the kidney and completely excised.Stage II: Tumor that extends beyond the kidney but is completely excised. This includes penetration of the renal

1	39-3Staging of Wilms’ tumorStage I: Tumor limited to the kidney and completely excised.Stage II: Tumor that extends beyond the kidney but is completely excised. This includes penetration of the renal capsule, invasion of the soft tissues of the renal sinus, or blood vessels within the nephrectomy specimen outside the renal parenchyma containing tumor. No residual tumor is apparent at or beyond the margins of excision.a Stage III: Residual nonhematogenous tumor confined to the abdomen. Lymph nodes in the abdomen or pelvis contain tumor. Peritoneal contamination by the tumor, such as by spillage or biopsy of tumor before or during surgery. Tumor growth that has penetrated through the peritoneal surface. Implants are found on the peritoneal surfaces. Tumor extends beyond the surgical margins either microscopically or grossly. Tumor is not completely resectable because of local infiltration into vital structures. The tumor was treated with preoperative chemotherapy with or without

1	margins either microscopically or grossly. Tumor is not completely resectable because of local infiltration into vital structures. The tumor was treated with preoperative chemotherapy with or without biopsy. Tumor is removed in greater than one piece.Stage IV: Hematogenous metastases or lymph node involvement outside the abdomino-pelvic region.Stage V: Bilateral renal involvement.International Neuroblastoma Staging SystemStage 1: Localized tumor with complete gross resection, with or without microscopic residual diseaseStage 2A: Localized tumor with incomplete gross excision; representative ipsilateral nonadherent lymph nodes negative for tumorStage 2B: Localized tumor with or without complete gross excision, with ipsilateral nonadherent lymph nodes positive for tumor. Enlarged contralateral lymph nodes must be negative microscopicallyStage 3: Unresectable unilateral tumor crossing midline, with or without regional lymph node involvement; or localized unilateral tumor with

1	contralateral lymph nodes must be negative microscopicallyStage 3: Unresectable unilateral tumor crossing midline, with or without regional lymph node involvement; or localized unilateral tumor with contralateral regional lymph node involvement; or midline tumorStage 4: Any primary tumor with dissemination to distant lymph nodes, bone, bone marrow, liver, skin, and/or other organsStage 4S: In infants <1 year of age; localized primary tumor with dissemination limited to skin, liver, and/or bone marrowInternational Neuroblastoma Risk Group Staging SystemL1 Localized tumor not involving vital structures as defined by the list of IDRFs and confined to one body compartmentL2 Locoregional tumor with the presence of one or more IDRFsM Distant metastatic disease (except MS)MS Metastatic disease in children <18 months confined to skin, liver, and bone marrow aRupture or spillage confined to the flank, including biopsy of the tumor, is no longer included in stage II and is now included in

1	in children <18 months confined to skin, liver, and bone marrow aRupture or spillage confined to the flank, including biopsy of the tumor, is no longer included in stage II and is now included in stage III.(SIOP), mainly involving European countries. Significant dif-ferences in the approach to patients with Wilms’ tumor have been highlighted by these studies. NWTSG supports a strat-egy of surgery followed by chemotherapy in most instances, whereas the SIOP approach is to shrink the tumor using preoper-ative chemotherapy. There are instances were preoperative che-motherapy is supported by both groups, including the presence of bilateral involvement or inferior vena cava involvement that extends above the hepatic veins and involvement of a solitary kidney by Wilms’ tumor. The NWTSG proponents argue that preoperative therapy in other instances results in a loss of impor-tant staging information, and therefore places patients at higher risk for recurrence; alternatively, it may lead to

1	argue that preoperative therapy in other instances results in a loss of impor-tant staging information, and therefore places patients at higher risk for recurrence; alternatively, it may lead to overly aggres-sive treatment in some cases and greater morbidity. However, the overall survival rates are not different between the NWTSG and SIOP approaches.The goal of surgery is complete removal of the tumor. It is crucial to avoid tumor rupture or injury to contiguous organs. A sampling of regional lymph nodes should be included, and all suspicious nodes should be sampled. Typically, a large transverse abdominal incision is made, and a transperitoneal approach is used. The opposite side is carefully inspected to ensure that there is no disease present. Although historically this involved the complete mobilization of the contralateral kidney, current evidence indicates that preoperative, high-resolution CT scanning is of sufficient accuracy for the detection of clinically significant

1	the complete mobilization of the contralateral kidney, current evidence indicates that preoperative, high-resolution CT scanning is of sufficient accuracy for the detection of clinically significant lesions if they are present. Provided only unilateral disease is present, a radical nephroureterectomy is then performed with control of the renal pedicle as an initial step. If there is spread above the hepatic veins, an intrathoracic approach may be required. If bilateral disease is encountered, both lesions are biopsied, and chemotherapy is administered followed by a nephron-sparing procedure.Chemotherapy. Following nephroureterectomy for Wilms’ tumor, the need for chemotherapy and/or radiation therapy are determined by the histology of the tumor and the clinical stage of the patient (Table 39-3). Essentially, patients who have dis-ease confined to one kidney completely excised surgically receive a short course of chemotherapy and can expect a 97% 4-year survival, with tumor relapse

1	39-3). Essentially, patients who have dis-ease confined to one kidney completely excised surgically receive a short course of chemotherapy and can expect a 97% 4-year survival, with tumor relapse rare after that time. Patients with more advanced disease or with unfavorable histol-ogy receive more intensive chemotherapy and radiation. Even in stage IV, high cure rates may be achieved. The survival rates are worse in the small percentage of patients considered to have unfavorable histology.NeuroblastomaClinical Presentation. Neuroblastoma is the third most com-mon pediatric malignancy and accounts for approximately 10% of all childhood cancers. The vast majority of patients have advanced disease at the time of presentation, and unlike Wilms’ tumor, in which cure is expected in the vast majority of patients, the overall survival of patients with neuroblastoma is significantly lower. Over 80% of cases present before the age of 4 years, and the peak incidence is two years of age.

1	vast majority of patients, the overall survival of patients with neuroblastoma is significantly lower. Over 80% of cases present before the age of 4 years, and the peak incidence is two years of age. Neuro-blastomas arise from the neural crest cells and show different levels of differentiation. The tumor originates most frequently in the adrenal glands, posterior mediastinum, neck, or pelvis but can arise in any sympathetic ganglion. The clinical presen-tation depends on the site of the primary and the presence of metastases.9Brunicardi_Ch39_p1705-p1758.indd 174812/02/19 11:27 AM 1749PEDIATRIC SURGERYCHAPTER 39Two-thirds of these tumors are first noted as an asymp-tomatic abdominal mass. The tumor may cross the midline, and a majority of patients will already show signs of metastatic disease. Occasionally, children may experience pain from the tumor mass or from bony metastases. Proptosis and perior-bital ecchymosis may occur due to the presence of retrobulbar metastasis. Because

1	disease. Occasionally, children may experience pain from the tumor mass or from bony metastases. Proptosis and perior-bital ecchymosis may occur due to the presence of retrobulbar metastasis. Because they originate in paraspinal ganglia, neuro-blastomas may invade through neural foramina and compress the spinal cord, causing muscle weakness or sensory changes. Rarely, children may have severe watery diarrhea due to the secretion of vasoactive intestinal peptide by the tumor, or with paraneoplastic neurologic findings including cerebellar ataxia or opsoclonus/myoclonus. The International Neuroblastoma Stag-ing System and the International Neuroblastoma Risk Group Staging System are provided in Table 39-3.Diagnostic Evaluation. Since these tumors derive from the sympathetic nervous system, catecholamines and their metabo-lites will be produced at increased levels. These include elevated levels of serum catecholamines (dopamine, norepinephrine) or urine catecholamine metabolites:

1	catecholamines and their metabo-lites will be produced at increased levels. These include elevated levels of serum catecholamines (dopamine, norepinephrine) or urine catecholamine metabolites: vanillylmandelic acid (VMA) or homovanillic acid (HVA). Measurement of VMA and HVMA in serum and urine aids in the diagnosis and in monitoring ade-quacy of future treatment and recurrence. The minimum criterion for a diagnosis of neuroblastoma is based on one of the following: (a) an unequivocal pathologic diagnosis made from tumor tissue by light microscopy (with or without immunohistology, electron microscopy, or increased levels of serum catecholamines or uri-nary catecholamine metabolites); (b) the combination of bone marrow aspirate or biopsy containing unequivocal tumor cells and increased levels of serum catecholamines or urinary catechol-amine metabolites as described earlier.The patient should be evaluated by abdominal computer-ized tomography, which may show displacement and

1	levels of serum catecholamines or urinary catechol-amine metabolites as described earlier.The patient should be evaluated by abdominal computer-ized tomography, which may show displacement and occasion-ally obstruction of the ureter of an intact kidney (Fig. 39-38). Prior to the institution of therapy, a complete staging workup should be performed. This includes radiograph of the chest, bone marrow biopsy, and radionuclide scans to search for metastases. Any abnormality on chest X-ray should be followed up with CT of the chest.Prognostic Indicators. A number of biologic variables have been studied in children with neuroblastoma. An open biopsy is required in order to provide tissue for this analysis. Hyperdip-loid tumor DNA is associated with a favorable prognosis, and Figure 39-38. Abdominal neuroblastoma arising from the right retroperitoneum (arrow).N-myc amplification is associated with a poor prognosis regard-less of patient age. The Shimada classification describes tumors as

1	neuroblastoma arising from the right retroperitoneum (arrow).N-myc amplification is associated with a poor prognosis regard-less of patient age. The Shimada classification describes tumors as either favorable or unfavorable histology based on the degree of differentiation, the mitosis-karyorrhexis index, and the pres-ence or absence of schwannian stroma. In general, children of any age with localized neuroblastoma and infants younger than 1 year of age with advanced disease and favorable disease char-acteristics have a high likelihood of disease-free survival. By contrast, older children with advanced-stage disease have a sig-nificantly decreased chance for cure despite intensive therapy. For example, aggressive multiagent chemotherapy has resulted in a 2-year survival rate of approximately 20% in older children with stage IV disease. Neuroblastoma in the adolescent has a worse long-term prognosis regardless of stage or site and, in many cases, a more prolonged course.Surgery. The

1	20% in older children with stage IV disease. Neuroblastoma in the adolescent has a worse long-term prognosis regardless of stage or site and, in many cases, a more prolonged course.Surgery. The goal of surgery is complete resection. However, this is often not possible at initial presentation due to the exten-sive locoregional spread of the tumor at the time of presenta-tion. Under these circumstances, a biopsy is performed, and preoperative chemotherapy is provided based upon the stage of the tumor. After neoadjuvant treatment has been administered, surgical resection is performed. The principal goal of surgery is to obtain at least 95% resection without compromising major structures. Abdominal tumors are approached through a trans-verse incision. Thoracic tumors may be approached through a posterolateral thoracotomy or through a thoracoscopic approach. These may have an intraspinal component. In all cases of intra-thoracic neuroblastoma, particularly those at the thoracic inlet, it

1	a posterolateral thoracotomy or through a thoracoscopic approach. These may have an intraspinal component. In all cases of intra-thoracic neuroblastoma, particularly those at the thoracic inlet, it is important to be aware of the possibility of a Horner’s syn-drome (anhidrosis, ptosis, meiosis) developing. This typically resolves, although it may take many months to do so.Neuroblastoma in Infants. Spontaneous regression of neu-roblastoma has been well described in infants, especially in those with stage 4S disease. Regression generally occurs only in tumors with a near triploid number of chromosomes that also lack N-myc amplification and loss of chromosome 1p. Recent studies indicate that infants with asymptomatic, small, low-stage neuroblastoma detected by screening may have tumors that spontaneously regress. These patients may be observed safely without surgical intervention or tissue diagnosis.RhabdomyosarcomaRhabdomyosarcoma is a primitive soft tissue tumor that arises from

1	that spontaneously regress. These patients may be observed safely without surgical intervention or tissue diagnosis.RhabdomyosarcomaRhabdomyosarcoma is a primitive soft tissue tumor that arises from mesenchymal tissues. The most common sites of origin include the head and neck (36%), extremities (19%), genitourinary tract (2%), and trunk (9%), although the tumor can arise virtually anywhere. The clinical presentation of the tumor depends on the site of origin. The diagnosis is confirmed with incisional or excisional biopsy after evaluation by MRI, CT scans of the affected area and the chest, and bone marrow biopsy. The tumor grows locally into surrounding structures and metastasizes widely to lung, regional lymph nodes, liver, brain, and bone marrow. The staging system for rhabdomyosarcoma is based upon the TNM system, as established by the Soft Tissue Sarcoma Committee of the Children’s Oncology Group. It is shown in Table 39-4. Surgery is an important component of the staging

1	is based upon the TNM system, as established by the Soft Tissue Sarcoma Committee of the Children’s Oncology Group. It is shown in Table 39-4. Surgery is an important component of the staging strategy and involves biopsy of the lesion and evaluation of lymphatics. Primary resection should be undertaken when complete excision can be performed without causing disability. If this is not possible, the lesion is biopsied, and intensive che-motherapy is administered. It is important to plan the biopsy so that it does not interfere with subsequent resection. After the Brunicardi_Ch39_p1705-p1758.indd 174912/02/19 11:27 AM 1750SPECIFIC CONSIDERATIONSPART IItumor has decreased in size, resection of gross residual disease should be performed. Radiation therapy is effective in achieving local control when microscopic or gross residual disease exists following initial treatment. Patients with completely resected tumors of embryonal histology do well without radiation ther-apy, but radiation

1	when microscopic or gross residual disease exists following initial treatment. Patients with completely resected tumors of embryonal histology do well without radiation ther-apy, but radiation therapy benefits patients with group I tumors with alveolar or undifferentiated histology.Prognosis. The prognosis for rhabdomyosarcoma is related to the site of origin, resectability, presence of metastases, number of metastatic sites, and histopathology. Primary sites with more favorable prognoses include the orbit and nonparameningeal head and neck, paratestis and vagina (nonbladder, nonprostate genitourinary), and the biliary tract. Patients with tumors less than 5 cm in size have improved survival compared to children with larger tumors, while children with metastatic disease at diagnosis have the poorest prognosis. Tumor histology influ-ences prognosis and the embryonal variant is favorable while the alveolar subtype is unfavorable.TeratomaTeratomas are tumors composed of tissue from all

1	the poorest prognosis. Tumor histology influ-ences prognosis and the embryonal variant is favorable while the alveolar subtype is unfavorable.TeratomaTeratomas are tumors composed of tissue from all three embry-onic germ layers. They may be benign or malignant, they may arise in any part of the body, and they are usually found in mid-line structures. Thoracic teratomas usually present as an anterior mediastinal mass. Ovarian teratomas present as an abdominal mass often with symptoms of torsion, bleeding, or rupture. Ret-roperitoneal teratomas may present as a flank or abdominal mass.Mature teratomas usually contain well-differentiated tis-sues and are benign, while immature teratomas contain vary-ing degrees of immature neuroepithelium or blastemal tissues. Immature teratomas can be graded from 1 to 3 based on the amount of immature neuroglial tissue present. Tumors of higher grade are more likely to have foci of yolk sac tumor. Malignant germ cell tumors usually contain frankly

1	graded from 1 to 3 based on the amount of immature neuroglial tissue present. Tumors of higher grade are more likely to have foci of yolk sac tumor. Malignant germ cell tumors usually contain frankly neoplastic tissues of germ cell origin (i.e., yolk sac carcinoma, embryonal carcinoma, germinoma, or choriocarcinoma). Yolk sac carci-nomas produce α-fetoprotein (AFP), while choriocarcinomas produce β-human chorionic gonadotropin (BHCG) resulting in elevation of these substances in the serum, which can serve as tumor markers. In addition, germinomas can also produce elevation of serum BHCG but not to the levels associated with choriocarcinoma.Table 39-4Staging of RhabdomyosarcomaSTAGESITESTSIZENM1Orbit, nonparameningeal head and neck, genitourinary (other than kidney, bladder, and prostate), and biliaryT1 or T2a or bAny NM02Bladder/prostate, extremity, cranial parameningeal, otherT1 or T2a N0 or NXM03Bladder/prostate, extremity, cranial parameningeal, otherT1 or T2aN1M0 bAny NM04AllT1

1	and biliaryT1 or T2a or bAny NM02Bladder/prostate, extremity, cranial parameningeal, otherT1 or T2a N0 or NXM03Bladder/prostate, extremity, cranial parameningeal, otherT1 or T2aN1M0 bAny NM04AllT1 or T2a or bAny NM1T1 = tumor confined to anatomic site of origin; T2 = tumor extension and/or fixed to surrounding tissues; a = ≤5 cm; b = >5 cm; N0 = regional nodes not clinically involved; N1 = regional nodes clinically involved; NX = regional node status unknown; M0 = no distant metastasis; M1 = metastasis present.Clinical group:Group 1: Localized disease, completely resected, no regional lymph node involvement.Group 2: Localized disease, gross total resection but microscopic residual disease; or regional lymph nodes involved.Group 3: Localized disease with gross residual disease after incomplete resection or biopsy only.Group 4: Metastatic disease at diagnosis.Figure 39-39. Sacrococcygeal teratoma in a 2-day-old boy.Sacrococcygeal Teratoma. Sacrococcygeal teratoma usually presents as a

1	resection or biopsy only.Group 4: Metastatic disease at diagnosis.Figure 39-39. Sacrococcygeal teratoma in a 2-day-old boy.Sacrococcygeal Teratoma. Sacrococcygeal teratoma usually presents as a large mass extending from the sacrum in the new-born period. Diagnosis may be established by prenatal US. In fetuses with evidence of hydrops and a large sacrococcygeal teratoma, prognosis is poor; thus, prenatal intervention has been advocated in such patients. The mass may be as small as a few centimeters in diameter or as massive as the size of the infant (Fig. 39-39). The tumor has been classified based upon the location and degree of intrapelvic extension. Lesions that grow predominantly into the presacral space often present later in childhood. The differential diagnosis consists of neural tumors, lipoma, and myelomeningoceles.Most tumors are identified at birth and are benign. Malig-nant yolk sac tumor histology occurs in a minority of these tumors. Complete resection of the tumor as

1	tumors, lipoma, and myelomeningoceles.Most tumors are identified at birth and are benign. Malig-nant yolk sac tumor histology occurs in a minority of these tumors. Complete resection of the tumor as early as possible is essential. The rectum and genital structures are often distorted by the tumor but usually can be preserved in the course of resection. Perioperative complications of hypothermia and hemorrhage can occur with massive tumors and may prove lethal. This is of particular concern in small, preterm infants with large tumors. The cure rate is excellent if the tumor is excised completely. Brunicardi_Ch39_p1705-p1758.indd 175012/02/19 11:27 AM 1751PEDIATRIC SURGERYCHAPTER 39The majority of patients who develop recurrent disease are sal-vageable with subsequent platinum-based chemotherapy.Liver TumorsMore than two-thirds of all liver tumors in children are malig-nant. There are two major histologic subgroups: hepatoblastoma and hepatocellular carcinoma. The age of onset of

1	TumorsMore than two-thirds of all liver tumors in children are malig-nant. There are two major histologic subgroups: hepatoblastoma and hepatocellular carcinoma. The age of onset of liver cancer in children is related to the histology of the tumor. Hepatoblastoma is the most common malignancy of the liver in children, with most of these tumors diagnosed before 4 years of age. Hepatocel-lular carcinoma is the next most common, with a peak age inci-dence between 10 and 15 years. Malignant mesenchymomas and sarcomas are much less common but constitute the remainder of the malignancies. The finding of a liver mass does not necessar-ily imply that a malignancy is present. Nearly 50% of all masses are benign, and hemangiomas are the most common lesion.Most children with a liver tumor present with an abdomi-nal mass that is usually painless, which the parents note while changing the child’s clothes or while bathing the child. The patients are rarely jaundiced but may complain of anorexia and

1	an abdomi-nal mass that is usually painless, which the parents note while changing the child’s clothes or while bathing the child. The patients are rarely jaundiced but may complain of anorexia and weight loss. Most liver function tests are normal. AFP levels are increased in 90% of children with hepatoblastomas but much less commonly in other liver malignancies. Radiographic evaluation of these children should include an abdominal CT scan to identify the lesion and to determine the degree of local invasiveness (Fig. 39-40). For malignant appearing lesions, a biopsy should be performed unless the lesion can be completely resected easily. Hepatoblastoma is most often unifocal, while hepatocellular carcinoma is often extensively invasive or multi-centric. If a hepatoblastoma is completely removed, the majority of patients survive, but only a minority of patients have lesions amenable to complete resection at diagnosis.A staging system based on postsurgical extent of tumor and surgical

1	removed, the majority of patients survive, but only a minority of patients have lesions amenable to complete resection at diagnosis.A staging system based on postsurgical extent of tumor and surgical resectability is shown in Table 39-5. The overall survival rate for children with hepatoblastoma is 70%, but it is only 25% for hepatocellular carcinoma. Children diagnosed with stage I and II hepatoblastoma have a cure rate of greater than 90% compared to 60% for stage III and approximately 20% for stage IV. In children diagnosed with hepatocellular carcinoma, those with stage I have a good outcome, whereas stages III and IV are usually fatal. The fibrolamellar variant of hepatocel-lular carcinoma may have a better prognosis.Surgery. The abdominal CT scan usually will determine the resectability of the lesion, although occasionally this can only Figure 39-40. Computed tomography of the abdomen showing a hepatocellular carcinoma in a 12-year-old boy.be determined at the time of

1	resectability of the lesion, although occasionally this can only Figure 39-40. Computed tomography of the abdomen showing a hepatocellular carcinoma in a 12-year-old boy.be determined at the time of exploration. Complete surgical resection of the tumor is the primary goal and is essential for cure. For tumors that are unresectable, preoperative chemother-apy should be administered to reduce the size of the tumor and improve the possibility for complete removal. Chemotherapy is more successful for hepatoblastoma than for hepatocellular carcinoma. Areas of locally invasive disease, such as the dia-phragm, should be resected at the time of surgery. For unre-sectable tumors, liver transplantation may be offered in select patients. The fibrolamellar variant of hepatocellular carcinoma may have a better outcome with liver transplantation than other hepatocellular carcinomas.TRAUMA IN CHILDRENInjury is the leading cause of death among children older than 1 year. In fact, trauma accounts for

1	a better outcome with liver transplantation than other hepatocellular carcinomas.TRAUMA IN CHILDRENInjury is the leading cause of death among children older than 1 year. In fact, trauma accounts for almost half of all pediatric deaths, more than cancer, congenital anomalies, pneumonia, heart disease, homicide, and meningitis combined. Death from unintentional injuries accounts for 65% of all injury-related deaths in children younger than 19 years. Motor vehicle colli-sions are the leading cause of death in people age 1 to 19 years, followed by homicide or suicide (predominantly with firearms) and drowning. Each year, approximately 20,000 children and teenagers die as a result of injury in the United States. For every child who dies from an injury, it is calculated that 40 others are hospitalized and 1120 are treated in emergency departments. An estimated 50,000 children acquire permanent disabilities each year, most of which are the result of head injuries. Thus, the problem of

1	hospitalized and 1120 are treated in emergency departments. An estimated 50,000 children acquire permanent disabilities each year, most of which are the result of head injuries. Thus, the problem of pediatric trauma continues to be one of the major threats to the health and well-being of children.Specific considerations apply to trauma in children that influence management and outcome. These relate to the mecha-nisms of injury, the anatomic variations in children compared to adults, and the physiologic responses.Mechanisms of InjuryMost pediatric trauma is blunt. Penetrating injuries are seen in the setting of gun violence, falls onto sharp objects, or penetra-tion by glass after falling through windows. Age and gender significantly influence the patterns of injury. Male children between 14 and 18 years of age are exposed to contact sports, gun violence, and in some jurisdictions drive motor vehicles. As a result, they have a different pattern of injury than younger children,

1	between 14 and 18 years of age are exposed to contact sports, gun violence, and in some jurisdictions drive motor vehicles. As a result, they have a different pattern of injury than younger children, characterized by higher injury severity scores. In the infant and toddler age group, falls are a 10Table 39-5Staging of pediatric liver cancerStage I: No metastases, tumor completely resectedStage II: No metastases, tumor grossly resected with microscopic residual disease (i.e., positive margins); or tumor rupture, or tumor spill at the time of surgeryStage III: No distant metastases, tumor unresectable or resected with gross residual tumor, or positive lymph nodesStage IV: Distant metastases regardless of the extent of liver involvementData from Douglass E, Ortega J, Feusner J, et al. Hepatocellular carcinoma (HCA) in children and adolescents: results from the Pediatric Intergroup Hepatoma Study (CCG 8881/POG 8945), Proc Am Soc Clin Oncol. 1994;13:A-1439.Brunicardi_Ch39_p1705-p1758.indd

1	carcinoma (HCA) in children and adolescents: results from the Pediatric Intergroup Hepatoma Study (CCG 8881/POG 8945), Proc Am Soc Clin Oncol. 1994;13:A-1439.Brunicardi_Ch39_p1705-p1758.indd 175112/02/19 11:27 AM 1752SPECIFIC CONSIDERATIONSPART IIcommon cause of severe injury. Injuries in the home are extremely common. These include falls, near-drownings, caustic ingestion, and nonaccidental injuries.Initial ManagementThe goals of managing the pediatric trauma patient are similar to those of adults and follow Advanced Trauma Life Support guidelines as established by the American College of Surgeons Committee on Trauma. Airway control is the first priority. In a child, respiratory arrest can proceed quickly to cardiac arrest. It is important to be aware of the anatomic differences between the airway of the child and the adult. The child has a large head, shorter neck, smaller and anterior larynx, floppy epiglottis, short trachea, and large tongue. The size of the endotracheal tube

1	the airway of the child and the adult. The child has a large head, shorter neck, smaller and anterior larynx, floppy epiglottis, short trachea, and large tongue. The size of the endotracheal tube can be estimated by the formula (age + 16)/4. It is important to use uncuffed endotracheal tubes in children younger than 8 years in order to minimize tracheal trauma. After evaluation of the airway, breathing is assessed. It is important to consider that gastric distention from aerophagia can severely compromise respirations. A nasogastric tube should therefore be placed early during the resuscitation if there is no head injury suspected, or an orogastric tube in cases of head injury. Pneumothorax or hemothorax should be treated promptly. When evaluating the circulation, it is important to recognize that tachycardia is usu-ally the earliest measurable response to hypovolemia. Other signs of impending hypovolemic shock in children include changes in mentation, delayed capillary refill, skin

1	that tachycardia is usu-ally the earliest measurable response to hypovolemia. Other signs of impending hypovolemic shock in children include changes in mentation, delayed capillary refill, skin pallor, and hypothermia. IV access should be rapidly obtained once the patient arrives in the trauma bay. The first approach should be to use the antecubital fossae. If this is not possible, a cut-down into the saphenous at the groin can be performed quickly and safely. Intraosseous cannulation can provide temporary access in children and young adults until IV access is established. US-guided central line placement in the groin or neck should be considered in patients in whom large bore peripheral IV access is not obtained. Blood is drawn for cross-match and evaluation of liver enzymes, lipase, amylase, and hematologic profile after the IV lines are placed.In patients who show signs of volume depletion, a 20 mL/kg bolus of saline or lactated Ringer’s should be promptly given. If the patient

1	and hematologic profile after the IV lines are placed.In patients who show signs of volume depletion, a 20 mL/kg bolus of saline or lactated Ringer’s should be promptly given. If the patient does not respond to three boluses, blood should be transfused (10 mL/kg). The source of bleeding should be established. Common sites include the chest, abdomen, pel-vis, extremity fractures, or large scalp wounds. These should be carefully sought. Care is taken to avoid hypothermia by infusing warmed fluids and by using external warming devices.Evaluation of InjuryAll patients should receive an X-ray of the cervical spine, chest, and abdomen with pelvis. All extremities that are suspicious for fracture should also be evaluated by X-ray. Plain cervical spine films are preferable to performing routine neck CT scans in the child, as X-rays provide sufficient anatomic detail. But if a head CT is obtained, it may be reasonable to obtain images down to C-2 since odontoid views in small children are

1	neck CT scans in the child, as X-rays provide sufficient anatomic detail. But if a head CT is obtained, it may be reasonable to obtain images down to C-2 since odontoid views in small children are difficult to obtain. In most children, it is possible to diagnose clinically sig-nificant cervical spine injuries using this approach while mini-mizing the degree of radiation exposure. Screening blood work that includes AST, ALT, and amylase/lipase is useful for the evaluation of liver and pancreatic injures. Significant elevation in these tests requires further evaluation by CT scanning. The child with significant abdominal tenderness and a mechanism of injury that could cause intra-abdominal injury should undergo abdominal CT scanning using IV and oral contrast in all cases. There is a limited role for diagnostic peritoneal lavage (DPL) in children as a screening test. However, this can be occasionally useful in the child who is brought emergently to the operating room for management of

1	role for diagnostic peritoneal lavage (DPL) in children as a screening test. However, this can be occasionally useful in the child who is brought emergently to the operating room for management of significant intracranial hemorrhage. At the time of craniotomy, a DPL, or alternatively, a diagnostic laparoscopy, can be performed concurrently to identify abdomi-nal bleeding. Although focused abdominal US (FAST exam) is extremely useful in the evaluation of adult abdominal trauma, it is not widely accepted in the management of pediatric blunt abdominal trauma. In part, this relates to the widespread use of nonoperative treatment for most solid-organ injuries. Thus, a positive abdominal US scan would not alter this approach in a hemodynamically stable patient.Injuries to the Central Nervous SystemThe central nervous system (CNS) is the most commonly injured organ system and is the leading cause of death among injured children. In the toddler age group, nonaccidental trauma is the most

1	SystemThe central nervous system (CNS) is the most commonly injured organ system and is the leading cause of death among injured children. In the toddler age group, nonaccidental trauma is the most common cause of serious head injury. Findings suggestive of abuse include the presence of retinal hemorrhage on fundo-scopic evaluation and intracranial hemorrhage without evidence of external trauma (indicative of a shaking injury) and fractures at different stages of healing on skeletal survey. In older children, CNS injury occurs most commonly after falls and bicycle and motor vehicle collisions. The initial head CT can often underesti-mate the extent of injury in children. Criteria for head CT include any loss of consciousness or amnesia to the trauma, or inabil-ity to assess the CNS status as in the intubated patient. Patients with mild, isolated head injury (GCS 14-15) and negative CT scans can be discharged if their neurologic status is normal after 6 hours of observation. Young

1	as in the intubated patient. Patients with mild, isolated head injury (GCS 14-15) and negative CT scans can be discharged if their neurologic status is normal after 6 hours of observation. Young children and those in whom there is multisystem involvement should be admitted to the hospital for observation. Any change in the neurologic status warrants neu-rosurgical evaluation and repeat CT scanning. In patients with severe head injury (GCS 8 or less), urgent neurosurgical consulta-tion is required. These patients are evaluated for intracranial pres-sure monitoring and for the need to undergo craniotomy.Thoracic InjuriesThe pediatric thorax is pliable due to incomplete calcification of the ribs and cartilages. As a result, blunt chest injury com-monly results in pulmonary contusion, although rib fractures are infrequent. Diagnosis is made by chest radiograph and may be associated with severe hypoxia requiring mechanical ventila-tion. Pulmonary contusion usually resolves with careful

1	rib fractures are infrequent. Diagnosis is made by chest radiograph and may be associated with severe hypoxia requiring mechanical ventila-tion. Pulmonary contusion usually resolves with careful venti-lator management and judicious volume resuscitation. Children who have sustained massive blunt thoracic injury may develop traumatic asphyxia. This is characterized by cervical and facial petechial hemorrhages or cyanosis associated with vascular engorgement and subconjunctival hemorrhage. Management includes ventilation and treatment of coexisting CNS or abdomi-nal injuries. Penetrating thoracic injuries may result in damage to the lung or to major disruption of the bronchi or great vessels.Abdominal InjuriesIn children, the small rib cage and minimal muscular coverage of the abdomen can result in significant injury after seemingly minor trauma. The liver and spleen in particular are relatively unprotected and are often injured after direct abdominal trauma. Duodenal injuries are

1	can result in significant injury after seemingly minor trauma. The liver and spleen in particular are relatively unprotected and are often injured after direct abdominal trauma. Duodenal injuries are usually the result of blunt trauma, which may arise from child abuse or injury from a bicycle handlebar. Duodenal hematomas usually resolve without surgery. Brunicardi_Ch39_p1705-p1758.indd 175212/02/19 11:27 AM 1753PEDIATRIC SURGERYCHAPTER 39Small intestinal injury usually occurs in the jejunum in the area of fixation by the ligament of Treitz. These injuries are usually caused by rapid deceleration in the setting of a lap belt. There may be a hematoma on the anterior abdominal wall caused by a lap belt, the so-called seat belt sign (Fig. 39-41A). This should alert the caregiver to the possibility of an underlying small bowel injury (Fig. 39-41B), as well as to a potential lumbar spine injury (Chance fracture).The spleen is injured relatively commonly after blunt abdominal trauma in

1	of an underlying small bowel injury (Fig. 39-41B), as well as to a potential lumbar spine injury (Chance fracture).The spleen is injured relatively commonly after blunt abdominal trauma in children. The extent of injury to the spleen is graded (Table 39-6), and the management is governed by the injury grade. Current treatment involves a nonoperative approach in most cases, even for grade 4 injuries, assuming the patient is hemodynamically stable. This approach avoids surgery in most cases. All patients should be placed in a monitored unit, and type-specific blood should be available for transfusion. When nonoperative management is successful, as it is in most cases, an extended period of bed rest is prescribed. This optimizes the chance for healing and minimizes the likelihood of reinjury. A typical guideline is to keep the children on extremely restricted activity for 2 weeks longer than the grade of spleen injury (i.e., a child with a grade 4 spleen injury receives 6 weeks of

1	A typical guideline is to keep the children on extremely restricted activity for 2 weeks longer than the grade of spleen injury (i.e., a child with a grade 4 spleen injury receives 6 weeks of restricted activity). In children who have an ongoing fluid requirement, BAFigure 39-41. Abdominal computed tomography of patient who sustained a lapbelt injury. A. Bruising is noted across the abdomen from the lapbelt. B. At laparotomy, a perforation of the small bowel was identified.or when a blood transfusion is required, exploration should not be delayed. At surgery, the spleen can often be salvaged. If a splenectomy is performed, prophylactic antibiotics and immuni-zations should be administered to protect against overwhelming post splenectomy sepsis. The liver is also commonly injured after blunt abdominal trauma. A grading system is used to character-ize hepatic injuries (Table 39-7), and nonoperative management is usually successful (Fig. 39-42). Recent studies have shown that associated

1	abdominal trauma. A grading system is used to character-ize hepatic injuries (Table 39-7), and nonoperative management is usually successful (Fig. 39-42). Recent studies have shown that associated injuries are more significant predictors of out-come in children with liver injuries than the actual injury grade. Criteria for surgery are similar to those for splenic injury and primarily involve hemodynamic instability. The intraoperative considerations in the management of massive hepatic injury are similar in children and adults. Renal contusions may occur after significant blunt abdominal trauma. Nonoperative management is usually successful, unless patients are unstable due to active renal bleeding. It is important to confirm the presence of a nor-mal contralateral kidney at the time of surgery.FETAL INTERVENTIONOne to the most exciting developments in the field of pediatric surgery has been the emergence of fetal surgery. In general terms, performance of a fetal intervention may be

1	surgery.FETAL INTERVENTIONOne to the most exciting developments in the field of pediatric surgery has been the emergence of fetal surgery. In general terms, performance of a fetal intervention may be justified in the setting where a defect is present that would cause devastating consequences to the infant if left uncorrected. For the vast majority of congenital anomalies, postnatal surgery is the preferred modality. However, in specific circumstances, fetal surgery may offer the best possibility for a successful outcome. Table 39-6Grading of splenic injuriesGrade I: Subcapsular hematoma, <10% surface area capsular tear, <1 cm in depthGrade II: Subcapsular hematoma, nonexpanding, 10%–50% surface area; intraparenchymal hematoma, nonexpanding, <2 cm in diameter; capsular tear, active bleeding, 1–3 cm, does not involve trabecular vesselGrade III: Subcapsular hematoma, >50% surface area or expanding; intraparenchymal hematoma, >2 cm or expanding; laceration >3 cm in depth or involving

1	1–3 cm, does not involve trabecular vesselGrade III: Subcapsular hematoma, >50% surface area or expanding; intraparenchymal hematoma, >2 cm or expanding; laceration >3 cm in depth or involving trabecular vesselsGrade IV: Ruptured intraparenchymal hematoma with active bleeding; laceration involving segmental or hilar vessels producing major devascularizatrion (>25% of spleen).Grade V: Shattered spleen; hilar vascular injury that devascularizes spleenTable 39-7Liver injury grading systemGrade I: Capsular tear <1 cm in depthGrade II: Capsular tear 1–3 cm in depth, <10 cm lengthGrade III: Capsular tear >3 cm in depthGrade IV: Parenchymal disruption 25%–75% of hepatic lobe or 1–3 Couinaud’s segmentsGrade V: Parenchymal disruption >75% of hepatic lobe or >3 Couinaud’s segments within a single lobe, injury to retrohepatic vena cavaReproduced with permission from Moore EE, Cogbill TH, Malangoni MA, et al: Organ injury scaling, Surg Clin North Am. 1995

1	>3 Couinaud’s segments within a single lobe, injury to retrohepatic vena cavaReproduced with permission from Moore EE, Cogbill TH, Malangoni MA, et al: Organ injury scaling, Surg Clin North Am. 1995 Apr;75(2):293-303.Brunicardi_Ch39_p1705-p1758.indd 175312/02/19 11:27 AM 1754SPECIFIC CONSIDERATIONSPART IIFigure 39-43. The EXIT procedure (ex utero intrapartum treat-ment) in a 34-week gestation age baby with a large cervical tera-toma. Intubation is being performed while the fetus is on placental support.Figure 39-42. Abdominal computed tomography in a child dem-onstrating a grade 3 liver laceration (arrows).Fetal Surgery for MyelomeningoceleMyelomeningocele refers to a spectrum of anomalies in which portions of the spinal cord are uncovered by the spinal column. This leaves the neural tissue exposed to the injurious effects of the amniotic fluid, as well as to trauma from contact with the uterine wall. Nerve damage ensues, resulting in varying degrees of lower extremity paralysis

1	tissue exposed to the injurious effects of the amniotic fluid, as well as to trauma from contact with the uterine wall. Nerve damage ensues, resulting in varying degrees of lower extremity paralysis as well as bowel and bladder dys-function. Initial observations indicated that the extent of injury progressed throughout the pregnancy, which provided the ratio-nale for fetal intervention. The current in utero approach for the fetus with myelomeningocele has focused on obtaining cover-age of the exposed spinal cord. The efficacy of in utero treat-ment versus postnatal repair was recently compared in a large multicenter trial as described earlier and showed that prenatal surgery for myelomeningocele reduced the need for shunting and improved motor outcomes at 30 months but was associ-ated with maternal and fetal risks. The results of this study have paved the way for the acceptance of in utero repair of myelome-ningocele in certain centers with the experience and expertise to perform this

1	maternal and fetal risks. The results of this study have paved the way for the acceptance of in utero repair of myelome-ningocele in certain centers with the experience and expertise to perform this procedure safely.The EXIT ProcedureThe EXIT procedure is an abbreviation for ex utero intrapar-tum treatment. It is utilized in circumstances where airway obstruction is predicted at the time of delivery due to the pres-ence of a large neck mass, such as a cystic hygroma or teratoma (Fig. 39-43), or congenital tracheal stenosis. The success of the procedure is dependent upon the maintenance of utero-placen-tal perfusion for a sufficient duration to secure the airway. To achieve this, deep uterine relaxation is obtained during a cae-sarian section under general anesthesia. Uterine perfusion with warmed saline also promotes relaxation and blood flow to the placenta. On average, between 20 and 30 minutes of placental perfusion can be achieved. The fetal airway is secured either by placement

1	warmed saline also promotes relaxation and blood flow to the placenta. On average, between 20 and 30 minutes of placental perfusion can be achieved. The fetal airway is secured either by placement of an orotracheal tube or performance of a tracheos-tomy. Once the airway is secured, the cord is cut, and a defini-tive procedure may be performed to relieve the obstruction in the postnatal period. In general terms, cystic neck masses such as lymphangiomas have a more favorable response to an EXIT procedure as compared to solid tumors, such as teratomas, par-ticularly in premature infants.The decision to perform a fetal intervention requires careful patient selection, as well as a multidisciplinary center that is dedicated to the surgical care of the fetus and the mother. Patient selection is dependent in part upon highly accurate prenatal imaging that includes US and MRI. Significant risks may be associated with the performance of a fetal surgical procedure, to both the mother and the

1	is dependent in part upon highly accurate prenatal imaging that includes US and MRI. Significant risks may be associated with the performance of a fetal surgical procedure, to both the mother and the fetus. From the maternal viewpoint, open fetal surgery may lead to uterine bleeding due to the uterine relaxation required during the procedure. The long-term effects on subsequent pregnancies remain to be established. For the fetus, in utero surgery carries the risk of premature labor and amniotic fluid leak. As a result, these procedures are performed only when the expected benefit of fetal intervention outweighs the risk to the fetus of standard postnatal care. Currently, open fetal intervention may be efficacious in certain instances of large congenital lung lesions with hydrops, large teratomas with hydrops, twin-twin transfusion syndrome, certain cases of congenital lower urinary tract obstruction, and myelomeningocele. The Management of Myelomeningocele Study, which was funded by

1	with hydrops, twin-twin transfusion syndrome, certain cases of congenital lower urinary tract obstruction, and myelomeningocele. The Management of Myelomeningocele Study, which was funded by the NIH, compared prenatal with postnatal repair of myelomeningocele, and determined that prenatal repair was associated with improved motor skills and independent walking. There are ongoing trials for the evaluation of fetal tracheal occlusion in the setting of severe congenital diaphragmatic hernia, from which early results are very promising. The field has undertaken a rigorous evaluation of the potential benefit of prenatal as compared to postnatal management of many of these conditions, given the significant risk that may be associated with fetal therapy.Fetal Surgery for Lower Urinary Tract ObstructionLower urinary tract obstruction refers to a group of diseases characterized by obstruction of the distal urinary system. Com-mon causes include the presence of posterior urethral valves and

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1	J Pediatr Surg. 1998;33:830-833.Hackam DJ, Potoka D, Meza M, et al. Utility of radiographic hepatic injury grade in predicting outcome for children after blunt abdominal trauma. J Pediatr Surg. 2002;37:386-389.Hackam DJ, Reblock K, Barksdale E, et al. The influence of Down’s syndrome on the management and outcome of children with Hirschsprung’s disease. J Pediatr Surg. 2003;38:946-949.Hackam DJ, Superina R, Pearl R, et al. Single-stage repair of Hirschsprung’s disease: a comparison of 109 patients over 5 years. J Pediatr Surg. 1997;32:1028-1031.Hamner CE, Groner JI, Caniano DA, Hayes JR, Kenney BD. Blunt intraabdominal arterial injury in pediatric trauma patients: injury distribution and markers of outcome. J Pediatr Surg. 2008;43:916-923.Harnoss JC, Zelienka I, Probst P, et al. Antibiotics versus surgical therapy for uncomplicated appendicitis: systematic review and meta-analysis of controlled trials (PROSPERO 2015: CRD42015016882). Ann Surg. 2016;265:889-900.Harrison MR. Fetal

1	versus surgical therapy for uncomplicated appendicitis: systematic review and meta-analysis of controlled trials (PROSPERO 2015: CRD42015016882). Ann Surg. 2016;265:889-900.Harrison MR. Fetal surgery: trials, tribulations, and turf. J Pediatr Surg. 2003;38:275-282.Harrison MR, Keller RL, Hawgood S, et al. A randomized trial of fetal endoscopic tracheal occlusion for severe fetal congenital diaphragmatic hernia. N Engl J Med. 2003;349:1916-1924.Harrison MR, Sydorak RM, Farrell J, et al. Fetoscopic temporary tracheal occlusion for congenital diaphragmatic hernia: prelude to a randomized, controlled trial. J Pediatr Surg. 2003;38:1012-1020.Heath JK. Transcriptional networks and signaling pathways that govern vertebrate intestinal development. Curr Top Dev Biol. 2010;90:159-192.Hedrick H, Flake A, Crombleholme T, et al. History of fetal diagnosis and therapy: Children’s Hospital of Philadelphia experience. Fetal Diagn Ther. 2003;18:65-82.Hilton EN, Manson FD, Urquhart JE, et al.

1	H, Flake A, Crombleholme T, et al. History of fetal diagnosis and therapy: Children’s Hospital of Philadelphia experience. Fetal Diagn Ther. 2003;18:65-82.Hilton EN, Manson FD, Urquhart JE, et al. Left-sided embryonic expression of the BCL-6 corepressor, BCOR, is required for vertebrate laterality determination. Hum Mol Genet. 2007;16:1773-1782.Hirschl RB, Philip WF, Glick L, et al. A prospective, randomized pilot trial of perfluorocarbon-induced lung growth in newborns with congenital diaphragmatic hernia. J Pediatr Surg. 2003;38:283-289.Huh JW, Raghupathi R. New concepts in treatment of pediatric traumatic brain injury. Anesthesiol Clin. 2009;27(2):213-240.Hutchings L, Willett K. Cervical spine clearance in pediatric trauma: a review of current literature. J Trauma. 2009;67(4):687-691.Jani J, Nicolaides KH, Keller RL, et al. Observed to expected lung area to head circumference ratio in the prediction of survival in fetuses with isolated diaphragmatic hernia. Ultrasound Obstet

1	J, Nicolaides KH, Keller RL, et al. Observed to expected lung area to head circumference ratio in the prediction of survival in fetuses with isolated diaphragmatic hernia. Ultrasound Obstet Gynecol. 2007;30:67-71.Jani JC, Peralta CF, Nicolaides KH. Lung-to-head ratio: a need to unify the technique. Ultrasound Obstet Gynecol. 2012;39:2-6.Johnigan RH, Pereira KD, Poole MD. Community-acquired methicillin-resistant Staphylococcus aureus in children and adolescents: changing trends. Arch Otolaryngol Head Neck Surg. 2003;129(10):1049-1052.Johnson MP, Sutton LN, Rintoul N, et al. Fetal myelomeningocele repair: short-term clinical outcomes. Am J Obstet Gynecol. 2003;189:482-487.Kalapurakal J, Li S, Breslow N, et al. Influence of radiation therapy delay on abdominal tumor recurrence in patients with favorable histology Wilms’ tumor treated on NWTS-3 and NWTS-4: a report from the National Wilms’ Tumor Study Group. Int J Radiat Oncol Biol Phys. 2003;57:495-499.Kamata S, Ishikawa S, Usui N, et

1	favorable histology Wilms’ tumor treated on NWTS-3 and NWTS-4: a report from the National Wilms’ Tumor Study Group. Int J Radiat Oncol Biol Phys. 2003;57:495-499.Kamata S, Ishikawa S, Usui N, et al. Prenatal diagnosis of abdominal wall defects and their prognosis. J Pediatr Surg. 1996;31:267-271.Kantarci S, Al-Gazali L, Hill RS, et al. Mutations in LRP2, which encodes the multiligand receptor megalin, cause Donnai-Barrow and facio-oculo-acoustico-renal syndromes. Nat Genet. 2007;39:957-959.Katzenstein HM, Krailo MD, Malogolowkin M, et al. Hepatocellular carcinoma in children and adolescents: results from the Pediatric Oncology Group and the Children’s Cancer Group Intergroup Study. J Clin Oncol. 2002;20:2789-2797.Kim HB, Fauza D, Garza J, Oh JT, Nurko S, Jaksic T. Serial transverse enteroplasty (STEP): a novel bowel lengthening procedure. J Pediatr Surg. 2003;38:425-429.Kim HB, Lee PW, Garza J, et al. Serial transverse enteroplasty for short bowel syndrome: a case report. J Pediatr

1	(STEP): a novel bowel lengthening procedure. J Pediatr Surg. 2003;38:425-429.Kim HB, Lee PW, Garza J, et al. Serial transverse enteroplasty for short bowel syndrome: a case report. J Pediatr Surg. 2003;38:881-885.Kim JR, Suh CH, Yoon HM, et al. Performance of MRI for suspected appendicitis in pediatric patients and negative appendectomy rate: a systematic review and meta-analysis. J Magn Reson Imaging. 2018;47(3):767-778.Brunicardi_Ch39_p1705-p1758.indd 175612/02/19 11:27 AM 1757PEDIATRIC SURGERYCHAPTER 39Kliegman RM. Models of the pathogenesis of necrotizing enterocolitis. J Pediatr. 1990;117:S2-S5.Kliegman RM, Fanaroff AA. Necrotizing enterocolitis. N Engl J Med. 1984;310:1093-1103.Koivusalo AI, Korpela R, Wirtavuori K, Piiparinen S, Rintala RJ, Pakarinen MP. A single-blinded, randomized comparison of laparoscopic versus open hernia repair in children. Pediatrics. 2009;123:332-337.Konkin D, O’hali W, Webber EM, Blair GK. Outcomes in esophageal atresia and tracheoesophageal

1	randomized comparison of laparoscopic versus open hernia repair in children. Pediatrics. 2009;123:332-337.Konkin D, O’hali W, Webber EM, Blair GK. Outcomes in esophageal atresia and tracheoesophageal fistula. J Pediatr Surg. 2003;38:1726-1729.Kosloske AM. Operative techniques for the treatment of neonatal necrotizing enterocolitis. Surg Gynecol Obstet. 1979;149:740-744.Kosloske AM. Indications for operation in necrotizing enterocolitis revisited. J Pediatr Surg. 1994;29:663-666.Kosloske AM, Lilly JR. Paracentesis and lavage for diagnosis of intestinal gangrene in neonatal necrotizing enterocolitis. J Pediatr Surg. 1978;13:315-320.Lacroix J, Hebert PC, Hutchison JS, et al. Transfusion strategies for patients in pediatric intensive care units. N Engl J Med. 2007;356:1609-1619.Langer J, Durrant A, de la Torre L, et al. One-stage transanal Soave pullthrough for Hirschsprung disease: a multicenter experience with 141 children. Ann Surg. 2003;238:569-583.Levitt MA, Ferraraccio D, Arbesman

1	A, de la Torre L, et al. One-stage transanal Soave pullthrough for Hirschsprung disease: a multicenter experience with 141 children. Ann Surg. 2003;238:569-583.Levitt MA, Ferraraccio D, Arbesman M, et al. Variability of inguinal hernia surgical technique: a survey of North American pediatric surgeons. J Pediatr Surg. 2002;37:745-751.Lille ST, Rand RP, Tapper D, Gruss JS. The surgical management of giant cervicofacial lymphatic malformations. J Pediatr Surg. 1996;31:1648-1650.Limmer J, Gortner L, Kelsch G, Schutze F, Berger D. Diagnosis and treatment of necrotizing enterocolitis. A retrospective evaluation of abdominal paracentesis and continuous postoperative lavage. Acta Paediatr Suppl. 1994;396:65-69.Lintula H, Kokki H, Vanamo K. Single-blind randomized clinical trial of laparoscopic versus open appendicectomy in children. Br J Surg. 2001;88:510-514.Lipshutz G, Albanese C, Feldstein V, et al. Prospective analysis of lung-to-head ratio predicts survival for patients with prenatally

1	open appendicectomy in children. Br J Surg. 2001;88:510-514.Lipshutz G, Albanese C, Feldstein V, et al. Prospective analysis of lung-to-head ratio predicts survival for patients with prenatally diagnosed congenital diaphragmatic hernia. J Pediatr Surg. 1997;32:1634-1636.Little D, Rescorla F, Grosfeld J, et al. Long-term analysis of children with esophageal atresia and tracheoesophageal fistula. J Pediatr Surg. 2003;38:852-856.Loeb DM, Thornton K, Shokek O. Pediatric soft tissue sarcomas. Surg Clin North Am. 2008;88:615-627.Luig M, Lui K. Epidemiology of necrotizing enterocolitis—part I: changing regional trends in extremely preterm infants over 14 years. J Paediatr Child Health. 2005;41:169-173.Lynch L, O’Donoghue D, Dean J, O’Sullivan J, O’Farrelly C, Golden-Mason L. Detection and characterization of hemopoietic stem cells in the adult human small intestine. J Immunol. 2006;176:5199-5204.Maheshwari A, Patel RM, Christensen RD. Anemia, red blood cell transfusions, and necrotizing

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1	correlates with extent of disease. J Pediatr Surg. 2001;36:1122-1129.Neville HL, Andrassy RJ, Lally K, et al. Lymphatic mapping with sentinel node biopsy in pediatric patients. J Pediatr Surg. 2000;35:961-964.Nino DF, Sodhi CP, Hackam DJ. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms. Nat Rev Gastroenterol Hepatol. 2016;13:590-600.Nio M, Ohi R, Miyano T, et al. Fiveand 10-year survival rates after surgery for biliary atresia: a report from the Japanese Biliary Atresia Registry. J Pediatr Surg. 2003;38:997-1000.O’Donovan DJ, Baetiong A, Adams K, et al. Necrotizing enterocolitis and gastrointestinal complications after indomethacin therapy and surgical ligation in premature infants with patent ductus arteriosus. J Perinatol. 2003;23: 286-290.Olutoye OO, Coleman BG, Hubbard A, et al. Prenatal diagnosis and management of congenital lobar emphysema. J Pediatr Surg. 2000;35:792-795.Ortega JA, Douglass EC, Feusner J, et al. Randomized comparison of

1	OO, Coleman BG, Hubbard A, et al. Prenatal diagnosis and management of congenital lobar emphysema. J Pediatr Surg. 2000;35:792-795.Ortega JA, Douglass EC, Feusner J, et al. Randomized comparison of cisplatin/vincristine/fluorouracil and cisplatin/continuous infusion doxorubicin for treatment of pediatric hepatoblastoma: a report from the Children’s Cancer Group and the Pediatric Oncology Group. J Clin Oncol. 2000;18:2665.Pandya S, Heiss K. Pyloric stenosis in pediatric surgery: an evidence based review. Surg Clin North Am. 2012;92:527-539, vii-viii.Panesar J, Higgins K, Daya H, et al. Nontuberculous mycobacterial cervical adenitis: a ten-year retrospective review. Laryngoscope. 2003;113:149-154.Pedersen A, Petersen O, Wara P, et al. Randomized clinical trial of laparoscopic versus open appendicectomy. Br J Surg. 2001;88:200-205.Pena A, Guardino K, Tovilla J, et al. Bowel management for fecal incontinence in patients with anorectal malformations. J Pediatr Surg.

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1	a common diagnostic error with significant consequences in girls with anorectal malformations. J Pediatr Surg. 2002;37:961-965.Rothenberg S. Laparoscopic Nissen procedure in children. Semin Laparosc Surg. 2002;9:146-152.Sandler A, Ein S, Connolly B, et al. Unsuccessful air-enema reduction of intussusception: is a second attempt worthwhile? Pediatr Surg Int. 1999;15:214-216.Sarioglu A, McGahren ED, Rodgers BM. Effects of carotid artery repair following neonatal extracorporeal membrane oxygenation. Pediatr Surg Int. 2000;16:15-18.Schier F, Montupet P, Esposito C. Laparoscopic inguinal herniorrhaphy in children: a three-center experience with 933 repairs. J Pediatr Surg. 2002;37:395-397.Schonfeld D, Lee LK. Blunt abdominal trauma in children. Curr Opin Pediatr. 2012;24:314-318.Shamberger R, Guthrie K, Ritchey M, et al. Surgery-related factors and local recurrence of Wilms tumor in National Wilms Tumor Study 4. Ann Surg. 1999;229:292-297.Shimada H, Ambros I, Dehner L, et al. The

1	R, Guthrie K, Ritchey M, et al. Surgery-related factors and local recurrence of Wilms tumor in National Wilms Tumor Study 4. Ann Surg. 1999;229:292-297.Shimada H, Ambros I, Dehner L, et al. The International Neuroblastoma Pathology Classification (the Shimada system). Cancer. 1999;86:364-372.Shivakumar P, Campbell KM, Sabla GE, et al. Obstruction of extrahepatic bile ducts by lymphocytes is regulated by IFNgamma in experimental biliary atresia. J Clin Invest. 2004;114:322-329.Simons SHP, van Dijk M, van Lingen R, et al. Routine morphine infusion in preterm newborns who received ventilatory support: a randomized controlled trial. JAMA. 2003;290:2419-2427.Soffer SZ, Rosen NG, Hong AR, et al. Cloacal exstrophy: a unified management plan. J Pediatr Surg. 2000;35:932-937.Spitz L, Kiely E, Morecroft J, et al. Oesophageal atresia: at-risk groups for the 1990s. J Pediatr Surg. 1994;29:723-725.Sun L, Rommens JM, Corvol H, et al. Multiple apical plasma membrane constituents are associated

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1	review of presenting symptoms, evaluation, management, and outcome in infants. Dig Dis Sci. 2003;48:1723-1729.Tsao K, St Peter SD, Sharp SW, et al. Current application of thoracoscopy in children. J Laparoendosc Adv Surg Tech A. 2008;18:131-135.Tulipan N, Sutton L, Bruner J, et al. The effect of intrauterine myelomeningocele repair on the incidence of shunt-dependent hydrocephalus. Pediatr Neurosurg. 2003;38:27-33.Vargas JV, Vlassov D, Colman D, Brioschi ML. A thermodynamic model to predict the thermal response of living beings during pneumoperitoneum procedures. J Med Eng Technol. 2005;29:75-81.Wang KS, Shaul DB. Two-stage laparoscopic orchidopexy with gubernacular preservation: preliminary report of a new approach to the intraabdominal testis. J Pediatr Endosurg Innovative Tech. 2004;8:252-255.Wenzler D, Bloom D, Park J. What is the rate of spontaneous testicular descent in infants with cryptorchidism? J Urol. 2004;171:849-851.Wildhaber B, Coran A, Drongowski R, et al. The Kasai

1	D, Bloom D, Park J. What is the rate of spontaneous testicular descent in infants with cryptorchidism? J Urol. 2004;171:849-851.Wildhaber B, Coran A, Drongowski R, et al. The Kasai portoenterostomy for biliary atresia: a review of a 27-year experience with 81 patients. J Pediatr Surg. 2003;38:1480-1485.Wood JH, Partrick DA, Johnston RB, Jr. The inflammatory response to injury in children. Curr Opin Pediatr. 2010;22:315-320.Xu J, Adams S, Liu YC, Karpelowsky J. Nonoperative management in children with early acute appendicitis: a systematic review. J Pediatr Surg. 2017;52:1409-1415.Yang EY, Allmendinger N, Johnson SM, Chen C, Wilson JM, Fishman SJ. Neonatal thoracoscopic repair of congenital diaphragmatic hernia: selection criteria for successful outcome. J Pediatr Surg. 2005;40:1369-1375.Brunicardi_Ch39_p1705-p1758.indd 175812/02/19 11:27 AM

1	UrologyAhmad Shabsigh, Michael Sourial, Fara F. Bellows, Christopher McClung, Rama Jayanthi, Stephanie Kielb, Geoffrey N. Box, Bodo E. Knudsen, and Cheryl T. Lee 40chapterANATOMYThe anatomic structures that generally require urologic man-agement include the kidneys, adrenal glands, ureters, bladder, prostate, seminal vesicles, vas deferens, penis, urethra, scrotum, and testes. These organs are located in retroperitoneal or extra-peritoneal spaces. However, a transperitoneal approach may be utilized to access the kidney, ureters, bladder, or retroperitoneal lymph nodes during certain urologic operations.Kidney and Adrenal GlandThe kidneys are paired retroperitoneal organs that are invested in a fibro-fatty layer of tissue known as Gerota’s fascia. This natural barrier helps to tamponade bleeding and thus may provide renal and hemodynamic protection in cases of renal trauma or spontaneous renal hemorrhage. It also may assist in preventing tumor invasion into surrounding struc-tures in

1	and thus may provide renal and hemodynamic protection in cases of renal trauma or spontaneous renal hemorrhage. It also may assist in preventing tumor invasion into surrounding struc-tures in the case of large renal masses. The kidneys are bor-dered posterolaterally by the quadratus lumborum muscle and posteromedially by the psoas muscle. Additionally, the diaphragm drapes across the posterior aspect of the superior pole of each kidney.The left kidney is bordered anterolaterally by the spleen and descending colon. The pancreatic tail borders the antero-medial left kidney. The right kidney is bordered anterolaterally by the liver and the ascending colon. The second portion of the duodenum may be encountered near the right renal vessels and thus sometimes requires anteromedial reflection, known as the Kocher maneuver, to achieve intraoperative vascular control during right renal surgery.The kidneys are end organs, which are responsible for their vulnerability to infarction. The renal

1	known as the Kocher maneuver, to achieve intraoperative vascular control during right renal surgery.The kidneys are end organs, which are responsible for their vulnerability to infarction. The renal arteries extend from the aorta and then branch into several segmental arteries and arterioles before becoming glomeruli. Each artery runs poste-rior to their respective renal vein. Occasionally, an accessory renal artery will arise, but in general, each kidney receives a single main renal artery. Each renal vein drains directly into the IVC and is located anteriorly to its respective renal artery when entering the kidney. The right renal vein is much shorter than the left and does not receive collateral venous drainage. The left renal vein passes anteriorly to the aorta and receives drainage from the left gonadal vein, the left inferior adrenal vein, and a lumbar vein.The collecting system of the kidney begins as minor caly-ces near the renal papillae and then coalesces into major calyces.

1	the left gonadal vein, the left inferior adrenal vein, and a lumbar vein.The collecting system of the kidney begins as minor caly-ces near the renal papillae and then coalesces into major calyces. Major calyces join to form the renal pelvis, which then tapers down to the ureteropelvic junction (UPJ), from which the ureter emanates. The pelvis is located posterior to its respective renal artery.The adrenal gland is superomedial to its respective kidney within Gerota’s fascia. Adrenal arterial supply arises from mul-tiple sources: the inferior phrenic artery, aortic branches, and renal arterial branches. Venous drainage mirrors arterial supply. On the right side, the adrenal gland drains directly into the IVC. The right adrenal vein can be quite short (<1 cm) and can be Anatomy 1759Kidney and Adrenal Gland / 1759Ureter / 1760Bladder and Prostate / 1760Penis / 1760Scrotum and Testes / 1760Infection 1761Cystitis / 1761Pyelonephritis / 1761Prostatitis / 1761Epididymo-Orchitis /

1	and Adrenal Gland / 1759Ureter / 1760Bladder and Prostate / 1760Penis / 1760Scrotum and Testes / 1760Infection 1761Cystitis / 1761Pyelonephritis / 1761Prostatitis / 1761Epididymo-Orchitis / 1761Balanitis and Balanoposthitis / 1762Urinary Tract Obstruction 1762Urolithiasis / 1762Benign Prostatic Hyperplasia / 1763Urethral Stricture / 1764Other Causes of Obstruction / 1764Genitourinary Trauma 1764Kidneys / 1765Ureters / 1765Bladder / 1766Urethral Injuries / 1766External Genital Injuries / 1767Emergencies 1768Acute Urinary Retention / 1768Testicular Torsion / 1769Fournier’s Gangrene / 1769Priapism / 1769Paraphimosis / 1770Emphysematous Pyelonephritis / 1770Urologic Malignancies 1770Bladder Cancer / 1770Testicular Cancer / 1771Kidney Cancer / 1772Prostate Cancer / 1774Urethral Cancer / 1775Common Urologic Conditions 1775Urinary Incontinence and Voiding Dysfunction / 1775Erectile Dysfunction / 1775Pediatric Urology 1776Hypospadias / 1776Urinary Tract Infections in Children / 1777Prenatal

1	Urologic Conditions 1775Urinary Incontinence and Voiding Dysfunction / 1775Erectile Dysfunction / 1775Pediatric Urology 1776Hypospadias / 1776Urinary Tract Infections in Children / 1777Prenatal Hydronephrosis / 1777Cryptorchidism / 1777Brunicardi_Ch40_p1759-p1782.indd 175901/03/19 6:34 PM 1760Key Points1 Most small ureteral calculi will pass spontaneously or with the use of medical expulsive therapy, but larger stones (>6 mm) are better treated with ureteral stenting or lithotripsy.2 Benign prostatic hyperplasia can be managed effectively with medical therapy or minimally invasive endoscopic and robotic surgical techniques depending on the urinary symp-toms, patient bother, prostate size, and patient’s therapeutic choice. 3 Patients with recurrent urethral stricture after endoscopic treatment are unlikely to derive sustained benefit from future endoscopic therapies and should be referred for urethral reconstruction.4 The vast majority of renal trauma can be treated

1	endoscopic treatment are unlikely to derive sustained benefit from future endoscopic therapies and should be referred for urethral reconstruction.4 The vast majority of renal trauma can be treated conserva-tively, with early surgical intervention reserved for persistent bleeding, renal vascular, or ureteral injuries. 5 Extraperitoneal bladder ruptures can be treated conserva-tively, but intraperitoneal ruptures typically require surgical repair.6 Testicular torsion is an emergency where successful testicu-lar salvage is inversely related to the delay in repair, so cases with a high degree of clinical suspicion should not wait for a radiologic diagnosis.7 Fournier’s gangrene is a rapidly progressive and potentially lethal condition that requires aggressive débridement and close follow-up due to the frequent need for repeat débridement.8 The management of early stage prostate cancer has changed significantly, with a much greater emphasis on risk stratifi-cation. Low risk patients are

1	due to the frequent need for repeat débridement.8 The management of early stage prostate cancer has changed significantly, with a much greater emphasis on risk stratifi-cation. Low risk patients are largely treated with active surveillance. 9 Treatments for urinary incontinence and voiding dysfunction are varied depending on the etiology, severity, and bother of the symptom. Behavior modification, bladder retraining, and medical therapies can all be effective in improving symptoms without the need for surgery.a source of significant bleeding if inadvertently injured during renal or adrenal surgery.UreterThe ureters are smooth muscle–based tubular structures that connect the renal pelvis to the bladder. The blood supply arises from the surrounding vasculature. The proximal blood supply inserts on the medial aspect of the ureter and arises from the aorta and renal artery, and the distal blood supply inserts lat-erally and arises from the surrounding iliac vessels and their branches.

1	inserts on the medial aspect of the ureter and arises from the aorta and renal artery, and the distal blood supply inserts lat-erally and arises from the surrounding iliac vessels and their branches. The arterial supply inserts via a fatty layer of tissue around the ureter, and thus surgical preservation of the periure-teral tissue is essential to maintain vascularization and achieve successful ureteral reconstruction.The ureters initially course along the psoas muscle and then run distally along the pelvic sidewall. They generally pass posterior to the uterine arteries, making them susceptible to injury during hysterectomy. The ureters enter the bladder lat-erally and pass through the bladder wall at an oblique angle, which helps prevent reflux of urine during bladder filling. The ureters propel urine into the bladder via the ureteral orifices.Bladder and ProstateThe bladder is located extraperitoneally in the pelvis and pos-terior to the pubis. A portion of the bladder dome is

1	propel urine into the bladder via the ureteral orifices.Bladder and ProstateThe bladder is located extraperitoneally in the pelvis and pos-terior to the pubis. A portion of the bladder dome is draped by peritoneum, and rupture or injury at this location can result in intraperitoneal urine leakage and subsequent chemical peritoni-tis. The average adult bladder holds approximately 500 mL of urine; however, in rare cases, capacity can reach up to or greater than 1000 mL, in which case the bladder extends towards the umbilicus. The sigmoid colon lies adjacent to the bladder and can fistulize to the lateral wall or dome of the bladder in cases of diverticulitis or colon cancer. The rectum lies posteriorly to the bladder in men, and the uterus and vagina lie posteriorly to the bladder in women.The prostate is a walnut-shaped gland that encircles the urethra and is located in males immediately beneath the blad-der neck. Smooth muscle fibers distribute throughout the gland, which can contract

1	prostate is a walnut-shaped gland that encircles the urethra and is located in males immediately beneath the blad-der neck. Smooth muscle fibers distribute throughout the gland, which can contract and facilitate bladder outlet obstruction. The average prostate measures approximately 30 mL in volume. Puboprostatic ligaments suspend the prostate to the pubis, and in the instance of pelvic trauma, shearing forces can cause disrup-tion of the posterior urethra (known as pelvic fracture urethral injury). The external urethral sphincter houses the membranous urethra and sits just below the apex of the prostate. Vasculature to the bladder and prostate arises from the superior and inferior vesical arteries, which branch from the internal iliac arteries.PenisThe penis is comprised of three bodies: two corpora cavernosa, which are responsible for erection, and the corpus spongiosum, which surrounds the urethra and gives rise to the glans penis. These three structures are all encased by skin and

1	two corpora cavernosa, which are responsible for erection, and the corpus spongiosum, which surrounds the urethra and gives rise to the glans penis. These three structures are all encased by skin and dartos fascia, as well as an inner investing layer of fascia called Buck’s fascia. The corpora cavernosa are spongy sinusoidal bodies that expand with parasympathetic neural stimulation to create an erection. Thick fascia, called tunica albuginea, assists in producing rigid-ity during erection. Each corpus cavernosum features a centrally located cavernosal artery, which arises from the penile artery. A porous septum separates the two corpora and allows for trans-corporal blood exchange. The corpus spongiosum is located on the ventrum of the penis. The corpus spongiosum lacks a tough fascia similar to tunica albuginea and thus does not exhibit the same rigidity during erection.Scrotum and TestesThe scrotum is a potential space that surrounds the testes, epididymis, and spermatic cords. The

1	to tunica albuginea and thus does not exhibit the same rigidity during erection.Scrotum and TestesThe scrotum is a potential space that surrounds the testes, epididymis, and spermatic cords. The scrotum is comprised of many layers aside from skin and dartos fascia, and each derives from a particular layer of the anterior abdominal wall. The external spermatic fascia arises from the external oblique fascia, the cremasteric fascia arises from the internal oblique fascia, and the internal spermatic fascia arises from the transver-sus abdominis fascia. The testes are separated from the scrotal layers by the visceral and parietal layers of the tunica vaginalis, between which hydroceles form. The spermatic cord contains Brunicardi_Ch40_p1759-p1782.indd 176001/03/19 6:34 PM 1761UROLOGYCHAPTER 40the vas deferens, the venous pampiniform plexus, and arterial blood supply to the superior pole of the testis via three separate sources. The testicular artery arises directly from the aorta; the

1	40the vas deferens, the venous pampiniform plexus, and arterial blood supply to the superior pole of the testis via three separate sources. The testicular artery arises directly from the aorta; the deferential artery, which supplies the vas deferens, arises from the internal iliac artery; and the cremasteric artery, which sup-plies the cremaster musculature, arises from the external iliac artery. The presence of multiple arterial sources provides col-lateral flow and prevents ischemia in the event of injury to a particular vascular branch. The venous pampiniform plexus can dilate to form a palpable or visible varicocele, which can serve as an etiology of chronic testicular pain or infertility.INFECTIONCystitisUncomplicated cystitis usually presents as new onset urinary frequency, urgency, and dysuria. Patients may also report lower back pain, suprapubic pain, foul-smelling urine, or gross hema-turia. Urinalysis with microscopy assists with diagnosis by confirming the presence of

1	and dysuria. Patients may also report lower back pain, suprapubic pain, foul-smelling urine, or gross hema-turia. Urinalysis with microscopy assists with diagnosis by confirming the presence of pyuria, hematuria, and bacteriuria. Office dipstick may be helpful, as the presence of nitrites reflects bacterial colonization and the presence of leukocyte esterase reflects pyuria. Risk factors for the development of uncompli-cated cystitis include female gender, sexual activity, and use of spermicides.1 Three days of antibiotics are generally sufficient for treatment of uncomplicated cystitis. Fluoroquinolones and trimethoprim-sulfamethoxazole are well tolerated and are eas-ily available. Nitrofurantoin, which is also commonly used for uncomplicated cystitis, requires 5 days of treatment. Men with uncomplicated cystitis should undergo 7 days of treatment.Complicated cystitis may arise in the setting of structural or functional urinary tract abnormalities, recent urinary tract

1	Men with uncomplicated cystitis should undergo 7 days of treatment.Complicated cystitis may arise in the setting of structural or functional urinary tract abnormalities, recent urinary tract instru-mentation, recent antimicrobial use, immunosuppressed states, pregnancy, or hospital-acquired infection. Symptoms may be simi-lar to uncomplicated cystitis but can progress to pyelonephritis if left untreated. Elderly or very young patients tend to exhibit leth-argy, change in mental status, or anorexia, which may confound the diagnosis of a urinary tract infection. Patients may require hos-pitalization if febrile or if symptoms are severe. Treatment consists of 10 to 14 days of antibiotics. Fluoroquinolones or trimethoprim-sulfamethoxazole are usually effective and should be administered based on culture results and/or regional bacteriograms. Asymp-tomatic bacteriuria does not require treatment unless detected dur-ing pregnancy or if urinary tract instrumentation is

1	be administered based on culture results and/or regional bacteriograms. Asymp-tomatic bacteriuria does not require treatment unless detected dur-ing pregnancy or if urinary tract instrumentation is planned.1PyelonephritisPyelonephritis arises when a bladder infection ascends proxi-mally along the ureters to the renal parenchyma. It may also result from hematogenous spread, such as in the case of intra-venous drug abuse or in patients with bacteremia from other sources. Patients with pyelonephritis may present with fevers, flank pain, nausea, vomiting, and lower urinary tract symptoms. Physical exam may reveal tenderness of the costovertebral angle. Patients may appear toxic, with poor oral intake. Labora-tory evaluation may reveal leukocytosis with elevated neutro-phils. Urinalysis usually demonstrates the presence of pyuria and bacteriuria, and urine culture should be sent prior to start-ing broad-spectrum antibiotics. Imaging should be considered to rule out obstruction, which could

1	demonstrates the presence of pyuria and bacteriuria, and urine culture should be sent prior to start-ing broad-spectrum antibiotics. Imaging should be considered to rule out obstruction, which could prolong the recovery period despite appropriate antimicrobial treatment.Acute pyelonephritis requires 7 to 14 days of antibiotic therapy. Mild or moderate cases, even if febrile, can safely be treated as an outpatient with oral antibiotics. Fluoroquino-lones and trimethoprim-sulfamethoxazole are ideal for treating pyelonephritis. Nitrofurantoin should not be used as it does not penetrate renal parenchyma. Patients with concern for sepsis or inability to tolerate oral intake may require hospitalization with IV antibiotics while awaiting culture results. Fevers may persist for up to 72 hours despite appropriate treatment. The presence of persistent fevers or symptoms after this time period warrants cross-sectional imaging to rule out renal or perinephric abscess. Treatment for renal or

1	despite appropriate treatment. The presence of persistent fevers or symptoms after this time period warrants cross-sectional imaging to rule out renal or perinephric abscess. Treatment for renal or perinephric abscess usually consists of percutaneous drainage and broad-spectrum IV antibiotics.ProstatitisAcute prostatitis is marked by fever, suprapubic or perineal pain, and new onset lower urinary tract symptoms, namely dys-uria, frequency, urgency, changes in stream caliber, or difficulty emptying the bladder. It is most often caused by urinary patho-gens. Digital rectal exam may reveal a tender and soft pros-tate. Bladder drainage with a Foley or suprapubic tube may be required if urinary retention is present. Treatment consists of a long-term course (4–6 weeks) of antibiotics. If not treated in a timely fashion, acute prostatitis can develop into severe sepsis or a prostatic abscess. Prostatic abscesses may require drainage via a transurethral approach or transrectal needle

1	not treated in a timely fashion, acute prostatitis can develop into severe sepsis or a prostatic abscess. Prostatic abscesses may require drainage via a transurethral approach or transrectal needle aspiration.Chronic prostatitis may be bacterial or abacterial. Symptoms in both cases include perineal, suprapubic, or penile pain, along with urinary frequency, urgency, or change in stream caliber. Men may also report pain in the groin, lower back, or testes. Fever is not observed in chronic prostatitis, and onset may occur over many months. Patients with chronic bacterial prostatitis may also report recurrent UTIs, with cultures consistently exhibiting the same bacteria. Differentiation between the two etiologies requires culture of expressed prostatic secretion to confirm the presence or absence of bacteria. Treatment of chronic bacterial prostatitis includes long-term antibiotics and α-blockers.Chronic abacterial prostatitis is also known as chronic pel-vic pain syndrome (CPPS).

1	or absence of bacteria. Treatment of chronic bacterial prostatitis includes long-term antibiotics and α-blockers.Chronic abacterial prostatitis is also known as chronic pel-vic pain syndrome (CPPS). Symptoms are similar to chronic bacterial prostatitis, but generally do not respond well to long-term antibiotics for treatment. It is generally somewhat more difficult to achieve symptomatic relief when treating CPPS, and options include α-blockers, NSAIDs, neuromodulators, and/or pelvic floor physical therapy.2Epididymo-OrchitisEpididymitis refers to inflammation of the epididymis. In most cases of bacterial infection, the testis is also affected, thus is encompassed by the term “epididymo-orchitis.” Common eti-ologies include sexually transmitted infection, especially in younger males, or urinary tract infection, which is more com-monly seen in older males. Other possible etiologies include underlying congenital urologic abnormality or incomplete blad-der emptying. Symptoms include pain

1	tract infection, which is more com-monly seen in older males. Other possible etiologies include underlying congenital urologic abnormality or incomplete blad-der emptying. Symptoms include pain and swelling of the epi-didymis and testis. Some men may report nausea or vomiting, which arises as a result of irritation of the spermatic cord. Uri-nary symptoms may be present, but absence of symptoms does not rule out bacterial epididymo-orchitis. Physical exam gen-erally reveals a tender, swollen epididymis and testis. Scrotal skin erythema or reactive hydrocele may be present as well. A complete blood count should be performed to rule out leukocy-tosis, and urinalysis with urine culture should be collected prior to initiation of antibiotics. Urethral swab should be performed if sexually transmitted infection is a possible etiology. The clini-cal presentation of testicular torsion can be quite similar to that of Brunicardi_Ch40_p1759-p1782.indd 176101/03/19 6:34 PM 1762SPECIFIC

1	transmitted infection is a possible etiology. The clini-cal presentation of testicular torsion can be quite similar to that of Brunicardi_Ch40_p1759-p1782.indd 176101/03/19 6:34 PM 1762SPECIFIC CONSIDERATIONSPART IIepididymo-orchitis. It may be quite difficult to clinically differenti-ate the two entities, but one should keep in mind that the onset of torsion tends to be slightly more acute (within 4–8 hours) than that of epididymo-orchitis (which generally arises over the course of 24–48 hours). Scrotal ultrasound can assist in diagnosis; how-ever, in cases of severe orchitis, testicular flow can be compro-mised, which may raise concern for torsion. Scrotal exploration should be considered in any equivocal case: a missed torsion can result in testicular loss secondary to necrosis.Treatment of epididymo-orchitis consists of single dose of ceftriaxone and azithromycin if there is concern for sexu-ally transmitted infection, as well as 14 days of oral antibiotic therapy, NSAIDs, and

1	of epididymo-orchitis consists of single dose of ceftriaxone and azithromycin if there is concern for sexu-ally transmitted infection, as well as 14 days of oral antibiotic therapy, NSAIDs, and scrotal support. If the patient exhibits fevers or toxic presentation, hospitalization with IV antibiotics may be required.Balanitis and BalanoposthitisBalanitis refers to inflammation of the glans penis. Balano-posthitis arises when the foreskin is also involved. Common etiologies include fungal infection, bacterial infection, contact dermatitis, or local trauma. Exam reveals a diffusely erythema-tous and warm glans penis, with inner preputial erythema as well if balanoposthitis is present. Treatment includes appropri-ate hygiene, topical antibiotics or antifungals, and occasionally topical steroids. If there is an inappropriate response to treat-ment, the differential diagnosis should include malignancy, pso-riasis, or infectious agents such as HPV.3URINARY TRACT OBSTRUCTIONUrolithiasisRenal

1	If there is an inappropriate response to treat-ment, the differential diagnosis should include malignancy, pso-riasis, or infectious agents such as HPV.3URINARY TRACT OBSTRUCTIONUrolithiasisRenal stone disease is a common problem that is a major health care burden to society today. The prevalence of stone disease in the United States has increased over the past several decades as reported by the National Health and Nutrition Examination Survey (NHANES), and was estimated at 8.8% for the period between 2007 and 2010.4 This prevalence has increased with factors such as global warming, poor diet choices, and the obesity trend. Overall, the total estimated annual expenditure for individuals with claims for a diagnosis of urolithiasis was almost $2.1 billion in 2000, representing a 50% increase since 1994.5 Risk factors for stone formation include dietary habits, family history, white race, geographical location or occupa-tional exposure to heat/dehydration, intestinal disease, and male

1	since 1994.5 Risk factors for stone formation include dietary habits, family history, white race, geographical location or occupa-tional exposure to heat/dehydration, intestinal disease, and male gender, although the gender gap is decreasing.6 More recently, stone formation has also been associated with obesity, metabolic syndrome, and diabetes mellitus.7,8Stones are most commonly composed of calcium oxa-late. Other stone compositions include calcium phosphate, uric acid, cystine, medication-related, and infectious stones (stru-vite or carbonate apatite) or a mix thereof. Stone composition can vary based on a number of underlying pathophysiological processes. For example, hyperoxaluria may be seen in patients who have undergone small bowel resection, particularly the ter-minal ileum. This can result in an increase in unabsorbed fatty acids and bile salts which undergo saponification by binding with calcium in the bowel. The increase in unbound oxalate is absorbed by the large

1	This can result in an increase in unabsorbed fatty acids and bile salts which undergo saponification by binding with calcium in the bowel. The increase in unbound oxalate is absorbed by the large intestine and subsequently excreted in the urine, favoring the formation of calcium oxalate stones. Uric acid stones will form in a context of acidic urinary pH, low uri-nary volume, and high oral intake of purines. Countering these factors by alkalinizing the urine and increasing urine output Figure 40-1. Struvite (infectious) stones are evident on a plain radiograph of the abdomen. The red arrows highlight a left Stag-horn calculus filling the renal pelvis and calyces and several stones in the right lower pole of the kidney.may lead to dissolution of uric acid stones and reduced fur-ther formation.9 Proteus species, Klebsiella species, and other urease-producing bacteria metabolize urea into ammonium and bicarbonate. The alkaline milieu (pH >7) predisposes to infec-tious (struvite) stones

1	Proteus species, Klebsiella species, and other urease-producing bacteria metabolize urea into ammonium and bicarbonate. The alkaline milieu (pH >7) predisposes to infec-tious (struvite) stones with the precipitation of magnesium, ammonium, and phosphate (Fig. 40-1).Evaluation for first-time stone formers should include a complete medical history and physical exam, basic metabolic panel, calcium, uric acid, urinalysis and culture, and radio-graphic imaging. A noncontrast computed tomography (CT) scan is the most sensitive (98%) and specific (97%) exam to detect urolithiasis10 and can provide additional anatomical infor-mation useful for surgical planning, although its use in recurrent stone formers should be balanced by cost and radiation expo-sure. Low-dose CT is currently the preferred imaging study for patients with a body mass index (BMI) <30. This imaging study uses less than one-third of the estimated effective ionizing radiation dose (3 mSv) compared to standard dose noncontrast

1	study for patients with a body mass index (BMI) <30. This imaging study uses less than one-third of the estimated effective ionizing radiation dose (3 mSv) compared to standard dose noncontrast CT (10 mSv),10 while maintaining excellent sensitivity (95%) and specificity (97%).11 Plain abdominal X-ray can be used to follow radiopaque stones such as calcium-containing stones or struvite stones, although at times struvite can be difficult to see on plain X-ray, especially when the fragments are small. Uric acid and triamterene stones are radiolucent on plain abdominal X-ray but will be visible on noncontrast CT. A full metabolic evaluation with a 24-hour urine collection is indicated in recur-rent stone formers, high-risk stone formers, or interested first-time stone formers.12The natural history of stones is variable and depends pri-marily on their size and location. Smaller and more distal stones are much more likely to pass spontaneously without the need for surgical

1	natural history of stones is variable and depends pri-marily on their size and location. Smaller and more distal stones are much more likely to pass spontaneously without the need for surgical intervention.13,14 Patients with ureteral stones ≤10 mm can be offered a period of observation if their pain is well controlled without signs of infection or renal insuf-ficiency. α-Blockers, which inhibit ureteral peristalsis, have been shown in meta-analyses to be particularly useful in patients with distal ureter stones ≤10 mm, improving the rate of stone passage from 54% to 77%,15,16 with shortened time to expulsion and fewer colic episodes.171Brunicardi_Ch40_p1759-p1782.indd 176201/03/19 6:34 PM 1763UROLOGYCHAPTER 40Patients who have not passed their stone after a 4to 6-week observation period, those with larger stones, or those who desire immediate intervention, may be offered one of three definitive surgical interventions: shockwave lithotripsy (SWL), ureteroscopy (URS), or

1	period, those with larger stones, or those who desire immediate intervention, may be offered one of three definitive surgical interventions: shockwave lithotripsy (SWL), ureteroscopy (URS), or percutaneous nephrolithotomy (PCNL). Open surgical management of stones has been relegated to historic interest for the most part with less than 1% of stone surgery needing to be done open with access to modern endou-rologic equipment. The choice of the procedure will depend primarily on stone-related factors (e.g., stone size, location, and composition/density), and patient-related factors (e.g., comor-bidities, coagulopathy, obesity, renal anatomy, and surrounding structures).Shockwave lithotripsy is the procedure associated with the least morbidity and the lowest complication rate but is also associated with a lower success rate at treating stones as a single procedure and requires the patient to pass the stone fragments afterwards.15,16 The modality can be used for stones in the prox-imal

1	with a lower success rate at treating stones as a single procedure and requires the patient to pass the stone fragments afterwards.15,16 The modality can be used for stones in the prox-imal ureter (particularly if <10 mm) or non–lower-pole renal stones <2 cm.15,16 The stone is located under fluoroscopic guid-ance, which is coupled to an extracorporeal lithotripter aimed at the stone. The stone is fragmented in a completely nonin-vasive manner. Complications associated with this procedure include subcapsular or perinephric renal hematoma and ureteral obstruction by stone fragments (“Steinstrausse”; Fig. 40-2). Ureteroscopy is the procedure of choice for patients with middle or distal ureteral stones. It also has a higher success rate than SWL in treating >10-mm proximal ureteral stones and renal stones.15,16 This procedure involves advancing a semi-rigid or flexible ureteroscope to the level of the stone and fragmenting it under direct visualization, often using a holmium:YAG laser.

1	renal stones.15,16 This procedure involves advancing a semi-rigid or flexible ureteroscope to the level of the stone and fragmenting it under direct visualization, often using a holmium:YAG laser. The surgeon is able to visualize the stone during fragmentation and thereby has some control over how small the fragments are. In addition, stone fragments may also be actively removed with a small nitinol stone basket. This is where the procedure may have an advantage over SWL. However, many patients have a ureteral stent placed after ureteroscopy, and, although tempo-rary, this remains a major source of morbidity for the patient. Specific complications of URS include ureteral injury or stric-ture. PCNL is reserved for patients with larger or more complex Figure 40-2. Ureteral obstruction in a patient with Steinstrausse. A plain abdominal radiograph (KUB) demonstrates Steinstrausse in the right distal ureter between the white arrows.stone burden, and requires a percutaneous tract into the

1	patient with Steinstrausse. A plain abdominal radiograph (KUB) demonstrates Steinstrausse in the right distal ureter between the white arrows.stone burden, and requires a percutaneous tract into the kidney. Most stones larger than 2 cm are treated with PCNL although there is a role for PCNL for smaller stones located in the lower pole of the collecting system.18,19 More powerful lithotripters (pneumatic, ultrasound) and larger instruments (stone graspers) can be used to fragment and remove these larger stones through the percutaneous tract. Complications include injury to adjacent organs, acute and delayed renal bleeding due to pseudoaneu-rysm or arteriovenous fistula formation, sepsis, or renal pelvis perforation.General preventative measures include correcting dietary habits, particularly increasing fluid intake to produce >2.5 liters of urine per day, limiting sodium, reducing animal protein intake, and monitoring foods high in oxalate. Medical therapy such as thiazide diuretics

1	increasing fluid intake to produce >2.5 liters of urine per day, limiting sodium, reducing animal protein intake, and monitoring foods high in oxalate. Medical therapy such as thiazide diuretics (helpful for hypercalciuria), urinary alkalization with potassium citrate, or allopurinol may also be indicated depending on the clinical situation.12Benign Prostatic HyperplasiaBenign prostatic hyperplasia (BPH) refers to the histological findings of smooth muscle and fibroblast/epithelial cell pro-liferation in the transition zone of the prostate. Lower urinary tract symptoms (LUTS) may be secondary to benign prostatic enlargement (BPE) causing progressive bladder outlet obstruc-tion but may also be due to numerous other conditions (e.g., urethral stricture, infection, overactive or neurogenic bladder, malignancy). Although some male patients with LUTS may have BPE, not all patients with an enlarged prostate have LUTS. The prevalence of LUTS attributed to BPH in men over the age of 50 is

1	bladder, malignancy). Although some male patients with LUTS may have BPE, not all patients with an enlarged prostate have LUTS. The prevalence of LUTS attributed to BPH in men over the age of 50 is estimated at 50% to 75% and increases with age with a prevalence of 80% in men over the age of 70.20 The treatment modalities have dramatically evolved over the past decades, with medical management typically used for first-line therapy. Endoscopic and minimally invasive techniques are used for those failing or intolerant of medical therapy.Men with BPH/LUTS are evaluated with a complete his-tory and physical exam including digital rectal exam. LUTS should be clearly defined, in addition to their severity and degree of bother. Validated questionnaires to quantify the patient’s symptoms and degree of bother include the American Urologi-cal Association Symptom Index (AUA-SI) and the International Prostate Symptom Score (IPSS).21,22 Complications of BPH such as urinary retention, incontinence,

1	of bother include the American Urologi-cal Association Symptom Index (AUA-SI) and the International Prostate Symptom Score (IPSS).21,22 Complications of BPH such as urinary retention, incontinence, renal failure, hema-turia, or recurrent infections should also be considered. Basic workup includes a urinalysis and culture to rule out infection. After an informative discussion about the risks and benefits of prostate cancer screening, a serum PSA is measured when life expectancy is >10 years and if the diagnosis of prostate can-cer will alter management.23 Other diagnostic testing such as cystoscopy, cytology, postvoid residual (PVR), urodynamics, and radiologic imaging of the prostate, although not done rou-tinely, may be required in patients with a definite indication (e.g., hematuria), uncertain diagnosis, poor response to therapy, or for surgical planning.24The first line of treatment is most commonly pharmaco-therapy for those men with bothersome symptoms. α-Blockers work by

1	uncertain diagnosis, poor response to therapy, or for surgical planning.24The first line of treatment is most commonly pharmaco-therapy for those men with bothersome symptoms. α-Blockers work by relaxing the smooth muscle of the prostate and bladder neck. All α-blocker agents are equally effective,25 and their side effects may include orthostatic hypo-tension, dizziness, asthenia, headache, nasal congestion, and retrograde ejaculation. Their effect is usually seen within days. Five-α reductase inhibitors (5-ARIs) block the conversion of 2Brunicardi_Ch40_p1759-p1782.indd 176301/03/19 6:34 PM 1764SPECIFIC CONSIDERATIONSPART IItestosterone to dihydrotestosterone (DHT), the hormone primar-ily responsible for BPH progression. These reduce prostatic size by 20% to 25%,26 but their effects are seen only after 4 to 6 months. Side effects include erectile dysfunction, decreased libido, and, rarely, gynecomastia. 5-ARIs, but not α-blockers, can alter disease progression as demonstrated by

1	are seen only after 4 to 6 months. Side effects include erectile dysfunction, decreased libido, and, rarely, gynecomastia. 5-ARIs, but not α-blockers, can alter disease progression as demonstrated by two landmark tri-als, the MTOPS27 and CombAT28 trials. These trials evaluated combination therapy using α-blockers and 5-ARIs. Patients on 5-ARIs, particularly those with larger prostates, had a reduced risk of both developing acute urinary retention and requiring surgical intervention. More recently, daily phosphodiesterase-5 inhibitors, which are most often used for erectile dysfunction (ED), have now been approved for treating patients with BPH. These can be particularly valuable in patients with concomitant ED.29,30Surgical modalities for BPH continue to evolve towards less invasive endoscopic procedures. Transurethral resection of the prostate (TURP) remains the mainstay of endoscopic pro-cedures, with low treatment failure and complication rates.31 TUR syndrome is associated with

1	procedures. Transurethral resection of the prostate (TURP) remains the mainstay of endoscopic pro-cedures, with low treatment failure and complication rates.31 TUR syndrome is associated with prolonged use of hypotonic irrigation fluid, resulting in fluid overload and dilutional hypo-natremia. Symptoms include nausea/vomiting, bradycardia and hypertension, pulmonary edema, mental status changes, and rarely death. Other endoscopic modalities used today include bipolar TURP and various laser procedures (e.g., Ho:YAG laser enucleation of the prostate, Ho:YAG laser ablation of the prostate, and photoselective vaporization of the prostate) with the goal of enucleating or vaporizing prostatic tissue. Normal saline is used for irrigation with these modalities, which greatly reduces the risk of TUR syndrome. Generally, laser procedures have been associated with shorter catheterization time and length of stay with comparable improvements in LUTS to open prostatectomy or TURP.32-34 Open, and

1	TUR syndrome. Generally, laser procedures have been associated with shorter catheterization time and length of stay with comparable improvements in LUTS to open prostatectomy or TURP.32-34 Open, and more recently laparo-scopic and robotic simple prostatectomy can also be performed for patients with moderate-severe, bothersome LUTS due to BPH. These are usually reserved for patients with larger pros-tatic volumes (>100 cc), or patients requiring concomitant blad-der surgery (e.g., bladder diverticulectomy or stones).23Urethral StrictureA urethral stricture is an area of scarring or fibrosis that causes concentric narrowing of the urethra, impeding the flow of urine as it drains from the bladder. Strictures occur at a prevalence of 0.9% of the population in the United States.35 Causes of urethral stricture disease include trauma (19%), iatrogenic causes (33%), inflammatory causes (15%), and idiopathic causes (33%).36 Symptoms of urethral stricture disease include incomplete emptying,

1	urethral stricture disease include trauma (19%), iatrogenic causes (33%), inflammatory causes (15%), and idiopathic causes (33%).36 Symptoms of urethral stricture disease include incomplete emptying, weak urinary stream, urinary urgency/frequency, and pain.37,38The anatomy of the urethra in men can be divided into the following segments proceeding from cephalad to caudad: prostatic, membranous, bulbous (the area between the pelvic floor and the penoscrotal junction), and penile. A stricture can occur in any segment of the urethra, but it is most common in the bulbar urethra.Options to treat urethral stricture disease can be divided into two general categories: endoscopic and surgical reconstruc-tion. Endoscopic treatments include a urethral dilation or stric-ture incision with a cystoscope. The latter is referred to as a direct vision internal urethrotomy. The success rate of one endo-scopic attempt to treat a urethral stricture is around 30%.39 The success of repeat endoscopic

1	The latter is referred to as a direct vision internal urethrotomy. The success rate of one endo-scopic attempt to treat a urethral stricture is around 30%.39 The success of repeat endoscopic treatments of a urethral stricture drops to 13%, and recurrent dilations have been associated with the need for more complex reconstructive surgeries for definitive management.39,40 For that reason, com-mon practice is to attempt one endoscopic intervention prior to referral for reconstructive surgery.Surgical reconstruction of the urethra, referred to as a ure-throplasty, can be divided into two general categories: excisional and tissue substitution. An excisional repair involves resection of the strictured segment of the urethra, and direct anastomo-sis of the two healthy urethral ends. This repair technique is generally reserved for membranous strictures and short bulbar strictures. Tissue substitution involves augmenting a narrowed urethral lumen with free tissue grafts. The most common tissue

1	is generally reserved for membranous strictures and short bulbar strictures. Tissue substitution involves augmenting a narrowed urethral lumen with free tissue grafts. The most common tissue substitute is buccal (oral) mucosal graft.Other Causes of ObstructionRetroperitoneal fibrosis (RPF) is a rare cause of ureteric obstruction secondary to an inflammatory and fibrotic pro-cess of the retroperitoneal structures. Most cases (>70%) are idiopathic. Identifiable causes in the remaining cases include periaortic inflammation due to aneurysms, medications (e.g., methysergide, ergot derivatives, β-blockers, phenacetin), infec-tions (e.g., tuberculosis, schistosomiasis), and malignancy (e.g., lymphoma, multiple myeloma, sarcoma). Symptoms are non-specific and may include general abdominal discomfort or back pain, flank pain due to ureteral obstruction, or lower extremity edema due to vena caval compression. Laboratory abnormali-ties such as normocytic anemia, an elevated C-reactive protein,

1	or back pain, flank pain due to ureteral obstruction, or lower extremity edema due to vena caval compression. Laboratory abnormali-ties such as normocytic anemia, an elevated C-reactive protein, or ESR are identified in about two-thirds of cases.41 The classic radiological findings consist of a well-defined retroperitoneal soft tissue mass encasing the great vessels with medialization of the ureters. Contrast enhancement on CT scan, magnetic reso-nance imaging (MRI), and positron emission tomography (PET) scan can also be used to monitor disease activity and assess response to treatment.42Patients with symptomatic renal obstruction, renal insuf-ficiency, or signs of infection should be decompressed with either ureteral stents or nephrostomy and monitored for postob-structive diuresis. Biopsy of the retroperitoneal mass to exclude malignancy should be considered prior to commencing treat-ment. Steroid therapy remains the mainstay of medical treat-ment, although other immunosuppressive

1	of the retroperitoneal mass to exclude malignancy should be considered prior to commencing treat-ment. Steroid therapy remains the mainstay of medical treat-ment, although other immunosuppressive agents have been described.43 If medical treatment fails, open or minimally inva-sive bilateral ureterolysis with intraperitonealization or omental wrapping of the ureters is indicated.Ureteral obstruction secondary to tumor (benign or malig-nant) is commonly encountered. Ureteral stenting can be tried initially, but it fails in approximately one-half of cases.44 Other strategies such as percutaneous nephrostomy, ureteral stenting in tandem, metallic, and metal-mesh stents have been described. Metallic stents may be more cost-effective due to less frequent stent exchanges,45-47 although cost savings may be offset by the limited life expectancy in this patient population.44GENITOURINARY TRAUMAGenitourinary (GU) trauma is rare. Approximately 10% of vic-tims of abdominal trauma will have a

1	savings may be offset by the limited life expectancy in this patient population.44GENITOURINARY TRAUMAGenitourinary (GU) trauma is rare. Approximately 10% of vic-tims of abdominal trauma will have a urologic injury.48 Any portion of the GU tract can be injured including the follow-ing: kidneys, ureters, bladder, urethra, and the external genita-lia including the testicles. Mechanisms of trauma parallel other injury mechanisms, the majority of which include blunt and penetrating injuries. This section will be divided into the man-agement of each organ involved in the GU system.3Brunicardi_Ch40_p1759-p1782.indd 176401/03/19 6:34 PM 1765UROLOGYCHAPTER 40Table 40-1The American Association for the Surgery of Trauma (AAST) renal trauma grading systemGRADEDESCRIPTIONMANAGEMENT1Contusion or nonenlarging subcapsular perirenal hematomaGenerally managed conservatively.2Perinephric hematoma without obvious parenchymal laceration on CT, or a <1 cm laceration into the cortex of the

1	or nonenlarging subcapsular perirenal hematomaGenerally managed conservatively.2Perinephric hematoma without obvious parenchymal laceration on CT, or a <1 cm laceration into the cortex of the kidneyGenerally managed conservatively in a stable patient.3>1 cm laceration into the cortex without involvement of the collecting systemGenerally managed conservatively in a stable patient.4A deep laceration into the collecting system with evidence of urinary extravasation on CT, or a segmental renal artery or vein injury with contained hematoma, or partial vessel laceration, or vessel thrombosisCan be observed expectantly in the stable patient, but may require subsequent urgent or delayed repair. Renal artery embolization may be an option for those who fail conservative therapy.5Renal pedicle injury or multiple deep renal lacerations (“shattered kidney”)Patients often require surgical exploration, but stable patients with only parenchymal injury may be safely treated conservatively.CT =

1	injury or multiple deep renal lacerations (“shattered kidney”)Patients often require surgical exploration, but stable patients with only parenchymal injury may be safely treated conservatively.CT = computed tomography.KidneysThe prime goal of renal trauma management is preservation of renal function. Renal trauma has become largely nonop-erative in modern times, especially in the setting of lowto intermediate-grade renal injuries from a blunt mechanism of action. The role of angioembolization through vascular and interventional radiology has further increased this nonopera-tive management.49,50The first goal of renal trauma is to accurately grade the renal injury. The gold standard test to diagnose and stage a renal injury includes a CT scan with IV contrast, with delayed images. In most centers, this is referred to as a “CT urogram,” in which delayed contrast imaging delineates the upper urinary tract collecting system. Criteria that would mandate renal imaging include the presence

1	centers, this is referred to as a “CT urogram,” in which delayed contrast imaging delineates the upper urinary tract collecting system. Criteria that would mandate renal imaging include the presence of gross hematuria, microscopic hematuria with hypotension, and mechanisms increasing the prevalence of renal injury (sudden deceleration injuries, flank contusion, etc). The American Association for the Surgery of Trauma (AAST) renal trauma grading system is described in Table 40-1.51,52The management of renal injuries depends not only on the grade but also on the injury mechanism and clinical symptoms. Absolute indications for surgical or radiological intervention on renal trauma include life-threatening hemorrhage, renal pedicle avulsion, or pulsatile/expanding retroperitoneal hematoma. Moreover, those suffering penetrating renal trauma with a ret-roperitoneal hematoma should undergo exploration when hemo-dynamic instability exists.In a hemodynamically stable patient with a renal

1	Moreover, those suffering penetrating renal trauma with a ret-roperitoneal hematoma should undergo exploration when hemo-dynamic instability exists.In a hemodynamically stable patient with a renal injury, renal trauma should be initially observed. Data suggests that this approach may even be feasible in the setting of isolated, penetrating renal injuries.53 Conservative management entails bed rest and hemodynamic monitoring. Patients with a grade 4 renal injury (Fig. 40-3A to D) should be treated in the same manner, and a repeat CT scan should be done to make certain that the urinary extravasation has resolved.54 Otherwise, urinoma and subsequent abscess formation may occur. If uri-nary extravasation is persistent, placement of a ureteral stent or nephrostomy tube should be considered.Across the board, the most common surgery for renal sur-gery in modern times is unfortunately a nephrectomy.55 Early renal vascular control may minimize nephrectomy rates.56 This is accomplished by

1	the board, the most common surgery for renal sur-gery in modern times is unfortunately a nephrectomy.55 Early renal vascular control may minimize nephrectomy rates.56 This is accomplished by isolating the renal vascular medially prior to opening the perinephric hematoma. If uncontrolled bleeding is encountered once the hematoma is opened, occlusion of the renal vasculature can be performed. At that time, a renorrhaphy can be safely done as can a nephrectomy in the setting of a grade 5 renal injury.UretersThere is no association between the magnitude of ureteral injury and the degree of hematuria that is present.57 A high index of suspicion is required. Diagnosis requires either a CT urogram, IVP, or a cystoscopy with a retrograde pyelogram. Unlike renal injury, the ureters more commonly are injured through iatro-genic mechanisms. Common surgical procedures in which the ureters are injured include gynecological, colorectal, and uro-logical surgeries. The repair of ureteric injuries

1	are injured through iatro-genic mechanisms. Common surgical procedures in which the ureters are injured include gynecological, colorectal, and uro-logical surgeries. The repair of ureteric injuries depends on the time of identification from initial injury, location, and length of the injured ureteral segment involved.Iatrogenic ureteral injuries should be initially managed with ureteral stent placement when possible. When stenting is not fea-sible, open repair may be attempted when the patient presents shortly after injury. When stent placement is not feasible or when presentation is delayed, nephrostomy tube placement should be considered until formal repair can be safely done.Ureteral injuries of traumatic origin (penetrating injuries, multiple intra-abdominal traumas) should be repaired during the index admission when possible. Hemodynamically stable patients undergoing laparotomy for other reasons in which a high index of suspicion of a ureteral injury is present should have

1	during the index admission when possible. Hemodynamically stable patients undergoing laparotomy for other reasons in which a high index of suspicion of a ureteral injury is present should have ureteral exploration. Stable patients in this same situation that are identified to have a ureteral injury should have primary repair at the time of exploration. If a patient is hemodynamically unstable, the ureter can be ligated with subsequent nephrostomy tube placement. Ureteral repair can then be delayed until the patient is stable for surgery.4Brunicardi_Ch40_p1759-p1782.indd 176501/03/19 6:34 PM 1766SPECIFIC CONSIDERATIONSPART IIABCDFigure 40-3. Grade 4 renal injury as demonstrated on abdominal computed tomography imaging with intravenous contrast. A. The yellow arrow points to extravasated contrast in the right perirenal fat. B and C. The right kidney has been fractured, as seen at the yellow arrow. Hematoma and extravasated contrast are seen in the mid-anterior pole of the kidney. D.

1	contrast in the right perirenal fat. B and C. The right kidney has been fractured, as seen at the yellow arrow. Hematoma and extravasated contrast are seen in the mid-anterior pole of the kidney. D. Coronal view. The yellow arrow reveals the upper pole renal fracture with disruption of the collecting system.The definitive operative management of a ureteral injury depends on the location and the extent of devitalization. It is important to debride devitalized ends of the ureter, whether it is from a contusion via a gunshot wound or an iatrogenic ther-mal injury. Upper ureteral injuries that are short can generally be resected and anastomosed primarily.58 Ureteral mobilization with preservation of ureteral adventitia to maintain vascular supply can aid in bridging short defects. In modern times, more aggressive maneuvers to directly anastomose more proximal ureteral injuries to the bladder are possible. Maneuvers used to bridge the defect of ureteral length for direct anastomosis to the

1	more aggressive maneuvers to directly anastomose more proximal ureteral injuries to the bladder are possible. Maneuvers used to bridge the defect of ureteral length for direct anastomosis to the bladder include the following: bladder mobilization with liga-tion of the contralateral bladder pedicles, psoas hitch (tacking the bladder down to the ipsilateral psoas tendon), and the Boari flap with downward nephropexy. Creation of a Boari flap uti-lizes a tubularized flap of anterior bladder wall to bridge long defects. Bridging defects as high as the proximal ureter have been reported in association with this technique.59 When blad-der-to-ureter anastomosis is not possible with these maneuvers, the remaining options include trans-ureteroureterostomy (anas-tomosing the injured ureter to the contralateral ureter), creation of an ileal ureter, or renal auto transplantation to the pelvis.BladderThe bladder can be injured through iatrogenic and classic trau-matic mechanisms. Indications for

1	contralateral ureter), creation of an ileal ureter, or renal auto transplantation to the pelvis.BladderThe bladder can be injured through iatrogenic and classic trau-matic mechanisms. Indications for bladder imaging include gross hematuria in the setting of injuries with a correlation for bladder injury. The most common clinical scenario is gross hematuria associated with a pelvic fracture, which is associated with a 29% chance of bladder laceration.60 Diagnosis of bladder injuries requires either a CT cystogram or a fluoroscopic cysto-gram. The sensitivities and specificities of these two modalities are similar.61,62 The bladder should be filled with approximately 300 cc of contrast for either of these imaging modalities. Contrast may be visible at the sight of injury, within the perito-neal space (Fig. 40-4A), or in the perivesical space (Fig. 40-4B). Simply capping the Foley catheter alone on a delayed excretory phase of abdominal CT imaging is insufficient to diagnose a bladder

1	space (Fig. 40-4A), or in the perivesical space (Fig. 40-4B). Simply capping the Foley catheter alone on a delayed excretory phase of abdominal CT imaging is insufficient to diagnose a bladder injury.61Two general categories of bladder injuries are extraperito-neal and intraperitoneal injuries. An intraperitoneal injury requires repair during the index admission after the patient has been resuscitated. Delayed repairs are associated with abdominal sepsis. Conversely, extra peritoneal injuries can gen-erally be managed with Foley catheter drainage alone. Situa-tions in which extraperitoneal bladder injuries should be treated with operative repair include complex injuries involving bone spicules from a pelvic fracture within the laceration and concur-rent rectal or bladder lacerations, which increase the possibility of fistula formation. Bladder neck injuries should also be treated operatively during the index admission as these injuries occa-sionally do no heal with Foley catheter

1	which increase the possibility of fistula formation. Bladder neck injuries should also be treated operatively during the index admission as these injuries occa-sionally do no heal with Foley catheter drainage alone. Repeat cystography should be done 7 to 14 days later prior to Foley removal to ensure that the laceration, or operative repair, has healed.63Urethral InjuriesCommon mechanisms of trauma of the urethra include pelvic fracture associated injuries and straddle injuries. Pelvic fracture associated injuries occur at the level of the membranous urethra, whereas straddle injuries occur at the level of the bulbar urethra. The clinical hallmark of a urethral injury is blood at the meatus. A retrograde urethrogram should be done when this clinical sign is present to diagnose an injury, prior to attempted Foley cath-eter placement (Fig. 40-5A).64,65The initial step in management of a urethral injury is bladder drainage to prevent urinoma formation and subsequent abscess formation. In

1	to attempted Foley cath-eter placement (Fig. 40-5A).64,65The initial step in management of a urethral injury is bladder drainage to prevent urinoma formation and subsequent abscess formation. In general, this is accomplished through 5Brunicardi_Ch40_p1759-p1782.indd 176601/03/19 6:35 PM 1767UROLOGYCHAPTER 40Figure 40-4. Intraperitoneal and extraperitoneal bladder injuries. A. During a computed tomography (CT) cystogram, intraperitoneal contrast is seen within the peritoneal space at the red arrow. B. During a CT cystogram, extravesical contrast is seen contained within the extraperitoneal space at the red arrow.ABFigure 40-5. A. Retrograde urethrogram showing an area of nar-rowing at the double white arrow. This indicates a bulbar urethral stricture. B. After urethroplasty, a retrograde urethrogram demon-strates a normal-appearing and patent bulbar urethra at the arrow.ABplacement of an SP tube. After stabilization, some centers per-form “primary urethral alignment.” This is a

1	urethrogram demon-strates a normal-appearing and patent bulbar urethra at the arrow.ABplacement of an SP tube. After stabilization, some centers per-form “primary urethral alignment.” This is a dual antegrade and retrograde endoscopic procedure utilizing fluoroscopy to bridge the urethral defect and to place a Foley catheter across the injury. Subsequent restructure rates are high, but the severity of stricture formation may be less when primary alignment is performed.66 If patients are managed with an SP tube alone, the site of disruption leaves the patient with a urethral stricture and subsequent restructure. This requires a treatment with a urethro-plasty after the patient’s period of convalescence has resolved (Fig. 40-5B).Penetrating injuries to the anterior urethra are rare. In a hemodynamically stable patient with an uncomplicated injury, it is expert opinion to perform exploration with primary repair during index admission. Complicated injuries with extensive tissue

1	In a hemodynamically stable patient with an uncomplicated injury, it is expert opinion to perform exploration with primary repair during index admission. Complicated injuries with extensive tissue devitalization should be managed with SP tube urinary diversion and delayed reconstruction.67External Genital InjuriesPenile fractures classically occur with excessive torqueing of the erect penis. This excessive torqueing results in rupture of the tunica albuginea, the fascial coating of the erectile bodies. Common symptoms include immediate detumescence with subsequent development of a hematoma. Clinical history and examination alone are sufficient to warrant surgical exploration with primary suture repair of the corporal body laceration. For equivocal cases, ultrasonography or an MRI may be done.68,69 Up to 10% of penile fractures are associated with urethral inju-ries. Blood at the meatus signifies the possibility of a coexisting urethral injury. This should be evaluated with either a

1	done.68,69 Up to 10% of penile fractures are associated with urethral inju-ries. Blood at the meatus signifies the possibility of a coexisting urethral injury. This should be evaluated with either a retrograde urethrogram or cystoscopy at the time of repair.Scrotal trauma generally occurs from a blunt mechanism. Injuries to the testis, epididymis, and spermatic cord may occur. Hematomas with subsequent ecchymosis are common with such injuries. Testicular rupture occurs with fracture of the fascial coating of the testicle, called the tunica albuginea. This may occur with blunt or penetrating mechanisms. The most spe-cific findings on ultrasonography are loss of testicular contour and heterogeneous echotexture of parenchyma. The highest reported sensitivity for testicular rupture on ultrasound is 93%.70 With diagnosis of a testicular rupture or when a high index of Brunicardi_Ch40_p1759-p1782.indd 176701/03/19 6:35 PM 1768SPECIFIC CONSIDERATIONSPART IIBladderUrethraPosterior

1	ultrasound is 93%.70 With diagnosis of a testicular rupture or when a high index of Brunicardi_Ch40_p1759-p1782.indd 176701/03/19 6:35 PM 1768SPECIFIC CONSIDERATIONSPART IIBladderUrethraPosterior prostateAnterior prostateABFigure 40-6. Coudé catheter. A. A schematic drawing of a lat-eral view of the prostatic urethra showing the upward angulation at the bladder neck, which a coudé catheter is helpful in negotiating. B. The tip of a coudé catheter. Note the curved tip, which should always point to 12 o’clock when inserted.suspicion is present (especially with penetrating trauma), explo-ration should be performed. Testicular salvage rates are high in modern times and involve suture repair of the site of rupture.70 When primary repair is not possible, a simple orchiectomy should be performed.EMERGENCIESAcute Urinary RetentionAcute urinary retention (AUR) can happen in men or women and results from a variety of causes, although it most commonly occurs in men with benign prostatic

1	Urinary RetentionAcute urinary retention (AUR) can happen in men or women and results from a variety of causes, although it most commonly occurs in men with benign prostatic hyperplasia (BPH).71,72 Other chronic causes of poor bladder emptying, such as diabetic neuropathy, urethral stricture, multiple sclerosis, or Parkinson’s disease, can result in episodes of complete urinary retention, often when the bladder becomes overdistended. This frequently occurs in the hospital setting when patients have limited mobil-ity and are receiving medications that decrease bladder con-tractility, including opiates or anticholinergics. Constipation, a common side effect of those medications, can itself worsen uri-nary retention. Significant hematuria can result in the formation of blood clots, which may block the urethra and cause retention.Although some patients receiving large doses of narcot-ics or those with chronically decompensated bladders may not experience discomfort, most patients with AUR

1	block the urethra and cause retention.Although some patients receiving large doses of narcot-ics or those with chronically decompensated bladders may not experience discomfort, most patients with AUR have significant pain. Untreated severe urinary retention (often accompanied by overflow incontinence) may result in acute renal failure. Treat-ment should include placement of a urethral catheter as quickly as possible. However, BPH or urethral strictures often make the placement of a catheter difficult. For men with BPH, a coude (French for curved) catheter is helpful in negotiating past the angulation in the prostatic urethra (Fig. 40-6A). The curved por-tion (which is angled in line with the balloon port) is maintained at the 12 o’clock position as it is passed through the urethra (Fig. 40-6B). A common mistake is to use a smaller catheter to bypass the enlarged prostate. However, a larger (18F to 20F) catheter is less flexible and is more likely to push into the blad-der rather than

1	A common mistake is to use a smaller catheter to bypass the enlarged prostate. However, a larger (18F to 20F) catheter is less flexible and is more likely to push into the blad-der rather than curl in the prostatic urethra.Smaller catheters, however, are quite useful for bypass-ing a urethral stricture. A urethral stricture should be suspected when the catheter meets resistance closer to the meatus, as many strictures occur in the distal urethra, which is narrower than the proximal portion. Using a 12F or 14F catheter often will allow the passage of the catheter into the bladder. If cath-eter placement is not successful, a urologic consultation should be requested. The urologist can either choose to (a) use a cys-toscope, guidewire, and urethral dilators to dilate the stricture and place a Council-tip catheter via Seldinger technique; or (b) place a suprapubic tube approximately two fingerbreadths above the pubic symphysis. With regard to the suprapubic tube, ultrasound-guidance or

1	a Council-tip catheter via Seldinger technique; or (b) place a suprapubic tube approximately two fingerbreadths above the pubic symphysis. With regard to the suprapubic tube, ultrasound-guidance or aspiration with a finder needle should be used first to localize the bladder and avoid intra-abdominal contents, although bowel injury is unlikely with a distended bladder filling the pelvis. If hematuria is the cause of retention, continuous bladder irrigation often is necessary to prevent clot formation. This is done through a large three-way catheter that has an additional port for fluid inflow. Fluid is infused by grav-ity only because the use of higher pressure may result in bladder rupture if outflow is occluded.Once the bladder is adequately drained, the cause of AUR should be addressed. For men with suspected BPH, an α-blocker such as tamsulosin should be started, and these have been shown to increase the likelihood of a successful trial without a catheter.73 Although finasteride

1	For men with suspected BPH, an α-blocker such as tamsulosin should be started, and these have been shown to increase the likelihood of a successful trial without a catheter.73 Although finasteride and dutasteride (5α-reductase inhibitors) have been shown to reduce the incidence of urinary retention by 50%, they require several months to take effect and are most beneficial in large prostates; therefore, they will not provide significant benefit in the short term. Narcotics should be tapered as tolerated, and constipation should be treated.Acute spinal cord compression, which is accompanied by saddle paresthesias, is a neurologic emergency that requires neurosurgical or orthopedic consultation. In most cases, except severe neurologic injuries, patients will be able to resume void-ing, and the catheter can be removed after 1 to 2 days. Postvoid residuals should be checked with a portable ultrasound device (bladder scanner) or by “straight” catheterization to determine the residual amount

1	catheter can be removed after 1 to 2 days. Postvoid residuals should be checked with a portable ultrasound device (bladder scanner) or by “straight” catheterization to determine the residual amount of urine left after the patient tries to empty his or her bladder. In patients with severe liver dysfunction, the bladder scanner may inadvertently misinterpret ascites for urine. The inability to void or the presence of a postvoid residual over 200 mL is concerning for development of another episode of AUR. Patients may be given the option of an indwelling cath-eter for another few days with a subsequent voiding trial or to perform clean intermittent catheterization (CIC), whereby, after Brunicardi_Ch40_p1759-p1782.indd 176801/03/19 6:35 PM 1769UROLOGYCHAPTER 40Figure 40-7. Fournier’s gangrene. A. Necrotic scrotal skin from Fournier’s gangrene. B. Debridement of gangrenous tissue. Note the extensive debridement, which is commonly required. The right testicle required removal in this

1	A. Necrotic scrotal skin from Fournier’s gangrene. B. Debridement of gangrenous tissue. Note the extensive debridement, which is commonly required. The right testicle required removal in this case (the left is wrapped in gauze), but typically, the testes are not involved with the necrotic process.predetermined intervals (4–6 hours) or after voiding attempts, the patient passes a catheter into the bladder and empties it. This is the preferred method because it reduces the likelihood of infections from indwelling catheters and may improve blad-der functionality. However, most patients are resistant to this approach.Testicular TorsionThe differential diagnosis of acute scrotal pain includes testicu-lar torsion.74 This usually occurs in neonates or adolescent boys but may be observed in other age groups. The blood supply to the testicle is compromised due to twisting of the spermatic cord within the tunica vaginalis, resulting in ischemia to the epididy-mis and the testis. In newborns, an

1	age groups. The blood supply to the testicle is compromised due to twisting of the spermatic cord within the tunica vaginalis, resulting in ischemia to the epididy-mis and the testis. In newborns, an extravaginal torsion also can occur with twisting of the tunica vaginalis and spermatic cord together. Risk factors for torsion include undescended testis, testicular tumor, and a “bell-clapper” deformity—poor guber-nacular fixation of the testicles to the scrotal wall.Clinical history is vital for diagnosis.75 Patients describe a sudden onset of pain at a distinct point in time, with subsequent swelling. Physical examination may demonstrate a swollen, asymmetric scrotum with a tender, high-riding testicle. Children normally have a brisk cremasteric reflex that usually is lost in the setting of torsion. The diagnosis is made by clinical history and examination but can be supported by a Doppler ultrasound, which typically shows decreased intratesticular blood flow rela-tive to the

1	setting of torsion. The diagnosis is made by clinical history and examination but can be supported by a Doppler ultrasound, which typically shows decreased intratesticular blood flow rela-tive to the contralateral testis. If an ultrasound is not promptly available, timely surgical exploration should be performed.Immediate surgical exploration can salvage an ischemic testis.76 At the time of surgery, the contralateral testes also must be explored and fixed to the dartos fascia due to the pos-sibility that the same anatomic defect allowing torsion exists on the contralateral side. Midline (along the median raphe) or bilateral transverse scrotal incisions are made. Once the testis is detorsed, it should be assessed for viability after being given time for normal blood flow to resume. One can assess the blood flow using intraoperative Doppler or by incis-ing the tunica vaginalis and observing tissue viability. The testes are fixed to the dartos fascia with a small, nonabsorbable suture on

1	assess the blood flow using intraoperative Doppler or by incis-ing the tunica vaginalis and observing tissue viability. The testes are fixed to the dartos fascia with a small, nonabsorbable suture on their medial, lateral, and dependent aspects, taking care to ensure that the spermatic cord is not twisted before doing so. An orchiectomy should be performed to avoid later risk of abscess formation only if the testis is clearly necrotic.Fournier’s GangreneFournier’s gangrene is a necrotizing fasciitis of the male geni-talia and perineum that can be rapidly progressive and fatal if not treated promptly (Fig. 40-7). The mortality rate has been reported to be as high as 67%.77 Risk factors for Fournier’s gangrene include perirectal abscesses, diabetes, obesity, and chronic alcoholism.78 The often polymicrobial infection spreads along dartos, Scarpa’s, and Colles’ fascia. Clinical signs include perineal and scrotal pain, inflammation, necrosis, and crepitus.78 The diagnosis is largely made

1	polymicrobial infection spreads along dartos, Scarpa’s, and Colles’ fascia. Clinical signs include perineal and scrotal pain, inflammation, necrosis, and crepitus.78 The diagnosis is largely made on clinical suspicion; however, radiographic findings on CT imaging often assist with the diag-nosis, including soft tissue air associated with fluid collections within the deep fascia.79Prompt and aggressive surgical debridement of nonvia-ble tissue and broad spectrum antibiotics are necessary to prevent further spread (Fig. 40-7A). Fecal diversion with endorectal tubes serve as an option for conservative fecal diversion.80 If there is damage to the external anal sphincter, 67patients may require a colostomy. Patients frequently require return trips to the operating room for further debridement. Negative pressure wound therapy systems have been shown to reduce hospitalization time by aiding in wound healing.81 Reconstructive strategies involving skin grafting are needed when large tissue

1	Negative pressure wound therapy systems have been shown to reduce hospitalization time by aiding in wound healing.81 Reconstructive strategies involving skin grafting are needed when large tissue defects result from extensive tissue damage.PriapismPriapism is a persistent erection for greater than 4 hours unre-lated to sexual stimulation.82 Priapism is divided into two types, based on the underlying pathophysiology. The most common type—low-flow/ischemic priapism—is a medical emergency. On examination, the penis is very tender, and both cavernosal bodies will be rigid while the glans will be flaccid. Decreased venous outflow with persistent inflow results in increased intracorporal pressure and tumescence, which is the normal process of erection. Diminished arterial inflow due to elevated Brunicardi_Ch40_p1759-p1782.indd 176901/03/19 6:35 PM 1770SPECIFIC CONSIDERATIONSPART IIintrapenile pressure usually is brief under normal circumstances. Priapism is essentially a compartment

1	Brunicardi_Ch40_p1759-p1782.indd 176901/03/19 6:35 PM 1770SPECIFIC CONSIDERATIONSPART IIintrapenile pressure usually is brief under normal circumstances. Priapism is essentially a compartment syndrome. With pro-longed erection (priapism), the sustained decrease in arterial inflow ultimately causes tissue hypoxia, acidosis, and edema and results in long-term fibrosis and impotence, and sometimes frank necrosis. Risk factors include sickle cell disease or trait, malignancy, medications, cocaine abuse, certain antidepres-sants, and total parenteral nutrition.82-84 If a cause is not identi-fied, a hematologic workup is necessary to rule out malignancy or blood dyscrasias.The management of priapism is rapid detumescence with the goal of preservation of future erectile function. The ability to achieve normal erections is directly related to the length of the episode of priapism. Ischemic priapism can be confirmed with a penile blood gas from the cavernosal bodies demonstrating hypoxic,

1	to achieve normal erections is directly related to the length of the episode of priapism. Ischemic priapism can be confirmed with a penile blood gas from the cavernosal bodies demonstrating hypoxic, acidotic blood. Initial management can include sys-temic treatment of the underlying disorder (fluid and oxygen for sickle cell patients) but this should be done concurrently with an active treatment to reduce the priapism.82 The initial intervention may be therapeutic aspiration or injection of sym-pathomimetics (phenylephrine). Insertion of a large-gauge needle (16–21 gauge) into the lateral aspect of one corporal body allows thorough aspiration and irrigation of both corporal bod-ies because of widely communicating intercavernosal channels. Injection of phenylephrine (diluted 100–500 mcg/mL and given in 1 mL increments every 3–5 minutes for up to 1 hour before determining failure) into the corporal bodies works to cause vasoconstriction, but the patient should be monitored for acute

1	and given in 1 mL increments every 3–5 minutes for up to 1 hour before determining failure) into the corporal bodies works to cause vasoconstriction, but the patient should be monitored for acute hypertension and reflex bradycardia especially in patients with high cardiovascular risk.A surgical shunt is sometimes necessary to resolve the episode if phenylephrine fails. Distal (corporoglanular) shunts should be performed first because they are the easiest to perform and the lowest amount of complications. A Winter shunt uses a large biopsy needle to create holes between the glans and cor-pora; however, if this fails, an operative procedure can be per-formed to remove the distal tips from each corpora (Al-Ghorab). Proximal shunts such as Grayhack (corporal-saphenous vein) or Quackel (proximal cavernosumspongiosum) shunts may be required in refractory cases.The other form of priapism (high-flow/traumatic priapism) is rare and is related to penile or perineal trauma resulting in a

1	(proximal cavernosumspongiosum) shunts may be required in refractory cases.The other form of priapism (high-flow/traumatic priapism) is rare and is related to penile or perineal trauma resulting in a cavernous artery–corporal body fistula. This form is not painful because it is not related to ischemia and can be managed con-servatively with observation. Many cases will resolve with time; those that do not can undergo selective arterial embolization.82ParaphimosisParaphimosis is a common problem that represents a true medi-cal emergency for uncircumcised men. When the foreskin is retracted for prolonged periods, constriction of the glans penis may ensue. This is particularly likely in hospitalized patients who are confined to bed or who have altered mental status and are unable to respond to pain. Delay can be catastrophic as penile necrosis may occur due to ischemia. Penile blocks, pain medication, and sedation are sometimes necessary before manual reduction. It is useful to apply

1	to pain. Delay can be catastrophic as penile necrosis may occur due to ischemia. Penile blocks, pain medication, and sedation are sometimes necessary before manual reduction. It is useful to apply firm pressure to the edematous distal penis for several minutes.85 Although painful, this reduction in penile edema can be the key to success. With the fingers pulling the constricting band distally, the thumbs can push the glans penis back into normal location. Compres-sion wraps have shown some benefit without the need for the physician to use hand compression.86 If the foreskin cannot be manually reduced, surgical intervention is required.Emphysematous PyelonephritisEmphysematous pyelonephritis is a life-threatening infection that results from complicated pyelonephritis by gas-producing organisms. It is an acute necrotizing infection of the kidney that occurs predominantly in diabetic patients.87 Patients frequently present with sepsis and ketoacidosis. Escherichia coli appears to be the

1	It is an acute necrotizing infection of the kidney that occurs predominantly in diabetic patients.87 Patients frequently present with sepsis and ketoacidosis. Escherichia coli appears to be the most frequent organism responsible for this infection. Patients require supportive care, IV antibiotics, and relief of any urinary tract obstruction. Third-generation cephalosporins have been suggested as the initial antibiotic of choice and fluoroqui-nolones avoided due to high rates of resistance.88 Emphysema-tous pyelonephritis can be subdivided based on the extent of infection. Cases where gas is isolated to the kidney frequently can be managed conservatively with the placement of a neph-rostomy tube to allow drainage of purulent material. When there is extensive involvement of the perirenal tissue, conservative management may not be successful and strong consideration should be given to nephrectomy, particularly if the patient is displaying signs of sepsis.89,90UROLOGIC MALIGNANCIESBladder

1	conservative management may not be successful and strong consideration should be given to nephrectomy, particularly if the patient is displaying signs of sepsis.89,90UROLOGIC MALIGNANCIESBladder CancerEpidemiology and Presentation. In 2018, 81,190 men and women will be diagnosed with bladder cancer, and 17, 240 will die from their disease.91 The disease is highly prevalent, with over 700,000 patients living with the disease in the United States as of 2016. Men have nearly three times the incidence of women. Tobacco use is the most frequent risk factor, followed by occupational exposure to various carcinogenic materials such as industrial solvents (e.g., aromatic amines). Other risk fac-tors include arsenic, radiation, cyclophosphamide, and chronic exposure to foreign bodies (stones and catheters) and specific urinary parasites. The most common bladder cancer histology in the United States is urothelial carcinoma (UC), accounting for 90% of tumors, which tends towards a better

1	and catheters) and specific urinary parasites. The most common bladder cancer histology in the United States is urothelial carcinoma (UC), accounting for 90% of tumors, which tends towards a better prognosis as compared to the rarer forms, including squamous cell carcinoma (<10%), adenocarcinoma (1–2%), and small cell cancer (<1%). Unfortunately, there is no reliable screening test for bladder cancer, although patients felt to be at high risk may undergo urine sampling for microhematuria or abnormal cytology. Smoking cessation should be advised in all tobacco users as a preventive measure. The most common symptoms at presentation are hema-turia (gross or microscopic) and/or irritable voiding (urgency, frequency, and dysuria). Office cystoscopy is an effective means to diagnose bladder cancer.Staging. Clinical staging is completed with CT or MRI to assess intraabdominal nodal and visceral sites of metastasis. The upper tracts should be evaluated with CT urography or retro-grade

1	cancer.Staging. Clinical staging is completed with CT or MRI to assess intraabdominal nodal and visceral sites of metastasis. The upper tracts should be evaluated with CT urography or retro-grade pyelography. Chest radiograph provides initial evaluation of the thorax and mediastinum. A bone scan should be obtained if the patient complains of bone pain, has known locally advanced or metastatic disease, or an unexplained elevation in the serum alkaline phosphatase level. Pathologic staging has been outlined by the American Joint Committee on Cancer.92Transurethral resection of bladder tumor (TURBT) should include an examination under anesthesia (EUA) and sampling of the bladder muscular wall to fully assess depth of invasion. The presence of induration or a mass on EUA denotes extravesical Brunicardi_Ch40_p1759-p1782.indd 177001/03/19 6:35 PM 1771UROLOGYCHAPTER 40tumor extension and may alter the patient’s treatment plan. It may also be appropriate to biopsy multiple areas of mucosa

1	177001/03/19 6:35 PM 1771UROLOGYCHAPTER 40tumor extension and may alter the patient’s treatment plan. It may also be appropriate to biopsy multiple areas of mucosa to identify multifocal carcinoma in situ (CIS). Restaging TURBT within 2 to 6 weeks is recommended in the patient with incom-plete, under-sampled, or uncertain resection. This is especially important in the patient with Tis, Ta, or T1 disease, as well as the patient with suspected T2 disease who is being considered for a bladder preservation treatment strategy. Invasion into the lamina propria and certainly the muscular wall demonstrates increased potential for distant metastases; muscle invasion is rarely treated completely with TURBT and requires additional therapy for adequate local control.Recurrence rates of non–muscle-invasive bladder can-cers are high, ranging from 50% to 70%.93 Adjuvant treatment strategies have thus been adopted after TURBT to reduce these rates. Intravesical chemotherapy used in conjunction

1	bladder can-cers are high, ranging from 50% to 70%.93 Adjuvant treatment strategies have thus been adopted after TURBT to reduce these rates. Intravesical chemotherapy used in conjunction with TURBT can reduce the risk of recurrence by 44% to 73% in patients with primary Ta and T1 tumors and by 38% to 65% in patients with recurrent Ta, T1, and Tis tumors when compared to TURBT alone.94 Intravesical immunotherapy using bacil-lus Calmette-Guérin (BCG) also provides a significant reduc-tion in recurrence that is greater than 50% in this population. Despite improved rates of disease-free survival, standard induc-tion courses of intravesical chemotherapy and immunotherapy do not improve disease-specific survival.94 However, when an induction course of BCG is followed by a series of maintenance doses consisting of weekly BCG given for 3 weeks at 3, 6, 12, 18, 24, 30, and 36 months after induction, disease-free and over-all survival can be prolonged.95 In patients who fail an initial or

1	doses consisting of weekly BCG given for 3 weeks at 3, 6, 12, 18, 24, 30, and 36 months after induction, disease-free and over-all survival can be prolonged.95 In patients who fail an initial or maintenance course of intravesical therapy, it may be rea-sonable to try another agent; however, one must consider the risk of progression and not delay definitive treatment. Roughly 15% to 30% of patients presenting with non–muscle-invasive tumors will eventually progress to muscle invasion. Radical cys-tectomy remains the most effective single-modality treatment for patients with muscle-invasive bladder cancer, refractory high-risk non–muscle-invasive disease, and especially lymph node–negative disease with a reported 10-year recurrence-free survival of organ-confined lymph node–negative (<pT2N0) dis-ease between 69% and 87%.94,96,97Surgical Considerations. Cystectomy is indicated in the treat-ment of refractory NMIBC or to assert local control for muscle invasive bladder cancer (MIBC).98

1	dis-ease between 69% and 87%.94,96,97Surgical Considerations. Cystectomy is indicated in the treat-ment of refractory NMIBC or to assert local control for muscle invasive bladder cancer (MIBC).98 Effective local control in the pelvis is achieved in 93% of cases with cystectomy. Indica-tions for partial cystectomy are limited and generally apply to isolated tumors or those within diverticulum. Classic teaching suggests that patients with CIS should not be candidates, though the use of intravesical BCG to treat CIS may have broadened this application. For patients with MIBC, neoadjuvant systemic chemotherapy with M-VAC or gemcitabine and cisplatin (prior to cystectomy) offers a survival advantage when compared to radical cystectomy alone.98Robotic approaches for cystectomy are increasingly used, but the urinary diversion is still usually performed through an open incision. The benefits of the robotic portion are decreased blood loss during the pelvic dissection (due to the

1	increasingly used, but the urinary diversion is still usually performed through an open incision. The benefits of the robotic portion are decreased blood loss during the pelvic dissection (due to the pneumoperi-toneum). However, recent evidence (randomized controlled trials of open vs. robot-assisted radical cystectomy) did not demonstrate any difference in oncologic efficacy or complica-tion rates.Complications of bladder cancer surgery involve bladder perforation during transurethral resection of the bladder tumor, which require catheter drainage for several days if small (com-mon) or open repair if large and intraperitoneal (rare). Cys-tectomy and urinary diversion may result in prolonged ileus, bowel obstruction, intestinal anastomotic leak, urine leak, or rectal injury. A urine leak from the ureteroileal anastomoses is a common cause of ileus, intra-abdominal urinoma, abscess formation, and wound dehiscence. Deep venous thrombosis is common after cystectomy due to the advanced

1	from the ureteroileal anastomoses is a common cause of ileus, intra-abdominal urinoma, abscess formation, and wound dehiscence. Deep venous thrombosis is common after cystectomy due to the advanced age of most patients, proximity of the iliac veins to the resection and lymph node dissection, and the presence of malignancy. The utility of subcutaneous heparin in the perioperative period can minimize the risk of venous thromboembolism. Contemporary series from high volume centers report readmission rates of 25%, complica-tion rates of 50% to 60%, and perioperative mortality in the first 90 days at 5% to 10%.99,100Urinary diversion can be accomplished using an incon-tinent or continent abdominal stoma or orthotopic continent reconstruction. The evolution of patient selection and surgical technique has led to improved outcomes for orthotopic diver-sion, although there are still patients who are better served with an ileal conduit. Motivated patients are considered for ortho-topic

1	technique has led to improved outcomes for orthotopic diver-sion, although there are still patients who are better served with an ileal conduit. Motivated patients are considered for ortho-topic neobladder diversion if they have a preoperative serum creatinine less than 2.0 mg/mL, normal preoperative bowel function, a negative urethral margin based on intraoperative frozen section at the time of cystectomy, and an intact sphincter after complete tumor resection.Alternatives to cystectomy include observation, systemic chemotherapy, radiation therapy, or a combination of chemo-therapy and radiation. These modalities may be required in patients who are a poor surgical risk, who refuse surgery, or who are elderly.Bladder preservation using radiation as the definitive ther-apy may be feasible in selected patients. In this context, trimo-dality therapy is preceded by aggressive TURBT and offers an improved rate of survival when performed in conjunction with chemotherapy. Up to 42% 5-year

1	in selected patients. In this context, trimo-dality therapy is preceded by aggressive TURBT and offers an improved rate of survival when performed in conjunction with chemotherapy. Up to 42% 5-year disease-specific survival can be achieved in patients with preserved bladders, with the best overall survival outcome in younger patients with lower stage tumors without lymphovascular or nodal involvement.More recently, immunotherapeutic treatments have shown significant promise in the treatment of locally advanced and metastatic bladder cancer. Five agents have recently been approved for patients who have progressed on or after platinum-based chemotherapy or have progressed within 12 months of neoadjuvant or adjuvant treatment. These agents include PD-L1 inhibitors (atezolizumab, avelumab, durvalumab) and PD-1 inhibitors (nivolumab and pembrolizumab). Response rates for these agents are ∼15% to 20% but may have extended median overall survival as much as 10.3 months when compared to

1	durvalumab) and PD-1 inhibitors (nivolumab and pembrolizumab). Response rates for these agents are ∼15% to 20% but may have extended median overall survival as much as 10.3 months when compared to chemotherapy.101,102Testicular CancerTesticular cancer is the most common cancer in men age 20 to 40 years and the second most common cancer in young men age 15 to 19 years. Metastases to the testis (usually lymphoma in older men) are rare. In 2018 there were 9310 new cases and 400 deaths from the disease.91 The incidence of testis cancer varies around the world.103 It contains a heterogeneous group of tumors, of which 95% are germ cell tumors; the rest originate from stromal cells (Leydig or Sertoli cells). Germ cell tumors can be classified as either seminomatous or nonseminomatous. Seminoma constitutes more than 50% of all testis cancer. The Brunicardi_Ch40_p1759-p1782.indd 177101/03/19 6:35 PM 1772SPECIFIC CONSIDERATIONSPART IIFigure 40-8. Scrotal ultrasound of the right testis. A

1	constitutes more than 50% of all testis cancer. The Brunicardi_Ch40_p1759-p1782.indd 177101/03/19 6:35 PM 1772SPECIFIC CONSIDERATIONSPART IIFigure 40-8. Scrotal ultrasound of the right testis. A heteroge-neous echoic mass is seen.incidence of bilateral GCT is approximately 2.5%.104,105 There are four established risk factors for testis cancer: cryptorchidism, family history of testis cancer, a personal history of testis can-cer, and intratubular germ cell neoplasia. Most patients present with testicular pain or a testicular mass. Respiratory symptoms, back pain, weight loss, or gynecomastia may indicate metastatic disease (10–20%). A testicular mass is considered malignant till proven otherwise. Similarly, retroperitoneal lymphadenopathy in young men should be considered metastatic testicular cancer.Standard initial workup includes scrotal ultrasound (Fig. 40-8) and serum tumor markers (α-fetoprotein, quanti-tative human chorionic gonadotropin, and lactate dehydroge-nase). Most

1	testicular cancer.Standard initial workup includes scrotal ultrasound (Fig. 40-8) and serum tumor markers (α-fetoprotein, quanti-tative human chorionic gonadotropin, and lactate dehydroge-nase). Most consider percutaneous biopsy contraindicated due to the rare but historical risk of disturbing the natural lymphatic drainage to the retroperitoneum and possible seeding of the scrotum.106 Radical inguinal orchiectomy is the gold standard treatment for excision of the primary tumor. Partial orchiec-tomy through an inguinal approach may be considered in some cases, including a suspected diagnosis of lymphoma. Chest and abdominal axial imaging are the main staging tools. Testicular cancer has a very predictable pattern of spread. Right testicular cancer tends to metastasize to the interaortocaval lymph node, followed by paracaval and paraaortic lymph nodes. Left-sided testicular cancer rarely crosses to the paracaval lymph nodes.107 Clinical TNM staging includes local stage, distant

1	lymph node, followed by paracaval and paraaortic lymph nodes. Left-sided testicular cancer rarely crosses to the paracaval lymph nodes.107 Clinical TNM staging includes local stage, distant metastasis, and tumor markers.Depending on the stage and histology of the primary tumor, multiple treatment options are available. These include active surveillance, retroperitoneal lymph node dissection (RPLND), and adjuvant chemotherapy or radiation therapy. Active surveillance for localized disease follows a tight sched-ule of physical exams, tumor markers and imaging studies. The cancer recurs in 20% to 30% of patients on active surveillance. The presence of embryonal carcinoma and vascular invasion seem to be interrelated predictors of recurrence.108 Recurrence usually occurs within the first 2 years and in the retroperito-neum.109 Pure seminoma is radiosensitive; stages I, IIa, and IIb disease can be treated with external-beam radiation to the retro-peritoneal nodes. Alternatively, a single

1	and in the retroperito-neum.109 Pure seminoma is radiosensitive; stages I, IIa, and IIb disease can be treated with external-beam radiation to the retro-peritoneal nodes. Alternatively, a single dose of carboplatin for stage I seminoma was found to be just as effective as radiation therapy.110 More advanced seminoma is treated with platinum-based systemic chemotherapy.Stages I to IIA nonseminomatous testis cancer is poten-tially cured with RPLND or chemotherapy.111 Persistently high tumor markers after radical orchiectomy or high-stage meta-static germ cell tumors warrant systemic chemotherapy. Due to the high rates of teratoma or viable germ cell tumor, postchemo-therapy bulky masses are resected by RPLND or other surgi-cal procedures. The overall survival rate of localized disease is outstanding (99% at 5 years). Patients with more advanced distant metastatic disease (stage III) have 75% survival rates. The overall prognosis is generally better for seminomatous than nonseminomatous

1	(99% at 5 years). Patients with more advanced distant metastatic disease (stage III) have 75% survival rates. The overall prognosis is generally better for seminomatous than nonseminomatous germ cell tumors.112Surgical Considerations. Radical orchiectomy is done through an inguinal incision extending from the external inguinal ring to the internal inguinal ring. The spermatic cord is ligated at the internal ring with long silk sutures for easier identification during a future RPLND. Integrity of the scrotal skin during orchiectomy is important. Complications of radi-cal orchiectomy include scrotal hematoma, chronic pain, and hernia.For RPLND, a midline incision is usually made from the xiphoid process to the pubic symphysis. All the lymphatic tissue is removed from the targeted areas using the classical split and roll technique, and all lumbar vessels are tied. Postganglionic sympathetic nerve sparing is possible in most cases for pres-ervation of ejaculatory function.113

1	areas using the classical split and roll technique, and all lumbar vessels are tied. Postganglionic sympathetic nerve sparing is possible in most cases for pres-ervation of ejaculatory function.113 Robotic-assisted RPLND is growing, with faster recovery time and similar short term oncologic results.114 Complications after RPLND include bowel obstruction, excessive bleeding, chylous ascites, and ejaculatory dysfunction.Kidney CancerRenal cell carcinoma (RCC) results in approximately 3.8% of all new cancers, with an estimated 65,340 new cases and 14,970 deaths related to kidney cancer in 2018.91 Despite several advancements with immune-based and targeted molecular ther-apies demonstrating durable clinic responses, RCC still remains primarily a surgical disease and classically does not respond to conventional chemotherapy regimens or radiation therapy.Most patients diagnosed with RCC in the modern era typically present with an incidentally discovered renal mass on abdominal radiographic

1	to conventional chemotherapy regimens or radiation therapy.Most patients diagnosed with RCC in the modern era typically present with an incidentally discovered renal mass on abdominal radiographic imaging. Differential diagnosis of a renal mass includes malignant tumors (e.g., RCC, urothelial carcinoma, sarcomas, lymphoma, metastasis), benign tumors (e.g., cysts, angiomyolipoma, oncocytoma), and inflammatory lesions (e.g. abscesses, xanthogranulomatous pyelonephritis, tuberculosis). Renal CT imaging with intravenous contrast remains the single most important radiographic test to delineate the nature of the mass. In general, any solid renal mass that enhances by more than 15 Hounsfield units is an RCC until proven otherwise. However, even if there is contrast enhance-ment on axial imaging, approximately 15% to 30% of solid renal masses are benign on final surgical pathology.115 Renal tumor biopsy can help distinguish between malignant or benign tumors, but this has not been widely

1	approximately 15% to 30% of solid renal masses are benign on final surgical pathology.115 Renal tumor biopsy can help distinguish between malignant or benign tumors, but this has not been widely adopted by the urological community, despite series showing their high diagnostic yield, concordance with surgical pathology, and safety.116-118 Biopsy remains particularly useful in patients considering surveillance or thermoablative therapy, or in patients with suspicion of metastasis or lymphoma.Major recognized risk factors for RCC include smoking, obesity, and hypertension. Although most RCCs are discovered incidentally, some patients present with signs or symptoms Brunicardi_Ch40_p1759-p1782.indd 177201/03/19 6:35 PM 1773UROLOGYCHAPTER 40which may be the result of local tumor growth (e.g., flank pain, hematuria, perirenal hematoma), paraneoplastic syndromes (e.g., hypertension, weight loss, hypercalcemia, polycythemia/anemia, abnormal liver function tests), or metastatic disease. RCC

1	flank pain, hematuria, perirenal hematoma), paraneoplastic syndromes (e.g., hypertension, weight loss, hypercalcemia, polycythemia/anemia, abnormal liver function tests), or metastatic disease. RCC metastasizes primarily to the lungs, lymph nodes, bone, liver, adrenal glands, and brain. Familial RCC subtypes with classical clinical manifestations are also well described. The von Hippel-Lindau disease, occurring as a result of a mutation in the tumor suppressor gene VHL (3p25-26), commonly mani-fests itself with clear cell RCC, pheochromocytomas, retinal angiomas, central nervous system hemangioblastomas, pancre-atic cysts, and other tumors. Other familial syndromes include hereditary papillary RCC (papillary type 1 RCC), familial leiomyomatosis (papillary type 2 RCC), and Birt-Hogg-Dube syndrome (chromophobe RCC, hybrid oncocytic tumors, and oncocytoma). Familial RCC syndromes should be suspected in younger patients and patients with multicentric and/or bilateral tumors.Clear cell RCC

1	(chromophobe RCC, hybrid oncocytic tumors, and oncocytoma). Familial RCC syndromes should be suspected in younger patients and patients with multicentric and/or bilateral tumors.Clear cell RCC is the most common subtype, accounting for 70% to 80% of all RCCs. Papillary RCC occurs in 10% to 15%, type 1 being associated with a better prognosis, and type 2 a worse prognosis. Other subtypes include chromophobe RCC, collecting duct carcinoma, and unclassified type.RCC may locally progress and cause invasion of the renal capsule and perirenal fat or the collecting system. RCC may also directly progress into the venous system in the form of a tumor thrombus that can extend into the IVC and into the right atrium. Staging is the single most important prognostic factor for RCC.119,120 Studies demonstrate a 70% to 90% 5-year survival rate for organ confined disease (stages I–II), compared to 0% to 10% for patients with systemic metastases (stage IV).119 Other important prognostic factors include

1	a 70% to 90% 5-year survival rate for organ confined disease (stages I–II), compared to 0% to 10% for patients with systemic metastases (stage IV).119 Other important prognostic factors include histological subtype,121 tumor size, lymph node involvement, and site of metastases.122Management options for small renal masses (<4 cm) includes active surveillance, thermoablative techniques, or sur-gical excision (Fig. 40-9). Percutaneous or laparoscopic ther-moablative techniques (cryoablation, radiofrequency ablation, high-intensity focused ultrasound) have been used to treat small renal masses, but they are associated with an increased risk of local recurrence.123,124Since the first laparoscopic radical nephrectomy described by Clayman et al in 1991,125 minimally invasive Figure 40-9. Computed tomography scan of the abdomen with intravenous and oral contrast. A small mid right posterior mass is seen.Figure 40-10. Intraoperative image of a small renal mass in prepa-ration for partial

1	tomography scan of the abdomen with intravenous and oral contrast. A small mid right posterior mass is seen.Figure 40-10. Intraoperative image of a small renal mass in prepa-ration for partial nephrectomy.surgical approaches, including laparoscopy with robotic assis-tance, have virtually supplanted open procedures for localized RCC (Fig. 40-10). Partial nephrectomy is most appropriate for patients with small tumors, solitary kidney, bilateral tumors, or familial RCC. Some tumors may not be amenable to abla-tive therapies or partial nephrectomy, in which case radical nephrectomy would be employed.126Radical nephrectomy involves removal of the entire kidney with dissection external to Gerota’s fascia. The colon is retracted medially after incising the white line of Toldt, fol-lowed by meticulous hilar dissection with ligation of the renal artery and vein. The adrenal gland is usually spared unless the tumor involves the gland or is immediately adjacent to it. Lymphadenectomy remains

1	hilar dissection with ligation of the renal artery and vein. The adrenal gland is usually spared unless the tumor involves the gland or is immediately adjacent to it. Lymphadenectomy remains controversial, and it is usually per-formed in patients with adenopathy on preoperative imaging or in patients with palpable lymph nodes intraoperatively. In partial nephrectomy, renal artery clamping is often performed to minimize blood loss while the tumor is excised. The goal is to remove the tumor with negative surgical margins while minimizing warm ischemia time to preserve as many func-tional nephrons as possible. With increasing experience, partial nephrectomy is now also performed on much more complex renal masses, including completely endophytic, central, and hilar tumors. Very large tumors or tumors with vena-caval thrombi can be removed robotically in experienced hands,127,128 but most are still removed using an open approach.In minimally invasive surgery, both partial and radical

1	or tumors with vena-caval thrombi can be removed robotically in experienced hands,127,128 but most are still removed using an open approach.In minimally invasive surgery, both partial and radical nephrectomy can be done via either a transperitoneal or retro-peritoneal approach. In open cases, a subcostal flank approach provides direct access to the retroperitoneum and is preferred for lower pole exposure, but it can limit access to the hilum, particularly with large renal masses. The anterior subcostal approach is preferred for larger renal masses. Bilateral ante-rior subcostal incisions (chevron incision) provides excellent vascular exposure (e.g., IVC thrombectomy, bilateral tumors). Midline incisions are usually reserved for renal trauma and for reconstructive procedures. Less commonly performed, the thoracoabdominal approach involves access usually above the 10th rib and is used for large upper pole or adrenal masses, IVC thrombectomy, or tumors involving adjacent structures.

1	performed, the thoracoabdominal approach involves access usually above the 10th rib and is used for large upper pole or adrenal masses, IVC thrombectomy, or tumors involving adjacent structures. Complications include injury to adjacent organs, and for partial nephrectomy, pseudoaneurysms/arteriovenous fistula formation and delayed urinary leak.Brunicardi_Ch40_p1759-p1782.indd 177301/03/19 6:35 PM 1774SPECIFIC CONSIDERATIONSPART IIProstate CancerProstate cancer is the most common noncutaneous cancer in men; 164,690 new cases of prostate cancer were diagnosed in 2018 and 29,430 men died from their disease.91 Screening for prostate cancer with detailed history, digital rectal examination, and serum prostate specific antigen (PSA) tests have changed the natural history of the disease. Since the introduction of pros-tate cancer screening in the mid-1980s, the incidence of meta-static prostate cancer has decreased by half. Currently 99% of newly diagnosed patients will survive more than

1	the introduction of pros-tate cancer screening in the mid-1980s, the incidence of meta-static prostate cancer has decreased by half. Currently 99% of newly diagnosed patients will survive more than 10 years.91While early screening for African American patients or patients with a family history of prostate cancer is widely accepted, screening for all men is more controversial. Despite data from large randomized clinical trials showing a decrease in mortality after prostate cancer screening, the U.S. Preventive Services Task Force recommended against the routine use of prostate cancer screening.129 Its recommendation was based on the harm and toxicity of overtreatment of nonlethal disease.130 The American Urologic Association subsequently recom-mended informed and shared decision-making and screening for high-risk disease for men between the ages of 55 and 69 with a life expectancy more than 10 years.131If the digital rectal examination is abnormal or if the PSA level is above expected

1	screening for high-risk disease for men between the ages of 55 and 69 with a life expectancy more than 10 years.131If the digital rectal examination is abnormal or if the PSA level is above expected for patients’ age and size of the prostate, a prostate biopsy is usually performed. Newer tests such as the 4K score, prostate health index, and PCA3 are sometimes used to inform the decision to proceed with biopsy. Recently, MRI fusion transrectal ultrasound-guided biopsy improved the accu-racy of prostate biopsy.Since most patients survive the disease, risk stratification systems are routinely utilized to guide staging and treatment. Clinical TNM stage, serum PSA levels, and the Gleason grading system are utilized in clinical practice. More recently, genetic testing on biopsy specimen was included in national guidelines. Historically, the Gleason scoring (GS) system included a pri-mary and secondary score based on the most common and sec-ond most common histologic patterns. Grades range

1	in national guidelines. Historically, the Gleason scoring (GS) system included a pri-mary and secondary score based on the most common and sec-ond most common histologic patterns. Grades range from 1 for the most differentiated to 5 for the least. The grades are added to create a resultant Gleason score.132 However, since no patients are assigned a score of less than 5 anymore, the grading system has been modified to a scale from 1 to 5. Grade one includes a GS of 3 + 3 = 6 or less, grade 2 for GS 3 + 4, grade 3 for GS 4 + 3, grade 4 for GS 4 + 4 and grade 5 for Gleason score of 9 or 10.133 Imaging studies like CT and bone scans are used to rule out metastatic disease in high-risk patients. The two most common sites of metastatic disease are pelvic/retroperitoneal lymph nodes and boney structures. Modern CT PET scans have a limited role at this point.Treatment for localized prostate cancer is guided by can-cer aggressiveness and patient’s preferences. Active sur-veillance is

1	boney structures. Modern CT PET scans have a limited role at this point.Treatment for localized prostate cancer is guided by can-cer aggressiveness and patient’s preferences. Active sur-veillance is recommended for patients with low-risk disease grade 1–2, early-stage disease (cT1c), and small volume disease as determined by biopsy. Large prospective cohorts and randomized clinical trials have established the safety of this approach.134,135 The risk of progression to metastatic disease with close follow-up and repeat prostate biopsies is less than 2% in over 12 years. Radical prostatectomy and pelvic lymph node dissection (robotic, laparoscopic, or open), image modulated radiation therapy (IMRT), and brachytherapy are the standard of care for curative treatments. All provide equal cancer specific survival for low and intermediate risk cancers. For higher risk prostate cancer patients, both surgery and IMRT with androgen 8Figure 40-11. The da Vinci Surgical System used commonly for

1	specific survival for low and intermediate risk cancers. For higher risk prostate cancer patients, both surgery and IMRT with androgen 8Figure 40-11. The da Vinci Surgical System used commonly for radical prostatectomy.deprivation therapy provide excellent cancer control. Cryother-apy, or high intensity focused ultrasound (HIFU) and focal therapy are emerging options that may be acceptable for some patients with low-risk disease.Level I evidence has established the role of adjuvant radia-tion therapy after radical prostatectomy for patients with posi-tive surgical margins, extracapsular extension, and high-grade disease.136,137 After definitive treatment of localized prostate cancer, rising PSA is an extremely reliable indicator of recur-rence or progression. However, it may take over 10 years for metastasis to appear on imaging studies.138 Once prostate cancer metastasizes, it is no longer curable. Medications that lower serum testosterone or androgen receptor blockers are able to

1	years for metastasis to appear on imaging studies.138 Once prostate cancer metastasizes, it is no longer curable. Medications that lower serum testosterone or androgen receptor blockers are able to control the disease, often for years. In addition, chemotherapy, immunotherapy, and radioisotope therapy at different stages of the disease increase the life expectancy of the patients or improve the quality of life. The cancer inevitably becomes resis-tant to these treatments. Nevertheless, patients with incurable prostate cancer can live many years, and a large number die of causes other than prostate cancer.Over the past few years, we have witnessed major devel-opments in the management of metastatic castrate resistant prostate cancer (mCRPC). New agents that interrupt androgen synthesis (e.g., abiraterone acetate)139,140 and new modulators of androgen receptors (e.g., enzalutamide)141,142 have significantly improved the life expectancy of patients with both androgen sensitive and

1	(e.g., abiraterone acetate)139,140 and new modulators of androgen receptors (e.g., enzalutamide)141,142 have significantly improved the life expectancy of patients with both androgen sensitive and resistant metastatic prostate cancer. Similarly, innovations in immunotherapy and chemotherapy delivery have advanced the management of advanced prostate cancer.Surgical Considerations. Open radical retropubic prostatec-tomy is done through a lower midline incision from below the umbilicus to the pubic symphysis. After entering the space of Retzius, the external iliac, obturator, and internal iliac lymph nodes are removed. The cavernosal nerves located on the pos-terolateral surface of the prostate capsule are usually spared on the side(s) with low risk of extracapsular extension of the dis-ease. Then the prostate is removed in a retrograde fashion, and the urethrovesical anastomosis is completed in an interrupted fashion.Robotic radical prostatectomy using the da Vinci robotic surgical

1	Then the prostate is removed in a retrograde fashion, and the urethrovesical anastomosis is completed in an interrupted fashion.Robotic radical prostatectomy using the da Vinci robotic surgical system (Fig. 40-11) is now the most common tech-nique (over 90% of all patients in the United States) for the Brunicardi_Ch40_p1759-p1782.indd 177401/03/19 6:35 PM 1775UROLOGYCHAPTER 40surgical treatment of localized prostate cancer. Robotic surgery has lower blood loss and faster convalescence, less bladder neck contracture, and lower early postoperative complications. Some data show a faster return of continence and lower rates of erectile dysfunction. The most common postoperative compli-cations include infection, urine leaks, ileus, lymphocele, and, very rarely, rectal or ureteral injury.To minimize the impact of these side effects, researchers have used different ablative techniques to obliterate the areas of significant cancer. By avoiding the need for whole gland radia-tion or

1	minimize the impact of these side effects, researchers have used different ablative techniques to obliterate the areas of significant cancer. By avoiding the need for whole gland radia-tion or removal, these focal ablative therapies aim to balance the long-term impact on quality of life with survival. Laser, high-focused ultrasound, cryotherapy, and photodynamic ablations have showed similar results in early studies.Urethral CancerUrethral carcinoma (UC) is a rare disease, the true incidence of which is unknown. It accounts for less than 1% of genito-urinary cancers.143,144 It is a disease of the older adult. Risk fac-tors include chronic inflammation from sexually transmitted diseases (human papillomavirus 16 and 18 in squamous cell carcinoma),145 chronic urethral stricture, and indwelling cath-eterization. Furthermore, urethral diverticulum and recurrent urinary tract infections increase the risk for women.The majority of patients present with irritative and obstructive voiding

1	cath-eterization. Furthermore, urethral diverticulum and recurrent urinary tract infections increase the risk for women.The majority of patients present with irritative and obstructive voiding symptoms, bleeding, or a palpable mass. Urothelial carcinoma is the most common histology; 29% of women have adenocarcinoma, and both genders can have squamous cell carcinoma. Untreated or refractory UC typically metastasizes through lymphatic channels to the inguinal and pelvic lymph nodes and hematologically to distant organs. Cys-toscopic biopsy establishes the diagnoses. An MRI of the pelvis is extremely helpful for defining local extension of the disease while CT scans of the chest, abdomen, and pelvis identify meta-static disease. Finally, it is also important to evaluate the entire urinary tract.The 5-year overall survival rates for distal urethral tumors is significantly better than for proximal cancers, 68% versus 40%, respectively.143,146 The median 5-year cancer-specific sur-vival is

1	5-year overall survival rates for distal urethral tumors is significantly better than for proximal cancers, 68% versus 40%, respectively.143,146 The median 5-year cancer-specific sur-vival is approximately 46%.144 Prognosis is dictated by patients’ age, race, clinical stage, and location of the tumor.If feasible, local endoscopic resection for low-volume, low-stage disease is preferable. Adjuvant intravesical instilla-tion of Bacillus Calmette-Guérin (BCG) should be considered for patients with proximal noninvasive disease.147 Due to the paucity of robust data, management of locally advanced dis-ease is more challenging. Either radical cystectomy or radiations are acceptable options. Unfortunately, local recurrence rates are high after aggressive monotherapy (63%).148 More recent data support the use of multimodal therapy.149,150 Small series of combinations of perioperative chemotherapy, surgery, and radiation indicate the best cancer control.151COMMON UROLOGIC CONDITIONSUrinary

1	support the use of multimodal therapy.149,150 Small series of combinations of perioperative chemotherapy, surgery, and radiation indicate the best cancer control.151COMMON UROLOGIC CONDITIONSUrinary Incontinence and Voiding DysfunctionUrinary incontinence is defined as the involuntary loss of urine. This is more common in women than men for a variety of rea-sons, including anatomic differences such as a shorter urethra and risk factors such as childbirth. Many patients may also suf-fer from bothersome symptoms without leakage of urine such as overactive bladder (frequency and urgency of urination and often nocturia), or obstructive symptoms such as hesitancy, weak stream, and incomplete bladder emptying. These condi-tions can have a negative impact on quality of life,152-154 but they are also associated with serious health issues, including depres-sion, anxiety, social isolation,155 and even falls and fractures in the elderly.156Urinary incontinence can be divided into several

1	are also associated with serious health issues, including depres-sion, anxiety, social isolation,155 and even falls and fractures in the elderly.156Urinary incontinence can be divided into several catego-ries, although patients (particularly women) may suffer from more than one type.157 Urge incontinence is the involuntary loss of urine associated with an urge to void. Stress leakage occurs with increases in intra-abdominal pressure, such as coughing or sneezing, and may relate to loss of sphincteric function, urethral hypermobility from pelvic floor laxity (often related to parity), or following prostate surgery in men. Overflow incontinence occurs in the setting of obstruction, with urine leakage occurring with movement causing over-flow of urine from a distended bladder. Genitourinary fistulas typically result in the most severe form of incontinence with constant leakage of urine regardless of presence or absence of activity or movement. Examples include vesicovaginal or

1	fistulas typically result in the most severe form of incontinence with constant leakage of urine regardless of presence or absence of activity or movement. Examples include vesicovaginal or ureterovaginal fistulae most often due to gynecologic surgery, or rectourethral fistulae in men from cancer, radiation, or sur-gical intervention.Treatments for urinary incontinence and voiding dysfunc-tion are varied depending on the etiology, severity, and bother of the symptom. Urge leakage and overactive blad-der can be treated by (a) behavioral modification (timed void-ing, adjustment to fluid intake, timing of diuretic medication, and improved constipation); (b) bladder retraining (pelvic floor physical therapy158); (c) medications (anticholinergics159 and β-3 agonists160); or (d) minimally invasive procedures (sacral neuro-modulation,161 percutaneous tibial nerve stimulation,162 or blad-der chemodenervation with detrusor botulinum toxin injection163).Stress incontinence in women can be

1	invasive procedures (sacral neuro-modulation,161 percutaneous tibial nerve stimulation,162 or blad-der chemodenervation with detrusor botulinum toxin injection163).Stress incontinence in women can be addressed by pelvic floor strengthening exercises, vaginally placed removable sup-port with a pessary, injection of urethral bulking agent, or sling procedures using polypropylene mesh or autologous tissue. In men, stress leakage is due to either iatrogenic causes or neuro-logic disease. Treatments include strengthening exercises as in women, slings, or implantation of an artificial urinary sphincter. Overflow incontinence treatment is directed at the cause of obstruction, often benign prostatic enlargement in men, with bladder drainage, medications such as α-blockers or 5-α reduc-tase inhibitors, or surgical removal of the obstructing gland. When fistulas are present, adherence to surgical principles such as tension-free multilayer closure, nonoverlapping suture lines, and tissue

1	inhibitors, or surgical removal of the obstructing gland. When fistulas are present, adherence to surgical principles such as tension-free multilayer closure, nonoverlapping suture lines, and tissue interposition when possible offers the highest likeli-hood for success.Erectile DysfunctionErectile dysfunction (ED) is defined as the inability to achieve and maintain an erection adequate for sexual intercourse. For-merly, this was known as a type of sexual dysfunction, but it is now understood that ED may be an early symptom of cardiovas-cular disease due to endothelial dysfunction. ED is a common disease for men later in life with a prevalence rate believed to range anywhere from 30% to 50% depending on age. Two large population-based studies, the Massachusetts Male Aging Study (MMAS) and the European Male Aging Study (EMAS), exam-ined men age 40 to 79 years and found that ED rates increased with age.164,1659Brunicardi_Ch40_p1759-p1782.indd 177501/03/19 6:35 PM 1776SPECIFIC

1	and the European Male Aging Study (EMAS), exam-ined men age 40 to 79 years and found that ED rates increased with age.164,1659Brunicardi_Ch40_p1759-p1782.indd 177501/03/19 6:35 PM 1776SPECIFIC CONSIDERATIONSPART IIErections are triggered via sexual stimulation setting off a cascade of events. Nitric oxide is released from nerve fibers and activating guanylyl cyclase leading to an increase in cyclic guanosine monophosphate (cGMP). The cGMP pathway leads to smooth muscle relaxation within the corpora cavernosa allow-ing blood to fill the lacunar spaces. Once the lacunar spaces are full, the expanded tissue compresses the subtunical venules thereby trapping blood within the penis and blocking venous out-flow. Phosphodiesterase type-5 hydrolyzes cGMP to reverse the process.166There are multiple mechanisms leading to ED including vasculogenic, neurogenic, iatrogenic, and psychologic, but often it is multifactorial. Vasculogenic ED can be a result of cardiovascular disease and

1	are multiple mechanisms leading to ED including vasculogenic, neurogenic, iatrogenic, and psychologic, but often it is multifactorial. Vasculogenic ED can be a result of cardiovascular disease and endothelial dysfunction leading to cavernosal artery insufficiency. Diseases such as hypertension (odds ratio [OR] 1.35–3.04), diabetes (OR 2.57), dyslipidemia (OR 1.83), and tobacco abuse (OR 1.4) all may increase the risk for ED.167 Nerve injuries due to diseases (diabetes, Parkin-son’s, multiple sclerosis, spinal cord injury) or surgery (radical prostatectomy, abdominoperineal resection, and other radical pelvic procedures) can lead to interruptions in the nerve signal-ing that causes nitric oxide release and therefore lead to ED. Iatrogenic causes may be a result of surgery (described earlier) or medication use, as in some antihypertensives, opiates, anti-androgens, and psychotherapeutics.168 Psychogenic ED, a com-mon reaction to stress and anxiety, is a result of noradrenaline release

1	or medication use, as in some antihypertensives, opiates, anti-androgens, and psychotherapeutics.168 Psychogenic ED, a com-mon reaction to stress and anxiety, is a result of noradrenaline release causing smooth muscle contraction and thereby inhibit-ing erections.169Treatment for ED begins with lifestyle modification by identifying any reversible risk factors such as stress/anxiety, medications, unhealthy diets, lack of exercise, and tobacco abuse.170 Medical therapy then begins with the use of phospho-diesterase type-5 inhibitors (PDE5i). These work by prolonging the activity of cGMP, leading to continued smooth muscle relax-ation allowing more blood inflow into the penis. Common drugs include sildenafil, tadalafil, vardenafil, and avanafil. They differ in time to peak concentration (lowest in avanafil, sildenafil, and vardenafil), half-life (highest in tadalafil), and the impact of lip-ids in foods (sildenafil and vardenafil must be taken on an empty stomach). Common side effects

1	in avanafil, sildenafil, and vardenafil), half-life (highest in tadalafil), and the impact of lip-ids in foods (sildenafil and vardenafil must be taken on an empty stomach). Common side effects include a headache, heartburn, facial flushing, nasal congestion, and myalgias.171 Patients on nitrate-containing medications should not be given PDE5i due to the risk of severe hypotension. Vision related conditions like macular degeneration, retinitis pigmentosa, and nonarter-itic anterior ischemic optic neuropathy are cause for increased awareness and possible ophthalmologic consult.172Second-line options for ED include vacuum erection devices (VED), intracavernosal injections (ICI), and intraure-thral suppositories. The VED is a mechanical device composed of a cylinder placed around the penis which then uses a vacuum to create negative pressure and pull blood into the penis. In order for blood to stay in the penis after the vacuum is released, a tight constriction band must be placed at the

1	then uses a vacuum to create negative pressure and pull blood into the penis. In order for blood to stay in the penis after the vacuum is released, a tight constriction band must be placed at the base of the penis. There is poor compliance due to difficulty with use and the common reactions of petechia, temporary paresthesia, color changes, and the penis being cold to touch.173,174 Alternatively, ICI uses vasoactive substances (prostaglandin E1 [alprostadil], papaverine, and phentolamine) either alone or in combination to trigger the erection cascade.168 Patients are trained to give themselves a self-injection when they want an erection, and it takes approximately 5 to 15 minutes until they are fully rigid if they respond. With ICI, there is greater concern for prolonged Figure 40-12. A three-piece penile implant for the treatment of erectile dysfunction. The prosthesis is composed of two cylinders placed in the penis, a fluid reservoir placed in the pelvis (upper left), and a pump

1	three-piece penile implant for the treatment of erectile dysfunction. The prosthesis is composed of two cylinders placed in the penis, a fluid reservoir placed in the pelvis (upper left), and a pump placed within the scrotum (bottom left).erection or priapism, so dose titration must be closely moni-tored. Intraurethral suppositories are composed of alprostadil in the form of a pellet which is then placed in the urethra and mas-saged for absorption. With suppository use, there are concerns about efficacy (only 46–65%) and compliance due to a burning sensation that limits the interest of some users.175,176Third-line treatment of ED is with surgery placement of a penile prosthesis. There are three main types (malleable, two-piece, and three-piece). The malleable device does not inflate/deflate and merely bends in and out of position for intercourse. The two-piece and three-piece devices are inflatable and dif-fer on the presence of a separate fluid reservoir. The two-piece device has the

1	and merely bends in and out of position for intercourse. The two-piece and three-piece devices are inflatable and dif-fer on the presence of a separate fluid reservoir. The two-piece device has the fluid maintained in the lower half of the penile cylinders, whereas, the three-piece device has a fluid reservoir placed in the pelvis or abdominal wall (Fig. 40-12). Overall, the inflatable prosthesis has high patient and partner satisfaction rates, >92% and >91%, respectively.177PEDIATRIC UROLOGYHypospadiasHypospadias, a condition which may be considered a form of incomplete maturation of the genitalia, is a common abnormal-ity that occurs in 1 out of 250 to 300 newborn boys. The most obvious aspect of hypospadias is a urethral opening that is not at the tip of the glans, and 70% to 80% of affected babies will have a meatus on the mid to distal shaft or proximal glans. A lesser number will have more proximal openings, whether penoscrotal, scrotal, or perineal. In addition to an abnormally

1	babies will have a meatus on the mid to distal shaft or proximal glans. A lesser number will have more proximal openings, whether penoscrotal, scrotal, or perineal. In addition to an abnormally located meatus, boys usually have deficient ventral foreskin. Associated penile Brunicardi_Ch40_p1759-p1782.indd 177601/03/19 6:35 PM 1777UROLOGYCHAPTER 40curvature, more common in the severe varieties, is referred to as chordee.No diagnostic studies are needed for the majority of boys with hypospadias as there is typically no increased risk of renal or bladder anomalies. Children with associated cryptorchidism, especially with proximal hypospadias and a nonpalpable tes-tis, have an increased risk of a having a coexisting disorder of sexual differentiation (DSD) and need to undergo a thorough evaluation including hormonal studies, karyotype, and pelvic ultrasonography.178Distal hypospadias can usually be repaired in one stage with success rates of greater than 95%. Most would advocate a

1	evaluation including hormonal studies, karyotype, and pelvic ultrasonography.178Distal hypospadias can usually be repaired in one stage with success rates of greater than 95%. Most would advocate a staged approach to proximal hypospadias with correction of penile curvature at the first stage and formal urethral reconstruc-tion at the second.179 Adults with corrected hypospadias usually have normal sexual function and fertility.Urinary Tract Infections in ChildrenUrinary tract infections (UTI) are common in children, and there is a greater chance of underlying anatomic abnormalities. Children may have conditions such as vesicoureteral reflux, ureteropelvic junction obstruction, ureteroceles, or ectopic ure-ters as causes of these infections. Because of this association, in the past all children with febrile infections would undergo complete evaluations including renal ultrasonography (US) as well as invasive studies such as voiding cystourethrogra-phy (VCUG). However, defining

1	all children with febrile infections would undergo complete evaluations including renal ultrasonography (US) as well as invasive studies such as voiding cystourethrogra-phy (VCUG). However, defining pyelonephritis as having a positive renal cortical scan, only 30% to 40% of children with febrile UTI will have reflux. Thus the majority of children with febrile infections, and a greater percentage of those with afe-brile infections (cystitis), will be anatomically normal.180 These data have led to a change in imaging guidelines for children with UTI.Guidelines put out by the American Academy of Pediat-rics have markedly changed the way children with infections are evaluated.181 These guidelines suggest that infants less than 2 months of age with febrile infections should undergo both a renal US and VCUG. Children between 2 months and 2 years who have their first documented infection only need have a renal ultrasound performed. A VCUG is only needed if there are abnormalities detected on

1	US and VCUG. Children between 2 months and 2 years who have their first documented infection only need have a renal ultrasound performed. A VCUG is only needed if there are abnormalities detected on the ultrasound such as hydrone-phrosis, scarring, or other evidence of anatomic abnormality. A VCUG may also be performed if a child has recurrent infections despite empirical treatment. These guidelines do not address children older than 2 years of age but one can assume that simi-lar algorithms of treatment would be appropriate.There is now greater understanding that most children with UTIs, whether pyelonephritis or cystitis, have some ele-ment of bladder and/or bowel dysfunction as the major factor in the development of the infection. Thus, all children with UTIs need to have a thorough assessment of daily bladder and bowel habits. The latter may be difficult to ascertain in younger children, but bowel dysfunction, even subclinical, may be the most important factor in the development

1	of daily bladder and bowel habits. The latter may be difficult to ascertain in younger children, but bowel dysfunction, even subclinical, may be the most important factor in the development of UTIs. Behavioral therapies such as regular and complete voiding in conjunction with a bowel program should be considered the mainstay of the prevention of infections as opposed to prophylactic antibiotics.Prenatal HydronephrosisAntenatal imaging will show hydronephrosis in nearly 1% of all babies. Though the majority of children will have benign hydro-nephrosis of no clinical significance, it may also be related to vesicoureteral reflux, ureteropelvic junction obstruction, ectopic ureter/ureteroceles, and other upper tract abnormalities. Typi-cally, nothing needs to be done for these children until after birth, at which point a baseline renal ultrasound can be per-formed. Other studies such as a VCUG or Lasix renal scans can then be done depending on the degree of dilation. Diagnosis of upper

1	after birth, at which point a baseline renal ultrasound can be per-formed. Other studies such as a VCUG or Lasix renal scans can then be done depending on the degree of dilation. Diagnosis of upper tract obstruction is usually based on progressive worsen-ing of dilation or renal function on serial examinations.Special consideration must be given for children with bilateral hydronephrosis or hydronephrosis associated with a solitary kidney, especially if linked to oligohydramnios. Since fetal urine production accounts for much of the amniotic fluid, low levels can be a sign of a severe abnormality of the urinary tract. Reduced amniotic fluid is of great consequence since nor-mal lung development is dependent on normal amniotic fluid volumes and children with oligohydramnios can be born with significant pulmonary insufficiency. Boys with bilateral hydro-nephrosis and low amniotic fluid are at high risk for having posterior urethral valves (PUV). Boys with PUV have as much as a 25% risk

1	significant pulmonary insufficiency. Boys with bilateral hydro-nephrosis and low amniotic fluid are at high risk for having posterior urethral valves (PUV). Boys with PUV have as much as a 25% risk of developing end stage renal disease at some point in their lives.182 Prenatal intervention such as placement of vesicoamniotic shunts have not been shown to reduce the risk of renal failure.CryptorchidismCryptorchidism or undescended testes (UDT) is a common condition occurring in 3% of full term and 30% of premature babies. Many of these testes will descend spontaneously due to the normal gonadotropin release that occurs in the first few months of life, so the true incidence is roughly 1% of boys. Untreated cryptorchidism will lead to testis damage, and there is evidence that permanent changes may occur by 3 years of age. Ideally, surgical treatment should occur prior to this age. UDT is usually an isolated finding, but it may occur as a part of a systemic condition such as Prader-Willi,

1	may occur by 3 years of age. Ideally, surgical treatment should occur prior to this age. UDT is usually an isolated finding, but it may occur as a part of a systemic condition such as Prader-Willi, Eagle-Barrett, or other such complex multisystem syndrome. Surgery is the treatment of choice; hormonal treatment has no role.The consequences of untreated cryptorchidism include infertility and malignant degeneration. One study on fertility suggested that men with a history of unilateral cryptorchidism will have no difference in paternity rates compared to normal controls. In contrast, men with bilateral cryptorchidism have up to a 50% rate of infertility.183 There is data to suggest that orchidopexy in the first year of life is associated with better total sperm counts in adulthood.184 With regard to malignancy, untreated UDT has a fivefold increase risk of tumor develop-ment compared to the normal population. However, there is data to suggest that prepubertal orchidopexy is protective

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1	Task Force recommendation statement. Ann Intern Med. 2012;157(2):120-134. 131. Carter HB, Albertsen PC, Barry MJ, et al. Early detection of prostate cancer: AUA Guideline. J Urol. 2013;190(2): 419-426. 132. Gleason DF, Mellinger GT. Prediction of prognosis for pros-tatic adenocarcinoma by combined histological grading and clinical staging. J Urol. 1974;111(1):58-64. 133. Epstein JI, Zelefsky MJ, Sjoberg DD, et al. A contemporary prostate cancer grading system: a validated alternative to the gleason score. Eur Urol. 2016;69(3):428-435. 134. Hamdy FC, Donovan JL, Lane JA, et al. 10-year outcomes after monitoring, surgery, or radiotherapy for localized prostate cancer. N Engl J Med. 2016;375(15):1415-1424. 135. Chung MS, Lee SH. Current status of active surveillance in prostate cancer. Investig Clin Urol. 2016;57(1):14-20. 136. Wiegel T, Bartkowiak D, Bottke D, et al. Adjuvant radiother-apy versus wait-and-see after radical prostatectomy: 10-year follow-up of the ARO 96-02/AUO AP 09/95

1	Clin Urol. 2016;57(1):14-20. 136. Wiegel T, Bartkowiak D, Bottke D, et al. Adjuvant radiother-apy versus wait-and-see after radical prostatectomy: 10-year follow-up of the ARO 96-02/AUO AP 09/95 trial. Eur Urol. 2014;66(2):243-250. 137. Thompson IM, Tangen CM, Paradelo J, et al. Adjuvant radiotherapy for pathological T3N0M0 prostate cancer significantly reduces risk of metastases and improves sur-vival: long-term followup of a randomized clinical trial. J Urol. 2009;181(3):956-962. 138. Pound CR, Partin AW, Eisenberger MA, Chan DW, Pear-son JD, Walsh PC. Natural history of progression after PSA elevation following radical prostatectomy. JAMA. 1999;281(17):1591-1597. 139. Ryan CJ, Smith MR, Fizazi K, et al. Abiraterone acetate plus prednisone versus placebo plus prednisone in chemother-apy-naive men with metastatic castration-resistant prostate cancer (COU-AA-302): final overall survival analysis of a randomised, double-blind, placebo-controlled phase 3 study. Lancet Oncol.

1	men with metastatic castration-resistant prostate cancer (COU-AA-302): final overall survival analysis of a randomised, double-blind, placebo-controlled phase 3 study. Lancet Oncol. 2015;16(2):152-160. 140. Logothetis CJ, Basch E, Molina A, et al. Effect of abiraterone acetate and prednisone compared with placebo and predni-sone on pain control and skeletal-related events in patients with metastatic castration-resistant prostate cancer: explor-atory analysis of data from the COU-AA-301 randomised trial. Lancet Oncol. 2012;13(12):1210-1217. 141. Loriot Y, Miller K, Sternberg CN, et al. Effect of enzalutamide on health-related quality of life, pain, and skeletal-related events in asymptomatic and minimally symptomatic, che-motherapy-naive patients with metastatic castration-resistant prostate cancer (PREVAIL): results from a randomised, phase 3 trial. Lancet Oncol. 2015;16(5):509-521. 142. Sternberg CN, de Bono JS, Chi KN, et al. Improved outcomes in elderly patients with metastatic

1	cancer (PREVAIL): results from a randomised, phase 3 trial. Lancet Oncol. 2015;16(5):509-521. 142. Sternberg CN, de Bono JS, Chi KN, et al. Improved outcomes in elderly patients with metastatic castration-resistant prostate cancer treated with the androgen receptor inhibitor enzalu-tamide: results from the phase III AFFIRM trial. Ann Oncol. 2014;25(2):429-434. 143. Dalbagni G, Zhang ZF, Lacombe L, Herr HW. Male ure-thral carcinoma: analysis of treatment outcome. Urology. 1999;53(6):1126-1132. 144. Swartz MA, Porter MP, Lin DW, Weiss NS. Incidence of primary urethral carcinoma in the United States. Urology. 2006;68(6):1164-1168. 145. Cupp MR, Malek RS, Goellner JR, Espy MJ, Smith TF. Detection of human papillomavirus DNA in primary squa-mous cell carcinoma of the male urethra. Urology. 1996;48(4): 551-555. 146. Dinney CP, Johnson DE, Swanson DA, Babaian RJ, von Eschenbach AC. Therapy and prognosis for male ante-rior urethral carcinoma: an update. Urology. 1994;43(4):

1	Urology. 1996;48(4): 551-555. 146. Dinney CP, Johnson DE, Swanson DA, Babaian RJ, von Eschenbach AC. Therapy and prognosis for male ante-rior urethral carcinoma: an update. Urology. 1994;43(4): 506-514. 147. Gofrit ON, Pode D, Pizov G, Zorn KC, Katz R, Shapiro A. Prostatic urothelial carcinoma: is transurethral prostatectomy necessary before bacillus Calmette-Guerin immunotherapy? BJU Int. 2009;103(7):905-908. 148. Gakis G, Witjes JA, Comperat E, et al. EAU guidelines on primary urethral carcinoma. Eur Urol. 2013;64(5):823-830. 149. Gakis G, Morgan TM, Daneshmand S, et al. Impact of perioperative chemotherapy on survival in patients with advanced primary urethral cancer: results of the international collaboration on primary urethral carcinoma. Ann Oncol. 2015;26(8):1754-1759. 150. Kent M, Zinman L, Girshovich L, Sands J, Vanni A. Com-bined chemoradiation as primary treatment for invasive male urethral cancer. J Urol. 2015;193(2):532-537. 151. Dayyani F, Pettaway CA, Kamat AM, Munsell

1	Zinman L, Girshovich L, Sands J, Vanni A. Com-bined chemoradiation as primary treatment for invasive male urethral cancer. J Urol. 2015;193(2):532-537. 151. Dayyani F, Pettaway CA, Kamat AM, Munsell MF, Sircar K, Pagliaro LC. Retrospective analysis of survival outcomes and the role of cisplatin-based chemotherapy in patients with ure-thral carcinomas referred to medical oncologists. Urol Oncol. 2013;31(7):1171-1177. 152. Aguilar-Navarro S, Navarrete-Reyes AP, Grados-Chavarria BH, Garcia-Lara JM, Amieva H, Avila-Funes JA. The sever-ity of urinary incontinence decreases health-related quality of life among community-dwelling elderly. J Gerontol A Biol Sci Med Sci. 2012;67(11):1266-1271. 153. Bettez M, Tu le M, Carlson K, et al. 2012 update: guidelines for adult urinary incontinence collaborative consensus docu-ment for the Canadian urological association. Can Urol Assoc J. 2012;6(5):354-363. 154. Tennstedt SL, Chiu GR, Link CL, Litman HJ, Kusek JW, McKinlay JB. The effects of severity

1	consensus docu-ment for the Canadian urological association. Can Urol Assoc J. 2012;6(5):354-363. 154. Tennstedt SL, Chiu GR, Link CL, Litman HJ, Kusek JW, McKinlay JB. The effects of severity of urine leakage on qual-ity of life in Hispanic, white, and black men and women: the Boston community health survey. Urology. 2010;75(1):27-33. 155. Ramage-Morin PL, Gilmour H. Urinary incontinence and loneliness in Canadian seniors. Health Rep. 2013;24(10): 3-10. 156. Brown JS, Vittinghoff E, Wyman JF, et al. Urinary inconti-nence: does it increase risk for falls and fractures? Study of Osteoporotic Fractures Research Group. J Am Geriatr Soc. 2000;48(7):721-725. 157. Abrams P, Cardozo L, Fall M, et al. The standardisation of terminology in lower urinary tract function: report from the standardisation sub-committee of the International Conti-nence Society. Urology. 2003;61(1):37-49. 158. Dumoulin C, Hunter KF, Moore K, et al. Conservative management for female urinary incontinence and pelvic

1	sub-committee of the International Conti-nence Society. Urology. 2003;61(1):37-49. 158. Dumoulin C, Hunter KF, Moore K, et al. Conservative management for female urinary incontinence and pelvic Brunicardi_Ch40_p1759-p1782.indd 178101/03/19 6:35 PM 1782SPECIFIC CONSIDERATIONSPART IIorgan prolapse review 2013: Summary of the 5th Interna-tional Consultation on Incontinence. Neurourol Urodyn. 2016;35(1):15-20. 159. Chapple C, Khullar V, Gabriel Z, Dooley JA. The effects of antimuscarinic treatments in overactive bladder: a systematic review and meta-analysis. Eur Urol. 2005;48(1):5-26. 160. Chapple CR, Cardozo L, Nitti VW, Siddiqui E, Michel MC. Mirabegron in overactive bladder: a review of efficacy, safety, and tolerability. Neurourol Urodyn. 2014;33(1):17-30. 161. van Kerrebroeck PE, van Voskuilen AC, Heesakkers JP, et al. Results of sacral neuromodulation therapy for urinary voiding dysfunction: outcomes of a prospective, worldwide clinical study. J Urol.

1	Kerrebroeck PE, van Voskuilen AC, Heesakkers JP, et al. Results of sacral neuromodulation therapy for urinary voiding dysfunction: outcomes of a prospective, worldwide clinical study. J Urol. 2007;178(5):2029-2034. 162. Burton C, Sajja A, Latthe PM. Effectiveness of percutane-ous posterior tibial nerve stimulation for overactive bladder: a systematic review and meta-analysis. Neurourol Urodyn. 2012;31(8):1206-1216. 163. Rovner E, Kennelly M, Schulte-Baukloh H, Zhou J, Haag-Molkenteller C, Dasgupta P. Urodynamic results and clinical outcomes with intradetrusor injections of onabotulinum-toxinA in a randomized, placebo-controlled dose-finding study in idiopathic overactive bladder. Neurourol Urodyn. 2011;30(4):556-562. 164. Johannes CB, Araujo AB, Feldman HA, Derby CA, Kleinman KP, McKinlay JB. Incidence of erectile dysfunction in men 40 to 69 years old: longitudinal results from the Massachusetts male aging study. J Urol. 2000;163(2):460-463. 165. Corona G, Lee DM, Forti G, et al.

1	JB. Incidence of erectile dysfunction in men 40 to 69 years old: longitudinal results from the Massachusetts male aging study. J Urol. 2000;163(2):460-463. 165. Corona G, Lee DM, Forti G, et al. Age-related changes in gen-eral and sexual health in middle-aged and older men: results from the European Male Ageing Study (EMAS). J Sex Med. 2010;7(4 Pt 1):1362-1380. 166. Lue TF. Erectile dysfunction. N Engl J Med. 2000;342(24): 1802-1813. 167. Francis ME, Kusek JW, Nyberg LM, Eggers PW. The con-tribution of common medical conditions and drug exposures to erectile dysfunction in adult males. J Urol. 2007;178(2): 591-596; discussion 596. 168. Yafi FA, Jenkins L, Albersen M, et al. Erectile dysfunction. Nat Rev Dis Primers. 2016;2:16003. 169. McCabe MP, Althof SE. A systematic review of the psycho-social outcomes associated with erectile dysfunction: does the impact of erectile dysfunction extend beyond a man’s inability to have sex? J Sex Med. 2014;11(2):347-363. 170. The process of care

1	outcomes associated with erectile dysfunction: does the impact of erectile dysfunction extend beyond a man’s inability to have sex? J Sex Med. 2014;11(2):347-363. 170. The process of care model for evaluation and treatment of erectile dysfunction. The Process of Care Consensus Panel. Int J Impot Res. 1999;11(2):59-70; discussion 70-54. 171. Yuan J, Zhang R, Yang Z, et al. Comparative effective-ness and safety of oral phosphodiesterase type 5 inhibitors for erectile dysfunction: a systematic review and network meta-analysis. Eur Urol. 2013;63(5):902-912. 172. Giuliano F, Jackson G, Montorsi F, Martin-Morales A, Raillard P. Safety of sildenafil citrate: review of 67 double-blind placebo-controlled trials and the postmarket-ing safety database. Int J Clin Pract. 2010;64(2):240-255. 173. Baltaci S, Aydos K, Kosar A, Anafarta K. Treating erectile dysfunction with a vacuum tumescence device: a retro-spective analysis of acceptance and satisfaction. Br J Urol. 1995;76(6):757-760. 174. Ganem

1	S, Aydos K, Kosar A, Anafarta K. Treating erectile dysfunction with a vacuum tumescence device: a retro-spective analysis of acceptance and satisfaction. Br J Urol. 1995;76(6):757-760. 174. Ganem JP, Lucey DT, Janosko EO, Carson CC. Unusual complications of the vacuum erection device. Urology. 1998;51(4):627-631. 175. Guay AT, Perez JB, Velasquez E, Newton RA, Jacobson JP. Clinical experience with intraurethral alprostadil (MUSE) in the treatment of men with erectile dysfunction. A retrospec-tive study. Medicated urethral system for erection. Eur Urol. 2000;38(6):671-676. 176. Fulgham PF, Cochran JS, Denman JL, et al. Disappointing initial results with transurethral alprostadil for erectile dys-function in a urology practice setting. J Urol. 1998;160 (6 pt 1):2041-2046. 177. Rajpurkar A, Dhabuwala CB. Comparison of satisfaction rates and erectile function in patients treated with silde-nafil, intracavernous prostaglandin E1 and penile implant surgery for erectile dysfunction in

1	A, Dhabuwala CB. Comparison of satisfaction rates and erectile function in patients treated with silde-nafil, intracavernous prostaglandin E1 and penile implant surgery for erectile dysfunction in urology practice. J Urol. 2003;170(1):159-163. 178. Kaefer M, Diamond D, Hendren WH, et al. The incidence of intersexuality in children with cryptorchidism and hypospa-dias: stratification based on gonadal palpability and meatal position. J Urol. 1999;162(3 pt 2):1003-1006; discussion 1006-1007. 179. Keays MA, Dave S. Current hypospadias management: Diag-nosis, surgical management, and long-term patient-centred outcomes. Can Urol Assoc J. 2017;11(1-2 suppl 1):S48-S53. 180. Preda I, Jodal U, Sixt R, Stokland E, Hansson S. Normal dimercaptosuccinic acid scintigraphy makes voiding cystoure-thrography unnecessary after urinary tract infection. J Pediatr. 2007;151(6):581-584, 584.e581. 181. Roberts KB. Urinary tract infection: clinical practice guide-line for the diagnosis and management of the

1	unnecessary after urinary tract infection. J Pediatr. 2007;151(6):581-584, 584.e581. 181. Roberts KB. Urinary tract infection: clinical practice guide-line for the diagnosis and management of the initial UTI in febrile infants and children 2 to 24 months. Pediatrics. 2011;128(3):595-610. 182. Bilgutay AN, Roth DR, Gonzales ET, Jr., et al. Posterior urethral valves: risk factors for progression to renal failure. J Pediatr Urol. 2016;12(3):179 e171-e177. 183. Lee PA. Fertility after cryptorchidism: epidemiology and other outcome studies. Urology. 2005;66(2):427-431. 184. Feyles F, Peiretti V, Mussa A, et al. Improved sperm count and motility in young men surgically treated for cryptorchidism in the first year of life. Eur J Pediatr Surg. 2014;24(5):376-380. 185. Pettersson A, Richiardi L, Nordenskjold A, Kaijser M, Akre O. Age at surgery for undescended testis and risk of testicular cancer. N Engl J Med. 2007;356(18):1835-1841.Brunicardi_Ch40_p1759-p1782.indd 178201/03/19 6:35 PM

1	GynecologySarah M. Temkin, Thomas Gregory, Elise C. Kohn, and Linda Duska 41chapterPATHOPHYSIOLOGY AND MECHANISMS OF DISEASEThe female reproductive system includes the external (vulva including the labia, clitoris, and vaginal opening) sex organs as well as the internal organs (uterus and cervix, fallopian tubes, and ovaries) that function in human reproduction. The female reproductive tract has a multitude of tightly regulated functions. The ovaries produce the ova (egg cells) and hormones necessary for maintenance of reproductive function. The fallopian tubes accommodate transit of an ovum to the uterus and provide a location for fertilization. The uterus accommodates an embryo that develops into the fetus. The cervix provides a barrier between the external and internal genital tract. Ongoing activities, such as angiogenesis and physiologic invasion, are necessary in order for the reproductive organs to fulfill their purpose and are usurped in disease. Immune surveillance is

1	Ongoing activities, such as angiogenesis and physiologic invasion, are necessary in order for the reproductive organs to fulfill their purpose and are usurped in disease. Immune surveillance is regulated in a fashion that allows implantation, placentation, and development of the fetus.Because the pelvis contains a multitude of spatially and temporally varied functions, pathologies range from mechanical events, such as ovarian torsion or ruptured ectopic pregnancy, to infection, such as pelvic inflammatory disease, to mass effects, including leiomyomata and malignancy, that can present with similar and even overlapping symptoms and signs. An acute abdomen presentation in a woman of child bearing potential can range from pregnancy-related catastrophes, to appendicitis, to a hemorrhagic ovarian cyst.The ongoing rupture, healing, and regrowth of the ovarian capsule and endometrium during the menstrual cycle use the same series of biologic and biochemic events that are also active in

1	ovarian cyst.The ongoing rupture, healing, and regrowth of the ovarian capsule and endometrium during the menstrual cycle use the same series of biologic and biochemic events that are also active in pathologic events such as endometriosis and endometriomas, mature teratomas, dysgerminomas, and progression to malig-nancy. Genetic abnormalities, both germ line and somatic, that may cause competence and/or promote disease are increasingly well understood. Incorporation of genetic and genomic infor-mation in disease diagnosis and assessment has altered how we diagnose and follow disease, in whom we increase our diligence in searching for disease, and ultimately how we use the drug and other therapeutic armamentarium available to the treating physician.These points will be incorporated with surgical approaches into discussions of anatomy, diagnostic workup, infection, sur-gical and medical aspects of the obstetric patient, pelvic floor dysfunction, and neoplasms.ANATOMYClinical gynecologic

1	approaches into discussions of anatomy, diagnostic workup, infection, sur-gical and medical aspects of the obstetric patient, pelvic floor dysfunction, and neoplasms.ANATOMYClinical gynecologic anatomy centers on the pelvis (L. basin). Aptly named, the bowl-shaped pelvis houses the confluence and intersection of multiple organ systems. Understanding 1Pathophysiology and Mechanisms of Disease 1783Anatomy 1783Structure and Support of the Pelvis and Genitalia / 1784Vulva / 1785Vagina / 1785Uterus / 1785Cervix / 1785Fallopian Tubes / 1786Ovaries / 1786Fibrovascular Ligaments and Avascular Tissue Planes / 1786Vasculature and Nerves of the Pelvis / 1787Evaluation and Diagnosis 1787Elements of a Gynecologic History / 1787The Gynecologic Examination / 1787Commonly Used Testing / 1789Common Office Procedures for Diagnosis / 1790Benign Gynecologic Conditions 1791Vulvar Lesions / 1791Vaginal Lesions / 1793Cervical Lesions / 1794Uterine Corpus / 1794Procedures Performed for Structural Causes

1	Procedures for Diagnosis / 1790Benign Gynecologic Conditions 1791Vulvar Lesions / 1791Vaginal Lesions / 1793Cervical Lesions / 1794Uterine Corpus / 1794Procedures Performed for Structural Causes of Abnormal Uterine Bleeding / 1796Benign Ovarian and Fallopian Tube Lesions / 1801Other Benign Pelvic Pathology / 1802Pregnancy-Related Surgical Conditions 1804Conditions and Procedures Performed Before Viability / 1804Conditions and Procedures Performed After Viability / 1805Pelvic Floor Dysfunction 1807Evaluation / 1807Surgery for Pelvic Organ Prolapse / 1807Surgery for Stress Urinary Incontinence / 1808Gynecologic Cancer 1809Vulvar Cancer / 1809Vaginal Cancer / 1810Cervical Cancer / 1811Uterine Cancer / 1813Ovarian Cancer / 1815Minimally Invasive Gynecologic Surgery 1820Hysteroscopy / 1820Laparoscopy / 1820Robotic Surgery / 1820Complications Pertinent to Gynecologic Surgery / 1821Brunicardi_Ch41_p1783-p1826.indd 178318/02/19 4:33 PM 1784those structural and functional

1	/ 1820Laparoscopy / 1820Robotic Surgery / 1820Complications Pertinent to Gynecologic Surgery / 1821Brunicardi_Ch41_p1783-p1826.indd 178318/02/19 4:33 PM 1784those structural and functional relationships is essential for the surgeon and allows an appreciation for the interplay of sexual function and reproduction as well as a context for understanding gynecologic pathology.Structure and Support of the Pelvis and GenitaliaThe bony pelvis is comprised by the sacrum posteriorly and the ischium, ilium, and pubic bones anteromedially. It supports the upper body and transmits the stresses of weight bearing to the lower limbs in addition to providing anchors for the supporting tissues of the pelvic floor.1 The opening of the pelvis is spanned by the muscles of the pelvic diaphragm (Fig. 41-1). The muscles of the pelvic sidewall include the iliacus, the psoas, and the obturator internus muscle (Fig. 41-2). These muscles contract tonically and include, from anterior to posterior,

1	41-1). The muscles of the pelvic sidewall include the iliacus, the psoas, and the obturator internus muscle (Fig. 41-2). These muscles contract tonically and include, from anterior to posterior, bilaterally, the pubococcygeus, puborectalis, iliococcygeus, and coccygeus muscles. The first two of these muscles contribute fibers to the fibromuscular perineal body. The urogenital hiatus is bordered laterally by the pubococcygeus muscles and anteriorly by the symphysis pubis. It is through this muscular defect that the urethra and vagina pass, and it is the focal point for the study of disorders of pelvic support such as cystocele, rectocele, and uterine prolapse.Pudendal nerveand arterySuperficial transverseperineii muscleIschiocavernosusmuscleVestibularbulbClitorisPubicramusUrethralmeatusBulbocavernosusmuscleBartholin’sglandPerinealmembranePerinealbodyExternal analsphincterGluteusmaximusAnusVaginalintroitusLevator animusclesFigure 41-1. Deeper muscles of the pelvic floor.Key

1	analsphincterGluteusmaximusAnusVaginalintroitusLevator animusclesFigure 41-1. Deeper muscles of the pelvic floor.Key Points1 Gynecologic causes of acute abdomen include PID and tubo-ovarian abscess, ovarian torsion, ruptured ectopic pregnancy, septic abortion. Pregnancy must be ruled out early in assessment of reproductive age patients presenting with abdominal or pelvic pain.2 The general gynecology exam must incorporate the whole physical examination in order to adequately diagnosis and treat gynecologic disorders.3 Benign gynecologic pathologies that are encountered at the time of surgery include endometriosis, endometriomas, fibroids, and ovarian cysts.4 It is critical that abnormal lesions of vulva, vagina, and cervix are biopsied for diagnosis before any treatment is planned; postmenopausal bleeding should always be investigated to rule out malignancy.5 Pelvic floor dysfunction (pelvic organ prolapse, urinary and fecal incontinence) is common; 11% of women will undergo a

1	postmenopausal bleeding should always be investigated to rule out malignancy.5 Pelvic floor dysfunction (pelvic organ prolapse, urinary and fecal incontinence) is common; 11% of women will undergo a reconstructive surgical procedure at some point in their lives.6 Pregnancy confers important changes to both the cardio-vascular system and the coagulation cascade. Trauma in pregnancy must be managed with these changes in mind.7 Early-stage cervical cancer is managed surgically, whereas chemoradiation is preferred for stages Ib2 and above.8 Risk-reducing salpingo-oopherectomy is recommended in women with BRCA1 or BRCA2 mutations.9 Optimal debulking for epithelial ovarian cancer is a criti-cal element in patient response and survival. The preferred postoperative therapy for optimally debulked advanced-stage ovarian epithelial ovarian cancer is intraperitoneal chemotherapy.10 Long-term sequelae of intestinal and urologic injury can be avoided by intraoperative

1	for optimally debulked advanced-stage ovarian epithelial ovarian cancer is intraperitoneal chemotherapy.10 Long-term sequelae of intestinal and urologic injury can be avoided by intraoperative identification.Brunicardi_Ch41_p1783-p1826.indd 178418/02/19 4:33 PM 1785GYNECOLOGYCHAPTER 41VulvaThe labia majora form the cutaneous boundaries of the lateral vulva and represent the female homologue of the male scrotum (Fig. 41-4). The labia majora are fatty folds covered by hair-bearing skin in the adult. They fuse anteriorly over the ante-rior prominence of the symphysis pubis, the mons pubis. The deeper portions of the adipose layers are called Colles fascia and insert onto the inferior margin of the perineal membrane, limiting spread of superficial hematomas inferiorly. Adjacent and medial to the labia majora are the labia minora, smaller folds of connective tissue covered laterally by non–hair-bearing skin and medially by vaginal mucosa. The anterior fusion of the labia minora forms

1	to the labia majora are the labia minora, smaller folds of connective tissue covered laterally by non–hair-bearing skin and medially by vaginal mucosa. The anterior fusion of the labia minora forms the prepuce and frenulum of the clitoris; posteriorly, the labia minora fuse to create the fossa navicularis and posterior fourchette. The term vestibule refers to the area medial to the labia minora bounded by the fossa navicularis and the clitoris. Both the urethra and the vagina open into the vestibule. Skene’s glands lie lateral and inferior to the urethral meatus. Cysts, abscesses, and neoplasms may arise in these glands.Erectile tissues and associated muscles are in the space between the perineal membrane and the vulvar subcutaneous tissues (see Fig. 41-1). The clitoris is formed by two crura and is suspended from the pubis. Overlying the crura are ischio-cavernosus muscles, which run along the inferior surfaces of the ischiopubic rami. Extending medially from the inferior end of the

1	and is suspended from the pubis. Overlying the crura are ischio-cavernosus muscles, which run along the inferior surfaces of the ischiopubic rami. Extending medially from the inferior end of the ischiocavernosus muscles are the superficial transverse perinei muscles. These terminate in the midline in the perineal body, caudal and deep to the posterior fourchette. Vestibular bulbs lie just deep to the vestibule and are covered laterally by bulbocavernosus muscles. These originate from the perineal body and insert into the body of the clitoris. At the inferior end of the vestibular bulbs are Bartholin’s glands, which connect to the vestibular skin by ducts.VaginaThe vagina is an elastic fibromuscular tube opening from the vestibule running superiorly and posteriorly, passing through the perineal membrane. The lower third is invested by the superficial and deep perineal muscles; it incorporates the ure-thra in its anterior wall and has a rich blood supply from the vaginal branches of the

1	membrane. The lower third is invested by the superficial and deep perineal muscles; it incorporates the ure-thra in its anterior wall and has a rich blood supply from the vaginal branches of the external and internal pudendal arteries. The upper two-thirds of the vagina are not invested by muscles. This portion lies in opposition to the bladder base anteriorly and the rectum and posterior pelvic cul-de-sac superiorly. The cervix opens into the posterior vaginal wall bulging into the vaginal lumen.UterusThe typically pear-shaped uterus consists of a fundus, cornua, body, and cervix. It lies between the bladder anteriorly and the rectosigmoid posteriorly. The endometrium lines the inside cavity and has a superficial functional layer that is shed with menstruation and a basal layer from which the new functional layer is formed. Sustained estrogenic stimulation without asso-ciated progestin maturation can lead to hyperplastic changes or carcinoma. Adenomyosis is a condition in which

1	which the new functional layer is formed. Sustained estrogenic stimulation without asso-ciated progestin maturation can lead to hyperplastic changes or carcinoma. Adenomyosis is a condition in which benign endo-metrial glands infiltrate into the muscle or myometrium of the uterus. The myometrium is composed of smooth muscle and the contraction of myometrium is a factor in menstrual pain and is essential in childbirth. The myometrium can develop benign smooth muscle neoplasms known as leiomyoma or fibroids.CervixThe cervix connects the uterus and vagina and projects into the upper vagina. The vagina forms an arched ring around the cervix described as the vaginal fornices—lateral, anterior, and posterior. The cervix is about 2.5-cm long with a fusiform endo-cervical canal lined by columnar epithelium lying between an internal and external os, or opening. The vaginal surface of the cervix is covered with stratified squamous epithelium, similar to that lining the vagina. The

1	by columnar epithelium lying between an internal and external os, or opening. The vaginal surface of the cervix is covered with stratified squamous epithelium, similar to that lining the vagina. The squamo-columnar junction, also referred to as the transformation zone, migrates at different stages of life and is influenced by estrogenic stimulation. The transformation zone develops as the columnar epithelium is replaced by squamous metaplasia. This transformation zone is Internal iliac arteryLateral sacralarterySuperiorglutealarteryInferior gluteal arteryCoccygeus muscleInternal pudendalarteryUterine arteryMiddle rectal arteryObturator internusmuscleObturator arterySuperior vesical arteryExternal iliac arteryCommon iliac arteryFigure 41-2. The muscles and vasculature of the pelvis.Hypogastric plexusObturator nerveVesical plexusUterovaginal plexus Rectal plexusLeft pelvic plexusSacral plexusSympathetic ganglionFigure 41-3. The nerve supply of the female

1	of the pelvis.Hypogastric plexusObturator nerveVesical plexusUterovaginal plexus Rectal plexusLeft pelvic plexusSacral plexusSympathetic ganglionFigure 41-3. The nerve supply of the female pelvis.Brunicardi_Ch41_p1783-p1826.indd 178518/02/19 4:33 PM 1786SPECIFIC CONSIDERATIONSPART IIvulnerable to human papilloma virus (HPV) infection and resul-tant premalignant changes. These changes can be detected by microscopic assessment of cervical cytological (or Pap) smear. If the duct of a cervical gland becomes occluded, the gland dis-tends to form a retention cyst or Nabothian follicle.Fallopian TubesThe bilateral fallopian tubes arise from the upper lateral cornua of the uterus and course posterolaterally within the upper border of the broad ligament. The tubes can be divided into four parts. The interstitial part forms a passage through the myometrium. The isthmus is the narrow portion extending out about 3 cm from the myometrium. The ampulla is thin-walled and tortuous with its

1	parts. The interstitial part forms a passage through the myometrium. The isthmus is the narrow portion extending out about 3 cm from the myometrium. The ampulla is thin-walled and tortuous with its lateral end free of the broad ligament. The infundibulum is the distal end fringed by a ring of delicate fronds or fimbriae. The fallopian tubes receive the ovum after ovulation. Peristal-sis carries the ovum to the ampulla where fertilization occurs. The zygote transits the tube over the course of 3 to 4 days to the uterus. Abnormal implantation in the fallopian tube is the most common site of ectopic pregnancies. The tubes may also be infected by ascending organisms, resulting in tubo-ovarian abscesses. Scarring of the fallopian tubes can lead to hydrosal-pinx. Recent evidence suggests most high-grade serous ovarian cancer originates in the fallopian tubes.OvariesThe ovaries are attached to the uterine cornu by the proper ovarian ligaments, or the utero-ovarian ligaments. The ovaries are

1	serous ovarian cancer originates in the fallopian tubes.OvariesThe ovaries are attached to the uterine cornu by the proper ovarian ligaments, or the utero-ovarian ligaments. The ovaries are sus-pended from the lateral pelvis by their vascular pedicles, the infundibulopelvic ligaments (IP) or ovarian arteries. These are also called the suspensory ligaments of the ovaries, and cor-respond to the genital vessels in the male. The IP’s are paired branches from the abdominal aorta arising just below the renal arteries. They merge with the peritoneum over the psoas major muscle and pass over the pelvic brim and the external iliac ves-sels. The ovarian veins ascend at first with the ovarian arteries, then track more laterally. The right ovarian vein ascends to drain BladderUterusRound ligamentExternal iliacartery and veinFallopian tubeOvarianvesselsOvarian ligamentBroad ligamentUterosacral ligamentSigmoid colonUreterOvaryFigure 41-5. Internal pelvic anatomy, from above.Figure 41-4. External

1	iliacartery and veinFallopian tubeOvarianvesselsOvarian ligamentBroad ligamentUterosacral ligamentSigmoid colonUreterOvaryFigure 41-5. Internal pelvic anatomy, from above.Figure 41-4. External genitalia. (Reproduced with permission from Rock J, Jones HW: TeLinde’s Operative Gynecology, 9th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2003.)ClitorisLabiumminusLabiummajusMouth ofBartholin’s glandFossa navicularisFourchetteAnusHymenVaginaSkene’sductsUrethralorificePrepuce ofclitorisdirectly into the inferior vena cava while the left vein drains into the left renal vein. Lymphatic drainage follows the arteries to the para-aortic lymph nodes. The ovaries are covered by a single layer of cells that is continuous with the mesothelium of the peritoneum. Beneath this is a fibrous stroma within which are embedded germ cells. At ovulation, an ovarian follicle ruptures through the ovarian epithelium.Fibrovascular Ligaments and Avascular Tissue PlanesFigure 41-5 is a view of the internal

1	which are embedded germ cells. At ovulation, an ovarian follicle ruptures through the ovarian epithelium.Fibrovascular Ligaments and Avascular Tissue PlanesFigure 41-5 is a view of the internal genitalia and deep pelvis as one would approach the pelvis from a midline abdominal incision. The central uterus and uterine cervix are supported by the pelvic floor muscles (Fig. 41-5). They are suspended by Brunicardi_Ch41_p1783-p1826.indd 178618/02/19 4:34 PM 1787GYNECOLOGYCHAPTER 41the lateral fibrous cardinal, or Mackenrodt’s ligament, and the uterosacral ligaments, which insert into the paracervical fascia medially and into the muscular sidewalls of the pelvis laterally. Posteriorly, the uterosacral ligaments provide support for the vagina and cervix as they course from the sacrum lateral to the rectum and insert into the paracervical fascia. Emanating from the uterine cornu and traveling through the inguinal canal are the round ligaments, eventually attaching to the subcutaneous

1	to the rectum and insert into the paracervical fascia. Emanating from the uterine cornu and traveling through the inguinal canal are the round ligaments, eventually attaching to the subcutaneous tissue of the mons pubis. The peritoneum enfolding the adnexa (tube, round ligament, and ovary) is referred to as the broad ligament, which separates the pelvic cavity into an anterior and posterior component.The peritoneal reflections in the pelvis anterior and pos-terior to the uterus are referred to as the anterior and posterior cul-de-sacs. The latter is also called the pouch or cul-de-sac of Douglas. On transverse section, seven avascular, and therefore important, surgical planes can be identified (Fig. 41-6). These include the right and left lateral paravesical and right and left pararectal spaces, and from anterior to posterior, the retropubic or prevesical space of Retzius and the vesicovaginal, rectovagi-nal, and retrorectal or presacral spaces.These avascular tissue planes are often

1	spaces, and from anterior to posterior, the retropubic or prevesical space of Retzius and the vesicovaginal, rectovagi-nal, and retrorectal or presacral spaces.These avascular tissue planes are often preserved and provide safe surgical access when the intraperitoneal pelvic anatomy is distorted by tumor, endometriosis, adhesions, or infection. Utilizing the avascular retroperitoneal planes, the ure-ter can be traced into the pelvis as it crosses the distal common iliac arteries laterally into the pararectal space and then courses inferior to the ovarian arteries and veins until crossing under the uterine arteries into the paravesical space just lateral to the cervix. After traveling to the cervix, the ureters course down-ward and medially over the anterior surface of the vagina before entering the base of the bladder in the vesicovaginal space.Vasculature and Nerves of the PelvisThe rich blood supply to the pelvis arises largely from the internal iliac arteries except for the middle

1	the base of the bladder in the vesicovaginal space.Vasculature and Nerves of the PelvisThe rich blood supply to the pelvis arises largely from the internal iliac arteries except for the middle sacral artery originating at the aortic bifurcation and the ovarian arteries originating from the abdominal aorta. There is also collateral flow and anastomo-ses to the pelvic vessels from the inferior mesenteric artery. The internal iliac, or hypogastric, arteries divide into anterior and pos-terior branches. The latter supply lumbar and gluteal branches. From the anterior division of the hypogastric arteries arise the Prevesical spaceParavesical spaceVesicovaginalspaceVesicouterine ligamentCardinal ligamentUterosacralligamentRetrovaginal spaceRetrorectal spaceSacrumRectumPararectal spaceCervicalfasciaCervixVesicalfasciaBladderPubovesical ligamentFigure 41-6. The avascular spaces of the female pelvis.obturator, uterine, pudendal, middle rectal, inferior gluteal, along with superior and middle

1	ligamentFigure 41-6. The avascular spaces of the female pelvis.obturator, uterine, pudendal, middle rectal, inferior gluteal, along with superior and middle vesical arteries (see Fig. 41-2).The major motor nerves found in the pelvis are the sci-atic, obturator, and femoral nerves (Fig. 41-3). Also important to the pelvic surgeon are the ilioinguinal, iliohypogastric and genitofemoral nerves, which arise as upper abdominal nerves, but are encountered on the most caudal portion of the anterior abdominal wall and the ventral portion of the external genitalia. Sympathetic fibers course along the major arteries and para-sympathetics form the superior and inferior pelvic plexus. The pudendal nerve arises from S2–S4 and travels laterally, exiting the greater sciatic foramen, hooking around the ischial spine and sacrospinous ligament, and returning via the greater sciatic foramen. It travels through Alcock’s canal and becomes the sen-sory and motor nerve of the perineum (see Figs. 41-1 and

1	ischial spine and sacrospinous ligament, and returning via the greater sciatic foramen. It travels through Alcock’s canal and becomes the sen-sory and motor nerve of the perineum (see Figs. 41-1 and 41-3). The motor neurons serve the tonically contracting urethral and anal sphincter, and direct branches from the S2–S4 nerves serve the levator ani muscles. During childbirth and other excessive straining, this tethered nerve (along with the levator ani muscles) is subject to stretch injury and is at least partially responsible for many female pelvic floor disorders.EVALUATION AND DIAGNOSISElements of a Gynecologic HistoryA complete history is a seminal part of any assessment (Table 41-1). Many gynecologic diseases can present with broad constitutional symptoms, occur secondary to other conditions, or be related to medications. A full history should include particular attention to family history, organ system history, including breast, gastrointestinal, and urinary tract symptoms, and

1	or be related to medications. A full history should include particular attention to family history, organ system history, including breast, gastrointestinal, and urinary tract symptoms, and a careful medication, anesthesia, and surgical history. The key elements of a focused gynecologic history include the following:• Date of last menstrual period• History of contraceptive and postmenopausal hormone use• Obstetrical history• Age at menarche and menopause (method of menopause, [e.g., drug, surgical])• Menstrual bleeding pattern• History of pelvic assessments, including cervical smear and HPV DNA results• History of pelvic infections, including HPV and HIV status• Sexual history• Prior gynecologic surgery(s)The Gynecologic ExaminationFor many young women, their gynecologist is their primary care physician. When that is the case, it is necessary that a full medical and surgical history be taken and that, in addition to the pelvic examination, the minimum additional examination should

1	care physician. When that is the case, it is necessary that a full medical and surgical history be taken and that, in addition to the pelvic examination, the minimum additional examination should include assessment of the thyroid, breasts, and cardiopul-monary system. Screening, reproductive counseling, and age-appropriate health services should be available to women of all ages with or without a routine pelvic examination, but the deci-sion to proceed with regular, annual pelvic examinations in oth-erwise healthy women is controversial.2,3 The U.S. Preventive Services Task Force recently evaluated the current evidence regarding the balance of benefits and harms of performing screening pelvic examinations in asymptomatic, nonpregnant adult women and concluded that the evidence is insufficient.32Brunicardi_Ch41_p1783-p1826.indd 178718/02/19 4:34 PM 1788SPECIFIC CONSIDERATIONSPART IIThe pelvic examination starts with a full abdominal exam-ination. Inguinal node evaluation is

1	178718/02/19 4:34 PM 1788SPECIFIC CONSIDERATIONSPART IIThe pelvic examination starts with a full abdominal exam-ination. Inguinal node evaluation is performed before placing the patient’s legs in the dorsal lithotomy position (in stirrups). A flexible, focused light source is essential, and vaginal instru-ments including speculums of variable sizes and shapes (Graves and Pederson), including pediatric sizes, are required to assure that the patient’s anatomy can be fully and comfortably viewed.The external genitalia are inspected first, noting the distri-bution of pubic hair, the skin color and contour, the Bartholin and Skene’s glands, and perianal area. Abnormalities are docu-mented and a map with measurements of abnormalities drawn. A warmed lubricated speculum is inserted into the vagina and gently opened to identify the cervix if present or the vaginal apex if not. To avoid confounding the location of pelvic pain with immediate speculum exam, or if there is a concern that a

1	vagina and gently opened to identify the cervix if present or the vaginal apex if not. To avoid confounding the location of pelvic pain with immediate speculum exam, or if there is a concern that a malignancy is present, careful digital assessment of a vaginal mass and location may be addressed prior to speculum place-ment in order to avoid abrading a vascular lesion and inducing hemorrhage. The speculum would then be inserted just short of the length to the mass in order to view that area directly before advancing. An uncomplicated speculum exam includes examination of the vaginal sidewalls, assessment of secretions, including culture if necessary, and collection of the cervical cytologic specimen and HPV test if indicated (see “Common Screening”).A bimanual examination is performed by placing two fin-gers in the vaginal canal; one finger may be used if patient has significant vaginal atrophy or has had prior radiation with ste-nosis (Fig. 41-7). Carefully and sequentially assess the

1	two fin-gers in the vaginal canal; one finger may be used if patient has significant vaginal atrophy or has had prior radiation with ste-nosis (Fig. 41-7). Carefully and sequentially assess the size and shape of the uterus by moving it against the abdominal hand, and the adnexa by carefully sweeping the abdominal hand down the side of the uterus. The rectovaginal examination, consisting of one finger in the vagina and one in the rectal vault, is used to further examine and characterize the location, shape, fixation, size, and complexity of the uterus, adnexa, cervix, and anterior and posterior cul-de-sacs. The rectovaginal exam also allows examination of the uterosacral ligaments from the back of the uterus sweeping laterally to the rectal finger and the sacrum, as well as assessment of the rectum and anal canal for masses.It is critical that presurgical assessments include a full gen-eral examination. This is particularly important with potential oncologic diagnoses or infectious

1	the rectum and anal canal for masses.It is critical that presurgical assessments include a full gen-eral examination. This is particularly important with potential oncologic diagnoses or infectious issues in order to assure that the proposed surgery is both safe and appropriate. Issues such as sites of metastatic cancer or infection, associated bleeding and/Table 41-1Key elements of the gynecologic historyISSUEELEMENTS TO EXPLOREASSOCIATED ISSUESMenstrual historyAge at menarche, menopause.Bleeding pattern, postmenopausal bleeding, spotting between periods.Any medications (warfarin, heparin, aspirin, herbals, others) or personal or family history that might lead to prolonged bleeding timesIdentifies abnormal patterns related to endocrine, structural, infectious, and oncologic etiologiesObstetrical historyNumber of pregnancies, dates, type of deliveries, pregnancy loss, abortion, complicationsIdentifies predisposing pregnancy for GTD, possible surgical complicationsSexual

1	etiologiesObstetrical historyNumber of pregnancies, dates, type of deliveries, pregnancy loss, abortion, complicationsIdentifies predisposing pregnancy for GTD, possible surgical complicationsSexual historyPartners, practices, protection; pregnancy intentionGuide the assessment of patient risk, risk-reduction strategies, the determination of necessary testing, and the identification of anatomical sites from which to collect specimens for STD testingInfectious diseasesSexually transmitted diseases and treatment and/or testing for theseAlso need to explore history of other GI diseases that may mimic STD (Crohn’s, diverticulitis)Contraceptive historyPresent contraception if appropriate, prior use, type and durationConcurrent pregnancy with procedure or complications of contraceptivesCytologic screeningFrequency, results (normal, prior abnormal Pap), any prior surgery or diagnoses, HPV testing historyProlonged intervals increase risk of cervical cancerRelationship to anal, vaginal, vulvar

1	screeningFrequency, results (normal, prior abnormal Pap), any prior surgery or diagnoses, HPV testing historyProlonged intervals increase risk of cervical cancerRelationship to anal, vaginal, vulvar cancersPrior gynecologic surgeryType (laparoscopy, vaginal, abdominal); diagnosis (endometriosis? ovarian cysts? tubo-ovarian abscess?); actual pathology if possibleAssess present history against this background (for example, granulosa cell pathology, is it now recurrent?)Pain historySite, location, relationship (with urination, with menses, with intercourse at initiation or deep penetration, with bowel movements), referralAssesses relationship to other organ systems, and potential involvement of these with process. Common examples presenting as pelvic pain, ureteral stone, endometriosis with bowel involvement, etcBrunicardi_Ch41_p1783-p1826.indd 178818/02/19 4:34 PM 1789GYNECOLOGYCHAPTER 41or clotting issues and history, and drug exposure, allergies, and current medications must be

1	bowel involvement, etcBrunicardi_Ch41_p1783-p1826.indd 178818/02/19 4:34 PM 1789GYNECOLOGYCHAPTER 41or clotting issues and history, and drug exposure, allergies, and current medications must be addressed.Commonly Used Testinga-Human Chorionic Gonadotropin Testing. Qualitative uri-nary pregnancy tests for human chorionic gonadotropin (b-hCG) are standard prior to any surgery in a woman of reproductive age and potential, regardless of contraception history. In addition, serum quantitative b-hCG testing is appropriate for evaluation of suspected ectopic pregnancy, gestational trophoblastic dis-ease, or ovarian mass in a young woman. In the case of ectopic pregnancy, serial levels are required when a pregnancy cannot be identified in the uterine cavity by imaging. As a general rule, 85% of viable, very early intrauterine pregnancies will have at least a 66% rise in the b-hCG level over 48 hours.Table 41-2Features of common causes of vaginitis BACTERIAL VAGINOSISVULVOVAGINAL

1	85% of viable, very early intrauterine pregnancies will have at least a 66% rise in the b-hCG level over 48 hours.Table 41-2Features of common causes of vaginitis BACTERIAL VAGINOSISVULVOVAGINAL CANDIDIASISTRICHOMONIASISPathogenAnaerobic organismsCandida albicansTrichomonas vaginalis% of vaginitis403020pH>4.5<4.5>4.5Signs and symptomsMalodorous, adherent dischargeWhite discharge, vulvar erythema, pruritus, dyspareuniaMalodorous purulent discharge, vulvovaginal erythema, dyspareuniaWet mountClue cellsPseudohyphae or budding yeasts in 40% of casesMotile trichomonadsKOH mount Pseudohyphae or budding yeasts in 70% of cases Amine test+−−TreatmentMetronidazole 500 mg twice a day for 7 d or 2 g single dose, metronidazole or clindamycin vaginal creamOral fluconazole 150 mg single dose, vaginal antifungal preparationsMetronidazole 2 g single dose and treatment of partner+ = positive; − = negative; KOH = potassium hydroxide.Figure 41-7. Bimanual abdominovaginal palpation of the

1	dose, vaginal antifungal preparationsMetronidazole 2 g single dose and treatment of partner+ = positive; − = negative; KOH = potassium hydroxide.Figure 41-7. Bimanual abdominovaginal palpation of the uterus.Microscopy of Vaginal Discharge. During a speculum exam, a cotton-tipped applicator is used to collect the vaginal dis-charge; it is smeared on a slide with several drops of 0.9% nor-mal saline to create a saline wet mount. A cover slide is placed and the slide is evaluated microscopically for the presence of mobile trichomonads (Trichomonas vaginalis) or clue cells (epithelial cells studded with bacteria, seen in bacterial vagi-nosis; Table 41-2). A potassium hydroxide (KOH) wet mount is the slide application of the collected vaginal discharge with 10% KOH; this destroys cellular elements. The test is posi-tive for vaginal candidiasis when pseudohyphae are seen (see Table 41-2).Chlamydia/Gonorrhea Testing. Nucleic acid amplification testing (NAAT) has emerged as the diagnostic

1	The test is posi-tive for vaginal candidiasis when pseudohyphae are seen (see Table 41-2).Chlamydia/Gonorrhea Testing. Nucleic acid amplification testing (NAAT) has emerged as the diagnostic test of choice for N gonorrhea and C trachomatis. A vaginal swab, endocervical swab, and/or urine sample, can be used for this test.Cervical Cancer Screening and Prevention. HPV infection is required for the development of epithelial cervical carcino-mas (squamous and adenocarcinomas), and HPV DNA can be identified in virtually all primary cervical malignancies. HPV is a ubiquitous double-stranded DNA virus commonly acquired in the female lower genital tract through sexual contact. After entry into the cell, the HPV protein E6 degrades the tumor sup-pressor p53, resulting in deregulation of cell cycle arrest. E7 inactivates the tumor suppressor RB and releases E2F transcrip-tion factors, causing cellular hyperproliferation. More than 100 HPV types have been identified, and up to 40 of these

1	cycle arrest. E7 inactivates the tumor suppressor RB and releases E2F transcrip-tion factors, causing cellular hyperproliferation. More than 100 HPV types have been identified, and up to 40 of these subtypes infect the anogenital region. At least 12 are considered high-risk or oncogenic, and HPV genotypes 16 and 18 cause approxi-mately 70% of cervical cancers worldwide.4Recent cervical cytology guidelines have increased the intervals between screenings for most women given the known natural history of HPV-related cervical dysplasia progression to cancer and the high negative predictive value of a negative HPV test.6 The current recommendations call for cervical smear screening every 3 to 5 years in women ages 21 to 65 years. If an Brunicardi_Ch41_p1783-p1826.indd 178918/02/19 4:34 PM 1790SPECIFIC CONSIDERATIONSPART IIHPV test performed at the same time also is negative, test-ing should be repeated every 5 years for women ages 30 to 65 years. Screening is not recommended for women

1	1790SPECIFIC CONSIDERATIONSPART IIHPV test performed at the same time also is negative, test-ing should be repeated every 5 years for women ages 30 to 65 years. Screening is not recommended for women age older than 65 or without a cervix (prior hysterectomy) unless they have a history of high-grade precancerous lesions. Women with a history of cervical dysplasia, HPV infection, or cervical cancer need more frequent screening based on their diagnosis. Primary high-risk HPV (hrHPV) screening is also an acceptable alterna-tive to cytologic screening for women ages 30-65 because of an increased detection of high-grade squamous intraepithelial lesion (HSIL) and increased negative predictive value.6HPV Vaccine. Three HPV vaccines have been approved by the U.S. Food and Drug Administration (FDA).7 In 2006, a quad-rivalent (4vHPV) vaccine was approved that targets HPV 16 and 18, which cause 70% of cervical cancers, and HPV geno-types 6 and 11, which cause 90% of genital warts. In Decem-ber

1	In 2006, a quad-rivalent (4vHPV) vaccine was approved that targets HPV 16 and 18, which cause 70% of cervical cancers, and HPV geno-types 6 and 11, which cause 90% of genital warts. In Decem-ber 2014, a nine-valent vaccine (9cHPV) was introduced to replace the 4vHPV vaccine, which includes protection against the HPV strains covered by the first generation of 4vHPV as well as five other HPV strains responsible for 20% of cervical cancers (HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58).7 The 9vHPV may be used to continue or complete a series started with a different HPV vaccine product. Vaccination with 9vHPV after completion of 4vHPV at least 12 months earlier is safe and may provide protection against additional HPV strains. A biva-lent vaccine that targets HPV genotypes 16 and 18 with a dif-ferent adjuvant that may have led to higher immunogenicity was approved in 2009 but is no longer marketed in the United States.Vaccination generates high concentrations of neutralizing antibodies to

1	adjuvant that may have led to higher immunogenicity was approved in 2009 but is no longer marketed in the United States.Vaccination generates high concentrations of neutralizing antibodies to HPV L1 protein, the antigen in all HPV vaccines. The vaccines are highly immunogenic, activating both humoral and cellular immune responses. Multiple randomized clinical trials have demonstrated nearly 100% efficacy in the preven-tion of the HPV subtype-specific precancerous cervical cell changes.7,8 These major clinical trials have used prevention of HSIL as the efficacy endpoints. Vaccination does not protect women who are already infected with HPV-16 or -18 at the time of vaccination.Current recommendations include HPV vaccination for boys and girls at age 11 or 12 years. (Vaccination can be started at age 9.) The Advisory Committee on Immunization Prac-tices (ACIP) also recommends vaccination for females aged 13 through 26 years and males aged 13 through 21 years not adequately vaccinated

1	at age 9.) The Advisory Committee on Immunization Prac-tices (ACIP) also recommends vaccination for females aged 13 through 26 years and males aged 13 through 21 years not adequately vaccinated previously. Catch-up vaccination is also recommended through age 26 years for gay, bisexual, and other men who have sex with men, transgender people, and for immu-nocompromised persons (including those with HIV infection) not adequately vaccinated previously.8 Two doses are given 6 to 12 months apart for patients with an intact immune system, age less than 15 years; three doses are recommended for those ages 15 to 26 years and immunocompromised persons.10 Cervical cancer screening continues to play an important role in detection and treatment of premalignant cervical lesions and prevention of cervical cancer in these high-risk patients and is currently recommended following HPV vaccination.Serum Cancer Antigen 125. Cancer antigen (CA) 125 is a large membrane glycoprotein belonging to the wide

1	cancer in these high-risk patients and is currently recommended following HPV vaccination.Serum Cancer Antigen 125. Cancer antigen (CA) 125 is a large membrane glycoprotein belonging to the wide mucin family commonly used as a tumor marker in patients known to have ovarian cancer. An elevated CA-125 in the patient without known ovarian cancer should be interpreted in conjunction with patient information and symptoms as well as imaging. In the setting of an adnexal mass, the serum CA-125 test may help with triage of a patient to the appropriate surgical management. The test should be used with caution as it is a nonspecific test and may be elevated with multiple benign conditions including endometriosis, fibroids, infection, and pregnancy and may even vary with the menstrual cycle. For these reasons, the CA-125 test is less useful in the premenopausal woman for triaging an adnexal mass. In the postmenopausal woman, a CA-125 greater than 35 in the setting of a complex adnexal mass

1	these reasons, the CA-125 test is less useful in the premenopausal woman for triaging an adnexal mass. In the postmenopausal woman, a CA-125 greater than 35 in the setting of a complex adnexal mass merits referral of the patient to a gynecologic oncologist.10Common Office Procedures for DiagnosisVulvar/Vaginal Biopsy. Any abnormal vulvar or vaginal lesion including skin color changes, raised lesions, or ulcer-ations should be biopsied. Local infiltration with local anes-thetic is followed by a 3to 5-mm punch biopsy appropriate to the lesion. The specimen is elevated with Adson forceps and cut from its base with scissors. The vaginal biopsy can sometimes be difficult to perform because of the angle of the lesion. After injection with local anesthetic, traction of the area with Allis forceps and direct resection of the lesion with scissors or cervi-cal biopsy instrument (Schubert, Kevorkian, etc) can achieve an adequate biopsy.Colposcopy and Cervical Biopsy. In cases of an abnormal Pap

1	and direct resection of the lesion with scissors or cervi-cal biopsy instrument (Schubert, Kevorkian, etc) can achieve an adequate biopsy.Colposcopy and Cervical Biopsy. In cases of an abnormal Pap smear cytology or positive HPV testing, a colposcopy is performed for a histologic evaluation. A colposcope is used to achieve 2x to 15x magnification of the cervix. Once the cer-vix is visualized, cervical mucus, if present, is removed, and then 3% acetic acid is applied to the cervix for one minute. This application dehydrates cells and causes dysplastic cells with dense nuclei to appear white. The lining of the cervix consists of squamous epithelium on the ectocervix, whereas columnar epithelium lines the endocervical canal. The ectocervix there-fore appears smooth and pale pink in color while the endocervix forms epithelial fronds or “grape-like” structures visible through the colposcope. The junction between columnar and squamous cell types is called the squamocolumnar junction (SCJ),

1	the endocervix forms epithelial fronds or “grape-like” structures visible through the colposcope. The junction between columnar and squamous cell types is called the squamocolumnar junction (SCJ), which in younger women is usually visible on the ectocervix. When columnar epithelium extends onto the ectocervix, it appears as a red zone surrounding the os and is called ectropion or ectopy. The transformation zone (TZ) is the area between mature squa-mous epithelium distally and columnar epithelium proximally, and it is the site of active squamous metaplasia. For colposcopy to be deemed adequate, the entire SCJ must be visualized dur-ing an adequate colposcopy. Areas with acetowhite, punctation, mosaicism, or atypical blood vessels seen during colposcopy may represent dysplasia or cancer and should be biopsied. A green filter enhances visualization of blood vessels by making them appear darker in contrast to the surrounding epithelium.An alternative to dilute acetic acid is Lugol’s

1	and should be biopsied. A green filter enhances visualization of blood vessels by making them appear darker in contrast to the surrounding epithelium.An alternative to dilute acetic acid is Lugol’s solution—a concentrated solution of iodine that reacts with the glycogen in normal squamous epithelium to make it appear dark brown. High-grade CIN lesions have low amounts of glycogen because the epithelium is poorly differentiated, and hence they do not turn brown with Lugol’s solution. This is termed Lugol’s nonstaining or Lugol’s negative. Historically, this used to be referred to as the Schiller’s test. Lugol’s can be useful for determining whether a colposcopically equivocal area warrants biopsy: Lugol’s staining areas are most likely normal epithelium, whereas Lugol’s nonstaining areas may be CIN, metaplasia, or inflammation.Brunicardi_Ch41_p1783-p1826.indd 179018/02/19 4:34 PM 1791GYNECOLOGYCHAPTER 41Endometrial Biopsy. Endometrial sampling should be per-formed before planned

1	may be CIN, metaplasia, or inflammation.Brunicardi_Ch41_p1783-p1826.indd 179018/02/19 4:34 PM 1791GYNECOLOGYCHAPTER 41Endometrial Biopsy. Endometrial sampling should be per-formed before planned hysterectomy if there is a history of bleeding between periods, heavy and/or frequent menstrual peri-ods, or postmenopausal bleeding. A patient with the potential for pregnancy should have a pregnancy test before the procedure. A pipelle endometrial biopsy can be performed in the office and is a cost-effective and safe procedure that is generally well tolerated by patients. The pipelle is a flexible polypropylene suction cannula with an outer diameter of 3.1 mm. The pipelle is inserted through the endocervix after cervical cleaning, and the depth of the uterine cavity is noted. If difficulty in entering the endometrium with the pipelle is encountered, a tenaculum may be used to straighten the cervix and/or an OS-finder may be use-ful in overcoming resistance within the endocervix. The

1	in entering the endometrium with the pipelle is encountered, a tenaculum may be used to straighten the cervix and/or an OS-finder may be use-ful in overcoming resistance within the endocervix. The endo-metrial specimen is obtained by pulling on the plunger within the pipelle, creating a small amount of suction. The pipelle is rotated and pulled back from the fundus to the lower uterine segment within the cavity to access all sides.11 Additional passes may be needed in order to acquire an adequate amount of tis-sue. If office biopsy is not possible due to patient discomfort or cervical stenosis, a dilatation and curettage in the operating room may be indicated depending on the clinical circumstances.Evaluation for Fistula. When a patient presents with copi-ous vaginal discharge, the provider should be concerned about a fistula with the urinary or gastrointestinal tract. A simple office procedure can be performed when there is a concern for a vesi-covaginal fistula. A vaginal tampon is

1	should be concerned about a fistula with the urinary or gastrointestinal tract. A simple office procedure can be performed when there is a concern for a vesi-covaginal fistula. A vaginal tampon is placed followed by instil-lation of sterile blue dye through a transurethral catheter into the bladder; a positive test is blue staining of the tampon. If the test is negative, one can evaluate for a ureterovaginal fistula. The patient is given phenazopyridine, which changes the color of urine to orange. If a tampon placed in the vagina stains orange, the test is positive. Alternatively, the patient can be given an intravenous injection of indigo carmine.Rectal fistula must be considered when a patient reports stool evacuation per vagina. It can be identified in a similar fashion using a large Foley catheter placed in the distal rectum through which dye may be injected, or with the use of an oral charcoal slurry and timed examination. Common areas for fis-tulae are at the vaginal apex, at

1	Foley catheter placed in the distal rectum through which dye may be injected, or with the use of an oral charcoal slurry and timed examination. Common areas for fis-tulae are at the vaginal apex, at the site of a surgical incision, or around the site of a prior episiotomy or perineal repair after a vaginal delivery.BENIGN GYNECOLOGIC CONDITIONSVulvar LesionsPatients presenting with vulvar symptoms should be carefully interviewed and examined, and a vulvar biopsy should be obtained whenever the diagnosis is in question, the patient does not respond to treatment, or premalignant and malignant disease is suspected. Vulvar conditions such as contact derma-titis, atrophic vulvovaginitis, lichen sclerosis, lichen planus, lichen chronicus simplex, Paget’s disease, Bowen’s disease, and invasive vulvar cancer are common particularly in postmeno-pausal women. Systemic diseases like psoriasis, eczema, Crohn’s disease, Behçet’s disease, vitiligo, and seborrheic der-matitis may also involve the

1	vulvar cancer are common particularly in postmeno-pausal women. Systemic diseases like psoriasis, eczema, Crohn’s disease, Behçet’s disease, vitiligo, and seborrheic der-matitis may also involve the vulvar skin.Leukoplakias. There are three types of leukoplakia, a flat white abnormality. Lichen sclerosis is the most common cause of leukoplakia.12 There are two peaks of onset: prepubertal girls and perimenopausal or postmenopausal women.13 Classically, it results in a figure-of-eight pattern of white epithelium around the anus and vulva resulting in variable scarring and itching, and less commonly pain. Diagnosis is confirmed with biopsy, and treatment consists of topical steroids. An established association between lichen sclerosis and vulvar squamous cell carcinoma estimates risk of malignant transformation up to 5%.13Lichen planus is a cause of leukoplakia with an onset in the fifth and sixth decade of life. Lichen planus, in contrast to lichen sclerosis which is limited to the

1	malignant transformation up to 5%.13Lichen planus is a cause of leukoplakia with an onset in the fifth and sixth decade of life. Lichen planus, in contrast to lichen sclerosis which is limited to the vulva and perianal skin, can involve the vagina and oral mucosa, and erosions occur in the majority of patients leading to a variable degree of scarring. Patients usually have a history and dysuria and dyspareunia, and complain of a burning vulvar pain. Histology is not specific, and biopsy is recommended. Treatment is with topical steroids. Systemic steroids are indicated for severe and/or unresponsive cases.Lichen simplex chronicus is the third cause of leukoplakia, but is distinguished from the other lichen diseases by epidermal thickening, absence of scarring, and a severe intolerable itch.13 Intense scratching is common, and contributes to the severity of the symptoms and predisposes the cracked skin to infections. Treatment consists of cessation of the scratching which some-times

1	Intense scratching is common, and contributes to the severity of the symptoms and predisposes the cracked skin to infections. Treatment consists of cessation of the scratching which some-times requires sedation, elimination of any allergen or irritant, suppression of inflammation with potent steroid ointments, and treatment of any coexisting infections.Bartholin’s Cyst or Abscess. Bartholin’s glands, great ves-tibular glands, are located at the vaginal orifice at the four and eight o’clock positions; they are rarely palpable in normal patients. They are lined with cuboidal epithelium and secrete mucoid material to keep the vulva moist. Their ducts are lined with transitional epithelium, and their obstruction secondary to inflammation may lead to the development of a Bartholin’s cyst or abscess. Bartholin’s cysts or abscesses are usually symptom-atic and are easily diagnosed on examination. Infections are usu-ally polymicrobial. Treatment consists of incision and drainage and placement

1	Bartholin’s cysts or abscesses are usually symptom-atic and are easily diagnosed on examination. Infections are usu-ally polymicrobial. Treatment consists of incision and drainage and placement of a Word catheter, a small catheter with a bal-loon tip, for 2 to 3 weeks to allow for formation and epitheliali-zation of a new duct. Recurrent cysts or abscesses may require marsupialization, but on occasion these necessitate excision of the whole gland. Marsupialization is performed by incising the cyst or abscess wall and securing its lining to the skin edges with interrupted sutures.14 Cysts or abscesses that fail to resolve after drainage and those occurring in patients over 40 years old should be biopsied to exclude malignancy.Molluscum Contagiosum. Molluscum contagiosum presents with dome-shaped papules and are caused by the poxvirus. The papules are usually 2 to 5 mm in diameter and classically have a central umbilication. They are spread by direct skin contact, and present on the

1	papules and are caused by the poxvirus. The papules are usually 2 to 5 mm in diameter and classically have a central umbilication. They are spread by direct skin contact, and present on the vulva, as well as abdomen, trunk, arms, and thighs. Lesions typically clear in several months, but they can be treated with cryotherapy, curettage, or cantharidin, a topical blistering agent.Genital Ulcers. The frequency of the infectious etiologies of genital ulcers varies by geographic location. The most common causes of sexually transmitted genital ulcers in young adults in the United States are, in descending order of prevalence, herpes simplex virus (HSV), syphilis, and chancroid.15 Other infec-tious causes of genital ulcers include lymphogranuloma vene-reum and granuloma inguinale. Noninfectious etiologies include Behçet’s disease, neoplasms, and trauma. Table 41-3 outlines a rational approach to their evaluation and diagnosis.3Brunicardi_Ch41_p1783-p1826.indd 179118/02/19 4:34 PM

1	etiologies include Behçet’s disease, neoplasms, and trauma. Table 41-3 outlines a rational approach to their evaluation and diagnosis.3Brunicardi_Ch41_p1783-p1826.indd 179118/02/19 4:34 PM 1792SPECIFIC CONSIDERATIONSPART IIVulvar Condyloma. Condylomata acuminata (anogenital warts) are viral infections caused by HPV.16 Genital infection with HPV is the most common sexually transmitted infection in the United States today. HPV 6 and 11 are the most common low-risk types and are implicated in 90% of cases of genital warts.17 Women with immunosuppression due to HIV or solid organ transplant are at higher risk of vulvar condyloma than immunocompetent women.18,19 Genital warts are skin-colored or pink and range from smooth flattened papules to verrucous papilliform lesions. Lesions may be single or multiple and extensive. Diagnosis should be confirmed with biopsy as verru-cous vulvar cancers can be mistaken for condylomata.20 If small, self-administered topical imiquimod 5% cream or

1	single or multiple and extensive. Diagnosis should be confirmed with biopsy as verru-cous vulvar cancers can be mistaken for condylomata.20 If small, self-administered topical imiquimod 5% cream or trichloroace-tic acid for in-office applications may be tried. Extensive lesions may require surgical modalities that include cryotherapy, laser ablation, cauterization, and surgical excision.Paget’s Disease of the Vulva. Paget’s disease of the vulva is an intraepithelial disease of unknown etiology that affects Table 41-3Clinical features of genital ulcers syndromes HERPESSYPHILISCHANCROIDLYMPHOGRANULOMA VENEREUMGRANULOMA INGUINALE (DONOVANOSIS)PathogenHSV type 2 and less commonly HSV type 1Treponema palladiumHaemophilus ducreyiChlamydia trachomatis L1-L3Calymmato-bacterium granulomatisIncubation period2–7 days2–4 weeks (1–12 weeks)1–14 days3 days–6 weeks1–4 weeks (up to 6 months)Primary lesionVesiclePapulePapule or pustulePapule, pustule, or vesiclePapuleNumber of lesionsMultiple, may

1	period2–7 days2–4 weeks (1–12 weeks)1–14 days3 days–6 weeks1–4 weeks (up to 6 months)Primary lesionVesiclePapulePapule or pustulePapule, pustule, or vesiclePapuleNumber of lesionsMultiple, may coalesceUsually oneUsually multiple, may coalesceUsually oneVariableDiameter (mm)1–25–152–202–10VariableEdgesErythematousSharply demarcated, elevated, round, or ovalUndermined, ragged, irregularElevated, round, or ovalElevated, irregularDepthSuperficialSuperficial or deepExcavatedSuperficial or deepElevatedBaseSerous, erythematousSmooth, nonpurulentPurulentVariableRed and rough (“beefy”)IndurationNoneFirmSoftOccasionally firmFirmPainCommonUnusualUsually very tenderVariableUncommonLymph-adenopathyFirm, tender, often bilateralFirm, nontender, bilateralTender, may suppate, usually unilateralTender, may suppurate, loculated, usually unilateralPseudo-adenopathyTreatmentacyclovir (ACV) 400 mg POI three times a day for 7–10 days for primary infection and 400 mg PO three times a day for 5 days for

1	may suppurate, loculated, usually unilateralPseudo-adenopathyTreatmentacyclovir (ACV) 400 mg POI three times a day for 7–10 days for primary infection and 400 mg PO three times a day for 5 days for episodic managementPrimary, secondary, and early latent (<1 year): benzathine PCN-G 2.4 million U IM × 1Late latent (>1 year) and latent of unknown duration: benzathine PCN-G 2.4 million units IM every week × 3azithromycin 1 g po or ceftriaxone 250 mg IM × 1 OR Ciprofloxacin 500 mg po twice a day for 3 daysErythromycin base 500 mg po three times a day for 7 daysDoxycycline 100 mg po twice a day × 21 days ORErythromycin base 500 mg po four times a day for 21 daysDoxycycline 100 mg po twice a day for 3 weeks until all lesions have healedSuppressionacyclovir 400 mg po twice a day for those with frequent outbreaks Data from Stenchever M, Droegemueller W, Herbst A, et al: Comprehensive Gynecology, 4th ed. St Louis, MO: Elsevier/Mosby; 2001.Brunicardi_Ch41_p1783-p1826.indd

1	for those with frequent outbreaks Data from Stenchever M, Droegemueller W, Herbst A, et al: Comprehensive Gynecology, 4th ed. St Louis, MO: Elsevier/Mosby; 2001.Brunicardi_Ch41_p1783-p1826.indd 179218/02/19 4:34 PM 1793GYNECOLOGYCHAPTER 41mostly postmenopausal women in their sixth decade of life. It causes chronic vulvar itching and is sometimes associated with an underlying invasive vulvar adenocarcinoma or invasive cancers of the breast, cervix, or gastrointestinal tract. Grossly, the lesion is variable but usually confluent, raised, erythema-tous to violet, and waxy in appearance. Biopsy is required for diagnosis; the disease is intraepithelial and characterized by Paget’s cells with large pale cytoplasm. Treatment is assess-ment for other potential concurrent adenocarcinomas and then surgical removal by wide local resection of the involved area with a 2-cm margin. Free margins are difficult to obtain because the disease usually extends beyond the clinically visible area.21

1	then surgical removal by wide local resection of the involved area with a 2-cm margin. Free margins are difficult to obtain because the disease usually extends beyond the clinically visible area.21 Intraoperative frozen section of the margins can be done; how-ever, Paget’s vulvar lesions have a high likelihood of recurrence even after securing negative resection margins.Vulvar Intraepithelial Neoplasia. Two pathologically dis-tinct premalignant lesions of the vulva are currently recog-nized. Vulvar intraepithelial neoplasia (VIN) of usual type (uVIN) is caused by the HPV virus, tends to occur in younger women, and presents as multifocal disease. VIN of differenti-ated type (dVIN) develops independently of HPV and is typi-cally unifocal and seen in postmenopausal women. VIN is similar to its cervical intraepithelial neoplasia (CIN) counterpart in the cervix. In 2012, the pathologic terminology of HPV-related disease in the anogenital region was harmonized into a two-tier system where

1	its cervical intraepithelial neoplasia (CIN) counterpart in the cervix. In 2012, the pathologic terminology of HPV-related disease in the anogenital region was harmonized into a two-tier system where LSIL is equivalent to uVIN 1 and HSIL encompasses uVIN 2 and uVIN 3.22 Additional risk factors for the development of VIN include HIV infection, immunosup-pression, smoking, vulvar dermatoses such as lichen sclerosis, CIN, and a history of cervical cancer. Vulvar pruritus is the most common complaint in women with symptoms. Lesions may be vague or raised, and they may be velvety with sharply demar-cated borders. Diagnosis is made with a vulvar skin biopsy and multiple biopsies are sometimes necessary. Evaluation of the perianal and anal area is important as the disease may involve these areas. Once invasive disease is ruled out, treatment usually involves wide surgical excision; however, the treatment approaches may also include 5% imiquimod cream, CO2 laser ablation, or cavitational

1	Once invasive disease is ruled out, treatment usually involves wide surgical excision; however, the treatment approaches may also include 5% imiquimod cream, CO2 laser ablation, or cavitational ultrasonic surgical aspiration (CUSA), and depends on the number of lesions and their severity. When laser ablation is used, a 1-mm depth in hair-free areas is usually sufficient, while hairy lesions require ablation to a 3-mm depth because the hair follicles’ roots can reach a depth of 2.5 mm. Unfortunately, VIN tends to recur in up to 30% of cases, and high-grade lesions will progress to invasive disease in approxi-mately 10% of patients if left untreated.23Vaginal LesionsVaginitis (see Table 41-2). Vulvovaginal symptoms are extremely common, accounting for over 10 million office visits per year in the United States. The causes of vaginal complaints are commonly infectious in origin, but they include a number of noninfectious causes, such as chemicals or irritants, hormone deficiency, foreign

1	the United States. The causes of vaginal complaints are commonly infectious in origin, but they include a number of noninfectious causes, such as chemicals or irritants, hormone deficiency, foreign bodies, systemic diseases, and malignancy. Symptoms include abnormal vaginal discharge, pruritus, irrita-tion, burning, odor, dyspareunia, bleeding, and ulcers. A puru-lent discharge from the cervix should always raise suspicion of upper genital tract infection even in the absence of pelvic pain or other signs.Normal vaginal discharge is white or transparent, thick, and mostly odorless. It increases during pregnancy, with use of estrogen-progestin contraceptives, or at mid-cycle around the time of ovulation. Complaints of foul odor and abnormal vaginal discharge should be investigated. Candidiasis, bacte-rial vaginosis, and trichomoniasis account for 90% of vaginitis cases. The initial workup includes pelvic examination, vagi-nal pH testing, microscopy, vaginal cultures if microscopy is

1	bacte-rial vaginosis, and trichomoniasis account for 90% of vaginitis cases. The initial workup includes pelvic examination, vagi-nal pH testing, microscopy, vaginal cultures if microscopy is normal, and gonorrhea/Chlamydia NAAT (see earlier section, “Common Screening and Testing”).24 The pH of normal vaginal secretions is 3.8 to 4.4, which is hostile to growth of pathogens, and pH greater than or equal to 4.9 is indicative of a bacterial or protozoal infection. Treatment of vaginal infection before anticipated surgery is appropriate, particularly for BV, which may be associated with a higher risk for vaginal cuff infections (Fig. 41-8).Bacterial Vaginosis Bacterial vaginosis (BV) accounts for 50% of vaginal infections. It results from reduction in concentration of the normally dominant lactobacilli and increase in concentration of anaerobic organisms like Gardnerella vaginalis, M hominis, Bacteroides species, and others.25 Diagnosis is made by microscopic demonstration of clue cells.

1	and increase in concentration of anaerobic organisms like Gardnerella vaginalis, M hominis, Bacteroides species, and others.25 Diagnosis is made by microscopic demonstration of clue cells. The discharge typically produces a fishy odor upon addition of KOH (amine or Whiff test). Initial treatment is usually a 7-day course of metronidazole.Vulvovaginal Candidiasis Vulvovaginal candidiasis (VVC) is the most common cause of vulvar pruritus. It is generally caused by C albicans and occasionally by other Candida species. It is common in pregnancy, diabetics, patients taking antibiotics, and in immunocompromised hosts. Initial treatment is usually with topical antifungals, although one dose oral antifungal treatments is also effective.Trichomonas Vaginalis Trichomoniasis is a sexually transmit-ted infection of a flagellated protozoan and can present with malodorous, purulent discharge. It is typically diagnosed with visualization of the trichomonads during saline wet mount microscopy.

1	infection of a flagellated protozoan and can present with malodorous, purulent discharge. It is typically diagnosed with visualization of the trichomonads during saline wet mount microscopy. Initial treatment is usually a 7-day course of metronidazole.Gartner’s Duct Cyst. A Gartner’s duct cyst is a remnant of the Wolffian tract; it is typically found on the lateral vaginal walls. Patients can be asymptomatic or present with complaints of dyspareunia or difficulty inserting a tampon. If symptom-atic, these cysts may be surgically excised or marsupialized. If surgery is planned, preoperative magnetic resonance imaging (MRI) should be obtained to determine the extent of the cyst and verify the diagnosis.Vaginal Condyloma. The etiology and treatment of vaginal condyloma is similar to vulvar condyloma (see earlier section, “Vulvar Condyloma”).Vaginal Intraepithelial Neoplasia. Vaginal intraepithelial neoplasia, or VaIN, is similar to VIN and is classified based on the degree of epithelial

1	(see earlier section, “Vulvar Condyloma”).Vaginal Intraepithelial Neoplasia. Vaginal intraepithelial neoplasia, or VaIN, is similar to VIN and is classified based on the degree of epithelial involvement as mild (I), moderate (II), severe (III), or carcinoma in situ.26 Upwards of 65% to 80% of VaIN or vaginal cancers are associated with HPV infection. Typically, a patient will have a history of cervical dysplasia and a prior hysterectomy. The majority of lesions are located in the upper one-third of the vagina. Lesions are usually asymptomatic and found incidentally on cytological screening. Biopsy at the time of colposcopy is diagnostic and rules out invasive disease. VaIN is treated with laser ablation, surgical excision, or topical 5-FU therapy.4Brunicardi_Ch41_p1783-p1826.indd 179318/02/19 4:34 PM 1794SPECIFIC CONSIDERATIONSPART IICervical LesionsBenign Cervical Lesions. Benign lesions of the cervix include endocervical polyps, nabothian cysts (clear, fluid filled cysts with

1	4:34 PM 1794SPECIFIC CONSIDERATIONSPART IICervical LesionsBenign Cervical Lesions. Benign lesions of the cervix include endocervical polyps, nabothian cysts (clear, fluid filled cysts with smooth surfaces), trauma (such as delivery-related cervi-cal tear or prior cervical surgery), malformation of the cervix, and cervical condyloma. For endocervical polyps, exploration of the base of the polyp with a cotton swab tip to identify that it is cervical and not uterine and to identify the stalk characteris-tics can help identify the appropriate surgical approach. Small polyps with identifiable base can be removed by grasping the polyp with ring forceps and slowly rotating it until separated from its base. Use of loop electroexcisional procedure (LEEP) is appropriate for larger lesions. Laser or other ablative procedures are appropriate for condyloma proven by biopsy.Cervical Intraepithelial Neoplasia. Following HPV expo-sure, dysplastic changes are common. Low grade dysplasia (cer-vical

1	or other ablative procedures are appropriate for condyloma proven by biopsy.Cervical Intraepithelial Neoplasia. Following HPV expo-sure, dysplastic changes are common. Low grade dysplasia (cer-vical intraepithelial neoplasia [CIN] I) can be observed and will most often regress to normal within 2 years. However, for girls or women in whom HPV infection is persistent, progression to high-grade cervical dysplasia (CIN II or III) usually require additional treatment due to the high risk of transformation to malignancy. Excisional procedures serve the therapeutic pur-pose of removal of dysplastic cells, and a diagnostic purpose as histologic review to rule out concomitant early stage cervical cancer can be performed. Either a LEEP or cold knife conization (CKC) may be used for surgical excision of the squamocolum-nar junction (SCJ) and outer endocervical canal. Risks of both procedures include bleeding, postprocedure infection, cervical stenosis, and risk of preterm delivery with

1	excision of the squamocolum-nar junction (SCJ) and outer endocervical canal. Risks of both procedures include bleeding, postprocedure infection, cervical stenosis, and risk of preterm delivery with subsequent pregnan-cies. The benefit of a LEEP is that it can be performed in the office under local anesthesia. A looped wire attachment for a standard monopolar electrosurgical unit is used to perform a LEEP excision. Loops range in a variety of shapes and sizes to accommodate different sizes of cervix. Optimally, one pass of the loop should excise the entire SCJ. Hemostasis of the remain-ing cervix is achieved with the ball electrode and ferrous sulfate paste (Monsel’s solution).A cervical cold knife conization allows for an excision where the margin status is not obscured by cauterized artifact. This may be particularly useful when the endocervical margin is of interest, or in cases of adenocarcinoma in situ and microin-vasive squamous cell carcinoma, where margin status dictates the

1	This may be particularly useful when the endocervical margin is of interest, or in cases of adenocarcinoma in situ and microin-vasive squamous cell carcinoma, where margin status dictates the type and need for future therapy. After injection with dilute vasopressin and the placement of stay sutures at three and nine o’clock on the cervix, a #11 blade is used to circumferentially excise the conical biopsy. Hemostasis is achieved with the cau-tery or Monsel’s solution.Uterine CorpusThe average age of menarche, or first menstrual period, in the United States is 12 years and 5 months. Duration of normal menstruation is between 2 to 7 days, with a flow of less than 80 mL, cycling every 21 to 35 days.27 Nonpregnant patients, who present with heavy bleeding and are 35 years of age and older or have risk factors for endometrial cancer, must be ruled out for malignancy as the first step in their management (see earlier section, “Endometrial Biopsy”).Abnormal Uterine Bleeding. The

1	older or have risk factors for endometrial cancer, must be ruled out for malignancy as the first step in their management (see earlier section, “Endometrial Biopsy”).Abnormal Uterine Bleeding. The classification of abnormal uterine bleeding (AUB) has been recently updated.28 Abnormal uterine bleeding may be heavy (AUB/HMB) or intermenstrual (AUB/IMB) and is further divided into acute and chronic cat-egories. Acute AUB is an episode of heavy bleeding that is of sufficient quantity to require immediate intervention to pre-vent further blood loss. Acute AUB may occur in the setting of chronic AUB. Women with acute AUB should be assessed Vaginal dischargeand/or pruritusInterviewExamWet & KOH mountsVaginal pHMetronidazoleorClindamycinCandidiasisAntifungalsTrichomoniasispH <4.5HyphaeBudding yeastspH >4.5TrichomonadspH >4.5Clue cellsPositive whiff testUlcersPruritic lesionsVaginalatrophyAtrophic vaginitisTopical estrogenBiopsyOral metronidazoleBacterialvaginosisFigure 41-8. Treatment

1	yeastspH >4.5TrichomonadspH >4.5Clue cellsPositive whiff testUlcersPruritic lesionsVaginalatrophyAtrophic vaginitisTopical estrogenBiopsyOral metronidazoleBacterialvaginosisFigure 41-8. Treatment algorithm for vulvovaginitis.Brunicardi_Ch41_p1783-p1826.indd 179418/02/19 4:34 PM 1795GYNECOLOGYCHAPTER 41rapidly to determine acuity, determine most the likely etiol-ogy of bleeding, and choose the appropriate treatment. Chronic AUB is abnormal uterine bleeding present for most of the previ-ous 6 months.The many causes of AUB are further divided into two cat-egories: structural causes and nonstructural causes. Structural causes include polyps, adenomyosis, leiomyomata, and malig-nancy. Nonstructural causes can include coagulopathy, ovulatory dysfunction, endometrial effects, and iatrogenic causes. Clini-cal screening for underlying disorders of hemostasis is recom-mended in women with heavy menses since menarche, and other risk factors such as bleeding with dental work, epistaxis one or

1	Clini-cal screening for underlying disorders of hemostasis is recom-mended in women with heavy menses since menarche, and other risk factors such as bleeding with dental work, epistaxis one or more times per month, or a family history of bleeding symptoms. Poly-, oligo-, and amenorrhea are menstrual cycles of less than 21 days, longer than 35 days, or the absence of uterine bleeding for 6 months or a period equivalent to three missed cycles.Endometrial Polyps. Endometrial polyps are localized hyper-plastic growth of endometrial glands and stroma around a vas-cular core forming sessile or pedunculated projections from the surface of the endometrium.29 Endometrial polyps are rarely neo-plastic (<1%) and may be single or multiple. Many are asymp-tomatic; however, they are responsible for about 25% of cases of abnormal uterine bleeding, usually metrorrhagia. Polyps are common in patients on tamoxifen therapy and in periand post-menopausal women. Up to 2.5% of patients with a polyp may

1	about 25% of cases of abnormal uterine bleeding, usually metrorrhagia. Polyps are common in patients on tamoxifen therapy and in periand post-menopausal women. Up to 2.5% of patients with a polyp may harbor foci of endometrial carcinoma.30 Diagnosis can be made with saline-infused hysterosonography, hysterosalpingogram, or by direct visualization at the time of hysteroscopy. Defini-tive treatment, in the absence of malignancy, involves resection with operative hysteroscopy or by sharp curettage.Adenomyosis. Adenomyosis refers to ectopic endometrial glands and stroma situated within the myometrium. When dif-fuse, it results in globular uterine enlargement secondary to hyperplasia and hypertrophy of the surrounding myometrium. Adenomyosis is very common, tends to occur in parous women, and is frequently an incidental finding at the time of surgery. Symptoms include menorrhagia, dysmenorrhea, and diffuse globular uterine enlargement. MRI typically reveals islands within the myometrium

1	is frequently an incidental finding at the time of surgery. Symptoms include menorrhagia, dysmenorrhea, and diffuse globular uterine enlargement. MRI typically reveals islands within the myometrium with increased signal intensity.31 Defini-tive diagnosis is obtained via hysterectomy and pathologic examination.Uterine Leiomyomas. Leiomyomas, also known colloqui-ally as fibroids, are the most common female pelvic tumor and occurs in response to growth of the uterine smooth muscle cells (myometrium). They are common in the reproductive years, and by age 50. Leiomyomas are described according to their anatomic location (Fig. 41-9) as intramural, subserosal, submu-cosal, pedunculated, and cervical. Rarely, they can be ectopic.27 Most are asymptomatic; however, abnormal uterine bleeding caused by leiomyomas is the most common indication for hys-terectomy in the United States. Other manifestations include pain, pregnancy complications, and infertility. Pain may result from degenerating

1	by leiomyomas is the most common indication for hys-terectomy in the United States. Other manifestations include pain, pregnancy complications, and infertility. Pain may result from degenerating myomas that outgrow their blood supply or from compression of other pelvic organs such as the bowel, bladder, and ureters. Hormonal changes during pregnancy can cause significant enlargement of preexisting myomas, which may lead to significant distortion of the uterine cavity resulting in recurrent miscarriages, fetal malpresentations, intrauterine growth restriction, obstruction of labor or abnormal placenta-tion, and the subsequent need for cesarean delivery, abruption, preterm labor, and pain from degeneration.SubserousPedunculatedSubmucousProlapsedIntercavitaryIntramuralFigure 41-9. Types of uterine myomas.Menorrhagia resulting from leiomyomas can be severe at times, requiring hospitalization or transfusion. Examination typically reveals an enlarged and irregular uterus. Diagnosis is

1	of uterine myomas.Menorrhagia resulting from leiomyomas can be severe at times, requiring hospitalization or transfusion. Examination typically reveals an enlarged and irregular uterus. Diagnosis is usually made by transvaginal ultrasonography. Other diagnos-tic modalities, including MRI, computed tomography (CT), and hysterosalpingogram or saline-infused hysterosalpingography, are especially useful in the cases of submucosal and intrauterine myomas. Management options of leiomyomas are tailored to the individual patient depending on her age and desire for fertil-ity and the size, location, and symptoms of the myomas. Con-servative management options include oral contraceptive pills (OCPs), medroxyprogesterone acetate, GnRH agonists, uterine artery embolization, myomectomy, and hysterectomy.32-34 Uter-ine artery embolization is contraindicated in patients planning future pregnancy and may result in acute degeneration of myo-mas requiring hospitalization for pain control. Myomectomy is

1	Uter-ine artery embolization is contraindicated in patients planning future pregnancy and may result in acute degeneration of myo-mas requiring hospitalization for pain control. Myomectomy is indicated in patients with infertility thought secondary to fibroids and for those with symptomatic fibroids who wish to preserve their reproductive capacity. Hysterectomy is the only definitive therapy. Treatment with GnRH agonists for 3 months prior to surgery may be administered in anemic patients, and it may allow them time to normalize their hematocrit, avoiding transfusions; GnRH also decreases blood loss at hysterectomy and shrinks the myomas by an average of 30%. The latter may make the preferred vaginal surgical approach more feasible.Endometrial Hyperplasia. Endometrial hyperplasia is caused by chronic unopposed hyperestrogenic state (relative absence of progesterone) and is characterized by proliferation of endo-metrial glands resulting in increased gland-to-stroma ratio. It can be

1	by chronic unopposed hyperestrogenic state (relative absence of progesterone) and is characterized by proliferation of endo-metrial glands resulting in increased gland-to-stroma ratio. It can be asymptomatic or, more commonly, result in abnormal vaginal bleeding. Hyperplasia can be either simple or complex, based on the architecture of the glands. Of greater importance is the presence or absence of nuclear atypia, described by the WHO classification.35 A classic retrospective review suggested that untreated endometrial hyperplasia progresses to malig-nancy in 1%, 3%, 8%, and 29% of cases of simple, complex, simple with atypia, and complex hyperplasia with atypia, respectively.36 A more modern prospective study noted that of patients who had complex atypical hyperplasia on endometrial biopsy performed prior to hysterectomy, 42.5% had cancer at the time of hysterectomy.37 Simple and complex hyperplasias can be treated with progestins, and women should have repeat

1	on endometrial biopsy performed prior to hysterectomy, 42.5% had cancer at the time of hysterectomy.37 Simple and complex hyperplasias can be treated with progestins, and women should have repeat Brunicardi_Ch41_p1783-p1826.indd 179518/02/19 4:34 PM 1796SPECIFIC CONSIDERATIONSPART IIendometrial sampling in 3 to 6 months. Atypical hyperplasia is considered a premalignant condition and is treated ideally with simple hysterectomy. If preservation of fertility is desired or surgery is contraindicated, treatment with high-dose progestins such as megesterol acetate 40 to 160 mg per day or with a pro-gesterone IUD usually reverses these lesions. Close follow-up and repeated sampling are necessary.The reliability of the pathologic diagnosis of complex atypical hyperplasia is poor, and better and more objective clas-sifications predictive of malignant endometrial behavior are needed.38 These observations led to the new classification of endometrial intraepithelial neoplasia (EIN). In 2014,

1	more objective clas-sifications predictive of malignant endometrial behavior are needed.38 These observations led to the new classification of endometrial intraepithelial neoplasia (EIN). In 2014, the WHO Classification system introduced the diagnosis of EIN into a binary system that aligns with clinical options: hyperplasias are divided into hyperplasia without atypia, and EIN. The new clas-sification is intended to have clinical implications: hyperplasia without atypia may be managed with hormonal therapy, while EIN should be considered a premalignant lesion.The new classification moves the focus away from cyto-logic atypia and puts more emphasis on glandular crowding and complexity. While atypia is still important, proliferations can get to EIN without it. For example, the diagnosis of EIN includes cases that lack overt cytologic atypia but show a distinct popu-lation from the background epithelium. Morphometric data is utilized to calculate the so-called D-score, which takes into

1	EIN includes cases that lack overt cytologic atypia but show a distinct popu-lation from the background epithelium. Morphometric data is utilized to calculate the so-called D-score, which takes into account percentage of stroma, glandular complexity, and gland pleomorphism in an objective manner. A D-score of less than 1 connotes a high rate of progression to endometrial cancer and therefore a diagnosis of EIN. EIN is more predictive than CAH of underlying endometrial malignancy.39 Most pathology reports are provided with both diagnoses as the transition is made.Clinicians should be careful to not confuse EIN with endometrial intraepithelial carcinoma (EIC). EIC is a precursor lesion for serous endometrial cancer, and women with a preop-erative diagnosis of EIC should always have hysterectomy and appropriate surgical staging performed.Procedures Performed for Structural Causes of Abnormal Uterine BleedingDilation and Curettage. The patient is placed on the operat-ing table in a

1	and appropriate surgical staging performed.Procedures Performed for Structural Causes of Abnormal Uterine BleedingDilation and Curettage. The patient is placed on the operat-ing table in a lithotomy position, and the vagina and cervix are prepared as for any vaginal operation. The cervix is grasped on the anterior lip with a tenaculum. Some traction on the cervix is necessary to straighten the cervical canal and the uterine cavity. A uterine sound is inserted into the uterine cavity, and the depth of the uterus is noted. The cervical canal is then systematically dilated beginning with a small cervical dilator. Most operations can be performed after the cervix is dilated to accommodate a number 8 or 9 Hegar dilator or its equivalent. Dilatation is accomplished by firm, constant pressure with a dilator directed in the axis of the uterus (Fig. 41-10). The endometrial cavity is then systemically scraped with a uterine curette. Using the larg-est curette available or suction curettage is a

1	a dilator directed in the axis of the uterus (Fig. 41-10). The endometrial cavity is then systemically scraped with a uterine curette. Using the larg-est curette available or suction curettage is a safer choice than a small curette, which tends to cause perforation with less pres-sure. Uterine perforation is the major complication of dilatation and curettage, diagnosed when the operator finds no resistance to a dilator or curette. Laparoscopy can identify any damage to vessels or bowel if clinically indicated. A uterine perforation through the fundus of the uterus with a dilator or uterine sound is low risk for injury and may be observed without laparoscopy if there is no significant vaginal bleeding noted.CommonductstonesearcherBACFigure 41-10. Dilatation and curettage of the uterus.Brunicardi_Ch41_p1783-p1826.indd 179618/02/19 4:34 PM 1797GYNECOLOGYCHAPTER 41Hysteroscopy. Hysteroscopy, like laparoscopy, has gained widespread support for use both for diagnosis and treatment of

1	179618/02/19 4:34 PM 1797GYNECOLOGYCHAPTER 41Hysteroscopy. Hysteroscopy, like laparoscopy, has gained widespread support for use both for diagnosis and treatment of intrauterine pathology and for ablation of the endometrium as an alternative to hysterectomy for the treatment of abnormal uterine bleeding. Hysteroscopes can have an objective lens that is offset from the long axis from 0° to 30°.Diagnostic Hysteroscopy The diagnostic hysteroscope usu-ally has an external diameter of 5 mm. Some diagnostic sheaths allow passage of flexible instruments for biopsy and cutting. Following dilation of the cervix, a diagnostic hysteroscope is placed, and the uterine cavity is distended with the media of choice. Inspection of the cavity includes identifying the uter-ine fundus, cornua, and any other anomalies to include polyps, leiomyomas, or uterine septum. A dilation and curettage or directed polypectomy with forceps can be performed following identification.Newer office hysteroscopes can

1	anomalies to include polyps, leiomyomas, or uterine septum. A dilation and curettage or directed polypectomy with forceps can be performed following identification.Newer office hysteroscopes can be used to perform hyster-oscopy in the office. A paracervical block is placed, and a flex-ible 3-mm hysteroscope is used. Generally, office hysteroscopy is performed only for diagnostic purposes.Operative Hysteroscopy An operative hysteroscope is wider than a diagnostic hysteroscope and usually has an inte-gral unipolar or bipolar resecting loop identical to a urologic resectoscope. Electrolyte contacting media are incompatible with conventional monopolar resectocopic instruments, but electrolyte-free isotonic solutions such as 5% mannitol, 1.5% glycine and 3% sorbitol are acceptable. Large volume deficits have been associated with secondary hyponatremic hypervol-emia due to their metabolism to free water after intravasation. Fluid-management systems are available to monitor the amount of

1	deficits have been associated with secondary hyponatremic hypervol-emia due to their metabolism to free water after intravasation. Fluid-management systems are available to monitor the amount of distension media lost during hysteroscopy in order to prevent fluid overload. When fluid deficits reach 1000 to 1500 mL, the procedure should be terminated, and the patient’s serum elec-trolytes should be assessed.40 If bipolar instruments are used, resectoscopic instruments can be used without the unique issues related to electrolyte-free hypotonic solutions.43Hysteroscopic Polypectomy Removal of an intrauterine polyp can be performed following diagnostic hysteroscopy through grasping with a polyp forceps. Alternatively, using operative hysteroscopy the base of the polyp is incised with hysteroscopic scissors. The hysteroscope, sleeve, and polyp are removed simultaneously because most polyps will not fit through the operating channel. Extremely large polyps may have to be removed piecemeal.

1	scissors. The hysteroscope, sleeve, and polyp are removed simultaneously because most polyps will not fit through the operating channel. Extremely large polyps may have to be removed piecemeal. Any residual base of the polyp may be removed with biopsy forceps.Endometrial Ablation A common treatment for abnormal uterine bleeding in the absence of endometrial hyperplasia is ablation of the endometrium. Historically, this was performed with an operative hysteroscope using an electrosurgical “roller ball,” where the endometrium was destroyed down to the myo-metrium in a systematic fashion. Currently, hysteroscopic endo-metrial ablation has been widely supplanted by various devices, including heated free fluid, cryotherapy, thermal balloon, microwave, and radiofrequency electricity. Most ablation tech-niques result in amenorrhea in approximately half the patients and decreased menstruation in another third of the patients over the first year of therapy.42 Subsequent hysterectomy fol-lowing

1	tech-niques result in amenorrhea in approximately half the patients and decreased menstruation in another third of the patients over the first year of therapy.42 Subsequent hysterectomy fol-lowing endometrial ablation is common with rates as high as 40%.43Ablation is not recommended in postmenopausal women.Myomectomy Myomectomy (Fig. 41-11) is the removal of fibroids, and it can be treatment for abnormal uterine bleeding, bulk symptoms, or infertility. Hemostasis during myomectomy can be aided medically by direct injection of dilute vasopressin. Submucosal leiomyoma can be removed safely hysteroscopi-cally. Because myoma tissue is relatively dense, a power cut-ting instrument is required. The most common method is use of electrosurgery. Both pedunculated and submucosal fibroids are shaved into small pieces with the hysteroresectoscope. Stalk resection should only be done to release a pedunculated fibroid if it is 10 mm or less in size; larger fibroids are difficult to remove in one

1	into small pieces with the hysteroresectoscope. Stalk resection should only be done to release a pedunculated fibroid if it is 10 mm or less in size; larger fibroids are difficult to remove in one piece without excessive cervical dilatation.44Subserosal, or pedunculated fibroids may require an open or laparoscopic approach depending on the size and location or the leiomyoma. In addition to vasopressin, hemostasis can be further managed through the placement of a Penrose drain around the base of the uterus, pulled through small perforations in the broad ligament lateral to the uterine blood supply on either side and clamped to form a tourniquet for uterine blood flow. An incision is then made through the uterine serosa into the myoma. The pseudocapsule surrounding the tumor is identified, and the tumor is bluntly dissected out with scissors, or bluntly if open. Vessels to the myoma are dessicated with the electrosurgical unit. Several myomas may be removed through a single incision,

1	and the tumor is bluntly dissected out with scissors, or bluntly if open. Vessels to the myoma are dessicated with the electrosurgical unit. Several myomas may be removed through a single incision, depending upon size. The uterine incisions are then closed with absorbable sutures to obliterate the dead space and provide hemostasis. The uterine serosa is closed with a 3-0 absorbable suture, placed subserosally if possible. Because myomectomies are associated with considerable postoperative adhesion formation, barrier techniques are used to decrease adhesion formation.During a laparoscopic myomectomy, hemostasis is assisted by intrauterine injection of dilute vasopressin (10 U in 50 mL) at the site of incision, similar to an open procedure. This is usually performed percutaneously with a spinal needle. Pedunculated leiomyomas can be excised at the base using scissors or a power instrument. Intramural leiomyomas require deep dissection into the uterine tissue, which must be closed

1	a spinal needle. Pedunculated leiomyomas can be excised at the base using scissors or a power instrument. Intramural leiomyomas require deep dissection into the uterine tissue, which must be closed subsequently with laparoscopic suturing techniques. Removing the specimen may require morcellation; this should be performed after placement of the specimen in a bag. Although power morcellators were previously used for this purpose, an FDA warning in 2014 has virtually eliminated their use. Severe complications including damage to surrounding bowels and vascular structures caused by the spinning blade of the morcellator were reported. Multiple reports of benign tissues such as leiomyoma and endometriosis scattering and dispersing onto abdominal organ surfaces lead-ing to inflammation, infection, and intestinal obstruction often requiring additional surgical interventions and treatments were made. The unintentional dissemination of malignant cells wors-ens prognosis if an undiagnosed

1	and intestinal obstruction often requiring additional surgical interventions and treatments were made. The unintentional dissemination of malignant cells wors-ens prognosis if an undiagnosed malignancy (most frequently leiomyosarcoma) was morcellated. Although contained morcel-lation (in a bag) may reduce these risks, informed consent to the patient is prudent.45Total Abdominal Hysterectomy (Fig. 41-12) After the abdomen is entered, the upper abdomen is examined for evi-dence of extrapelvic disease, and a suitable retractor is placed in the abdominal incision. The uterus is grasped at either cornu with clamps and pulled up into the incision. The round ligament is identified and divided. The peritoneal incision is extended from the round ligament to just past the ovarian hilum, lat-eral the infundibulopelvic ligament, if the ovaries are to be removed. The retroperitoneal space is bluntly opened, the ure-ter identified on the medial leaf of the broad ligament, and the

1	hilum, lat-eral the infundibulopelvic ligament, if the ovaries are to be removed. The retroperitoneal space is bluntly opened, the ure-ter identified on the medial leaf of the broad ligament, and the Brunicardi_Ch41_p1783-p1826.indd 179718/02/19 4:34 PM 1798SPECIFIC CONSIDERATIONSPART IIinfundibulopelvic ligament isolated, clamped, cut, and suture-ligated; a similar procedure is carried out on the opposite side. If the ovaries are to be left in situ, the ureter is identified and an opening below the utero-ovarian ligament and fallopian tube created. The fallopian tube and utero-ovarian ligament are clamped, cut, and ligated. The bladder is mobilized by sharply dissecting it free of the anterior surface of the uterus and cervix. Clamps are placed on the uterine vessels at the cervicouterine junction, and the vessels are cut and suture-ligated. The cardinal ligaments are then serially clamped, cut, and ligated. Follow-ing division of the remaining cardinal ligaments, the uterus is

1	junction, and the vessels are cut and suture-ligated. The cardinal ligaments are then serially clamped, cut, and ligated. Follow-ing division of the remaining cardinal ligaments, the uterus is elevated and the vagina clamped. The cervix is amputated from the vagina with scissors or a knife. Sutures are placed at each lateral angle of the vagina, and the remainder of the vagina is closed with a running or interrupted absorbable suture. Pelvic reperitonealization is not necessary.Transvaginal Hysterectomy (Fig. 41-13) Vaginal hysterectomy is the preferred approach in patients in whom the uterus descends and the pubic arch allows enough space for a vaginal operation. A bladder catheter can be placed before the procedure and the patient is placed in a lithotomy position. A weighted vaginal speculum is placed in the vagina, and the cervix is grasped with a tenaculum and pulled in the axis of the vagina. Injection of the cervix and paracervical tissue with analgesic with epinephrine may be

1	is placed in the vagina, and the cervix is grasped with a tenaculum and pulled in the axis of the vagina. Injection of the cervix and paracervical tissue with analgesic with epinephrine may be helpful in defining planes and decreasing obscuring bleeding. A circumferential incision may be made with a scalpel or scissors. The posterior cul-de-sac is identified and entered with scissors. A long, weighted speculum is then placed through this opening into the peritoneal cavity. Metzenbaum scissors are used to dissect anteriorly on the cervix down to the pubocervical-vesical fascia, reflecting the bladder off the lower uterine segment. When the peritoneum of the anterior cul-de-sac is identified, it is entered with the scissors, and a retractor is placed in the defect. The uterosacral ligaments are identified, doubly clamped, cut, and ligated. Serial clamps are placed on the parametrial structures above the uterosacral ligament; these pedicles are cut and ligated. At the cornu of the

1	are identified, doubly clamped, cut, and ligated. Serial clamps are placed on the parametrial structures above the uterosacral ligament; these pedicles are cut and ligated. At the cornu of the uterus, the tube, round ligament, and utero-ovarian ligament of the ovary are doubly clamped and cut. The procedure is carried out usually concurrently on the opposite side, and the uterus is removed. The pelvis is inspected for hemostasis; all bleeding must be meticulously controlled at this point.The pelvic peritoneum is closed with a running purse-string suture incorporating the uterosacral and ovarian pedicles, those that were held. This exteriorizes those areas that might tend to bleed. The sutures attached to the ovarian pedicles are cut. The vagina may be closed with interrupted mattress stitches, ABCDEFFigure 41-11. Myomectomy.Brunicardi_Ch41_p1783-p1826.indd 179818/02/19 4:34 PM 1799GYNECOLOGYCHAPTER 41Figure 41-12. Hysterectomy.BladderBladderRound ligamentRound ligamentFallopian

1	ABCDEFFigure 41-11. Myomectomy.Brunicardi_Ch41_p1783-p1826.indd 179818/02/19 4:34 PM 1799GYNECOLOGYCHAPTER 41Figure 41-12. Hysterectomy.BladderBladderRound ligamentRound ligamentFallopian tubeFallopian tubeOvaryBADCFEOvarian ligamentUterinevesselsUreterUreterCardinalligamentUterusBrunicardi_Ch41_p1783-p1826.indd 179918/02/19 4:34 PM 1800SPECIFIC CONSIDERATIONSPART IIincorporating the uterosacral ligaments into the corner of the vagina with each lateral stitch. On occasion, the uterus, which is initially too large to remove vaginally, may be reduced in size by morcellation (Fig. 41-14). After the uterine vessels have been clamped and ligated, serial wedges are taken from the central portion of the uterus in order to reduce the uterine mass. This procedure will allow the vaginal delivery of even very large uterine leiomyomas.Laparoscopic Hysterectomy The advantages of laparoscopy over laparotomy include decreased postoperative pain, shorter hospital stays, and reduced blood

1	delivery of even very large uterine leiomyomas.Laparoscopic Hysterectomy The advantages of laparoscopy over laparotomy include decreased postoperative pain, shorter hospital stays, and reduced blood loss. Laparoscopy has been used to augment vaginal hysterectomy to avoid laparotomy in patients with known pelvic adhesions, endometriosis, or to ensure removal of the entire ovary if oophorectomy is planned or an adnexal mass is present. Over 20% of benign hysterec-tomies performed in the United States are estimated to be per-formed laparoscopically.46Although multiple variations in technique exist, there are three basic laparoscopic approaches for hysterectomy: lapa-roscopic-assisted vaginal hysterectomy (LAVH), total lapa-roscopic hysterectomy (TLH), and laparoscopic supracervical hysterectomy (LSH). The technically simplest is the LAVH. A multiple-port approach is used to survey the peritoneal cavity, and any pelvic adhesions are lysed. The round ligaments are then occluded and

1	(LSH). The technically simplest is the LAVH. A multiple-port approach is used to survey the peritoneal cavity, and any pelvic adhesions are lysed. The round ligaments are then occluded and divided, and the uterovesical peritoneum and peritoneum lateral to the ovarian ligament are incised. The course of the ureter and any adhesions or implants, such as endometriosis that might place the ureter in the way of the surgical dissection, are carefully dissected. Next, the proximal uterine blood supply is dissected for identification and then occluded with a laparoscopic energy device. When the ova-ries are removed, the infundibulopelvic ligaments containing the ovarian vessels are divided. If the ovaries are conserved, the utero-ovarian ligament and blood vessels are divided and occluded. In many cases, the posterior cul-de-sac is also incised laparoscopically and the uterosacral ligaments separated with an energy device. The amount of dissection that is done prior to the vaginal portion

1	many cases, the posterior cul-de-sac is also incised laparoscopically and the uterosacral ligaments separated with an energy device. The amount of dissection that is done prior to the vaginal portion depends on individual patient characteristics and operator comfort with the vaginal approach, and it may include as little as ovarian and adhesion management to full dissection, including bladder dissection, with only the last vaginal incision done by the vaginal approach. During a TLH, the vaginal inci-sion is performed laparoscopically, and the vaginal incision may be closed with laparoscopic suturing. This procedure is used for the indications listed earlier and also when lack of uterine descent makes the vaginal approach impossible.VaginaVaginaGIHCardinalligamentVaginaFigure 41-12. (Continued)Brunicardi_Ch41_p1783-p1826.indd 180018/02/19 4:34 PM 1801GYNECOLOGYCHAPTER 41During an LSH, the uterine vessels are divided after the bladder is dissected from the anterior uterus. The

1	180018/02/19 4:34 PM 1801GYNECOLOGYCHAPTER 41During an LSH, the uterine vessels are divided after the bladder is dissected from the anterior uterus. The ascending branches of the uterine arteries are occluded, and the entire uterine fundus is amputated from the cervix. The endocervix is either cauterized or cored out. The fundus is then morcellated and removed an abdominal port. The end result is an intact cer-vix, with no surgical dissection performed below the uterine artery. This approach avoids both a large abdominal incision and a vaginal incision. The risks of LSH including subsequent bothersome bleeding from the remaining endometrium or endo-cervix and cancer risk from the residual cervical stump combin-ing with concerns about power morcellation (see earlier section, “Myomectomy”) have made this procedure less attractive.Benign Ovarian and Fallopian Tube LesionsThe most common ovarian benign findings include functional follicular cysts, endometriomas (due to ovarian

1	have made this procedure less attractive.Benign Ovarian and Fallopian Tube LesionsThe most common ovarian benign findings include functional follicular cysts, endometriomas (due to ovarian endometriosis), and serous cystadenomas or cystadenofibromas. These can present with varying degrees or pelvic pain, or sometimes be completely asymptomatic. Ultrasound is the best initial imaging modality for evaluating ovarian abnormalities.Ovarian Cystectomy. When a cystic lesion persists or causes pelvic pain, surgical intervention is usually justified. Perform-ing a cystectomy with ovarian preservation is recommended in women who desire future fertility. Whether the cystectomy is performed laparoscopically or by laparotomy, the procedure is Figure 41-13. Vaginal hysterectomy.Brunicardi_Ch41_p1783-p1826.indd 180118/02/19 4:34 PM 1802SPECIFIC CONSIDERATIONSPART IIinitiated with inspection of the peritoneal cavity, peritoneum, diaphragm, liver, and pelvis. In the absence of signs of

1	180118/02/19 4:34 PM 1802SPECIFIC CONSIDERATIONSPART IIinitiated with inspection of the peritoneal cavity, peritoneum, diaphragm, liver, and pelvis. In the absence of signs of malig-nancy, pelvic washings are obtained, and the ovarian capsule is incised superficially sharply or with the electrosurgical unit. The cyst is shelled out carefully through the incision. During laparos-copy, it is placed in a bag, intact if possible, and the bag opening is brought through a 10-mm port. If a cyst should rupture before removal, contents are aspirated thoroughly, and the cyst wall is removed and sent for pathologic evaluation. The peritoneal cavity is copiously rinsed with Ringer’s lactate solution. This is especially important when a dermoid cyst is ruptured because the sebaceous material can cause a chemical peritonitis unless all the visible oily substance is carefully removed. A cyst may need to be drained to facilitate removal, but only after bag edges are completely out of the abdomen

1	a chemical peritonitis unless all the visible oily substance is carefully removed. A cyst may need to be drained to facilitate removal, but only after bag edges are completely out of the abdomen assuring no leakage within the abdomen. Hemostasis of the ovary is achieved with bipolar electrocoagulation, but the ovary is usually not closed. If there are solid growths within the cyst, it should be sent for frozen section to verify the absence of the malignancy. If malignancy is detected, immediate definitive surgery is recommended.Removal of Adnexa. Indications for removal of adnexae include persistent ovarian cyst, pelvic pain, concern for malig-nancy, and risk reduction surgery in women with genetic predis-position for ovarian or endometrial cancers (BRCA1/2 mutation carrier, Lynch syndrome). In general, the peritoneum lateral to the infundibulopelvic (IP) ligament is incised in a parallel fashion to allow retroperitoneal dissection and identification of the ureter. Once this has been

1	In general, the peritoneum lateral to the infundibulopelvic (IP) ligament is incised in a parallel fashion to allow retroperitoneal dissection and identification of the ureter. Once this has been accomplished, the IP ligament is ligated with suture or an energy source (ultrasonic or bipolar). The remaining posterior leaf of the broad ligament is incised toward the uterus in a direction parallel to the utero-ovarian liga-ment to avoid ureteral injury. The fallopian tube and utero-ovarian ligaments are then ligated with either suture or an energy source. If performed laparoscopically, the specimen(s) is/are removed in a bag as described earlier.Tubal Sterilization. As in diagnostic laparoscopy, a oneor two-port technique can be used. Fallopian tubes are occluded in the mid-isthmic section, approximately 3 cm from the cornua, using clips, elastic bands, or bipolar electrosurgery. With elec-trosurgery, approximately 2 cm of tube should be desiccated. Pregnancy rates after any of these

1	approximately 3 cm from the cornua, using clips, elastic bands, or bipolar electrosurgery. With elec-trosurgery, approximately 2 cm of tube should be desiccated. Pregnancy rates after any of these techniques have been reported Figure 41-14. Uterine morcellation through the vagina.in the range of 3 per 1000 women. Complete removal of the fal-lopian tube (salpingectomy) at the time of tubal sterilization for the purposes of ovarian cancer prevention has recently become more common.47A transvaginal tubal occlusion technique may also be used for tubal sterilization. A routine hysteroscopy is first performed to inspect the cavity and identify the tubal ostia. The tubal insert introducer sheath is then placed into the working channel of the hysteroscope. The insert is then threaded into the fallopian tube. Following this procedure, the patient must undergo a hys-terosalpingogram to confirm tubal occlusion at 3 months post procedure. Prior to the hysterosalpingogram, the patient is

1	the fallopian tube. Following this procedure, the patient must undergo a hys-terosalpingogram to confirm tubal occlusion at 3 months post procedure. Prior to the hysterosalpingogram, the patient is coun-seled to use a reliable birth control method. Transvaginal tubal sterilization has been associated with perforation of the uterus and/or fallopian tubes, identification of inserts in the abdominal or pelvic cavity, persistent pain, and suspected allergic or hyper-sensitivity reactions.Other Benign Pelvic PathologyChronic Pelvic Pain. Chronic pelvic pain is defined as pain below the umbilicus that has lasted at least 6 months or causes functional disability, requiring treatment. While there can be gastrointestinal and urologic causes of chronic pelvic pain, gynecologic causes are frequently identified. Oftentimes, a surgical evaluation is needed for diagnosis and/or intervention. The most common gynecologic causes of chronic pelvic pain include endometriosis, adenomyosis, uterine

1	identified. Oftentimes, a surgical evaluation is needed for diagnosis and/or intervention. The most common gynecologic causes of chronic pelvic pain include endometriosis, adenomyosis, uterine leiomyomas, and adhesive disease.Endometriosis Endometriosis is the finding of ectopic endo-metrial glands and stroma outside the uterus. It affects 10% of the general population, and it is an incidental finding at the time of laparoscopy in more than 20% of asymptomatic women. Chronic pelvic pain (80%) and infertility (20–50%) are the two most common symptoms.27 The pathophysiology of endometrio-sis is poorly understood; etiologic theories explaining dissemi-nation of endometrial glands include retrograde menstruation, lymphatic and vascular spread of endometrial glands, and coe-lomic metaplasia. Endometriosis commonly involves the ova-ries, pelvic peritoneal surfaces, and uterosacral ligaments. Other possible sites include the rectovaginal septum, sigmoid colon, intraperitoneal organs,

1	Endometriosis commonly involves the ova-ries, pelvic peritoneal surfaces, and uterosacral ligaments. Other possible sites include the rectovaginal septum, sigmoid colon, intraperitoneal organs, retroperitoneal space, ureters, incisional scars, umbilicus, and even the thoracic cavity. Involvement of the fallopian tubes may lead to scarring, blockage, and subse-quent infertility. Ovarian involvement varies from superficial implants to large complex ovarian masses called endometriomas or “chocolate cysts.” Endometriomas are found in approximately one-third of women with endometriosis and are often bilateral.While endometriosis can be totally asymptomatic, com-plaints vary from mild dyspareunia and cyclic dysmenorrhea, to debilitating chronic pelvic pain with dysmenorrhea. Less com-mon manifestations include painful defecation, hematochezia, and hematuria if there is bowel and/or bladder involvement. Catamanial pneumothorax has been reported from endometrio-sis implanted in the pleura.

1	include painful defecation, hematochezia, and hematuria if there is bowel and/or bladder involvement. Catamanial pneumothorax has been reported from endometrio-sis implanted in the pleura. Pelvic examination in symptomatic patients typically demonstrates generalized pelvic tenderness, nodularity of the uterosacral ligaments, and at times a pelvic mass may be appreciated if an endometrioma is present. The severity of symptoms does not correlate with the degree of clini-cal disease present. Endometriosis commonly causes of eleva-tions in serum CA-125. Definitive diagnosis usually requires laparoscopy and visualization of the pathognomonic endome-triotic implants. These appear as blue, brown, black, white, or yellow lesions that can be raised and at times puckered giving Brunicardi_Ch41_p1783-p1826.indd 180218/02/19 4:34 PM 1803GYNECOLOGYCHAPTER 41Table 41-4Centers for Disease Control and Prevention recommended treatment of pelvic inflammatory disease (2015)RECOMMENDED

1	180218/02/19 4:34 PM 1803GYNECOLOGYCHAPTER 41Table 41-4Centers for Disease Control and Prevention recommended treatment of pelvic inflammatory disease (2015)RECOMMENDED INTRAMUSCULAR/ORAL REGIMENSCeftriaxone 250 mg IM in a single dosePLUSDoxycycline 100 mg orally twice a day for 14 dayswith* or withoutMetronidazole 500 mg orally twice a day for 14 daysORCefoxitin 2 g IM in a single dose and Probenecid, 1 g orally administered concurrently in a single dosePLUSDoxycycline 100 mg orally twice a day for 14 dayswith or withoutMetronidazole 500 mg orally twice a day for 14 daysOROther parenteral third-generation cephalosporin (e.g., ceftizoxime or cefotaxime)PLUSDoxycycline 100 mg orally twice a day for 14 dayswith* or withoutMetronidazole 500 mg orally twice a day for 14 daysRECOMMENDED PARENTERAL REGIMENSCefotetan 2 g IV every 12 hoursPLUSDoxycycline 100 mg orally or IV every 12 hoursORCefoxitin 2 g IV every 6 hoursPLUSDoxycycline 100 mg orally or IV every 12 hoursORClindamycin 900

1	PARENTERAL REGIMENSCefotetan 2 g IV every 12 hoursPLUSDoxycycline 100 mg orally or IV every 12 hoursORCefoxitin 2 g IV every 6 hoursPLUSDoxycycline 100 mg orally or IV every 12 hoursORClindamycin 900 mg IV every 8 hoursPLUSGentamicin loading dose IV or IM (2 mg/kg), followed by a maintenance dose (1.5 mg/kg) every 8 hours. Single daily dosing (3–5 mg/kg) can be substituted.ALTERNATIVE PARENTERAL REGIMENAmpicillin/Sulbactam 3 g IV every 6 hoursPLUSDoxycycline 100 mg orally or IV every 12 hours*The addition of metronidazole to treatment regimens with third-generation cephalosporins should be considered until the need for extended anaerobic coverage is ruled out.Data from Centers for Disease Control and Prevention. 2015 Sexually Transmitted Diseases Treatment Guidelines: Pelvic Inflammatory Disease.them a “gunpowder” appearance. Biopsy is not routinely done but should be obtained if the diagnosis is in doubt.Treatment is guided by severity of the symptoms and whether preservation of

1	Disease.them a “gunpowder” appearance. Biopsy is not routinely done but should be obtained if the diagnosis is in doubt.Treatment is guided by severity of the symptoms and whether preservation of fertility is desired and varies from expectant, to medical, to surgical.48,49 Expectant management is appropriate in asymptomatic patients. Those with mild symp-toms can be managed with oral contraceptive pills and/or non-steroidal anti-inflammatory analgesia; moderate symptoms are treated with medroxyprogesterone acetate. Severe symptoms are treated with gonadotropin releasing hormone (GnRH) ago-nists to induce medical pseudomenopause.Surgical management for endometriosis varies depend-ing on the age and fertility desires of the patient. A diagnos-tic laparoscopy with biopsies may be indicated to confirm the diagnosis of endometriosis. If endometriosis is suspected, an operative laparoscopy with ablation of endometriotic implants usually decreases the severity of pelvic pain. Ablation of

1	to confirm the diagnosis of endometriosis. If endometriosis is suspected, an operative laparoscopy with ablation of endometriotic implants usually decreases the severity of pelvic pain. Ablation of endo-metriotic implants can be performed with CO2 laser or elec-trocautery, and/or resection of deep endometriotic implants.48 Endometriomas can cause pain and if found should be treated by ovarian cystectomy. Complete resection of the cyst wall is required as recurrence of the endometrioma is common after partial removal. Unfortunately, endometriosis is a chronic dis-ease, and conservative therapy, medical or surgical, provides only temporary relief, with the majority of patients relapsing with 1 to 2 years. For patients with severe debilitating symp-toms who do not desire future fertility and have not responded to conservative management extirpative surgery to remove the uterus, ovaries, and fallopian tubes; this intervention is curative and should be considered.Although endometriosis is

1	have not responded to conservative management extirpative surgery to remove the uterus, ovaries, and fallopian tubes; this intervention is curative and should be considered.Although endometriosis is not generally thought to be a premalignant lesion, there is an increased risk of type I ovar-ian cancer in women with a history of endometriosis.50 Molecu-lar evidence that endometriosis is likely a precursor lesion to clear cell carcinoma and endometrioid carcinomas includes the presence of mutations in both PIK3CA and ARID1A in benign endometriotic lesions in close proximity, suggesting that loss of expression of these genes likely occurs early in the development of endometrioid carcinomas.51,52Pelvic Adhesive Disease Pelvic adhesions usually are related to previous surgery, endometriosis, or infection, the latter of which can be either genital (i.e., pelvic inflammatory disease) or extragenital (e.g., ruptured appendix) in origin. Adhesions can be lysed mechanically and preferably with

1	infection, the latter of which can be either genital (i.e., pelvic inflammatory disease) or extragenital (e.g., ruptured appendix) in origin. Adhesions can be lysed mechanically and preferably with minimal cautery.Pelvic Inflammatory Disease. Pelvic inflammatory disease (PID) is an inflammatory disorder of the upper female genital tract, including any combination of endometritis, salpingitis, tubo-ovarian abscess, and pelvic peritonitis. Sexually transmitted organisms, especially N gonorrhoeae and C trachomatis, are implicated in many cases although microorganisms that comprise the vaginal flora (e.g., anaerobes, G vaginalis, Haemophilus influenzae, enteric Gram-negative rods, and Streptococcus agalactiae) have been implicated as well. PID can additionally result from extension of other pelvic and abdominal infections, such as appendicitis and diverticulitis, or may be precipitated by medical procedure, such as hysterosalpingography, endometrial biopsy, or dilation and

1	of other pelvic and abdominal infections, such as appendicitis and diverticulitis, or may be precipitated by medical procedure, such as hysterosalpingography, endometrial biopsy, or dilation and curettage.53,54The presentation of PID can be subtle. Differential diagnosis includes appendicitis, cholecystitis, inflammatory bowel disease, pyelonephritis, nephrolithiasis, ectopic pregnancy, and ovarian torsion. Long-term sequelae can include infertility, chronic pelvic pain, and increased risk of ectopic pregnancy. Because of the severity of these sequelae, presumptive treatment is recommended in young, sexually active women experiencing pelvic or lower abdominal pain, when no cause for the illness other than PID can be identified and if cervical motion tenderness, uterine tenderness, or adnexal tenderness is present on examination. Because of the psychosocial complexity associated with a diagnosis of PID, additional criteria should be used to enhance the specificity of the minimum

1	adnexal tenderness is present on examination. Because of the psychosocial complexity associated with a diagnosis of PID, additional criteria should be used to enhance the specificity of the minimum clinical criteria when possible. These include the following: oral temperature >101°F (>38.3°C); abnormal cervical mucopurulent discharge or cervical friability; presence Brunicardi_Ch41_p1783-p1826.indd 180318/02/19 4:34 PM 1804SPECIFIC CONSIDERATIONSPART IIof abundant numbers of white blood cells on saline microscopy of vaginal fluid; elevated erythrocyte sedimentation rate; elevated C-reactive protein; and laboratory documentation of cervical infection with N gonorrhoeae or C trachomatis. Laparoscopy can be used to obtain a more accurate diagnosis of salpingitis and a more complete bacteriologic diagnosis and is often useful in ruling out other causes of peritonitis. Laparoscopic findings may include swollen erythematous tubes with purulent exudates.55Several outpatient parenteral

1	diagnosis and is often useful in ruling out other causes of peritonitis. Laparoscopic findings may include swollen erythematous tubes with purulent exudates.55Several outpatient parenteral and oral antimicrobial regi-mens have been effective in achieving clinical and microbio-logic cure. Hospitalization for intravenous antibiotics may be necessitated in cases of where surgical emergencies cannot be ruled out, tubo-ovarian abscess is identified, pregnancy, severe illness (nausea and vomiting, or high fever), inability to follow or tolerate an outpatient oral regimen; or failure of outpatient oral antimicrobial therapy. Treatment of a tubo-ovarian abscess may include placement of a percutaneous drain in addition to intravenous antibiotics.55Surgical intervention becomes necessary if medical therapy fails or if the patient becomes unstable. Hysterec-tomy and bilateral salpingo-oophorectomy is the procedure of choice; however, conservative surgery must be considered in young patients

1	therapy fails or if the patient becomes unstable. Hysterec-tomy and bilateral salpingo-oophorectomy is the procedure of choice; however, conservative surgery must be considered in young patients desiring future fertility. The abdomen should be explored for metastatic abscesses, and special attention must be paid to bowel, bladder, and ureteral safety due to the friabil-ity of the infected tissue and the adhesions commonly encoun-tered at the time of surgery. Placement of an intraperitoneal drain and mass closure of the peritoneum, muscle, and fascia with delayed-absorbable sutures is advised. Conservative sur-gery, when feasible, may be attempted by laparoscopy and may involve unilateral salpingo-oophorectomy or drainage of the abscess and liberal irrigation of the abdomen and pelvis.53PREGNANCY-RELATED SURGICAL CONDITIONSMany pregnant women will undergo invasive diagnostic proce-dures for prenatal diagnosis, and in the United States, nearly one-third of all births are cesarean

1	SURGICAL CONDITIONSMany pregnant women will undergo invasive diagnostic proce-dures for prenatal diagnosis, and in the United States, nearly one-third of all births are cesarean deliveries.56 About 1 in 500 pregnant women will require surgery for nonob-stetrical issues.57,58 Diagnostic challenges and physiologic changes due to pregnancy, as well as the unique anesthesia risks and potential risks to the pregnancy, should be kept in mind whether the primary surgeon is an obstetrician, gynecologist, or a general surgeon (Table 41-5).58Trauma in the obstetric patient requires stabilization of the mother while considering the fetal compartment.58,59 Trauma-related hypovolemia may be compounded by pregnancy-induced decreases in systemic vascular resistance, and when supine, the weight of the gravid uterus on the vena cava. When feasible, a left lateral tilt should be instituted to improve venous return to the right heart. Later in pregnancy, the small bowel is dis-placed into the upper

1	gravid uterus on the vena cava. When feasible, a left lateral tilt should be instituted to improve venous return to the right heart. Later in pregnancy, the small bowel is dis-placed into the upper abdomen, making it vulnerable to complex injury from penetrating upper abdominal trauma. Though small bowel is displaced from the pelvis, the dramatic increase in pel-vic blood flow can lead to rapid blood loss due to penetrating pelvic trauma, fractures, or avulsion of pelvic vessels. Gastric motility is decreased increasing the risk of aspiration. Peritoneal signs may be attenuated by the stretching of the abdominal wall. Several coagulation factors are also increased in pregnancy, increasing the likelihood for thromboembolic events, but also giving the unsuspecting surgeon false security when low-normal levels are observed during resuscitative efforts. Only the third 5Table 41-5Physiologic changes due to pregnancyCardiovascular changes Increased cardiac output Increased blood

1	when low-normal levels are observed during resuscitative efforts. Only the third 5Table 41-5Physiologic changes due to pregnancyCardiovascular changes Increased cardiac output Increased blood volume Increased heart rate Decreased blood pressure Decreased systemic vascular resistance Decreased venous return from lower extremitiesRespiratory changes Increased minute ventilation Decreased functional residual capacityGastrointestinal changes Decreased gastric motility Delayed gastric emptyingCoagulation changes Increased clotting factors (II, VII, VIII, IX, X) Increased fibrinogen Increased risk for venous thromboembolismRenal changes Increased renal plasma flow and GFR Ureteral dilationReproduced with permission from Gabbe S NJ, Simpson J: Obstetrics: Normal and Problem Pregnancies, 6th ed. Philadelphia, PA: Elsevier/Saunders; 2012.trimester fetus has any ability to autoregulate in the context of decreased uterine blood flow and oxygen delivery. In the third trimester, perimortem

1	ed. Philadelphia, PA: Elsevier/Saunders; 2012.trimester fetus has any ability to autoregulate in the context of decreased uterine blood flow and oxygen delivery. In the third trimester, perimortem cesarean delivery should be considered as part of maternal resuscitation in cases of maternal hemodynamic collapse. Though treating the maternal compartment is the pri-mary concern, it should also be recognized that the fetus will be impacted significantly by maternal hypotension, as blood may be shunted away from the uterus.Conditions and Procedures Performed Before ViabilityAmniocentesis/Chorionic Villus Sampling. Noninvasive prenatal testing has for the most part replaced invasive fetal testing. Amniocentesis is a procedure in which amniotic fluid is aspirated from the uterine cavity and sent for genetic or labora-tory testing typically under ultrasound guidance with a 20to 22-gauge needle. This procedure may be used to confirm abnor-mal noninvasive testing.Miscarriage and Pregnancy

1	sent for genetic or labora-tory testing typically under ultrasound guidance with a 20to 22-gauge needle. This procedure may be used to confirm abnor-mal noninvasive testing.Miscarriage and Pregnancy Terminations. Spontaneous pregnancy loss is common. Although the miscarriage rate among women who know they are pregnant is roughly 10% to 20%, if the start of pregnancy is set to fertilization, rates are as high as 50%. Chromosomal abnormalities are the underlying cause of miscarriage and are present in over half of cases. Patient may report cramping, bleeding and passage of tissue. If products of conception are not passed, diagnosis can be made by transvagi-nal ultrasound if an empty gestational sac is identified or an embryo is noted to not have a heartbeat. Treatment can include expectant management, medical management with misoprostol, or surgical management with dilation and curettage.60Half of all pregnancies in the United States are unintended, and many of these are undesired.

1	management, medical management with misoprostol, or surgical management with dilation and curettage.60Half of all pregnancies in the United States are unintended, and many of these are undesired. Additional reasons for termi-nation of pregnancy include fetal anomalies such as trisomies, fetal infections, and maternal health. Medical terminations are Brunicardi_Ch41_p1783-p1826.indd 180418/02/19 4:34 PM 1805GYNECOLOGYCHAPTER 41available up to 10 weeks of gestation, and surgical terminations can be performed to viability. Rates of pregnancy termination have been declining due decreasing access to abortion ser-vices and widespread availability of long-acting contraceptives (LARC). LARCs are safe, effective, easy to use and protect against unintended pregnancy for up to 10 years.61Up to 15 weeks’ gestation, manual vacuum aspiration can be used following cervical dilation to mechanically evacuate the fetus or embryo, placenta, and membranes by suction using a manual syringe.

1	to 15 weeks’ gestation, manual vacuum aspiration can be used following cervical dilation to mechanically evacuate the fetus or embryo, placenta, and membranes by suction using a manual syringe. Alternatively, cervical dilation and suction curettage can be performed. The uterine cervix is grasped with a tenaculum, then mechanically dilated occasionally using adjunc-tive prostaglandins, and an appropriately sized vacuum cannula is inserted into the uterus and rotated on its axis to remove the products of conception. Dilation and extraction is performed for pregnancies in the second trimester. The additional cervical dilation required at greater gestational ages is usually a two-step (often over 2 days) process. Osmotic dilators are placed within the cervix a day prior to the procedure and expand as water is absorbed, passively dilating the endocervical canal. These are removed immediately prior to the procedure and mechanical dilation is then performed as needed. Forceps are then used

1	expand as water is absorbed, passively dilating the endocervical canal. These are removed immediately prior to the procedure and mechanical dilation is then performed as needed. Forceps are then used to remove fetal parts. Curettage of the postabortal uterus must be approached carefully because the uterus is extremely soft and perforation can occur with very little warning. Complications are rare (particularly when contrasted to the risks of pregnancy and term delivery) but include infection, hemorrhage due to uterine atony, cervical lacerations, uterine perforations, and inadvertent bowel injury from the vacuum cannula or forceps.Cerclage. Cervical insufficiency is defined as painless cervical dilation leading to recurrent second trimester pregnancy loss, or shortened cervical length as determined by transvaginal ultra-sound, or advanced cervical change before 24 weeks’ gestation in a woman with either prior preterm birth/loss or significant risk factors for insufficiency. A cervical

1	by transvaginal ultra-sound, or advanced cervical change before 24 weeks’ gestation in a woman with either prior preterm birth/loss or significant risk factors for insufficiency. A cervical cerclage refers to a procedure in which suture or synthetic tape is used to circum-ferentially reinforce the cervix to improve pregnancy outcome in at-risk patients.62 Shirodkar and McDonald techniques have been described63,64; both involve transvaginally placing a non-absorbable suture at the uterocervical junction to lengthen and close the cervix. An abdominal cerclage of the lower uterine segment performed laparoor by laparotomy can be considered for a patient with a severely shortened or absent cervix who has previously failed a transvaginal cerclage.Ectopic Pregnancies. Extrauterine pregnancies are most com-monly located along the fallopian tubes but can also implant on the ovary. Rarely, implantation can occur primarily on other abdominal organs or peritoneal surfaces. A high index of

1	are most com-monly located along the fallopian tubes but can also implant on the ovary. Rarely, implantation can occur primarily on other abdominal organs or peritoneal surfaces. A high index of suspi-cion and early diagnosis typically includes an abnormal rise in b-hCG assays and presence of an adnexal mass on transvaginal ultrasound. Early ectopic pregnancies can be managed medi-cally with a methotrexate injection; however, close follow-up with twice-weekly b-hCG testing is required. Laparoscopy is the definitive management and can be used either as primary treatment or when medical management fails. The tube should be removed (salpingectomy) in its entirety if the ectopic is iden-tified within the fallopian tube. This can be performed using a vessel sealing device or even an endo-loop and endo-shears. Laparotomy is reserved for unstable patients with a known hemoperitoneum where Kelly clamps can be placed along the mesosalpinx to control bleeding. Cornual ectopic pregnancies may

1	and endo-shears. Laparotomy is reserved for unstable patients with a known hemoperitoneum where Kelly clamps can be placed along the mesosalpinx to control bleeding. Cornual ectopic pregnancies may require wedge resection of the uterine serosa and myo-metrium, which is then closed in two layers.65 Linear salpin-gostomy along the antimesenteric border and removal of the products of conception is now rarely used due to low rates of postoperative tubal function and high recurrent ectopic pregnan-cies presumably due to scarring.Conditions and Procedures Performed After ViabilityObstetric Lacerations and Repair. At the time of vaginal delivery, perineal lacerations are common. These lacerations involve, in varying degrees, the vaginal mucosa, the muscular elements inserting onto the perineal body, the levator ani, and in 4% to 5% of vaginal deliveries, the anal sphincter or anorectal mucosa. Although episiotomies were historically cut prophy-lactically to prevent unstructured tearing of

1	body, the levator ani, and in 4% to 5% of vaginal deliveries, the anal sphincter or anorectal mucosa. Although episiotomies were historically cut prophy-lactically to prevent unstructured tearing of the perineum, this practice has fallen out of favor as the benefit of episiotomy has not been demonstrated.Perineal Laceration First-degree tears involve only the perineal skin and may or may not need to be reapproximated. Second-degree tears involve the perineal body and can gener-ally be repaired with some variation using a single continuous, nonlocking suture technique, typically a 2-0 or 3-0 synthetic delayed absorbable suture. The apex of the vaginal epithelial is approximated first including epithelium and underlying tissue to build up the rectovaginal septum. Upon reaching the hymenal ring, the perineal body and bulbocavernosus muscle are reap-proximated, and a transition stitch is placed from the vaginal mucosa, which was repaired along a horizontal plane, to the deep perineal

1	ring, the perineal body and bulbocavernosus muscle are reap-proximated, and a transition stitch is placed from the vaginal mucosa, which was repaired along a horizontal plane, to the deep perineal layer, which lies in a vertically-oriented plane. A running closure is then completed incorporating the deep peri-neal tissues from the introitus to the extent of the perineal defect. At this point, the perineal skin is closed from inferior to superior in a subcuticular fashion and tied just inside the introitus.Third-degree lacerations extend through the perineal body and involve the external anal sphincter, while fourth-degree lac-erations involve the internal anal sphincter and rectal mucosa. When present, thirdand fourth-degree lacerations should be repaired first before proceeding with the second-degree repair. This is accomplished by first closing the anal mucosa, and then identifying and closing the internal anal sphincter in a second layer. The external anal sphincter is then

1	the second-degree repair. This is accomplished by first closing the anal mucosa, and then identifying and closing the internal anal sphincter in a second layer. The external anal sphincter is then identified, and the muscular cylinder is reconstructed by suturing the severed ends together using either an end-to-end or overlapping technique. Although these are typically straightforward layered closures, knowledge of the anatomy is important. Incomplete reconstruc-tion, particularly of thirdor fourth-degree lacerations, can contribute to future pelvic floor disorders, as well as the devel-opment of fistulae or incontinence.Cervical and Vaginal Lacerations Significant lacerations to the cervix or vagina may also occur during childbirth, particu-larly with instrumented deliveries or macrosomic infants. These lacerations may present as persistent bleeding, not readily rec-ognized due to their location, and often in association with a firmly contracted uterus. Vaginal lacerations may be

1	infants. These lacerations may present as persistent bleeding, not readily rec-ognized due to their location, and often in association with a firmly contracted uterus. Vaginal lacerations may be repaired primarily but should only be closed after deeper tissues are inspected to insure no active bleeding. Cervical lacerations can be repaired in a running, locking fashion, insuring that the apex of the laceration is incorporated in the closure. If the apex is challenging to reach, the closure can be started more distally using the suture to apply traction so that the apex may be closed.Brunicardi_Ch41_p1783-p1826.indd 180518/02/19 4:34 PM 1806SPECIFIC CONSIDERATIONSPART IIPuerperal Hematoma Trauma during childbirth can occasion-ally result in significant hematoma formation with or without a visible laceration. These hematomas may hide significant blood loss and most commonly occur in the vulva, paravaginal, and pelvic retroperitoneum. Typical presentation is pain and mass effect.

1	a visible laceration. These hematomas may hide significant blood loss and most commonly occur in the vulva, paravaginal, and pelvic retroperitoneum. Typical presentation is pain and mass effect. Small hematomas can be managed conservatively with close observation and patient monitoring. Though there are no evidence-based size criteria, an unstable patient or expand-ing hematomas should prompt surgical intervention. After the hematoma is incised and drained, diffuse venous oozing is usu-ally encountered rather than a single bleeding vessel. Hemo-stasis can be achieved using electrosurgery or fine absorbable suture, though caution must be used due to the proximity of bowel, bladder, and ureters to some hematomas. Pressure on the vulva or packing the vagina, rather than the hematoma cavity, may prevent further bleeding.Cesarean Deliveries. Typical indications for cesarean deliv-ery include nonreassuring fetal status, breech or other malpre-sentations, triplet and higher order gestations,

1	prevent further bleeding.Cesarean Deliveries. Typical indications for cesarean deliv-ery include nonreassuring fetal status, breech or other malpre-sentations, triplet and higher order gestations, cephalopelvic disproportion, failure to progress in labor, placenta previa, and active genital herpes. Previous low transverse cesarean deliv-ery is not a contraindication to subsequent vaginal birth after cesarean; however, much of the increase in cesarean delivery in the past two decades is attributable to planned repeat cesareans. Cesarean deliveries typically are performed via a lower anterior (caudal) uterine transverse incision because there is decreased blood loss, and the uterine rupture rate with future pregnancies is about 0.5% (Fig. 41-15). A prior classical cesarean delivery is an absolute indication for a planned repeat cesarean delivery because of a high rate of uterine rupture during labor, unlike with the lower anterior uterine transverse incision. Abdominal access is

1	an absolute indication for a planned repeat cesarean delivery because of a high rate of uterine rupture during labor, unlike with the lower anterior uterine transverse incision. Abdominal access is obtained by a Pfannenstiel, Maylard or vertical inci-sion. Once the abdomen is entered, a vesicouterine reflection is created if a low transverse uterine incision is planned. The uter-ine incision is then made and extended laterally, avoiding the uterine vessels. After amniotomy, the baby is delivered, and the uterus is closed. Approximately 1000 mL of blood is typically lost during a cesarean delivery. Along with rapid closure of the uterine incision, uterotonics, such as intravenous oxytocin, are administered. A classical, vertical, uterine incision is made in EDABCFigure 41-15. Uterine incisions for cesarean delivery. (Reproduced with permission from Gabbe S, Niebyl J, Simpson J: Obstetrics: Normal and Problem Pregnancies, 5th ed. Philadelphia, PA: Elsevier/ Churchill Livingstone;

1	incisions for cesarean delivery. (Reproduced with permission from Gabbe S, Niebyl J, Simpson J: Obstetrics: Normal and Problem Pregnancies, 5th ed. Philadelphia, PA: Elsevier/ Churchill Livingstone; 2007.)certain very early viable gestations, or in the case of certain transverse lies or abnormal placentation. Infection, excessive blood loss due to uterine atony, and urinary tract and bowel inju-ries are potential complications at the time of cesarean delivery. The risk of those injuries, as well as abnormal placentation (pla-centa accreta, increta, and percreta) rises with each subsequent cesarean delivery. Bleeding can only be controlled in some instances by performing a cesarean hysterectomy.Postpartum Hemorrhage. Postpartum hemorrhage is an obstetrical emergency that can follow either vaginal or cesarean delivery. Hemorrhage is usually caused by uterine atony, trauma to the genital tract, or rarely, coagulation disorders. Hemorrhage may also be caused by abnormal placentation (also

1	or cesarean delivery. Hemorrhage is usually caused by uterine atony, trauma to the genital tract, or rarely, coagulation disorders. Hemorrhage may also be caused by abnormal placentation (also called mor-bidly adherent placenta). Management consists of mitigating potential obstetric causes while simultaneously acting to avert or treat hypovolemic shock. In the absence of atony, the genital tract should be thoroughly evaluated for trauma. Atony is the most common cause of postpartum hemorrhage. It is typically treated with fundal massage and uterotonics such as oxytocin, methylergonovine, carboprost tromethamin, and misoprostol. When aggressive medical management fails, surgical manage-ment may be necessary and life-saving.66Uterine Curettage Retained products of conception may result in uterine atony. It may be possible to remove retained prod-ucts via manual extraction or with ring forceps. Bedside ultra-sound may be helpful in localization. When clinical suspicion is high, uterine

1	uterine atony. It may be possible to remove retained prod-ucts via manual extraction or with ring forceps. Bedside ultra-sound may be helpful in localization. When clinical suspicion is high, uterine curettage is indicated. A blunt, large curette, banjo curette, is introduced and removal of retained tissue typi-cally results in contraction of the myometrium and cessation of bleeding.Procedures Short of Hysterectomy As bleeding from post-partum hemorrhage becomes increasingly acute, interventions short of hysterectomy should be carried out expeditiously while supporting the hemodynamic status of the patient and prepar-ing for possible definitive surgery. A number of techniques for packing and tamponade of the uterus have been described, including a balloon device reported by Bakri and colleagues.67 These are typically left in place for 24 to 36 hours and appear to be safe and often effective conservative measures short of laparotomy and hysterectomy. The B-Lynch compression suture may

1	These are typically left in place for 24 to 36 hours and appear to be safe and often effective conservative measures short of laparotomy and hysterectomy. The B-Lynch compression suture may control bleeding of atony at the time of cesarean section. A suture is placed through the hysterotomy, around the fundus of the uterus anterior to posterior, and then through the posterior lower uterine segment, to the contralateral side. At this point, the steps are reversed with the suture brought around the fundus posterior to anterior, through the contralateral side of the hys-terotomy, and then tied in the midline to compress the uterus. Additional procedures described include the O’Leary uterine artery ligation and the hypogastric artery ligation. “O’Leary stitches” are a series of sutures placed around the branches of the uterine artery and through the myometrium, resulting in compression of the vessels against the uterus. Hypogastric artery ligation entails the isolation of the internal

1	around the branches of the uterine artery and through the myometrium, resulting in compression of the vessels against the uterus. Hypogastric artery ligation entails the isolation of the internal iliac artery at its bifurcation with the external iliac artery. The hypogastric artery is ligated at least 3 cm distal to the bifurcation to avoid compromising the posterior division.Postpartum/Cesarean Hysterectomy A cesarean or postpar-tum (absent a prior cesarean delivery) hysterectomy involves the same steps as in a nonpregnant patient, but it is distinctly different due to the engorged vessels and the pliability of the tis-sues. If a cesarean section has been performed, occasionally the Brunicardi_Ch41_p1783-p1826.indd 180618/02/19 4:34 PM 1807GYNECOLOGYCHAPTER 41incision can be used for traction to keep the vessels and tissues attenuated. Vascular pedicles should be secured with clamps, but not ligated until both uterine arteries have been secured, to fully control bleeding. Lack of

1	traction to keep the vessels and tissues attenuated. Vascular pedicles should be secured with clamps, but not ligated until both uterine arteries have been secured, to fully control bleeding. Lack of typical anatomic landmarks requires careful identification of the ureters and the dilated cervix visu-ally or by palpation, to separate from the bladder and vagina (Fig. 41-16). This procedure is often done for life-threatening hemorrhage, thus appropriate blood products, including packed red blood cells, fresh frozen plasma, platelets, and fibrinogen should be on call and are usually required. Fibrinogen is typi-cally elevated in a pregnant woman, such that a low-normal fibrinogen level can be cause for alarm, and further fibrinogen may be required before consumptive coagulopathy reverses. A massive transfusion protocol is helpful.Abnormal Placentation. Placenta accreta describes the clinical condition when the placenta invades and is inseparable from the uterine wall. When the chorionic

1	massive transfusion protocol is helpful.Abnormal Placentation. Placenta accreta describes the clinical condition when the placenta invades and is inseparable from the uterine wall. When the chorionic villi invades the myometrium, the term placenta increta is used; whereas placenta percreta describes invasion through the myometrium and serosa, and even into adjacent organs such as the bladder. Abnormal placentation has increased in parallel to the cesarean section rate in the United States. When cytotrophoblasts invade decidualized endometrium and encounter a uterine scar, they do not encounter the normal myometrial signals to stop invasion. In the setting of a placenta previa, the presence of a uterine scare is a particular risk for placenta accreta with rates of 11%, 40%, and 61% for one, two, or three prior cesarean deliveries, respectively.68 Ultrasound or MRI can assist in the diagnosis, depending on the experience and comfort of the imager.69,70Women at risk for abnormal

1	for one, two, or three prior cesarean deliveries, respectively.68 Ultrasound or MRI can assist in the diagnosis, depending on the experience and comfort of the imager.69,70Women at risk for abnormal placentation should ideally be identified during pregnancy and be prepared for cesarean sec-tion followed by cesarean hysterectomy. Since the blood supply to the gravid uterus is 500 cc per minute, these surgeries have the potential to have very high blood loss, which can then lead to the development of disseminated intravascular coagulation. Over 50% of cases require more than 4 units of blood transfused. BladderUreter identifiedClamps on uterine vesselsFigure 41-16. Demonstration of location of distal ureter and bladder, and their relationship to uterine vessels. (Reproduced with permission from Nichols DH: Gynecologic and Obstetric Surgery, Vol. 1. Philadelphia, PA: Elsevier; 1993.)Unintentional bladder or ureteral injuries are common as well due to impaired visualization and poor

1	from Nichols DH: Gynecologic and Obstetric Surgery, Vol. 1. Philadelphia, PA: Elsevier; 1993.)Unintentional bladder or ureteral injuries are common as well due to impaired visualization and poor dissection planes. For these reasons, patients with suspected placenta accreta should be delivered in a tertiary care center with a multidisciplinary team that has the capacity for massive blood transfusion pro-tocol. While some sites have implemented protocols involving interventional radiology with placement of occlusive balloons in the uterine arteries prior to delivery, these protocols have not been shown to decrease morbidity or overall blood loss. Postop-erative embolization should be available. Even with scheduled delivery in a well-resourced setting with a highly experienced and prepared multidisciplinary team, the morbidity of abnormal placentation is high. ICU stays are common, and maternal mor-tality as high as 7% has been reported.69Delayed hysterectomy where the placenta is left

1	multidisciplinary team, the morbidity of abnormal placentation is high. ICU stays are common, and maternal mor-tality as high as 7% has been reported.69Delayed hysterectomy where the placenta is left in situ after delivery of the baby if there is not significant bleeding and the mother is stable is advocated by certain centers but remains controversial.71 The risks of leaving the placenta in utero include later hemorrhage, infection, and sepsis. Planned hysterectomy at 6 to 12 weeks postpartum is recommended unless subsequent fertility is strongly desire.69-71PELVIC FLOOR DYSFUNCTIONPelvic floor disorders can be categorized, from a urogyneco-logic perspective, into three main topics: female urinary incontinence and voiding dysfunction, pelvic organ pro-lapse, and disorders of defecation.72 Approximately 11% of women will undergo surgery for incontinence or prolapse.73 The normal functions of support, storage, and evacuation can be altered by derangements in neuromuscular function both

1	Approximately 11% of women will undergo surgery for incontinence or prolapse.73 The normal functions of support, storage, and evacuation can be altered by derangements in neuromuscular function both cen-trally and peripherally and through acquired changes in connec-tive tissue. Reconstructive surgeons aim to repair or compensate for many of these losses.EvaluationDiagnostic evaluations, in addition to the history and examina-tions previously described, can aid in the diagnosis of many pel-vic floor disorders. Cystoscopy, multichannel urodynamics, and/or fluoroscopic evaluation of the urinary tract can be obtained for patients with urinary incontinence or voiding dysfunction.74 Defecography, anal manometry, and endorectal ultrasound may be useful for diagnosis of defecatory dysfunction. A standard-ized examination called the pelvic organ prolapse quantifica-tion (POP-Q)74 helps to clarify which vaginal compartment, and therefore which specific structure, has lost its anatomic integrity

1	examination called the pelvic organ prolapse quantifica-tion (POP-Q)74 helps to clarify which vaginal compartment, and therefore which specific structure, has lost its anatomic integrity in women with uterovaginal prolapse. Finally, dynamic MRI and pelvic floor electromyography has growing utility for all three disorders.Surgery for Pelvic Organ ProlapseMany factors are important in determining which reconstruc-tive operation is optimal for a given patient with pelvic organ prolapse. Surgical decisions are often based on case series and expert opinions that may not have universal applicability. How-ever, the few reports with the highest level of evidence sug-gests that failure rates for prolapse reconstruction may be twice as high using the vaginal approach when compared with the abdominal route.75,76Colporrhaphy. Anterior colporrhaphy, also known as an “anterior repair,” is performed for a symptomatic cystocele. The procedure begins with incision of the anterior vaginal epithelium

1	route.75,76Colporrhaphy. Anterior colporrhaphy, also known as an “anterior repair,” is performed for a symptomatic cystocele. The procedure begins with incision of the anterior vaginal epithelium 6Brunicardi_Ch41_p1783-p1826.indd 180718/02/19 4:34 PM 1808SPECIFIC CONSIDERATIONSPART IIin a midline sagittal direction. The epithelium is dissected away from the underlying vaginal muscularis. The vaginal muscularis is plicated with interrupted delayed absorbable stitches, after which the epithelium is trimmed and reapproximated. The vaginal canal is therefore shortened and narrowed proportionate to the amount of removed epithelium. Posterior colporrhaphy is performed for a symptomatic rectocele. This procedure is performed in a similar manner, often including the distal pubococcygeus muscles in the plication. Recently, in attempts to decrease surgical failures alluded to previously, many surgeons have opted to utilize grafts and meshes to augment these vaginally performed procedures.

1	in the plication. Recently, in attempts to decrease surgical failures alluded to previously, many surgeons have opted to utilize grafts and meshes to augment these vaginally performed procedures. Unfortunately, the apparent number of postoperative complications, including mesh erosion, pelvic pain, and dyspareunia, prompted the FDA to publish a warning encouraging a much more limited use of vaginal mesh for prolapse repair until greater surveillance and more rigorous studies could be completed.77Sacrospinous and Uterosacral Ligament Fixations. Both the sacrospinous ligament fixation (SSLF) and uterosacral ligament fixation (USLF) procedures are vaginal procedures that suspend the apex of the vagina using native tissue for treatment of apical prolapse. The sacrospinous ligament is found embedded in and continuous with the coccygeus muscle, which extends from the ischial spine to the lateral surface of the sacrum. The procedure begins with entry into the rectovaginal space, usually by

1	in and continuous with the coccygeus muscle, which extends from the ischial spine to the lateral surface of the sacrum. The procedure begins with entry into the rectovaginal space, usually by incising the posterior vaginal wall at its attachment to the perineal body. The space is developed to the level of the vaginal apex and the rectal pillar is penetrated to gain access to the pararectal space. A long-ligature carrier is used to place sutures medial to the ischial spine, through the substance of the ligament-muscle complex. Structures at risk in this procedure include the pudendal neurovascular bundle, the inferior gluteal neurovascular bundle, lumbosacral plexus, and sciatic nerve. After the stitches are placed, the free ends are sewn to the undersurface of the vaginal cuff. The sacrospinous stitches are tied to firmly approximate the vagina to the ligament without suture bridging.When using the uterosacral ligaments for repair of prolapse, it is important to recall that these

1	stitches are tied to firmly approximate the vagina to the ligament without suture bridging.When using the uterosacral ligaments for repair of prolapse, it is important to recall that these structures are not “ligaments” in the true sense of the word, but rather condensations of smooth muscle, collagen, and elastin. Several support sutures are placed from the lateral-most portion of the vaginal cuff to the distal-most part of the ligament, and the medial vaginal cuff to the proximal ligament. Intraoperative evaluation of the lower urinary tract is important to confirm the absence of ureteral compromise.Colpocleisis. Colpocleisis is reserved for patients who are elderly, who do not wish to retain coital ability, and for whom there is good reason not to perform a more extensive recon-structive operation. A colpocleisis removes of part or all of the vaginal epithelium, obliterating the vaginal vault and leaving the external genitalia unchanged. The procedure can be performed with or

1	operation. A colpocleisis removes of part or all of the vaginal epithelium, obliterating the vaginal vault and leaving the external genitalia unchanged. The procedure can be performed with or without a hysterectomy. Successive purse-string sutures through the vaginal muscularis are used to reduce the prolapsed organs to above the level of the levator plate.Sacrocolpopexy. The procedure with the lowest risk of recurrence for patients with prolapse of the vaginal apex is an abdominal sacral colpopexy. In these patients, the natural apical support structure, the cardinal–uterosacral ligament complex, is often damaged and attenuated. The abdominal placement, as opposed to vaginal placement, of graft material to compensate for defective vaginal support structures is well described.78 Api-cal support defects rarely exist in isolation, and the sacrocol-popexy may be modified to include the anterior and posterior vaginal walls as well as the perineal body in the suspension. Sacrocolpopexies

1	support defects rarely exist in isolation, and the sacrocol-popexy may be modified to include the anterior and posterior vaginal walls as well as the perineal body in the suspension. Sacrocolpopexies can be performed via laparotomy as well as via laparoscopy or robotically. Like rectopexies and low anterior resections, deep pelvic access is needed. Significant suturing at varied angles is required. The advent of the DaVinci robotic laparoscopic system has made visualization and adequate place-ment of the mesh and sutures easier to perform when using the minimally invasive approach.During a sacrocolpopexy, a rigid stent (usually an EEA sizer) is placed into the vagina to facilitate its dissection from the overlying bladder and rectum and to allow the graft material to be spread evenly over its surface. A strip of synthetic mesh is fixed to the anterior and posterior vaginal walls. The peritoneum overlying the presacral area is opened, extending to the poste-rior cul-de-sac. The sigmoid

1	its surface. A strip of synthetic mesh is fixed to the anterior and posterior vaginal walls. The peritoneum overlying the presacral area is opened, extending to the poste-rior cul-de-sac. The sigmoid colon is retracted medially, and the anterior surface of the sacrum is skeletonized. Two to four permanent sutures are placed through the anterior longitudinal ligament in the midline, starting at the S2 level and proceeding distally. The sutures are passed through the graft at an appropri-ate location to support the vaginal vault without tension. The peritoneum is then closed with an absorbable running suture. The most dangerous potential complication of sacrocolpopexy is sacral hemorrhage.Surgery for Stress Urinary IncontinenceStress incontinence is believed to be caused by lack of urethro-vaginal support (urethral hypermobility) or intrinsic sphincter deficiency (ISD). ISD is a term applied to a subset of stress-incontinent patients who have particularly severe symptoms, including

1	support (urethral hypermobility) or intrinsic sphincter deficiency (ISD). ISD is a term applied to a subset of stress-incontinent patients who have particularly severe symptoms, including urine leakage with minimal exertion. This condition is often recognized clinically as the low pressure or “drainpipe” urethra. The urethral sphincter mechanism in these patients is severely damaged, limiting coaptation of the urethra. Standard surgical procedures used to correct stress incontinence share a common feature: partial urethral obstruction that achieves ure-thral closure under stress.Burch Procedure. Despite the wide acceptance of midurethral sling procedures, a retropubic urethropexy procedure called the Burch procedure is still performed for stress incontinence.79 The space of Retzius is approached extraperitoneally, from an abdominal approach, allowing the bladder to be mobilized from the surrounding adipose tissue and lateral pelvis. Two pairs of large-caliber nonabsorbable sutures are

1	extraperitoneally, from an abdominal approach, allowing the bladder to be mobilized from the surrounding adipose tissue and lateral pelvis. Two pairs of large-caliber nonabsorbable sutures are placed through the peri-urethral vaginal wall, one pair at the midurethra and one at the urethrovesical junction. Each stitch is then anchored to the ipsi-lateral Cooper’s (iliopectineal) ligament. The sutures are tied to give preferential support to the urethrovesical junction relative to the anterior vaginal wall without overcorrection. Long-term outcome studies up to 10 years have shown the Burch procedure yields cure rates of 80% to 85%.Tensionless Sling. The tension-free vaginal tape (TVT) is a modified sling that uses a strip of polypropylene mesh. Unlike traditional sling procedures, the mesh is positioned at the midurethra, not the urethrovesical junction, and it is not sutured or otherwise fixed into place. Advantages of TVT include the ability to perform the procedure under local

1	mesh is positioned at the midurethra, not the urethrovesical junction, and it is not sutured or otherwise fixed into place. Advantages of TVT include the ability to perform the procedure under local anesthesia on an outpatient basis. Small subepithelial tunnels are made bilater-ally to the descending pubic rami through an anterior vaginal wall incision. A specialized conical metal needle coupled to a handle is used to drive one end of the sling through the peri-neal membrane, space of Retzius, and through one of two small suprapubic stab incisions. The tape is set in place without any Brunicardi_Ch41_p1783-p1826.indd 180818/02/19 4:34 PM 1809GYNECOLOGYCHAPTER 41tension after bringing up the other end of the tape through the other side. Recently, multiple modifications have been made to carry the tape through the bilateral medial portions of the obtu-rator space (TVT-O). Risks of the procedure include visceral injury from blind introduction of the needle, bleeding, and nerve and

1	to carry the tape through the bilateral medial portions of the obtu-rator space (TVT-O). Risks of the procedure include visceral injury from blind introduction of the needle, bleeding, and nerve and muscle injury in the obturator space. Additionally, voiding dysfunction and delayed erosion of mesh into the bladder or urethra has been seen.Urethral Bulking Injections. A transurethral or periurethral injection of bulking agents is indicated for patients with intrin-sic sphincter deficiency. Several synthetic injectable agents, such as polydimethylsiloxane and calcium hydroxylapatite are now used, as glutaraldehyde cross-linked (GAX) bovine dermal collagen is no longer commercially available.80 Anesthesia is easily obtained by using intraurethral 2% lidocaine jelly and/or transvaginal injection of the periurethral tissues with 5 mL of 1% lidocaine. The material is injected underneath the urethral mucosa at the bladder neck and proximal urethra at multiple positions, until mucosal bulk

1	of the periurethral tissues with 5 mL of 1% lidocaine. The material is injected underneath the urethral mucosa at the bladder neck and proximal urethra at multiple positions, until mucosal bulk has improved. Patients must dem-onstrate a negative reaction to a collagen skin test prior to injec-tion. The long-term cure rate is 20% to 30%, with an additional 50% to 60% of patients demonstrating improvement.72 Repeat injections are frequently necessary because of migration and dissolution of the collagen material.Mesh in Reconstructive Pelvic Surgery. As noted earlier, pelvic reconstructive surgery frequently uses polypropylene mesh to augment procedures in the hopes of providing long-lasting repair. However, use of permanent mesh is associated with complications, most notably mesh erosion. In 2011, the FDA issued an updated statement to stipulate the risks when using transvaginally inserted mesh for prolapse.81 Ultimately, this has led to categorizing transvaginal mesh products as class

1	2011, the FDA issued an updated statement to stipulate the risks when using transvaginally inserted mesh for prolapse.81 Ultimately, this has led to categorizing transvaginal mesh products as class III devices in 2016. In addition to appropriate patient selection, and extensive informed consent, the American Urogynecologic Society recommends appropriate training to perform the proce-dures and manage the complications.82,83GYNECOLOGIC CANCERVulvar CancerVulvar cancer is the fourth most common gynecologic cancer. The mean age at diagnosis is 65, though this has trended down over the last several decades.84 Evidence supports an HPV-dependent pathway of carcinogenesis with risk factors similar to VIN in approximately 60% of cases. A second pathway inde-pendent of HPV is associated with chronic inflammation, vul-var dystrophy.85 Patients usually present with a vulvar ulcer or mass. Pruritus is a common complaint, and vulvar bleeding or enlarged inguinal lymph nodes are signs of advanced

1	inflammation, vul-var dystrophy.85 Patients usually present with a vulvar ulcer or mass. Pruritus is a common complaint, and vulvar bleeding or enlarged inguinal lymph nodes are signs of advanced disease. Careful evaluation of the patient is necessary to rule out con-current lesions of the vagina and cervix. Biopsy is required and should be sufficiently deep to allow evaluation of the extent of stromal invasion. Vulvar carcinomas are squamous in 90% of cases. Other less common histologies include melanoma (5%), basal cell carcinoma (2%), and soft tissue sarcomas (1–2%).Spread of vulvar carcinoma is by direct local extension and via lymphatic microembolization. Hematogenous spread is uncommon except for vulvar melanoma. Lymphatic spread seems to follow a stepwise, predictable pattern traveling from superficial, above the cribriform fascia, to deep inguinofemo-ral nodes and ultimately the pelvic, external iliac, nodal basin Superficial inferiorepigastric v.Superficialexternalpudendal

1	from superficial, above the cribriform fascia, to deep inguinofemo-ral nodes and ultimately the pelvic, external iliac, nodal basin Superficial inferiorepigastric v.Superficialexternalpudendal v.Superficial femorallymph nodesGreat saphenous v.Fossa ovalisSuperficialcircumflex iliac v.Superficial inguinallymph nodesInguinal ligamentExternalinguinal ringRound ligamentFigure 41-17. Lymphatic drainage of the vulva delineated by Stanley Way.(Fig. 41-17).86,87 The node of Cloquet is an important sentinel node situated in the route of spread to the pelvic lymph nodes.Staging and primary surgical treatment are typically pre-formed as a single procedure and tailored to the individual patient (Table 41-6). Surgical staging accounts for the most important prognostic factors including tumor size, depth of invasion, inguinofemoral node status, and distant spread. The most conservative procedure should be performed in view of the high morbidity of aggressive surgical management. This typi-cally

1	depth of invasion, inguinofemoral node status, and distant spread. The most conservative procedure should be performed in view of the high morbidity of aggressive surgical management. This typi-cally involves radical resection of the vulvar tumor targeting a 1 to 2 cm margin around the lesion, and carried to the deep perineal fascia of the urogenital diaphragm with and ipsilateral or bilateral inguinofemoral lymphadenectomy (Fig. 41-18). For tumors ≤2 cm in size with ≤1 mm invasion (FIGO stage IA), lymphadenectomy may be safely omitted, and wide local or Table 41-62009 FIGO staging of vulvar carcinomaIATumor confined to the vulva or perineum, ≤2 cm in size with stromal invasion ≤1 mm, negative nodes1BTumor confined to the vulva or perineum, >2 cm in size or with stromal invasion >1 mm, negative nodesIITumor of any size with adjacent spread (1/3 lower urethra, 1/3 lower vagina, anus), negative nodesIIIATumor of any size with positive inguino-femoral lymph nodes(i) 1 lymph node

1	mm, negative nodesIITumor of any size with adjacent spread (1/3 lower urethra, 1/3 lower vagina, anus), negative nodesIIIATumor of any size with positive inguino-femoral lymph nodes(i) 1 lymph node metastasis ≥5 mm(ii) 1–2 lymph node metastasis(es) of <5 mmIIIB(i) 2 or more lymph nodes metastases ≥5 mm(ii) 3 or more lymph nodes metastases <5 mmIIICPositive node(s) with extracapsular spreadIVA(i) Tumor invades other regional structures (2/3 upper urethra, 2/3 upper vagina), bladder mucosa, rectal mucosa, or fixed to pelvic bone(ii) Fixed or ulcerated inguino-femoral lymph nodesIVBAny distant metastasis including pelvic lymph nodesModified with permission from Pecorelli S: Revised FIGO staging for carcinoma of the vulva, cervix, and endometrium, Int J Gynaecol Obstet. 2009 May;105(2):103-104.Brunicardi_Ch41_p1783-p1826.indd 180918/02/19 4:34 PM 1810SPECIFIC CONSIDERATIONSPART IIradical local excision are adequate. Patients with IB tumors have deeper invasion but negative nodes and

1	180918/02/19 4:34 PM 1810SPECIFIC CONSIDERATIONSPART IIradical local excision are adequate. Patients with IB tumors have deeper invasion but negative nodes and therefore carry an excellent prognosis. Stage II includes patients with local exten-sion and negative nodes and therefore carry a prognosis similar to other node-negative patients.Stage III disease includes patients with lymph node metas-tases, and stage IV disease is either locally advanced or distant metastasis. Treatment options for stage III and stage IV dis-ease include (a) chemoradiation followed by limited resection if needed; (b) radical vulvectomy; and (c) radical vulvectomy coupled with pelvic exenteration. External beam radiotherapy combined with radiosensitizing chemotherapy of cisplatin and 5-fluorouracil (5-FU) is emerging as the preferred initial management of advanced disease, followed by limited surgical resection of residual disease.88-90 Reconstruction of the vulva and groin, if needed, can be

1	(5-FU) is emerging as the preferred initial management of advanced disease, followed by limited surgical resection of residual disease.88-90 Reconstruction of the vulva and groin, if needed, can be accomplished using grafts and rota-tional or myocutaneous flaps depending on the size and type of defect.Inguinofemoral lymphadenectomy is indicated beyond clinical stage IA. Unilateral lymphadenectomy is recom-mended for lateralized lesions or bilateral for central lesions that cross the midline, or those involving the periclitoral area (Figs. 41-19 and 41-20). Complications of complete inguino-femoral lymphadenectomy include wound dehiscence or infec-tion and lymphedema. Sentinel lymph node biopsy (SLNB) is an alternative to inguinofemoral lymphadenectomy for selected patients with stage I or II disease and no palpable inguinofemo-ral nodes. SLNB appears to be effective in detecting inguino-femoral lymph node metastases without increasing the risk of groin recurrence while avoiding the

1	or II disease and no palpable inguinofemo-ral nodes. SLNB appears to be effective in detecting inguino-femoral lymph node metastases without increasing the risk of groin recurrence while avoiding the morbidities associated with complete inguinofemoral lymphadenectomy. Several prospec-tive studies support this approach.91,92 However, it is recognized that successful SLNB depends on operator experience. Surgeons with limited experience in SLNB (have performed fewer than 10 of these procedures) may choose to perform complete groin node dissection or use this procedure only for tumors that are less than 2 cm in size.Nodal failure in the groin and pelvis is difficult to treat successfully, and attention to primary management of these areas is key. Postoperative adjuvant inguinal and pelvic radio-therapy is indicated when inguinal lymph nodes are positive and is superior to pelvic lymphadenectomy, which has been largely abandoned. It is also indicated when the vulvectomy margins are

1	radio-therapy is indicated when inguinal lymph nodes are positive and is superior to pelvic lymphadenectomy, which has been largely abandoned. It is also indicated when the vulvectomy margins are positive or close positive for disease and further surgical management is not anatomically feasible.Vaginal CancerVaginal carcinoma is a rare gynecologic malignancy and accounts for about 3% of cancers affecting the female repro-ductive system.84 Squamous cell carcinomas account for 85% to 90% of cases; more than two-thirds of vaginal cancers are diagnosed in women 60 years of age or older. Risk factors are similar to other HPV-related cervical and vulvar cancers. Rare clear cell carcinoma of the vagina is associated to in utero expo-sure to diethylstilbestrol (DES), which is now largely of his-torical interest due to aging of the exposed cohort.93 Patients with vaginal cancer usually present with postmenopausal and/or postcoital bleeding and may also complain of vaginal discharge, vaginal

1	interest due to aging of the exposed cohort.93 Patients with vaginal cancer usually present with postmenopausal and/or postcoital bleeding and may also complain of vaginal discharge, vaginal mass, dysuria, hematuria, rectal bleeding, or pelvic pain, which may be indicative of advanced disease. Diagnosis is made via biopsy of suspicious lesions, which may require colposcopic guidance.85Figure 41-18. Extent of modified radical hemivulvectomy for stages I and II squamous cancer of the vulva.Superficial femoral nodesCribriformfasciaDeep femoral nodesFemoral a.Femoral n.Sartorius m.Iliopsoas m.FemurEpidermuslateralmedialAdductor longusPectineus m.Femoral v.Camper’s fasciaFigure 41-19. The anatomy of the inguinal triangle by cross-section.Pubic tubercleFemoral v.Sapheno-femoraljunctionFigure 41-20. Landmarks for choosing an incision for an inguinal lymphadenectomy.Brunicardi_Ch41_p1783-p1826.indd 181018/02/19 4:34 PM 1811GYNECOLOGYCHAPTER 41Vaginal cancer is staged clinically by pelvic

1	for choosing an incision for an inguinal lymphadenectomy.Brunicardi_Ch41_p1783-p1826.indd 181018/02/19 4:34 PM 1811GYNECOLOGYCHAPTER 41Vaginal cancer is staged clinically by pelvic exam, chest X-ray, cystoscopy, and proctoscopy (Table 41-7).94 Vaginal cancer spreads by local extension to adjacent pelvic structures, by lymphatic embolization to regional lymph nodes, and, less commonly, via the hematogenous route. Lymphatic drainage is complex, but in general, lesions in the upper vagina drain to the pelvic lymph nodes while lesions involving the lower third drain to the inguinofemoral lymph nodes.Stage I disease, involving the upper vagina, may be treated surgically or with intracavitary radiation therapy.86,87,95 Surgery consists of a radical hysterectomy, upper vaginectomy, and bilateral pelvic lymphadenectomy. Stage I disease in the mid to lower vagina is treated with radiation and concurrent chemo-therapy. External beam pelvic radiation is the mainstay of treat-ment for stages

1	pelvic lymphadenectomy. Stage I disease in the mid to lower vagina is treated with radiation and concurrent chemo-therapy. External beam pelvic radiation is the mainstay of treat-ment for stages II to IV and may be followed by intracavitary Table 41-7FIGO staging of vaginal carcinoma0Carcinoma in situ; intraepithelial neoplasia grade 3ITumor limited to the vaginal wallIITumor has involved the subvaginal tissue but has not extended to the pelvic wallIIITumor extends to the pelvic wallIVTumor has extended beyond the true pelvis or has involved the mucosa of the bladder or rectumIVATumor invades bladder and/or rectal mucosa and/or direct extension beyond the true pelvisIVBDistant metastasisand/or interstitial brachytherapy. Prognosis for treated early stage disease is excellent with more than 90% 5-year survival rates. Advanced stage disease, however, carries a poor progno-sis with only 15% to 40% 5-year survival rates.Cervical CancerGeneral Principles. There are over 12,000 new cases

1	90% 5-year survival rates. Advanced stage disease, however, carries a poor progno-sis with only 15% to 40% 5-year survival rates.Cervical CancerGeneral Principles. There are over 12,000 new cases of cervical cancer and over 4000 cervical cancer deaths annually in the United States.96 It is a major killer worldwide causing 275,000 deaths annually.97 Risk factors for cervical squamous cell and adenocarcinoma, the two most common histologies, are largely related to acquisition of and immune response to carcinogenic subtypes of the HPV virus. Cervical screening is correlated with early identification and treatment of preinvasive disease.98 Cervical cancer is most commonly identified in women with long intervals between screenings, or with no prior screening. It is also associated with early age at first intercourse, multiple sexual partners, smoking, and oral contraceptive use.Early cervical cancer is usually asymptomatic, though irregu-lar or postcoital bleeding may be present,

1	early age at first intercourse, multiple sexual partners, smoking, and oral contraceptive use.Early cervical cancer is usually asymptomatic, though irregu-lar or postcoital bleeding may be present, particularly in more advanced disease. The diagnosis of cervical cancer is made by cervical biopsy, either of a gross lesion or a colposcopically-identified lesion. Cervical cancer is staged clinically due to the high disease burden in the developing world.99 Despite the prog-nostic value of clinical staging, in the developed world, surgical and radiologic staging is used to determine the extent of tumor spread and identify lymph node involvement. Lymph node metastasis is common and one of the most important prognostic factors in this disease, and positron emission tomography scans are useful in pretreatment planning and determination of radia-tion fields for women with locally advanced disease. Staging and management options are outlined in Table 41-8.7Table 41-82009 FIGO cervical cancer

1	in pretreatment planning and determination of radia-tion fields for women with locally advanced disease. Staging and management options are outlined in Table 41-8.7Table 41-82009 FIGO cervical cancer staging and management optionsSTAGEDESCRIPTIONOPTIONS FOR MANAGEMENT0Carcinoma in situAdenocarcinoma in situ: simple hysterectomy, may be followed for fertility preservation if all margins negative on coneSquamous cell carcinoma in situ: local excision with LEEP or cone or laser ablationIConfined to the cervixA1: Confined to the cervix, diagnosed only by microscopy with invasion of ≤3 mm in depth and lateral spread ≤7 mmA2: Confined to the cervix, diagnosed with microscopy with invasion of >3 mm and <5 mm with lateral spread ≤7 mmB1: Clinically visible lesion or greater than A2, ≤4 cm in greatest dimensionB2: Clinically visible lesion, >4 cm in greatest dimensionA1 and some A2: fertility preservation through large cone followed by close monitoring, followed by hysterectomyB1 and B2:

1	greatest dimensionB2: Clinically visible lesion, >4 cm in greatest dimensionA1 and some A2: fertility preservation through large cone followed by close monitoring, followed by hysterectomyB1 and B2: radical hysterectomy or chemoradiation; radical trachelectomy with uterine preservation for childbearing is under investigation for highly selected patients with small lesionsIIA1: Involvement of the upper two-thirds of the vagina, without parametrial invasion, ≤4 cm in greatest dimensionA2: >4 cm in greatest dimensionB: Parametrial involvementFor some IIA radical hysterectomy may be consideredIIA and B: chemoradiation is preferredIIIA. Involvement of the lower third of the vaginaB. Involvement of a parametria to the sidewall or obstruction of one or both ureters on imagingChemoradiationIVA. Local involvement of the bladder or rectumB. Distant metastasesA. ChemoradiationB. Chemotherapy with palliative radiation as indicatedData from Pecorelli S: Revised FIGO staging for carcinoma of the

1	Local involvement of the bladder or rectumB. Distant metastasesA. ChemoradiationB. Chemotherapy with palliative radiation as indicatedData from Pecorelli S: Revised FIGO staging for carcinoma of the vulva, cervix, and endometrium, Int J Gynaecol Obstet. 2009 May;105(2):103-104.Brunicardi_Ch41_p1783-p1826.indd 181118/02/19 4:34 PM 1812SPECIFIC CONSIDERATIONSPART IIProcedures for Cervical Cancer Treatment. Certain cervical cancers that are confined to the cervix may be treated surgically. Very small lesions (less than 7 mm wide, less than 3 mm deep) with no LVSI may be treated with simple hysterectomy. In a woman who desires future fertility, a cone biopsy with negative surgical margins may be an acceptable alternative. Any tumor larger than this (larger than stage IA1) should be treated with radical hysterectomy or in special cases radical trachelectomy for fertility preservation. Some authors advocate a large cone biopsy with lymph node dissection for stage IA2 tumors in patients

1	with radical hysterectomy or in special cases radical trachelectomy for fertility preservation. Some authors advocate a large cone biopsy with lymph node dissection for stage IA2 tumors in patients who desire future fertility, though this recommenda-tion is somewhat controversial. Tumors that are greater than 4 cm in size are most often treated with chemoRT even if they Figure 41-21. Radical hysterectomy.BAUterusOvaryFallopian tubeCRound ligamentVesicouterinefoldUterinevesselsEDPararectalspaceLymphnodesParavesical spaceFExternal iliac vesselsInternal iliac arteryGHISuperior vesicalarteryUterine arteryare confined to the cervix, given the high likelihood of need for postoperative radiotherapy due to cervical risk factors.Radical Hysterectomy This procedure may be performed via laparotomy, or increasingly via a minimally invasive (laparo-scopic or robotic) approach.100 The key elements are dissection of the pelvic and periaortic nodes and the dissection of the para-metrium from the

1	or increasingly via a minimally invasive (laparo-scopic or robotic) approach.100 The key elements are dissection of the pelvic and periaortic nodes and the dissection of the para-metrium from the pelvic sidewall to allow en bloc removal with the uterus. The principle steps of an open procedure are demon-strated in Fig. 41-21. In contrast to a typical simple hysterectomy, the radical hysterectomy involves dissection much closer to the bowel, bladder, ureters, and great vessels, resulting in a higher complication rate to these organs. Additionally, disruption of the Brunicardi_Ch41_p1783-p1826.indd 181218/02/19 4:35 PM 1813GYNECOLOGYCHAPTER 41MUreterVaginaJKOvary and ligamentFallopian tubeUreterLUterosacralligamentFigure 41-21. (Continued)nerves supplying the bladder and the rectum, which traverse the cardinal and uterosacral ligaments, may result in temporary or long-term bladder and bowel dysfunction. Radical hysterecto-mies allow for the maintenance of the ovaries since the

1	which traverse the cardinal and uterosacral ligaments, may result in temporary or long-term bladder and bowel dysfunction. Radical hysterecto-mies allow for the maintenance of the ovaries since the incidence of metastases to this area is very low, providing a clear advantage of surgery over radiation therapy in the younger patient.Radical Trachelectomy Interest in fertility preservation with stages IA1 and 2, and stage IB1 lesions has led to the develop-ment of methods of radical trachelectomy with uterine preserva-tion. This procedure depends on an adequate blood supply to the uterus from the ovarian anastamoses, as the cervical portion is removed. The lower uterine segment closed with a cerclage and attached directly to the vaginal cuff. The rates of recurrence, pregnancy outcomes, and the best surgical candidates for this surgery are still under study,101 but there are sufficient numbers and experience, both obstetric and surgical, to suggest that this procedure is oncologically

1	the best surgical candidates for this surgery are still under study,101 but there are sufficient numbers and experience, both obstetric and surgical, to suggest that this procedure is oncologically safe and allows live births.Pelvic Exenteration for Recurrent Disease (Fig. 41-22) Cervical cancer recurrences after primary surgical management are treated with radiation. Surgery may be a consideration in selected patients with recurrent cervical cancer who have received maximal radiation therapy. If the recurrence is locally confined with no evidence of spread or metastatic disease, then pelvic exenteration may be considered. Attempted exenteration procedures are aborted intraoperatively if metastatic disease is found. Exenteration is tailored for the disease size and location and may be supralevator or extend below the levator ani muscle and require vulvar resection. Reconstruction of the pelvis may require a continent urinary pouch (if radiation enteritis is limited) or ileal conduit

1	or extend below the levator ani muscle and require vulvar resection. Reconstruction of the pelvis may require a continent urinary pouch (if radiation enteritis is limited) or ileal conduit and colostomy, as well as rebuilding of the pelvic floor and vagina with grafts or myocutaneous flaps.Uterine CancerEndometrial Cancer. Endometrial cancer is the most com-mon gynecologic malignancy and fourth most common cancer in women.96 It is most common in menopausal women in the fifth decade of life; up to 15% to 25% of cases occur prior to menopause, and 1% to 5% occur before age 40. Risk factors for the most common type of endometrial cancer include increased exposure to estrogen without adequate opposition by progester-one, either endogenous (obesity, chronic anovulation) or exog-enous (hormone replacement). Additional risk factors include diabetes, Lynch II syndrome (hereditary nonpolyposis coli syn-drome), and prolonged use of tamoxifen. Tamoxifen is a mixed agonist/antagonist ligand for

1	replacement). Additional risk factors include diabetes, Lynch II syndrome (hereditary nonpolyposis coli syn-drome), and prolonged use of tamoxifen. Tamoxifen is a mixed agonist/antagonist ligand for the estrogen receptor. It is an ago-nistic in the uterus and an antagonistic to the breast and ovary. Protective factors for endometrial cancer include smoking and use of combination oral contraceptive pills. Adenocarcinomas are the most prevalent histologic type.Endometrial adenocarcinomas have historically been divided into type I and type II tumors with five classic histologic subtypes. Type I tumors are estrogen-dependent endometrioid Brunicardi_Ch41_p1783-p1826.indd 181318/02/19 4:35 PM 1814SPECIFIC CONSIDERATIONSPART IIFigure 41-22. Pelvic exenteration.histology and have a relatively favorable prognosis; they can be broken down further by presence or absence of microsatellite instability. Type II endometrial cancers are estrogen-independent, aggressive, and characterized by

1	favorable prognosis; they can be broken down further by presence or absence of microsatellite instability. Type II endometrial cancers are estrogen-independent, aggressive, and characterized by nonendometrioid, serous or clear cell, histology, or carcinosarcoma.102 Emerging data, however, suggest that the molecular features could provide reproducible subtypes that have the potential to guide and refine treatment. The most comprehensive molecular study of endometrial cancer to date has been The Cancer Genome Atlas, which included a combination of whole genome sequencing, exome sequencing, microsatellite instability assays, copy number analysis, and proteomics.103 Molecular information was used to classify 232 endometrial cancer patients into four groups: POLE ultramutated, MSI hypermutated, copy number low, and copy number high that correlated with progression-free survival.103 Two practical pared-down classification systems to identify four molecular subgroups with distinct prognostic

1	copy number low, and copy number high that correlated with progression-free survival.103 Two practical pared-down classification systems to identify four molecular subgroups with distinct prognostic outcomes have been described.104,105Postmenopausal bleeding is the most common presenta-tion of endometrial cancer and often permits early stage diag-nosis, resulting in a favorable prognosis. Abnormal bleeding should prompt endometrial evaluation and sampling, which is usually done with an office endometrial biopsy, though at times requires operative curettage or diagnostic hysteroscopy. Transvaginal ultrasonography (TVUS) often reveals a thickened endometrial stripe. An endometrial stripe measuring 5 mm or more in a postmenopausal patient with vaginal bleeding raises concern and should be followed by endometrial sampling; patients with stripe of 4 mm or less rarely have occult malig-nancy, and TVUS may thus be used to triage patients before invasive endometrial sampling. Even with a

1	followed by endometrial sampling; patients with stripe of 4 mm or less rarely have occult malig-nancy, and TVUS may thus be used to triage patients before invasive endometrial sampling. Even with a normal endometrial stripe, endometrial sampling should be performed for persistent postmenopausal bleeding. Uterine cancer is surgically staged and is graded based on the degree of histologic differentiation of the glandular components (Table 41-9).99 Grade is an important prognostic factor, independent of stage.Treatment is surgical, and most commonly involves hysterectomy, bilateral salpingo-oophorectomy, peritoneal cytology, and resection of any gross disease.87 Evidence supports equivalent oncologic outcomes with minimally invasive approaches.106 The inclusion and utility of lymphadenectomy remains an area of controversy. If a lymph node dissection is performed, it may be performed via laparotomy or laparoscopy. Generally, the bilateral pelvic and para-aortic lymph nodes are removed.

1	remains an area of controversy. If a lymph node dissection is performed, it may be performed via laparotomy or laparoscopy. Generally, the bilateral pelvic and para-aortic lymph nodes are removed. The pelvic node dissection includes: bilateral removal of nodal tissue from the distal one-half of each common iliac artery, the anterior and medial aspect of the proximal half of the external iliac artery and vein, and the distal half of the obturator fat pad anterior to the obturator nerve. Most of the pelvic lymph nodes lie anterior, medially, and posteriorly to the external and internal iliac vessels and the obturator nerve. There are a few nodes that lie lateral to these structures, between the vessels and the pelvic sidewall, and these are generally removed in a complete dissection. The para-aortic lymph nodes include resection of nodal tissue over the distal vena cava from the level of the inferior mesenteric artery to the mid right common iliac artery and between the aorta and

1	The para-aortic lymph nodes include resection of nodal tissue over the distal vena cava from the level of the inferior mesenteric artery to the mid right common iliac artery and between the aorta and the left ureter from the inferior mesenteric artery to the left mid common iliac artery. Some also advocate resection of lymph nodes between the IMA and the gonadal vessels, as some uterine fundal tumors may drain directly into these lymph nodes.107The need for postoperative intervention is individualized based on the histology, stage, and risk factors such as age, lym-phvascular space invasion, and histology. Early-stage patients Table 41-92009 International Federation of Gynecology and Obstetrics staging of carcinoma of the uterine corpusI ATumor confined to the uterus, no or <½ myometrial invasionI BTumor confined to the uterus, >½ myometrial invasionIICervical stromal invasion, but not beyond uterusIII ATumor invades serosa or adnexaIII BVaginal and/or parametrial involvementIII

1	invasionI BTumor confined to the uterus, >½ myometrial invasionIICervical stromal invasion, but not beyond uterusIII ATumor invades serosa or adnexaIII BVaginal and/or parametrial involvementIII C1Pelvic-node involvementIII C2Para-aortic involvementIV ATumor invasion bladder and/or bowel mucosaIV BDistant metastases including abdominal metastases and/or inguinal lymph nodesData from Pecorelli S: Revised FIGO staging for carcinoma of the vulva, cervix, and endometrium, Int J Gynaecol Obstet. 2009 May;105(2):103-104.Brunicardi_Ch41_p1783-p1826.indd 181418/02/19 4:35 PM 1815GYNECOLOGYCHAPTER 41are typically cured with surgery alone, while patients with high-intermediate risk factors, as defined by collaborative tri-als groups, commonly receive intracavitary brachytherapy to decrease local recurrence.108,109 Patients with advanced disease and high-grade histologies commonly receive platinum-based chemotherapy with or without radiation.Similar to the case with vulvar cancer described

1	recurrence.108,109 Patients with advanced disease and high-grade histologies commonly receive platinum-based chemotherapy with or without radiation.Similar to the case with vulvar cancer described earlier, sentinel node biopsy is becoming more prevalent in endome-trial cancer. A sentinel lymph node biopsy may be considered in apparent uterine-confined malignancy when there is no metasta-sis demonstrated by imaging studies or no obvious extrauterine disease at exploration. For this procedure, most frequently the cervix is injected with ICG dye, and the immunofluorescence detecting camera is used either robotically or laparoscopically to identify the sentinel node. If no node is mapped, a full lymph-adenectomy is generally advised.110Lynch Syndrome. Lynch syndrome, a cancer family syn-drome also known as hereditary nonpolyposis colorectal cancer (HNPCC), is an autosomal dominant inherited predisposition to develop colorectal carcinoma and extracolonic cancers, pre-dominantly including

1	also known as hereditary nonpolyposis colorectal cancer (HNPCC), is an autosomal dominant inherited predisposition to develop colorectal carcinoma and extracolonic cancers, pre-dominantly including tumors of the uterus and ovaries, now also including breast cancer.111 Genes involved in HNPCC are those required for proper single-strand DNA repair via the mismatch repair pathway; most commonly involved are MLH1, MSH2, MSH6, and PMS2. The risk of colorectal carcinoma is as high as 75% by age 75 years. Affected women have a 40% and 10% lifetime risk of developing uterine and ovarian cancers, respec-tively. Surveillance has not been proven to identify disease in early stage for these patients, though it is recommended and should include annual cervical cytology, mammography, trans-vaginal ultrasonography, CA-125 measurements, and an endo-metrial biopsy. Risk-reducing salpingo-oophorectomy with hysterectomy is now being recommended for women who have completed childbearing, ideally 5 to 10

1	CA-125 measurements, and an endo-metrial biopsy. Risk-reducing salpingo-oophorectomy with hysterectomy is now being recommended for women who have completed childbearing, ideally 5 to 10 years earlier than the first case of endometrial or ovarian cancer in the family. Dys-regulation of the mismatch repair pathway leads to the micro-satellite instability phenotype, now known be associated with susceptibility to select immunotherapy agents.Uterine Sarcomas. Uterine sarcomas arise from the uterine muscle and connective tissue elements and are typically aggres-sive tumors with a poorer prognosis compared to the more common endometrial carcinomas. The most common histopath-ologic types are endometrial stromal sarcomas, undifferentiated endometrial sarcomas, and leiomyosarcomas. Risk factors are challenging to assess but may include prior pelvic radiation and tamoxifen exposure. Patients typically present with bleeding or mass effects, although some are discovered incidentally at the time

1	challenging to assess but may include prior pelvic radiation and tamoxifen exposure. Patients typically present with bleeding or mass effects, although some are discovered incidentally at the time of hysterectomy for other indications. Leiomyosarcoma is the most common uterine sarcoma, and hysterectomy with salpingoophorectomy is the treatment of choice. Lymph node metastases are rare in sarcomas in general, and in the absence of palpable nodes or extrauterine disease. There are limited data to support cytoreduction when extrauterine disease is present. The benefits of adjuvant therapy are unknown. Advanced disease is typically treated with systemic chemotherapy.112Ovarian CancerEpithelial Ovarian, Tubal, and Primary Peritoneal Cancer. Ovarian cancer is a rare disease affecting 1 in 70 women with a median age at diagnosis of 62 years.96 Epithelial malignancies make up the vast majority of ovarian cancers. The majority of women (70%) are diagnosed at with advanced staged disease

1	70 women with a median age at diagnosis of 62 years.96 Epithelial malignancies make up the vast majority of ovarian cancers. The majority of women (70%) are diagnosed at with advanced staged disease leading to the poor survival associated with this malignancy. Survival in advanced disease is due both to late diagnosis and lack of effective second-line cytotoxic therapy for the major-ity of patients who relapse following initial clinical complete response to platinum-based chemotherapy. Despite multiple pro-spective population based trials evaluating the use of CA-125, ultrasound, or combinations of these tests for early detection of disease, a mortality benefit to screening programs has not been demonstrated.113-116 Symptoms for either benign or malignant ovarian tumors are nonspecific but frequent, and they include bloating, pelvic or abdominal pain, difficulty eating or feeling full quickly, and urinary symptoms of urgency or frequency,117 which form the basis of an ovarian cancer

1	frequent, and they include bloating, pelvic or abdominal pain, difficulty eating or feeling full quickly, and urinary symptoms of urgency or frequency,117 which form the basis of an ovarian cancer symptom index (Table 41-10). When newly developed and persistent, these symptoms should prompt an evaluation specifically targeted for identification of gynecologic malignancy.The histologic heterogeneity of ovarian cancer has long been recognized, but with the emergence of more robust clini-copathologic, molecular, and genetic data over the past decade these distinctions have become more clearly defined. Type I tumors consist of low-grade serous (LGS), low-grade endome-trioid, clear cell carcinomas (CCC), and mucinous carcinomas and are characterized by mutations in KRAS, BRAF, PTEN, PIK3CA, CTNNB1, ARID1A, and PPP2R1A. Type II ovarian cancers are the most common of the ovarian cancer histotypes, consisting of high-grade serous (70%), high-grade endometri-oid, carcinosarcoma, and

1	PIK3CA, CTNNB1, ARID1A, and PPP2R1A. Type II ovarian cancers are the most common of the ovarian cancer histotypes, consisting of high-grade serous (70%), high-grade endometri-oid, carcinosarcoma, and undifferentiated carcinomas. Type II tumors are defined by TP53 mutations, which are rare in type I cancers.118-121 Each of these types have distinct risk factors and potential precursor lesions.121Risk factors for development of ovarian cancer include hormonal factors such as early menarche, late menopause, and nulliparity. The use of oral contraceptives reduces risk of ovar-ian carcinoma—this risk reduction persists for up to 30 years after cessation of use.122 Additionally, tubal ligation and hyster-ectomy decrease population level epithelial ovarian cancer risk. Genetic predisposition to breast or ovarian cancer is the most important known risk for the development of ovarian cancer, and 18% to 24% of ovarian carcinomas may arise in conjunction with a hereditary predisposition.123-128

1	breast or ovarian cancer is the most important known risk for the development of ovarian cancer, and 18% to 24% of ovarian carcinomas may arise in conjunction with a hereditary predisposition.123-128 Germline genetic muta-tions are far more common among type II ovarian cancers, while endometriosis and hormonal factors predispose to type I ovarian malignancies.121,126,129Since 2007, the National Comprehensive Cancer Network guidelines began recommending that all women diagnosed with ovarian cancer receive genetic testing as up to 20% of ovarian cancer patients are BRCA1/2 mutation carriers.127,130-134 Although family history of breast and/or epithelial ovarian cancer is one of the strongest factors for lifetime risk of having breast or epithelial ovarian cancer, up to 50% of women with ovarian cancer who test positive for a BRCA mutation have no fam-ily history of either malignancy, supporting the importance of testing all women with a personal diagnosis of ovarian cancer, regardless

1	cancer who test positive for a BRCA mutation have no fam-ily history of either malignancy, supporting the importance of testing all women with a personal diagnosis of ovarian cancer, regardless of family history. The identification of deleterious mutations allows for cascade testing. Relatives of the affected patient are referred for genetic testing limited to the identified mutation. The lifetime risk for the development of ovarian can-cer for carriers of mutations in the BRCA1 and BRCA2 genes Brunicardi_Ch41_p1783-p1826.indd 181518/02/19 4:35 PM 1816SPECIFIC CONSIDERATIONSPART IIis estimated to be between 20% and 45% and 10% and 20%, respectively.123,130,135One of the challenges associated with early detection of ovarian cancer has historically been the lack of an identifiable precursor lesion. In 2001, however, “dysplastic changes” in the fallopian tubes removed from women with increased risk of developing ovarian carcinoma were first described.136 Subse-quent careful

1	precursor lesion. In 2001, however, “dysplastic changes” in the fallopian tubes removed from women with increased risk of developing ovarian carcinoma were first described.136 Subse-quent careful microscopic examination using a newly developed “sectioning and extensively examining of the fimbriated end” protocol (SEE-FIM) of the grossly normal fallopian tubes and ovaries from women with BRCA1/2 mutations revealed occult tubal cancer and precancers designated as serous tubal intraepi-thelial carcinoma. The relationship between serous tubal intraep-ithelial carcinomas and high-grade serous and endometrioid cancers is supported by the ubiquitous presence of TP53 muta-tions and their typical location within the fimbriated end of the fallopian tube.118,121,137 High-grade, serous epithelial cancers of the ovary, fallopian tube, and peritoneum are now recognized to have a common fallopian tubal precursor lesion and often com-bined under the rubric of epithelial ovarian cancer (HGSOC).For

1	cancers of the ovary, fallopian tube, and peritoneum are now recognized to have a common fallopian tubal precursor lesion and often com-bined under the rubric of epithelial ovarian cancer (HGSOC).For women at increased risk of ovarian cancer, the only confirmed prevention strategy is risk-reducing salpingo-oopherectomy.138,139 The lifetime risk of HGSOC is reduced to under 3% with risk-reducing salpingo-oopherectomy. A modern understanding of the fallopian tube as the site of origin for many ovarian cancers has led to the suggestion that opportunistic salpingectomy could be implemented as a potential cancer prevention strategy in the general population. Scandinavian population-based cohort studies have demon-strated a significant decrease in epithelial ovarian cancer following salpingectomy.140,141 Opportunistic salpingectomy is feasible among women undergoing tubal ligation, hysterectomy, or other pelvic surgery.142 Early Staged Ovarian Cancer. Early stage epithelial ovarian cancer

1	Opportunistic salpingectomy is feasible among women undergoing tubal ligation, hysterectomy, or other pelvic surgery.142 Early Staged Ovarian Cancer. Early stage epithelial ovarian cancer has an excellent outcome. Low grade, stages IA and B disease can be cured in up to 90% to 95% of cases by a complete surgical procedure. The prevailing position in the United States is that such patients do not benefit from chemotherapy.143 8The standard of care for women with stages IC and II, and all women with grade 3 or clear cell histology, is adjuvant che-motherapy with 3 to 6 cycles of platinumand taxane-based chemotherapy.144Advanced Ovarian Cancer. A pelvic mass with ascites, an omental cake, and an elevated CA-125 is pathognomonic for advanced ovarian cancer. CT scan is the imaging modality of choice to evaluate the upper abdomen and potential resect-ability of disease. Concerning physical or radiographic exam findings should prompt referral to a gynecologic oncologist (Table 41-10), as

1	of choice to evaluate the upper abdomen and potential resect-ability of disease. Concerning physical or radiographic exam findings should prompt referral to a gynecologic oncologist (Table 41-10), as studies demonstrate inferior patient outcome for women who have had primary surgery by nongynecologic oncologists.The objectives of surgery in ovarian cancer are threefold. The first is to make the histologic diagnosis. The second is to assess the extent of disease through complete surgical staging (Tables 41-11 and 41-12). When epithelial ovarian cancer is identified on frozen section and disease is grossly limited to the pelvis, complete staging with node dissection will upstage nearly one-third of patients.145 The third objective is (when feasible) surgical cytoreduction or debulking. The extent of disease upon entering the abdomen and the residual disease upon completion of the debulking surgery are independent prognostic variables for patient outcome. The Gynecologic Oncology Group

1	extent of disease upon entering the abdomen and the residual disease upon completion of the debulking surgery are independent prognostic variables for patient outcome. The Gynecologic Oncology Group has defined optimal residual disease as residual tumor ≤1 cm in the largest diameter. However, more contemporary data suggest that the most favorable survival outcomes are associated with complete cytoreduction to no gross residual disease.146 Decisions about the benefits and risks of radical debulking for individual presentations and diverse pathology depend on the age and medical stability of the patient, as well as the pathologic type of the cancer.The publication of two randomized prospective trials of neoadjuvant chemotherapy (NACT) for ovarian cancer has led to a questioning of the dogma of maximum surgical effort. Both trials revealed no survival difference compared to primary deb-ulking.147,148 In a patient who is medically compromised or in whom complete primary cytoreduction is

1	of maximum surgical effort. Both trials revealed no survival difference compared to primary deb-ulking.147,148 In a patient who is medically compromised or in whom complete primary cytoreduction is unlikely, neoadjuvant Table 41-10Ovarian cancer symptom index (2007) and ACOG guidelines for patient referral to gynecologic oncologyOVARIAN CANCER SYMPTOM INDEXACOG GUIDELINES FOR REFERRAL OF PREMENOPAUSAL WOMEN WITH MASS SUSPICIOUS FOR OVCAACOG GUIDELINES FOR REFERRAL OF POSTMENOPAUSAL WOMEN WITH MASS SUSPICIOUS FOR OVCADevelopment of, change in, and/or persistence in:1 or more of:1 or more of:BloatingCA-125 >200 U/mLElevated CA-125Pelvic or abdominal painAscitesAscitesDifficulty eating or feeling full quicklyEvidence of abdominal or distant metastasisNodular or fixed pelvic massUrinary symptoms of urgency or frequencyFamily history of 1 or more first degree relatives with ovarian or breast cancerEvidence of abdominal or distant metastasisFamily history of one or more first-degree

1	symptoms of urgency or frequencyFamily history of 1 or more first degree relatives with ovarian or breast cancerEvidence of abdominal or distant metastasisFamily history of one or more first-degree relatives with ovarian or breast cancer ACOG = American Congress of Obstetricians and Gynecologists.Data from Goff BA, Mandel LS, Drescher CW, et al. Development of an ovarian cancer symptom index: possibilities for earlier detection. Cancer. 2007;109:221-227; Dearking AC, Aletti GD, McGree ME, Weaver AL, Sommerfield MK, Cliby WA. How relevant are ACOG and SGO guidelines for referral of adnexal mass? Obstet Gynecol. 2007;110:841-848.Brunicardi_Ch41_p1783-p1826.indd 181618/02/19 4:35 PM 1817GYNECOLOGYCHAPTER 41Table 41-112014 International Federation of Gynecology and Obstetrics staging of epithelial ovarian cancerITumor confined to ovaries or fallopian tube(s)T1IATumor limited to one ovary (capsule intact) or fallopian tubeNo tumor on ovarian or fallopian tube surfaceNo malignant

1	of epithelial ovarian cancerITumor confined to ovaries or fallopian tube(s)T1IATumor limited to one ovary (capsule intact) or fallopian tubeNo tumor on ovarian or fallopian tube surfaceNo malignant cells in the ascites or peritoneal washingsT1aIBTumor limited to both ovaries (capsules intact) or fallopian tubesNo tumor on ovarian or fallopian tube surfaceNo malignant cells in the ascites or peritoneal washingsT1bICTumor limited to one or both ovaries or fallopian tubes, with any of the following:IC1 Surgical spill intraoperativelyIC2 Capsule ruptured before surgery or tumor on ovarian or fallopian tube surfaceIC3 Malignant cells present in the ascites or peritoneal washingsT1cIITumor involves one or both ovaries or fallopian tubes with pelvic extension (below pelvic brim) or peritoneal cancer (Tp)T2IIAExtension and/or implants on the uterus and/or fallopian tubes/and/or ovariesT2aIIBExtension to other pelvic intraperitoneal tissuesT2bIIITumor involves one or both ovaries, or

1	cancer (Tp)T2IIAExtension and/or implants on the uterus and/or fallopian tubes/and/or ovariesT2aIIBExtension to other pelvic intraperitoneal tissuesT2bIIITumor involves one or both ovaries, or fallopian tubes, or primary peritoneal cancer, with cytologically or histologically confirmed spread to the peritoneum outside the pelvis and/or metastasis to the retroperitoneal lymph nodesT3IIIAMetastasis to the retroperitoneal lymph nodes with or without microscopic peritoneal involvement beyond the pelvisT1, T2, T3aN1IIIA1Positive retroperitoneal lymph nodes only (cytologically or histologically proven) IIIA1(i)Metastasis ≤10 mm in greatest dimension (note this is tumor dimension and not lymph node dimension)T3a/T3aN1IIIA1(ii)Metastasis >10 mm in greatest dimension IIIA 2Microscopic extrapelvic (above the pelvic brim) peritoneal involvement with or without positive retroperitoneal lymph nodesT3a/T3aN1IIIBMacroscopic peritoneal metastases beyond the pelvic brim ≤2 cm in greatest dimension,

1	(above the pelvic brim) peritoneal involvement with or without positive retroperitoneal lymph nodesT3a/T3aN1IIIBMacroscopic peritoneal metastases beyond the pelvic brim ≤2 cm in greatest dimension, with or without metastasis to the retroperitoneal lymph nodesT3b/T3bN1III CMacroscopic peritoneal metastases beyond the pelvic brim >2 cm in greatest dimension, with or without metastases to the retroperitoneal nodes (Note 1)T3c/T3cN1IVDistant metastasis excluding peritoneal metastases Stage IV A: Pleural effusion with positive cytologyStage IV B: Metastases to extra-abdominal organs (including inguinal lymph nodes and lymph nodes outside of abdominal cavity) (Note 2)Any T, any N, M1Reproduced with permission from Mutch DG, Prat J: 2014 FIGO staging for ovarian, fallopian tube and peritoneal cancer, Gynecol Oncol. 2014 Jun; 133(3):401-404.Table 41-12Components of comprehensive surgical staging and debulking of epithelial ovarian cancerVertical abdominal incision adequate to visualize the

1	cancer, Gynecol Oncol. 2014 Jun; 133(3):401-404.Table 41-12Components of comprehensive surgical staging and debulking of epithelial ovarian cancerVertical abdominal incision adequate to visualize the diaphragmsEvacuation of ascitesPeritoneal washings of each pelvic gutter and diaphragmEn bloc hysterectomy and bilateral salpingo-oopherectomyInfragastric omentectomyRetroperitoneal and pelvic lymph node dissectionExamination of the entire bowelRandom biopsies of apparently uninvolved areas of peritoneum, pericolic gutters, diaphragmchemotherapy followed by interval debulking may be more appropriate and is supported by recent randomized controlled trials. Typically, treatment with NACT includes three cycles of platinum-based chemotherapy prior to open debulking, then three additional cycles after surgery. Diagnostic laparoscopic evaluation prior to cytoreductive surgery has been suggested as a means to avoid unnecessary laparotomy, resulting in subop-timal cytoreduction. Patients deemed

1	after surgery. Diagnostic laparoscopic evaluation prior to cytoreductive surgery has been suggested as a means to avoid unnecessary laparotomy, resulting in subop-timal cytoreduction. Patients deemed not to be candidates for cytoreduction could proceed immediately to NACT at the time of tissue collection for definitive diagnosis. A Fagotti predictive index ≥8 (Table 41-13) is a predictor of suboptimal cytoreduc-tion in advanced ovarian cancer with reasonable sensitivity and high specificity.149 These recommendations currently apply to HGSOC, clear cell cancer, and high-grade endometrioid ovarian Brunicardi_Ch41_p1783-p1826.indd 181718/02/19 4:35 PM 1818SPECIFIC CONSIDERATIONSPART IIcancers. Low-grade tumors are less chemotherapy sensitive, and primary surgical resection is recommended when feasible. Standard of care adjuvant therapy of advanced stage epithe-lial ovarian cancer remains intravenous platinumand tax-ane-based chemotherapy.150 In 2006, the National Cancer Institute

1	when feasible. Standard of care adjuvant therapy of advanced stage epithe-lial ovarian cancer remains intravenous platinumand tax-ane-based chemotherapy.150 In 2006, the National Cancer Institute issued a clinical alert indicating that combination intrave-nous/intraperitoneal platinum/taxane postoperative chemotherapy should be considered first line for women with optimally cytore-duced EOC. This was the result of completion and analysis of three independent randomized clinical trials showing a significant survival advantage for intraperitoneal therapy.151,152 Intraperitoneal (IP) therapy is administered via an implanted 9.6 French venous port catheter with the port placed over the right or left costal 9margin. The catheter is tunneled caudad with insertion through the fascia in the lower abdomen and the tip in the pelvis. The IP cath-eter may be placed at the time of surgical debulking via an open laparotomy approach or prior to initiating chemotherapy via a laparoscopic approach. In

1	abdomen and the tip in the pelvis. The IP cath-eter may be placed at the time of surgical debulking via an open laparotomy approach or prior to initiating chemotherapy via a laparoscopic approach. In some centers, the IP catheter may be placed by interventional radiology with CT guidance.Patients who have suboptimally debulked advanced stage disease and/or who are not candidates for intraperitoneal ther-apy should receive intravenous adjuvant chemotherapy. Interest has increased in both dose dense IV chemotherapy dosing as well as incorporation of biologic agents.Secondary cytoreduction upon recurrence can be con-sidered (Table 41-14). Patients who have had a disease-free Table 41-13Laparoscopic assessment of advanced ovarian cancer to predict surgical resectabilityLAPAROSCOPIC FEATURESCORE 0SCORE 2Peritoneal carcinomatosisCarcinomatosis involving a limited area (along the paracolic gutter or the pelvic peritoneum) and surgically removable by peritonectomyUnresectable massive

1	0SCORE 2Peritoneal carcinomatosisCarcinomatosis involving a limited area (along the paracolic gutter or the pelvic peritoneum) and surgically removable by peritonectomyUnresectable massive peritoneal involvement as well as with a miliary pattern of distributionDiaphragmatic diseaseNo infiltrating carcinomatosis and no nodules confluent with the most part of the diaphragmatic surfaceWidespread infiltrating carcinomatosis or nodules confluent with the most part of the diaphragmatic surfaceMesenteric diseaseNo large infiltrating nodules and no involvement of the root of the mesentery as would be indicated by limited movement of the various intestinal segmentsLarge infiltrating nodules or involvement of the root of the mesentery indicated by limited movement of the various intestinal segmentsOmental diseaseNo tumor diffusion observed along the omentum up to the large stomach curvatureTumor diffusion observed along the omentum up to the large stomach curvatureBowel infiltrationNo bowel

1	diseaseNo tumor diffusion observed along the omentum up to the large stomach curvatureTumor diffusion observed along the omentum up to the large stomach curvatureBowel infiltrationNo bowel resection was assumed and no miliary carcinomatosis on the ansae observedBowel resection assumed or miliary carcinomatosis on the ansae observedStomach infiltrationNo obvious neoplastic involvement of the gastric wallObvious neoplastic involvement of the gastric wallLiver metastasesNo surface lesionsAny surface lesionTable 41-14Guidelines for secondary therapy of epithelial ovarian cancerTIME FROM COMPLETION OF PRIMARY THERAPYDEFINITIONINTERVENTIONProgression on therapyPlatinum-refractoryNo value of secondary debulking unless remediating complication such as bowel obstructionNon–platinum-based chemotherapyConsider clinical trialProgression within 6 months of completion of primary therapyPlatinum-resistantNo value of secondary debulking unless remediating complication such as bowel

1	chemotherapyConsider clinical trialProgression within 6 months of completion of primary therapyPlatinum-resistantNo value of secondary debulking unless remediating complication such as bowel obstructionNon–platinum-based chemotherapy consider adding bevacizumabConsider clinical trialProgression after 6 months post completion of primary therapyPlatinum-sensitiveConsider secondary debulking if greater than 12 months intervalConsider platinum +/− taxane +/− bevacizumab, +/− pegylated liposomal doxorubicin, +/− gemcitabineConsider maintenance PARP inhibitorConsider clinical trialBrunicardi_Ch41_p1783-p1826.indd 181818/02/19 4:35 PM 1819GYNECOLOGYCHAPTER 41period of at least 12 months following an initial complete clini-cal response to surgery and initial chemotherapy, who have no evidence of carcinomatosis on imaging, and who have disease that can be completely resected are considered optimal candi-dates. A randomized controlled trial reported in abstract form demonstrated a benefit

1	of carcinomatosis on imaging, and who have disease that can be completely resected are considered optimal candi-dates. A randomized controlled trial reported in abstract form demonstrated a benefit of secondary cytoreduction under strict entry criteria (DESKTOP3); the GOG-0213 study of secondary cytoreduction is maturing. Debulking surgery done after subse-quent relapses or in women with early recurrence has not been shown to result in an outcome benefit and should be used only to palliate disease complications.The most common cause of palliative surgery is bypass of bowel obstruction. The majority of women with advanced ovarian cancer will eventually develop and potentially die from malignant bowel obstruction. While management of these cases is controversial, in some cases surgical correction has been shown to prolong life and improve quality of life.153 Nonsurgical options include placement of a venting gastrostomy tube, per-formed endoscopically or surgically. Management of

1	correction has been shown to prolong life and improve quality of life.153 Nonsurgical options include placement of a venting gastrostomy tube, per-formed endoscopically or surgically. Management of malignant bowel obstruction in women with recurrent advanced disease should be individualized.Chemotherapy is the mainstay of therapy for recurrent EOC. Treatment approaches are based upon platinum sensitivity.154 Referral to an oncologist with specific expertise in chemothera-peutic treatment of ovarian cancer and access to clinical trials is important. In determining secondary and subsequent ther-apy, consideration of prior therapies, sites of disease, organs at risk from cancer, organs sustaining injury from prior ther-apy, and quality of life desires of patient should be taken into consideration.Ovarian Germ Cell Tumors. Ovarian germ cell tumors occur most commonly in women under age 30. The most common benign germ cell neoplasm is the mature cystic teratoma; approximately 1% of

1	Germ Cell Tumors. Ovarian germ cell tumors occur most commonly in women under age 30. The most common benign germ cell neoplasm is the mature cystic teratoma; approximately 1% of teratomas contain a secondary malig-nancy arising from one of the components, most commonly squamous cell cancer and most commonly in postmenopausal women. Malignant germ cell tumors often grow and dissemi-nate rapidly and are symptomatic. The rapid growth may be accompanied by torsion or rupture, producing an acute abdo-men and the need for emergent intervention. Because they are derived from primordial germ cells, many produce charac-teristic tumor markers. Immature teratomas comprise a sig-nificant proportion of malignant germ cell tumors and may be associated with elevated lactate dehydrogenase (LDH) or α-fetoprotein (AFP). Excluding teratomas, the most common malignant germ cell tumor is dysgerminoma, made up of pure undifferentiated germ cells. Bilaterality occurs in up to 15% of patients; lactate

1	α-fetoprotein (AFP). Excluding teratomas, the most common malignant germ cell tumor is dysgerminoma, made up of pure undifferentiated germ cells. Bilaterality occurs in up to 15% of patients; lactate dehydrogenase is commonly elevated, and elevated b-hCG may occur.Less common malignant germ cell tumors include endo-dermal sinus or yolk sac tumors, embyronal carcinomas, mixed germ cell neoplasms, polyembryomas, and choriocarcinomas. Endodermal sinus tumors may have elevated AFP levels in the blood while embryonal and mixed germ cell tumors may have elevated b-hCG, LDH, or AFP. Tumor markers are useful to fol-low during surveillance and definitive therapy. Other than com-pletely resected stage I, grade I immature teratoma, adjuvant chemotherapy with a platinum-containing regimen has been his-torically recommended.155 Because of the high response rates to chemotherapy and the long-term toxicity of treatment, a “watch and wait” approach with treatment only upon recurrence has been

1	been his-torically recommended.155 Because of the high response rates to chemotherapy and the long-term toxicity of treatment, a “watch and wait” approach with treatment only upon recurrence has been suggested as safe for selected, well-staged patients with germ cell tumors.156 The cure rate remains high, near 90% even when metastatic disease is present; recurrent disease is more difficult to eradicate.155Fertility preservation is the standard surgical approach for ovarian germ cell tumors as disease tends to be diagnosed at stage I, and salvage chemotherapy is overall extremely suc-cessful. Staging should include removal of the involved ovary, biopsy of any suspicious areas, pelvic and para-aortic node dis-section, and omentectomy. Hysterectomy or removal of the sec-ond ovary is rarely indicated.Growing teratoma syndrome is a rare sequela of germ cell malignancies. Characteristically, during or after chemotherapy slow-growing tumors will increase in size and may even com-press

1	indicated.Growing teratoma syndrome is a rare sequela of germ cell malignancies. Characteristically, during or after chemotherapy slow-growing tumors will increase in size and may even com-press surrounding organs. Malignant transformation within these masses has been described. Treatment is with surgical resection.157Ovarian Sex Cord-Stromal Tumors. Sex cord-stromal cell tumors, rare tumors, are derived from cells that support and surround the oocyte and can present with symptoms referable to endocrine activity of the tumor. These include granulosa cell tumors (female differentiated), fibroma-thecomas, and Sertoli-Leydig cell tumors (male differentiated). Granulosa cell tumors are the most common in this group and are a low-grade malignancy with fewer than 3% bilaterality. They are treated with conservative surgery, similar to germ cell tumors in young women.155 Hysterectomy and bilateral salpingo-oophorectomy is recommended for women who have completed childbearing. Nodal staging

1	with conservative surgery, similar to germ cell tumors in young women.155 Hysterectomy and bilateral salpingo-oophorectomy is recommended for women who have completed childbearing. Nodal staging can be safely omitted in the absence of grossly involve nodes and fertility preservation is possible in disease limited to one ovary, the most common presentation. Debulking surgery is recommended for more extensive disease. These tumors and the thecomas in the same class often stimulate estrogen production and can be found in association with endometrial hyperplasia and cancer (5%). Granulosa cell tumors can recur over a prolonged period given their low rate of proliferation and tendency for local or intraperitoneal recurrence. Inhibin has been shown to be elaborated by these tumors and often is followed to identify recurrence of the disease. The Sertoli/Leydig cell tumors can present with virilization as a primary symptom. Evaluation of the ovary when this symptom is found is always of

1	is followed to identify recurrence of the disease. The Sertoli/Leydig cell tumors can present with virilization as a primary symptom. Evaluation of the ovary when this symptom is found is always of value.Gestational Trophoblastic Disease. Gestational trophoblas-tic disease (GTD) is a spectrum of abnormal pregnancy–related trophoblastic proliferations. Premalignant histologic types include partial and complete hydatidiform moles. Primary sur-gery for diagnosis and initial therapy is a suction dilatation and curettage. Clinically, partial moles present as missed abortions and usually resolve with observation. Partial moles are triploid, usually XXY, which can result from dispermic fertilization of an egg. A previously described classical presentation of hyper-emesis gravidarum, hyperthyroidism, preeclampsia, pulmonary trophoblastic embolization, and uterine size larger than dates is rarely seen today because of routine ultrasound assessments during early pregnancy. Even in the first

1	preeclampsia, pulmonary trophoblastic embolization, and uterine size larger than dates is rarely seen today because of routine ultrasound assessments during early pregnancy. Even in the first trimester, however, a characteristic “snow storm” appearance may be seen on ultra-sound. Pathologic examination will demonstrate no fetal tissue and have a diploid karyotype resulting from paternal duplication occurring after loss of maternal genetic material, or occasionally Brunicardi_Ch41_p1783-p1826.indd 181918/02/19 4:35 PM 1820SPECIFIC CONSIDERATIONSPART IIwith dispermic fertilization of an empty egg. Often associated theca lutein ovarian cysts, which can be greater than 6 cm in diameter, are seen on ultrasound. They should be followed without surgical intervention as they resolve with removal or treatment of the GTD. Following uterine evacuation, patients with molar pregnancies must be followed closely with weekly b-hCGs until normal for 3 weeks and then monthly for at least 6 months.

1	or treatment of the GTD. Following uterine evacuation, patients with molar pregnancies must be followed closely with weekly b-hCGs until normal for 3 weeks and then monthly for at least 6 months. Contraception should be provided to allow for sur-veillance. Any increase in b-hCG should trigger further evalua-tion and consideration of chemotherapy.158,159Invasive moles, choriocarcinoma, and placental site tro-phoblastic tumors are malignant disorders. Invasive moles are diagnosed following the diagnosis of a molar pregnancy if any of the following are demonstrated: (a) a plateau of b-hCG lasts for four measurements over a period of 3 weeks or longer; (b) a rise in b-hCG for three consecutive weekly measurements over at least a period of 2 weeks or more; or (c) b-hCG level remains elevated for 6 months or more. Metastatic GTD can present on the cervix, vagina, liver, lung, or brain and should not be man-aged surgically. In a woman of reproductive age, a diagnosis of metastatic GTN can be

1	6 months or more. Metastatic GTD can present on the cervix, vagina, liver, lung, or brain and should not be man-aged surgically. In a woman of reproductive age, a diagnosis of metastatic GTN can be made without biopsy if a b-hCG is found to be elevated in the setting of widespread metastatic disease. In fact, given the incidence of bleeding complications biopsy is not recommend.Chemotherapy is the primary recommended therapy. Per 2000 FIGO staging and classification, a risk score of 6 and below is classified as low risk and above 6 is considered high risk (Table 41-15). Low-risk patients are treated with single agent chemotherapy (methotrexate or actinomycin-D); high-risk patients receive multiagent chemotherapy. In either case, chemotherapy continues until b-hCG levels have normalized. Modern salvage and cure rates are high, with 5-year survival of high-risk patients reported as high as 90%.160 Twelve months of surveillance with contraception is recommended following treatment in

1	Modern salvage and cure rates are high, with 5-year survival of high-risk patients reported as high as 90%.160 Twelve months of surveillance with contraception is recommended following treatment in order to allow complete surveillance for relapse.Beyond dilation and curettage, surgery may have a role in the management of GTD. Hysterectomy is recommended for placental site trophoblastic tumors for which metastasis is rare. Laparotomy may be indicated in the cases of uncontrolled intra-abdominal or uterine bleeding. Neurosurgery may be required if there is intracranial bleeding or increased intracranial pressure due to metastatic disease.159MINIMALLY INVASIVE GYNECOLOGIC SURGERYHysteroscopySee earlier section, “Hysteroscopy” under “Procedures Per-formed for Structural Causes of Abnormal Uterine Bleeding.”LaparoscopyThe standard method for gynecologic laparoscopy follows the same methods as all minimally invasive surgery. In general, a camera port is placed near the umbilicus. Sometimes

1	Bleeding.”LaparoscopyThe standard method for gynecologic laparoscopy follows the same methods as all minimally invasive surgery. In general, a camera port is placed near the umbilicus. Sometimes it must be placed more cephalad if the patient has a larger fibroid uterus. Two additional ports are placed laterally, usually just superior and medial to the anterior superior iliac spines. Single site lapa-roscopic procedures may improve cosmesis and reduce post-operative pain, but challenges including lack of triangulation and instrument crowding at the umbilicus make this technique challenging to apply to more complex procedures.161Robotic SurgeryOver the last decade, there has been increased use of robot-ics for gynecologic surgery. With the DaVinci robotic system, the surgeon sits at a console and visualizes the operative field with three-dimensional optics. The use of robotic surgery has been described for virtually every gynecologic procedure that has been performed abdominally or

1	and visualizes the operative field with three-dimensional optics. The use of robotic surgery has been described for virtually every gynecologic procedure that has been performed abdominally or laparoscopically. The lapa-roscopic instruments are “wristed” and move as the surgeon’s hands/fingers move the actuators at the console. Robotic surgery Table 41-15International Federation of Gynecology and Obstetrics/World Health Organization scoring system for gestational trophoblastic disease based on prognostic factors SCORE 0124Age<40>40––Antecedent pregnancyMoleAbortionTermInterval from index pregnancy, months<44–67–12>12Pretreatment hCG mIU/mL<103>103–104>104–105>105Largest tumor size including uterus, cm–3–4≥5–Site of metastases including uterusLungSpleen, kidneyGastrointestinal tractBrain, liverNumber of metastases identified–1–45–8>8Previous failed chemotherapy––Single drugTwo or more drugsBrunicardi_Ch41_p1783-p1826.indd 182018/02/19 4:35 PM 1821GYNECOLOGYCHAPTER 41uses a camera

1	liverNumber of metastases identified–1–45–8>8Previous failed chemotherapy––Single drugTwo or more drugsBrunicardi_Ch41_p1783-p1826.indd 182018/02/19 4:35 PM 1821GYNECOLOGYCHAPTER 41uses a camera port, two to three robotic ports, and an accessory port. More meticulous dissection, improved visualization, and ability to operate with lower intra-abdominal pressures make the robotic platform advantageous, especially in obese patients. Longer set-up time and increased cost, however, are distinct disadvantages. The robotic unit costs up to $2.3 million and is associated with annual maintenance costs of $180,000 a year.162There is significant data to support robotic surgery in gynecologic malignancy; however, most procedures can be per-formed successfully with either robotic or laparoscopic platform depending on operator comfort and skill set. One large study sug-gested a lower conversion to laparotomy rate for robotic versus laparoscopic hysterectomy, but this was not statistically

1	platform depending on operator comfort and skill set. One large study sug-gested a lower conversion to laparotomy rate for robotic versus laparoscopic hysterectomy, but this was not statistically signifi-cant: conversion to laparotomy for laparoscopic hysterectomy was 9.9% compared with 4.9% for robotic cases (P =.06).163Complications Pertinent to Gynecologic SurgeryAbdominal Wall Vessels. The vessel at greatest risk of injury during the lateral trocar placement is the inferior epigastric artery. The superficial epigastric vessels and the superficial circumflex iliac vessels can be injured as well (Fig. 41-23). The primary methods to avoid vessel injury are knowledge of the vessels at risk and their visualization prior to trocar placement, when possible. The superficial vessels often can be seen and avoided by transillumination of the abdominal wall with the laparoscope. In contrast, the larger inferior epigastric vessels cannot be seen by transillumination because of their deeper

1	be seen and avoided by transillumination of the abdominal wall with the laparoscope. In contrast, the larger inferior epigastric vessels cannot be seen by transillumination because of their deeper location; these vessels often can be seen laparoscopically and avoided as they course along the peritoneum between the lateral umbilical fold of the bladder and the insertion of the round ligament into the inguinal canal. Anatomic variation and anastomoses between vessels make it impossible to know the exact location of all the abdominal wall vessels. For this reason, other strategies also should be used to avoid vessel injury, including the use of trocars with conical tips rather than pyramid tips and the use of the smallest trocars possible lateral to the midline.Intestinal Injury. Another potentially serious complication of laparoscopic surgery is injury to either small or large intestines. 10An estimated incidence of bowel injury during laparoscopic gynecologic surgery is estimated to be

1	serious complication of laparoscopic surgery is injury to either small or large intestines. 10An estimated incidence of bowel injury during laparoscopic gynecologic surgery is estimated to be 0.13%, 41% of which had a delayed diagnosis.164 Bowel injury can occur at the time of trocar insertion, especially if the patient has had previous abdominal procedures that often result in bowel adhesions to the anterior abdominal wall peritoneum, but rates appear simi-lar regardless of entry technique. Due to the proximity of sur-gery to the bowel, thermal injury due to electrosurgery is also frequently implicated in intestinal injury. Time to diagnosis in these cases is typically several days postoperatively as a thermal injury takes time to mature and necrose.Urologic Injuries. A risk of injury to the urogenital tract is inherent to gynecologic surgery due to proximity. Prevention of injury and intraoperative recognition and repair are crucial to avoiding long-term sequelae. Most urogenital

1	to the urogenital tract is inherent to gynecologic surgery due to proximity. Prevention of injury and intraoperative recognition and repair are crucial to avoiding long-term sequelae. Most urogenital fistulae are the result of unrecognized injuries to the urogenital tract at the time of surgery.Bladder Injury. Placement of a Foley catheter prior to gyne-cologic surgery is critical to reducing risk of bladder injuries. Bladder injury during open or laparoscopic surgery results from retroperitoneal perforation during lower trocar placement or during sharp dissection of the bladder from the lower uterine segment during hysterectomy. The latter of these two situa-tions is usually recognized intraoperatively; the first sign of the former may be postoperative hematuria, lower-port incisional drainage, or pneumoturia during laparoscopy. Once diagnosed, large defects require layered closure, whereas smaller defects usually close spontaneously within days or weeks with the aid of transurethral

1	or pneumoturia during laparoscopy. Once diagnosed, large defects require layered closure, whereas smaller defects usually close spontaneously within days or weeks with the aid of transurethral catheter drainage.Ureteral Injury. Although ureteral injury is rare, occurring in less than 1% of gynecologic procedures, it is the most serious of the complications related to gynecologic surgery, particularly if unrecognized.165,166 There are three anatomic locations where the ureter is at risk during gynecologic procedures (see Fig. 41-5): (a) the ureter descends over the pelvic brim as it courses over the bifurcation of the common iliac artery into the external and internal iliac arteries just below the ovarian vessels; (b) in the pelvis, the ureter courses along the lateral aspect of the broad ligament to enter the base of the broad ligament; and (c) the ure-ter is found less than 2 cm lateral to the cervix, passing under the uterine artery and then medially over the anterior vaginal

1	broad ligament to enter the base of the broad ligament; and (c) the ure-ter is found less than 2 cm lateral to the cervix, passing under the uterine artery and then medially over the anterior vaginal for-nix before entering the trigone of the bladder—this is the most common location of ureteral injury. Ureteral injuries, including complete ligation, partial resection, or thermal injuries, usually will manifest within hours to days of surgery. Complete obstruc-tion most often manifests as flank pain, whereas the first sign of partial or complete transection may be symptoms of intra-abdominal irritation caused by urine leakage. Transperitoneal thermal injuries resulting from fulguration of endometriosis may be similar to those after transection, but the appearance of symp-toms may be delayed several days until tissue necrosis occurs.Routine cystoscopy following hysterectomy is advocated by some gynecologists. For procedures performed for prolapse or incontinence where injury to the

1	several days until tissue necrosis occurs.Routine cystoscopy following hysterectomy is advocated by some gynecologists. For procedures performed for prolapse or incontinence where injury to the urinary tract is highest, rou-tine cystoscopy is recommended. Consideration of a surgeon’s individual complication rate and the difficulty of an individ-ual procedure are considerations for the provision of routine cystoscopy.166Vaginal Vault Dehiscence. This complication of hysterec-tomy seems to be more common in laparoscopic and robotic DeepvesselsSuperficial vessels Inferiorepigastric DeepcircumflexiliacSuperficial epigastricSuperficialcircumflex iliacFigure 41-23. Location of anterior abdominal wall blood vessels.Brunicardi_Ch41_p1783-p1826.indd 182118/02/19 4:35 PM 1822SPECIFIC CONSIDERATIONSPART IIsurgeries. This may be due to the use of cautery in dividing the vaginal cuff or in the method of vaginal closure when done mini-mally invasively. Vaginal closure of the cuff

1	CONSIDERATIONSPART IIsurgeries. This may be due to the use of cautery in dividing the vaginal cuff or in the method of vaginal closure when done mini-mally invasively. Vaginal closure of the cuff appears to decrease the rate of vaginal cuff dehiscence in MIS hysterectomy.Hemodynamically stable women without bowel eviscera-tion may be candidates for transvaginal repair without abdomi-nal exploration. Vaginal approach may also be appropriate in select cases of evisceration in which the bowel can be com-pletely evaluated vaginally. Since bowel evisceration can lead to peritonitis and sepsis, all women with bowel eviscerations are considered to have a surgical emergency, and surgery should not be delayed for imaging. In most cases of bowel eviscera-tion, evaluation of the bowel by laparoscopy or laparotomy is indicated to ensure bowel integrity.REFERENCES 1. Anson B. Atlas of Human Anatomy. Philadelphia: WB Saunders, 1950. 2. Force USPST. Screening for gynecologic conditions with pel-vic

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1	versus primary surgery for newly diagnosed advanced ovar-ian cancer (CHORUS): an open-label, randomised, controlled, non-inferiority trial. Lancet. 2015;386:249-257. 149. Gómez-Hidalgo NR, Martinez-Cannon BA, Nick AM, et al. Predictors of optimal cytoreduction in patients with newly diagnosed advanced-stage epithelial ovarian cancer: time to incorporate laparoscopic assessment into the standard of care. Gynecol Oncol. 2015;137:553-558. 150. McGuire WP, Hoskins WJ, Brady MF, et al. Cyclophospha-mide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer [see com-ments]. N Engl J Med. 1996;334:1-6. 151. Armstrong DK, Bundy BN, Wenzel L, et al. Intraperitoneal cisplatin and paclitaxel in ovarian cancer. N Engl J Med. 2006;354:34-43. 152. Walker JL, Armstrong DK, Huang HQ, et al. Intraperitoneal catheter outcomes in a phase III trial of intravenous versus intraperitoneal chemotherapy in optimal stage III ovarian and primary peritoneal

1	JL, Armstrong DK, Huang HQ, et al. Intraperitoneal catheter outcomes in a phase III trial of intravenous versus intraperitoneal chemotherapy in optimal stage III ovarian and primary peritoneal cancer: a Gynecologic Oncology Group Study. Gynecol Oncol. 2006;100:27-32. 153. Chi DS, Phaeton R, Miner TJ, et al. A prospective outcomes analysis of palliative procedures performed for malignant intestinal obstruction due to recurrent ovarian cancer. Oncolo-gist. 2009;14:835-839. 154. Markman M, Reichman B, Hakes T, Jones W. Responses to second-line cisplatin-based intraperitoneal therapy in ovarian cancer: influence of a prior response to intravenous cisplatin. J Clin Oncol. 1991;9:1801-1805. 155. Gershenson DM. Treatment of ovarian cancer in young women. Clin Obstet Gynecol. 2012;55:65-74. 156. Mangili G, Sigismondi C, Lorusso D, et al. The role of stag-ing and adjuvant chemotherapy in stage I malignant ovarian Brunicardi_Ch41_p1783-p1826.indd 182518/02/19 4:35 PM 1826SPECIFIC

1	G, Sigismondi C, Lorusso D, et al. The role of stag-ing and adjuvant chemotherapy in stage I malignant ovarian Brunicardi_Ch41_p1783-p1826.indd 182518/02/19 4:35 PM 1826SPECIFIC CONSIDERATIONSPART IIgerm cell tumors (MOGTs): the MITO-9 study. Ann Oncol. 2017;28:333-338. 157. Merard R, Ganesan R, Hirschowitz L. Growing teratoma syn-drome: a report of 2 cases and review of the literature. Int J Gynecol Pathol. 2015;34:465-472. 158. Lurain JR. Gestational trophoblastic disease II: classification and management of gestational trophoblastic neoplasia. Am J Obstet Gynecol. 2011;204:11-18. 159. Ngan HYS, Seckl MJ, Berkowitz RS, et al. Update on the diagnosis and management of gestational trophoblastic dis-ease. Int J Gynecol Obstet. 2015;131:S123-S126. 160. Seckl MJ, Sebire NJ, Berkowitz RS. Gestational trophoblastic disease. Lancet. 2010;376:717-729. 161. Sinha R, Sundaram M, Mahajan C, et al. Single-incision total laparoscopic hysterectomy. J Minim Access Surg. 2011;7:78-82. 162. Sinha

1	Gestational trophoblastic disease. Lancet. 2010;376:717-729. 161. Sinha R, Sundaram M, Mahajan C, et al. Single-incision total laparoscopic hysterectomy. J Minim Access Surg. 2011;7:78-82. 162. Sinha RY, Raje SR, Rao GA. Three-dimensional lapa-roscopy: principles and practice. J Minim Access Surg. 2017;13:165-169. 163. Gaia G, Holloway RW, Santoro L, Ahmad S, Di Silverio E, Spinillo A. Robotic-assisted hysterectomy for endome-trial cancer compared with traditional laparoscopic and laparotomy approaches: a systematic review. Obstet Gynecol. 2010;116:1422-1431. 164. Llarena NC, Shah AB, Milad MP. Bowel injury in gyneco-logic laparoscopy: a systematic review. Obstet Gynecol. 2015;125:1407-1417. 165. Sharp HT, Adelman MR. Prevention, recognition, and man-agement of urologic injuries during gynecologic surgery. Obstet Gynecol. 2016;127:1085-1096. 166. Teeluckdharry B, Gilmour D, Flowerdew G. Urinary tract injury at benign gynecologic surgery and the role of cystos-copy: a systematic review

1	surgery. Obstet Gynecol. 2016;127:1085-1096. 166. Teeluckdharry B, Gilmour D, Flowerdew G. Urinary tract injury at benign gynecologic surgery and the role of cystos-copy: a systematic review and meta-analysis. Obstet Gynecol. 2015;126:1161-1169. 167. Centers for Disease Control and Prevention. Sexually Trans-mitted Diseases Treatment Guidelines: Pelvic Inflammatory Disease. Available: https://www.cdc.gov/std/tg2015/pid.htm. Accessed August 11, 2018. 168. Dearking AC, Aletti GD, McGree ME, Weaver AL, Som-merfield MK, Cliby WA. How relevant are ACOG and SGO guidelines for referral of adnexal mass? Obstet Gynecol. 2007;110:841-848. 169. Mutch DG, Prat J. 2014 FIGO staging for ovarian, fallopian tube and peritoneal cancer. Gynecol Oncol. 2014;133:401-404.Brunicardi_Ch41_p1783-p1826.indd 182618/02/19 4:35 PM

1	NeurosurgeryAshwin G. Ramayya, Saurabh Sinha, and M. Sean Grady 42chapterOverview 1827Neuroanatomy 1827Neurologic Examination 1829Diagnostic Studies / 1829Neurologic and Neurosurgical Emergencies 1831Raised Intracranial Pressure / 1831Brain Stem Compression / 1833Stroke / 1833Seizure / 1833Trauma 1833Head Trauma / 1833Spine Trauma / 1842Peripheral Nerve Trauma / 1847Cerebrovascular Disease 1849Ischemic Diseases / 1849Thrombotic Disease / 1849Embolic Disease / 1849Hemorrhagic Diseases / 1850Tumors of the Central Nervous System 1854Intracranial Tumors / 1854Metastatic Tumors / 1854Glial Tumors / 1855Neural Tumors and Mixed Tumors / 1857Neural Crest Tumors / 1857Miscellaneous Tumors / 1857Embryologic Tumors / 1858Spinal Tumors / 1859Future Directions / 1861Spine: Basic Concepts 1861Stability / 1861Neural Compression / 1862Patterns of Disease / 1862Spine Fusion Surgery / 1865Spinal Instrumentation / 1865Arthrodesis / 1866Peripheral Nerve 1866Peripheral Nerve Tumors / 1866Entrapment

1	/ 1861Neural Compression / 1862Patterns of Disease / 1862Spine Fusion Surgery / 1865Spinal Instrumentation / 1865Arthrodesis / 1866Peripheral Nerve 1866Peripheral Nerve Tumors / 1866Entrapment Neuropathies / 1867Autoimmune and Inflammatory Disorders / 1867Infection 1867Cranial / 1867Spine / 1868Functional Neurosurgery 1869Epilepsy Surgery / 1869Deep Brain Stimulation / 1869Trigeminal Neuralgia / 1871Stereotactic Radiosurgery 1871Arteriovenous Malformations / 1872Vestibular Schwannomas / 1872Intracranial Metastases / 1872Congenital and Developmental Anomalies 1872Dysraphism / 1872Spina Bifida Occulta / 1872Spina Bifida With Myelomeningocele / 1872Encephalocele / 1872Craniosynostosis / 1872Hydrocephalus / 1873Chiari I Malformation / 1873OVERVIEWNeurologic surgery provides the operative and nonoperative man-agement (i.e., prevention, diagnosis, evaluation, treatment, criti-cal care, and rehabilitation) of disorders of the central, peripheral, and autonomic nervous systems (ANSs).

1	and nonoperative man-agement (i.e., prevention, diagnosis, evaluation, treatment, criti-cal care, and rehabilitation) of disorders of the central, peripheral, and autonomic nervous systems (ANSs). Such disorders include those of the brain, meninges, skull and skull base, and their blood supply, including surgical and endovascular treat-ment of disorders of the intracranial and extracranial vasculature supplying the brain and spinal cord; disorders of the pituitary gland; disorders of the spinal cord, meninges, and vertebral col-umn, including those that may require treatment by fusion, instru-mentation, or endovascular techniques; and disorders of the cranial and spinal nerves throughout their distribution.An accurate history is the first step toward neurologic diagnosis. A history of trauma or of neurologic symptoms is of obvious interest, but general constitutional symptoms are also important. Neurologic disease may have systemic effects, while diseases of other systems may affect

1	or of neurologic symptoms is of obvious interest, but general constitutional symptoms are also important. Neurologic disease may have systemic effects, while diseases of other systems may affect neurologic function. The patient’s general medical ability to withstand the physi-ologic stress of anesthesia and surgery should be understood. A detailed history from the patient and/or family, along with a reliable physical examination, will clarify these issues.NEUROANATOMYAn understanding of neuroanatomy is the foundation of com-prehensive neurologic examination and diagnosis. Salient fea-tures will be considered, from cephalad to caudad. The cerebral hemispheres (or telencephalon) consist of the cerebral cortex, underlying white matter, the basal ganglia, hippocampus, and amygdala. The cerebral cortex is the most recently evolved part of the nervous system. Its functions are mapped to discrete ana-tomic areas. The frontal areas are involved in executive function, decision making, and

1	cortex is the most recently evolved part of the nervous system. Its functions are mapped to discrete ana-tomic areas. The frontal areas are involved in executive function, decision making, and restraint of emotions. The motor strip, or precentral gyrus, is the most posterior component of the frontal lobes, and is arranged along a homunculus with the head inferior and lateral to the lower extremities superiorly and medially. The motor speech area (Broca’s area) lies in the left posterior inferior frontal lobe in almost all right-handed people and in up to 90% of left-handed people. The parietal lobe lies between the central sulcus anteriorly and the occipital lobe posteriorly. The postcen-tral gyrus is the sensory strip, also arranged along a homunculus. The rest of the parietal lobe is involved with awareness of one’s body in space and relative to the immediate environment, body orientation, and spatial relationships. The occipital lobes are most posterior. The visual cortex is

1	is involved with awareness of one’s body in space and relative to the immediate environment, body orientation, and spatial relationships. The occipital lobes are most posterior. The visual cortex is arrayed along the apposing 1Brunicardi_Ch42_p1827-p1878.indd 182701/03/19 7:16 PM 1828medial surfaces of the occipital lobes. The left occipital lobe receives and integrates data from the left half of each retina. A left occipital lesion would therefore result in an inability to see objects right of center. The temporal lobes lie below the Sylvian fissures. The hippocampus, amygdala, and lower optic radia-tions (Meyer’s loops) are important components of the temporal lobe and are involved in memory, emotion, and vision, respec-tively. The receptive speech area (Wernicke’s area) typically is found in the area of the left posterior superior temporal lobe and inferior parietal lobe. The basal ganglia include the caudate, putamen, globus pallidus, subthalamic nucleus, substantia nigra, and

1	found in the area of the left posterior superior temporal lobe and inferior parietal lobe. The basal ganglia include the caudate, putamen, globus pallidus, subthalamic nucleus, substantia nigra, and nucleus accumbens. These structures are involved in the selection, activation and termination of movement, and facilitate learning of appropriate context-dependent motor behaviors.Lying deep to the cerebral hemispheres is the diencepha-lon, which includes the thalamus and hypothalamus. The thala-mus is a key processor and relay circuit for most motor and sensory information traveling to or from cortex. The hypothala-mus regulates homeostasis via the autonomic and neuroendo-crine systems.The brain stem consists of the midbrain (mesencephalon), pons (metencephalon), and medulla (myelencephalon). Longi-tudinal fibers run through the brain stem, carrying motor and sensory information between the cerebral hemispheres and spi-nal cord. The corticospinal tract is the major motor tract, while the

1	Longi-tudinal fibers run through the brain stem, carrying motor and sensory information between the cerebral hemispheres and spi-nal cord. The corticospinal tract is the major motor tract, while the medial lemniscus and spinothalamic tracts are the major sensory tracts. The nuclei of cranial nerves III through XII are also located within the brain stem. These nerves relay the motor, sensory, and special sense functions of the eye, face, mouth, and throat.The cerebellum arises from the dorsal aspect of the brain stem. It integrates somatosensory, vestibular, and motor infor-mation for coordination and timing of movement. Midline, or vermian, lesions lead to truncal ataxia. Lateral, or hemispheric, lesions lead to tremor and dyscoordination in the extremities.The ventricular system is the cerebrospinal fluid (CSF)–containing contiguous space inside the brain, continuous with the subarachnoid space outside the brain. The paired lateral ventricles consist of temporal, occipital, and

1	the cerebrospinal fluid (CSF)–containing contiguous space inside the brain, continuous with the subarachnoid space outside the brain. The paired lateral ventricles consist of temporal, occipital, and frontal horns, as well as the main body. CSF travels from each lateral ventricle through the foramina of Monroe to the third ventricle, located between the left and right thalami. CSF then drains through the cerebral aqueduct to the fourth ventricle within the brain stem. The foramen of Magendie (midline) and paired foram-ina of Luschka (lateral) drain to the subarachnoid space. The approximate CSF volume in an average adult is 150 mL, and the choroid plexus produces approximately 500 mL of CSF per day.The spinal cord starts at the bottom of the medulla and extends caudally through the spinal canal to the first lumbar ver-tebra, approximately. Motor tracts (efferent pathways) continue from the brain stem down via the lateral and anterior corticospi-nal tracts to anterior horn cells, and

1	canal to the first lumbar ver-tebra, approximately. Motor tracts (efferent pathways) continue from the brain stem down via the lateral and anterior corticospi-nal tracts to anterior horn cells, and then exit via ventral nerve roots. Sensory information (afferent pathways) enters via dorsal nerve roots, travels cranially via the dorsal columns (proprio-ception and fine touch) or spinothalamic tract (pain and tem-perature), and into the brain stem. Paired nerves exit the spinal cord at each level. There are 31 pairs: 8 cervical, 12 thoracic, 5 lumbar, 5 sacral, and 1 coccygeal.The dorsal and ventral nerve roots at each level fuse to form mixed motor-sensory spinal nerves and spread through the body to provide innervation to muscles and sensory organs. The C5–T1 spinal nerves intersect in the brachial plexus and divide to form the main nerve branches to the arm, including the median, ulnar, and radial nerves. The L2–S4 spinal nerves intersect in the lumbosacral plexus, and divide to

1	the brachial plexus and divide to form the main nerve branches to the arm, including the median, ulnar, and radial nerves. The L2–S4 spinal nerves intersect in the lumbosacral plexus, and divide to form the main nerve branches to the leg, including the femoral and sciatic nerves.The principal motor tract of the spinal cord is the cortico-spinal tract. It is a two-neuron path, including an upper motor neuron and a lower motor neuron. The upper motor neuron cell body is located within the motor strip of the cerebral cortex. The axon travels through the internal capsule to the brain stem, decussates at the brain stem–spinal cord junction, and travels down the contralateral corticospinal tract to the lower motor Key Points1 Neurologic surgery specializes in primarily surgical manage-ment of central, peripheral, and autonomic nervous system disorders.2 Although clinical examination is paramount, neurosurgical diagnosis and treatment are aided largely by a variety of modalities, such as

1	central, peripheral, and autonomic nervous system disorders.2 Although clinical examination is paramount, neurosurgical diagnosis and treatment are aided largely by a variety of modalities, such as magnetic resonance imaging and intra-cranial pressure monitoring.3 The common treatment goals for traumatic brain and spinal injury are aimed at preventing secondary insults of hypoxia and hypotension.4 Aneurysmal subarachnoid hemorrhage remains one of the most morbid and intensive neurosurgical diseases. Endovas-cular therapy is a growing technology that allows for safer securing of ruptured aneurysms.5 Brain tumors can arise from primary or metastatic tissues. Treatment typically involves resection, followed by radia-tion and/or chemotherapy, depending on the type and grade of tumor.6 Spinal instrumentation is used for surgical stabilization of many types of spinal instability, including traumatic, infec-tious, oncologic, and degenerative.7 Infection of the nervous system is a serious and

1	is used for surgical stabilization of many types of spinal instability, including traumatic, infec-tious, oncologic, and degenerative.7 Infection of the nervous system is a serious and prevalent medical problem. Operative management is indicated for most conditions in which there is symptomatic compression of neural structures.8 Functional neurosurgery via device implantation is a rapidly evolving discipline that has already become the standard of care in treating medically refractory Parkinson’s disease and essential tremor. A wider variety of deep brain stimulation targets will treat additional neuropsychiatric diseases.9 Stereotactic radiosurgery is a powerful treatment option for intracranial disease, whether it is primary or adjunct. Gamma knife surgery can be used to treat tumors, vascular malfor-mations, and cranial neuralgias.Brunicardi_Ch42_p1827-p1878.indd 182801/03/19 7:16 PM 1829NEUROSURGERYCHAPTER 42neuron in the anterior horn at the appropriate level. The lower motor

1	malfor-mations, and cranial neuralgias.Brunicardi_Ch42_p1827-p1878.indd 182801/03/19 7:16 PM 1829NEUROSURGERYCHAPTER 42neuron in the anterior horn at the appropriate level. The lower motor neuron axon then travels via peripheral nerves to its tar-get muscle. Damage to upper motor neurons typically results in hyperreflexia and mild atrophy. Damage to lower motor neurons results in flaccidity and significant atrophy.The two major sensory tracts are three-neuron pathways. Fine touch and proprioceptive signals enter the spinal cord via the dorsal root ganglia and then ascend ipsilaterally via the dorsal columns. Then they synapse and decussate in the lower medulla, travel up the contralateral medial lemniscus to make a second synapse in the thalamus, and then finally ascend to the sensory cortex. Pain and temperature fibers first synapse in the dorsal horn of the spinal cord at their entry level, decussate, and then travel up the contralateral spinothalamic tracts to the thalamus. The

1	cortex. Pain and temperature fibers first synapse in the dorsal horn of the spinal cord at their entry level, decussate, and then travel up the contralateral spinothalamic tracts to the thalamus. The second synapse occurs in the thalamus, and the output axons ascend to the sensory cortex.The aforementioned motor and sensory tracts together constitute the somatic nervous system. In addition to this sys-tem, the ANS is the other constituent of the nervous system. The ANS carries messages for homeostasis and visceral regu-lation from the central nervous system (CNS) to target struc-tures such as arteries, veins, the heart, sweat glands, and the digestive tract.1 CNS control of the ANS arises particularly from the hypothalamus and the nucleus of the tractus solitarius. The ANS is divided into the sympathetic, parasympathetic, and enteric systems. The sympathetic system drives the “fight or flight” response, using epinephrine to increase heart rate, blood pressure, blood glucose, and

1	into the sympathetic, parasympathetic, and enteric systems. The sympathetic system drives the “fight or flight” response, using epinephrine to increase heart rate, blood pressure, blood glucose, and temperature, as well as to dilate the pupils. It arises from the thoracolumbar spinal segments. The parasympathetic system promotes the “rest and digest” state and uses acetylcholine to maintain basal metabolic func-tion under nonstressful conditions. Parasympathetic fibers arise from cranial nerves III, VII, IX, and X, and from the second to fourth sacral segments. The enteric nervous system controls the complex synchronization of the digestive tract, especially the pancreas, gallbladder, and small and large bowels. It can run autonomously but is regulated by the sympathetic and parasym-pathetic systems.NEUROLOGIC EXAMINATIONThe neurologic examination is divided into several components and generally is done from head to toe. First, one must assess men-tal status. A patient may be awake,

1	systems.NEUROLOGIC EXAMINATIONThe neurologic examination is divided into several components and generally is done from head to toe. First, one must assess men-tal status. A patient may be awake, lethargic (will follow com-mands and answer questions, but then returns to sleep), stuporous (difficult to arouse), or comatose (no purposeful response to voice or pain). Cranial nerves may be thoroughly tested in the awake patient, but pupil reactivity, eye movement, facial symmetry, and gag are the most relevant measures when mental status is impaired. Motor testing is based on maximal effort of major muscle groups in those able to follow commands, while assessing for amplitude and symmetry of movement to deep central pain may be all that is possible for stuporous patients. Table 42-1 details scoring for motor assessment tests. Characteristic motor reactions to pain in patients with depressed mental status include withdrawal from stimulus, localization to stimulus, flexor (decorticate)

1	scoring for motor assessment tests. Characteristic motor reactions to pain in patients with depressed mental status include withdrawal from stimulus, localization to stimulus, flexor (decorticate) posturing, extensor (decerebrate) posturing, or no reaction (in order of wors-ening pathology). Figure 42-1 diagrams the clinical patterns of posturing. This forms the basis of determining the Glasgow Coma Scale (GCS) motor score, as detailed in Table 42-2. Light touch, proprioception, temperature, and pain testing may be useful in awake patients but is often impossible without good cooperation. It is critical to document sensory patterns in spinal cord injury (SCI) patients. Muscle stretch reflexes should be examined. Often com-paring left to right or upper extremity to lower extremity reflexes for symmetry is the most useful for localizing a lesion. Check for ankle-jerk clonus or up-going toes (Babinski’s test). Presence of either is pathologic and signifies upper motor neuron

1	reflexes for symmetry is the most useful for localizing a lesion. Check for ankle-jerk clonus or up-going toes (Babinski’s test). Presence of either is pathologic and signifies upper motor neuron disease.Diagnostic StudiesPlain Films. Plain X-rays of the skull may demonstrate frac-tures, osteolytic or osteoblastic lesions, radiolucent foreign bodies, or pneumocephaly (air in the head). Plain films of the cervical, thoracic, and lumbar spine are used to assess for evi-dence of bony trauma or soft tissue swelling suggesting fracture. Spinal deformities and osteolytic or osteoblastic pathologic pro-cesses also will be apparent. However, the use of plain films has decreased given the rapid availability and significantly increased detail of computed tomography (CT) scans. They are typically used for assessing alignment in patients with known fractures, for intraoperative localization, and postoperative assessment of spinal instrumentation.Computed Tomography. The noncontrast CT scan of the

1	for assessing alignment in patients with known fractures, for intraoperative localization, and postoperative assessment of spinal instrumentation.Computed Tomography. The noncontrast CT scan of the head is an extremely useful diagnostic tool in the setting of new focal neurologic deficit, decreased mental status, or trauma. It is rapid and almost universally available in hospitals in the United States. Its sensitivity allows for the detection of acute hemorrhage. Fine-slice CT scanning of the spine is helpful for defining bony anatomy and pathology and is the method of choice for iden-tifying fractures of the spine. By providing an assessment of spinal alignment, CT scans can provide an indirect assessment of ligamentous injury, for example, “Rule of Spence” for assess-ing transverse ligament injury during Jefferson fractures (see “Spine Trauma” section later in this chapter). Conventional con-trast-enhanced CT scan will help show neoplastic or infectious processes. In the current

1	injury during Jefferson fractures (see “Spine Trauma” section later in this chapter). Conventional con-trast-enhanced CT scan will help show neoplastic or infectious processes. In the current era, contrast CT generally is used for those patients who cannot undergo magnetic resonance imaging (MRI) scanning due to pacemakers or metal in the orbits (see following section for discussion of CT angiography, venogra-phy, and perfusion).Magnetic Resonance Imaging. Magnetic resonance imaging (MRI) provides excellent imaging of soft tissue structures in the head and spine. It is a complex and evolving science. Several of the most clinically useful MRI sequences are worth describing. T1 sequences made before and after gadolin-ium administration are useful for detecting neoplastic and infec-tious processes. T2 sequences facilitate assessment of 2Table 42-1Motor scoring systemGRADEDESCRIPTION0No muscle contraction1Visible muscle contraction without movement across the joint2Movement in the

1	processes. T2 sequences facilitate assessment of 2Table 42-1Motor scoring systemGRADEDESCRIPTION0No muscle contraction1Visible muscle contraction without movement across the joint2Movement in the horizontal plane, unable to overcome gravity3Movement against gravity4Movement against some resistance5Normal strengthBrunicardi_Ch42_p1827-p1878.indd 182901/03/19 7:16 PM 1830SPECIFIC CONSIDERATIONSPART IITable 42-2The Glasgow Coma Scale scoreaMOTOR RESPONSE VERBAL RESPONSE EYE-OPENING RESPONSE Obeys commands6Oriented5Opens spontaneously4Localizes to pain5Confused4Opens to speech3Withdraws from pain4Inappropriate words3Opens to pain2Flexor posturing3Unintelligible sounds2No eye opening1Extensor posturing2No sounds1 No movement1 aAdd the three scores to obtain the Glasgow Coma Scale (GCS) score, which can range from 3 to 15. Add “T” after the GCS if intubated and no verbal score is possible. For these patients, the GCS can range from 3T to 10T.lesion-associated edema in the brain and

1	score, which can range from 3 to 15. Add “T” after the GCS if intubated and no verbal score is possible. For these patients, the GCS can range from 3T to 10T.lesion-associated edema in the brain and neural compression in the spine by the presence or absence of bright T2 CSF signals. Fluid-attenuated inversion recovery (FLAIR) imaging is a T2 sequence with suppression of the CSF signal, so as to emphasize lesions and edema, particularly adjacent to the ventricles. Diffu-sion-weighted images is the gold-standard for identifying isch-emic stroke within 12 hours of symptom onset.2 Gradient echo sequences (GRE) can be used to identify acute-subacute blood products and are used to assess for micro-hemorrhages in trau-matic diffuse axonal injury (DAI), amyloid angiopathy, and also in the diagnosis of cavernous malformations. In the spine, short-tau inversion recovery (STIR) or fat-suppressed T2 sequences are useful for assessing for the acuity of fractures (by measuring bone edema), and

1	of cavernous malformations. In the spine, short-tau inversion recovery (STIR) or fat-suppressed T2 sequences are useful for assessing for the acuity of fractures (by measuring bone edema), and identifying ligamentous injury.CT and MR Angiography. Recent advances in CT technol-ogy such as short acquisition times with multidetector technol-ogy has allowed for imaging of vascular anatomy. Fine-slice CT scans can be combined with a timed-bolus of intravenous contrast in the arterial phase (angiography, CTA) and venous phase (venography, CTV) to assess arterial and venous vascu-lature, respectively. Although traditional catheter-based angiog-raphy still serves as the gold-standard, CTA and CTV provide Figure 42-1. Patterns of motor responses associated with various lesions. A. Left hemispheric lesion with right hemiplegia and left local-ization. B. Deep cerebral/thalamic lesion with bilateral flexor posturing. C. Midbrain or pontine lesion with bilateral extensor posturing. D. Medullary

1	lesion with right hemiplegia and left local-ization. B. Deep cerebral/thalamic lesion with bilateral flexor posturing. C. Midbrain or pontine lesion with bilateral extensor posturing. D. Medullary lesion with general flaccidity. (Adapted with permission from Rengachary SS, Ellenbogen RG: Principles of Neurosurgery, 2nd ed. New York, NY: Elsevier/Mosby; 2005.)Brunicardi_Ch42_p1827-p1878.indd 183001/03/19 7:16 PM 1831NEUROSURGERYCHAPTER 42noninvasive alternative for the initial screening assessment and follow-up of patients with suspected or known vascular lesions, as well as the evaluation of vasospasm. Similarly, fine-slice time-of-flight axial images can be reformatted in three dimen-sions to build MRI angiograms and MRI venograms. MRI angiograms can detect stenosis of the cervical carotid arteries or intracranial aneurysms >3 mm in diameter. MRI venograms can assess the dural venous sinuses for patency or thrombosis. Two-dimensional time of flight imaging performs vascular

1	carotid arteries or intracranial aneurysms >3 mm in diameter. MRI venograms can assess the dural venous sinuses for patency or thrombosis. Two-dimensional time of flight imaging performs vascular reconstructions purely based on flow and does not require gado-linium contrast administration.CT and MR Perfusion. Perfusion scans have recently emerged as a method to a global assessment of the vascular integrity in the cerebral hemispheres, which is very important in the assess-ment of ischemic stroke (see “Stroke”). CT perfusion scans gen-erate quantitative color maps that indicate various physiologic parameters such as cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) through quan-titative analysis of rapidly acquired image sequences during intravenous contrast administration. Similar to perfusion CT, perfusion MRI can be used to generate quantitative color maps of relative cerebral CBV and MTT. These perfusion-based mea-sures can be used along with

1	administration. Similar to perfusion CT, perfusion MRI can be used to generate quantitative color maps of relative cerebral CBV and MTT. These perfusion-based mea-sures can be used along with diffusion-weighted imaging in the evaluation of ischemic stroke, particularly to identify an isch-emic “penumbra” or tissue that is ischemic but not yet infarcted, and may be salvageable with intervention.3,4Angiography. Transarterial catheter-based angiography remains the gold standard for evaluation of vascular pathology of the brain and spine. The current state of the art is biplanar imaging to reduce dye load and facilitate interventional proce-dures. Digital subtraction technologies minimize bony inter-ference in the resultant images. Bilateral carotid arteries and bilateral vertebral arteries may be injected and followed through arterial, capillary, and venous phases for a complete cerebral angiogram.Electroencephalography. Electroencephalography (EEG) involves measuring weak electrical

1	may be injected and followed through arterial, capillary, and venous phases for a complete cerebral angiogram.Electroencephalography. Electroencephalography (EEG) involves measuring weak electrical signals from the brain that are transmitted through the skull through electrodes that are applied to the scalp. The voltage fluctuations detected by EEG are thought to reflect summed membrane potentials from under-lying brain tissue. Clinically, EEG is useful for detecting sei-zures, interictal markers of epileptogenic tissue, and widespread abnormalities in brain function, such as diffuse encephalopathy. EEG is also used in concert with electrical stimulation to detect sensory evoked potentials that can be useful for intraoperative mapping during cranial and spine surgery.Electromyography and Nerve Conduction Studies. Electromyography and nerve conduction studies (EMG/NCS) are useful for assessing the function of peripheral nerves. EMG records muscle activity in response to a proximal

1	Nerve Conduction Studies. Electromyography and nerve conduction studies (EMG/NCS) are useful for assessing the function of peripheral nerves. EMG records muscle activity in response to a proximal stimulation of the motor nerve. NCS record the velocity and amplitude of the nerve action potential. EMG/NCS typically is performed approximately 3 to 4 weeks after an acute injury, as nerves distal to the injury continue to transmit electrical impulses normally until degeneration of the distal nerve progresses.Invasive Monitoring. The most reliable monitor, always, is an alert patient with a reliable neurologic examination. If a reliable neurologic examination is not possible due to the pres-ence of brain injury, sedatives, or paralytics, or if there is active and unstable intracranial pathology, invasive monitoring is required. There are several methods of monitoring intracranial physiology. The methods described in the following sections are bedside intensive care unit (ICU) procedures

1	invasive monitoring is required. There are several methods of monitoring intracranial physiology. The methods described in the following sections are bedside intensive care unit (ICU) procedures that allow for continuous monitoring. Both procedures involve making a small hole in the skull with a hand-held drill. They generally are placed in the right frontal region to minimize the neurologic impact of possible complications such as hemorrhage.External Ventricular Drain. An external ventricular drain is also known as a ventriculostomy. A perforated plastic catheter is inserted into the frontal horn of the lateral ventricle. An uninter-rupted fluid column through a rigid tube allows transduction of intracranial pressure (ICP). CSF also can be drained to reduce ICP or sampled for laboratory studies.Intraparenchymal Physiologic Monitoring. Intraparenchy-mal monitors can be inserted into the brain through a threaded post locked securely into a burr hole, commonly referred to as a bolt. A

1	studies.Intraparenchymal Physiologic Monitoring. Intraparenchy-mal monitors can be inserted into the brain through a threaded post locked securely into a burr hole, commonly referred to as a bolt. A bolt allows ICP monitoring with a fiber-optic pressure transducer, but it is smaller and less invasive than a ventriculos-tomy and may be associated with fewer complications, although the data do not clearly support this. Furthermore, a bolt can also be used to introduce probes to measure brain tissue oxygenation, brain temperature, and to perform microdialysis of parenchymal samples; however, the utility of these latter measures in clinical practice is still under investigation. Patients with severe brain injury due to trauma or aneurysmal hemorrhage may benefit from placement of these sensors in addition to a ventriculos-tomy to drain CSF for control of ICP. Such monitoring requires two twist-drill holes, which may be placed on adjacent or oppo-site sides of the head.NEUROLOGIC AND

1	in addition to a ventriculos-tomy to drain CSF for control of ICP. Such monitoring requires two twist-drill holes, which may be placed on adjacent or oppo-site sides of the head.NEUROLOGIC AND NEUROSURGICAL EMERGENCIESRaised Intracranial PressureICP normally varies between 4 and 14 mmHg. Sustained ICP levels above 20 mmHg can injure the brain. The Monro-Kellie doctrine states that the cranial vault is a rigid structure, and therefore, the total volume of the contents determines ICP. The three normal contents of the cranial vault are brain tis-sue, blood, and CSF. The brain’s contents can expand due to swelling from traumatic brain injury (TBI), stroke, or reactive edema. Blood volume can increase by extravasation to form a hematoma, or by reactive vasodilation in a hypoventilating, hypercarbic patient. CSF volume increases in the setting of hydrocephalus. Figure 42-2 demonstrates the classic CT find-ings of hydrocephalus. The addition of a lesion, such as a tumor or abscess, also will

1	patient. CSF volume increases in the setting of hydrocephalus. Figure 42-2 demonstrates the classic CT find-ings of hydrocephalus. The addition of a lesion, such as a tumor or abscess, also will increase ICP. The pressure-volume curve depicted in Fig. 42-3 demonstrates a compensated region with a small ΔP/ΔV, and an uncompensated region with large ΔP/ΔV. In the compensated region, increased volume is offset by decreased volume of CSF and blood.Increased ICP can injure the brain in several ways. Focal mass lesions cause shift and herniation. Temporal lesions push the uncus medially and compress the midbrain. This phenom-enon is known as uncal herniation. The posterior cerebral artery (PCA) passes between the uncus and midbrain and may be occluded, leading to an occipital infarct. Masses higher up in the hemisphere can push the cingulate gyrus under the falx cerebri. This process is known as subfalcine herniation. The anterior cerebral artery (ACA) branches run along the medial surface

1	up in the hemisphere can push the cingulate gyrus under the falx cerebri. This process is known as subfalcine herniation. The anterior cerebral artery (ACA) branches run along the medial surface of the cingulate gyrus and may be occluded in this case, leading to medial frontal and parietal infarcts. Diffuse increases in pressure Brunicardi_Ch42_p1827-p1878.indd 183101/03/19 7:16 PM 1832SPECIFIC CONSIDERATIONSPART IIin the cerebral hemispheres can lead to central, or transtento-rial, herniation. Increased pressure in the posterior fossa can lead to upward central herniation or downward tonsillar hernia-tion through the foramen magnum. Uncal, transtentorial, and tonsillar herniation can cause direct damage to the brain stem. Figure 42-4 diagrams patterns of herniation.Patients with increased ICP, or intracranial hypertension, often will present with headache, nausea, vomiting, and progres-sive mental status decline. Cushing’s triad is the classic presen-tation of hypertension,

1	increased ICP, or intracranial hypertension, often will present with headache, nausea, vomiting, and progres-sive mental status decline. Cushing’s triad is the classic presen-tation of hypertension, bradycardia, and irregular respirations. Focal neurologic deficits such as hemiparesis may be present if there is a focal mass lesion causing the problem. Patients with these symptoms should undergo an immediate head CT and rapid neurosurgical evaluation.Initial management of intracranial hypertension includes airway protection and adequate ventilation. A bolus of man-nitol up to 1 g/kg causes free water diuresis, increased serum osmolality, and extraction of water from the brain. The effect is delayed by about 20 minutes and has a transient benefit. Driv-ing serum osmolality above 300 mOsm/L is of indeterminate benefit and can have deleterious cardiovascular side effects, such as hypovolemia that leads to hypotension and decreased brain perfusion. A ventriculostomy and/or craniectomy may

1	is of indeterminate benefit and can have deleterious cardiovascular side effects, such as hypovolemia that leads to hypotension and decreased brain perfusion. A ventriculostomy and/or craniectomy may be needed for definitive decompression.It is critical to note that lethargic or obtunded patients often have decreased respiratory drive. This causes the partial pressure of arterial carbon dioxide (Paco2) to increase, resulting in cerebral vasodilation and worsening of intracranial hyperten-sion. This cycle causes a characteristic “crashing patient,” who rapidly loses airway protection, becomes apneic, and herniates. Emergent intubation and ventilation to reduce Paco2 to roughly 35 mmHg can reverse this process.Figure 42-2. Head computed tomography scan demonstrating hydrocephalus. The third ventricle (3rd) is widened and rounded, the anterior horns of the lateral ventricles are plump, and pressure-driven flow of cerebrospinal fluid into brain parenchyma adjacent to the ventricles is

1	ventricle (3rd) is widened and rounded, the anterior horns of the lateral ventricles are plump, and pressure-driven flow of cerebrospinal fluid into brain parenchyma adjacent to the ventricles is seen (arrowhead). This is known as transepen-dymal flow of cerebrospinal fluid.Intracranial volume (arbitrary units)Intracranial pressure (mmHg)0102030405060708090100110120130102030405060708090100˜V˜P˜V˜P˜V˜PLow complianceecnailpmoc oNecnailpmoc hgiHFigure 42-3. Pressure-volume curve demonstrating the effect of changing the volume of intracranial contents on intracranial pressure. Note the compensated zone, with little change of pressure with change of volume, and the uncompensated zone, with significant change of pres-sure with change of volume. (Adapted with permission from Ellenbogen RG, Abdulrauf SI, Sekhar LN: Principles of Neurosurgery, 3rd ed. Philadelphia, PA: Elsevier/Saunders; 2012.)Brunicardi_Ch42_p1827-p1878.indd 183201/03/19 7:16 PM 1833NEUROSURGERYCHAPTER 42Brain Stem

1	Abdulrauf SI, Sekhar LN: Principles of Neurosurgery, 3rd ed. Philadelphia, PA: Elsevier/Saunders; 2012.)Brunicardi_Ch42_p1827-p1878.indd 183201/03/19 7:16 PM 1833NEUROSURGERYCHAPTER 42Brain Stem CompressionThe posterior fossa (brain stem and cerebellum) requires special consideration because the volume of the posterior fossa within the cranial vault is small. Posterior fossa lesions such as tumors, hemorrhage, or stroke can cause mass effect that can rapidly kill the patient in two ways. Occlusion of the fourth ventricle can lead to acute obstructive hydrocephalus, raised ICP, herniation, and eventually death. This mass effect can also lead directly to brain stem compression (Fig. 42-5). Symptoms of brain stem compression include hypertension, agitation, and progressive obtundation, followed rapidly by brain death. A patient exhibit-ing any of these symptoms needs an emergent neurosurgical evaluation for possible ventriculostomy or suboccipital crani-ectomy (removal of the bone

1	followed rapidly by brain death. A patient exhibit-ing any of these symptoms needs an emergent neurosurgical evaluation for possible ventriculostomy or suboccipital crani-ectomy (removal of the bone covering the cerebellum). This situation is especially critical, as expeditious decompression can lead to significant functional recovery.StrokePatients presenting with acute focal neurologic deficits at a clearly defined time of onset (i.e., when the patient was last seen in a normal state of health) must be evaluated as rapidly as possible. An emergent head CT scan should be done. The study is often normal because CT changes from ischemic stroke may take up to 24 hours to appear (Fig. 42-6). A patient with a clinical diagnosis of acute stroke <4.5 hours old, without hemor-rhage on CT, may be a candidate for thrombolytic therapy with tissue plasminogen activator (tPA). When a proximal large-vessel obstruction is suspected, patients should be evaluated for endovascular mechanical

1	may be a candidate for thrombolytic therapy with tissue plasminogen activator (tPA). When a proximal large-vessel obstruction is suspected, patients should be evaluated for endovascular mechanical thrombectomy if therapy can be initiated within 6 to 8 hours of symptom onset. Intravenous tPA should be given regardless, but a noninvasive intracranial vas-cular study such as CT angiography should also be obtained in these cases. An emergent MRI is helpful but not always diag-nostically necessary.SeizureA seizure is defined as an uncontrolled synchronous organiza-tion of neuronal electrical activity. A new-onset seizure often signifies an irritative mass lesion in the brain, particularly in adults, in whom tumors commonly present with seizure. Patients with traumatic intracranial hemorrhage are at risk for seizure. In addition to airway and ventilatory problems, a seizing patient is also at risk for neural excitotoxicity if the activity is prolonged, such as in status epilepticus. Any

1	are at risk for seizure. In addition to airway and ventilatory problems, a seizing patient is also at risk for neural excitotoxicity if the activity is prolonged, such as in status epilepticus. Any patient with a new-onset sei-zure should have imaging of the brain after the seizure is con-trolled and the patient is resuscitated.TRAUMATrauma is the leading cause of death in children and young adults; however, the incidence of death and disability from trauma has been slowly decreasing. This decline is partly attrib-utable to increased awareness of safety devices such as seat belts and motorist helmets. Nonetheless, trauma remains a major cause of morbidity and mortality, and it can affect every major organ system in the body. The three main areas of neurosurgical focus are: traumatic brain injury (TBI), spinal cord injury (SCI), and peripheral nerve injury.Head TraumaGlasgow Coma Scale Score. The initial assessment of the trauma patient includes the primary survey, resuscitation,

1	brain injury (TBI), spinal cord injury (SCI), and peripheral nerve injury.Head TraumaGlasgow Coma Scale Score. The initial assessment of the trauma patient includes the primary survey, resuscitation, 1324Figure 42-4. Schematic drawing of brain herniation patterns. 1. Subfalcine herniation. The cingulate gyrus shifts across midline under the falx cerebri. 2. Uncal herniation. The uncus (medial tem-poral lobe gyrus) shifts medially and compresses the midbrain and cerebral peduncle. 3. Central transtentorial herniation. The dien-cephalon and midbrain shift caudally through the tentorial inci-sura. 4. Tonsillar herniation. The cerebellar tonsil shifts caudally through the foramen magnum. (Reproduced with permission from Wilkins RH, Rengachary SS: Neurosurgery, 2nd ed. New York, NY: McGraw Hill Education; 1996.)Figure 42-5. Maturing cerebellar stroke seen as a hypodense area in the right cerebellar hemisphere (arrowhead) on head computed tomography in a patient with rapidly progressing

1	Hill Education; 1996.)Figure 42-5. Maturing cerebellar stroke seen as a hypodense area in the right cerebellar hemisphere (arrowhead) on head computed tomography in a patient with rapidly progressing obtundation 2 days after the initial onset of symptoms. Swelling of the infarcted tissue causes posterior fossa mass effect. The fourth ventricle is obliterated and not visible, and the brain stem is being compressed.Brunicardi_Ch42_p1827-p1878.indd 183301/03/19 7:16 PM 1834SPECIFIC CONSIDERATIONSPART IIsecondary survey, and definitive care. Neurosurgical evalua-tion begins during the primary survey with the determination of the GCS score (usually referred to simply as the GCS) for the patient. The GCS is determined by adding the scores of the best responses of the patient in each of three categories. The motor score ranges from 1 to 6, verbal from 1 to 5, and eyes from 1 to 4. The GCS therefore ranges from 3 to 15, as detailed in Table 42-2. Tracheal intubation or severe facial or

1	categories. The motor score ranges from 1 to 6, verbal from 1 to 5, and eyes from 1 to 4. The GCS therefore ranges from 3 to 15, as detailed in Table 42-2. Tracheal intubation or severe facial or eye swelling can impede verbal and eye responses. In these circumstances, the patient is given the score of 1 with a modifier, such as verbal “1T” where T = tube.Scalp Injury. Blunt or penetrating trauma to the head can cause injury to the densely vascularized scalp, and significant blood loss can result. Direct pressure initially controls the bleeding, allowing close inspection of the injury. If a simple laceration is found, it should be copiously irrigated and closed primarily. If the laceration is short, a single-layer, percutaneous suture clo-sure will suffice. If the laceration is long or has multiple arms, the patient may need debridement and closure in the operating room, with its superior lighting and wider selection of instru-ments and suture materials. Careful reapproximation of the

1	multiple arms, the patient may need debridement and closure in the operating room, with its superior lighting and wider selection of instru-ments and suture materials. Careful reapproximation of the galea will provide a more secure closure and better hemostasis. ACBFigure 42-6. A. Head computed tomography scan of a patient with a 4-day-old stroke that occluded the right middle cerebral and posterior cerebral arteries. The infarcted tissue is the hypodense (dark) area indicated by the arrowheads. The patient presented with left-sided weakness and left visual field loss, but then became less responsive, prompting this head computed tomography. Note the right-to-left midline shift. B. Same patient status post decompressive right hemicraniectomy. Note the free expansion of swollen brain outside the normal confines of the skull. C. Patient with a right middle cerebral artery ischemic stroke with areas of hemorrhagic conversion, seen as hyperdense (bright) areas within the infarcted

1	outside the normal confines of the skull. C. Patient with a right middle cerebral artery ischemic stroke with areas of hemorrhagic conversion, seen as hyperdense (bright) areas within the infarcted tissue. This patient also required hemicraniectomy for severe mass effect. Note the lack of midline shift postoperatively.Brunicardi_Ch42_p1827-p1878.indd 183401/03/19 7:16 PM 1835NEUROSURGERYCHAPTER 42Blunt trauma also can cause crush injury with subsequent tissue necrosis. These wounds require debridement and consideration of advancement flaps to cover the defect.Skull Fractures. The usual classification system for bony fractures may be applied to the skull. The fracture may be characterized by skull X-rays or head CT.5 A closed fracture is covered by intact skin. An open, or compound, fracture is associated with disrupted overlying skin. The fracture lines may be single (linear); multiple and radiating from a point (stellate); or multiple, creating fragments of bone (comminuted).

1	fracture is associated with disrupted overlying skin. The fracture lines may be single (linear); multiple and radiating from a point (stellate); or multiple, creating fragments of bone (comminuted). Closed skull fractures do not normally require specific treatment. Open fractures require repair of the scalp and operative debridement. Indications for craniotomy include depression greater than the cranial thickness, intracranial hematoma, and frontal sinus involvement.6 Skull fractures generally indicate that a signifi-cant amount of force was transmitted to the head and should increase the suspicion for intracranial injury. Fractures that cross meningeal arteries can cause rupture of the underlying vessels and subsequent epidural hematoma (EDH) formation.Depressed skull fractures may result from a focal injury of significant force. The inner and outer cortices of the skull are disrupted, and a fragment of bone is pressed in toward the brain in relation to adjacent intact skull. The

1	from a focal injury of significant force. The inner and outer cortices of the skull are disrupted, and a fragment of bone is pressed in toward the brain in relation to adjacent intact skull. The fragment may overlap the edge of intact bone, or it may plunge completely below the level of adjacent normal skull. The inner cortex of the bone fragments often has multiple sharp edges that can lacerate dura, brain, and vessels. Craniotomy is required to ele-vate the fracture, repair dural disruption, and obtain hemostasis in these cases (Fig. 42-7). However, fractures overlying dural venous sinuses require restraint. Surgical exploration can lead to life-threatening hemorrhage from the lacerated sinus.Fractures of the skull base are common in head-injured patients, and they indicate significant impact. They are gener-ally apparent on routine head CT, but they should be evaluated with dedicated fine-slice coronal-section CT scan to document and delineate the extent of the fracture and

1	impact. They are gener-ally apparent on routine head CT, but they should be evaluated with dedicated fine-slice coronal-section CT scan to document and delineate the extent of the fracture and involved structures. If asymptomatic, they require no treatment. Skull base fractures requiring intervention include those with an associated cranial nerve deficit or CSF leak. A fracture of the temporal bone, for instance, can damage the facial or vestibulocochlear nerve, resulting in vertigo, ipsilateral deafness, or facial paralysis. A communication may be formed between the subarachnoid space and the middle ear, allowing CSF drainage into the pharynx via the Eustachian tube or from the ear (otorrhea). Extravasation of blood results in ecchymosis behind the ear, known as Battle’s sign. A fracture of the anterior skull base can result in anos-mia (loss of smell from damage to the olfactory nerve), CSF drainage from the nose (rhinorrhea), or periorbital ecchymosis, known as raccoon eyes.Copious

1	of the anterior skull base can result in anos-mia (loss of smell from damage to the olfactory nerve), CSF drainage from the nose (rhinorrhea), or periorbital ecchymosis, known as raccoon eyes.Copious clear drainage from the nose or ear makes the diagnosis of CSF leakage obvious. Often, however, the drain-age may be discolored with blood or small in volume if some drains into the throat. In indeterminate cases, it is important to consider radiographic findings on the CT scan near the fracture that suggest CSF leak, such as pneumocephalus, subarachnoid, or intraparenchymal blood at the fracture site. The “halo” test assesses for a double ring when a drop of the fluid is allowed to fall on an absorbent surface, but it has been shown to have poor clinical utility.7 The fluid can be sent for β-2 transferrin testing, a carbohydrate-free isoform of transferrin exclusively found in the CSF; however, these tests often take 1 to 2 weeks to result and also can be difficult to incorporate into

1	β-2 transferrin testing, a carbohydrate-free isoform of transferrin exclusively found in the CSF; however, these tests often take 1 to 2 weeks to result and also can be difficult to incorporate into clinical practice.BAFigure 42-7. A. Bone-window axial head computed tomography (CT) of a patient who presented aphasic after being struck with the bot-tom of a beer bottle. CT demonstrates a depressed skull fracture in the left posterior temporoparietal area. B. Brain-window axial head CT demonstrating intraparenchymal hematoma caused by laceration of cortical vessels by the edge of the fractured bone. Arrowhead indicates traumatic subarachnoid hemorrhage in the sylvanian fissure.Brunicardi_Ch42_p1827-p1878.indd 183501/03/19 7:16 PM 1836SPECIFIC CONSIDERATIONSPART IIMany CSF leaks will heal with elevation of the head of the bed for several days. An elevation of the head of the bed reduces the hydrostatic pressure of the CSF fluid column in the cranial vault, near the site of the

1	heal with elevation of the head of the bed for several days. An elevation of the head of the bed reduces the hydrostatic pressure of the CSF fluid column in the cranial vault, near the site of the defect. As such, when the CSF leak is in the lumbar thecal sac, the head of the bed should be flat so as to maximize hydrostatic pressure of the CSF fluid column at the cranial vault, away from the site of the defect. In addi-tion, lumbar drain can be used to reduce CSF pressure. When there is a contraindication, to lumbar drain placement (such as an intracranial mass lesion or hematoma), an extraventricular drain should be used for CSF diversion. Although persistent CSF leaks have been shown to increase the risk of meningitis,8 there is no evidence supporting the use of prophylactic antibi-otic use for preventing meningitis in patients with CSF leaks.9Traumatic cranial neuropathies generally can be managed conservatively, with documentation of the extent of impairment and signs of recovery.

1	use for preventing meningitis in patients with CSF leaks.9Traumatic cranial neuropathies generally can be managed conservatively, with documentation of the extent of impairment and signs of recovery. Patients with traumatic facial nerve pal-sies may benefit from a course of steroids, although their benefit is unproven. Patients with facial nerve palsy of abrupt onset, who do not respond to steroids within 48 to 72 hours, may be considered for surgical decompression of the petrous portion of the facial nerve. Patients also may present with delayed-onset facial nerve palsy. Again, steroids are used and surgery can be considered, with mixed results.Closed Head Injury. Closed head injury (CHI) is the most common type of TBI and a significant cause of morbidity and mortality in the United States. There are two important factors that affect the outcome of CHI in general. The initial impact causes the primary injury, defined as the immediate injury to neurons from transmission of the force

1	There are two important factors that affect the outcome of CHI in general. The initial impact causes the primary injury, defined as the immediate injury to neurons from transmission of the force of impact. The long, delicate axons of the neurons can shear as they undergo differ-ential acceleration or deceleration along their projecting path-ways. Prevention strategies, such as wearing helmets, remain the best means to decrease disability from primary injury. Sub-sequent neuronal damage due to the sequelae of trauma is referred to as secondary injury. Hypoxia, hypotension, hydro-cephalus, intracranial hypertension, thrombosis, and intracranial hemorrhage may all be mechanisms of secondary injury. One focus of basic research in TBI, critical care medicine, and neurosurgical intervention is to decrease the effects of sec-ondary injury.The Brain Trauma Foundation’s most recent summary of management recommendations for TBI patients was published in 2016 and is endorsed by the American

1	is to decrease the effects of sec-ondary injury.The Brain Trauma Foundation’s most recent summary of management recommendations for TBI patients was published in 2016 and is endorsed by the American Association of Neuro-logical Surgeons, Congress of Neurological Surgeons, and the World Health Organization.10 The guidelines standardize the care of these patients with the hope of improving outcomes. Level I recommendations are based on a body of high-quality evidence, such as large, well-received randomized controlled trials. Level II and III recommendations are based on moderate and low quality evidence, respectively. Some of the common patterns of CHI, including concussion, contusion, and diffuse axonal injury, are discussed in “Types of Closed Head Injury.”11Initial Assessment The initial evaluation of a trauma patient remains the same whether or not the primary surveyor suspects head injury. The first three elements of the ABCDs of resus-citation—airway, breathing, and

1	initial evaluation of a trauma patient remains the same whether or not the primary surveyor suspects head injury. The first three elements of the ABCDs of resus-citation—airway, breathing, and circulation—must be assessed and stabilized. Hypoxia and hypotension are known to worsen outcome in TBI (due to secondary injury), making cardiopul-monary stabilization critical. Patients who cannot follow com-mands require intubation for airway protection and ventilatory control. The fourth element, assessment of “D,” for disability, is undertaken next. Motor activity, speech, and eye opening can be assessed in a few seconds and a GCS score assigned.The following is an example of how a primary surveyor may efficiently assess disability and GCS: Approach the patient and enter his or her field of view. Observe whether the patient is visually attentive. Clearly command: “Tell me your name.” Then ask the patient to lift up two fingers on each side sequen-tially, and wiggle the toes. A visually or

1	Observe whether the patient is visually attentive. Clearly command: “Tell me your name.” Then ask the patient to lift up two fingers on each side sequen-tially, and wiggle the toes. A visually or verbally unresponsive patient should be assessed for response to peripheral stimuli such as nail-bed pressure, or deep central painful stimulation, such as a firm, twisting pinch of the sensitive supraclavicular skin. Watch for eye opening and movement of the extremities, whether purposeful or reflexive. Assess the verbal response. The motor, verbal, and eye-opening scores may be correctly assigned using this rapid examination. An initial assessment of the probability of significant head injury can be made, assuming that pharmacologic and toxic elements have not obscured the examination. The surveyor must also take note of any external signs of head injury, including bleeding from the scalp, nose, or ear, or deformation of the skull or face.Classification TBI can be classified as mild,

1	surveyor must also take note of any external signs of head injury, including bleeding from the scalp, nose, or ear, or deformation of the skull or face.Classification TBI can be classified as mild, moderate, or severe. For patients with a history of head trauma, classifica-tion is as follows: severe head injury if the GCS score is 3 to 8, moderate head injury if the GCS score is 9 to 12, and mild head injury if the GCS score is 13 to 15. Many patients present to emergency rooms and trauma bays with a history of TBI. A tri-age system must be used to maximize resource utilization while minimizing the chance of missing occult or progressing injuries.TBI patients who are asymptomatic, who have only headache, dizziness, or scalp lacerations, and who did not lose consciousness, have a low risk for intracranial injury and may be discharged home without a head CT scan.12,13 Head-injured patients who are discharged should be sent home with reliable family or friends who can observe the patient

1	intracranial injury and may be discharged home without a head CT scan.12,13 Head-injured patients who are discharged should be sent home with reliable family or friends who can observe the patient for the first postin-jury day. Printed discharge instructions, which describe moni-toring for confusion, persistent nausea, weakness, or speech difficulty, should be provided to the caretaker. The patient should return to the emergency department for evaluation of such symptoms.Patients with a history of altered consciousness, amne-sia, progressive headache, skull or facial fracture, vomiting, or seizure have a moderate risk for intracranial injury and should undergo a prompt head CT. If the CT is normal, and the neuro-logic examination has returned to baseline (excluding amnesia of the event), then the patient can be discharged to the care of a responsible adult, again with printed criteria for returning to the emergency room. Otherwise the patient must be admitted for a 24-hour observation

1	the patient can be discharged to the care of a responsible adult, again with printed criteria for returning to the emergency room. Otherwise the patient must be admitted for a 24-hour observation period.Patients with depressed consciousness, focal neurologic deficits, penetrating injury, depressed skull fracture, or changing neurologic examination have a high risk for intracranial injury. These patients should undergo immediate head CT and admis-sion for observation or intervention as needed.Types of Closed Head Injury Concussion A concussion is defined as temporary neuronal dysfunction following nonpenetrating head trauma. The head CT is normal, and deficits resolve over minutes to hours. Defini-tions vary; some require transient loss of consciousness, while others include patients with any alteration of mental status. Memory difficulties, especially amnesia of the event, are very 3Brunicardi_Ch42_p1827-p1878.indd 183601/03/19 7:16 PM 1837NEUROSURGERYCHAPTER 42common. Concussions

1	alteration of mental status. Memory difficulties, especially amnesia of the event, are very 3Brunicardi_Ch42_p1827-p1878.indd 183601/03/19 7:16 PM 1837NEUROSURGERYCHAPTER 42common. Concussions may be graded. One method is the Col-orado grading system.14 Head trauma patients with confusion only are grade 1, patients with amnesia are grade 2, and patients who lose consciousness are grade 3. Studies have shown that the brain remains in a hypermetabolic state for up to a week after injury. The brain is also much more susceptible to injury from even minor head trauma in the first 1 to 2 weeks after concus-sion. This is known as second-impact syndrome, and patients should be informed that, even after mild head injury, they might experience memory difficulties or persistent headaches. Return to play guidelines after sports-related concussions are contro-versial and are under active debate.15Contusion A contusion is a bruise of the brain, and occurs when the force from trauma is

1	Return to play guidelines after sports-related concussions are contro-versial and are under active debate.15Contusion A contusion is a bruise of the brain, and occurs when the force from trauma is sufficient to cause breakdown of small vessels and extravasation of blood into the brain. The contused areas appear bright on CT scan, as seen in Fig. 42-8. The frontal, occipital, and temporal poles are most often involved. The brain sustains injury as it collides with rough, bony surfaces. Contu-sions themselves rarely cause significant mass effect as they represent small amounts of blood in injured parenchyma rather than coherent blood clots. Edema may develop around a contu-sion, causing mass effect. Contusions may enlarge or progress to frank hematoma, particularly during the first 24 hours. Contu-sions also may occur in brain tissue opposite the site of impact. This is known as a contre-coup injury. These contusions result from deceleration of the brain against the skull.Diffuse Axonal

1	Contu-sions also may occur in brain tissue opposite the site of impact. This is known as a contre-coup injury. These contusions result from deceleration of the brain against the skull.Diffuse Axonal Injury Diffuse axonal injury (DAI) is caused by damage to axons throughout the brain, due to rotational acceleration and then deceleration. Axons may be completely disrupted and then retract, forming axon balls. Small hemor-rhages can be seen in more severe cases, especially on MRI. Hemorrhage is classically seen in the corpus callosum and the dorsolateral midbrain. DAI can be considered to be a severe form of a concussion, often with irreversible consequence. It can often explain a poor neurological examination (such as impaired arousal) in cases without clear radiographic signs of global bran injury, particularly when there is damage in struc-tures, such as the pontine reticular activating system or bilateral thalami, that are necessary for arousal. In these cases, alternative

1	global bran injury, particularly when there is damage in struc-tures, such as the pontine reticular activating system or bilateral thalami, that are necessary for arousal. In these cases, alternative explanations of poor arousal, such as a basilar thrombus, must also be investigated.Penetrating Injury These injuries are complex and must be evaluated individually. The two main subtypes are missile (e.g., due to bullets or fragmentation devices) and nonmissile (e.g., due to knives or ice picks). Some general principles apply. If available, skull X-rays and CT scans are useful in assessing the nature of the injury. Cerebral angiography must be considered if the object passes near a major artery or dural venous sinus. Operative exploration is necessary to remove any object extend-ing out of the cranium, as well as for debridement, irrigation, hemostasis, and definitive closure. Small objects contained within brain parenchyma are often left in place to avoid iat-rogenic secondary brain

1	the cranium, as well as for debridement, irrigation, hemostasis, and definitive closure. Small objects contained within brain parenchyma are often left in place to avoid iat-rogenic secondary brain injury. High-velocity missile injuries (from high-powered hunting rifles or military weapons) are especially deadly, because the associated shock wave causes cavitary tissue destruction of an area that is much larger than the projectile itself. Projectiles that penetrate both hemispheres or traverse the ventricles are almost universally fatal. Antibiot-ics are given to decrease the chances of meningitis or abscess formation; however, the evidence supporting the use of antibi-otics following missile injury is weak and largely comes from retrospective case studies and expert opinion. Recent guidelines published in regard to preventing combat-related infections recommend antimicrobial therapy for 5 days or until resolu-tion of the associated CSF leak, albeit with limited supporting

1	guidelines published in regard to preventing combat-related infections recommend antimicrobial therapy for 5 days or until resolu-tion of the associated CSF leak, albeit with limited supporting evidence.16Traumatic Intracranial Hematomas. The various traumatic intracranial hematomas contribute to death and disability sec-ondary to head injury. Hematomas can expand rapidly and cause brain shift and subsequent herniation. Emergent neurosurgical evaluation and intervention often are necessary.Epidural Hematoma EDH is the accumulation of blood between the skull and the dura. EDH usually results from arte-rial disruption, especially of the middle meningeal artery. The dura is adherent to bone, and some pressure is required to dis-sect between the two. On head CT, the blood clot is bright, biconvex in shape (lentiform), and has a well-defined border that usually respects cranial suture lines. An EDH is typically found over the convexities but may rarely occur in the posterior fossa as well.

1	in shape (lentiform), and has a well-defined border that usually respects cranial suture lines. An EDH is typically found over the convexities but may rarely occur in the posterior fossa as well. EDH has a classic, three-stage clinical presenta-tion that is probably seen in only 20% of cases. The patient is initially unconscious from the concussive aspect of the head trauma. The patient then awakens and has a “lucid interval,” while the hematoma subclinically expands. As the volume of the hematoma grows, the decompensated region of the pressure-volume curve is reached, ICP increases, and the patient rapidly becomes lethargic and herniates. Uncal herniation from an EDH classically causes ipsilateral third nerve palsy and contralateral hemiparesis.Open craniectomy for evacuation of the congealed clot and hemostasis generally is indicated for EDH. In some cases, EDH can be caused from bony venous bleeding that is self-limited Figure 42-8. Severe bilateral contusions in the basal aspect

1	clot and hemostasis generally is indicated for EDH. In some cases, EDH can be caused from bony venous bleeding that is self-limited Figure 42-8. Severe bilateral contusions in the basal aspect of the frontal lobes, caused by the brain moving over the rough, irregular skull base during sudden cranial acceleration.Brunicardi_Ch42_p1827-p1878.indd 183701/03/19 7:16 PM 1838SPECIFIC CONSIDERATIONSPART IIand may not require surgical intervention. Generally, patients who meet all of the following criteria may be managed conser-vatively: clot volume <30 cm3, maximum thickness <1.5 cm, and GCS score >8.10 Prognosis after successful evacuation is better for EDH than subdural hematoma (SDH). EDHs are associ-ated with lower-energy trauma with less resultant primary brain injury. Good outcomes may be seen in 85% to 90% of patients, with rapid CT scan and intervention.11 In some cases, EDH can also be caused by dural venous sinus tears that rapidly expand and are typically associated with a

1	may be seen in 85% to 90% of patients, with rapid CT scan and intervention.11 In some cases, EDH can also be caused by dural venous sinus tears that rapidly expand and are typically associated with a high degree of morbidity when treated surgically.Acute Subdural Hematoma An acute SDH is the result of an accumulation of blood between the arachnoid membrane and the dura. Acute SDH usually results from venous bleed-ing, typically from tearing of a bridging vein running from the cerebral cortex to the dural sinuses. The bridging veins are sub-ject to stretching and tearing during acceleration/deceleration of the head because the brain shifts in relation to the dura, which firmly adheres to the skull. Elderly and alcoholic patients are at higher risk for acute SDH formation after head trauma due to brain atrophy.On head CT scan, the clot is bright or mixed-density, cres-cent-shaped (lunate), may have a less distinct border, and does not cross the midline due to the presence of the falx.

1	due to brain atrophy.On head CT scan, the clot is bright or mixed-density, cres-cent-shaped (lunate), may have a less distinct border, and does not cross the midline due to the presence of the falx. Most SDHs occur over the cerebral hemispheres, but they may also occur between the hemispheres or layer over the tentorium.Open craniotomy for evacuation of acute SDH is indicated for any of the following: thickness >1 cm, midline shift >5 mm, or GCS drop by two or more points from the time of injury to hospitalization. Nonoperatively managed hematomas may sta-bilize and eventually reabsorb, or evolve into chronic SDHs.17 This management requires frequent neurologic examinations until the clot stabilizes based on serial head CT scans.The prognosis for functional recovery is significantly worse for acute SDH than EDH because it is associated with greater primary injury to brain parenchyma from high-energy impacts. Prompt recognition and intervention minimizes sec-ondary injury. The elderly

1	for acute SDH than EDH because it is associated with greater primary injury to brain parenchyma from high-energy impacts. Prompt recognition and intervention minimizes sec-ondary injury. The elderly patients with low admission GCS, or high postoperative ICP do poorly, with as few as 5% attaining functional recovery.18Chronic Subdural Hematoma Chronic SDH is a collection of blood breakdown products that is at least 2 to 3 weeks old. Acute hematomas are bright white (hyperdense) on CT scan for approximately 3 days, after which they fade to isodensity with brain, and then to hypodensity after 2 to 3 weeks. A true chronic SDH will be nearly as dark as CSF on CT. Traces of white are often seen due to small, recurrent hemorrhages into the col-lection. These small bleeds may expand the collection enough to make it symptomatic. This phenomenon is referred to as an acute-on-chronic SDH. Figure 42-9 demonstrates the CT appearance of an acute-on-chronic SDH. Vascularized mem-branes form within

1	enough to make it symptomatic. This phenomenon is referred to as an acute-on-chronic SDH. Figure 42-9 demonstrates the CT appearance of an acute-on-chronic SDH. Vascularized mem-branes form within the hematoma as it matures. These mem-branes may be the source of acute hemorrhage.Chronic SDHs often occur in patients without a clear his-tory of head trauma as they may arise from minor head injury. Alcoholics, the elderly, and patients on anticoagulation are at higher risk for developing chronic SDH. Patients may present with headache, seizure, confusion, contralateral hemiparesis, or coma.A chronic SDH >1 cm or any symptomatic SDH should be surgically drained. Unlike acute SDH, which consists of a thick, congealed clot, chronic SDH typically consists of a viscous fluid with the texture and dark brown color reminiscent of motor oil. A simple burr hole can effectively drain most chronic SDHs. However, the optimal treatment of chronic SDH remains con-troversial.19 Recent data suggest that

1	dark brown color reminiscent of motor oil. A simple burr hole can effectively drain most chronic SDHs. However, the optimal treatment of chronic SDH remains con-troversial.19 Recent data suggest that open craniotomy is effec-tive at reducing recurrence, but may be associated with more short-term complications.20 Most authorities agree that burr hole drainage should be attempted first to obviate the risks of formal craniotomy.21 A single burr hole placed over the dependent edge of the collection can be made, and the space is copiously irri-gated until the fluid is clear. A second, more anterior burr hole can then be placed if the collection does not drain satisfactorily due to containment by membranes. The procedure is converted to open craniotomy if the SDH is too congealed for irrigation drainage, the complex of membranes prevents effective drain-age, or persistent hemorrhage occurs that cannot be reached with bipolar cautery through the burr hole. The required surgi-cal prepping and

1	drainage, the complex of membranes prevents effective drain-age, or persistent hemorrhage occurs that cannot be reached with bipolar cautery through the burr hole. The required surgi-cal prepping and draping are always performed to allow simple conversion to craniotomy, and the scalp incision and burr holes are placed to allow easy incorporation into larger skin flaps.There are various strategies to prevent reaccumulation of blood. Subdural or subgaleal drains may be left in place for 1 to 2 days. Subdural drains have been shown to reduce the risk of recurrence, whereas corticosteroid use in this patient popula-tion has been associated with higher morbidity without benefit.20 Mild hydration and bedrest with the head of the bed flat may Figure 42-9. Head computed tomography scan of an elderly patient with progressing left hemiplegia and lethargy, demonstrat-ing an acute-on-chronic subdural hematoma. History revealed that the patient sustained a fall 4 weeks before presentation.

1	an elderly patient with progressing left hemiplegia and lethargy, demonstrat-ing an acute-on-chronic subdural hematoma. History revealed that the patient sustained a fall 4 weeks before presentation. Arrowheads outline the hematoma. The acute component is slightly denser and is seen as the hyperdense area in the dependent portion.Brunicardi_Ch42_p1827-p1878.indd 183801/03/19 7:16 PM 1839NEUROSURGERYCHAPTER 42encourage brain expansion. High levels of inspired oxygen may help draw nitrogen out of the cavity. Regardless of the strategy used, follow-up head CT scans are required postoperatively and approximately 1 month later to document resolution.Intraparenchymal Hemorrhage Isolated hematomas within the brain parenchyma are most often associated with hyper-tensive hemorrhage or arteriovenous malformations (AVMs). Bleeding may occur in a contused area of brain. Mass effect from developing hematomas may present as a delayed neuro-logic deficit. Delayed traumatic intracerebral

1	arteriovenous malformations (AVMs). Bleeding may occur in a contused area of brain. Mass effect from developing hematomas may present as a delayed neuro-logic deficit. Delayed traumatic intracerebral hemorrhage is most likely to occur within the first 24 hours. Patients with contusion on the initial head CT scan should be reimaged 24 hours after the trauma to document stable pathology. Indica-tions for craniotomy include: any clot volume >50 cm3 or a clot volume >20 cm3 with referable neurologic deterioration (GCS 6–8) and associated midline shift >5 mm or basal cistern compression.22Pneumocephalus Pneumocephalus, or air in the intracranial cavity, is commonly seen in neurosurgical patients following head trauma or following intracranial surgery.23 Pneumoceph-alus requires a defect in the skull that allows air to enter the intracranial cavity. This may occur following may represent an iatrogenic defect created following cranial surgery or follow-ing head trauma. Approximately 66% of

1	skull that allows air to enter the intracranial cavity. This may occur following may represent an iatrogenic defect created following cranial surgery or follow-ing head trauma. Approximately 66% of postcraniotomy CT scans demonstrate some extent of pneumocephalus.24 The iden-tification of pneumocephalus following head trauma can offer important clues about the extent of injury, such as the presence of skull based fractures or a CSF leak. In rare cases, pneumo-cephalus can also be seen in association with skull based tumors or infections.A tension pneumocephalus occurs when the intracranial air pocket is under tension which can result in life threatening herniation if left untreated. This is a neurosurgical emergency and requires an urgent neurosurgical consultation. Two radio-graphic features have been associated with a tension pneumo-cephalus25 (Fig. 42-10). First, the “Mount Fuji” sign, where the air pocket separates the frontal lobes and widens the inter-hemi-spheric fissure,

1	features have been associated with a tension pneumo-cephalus25 (Fig. 42-10). First, the “Mount Fuji” sign, where the air pocket separates the frontal lobes and widens the inter-hemi-spheric fissure, mimicking the silhouette of Mount Fuji. Second, the “air bubble” sign, where there are multi-focal pockets of air throughout the subarachnoid cisterns, putatively within the sub-arachnoid space. These radiographic findings are helpful clues; however, the diagnosis of tension pneumocephalus also requires a worsening neurological exam consistent with increased intra-cranial pressure and impending herniation. A burr hole may be used to relieve intracranial pressure per the discretion of the neurosurgical team.When not associated with tension dynamics, the clinical significance and management of pneumocephalus depends on the underlying mechanism. There are thought to be two major mechanisms by which pneumocephalus develops.24 First, the “ball valve” mechanism involves the passage of air into

1	pneumocephalus depends on the underlying mechanism. There are thought to be two major mechanisms by which pneumocephalus develops.24 First, the “ball valve” mechanism involves the passage of air into the intracranial cavity during periods of positive pressure, whereby the defect in the skull acts as a one-way valve. In these cases, management involves avoiding positive pressure ventilation, and laying the head of the bed flat to minimize air traveling upwards into the cranial cavity. Second, the “inverted bottle” mechanism involves air entering the intracranial space due to a negative pressure gradient created by the drainage of CSF. In most cases, drainage occurs through a traumatic or iatrogenic CSF leak, but it may also occur through ventricular or lumbar drainage. In these cases, management should be focused on minimizing CSF drainage through the defect. If the CSF leak is at the skull base, as is the case following basilar skull fractures, or those involving the mastoid air

1	management should be focused on minimizing CSF drainage through the defect. If the CSF leak is at the skull base, as is the case following basilar skull fractures, or those involving the mastoid air cells, then the head of bed must be elevated so as to reduce hydrostatic pressure in the ven-tricular CSF fluid column, and controlled CSF diversion can be performed using an extraventricular or lumbar drain (see “Skull Fractures” for further discussion). Definitive repair of the skull-based defect can also be considered, but this is often done on an elective basis. In general, nontension pneumocephalus will resolve on its own with time as it is resorbed into the blood stream. Supplemental 100% oxygen may be used to increase the rate of resorption by increasing the diffusion gradient of nitro-gen-predominant intracranial air pocket and the blood stream.26Management of Traumatic Brain Injury General Medical Management Several medical steps may be taken to minimize secondary injury and the

1	intracranial air pocket and the blood stream.26Management of Traumatic Brain Injury General Medical Management Several medical steps may be taken to minimize secondary injury and the systemic con-sequences of head injury. Patients with a documented CHI and evidence of intracranial hemorrhage or a depressed skull fracture should receive a 1 g Keppra loading dose, followed by 1 week of therapeutic maintenance Keppra, typically 500 mg twice a day. Antiseizure prophylaxis has been shown to decrease the incidence of early posttraumatic seizures.27 There is no evi-dence to support long-term use of prophylactic antiepileptic agents. Even though the clinical studies supporting early anti-seizure prophylaxis used phenytoin, Keppra is typically used in clinical practice due to a more favorable side effect profile. Blood glucose levels should be closely monitored by free blood sugar checks and controlled with sliding scale insulin. Fevers also should be evaluated and controlled with

1	side effect profile. Blood glucose levels should be closely monitored by free blood sugar checks and controlled with sliding scale insulin. Fevers also should be evaluated and controlled with antipyretics, as well as source-directed therapy when possible. Hyperglycemia and hyperthermia are toxic to injured neurons and contribute to secondary injury. Head-injured patients have an increased prevalence of peptic ulceration and GI bleeding. Peptic ulcers occurring in patients with head injury or high ICP are referred to as Cushing’s ulcers. Ulcer prophylaxis should be used. Com-pression stockings or athrombic pumps should be used when the patient cannot be mobilized rapidly for prophylaxis of deep venous thrombosis.Steroids and Traumatic Brain Injury Per a level 1 recom-mendation (high-quality evidence) from the Brain Trauma Foundation,10 steroids are not recommended for the management of TBI or reduction of elevated ICP. Also, high-dose methyl-prednisolone is contraindicated in severe

1	evidence) from the Brain Trauma Foundation,10 steroids are not recommended for the management of TBI or reduction of elevated ICP. Also, high-dose methyl-prednisolone is contraindicated in severe TBI. A large random-ized controlled trial (CRASH; n = 9673, 6-month follow-up,28 Figure 42-10. CT image on left illustrates “Mt. Fuji sign” (arrow-head) and intraparnchymal air (arrow). CT image on R illustrates brain herniation into the ethmoid sinus (arrowhead).Brunicardi_Ch42_p1827-p1878.indd 183901/03/19 7:16 PM 1840SPECIFIC CONSIDERATIONSPART IIdemonstrated an increased risk of 6-month mortality in severe TBI (GCS 3–8) that received methylprednisolone (47%), as compared to placebo (42%, P = 0.0024). This effect was also present when analyzing TBI of all severity levels (25.7% meth-ylprednisolone vs. 22.3% placebo, P = 0.0001).Blood Pressure Management Blood pressure management in TBI is a complex issue. On one hand, hypotension results in hypoperfusion that may worsen brain injury

1	vs. 22.3% placebo, P = 0.0001).Blood Pressure Management Blood pressure management in TBI is a complex issue. On one hand, hypotension results in hypoperfusion that may worsen brain injury that occurs follow-ing TBI. On another hand, hypertension may result in expansion of intracranial hematomas that are often seen in TBI.There is clear evidence from retrospective studies, that frank hypotension (SBP <90 mmHg) is associated with increased mortality in TBI, particularly in the prehospital setting and during resuscitation.29 A large retrospective cohort study (n = 15,733) identified hypotension thresholds that were associ-ated with an increased risk of mortality in patients with TBI of varying age.30 Based on these data, the Brain Trauma Founda-tion guidelines10 provide a level III (low-quality) recommenda-tion that maintaining systolic blood pressures >100 mmHg (ages 50–69 years), or >110 mmHg (ages 15–49 years or >70 years) may be considered to reduce mortality and improve outcomes.

1	recommenda-tion that maintaining systolic blood pressures >100 mmHg (ages 50–69 years), or >110 mmHg (ages 15–49 years or >70 years) may be considered to reduce mortality and improve outcomes. More recently, a large retrospective study31 demonstrated a dose-dependent relation between the duration of prehospital hypotension and increased mortality in patients with TBI, such that a 10-point increase in systolic blood pressure across a broad range (40–119 mmHg) was associated with an 18.8% decrease in adjusted odds of inhospital mortality. These results suggest that having a single “hypotension threshold” may not be suf-ficient in management of TBI and may require more aggressive management than currently employed. Furthermore, an impor-tant and underappreciated consideration in blood pressure man-agement is the baseline blood pressure of the patient. Future studies should assess blood pressure management goals that are tailored to each individual patient’s baseline blood pressure.On the

1	man-agement is the baseline blood pressure of the patient. Future studies should assess blood pressure management goals that are tailored to each individual patient’s baseline blood pressure.On the other hand, hypertension in TBI may have impli-cations for intracranial hematoma expansion. It is common in clinical practice to recommend that systolic blood pressures are maintained <160 mmHg to mitigate the risk of hematoma expan-sion. Evidence supporting this practice is largely extrapolated from non-TBI patients. A small retrospective study (n = 69)32 demonstrated an increased risk of postcraniotomy intracranial hematoma in patients with intraoperative hypertension (62% vs. 34% controls, P <0.001), and postoperative hyperten-sion in the first 12 hours after surgery (62% vs. 25% controls, P <0.001). A recent large retrospective study in patients with anti-coagulant-associated intracranial hematoma demonstrated that lowering SBP to less than 160 mmHg within 4 hours of admis-sion was

1	P <0.001). A recent large retrospective study in patients with anti-coagulant-associated intracranial hematoma demonstrated that lowering SBP to less than 160 mmHg within 4 hours of admis-sion was associated with a reduced risk of hematoma expan-sion (n = 691, 33.1% <160 mmHg vs. 52.4 % in ≥160 mmHg; P <.001).33 However, there are no specific recommendations from the Brain Trauma Foundation on a hypertension threshold to avoid in patients with traumatic intracranial hematoma.Anticoagulation Reversal and Prophylaxis Patients with intracranial hematoma who are on anticoagulation for car-diovascular indications (atrial fibrillation, cardiac stents, or mechanical valves) or stroke prevention present a challenging population. Anticoagulation reversal is important to reduce the risk of hematoma expansion; however, anticoagulant reversal is also associated with thrombotic cardiovascular complications. A recent retrospective study in patients with nontraumatic, oral-anticoagulant–associated

1	expansion; however, anticoagulant reversal is also associated with thrombotic cardiovascular complications. A recent retrospective study in patients with nontraumatic, oral-anticoagulant–associated intracranial hematoma showed that lowering the INR to <1.3 within 4 hours of admission was an independent predictor of hematoma expansion (n = 853; 19.8% vs. 41.5% in INR of ≥1.3; P <.001). Furthermore, this study showed that the risk of ischemic complications was greater in patients that were not restarted on oral anticoagulation as com-pared to those that were subsequently restarted (n = 719; 5.2% vs. 15%, no restart, P <0.001); however, they did not observe a significant increase in the risk of hemorrhage with anticoagula-tion restart (n = 719; 8.1%, vs. 6.6%, P = 0.48). The median time to anticoagulation restart was 30 days after discharge (inter-quartile range 18–65), as such, these data do not speak to risks and benefits of restarting anticoagulation in the acute post-bleed interval.

1	restart was 30 days after discharge (inter-quartile range 18–65), as such, these data do not speak to risks and benefits of restarting anticoagulation in the acute post-bleed interval. It is important to note that the risks and benefits of restarting anticoagulation will vary based on the individual patient and the patient’s indications for anticoagulation (e.g., mechanical heart valve vs. atrial fibrillation). As such, close collaboration between the neurosurgery and cardiology teams are important in optimizing a management strategy for these patients.Anticoagulation prophylaxis for prevention of venous thrombosis also involves a risk-benefit analysis. Per a level III (low-quality) recommendation of Brain Trauma Foundation Guidelines,10 anticoagulation prophylaxis with low-molecular-weight heparin or low-dose unfractionated heparin may be used to reduce the risk of venous thrombosis, even though it is associ-ated with an increased risk of intracranial hematoma expansion. It may be

1	heparin or low-dose unfractionated heparin may be used to reduce the risk of venous thrombosis, even though it is associ-ated with an increased risk of intracranial hematoma expansion. It may be reasonable to initiate prophylactic anticoagulation 24 hours after an intracranial hematoma is deemed to be stable. A single-center retrospective study (n = 236) found that such a strategy was associated with a decreased risk of DVT (0% vs. 5.6% (n = 6), P <0.001), but did not observe significant differ-ences in the rates of pulmonary embolism (0.78% (n = 1) vs. 3.74% (n = 4), P = 0.18) or intracranial hematoma expansion (0.7% [1] vs. 2.8% [3], P = 0.3). However, because of the low rate of clinical events observed in this series, the study may have been underpowered to identify small differences in pulmonary embolism or hematoma expansion.Indications for Invasive Intracranial Monitoring In patients with severe TBI (GCS <8), the Brain Trauma Founda-tion guidelines endorse a level IIB

1	in pulmonary embolism or hematoma expansion.Indications for Invasive Intracranial Monitoring In patients with severe TBI (GCS <8), the Brain Trauma Founda-tion guidelines endorse a level IIB recommendation (low-quality of evidence) for ICP and cerebral perfusion pressure (CPP) to reduce short-term mortality (within 2 weeks of hospitalization). They also provide level IIB recommendations for treating ICP >22 mmHg and treating CPP level between 60 and 70 mmHg to optimize outcomes. These recommendations are supported by a recent retrospective cohort study (n = 459)34 that identified ICP and CPP thresholds that best discriminated between survi-vors and nonsurvivors in severe TBI, and also between survivors with “poor” and “favorable’ outcomes (Glasgow Outcome Scale 1–3 vs. 4–6). A large, multicenter randomized controlled trial performed in 6 hospitals in Equador and Bolivia did not sup-port the claim that intracranial monitoring in severe TBI results in improved clinical outcomes.

1	multicenter randomized controlled trial performed in 6 hospitals in Equador and Bolivia did not sup-port the claim that intracranial monitoring in severe TBI results in improved clinical outcomes. Chestnut et al in 2012 did not observe a significant difference in mortality or favorable out-comes (as assessed by the Glasgow Outcome Scale) when severe TBI patients were managed with an intracranial monitor (n = 56), or with imaging and clinical exam (n = 53, P = 0.43). Advanced multimodal monitoring such as brain tissue oxygen (PbrO2) mon-itoring, jugular bulb monitoring of arteriovenous oxygen content difference (AVDO2), cerebral autoregulation with TCD, and micro dialysis are under active investigation. Only jugular bulb monitoring of AVDO2 is associated with a level III (poor-quality evidence) recommendation to guide management in severe TBI. Brunicardi_Ch42_p1827-p1878.indd 184001/03/19 7:16 PM 1841NEUROSURGERYCHAPTER 42The Brain Tissue Oxygen Monitoring in TBI (BOOST) trials are

1	recommendation to guide management in severe TBI. Brunicardi_Ch42_p1827-p1878.indd 184001/03/19 7:16 PM 1841NEUROSURGERYCHAPTER 42The Brain Tissue Oxygen Monitoring in TBI (BOOST) trials are actively investigating the added benefit of brain tissue oxygen-ation beyond intracranial pressure monitoring in severe TBI.Decompressive Craniectomy for Severe TBI Decompressive craniectomy can be performed to relieve intracranial pressure associated with diffuse cerebral edema in cases of severe TBI without mass lesions (e.g., extra-axial hematoma). This is a controversial issue as there is a paucity of high-quality evi-dence providing clear support for or against this intervention. The DECRA trial (a multicenter, randomized, controlled trial, n = 155) compared bifrontal decompressive craniectomy to medical management for the treatment of patients with severe TBI and elevated intracranial pressure refractory to first-tier therapies (ICP >20 mmHg for at least 15 minutes within an hour). They

1	to medical management for the treatment of patients with severe TBI and elevated intracranial pressure refractory to first-tier therapies (ICP >20 mmHg for at least 15 minutes within an hour). They found no significant difference in mortality at six months, and found that functional outcomes (as measured by the Extended Glasgow Outcome Scale) were worse in patients who underwent surgery. They found a clear improvement in ICP and number of days in the ICU in patients that underwent surgery as compared to medical management. Of note, they used an intention-to-treat analysis, such that 18% of patients in the medical management group underwent a delayed cra-niotomy as a life-saving procedure. More recently, the RES-CUE-ICP trial36 (a multicenter, randomized, controlled trial, n = 408) compared decompressive craniotomy and ongoing medical care in patients with severe TBI (without mass lesions) with elevated ICP (>25 mmHg) refractory to firstand second-tier interventions (medical management

1	decompressive craniotomy and ongoing medical care in patients with severe TBI (without mass lesions) with elevated ICP (>25 mmHg) refractory to firstand second-tier interventions (medical management and ventriculostomy). Patients were randomized to either receive a barbiturate infu-sion (medical group) or undergo decompressive craniotomy (surgery group; unilateral hemicraniectomy vs. bifrontal crani-otomy depending on degree of bilateral swelling and surgeon discretion). Again, they used an intention-to-treat analysis such that 37% of patients of the medical group underwent decom-pressive hemicraniectomy. At 6 months, decompressive crani-ectomy in patients with traumatic brain injury and refractory intracranial hypertension resulted in lower mortality and higher rates of vegetative state, lower severe disability, and upper severe disability than medical care. The rates of moderate dis-ability and good recovery were similar in the two groups. The recent Brain Trauma Foundation

1	state, lower severe disability, and upper severe disability than medical care. The rates of moderate dis-ability and good recovery were similar in the two groups. The recent Brain Trauma Foundation Guidelines offer a level II (moderate-quality) recommendation against performing a bifrontal decompressive hemicraniectomy to improve func-tional outcomes at 6 months in patients with severe TBI with diffuse injury and no mass lesions, and with elevated ICP that is medically refractory. They note that this procedure has been demonstrated to reduce time in the ICU and ICP. However, they have not made an updated recommendation since the results of the RESCUE-ICP trial have been published.The results of the DECRA and RESCUE-ICP trials suggest caution and careful consideration prior to perform-ing decompressive craniotomy in treating severe TBI without mass lesions. There is now evidence that this procedure can be lifesaving and reduce mortality at 6 months; however, it is not clear that the

1	decompressive craniotomy in treating severe TBI without mass lesions. There is now evidence that this procedure can be lifesaving and reduce mortality at 6 months; however, it is not clear that the survivors have a favorable functional outcome (as grossly measured by the Extended Glasgow Outcome Scale). By improving ICP and reducing time in the ICU, it may hasten the recovery process by allowing patients to begin rehabilita-tion earlier. Also, several unanswered questions remain. For example, might outcomes be improved if decompressive cra-niectomy was performed earlier, prior to the patient develop-ing refractory ICP, and presumably secondary brain injury? As such, the decision of whether or not to perform decompressive craniotomy must be carefully considered within the context of each individual patient’s clinical scenario, the patient’s avail-able social support system, and the family’s disposition and goals of care.Vascular Injury. Trauma to the head or neck may cause damage to

1	individual patient’s clinical scenario, the patient’s avail-able social support system, and the family’s disposition and goals of care.Vascular Injury. Trauma to the head or neck may cause damage to the carotid or vertebrobasilar systems. Generally, dissection refers to violation of the vessel wall intima. Blood at arterial pressures can then open a plane between the intima and media, within the media, or between the media and adventitia. The newly created space within the vessel wall is referred to as the false lumen. Tissue or organs supplied by dissected ves-sels may subsequently be injured in several ways. Expansion of the hematoma within the vessel wall can lead to narrowing of the true vessel lumen and reduction or cessation of distal blood flow. Slow-flowing or stagnant blood within the false lumen exposed to thrombogenic vessel wall elements may thrombose. Pieces of thrombus may then detach and cause distal embolic arterial occlusion. Also, the remaining partial-thickness

1	the false lumen exposed to thrombogenic vessel wall elements may thrombose. Pieces of thrombus may then detach and cause distal embolic arterial occlusion. Also, the remaining partial-thickness vessel wall may rupture, damaging adjacent structures.Traumatic dissection may occur in the carotid artery (ante-rior circulation) or the vertebral or basilar arteries (posterior cir-culation). Dissections may be extradural or intradural. Intradural dissection can present with subarachnoid hemorrhage (SAH). Traditional angiography remains the basis of diagnosis and characterization of arterial dissection. Angiographic abnormali-ties include stenosis of the true lumen, or “string-sign,” visible intimal flaps, and the appearance of contrast in the false lumen. Four-vessel cerebral angiography should be performed when suspicion of dissection exists.Historically, patients with documented arterial dissec-tion have been anticoagulated with heparin and then warfarin to prevent thromboembolic stroke.

1	be performed when suspicion of dissection exists.Historically, patients with documented arterial dissec-tion have been anticoagulated with heparin and then warfarin to prevent thromboembolic stroke. Trauma patients often have concomitant absolute or relative contraindications to anticoagu-lation, complicating management. Antiplatelet therapy is often implemented in lieu of full anticoagulation, however, there is no randomized clinical trial comparing the two therapies.37 Consider surgical or interventional techniques for persisting embolic disease and for vertebral dissections presenting with SAH. Surgical options include vessel ligation and bypass graft-ing. Interventional radiology techniques include stenting and vessel occlusion. Occlusion techniques require sufficient col-lateral circulation to perfuse the vascular territory previously supplied by the occluded vessel.Carotid Dissection Carotid dissection may result from neck extension combined with lateral bending to the opposite

1	to perfuse the vascular territory previously supplied by the occluded vessel.Carotid Dissection Carotid dissection may result from neck extension combined with lateral bending to the opposite side, or trauma from an incorrectly placed shoulder belt tightening across the neck in a motor vehicle accident. Extension or bend-ing stretches the carotid over the bony transverse processes of the cervical vertebrae, while seat belt injuries cause direct trauma. Symptoms of cervical carotid dissection include con-tralateral neurologic deficit from brain ischemia, headache, and ipsilateral Horner’s syndrome from disruption of the sympa-thetic tracts ascending from the stellate ganglion on the surface of the carotid artery. The patient may complain of a bruit.Traumatic vessel wall injury to the portion of the carotid artery running through the cavernous sinus may result in a carotid-cavernous fistula (CCF). This creates a high-pressure, high-flow pathophysiologic blood flow pattern. CCFs

1	portion of the carotid artery running through the cavernous sinus may result in a carotid-cavernous fistula (CCF). This creates a high-pressure, high-flow pathophysiologic blood flow pattern. CCFs clas-sically present with pulsatile proptosis (the globe pulses out-ward with arterial pulsation), retro-orbital pain, and decreased visual acuity or loss of normal eye movement (due to damage Brunicardi_Ch42_p1827-p1878.indd 184101/03/19 7:16 PM 1842SPECIFIC CONSIDERATIONSPART IIto cranial nerves III, IV, and VI as they pass through the cav-ernous sinus). Symptomatic CCFs should be treated to preserve eye function. Fistulae may be closed by balloon occlusion using interventional neuroradiology techniques. Fistulae with wide necks are difficult to treat and may require total occlusion of the parent carotid artery.Vertebrobasilar Dissection Vertebrobasilar dissection may result from sudden rotation or flexion/extension of the neck, chiropractic manipulation, or a direct blow to the neck.

1	parent carotid artery.Vertebrobasilar Dissection Vertebrobasilar dissection may result from sudden rotation or flexion/extension of the neck, chiropractic manipulation, or a direct blow to the neck. Com-mon symptoms are neck pain, headache, and brain stem stroke or SAH. The risks and benefits of aspirin therapy are unclear when a vertebral dissection extends intracranially. The theoreti-cally increased friability of the vessel wall may increase the risk of SAH when coupled with an antiplatelet agent. Consultation of a stroke neurologist is recommended in this situation.Brain Death. Brain death occurs when there is an absence of signs of brain stem function or motor response to deep central pain in the absence of pharmacologic or systemic medical con-ditions that could impair brain function.Clinical Examination A neurologist, neurosurgeon, or inten-sivist generally performs the clinical brain death examination. Two examinations consistent with brain death 12 hours apart, or one

1	function.Clinical Examination A neurologist, neurosurgeon, or inten-sivist generally performs the clinical brain death examination. Two examinations consistent with brain death 12 hours apart, or one examination consistent with brain death followed by a consistent confirmatory study generally is sufficient to declare brain death (see following paragraphs). Hospital regulations and local laws regarding documentation should be followed closely.Establish the absence of complicating conditions before beginning the examination. The patient must be normotensive, euthermic, and oxygenating well. The patient may not be under the effects of any sedating or paralytic drugs.Documentation of no brain stem function requires the fol-lowing: nonreactive pupils; lack of corneal blink, oculocephalic (doll’s eyes), oculovestibular (cold calorics) reflexes; and loss of drive to breathe (apnea test). The apnea test demonstrates no spontaneous breathing even when Paco2 is allowed to rise above 60

1	(doll’s eyes), oculovestibular (cold calorics) reflexes; and loss of drive to breathe (apnea test). The apnea test demonstrates no spontaneous breathing even when Paco2 is allowed to rise above 60 mmHg.Deep central painful stimuli are provided by bilateral forceful twisting pinch of the supraclavicular skin and pressure to the medial canthal notch. Pathologic responses such as flexor or extensor posturing are not compatible with brain death. Spi-nal reflexes to peripheral pain, such as triple flexion of the lower extremities, are compatible with brain death.Confirmatory Studies Confirmatory studies are performed after a documented clinical examination consistent with brain death. A study consistent with brain death may obviate the need to wait 12 hours for a second examination. This is especially important when the patient is a potential organ donor, as brain-dead patients often have progressive hemodynamic instability. Lack of cerebral blood flow consistent with brain death may be

1	especially important when the patient is a potential organ donor, as brain-dead patients often have progressive hemodynamic instability. Lack of cerebral blood flow consistent with brain death may be documented by cerebral angiography or technetium radio-nuclide study. A “to-and-fro” pattern on transcranial Doppler ultrasonography indicates no net forward flow through the cere-bral vasculature, consistent with brain death. An electroenceph-alogram (EEG) documenting electrical silence has been used but generally is not favored because there is often significant artifact which impairs interpretation.Spine TraumaThe spine is a complex biomechanical structure. The spine pro-vides structural support for the body as the principal compo-nent of the axial skeleton, while protecting the spinal cord and nerve roots. Trauma may fracture bones or cause ligamentous disruption. Often, bone and ligament damage occur together. Damage to these elements reduces the strength of the spine and may cause

1	and nerve roots. Trauma may fracture bones or cause ligamentous disruption. Often, bone and ligament damage occur together. Damage to these elements reduces the strength of the spine and may cause instability, which compromises both supportive and protective functions. Spine trauma may occur with or without neurologic injury.Neurologic injury from spine trauma is classified as either incomplete or complete. If there is some residual motor or sensory neurologic function below the level of the lesion, as assessed by clinical examination, the injury is defined as incom-plete.38 A patient with complete neurologic dysfunction persist-ing 24 hours after injury has a very low probability of return of function in the involved area.Neurologic injury from spine trauma may occur immedi-ately or in delayed fashion. Immediate neurologic injury may be due to direct damage to the spinal cord or nerve roots from pen-etrating injuries, especially from stab wounds or gunshots. Blunt trauma may transfer

1	fashion. Immediate neurologic injury may be due to direct damage to the spinal cord or nerve roots from pen-etrating injuries, especially from stab wounds or gunshots. Blunt trauma may transfer sufficient force to the spine to cause acute disruption of bone and ligament, leading to subluxation, which is a shift of one vertebral element in relation to the adjacent level. Subluxation decreases the size of the spinal canal and neu-ral foramina and causes compression of the cord or roots. Such neural impingement can also result from retropulsion of bone fragments into the canal during a fracture. Transection, crush injury, and cord compression impairing perfusion are mecha-nisms leading to SCI. Delayed neurologic injury may occur dur-ing transportation, examination of an improperly immobilized patient, or during a hypotensive episode.The Mechanics of Spine Trauma. Trauma causes a wide variety of injury patterns in the spine due to its biomechanical complexity. A mechanistic approach

1	patient, or during a hypotensive episode.The Mechanics of Spine Trauma. Trauma causes a wide variety of injury patterns in the spine due to its biomechanical complexity. A mechanistic approach facilitates an understand-ing of the patterns of injury, as there are only a few types of forces that can be applied to the spine. Although these forces are discussed individually, they often occur in combination. Several of the most common injury patterns are then presented to illus-trate the clinical results of these forces applied at pathologically high levels.Flexion/Extension Bending the head and body forward into a fetal position flexes the spine. Flexion loads the spine anteriorly (the vertebral bodies) and distracts the spine posteriorly (the spi-nous process and interspinous ligaments). High flexion forces occur during front-end motor vehicle collisions, and backward falls when the head strikes first. Arching the neck and back extends the spine. Extension loads the spine posteriorly and

1	flexion forces occur during front-end motor vehicle collisions, and backward falls when the head strikes first. Arching the neck and back extends the spine. Extension loads the spine posteriorly and distracts the spine anteriorly. High extension forces occur dur-ing rear-end motor vehicle collisions (especially if there is no headrest), frontward falls when the head strikes first, or diving into shallow water.Compression/Distraction Force applied along the spinal axis (axial loading) compresses the spine. Compression loads the spine anteriorly and posteriorly. High compression forces occur when a falling object strikes the head or shoulders, or when landing on the feet, buttocks, or head after a fall from height. A pulling force in line with the spinal axis distracts the spine. Dis-traction unloads the spine anteriorly and posteriorly. Distraction forces occur during a hanging, when the chin or occiput strikes an object first during a fall, or when a passenger submarines under a loose

1	unloads the spine anteriorly and posteriorly. Distraction forces occur during a hanging, when the chin or occiput strikes an object first during a fall, or when a passenger submarines under a loose seat belt during a front-end motor vehicle collision.Rotation Force applied tangential to the spinal axis rotates the spine. Rotation depends on the range of motion of interverte-bral facet joints. High rotational forces occur during off-center Brunicardi_Ch42_p1827-p1878.indd 184201/03/19 7:16 PM 1843NEUROSURGERYCHAPTER 42impacts to the body or head or during glancing automobile accidents.Patterns of Injury. Certain patterns of injury resulting from combinations of the previously mentioned forces occur com-monly and should be recognized during plain film imaging of the spine. Always completely evaluate the spine. A patient with a spine injury at one level has a significant risk for additional injuries at other levels.Cervical The cervical spine is more mobile than the thoraco-lumbar

1	evaluate the spine. A patient with a spine injury at one level has a significant risk for additional injuries at other levels.Cervical The cervical spine is more mobile than the thoraco-lumbar spine. Stability comes primarily from the multiple liga-mentous connections of adjacent vertebral levels. Disruption of the cervical ligaments can lead to instability in the absence of fracture. The mass of the head transmits significant forces to the cervical spine during abrupt acceleration or deceleration, increasing risk for injury.Jefferson Fracture A Jefferson fracture is a bursting fracture of the ring of C1 (the atlas) due to compression forces. There are usually two or more fractures through the ring of C1. The open-mouth odontoid view may show lateral dislocation of the lateral masses of C1. The rule of Spence states that 7 mm or greater combined dislocation indicates disruption of the transverse liga-ment. The transverse ligament stabilizes C1 with respect to C2. Jefferson fractures

1	C1. The rule of Spence states that 7 mm or greater combined dislocation indicates disruption of the transverse liga-ment. The transverse ligament stabilizes C1 with respect to C2. Jefferson fractures dislocated <7 mm usually are treated with a rigid collar, while those dislocated 7 mm or greater usually are treated with a halo vest. Surgical intervention is not indicated.Odontoid Fractures The odontoid process, or dens, is the large ellipse of bone arising anteriorly from C2 (the axis) and projecting up through the ring of C1 (the atlas). Several strong ligaments connect the dens to C1 and to the base of the skull. Odontoid fractures usually result from flexion forces. Odontoid fractures are classified as type I, II, or III. A type I fracture involves the tip only. A type II fracture passes through the base of the odontoid process. A type III fracture passes through the body of C2. Types II and III are considered unstable and should be externally immobilized or fused surgically.

1	passes through the base of the odontoid process. A type III fracture passes through the body of C2. Types II and III are considered unstable and should be externally immobilized or fused surgically. Surgery often is undertaken for widely displaced fractures (poor chance of fus-ing) and for those that fail external immobilization. Type I frac-tures usually fuse with external immobilization only.Hangman’s Fracture Traditionally considered a hyperexten-sion/distraction injury from placement of the noose under the angle of the jaw, hangman’s fractures also may occur with hyperextension/compression, as with diving accidents, or hyper-flexion. The injury is defined by bilateral C2 pars interarticularis fractures. The pars interarticularis is the bone between superior and inferior facet joints. Thus, the posterior bony connection between C1 and C3 is lost. Hangman’s fractures heal well with external immobilization. Surgery is indicated if there is spinal cord compression or after failure of

1	Thus, the posterior bony connection between C1 and C3 is lost. Hangman’s fractures heal well with external immobilization. Surgery is indicated if there is spinal cord compression or after failure of external immobilization.Jumped Facets—Hyperflexion Injury The facet joints of the cervical spine slope forward. In a hyperflexion injury, the supe-rior facet can “jump” over the inferior facet of the level above if the joint capsule is torn. Hyperflexion/rotation can cause a unilateral jumped facet, whereas hyperflexion/distraction leads to bilateral jumped facets. Patients with unilateral injury usually are neurologically intact. Those with bilateral injury, however, typically suffer from spinal cord damage, since the anteropos-terior diameter of the spinal canal is compromised by bilateral injury, leading to spinal cord compression (Fig. 42-11).Thoracolumbar The thoracic spine is stabilized significantly by the rib cage. The lumbar spine has comparatively large vertebrae. Thus, the

1	injury, leading to spinal cord compression (Fig. 42-11).Thoracolumbar The thoracic spine is stabilized significantly by the rib cage. The lumbar spine has comparatively large vertebrae. Thus, the thoracolumbar spine has a higher thresh-old for injury than the cervical spine. A three-column model is useful for categorizing thoracolumbar injuries.39 The anterior longitudinal ligament and the anterior half of the vertebral body constitute the anterior column. The posterior half of the ver-tebral body and the posterior longitudinal ligament constitute the middle column. The pedicles, facet joints, laminae, spinous processes, and interspinous ligaments constitute the posterior column.Compression Fracture Compression fracture is a compression/flexion injury causing failure of the anterior column only. It is stable and not associated with neurologic deficit, although the patient may still have significant pain (Fig. 42-12).Burst Fracture Burst fracture is a pure axial compression injury

1	only. It is stable and not associated with neurologic deficit, although the patient may still have significant pain (Fig. 42-12).Burst Fracture Burst fracture is a pure axial compression injury causing failure of the anterior and middle columns. It is unstable, and perhaps half of patients have neurologic deficit due to compression of the cord or cauda equina from bone frag-ments retropulsed into the spinal canal.Chance Fracture Chance fracture is a flexion-distraction injury causing failure of the middle and posterior columns, sometimes with anterior wedging. Typical injury is from a lap seat-belt hyperflexion with associated abdominal injury. It often is unsta-ble and associated with neurologic deficit.Fracture-Dislocation Fracture-dislocation is failure of the anterior, middle, and posterior columns caused by flexion/dis-traction, shear, or compression forces. Neurologic deficit can result from retropulsion of middle column bone fragments into the spinal canal, or from subluxation

1	columns caused by flexion/dis-traction, shear, or compression forces. Neurologic deficit can result from retropulsion of middle column bone fragments into the spinal canal, or from subluxation causing decreased canal diameter (Fig. 42-13).Initial Assessment and Management. The possibility of a spine injury must be considered in all trauma patients. A patient with no symptoms referable to neurologic injury, a normal neu-rologic examination, no neck or back pain, and a known mecha-nism of injury unlikely to cause spine injury is at minimal risk for significant injury to the spine. Victims of moderate or severe trauma, especially those with injuries to other organ systems, usually fail to meet these criteria or cannot be assessed ade-quately. The latter often is due to impaired sensorium or signifi-cant pain. Because of the potentially catastrophic consequences of missing occult spine instability in a neurologically intact patient, a high level of clinical suspicion should govern patient

1	pain. Because of the potentially catastrophic consequences of missing occult spine instability in a neurologically intact patient, a high level of clinical suspicion should govern patient care until completion of clinical and radiographic evaluation.The trauma patient should be kept on a hard, flat board with straps and pads used for immobilization. A hard cervical collar is kept in place. These steps minimize forces transferred through the spine and therefore decrease the chance of causing dislocation, subluxation, or neural compression during transport to the trauma bay. The patient is then moved from the board to a flat stretcher. The primary survey and resuscitation are com-pleted. Physical examination and initial X-rays follow.For the examination, approach the patient as described in “Neurologic Examination” earlier in this chapter. Evaluation for spine or SCI is easier and more informative in awake patients. If the patient is awake, ask if he or she recalls details of the nature

1	“Neurologic Examination” earlier in this chapter. Evaluation for spine or SCI is easier and more informative in awake patients. If the patient is awake, ask if he or she recalls details of the nature of the trauma, and if there was loss of consciousness, numbness, or inability to move any or all limbs. Assess motor function by response to commands or pain, as appropriate. Assess pinprick, light touch, and joint position, if possible. Determining the ana-tomically lowest level of intact sensation can pinpoint the level of the lesion along the spine. Testing sensation in an ascending fashion will allow the patient to better discern the true stimulus Brunicardi_Ch42_p1827-p1878.indd 184301/03/19 7:16 PM 1844SPECIFIC CONSIDERATIONSPART IIas opposed to determine when it is extinguished. Document muscle stretch reflexes, lower sacral reflexes (i.e., anal wink and bulbocavernosus), and rectal tone.American Spinal Injury Association Classification The American Spinal Injury Association

1	Document muscle stretch reflexes, lower sacral reflexes (i.e., anal wink and bulbocavernosus), and rectal tone.American Spinal Injury Association Classification The American Spinal Injury Association provides a method of clas-sifying patients with spine injuries. The classification indicates completeness and level of the injury and the associated deficit. A form similar to that shown in Fig. 42-14 should be available in the trauma bay and completed for any spine injury patient. The association also has worked to develop recommendations and guidelines to standardize the care of SCI patients in an effort to improve the quality of care.Neurologic Syndromes. Penetrating, compressive, or isch-emic cord injury can lead to several characteristic presentations Figure 42-11. A. Lateral cervical spine X-ray of an elderly woman who struck her head during a backward fall. Arrowhead points to jumped facets at C5–C6. Note the anterior displace-ment of the C5 body with respect to the C6 body. B.

1	spine X-ray of an elderly woman who struck her head during a backward fall. Arrowhead points to jumped facets at C5–C6. Note the anterior displace-ment of the C5 body with respect to the C6 body. B. Sagittal T2-weighted magnetic resonance imaging of the same patient, revealing compromise of the spinal canal and compression of the cord. Note the bright signal within the cord at the level of compression, indicating spinal cord injury. C. Lateral cervical spine X-ray of same patient after application of cervical trac-tion and manual reduction. Note restoration of normal alignment. D. Lateral cervical spine X-ray after posterior cervical fusion to restabilize the C5–C6 segment of the spine.ABCDBrunicardi_Ch42_p1827-p1878.indd 184401/03/19 7:16 PM 1845NEUROSURGERYCHAPTER 42Figure 42-12. A. Lateral lumbar spine X-ray showing a compres-sion fracture of L2. Arrowhead points to anterior wedge deformity. Note the posterior wall of the vertebral body has retained normal height and alignment.

1	Lateral lumbar spine X-ray showing a compres-sion fracture of L2. Arrowhead points to anterior wedge deformity. Note the posterior wall of the vertebral body has retained normal height and alignment. B. Axial computed tomography scan through the same fracture. Arrowhead demonstrates a transverse discontinu-ity in the superior endplate of the L2 body.ABFigure 42-13. Sagittal reconstruction of an axial fine-slice com-puted tomography scan through the lumbar spine demonstrating a severe fracture-dislocation through the body of L2.based on the anatomy of injury. The neurologic deficits may be deduced from the anatomy of the long sensory and motor tracts and understanding of their decussations (Fig. 42-15). Four pat-terns are discussed. First, injury to the entire cord at a given level results in anatomic or functional cord transection with total loss of motor and sensory function below the level of the lesion. The typical mechanism is severe traumatic vertebral subluxation reducing spinal

1	in anatomic or functional cord transection with total loss of motor and sensory function below the level of the lesion. The typical mechanism is severe traumatic vertebral subluxation reducing spinal canal diameter and crushing the cord. Second, injury to half the cord at a given level results in Brown-Séquard syndrome, with loss of motor control and proprioception ipsilat-erally and loss of nociception and thermoception contralaterally. The typical mechanism is a stab or gunshot wound. Third, injury to the interior gray matter of the cord in the cervical spine results in a central cord syndrome, with upper extremity worse than lower extremity weakness and various degrees of numbness. The typical mechanism is transient compression of the cervical cord by the ligamentum flavum buckling during traumatic neck hyperextension. This syndrome occurs in patients with preex-isting cervical stenosis. Fourth, injury to the ventral half of the cord results in the anterior cord syndrome, with

1	during traumatic neck hyperextension. This syndrome occurs in patients with preex-isting cervical stenosis. Fourth, injury to the ventral half of the cord results in the anterior cord syndrome, with paralysis and loss of nociception and thermoception bilaterally. The typical mechanism is an acute disc herniation or ischemia from anterior spinal artery occlusion.Studies. Anteroposterior and lateral plain films provide a rapid survey of the bony spine. Plain films detect fractures and dislo-cations well. Adequate visualization of the lower cervical and upper thoracic spine often is impossible because of the shoul-der girdle. Complete plain film imaging of the cervical spine includes an open-mouth view to assess the odontoid process and the lateral masses of C1. Fine-slice CT scan with sagittal and coronal reconstructions provides good detail of bony anatomy and is good for characterizing fractures seen on plain films, as well as visualizing C7–T1 when not well seen on plain films. MRI

1	and coronal reconstructions provides good detail of bony anatomy and is good for characterizing fractures seen on plain films, as well as visualizing C7–T1 when not well seen on plain films. MRI provides the best soft tissue imaging. Canal compromise from subluxation, acute disc herniations, or ligamentous disrup-tion is clearly seen. MRI also may detect EDHs or damage to the spinal cord itself, including contusions or areas of ischemia.Brunicardi_Ch42_p1827-p1878.indd 184501/03/19 7:16 PM 1846SPECIFIC CONSIDERATIONSPART IIIndications for Screening for Vascular Injury With Cer-vical Spine Trauma It is important to consider the pres-ence of blunt cerebrovascular injury in patents with cervical spine trauma; however, the specific indications for obtaining a screening CT angiography study are controversial. Many trauma centers rely on the Denver Criteria.40 These criteria indicate that screening should be employed for a cervical ver-tebral body or transverse foramen fracture,

1	study are controversial. Many trauma centers rely on the Denver Criteria.40 These criteria indicate that screening should be employed for a cervical ver-tebral body or transverse foramen fracture, subluxation, or ligamentous injury at any level, or any fracture at the level of C1–C3, among other indications such as concerning mecha-nism, Lefort mid-face fractures, or basilar fractures through the carotid canal. A recent single-center retrospective study41 (n = 1717 cervical spine fractures) found a higher risk of ver-tebral artery injury only in the setting of fractures of C1 and C2 (combined), those that involve the transverse foramen, or had significant subluxation. They did not find that “high-risk” cervical spine fractures as defined by the Denver Criteria were associated with an increased risk of blunt cerebrovascular injury. Based on these data, we feel that it is appropriate to use a more defined set of screening criteria as outlined by Lock-wood et al to reduce cost and

1	increased risk of blunt cerebrovascular injury. Based on these data, we feel that it is appropriate to use a more defined set of screening criteria as outlined by Lock-wood et al to reduce cost and contrast-exposure in patients with cervical spine fractures.Figure 42-14. The American Spinal Injury Association system for categorizing spinal cord injury patients according to level and degree of neurologic deficit.Dorsal column(touch, vibration)Corticospinal tract(upper motor)Anterior horn(lower motor)Spinothalmic tract(pain, temperature)TransectionCentral cordBrown-SequardAnterior spinal a.Figure 42-15. Spinal cord injury patterns. a. = artery. (Adapted with permission from Hoff J, Boland M: Neurosurgery, in Schwartz SI: Principles of Surgery, 7th ed., New York, NY: McGraw-Hill Education; 1999.)Brunicardi_Ch42_p1827-p1878.indd 184601/03/19 7:16 PM 1847NEUROSURGERYCHAPTER 42Definitive Management Spinal-Dose Steroids Several studies have investigated the use of methylprednilosone in

1	1999.)Brunicardi_Ch42_p1827-p1878.indd 184601/03/19 7:16 PM 1847NEUROSURGERYCHAPTER 42Definitive Management Spinal-Dose Steroids Several studies have investigated the use of methylprednilosone in acute spinal cord injury. The National Acute Spinal Cord Injury studies (NASCIS I, II, and III) provided some support for the view that administration of high-dose methylprednilosone in acute spinal cord injury results in improved neurologic outcomes. A post-hoc analy-sis performed as part of NASCIS II demonstrated improved neurologic outcomes if methylprednilosone was administered within 8 hours of injury. A post-hoc analysis in NASCIS III showed improved outcomes at 6 weeks and 6 months, but not 1 year when methylprednilosone was administered within 3 and 8 hours of injury.42,43,44 However, these findings are tempered by the fact that these benefits were modest and only demonstrated in post-hoc analyses, and by the high rate of medical compli-cations associated with corticosteroid

1	these findings are tempered by the fact that these benefits were modest and only demonstrated in post-hoc analyses, and by the high rate of medical compli-cations associated with corticosteroid administration. All three NASCIS trials showed that methylprednilosone was associated with a higher rate of complications such as pneumonia, severe sepsis, and poor wound healing. A recent Cochrane review did not observe a significant increase in complications or mortal-ity associated with methylprednilosone administration in acute spinal cord injury, but did observe a trend towards this effect.45 Despite the lack of clear evidence on this issue, the most recent acute spinal cord injury guidelines provide a controversial level I recommendation against the use of corticosteroids in acute spinal cord injury.46 Some authors have argued for the use of methylprednilosone within 8 hours of acute spinal cord injury in carefully selected patients (e.g., young males that are less prone to medical

1	cord injury.46 Some authors have argued for the use of methylprednilosone within 8 hours of acute spinal cord injury in carefully selected patients (e.g., young males that are less prone to medical complications associated with corticosteroids47). Thus, clear consensus on the use of spinal-dose steroids does not exist.48 A decision to use or not use spinal-dose steroids may be dictated by local or regional practice patterns, especially given the legal liability issues surrounding SCI. Patients with gunshot or nerve root (cauda equina) injuries, or those who are pregnant, <14 years old, or on chronic steroids were excluded from the NASCIS studies and should not receive spinal-dose steroids. In addition to steroids, hypothermia for SCI has also received attention. There is even less evidence supporting the use of this treatment, and thus, it is not currently recommended.49Orthotic Devices Rigid external orthotic devices can stabilize the spine by decreasing range of motion and

1	evidence supporting the use of this treatment, and thus, it is not currently recommended.49Orthotic Devices Rigid external orthotic devices can stabilize the spine by decreasing range of motion and minimizing stress transmitted through the spine. Commonly used rigid cervical orthoses include Philadelphia and Miami-J collars. Cervical collars are inadequate for C1, C2, or cervicothoracic instability. Cervicothoracic orthoses brace the upper thorax and the neck, improving stabilization over the cervicothoracic region. Minerva braces improve high cervical stabilization by brac-ing from the upper thorax to the chin and occiput. Halo vest assemblies provide the most external cervical stabilization. Four pins are driven into the skull to lock the halo ring in position. Four posts arising from a tight-fitting rigid plastic vest immobilize the halo ring. Lumbar stabilization may be provided by thoracolumbosacral orthoses. A variety of compa-nies manufacture lines of spinal orthotics. A

1	from a tight-fitting rigid plastic vest immobilize the halo ring. Lumbar stabilization may be provided by thoracolumbosacral orthoses. A variety of compa-nies manufacture lines of spinal orthotics. A physician familiar with the technique should fit a halo vest. Assistance from a trained orthotics technician improves fitting and adjustment of the other devices.Surgery Neurosurgical intervention has two goals: decompres-sion of the spinal cord and nerve roots, and stabilization of the spine. When spinal cord injury is caused by a hyperflexiondistraction injury that results in cord compression and an unstable spine, both surgical decompression and fusion are typically required. However, in cases of a hyperextension injury causing central cord syndrome due to chronic cervical stenosis, surgi-cal decompression may be needed without the need for inter-nal fixation. In cases where there significant anterolisthesis (subaxial cervical jumped or perched facets), reduction of the fracture may be

1	decompression may be needed without the need for inter-nal fixation. In cases where there significant anterolisthesis (subaxial cervical jumped or perched facets), reduction of the fracture may be important for both decompression and stabili-zation. However, in some cases, reduction of the fracture may not be sufficient for decompression, and further decompressive surgery may still be needed.50Several controversial topics require consideration here. First, the timing of surgery has been a controversial topic. In the past, it has been suggested that patients with incomplete injury, or a deteriorating exam warrant emergent decompres-sion, whereas patients with complete injuries can undergo sur-gery in a nonemergent manner. It is important to appreciate the risks of taking a medically unstable patient to surgery, such as a polytrauma patient with hemorrhagic shock or a complete spinal cord injury patient in neurogenic shock. However, there are clear benefits to early surgical

1	unstable patient to surgery, such as a polytrauma patient with hemorrhagic shock or a complete spinal cord injury patient in neurogenic shock. However, there are clear benefits to early surgical decompression: it can allow early mobilization, aggressive nursing care, and physical ther-apy. Furthermore, a recent prospective cohort study found that the odds of observing a two-point increase in ASIA grade at 6 months was higher in patients that underwent surgery within 24 hours, as compared to those that underwent surgery after 24 hours.51 These data suggest that a subpopulation of patients may significantly benefit from early surgery; however, the char-acteristics of these patients were not described, suggesting a het-erogeneous population in terms of preoperative ASIA grade and imaging.In general, spine trauma patients with complete neurologic deficit, without any signs of recovery, or those without any neu-rologic deficits who have bony or ligamentous injury requiring open fixation,

1	general, spine trauma patients with complete neurologic deficit, without any signs of recovery, or those without any neu-rologic deficits who have bony or ligamentous injury requiring open fixation, may be medically stabilized before undergoing surgery. Surgical stabilization may be indicated for some inju-ries that would eventually heal with conservative treatment. Solid surgical stabilization may also allow a patient to be man-aged with a rigid cervical collar who would otherwise require halo-vest immobilization.Continued Care. Regional SCI centers with nurses, respira-tory therapists, pulmonologists, physical therapists, physiat-rists, and neurosurgeons specifically trained in caring for these patients may improve outcomes. Frequently encountered ICU issues include hypotension due to neurogenic shock (due to loss of sympathetic tone) and aspiration pneumonia. The recent guidelines recommend maintaining MAPs >85 for 7 days after injury.46 Chronically, prevention and treatment of

1	neurogenic shock (due to loss of sympathetic tone) and aspiration pneumonia. The recent guidelines recommend maintaining MAPs >85 for 7 days after injury.46 Chronically, prevention and treatment of deep venous thrombosis, autonomic hyperreflexia, and decubitus ulcer for-mation are important. Many patients with cervical or high tho-racic cord injuries require prolonged ventilatory support until the chest wall becomes stiff enough to provide resistance for diaphragmatic breathing. Patients with high cervical cord inju-ries (C4 or above) will often require permanent ventilatory sup-port. Patients should be transferred to SCI rehabilitation centers after stabilization of medical and surgical issues.Peripheral Nerve TraumaThe peripheral nervous system extends throughout the body and is subject to injury from a wide variety of trauma. Periph-eral nerves transmit motor and sensory information from the CNS to the body. An individual nerve may have pure motor, pure sensory, or mixed motor and

1	to injury from a wide variety of trauma. Periph-eral nerves transmit motor and sensory information from the CNS to the body. An individual nerve may have pure motor, pure sensory, or mixed motor and sensory functions. The key Brunicardi_Ch42_p1827-p1878.indd 184701/03/19 7:16 PM 1848SPECIFIC CONSIDERATIONSPART IIinformation-carrying structure of the nerve is the axon. The axon transmits information from the neuronal cell body and may measure from <1 mm to >1 m in length. Axons that travel a significant distance are often covered with myelin, which is a lipid-rich, electrically insulating sheath formed by Schwann cells. Myelinated axons transmit signals much more rapidly than unmyelinated axons because the voltage shifts and currents that define action potentials effectively jump from gap to gap over the insulated lengths of the axon.Axons, whether myelinated or unmyelinated, travel through a collagenous connective tissue known as endoneurium. Groups of axons and their endoneurium

1	gap to gap over the insulated lengths of the axon.Axons, whether myelinated or unmyelinated, travel through a collagenous connective tissue known as endoneurium. Groups of axons and their endoneurium form bundles known as fascicles. Fascicles run through a tubular collagenous tissue known as perineurium. Groups of fascicles are suspended in mesoneurium. Fascicles and their mesoneurium run through another tubular collagenous tissue known as epineurium. The epineurium and its contents form the nerve.There are four major mechanisms of injury to peripheral nerves. Nerves may be lacerated, stretched, compressed, or contused. Knives, passing bullets, or jagged bone fractures may lacerate nerves. Adjacent expanding hematomas or dislocated fractures may stretch nerves. Expanding hematomas, external orthoses such as casts or braces, or blunt trauma over a super-ficial nerve may compress or crush nerves. Shock waves from high-velocity bullets may contuse nerves. These mechanisms of injury cause

1	orthoses such as casts or braces, or blunt trauma over a super-ficial nerve may compress or crush nerves. Shock waves from high-velocity bullets may contuse nerves. These mechanisms of injury cause damage to the various anatomic components of the nerve. The patterns of damage are categorized in “Types of Injury.”Certain nerve segments are particularly vulnerable to injury. The following four characteristics make a nerve seg-ment more vulnerable: proximity to a joint, superficial course, passage through a confined space, and being fixed in position.Types of Injury. The traditional classification system for peripheral nerve injury is the Seddon classification. Seddon described three injury patterns as defined in the “Neurapraxia,” “Axonotmesis,” and “Neurotmesis” sections. The Seddon clas-sification provides a simple, anatomically based approach to peripheral nerve injury.52Neurapraxia Neurapraxia is defined as the temporary failure of nerve function without physical axonal disruption.

1	provides a simple, anatomically based approach to peripheral nerve injury.52Neurapraxia Neurapraxia is defined as the temporary failure of nerve function without physical axonal disruption. Axon degeneration does not occur. Return of normal axonal function occurs over hours to months, often in the 2to 4-week range.Axonotmesis Axonotmesis is the disruption of axons and myelin. The surrounding connective tissues, including endo-neurium, are intact. The axons degenerate proximally and dis-tally from the area of injury. Distal degeneration is known as Wallerian degeneration. Axon regeneration within the con-nective tissue pathways can occur, leading to restoration of function. Axons regenerate at a rate of 1 mm per day. Significant functional recovery may occur for up to 18 months. Scarring at the site of injury from connective tissue reaction can form a neuroma and interfere with regeneration.Neurotmesis Neurotmesis is the disruption of axons and endoneurial tubes. Peripheral collagenous

1	the site of injury from connective tissue reaction can form a neuroma and interfere with regeneration.Neurotmesis Neurotmesis is the disruption of axons and endoneurial tubes. Peripheral collagenous components, such as the epineurium, may or may not be intact. Proximal and distal axonal degeneration occurs. The likelihood of effective axonal regeneration across the site of injury depends on the extent of neuroma formation and on the degree of persisting anatomic alignment of the connective tissue structures. For instance, an injury may damage axons, myelin, and endoneurium, but leave perineurium intact. In this case, the fascicle sheath is intact, and appropriate axonal regeneration is more likely to occur than if the sheath is interrupted.Management of Peripheral Nerve Injury. The sensory and motor deficits should be accurately documented. Deficits are usually immediate. Progressive deficit suggests a process such as an expanding hematoma and may warrant early sur-gical exploration.

1	and motor deficits should be accurately documented. Deficits are usually immediate. Progressive deficit suggests a process such as an expanding hematoma and may warrant early sur-gical exploration. Clean, sharp injuries may also benefit from early exploration and reanastomosis. Most other peripheral nerve injuries should be observed. EMG/NCS studies should be done 3-6 to weeks postinjury if deficits persist. Axon seg-ments distal to the site of injury will conduct action potentials normally until Wallerian degeneration occurs, rendering EMG/NCS before 3 weeks uninformative. Continued observation is indicated if function improves. Surgical exploration of the nerve may be undertaken if no functional improvement occurs over 3 months. If intraoperative electrical testing reveals conduction across the injury, continue observation. In the absence of con-duction, the injured segment should be resected and end-to-end primary anastomosis attempted. However, anastomoses under tension will not

1	the injury, continue observation. In the absence of con-duction, the injured segment should be resected and end-to-end primary anastomosis attempted. However, anastomoses under tension will not heal. A nerve graft may be needed to bridge the gap between the proximal and distal nerve ends. The sural nerve often is harvested, as it carries only sensory fibers and leaves a minor deficit when resected. The connective tissue structures of the nerve graft may provide a pathway for effective axonal regrowth across the injury.Patterns of Injury Brachial Plexus The brachial plexus may be injured in a variety of ways. Parturition or a motorcycle accident can lead to plexus injury due to dislocation of the glenohumeral joint. Attempting to arrest a fall with one’s hands can lead to a stretch injury of the plexus due to abrupt movement of the shoulder girdle. An api-cal lung (Pancoast) tumor can cause compression injury to the plexus. There are many patterns of neurologic deficits possible with

1	of the plexus due to abrupt movement of the shoulder girdle. An api-cal lung (Pancoast) tumor can cause compression injury to the plexus. There are many patterns of neurologic deficits possible with injury to the various components of the brachial plexus, and understanding them all would require extensive neuroana-tomic discussion. Two well-known eponymous syndromes are Erb’s palsy and Klumpke’s palsy. Injury high in the plexus to the C5 and C6 roots resulting from glenohumeral dislocation causes Erb’s palsy with the characteristic “bellhop’s tip” posi-tion. The arm hangs at the side, internally rotated. Hand move-ments are not affected. Injury low in the plexus, to the C8 and T1 roots, resulting from stretch or compression injury, causes Klumpke’s palsy with the characteristic “claw hand” deformity. There is weakness of the intrinsic hand muscles, similar to that seen with ulnar nerve injury.Radial Nerve The radial nerve courses through the axilla, then laterally and posteriorly in

1	deformity. There is weakness of the intrinsic hand muscles, similar to that seen with ulnar nerve injury.Radial Nerve The radial nerve courses through the axilla, then laterally and posteriorly in the spiral groove of the humerus. Improper crutch use can cause damage to the axillary portion. The section of the nerve traversing the spiral groove can be damaged by humerus fractures or pressure from improper posi-tioning during sleep. This classically occurs when the patient is intoxicated and is called “Saturday night palsy.” The key find-ing is wrist drop (i.e., weakness of hand and finger extensors). Axillary (proximal) injury causes triceps weakness in addition to wrist drop.Common Peroneal Neuropathy The common peroneal nerve forms the lateral half of the sciatic nerve (the medial half being the tibial nerve). It receives contributions from L4, L5, S1, and S2. It emerges as a separate nerve in the popliteal fossa and laterally wraps around the fibular neck, after which it splits to

1	being the tibial nerve). It receives contributions from L4, L5, S1, and S2. It emerges as a separate nerve in the popliteal fossa and laterally wraps around the fibular neck, after which it splits to Brunicardi_Ch42_p1827-p1878.indd 184801/03/19 7:16 PM 1849NEUROSURGERYCHAPTER 42form the deep and superficial peroneal nerves. The superficial, fixed location at the fibular neck makes the common peroneal nerve susceptible to compression. The classic cause of traumatic peroneal neuropathy is crush injury from a car bumper striking the lateral aspect of the leg at the knee. Symptoms of common peroneal neuropathy include foot drop (weakness of the tibialis anterior), eversion weakness, and numbness over the anterolat-eral surface of the lower leg and dorsum of the foot. In contrast, a foot drop due to L5 radiculopathy spares eversion because the S1 fibers are intact. Surgical exploration of a common peroneal crush lesion is typically a low yield endeavor. Rare cases may be due to

1	foot drop due to L5 radiculopathy spares eversion because the S1 fibers are intact. Surgical exploration of a common peroneal crush lesion is typically a low yield endeavor. Rare cases may be due to compressive fibers or adhesions that may be lysed, with the possibility of return of function.CEREBROVASCULAR DISEASECerebrovascular disease is the most frequent cause of new, rapid-onset, nontraumatic neurologic deficit. It is far more common than seizures or tumors. Vascular structures are subject to a vari-ety of chronic pathologic processes that compromise vessel wall integrity. Diabetes, high cholesterol, high blood pressure, and smoking – common comorbidities in the general population – are important risk factors for vascular disease. These conditions can lead to vascular damage by such mechanisms as atheroma deposition causing luminal stenosis, endothelial damage promot-ing thrombogenesis, and weakening of the vessel wall result-ing in aneurysm formation or dissection. These

1	such mechanisms as atheroma deposition causing luminal stenosis, endothelial damage promot-ing thrombogenesis, and weakening of the vessel wall result-ing in aneurysm formation or dissection. These processes may coexist. For instance, a vessel containing an atheromatous plaque will have a decreased luminal diameter. The plaque also may have compromised endothelium, providing the opportunity for thrombus formation, which can lead to acute total occlusion of the remaining lumen. Aneurysms and dissection often occur in atheromatous vessels. Specific patterns of disease relevant to the cerebrovascular system include atheromatous and thrombotic carotid occlusion, brain ischemia from proximal embolic disease, vessel wall rupture leading to hemorrhage, and rupture of abnor-mal, thin-walled structures, specifically aneurysms and AVMs.Ischemic DiseasesIschemic stroke accounts for approximately 85% of acute cerebrovascular events. Symptoms of acute ischemic stroke vary based on the functions of

1	specifically aneurysms and AVMs.Ischemic DiseasesIschemic stroke accounts for approximately 85% of acute cerebrovascular events. Symptoms of acute ischemic stroke vary based on the functions of the neural tissues supplied by the occluded vessel, and the presence or absence of collateral circulation. The circle of Willis provides extensive collateral circulation, as it connects the right and left carotid arteries to each other and each to the vertebrobasilar system. Patients with complete occlusion of the carotid artery proximal to the circle of Willis may be asymptomatic if the blood flow patterns can shift and provide sufficient circulation to the ipsilateral cerebral hemisphere from the contralateral carotid and the basilar artery. However, the anatomy of the circle of Willis is highly variable. Patients may have a congenitally hypoplastic or missing com-municating artery with resultant bilateral ACA supply by one carotid, or the PCA may be supplied by the carotid artery rather than

1	Patients may have a congenitally hypoplastic or missing com-municating artery with resultant bilateral ACA supply by one carotid, or the PCA may be supplied by the carotid artery rather than the basilar. Similarly, one vertebral artery is often domi-nant and the other is hypoplastic. These variations may make disease in a particular vessel more neurologically devastating than in a patient with full collateral circulation. Occlusion distal to the circle of Willis generally results in a stroke in the territory supplied by that particular artery.Neurologic deficit from occlusive disease may be tempo-rary or permanent. A patient with sudden-onset focal neurologic deficit that resolves within 24 hours has had a transient ischemic attack (TIA). A patient with permanent deficit has had a com-pleted stroke.Thrombotic DiseaseThe most common area of neurologically significant vessel thrombosis is the carotid artery in the neck. Disease occurs at the carotid bifurcation. Thrombosis of a carotid

1	stroke.Thrombotic DiseaseThe most common area of neurologically significant vessel thrombosis is the carotid artery in the neck. Disease occurs at the carotid bifurcation. Thrombosis of a carotid artery chronically narrowed by atheroma can lead to acute carotid occlusion. As discussed previously, this can be asymptomatic due to sufficient collateralization. The more common concern is thromboem-bolus. Intracranial arterial occlusion by local thrombus formation may occur, but it is rare compared to embolic occlusion.Management. Complete occlusion of the carotid artery with-out referable neurologic deficit requires no treatment. A patient with new neurologic deficit and an angiographically confirmed complete carotid occlusion contralateral to the symptoms should be considered for emergent carotid endarterectomy.53 Surgery should be performed within 2 hours of symptom onset and should not be performed on obtunded or comatose patients. These restrictions significantly reduce the number of

1	endarterectomy.53 Surgery should be performed within 2 hours of symptom onset and should not be performed on obtunded or comatose patients. These restrictions significantly reduce the number of opera-tive candidates. In nonemergent cases, the results of the large-scale North American Symptomatic Carotid Stenosis Trial (NASCET) demonstrated a stroke reduction benefit to surgical revascularization in patients with severe stenosis, defined as occlusion of 70% to 99% of the carotid.54 Practice guidelines recommend revascularization at this level of stenosis even if asymptomatic. Surgical options for these patients include both carotid endarterectomy and carotid stenting, which have been shown to produce equal outcomes over long-term follow-up.55Embolic DiseaseEmboli causing strokes may originate from a number of sources, including: the left atrium in atrial fibrillation, a hypokinetic left ventricular wall segment, valvular vegetations, an atheromatous aortic arch, stenotic/atheromatous

1	from a number of sources, including: the left atrium in atrial fibrillation, a hypokinetic left ventricular wall segment, valvular vegetations, an atheromatous aortic arch, stenotic/atheromatous carotid bifurcations, or from the systemic venous system in the presence of a right-to-left shunt, such as a patent foramen ovale. The majority of emboli enter the anterior (carotid) circulation rather than the posterior (vertebro-basilar) circulation. Characteristic clinical syndromes result from embolic occlusion of various vessels within these circulations.Common Types of Strokes Anterior Cerebral Artery Stroke The ACA supplies the medial frontal and parietal lobes as it courses into the inter-hemispheric fissure. Due to its vascular supply of the motor cortex, ACA stroke characteristically results in contralateral leg weakness.Middle Cerebral Artery Stroke The MCA supplies the lateral frontal and parietal lobes and the temporal lobe. MCA stroke results in contralateral face and arm

1	results in contralateral leg weakness.Middle Cerebral Artery Stroke The MCA supplies the lateral frontal and parietal lobes and the temporal lobe. MCA stroke results in contralateral face and arm weakness. Dominant-hemisphere MCA stroke causes language deficits due to its sup-ply of Broca’s area, Wernicke’s area, and the white matter tracts that connect the two. Proximal MCA occlusion with ischemia and swelling in the entire MCA territory can lead to significant intracranial mass effect and midline shift (see Fig. 42-6), termed malignant MCA stroke.Posterior Cerebral Artery Stroke The PCA supplies the occipital lobe. PCA stroke results in a contralateral homony-mous hemianopsia (see Fig. 42-6).Posterior Inferior Cerebellar Artery Stroke The PICA sup-plies the lateral medulla and the inferior half of the cerebellar Brunicardi_Ch42_p1827-p1878.indd 184901/03/19 7:16 PM 1850SPECIFIC CONSIDERATIONSPART IIhemispheres. PICA stroke results in nausea, vomiting, nystag-mus, dysphagia,

1	half of the cerebellar Brunicardi_Ch42_p1827-p1878.indd 184901/03/19 7:16 PM 1850SPECIFIC CONSIDERATIONSPART IIhemispheres. PICA stroke results in nausea, vomiting, nystag-mus, dysphagia, ipsilateral Horner’s syndrome, and ipsilateral limb ataxia. The constellation of symptoms resulting from PICA occlusion is referred to as the lateral medullary or Wallenberg’s syndrome.Management. Ischemic stroke management has two goals: reopen the occluded vessel and maintain blood flow to ischemic “penumbra” tissues bordering the vascular territory. Reopen-ing the vessel has historically been attempted with recombinant tPA.56 tPA administration within 3 hours of the onset of neu-rologic deficit improves outcome at 3 months. In the setting of suspected ischemic stroke, a head CT must be performed immediately to differentiate ischemic from hemorrhagic stroke. Intracranial hemorrhage, major surgery within the previous 2 weeks, GI or genitourinary hemorrhage in the previous 3 weeks, platelet

1	immediately to differentiate ischemic from hemorrhagic stroke. Intracranial hemorrhage, major surgery within the previous 2 weeks, GI or genitourinary hemorrhage in the previous 3 weeks, platelet count less than 100,000/µL, and systolic blood pressure >185 mmHg are among the contraindications to tPA therapy.In recent years, a paradigm shift in ischemic stroke man-agement has occurred with the advent of endovascular mechani-cal thrombectomy. Though tPA can be effective for strokes of smaller vessels, it produces recanalization in only 20% of large vessel ischemic strokes, and even less for internal carotid artery occlusion.57 In mechanical thrombectomy, the intracra-nial circulation is accessed endovascularly, and stent-retriever devices can be deployed to definitively remove clot and stent open involved vessels. Initial investigations into the technol-ogy began in 1999 with varied success. Technological advances proceeded, and in 2015, large-scale clinical trials investigating

1	stent open involved vessels. Initial investigations into the technol-ogy began in 1999 with varied success. Technological advances proceeded, and in 2015, large-scale clinical trials investigating mechanical thrombectomy for large vessel occlusion were pub-lished. The MR-CLEAN trial was one of these. In this trial, 500 patients were randomized to tPA and medical therapy vs. mechanical thrombectomy. The latter group had a significantly better 90-day outcome. A domino effect ensued in which four other similarly large-scale trials (e.g., REVASCAT)58 were ter-minated early.Benefits to mechanical thrombectomy include less strin-gent criteria than tPA administration, focused therapy, and time windows between 6 and 12 hours in trials. Currently, mechani-cal thrombectomy is being used for large vessel occlusion within 6 hours of symptom onset for those patients not eligible for tPA, which accounts for about 10% of ischemic stroke. Its indica-tions and associated technology continue to evolve.

1	occlusion within 6 hours of symptom onset for those patients not eligible for tPA, which accounts for about 10% of ischemic stroke. Its indica-tions and associated technology continue to evolve. It should be noted that current guidelines still support tPA as first-line therapy even for those eligible for mechanical thrombectomy.Patients not eligible for tPA or mechanical thrombectomy require hemodynamic optimization and neurologic monitoring. Admit such patients to the ICU stroke service for blood pres-sure management and frequent neurologic checks. Permissive hypertension allows for maximal cerebral perfusion. Systolic blood pressure >180 mmHg may require treatment, but the opti-mal mean arterial pressure goal is between 100 and 140 mmHg. Give normal saline solution without glucose (which could injure neurons in the penumbra due to osmotic fluid shift), and aim for normovolemia. A stroke patient who worsens clinically should undergo repeat head CT to evaluate for hemorrhage or

1	(which could injure neurons in the penumbra due to osmotic fluid shift), and aim for normovolemia. A stroke patient who worsens clinically should undergo repeat head CT to evaluate for hemorrhage or increas-ing mass effect from swelling, which typically peaks 3 to 5 days after the stroke. Significant swelling from an MCA or cerebellar stroke may cause herniation and brain stem injury. A decom-pressive hemicraniectomy or suboccipital craniectomy can be a life-saving intervention for these select stroke patients. In stud-ies of malignant MCA syndrome, decompressive hemicraniec-tomy showed favorable mortality and functional outcomes.59 This treatment option, however, should be considered with the understanding of potentially poor functional recovery regardless of therapy. One study showed that less than half of malignant MCA patients who underwent decompressive hemicraniectomy returned home following rehabilitation, which is even fewer for those undergoing medical therapy.60Hemorrhagic

1	less than half of malignant MCA patients who underwent decompressive hemicraniectomy returned home following rehabilitation, which is even fewer for those undergoing medical therapy.60Hemorrhagic DiseasesIntracranial hemorrhage from abnormal or diseased vascular structures accounts for approximately 15% of acute cerebrovas-cular events. Hypertension and amyloid angiopathy account for most intraparenchymal hemorrhages, although AVMs, aneu-rysms, venous thrombosis, tumors, hemorrhagic conversion of ischemic infarct, and fungal infections also may be the cause. The term intracranial hemorrhage is frequently used to signify intraparenchymal hemorrhage and will be used here. Intracra-nial hemorrhage causes local neuronal injury and dysfunction and also may cause global dysfunction due to mass effect if sufficiently large. The Intracerebral Hemorrhage (ICH Score) is commonly used to risk-stratify these patients, and it takes into account GCS, age, hemorrhage volume, presence of

1	due to mass effect if sufficiently large. The Intracerebral Hemorrhage (ICH Score) is commonly used to risk-stratify these patients, and it takes into account GCS, age, hemorrhage volume, presence of intraven-tricular hemorrhage, and location to predict mortality. AVM or aneurysm rupture (along with trauma, discussed previously in this chapter) result in subarachnoid hemorrhage (SAH) because the major cerebral and cortical blood vessels travel in the sub-arachnoid space, between the pia and the arachnoid membranes. SAH can cause immediate concussive-like neuronal dysfunc-tion by exposure of the brain to intra-arterial pressure pulsa-tions during the hemorrhage. Moreover, it can cause delayed ischemia from cerebral arterial vasospasm, which can present as acute worsening of the patient’s neurological status days to weeks after the injury. Patients presenting with intracranial hemorrhages that do not follow typical patterns should undergo cerebral angiography or MRI to evaluate for

1	neurological status days to weeks after the injury. Patients presenting with intracranial hemorrhages that do not follow typical patterns should undergo cerebral angiography or MRI to evaluate for possible underlying lesions, such as AVM or tumor.Hemorrhagic stroke most commonly occurs within the basal ganglia or cerebellum. The patient is usually hypertensive on admission and has a history of poorly controlled hyperten-sion. Such patients are more likely to present with lethargy or obtundation compared to those who suffer an ischemic stroke. Depressed mental status results from mass effect from the hema-toma in deep structures, which can produce midline shift or herniation. Ischemic stroke does not cause mass effect acutely. Therefore, patients are more likely to present with normal con-sciousness and a focal neurologic deficit. Hemorrhagic strokes tend to present with a relatively gradual decline in neurologic function as the hematoma expands, rather than the immediately maximal

1	and a focal neurologic deficit. Hemorrhagic strokes tend to present with a relatively gradual decline in neurologic function as the hematoma expands, rather than the immediately maximal symptoms caused by ischemic stroke. Table 42-3 pro-vides a listing of relative incidences of intracranial hemorrhage by anatomic distribution.Hypertension. Hypertension increases the relative risk of intracranial hemorrhage by approximately fourfold, likely due to chronic degenerative vasculopathy. Hypertensive hemor-rhages often present in the basal ganglia, thalamus, or pons, and result from breakage of small perforating arteries that branch off of much larger parent vessels (Fig. 42-16).Most hypertensive hemorrhages should be medically man-aged. The hematoma often contains intact, salvageable axons because the blood dissects through and along neural tracts, and surgical clot evacuation destroys these axons. Factors potentially favoring surgery include: superficial clot location, young age,

1	because the blood dissects through and along neural tracts, and surgical clot evacuation destroys these axons. Factors potentially favoring surgery include: superficial clot location, young age, nondominant hemisphere, rapid deterioration, and significant Brunicardi_Ch42_p1827-p1878.indd 185001/03/19 7:16 PM 1851NEUROSURGERYCHAPTER 42Table 42-3Anatomic distribution of intracranial hemorrhages and correlated symptoms% OF INTRACRANIAL HEMORRHAGESLOCATIONCLASSIC SYMPTOMS50Basal ganglia (putamen, globus pallidus), internal capsuleContralateral hemiparesis15ThalamusContralateral hemisensory loss10–20Cerebral white matter (lobar)Depends on location (weakness, numbness, partial loss of visual field)10–15PonsHemiparesis; may be devastating10CerebellumLethargy or coma due to brain stem compression and/or hydrocephalus1–6Brain stem (excluding pons)Often devastatingFigure 42-16. A. Head computed tomography scan of a patient with left-sided weakness and progressive lethargy reveals a right

1	and/or hydrocephalus1–6Brain stem (excluding pons)Often devastatingFigure 42-16. A. Head computed tomography scan of a patient with left-sided weakness and progressive lethargy reveals a right basal ganglia hemorrhage (arrowhead). The blood clot is bright white. Hypodensity around the clot represents cerebral edema. There is blood within the ventricular system. B. Another patient with intraventricular extension of a basal ganglia hemorrhage. The patient developed right-sided weak-ness and then lethargy. Head computed tomography indicated hydrocephalus. A ventriculostomy was placed for cerebrospinal fluid drainage (arrowhead indicates cross-sectional view of the catheter entering the anterior horn of the right lateral ventricle).mass effect. However, the most comprehensive randomized clinical trials to date did not show an overall improved outcome in surgically evacuated intracranial hemorrhage, except for the subgroup of patients with clot <1 cm from the cortical surface.61 More

1	clinical trials to date did not show an overall improved outcome in surgically evacuated intracranial hemorrhage, except for the subgroup of patients with clot <1 cm from the cortical surface.61 More recent studies have assessed the role of minimally-invasive catheter evacuation of clot; these investigations are ongoing.62 Medical management remains the gold standard, however, and includes moderate blood pressure control, normalizing platelet and clotting function, phenytoin or levetiracetam for seizure prophylaxis, and electrolyte management. Intubate patients who cannot clearly follow commands to prevent aspiration and hypercarbia. Follow and document the neurologic examination and communicate with the family regarding appropriateness for rehabilitation vs. withdrawal of care.Amyloid Angiopathy. The presence of pathologic amyloid deposition in the media of small cortical vessels compromises vessel integrity and tends to cause more superficial (lobar) hemorrhages than hypertensive

1	Angiopathy. The presence of pathologic amyloid deposition in the media of small cortical vessels compromises vessel integrity and tends to cause more superficial (lobar) hemorrhages than hypertensive intracranial hemorrhage. Amy-loid laden vessels may hemorrhage multiple times. The super-ficial location of amyloid hemorrhages may make surgical evacuation less morbid compared to typical deep hypertensive hemorrhages. Nonetheless, medical management and family counseling should be approached similarly to patients with hypertensive hemorrhages.Brunicardi_Ch42_p1827-p1878.indd 185101/03/19 7:16 PM 1852SPECIFIC CONSIDERATIONSPART IITable 42-5The Hunt-Hess clinical grading system for subarachnoid hemorrhageHUNT-HESS GRADECLINICAL PRESENTATION0Asymptomatic; unruptured aneurysm1Awake; asymptomatic or mild headache; mild nuchal rigidity2Awake; moderate to severe headache, cranial nerve palsy (e.g., cranial nerve III or IV), nuchal rigidity3Lethargic; mild focal neurologic deficit (e.g.,

1	or mild headache; mild nuchal rigidity2Awake; moderate to severe headache, cranial nerve palsy (e.g., cranial nerve III or IV), nuchal rigidity3Lethargic; mild focal neurologic deficit (e.g., pronator drift)4Stuporous; significant neurologic deficit (e.g., hemiplegia)5Comatose; posturingTable 42-4Prevalence of cerebral aneurysm by locationPREVALENCEANEURYSM LOCATION (VERNACULAR NAME)Anterior circulation 85% 30% Anterior communicating artery (A-Comm)25% Posterior communicating artery (P-Comm)20% Middle cerebral artery bifurcation10% OtherPosterior circulation 15% 10% Basilar artery, most frequently at the basilar tip5% Vertebral artery, usually at the posterior inferior cerebellar arteryCerebral Aneurysm. An aneurysm is a focal dilatation of the vessel wall and is most often a balloon-like outpouching, but may also be fusiform. Aneurysms usually occur at branch points of major vessels (e.g., internal carotid artery bifurca-tion), or at the origin of smaller vessels (e.g.,

1	balloon-like outpouching, but may also be fusiform. Aneurysms usually occur at branch points of major vessels (e.g., internal carotid artery bifurca-tion), or at the origin of smaller vessels (e.g., posterior com-municating artery or ophthalmic artery). Approximately 85% of aneurysms arise from the anterior circulation (carotid) and 15% from the posterior circulation (vertebrobasilar). Table 42-4 shows the percentage distribution of cerebral aneurysms by location. Aneurysms are thin walled and at risk for rupture. The major cerebral vessels, and therefore aneurysms, lie in the subarachnoid space. Rupture results in SAH. The aneurys-mal tear may be small and seal quickly, or it may not. SAH may consist of a thin layer of blood in the CSF spaces, or thick layers of blood around the brain and extending into brain parenchyma, resulting in a clot with mass effect. Because the meningeal linings of the brain are sensitive with free nerve endings, SAH usually results in a sudden, severe

1	and extending into brain parenchyma, resulting in a clot with mass effect. Because the meningeal linings of the brain are sensitive with free nerve endings, SAH usually results in a sudden, severe “thunderclap” headache. A patient will classically describe “the worst head-ache of my life.” Presenting neurologic symptoms may range from mild headache to coma to sudden death. The Hunt-Hess grading system categorizes patients clinically (Table 42-5). The World Federation of Neurological Surgery (WFNS) SAH Grading Scale is also used for this purpose. The Fisher Scale and a more recent modified form use head CT characteristics, described in the following section, to stratify patients based on risk of vasospasm-induced delayed cerebral ischemia. Both scales are used in conjunction as a quick way to communicate severity of aneurysmal SAH.Patients with symptoms suspicious for SAH should have a head CT immediately. Acute SAH appears as a bright signal in the fissures and CSF cisterns around the

1	to communicate severity of aneurysmal SAH.Patients with symptoms suspicious for SAH should have a head CT immediately. Acute SAH appears as a bright signal in the fissures and CSF cisterns around the base of the brain, as shown in Fig. 42-17. CT is rapid, noninvasive, and approxi-mately 95% sensitive. In patients with suspicious symptoms but negative head CT, a lumbar puncture (LP) should be per-formed. An LP with xanthochromia and high red blood cell counts (usually 100,000/mL), which do not decrease between tubes 1 and 4, is consistent with SAH. Negative CT and LP essentially rules out SAH. Patients diagnosed with SAH require four-vessel cerebral angiography within 24 hours to assess for aneurysm or other vascular malformation. Cathe-ter angiography remains the gold standard for assessing the patient’s cerebral vasculature, relevant anomalies, and pres-ence, location, and morphology of the cerebral aneurysms. Figure 42-18A demonstrates the typical anteroposterior digital subtraction

1	the patient’s cerebral vasculature, relevant anomalies, and pres-ence, location, and morphology of the cerebral aneurysms. Figure 42-18A demonstrates the typical anteroposterior digital subtraction angiographic view of a cerebral aneurysm. Figure 42-18B shows the anatomy of the circle of Willis in a simplified graphic representation to assist in visualizing the locations of various cerebral aneurysms.SAH patients should be admitted to the neurologic ICU. Hunt-Hess grade 4 and 5 patients require intubation and hemo-dynamic monitoring and stabilization. The current standard of care for ruptured aneurysms requires early aneurysmal occlu-sion. There are two options for occlusion. The patient may undergo craniotomy with microsurgical dissection and place-ment of a titanium clip across the aneurysm neck to exclude the aneurysm from the circulation and reconstitute the lumen of the parent vessel. The second option is to utilize an endovascular approach for treatment, which has traditionally

1	neck to exclude the aneurysm from the circulation and reconstitute the lumen of the parent vessel. The second option is to utilize an endovascular approach for treatment, which has traditionally taken the form of “coiling.” The patient is taken to the interventional neuro-radiology suite for placement of looped titanium coils inside the aneurysm dome. The coils support thrombosis and prevent Figure 42-17. Non contrast CT scan on the left shows diffuse subarachnoid blood in the cisterns with some concentration in the region of the anterior cerebral arteries. This is confirmed by the image on the right, a CT angiogram, where the arrow indicates an anterior communicating artery aneurysm.Brunicardi_Ch42_p1827-p1878.indd 185201/03/19 7:16 PM 1853NEUROSURGERYCHAPTER 42Figure 42-18. A. Anteroposterior view after injection of contrast dye in the right internal carotid artery demonstrates an aneurysm of the middle cerebral artery bifurcation. B. Figure depicting the anatomy of the circle

1	view after injection of contrast dye in the right internal carotid artery demonstrates an aneurysm of the middle cerebral artery bifurcation. B. Figure depicting the anatomy of the circle of Willis and the common sites for aneu-rysms. ICA = internal cerebral artery; MCA = middle cerebral artery. (Reproduced with permission from Osborn AG: Handbook of Neuroradiology: Brain and Skull. St. Louis, MO: Mosby-Year Book, Inc; 1991.)AIntracranial aneurysumsAnteriorcommunicating artery30% – 35%MCA bifurcation20%Basilar5%Posterior fossaMiscellaneous sites distalto circle of Willis1% – 3%ICA/posteriorcommunicating30% – 35%Bblood flow into the aneurysm. Newer endovascular options include flow diversion and stent-assisted coiling. Factors favor-ing craniotomy and clipping include young age, good medical condition, and broad aneurysm necks. Factors favoring coil-ing include age, medical comorbidities, and narrow aneurysm necks. Due to coil migration or compaction over time, surgical clipping is

1	condition, and broad aneurysm necks. Factors favoring coil-ing include age, medical comorbidities, and narrow aneurysm necks. Due to coil migration or compaction over time, surgical clipping is believed to result in a more definitive cure. The decision to clip or coil is complex and should be fully explored. Practice standards have changed in recent years with the advent of more nuanced endovascular techniques and treatment options, as described earlier. Current guidelines favor endovas-cular therapy as the preferred first-line approach. The Interna-tional Subarachnoid Aneurysm Trial researchers suggested that endovascular occlusion resulted in better mortality outcomes for certain types of cerebral aneurysms, although this trial was marred by poor selection and randomization techniques, and the validity of its conclusions have been questioned.63 Long-term outcomes may be better in younger patients with clipped aneurysms, as demonstrated in the Barrow Ruptured Aneurysm Trial (BRAT).64

1	the validity of its conclusions have been questioned.63 Long-term outcomes may be better in younger patients with clipped aneurysms, as demonstrated in the Barrow Ruptured Aneurysm Trial (BRAT).64 However, this trial has a number of similar criticisms as well. Debate also continues regarding optimal care for unruptured intracranial aneurysms, with a recent large-scale study showing no clear benefit to open surgical vs. endovascu-lar approaches.65SAH patients often require 1 to 3 weeks of ICU care after aneurysm occlusion for medical complications that accompany neurologic injury. In addition to routine ICU concerns, SAH patients are also at risk for cerebral vasospasm. In vasospasm, cerebral arteries constrict pathologically and can cause isch-emia or stroke from 4 to 21 days after SAH. Current vasospasm prophylaxis includes maintenance of optimal perfusion with hypertension and mild hypervolemia, as well as administration of nimodipine, a calcium channel blocker that may decrease the

1	vasospasm prophylaxis includes maintenance of optimal perfusion with hypertension and mild hypervolemia, as well as administration of nimodipine, a calcium channel blocker that may decrease the incidence and degree of spasm, though its mechanism is debated. Neurointerventional options for treating symptomatic vasospasm include intra-arterial papaverine or nicardipine, and balloon angioplasty for larger caliber vessels.Aneurysmal SAH has an approximate mortality rate of 50% in the first month. Approximately one-third of survivors return to pre-SAH function, and the remaining two-thirds have mild to severe disability. Most require rehabilitation after hospitalization.Arteriovenous Malformations. AVMs are abnormal, dilated arteries and veins without an intervening capillary bed. The nidus of the AVM contains a tangled mass of vessels but no neural tissue. AVMs may be asymptomatic or present with SAH, intra-parenchymal hemorrhage, or seizures. Small AVMs present with hemorrhage more often

1	the AVM contains a tangled mass of vessels but no neural tissue. AVMs may be asymptomatic or present with SAH, intra-parenchymal hemorrhage, or seizures. Small AVMs present with hemorrhage more often than large AVMs, which tend to present with seizures. Headache, bruit, or focal neurologic deficits are less common symptoms. AVMs hemorrhage at an average rate of 2% to 4% a year. Figure 42-19 demonstrates the angiographic appearance of an AVM in arterial and venous phases.For unruptured AVMs, recent evidence from over two hundred patients supports medical management alone rather than intervention due to risk of stroke.66 Because AVM rupture can present radiographically as SAH, it is important to consider several management differences as compared to aneurysmal SAH. Definitive therapy for the AVM usually is delayed 3 to 4 weeks to allow the brain to recover from acute injury. There is less risk of devastating early rebleeding from AVMs, and vasospasm is much less common. Three

1	for the AVM usually is delayed 3 to 4 weeks to allow the brain to recover from acute injury. There is less risk of devastating early rebleeding from AVMs, and vasospasm is much less common. Three therapeutic modalities for AVMs are currently in common use: microsurgical exci-sion, interventional radiology or endovascular embolization, and stereotactic radiosurgery (SRS). AVMs that are large, near eloquent cortex, or that drain to deep venous structures are con-sidered high grade and more difficult to surgically resect with-out causing a significant neurologic deficit. Radiosurgery can treat these lesions, although it is limited to lesions <3 cm in diameter and has a 2-year lag time (i.e., the AVM may bleed in the interval). Embolization reduces flow through the AVM. It is usually considered adjunctive therapy, but it may serve as the sole treatment for deep, inaccessible lesions.4Brunicardi_Ch42_p1827-p1878.indd 185301/03/19 7:16 PM 1854SPECIFIC CONSIDERATIONSPART IITUMORS OF THE

1	adjunctive therapy, but it may serve as the sole treatment for deep, inaccessible lesions.4Brunicardi_Ch42_p1827-p1878.indd 185301/03/19 7:16 PM 1854SPECIFIC CONSIDERATIONSPART IITUMORS OF THE CENTRAL NERVOUS SYSTEMA wide variety of tumors affect the brain and spine. Primary benign and malignant tumors arise from the various elements of the CNS, including neurons, glia, and meninges. Tumors metas-tasize to the CNS from many primary sources. Presenta-tion varies widely depending on relevant neuroanatomy. Prognosis depends on histology and anatomy. Modern brain tumor centers use team approaches to CNS tumors, as patients may require a combination of surgery (including newer, more minimally invasive approaches), radiation therapy, chemother-apy, SRS, and research protocol enrollment for studies assessing the efficacy of newer approaches such as immunotherapy. Tumors affecting the peripheral nervous system are discussed in the “Peripheral Nerve” section.Intracranial TumorsIntracranial

1	assessing the efficacy of newer approaches such as immunotherapy. Tumors affecting the peripheral nervous system are discussed in the “Peripheral Nerve” section.Intracranial TumorsIntracranial tumors can cause brain injury from mass effect, dys-function or destruction of adjacent neural structures, swelling, or abnormal electrical activity (seizures). Supratentorial tumors commonly present with focal neurologic deficit, such as contra-lateral limb weakness, visual field deficit, headache, or seizure. Infratentorial tumors often cause increased ICP due to hydro-cephalus from compression of the fourth ventricle, leading to headache, nausea, vomiting, or diplopia. Cerebellar hemisphere or brain stem dysfunction can result in ataxia, nystagmus, or cranial nerve palsies. Infratentorial tumors rarely cause seizures.All patients with symptoms concerning for brain tumor should undergo MRI with and without gadolinium. Gadolinium-based contrast can identify locations of blood-brain barrier

1	rarely cause seizures.All patients with symptoms concerning for brain tumor should undergo MRI with and without gadolinium. Gadolinium-based contrast can identify locations of blood-brain barrier break-down of tumors and, when used in conjunction with other MRI sequences, is essential in narrowing the differential diagnosis. Ini-tial management of a patient with a symptomatic brain tumor gen-erally includes dexamethasone for reduction of vasogenic edema, and phenytoin or levetiracetam if the patient has seized. Patients with significant weakness, lethargy, or hydrocephalus should be admitted for observation until definitive care is administered.Metastatic TumorsCerebral metastases are the most common type of intracranial tumor. Prolonged cancer patient survival and improved CNS imaging have increased the likelihood of diagnosing cerebral metastases. The sources of most cerebral metastases are (in decreasing frequency): lung, breast, kidney, GI tract, and mela-noma. Lung and breast

1	have increased the likelihood of diagnosing cerebral metastases. The sources of most cerebral metastases are (in decreasing frequency): lung, breast, kidney, GI tract, and mela-noma. Lung and breast cancers account for more than half of cerebral metastases. Metastatic cells usually travel to the brain hematogenously and frequently seed the gray-white junction due to characteristic blood vessel caliber change. Other com-mon locations are the cerebellum and the meninges. Menin-geal involvement may result in carcinomatous meningitis, also known as leptomeningeal carcinomatosis. MRI preand post-contrast administration is the study of choice for evaluation. Figure 42-20 demonstrates bilateral cerebellar metastases. These lesions are typically well circumscribed, round, and multiple. Such findings should prompt a metastatic work-up, including CT scan of the chest, abdomen, and pelvis, and a bone scan.Management largely depends upon the primary tumor, overall tumor burden, patient’s medical

1	should prompt a metastatic work-up, including CT scan of the chest, abdomen, and pelvis, and a bone scan.Management largely depends upon the primary tumor, overall tumor burden, patient’s medical condition, and location and number of metastases. The beliefs of the patient and family regarding aggressive care must be considered, with the primary goal of optimizing survival time while maintaining or improv-ing neurological function. Neurosurgical intervention can be indicated for a number of reasons. Biopsy can be obtained to provide a tissue diagnosis and further direct therapy. Hydro-cephalus from increased intracranial pressure due to intracranial tumor burden can be temporized via placement of a ventricu-loperitoneal shunt. Craniotomy can be used for debulking of intracranial tumor burden or resection. Data from random-ized controlled trials have supported the use of craniotomy for tumor resection plus whole-brain radiation therapy (WBRT) or stereotactic radiosurgery (SRS) for

1	burden or resection. Data from random-ized controlled trials have supported the use of craniotomy for tumor resection plus whole-brain radiation therapy (WBRT) or stereotactic radiosurgery (SRS) for patients with a single surgically accessible metastatic lesion, compared to radiation therapy alone. In one randomized trial assessing surgery and WBRT vs. WBRT alone, local recurrence decreased and median 5ABFigure 42-19. A. Lateral view after injection of contrast dye in the left internal carotid artery demonstrates a 3 × 4 cm left frontal arteriove-nous malformation indicated by arrowheads. This image was taken 1.06 seconds after dye injection, and is referred to as an arterial phase image. B. Same view taken 4.10 seconds after dye injection, providing a venous phase image. The arrow points to the arteriovenous malfor-mation nidus. The arrowheads indicate two pathologically enlarged draining veins. ACA = anterior cerebral artery; ICA = internal carotid artery; MCA = middle cerebral

1	to the arteriovenous malfor-mation nidus. The arrowheads indicate two pathologically enlarged draining veins. ACA = anterior cerebral artery; ICA = internal carotid artery; MCA = middle cerebral artery.Brunicardi_Ch42_p1827-p1878.indd 185401/03/19 7:16 PM 1855NEUROSURGERYCHAPTER 42survival increased from 15 to 40 weeks.67 For multiple metas-tases, it should be noted that craniotomy primarily for resec-tion is typically not indicated unless all detectable metastases can be resected. It may however still be useful for symptomatic relief from a primary lesion. Recent data suggest that SRS (e.g. Gamma Knife) may be applied to multiple metastases in one session with improved outcome.68Glial TumorsGlial cells provide the anatomic and physiologic support for neurons and their processes in the brain. Tumors arising from glial cells are termed gliomas, and they represent the most com-mon primary brain tumor. The several types of glial cells give rise to distinct primary CNS

1	processes in the brain. Tumors arising from glial cells are termed gliomas, and they represent the most com-mon primary brain tumor. The several types of glial cells give rise to distinct primary CNS neoplasms.Astrocytoma. Astrocytoma is the most common primary CNS neoplasm. The term glioma often is used to refer to astrocyto-mas specifically, excluding other glial tumors. Astrocytomas are graded from I to IV. Grades I and II are referred to as low-grade astrocytoma or low grade glioma, grade III as anaplastic astro-cytoma, and grade IV as glioblastoma multiforme (GBM). Prog-nosis varies significantly between grades I/II, III, and IV, but not between I and II. Median survival is 8 years after diagnosis with a low-grade tumor, 2 to 3 years with an anaplastic astrocytoma, and roughly 1 year with a GBM. GBMs account for almost two-thirds of all astrocytomas, anaplastic astrocytomas account for two-thirds of the rest, and low-grade astrocytomas the remainder. Fig. 42-21 demonstrates the

1	with a GBM. GBMs account for almost two-thirds of all astrocytomas, anaplastic astrocytomas account for two-thirds of the rest, and low-grade astrocytomas the remainder. Fig. 42-21 demonstrates the typical appearance of a GBM.The great majority of astrocytomas infiltrate adjacent brain. Juvenile pilocytic astrocytomas and pleomorphic xantho-astrocytomas are exceptions. These tumors are circumscribed, low grade, and associated with a good prognosis. Histologic features associated with higher grade include hypercellularity, nuclear atypia, and endovascular hyperplasia. Necrosis is pres-ent only with GBMs; it is required for the diagnosis.Gross total resection should be attempted for suspected astrocytomas. Motor cortex, language centers, deep or midline structures, or brainstem location may make this impossible without unacceptable, devastating neurologic deficit. Advanced imaging, such as diffusion tensor imaging (DTI) and functional MRI (fMRI), are seeing increased use as means of

1	may make this impossible without unacceptable, devastating neurologic deficit. Advanced imaging, such as diffusion tensor imaging (DTI) and functional MRI (fMRI), are seeing increased use as means of assessing peritumoral structure and function to guide surgical decision-making. However, some lesions may be in such precarious regions as to be limited to stereotactic needle biopsy specimen. Gross total resection followed by fractioned radiotherapy (FRT) improves survival for all grades, although radiation therapy may be delayed until recurrence in low-grade tumors. Alongside FRT, adjuvant chemotherapy with temozolomide was demon-strated in a randomized controlled trial to increase short-term survival rate.69 Bevacizumab, an anti-VEGF antibody, is another treatment option under investigation. There are various ongoing research studies for GBM adjuvant therapy; these should be dis-cussed with the patient and family. Other options include Iotrex-containing balloons for conformal radiation

1	There are various ongoing research studies for GBM adjuvant therapy; these should be dis-cussed with the patient and family. Other options include Iotrex-containing balloons for conformal radiation brachytherapy (Glia-Site), placed in the resection cavity at the time of surgery ABFigure 42-20. A. Precontrast T1-weighted axial magnetic resonance imaging demonstrating bilateral hemorrhagic cerebellar metastases. Patient presented with ataxia and then lethargy progressing to deep coma. This patient has total effacement of the fourth ventricle and severe brain stem compression. The fourth ventricle cerebrospinal fluid space should be at the tip of the arrowhead. Patient recovered to normal mental status after emergent posterior fossa craniotomy. B. Postcontrast T1-weighted axial magnetic resonance imaging demonstrating a ring-enhancing lesion in the lateral left temporal lobe with moderate edema. The uncus (U) is compressing the left cerebral peduncle (CP) and displacing the brain stem to

1	imaging demonstrating a ring-enhancing lesion in the lateral left temporal lobe with moderate edema. The uncus (U) is compressing the left cerebral peduncle (CP) and displacing the brain stem to the right.Brunicardi_Ch42_p1827-p1878.indd 185501/03/19 7:17 PM 1856SPECIFIC CONSIDERATIONSPART IIfor recurrence. Adjuvant therapy remains marginally effective; survival has changed little over the last several decades.Oligodendroglioma. Oligodendroglioma accounts for approx-imately 10% of gliomas, arising from the oligodendrocytes that create myelin in the CNS. They often present with seizures. Calcifications and hemorrhage on CT or MRI suggest the diag-nosis. Oligodendrogliomas are also graded from I to IV; grade portends prognosis. Prognosis is better overall than for astro-cytomas. Median survival ranges from 2 to 7 years for high-est and lowest grade tumors, respectively. Aggressive resection improves survival. Many oligodendrogliomas will respond to procarbazine, lomustine (CCNU),

1	survival ranges from 2 to 7 years for high-est and lowest grade tumors, respectively. Aggressive resection improves survival. Many oligodendrogliomas will respond to procarbazine, lomustine (CCNU), and vincristine (PCV) che-motherapy. A particular chromosomal deletion, 1p19q, has been associated with robust response to the chemotherapeutic agent temozolomide. Radiation has not been clearly shown to prolong survival.Recent updates to brain tumor classification by the WHO (discussed in the following section) note that high-grade (at least II or III) oligodendrogliomas and astrocytomas are classi-fied together as diffuse gliomas. In fact, more similarity is seen between high-grade astrocytomas and oligodendrolgiomas than between high-grade astrocytomas and low-grade astrocytomas. Further discussion of this nosology is beyond the scope of this chapter, but implications for the future of neuro-oncology are discussed here.Ependymoma. The lining of the ventricular system consists of

1	discussion of this nosology is beyond the scope of this chapter, but implications for the future of neuro-oncology are discussed here.Ependymoma. The lining of the ventricular system consists of cuboidal/columnar ependymal cells from which ependy-momas may arise. Although most pediatric ependymomas are supratentorial, two-thirds of adult ependymomas are infratento-rial. Supratentorial ependymomas arise from the lateral or third ventricles. The infratentorial tumors arise from the floor of the fourth ventricle (i.e., off the posterior brainstem). The most com-mon symptoms are headache, nausea, vomiting, or vertigo, sec-ondary to increased ICP from obstruction of CSF flow through the fourth ventricle. The tumors may grow out the foramina of Luschka to form a cerebellopontine angle mass. They may also spread through the CSF to form “drop mets” in the spinal canal. The two main histologic subtypes are papillary and anaplastic, the latter characterized by increased mitotic activity and

1	They may also spread through the CSF to form “drop mets” in the spinal canal. The two main histologic subtypes are papillary and anaplastic, the latter characterized by increased mitotic activity and areas of necrosis. Gross total resection often is impossible because the tumor arises from the brain stem. The goal of surgery is to achieve maximal resection without injuring the very delicate brainstem. Suboccipital craniotomy and midline separation of the cerebellar hemispheres allows access to tumors in the fourth ventricle. Postoperative radiation therapy significantly improves survival. Patients with CSF spread documented by LP or con-trast MRI should also have whole-spine radiation plus focused doses to visualized metastases.Choroid Plexus Papilloma. The choroid plexus is composed of many small vascular tufts covered with cuboidal epithelium. It represents part of the interface between blood and brain. The choroid cells create CSF from blood via ultrafiltration and release it into

1	small vascular tufts covered with cuboidal epithelium. It represents part of the interface between blood and brain. The choroid cells create CSF from blood via ultrafiltration and release it into the ventricular system. Choroid plexus papil-lomas and choroid plexus carcinomas (rare, mostly pediatric) may arise from these cells. Papillomas usually occur in infants (typically supratentorial in the lateral ventricle) but also occur in adults (usually infratentorial in the fourth ventricle). Papillomas ABFigure 42-21. A. Postcontrast T1-weighted axial magnetic resonance imaging demonstrating a ring-enhancing lesion in the anteromedial right temporal lobe with central necrosis (dark area) consistent with glioblastoma multiforme. B. T2-weighted axial magnetic resonance imaging with extensive bright signal signifying peritumoral edema seen with glioblastoma multiformes.Brunicardi_Ch42_p1827-p1878.indd 185601/03/19 7:17 PM 1857NEUROSURGERYCHAPTER 42are well circumscribed and vividly

1	bright signal signifying peritumoral edema seen with glioblastoma multiformes.Brunicardi_Ch42_p1827-p1878.indd 185601/03/19 7:17 PM 1857NEUROSURGERYCHAPTER 42are well circumscribed and vividly enhance due to extensive vasculature. Like ependymomas, adult choroid plexus papillo-mas usually present with symptoms of increased ICP. Treatment is surgical excision. Total surgical excision is curative; recurrent papillomas should be re-resected. Radiation and chemotherapy are not indicated for papillomas. Radiation is adjunctive to aggressive surgery for carcinomas, but the results are generally poor.Neural Tumors and Mixed TumorsNeural and mixed tumors are a diverse group that includes tumors variously containing normal or abnormal neurons and/or normal or abnormal glial cells. Primitive neuroectodermal tumors arise from bipotential cells, capable of differentiating into neurons or glial cells.Medulloblastoma. Medulloblastoma is classically described as the most common type of primitive

1	tumors arise from bipotential cells, capable of differentiating into neurons or glial cells.Medulloblastoma. Medulloblastoma is classically described as the most common type of primitive neuroectodermal tumor (PNET), although this term has been removed in the latest WHO classification of central nervous system tumors. Most occur in the first decade of life, but there is a second peak around age 30. Medulloblastoma is the most common malignant pediatric brain tumor. They are usually midline. Most occur in the cerebellum and present with symptoms of increased ICP. Histologic char-acteristics include densely packed small round cells with large nuclei and scant cytoplasm. They are generally not encapsulated, frequently disseminate within the CNS, and should undergo sur-gical resection followed by radiation therapy and chemotherapy.Ganglioglioma. Ganglioglioma is a mixed tumor in which both neurons and glial cells are neoplastic. They occur in the first three decades of life, often in the

1	by radiation therapy and chemotherapy.Ganglioglioma. Ganglioglioma is a mixed tumor in which both neurons and glial cells are neoplastic. They occur in the first three decades of life, often in the medial temporal lobe, as circumscribed masses that may contain cysts or calcium and may enhance. The presenting symptom is usually a seizure, due to the medial temporal location. Patients have a good prognosis after complete surgical resection.Neural Crest TumorsMultipotent neural crest cells develop into a variety of dispa-rate cell types, including smooth muscle cells, sympathetic and parasympathetic neurons, melanocytes, Schwann cells, and arachnoid cap cells. They migrate in early development from the primitive neural tube throughout the body.Miscellaneous TumorsMeningioma. Meningiomas are derived from arachnoid cap cells of the arachnoid mater. They appear to arise from the dura mater grossly and on MRI and are commonly referred to as dural-based tumors. The most common intracranial

1	derived from arachnoid cap cells of the arachnoid mater. They appear to arise from the dura mater grossly and on MRI and are commonly referred to as dural-based tumors. The most common intracranial locations are along the falx (Fig. 42-22), the convexities (i.e., over the cerebral hemispheres), and the sphenoid wing. Less common locations include the foramen magnum, olfactory groove, and inside the lateral ventricle. Most are slow growing, encapsu-lated, benign tumors. Aggressive atypical or malignant menin-giomas may invade adjacent bone or cerebral cortex. Previous cranial irradiation increases the incidence of meningiomas. Approximately 10% of patients with a meningioma have multiple meningiomas. Total resection is curative, although involvement with small perforating arteries or cranial nerves may make total resection of skull base tumors impossible with-out significant neurologic deficit. The Simpson grading scale is used to characterize the extent of resection. Small,

1	or cranial nerves may make total resection of skull base tumors impossible with-out significant neurologic deficit. The Simpson grading scale is used to characterize the extent of resection. Small, asymptom-atic meningiomas can be followed until symptomatic or until significant growth is documented on serial imaging studies. Atypical and malignant meningiomas may require postoperative radiation. Patients may develop recurrences from the surgical bed or distant de novo tumors.Vestibular Schwannoma (Acoustic Neuroma). Vestibular schwannomas predominantly arise from the superior half of the vestibular portion of the vestibulocochlear nerve (cranial nerve VIII) (Fig. 42-23). Commonly, patients present with progressive hearing loss, tinnitus, or balance difficulty. Large tumors may cause brain stem compression and obstructive hydrocephalus. Bilateral acoustic neuromas are pathognomonic for neurofibro-matosis type 2 (NF2), a syndrome resulting from mutation of chromosome 22. NF2 patients

1	stem compression and obstructive hydrocephalus. Bilateral acoustic neuromas are pathognomonic for neurofibro-matosis type 2 (NF2), a syndrome resulting from mutation of chromosome 22. NF2 patients have an increased incidence of spinal and cranial meningiomas and gliomas.Vestibular schwannomas may be treated with microsur-gical resection or SRS (Gamma Knife radiosurgery or linear accelerator technology). The main complication with treatment is damage to the facial nerve (cranial nerve VII), which runs through the internal auditory canal with the vestibulocochlear nerve. Risk of facial nerve dysfunction increases with increasing tumor diameter. SRS is preferred for tumors <3 cm, and micro-surgical resection for those >3 cm (SRS can be supplemented for any residual tumor following resection).Pituitary Adenoma. Pituitary adenomas arise from the anterior pituitary gland (adenohypophysis). Tumors <1 cm diameter are considered microadenomas; larger tumors are macroadenomas. Figure

1	resection).Pituitary Adenoma. Pituitary adenomas arise from the anterior pituitary gland (adenohypophysis). Tumors <1 cm diameter are considered microadenomas; larger tumors are macroadenomas. Figure 42-22. Postcontrast T1-weighted coronal magnetic reso-nance imaging demonstrating a brightly enhancing lesion arising from the falx cerebri with moderate edema and mass effect on the right lateral ventricle. This is a falcine meningioma. Note also the small separate meningioma arising from the dura over the cerebral convexity.Brunicardi_Ch42_p1827-p1878.indd 185701/03/19 7:17 PM 1858SPECIFIC CONSIDERATIONSPART IIPituitary tumors may be functional (i.e., secrete endocrinologi-cally active compounds at pathologic levels) or nonfunctional (i.e., secrete nothing or inactive compounds). Functional tumors are often diagnosed when quite small, due to endocrine dysfunc-tion. The most common endocrine syndromes are Cushing’s disease, due to adrenocorticotropic hormone secretion, ForbesAlbright

1	tumors are often diagnosed when quite small, due to endocrine dysfunc-tion. The most common endocrine syndromes are Cushing’s disease, due to adrenocorticotropic hormone secretion, ForbesAlbright syndrome, due to prolactin secretion, and acromegaly, due to growth hormone secretion. Nonfunctional tumors are typically diagnosed as larger lesions causing mass effects such as visual field deficits due to compression of the optic chiasm or panhypopituita-rism due to compression of the gland. Figure 42-24 demonstrates a large pituitary adenoma. Hemorrhage into a pituitary tumor causes abrupt symptoms of headache, visual disturbance, decreased men-tal status, and endocrine dysfunction. This is known as pituitary apoplexy.Symptomatic pituitary tumors should be decompressed surgically to eliminate mass effect and/or to attempt an endo-crine cure. However, prolactin-secreting tumors (prolactinomas) usually shrink with dopaminergic therapy alone. First-line phar-macotherapy for small

1	eliminate mass effect and/or to attempt an endo-crine cure. However, prolactin-secreting tumors (prolactinomas) usually shrink with dopaminergic therapy alone. First-line phar-macotherapy for small prolactinomas is cabergoline, a dopamine agonist that inhibits production and secretion of prolactin, and is preferred over bromocriptine for its superior side effect profile. Consider surgery for prolactinomas with persistent mass effect or endocrinologic dysfunction in spite of adequate dopamine agonist therapy. Most pituitary tumors are approached transna-sally via the transsphenoidal approach, and minimally invasive, endoscopic surgical techniques are being used increasingly.Hemangioblastoma. Hemangioblastomas occur almost exclusively in the posterior fossa, with about 20% occurring in patients with von Hippel-Lindau (VHL) disease, a multisys-tem neoplastic disorder. Other tumors associated with VHL are renal cell carcinoma, pheochromocytoma, and retinal angio-mas. Many appear as cystic

1	with von Hippel-Lindau (VHL) disease, a multisys-tem neoplastic disorder. Other tumors associated with VHL are renal cell carcinoma, pheochromocytoma, and retinal angio-mas. Many appear as cystic tumors with an enhancing tumor on the cyst wall known as the mural nodule. Surgical resection is curative for sporadic (non-VHL associated) tumors. Pathology reveals abundant thin-walled vascular channels; internal debulk-ing may be bloody. En bloc resection of the mural nodule alone, leaving the cyst wall, is sufficient.Lymphoma. CNS lymphoma may arise either primarily in the CNS or secondarily from systemic disease. Recent rising inci-dence may be due to growing transplant and AIDS populations. Presenting symptoms include mental status changes, headache due to increased ICP, and cranial nerve palsy due to lymphoma-tous meningitis (analogous to carcinomatous meningitis). Often, lymphoma appears hyperdense on CT due to dense cellularity. Most lesions typically enhance with contrast on MRI,

1	palsy due to lymphoma-tous meningitis (analogous to carcinomatous meningitis). Often, lymphoma appears hyperdense on CT due to dense cellularity. Most lesions typically enhance with contrast on MRI, and can be differentiated by diffusion restriction on diffusion-weighted sequences. Surgical excision is not indicated. A stereotactic needle biopsy specimen usually confirms the diagnosis. Sub-sequent treatment includes steroids, whole-brain radiation, and chemotherapy. Intrathecal methotrexate is an additional treat-ment option.Embryologic TumorsEmbryologic tumors result from embryonal remnants that fail to involute completely or differentiate properly during development.Craniopharyngioma. Craniopharyngiomas are benign cystic lesions that occur in the sellar region that occur most frequently in children. A second peak of incidence also exists around 50 years of age. Craniopharyngiomas arise from remnant embry-onic tissue in the pituitary stalk. Calcification occurs in all pedi-atric and

1	in children. A second peak of incidence also exists around 50 years of age. Craniopharyngiomas arise from remnant embry-onic tissue in the pituitary stalk. Calcification occurs in all pedi-atric and roughly half of adult craniopharyngiomas. Symptoms result from compression of adjacent structures, especially the optic chiasm. Pituitary or hypothalamic dysfunction or hydro-cephalus may develop. Treatment is primarily surgical. Excision ABFigure 42-23. A. Postcontrast T1-weighted axial magnetic resonance imaging demonstrating a brightly enhancing mass on the right ves-tibular nerve with an enhancing tail going into the internal auditory canal (arrowhead). Pathology demonstrated vestibular schwannoma. B. Postcontrast T1-weighted sagittal magnetic resonance imaging of the same lesion, indicated by the arrowhead. Note small incidental meningioma at the top of the scan.Brunicardi_Ch42_p1827-p1878.indd 185801/03/19 7:17 PM 1859NEUROSURGERYCHAPTER 42is somewhat easier in children, as the

1	by the arrowhead. Note small incidental meningioma at the top of the scan.Brunicardi_Ch42_p1827-p1878.indd 185801/03/19 7:17 PM 1859NEUROSURGERYCHAPTER 42is somewhat easier in children, as the tumor is often soft and easily suctioned. Adult tumors are often firm and adherent to adjacent vital structures. Visual loss, pituitary endocrine hypo-function, diabetes insipidus, and cognitive impairment from basal frontal injury are common complications.Epidermoid. Epidermoid tumors are cystic lesions with strati-fied squamous epithelial walls from trapped ectodermal cell rests that grow slowly and linearly by desquamation into the cyst cavity. The cysts contain keratin, cholesterol, and cellu-lar debris (Fig. 42-25). They occur most frequently in the cer-ebellopontine angle and may cause symptoms due to brainstem compression. Recurrent bouts of aseptic meningitis may occur due to release of irritative cyst contents into the subarachnoid space (Mollaret’s meningitis). Treatment is

1	symptoms due to brainstem compression. Recurrent bouts of aseptic meningitis may occur due to release of irritative cyst contents into the subarachnoid space (Mollaret’s meningitis). Treatment is surgical drainage and removal of the cyst wall. Intraoperative spillage of cyst contents may lead to severe chemical meningitis and must be avoided by containment and aspiration.Dermoid. Dermoids are less common than epidermoid tumors. They contain hair follicles and sebaceous glands in addition to a squamous epithelium. Dermoids may be found anywhere along the craniospinal axis. They are more commonly midline structures and are associated with more anomalies than epidermoids. They may be associated with trauma, as from a lumbar puncture that drags skin structures into the spine. Bacterial meningitis may occur when dermoids are associated with a dermal sinus tract. Treatment of symptomatic lesions is surgical resection, again with care to control cyst contents.Teratoma. Teratomas are germ

1	may occur when dermoids are associated with a dermal sinus tract. Treatment of symptomatic lesions is surgical resection, again with care to control cyst contents.Teratoma. Teratomas are germ cell tumors that arise in the midline, often in the pineal region (the area behind the third ventricle, above the midbrain and cerebellum). They contain elements from all three embryonal layers: ectoderm, meso-derm, and endoderm. Teratomas may contain skin, cartilage, GI glands, and teeth. Teratomas with more primitive features are more malignant, while those with more differentiated tissues are more benign. Surgical excision may be attempted. However, the prognosis for malignant teratomas is very poor.Spinal TumorsApproximately 20% of CNS tumors occur in the spine, and a wide variety of spinal tumors exist. Unlike cranial tumors, the majority of spinal tumors are histologically benign. Under-standing two major spinal concepts—stability and neural com-pression—facilitates an understanding of the

1	exist. Unlike cranial tumors, the majority of spinal tumors are histologically benign. Under-standing two major spinal concepts—stability and neural com-pression—facilitates an understanding of the effects of spinal tumors. Destruction of bones or ligaments can cause spinal instability, leading to deformities such as compression, kyphosis, subluxation, all of which harbor the potential for subsequent neural compression. Tumor growth in the spinal canal or neu-ral foramina can cause direct compression of the spinal cord or nerve roots and cause pain and loss of function. Classically, the pain is worse at night. Anatomic categorization provides the most logical approach to these tumors. Certain tumors present in characteristic locations. An understanding of the anatomy leads to an understanding of the clinical presentation and possible therapeutic options.Extradural Tumors. Extradural tumors account for approxi-mately 55% of spinal tumors. This category includes tumors Figure

1	understanding of the clinical presentation and possible therapeutic options.Extradural Tumors. Extradural tumors account for approxi-mately 55% of spinal tumors. This category includes tumors Figure 42-24. Postcontrast T1-weighted sagittal magnetic reso-nance imaging demonstrating a large sellar/suprasellar lesion (arrowheads) involving the third ventricle superiorly and abut-ting the midbrain and pons posteriorly. The patient presented with progressive visual field and acuity loss. Pathology and lab work revealed a nonfunctioning pituitary adenoma.Figure 42-25. Postcontrast T1-weighted axial magnetic resonance imaging demonstrating a nonenhancing mass in the left cerebello-pontine angle with brain stem compression. White arrowhead indi-cates interface of tumor and brain stem. Black arrowhead indicates deformed fourth ventricle. Pathology revealed epidermoid tumor.Brunicardi_Ch42_p1827-p1878.indd 185901/03/19 7:17 PM 1860SPECIFIC CONSIDERATIONSPART IIarising within the bony

1	arrowhead indicates deformed fourth ventricle. Pathology revealed epidermoid tumor.Brunicardi_Ch42_p1827-p1878.indd 185901/03/19 7:17 PM 1860SPECIFIC CONSIDERATIONSPART IIarising within the bony vertebral structures and from within the epidural space. Destruction of the bone can lead to instability and fractures, causing pain and/or deformity. Epidural expan-sion can lead to spinal cord or nerve root compression with myelopathy, radiculopathy, or a combination thereof.Metastatic Tumors Metastatic tumors are the most common extradural tumors. Spinal metastases most commonly occur in the thoracic and lumbar vertebral bodies because the greatest volume of red bone marrow is found in these regions. The most common primary sources of spine metastasis are lymphoma, lung, breast, and prostate. Other sources include renal, colon, thyroid, sarcoma, and melanoma. Most spinal metastases create osteolytic lesions. Osteoblastic, sclerotic lesions suggest pros-tate cancer in men and breast

1	Other sources include renal, colon, thyroid, sarcoma, and melanoma. Most spinal metastases create osteolytic lesions. Osteoblastic, sclerotic lesions suggest pros-tate cancer in men and breast cancer in women.Patients with progressive neurologic dysfunction due to a metastatic lesion should undergo urgent surgery followed by radiation therapy.70 Patients with debilitating pain may undergo radiation therapy with close observation for neurologic deterio-ration. Preoperative neurologic function correlates with postop-erative function. Patients may lose function over hours. These patients should be given high-dose IV dexamethasone, taken immediately to MRI, and then to the OR or radiation therapy suite. Indications for surgery include failure of radiation ther-apy, spinal instability, recurrence after radiation therapy, and the need for diagnosis in cases of unknown primary tumors. Most cases with significant bone involvement require both decom-pression and fusion. Bony fusion usually

1	after radiation therapy, and the need for diagnosis in cases of unknown primary tumors. Most cases with significant bone involvement require both decom-pression and fusion. Bony fusion usually takes 2 to 3 months. Prognosis governs operative decisions. Surgery is unlikely to improve quality of life for patients with a life expectancy of 3 months or less, but it is likely to improve quality of life for patients with life expectancy of 6 months or more. Benefit for patients with 3to 6-months’ life expectancy is unclear and requires frank discussion with the patient and family. Patients who are unlikely to tolerate general anesthesia, are already completely paralyzed, or who have very radiosensitive tumors such as multiple myeloma and lymphoma, should not generally undergo surgery.Management Principles of Spinal Cord Compression in Metastatic Cancer of the Spine Spinal cord compression due to tumor burden is important to distinguish because it can, as with any other form of cord

1	Principles of Spinal Cord Compression in Metastatic Cancer of the Spine Spinal cord compression due to tumor burden is important to distinguish because it can, as with any other form of cord compression, cause paralysis and loss of bowel and bladder function. A randomized controlled trial demonstrated that patients with spinal cord compression from metastatic tumor have better outcomes with decompres-sive surgery and radiotherapy compared to radiotherapy alone.70 Patients with radiosensitive tumors, such as multiple myeloma and lymphoma, are excluded from this group. Another impor-tant tool in assessing these patients is the SINS (Spinal Instabil-ity Neoplastic Score). This scale grades the utility of operative intervention in metastatic cancer to the spine on the basis of pain, deformity, location, type of bone lesion, integrity of verte-bral body, and posterior element involvement.71Primary Tumors Hemangiomas are benign tumors found in 10% of people at autopsy. They occur in the

1	location, type of bone lesion, integrity of verte-bral body, and posterior element involvement.71Primary Tumors Hemangiomas are benign tumors found in 10% of people at autopsy. They occur in the vertebral bodies of the thoracolumbar spine and are frequently asymptomatic. They are often vascular and may hemorrhage, causing pain or neurologic deficit. Large hemangiomas can destabilize the spine and predispose to fracture. Osteoblastic lesions include osteoid osteoma and osteoblastoma. The latter tends to be larger and more destructive. Aneurysmal bone cysts are nonneoplastic, expansile, lytic lesions containing thin-walled blood cavities that usually occur in the lamina or spinous processes of the cer-vicothoracic spine. They may cause pain or sufficiently weaken the bone to cause a fracture. Cancers arising primarily in the bony spine include Ewing’s sarcoma, osteosarcoma, chondro-sarcoma, and plasmacytoma.Intradural Extramedullary Tumors Intradural extramed-ullary tumors constitute

1	Cancers arising primarily in the bony spine include Ewing’s sarcoma, osteosarcoma, chondro-sarcoma, and plasmacytoma.Intradural Extramedullary Tumors Intradural extramed-ullary tumors constitute approximately 40% of spinal tumors and arise from the meninges or nerve root elements. They may compress the spinal cord, causing myelopathy, or the nerve roots, causing radiculopathy. The most common intradural extramedullary tumors are typically benign, slow growing, and well circumscribed. Rare benign epidural masses include arach-noid cysts, dermoids, and epidermoids. Rare malignant epidural tumors include metastases and high-grade gliomas, or “drop” metastases from posterior fossa gliomas.Meningioma Meningiomas arise from the arachnoidea mater. They appear to be dural based and enhance on MRI. An enhanc-ing “dural tail” may be seen. They occur most commonly in the thoracic spine (Fig. 42-26) but also arise in the cervical and lumbar regions. Some spinal meningiomas grow into the Figure

1	An enhanc-ing “dural tail” may be seen. They occur most commonly in the thoracic spine (Fig. 42-26) but also arise in the cervical and lumbar regions. Some spinal meningiomas grow into the Figure 42-26. T2-weighted sagittal magnetic resonance imaging of the midthoracic spine demonstrating a well-encapsulated tumor arising from the dura posteriorly and compressing the spinal cord. Arrowhead points to dorsal location of the mass. The patient pre-sented with worsening gait and lower extremity spasticity. Pathol-ogy demonstrated meningioma.Brunicardi_Ch42_p1827-p1878.indd 186001/03/19 7:17 PM 1861NEUROSURGERYCHAPTER 42epidural space. Growth causes cord compression and progres-sive myelopathy with hyperreflexia, spasticity, and gait difficul-ties. Surgical excision is the treatment of choice. The surgeon often finds a clean margin between the tumor, dura, and spinal cord, allowing en bloc resection without damage to the cord.Schwannoma Schwannomas are derived from peripheral nerve

1	choice. The surgeon often finds a clean margin between the tumor, dura, and spinal cord, allowing en bloc resection without damage to the cord.Schwannoma Schwannomas are derived from peripheral nerve sheath Schwann cells. They are benign, encapsulated tumors that rarely undergo malignant degeneration. While two-thirds are entirely intradural, one-sixth are entirely extradural, and one-sixth have a classic “dumbbell” shape from intradural and extra-dural components. Symptoms result from radiculopathy, often presenting as pain or myelopathy. Symptomatic lesions should be surgically resected. The parent nerve root usually can be pre-served. Patients with multiple schwannomas likely have NF2. In these patients, a careful neurologic examination is needed to determine which lesions are symptomatic and require resection.Neurofibroma In contrast to schwannomas, neurofibromas tend to appear more fusiform and to grow within the parent nerve, rather than forming an encapsulated mass branching

1	and require resection.Neurofibroma In contrast to schwannomas, neurofibromas tend to appear more fusiform and to grow within the parent nerve, rather than forming an encapsulated mass branching off the nerve. Neurofibromas are benign but not encapsulated. They present similarly to schwannomas, and the two may be difficult to differentiate on imaging. Salvage of the parent nerve is more challenging with neurofibromas. To improve the likelihood of total resection, thoracic and high cervical nerve roots may be sacrificed with minimal deficit. Patients with multiple neurofi-bromas likely have NF1, also known as von Recklinghausen’s neurofibromatosis. Resection for symptomatic lesions should be offered.Intramedullary Tumors. Intramedullary tumors constitute approximately 5% of spinal tumors. They arise from within the parenchyma of the spinal cord. Common presenting symptoms are local dysesthesia, burning pain, radicular pain, sensory loss, weakness, or sphincter dysfunction. Patients with

1	arise from within the parenchyma of the spinal cord. Common presenting symptoms are local dysesthesia, burning pain, radicular pain, sensory loss, weakness, or sphincter dysfunction. Patients with such symp-toms should undergo MRI of the entire spine with and without enhancement.Ependymoma Ependymomas are the most common intramed-ullary tumors in adults. There are several histologic variants. The myxopapillary type occurs in the conus medullaris or the filum terminale in the lumbar region and has the best progno-sis after resection. The cellular type occurs more frequently in the cervical cord. Many spinal ependymomas have cystic areas and may contain hemorrhage. Surgical removal can improve function. A distinct tumor margin often exists, allowing safer excision. Postoperative radiation therapy after subtotal resection may prolong disease control.Astrocytoma Astrocytomas are the most common intramedul-lary tumors in children, although they also occur in adults. They may occur at all

1	therapy after subtotal resection may prolong disease control.Astrocytoma Astrocytomas are the most common intramedul-lary tumors in children, although they also occur in adults. They may occur at all levels, although more often in the cervical cord. The tumor may interfere with the CSF-containing central canal of the spinal cord, leading to a dilated central canal, referred to as syringomyelia (syrinx). Spinal astrocytomas are usually low grade, but complete excision is rarely possible due to the nonencapsulated, infiltrative nature of the tumor. As a result, patients with astrocytomas fare worse overall than patients with ependymomas.Other Tumors. Other types of rare tumors include high-grade astrocytomas, dermoids, epidermoids, teratomas, hemangiomas, hemangioblastomas, and metastases. Patients usually present with pain. Prognosis generally depends on preoperative func-tion and the histologic characteristics of the lesion.Future DirectionsFuture directions in the neurosurgical

1	Patients usually present with pain. Prognosis generally depends on preoperative func-tion and the histologic characteristics of the lesion.Future DirectionsFuture directions in the neurosurgical management of brain tumors are related to improved genetic characterization of brain tumors and technological advances. Regarding the former, the recent update of the World Health Organization classification of central nervous system tumors, alluded to earlier in this chap-ter, emphasizes an integrated classification approach utilizing both histologic and molecular characteristics.72 The addition of the latter feature underscores significant advances in the genet-ics of CNS tumors, which are being translated to treatment. Immunotherapy, for example, is an active area of research in the treatment of GBM. In this approach, immune cells such as T cells and dendritic cells are leveraged to target tumor-specific tissue. These approaches will play an important role as adjuvant therapy to

1	treatment of GBM. In this approach, immune cells such as T cells and dendritic cells are leveraged to target tumor-specific tissue. These approaches will play an important role as adjuvant therapy to neurosurgical approaches.Recent advances in neurosurgical technology are being used to address previous operative limitations. Intraoperative fluorescein is being studied as a means of marking abnor-mal tissue that appears grossly normal in order to maximize resection and minimize recurrence. Indications for SRS are expanding and provide a noninvasive option that can be used as monotherapy or as supplemental therapy to operative inter-vention. Moreover, new minimally invasive techniques, such as MRI-guided laser interstitial thermal therapy (MRgLITT), are actively being studied for less morbid access to otherwise difficult-to-reach tumors.SPINE: BASIC CONCEPTSThe spine is a complex structure and is subject to an extensive array of pathologic processes, including degeneration,

1	less morbid access to otherwise difficult-to-reach tumors.SPINE: BASIC CONCEPTSThe spine is a complex structure and is subject to an extensive array of pathologic processes, including degeneration, inflam-mation, infection, neoplasia, and trauma. Discussions of spine trauma, tumor, and infection are addressed separately in this chapter in the “Infection—Spine,” “Spinal Tumors,” and “Spine Trauma” sections. General concepts, common patterns of dis-ease, and basic operative interventions are presented here.The spine consists of a series of stacked vertebrae, inter-vening discs, and longitudinal ligaments. The vertebrae consist of the vertebral body anteriorly and the pedicles, articular facets, laminae, and spinous processes posteriorly. The intervertebral discs have two components. The tough, fibrous ring that runs around the outer diameter of the two adjacent vertebral bodies is known as the annulus fibrosus. The spongy material inside the ring of the annulus is known as the nucleus

1	fibrous ring that runs around the outer diameter of the two adjacent vertebral bodies is known as the annulus fibrosus. The spongy material inside the ring of the annulus is known as the nucleus pulposus. The annu-lus and the nucleus provide a cushion between adjacent verte-bral bodies, absorb forces transmitted to the spine, and allow some movement between the vertebral bodies. The ligaments stabilize the spine by limiting the motion of adjacent vertebrae.Stability and neural compression are the two concepts crit-ical to understanding the mechanics and pathologic processes affecting the spine.StabilityThe spinal column is the principal structural component of the axial spine, and it must bear significant loads. The vertebrae increase in size from the top to the bottom of the spine, cor-relating with the increased total loads that the more caudal ele-ments must bear. The cervical spine is the most mobile. Cervical stability depends greatly on the integrity of the ligaments that run

1	with the increased total loads that the more caudal ele-ments must bear. The cervical spine is the most mobile. Cervical stability depends greatly on the integrity of the ligaments that run from level to level. The thoracic spine is the least mobile, due to the stabilizing effect of the rib cage. The lumbar spine has relatively massive vertebrae, supports heavy loads, and has Brunicardi_Ch42_p1827-p1878.indd 186101/03/19 7:17 PM 1862SPECIFIC CONSIDERATIONSPART IIintermediate mobility. The sacral spine is fused together and has no intrinsic mobility. The load borne by the lumbar spine is transmitted to the sacrum, and then the pelvis through the sacro-iliac joints. The coccyx is the most inferior segment of the spine and has no significant contribution to load bearing or mobility.A stable spine is one that can bear normally experienced forces resulting from body mass, movement, and muscle con-traction, while maintaining normal structure and alignment. An unstable spine will shift

1	spine is one that can bear normally experienced forces resulting from body mass, movement, and muscle con-traction, while maintaining normal structure and alignment. An unstable spine will shift or sublux under these forces. The determinants of spinal stability vary throughout the cervical, thoracic, and lumbar portions. In elementary form, stability depends on the structural integrity of the hard, bony elements of the vertebral column, as well as the tensile integrity and security of the supporting ligamentous attachments. Plain X-rays and CT scans are sensitive for detecting bony defects such as frac-tures or subluxation, while MRI better detects disruptions of the soft tissues, including ligaments and intervertebral discs. Specific patterns of abnormalities seen on imaging studies may suggest or diagnose spinal instability.A common form of nontraumatic spinal instability is lum-bar spondylolisthesis, which is typically a forward slippage of a lumbar vertebra relative to the lower

1	suggest or diagnose spinal instability.A common form of nontraumatic spinal instability is lum-bar spondylolisthesis, which is typically a forward slippage of a lumbar vertebra relative to the lower vertebra on which it rests. This results from congenital or degenerative disruption of the pars interarticularis, the critical bridge of bone that spans adja-cent facet joints. In the setting of a pars defect, there is no solid bony connection between the adjacent vertebrae. The spine is unstable and anterior listhesis (slippage) may result. Patients typically present with severe low back pain that is exacerbated with movement and load bearing (mechanical low back pain). Radiculopathy in this setting indicates neuroforaminal compres-sion. Figure 42-27 demonstrates an L4 and L5 spondylolisthesis.Neural CompressionBesides providing a stable, central element of the body’s support structure, the spine also protects the spinal cord and nerve roots as they pass through the neural foramina to

1	CompressionBesides providing a stable, central element of the body’s support structure, the spine also protects the spinal cord and nerve roots as they pass through the neural foramina to form the peripheral nervous system. In a healthy spine, the spinal cord and nerve roots are suspended in CSF, free of mechanical compression. Patho-logic processes that can lead to CSF space impingement and neural compression include: hypertrophic degenerative changes in the intervertebral discs and facet joints, expansion of epidural masses such as tumors or abscesses, and subluxation (i.e., slip-page) of adjacent vertebral bodies. Subluxation may be due to trauma that exceeds the spine’s load-bearing capabilities and leads to structural failure, or chronic structural degradation by degenerative disease, infection, or tumor. Subluxation reduces the cross-sectional area of the central canal and the neural foramina (see Fig. 42-10B). Reduced central canal area can lead to myelop-athy. Reduced neural

1	infection, or tumor. Subluxation reduces the cross-sectional area of the central canal and the neural foramina (see Fig. 42-10B). Reduced central canal area can lead to myelop-athy. Reduced neural foraminal area can lead to radiculopathy.Myelopathy. Compression of the spinal cord can cause distur-bance of function known as myelopathy. This dysfunction may be secondary to the direct effects of compression, cord isch-emia due to reduced perfusion, or pathologic changes due to repeated cord trauma. These mechanisms lead to demyelination of the corticospinal tracts, which are long descending motor tracts. Corticospinal tract damage leads to upper motor neuron signs and symptoms, including hyperreflexia, spasticity, and weakness. These mechanisms also cause damage to the dorsal columns, which carry ascending proprioception, vibration, and two-point discrimination information. Loss of proprioception makes fine motor tasks and ambulation difficult.Radiculopathy. Compression of the nerve

1	carry ascending proprioception, vibration, and two-point discrimination information. Loss of proprioception makes fine motor tasks and ambulation difficult.Radiculopathy. Compression of the nerve roots causes distur-bance of root function, known as radiculopathy. Characteristic features of radiculopathy include lower motor neuron signs and symptoms (hyporeflexia, atrophy, and weakness) and sensory dis-turbances such as numbness or tingling sensations (paresthesias), burning sensations (dysesthesias), and shooting (radicular) pain. Myelopathy and radiculopathy often present together in diseases that involve the central canal and the neural foramina. This com-bination can lead to lower motor neuron dysfunction at the level of disease, and upper motor neuron dysfunction below that level.Patterns of DiseaseCervical Radiculopathy. The cervical nerve roots exit the central canal above the pedicle of the same-numbered verte-bra and at the level of the higher adjacent intervertebral disc. For

1	of DiseaseCervical Radiculopathy. The cervical nerve roots exit the central canal above the pedicle of the same-numbered verte-bra and at the level of the higher adjacent intervertebral disc. For example, the C6 nerve root passes above the C6 pedicle at the level of the C5–C6 discs. The cervical nerve roots may be compressed acutely by disc herniation, or chronically by hypertrophic degenerative changes of the discs, facets, and liga-ments. Table 42-6 summarizes the effects of various disc her-niations. Most patients with acute disc herniations will improve without surgery. NSAIDs or cervical traction may help alle-viate symptoms. Patients whose symptoms do not resolve or who have significant weakness should undergo decompressive surgery. The two main options for nerve root decompression are anterior cervical discectomy and fusion (ACDF) and posterior cervical foraminotomy (keyhole foraminotomy). ACDF allows more direct access to and removal of the pathology (anterior to the nerve

1	are anterior cervical discectomy and fusion (ACDF) and posterior cervical foraminotomy (keyhole foraminotomy). ACDF allows more direct access to and removal of the pathology (anterior to the nerve root). However, the procedure requires fusion because discectomy causes a collapse of the interbody space and instabil-ity will likely occur. Figure 42-28 demonstrates a C6–C7 ACDF with the typical interposed graft and plating system. Keyhole foraminotomy allows for decompression without requiring Figure 42-27. Lateral lumbar spine X-ray demonstrates a 25% anterior slippage of L4 on L5 due to a defect in the L4 pars interar-ticularis. This is called spondylolisthesis.Brunicardi_Ch42_p1827-p1878.indd 186201/03/19 7:17 PM 1863NEUROSURGERYCHAPTER 42Table 42-6Cervical disc herniations and symptoms by levelLEVELFREQUENCY (%)ROOT INJUREDREFLEXWEAKNESSNUMBNESSC4–C52C5—DeltoidShoulderC5–C619C6BicepsBiceps brachiiThumbC6–C769C7TricepsWrist extensors (wrist drop)Second and third

1	and symptoms by levelLEVELFREQUENCY (%)ROOT INJUREDREFLEXWEAKNESSNUMBNESSC4–C52C5—DeltoidShoulderC5–C619C6BicepsBiceps brachiiThumbC6–C769C7TricepsWrist extensors (wrist drop)Second and third digitsC7–T110C8—Hand intrinsicsFourth and fifth digitsAdapted with permission from Greenberg MS. Handbook of Neurosurgery, 7th ed. New York, NY: Thieme; 2010.BAFigure 42-28. A. Anteroposterior cervical spine X-ray showing the position of an anterior cervical plate used for stabilization after C6–C7 discectomy. Patient presented with right triceps weakness and dysesthesias in the right fifth digit. Magnetic resonance imaging revealed a right paracentral C6–C7 herniated disc compressing the exiting C7 nerve root. B. Lateral cervical spine X-ray of the same patient clearly demonstrates the position of the plate and screws. The allograft bone spacer placed in the drilled-out disc space is also apparent.fusion, but it is less effective for removing centrally located canal pathology.Cervical

1	of the plate and screws. The allograft bone spacer placed in the drilled-out disc space is also apparent.fusion, but it is less effective for removing centrally located canal pathology.Cervical Spondylotic Myelopathy. The term spondylosis refers to diffuse degenerative and hypertrophic changes of the discs, intervertebral joints, and ligaments, which collectively result in spinal stenosis. Spinal cord dysfunction (myelopathy) due to cord compression from cervical spinal degenerative dis-ease is therefore referred to as cervical spondylotic myelopathy (CSM). Classically CSM presents with spasticity and hyper-reflexia due to corticospinal tract dysfunction, upper extremity weakness and atrophy from degeneration of the motor neu-rons in the anterior horns of the spinal gray matter, and loss of lower extremity proprioception due to dorsal column injury. Figure 42-29 demonstrates typical findings. Some patients com-plain of difficulty buttoning shirts, using utensils, and ambulating.

1	of lower extremity proprioception due to dorsal column injury. Figure 42-29 demonstrates typical findings. Some patients com-plain of difficulty buttoning shirts, using utensils, and ambulating. Spondylosis is usually diffuse, so the usual treatment for CSM is multilevel (usually C3–C7) cervical laminectomy, although patients with disease localized over one to three levels may be can-didates for anterior decompression and fusion. Fig. 42-30 demon-strates the postoperative appearance of a vertebral corpectomy and fusion for CSM. Thorough cervical laminectomy decompresses the cord posteriorly. Patients often have slow recovery due to the extensive chronic changes in the cervical cord and may benefit from rehabilitation programs. The other disease that classically presents with combined upper and lower motor neuron symp-toms is amyotrophic lateral sclerosis (ALS). Care must be taken Brunicardi_Ch42_p1827-p1878.indd 186301/03/19 7:17 PM 1864SPECIFIC CONSIDERATIONSPART IIto avoid

1	upper and lower motor neuron symp-toms is amyotrophic lateral sclerosis (ALS). Care must be taken Brunicardi_Ch42_p1827-p1878.indd 186301/03/19 7:17 PM 1864SPECIFIC CONSIDERATIONSPART IIto avoid offering cervical laminectomy to a patient with undi-agnosed ALS. Two findings help differentiate CSM from ALS: cranial nerve dysfunction such as dysphagia (not typically caused by cervical spine disease) and sensory disturbance (not found in ALS).Thoracic Disc Herniation. Thoracic disc herniation accounts for <1% of herniated discs. A patient may present with radicular pain or sensorimotor changes in the lower extremities due to cord compression. A posterior approach via midline incision and lami-nectomy should be avoided because of the high incidence of cord injury from manipulation and retraction. Anterior approaches via thoracotomy minimize risk to the cord and allow excellent access to the disc. The radicular arteries running from the aorta to the thoracic cord should be spared, when

1	Anterior approaches via thoracotomy minimize risk to the cord and allow excellent access to the disc. The radicular arteries running from the aorta to the thoracic cord should be spared, when possible, to avoid ischemia. Alternatively, a posterolateral approach is possible via resection of the rib head and facet joint. Finally, a transpedicular approach may be attempted for lateral disc herniations.73Lumbar Radiculopathy. Lumbar nerve roots exit the thecal sac, pass over the higher adjacent disc space, and exit the canal under the pedicle of the same-numbered vertebra. Therefore, the L5 nerve root passes over the L4–L5 disc space and exits under the L5 pedicle (Fig. 42-31). Lumbar discs may herniate with or without a history of trauma or straining. Normally they cause lancinating (radicular) pain down the leg (Table 42-7). Most acute herniated lumbar discs improve symptomatically without surgery. Surgery is indicated for symptoms persisting more than 6 to 8 weeks, progressive motor

1	pain down the leg (Table 42-7). Most acute herniated lumbar discs improve symptomatically without surgery. Surgery is indicated for symptoms persisting more than 6 to 8 weeks, progressive motor deficit (e.g., foot drop), or for patients with incapacitating pain not manage-able with analgesics. A recent randomized control trial (Spine Patients Outcome Research Trial [SPORT])74 did not observe significant differences between patients randomized to undergo surgery vs. conservative management when using an intent-to-treat analysis. Because of the high amount of bi-directional cross-over between the surgery and conservative group, a sub-sequent reanalysis of the data as observational cohort analysis, demonstrated improved functional outcomes in terms of pain and physical function, more so at 3 months than at 2 years.74Discectomy is performed using a midline incision, partial removal of the overlying laminae (hemilaminectomy or lami-notomy), identification of the thecal sac and nerve root,

1	months than at 2 years.74Discectomy is performed using a midline incision, partial removal of the overlying laminae (hemilaminectomy or lami-notomy), identification of the thecal sac and nerve root, and extraction of disc fragments. Free-floating disc fragments may be found. Often, however, the herniated disc material is still contained within the annulus, requiring incision of the poste-rior longitudinal ligament and curettage of the disc space. After lumbar discectomy, approximately two-thirds of patients will have complete relief of pain, and up to 85% will have significant improvement.Neurogenic Claudication. Neurogenic claudication is char-acterized by low back and leg pain that occurs while walking and is relieved by stopping, leaning forward, or sitting. It is normally caused by degenerative lumbar stenosis causing com-pression of the cauda equina. Neurogenic claudication must be distinguished from vascular claudication, which tends to resolve quickly with cessation of walking.

1	lumbar stenosis causing com-pression of the cauda equina. Neurogenic claudication must be distinguished from vascular claudication, which tends to resolve quickly with cessation of walking. There is typically no need to change position, and the pain follows a stocking distribution rather than a dermatomal distribution. Pallor and coldness of the feet, and normal neurologic examination are also typical, though diabetic patients may present a challenge with microvascular Figure 42-29. T2-weighted sagittal magnetic resonance imaging of the cervical spine showing multilevel degenerative changes caus-ing spinal stenosis that is worst at C5–C6. Note the bright signal within the cord at that level, consistent with myelopathy.Figure 42-30. Lateral cervical spine X-ray status post C5 corpec-tomy for cervical spondylotic myelopathy. This involves removal of the C4–C5 disc, C5 vertebral body, and C5–C6 disc, decom-pressing at two levels. A bone strut is visible bridging C4 to C6. The plate and

1	for cervical spondylotic myelopathy. This involves removal of the C4–C5 disc, C5 vertebral body, and C5–C6 disc, decom-pressing at two levels. A bone strut is visible bridging C4 to C6. The plate and screws stabilize the segments.Brunicardi_Ch42_p1827-p1878.indd 186401/03/19 7:17 PM 1865NEUROSURGERYCHAPTER 42neuropathy. Patients with neurogenic claudication have a slowly progressive course and may be surgical candidates when their pain interferes with their lifestyle. The usual surgery is an L3 to L5 lumbar laminectomy to decompress the nerve roots. A recent randomized control trial did not observe a significant difference in outcomes between surgical and conservative management of lumbar stenosis at 1 year.75 Thus, surgical management of lum-bar stenosis should be reserved for patients that do not improve with physical therapy.Cauda Equina Syndrome. Cauda equina syndrome is due to compression of the cauda equina and may result from massive disc herniation, EDH, epidural abscess,

1	that do not improve with physical therapy.Cauda Equina Syndrome. Cauda equina syndrome is due to compression of the cauda equina and may result from massive disc herniation, EDH, epidural abscess, tumor, or subluxation from trauma. Patients with cauda equina compression often present with urinary retention, saddle anesthesia, or progressing leg weakness. Saddle anesthesia is numbness in the perineum, genitals, buttocks, and upper inner thighs. Patients with sus-pected cauda equina syndrome should undergo immediate MRI of the lumbar spine to evaluate for a surgical lesion. Mass lesions should be removed urgently via laminectomy to preserve sphincter function and ambulation.Spine Fusion SurgeryFusion surgery is often required for patients with spinal instabil-ity resulting from disease, surgical intervention, or both. Fusion procedures lock adjacent vertebrae together. Fusion occurs when the body forms a solid mass of bone incorporating the adjacent vertebrae, eliminating normal

1	surgical intervention, or both. Fusion procedures lock adjacent vertebrae together. Fusion occurs when the body forms a solid mass of bone incorporating the adjacent vertebrae, eliminating normal intervertebral move-ment. Stabilization and immobilization promote bony fusion. Internal instrumentation and external orthoses are often used to stabilize and immobilize the fused spinal segments.Spinal InstrumentationInternal fixation devices for spinal segmental immobilization have been developed for all levels of the spine. Most spinal instrumentation constructs have two elements. The first element is a device that solidly attaches to the vertebral bodies. Options include wires wrapped around laminae or spi-nous processes, hooks placed under the lamina or around the pedicles, or screws placed in the pedicles or the vertebral bodies. The second element is a device that traverses vertebral seg-ments. Options include rods and plates that lock directly to the wires, hooks, or screws at each

1	in the pedicles or the vertebral bodies. The second element is a device that traverses vertebral seg-ments. Options include rods and plates that lock directly to the wires, hooks, or screws at each vertebral level. Spinal instru-mentation devices are available for anterior and posterior fusion in the cervical, thoracic, and lumbar regions. Most modern spi-nal instrumentation devices are made of titanium to minimize problems with future MRI scanning (Fig. 42-32). All spinal instrumentation constructs will eventually fail by loosening or breaking if bony fusion does not occur.6ABFigure 42-31. A. T2-weighted sagittal magnetic resonance imaging shows an L5–S1 disc herniation causing significant canal compromise and displacement of nerve roots. B. T2-weighted axial magnetic resonance imaging of the same patient shows the large left paracentral disc herniation at L5–S1. Arrowheads delineate the extent of the herniation. The arrow indicates the right S1 nerve root passing through free of

1	of the same patient shows the large left paracentral disc herniation at L5–S1. Arrowheads delineate the extent of the herniation. The arrow indicates the right S1 nerve root passing through free of compression. The left S1 nerve root is under severe compression and is not seen.Table 42-7Lumbar disc herniations and symptoms by levelLEVELFREQUENCY (%)ROOT INJUREDREFLEXWEAKNESSNUMBNESSL3–L45L4PatellarQuadricepsAnterior thighL4–L545L5—Tibialis anterior (foot drop)Great toeL5–S150S1AchillesGastrocnemiusLateral footAdapted with permission from Greenberg MS. Handbook of Neurosurgery, 7th ed. New York, NY: Thieme; 2010.Brunicardi_Ch42_p1827-p1878.indd 186501/03/19 7:17 PM 1866SPECIFIC CONSIDERATIONSPART IIFigure 42-32. A. Lateral lumbar spine X-ray showing pedicle screws and connecting rods used to stabilize L4 with respect to L5. This instrumentation was placed as part of a fusion operation to stabilize progressive L4–L5 spondylolisthesis with intractable low back pain. B.

1	rods used to stabilize L4 with respect to L5. This instrumentation was placed as part of a fusion operation to stabilize progressive L4–L5 spondylolisthesis with intractable low back pain. B. Anteroposterior lumbar spine X-ray showing L3 to L5 instrumentation with pedicle screws and connecting rods. The patient had previously sustained an L4 burst fracture. Note the sig-nificant loss of height of the L4 body compared to adjacent levels. The small row of staples to the right delineates the incision over the iliac crest used to harvest cancellous bone as a nonstructural osteoinductive autograft fusion designed to induce formation of a solid bone bridge from L3 to L5 (arthrodesis).ArthrodesisArthrodesis refers to the obliteration of motion or instability by incorporating the relevant components into a solid mass of bone. Arthrodesis must occur in any fused segment to have long-term stability. Failure of arthrodesis results in failed fusion, often in the form of a fibrous nonunion. The

1	into a solid mass of bone. Arthrodesis must occur in any fused segment to have long-term stability. Failure of arthrodesis results in failed fusion, often in the form of a fibrous nonunion. The rates of successful fusion are higher in the cervical spine than the lum-bar spine. Arthrodesis requires ingrowth of new bone formed by the patient’s osteoblasts across the unstable defect. Insert-ing graft material, such as autograft or allograft, into the defect provides a bridge for osteoblasts and promotes fusion. The term autograft refers to the patient’s own bone, often harvested from the iliac crest. Iliac crest bone graft is a source of both cortical and cancellous bone. Cortical bone provides structural support, while cancellous bone provides a matrix for bony ingrowth. The term allograft refers to sterilized bone from human tissue banks. Allografts also may be cortical, cancellous, or both. Allograft lacks the array of osteoinductive endogenous compounds intrinsic to autograft,

1	refers to sterilized bone from human tissue banks. Allografts also may be cortical, cancellous, or both. Allograft lacks the array of osteoinductive endogenous compounds intrinsic to autograft, although supplemental products such as demineralized bone matrix paste can be added to encourage new bone formation. Other techniques for increasing the rates of suc-cessful fusion are being developed, including the integration of osteoinductive bone morphogenetic proteins, known as BMPs, into the fusion constructs.Dynamic stabilization refers to the creation of spinal sta-bility without achieving a bony fusion. The concept applies to both cervical and lumbar motion segments. Artificial lumbar and cervical disc replacement therapies are recent developments in degenerative spine disease that address this concept. How-ever, their use is limited to very select cases. Another motion preservation technique that may hold promise is segmental “soft” stabilization.77 In cases of degenerative

1	address this concept. How-ever, their use is limited to very select cases. Another motion preservation technique that may hold promise is segmental “soft” stabilization.77 In cases of degenerative spondylolisthe-sis, such systems in the lumbar spine allow for decompressive laminectomy without increasing slippage. In theory, adjacent level facets and discs are spared the stresses of a neighboring bony fusion moment arm.PERIPHERAL NERVECommon pathologic processes that compromise function of the peripheral nervous system include mechanical compression, ischemia, inflammation, and neoplasia.Peripheral Nerve TumorsMost peripheral nerve tumors are benign and grow slowly. Significant pain increases the likelihood that the patient has a malignant tumor. Treatment for peripheral nerve tumors is sur-gical resection to establish diagnosis and evaluate for signs of malignancy. These tumors have various degrees of involvement with the parent nerve. Some can be resected with minimal or no damage to

1	resection to establish diagnosis and evaluate for signs of malignancy. These tumors have various degrees of involvement with the parent nerve. Some can be resected with minimal or no damage to the nerve. Tumors that grow within the nerve often contain functioning fascicles. Total excision of these tumors requires sacrifice of the parent nerve. The choice of subtotal resection, nerve preservation, and observation, vs. total resec-tion with nerve sacrifice depends on tumor histology and the function of the parent nerve.Schwannoma. Schwannomas are the most common peripheral nerve tumors, also referred to as neurilemomas or neurinomas. Most occur in the third decade of life. These benign tumors arise from Schwann cells, which form myelin in peripheral nerves. The most characteristic presentation is a mass lesion with point tenderness and shooting pains on direct palpation. Spontaneous ABBrunicardi_Ch42_p1827-p1878.indd 186601/03/19 7:17 PM 1867NEUROSURGERYCHAPTER 42or continuous pain

1	is a mass lesion with point tenderness and shooting pains on direct palpation. Spontaneous ABBrunicardi_Ch42_p1827-p1878.indd 186601/03/19 7:17 PM 1867NEUROSURGERYCHAPTER 42or continuous pain suggests malignancy. Schwannomas tend to grow slowly and eccentrically on parent nerves. The eccentric location and discrete encapsulated nature of these tumors often allow total resection without significant damage to the parent nerve. Subtotal resection and observation is reasonable for schwannomas entwined in important nerves, as the incidence of malignant transformation is extremely low.Neurofibroma. Neurofibromas arise within the nerve and tend to be fusiform masses, unlike schwannomas, which tend to grow out of the nerve. Neurofibromas often present as a mass that is tender to palpation. They usually lack the shooting pains char-acteristic of schwannomas. Neurofibromas are often difficult to resect completely without sacrifice of the parent nerve. Neuro-fibromas have a higher incidence

1	usually lack the shooting pains char-acteristic of schwannomas. Neurofibromas are often difficult to resect completely without sacrifice of the parent nerve. Neuro-fibromas have a higher incidence of malignant transformation; therefore, patients with known residual tumors require close observation. Patients with NF1 often have multiple neurofibro-mas. These patients should be offered resection for symptomatic tumors. Risk of malignant degeneration is up to 10%. Malignant neurofibromas have the histologic characteristics of sarcoma.Malignant Nerve Sheath Tumors. Malignant nerve sheath tumors include solitary sarcomas, degenerated neurofibromas, and neuroepitheliomas. Patients with malignant peripheral nerve tumors typically complain of constant pain, rather than pain only on palpation, and are more likely to have motor and sensory deficits in the distribution of the parent nerve. Treat-ment for these tumors is radical excision. This often requires sacrifice of the parent nerve.

1	and are more likely to have motor and sensory deficits in the distribution of the parent nerve. Treat-ment for these tumors is radical excision. This often requires sacrifice of the parent nerve. Invasion of nearby soft tissues may occur and necessitate wide resection or amputation in an attempt to prevent systemic metastasis.Entrapment NeuropathiesEntrapment neuropathy presents as neurologic dysfunction in nerves passing through a pathologically small, fixed space. Nerve dysfunction may result directly from chronic, repetitive pressure on the nerve, or from ischemic damage due to impaired perfusion.77 Entrapment causing dysfunction of nerve signaling may be associated with numbness, paresthesias, weakness, or muscle atrophy. The two most common sites of entrapment neu-ropathy are the ulnar nerve at the medial aspect of the elbow and the median nerve at the wrist. Usually EMG/NCS demon-strate slowing across the entrapped segment of nerve. Mechani-cal peripheral nerve disorders

1	the ulnar nerve at the medial aspect of the elbow and the median nerve at the wrist. Usually EMG/NCS demon-strate slowing across the entrapped segment of nerve. Mechani-cal peripheral nerve disorders resulting from trauma (brachial plexus disruption, radial nerve damage from humerus fractures, and common peroneal nerve crush injuries) are discussed in the section “Trauma.”Ulnar Neuropathy. The ulnar nerve has contributions from the C7, C8, and T1 nerve roots, arises from the medial cord of the brachial plexus, and supplies most of the intrinsic hand mus-cles (interossei and third and fourth lumbricals) and sensation to the fourth and fifth digits. It passes posteriorly to the medial epicondyle at the elbow in the condylar groove. This segment is superficial and subject to external compression and repeti-tive minor impacts. Patients with ulnar entrapment at the elbow present with numbness and tingling in the medial palm, as well as the fourth and fifth digits. Motor deficits include

1	and repeti-tive minor impacts. Patients with ulnar entrapment at the elbow present with numbness and tingling in the medial palm, as well as the fourth and fifth digits. Motor deficits include weakness and wasting of the intrinsic hand muscles. Treatment for symp-tomatic ulnar entrapment neuropathy is surgical exploration and incision of the fibrous aponeurotic arch that overlies the nerve. A 6-cm curvilinear incision centered between the medial epi-condyle and the olecranon allows exploration of up to 10 cm of nerve and lysis of compressive tissues.Carpal Tunnel Syndrome. The median nerve has contribu-tions from the C5 to T1 nerve roots, arises from the medial and lateral cords of the brachial plexus, and supplies the muscles of wrist and finger flexion and sensation to the palmar aspect of the first, second, and third digits. The median nerve passes through the carpal tunnel in the wrist, lying superficial to the four deep and four superficial flexor tendons. The transverse carpal

1	of the first, second, and third digits. The median nerve passes through the carpal tunnel in the wrist, lying superficial to the four deep and four superficial flexor tendons. The transverse carpal liga-ment is a tough, fibrous band that forms the roof of the carpal tunnel. The ligament attaches to the pisiform and hamate medi-ally and the trapezium and scaphoid laterally. Patients complain of numbness and tingling in the supplied digits, clumsiness, and worsening with sleep or repetitive wrist movement. Patients may notice wasting of the thenar eminence. Treatment for symptomatic carpal tunnel syndrome unresponsive to splinting, analgesics, and rest is surgical division of the flexor retinacu-lum. This often provides prompt relief of pain symptoms and slow recovery of numbness and strength.Autoimmune and Inflammatory DisordersThese are not surgical diseases, but they merit brief mention as they are included in the differential diagnosis for new-onset weakness. Their characteristic

1	and Inflammatory DisordersThese are not surgical diseases, but they merit brief mention as they are included in the differential diagnosis for new-onset weakness. Their characteristic presentations help distinguish them from weakness due to structural lesions.Guillain-Barré Syndrome. Guillain-Barré syndrome is an acute inflammatory demyelinating polyradiculopathy often occurring after viral infection, surgery, inoculations, or myco-plasma infections. Patients classically present with weakness ascending from the legs to the body, arms, and even cranial nerves. Symptoms usually progress over 2 to 4 weeks and then resolve. Care is supportive. Respiratory weakness may require ventilatory support.Myasthenia Gravis. Myasthenia gravis is an autoimmune process in which antibodies form to the acetylcholine recep-tors of muscles, leading to fluctuating weakness. Most patients have either thymic hyperplasia or thymoma. The most common symptoms are diplopia, ptosis, dysarthria, and dysphagia.

1	recep-tors of muscles, leading to fluctuating weakness. Most patients have either thymic hyperplasia or thymoma. The most common symptoms are diplopia, ptosis, dysarthria, and dysphagia. More severe cases have limb or respiratory involvement. Weakness worsens with repetitive movement. Treatment is with acetylcho-linesterase inhibitors and possible thymectomy.Eaton-Lambert Syndrome. Eaton-Lambert syndrome is an autoimmune process with antibodies to the presynaptic calcium channels. This is a paraneoplastic syndrome most commonly associated with oat cell carcinoma. Patients have weakness of proximal limb muscles that improves with repetitive movement. This diagnosis must prompt oncologic evaluation.INFECTIONCNS infections of interest to neurosurgeons include those that cause focal neurologic deficit due to mass effect, require surgi-cal aspiration or drainage because antibiotic therapy alone is insufficient, cause mechanical instability of the spine, or occur after neurosurgical

1	deficit due to mass effect, require surgi-cal aspiration or drainage because antibiotic therapy alone is insufficient, cause mechanical instability of the spine, or occur after neurosurgical procedures.CranialOsteomyelitis. The skull is highly vascular and resistant to infections. Osteomyelitis of the skull may develop by contigu-ous spread from pyogenic sinus disease or from contamination by penetrating trauma. Staphylococcus aureus and S epidermidis are the most frequent causative organisms. Patients usually present with redness, swelling, and pain. Contrast head CT aids 7Brunicardi_Ch42_p1827-p1878.indd 186701/03/19 7:17 PM 1868SPECIFIC CONSIDERATIONSPART IIdiagnosis and shows the extent of involved bone, along with associated abscesses or empyema. Osteomyelitis treatment entails surgical debridement of involved bone followed by 2 to 4 months of antibiotics. Craniotomy wound infections are a special concern because performing a craniotomy creates a devascularized free bone

1	surgical debridement of involved bone followed by 2 to 4 months of antibiotics. Craniotomy wound infections are a special concern because performing a craniotomy creates a devascularized free bone flap susceptible to infection and not penetrated by antibiotics. These wounds must be debrided and the bone flaps removed and discarded. Subsequent care involves appropriate antibiotic therapy, observation for signs of recurrent infection off antibiotics, and return to the OR for titanium or methylmethacrylate cranioplasty 6 to 12 months later.Subdural Empyema. Subdural empyema is a rapidly progres-sive pyogenic infection. The subdural space lacks significant barriers to the spread of the infection, such as compartmental-ization or septations. Subdural empyemas usually occur over the cerebral convexities. Potential infectious sources include sinus disease, penetrating trauma, and otitis. Streptococci and staphylococci are the most frequent sources. Presenting symp-toms include fever,

1	convexities. Potential infectious sources include sinus disease, penetrating trauma, and otitis. Streptococci and staphylococci are the most frequent sources. Presenting symp-toms include fever, headache, neck stiffness, seizures, or focal neurologic deficit. Neurologic deficit results from inflammation of cortical blood vessels, leading to thrombosis and stroke. The most common deficit is contralateral hemiparesis. Patients with suggestive symptoms should undergo rapid contrast CT scan. LP frequently fails to yield the offending organism and risks herniation due to mass effect. Typical treatment is wide hemi-craniectomy, dural opening, and lavage. The pus may be thick or septated, making burr hole drainage or small craniotomy insufficient. Patients then require 1 to 2 months of antibiotics. Subdural empyema has 10% to 20% mortality risk and common chronic sequelae, including development of a seizure disorder and residual hemiparesis. However, many patients do make a good

1	of antibiotics. Subdural empyema has 10% to 20% mortality risk and common chronic sequelae, including development of a seizure disorder and residual hemiparesis. However, many patients do make a good recovery.Brain Abscess. Brain abscess is encapsulated infection within the brain parenchyma. It may spread hematogenously in patients with endocarditis or intracardiac or intrapulmonary right-to-left shunts, by migration from the sinuses or ear, or via direct seed-ing by penetrating trauma. Disorganized cerebritis often pre-cedes formation of the organized, walled-off abscess. Patients may present with nonspecific symptoms such as headache, nau-sea, or lethargy, or with focal neurologic deficit such as hemi-paresis. Alternatively, patients may present in extremis if the abscess ruptures into the ventricular system. Abscesses appear as well-demarcated, ring-enhancing, thin-walled lesions on CT scan and MRI, and often have associated edema and mass effect. Patients require antibiotic

1	into the ventricular system. Abscesses appear as well-demarcated, ring-enhancing, thin-walled lesions on CT scan and MRI, and often have associated edema and mass effect. Patients require antibiotic therapy after needle aspira-tion or surgical evacuation. Antibiotic therapy without surgical evacuation may be considered for patients with small, multiple, or critically located abscesses. Abscesses that are large, cause mass effect, decreased mental status, or that fail to decrease in size after 1 week of antibiotics, should be evacuated. Nonsurgical management still requires aspiration or biopsy specimen for organism culture and sensitivities. Blood and CSF cultures rarely give definitive diagnosis. Removal of an encapsulated abscess significantly shortens the length of antibiotic therapy required to eliminate all organisms. Common chronic sequelae after suc-cessful treatment include seizures or focal neurologic deficit.SpinePyogenic Vertebral Osteomyelitis. Pyogenic vertebral

1	therapy required to eliminate all organisms. Common chronic sequelae after suc-cessful treatment include seizures or focal neurologic deficit.SpinePyogenic Vertebral Osteomyelitis. Pyogenic vertebral osteomyelitis is a destructive bacterial infection of the vertebrae, usually of the vertebral body. Vertebral osteomyelitis frequently results from hematogenous spread of distant disease, but may occur as an extension of adjacent disease, such as psoas abscess or perinephric abscess. S aureus and Enterobacter spp. are the most frequent etiologic organisms. Patients usually present with fever and back pain. Diabetics, IV drug abusers, and dialysis patients have increased incidence of vertebral osteomyelitis. Epidural extension may lead to compression of the spinal cord or nerve roots with resultant neurologic deficit. Osteomyelitis presents a lytic picture on imaging and must be distinguished from neoplastic disease. Adjacent intervertebral disc involve-ment occurs frequently with pyogenic

1	neurologic deficit. Osteomyelitis presents a lytic picture on imaging and must be distinguished from neoplastic disease. Adjacent intervertebral disc involve-ment occurs frequently with pyogenic osteomyelitis, but rarely with neoplasia. Plain films and CT help assess the extent of bony destruction or deformity such as kyphosis. MRI shows adjacent soft tissue or epidural disease. Most cases can be treated suc-cessfully with antibiotics alone, although the organism must be isolated to steer antibiotic choice. Blood cultures may be positive. Surgical intervention may be required for debridement when antibiotics alone fail, or for stabilization and fusion in the setting of instability and deformity.Tuberculous Vertebral Osteomyelitis. Tuberculous verte-bral osteomyelitis, also known as Pott’s disease, occurs most commonly in underdeveloped countries and in the immuno-compromised. Several features differentiate tuberculous osteo-myelitis from bacterial osteomyelitis. The infection is

1	disease, occurs most commonly in underdeveloped countries and in the immuno-compromised. Several features differentiate tuberculous osteo-myelitis from bacterial osteomyelitis. The infection is indolent and symptoms often progress slowly over months. Tuberculosis rarely involves the intervertebral disc. The involved bodies may have sclerotic rather than lytic changes. Multiple nonadjacent vertebrae may be involved. The upper lumbar and lower tho-racic vertebrae are most commonly affected. Diagnosis requires documentation of acid-fast bacilli. Treatment involves long-term antimycobacterial drugs. Patients with spinal instability or neural compression from epidural inflammatory tissue should undergo debridement and fusion as needed.Discitis. Primary infection of the intervertebral disc space, or discitis, is most commonly secondary to postoperative infec-tions. Spontaneous discitis occurs more commonly in children. S aureus and S epidermidis account for most cases. The pri-mary symptom

1	or discitis, is most commonly secondary to postoperative infec-tions. Spontaneous discitis occurs more commonly in children. S aureus and S epidermidis account for most cases. The pri-mary symptom is back pain. Other signs and symptoms include radicular pain, fevers, paraspinal muscle spasm, and localized tenderness to palpation. Many cases will resolve without anti-biotics, which generally are given for positive blood or biopsy specimen cultures or persistent constitutional symptoms. Most patients will have spontaneous fusion across the involved disc and do not need debridement or fusion.Epidural Abscess. Epidural abscesses may arise from or spread to the adjacent bone or disc, so distinguishing between vertebral osteomyelitis or discitis and a spinal epidural abscess may be difficult. The most common presenting signs and symp-toms are back pain, fever, and tenderness to palpation of the spine. The most significant risk of epidural abscess is weakness progressing to paralysis due to

1	most common presenting signs and symp-toms are back pain, fever, and tenderness to palpation of the spine. The most significant risk of epidural abscess is weakness progressing to paralysis due to spinal cord or nerve root dam-age. Cord and root damage may be due to direct compression or to inflammatory thrombosis resulting in venous infarction. S aureus and Streptococcus spp. are the most common organisms. Methicillin-resistant S aureus now constitutes a significant pro-portion of these infections, as high as 40%.78 The source may be hematogenous spread, local extension, or operative contamina-tion. MRI best demonstrates the epidural space and degree of neural compromise. Patients with spinal epidural abscess and neurologic compromise should undergo surgical debridement for decompression and diagnosis, followed by culture-directed Brunicardi_Ch42_p1827-p1878.indd 186801/03/19 7:17 PM 1869NEUROSURGERYCHAPTER 42antibiotic therapy. Relative contraindications to surgery include

1	and diagnosis, followed by culture-directed Brunicardi_Ch42_p1827-p1878.indd 186801/03/19 7:17 PM 1869NEUROSURGERYCHAPTER 42antibiotic therapy. Relative contraindications to surgery include prohibitive comorbidities or total lack of neurologic function below the involved level. Patients with no neurologic deficits and an identified organism may be treated with antibiotics alone and very close observation. However, this management strat-egy remains somewhat controversial because these patients can undergo rapid and irreversible neurologic decline. Most epidural abscesses can be accessed via laminectomy without fusion. Col-lections predominantly anterior to the cervical or thoracic cord may require anterior approach and fusion.FUNCTIONAL NEUROSURGERYEpilepsy SurgerySeizures result from uncontrolled neuronal electrical activity. Seizures may result from irritative lesions in the brain, such as tumors or hematomas, or from physiologic or structural abnor-malities. Seizures may involve

1	neuronal electrical activity. Seizures may result from irritative lesions in the brain, such as tumors or hematomas, or from physiologic or structural abnor-malities. Seizures may involve a part of the brain (focal) or the entire brain (generalized). Focal seizures may be associated with normal consciousness (simple) or decreased conscious-ness (complex). All generalized seizures cause loss of con-sciousness. Focal seizures may secondarily generalize. Patients with multiple unprovoked seizures over time are considered to have epilepsy. The type of epilepsy depends on such factors as type of seizures, electroencephalographic (EEG) findings, associated syndromes, and identifiable etiologies. All patients with unexplained seizures (i.e., no obvious cause such as head trauma or alcohol withdrawal) require thorough neurologic evaluation, including imaging to evaluate for a mass lesion. Antiepileptic drugs (AEDs) form the first line of therapy for epilepsy, initially as monotherapy, then as

1	require thorough neurologic evaluation, including imaging to evaluate for a mass lesion. Antiepileptic drugs (AEDs) form the first line of therapy for epilepsy, initially as monotherapy, then as combination ther-apy. Epilepsy patients who have failed satisfactory trials of sev-eral AED combination regimens may be candidates for surgical intervention. Lack of seizure control or patient intolerance of the medications may constitute failure. Epilepsy surgery can decrease the frequency of seizures by resection of the electrical source of the seizures, or decrease the severity of seizures by disconnecting white matter tracts through which the abnormal electrical activity spreads. Four types of epilepsy surgery are discussed in sections that follow. Epilepsy surgery appears to be extremely underused, given the relatively low risk of the procedures, and the crippling social and economic effects of uncontrolled or partially controlled epilepsy.79 Patients with symptoms, imaging abnormalities,

1	given the relatively low risk of the procedures, and the crippling social and economic effects of uncontrolled or partially controlled epilepsy.79 Patients with symptoms, imaging abnormalities, and EEG analysis compat-ible with a specific seizure focus are most likely to have good results from epilepsy surgery.Anterior Temporal Lobectomy. Medial temporal lobe struc-tural abnormalities can lead to complex partial seizures (CPS). Many patients with CPS have poor seizure control on medi-cations. Patients with CPS may have significant reduction in seizure frequency or cessation of seizures after resection of the anterior temporal lobe. The amygdala and the head of the hip-pocampus are removed as part of the lobectomy. Resection may be taken back approximately 4.5 cm from the temporal tip in the language-dominant hemisphere, and 6 cm from the temporal tip in the language nondominant hemisphere, with low risk of significant neurologic deficits.80 The two main risks of anterior temporal

1	in the language-dominant hemisphere, and 6 cm from the temporal tip in the language nondominant hemisphere, with low risk of significant neurologic deficits.80 The two main risks of anterior temporal lobectomy are memory impairment and visual loss. Removal of the hippocampus in a patient with an atrophied or nonfunctional contralateral hippocampus causes a global mem-ory deficit. Interruption of the optic radiations, which carry visual signals from the contralateral superior visual quadrants of both eyes, causes a contralateral superior quadrantanopia, known as a pie in the sky field cut.Corpus Callosotomy. Patients with generalized seizures, atonic seizures associated with drop attacks, or absence sei-zures, who are found to have bilaterally coordinated patho-logic cortical discharges on EEG and who fail AED therapy, may be candidates for corpus callosotomy. The corpus cal-losum is a large white matter tract that connects the cerebral hemispheres. Loss of consciousness requires

1	on EEG and who fail AED therapy, may be candidates for corpus callosotomy. The corpus cal-losum is a large white matter tract that connects the cerebral hemispheres. Loss of consciousness requires simultaneous seizure activity in both hemispheres. Focal or partial seizures may spread via the corpus callosum to the contralateral hemi-sphere, causing generalization and loss of consciousness. Divi-sion of the corpus callosum can interrupt this spread. Patients may have decreased numbers of seizures and/or fewer epi-sodes of lost consciousness. Usually only the anterior half or two-thirds of the corpus callosum is divided, as more extensive division increases the risk of disconnection syndrome. Patients with disconnection syndrome are unable to match objects in the opposite visual hemifields, to identify objects held in one hand with the other hemifield, and to write with the left hand or name objects held in the left hand (in left hemisphere–dominant patients).Hemispherectomy. Children

1	to identify objects held in one hand with the other hemifield, and to write with the left hand or name objects held in the left hand (in left hemisphere–dominant patients).Hemispherectomy. Children with intractable epilepsy, struc-tural anomalies in one hemisphere, and contralateral hemiple-gia, may have improved seizure control after resection of the hemisphere (anatomic hemispherectomy) or disruption of all connections to the hemisphere (functional hemispherectomy). Functional hemispherectomy often is preferred over anatomic hemispherectomy because of the high incidence of complica-tions such as hematoma formation and ventriculoperitoneal shunt dependence associated with the latter.Vagus Nerve Stimulation. Neuromodulatory treatments like vagus nerve stimulation (VNS), approved by the U.S. Food and Drug Administration (FDA) in 1997, are less invasive and offer some titratability in addition to reversibility unlike the resec-tive surgical options previously described. Since first

1	U.S. Food and Drug Administration (FDA) in 1997, are less invasive and offer some titratability in addition to reversibility unlike the resec-tive surgical options previously described. Since first reported in 1985, VNS has proven to be efficacious in certain patient populations for several disorders such as treatment-resistant major depressive disorder, bipolar disorder, and epilepsy. In VNS, a pulse generator is placed under the skin in the chest and is connected to the vagus nerve by an electrical lead. Chronic, intermittent VNS has been proven to be an effective option for patients suffering from medically refractory seizures who are not candidates for surgical resection. Although only a small minority of patients will be entirely seizure-free, three blinded, randomized-controlled trials have examined VNS and demon-strated significant clinical improvement compared to sham.81-83 Generally VNS is well-tolerated and safe, as device implantation is associated with a low rate of

1	trials have examined VNS and demon-strated significant clinical improvement compared to sham.81-83 Generally VNS is well-tolerated and safe, as device implantation is associated with a low rate of perioperative complications. Addi-tionally, the majority of side effects are stimulation-dependent and thus, reversible. For the most part, VNS is limited in its appli-cation because it can only exert its effects by altering neural activ-ity via the vagus nerve. Procedures with brain region-specificity are being investigated.Deep Brain StimulationThe following summary of deep brain stimulation (DBS) will include a review of the current FDA-approved indications, as well the expanding applications of this therapy, currently being investigated preclinically and in clinical trials. While the Brunicardi_Ch42_p1827-p1878.indd 186901/03/19 7:17 PM 1870SPECIFIC CONSIDERATIONSPART IImechanism of action of DBS continues to elude our understand-ing, it is well established that administering

1	Brunicardi_Ch42_p1827-p1878.indd 186901/03/19 7:17 PM 1870SPECIFIC CONSIDERATIONSPART IImechanism of action of DBS continues to elude our understand-ing, it is well established that administering electrical stimula-tion to a nucleus in the brain known to be involved in a given disease can disrupt the pathologic signals emanating to or from this brain region. A fine electrical lead is placed in a deep brain nucleus and connected to pulse generators placed in the chest in a manner similar to cardiac pacemakers. Connector wires travel from the generators in the subcutaneous space up the neck and in the subgaleal space in the head, to connect the pulse gen-erators to the electrical leads. Proper lead placement is accom-plished with stereotactic guidance. A frame is rigidly fixed to the patient’s head, and an MRI is obtained with the frame in place. Calculation of the coordinates of the millimeter-sized deep brain nuclei is performed in relation to the three-dimensional space defined

1	head, and an MRI is obtained with the frame in place. Calculation of the coordinates of the millimeter-sized deep brain nuclei is performed in relation to the three-dimensional space defined by the fixed frame, allowing for accurate targeting of the nucleus (Fig. 42-33). Postoperatively, the pulse generators can be interrogated and adjusted with hand-held, transcutane-ous, noninvasive devices as needed for symptom control.Essential Tremor. Essential tremor is the most common movement disorder in the western world and is characterized by action tremor (4–8 Hz rhythmic oscillations) of the hands, forearms, head, and voice. Essential tremor often starts in the third or fourth decade of life and increases in frequency and amplitude with age. β-Blockers can decrease symptoms, but patients with poor medical control and significant functional impairment significantly benefit from placement of a deep brain stimulator in the contralateral ventralis intermediate nucleus of the thalamus. In

1	with poor medical control and significant functional impairment significantly benefit from placement of a deep brain stimulator in the contralateral ventralis intermediate nucleus of the thalamus. In properly selected patients, DBS of this region of the thalamus appears to provide robust and durable symptom control.84,85Parkinson’s Disease. Parkinson’s disease is a progressive dis-order characterized by rigidity, bradykinesia, and resting tremor, due to loss of dopamine-secreting neurons in the substantia nigra. Dopaminergic agents such as levodopa/carbidopa and anticholinergic agents such as amantadine and selegiline form the basis of medical therapy. Patients with poor medical control or significant drug side effects may benefit significantly from placement of bilateral deep brain stimulators in the subthalamic nuclei. Although the globus pallidus interna has also been a widely targeted area, the subthalamic nuclei is now the most accepted target in deep brain stimulation for

1	stimulators in the subthalamic nuclei. Although the globus pallidus interna has also been a widely targeted area, the subthalamic nuclei is now the most accepted target in deep brain stimulation for Parkinson’s disease.86 Deep brain stimulation provides durable symp-tom relief with good postoperative neuropsychologic function in properly selected patients.87Recently, a large randomized controlled trial compared bilat-eral DBS (n = 121) to best medical therapy in advanced Parkin-son’s disease (n = 134).88 The DBS group did significantly better in both motor function and quality of life. While adverse events were 3.8 times more likely in the DBS group, 99% of these events had resolved by 6 months. There was a 0.8% risk of death due to the procedure, and there was no difference in risk of adverse events when comparing older (≥70 years) to younger patients (<70 years). Thus, the benefits of DBS over medical therapy are clear, especially when considering quality of life measures.Another

1	adverse events when comparing older (≥70 years) to younger patients (<70 years). Thus, the benefits of DBS over medical therapy are clear, especially when considering quality of life measures.Another recent randomized controlled trial focused on defining the optimal targets for DBS in Parkinson’s disease.89 While the subthalamic nucleus (STN) and the globus pallidus interna (GPi) have been successfully targeted in the past, a direct comparison of the two was lacking. In this study, 299 subjects were randomized to receive either bilateral STN or GPi stimula-tors and were evaluated for 2 years. The primary outcome was motor function, as assessed by part III of the Unified Parkin-son’s Disease Rating Scale (UPDRS). The study found no sig-nificant difference in motor improvement between target sites. However, a significant difference was found in a secondary outcome measuring depression. On the Beck Depression Inven-tory, the pallidal stimulation group improved slightly compared with the

1	sites. However, a significant difference was found in a secondary outcome measuring depression. On the Beck Depression Inven-tory, the pallidal stimulation group improved slightly compared with the STN group, which actually worsened slightly. Never-theless, the actual incidence of depressive episodes requiring prolonged or new hospitalization was 2.6% and 0.7% in GPi and STN, respectively, which was not significantly different. On the other hand, the STN group was found to require less adjunctive dopaminergic pharmacotherapy than the GPi group. In terms of overall severe adverse events, there was no difference between groups. The investigators concluded that both target sites are effective and that nonmotor factors such as psychiatric symp-toms may be a consideration in DBS target selection.Dystonia. The FDA humanitarian device exemption has been made for DBS for dystonia but is limited to patients ≥7 years of age with primary dystonia, including generalized and/or seg-mental

1	selection.Dystonia. The FDA humanitarian device exemption has been made for DBS for dystonia but is limited to patients ≥7 years of age with primary dystonia, including generalized and/or seg-mental dystonia, hemidystonia, or cervical dystonia (torticollis). Dystonia is characterized by sustained muscle contractions that cause repetitive movements and involuntary postures. Cognitive function is typically spared, and pharmacological therapy is fre-quently inadequate. The positive impact of DBS on Parkinson’s and essential tremor has led neurologists and neurosurgeons to direct their attention to DBS for treatment of idiopathic focal and generalized dystonia.Although the pathophysiology of idiopathic dystonia is unclear, positron emission tomography studies have shown disturbed glucose metabolism in the GPi, suggesting secondary pathologic activation of the motor cortex. Indeed, the GPi is cur-rently considered the most efficacious target for dystonia, and controlled trials indicate

1	in the GPi, suggesting secondary pathologic activation of the motor cortex. Indeed, the GPi is cur-rently considered the most efficacious target for dystonia, and controlled trials indicate approximately a 50% improvement in motor function and disability.90 Since many patients undergoing 8Figure 42-33. Fast spin echo coronal magnetic resonance imaging demonstrating position of deep brain stimulator leads in the subtha-lamic nuclei bilaterally. The electrodes appear thick and wavy due to magnetic susceptibility artifact.Brunicardi_Ch42_p1827-p1878.indd 187001/03/19 7:17 PM 1871NEUROSURGERYCHAPTER 42surgery for dystonia are children and young adults, DBS is an attractive surgical option because it can be titrated, revised, and reversed according to individual needs and growth patterns.Obsessive-Compulsive Disorder. The safety and efficacy of DBS, as well as its titratability and reversibility, that have been demonstrated for the treatment of movement disorders in the 1990s and 2000s

1	Disorder. The safety and efficacy of DBS, as well as its titratability and reversibility, that have been demonstrated for the treatment of movement disorders in the 1990s and 2000s has spawned an increasing interest and awareness of the capabilities of nonlesional surgical treatments for diseases of the brain. An obvious outgrowth of DBS for movement disorders has been the treatment of medically refrac-tory psychiatric disorders. Despite the dark history of frontal leucotomy procedures that dominated the early 20th century, nonlesional DBS for psychiatric disorders are now considered potential treatment strategies.Functional neuroimaging has implicated certain brain regions in the pathogenesis of a variety of psychiatric disorders. The FDA has approved a humanitarian device exemption for DBS targeting the ventral capsule/ventral striatum for severe obsessive-compulsive disorder (OCD). Recent case reports and pilot studies have reported remission in patients suffering from refractory

1	DBS targeting the ventral capsule/ventral striatum for severe obsessive-compulsive disorder (OCD). Recent case reports and pilot studies have reported remission in patients suffering from refractory OCD following DBS. A pilot study using a blinded, staggered-onset design found that four (66.7%) of six patients met a stringent criterion as “responders” (≥35% improvement), according to the Yale-Brown Obsessive Compulsive Scale after 12 months of stimulation.91 In this study, patients did not improve during the sham phase. Adverse events were generally mild and modifiable with setting changes, and stimulation inter-ruption led to rapid yet reversible development of depressive symptoms in two cases. Thus, DBS has promise as a therapy of last resort for carefully selected cases of severe OCD.Expanding Indications of Deep Brain Stimulation. There are multiple disorders, both psychiatric and neurologic, that have exhibited significant promise as potential indications for DBS in large-scale

1	Indications of Deep Brain Stimulation. There are multiple disorders, both psychiatric and neurologic, that have exhibited significant promise as potential indications for DBS in large-scale trials. Recently, there have been reports of significant improvements in refractory depression with DBS. Lozano and colleagues performed an open label study with extended follow-up on 20 patients targeting an area within the subcallosal cin-gulate gyrus (SCG) with bilateral DBS.92 At the last follow-up visit in this study (range: 3–6 years), the average response rate was 64%, according to the Hamilton Rating Scale for Depres-sion. Of note, impairment in social functioning was improved, and no significant adverse events were reported. Because two patients died by suicide during depressive relapses, it remains unclear if DBS can only improve quality of life or significantly suppress relapses and extend life-span in this extremely delicate patient population. Of note, as seen in OCD, the ventral

1	it remains unclear if DBS can only improve quality of life or significantly suppress relapses and extend life-span in this extremely delicate patient population. Of note, as seen in OCD, the ventral capsule/ventral striatum has also been targeted for depression, as well as the nucleus accumbens directly, which lies within the ventral striatum. Studies of DBS in this region report an approximate 40% to 60% response rate, and results from a recent, multicenter randomized controlled trial are pending.93DBS as a potential therapy for epilepsy targeting the ante-rior nucleus of the thalamus has been investigated in a multi-center, double-blind, randomized trial (SANTE).94 In this trial, the group receiving DBS showed a 29% greater reduction in seizure frequency in relation to the sham group in the last month of the blinded phase. Complex partial and the “most severe” seizures were significantly reduced in the cohort who had the stimulator on DBS-on group. After the blinded phase of the

1	in the last month of the blinded phase. Complex partial and the “most severe” seizures were significantly reduced in the cohort who had the stimulator on DBS-on group. After the blinded phase of the trial was complete, 54% of patients had a seizure reduction of at least 50%. Fourteen patients were seizure-free for at least 6 months; eight were seizure-free for at least one year, four for at least two years, and one patient for more than four years. Because of the modest benefit during the blinded phase of this trial, FDA-approval has yet to be granted to DBS for epilepsy targeting the thalamus in the United States, though approval has been given in Europe and Canada.The region-specific, neuromodulatory capabilities of DBS have inspired the open label use of this technique in many other neurologic and psychiatric disorders, including but not limited to Tourette syndrome, Huntington’s disease, and Alzheimer dis-ease. Preclinical studies of both substance abuse and obesity have also

1	neurologic and psychiatric disorders, including but not limited to Tourette syndrome, Huntington’s disease, and Alzheimer dis-ease. Preclinical studies of both substance abuse and obesity have also shown promise.95,96 The opportunity to model reward-seeking behaviors associated with these disorders in animals provides the ability to not only test safety but also study mecha-nisms and inform the design of future clinical trials.Trigeminal NeuralgiaTrigeminal neuralgia, also known as tic douloureux, is charac-terized by repetitive, unilateral, sharp, and lancinating pains in the distribution of, typically, the second, but sometimes third, branch of cranial nerve V, the trigeminal nerve. The patient may describe a “trigger point,” an area on the face that elicits the pain when touched. A current leading etiologic hypothesis for trigeminal neuralgia is irritation and pulsatile compression of the root entry zone of the nerve by an artery in the posterior fossa, usually a loop of the

1	A current leading etiologic hypothesis for trigeminal neuralgia is irritation and pulsatile compression of the root entry zone of the nerve by an artery in the posterior fossa, usually a loop of the superior cerebellar artery. The pain is excruciating and can be debilitating. Medical therapy, including carbamazepine and amitriptyline, may reduce the frequency of events. Options for medically refractory cases include percuta-neous injection of glycerol into the path of the nerve, periph-eral transection of the nerve branches, SRS, and microvascular decompression (MVD).MVD involves performing a small posterior fossa crani-otomy on the side of the symptoms, retraction of the cerebellar hemisphere, and exploration of cranial nerve V. If an artery is found near the nerve, the vessel is freed of any adhesions and nonabsorbable material is placed between the nerve root and the artery. MVD remains the first definitive management option because SRS is associated with a substantial incidence of

1	any adhesions and nonabsorbable material is placed between the nerve root and the artery. MVD remains the first definitive management option because SRS is associated with a substantial incidence of facial numbness.97,98STEREOTACTIC RADIOSURGERYThe term stereotactic radiosurgery (SRS) refers to techniques that allow delivery of high-dose radiation that conforms to the shape of the target and has rapid isodose fall-off, minimiz-ing damage to adjacent neural structures. The two most common devices used for conformal SRS for intracranial lesions are the LINAC (linear accelerator) and the gamma knife. LINAC delivers a focused beam of x-ray radiation from a port that arcs part way around the patient’s head. Linear accelerators are commonly used to provide fractionated radiation for lesions outside the CNS. They are found in most radiation oncology departments. After upgrades to the software and collimators, SRS can be performed with these existing units. The gamma knife delivers 201

1	outside the CNS. They are found in most radiation oncology departments. After upgrades to the software and collimators, SRS can be performed with these existing units. The gamma knife delivers 201 focused beams of gamma radiation from cobalt sources through a specially designed colander-like helmet. Gamma knife units are used only for intracranial disease and cost up to $5 million; thus, they are most appropriate in high patient–volume centers. There is ongoing debate in the literature 9Brunicardi_Ch42_p1827-p1878.indd 187101/03/19 7:17 PM 1872SPECIFIC CONSIDERATIONSPART IIregarding the two technologies.99-101 Both continue to evolve, allowing more precise and complex isodose conformation to complex lesions. Most lesions can be treated equally well with either technology. Lesions abutting the medulla or the spinal cord should not be treated with SRS because these structures do not tolerate the radiation dose delivered to structures within mil-limeters of the target. Also,

1	abutting the medulla or the spinal cord should not be treated with SRS because these structures do not tolerate the radiation dose delivered to structures within mil-limeters of the target. Also, medullary or spinal cord compres-sion can result from swelling of the lesion after the radiosurgery dose, resulting in devastating neurologic deficit.Proton beam is an evolving SRS technology that may play a specialized role in treatment of lesions where posttarget exit-ing radiation limits photon-based therapies.102 For example, the physical properties of photons cause destruction upon entry and exit from tissue, which can be particularly harmful to skull-base or clival lesions such as chordoma, in which the exiting pathway travels through the brain stem. Proton beam therapy uses accel-erated protons, which dissipate energy upon impact and do not cause additional exiting damage. Currently, there are very few centers using this technology.CyberKnife is another radiosurgery system that has

1	protons, which dissipate energy upon impact and do not cause additional exiting damage. Currently, there are very few centers using this technology.CyberKnife is another radiosurgery system that has neu-rosurgical application. It is a frameless, robotic, LINAC-based system that allows for targeting of spinal neoplasms with higher resolution than conventional external beam radiotherapy.103 Using imaging tracking in real time, the CyberKnife is able to adjust to breathing artifact and patient movement. The applica-tion of this technology is rapidly growing.Arteriovenous MalformationsSRS has been found to be an effective stand-alone therapy for AVMs up to 3 cm in diameter. SRS is best for lesions that are dif-ficult to access surgically due to high likelihood of postoperative neurologic deficit. However, SRS is not effective for lesions >3 cm. Effective obliteration and elimination of the risk of hemorrhage takes 2 to 3 years. Overall, there is an approximately 2% annual incidence of AVM

1	However, SRS is not effective for lesions >3 cm. Effective obliteration and elimination of the risk of hemorrhage takes 2 to 3 years. Overall, there is an approximately 2% annual incidence of AVM hemorrhage,104 although one study found a 50% decrease in hemorrhage rate during the latency period before angiographic obliteration.105 Nonetheless, surgical excision remains the preferred therapeutic modality, while SRS is reserved for cases deemed very high risk for surgery due to location or patient factors.106 Some patients with large AVMs who undergo surgery will have unresectable residual lesions. In these patients, SRS may be used as an effective adjunctive therapy.Vestibular SchwannomasSRS has been introduced as a therapeutic alternative to micro-surgical resection for vestibular schwannomas up to 2.5 cm in maximum diameter. SRS provides high rates of tumor growth arrest and possible reduction in size with low rates of facial nerve palsy. Patients with functional ipsilateral

1	schwannomas up to 2.5 cm in maximum diameter. SRS provides high rates of tumor growth arrest and possible reduction in size with low rates of facial nerve palsy. Patients with functional ipsilateral preprocedure hearing may be more likely to retain functional hearing postpro-cedure than with microsurgery. The limitations of SRS include inability to treat tumors >2.5 cm, the possibility of radiation-induced malignant transformation of these benign tumors, and lack of long-term follow-up. SRS centers are accumulating experience with these tumors and accumulating data on long-term results.107,108 The indications for microsurgery and SRS will continue to evolve. Either approach should be undertaken at a high-volume center, as studies show the patient outcomes improve with increased surgeon experience.109Intracranial MetastasesPatients with solitary or multiple intracranial metastases may be treated primarily with SRS.110 Patients have improved survival after SRS compared to no treatment

1	MetastasesPatients with solitary or multiple intracranial metastases may be treated primarily with SRS.110 Patients have improved survival after SRS compared to no treatment or WBRT, and similar sur-vival to patients undergoing total surgical resection. Patients with lesions >3 cm in diameter or evidence of ICH should undergo surgical decompression rather than SRS. Some studies show improved survival with up to seven intracranial masses. Patients with multiple intracranial masses have almost zero long-term survival, and most will die of their intracranial dis-ease. Patients with intracranial metastases live 3 to 6 months on average with medical care and WBRT. This can be extended to 9 to 16 months with SRS or surgery, depending on tumor type, age, and patient condition.111CONGENITAL AND DEVELOPMENTAL ANOMALIESDysraphismDysraphism describes defects of fusion of the neural tube involving the neural tube itself, or overlying bone or skin. Dys-raphism may occur in the spine or head.

1	AND DEVELOPMENTAL ANOMALIESDysraphismDysraphism describes defects of fusion of the neural tube involving the neural tube itself, or overlying bone or skin. Dys-raphism may occur in the spine or head. Neural tube defects are among the most common congenital abnormalities. Prenatal vitamins, especially folic acid, reduce the incidence of neural tube defects.Spina Bifida OccultaSpina bifida occulta is congenital absence of posterior vertebral elements. The spinous process is always missing, the laminae may be missing to various degrees, but the underlying neural tissues are not involved. Spina bifida occulta is found in 25% of the general population, and it is asymptomatic unless associated with other developmental abnormalities.Spina Bifida With MyelomeningoceleSpina bifida with myelomeningocele describes the congenital absence of posterior vertebral elements with protrusion of the meninges through the defect, with underlying neural structural abnormalities. Common findings include

1	describes the congenital absence of posterior vertebral elements with protrusion of the meninges through the defect, with underlying neural structural abnormalities. Common findings include weakness and atrophy of the lower extremities, gait disturbance, urinary incontinence, constipation, and deformities of the foot. Myelomeningoceles arising from the high lumbar cord usually cause total paraly-sis and incontinence, while those arising from the sacral cord may have only clawing of the foot and partial urinary function loss. Myelomeningocele patients often have hydrocephalus and a Chiari II malformation, an abnormal downward herniation of the cerebellum and brain stem through the foramen magnum. Patients with abnormal protrusion of meninges through the bony defect without abnormalities of the underlying neural tissue have a meningocele. Most of these patients are neurologically normal.EncephaloceleHerniation of brain encased in meninges through the skull that forms an intracranial

1	of the underlying neural tissue have a meningocele. Most of these patients are neurologically normal.EncephaloceleHerniation of brain encased in meninges through the skull that forms an intracranial mass is referred to as encephalocele. Herniation of meninges without brain tissue is referred to as a meningocele. Most occur over the convexity of the skull. More rarely, the tissue protrudes through the skull base into the sinuses. Treatment involves excision of the herniated tissue and closure of the defect. Most patients with encephaloceles and meningoceles have impaired cognitive development. Patients with greater amounts of herniated neural tissue tend to have more severe cognitive deficits.CraniosynostosisCraniosynostosis is the abnormal early fusion of a cranial suture line with resultant restriction of skull growth in the affected area Brunicardi_Ch42_p1827-p1878.indd 187201/03/19 7:17 PM 1873NEUROSURGERYCHAPTER 42Figure 42-34. A. Axial head computed tomography scan revealing

1	restriction of skull growth in the affected area Brunicardi_Ch42_p1827-p1878.indd 187201/03/19 7:17 PM 1873NEUROSURGERYCHAPTER 42Figure 42-34. A. Axial head computed tomography scan revealing dilated ventricular system. Note dilated atria of the lateral ventricles (arrowheads) and rounded third ventricle (arrow). The large size of the ventricles and lack of transependymal flow indicate a chronic process (contrast to Fig. 42-2). The patient had normal-pressure hydrocephalus and had improved ambulation after placement of a ventriculoperitoneal shunt. B. Higher cut from same scan showing ventricular catheter in place in the frontal horn of the right lateral ventricle.ABand compensatory bulging at the other sutures. Skull growth occurs at the cranial sutures for the first 2 years of life, at the end of which the skull has achieved >90% of its eventual adult size. Fusion of the sagittal suture, or sagittal synostosis, results in a boat-shaped head, known as scaphocephaly. Unilateral

1	at the end of which the skull has achieved >90% of its eventual adult size. Fusion of the sagittal suture, or sagittal synostosis, results in a boat-shaped head, known as scaphocephaly. Unilateral coronal synostosis results in ipsilateral forehead flattening and outward deviation of the orbit, known as plagiocephaly. The contralat-eral normal forehead appears to bulge by comparison. Bilat-eral coronal synostosis results in a broad, flattened forehead, known as brachycephaly, and is often associated with maxil-lary hypoplasia and proptosis. Unilateral or bilateral lambdoid synostosis results in flattening of the occiput. Occipital flat-tening can result from abnormal suture fusion (synostosis), or from physical remolding of the skull caused by always placing the baby in the supine position for sleep (known as positional plagiocephaly). Placing the baby in the prone position or tilted onto the contralateral side may restore near-normal skull shape in most cases of lambdoid synostosis,

1	for sleep (known as positional plagiocephaly). Placing the baby in the prone position or tilted onto the contralateral side may restore near-normal skull shape in most cases of lambdoid synostosis, avoiding surgery. Treat-ment for synostoses in general is surgical, involving resection of the fused suture, or more complex reconstructive techniques for severe or refractory cases.HydrocephalusExcess CSF in the brain that results in enlarged ventricles is known as hydrocephalus. CSF flows from the ventricles to the subarachnoid space and is then absorbed into the venous blood through the arachnoid granulations. Hydrocephalus may be classified as communicating or obstructive (outlined in the next two sections), and congenital or acquired. Congenital lesions associated with or causing hydrocephalus include stenosis of the cerebral aqueduct, Chiari malformation, myelomeningo-cele, and intrauterine infection. Acquired hydrocephalus may result from occlusion of arachnoid granulations by

1	include stenosis of the cerebral aqueduct, Chiari malformation, myelomeningo-cele, and intrauterine infection. Acquired hydrocephalus may result from occlusion of arachnoid granulations by meningitis, germinal matrix hemorrhage, or SAH. CSF pathways may be occluded by adjacent tumors (Fig. 42-34).Communicating Hydrocephalus. Obstruction at the level of the arachnoid granulations constitutes communicating hydro-cephalus. This usually causes dilation of the lateral, third, and fourth ventricles equally. The most common causes in adults are meningitis and SAH. Hydrocephalus may be transient after SAH, with reestablishment of normal CSF absorption after the protein content of the CSF returns to normal and the granula-tions reopen.Obstructive Hydrocephalus. Obstruction of CSF pathways is known as obstructive hydrocephalus. Ventricles proximal to the obstruction dilate, while those distal to the obstruction remain normal in size. Typical patterns include dilation of the lateral ventricles

1	as obstructive hydrocephalus. Ventricles proximal to the obstruction dilate, while those distal to the obstruction remain normal in size. Typical patterns include dilation of the lateral ventricles due to a colloid cyst occluding the foramen of Monro, dilation of the lateral and third ventricles due to a tectal (midbrain) glioma or pineal region tumor occluding the cerebral aqueduct, or dilation of the lateral and third ventricles with obliteration of the fourth ventricle by an intraventricular tumor of the fourth ventricle. Obstructive hydrocephalus may present precipitously and require urgent shunting to prevent herniation.Chiari I MalformationChiari I malformation is the caudal displacement of the cer-ebellar tonsils below the foramen magnum. It may be seen as an incidental finding on MRI scans in asymptomatic patients. Symptomatic patients usually present with headache, neck pain, or symptoms of myelopathy, including numbness or weakness in the extremities. A syrinx may be

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1	187601/03/19 7:17 PM 1877NEUROSURGERYCHAPTER 42 104. Karlsson B, Lax I, Soderman M. Risk for hemorrhage during the 2-year latency period following gamma knife radiosurgery for arteriovenous malformations. Int J Radiat Oncol Biol Phys. 2001;49:1045-1051. 105. Maruyama K, Kawahara N, Shin M, et al. The risk of hemor-rhage after radiosurgery for cerebral arteriovenous malforma-tions. N Engl J Med. 2005;352:146-153. 106. Pan DH, Guo WY, Chung WY, et al. Gamma knife radiosur-gery as a single treatment modality for large cerebral arterio-venous malformations. J Neurosurg. 2000;93:113-119. 107. Regis J, Pellet W, Delsanti C, et al. Functional outcome after gamma knife surgery or microsurgery for vestibular schwan-nomas. J Neurosurg. 2002;97:1091-1100. 108. Shin M, Ueki K, Kurita H, et al. Malignant transformation of a vestibular schwannoma after gamma knife radiosurgery. Lancet. 2002;360:309-310. 109. Elsmore AJ, Mendoza ND. The operative learning curve for vestibular schwannoma excision

1	transformation of a vestibular schwannoma after gamma knife radiosurgery. Lancet. 2002;360:309-310. 109. Elsmore AJ, Mendoza ND. The operative learning curve for vestibular schwannoma excision via the retrosigmoid approach. Br J Neurosurg. 2002;16:448-455. 110. Gerosa M, Nicolato A, Foroni R, et al. Gamma knife radio-surgery for brain metastases: a primary therapeutic option. J Neurosurg. 2002;97:515-524. 111. Pollock BE, Brown PD, Foote RL, et al. Properly selected patients with multiple brain metastases may benefit from aggressive treatment of their intracranial disease. J Neurooncol. 2003;61:73-80.Brunicardi_Ch42_p1827-p1878.indd 187701/03/19 7:17 PM

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1	Orthopedic SurgeryNabil A. Ebraheim, Bert J. Thomas, Freddie H. Fu, Bart Muller, Dharmesh Vyas, Matt Niesen, Jonathan Pribaz, and Klaus Draenert 43chapterIntroduction 1880Orthopedic Trauma 1881Introduction / 1881Open Fractures / 1881Compartment Syndrome / 1883Treatment of Fractures and Dislocations 1883Clavicle Fractures / 1883Scapula Fractures / 1883Shoulder Dislocations / 1884Proximal Humerus Fractures / 1884Humeral Shaft Fractures / 1884Distal Humerus Fractures / 1885Elbow Dislocations / 1885Radial Head Fractures / 1886Olecranon Fractures / 1886Forearm Fractures / 1886Distal Radius Fractures / 1886Scaphoid Fractures / 1887Pelvic Fractures / 1887Acetabular Fractures / 1888Hip Dislocations / 1888Hip Fractures / 1888Femoral Shaft Fractures / 1890Distal Femur Fractures / 1890Knee Dislocations / 1890Patella/Extensor Mechanism Injuries / 1891Tibial Plateau Fractures / 1891Tibial Shaft Fractures / 1891Tibial Plafond (Pilon) Fractures / 1892Ankle and Subtalar Dislocations / 1892Ankle

1	/ 1890Patella/Extensor Mechanism Injuries / 1891Tibial Plateau Fractures / 1891Tibial Shaft Fractures / 1891Tibial Plafond (Pilon) Fractures / 1892Ankle and Subtalar Dislocations / 1892Ankle Fractures / 1893Maisonneuve Fractures / 1893Calcaneal Fractures / 1893Talus Fractures / 1894Foot Fractures / 1894Sports Medicine 1895Introduction / 1895Shoulder 1895Rotator Cuff / 1895Shoulder Instability / 1896Posterior Dislocation of the Shoulder / 1896Superior Labrum and Biceps Tendon / 1896Impingement Syndromes / 1896The Acromioclavicular Joint / 1897Knee 1897Menisci / 1897Collateral Ligaments / 1898Cruciate Ligaments / 1898Posterolateral Corner / 1899Hip 1899Femoroacetabular Impingement / 1899Spine 1900Spinal Trauma / 1900Occipital Cervical Dislocation / 1900Fractures of C1 (Jefferson Fracture) / 1900Fractures of C2 (Odontoid Fracture) / 1900Hangman’s Fractures of C2 / 1901Compression Fracture of the Cervical Spine / 1901Burst Fractures of the Cervical Spine / 1901Unilateral and

1	/ 1900Fractures of C2 (Odontoid Fracture) / 1900Hangman’s Fractures of C2 / 1901Compression Fracture of the Cervical Spine / 1901Burst Fractures of the Cervical Spine / 1901Unilateral and Bilateral Facet Dislocation / 1901Clay-Shoveler’s Injury / 1902Fractures of the Thoracic and Lumbar Spine 1902Thoracic Lumbar Spine Injury / 1902Compression Fracture / 1902Burst Fracture / 1902Seatbelt Injuries (Flexion Distraction Injuries) / 1902Fracture Dislocations of the Spine / 1903Disc Herniation / 1903Cauda Equina Syndrome / 1903Spinal Stenosis / 1903Back Pain and Degenerative Disc Disease / 1904Scoliosis / 1904Idiopathic Scoliosis / 1904Neuromuscular Scoliosis / 1904Joint Reconstruction 1904Introduction to Arthritis / 1904Examination of the Patient / 1904Nonoperative Management and Prevention of Arthritis / 1905Injections / 1905Surgical Management of Arthritis / 1906Computer Navigation, Robotics, and Joint Arthroplasty / 1908Fixation Options in Joint Arthroplasty / 1910Complications

1	of Arthritis / 1905Injections / 1905Surgical Management of Arthritis / 1906Computer Navigation, Robotics, and Joint Arthroplasty / 1908Fixation Options in Joint Arthroplasty / 1910Complications in Joint Arthroplasty / 1910Orthopedic Pathology and Oncology 1910Diagnosis of Malignant Bone Tumors / 1910Osteosarcoma 1911Intramedullary Osteosarcoma / 1911Parosteal Osteosarcoma / 1912Periosteal Osteosarcoma / 1912Paget’s Sarcoma / 1912Radiation-Induced Sarcoma / 1912Ewing’s Sarcoma 1912Cartilage-Forming Tumors 1912Chondrosarcomas / 1912Fibrous Lesions of Bone 1912Desmoplastic Fibroma / 1912Malignant Fibrous Histiocytoma of Bone / 1913Malignant Vascular Tumors / 1913Miscellaneous Tumors 1913Giant Cell Tumor of Bone / 1913Adamantinoma and Osteofibrous Dysplasia / 1913Primary Lymphoma of Bone / 1914Chordoma / 1914Multiple Myeloma / 1914Metastatic Bone Tumors 1914Pediatric Orthopedics 1915Birth Injuries / 1915Skeletal Growth / 1915Pediatric Fractures / 1916Classification of Growth Plate

1	/ 1914Chordoma / 1914Multiple Myeloma / 1914Metastatic Bone Tumors 1914Pediatric Orthopedics 1915Birth Injuries / 1915Skeletal Growth / 1915Pediatric Fractures / 1916Classification of Growth Plate Injuries / 1916Diaphyseal Injuries in a Pediatric Patient / 1916Fractures of the Pediatric Hip / 1916Fractures of the Femoral Shaft / 1916Pediatric Ankle Fractures / 1917Pediatric Elbow Fractures / 1917Brunicardi_Ch43_p1879-p1924.indd 187922/02/19 10:40 AM 1880Key Points1 The main principle of internal fixation for fracture care (most commonly intramedullary nails or plate and screw fixation) is to create a stable construct that will allow the fracture to heal in proper length, alignment, and rotation.2 In open fractures, early administration of intravenous antibiotics is important to avoid infection. An external fixator is used when the wound is grossly contaminated, and definitive treatment of the wound is delayed until the wound is sufficiently clean. Early wound coverage is

1	to avoid infection. An external fixator is used when the wound is grossly contaminated, and definitive treatment of the wound is delayed until the wound is sufficiently clean. Early wound coverage is important.3 Early diagnosis and treatment of compartment syndrome is important in order to prevent irreversible damage to the muscles and the nerves, which is time sensitive. Emergency fasciotomy by releasing the tight fascia is a limb-saving procedure.4 Fractures of the scapula are typically the result of a high-energy trauma with a high incidence of associated injuries that typically involve the ribs and the lungs.5 The shoulder is one of the most commonly dislocated joints, and most dislocations are anterior. Posterior dislo-cations may be missed and are typically associated with seizures or electric shock. Anterior-posterior and axillary views of the shoulder are necessary for the diagnosis.6 Isolated humeral shaft fractures are usually treated conser-vatively. The radial nerve

1	or electric shock. Anterior-posterior and axillary views of the shoulder are necessary for the diagnosis.6 Isolated humeral shaft fractures are usually treated conser-vatively. The radial nerve spirals around the humeral shaft and is at risk for injury; therefore, a careful neurovascular exam is important. The patient should be checked for wrist drop.7 Hemorrhage from pelvic trauma can be life-threatening. An important first line of treatment in the emergency department is resuscitation of the patient with fluids, including blood, and the application of a pelvic binder or sheet that is wrapped tightly around the pelvis to control bleeding.8 In spinal injury, spinal stability must be assessed, and the patient should be immobilized until there is further under-standing of the injury. A computed tomography scan is more reliable in assessing spine injury than plain radiographs.9 Spinal cord injuries can be complete or incomplete, and every attempt should be made to avoid further injury.

1	tomography scan is more reliable in assessing spine injury than plain radiographs.9 Spinal cord injuries can be complete or incomplete, and every attempt should be made to avoid further injury. Patients should be triaged to trauma centers since trauma center care is associated with reduced paralysis and improved outcome.10 According to the Centers for Disease Control and Prevention and the National Health Interview Survey, approximately 55 million adults have been diagnosed with some form of arthritis. This number is projected to grow substantially in the future.11 A combination of nonsteroidal anti-inflammatory medica-tions, physiotherapy, and weight loss with the help of a dietary consultation, and physical therapy are typically the first line of treatment for knee osteoarthritis. Weight loss of as little as 11 lbs (5 kg) has been shown to decrease the risk of developing knee osteoarthritis in women by 50%.12 Minimally invasive surgical techniques improve cosmesis and allow for

1	loss of as little as 11 lbs (5 kg) has been shown to decrease the risk of developing knee osteoarthritis in women by 50%.12 Minimally invasive surgical techniques improve cosmesis and allow for early rehabilitation. However, they may be associated with decreased visualization intraoperatively, associated risks of component malposition, intraoperative fracture, and nerve or vascular injury.Developmental Disease 1917Developmental Dysplasia of the Hip / 1917Treatment of DDH / 1918Legg-Calvé-Perthes Disease / 1918Slipped Capital Femoral Epiphysis / 1918Lower Extremity Rotational Abnormalities / 1918Congenital Talipes Equinovarus (Clubfoot) / 1918Osgood-Schlatter Disease / 1918INTRODUCTIONEvery physician should be familiar with orthopedics and ortho-pedic surgery. Anyone who cares for patients in an outpatient or emergency room setting will find that the majority of presenting complaints involve the musculoskeletal system. A basic under-standing of the principles of care for

1	for patients in an outpatient or emergency room setting will find that the majority of presenting complaints involve the musculoskeletal system. A basic under-standing of the principles of care for musculoskeletal conditions is essential for the health care profession.For physicians, the field of orthopedics offers an array of subspecialties with such diversity that it seems that “there is something for everyone.” Trauma specialists have the satis-faction of physically putting complex fractures back together. Sports medicine offers remarkably rapid recovery in athletes who have suffered fibrocartilage and ligament tears with ever-improving arthroscopic techniques and instrumentation. Spine surgeons see remarkable results from their minimally inva-sive microscopic techniques, while also managing massive deformities with new instrumentation and open surgery. Joint reconstruction is one of our most exciting subspecialties, work-ing with orthopedic bioengineers to develop improved

1	also managing massive deformities with new instrumentation and open surgery. Joint reconstruction is one of our most exciting subspecialties, work-ing with orthopedic bioengineers to develop improved designs, biomaterials, and minimally invasive surgical approaches for faster return to function for patients debilitated by arthritis and injury. Musculoskeletal oncology offers the intellectual chal-lenge of arriving at appropriate differential diagnoses as well as the technical challenge of limb salvage and major reconstruc-tive surgery. Pediatric orthopedics is an especially challenging and rewarding subspecialty because of the remarkable ability of children to heal their fractures quickly and remodel their bones. The incredible array of congenital and developmental disorders makes pediatrics a uniquely intellectually challenging field as well. The authors hope that our readers will share our enthusi-asm for orthopedic surgery and all of its subspecialties: trauma, sports, spine, joint

1	a uniquely intellectually challenging field as well. The authors hope that our readers will share our enthusi-asm for orthopedic surgery and all of its subspecialties: trauma, sports, spine, joint replacement, musculoskeletal oncology, foot and ankle, hand, and pediatric orthopedics.Brunicardi_Ch43_p1879-p1924.indd 188022/02/19 10:40 AM 1881ORTHOPEDIC SURGERYCHAPTER 43ORTHOPEDIC TRAUMAIntroductionMusculoskeletal injuries resulting from trauma include frac-tures of bones, damage to joints, and injuries to soft tissues. Long bone fractures can be described as transverse, oblique, spiral, segmental, or comminuted (Fig. 43-1). The goals of treat-ing musculoskeletal injuries are to restore the normal anatomy, stabilize fractures to allow early mobility and minimize com-plications related to multiple system trauma, and to repair or reconstruct these injuries to restore function.Fractures frequently result from high-energy trauma as well as from falls onto an extremity (Fig. 43-2). The

1	to multiple system trauma, and to repair or reconstruct these injuries to restore function.Fractures frequently result from high-energy trauma as well as from falls onto an extremity (Fig. 43-2). The majority of fractures can heal well with immobilization, which stabilizes the fracture while new bone forms at the fracture site. Methods of immobilization can vary and depend on the fracture being treated. The most common tool used in orthopedics to treat fractures is immobilization with a splint, cast, or braces, and their proper application is important to successfully treat the injury without causing additional problems. A successful splint contains adequate padding on the underlying skin, particularly over bony prominences, to prevent pressure or burns that can be caused by plaster. Splints, which are not circumferential, are preferred for acute injuries because they allow room for swell-ing that inevitably occurs after a fracture. The splint may later be changed to a cast as the

1	which are not circumferential, are preferred for acute injuries because they allow room for swell-ing that inevitably occurs after a fracture. The splint may later be changed to a cast as the swelling subsides.Fractures that are displaced or angulated require closed reduction to properly realign the bone. This is done using anal-gesia, local or general anesthesia, and often muscle relaxation. Reduction is performed with axial traction and reversal of the mechanism of injury in order to restore length, rotation, and angu-lation. A splint is then applied and can be gently molded to help hold the reduction in place. It is important to obtain X-rays after a closed reduction to verify acceptable alignment of the fracture, and to perform a neurovascular exam to ensure the splint is not too tight or that manipulation did not change the neurovascular status. Careful monitoring with timely clinical and radiological examination is necessary in the outpatient setting.For certain fractures,

1	tight or that manipulation did not change the neurovascular status. Careful monitoring with timely clinical and radiological examination is necessary in the outpatient setting.For certain fractures, splint or cast immobilization is inad-equate, and in these instances internal fixation or external fixa-tion is used. A variety of implants can be used to stabilize the fracture such as screws, plates, rods, and external fixators. The main principle of orthopedic implants for fracture care is to cre-ate a stable construct that will allow the fracture to heal in proper length, alignment, and rotation. Screws can be placed across a fracture to create compression at the fracture site, which promotes healing. Plates can be placed on the cortex of bones and held with screws, which creates a long area of fixa-tion to stabilize the fracture. Intramedullary rods are commonly used for long bone fractures, such as the femur and tibia (Fig. 43-3A,B). Usually, the fracture is reamed prior to the

1	area of fixa-tion to stabilize the fracture. Intramedullary rods are commonly used for long bone fractures, such as the femur and tibia (Fig. 43-3A,B). Usually, the fracture is reamed prior to the insertion of the rod into the intramedullary canal. Screws can then be placed across the cortices of the bone through holes in the rod proximal and distal to the fracture to create a locked construct that further stabilizes the rod. When the fracture is locked proximally and distally, this is called static locking. In situations where patients are severely injured and cannot safely undergo more invasive open surgery, damage control orthope-dics is done by utilizing an external fixator. External fixators are also used when the soft tissues are too swollen or injured to allow for surgical incisions to be safely made. The wrinkle test is helpful in guiding the most suitable time for definitive sur-gery. An external fixation device can be used to temporarily immobilize the fracture, especially

1	to be safely made. The wrinkle test is helpful in guiding the most suitable time for definitive sur-gery. An external fixation device can be used to temporarily immobilize the fracture, especially if the fracture is open and contaminated. External fixators involve pins placed into bone proximal and distal to the fracture (through healthy tissues), which are then connected by strong rods on the outside of the extremity, creating a stable construct.Open FracturesAn open fracture occurs when the bone breaks through the skin. These typically result from high-energy injuries and are often associated with significant damage to the surrounding soft tissues and contamination of the wound. These injuries are classified into three types according to the Gustillo-Anderson Classification.• Type I injuries are low energy and wounds are usually less than 1 cm.• Type II injuries have a wound length of 2 to 10 cm with mod-erate soft tissue damage and wound contamination.1ABCDEFFigure 43-1. Types of

1	are low energy and wounds are usually less than 1 cm.• Type II injuries have a wound length of 2 to 10 cm with mod-erate soft tissue damage and wound contamination.1ABCDEFFigure 43-1. Types of fractures. A. Normal bone. B. Transverse. C. Oblique. D. Spiral. E. Segmental. F. Comminuted.Figure 43-2. Transverse tibia fracture and segmental fibula fracture.Brunicardi_Ch43_p1879-p1924.indd 188122/02/19 10:40 AM 1882SPECIFIC CONSIDERATIONSPART II• Type III injuries are high-energy wounds usually greater than 10 cm in length with extensive muscle devitalization. The wound is highly contaminated with extensive soft tissue damage.These injuries require immediate administration of antibi-otics and irrigation and debridement of the wound. The goal of the treatment is to achieve fracture healing and to prevent wound infections and osteomyelitis. They are frequently associated with injuries to surrounding vessels and nerves, which must be addressed as well. When the wound is contami-nated, an

1	and to prevent wound infections and osteomyelitis. They are frequently associated with injuries to surrounding vessels and nerves, which must be addressed as well. When the wound is contami-nated, an external fixator is initially used (Fig. 43-4A,B). Often, definitive treatment of the fracture is delayed until the wound is 2ABFigure 43-3. A. Transverse femur fracture. B. Intramedullary rod stabilizes femur fracture.ABFigure 43-4. A. Gustillo-Anderson fracture type III open fracture. B. Image of external fixator of the tibia.Brunicardi_Ch43_p1879-p1924.indd 188222/02/19 10:40 AM 1883ORTHOPEDIC SURGERYCHAPTER 43sufficiently cleaned and healthy soft tissue is available to cover the fracture. Early coverage of the wound is important to avoid infection. Usually a large wound in the proximal or middle third of the tibia can be covered using local muscle flaps, while the distal third of the tibia will require a free flap. In general, an increase in Gustillo grade is associated with an

1	or middle third of the tibia can be covered using local muscle flaps, while the distal third of the tibia will require a free flap. In general, an increase in Gustillo grade is associated with an increase in infec-tion risk.Compartment SyndromeCompartment syndrome is an orthopedic emergency caused by significant swelling within a compartment of an injured extrem-ity that jeopardizes blood flow and microcirculation to the limb. Increased pressure within the compartment compromises perfu-sion to muscles and nerves and can cause ischemia or necrosis. Patients complain of pain that is greater than expected for the injury or surgery. There may be an increase in analgesic require-ments. Early high index of suspicion is necessary for timely diagnosis and treatment of compartment syndrome. The usual clinical findings are pain, swelling, and pain with passive stretch of the compartment muscles. Numbness, paralysis, and the absence of a pulse are late findings. While the diagnosis is usu-ally

1	clinical findings are pain, swelling, and pain with passive stretch of the compartment muscles. Numbness, paralysis, and the absence of a pulse are late findings. While the diagnosis is usu-ally based on clinical exam, compartment pressures can be mea-sured with needles placed into the compartment, which is necessary in unconscious patients and those who will not coop-erate with the exam. Compartment pressure within 30 mmHg of the diastolic pressure is diagnostic of compartment syndrome. When compartment syndrome is suspected, emergent fasciot-omy (Fig. 43-5A,B) must be performed in which the overlying tight fascia is released through long incisions. Fasciotomy must be done as soon as possible to prevent damage to muscles and nerves that will result in irreversible necrosis and Volkmann’s ischemic contractures with severe loss of function.TREATMENT OF FRACTURES AND DISLOCATIONSClavicle FracturesFractures of the clavicle are one of the most common fractures in orthopedics. They

1	ischemic contractures with severe loss of function.TREATMENT OF FRACTURES AND DISLOCATIONSClavicle FracturesFractures of the clavicle are one of the most common fractures in orthopedics. They typically occur following a fall onto the shoulder. The majority of clavicle fractures occur in the middle third of the clavicle. Since the clavicle is subcutaneous, the frac-ture is often evident on inspection. Most clavicle fractures can be treated nonoperatively with a sling, early range of motion exercises, and gradual return to normal activities. Fractures that are significantly displaced and shortened or that penetrate or tent the skin are treated with open reduction and internal fixa-tion, typically with plate and screw fixation.Distal clavicle fractures are less common and may occur with coracoclavicular ligament ruptures. These injuries can be more troublesome and are at risk for nonunion if the bone ends are not in contact. If there is displacement of the fracture and the fracture is

1	ligament ruptures. These injuries can be more troublesome and are at risk for nonunion if the bone ends are not in contact. If there is displacement of the fracture and the fracture is proximal to the coracoclavicular ligament, surgical management is often recommended.Acromioclavicular (AC) joint injuries occur from either a fall directly onto the shoulder or onto an outstretched hand and can result in tears of the acromioclavicular and cora-coclavicular ligaments. A step-off, or separation, of the AC joint may be apparent on radiographs. The majority of these injuries can be treated with a sling and gentle range of motion. Although controversial, injuries resulting in severe displacement of the clavicle at the AC joint usually require open reduction and surgical repair, especially in athletes and manual workers.The sternoclavicular (SC) joint is the only articulation between the upper extremity and the axial skeleton. Injuries to this joint are rare. Anterior dislocations occur more

1	and manual workers.The sternoclavicular (SC) joint is the only articulation between the upper extremity and the axial skeleton. Injuries to this joint are rare. Anterior dislocations occur more frequently, and although closed reduction can be attempted, recurrence of the dislocation is typical. Patients are given a sling and the out-come is usually good, despite the visible bump and swelling. Posterior SC joint dislocations are rare and not grossly visible and can be easily missed. They can be dangerous injuries, result-ing in pulmonary or neurovascular compromise. Therefore, closed or open reduction under general anesthesia is recom-mended with a cardiac surgeon back-up.Scapula FracturesFractures of the scapula often result from significant high-energy trauma (Fig. 43-6) with about 80% associated injuries, 3Compartmentsyndrome ofthe forearmABFigure 43-5. A. Image showing compartment syndrome of the forearm. B. Fasciotomy of the leg. Notice gaping of the wound and bulging of the

1	80% associated injuries, 3Compartmentsyndrome ofthe forearmABFigure 43-5. A. Image showing compartment syndrome of the forearm. B. Fasciotomy of the leg. Notice gaping of the wound and bulging of the muscles.Brunicardi_Ch43_p1879-p1924.indd 188322/02/19 10:40 AM 1884SPECIFIC CONSIDERATIONSPART IImost commonly involving the head, ribs, and lungs. Pulmonary injuries occur in over one-third of patients. Most scapula fractures are treated nonoperatively with a sling and early range of motion. Surgery is performed when there is involve-ment of the glenoid with a major articular step-off or if there is a glenoid rim fracture with subluxation of the joint.Shoulder DislocationsThe shoulder is the most commonly dislocated large joint. Most dislocations are anterior. They are often associated with injuries to the anterior inferior glenoid labrum (Bankart lesion), impac-tion fractures of the humeral head (Hill-Sachs lesion) (Fig. 43-7), and rotator cuff tears in the elderly. The axillary

1	with injuries to the anterior inferior glenoid labrum (Bankart lesion), impac-tion fractures of the humeral head (Hill-Sachs lesion) (Fig. 43-7), and rotator cuff tears in the elderly. The axillary nerve is at risk of being injured in shoulder dislocation. If the patient is unable to raise the arm after reduction of shoulder dislocation, then it is most likely due to a rotator cuff tear in the elderly and axillary nerve injury in the young.There is a high recurrence rate that correlates with the age of the patient at the time of dislocation. There is a 90% redislocation rate if the patient is younger than 20 years of age. Posterior dislocations are associated with seizures or electric shock. Adequate radiographs are required to diagnose a shoul-der dislocation, with the axillary view being the most important. The patient’s shoulder is usually locked in internal rotation with limitation of external rotation and axillary view will show the posterior dislocation (Fig. 43-8A,B). If proper

1	the most important. The patient’s shoulder is usually locked in internal rotation with limitation of external rotation and axillary view will show the posterior dislocation (Fig. 43-8A,B). If proper X-rays are not performed, then dislocations can be missed and can result in significant disability to the patient. A computed tomography (CT) scan should be performed if an axillary view is unable to be obtained. In general, dislocation of the shoulders can be managed with closed reduction followed by a short period of sling immobilization.Proximal Humerus FracturesProximal humerus fractures occur most frequently in elderly female patients following a fall onto the shoulder, though they can also occur following high-energy trauma in young patients. They have historically been classified by the number of fracture fragments using Neer’s classification (Fig. 43-9), which divides the proximal humerus into four parts: the humeral head, greater tuberosities, lesser tuberosities, and the humeral

1	number of fracture fragments using Neer’s classification (Fig. 43-9), which divides the proximal humerus into four parts: the humeral head, greater tuberosities, lesser tuberosities, and the humeral shaft. Treat-ment is determined by the displacement of the fracture frag-ments, the amount of angulation of the fracture, and the amount of comminution (which means multiple fracture fragments). If there is suspicion of an intra-articular fracture, CT scan is often indicated. The majority of proximal humerus fractures are mini-mally displaced and can be treated with sling immobilization, followed by early shoulder motion and pendulum exercises. Physiotherapy should be started within 2 weeks of the injury to prevent stiffness, especially in the elderly. Displaced fractures and fractures involving the humeral head are at increased risk for osteonecrosis, and therefore surgery is often recommended. If there is adequate bone stock and the fracture can be success-fully reduced, open reduction

1	the humeral head are at increased risk for osteonecrosis, and therefore surgery is often recommended. If there is adequate bone stock and the fracture can be success-fully reduced, open reduction internal fixation with plate and screw fixation is the treatment of choice. Older patients with osteoporosis, comminuted fractures, head-splitting fractures, and four-part fractures or fracture dislocations are typically treated with a prosthetic replacement of the humeral head or a hemiarthroplasty. Reverse shoulder arthroplasty is gaining popularity in the elderly as well.Humeral Shaft FracturesThe majority of humeral shaft fractures can heal with nonsurgi-cal management if they are within an acceptable degree of angulation. The radial nerve spirals around the humeral shaft and is at risk for injury; therefore, a careful neurovascular exam is important. If you have a patient with a humeral shaft fracture, check the patient for wrist drop (Fig. 43-10). Most radial nerve injuries are

1	for injury; therefore, a careful neurovascular exam is important. If you have a patient with a humeral shaft fracture, check the patient for wrist drop (Fig. 43-10). Most radial nerve injuries are neurapraxias, or stretching of the nerve, and function typically returns within 3 to 4 months. A spiral fracture of the distal one-third of the humeral shaft is commonly associated with neurapraxia of the radial nerve, and this fracture is called a Holstein-Lewis fracture. Humeral shaft fractures are typically treated with a coaptation splint or functional bracing, which consists of a plastic clamshell brace with Velcro straps. Criteria for acceptable alignment are less than 20° anterior angulation, less than 30° varus/valgus angulation, and less than 3 cm shortening. Radial nerve palsy is not a contraindication to conservative treatment. Close follow-up with serial radiographs 456Figure 43-6. Scapula fracture. Notice the body and the glenoid are involved.Figure 43-7. Hill-Sachs humeral head

1	contraindication to conservative treatment. Close follow-up with serial radiographs 456Figure 43-6. Scapula fracture. Notice the body and the glenoid are involved.Figure 43-7. Hill-Sachs humeral head impaction fracture and Bankart lesion, which is an avulsion of the anterior inferior labrum.Brunicardi_Ch43_p1879-p1924.indd 188422/02/19 10:40 AM 1885ORTHOPEDIC SURGERYCHAPTER 43is important to verify healing of the fracture, and gentle motion exercises are begun within 1 to 2 weeks. Fractures with signifi-cant angulation are most commonly treated with open reduc-tion and plate fixation, with care to protect the radial nerve as it often lies close to the fracture site. Intramedullary nailing can also be performed, though it carries the risk of shoulder pain from the nail insertion. A plate is usually more stable than a nail and allows early weight-bearing through the humerus. Sponta-neous recovery of radial nerve palsy can occur up to 6 months after injury. The patient should have an

1	is usually more stable than a nail and allows early weight-bearing through the humerus. Sponta-neous recovery of radial nerve palsy can occur up to 6 months after injury. The patient should have an EMG to monitor recov-ery of the nerve. In an open fracture of the humeral shaft with radial nerve palsy, the nerve should be explored for the possibil-ity of a significant nerve injury or laceration.Distal Humerus FracturesFractures of the distal humerus result from falls onto the elbow or onto an outstretched arm. Supracondylar fractures occurring above the elbow joint are most common and do not involve the articular surface. Minimally displaced fractures can occasion-ally be treated with a posterior long arm splint, with the elbow typically flexed to 90°. However, fixation is often recommended to allow early range of motion and prevent stiffness. Fractures involving the articular surface are treated with plate fixation, and depending on the fracture pattern they may require more than one

1	to allow early range of motion and prevent stiffness. Fractures involving the articular surface are treated with plate fixation, and depending on the fracture pattern they may require more than one (usually anatomically contoured) plate. As with other intra-articular fractures, the goals of treatment are anatomic reduction of the joint surface with stable fixation, restoration of the anatomic alignment of the joint, and early range of motion. Severely comminuted fractures, especially in the elderly, may be treated with a total elbow replacement. Fractures about the elbow are notorious for developing stiffness and therefore early motion of the elbow is paramount to a successful outcome. Range of motion should be started as soon as the patient can tolerate therapy.Elbow DislocationsDislocations of the elbow are common and typically occur posteriorly after a fall on an outstretched hand. A dislocation results in injury to the joint capsule and rupture of the lateral collateral ligament,

1	of the elbow are common and typically occur posteriorly after a fall on an outstretched hand. A dislocation results in injury to the joint capsule and rupture of the lateral collateral ligament, with possible involvement of the medial collateral ligament, as well as possible fractures of the radial head and coronoid. This combination of injuries is called the “terrible triad,” which is a challenging injury and carries the Figure 43-8. A. Posterior shoulder dislocation showing limitation of external rotation. B. Axillary view sowing posterior dislocation of the shoulder.Figure 43-9. Four-part proximal humeral head fracture.Figure 43-10. Radial nerve palsy due to humeral shaft fracture causing wrist drop.Brunicardi_Ch43_p1879-p1924.indd 188522/02/19 10:40 AM 1886SPECIFIC CONSIDERATIONSPART IIworst prognosis. Simple elbow dislocations should be urgently reduced with the patient under sedation and treated with a short period of immobilization, utilizing a posterior splint. Stiffness

1	prognosis. Simple elbow dislocations should be urgently reduced with the patient under sedation and treated with a short period of immobilization, utilizing a posterior splint. Stiffness of the elbow is a common complication following elbow dislocations and therefore only short-term immobiliza-tion (about 7–10 days) followed by early range of motion is recommended.Dislocations associated with fractures may be treated sur-gically if there is any instability of the elbow joint. The “terrible triad” is an unstable injury comprising of an elbow disloca-tion as well as fractures to the radial head and coronoid, which requires surgery. Surgery includes repair of the torn lateral col-lateral ligament, fixation or replacement of the radial head, and possible fixation of the coronoid, depending on the size of this fracture fragment.Radial Head FracturesMost fractures of the radial head can be treated nonoperatively, simply with a sling for 1 to 2 days followed by motion exercises. Surgery is

1	the size of this fracture fragment.Radial Head FracturesMost fractures of the radial head can be treated nonoperatively, simply with a sling for 1 to 2 days followed by motion exercises. Surgery is recommended if there is a displaced fracture, if the fracture blocks pronation or supination of the forearm, if there is an associated dislocation of the elbow, or if the patient has associated wrist pain (Essex-Lopresti fracture). Surgery can be fixation or replacement. If the fracture can be well reduced, it is fixed with 1 or 2 screws. If the radial head is fractured into multiple pieces, the treatment of choice is a radial head replace-ment with a metallic implant. Simple excision of the radial head can also be performed in low demand patients with an isolated radial head fracture; otherwise, it may lead to instability of the elbow and the wrist over time.Olecranon FracturesOlecranon fractures usually occur following a fall directly onto a flexed elbow (Fig. 43-11). Nondisplaced

1	it may lead to instability of the elbow and the wrist over time.Olecranon FracturesOlecranon fractures usually occur following a fall directly onto a flexed elbow (Fig. 43-11). Nondisplaced fractures are treated with a splint in 45° to 90° of flexion for a short time followed by range of motion exercises to prevent stiffness. Because the triceps inserts on the olecranon, the pull of the muscle often causes active extension of the elbow and displacement of the fracture, and therefore the olecranon fracture should be fixed surgically. Simple transverse fractures can be fixed with a tension band construct, which consists of wire passing through the ulna, distal to the fracture, and wrapped in a figure-of-8 fashion around two or more pins placed proximally into the olecranon, crossing and stabilizing the fracture. This tension band construct creates a compressive force across the articular aspect of the fracture that will promote healing. Fractures that are comminuted or have large

1	stabilizing the fracture. This tension band construct creates a compressive force across the articular aspect of the fracture that will promote healing. Fractures that are comminuted or have large fragments are usually treated with plate and screw fixation. Excision of the olecranon with advancement of the triceps can be done in elderly patients when the fracture involves less than 50% of the joint surface. Because of the subcutaneous location of the olecranon, symp-tomatic hardware is a frequent complication, causing irritation to the patient; it may need to be removed after the fracture has healed. Stiffness of the elbow is another complication seen in a large number of patients.Forearm FracturesForearm fractures are common injuries that result from high-energy trauma or from falls onto an outstretched arm. Both bone forearm fractures generally require surgery with plate and screw fixation. The radius has a bow and rotates around the straight ulna for proper pronation and supination

1	an outstretched arm. Both bone forearm fractures generally require surgery with plate and screw fixation. The radius has a bow and rotates around the straight ulna for proper pronation and supination of the forearm, and therefore this anatomic relationship needs to be restored to maintain function. An isolated fracture of the ulna shaft, or a “nightstick fracture,” occurs from a direct blow to the side of the forearm. These can usually be treated in a cast, splint, or brace. Fractures that are angulated or displaced can be treated with open reduction and plate fixation. A Monteggia fracture is a fracture of the proximal third of the ulna associated with a radial head dislocation. The radial head dislocation may be missed. Careful evaluation of the radiograph, especially the relation-ship with the radial head to the capitellum is necessary for the diagnosis of this injury. These fractures are common in children and rare in adults. These injuries require surgery to fix the ulna fracture

1	the radial head to the capitellum is necessary for the diagnosis of this injury. These fractures are common in children and rare in adults. These injuries require surgery to fix the ulna fracture with plate and screw fixation and to reduce the radial head dislocation. A Galeazzi fracture is a fracture of the distal third radial shaft associated with distal radioulnar joint (DRUJ) injury at the wrist. If the fracture of the radius is less than 7.5 cm from the joint, the distal radioulnar joint is injured in a large number of cases. After the radius is fixed with plate and screw fixation, the DRUJ is assessed for stability and may need wires placed across the joint temporarily.Distal Radius FracturesDistal radius fractures commonly occur in older patients due to a fall or osteoporosis. In younger patients, these fractures usu-ally occur due to high-energy trauma. A Colles fracture is a low energy fracture that is extra-articular and usually dorsally displaced. It has a characteristic

1	younger patients, these fractures usu-ally occur due to high-energy trauma. A Colles fracture is a low energy fracture that is extra-articular and usually dorsally displaced. It has a characteristic appearance of a fork, naming the fracture the “dinner-fork” deformity. A Smith’s fracture is a reverse Colles fracture, usually extra-articular and volarly displaced. A Chauffer’s fracture involves the radial styloid process and may cause occult carpal disruption. A Barton’s fracture can be either volar or dorsal. It is a fracture dislocation of the radiocarpal joint, with an intra-articular volar or dorsal fracture.Every attempt should be made to rule out fractures that extend intra-articularly into the wrist joint or involve the DRUJ. Patients should be evaluated for a median nerve injury and osteoporosis if suspected. Loss of thumb extension from extensor pollicus longus tendon rupture can occur especially in nondisplaced distal radius fractures. Treatment is often a closed Figure

1	and osteoporosis if suspected. Loss of thumb extension from extensor pollicus longus tendon rupture can occur especially in nondisplaced distal radius fractures. Treatment is often a closed Figure 43-11. Displaced olecranon fracture.Brunicardi_Ch43_p1879-p1924.indd 188622/02/19 10:40 AM 1887ORTHOPEDIC SURGERYCHAPTER 43reduction and immobilization. Surgery utilizing a variety of sur-gical techniques is done for unstable fractures as well as those with significant intra-articular involvement.Scaphoid FracturesScaphoid fracture is the most common fracture of the carpal bone. Its diagnosis can be easily missed, and the fracture can lead to nonunion and avascular necrosis. It usually occurs in the waist of the scaphoid but can occur in the proximal or distal pole. Proximal scaphoid fracture will have a higher inci-dence of avascular necrosis due to interruption of the retro-grade blood supply. Tenderness in the anatomic snuffbox after trauma should be considered a scaphoid fracture

1	will have a higher inci-dence of avascular necrosis due to interruption of the retro-grade blood supply. Tenderness in the anatomic snuffbox after trauma should be considered a scaphoid fracture until proven otherwise. Magnetic resonance imaging (MRI) will be help-ful in early diagnosis if no fracture is visible on an X-ray. A thumb spica cast is used for stable nondisplaced fracture, while reduction and screw fixation of the fracture is usually done for displaced fractures. The dorsal approach is used for proximal fractures, and the volar approach is used for the majority of other fractures.Pelvic FracturesPelvic fractures are indicative of high-energy trauma and are associated with head, chest, abdominal, and urogenital injuries. Hemorrhage from pelvic trauma can be life-threatening and patients can present with hemodynamic instability, requiring significant fluid resuscitation and blood transfusions. The bleed-ing that occurs is often due to injury to the venous plexus in the

1	and patients can present with hemodynamic instability, requiring significant fluid resuscitation and blood transfusions. The bleed-ing that occurs is often due to injury to the venous plexus in the posterior pelvis or from the fracture itself. It can also be due to a large vessel injury such as the superior gluteal artery at the greater sciatic notch. Immediate resuscitation with fluids and blood is critical. In hemodynamically unstable patients, blood, fresh frozen plasma, and platelets are given in a 1:1:1 ratio. These patients may require surgical exploration or interven-tional radiology embolization to stop the bleeding. An important first-line treatment in the emergency department is the application of a pelvic binder or sheet that is wrapped tightly around the pelvis to help control bleeding. This is important when there is an increase in the volume of the pelvis by the anteroposterior compression mechanism (an open book mechanism). The pelvic binder is clearly the initial

1	bleeding. This is important when there is an increase in the volume of the pelvis by the anteroposterior compression mechanism (an open book mechanism). The pelvic binder is clearly the initial management of an unstable open book fracture of the pelvis with bleeding. Traction pins may be applied in the emergency department if there is vertical migration of the hemipelvis. An external fixator may also be placed in the operating room, but it is less fre-quently used. Other associated injuries are bladder and urethral injuries that manifest with bleeding from the urethral meatus or blood in the urinary catheter, and these need to be assessed with a retrograde urethrogram.The pelvis is a ring structure made up of the sacrum and the two innominate bones that are held together by strong liga-ments. Because it is a ring, displacement can only occur if the ring is disrupted in two places. This may occur either from frac-tures of the bones or tears of the ligaments that can cause dis-location.

1	Because it is a ring, displacement can only occur if the ring is disrupted in two places. This may occur either from frac-tures of the bones or tears of the ligaments that can cause dis-location. When you see an anterior fracture of the ring, check for a posterior injury (Fig. 43-12). There are three main fracture patterns that occur from trauma to the pelvis. An anteroposterior force to the pelvis causes an “open book” injury pattern in which the pelvis springs open, hinged on the intact posterior ligaments with widening of the pubic symphysis. A lateral compression pattern results from a crush injury that causes fractures to the ilium, sacrum, and pubic rami. Vertical shear injuries are very unstable since they result from disruption of the strong posterior pelvic ligaments and are associated with significant blood loss and visceral injuries. Fractures of the sacrum may be difficult to see on X-ray, and therefore CT scans are often needed to completely visualize the fracture

1	associated with significant blood loss and visceral injuries. Fractures of the sacrum may be difficult to see on X-ray, and therefore CT scans are often needed to completely visualize the fracture pattern. The sacral nerves pass through foramen in the sacrum, and therefore fractures that are close to the foramen can result in nerve injuries. Fractures that involve the sacral canal have a high incidence of nerve injuries and cauda equina syndrome. Fractures that involve the ala of the sacrum may involve the L5 nerve root. Vertical fractures of the 7ABFigure 43-12. A. Pelvic fracture showing anterior and posterior disruption of the pelvis. B. Image depicting a vertical shear fracture with cephalad migration of the hemi-pelvis.Brunicardi_Ch43_p1879-p1924.indd 188722/02/19 10:40 AM 1888SPECIFIC CONSIDERATIONSPART IIsacrum can be highly unstable even after fixation and may be associated with sacral nerve root injuries.Treatment of pelvic fractures depends on the fracture pat-tern.

1	CONSIDERATIONSPART IIsacrum can be highly unstable even after fixation and may be associated with sacral nerve root injuries.Treatment of pelvic fractures depends on the fracture pat-tern. Stable, minimally displaced fractures such as many lower energy lateral compression fractures can be treated nonopera-tively with protected weight-bearing. Open book injuries in which the pubic symphysis is widened more than 2.5 cm may require an anterior plate, and if the posterior pelvic ligaments are also injured, the patient will need posterior fixation. Pos-terior stabilization is typically performed with screws placed percutaneously through the ilium into the sacrum to stabilize the pelvis posteriorly, and a plate is applied over the pubic symphy-sis for anterior stabilization. Displaced sacral fractures and iliac wing fractures are treated with screws or plates, while pubic rami fractures can usually be managed nonoperatively. While most pelvic fractures are caused by high-energy trauma,

1	fractures and iliac wing fractures are treated with screws or plates, while pubic rami fractures can usually be managed nonoperatively. While most pelvic fractures are caused by high-energy trauma, elderly patients with osteoporotic bone can also suffer pelvic fractures after a fall, usually fracturing the pubic rami. Since these are stable injuries, they can be managed nonoperatively with pro-tected weight-bearing.Acetabular FracturesThe acetabulum forms the socket of the hip joint, and fractures occur when the femoral head is driven into the acetabulum in the setting of high-energy trauma. Sciatic nerve function should be examined carefully after an acetabulum fracture. It is impor-tant to rule out dislocation of the hip, which should be reduced immediately to prevent avascular necrosis of the femoral head. Usually 45° oblique views, called Judet views, are utilized. CT scans are very important to visualize the fracture pattern. According to Judet and Letournel, there are ten

1	of the femoral head. Usually 45° oblique views, called Judet views, are utilized. CT scans are very important to visualize the fracture pattern. According to Judet and Letournel, there are ten acetabular fracture patterns: five simple and five complex fracture types (Fig. 43-13). These fractures often require surgery in order to obtain anatomic reduction and to minimize the development of degenerative arthritis.Hip DislocationsHip dislocations almost always result from high-energy trauma; they most commonly occur posteriorly and less commonly ante-riorly (Fig. 43-14). They can cause injury to the sciatic nerve, which runs directly posterior to the hip joint. Examine the patient for foot drop and numbness at the top of the foot. Hip dislocation can be simple, or it may be associated with a fracture of the acetabulum or femoral head. Hip dislocations need to be emergently reduced because of the risk of osteonecrosis of the femoral head if the reduction is delayed. Closed reduction is

1	a fracture of the acetabulum or femoral head. Hip dislocations need to be emergently reduced because of the risk of osteonecrosis of the femoral head if the reduction is delayed. Closed reduction is usually successful with adequate sedation or under general anes-thesia. Once reduction is done, a CT scan is ordered to define the extent of the injury. A CT scan will show associated frac-tures, trapped intraarticular fracture fragments, and the congru-ity of the reduction. If the reduction is unsuccessful, or if there is a fracture fragment inside the joint, then an open reduction is indicated. Hip dislocations that are associated with a femoral head fracture are at increased risk for osteonecrosis of the femo-ral head and posttraumatic osteoarthritis. The femoral head frac-ture associated with hip dislocation is called a Pipkin fracture. If the dislocation is associated with posterior wall fractures, the stability of the hip joint should be assessed carefully, even if the fragment is

1	with hip dislocation is called a Pipkin fracture. If the dislocation is associated with posterior wall fractures, the stability of the hip joint should be assessed carefully, even if the fragment is small. This is usually done by an examination of the patient under anesthesia.Hip FracturesHip fractures are an extremely common injury seen in orthope-dics and are associated with significant morbidity and mortal-ity. They most often occur in elderly patients after ground level falls, are much more common in women than men, and occur more commonly in patients with osteoporosis. The three most common fractures in the elderly are those of the wrist, spine, and hip. Patients who suffer hip fractures are at increased risk for many complications, including deep vein thrombosis, pul-monary embolism, pneumonia, deconditioning, pressure sores, and even death. The mortality rate in the first year following a hip fracture is around 25%. One of the most important rea-sons for performing surgery is

1	pneumonia, deconditioning, pressure sores, and even death. The mortality rate in the first year following a hip fracture is around 25%. One of the most important rea-sons for performing surgery is to prevent these complications because getting patients out of bed and walking as soon as pos-sible diminishes their risk for many of these adverse events. Performing early surgery also decreases the complications in these patients. Therefore, surgery is almost always the treat-ment of choice for hip fractures. The type of surgery performed is determined by the anatomic location of the fracture and the fracture pattern. Surgery should be performed as soon as pos-sible, typically within 24 to 48 hours; however, since many of these patients suffer other comorbidities, they must be properly medically optimized before surgery. The goals of surgery are to minimize pain, restore hip function, and allow early mobi-lization, the importance of which cannot be overemphasized. The functional outcome

1	optimized before surgery. The goals of surgery are to minimize pain, restore hip function, and allow early mobi-lization, the importance of which cannot be overemphasized. The functional outcome for patients following a hip fracture is largely based on their level of mobility and independence Figure 43-13. Types of acetabular fractures.Figure 43-14. Posterior and anterior dislocation.Brunicardi_Ch43_p1879-p1924.indd 188822/02/19 10:40 AM 1889ORTHOPEDIC SURGERYCHAPTER 43before their injury. Many patients become less independent, may require assistive devices to help them walk, and some may require a long-term nursing or rehabilitation facility. Hip frac-tures can be femoral neck fractures, intertrochanteric fractures, or subtrochanteric fractures (Fig. 43-15).Femoral Neck Fractures. Femoral neck fractures occur within the capsule of the hip joint. The main blood supply to the femo-ral neck and head comes from the deep branches of the medial femoral circumflex arteries, which run

1	neck fractures occur within the capsule of the hip joint. The main blood supply to the femo-ral neck and head comes from the deep branches of the medial femoral circumflex arteries, which run along the femoral neck, and when the fracture is displaced, there is an interruption in the blood supply of the femoral head, which can lead to osteonecro-sis. Femoral neck fractures that are nondisplaced have a low risk of disruption of blood flow and therefore can be treated with in situ internal fixation. Three partially threaded cancellous screws are placed through a small incision over the lateral proximal femur, directed through the femoral neck and into the femoral head. Patients can usually begin protected weight-bearing imme-diately after surgery. Displaced femoral neck fractures will likely disrupt the blood supply and therefore need to be treated with a prosthetic replacement in older adults. Most commonly a hemi-arthroplasty is performed in which the femoral head and neck are replaced

1	disrupt the blood supply and therefore need to be treated with a prosthetic replacement in older adults. Most commonly a hemi-arthroplasty is performed in which the femoral head and neck are replaced with a metal head and neck into the femoral canal. Higher demand patients and those who have osteoarthritis of the hip joint and hip pain before their fracture may receive a total hip replacement, in which the acetabulum is also replaced with a prosthesis, typically a plastic cup inside a metal shell. Patients can begin weight-bearing immediately after surgery. Displaced femoral neck fractures in young patients are the result of a high-energy trauma and are usually treated by reduction with screw fixation. The reduction may be closed or open.Intertrochanteric Hip Fractures. Intertrochanteric hip frac-tures occur between the greater and lesser trochanters of the proximal femur. Because the blood supply to this area is abun-dant, osteonecrosis is uncommon, and therefore these fractures can

1	hip frac-tures occur between the greater and lesser trochanters of the proximal femur. Because the blood supply to this area is abun-dant, osteonecrosis is uncommon, and therefore these fractures can be treated with reduction and internal fixation. Displaced fractures need to be realigned, and this often involves placing the patient on a fracture table where traction and rotation can be applied to the affected leg to reduce the fracture. There are two devices that can be used. In stable fractures, a sliding hip screw includes a large screw placed from the lateral cortex of the proximal femur across the fracture and into the femoral neck and head, followed by a side plate along the lateral cortex of the femur, which is then fixed to the shaft with screws. A cephalom-edullary nail includes a nail placed down the medullary canal of the femur and a large screw that engages the nail as it is passed from the lateral cortex up into the neck and head. Nails are usually used in unstable

1	a nail placed down the medullary canal of the femur and a large screw that engages the nail as it is passed from the lateral cortex up into the neck and head. Nails are usually used in unstable fractures and allow protected weight-bearing postoperatively. The reverse oblique intertrochanteric fracture is a specific type of fracture that exits on the lateral cortex (Fig. 43-16). This is best treated with a cephalomedullary Figure 43-15. Types of hip fractures.Figure 43-16. Classic intertrochanteric fracture and reverse oblique fracture. Notice that the fracture line of the reverse oblique fracture exits on the lateral cortex.Brunicardi_Ch43_p1879-p1924.indd 188922/02/19 10:40 AM 1890SPECIFIC CONSIDERATIONSPART IInail; a dynamic hip screw is the wrong device to be used in reverse oblique fractures because it will lead to sliding, shorten-ing, and medial displacement of the fracture.Subtrochanteric Hip Fractures. Subtrochanteric hip frac-tures occur in the proximal femoral shaft just

1	fractures because it will lead to sliding, shorten-ing, and medial displacement of the fracture.Subtrochanteric Hip Fractures. Subtrochanteric hip frac-tures occur in the proximal femoral shaft just distal to the lesser trochanter in an area of high biomechanical stresses. While they can occur in older adult patients after a fall, they are also seen in high-energy trauma. Because of the forces of muscles attached to the fractured segments, they tend to be significantly displaced (Fig. 43-17) and may be difficult to reduce. They are most often treated with a long cephalomedullary nail that includes a screw distally to lock the nail in place and prevent rotation of the femur. Fixed angle plates or blade plates are sometimes used in the treatment of subtrochanteric fractures. In most cases, pro-tected weight-bearing can begin soon after surgery. Complica-tions usually include malunion and nonunion of the fracture.Bisphosphonate-related subtrochanteric fractures are an example of

1	cases, pro-tected weight-bearing can begin soon after surgery. Complica-tions usually include malunion and nonunion of the fracture.Bisphosphonate-related subtrochanteric fractures are an example of insufficiency fractures that may be related to the long-term use of bisphosphonates. These fractures have been recently identified. An intramedullary nail is the treatment of choice for this fracture.Femoral Shaft FracturesFractures of the femoral shaft are caused by high-energy trauma and may be associated with other severe injuries. Long bone fractures, such as femoral shaft fractures, put these patients at risk for complications such as thromboembolic events and acute respiratory distress syndrome (ARDS), and therefore it is important to fix these quickly, typically within 24 hours. They are most commonly fixed with an intramedullary nail that can be placed antegrade (from the piriformis fossa or greater tro-chanter down the canal) or retrograde (through an incision into the knee joint

1	most commonly fixed with an intramedullary nail that can be placed antegrade (from the piriformis fossa or greater tro-chanter down the canal) or retrograde (through an incision into the knee joint and up the canal), with screws placed through proximal and distal holes to lock the nail in place, creating a stable construct to allow weight-bearing. Trauma patients who are hemodynamically unstable or who have other life-threatening injuries are treated temporarily with an external fixator until they can safely undergo surgery. This is called “damage control orthopedics.” The base deficit and lactic acid levels are moni-tored and used as guides to indicate if the patient is adequately resuscitated. When their levels are normal, it means the tissue is adequately oxygenated and the patient can undergo definitive fixation of the femur.Distal Femur FracturesDistal femur fractures are the result of a fall from a height or from high-energy trauma. They can also occur in elderly patients with

1	undergo definitive fixation of the femur.Distal Femur FracturesDistal femur fractures are the result of a fall from a height or from high-energy trauma. They can also occur in elderly patients with osteoporotic bone after a fall onto the knee. While nondisplaced fractures in the elderly may be treated nonoper-atively with a hinged knee brace and early motion exercises, most require surgery. These fractures can involve the articu-lar surface of the knee joint, so anatomic reduction of the joint surface is crucial. They are fixed with plates and screws, often utilizing a locking construct. The plate is placed over the lateral, or rarely the medial cortex depending on the fracture pattern. A retrograde intramedullary rod inserted through the knee can also be used, especially in extraarticular fracture patterns. The goal of surgery is to achieve anatomic reduction, stable fixation, and allow early knee range of motion. Intra-articular fractures require the patient to be non–weight-bearing

1	fracture patterns. The goal of surgery is to achieve anatomic reduction, stable fixation, and allow early knee range of motion. Intra-articular fractures require the patient to be non–weight-bearing until the frac-ture shows signs of healing. Complications of these fractures include nonunion, malunion, and stiffness of the knee. Be aware of Hoffa fractures, a coronal fractures that usually involve the lateral femoral condyle. They can be missed on X-rays, but they are easily diagnosed by CT scan. It may need a different fixa-tion than that required for the associated supracondylar fracture component.Knee DislocationsDislocation of the knee is a rare but devastating injury that can be limb-threatening. Some dislocations spontaneously reduce and can be underdiagnosed. When the knee dislocates, the anterior cruciate ligament (ACL) and posterior cruciate liga-ment (PCL) are torn, and various degrees of injury occur to the lateral collateral ligament (LCL), medial collateral ligament

1	the anterior cruciate ligament (ACL) and posterior cruciate liga-ment (PCL) are torn, and various degrees of injury occur to the lateral collateral ligament (LCL), medial collateral ligament (MCL), posterolateral corner, joint capsule, and menisci. How-ever, the danger is due to the close proximity of the popliteal artery, which runs directly behind the knee and may kink or sustain a tear of the intimal wall when the knee dislocates. A neurovascular exam is extremely important, focusing on the common peroneal nerve and the vascular status of the extrem-ity, followed by immediate reduction of the knee and repeat neurovascular exam. If the pulses are normal, the ankle brachial index (ABI) should be measured. If the ABI is more than 0.9, then the patient should be monitored with serial examination. If the ABI is less than 0.9, then a CTA or an arterial duplex ultra-sound should be performed. If there is evidence of diminished pulses after reduction, an angiogram must be performed. If the

1	If the ABI is less than 0.9, then a CTA or an arterial duplex ultra-sound should be performed. If there is evidence of diminished pulses after reduction, an angiogram must be performed. If the pulses are absent after reduction, immediate surgical explora-tion and/or repair should be done by a vascular surgeon. Pro-phylactic fasciotomy of the leg is usually done. Time is critical to reestablish the circulation of the limb. If ischemia time is more than 8 hours, then there is a very high rate of amputation. With regard to the ligamentous injuries, an external fixator may be initially used to stabilize the unstable knee and protect the reduction. Subsequently, an MRI will identify what structures have been torn. Because a dislocation causes so much damage to the knee, a delayed multiligamentous reconstruction is recom-mended on an elective basis in order to stabilize the knee joint. Figure 43-17. Illustration showing subtrochanteric fracture with the deforming forces of the

1	reconstruction is recom-mended on an elective basis in order to stabilize the knee joint. Figure 43-17. Illustration showing subtrochanteric fracture with the deforming forces of the muscle.Brunicardi_Ch43_p1879-p1924.indd 189022/02/19 10:40 AM 1891ORTHOPEDIC SURGERYCHAPTER 43Stiffness and instability of the knee are common complications after this injury.Patella/Extensor Mechanism InjuriesThe extensor mechanism is comprised of the quadriceps ten-don, the patella, and the patella ligament. This mechanism func-tions to extend the knee. Injuries can result after a fall directly onto the knee or from forcible contraction of the quadriceps. It is important to examine the knee for the ability to actively extend the knee. Quadriceps tendon ruptures, patella fractures, or patella ligament ruptures can result in a loss of active knee extension requiring surgery. Nondisplaced patella fractures with intact active knee extension can be treated nonoperatively with a cast or knee immobilizer,

1	can result in a loss of active knee extension requiring surgery. Nondisplaced patella fractures with intact active knee extension can be treated nonoperatively with a cast or knee immobilizer, holding the knee in full extension, and weight-bearing is permitted. Displaced or comminuted frac-tures require surgery with tension band wiring and/or screws. Symptomatic hardware is a common complication. Acute osteo-chondral fractures can be managed with internal fixation. Quad-riceps tendon and patella tendon ruptures with loss of active knee extension are treated with suture repair. After surgery, the knee is held in extension, and knee flexion is slowly increased over several weeks using a hinged knee brace.Patella dislocations are common injuries that occur when the femur is forcibly internally rotated on an externally rotated tibia while the foot is planted on the ground. They typically dislocate laterally and often relocate spontaneously. The medial patellofemoral ligament is the

1	rotated on an externally rotated tibia while the foot is planted on the ground. They typically dislocate laterally and often relocate spontaneously. The medial patellofemoral ligament is the primary stabilizer of the patella. Patients present with a significant knee effusion and medial-sided tenderness. During the physical exam, these patients may elicit a positive apprehension test, in which a lateral force to the patella elicits pain and the sensation of an impending dis-location. Dislocated patellas can be reduced by extending the knee and manual reduction and are treated with temporary knee immobilization. Make sure that there is no fracture or loose bod-ies, which would be an indication for surgery. MRIs will show the classic bone bruise and edema involving the medial facet of the patella and the lateral condyle of the femur. There is a high rate of recurrent dislocation with nonoperative treatment, which may require surgical intervention.Tibial Plateau FracturesThe tibial

1	the patella and the lateral condyle of the femur. There is a high rate of recurrent dislocation with nonoperative treatment, which may require surgical intervention.Tibial Plateau FracturesThe tibial plateau is comprised of the articular surfaces and underlying cancellous bone of the medial and lateral plateaus of the proximal tibia. Fractures of the plateau result from axial loads sustained in falls from a height or high-energy trauma, and they are often associated with injuries to the menisci and cartilage of the knee. Fractures can involve the medial, lat-eral, or both plateaus with significant comminution, angula-tion, and depression, creating a challenging injury to fix. The Schatzker classification is commonly used in tibial plateau fractures (Fig. 43-18).• Type I: Lateral split fracture• Type II: Lateral split-depressed fracture• Type III: Lateral pure depression fracture• Type IV: Medial plateau fracture• Type V: Bicondylar fracture• Type VI: Metaphyseal-diaphyseal

1	split fracture• Type II: Lateral split-depressed fracture• Type III: Lateral pure depression fracture• Type IV: Medial plateau fracture• Type V: Bicondylar fracture• Type VI: Metaphyseal-diaphyseal disassociationMeniscal tears occur more on the lateral side and tend to be peripheral tears, especially if there is more than 6 mm depres-sion or separation of the joint. Type IV, which is the medial tibial plateau fracture, could be a variant of a knee dislocation. The ankle brachial index (ABI) should be used in this situation and in more complex types of tibial plateau fractures. Clinically, laxity of more than 10° may indicate instability of the fracture; however, the test may be painful and hard to perform. A CT scan is important to visualize the intra-articular involvement of the fracture. Minimally displaced fractures may be treated nonoperatively with strict non–weight-bearing until the fracture heals. Fractures associated with displaced articular fragments require surgery in order

1	Minimally displaced fractures may be treated nonoperatively with strict non–weight-bearing until the fracture heals. Fractures associated with displaced articular fragments require surgery in order to restore the smooth contour of the articular surface. They are treated with plates and screws placed medially, laterally, or both. Stabilization of a posteromedial fragment may require a separate posteromedial approach. Since there is often a depression of the cancellous bone, bone graft or bone substitutes, particularly calcium phosphate which resists compression, may be needed to buttress the articular surface and restore the anatomic alignment of the tibia. Patients are kept strictly non–weight-bearing for several months until the fracture begins to heal, though early range of motion is encouraged to prevent stiffness. Repair of ligament or meniscus injuries may also be indicated at the time of surgery. Knee stiffness and osteoarthritis are common complications of these injuries. The

1	to prevent stiffness. Repair of ligament or meniscus injuries may also be indicated at the time of surgery. Knee stiffness and osteoarthritis are common complications of these injuries. The goal of the surgery is to restore joint stability and alignment.Tibial Shaft FracturesTibial shaft fractures are the most common long bone frac-tures and occur following high-energy trauma, direct blows, and severe twisting injuries. Trauma and direct blows to the Figure 43-18. Tibial plateau fracture classification.Brunicardi_Ch43_p1879-p1924.indd 189122/02/19 10:40 AM 1892SPECIFIC CONSIDERATIONSPART IItibia result in transverse or comminuted fracture patterns, while torsional injuries cause spiral fractures. Fractures with minimal angulation can be treated with reduction and casting, followed by transition to a functional brace and slow return to weight-bearing. Such fractures may need to be immobi-lized for several months since these fractures can be slow to heal. Most tibial shaft

1	by transition to a functional brace and slow return to weight-bearing. Such fractures may need to be immobi-lized for several months since these fractures can be slow to heal. Most tibial shaft fractures, especially comminuted and angulated fractures, are treated with an intramedullary nail placed down the tibial canal, with interlocking screws placed proximally and distally. Weight-bearing can begin soon after surgery. Proximal third tibial fractures are challenging and can result in malalignment, usually valgus and apex anterior angulation. Knee pain is common after intramedullary rod placement. Plate and screw fixation can also be used; how-ever, since the tibia is subcutaneous, hardware placed along the shaft can increase the risk of wound complications, mak-ing intramedullary nailing the preferred treatment. Fibula shaft fractures often occur along with tibial shaft fractures, though they usually heal well without surgery. Tibial frac-tures, both closed and open, can be

1	nailing the preferred treatment. Fibula shaft fractures often occur along with tibial shaft fractures, though they usually heal well without surgery. Tibial frac-tures, both closed and open, can be associated with compart-ment syndrome. Patients usually have pain out of proportion with swelling of the leg and pain with passive stretch. Com-partment pressure within 30 mmHg of the diastolic pressure is diagnostic of compartment syndrome.Tibial Plafond (Pilon) FracturesThe tibial plafond is the distal tibial articular surface of the ankle joint. Pilon fractures are typically high-energy injuries that usu-ally result from axial compression. These injuries can cause significant soft tissue injury, severely comminuted intra-articular and metaphyseal fragments (Fig. 43-19A,B), and wound heal-ing problems, making these fractures very difficult to treat. Due to the soft tissue injury, these fractures are initially treated with external fixation until the swelling subsides, which may take

1	problems, making these fractures very difficult to treat. Due to the soft tissue injury, these fractures are initially treated with external fixation until the swelling subsides, which may take several days to weeks. The wrinkle test is helpful in this situation to assess when the soft tissues are amenable to defini-tive fixation. A CT scan is usually obtained after the fracture is stabilized by an external fixator. The CT scan will clearly define the fracture fragments and helps in planning the surgical approach and fixation. Minimal incision techniques and mini-mal fixation are becoming popular in some situations. The main goal of surgery is to restore the articular surface. Fixation of the fibula in order to maintain and establish anatomic length is done in some cases. Bone grafts or bone substitutes may be used to fill the void in the metaphyseal region. A variety of fixation techniques may be used including plates to stabilize the metaphysis to the diaphysis. Patients are kept

1	bone substitutes may be used to fill the void in the metaphyseal region. A variety of fixation techniques may be used including plates to stabilize the metaphysis to the diaphysis. Patients are kept non–weight-bearing for many months until the fracture heals. Despite best efforts, patients may suffer from ankle pain and stiffness, arthri-tis, wound healing problems, infection, nonunion, and some patients may eventually require ankle fusion. Early fixation of pilon fractures with plates can increase the incidence of wound complications significantly.Ankle and Subtalar DislocationsAnkle Dislocations. The ankle joint is a complex hinge joint comprised of the distal tibial plafond, medial malleolus, and lateral malleolus and their articulation with the talus. Several ligaments also contribute to the stability of the ankle joint, including the deltoid ligament medially, the syndesmotic liga-ments between the tibia and fibula, and the anterior talofibular, posterior talofibular, and

1	to the stability of the ankle joint, including the deltoid ligament medially, the syndesmotic liga-ments between the tibia and fibula, and the anterior talofibular, posterior talofibular, and calcaneofibular ligaments laterally. Dislocations of the ankle joint result from a severe twisting injury and often occur with fractures. At times, dislocations FibulaTibiaTalusABFigure 43-19. A. Tibial pilon fracture with comminution. B. Pilon fracture and its main fracture fragments.Brunicardi_Ch43_p1879-p1924.indd 189222/02/19 10:40 AM 1893ORTHOPEDIC SURGERYCHAPTER 43can place significant pressure on the overlying skin and can cause neurovascular compromise; therefore, prompt reduction is extremely important followed by splinting.Subtalar Dislocations. Subtalar dislocations can be medial or lateral, depending on the position of the foot. The medial dislocation is more common. Lateral dislocations are less com-mon, can be open, and are more likely to be associated with fractures.

1	or lateral, depending on the position of the foot. The medial dislocation is more common. Lateral dislocations are less com-mon, can be open, and are more likely to be associated with fractures. Irreducible lateral subtalar dislocations may occur from a trapped tibialis posterior tendon, which will block the reduction. The main complication of subtalar dislocations is subtalar arthritis.Ankle FracturesAnkle fractures are very common and result from a twisting injury to the ankle. The patterns of ankle fractures depend on the direction of force and the position of the foot and ankle at the time of injury. The goals of treating ankle fractures are to restore the anatomy of the ankle joint and to restore the length and rota-tion of the fibula. Initial treatment includes closed reduction and placement of a well-padded splint in order to protect the skin. Swelling can be a significant problem, so elevation of the foot is encouraged. Surgery may be delayed until the skin condition permits.

1	placement of a well-padded splint in order to protect the skin. Swelling can be a significant problem, so elevation of the foot is encouraged. Surgery may be delayed until the skin condition permits. Fractures of the ankle may be:1. Isolated malleolar fractures, usually the lateral malleolus or the medial malleolus.2. Bimalleolar fractures that involve the lateral and medial malleolus. Please note that the deltoid ligament may be in-jured instead of the medial malleolus.3. Trimalleolar fractures that involve the lateral malleolus, medial malleolus, and posterior malleolus.Lateral Malleolus Fractures. Isolated fractures of the lateral malleolus with less than 3 mm displacement and no talar shift may be stable. An external rotation stress radiograph or a grav-ity test is used to assess the competency of the deltoid ligament, with the goal of the test to exclude deltoid injury. If the patient has a deltoid injury, in addition to the fibular fracture, then the patient will need surgery.

1	competency of the deltoid ligament, with the goal of the test to exclude deltoid injury. If the patient has a deltoid injury, in addition to the fibular fracture, then the patient will need surgery. The fracture will require anatomic reduction in order to restore normal ankle joint congruity. The talus can sublux laterally following lateral malleolus fractures, and even 1 millimeter of talar shift decreases the surface contact between the talus and the tibia by 40%, increasing the risk of developing arthritis. Open reduction and internal fixation of the fibula is usually done with plate and screws.Medial Malleolar Fractures. An isolated fracture of the medial malleolus is usually an avulsion-type injury. Minimally displaced fractures can be treated with a cast or walking boot, while displaced fractures are usually fixed with screws.Bimalleolar Fractures. Fractures to both the medial and lat-eral malleoli usually require surgery. These injuries are more unstable, and the talus will

1	fractures are usually fixed with screws.Bimalleolar Fractures. Fractures to both the medial and lat-eral malleoli usually require surgery. These injuries are more unstable, and the talus will often sublux or completely dislo-cate laterally. They are treated by reducing and fixing both malleoli during surgery. Occasionally, the posterior articular surface of the distal tibia, or posterior malleolus, can be frac-tured as well, resulting in a trimalleolar ankle fracture. Often it is a small fragment and does not need to be fixed; however, if it involves a significant amount of the articular surface, the posterior malleolus should be fixed with screws placed either anteriorly, posteriorly, or with an antiglide plate. In all ankle fractures, especially in ones associated with deltoid rupture, a syndesmotic injury should be considered. After the fixation of the fractures, an intraoperative external rotation stress test will diagnose syndesmotic injury.Syndesmosis Injuries. The syndesmosis

1	a syndesmotic injury should be considered. After the fixation of the fractures, an intraoperative external rotation stress test will diagnose syndesmotic injury.Syndesmosis Injuries. The syndesmosis is comprised of several ligaments between the distal tibia and fibula that pro-vide stability to the ankle joint by resisting axial, rotational, and translational forces. The syndesmosis can be disrupted at the time of ankle fractures and requires special attention (Fig. 43-20). Widening of the space between the distal tibia and fibula after fixing the fractures is indicative of a syndesmo-sis injury, and an intraoperative external rotation stress radio-graph can be helpful for evaluation. Such injuries are treated with one or two screws placed laterally from the fibula into the tibia, parallel to the ankle joint. Patients are kept non–weight-bearing for several weeks. The screws are often removed after 3 to 6 months, though they can be left in place and are typically

1	the tibia, parallel to the ankle joint. Patients are kept non–weight-bearing for several weeks. The screws are often removed after 3 to 6 months, though they can be left in place and are typically asymptomatic.Maisonneuve FracturesA Maisonneuve fracture is a fracture of the proximal fibula associated with fracture of the medial malleolus or rupture of the deltoid ligament. There is always an associated syndesmotic injury (Fig. 43-21). Diagnosis may be difficult, and the injury may be missed. These injuries require surgical treatment with fixation of the syndesmosis by screws from the fibula to the tibia.Calcaneal FracturesCalcaneal fractures usually occur following a fall from a height and are often associated with other injuries, including lum-bar spine fractures. There is a high incidence of compartment Figure 43-20. Ankle fracture with syndesmotic injury (white arrow).Brunicardi_Ch43_p1879-p1924.indd 189322/02/19 10:40 AM 1894SPECIFIC CONSIDERATIONSPART IIsyndrome of the foot

1	of compartment Figure 43-20. Ankle fracture with syndesmotic injury (white arrow).Brunicardi_Ch43_p1879-p1924.indd 189322/02/19 10:40 AM 1894SPECIFIC CONSIDERATIONSPART IIsyndrome of the foot associated with calcaneal fractures. These injuries are often intra-articular and can result in collapse of the weight-bearing posterior facet of the calcaneus. The Bohler angle, which is normally between 20° and 40°, would be reduced or even flattened. CT scans are useful to better visualize the fracture pattern. Some fractures can be treated nonoperatively in a well-padded splint with patients being kept non–weight-bearing for up to 12 weeks. Displaced intraarticular fractures can be treated surgically once the swelling subsides and the wrinkle test is positive. Surgery can be done with lag screws or with plate and screw fixation. Despite adequate treatment, calcaneal fractures can be debilitating injuries, leading to significant heel pain and arthritis. The outcome of surgery depends on

1	or with plate and screw fixation. Despite adequate treatment, calcaneal fractures can be debilitating injuries, leading to significant heel pain and arthritis. The outcome of surgery depends on the com-minution of the fracture and degree of articular involvement. The more fragments seen on a CT scan, the worse the outcome for the patient. Wound complications are a problem for calca-neal fractures with the risk being even higher in diabetics, smok-ers, and in open fractures. Open calcaneal fractures have a high rate of amputation.Talus FracturesFractures of the talus commonly result from forced dorsiflexion of the ankle, causing the talar neck to impact on the anterior distal tibia. The dominant blood supply is the artery of the tarsal canal. The blood supply to the talus can be jeopardized after a displaced fracture and may lead to osteonecrosis (Fig. 43-22), which is an unfortunately common complication following talus fractures. The incidence of osteonecrosis depends on the degree

1	after a displaced fracture and may lead to osteonecrosis (Fig. 43-22), which is an unfortunately common complication following talus fractures. The incidence of osteonecrosis depends on the degree of displacement of the fracture. The Hawkin’s sign is a sub-chondral lucency that is seen on the mortise X-ray at 6 weeks and indicates that there is vascularity of the talus. This indicates that there is no avascular necrosis. Nondisplaced fractures are treated with a cast and have a 15% risk of osteonecrosis, while displaced fractures are often treated surgically with screw fixa-tion. There is a high risk of osteonecrosis, ranging from 30% to 100%. Subtalar arthritis is the most common complication from this injury. Varus malunion that results from inadequate reduc-tion is the most preventable complication. This complication leads to a decreased subtalar range of motion and eversion. With varus malunion, the patient will walk with the foot internally rotated.Foot FracturesThe tarsal bones,

1	complication. This complication leads to a decreased subtalar range of motion and eversion. With varus malunion, the patient will walk with the foot internally rotated.Foot FracturesThe tarsal bones, including the navicular, the cuboid, and the three cuneiform bones, link the hind foot to the metatarsals and provide mechanical stability to the arch of the foot. Isolated fractures to these bones are rare and are often treated nonopera-tively with a cast or boot. Cuboid fractures are also known as “nutcracker fractures” and may indicate a Lisfranc injury. Stress fractures of the navicular can be occult, and a diagnosis may be challenging to make in a patient that complains of foot pain. An MRI may be needed for diagnosis. Treatment is often a short leg non–weight-bearing cast. The Lisfranc ligament, which con-nects the second metatarsal base to the medial cuneiform, is an important stabilizer of the midfoot. Lisfranc injuries can be seen following torsional forces to the foot or from

1	ligament, which con-nects the second metatarsal base to the medial cuneiform, is an important stabilizer of the midfoot. Lisfranc injuries can be seen following torsional forces to the foot or from crush injuries. These injuries can be missed and often require surgery because anatomic reduction is extremely important for a successful outcome. Open reduction and internal fixation is the technique used for Lisfranc fractures. Primary arthrodesis is often used for purely ligamentous injuries. The main complication of Lisfranc injuries is posttraumatic arthritis. Metatarsal fractures similarly result from twisting or crush injuries and most can be treated Figure 43-21. Maisonneuve fracture showing proximal fibular fracture and associated ankle injury with disruption of syndesmosis.Figure 43-22. Displaced talar neck fracture. Notice the interrup-tion of the blood supply in the talus.Brunicardi_Ch43_p1879-p1924.indd 189422/02/19 10:40 AM 1895ORTHOPEDIC SURGERYCHAPTER 43nonoperatively

1	talar neck fracture. Notice the interrup-tion of the blood supply in the talus.Brunicardi_Ch43_p1879-p1924.indd 189422/02/19 10:40 AM 1895ORTHOPEDIC SURGERYCHAPTER 43nonoperatively with a hard-soled shoe and weight-bearing as tolerated. The base of the fifth metatarsal, however, warrants close attention. Fifth metatarsal fractures at the metaphyseal-diaphyseal junction (fourth and fifth metatarsal articulation) are called Jones fractures. These fractures can jeopardize blood flow, are at risk for nonunion, and may be associated with cav-ovarus hindfoot. Jones fractures need close follow-up to assess for healing if treated by short-leg cast and non–weight-bearing. In athletes and active young patients, screw fixation is usually used to stabilize the fracture. Injuries to the metatarsal-phalangeal joints and phalangeal fractures can be treated symptomatically or with buddy taping with weight-bearing as tolerated in a hard-soled shoe.SPORTS MEDICINEIntroductionSports medicine deals

1	joints and phalangeal fractures can be treated symptomatically or with buddy taping with weight-bearing as tolerated in a hard-soled shoe.SPORTS MEDICINEIntroductionSports medicine deals with the prevention and treatment of inju-ries related to sports and exercise. These injuries encompass various areas in the musculoskeletal system. In recent years, sports-related injuries have increased, and the sports medicine field has been expanding. There are multiple factors leading to this increase in sports-related injuries. They include athletes participating in one sport year-round, more “weekend warriors” participating in sporting activity, and increased expectations for higher performance.The orthopedic subspecialty of sports medicine treats a broad spectrum of patients, ranging from children who have just started participating in their first sports to the specialized care of professional athletes. Medical treatment of athletes, recreational or professional, can be complex as shortand

1	who have just started participating in their first sports to the specialized care of professional athletes. Medical treatment of athletes, recreational or professional, can be complex as shortand long-term outcomes are influenced by the higher demand that athletes put on their bodies. Additionally, the orthopedic sports medicine specialist does not only treat the patient’s injuries but also has to consider the patient’s attempted return to his or her previous level of activity. “Getting back in the game” is sometimes subject to pressure and competing interests from third parties (e.g., team members, coaches, parents, fans). This can make the athlete’s treatment and the rehabilitation a chal-lenging process.Surgical intervention for ligament and cartilage injuries in sports medicine patients is usually done using arthroscopic techniques. The most frequently injured joints are the shoulder, knee, and hip. Therefore, treatment of common injuries in these joints will be the scope of this

1	is usually done using arthroscopic techniques. The most frequently injured joints are the shoulder, knee, and hip. Therefore, treatment of common injuries in these joints will be the scope of this section.SHOULDERRotator CuffRotator cuff injuries are among the most common reasons to visit an orthopedic sports specialist. Often, these injuries are associated with forceful or repeated overhead and pulling movements. The rotator cuff provides shoulder movement and glenohumeral joint stability, and injuries can typically lead to pain, weakness, and restricted movement of the arm. Over recent years, improvement of surgical indications, operative techniques, and rehabilitation protocols has led to better out-comes. Studies suggest that arthroscopic techniques are equal or superior to open techniques for most indications. Contro-versies surrounding rotator cuff repair remain and include use of acromioplasty, enhancement of healing with orthobiologics (Fig. 43-23), singlevs. double-row

1	techniques for most indications. Contro-versies surrounding rotator cuff repair remain and include use of acromioplasty, enhancement of healing with orthobiologics (Fig. 43-23), singlevs. double-row fixation, and the treatment ABrotator cuff teargreater tuberosity/RC attachment siterotator cuff repairCFigure 43-23. Imaging and treatment of rotator cuff tears. A. Magnetic resonance imaging coronal T2 image showing a full-thickness and moderately retracted tear (arrow) of the supraspinatus tendon. B. Arthroscopic image showing the supraspinatus tendon tear as viewed from a posterior portal during the surgery. C. Arthroscopic image showing completion of repair of the supraspinatus tendon tear using suture anchors imbedded in the greater tuberosity of the humerus and attached sutures that capture and reduce the torn tendon to its native insertion site.Brunicardi_Ch43_p1879-p1924.indd 189522/02/19 10:40 AM 1896SPECIFIC CONSIDERATIONSPART IIof massive or large tears. Rehabilitation

1	capture and reduce the torn tendon to its native insertion site.Brunicardi_Ch43_p1879-p1924.indd 189522/02/19 10:40 AM 1896SPECIFIC CONSIDERATIONSPART IIof massive or large tears. Rehabilitation after surgery plays an important role to restore strength, motion, and function and, ultimately, to return the patient to his or her previous level of activity. The standard rehabilitation protocol is made up of three consecutive stages: immobilization, passive exercise, and active exercise. Immobilization can be established by using a sling, and passive exercises should be initiated by the therapist in the first 4 to 6 weeks after surgery. The therapist moves the arm in different positions to improve range of motion (ROM) while providing support. After 4 to 6 weeks, active exercises can be gradually introduced. At 8 to 12 weeks, muscle strength and improvement of arm control are increased by starting a strength-ening exercise program.Shoulder InstabilityThe most common etiology for

1	be gradually introduced. At 8 to 12 weeks, muscle strength and improvement of arm control are increased by starting a strength-ening exercise program.Shoulder InstabilityThe most common etiology for shoulder instability is related to trauma, especially shoulder dislocation. After a shoulder has dislocated, it becomes vulnerable to repeat episodes of instability and may develop into a chronic problem. Most of the shoulder’s stability is provided dynamically by the rotator cuff and stati-cally by the shoulder capsule and ligaments. The most com-mon dislocation is in the anterior-inferior direction. Typically, patients with an anterior dislocation present with pain and an externally rotated shoulder. Younger patients are more sus-ceptible to suffer from repeat dislocations than older patients. The position of the humeral head with respect to the glenoid and other bony pathology can be identified with radiographs. Views from different angles should be obtained to thoroughly evaluate the

1	The position of the humeral head with respect to the glenoid and other bony pathology can be identified with radiographs. Views from different angles should be obtained to thoroughly evaluate the patient; an anterior-posterior (AP) view, a glenoid (axillary) view, and a “Y” view of the shoulder are recom-mended in assessing this injury. Immediate reduction of the glenohumeral joint is paramount to the initial treatment of this injury. Repeat radiographs should be attained to ensure that the humeral head is appropriately positioned. As soft tissue struc-tures are typically damaged in these injuries, an MRI can be obtained to evaluate these structures.Relocation of the shoulder is generally accomplished with the patient in supine position and the arm under gentle traction and slight abduction. Some sedation is helpful as it relaxes the patient’s musculature. Whether or not to immobilize a first-time-dislocated shoulder remains controversial, as does the position of immobilization.

1	Some sedation is helpful as it relaxes the patient’s musculature. Whether or not to immobilize a first-time-dislocated shoulder remains controversial, as does the position of immobilization. Additionally, some surgeons argue that early surgical repair of capsulolabral structures is appropri-ate as the recurrence rate in the young population is high and may lead to more extensive bony involvement and ultimately more invasive open procedures.Prolonged immobilization is not recommended because this will often lead to substantial stiffness in the shoulder and does not appreciably decrease the redislocation rate. Unfor-tunately, many patients experience recurrent dislocations, in which case surgical stabilization of the shoulder should be con-sidered. Arthroscopic stabilization procedures have been the gold standard treatment for the majority of injuries related to shoulder dislocations, typically a tear of the anteroinferior cap-sulolabral complex (Bankart lesion). There are a subset of

1	been the gold standard treatment for the majority of injuries related to shoulder dislocations, typically a tear of the anteroinferior cap-sulolabral complex (Bankart lesion). There are a subset of inju-ries, typically involving large glenoid bony lesions, that require more extensive intervention with a Latarjet procedure or bone grafting. After surgery, the shoulder is temporarily immobi-lized with a sling. When the sling is removed, exercises will be started to rehabilitate the shoulder, improve ROM, and prevent scarring. Strengthening exercises will gradually be added to the rehabilitation plan.Posterior Dislocation of the ShoulderPosterior dislocations of the shoulder are rare and could be missed. This dislocation can occur due to electric shock or sei-zures. Examination of the patient will show limitation in exter-nal rotation of the shoulder. The shoulder will be locked in an internally rotated position. The posterior dislocation could be missed on the AP view of the shoulder,

1	will show limitation in exter-nal rotation of the shoulder. The shoulder will be locked in an internally rotated position. The posterior dislocation could be missed on the AP view of the shoulder, and an axillary view should be obtained to avoid missing the injury.Superior Labrum and Biceps TendonThe labrum is a structure that helps to deepen the shoulder socket and stabilize the glenohumeral joint. Additionally, it serves as an attachment point for many of the shoulder ligaments, as well as the long head of the biceps tendon. A superior labrum ante-rior and posterior (SLAP) lesion may occur in the superior part of the labrum, usually anterior and posterior to the attachment of the biceps tendon, with occasional involvement of the biceps tendon in certain cases. Injuries to the superior labrum can be caused by trauma or by repetitive shoulder motion, such as in throwing athletes. Radiographs are generally obtained to evalu-ate for concomitant bony injuries or osteoarthritic changes.

1	labrum can be caused by trauma or by repetitive shoulder motion, such as in throwing athletes. Radiographs are generally obtained to evalu-ate for concomitant bony injuries or osteoarthritic changes. The labrum itself, and other soft tissue, is better visualized with MRI with addition of a gadolinium arthrogram adding sensitivity for labral injury detection (Fig. 43-24). A coronal view MRI will clearly show the condition of the superior labrum.Conservative and operative treatments have had mixed results depending on the patient’s age, activity level, type of tear, and presence of concomitant injuries. If symptoms do not improve with adequate physical therapy and/or nonsteroidal anti-inflammatory drugs (NSAIDs), surgical intervention is usu-ally indicated. Some SLAP injuries involve the biceps tendon, which may require either tenotomy or tenodesis.After surgical repair, the shoulder needs to be immobilized to protect the repair and allow for healing. Usually a sling is used for 4 weeks

1	tendon, which may require either tenotomy or tenodesis.After surgical repair, the shoulder needs to be immobilized to protect the repair and allow for healing. Usually a sling is used for 4 weeks after surgery. Then a physical therapy program will gradually start improving range of motion and prevent scar formation and stiffness from developing. As healing progresses, exercises to strengthen the shoulder muscles and the rotator cuff will gradually be added to the program typically around 4 to 6 weeks after surgery. Return to early interval throwing can generally be allowed around 3 to 4 months after surgery.Impingement SyndromesAfter minor trauma or repetitive injury, patients may experi-ence pain and discomfort which can be due to irritation of the tissues in the subacromial space. In many cases these shoulder impingement syndromes are caused by simple bursitis or ten-donitis of the long head of the biceps or supraspinatus tendon. Occasionally, impingement syndromes can progress to

1	cases these shoulder impingement syndromes are caused by simple bursitis or ten-donitis of the long head of the biceps or supraspinatus tendon. Occasionally, impingement syndromes can progress to tears of the supraspinatus tendon, which can be confirmed by MRI or ultrasound.The goal of treatment is to reduce pain and restore func-tion. Initial treatment is generally nonsurgical and involves rest, NSAIDs, and physical therapy. If pain is not adequately relieved, an injection of a local anesthetic and corticosteroid may be helpful, for both therapeutic and diagnostic purposes.If conservative treatment does not relieve pain, surgery is recommended, with the goal to excise the bursa and create more subacromial space. Generally, surgery is performed arthroscopi-cally and encompasses bursectomy and subacromial decompres-sion via acromioplasty. If the rotator cuff (supraspinatus tendon) is also injured, arthroscopic repair is usually indicated to restore Brunicardi_Ch43_p1879-p1924.indd

1	and subacromial decompres-sion via acromioplasty. If the rotator cuff (supraspinatus tendon) is also injured, arthroscopic repair is usually indicated to restore Brunicardi_Ch43_p1879-p1924.indd 189622/02/19 10:40 AM 1897ORTHOPEDIC SURGERYCHAPTER 43function and can be accompanied by a bony resection of the inferior portion of the acromion.The Acromioclavicular JointThe acromioclavicular joint is a gliding synovial joint com-prised of the lateral end of the clavicle and medial facet of the acromion, and it has limited mobility. The joint is stabilized by three ligaments: the superior acromioclavicular ligament, the inferior acromioclavicular ligament, and the coracoclavicular ligament. Injuries to these ligaments are commonly sustained by a lateral blow to the shoulder while playing contact sports such as football and ice hockey and may cause displacement of the joint. An acromioclavicular sprain is also referred to as a shoul-der separation. The least severe, types I and II, are

1	sports such as football and ice hockey and may cause displacement of the joint. An acromioclavicular sprain is also referred to as a shoul-der separation. The least severe, types I and II, are typically treated conservatively. Treatment of type III injuries, where the clavicle is displaced up to 100%, is controversial. Some advo-cate for early surgical intervention, while others recommend symptomatic treatment followed by ligament reconstruction if symptoms persist. Types IV to VI, where the coracoclavicular ligaments are completely torn and the clavicle is significantly displaced, are often treated surgically.KNEEThe knee is the largest joint in the human body and is a pivotal hinge joint, which allows flexion and extension as well as some medial and lateral rotation. The knee bears tremendous axial loads as well as torsional and sheer forces, making it vulnerable to both acute injury and the development of osteoarthritis. In sports, the major stabilizing structures such as the ACL

1	axial loads as well as torsional and sheer forces, making it vulnerable to both acute injury and the development of osteoarthritis. In sports, the major stabilizing structures such as the ACL and the medial collateral ligament (MCL) are frequently injured. Other common knee injuries involve the menisci, posterolateral corner (PLC), posterior cruciate ligament (PCL), and patellofemoral joint.MenisciThe menisci are crescent-shaped pieces of fibrocartilage that provide joint stability, shock absorption, load distribution, and proprioception to the knee. Sudden meniscal tears often happen during sports, usually during contact or twisting injuries to the knee. Symptoms associated with a meniscus tear include pain, swelling, stiffness, catching, and locking of the knee. Radio-graphs are typically obtained to assess for a concomitant bony injury, the presence of (early) osteoarthritis, and leg alignment abnormalities. However, since menisci are radiolucent and are not seen on radiographs, an

1	obtained to assess for a concomitant bony injury, the presence of (early) osteoarthritis, and leg alignment abnormalities. However, since menisci are radiolucent and are not seen on radiographs, an MRI is obtained to assess the status of the menisci and the soft tissue surrounding the knee joint (Fig. 43-25). Small tears on the outer edge of the meniscus may not cause symptoms, and provided the knee is stable, nonsurgi-cal treatment may be sufficient.The most commonly performed surgical procedure for meniscus tears is partial (subtotal) meniscectomy. However, it has become increasingly clear over recent years that pres-ervation of the load-distributing function of the meniscus is important in preventing the development of early osteoar-thritis. Research into the use of orthobiologics (e.g., micro-fracture of the notch, fibrin clot) for meniscal repairs has expanded the indications for repair rather than excising the torn fragment, especially if the fragment is large. Tears have been

1	micro-fracture of the notch, fibrin clot) for meniscal repairs has expanded the indications for repair rather than excising the torn fragment, especially if the fragment is large. Tears have been reported in virtually all portions of the meniscus, with radial and longitudinal tears being the most common. Menis-cal root tears are less common, but they are increasingly being recognized as devastating injuries that cause serious alterations of knee contact forces. Surgical techniques are developing to repair the root to restore its function. Meniscus transplantation may be an option for young patients with a largely deficient meniscus.ABLIGHTlabral repairglenoidhumeralheadCFigure 43-24. Imaging and treatment of a shoulder glenoid labrum tear. A. Magnetic resonance imaging axial T1 image showing a tear of the posterior superior labrum (arrow). B. Arthroscopic image with the patient in the lateral decubitus position showing detachment of the torn labrum away from the glenoid. C.

1	image showing a tear of the posterior superior labrum (arrow). B. Arthroscopic image with the patient in the lateral decubitus position showing detachment of the torn labrum away from the glenoid. C. Arthroscopic image demonstrating repair of the labrum to its attachment site using anchors in the glenoid and sutures that fixes the labrum to the glenoid.Brunicardi_Ch43_p1879-p1924.indd 189722/02/19 10:41 AM 1898SPECIFIC CONSIDERATIONSPART IIThe paradigm of treatment of torn menisci is shifting thanks to the development of superior surgical techniques, use of orthobiologics, and promising first results with root repair and meniscus transplantations. Nowadays, physicians are well informed on the significance of meniscal preservation when there is potential for healing.Directly after surgery, the knee is immobilized with a brace and weight-bearing is protected to allow the meniscus to heal. When healing is complete, range of motion and strength will need to be regained. Physical

1	surgery, the knee is immobilized with a brace and weight-bearing is protected to allow the meniscus to heal. When healing is complete, range of motion and strength will need to be regained. Physical therapy is an integral com-ponent of healing and return to play, which usually is allowed between 4 and 6 months after surgery.Collateral LigamentsThe MCL is the most frequently injured knee ligament, which usually occurs after excessive valgus stress of the knee. In more severe injuries, tearing of the meniscus and ACL can also occur, which is known as the “unhappy triad.” This is most often seen in contact sports with a lateral blow to a planted leg, causing a significant valgus force.The MCL has good healing potential, and grade I and II injuries usually improve with bracing and activity modifica-tion. Grade III injuries may also improve with conservative treatment, and often these injuries are initially treated non-operatively. The majority of MCL injuries occur in the mid-substance or

1	Grade III injuries may also improve with conservative treatment, and often these injuries are initially treated non-operatively. The majority of MCL injuries occur in the mid-substance or at the femoral insertion side. There is a small subset of tibial-sided grade III tears that are associated with worse clinical outcome following conservative treatment, and therefore surgical repair is often recommended. Recon-struction is rare because surgical repair is usually effective in restoring the MCL. LCL injuries are much less common than MCL ligament injuries, but, similarly, most are man-aged conservatively.With return of range of motion and normal gait pattern, patients are functionally progressed towards return to sports. A functional brace during sports is often advised.Cruciate LigamentsThe cruciate ligaments are situated centrally within the intercon-dylar notch of the knee. The biomechanical function of both the ACL and the PCL is complex and three-dimensional, but both play an

1	cruciate ligaments are situated centrally within the intercon-dylar notch of the knee. The biomechanical function of both the ACL and the PCL is complex and three-dimensional, but both play an important role in providing anteroposterior and rota-tional stability of the knee.ACL tears are a common sports injury, especially in sports involving sudden stopping and cutting (e.g., soccer, basketball) or contact (e.g., football). A torn ACL will result in altered knee biomechanics and kinematics and thus can potentially lead to the early development of degenerative changes. Since a torn ACL will not heal on its own, surgical ACL reconstruction is generally the treatment of choice in patients who are young and active. Patients with a more sedentary lifestyle and who experi-ence no persisting or disabling instability in daily life may be effectively treated with conservative management (i.e., bracing and physical therapy).ABCFigure 43-25. Imaging and treatment of a knee lateral and meniscus

1	disabling instability in daily life may be effectively treated with conservative management (i.e., bracing and physical therapy).ABCFigure 43-25. Imaging and treatment of a knee lateral and meniscus tear. A. Magnetic resonance imaging sagittal T2 image of the knee showing a displaced bucket-handle lateral meniscus tear (arrow). B. Arthroscopic image showing the remnant rim of the lateral meniscus prior to reduction and fixation of the torn bucket-handle fragment. C. Arthroscopic image after the torn segment is reduced and fixed to the remaining meniscus and the lateral capsule using suture.Brunicardi_Ch43_p1879-p1924.indd 189822/02/19 10:41 AM 1899ORTHOPEDIC SURGERYCHAPTER 43A patient with an ACL tear typically presents with pain and swelling, instability, loss of ROM, joint line tenderness (if there is an associated meniscus injury), and discomfort while walking. The Lachman’s exam is the best clinical test for an ACL tear. Radiographs are obtained to evaluate joint condi-tion

1	(if there is an associated meniscus injury), and discomfort while walking. The Lachman’s exam is the best clinical test for an ACL tear. Radiographs are obtained to evaluate joint condi-tion and possible associated osseous injuries. To visualize the ACL and other soft tissue in the knee, an MRI should be obtained. Although an MRI is not required to make the diag-nosis, the information it provides is invaluable with regard to objectifying anatomic characteristics by taking measurements, assessing concomitant injuries, and presurgical planning in gen-eral (Fig. 43-26).Reconstruction is performed with use of a tendon-graft that will replace the native ACL. Commonly used graft sources include the patellar, hamstrings, and quadriceps tendons. These tendons can be harvested from the same knee (i.e., autografts) during the same procedure. Alternatively, a cadaver graft (i.e., allograft) can be used. Both have their associated benefits, including the absence of donor site morbidity with an

1	(i.e., autografts) during the same procedure. Alternatively, a cadaver graft (i.e., allograft) can be used. Both have their associated benefits, including the absence of donor site morbidity with an allograft and better healing potential with an autograft. As such, it is important to have a discussion with the patient and provide the necessary information for them to make an informed decision regarding graft type.Injuries of the PCL are less common than other knee ligaments. Frequently seen causes are a bent knee hitting a dashboard in a car accident or falling on a knee that is bent during running. A rupture of the PCL is usually better tolerated than ACL rupture, since many tears (i.e., grades I and II) have the potential to heal on their own and do not result in much knee instability. Most grade I and II injuries are treated non-operatively. Combined PCL/PLC, PCL/MCL, and grade III PCL injuries do present a challenge with regard to appropriate management. Chronic PCL-deficient

1	Most grade I and II injuries are treated non-operatively. Combined PCL/PLC, PCL/MCL, and grade III PCL injuries do present a challenge with regard to appropriate management. Chronic PCL-deficient (grade III) knees have an increased incidence of osteoarthrosis, particularly in the patel-lofemoral and medial knee compartments. Indication for sur-gery is influenced by age, activity level, and the presence of concomitant injuries. Different surgical techniques have been proposed; the most common are the “inlay” technique and the transtibial technique.The goal of cruciate ligament (both ACL and PCL) reconstruction is to restore native knee kinematics, provide the patient with the best potential for a successful outcome, and to prevent the development of long-term complications, such as osteoarthrosis.Posterolateral CornerCritical structures of the posterolateral corner are the LCL, pop-liteus tendon, and popliteofibular ligament. These structures each contribute to the static and dynamic

1	CornerCritical structures of the posterolateral corner are the LCL, pop-liteus tendon, and popliteofibular ligament. These structures each contribute to the static and dynamic stability of the knee and are commonly seen in combination with other ligamentous injuries, most notably the ACL. It is important to evaluate the PLC after any knee injury as a deficient PLC causes altered knee biomechanics and subsequently increases the stress on sur-rounding stabilizing structures. As such, it has been shown that a deficient PLC is a primary cause of graft failure in cruciate ligament reconstruction.Acute high-grade injuries of the PLC with obvious defi-cient structures require surgical intervention. Since primary repair becomes increasingly difficult as time between injury and surgery increases, a cut-off of 2 to 3 weeks is usually the limit to repair the deficient structures. With more chronic PLC injuries or midsubstance tears, reconstruction is recommended to restore knee

1	increases, a cut-off of 2 to 3 weeks is usually the limit to repair the deficient structures. With more chronic PLC injuries or midsubstance tears, reconstruction is recommended to restore knee stability.HIPFemoroacetabular ImpingementFemoroacetabular impingement (FAI) is a pathologic condi-tion that refers to impingement of the anterior femoral head-neck junction against the anterosuperior acetabular labrum. This is frequently caused by abnormal bony offset at the femoral head-neck junction and is called CAM impingement, which usually affects young males. On the other hand, a Pincer lesion usually occurs due to abnormal acetabular version and excessive anterolateral acetabular bony rim coverage, or a combination of these, which usually occurs in females. Recognition of FAI can be clinically and radiologically dif-ficult. However, familiarity with this disorder is essential AxyzBCDEFigure 43-26. Magnetic resonance imaging of a torn anterior cruciate ligament (ACL). A–C. Proton

1	clinically and radiologically dif-ficult. However, familiarity with this disorder is essential AxyzBCDEFigure 43-26. Magnetic resonance imaging of a torn anterior cruciate ligament (ACL). A–C. Proton density sagittal cuts, showing antero-posterior tibial insertion site length measurement “x,” intra-articular ligament length measurement “y,” and ACL inclination angle measure-ment “z.” Oblique (in the same plane as the ACL runs) coronal cuts showing a complete ACL tear with separate images of a PL bundle tear (D) and an anteromedial bundle tear (E).Brunicardi_Ch43_p1879-p1924.indd 189922/02/19 10:41 AM 1900SPECIFIC CONSIDERATIONSPART IIbecause FAI can lead to labral tears, cartilage delamination, and, if untreated, osteoarthritis.Commonly, patients present with anterior groin pain exacerbated by activities involving hip flexion or pain over the greater trochanter, as well as grinding or popping. Patients report pain with flexion and internal rotation, and after pro-longed sitting.

1	by activities involving hip flexion or pain over the greater trochanter, as well as grinding or popping. Patients report pain with flexion and internal rotation, and after pro-longed sitting. On examination, there is a decrease in internal rotation that appears out of proportion to the loss of the other ranges of motion, and flexion can also be limited. The impinge-ment test, elicited by 90° of flexion and adduction and internal rotation of the hip, is almost always positive, signified by pain in the groin region.The imaging findings of FAI can be seen on plain radio-graphs, CT scan, MRI, and magnetic resonance angiography. Some of the abnormalities seen include abnormal lateral femo-ral head/neck offset seen as a lateral femoral neck bump, os acetabuli, synovial herniation pits, acetabular over-coverage, hyaline cartilage abnormalities, and labral tears.Treatment of FAI has traditionally been surgical and has evolved from open surgical treatment with acetabuloplasty, to combined

1	over-coverage, hyaline cartilage abnormalities, and labral tears.Treatment of FAI has traditionally been surgical and has evolved from open surgical treatment with acetabuloplasty, to combined open-arthroscopic–assisted techniques, to all arthroscopic techniques. Hip arthroscopy is becoming increas-ingly popular and is being more frequently applied for this indi-cation. This popularity is largely the result of studies reporting on improvement of functional outcome measures with follow-up of 10 years in some studies and with relatively low compli-cation rates.SPINESpinal TraumaIn spinal injury, spinal stability must be assessed and the patient immobilized until the spine is cleared. CT scan is more reliable in assessing spine injury than plain radiographs. In patients with ankylosing spondylitis, an MRI is the best study to rule out occult fracture and epidural bleeding. When neurologic deficits are present a decompressive procedure may be indi-cated. In spinal cord compression, prompt

1	an MRI is the best study to rule out occult fracture and epidural bleeding. When neurologic deficits are present a decompressive procedure may be indi-cated. In spinal cord compression, prompt decompression should be performed. Spinal cord injuries should be triaged to trauma centers since trauma center care is associated with reduced paralysis.Occipital Cervical DislocationMotor vehicle accidents can cause dislocation of the occiput on the condyles of the atlas (C1). Most patients with this injury suffer cervical cord injury and do not survive. Traction on the spine is contraindicated. Treatment consists of stabilization and fusion in situ using a screw plate from the mid cervical spine to the occiput.Fractures of C1 (Jefferson Fracture)Fracture of the C1 ring was described by Jefferson in 1920. The thin anterior and posterior rings of the C1 vertebra fracture with axial loads. C1 fracture causes the lateral masses of C1 to spread, which can be visible on an open-mouth view. A

1	in 1920. The thin anterior and posterior rings of the C1 vertebra fracture with axial loads. C1 fracture causes the lateral masses of C1 to spread, which can be visible on an open-mouth view. A lateral view of the C-spine may show the fracture; however, this injury could be missed due to inadequate visualization of the occipitocervical junction. CT scan is the ideal study for a Jefferson Fracture. The transverse ligament may be ruptured with a Jefferson fracture, and this will render the fracture unstable, which can cause injury to the spinal cord (Fig. 43-27). Jefferson fractures may be associ-ated with other spine fractures. This injury is rarely associated with neurologic injury. The treatment of a Jefferson fracture is based on the integrity of the transverse ligament. The integrity of the transverse ligament is assessed by the amount of C1 lat-eral mass displacement determined by open-mouth radiograph and CT scan. Significantly displaced fractures (less than 7 mm) indicate

1	of the transverse ligament is assessed by the amount of C1 lat-eral mass displacement determined by open-mouth radiograph and CT scan. Significantly displaced fractures (less than 7 mm) indicate disruption of the transverse ligament. An increase in the atlanto-dense interval (ADI) may indicate a transverse ligament injury. Normally, the ADI is less than 3 mm, as seen on the lat-eral view. An unstable injury with a rupture of the transverse ligament may need a posterior C1-C2 fusion.Bracing with a cervicothoracic orthosis or a halo ring and vest is the recommended treatment for nondisplaced and mini-mally displaced fractures; significantly displaced unstable frac-tures require more definitive surgical treatment.Fractures of C2 (Odontoid Fracture)Half of normal cervical rotation occurs at the atlantoaxial joint. The odontoid (Dens) is a small bony process which arises from the body of C2, and articulates with the body of C1 (the Atlas). There are three types of odontoid fractures (Fig.

1	atlantoaxial joint. The odontoid (Dens) is a small bony process which arises from the body of C2, and articulates with the body of C1 (the Atlas). There are three types of odontoid fractures (Fig. 43-28). Type I fractures are the most common and are avulsion fractures off the tip of the dens. Type I fractures occur when there is tension applied to the alar ligaments (which span from the tip of the odontoid to the skull bypassing the C1 vertebra). Type I frac-tures are stable and managed nonoperatively.Type II fractures, at the base of the odontoid, results from lateral loading forces. Operative stabilization in patients with a high risk of fracture nonunion is the preferred treatment since immobilization in a halo vest results in nonunion rates ranging 89Figure 43-27. Jefferson fracture with and without ligamentous injury. Note the increased ADI and the potential compression of the spinal cord (orange color).Brunicardi_Ch43_p1879-p1924.indd 190022/02/19 10:41 AM 1901ORTHOPEDIC

1	with and without ligamentous injury. Note the increased ADI and the potential compression of the spinal cord (orange color).Brunicardi_Ch43_p1879-p1924.indd 190022/02/19 10:41 AM 1901ORTHOPEDIC SURGERYCHAPTER 43from 20% to 80%. The risk of nonunion includes displacement greater than 5 mm, angulation greater than 10 degrees, age over 50 years, smoking, and delayed diagnosis more than 4 weeks. Nonunion occurs due to interruption of the blood supply. Trans-fixing the odontoid fracture with a screw maintains rotational movement. Posterior fusion of C1 on C2 is another option, but this results in decreased cervical spine rotation; 50% of rotation of the cervical spine comes from C1 and C2 joint.Type III fractures extend into the body of C2, below the origin of the odontoid process. The cancellous bone is rich in blood supply and usually heals well. Type III fractures are gen-erally treated with a halo brace.Hangman’s Fractures of C2Hangman’s Fractures are a bilateral fracture of the

1	bone is rich in blood supply and usually heals well. Type III fractures are gen-erally treated with a halo brace.Hangman’s Fractures of C2Hangman’s Fractures are a bilateral fracture of the pars inter-articularis (Fig. 43-29). The spinal canal is usually widened, and neurological deficits rarely occur (Fig. 43-30). It results from sudden extension forces on the neck. Treatment is simple immobilization in a halo vest. Higher energy injuries causing severe extension forces can dislocate the C2-3 facet complex and damage the C2-3 disc. Significantly displaced Hangman’s fractures are managed by internal fixation and bone grafting between C2 and C3. When the fracture is severely angulated, it may indicate a flexion distraction injury, and traction on the C-spine may exacerbate the injury.Compression Fracture of the Cervical SpineIn C3 to C7 an axial load can cause fracture of the endplate while preserving the posterior cortex of the vertebral body. These fractures generally heal well and

1	Fracture of the Cervical SpineIn C3 to C7 an axial load can cause fracture of the endplate while preserving the posterior cortex of the vertebral body. These fractures generally heal well and are treated nonopera-tively with analgesics and a cervical brace.Burst Fractures of the Cervical SpineBurst Fractures of the cervical spine usually result from axial loads such as in diving and motor vehicle accidents. The injury results in displacement of bony fragments into the canal, injur-ing the spinal cord. Burst fractures are treated surgically by anterior decompression (corpectomy) and reconstruction using a bone graft strut stabilized with a plate and screws.Unilateral and Bilateral Facet DislocationThis injury is usually associated with motor vehicle accidents. A restrained passenger can suffer forced flexion with distrac-tion resulting in dislocation of the facets. The diagnosis can be made on lateral radiographs. Unilateral facet dislocation can be missed on an X-ray. It usually shows

1	forced flexion with distrac-tion resulting in dislocation of the facets. The diagnosis can be made on lateral radiographs. Unilateral facet dislocation can be missed on an X-ray. It usually shows less than 25% sublux-ation on an X-ray, and it affects the nerve roots. Bilateral facet dislocation will have more than 50% subluxation on an X-ray and may cause severe spinal cord injury. Treatment consists of closed reduction with axial traction utilizing cranial tongs, grad-uated application of weight, and periodic X-rays. The patient is kept awake for safety concerns. A closed reduction should not be done if the patient is not awake. Facet dislocations could be associated with disc herniation. An MRI is the study of choice to rule out disc herniations and should be done prior to reduc-tion in an unconscious patient or prior to open reduction and/or surgical fixation. When a reduction is obtained, the patient is taken to surgery for fusion, which may be performed anteriorly Figure

1	in an unconscious patient or prior to open reduction and/or surgical fixation. When a reduction is obtained, the patient is taken to surgery for fusion, which may be performed anteriorly Figure 43-28. The three types of Odontoid fractures.Figure 43-29. Lateral view shows displaced hangman’s fracture.Figure 43-30. Hangman’s fracture cross-section view shows wid-ening of the canal, and therefore the spinal cord is not affected.Brunicardi_Ch43_p1879-p1924.indd 190122/02/19 10:41 AM 1902SPECIFIC CONSIDERATIONSPART IIor posteriorly. Anterior surgery is necessary if the patient has an associated herniated disc.Clay-Shoveler’s InjuryClay-shoveler’s injury can result from a motor vehicle accident or from shoveling soil or clay. The injury (of C6, C7, T1, and T2) is the result of avulsion fracture of the spinous process by the paraspinal muscle forces (Fig. 43-31). The fracture is treated nonoperatively with analgesics and a soft collar.FRACTURES OF THE THORACIC AND LUMBAR SPINEThoracic

1	of the spinous process by the paraspinal muscle forces (Fig. 43-31). The fracture is treated nonoperatively with analgesics and a soft collar.FRACTURES OF THE THORACIC AND LUMBAR SPINEThoracic Lumbar Spine InjuryThe ribs stabilize fractures of the thoracic spine, making these fractures more stable than similar fractures of the lumbar spine. Neurologic injuries are more common in the thoracic and proxi-mal lumbar spine because of the presence of the spinal cord, which ends at the L2 level, as well as the small spinal canal diameter of the thoracic spine.Compression FractureCompression fractures result from osteoporosis as well as trauma. Compression fractures involve a fracture of the anterior part of the vertebral body without associated posterior cortex fracture. Thoracolumbar compression fractures are treated non-operatively with braces and analgesics.Burst FractureBurst fractures are caused by falls and high-energy automo-bile accidents. The posterior cortex fracture (middle

1	fractures are treated non-operatively with braces and analgesics.Burst FractureBurst fractures are caused by falls and high-energy automo-bile accidents. The posterior cortex fracture (middle column involvement) differentiates the burst fracture from a compres-sion fracture. The injury may be associated with neurological deficits due to retropulsion of bone into the canal. A vertical lamina fracture may contain an invaginated segment of the dura mater with accompanying nerve root injury and dural tear. Wid-ening of the pedicle in an AP view of the spine will indicate a burst fracture. CT scan will define the bony injury, and an MRI will show compression of the neural elements and any injury to the posterior ligaments.Treatment is nonoperative with an orthoses and mobiliza-tion of the patient if the fracture is stable. Surgery is done for decompression and destabilization of the spine if the patient has neurologic deficits or if the fracture is unstable.Seatbelt Injuries (Flexion

1	patient if the fracture is stable. Surgery is done for decompression and destabilization of the spine if the patient has neurologic deficits or if the fracture is unstable.Seatbelt Injuries (Flexion Distraction Injuries)A seatbelt injury occurs when there is acute forward flexion of the trunk and anterior (i.e., seatbelt) restraint. The pelvis and upper torso move forward, and failure of the spine under tension begins with the posterior elements. Tearing of the dorsal fascia, the interspinous ligament, dislocation of the facets, and tearing of the discs occurs. Seatbelt injury may be bony or ligamen-tous (Fig. 43-32). The bone of the spinous process, the lamina, the pedicles, and the vertebral body fail in tension (“chance Figure 43-32. Seatbelt injuries (flexion-distraction injuries) can be bony or ligamentous.Figure 43-31. Fracture of the transverse process of C6 or C7.Brunicardi_Ch43_p1879-p1924.indd 190222/02/19 10:41 AM 1903ORTHOPEDIC SURGERYCHAPTER 43fracture”). The bony

1	can be bony or ligamentous.Figure 43-31. Fracture of the transverse process of C6 or C7.Brunicardi_Ch43_p1879-p1924.indd 190222/02/19 10:41 AM 1903ORTHOPEDIC SURGERYCHAPTER 43fracture”). The bony injury could be stable. Flexion distraction injuries involving the soft tissue, with injury to the posterior spine elements, are usually unstable. This unstable injury will require internal fixation and fusion with bone grafting. This spine injury may be associated with a colon injury, especially in children.Fracture Dislocations of the SpineFracture dislocations of the spine displace the bony elements by translation or rotation, resulting in canal narrowing and nerve injury.Reduction of the displaced bones is the best way to improve the canal dimensions.Patients with fracture dislocations of the spine and par-tial nerve function can recover. Fracture dislocations are treated operatively with surgical stabilization.Disc HerniationDisc herniation, most common between ages of 20 and 50, can

1	the spine and par-tial nerve function can recover. Fracture dislocations are treated operatively with surgical stabilization.Disc HerniationDisc herniation, most common between ages of 20 and 50, can occur in the cervical, thoracic, or the lumbar spine, and consists of a tear of the annulus allowing the nucleus pulposus material to extrude through the annulus and enter the canal, pressing on the exiting nerve or the “traversing” nerve roots. In the cervical spine, spinal cord compression can occur.Symptoms of most disc herniations resolve within 8 weeks as the nerve root accommodates and inflammation recedes. The bulk of the extruded nucleus pulposus resorbs over time. When symptoms persist beyond 6 to 8 weeks, surgery with excision of the involved disc and decompression of the nerve roots may be indicated.In cervical disc herniation, an anterior approach to the spine is performed with dissection through a transverse incision on the neck. Dissection is carried between the trachea,

1	may be indicated.In cervical disc herniation, an anterior approach to the spine is performed with dissection through a transverse incision on the neck. Dissection is carried between the trachea, esopha-gus medially, and the carotid sheath laterally. The disc is then removed. The disc space is usually filled with bone graft to fuse the vertebrae. A locking screw low profile titanium plate is then attached to the vertebrae.Posterior decompression and laminotomy exposes the pos-terior elements of the spine. A portion of the lamina is removed to allow access to the canal to correct foraminal impingement or to remove lateral disc herniations. While the posterior approach does not require fusion with plates and screws, central disc her-niation cannot be managed through a posterior approach since the spinal cord cannot be safely retracted.In thoracic spine disc herniation, the posterior approach is contraindicated because it may lead to paralysis.For lumbar disc herniation, a midline

1	since the spinal cord cannot be safely retracted.In thoracic spine disc herniation, the posterior approach is contraindicated because it may lead to paralysis.For lumbar disc herniation, a midline incision is used, and laminotomy allows visualization of the lateral recess. Retraction of the dura allows visualization of the traversing nerve roots as well as of the disc fragment.Cauda Equina SyndromeCauda equina syndrome is uncommon and occurs from a central disc herniation (Fig. 43-33). This can be a difficult diagnosis to make; however, it is a true emergency, and a delay in diagnosis can lead to permanent impairment. The patient will complain of back pain with bilateral leg pain. Bladder and bowel diffi-culty such as incontinence and frequency, saddle anesthesia, decreased perianal sensation, impotence, diminished rectal tone, and motor deficits. MRI will show a central disc herniation. Treatment is with urgent diagnosis and urgent surgical decom-pression. The results are better if

1	impotence, diminished rectal tone, and motor deficits. MRI will show a central disc herniation. Treatment is with urgent diagnosis and urgent surgical decom-pression. The results are better if the decompression is done within 48 hours of onset of symptoms. A central disc herniation causing cauda equina should be differentiated from a postero-lateral disc herniation. The posterolateral disc herniation usu-ally affects a nerve root and can be treated conservatively, at least initially. In the case of central disc herniation, it affects the cauda equina (the lumbosacral nerve roots), and this is a surgical emergency. Spontaneous recovery does not occur, and the outcome is catastrophic, including permanent loss of bowel and bladder control as well as the ability to have an erection if treatment is delayed.Spinal StenosisA loss of hydration of the discs causes loss of disc height and bulging of annular tissue and the ligamentum flavum, which effectively narrows the canal (spinal stenosis).

1	is delayed.Spinal StenosisA loss of hydration of the discs causes loss of disc height and bulging of annular tissue and the ligamentum flavum, which effectively narrows the canal (spinal stenosis). Osteophyte for-mation on the facet joints can also cause nerve impingement. Cervical stenosis can cause myelopathic symptoms (hyper-reflexia, problems with fine hand dexterity, balance problems resulting in gait disturbance, weakness, and pain). In patients with low back pain and gait disturbance, obtain an MRI of the cervical spine to rule out cervical myelopathy. Pathology of the lumbo-sacral spine does not cause gait disturbances.Lumbar stenosis causes neurogenic claudication (pro-gressive pain, weakness, and numbness in the legs). The clau-dication symptoms result from standing and walking, which increases lumbar lordosis. Extension of the spine decreases the spinal canal diameter as well as the foramen size and worsens the condition. The symptoms resolve with sitting and bending

1	which increases lumbar lordosis. Extension of the spine decreases the spinal canal diameter as well as the foramen size and worsens the condition. The symptoms resolve with sitting and bending forward (i.e., over a shopping cart) (Fig. 43-34). Flexion of the spine increases the spinal canal diameter as well as the foramen size and decrease the symptoms. The patient may have a normal neurologic exam, and it is important to study the vascular status of the patient and differentiate between neurologic and vascular claudication. Examine the pulses, prescribe a noninvasive vas-cular study if necessary. In general, walking causes the symp-toms for both conditions, and standing relieves the vascular claudication symptoms. Spinal stenosis is treated with NSAIDs, epidural steroid injections, and physical therapy. Resistant cases may require surgical decompression.Spinal stenosis usually occurs in patients over 50 years of age. With degenerative spondylolisthesis or scoliosis, fusion with

1	and physical therapy. Resistant cases may require surgical decompression.Spinal stenosis usually occurs in patients over 50 years of age. With degenerative spondylolisthesis or scoliosis, fusion with instrumentation is usually required to prevent progression of the deformity.Figure 43-33. Image showing central disc herniation affecting the cauda equina and the classic posterolateral disc herniation affecting a nerve root.Brunicardi_Ch43_p1879-p1924.indd 190322/02/19 10:41 AM 1904SPECIFIC CONSIDERATIONSPART IIBack Pain and Degenerative Disc DiseaseBack pain occurs in the majority of adults but is usually self-limited, resolving in 1 to 2 weeks. Chronic unremitting back pain may suggest the possibility of infection, malignancy, or metastatic disease.While radiographs are one option in the management of disabling low back pain, they are ineffective at ruling out malig-nancy, and radiographic findings correlate poorly with symp-toms. Patients with severe degenerative symptoms may have

1	management of disabling low back pain, they are ineffective at ruling out malig-nancy, and radiographic findings correlate poorly with symp-toms. Patients with severe degenerative symptoms may have no pain, while others with mild degenerative findings complain of severe pain. The potential for secondary gain and psychiatric problems and the unpredictable results of spine fusion add to the difficulty of diagnosis and choosing a treatment plan.Intervertebral disc replacement prostheses are experimen-tal in the treatment of degenerative disc disease. The potential for loosening, creation of wear debris, and bone loss complicat-ing revision surgery are concerns, as are the proximity of the device to the spinal canal and the great vessels.ScoliosisScoliosis is a lateral curvature of the spine. Lateral bending of the spine is always accompanied by rotational deformity (coupling).In order to measure the severity of scoliosis, lines are drawn along the endplates of the vertebral bodies at

1	Lateral bending of the spine is always accompanied by rotational deformity (coupling).In order to measure the severity of scoliosis, lines are drawn along the endplates of the vertebral bodies at either end of the curve, and the angle formed when these lines intersect determines the magnitude of the curve.Scoliotic curves are classified as congenital, degenera-tive, metabolic (mucopolysaccharidoses), neurogenic (cerebral palsy), and myogenic curves (muscular dystrophy). Idiopathic scoliosis is the most common form and represents a spectrum of genetic disease.Adults with scoliosis may present with axial pain and imbalance in posture. Treatment for scoliosis may include anti-inflammatory medications, therapy, and activity modification. In severe cases with objective deformity, surgical correction of the deformity may be indicated.Idiopathic ScoliosisThe majority of idiopathic scoliosis curves become apparent during adolescence and progress during skeletal growth. Ini-tial management

1	of the deformity may be indicated.Idiopathic ScoliosisThe majority of idiopathic scoliosis curves become apparent during adolescence and progress during skeletal growth. Ini-tial management consists of observation. Rapidly progressing curves are treated with braces. Brace treatment is recommended for curves between 20 and 40 degrees. For patients with large curves, surgical intervention may be needed using rods with grafting and fusion.Neuromuscular ScoliosisNeurologic conditions such as polio and cerebral palsy can lead to “uncompensated” scoliosis curves where the patient is unable to lean with his upper body to restore balance. Scoliosis correc-tion surgery may be needed to facilitate sitting balance and to avoid skin breakdown caused by pelvic obliquity.JOINT RECONSTRUCTIONIntroduction to ArthritisArthritis refers to a large number of medical conditions, includ-ing osteoarthritis, rheumatoid arthritis, septic arthritis, and post-traumatic arthritis. Each has the potential to lead

1	to ArthritisArthritis refers to a large number of medical conditions, includ-ing osteoarthritis, rheumatoid arthritis, septic arthritis, and post-traumatic arthritis. Each has the potential to lead to loss of articular cartilage lining the joints. According to the CDC and the National Health Interview Survey, approximately 55 million adults (22% of the U.S. population) have been diag-nosed with some form of arthritis. This number is pro-jected to grow to an astounding 67 million adults by 2030 (or 25% of the U.S. population).Arthritis causes pain, loss of range of motion, decreased ability to perform work duties or participate in social functions, and decreased quality of life. The number of individuals suffer-ing from arthritic conditions will continue to rise as the “baby boomer” generation enters old age and the prevalence of obesity rises in the U.S. population, as age and obesity are two major factors in the onset of arthritis.Examination of the PatientA thorough history and

1	enters old age and the prevalence of obesity rises in the U.S. population, as age and obesity are two major factors in the onset of arthritis.Examination of the PatientA thorough history and physical examination is indicated for all orthopedic patients. Patient history should include loca-tion, quality, severity, timing, and radiation of pain along with any referred pain, associated signs and symptoms, modify-ing factors, or prior treatments, including both conservative and surgical measures. Other details within the history and physical examination are equally important in establishing a diagnosis and successfully developing a treatment plan. If you listen carefully to your patients, they will often tell you their diagnosis.For example, location of “hip pain” can narrow a dif-ferential diagnosis. Patients with activity-related groin pain often are found to have hip arthritis, whereas patients with 10Figure 43-34. Person is seen bending over shopping cart to improve

1	a dif-ferential diagnosis. Patients with activity-related groin pain often are found to have hip arthritis, whereas patients with 10Figure 43-34. Person is seen bending over shopping cart to improve symptoms.Brunicardi_Ch43_p1879-p1924.indd 190422/02/19 10:41 AM 1905ORTHOPEDIC SURGERYCHAPTER 43peritrochanteric pain (lateral hip pain) may be suffering from trochanteric bursitis. The importance of listening and focusing on the patient’s description of location and type of pain cannot be overemphasized.Physical examination should begin by observing the patient’s gait, both with and without assistive devices if pos-sible. This demonstrates the extent of the patient’s functional deficit and the effect of the patient’s pain. Typical gait pat-terns include antalgic gait due to pain, or a “Trendelenburg gait” (Fig. 43-35) where abductor weakness may lead to a poor outcome following total hip arthroplasty. Other aspects of the exam include assessment of leg length discrepancy, joint

1	a “Trendelenburg gait” (Fig. 43-35) where abductor weakness may lead to a poor outcome following total hip arthroplasty. Other aspects of the exam include assessment of leg length discrepancy, joint con-tractures, skin changes, assessment for prior surgical incisions to identify prior treatments or plan future surgical approaches, neurovascular status, and strength, as well as range of motion. These details document functional status and help to formulate a differential diagnosis. Patients with “hip pain” may have lumbar spinal stenosis, radiculopathy, or vascular disease that may play a large role in their presentation. Once an appropriate physi-cal examination is performed, weight-bearing radiographs are needed. Advanced imaging, including CT and MRI, are rarely indicated in the initial workup. Once a diagnosis is made, spe-cific treatment directed towards the patient’s condition can be initiated. The goals of treatment are to improve pain, preserve motion, and maximize patient

1	workup. Once a diagnosis is made, spe-cific treatment directed towards the patient’s condition can be initiated. The goals of treatment are to improve pain, preserve motion, and maximize patient function, independence, and qual-ity of life.Nonoperative Management and Prevention of ArthritisNonoperative measures to treat arthritis include weight loss, activity modification, rest, physical therapy, NSAIDs, bracing, and assistive devices such a cane or walker. These treatments have the potential to decrease symptoms and improve function and quality of life. For example, holding a cane on the opposite side of the symptomatic extremity reduces the forces across the hip joint and subsequently decreases hip pain (Fig. 43-36). In nearly all cases, it is best to treat patients nonoperatively prior to recommending surgery.Health and exercise can also play a role in the prevention of arthritis. Weight loss of as little as 11 lbs (5 kg) has been shown to decrease the risk of developing knee

1	prior to recommending surgery.Health and exercise can also play a role in the prevention of arthritis. Weight loss of as little as 11 lbs (5 kg) has been shown to decrease the risk of developing knee osteoarthritis in 11women by 50%. Similarly, patients who engage in regu-lar physical activity have a lower incidence of arthritis. However, despite nonoperative treatment, surgical intervention may be required to effectively manage patient symptoms.InjectionsJoint injections are commonly performed into the knee and shoulder. Common injections into the knee include corticoste-roids and hyaluronic-acid gels. Corticosteroid injections can decrease inflammation within the joint. These injections are usually administered in combination with a local anesthetic, such as lidocaine, in order to provide more immediate relief for both diagnostic and therapeutic purposes. If the patient has immediate relief of pain with injection of the joint, this localizes the source of the patient’s pain to the

1	more immediate relief for both diagnostic and therapeutic purposes. If the patient has immediate relief of pain with injection of the joint, this localizes the source of the patient’s pain to the joint and may assist with diagnosis. Diagnostic hip injections are particularly helpful in distinguishing pain resulting from hip versus lumbar spine pathology. Any benefit received is therapeutic for the patient. Hyaluronic acid injections in the knee are frequently used and are commonly referred to as “viscosupplementation.” The viscosity of the synovial fluid is increased by hyaluronic acid, but its role and mechanism are not well defined yet. There is a risk of joint infection, cartilage injury from the needle, hemarthrosis, and failure to receive benefit. Short-term altered glucose metabolism in diabetic patients is common with cor-ticosteroid injections. The efficacy of hyaluronic acid injec-tions has been questioned by recent evidence summarized in the American Academy of Orthopaedic

1	in diabetic patients is common with cor-ticosteroid injections. The efficacy of hyaluronic acid injec-tions has been questioned by recent evidence summarized in the American Academy of Orthopaedic Surgeons’ Clinical Practice Guidelines.Figure 43-35. Trendelenburg Gait resulting from weakness of abductor muscles.Figure 43-36. Holding the cane on the opposite side of the pathol-ogy is beneficial in decreasing arthritis pain in the hip.Brunicardi_Ch43_p1879-p1924.indd 190522/02/19 10:41 AM 1906SPECIFIC CONSIDERATIONSPART IISurgical Management of ArthritisThe most commonly performed procedure for arthritis of a major joint is arthroplasty, or joint replacement. Joint replace-ments, including hip and knee arthroplasty, are considered two of the most successful procedures performed in all of surgery. However, nonarthroplasty options exist and are typically per-formed for certain indications and goals.Osteotomy. Osteotomy is cutting of the bone to change the position of the fragments,

1	of surgery. However, nonarthroplasty options exist and are typically per-formed for certain indications and goals.Osteotomy. Osteotomy is cutting of the bone to change the position of the fragments, thereby improving rotation, align-ment, or angulation. Osteotomy can be performed for both con-genital and acquired deformities that contribute to the patient’s pain or development or progression of disease. Pelvic and femo-ral osteotomy can be utilized in the treatment of developmen-tal dysplasia of the hip. The position of the acetabulum can be altered with pelvic osteotomies in order to provide more appro-priate coverage of the femoral head, which is typically deficient anteriorly and laterally. Femoral osteotomies can be performed to correct version and varus/valgus deformity of the femoral neck. Osteotomies are performed to obtain more normal align-ment and coverage of the femoral head within the acetabulum to prevent or delay future disease.An osteotomy commonly used in the knee is a

1	neck. Osteotomies are performed to obtain more normal align-ment and coverage of the femoral head within the acetabulum to prevent or delay future disease.An osteotomy commonly used in the knee is a proxi-mal tibia osteotomy. An adult patient who presents with iso-lated medial compartment knee arthritis and associated varus deformity would be a candidate for a valgus-producing (high tibial) osteotomy. An osteotomy that realigns the knee into slight valgus has the potential to off-load the medial com-partment, slow disease progression, and prevent or delay the need for further procedures (unicompartmental or total knee arthroplasties).Arthrodesis. Arthrodesis is a treatment option for severe arthritis where the overlying articular cartilage is removed and two opposing bones heal together with the use of hardware (internal or temporary external fixation) often supplemented by bone graft. After successful arthrodesis, no motion is possible through the joint and the source of pain is

1	with the use of hardware (internal or temporary external fixation) often supplemented by bone graft. After successful arthrodesis, no motion is possible through the joint and the source of pain is removed. Arthrodesis of large joints, such as the knee, shoulder, or hip, are typically explored as an option in the face of infection, in older adult, low-demand patients or in young, active patients who are con-sidered too young for a joint replacement (out of concern for component wear and the need for early revision). Arthrodesis can also serve as a “last resort” procedure in orthopedics when joint preserving treatments fail due to fracture or infection. Ankle arthrodesis is the primary procedure performed in adult patients with traumatic arthritis of the ankle.Joint Arthroplasty/Joint Replacement. Joint arthroplasty is the most common option for patients suffering from pain associated with arthritis in a joint. The surfaces of the bones are replaced after removing the damaged articular

1	arthroplasty is the most common option for patients suffering from pain associated with arthritis in a joint. The surfaces of the bones are replaced after removing the damaged articular cartilage. The amount of bone and the determination of how to make the bone cuts is made based on preoperative radiographs and templating, cutting guides, anatomic measurements, and soft tissue/ligament balancing. The cut bony surfaces are covered with new compo-nents, usually made of metal, ceramic, or polyethylene. These new components are sized to appropriately match the patient, based on templated preoperative radiographs, intraoperative measurements, and examination for stability, leg length, align-ment, and range of motion.If all compartments or surfaces of the joint are replaced, the arthroplasty is referred to as a total joint arthroplasty. In comparison, if only one surface or compartment of the joint is replaced, it is referred to as hemiarthroplasty (hip, shoulder) or unicompartmental

1	is referred to as a total joint arthroplasty. In comparison, if only one surface or compartment of the joint is replaced, it is referred to as hemiarthroplasty (hip, shoulder) or unicompartmental arthroplasty (knee). Total hip and knee arthroplasties are considered among the most successful of all surgical procedures performed in terms of patient outcome and improvement in pain.Hip Arthroplasty Background Hip arthroplasty is utilized for end stage arthri-tis in the hip that has failed a reasonable trial of nonoperative measures (Fig. 43-37). Conventional hip arthroplasty commonly refers to total hip arthroplasty where both the femoral head and acetabulum are replaced or resurfaced, respectively. Finally, hemiarthroplasty describes the replacement of the femoral head and neck with a stemmed femoral component in isolation. The acetabulum is not addressed surgically.History of Hip Arthroplasty The history of hip arthroplasty (hip replacement) may be broken down into “Pre-Charnley” and

1	femoral component in isolation. The acetabulum is not addressed surgically.History of Hip Arthroplasty The history of hip arthroplasty (hip replacement) may be broken down into “Pre-Charnley” and “Post-Charnley” eras, referring to the significant contri-butions of Sir John Charnley to the evolution of hip arthro-plasty. Prior to Charnley’s contributions, hip arthroplasty consisted of a variety of procedures with highly variable results. Early attempts at relieving hip pain were made with interpositional arthroplasty, where tissue layers, plastic, or metal were placed between the worn articular surfaces. Frac-ture of the interposed material or loosening of components often led to failure.Later attempts introduced stemmed components to improve fixation. One of the earliest femoral components was designed by Austin-Moore. This prosthesis replaced the femoral head and neck with a metal component secured into the femoral shaft with a stem extending down the diaphysis. This prosthesis was

1	was designed by Austin-Moore. This prosthesis replaced the femoral head and neck with a metal component secured into the femoral shaft with a stem extending down the diaphysis. This prosthesis was utilized in hemiarthroplasty for many years and served as Figure 43-37. Osteoarthritis femoral head. Note erosion of weight-bearing cartilage and peripheral osteophytes.Brunicardi_Ch43_p1879-p1924.indd 190622/02/19 10:41 AM 1907ORTHOPEDIC SURGERYCHAPTER 43a step in the development of total hip arthroplasty with the later addition of the acetabular component.Surgical Approaches to the Hip A variety of approaches to the hip joint have been utilized in joint arthroplasty, includ-ing anterior approach (Smith Petersen), anterolateral approach (Watson-Jones), lateral approach (Hardinge), and posterior approach (Kocher Langenbach). Each approach contains a unique set of advantages and disadvantages. The following is a brief summary of the most common approaches that are utilized in total hip

1	approach (Kocher Langenbach). Each approach contains a unique set of advantages and disadvantages. The following is a brief summary of the most common approaches that are utilized in total hip arthroplasty.Anterior approach (Smith Petersen): This approach is an internervous and intermuscular approach. It utilizes the inter-nervous plane between the femoral nerve and superior gluteal nerve. Superficially, the plane between the sartorius (femo-ral nerve) and tensor fasciae lata (superior gluteal nerve) is dissected in the deep layer and the plane between the rectus femoris (femoral nerve) and gluteus medius (superior glu-teal nerve) is dissected. Advantages to this approach include supine positioning, use of intraoperative fluoroscopy for acetabular component positioning, and discontinuation of all ambulatory assistive devices 1 week earlier than other approached. Downsides include difficult preparation and placement of the femoral component with higher rate of femoral fracture/femoral

1	of all ambulatory assistive devices 1 week earlier than other approached. Downsides include difficult preparation and placement of the femoral component with higher rate of femoral fracture/femoral component revision, higher rate of wound complications, and lack of a true extensile approach.Posterior approach (Kocher–Langenbach): The posterior approach is a muscle-splitting approach without an interner-vous plane. After incising the skin and subcutaneous fat, the fascia lata is incised along with the gluteus maximus. The short external rotators are exposed and dissected, includ-ing the piriformis, superior and inferior gemelli, obturator internus and externus, and quadratus femoris. This allows internal rotation of the hip along with flexion and adduction to dislocate the hip. The posterior approach with posterior soft tissue repair has no increased rate of dislocation com-pared to the anterior approach. The posterior approach is extensile and provides excellent exposure of both the

1	approach with posterior soft tissue repair has no increased rate of dislocation com-pared to the anterior approach. The posterior approach is extensile and provides excellent exposure of both the femur and acetabulum for complex and revision cases.Lateral approach (Hardinge): While there have been many modifications to the original Hardinge approach, first described in 1982, most involve releasing the anterior one-third of the gluteus medius, underlying minimus, abductor tendon, and vastus lateralis distally in one sleeve off of the greater trochanter. The capsule is then incised to expose the hip joint. Care must be taken to protect the superior gluteal nerve during this exposure, which lies 5 cm proximal to the tip of the greater trochanter.Exposure of the acetabulum is excellent with the modified Hardinge approach, which is extensile. However, access to the posterior column is limited compared to the posterior approach. The increased risk of postoperative Trendelenburg gait, other

1	modified Hardinge approach, which is extensile. However, access to the posterior column is limited compared to the posterior approach. The increased risk of postoperative Trendelenburg gait, other pathologic gait, and heterotopic ossification compared to all other approaches to the hip have made it far less commonly performed than the posterior approach. Minimally invasive total hip arthroplasty is associated with decreased visualiza-tion intraoperatively and associated risks of component malposition, intraoperative fracture, and nerve or vascular injury. In fact, the only documented benefit of minimally inva-sive techniques appears to be a smaller incision, but with increased soft tissue tension intraoperatively comes the risk of compromised wound healing and periprosthetic joint infection.Bearing Surfaces in Hip Arthroplasty The most common combination of bearing surfaces used in total hip arthroplasty is a metal (generally cobalt chrome) or ceramic prosthetic head, articulating

1	Surfaces in Hip Arthroplasty The most common combination of bearing surfaces used in total hip arthroplasty is a metal (generally cobalt chrome) or ceramic prosthetic head, articulating with a polyethylene liner. Metal on metal (MOM) articulations have largely been abandoned in total hip arthro-plasty as they are associated with production of metal ions that deposit in solid organs, pseudotumors that are locally destruc-tive to soft tissue/bone, and risk of early failure (Fig. 43-38). Ceramic on ceramic articulations have the lowest friction of all current bearing combinations. However, ceramic may fracture or squeak in ceramic on ceramic total hip arthroplasties.Alignment of Hip Arthroplasty Components Proper align-ment of hip arthroplasty components is vital to a successful procedure and patient outcome. Surgeons aim for appropriate alignment of components to restore a functional and stable range of motion. This is accomplished with combined version of the femoral and acetabular

1	and patient outcome. Surgeons aim for appropriate alignment of components to restore a functional and stable range of motion. This is accomplished with combined version of the femoral and acetabular components, appropriate abduc-tion of the acetabular components, and staying true to Sir John Charnley’s principles: establishing a low friction articulation, 12Figure 43-38. Failed ceramic on metal hip arthroplasty components. Note the metallic staining on the ceramic femoral head.Brunicardi_Ch43_p1879-p1924.indd 190722/02/19 10:41 AM 1908SPECIFIC CONSIDERATIONSPART IImedializing the acetabular component and center of rotation and restoring abductor length and tension with restoration of appropriate length and femoral offset. Inappropriate placement of components can lead to early failure, accelerated component wear, dislocation, need for revision surgery, as well as poor patient outcomes and satisfaction.Knee Arthroplasty Background Knee arthroplasty is indicated for end-stage

1	accelerated component wear, dislocation, need for revision surgery, as well as poor patient outcomes and satisfaction.Knee Arthroplasty Background Knee arthroplasty is indicated for end-stage arthritis that has failed a reasonable trial of nonoperative mea-sures (Figs. 43-39 and 43-40). Knee arthroplasty commonly refers to total knee arthroplasty where the distal femur, tibia, and patella are resurfaced after any remaining articular cartilage and a layer of subchondral bone are resected. A unicompartmental knee arthroplasty consists of replacing one compartment of the knee, most commonly the medial compartment.Surgical Approach to the Knee Total knee arthroplasty is generally accomplished through a medial parapatellar approach. This approach utilizes a longitudinal skin incision extending, on average, 5 cm proximal to the patella to the medial aspect of the tibial tubercle distally. Dissection is carried down to the capsule. To gain access to the joint, an arthrotomy is performed

1	on average, 5 cm proximal to the patella to the medial aspect of the tibial tubercle distally. Dissection is carried down to the capsule. To gain access to the joint, an arthrotomy is performed medial to the patella extending proximally along the most medial aspect of the quadriceps tendon and distally just medial to the patellar tendon. This approach provides excellent exposure to all three compartments of the knee after patellar dislocation.Once the joint surfaces are adequately exposed, remain-ing articular cartilage and a thin layer of underlying bone are removed prior to placement of prosthetic components. Bone cuts are made based on preoperative templating, cutting guides, ligament balancing, and anatomic measurements (Figs. 43-41 and 43-42).Bearing Surfaces in Knee Arthroplasty The femoral com-ponent consists of a metal prosthetic cap sized to fit the normal shape of the distal femur. The tibia is cut perpendicular to the anatomic and mechanical axis, and a flat, stemmed, metal

1	femoral com-ponent consists of a metal prosthetic cap sized to fit the normal shape of the distal femur. The tibia is cut perpendicular to the anatomic and mechanical axis, and a flat, stemmed, metal tray is placed that serves as a base plate for a polyethylene bearing surface. The patella is usually resurfaced with a polyethylene component.Two types of primary total knee arthroplasty systems exist, including cruciate retaining and posterior stabilized systems. As the name implies, with cruciate retaining systems, the PCL is retained in hopes of preserving more normal knee structures and minimizing bone loss, while in posterior stabilized systems the ligament is sacrificed and the components are designed to accommodate for the loss. These two systems have equivalent results in knee arthroplasty.Alignment and Balancing in Knee Arthroplasty Appropri-ate sizing and positioning of the components and balancing of the size and geometry of bony gaps in flexion and extension are essential for

1	and Balancing in Knee Arthroplasty Appropri-ate sizing and positioning of the components and balancing of the size and geometry of bony gaps in flexion and extension are essential for a successful knee arthroplasty. Inappropriate com-ponent position can lead to early wear and failure, instability, pain, and stiffness.Computer Navigation, Robotics, and Joint ArthroplastyComputer-navigated joint arthroplasty has the theoretical ben-efit of more accurate and consistent placement of arthroplasty components through intraoperative feedback to the surgeon regarding component position, planned bone cuts, and align-ment. Disadvantages include increased costs of the technology, prolonged operative times, and risk of infection/fracture at the sites of intraoperative sensor placement within bone. Use of Figure 43-39. Valgus deformity. Osteoarthritis of lateral compart-ment right knee.Figure 43-40. Osteoarthritis of both knees. Note varus alignment of right knee and valgus alignment of left knee

1	of Figure 43-39. Valgus deformity. Osteoarthritis of lateral compart-ment right knee.Figure 43-40. Osteoarthritis of both knees. Note varus alignment of right knee and valgus alignment of left knee (windswept deformity).Brunicardi_Ch43_p1879-p1924.indd 190822/02/19 10:41 AM 1909ORTHOPEDIC SURGERYCHAPTER 43ABFigure 43-41. A. Varus knee with osteoarthritis. B. Right total knee replacement.Figure 43-42. Computer-assisted robotic targeting arm for total knee replacement.Brunicardi_Ch43_p1879-p1924.indd 190922/02/19 10:41 AM 1910SPECIFIC CONSIDERATIONSPART IIcomputer navigation in total joint arthroplasty has been shown to minimize outliers in alignment, but there has been no proven benefit in survival or function secondary to computer-navigated or robotic-assisted joint replacement.Fixation Options in Joint ArthroplastyComponents in hip and knee arthroplasty can be secured with cement or biologic fixation. The cement most commonly used is polymethylmethacrylate (PMMA). PMMA serves

1	Options in Joint ArthroplastyComponents in hip and knee arthroplasty can be secured with cement or biologic fixation. The cement most commonly used is polymethylmethacrylate (PMMA). PMMA serves as a grout between the component and the bone surface. Components secured without cement are grit blasted or porous coated to allow bony on growth or ingrowth, respectively. Hydroxyapatite can also be utilized on implant surfaces to promote bone ingrowth or ongrowth through osteoconductive properties. A majority of hip joint arthroplasty components are now secured without cement, where initial fixation of components is accomplished through press fit techniques. In knee arthroplasty, cement utilization is generally preferred. In hip replacement patients where biologic fixation is unreliable, such as older adults, osteoporotic or previ-ously irradiated cement may be a better option. With revision total hip arthroplasty, cement fixation of components has been shown to lead to earlier mechanical

1	as older adults, osteoporotic or previ-ously irradiated cement may be a better option. With revision total hip arthroplasty, cement fixation of components has been shown to lead to earlier mechanical failure.Osteolysis and Aseptic Loosening. Osteolysis is a term used to describe abnormal resorption of bone. Osteolysis can be caused by underlying infection, metastatic disease, or in case of joint replacement, the production of wear debris. Even with appropriately positioned components, some wear of the bear-ing surfaces is expected over time. However, wear rates as well as the size and amount of wear debris differs with the bearing surface. Friction in ceramic on ceramic articulations is the low-est of all bearing surfaces; however, there is increased risk of component fracture and postoperative “squeaking.” In metal or ceramic on polyethylene articulations, wear debris is produced, and polyethylene particles are phagocytized by local macro-phages. Activated macrophages lead to an

1	“squeaking.” In metal or ceramic on polyethylene articulations, wear debris is produced, and polyethylene particles are phagocytized by local macro-phages. Activated macrophages lead to an osteolytic process and bone resorption. Particulate methylmethacrylate cement debris can also play a role in osteolysis by damaging the polyethylene bearing surface. Osteolysis has been shown to be significantly decreased with the advent and use of highly cross-linked poly-ethylene. Improperly positioned components or patient-related factors such as high impact activities can lead to increased wear. A substantial osteolytic response may occur and lead to compo-nent micromotion and aseptic loosening. Patients who present to clinic with pain following joint arthroplasty and an increas-ing zone of osteolysis in the periprosthetic region frequently need revision surgery (Fig. 43-43). Alternative bearing surfaces continue to be explored in hopes of decreasing component wear, associated osteolysis, and

1	in the periprosthetic region frequently need revision surgery (Fig. 43-43). Alternative bearing surfaces continue to be explored in hopes of decreasing component wear, associated osteolysis, and aseptic loosening.Complications in Joint ArthroplastyThe risk of any complication following joint arthroplasty proce-dures falls in the range of 5% to 10%. Risks shared by hip and knee arthroplasties include infection, intraoperative or postop-erative fracture, vascular injury, need for intraoperative or post-operative blood transfusion, nerve injury or nerve palsy (most commonly involving the deep peroneal nerve and loss of ankle dorsiflexion), stress shielding, component fracture or wear, and medical complications, including venous thromboembolic dis-ease (DVT and PE), myocardial infarction, or cerebrovascular accident. Complications unique to total hip arthroplasty include dislocation, leg length discrepancy, and iliopsoas impingement or tendonitis.Dislocation Following Hip

1	infarction, or cerebrovascular accident. Complications unique to total hip arthroplasty include dislocation, leg length discrepancy, and iliopsoas impingement or tendonitis.Dislocation Following Hip Arthroplasty. Dislocation can result from malpositioned components (inadequate com-bined version of the femoral stem and acetabular component; extremes of inclination of the acetabular component), noncom-pliance, cognitive or neuromuscular disorders, compromised soft tissue envelope from revision surgery, fracture, or insuf-ficient restoration of length and/or offset. Comparable disloca-tion rates have been found with anterolateral, lateral, anterior, and posterior with soft tissue repair (approximately 0.5%) approaches. History, physical examination, and radiographs are vital to proper treatment of dislocation. Closed reduction can usually be performed with conscious sedation and gentle traction or manipulation. Rarely, open reduction may be neces-sary. Component position should be

1	treatment of dislocation. Closed reduction can usually be performed with conscious sedation and gentle traction or manipulation. Rarely, open reduction may be neces-sary. Component position should be assessed in patients with multiple dislocations. Patients with recurrent dislocations and suboptimally positioned components may require component revision. Patients with recurrent dislocations and properly posi-tioned components should be considered for conversion to a device with a larger prosthetic head (dual mobility construct) or a constrained total hip arthroplasty implant that provides improved stability.ORTHOPEDIC PATHOLOGY AND ONCOLOGYDiagnosis of Malignant Bone TumorsHistory. Diagnosis of musculoskeletal tumors begins with a thorough patient history. A history of unremitting pain unre-lated to activity or pain that interferes with sleep suggests malig-nancy. Patient age can help in establishing a differential. Round Figure 43-43. Failed total knee replacement. Note subsided,

1	unre-lated to activity or pain that interferes with sleep suggests malig-nancy. Patient age can help in establishing a differential. Round Figure 43-43. Failed total knee replacement. Note subsided, loose, tibial component.Brunicardi_Ch43_p1879-p1924.indd 191022/02/19 10:41 AM 1911ORTHOPEDIC SURGERYCHAPTER 43blue cell lesions are most likely neuroblastoma in a 5-year-old, Ewing’s sarcoma in a 10-year-old, lymphoma in a 20-year-old, and myeloma in a 60-year-old. Gender also aids in the differen-tial. For instance, giant cell tumor is more common in females, while osteosarcoma is more common in males. Multiple bone involvement may suggest enchondromas (Ollier disease, Maffucci’s syndrome) or osteochondromas (multiple hereditary exostoses).Laboratory Tests. Laboratory tests determine the level of cellular turnover (lactate dehydrogenase [LDH]) or of bone destruction (calcium, alkaline phosphatase). Elevated prostate-specific antigen (PSA) suggests prostate

1	tests determine the level of cellular turnover (lactate dehydrogenase [LDH]) or of bone destruction (calcium, alkaline phosphatase). Elevated prostate-specific antigen (PSA) suggests prostate cancer.Imaging. Radiographic studies are critical in the diagnosis of bony tumors. Radiographs can help assess the aggressive-ness of the tumor. Four questions should be addressed when assessing radiographs: (a) Where is the tumor—in which bone (Table 43-1) and in which part of the bone is the lesion? (Table 43-2) (b) What is the tumor doing to the bone (clinical behavior)? (c) What is the bone doing to the tumor (biologic response)? and (d) What is the matrix pattern? Matrix is the acellular interstitial substance produced by tumor cells. Particu-lar attention should be paid to the junction between the tumor and the host bone since this margin can also indicate the aggres-siveness of the tumor. Ewing’s sarcoma has a characteristic “onion skin” periosteal reaction pattern. This reaction pattern

1	the tumor and the host bone since this margin can also indicate the aggres-siveness of the tumor. Ewing’s sarcoma has a characteristic “onion skin” periosteal reaction pattern. This reaction pattern also occurs in other tumors and infections.OSTEOSARCOMAThe most common primary malignant bone tumor is osteosar-coma (Fig. 43-44). Osteosarcomas are classified as osteoblas-tic, chondroblastic, fibroblastic, telangiectatic, round cell, or MFH-like, according to the predominant cell type. Most osteo-sarcomas present in patients between 10 and 20 years of age. Secondary osteosarcomas occur in older patients in abnormal bone affected by Paget’s disease, radiation, or bone infarct.Intramedullary OsteosarcomaThis is the most common primary sarcoma of the bone. It usu-ally occurs in the distal femur or the proximal tibia in young people. This condition may also occur at the proximal humerus, proximal femur, or pelvis. It usually presents itself as a high-grade extracompartmental disease. It can

1	or the proximal tibia in young people. This condition may also occur at the proximal humerus, proximal femur, or pelvis. It usually presents itself as a high-grade extracompartmental disease. It can metastasize to the bone, which is called a “skip lesion,” but the lung is the primary site of metastases. Long term survival is 75% with adequate treatment. The response to chemotherapy (98% necrosis of the Table 43-1Common locations of bone tumorsFEMURDistal posteriorParosteal osteosarcomaDistal anteriorPeriosteal osteosarcoma, periosteal chondroma or chondrosarcoma, myositis ossificansTIBIAAdamantinoma, chondromyxoid, fibromaHANDS AND FEETEnchondroma, exostosisCalcaneusUnicameral bone cyst, lipoma, chondroblastoma, osteosarcomaSPINEAnteriorMetastatic, myeloma, Paget’s disease, vascular malformation, giant cell tumorPosteriorOsteoid osteoma, osteoblastoma; aneurysmal bone cystPELVISMetastatic, myeloma, chondrosarcoma, giant cell tumor, aneurysmal bone cyst, Paget’s disease, Ewing’s

1	malformation, giant cell tumorPosteriorOsteoid osteoma, osteoblastoma; aneurysmal bone cystPELVISMetastatic, myeloma, chondrosarcoma, giant cell tumor, aneurysmal bone cyst, Paget’s disease, Ewing’s SarcomaSACRUMChordoma (midline), chondrosarcoma, giant cell tumor, aneurysmal bone cyst, lymphomaRIBSMetastatic, myeloma, fibrous dysplasia, chondrosarcomaTable 43-2Tumor location in boneEpiphysisChondroblastoma, clear cell chondrosarcoma, giant cell tumor (GCT), infection, dysplasia epiphysealis hemimelica (DEH)MetaphysisMost common site of involvementDiaphysisF-Fibrous dysplasia, EG-Eosinophilic Granuloma, N-Nonossifying Fibroma, O-Osteoid osteoma, M-Myeloma, A-Adamantinoma, S-Simple Bone Cyst, H-Histiocytosis, I-InfectionFigure 43-44. Osteosarcoma.Brunicardi_Ch43_p1879-p1924.indd 191122/02/19 10:41 AM 1912SPECIFIC CONSIDERATIONSPART IItumor after chemotherapy is a good sign) and the stage of the disease determines the prognosis. Young patients may present with pain and swelling,

1	10:41 AM 1912SPECIFIC CONSIDERATIONSPART IItumor after chemotherapy is a good sign) and the stage of the disease determines the prognosis. Young patients may present with pain and swelling, with X-rays showing plastic lesions in some areas of destruction with periosteal reaction called “Codman’s Triangle.” X-rays may show bone formation with a sunburst appearance. MRI should involve the entire bone to diagnose the skip metastasis. CT scans of the chest are usually done to find primary metastases. Alkaline phosphatase is usu-ally high. Diagnosis is typically confirmed with a biopsy, which is done after staging the tumor. Proper biopsy technique should be employed, which includes longitudinal incisions. As a gen-eral rule, the biopsy should be done by the same surgeon who will provide the definitive treatment for the patient. Treatment of osteosarcoma will be preoperative chemotherapy and wide resection, followed by postoperative chemotherapy.Parosteal OsteosarcomaParosteal

1	the definitive treatment for the patient. Treatment of osteosarcoma will be preoperative chemotherapy and wide resection, followed by postoperative chemotherapy.Parosteal OsteosarcomaParosteal osteosarcoma is a low-grade surface osteosarcoma that appears as if it were stuck on the bone, especially in the pos-terior distal femoral metaphysis (80%). The differential diagno-sis includes osteochondroma and myositis ossificans. Treatment consists of wide excision. The prognosis is 95% 5-year survival as it is a low-grade tumor.Periosteal OsteosarcomaPeriosteal osteosarcoma is a high-grade tumor. It occurs on the anterior surface of the distal femur or proximal tibia. The lesion appears chondroblastic on histology. Radiographs show scalloping of the underlying cortex with a “sunburst” periosteal reaction. Treatment is chemotherapy and wide surgical excision. The 5-year survival rate is 80%.Paget’s SarcomaPaget’s sarcoma is a rare complication of Paget’s disease. In Paget’s disease with

1	reaction. Treatment is chemotherapy and wide surgical excision. The 5-year survival rate is 80%.Paget’s SarcomaPaget’s sarcoma is a rare complication of Paget’s disease. In Paget’s disease with multiple bone involvement, osteogenic sar-coma, fibrosarcoma, chondrosarcoma, and MFH have occurred, most often in the pelvis, but also in the femur, humerus, spine, and skull. This malignant transformation occurs in less than 1% of patients. The patient will complain of new onset pain and swelling. The physician must have a high index of suspicion in patients with Paget’s who previously had no pain. Imaging may demonstrate osteolytic areas and loss of normal fatty marrow and multifocal lesions. Treatment of Paget’s sarcoma is chemo-therapy and wide surgical excision. The prognosis is poor, and the 5-year survival rate is less than 10%.Radiation-Induced SarcomaThe three criteria for diagnosis of radiation-induced sarcoma are (a) histology different from the original lesion, (b) sarcoma develops

1	5-year survival rate is less than 10%.Radiation-Induced SarcomaThe three criteria for diagnosis of radiation-induced sarcoma are (a) histology different from the original lesion, (b) sarcoma develops in the irradiated field, and (c) a 3to 5-year latent period between radiation and sarcoma development. Radiation for carcinoma of the breast and cervix can result in osteosar-coma, chondrosarcoma, fibrosarcoma, or MFH. Treatment is a combination of chemotherapy and surgery.EWING’S SARCOMAEwing’s sarcoma is the second most common primary bone tumor in patients under 25 years of age. The typical presenta-tion is a tumor in the diaphysis of long bones, especially the femur. It can also be seen in the pelvis, the proximal tibia, and proximal humerus, and it is usually seen in young white males. It has a t(11:22) translocation and positive CD99. The patient may have pain and fever with an elevated sedimentation rate and WBC count; the condition may be confused with an infection. An “onion

1	It has a t(11:22) translocation and positive CD99. The patient may have pain and fever with an elevated sedimentation rate and WBC count; the condition may be confused with an infection. An “onion skin” periosteal reaction may be seen on radiographs. A large soft-tissue extension from the primary bone tumor may be seen, and histology reveals a small, round, blue cell tumor (Fig. 43-45). Diagnosis is confirmed with bone marrow biopsy specimen. Bone scan can identify multiple lesions. Treatment is chemotherapy and surgery or radiation therapy for spine or pelvic lesions.CARTILAGE-FORMING TUMORSChondrosarcomasChondrosarcomas typically occur in male patients over 40 years of age, and they are the third most common primary bone malignancies. Primary chondrosarcomas can form clear cell, mesenchymal, or dedifferentiated neoplastic cartilage. Secondary chondrosarcomas may also develop in preexisting lesions such as exostoses or enchondromas. Pelvis, shoulder, and ribs are common locations.

1	or dedifferentiated neoplastic cartilage. Secondary chondrosarcomas may also develop in preexisting lesions such as exostoses or enchondromas. Pelvis, shoulder, and ribs are common locations. Chondroid or “popcorn” cal-cifications are typical on radiographs. Clear cell chondrosar-coma and mesenchymal chondrosarcoma occur in younger patients (second to fifth decades of life). Clear cell chondro-sarcomas are low-grade lesions that often affect the epiphyses. The dedifferentiated chondrosarcoma is a high-grade chondro-sarcoma with a less than 10% survival rate. It has a biomor-phic histology, with a chondroid component and a high-grade spindle cell component.The treatment of chondrosarcoma is surgical excision, since cells are not chemosensitive or radiosensitive. For high-grade lesions, wide or radical resection is recommended. Pelvic and scapular chondrosarcomas have a high recurrence rate, and adjuvant chemotherapy does not improve survival rates.FIBROUS LESIONS OF BONEDesmoplastic

1	or radical resection is recommended. Pelvic and scapular chondrosarcomas have a high recurrence rate, and adjuvant chemotherapy does not improve survival rates.FIBROUS LESIONS OF BONEDesmoplastic FibromaDesmoplastic fibroma is a rare tumor occurring in the mandible, femur, pelvis, radius, or tibia in young adults. It presents as a painful lesion. Radiographs show a metadiaphyseal “soap bubble” appearance and endosteal scalloping. Histology resembles Figure 43-45. Ewing’s sarcoma.Brunicardi_Ch43_p1879-p1924.indd 191222/02/19 10:41 AM 1913ORTHOPEDIC SURGERYCHAPTER 43desmoid tumors or fibromatosis. Recommended treatment is wide excision to avoid recurrence.Malignant Fibrous Histiocytoma of BoneMFH occurs in the metadiaphysis of long bones after condi-tions like nonossifying fibromas and bone infarcts. It may pres-ent with pain or by a pathologic fracture. Radiographs typically show destructive lesions with soft-tissue extension. Histology resembles osteosarcoma with pleomorphic

1	and bone infarcts. It may pres-ent with pain or by a pathologic fracture. Radiographs typically show destructive lesions with soft-tissue extension. Histology resembles osteosarcoma with pleomorphic spindle cells, his-tiocytes, and giant cells, but no neoplastic osteoid formation. Treatment is chemotherapy and wide surgical excision.Malignant Vascular TumorsHemangioendothelioma. Hemangioendothelioma is a malig-nant neoplasm arising from vascular endothelium in long bones and most often occurs in the lower extremity. Radiographs show a metadiaphyseal lytic lesion with a “soap bubble” appearance. Histology reveals eosinophilic cells in a basophilic stroma. Lesions may be multifocal. Treatment consists of curettage for low-grade lesions and wide excision +/radiation therapy for high-grade lesions.Hemangiopericytoma. Hemangiopericytoma is usually a solitary lesion occurring in the soft tissues or the axial skeleton and proximal long bones in middle-aged or older adult males. Histology

1	lesions.Hemangiopericytoma. Hemangiopericytoma is usually a solitary lesion occurring in the soft tissues or the axial skeleton and proximal long bones in middle-aged or older adult males. Histology reveals branching “staghorn” vascular spaces. The tumor cells resemble cells normally seen adjacent to capillaries. Treatment is wide excision.Angiosarcoma of Bone. Angiosarcoma is a soft tissue malig-nancy usually seen in older adult males; chronic vascular stasis is a risk factor. Histology reveals vascular channels with ana-plasia. Treatment is wide excision, or if the tumor is surgically inaccessible, radiation.MISCELLANEOUS TUMORSGiant Cell Tumor of BoneGiant cell tumor is a benign aggressive tumor. Fifty per-cent of these tumors occur around the knee, especially at the distal femur and the proximal tibial. Giant cell tumors may also occur in the distal radius, proximal humerus, and pelvis (especially the sacrum ala) in women 20 to 40 years of age. Presenting complaints include pain

1	the proximal tibial. Giant cell tumors may also occur in the distal radius, proximal humerus, and pelvis (especially the sacrum ala) in women 20 to 40 years of age. Presenting complaints include pain and pathologic fracture. Imaging reveals eccentric, epimetaphyseal lytic lesions erod-ing the subchondral bone. Histology reveals multinucleate giant cells and mononuclear stromal cells. An abundance of giant cells in the field can help establish the diagnosis, and the nuclei of giant cells appear the same as the stroma cells (all nuclei look similar) (Fig. 43-46). Giant cell tumors must be differentiated from the Brown tumor of hyperparathy-roidism. While both have giant cells, hyperparathyroidism affects multiple areas, and the serum calcium is not normal. Epiphyseal lesions such as chondroblastoma should also be part of the differential diagnosis. These tumors can occasion-ally metastasize to the chest. Primary malignant giant cell tumor has a poor prognosis. Treatment of giant cell

1	should also be part of the differential diagnosis. These tumors can occasion-ally metastasize to the chest. Primary malignant giant cell tumor has a poor prognosis. Treatment of giant cell tumors is with curettage and high-speed burr. Recurrence rates are high with simple curettage, and the use of adjuvants such as cryosurgery, phenol, or polymethylmethacrylate bone cement may help decrease recurrence rates. After pathologic fractures, wide excision with reconstruction or amputation may be required.Adamantinoma and Osteofibrous DysplasiaAdamantinomas are low-grade malignant tumors usually seen in the tibia (Fig. 43-47). Adamantinomas are capable of metas-tasizing to the lung. The patient may present with pain and/or bowing of the tibia. X-ray reveals multiple lucent lesions on the cortex of the tibia. Histology reveals a biphasic tumor with nests of epithelial cells and fibrous stroma (see Fig. 43-46). Osteofibrous dysplasia is considered the precursor to ada-mantinoma and should be

1	of the tibia. Histology reveals a biphasic tumor with nests of epithelial cells and fibrous stroma (see Fig. 43-46). Osteofibrous dysplasia is considered the precursor to ada-mantinoma and should be part of the differential diagnosis. Osteofibrous dysplasia is a benign lesion, usually occurring in children, at the anterior tibia, which is treated with obser-vation. The treatment of adamantinoma is with wide surgical excision.Figure 43-46. Giant cell tumor.Figure 43-47. Typical location of adamantinoma.Brunicardi_Ch43_p1879-p1924.indd 191322/02/19 10:41 AM 1914SPECIFIC CONSIDERATIONSPART IIPrimary Lymphoma of BonePrimary lymphoma accounts for about 5% of all neoplasms of bone. Long bone involvement is more frequent than spine. Lym-phoma of bone typically occurs in males in their forties. Histol-ogy reveals large B cell lymphomas. Treatment is a combination of chemotherapy and radiation. Surgery may be required for stabilization of pathologic fractures.ChordomaChordoma arises from

1	Histol-ogy reveals large B cell lymphomas. Treatment is a combination of chemotherapy and radiation. Surgery may be required for stabilization of pathologic fractures.ChordomaChordoma arises from notochordal remnants in the sacrum. It is usually midline in location. These tumors are found in middle-aged to older men and presents with bladder and bowel symptoms due to involvement of the cauda equina. Visual-ization of the lesion may be difficult because of the bowel gas shadow. Diagnosis may be delayed. An MRI shows a destructive extensile midline lesion with a large soft tissue mass. Histology shows epithelioid cells arranged in cords with vacuolated foamy physaliferous cells. These cells are keratin positive. Treatment is surgical excision and muscle flaps and a mesh for reconstruction. Urinary diversion and colostomy may be needed for loss of bladder and bowel control. Local recurrence is common.Multiple MyelomaMyeloma, the most common primary bone malignancy, is a pro-liferative

1	Urinary diversion and colostomy may be needed for loss of bladder and bowel control. Local recurrence is common.Multiple MyelomaMyeloma, the most common primary bone malignancy, is a pro-liferative disorder of B cells with plasma cells producing immu-noglobins. These plasma cells have a classic eccentric nucleus giving a “signet ring” appearance (Fig. 43-48). Evidence of monoclonal protein in the serum and/or urine (Bence Jones proteinuria), and hypercalcemia, renal insufficiency, anemia, or bone disease are usually present.Presenting symptoms in myeloma range from bone pain and osteopenia to focal lytic lesions with pathologic fractures and hypercalcemia. Myeloma protein 1-α stimulates osteo-clast formation. Osteoclast activating factors increase recep-tor activator of nuclear factor κB ligand (RANKL) in the bone marrow. RANKL induces osteoclast differentiation and acti-vation. Myeloma cells inhibit osteoblast differentiation and activity. Serum and urine electrophoresis detect the M

1	(RANKL) in the bone marrow. RANKL induces osteoclast differentiation and acti-vation. Myeloma cells inhibit osteoblast differentiation and activity. Serum and urine electrophoresis detect the M protein. Workup also includes complete blood cell count, erythrocyte sedimentation rate, calcium levels, renal function assessment, β2-microglobulin levels, and a skeletal survey. X-ray will show multiple punched out lytic lesions. Bone scans may be cold in about 30% of cases. The SPEP, UPEP, and bone marrow biopsy are helpful in diagnosis. Histology will show atypical plasma cells with eccentric nuclei, its appearance resembles a “signet ring (Fig. 43-49).” Plasmacytoma is a solitary tumor with a negative bone marrow biopsy, usually treated with radiation to the lesion. Myeloma is treated with bisphosphonates, chemo-therapy, stem cell transplantation, and radiation therapy. Surgi-cal stabilization and irradiation is done for pathologic fractures or impending fractures. Many patients with

1	bisphosphonates, chemo-therapy, stem cell transplantation, and radiation therapy. Surgi-cal stabilization and irradiation is done for pathologic fractures or impending fractures. Many patients with myeloma develop a vertebral compression fracture. Kyphoplasty can be useful in providing pain relief. The risks of cement extravasation and related complications are lower with kyphoplasty than with ver-tebroplasty. If there is instability or if there is neural compres-sion, surgical stabilization may be required.METASTATIC BONE TUMORSMetastatic bone tumors are more common than primary bone tumors. Metastatic tumors affect the lung, liver, and bone. Cancers that commonly metastasize to bone are breast, lung, thyroid, kidney, and prostate. In patients older than 40 years of age, metastases and myeloma are the most common causes of destructive lesions in bone. The most common site of involve-ment is the axial skeleton, especially the thoracic spine, and proximal ends of long bones, especially

1	are the most common causes of destructive lesions in bone. The most common site of involve-ment is the axial skeleton, especially the thoracic spine, and proximal ends of long bones, especially the proximal femur. Lung and renal cell carcinomas can metastasize distal to the knee and elbow. Malignant cells are able to detach from one location and set up a focus at a distant site. The tumor activates osteoclasts and causes destruction of the bone, a mechanism that involves the RANK/RANKL pathway. The patient may present with pain, pathologic fractures, or the manifestation of hypercalcemia. Workup of a patient with a suspected metastatic disease to bone and an unknown primary tumor should include CT of the chest, abdomen, and pelvis. The extent of the disease is evaluated by bone scans (myeloma and thyroid are usually cold in bone scans), mammography, tumor markers, serum, and urine electrophoresis (SPEP and UPEP). A biopsy may be nec-essary to rule out primary bone lesions if the

1	(myeloma and thyroid are usually cold in bone scans), mammography, tumor markers, serum, and urine electrophoresis (SPEP and UPEP). A biopsy may be nec-essary to rule out primary bone lesions if the primary site is not identified. Treatment of bone tumors depends on the diagnosis, as metastatic tumors are treated differently than primary bone tumors. Metastatic tumors are usually treated by bisphospho-nates and by surgical stabilization with postoperative radiation if warranted. Primary bone tumors are usually treated by wide excision with chemotherapy in high-grade tumors (chondrosar-coma are treated only with wide excision). Radiation therapy can be used in Ewing’s.Multiple myelomaEccentricnucleusSignet ring appearanceFigure 43-48. Signet ring.Figure 43-49. Multiple Myeloma showing the eccentric nuclei and the signet appearance of cells.Brunicardi_Ch43_p1879-p1924.indd 191422/02/19 10:41 AM 1915ORTHOPEDIC SURGERYCHAPTER 43PEDIATRIC ORTHOPEDICSBirth InjuriesNeonatal Brachial

1	the eccentric nuclei and the signet appearance of cells.Brunicardi_Ch43_p1879-p1924.indd 191422/02/19 10:41 AM 1915ORTHOPEDIC SURGERYCHAPTER 43PEDIATRIC ORTHOPEDICSBirth InjuriesNeonatal Brachial Plexus Palsy. Injury of the brachial plexus during delivery occurs in 2 births in every 1000. Large birth weight, forceps delivery, breech presentation, and pro-longed second stage of labor with shoulder dystocia are risk factors. Brachial plexus injury usually represents a stretch injury on the nerve roots of the upper or lower plexus.Upper plexus injuries (Erb-Duchenne) are lesions mani-fested by weakness of shoulder abductors and external rota-tors as well as the elbow flexors (Fig. 43-50). The hand is not involved. It has a good prognosis, if the biceps function is pres-ent early.In lower plexus injury, the hand is involved, with defor-mity of the fingers. An ipsilateral Horner’s Syndrome consist-ing of ptosis, myosis, anhidrosis, and enophthalmos may occur indicating a preganglionic

1	plexus injury, the hand is involved, with defor-mity of the fingers. An ipsilateral Horner’s Syndrome consist-ing of ptosis, myosis, anhidrosis, and enophthalmos may occur indicating a preganglionic injury of the T1 cervical sympathetic nerve. This condition has a poor prognosis.Management is therapy and gentle, passive range-of-motion exercises to preserve motion in the shoulder and prevent muscle contractures and joint incongruency in the early neonatal period while awaiting return of neurologic function and motor reinnervation. Early surgical intervention for the brachial plexus is indicated in infants who did not recover elbow flexion by 3 months of age, as they are anticipated to have a poor chance of full recovery.Surgical intervention includes microsurgical repair proce-dures in the form of neurolysis, nerve transfer, or nerve grafts. Later orthopedic reconstruction such as muscle rebalancing procedures may be considered to improve function around the shoulder.Cerebral

1	in the form of neurolysis, nerve transfer, or nerve grafts. Later orthopedic reconstruction such as muscle rebalancing procedures may be considered to improve function around the shoulder.Cerebral Palsy. Cerebral palsy results from an injury to the brain, which may be associated with mental impairment. Cere-bral palsy is classified as spastic, athetotic, or ataxic and may present with spasticity, hemiplegia, diplegia, or scoliosis. The typical cerebral palsy patient is hyperreflexic with increased muscle tone and spasm. Treatment includes tendon lengthen-ing procedures, release of contractures, and tendon transfers to maintain motion and function.Figure 43-50. Erb’s point.Reserve zone(resting zone)Proliferative zoneHypertrophic zoneZone ofmaturationZone ofdegenerationWWeaWeWeaWeaWeaWeaWeaWWWWWWWWWWWWWWWkkzkzkzkzkkzkzkzkzkzkkkkkkkk zkkkkkzkkoWeak zoneZone ofprovisionalcalcificationFigure 43-51. Different zones of the growth plate.Hip dislocation or subluxation results from unbalanced

1	zkkkkkzkkoWeak zoneZone ofprovisionalcalcificationFigure 43-51. Different zones of the growth plate.Hip dislocation or subluxation results from unbalanced muscle forces in many cerebral palsy patients. Early treatment consists of soft tissue releases in the form of adductor tendon releases, iliopsoas releases, and immobilization in an abduction brace.In older children with severe deformity, bony procedures in the form of open reduction and femoral or acetabular osteoto-mies are usually required. Femoral head resection is considered to be a salvage procedure in nonambulatory patients with pain-ful dislocated hips.Knee flexion contractures are treated with hamstring muscle lengthenings and immobilization in knee extension braces.Foot and ankle deformities are treated even in nonambu-latory patients to facilitate shoe wear. The most common foot deformity in cerebral palsy is an equinovalgus foot caused by heel cord contracture and peroneal spasm. Tendon balancing is usually necessary,

1	patients to facilitate shoe wear. The most common foot deformity in cerebral palsy is an equinovalgus foot caused by heel cord contracture and peroneal spasm. Tendon balancing is usually necessary, and bony reconstruction may also be needed in severe cases.Skeletal GrowthInjury, inflammatory disease, and developmental disorders in actively growing bones requires special attention to preserve the growth plates. The pediatric skeleton is incompletely ossi-fied making the diagnosis of an injury difficult, since signifi-cant portions of the skeleton are invisible on radiographs. The epiphysis, generally containing an articular surface, is found at the ends of the long bone. The physis, or growth plate, is found beneath the epiphysis. The physis is divided into spe-cific zones: the reserve zone, the zone of proliferation, and the hypertrophic zone. The hypertrophic zone has three phases: the maturation zone, the degenerative zone, and the zone of calcification (Fig. 43-51).Injury or insult

1	the zone of proliferation, and the hypertrophic zone. The hypertrophic zone has three phases: the maturation zone, the degenerative zone, and the zone of calcification (Fig. 43-51).Injury or insult to the growth plate can lead to premature growth arrest or angular deformity of the limb. Surrounding the metaphyseal and diaphyseal bone is the periosteum. This meta-bolically active layer of tissue synthesizes new bone onto the diaphyseal and metaphyseal bone and provides circumferential growth of the bones.Ossification centers in the epiphysis appear in a predict-able order and can help determine “bone age.”Brunicardi_Ch43_p1879-p1924.indd 191522/02/19 10:41 AM 1916SPECIFIC CONSIDERATIONSPART IIPediatric FracturesIn a pediatric patient, the epiphyseal growth plate is unossified and weak and is at risk of fracture. Reduction and stabiliza-tion of epiphyseal fractures is critical to minimize permanent growth disturbances and deformity. Fractures near the growth plate have significant

1	and is at risk of fracture. Reduction and stabiliza-tion of epiphyseal fractures is critical to minimize permanent growth disturbances and deformity. Fractures near the growth plate have significant potential to remodel. For example, 80% of the growth of the humerus occurs from the proximal humeral growth plate; therefore, severely displaced proximal humeral fracture can remodel in the younger age group.Classification of Growth Plate InjuriesSalter and Harris described a useful classification for epiphyseal fractures (Fig. 43-52). A Salter-Harris type I injury is a simple transverse fracture through the physis. A Salter-Harris type II fracture contains a component of fracture through the growth plate in continuity with a fracture of the metaphysis. A Salter-Harris type III fracture occurs through the epiphysis and exits through the growth plate, while a Salter-Harris type IV fracture extends through the physis from the metaphysis into the epiphy-sis. A Salter-Harris type V fracture is

1	through the epiphysis and exits through the growth plate, while a Salter-Harris type IV fracture extends through the physis from the metaphysis into the epiphy-sis. A Salter-Harris type V fracture is a crushing injury to the physis. Type III and type IV involve the joint.Treatment of growth plate fracture requires anatomic reduction of the fragments, closed or open. If internal fixation is used, avoid placing the hardware across the growth plate to minimize the chance of injury and premature growth plate clo-sure. When hardware needs to be placed across the physis, it should be limited to smooth K-wires. The most common com-plication is a physeal arrest resulting in leg length discrepancy (LLD) and/or angular deformity. Complete arrest will lead to LLD. Partial arrest will result in angulation in the area of the bar, bridge, and fusion. If less than 50% of the physis is involved and the patient has two years of growth remaining, the bar is usually resected with interposition of fat

1	in the area of the bar, bridge, and fusion. If less than 50% of the physis is involved and the patient has two years of growth remaining, the bar is usually resected with interposition of fat graft. If the bar (fusion area) is more than 50%, the surgeon will complete the arrest on the same side and will do a contralateral epiphysiodesis on the other extremity.Distal femur physeal fractures are known to have a high rate of leg length discrepancy and angular deformity. The injury will need an anatomical reduction and close follow-up. Parents need to be counseled about the poor prognosis associated with these fractures.Diaphyseal Injuries in a Pediatric PatientLong bone diaphyseal fractures are generally treated closed. Pediatric patients are capable of extensive remodeling so that an angular deformity within the plane of an adjacent joint is often completely remodeled by the growth of the child. Older children do not remodel as well. A 10° angulation in both bones of the forearm in a

1	deformity within the plane of an adjacent joint is often completely remodeled by the growth of the child. Older children do not remodel as well. A 10° angulation in both bones of the forearm in a child over an age of 10 years may cause significant limitation of rotation of the forearm. When internal fixation of a diaphyseal fracture is required, fixation through the physis is avoided.Fractures of the Pediatric HipFractures of the pediatric hip can occur with high-energy trauma, and there is a high rate of avascular necrosis. Pedi-atric patients with hip fractures may be treated with a spica cast. The spica cast includes the abdomen, lower back, pelvis, and lower limb, and derives its name from the resemblance of the plaster wrap over the hip to wheat “spica.” Closed or open reduction and internal fixation is done in fractures with severe displacement. Avascular necrosis is the most common complication after hip fractures. The incidence depends on the age of the patient and the type of

1	fixation is done in fractures with severe displacement. Avascular necrosis is the most common complication after hip fractures. The incidence depends on the age of the patient and the type of the fracture. Children between 3 and 8 years old with very proximal fractures such as transphyseal fractures have the highest incidence of avas-cular necrosis.Fractures of the Femoral ShaftConsider child abuse if a femoral shaft fracture occurs before the walking age. Femoral shaft fractures in a child younger than 6 months are usually treated by a Pavlik harness or spica cast. A child between 6 months and 5 years with an acceptable shortening of the femur is usually treated by an immediate spica cast. The child between 5 years and 11 years is usu-ally treated by surgery. If the fracture is transverse, flexible IM nails may be used, especially if the child weighs less than 100 lbs (45 kg). If the fracture is too proximal or too distal, or if the fracture is comminuted and unstable, a submuscu-lar

1	flexible IM nails may be used, especially if the child weighs less than 100 lbs (45 kg). If the fracture is too proximal or too distal, or if the fracture is comminuted and unstable, a submuscu-lar bridge plate is usually used; alternatively, an external fix-ator may also be used, especially in multiple trauma patients. If the patient is older than 11 years, an interlocking IM rod with a lateral trochanteric entry is used. Insertion of IM rod in younger children can cause avascular necrosis of the femoral head due to interruption of the blood supply. Refracture of the femur is a risk after using an external fixator. Overgrowth of the injured femur with leg length discrepancy can occur in children between 2 and 10 years of age.Figure 43-52. Classification of growth plate injuries.Brunicardi_Ch43_p1879-p1924.indd 191622/02/19 10:41 AM 1917ORTHOPEDIC SURGERYCHAPTER 43Pediatric Ankle FracturesPediatric ankle fractures include several types. Salter-Harris type I and type II usually

1	191622/02/19 10:41 AM 1917ORTHOPEDIC SURGERYCHAPTER 43Pediatric Ankle FracturesPediatric ankle fractures include several types. Salter-Harris type I and type II usually involve the fibula, and the fracture may not be apparent. The patient may present with pain and swelling. Salter-Harris type III usually involves fracture of the medial malleolus or avulsion of the anterior inferior tibiofibular ligament from the tibia. It is called a Tillaux fracture. Tillaux fractures occur because the lateral part of the ankle is not fused and it is weak. Triplane fractures are complex ankle fractures in older children as a result of partial closure of the growth plate, and they appear as a Salter II in the lateral view and as a Salter III in an AP view (Fig. 43-53). Salter-Harris I and II fractures are usually managed with casting. Salter-Harris III or IV fractures are usually managed by closed or open reduction and internal fixation. Smooth percutaneous pins or screws are utilized, avoiding

1	are usually managed with casting. Salter-Harris III or IV fractures are usually managed by closed or open reduction and internal fixation. Smooth percutaneous pins or screws are utilized, avoiding the physis.Pediatric Elbow FracturesManagement of pediatric elbow fractures is complex. Famil-iarity with the timing of the ossification centers’ appearance aids in diagnosis. Distal humeral physeal separation can occur from child abuse and can be mistaken for an elbow dislocation. A lateral condylar fracture of the elbow is a significant injury, and when it is displaced it will need anatomical surgical reduc-tion. Medial epicondyle fractures of the elbow are usually treated conservatively unless they are severely displaced. It is associated with elbow dislocation in 50% of cases. When the elbow is reduced, the fragment may lodge in the joint itself and must be removed and fixed. In supracondylar fractures of the humerus (Fig. 43-54), the neurovascular status of the extrem-ity must be

1	elbow is reduced, the fragment may lodge in the joint itself and must be removed and fixed. In supracondylar fractures of the humerus (Fig. 43-54), the neurovascular status of the extrem-ity must be assessed carefully before, during, and after treat-ment. The anterior interosseous nerve could be injured, and the patient may not be able to make an “OK sign” (Fig. 43-55). The brachial artery may also be injured. Closed reduction, pos-sible open reduction, and percutaneous pinning is usually done for these fractures. The procedure should be done emergently if there is concern about the vascular status of the extremity. Close follow-up for maintenance of reduction and neurovas-cular status is needed.DEVELOPMENTAL DISEASEDevelopmental Dysplasia of the HipDevelopmental dysplasia of the hip (DDH) involves a spectrum of disease that includes dysplasia, subluxation, or dislocation of the hip. Teratologic hip dislocation is a different entity in which the hip is dislocated in utero and

1	hip (DDH) involves a spectrum of disease that includes dysplasia, subluxation, or dislocation of the hip. Teratologic hip dislocation is a different entity in which the hip is dislocated in utero and irreducible on neonatal examination, usually associated with neuromuscular conditions and genetic syndromes. Developmental dysplasia of the hip is most often seen in firstborn females with a positive family history or with breech birth.Untreated hip dislocations can lead to a dysplastic acetab-ulum, and they should be recognized and treated early. New-borns are examined for hip instability within the first 72 hours HumerusFigure 43-54. Supracondylar fracture of the humerus.Figure 43-55. Unable to make the “OK” sign due to interosseous nerve injury.Figure 43-53. Triplane fracture of the ankle in children.Brunicardi_Ch43_p1879-p1924.indd 191722/02/19 10:42 AM 1918SPECIFIC CONSIDERATIONSPART IIof life. Ortolani’s test consists of gentle elevation and abduction of the femur causing a

1	in children.Brunicardi_Ch43_p1879-p1924.indd 191722/02/19 10:42 AM 1918SPECIFIC CONSIDERATIONSPART IIof life. Ortolani’s test consists of gentle elevation and abduction of the femur causing a palpable click in the relocation of a dis-located hip. Barlow’s test is gentle adduction and depression of the femur, which causes a palpable click as the hip slips into a dislocated position. In older infants (older than 3 months), limited abduction of the involved hip is an important finding. Infants with a dislocated or dislocatable hip will have apparent length discrepancies of the femur when the hip is positioned at 90° (Galeazzi test).Since the bones are not ossified at birth, X-ray images of the acetabulum and femoral head are not reliable for diag-nosis. Ultrasound is the imaging modality of choice in the neonatal period and can often demonstrate a dislocated or dislocatable hip.Treatment of DDHThe main goal in the treatment of DDH is to achieve stable concentric reduction of the

1	of choice in the neonatal period and can often demonstrate a dislocated or dislocatable hip.Treatment of DDHThe main goal in the treatment of DDH is to achieve stable concentric reduction of the hip.• Neonate to 6 months: Early treatment with abduction and flexion in a Pavlik harness for 6 to 12 weeks is usually suf-ficient. Avoid severe abduction and flexion in the Pavlik har-ness to avoid the risk of avascular necrosis of the femoral head and femoral nerve palsy.• Children 6 to 18 months: Closed reduction and application of hip spica cast is indicated in this age group and in those children who failed Pavlik harness treatment.• Children older than 18 months: Open reduction and cap-sulorrhaphy is indicated in this age group. A variety of procedures, including femoral shortening and pelvic osteoto-mies, are done in older age groups and in more severe cases. Osteonecrosis of the femoral head is a possible complication of treatment and can result in pain and decreased range of

1	and pelvic osteoto-mies, are done in older age groups and in more severe cases. Osteonecrosis of the femoral head is a possible complication of treatment and can result in pain and decreased range of motion.Legg-Calvé-Perthes DiseaseOsteonecrosis of the proximal femoral epiphysis can cause flattening of the femoral head called Legg-Calvé Perthes disease. The age at presentation is between 4 and 8 years of age and occurs more in males, usually affecting one side. Younger age at presentation (less than 6 years old) will have a better prognosis. The patient presents with groin or knee pain, decreased hip motion, and a limp. Treatment includes traction, physical therapy, abduction exercises, and crutches. Restoration of range of motion is important. Femoral and pelvic osteotomies may be needed in extreme cases and in older children.Slipped Capital Femoral EpiphysisChildren ages 10 to 16 years can develop displacement of the epiphysis on the femoral neck with no history of injury. The

1	in extreme cases and in older children.Slipped Capital Femoral EpiphysisChildren ages 10 to 16 years can develop displacement of the epiphysis on the femoral neck with no history of injury. The slippage occurs through the weak zone (hypertrophic zone) of the growth plate. When slippage occurs in young patients, check for endocrine disorders such as hypothyroid-ism, renal osteodystrophy, and growth hormone deficiency. Slipped capital femoral epiphysis (SCFE) is associated with African-American heritage and obesity, and it is more com-mon in boys than in girls. One-quarter of cases are bilateral. In patients with endocrine etiology, the condition is usually bilateral. Patients generally present with groin and anterior thigh pain, and the patient may have antalgic gait and a limp. Patient may present with knee pain that can lead to missing the diagnosis. In pediatric patients with knee pain, the ipsi-lateral hip should be assessed as well.Examination of the patient will show obligatory

1	present with knee pain that can lead to missing the diagnosis. In pediatric patients with knee pain, the ipsi-lateral hip should be assessed as well.Examination of the patient will show obligatory external rotation with flexion and loss of internal rotation of the hip. Obtain AP and frog leg lateral views of both the hips.Slipped epiphysis is classified as either stable or unstable on the basis of the patient’s ability to bear weight. It is classi-fied as stable if the patient is able to bear weight and the risk of osteonecrosis is less than 10%. It is classified as unstable if the patient is unable to bear weight even with crutches, and the incidence of avascular necrosis is high.Treatment for slipped capital femoral epiphysis patients is percutaneous screw fixation through the femoral neck to engage the epiphysis, causing the growth plate to close. Reduc-tion of the slipped epiphysis is not recommended because of an increased risk of avascular necrosis. One screw is usually adequate

1	to engage the epiphysis, causing the growth plate to close. Reduc-tion of the slipped epiphysis is not recommended because of an increased risk of avascular necrosis. One screw is usually adequate to prevent further slip.Lower Extremity Rotational AbnormalitiesIntoeing can result from femoral anteversion, tibial torsion, and metatarsus adductus. Mild degree of intoeing is normal in young children 3 to 5 years of age.Excessive internal rotation of the femur will usually cor-rect by age 8. Severe rotation with functional impairment that does not correct by age 10 or 11 may require rotational femoral osteotomy.Tibial torsion is the most common cause of intoeing in toddlers and could be bilateral. The condition usually resolves without treatment.Metatarsus adductus in infants will also resolve spontane-ously in most cases.Congenital Talipes Equinovarus (Clubfoot)Clubfoot is a congenital disorder, and its etiology is not known. Clubfoot is a common problem associated with con-tractures of

1	spontane-ously in most cases.Congenital Talipes Equinovarus (Clubfoot)Clubfoot is a congenital disorder, and its etiology is not known. Clubfoot is a common problem associated with con-tractures of the medial tendons of the foot, a tight Achilles tendon, and contractures of the ankle, hindfoot, and midfoot. The foot is usually small, and it is in the equinus, varus, cavus, and adduction position. Talipes equinovarus can be corrected by sequential corrective casting of the foot. The serial manip-ulation and the casting technique is called the Ponseti tech-nique, and it has a high success rate. A successful program of casting may be complete in 1 to 5 months. In patients with severe disease or who initiate treatment after 9 months of age, surgical release of contracted soft tissues may be necessary. The procedure is called posteromedial soft tissue release and tendon lengthening.Osgood-Schlatter DiseaseOsgood-Schlatter disease is a common problem most often seen in athletically active

1	necessary. The procedure is called posteromedial soft tissue release and tendon lengthening.Osgood-Schlatter DiseaseOsgood-Schlatter disease is a common problem most often seen in athletically active adolescents, especially in sprint-ers and jumpers. It is a traction apophysitis of tibial tubercule (Figs. 43-56 and 43-57). One must know the difference between the epiphysis, apophysis, and physis. This disorder is charac-terized by ossification in the distal patellar tendon at the point of its tibial insertion, and it is thought to result from mechani-cal stress on the tendinous insertion. The disease presents with severe local pain and tenderness in the area of the tibial tubercle. Radiographs may show calcified ossicles within the tendon at its insertion.Brunicardi_Ch43_p1879-p1924.indd 191822/02/19 10:42 AM 1919ORTHOPEDIC SURGERYCHAPTER 43Treatment for the disease is activity restriction and anti-inflammatory drugs. The majority of patients improve with con-servative treatment,

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1	Mahar AT, Miesen D, Newton PO. Displaced pediat-ric supracondylar humerus fractures: biomechanical analy-sis of percutaneous pinning techniques. J Pediatr Orthop. 2002;22(4):440-443.Lincoln TL, Suen PW. Common rotational variations in children. J Am Acad Orthop Surg. 2003;11(5):312-320.Loder RT, Aronsson DD, Weinstein SL, Breur GJ, Ganz R, Leunig M. Slipped capital femoral epiphysis. Instr Course Lect. 2008;57:473-498.Mahan ST, Katz JN, Kim YJ. To screen or not to screen? A decision analysis of the utility of screening for developmental dysplasia of the hip. J Bone Joint Surg Am. 2009;91(7):1705-1719.Murray AW, Wilson NI. Changing incidence of slipped capital fem-oral epiphysis: a relationship with obesity? J Bone Joint Surg Br. 2008;90(1):92-94.O’Donnell S. Pediatric ankle fractures. In: The Orthopedic Consult Survival Guide. New York: Springer; 2017:265-269.Palocaren T, Holmes L, Rogers K, Kumar SJ. Outcome of in situ pinning in patients with unstable slipped capital femoral

1	In: The Orthopedic Consult Survival Guide. New York: Springer; 2017:265-269.Palocaren T, Holmes L, Rogers K, Kumar SJ. Outcome of in situ pinning in patients with unstable slipped capital femoral Brunicardi_Ch43_p1879-p1924.indd 192322/02/19 10:42 AM 1924SPECIFIC CONSIDERATIONSPART IITokmakova KP, Stanton RP, Mason DE. Factors influenc-ing the development of osteonecrosis in patients treated for slipped capital femoral epiphysis. J Bone Joint Surg Am. 2003;85(5):798-801.Trousdale RT. Acetabular osteotomy: indications and results. Clin Orthop Relat Res. 2004;429:182-187.Wainwright AM, Auld T, Benson MK, Theologis TN. The clas-sification of congenital talipes equinovarus. Bone Joint J. 2002;84(7):1020-1024.Weiler R, Ingram M, Wolman R. Osgood-Schlatter disease. Br Med J (Online). 2011;1:343.epiphysis: assessment of risk factors associated with avascular necrosis. J Pediatr Orthop. 2010;30(1):31-36.Parsch K, Weller S, Parsch D. Open reduction and smooth Kirschner wire fixation for

1	assessment of risk factors associated with avascular necrosis. J Pediatr Orthop. 2010;30(1):31-36.Parsch K, Weller S, Parsch D. Open reduction and smooth Kirschner wire fixation for unstable slipped capital femoral epiphysis. J Pediatr Orthop. 2009;29(1):1-8.Rosenbaum P, Paneth N, Leviton A, et al. A report: the definition and classification of cerebral palsy April 2006. Dev Med Child Neurol Suppl. 2007;109(suppl 109):8-14.Sjöberg I, Erichs K, Bjerre I. Cause and effect of obstetric (neonatal) brachial plexus palsy. Acta Paediatrica. 1988;77(3): 357-364.Stevenson DA, Mineau G, Kerber RA, Viskochil DH, Schaefer C, Roach JW. Familial predisposition to developmental dysplasia of the hip. J Pediatr Orthop. 2009;29(5):463-466.Brunicardi_Ch43_p1879-p1924.indd 192422/02/19 10:42 AM

1	Surgery of the Hand and WristScott D. Lifchez and Brian H. Cho 44chapterINTRODUCTIONThe highly mobile, functional, and strong hand is a major dis-tinguishing point between humans and the nonhuman primates. The hand is an essential participant for activities of daily living, vocation, and recreational activities. The hand is even adaptable enough to read for the blind and speak for the mute. The under-lying goal of all aspects of hand surgery is to maximize mobil-ity, sensibility, stability, and strength while minimizing pain. These goals are then maximized to the extent possible given the patient’s particular pathology. Hand surgery is a regional specialty.Hand surgeons integrate components of neurologic, ortho-pedic, plastic, and vascular surgery in the care of patients with disorders of the upper extremities.1ANATOMY OF THE HAND AND WRISTIn order to understand any disorder of the hand, one must under-stand the anatomy of the underlying structures. Examina-tion of the hand is based

1	the upper extremities.1ANATOMY OF THE HAND AND WRISTIn order to understand any disorder of the hand, one must under-stand the anatomy of the underlying structures. Examina-tion of the hand is based on demonstrating the function or lack thereof of each of these structures.BonesThe hand is highly mobile in space to allow maximum flex-ibility in function. As such, a number of directions particular to the hand are necessary in order to properly describe posi-tion, motion, and so on.1 Palmar (or volar) refers to the anterior surface of the hand in the anatomic position; dorsal refers to the posterior surface in the anatomic position. The hand can rotate at the wrist level; rotation to bring the palm down is called 2Introduction 1925Anatomy of the Hand and Wrist 1925Bones / 1925Muscles Affecting the Hand and Wrist / 1926Tendons and Pulleys / 1929Vascular / 1929Nerve / 1930Hand Examination 1931Emergency Department/Inpatient Consultation / 1931Hand Imaging 1932Plain X-Rays / 1932Computed

1	the Hand and Wrist / 1926Tendons and Pulleys / 1929Vascular / 1929Nerve / 1930Hand Examination 1931Emergency Department/Inpatient Consultation / 1931Hand Imaging 1932Plain X-Rays / 1932Computed Tomography / 1932Ultrasonography / 1932Magnetic Resonance Imaging / 1933Angiography / 1933Trauma 1933Fractures and Dislocations / 1934Tendons / 1935Nerve Injuries / 1936Vascular Injuries / 1936Anesthesia 1936Local Anesthesia / 1936Hand Surgery Under Local Anesthesia / 1938Postoperative Pain Management / 1938Special Considerations 1938Amputations and Replantation / 1938Fingertip Injuries / 1938High-Pressure Injection Injuries / 1939Compartment Syndrome / 1939Complications 1943Nonunion / 1943Stiffness / 1943Neuroma / 1943Regional Pain Syndromes / 1943Nerve Compression 1943Carpal Tunnel Syndrome / 1944Cubital Tunnel Syndrome / 1944Other Sites of Nerve Compression / 1945Degenerative Joint Disease 1945Small Joints (Metacarpophalangeal and Interphalangeal) 1945Wrist / 1945Rheumatoid Arthritis /

1	/ 1944Cubital Tunnel Syndrome / 1944Other Sites of Nerve Compression / 1945Degenerative Joint Disease 1945Small Joints (Metacarpophalangeal and Interphalangeal) 1945Wrist / 1945Rheumatoid Arthritis / 1946Dupuytren’s Contracture 1947Infections 1947Cellulitis / 1947Abscess / 1948Collar-Button Abscess / 1948Osteomyelitis / 1949Pyogenic Arthritis / 1949Necrotizing Infections / 1949Infectious Flexor Tenosynovitis / 1950Felon / 1951Paronychia / 1951Tumors 1952Benign Soft Tissue Tumors / 1953Malignant Soft Tissue Tumors— Cutaneous / 1955Malignant Soft Tissue Tumors—Noncutaneous / 1956Benign Bone Tumors / 1956Malignant Bone Tumors / 1957Secondary Metastatic Tumors / 1958Burns 1958Acute Management / 1958Surgical Management / 1959Reconstruction / 1959Special Considerations / 1960Vascular Disease 1960Progressive Thrombotic Disease / 1960Systemic Vasculopathy / 1960Vasospastic Disorders / 1961Congenital Differences 1961Failure of Formation / 1961Failure of Differentiation / 1961Duplication /

1	Thrombotic Disease / 1960Systemic Vasculopathy / 1960Vasospastic Disorders / 1961Congenital Differences 1961Failure of Formation / 1961Failure of Differentiation / 1961Duplication / 1961Overgrowth / 1961Constriction Band Syndrome / 1961Generalized Skeletal Anomalies and Syndromes / 1961Reconstructive Transplantation of the Upper Extremity 1962Brunicardi_Ch44_p1925-p1966.indd 192520/02/19 2:48 PM 1926pronation, and rotation to bring the palm up is called supina-tion. Because the hand can rotate in space, the terms medial and lateral are avoided. Radial and ulnar are used instead as these terms do not vary with respect to the rotational position of the hand. Abduction and adduction, when used on the hand, refer to movement of the digits away from and toward the middle finger, respectively (Fig. 44-1).The hand is comprised of 19 bones arranged in five rays.2 A ray is defined as a digit (finger or thumb) from the metacarpal base to the tip of the digit (Fig. 44-2A). The rays are

1	(Fig. 44-1).The hand is comprised of 19 bones arranged in five rays.2 A ray is defined as a digit (finger or thumb) from the metacarpal base to the tip of the digit (Fig. 44-2A). The rays are numbered 1 to 5, beginning with the thumb. By convention, however, they are referred to by name: thumb, index, middle, ring, and small. There are five metacarpals, comprising the visible palm of the hand. Each digit has a proximal and a distal phalanx, but only the fingers have a middle phalanx as well. The metacarpopha-langeal (MP) joint typically allows 90° of flexion with a small amount of hyperextension. In addition, the fingers can actively abduct (move away from the middle finger) and adduct (move toward the middle finger). The thumb, in contrast, moves prin-cipally in the flexion-extension arc at the MP joint. Although there can be laxity in the radial and ulnar direction, the thumb cannot actively move in these directions at the MP level. The proximal interphalangeal joint (PIP) is the

1	at the MP joint. Although there can be laxity in the radial and ulnar direction, the thumb cannot actively move in these directions at the MP level. The proximal interphalangeal joint (PIP) is the critical joint for finger mobility. Normal motion is 0° to 95° (full extension to flexion). The distal interphalangeal joint (DIP) also moves only in a flexion-extension plane from 0° to 90° on average. The thumb interphalangeal joint (IP) also moves only in a flexion-extension plane. Its normal motion is highly variable between individuals, but averages 0° to 80°.Each of the MP and IP joints has a radial and ulnar col-lateral ligament to support it. The IP joint collateral ligaments are on tension with the joint fully extended. For the fingers, the MP joint collateral ligaments are on tension with the joint bent 90°. Collateral ligaments have a tendency to contract when not placed on tension; this becomes relevant when splinting the hand (see later “Trauma” section on splinting).The wrist

1	with the joint bent 90°. Collateral ligaments have a tendency to contract when not placed on tension; this becomes relevant when splinting the hand (see later “Trauma” section on splinting).The wrist consists of eight carpal bones divided into two rows (see Fig. 44-2B).2 The proximal row consists of the scaph-oid, lunate, and triquetrum. The lunate is the principle axis of motion of the hand onto the forearm. It bears approximately 35% of the load of the wrist onto the forearm. The scaphoid is shaped like the keel of a boat and bears 55% of the load of the hand onto the forearm, but it also serves as the principle link between the proximal and distal rows, allowing for motion while maintaining stability. Both the scaphoid and the lunate articulate with the radius. The triquetrum resides ulnar to the lunate. It does not interact with the ulna proximally; rather, it interacts with a cartilage suspended between the ulnar styloid and the distal radius called with triangular fibrocartilage

1	to the lunate. It does not interact with the ulna proximally; rather, it interacts with a cartilage suspended between the ulnar styloid and the distal radius called with triangular fibrocartilage com-plex (TFCC) (see Fig. 44-2B). The remaining 10% of load of the hand onto the forearm is transmitted through the TFCC.3The distal row consists of four bones. The trapezium resides between the scaphoid and the thumb metacarpal. Dis-tally, it has a saddle-shaped surface, which interacts with a reciprocally saddle-shaped base of the thumb metacarpal to allow for high mobility of the thumb carpometacarpal (CMC) joint in radial-ulnar and palmar-dorsal directions and opposition (Fig. 44-1B). The trapezoid rests between the scaphoid and the index finger metacarpal. The capitate, the largest carpal bone and first to ossify in a child, lies between the lunate and the middle finger metacarpal, but it also interacts with the scaph-oid on its proximal radial surface. The index and middle finger CMC

1	bone and first to ossify in a child, lies between the lunate and the middle finger metacarpal, but it also interacts with the scaph-oid on its proximal radial surface. The index and middle finger CMC joints are highly stable and have minimal mobility. The hamate is the ulnar-most bone in the distal row, sitting between the triquetrum proximally and the ring and small finger metacar-pals distally. The ring and small finger CMC joints are mobile, principally in the flexion-extension direction.The pisiform is a carpal bone only by geography. It is a sesamoid bone within the FCU tendon (see following section). It does not bear load and can be excised, when necessary, without consequence.Muscles Affecting the Hand and WristThe wrist is moved by multiple tendons that originate from the forearm and elbow. The digits of the hand are moved by both intrinsic (originating within the hand) and extrinsic (originating in the forearm) muscles. All of these muscles are innervated by the median,

1	and elbow. The digits of the hand are moved by both intrinsic (originating within the hand) and extrinsic (originating in the forearm) muscles. All of these muscles are innervated by the median, radial, or ulnar nerves (or their branches) (Fig. 44-3).Three muscles flex the wrist, all of which originate from the medial epicondyle of the humerus. The flexor carpi radialis (FCR, median nerve) inserts on the volar base of the index fin-ger metacarpal. The flexor carpi ulnaris (FCU, ulnar nerve) also originates from the proximal ulna and inserts on the volar base of the small finger metacarpal. The palmaris longus (PL) tendon does not insert on a bone; it inserts on the palmar fascia, located deep to the skin in the central proximal palm, and is absent in up to 15% of patients. The FCR also deviates the wrist radially, whereas the FCU deviates the wrist ulnarly.All three wrist extensors are innervated by the radial nerve or its branches. The extensor carpi radialis longus (ECRL) Key

1	deviates the wrist radially, whereas the FCU deviates the wrist ulnarly.All three wrist extensors are innervated by the radial nerve or its branches. The extensor carpi radialis longus (ECRL) Key Points1 Surgery of the hand is a regional specialty, integrating com-ponents of neurologic, orthopedic, plastic, and vascular surgery.2 Understanding hand anatomy is the key to proper diagnosis of injury, infection, and degenerative disease of the hand.3 After evaluation and/or treatment, patients should be splinted to protect the injured digits and keep the collateral ligaments of the injured joints on tension (metacarpophalangeal joints flexed, interphalangeal joints extended).4 Healing of an injured or diseased structure in the hand is not the endpoint of treatment; the goal of any intervention must be to obtain structure healing, relief of pain, and maximiza-tion of function.5 If a patient managed conservatively for cellulitis does not improve within 24 to 48 hours of appropriate

1	must be to obtain structure healing, relief of pain, and maximiza-tion of function.5 If a patient managed conservatively for cellulitis does not improve within 24 to 48 hours of appropriate intravenous antibiotics, abscess must be suspected.6 Clinical examination, particularly noting the area of greatest tenderness and/or inflammation, is the most useful diagnos-tic tool for hand infections.Brunicardi_Ch44_p1925-p1966.indd 192620/02/19 2:48 PM 1927SURGERY OF THE HAND AND WRISTCHAPTER 44originates from the distal shaft of the humerus and inserts on the dorsal base of the index finger metacarpal. The extensor carpi radialis brevis (ECRB) originates from the lateral epicondyle of the humerus and inserts on the dorsal base of the middle finger metacarpal. The extensor carpi ulnaris (ECU) also originates from the lateral epicondyle of the humerus and inserts on the dorsal base of the small finger metacarpal. The ECRL deviates the wrist radially, whereas the ECU deviates the wrist

1	also originates from the lateral epicondyle of the humerus and inserts on the dorsal base of the small finger metacarpal. The ECRL deviates the wrist radially, whereas the ECU deviates the wrist ulnarly.The long flexors of the fingers all originate from the medial epicondyle of the humerus. The flexor digitorum super-ficialis (FDS) inserts on the base of the middle phalanx of each finger and primarily flexes the PIP joint. The flexor digitorum profundus (FDP) inserts on the base of the distal phalanx and primarily flexes the DIP joint. The flexor pollicis longus (FPL) originates more distally, from the ulna, radius, and interosseous membrane between them in the forearm. It inserts on the base of the distal phalanx of the thumb and primarily flexes the IP joint. All of these tendons can also flex the more proximal joint(s) in their respective rays. All of these muscles are innervated by the median nerve (or its branches) except the FDP to the ring and small fingers, which are

1	can also flex the more proximal joint(s) in their respective rays. All of these muscles are innervated by the median nerve (or its branches) except the FDP to the ring and small fingers, which are innervated by the ulnar nerve.The extrinsic extensors of the fingers and thumb are all innervated by the posterior interosseous nerve (PIN, branch of the radial nerve). The extensor digitorum communis (EDC) originates from the lateral epicondyle of the humerus and extends the MP joints of the fingers. Unlike most tendons that attach directly into a bone, the EDC tendons do not insert on the dorsal base of the proximal phalanx, but rather into a soft tissue sling called the sagittal hood, which surrounds the proximal phalanx base and pulls up on the volar surface in a ABCDFigure 44-1. Directions of finger, hand, and wrist motion. A. Finger abduction (white arrows) and adduction (black arrows). B. Thumb radial (black arrow) and palmar (white arrow) abduction. C. Thumb and small finger

1	of finger, hand, and wrist motion. A. Finger abduction (white arrows) and adduction (black arrows). B. Thumb radial (black arrow) and palmar (white arrow) abduction. C. Thumb and small finger opposition. D. Hand/wrist pronation (black arrow) and supination (white arrow).Brunicardi_Ch44_p1925-p1966.indd 192720/02/19 2:48 PM 1928SPECIFIC CONSIDERATIONSPART IIhammock-like manner. More distally in the dorsal forearm, the extensor indices proprius (EIP) and extensor digiti quinti (EDQ) originate from the ulna, radius, and posterior interosseous mem-brane and insert on the sagittal hood of the index and small fingers, respectively.The thumb has three separate extrinsic extensors. All of these originate from the dorsal ulna in the mid-forearm and are innervated by the PIN. The abductor pollicis longus (APL) inserts on the radial base of the thumb metacarpal to produce some extension, but mostly abduction. The extensor pollicis ECRL/ECRBEPLEDQECUTCL23455432Radial AANUlnarSCHMedian

1	pollicis longus (APL) inserts on the radial base of the thumb metacarpal to produce some extension, but mostly abduction. The extensor pollicis ECRL/ECRBEPLEDQECUTCL23455432Radial AANUlnarSCHMedian NAPLEPBFPLPFCREIP/EDCFigure 44-3. Cross-section of the wrist at the midcarpal level. The relative geography of the neurologic and tendinous structures can be seen. The transverse carpal ligament (TCL) is the roof of the carpal tunnel, passing volar to the median nerve and long flexor tendons. The TCL is also the floor of the ulnar tunnel, or Guyon’s canal, passing dorsal to the ulnar artery and nerve. The wrist and digital extensor tendons are also seen, distal to their compartments on the distal radius and ulna. Bones: C = capitate; H = hamate; P = pisiform; S = scaphoid. Tendons (flexor digitorum superficialis is volar to flexor digitorum profundus within the carpal tunnel): 2 = index finger; 3 = middle finger; 4 = ring finger; 5 = small finger. A = artery; APL = abductor pollicis longus;

1	superficialis is volar to flexor digitorum profundus within the carpal tunnel): 2 = index finger; 3 = middle finger; 4 = ring finger; 5 = small finger. A = artery; APL = abductor pollicis longus; ECRB = extensor carpi radialis brevis; ECRL = extensor carpi radialis longus; ECU = extensor carpi ulnaris; EDC = extensor digitorum communis; EDQ = extensor digiti quinti; EIP = extensor indices proprius; EPB = extensor pollicis brevis; EPL = extensor pollicis longus; FCR = flexor carpi radialis; FPL = flexor pollicis longus; N = nerve.ABFigure 44-2. Bony architecture of the hand and wrist. A. Bones of the hand and digits. All rays have metacarpophalangeal (MP) joints. The fingers have proximal and distal interphalangeal joints (PIP and DIP), but the thumb has a single interphalangeal (IP) joint. B. Bones of the wrist. The proximal row consists of the scaphoid, lunate, and capitate. The distal row bones articulate with the metacarpals: the trapezium with the thumb, the trapezoid with the

1	B. Bones of the wrist. The proximal row consists of the scaphoid, lunate, and capitate. The distal row bones articulate with the metacarpals: the trapezium with the thumb, the trapezoid with the index, the capitate with the middle, and the hamate with the ring and small. The pisiform bone is a sesamoid within the flexor carpi ulnaris tendon. It overlaps the triquetrum and hamate but does not contribute to a carpal row. CMC = carpometacarpal; TFCC = triangular fibrocartilage complex.Brunicardi_Ch44_p1925-p1966.indd 192820/02/19 2:48 PM 1929SURGERY OF THE HAND AND WRISTCHAPTER 44brevis (EPB) inserts on the base of the thumb proximal pha-lanx. The extensor pollicis longus (EPL) inserts on the base of the thumb distal phalanx.The intrinsic muscles of the hand are what allow humans fine, subtle movements of the hand. Microsurgery, typing, and even video gaming would be difficult, if not impossible, without them.The thenar muscles originate from the volar radial surface of the scaphoid

1	movements of the hand. Microsurgery, typing, and even video gaming would be difficult, if not impossible, without them.The thenar muscles originate from the volar radial surface of the scaphoid and trapezium and the flexor retinaculum. The abductor pollicis brevis (APB) inserts on the radial base of the thumb proximal phalanx and abducts the thumb in a radial and volar direction. The opponens pollicis (OP) inserts on the radial distal aspect of the thumb metacarpal and draws the thumb across the palm toward the small finger. The flexor pollicis bre-vis (FPB) inserts on the base of the thumb proximal phalanx and flexes the thumb MP joint. The APB, OP, and superficial head of the FPB are all innervated by the thenar motor branch of the median nerve.The lumbrical muscles are unique in the body in that they originate from a tendon. Each finger’s lumbrical originates from the FDP tendon in the palm. The lumbrical tendon passes along the radial aspect of the digit to flex the MP and extend

1	in that they originate from a tendon. Each finger’s lumbrical originates from the FDP tendon in the palm. The lumbrical tendon passes along the radial aspect of the digit to flex the MP and extend the IP joints. The index and middle lumbricals are median nerve inner-vated, and the ring and small finger lumbricals are ulnar nerve innervated.The hypothenar muscles originate from the pisiform, hamate, and flexor retinaculum and insert on the ulnar base of the small finger proximal phalanx. The abductor digiti quinti (ADQ) abducts the small finger. The opponens digiti quinti (ODQ) brings the small finger across the palm in reciprocal motion to the OP. The flexor digiti quinti (FDQ) flexes the small finger metacarpal. All of these muscles are innervated by the ulnar nerve.The interosseous muscles occupy the space between the metacarpal bones. Their tendons insert on the bases of the proxi-mal phalanges. All act to flex the MP joints and extend the IP joints. The three palmar interosseous

1	occupy the space between the metacarpal bones. Their tendons insert on the bases of the proxi-mal phalanges. All act to flex the MP joints and extend the IP joints. The three palmar interosseous muscles adduct the fin-gers. The four dorsal interosseous muscles abduct the fingers. The adductor pollicis originates from the middle finger metacar-pal and inserts on the ulnar base of the thumb proximal phalanx. It acts to adduct the thumb. All of these muscles, as well as the deep head of the FPB, are innervated by the ulnar nerve.Tendons and PulleysMultiple pulleys pass over or surround the extrinsic tendons en route to or within the hand. Their purpose is to maintain tendon position near the bone, allowing maximal translation of tendon excursion into joint motion.The most well known of the wrist-level pulleys is the flexor retinaculum, also known as the transverse carpal liga-ment. It attaches to the scaphoid tubercle and trapezium radially and the hook of the hamate bone and pisiform

1	wrist-level pulleys is the flexor retinaculum, also known as the transverse carpal liga-ment. It attaches to the scaphoid tubercle and trapezium radially and the hook of the hamate bone and pisiform ulnarly. Deep to this ligament, between the scaphoid (radially) and the hamate (ulnarly), pass the FDS, FDP, and FPL tendons as well as the median nerve. This area is also known as the carpal tunnel (see Fig. 44-3).On the dorsum of the wrist, the extensor retinaculum is divided into six compartments. Beginning on the radial aspect of the radius, the first compartment contains the APL and EPB tendons. The second holds the ECRL and ECRB tendons. The EPL passes through the third compartment. The fourth com-partment contains the EIP and EDC tendons, the fifth the EDQ, and the sixth the ECU. The sixth compartment is located on the ulnar aspect of the distal ulna. Although the compartments end at the radiocarpal/ulnocarpal joints, the relative geography of the tendons is preserved over the

1	sixth compartment is located on the ulnar aspect of the distal ulna. Although the compartments end at the radiocarpal/ulnocarpal joints, the relative geography of the tendons is preserved over the carpal bones (see Fig. 44-3).In the hand, the pulleys maintain the long flexor tendons in close apposition to the fingers and thumb. There are no extensor pulleys within the hand. Each finger has five annular and three cruciate pulleys (Fig. 44-4). The second and fourth (A2 and A4) pulleys are the critical structures to prevent bowstringing of the finger.3 The remaining pulleys can be divided as needed for sur-gical exposure or to relieve a stricture area.VascularTwo major arteries serve the hand. The radial artery travels under the brachioradialis muscle in the forearm. At the junc-tion of the middle and distal thirds of the forearm, the artery becomes superficial and palpable, passing just radial to the FCR tendon. At the wrist level, the artery splits into two branches. The smaller,

1	the middle and distal thirds of the forearm, the artery becomes superficial and palpable, passing just radial to the FCR tendon. At the wrist level, the artery splits into two branches. The smaller, superficial branch passes volarly into the palm to contribute to the superficial palmar arch. The larger branch passes dorsally over the scaphoid bone, under the EPL and EPB tendons (known as the anatomic snuffbox) and back volarly between the proximal thumb and index finger metacarpals to form the superficial palmar arch.The ulnar artery travels deep to the FCU muscle in the forearm. When the FCU becomes tendinous, the ulnar artery resides deep and slightly radial to it. At the wrist, the artery travels between the hamate and pisiform bones superficial to the transverse carpal ligament (known as Guyon’s canal) into the palm. The larger, superficial branch forms the superficial A5C3A4C2A3C1A2A1Figure 44-4. Drawing of anteroposterior and lateral view of the pulley

1	carpal ligament (known as Guyon’s canal) into the palm. The larger, superficial branch forms the superficial A5C3A4C2A3C1A2A1Figure 44-4. Drawing of anteroposterior and lateral view of the pulley system.Brunicardi_Ch44_p1925-p1966.indd 192920/02/19 2:48 PM 1930SPECIFIC CONSIDERATIONSPART IIpalmar arch. The deeper branch contributes to the deep palmar arch (Fig. 44-5A). In 97% of patients, at least one of the deep or superficial palmar arches is intact, allowing for the entire hand to survive on the radial or ulnar artery.5Each digit receives a radial and ulnar digital artery. For the thumb, the radial digital artery may come from the deep palmar arch or the main body of the radial artery. The larger ulnar digi-tal artery comes off the deep arch as either a discrete unit, the princeps pollicis artery, or less frequently as the first common digital artery, which then splits into the radial digital artery to the index finger and the ulnar digital artery to the thumb. The second,

1	pollicis artery, or less frequently as the first common digital artery, which then splits into the radial digital artery to the index finger and the ulnar digital artery to the thumb. The second, third, and fourth digital arteries typically branch off the superficial palmar arch and pass over the similarly named inter-osseous spaces respectively, ultimately dividing into two proper digital arteries each. The ulnar digital artery of the small finger comes off as a separate branch from the superficial arch. Within the finger, the proper digital arteries travel lateral to the bones and tendons, just palmar to the midaxis of the digit, but dorsal to the proper digital nerves (Fig. 44-5B).NerveThree principal nerves serve the forearm, wrist, and hand: the median, radial, and ulnar nerves. The most critical of these from a sensory standpoint is the median nerve. The median nerve begins as a terminal branch of the medial and lateral cords of the brachial plexus. It receives fibers from

1	The most critical of these from a sensory standpoint is the median nerve. The median nerve begins as a terminal branch of the medial and lateral cords of the brachial plexus. It receives fibers from C5–T1. The palmar cuta-neous branch of the median nerve separates from the main body of the nerve 6 cm proximal to the volar wrist crease and serves the proximal, radial-sided palm. The main body of the median nerve splits into several branches after the carpal tunnel: a radial digital branch to the thumb, an ulnar digital nerve to the thumb, and a radial digital nerve to the index finger (sometimes begin-ning as a single first common digital nerve); the second common digital nerve that branches into the ulnar digital nerve to the index finger and the radial digital nerve to the middle finger; and a third common digital nerve that branches into the ulnar digital nerve to the middle finger and a radial digital nerve to the ring finger. The digital nerves provide volar-sided sensation from

1	and a third common digital nerve that branches into the ulnar digital nerve to the middle finger and a radial digital nerve to the ring finger. The digital nerves provide volar-sided sensation from the metacarpal head level to the tip of the digit. They also, through their dorsal branches, provide dorsal-sided sensation to the dig-its from the midportion of the middle phalanx distally via dorsal branches. The thenar motor branch of the median nerve most commonly passes through the carpal tunnel and then travels in a recurrent fashion back to the thenar muscles. Less commonly, the nerve passes through or proximal to the transverse carpal ligament en route to its muscles.In the forearm, the median nerve gives motor branches to all of the flexor muscles except the FCU, and the ring and small finger portions of the FDP. Distal median motor fibers (with the exception of those to the thenar muscles) are carried through a large branch called the anterior interosseous nerve.The ulnar nerve is

1	finger portions of the FDP. Distal median motor fibers (with the exception of those to the thenar muscles) are carried through a large branch called the anterior interosseous nerve.The ulnar nerve is a terminal branch of the medial cord of the brachial plexus. It receives innervation from C8 and T1 roots. The FCU and FDP (ring/small) receive motor fibers from the ulnar nerve. In the distal forearm, 5 cm above the head of the ulna, the nerve gives off a dorsal sensory branch. Once in the hand, the nerve splits into the motor branch and sensory branches. The motor branch curves radially at the hook of the hamate bone to innervate the intrinsic muscles, as described ear-lier. The sensory branches become the ulnar digital nerve to the small finger and the fourth common digital nerve, which splits into the ulnar digital nerve to the ring finger and the radial digi-tal nerve to the small finger. The sensory nerves provide distal dorsal sensation similar to the median nerve branches.The

1	splits into the ulnar digital nerve to the ring finger and the radial digi-tal nerve to the small finger. The sensory nerves provide distal dorsal sensation similar to the median nerve branches.The radial nerve is the larger of two terminal branches of the posterior cord of the brachial plexus. It receives fibers from C5–T1 nerve roots. It innervates all of the extensor muscles of the forearm and wrist through the PIN branch except for the ECRL, which is innervated by the main body of the radial nerve in the distal upper arm. There is no ulnar nerve contribution to extension of the wrist, thumb, or finger MP joints. As noted ear-lier, the ulnar innervated intrinsic hand muscles are the principle ABFigure 44-5. Arteries of the hand and finger. A. Relative position of the superficial and deep palmar arches to the bony structures and each other; note the radial artery passes dorsal to the thumb metacarpal base, through the first web space, and anterior to the index metacarpal base as it

1	palmar arches to the bony structures and each other; note the radial artery passes dorsal to the thumb metacarpal base, through the first web space, and anterior to the index metacarpal base as it forms the deep arch. B. The neurovascular bundles lay volar to the midaxis of the digit with the artery dorsal to the nerve; Grayson’s ligament (volar) and Cleland’s ligament (dorsal) connect the bone to the skin surrounding the bundle.Brunicardi_Ch44_p1925-p1966.indd 193020/02/19 2:48 PM 1931SURGERY OF THE HAND AND WRISTCHAPTER 44extensors of the finger IP joints, although the long finger exten-sors (EDC, EIP, EDQ) make a secondary contribution to this function.In the proximal dorsal forearm, the superficial radial nerve (SRN) is the other terminal branch of the radial nerve. It travels deep to the brachioradialis muscle until 6 cm proximal to the radial styloid, where it becomes superficial. The SRN provides sensation to the dorsal hand and the radial three and a half dig-its up to the

1	to the brachioradialis muscle until 6 cm proximal to the radial styloid, where it becomes superficial. The SRN provides sensation to the dorsal hand and the radial three and a half dig-its up to the level of the mid-middle phalanx (where the dorsal branches of the proper digital nerves take over, as described earlier). The dorsal branch of the ulnar nerve provides sensation to the ulnar one and a half digits and dorsal hand in complement to the SRN.HAND EXAMINATIONEmergency Department/Inpatient ConsultationA common scenario in which the hand surgeon will be intro-duced to the patient is in trauma or other acute situations. The patient is evaluated by inspection, palpation, and provocative testing.On inspection, one should first note the position of the hand. The resting hand has a normal cascade of the fingers, with the small finger flexed most and the index finger least (Fig. 44-6). Disturbance of this suggests a tendon or skeletal problem. Also note any gross deformities or wounds

1	cascade of the fingers, with the small finger flexed most and the index finger least (Fig. 44-6). Disturbance of this suggests a tendon or skeletal problem. Also note any gross deformities or wounds and what deeper structures, if any, are visible in such wounds. Observe for abnormal coloration of a portion or all of the hand (this can be confounded by ambient temperature or other injuries), edema, and/or clubbing of the fingertips.Palpation typically begins with the radial and ulnar artery pulses at the wrist level. Pencil Doppler examination can sup-plement this and evaluate distal vessels. A pulsatile signal is normally detectable by pencil Doppler in the pad of the finger at the center of the whorl of creases. Discrepancies between digits should be noted. If all other tests are inconclusive, pricking the involved digit with a 25-gauge needle should produce bright red capillary bleeding. If an attached digit demonstrates inadequate or absent blood flow (warm ischemia), the urgency

1	pricking the involved digit with a 25-gauge needle should produce bright red capillary bleeding. If an attached digit demonstrates inadequate or absent blood flow (warm ischemia), the urgency of complet-ing the evaluation and initiating treatment markedly increases.Sensation must be evaluated prior to any administration of local anesthetic. At a minimum, light and sharp touch sensation should be documented for the radial and ulnar aspects of the tip of each digit. Beware of writing “sensation intact” at the con-clusion of this evaluation. Rather, one should document what was tested (e.g., “light and sharp touch sensation present and symmetric to the tips of all digits of the injured hand”). For a more detailed evaluation of hand sensation, two-point discrimi-nation may be assessed using a bent paperclip or monofilament. In the setting of a sharp injury, sensory deficit implies a lacer-ated structure until proven otherwise. Once sensation has been evaluated and documented, the injured

1	bent paperclip or monofilament. In the setting of a sharp injury, sensory deficit implies a lacer-ated structure until proven otherwise. Once sensation has been evaluated and documented, the injured hand can be anesthetized for patient comfort during the remainder of the examination (see below).Ability to flex and extend the wrist and digital joints is typically examined next. At the wrist level, the FCR and FCU tendons should be palpable during flexion. The wrist exten-sors are not as readily palpated due to the extensor retinaculum. Ability to flex the DIP joint (FDP) is tested by blocking the finger at the middle phalanx level. To test the FDS to each finger, hold the remaining three fingers in slight hyperextension and ask the patient to flex the involved digit (Fig. 44-7). This maneuver makes use of the fact that the FDP tendons share a common muscle belly. Placing the remaining fingers in exten-sion prevents the FDP from firing, and allows the FDS, which has a separate muscle

1	makes use of the fact that the FDP tendons share a common muscle belly. Placing the remaining fingers in exten-sion prevents the FDP from firing, and allows the FDS, which has a separate muscle belly for each tendon, to fire. Strength in grip, finger abduction, and thumb opposition is tested and compared to the uninjured side. Range of motion for the wrist, MP, and IP joints should be noted and compared to the opposite side.If there is suspicion for closed space infection, the hand should be evaluated for erythema, swelling, fluctuance, and localized tenderness. The dorsum of the hand does not have fascial septae; thus, dorsal infections can spread more widely than palmar ones. The epitrochlear and axillary nodes should be palpated for enlargement and tenderness. Findings for spe-cific infectious processes will be discussed in the “Infections” section.ABFigure 44-6. In the normal resting hand, the fingers assume a slightly flexed posture from the index finger (least) to the small

1	infectious processes will be discussed in the “Infections” section.ABFigure 44-6. In the normal resting hand, the fingers assume a slightly flexed posture from the index finger (least) to the small finger (most). A. Anteroposterior view. B. Lateral view.Brunicardi_Ch44_p1925-p1966.indd 193120/02/19 2:48 PM 1932SPECIFIC CONSIDERATIONSPART IIAdditional exam maneuvers and findings, such as those for office consultations, will be discussed with each disease pro-cess covered later in this chapter.HAND IMAGINGPlain X-RaysAlmost every hand evaluation should include plain X-rays of the injured or affected part. A standard, anteroposterior, lateral, and oblique view of the hand or wrist (as appropriate) is rapid, inexpensive, and usually provides sufficient information about the bony structures to achieve a diagnosis in conjunction with the symptoms and findings.6Lucencies within the bone should be noted. Most com-monly, these represent fractures, but they can on occasion rep-resent

1	to achieve a diagnosis in conjunction with the symptoms and findings.6Lucencies within the bone should be noted. Most com-monly, these represent fractures, but they can on occasion rep-resent neoplastic or degenerative processes. Great care should be taken to evaluate the entire X-ray, typically beginning away from the area of the patient’s complaint. Additional injuries can be missed, which might affect the treatment plan selected and eventual outcome.Congruency of adjacent joints should also be noted. The MP and IP joints of the fingers should all be in the same plain on any given view. Incongruency of the joint(s) of one finger implies fracture with rotation. At the wrist level, the proxi-mal and distal edge of the proximal row and proximal edge of the distal row should be smooth arcs, known as Gilula’s arcs (Fig. 44-8A). Disruption of these implies ligamentous injury or possibly dislocation (Fig. 44-8B).7Computed TomographyComputed tomography (CT) scanning of the hand and wrist

1	known as Gilula’s arcs (Fig. 44-8A). Disruption of these implies ligamentous injury or possibly dislocation (Fig. 44-8B).7Computed TomographyComputed tomography (CT) scanning of the hand and wrist can provide additional bony information when plain X-rays are insufficient. Comminuted fractures of the distal radius can be better visualized for number and orientation of fragments. Scaphoid fractures can be evaluated for displacement and com-minution preoperatively as well as for the presence of bony bridging postoperatively (Fig. 44-9). Recent studies have sug-gested that in the setting of suspected scaphoid fractures with negative radiographs, the use of CT scans may decrease the healthcare costs and patient morbidity.8 CT scans are also useful for CMC fractures of the hand where overlap on a plain X-ray lateral view may make diagnosis difficult.Unlike the trunk and more proximal extremities, CT scans with contrast are less useful to demonstrate abscess cavities due to the small area

1	on a plain X-ray lateral view may make diagnosis difficult.Unlike the trunk and more proximal extremities, CT scans with contrast are less useful to demonstrate abscess cavities due to the small area of these spaces.UltrasonographyUltrasonography has the advantages of being able to demon-strate soft tissue structures and being available on nights and weekends. Unfortunately, it is also highly operator dependent. In the middle of the night when magnetic resonance imaging (MRI) is not available, ultrasound may be able to demonstrate a Figure 44-7. The examiner holds the untested fingers in full exten-sion, preventing contracture of the flexor digitorum profundus. In this position, the patient is asked to flex the finger, and only the flexor digitorum superficialis will be able to fire.ABFigure 44-8. Gilula’s arcs are seen shown in this normal patient (A) and in a patient with a scaphoid fracture and perilunate dislocation (B).Brunicardi_Ch44_p1925-p1966.indd 193220/02/19 2:48 PM

1	44-8. Gilula’s arcs are seen shown in this normal patient (A) and in a patient with a scaphoid fracture and perilunate dislocation (B).Brunicardi_Ch44_p1925-p1966.indd 193220/02/19 2:48 PM 1933SURGERY OF THE HAND AND WRISTCHAPTER 44Figure 44-9. A. Preoperative images demonstrate a nonunion of a scaphoid fracture sustained 4 years earlier. B. Postoperatively, cross-sectional imaging with a computed tomography scan in the coronal plan demonstrates bone crossing the previous fracture line. This can be difficult to discern on plain X-rays due to overlap of bone fragments.ABlarge deep infection in the hand but is rarely more useful than a thorough clinical examination. Additionally, the use of dynamic ultrasound may be used to evaluate tendon motion and aid in the diagnosis of tendon pathology or injury.9Magnetic Resonance ImagingMRI provides the best noninvasive visualization of the soft tis-sue structures. With contrast, MRI can demonstrate an occult abscess. Unfortunately, it is

1	or injury.9Magnetic Resonance ImagingMRI provides the best noninvasive visualization of the soft tis-sue structures. With contrast, MRI can demonstrate an occult abscess. Unfortunately, it is often not available on an urgent basis for hand issues when this information is often needed. MRI can also demonstrate soft tissue injuries such as cartilage or ligament tears or tendonitis (usually by demonstrating edema in the area in question). It can demonstrate occult fractures that are not sufficiently displaced to be seen on X-ray or CT (again, by demonstrating edema). MRI can also demonstrate vascular disturbance of a bone, as in a patient with avascular necrosis of the scaphoid (Fig. 44-10).AngiographyAngiography of the upper extremity is rarely used. In many cen-ters, MRI and CT angiography provide sufficient resolution of the vascular structures to make traditional angiography unnec-essary. Also, primary vascular disease of the upper extremity is relatively uncommon. In the trauma

1	provide sufficient resolution of the vascular structures to make traditional angiography unnec-essary. Also, primary vascular disease of the upper extremity is relatively uncommon. In the trauma setting, vascular distur-bance usually mandates exploration and direct visualization of the structures in question, and angiography is thus obviated.For a patient with vascular disease of the upper extrem-ity, angiography of the upper extremity is usually performed through a femoral access much like with the leg. An arterial catheter can be used to deliver thrombolytic drugs to treat a thrombotic process.TRAUMAThe upper extremity–injured patient may have additional inju-ries to other parts of the body. All injured patients should receive an appropriate trauma survey to look for additional injuries.The patient with upper extremity trauma is evaluated as described in the “Hand Examination” section. Sensory exami-nation should be performed early. Once sensory status has been documented,

1	injuries.The patient with upper extremity trauma is evaluated as described in the “Hand Examination” section. Sensory exami-nation should be performed early. Once sensory status has been documented, administration of local anesthesia can provide comfort to the patient during the remainder of the evaluation Figure 44-10. T1-weighted magnetic resonance imaging shows perfused bone as white. In this patient, there is the absence of white-ness where the scaphoid should be (dashed circle), consistent with avascular necrosis.Brunicardi_Ch44_p1925-p1966.indd 193320/02/19 2:48 PM 1934SPECIFIC CONSIDERATIONSPART IIand subsequent treatment. Patients with nonclean wounds who received fewer than three prior doses of tetanus toxoid (or more than 5 years since last tetanus vaccination) or have an unknown history of prior doses should receive tetanus immunoglobulin as well as tetanus vaccination.10Fractures and DislocationsFor dislocations and displaced fractures, a visible deformity is often

1	unknown history of prior doses should receive tetanus immunoglobulin as well as tetanus vaccination.10Fractures and DislocationsFor dislocations and displaced fractures, a visible deformity is often present. Nondisplaced fractures may not show a gross deformity but will have edema and tenderness to palpation at the fracture site. A fracture is described by its displacement, rotation, and angulation. A fracture is also described in terms of comminution and the number and complexity of fracture fragments. Displacement is described as a percentage of the diameter of the bone; rotation is described in degrees of supina-tion or pronation with respect to the rest of the hand; angula-tion is described in degrees. To avoid confusion, it is useful to describe which direction the angle of the fracture points. All injuries should be evaluated for nearby wounds (open) that may introduce bacteria into the fracture site or joint space.Once the initial force on the fracture ceases, the tendons

1	fracture points. All injuries should be evaluated for nearby wounds (open) that may introduce bacteria into the fracture site or joint space.Once the initial force on the fracture ceases, the tendons passing beyond the fracture site provide the principal deforming force. Their force is directed proximally and, to a lesser extent, volarly. Based on this, the stability of a fracture can be deter-mined by the orientation of the fracture with respect to the shaft of the bone. Transverse fractures are typically stable. Oblique fractures typically shorten. Spiral fractures typically rotate as they shorten and thus require surgical treatment.Fractures of the tuft of the distal phalanx are common. Catching of a finger in a closing door is a common causative mechanism. These fractures are often nondisplaced and do not require treatment beyond protection of the distal phalanx from additional trauma while the fracture heals.Displaced transverse fractures of the phalanges can usu-ally be reduced

1	and do not require treatment beyond protection of the distal phalanx from additional trauma while the fracture heals.Displaced transverse fractures of the phalanges can usu-ally be reduced with distraction. The distal part is pulled away from the main body of the hand and then pushed in the direc-tion of the proximal shaft of the finger, and then distraction is released. Postreduction X-rays should routinely be performed to document satisfactory reduction. Oblique and spiral frac-tures usually are unstable after reduction. The involved digit(s) should be splinted until appropriate surgical intervention can be performed.Articular fractures of the IP and MP joints are worrisome because they may compromise motion. Chip fractures must be evaluated for instability of the collateral ligaments. If the joint is stable, the patient should initially be splinted for comfort. Motion therapy should be instituted early (ideally within the first week) to prevent stiffness. For larger fractures, the

1	If the joint is stable, the patient should initially be splinted for comfort. Motion therapy should be instituted early (ideally within the first week) to prevent stiffness. For larger fractures, the patient should be splinted until surgical treatment can be performed. In surgery, the fracture is typically internally fixated to allow for early motion, again with the goal of preventing stiffness.11,12Dislocations of the PIP joints produce traction on the neurovascular structures but usually do not lacerate them. In general, the patient should not be sent home with a joint that remains dislocated. Most commonly, the distal part is dorsal to the proximal shaft and sits in a hyperextended position. For this patient, the examiner gently applies pressure to the base of the distal part until it passes beyond the head of the proximal phalanx. Once there, the relocated PIP joint is gently flexed, confirming the joint is in fact reduced. The joint is splinted in slight flexion to prevent

1	it passes beyond the head of the proximal phalanx. Once there, the relocated PIP joint is gently flexed, confirming the joint is in fact reduced. The joint is splinted in slight flexion to prevent redislocation. On occasion, the head of the proximal phalanx may pass between the two slips of the FDS tendon. For these patients, the joint may not be reducible in a closed fashion.Angulated fractures of the small finger metacarpal neck (“boxer’s fracture”) are another common injury seen in the ER. Typical history is that the patient struck another individual or rigid object with a hook punch. These are often stable after reduction using the Jahss maneuver (Fig. 44-11).13Fractures of the thumb metacarpal base are often unstable. The Bennett fracture displaces the volar-ulnar base of the bone. The remainder of the articular surface and the shaft typically dislocate dorsoradially and shorten. The thumb often appears grossly shortened, and the proximal shaft of the metacarpal may reside at the

1	remainder of the articular surface and the shaft typically dislocate dorsoradially and shorten. The thumb often appears grossly shortened, and the proximal shaft of the metacarpal may reside at the level of the trapezium or even the scaphoid on X-ray. In a Rolando fracture, a second fracture line occurs between the remaining articular surface and the shaft. These fractures nearly always require open reduction and internal fixation.Most nondisplaced fractures do not require surgical treat-ment. The scaphoid bone of the wrist is a notable exception to this rule. Due to peculiarities in its vascular supply, particularly vulnerable at its proximal end, nondisplaced scaphoid fractures can fail to unite in up to 20% of patients even with appropriate immobilization. Recent developments in hardware and surgi-cal technique have allowed stabilization of the fracture with minimal surgical exposure. One prospective randomized series of scaphoid wrist fractures demonstrated shortening of time to

1	and surgi-cal technique have allowed stabilization of the fracture with minimal surgical exposure. One prospective randomized series of scaphoid wrist fractures demonstrated shortening of time to union by up to 6 weeks in the surgically treated group, but no difference in rate of union.14 Surgery may be useful in the younger, more active patient who would benefit from an earlier return to full activity.Ligament injuries of the wrist can be difficult to recognize. Patients often present late and may not be able to localize their pain. In severe cases, the ligaments of the wrist can rupture to the point of dislocation of the capitate off the lunate or even the lunate off the radius. Mayfield and colleagues classified the progression of this injury into four groups.15 In the most severe group, the lunate dislocates off the radius into the carpal tunnel. In some circumstances, the scaphoid bone may break rather than Figure 44-11. The Jahss maneuver. The surgeon fully flexes the patient’s

1	the lunate dislocates off the radius into the carpal tunnel. In some circumstances, the scaphoid bone may break rather than Figure 44-11. The Jahss maneuver. The surgeon fully flexes the patient’s small finger into the palm and secures it in his distal hand. The proximal hand controls the wrist and places the thumb on the patient’s fracture apex (the most prominent dorsal point). The examiner distracts the fracture, pushes dorsally with the distal hand (up arrow), and resists dorsal motion with the proximal hand (down arrow).Brunicardi_Ch44_p1925-p1966.indd 193420/02/19 2:48 PM 1935SURGERY OF THE HAND AND WRISTCHAPTER 44the scapholunate ligament rupturing. Attention to the congru-ency or disruption of Gilula’s arcs will help the examiner to recognize this injury. For patients with type 4 (most severe) and some with type 3 injury, the examiner should also evaluate for sensory disturbance in the median nerve distribution because this may indicate acute carpal tunnel syndrome and

1	type 4 (most severe) and some with type 3 injury, the examiner should also evaluate for sensory disturbance in the median nerve distribution because this may indicate acute carpal tunnel syndrome and necessitate more urgent intervention. Although the Mayfield pattern of injury is most common, force can also transmit along alternate paths through the carpus.16After reduction of fractures and dislocations (as well as after surgical repair of these and many other injuries), the hand must be splinted in a protected position. For the fingers, MP joints should be splinted 90°, and the IP joints at 0° (called the intrinsic plus position). The wrist is generally splinted at 20° extension because this puts the hand in a more functional posi-tion. This keeps the collateral ligaments on tension and helps prevent secondary contracture. In general, one of three splints should be used for the emergency department (ED) patient (Fig. 44-12). The ulnar gutter splint uses places plaster around the

1	and helps prevent secondary contracture. In general, one of three splints should be used for the emergency department (ED) patient (Fig. 44-12). The ulnar gutter splint uses places plaster around the ulnar border of the hand. It is generally appropriate for small finger injuries only. Dorsal plaster splints can be used for injuries of any of the fingers. Plaster is more readily con-toured to the dorsal surface of the hand than the volar surface, particularly in the setting of trauma-associated edema. For thumb injuries, the thumb spica splint is used to keep the thumb radially and palmarly abducted from the hand. Lastly, sugar tong splints include a volar and dorsal slab that includes the elbow in order to prevent supination and pronation. Sugar tong splints are most often used in the setting of acute distal radius or ulna fractures.TendonsInjuries to the flexor and extensor tendons compromise the mobility and strength of the digits. On inspection, injury is nor-mally suspected by

1	setting of acute distal radius or ulna fractures.TendonsInjuries to the flexor and extensor tendons compromise the mobility and strength of the digits. On inspection, injury is nor-mally suspected by loss of the normal cascade of the fingers. The patient should be examined as described earlier to evaluate for which tendon motion is deficient. If the patient is unable to cooperate, extension of the wrist will produce passive flexion of the fingers and also demonstrate a deficit. This is referred to at the tenodesis maneuver.Flexor tendon injuries are described based on zones (Fig. 44-13). Up until 40 years ago, zone 2 injuries were always reconstructed and never repaired primarily due to concern that the bulk of repair within the flexor sheath would prevent tendon glide. The work of Dr. Kleinert and colleagues at the University of Lou-isville changed this “axiom” and established the principle of pri-mary repair and early controlled mobilization postoperatively.17 Flexor tendon

1	Dr. Kleinert and colleagues at the University of Lou-isville changed this “axiom” and established the principle of pri-mary repair and early controlled mobilization postoperatively.17 Flexor tendon injuries should always be repaired in the operat-ing room. Although they do not need to be repaired on the day 3Figure 44-12. Commons splints used for hand injuries/surgeries. A. Ulnar gutter splint. The ring and small fingers are included and maintain an interphalangeal (IP) joint extension and metacarpopha-langeal (MP) joint flexion to 90°. B. Dorsal four-finger splint. As with the ulnar gutter splint, finger MP joints are flexed to 90° with IP joints kept fully extended. C. Thumb spica splint. One easy method to fabricate is to place one slab of plaster radially over the wrist and thumb with a second square of plaster over the thenar eminence, which joins the first. D. Sugar tong splint. This dorsal and volar slab splints immobilizes the wrist and elbow in neutral and 90° positions,

1	with a second square of plaster over the thenar eminence, which joins the first. D. Sugar tong splint. This dorsal and volar slab splints immobilizes the wrist and elbow in neutral and 90° positions, respectively.Figure 44-13. The zones of flexor tendon injury. I. Flexor digito-rum superficialis insertion to the flexor digitorum profundus inser-tion. II. Start of the A1 pulley to the flexor digitorum superficialis insertion. III. End of the carpal tunnel to the start of the A1 pulley. IV. Within the carpal tunnel. V. Proximal to the carpal tunnel.Brunicardi_Ch44_p1925-p1966.indd 193520/02/19 2:48 PM 1936SPECIFIC CONSIDERATIONSPART IIof injury, the closer to the day of injury they are repaired, the easier it will be to retrieve the retracted proximal end in surgery. The laceration should be washed out and closed at the skin level only using permanent sutures. The hand should be splinted as described earlier; one notable difference is that the wrist should be splinted at slight

1	should be washed out and closed at the skin level only using permanent sutures. The hand should be splinted as described earlier; one notable difference is that the wrist should be splinted at slight flexion (about 20°) to help decrease the retracting force on the proximal cut tendon end.Extensor tendons do not pass through a sheath in the fin-gers. As such, bulkiness of repair is less of a concern. With proper supervision/experience and equipment, primary extensor tendon repair can be performed in the ED.Very distal extensor injuries near the insertion on the dor-sal base of the distal phalanx may not have sufficient distal ten-don to hold a suture. Closed injuries, called mallet fingers, can be treated with extension splinting of the DIP joint for 6 contin-uous weeks. For patients with open injuries, a dermatotenodesis suture is performed. A 2-0 or 3-0 suture is passed through the distal skin, tendon remnant, and proximal tendon as a mattress suture. Using a suture of a different

1	open injuries, a dermatotenodesis suture is performed. A 2-0 or 3-0 suture is passed through the distal skin, tendon remnant, and proximal tendon as a mattress suture. Using a suture of a different color than the skin clos-ing sutures will help prevent removing the dermatotenodesis suture(s) too soon. The DIP joint is splinted in extension.More proximal injuries are typically repaired with a 3-0 braided permanent suture. Horizontal mattress or figure-of-eight sutures should be used, two per tendon if possible. Great care should be used to ensure matching the appropriate proximal and distal tendon ends. The patient is splinted with IP joints in extension and the wrist in extension per usual. MP joints should be splinted in 45° flexion, sometimes less. Although this posi-tion is not ideal for MP collateral ligaments, it is important for taking tension off of the tendon repairs.Nerve InjuriesIn the setting of a sharp injury, a sensory deficit implies a nerve laceration until proven

1	for MP collateral ligaments, it is important for taking tension off of the tendon repairs.Nerve InjuriesIn the setting of a sharp injury, a sensory deficit implies a nerve laceration until proven otherwise. For blunt injuries, even dis-placed fractures and dislocations, nerves are often contused but not lacerated and are managed expectantly. Nerve repairs require appropriate microsurgical equipment and suture; they should not be performed in the ED. As with tendons, nerve injuries do not require immediate exploration. However, earlier exploration will allow for easier identification of structures and less scar tissue to be present. The nerve must be resected back to healthy nerve fascicle prior to repair. Delay between injury and repair can thus make a difference between the ability to repair a nerve primarily or the need to use a graft. The injured hand should be splinted with MPs at 90° and IPs at 0°, as described earlier.Vascular InjuriesVascular injuries have the potential to be

1	a nerve primarily or the need to use a graft. The injured hand should be splinted with MPs at 90° and IPs at 0°, as described earlier.Vascular InjuriesVascular injuries have the potential to be limb or digit threaten-ing. A partial laceration of an artery at the wrist level can poten-tially cause exsanguinating hemorrhage. Consultations for these injuries must be evaluated urgently.Initial treatment for an actively bleeding wound should be direct local pressure for no less than 10 continuous minutes. If this is unsuccessful, an upper extremity tourniquet inflated to 100 mmHg above the systolic pressure should be used. One should keep this tourniquet time to less than 2 hours to avoid tissue necrosis. Once bleeding is controlled well enough to evaluate the wound, it may be cautiously explored to evaluate for bleeding points. One must be very cautious if attempting to ligate these to ensure that adjacent structures such as nerves are not included in the ligature.The hand must be

1	explored to evaluate for bleeding points. One must be very cautious if attempting to ligate these to ensure that adjacent structures such as nerves are not included in the ligature.The hand must be evaluated for adequacy of perfusion to the hand as a whole as well as the individual digits. Capillary refill, turgor, Doppler signal, and bleeding to pinprick all pro-vide useful information regarding vascular status. The finger or hand with vascular compromise requires urgent operative explo-ration. Unlike the complete amputation, in which the amputated part can be cold preserved (see later section, “Amputation and Replantation”), devascularization without amputation produces warm ischemia, which is tolerated only for a matter of hours.For the noncritical vascular injury, two treatment options exist. Simple ligation will control hemorrhage. At least one of the palmar arterial arches is intact in 97% of patients, so this will usually not compromise hand perfusion.5 Each digit also has two

1	exist. Simple ligation will control hemorrhage. At least one of the palmar arterial arches is intact in 97% of patients, so this will usually not compromise hand perfusion.5 Each digit also has two arterial inflows and can survive on one (see “Amputations and Replantation” section). In the academic hospital setting, however, consideration should be given to repairing all vascular injuries. Instructing a resident in vascular repair in the noncriti-cal setting will produce a more skilled and prepared resident for when a critical vascular injury does arise.ANESTHESIALocal AnesthesiaAnesthetic blockade can be administered at the wrist level, digi-tal level, or with local infiltration as needed. Keep in mind that all local anesthetics are less effective in areas of inflammation.The agents most commonly used are lidocaine and bupiva-caine. Lidocaine has the advantage of rapid onset, whereas bupi-vacaine has the advantage of long duration (average 6–8 hours).18 Although bupivacaine can

1	most commonly used are lidocaine and bupiva-caine. Lidocaine has the advantage of rapid onset, whereas bupi-vacaine has the advantage of long duration (average 6–8 hours).18 Although bupivacaine can produce irreversible heart block in high doses, this is rarely an issue with the amounts typically used in the hand. For pediatric patients, the tolerated dose is 2.5 mg/kg. This can be easily remembered by noting that when using 0.25% bupivacaine, 1 mL/kg is acceptable dosing.A commonly held axiom is that epinephrine is unaccept-able to be used in the hand. Several recent large series have dispelled this myth.19 Epinephrine should not be used in the fingertip and not in concentrations higher than 1:100,000 (i.e., what is present in commercially available local anesthetic with epinephrine). Beyond that, its use is acceptable and may be use-ful in an ED where tourniquet control may not be available. Also, because most ED procedures are done under pure local anesthesia, many patients will

1	Beyond that, its use is acceptable and may be use-ful in an ED where tourniquet control may not be available. Also, because most ED procedures are done under pure local anesthesia, many patients will not tolerate the discomfort of the tourniquet beyond 30 minutes.20 Epinephrine will provide hemostasis and also prolong the effect of the local anesthetic.Studies have reported that the addition of sodium bicar-bonate (NaHCO3) in order to buffer local anesthetic solutions and decrease the pain experienced during the administration of local anesthetic.21 This decrease in pain has been attributed to decreasing the acidity of local anesthetic solutions. In the clinical setting, the mixing of 8.4% sodium bicarbonate with 1% lidocaine with 1:100,000 epinephrine in a 1:9 ratio is ade-quate to provide a decrease in pain during the injection of local anesthetic.22Simple lacerations, particularly on the dorsum of the hand, can be anesthetized with local infiltration. This is performed in the

1	provide a decrease in pain during the injection of local anesthetic.22Simple lacerations, particularly on the dorsum of the hand, can be anesthetized with local infiltration. This is performed in the standard fashion.Blocking of the digital nerves at the metacarpal head level is useful for volar injuries distal to this point and for dorsal injuries beyond the midpoint of the middle phalanx (via dor-sal branches of the proper digital nerves). Fingertip injuries are particularly well anesthetized by this technique. A digit can be anesthetized via a flexor sheath approach or via the dorsal web space (Fig. 44-14A,B).Brunicardi_Ch44_p1925-p1966.indd 193620/02/19 2:48 PM 1937SURGERY OF THE HAND AND WRISTCHAPTER 44Figure 44-14. Local anesthesia can be administered at the digital or the wrist level. A. A single injection into the flexor tendon sheath at the metacarpal head level provides complete anesthesia for the digit. B. Alternatively, one can inject from a dorsal approach into the

1	level. A. A single injection into the flexor tendon sheath at the metacarpal head level provides complete anesthesia for the digit. B. Alternatively, one can inject from a dorsal approach into the web space on either side. C. The superficial radial nerve is blocked by infiltrating subcutaneously over the distal radius from the radial artery pulse to the distal radioulnar joint. The dorsal sensory branch of the ulnar nerve is blocked in similar fashion over the distal ulna. D. To block the ulnar nerve, insert the needle parallel to the plane of the palm and deep to the flexor carpi ulnaris tendon; aspirate to confirm the needle is not in the adjacent ulnar artery. E. To block the median nerve, insert the needle just ulnar to the palmaris longus tendon into the carpal tunnel. One should feel two points of resistance: one when piercing the skin, the second when piercing the antebrachial fascia.Brunicardi_Ch44_p1925-p1966.indd 193720/02/19 2:48 PM 1938SPECIFIC

1	tunnel. One should feel two points of resistance: one when piercing the skin, the second when piercing the antebrachial fascia.Brunicardi_Ch44_p1925-p1966.indd 193720/02/19 2:48 PM 1938SPECIFIC CONSIDERATIONSPART IIBlocking one or more nerves as they cross the wrist can provide several advantages: anesthesia for multiple injured dig-its, avoiding areas of inflammation where the local anesthetic agent may be less effective, and avoiding injection where the volume of fluid injected may make treatment harder (such as fracture reduction). Four major nerves cross the wrist: the median nerve, SRN, ulnar nerve, and dorsal sensory branch of the ulnar nerve (Fig. 44-14C–E). When blocking the median and ulnar nerves, beware of intraneural injection, which can cause irreversible neural scarring. If the patient complains of severe paresthesias with injection or high resistance is encountered, the needle should be repositioned.Hand Surgery Under Local AnesthesiaWide awake hand surgery is

1	If the patient complains of severe paresthesias with injection or high resistance is encountered, the needle should be repositioned.Hand Surgery Under Local AnesthesiaWide awake hand surgery is surgery that is performed under sur-geon-administered local anesthesia with field sterility but with-out the use of sedation or a tourniquet. A major benefit of this approach is the reduction of healthcare costs due to the elimination of an anesthesia provider and postoperative monitoring because only local anesthesia is used. Further benefits of sedation-free sur-gery include decreased time spent in the hospital for surgery and the ability of patients to follow instructions during surgery. This advantage is evident during flexor tendon repairs, where intra-operative active movement allows direct visualization of the tendon repair under active movement.23 Perceived weaknesses of sedation-free surgery include patient intraoperative anxiety and fear of pain during the administration of local

1	visualization of the tendon repair under active movement.23 Perceived weaknesses of sedation-free surgery include patient intraoperative anxiety and fear of pain during the administration of local anesthetic. A study by Davison et al, however, found that patients undergoing carpal tunnel release under wide awake local had no difference in anxiety or pain compared to patients undergoing carpal tunnel release with sedation.24Postoperative Pain ManagementSince the recognition of pain as the fifth vital sign in the early 2000s, the number of opioid prescriptions has risen dramati-cally. Accordingly, over the last decade, the United States has seen an increase the number of deaths due to prescription opi-oid overdose. Deaths due to opioid overdose now exceeds the number of deaths caused by heroin and cocaine combined. As healthcare providers, it is essential that we adequately treat post-operative pain with the minimal amount of narcotics necessary. A recent study by Rodgers et al

1	by heroin and cocaine combined. As healthcare providers, it is essential that we adequately treat post-operative pain with the minimal amount of narcotics necessary. A recent study by Rodgers et al identified that the majority of patients undergoing elective hand surgery used prescription pain medication for only 2 or fewer days after surgery. Many patients achieved adequate pain control with over-the-counter pain med-ication and were often left with unused opioid analgesics.25Accordingly, there has been increased emphasis on educat-ing prescribers on the recognition of opioid abuse and guide-lines for appropriate opioid prescribing. Approaches such as multimodal pain management and opioid prescription protocols have shown to achieve adequate pain control while also reduc-ing excess opioid prescriptions.26SPECIAL CONSIDERATIONSAmputations and ReplantationAfter replantation was first reported, replantation was attempted for nearly all amputations.27 Over the ensuing decades, more

1	opioid prescriptions.26SPECIAL CONSIDERATIONSAmputations and ReplantationAfter replantation was first reported, replantation was attempted for nearly all amputations.27 Over the ensuing decades, more stringent guidelines have been established regarding what should be replanted. Indications for replantation include ampu-tations of the thumb, multiple digit amputations, and amputa-tions in children. Relative contraindications to replantation include crush injuries, injuries to a single digit distal to the PIP joint, and patients who are unable to tolerate a long surgical procedure. As with all guidelines, one should evaluate the par-ticular needs of the injured patient.In preparation for replantation, the amputated part and proximal stump should be appropriately treated. The ampu-tated part should be wrapped in moistened gauze and placed in a sealed plastic bag. This bag should then be placed in an ice water bath. Do not use dry ice, and do not allow the part to contact ice directly;

1	should be wrapped in moistened gauze and placed in a sealed plastic bag. This bag should then be placed in an ice water bath. Do not use dry ice, and do not allow the part to contact ice directly; frostbite can occur in the amputated part, which will decrease its chance of survival after replantation. Bleeding should be controlled in the proximal stump by as mini-mal a means necessary, and the stump should be dressed with a nonadherent gauze and bulky dressing.For digital amputations deemed unsalvageable, revision amputation can be performed in the ED if appropriate equip-ment is available. Bony prominences should be smoothed off with a rongeur and/or rasp. Great care must be taken to identify the digital nerves and resect them back as far proximally in the wound as possible; this helps decrease the chance of painful neuroma in the skin closure. Skin may be closed with perma-nent or absorbable sutures; absorbable sutures will spare the patient the discomfort of suture removal several

1	the chance of painful neuroma in the skin closure. Skin may be closed with perma-nent or absorbable sutures; absorbable sutures will spare the patient the discomfort of suture removal several weeks later. For more proximal unsalvageable amputations, revision should be performed in the operating room to maximize vascular and neural control.Prostheses can be made for amputated parts. The more proximal the amputation, the more important to function the prosthesis is likely to be. Although finger-level prostheses are generally considered cosmetic, patients with multiple finger amputations proximal to the DIP have demonstrable functional benefit from their prosthesis as well.28Fingertip InjuriesFingertip injuries are among the most common pathologies seen in an ED. The usual history is that a door closed on the finger (commonly the middle, due to its increased length) or something heavy fell on the finger.Initial evaluation should include: wound(s) including the nail bed, perfusion,

1	a door closed on the finger (commonly the middle, due to its increased length) or something heavy fell on the finger.Initial evaluation should include: wound(s) including the nail bed, perfusion, sensation, and presence and severity of fractures. For the common scenario, complex lacerations with minimally displaced fracture(s) and no loss of perfusion, the wound is cleansed, sutured, and splinted in the ED. To properly assess the nail bed, the nail plate (hard part of the nail) should be removed. A Freer periosteal elevator is well suited for this purpose. Lacerations are repaired with 6-0 fast gut suture. Great care must be taken when suturing because excessive traction with the needle can further lacerate the tissue. After repair, the nail folds are splinted with the patient’s own nail plate (if avail-able) or with aluminum foil from the suture pack. This is done to prevent scarring from the nail folds down to the nail bed that would further compromise healing of the nail.In some

1	plate (if avail-able) or with aluminum foil from the suture pack. This is done to prevent scarring from the nail folds down to the nail bed that would further compromise healing of the nail.In some situations, tissue may have been avulsed in the injury and be unavailable for repair. Choice of treatment options depends on the amount and location of tissue loss (Fig. 44-15). Historically, wounds less than 1 cm2 with no exposed bone can be treated with local wound care and secondary intention. Recently, studies have reported that wounds with an average size of 1.75 cm2 have healed well with excellent functional and aesthetic results.29 For larger wounds or wounds or with bone exposed, one must decide if the finger is worth preserving at the current length or if shortening to allow for primary closure is a Brunicardi_Ch44_p1925-p1966.indd 193820/02/19 2:48 PM 1939SURGERY OF THE HAND AND WRISTCHAPTER 44better solution. A useful guideline is the amount of fingernail still present; if

1	closure is a Brunicardi_Ch44_p1925-p1966.indd 193820/02/19 2:48 PM 1939SURGERY OF THE HAND AND WRISTCHAPTER 44better solution. A useful guideline is the amount of fingernail still present; if greater than 50% is present, local or regional flap coverage may be a good solution.If sufficient local tissue is present, homodigital flaps can be considered. A wide range of antegrade and retrograde homodig-ital flaps can be mobilized to cover the defect. Some carry sen-sation or can receive nerve coaptation to recover sensation over time.30 For the thumb only, the entire volar skin including both neurovascular bundles can be raised and advanced distally up to 1.5 cm2.31 The thumb receives separate vascularity to its dorsal skin from the radial artery. This flap is not appropriate for the fingers. Patients retain full sensibility in the advanced skin and can be mobilized within days of surgery (Fig. 44-16A–C).For wounds too large to cover with homodigital tissue, regional flaps can be

1	fingers. Patients retain full sensibility in the advanced skin and can be mobilized within days of surgery (Fig. 44-16A–C).For wounds too large to cover with homodigital tissue, regional flaps can be considered. The skin from the distal radial thenar eminence can be raised as a random pattern flap (Fig. 44-16D–F). The finger is maintained in flexion for 14 to 21 days until division of the flap pedicle and inset of the flap. Some authors have reported prolonged stiffness in patients over 30 years old, but careful flap design helps minimize this complication.32 Alternatively, the skin from the dorsum of the middle phalanx of an adjacent digit can be raised as a flap to cover the volar P3 (Fig. 44-16G–I). The flap is inset at 14 to 21 days. Long-term studies have shown this flap develops sen-sation over time.33Patients with fingertip injures must be assessed for the possibility of salvage of the injured digit(s) taken within the context of the patient’s recovery needs and goals. The

1	sen-sation over time.33Patients with fingertip injures must be assessed for the possibility of salvage of the injured digit(s) taken within the context of the patient’s recovery needs and goals. The surgeon then matches the available options to the particular patient needs.High-Pressure Injection InjuriesHigh-pressure devices are commonly used for cleaning and applications of liquids such as lubricants and paint. Most commonly, the inexperienced worker accidentally discharges the device into his nondominant hand at the base of the digit. Severity of injury depends on the amount and type of liquid injected; hydrophobic compounds cause greater damage.34These injuries are typically quite innocuous to inspection. They are, however, digit-threatening emergencies. The patient should be informed of the severity of the injury, and explora-tion is ideally performed within 6 hours of injury. Up to 50% of such injuries result in loss of the digit, but early recogni-tion and treatment are

1	of the severity of the injury, and explora-tion is ideally performed within 6 hours of injury. Up to 50% of such injuries result in loss of the digit, but early recogni-tion and treatment are associated with increased chance of digit survival.35 Early frank discussion with the patient and initiation of appropriate treatment produce the best results and medicole-gal protection.Compartment SyndromeCompartment syndromes can occur in the forearm and/or the hand. As in other locations, these are potentially limb-threat-ening issues. Principle symptoms are pain in the affected com-partments, tense swelling, tenderness to palpation over the compartment, and pain with passive stretch of the muscles of the compartment.36 Pulse changes are a late finding; normal pulses do not rule out compartment syndrome.There are three compartments in the forearm and four groups of compartments in the hand. The volar forearm is one compartment. On the dorsum of the forearm, there is the dorsal compartment as

1	are three compartments in the forearm and four groups of compartments in the hand. The volar forearm is one compartment. On the dorsum of the forearm, there is the dorsal compartment as well as the mobile wad compartment, begin-ning proximally over the lateral epicondyle. In the hand, the thenar and hypothenar eminences each represent a compart-ment. The seven interosseous muscles each behave as a separate compartment.Compartment syndrome can be caused by intrinsic and extrinsic causes. Intrinsic causes include edema and hematoma due to fracture. Extrinsic causes include splints and dressings that are circumferentially too tight and intravenous infiltrations. Infiltrations with hyperosmolar fluids such as X-ray contrast are particularly dangerous, because additional water will be drawn in to neutralize the hyperosmolarity.Measurement of compartment pressures can be a useful adjunct to assessment of the patient. The Stryker pressure mea-surement device or similar device is kept in many

1	to neutralize the hyperosmolarity.Measurement of compartment pressures can be a useful adjunct to assessment of the patient. The Stryker pressure mea-surement device or similar device is kept in many operating rooms for this purpose. The needle is inserted into the compart-ment in question, a gentle flush with 0.1 to 0.2 cc of saline clears the measurement chamber, and a reading is obtained. Studies have disagreed about whether the criterion is a measured pres-sure (30–45 mmHg, depending on the series) or within a certain amount of the diastolic blood pressure.37Compartment releases are performed in the operating room under tourniquet control. Release of the volar forearm compartment includes release of the carpal tunnel. As the inci-sion travels distally, it should pass ulnar and then curve back radially just before the carpal tunnel. This avoids a linear inci-sion across a flexion crease and also decreases the chance of injury to the palmar cutaneous branch of the median nerve. One

1	back radially just before the carpal tunnel. This avoids a linear inci-sion across a flexion crease and also decreases the chance of injury to the palmar cutaneous branch of the median nerve. One dorsal forearm incision can release the dorsal compartment and the mobile wad. In the hand, the thenar and hypothenar com-partments are released each with a single incision. The interos-seous compartments are released with incisions over the index and ring metacarpal shafts. Dissection then continues radial and ulnar to each of these bones and provides release of all the mus-cle compartments. Any dead muscle is debrided. Incisions are left open and covered with a nonadherent dressing. The wounds are reexplored in 2 to 3 days to assess for muscle viability. Often the incisions can be closed primarily, but a skin graft may be needed for the forearm.Fingertip injuryGreater than 50%nailbed remainingHeal by secondaryintentionSufficient same digittissueVolar V-YNoNoNoNoYesYesYesYesCross-finger

1	but a skin graft may be needed for the forearm.Fingertip injuryGreater than 50%nailbed remainingHeal by secondaryintentionSufficient same digittissueVolar V-YNoNoNoNoYesYesYesYesCross-finger flapBilateral V-YMoberg flap(Thumb only)Shorten bone forprimary stumpclosureTissue lossThenar flapWound <1 cm2 andno exposed bonePrimary repairFigure 44-15. Treatment algorithm for management of fingertip injuries. See text for description of flaps.Brunicardi_Ch44_p1925-p1966.indd 193920/02/19 2:48 PM 1940SPECIFIC CONSIDERATIONSPART IIFigure 44-16. Local flaps for digital tip coverage. A–C. For thumb injuries, Moberg described elevation of the entire volar skin with both neurovascular bundles for distal advancement. Sensation to the advanced skin is maintained. D–F. An 8-year-old girl underwent fingertip replantation that did not survive. A thenar flap was transferred to cover the defect. Some authors advise against its use in patients over 30 years old. G–I. In this 45-year-old man, the

1	fingertip replantation that did not survive. A thenar flap was transferred to cover the defect. Some authors advise against its use in patients over 30 years old. G–I. In this 45-year-old man, the entire skin of P3 of the long finger was avulsed and unrecoverable. A cross-finger flap was transferred and provides excellent, durable coverage. The border of the flap and surrounding skin is still apparent 4.5 months after surgery.Brunicardi_Ch44_p1925-p1966.indd 194020/02/19 2:49 PM 1941SURGERY OF THE HAND AND WRISTCHAPTER 44Figure 44-16. (Continued)Brunicardi_Ch44_p1925-p1966.indd 194120/02/19 2:49 PM 1942SPECIFIC CONSIDERATIONSPART IIFigure 44-16. (Continued)Brunicardi_Ch44_p1925-p1966.indd 194220/02/19 2:49 PM 1943SURGERY OF THE HAND AND WRISTCHAPTER 44If the examiner feels the patient does not have a compart-ment syndrome, elevation and serial examination are manda-tory. When in doubt, it is safer to release an early compartment syndrome than wait to release and risk

1	the patient does not have a compart-ment syndrome, elevation and serial examination are manda-tory. When in doubt, it is safer to release an early compartment syndrome than wait to release and risk muscle necrosis. Pro-gression of compartment syndrome can lead to Volkmann’s ischemic contracture with muscle loss and scarring that may compress nerves and other critical structures. Medicolegally, it is far easier to defend releasing an early compartment syn-drome than delaying treatment until the process has progressed to necrosis and/or deeper scarring.COMPLICATIONSNonunionAny fractured bone has the risk of failing to heal. Fortunately, in the fingers and hand, this is a rare problem. Tuft injuries, where soft tissue interposes between the fracture fragments, have rela-tively higher risk of this problem. The nonunited tuft can be treated with debridement and bone grafting or revision amputa-tion depending on the needs and goals of the patient. Phalan-geal and metacarpal nonunions are

1	of this problem. The nonunited tuft can be treated with debridement and bone grafting or revision amputa-tion depending on the needs and goals of the patient. Phalan-geal and metacarpal nonunions are also quite rare. They can similarly be treated with debridement of the nonunion, grafting, and rigid fixation.38 More proximally, the scaphoid bone of the wrist has a significant risk of nonunion even if nondisplaced (see Fig. 44-9A). Any patient suspected of a scaphoid injury, namely those with tenderness at the anatomic snuffbox, should be placed in a thumb spica splint and reevaluated within 2 weeks even if initial X-rays show no fracture. Scaphoid nonunions can be quite challenging to repair, and immobilization at the time of injury in a thumb spica splint is essentially always warranted.39StiffnessThe desired outcome of any hand injury is a painless, mobile, functional hand. Multiple factors can contribute to decreased mobility, including complex injuries of soft tissue and bone,

1	desired outcome of any hand injury is a painless, mobile, functional hand. Multiple factors can contribute to decreased mobility, including complex injuries of soft tissue and bone, noncompliance of the patient with postoperative therapy, and inappropriate splinting. The surgeon performing the initial eval-uation can greatly impact this last factor. The goal of splinting is to keep the collateral ligaments on tension (MPs at 90°, IP joints straight). For severe cases of stiffness, mobilization sur-geries such as tenolysis and capsulotomies can be performed, but these rarely produce normal range of motion.40 Prevention of joint contractures with appropriate splinting and early, pro-tected mobilization is the best option to maximize mobility at the end of healing. Healing of an injured or diseased structure in the hand is not the endpoint of treatment; the goal of any inter-vention must be to obtain structure healing, relief of pain, and maximization of function.NeuromaAny lacerated

1	diseased structure in the hand is not the endpoint of treatment; the goal of any inter-vention must be to obtain structure healing, relief of pain, and maximization of function.NeuromaAny lacerated nerve will form a neuroma. A neuroma consists of a ball of scar and axon sprouts at the end of the injured nerve.41 In unfavorable circumstances, this neuroma can become painful. The SRN is particularly notorious for this problem. By provid-ing proximal axon sprouts a target, nerve repair is an excellent preventive technique. In some circumstances, such as injuries requiring amputation, this is not possible. As mentioned earlier, the surgeon should resect the nerve stump as far proximally in the wound as possible to avoid the nerve stump healing in the cutaneous scar to minimize this risk.For the patient who develops a painful neuroma, nonsurgi-cal treatments are initiated first. The neuroma can be identified by the presence of a Tinel’s sign. Therapy techniques of desen-sitization,

1	the patient who develops a painful neuroma, nonsurgi-cal treatments are initiated first. The neuroma can be identified by the presence of a Tinel’s sign. Therapy techniques of desen-sitization, ultrasound, and electrical stimulation have all proven useful. Corticosteroid injection to the neuroma has also proven useful in some hands.When these techniques fail, surgery is contemplated. The neuroma can be resected, but a new one will form to replace it. The nerve ending can be buried in muscle or even bone to pre-vent the neuroma from residing in a superficial location where it may be impacted frequently.Regional Pain SyndromesInjuries to the upper extremity can occasionally result in the patient experiencing pain beyond the area of initial injury. Reflex sympathetic dystrophy and sympathetic mediated pain are two terms that have been used in the past to describe this phenomenon. Both are inaccurate, as the sympathetic nervous system is not always involved. Current terminology for this

1	mediated pain are two terms that have been used in the past to describe this phenomenon. Both are inaccurate, as the sympathetic nervous system is not always involved. Current terminology for this condition is complex regional pain syndrome (CRPS). Type I occurs in the absence of a documented nerve injury; type II occurs in the presence of one.42CRPSs manifest as pain beyond the area of initial inju-ries. There is often associated edema and changes in hair and/or sweat distribution. Comparison to the unaffected side is useful to better appreciate these findings. There are currently no imag-ing studies that can be considered diagnostic for CRPS.43For the patient in whom the diagnosis of CRPS is not clear, no definitive diagnostic study exists. Patients suspected of CRPS should be referred for aggressive hand therapy. Brief trials of oral corticosteroids have been successful in some series. Referral to a pain management specialist including a trial of stel-late ganglion blocks is also

1	for aggressive hand therapy. Brief trials of oral corticosteroids have been successful in some series. Referral to a pain management specialist including a trial of stel-late ganglion blocks is also frequently employed.NERVE COMPRESSIONNerves conduct signals along their axonal membranes toward their end organs. Sensory axons carry signals from distal to proximal; motor axons from proximal to distal. Myelin from Schwann cells allows faster conduction of signals. Signals jump from the start of one Schwann cell to the end of the cell (a loca-tion called a gap junction) and only require the slower mem-brane depolarization in these locations.Nerve compression creates a mechanical disturbance of the nerve.44 In early disease, the conduction signal is slowed across the area of compression. When compression occurs to a sufficient degree for a sufficient time, individual axons may die. On a nerve conduction study, this manifests as a decrease in amplitude. Muscles receiving motor axons may

1	compression occurs to a sufficient degree for a sufficient time, individual axons may die. On a nerve conduction study, this manifests as a decrease in amplitude. Muscles receiving motor axons may show electri-cal disturbance on electromyogram (EMG) when sufficiently deprived of their axonal input.Compression of sensory nerves typically produces a com-bination of numbness, paresthesias (pins and needles), and pain. Knowledge of the anatomic distribution of the peripheral nerves can aid in diagnosis. Sensory disturbance outside an area of dis-tribution of a particular nerve (e.g., volar and dorsal radial-sided hand numbness for median nerve) makes compression of that nerve less likely. Diseases that cause systemic neuropathy (e.g., diabetes) can make diagnosis more difficult.Nerve compression can theoretically occur anywhere along a peripheral nerve’s course. The most common sites of nerve compression in the upper extremity are the median nerve at the carpal tunnel, ulnar nerve at the

1	can theoretically occur anywhere along a peripheral nerve’s course. The most common sites of nerve compression in the upper extremity are the median nerve at the carpal tunnel, ulnar nerve at the cubital tunnel, and ulnar nerve at Guyon’s canal. Other, less common locations of nerve 4Brunicardi_Ch44_p1925-p1966.indd 194320/02/19 2:49 PM 1944SPECIFIC CONSIDERATIONSPART IIcompression are described as well. In addition, a nerve can become compressed in scar due to a previous trauma.Carpal Tunnel SyndromeThe most common location of upper extremity nerve compres-sion is the median nerve at the carpal tunnel, called carpal tunnel syndrome (CTS). The carpal tunnel is bordered by the scaphoid bone radially, the lunate and capitate bones dorsally, and the hook of the hamate bone ulnarly (see Fig. 44-3). The transverse carpal ligament, also called the flexor retinaculum, is its super-ficial border. The FPL, four FDS, and four FDP tendons pass through the carpal tunnel along with the median

1	44-3). The transverse carpal ligament, also called the flexor retinaculum, is its super-ficial border. The FPL, four FDS, and four FDP tendons pass through the carpal tunnel along with the median nerve. Of these 10 structures, the median nerve is relatively superficial and radial to the other nine.An estimated 53 per 10,000 working adults have evidence of CTS. The National Institute for Occupational Safety and Health website asserts, “There is strong evidence of a positive association between exposure to a combination of risk factors (e.g., force and repetition, force and posture) and CTS.”45 There is disagreement among hand surgeons regarding whether occur-rence of CTS in a patient who does repetitive activities at work represents a work-related injury.Initial evaluation of the patient consists of symptom inven-tory: location and character of the symptoms, sleep disturbance due to symptoms, history of dropping objects, and difficulty manipulating small objects such as buttons, coins,

1	of symptom inven-tory: location and character of the symptoms, sleep disturbance due to symptoms, history of dropping objects, and difficulty manipulating small objects such as buttons, coins, or jewelry clasps.46Physical examination should begin with inspection. Look for evidence of wasting of the thenar muscles. Tinel’s sign should be tested over the median nerve from the volar wrist flexion crease to the proximal palm, although this test has significant interexam-iner variability.47 Applying pressure over the carpal tunnel while flexing the wrist has been shown in one series to have the high-est sensitivity when compared to Phalen’s and Tinel’s signs.48 Strength of the thumb in opposition should also be tested.Early treatment of CTS consists of conservative man-agement. The patient is given a splint to keep the wrist at 20° extension worn at nighttime. Many patients can have years of symptom relief with this management. As a treatment and diag-nostic modality, corticosteroid

1	is given a splint to keep the wrist at 20° extension worn at nighttime. Many patients can have years of symptom relief with this management. As a treatment and diag-nostic modality, corticosteroid injection of the carpal tunnel is often employed. Mixing local anesthetic into the solution pro-vides the benefit of early symptom relief (corticosteroids often take 3–7 days to provide noticeable benefit), and report of postin-jection anesthesia in the median nerve distribution confirms the injection went into the correct location. Multiple authors have shown a strong correlation to relief of symptoms with cortico-steroid injection and good response to carpal tunnel release.49When lesser measures fail or are no longer effective, carpal tunnel release is indicated. Open carpal tunnel release is a time-tested procedure with documented long-term relief of symptoms. A direct incision is made over the carpal tun-nel, typically in line with where the ring finger pad touches the proximal palm in

1	a time-tested procedure with documented long-term relief of symptoms. A direct incision is made over the carpal tun-nel, typically in line with where the ring finger pad touches the proximal palm in flexion. Skin is divided followed by palmar fascia. The carpal tunnel contents are visualized as they exit the carpal tunnel. The transverse carpal ligament is divided with the median nerve visualized and protected at all times. Improve-ment in symptoms is typically noted by the first postoperative visit, although symptom relief may be incomplete for patients with long-standing disease or systemic nerve-affecting diseases such as diabetes.Endoscopic techniques have been devised to address CTS. All involve avoidance of incising the skin directly over the carpal tunnel. In experienced hands, endoscopic carpal tunnel release provides the same relief of CTS with less intense and shorter lasting postoperative pain. After 3 months, however, the results are equivalent to open release.50 In

1	endoscopic carpal tunnel release provides the same relief of CTS with less intense and shorter lasting postoperative pain. After 3 months, however, the results are equivalent to open release.50 In inexperienced hands, there may be a higher risk of injury to the median nerve with the endoscopic techniques; this procedure is not for the occasional carpal tunnel surgeon.Cubital Tunnel SyndromeThe second most common location of upper extremity nerve compression is the ulnar nerve where it passes behind the elbow at the cubital tunnel. The cubital tunnel retinaculum passes between the medial epicondyle of the humerus and the olec-ranon process of the ulna. It stabilizes the ulnar nerve in this location during elbow motion. Over time, or sometimes after trauma, the ulnar nerve can become less stabilized in this area. Motion of the elbow then produces trauma to the nerve as it impacts the retinaculum and medial epicondyle.Cubital tunnel syndrome may produce sensory and motor symptoms.51 The

1	in this area. Motion of the elbow then produces trauma to the nerve as it impacts the retinaculum and medial epicondyle.Cubital tunnel syndrome may produce sensory and motor symptoms.51 The small finger and ulnar half of the ring fin-gers may have numbness, paresthesias, and/or pain. The ulnar nerve also innervates the dorsal surface of the small finger and ulnar side of the ring finger, so numbness in these areas can be explained by cubital tunnel syndrome. The patient may also report weakness in grip due to effects on the FDP tendons to the ring and small fingers and the intrinsic hand muscles. Patients with advanced disease may complain of inability to fully extend the ring and small finger IP joints.Physical examination for cubital tunnel syndrome begins with inspection. Look for wasting in the hypothenar eminence and the interdigital web spaces. When the hand rests flat on the table, the small finger may rest in abduction with respect to the other fingers; this is called

1	for wasting in the hypothenar eminence and the interdigital web spaces. When the hand rests flat on the table, the small finger may rest in abduction with respect to the other fingers; this is called Wartenberg’s sign. Tinel’s sign is often present at the cubital tunnel. Elbow flexion and the shoulder internal rotation tests are affective maneuvers to aid in the diagnosis of cubital tunnel syndrome.52 Grip strength and finger abduction strength should be compared to the unaffected side. Froment’s sign can be tested by placing a sheet of paper between the thumb and index finger and instructing the patient to hold on to the paper while the examiner pulls it away without flexing the finger or thumb (this tests the strength of the adduc-tor pollicis and first dorsal interosseous muscles). If the patient must flex the index finger and/or thumb (FDP-index and FPL, both median nerve supplied) to maintain traction on the paper, this is a positive response.Early treatment of cubital tunnel

1	If the patient must flex the index finger and/or thumb (FDP-index and FPL, both median nerve supplied) to maintain traction on the paper, this is a positive response.Early treatment of cubital tunnel syndrome begins with avoiding maximal flexion of the elbow. Splints are often used for this purpose. Corticosteroid injection is rarely done for this condition; unlike in the carpal tunnel, there is very little space within the tunnel outside of the nerve. Injection in this area runs a risk of intraneural injection, which can cause permanent scar-ring of the nerve and dysfunction.When conservative management fails, surgery has been contemplated. Treatment options include releasing the cubital tunnel retinaculum with or without transposing the nerve ante-rior to the elbow. While some authors advocate anterior trans-position into the flexor-pronator muscle group with the goal of maximizing nerve recovery, recent studies have demonstrated equivalent results between transposition and in situ

1	anterior trans-position into the flexor-pronator muscle group with the goal of maximizing nerve recovery, recent studies have demonstrated equivalent results between transposition and in situ release of the nerve even in advanced cases. For this reason, the simpler in situ release, either open or endoscopic, is preferred by many surgeons.53Brunicardi_Ch44_p1925-p1966.indd 194420/02/19 2:49 PM 1945SURGERY OF THE HAND AND WRISTCHAPTER 44Other Sites of Nerve CompressionAll nerves crossing the forearm have areas described where compression can occur.51 The median nerve can be compressed as it passes under the pronator teres. The ulnar nerve can be compressed as it passes through Guyon’s canal. The radial nerve, or its posterior interosseous branch, can be compressed as it passes through the radial tunnel (distal to the elbow where the nerve divides and passes under the arch of the supinator muscle). The SRN can be compressed distally in the forearm as it emerges from under the

1	the radial tunnel (distal to the elbow where the nerve divides and passes under the arch of the supinator muscle). The SRN can be compressed distally in the forearm as it emerges from under the brachioradialis tendon, called Wartenberg’s syndrome. As mentioned previously, any nerve can become compressed in scar at the site of a previous trauma.DEGENERATIVE JOINT DISEASEAs with other joints in the body, the joints of the hand and wrist can develop degenerative changes. Symptoms typically begin in the fifth decade of life. Symptoms consist of joint pain and stiffness and often are exacerbated with changes in the weather. Any of the joints can become involved. As the articular carti-lage wears out, pain typically increases and range of motion decreases. The patient should always be asked to what degree symptoms are impeding activities.Physical findings are documented in serial fashion from the initial visit and subsequent visits. Pain with axial loading of the joint may be present.

1	to what degree symptoms are impeding activities.Physical findings are documented in serial fashion from the initial visit and subsequent visits. Pain with axial loading of the joint may be present. Decreased range of motion may be a late finding. Instability of the collateral ligaments of the joint is uncommon in the absence of inflammatory arthritis.Plain X-rays are typically sufficient to demonstrate arthri-tis. Initially, the affected joint has a narrower radiolucent space between the bones. As joint degeneration progresses, the joint space further collapses. Bone spurs, loose bodies, and cystic changes in the bone adjacent to the joint all may become appar-ent. X-ray findings do not always correlate with patient symp-toms. Patients with advanced X-ray findings may have minimal symptoms, and vice versa. Treatment is initiated and progressed based on the patient’s symptoms regardless of imaging findings.Initial management begins with rest of the painful joint. Splints are often

1	and vice versa. Treatment is initiated and progressed based on the patient’s symptoms regardless of imaging findings.Initial management begins with rest of the painful joint. Splints are often useful, but may significantly impair the patient in activities and thus are frequently used at nighttime only. Oral nonsteroidal anti-inflammatory medications such as ibuprofen and naproxen are also useful. Patients on anticoagulants and antiplatelet medications may not be able to take these, and some patients simply do not tolerate the gastric irritation side effect even if they take the medication with food.For patients with localized disease affecting only one or a few joints, corticosteroid injection may be contemplated. Nee-dle insertion can be difficult since these joint spaces are quite narrow even before degenerative disease sets in. Also, many corticosteroid injections are suspensions, not solutions; injected corticosteroid will remain in the joint space and can be seen as a white paste

1	even before degenerative disease sets in. Also, many corticosteroid injections are suspensions, not solutions; injected corticosteroid will remain in the joint space and can be seen as a white paste if surgery is performed on a joint that has been previously injected.Small Joints (Metacarpophalangeal and Interphalangeal)When conservative measures fail, two principal surgical options exist: arthrodesis and arthroplasty. The surgeon and patient must decide together as to whether conservative measures have failed. Surgery for arthritis, whether arthrodesis or arthroplasty, is performed for the purpose of relieving pain. Arthrodesis, fusion of a joint can be performed with a tension band or axial compression screw techniques.54 Both methods provides excel-lent relief of pain and is durable over time. However, it comes at the price of total loss of motion.Silicone implant arthroplasty has been available for over 40 years.55 Rather than a true replacement of the joint, the silicone implant

1	time. However, it comes at the price of total loss of motion.Silicone implant arthroplasty has been available for over 40 years.55 Rather than a true replacement of the joint, the silicone implant acts as a spacer between the two bones adja-cent to the joint. This allows for motion without bony contact that would produce pain. Long-term studies have shown that all implants fracture over time, but usually continue to preserve motion and pain relief.56In the past 15 years, resurfacing implant arthroplasties have become available for the small joints of the hand. Multiple different materials have been used to fabricate such implants. These are designed to behave as a true joint resurfacing (as knee and hip arthroplasty implants are) and have shown promising outcomes in shortand intermediate-term studies.56 Neither the silicone nor the resurfacing arthroplasties preserve (or restore) full motion of the MP or PIP joints.WristThe CMC joint of the thumb, also called the basilar joint, is

1	studies.56 Neither the silicone nor the resurfacing arthroplasties preserve (or restore) full motion of the MP or PIP joints.WristThe CMC joint of the thumb, also called the basilar joint, is another common location of arthritis pain. Pain in this joint par-ticularly disturbs function because the CMC joint is essential for opposition and cylindrical grasp. Patients will typically com-plain of pain with opening a tight jar or doorknob and strong pinch activities such as knitting. Conservative management is used first, as described earlier. Prefabricated, removable thumb spica splinting can provide excellent relief of symptoms for many patients.Multiple surgical options exist for thumb CMC arthritis. Many resurfacing implants have been used in the past; often they have shown good shortand intermediate-term results and poor long-term results. Resection of the arthritic trapezium provides excellent relief of pain; however, many authors feel that stabi-lization of the thumb metacarpal base

1	results and poor long-term results. Resection of the arthritic trapezium provides excellent relief of pain; however, many authors feel that stabi-lization of the thumb metacarpal base is necessary to prevent shortening and instability.57 Some surgeons have demonstrated excellent long-term results from resection of the trapezium without permanent stabilization of the metacarpal base.58 For both of these operations, the thumb base may not be sufficiently stable to withstand heavy labor. For these patients, fusion of the thumb CMC in mild opposition provides excellent pain relief and durability. The patient must be warned preoperatively that he will not be able to lay his hand flat after the surgery. This loss of motion can be problematic when the patient attempts to tuck in clothing or reach into a narrow space.59Degenerative change of the radiocarpal and midcarpal joints is often a consequence of scapholunate ligament injury. Often the initial injury goes untreated, with the patient

1	into a narrow space.59Degenerative change of the radiocarpal and midcarpal joints is often a consequence of scapholunate ligament injury. Often the initial injury goes untreated, with the patient believ-ing it is merely a “sprain”; the patient is first diagnosed with the initial injury when he presents years later with degenerative changes.Degenerative wrist changes associated with the scaph-olunate ligament follow a predictable pattern over many years, called scapholunate advanced collapse or SLAC wrist.60 Because of this slow progression (Fig. 44-17A), patients can usually be treated with a motion-sparing procedure. If there is truly no arthritic change present, the scapholunate ligament can be reconstructed.If arthritis is limited to the radiocarpal joint, two motion-sparing options are available. The proximal carpal row (scaphoid, lunate, and triquetrum) can be removed (proximal row carpectomy [PRC]). The lunate facet of the radius then Brunicardi_Ch44_p1925-p1966.indd

1	are available. The proximal carpal row (scaphoid, lunate, and triquetrum) can be removed (proximal row carpectomy [PRC]). The lunate facet of the radius then Brunicardi_Ch44_p1925-p1966.indd 194520/02/19 2:49 PM 1946SPECIFIC CONSIDERATIONSPART IIarticulates with the base of the capitate, whose articular surface is similar in shape to that of the base of the lunate. Studies have shown maintenance of approximately 68% of the wrist flexion-extension arc and 72% of hand strength compared to the con-tralateral side.61 Alternatively, the scaphoid can be excised, and four-bone fusion (lunate, capitate, hamate, and triquetrum) can be performed. This maintains the full length of the wrist and the lunate in the lunate facet of the radius. Some series have shown better strength but less mobility with this technique, oth-ers have shown equivalent results to the PRC.62 The four-bone fusion does appear to be more durable for younger patients and/or those who perform heavy labor.If the patient

1	with this technique, oth-ers have shown equivalent results to the PRC.62 The four-bone fusion does appear to be more durable for younger patients and/or those who perform heavy labor.If the patient presents with pancarpal arthritis or motion-sparing measures have failed to alleviate pain, total wrist fusion is the final surgical option. The distal radius is fused, through the proximal and distal carpal rows to the third metacarpal, typi-cally with a dorsal plate and screws. Multiple long-term studies have shown excellent pain relief and durability; this comes at the cost of total loss of wrist motion. This is surprisingly well tolerated in most patients, especially if the other hand/wrist is unaffected. The only activity of daily living that cannot be done with a fused wrist is personal toileting.Rheumatoid ArthritisRheumatoid arthritis (RA) is an inflammatory arthritis that can affect any joint in the body. Inflamed synovium causes articular cartilage breakdown with pain and

1	toileting.Rheumatoid ArthritisRheumatoid arthritis (RA) is an inflammatory arthritis that can affect any joint in the body. Inflamed synovium causes articular cartilage breakdown with pain and decreased range of motion. The goals of hand surgery for the RA patient are relief of pain, improvement of function, slowing progression of disease, and improvement in appearance.63 In addition, swelling of the joint due to the inflammation can cause laxity and even failure of the collateral ligaments supporting the joints. Recent advances in the medical care of RA have made the need for surgical care of these patients far less common than in previous decades.MP joints of the fingers are commonly affected. The base of the proximal phalanx progressively subluxates and eventu-ally dislocates volarly with respect to the metacarpal head. The collateral ligaments, particularly on the radial side, stretch out and cause the ulnar deviation of the fingers characteristic of the rheumatoid hand. In more

1	with respect to the metacarpal head. The collateral ligaments, particularly on the radial side, stretch out and cause the ulnar deviation of the fingers characteristic of the rheumatoid hand. In more advanced cases, the joint may not be salvageable (Fig. 44-17B). For these patients, implant arthro-plasty is the mainstay of surgical treatment. Silicone implants have been used for over 40 years with good results.64 The sili-cone implant acts as a spacer between proximal and distal bone, rather than as a true resurfacing arthroplasty. The radial col-lateral ligament must be repaired to appropriate length to cor-rect the preoperative ulnar deviation of the MP joint. Extensor tendon centralization is then performed, as needed, at the end of the procedure.For MP joint and PIP joint disease, fusion is an option. However, since RA usually affects multiple joints, fusion is typically avoided due to impaired function of adjacent joints, which would leave a severe motion deficit to the

1	fusion is an option. However, since RA usually affects multiple joints, fusion is typically avoided due to impaired function of adjacent joints, which would leave a severe motion deficit to the finger.Failure of the support ligaments of the distal radioulnar joint (DRUJ) leads to the caput ulnae posture of the wrist with the ulnar head prominent dorsally. As this dorsal prominence becomes more advanced, the ulna head, denuded of its cartilage to act as a buffer, erodes into the overlying extensor tendons. Extensor tenosynovitis, followed ultimately by tendon rupture, begins ulnarly and proceeds radially. Rupture of the ECU ten-don may go unnoticed due to the intact ECRL and ECRB ten-dons to extend the wrist. EDQ rupture may go unnoticed if a sufficiently robust EDC tendon to the small finger exists. Once the fourth compartment (EDC) tendons begin to fail, the motion deficit is unable to be ignored by the patient.Surgical solutions must address the tendon ruptures as well as the DRUJ

1	exists. Once the fourth compartment (EDC) tendons begin to fail, the motion deficit is unable to be ignored by the patient.Surgical solutions must address the tendon ruptures as well as the DRUJ synovitis and instability and ulna head break-down that led to them.65 Excision of the ulna head removes the bony prominence. The DRUJ synovitis must also be resected. Figure 44-17. Arthritis of the hand and wrist. A. This patient injured her scapholunate ligament years prior to presentation. The scapholunate interval is widened (double arrow), and the radioscaphoid joint is degenerated (solid oval), but the radiolunate and lunocapitate joint spaces are well preserved (dashed ovals). B. This patient has had rheumatoid arthritis for decades. The classic volar subluxation of the metacarpophalangeal joints of the fingers (dashed oval) and radial deviation of the fingers are apparent.Brunicardi_Ch44_p1925-p1966.indd 194620/02/19 2:49 PM 1947SURGERY OF THE HAND AND WRISTCHAPTER 44Alternatively,

1	of the fingers (dashed oval) and radial deviation of the fingers are apparent.Brunicardi_Ch44_p1925-p1966.indd 194620/02/19 2:49 PM 1947SURGERY OF THE HAND AND WRISTCHAPTER 44Alternatively, the DRUJ can be fused and the ulna neck resected to create a pseudoarthrosis to allow for rotation. For both pro-cedures, the remaining distal ulna must be stabilized. Multiple techniques have been described using portions of FCU, ECU, wrist capsule, and combinations thereof.The ruptured extensor tendons are typically degenerated over a significant length. Primary repair is almost never pos-sible, and the frequent occurrence of multiple tendon ruptures makes repair with graft less desirable due to the need for mul-tiple graft donors.Strict compliance with postoperative therapy is essential to maximizing the surgical result. Due to the chronic inflam-mation associated with RA, tendon and ligament repairs will be slower to achieve maximal tensile strength. Prolonged night-time splinting, usually

1	the surgical result. Due to the chronic inflam-mation associated with RA, tendon and ligament repairs will be slower to achieve maximal tensile strength. Prolonged night-time splinting, usually for months, helps prevent recurrence of extensor lag. Finally, the disease may progress over time. Reconstructions that were initially adequate may stretch out or fail over time. Medical management is the key to slowing dis-ease progression and maximizing the durability of any surgical reconstruction.DUPUYTREN’S CONTRACTUREIn 1614, a Swiss surgeon named Felix Plater first described con-tracture of multiple fingers due to palpable, cord-like structures on the volar surface of the hand and fingers. The disease state he described would ultimately come to be known as Dupuytren’s contracture. Dupuytren’s name came to be associated with the disease after he performed an open fasciotomy of a contracted cord before a class of medical students in 1831.66The palmar fascia consists of collagen bundles in

1	name came to be associated with the disease after he performed an open fasciotomy of a contracted cord before a class of medical students in 1831.66The palmar fascia consists of collagen bundles in the palm and fingers. These are primarily longitudinally oriented and reside as a layer between the overlying skin and the underlying tendons and neurovascular structures. There are also connections from this layer to the deep structures below and the skin above. Much is known about the progression of these structures from their normal state (called bands) to their contracted state (called cords), but little is known on how or why this process begins.Increased collagen deposition leads to a palpable nodule in the palm. Over time, there is increased deposition distally into the fingers. This collagen becomes organized and linearly ori-ented. These collagen bundles, with the aid of myofibroblasts, contract down to form the cords, which are the hallmark of the symptomatic patient. Detail of

1	becomes organized and linearly ori-ented. These collagen bundles, with the aid of myofibroblasts, contract down to form the cords, which are the hallmark of the symptomatic patient. Detail of the molecular and cell biology of Dupuytren’s disease is beyond the scope of this chapter but is available in multiple hand surgery texts.67Most nonoperative management techniques will not delay the progression of disease. Corticosteroid injections may soften nodules and decrease the discomfort associated with them but are ineffective against cords. Splinting has similarly been shown not to retard disease progression.Recently, several minimally invasive treatment approaches have been described for the treatment of Dupuytren’s disease.68 Disruption of the cord with a needle is an effective means of releasing contractures, particularly at the MP joint level. Long-term studies have demonstrated more rapid recovery from needle fasciotomy, as the procedure is called, but more durable results with

1	releasing contractures, particularly at the MP joint level. Long-term studies have demonstrated more rapid recovery from needle fasciotomy, as the procedure is called, but more durable results with fasciectomy.69 Injectable clostridial collagenase was approved by the U.S. Food and Drug Administration in 2009, and although it has shown good early results, treatment costs remain high.70For patients with advanced disease including contrac-tures of the digits that limit function, surgery is the mainstay of therapy. Although rate of progression should weigh heavily in the decision of whether or not to perform surgery, general guidelines are MP contractures greater than or equal to 30° and/or PIP contractures greater than or equal to 20°.71Surgery consists of an open approach through the skin down to the involved cords. Skin is elevated off of the under-lying cords. Great care must be taken to preserve as much of the subdermal vascular plexus with the elevated skin flaps to minimize

1	skin down to the involved cords. Skin is elevated off of the under-lying cords. Great care must be taken to preserve as much of the subdermal vascular plexus with the elevated skin flaps to minimize postoperative skin necrosis. All nerves, tendons, and blood vessels in the operative field should be identified. Once this is done, the involved cord is resected while keeping the critical deeper structures under direct vision. The skin is then closed, with local flap transpositions as needed, to allow for full extension of the fingers that have been released (Fig. 44-18).Alternative cord resection techniques include removal of the skin over the contracture (dermatofasciectomy). This requires a skin graft to the wound and should only be done if skin cannot be separated from the cords and local tissue cannot be rearranged with local flaps to provide closure of the wound.Complications of surgical treatment of Dupuytren’s dis-ease occur in as many as 24% of cases.72 Problems include digi-tal

1	tissue cannot be rearranged with local flaps to provide closure of the wound.Complications of surgical treatment of Dupuytren’s dis-ease occur in as many as 24% of cases.72 Problems include digi-tal nerve laceration, digital artery laceration, buttonholing of the skin, hematoma, swelling, and pain, including some patients with CRPS (see earlier section on CRPS). Digital nerve injury can be quite devastating, producing annoying numbness at best or a painful neuroma in worse situations.Hand therapy is typically instituted within a week of sur-gery to begin mobilization of the fingers and edema control. The therapist can also identify any early wound problems because he or she will see the patient more frequently than the surgeon. Extension hand splinting is maintained for 4 to 6 weeks, with nighttime splinting continued for an additional 6 to 8 weeks. After this point, the patient is serially followed for evidence of recurrence or extension of disease.INFECTIONSTrauma is the most common

1	nighttime splinting continued for an additional 6 to 8 weeks. After this point, the patient is serially followed for evidence of recurrence or extension of disease.INFECTIONSTrauma is the most common cause of hand infections. Other predisposing factors include diabetes, neuropathies, and immu-nocompromised patients. Proper treatment consists of incision and drainage of any collections followed by debridement, obtain-ing wound cultures, antibiotic therapy, elevation, and immobi-lization. Staphylococcus and Streptococcus are the offending pathogens in about 90% of hand infections. Infections caused by intravenous drug use or human bites and those associated with diabetes will often be polymicrobial, including gram-positive and gram-negative species. Heavily contaminated injuries require anaerobic coverage. Although α-hemolytic Streptococcus and Staphylococcus aureus are the most commonly encountered pathogens in human bites, Eikenella corrodens is isolated in up to one-third of cases

1	coverage. Although α-hemolytic Streptococcus and Staphylococcus aureus are the most commonly encountered pathogens in human bites, Eikenella corrodens is isolated in up to one-third of cases and should be considered when choosing antimicrobial therapy. Ziehl-Neelsen staining and cultures at 28°C to 32°C in Lowenstein-Jensen medium must be performed if there is a suspicion for atypical mycobacteria.73CellulitisCellulitis is characterized by a nonpurulent diffuse spreading of inflammation characterized by erythema, warmth, pain, swell-ing, and induration. Skin breakdown is a frequent cause, but Brunicardi_Ch44_p1925-p1966.indd 194720/02/19 2:49 PM 1948SPECIFIC CONSIDERATIONSPART IIFigure 44-18. Dupuytren’s disease. A. This patient has cords affecting the thumb, middle, ring, and small fingers. B. The resected specimens are shown. C. Postoperatively, the patient went on to heal all his incisions and, with the aid of weeks of hand therapy, recover full motion.often no inciting factor

1	B. The resected specimens are shown. C. Postoperatively, the patient went on to heal all his incisions and, with the aid of weeks of hand therapy, recover full motion.often no inciting factor is identified. Group A α-hemolytic Streptococcus is the most common offending pathogen and causes a more diffuse spread of infection. S aureus is the second most common offending pathogen and will cause a more local-ized cellulitis. The diagnosis of cellulitis is clinical. Septic arthritis, osteomyelitis, an abscess, a deep-space infection, and necrotizing fasciitis are severe infectious processes that may initially mimic cellulitis. These must be ruled out appropriately before initiating treatment, and serial exams should be con-ducted to ensure proper diagnosis. Treatment of cellulitis con-sists of elevation, splint immobilization, and antibiotics that cover both Streptococcus and Staphylococcus. Intravenous antibiotics are usually initiated for patients with severe comorbidities and those who

1	elevation, splint immobilization, and antibiotics that cover both Streptococcus and Staphylococcus. Intravenous antibiotics are usually initiated for patients with severe comorbidities and those who fail to improve on oral antibiotics after 24 to 48 hours. Failure to improve after 24 hours indicates a need to search for an underlying abscess or other infectious cause.735AbscessAn abscess will present much like cellulitis, but they are two clinically separate entities. The defining difference is an area of fluctuance. Skin-puncturing trauma is the most common cause. S aureus is the most common pathogen, followed by Streptococcus. Treatment consists of incision and drainage with appropriate debridement, wound cultures, wound packing, elevation, immo-bilization, and antibiotics. The packing should be removed in 12 to 24 hours or sooner if there is clinical concern, and warm soapy water soaks with fresh packing should be initiated. Most should be allowed to heal secondarily. Delayed

1	should be removed in 12 to 24 hours or sooner if there is clinical concern, and warm soapy water soaks with fresh packing should be initiated. Most should be allowed to heal secondarily. Delayed primary clo-sure should only be performed after repeat washouts for larger wounds where complete infection control has been achieved.Collar-Button AbscessThis is a subfascial infection of a web space and is usually caused by skin trauma that becomes infected; it often occurs in Brunicardi_Ch44_p1925-p1966.indd 194820/02/19 2:49 PM 1949SURGERY OF THE HAND AND WRISTCHAPTER 44laborers. The adherence of the palmar web space skin to the pal-mar fascia prevents lateral spread, so the infection courses dor-sally, resulting in both palmar web space tenderness and dorsal web space swelling and tenderness. The adjacent fingers will be held in abduction with pain on adduction (Fig. 44-19). Incision and drainage, often using separate volar and dorsal incisions, is mandatory, and follows the same

1	The adjacent fingers will be held in abduction with pain on adduction (Fig. 44-19). Incision and drainage, often using separate volar and dorsal incisions, is mandatory, and follows the same treatment as for any abscess or deep-space infection.OsteomyelitisOsteomyelitis in the hand usually occurs due to an open fracture with significant soft tissue injury. The presence of infected hard-ware, peripheral vascular disease, diabetes, and alcohol or drug abuse are also predisposing factors. Presentation includes per-sistent or recurrent swelling with pain, erythema, and possible drainage. It will take 2 to 3 weeks for periosteal reaction and osteopenia to be detected on radiographs. Bone scans and MRI Figure 44-19. Collar-Button abscess A. The fingers surround-ing the involved (second) web space rest in greater abduction than the other fingers. B. Dorsal and volar drainage incisions are made, separated by a bridge of intact web skin; a Penrose drain prevents the skin from closing too

1	space rest in greater abduction than the other fingers. B. Dorsal and volar drainage incisions are made, separated by a bridge of intact web skin; a Penrose drain prevents the skin from closing too early.are useful modalities to aid in diagnosis. Erythrocyte sedimenta-tion rate (ESR) and C-reactive protein (CRP) have low specific-ity but are useful for monitoring the progress of treatment, with CRP being more reliable. Treatment consists of antibiotics alone in the early stage as long as there is favorable response. All necrotic bone and soft tissue, if present, must be debrided. Initial intravenous antibiotic therapy should cover S aureus, the most common pathogen, and should then be adjusted according to bone cultures. Antibiotic therapy is continued for 4 to 6 weeks once the patient clinically improves and there is no further need for debridement. For osteomyelitis in the setting of an acute fracture with internal fixation in place, the hardware should be left in place as long as

1	improves and there is no further need for debridement. For osteomyelitis in the setting of an acute fracture with internal fixation in place, the hardware should be left in place as long as it is stable and the fracture has not yet healed. If the hardware is unstable, it must be replaced. An external fixation device may be useful in this setting. If osteo-myelitis occurs in a healed fracture, all hardware and necrotic bone and soft tissue must be removed.74Pyogenic ArthritisInfection of a joint will progress quickly to severe cartilage and bony destruction if not addressed quickly. Direct trauma and local spread of an infection are the most common causes. Hema-togenous spread occurs most commonly in patients who are immunocompromised. S aureus is the most common pathogen, followed by Streptococcus species. Neisseria gonorrhoeae is the most common cause of atraumatic septic arthritis in an adult less than 30 years of age. Presentation includes exacerbation of pain with any joint

1	Streptococcus species. Neisseria gonorrhoeae is the most common cause of atraumatic septic arthritis in an adult less than 30 years of age. Presentation includes exacerbation of pain with any joint movement, severe pain on axial load, swell-ing, erythema, and tenderness. Radiographs may show a foreign body or fracture, with widened joint space early in the process and decreased joint space late in the process due to destruc-tion. Joint aspiration with cell count, Gram stain, and culture is used to secure the diagnosis. Treatment of nongonococcal septic arthritis includes open arthrotomy, irrigation, debridement, and packing the joint or leaving a drain in place. Intravenous antibi-otics are continued until there is clinical improvement, followed by 2 to 4 weeks of additional oral or intravenous antibiotics. Gonococcal septic arthritis is usually treated nonoperatively. Intravenous ceftriaxone is first-line therapy. Joint aspiration may be used to obtain cultures and decrease joint

1	antibiotics. Gonococcal septic arthritis is usually treated nonoperatively. Intravenous ceftriaxone is first-line therapy. Joint aspiration may be used to obtain cultures and decrease joint pressure.75Necrotizing InfectionsNecrotizing soft tissue infections occur when the immune system is unable to contain an infection, leading to extensive spread with death of all involved tissues. This is different from an abscess, which forms when a functioning immune system is able to “wall off” the infectious focus. Necrotizing infections can result in loss of limb or life, even with prompt medical care.Bacteria spread along the fascial layer, resulting in the death of soft tissues, which is in part due to the extensive blood vessel thrombosis that occurs. An inciting event is not always identified. Immunocompromised patients and those who abuse drugs or alcohol are at greater risk, with intravenous drug users having the highest increased risk. The infection can by monoor polymicrobial, with

1	Immunocompromised patients and those who abuse drugs or alcohol are at greater risk, with intravenous drug users having the highest increased risk. The infection can by monoor polymicrobial, with group A β-hemolytic Streptococcus being the most common pathogen, followed by α-hemolytic Streptococcus, S aureus, and anaerobes. Prompt clinical diag-nosis and treatment are the most important factors for salvag-ing limbs and saving life. Patients will present with pain out of proportion with findings. Appearance of skin may range from normal to erythematous or maroon with edema, induration, and blistering. Crepitus may occur if a gas-forming organism Brunicardi_Ch44_p1925-p1966.indd 194920/02/19 2:49 PM 1950SPECIFIC CONSIDERATIONSPART IIis involved. “Dirty dishwater fluid” may be encountered as a scant grayish fluid, but often there is little to no discharge. There may be no appreciable leukocytosis. The infection can progress rapidly and can lead to septic shock and disseminated

1	as a scant grayish fluid, but often there is little to no discharge. There may be no appreciable leukocytosis. The infection can progress rapidly and can lead to septic shock and disseminated intravas-cular coagulation. Radiographs may reveal gas formation, but they must not delay emergent debridement once the diagnosis is suspected. Intravenous antibiotics should be started imme-diately to cover gram-positive, gram-negative, and anaerobic bacteria. Patients will require multiple debridements, and the spread of infection is normally wider than expected based on initial assessment.73Necrotizing myositis, or myonecrosis, is usually caused by Clostridium perfringens due to heavily contaminated wounds. Unlike necrotizing fasciitis, muscle is universally involved and found to be necrotic. Treatment includes emergent debride-ment of all necrotic tissue along with empirical intravenous antibiotics.Wet gangrene is most common in diabetics with renal failure and an arteriovenous shunt. It is

1	includes emergent debride-ment of all necrotic tissue along with empirical intravenous antibiotics.Wet gangrene is most common in diabetics with renal failure and an arteriovenous shunt. It is usually polymicrobial. Patients will present with a necrotic digit that is purulent and very malodorous, with rapidly evolving pain, swelling, skin discoloration, and systemic collapse. Emergent treatment is the same as for other necrotizing infections, and amputation of the involved digit or extremity must often be performed.Infectious Flexor TenosynovitisFlexor tenosynovitis (FTS) is a severe pathophysiologic state causing disruption of normal flexor tendon function in the hand. A variety of etiologies are responsible for this process. Most acute cases of FTS are due to purulent infection. FTS also can occur secondary to chronic inflammation as a result of diabetes, RA, crystalline deposition, overuse syndromes, amyloidosis, psoriatic arthritis, systemic lupus erythematosus, and

1	FTS also can occur secondary to chronic inflammation as a result of diabetes, RA, crystalline deposition, overuse syndromes, amyloidosis, psoriatic arthritis, systemic lupus erythematosus, and sarcoidosis.The primary mechanism of infectious FTS usually is penetrating trauma. Most infections are caused by skin flora, including both Staphylococcus and Streptococcus species. Bac-teria involved vary by etiology of the infection: bite wounds (Pasteurella multocida—cat, E corrodens—human); diabetic patients (Bacteroides, Fusobacterium, Haemophilus species, gram-negative organisms); hematogenous spread (Mycobacte-rium tuberculosis, N gonorrhoeae); or water-related punctures (Vibrio vulnificus, Mycobacterium marinum). Infection in any of the fingers may spread proximally into the wrist, carpal tun-nel, and forearm, also known as Parona’s space.76Suppurative FTS has the ability to rapidly destroy a finger’s functional capacity and is considered a surgical emer-gency. Suppurative FTS results

1	and forearm, also known as Parona’s space.76Suppurative FTS has the ability to rapidly destroy a finger’s functional capacity and is considered a surgical emer-gency. Suppurative FTS results from bacteria multiplying in the closed space of the flexor tendon sheath and culture-rich synovial fluid medium causing migration of inflammatory cells and subsequent swelling. The inflammatory reaction within the closed tendon sheath quickly erodes the paratenon, leading to adhesions and scarring, as well as increase in pressures within the tendon sheath that may lead to ischemia. The ultimate con-sequences are tendon necrosis, disruption of the tendon sheath, and digital contracture.Patients with infectious FTS present with pain, redness, and fever (Fig. 44-20). Physical examination reveals Kanavel’s “cardinal” signs of flexor tendon sheath infection: finger held in slight flexion, fusiform swelling, tenderness along the flexor ten-don sheath, and pain over the flexor sheath with passive

1	“cardinal” signs of flexor tendon sheath infection: finger held in slight flexion, fusiform swelling, tenderness along the flexor ten-don sheath, and pain over the flexor sheath with passive exten-sion of the digit.77 Kanavel’s signs may be absent in patients who are immunocompromised, have early manifestations of Figure 44-20. Suppurative flexor tenosynovitis of the ring finger. A. The finger demonstrates fusiform swelling and flexed posture. B. Proximal exposure for drainage. C. Distal drainage incision.Brunicardi_Ch44_p1925-p1966.indd 195020/02/19 2:49 PM 1951SURGERY OF THE HAND AND WRISTCHAPTER 44infection, have recently received antibiotics, or have a chronic, indolent infection.If a patient presents with suspected infectious FTS, empiric intravenous antibiotics should be initiated. Prompt medical ther-apy in early cases may prevent the need for surgical drainage. For healthy individuals, empiric antibiotic therapy should cover Staphylococcus and Streptococcus. For

1	be initiated. Prompt medical ther-apy in early cases may prevent the need for surgical drainage. For healthy individuals, empiric antibiotic therapy should cover Staphylococcus and Streptococcus. For immunocompromised patients (including diabetics) or infections associated with bite wounds, empiric treatment should include coverage of gram-negative organisms as well.78Adjuncts to antibiotics include splint immobilization (intrinsic plus position preferred) and elevation until infec-tion is under control. Hand rehabilitation (i.e., range-of-motion exercises and edema control) should be initiated once pain and inflammation are under control.If medical treatment alone is attempted, then initial inpa-tient observation is indicated. Surgical intervention is necessary if no obvious improvement has occurred within 12 to 24 hours.Several surgical approaches can be used to drain infectious FTS. The method used is based on the extent of the infection. Michon developed a classification scheme

1	has occurred within 12 to 24 hours.Several surgical approaches can be used to drain infectious FTS. The method used is based on the extent of the infection. Michon developed a classification scheme that can be use-ful in guiding surgical treatment (Table 44-1).79 Figure 44-20 (B and C) demonstrates drainage of a stage II FTS. A Brunner incision allows better initial exposure but may yield difficul-ties with tendon coverage if skin necrosis occurs. A 16-gauge catheter or 5-French pediatric feeding tube then is inserted into the tendon sheath through the proximal incision. The sheath is copiously irrigated with normal saline. Avoid excessive fluid extravasation into the soft tissue because the resulting increase in tissue pressure can lead to necrosis of the digit. The catheter is removed after irrigation. The incisions are left open. Some surgeons prefer a continuous irrigation technique for a period of 24 to 48 hours. The catheter is sewn in place, and a small drain is placed at the

1	after irrigation. The incisions are left open. Some surgeons prefer a continuous irrigation technique for a period of 24 to 48 hours. The catheter is sewn in place, and a small drain is placed at the distal incision site. Continuous or intermittent irrigation every 2 to 4 hours with sterile saline can then be per-formed through the indwelling catheter.After surgery, an intrinsic plus splint is applied, the hand is elevated, and the appropriate empiric antibiotic coverage is instituted while awaiting culture results. The hand is reexamined the following day. Whirlpool therapy and range of motion are begun. Drains are removed before discharge from the hospital. The wounds are left open to heal by secondary intention. In severe cases, repeat irrigation and operative debridement may be required.Antibiotic therapy is guided by culture results as well as clinical improvement. Once there is no further need for debride-ment, a 7to 14-day course of oral antibiotics is generally prescribed.

1	therapy is guided by culture results as well as clinical improvement. Once there is no further need for debride-ment, a 7to 14-day course of oral antibiotics is generally prescribed. Consultation with an infectious disease specialist should be considered early in order to maximize efficiency and efficacy of therapy.FelonA felon is a subcutaneous abscess of the fingertip and is most commonly caused by penetrating trauma. S aureus is the most common pathogen. The fingertip contains multiple septa con-necting the distal phalanx to the skin. These septa are poorly compliant, and presence of an abscess will increase pressure and lead to severe pain and tissue death. Patients will experience erythema, swelling, and tenderness of the volar digital pad. Oral antibiotics may resolve the infection if diagnosed very early, but incision and drainage is indicated when fluctuance is identified. A digital block should be performed, followed by a longitudi-nal incision over the point of maximal

1	if diagnosed very early, but incision and drainage is indicated when fluctuance is identified. A digital block should be performed, followed by a longitudi-nal incision over the point of maximal fluctuance (Fig. 44-21). Transverse and lateral incisions should be avoided, and the incision should never extend across the distal phalangeal joint crease. Deep incision should not be performed as this may cause seeding of bacteria into the flexor tendon sheath. The wound is irrigated and packed, with warm soapy water soaks and packing changes initiated within 24 hours and performed two to three times daily until secondarily healed. Antibiotic coverage should cover for Staphylococcus and Streptococcus species.73ParonychiaParonychia is an infection beneath the nail fold. The nail plate can be viewed as an invagination into the dorsal skin extend-ing down to the distal phalanx periosteum. Predisposing factors include anything that causes nail trauma, such as manicures, artificial nails, or nail

1	as an invagination into the dorsal skin extend-ing down to the distal phalanx periosteum. Predisposing factors include anything that causes nail trauma, such as manicures, artificial nails, or nail biting. The infection may spread around Table 44-1Michon’s stages of suppurative flexor tenosynovitis and appropriate treatmentSTAGEFINDINGSTREATMENTIIncreased fluid in sheath, mainly a serous exudateCatheter irrigationIIPurulent fluid, granulomatous synoviumMinimal invasive drainage ± indwelling catheter irrigationIIINecrosis of the tendon, pulleys, or tendon sheathExtensive open debridement and possible amputationBAFigure 44-21. Felon. A. Lateral view of the digit showing fluctu-ance between the skin of the pad and the underlying distal phalanx bone. B. The authors prefer to drain felons with a longitudinal inci-sion (dashed line) directly over the area of maximal fluctuance.Brunicardi_Ch44_p1925-p1966.indd 195120/02/19 2:49 PM 1952SPECIFIC CONSIDERATIONSPART IIthe nail plate from

1	a longitudinal inci-sion (dashed line) directly over the area of maximal fluctuance.Brunicardi_Ch44_p1925-p1966.indd 195120/02/19 2:49 PM 1952SPECIFIC CONSIDERATIONSPART IIthe nail plate from one side to the other, or it may extend into the pulp and result in a felon. An acute paronychia is usually caused by S aureus or Streptococcal species. Patients report pain, ery-thema, swelling, and possibly purulent drainage involving the periungual tissue. Treatment consists of warm water soaks and oral antibiotics if diagnosed early. If purulence or fluctu-ance is present, then a freer elevator or 18-gauge needle can be passed along the involved nail fold to decompress the collection (Fig. 44-22). If the infection involves the eponychial fold, a small proximally based flap of eponychium is created by using a scalpel, followed by irrigation and packing. The nail plate must be removed if the infection extends beneath the nail plate. Packing is kept in place for 24 to 48 hours, followed by

1	by using a scalpel, followed by irrigation and packing. The nail plate must be removed if the infection extends beneath the nail plate. Packing is kept in place for 24 to 48 hours, followed by warm water soaks and local wound care. Usually, the wound cannot be repacked once the dressing is removed.73A chronic paronychia is most commonly caused by Can-dida species and is most often found in patients who perform jobs involving the submersion of their hands in water or other moist environments. These develop into thickened nails with callus-like formation along the nail folds and may occasion-ally become red and inflamed. They do not respond to antibi-otic treatment, and nail plate removal with marsupialization of the skin proximal to the eponychial fold will allow the wound to heal secondarily. The environmental factors leading to the chronic paronychia must also be corrected in order for treatment to be successful.All hand infections other than cellulitis will require surgi-cal

1	The environmental factors leading to the chronic paronychia must also be corrected in order for treatment to be successful.All hand infections other than cellulitis will require surgi-cal management. Clinical examination, particularly noting the area of greatest tenderness and/or inflammation, is the single most useful diagnostic tool to localize any puru-lence requiring drainage. Specific recommendations for differ-entiating among the possible locations of hand infection are included in the diagnostic algorithm shown in Fig. 44-23.TUMORSTumors of the hand and upper extremity can be classified as benign soft tissue tumors; malignant soft tissue tumors (subclas-sified into cutaneous and noncutaneous malignancies); benign bony tumors; malignant bony tumors; and secondary metastatic tumors. Initial investigation for any mass starts with a complete 6ABAFigure 44-22. Paronychia. A. Fluctuance in the nail fold is the hallmark of this infection. B. The authors prefer to drain a paro-nychia

1	Initial investigation for any mass starts with a complete 6ABAFigure 44-22. Paronychia. A. Fluctuance in the nail fold is the hallmark of this infection. B. The authors prefer to drain a paro-nychia using the bevel of an 18-gauge needle inserted between the nail fold and the nail plate at the location of maximal fluctuance.NondiagnosticFractureForeign bodyCellulitisadmit, IV Abxserial examSite of fluctuanceEntire fingerseYoNPyogenic FTSKanavel’ssigns presentMRI if nofluctuanceSubcutaneousabscessThenarabscessMidpalmabscessHypothenarabscessDistalLoss ofpalmarconcavityRadial toIF MCUlnar toSF MCWeb spaceabscessPalmPain withaxial loadingof jointPyogenic vs.crystallinearthritisConsiderarthrocentesisNo improvementin 48 hoursHand inflammationPlain X-raysPartial fingerDorsalCenteredon jointBetweendigitsLocalized fluctuanceFigure 44-23. Diagnostic algorithm. Diagnostic workup for a patient with hand inflammation to evaluate for infection. See text for details about particular infectious

1	fluctuanceFigure 44-23. Diagnostic algorithm. Diagnostic workup for a patient with hand inflammation to evaluate for infection. See text for details about particular infectious diagnoses. Abx = antibiotics; FTS = flexor tenosynovitis; IF MC = index finger metacarpal; MRI = magnetic resonance imaging; SF MC = small finger metacarpal.Brunicardi_Ch44_p1925-p1966.indd 195220/02/19 2:49 PM 1953SURGERY OF THE HAND AND WRISTCHAPTER 44history and physical exam. Hand and/or wrist X-rays should be obtained in every patient presenting with a mass unless clearly not indicated (e.g., a superficial skin lesion with no aggressive/malignant features). The workup proceeds in an orderly fashion until a diagnosis is obtained. Once a benign diagnosis is secured (by strong clinical suspicion in an experienced hand surgeon, radiographic evidence, or tissue biopsy), further workup is not needed; this may occur at any point in the workup of a mass.Most hand masses are benign and can be readily diagnosed

1	hand surgeon, radiographic evidence, or tissue biopsy), further workup is not needed; this may occur at any point in the workup of a mass.Most hand masses are benign and can be readily diagnosed without advanced imaging or tissue biopsy. When necessary, additional workup may include baseline laboratory studies, CT and/or MRI of the involved region, and a bone scan or positron emission tomography (PET) scan. Staging of a malignant tumor may occur before biopsy if a malignancy is strongly suspected, or it may occur after formal biopsy. Staging includes a chest X-ray and CT with intravenous contrast of the chest, abdomen, and pelvis to detect possible metastasis. Biopsy of the mass is always the last step of a workup and should occur only after all other available information has been gathered. Any mass that is over 5 cm in size, is rapidly increasing in size (as judged by an experienced surgeon or oncologist), is symptomatic or painful, or has an aggressive clinical or radiographic

1	Any mass that is over 5 cm in size, is rapidly increasing in size (as judged by an experienced surgeon or oncologist), is symptomatic or painful, or has an aggressive clinical or radiographic appearance war-rants workup and biopsy to rule out malignancy.CT scans are useful for detecting bony tumor extension across planes and identifying tumors of small bones, such as the carpal bones. MRI is useful for evaluating soft tissue tumor involvement (e.g., which muscle compartments are involved) as well as intramedullary lesions. Most soft tissue tumors will appear dark on T1-weighted images and bright on T2-weighted images. Hematomas, hemangiomas, lipomas, liposarcomas, and adipose tissue will appear bright on T1-weighted images and dark on T2-weighted images. Scintigraphy uses methylene diphosphonate attached to technetium-99m. This complex will attach to hydroxyapatite. Immediate uptake is seen in areas of increased vascularity, such as infection, trauma, and neoplasia. Increased uptake 2

1	attached to technetium-99m. This complex will attach to hydroxyapatite. Immediate uptake is seen in areas of increased vascularity, such as infection, trauma, and neoplasia. Increased uptake 2 to 3 hours later is seen in “pooled” areas where new bone formation has occurred. This modality is useful for detecting areas of tumor invasion or metastases not other-wise seen on prior CT, MRI, or radiographs.Biopsy is reserved for masses that cannot be diagnosed as benign based on prior clinical and radiographic exams. Needle biopsy is not reliable for primary diagnosis, but it can be use-ful for recurrent or metastatic disease. Open excisional (if mass is less than 5 cm in size) or incisional (if mass is greater than 5 cm in size) biopsy is the most common biopsy method. Proper surgical oncologic technique is strictly adhered to in order to prevent tumor spread into uninvolved tissues or compartments. This includes making all incisions longitudinally using sharp dissection and meticulous

1	technique is strictly adhered to in order to prevent tumor spread into uninvolved tissues or compartments. This includes making all incisions longitudinally using sharp dissection and meticulous hemostasis; carrying the incision directly down to the tumor with no development of tissue planes (i.e., making a straight-line path from skin to tumor); incising through the fewest number of muscle compartments; and avoid-ing critical neurovascular structures. The CT or MRI images will help determine the best surgical approach for biopsy or resection in order to avoid uninvolved compartments and criti-cal structures.80Benign Soft Tissue TumorsGanglion Cyst. This is the most common soft tissue tumor of the hand and wrist, comprising 50% to 70% of all soft tis-sue tumors in this region. They can occur at any age but are most common in the second to fourth decades with a slight predilection toward females. Patients may report a slowgrowing soft mass that may fluctuate in size and can sometimes

1	at any age but are most common in the second to fourth decades with a slight predilection toward females. Patients may report a slowgrowing soft mass that may fluctuate in size and can sometimes be associated with mild pain. Compressive neuropathies may be seen if they occur in Guyon’s canal or the carpal tunnel, but they are uncommon. There are no reports of malignant degeneration. History and physical exam are usually sufficient to establish a diagnosis. Occurrence by location is as follows: 60% to 70% occur on the dorsal wrist between the third and fourth exten-sor compartments and are connected by a stalk to the scaph-olunate ligament (Fig. 44-24); 18% to 20% occur on the volar wrist; and 10% to 12% occur in the digits as volar retinacular or flexor tendon sheath cysts. The cyst transilluminates. There is always a stalk that communicates with the underlying joint or tendon sheath. The cyst wall is composed of compressed col-lagen fibers with no epithelial or synovial cells

1	transilluminates. There is always a stalk that communicates with the underlying joint or tendon sheath. The cyst wall is composed of compressed col-lagen fibers with no epithelial or synovial cells present. Clear viscous mucin fills the cyst and is composed of glucosamine, albumin, globulin, and hyaluronic acid. The etiology is unclear. The most accepted theory currently is Angelides’ who proposed that repeated stress of a joint, ligament, or tendon sheath causes an increase of mucin-producing cells and subsequent mucin pro-duction. The increased mucin production dissects superficially and coalesces into a cyst. The successful treatment of dorsal ganglion cysts by excising only the stalk supports this theory.80Treatment consists of observation if asymptomatic. If symptoms exist or the patient desires removal for cosmetic appearance, aspiration of the cyst may be performed with a Figure 44-24. Dorsal wrist ganglion cyst. These typically occur between the third and fourth dorsal

1	the patient desires removal for cosmetic appearance, aspiration of the cyst may be performed with a Figure 44-24. Dorsal wrist ganglion cyst. These typically occur between the third and fourth dorsal extensor compartments and have a stalk connecting the base of the cyst to the scapholunate ligament.Brunicardi_Ch44_p1925-p1966.indd 195320/02/19 2:49 PM 1954SPECIFIC CONSIDERATIONSPART IIsuccessful cure rate ranging from 15% to 89%. The benefit of injected steroids is inconclusive. Aspiration of a volar wrist ganglion cyst can be dangerous due to the potential of injur-ing neurovascular structures. Open excision and arthroscopic excision of the cyst stalk are surgical options for cysts that are not amendable to aspiration. A recent meta-analysis reported recurrence rates after either needle aspiration, open excision, and arthroscopic excision as 59%, 21%, and 6%, respectively.81Mucous Cyst. A mucous cyst is a ganglion cyst of the DIP joint. They occur most commonly in the fifth to

1	aspiration, open excision, and arthroscopic excision as 59%, 21%, and 6%, respectively.81Mucous Cyst. A mucous cyst is a ganglion cyst of the DIP joint. They occur most commonly in the fifth to seventh decades, and the underlying cause is associated osteoarthritis of the DIP joint. They are slow growing and usually occur on one side of the ter-minal extensor tendon between the DIP joint and the eponych-ium. The earliest clinical sign is often longitudinal grooving of the involved nail plate followed by a small enlarging mass and then attenuation of overlying skin. X-rays will show signs of osteoarthritis within the DIP joint. Heberden nodes (osteophytes within the DIP joint) are often seen on X-ray.Possible treatment includes observation, aspiration, or excision. If the cyst is not draining and the overlying skin is intact, the patient may be offered reassurance. A draining cyst poses risk of DIP joint infection due to the tract communicating with the DIP joint and should be excised.

1	and the overlying skin is intact, the patient may be offered reassurance. A draining cyst poses risk of DIP joint infection due to the tract communicating with the DIP joint and should be excised. If the cyst is symp-tomatic, painful, or the patient desires removal for cosmetic pur-poses, excision should be performed. Any osteophytes in the DIP joint must be removed to reduce recurrence. Aspiration is an option for treatment, but this poses the risk of DIP joint infec-tion through seeding of bacteria into the joint or by the devel-opment of a draining sinus tract. It is generally not performed.Giant Cell Tumor of the Tendon Sheath. Also known as a xanthosarcoma, fibrous xanthoma, localized nodular synovitis, sclerosing hemangioma, or pigmented villonodular tenosynovi-tis, giant cell tumor of the tendon sheath is the second most com-mon soft tissue mass of the hand and wrist. It is a benign lesion with no clear pathogenesis. The tumor is a growth of polyclonal cells with no risk of

1	of the tendon sheath is the second most com-mon soft tissue mass of the hand and wrist. It is a benign lesion with no clear pathogenesis. The tumor is a growth of polyclonal cells with no risk of malignant transformation. Despite the simi-larity in name, it is not histopathologically related to giant cell tumor of the bone.82Giant cell tumor of the tendon sheath occurs as a firm slow-growing painless mass over months to years and will often feel bumpy or nodular, which is a distinguishing characteristic helpful for diagnosis. It has a predilection for occurring in close proximity to joints along flexor surfaces of the wrist, hands, and digits (especially the PIP joints of the radial digits) and occurs most commonly between the second and fifth decades (Fig. 44-25A). These tumors do not transilluminate. Direct extension into joints and ligaments can make complete exci-sion difficult. Gross appearance of the tumor will show a wellcircumscribed nodular firm mass with a deep brown color

1	Direct extension into joints and ligaments can make complete exci-sion difficult. Gross appearance of the tumor will show a wellcircumscribed nodular firm mass with a deep brown color due to the large amount of hemosiderin content, which is easily detected on histologic staining (Fig. 44-25B). Multinucleated giant cells and hemosiderin-laden macrophages are characteristic.80This tumor is not visible on radiographs. Approximately 20% will show extrinsic cortical erosion on X-ray. This is a risk factor for recurrence, and removal of the cortical shell should be considered. MRI is useful for delineating involvement with tendons, ligaments, and joints.The standard treatment is marginal excision. These tumors will often grow next to or around neurovascular bundles, and an Allen’s test should always be performed preoperatively to con-firm adequate blood supply by both ulnar and radial arteries as Figure 44-25. Giant cell tumor of tendon sheath. A. The mass pro-duces lobulated enlargement of

1	be performed preoperatively to con-firm adequate blood supply by both ulnar and radial arteries as Figure 44-25. Giant cell tumor of tendon sheath. A. The mass pro-duces lobulated enlargement of the external finger. B. The excised giant cell tumor has a multilobulated, tan-brown appearance.ABwell as dual blood supply to an involved digit via the ulnar and radial proper digital arteries. It is important to completely excise the stalk because this will greatly reduce tumor recurrence even in the setting of residual tumor. If tumor is suspected to have extended into the joint, the joint must be opened and all tumor removed. Despite this being a benign lesion, local recurrence is varies widely from 4% to 44%. Some variants can mimic more aggressive processes, and malignancy must be considered if aggressive features are identified, such as direct bony invasion.82Lipoma. Lipomas of the hand and wrist may occur in multiple anatomic locations, including subcutaneous tissues; intramus-cularly

1	if aggressive features are identified, such as direct bony invasion.82Lipoma. Lipomas of the hand and wrist may occur in multiple anatomic locations, including subcutaneous tissues; intramus-cularly (especially thenar or hypothenar muscles); deep spaces; carpal tunnel or Guyon’s canal; and rarely bone or nerve. They typically present as a painless, slow-growing, soft, and mobile mass over a period of months to years. Painful findings sug-gest close approximation to a neurovascular structure or, less commonly, a malignant lesion such as liposarcoma. Lipomas do not transilluminate. They resemble mature fat histologically. X-rays typically reveal no abnormality. MRI is a helpful imag-ing modality to evaluate a lipoma and will show signal charac-teristics that are suggestive of adipose tissue.80Asymptomatic lesions with no aggressive findings may be observed. Marginal excision is recommended for symptomatic, painful, or enlarging lipomas or those that cause dysfunction. MRI is recommended

1	lesions with no aggressive findings may be observed. Marginal excision is recommended for symptomatic, painful, or enlarging lipomas or those that cause dysfunction. MRI is recommended for deep lipomas to evaluate proxim-ity or involvement of critical structures, followed by marginal excision if MRI findings are consistent with a lipoma. If MRI findings are not consistent with a lipoma, incisional biopsy is warranted. Recurrence after marginal excision is rare.80Brunicardi_Ch44_p1925-p1966.indd 195420/02/19 2:50 PM 1955SURGERY OF THE HAND AND WRISTCHAPTER 44Schwannoma. A schwannoma, also known as a neurilem-moma, is a type of benign peripheral nerve sheath tumor. It is the most common benign peripheral nerve sheath tumor of the upper extremity.83 The majority occur as single solitary masses. Patients with neurofibromatosis type 1 (NF1) or 2 (NF2) may develop multiple schwannomas involving large peripheral nerve trunks or bilateral acoustic schwannomas, respectively. These tumors

1	masses. Patients with neurofibromatosis type 1 (NF1) or 2 (NF2) may develop multiple schwannomas involving large peripheral nerve trunks or bilateral acoustic schwannomas, respectively. These tumors arise from the Schwann cell and occur most often in the middle decades of life. They grow as painless, slow-growing, firm, round, well-encapsulated masses with a predilection toward flexor surfaces of the forearm and palm (given their presence of large nerves). Schwannomas grow from the peripheral nerve sheath and are usually connected by a pedicled stalk. The tumor is well demar-cated and can be readily separated from the nerve fascicles (Fig. 44-26). Unlike neurofibromas, they do not grow within the nerve. Paresthesias or other neurologic findings may occur, but they are usually absent, as is the Tinel’s sign. Findings such as pain, paresthesias, or numbness should raise concern for a tumor causing a compressive neuropathy or a tumor that is malignant.83Histologic exam reveals Antoni

1	is the Tinel’s sign. Findings such as pain, paresthesias, or numbness should raise concern for a tumor causing a compressive neuropathy or a tumor that is malignant.83Histologic exam reveals Antoni type A palisades of spindle cells with large oval nuclei with interlacing fascicles. Less cellular regions appear as Antoni type B areas. Mutations of the schwanomin gene on chromosome 22 are found in 50% of sporadic cases and 100% of acoustic schwannomas in patients with NF2.84Surgical treatment is reserved for symptomatic tumors and those that require biopsy to rule out a malignant process. An MRI should be obtained prior to surgery to confirm that the tumor is not located within the nerve (i.e., a neurofibroma) and that it is consistent with a schwannoma. Operative treatment involves excisional biopsy. If the tumor is adherent to adjacent soft tissue or not encapsulated, incisional biopsy is performed and excision is delayed pending pathology results. Malignant degeneration is

1	excisional biopsy. If the tumor is adherent to adjacent soft tissue or not encapsulated, incisional biopsy is performed and excision is delayed pending pathology results. Malignant degeneration is exceedingly rare.83Malignant Soft Tissue Tumors—CutaneousSquamous Cell Carcinoma. Squamous cell carcinoma (SCC) is the most common primary malignant tumor of the hand, accounting for 75% to 90% of all malignancies of the hand. Eleven percent of all cutaneous SCC occurs in the hand.85 It is the most common malignancy of the nail bed. Risk factors include sun exposure, radiation exposure, chronic ulcers, immu-nosuppression, xeroderma pigmentosa, and actinic keratosis. Marjolin’s ulcers represent malignant degeneration of old burn or traumatic wounds into an SCC and are a more aggressive type. Transplant patients on immunosuppression have a fourfold increased risk, and patients with xeroderma pigmentosa have a 65 to 200–fold increased risk of developing an SCC.86 They often develop as small,

1	patients on immunosuppression have a fourfold increased risk, and patients with xeroderma pigmentosa have a 65 to 200–fold increased risk of developing an SCC.86 They often develop as small, firm nodules or plaques with indistinct margins and surface irregularities ranging from smooth to ver-ruciform or ulcerated (Fig. 44-27). They are locally invasive, with 2% to 5% lymph node involvement. Metastasis rates of up to 20% have been reported in radiation or burn wounds. Stan-dard treatment is excision with 0.5to 1.0-cm margins. Other treatment options include curettage and electrodessication, cryotherapy, and radiotherapy.85Basal Cell Carcinoma. Basal cell carcinoma (BCC) is the sec-ond most common primary malignancy of the hand, accounting for 3% to 12%; 2% to 3% of all BCCs occur on the hand. Risk fac-tors are similar for SCC and include chronic sun exposure, light complexion, immunosuppression, inorganic arsenic exposure, and Gorlin’s syndrome. Presentation includes a small,

1	on the hand. Risk fac-tors are similar for SCC and include chronic sun exposure, light complexion, immunosuppression, inorganic arsenic exposure, and Gorlin’s syndrome. Presentation includes a small, well-defined nodule with a translucent, pearly border and overlying telangi-ectasias (Fig. 44-28). Metastasis is very rare. Standard treatment is excision with 5-mm margins. Other treatment options include curettage and electrodessication, cryotherapy, and radiotherapy.Melanoma. Melanoma accounts for approximately 4% of skin cancers and is responsible of 80% of all deaths from skin cancer. Approximately 2% of all cutaneous melanomas occur in the hand.87 Risk factors include sun exposure (especially blis-tering sunburns as a child), dysplastic nevi, light complexion, family history of melanoma, immunosuppression, and congenital Figure 44-26. Schwannomas grow as a firm, round, well-encapsulated mass within the epineurium of a peripheral nerve. Schwannomas are able to be separated from the

1	immunosuppression, and congenital Figure 44-26. Schwannomas grow as a firm, round, well-encapsulated mass within the epineurium of a peripheral nerve. Schwannomas are able to be separated from the nerve fascicles relatively easily because they do not infiltrate between them (unlike neurofibromas).Figure 44-27. Squamous cell carcinoma involving the nail fold and nail bed. Note the wart-like and ulcerated appearance.Brunicardi_Ch44_p1925-p1966.indd 195520/02/19 2:50 PM 1956SPECIFIC CONSIDERATIONSPART IInevi. Pigmented lesions with irregular borders, color changes, increase in growth, or change in shape are suggestive of mela-noma. Breslow thickness is the most important factor in predicting survival for a primary melanoma. Melanoma in situ lesions should be surgically excised with 0.5 cm margins. For lesions up to 1 mm in thickness, 1-cm margins should be used. Two centimeter mar-gins should be used for lesions over 1 mm in thickness.88 Sentinel lymph node biopsy is done for lesions

1	margins. For lesions up to 1 mm in thickness, 1-cm margins should be used. Two centimeter mar-gins should be used for lesions over 1 mm in thickness.88 Sentinel lymph node biopsy is done for lesions over 1 mm in thickness or for any lesion that is over 0.76 mm in thickness and exhibits ulcer-ation or high mitotic rate.89 Any clinically palpable lymph node requires a formal lymph node dissection of the involved basin, as do sentinel lymph nodes positive for melanoma. Lymph node dis-section has not been shown to offer any long-term survival ben-efit, but the information gained from sentinel lymph node biopsy (or lymph node dissection) does offer valuable staging informa-tion that is important for prognosis. For cases of subungual mela-nomas, DIP amputation is the current standard of care. A recent study reported similar recurrence and survival rates when com-paring patients treated with either DIP amputations or wide local excision; however, there was insufficient evidence to conclude

1	A recent study reported similar recurrence and survival rates when com-paring patients treated with either DIP amputations or wide local excision; however, there was insufficient evidence to conclude if one treatment was superior to another.90Malignant Soft Tissue Tumors—NoncutaneousPrimary soft tissue sarcomas of the upper extremity are very rare. Approximately 12,000 new cases of sarcomas are diag-nosed each year and of those, only 15% occur in upper extremity.80 Statistical inference is limited due to the rare occur-rence of these tumors, but mortality rate is very high despite the aggressive treatments. Fewer than 5% of soft tissue sarcomas of the upper extremity will develop lymph node metastasis. Cutaneous malignancies must be considered in the differential diagnosis for any patient with palpable lymph nodes in the setting of any upper extremity mass. Any lesion of the upper extremity that is over 5 cm in diameter, rapidly enlarges, or is painful should be considered malignant

1	with palpable lymph nodes in the setting of any upper extremity mass. Any lesion of the upper extremity that is over 5 cm in diameter, rapidly enlarges, or is painful should be considered malignant until proven otherwise.91Treatment for soft tissue sarcomas can range from pallia-tive debulking to attempted curative resection. Many muscles of the upper extremity and their compartments cross joints (e.g., forearm flexors). Any malignancy within a compartment mandates complete resection of that compartment, and there-fore, amputations must often be performed at levels much more proximal than the level of the actual tumor. Many soft tissue sarcomas are not responsive to radiation or chemotherapy, and use of these adjuvant treatments must be decided upon after discussion with medical and radiation oncologists in a multi-disciplinary team. Several studies have shown higher mortality rates in patients who undergo initial tumor biopsy of sarcomas at institutions from which they do not

1	radiation oncologists in a multi-disciplinary team. Several studies have shown higher mortality rates in patients who undergo initial tumor biopsy of sarcomas at institutions from which they do not ultimately receive treatment. These studies recommend biopsy be performed at the institution at which definitive treatment will be provided.92 Institutions best suited for such treatment should have pathologists familiar with soft tissue sarcomas, medical and radiation oncologists, surgical oncologists, and a multidisciplinary tumor board.An in-depth review of each type of soft tissue sarcoma is beyond the scope of this chapter. Epithelioid sarcoma is the most common primary soft tissue sarcoma of the upper extremity and usually presents as a benign-like slow-growing mass during the third or fourth decades. It has a propensity for the forearm, palm, and digits. Spread to lymph nodes has been reported. It typically spreads along fascial planes.80 Synovial sarcoma is argued by some to be the

1	decades. It has a propensity for the forearm, palm, and digits. Spread to lymph nodes has been reported. It typically spreads along fascial planes.80 Synovial sarcoma is argued by some to be the most common primary soft tissue sarcoma of the hand and wrist, but the paucity of case reports is inconclusive. It is a high-grade malignancy that is painless and slow-growing and usually occurs adjacent to, but not involving, joints. It is most common in the second to fifth decades of life. Tumor size (greater than 5 cm) is positively correlated with mortality. Other sarcomas include malignant fibrous histiocytoma, liposarcoma, fibrosarcoma, dermatofibrosarcoma protuberans, and malignant peripheral nerve sheath tumors, and more information can be found in further selected reading.93 The majority of metastases to the hand involve secondary bone tumors and are discussed later in the section, “Secondary Metastatic Tumors.”Benign Bone TumorsPrimary benign bone tumors of the hand and wrist make up

1	metastases to the hand involve secondary bone tumors and are discussed later in the section, “Secondary Metastatic Tumors.”Benign Bone TumorsPrimary benign bone tumors of the hand and wrist make up a total of 7% of all primary benign bone tumors in the body. Benign tumors of cartilage origin comprise 79% of all primary benign bone tumors of the hand and wrist.94Enchondroma. This is the most common primary benign bone tumor of the hand and wrist and is of cartilage origin. Up to 90% of all bone tumors in the hand and wrist are enchondromas, with 35% to 54% of all enchondromas occurring in the hand and wrist. They are often found incidentally on X-rays taken for other reasons (e.g., hand trauma). They are usually solitary and favor the diaphysis of small tubular bones and are most com-mon in the second and third decades of life. The most common location is in the proximal phalanges, followed by the metacar-pals and then middle phalanges. Enchondroma has never been reported in the

1	in the second and third decades of life. The most common location is in the proximal phalanges, followed by the metacar-pals and then middle phalanges. Enchondroma has never been reported in the trapezoid. Presentation is usually asymptomatic, but pain may occur if there is a pathologic fracture or impending fracture. The etiology is believed to be from a fragment of carti-lage from the central physis. Histology shows well-differentiated hyaline cartilage with lamellar bone and calcification.94Figure 44-28. Basal cell carcinoma of the dorsal hand with sur-rounding telangiectasia.Brunicardi_Ch44_p1925-p1966.indd 195620/02/19 2:50 PM 1957SURGERY OF THE HAND AND WRISTCHAPTER 44Figure 44-29. Enchondroma. A. X-ray of the phalanx demon-strates a well-defined central lucency. Surrounding cortex may thin or thicken. Thinning of the cortex contributes to risk of pathologic fracture. B. Intraoperative fluoroscopy after curettage of the tumor. A radiopaque ribbon is used to occupy the defect

1	may thin or thicken. Thinning of the cortex contributes to risk of pathologic fracture. B. Intraoperative fluoroscopy after curettage of the tumor. A radiopaque ribbon is used to occupy the defect to help ensure that there is no tumor (similarly radiolucent to the defect after curettage) left behind prior to bone grafting.BATwo variants of enchondroma include Ollier’s disease (multiple enchondromatosis) and Maffucci’s syndrome (multi-ple enchondromatosis associated with multiple soft tissue hem-angiomas). Malignant transformation is very rare in the solitary form, but there is a 25% incidence by age 40 in Ollier’s patients and a 100% life-time incidence in Maffucci’s patients. When malignant transformation does occur, it is almost uniformly a chondrosarcoma with pain and rapid growth.95Diagnosis is usually made based on history, physical exam, and X-rays. There is a well-defined, multilobulated cen-tral lucency in the metaphysis or diaphysis that can expand caus-ing cortical thinning

1	is usually made based on history, physical exam, and X-rays. There is a well-defined, multilobulated cen-tral lucency in the metaphysis or diaphysis that can expand caus-ing cortical thinning or, sometimes, thickening (Fig. 44-29A). Further imaging is seldom needed, but a CT would be the study of choice.Observation is indicated for asymptomatic enchondromas with no risk of impending fracture, followed by annual X-rays for 2 years. If a pathologic fracture is found, it is treated with immobilization until fracture union and then surgically treated. If there is any uncertainty as to whether it is an enchondroma, incisional biopsy is indicated, and definitive treatment is postponed pending final pathology. Symptomatic lesions and those with impending fracture are treated surgically. Surgical treatment consists of an open incisional biopsy and confirmation by frozen section that it is well-differentiated hyaline cartilage. Curettage and high-speed burring are used to ablate the tumor.

1	treatment consists of an open incisional biopsy and confirmation by frozen section that it is well-differentiated hyaline cartilage. Curettage and high-speed burring are used to ablate the tumor. Intraoperative fluoroscopy is used to confirm complete ablation (Fig. 44-29B). The defect is then packed with bone graft or bone substitute. Recurrence ranges from 2% to 15%. X-rays should be obtained serially after surgery.94Periosteal Chondroma. Periosteal chondromas are benign bone tumors of cartilage origin that arise most commonly within or adjacent to periosteum at the metaphyseal-diaphyseal junc-tion in phalanges. They occur usually in the second or third decade as solitary lesions with pain, swelling, deformity, and possible pathologic fracture. X-rays reveal a subperiosteal lytic, unilobular lesion with erosion into adjacent cortex. There is often a rim of sclerosis. Histologically, they appear as aggres-sive cartilage with atypia, and it can be difficult to differentiate these from

1	lesion with erosion into adjacent cortex. There is often a rim of sclerosis. Histologically, they appear as aggres-sive cartilage with atypia, and it can be difficult to differentiate these from chondrosarcomas.94Diagnosis involves X-rays with incisional biopsy to con-firm the benign diagnosis and avoid unnecessary amputation. Treatment includes en bloc resection of periosteum and cortico-cancellous bone. Recurrence is less than 4%.Osteoid Osteoma. This is a tumor of bone origin. Approxi-mately 5% to 15% of all osteoid osteomas occur in the hand and wrist and are most often found in the proximal phalanx or car-pus. They usually occur in the second or third decade and pres-ent with a deep, dull ache that is classically worse at night and relieved by nonsteroidal anti-inflammatory drugs (NSAIDs). X-rays reveal a central lucency that is usually less than 1 cm in diameter surrounded by reactive sclerosis. Bone scan or CT is helpful to secure the diagnosis.96Treatment consists of NSAID

1	(NSAIDs). X-rays reveal a central lucency that is usually less than 1 cm in diameter surrounded by reactive sclerosis. Bone scan or CT is helpful to secure the diagnosis.96Treatment consists of NSAID therapy only, and resolu-tion occurs at an average of 33 months. If the patient does not wish to undergo prolonged discomfort with conservative ther-apy, curettage or percutaneous ablation of the nucleus may be performed.96Giant Cell Tumor of Bone. Giant cell tumors of bone make up only 4% to 5% of all benign bone tumors in the body, and only 12% of these occur in the hand or wrist. Although its name is similar to that of “giant cell tumor of tendon sheath,” they are two separate tumors and do not share the same clinical or histo-pathologic characteristics. Approximately 2% occur in the hand and 10% occur in the distal radius; those within the distal radius are more aggressive. They usually occur in the fourth decade with pain and swelling and possibly pathologic fracture.97Giant cell

1	and 10% occur in the distal radius; those within the distal radius are more aggressive. They usually occur in the fourth decade with pain and swelling and possibly pathologic fracture.97Giant cell tumor of the bone is unique in that it is benign on histology but does have metastatic potential and can cause death. It should be considered a low-grade malignancy.97 Workup includes a CT of the chest and total-body scintigra-phy to evaluate for metastases and multifocal lesions and MRI to evaluate the extent of local tissue involvement. The recom-mended treatment consists of surgical resection of the involved phalanges or metacarpals and wide excision of entire carpal rows. Treatment with curettage and adjuvant treatments only results in a high rate of recurrence. Local and systemic surveil-lance must be done for at least 10 years because metastasis has been reported to occur as late as 10 years postoperatively.97,98Malignant Bone TumorsMalignant primary and secondary bone tumors of the

1	must be done for at least 10 years because metastasis has been reported to occur as late as 10 years postoperatively.97,98Malignant Bone TumorsMalignant primary and secondary bone tumors of the hand, like soft tissue malignancies, are exceedingly rare. An in-depth Brunicardi_Ch44_p1925-p1966.indd 195720/02/19 2:50 PM 1958SPECIFIC CONSIDERATIONSPART IIreview is beyond the scope of this chapter. The same principles for soft tissue sarcomas of the upper extremity apply here with regard to evaluation, biopsy, and treatment.Chondrosarcoma comprises 41% of all primary malignant bone tumors of the hand and wrist but only 1.5% of all chon-drosarcomas overall. It is most likely to occur from malignant degeneration from a preexisting lesion, with enchondromatosis and osteochondromatosis being the most common. It usually presents as a slow-growing, painless mass in the fourth to sixth decades and can be difficult to differentiate from its benign counterparts. X-ray reveals endosteal erosion,

1	most common. It usually presents as a slow-growing, painless mass in the fourth to sixth decades and can be difficult to differentiate from its benign counterparts. X-ray reveals endosteal erosion, cortical expan-sion, cortical destruction, and calcification. Metastasis has never been reported for chondrosarcomas of the hand. Chondrosarco-mas are not responsive to chemotherapy or radiation.99Osteosarcoma of the hand is exceedingly rare; only 0.18% of osteosarcomas occur in the hand. It usually presents as a painful swelling with pathologic fracture in the fifth to eighth decades of life. Radiation exposure is believed to be a possible risk factor. X-ray findings vary widely, with 90% of tumors occurring at a metaphyseal location. Findings include an osteo-blastic or osteolytic lesion, cortical breakthrough with soft tissue extension, a “sunburst” pattern radially, or periosteal elevation (Codman’s triangle). The presence or absence of metastasis is the most important prognostic

1	cortical breakthrough with soft tissue extension, a “sunburst” pattern radially, or periosteal elevation (Codman’s triangle). The presence or absence of metastasis is the most important prognostic factor, with a 5-year survival of 70% in the absence of metastases and a 5-year survival of 10% if present. Preoperative chemotherapy is usually given, but radi-ation therapy plays no role.100Secondary Metastatic TumorsMetastases to the hand or wrist are rare, with only 0.1% of skel-etal metastases occurring in the hand. The majority of metas-tases to the hand are bone lesions, but soft tissue metastases have been reported. The most common primary site is the lung (40%), followed by the kidney (13%) and the breast (11%). Approximately 16% will have no known diagnosis of cancer.101 The most common sites are the distal phalanges, followed by the proximal and middle phalanges, metacarpals, and carpus. Patients will present with pain, swelling, and erythema. Dif-ferential diagnosis includes

1	sites are the distal phalanges, followed by the proximal and middle phalanges, metacarpals, and carpus. Patients will present with pain, swelling, and erythema. Dif-ferential diagnosis includes felon, gout, osteomyelitis, trauma, RA, or skin cancer. Treatment of a hand or wrist metastatic lesion must not interfere with treatment of the primary cancer. Treatment is usually palliative (simple excision or amputa-tion). The average life expectancy for these patients is less than 6 months.101BURNSThe palm of the hand makes up approximately 1% of the total body surface area. A burn involving the entire hand and digits is unlikely to cause life-threatening injury or shock, but seem-ingly small burns to the hand may cause severe permanent loss of function if not treated appropriately. Burns to the hand can cause serious shortand long-term disability. All burns to the hand are considered severe injuries that warrant transfer to a dedicated burn center for specialized treatment. This

1	to the hand can cause serious shortand long-term disability. All burns to the hand are considered severe injuries that warrant transfer to a dedicated burn center for specialized treatment. This manage-ment will include a multidisciplinary team consisting of hand surgeons, burn surgeons, burn-specialized nurses, occupational therapists, case managers, and social workers.Superficial burns involve damage to the epidermis only and present with erythema, no blistering, and full sensation with blanching of skin. These will heal without scarring. Super-ficial partial-thickness burns involve damage to the papillary dermis; all skin appendages are preserved, and therefore, these readily reepithelialize with minimal to no scarring. Superficial partial-thickness burns are sensate and present with pain, ery-thema, blistering, and blanching of skin. Topical dressings are the mainstay of treatment. Deep partial-thickness burns involve damage to the reticular dermis with damage to skin appendages,

1	ery-thema, blistering, and blanching of skin. Topical dressings are the mainstay of treatment. Deep partial-thickness burns involve damage to the reticular dermis with damage to skin appendages, as well as the dermal plexus blood vessels and nerves. These have decreased sensation and no cap refill and appear pale or white. Blistering may be present. Damage to the skin append-ages and blood supply in the dermal plexus precludes spontane-ous healing without scar. Excision with skin grafting is needed. Third-degree burns involve full-thickness damage through the dermis and are insensate with no blistering. They appear dry, leathery, and even charred.Acute ManagementAdvanced trauma life support guidelines should be followed. After primary survey, circulation to the hand should be assessed. Palpation and Doppler ultrasound should be used to evaluate blood flow within the radial and ulnar arteries, the pal-mar arches, and digital blood flow at the radial and ulnar aspect of each volar

1	Palpation and Doppler ultrasound should be used to evaluate blood flow within the radial and ulnar arteries, the pal-mar arches, and digital blood flow at the radial and ulnar aspect of each volar digital pad. A sensorimotor exam should be per-formed. Objective evidence of inadequate perfusion (i.e., deteri-orating clinical exam with changes in or loss of pulse or Doppler signal) indicates the need for escharotomy, especially in the set-ting of circumferential burns. Escharotomy may be performed at bedside with scalpel or electrocautery under local anesthesia or intravenous sedation. In the forearm, axially oriented midra-dial and midulnar incisions are made for the entire extent of the burn. Escharotomy should proceed as distally as necessary into the wrist and hand to restore perfusion. Digital escharotomies are made via a midaxial (the middle of the longitudinal axis on sagittal view) incision over the radial aspects of the thumb and small finger and the ulnar aspects of the index,

1	Digital escharotomies are made via a midaxial (the middle of the longitudinal axis on sagittal view) incision over the radial aspects of the thumb and small finger and the ulnar aspects of the index, middle, and ring fingers.102 These locations for digital escharotomies avoid pain-ful scars on the heavy-contact surfaces of each respective digit. After primary survey, vascular, and sensorimotor exams are complete, careful documentation should be made of all burns. This is best done with a Lund and Browder chart and includes location, surface area, and initial depth of burn.The burns should be dressed as soon as examination is complete. Gauze moistened with normal saline is a good initial dressing because it is easy, readily available, and will not leave ointment or cream on the wounds, which can hinder frequent examinations in the initial period. It is critical that no dressing is wrapped in a circumferential manner around any body part. Edema and swelling can lead to extremity

1	which can hinder frequent examinations in the initial period. It is critical that no dressing is wrapped in a circumferential manner around any body part. Edema and swelling can lead to extremity ischemia if a circum-ferential dressing is in place. It is important to maintain body temperature above 37°C, especially in burn patients who have lost thermoregulatory function of the skin and now have moist dressings in place. The hands should be elevated above heart level to decrease edema formation, which can hinder motion and lead to late scar contracture. The hand should be splinted in the intrinsic plus position with the MPs flexed to 90° (placing MP collateral ligaments under tension), the IPs in straight extension (prevents volar plate adhesion), and the wrist in approximately 15° of extension.103 In rare cases, Kirschner wires or heavy steel wires/pins are needed to keep a joint in proper position. These are placed percutaneously through the involved joint and serve as a temporary

1	In rare cases, Kirschner wires or heavy steel wires/pins are needed to keep a joint in proper position. These are placed percutaneously through the involved joint and serve as a temporary joint stabilizer.After the primary and secondary surveys are complete, the wound should be evaluated again. Devitalized tissue should be Brunicardi_Ch44_p1925-p1966.indd 195820/02/19 2:50 PM 1959SURGERY OF THE HAND AND WRISTCHAPTER 44debrided. Wounds should be cleansed twice daily, typically with normal saline. Second-degree superficial burns may be dressed with Xeroform gauze and bacitracin. Silver sulfadiazine cream is another option for any secondor third-degree wound. It cov-ers gram-positive and gram-negative microbes, but it does not penetrate eschar. It should be applied at least one-sixteenth of an inch thick. Sulfamylon can be used in conjunction with silver sulfadiazine or alone. It deeply penetrates eschar and tissues and has good gram-positive coverage.Surgical ManagementAny burn

1	of an inch thick. Sulfamylon can be used in conjunction with silver sulfadiazine or alone. It deeply penetrates eschar and tissues and has good gram-positive coverage.Surgical ManagementAny burn wound will eventually heal with proper wound care. However, this may involve unacceptable scarring, deformity, contractures, pain, and unstable wounds that are prone to breakdown. The goal is to restore preinjury function as much as possible with a wound that is durable, supple, nonpainful, and allows the patient to return to society as an active member. Local wound care is the ideal treatment for wounds that can heal completely within 14 days while not sacrificing function. For deep partial-thickness or full-thickness burns, early surgical excision and skin grafting is necessary.103Considerable controversy surrounds the need, timing, and method of grafting burns. Careful consideration must be given to the patient’s overall status, their preinjury state, and the type of work and recreational

1	controversy surrounds the need, timing, and method of grafting burns. Careful consideration must be given to the patient’s overall status, their preinjury state, and the type of work and recreational activities they enjoyed in order to have a better understanding of which issues should be addressed. Tangential excision of the wounds should be performed under tourniquet to minimize blood loss and is carried down to viable tissue. Avoid excising through fascia (epimysium) overlying muscles or exposing tendons, bone, joint capsules, or neurovascular structures. Tissues capable of receiv-ing a skin graft include well-vascularized fat, muscle, perineu-rium, paratenon, perichondrium, and periosteum. Exposure of deep structures without an adequately graftable bed mandates further coverage before skin grafting can occur (discussed later in “Reconstruction”).Once there is an adequate bed, grafting is the next step. If there is any doubt as to whether the wound bed can support a skin graft, a

1	skin grafting can occur (discussed later in “Reconstruction”).Once there is an adequate bed, grafting is the next step. If there is any doubt as to whether the wound bed can support a skin graft, a temporary dressing such as Allograft (human cadaver skin) should be placed and the patient reexamined fre-quently for signs of granulation tissue and wound bed viability. It can remain in place for up to 14 days before rejection and can serve as a way of “testing” if a wound is ready to receive a skin graft. Skin grafts to the dorsum of the hand are typi-cally split-thickness sheet grafts (not meshed), as sheet grafts have a superior aesthetic appearance. Skin grafts to the palmar aspects of the hand should be full-thickness in order to provide the dermal durability needed for daily functions. Skin grafts are secured with staples, sutures, fibrin glue, or even skin glue. It is important to bolster every skin graft. This prevents shearing loss and also keeps the skin graft in contact with

1	Skin grafts are secured with staples, sutures, fibrin glue, or even skin glue. It is important to bolster every skin graft. This prevents shearing loss and also keeps the skin graft in contact with the wound bed, preventing fluid collections that can lead to graft loss. A bol-ster may consist of a tie-over bolster and a splint or a negativepressure dressing. The hand should be splinted in intrinsic plus for 7 days after skin grafting. Once the graft is adherent, hand therapy should begin, consisting of active and passive range-of-motion exercises and modalities.103ReconstructionReconstruction of burn wounds can begin as early as the acute setting and continue into the subacute and late stages. Burns may initially be superficial but later convert to deep burns (especially with grease, oil, and alkali burns) due to infection, tissue desiccation, or continued trauma, or they may be deep from the outset of injury. Debridement or excision of burns may result in exposure of viable muscle,

1	and alkali burns) due to infection, tissue desiccation, or continued trauma, or they may be deep from the outset of injury. Debridement or excision of burns may result in exposure of viable muscle, bone, tendon, cartilage, joints, and neurovascular structures, as well as loss of fascial layers that are required for overlying soft tissue to glide during movement. Simply skin grafting these exposed structures will result in unstable wounds that are prone to chronic breakdown. Soft tissue contractures will develop as the skin grafts adhere to the structures, effectively anchoring them in static position. This is especially true for tendons, where gliding capability is paramount for function. Flap coverage is required in these situ-ations. The reversed radial forearm flap is a local flap and is often the first choice for flap coverage of the hand. If the zone of injury or size of defect precludes its use, other skin and fat flaps, including the free lateral arm, free anterolateral thigh,

1	is often the first choice for flap coverage of the hand. If the zone of injury or size of defect precludes its use, other skin and fat flaps, including the free lateral arm, free anterolateral thigh, or even free parascapular flaps, may be useful, provided the patient can tolerate a free tissue transfer (see Chapter 45) operation (Fig. 44-30). The digits may also be buried subcutaneously in the lower abdominal skin or groin crease. Vascular ingrowth from the digits into the abdominal or groin skin occurs over 2 to 3 weeks, allowing division of the flap(s) and achieving full-thickness coverage of the wounds.104An acellular dermal regenerative substitute (e.g., Integra) may be used for wounds that have exposed structures and require more durability than is offered by a skin graft such as full-thickness loss overlying the extensor tendons of the wrist and hand.105 Dermal substitute is a good option for wounds that are not extensive enough to warrant a flap and for patients who are poor

1	loss overlying the extensor tendons of the wrist and hand.105 Dermal substitute is a good option for wounds that are not extensive enough to warrant a flap and for patients who are poor candidates for an extensive surgery. Integra is com-posed of acellular cross-linked bovine tendon collagen and gly-cosaminoglycan with an overlying silicone sheet. It is applied much like a skin graft. After incorporation in 14 to 21 days, it is capable of accepting a skin graft (after removing the silicone sheet). Conceptually, it works by replacing the lost dermis and adds durability to a wound bed. It may be reapplied multiple times to the same area if thicker neodermis is desired. Although cultured autologous keratinocytes have been used, they are expensive, time-consuming, and do not provide prompt or durable coverage.Web space contractures are the most common deformity resulting after hand burns. They may occur late despite the best efforts. In the normal web space, the leading edge of the volar

1	durable coverage.Web space contractures are the most common deformity resulting after hand burns. They may occur late despite the best efforts. In the normal web space, the leading edge of the volar Figure 44-30. Free anterolateral thigh flap reconstruction of a large dorsal hand wound. Once wound coverage is stable, this flap will need to be surgically revised to achieve proper contour.Brunicardi_Ch44_p1925-p1966.indd 195920/02/19 2:50 PM 1960SPECIFIC CONSIDERATIONSPART IIaspect of the web is distal to the dorsal aspect. This is reversed in web space contractures and limits digit abduction. Local modified Z-plasty (double-opposing Z-plasty) is the preferred treatment (Fig. 44-31).Special ConsiderationsChemical burns pose a risk to healthcare providers and should be considered hazardous material. They must also be removed from the patient or continued burn injury will occur. A complete discussion of all chemicals causing burns is beyond the scope of this chapter. Hydrofluoric acid

1	material. They must also be removed from the patient or continued burn injury will occur. A complete discussion of all chemicals causing burns is beyond the scope of this chapter. Hydrofluoric acid produces a slow onset of severe pain and continues to penetrate deeper structures. It avidly binds tissue and circulating calcium and can lead to hypocalcemia and cardiac arrest. The wound should be irrigated copiously with water followed by topical or intra-arterial injection of calcium gluconate. Chromic acid burns should be treated with immediate lavage, phosphate buffer soaks and immediate surgical excision. Cement can result in chemical burns and should be treated with immediate irrigation and topical antibacterial ointments. Alka-line and acid burns require copious irrigation with water, with alkali burns often requiring hours of irrigation. Phenol burns should be irrigated with dilute polyethylene glycol wash fol-lowed by high-flow water lavage.106VASCULAR DISEASEVascular disease

1	with alkali burns often requiring hours of irrigation. Phenol burns should be irrigated with dilute polyethylene glycol wash fol-lowed by high-flow water lavage.106VASCULAR DISEASEVascular disease encompasses a broad spectrum of disorders leading to compromised perfusion to the hand and digits and may potentially cause ischemia and necrosis. Chronic vascular disorders tend to develop slowly and are typically seen in older patients. This includes progressive thrombosis, aneurysms, sys-temic vasculopathy, and vasospastic disorders. Disorders unique or common to the hand are discussed in the following sections.Progressive Thrombotic DiseaseHypothenar hammer syndrome involves occlusion of the ulnar artery at the wrist and is the most common occlusive vascular disorder of the upper extremity. The etiology is believed to be chronic trauma to the ulnar artery as it exits Guyon’s canal. The classic example is a construction worker who frequently uses heavy equipment, such as jackhammers, that

1	etiology is believed to be chronic trauma to the ulnar artery as it exits Guyon’s canal. The classic example is a construction worker who frequently uses heavy equipment, such as jackhammers, that cause prolonged vibration and repetitive impact on the ulnar aspect of the palm. This causes periadventitial arterial damage that results in scar-ring and eventual compression, as well as medial and intimal damage.107 The artery then becomes weakened and prone to aneurysm and/or thrombosis. If a thrombus forms, it may embo-lize, producing digital ischemia. Symptoms may be chronic or acute and include pain, numbness and tingling, weakness of grip, discoloration of the fingers, and even gangrene or ulcers of the fingertips.If acute in onset, proximal occlusions may be extracted with a balloon catheter or, sometimes, under direct vision via an arteriotomy. Very distal embolism may require infusion of thrombolytics to dissolve clots and allow reperfusion. Large-vessel acute embolism and

1	catheter or, sometimes, under direct vision via an arteriotomy. Very distal embolism may require infusion of thrombolytics to dissolve clots and allow reperfusion. Large-vessel acute embolism and reperfusion may result in edema and compartment syndrome, requiring fasciotomy. A high index of suspicion must be maintained.For the more common scenario of chronic, progres-sive occlusion, the involved segment of ulnar artery should be resected. There is disagreement in the literature regarding whether simple ligation and excision is sufficient for patients with sufficient distal flow or if all patients should undergo vas-cular reconstruction.108 The authors’ personal preference is to reconstruct all patients.Systemic VasculopathyBuerger’s disease (thromboangiitis obliterans) is an inflamma-tory occlusive disease affecting small and medium-sized arter-ies and veins. It is strongly influenced by smoking and will often resolve upon smoking cessation. The disease is classified into acute,

1	occlusive disease affecting small and medium-sized arter-ies and veins. It is strongly influenced by smoking and will often resolve upon smoking cessation. The disease is classified into acute, intermediate, and chronic, depending on histologic progression of the disease. Migratory phlebitis occurs distal to the elbow, resulting in ischemia, rest pain, and ulceration and necrosis of the digits. It can continue to cause more proximal ischemia and ultimately lead to loss of the hands. Treatment must start with smoking cessation. Failure to stop smoking will make any surgical intervention unsuccessful. Arteriography is useful to determine arterial flow and whether bypass is possible. ABFigure 44-31. Z-plasty release of web space contracture. A. First web space burn contracture. B. Immediate postoperative result.Brunicardi_Ch44_p1925-p1966.indd 196020/02/19 2:50 PM 1961SURGERY OF THE HAND AND WRISTCHAPTER 44If direct bypass is not possible, alternatives include arteriali-zation of the

1	result.Brunicardi_Ch44_p1925-p1966.indd 196020/02/19 2:50 PM 1961SURGERY OF THE HAND AND WRISTCHAPTER 44If direct bypass is not possible, alternatives include arteriali-zation of the venous system by connecting the dorsal venous network to the brachial artery or possible free microvascular omental transfer beneath the dorsal forearm or hand for indirect revascularization.109Vasospastic DisordersRaynaud’s syndrome results from excessive sympathetic ner-vous system stimulation. Perfusion is diminished and fingers often become cyanotic. Although the onset of the symptoms is benign, chronic episodes can result in atrophic changes and painful ulceration or gangrene of the digits. Raynaud’s disease occurs without another associated disease. This disease predom-inately affects young women and is often bilateral. The vascular system is structurally intact without any obstructions. There is no ulceration, gangrene, or digit loss. In contrast, Raynaud’s phenomenon is associated with an

1	and is often bilateral. The vascular system is structurally intact without any obstructions. There is no ulceration, gangrene, or digit loss. In contrast, Raynaud’s phenomenon is associated with an underlying connective tissue disorder, such as scleroderma. Arterial stenosis is present due to disease changes in blood vessels as a result of the specific medical disorder.110Scleroderma is an autoimmune connective tissue disorder resulting in fibrosis and abnormal collagen deposition in tissue. Many organs can be affected, with the skin most commonly and noticeably involved. In this disease, blood vessels are injured by intimal fibrosis leading to microvascular disease. The ves-sels become subject to Raynaud’s phenomenon, and patients develop painful, ulcerated, and sometimes necrotic digits.109,110Sympathectomy can provide pain relief and healing of ulcers for patients with scleroderma and Raynaud’s phenom-enon. In this procedure, adventitia is stripped from the radial artery, ulnar

1	can provide pain relief and healing of ulcers for patients with scleroderma and Raynaud’s phenom-enon. In this procedure, adventitia is stripped from the radial artery, ulnar artery, superficial palmar arch, and digital arter-ies in various combinations based on the affected digits being treated. The decrease in sympathetic tone allows for vasodila-tion and increased blood flow. If the patient notes significant distal pain relief and/or previously ischemic tissue improves in color after a test administration of local anesthetic, sympathec-tomy may provide the same results in a long-term fashion.111 Recently, several studies have investigated the use of botulinum toxin on improving digital perfusion in patients with Raynaud’s. Reports have shown improved objective measurements of hand function 8-12 weeks after injection.112CONGENITAL DIFFERENCESCongenital differences in a newborn can be particularly dis-abling as the child learns to interact with the environment by using the hands. The

1	8-12 weeks after injection.112CONGENITAL DIFFERENCESCongenital differences in a newborn can be particularly dis-abling as the child learns to interact with the environment by using the hands. The degree of anomaly can range from minor, such as a digital disproportion, to severe, such as total absence of a forearm bone. In recent years, increasing knowledge of the molecular basis of embryonic limb development has sig-nificantly enhanced the understanding of congenital differences. Congenital hand differences have an incidence of 1:1500 births. The two most common differences encountered are syndactyly and polydactyly.113There are numerous classification systems for hand dif-ferences. The Swanson classification, adopted by the American Society for Surgery of the Hand, delineates seven groups orga-nized based on anatomic parts affected by types of embryonic failures.114,115Failure of FormationThe failure of the formation of parts is a group of congenital differences that forms as a

1	groups orga-nized based on anatomic parts affected by types of embryonic failures.114,115Failure of FormationThe failure of the formation of parts is a group of congenital differences that forms as a result of a transverse or longitudinal arrest of development. Conditions in this group include radial club hand, a deformity that involves some or all of the tissues on the radial side of the forearm and hand, and ulnar club hand, which involves underdevelopment or absence of the ulnar-sided bones.Failure of DifferentiationThe failure of the differentiation of parts comprises conditions where the tissues of the hand fail to separate during embryo-genesis. Syndactyly, in which two or more fingers are fused together, is the most common congenital hand deformity and occurs in 7 out of every 10,000 live births. There is a famil-ial tendency to develop this deformity. This deformity often involves both hands, and males are more often affected than females. Syndactyly is classified as either

1	live births. There is a famil-ial tendency to develop this deformity. This deformity often involves both hands, and males are more often affected than females. Syndactyly is classified as either simple (soft tissue only) or complex (bone and/or cartilage also involved), and complete (full length of the digits) or incomplete (less than the full length).Surgical release of syndactyly requires the use of local flaps to create a floor for the interdigital web space and to partially surface the adjacent sides of the separated digits (Fig. 44-32). Residual defects along the sides of the separated fingers are covered with full-thickness skin grafts. Surgery usu-ally is performed at 6 to 12 months of age.DuplicationDuplication of digits is also known as polydactyly. Radial polydactyly is usually manifests as thumb duplication. Wassel described a classification system for thumb duplications based on the level of bifurcation.116 When two thumbs are present in the same hand, they are rarely

1	manifests as thumb duplication. Wassel described a classification system for thumb duplications based on the level of bifurcation.116 When two thumbs are present in the same hand, they are rarely both normal in size, alignment, and mobility. In the most common form of thumb duplication, a single broad metacarpal supports two proximal phalanges, each of which supports a distal phalanx. Optimal reconstruction requires merging of elements of both component digits. Usually the ulnar thumb is maintained. If the duplication occurs at the MP joint, the radial collateral ligament is preserved with the metacarpal and attached to the proximal phalanx of the retained ulnar thumb. Surgery is usually performed at 6 to 12 months of age. Ulnar-sided polydactyly may often be treated by simple excision of the extra digit.OvergrowthOvergrowth of digits is also known as macrodactyly, which causes an abnormally large digit. In this situation, the hand and the forearm also may be involved. In this rare

1	of the extra digit.OvergrowthOvergrowth of digits is also known as macrodactyly, which causes an abnormally large digit. In this situation, the hand and the forearm also may be involved. In this rare condition, all parts of a digit are affected; however, in most cases, only one digit is involved, and it is usually the index finger. This condition is more commonly seen in males. Surgical treatment of this condi-tion is complex, and the outcomes may be less than desirable. Sometimes, amputation of the enlarged digit provides the best functional result.Constriction Band SyndromeUnderdeveloped fingers or thumbs are associated with many congenital hand deformities. Surgical treatment is not always required to correct these deformities. Underdeveloped fingers may include the following: small digits (brachydactyly), miss-ing muscles, underdeveloped or missing bones, or absence of a digit.Generalized Skeletal Anomalies and SyndromesThis is a rare and complex group of unclassified

1	small digits (brachydactyly), miss-ing muscles, underdeveloped or missing bones, or absence of a digit.Generalized Skeletal Anomalies and SyndromesThis is a rare and complex group of unclassified problems.Brunicardi_Ch44_p1925-p1966.indd 196120/02/19 2:50 PM 1962SPECIFIC CONSIDERATIONSPART IIRECONSTRUCTIVE TRANSPLANTATION OF THE UPPER EXTREMITYHand transplantation was first performed in humans in the late 1990s both in Louisville, Kentucky, and Lyon, France.117 The treating surgeons were able to successfully remove an upper extremity from a brain-dead donor, attach it to an upper extrem-ity amputee, and have the tissue survive. In the subsequent 15 years, many additional centers have achieved technical suc-cess with upper extremity transplantation as well.The technical considerations of hand transplantation have proven to be only the beginning of challenges in bring-ing this treatment option to the general public. Replantation of an amputated limb was first reported by Malt in

1	of hand transplantation have proven to be only the beginning of challenges in bring-ing this treatment option to the general public. Replantation of an amputated limb was first reported by Malt in 1962.118 In a limb replantation, there is a zone of injury, and cold preser-vation of the amputated part does not begin immediately. In a limb transplant, the harvest can be done as proximally as neces-sary to ensure that only healthy tissue is present on both sides of the repair and to obviate the need for limb shortening, and cold preservation of the amputated part can begin immediately after harvest.A major concern regarding the use of limb transplanta-tion is the immunosuppression medications required to prevent rejection of the transplanted limb. Unlike organ transplantation, which provides a critical organ without which the recipient could not survive or would require chronic mechanical support (e.g., hemodialysis), the absence of one or even multiple limbs does not represent an

1	a critical organ without which the recipient could not survive or would require chronic mechanical support (e.g., hemodialysis), the absence of one or even multiple limbs does not represent an immediate threat to a patient’s survival. Multiple studies have documented the nephrotoxic and other side effects of tacrolimus (FK 506), the principle antirejection agent used in transplant immunomodulation protocols.119,120Due to these concerns, much research has been directed at minimizing the amount of antirejection medication as well as promoting tolerance or even chimerism. Donor bone mar-row transplantation to the limb transplant recipient has been shown to be beneficial toward this purpose and is part of the limb transplant protocol in some centers.121,122 Recent research with donor bone marrow infusions has shown that lower lev-els of immunosuppressive drugs may be possible, as well as fewer immunosuppressive agents.121 Further research is needed in order to determine the efficacy and

1	infusions has shown that lower lev-els of immunosuppressive drugs may be possible, as well as fewer immunosuppressive agents.121 Further research is needed in order to determine the efficacy and utility of donor bone mar-row transfusions and how they impact transplant recipients in the short and long term.The final challenge in consideration of a patient for limb transplantation is selection of an appropriate candidate. There are multiple patient factors that need to be considered to deter-mine if a patient is an appropriate candidate for hand transplan-tation. These include medical concerns, such as immunologic issues (both antibodies and the presence of occult neoplasms or indolent viruses such as cytomegalovirus), hematologic issues including coagulopathies, and anatomic issues such as quality of skin envelope and amputation level of the bone and neuro-muscular structures. Psychological and social factors must also be considered related to the recipient’s ability to comply with

1	as quality of skin envelope and amputation level of the bone and neuro-muscular structures. Psychological and social factors must also be considered related to the recipient’s ability to comply with postoperative medication and therapy protocols as well as to cope with a continuous visible presence of a limb originating from another person.123The promise of upper limb transplantation as a recon-structive technique remains high. Both civilian and military amputees stand to receive a marked functional benefit from this treatment. With the number of transplants performed worldwide ABCFigure 44-32. Syndactyly. A. Hand of a 1-year-old patient with complex syndactyly between the long and ring fingers. Complex syndactyly refers to fingers joined by bone or cartilaginous union, usually in a side-to-side fashion at the distal phalanges. B. Antero-posterior radiograph. C. The syndactyly is divided with interdigitat-ing full-thickness flaps, a dorsal trapezoidal-shaped flap to resurface the

1	fashion at the distal phalanges. B. Antero-posterior radiograph. C. The syndactyly is divided with interdigitat-ing full-thickness flaps, a dorsal trapezoidal-shaped flap to resurface the floor of the web space, and full-thickness skin grafts. Note the skin grafts on the ulnar and radial sides of the new web space.Brunicardi_Ch44_p1925-p1966.indd 196220/02/19 2:50 PM 1963SURGERY OF THE HAND AND WRISTCHAPTER 44approaching 100 as well as decades of animal research, under-standing of how best to use this technique from functional, patient safety, and cost-effectiveness standpoints continues to grow.REFERENCESEntries highlighted in bright blue are key references. 1. American Society for Surgery of the Hand. The Hand: Examination and Diagnosis. 3rd ed. New York: Churchill Livingstone; 1990:5-13. 2. Moore KL. The Upper Limb. Clinically Oriented Anatomy. Baltimore: Williams & Wilkins; 1992:501-635. 3. Schuind F, Cooney WP, Linscheid RL, An KN, Chao EY. Force and pressure transmission

1	KL. The Upper Limb. Clinically Oriented Anatomy. Baltimore: Williams & Wilkins; 1992:501-635. 3. Schuind F, Cooney WP, Linscheid RL, An KN, Chao EY. Force and pressure transmission through the normal wrist. A theoretical two-dimensional study in the posteroanterior plane. J Biomech. 1995;28(5):587-601. 4. Gordon JA, Stone L, Gordon L. Surface markers for locating the pulleys and flexor tendon anatomy in the palm and fingers with reference to minimally invasive incisions. J Hand Surg Am. 2012;37:913-918. 5. Dumanian GA, Segalman K, Buehner JW, Koontz CL, Hendrickson MF, Wilgis EF. Analysis of digital pulse-volume recordings with radial and ulnar artery compression. Plast Reconstr Surg. 1998;102:1993-1998. 6. Green DP. General principles. In: Green DP, Hotchkiss RN, Pedersen WC, Wolfe SW, eds. Green’s Operative Hand Sur-gery. 5th ed. Philadelphia: Churchill Livingstone; 2005:3-24. 7. Gilula LA. Carpal injuries: analytic approach and case exer-cises. AJR Am J Roentgenol.

1	WC, Wolfe SW, eds. Green’s Operative Hand Sur-gery. 5th ed. Philadelphia: Churchill Livingstone; 2005:3-24. 7. Gilula LA. Carpal injuries: analytic approach and case exer-cises. AJR Am J Roentgenol. 1979;133:503-517. 8. Karl JW, Swart E, Strauch RJ. Diagnosis of occult scaphoid fractures: a cost-effectiveness analysis. J Bone Joint Surg Am. 2015;97(22):1860-1868. 9. Dezfuli B, Taljanovic MS, Melville DM, Krupinski EA, Sheppard JE. Accuracy of high-resolution ultrasonography in the detection of extensor tendon lacerations. Ann Plast Surg. 2016;76(2):187-192. 10. Kretsinger K, Broder KR, Cortese MM, et al. Preventing teta-nus, diphtheria, and pertussis among adults: use of tetanus tox-oid, reduced diphtheria toxoid and acellular pertussis vaccine recommendations of the Advisory Committee on Immuni-zation Practices (ACIP) and recommendation of ACIP, sup-ported by the Healthcare Infection Control Practices Advisory Committee (HICPAC), for use of Tdap among health-care personnel. MMWR

1	on Immuni-zation Practices (ACIP) and recommendation of ACIP, sup-ported by the Healthcare Infection Control Practices Advisory Committee (HICPAC), for use of Tdap among health-care personnel. MMWR Recomm Rep. 2006;55(Rr-17):1-37. 11. Hastings H 2nd, Carroll C 4th. Treatment of closed articu-lar fractures of the metacarpophalangeal and interphalangeal joints. Hand Clin. 1988;4:203-227. 12. Liodaki E, Xing SG, Mailaender P, Stang F. Management of difficult intra-articular fractures or fracture dislocations of the proximal interphalangeal joint. J Hand Surg Eur Vol. 2015;40(1):16-23. 13. Jahss SA. Fractures of the metacarpals: a new method of reduction and immobilization. J Bone Joint Surg. 1938;20(1):178-186. 14. Bond CD. Percutaneous screw fixation or cast immobilization for nondisplaced scaphoid fractures. J Bone Joint Surg Am. 2001;83-a(4):483-488. 15. Mayfield JK, Johnson RP, Kilcoyne RF. The ligaments of the human wrist and their functional significance. Anat Rec.

1	nondisplaced scaphoid fractures. J Bone Joint Surg Am. 2001;83-a(4):483-488. 15. Mayfield JK, Johnson RP, Kilcoyne RF. The ligaments of the human wrist and their functional significance. Anat Rec. 1976;186(3):417-428. 16. Apostolides JG, Lifchez SD, Christy MR. Complex and rare fracture patterns in perilunate dislocations. Hand (N Y). 2011;6(3):287-294. 17. Kleinert HE, Kutz JE, Atasoy E, Stormo A. Primary repair of flexor tendons. Orthop Clin North Am. 1973;4(4): 865-876. This key manuscript changed the “axiom” and established that zone two flexor tendon injuries could be immediately repaired primarly. 18. Vinycomb TI, Sahhar LJ. Comparison of local anesthetics for digital nerve blocks: a systematic review. J Hand Surg Am. 2010;39(4):744-751.e5. 19. Lalonde D, Bell M, Benoit P, Sparkes G, Denkler K, Chang P. A multicenter prospective study of 3110 consecutive cases of elective epinephrine use in the fingers and hand: the Dalhousie Project clinical phase. J Hand Surg Am.

1	P, Sparkes G, Denkler K, Chang P. A multicenter prospective study of 3110 consecutive cases of elective epinephrine use in the fingers and hand: the Dalhousie Project clinical phase. J Hand Surg Am. 2005;30:1061-1067. This large case series supports that the use of lidocaine with epinephrine is safe to use in the hand. 20. Yousif NJ, Grunert BK, Forte RA, Matloub HS, Sanger JR. A comparison of upper arm and forearm tourniquet tolerance. J Hand Surg Br. 1993;18:639-641. 21. Lee HJ, Cho YJ, Gong HS, Rhee SH, Park HS, Baek GH. The effect of buffered lidocaine in local anesthesia: a pro-spective, randomized, double-blind study. J Hand Surg Am. 2013;38(5):971-975. 22. Best CA, Best AA, Best TJ, Hamilton DA. Buffered lidocaine and bupivacaine mixture—the ideal local anesthetic solution? Plast Surg (Oakv). 2015;23(2):87-90. 23. Higgins A, Lalonde DH, Bell M, McKee D, Lalonde JF. Avoiding flexor tendon repair rupture with intraoperative total active movement examination. Plast Reconstr

1	Surg (Oakv). 2015;23(2):87-90. 23. Higgins A, Lalonde DH, Bell M, McKee D, Lalonde JF. Avoiding flexor tendon repair rupture with intraoperative total active movement examination. Plast Reconstr Surg. 2010; 126(3):941-945. 24. Davison PG, Cobb T, Lalonde DH. The patient’s perspective on carpal tunnel surgery related to the type of anesthesia: a prospective cohort study. Hand (N Y). 2013;8(1):47-53. 25. Rodgers J, Cunningham K, Fitzgerald K, Finnerty E. Opioid consumption following outpatient upper extremity surgery. J Hand Surg Am. 2012;37(4):645-650. 26. Stanek JJ, Renslow MA, Kalliainen LK. The effect of an educational program on opioid prescription patterns in hand surgery: a quality improvement program. J Hand Surg Am. 2015;40(2):341-346. 27. Komatsu S, Tamai S. Successful replantation of a com-pletely cut-off thumb: case report. Plast Reconstr Surg. 1968;42:374-377. 28. Lifchez SD, Marchant-Hanson J, Matloub HS, Sanger JR, Dzwierzynski WW, Nguyen HH. Functional improvement with

1	of a com-pletely cut-off thumb: case report. Plast Reconstr Surg. 1968;42:374-377. 28. Lifchez SD, Marchant-Hanson J, Matloub HS, Sanger JR, Dzwierzynski WW, Nguyen HH. Functional improvement with digital prosthesis use after multiple digit amputations. J Hand Surg Am. 2005;30:790-794. 29. Weichman KE, Wilson SC, Samra F, Reavey P, Sharma S, Haddock NT. Treatment and outcomes of fingertip injuries at a large metropolitan public hospital. Plast Reconstr Surg. 2013;131(1):107-112. 30. Bickel KD, Dosanjh A. Fingertip reconstruction. J Hand Surg Am. 2008;33(8):1417-1419. 31. Moberg E. The treatment of mutilating injuries of the upper limb. Surg Clin North Am. 1964;44:1107-1113. 32. Melone CP, Jr, Beasley RW, Carstens JH, Jr. The thenar flap—an analysis of its use in 150 cases. J Hand Surg Am. 1982;7(3):291-297. 33. Johnson RK, Iverson RE. Cross-finger pedicle flaps in the hand. J Bone Joint Surg Am. 1971;53(5):913-919. 34. Cannon TA. High-pressure injection injuries of the hand. Orthop

1	1982;7(3):291-297. 33. Johnson RK, Iverson RE. Cross-finger pedicle flaps in the hand. J Bone Joint Surg Am. 1971;53(5):913-919. 34. Cannon TA. High-pressure injection injuries of the hand. Orthop Clin North Am. 2016;47(3):617-624. 35. Bekler H, Gokce A, Beyzadeoglu T, Parmaksizoglu F. The sur-gical treatment and outcomes of high-pressure injection inju-ries of the hand. J Hand Surg Eur Vol. 2007;32(4):394-399. 36. Kalyani BS et al. Compartment syndrome of the forearm: a systematic review. J Hand Surg Am. 2011;36(3):535-543. 37. Staudt JM, Smeulders MJ, van der Horst CM. Normal com-partment pressures of the lower leg in children. J Bone Joint Surg Br. 2008;90(2):215-219. 38. Al-Qattan MM, Abou Al-Shaar H, Al Mugaren FM. Non-union without avascular necrosis of finger phalangeal neck Brunicardi_Ch44_p1925-p1966.indd 196320/02/19 2:50 PM 1964SPECIFIC CONSIDERATIONSPART IIfractures in children: report of 4 cases. J Hand Surg Am. 2014;39(8):1529-1534. 39. Munk B, Larsen CF. Bone

1	Brunicardi_Ch44_p1925-p1966.indd 196320/02/19 2:50 PM 1964SPECIFIC CONSIDERATIONSPART IIfractures in children: report of 4 cases. J Hand Surg Am. 2014;39(8):1529-1534. 39. Munk B, Larsen CF. Bone grafting the scaphoid nonunion: a systematic review of 147 publications including 5,246 cases of scaphoid nonunion. Acta Orthop Scand. 2004;75(5):618-629. 40. Curtis RM. Capsulectomy of the interphalangeal joints of the fingers. J Bone Joint Surg Am. 1954;36-a(6):1219-1232. 41. Brogan DM, Kakar S. Management of neuromas of the upper extremity. Hand Clin. 2013;29(3):409-420. 42. Zimmerman RM, Astifidis RP, Katz RD. Modalities for complex regional pain syndrome. J Hand Surg Am. 2015;40(7):1469-1472. 43. Schurmann M, Zaspel J, Löhr P, et al. Imaging in early post-traumatic complex regional pain syndrome: a comparison of diagnostic methods. Clin J Pain. 2007;23(5):449-457. 44. Mackinnon SE. Pathophysiology of nerve compression. Hand Clin. 2002;18(2):231-241. 45. US Department of Health and

1	syndrome: a comparison of diagnostic methods. Clin J Pain. 2007;23(5):449-457. 44. Mackinnon SE. Pathophysiology of nerve compression. Hand Clin. 2002;18(2):231-241. 45. US Department of Health and Human Services. Hand/wrist musculoskeletal disorders (carpal tunnel syndrome, hand/wrist tendonitis, and hand-arm vibration syndrome): evidence for work-relatedness. Available at: https://www.cdc.gov/niosh/docs/97-141/pdfs/97-141.pdf. Accessed August 16, 2018. 46. American Academy of Orthopedic Surgeons. Management of Carpal Tunnel Syndrome Evidence-Based Clinical Practice Guideline. Available at: https://www.aaos.org/uploadedFiles/PreProduction/Quality/Guidelines_and_Reviews/guidelines/CTS%20CPG_2.29.16.pdf. Accessed August 16, 2018. 47. Lifchez SD, Means KR, Jr, Dunn RE, Williams EH, Dellon AL. Intraand inter-examiner variability in performing Tinel’s test. J Hand Surg Am. 2010;35(2):212-216. 48. Williams TM, Mackinnon SE, Novak CB, McCabe S, Kelly L. Verification of the pressure

1	AL. Intraand inter-examiner variability in performing Tinel’s test. J Hand Surg Am. 2010;35(2):212-216. 48. Williams TM, Mackinnon SE, Novak CB, McCabe S, Kelly L. Verification of the pressure provocative test in carpal tunnel syndrome. Ann Plast Surg. 1992;29(1):8-11. 49. Marshall S, Tardif G, Ashworth N. Local corticosteroid injec-tion for carpal tunnel syndrome. Cochrane Database Syst Rev. 2007(2):Cd001554. 50. Trumble TE, Diao E, Abrams RA, Gilbert-Anderson MM. Single-portal endoscopic carpal tunnel release compared with open release : a prospective, randomized trial. J Bone Joint Surg Am. 2002;84-a(7):1107-1115. Carpal tunnel release is one of the most common procedures performed by hand sur-geons. This study by Trumble highlights that although patients undergoing endoscopic carpal tunnel release have less pain in the immediate postoperative period, clinical outcomes after 3 months show no difference compared to traditional open approaches. 51. Mackinnon SE, Novak CB.

1	carpal tunnel release have less pain in the immediate postoperative period, clinical outcomes after 3 months show no difference compared to traditional open approaches. 51. Mackinnon SE, Novak CB. Compression neuropathies. In: Wolfe SW, Hotchkiss RN, Kozin SH, Cohen MS, eds. Green’s Operative Hand Surgery. 7th ed. Amsterdam: Elsevier; 2016:921-958. This chapter does well to explain the mechanism, pathophysiology, and treatment for compression neuropathies in the upper extremity. 52. Ochi K, Horiuchi Y, Tanabe A, Morita K, Takeda K, Ninomiya K. Comparison of shoulder internal rotation test with the elbow flexion test in the diagnosis of cubital tunnel syndrome. J Hand Surg Am. 2011;36(5):782-787. 53. Goldfarb CA, Sutter MM, Martens EJ, Manske PR. Incidence of re-operation and subjective outcome following in situ decompression of the ulnar nerve at the cubital tunnel. J Hand Surg Eur Vol. 2009;34:379-383. 54. Kocak E, Carruthers KH, Kobus RJ. Distal interphalangeal joint arthrodesis

1	outcome following in situ decompression of the ulnar nerve at the cubital tunnel. J Hand Surg Eur Vol. 2009;34:379-383. 54. Kocak E, Carruthers KH, Kobus RJ. Distal interphalangeal joint arthrodesis with the Herbert headless compression screw: outcomes and complications in 64 consecutively treated joints. Hand (N Y). 2011;6(1):56-59. 55. Swanson AB. Implant resection arthroplasty of the proximal interphalangeal joint. Orthop Clin North Am. 1973;4:1007-1029. 56. Adkinson JM, Chung KC. Advances in small joint arthroplasty of the hand. Plast Reconstr Surg. 2014;134(6):1260-1268. 57. Naram A, Lyons K, Rothkopf DM, et al. Increased complica-tions in trapeziectomy with ligament reconstruction and ten-don interposition compared with trapeziectomy alone. Hand (N Y). 2016;11(1):78-82. 58. Gray KV, Meals RA. Hematoma and distraction arthroplasty for thumb basal joint osteoarthritis: minimum 6.5-year follow-up evaluation. J Hand Surg Am. 2007;32(1):23-29. 59. Kenniston JA, Bozentka DJ. Treatment

1	Meals RA. Hematoma and distraction arthroplasty for thumb basal joint osteoarthritis: minimum 6.5-year follow-up evaluation. J Hand Surg Am. 2007;32(1):23-29. 59. Kenniston JA, Bozentka DJ. Treatment of advanced carpo-metacarpal joint disease: arthrodesis. Hand Clin. 2008;24(3): 285-294, vi-vii. 60. Watson HK, Ballet FL. The SLAC wrist: scapholunate advanced collapse pattern of degenerative arthritis. J Hand Surg Am. 1984;9(3):358-365. 61. Wall LB, Didonna ML, Kiefhaber TR, Stern PJ. Proximal row carpectomy: minimum 20-year follow-up. J Hand Surg Am. 2013;38(8):1498-1504. 62. Goldfarb CA, Stern PJ, Kiefhaber TR. Palmar midcarpal instability: the results of treatment with 4-corner arthrodesis. J Hand Surg Am. 2004;29(2):258-263. 63. Chung KC, Pushman AG. Current concepts in the man-agement of the rheumatoid hand. J Hand Surg Am. 2011;36(4):736-747; quiz 747. Surgical treatment for rheu-matoid arthritis of the hand has decreased due to the advances in medical management. This article

1	of the rheumatoid hand. J Hand Surg Am. 2011;36(4):736-747; quiz 747. Surgical treatment for rheu-matoid arthritis of the hand has decreased due to the advances in medical management. This article serves as thorough review for hand surgeons on the treatment of rheumatoid hand. 64. Swanson AB. Silicone rubber implants for replacement of arthritis or destroyed joints in the hand. Surg Clin North Am. 1968;48(5):1113-1127. 65. Fujita S, Masada K, Takeuchi E, Yasuda M, Komatsubara Y, Hashimoto H. Modified Sauve-Kapandji procedure for disorders of the distal radioulnar joint in patients with rheu-matoid arthritis. Surgical technique. J Bone Joint Surg Am. 2006;88(Suppl 1 Pt 1):24-28. 66. Elliot D, Ragoowansi R. Dupuytren’s disease secondary to acute injury, infection or operation distal to the elbow in the ipsilateral upper limb—a historical review. J Hand Surg Br. 2005;30(2):148-156. 67. Eaton C. Dupuytren disease. In: Wolfe SW, Hotchkiss RN, Kozin SH, Cohen MS, eds. Green’s Operative

1	the elbow in the ipsilateral upper limb—a historical review. J Hand Surg Br. 2005;30(2):148-156. 67. Eaton C. Dupuytren disease. In: Wolfe SW, Hotchkiss RN, Kozin SH, Cohen MS, eds. Green’s Operative Hand Surgery. 7th ed. Amsterdam: Elsevier; 2016. 68. Murphy A, Lalonde DH, Eaton C, et al. Minimally inva-sive options in Dupuytren’s contracture: aponeurotomy, enzymes, stretching, and fat grafting. Plast Reconstr Surg. 2014;134(5):822e-829e. 69. van Rijssen AL, ter Linden H, Werker PM. Five-year results of a randomized clinical trial on treatment in Dupuytren’s disease: percutaneous needle fasciotomy versus limited fas-ciectomy. Plast Reconstr Surg. 2012;129:469-477. Although percutaneous needle fasciotomy is less invasive than limited fasciectomy, this study showed that fasciectomy provided more durable and lasting results. 70. Hurst LC, Badalamente MA, Hentz VR, et al. Injectable colla-genase clostridium histolyticum for Dupuytren’s contracture. N Engl J Med.

1	that fasciectomy provided more durable and lasting results. 70. Hurst LC, Badalamente MA, Hentz VR, et al. Injectable colla-genase clostridium histolyticum for Dupuytren’s contracture. N Engl J Med. 2009;361:968-979. 71. Saar JD, Grothaus PC. Dupuytren’s disease: an overview. Plast Reconstr Surg. 2000;106:125-134. 72. Crean SM, Gerber RA, Le Graverand MP, Boyd DM, Cappelleri JC. The efficacy and safety of fasciectomy and fas-ciotomy for Dupuytren’s contracture in European patients: a structured review of published studies. J Hand Surg Eur Vol. 2011;36:396-407. 73. McDonald LS, Bavaro MF, Hofmeister EP, Kroonen LT. Hand infections. J Hand Surg Am. 2011;36(8):1403-1412.Brunicardi_Ch44_p1925-p1966.indd 196420/02/19 2:50 PM 1965SURGERY OF THE HAND AND WRISTCHAPTER 44 74. Honda H, McDonald JR. Current recommendations in the management of osteomyelitis of the hand and wrist. J Hand Surg Am. 2009;34(6):1135-1136. 75. Murray PM. Septic arthritis of the hand and wrist. Hand Clin.

1	McDonald JR. Current recommendations in the management of osteomyelitis of the hand and wrist. J Hand Surg Am. 2009;34(6):1135-1136. 75. Murray PM. Septic arthritis of the hand and wrist. Hand Clin. 1998;14(4):579-587, viii. 76. Boles SD, Schmidt CC. Pyogenic flexor tenosynovitis. Hand Clin. 1998;14(4):567-578. 77. Kanavel AB. The treatment of acute suppurative tenosynovi-tis—discussion of technique. In: Infections of the Hand; A Guide to the Surgical Treatment of Acute and Chronic Sup-purative Processes in the Fingers, Hand, and Forearm. 5th ed. Philadelphia: Lea and Febiger; 1925:985. 78. Giladi AM, Malay S, Chung KC. A systematic review of the management of acute pyogenic flexor tenosynovitis. J Hand Surg Eur Vol. 2015;40(7):720-728. 79. Michon J. Phlegmon of the tendon sheaths (in French). Ann Chir. 1974;28(4):277-280. 80. Athanasian E. Bone and soft tissue tumors. In: Wolfe SW, Hotchkiss RN, Kozin SH, Cohen MS, eds. Green’s Operative Hand Surgery. 7th ed. Amsterdam: Elsevier;

1	French). Ann Chir. 1974;28(4):277-280. 80. Athanasian E. Bone and soft tissue tumors. In: Wolfe SW, Hotchkiss RN, Kozin SH, Cohen MS, eds. Green’s Operative Hand Surgery. 7th ed. Amsterdam: Elsevier; 2016. 81. Head L, Gencarelli JR, Allen M. Wrist ganglion treatment: systematic review and meta-analysis. J Hand Surg Am. 2015;40(3):546-553.e8. 82. Lanzinger WD, Bindra R. Giant cell tumor of the tendon sheath. J Hand Surg Am. 2013;38(1):154-157; quiz 157. 83. Phalen GS. Neurilemomas of the forearm and hand. Clin Orthop. 1976;114:219-222. 84. Lekanne Deprez RH, Bianchi AB, Groen NA, et al. Fre-quent NF2 gene transcript mutations in sporadic menin-giomas and vestibular schwannomas. Am J Hum Genet. 1994;54:1022-1029. 85. TerKonda SP, Perdikis G. Non-melanotic skin tumors of the upper extremity. Hand Clin. 2004;20:293-301. 86. Webber T, Wolf JM. Squamous cell carcinoma of the hand in solid organ transplant patients. J Hand Surg Am. 2014;39(3):567-570. 87. English C, Hammert WC. Cutaneous

1	Hand Clin. 2004;20:293-301. 86. Webber T, Wolf JM. Squamous cell carcinoma of the hand in solid organ transplant patients. J Hand Surg Am. 2014;39(3):567-570. 87. English C, Hammert WC. Cutaneous malignancies of the upper extremity. J Hand Surg Am. 2012;37(2):367-377. 88. Coit DG, Thompson JA, Andtbacka R, et al. Melanoma, version 2.2016. J Natl Compr Canc Netw. 2016;14(4): 450-473. 89. Dummer RA, Hauschild A, Lindenblatt N, et al. Cutane-ous malignant melanoma: ESMO clinical recommenda-tions for diagnosis, treatment and follow-up. Ann Oncol. 2009;20(Suppl 4):129-131. 90. Cochran AM. Subungual melanoma: a review of current treat-ment. Plast Reconstr Surg. 2014;134(2):259-273. 91. Mahajan A. The contemporary role of the use of radiation therapy in the management of sarcoma. Surg Oncol Clin N Am. 2000;9(3):503-524, ix. 92. Mankin HJ, Mankin CJ, Simon MA. The hazards of the biopsy, revisited. Members of the Musculoskeletal Tumor Society. J Bone Joint Surg Am.

1	of sarcoma. Surg Oncol Clin N Am. 2000;9(3):503-524, ix. 92. Mankin HJ, Mankin CJ, Simon MA. The hazards of the biopsy, revisited. Members of the Musculoskeletal Tumor Society. J Bone Joint Surg Am. 1996;78(5):656-663. 93. Murray PM. Soft tissue sarcoma of the upper extremity. Hand Clin. 2004;20(3):325-333, vii. The subject of soft tissue sarcomas is very broad and specific. This article by Murray provides a concise and accurate summary of soft tissue sarco-mas of the upper extremity. 94. Unni KK, Dahlin DC. Dahlin’s Bone Tumors: General Aspects and Data on 11,087 Cases. 5th ed. Philadelphia: Lippincott-Raven; 1996. 95. Henderson M, Neumeister MW, Bueno RA, Jr. Hand tumors: II. Benign and malignant bone tumors of the hand. Plast Reconstr Surg. 2014;133(6):814e-821e. 96. Marcuzzi A, Acciaro AL, Landi A. Osteoid osteoma of the hand and wrist. J Hand Surg Br. 2002;27(5):440-443. 97. Maloney WJ, Vaughan LM, Jones HH, Ross J, Nagel DA. Benign metastasizing giant-cell tumor of bone. Report

1	AL, Landi A. Osteoid osteoma of the hand and wrist. J Hand Surg Br. 2002;27(5):440-443. 97. Maloney WJ, Vaughan LM, Jones HH, Ross J, Nagel DA. Benign metastasizing giant-cell tumor of bone. Report of three cases and review of the literature. Clin Orthop Relat Res. 1989(243):208-215. 98. Oliveira VC, van der Heijden L, van der Geest IC, et al. Giant cell tumours of the small bones of the hands and feet: long-term results of 30 patients and a systematic literature review. Bone Joint J. 2013;95-b(6):838-845. 99. Ogose A, Unni KK, Swee RG, et al. Chondrosarcoma of small bones of the hands and feet. Cancer. 1997;80:50-59. 100. Okada K, Wold LE, Beabout JW, et al. Osteosarcoma of the hand: a clinicopathologic study of 12 cases. Cancer. 1993;72:719-725. 101. Amadio PC, Lombardi RM. Metastatic tumors of the hand. J Hand Surg Am. 1987;12:311-316. 102. Sheridan RL. Acute hand burns in children: management and long-term outcome based on a 10-year experience with 698 injured hands. Ann Surg.

1	tumors of the hand. J Hand Surg Am. 1987;12:311-316. 102. Sheridan RL. Acute hand burns in children: management and long-term outcome based on a 10-year experience with 698 injured hands. Ann Surg. 1999;229:558-564. 103. Pan BS, Vu AT, Yakuboff KP. Management of the acutely burned hand. J Hand Surg Am. 2015;40(7):1477-1484; quiz 1485. 104. Herndon D. Total Burn Care. 2nd ed. London: WB Saunders; 2002. 105. Haslik W, Kamolz LP, Nathschläger G, et al. First experi-ences with the collagen-elastin matrix Matriderm as a der-mal substitute in severe burn injuries of the hand. Burns. 2007;33:364-368. 106. Robinson EP, Chhabra AB. Hand chemical burns. J Hand Surg Am. 2015;40(3):605-612; quiz 613. 107. Conn J Jr, Bergan JJ, Bell JL. Hypothenar hammer syndrome: posttraumatic digital ischemia. Surgery. 1970;68(6):1122-1128. 108. Lifchez SD, Higgins JP. Long-term results of surgical treat-ment for hypothenar hammer syndrome. Plast Reconstr Surg. 2009;124(1):210-216. 109. Michelotti BM, Rizzo M,

1	1970;68(6):1122-1128. 108. Lifchez SD, Higgins JP. Long-term results of surgical treat-ment for hypothenar hammer syndrome. Plast Reconstr Surg. 2009;124(1):210-216. 109. Michelotti BM, Rizzo M, Moran SL. Connective tissue disor-ders associated with vasculitis and vaso-occlusive disease of the hand. Hand Clin. 2015;31(1):63-73. 110. Hotchkiss R, Marks T. Management of acute and chronic vas-cular conditions of the hand. Curr Rev Musculoskelet Med. 2014;7(1):47-52. 111. Ruch DS, Holden M, Smith BP, et al. Periarterial sympathec-tomy in scleroderma patients: intermediate-term follow-up. J Hand Surg Am. 2002;27:258-264. 112. Uppal L, Dhaliwal K, Butler PE. A prospective study of the use of botulinum toxin injections in the treatment of Raynaud’s syndrome associated with scleroderma. J Hand Surg Eur Vol. 2014;39(8):876-880. 113. Ekblom AG, Laurell T, Arner M. Epidemiology of congenital upper limb anomalies in 562 children born in 1997 to 2007: a total population study from Stockholm,

1	Surg Eur Vol. 2014;39(8):876-880. 113. Ekblom AG, Laurell T, Arner M. Epidemiology of congenital upper limb anomalies in 562 children born in 1997 to 2007: a total population study from Stockholm, Sweden. J Hand Surg Am. 2010;35(11):1742-1754. 114. Swanson AB. A classification for congenital limb malfor-mations. J Hand Surg Am. 1976;1:8-22. Swanson developed the seven key categories for the organization of congenital limb malformations later adopted by the American Society for Surgery of the Hand. 115. Bates SJ, Hansen SL, Jones NF. Reconstruction of congeni-tal differences of the hand. Plast Reconstr Surg. 2009;124 (1 Suppl):128e-143e. 116. Wassel HD. The results of surgery for polydactyly of the thumb. A review. Clin Orthop Relat Res. 1969;64: 175-193. 117. Lee WP, Mathes DW. Hand transplantation: pertinent data and future outlook. J Hand Surg Am. 1999;24:906-913. 118. Malt RA, McKhann CF. Replantation of severed arms. JAMA. 1964;189:716.Brunicardi_Ch44_p1925-p1966.indd

1	Hand transplantation: pertinent data and future outlook. J Hand Surg Am. 1999;24:906-913. 118. Malt RA, McKhann CF. Replantation of severed arms. JAMA. 1964;189:716.Brunicardi_Ch44_p1925-p1966.indd 196520/02/19 2:50 PM 1966SPECIFIC CONSIDERATIONSPART II 119. Starzl TE, Fung J, Jordan M, et al. Kidney transplantation under FK 506. JAMA. 1990;264:63-67. 120. Gorantla VS, Brandacher G, Schneeberger S, et al. Favoring the risk-benefit balance for upper extremity transplantation: the Pittsburgh Protocol. Hand Clin. 2011;27:511-520. 121. Schneeberger S, Gorantla VS, Brandacher G, et al. Upperex-tremity transplantation using a cell-based protocol to mini-mize immunosuppression. Ann Surg. 2013;257:345-351. 122. Brandacher G, Lee WP, Schneeberger S. Minimizing immu-nosuppression in hand transplantation. Expert Rev Clin Immu-nol. 2012;8(7):673-683; quiz 684. 123. Shores JT. Recipient screening and selection: who is the right candidate for hand transplantation. Hand Clin.

1	in hand transplantation. Expert Rev Clin Immu-nol. 2012;8(7):673-683; quiz 684. 123. Shores JT. Recipient screening and selection: who is the right candidate for hand transplantation. Hand Clin. 2011;27:539-543.Brunicardi_Ch44_p1925-p1966.indd 196620/02/19 2:50 PM

1	Plastic and Reconstructive SurgeryRajiv Y. Chandawarkar, Michael J. Miller, Brian C. Kellogg, Steven A. Schulz, Ian L. Valerio, and Richard E. Kirschner 45chapterINTRODUCTIONPlastic and reconstructive surgery is a unique subspecialty of surgery that consists of a set of techniques intended to mod-ify the amount, position, quality, or organization of tissues in order to restore function and appearance. The name of the field is derived from the Greek word plastikos, which means “to mold.” An object is considered plastic if its shape can be modi-fied without destruction. In this sense, all human tissues have some degree of plasticity. They can be nondestructively modi-fied if the surgeon adheres to certain principles. Understanding and applying these principles to solve clinical problems is the essence of plastic and reconstructive surgery. Although informal references to this type of surgery can be found in the modern literature as early as the 17th century, American surgeon John

1	is the essence of plastic and reconstructive surgery. Although informal references to this type of surgery can be found in the modern literature as early as the 17th century, American surgeon John Staige Davis published the first textbook dedicated to the field in 1919, entitled Plastic Surgery—Its Principles and Practice. He coined the term that we have used to refer to the specialty ever since. Science has always evolved in a nonlinear fashion: seminal discoveries in different parts of the world have all col-lectively fueled progress and addressed an unmet need. The evolution of plastic and reconstructive surgery has followed the same path: the Edwin Smith Papyrus1 (Egypt, 1600 b.c.) (Fig. 45-1) described facial reconstruction; the Shushruta Samhita (India, 1500 b.c.) (Fig. 45-2) described nasal reconstruction; and Aulus Cornelius Celsus (Rome, 1 a.d.) described opera-tions for facial reconstruction. The underlying impetus for this evolution is the common unmet need for restoring

1	nasal reconstruction; and Aulus Cornelius Celsus (Rome, 1 a.d.) described opera-tions for facial reconstruction. The underlying impetus for this evolution is the common unmet need for restoring defects, be they congenital, traumatic, or functional.This strong thread of advances in reconstructive surgery continues even today. What does seem under-recognized is that the clinical practice of plastic and reconstructive surgery touches on every other area of surgery. Enhanced reconstructive capabilities strengthen all other specialties significantly, such as the ability to safely perform radical cancer operations, sal-vage traumatic limbs, or extend the reach of neonatal medicine by congenital reconstruction. Each surgical specialty encoun-ters problems that might be addressed by some form of tissue repair, modification, rearrangement, transfer, or replacement. Since its inception, plastic surgeons have routinely responded to the medical needs of the society and helped restore form and

1	of tissue repair, modification, rearrangement, transfer, or replacement. Since its inception, plastic surgeons have routinely responded to the medical needs of the society and helped restore form and function. One of the most powerful examples of this response is the advances that occurred as a result of World Wars I and II. Walter Yeo, a sailor injured at the Battle of Jutland, is assumed to have received plastic surgery in 1917. The photograph shows him before (Fig. 45-3, left) and after (right) receiving a flap surgery performed by Gillies.The Gulf war and the conflicts in the Middle East have prompted several revolutionary reconstructive surgical advances in limb salvage, microsurgery, supermicrosurgery, hand, face, and abdominal wall transplantation. Plastic surgeons have also targeted muscle reinnervation, tissue engineering, and regenera-tive medicine.When society calls, plastic surgeons rise to the challenge and create novel methods to address its needs. For example,

1	targeted muscle reinnervation, tissue engineering, and regenera-tive medicine.When society calls, plastic surgeons rise to the challenge and create novel methods to address its needs. For example, neurosurgeons at times must replace or stabilize bone in the cranium or spine, and healthy soft tissue coverage is essen-tial for optimal healing. Head and neck surgeons face tissue replacement problems in order to restore normal function and appearance after major tumor ablation. Thoracic surgeons must manage bronchopleural fistulae, esophageal defects, or loss of chest wall integrity after trauma or tumor resection. Cardiolo-gists and cardiac surgeons at times face complicated wound Introduction 1967Purpose 1969General Principles 1969Skin Incisions / 1969Incision Repair / 1970Wound Healing / 1971Phases of Wound Healing / 1971Reconstructive Surgery 1974Reconstructive Strategies and Methods 1974Skin Grafts and Skin Substitutes / 1975Pediatric Plastic Surgery 1981Congenital Craniofacial

1	/ 1971Phases of Wound Healing / 1971Reconstructive Surgery 1974Reconstructive Strategies and Methods 1974Skin Grafts and Skin Substitutes / 1975Pediatric Plastic Surgery 1981Congenital Craniofacial Anomalies / 1981Reconstructive Surgery in Adults 2001Maxillofacial injuries and Fractures / 2002Mandible Fractures / 2002Frontal Sinus Fractures / 2003Orbital Fractures / 2004Zygomaticomaxillary Complex Fractures / 2004Nasoorbitalethmoid and Panfacial Fractures / 2005Posttraumatic Extremity Reconstruction / 2005Oncologic Reconstructive Surgery / 2008Breast Reconstruction / 2009Oncoplastic Breast Reconstruction / 2009Implant-based Reconstruction / 2009Tissue Flaps and Breast Implants / 2010Autologous Tissue Reconstruction / 2010Accessory Procedures / 2011Trunk and Abdominal Reconstruction / 2011Pelvic Reconstruction / 2012Other Clinical Circumstances / 2012Aesthetic Surgery and Medicine 2016Aesthetic Surgery of the Face / 2017Aesthetic Surgery of the Breast / 2018Aesthetic Surgery of

1	/ 2011Pelvic Reconstruction / 2012Other Clinical Circumstances / 2012Aesthetic Surgery and Medicine 2016Aesthetic Surgery of the Face / 2017Aesthetic Surgery of the Breast / 2018Aesthetic Surgery of the Body / 2018Suction Lipectomy / 2022Autologous Fat Grafting / 2024Brunicardi_Ch45_p1967-p2026.indd 196701/03/19 6:26 PM 1968Figure 45-1. The Edwin Smith papyrus (Egypt, 1600 b.c.).Figure 45-2. Statue of Shushruta, considered the “founding father of surgery” in India.Key Points1 It is critical to understand the physiologic basis and ratio-nale of wound healing in order to further assimilate surgi-cal and nonsurgical care of wounds and methods of wound care.2 Understanding the reconstructive choices in tissue repair cases is critical for any surgeon. The principles of soft tis-sue and skin repair are important for the reconstruction of defects, whether in a trauma situation of after excision of lesions.3 Children with cleft and craniofacial differences have com-plex medical, surgical,

1	repair are important for the reconstruction of defects, whether in a trauma situation of after excision of lesions.3 Children with cleft and craniofacial differences have com-plex medical, surgical, and social needs. Coordinated, interdisciplinary team care is crucial to success.4 Robin sequence, characterized by micrognathia, glossop-tosis, and airway obstruction, can be managed with prone positioning, tongue-lip adhesion, mandibular distraction osteogenesis, or tracheostomy.5 The first-line treatment for high-risk hemangiomas is oral propranolol, which can induce rapid involution and has a more favorable side effect profile than systemic steroids.6 The coordination of care for patients in a trauma depart-ment is an important part of a surgeon’s role, whether that role be as a trauma emergency department surgeon or a surgeon in practice.7 The careful evaluation of a patient in a polytrauma involves a thorough assessment of internal and soft tissue injuries, planning of care, and the

1	department surgeon or a surgeon in practice.7 The careful evaluation of a patient in a polytrauma involves a thorough assessment of internal and soft tissue injuries, planning of care, and the appropriate triage of reconstructive procedures. As a leader in a trauma bay of the trauma service, the surgeon typically assumes a cap-tain’s role in decision-making.8 Principles of oncologic reconstruction have evolved sig-nificantly, and a deeper understanding of these reconstruc-tive choices is essential for a surgeon who is often the first point of contact for cancer patients and responsible for making critical referrals.9 The combined work of general surgeons and reconstruc-tive plastic surgeons has revolutionized the care of abdom-inal wall defects, including ventral hernias, repair after tumor ablation, and bariatric surgery.10 Any critical care unit or a medical surgical team that takes care of debilitated patients needs a detailed understanding of pressure sores, including their

1	tumor ablation, and bariatric surgery.10 Any critical care unit or a medical surgical team that takes care of debilitated patients needs a detailed understanding of pressure sores, including their etiology and the recon-structive options that are available to these patients.infections, sternal osteomyelitis, or failure of soft tissue cov-erage that leads to exposure and contamination of implanted devices such as left ventricular assist devices or cardiac pace-makers. Orthopedic surgeons managing segmental bone defects in the extremities at times require replacement by surgical transfer of vascularized bone segments rather than conventional bone grafts or alloplastic substitutes. Urologists, colorectal sur-geons, and gynecologists who commonly perform surgery in the perineum encounter nonhealing wounds or fistulae. All of these problems may be managed or potentially prevented by judicious application of tissue methods developed and practiced by plastic and reconstructive

1	encounter nonhealing wounds or fistulae. All of these problems may be managed or potentially prevented by judicious application of tissue methods developed and practiced by plastic and reconstructive surgeons.Plastic and reconstructive surgery is field characterized by innovation, and it has yielded important contributions to other surgical specialties. These include notable advances in hand and upper extremity surgery, craniofacial surgery, peripheral nerve surgery, and reconstructive microsurgery. Entirely new fields of have emerged from plastic surgery research. Joseph E. Murray, a Boston plastic surgeon, and his team performed the first renal transplantation procedures and laid the foundation for modern organ transplantation, an achievement for which he was awarded the Nobel Prize in Medicine in 1990 (Fig. 45-4). This spirit of innovation continues with ongoing active research by plastic surgeons in composite tissue allotransplantation, tis-sue engineering, biomaterials, cell

1	in Medicine in 1990 (Fig. 45-4). This spirit of innovation continues with ongoing active research by plastic surgeons in composite tissue allotransplantation, tis-sue engineering, biomaterials, cell transplantation, regenerative medicine, computer-assisted surgical planning, medical appli-cation of three-dimensional manufacturing methods, infection control, and outcomes research. Plastic and reconstructive sur-gery is a vibrant field that brings tremendous value to people’s health and quality of life through life-changing reconstructive, restorative, and transformative surgeries.Brunicardi_Ch45_p1967-p2026.indd 196801/03/19 6:26 PM 1969PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-3. Walter Yeo, a sailor injured at the Battle of Jutland in 1917.Figure 45-4. Joseph E. Murray, MD, awarded the Nobel Prize in Medicine in 1990.PURPOSEThe purpose of this chapter is to inform about the general prin-ciples of plastic and reconstructive surgery, which apply to all areas of surgery,

1	awarded the Nobel Prize in Medicine in 1990.PURPOSEThe purpose of this chapter is to inform about the general prin-ciples of plastic and reconstructive surgery, which apply to all areas of surgery, and to provide current examples of practice. Studying this chapter will help the reader to understand (a) the principles of plastic surgery that translate into other surgi-cal specialties; (b) the kind of clinical problems that may be addressed using plastic surgery techniques; and (c) the types of research found in plastic and reconstructive surgery. It will make clearer the nature of the field and its role in the multidis-ciplinary care environment of modern healthcare.GENERAL PRINCIPLESGeneral principles of plastic surgery relate to technical aspects of incision planning and wound repair. These principles apply to all surgical disciplines. As such, every surgeon can benefit from learning and applying them. Previously, tremendous emphasis was placed on simply understanding the nature of

1	principles apply to all surgical disciplines. As such, every surgeon can benefit from learning and applying them. Previously, tremendous emphasis was placed on simply understanding the nature of skin, which is completely justified; however, over the past few years plastic surgical focus has expanded to include the entire integument. Muscles, fascia, fat, skeletal framework, nerves, vascular net-works, and their dynamic interactions have become far more important factors that are choreographed in most reconstructive processes.Skin IncisionsFrom a surgical viewpoint, the skin is a multilayered tissue formed by dermis and epidermis. It is the largest organ in the human body and exists in a state of dynamic equilibrium from the balance of tension created by external and internal factors. Externally, skin and underlying subcutaneous tissue are acted on by gravity and clothing. Internal factors include skin elasticity, which is simply the ability to stretch and return to prestretch

1	Externally, skin and underlying subcutaneous tissue are acted on by gravity and clothing. Internal factors include skin elasticity, which is simply the ability to stretch and return to prestretch architecture upon removal of the stretch. The dermis is com-posed of different types of collagen and elastic protein fibers (elastin), and epidermis, composed primarily of cells anchored together in various stages of maturation. The skin serves impor-tant functions of thermoregulation, affording tactile sensation, and protection from foreign materials and microorganisms. Areas of skin exposed to view in normal clothing play a sig-nificant role in personal appearance and social interaction. As a result, even favorable scars from surgical incisions can have an undesirable effect on personal appearance. Thoughtful place-ment and performance of a surgical incision will minimize the risk of adverse consequences that can result in shortand long-term morbidity.Human skin exists in a resting state of

1	Thoughtful place-ment and performance of a surgical incision will minimize the risk of adverse consequences that can result in shortand long-term morbidity.Human skin exists in a resting state of tension caused by gravity and its conformation over underlying structures between sites that are tethered by subcutaneous fibrous tissue, which secure the deep surface of the dermis to underlying points of fixation. When the skin is incised linearly, the wound edges separate in a predicable fashion forming an ellipse with the long axis perpendicular to the lines of greatest tension. These tension lines are often called “Langer’s lines,” after Carl Langer, a 19th century anatomist from Vienna who first described them based on studies in fresh cadavers (Fig. 45-5). Later, Borges described relaxed skin tension lines, which follow furrows formed when the skin is relaxed and are produced by pinching the skin. Inci-sions placed parallel to these lines often heal with less conspicu-ous scar because

1	tension lines, which follow furrows formed when the skin is relaxed and are produced by pinching the skin. Inci-sions placed parallel to these lines often heal with less conspicu-ous scar because the skin often has natural wrinkles following these lines and there is less tension perpendicular to the orien-tation of the wound1 (Fig. 45-6). Based on these principles,2 a recommended pattern for incisions can be made (Fig. 45-7).Using the proper technique for creating and repairing skin incisions ensures uncomplicated wound healing with few distorting surface scars. The epidermis and superficial dermis should be incised sharply with a scalpel. The incision is then continued through the deep dermis and subdermal plexus of blood vessels with electrocautery. This technique helps to mini-mize collateral tissue injury along the wound margins to facili-tate prompt and reliable healing. It is essential to maintain the orientation of the scalpel or electrocautery blade perpendicular to the

1	collateral tissue injury along the wound margins to facili-tate prompt and reliable healing. It is essential to maintain the orientation of the scalpel or electrocautery blade perpendicular to the surface of the skin in order to facilitate accurate reap-proximation during wound closure. As the incision is deepened through the subcutaneous tissue to expose underlying structures, it is important to avoid creating multiple pathways through the tissue, which can create focal areas of devitalized tissue that form a nidus of infection or lead to delayed wound healing. The Brunicardi_Ch45_p1967-p2026.indd 196901/03/19 6:26 PM 1970SPECIFIC CONSIDERATIONSPART IIFigure 45-5. “Langer’s lines,” named after Carl Langer, a 19th century anatomist from Vienna.Figure 45-6. Lines of relaxed skin tension.Figure 45-7. Planning of incisions based on lines of skin tension.surgeon should extend the incision through the subcutaneous fat by tracing the same line each time with the scalpel or

1	skin tension.Figure 45-7. Planning of incisions based on lines of skin tension.surgeon should extend the incision through the subcutaneous fat by tracing the same line each time with the scalpel or electrocau-tery in order to reach the deeper structures.Traumatic wounds do not permit the same careful plan-ning that is possible with incisions made in undamaged skin. Nevertheless, optimum repair of traumatic lacerations involves similar principles applicable in nontraumatic circumstances. The surgeon must remove as much traumatized tissue as pos-sible from the wound edges, converting the uncontrolled trau-matic wound into a controlled surgical wound. All devitalized tissue is excised. The same principles of making incisions perpendicular to the skin surface and avoiding creating mul-tiple pathways through the subcutaneous tissues apply. In this process, an attempt can be made to reorient the wound into a more favorable direction. A variety of methods are available to perform this

1	pathways through the subcutaneous tissues apply. In this process, an attempt can be made to reorient the wound into a more favorable direction. A variety of methods are available to perform this reorientation, and they often involve creating small local flaps of undamaged tissue using geometric tissue rearrangements. These techniques will be considered later in this chapter. Following these principles increases the likelihood of uncomplicated wound healing and reduces the need for later treatment of unfavorable scars. However, there are situations in which the direction of the incision has been preestablished, as in acute lacerations, burns, or old contracted and distorting scars. In these circumstances, the principles of proper incision placement can be combined with simple surgical techniques to reorient the scar and lessen the deformity.When making an incision in an area of previous scar-ring, such as in a scar revision or a reoperation, it is preferable to completely excise the

1	to reorient the scar and lessen the deformity.When making an incision in an area of previous scar-ring, such as in a scar revision or a reoperation, it is preferable to completely excise the scar when making the skin incision and not simply make the incision through the old scar. Closing scarred wound edges increases the likelihood of delayed wound healing, infections, and unfavorable new scars. It only takes a few moments to make the skin incision outside of the area of scarring through unscarred skin. Once the skin incisions on each side of the previous scar reach into the subcutaneous tissue, then the surface scar can be removed completely at the subder-mal level. This approach ensures that the final repair relies on undamaged tissues, thus facilitating uncomplicated healing and lowering the risk of an unfavorable scar.Incision RepairA well-performed skin incision sets the stage for an accurate repair that minimizes the risk of unfavorable scarring. An unfa-vorable scar is

1	lowering the risk of an unfavorable scar.Incision RepairA well-performed skin incision sets the stage for an accurate repair that minimizes the risk of unfavorable scarring. An unfa-vorable scar is characterized by excessive amount of collagen Brunicardi_Ch45_p1967-p2026.indd 197001/03/19 6:26 PM 1971PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45deposition,4 leading to hypertrophic scarring or keloid formation (Fig. 45-8). The difference between them is that a hypertrophic scar stops growing 6 months after the injury, whereas a keloid continues to grow, even growing well beyond its borders. Accu-rate approximation and stabilization of the skin edges helps to minimize the amount of collagen deposition required for skin healing. The most important layer to approximate is the dermis because this layer contains the healing elements such as blood supply and cellular elements that create the extracellular matrix necessary for healing. Optimal wound closure involves placing deep dermal

1	this layer contains the healing elements such as blood supply and cellular elements that create the extracellular matrix necessary for healing. Optimal wound closure involves placing deep dermal sutures followed by superficial sutures that incorpo-rated the upper layers of the dermis and epidermis. Absorbable deep dermal sutures have the advantage of disappearing over time; however, they can promote prolonged inflammation dur-ing this process. Nonabsorbable sutures minimize inflammation and might be indicated in individuals who are particularly prone to scar formation. A step-off between each side of the wound should be avoided because an uneven surface on each side of the wound can cause a shadow that accentuates the presence of the scar. Stability between the two wound edges is important because motion between the two sides of the wound prolongs the inflammatory phase of healing and requires additional col-lagen to be deposited. The timing of suture removal depends on the type of

1	because motion between the two sides of the wound prolongs the inflammatory phase of healing and requires additional col-lagen to be deposited. The timing of suture removal depends on the type of suture placed in the superficial closure. Sutures placed at the surface that go deep into the dermis can leave addi-tional scarring at the entry and exit points of the suture mate-rial in addition to the incisional scar. Sutures like this should be removed within the first week. If the superficial sutures are placed more shallowly in the dermis, there is a reduced tendency to form additional scarring. A subcuticular suture may be used instead of simple sutures. This type of technique avoids the risk of additional scarring along the wound edge; however, it can be more difficult to accurately reapproximate the skin edges with-out a step-off between the two sides.Wound HealingIn the United States, nonhealing wounds affect about 3 to 6 mil-lion people, with persons 65 years and older accounting

1	the skin edges with-out a step-off between the two sides.Wound HealingIn the United States, nonhealing wounds affect about 3 to 6 mil-lion people, with persons 65 years and older accounting for 85% of these events. The annual cost of this problem is estimated to be as high as $25 billion for hospital admissions, antibiotics, and local wound care.3Normal wound healing is achieved through four highly choreographed, overlapping biophysiologic phases: hemostasis, inflammation, proliferation, and tissue remodeling or resolu-tion. Each phase initiates a cascading set of processes critical to the desired result of a healed wound.1Figure 45-8. Hypertrophic scar (left) and keloid (right).Figure 45-9. Phases of wound healing.Hypertrophic ScarKeloidBlood clotBlood vesselScabFibroblastFibroblastsproliferatingFreshlyhealedepidermisFreshlyhealeddermisMacrophageSubcutaneousfatBleedingInflammatoryProliferativeRemodelingSeveral factors impede wound healing and need to be understood so that they can be

1	factors impede wound healing and need to be understood so that they can be mitigated. Successful mitiga-tion of these adverse factors requires precise, least-traumatic surgical technique that incorporates new methods of prevention and treatment of infection and an understanding of the role of microbial behavior, including the formation of biofilm. Because chronic diseases such as diabetes, vascular insufficiency, and obesity are on the rise, there must be a better understanding of chronic versus acute wounds and how comorbid conditions affect wound healing. Lastly, the impact of age, gender, and nutrition becomes more important as the population of aging patients increases.Phases of Wound HealingThere are different processes that characterize healing in sev-eral types of tissue, such as skin, muscle, or bone, and there is a strong underlying mechanism that is best understood in terms of a simple skin injury. The process of wound healing is com-prised of four integrated processes that

1	muscle, or bone, and there is a strong underlying mechanism that is best understood in terms of a simple skin injury. The process of wound healing is com-prised of four integrated processes that overlap: (a) bleeding and hemostasis, (b) inflammation, (c) proliferation, and (d) tissue modeling or resolution (Fig. 45-9).These processes occur in sequence over a 1-year duration, but they also significantly overlap and work in terms of a “con-tinuum of processes” rather than discrete “stop-and-go” phases. As shown in Fig. 45-9, each phase is characterized by several Brunicardi_Ch45_p1967-p2026.indd 197101/03/19 6:26 PM 1972SPECIFIC CONSIDERATIONSPART IIwell-defined processes that are dominated by cellular as well as noncellular elements, such as platelets, macrophages, and cyto-kines, that act in concert.Hemostasis. This phase of healing occurs immediately after tissue injury. The most important cells that play a role in the hemostatic process are platelets that degranulate and result

1	act in concert.Hemostasis. This phase of healing occurs immediately after tissue injury. The most important cells that play a role in the hemostatic process are platelets that degranulate and result in the formation of a clot. The extracellular matrix that supports the tissue framework and otherwise acts as a barrier is now open to the vascular compartment, resulting in the release of several factors into the wound. In addition, the release of proteins— otherwise stored within the extracellular matrix—and the presi-dent cells act as further stimulants that start the hemostatic pro-cess. Inflammatory plasma proteins and leukocytes also migrate into the wound. On the cellular level, the plasma membrane of each platelet contains several receptors for collagen (glycopro-tein 1A and 2A). Once these receptors are activated, glycolated granules holding multiple factors that activate hemostasis and inflammation are disrupted, releasing bioactive factors that stimulate platelet aggregation,

1	these receptors are activated, glycolated granules holding multiple factors that activate hemostasis and inflammation are disrupted, releasing bioactive factors that stimulate platelet aggregation, vasoconstriction, and the subse-quent activation of the clotting cascade. As these initial platelet activation factors are released, there is a subsequent push that influences angiogenesis inflammation. These systemic immune response platelet-derived factors include biologically active proteins, such as PDGF, TGF-β, and VEGF, as well as other cytokines, such as PF4 and CD40L.In addition to the release of these factors, the binding of selected proteins within the already developed fibroblasts and the combination of two elements within the extracellular matrix create a chemotactic gradient that activates cell recruitment, cell migration, and cell differentiation and promotes tissue repair. This has been demonstrated clinically in several instances, including orthopedic surgery, cardiac

1	that activates cell recruitment, cell migration, and cell differentiation and promotes tissue repair. This has been demonstrated clinically in several instances, including orthopedic surgery, cardiac surgery, and certain types of skin repair, where autologous platelet transfusions have shown to accelerate the healing process.The subsequent fate of the platelet plug is determined by the amount of circulating fibrinogen. The vascular system interacts with the sympathetic nervous system by eliciting vasoconstriction from the actions of cytokines, prostaglandins, and catecholamines. There is also an alteration of capillary permeability caused by histaminic responses and the mediation of VEGF, which is released from micelles and the damaged endothelium. This highly interactive process results in decreasing blood loss while simultaneously delivering bioactive proteins and cells into the wound environment that kick start the inflammatory process.Inflammation. This is the second phase of

1	in decreasing blood loss while simultaneously delivering bioactive proteins and cells into the wound environment that kick start the inflammatory process.Inflammation. This is the second phase of wound healing and arguably overlaps the hemostatic face. Polymorphonuclear leu-kocytes (PMNs) and macrophages appear in the wound right after platelets, and their primary role is mainly to act as scav-engers. They clear the wound environment of debris, foreign material, bacteria, dead tissue cells and any other devitalized issues that would otherwise impede the healing process. Both macrophages and PMNs aid in phagocytosis and the secretion of free articles that kill bacteria and reduce the bioburden. Cel-lular migration into the wound is highly controlled by bioactive agents within the wound and within the vascular compart-ment. These include cytokines, integrins, selection, and other collagen-derived substances that act in concert. Through anti-body activation, polymorphonuclear cells also

1	within the vascular compart-ment. These include cytokines, integrins, selection, and other collagen-derived substances that act in concert. Through anti-body activation, polymorphonuclear cells also interact with the humoral system to facilitate the key functions of cell activation, recruitment, and proliferation, as well as migration from the intravascular compartment to the extracellular matrix. Within 48 hours of tissue injury, PMNs and macrophages are recruited to the wound in very large numbers, heralding the inflamma-tory response. As described in other chapters in this text, macro-phages possess a very large repertoire of functions, all of which are geared towards removing the nonviable elements in the wound and recruiting other cell types into the wound that facili-tate angiogenesis, fibroblast function, and subsequent repair. A summary of various macrophage-related functions is broadly classified into 7 major categories:1. Phagocytosis2. Release of reactive oxygen species

1	fibroblast function, and subsequent repair. A summary of various macrophage-related functions is broadly classified into 7 major categories:1. Phagocytosis2. Release of reactive oxygen species that result in cellular kill-ing specifically related towards bacterial lysis3. Release of nitric oxide that is deadly to several otherwise antibody-resistant bacteria4. Cytokine release of interleukins (IL1, IL2, IL4, and IL12)5. Angiogenesis via VEGF that promotes capillary budding6. Recruitment of other cells into the wound that continue the healing process7. Different homeostatic roles of macrophages and Langerhans cells, including wound repair, follicle regeneration, salt bal-ance, and cancer regression and progression in the skinInterestingly, the inflammatory phase determines the dif-ference between chronic and acute wounds. Uncomplicated wounds heal within 4 to 6 weeks. If they continue to remain nonhealing beyond this time, they are termed chronic. Several local and systemic factors

1	between chronic and acute wounds. Uncomplicated wounds heal within 4 to 6 weeks. If they continue to remain nonhealing beyond this time, they are termed chronic. Several local and systemic factors affect the inflammatory phase of wound healing directly. These include pressure, tissue hypoxia, infection, tissue contamination, desiccation, and maceration. Systemic factors include age, stress, and comorbid conditions such as diabetes, vascular insufficiency, immunocompromise, malnourishment, obesity, and smoking. The common thread, however, in all nonhealing chronic wounds is the persistence of proinflammatory conditions. These specific tissue deficits result in a chronic cycle of chronically migrating inflammatory cells (PMNs, macrophages) that scavenge early healing tissue, degrade the newly formed matrix proteins, and then cyclically recover only to restart the inflammatory phase. This cycle leads to a chronically unstable wound that is unable to progress to the next phases of

1	the newly formed matrix proteins, and then cyclically recover only to restart the inflammatory phase. This cycle leads to a chronically unstable wound that is unable to progress to the next phases of healing: cell proliferation, tissue remodeling, and resolution.Biofilm One of the recent discoveries in the area of biofilm is an important microbial factor that impedes healing by affecting inflammatory processes in the wound-healing continuum. Biofilm comprises a colony of microorganisms enveloped with a matrix of extracellular polymers also known as extracellular polymeric substance (EPS) (Fig. 45-10). EPS affects chronic and acute dermal wounds. Its life cycle and effects on the bacterial colonies it protects are shown in Figs. 45-11 and 45-12. These include antibiotic resistance; latency (the ability to enter into latent states during inhospitable conditions); increasing species diversity; and quorum sensing (bacteria in the biofilm engage in a type of decision-making process in

1	(the ability to enter into latent states during inhospitable conditions); increasing species diversity; and quorum sensing (bacteria in the biofilm engage in a type of decision-making process in which behavior is coordinated through a “chemical” vocabulary).Proliferation. This phase is arguably the first step towards restoration of tissue continuity. It is characterized by the pro-duction of extracellular matrix by the fibroblast, the most prominent cell type in the proliferative phase. Fibroblasts are Brunicardi_Ch45_p1967-p2026.indd 197201/03/19 6:26 PM 1973PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-10. Slough that also comprises biofilm.Figure 45-11. The lifecycle of biofilm.Figure 45-12. Biofilm is a barrier to wound healing.V. choleraebiofilmPhytoplanktonMetabolicallyactive cellMetabolicallyquiescent cellPlanktonic V. choleraeMSHA pilusAquatic environmentFlagellumDetritusZooplanktonSmall intestineTCPSheddingIngestionReleaseTCPbundlingMucusHuman hostStoolthe

1	cellMetabolicallyquiescent cellPlanktonic V. choleraeMSHA pilusAquatic environmentFlagellumDetritusZooplanktonSmall intestineTCPSheddingIngestionReleaseTCPbundlingMucusHuman hostStoolthe architects of wound healing and appear in the wound right at the end of the inflammatory phase. Collectively, fibroblasts support several major functions that lead to tissue repair, includ-ing the formation of collagen and the structural creation of the extracellular matrix. The formation of fibrin and fibronectin that is precipitated from the blood clot results in the formation of a provisional extracellular matrix that serves as a scaffold. Typically, this matrix can be compared to the framework of a building without any walls or windows. The protein scaf-fold serves as a solid framework that subsequently hosts cells including human macrophages and fibroblasts. Simultane-ous VEGF-derived angiogenesis promotes the formation of small vascular loops, known as capillary buds, that proliferate within

1	hosts cells including human macrophages and fibroblasts. Simultane-ous VEGF-derived angiogenesis promotes the formation of small vascular loops, known as capillary buds, that proliferate within the fibroblast matrix. Paradoxically, the major activat-ing factor responsible for the formation of capillary buds is low oxygen tension. Poor oxygenation of the tissues increases Brunicardi_Ch45_p1967-p2026.indd 197301/03/19 6:26 PM 1974SPECIFIC CONSIDERATIONSPART IIthe expression of hypoxia inducible factor (HIF) by endothe-lial cells. Specific DNA sequences of cells that regulate angio-genesis are turned on by HIF. This paradoxical negative loop is directly related to a low oxygen tension within the tissues. Subsequent release of the epidermal growth factor EGF and the transforming growth factor TGF-α by several cell types, including macrophages, platelets, and keratinocytes, strengthen the newly formed extracellular matrix. Once a robust scaffold is built, the epidermal cells from the

1	TGF-α by several cell types, including macrophages, platelets, and keratinocytes, strengthen the newly formed extracellular matrix. Once a robust scaffold is built, the epidermal cells from the edges of the wound on all sides migrate towards the center of the wound. This process is facilitated by several factors, including angiogenesis, neovas-cularization, and the release of fibroblast growth factor TGF-β and epidermal growth factor. The formation of the extracellular matrix is the key process that leads to subsequent reepithelial-ization. The extracellular matrix is primarily made of collagen. The different types of collagen that occur more predominantly in different types of tissues characterize the type of healing that occurs. Specifically, type I is present in scar tissues. After the formation of collagen, the fibers are now attached to form a provisional fibrin matrix. After a variety of complicated signal-ing that includes the transcription and processing of collagen messenger

1	formation of collagen, the fibers are now attached to form a provisional fibrin matrix. After a variety of complicated signal-ing that includes the transcription and processing of collagen messenger RNA, the collagen gets attached to hydroxylation of protein and lysine. The hydroxyproline in the collagen is responsible for the stable helical confirmation that is critical for the formation of a robust strong scar. It then transforms itself into a classical triple helical structure that is subsequently modified through glycosylation. It is important to realize that increased collagen stability is directly related to the degree of hydroxylation of the collagen and that fragile forms of colla-gen (which result in a fragile scar) are largely due to increases in nonhydroxylated collagen forms. Certain diseases including scurvy (vitamin C deficiency) or other diseases that are pre-dominantly anaerobic in their nature can cause the formation of week nonhydroxylated collagen, which is fragile

1	Certain diseases including scurvy (vitamin C deficiency) or other diseases that are pre-dominantly anaerobic in their nature can cause the formation of week nonhydroxylated collagen, which is fragile and can easily undergo denaturation and lysis.The next step is the cleavage of the procollagen N and C terminal peptides. A very important extracellular enzyme called lysyl oxidase is responsible for the strengthening of collagen by the formation of strong, stable cross-linkages. Microscopic examination of stable mature scars reveals that strong cross-linkages present in the intramolecular and the intermolecular compartments directly correlate with strength and stability. Epi-dermal cells migrate over the scaffold, and after the epithelial bridge is completed, enzymes are released to dissolve the attach-ment at the base of the overlying scab that falls off.Contraction is one of the key end phases of proliferation. Typically, contraction starts approximately 7 days from tissue injury, when

1	the attach-ment at the base of the overlying scab that falls off.Contraction is one of the key end phases of proliferation. Typically, contraction starts approximately 7 days from tissue injury, when the fibroblasts differentiate into myofibroblasts. Myofibroblasts are similar to smooth muscle cells, have the same amount of actin (responsible for mobility), and are responsible for contraction it peaks at around 10 days post injury but can continue for several weeks. Myofibroblasts attach to the extra cellular matrix (ECM) at the wound edges and to each other as well as to the wound edges via desmosomes and the fibronexus, through which actin in the myofibroblast is linked across the cell membrane to molecules in the extracellular matrix like fibro-nectin and collagen. This in turn facilitates the myofibroblasts to pull the ECM when they contract, thus reducing the wound size. Wounds contract at the rate of 0.75 mm to 1 mm daily. The formation of a strong, contracted, cross-linked

1	the myofibroblasts to pull the ECM when they contract, thus reducing the wound size. Wounds contract at the rate of 0.75 mm to 1 mm daily. The formation of a strong, contracted, cross-linked collagen scar with reepithelization heralds the end of the proliferative phase. Contraction usually does not occur symmetrically; instead, most wounds have an “axis of contraction” that allows for greater organization and alignment of cells with collagen.Remodeling/Maturation. The remodeling phase is also termed the maturation phase. It is primarily characterized by the remodeling of collagen through a balance between collagen for-mation and collagen lysis that results in the formation of a strong scar. Biochemically, the collagen is remodeled from type III to type I and is also accompanied by complete reepithelialization of the wound. The lysis of collagen is mediated by collagenases that are secreted by various cells—fibroblasts, neutrophils, and macrophages—each of which can cleave the collagen

1	reepithelialization of the wound. The lysis of collagen is mediated by collagenases that are secreted by various cells—fibroblasts, neutrophils, and macrophages—each of which can cleave the collagen molecule at different but specific locations on all three chains and break it down to characteristic three-quarter and one-quarter pieces. These collagen fragments undergo further denaturation and digestion by other proteases. There is significant remodeling of the collagen during this process. It is aligned along tension lines, and significant reabsorption of water from the collagen fibers result in a denser alignment and stronger cross-linking. The remodeling phase establishes a new equilibrium with the forma-tion of an organized scar. Several molecules, including TGF-β, which induces intracellular signaling of SMAD proteins, play an important role in the remodeling phase. Using SM 80 knockout mice and transgenic animals, a critical role of the SMAD path-way in the formation of scar has

1	signaling of SMAD proteins, play an important role in the remodeling phase. Using SM 80 knockout mice and transgenic animals, a critical role of the SMAD path-way in the formation of scar has been delineated. This process is also facilitated by apoptosis and programmatic cell death, which helps to former a thinner scar that is stronger and more cosmeti-cally appealing. This phase begins 3 weeks after the injury and continues for over 1 year. One must realize that despite the best cross-linking, scar tissue is weaker than injured skin and regains only 80% of its uninjured tensile strength. As it matures fur-ther, it becomes less red and less vascular because the reduced biologic activity within the scar renders the vascular capillaries redundant and they apoptose.RECONSTRUCTIVE SURGERYReconstructive surgery restores normal anatomy and function using plastic surgery methods of tissue repair, rearrangement, and replacement. Tissues can be missing or damaged as a con-sequence of trauma,

1	surgery restores normal anatomy and function using plastic surgery methods of tissue repair, rearrangement, and replacement. Tissues can be missing or damaged as a con-sequence of trauma, cancer, degeneration, congenital abnor-malities, and aging. The primary adverse consequence of lost or impaired tissue is functional disability, which includes physical, psychologic, or social dysfunction. The clinical objective is to reestablish normal anatomy, function, and appearance in order to restore the patient as closely as possible to normal health. The most useful techniques transfer and modify tissues in the form of tissue grafts and surgical flaps.RECONSTRUCTIVE STRATEGIES AND METHODSThe main aim of wound healing is to achieve a closed wound. Ordinarily, wounds heal via three main mechanisms:1. Primary intention: This type of healing occurs in a clean wound without any apparent tissue loss. Mostly seen in surgical incisions that have been approximated (primary closure), healing by primary

1	intention: This type of healing occurs in a clean wound without any apparent tissue loss. Mostly seen in surgical incisions that have been approximated (primary closure), healing by primary intention can only be imple-mented when the closure of the wound is precise and there is minimal disruption to the local tissue or the epithelial basement membrane. Typically, this wound seals off within 24 hours. Healing is faster than healing by secondary inten-tion, and there is the least amount of scarring.2Brunicardi_Ch45_p1967-p2026.indd 197401/03/19 6:26 PM 1975PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 452. Secondary intention: Tissue loss following major trauma results in the formation of granulation tissue, which results in a broader scar (see earlier section, “Phases of Wound Healing”).3. Tertiary intention (delayed primary closure or second-ary suture): The wound is initially cleaned, debrided, and observed, typically 4 or 5 days before closure. Examples of this type of healing

1	intention (delayed primary closure or second-ary suture): The wound is initially cleaned, debrided, and observed, typically 4 or 5 days before closure. Examples of this type of healing include healing through the use of tissue grafts, including skin grafts and substitutes.Skin Grafts and Skin SubstitutesSkin grafting methods date back millennia to ancient India, where they were used to resurface nasal defects. They were introduced in the modern era by Guiseppe Baronio, an Italian physician who studied skin grafting techniques in sheep and published his work entitled Degli Innesti Animali (On Grafting in Animals) in 1804.4It is important to know the basic anatomic structure of skin in order to understand the principles of skin grafting. Skin is comprised of the epidermis, the dermis, specialized sensory nerve endings, and various skin appendages that lubricate and protect the skin as well as contribute to functions such as ther-moregulation. The epidermis is a layer of cells that

1	specialized sensory nerve endings, and various skin appendages that lubricate and protect the skin as well as contribute to functions such as ther-moregulation. The epidermis is a layer of cells that affords pri-mary barrier function. It begins with a layer of cells called the basal layer. These are cuboidal-shaped cells that multiply and differentiate into flattened, keratinized squamous cells, which progressively migrate from the basal layers until they are finally released from the surface in a process known as desquamation. The junction between the dermis and the epidermis is composed of projections from the dermis into the epidermis, which are called dermal papillae. This feature secures the epidermis to the dermis by resisting sheer forces transmitted from the skin surface, helping to prevent separation of the epidermis from the dermis. The dermis contains sebaceous glands, whereas sweat glands and hair follicles are actually located below the dermis in the subcutaneous tissue

1	to prevent separation of the epidermis from the dermis. The dermis contains sebaceous glands, whereas sweat glands and hair follicles are actually located below the dermis in the subcutaneous tissue and traverse the dermis and epithe-lium to reach the body surface. The dermal thickness and con-centration of skin appendages vary widely from one location to another on the body. The blood supply to the skin occurs in a variety of patterns that form the basis for transferring tissue-containing skin, which will be discussed later in this chapter. Regardless of the pattern, there is a network of vessels just below the dermis called the subdermal plexus that supplies the skin immediately above and is important in thermoregulation. Finally, terminal vessels and capillaries fill the dermis and pen-etrate the dermal papillae to perfuse the cellular elements of the dermis and epidermis.Skin grafting methods include split-thickness skin grafts (STSG), full-thickness skin grafts (FTSG), and

1	pen-etrate the dermal papillae to perfuse the cellular elements of the dermis and epidermis.Skin grafting methods include split-thickness skin grafts (STSG), full-thickness skin grafts (FTSG), and composite tissue grafts. Each has its advantages and disadvantages, and select-ing the best technique for a given circumstance depends on the reconstructive requirements, the quality of the recipient wound bed, and the availability of donor site tissue.Split-Thickness Grafts. An STSG is the simplest method of tissue transfer. The name is derived from how these grafts are harvested by cutting through (i.e., splitting) the dermis at various levels. Thin STSGs are harvested through the superficial levels of the dermis. Thick grafts are harvested through deeper layers and include a larger amount of dermal tissue. The impor-tant characteristics of STSGs are determined by the thickness of dermis present in the graft. Thin grafts undergo less primary contraction after harvest because they contain

1	dermal tissue. The impor-tant characteristics of STSGs are determined by the thickness of dermis present in the graft. Thin grafts undergo less primary contraction after harvest because they contain fewer elements of the dermal extracellular matrix such as elastic fibers. Thick grafts undergo greater amounts of primary contraction. This is important to remember when harvesting the graft because it is necessary to obtain sufficient tissue in order to restore the defect. On the other hand, thin grafts allow the wound to undergo a greater amount of contraction in a process traditionally referred to secondary contraction of the graft. This becomes important if the wound is adjacent to a mobile structure such as the oral commissure, which might be distorted as healing progresses. Thin grafts also have improved chances of complete engraft-ment, or “taking,” as they contain mostly epidermis, which has low metabolic demands, in contrast to thicker grafts that contain more dermis with greater

1	have improved chances of complete engraft-ment, or “taking,” as they contain mostly epidermis, which has low metabolic demands, in contrast to thicker grafts that contain more dermis with greater metabolic needs.A variety of techniques have been described to maximize the surface area that can be covered by harvested skin amount while minimizing the size of the donor site.5 One approach is to process the harvested skin into micrografts using devices spe-cially designed for this purpose in the operating room. Another method is fractional skin harvesting, which involves harvesting a large number of full-thickness skin tissue columns that are then seeded onto the wound surface. The traditional method, however, is to mesh the graft. Meshed grafts usually also have enhanced reliability of engraftment because the fenestrations allow for egress of wound fluid and excellent contour match-ing of the wound bed by the graft. The fenestrations in meshed grafts must epithelialize by secondary

1	engraftment because the fenestrations allow for egress of wound fluid and excellent contour match-ing of the wound bed by the graft. The fenestrations in meshed grafts must epithelialize by secondary intention from the sur-rounding graft skin. The major drawbacks of meshed grafts are poor cosmetic appearance and high rates of secondary contrac-tion. Meshing ratios used usually range from 1:1.5 to 1:6, with higher ratios associated with magnified drawbacks related to meshing. For any case, a decision to mesh the graft must be balanced against the disadvantages. Other differences between thin and thick STSGs include final durability, pigmentation, and tendency to desiccation of the final result. The distinguishing characteristics of skin grafts types based on thickness are sum-marized in Fig. 45-13.STSG donor sites heal by regeneration from dermal and epidermal elements remaining in the harvest site. Recesses between dermal papillae projecting into the dermis are lined by basal cells.

1	45-13.STSG donor sites heal by regeneration from dermal and epidermal elements remaining in the harvest site. Recesses between dermal papillae projecting into the dermis are lined by basal cells. These cells migrate across the wound surface and Figure 45-13A. Skin grafts categorized based on thickness.ThinIntermediateSplit skinThickFull thicknessskinABrunicardi_Ch45_p1967-p2026.indd 197501/03/19 6:26 PM 1976SPECIFIC CONSIDERATIONSPART IIDermal content1° contraction2° contractionEngraftmentDurabilityPigmentationResist desiccationRecipient bedAppearanceSTSG(thin) ++++++++++++++++++++++++++++++++++++++++++++++++++++++STSG(thick)FTSGBFigure 45-13B. Characteristics of skin grafts.reepithelialize it. During this process, the donor site must be kept moist and free of bacterial contamination. Depending on the thickness of the graft, uncomplicated donor site epitheliali-zation typically is complete in 2 weeks. In most cases, it should be protected from mechanical shear and drying until the

1	Depending on the thickness of the graft, uncomplicated donor site epitheliali-zation typically is complete in 2 weeks. In most cases, it should be protected from mechanical shear and drying until the new skin matures with epidermal and dermal thickening and reac-tivation of sebaceous and sweat glands. Part of managing the donor site includes minimizing pain. Some recommended treat-ments include (a) subcutaneous anesthetic injection of adren-aline-lidocaine; (b) ice application; (c) topical agents such as lidocaine and bupivacaine; and (d) hydrocolloidand polyure-thane-based wound dressings accompanied with fibrin sealant.6 Maintaining air-tight coverage using transparent adhesive film dressing can protect the donor site during reepithelialization and minimize pain.Full-Thickness Grafts. By definition, full-thickness skin grafts include the epidermis and the complete dermis. When harvesting and preparing this type of skin graft, the surgeon must carefully remove any retained

1	Grafts. By definition, full-thickness skin grafts include the epidermis and the complete dermis. When harvesting and preparing this type of skin graft, the surgeon must carefully remove any retained subcutaneous tissue from the deep surface of the dermis in order to maximize the poten-tial for engraftment. Full-thickness grafts are associated with the greatest amount of primary contraction, the least amount of secondary contraction, the highest durability, and ultimately the best cosmetic appearance. As a result, they are frequently used in reconstructing superficial wounds of the face and the hands. These grafts require clean, well-vascularized recipient beds free of bacterial colonization, previous irradiation, or fibrous wound tissue. They also work poorly in wounds associated with previ-ous radiation treatments in cancer patients. The harvest site for an FTSG must be closed primarily because no skin elements remain in the area of harvest.Skin Substitutes. Skin substitutes are

1	previ-ous radiation treatments in cancer patients. The harvest site for an FTSG must be closed primarily because no skin elements remain in the area of harvest.Skin Substitutes. Skin substitutes are typically types of extra-cellular matrices that are often acellular in nature and are either human-derived (allografts), animal-derived (xenografts), tissue engineered, or a combination of the three.7 These substitutes most often are employed to replace lost dermal and/or epider-mal skin layers resulting from burns, trauma, and other super-ficial injuries to the outer skin layers. While a complete review of all of these commercially available materials is beyond the scope of this chapter, the benefits and applications of these use-ful adjuncts is growing, and they been have shown to play an important role in current as well as future reconstructive, regen-erative, and restorative measures for tissue and skin replace-ment. Essentially, they act similarly to grafts as they rely on

1	play an important role in current as well as future reconstructive, regen-erative, and restorative measures for tissue and skin replace-ment. Essentially, they act similarly to grafts as they rely on revascularization and autologous cell repopulation of the con-struct in order to “take” and become part of the lost anatomic structure they are acting to restore.Graft Take. Skin graft healing, or “take,” occurs in three phases: imbibition, inosculation, and revascularization. Plas-matic imbibition takes place during the first 24 to 48 hours after placement of the graft onto the defect. During this time, the graft is held in place by a thin film of fibrin, and the cellular elements survive by diffusion of oxygen and substrate from plasma pres-ent in the open wound. After 48 hours, a fine vascular network forms from capillaries and small blood vessels in the wound bed and advances through the fibrin layer toward the graft. These new vascular buds encounter open, cut end vessels on the deep

1	network forms from capillaries and small blood vessels in the wound bed and advances through the fibrin layer toward the graft. These new vascular buds encounter open, cut end vessels on the deep surface of the dermis of the graft and line up, forming loose anastomoses that begin to allow blood flow and the transfer of some nutrients and oxygen. This phase is called inosculation and is the period during which the graft is most at risk for fail-ure. If the tenuous alignment of vessels between the wound bed and the graft are disrupted, then the final phase of healing will not occur. Events that can cause graft failure at this time include mechanical shear, formation of a seroma or hematoma, or bac-terial contamination. The final phase of engraftment is called revascularization. During this phase, firmer vascular anastomo-ses are formed as the vessels heal, and the graft becomes per-fused from the wound bed. Signs of perfusion, such as improved coloration and evidence of capillary

1	this phase, firmer vascular anastomo-ses are formed as the vessels heal, and the graft becomes per-fused from the wound bed. Signs of perfusion, such as improved coloration and evidence of capillary refill, confirm engraftment and graft take. In most circumstances, these phases are complete by 4 to 5 days after graft placement. The dressing used after placing the skin graft is a critical part of success. It must prevent desiccation and shear stress from disrupting the graft, especially during the critical period of inosculation. Tie-over bolster dress-ings are a traditional method. Topical negative pressure wound dressings have been demonstrated to increase quantity and qual-ity of split-thickness skin graft take compared to traditional bol-ster dressings. The benefits are particularly evident in wounds with irregular surface contours in areas that might be difficult to avoid motion.8After skin graft take, the graft remains subject to late fail-ure due to mechanical shear,

1	evident in wounds with irregular surface contours in areas that might be difficult to avoid motion.8After skin graft take, the graft remains subject to late fail-ure due to mechanical shear, desiccation, or bacterial infection. Depending on the location and clinical setting, the graft should continue to be protected using dressings, topical moisturizing creams, or antibacterial medications as indicated until stable healing obtains in up to 2 weeks.Composite Grafts. Composite grafts contain other types of tissue besides skin. Additional elements must have low met-abolic requirements in order to survive the time required for revascularization. Composite grafts might include subcutane-ous fat, cartilage, perichondrium, and small amounts of muscle. Indications for composite grafts are limited to small areas with specialized tissue requirements such as nasal reconstruction. For example, excision of a skin cancer involving the nasal lobule may create a composite defect that involves

1	limited to small areas with specialized tissue requirements such as nasal reconstruction. For example, excision of a skin cancer involving the nasal lobule may create a composite defect that involves internal nasal lin-ing, supporting nasal cartilage, and external skin. The ear is a good donor site for a composite graft of tissue with a good color match for the face and small amounts of tissue configured natu-rally to simulate the contours of the nose. For example, harvest of tissue from the root of the helix of the ear causes a relatively inconspicuous donor site. The donor site for composite tissue grafts must be repaired with primary closure.Surgical Flaps. A surgical flap is a unit of tissue harvested from a donor site and transferred to another location for Brunicardi_Ch45_p1967-p2026.indd 197601/03/19 6:26 PM 1977PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45reconstructive purposes. The term “flap” is derived from tech-niques of adjacent skin tissue transfers fashioned as

1	197601/03/19 6:26 PM 1977PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45reconstructive purposes. The term “flap” is derived from tech-niques of adjacent skin tissue transfers fashioned as flaps of skin that were elevated and folded into the defect. The distinguishing feature of a surgical flap is having a blood supply independent of the injured area. A graft must go through the phases of heal-ing described previously as it derives a new blood supply from the wound bed. A flap is brought to the wound with its own blood supply. This allows restoring tissue in areas of poor blood supply or with tissue requirements greater than what can be sup-ported through a period of diffusion only.There are a tremendous variety of surgical flaps that can be created depending on the individual patient’s reconstructive needs and available tissues. The challenge of reconstructive sur-gery is to design an appropriate flap to restore the defect with a minimal amount of morbidity related to the flap donor

1	needs and available tissues. The challenge of reconstructive sur-gery is to design an appropriate flap to restore the defect with a minimal amount of morbidity related to the flap donor site. The different kinds of flaps can be broadly classified by three distinct characteristics: (a) the types of tissue contained, (b) the proximity to the defect, and (c) the pattern of blood supply.The first way to classify different types of surgical flaps is by what tissue they contain. Nearly any type of vascularized tissue can be transferred as a surgical flap. One of the most com-mon is a cutaneous flap, which contains skin and subcutaneous tissue. Another versatile type is a muscle flap, which contains only muscle. Musculocutaneous flaps contain a portion of mus-cle along with the overlying skin and all the intervening tissues. An osseous flap contains a segment of bone, and an osteocuta-neous flap includes skin as well as the bone. Flaps can also be designed to include fascia and peripheral

1	and all the intervening tissues. An osseous flap contains a segment of bone, and an osteocuta-neous flap includes skin as well as the bone. Flaps can also be designed to include fascia and peripheral nerves. Visceral flaps contain segments of jejunum, stomach, colon, or the greater omentum. The choice of flap depends upon the reconstructive needs and availability of tissue.The second way to classify surgical flaps is by their prox-imity to the defect. The location and distance between the flap donor site and the defect usually dictate the method required to transfer the tissue with preservation of the blood supply. Local flaps have a donor site located immediately adjacent to the defect.9 Regional flaps are harvested from the same anatomic region as the defect. Distant flaps are harvested and trans-ferred from outside the anatomic region of the defect. Dur-ing the transfer of all of these flaps, the blood supply remains attached to the source anatomic region. The tissue transmitting

1	and trans-ferred from outside the anatomic region of the defect. Dur-ing the transfer of all of these flaps, the blood supply remains attached to the source anatomic region. The tissue transmitting the blood supply is called the flap pedicle. When the blood supply is not divided during the transfer, it is referred to as a pedicled flap. If the distance between the donor site and the defect exceeds the length of the pedicle, the vessels can Figure 45-14. Limberg flap.be divided and then reattached to uninjured vessels within or adjacent to the defect after the tissue is placed there. This technique is called a free tissue transfer, and flaps transferred in this fashion are called free flaps because for some period of time during the procedure the tissue of the flap is completely separated, or free, of the patient. The diameter of the blood vessels that supply common surgical flaps is usually less than 5 mm. Repairing blood vessels of this caliber is considered microvascular surgery,

1	or free, of the patient. The diameter of the blood vessels that supply common surgical flaps is usually less than 5 mm. Repairing blood vessels of this caliber is considered microvascular surgery, and techniques for doing this are part of reconstructive microsurgery.The third and perhaps most important way to classify dif-ferent surgical flaps is by the pattern of their blood supply.10 Using this criterion, flaps are traditionally divided into random pattern flaps, axial pattern flaps, musculocutaneous flaps, fas-ciocutaneous flaps, direct cutaneous flaps, perforator flaps, and free flaps. These designations are based on how vessels reach from the deeper, usually named, arteries and veins to the super-ficial tissues and skin. These are described in greater detail in the following section.Random Pattern Flaps. The simplest flap designs are random pattern flaps, so named because the blood supply is based on unnamed vessels in the attached base of the flap that perfuse through the

1	Pattern Flaps. The simplest flap designs are random pattern flaps, so named because the blood supply is based on unnamed vessels in the attached base of the flap that perfuse through the subdermal plexus.11 Random flaps are typically used to reconstruct relatively small, full-thickness defects, and they are designed following geometric principles of skin rearrange-ment with a traditional length-to-width ratio of 3:1. Exceptions to this principle regarding reliable dimensions abound, however, because of the variability in the patterns of perfusion and the density of the subdermal plexus in different regions of the body.Random pattern flaps can be further subdivided based on the geometry of the transfer. Examples of this are transposition flaps, advancement flaps, and interpolated flaps. A transposition flap is fashioned adjacent to an area needing reconstruction and rotated into the defect. Large transposition flaps can require a skin graft to close the donor site. To avoid this

1	A transposition flap is fashioned adjacent to an area needing reconstruction and rotated into the defect. Large transposition flaps can require a skin graft to close the donor site. To avoid this problem, spe-cialized types of transposition flaps have been devised. One that is particularly useful is called a Z-plasty. In this technique, two flaps are rotated, each into the donor site of the other, to rearrange the tissues in a way that redirects the lines of tension and lengthens the central limb. Another is the rhomboid (Lim-berg) flap (Fig. 45-14). In this technique, a skin flap is precisely designed with opposing 60° and 120° angles at the corners of a rhomboid designed immediately adjacent to the defect. This design can be modified to allow the flap to rotate into the defect Area withmaximum laxityABCD120°60°Brunicardi_Ch45_p1967-p2026.indd 197701/03/19 6:26 PM 1978SPECIFIC CONSIDERATIONSPART IIwith primary closure of the donor site with minimal distortion of the surrounding

1	laxityABCD120°60°Brunicardi_Ch45_p1967-p2026.indd 197701/03/19 6:26 PM 1978SPECIFIC CONSIDERATIONSPART IIwith primary closure of the donor site with minimal distortion of the surrounding tissues as shown in the case of a gluteal repair (Fig. 45-15A–B, by complex closure; Fig. 45-15C–E, by modi-fied Limberg flap). Modifications on the angle, including the Dufourmental modification, cause the parametric configuration to be optimized based on the defect12 (Fig. 45-16). Rotational flaps are a type of transposition that is semicircular in design, allowing the tissue to be rotated and permitting primary closure. Advancement flaps differ from transposition flaps because the tissue is moved forward from the donor site along the flap’s long axis rather than being rotated about a point. Two common vari-ants include the rectangular advancement flap (Fig. 45-17) and the V-Y advancement flap (Fig. 45-18). Finally, interpolation flaps rotate about a pivot point but are used to transfer tissue

1	vari-ants include the rectangular advancement flap (Fig. 45-17) and the V-Y advancement flap (Fig. 45-18). Finally, interpolation flaps rotate about a pivot point but are used to transfer tissue ABCDEFigure 45-15. Reconstruction of a gluteal defect using complex closure and reconstruction of a gluteal defect using a modified Limberg flap.Brunicardi_Ch45_p1967-p2026.indd 197801/03/19 6:26 PM 1979PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-16. Dufourmental modification.Figure 45-17. Rectangular advancement flap.Figure 45-18. V-Y flap closure.BABYXZCADEF˜1˜2°Advancement flapABCDinto a nonadjacent area with an intervening portion of undam-aged tissue between the donor site and the defect (Fig. 45-19).Axial Pattern Flaps. Historically, surgeons made an increas-ing variety of surgical flaps to address a greater assortment of reconstructive problems. In the process, they noticed that some of these flaps routinely violated the strict limitations of accepted length-to-width

1	surgical flaps to address a greater assortment of reconstructive problems. In the process, they noticed that some of these flaps routinely violated the strict limitations of accepted length-to-width ratio. Further investigation demon-strated that these flaps had significant arteries running parallel to the long axis of the flap. These flaps became known as axial pattern flaps.12 The earliest example of this type of flap is the deltopectoral flap, originally described in 1971 by Bakamjian (Fig. 45-20A,B). This flap is based on cutaneous vessels perfo-rating from inside the chest from the internal mammary artery and vein. After entering the subcutaneous tissues, they travel obliquely from the sternal border toward the deltoid area of the arm. Long flaps can be designed based on these vessels, which can reach into the head and neck to provide thin tissue from the upper chest to restore defects, especially after tumor ablation. Other important and useful axial pattern flaps are the groin

1	which can reach into the head and neck to provide thin tissue from the upper chest to restore defects, especially after tumor ablation. Other important and useful axial pattern flaps are the groin flap and the posterior thigh flap.Musculocutaneous Flaps. The vascular pattern of musculo-cutaneous flaps arises from major vessels that primarily supply a muscle and then secondarily supply the skin through multiple small vessels traversing between the superficial surface of the muscle and the subdermal plexus. The discovery of this pat-tern of cutaneous blood supply was a major breakthrough in reconstructive surgery because it made it possible to transfer units of tissue much larger than was possible with random or axial pattern flaps, enabling plastic surgeons to restore a greater range of deformities. Mathes and Nahai classified individual muscles into five types (I–V) according to the number and dom-inance of the vascular pedicles supplying each13 (Table 45-1). There may be advantages

1	Mathes and Nahai classified individual muscles into five types (I–V) according to the number and dom-inance of the vascular pedicles supplying each13 (Table 45-1). There may be advantages to including muscle in a surgical flap besides ensuring adequate blood supply to the overlying skin. The classic example is breast reconstruction using a latissimus dorsi myocutaneous flap (Fig. 45-21A–C). Here, the latissimus muscle is harvested pedicled on the thoracodorsal vessels and transposed anteriorly onto the chest wall. Muscle is a highly vascularized tissue that is bulky and deformable. It can help to repair visible surface contour deformities by increasing the pro-jection of tissue in the defect to reach the level of the surround-ing undamaged tissues. It can also easily contour to fill spaces in a complicated wound surface, thus helping to prevent small fluid collections in recesses, which can be a harbor bacteria and become a nidus of infection. It is also possible to provide

1	fill spaces in a complicated wound surface, thus helping to prevent small fluid collections in recesses, which can be a harbor bacteria and become a nidus of infection. It is also possible to provide func-tional restoration using musculocutaneous flaps by coapting the motor nerve of the muscle in the flap to a corresponding motor nerve in the defect. This method can be used to restore motor function in patients with motor loss in the extremities or face.Fasciocutaneous Flaps. Rather than having a blood supply primarily from underlying muscle, the skin and subcutaneous tissues of some anatomic regions are supplied from vessels communicating with the underlying superficial or deep fascia. Such flaps are referred to as fasciocutaneous flaps. The artery and vein of the flap pedicle passes between rather than through muscles, form a plexus of vessels within the fascia, and then send multiple small vessels to the subdermal plexus to perfuse the skin. There are clinical circumstances when a

1	rather than through muscles, form a plexus of vessels within the fascia, and then send multiple small vessels to the subdermal plexus to perfuse the skin. There are clinical circumstances when a fasciocutane-ous flap might have advantages over a musculocutaneous flap. Fasciocutaneous flaps are usually thinner compared to muscu-locutaneous flaps. They also do not create a functional loss of muscle in the donor site. Mathes and Nahai classified fasciocu-taneous flaps into types A, B, and C (Table 45-2) based on how the vascular pedicle reaches the fascia from the major vessels deep to the fascia and muscles. Sural perforator fasciocutaneous flaps (Fig. 45-22A–D) are a modern example of reconstructing lower extremity defects that would be difficult to reconstruct without microvascular surgery.Direct Cutaneous Flaps. Some surgical flaps have a vascu-lar pedicle that reaches directly to the superficial tissues and subdermal plexus without passing through a muscle or fascia plexus. These

1	Cutaneous Flaps. Some surgical flaps have a vascu-lar pedicle that reaches directly to the superficial tissues and subdermal plexus without passing through a muscle or fascia plexus. These are called direct cutaneous flaps.Perforator Flaps. The final kind of surgical flap classified by the pattern of blood supply is the perforator propeller flap.14,15 The geometric measurements that are critical to its success are summarized in Fig. 45-23. Reconstructive procedures based Brunicardi_Ch45_p1967-p2026.indd 197901/03/19 6:27 PM 1980SPECIFIC CONSIDERATIONSPART IIFigure 45-19. Forehead flap for nasal reconstruction.ADBECFon these flaps are the result of complementary advances in our understanding of cutaneous blood supply and improved surgical techniques.Ian Taylor and a team of investigators from Melbourne, Australia, discovered that the blood supply to all portions of the skin was organized into discreet units, which they called angiosomes18. Analogous to dermatomes that describe the

1	from Melbourne, Australia, discovered that the blood supply to all portions of the skin was organized into discreet units, which they called angiosomes18. Analogous to dermatomes that describe the patterns of cutaneous sensation supplied by single sensory nerves, the cutaneous perfusion is organized into angiosomes supplied by a single arteries. These arteries arise from source blood vessels located deep to other structures like muscle and fascia and penetrate through as perforating vessels. Often the artery is accompanied by two venae commitantes, but in many regions an additional venous drainage system is present in the superficial planes. The territories of adjacent angiosomes over-lap similarly to how dermatomes overlap. An angiosome is defined by the limits of an artery’s terminal branching. At the borders, these arterioles form anastomoses with the neighbor-ing angiosome. The vessels that pass between these anatomic angiosomes are called choke vessels. In life, these may open or

1	At the borders, these arterioles form anastomoses with the neighbor-ing angiosome. The vessels that pass between these anatomic angiosomes are called choke vessels. In life, these may open or close in response to physiologic changes in order to increase or decrease, respectively, an artery’s dynamic angiosome momen-tarily. Accordingly, at any given time point, the dynamic angio-some of an artery may be approximated by the volume of tissue stained by an intravascular administration of fluorescein into that artery (indicating the reach of blood flow from that artery into tissues). The potential angiosome of an artery is the vol-ume of tissue that can be included in a flap that has undergone conditioning (see the following section). Both the dynamic and potential angiosomes extend beyond the anatomic angiosome of an artery. Although the angiosome concept provides some guidance to the size and volume limits of a flap harvest, there remains no quantifiable method to predict safe flap

1	the anatomic angiosome of an artery. Although the angiosome concept provides some guidance to the size and volume limits of a flap harvest, there remains no quantifiable method to predict safe flap harvest lim-its with precision.Brunicardi_Ch45_p1967-p2026.indd 198001/03/19 6:27 PM 1981PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-20A, B. Deltopectoral flap for cheek reconstruction.Table 45-1Mathes-Nahai classification of muscular flapsCLASSIFICATIONVASCULAR SUPPLYEXAMPLEType IOne vascular pedicleGastrocnemiusType IIDominant and minor pedicles (the flap cannot survive based only on the minor pedicles)GracilisType IIITwo dominant pediclesRectus abdominisType IVSegmental pediclesSartoriusType VOne dominant pedicle with secondary segmental pedicles (the flap can survive based only on the secondary pedicles)Pectoralis majorALimit of areatubed ondeep aspectSkinGraftsBTissue Expansion. Tissue expansion is a technique that increases the amount of tissue in a surgical flap by

1	only on the secondary pedicles)Pectoralis majorALimit of areatubed ondeep aspectSkinGraftsBTissue Expansion. Tissue expansion is a technique that increases the amount of tissue in a surgical flap by first plac-ing an inflatable device into the tissue beneath the planned flap and gradually expanding the tissue by regular inflation. Staged reconstruction using tissue expansion can significantly increase the amount of local, well-matched tissue for transfer while decreasing donor site morbidity. The most common method of skin expansion involves the placement of an inflatable silicon elastomer similar to a balloon with a filling port that is gener-ally positioned in an easily accessible location beneath the skin. After wound healing, the device is gradually inflated by serial injections of sterile saline solution into the filling port. The process can require several weeks, depending on the amount of expansion and compliance of the tissues. When expansion is complete, the expander is

1	sterile saline solution into the filling port. The process can require several weeks, depending on the amount of expansion and compliance of the tissues. When expansion is complete, the expander is removed, and the resulting expanded tissue is transferred into the defect.The process of expanding flaps confers physiologic bene-fits that increase the reliability of the flap tissue. Histologically, expanded skin demonstrates thickened dermis with enhanced vasculature and diminished subcutaneous fat. Studies have shown that the increased amount of skin is the result of actual generation of new tissue. Also, the blood supply to an expanded flap is improved because of the period of delay associated with expansion process and the capsule formed around the device is highly vascular and contributes to the quality of blood supply.16The disadvantages of tissue expansion have to do with pos-sible complications, which include infection, hematoma, seroma, expander extrusion, implant failure, skin

1	to the quality of blood supply.16The disadvantages of tissue expansion have to do with pos-sible complications, which include infection, hematoma, seroma, expander extrusion, implant failure, skin necrosis, pain, and peripheral nerve injury. Furthermore, an inflated expander is vis-ible, and the temporary deformity may cause patients distress.Tissue expansion has found particular usefulness in man-aging giant congenital nevi, secondary reconstruction of exten-sive burn scars, scalp reconstruction, and breast reconstruction. Expanders are available in a multitude of shapes and sizes, depending on the reconstructive needs. The technique permits reconstruction with tissue of similar color, texture, and thick-ness, with minimal donor site morbidity.PEDIATRIC PLASTIC SURGERYCongenital Craniofacial AnomaliesIn 1981, Whitaker et al introduced a simple classification sys-tem to help conceptualize the vast array of congenital pathology involving the craniofacial region.17 Based on anatomy,

1	AnomaliesIn 1981, Whitaker et al introduced a simple classification sys-tem to help conceptualize the vast array of congenital pathology involving the craniofacial region.17 Based on anatomy, etiology, and current treatment principles, most cra-niofacial anomalies can be classified into one of four categories: clefts, synostoses, atrophy-hypoplasia, or hypertrophy-hyper-plasia-neoplasia (Table 45-3).Clefts. Arguably, no operation in plastic surgery is more demanding of reconstructive principle and aesthetic intuition 3Brunicardi_Ch45_p1967-p2026.indd 198101/03/19 6:27 PM 1982SPECIFIC CONSIDERATIONSPART IIFigure 45-21. Breast reconstruction (right side) with a latissimus flap.B Preop, right mastectomy and left previous implant reconstructionC Postoperative: bilateral latissimus flap with implantSkin usedfor flapLatissimusdorsimuscleClosedincisionImplantundermusclesLatissimusdorsi flapin placeATable 45-2Nahai-Mathes classification of fasciocutaneous flapsCLASSIFICATIONVASCULAR

1	with implantSkin usedfor flapLatissimusdorsimuscleClosedincisionImplantundermusclesLatissimusdorsi flapin placeATable 45-2Nahai-Mathes classification of fasciocutaneous flapsCLASSIFICATIONVASCULAR SUPPLYEXAMPLEType ADirect cutaneous vessel that penetrates the fasciaTemporoparietal fascial flapType BSeptocutaneous vessel that penetrates the fasciaRadial artery forearm flapType CMusculocutaneous vessel that penetrates the fasciaTransverse rectus abdominis myocutaneous flapthan a cleft lip repair. Orofacial clefting is the most common birth defect in the world. Cleft lip, with or without cleft palate (CL/P), occurs spontaneously among Caucasian populations in approximately 1 out of every 1000 births. It is over twice as common (1 in 450) among Asians and Native Americans and half as common (1 in 2000) in African Americans. There is a predilection among males, who are twice as likely to be affected as females. Left-sided cleft lip is twice as common as right and nine times as common as

1	(1 in 2000) in African Americans. There is a predilection among males, who are twice as likely to be affected as females. Left-sided cleft lip is twice as common as right and nine times as common as bilateral. Of patients born with CL/P, 29% have associated anomalies, which can range from minor physical differences to major organ involvement. While a fam-ily history of CL/P remains the strongest known predictive factor, other extrinsic risk factors include maternal smoking or early exposure to the anticonvulsant drug phenytoin.18Epidemiologically, isolated cleft palate (CP) appears to be distinctly different from CL/P. CP occurs in 1 of every 2000 live births. It is twice as common in females, and it demonstrates no racial or ethnic preponderance. Nearly half of patients with iso-lated CP have a diagnosable syndrome and additional congeni-tal anomalies. Evaluation by a geneticist is therefore indicated in all babies born with isolated CP. Like CL/P, isolated CP is multifactorial.

1	CP have a diagnosable syndrome and additional congeni-tal anomalies. Evaluation by a geneticist is therefore indicated in all babies born with isolated CP. Like CL/P, isolated CP is multifactorial. Known environmental risk factors include mater-nal smoking or alcohol consumption, folate deficiency, use of steroids or anticonvulsant medications, or retinoid (vitamin A) excess.Some familial patterns of orofacial clefting have been linked to specific genetic mutations. Van der Woude syndrome, an autosomal dominant form of CL/P associated with lower lip pits, is caused by an IRF6 gene mutation (Fig. 45-24).23 Stick-ler syndrome should be suspected in patients with isolated CP, Brunicardi_Ch45_p1967-p2026.indd 198201/03/19 6:27 PM 1983PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-22. Reconstruction of a lateral malleolar defect using a reverse sural perforator flap.Figure 45-23. Geometric considerations for a propeller flap.ABCDABaDefectPerforatorbc+CDwith associated eye

1	45-22. Reconstruction of a lateral malleolar defect using a reverse sural perforator flap.Figure 45-23. Geometric considerations for a propeller flap.ABCDABaDefectPerforatorbc+CDwith associated eye defects, sensorineural hearing loss, and joint abnormalities. This constellation of findings is due to an autosomal dominant mutation in a procollagen gene. Stickler is also the most common syndrome associated with Pierre Robin sequence (micrognathia, glossoptosis, and respiratory distress).19 These examples help emphasize the importance of early genetic workup for patients in whom a syndrome is suspected.Embryology of the Lip and Palate The “primary palate,” which includes the nostril sill, upper lip, alveolus, and hard pal-ate anterior to the incisive foramen, forms from fusion between the medial nasal and maxillary prominences during weeks 4 through 7 of gestation.20,24 Development of the hard palate pos-terior to the incisive foramen and the soft palate, which are col-lectively known as

1	nasal and maxillary prominences during weeks 4 through 7 of gestation.20,24 Development of the hard palate pos-terior to the incisive foramen and the soft palate, which are col-lectively known as the “secondary palate,” occurs during weeks Brunicardi_Ch45_p1967-p2026.indd 198301/03/19 6:27 PM 1984SPECIFIC CONSIDERATIONSPART IIFigure 45-24. Van der Woude syndrome.Table 45-3Classification of craniofacial anomalies211. Clefts2. Synostoses3. Atrophy–hypoplasia4. Hypertrophy–hyperplasia–neoplasia6 through 12 of gestation. The lateral palatine processes initially hang vertically on either side of the developing tongue. Around week 8, these palatal shelves rotate into a horizontal orientation, bringing their free edges into close proximity with the nasal septum. Midline fusion then commences, proceeding posteriorly from the incisive foramen (Fig. 45-25).23Normal and Cleft Anatomy There are several key defining characteristics of the lip that make its surgical repair so chal-lenging. On

1	proceeding posteriorly from the incisive foramen (Fig. 45-25).23Normal and Cleft Anatomy There are several key defining characteristics of the lip that make its surgical repair so chal-lenging. On the surface, the philtrum of the upper lip is com-prised of paired philtral columns and a central philtral dimple. The white roll passes along the vermilion-cutaneous junction, peaking at the base of the philtral columns and dipping centrally to form Cupid’s bow. Deep to the surface, the paired orbicularis oris muscles originate lateral to the oral commissures and encir-cle the mouth, decussating in the midline and sending off dermal insertions to the philtrum. This intrinsic muscle of the lip pro-vides oral competence and assists with speech production and facial expression. Continuity of the orbicularis oris muscle is disrupted in babies born with a cleft lip. Aberrant muscle inser-tion into the piriform aperture laterally and the anterior nasal spine medially contributes to the hallmark

1	orbicularis oris muscle is disrupted in babies born with a cleft lip. Aberrant muscle inser-tion into the piriform aperture laterally and the anterior nasal spine medially contributes to the hallmark appearance of cleft lip and nasal deformity (Fig. 45-26).20,25Clefts of the lip can be described as unilateral or bilateral and microform, incomplete, or complete. Microform cleft lip is the most minor variant and may manifest as subtly as a small notch in the vermilion. An incomplete cleft lip, by definition, requires an intact nasal sill. The term can otherwise be applied to a wide spectrum of anomaly, ranging from a partial cleft of the lip alone (Fig. 45-27A) to a near-complete cleft of the entire primary palate. A complete cleft lip involves all structures of the primary palate in their entirety, extending through the nasal sill and opening into the anterior nasal floor (Fig. 45-27B).20,26The normal palate functions primarily as a speech organ, but it is also intimately involved in

1	entirety, extending through the nasal sill and opening into the anterior nasal floor (Fig. 45-27B).20,26The normal palate functions primarily as a speech organ, but it is also intimately involved in feeding, swallowing, and breathing. The soft palate, or velum, together with lateral and posterior pharyngeal walls, can be conceptualized as a valve that regulates the passage of air through the nasopharynx. The paired levator veli palatini muscles descend from the cranial base and decussate in the midline to form a sling within the soft palate. This sling acts to elevate the velum against the posterior pharyngeal wall, effectively closing the velopharyngeal port. In patients with cleft palate, the levator muscles are unable to cross the midline. Instead, they run parallel to the cleft margin and insert aberrantly into the posterior edge of the hard palate (Fig. 45-28A,B). Air is allowed to leak through the nose dur-ing attempts to suck or speak. This inability to build negative or

1	margin and insert aberrantly into the posterior edge of the hard palate (Fig. 45-28A,B). Air is allowed to leak through the nose dur-ing attempts to suck or speak. This inability to build negative or positive intraoral pressure makes either task difficult, if not impossible. The tensor veli palatini muscles, which normally function to vent and drain the Eustachian tubes, are also dis-rupted in cleft anatomy. Eustachian tube dysfunction predis-poses patients to frequent bouts of otitis media, which can lead to permanent hearing loss if left untreated.20The most clinically useful system to describe cleft pal-ate morphology is the Veau classification. A Veau I cleft is midline and limited to the soft palate alone, whereas a Veau II cleft may extend further anteriorly to involve the midline of the posterior hard palate (the “secondary palate”). A Veau III cleft is a complete unilateral cleft of primary and secondary pal-ates, in which the cleft extends through the lip, the alveolus, the

1	of the posterior hard palate (the “secondary palate”). A Veau III cleft is a complete unilateral cleft of primary and secondary pal-ates, in which the cleft extends through the lip, the alveolus, the entire length of the nasal floor on the cleft side, and the midline of the soft palate. Veau IV clefts are bilateral complete clefts of the primary palate that converge at the incisive foramen and continue posteriorly through the entire secondary palate (Fig. 45-29A,B). Not included in the Veau classification is the submucous cleft palate, which occurs when there is clefting of the soft palate musculature beneath intact mucosa. Submucous cleft palate classically presents as the triad of a bifid uvula, a midline translucency called the “zona Pellucida” and a palpable notch of the posterior hard palate.21Presurgical Infant Orthopedics Current literature suggests aesthetic outcomes in patients with complete unilateral or bilateral clefts may be improved by reestablishing more nor-mal

1	hard palate.21Presurgical Infant Orthopedics Current literature suggests aesthetic outcomes in patients with complete unilateral or bilateral clefts may be improved by reestablishing more nor-mal skeletal, cartilaginous, and soft tissue relationships prior to definitive lip repair. Presurgical infant orthopedics (PSIO) can help to narrow wide clefts and align dental arches in prepara-tion for surgery. Some methods of PSIO, such as nasoalveolar molding (NAM), provide the added benefits of elongating the columella and improving nasal tip asymmetry.22 The most com-mon barrier to PSIO implementation is its imposition on fami-lies, who must be willing and able to keep frequent follow-up appointments for appliance adjustment. An excellent alternative to PSIO is a lip adhesion procedure, in which a complete cleft is surgically converted to an incomplete cleft. This preliminary stage of lip repair restores soft tissue continuity at the nasal sill, which helps to realign the underlying dental

1	a complete cleft is surgically converted to an incomplete cleft. This preliminary stage of lip repair restores soft tissue continuity at the nasal sill, which helps to realign the underlying dental arches and reap-proximate the soft tissues. In addition, the nasal deformity can be improved, both by repositioning of the cleft side alar base and placement of nasal conformers.23Cleft Lip Repair Although cleft lip surgery can be traced to antiq-uity, it was not until the first half of the 20th century that sur-geons began to realize the inadequacy of a straight-line repair. In 1955, Ralph Millard pioneered his “rotation-advancement” tech-nique, which was the first to address upper lip length deficiency while preserving intricate philtral anatomy (Fig. 45-29C).24 The back-cut is designed high on the medial lip element just beneath the columella, enabling a downward rotation and leveling of Cupid’s bow, while the lateral lip element is advanced into the Brunicardi_Ch45_p1967-p2026.indd

1	on the medial lip element just beneath the columella, enabling a downward rotation and leveling of Cupid’s bow, while the lateral lip element is advanced into the Brunicardi_Ch45_p1967-p2026.indd 198401/03/19 6:27 PM 1985PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-25. Facial prominences and their contributions to facial development. Cleft lip results from failure of fusion between maxillary and medial nasal (a component of frontonasal) prominences.ACDEBrotation defect. Although other techniques exist, most lip repairs performed today are minor modifications of Millard’s original rotation-advancement principle.20Bilateral cleft lip presents an even greater set of challenges to the reconstructive surgeon. With no overlying orbicularis oris muscle, an unrestrained premaxilla rotates anteriorly, com-pletely displacing the incisor-bearing portion of the alveolus from the maxillary dental arch. Orbicularis continuity must be restored over an often protuberant premaxilla. The

1	anteriorly, com-pletely displacing the incisor-bearing portion of the alveolus from the maxillary dental arch. Orbicularis continuity must be restored over an often protuberant premaxilla. The surgeon must carefully recreate the appearance of a symmetrical philtrum and median labial tubercle. Prototypical markings for bilateral cleft lip repair are demonstrated in Fig. 45-30A,B.20Any surgical approach to bilateral cleft lip repair would be incomplete without addressing the nasal stigmata, which include a short or absent columella, a poorly defined and underprojected nasal tip, and malpositioned lower lateral cartilages.25 Primary nasoplasty at the time of lip repair has become an increasingly common practice. Nasal skin and soft tissue are dissected free from the underlying cartilaginous framework, allowing for suture manipulation of lower lateral cartilages to improve tip symmetry, support, and projection.20Cleft Palate Repair The primary goal of palatoplasty is to enable normal

1	framework, allowing for suture manipulation of lower lateral cartilages to improve tip symmetry, support, and projection.20Cleft Palate Repair The primary goal of palatoplasty is to enable normal speech development. A successful palate repair is one that results in a robust, layered reconstruction of the cleft and restoration of functional velar anatomy. The two most com-mon techniques employed for soft palate repair are intravelar veloplasty (IVV) and Furlow double-opposing Z-plasty. Para-mount to each technique is the complete release of aberrant levator muscle insertions from the posterior edge of the hard palate. This maneuver untethers the velum anteriorly, enabling maximal levator muscle excursion in the superior and posterior directions postoperatively.21Brunicardi_Ch45_p1967-p2026.indd 198501/03/19 6:27 PM 1986SPECIFIC CONSIDERATIONSPART IIFigure 45-27. Variations in unilateral cleft lip morphology. Left unilateral incomplete cleft lip.Figure 45-26. Hallmarks of unilateral

1	198501/03/19 6:27 PM 1986SPECIFIC CONSIDERATIONSPART IIFigure 45-27. Variations in unilateral cleft lip morphology. Left unilateral incomplete cleft lip.Figure 45-26. Hallmarks of unilateral cleft lip deformity include depression of the nasal tip and flaring of the alar base on the cleft side, deviation of the caudal septum and columella toward the non-cleft side, and deficient lip height (short philtral column) on the cleft side with cephalad rotation of the cleft side of cupid’s bow.ABIntravelar veloplasty requires meticulous dissection of the levator muscles with retropositioning and reconstruction of the sling mechanism in the posterior aspect of the soft palate. A Furlow double-opposing Z-plasty involves cleverly designed mirror image Z-plasties on the oral and nasal sides of the soft palate where the central limb of each Z-plasty is the cleft. The posteriorly based flap of mucosa on each surface of the palate incorporates the underlying levator muscle. Transposition of these

1	soft palate where the central limb of each Z-plasty is the cleft. The posteriorly based flap of mucosa on each surface of the palate incorporates the underlying levator muscle. Transposition of these flaps across the cleft lengthens the palate and, in a man-ner similar to IVV, corrects levator malposition. Lateral relax-ing incisions can be utilized to relieve tension on the closure, if necessary (Fig. 45-31A–C).21,31 In experienced hands, both techniques have demonstrated excellent speech outcomes and low fistula rates. However, direct comparison between the two methods has been difficult due to ongoing evolution of the IVV technique and wide variability in the extent of dissection between performing surgeons.26Clefts involving the hard palate (Veau II–IV) often require additional maneuvers for reconstruction. Wide undermining of the nasal floor mucosa in the subperiosteal plane facilitates the nasal-side repair. As palatal mucoperiosteum is thicker and less pliable, the oral-side

1	for reconstruction. Wide undermining of the nasal floor mucosa in the subperiosteal plane facilitates the nasal-side repair. As palatal mucoperiosteum is thicker and less pliable, the oral-side closure generally requires the use of relax-ing incisions along the lingual side of the alveolar ridge. Addi-tional medialization of the palatal soft tissue can be obtained by increasing isolation of the greater palatine neurovascular pedicle, which emerges from its foramen near the posterolateral aspect of the hard palate. Narrow Veau II clefts may be closed on the oral side by medialization of bilateral bipedicled muco-periosteal flaps (von Langenbeck palatoplasty), while wider clefts may require detachment of one or both flaps anteriorly for additional medialization (Bardach two-flap palatoplasty). Lateral relaxing incisions are left open, and typically heal by secondary intention within two weeks (Fig. 45-32).21,27Complications of palate repair include oronasal fistula, velopharyngeal

1	Lateral relaxing incisions are left open, and typically heal by secondary intention within two weeks (Fig. 45-32).21,27Complications of palate repair include oronasal fistula, velopharyngeal dysfunction, obstructive sleep apnea, and mid-face growth deficiency. Reported fistula rates vary widely in the literature, but increased incidence has been correlated with less experienced surgeons, wider clefts, and bilateral clefts.21,22 Few oronasal fistulae are amenable to closure with simple local tissue rearrangement. More commonly, a complete reelevation of palatal mucosa is required in order to obtain a tension-free layered closure. In the case of large or recurrent fistulae, there may be insufficient tissue available locally, and recruitment of regional healthy tissue from the buccal mucosa or tongue may be necessary.32Velopharyngeal dysfunction (VPD) is caused by incom-plete closure of the velopharyngeal port, which results in air leaking through the nose during speech. Approximately

1	or tongue may be necessary.32Velopharyngeal dysfunction (VPD) is caused by incom-plete closure of the velopharyngeal port, which results in air leaking through the nose during speech. Approximately 20% of patients develop VPD after primary palatoplasty. After insuring complete release and proper orientation of levator muscles, a posterior pharyngeal flap or a sphincter pharyngoplasty may be required to decrease the size of the velopharyngeal gap, allowing Brunicardi_Ch45_p1967-p2026.indd 198601/03/19 6:27 PM 1987PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-28. Left unilateral complete cleft lip.AponeurosisAHamulusTensor muscleLevator muscleUvulus muscleAponeurosisBHamulusTensor muscleAccessory muscleLevator muscleFigure 45-29. A. Normal anatomy: the levator veli palatini muscle forms a muscular sling in the posterior aspect of the soft palate. B. Cleft anatomy: the levator veli palatini muscles turn anteriorly, run along the cleft margin, and insert aberrantly into the

1	forms a muscular sling in the posterior aspect of the soft palate. B. Cleft anatomy: the levator veli palatini muscles turn anteriorly, run along the cleft margin, and insert aberrantly into the posterior edge of the hard palate. C. Rotation-advancement markings and repair for a unilateral complete cleft lip.ABCnasal air escape during speech.21 These operations carry a risk of obstructive sleep apnea, so preoperative polysomnography is indicated to rule out significant sleep-disordered breathing at baseline.Timeline for Repair The longstanding debate regarding opti-mal timing for lip and palate repair is ongoing. Central to this controversy is the impact of early surgical intervention on speech outcomes and midface growth. Current evidence sug-gests earlier palate repair is better for speech but more detri-mental to midface growth.21 Cleft care algorithms represent a compromise. Most experts perform lip repair between 3 and 6 months of age.33,34 Palate repair should be completed

1	speech but more detri-mental to midface growth.21 Cleft care algorithms represent a compromise. Most experts perform lip repair between 3 and 6 months of age.33,34 Palate repair should be completed prior to the onset of speech development, usually around 10 to 12 months of age. The alveolar cleft is often repaired secondarily with a can-cellous bone graft from the iliac crest. This operation provides bony support for the permanent teeth that will erupt adjacent to the cleft, and it is usually performed around 7 to 9 years of age. Orthognathic surgery and secondary rhinoplasty, if necessary, are delayed until skeletal maturity. The treatment timeline used at Nationwide Children’s Hospital can be seen in Fig. 45-33.Brunicardi_Ch45_p1967-p2026.indd 198701/03/19 6:28 PM 1988SPECIFIC CONSIDERATIONSPART IIABFigure 45-30. A. Bilateral cleft lip repair diagram. B. Bilateral cleft lip repair.ABCFigure 45-31. Furlow double opposing Z-plasty. A. Oral side markings. B. Nasal side markings.

1	45-30. A. Bilateral cleft lip repair diagram. B. Bilateral cleft lip repair.ABCFigure 45-31. Furlow double opposing Z-plasty. A. Oral side markings. B. Nasal side markings. Note that the levator veli pala-tini muscle remains attached to the posteriorly based flap on each surface. C. Flap transposition and closure. The levator veli pala-tini muscle bundles, being attached to the posteriorly based flaps, are reoriented transversely and retrodisplaced as a result of flap transposition.Brunicardi_Ch45_p1967-p2026.indd 198801/03/19 6:28 PM 1989PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-34. The Tessier classification of craniofacial clefts. Numbered lines designate soft tissue manifestations (above) of the underlying skeletal clefts (below).Lip adhesion(1–2 months)Lip and primarynose repair(3–6 months)Orthognathicsurgery*(skeletal maturity)Definitiverhinoplasty*(after jaw surgery)Palate repair(10–12 months)Lip or noserevision*(> 6 years)VPD surgery*(4–7 years)Alveolar

1	primarynose repair(3–6 months)Orthognathicsurgery*(skeletal maturity)Definitiverhinoplasty*(after jaw surgery)Palate repair(10–12 months)Lip or noserevision*(> 6 years)VPD surgery*(4–7 years)Alveolar bonegrafting(7–11 years)Figure 45-32. Traditional von Langenbeck palatal repair with bilateral bipedicled mucoperiosteal flap.Figure 45-33. The treatment timeline used at Nationwide Children’s Hospital.The Importance of Team in Cleft Care Children born with CL/P require expertise of medical professionals from many different disciplines. In addition to experienced craniofacial surgeons, cleft teams typically consist of otolaryngologists, pediatricians, speech pathologists, feeding specialists, pediatric dentists, orthodontists, geneticists, psychologists, nurses, and social workers. Each member is an integral part of the team and absolutely essential for the delivery of comprehensive cleft care.21Atypical Craniofacial Clefts Beyond the familiar scope of clefts confined to the lip and

1	member is an integral part of the team and absolutely essential for the delivery of comprehensive cleft care.21Atypical Craniofacial Clefts Beyond the familiar scope of clefts confined to the lip and palate, there exist myriad forms of clefting that may affect the craniofacial skeleton. Sound epide-miologic studies of these atypical craniofacial clefts have been precluded by their extreme rarity, but rough estimates place them on the order of 100 times less common than CL/P. As a result, definitive causality has not been established. With the exception of some well-defined syndromes that include atypical craniofacial clefts, genetics does not appear to play a significant part in their pathogenesis. Some extrinsic factors that have been implicated include radiation, prenatal infections, early gesta-tional exposure to teratogenic drugs or chemicals, and amniotic bands. Metabolic derangements and vascular disturbances have also been hypothesized to play a role.27While CL/P can be

1	early gesta-tional exposure to teratogenic drugs or chemicals, and amniotic bands. Metabolic derangements and vascular disturbances have also been hypothesized to play a role.27While CL/P can be logically explained as an embryologic failure of fusion between facial processes, the location of the atypical craniofacial clefts is not well-accounted for by this theory. In the 1960s, Weston and Johnston used animal mod-els to demonstrate the vast contributions of neural crest cells to mesynchymal development of the face. They postulated that failure of these cells to penetrate into the developing face could lead to breakdown of the surrounding epithelia and result in atypical craniofacial clefts. The last 30 years has seen contin-ued refinement of this theory. Most recent evidence suggests that neural crest cells form developmental rests or ossification centers within the well-known facial processes. An abnormal number or impaired differentiation of these ossification centers may better

1	neural crest cells form developmental rests or ossification centers within the well-known facial processes. An abnormal number or impaired differentiation of these ossification centers may better explain the locations of clefts that seem to follow no known embryologic fusion plane.33In 1974, Paul Tessier published detailed anatomic obser-vations of a large series of children with atypical craniofacial clefts. He introduced a simple numbering system to classify these clefts based strictly on involved anatomy.28 Clefts were assigned numbers 0 to 14 as they radiate around the orbit. Num-bers 0 to 7 describe facial clefts, while 8 to 14 described cranial clefts. Fig. 45-34 illustrates the paths of soft tissue clefts (above) and their corresponding skeletal clefts (below).33,35A number 0 facial cleft and its number 14 cranial extension are midline clefts, which may be characterized by tissue defi-ciency or excess. Holoprosencephaly, a term used to describe a

1	(below).33,35A number 0 facial cleft and its number 14 cranial extension are midline clefts, which may be characterized by tissue defi-ciency or excess. Holoprosencephaly, a term used to describe a 10234568910111213141413121110987665432130334301122347Brunicardi_Ch45_p1967-p2026.indd 198901/03/19 6:28 PM 1990SPECIFIC CONSIDERATIONSPART IIfailed cleavage of the prosencephalon into two separate cere-bral hemispheres, presents as a midline tissue deficiency that causes variable degrees of hypotelorism and upper lip and nasal deformity. Mildly affected patients may have near-normal intel-ligence, while severely affected cases are incompatible with life. Representing the opposite end of the spectrum, patients with median cleft face dysmorphism typically present with a median clefts of the lip and/or premaxilla midline tissue excess, hypertelorism, bifid cranium, and a normal underlying CNS (Fig. 45-35A,B).33Tessier clefts 1, 2, and 3 originate at the cupids bow. All proceed cephalad

1	lip and/or premaxilla midline tissue excess, hypertelorism, bifid cranium, and a normal underlying CNS (Fig. 45-35A,B).33Tessier clefts 1, 2, and 3 originate at the cupids bow. All proceed cephalad through the piriform aperture and affect the nose. While number 1 and 2 clefts spare the orbit, number 3 clefts create continuity between the orbit, maxillary sinus, nasal and oral cavities. Clefts 4, 5, and 6 begin lateral to cupids bow, spare the nose, and pass cephalad to affect the orbit and lower eyelid. The number 7 cleft, otherwise known as craniofacial microsomia, extends transversely along a line from the oral com-missure to the auricular tragus. Underlying skeletal clefts can involve the mandible, maxilla, orbit, and cranium. Tessier clefts 8 through 10 continue to radiate laterally and superiorly around the orbit. Cranial extensions are numbered such that the sum of the facial cleft and its corresponding cranial extension is always 14. For example, the number 1 facial cleft

1	and superiorly around the orbit. Cranial extensions are numbered such that the sum of the facial cleft and its corresponding cranial extension is always 14. For example, the number 1 facial cleft continues as the number 13 cranial cleft, and the number 5 facial cleft continues as the number 9 cranial cleft.33,35 Clefts can be unilateral or bilateral and ABFigure 45-35. Tessier 0-14 clefts. A. Holoprosencephaly. Note the midline tissue deficiency, hypotelorism, and the rudimentary nose known as a “proboscis.” The degree of facial deformity in patients with holoprosencephaly typically reflects the degree to which the underlying CNS is affected. B. Median cleft face dysmorphism. Note the marked midline tissue excess and hypertelorism. Although this patient exhibits an obvious encephalocele, CNS function is usually normal.may occur in any combination. The constellation of bilateral Tes-sier clefts 6, 7, and 8 has been well-described within the context of Treacher Collins syndrome, in

1	CNS function is usually normal.may occur in any combination. The constellation of bilateral Tes-sier clefts 6, 7, and 8 has been well-described within the context of Treacher Collins syndrome, in which patients exhibit malar hypoplasia, lower eyelid colobomas, and downward-slanting palpebral fissures (Fig. 45-36A–C).33Treatment of atypical craniofacial clefts varies widely with each unique patient. Classical approaches to surgical man-agement involved excision of atrophic soft tissue along cleft margins with reconstruction by local tissue rearrangement, with or without underlying bone grafting. Unfortunately, this meth-odology gives little consideration to the aesthetic units of the face, and the resulting scars often cause postoperative deformi-ties of their own. Ortiz-Monasterio and Taylor proposed a new treatment philosophy based on the following tenants:1. Restoration of the craniofacial skeleton2. Reconstruction with skin and soft tissue with like color and texture3. Generous use

1	proposed a new treatment philosophy based on the following tenants:1. Restoration of the craniofacial skeleton2. Reconstruction with skin and soft tissue with like color and texture3. Generous use of tissue expanders4. Aesthetic unit and subunit reconstruction5. Scar location at limits of aesthetic subunits6. Symmetrical repositioning of key facial landmarksFig. 45-37 demonstrates the dramatic improvement in aes-thetic outcome that is attainable when abiding by this treatment philosophy.29Brunicardi_Ch45_p1967-p2026.indd 199001/03/19 6:28 PM 1991PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45ABCFigure 45-36. A child with Treacher Collins syndrome and the hallmark combination of Tessier clefts 6, 7, and 8. Note the downward-slanting palpebral fissures and profound malar hypoplasia due to complete absence of zygomas.Barring immediate danger to vital structures such as the eye, the timing of reconstruction can be determined on a case-by-case basis. Soft tissue clefts can be excised and

1	complete absence of zygomas.Barring immediate danger to vital structures such as the eye, the timing of reconstruction can be determined on a case-by-case basis. Soft tissue clefts can be excised and closed by classical measures within the first year of life. However, bony reconstruction should be delayed until at least 5 to 6 years of age to minimize iatrogenic impairment of facial growth. Serial tissue expansion of the cheek prior to this time may be necessary to excise unfavorable scars and reorient them along aesthetic subunit boundaries. Preoperative imaging, such as computed tomography (CT) or magnetic resonance imaging (MRI), is necessary to fully characterize the defects and plan the opera-tion. Additional preoperative workup should include anesthe-sia evaluation and labs, as these operations can be lengthy and accompanied by significant blood loss. Preparedness for blood transfusion is imperative.33,34Craniofacial clefts are typically approached through a combination of

1	these operations can be lengthy and accompanied by significant blood loss. Preparedness for blood transfusion is imperative.33,34Craniofacial clefts are typically approached through a combination of bicoronal and oral vestibular incisions. Various osteotomies have been described to reposition components of Brunicardi_Ch45_p1967-p2026.indd 199101/03/19 6:28 PM 1992SPECIFIC CONSIDERATIONSPART IIFigure 45-37. (left) Eight-year-old girl with significant deformity from local tissue rearrangement to reconstruct a right Tessier no. 4 cleft. (center) Schematic depicting current scars with a solid line and proper scars with a dotted line. (right) Same patient after serial tissue expan-sion and relocation of scars along borders of aesthetic units.the craniofacial skeleton, such as the orbits, maxilla, and man-dible. These may be used in conjunction with bone grafts from the calvarium, ribs or iliac crest, and fixation can be achieved with standard techniques using bioresorbable plates or

1	maxilla, and man-dible. These may be used in conjunction with bone grafts from the calvarium, ribs or iliac crest, and fixation can be achieved with standard techniques using bioresorbable plates or sutures.33Craniosynostosis. The term “craniosynostosis” refers to pre-mature fusion of one or more calvarial sutures. It occurs in up to 1 out of every 2000 live births, and single-suture, nonsyndromic patients account for 85% of cases. Of these, isolated sagittal cra-niosynostosis is the most common form, while lamdoidal is the least common. Normal suture maintenance is driven by underly-ing brain growth and a complex biochemical interplay between the suture and the underlying dura mater.30 Multiple genes have been implicated in the development of craniosynostosis, the most notable of which being FGFR and TWIST. Fifty percent of these present as de novo mutations, and most exhibit an autoso-mal dominant inheritance pattern. Environmental associations, such as maternal smoking, have been

1	being FGFR and TWIST. Fifty percent of these present as de novo mutations, and most exhibit an autoso-mal dominant inheritance pattern. Environmental associations, such as maternal smoking, have been postulated, but definitive causality has not been proven.31According to Virchow’s law, patients with craniosynosto-sis exhibit a predictable pattern of deformity that results from an arrest of cranial growth perpendicular to the prematurely fused suture, with a compensatory increase in growth parallel to the affected suture (Fig. 45-38). Isolated sagittal craniosynostosis, Patent suturesFused midline sutureFigure 45-38. (left) Patent sutures permit normal cranial growth in all directions. (right) Craniosynostosis results in restricted cranial growth across the synostotic suture with a compensatory increased growth parallel to the synostotic suture (Virchow’s law).for example, results in restricted cranial growth in the transverse direction and a compensatory increase in the

1	with a compensatory increased growth parallel to the synostotic suture (Virchow’s law).for example, results in restricted cranial growth in the transverse direction and a compensatory increase in the anterior-posterior diameter of the head with frontal and/or occipital bossing. This head shape is commonly referred to as “scaphocephaly.” Fig. 45-39 depicts various other isolated craniosynostoses and the patterns of deformity that ensue.36All patients with craniosynostosis should be screened for intracranial hypertension. It has been estimated that up to 17% of patients with single-suture involvement may develop elevated intracranial pressure (ICP). This risk approaches 50% in patients with multisuture craniosynostosis.36 Signs and symptoms of increased ICP may include headache, inconsolability, nausea, vomiting, lethargy, sleep apnea, developmental delay, bulging fontanelles, hydrocephalus, papilledema, or loss of vision.36,38 Facial dysmorphism and a strong family history should

1	nausea, vomiting, lethargy, sleep apnea, developmental delay, bulging fontanelles, hydrocephalus, papilledema, or loss of vision.36,38 Facial dysmorphism and a strong family history should raise suspicion for syndromic etiology, as seen in Apert, Crouzon, Pfeiffer, and Saethre-Chotzen syndromes, among others.Diagnosis of craniosynostosis begins with physical exam. A recent prospective multicenter study suggests 98% accu-racy of diagnosis based upon physical exam findings alone. Palpable ridges may be present on the cranium but are not pathognomonic for craniosynostosis. The much more reliable physical exam finding involves recognition of the distinct pat-terns of cranial growth that result from premature fusion of one or more sutures. Dysmorphic facies, suspicion for multisuture involvement, or any degree of uncertainty in the diagnosis can be clarified with adjunctive imaging. While skull plain films can provide useful information, 3D computed tomography has emerged as the new gold

1	or any degree of uncertainty in the diagnosis can be clarified with adjunctive imaging. While skull plain films can provide useful information, 3D computed tomography has emerged as the new gold standard imaging modality for diag-nosing craniosynostosis.37The goals of treatment for craniosynostosis are to achieve a more normalized head shape and to treat or prevent nega-tive impacts on development that may result from increased ICP.37 In general, two approaches exist: (a) strip craniectomy procedures and (b) remodeling procedures. Simply put, strip craniectomy procedures remove the synostotic suture in order to disinhibit cranial growth across the affected suture. Adjunc-tive techniques, such as cranial spring or distractor placement versus postoperative helmet therapy are frequently combined with strip craniectomies to improve aesthetic outcomes. Many surgeons who perform these procedures will do so as early as Brunicardi_Ch45_p1967-p2026.indd 199201/03/19 6:28 PM 1993PLASTIC

1	with strip craniectomies to improve aesthetic outcomes. Many surgeons who perform these procedures will do so as early as Brunicardi_Ch45_p1967-p2026.indd 199201/03/19 6:28 PM 1993PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45NormocephalyScaphocephalyTrigonocephaly AnteriorplagiocephalyBrachycephalyPosteriorplagiocephalyFigure 45-39. Patterns of single-suture cranio-synostosis. Scaphocephaly results from sagittal synostosis. Trigonocephaly results from metopic synostosis. Anterior plagiocephaly results from unilateral coronal synostosis. Brachycephaly results from bilateral coronal synostosis. Posterior plagiocephaly results from unilateral lambdoidal synostosis.6 to 12 weeks of life to take advantage of early rapid brain growth, which helps drive cranial expansion after release of the synostotic suture. In addition, younger patients have a better capacity to heal the resulting cranial defects due to the high osteogeneticity of the underlying dura, which decreases substan-tially

1	synostotic suture. In addition, younger patients have a better capacity to heal the resulting cranial defects due to the high osteogeneticity of the underlying dura, which decreases substan-tially with age.37 Remodeling procedures go further to normalize head shape by complete removal, rearrangement, and replace-ment of abnormal areas of the calvarium. Given the limited efficacy of the aforementioned strip craniectomy techniques in patients older than 6 months of age, cranial vault remodeling is generally accepted as the definitive treatment for craniosynos-tosis in this age group.36Advantages of strip craniectomy procedures include shorter operative times, less blood loss, and shorter hospital stays, while disadvantages include an inability to treat complex deformities from multisuture involvement, inability to treat areas of compensatory increased cranial growth, and the neces-sity for secondary hardware removal procedures. Remodeling procedures offer a more definitive correction of

1	inability to treat areas of compensatory increased cranial growth, and the neces-sity for secondary hardware removal procedures. Remodeling procedures offer a more definitive correction of head shape in a single surgical procedure at the cost of increased operative times, higher rate of blood transfusions, and increased length of hospital stays.37The complexity of patients with syndromic craniosynosto-ses, such as Crouzon or Apert syndrome, mandates multidisci-plinary care from an experienced team of subspecialists. These patients may present with urgent airway obstruction, danger-ously elevated ICP, and/or vision-threatening globe protrusion (Fig. 45-40A–C).23 Early surgical interventions, such as strip craniectomy or posterior cranial vault distraction, are designed to increase cranial volume and therefore decrease ICP. Although optimal timing of definitive reconstruction is debatable, results of cranial vault remodeling and midface advancement surgeries appear more stable and

1	volume and therefore decrease ICP. Although optimal timing of definitive reconstruction is debatable, results of cranial vault remodeling and midface advancement surgeries appear more stable and demonstrate less relapse when delayed.32 Hearing, speech, and feeding difficulties are common among patients with syndromic craniosynostoses. As always, the psy-chosocial implications of such profound facial differences make social workers and psychologists indispensable members of the team.23Atrophy and Hypoplasia. Two conditions that exemplify the atrophy and hypoplasia class of craniofacial anomalies are progressive hemifacial atrophy and Robin sequence. Progres-sive hemifacial atrophy, otherwise known as Parry-Romberg syndrome, is a rare, acquired, idiopathic atrophy of the skin, subcutaneous tissue, muscle, and occasionally bone affecting one side of the face (Fig. 45-41). With a typical onset during the first or second decade of life, this self-limiting condition progresses with an

1	tissue, muscle, and occasionally bone affecting one side of the face (Fig. 45-41). With a typical onset during the first or second decade of life, this self-limiting condition progresses with an indolent course for 2 to 10 years before sta-bilizing, or “burning out.” The pathogenesis of Parry-Romberg syndrome is not well understood. Autoimmune processes such as scleroderma, chronic neurotropic viral infections, trigeminal neuritis, intracerebral vascular malformations, and increased sympathetic nerve activity have all been postulated to play a role. After progression of atrophy ceases, the mainstay of treat-ment is volume and contour restoration with autologous fat grafting. More severe cases may require microvascular transfer of free tissue, such as the parascapular fasciocutaneous flap.33Robin sequence is defined as the triad of micrognathia, glossoptosis, and airway obstruction (Fig. 45-42).23 Cleft palate is present in up to 90% of affected patients, though it is not an obligatory

1	sequence is defined as the triad of micrognathia, glossoptosis, and airway obstruction (Fig. 45-42).23 Cleft palate is present in up to 90% of affected patients, though it is not an obligatory component of the diagnosis. The cause of this condi-tion is not known, but many believe mandibular hypoplasia to be the inciting event. According to this theory, micrognathia (small jaw) prevents forward migration of the tongue during gestational development. Glossoptosis results, where the tongue remains flipped dorsally into an obstructive position within the oropharyngeal airway. The first step in management is prone positioning, which utilizes gravity to bring the mandible and tongue base forward and alleviate the upper airway obstruction. More severely affected babies may require emergent endotra-cheal intubation at the time of delivery in order to secure the airway.34A diagnosable syndrome can be expected in over 50% of patients born with Robin sequence. Stickler syndrome (congeni-tal

1	intubation at the time of delivery in order to secure the airway.34A diagnosable syndrome can be expected in over 50% of patients born with Robin sequence. Stickler syndrome (congeni-tal ocular, orofacial, auditory, and articular anomalies), which is the leading cause of childhood blindness due to retinal detach-ment, is the most commonly associated syndrome. For this reason, ophthalmology and genetics evaluations are indicated in all patients with Robin sequence. Additionally, a thorough airway evaluation by an otolaryngologist is necessary to con-firm obstruction at the level of the tongue base and to rule out intrinsic airway anomalies or obstruction at lower levels of the respiratory tract.41Babies who are mildly affected can often be managed nonsurgically with prone positioning alone. Close monitoring is required because obstructive symptoms do not always fol-low a linear course to resolution. High caloric expenditure on Brunicardi_Ch45_p1967-p2026.indd 199301/03/19 6:28 PM

1	Close monitoring is required because obstructive symptoms do not always fol-low a linear course to resolution. High caloric expenditure on Brunicardi_Ch45_p1967-p2026.indd 199301/03/19 6:28 PM 1994SPECIFIC CONSIDERATIONSPART IIABCFigure 45-40. A and B. Frontal and lateral views of a young girl affected by Crouzon syndrome. Brachycephaly is appreciable on the lateral view, which results from bicoronal craniosynostosis. This patient also exhibits exorbitism and significant midface hyposplasia. C. A patient with Crouzon syndrome whose severe exorbitism has led to exposure keratitis.Brunicardi_Ch45_p1967-p2026.indd 199401/03/19 6:29 PM 1995PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-41. Child with progressive hemifacial atrophy, other-wise known as Parry-Romberg syndrome.Figure 45-42. An infant with Robin sequence. Marked microgna-thia and glossoptosis cause respiratory distress due to upper airway obstruction at the level of the tongue base. Note the presence of sternal

1	45-42. An infant with Robin sequence. Marked microgna-thia and glossoptosis cause respiratory distress due to upper airway obstruction at the level of the tongue base. Note the presence of sternal retraction during inspiration.increased work of breathing, in combination with reflux and feeding difficulties that are ubiquitous in this population, may manifest as poor weight gain over time. Persistent failure to thrive indicates a failure of conservative management.41Robin sequence patients with single-level obstruction at the tongue base who have failed conservative measures should be considered for surgical airway management.41 Tongue-lip adhesion (TLA) is designed to bring the tongue base forward and out of the airway by temporarily sewing the under-surface of the tongue to the mucosal surface of the lower lip. Adhesions are typically reversed within the first year of life as significant mandibular growth and improved muscle tone of the tongue result in a stable airway.35Another

1	surface of the lower lip. Adhesions are typically reversed within the first year of life as significant mandibular growth and improved muscle tone of the tongue result in a stable airway.35Another option to treat upper airway obstruction in patients with Robin sequence is mandibular distraction osteogenesis (MDO). In this procedure, osteotomies are made in bilateral mandibular rami, and distractor devices are applied that enable a gradual (1–2 mm/day) lengthening of the mandible. As the mandible is brought forward, the tongue base follows, result-ing in enlargement of the oropharyngeal airway. Specific risks include injury to tooth buds, inferior alveolar or marginal man-dibular nerves, and disruption of mandibular growth potential.41In Robin sequence, patients who fail or are not candidates for less invasive surgical maneuvers, tracheostomy remains the definitive option for airway control. Figure 45-43 represents an algorithm for management of children with Robin sequence proposed on

1	for less invasive surgical maneuvers, tracheostomy remains the definitive option for airway control. Figure 45-43 represents an algorithm for management of children with Robin sequence proposed on the basis that TLA is less invasive and does not preclude subsequent MDO in the event of failure.42 However, 4one option has not been proven to be significantly better than the other, and many surgeons prefer MDO as a first-line intervention.Hypertrophy, Hyperplasia, and Neoplasia. Numerous hypertrophic, hyperplastic, or neoplastic processes can affect the craniofacial region. The presence of certain vascular anomalies in the face can result in hypertrophy of surrounding bone or soft tissue.19 Patients with neurofibromatosis-1 may similarly present with hemifacial hypertrophy related to the presence of an underlying plexiform neurofibroma.36 Fibrous dysplasia is a focal error in osteoblast differentiation that leads to replacement of normal bone with a disorganized mass of bony trabeculae

1	of an underlying plexiform neurofibroma.36 Fibrous dysplasia is a focal error in osteoblast differentiation that leads to replacement of normal bone with a disorganized mass of bony trabeculae and fibrous tissue. Seventy percent of lesions are monostotic, and MandibulardistractionosteogenesisLaryngotrachealanomaly?Treat anomaly +/– tracheostomyPronepositioningObservationTongue-lip adhesionObservationFigure 45-43. Algorithm for management of children with Robin sequence.Brunicardi_Ch45_p1967-p2026.indd 199501/03/19 6:29 PM 1996SPECIFIC CONSIDERATIONSPART IIthe remaining 30% are polyostotic. In the craniofacial region, fibrous dysplasia typically presents in childhood with pain and progressive asymmetry. Patients with McCune-Albright syn-drome have polyostotic fibrous dysplasia, café au lait spots, and hyperfunctioning endocrinopathies, which classically manifest as precocious puberty. Lesions have a distinct “ground glass” appearance on CT scan. Small, monostotic fibrous dysplasia

1	lait spots, and hyperfunctioning endocrinopathies, which classically manifest as precocious puberty. Lesions have a distinct “ground glass” appearance on CT scan. Small, monostotic fibrous dysplasia lesions can occasionally be resected completely and recon-structed with bone grafts. More commonly, surgical debulking and contouring is the treatment of choice.37Vascular Anomalies. Vascular anomalies affect approxi-mately 5.5% of the population. They can be broadly categorized as either tumors or malformations.38 Vascular tumors are char-acterized histologically by endothelial cell proliferation, with or without luminal structure. In contrast, vascular malformations are collections of abnormally developed vessels without signifi-cant endothelial cell turnover.39Hemangiomas Hemangiomas are the most common vascular tumor in children, presenting in up to 20% of premature infants. Females are four times as likely to be affected as males, and darker-skinned individuals are rarely affected.

1	the most common vascular tumor in children, presenting in up to 20% of premature infants. Females are four times as likely to be affected as males, and darker-skinned individuals are rarely affected. These benign tumors are believed to be collections of primitive blood vessels formed from angioblasts. Hemangiomas can occur anywhere throughout the body, with the liver being the most common extracutaneous site.46The natural history of hemangiomas is highly predict-able depending on the timing of presentation and early clinical course. Infantile hemangiomas appear shortly after birth, usu-ally between 2 weeks and 2 months of life. Cutaneous infantile hemangiomas may initially resemble a red scratch or bruise, while subcutaneous or visceral lesions go unnoticed. Rapid growth ensues over the next 9 to 12 months (“the proliferative phase”). During this time, cutaneous lesions become bright red and tense, while subcutaneous lesions may present as deep soft tissue masses with a

1	over the next 9 to 12 months (“the proliferative phase”). During this time, cutaneous lesions become bright red and tense, while subcutaneous lesions may present as deep soft tissue masses with a bluish/purplish hue. After plateau of the proliferative phase, infantile hemangiomas reliably undergo a slow regression (“involution”), which is usually complete by 4 years of age. History alone can help differentiate a congenital hemangioma, which is fully formed at birth, from an infantile one. Congenital hemangiomas may exhibit rapidly involuting (RICH), noninvoluting (NICH), or partially involuting (PICH) clinical courses. History and physical is often sufficient to diagnose a hemangioma. Doppler ultrasound has become the imaging modality of choice, while MRI is typically reserved to confirm the diagnosis in cases of uncertainty.40Most hemangiomas can be observed and allowed to invo-lute spontaneously. High-risk lesions that may require early intervention include ulcerated and bleeding

1	the diagnosis in cases of uncertainty.40Most hemangiomas can be observed and allowed to invo-lute spontaneously. High-risk lesions that may require early intervention include ulcerated and bleeding hemangiomas; periocular hemangiomas, which can occlude the visual axis and lead to blindness; hemangiomas in the beard distribution, which place the patient at risk for upper airway obstruction (Fig. 45-44); and posterior midline lumbosacral hemangiomas, which may indicate underlying spinal dysraphism and cause cord compression. Patients with three or more hemangiomas should be screened by ultrasound for involvement of abdomi-nal viscera, as large hepatic lesions may lead to high-output heart failure. Large segmental hemangiomas in the cranial nerve V distribution (Fig. 45-45) should raise suspicion for PHACES association (Posterior fossa malformations, Heman-giomas, Arterial anomalies, Cardiac defects, Eye anomalies, Sternal defects).46 The LUMBAR association (Lower body Figure

1	raise suspicion for PHACES association (Posterior fossa malformations, Heman-giomas, Arterial anomalies, Cardiac defects, Eye anomalies, Sternal defects).46 The LUMBAR association (Lower body Figure 45-44. Hemangiomas in the beard distribution.hemangiomas, Urogenital anomalies, Myelopathy, Bony defor-mities, Anorectal/Arterial malformations, Renal anomalies) should be considered in patients with large infantile hemangio-mas of the lumbosacral region or lower extremities.41Oral propranolol therapy has emerged as the first-line treatment for complicated or high-risk infantile hemangio-mas. When administered during the proliferative phase, this nonselective beta adrenergic receptor blocker causes rapid invo-lution of the hemangioma. Several randomized, controlled trials have demonstrated oral propranolol to cause a greater decrease in lesion size compared to placebo and steroid therapy.42 In addition, many clinicians believe the side effect profile of pro-pranolol (hypoglycemia, sleep

1	propranolol to cause a greater decrease in lesion size compared to placebo and steroid therapy.42 In addition, many clinicians believe the side effect profile of pro-pranolol (hypoglycemia, sleep disturbances, hypotension, bra-dycardia, bronchospasm) to be more favorable than that of systemic steroids.43While hemangioma involution may result in no visible sequelae, up to 50% of patients are left with a residual fibrofatty mass with atrophic, hypopigmented and/or telangiectatic over-lying skin (Fig. 45-46A,B). If the residual deformity is troubling to the patient, surgical excision may be indicated.46Vascular Malformations Vascular malformations are collec-tions of abnormally formed vessels that demonstrate minimal endothelial cell turnover. They are present at birth and grow slowly in proportion with the patient. Vascular malformations are classified on the basis of anatomic origin of the abnormal vessels: capillary malformations (CM), venous malformations (VM), lymphatic

1	in proportion with the patient. Vascular malformations are classified on the basis of anatomic origin of the abnormal vessels: capillary malformations (CM), venous malformations (VM), lymphatic malformations (LM), and arteriovenous mal-formations (AVM). These classes can be further categorized into “slow-flow” or “fast-flow” lesions (Table 45-4).46Capillary malformations, formerly known as “port wine stains,” present at birth as flat, pink patches of skin. They typi-cally darken with age and may develop a thickened or “cob-blestoned” appearance. CMs may be found anywhere on the body, and overgrowth of underlying soft tissue or bone can occur. History and physical is sufficient to diagnose isolated CMs, but syndromic associations do exist that would warrant 5Brunicardi_Ch45_p1967-p2026.indd 199601/03/19 6:29 PM 1997PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-45. Large segmental hemangiomas in the cranial nerve V distribution.Figure 45-46. Twenty-year-old female with a

1	199601/03/19 6:29 PM 1997PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-45. Large segmental hemangiomas in the cranial nerve V distribution.Figure 45-46. Twenty-year-old female with a capillary malformations of the right cheek. A. Before and (B) after pulsed-dye laser treatment.ABTable 45-4Classification of vascular malformationsSLOW FLOWFAST FLOWCapillary malformationsVenous malformationsLymphatic malformationsArteriovenous malformationsfurther work-up.46 Sturge-Weber syndrome often presents with CMs in the V1/V2 nerve distributions of the face and may be accompanied by vascular malformations of the underlying lep-tomeninges or globe. Patients are at high risk for seizure, stroke, and glaucoma, for which pharmacologic prophylaxis may be indicated.44 The mainstay of treatment of CMs is pulsed-dye laser therapy (Fig. 45-47A, pre procedure; Fig. 45-47B post pro-cedure). Other surgical interventions, if necessary, are aimed at addressing soft tissue or bony overgrowth.46Venous

1	CMs is pulsed-dye laser therapy (Fig. 45-47A, pre procedure; Fig. 45-47B post pro-cedure). Other surgical interventions, if necessary, are aimed at addressing soft tissue or bony overgrowth.46Venous malformations are lobulated collections of dilated veins that typically involve skin, mucosa, or subcutaneous tis-sue, although 50% demonstrate deeper involvement. Lesions may or may not be noted at the time of birth. VMs generally grow in proportion to the patient but may undergo accelerated growth during puberty or pregnancy. Swelling of the mass may occur with dependent positioning or Valsalva maneuvers, such as crying. On exam, superficial VMs are soft, compressible masses with a bluish hue. Firm, tender nodules may be present, which represent calcifications known as phleboliths. Deeper, intramuscular VMs may present with pain or increased extrem-ity circumference, while lesions of the GI tract may simply pres-ent with bleeding. MRI with contrast is the imaging modality of choice,

1	intramuscular VMs may present with pain or increased extrem-ity circumference, while lesions of the GI tract may simply pres-ent with bleeding. MRI with contrast is the imaging modality of choice, although ultrasound can be used in infants and young children to avoid sedation. Observation is indicated for asymp-tomatic lesions. Compression of involved extremities helps alleviate pain and swelling and prevent thrombosis and phlebo-lith formation. Due to the high risk of recurrence after surgi-cal excision, the first line of treatment for symptomatic VMs is sclerotherapy. Surgery is reserved for small, well-localized lesions amenable to complete resection; extremity lesions near major peripheral nerves; or residual deformities after sclero-therapy (Fig. 45-48A, before laser; Fig. 45-48B, after laser; and Fig. 45-48C, after limited resection).46Brunicardi_Ch45_p1967-p2026.indd 199701/03/19 6:29 PM 1998SPECIFIC CONSIDERATIONSPART IIABABCFigure 45-47. A. A 3-year-old patient with an

1	laser; and Fig. 45-48C, after limited resection).46Brunicardi_Ch45_p1967-p2026.indd 199701/03/19 6:29 PM 1998SPECIFIC CONSIDERATIONSPART IIABABCFigure 45-47. A. A 3-year-old patient with an involuting hem-angioma of the right cheek. B. The same patient at 8 years of age showing minimal sequelae after completion of involution.Figure 45-48. A 5-year-old boy with venous malformation of the lower lip. A. Initial presentation. B. After three sclerotherapy treat-ments. C. Six weeks after surgical debulking of residual fibrotic tissue.Brunicardi_Ch45_p1967-p2026.indd 199801/03/19 6:29 PM 1999PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-49. A. Lymphatic malformation of the neck. B. After sclerotherapy with significant skin excess. C. Seven months after resection of excess skin.Lymphatic malformations, previously referred to as “cys-tic hygromas,” are collections of abnormal lymph channels that may cross multiple tissue planes and cause swelling, pain, bleeding, or bony

1	malformations, previously referred to as “cys-tic hygromas,” are collections of abnormal lymph channels that may cross multiple tissue planes and cause swelling, pain, bleeding, or bony overgrowth. LMs are classified as macrocys-tic, microcystic or combined. Large, macrocystic lesions can alter form and impair function locally through mass effect. Cuta-neous components of LMs present as vesicles that may bleed or become infected. While superficial lesions can be diagnosed by history and physical exam alone, deeper lesions require MRI ABCto confirm the diagnosis and assess the extent of the disease. Asymptomatic LMs can be observed. Sclerotherapy is the treat-ment of choice for all macrocysts. Symptomatic microcystic LMs have been treated with oral sirolimus, and draining cutane-ous vesicles have been successfully ablated with CO2 laser ther-apy. Recurrence after surgery is common; therefore, excision is reserved for severely symptomatic lesions no longer amenable to sclerotherapy or

1	have been successfully ablated with CO2 laser ther-apy. Recurrence after surgery is common; therefore, excision is reserved for severely symptomatic lesions no longer amenable to sclerotherapy or small, well-localized lesions where excision can be curative (Fig. 45-49A–C).46Brunicardi_Ch45_p1967-p2026.indd 199901/03/19 6:30 PM 2000SPECIFIC CONSIDERATIONSPART IIArteriovenous malformations are abnormal vascular con-nections between arteries and veins without intervening capil-lary beds. AVMs involving the skin appear pink and are warm to the touch. A palpable pulse or thrill may be present from the fast-flow shunting of blood from arterial to venous circu-lation. Lack of local capillaries can cause a painful, ischemic ulceration of the skin. Patients with large AVMs are at risk for development of congestive heart failure. Doppler ultrasound is the imaging modality of choice, but MRI is often obtained to provide additional information on the extent of the lesion. Observation is

1	development of congestive heart failure. Doppler ultrasound is the imaging modality of choice, but MRI is often obtained to provide additional information on the extent of the lesion. Observation is appropriate for asymptomatic AVMs. For symp-tomatic AVMs, embolization is frequently employed 24 to 72 hours prior to excision to minimize operative blood loss. Excision or embolization alone is rarely curative and highly likely to recur. Indications for surgery include small, well-localized AVMs; focal deformities that result from an AVM; or symptomatic AVMs not amenable to embolization.46When multiple types of vascular malformations cohabi-tate, they are collectively referred to as combined malforma-tions. Patients with Klippel-Trenaunay syndrome demonstrate a combined capillary, venous, and lymphatic malformation of an extremity resulting in bony and/or soft tissue overgrowth (Fig. 45-50).45Figure 45-50. A patient with Klippel-Trenaunay syndrome involv-ing the right lower extremity.

1	lymphatic malformation of an extremity resulting in bony and/or soft tissue overgrowth (Fig. 45-50).45Figure 45-50. A patient with Klippel-Trenaunay syndrome involv-ing the right lower extremity. The combined capillary, venous, and lymphatic malformations result in generalized overgrowth of the extremity.Table 45-5Classification of CMN’sPROJECTED ADULT DIAMETERCMN CLASSIFICATION<1.5 cmSmall≥1.5 cm and <11 cmMedium≥11 cm and ≤20 cmLarge>20 cmGiantCongenital Melanocytic Nevi. Congenital melanocytic nevi (CMN) are hyperpigmented lesions present at birth that result from ectopic rests of melanocytes within the skin. They can be distinguished histologically from acquired nevi by their exten-sion into the deep dermis, subcutaneous tissue, or muscle.46 Depending on their size and location, CMNs may cause severe disfigurement and accompanying psychologic distress. Classi-fication is based on projected diameter of the largest dimension on the fully-grown adult (Table 45-5)47. While CMNs are

1	may cause severe disfigurement and accompanying psychologic distress. Classi-fication is based on projected diameter of the largest dimension on the fully-grown adult (Table 45-5)47. While CMNs are gener-ally common (1% incidence), only 1 in 20,000 children are born with a giant lesion. At birth, CMNs often appear flat, brown and hairless. They grow in proportion with the patient and may develop color variegation, verrucous thickening, hypertrichosis, erosions, or ulcerations over time. CMNs carry an estimated 0.7% to 2.9% lifetime risk of melanoma, with the majority of cases presenting before puberty. Patients with giant CMNs, multiple satellite lesions, or trunk lesions appear to be at higher risk for malignancy. Melanomas can develop within the CMN itself, but they may also present as primary cancers at distant, extra-cutaneous sites, such as the GI tract or the central nervous system. Patients with CMNs require regular skin surveillance by a dermatologist. A biopsy is indicated

1	primary cancers at distant, extra-cutaneous sites, such as the GI tract or the central nervous system. Patients with CMNs require regular skin surveillance by a dermatologist. A biopsy is indicated for concerning changes in color or shape, nodularity, or ulceration. If melanoma is diag-nosed, management should proceed in accordance with standard melanoma treatment guidelines.55CMNs with multiple (>20) satellite lesions or midline CMNs over the trunk or calvaria should raise suspicion for neu-rocutaneous melanosis, a condition resulting from melanoblast proliferation in the central nervous system (CNS). In addition to the risk of CNS melanoma, patients with neurocutaneous melanosis may suffer from developmental delay, seizures, intracranial hemorrhages, hydrocephalus, cranial nerve palsies, or tethered spinal cord. High-risk patients should be evaluated by MRI between 4 and 6 months of age. While asymptomatic patients may be followed with serial MRI, patients with symp-tomatic

1	palsies, or tethered spinal cord. High-risk patients should be evaluated by MRI between 4 and 6 months of age. While asymptomatic patients may be followed with serial MRI, patients with symp-tomatic neurocutaneous melanosis often succumb to their dis-ease within 2 to 3 years of diagnosis.54The goals in surgical management of CMN are (a) to decrease cancer risk, (b) to reduce symptoms, (c) to improve appearance, (d) to improve psychosocial health, and (e) to maintain function.54 It is important to note that the risk of mela-noma is not eliminated even with complete excision of a CMN. Indeed, a definitive cancer risk reduction from surgical excision of CMNs has yet to be proven. Management paradigms have therefore shifted from complete excision and reconstruction to maximal excision and reconstruction without compromis-ing function or aesthetic outcome.55 From serial excisions or skin grafting, to tissue expansion or free tissue transfer, plastic surgeons have drawn from the entire

1	reconstruction without compromis-ing function or aesthetic outcome.55 From serial excisions or skin grafting, to tissue expansion or free tissue transfer, plastic surgeons have drawn from the entire armamentarium in meet-ing the substantial reconstructive challenges posed by giant CMNs. Treatment plans must be grounded in principle: “tissue Brunicardi_Ch45_p1967-p2026.indd 200001/03/19 6:30 PM 2001PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45losses should be replaced in kind,” and “reconstruct by units.”48 Figure 45-51A–C shows an infant with a giant CMN of the pos-terior trunk and right flank preoperatively; at end of the first round of tissue expansion; and at the culmination of three rounds of tissue expansion, excision, and closure by local tissue rearrangement.49Figure 45-51. A. An infant with a giant CMN of the posterior trunk and right flank. B. Tissues expanders were placed under adjacent normal skin in preparation for first-stage excision. C. The same patient at 11 years

1	An infant with a giant CMN of the posterior trunk and right flank. B. Tissues expanders were placed under adjacent normal skin in preparation for first-stage excision. C. The same patient at 11 years of age after three rounds of tissue expansion and excision.ABCRECONSTRUCTIVE SURGERY IN ADULTSReconstructive surgery applies techniques that modify tissues in order to restore a normal function and appearance in a patient with congenital or acquired deformities. The most common causes of acquired deformities are traumatic injuries and cancer.Brunicardi_Ch45_p1967-p2026.indd 200101/03/19 6:30 PM 2002SPECIFIC CONSIDERATIONSPART IIWe will focus first on trauma. Although any anatomic region can be subjected to injuries that might require reconstruc-tive surgery, traumatic fractures, and soft tissue damage in the head and neck and extremities are most common. The manner in which these reconstructive steps are conducted is criti-cal. Reconstructive surgery involves the coordination of many

1	damage in the head and neck and extremities are most common. The manner in which these reconstructive steps are conducted is criti-cal. Reconstructive surgery involves the coordination of many specialties and must occur according to a particular time-line, involving complex system-based practice.Maxillofacial Injuries and FracturesManagement of maxillofacial injuries typically occurs in the context of multiple trauma. Concomitant injuries beyond the face are the rule rather than the exception. The first phase of care is activation of the advanced trauma life support proto-cols. The most common life-threatening considerations in the facial trauma patient are airway maintenance, control of bleed-ing, identification and treatment of aspiration, assessment for closed head injuries, and identification of other injuries. Once the patient’s condition has been stabilized and life-threatening injuries managed, attention is directed to diagnosis and manage-ment of craniofacial

1	and identification of other injuries. Once the patient’s condition has been stabilized and life-threatening injuries managed, attention is directed to diagnosis and manage-ment of craniofacial injuries.Physical examination of the face focuses first on assess-ment of soft tissue injuries as manifested by surface contusions and lacerations. Part of this process is intranasal and intraoral examination. Associated injuries to the underlying facial skel-eton are determined by observation, palpation, and digital bone examination through open lacerations. Signs of a facial frac-ture include contour abnormalities, irregularities of normally smooth contours such as the orbital rims or inferior border of the mandible, instability, tenderness, ecchymosis, facial asym-metry, or displacement of facial landmarks. Traditional plain radiographs have largely been replaced by high-resolution CT, which is widely available at emergency centers that typically receive these patients. Reformatting raw scans

1	landmarks. Traditional plain radiographs have largely been replaced by high-resolution CT, which is widely available at emergency centers that typically receive these patients. Reformatting raw scans into coronal, sag-ittal, and 3D views is a valuable method to elucidate and plan treatment for complex injuries.The facial skeleton can be divided into the upper third, middle third, and lower third. The upper third is comprised bounded inferiorly by the superior orbital rim and is formed by the frontal bone. The middle third is the most complex and is formed primarily by the maxilla, nasal bones, and zygoma. The lower third is inferior to the oral cavity and is formed by the mandible. The functional structure of the midface may be understood as a system of buttresses formed by the frontal, maxillary, zygomatic, and sphenoid bones. These buttresses are oriented vertically and horizontally and distribute forces applied to the bones in order to maintain their shape and position with-out

1	maxillary, zygomatic, and sphenoid bones. These buttresses are oriented vertically and horizontally and distribute forces applied to the bones in order to maintain their shape and position with-out fracturing. There are three paired vertical buttresses called the nasomaxillary, zygomaticomaxillary, and pterygomaxillary buttresses. The horizontal buttresses of the midface pass through the superior and inferior orbital rims and hard palate. A guiding principle of facial facture management is to restore the integrity of these buttresses.Mandible FracturesMandibular fractures are common injuries that may lead to permanent disability if not diagnosed and properly treated. The mandibular angle, ramus, coronoid process, and condyle are points of attachment for the muscles of mastication, including the masseter, temporalis, lateral pterygoid, and medial pterygoid muscles (Fig. 45-52). Fractures are frequently multiple. Altera-tions in dental occlusion usually accompany mandible fractures.

1	the masseter, temporalis, lateral pterygoid, and medial pterygoid muscles (Fig. 45-52). Fractures are frequently multiple. Altera-tions in dental occlusion usually accompany mandible fractures. Malocclusion is caused by forces exerted on the mandible of the 6CoronoidprocessRamusAngleBodySymphysisCondyleFigure 45-52. Mandibular anatomy.many muscles of mastication on the fracture segments. Den-tal occlusion is perhaps the most important basic relationship to understand about fracture of the midface and mandible. The Angle classification system describes the relationship of the maxillary teeth to the mandibular teeth. Class I is normal occlu-sion, with the mesial buccal cusp of the first maxillary molar fitting into the intercuspal groove of the mandibular first molar. Class II malocclusion is characterized by anterior (mesial) posi-tioning, and class III malocclusion is posterior (distal) posi-tioning of the maxillary teeth with respect to the mandibular teeth (Fig. 45-53). These

1	is characterized by anterior (mesial) posi-tioning, and class III malocclusion is posterior (distal) posi-tioning of the maxillary teeth with respect to the mandibular teeth (Fig. 45-53). These occlusal relationships guide clinical management.The goals of surgical treatment include restoration of den-tal occlusion, fracture reduction and stable fixation, and soft Figure 45-53. Angle classification. Class I: The mesial buccal cusp of the maxillary first molar fits into the intercuspal groove of the mandibular first molar. Class II: The mesial buccal cusp of the maxillary first molar is mesial to the intercuspal groove of the mandibular first molar. Class III: The mesial buccal cusp of the maxillary first molar is distal to the intercuspal groove of the man-dibular first molar.IIIIIIBrunicardi_Ch45_p1967-p2026.indd 200201/03/19 6:30 PM 2003PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45tissue repair. Nonsurgical treatment may be used in situations in which there is minimal

1	200201/03/19 6:30 PM 2003PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45tissue repair. Nonsurgical treatment may be used in situations in which there is minimal displacement, preservation of the pretraumatic occlusive relationship, normal range of motion, and no significant soft tissue injury. Operative repair involves first establishing and stabilizing dental occlusion and holding in place with maxillomandibular fixation to stabilize the relation-ships between the mandible and maxilla. The simplest method for this is to apply arch bars to the maxillary and mandibular teeth then use secure them together using interdental wires. Alternatives are sometimes indicated (e.g., screws placed into the bone of the maxilla and mandible that serve as posts for spanning the maxilla and mandible with wires), especially for patients with poor dentition. Once the dental relationships are established, then the fractures can then be reduced and fixed using wire or plates and screws that are specially

1	wires), especially for patients with poor dentition. Once the dental relationships are established, then the fractures can then be reduced and fixed using wire or plates and screws that are specially designed for this purpose. The fracture is surgically exposed using multiple incisions, depending on the location of the fracture and condi-tion of the soft tissues. The fracture is visualized and manually reduced. Fixation may be accomplished using traditional inter-fragment wires, but plating systems are generally superior. The mandibular plating approach follows two schools of thought: rigid fixation as espoused by the Association for Osteosynthe-sis/Association for the Study of Internal Fixation and less rigid but functionally stable fixation (Champy technique). Regardless of the approach, it is important to release maxillomandibular fixation and begin range of motion as soon as possible to pre-vent temporomandibular joint ankylosis. Fractures immediately inferior to the mandibular

1	it is important to release maxillomandibular fixation and begin range of motion as soon as possible to pre-vent temporomandibular joint ankylosis. Fractures immediately inferior to the mandibular condyles, called subcondylar frac-tures, are unique in that there is ordinarily minimal displace-ment because the fragments are less subject to displacement from muscle forces and there is little bone available across the ClosedOpenYesYesNoNoAnteriortable onlyAnterior andposteriortables ObservationAnterior ORIFAnterior ORIFAnterior ORIFCranialization of sinusObliteration of NF ductbone grafting orificefat/fascial grafting orificeflap coverage of cavityremoval of posterior tableburring of mucosa-----ExplorationEstablish DiagnosisPhysical examCT scanDepressed?CSF leak ordisplacedposterior wall?Figure 45-54. Algorithm for the treatment of frontal sinus fracture. CSF = cerebrospinal fluid; CT = computed tomography; NF = nasofrontal; ORIF = open reduction, internal fixation.fracture line to permit

1	45-54. Algorithm for the treatment of frontal sinus fracture. CSF = cerebrospinal fluid; CT = computed tomography; NF = nasofrontal; ORIF = open reduction, internal fixation.fracture line to permit fixation. These are most often treated with maxillomandibular fixation alone.Important considerations in postoperative management are release from maxillary-mandibular fixation and resumption of range of motion as soon as possible to minimize the risk of tem-poromandibular joint ankylosis. Complications to be avoided include infection, nonunion, malunion, malocclusion, facial nerve injury, mental nerve injury, and dental fractures.Frontal Sinus FracturesThe frontal sinus is located in the upper third of the face. It is actually a paired structure ordinarily fused in the midline imme-diately superior to the orbital rims. It has an anterior bony table that defines the contour of the forehead and a posterior table that separates the sinus cavity from the underlying dura of the intra-cranial

1	to the orbital rims. It has an anterior bony table that defines the contour of the forehead and a posterior table that separates the sinus cavity from the underlying dura of the intra-cranial frontal fossa. The anterior table is a relatively weak and subject to fracture when it sustains a direct forceful blow, mak-ing frontal sinus fractures relatively common in facial trauma. Each sinus drains through the medial floor into its frontonasal duct, which empties into the middle meatus within the nose.Treatment of a frontal sinus fracture depends on the frac-ture characteristics as shown in the algorithm (Fig. 45-54). The diagnosis is established by physical examination and confirmed by CT scan. Closed fractures that are not depressed and caus-ing a visible deformity may be observed. Depressed or open fractures must be explored. Fractures that involve only the anterior table are reduced and fixed using interosseous wires or miniature plates and screws. Fractures of the posterior table

1	or open fractures must be explored. Fractures that involve only the anterior table are reduced and fixed using interosseous wires or miniature plates and screws. Fractures of the posterior table without disruption of the dura evidenced by leaking cerebro-spinal fluid can be treated in similar fashion. When the dura is disrupted, excising the bone and mucosa or the posterior table Brunicardi_Ch45_p1967-p2026.indd 200301/03/19 6:30 PM 2004SPECIFIC CONSIDERATIONSPART IIand obliterating the nasofrontal duct with a local graft or flap converts with frontal sinus into the anterior frontal fossa of the cranial vault, “cranializing” it.Orbital FracturesTreatment of all orbital injuries begins with a careful examina-tion of the globe, which often is best completed by a specialist to assess visual acuity and ocular mobility and to rule out globe injury. Fractures may involve the orbital roof, the orbital floor, or the lateral or medial walls (Fig. 45-55). The most common fracture involves

1	acuity and ocular mobility and to rule out globe injury. Fractures may involve the orbital roof, the orbital floor, or the lateral or medial walls (Fig. 45-55). The most common fracture involves the floor because this is the weakest bone. This type of fracture is referred to as an orbital a “blow-out” frac-ture because the cause is usually direct impact to the globe that results in a sudden increase in intraorbital pressure with failure of the orbital floor. The typical history is either a direct blow Figure 45-55. Facial bone anatomy.FrontalTemporalSphenoidZygomaMaxillaSphenoidFrontalZygomaMaxillaTemporalABduring an altercation or a sports-related event with a small ball directly striking the orbit. Because the medial floor and inferior medial wall are made of the thinnest bone, fractures occur most frequently at these locations. These injuries may be treated with observation only if they are isolated and small without signs of displacement or limitation of mobility of the globe.

1	occur most frequently at these locations. These injuries may be treated with observation only if they are isolated and small without signs of displacement or limitation of mobility of the globe. However, surgical treatment is generally indicated for large fractures or ones associated with enophthalmos (retrusion of the globe), which suggests increased intraorbital volume and restriction of upward gaze on the injured side, with entrapment of inferior orbital tissues or double vision (diplopia) persisting greater than 2 weeks.28 There are a variety of options for surgical exposure of the orbital floor, including the transconjunctival, subciliary, and lower blepharoplasty incisions. All provide good access for accurate diagnosis and treatment, which involves reducing orbital contents and repairing the floor with either autologous bone or synthetic materials. Late complications include per-sistent diplopia, enophthalmos, or displacement of the lower eyelid ciliary margin inferiorly

1	repairing the floor with either autologous bone or synthetic materials. Late complications include per-sistent diplopia, enophthalmos, or displacement of the lower eyelid ciliary margin inferiorly (ectropion) or rolling inward (entropion). Entropion causes the eyelashes to brush constantly against the cornea and is very uncomfortable. Each of these sequelae has procedures for repair should they occur.Orbital floor fractures can be associated with fractures of the lateral or inferior orbital rim. These are typically a compo-nent of facial fractures that extend beyond the orbit involving the zygomatic and maxillary bones and are discussed in more detail in the next section.It is important to be aware of two adverse associated con-ditions seen at times in patients with orbital fractures. The first is superior orbital fissure syndrome. Cranial nerves III (oculo-motor nerve), IV (trochlear nerve), and VI (abducens nerve), and the first division of cranial nerve V (VI, trigeminal nerve)

1	The first is superior orbital fissure syndrome. Cranial nerves III (oculo-motor nerve), IV (trochlear nerve), and VI (abducens nerve), and the first division of cranial nerve V (VI, trigeminal nerve) pass into the orbit from the base of the skull and into the orbit through the superior orbital fissure. Direct fractures of the pos-terior orbit or localized swelling caused by a fracture nearby can cause compression of these nerves. Symptoms include eyelid ptosis, protrusion of the globe (proptosis), paralysis of the extra-ocular muscles, and anesthesia supraorbital and trochlear nerve distributions. The second condition to remember is orbital apex syndrome. This is the most severe circumstance in which supe-rior orbital fissure syndrome is combined with signs of optic nerve (cranial nerve II) compression manifested visual changes ranging up to complete blindness. This is a medical emergency that requires immediate treatment to prevent permanent loss of function.Zygomaticomaxillary

1	II) compression manifested visual changes ranging up to complete blindness. This is a medical emergency that requires immediate treatment to prevent permanent loss of function.Zygomaticomaxillary Complex FracturesThe zygoma defines the lateral contour of the middle third of the face and forms the lateral and inferior borders of the orbit. It articulates with the sphenoid bone in the lateral orbit, the maxilla medially and inferiorly, the frontal bone superiorly, and the temporal bone laterally. It forms the anterior portion of the zygomatic arch, articulating with the zygomatic projection of the temporal bone. The temporalis muscle, a major muscle of mastication, passes beneath the zygomatic arch and inserts on the coronoid process of the mandible.Fractures of the zygomatic bone may involve the zygo-matic arch alone or any of its other portions and bony relation-ships. Isolated arch fractures manifest as a flattened, wide facial appearance with edema and ecchymosis. Typically, they

1	the zygo-matic arch alone or any of its other portions and bony relation-ships. Isolated arch fractures manifest as a flattened, wide facial appearance with edema and ecchymosis. Typically, they are also associated with pain or limited mobility of the mandible. Nondisplaced fractures may be treated without surgery, but Brunicardi_Ch45_p1967-p2026.indd 200401/03/19 6:30 PM 2005PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45displaced or comminuted fractures should be reduced and stabi-lized. This can be accomplished using an indirect approach from above the hairline in the temporal scalp, the so-called “Gilles approach,” or directly through a coronal incision in severe fractures.A common fracture pattern is called the zygomaticomaxil-lary complex (ZMC) fracture. This involves the zygomatic arch, the inferior orbital rim, the zygomaticomaxillary buttress, the lateral orbital wall, and the zygomaticofrontal buttress. Muscle forces acting on the fracture segment tend to rotate it

1	arch, the inferior orbital rim, the zygomaticomaxillary buttress, the lateral orbital wall, and the zygomaticofrontal buttress. Muscle forces acting on the fracture segment tend to rotate it laterally and inferiorly, thereby expanding the orbital volume, limiting mandibular excursion, creating an inferior cant to the palpebral fissure, and flattening the malar eminence. ZMC fractures are almost always accompanied on physical examination by altered sensation in the infraorbital nerve distribution and a subconjunc-tival hematoma.Treatment of displaced ZMC fractures is surgical. Each fracture site is exposed through incisions strategically placed to gain access but minimize disfiguring facial scars afterwards. These include an incision in the upper eyelid, exposing the zygomaticofrontal buttress and lateral orbital wall; a subtarsal or transconjunctival incision in the lower eyelid, exposing the orbital floor and infraorbital rim; and a maxillary gingivobuc-cal sulcus incision, exposing

1	and lateral orbital wall; a subtarsal or transconjunctival incision in the lower eyelid, exposing the orbital floor and infraorbital rim; and a maxillary gingivobuc-cal sulcus incision, exposing the zygomaticomaxillary buttress. Severe fractures involving the arch require wide exposure through a coronal incision.Nasoorbitalethmoid and Panfacial FracturesNasoorbitalethmoid (NOE) fractures are defined anatomically by a combination of injuries that involve the medial orbits, the nasal bones, the nasal processes of the frontal bone, and the frontal processes of the maxilla. If improperly treated, these injuries cause severe disfigurement and functional deficits from nasal airway collapse, medial orbital disruption, displacement of medial canthus of the eyelids, and nasolacrimal apparatus dysfunction. Telecanthus is abnormally wide separation of the medical canthus of the eyelids and is produced by a splaying apart of the nasomaxillary buttresses to which the medial can-thal ligaments are

1	Telecanthus is abnormally wide separation of the medical canthus of the eyelids and is produced by a splaying apart of the nasomaxillary buttresses to which the medial can-thal ligaments are attached. NOE fractures require surgical man-agement with open reduction and internal fixation. At times, the thin bones are so comminuted that they are not salvageable and must be replaced or augmented using autologous bone grafts or synthetic materials. Each fragment is carefully identified, returned to a normal anatomic position, and fixed in place using plates and screws or interosseous wiring all bone fragments meticulously, potentially with primary bone grafting, to restore their normal configuration. The key to the successful repair of NOE fractures is to carefully reestablish the nasomaxillary buttress and to restore the normal points of attachment of the medial canthal ligaments.NOE fractures are typically caused by such extreme forces that they are frequently associated with intracranial

1	buttress and to restore the normal points of attachment of the medial canthal ligaments.NOE fractures are typically caused by such extreme forces that they are frequently associated with intracranial injuries and multiple other facial bone fractures in a presentation referred to as a panfacial fracture. These may involve any combination of the fractures described previously. The challenge of these injuries is to reestablish normal relationships of key anatomic landmarks. A combination of salvable bone fragments, autolo-gous bone grafting, and synthetic materials accomplishes this.Posttraumatic Extremity ReconstructionThe primary goal in posttraumatic extremity reconstruction is to maximize function. When structural integrity, motor function, and sensation can be reasonably preserved, then extremity salvage may be attempted. Otherwise, severe injuries require amputation best performed following reconstructive surgery principals that set the stage for maximizing function with

1	then extremity salvage may be attempted. Otherwise, severe injuries require amputation best performed following reconstructive surgery principals that set the stage for maximizing function with pros-thetics and minimizing chronic pain and risk of tissue break-down. Microvascular surgical techniques are an essential part of extremity trauma surgery, allowing replantation of amputated parts or transfer of vascularized bone and soft tissue when tis-sue in zone of injury cannot be salvaged. Soft tissue techniques combined with advances in bone fixation and regeneration with distraction have proven tremendous benefit for patients with severe limb-threatening extremity trauma. Current state-of-the-art techniques require multidisciplinary cooperation between orthopedic, vascular, and plastic surgeons as presented in the algorithm (Fig. 45-56). Reconstructive techniques include the use of vascularized bone, bone distraction techniques, external fixation, nerve grafts and transfers, composite

1	as presented in the algorithm (Fig. 45-56). Reconstructive techniques include the use of vascularized bone, bone distraction techniques, external fixation, nerve grafts and transfers, composite tissue flaps, and functioning muscle transfers tailored to the given defect. The future promises further advances with routine application of vascularized composite allografts, engineered tissue replace-ments, and computer animated prosthetics controlled intuitively by patients via sensors that are placed on the amputation stump and able to detect impulses transmitted through undamaged peripheral nerves remaining in the extremity.Common causes of high-energy lower extremity trauma include road traffic accidents, falls from a height, direct blows, sports injuries, and gunshots. As with maxillofacial trauma, the first phase of care is activation of the advanced trauma life support protocols. The most common life-threatening consider-ations are airway maintenance, control of bleeding, and

1	trauma, the first phase of care is activation of the advanced trauma life support protocols. The most common life-threatening consider-ations are airway maintenance, control of bleeding, and identi-fication of other injuries. Once the patient’s condition has been stabilized and life-threatening injuries managed, attention is directed to diagnosis and management of the extremity. Tetanus vaccine and antibiotics should be provided as soon as possible for open wounds.Systematic evaluation of the traumatized extremity helps to ensure no important findings are missed. Physical examina-tion to assess the neurovascular status, soft tissue condi-tion, and location of bone fractures forms the foundation of ordering imaging studies to provide details of bone and vas-cular injuries. Evidence of absent pulses is an indication to con-sider Doppler ultrasound examination followed by angiography to detail the exact nature of the injury. The blood supply must be immediately restored to devascularized

1	pulses is an indication to con-sider Doppler ultrasound examination followed by angiography to detail the exact nature of the injury. The blood supply must be immediately restored to devascularized extremities. Crush injuries might be associated with compartment syndrome, in which tissue pressure due to swelling in the constricted facial compartments exceeds capillary perfusion pressure and causes nerve and muscle ischemia. In the early stages of compartment syndrome, findings include pain on passive stretch of the com-partment’s musculature in a pale, pulseless extremity without evidence of direct vascular injury. Neurologic changes consist-ing of paresthesias followed by motor paralysis are late signs. Once recognized, decompressive fasciotomies must be per-formed as soon as possible to prevent permanent tissue loss. Compartment syndrome can be a late event after fracture reduc-tion and fixation (either internal or external), so the extremity must be reevaluated regularly in the

1	to prevent permanent tissue loss. Compartment syndrome can be a late event after fracture reduc-tion and fixation (either internal or external), so the extremity must be reevaluated regularly in the early postoperative period. This is especially true in situations where there has been a period of ischemia prior to successful revascularization.Several scoring systems for extremity trauma severity have been suggested to aid in treatment planning. Open fractures can be classified according to a system devised by Gustilo and 7Brunicardi_Ch45_p1967-p2026.indd 200501/03/19 6:30 PM 2006SPECIFIC CONSIDERATIONSPART IIReconstructableKnee functionalAdequate soft tissueDirty woundDirty woundClean woundFoot availableFoot not availableClean woundInadequate soft tissueKnee irreparableUnreconstructableTraumaticbelow kneeinjuryAmputationLimbreconstruction/replantationDelayedclosurePrimaryclosureFoot filetfree flapParascapularfree flapImmediatefree flapDelayedfree flapPrimaryreconstructionBelow

1	kneeinjuryAmputationLimbreconstruction/replantationDelayedclosurePrimaryclosureFoot filetfree flapParascapularfree flapImmediatefree flapDelayedfree flapPrimaryreconstructionBelow kneesalvageBelow kneesalvageAbove kneeamputationFigure 45-56. Algorithm of posttraumatic extremity reconstruction.colleagues. Grades I and II are open fractures with minimal soft tissue disruption. Grade III injuries most often require consider-ation of soft tissue reconstruction. Grade IIIA are open fractures with severe soft tissue injury but adequate soft tissues to repair. Grade IIIB involves a loss of soft tissue that will require some technique for tissue replacement. Grade IIIC involves a vascular injury requiring reconstruction. For the most severe injuries, the most important decision is whether to attempt extremity salvage or proceed with amputation. Patients with extensive fracture comminution, bone or soft tissue loss, wound contamination, and devascularization have a poor prognosis. Extremity

1	extremity salvage or proceed with amputation. Patients with extensive fracture comminution, bone or soft tissue loss, wound contamination, and devascularization have a poor prognosis. Extremity salvage requires multiple operations and a prolonged period of rehabili-tation and physical therapy. The loss of plantar sensation histori-cally favored below-knee amputation, but this is no longer an absolute recommendation. A final decision to attempt salvage must be made within the context of comorbidities, socioeco-nomic considerations, patient motivation, and overall rehabilita-tive potential.The first step in surgical management is complete debride-ment of all devitalized tissue. Early one-stage wound coverage and bony reconstruction is generally advocated and should be performed jointly by extremity trauma orthopedic and plastic surgical teams.50 It is acceptable for reconstruction to be deferred briefly if the adequacy of debridement is certain. Negative pres-sure wound therapy is

1	by extremity trauma orthopedic and plastic surgical teams.50 It is acceptable for reconstruction to be deferred briefly if the adequacy of debridement is certain. Negative pres-sure wound therapy is useful between debridement and defini-tive reconstruction to control the wound drainage and prevent bacterial contamination. When there is segmental bone loss, it is advisable to achieve soft tissue closure prior to performing osse-ous reconstruction. Preparation for later restoration of the bone requires steps to prevent the soft tissue from collapsing into the space where bone is needed. A common technique for this is to fill the space with antibiotic-impregnated beads or an antibiotic spacer at the time of soft tissue restoration until definitive bony reconstruction is possible. An external fixation may be needed, if there is segmental bone loss (Fig. 45-57A,B).The sequence for reconstruction is meticulous debride-ment of nonviable tissue, fracture reduction and stabilization, vascular

1	fixation may be needed, if there is segmental bone loss (Fig. 45-57A,B).The sequence for reconstruction is meticulous debride-ment of nonviable tissue, fracture reduction and stabilization, vascular repair if necessary, and finally restoration of the soft tissue coverage. A multidisciplinary team of specialists works together to perform these procedures in order to obtain the best outcomes. Orthopedic and plastic surgeons perform wound debridement. Orthopedic surgeons then reduce and stabilize the fractures. Vascular surgeons reconstruct damage major vessels. Finally, plastic and reconstructive surgeons perform soft tissue coverage. Ideally, each operating team completes their part of the procedure sequentially during the same anesthetic.Choices for soft tissue coverage of open fractures include split-thickness skin grafts, temporary skin substitutes fol-lowed later by skin grafting, local rotation flaps, or free tissue transfers. Selecting the most appropriate option depends on the

1	include split-thickness skin grafts, temporary skin substitutes fol-lowed later by skin grafting, local rotation flaps, or free tissue transfers. Selecting the most appropriate option depends on the quality of the local tissues and location of the soft tissue defect relative to the underlying fracture and fixation hard-ware. The guiding principle is to be certain that the source of tissue transferred into the defect is outside of the zone of injury. When flaps are selected, either fasciocutaneous or muscular flaps may be indicated depending on tissue avail-ability, wound bed contours, and surgeon preferences. Uneven wound surface contours are more reliably obliterated with a Brunicardi_Ch45_p1967-p2026.indd 200601/03/19 6:30 PM 2007PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-57A, B. An external fixation for segmental bone loss.Figure 45-58. A. Defect ulnar side of the forearm, with an external fixator. B. Propeller flap. C. Flap is inset. D. Six weeks post

1	45Figure 45-57A, B. An external fixation for segmental bone loss.Figure 45-58. A. Defect ulnar side of the forearm, with an external fixator. B. Propeller flap. C. Flap is inset. D. Six weeks post operation.ABpliable muscle flap. Fasciocutaneous flaps may provide more durable coverage in areas subject to abrasion or pressure from footwear, for example, on the foot or around the ankle. Some defects can be covered with flaps containing both skin and muscle if indicated. Ideal coverage for weight-bearing areas should be able to resist pressure and shear and provide sensa-tion. Split-thickness skin grafts are reasonable for coverage of exposed healthy muscle or soft tissue. Local flaps may be used to cover smaller defects as long as uninjured tissue is located nearby. These may be designed as traditional random or axial ABCDflaps, but the most advanced techniques are based on under-lying perforators that allow extremely versatile flap designs customized to the defect. These flaps are

1	as traditional random or axial ABCDflaps, but the most advanced techniques are based on under-lying perforators that allow extremely versatile flap designs customized to the defect. These flaps are designed with a per-forating vessel at the base near to the defect and a long axis extending an equal distance opposite. The flap is elevated and rotated into the defect in a motion reminiscent of an airplane propeller, which gives rise to the designation “propeller flap” for this kind of reconstruction (Fig. 45-58A, defect ulnar side of the forearm, with an external fixator; Fig. 45-58B, propel-ler flap; Fig. 45-58C, flap is inset; Fig. 45-58D, 6 weeks after Brunicardi_Ch45_p1967-p2026.indd 200701/03/19 6:31 PM 2008SPECIFIC CONSIDERATIONSPART IIthe operation). The advantages of this technique are that it does not impair muscle function and it can often complete a complex reconstruction without the need for microvascular surgery.When requirements exceed the potential for skin grafts or

1	are that it does not impair muscle function and it can often complete a complex reconstruction without the need for microvascular surgery.When requirements exceed the potential for skin grafts or local flaps, tissue must be transferred from distant sites. The reconstructive choices differ based on the anatomic location of the defect and the extent of damage. This is often the case for major injuries in the middle or lower third of the leg where bones are covered with thin soft tissue and less donor tissue is available. A traditional method is to obtain tissue by creating a pedicled flap from the opposite, uninjured extremity. Cross-leg flaps remain effective, but indications are limited to circum-stances where microsurgery is not possible or in young children who are less prone to risks associated with prolonged immobi-lization necessary for these flaps, such as joint stiffness or deep vein thrombosis. Free tissue transfer is the preferred alternative. The general principles of

1	associated with prolonged immobi-lization necessary for these flaps, such as joint stiffness or deep vein thrombosis. Free tissue transfer is the preferred alternative. The general principles of reconstructive microsurgery in lower extremity trauma are to select recipient vessels outside of the zone of injury, select donor tissue suitable for the defect with minimal risk of donor site morbidity, and ensure there is bone stability before reconstruction using either internal or external fixation. For example, a latissimus dorsi muscle flap provides a large amount of tissue for reconstruction, but loss of the latis-simus function can make it more difficult for the patient to use crutches for ambulation during rehabilitation. Muscle or fascio-cutaneous flaps each have a role in selected circumstances.51 Bone can also be added to help fracture repair.52 Free flaps can also be designed as “flow-through” flaps, which reconstruct missing segments of major vessels and provide soft tissue or

1	Bone can also be added to help fracture repair.52 Free flaps can also be designed as “flow-through” flaps, which reconstruct missing segments of major vessels and provide soft tissue or bone coverage.53After wound healing, proper physical and/or occupational therapy and rehabilitation is essential for the best long-term out-comes. This often requires many months of consistent retrain-ing and conditioning in order to return to the functional status enjoyed by the patient before injury. Properly fitted orthotic appliances and footwear provide essential protection against pressure-related complications and can improve function. Late complications such as osteomyelitis may appear, evidenced by signs of infection months or even years after reconstruction. Very often this is caused by inadequate debridement at the time of initial surgery.Tumor locationPrimaryreconstructive optionSecondaryreconstructive optionLower-extremity bone sarcomacomposite resectionDistal femur/proximal tibiaPedicled

1	debridement at the time of initial surgery.Tumor locationPrimaryreconstructive optionSecondaryreconstructive optionLower-extremity bone sarcomacomposite resectionDistal femur/proximal tibiaPedicled gastrocnemius ±soleusDistally-based pedicledALT; anterior bipedicledfasciocutaneous flap; pedicledsural artery flap; free flapMid/distal tibiaPrimary closurePedicled gastrocneumius± soleus; propeller,keystone flaps; free flapProximal/mid-femurPrimary closurePedicled ALT;Pedicled rectusabdominis; free flapWhen limb salvage either is not possible or is not in the best interest of the patient, amputation is indicated. Maxi-mizing limb length, providing durable soft tissue coverage, and managing peripheral nerves to avoid chronic pain help to ensure good functional recovery using extremity prosthet-ics. Ideally, local tissues are used; however, when they are unavailable or inadequate, the amputated part can be a use-ful source of skin grafts or tissues for microvascular free transfers to the

1	Ideally, local tissues are used; however, when they are unavailable or inadequate, the amputated part can be a use-ful source of skin grafts or tissues for microvascular free transfers to the stump, which preserves length and avoids a more proximal amputation. Transected nerves from ampu-tation procedures can be managed using a technique called targeted muscle reinnervation (TMR). TMR surgery takes the transected peripheral nerves resulting from the amputation procedure, and a nerve transfer is then performed to freshly deinnervated motor nerves within the residual limb or stump. By performing these nerve transfers, the sensory and mixed-motor sensory nerves typically transected during amputation are given fresh motor nerves to rapidly reinnervate, which can directly aid in bioprosthetic function and improve pain control. The improvement in pain is a result of reducing phantom limb pain and symptomatic neuroma formation. This technique has shown to be a major advance over traditional

1	function and improve pain control. The improvement in pain is a result of reducing phantom limb pain and symptomatic neuroma formation. This technique has shown to be a major advance over traditional traction neurec-tomy techniques, which often contribute to increased phan-tom and residual limb pain rates and a much higher chance of symptomatic neuroma formation compared to TMR.54Oncologic Reconstructive SurgeryOncology-related reconstructive surgery has broad applica-tions in specialty of plastic and reconstructive surgery. Solid tumors necessarily destroy normal tissues, and surgical treat-ment involves excising the tumor with a margin of uninvolved normal tissue, which adds to the extent of tissue loss. As is illustrated in the case of a lower extremity sarcoma, recon-structive strategies are meticulously designed as an algorithm for effective functional and cosmetic restoration (Fig. 45-59) . Chemotherapy and radiation have side effects and com-plications that can cause tissue

1	are meticulously designed as an algorithm for effective functional and cosmetic restoration (Fig. 45-59) . Chemotherapy and radiation have side effects and com-plications that can cause tissue loss, leading to functional and cosmetic deformities that can be improved with recon-structive surgery. The goal of comprehensive cancer treatment is to restore the patient to full health, which includes normal function and appearance.8Figure 45-59. Algorithm for effective functional and cosmetic restoration after resection of a lower extremity sarcoma.Brunicardi_Ch45_p1967-p2026.indd 200801/03/19 6:31 PM 2009PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Reconstructive surgery in the context of oncology has sev-eral distinctive aspects compared to the larger field of recon-structive surgery in general. The procedure must be highly reliable in order to avoid surgical complications that might interfere with adjuvant therapies.Breast ReconstructionBreast cancer is the most common malignancy

1	general. The procedure must be highly reliable in order to avoid surgical complications that might interfere with adjuvant therapies.Breast ReconstructionBreast cancer is the most common malignancy besides skin can-cer in women and the second leading cause of cancer-related death for women in the United States. Breast reconstruction is an important part of comprehensive cancer treatment. A number of studies have shown that breast reconstruction, both imme-diate and delayed, does not impede standard oncologic treat-ment, does not delay detection of recurrent cancer, and does not change the overall mortality associated with the disease.46-48Preoperative counseling of the breast cancer patient regarding reconstruction options should include discussion of the timing and technique of reconstruction. It is important to ensure that the patient has realistic expectations of outcome and an understanding of the number of procedures that might be necessary to perform in order to obtain the best

1	It is important to ensure that the patient has realistic expectations of outcome and an understanding of the number of procedures that might be necessary to perform in order to obtain the best outcome. The plastic surgeon and surgical oncologist must maintain close communication to achieve optimal results.Delayed breast reconstruction occurs any time after the mastectomy is performed, usually 3 to 6 months after the opera-tion, depending on the patient’s circumstances and reasons for not electing immediate reconstruction. Although good out-comes can be obtained, it is more difficult to achieve a result that is similar to the preoperative breast shape and size because of established scarring of the chest wall. Nevertheless, it is a good option for patients who are undecided or not candidates for immediate reconstruction because of advanced disease or comorbidities.Immediate reconstruction is defined as initiation of the breast reconstructive process at the time of the ablative

1	candidates for immediate reconstruction because of advanced disease or comorbidities.Immediate reconstruction is defined as initiation of the breast reconstructive process at the time of the ablative sur-gery. Patients are considered candidates for immediate recon-struction who are in general good health and have stage I or stage II disease determined primarily by the size and location of the tumor. There are selected exceptions, such as when an extensive resection requires chest wall coverage. Breast recon-struction might be performed in these cases, but it is really incidental to achieving chest wall coverage. Disadvantages of immediate reconstruction include the potential delay of adju-vant therapy in the event of postoperative complications. Also, if there is uncertainty regarding the need to adjuvant radiation therapy, decision-making regarding immediate reconstruction is a challenge. Breast reconstructions by all techniques are adversely affected by radiation therapy, and many

1	need to adjuvant radiation therapy, decision-making regarding immediate reconstruction is a challenge. Breast reconstructions by all techniques are adversely affected by radiation therapy, and many surgeons feel reconstruction should be delayed until at least 6 months after treatment.Once the patient chooses to have immediate reconstruction, she must select a reconstructive technique. In patients selected for breast conservation, oncoplastic tissue rearrangement can be performed to minimize adverse effects of lumpectomy on breast appearance. For patients electing total mastectomy there are essentially three options: (a) tissue expansion followed by breast implant placement, (b) combined tissue flaps with breast implants, and (c) autologous tissue flaps only. After examining the patient, the surgeon then should describe those methods for which the patient is a satisfactory candidate. The patient should then be encouraged to choose based on her goals and an under-standing of the

1	the surgeon then should describe those methods for which the patient is a satisfactory candidate. The patient should then be encouraged to choose based on her goals and an under-standing of the advantages and disadvantages of each technique.Oncoplastic Breast ReconstructionBreast conservation therapy (BCT) consists of excision of the breast tumor with a surrounding margin of normal tissue com-bined with postoperative whole-breast irradiation. Although the overall survival for properly selected patients is shown to be comparable to total mastectomy and reconstruction, the breast can often be distorted and unnatural appearing after treatment. The area of the lumpectomy may create a depression with con-tour deformity, and contraction of the lumpectomy space over time can distract the nipple out of alignment and create an asym-metry with the contralateral breast. This is especially true for women with small breasts in whom a high percentage of breast volume is removed with the

1	the nipple out of alignment and create an asym-metry with the contralateral breast. This is especially true for women with small breasts in whom a high percentage of breast volume is removed with the lumpectomy.Oncoplastic surgery refers to the set of techniques devel-oped to lessen breast deformity from a partial mastectomy. One of the most common methods of minimizing adverse effects on breast appearance of is to rearrange the skin, parenchyma, and nipple location of the breast at the time of tumor extirpation using surgical techniques developed for breast aesthetic surgery. This procedure involves elevating the skin from the underlying glandular tissue, mobilizing the nipple on a vascular pedicle, and preserving as much of the vascularized glandular tissue as possible. After lumpectomy, the tissue is rearranged to shift glandular tissue into the defect and redrape the skin and nipple onto the new breast mound. After healing and completion of radiotherapy, a contralateral

1	lumpectomy, the tissue is rearranged to shift glandular tissue into the defect and redrape the skin and nipple onto the new breast mound. After healing and completion of radiotherapy, a contralateral conventional mastopexy or breast reduction can be performed on the contralateral side to achieve symmetry.Implant-Based ReconstructionImmediate breast reconstruction based entirely on the use of implanted devices is initially the most expedient technique. Sometimes it is possible to place a full-size implant at the time of mastectomy when the breasts are small (volume <400 cc) and the patient is a young nonsmoker with good chest wall muscula-ture. In most patients, however, a period of tissue expansion is required. The tissue expander is inserted beneath the pectoralis major and serratus anterior muscles at the time of the mastec-tomy and partially inflated. Alternatively, the tissue expander can be placed only under the pectoralis major muscle or even completely on top of the chest wall

1	muscles at the time of the mastec-tomy and partially inflated. Alternatively, the tissue expander can be placed only under the pectoralis major muscle or even completely on top of the chest wall muscles then covered with acellular dermal matrix directly beneath the mastectomy skin flaps. Total muscle coverage is the traditional approach, but these alternatives may be suitable only for well-selected patients. Expansion usually requires 6 to 8 weeks to complete, and an implant exchange is performed typically 3 months later. The advantages of this technique are that it involves minimum additional surgery at the time of the mastectomy, has a recovery period essentially the same of that of the mastectomy alone, and creates no additional scarring. The disadvantages of this technique are the length of time necessary to complete the entire reconstruction (up to 1 year), the requirement for a minimum of two operative procedures, and a less predictable cosmetic result due to complete reliance

1	of time necessary to complete the entire reconstruction (up to 1 year), the requirement for a minimum of two operative procedures, and a less predictable cosmetic result due to complete reliance on devices. Also, the patient awak-ens from surgery without a full-size breast and during the time of expansion must accept a breast of abnormal size and shape. Although the final shape of the breast may be satisfactory, it may lack a natural consistency due to the superficial placement of the device, especially when saline-filled implants are used. Finally, breast implants may develop late complications such as capsular contracture, infection, or extrusion. This method is ideal for a slender, small-breasted woman with minimal ptosis Brunicardi_Ch45_p1967-p2026.indd 200901/03/19 6:31 PM 2010SPECIFIC CONSIDERATIONSPART IIwho wish to avoid additional scarring and time for convales-cence. It may also be suitable for women undergoing bilateral reconstruction because symmetry is more easily

1	CONSIDERATIONSPART IIwho wish to avoid additional scarring and time for convales-cence. It may also be suitable for women undergoing bilateral reconstruction because symmetry is more easily achieved if both breasts are restored using the same technique. Women who elect this type of immediate reconstruction must understand that breast implants do not have an unlimited service life and that additional surgery will be likely be required to replace the breast implant at some time in the future.Tissue Flaps and Breast ImplantsThe latissimus dorsi musculocutaneous flap is the most com-mon transfer used in combination with breast implants. Other flaps may also be used, depending on patient preference and tissue availability. The principal advantage in using a tissue flap is immediate replacement of missing skin and soft tissue. In cases where there is already adequate breast skin, then a muscle only may be transferred to provide suitable implant coverage. The implant allows the final breast

1	of missing skin and soft tissue. In cases where there is already adequate breast skin, then a muscle only may be transferred to provide suitable implant coverage. The implant allows the final breast volume to be accurately reproduced to match the contralateral breast or, in bilateral reconstruction, adjust the breast size according to the patient’s desires. The advantages of this technique are that the implant is protected by abundant tissue, a period of tissue expansion is avoided, and the full benefit of preserving the breast skin is realized to achieve a natural-appearing breast. The disadvantage of this technique compared to implants alone is that it results in additional scarring and requires a longer period of recovery. For many patients, this approach represents an acceptable com-promise between implant-only reconstruction and autologous tissue reconstruction, incorporating some of the advantages and disadvantages of each.Autologous Tissue ReconstructionImmediate reconstruction

1	between implant-only reconstruction and autologous tissue reconstruction, incorporating some of the advantages and disadvantages of each.Autologous Tissue ReconstructionImmediate reconstruction using only autologous tissue is the most elaborate method of breast reconstruction but consis-tently yields the most durable, natural-appearing results. Breast implants cannot match the ability of the autologous tissue to conform to the breast skin and envelop and simulate natural breast parenchyma. The most useful flap is the transverse rec-tus abdominis musculocutaneous (TRAM) flap, although other ABPreoperativePostoperativeImmediate right DIEP FlapFigure 45-60. A. Preoperation right breast cancer. B. After mastectomy and immediate reconstruction with a DIEP flap.donor areas are also possibilities in selected cases. Autologous reconstruction is usually the best option in patients who require adjuvant radiation therapy.55The TRAM flap may be transferred to the chest using a variety of methods,

1	in selected cases. Autologous reconstruction is usually the best option in patients who require adjuvant radiation therapy.55The TRAM flap may be transferred to the chest using a variety of methods, depending on the circumstances of the individual patient. As a pedicled flap, it is transferred based on the superior epigastric vessels and tunneled beneath the skin to reach the mastectomy defect. As a free flap, it is based on the inferior epigastric vessels that are revascularized by micro-vascular anastomosis to vessels on the chest wall nearby the mastectomy defect. Often the microvascular technique using the deep inferior epigastric perforator (DIEP) flap is preferred because there is less risk of partial flap loss or localized areas of fat necrosis due to a more reliable blood supply (Fig. 45-60A, before operation on right breast; Fig. 45-60B, after mastectomy and immediate reconstruction with a DIEP flap). In immediate reconstruction with an axillary dissection, the axillary

1	(Fig. 45-60A, before operation on right breast; Fig. 45-60B, after mastectomy and immediate reconstruction with a DIEP flap). In immediate reconstruction with an axillary dissection, the axillary vessels are completely exposed and free of scar following the lymph node dissection in patients without previous surgery and radiation. In women being treated for recurrence with previous axillary sur-gery, the axillary vessels are less reliable, and plans should be made for the possibility of using the internal mammary vessels. The internal mammary vessels have become the most common recipient vessels for free tissue transfer in breast reconstruction in the contemporary era of sentinel lymph node biopsy that is used as a technique to perform axillary lymph node dissection in a more limited number of patients. Regardless of the technique used to transfer the tissue, the donor site is closed in a similar manner as an abdominoplasty, by repairing the abdominal wall and advancing the upper

1	number of patients. Regardless of the technique used to transfer the tissue, the donor site is closed in a similar manner as an abdominoplasty, by repairing the abdominal wall and advancing the upper abdominal skin downward. The umbi-licus is preserved on its vascular stalk brought to the surface through a small incision immediately above its location on the abdominal wall (Fig. 45-61A,B). Other donor sites including the buttock may be used in transferring the skin and fat supplied by the inferior gluteal artery perforator (IGAP) or the superior gluteal perforator as the main blood supply.The advantages of using this technique are complete res-toration of the breast mound in a single stage, avoidance of Brunicardi_Ch45_p1967-p2026.indd 201001/03/19 6:31 PM 2011PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-61A, B. Preand postoperative images following IGAP flap.Figure 45-62A, B. Preand postoperative images following IGAP flap, nipple reconstruction, and

1	AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-61A, B. Preand postoperative images following IGAP flap.Figure 45-62A, B. Preand postoperative images following IGAP flap, nipple reconstruction, and tattooing.ABPreoperativePostoperativeDelayed right IGAP Flappotential problems associated with breast implants, and con-sistently superior cosmetic results. The disadvantages are the magnitude of the operation, additional scarring, risks of devel-opment of abdominal bulges, and a longer period of convales-cence. Although the initial cost is greater, over the long term the total cost appears to be less because of less need for second-ary procedures to exchange implants, achieve suitable cosmetic appearance, or care for implant-related problems. This is the best operation for patients who want the most natural breast res-toration possible and who are less concerned about the amount of surgery, scarring, and recovery period.Accessory ProceduresAfter complete healing of the breast mound from the

1	most natural breast res-toration possible and who are less concerned about the amount of surgery, scarring, and recovery period.Accessory ProceduresAfter complete healing of the breast mound from the initial stages of reconstruction, refinements and accessory procedures may be performed at a later time to optimize the natural appear-ance of the reconstructed breast. These may include soft tissue ABBefore nipple reconstructionPostoperativeBilateral IGAP Flapmodifications of the breast mound revision, repositioning or the breast implant, scar revisions, autologous fat grafting, and nip-ple-areola complex reconstruction. A variety of methods have been described for nipple reconstruction. They are all based on local tissue rearrangements or skin grafts to create a projecting piece of skin and subcutaneous tissue that simulates the natural nipple (Fig. 45-62A,B). The pigmentation of the areola may be simulated with tattooing of colored pigments selected to match the normal coloration of

1	subcutaneous tissue that simulates the natural nipple (Fig. 45-62A,B). The pigmentation of the areola may be simulated with tattooing of colored pigments selected to match the normal coloration of the patient’s original anatomy.Trunk and Abdominal ReconstructionIn the torso, as in most areas of the body, the location and size of the defect and the properties of the deficient tissue determine choice of reconstructive method. A distinction is made between partial-thickness and full-thickness defects when deciding between grafts, flaps, synthetic materials, or a combina-tion of techniques. Unlike the head and the lower leg, the trunk 9Brunicardi_Ch45_p1967-p2026.indd 201101/03/19 6:31 PM 2012SPECIFIC CONSIDERATIONSPART IIharbors a relative wealth of regional transposable axial pattern flaps that allow sturdy reconstruction, only rarely requiring dis-tant free tissue transfer. Indeed, the trunk serves as the body’s arsenal, providing its most robust flaps to rebuild its largest

1	flaps that allow sturdy reconstruction, only rarely requiring dis-tant free tissue transfer. Indeed, the trunk serves as the body’s arsenal, providing its most robust flaps to rebuild its largest defects.The chest wall is a rigid framework designed to resist both the negative pressure associated with respiration and the positive pressure from coughing and from transmitted intra-abdominal forces. Furthermore, it protects the heart, lungs, and great vessels from external trauma. Reconstructions of chest wall defects must restore these functions. When a full-thick-ness defect of the chest wall involves more than four, this is usually an indication for the need for rigid chest wall recon-struction usually using synthetic meshes made of polypropyl-ene, polyethylene, or polytetrafluoroethylene, which may be reinforced with polymethylmethacrylate acrylic. In contami-nated wounds, biologic materials are preferred, such as acel-lular dermal matrix allografts. For soft tissue restoration, the

1	which may be reinforced with polymethylmethacrylate acrylic. In contami-nated wounds, biologic materials are preferred, such as acel-lular dermal matrix allografts. For soft tissue restoration, the pectoralis major muscle is commonly used as a pedicled flap for coverage of the sternum, upper chest, and neck. It may be mobilized and transferred on a vascular pedicle based on the pectoral branch of the thoracoacromial artery or a vascular supply based on perforators from the internal mammary ves-sels. Either flap design is useful in covering the sternum after dehiscence or infection occurring as a complication of median sternotomy or with sternal resection for tumor extirpation. For the lower third of the sternum, a rectus abdominis muscle flap based on the superior epigastric vessels or the deep inferior epigastric vessels is useful. If based on the inferior blood sup-ply, it must be transferred as a free flap with recipient vessels outside of the zone in injury. The latissimus dorsi

1	deep inferior epigastric vessels is useful. If based on the inferior blood sup-ply, it must be transferred as a free flap with recipient vessels outside of the zone in injury. The latissimus dorsi musculocu-taneous flap is useful for chest wall reconstructions in places other than the anterior midline. Similar to the pectoralis major muscle, it may be transferred on either a single blood supply that is based on the thoracodorsal vessels from the subscapular system or on vessels perforating from deeper source vessels near to the posterior midline. The serratus anterior muscle can be included on the same vascular pedicle to further increase its surface area. Finally, the trapezius muscle flap, based on the transverse cervical vessels, is generally used as a pedicled flap to cover the upper midback, base of neck, and shoulder. The superior portion of the muscle along with the acromial attach-ment and spinal accessory nerve must be preserved to maintain normal shoulder elevation

1	upper midback, base of neck, and shoulder. The superior portion of the muscle along with the acromial attach-ment and spinal accessory nerve must be preserved to maintain normal shoulder elevation function.The abdominal wall also protects the internal vital organs from trauma, but with layers of strong torso-supporting mus-cles and fascia rather than with osseous structures. The goals of reconstruction are restoration of structural integrity, prevention of visceral herniation, and provision of dynamic muscular sup-port. Although abdominal wall defects may occur in association with oncologic tumor resections, the most common etiology is fascial dehiscence after laparotomy. When a reconstruction plan is being formulated, careful physical examination and review of the medical history will help prevent selection of an otherwise sound strategy that, because of previous incisions and trauma, is destined for failure.Superficial defects of the abdominal skin and subcutane-ous tissue are

1	help prevent selection of an otherwise sound strategy that, because of previous incisions and trauma, is destined for failure.Superficial defects of the abdominal skin and subcutane-ous tissue are usually easily controlled with skin grafts, local advancement flaps, or tissue expansion. Defects of the under-lying musculofascial structures are more difficult to manage. The abdominal wall fascia requires a minimal-tension closure to avoid dehiscence, recurrent incisional hernia formation, or abdominal compartment syndrome. Prosthetic meshes are frequently used to replace the fascia in clean wounds and in operations that create myofascial defects. When the wound is contaminated, as in infected mesh reconstructions, enterocuta-neous fistulas, or viscus perforations, prosthetic mesh is avoided because of the risk of infection. The technique of component separation procedure has proven beneficial for closing large midline defects with autologous tissue and avoiding prosthetic materials. This

1	of the risk of infection. The technique of component separation procedure has proven beneficial for closing large midline defects with autologous tissue and avoiding prosthetic materials. This procedure involves advancement of bilateral flaps composed of the anterior rectus fascia rectus and oblique muscles after lateral release. Midline defects measuring up to 10 cm superiorly, 18 cm centrally, and 8 cm inferiorly can be closed using this method.Techniques based on rearranging and reinforcing abdomi-nal wall elements might be inadequate for extremely large or full-thickness abdominal wall defects. For these defects, regional flaps or free flaps are required. Pedicled flaps from the thigh are useful, such as the tensor fasciae latae pedicled flap, based on the ascending branch of the lateral circumflex femoral vessels, or the anterolateral thigh flap, based on the descending branch of the lateral circumflex vessels. Bilateral flaps might be required.Pelvic ReconstructionAnother

1	lateral circumflex femoral vessels, or the anterolateral thigh flap, based on the descending branch of the lateral circumflex vessels. Bilateral flaps might be required.Pelvic ReconstructionAnother important area for consideration of reconstructive surgical procedures is in the perineum.56 The perineal region is part of the specialized part of the trunk that supports the pelvic outlet lying between the pubic symphysis, the coccyx, the inferior rami of the pubis, and the ischial tuberosities. Sup-port is provided by the urogenital diaphragm, the deep and superficial fasciae, and the skin. Specialized anatomic struc-tures pass through the perineum. Posteriorly is the anus, and anteriorly are the genitalia and urethra. Treatment of tumors involving this area often require a combination of surgery and radiation. The resulting loss of tissue and healing impairment coupled with the nonyielding nature of the bony pelvic outlet can result in unique reconstructive requirements that often are

1	and radiation. The resulting loss of tissue and healing impairment coupled with the nonyielding nature of the bony pelvic outlet can result in unique reconstructive requirements that often are best addressed with tissue transfer. The reconstruction must achieve wound healing and restore support to the pelvic con-tents, accommodate urinary and bowel function, and finally restore the penis in men and the vagina and vulva in women. Local flaps, regional flaps, or free tissue transfer all have pos-sible application depending on the extent of the resection and local tissue compromise.Other Clinical CircumstancesBesides trauma and cancer, other etiologies can cause functional and cosmetic deformities due to tissue impairment for which reconstructive surgery has value. These include pressure sores, diabetic foot ulcers, and lymphedema.Pressure Sores. A pressure ulcer is defined as tissue injury caused by physical pressure applied to the tissues from an exter-nal source at a magnitude that

1	diabetic foot ulcers, and lymphedema.Pressure Sores. A pressure ulcer is defined as tissue injury caused by physical pressure applied to the tissues from an exter-nal source at a magnitude that exceeds capillary perfusion pres-sure. Prolonged tissue ischemia leads to local tissue necrosis. Pressure ulcers tend to occur in people debilitated by advanced age, chronic illness, poor nutrition, prolonged immobilization, motor paralysis, or inadequate sensation. Spinal cord injury patients are especially prone to developing pressure sores. Pres-sure sores can also occur in healthy individuals who undergo prolonged surgical operations and parts of the body support-ing the weight of the patient on the operating table (e.g., the occiput, the sacral prominence, the heels of the feet) are improp-erly padded.57Brunicardi_Ch45_p1967-p2026.indd 201201/03/19 6:31 PM 2013PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Pressure sores are an important contributor to morbidity in patients suffering from

1	padded.57Brunicardi_Ch45_p1967-p2026.indd 201201/03/19 6:31 PM 2013PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Pressure sores are an important contributor to morbidity in patients suffering from limited mobility. Most can be prevented by diligent nursing care in an attentive, cooperative patient. Preventing pressure ulcers requires recognition of susceptible and utilizing appropriate measures to reduce pres-sure on areas of the body at risk. This involves frequent position changes while sitting or supine and the use of pressure-reducing medical equipment such as low-air-loss mattresses and seat cushions and heel protectors. Malnourishment, poor glucose control in diabetics, poor skin hygiene, urinary or bowel incon-tinence, muscle spasms, and joint contractures all increase the risk of pressure sore formation. Mitigating these factors is essential before embarking on a complex reconstructive treat-ment plan. Successful reconstruction also requires a cooperative and motivated

1	of pressure sore formation. Mitigating these factors is essential before embarking on a complex reconstructive treat-ment plan. Successful reconstruction also requires a cooperative and motivated patient with good social support.Surgical treatment of pressure ulcers is based on wound depth. The staging system is summarized in Fig. 45-63.58 Stage I and II ulcers are treated nonsurgically with local wound care and interventions to relieve pressure on the affected area. Patients with stage III or IV ulcers should be evaluated for surgery. Important features for preoperative assessment include the extent of soft tissue infection, the presence of con-taminated fluid collection or abscess, osteomyelitis, and com-munication with deep spaces (e.g., joint space, urethra, colon, or spinal canal). Laboratory blood tests and imaging studies help establish whether soft tissue or bone infection is present. Plain radiographs are usually adequate to rule out osteomyeli-tis; CT and MRI are helpful

1	Laboratory blood tests and imaging studies help establish whether soft tissue or bone infection is present. Plain radiographs are usually adequate to rule out osteomyeli-tis; CT and MRI are helpful when plain films are equivocal. Necrotic tissue and abscesses should be surgically debrided without delay to prevent or treat systemic sepsis. Bone must also be excised if it appears involved, as evidenced by poor bleeding, softness, or frank purulence. Patients with high spinal cord injuries at or above the level of the fifth thoracic vertebra may experience sudden extreme elevation of blood pressure, an 10Stage 1Observable pressure related alteration of intact skin whose indicators as compared to the adjacent or opposite area of the body may include changes in one or more of the following: skin temperature (warmth or coolness), tissue consistency (firm or boggy feel), and/or sensation (pain, itching). The ulcer appears as a defined area of persistent redness in lightly pigmented skin,

1	skin temperature (warmth or coolness), tissue consistency (firm or boggy feel), and/or sensation (pain, itching). The ulcer appears as a defined area of persistent redness in lightly pigmented skin, whereas in darker skin tones the ulcer may appear with persistent red, blue of purple hues.Stage 2Partial thickness skin loss involving epidermis and/or dermis. The ulcer is superficial and presents clinically as an abrasion, blister, or shallow crater.Stage 3Full thickness skin loss involving damage or necrosis of subcutaneous tissue that may extend down to but not through underlaying fascia. The ulcer presents clinically as a deep crater with or without undermining of adjacent tissue.Stage 4Full thickness skin loss with extensive destruction, tissue necrosis or damage to muscle, bone, or supporting structures (for example, tendon or joint capsule). Undermining and sinus tracts may also be associated with Stage 4 pressure ulcers.ABCD Figure 45-63. The staging system for pressure

1	or supporting structures (for example, tendon or joint capsule). Undermining and sinus tracts may also be associated with Stage 4 pressure ulcers.ABCD Figure 45-63. The staging system for pressure sores.autonomic-mediated event called hyperreflexia. This condition must be immediately recognized and treated to prevent intra-cranial and retinal hemorrhage, seizures, cardiac irregularities, and death.After adequate debridement, the pressure ulcer can be treated nonsurgically in patients who have shallow wounds with healthy surrounding tissues capable of healing secondarily with offloading pressure. Nonsurgical treatment is also best in patients for whom surgery is contraindicated because of previ-ous surgery or comorbidities. For surgical candidates, primary closure is rarely performed because an inadequate amount of quality surrounding tissue prevents closure without tension, making the repair predisposed to failure. Split-thickness skin grafting can be useful for shallow ulcers with

1	an inadequate amount of quality surrounding tissue prevents closure without tension, making the repair predisposed to failure. Split-thickness skin grafting can be useful for shallow ulcers with well-vascularized wound beds on which shear forces and pressure can be avoided after repair, a rare circumstance in most patients with pressure ulcers.The mainstay of surgical treatment is tissue transfer fol-lowing several guiding principles. Local muscle or musculocu-taneous flaps are suitable for areas of heavy contamination and complex wound surface contours. Durability requires the ability to consistently off-load of the area of reconstruction postopera-tively. Fasciocutaneous flaps afford more durable reconstruc-tion when off-loading is not possible. The anatomic location is an important determinant of flap choice. Once a donor site is selected, a flap of adequate size is designed and transferred in a way that avoids suture lines in the area under pressure. Large flaps also permit

1	determinant of flap choice. Once a donor site is selected, a flap of adequate size is designed and transferred in a way that avoids suture lines in the area under pressure. Large flaps also permit readvancement if the patient experiences a recurrent ulcer in the same area. Sacral pressure sores may be managed with fasciocutaneous or musculocutaneous flaps based on the gluteal vessels. Ischial pressure sores may be man-aged with gluteal flaps or flaps transferred from the posterior thigh, such as the posterior thigh flap based on the descend-ing branch of the inferior gluteal artery. Trochanteric ulcers Brunicardi_Ch45_p1967-p2026.indd 201301/03/19 6:31 PM 2014SPECIFIC CONSIDERATIONSPART IIFigure 45-64. Flap reconstruction of pressure ulcers. Top row: Preoperative and 1-month postoperative photos of a stage IV sacral decubitus ulcer treated with a myocutaneous gluteus maximus flap. Bottom row: Preoperative and 1-month postoperative photos of a stage IV trochan-teric ulcer treated

1	photos of a stage IV sacral decubitus ulcer treated with a myocutaneous gluteus maximus flap. Bottom row: Preoperative and 1-month postoperative photos of a stage IV trochan-teric ulcer treated with a myocutaneous V-Y tensor fasciae latae flap.may be managed with musculocutaneous flaps based on the tensor fasciae latae, rectus femoris, or vastus lateralis muscles (Fig. 45-64). The obligatory loss of motor function associated with using these flaps adds no additional functional impairment in patients already paralyzed as a result of strokes or spinal cord injuries.Proper postoperative care after flap reconstruction of pressure ulcers is critical for success. Low-pressure, air fluid-ized beds help to off-load the affected area and prevent new areas of involvement during the first 7 to 10 days of healing. Other important measures are adequate nutritional support and medications to prevent muscle spasms. Careful coordination with patient care providers is planned preoperatively in order

1	of healing. Other important measures are adequate nutritional support and medications to prevent muscle spasms. Careful coordination with patient care providers is planned preoperatively in order to avoid gaps in care that can lead to early recurrent ulceration. Care of the pressure ulcer patient is a labor-intensive process that requires attention to detail by the surgeon, nurses, thera-pists, caseworkers, and family.Diabetic Foot Ulceration. The pathophysiology of primary diabetic lower limb complications has three main components: (a) peripheral neuropathy (motor, sensory, and autonomic), (b) peripheral vascular disease, and (c) immunodeficiency. Altered foot biomechanics and gait caused by painless col-lapse of ligamentous support, foot joints, and foot arches change weight-bearing patterns. Blunted pain allows cutane-ous ulceration to begin. With breakdown of the skin barrier function, polymicrobial infections become established. Bac-terial invasion is often fostered by poor

1	patterns. Blunted pain allows cutane-ous ulceration to begin. With breakdown of the skin barrier function, polymicrobial infections become established. Bac-terial invasion is often fostered by poor blood supply due to peripheral vascular disease coupled with microangiopathy. Finally, local host defenses may be less effective in resisting bacteria because of poor blood supply and impaired cellular function. Cutaneous ulcerations may progress painlessly to involve deeper soft tissues and bone. The ultimate endpoint of this process is such severe tissue damage that extremity amputation is the only treatment remaining. More than 60% of nontraumatic lower extremity amputations occur in diabetics. The age-adjusted lower extremity amputation rate in diabet-ics (5.0 per 1000 diabetics) was approximately 28 times that of people without diabetes (0.2 per 1000 people).59 Improved patient education and medical management, early detection of foot problems, and prompt intervention play important

1	28 times that of people without diabetes (0.2 per 1000 people).59 Improved patient education and medical management, early detection of foot problems, and prompt intervention play important roles in improving the chances of limb preservation.60The best approach to managing diabetic patients with lower extremity wounds is to involve a multidisciplinary team composed of a plastic and reconstructive surgeon, a vascular surgeon, an orthopedic surgeon, a podiatrist, an endocrinolo-gist specializing in diabetes, a nutritionist, and a physical or Brunicardi_Ch45_p1967-p2026.indd 201401/03/19 6:31 PM 2015PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45occupational therapist. This brings together the greatest level of expertise to manage bone and soft tissue issues as well as the underlying disease and medical comorbidities. Treatment begins with rigorous control of blood glucose levels and a thor-ough assessment of comorbidities. In addition to careful detail-ing of the extent of the wound

1	and medical comorbidities. Treatment begins with rigorous control of blood glucose levels and a thor-ough assessment of comorbidities. In addition to careful detail-ing of the extent of the wound and the tissues involved, physical examination documents sensory deficits and vascular status. Plain radiographs, MRI, bone scintigraphy, and angiography or duplex Doppler ultrasound imaging may be indicated. A patient with significant vascular disease may be a candidate for lower extremity endovascular revascularization or open bypass.61 Nerve conduction studies may diagnose surgically reversible neuropathies at compressive sites and aid in decisions about whether to perform sensory nerve transfers to restore plantar sensibility.60 Antibiotic and fungal therapies should be guided by tissue culture results.Surgical management starts with debridement of devital-ized tissues. Methods of wound closure are dictated by the extent and location of the remaining defect. Negative pressure wound

1	culture results.Surgical management starts with debridement of devital-ized tissues. Methods of wound closure are dictated by the extent and location of the remaining defect. Negative pressure wound dressings may be appropriate for superficial defects in an effort to allow secondary healing or as a temporizing measure until definitive wound closure can be achieved. Skin grafts might be indicated at times but cannot be expected to provide durable cov-erage in weight-bearing or high-shear areas. Local and regional flaps can be considered if the extremity is free of significant occlusive peripheral vascular or combined with vascular bypass. Microvascular free tissue transfers are appropriate when defects are large or when local flaps are not available. Combination lower extremity bypass and free flap coverage has proved benefi-cial for the treatment of the diabetic foot in terms of healing and reduction of disease progression (Table 45-6). Consultation with a podiatrist or an orthopedic

1	free flap coverage has proved benefi-cial for the treatment of the diabetic foot in terms of healing and reduction of disease progression (Table 45-6). Consultation with a podiatrist or an orthopedic surgeon who specializes in foot and ankle problems can be considered to improve foot biomechanics and manage bony prominences that act as pressure points on the soft tissue to reduce the risk of recurrent ulceration. Proper foot-wear (including orthotic devices and off-loading shoe inserts), hygiene, and toenail and skin care are essential.60Lymphedema. Lymphedema is the abnormal accumulation of protein-rich fluid in the interstitial spaces of the tissues. It is a complex disorder with both congenital and acquired causes. No universally effective remedy has been devised, but a variety of treatment methods including reconstructive surgery have been effective in carefully selected patients.It is important to be familiar with the fundamentals of lymph physiology in order to understand the

1	methods including reconstructive surgery have been effective in carefully selected patients.It is important to be familiar with the fundamentals of lymph physiology in order to understand the rationale for the various forms of lymphedema treatment. Lymph fluid is formed at the capillary level where there is a net outflow of fluid and serum proteins from the intravascular space into the intersti-tium. In the average adult, this amounts to approximately 3 liters of fluid daily. Open-ended lymph capillaries collect this fluid where the lymphatic endothelial cells form loose intercellular connections that freely allow fluid to enter. From here, the net-work of specialized vascular structures gathers the extravasated fluid and transports it back into central circulation. The system is a high-volume transport mechanism that clears proteins and lipids from the interstitial space primarily by means of differ-ential pressure gradients. Lymph fluid enters the lymph vessels driven by colloid and

1	transport mechanism that clears proteins and lipids from the interstitial space primarily by means of differ-ential pressure gradients. Lymph fluid enters the lymph vessels driven by colloid and solute concentration gradients at the capil-lary level. Flow is sustained in the larger vessels through direct contractility of the lymph vessel walls and by indirect compres-sion from surrounding skeletal muscle activity. Throughout the system, one-way valves prevent reverse flow. The lymphatic vessels course throughout the body alongside the venous sys-tem, into which they eventually drain via the major thoracic and cervical ducts at the base of the neck.Under normal conditions, there is a balance between fluid formation and lymph transport capacity. With congenital hypo-plasia or acquired obstruction, there is a reduction in transport capacity resulting in accumulation of fluid and protein in the interstitium. Localized fluid stagnation, hypertension, and valvu-lar incompetence further

1	there is a reduction in transport capacity resulting in accumulation of fluid and protein in the interstitium. Localized fluid stagnation, hypertension, and valvu-lar incompetence further degrade transport capacity and acceler-ate lymph fluid accumulation edema. Dissolved and suspended serum proteins, cellular debris, and waste products of metabolism elicit an inflammatory response with associated with fibrovas-cular proliferation and collagen deposition leading to firm, non-pitting swelling characteristic of chronic, long-standing edema. Lymphoscintigraphy can help detail the lymphatic anatomy and quantify lymphatic flow. MRI can provide additional informa-tion about the larger caliber lymphatic vessels, possibly helping to identify specific points of obstruction.Primary lymphedema is caused by congenital hypopla-sia and is classified clinically based on the age of the affected individual when swelling first appears. Lymphedema present at birth is an autosomal dominant disorder

1	is caused by congenital hypopla-sia and is classified clinically based on the age of the affected individual when swelling first appears. Lymphedema present at birth is an autosomal dominant disorder sometimes referred to as Milroy’s disease. Lymphedema praecox occurs near the time of puberty but can appear up to age 35. This form tends to occur in females and usually affects the lower extremity. It accounts for more than 90% of cases. Finally, lymphedema tarda appears after the age of 35 years and is relatively rare.Secondary lymphedema is the acquired form of the dis-order and is more common than congenital causes. Worldwide the most common etiology is parasitic infestation with filarial, a highly specialized nematode transmitted by blood-eating insects Table 45-6Some reconstructive options for the diabetic footAREA OF DEFECTRECONSTRUCTIVE OPTIONSForefootV-Y advancementToe island flapSingle toe amputationLisfranc’s amputationMidfootV-Y advancementToe island flapMedial plantar artery

1	for the diabetic footAREA OF DEFECTRECONSTRUCTIVE OPTIONSForefootV-Y advancementToe island flapSingle toe amputationLisfranc’s amputationMidfootV-Y advancementToe island flapMedial plantar artery flapFree tissue transferTransmetatarsal amputationHindfootLateral calcaneal artery flapReversed sural artery flapMedial plantar artery flap ± flexor digitorum brevisAbductor hallucis muscle flapAbductor digiti minimi muscle flapFree tissue transferSyme’s amputationFoot dorsumSupramalleolar flapReversed sural artery flapThinner free flaps (e.g., temporoparietal fascia, radial forearm, groin, thinned anterolateral thigh flaps)Brunicardi_Ch45_p1967-p2026.indd 201501/03/19 6:31 PM 2016SPECIFIC CONSIDERATIONSPART IIFigure 45-65. Algorithm for lymphedema management.YesNoYesNoYesNoSymptomatic LymphedemaAmenable to physiologic lymphatic procedures?Suitable lymphatic vessels on MRL or ICGL for LVA?Secondary to surgery and/or XRT?LVA ±VLNTLiposuction ±excisionLVAonlyVLNTonlyConsider furtherLVA or

1	to physiologic lymphatic procedures?Suitable lymphatic vessels on MRL or ICGL for LVA?Secondary to surgery and/or XRT?LVA ±VLNTLiposuction ±excisionLVAonlyVLNTonlyConsider furtherLVA or VLNTInadequate response?Secondary to surgery and/or XRT?Severe functional impairment?Excess soft tissue? Skin changes?Yes• Responsive to nonsurgical therapy, but symptoms plateaued or worsening• Significant pitting edemaNo• Minimal or no improvement with nonsurgical therapy• Minimal to absent pitting edemafound mostly in developing countries. In nonaffected areas of the world, the most common cause of secondary lymphedema is regional lymphatic vessel destruction associated with can-cer treatment. It often occurs in the upper extremity of women treated with surgery and radiation therapy for breast cancer. In the lower extremities, it is associated with neoplasms treated with inguinal or retroperitoneal lymph node dissection.The goal of lymphedema treatment is to minimize func-tional and cosmetic

1	In the lower extremities, it is associated with neoplasms treated with inguinal or retroperitoneal lymph node dissection.The goal of lymphedema treatment is to minimize func-tional and cosmetic disability caused by chronic enlargement and to prevent infection of the involved extremity. The foun-dations of management are patient education and nonsurgical interventions, which include limb elevation, external compres-sive garments and devices, and manual lymphatic massage, sometimes referred to as complex decongestive physiother-apy. The patient must use protective gloves or garments when engaged in activities that might cause minor skin injury, such as gardening, smoking cigarettes, and cooking. Interstitial lymph fluid is prone to infection. When signs of infection appear, prompt treatment that often includes hospitalization with intravenous antibiotics is essential to prevent severe infection and further destruction of remaining lymphatic sys-tem and worsening of lymphedema.When

1	that often includes hospitalization with intravenous antibiotics is essential to prevent severe infection and further destruction of remaining lymphatic sys-tem and worsening of lymphedema.When nonsurgical methods fail, surgery can be consid-ered as a treatment option. Surgical operations for lymphedema are either ablative, designed to remove excess lymphedematous tissues, or reconstructive, intended to restore lymph function and improve transport capacity. These choices are presented in Fig. 45-65. Ablative procedures range from minimally invasive measures such as suction lipectomy to complete excision of skin and subcutaneous tissue down to muscle fascia with split-thickness skin grafting. Contemporary reconstructive procedures establish new connections between the venous and lymphatic systems somewhere proximal to the point of obstruction. A variety of methods have been described, including lympholymphatic, lym-phovenous, lymph node venous anastomoses, and vascularized lymph node

1	systems somewhere proximal to the point of obstruction. A variety of methods have been described, including lympholymphatic, lym-phovenous, lymph node venous anastomoses, and vascularized lymph node transfer. Each of these procedures can yield suc-cess, and it has become clear that patient selection is perhaps the most important aspect of surgical care because the patient must be matched to the procedure most likely to yield improved con-trol of swelling and prevent infection. Reconstructive surgery is not generally a cure for the condition, but rather it is intended to ease management challenges and reduce the risks of infection. After surgery, continued use of nonsurgical techniques is still required for optimal results.AESTHETIC SURGERY AND MEDICINEAesthetic, or cosmetic, surgery is an important part of the spe-cialty of plastic surgery. The American Medical Association defines cosmetic surgery as “surgery performed to reshape normal structures of the body to improve the patient’s

1	important part of the spe-cialty of plastic surgery. The American Medical Association defines cosmetic surgery as “surgery performed to reshape normal structures of the body to improve the patient’s appear-ance and self-esteem.” It is a natural extension of surgical tech-niques for tissue modification traditionally developed for other reasons. Because aesthetic surgery primarily relates to personal appearance and attractiveness and not a particular disease pro-cess, there has been a tendency to dismiss the health value of Brunicardi_Ch45_p1967-p2026.indd 201601/03/19 6:31 PM 2017PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45aesthetic surgery. Nevertheless, personal appearance plays an important role in psychosocial health. Physical attractiveness plays a role in the marketplace with well-documented influence on employment opportunities, advancement, and earnings.62 The multibillion industry of products and services designed to opti-mize appearance, which spans a wide spectrum

1	well-documented influence on employment opportunities, advancement, and earnings.62 The multibillion industry of products and services designed to opti-mize appearance, which spans a wide spectrum between simple cosmetics to elaborate surgical procedures, bears testament to the perceived value by the general population.Important work demonstrates a link between aesthetic sur-gery and psychosocial health. Surgery performed on the face,63 nose,64 ears,65 breast,66 and body67 can positively affect quality of life on multiple scales. There is a clear association between one’s personal appearance and success in the marketplace. As the primary benefits of aesthetic surgery are related to the psy-chosocial outcomes, it is important to assess the state of psycho-logical health prior to offering aesthetic surgery. A variety of preoperative psychological comorbidities can adversely affect outcomes, most notably a syndrome known as body dysmor-phic disorder,68 present in individuals who manifest

1	surgery. A variety of preoperative psychological comorbidities can adversely affect outcomes, most notably a syndrome known as body dysmor-phic disorder,68 present in individuals who manifest a preoccu-pation with one or more perceived defects or flaws in physical appearance that are not observable or appear slight to others.69 Performing a surgical procedure to modify personal appearance in such an individual is associated with a high risk of a poor outcome.It is important for all surgeons to have an appreciation of the methods of patient evaluation, surgical techniques, and typical outcomes that might be anticipated in aesthetic sur-gery. Patients seek aesthetic surgery when they are unable to achieve a personal standard of physical appearance without sur-gical modification of various body parts that most affect their appearance. This is especially true for features that are visible in public and strong determinants of appearance, such as the face, breasts, abdomen, and buttocks.

1	body parts that most affect their appearance. This is especially true for features that are visible in public and strong determinants of appearance, such as the face, breasts, abdomen, and buttocks. The etiology of undesir-able characteristics of form or skin quality can be familial or acquired through natural processes of aging, injury, cancer, or degeneration. Unwanted changes in appearance that result from these processes may still fall within the range of normal appearance yet fall short of the patient’s personal aesthetic ideal. Patient assessment requires an understanding of personal and cultural ideals of appearance. The surgeon must be knowledge-able about the various surgical and nonsurgical techniques that might be considered to address the patient’s concerns.In practical terms, there are both reconstructive and cos-metic elements to almost every plastic surgery case, and the def-inition of “normal” structure is sometimes very subjective and difficult to quantify.

1	terms, there are both reconstructive and cos-metic elements to almost every plastic surgery case, and the def-inition of “normal” structure is sometimes very subjective and difficult to quantify. Nevertheless, there are patients for whom it is a priority to make surgical changes to their bodies in the clear absence of a functional deformity. Aesthetic surgery patients present a unique challenge to the plastic surgeon because the most important outcome parameter is not truly appearance, but patient satisfaction. Optimally, a good cosmetic outcome will be associated with a high level of patient satisfaction. For this to be the case, the plastic surgeon must do a careful analysis of the patient’s motivations for wanting surgery, along with the patient’s goals and expectations. The surgeon must make a rea-sonable assessment that the improvements that can be achieved through surgery will meet the patient’s expectations. The sur-geon must appropriately counsel the patient about the

1	must make a rea-sonable assessment that the improvements that can be achieved through surgery will meet the patient’s expectations. The sur-geon must appropriately counsel the patient about the magni-tude of the recovery process, the exact location of scars, and potential complications. If complications do occur, the surgeon must manage these in a manner that preserves a positive doctor-patient relationship.Figure 45-66. Incisions for cervicofacial rhytidectomy.Aesthetic Surgery of the FaceA thorough evaluation of the patient who presents for facial aes-thetic surgery begins with acquiring a clear understanding of the patient’s primary concern regarding appearance. Examination focuses on that region but takes into consideration overall facial appearance that might be contributing to the patient’s concerns but of which the patient is unaware. The skin quality is care-fully assessed as well as the location, symmetry, and position of each critical feature of facial appearance such as

1	patient’s concerns but of which the patient is unaware. The skin quality is care-fully assessed as well as the location, symmetry, and position of each critical feature of facial appearance such as scalp hairline, forehead length, eyebrow shape and position, eyelid configu-ration, nasal proportions, and shape of the lips. Overall facial proportions are assessed, such as the prominence of the orbital rims and malar areas, the chin projection, and contours along the margin of the mandible. An appropriately performed facelift can yield an aesthetically pleasing result (Fig. 45-66).A variety of procedures have been described for modify-ing facial appearance. Nonsurgical interventions topical treat-ments of the skin surface include chemical and laser facial peels. Injections of biocompatible materials made of processed biologic proteins (e.g., collagen, hyaluronic acid) or synthetic materials such as polymethylmethacrylate can modify the depth of facial wrinkles and fullness of facial

1	materials made of processed biologic proteins (e.g., collagen, hyaluronic acid) or synthetic materials such as polymethylmethacrylate can modify the depth of facial wrinkles and fullness of facial structures such as the lips. Appearance can also be modified using neuromodulators to block facial muscle function to reduce undesirable move-ments of facial landmarks or deepening of facial wrinkles. Sur-gical interventions may be employed when the structure and position of facial features require modifications greater than what may be achieved with nonsurgical procedures. Browlift operations raise the position of the eyebrows (Fig. 45-67). Blepharoplasty is a set of procedures that modify the shape and position of the upper and lower eyelids. Facelift modifies the configuration and amount of facial skin and subcutaneous Brunicardi_Ch45_p1967-p2026.indd 201701/03/19 6:31 PM 2018SPECIFIC CONSIDERATIONSPART IIstructures to correct features such as deep nasolabial folds, skin redundancy

1	facial skin and subcutaneous Brunicardi_Ch45_p1967-p2026.indd 201701/03/19 6:31 PM 2018SPECIFIC CONSIDERATIONSPART IIstructures to correct features such as deep nasolabial folds, skin redundancy along the inferior border of the mandible, and loss of definition of neck contours. Rhinoplasty involves a complex set of procedures to modify the size, shape, and airway function of the nose (Fig. 45-68).Aesthetic Surgery of the BreastSurgery to modify the shape, volume, and nipple position of the breast are among the most common aesthetic procedures. Figure 45-67. Facelift. A. Preoperative appearance. B. Postopera-tive appearance.ABBreast reduction surgery reduces the amount of both skin and breast tissue volume and modifies the position of the nipple on the breast mound (Fig. 45-69). The most common indication is to treat symptoms of large breasts known as macromastia, which is associated with a symptomatic triad of upper back pain, bra strap grooving, and skin rashes under the fold of

1	common indication is to treat symptoms of large breasts known as macromastia, which is associated with a symptomatic triad of upper back pain, bra strap grooving, and skin rashes under the fold of the breasts. Unilateral breast reduction is often performed to achieve breast symmetry after contralateral postmastectomy breast reconstruc-tion. As with all breast surgery, achieving a natural and cos-metically acceptable appearance is essential to a satisfactory outcome. Mastopexy techniques share many aspects with breast reduction except that breast volume is preserved and only the amount of skin and location of the nipple are modified. Funda-mental to the success of the procedure is the establishment of symmetric and proper nipple position. Nipple ptosis is graded by the nipple position relative to the inframammary fold.Many patients seek surgical intervention to increase breast size in a procedure known as augmentation mammoplasty (Fig. 45-70). Breast volume is increased by insertion

1	to the inframammary fold.Many patients seek surgical intervention to increase breast size in a procedure known as augmentation mammoplasty (Fig. 45-70). Breast volume is increased by insertion of a syn-thetic implant specifically designed for this purpose. Modern breast implants are manufactured from various formulations of silicone polymers. The implant shell, which is on contact with the tissues, is always made from silicone elastomer. The filling material can be either silicone or saline, depending on the patient and surgeon preference. As with any surgical proce-dure that involves implanting synthetic materials, the surgeon must fully understand the nature of the materials and be able to inform the patient of all known risks and benefits.The pervasive risk of breast cancer among women man-dates careful consideration of the impact of any breast surgery on cancer screening, diagnosis, and treatment. Preoperative breast cancer screening consistent with current American Can-cer

1	women man-dates careful consideration of the impact of any breast surgery on cancer screening, diagnosis, and treatment. Preoperative breast cancer screening consistent with current American Can-cer Society guidelines should be performed for all patients undergoing elective breast reshaping surgery. After breast augmentation surgery, routine screening mammograms are no longer considered adequate. Patients with breast implants must have diagnostic mammograms where a radiologist studies the images at the time of the study to ensure they completely visual-ize the breast tissue.Gynecomastia is a condition of excess breast tissue in males. It can be caused by a wide range of medical disorders, including liver dysfunction, endocrine abnormalities, genetic syndromes (e.g., Klinefelter’s syndrome), renal disease, tes-ticular tumors, adrenal or pituitary adenomas, secreting lung carcinomas, and male breast cancer. Pharmacologic agents associated with the potential side effect of breast

1	renal disease, tes-ticular tumors, adrenal or pituitary adenomas, secreting lung carcinomas, and male breast cancer. Pharmacologic agents associated with the potential side effect of breast enlargement include marijuana use, digoxin, spironolactone, cimetidine, the-ophylline, diazepam, and reserpine. Although all of these pos-sible causes must be considered in any patient presenting with gynecomastia, the majority of patients have idiopathic enlarge-ment of the breast parenchyma, often occurring in teenagers. Surgical correction of this condition as often indicated.Aesthetic Surgery of the BodyAesthetic surgery may be applied to the torso and extremities. The most common circumstance is following massive weight loss, typically as a result of bariatric surgery. Morbid obesity stretches the skin and supporting ligaments that tether it to the underlying fascial framework. Decreasing the amount of sub-cutaneous fat often results in significant skin laxity that creates body contour

1	the skin and supporting ligaments that tether it to the underlying fascial framework. Decreasing the amount of sub-cutaneous fat often results in significant skin laxity that creates body contour deformities. Improvement can be achieved only through skin excision. Therefore, all body-contouring surgery Brunicardi_Ch45_p1967-p2026.indd 201801/03/19 6:31 PM 2019PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45ANaso-frontal angleNaso-labial angleTip-columellar angleLower lateral cartilageUpper lateral cartilageBCFigure 45-68. A. Rhinoplasty anatomy. B. Preoperative appear-ance. C. Postoperative appearance.Brunicardi_Ch45_p1967-p2026.indd 201901/03/19 6:31 PM 2020SPECIFIC CONSIDERATIONSPART IIFigure 45-69. Inferior pedicle reduction mammaplasty.De-epithelializedareaExcised arearepresents a trade of excess skin for scar, and this must be emphasized during patient consultation. The patient willing to accept scars in exchange for improved contour is likely to be satisfied with the

1	a trade of excess skin for scar, and this must be emphasized during patient consultation. The patient willing to accept scars in exchange for improved contour is likely to be satisfied with the procedures. With the increased number of bar-iatric surgery procedures over the past decade, body-contouring surgery has become very popular and is emerging as a new sub-specialty of plastic surgery.Excess skin and subcutaneous tissue on the anterior abdominal wall creates a redundancy that can hang over the pubic area called an abdominal wall pannus. It can cause dif-ficulty dressing and maintaining proper personal hygiene. A panniculectomy is a procedure that removes the redundant skin and subcutaneous tissue of the pannus. If additional contouring of the abdominal wall is performed, the procedure is known as abdominoplasty. During this procedure, not only is the pannus excised but the maximum amount of skin is excised to tighten the abdominal wall. Optimum contouring typically requires

1	is known as abdominoplasty. During this procedure, not only is the pannus excised but the maximum amount of skin is excised to tighten the abdominal wall. Optimum contouring typically requires tightening of the underlying abdominal wall by suturing the midline and transposing the umbilicus as the upper abdominal skin is advanced inferiorly. At times additional skin must be excised transversely, requiring a concurrent vertical incision to remove skin in two vectors (Fig. 45-71). Possible complications include skin necrosis, persistent paresthesias of the abdominal wall, seroma, and wound separation. Necrosis of the umbili-cus may complicate preservation of that structure if the stalk is excessively long or an umbilical hernia is repaired. Adding a Brunicardi_Ch45_p1967-p2026.indd 202001/03/19 6:32 PM 2021PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-70. Placement of breast implant. A. Subglandular. B. Subpectoral.Figure 45-69. (Continued)ImplantBAPectoralis

1	202001/03/19 6:32 PM 2021PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45Figure 45-70. Placement of breast implant. A. Subglandular. B. Subpectoral.Figure 45-69. (Continued)ImplantBAPectoralis majormusclevertical resection increases the incidence of skin necrosis, espe-cially at the confluence of scars in the lower abdomen.Brachioplasty, or arm lift, excises excess skin and subcu-taneous tissue from the arms. It results in improved contour but leaves a visible longitudinal scar on the medial aspect of the arm. Therefore, it is reserved for patients with excessive skin in that region. The patient willing to accept the scar can be happy with the results. Complications include distal seroma and wound separation. Paresthesias in the upper arm and forearm may occur secondary to injury of sensory nerves passing through the resec-tion area, though this rarely affects function. Incisions that cross the axilla must be designed to avoid axillary contractures that limit shoulder mobility.Thigh

1	nerves passing through the resec-tion area, though this rarely affects function. Incisions that cross the axilla must be designed to avoid axillary contractures that limit shoulder mobility.Thigh and buttock lifts treat loose skin on the thighs and buttocks. A variety of methods have been described, and applica-tion requires proper patient selection in order to obtain the best outcome. The lateral thighs can be lifted simultaneously during abdominoplasty with one scar along the belt line. If the lift is continued on the posterior torso, a buttocks lift can be performed as well. This procedure is referred to as a circumferential lower body lift. Contouring the medial thighs typically requires an inci-sion in the groin crease. Firmly anchoring the deep thigh fascia to Colles’ fascia is essential to help prevent spreading of the labia. In cases of severe excess skin on the inner thighs, a long verti-cal incision is necessary. Complications of thigh and buttock lift include seroma, wound

1	to help prevent spreading of the labia. In cases of severe excess skin on the inner thighs, a long verti-cal incision is necessary. Complications of thigh and buttock lift include seroma, wound separation, skin necrosis, and change in the shape of the genital region (with possible sexual dysfunction).Brunicardi_Ch45_p1967-p2026.indd 202101/03/19 6:32 PM 2022SPECIFIC CONSIDERATIONSPART IIABFigure 45-71. A. Preoperative photo of 35-year-old woman after gastric bypass and massive weight loss. B. Patient 12 months after a fleurde-lis abdominoplasty.Suction LipectomyLiposuction is a technique that involves the removal of adipose tissue through minimal incisions using a hollow suction can-nula system. The key consideration in determining acceptable candidates for this body contouring technique directly relies on the patient’s inherent skin elasticity, which provides the sought-after retraction of skin over the lipoaspirated adipose depot to improve area contour. Thus, assessment of skin

1	directly relies on the patient’s inherent skin elasticity, which provides the sought-after retraction of skin over the lipoaspirated adipose depot to improve area contour. Thus, assessment of skin tone is a vital part of the preoperative patient evaluation. If there is excessive skin laxity in the body area to be treated, it may worsen after liposuction and contribute to contour irregularities, voids, and abnormal appearance.This technique can be highly effective in the correctly chosen patient as the access port sites provide minimally vis-ible scars and can remove significant amounts of fatty tissue to improve contour. However, it is worth mentioning that liposuc-tion is not considered a weight-loss treatment; rather, it is a tool for addressing unwanted and troublesome adipose depots. Typi-cally, the best candidates for liposuction are individuals who are close to their goal weight and have focal adipose deposits that are resistant to diet and exercise (Fig. 45-72). The suction

1	Typi-cally, the best candidates for liposuction are individuals who are close to their goal weight and have focal adipose deposits that are resistant to diet and exercise (Fig. 45-72). The suction cannula system removes adipose tissue by avulsing fat into the small holes located within the cannula tip. As the cannula is repeatedly passed throughout the adipose planes to remove the fat, one can often visualize and feel the reduction in the fat depot area treated. In general, larger-diameter cannulas remove adi-pose tissue at a faster rate yet carry a higher risk of causing contour irregularities such as grooving and/or uneven removal of fat. Newer liposuction technologies employing ultrasonic or laser probes to heat and emulsify fat via cavitation before suc-tion are gaining increasing application because they also aid in better tightening of the overlying skin envelope. However, use of these technologies also increases the chance and incidence of tissue damage and injury from the heat

1	because they also aid in better tightening of the overlying skin envelope. However, use of these technologies also increases the chance and incidence of tissue damage and injury from the heat of the cannula and can cause burn injury to skin and underlying structures.A major advance in the field of liposuction involves appli-cation of tumescent local anesthesia. This method involves the infiltration of very dilute lidocaine and epinephrine (lidocaine 0.05% and epinephrine 1:1,000,000) in large volumes through-out the subcutaneous tissues prior to suction removal of fatty tissue. Tumescent volumes can range from one to three times the anticipated aspirate volume. The dilute lidocaine provides sufficient anesthesia to allow the liposuction to be performed without additional agents in some instances. However, in cases where large volumes of fat are to be removed or in cases where multiple sites are to be addressed, then sedation and/or general anesthesia is often preferred. With tumescent

1	However, in cases where large volumes of fat are to be removed or in cases where multiple sites are to be addressed, then sedation and/or general anesthesia is often preferred. With tumescent anesthesia, the absorption of the dilute lidocaine from the subcutaneous tissue is very slow, with peak plasma concentrations occurring approx-imately 10 hours after the procedure. Therefore, the standard lidocaine dosing limit of 7 mg/kg may be safely exceeded. Cur-rent recommendations suggest a limit of 35 mg/kg of lidocaine with tumescent anesthesia. A very important component of the tumescent anesthetic solution is diluted epinephrine, which has proved to limit blood loss during the procedure.Safety issues are paramount for liposuction because of potential fluid shifts postoperatively and hypothermia. If ≥5000 mL of aspirate is to be removed, the procedure should be Brunicardi_Ch45_p1967-p2026.indd 202201/03/19 6:32 PM 2023PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45ABCFigure 45-72. A and

1	If ≥5000 mL of aspirate is to be removed, the procedure should be Brunicardi_Ch45_p1967-p2026.indd 202201/03/19 6:32 PM 2023PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45ABCFigure 45-72. A and B. Preoperative photos of a 22-year-old woman with focal adipose deposits on the trunk and extremities. C. Patient 3 months after surgery.Brunicardi_Ch45_p1967-p2026.indd 202301/03/19 6:32 PM 2024SPECIFIC CONSIDERATIONSPART IIperformed in an accredited acute care hospital facility. After the procedure, vital signs and urinary output should be monitored overnight in an appropriate facility by qualified and competent staff familiar with perioperative care of the liposuction patient.Autologous Fat GraftingThe concept of reinjecting fat tissue harvested by liposuction has been put into practice for decades. Key to the technique is a pro-cessing step in which the sterilely collected fat is separated from the aqueous (primarily tumescent fluid) and free lipid fractions. This can be done by

1	for decades. Key to the technique is a pro-cessing step in which the sterilely collected fat is separated from the aqueous (primarily tumescent fluid) and free lipid fractions. This can be done by centrifugation and/or filtering. Ideally, the prepared adipose grafts are then injected into the tissues using specially designed blunt-tipped cannulas that provide for micro-graft injection. Small aliquots of fat grafts are injected with each cannula pass to deposit the grafts within the vascularized tissues of the recipient bed. Autologous fat grafting has gained increased interest and has been applied to various areas of aesthetic and reconstructive surgery. Specific applications include fat grafting to augment areas where fat atrophy is commonplace (aging of the face or hands), to enhance breast aesthetics and/or other breast reconstruction techniques, gluteal augmentation, or to address contour deformities or irregularities caused by iatrogenic, trau-matic, oncologic, or congenital

1	breast aesthetics and/or other breast reconstruction techniques, gluteal augmentation, or to address contour deformities or irregularities caused by iatrogenic, trau-matic, oncologic, or congenital processes.REFERENCESEntries highlighted in bright blue are key references. 1. Martin, Andrew J. (2005-07-27). “Academy Papyrus to be Exhibited at the Metropolitan Museum of Art” (Press release). The New York Academy of Medicine. Archived from the origi-nal on November 27, 2010. 2. Borges AF, Alexander JE. Relaxed skin tension lines, Z-plasties on scars, and fusiform excision of lesions. Br J Plast Surg. 1962;15:242-254. 3. Wilhelmi BJ, Blackwell SJ, Phillips LG. Langer’s lines: to use or not to use. Plast Reconstr Surg. 1999;104:208-214. 4. Staylor A. Wound care devices: growth amid uncertainty. Med Tech Insight. 2009;11(1):32-47. 5. Baronio G. On Grafting in Animals. Boston: Boston Medical Library; 1985. This is a modern publication of the classic 18th century work by Guiseppi Baronio who

1	Med Tech Insight. 2009;11(1):32-47. 5. Baronio G. On Grafting in Animals. Boston: Boston Medical Library; 1985. This is a modern publication of the classic 18th century work by Guiseppi Baronio who studied skin grafting in animals. Baronio’s work represents the first preclinical animal study of a surgical procedure. The logo of the most important professional organization dedicated to plastic surgery research, the Plastic Surgery Research Council, is based on Baronio’s illustration of a sheep with multiple grafted areas of skin on the back. 6. Singh M, Nuutila K, Kruse C, Robson MC, Caterson E, Eriksson E. Challenging the conventional therapy: emerging skin graft techniques for wound healing. Plast Reconstruct Surg. 2015;136(4):524e-530e. 7. Sinha S, Schreiner AJ, Biernaskie J, Nickerson D, Gabriel VA. Treating pain on skin graft donor sites: review and clini-cal recommendations. J Trauma Acute Care Surg. 2017;83(5): 954-964. 8. Kagan RJ, Peck MD, Ahrenholz DH, et al. Surgical

1	D, Gabriel VA. Treating pain on skin graft donor sites: review and clini-cal recommendations. J Trauma Acute Care Surg. 2017;83(5): 954-964. 8. Kagan RJ, Peck MD, Ahrenholz DH, et al. Surgical manage-ment of the burn wound and use of skin substitutes: an expert panel white paper. J Burn Care Res. 2013;34(2):e60-e79. A variety of skin substitutes are available for repairing areas of skin loss from injuries such as deep partial-thickness or full-thickness burns. This article provides a nice summary of con-temporary options. 9. Azzopardi EA, Boyce DE, Dickson WA, et al. Application of topical negative pressure (vacuum-assisted closure) to split-thickness skin grafts: a structured evidence-based review. Ann Plast Surg. 2013;70(1):23-29. 10. Maciel-Miranda A, Morris SF, Hallock GG. Local flaps, including pedicled perforator flaps: anatomy, technique, and applications. Plast Reconstruct Surg. 2013;131(6): 896e-911e. 11. Kunert P. Structure and construction: the system of skin flaps. Ann

1	including pedicled perforator flaps: anatomy, technique, and applications. Plast Reconstruct Surg. 2013;131(6): 896e-911e. 11. Kunert P. Structure and construction: the system of skin flaps. Ann Plast Surg. 1991;27(6):509-516; discussion 517-518. 12. McGregor IA, Morgan G. Axial and random pattern flaps. Br J Plastic Surg. 1973;26(3):202-213. 13. Rajabi A, Dolovich AT, Johnston JD. From the rhombic transpo-sition flap toward Z-plasty: an optimized design using the finite element method. J Biomech. 2015;48(13):3672-3678. 14. Bakamjian VY, Long M, Rigg B. Experience with the medially based deltopectoral flap in reconstructive surgery of the head and neck. Br J Plast Surg. 1971;24(2):174-183. 16. Geddes CR, Morris SF, Neligan PC. Perforator flaps: evo-lution, classification, and applications. Ann Plast Surg. 2003;50(1):90-99. 17. Sinna R, Boloorchi A, Mahajan AL, Qassemyar Q, Robbe M. What should define a “perforator flap”? Plast Reconstr Surg. 2010;126(6):2258-2263. 18. Taylor GI,

1	Ann Plast Surg. 2003;50(1):90-99. 17. Sinna R, Boloorchi A, Mahajan AL, Qassemyar Q, Robbe M. What should define a “perforator flap”? Plast Reconstr Surg. 2010;126(6):2258-2263. 18. Taylor GI, Palmer JH. The vascular territories (angiosomes) of the body: experimental study and clinical applications. Br J Plast Surg. 1987;40(2):113-141. This is the classic article studying blood supply to the skin that introduced the angiosome concept and transformed our under-standing of the anatomic basis of surgical flap design. The blood supply was shown to be a continuous three-dimensional network of vessels in all tissue layers. The anatomical territory of a source artery corresponded in both the skin and deep tissues and gave rise to the angiosome concept. 19. Buchanan PJ, Kung TA, Cederna PS. Evidence-based medicine: wound closure. Plast Reconstr Surg. 2014;134(6):1391-1404. This is an excellent summary of the basic principles of wound healing. It explains the physiologic basis and rationale

1	medicine: wound closure. Plast Reconstr Surg. 2014;134(6):1391-1404. This is an excellent summary of the basic principles of wound healing. It explains the physiologic basis and rationale for vari-ous wound care methods, including dressings, negative pressure wound therapy, skin and dermal substitutes, and tissue expan-sion. This is basic knowledge that is important for all surgeons to understand. 20. Whitaker LA, Pashayan H, Reichman J. A proposed new classification of craniofacial anomalies. Cleft Palate J. 1981;18(3):161-176. 21. Monson LA, Kirschner RE, Losee JE. Primary repair of cleft lip and nasal deformity. Plast Reconstr Surg. 2013;132(6): 1040e-1053e. 22. Fattah AY. Craniofacial syndromes: genetics, embryology, and clinical relevance. In: Bentz ML, Bauer BS, Zuker RM, eds. Principles & Practice of Pediatric Plastic Surgery. Boca Raton: CRC Press; 2016:393-452. 23. Hoffman WY, Fisher DM. Unilateral cleft lip repair. In: Bentz ML, Bauer BS, Zuker RM, eds. Principles &

1	Principles & Practice of Pediatric Plastic Surgery. Boca Raton: CRC Press; 2016:393-452. 23. Hoffman WY, Fisher DM. Unilateral cleft lip repair. In: Bentz ML, Bauer BS, Zuker RM, eds. Principles & Practice of Pediatric Plastic Surgery. Boca Raton: CRC Press; 2016: 453-478. 24. van Aalst JA, Kolappa KK, Sadove M. MOC-PSSM CME article: nonsyndromic cleft palate. Plast Reconstr Surg. 2008; 121(1 suppl):1-14. 25. Garfinkle JS, Grayson BH. Nasoalveolar molding and columella elongation in preparation for the primary repair of unilateral and bilateral cleft lip and palate. In: Losee JE, ed. Craniofacial, Head and Neck Surgery and Pediatric Plastic Surgery. Philadel-phia: Elsevier; 2013:1223-1251. 26. Kirschner REA, Losee JE. Lip adhesion. In: Losee J, Kirschner RE, eds. Comprehensive Cleft Care. Boca Raton, FL: CRC Press; 2016:781-792. This is the definitive textbook on pediatric plastic surgery that covers each aspect in depth. 27. Hoffman WY. Cleft palate. In: Losee JE, ed. Craniofacial,

1	Boca Raton, FL: CRC Press; 2016:781-792. This is the definitive textbook on pediatric plastic surgery that covers each aspect in depth. 27. Hoffman WY. Cleft palate. In: Losee JE, ed. Craniofacial, Head and Neck Surgery and Pediatric Plastic Surgery. Philadelphia: Elsevier; 2013:568-583.Brunicardi_Ch45_p1967-p2026.indd 202401/03/19 6:32 PM 2025PLASTIC AND RECONSTRUCTIVE SURGERYCHAPTER 45 28. Moe KS, Murr AH, Wester ST. Orbital Fractures. Facial Plast Surg Clin North Am. 2018 May;26(2):237-251. doi: 10.1016/j.fsc.2017.12.007. Review. PubMed PMID: 29636153. 29. Fattah AY. Craniofacial syndromes: genetics, embryology, and clinical relevance. In: Bentz ML, Bauer BS, Zuker RM, eds. Principles & Practice of Pediatric Plastic Surgery. Boca Raton: CRC Press; 2016:393-452. 30. Patel PK, Kawamoto HK, Jr. Atypical craniofacial clefts. In: Bentz ML, Bauer BS, Zuker RM, eds. Principles & Prac-tice of Pediatric Plastic Surgery. Boca Raton: CRC Press; 2016:663-723. 31. Tessier P. Anatomical

1	HK, Jr. Atypical craniofacial clefts. In: Bentz ML, Bauer BS, Zuker RM, eds. Principles & Prac-tice of Pediatric Plastic Surgery. Boca Raton: CRC Press; 2016:663-723. 31. Tessier P. Anatomical classification facial, cranio-facial and latero-facial clefts. J Maxillofac Surg. 1976;4(2):69-92. 32. Monasterio FO, Taylor JA. Major craniofacial clefts: case series and treatment philosophy. Plast Reconstr Surg. 2008;122(2):534-543. 33. Forrest CR, Nguyen PD, Smith DM. Craniosynostosis. In: Bentz ML, Bauer BS, Zuker RM, eds. Principles & Practice of Pedi-atric Plastic Surgery. Boca Raton: CRC Press; 2016:595-647. 34. Fearon JA. Evidence-based medicine: craniosynostosis. Plast Reconstr Surg. 2014;133(5):1261-1275. 35. Persing JA. MOC-PS(SM) CME article: management consider-ations in the treatment of craniosynostosis. Plast Reconstr Surg. 2008;121(4 suppl):1-11. 36. Taylor JA, Bartlett SP. What’s new in syndromic craniosynosto-sis surgery? Plast Reconstr Surg. 2017;140(1):82e-93e. 37. Vaienti

1	of craniosynostosis. Plast Reconstr Surg. 2008;121(4 suppl):1-11. 36. Taylor JA, Bartlett SP. What’s new in syndromic craniosynosto-sis surgery? Plast Reconstr Surg. 2017;140(1):82e-93e. 37. Vaienti L, Soresina M, Menozzi A. Parascapular free flap and fat grafts: combined surgical methods in morphological resto-ration of hemifacial progressive atrophy. Plast Reconstr Surg. 2005;116(3):699-711. 38. Evans KN, Sie KC, Hopper RA, Glass RP, Hing AV, Cunning-ham ML. Robin sequence: from diagnosis to development of an effective management plan. Pediatrics. 2011;127(5):936-948. 39. Kirschner RE, Low DW, Randall P, et al. Surgical airway man-agement in Pierre Robin sequence: is there a role for tongue-lip adhesion? Cleft Palate Craniofac J. 2003;40(1):13-18. 40. Overdiek A, Feifel H, Schaper J, Mayatepek E, Rosenbaum T. Diagnostic delay of NF1 in hemifacial hypertrophy due to plexiform neurofibromas. Brain Dev. 2006;28(5):275-280. 41. Ricalde P, Magliocca KR, Lee JS. Craniofacial fibrous

1	Mayatepek E, Rosenbaum T. Diagnostic delay of NF1 in hemifacial hypertrophy due to plexiform neurofibromas. Brain Dev. 2006;28(5):275-280. 41. Ricalde P, Magliocca KR, Lee JS. Craniofacial fibrous dyspla-sia. Oral Maxillofac Surg Clin North Am. 2012;24(3):427-441. 42. Mulliken JB, Glowacki J. Hemangiomas and vascular malfor-mations in infants and children: a classification based on endo-thelial characteristics. Plast Reconstr Surg. 1982;69(3):412-422. 43. Greene AK, Phillips JH. Vascular anomalies. In: Bentz ML, Bauer BS, Zuker RM, eds. Principles & Practice of Pediatric Plastic Surgery. Boca Raton: CRC Press; 2016:199-230. 44. Biswas A, Pan X, Meyer M, et al. Urinary excretion of microRNA-126 is a biomarker for hemangioma proliferation. Plast Reconstr Surg. 2017;139(6):1277e-1284e. 45. Iacobas I, Burrows PE, Frieden IJ, et al. LUMBAR: association between cutaneous infantile hemangiomas of the lower body and regional congenital anomalies. J Pediatr. 2010;157(5):

1	I, Burrows PE, Frieden IJ, et al. LUMBAR: association between cutaneous infantile hemangiomas of the lower body and regional congenital anomalies. J Pediatr. 2010;157(5): 795-801.e1-e7. 46. Taylor CW, Horgan K, Dodwell D. Oncological aspects of breast reconstruction. Breast. 2005 Apr;14(2):118-30. Review. PubMed PMID: 15767181. 47. Nicholas Zdenkowski, Butow P, Tesson S, Boyle F. A system-atic review of decision aids for patients making a decision about treatment for early breast cancer. Breast. 2016 Apr;26:31-45. doi: 10.1016/j.breast.2015.12.007. Epub 2016 Jan 8. Review. PubMed PMID: 27017240. 48. Cho BC, McCready DR. Oncologic principles in breast recon-struction. Clin Plast Surg. 2007 Jan;34(1):1-13; abstract v. Review. PubMed PMID: 17307067. 49. Jacob AG, Driscoll DJ, Shaughnessy WJ, Stanson AW, Clay RP, Gloviczki P. Klippel-Trenaunay syndrome: spectrum and man-agement. Mayo Clin Proc. 1998;73(1):28-36. 50. Arneja JS, Gosain AK. Giant congenital melanocytic nevi. Plast Reconstr

1	Stanson AW, Clay RP, Gloviczki P. Klippel-Trenaunay syndrome: spectrum and man-agement. Mayo Clin Proc. 1998;73(1):28-36. 50. Arneja JS, Gosain AK. Giant congenital melanocytic nevi. Plast Reconstr Surg. 2009;124(1 suppl):1e-13e. 51. Arad E, Zuker RM. The shifting paradigm in the management of giant congenital melanocytic nevi: review and clinical appli-cations. Plast Reconstr Surg. 2014;133(2):367-376. 52. Millard DR. Principlization of Plastic Surgery. 1st ed. Boston/Toronto: Little, Brown; 1986. 53. Corcoran J, Bauer BS. Cutaneous lesions. In: Bentz ML, Bauer BS, Zuker RM, eds. Principles & Practice of Pediatric Plastic Surgery. Boca Raton: CRC Press; 2016:453-478. 54. Bosse MJ et al. An analysis of outcomes of reconstruction or amputation after leg-threatening injuries. N Engl J Med. 2002;347(24):1924-1931. 55. Gustilo RB, Merkow RL, Templeman D. The management of open fractures. J Bone Joint Surg. 1990;72(2):299-304. 56. Crowley DJ, Kanakaris NK, Giannoudis PV. Debridement and

1	2002;347(24):1924-1931. 55. Gustilo RB, Merkow RL, Templeman D. The management of open fractures. J Bone Joint Surg. 1990;72(2):299-304. 56. Crowley DJ, Kanakaris NK, Giannoudis PV. Debridement and wound closure of open fractures: the impact of the time factor on infection rates. Injury. 2007;38(8):879-889. 57. Cho EH, Shammas RL, Carney MJ, et al. Muscle versus fascio-cutaneous free flaps in lower extremity traumatic reconstruc-tion: a multicenter outcomes analysis. Plast Reconstr Surg. 2018;141(1):191-199. 58. Yazar S, Lin CH, Wei FC. One-stage reconstruction of compos-ite bone and soft-tissue defects in traumatic lower extremities. Plast Reconstr Surg. 2004;114(6):1457-1466. 59. Gurney JK(1), Stanley J(2), York S(3), Rosenbaum D(4), Sar-fati D(2). Risk of lower limb amputation in a national preva-lent cohort of patients with diabetes. Diabetologia. 2018 Mar;61(3):626-635. doi: 10.1007/s00125-017-4488-8. Epub 2017 Nov 3. 60. Wukich DK, Raspovic KM. What Role Does Function Play in

1	preva-lent cohort of patients with diabetes. Diabetologia. 2018 Mar;61(3):626-635. doi: 10.1007/s00125-017-4488-8. Epub 2017 Nov 3. 60. Wukich DK, Raspovic KM. What Role Does Function Play in Deciding on Limb Salvage versus Amputation in Patients With Diabetes? Plast Reconstr Surg. 2016 Sep;138(3 Suppl):188S-95S. doi: 10.1097/PRS.0000000000002713. Review. PubMed PMID: 27556759. 61. Nelson JA, Disa JJ. Breast reconstruction and radiation therapy: an update. Plast Reconstr Surg. 2017;140:60S-68S. Radiation therapy has an adverse effect on all forms of breast reconstruction. The need for radiation therapy affects the opti-mal timing and technique for breast reconstructive surgery. It is helpful for all surgeons caring for breast cancer patients to have an understanding of the issues involved, and this paper provides an excellent summary of the issues surrounding breast reconstruction and radiation therapy. 62. Weichman KE, Matros E, Disa JJ. Reconstruction of peripelvic oncologic

1	and this paper provides an excellent summary of the issues surrounding breast reconstruction and radiation therapy. 62. Weichman KE, Matros E, Disa JJ. Reconstruction of peripelvic oncologic defects. Plast Reconstr Surg. 2017;140(4):601e-612e. General surgeons often encounter problems in the perineum. This article offers an excellent summary of how to manage surgical problems in this region. It provides a review of anat-omy, the types of problems encountered, and appropriate local, regional, or free-flap options based on the location of the defect and donor-site characteristics. 63. Cushing CA, Phillips LG. Evidence-based medicine: pres-sure sores. Plast Reconstr Surg. 2013;132(6):1720-1732. Pressure sores are a common problem affecting surgical patients of all types, and it is important for all surgeons to understand how to prevent and treat them. This paper provides an excellent overview of the problem, with emphasis on risk factors, patho-physiology, classification, and treatment

1	for all surgeons to understand how to prevent and treat them. This paper provides an excellent overview of the problem, with emphasis on risk factors, patho-physiology, classification, and treatment options. Most impor-tantly, it reviews steps for the prevention of pressure sores.64. Edsberg LE, Black JM, Goldberg M, McNichol L, Moore L, Sieggreen M. Revised National Pressure Ulcer Advisory Panel pressure injury staging system: revised pressure injury staging system. J Wound Ostomy Continence Nurs. 2016;43(6):585-597. 65. Centers for Disease Control and Prevention. 2017 National Diabetes Statistics Report, 2017. Available at: https://www.cdc.gov/diabetes/data/statistics/statistics-report.html. Accessed January 20, 2019.Brunicardi_Ch45_p1967-p2026.indd 202501/03/19 6:32 PM 2026SPECIFIC CONSIDERATIONSPART II 66. Clemens MW, Attinger CE, Colen LB. Foot reconstruction. In: Mathes SJ, ed. Plastic Surgery. 2nd ed. Philadelphia: Elsevier; 2006:1403. 67. Hinchliffe RJ, Andros G, Apelqvist

1	CONSIDERATIONSPART II 66. Clemens MW, Attinger CE, Colen LB. Foot reconstruction. In: Mathes SJ, ed. Plastic Surgery. 2nd ed. Philadelphia: Elsevier; 2006:1403. 67. Hinchliffe RJ, Andros G, Apelqvist J, et al. A systematic review of the effectiveness of revascularization of the ulcerated foot in patients with diabetes and peripheral arterial disease. Diabetes Metab Res Rev. 2012;28(suppl 1):179-217. 68. Johnson SK, Podratz KE, Dipboye RL, Gibbons E. Physi-cal attractiveness biases in ratings of employment suitability: tracking down the “beauty is beastly” effect. J Soc Psychol. 2010;150(3):301-318. 69. Jacono A, Chastant RP, Dibelius G. Association of patient self-esteem with perceived outcome after face-lift surgery. JAMA Facial Plast Surg. 2016;18(1):42-46. 70. Schwitzer JA, Sher SR, Fan KL, Scott AM, Gamble L, Baker SB. Assessing patient-reported satisfaction with appearance and quality of life following rhinoplasty using the FACE-Q appraisal scales. Plast Reconstr Surg.

1	SR, Fan KL, Scott AM, Gamble L, Baker SB. Assessing patient-reported satisfaction with appearance and quality of life following rhinoplasty using the FACE-Q appraisal scales. Plast Reconstr Surg. 2015;135(5):830e-837e. 71. Papadopulos NA, Niehaus R, Keller E, et al. The psychologic and psychosocial impact of otoplasty on children and adults. J Craniofac Surg. 2015;26(8):2309-2314. 72. McGrath MH. The psychological safety of breast implant sur-gery. Plast Reconstr Surg. 2007;120(7 suppl 1):103S-109S. 73. Papadopulos NA, Staffler V, Mirceva V, et al. Does abdomino-plasty have a positive influence on quality of life, self-esteem, and emotional stability? Plast Reconstr Surg. 2012;129(6):957e-962e. 74. Shridharani SM, Magarakis M, Manson PN, Rodriguez ED. Psychology of plastic and reconstructive surgery: a systematic clinical review. Plast Reconstr Surg. 2010;126(6):2243-2251. 75. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders. 5th ed. Arlington,

1	surgery: a systematic clinical review. Plast Reconstr Surg. 2010;126(6):2243-2251. 75. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders. 5th ed. Arlington, VA: American Psychiatric Association; 2013.Brunicardi_Ch45_p1967-p2026.indd 202601/03/19 6:32 PM

1	Anesthesia for Surgical PatientsJunaid Nizamuddin and Michael O’Connor 46chapterBRIEF HISTORY OF ANESTHESIAThe discovery of anesthesia is one of the seminal American con-tributions to the world. Along with infection control and blood transfusion, anesthesia has enabled surgery to occupy its fundamental place in medicine. Before the advent of anes-thesia in the 1840s, many substances and methods had been tried in the search for pain relief and better operating conditions. Patients were typically restrained by several attendants, and only the most stoic could tolerate the screams heard in the oper-ating theater.BeginningsHorace Wells (1815–1848), a dentist, first pursued using nitrous oxide for the relief of pain in surgical procedures in 1844.1 After experimenting on himself, Wells attempted to demonstrate the analgesic effects of nitrous oxide for a dental procedure at Harvard Medical School in 1845. The public demonstration was a failure, at least partially, due to improper

1	attempted to demonstrate the analgesic effects of nitrous oxide for a dental procedure at Harvard Medical School in 1845. The public demonstration was a failure, at least partially, due to improper administration of the gas. Wells never recovered from his humiliating experience and eventually committed suicide. However, he does hold a place in history as the first person to recognize and use the only anesthetic from the 1800s that is still in use today—nitrous oxide.Ether DayWilliam Morton (1819–1868) was a dentist and partner of Horace Wells. After taking a course in anesthesia from Wells, Morton left the partnership in Hartford, Connecticut, and established himself in Boston. He continued his interest in anesthesia, but using diethyl ether instead of nitrous oxide. Ether proved a good choice. He practiced the administration of ether on a 11dog and then used it when extracting teeth from patients in his office. On October 16, 1846, Morton gave the first pub-lic demonstration of ether

1	He practiced the administration of ether on a 11dog and then used it when extracting teeth from patients in his office. On October 16, 1846, Morton gave the first pub-lic demonstration of ether as an anesthetic for Johns Collins Warren, a distinguished surgeon and one of the founders of Massachusetts General Hospital. In attendance in the surgi-cal amphitheater were several surgeons, medical students, and a newspaper reporter. After induction of anesthesia, Warren successfully removed a vascular mass from the patient’s neck with no ill effects (Fig. 46-1). The description of this public demonstration of ether was published in the Boston Medical and Surgical Journal (now The New England Journal of Medicine).2 The stature of Warren lent considerable credence to the advent of surgical anesthesia. The news spread rapidly, and surgeons around the world were quick to adopt this new invention. Massachusetts General Hospital has restored and preserved the original amphi-theater where the

1	The news spread rapidly, and surgeons around the world were quick to adopt this new invention. Massachusetts General Hospital has restored and preserved the original amphi-theater where the demonstration took place, now called the Ether Dome. The description of the public demonstration of ether was voted as the most important article published in the history of The New England Journal of Medicine in its first 200 years.3The Modern EraAnesthesia has developed rapidly over the past century. Inhaled anesthetics, initially discovered fortuitously by observation, have been synthetically produced and remain the mainstay of anesthetic maintenance. The advent of the hollow syringe and needle and discovery of rapidly acting of intravenous anesthet-ics allowed for rapid induction of anesthesia. Development of endotracheal intubation and mechanical ventilation revolution-ized the delivery of inhaled anesthetics. The discovery of local anesthetics led to the development of peripheral nerve blocks

1	of endotracheal intubation and mechanical ventilation revolution-ized the delivery of inhaled anesthetics. The discovery of local anesthetics led to the development of peripheral nerve blocks Brief History of Anesthesia 2027Beginnings / 2027Ether Day / 2027The Modern Era / 2027Basic Pharmacology 2028Pharmacokinetics and Pharmacodynamics / 2028Administration, Distribution, Metabolism, and Elimination / 2029Pharmacodynamics / 2029Potency, Efficacy, Lethal Dose, and Therapeutic Index / 2029Anesthetic Agents 2029Inhaled Anesthetics / 2029Local Anesthetics / 2031Neuromuscular Blockers / 2031Anesthetic Monitoring 2032Perioperative Evaluation and Preparation 2033ASA Physical Status Assessment / 2034Airway Evaluation / 2034Cardiovascular Disease / 2034Pulmonary Disease / 2035Renal Disease / 2036Hepatic Disease / 2036Endocrine Disease / 2036Preoperative Fasting / 2036Patients With Advanced Directives / 2036Risk Estimation / 2036Intraoperative Management 2036General Anesthesia /

1	/ 2036Hepatic Disease / 2036Endocrine Disease / 2036Preoperative Fasting / 2036Patients With Advanced Directives / 2036Risk Estimation / 2036Intraoperative Management 2036General Anesthesia / 2036Monitored Anesthesia Care / 2039Regional Anesthesia/Acute Pain / 2039Recovery And Complications 2039The Postanesthesia Care Unit / 2039Enhanced Recovery After Surgery Pathways / 2039Acute Postoperative Pain / 2039Malignant Hyperthermia / 2040Cardiovascular Complications / 2040Respiratory Failure / 2040Neurologic and Psychiatric Complications / 2040Conclusion 2040Brunicardi_Ch46_p2027-p2044.indd 202701/03/19 11:03 AM 2028Figure 46-1. Robert Cutler Hinckley, The First Operation with Ether. (Reproduced with permission from Boston Medical Library in the Francis A. Countway Library of Medicine, Boston, Massachusetts.)Key Points1 The discovery of anesthesia was one of the most important advances and has enabled surgery to occupy its fundamental place in medicine.2 Advances in anesthetic

1	Boston, Massachusetts.)Key Points1 The discovery of anesthesia was one of the most important advances and has enabled surgery to occupy its fundamental place in medicine.2 Advances in anesthetic monitoring have made the admin-istration of anesthesia safer than ever. Types of cardiovas-cular monitors include arterial catheters, central venous and pulmonary artery catheters, and transesophageal echocardiography.3 A detailed preoperative evaluation should be performed on each patient when circumstances allow, with special atten-tion devoted to functional status. The American College of Cardiology/American Heart Association guidelines for pre-operative evaluation can guide workup.4 The American Society of Anesthesiologists has developed specific guidelines for preoperative fasting to mitigate the risk of aspiration of gastric contents; individual patients may need more stringent preoperative fasting periods and/or rapid sequence inductions.5 The American Society of Anesthesiologists has

1	the risk of aspiration of gastric contents; individual patients may need more stringent preoperative fasting periods and/or rapid sequence inductions.5 The American Society of Anesthesiologists has developed an algorithm for management of the difficult airway. Nota-bly, in patients in whom both intubation and ventilation are impossible, the algorithm calls for placement of a laryngeal mask airway as the next step.and spinal anesthesia. Concurrently, physiologic monitoring techniques have advanced to make the administration of anes-thesia safer than ever.Initially, anesthesia was given by medical students, nurses, and dentists, but eventually became a physician specialty of medicine of its own. The American Board of Anesthesiology was formed in 1938. Over the past 50 years, anesthesiology has increasingly specialized and also spread outside the operating room into critical care, pain management, and perioperative medicine.BASIC PHARMACOLOGYPharmacokinetics and

1	50 years, anesthesiology has increasingly specialized and also spread outside the operating room into critical care, pain management, and perioperative medicine.BASIC PHARMACOLOGYPharmacokinetics and PharmacodynamicsPharmacodynamics is the study of what a drug does to the body; pharmacokinetics is the study of what the body does to a drug. Brunicardi_Ch46_p2027-p2044.indd 202801/03/19 11:04 AM 2029ANESTHESIA FOR SURGICAL PATIENTSCHAPTER 46The conduct of anesthesia is predicated upon the pharmacody-namics and pharmacokinetics of the drugs used.4Administration, Distribution, Metabolism, and EliminationAdministration of a drug affects its pharmacokinetics, as there will be different rates of drug entry into the circulation. For example, medications administered via the oral route are subject to first-pass effect of the portal circulation; this can be bypassed with the IV, nasal, or sublingual route. Other routes of drug administration include transdermal, intramuscular, subcutaneous,

1	to first-pass effect of the portal circulation; this can be bypassed with the IV, nasal, or sublingual route. Other routes of drug administration include transdermal, intramuscular, subcutaneous, or inhalation.Distribution is the delivery of a drug from the systemic circulation to the tissues. Once a drug has entered the systemic circulation, the rate at which it will enter the tissues depends on the blood flow into that tissue, as well as the molecular size of the drug, lipid solubility, capillary permeability, polarity, plasma protein and tissue binding, and volume of distribution, the fluid volume in which the drug distributes. The distribution or redis-tribution of drugs can play a critical role in shaping their clinical use. For instance, clinically, the effect of propofol is terminated by its redistribution into fatty tissues and not metabolism of the drug.Metabolism is the permanent breakdown of original compounds into smaller metabolites. Drug elimination varies widely; some

1	by its redistribution into fatty tissues and not metabolism of the drug.Metabolism is the permanent breakdown of original compounds into smaller metabolites. Drug elimination varies widely; some drugs are excreted unchanged by the body, some decompose via plasma enzymes, and some are degraded in the liver. Many drugs rely on multiple pathways for metabolism and elimination (i.e., metabolized by liver enzymes and then excreted by the kidney).Context-sensitive half time is the time required for blood concentrations of a drug to decrease by 50% after its discontinu-ation, which is determined by the interaction of the duration of administration, distribution and accumulation, and metabolism and excretion. Fig. 46-2 illustrates the context sensitive half-time for commonly used anesthetics and opioids.4PharmacodynamicsPharmacodynamics, or how the plasma concentration of a drug translates into its effect on the body, depends on biologic vari-ability, receptor physiology, and clinical

1	and opioids.4PharmacodynamicsPharmacodynamics, or how the plasma concentration of a drug translates into its effect on the body, depends on biologic vari-ability, receptor physiology, and clinical evaluations of the actual drug. An agonist is a drug that causes a response (acti-vates a receptor). A full agonist produces the full receptor/tissue response, and a partial agonist elicits less than the maximum response induced by a full agonist. An antagonist is a drug that blocks agonist mediated responses. An additive effect means that a second drug acts with the first drug and will produce an effect that is equal to the algebraic summation of both drugs. A synergistic effect means that two drugs interact to produce an effect that is greater than expected from the two drugs’ alge-braic summation. Tolerance, desensitization, or tachyphylaxis occurs when a larger than expected dose is required to produce a response. Tolerance usually results from chronic drug exposure, either through

1	summation. Tolerance, desensitization, or tachyphylaxis occurs when a larger than expected dose is required to produce a response. Tolerance usually results from chronic drug exposure, either through enzyme induction (e.g., alcohol) or depletion of neurotransmitters (e.g., cocaine).4Potency, Efficacy, Lethal Dose, and Therapeutic IndexThe potency of a drug is the dose required to produce a given effect, such as pain relief or a change in heart rate. The aver-age sensitivity to a particular drug can be expressed through the calculation of the effective dose; ED50 would have the desired effect in 50% of the general population. The efficacy of any therapeutic agent is its power to produce a desired effect. Two drugs may have the same efficacy but different potencies. Dose-response curves show the relationship between the dose of a drug administered (or the resulting plasma concentration) and the pharmacologic effect of the drug. The lethal dose (LD50) of a drug produces death in 50% of

1	the relationship between the dose of a drug administered (or the resulting plasma concentration) and the pharmacologic effect of the drug. The lethal dose (LD50) of a drug produces death in 50% of animals to which it is given, and the toxic dose (TD50) is the dose that elicits a toxicity in 50% of humans to which it is given. The ratio of the toxic dose and effective dose, TD50/ED50, is the therapeutic index. A drug with a high therapeutic index is safer than a drug with a low or narrow therapeutic index.4ANESTHETIC AGENTSInhaled AnestheticsInhaled anesthetics have greatly advanced since the original dem-onstration with ether. Modern agents provide faster induction and emergence and provide all of the major characteristics of general anesthesia: unconsciousness, analgesia, and muscle relaxation.Minimum alveolar concentration (MAC) is a measure of anesthetic potency. It is the ED50 of an inhaled agent (i.e., the dose required to prevent movement in response to skin incision in 50% of

1	alveolar concentration (MAC) is a measure of anesthetic potency. It is the ED50 of an inhaled agent (i.e., the dose required to prevent movement in response to skin incision in 50% of patients). The higher the MAC, the less potent an agent is. Advantages and disadvantages of inhaled anesthetics are shown in Table 46-1.5Nitrous Oxide. Nitrous oxide has a low solubility and is a weak anesthetic agent, but it has the most rapid onset and offset. Because 600500400300Duration of infusion (min)2001000020406080100120140Time required for plasmalevels to drop by 50% (min)AEtomidateMidazolamPropofolSufentanilFentanylRemifentanil600500400300Duration of infusion (min)2001000020406080100120140Time required for plasmalevels to drop by 50% (min)BFigure 46-2. Context-sensitive half time for commonly used anesthetics (A) and opioids (B). The vertical axis represents the half-time, or the time required for plasma concentrations of the drug to drop by 50%. The horizontal axis represents the duration of

1	anesthetics (A) and opioids (B). The vertical axis represents the half-time, or the time required for plasma concentrations of the drug to drop by 50%. The horizontal axis represents the duration of a continuous infusion. (Reproduced with permission from Longnecker DE, Mackey SC, Newman MF, et al: Anesthesiology, 3rd ed. New York, NY: McGraw-Hill Education; 2018.)Brunicardi_Ch46_p2027-p2044.indd 202901/03/19 11:04 AM 2030SPECIFIC CONSIDERATIONSPART IITable 46-1Advantages and disadvantages of inhaled anestheticsANESTHETICADVANTAGESDISADVANTAGESNitrous OxideNo odor, taste, or pungencyRapid uptake and eliminationAnalgesic effectMinimal cardiovascular depressionMinimal biotransformationInexpensiveAirspace expansionIncreased nausea and vomitingInhibits methionine synthaseEnvironmental pollutantSupports combustionIsofluraneGood muscle relaxationBronchodilationStable heart rateInexpensiveSlow uptake and eliminationSevoflurane Rapid uptake and eliminationNot pungentBreakdown to compound A

1	combustionIsofluraneGood muscle relaxationBronchodilationStable heart rateInexpensiveSlow uptake and eliminationSevoflurane Rapid uptake and eliminationNot pungentBreakdown to compound A in circuitMore expensive than isofluraneDesflurane Rapid uptake and eliminationVery low biotransformationAirway irritantRequires electric/heated vaporizerExpensiveData from Longnecker DE, Brown DL, Newman MF, et al: Anesthesiology, 2nd ed. New York, NY: McGraw-Hill Ediucation; 2012.its MAC is 104%, it must be combined with other agents to pro-vide general anesthesia. A recent randomized controlled trial demonstrated that use of 70% nitrous oxide, given as part of a general anesthetic, did not increase the risk of death or major adverse cardiovascular events.6 Nitrous oxide has been shown to increase the rate of postoperative nausea and vomiting (PONV).Volatile Agents. The volatile inhaled anesthetics are gener-ally more soluble in blood than nitrous oxide, are more potent, and thus require lower

1	the rate of postoperative nausea and vomiting (PONV).Volatile Agents. The volatile inhaled anesthetics are gener-ally more soluble in blood than nitrous oxide, are more potent, and thus require lower inspired concentrations. Currently, iso-flurane, sevoflurane, and desflurane are the agents that are most commonly used. Older agents, including halothane, which was associated with hepatotoxicity, are no longer widely used.Isoflurane is the most inexpensive and widely available of the agents currently used. Sevoflurane has relatively rapid uptake and elimination. It is also not pungent and is therefore commonly used for inhalational induction. Desflurane has the most rapid uptake and elimination of the three most commonly used volatile agents. It is also the most expensive, requires a heated-electric vaporizer, and is an airway irritant. All of the volatile inhalational agents as well as the depolarizing neu-romuscular blocker succinylcholine are triggering agents for malignant

1	a heated-electric vaporizer, and is an airway irritant. All of the volatile inhalational agents as well as the depolarizing neu-romuscular blocker succinylcholine are triggering agents for malignant hyperthermia.5Intravenous Agents. Intravenous agents are used to produce unconsciousness, analgesia, muscle relaxation, and/or amnesia. They include barbiturates, propofol, benzodiazepines, ketamine, etomidate, opioids, and nonopioid analgesics.7Barbiturates Barbiturates used in anesthesia include thio-pental and methohexital. These drugs act as agonists at the γ-aminobutyric acid (GABA) receptor, which inhibit excitatory synaptic transmission. Clinically, they produce a rapid, smooth induction of general anesthesia and wear off quickly. They cause hypotension and myocardial depression in a dose-dependent manner. Barbiturates are seldom used in modern anesthesia, with the exception of methohexital, which is still commonly used dur-ing electroconvulsive therapy.Propofol Propofol is an

1	a dose-dependent manner. Barbiturates are seldom used in modern anesthesia, with the exception of methohexital, which is still commonly used dur-ing electroconvulsive therapy.Propofol Propofol is an alkylated phenol that inhibits synaptic transmission through its effects at the GABA receptor. It has a short duration, rapid recovery, and low incidence of nausea and vomiting. Consequently, it is the induction agent of choice. Propofol causes hypotension in a dose-dependent manner, and it should be used cautiously in patients with cardiac disease or hypovolemia. Continuous infusion of propofol is commonly used for sedation in the intensive care unit setting. Continuous infusions of propofol are also used for moderate-to-deep seda-tion for many procedures and are also commonly incorporated into total intravenous anesthetics (TIVA), most commonly for neurosurgical procedures. Propofol is an irritant and frequently causes pain on injection. Propofol also has anticonvulsant

1	incorporated into total intravenous anesthetics (TIVA), most commonly for neurosurgical procedures. Propofol is an irritant and frequently causes pain on injection. Propofol also has anticonvulsant properties.Benzodiazepines Benzodiazepines are most commonly used to reduce anxiety and produce amnesia. Midazolam, which has a rapid onset and relatively short duration of action, is by far the most commonly used benzodiazepine in anesthesia. Lorazepam and diazepam are still sometimes used as anxiolytics or amnes-tics. Benzodiazepines act as agonists at the GABAA receptor. They produce sedation, vasodilation, and respiratory depres-sion in a dose-dependent manner. They should be used with caution when given with opioids because a synergistic reaction causing respiratory depression is common. Oral midazolam is commonly used for anxiolysis in children. Benzodiazepines are excellent anticonvulsants and only rarely cause allergic reac-tions. Benzodiazepines should be administered cautiously in

1	midazolam is commonly used for anxiolysis in children. Benzodiazepines are excellent anticonvulsants and only rarely cause allergic reac-tions. Benzodiazepines should be administered cautiously in older adult patients due to the heightened risk of delayed awak-ening and postoperative delirium.Etomidate Etomidate is an imidazole derivative used for IV induction. Its rapid and almost complete hydrolysis to inactive metabolites results in rapid offset. Like the IV agents mentioned earlier, etomidate acts on the GABA receptor. Etomidate has little direct effect on cardiac output and heart rate; induction doses thus cause less reduction in blood pressure than seen with propofol. Etomidate is associated with pain on injection. Nota-bly, etomidate causes adrenal suppression,8,9 although whether a single dose of etomidate given at induction causes clinically relevant adrenal suppression remains controversial.10-13Dexmedetomidine Dexmedetomidine is an IV α2-adrenergic agonist, administered as

1	a single dose of etomidate given at induction causes clinically relevant adrenal suppression remains controversial.10-13Dexmedetomidine Dexmedetomidine is an IV α2-adrenergic agonist, administered as a continuous infusion, and has both sedative and analgesic properties. It is useful for sedation in an intensive care unit setting and as an adjunct to general anes-thesia, especially as part of a total intravenous anesthetic. Side effects include hypotension and bradycardia in a dose-dependent manner. It does not cause respiratory depression at commonly used doses and is thus particularly useful for procedural seda-tion for patients at high risk of respiratory complications. It is synergistic with opiates and thus can be used to facilitate an opiate-sparing anesthetic.14,15Ketamine Ketamine differs from the aforementioned IV agents in that it produces analgesia as well as amnesia. Its principal Brunicardi_Ch46_p2027-p2044.indd 203001/03/19 11:04 AM 2031ANESTHESIA FOR SURGICAL

1	differs from the aforementioned IV agents in that it produces analgesia as well as amnesia. Its principal Brunicardi_Ch46_p2027-p2044.indd 203001/03/19 11:04 AM 2031ANESTHESIA FOR SURGICAL PATIENTSCHAPTER 46action is on the N-methyl-d-aspartate (NMDA) receptor. It is a dissociative anesthetic, producing a cataleptic gaze with nystag-mus. Patients may experience delirium and hallucinations while regaining consciousness. The addition of benzodiazepines has been shown to reduce the incidence of these side effects. Ket-amine typically increases heart rate and blood pressure, which may cause myocardial ischemia in patients with coronary dis-ease. Ketamine is often used in acutely hypovolemic patients to maintain blood pressure via sympathetic stimulation. Impor-tantly, ketamine is a direct myocardial depressant in patients who are catecholamine depleted, and it can produce profound hypotension and low cardiac output in such patients. Ketamine is a bronchodilator and is sometimes used

1	myocardial depressant in patients who are catecholamine depleted, and it can produce profound hypotension and low cardiac output in such patients. Ketamine is a bronchodilator and is sometimes used as an induction agent in asthmatic patients. It can increase intracranial pressure and intraocular pressure, and thus its use in patients with trauma to the head and neck is controversial. Ketamine can be admin-istered intramuscularly to induce anesthesia in patients who would not tolerate an inhalational induction or IV placement, such as patients with developmental delay.Opioid Analgesics The commonly used opioids—morphine, codeine, hydromorphone, meperidine, and the fentanyl-based compounds—act on µ-receptors in the brain and spinal cord. The main side effects of opioids are euphoria, sedation, con-stipation, and respiratory depression, which also are mediated by µ-receptors in a dose-dependent fashion. Although opioids have differing potencies required for effective analgesia,

1	sedation, con-stipation, and respiratory depression, which also are mediated by µ-receptors in a dose-dependent fashion. Although opioids have differing potencies required for effective analgesia, equi-analgesic doses of opioids result in equal degrees of respiratory depression. Thus, there is no completely safe opioid analgesic, and no reason to suppose that one opioid is safer than another. The synthetic opioid fentanyl and its analogues sufentanil, alfentanil, and remifentanil are used in the operating room. They differ pharmacokinetically in their lipid solubility, tissue binding, and elimination profiles and thus have differing poten-cies and durations of action. Fentanyl, which is highly lipid-soluble, accumulates in tissues and exhibits a steep increase in its context-sensitive half-time with infusions. Remifentanil is remarkable in that it undergoes rapid hydrolysis that is unaf-fected by sex, age, weight, or renal or hepatic function, even after prolonged infusion. Alfentanil

1	with infusions. Remifentanil is remarkable in that it undergoes rapid hydrolysis that is unaf-fected by sex, age, weight, or renal or hepatic function, even after prolonged infusion. Alfentanil and sufentanil are seldom used, having largely been replaced by remifentanil in the mod-ern era. Morphine and meperidine have active metabolites that are renally excreted and thus should be used with caution or avoided in patients with renal insufficiency.Naloxone, an opioid antagonist, can be used to rapidly reverse the effects of opioids, and is commonly used to rescue patients from opioid-associated respiratory depression. Nalox-one is poorly absorbed orally and is also often combined with oral opioids to prevent abuse by injection use of the combined drug. Methylnaltrexone and alvimopan are both peripheral opi-oid antagonists and can reverse the opioid side effect of consti-pation without affecting analgesia.16,17Nonopioid Analgesics Ketorolac is a parenteral nonsteroidal anti-inflammatory

1	peripheral opi-oid antagonists and can reverse the opioid side effect of consti-pation without affecting analgesia.16,17Nonopioid Analgesics Ketorolac is a parenteral nonsteroidal anti-inflammatory drug (NSAID) that produces analgesia by reducing prostaglandin formation via inhibition of the enzyme cyclooxygenase (COX). Intraoperative use of ketorolac reduces postoperative need for opioids. Ketorolac along with other NSAIDs can cause major side effects, including bleeding, plate-let dysfunction, and acute kidney injury and should be used cau-tiously in elderly patients or patients with renal insufficiency.Acetaminophen is an analgesic drug and antipyretic; its site of action is in the central nervous system. Use of acetaminophen has been shown to reduce opioid requirements postoperatively. Long available in an orally administered form as well as a rec-tal suppository, an intravenous formulation of acetaminophen is now available which has become widely used in the postopera-tive

1	Long available in an orally administered form as well as a rec-tal suppository, an intravenous formulation of acetaminophen is now available which has become widely used in the postopera-tive setting.18Lidocaine is a local anesthetic commonly used for local infiltration, nerve blocks, or epidural infusions. Recently, intra-venous infusions of lidocaine have been shown to be beneficial in the perioperative period. A large meta-analysis of 42 trials with 2800 patients showed that intravenous lidocaine infusions modestly reduced postoperative pain, reduced opioid require-ments, and shortened time to recovery of bowel function for patients undergoing abdominal surgery.19Local AnestheticsLocal anesthetics act on sodium channels to block transmission of neural impulses. They are divided into two groups based on their chemical structure: the amides and the esters. In general, the amides are metabolized in the liver, and the esters are metabo-lized by plasma cholinesterases, which yield

1	two groups based on their chemical structure: the amides and the esters. In general, the amides are metabolized in the liver, and the esters are metabo-lized by plasma cholinesterases, which yield metabolites with slightly higher allergic potential than the amides. Amides include lidocaine, bupivacaine, mepivicaine, prilocaine, and ropivicaine. Lidocaine has a fairly rapid onset and is shorter acting. Ropivic-aine and bupivacaine have a slower onset and are longer lasting. All three are commonly used for local infiltration and regional nerve blocks. Amides are 95% metabolized in the liver, with a minority excreted unchanged in the kidneys. Prilocaine and mepivicaine are seldom used in anesthesia at present. Esters include cocaine, procaine, chloroprocaine, tetracaine, and ben-zocaine. Esters are hydrolyzed in the blood by plasma esterases.When used in large quantities over a short period of time, local anesthetic levels can rise in the blood and cause central nervous system (CNS)

1	Esters are hydrolyzed in the blood by plasma esterases.When used in large quantities over a short period of time, local anesthetic levels can rise in the blood and cause central nervous system (CNS) toxicity and cardiovascular toxicity. Symptoms of CNS toxicity include restlessness, tinnitus, and slurred speech and can progress to seizures and coma. Car-diovascular toxicity may manifest as hypotension, conduction abnormalities leading to heart block, and ventricular arrhyth-mias, and it may lead to cardiac arrest. The type of local anes-thetic used affects the risk of developing toxicity; bupivicaine is most often associated with cardiovascular toxicity. Other risk factors for local anesthetic systemic toxicity include cumulative dose, site of injection, and preexisting renal, hepatic, or cardiac disease in the patient.20 In addition to treating symptomatology, local anesthetic systemic toxicity can be treated with intrave-nous administration of lipid emulsion.21Neuromuscular

1	or cardiac disease in the patient.20 In addition to treating symptomatology, local anesthetic systemic toxicity can be treated with intrave-nous administration of lipid emulsion.21Neuromuscular BlockersWhile general anesthetics provide muscle relaxation, they usu-ally do so at a much deeper anesthetic depth than required for amnesia and hypnosis. For this reason, neuromuscular blockers are commonly administered to attain adequate relaxation at lev-els of anesthesia sufficient to produce hypnosis, amnesia, and analgesia. Neuromuscular blockers block conduction at the neu-romuscular junction of skeletal muscle.22The two categories of neuromuscular blockers in use are depolarizing and nondepolarizing blockers. Character-istics of neuromuscular blockers currently used are summa-rized in Table 46-2. Succinylcholine is the only depolarizing agent used currently. It binds to acetylcholine receptors on the postjunctional membrane in the neuromuscular junction and causes depolarization of

1	46-2. Succinylcholine is the only depolarizing agent used currently. It binds to acetylcholine receptors on the postjunctional membrane in the neuromuscular junction and causes depolarization of muscle fibers. The rapid onset (less than 60 seconds) and rapid offset (5–8 minutes) of succinyl-choline make it ideal for management of the airway in certain Brunicardi_Ch46_p2027-p2044.indd 203101/03/19 11:04 AM 2032SPECIFIC CONSIDERATIONSPART IITable 46-2Commonly used neuromuscular blockersAGENTTYPEINTUBATING DOSECONSIDERATIONSSuccinylcholineDepolarizer1 mg/kgCan cause severe hyperkalemiaContraindicated in burns, denervating conditionsExcessive or prolonged use can lead to phase II blockRocuroniumNondepolarizer0.6 mg/kg1.2 mg/kg for RSIPrimarily hepatic metabolismCan be reversed with suggamadex or acetylcholinesterase inhibitorVecuroniumNondepolarizer0.1 mg/kgPrimarily hepatic metabolismCan be reversed with suggamadex or acetylcholinesterase inhibitorCisatracuriumNondepolarizer0.1

1	or acetylcholinesterase inhibitorVecuroniumNondepolarizer0.1 mg/kgPrimarily hepatic metabolismCan be reversed with suggamadex or acetylcholinesterase inhibitorCisatracuriumNondepolarizer0.1 mg/kgHoffman degradationCan be reversed with an acetylcholinesterase inhibitorRSI = rapid sequence inductionsituations.23 Succinylcholine has several adverse effects includ-ing transient hyperkalemia, which can be severe or even fatal for patients with burns and denervating injuries. Succinylcholine can cause bradycardia, which can be severe in children. It is also associated with transient increases in intracranial and intraocular pressure. The depolarization caused by succinylcholine causes skeletal muscles to fasciculate, which in turn, can result in post-operative myalgias. Succinylcholine is a known trigger of malig-nant hyperthermia in susceptible individuals. Succinylcholine is broken down by psuedocholinesterase; patients who are homo-zygous for pseudocholinestrase deficiency will have

1	known trigger of malig-nant hyperthermia in susceptible individuals. Succinylcholine is broken down by psuedocholinesterase; patients who are homo-zygous for pseudocholinestrase deficiency will have prolonged neuromuscular blockade, typically lasting for several hours.There are several nondepolarizing neuromuscular block-ing agents in clinical use. Long-acting agents including pan-curonium are no longer widely used. Intermediate-duration neuromuscular blockers include the steroid-based drugs vecuronium and rocuronium, which are metabolized by the liver as well as by the kidney, and the benzylisoquinolone drugs atra-curium and cisatracurium, which undergo breakdown in plasma known as Hofmann elimination. All nondepolarizers reversibly bind to the postsynaptic terminal in the neuromuscular junc-tion and prevent acetylcholine from depolarizing the muscle. Muscle blockade occurs without fasciculation and without the subsequent side effects seen with succinylcholine. Neuromus-cular

1	junc-tion and prevent acetylcholine from depolarizing the muscle. Muscle blockade occurs without fasciculation and without the subsequent side effects seen with succinylcholine. Neuromus-cular blockade with nondepolarizing drugs is typically reversed. Failure to adequately reverse neuromuscular blockade is associ-ated with an increased risk of perioperative respiratory failure and death. Reversal agents include acetylcholinesterase inhibi-tors including neostigmine, edrophonium, or pyridostigmine that are given concurrently with muscarinic-anticholinergics, almost always atropine or glycopyrrolate. Recently, suggama-dex, a chelating agent, has been approved for use as a reversal agent for reversal of neuromuscular blockade by the steroid paralytics rocuronium and vecuronium. When given at a very high dose, suggamadex can even rapidly reverse the effect of an intubating dose of a steroid neuromuscular blocker.ANESTHETIC MONITORINGOver the past several decades, advancement in anesthetic

1	a very high dose, suggamadex can even rapidly reverse the effect of an intubating dose of a steroid neuromuscular blocker.ANESTHETIC MONITORINGOver the past several decades, advancement in anesthetic moni-toring has made administration of anesthesia safer than ever. The goal of anesthetic monitoring is to continuously moni-tor the patients’ cardiovascular status, pulmonary status, respiratory physiology, anesthetic depth, concentration of gases administered, and temperature. The American Society of Anesthesiology (ASA) has established standards for basic intra-operative monitoring that are listed in Table 46-3. Types of anesthetic monitoring are listed in Table 46-4.Cardiovascular monitoring includes continuous ECG monitoring as well as blood pressure monitoring, which is to be measured and recorded at least every 5 minutes. Blood pressure monitoring can be done using noninvasive blood pressure cuff measurements or invasively using an arterial catheter. Other car-diovascular monitors

1	recorded at least every 5 minutes. Blood pressure monitoring can be done using noninvasive blood pressure cuff measurements or invasively using an arterial catheter. Other car-diovascular monitors include monitoring of central venous pres-sure, pulmonary artery pressure, and cardiac output. In high-risk 22Table 46-3American Society of Anesthesiologists standards for basic intraoperative monitoringStandardsStandard 1: Qualified anesthesia personnel shall be present in the room throughout the conduct of all general anesthetics and regional and monitored anesthesia care.Standard 2: Oxygenation, ventilation, circulation, and temperature shall be continually evaluated Oxygenation Inspired gas oxygen analyzer Pulse oximetry Monitoring of patient clinical status Ventilation Auscultation Observation of the patient Observation of reservoir bag End-tidal carbon dioxide analysis Circulation Continuous electrocardiogram display Heart rate and blood pressure recorded at least every 5

1	of the patient Observation of reservoir bag End-tidal carbon dioxide analysis Circulation Continuous electrocardiogram display Heart rate and blood pressure recorded at least every 5 minutes Evaluation of circulation: auscultation of heart sounds, palpation of pulse, pulse oximetry, blood pressure monitoring with noninvasive means or intra-arterial catheter pressure measurement Temperature Core and/or skin temperatureReproduced with permission from Longnecker DE, Mackey SC, Newman MF, et al: Anesthesiology, 3rd ed. New York, NY: McGraw-Hill Education; 2018.Brunicardi_Ch46_p2027-p2044.indd 203201/03/19 11:04 AM 2033ANESTHESIA FOR SURGICAL PATIENTSCHAPTER 46Table 46-4Types of anesthesia monitors and their propertiesTYPE OF MONITORWHAT IS MEASUREDINVASIVENESSPOTENTIAL FOR COMPLICATIONSPhysical examinationHeart sounds, breath sounds, pulse, color, mental status, etcNoninvasive–Pulse oximetryArterial oxygen saturationNoninvasive–Arterial catheterBlood pressure, acid/base

1	examinationHeart sounds, breath sounds, pulse, color, mental status, etcNoninvasive–Pulse oximetryArterial oxygen saturationNoninvasive–Arterial catheterBlood pressure, acid/base statusInvasive++Noninvasive blood pressure measurementBlood pressureNoninvasive+/–ElectrocardiographyCardiac rhythm, rate, ST segmentsNoninvasive–CapnographyVentilatory, circulatory statusNoninvasive–Electroenceophalogram, bispectral index, etcBrain function, depth of anesthesiaNoninvasive+/–Temperature probeBody temperatureNoninvasive to invasive+/–Central venous pressure, pulmonary artery pressureCardiac function, volume statusInvasive+++Transesophageal echocardiogramCardiac function, volume statusInvasive+++Adapted with permission from Longnecker DE, Mackey SC, Newman MF, et al: Anesthesiology, 3rd ed. New York, NY: McGraw-Hill Education; 2018.anesthetics such as liver transplantation and cardiac surgery, transesophageal echocardiography (TEE) is employed to moni-tor myocardial function and volume status.

1	NY: McGraw-Hill Education; 2018.anesthetics such as liver transplantation and cardiac surgery, transesophageal echocardiography (TEE) is employed to moni-tor myocardial function and volume status. Intraoperative TEE can also be used to guide surgeons when performing complex cardiac surgeries, including cardiac valve replacements.Monitoring of oxygenation and ventilation includes use of continuous pulse oximetry, monitoring of exhaled end-tidal carbon dioxide (ETCO2), and monitoring of fraction of inspired oxygen. End tidal CO2 monitoring also provides important infor-mation about systemic perfusion. During cardiac arrest, there is no delivery of CO2 to the lungs, and the end-tidal CO2 is thus very low or zero; a sudden spike in end tidal CO2 during cardio-pulmonary resuscitation correlates with return of spontaneous circulation.24 Modern ventilators also measure peak and plateau inspiratory airway pressure and minute ventilation. Adequacy of oxygenation and ventilation can also be

1	with return of spontaneous circulation.24 Modern ventilators also measure peak and plateau inspiratory airway pressure and minute ventilation. Adequacy of oxygenation and ventilation can also be confirmed by arterial blood gas analysis.Temperature monitoring is performed using a temperature probe, usually inserted in the esophagus or nasopharynx. Core body temperature can be measured with temperature sensing Foley catheters. Temperature can also be measured at the skin.Several monitors exist that measure depth of anesthesia, including the bispectral index (BIS) monitor and the SedLine monitor. While these monitors were designed to prevent aware-ness under anesthesia, a multicenter trial of over 6000 patients showed that titrating anesthetic concentration to the BIS moni-tor was not superior to titrating anesthetic depth to end-tidal anesthetic concentration with goal MAC greater than 0.7.25Peripheral nerve stimulators should be used to moni-tor depth of neuromuscular blockade. A

1	to titrating anesthetic depth to end-tidal anesthetic concentration with goal MAC greater than 0.7.25Peripheral nerve stimulators should be used to moni-tor depth of neuromuscular blockade. A train-of-four monitor delivers four successive stimuli over 2 seconds. Presence of four twitches without fade with a ratio of the height of the first twitch to the height of the fourth twitch at least 0.9 suggests adequate reversal of neuromuscular blockade.26 The presence of one or two twitches (absence of the last two or three) is generally suffi-cient for the relaxation required for almost any kind of abdomi-nal or thoracic operation.PERIOPERATIVE EVALUATION AND PREPARATIONThe ASA has adopted basic standards for the evaluation of patients before surgery. These standards require the anesthe-siologist to evaluate the medical status of the patient, develop a plan of anesthetic care, and discuss this plan with the patient and/or the patient’s legal guardian.A preoperative evaluation includes an

1	to evaluate the medical status of the patient, develop a plan of anesthetic care, and discuss this plan with the patient and/or the patient’s legal guardian.A preoperative evaluation includes an appropriately detailed medical history, current drug therapy, appropriate physical examination, and review of laboratory and specific testing results. Based on these findings, the anesthesiologist may conclude that a patient is not in optimal medical condition to undergo elective surgery. These findings and opinions are then discussed with the patient’s primary physician or surgeon, and the surgery may be delayed (or cancelled) until the patient’s medical condition is further evaluated and optimized.The medical history obtained at the preoperative visit should include the patient’s previous exposure and experi-ence with anesthesia, as well as any family history of problems with anesthesia. History of atopy is an important aspect of this evaluation in that it may predispose patients to form

1	and experi-ence with anesthesia, as well as any family history of problems with anesthesia. History of atopy is an important aspect of this evaluation in that it may predispose patients to form antibodies against antigens that may be represented by agents administered during the perioperative period. Concurrent medications should be fully evaluated when circumstances allow, and adverse inter-actions with agents administered during the perioperative period need to be considered. A review of the function of major organ systems should also be performed. The physical examination is targeted primarily at the central nervous system, cardiovascular system, lungs, and airway.Laboratory testing should be based on the patient’s con-dition and the proposed procedure. Otherwise healthy patients usually do not need laboratory testing for minor procedures. Preoperative testing may be necessitated by findings on physi-cal examination; for example, an electrocardiogram should be obtained if an

1	usually do not need laboratory testing for minor procedures. Preoperative testing may be necessitated by findings on physi-cal examination; for example, an electrocardiogram should be obtained if an irregular heart rhythm is noted, and an echo-cardiogram may be indicated if a new murmur is observed on Brunicardi_Ch46_p2027-p2044.indd 203301/03/19 11:04 AM 2034SPECIFIC CONSIDERATIONSPART IIauscultation. Chest imaging or pulmonary function testing may be indicated if abnormalities are noted on pulmonary examina-tion and may be pertinent to the administration of the anesthetic or the recovery from anesthesia and surgery. Urine pregnancy testing is typically performed on the day of surgery in women of childbearing age.ASA Physical Status AssessmentThe ASA classification system is a scale used to risk-stratify patients for anesthesia and surgery. The scale, ranging from physical status I to VI, is shown in Table 46-5. Patients under-going emergent surgery are denoted by an “E”; for

1	used to risk-stratify patients for anesthesia and surgery. The scale, ranging from physical status I to VI, is shown in Table 46-5. Patients under-going emergent surgery are denoted by an “E”; for example, an otherwise healthy patient undergoing an appendectomy for appendicitis would be classified as ASA IE. Mortality has shown to increase with increasing ASA physical status, and it has been shown to be higher for patients undergoing emergency surgery.27Airway EvaluationAirway examination can identify most patients in whom management of the airway and conventional endotracheal CLASS 1: Soft palate, fauces, uvula, pillarsCLASS 2: Soft palate, fauces, portion of uvulaCLASS 3: Soft palate, base of uvulaCLASS 4: Hard palate onlyMALLAMPATI CLASSIFICATIONCLASS 1CLASS 2CLASS 3CLASS 4Figure 46-3. The Mallampati classification.Table 46-5American Society of Anesthesiologists physical status classificationASA PHYSICAL STATUSDESCRIPTIONIA healthy patient without systemic diseaseIIA patient with

1	Mallampati classification.Table 46-5American Society of Anesthesiologists physical status classificationASA PHYSICAL STATUSDESCRIPTIONIA healthy patient without systemic diseaseIIA patient with mild systemic diseaseIIIA patient with severe systemic diseaseIVA patient with severe systemic disease that is a constant threat to lifeVA moribund patient not expected to survive without the operationVIA declared brain-dead patient whose organs are being removed for donationData from American Society of Anesthesiologists: ASA Physical Status Classification System Developed. ASA House of Delegates/Executive Committee. Amended: October 15, 2014.intubation may be difficult. It is vitally important to recognize such patients before administering medications that induce apnea. The Mallampati classification (Fig. 46-3) is based on the structures visualized with maximal mouth opening and tongue protrusion in the sitting position.28 Patients with higher Mallampati classification, in combination with

1	(Fig. 46-3) is based on the structures visualized with maximal mouth opening and tongue protrusion in the sitting position.28 Patients with higher Mallampati classification, in combination with other airway abnormalities, can be difficult to intubate. Other predictors of difficult intubation include short neck, immobility of the neck,29 a large overbite, a small mandible, or the inability to shift the lower incisors in front of the upper incisors. The thyromental distance, the distance from the thyroid cartilage to the tip of the chin should be greater than 6 cm; thyromental distance of less than 6 cm has been associated with difficult intubation.30 Obesity is also a risk factor for difficult intubation, and neck circumference has been identified as a risk factor for both dif-ficult intubation as well as difficult mask ventilation.31Cardiovascular DiseaseCardiac risk is widely regarded as the most important risk associated with anesthesia and surgery, and it has been the focus of an

1	as well as difficult mask ventilation.31Cardiovascular DiseaseCardiac risk is widely regarded as the most important risk associated with anesthesia and surgery, and it has been the focus of an enormous amount of scholarship over the past four decades. The revised cardiac risk index incorporates six patient and surgical factors to assess a patient’s risk of major adverse cardiac events in the perioperative period: history of ischemic heart disease, congestive heart failure, cerebrovascular dis-ease, diabetes requiring insulin, chronic kidney disease with baseline creatinine greater than 2, and whether the surgery is in a high-risk area, namely major vascular, intraperitoneal, or intrathoracic. In 2014, the American College of Cardiology and the American Heart Association published guidelines for perioperative workup and management of patients with cardio-vascular disease; a simplified version is seen in Fig. 46-4 . Notably, this guideline stresses the importance of func-tional status

1	for perioperative workup and management of patients with cardio-vascular disease; a simplified version is seen in Fig. 46-4 . Notably, this guideline stresses the importance of func-tional status in determining need for further evaluation; patients with good functional status can typically proceed to surgery without additional evaluation. Functional capacity is measured in metabolic equivalents (METs), with patients unable to attain 4 METs considered to have poor functional status. Activities representing 4 METs including walking up a flight of stairs, climbing a hill, or walking on level ground at 3 to 4 miles per hour.3233Brunicardi_Ch46_p2027-p2044.indd 203401/03/19 11:04 AM 2035ANESTHESIA FOR SURGICAL PATIENTSCHAPTER 46These recent guidelines have minimized the role of routine screening. Preoperative electrocardiograms and stress testing are unnecessary for asymptomatic patients undergoing low-risk surgery.Special attention is required for patients with coronary stents.

1	screening. Preoperative electrocardiograms and stress testing are unnecessary for asymptomatic patients undergoing low-risk surgery.Special attention is required for patients with coronary stents. Elective surgery should be delayed a stent has been inserted, which allows time for it to stabilize and the risk of in-stent thrombosis to decrease. The ACC/AHA guidelines rec-ommend delaying elective surgery for 30 days after bare metal stent placement and for 1 year after drug eluting stent place-ment. Dual antiplatelet therapy should be continued for urgent or emergent procedures that take place before the minimum recommended waiting period.32 For semi-elective surgeries in patients with drug-eluting stents, where the risk of delaying sur-gery is greater than the risk of in-stent thrombosis, ACC/AHA guidelines recommend surgery be delayed for 180 days.Current recommendation is that β-blockers and statins should be continued in patients who are on them chronically. β-Blockers may be

1	ACC/AHA guidelines recommend surgery be delayed for 180 days.Current recommendation is that β-blockers and statins should be continued in patients who are on them chronically. β-Blockers may be started in the perioperative period for patients with multiple RCRI risk factors or who are at intermediate or high risk for myocardial ischemia. If started in the perioperative period, β-blockers should be started long enough before sur-gery to ascertain their safety, and not on the day of surgery.32,33 Recent large randomized trials have demonstrated excess risk of mortality and stroke simultaneously with decreased risk of myocardial events in moderateand high-risk patients who are newly treated with β-blockers in the periprocedural setting.34,35Implanted cardiac devices including pacemakers and implantable cardioverter-defibrillators also have important peri-operative implications. A 2011 ASA practice advisory stressed the importance of determining whether electromagnetic interfer-ence is

1	implantable cardioverter-defibrillators also have important peri-operative implications. A 2011 ASA practice advisory stressed the importance of determining whether electromagnetic interfer-ence is likely to occur during the planned procedure, determin-ing the current function and necessity of the implanted device, determining whether reprogramming or temporary disabling of the device is advantageous, having alternative therapy available for the time that the device is unavailable, and restoring device function in the postoperative period.36Pulmonary DiseaseChronic pulmonary disease is an increasingly recognized cause of morbidity and mortality in surgical patients. For patients with asthma or chronic obstructive pulmonary disease, exercise toler-ance and the frequency and severity of exacerbations should be evaluated. A focused history, including prior admissions and intubations for exacerbations, should be obtained. Treatment with bronchodilators in the perioperative setting is

1	exacerbations should be evaluated. A focused history, including prior admissions and intubations for exacerbations, should be obtained. Treatment with bronchodilators in the perioperative setting is appropri-ate, although there is no literature to either guide this care or to document a benefit from it. Most inhaled anesthetics act as bronchodilators.37 Desflurane can be an airway irritant, and it is often avoided in patients with reactive airway disease.The incidence of obstructive sleep apnea (OSA) has risen with the incidence of obesity. In 2014 the ASA published guide-lines for perioperative management of patients with OSA. These guidelines highlight the importance of identifying patients with obstructive sleep apnea during preoperative evaluation and obtaining a sleep study if appropriate. They also highlight the importance of the development of protocols by anesthesiologists and surgeons to manage OSA in the perioperative setting. There is consensus that these patients should

1	They also highlight the importance of the development of protocols by anesthesiologists and surgeons to manage OSA in the perioperative setting. There is consensus that these patients should not be extubated until they are completely awake, and that they should be treated with ACC/AHA algorithm of cardiac evaluation for noncardiac surgeryProceed to surgery with medical riskreduction and perioperative surveillancePostpone surgery until stabilized or correctedNo clinicalpredictorsProceed with surgeryProceed with surgery˜1 clinicalpredictorsIntermediate riskor vascular surgeryProceed with surgeryProceed to surgery with heart ratecontrol or consider noninvasive testingif it will change managementEmergency surgeryActive cardiac conditions• Unstable coronary syndromes (unstable or severe angina, recent MI)• Decompensated heart failure (HF; new onset, NYHA class IV)• Significant arrhythmias (Mobitz ll or third-degree heart block, supraventricular tachycardia or atrial fibrillation with

1	recent MI)• Decompensated heart failure (HF; new onset, NYHA class IV)• Significant arrhythmias (Mobitz ll or third-degree heart block, supraventricular tachycardia or atrial fibrillation with rapid ventricular rate (>100), symptomatic ventricular arrhythmia or bradycardia, new ventricular tachycardia)• Severe valvular disease (severe aortic or mitral stenosis)Step 1Step 2Low-risk surgery (risk <1%)• Superficial or endoscopic• Cataract, breast• Ambulatory surgeryStep 3Functional capacityGood; ≥4 METS (can walk flight of stairs without symptoms)Step 4Clinical predictors• Ischemic heart disease• Compensated or prior HF• Cerebrovascular disease (stroke, TIA)• Diabetes mellitus• Renal insufficiencyStep 5Figure 46-4. Simplified cardiac evaluation for noncardiac surgery. (Data from Eagle KA, Berger PB, Calkins H, et al: ACC/AHA guideline update for perioperative cardiovascular evaluation for noncardiac surgery–executive summary: a report of the American College of Cardiology/American

1	KA, Berger PB, Calkins H, et al: ACC/AHA guideline update for perioperative cardiovascular evaluation for noncardiac surgery–executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee to Update the 1996 Guidelines on Perioperative Cardiovascular Evaluation for Noncardiac Surgery), J Am Coll Cardiol. 2002 Feb 6;39(3):542-553.)Brunicardi_Ch46_p2027-p2044.indd 203501/03/19 11:04 AM 2036SPECIFIC CONSIDERATIONSPART IInoninvasive positive pressure ventilation in the postoperative period as indicated.38Renal DiseaseManagement of anesthesia in patients with chronic renal insuf-ficiency requires close attention to perioperative fluid man-agement and acid-base and electrolyte homeostasis. Doses of opioids and neuromuscular agents are typically reduced and dosing intervals increased to compensate for decreased renal excretion. Cisatracurium is often chosen as the muscle relax-ant in patients with severe renal

1	agents are typically reduced and dosing intervals increased to compensate for decreased renal excretion. Cisatracurium is often chosen as the muscle relax-ant in patients with severe renal insufficiency because its elimi-nation is unchanged by renal failure. Sugammadex, a reversal agent for steroid-based neuromuscular blockers, is not currently recommended for use in patients with advanced chronic kidney disease or end-stage renal disease.Hepatic DiseaseHepatic dysfunction has many causes, and it can compel significant changes in anesthetic care. First, anesthetic agents metabolized in the liver can accumulate in these patients and may have a longer duration of effect. To mitigate this, short-acting agents are strongly preferred in these patients. Hypoalbuminemia can paradoxically increase the free plasma levels of drugs, which can also exagger-ate their effects. In patients with substantial ascites, high intra-abdominal pressure may increase the risk of passive gastric reflux, and

1	the free plasma levels of drugs, which can also exagger-ate their effects. In patients with substantial ascites, high intra-abdominal pressure may increase the risk of passive gastric reflux, and thus many such patients are managed as if they have a full stomach, regardless of how long they have been NPO. In patients with significant hepatocellular dysfunction and/or portal hyperten-sion, the combination of thrombocytopenia and coagulation factor deficiency not only increase the risk of bleeding associated with surgery, they also are relative or absolute contraindications to a variety of anesthetic techniques, such as subarachnoid blocks and epidural anesthesia. Presence of esophageal varices increases the risk of gastric tubes and transesophageal echocardiography.Endocrine DiseasePerioperative management of the diabetic patient can be espe-cially challenging. A hemoglobin A1c level should be obtained if a recent level is not available, as an increased A1c level is asso-ciated with an

1	management of the diabetic patient can be espe-cially challenging. A hemoglobin A1c level should be obtained if a recent level is not available, as an increased A1c level is asso-ciated with an increase in perioperative complications including wound infections.39-41 Several institutions have implemented pro-tocols for glucose management for diabetic patients undergoing surgery, although recommendations differ on appropriate target glucose levels.39,42,43 Perhaps the most important thing for a prac-titioner to know and remember is that the difference between type 1 and type 2 diabetes is important, and that these two differ-ent diseases require different approaches to their management. In general, patients with type 2 diabetes have a lower risk of becom-ing hypoglycemic, tend to have higher blood sugars at baseline, and tolerate higher levels of serum glucose without significant acute hazard. Patients with type 1 diabetes, who are deficient in insulin production and thus require

1	higher blood sugars at baseline, and tolerate higher levels of serum glucose without significant acute hazard. Patients with type 1 diabetes, who are deficient in insulin production and thus require insulin administration to pre-vent ketosis, are far more likely to become hypoglycemic when subjected to stress, and they are also at risk for developing keto-acidosis with hyperglycemia. Patients with type 1 diabetes merit more careful monitoring of their blood sugars in the periopera-tive setting than patients with type 2 diabetes.44-47Preoperative FastingThe ASA has developed specific guidelines for preoperative fasting to mitigate the risk of aspiration of gastric contents. Table 46-6 shows guidelines for preoperative food and fluid intake for elective procedures. Individual patients may need lengthier fasting times than the guidelines indicate. Notably, a rapid sequence induction and intubation should be considered in patients who are at higher risk for aspiration such as those with

1	need lengthier fasting times than the guidelines indicate. Notably, a rapid sequence induction and intubation should be considered in patients who are at higher risk for aspiration such as those with very symptomatic gastroesophageal reflux, achala-sia, gastroparesis, or dysmotility, regardless of fasting status.48Patients With Advanced DirectivesPatients with do not resuscitate (DNR) and/or do not intu-bate (DNI) orders present a unique challenge. Patients or their power-of-attorney may choose to rescind these directives in the perioperative period, maintain them as originally ordered, or modify them to allow for a limited resuscitation. Both the ASA and the American College of Surgeons recommend that preoperative discussions with the patient and their family clarify the patient’s wishes, and both societies emphasize that policies that mandate uniform enforcement or disregarding of all DNR orders take away patients’ right to self-determination.49,50Risk EstimationSeveral risk

1	wishes, and both societies emphasize that policies that mandate uniform enforcement or disregarding of all DNR orders take away patients’ right to self-determination.49,50Risk EstimationSeveral risk calculators have been developed to estimate peri-operative morbidity and mortality. The American College of Surgeons National Surgical Quality Improvement Program (NSQIP) surgical risk calculator allows for estimation of risk of eight different adverse outcomes, including mortality.51 The Society of Thoracic Surgeons risk calculator estimates risk of morbidity and mortality after cardiac surgical procedures.52 While such risk estimators can provide widely differing esti-mates or may be inaccurate in certain situations,53 they are still invaluable aids for discussions with patients and their families about high-risk surgery.INTRAOPERATIVE MANAGEMENTGeneral AnesthesiaGeneral anesthesia remains the cornerstone of anesthesia prac-tice; many surgical procedures cannot be done under regional

1	families about high-risk surgery.INTRAOPERATIVE MANAGEMENTGeneral AnesthesiaGeneral anesthesia remains the cornerstone of anesthesia prac-tice; many surgical procedures cannot be done under regional techniques or monitored anesthesia care with sedation.The induction of general anesthesia can precipitate cata-strophic anesthetic complications. Many different techniques can be used to induce general anesthesia, each with significant advantages and disadvantages.Intravenous induction, used primarily in adults, quickly produces unconsciousness, and depending on the agent used, apnea as well. Propofol, the most common induction agent currently used, can cause hypotension due to its myocardial depressing and vasodilatory properties. Hypertension and 4Table 46-6Guidelines for food and fluid intake before elective surgeryTIME BEFORE SURGERYFOOD OR FLUID INTAKEUp to 8 hoursFood and fluids as desiredUp to 6 hoursaLight meal, infant formulaUp to 4 hoursBreast milkUp to 2 hoursClear liquids

1	intake before elective surgeryTIME BEFORE SURGERYFOOD OR FLUID INTAKEUp to 8 hoursFood and fluids as desiredUp to 6 hoursaLight meal, infant formulaUp to 4 hoursBreast milkUp to 2 hoursClear liquids onlyaLight meal refers to a limited amount of easily digestible food, such as toast or crackers. Individual patients may need lengthier fasting times than these guidelines indicate.Adapted with permission from Longnecker DE, Mackey SC, Newman MF, et al: Anesthesiology, 3rd ed. New York, NY: McGraw-Hill Education; 2018.Brunicardi_Ch46_p2027-p2044.indd 203601/03/19 11:04 AM 2037ANESTHESIA FOR SURGICAL PATIENTSCHAPTER 46tachycardia commonly occur during laryngoscopy or other sig-nificant airway stimulation.The goal of a rapid sequence induction (RSI) is to achieve secure protection of the airway with a cuffed endotracheal tube without ever mask ventilating a patient. It is intended to prevent vomiting and aspiration, and it is routinely employed in patients at heightened risk for

1	the airway with a cuffed endotracheal tube without ever mask ventilating a patient. It is intended to prevent vomiting and aspiration, and it is routinely employed in patients at heightened risk for aspiration. There are no randomized con-trolled trials that demonstrate any kind of outcome benefit of rapid sequence induction in such patients, but it is nevertheless routinely employed for this purpose in the United States.Pediatric patients are often not amenable to preoperative IV catheter placement. Hence, inhalation induction of anesthe-sia is commonly used in children, with IV placement occur-ring after induction. Even among children, however, patients at heightened risk for aspiration or with a full stomach may be best managed with preoperative IV placement and an IV induction. Patients with developmental delay may not be amenable to pre-operative IV placement or inhalational induction of anesthesia. In such patients, intramuscular administration of an agent such as ketamine is

1	with developmental delay may not be amenable to pre-operative IV placement or inhalational induction of anesthesia. In such patients, intramuscular administration of an agent such as ketamine is often required to induce anesthesia.Airway Management. Most anesthesiologists prefer to secure the airway of a patient undergoing general anesthesia, and this is usually accomplished immediately after anesthesia has been induced. The airway may be managed in several ways, includ-ing by face mask, with a laryngeal mask airway (LMA), or, most definitively, by endotracheal intubation with a cuffed endotracheal tube. Nasal and oral airways can help establish a patent airway in a patient being ventilated with a mask by creat-ing an air passage behind the tongue.The LMA is a cuffed supraglottic oral airway that is inserted through the oropharynx and ideally positioned just above the glottis opening. It is passed blindly, and the inflated cuff creates a seal around the laryngeal inlet. An LMA does

1	that is inserted through the oropharynx and ideally positioned just above the glottis opening. It is passed blindly, and the inflated cuff creates a seal around the laryngeal inlet. An LMA does not protect against aspiration and should generally not be used in patients with a high risk of aspiration.Tracheal intubation requires a skilled operator and proper equipment. In most elective anesthetics, attempts to intubate the trachea are facilitated by the administration of muscle relaxants in a patient who is already under a general anesthetic. Intubation is typically performed under direct visualization with a laryn-goscope, watching the endotracheal tube pass through the vocal cords into the trachea. To obtain a direct line of sight, the patient is placed in the sniffing position. The neck is flexed at the lower cervical spine and extended at the atlanto-occipital joint. This flexion and extension are amplified during laryngoscopy. Laryn-goscope blades can be curved (Macintosh) or

1	neck is flexed at the lower cervical spine and extended at the atlanto-occipital joint. This flexion and extension are amplified during laryngoscopy. Laryn-goscope blades can be curved (Macintosh) or straight (Miller) blades. Laryngoscopic views are typically reported in a classi-fication system developed by Cormack and Lehane (Fig. 46-5).Management of the Difficult Airway. Some patients have physical characteristics or a history suggestive of difficulty in placing an endotracheal tube. A short neck, limited neck mobil-ity, small interincisor distance, short thyromental distance, and high Mallampati classes may all represent a challenge to Figure 46-5. Laryngoscopic views obtained per Cormack and Lehane.Figure 46-6. Video laryngoscopy with the GlideScope.endotracheal intubation. Several tools have been developed to assist in management of the difficult airway.The Glidescope, a video laryngoscope, allows for visualiza-tion of the larynx on a video screen (Fig. 46-6). Having more of a

1	tools have been developed to assist in management of the difficult airway.The Glidescope, a video laryngoscope, allows for visualiza-tion of the larynx on a video screen (Fig. 46-6). Having more of a bend than a standard curved Macintosh blade, it can be advanta-geous for visualizing and intubating the trachea in patients with large tongues or relatively anterior glottis openings. Placement of the endotracheal tube once the larynx has been visualized can still be challenging. A recent study of ICU patients requiring intubation showed that video laryngoscopy did not improve first-pass orotracheal intubation success rate and was associated with higher rates of severe life-threatening complications.54The intubating laryngeal mask airway (ILMA) is an advanced form of LMA designed to maintain a patent airway and facilitate tracheal intubation. The ILMA can be placed in anticipated or unexpectedly difficult airways as an airway res-cue device and as a guide for intubating the trachea. The

1	a patent airway and facilitate tracheal intubation. The ILMA can be placed in anticipated or unexpectedly difficult airways as an airway res-cue device and as a guide for intubating the trachea. The device itself is substantially more rigid than other laryngeal mask airways, and includes a handle which the operator can use to displace the opening of the device. A specially manufactured endotracheal tube can be passed blindly through the ILMA into the larynx, or the ILMA can be used as a conduit for a flex-ible fiberoptic scope. Experience with airway management in general and the use of this device in particular is essential for its effective use in emergency situations; operators with little experience will enjoy little success with this device.The flexible fiberoptic intubation bronchoscope is the gold standard for difficult intubation. It is indicated in difficult or compromised airways where neck extension is not desirable or in cases with risk of dental damage. The flexible

1	is the gold standard for difficult intubation. It is indicated in difficult or compromised airways where neck extension is not desirable or in cases with risk of dental damage. The flexible bronchoscope allows excellent visualization of the airway and glottic open-ing. This technique can be used for oral and nasal intubation, for awake or asleep intubation, and for intubation in the awake, spontaneously ventilating patient whose airway has been treated with topical local anesthetic.The ASA has developed an algorithm for management of the difficult airway (Fig. 46-7).55 Notably, in patients in whom Brunicardi_Ch46_p2027-p2044.indd 203701/03/19 11:04 AM 2038SPECIFIC CONSIDERATIONSPART IIFigure 46-7. ASA difficult airway algorithm. (Reproduced with permission from Apfelbaum JL, Hagberg CA, Caplan RA, et al: Practice guidelines for management of the difficult airway: an updated report by the American Society of Anesthesiologists Task Force on Management of the Difficult Airway,

1	CA, Caplan RA, et al: Practice guidelines for management of the difficult airway: an updated report by the American Society of Anesthesiologists Task Force on Management of the Difficult Airway, Anesthesiology. 2013 Feb;118(2):251-270.)Brunicardi_Ch46_p2027-p2044.indd 203801/03/19 11:04 AM 2039ANESTHESIA FOR SURGICAL PATIENTSCHAPTER 46both intubation and ventilation are impossible, the algorithm calls for placement of an LMA with ventilation attempted through the LMA.Monitored Anesthesia CareMonitored anesthesia care (MAC) is when a patient under-goes a procedure under local anesthesia under the care of an anesthesiologist who can provide sedation as indicated. Seda-tion is administered to a level that allows the patient to main-tain airway reflexes and breath spontaneously. Advantages of MAC anesthesia include reduced invasiveness, as the airway is not manipulated, and faster recovery. ASA standard moni-tors must be used, including capnography, which allows for rapid detection of

1	of MAC anesthesia include reduced invasiveness, as the airway is not manipulated, and faster recovery. ASA standard moni-tors must be used, including capnography, which allows for rapid detection of apnea or hypoventilation. In some instances, most commonly for gastroenterology procedures, patients are administered an intravenous anesthetic that is often classified as a MAC, even though the patient is so deeply anesthetized that they have no significant response to significant airway stimula-tion; the airway is monitored by the anesthesia provider and secured if necessary.Regional Anesthesia/Acute PainRegional anesthesia allows for selective blockade and is an excellent anesthetic option for several different types of pro-cedures. Regional anesthesia can also provide excellent post-operative pain control. Regional techniques include neuraxial blockade, including spinal and epidural anesthetics, peripheral nerve blocks, and truncal blocks.Spinal anesthetics (also referred to as

1	pain control. Regional techniques include neuraxial blockade, including spinal and epidural anesthetics, peripheral nerve blocks, and truncal blocks.Spinal anesthetics (also referred to as subarachnoid blocks) can be used for lower extremity, lower abdominal, pelvic, and urologic and gynecologic procedures. A small caliber needle (typically 25-gauge or smaller) is inserted into the intrathecal space in the cauda aquina and below the conus medularis, and a small volume of local anesthetic is injected. Duration and level of the block in the spinal cord is affected by the anesthetic used, the dose employed, and baricity of solution injected. Complica-tions can include hypotension, bradycardia, postdural puncture headache, injury to local structures or nerves, and hematoma formation. The American Society of Regional Anesthesia pub-lishes guidelines regarding safe intervals to perform neuraxial anesthetics after the administration of anticoagulant and anti-platelet agents.An epidural

1	American Society of Regional Anesthesia pub-lishes guidelines regarding safe intervals to perform neuraxial anesthetics after the administration of anticoagulant and anti-platelet agents.An epidural catheter can be used as a primary anesthetic for a procedure, or it can be placed preoperatively and used in conjunction with a general anesthetic for postoperative pain control. Epidural catheters can be placed in the thoracic or lumbar spine and can remain in place for days after surgery. A dilute local anesthetic and/or opioid is administered through the catheter to provide analgesia. Complications of epidural anesthesia are similar to that of spinal anesthesia. In addition to improved pain control, benefits of epidural anesthesia include reduced pulmonary complications56 and decreased duration of postoperative ileus.57Peripheral nerve blockade can also be used to provide sur-gical anesthesia as well as postoperative analgesia, particularly for surgeries of the upper or lower

1	duration of postoperative ileus.57Peripheral nerve blockade can also be used to provide sur-gical anesthesia as well as postoperative analgesia, particularly for surgeries of the upper or lower extremities. The nerve or plexus of interest is located with ultrasound and/or peripheral nerve stimulator, and local anesthetic is injected around the nerve. Single-shot nerve blocks allow for surgical anesthesia and immediate postoperative analgesia and can last for several hours. Flexible catheters can also be placed in proximity to nerves to allow for continuous infusion and blockade that can continue for several days. Complications of peripheral nerve blocks include injury to nerves or nearby structures and local anesthetic systemic toxicity.Recently, truncal blocks have become more commonly and widely used for the treatment of postoperative pain. Trun-cal blocks include the transversus abdominis plane (TAP) block, the rectus sheath block, the pectoral nerve block, and the serra-tus

1	and widely used for the treatment of postoperative pain. Trun-cal blocks include the transversus abdominis plane (TAP) block, the rectus sheath block, the pectoral nerve block, and the serra-tus anterior plane block. These truncal blocks are usually done under ultrasound guidance with local anesthetic injected in the appropriate plane. Truncal blocks are typically performed as part of a multimodal approach to postoperative pain. Limited evidence suggests that use of truncal blocks decreases postop-erative opioid requirements.58,59RECOVERY AND COMPLICATIONSThe Postanesthesia Care UnitThe advent of the modern postanesthesia care unit represents a major advance in the safety of perioperative care, as the close monitoring that occurs there can prevent or expedite the man-agement of a variety of serious complications. Ventilation, oxygenation, hemodynamics, temperature, nausea, and pain are closely monitored in the PACU, with close attention also given to urine output, ongoing bleeding,

1	of serious complications. Ventilation, oxygenation, hemodynamics, temperature, nausea, and pain are closely monitored in the PACU, with close attention also given to urine output, ongoing bleeding, and drainage. To be dis-charge eligible, patients should have returned to their baseline mental status, be oxygenating and ventilating adequately, have adequate pain control, and have stable vital signs. There are multiple scoring systems that can be used assess suitability for discharge from PACU.60-62 Postoperative hemorrhage, hyper-tension or hypotension, myocardial ischemia, arrhythmias, and altered mental status commonly manifest in the postoperative care unit. Postoperative nausea and vomiting (PONV) occurs in 20% to 30% of surgical cases,63 and it is a common cause of increased PACU length of stay and increased cost of PACU stay.64 For this reason, many or most patients undergoing gen-eral anesthesia receive prophylactic antiemetics.Enhanced Recovery After Surgery PathwaysEnhanced

1	of stay and increased cost of PACU stay.64 For this reason, many or most patients undergoing gen-eral anesthesia receive prophylactic antiemetics.Enhanced Recovery After Surgery PathwaysEnhanced recovery after surgery (ERAS) pathways are multi-modal perioperative care pathways designed to hasten recovery after elective surgery. These pathways may include preoperative education and counseling, preoperative optimization, limiting preoperative bowel preparation, limiting preoperative fasting, providing multimodal analgesia (including regional anesthesia) as appropriate, minimizing intraoperative fluid administration, and early mobilization. ERAS pathways have been shown to reduce duration of hospitalization and reduce cost of periopera-tive care.65-67Acute Postoperative PainThe management of postoperative pain has changed dra-matically in the modern era, with multimodal approaches and regional techniques reducing the use of opiates. Regardless, opioids remain the mainstay of

1	of postoperative pain has changed dra-matically in the modern era, with multimodal approaches and regional techniques reducing the use of opiates. Regardless, opioids remain the mainstay of intraoperative and postoperative analgesia, especially for larger and more invasive procedures. Patients with chronic pain or opiate tolerance can present a unique challenge in the perioperative period, and they can ben-efit from regional and multimodal approaches. Such patients may benefit from early involvement of an acute pain medicine specialist.For the past decade, pain has been described as the fifth vital sign, and physicians have been strongly encouraged to 5Brunicardi_Ch46_p2027-p2044.indd 203901/03/19 11:04 AM 2040SPECIFIC CONSIDERATIONSPART IIaggressively treat pain in their patients. Generally speaking, physicians sought to accomplish this goal through the more liberal use of opiates. Opioid prescription has thus soared in recent years. Some studies suggest that 3% to 7% of surgical

1	speaking, physicians sought to accomplish this goal through the more liberal use of opiates. Opioid prescription has thus soared in recent years. Some studies suggest that 3% to 7% of surgical patients prescribed opioids in the postoperative period continue to use them for a prolonged period after surgery,68,69 suggesting that opioid abuse often begins in the postoperative period. The U.S. Centers for Disease Control and Prevention has recently declared that prescription drug abuse is an epidemic.70 Physi-cians and surgeons will have to continue to seek the fine line between adequate pain control and prescribing patterns that enable dependence.Malignant HyperthermiaMalignant hyperthermia (MH) is a hereditary, life-threatening, hypermetabolic disorder, developing during or after receiving general anesthesia. The clinical incidence of MH ranges from 1:10000 to 1:250,000.71 A genetic predisposition and expo-sure to one or more triggering agents are necessary to evoke MH. Triggering

1	general anesthesia. The clinical incidence of MH ranges from 1:10000 to 1:250,000.71 A genetic predisposition and expo-sure to one or more triggering agents are necessary to evoke MH. Triggering agents include all volatile anesthetics (e.g., isoflurane, sevoflurane, and desflurane) and the depolarizing neuromuscular blocker succinylcholine. Volatile anesthetics and/or succinylcholine cause a rise in the myoplasmic cal-cium concentration in susceptible patients, causing persistent muscle contraction, the production of large quantities of car-bon dioxide and lactic acid, and a relentless increase in body temperature.MH is often an autosomal dominant disorder associated with several gene loci, predominantly the ryanodine receptor gene RYR1. MH can be diagnosed with the caffeine-contracture halothane test, which requires a muscle biopsy. Genetic testing can be helpful after an episode of MH. There is no simple, reli-able blood screening test yet available for diagnosis.The classic MH

1	halothane test, which requires a muscle biopsy. Genetic testing can be helpful after an episode of MH. There is no simple, reli-able blood screening test yet available for diagnosis.The classic MH crisis entails a hypermetabolic state with tachycardia and increased end-tidal CO2. Relentless muscle con-traction causes respiratory and metabolic acidosis, as well as rhabdomyolysis, arrhythmias, hyperkalemia, and even sudden cardiac arrest. Hyperthermia typically occurs after the episode is well under way. Treatment must be aggressive and begin as soon as a case of MH is suspected. Volatile anesthetics should be stopped immediately and dantrolene given at an initial dose of 2.5 mg/kg intravenously. The national MH hotline should be contacted for help in managing any patient with MH. Patients should be monitored in the intensive care setting for possible recrudescence of MH.Cardiovascular ComplicationsHemodynamic perturbations are a common in the periopera-tive period. Arrhythmias may

1	should be monitored in the intensive care setting for possible recrudescence of MH.Cardiovascular ComplicationsHemodynamic perturbations are a common in the periopera-tive period. Arrhythmias may begin before, during, and after an anesthetic, and are particularly common after cardiothoracic and esophageal surgery. Hypotension may be due to anemia, hypovolemia, myocardial ischemia or dysfunction, or other less common events such as pulmonary embolism and ana-phylactic reactions. Hypertension is also common, particularly when antihypertensive regimens are altered in the perioperative period.Respiratory FailureRespiratory insufficiency and failure occur frequently in the postoperative period. Respiratory depression can occur as a con-sequence of residual neuromuscular blockade, residual inhaled anesthetics, or opioids. Mechanical airway obstruction can be ameliorated by rescue maneuvers, or insertion of an oral or nasal airway. It is imperative to evaluate and treat anesthesia-related

1	anesthetics, or opioids. Mechanical airway obstruction can be ameliorated by rescue maneuvers, or insertion of an oral or nasal airway. It is imperative to evaluate and treat anesthesia-related causes of respiratory insufficiency. The opioid antagonist nalox-one can be given for opioid related respiratory insufficiency, and an additional reversal agent can be given for residual neuromus-cular weakness. Respiratory failure can reasonably be managed with noninvasive positive pressure ventilation in many cases72; patients who fail this or are unlikely to benefit from it should be intubated. High-flow nasal cannula is increasingly being used for postextubation respiratory failure,73 and several studies have demonstrated its benefit in postoperative patients.74-77Neurologic and Psychiatric ComplicationsPerioperative neurologic and psychiatric complications include stroke, both ischemic and hemorrhagic, postoperative delirium, and postoperative cognitive dysfunction. Treatment of

1	ComplicationsPerioperative neurologic and psychiatric complications include stroke, both ischemic and hemorrhagic, postoperative delirium, and postoperative cognitive dysfunction. Treatment of periop-erative stroke may be difficult as initiation of anticoagulation or thrombolysis may not be safe after surgery. Postoperative delirium is common and transient. Treatment includes reorienta-tion, treatment of pain, workup for metabolic, hemodynamic, or respiratory perturbations, and consideration of the side effects of the anesthetics and analgesics administered. Antipsychotics such as haloperidol can be useful in the treatment of postopera-tive delirium.78 Postoperative cognitive dysfunction (POCD) is a decline in cognitive function that may last days or may persist for months. Risk factors for persistent POCD include advanced age, history of prior stroke, and lower educational level.79 POCD rates among older adult patients have been shown to be as high as 40% at hospital discharge and

1	for persistent POCD include advanced age, history of prior stroke, and lower educational level.79 POCD rates among older adult patients have been shown to be as high as 40% at hospital discharge and 12% 3 months after surgery. Notably, a causal link has not been established between administration of anesthesia and development of POCD, sug-gesting that it may be the physiologic stress of the perioperative experience that may lead to a decline in cognitive function in such patients.80CONCLUSIONThe practice of anesthesia has improved dramatically over the past century. Advances in training, pharmacology, anesthesia equipment, and monitoring have not only made anesthesia dra-matically safer but have also allowed ever sicker patients to benefit from surgery.REFERENCESEntries highlighted in bright blue are key references. 1. Haridas RP. Horace wells’ demonstration of nitrous oxide in Boston. Anesthesiology. 2013;119:1014-1022. 2. Bigelow HJ. insensibility during surgical operations produced

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1	AS, Chen YT, Taguchi A, et al. Postoperative nausea and vomiting following inpatient surgeries in a teaching hospital: a retrospective database analysis. Curr Med Res Opin. 2006;22: 1093-1099. 65. Eskicioglu C, Forbes SS, Aarts MA, et al. Enhanced recovery after surgery (ERAS) programs for patients having colorectal surgery: a meta-analysis of randomized trials. J Gastrointest Surg. 2009;13:2321-2329. 66. Sammour T, Zargar-Shoshtari K, Bhat A, et al. A programme of enhanced recovery after surgery (ERAS) is a cost-effective intervention in elective colonic surgery. N Z Med J. 2010; 123:61-70. 67. Reurings JC, Spanjersberg WR, Oostvogel HJ, et al. A prospec-tive cohort study to investigate cost-minimisation, of Traditional open, open fAst track recovery and laParoscopic fASt track mul-timodal management, for surgical patients with colon carcinomas (TAPAS study). BMC Surg. 2010;10:18. 68. Carroll I, Barelka P, Wang CK, et al. A pilot cohort study of the determinants of longitudinal

1	management, for surgical patients with colon carcinomas (TAPAS study). BMC Surg. 2010;10:18. 68. Carroll I, Barelka P, Wang CK, et al. A pilot cohort study of the determinants of longitudinal opioid use after surgery. Anesth Analg. 2012;115:694-702. 69. Clarke H, Soneji N, Ko DT, et al. Rates and risk factors for pro-longed opioid use after major surgery: population based cohort study. BMJ. 2014;348:g1251. 70. CDC grand rounds: prescription drug overdoses—a U.S. epi-demic. MMWR Morb Mortal Wkly Rep. 2012;61:10-13. 71. Rosenberg H, Pollock N, Schiemann A, et al. Malignant hyper-thermia: a review. Orphanet J Rare Dis. 2015;10:93. 72. Jaber S, Lescot T, Futier E, et al. Effect of noninvasive ventila-tion on tracheal reintubation among patients with hypoxemic respiratory failure following abdominal surgery: a randomized clinical trial. JAMA. 2016;315:1345-1353. 73. Hernandez G, Vaquero C, Gonzalez P, et al. Effect of postextu-bation high-flow nasal cannula vs conventional oxygen therapy

1	surgery: a randomized clinical trial. JAMA. 2016;315:1345-1353. 73. Hernandez G, Vaquero C, Gonzalez P, et al. Effect of postextu-bation high-flow nasal cannula vs conventional oxygen therapy on reintubation in low-risk patients: a randomized clinical trial. JAMA. 2016;315:1354-1361. 74. Corley A, Bull T, Spooner AJ, et al. Direct extubation onto high-flow nasal cannulae post-cardiac surgery versus standard treatment in patients with a BMI >/=30: a randomised con-trolled trial. Intensive Care Med. 2015;41:887-894. 75. Ansari BM, Hogan MP, Collier TJ, et al. A randomized con-trolled trial of high-flow nasal oxygen (optiflow) as part of an enhanced recovery program after lung resection surgery. Ann Thorac Surg. 2016;101:459-464.Brunicardi_Ch46_p2027-p2044.indd 204201/03/19 11:04 AM 2043ANESTHESIA FOR SURGICAL PATIENTSCHAPTER 46 76. Parke R, McGuinness S, Dixon R, et al. Open-label, phase II study of routine high-flow nasal oxygen therapy in cardiac sur-gical patients. Br J Anaesth.

1	FOR SURGICAL PATIENTSCHAPTER 46 76. Parke R, McGuinness S, Dixon R, et al. Open-label, phase II study of routine high-flow nasal oxygen therapy in cardiac sur-gical patients. Br J Anaesth. 2013;111:925-931. 77. Stephan F, Barrucand B, Petit P, et al. High-flow nasal oxygen vs noninvasive positive airway pressure in hypoxemic patients after cardiothoracic surgery: a randomized clinical trial. JAMA. 2015;313:2331-2339. 78. Vijayakumar B, Elango P, Ganessan R. Post-operative delirium in elderly patients. Indian J Anaesth. 2014;58:251-256. 79. Monk TG, Weldon BC, Garvan CW, et al. Predictors of cogni-tive dysfunction after major noncardiac surgery. Anesthesiology. 2008;108:18-30. 80. Crosby G, Culley DJ, Hyman BT. Preoperative cognitive assessment of the elderly surgical patient: a call for action. Anesthesiology. 2011;114:1265-1268.Brunicardi_Ch46_p2027-p2044.indd 204301/03/19 11:04 AM

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1	Surgical Considerations in Older AdultsAnne M. Suskind and Emily Finlayson 47chapterINTRODUCTIONAs the population ages, an increasing number of older adults will develop surgical diseases. The segment of the U.S. popu-lation age 65 and older is expected to double by 2050. Older adults present unique challenges in surgical management and decision-making. The accumulation of comorbid conditions and physiologic vulnerability that occurs with age put older adults at high risk for major morbidity and mortality after sur-gery. It is essential that surgeons approach this population with a new set of skills and knowledge to provide optimal care for this vulnerable population. A comprehensive understanding of the unique vulnerabilities of older adults—geriatric syndromes and risk factors—are required to accurately estimate surgical risk, inform surgical decision-making, and guide perioperative management. In this chapter, we will (a) discuss the physiologic conditions that are common in older

1	to accurately estimate surgical risk, inform surgical decision-making, and guide perioperative management. In this chapter, we will (a) discuss the physiologic conditions that are common in older adults that are essential for surgical risk assessment of older adults considering surgery, (b) describe best practices in perioperative care in the older adult, and (c) discuss special considerations and common pitfalls for surgical conditions that are common in the older adult.GERIATRIC SYNDROMESApproximately one-third of older individuals undergoing vascu-lar and urologic surgery suffer from geriatric syndromes.1,2 This term is used to describe clinical conditions that do not fit into discrete disease categories but that can substantially negatively impact quality of life and result in disability. Geriat-ric syndromes often involve multiple underlying factors and organ systems (i.e., multiple causation of a unified manifesta-tion3) and include frailty, falls, delirium, malnutrition,

1	Geriat-ric syndromes often involve multiple underlying factors and organ systems (i.e., multiple causation of a unified manifesta-tion3) and include frailty, falls, delirium, malnutrition, dizziness, syncope, urinary incontinence, and pressure ulcers among oth-ers. These syndromes can be present before surgery and/or 1develop as a result of surgery and hospitalization.1,2 Further-more, close attention to both the presence and development of geriatric syndromes among surgical candidates is important and often overlooked, making caring for older surgical patients unique compared to their younger and healthier counterparts.FrailtyFrailty is among the most widely studied geriatric syndrome in the surgical literature. Frail older individuals are at high risk for adverse events in the face of stressors such as surgery. They are more likely to experience surgical complications, delayed recovery, falls, and to develop functional impairment. Frailty is also associated with a higher risk of

1	stressors such as surgery. They are more likely to experience surgical complications, delayed recovery, falls, and to develop functional impairment. Frailty is also associated with a higher risk of death. It Is believed that frailty is a chronic, progressive condition that represents a spec-trum; less frail individuals may be responsive to strategies or interventions to ameliorate its clinical manifestations, while more frail individuals may demonstrate an irreversible predeath condition with limited life expectancy.4Frailty has been shown to be independently predictive of poor postoperative outcomes. Makary et al studied 594 older patients presenting for elective surgery at a university hospital and demonstrated that frail individuals were at increased risk of postoperative complications (OR 2.54; 95% CI 1.12–5.77), lon-ger length of stay (incidence rate ratio 1.69; 95% CI 1.28–2.23), and discharge to a skilled or assisted living facility after previ-ously living at home (OR 20.48;

1	(OR 2.54; 95% CI 1.12–5.77), lon-ger length of stay (incidence rate ratio 1.69; 95% CI 1.28–2.23), and discharge to a skilled or assisted living facility after previ-ously living at home (OR 20.48; 95% CI 5.54-75.68).5 Similar findings have been echoed throughout the surgical literature inclusive of vascular, colorectal, cardiac, urologic, and other types of procedures.6-11Definitions of frailty fall into two broad models; a pheno-typic model and a deficits accumulation model. The phenotypic model was originally described by Linda Fried using data from the Cardiovascular Health Study, which is an observational study of community-dwelling men and women age 65 years and Introduction 2045Geriatric Syndromes 2045Frailty / 2045Falls / 2047Delirium / 2047Preoperative Assessment 2047Best Practices: Preoperative Assessment / 2048Frailty Assessment / 2049Cognitive and Behavioral Assessment / 2049Medical Assessment / 2050Nutritional Assessment / 2051Psychosocial Considerations / 2052Medication

1	Preoperative Assessment / 2048Frailty Assessment / 2049Cognitive and Behavioral Assessment / 2049Medical Assessment / 2050Nutritional Assessment / 2051Psychosocial Considerations / 2052Medication Review / 2052Patient Counseling / 2052Preoperative Preparation 2052Patient Goals, Preferences, and Advance Directives / 2052Preoperative Fasting / 2053Antibiotic Prophylaxis and Venous Thromboembolism Prevention / 2053Surgical Prehabilitation / 2053Palliative Care Services for Older Surgical Patients / 2053Special Considerations 2054Functional Recovery / 2054Cancer Surgery / 2055Emergency Surgery / 2055Cardiovascular Surgery / 2055Valve Replacement / 2055Endovascular Aortic Surgery / 2056Palliative Surgery / 2056Summary 2056Brunicardi_Ch47_p2045-p2060.indd 204528/02/19 2:08 PM 2046Table 47-1Criteria used to define frailty• Weight loss: “In the last year, have you lost more than 10 pounds unintentionally (i.e., not due to dieting or exercise)?” If yes, then frail for weight loss

1	47-1Criteria used to define frailty• Weight loss: “In the last year, have you lost more than 10 pounds unintentionally (i.e., not due to dieting or exercise)?” If yes, then frail for weight loss criterion. At follow-up, weight loss was calculated as: (Weight in previous year – current measured weight)/(weight in previous year) = K. If K ≥0.05 and the subject does not report that he/she was trying to lose weight (i.e., unintentional weight loss of at least 5% of previous year’s body weight), then frail for weight loss = Yes.• Exhaustion: Using the CES-D Depression Scale, the following two statements are read. (a) I felt that everything I did was an effort; (b) I could not get going. The question is asked “How often in the last week did you feel this way?” 0 = rarely or none of the time (<1 day), 1 = some or a little of the time (1–2 days), 2 = a moderate amount of the time (3–4 days), or 3 = most of the time. Subjects answering “2” or “3” to either of these questions are categorized as

1	day), 1 = some or a little of the time (1–2 days), 2 = a moderate amount of the time (3–4 days), or 3 = most of the time. Subjects answering “2” or “3” to either of these questions are categorized as frail by the exhaustion criterion.• Physical Activity: Based on the short version of the Minnesota Leisure Time Activity questionnaire, asking about walking, chores (moderately strenuous), mowing the lawn, raking, gardening, hiking, jogging, biking, exercise cycling, dancing, aerobics, bowling, golf, singles tennis, doubles tennis, racquetball, calisthenics, swimming. Kcals per week expended are calculated using standardized algorithm. This variable is stratified by gender. Men: Those with Kcal of physical activity per week <383 are frail. Women: Those with Kcals per week <270 are frail.• Walk Time, stratified by gender and height (gender-specific cutoff a medium height). Men Height ≤173 cm Height >173 cmCutoff for time to walk 15 feet criterion for frailty≥7 seconds≥6

1	are frail.• Walk Time, stratified by gender and height (gender-specific cutoff a medium height). Men Height ≤173 cm Height >173 cmCutoff for time to walk 15 feet criterion for frailty≥7 seconds≥6 seconds Women Height ≤159 cm Height >159 cm≥7 seconds≥6 seconds• Grip Strength, stratified by gender and body mass index (BMI) quartiles: Men BMI ≤24 BMI 24.1–26 BMI 26.1–28 BMI >28Cutoff for grip strength (Kg) criterion for frailty≤29≤30≤30≤32 Women BMI ≤23 BMI 23.1–26 BMI 26.1–29 BMI >29≤17≤17.3≤18≤21Reproduced with permission from Fried LP, Tangen CM, Walston J, et al. Frailty in older adults: evidence for a phenotype, J Gerontol A Biol Sci Med Sci. 2001 Mar;56(3):M146-M156.older. Individuals in this cohort underwent baseline evaluations and had 4 to 7 years of follow-up with annual examinations and surveillance for the following outcomes: incident disease, hos-pitalization, falls, disability, and mortality. Based on observa-tions in these individuals over time, the following criteria were

1	and surveillance for the following outcomes: incident disease, hos-pitalization, falls, disability, and mortality. Based on observa-tions in these individuals over time, the following criteria were identified to define frailty: weight loss, exhaustion, physical activity, walk time, and grip strength (Table 47-1). The pres-ence of one or two of these factors is associated with intermedi-ate risk for poor outcomes, i.e., a “prefrail” phenotype, and the presence of three or more of these factors is associated with high risk for poor outcomes, i.e., a “frail” phenotype.12 This study additionally demonstrated that frailty is strongly associated Key Points1 Frailty, dementia, and functional impairment are significant contributors to morbidity and mortality after surgery. Assess-ment of these risk factors is essential in an older population.2 Geriatric perioperative pathways are effective in preventing delirium, a morbid and costly postoperative complication that is associated with cognitive

1	factors is essential in an older population.2 Geriatric perioperative pathways are effective in preventing delirium, a morbid and costly postoperative complication that is associated with cognitive decline.3 Population-based data demonstrate that mortality after emer-gency surgery and high-risk cancer surgery is substantially higher in older adults that in a younger population.4 Impaired cardiac function is responsible for more than half of the postoperative deaths in older adult patients, so careful attention must be paid to intravascular volume status in the perioperative period.5 Many frail elders experience significant and sustained func-tional decline after surgery.6 Older adults with acute abdominal pathology—appendicitis, cholecystitis—often do not have fever, elevated white blood cell count, or physical findings of peritonitis.Brunicardi_Ch47_p2045-p2060.indd 204628/02/19 2:08 PM 2047SURGICAL CONSIDERATIONS IN OLDER ADULTSCHAPTER 47with several major chronic diseases,

1	cell count, or physical findings of peritonitis.Brunicardi_Ch47_p2045-p2060.indd 204628/02/19 2:08 PM 2047SURGICAL CONSIDERATIONS IN OLDER ADULTSCHAPTER 47with several major chronic diseases, including cardiovascular disease, pulmonary disease, and diabetes; however, not all frail individuals demonstrated these associations. The same is true for disability. While there is some overlap between frailty and disability, not all frail individuals are disabled. These findings suggest that while there may be overlap between these three constructs in some individuals, frailty is a distinct process from both comorbidity and disability.12Alternatively, the deficit accumulation model, developed by Rockwood et al, suggests that frailty is defined by discrete failures of redundant physiologic systems. The more deficits that occur, the more likely it is that adverse outcomes will result. Using data from the Canadian Study of Health and Aging, a longitudinal study of individuals age 65 and

1	systems. The more deficits that occur, the more likely it is that adverse outcomes will result. Using data from the Canadian Study of Health and Aging, a longitudinal study of individuals age 65 and older, the authors developed a frailty index represented by the cumulative pro-portion of 92 accumulated deficits, which include symptoms, signs, functional impairments, and laboratory abnormalities. They demonstrated that deficits accumulated at a rate of 3% per year in their cohort, represented a gamma distribution, and increased with chronological age; they proposed that this model be used as a proxy for aging and mortality.13While these models are helpful to conceptualize frailty, no one model is all inclusive, and each may have applicability in different settings. For example, the frailty phenotype does not include items on cognition or mood and may not be easily appli-cable to the busy clinical setting.14 The deficits accumulation model is ideal for use in large databases, such as

1	phenotype does not include items on cognition or mood and may not be easily appli-cable to the busy clinical setting.14 The deficits accumulation model is ideal for use in large databases, such as the American College of Surgeons National Surgical Quality Improvement Project (ACS-NSQIP),15 and may be helpful for research and public health and policy purposes, but it is not practical for clini-cal care. Measurement of frailty in the clinical setting will be discussed later in this chapter.While frailty is often defined as a geriatric syndrome, it is also plausible that other geriatric syndromes (i.e., urinary incontinence, falls, pressure ulcers, delirium, and functional decline) may demonstrate shared risk factors that lead to frailty. In turn, frailty may also cause more risk factors and more geriat-ric syndromes.16 Regardless of the association and directionality between frailty and other geriatric syndromes, identification of each is essential in the preoperative setting in order

1	more geriat-ric syndromes.16 Regardless of the association and directionality between frailty and other geriatric syndromes, identification of each is essential in the preoperative setting in order to help risk stratify and potentially to mitigate risk for patients considering surgical intervention.FallsOlder adults are at markedly increased risk of falls, and one in three adults age 65 and older report falling in the last year. The incidence of falls increases with age, and close to 60% of individuals who have fallen in the last year will fall again within the following year. Falls are associated with subsequent declines in functional status, greater likelihood of nursing home placement, increased use of medical services, and development of a fear of falling. Approx-imately half of older individuals who fall are unable to get up, resulting in a “long lie,” which is further associated with lasting functional declines.17 In fact, falls can be so detrimental to older individuals that

1	individuals who fall are unable to get up, resulting in a “long lie,” which is further associated with lasting functional declines.17 In fact, falls can be so detrimental to older individuals that the Joint Commission (United States) established fall prevention as one of its national safety goals in 2015.18Causes of falls can be multifactorial, as with other geri-atric syndromes. Factors include age-related declines, chronic disease, medications, environmental factors, changing positions, routine activities, risk-taking behaviors, acute illness, or situ-ational hazards such as the unfamiliar setting of hospitals and long-term care facilities.17It has been shown that preoperative falls are associated with poor postoperative outcomes among patients undergoing elective surgery. One study looking at 7982 such patients found that a preoperative history of one, two, or three or more falls predicted postoperative falls at 30 days (adjusted OR 2.3, 3.6, 5.5, respectively) and 1 year (adjusted

1	at 7982 such patients found that a preoperative history of one, two, or three or more falls predicted postoperative falls at 30 days (adjusted OR 2.3, 3.6, 5.5, respectively) and 1 year (adjusted OR 2.3, 3.4, 6.9, respec-tively), in addition to predicting a decline in functional status at 30 days (adjusted OR 1.2, 2.4, 2.4, respectively) and 1 year (adjusted OR 1.3, 1.5, 3.2, respectively) and in-hospital compli-cations (adjusted OR 1.2, 1.3, 2.0, respectively).18 Furthermore, preoperative falls are a major predictor of poor postoperative outcomes and may be a valuable preoperative assessment tool as part of routine preoperative care.DeliriumDelirium is a disorder of attention and awareness that develops acutely and tends to fluctuate, as defined by the new Diagnos-tic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria. Delirium is so common among older adults that up to one third of patients age 70 and older admitted to the hospital experiences delirium, half

1	of Mental Disorders, Fifth Edition (DSM-5) criteria. Delirium is so common among older adults that up to one third of patients age 70 and older admitted to the hospital experiences delirium, half of which have delirium on admission and the other half of which develop delirium dur-ing the hospitalization itself.19 Rates of delirium among older patients undergoing surgery ranges from 4% to 5% in cataract and urologic procedures to 50% to 60% in infrarenal AAA repair or hip fracture surgery.20 Patients who develop postopera-tive delirium have a twoto threefold increased risk of mortality within the first year after surgery.21Preoperative assessment should focus on the identifi-cation of risk factors for delirium including age 70 years or older, cognitive impairment, limited physical function, history of alcohol abuse, abnormal serum sodium, potassium or glu-cose, intrathoracic surgery, and AAA surgery. Preoperative assessment and documentation of mental status is imperative in order to

1	history of alcohol abuse, abnormal serum sodium, potassium or glu-cose, intrathoracic surgery, and AAA surgery. Preoperative assessment and documentation of mental status is imperative in order to establish a baseline for postoperative comparison. Intraoperative blood loss is another risk factor for postopera-tive delirium, and patients with a postoperative hematocrit less than 30% are at increased risk, irrespective of baseline risk fac-tors. Postoperatively, undertreatment of pain is an important risk factor for delirium. Management of fluid, electrolyte and metabolic abnormalities, optimization of blood loss replace-ment, maintenance of circadian rhythms, and cautious prescrip-tion of medication and pain management are among the most important methods by which to minimize the risk of postop-erative delirium among surgical patients.20 Furthermore, many commonly used medications may induce delirium and should be avoided. They include drugs with anticholinergic properties,

1	risk of postop-erative delirium among surgical patients.20 Furthermore, many commonly used medications may induce delirium and should be avoided. They include drugs with anticholinergic properties, corticosteroids, merperidine, and sedative hypnotics. Finally, postoperative care bundles have been shown to successfully reduce the incidence of delirium. These strategies include sensory enhancement (glasses, hearing aids), early mobility, cognitive orientation and therapeutic activities, and sleep proto-cols. Treatment of postoperative delirium should focus on treat-able etiologies (Fig. 47-1), and pharmacologic treatment should be reserved for patients who are at risk of harming themselves or others.PREOPERATIVE ASSESSMENTPreoperative assessment in older adults is more complex than in younger individuals, as there are many unique characteris-tics that require consideration. The purpose of the assessment Brunicardi_Ch47_p2045-p2060.indd 204728/02/19 2:08 PM 2048SPECIFIC

1	than in younger individuals, as there are many unique characteris-tics that require consideration. The purpose of the assessment Brunicardi_Ch47_p2045-p2060.indd 204728/02/19 2:08 PM 2048SPECIFIC CONSIDERATIONSPART IIFigure 47-1. Useful algorithm to determine preoperative management of the acutely delirious patient.is not to “clear” the patient for surgery but rather to minimize risks and optimize good outcomes. The ACS NSQIP and the American Geriatrics Society (AGS) published best practice guidelines to help optimize this process. Preoperative planning should be pro-active, commencing at the time of surgical decision-making, if not sooner. To this end, the ACS NSQIP/AGS produced best practice guidelines and a preoperative management checklist to provide a framework for thinking about critical issues in this patient population (Table 47-2).22 In addition to these issues, they emphasize the importance of planning analgesia strategies, making efforts to minimize opioid use and to

1	critical issues in this patient population (Table 47-2).22 In addition to these issues, they emphasize the importance of planning analgesia strategies, making efforts to minimize opioid use and to prevent functional and cognitive decline, obtaining multidisciplinary consultation early and early involvement of allied health staff (i.e., physical or occupational therapy), and anticipating home health needs that may be required at discharge.23Best Practices: Preoperative AssessmentFunctional Assessment. As previously discussed, poor phys-ical function prior to surgery is associated with higher risk of major postoperative complications, increased need for intensive WORKUPPhysical exam• Check surgical wound• Check for tubes/lines/drains• Urinalysis• CBC, BMP• May consider: TSH, NH3, LFT, EtoH/tox• Blood/sputum/urine cultures-Vital signs, pulse ox, pain assessmentMedication reviewLaboratory evaluation:---Chest XRSubstance abuse history--PHARMACOLOGIC MANAGEMENT:ONLY FOR AGITATED DELIRIUM

1	Blood/sputum/urine cultures-Vital signs, pulse ox, pain assessmentMedication reviewLaboratory evaluation:---Chest XRSubstance abuse history--PHARMACOLOGIC MANAGEMENT:ONLY FOR AGITATED DELIRIUM AND PATIENT SAFETYAgent of choice: Low dose Haloperidol0.5 mg PO: 1–2 hours for effect, may redose in 60 m0.5 mg IM/IV: 20–40 min to take effect, may redosein 30 mMost patients respond to total of 1–2 mgCheck 12 lead EKG, hold if QTc >500----Use benzos only if there is a concern for withdrawalDIFFERENTIAL DIAGNOSIS DRUG!!! Electrolytes, environment change Lack of drugs (withdrawal), lack of sleep Infection, idiopathic Restraints, reduced sensory input (vision/hearing) Intracranial (CVA, bleed, post-ictal, meningitis) Urinary retention, or fecal impaction Metabolic, includes PE/MI, uremia, ammonia, thyroidDELIRIUMAlways check the medication list – there is acumulative effect burden, any new medicationor recent dose change is suspectAlgorithm for the acutelydelirious patient.For prevention in at

1	thyroidDELIRIUMAlways check the medication list – there is acumulative effect burden, any new medicationor recent dose change is suspectAlgorithm for the acutelydelirious patient.For prevention in at risk patientsplease refer to opposite side.Why are they delirious?Common Delirium Inducing MedicationsRisk FactorsPrecipitating Factors, in aaddition to SURGERYPhysical restraintsMalnutrition3 medication classes addedBladder catheterUncontrolled pain-----PREVENTION/CONSERVATIVE MANAGEMENTSensory enhancement: Hearing aids, glasses at bedsideMobilizationCognitive orientation and stimulationSleep enhancementMedication reviewNormalize environment• Get rid of tethers• Keep room calm and quiet• Encourage family/caregiver involvement------Address/remove precipitating factors or agentsIf MB consider hospitalist consult--Are they a danger tothemselves or others?Age >65Cognitive impairmentComorbidity burdenPoor functional statusHearing/vision impairmentDepression------Anticholinergics: Tricyclics,

1	hospitalist consult--Are they a danger tothemselves or others?Age >65Cognitive impairmentComorbidity burdenPoor functional statusHearing/vision impairmentDepression------Anticholinergics: Tricyclics, antihistamines,H2-blockers, antimuscarinics, antispasmodics,promethazine, olanzapine, paroxetineCorticosteroids: methylprednisone, prednisoneMeperidineSedative hypnotics: benzos, zolpidem----Nu-DESC Screen: ScoreEach item scored 0–2• Disorientation• Inappropriate behavior• Inappropriate communication• Illusions/hallucinations• Psychomotor retardationIs your patient acutely delirious?Yes?Differential and workupBrunicardi_Ch47_p2045-p2060.indd 204828/02/19 2:08 PM 2049SURGICAL CONSIDERATIONS IN OLDER ADULTSCHAPTER 47Table 47-2Immediate preoperative management checklist from the ACS NSQIP/AGS1. Confirm and document patient goals and treatment preferences, including advance directives2. Confirm and document patient’s health care proxy or surrogate decision-maker3. In patients with

1	and document patient goals and treatment preferences, including advance directives2. Confirm and document patient’s health care proxy or surrogate decision-maker3. In patients with existing advance directives, discuss new risks associated with the surgical procedure and an approach for potentially life-threatening problems consistent with the patient’s values and preferences4. Consider shortened fluid fast (clear liquids up to two hours before anesthesia)5. Adhere to existing best practices regarding antibiotic and venous thromboembolism prophylaxis6. Ensure nonessential medications have been stopped and essential medications have been takenAdapted with permission from Mohanty S, Rosenthal RA, Russell MM, et al: Optimal Perioperative Management of the Geriatric Patient: A Best Practices Guideline from the American College of Surgeons NSQIP and the American Geriatrics Society, J Am Coll Surg. 2016 May;222(5):930-947.rehabilitation services, increased rates of discharge to a skilled or

1	from the American College of Surgeons NSQIP and the American Geriatrics Society, J Am Coll Surg. 2016 May;222(5):930-947.rehabilitation services, increased rates of discharge to a skilled or assisted nursing facility, and higher mortality.24-26 Assessment of physical function and performance status in the preoperative setting are recommended by the ACS NSQIP/AGS best practice guidelines.22 There are several methods by which to measure physical function. Overall functional status may be ascertained by assessing the ability of an individual to perform activities of daily living (ADLs) and instrumental ADLs. ADLs include dressing, bathing, toileting, transferring, continence, and eating independently.25 Instrumental ADLs measure an individual’s ability to live independently and include the ability to perform the following tasks: shopping, laundry, mode of transportation, ability to handle finances, responsibility for won medications, food preparation, and housekeeping.27In addition to

1	ability to perform the following tasks: shopping, laundry, mode of transportation, ability to handle finances, responsibility for won medications, food preparation, and housekeeping.27In addition to assessment of ADLs and instrumental ADLs, the surgeon should also assess for deficits in vision, hearing, and swallowing, inquire about history of falls in the past year, evaluate for limitations in gait and mobility, and determine risk for falls, which can be performed via the timed up and go test (TUGT). This test measures gait and mobility impairment and is associated with increased risk for falls in ambulatory individu-als. All that is required to perform this test are a chair, a mark 10 feet in front of the chair, and a stopwatch. Individuals are instructed to do the following while being timed:1. Stand up from the chair (without using arm rests, if possible)2. Walk to the mark (10 feet in front of them)3. Turn4. Walk back to the chair5. Sit down in the chairA time of ≥15 seconds

1	timed:1. Stand up from the chair (without using arm rests, if possible)2. Walk to the mark (10 feet in front of them)3. Turn4. Walk back to the chair5. Sit down in the chairA time of ≥15 seconds indicates high risk of falls and should prompt referral to physical therapy for further assessment.In a prospective cohort of individuals age 65 and older undergoing surgery, the TUGT times were stratified into three groups that strongly correlated with varying risk for postop-erative complications and 1-year mortality. These groups were “slow” (≥15 seconds), “intermediate” (11–14 seconds), and “fast” (≤10 seconds). Postoperative complications and 1-year mortality in the slow group were significantly higher com-pared to those in the fast group, 52% to 77% versus 11% to 13% for complications, and 31% compared to 3% for mortality, respectively.9Frailty AssessmentAs stated earlier, frailty is an important consideration in preop-erative planning for older individuals. Measurement of frailty can

1	31% compared to 3% for mortality, respectively.9Frailty AssessmentAs stated earlier, frailty is an important consideration in preop-erative planning for older individuals. Measurement of frailty can take several forms. One method is to apply the operational definition put forth by Fried (see Table 47-1), which has been applied to surgical patients and shown to be an independent pre-dictor of postoperative adverse events, increased length of stay, and higher likelihood of discharge to a skilled or assisted living facility.5The frailty phenotype, however, may be cumbersome to apply in the busy clinical setting. To this end, Robinson proposed alternative definitions and methods for frailty mea-surement. One such method includes the following criteria: cog-nitive impairment (Mini-Cog score of ≤3), poor nutrition (serum albumin ≤3), history of falls (≥1 fall in the past 6 months), and low hematocrit (<35%).26 A second definition includes func-tional impairment (TUGT ≥15 seconds and

1	of ≤3), poor nutrition (serum albumin ≤3), history of falls (≥1 fall in the past 6 months), and low hematocrit (<35%).26 A second definition includes func-tional impairment (TUGT ≥15 seconds and dependence in any ADL) and comorbidity (Charlson index score ≥3).28Cognitive and Behavioral AssessmentPreoperative cognitive impairment is strongly linked to postop-erative delirium, worse surgical outcomes, longer hospital stays, increased risk of functional decline, and even mor-tality. History and cognitive assessment are important to con-sider early on in all surgical candidates age 65 years and older in the preoperative setting. The Mini-Cog, consisting of the three-item recall and clock draw tests, can be used to complete this assessment (Table 47-3). If possible, someone who knows the patient well (such as a spouse or family member) should be interviewed about the presence and evolution of any cognitive decline in the patient. If decline is present, the patient should be referred to a

1	well (such as a spouse or family member) should be interviewed about the presence and evolution of any cognitive decline in the patient. If decline is present, the patient should be referred to a primary care physician, geriatrician, or mental health specialist for further evaluation. Documentation of pre-operative cognitive status will further assist in the identification of any postoperative cognitive dysfunction.22 Risk factors for postoperative delirium should also be assessed in the preopera-tive period and are detailed in Table 47-4.In addition to measuring cognitive status, assessing the patient’s decision-making capacity is also important to deter-mine the patient’s ability to provide informed surgical consent. It is helpful to ask the patient to describe, in his/her own words, the important features of the discussion, the condition and indi-cations for surgery, and the risks, benefits, and alternatives to surgery. There are four legally-relevant criteria for decision-making

1	important features of the discussion, the condition and indi-cations for surgery, and the risks, benefits, and alternatives to surgery. There are four legally-relevant criteria for decision-making capacity22:1. The patient can clearly indicate his/her treatment choice.2. The patient understands the relevant information communi-cated by the physician.3. The patient acknowledges his/her medical condition, treat-ment options, and likely outcomes.4. The patient can engage in a rational discussion about the treatment options.Depression should also be screened for in the preoperative setting, as up to 11% of the population age 71 years and older in the United States suffer from this condition.29 Risk factors 2Brunicardi_Ch47_p2045-p2060.indd 204928/02/19 2:08 PM 2050SPECIFIC CONSIDERATIONSPART IITable 47-4Risk factors for postoperative deliriumCognitive and behavioral disorders:• Cognitive impairment and dementia• Untreated or inadequately controlled pain• Depression• Alcohol use• Sleep

1	47-4Risk factors for postoperative deliriumCognitive and behavioral disorders:• Cognitive impairment and dementia• Untreated or inadequately controlled pain• Depression• Alcohol use• Sleep deprivationDisease or illness related:• Severe illness or comorbidities• Renal insufficiency• Anemia• HypoxiaMetabolic:• Poor nutrition• Dehydration• Electrolyte abnormalitiesFunctional impairments:• Poor functional status• Immobilization• Hearing or vision impairmentOther:• Older age ≥70 years• Polypharmacy and use of psychotropic medications (benzodiazepines, anticholinergics, and antihistamines)• Risk of urinary retention or constipation, presence of urinary catheterReproduced with permission from Chow WB, Rosenthal RA, Merkow RP, et al. Optimal preoperative assessment of the geriatric surgical patient: a best practices guideline from the American College of Surgeons National Surgical Quality Improvement Program and the American Geriatrics Society, J Am Coll Surg. 2012 Oct;215(4):453-466.Table

1	a best practices guideline from the American College of Surgeons National Surgical Quality Improvement Program and the American Geriatrics Society, J Am Coll Surg. 2012 Oct;215(4):453-466.Table 47-3Cognitive assessment: three-item recall and clock draw1. Get the patient’s attention and say: “I am going to say three words that I want you to remember now and later. These words are banana, sunrise, chair. Please say them for me now.” Give the patient three tries to repeat the words. If unable after three tries, go to next item.2. Say all of the following phrases in the order indicated: “Please draw a clock in the space below. Start by drawing a large circle. Put all the numbers in the circle and set the hands to show 11:10 (10 past 11).” If the subject has not finished clock drawing in 3 minutes, discontinue and ask for recall items.3. Say: “What were the three words I asked you to remember?”Scoring:• 3 item recall (0-3 points): 1 point for each correct word• Clock draw (0-2

1	in 3 minutes, discontinue and ask for recall items.3. Say: “What were the three words I asked you to remember?”Scoring:• 3 item recall (0-3 points): 1 point for each correct word• Clock draw (0-2 points): 0 points for abnormal clock; 2 points for normal clock A normal clock has all of the following elements: 1. All numbers 1-12, each only once, are present in the correct order and direction (clockwise) inside the circle. 2. Two hands are present, one pointing to 11 and one pointing to 2. 3. Any clock missing any of these elements is scored abnormal. refusal to draw a clock is scored abnormalTotal score of 0, 1, or 2 suggests possible impairmentTotal score of 3, 4, or 5 suggests no impairmentReproduced with permission from Borson S, Scanlan J, Brush M, et al. The mini-cog: a cognitive ‘vital signs’ measure for dementia screening in multi-lingual elderly, Int J Geriatr Psychiatry. 2000 Nov;15(11):1021-1027.for depression in older adults include female gender, disability,

1	cognitive ‘vital signs’ measure for dementia screening in multi-lingual elderly, Int J Geriatr Psychiatry. 2000 Nov;15(11):1021-1027.for depression in older adults include female gender, disability, bereavement, and sleep disturbance. Poor health status, cogni-tive impairment, living alone, and new medical illness may also contribute to depression.30The Patient Health Questionnaire-2 (PHQ-2) can be used to screen for depression via the following two questions31:1. In the past 12 months, have you ever had a time when you felt sad, blue, depressed, or down for most of the time for at least two weeks?2. In the past 12 months, have you ever had a time, lasting at least two weeks, when you didn’t care about the tings things that you usually care about or when you didn’t enjoy the things that you usually enjoy?If a patient answers yes to either question, further revalu-ation with a primary care physician, geriatrician, or psychiatrist is recommended.Screening for alcohol and substance abuse

1	usually enjoy?If a patient answers yes to either question, further revalu-ation with a primary care physician, geriatrician, or psychiatrist is recommended.Screening for alcohol and substance abuse is also recom-mended, as alcohol use is common among older adults. Up to 13% of men and 8% of women ≥65 years consume at least 2 drinks per day and 14.5% of men and 3.3% of women con-sume 5 or more drinks per day.32 Alcohol and substance abuse are associated with increased rates of postoperative mortality and complications including pneumonia, sepsis, wound infec-tion and disruption, and prolonged length of stay.33,34 The ACS NSQIP/AGS recommend screening for alcohol and substance abuse among older individuals with the modified CAGE ques-tionnaire in combination with prescribing daily multivitamins. including folic acid and high dose (100 mg) oral or parenteral thiamine to patients who drink alcohol.22,35Medical AssessmentA thorough medical assessment should be performed in all older

1	including folic acid and high dose (100 mg) oral or parenteral thiamine to patients who drink alcohol.22,35Medical AssessmentA thorough medical assessment should be performed in all older operative candidates and should include a cardiac evaluation, pulmonary evaluation, nutritional assessment, and medication evaluation and management where appropriate.Cardiac adverse events are the most common cause of serious perioperative morbidity and mortality among patients undergoing noncardiac operations and occur more commonly in older adults.36,37 For these reasons, cardiac evalu-ation may be helpful to identify older patients with higher risk for cardiac complications who may be candidates for periopera-tive optimization. This evaluation should follow the American College of Cardiology and the American Heart Association (ACC/AHA) algorithm for cardiac evaluation and care. This is a step-wise approach that incorporates the following factors: (a) urgency of surgery (whether the procedure is

1	American Heart Association (ACC/AHA) algorithm for cardiac evaluation and care. This is a step-wise approach that incorporates the following factors: (a) urgency of surgery (whether the procedure is an emergency); the (b) presence of active major cardiac risk factors (i.e., unsta-ble coronary syndromes, decompensated heart failure, signifi-cant arrhythmias or severe valvular disease) that would necessitate referral to a cardiologist; (c) if risk factors for stable coronary artery disease are present, then calculation of risk for major adverse cardiac events using the ACS NSQIP calculator 3Brunicardi_Ch47_p2045-p2060.indd 205028/02/19 2:08 PM 2051SURGICAL CONSIDERATIONS IN OLDER ADULTSCHAPTER 47is recommended; (d) if the patient is at low risk for major car-diac events (<1%) then no further testing is needed; (e) if the patient is at elevated risk of major cardiac events, then determi-nation of functional capacity with an objective measure or scale may be helpful; (f) if functional

1	testing is needed; (e) if the patient is at elevated risk of major cardiac events, then determi-nation of functional capacity with an objective measure or scale may be helpful; (f) if functional capacity is poor, then additional testing such as pharmacological stress testing, may be helpful; and finally (g) if testing does not impact care, then one should proceed to surgery or consider alternative treatment strategies.38 Routine electrocardiograms are not indicated in older patients undergoing low-risk surgery in the absence of other risk factors.39-41The combined effect of depletion of intravascular volume, age-related impairment of response to catecholamines, and increased myocardial relaxation time adversely affects the cardiac function of an older adult patient under stress in the perioperative period. Aging has been demonstrated to cause a decrease in cardiac output by approximately 1% per year. Older individuals fail to augment heart rate to the same extent as younger

1	in the perioperative period. Aging has been demonstrated to cause a decrease in cardiac output by approximately 1% per year. Older individuals fail to augment heart rate to the same extent as younger individuals. More importantly, the ability to increase cardiac output with aging is dependent on ventricular dilatation, which is determined by preload. Therefore, careful attention must be paid to volume status in the perioperative period. Over one half of all postoperative deaths in older adult patients and 11% of postoperative complications are a result of impaired car-diac function under physiologic stress. Incomplete emptying of the ventricle at end systole and subsequent reduction in ejection fraction is characteristic of the aging heart. Reduced distensibil-ity, in addition to acute stressors, leads to impaired coronary perfusion and cardiac ischemia.An important predictor of surgical outcomes and cardiac complications in the older adult is congestive heart failure (CHF). CHF is

1	stressors, leads to impaired coronary perfusion and cardiac ischemia.An important predictor of surgical outcomes and cardiac complications in the older adult is congestive heart failure (CHF). CHF is present in approximately 10% of patients older than 65 years and is the leading cause of postoperative morbid-ity and mortality. This prevalence will likely increase as per-cutaneous interventions and medical therapy prolongs survival from myocardial ischemia and acute myocardial infarction. Therefore, identifying correctable and uncorrectable cardiovas-cular disease is critical before elective surgical interventions.Common pulmonary postoperative complications in older adults include atelectasis, pneumonia, and prolonged mechanical ventilation.42 These complications can contribute significantly to overall morbidity and mortality among older adults, affecting up to 15% of individuals ≥70 years in the postoperative setting.22,43 Risk factors for postoperative pulmonary complications

1	to overall morbidity and mortality among older adults, affecting up to 15% of individuals ≥70 years in the postoperative setting.22,43 Risk factors for postoperative pulmonary complications include both patient and procedure-related factors. Patient-related fac-tors include age >60, chronic obstructive pulmonary disease (COPD), American Society of Anesthesiologists (ASA) class II or greater, functional dependence, congestive heart failure, obstructive sleep apnea, pulmonary hypertension, current ciga-rette use, impaired sensorium, preoperative sepsis, weight loss >10% in 6 months, serum albumin <3.5 mg/dL, and blood urea nitrogen (BUN) ≥7.5 mmol/L (≥1.5 mg/dL). Surgical related fac-tors include prolonged operation of greater than 3 hours, surgi-cal site, emergency operation, general anesthesia, perioperative transfusion, and residual neuromuscular blockade after an opera-tion. Of note, obesity, well-controlled asthma, and diabetes are not risk factors for postoperative pulmonary

1	anesthesia, perioperative transfusion, and residual neuromuscular blockade after an opera-tion. Of note, obesity, well-controlled asthma, and diabetes are not risk factors for postoperative pulmonary complications. It is recommended that patients with COPD and asthma that is not well controlled undergo preoperative optimization of pulmonary function and other general recommendations include smoking cessation, preoperative intensive inspiratory muscle training, and selective chest radiograph and pulmonary function testing.22In general, the use of routine preoperative screening com-bined with the high cost of unnecessary testing dispute the use of a routine battery of preoperative screening tests in all patients. Instead, it is preferable to perform selected tests in high-risk patients based on history, physical exam, known comorbidities, and the type of procedure being planned.22Nutritional AssessmentNutritional status should also be performed in older adults prior to surgery, as poor

1	on history, physical exam, known comorbidities, and the type of procedure being planned.22Nutritional AssessmentNutritional status should also be performed in older adults prior to surgery, as poor nutrition is potentially modifiable and related to increased risk of postoperative complications. The most com-mon adverse events related to poor nutritional status are infec-tious complications (i.e., surgical site infections, pneumonia, urinary tract infections), wound complications (i.e., dehiscence and anastomotic leaks), and increased length of stay. The ACS NSQIP/AGS best practice guidelines recommend the following to screen for poor nutritional status:1. Document height and weight and calculate body mass index (BMI). A BMI <18.5 kg/m2 places an individual at risk and should prompt referral for full nutritional assessment.2. Measure baseline serum albumin and prealbumin levels. Serum albumin <3.0 g/dL (with no evidence of hepatic or renal dysfunction) should prompt referral for full

1	for full nutritional assessment.2. Measure baseline serum albumin and prealbumin levels. Serum albumin <3.0 g/dL (with no evidence of hepatic or renal dysfunction) should prompt referral for full nutritional assessment.3. Inquire about unintentional weight loss in the last year. Unintentional weight loss >10% to 15% in the past 6 months is associated with severe nutritional risk and should prompt assessment by a dietician.22The American Society for Parenteral and Enteral Nutrition (ASPEN) argues that measurement of serum albumin and pre-albumin reflect the severity of the inflammatory response rather than true poor nutritional status. Instead, they favor a more indi-vidualized approach whereby the presence of any of the follow-ing six factors would classify an individual with malnutrition:1. Insufficient energy intake. Severe malnutrition in the context of chronic illness is defined as <75% of estimated energy requirement for ≥1 month.2. Weight loss. Severe malnutrition in the context

1	energy intake. Severe malnutrition in the context of chronic illness is defined as <75% of estimated energy requirement for ≥1 month.2. Weight loss. Severe malnutrition in the context of chronic illness is defined as >5% weight loss in 1 month, >7.5% weight loss in 3 months, >10% weight loss in 6 months, and >20% weight loss in 1 year.3. Loss of muscle mass. Severe malnutrition is defined as severe muscle wasting of the temples, clavicles, shoulders, interosseous muscles, scapula, thigh, and calf.4. Loss of subcutaneous fat. Severe malnutrition is defined as loss of subcutaneous fat (e.g., orbital, triceps, fat overlying the ribs).5. Localized or generalized fluid accumulation that may sometimes mask weight loss. This can be demonstrated by fluid accumulation evident on exam (e.g., extremities, vulvar/scrotal edema, or ascites).6. Diminished functional status as measured by hand grip strength. Measurements for grip strength are based on nor-mative standards supplied by the

1	extremities, vulvar/scrotal edema, or ascites).6. Diminished functional status as measured by hand grip strength. Measurements for grip strength are based on nor-mative standards supplied by the manufacturer of the mea-surement device.44Regardless of the method used to measure nutritional sta-tus, attention to the diagnosis and management of malnutrition in the perioperative setting is imperative to optimize postopera-tive outcomes.4Brunicardi_Ch47_p2045-p2060.indd 205128/02/19 2:08 PM 2052SPECIFIC CONSIDERATIONSPART IIPsychosocial ConsiderationsAnxiety, depression, substance abuse, and social isolation are common, underdiagnosed conditions in older adults. Careful screening can identify these potential barriers to recovery, safe discharge after surgery, and maintenance of independence.Medication ReviewCareful review and documentation of the patient’s complete med-ication history is important in the preoperative setting. The review should include the use of nonprescription agents,

1	ReviewCareful review and documentation of the patient’s complete med-ication history is important in the preoperative setting. The review should include the use of nonprescription agents, including over-the-counter, nonsteroidal anti-inflammatory drugs (NSAIDs), vitamins, eye drops, topical agents, and herbal products.Additionally, it is important to discontinue medications that should be avoided prior to surgery in order to minimize potential adverse events and interactions. These include dis-continuation of all nonessential medications that are associated with increased surgical risk and medications with potential for drug interactions with anesthesia. Herbal medication should be stopped at least 7 days prior to surgery, and the Beers criteria should be reviewed. Medications with potential for withdrawal (e.g., selective serotonin reuptake inhibitor [SSRIs], tricyclic antidepressants, benzodiazepines, antipsychotics, monoamine oxidase inhibitors [MAOIs], β-blockers, clonidine,

1	for withdrawal (e.g., selective serotonin reuptake inhibitor [SSRIs], tricyclic antidepressants, benzodiazepines, antipsychotics, monoamine oxidase inhibitors [MAOIs], β-blockers, clonidine, statins, and corticosteroids) in addition to angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers should be con-tinued unless hypertension is well controlled. Medications that are associated with increased risk for postoperative delirium should also be avoided and replaced with safer medications. For example, benzodiazepines should be stopped or reduced, where possible. Pain should be adequately controlled and meperi-dine should be avoided for pain management. Caution should be used when prescribing antihistamine H1 antagonists (such as diphenhydramine/Benadryl) and medications with antihista-mine effects. Of note, there is no increased risk associated with neuroleptics (antipsychotics) and digoxin and no conclusive evi-dence against using H2 agonists, tricyclic

1	medications with antihista-mine effects. Of note, there is no increased risk associated with neuroleptics (antipsychotics) and digoxin and no conclusive evi-dence against using H2 agonists, tricyclic antidepressants, anti-Parkinson medications, steroids, NSAIDs, and antimuscarinics in the perioperative setting.Certain medications, such as β-blockers or statins, may be beneficial to start preoperatively in order to reduce risk of perioperative adverse events such as cardiac complications and stroke. There are ACC/AHA guidelines to support the use of perioperative β-blockers in patients who are already taking them, particularly those with independent cardiac indications such as arrhythmia or history of myocardial infarction and in patients who are undergoing intermediate risk or vascular surgery with no known coronary artery disease or with multiple clinical risk factors for ischemic heart disease. Statins should be started as soon as possible prior to surgery in patients who have known

1	with no known coronary artery disease or with multiple clinical risk factors for ischemic heart disease. Statins should be started as soon as possible prior to surgery in patients who have known vascular disease, elevated low-density lipoprotein cholesterol, or ischemia on thallium testing. They may also be considered in patients undergoing vascular and intermediate-risk surgery.Additionally, medications doses should be adjusted based on renal function. Older patients are at greater risk for renal impairment and chronic kidney disease; furthermore, adjustment of medications that are renally cleared is an important consider-ation. Glomerular filtration rate (GFR), rather than creatinine, is the best overall measure of renal function due to the fact that the ratio of GFR to creatinine decreases with increasing age.22Finally, medication use is very common among the older population, and older individuals should be monitored for polypharmacy and potential adverse interactions. This is of

1	with increasing age.22Finally, medication use is very common among the older population, and older individuals should be monitored for polypharmacy and potential adverse interactions. This is of real concern since 40% of individuals age 65 years and older are taking five or more medications prescribed by more than one doctor.45 Additionally, 68% of older adults use over-the-counter medications, dietary supplements, or both concurrently with prescription medications.46 Of note, polypharmacy has been associated with increased risk of cognitive impairment, morbidity, and mortality, and the risk of adverse drug reactions increases with a greater number of medications.45,47Patient CounselingDetermination of the patient’s preferences and expectations prior to surgery is an essential component to preoperative assessment and decision making. One study demonstrated that agreement between patients and their surrogates (primary care providers and close family members) was poor, despite patients

1	preoperative assessment and decision making. One study demonstrated that agreement between patients and their surrogates (primary care providers and close family members) was poor, despite patients predicting that their physicians (90%) and family members (87%) would accurately represent their wishes. Instead, they found that per-cent of agreement ranged from 59% to 88%, suggesting that sub-stituted judgement may not be a good proxy for an individual patient’s wishes.48 Another study looked at older patients with limited life expectancy due to cancer, congestive heart failure, or chronic obstructive pulmonary disease and found that 99% of patients would undergo a low-burden treatment to restore current health (with the alternative being death), but that 74% and 89% would forgo treatment if it resulted in severe functional or cognitive impairment, respectively.49 Furthermore, it is imperative that the surgeon have a substantive discussion with the patient prior to sur-gery to determine

1	it resulted in severe functional or cognitive impairment, respectively.49 Furthermore, it is imperative that the surgeon have a substantive discussion with the patient prior to sur-gery to determine their preferences and expectations and that fam-ily members and potential decision-making surrogates be involved. The ACS NSQIP/AGS Guidelines recommend that the following four points be included in these conversations:1. Ensure that the patient has an advance directive and desig-nated health care proxy.2. Discuss treatment goals and plans with the patient to ensure that the physician understands the patient’s preferences and expectations. This should be documented in the medical record.3. The surgeon should describe the expected postoperative course and possible complications, including the potential for functional decline and need for rehabilitation or nursing home care, if relevant.4. The physician should determine the patient’s family and social support systems. If support is

1	the potential for functional decline and need for rehabilitation or nursing home care, if relevant.4. The physician should determine the patient’s family and social support systems. If support is insufficient, then a referral to a social worker should be considered.22The American College of Surgeons has created a checklist for the optimal preoperative assessment of the geriatric surgical patient (see Table 47-2).PREOPERATIVE PREPARATIONIn the immediate preoperative period, careful planning is essen-tial to optimize the care of the frail older patient.Patient Goals, Preferences, and Advance DirectivesIt is important that surgeons have a good understanding of patients’ goals and wishes surrounding their medical care, par-ticularly towards the end of life. This should be established in the clinic setting prior to surgery and should be confirmed Brunicardi_Ch47_p2045-p2060.indd 205228/02/19 2:08 PM 2053SURGICAL CONSIDERATIONS IN OLDER ADULTSCHAPTER 47and documented throughout the

1	clinic setting prior to surgery and should be confirmed Brunicardi_Ch47_p2045-p2060.indd 205228/02/19 2:08 PM 2053SURGICAL CONSIDERATIONS IN OLDER ADULTSCHAPTER 47and documented throughout the process. Additionally, patients should be encouraged to designate a health care proxy to help with this process, should they be unable to make their own med-ical decisions. The healthcare team should also consider early palliative care consultation in individuals with poor prognoses who are electing to undergo surgery, particularly if they have a life expectancy of less than 6 months.Preoperative FastingHistorically, preoperative fasting began at midnight the night before elective surgery, whereby, patients were not permitted to have any oral intake of either liquids or solids. However, more recent literature suggests that there may be no clear benefit to extended periods of fasting greater than 6 hours.50 Based on the American Society of Anesthesiologists 2011 practice guidelines for all

1	literature suggests that there may be no clear benefit to extended periods of fasting greater than 6 hours.50 Based on the American Society of Anesthesiologists 2011 practice guidelines for all adults, fasting should take the form of stopping clear liq-uids at least 2 hours before elective procedures, stopping light food intake and/or nonhuman milk 6 hours before elective pro-cedures and stopping fried, fatty foods and meat at least 8 hours before elective procedures. Of note, patients with comorbidities or diseases that can affect gastric emptying (i.e., diabetes, hiatal hernia) may require additional periods of fasting.Antibiotic Prophylaxis and Venous Thromboembolism PreventionAntibiotic prophylaxis for older adults should comply with standard guidelines put forth by The Society for Healthcare Epidemiology of America/Surgical Infection Society/American Society of Health-System Pharmacists/Infectious Disease Society. Older adults who receive appropriate preoperative antibiotics

1	Healthcare Epidemiology of America/Surgical Infection Society/American Society of Health-System Pharmacists/Infectious Disease Society. Older adults who receive appropriate preoperative antibiotics demonstrate a mortality benefit at 60 days.51,52 Appropriate antibiotics should be administered within 60 minutes prior to surgical incision.Older adults are at higher risk for venous thromboem-bolism (VTE), making VTE risk stratification among this population essential. Older individuals undergoing orthopedic procedures (i.e., total hip or knee arthroplasty) or who have suffered a hip fracture should be treated with low molecular weight heparin (LMWH) (starting either 13 hours or more pre-operatively or 12 hours or more postoperatively) for a minimum of 10 to 14 days and up to 35 days. Older adults undergoing nonorthopedic surgery (i.e., general, abdominopelvic, bariatric, vascular plastic/reconstructive, and thoracic surgery) should have LMWH or low-dose unfractionated heparin (LDUH) and

1	undergoing nonorthopedic surgery (i.e., general, abdominopelvic, bariatric, vascular plastic/reconstructive, and thoracic surgery) should have LMWH or low-dose unfractionated heparin (LDUH) and mechanical prophylaxis with intermittent pneumatic compres-sion (IPC). Older adults undergoing craniotomy/spinal surgery or cardiac surgery should have IPC. Finally, older individuals who experienced major trauma and spinal cord injury should use LDUH/LMWH and IPC if not contraindicated.23Surgical PrehabilitationOne purpose of the preoperative assessment is to identify potentially modifiable risk factors in order to optimize surgi-cal outcomes. Several prehabilitation programs have emerged in order to help meet this need and have demonstrated prom-ising results. One of the first of such programs was referred to as the Proactive Care of Older People undergoing surgery (POPS) study in the United Kingdom’s National Health Service (NHS). This project was designed to decrease complications leading

1	referred to as the Proactive Care of Older People undergoing surgery (POPS) study in the United Kingdom’s National Health Service (NHS). This project was designed to decrease complications leading to increased hospital length of stay among at-risk older adults undergoing elective surgery. The authors performed a structured geriatric team intervention to identify at-risk patients and to then facilitate coordinated multidisciplinary care in the form of daily inpatient rounds, weekly multidisciplinary meet-ings, and biweekly ward rounds led by a POPS consultant/clini-cal nurse specialist. Outcomes in surgical patients undergoing the POPS intervention were compared to those among patients not undergoing the intervention. The POPS group had fewer postoperative complications, including lower rates of pneumo-nia (4% vs. 20%, P = 0.008) and delirium (4% vs. 19%, P = 0.028), better pain control (2% vs. 30%, P <0.001), lower rates of delayed mobilization (9% vs. 28%, P = 0.-12) and lower rates

1	of pneumo-nia (4% vs. 20%, P = 0.008) and delirium (4% vs. 19%, P = 0.028), better pain control (2% vs. 30%, P <0.001), lower rates of delayed mobilization (9% vs. 28%, P = 0.-12) and lower rates of inappropriate catheter use (7% vs. 37%, P = 0.046). They also demonstrated a reduction in hospital length of stay by 4.5 days.53The Michigan Surgical Home and Optimization Program (MSHOP) is another example of a successful prehabilitation program. This is a structured, home-based preoperative train-ing program that targets physical, nutritional, and psychological interventions. The intervention included the following four com-ponents: (a) a home-based walking program with daily remind-ers and feedback; (b) incentive spirometry instructions starting one week prior to surgery; (c) education on nutrition, stress management, and care planning; and (d) resources for smoking cessation, when appropriate. Eighty-two percent of individu-als enrolled in the study were actively engaged in the

1	on nutrition, stress management, and care planning; and (d) resources for smoking cessation, when appropriate. Eighty-two percent of individu-als enrolled in the study were actively engaged in the program. Compared to individuals who did not undergo the intervention, patients enrolled in the trial demonstrated a 31% reduction in hospital length of stay and a 28% reduction in cost.64 Collec-tively, the POPS study and the MSHOP studies are illustrative of the notion that attention to preand perioperative assessment in the older population result in improved postoperative out-comes that benefit the patient, hospital, and health care system.Palliative Care Services for Older Surgical PatientsAmong seriously ill individuals, palliative care services have the potential to increase quality of life, improve symptoms and patient satisfaction, and reduce caregiver burden.55-57 The role of palliative care in older surgical patients is not as well under-stood, and it is not widely understood in

1	improve symptoms and patient satisfaction, and reduce caregiver burden.55-57 The role of palliative care in older surgical patients is not as well under-stood, and it is not widely understood in the surgical population.58 Over a decade ago, the American College of Surgeons Pallia-tive Care Workgroup identified core competencies of surgical palliative care.59 The two key elements of palliative care—pain management and communication skills—are essential. For sur-geons who frequently care for individuals at high risk of mor-bidity and mortality, there are six additional core competency domains: patient care, medical knowledge, practice-based learn-ing, interpersonal skills, communication skills, and profession-alism (Table 47-5).Emerging data suggests that surgical patients benefit from the addition of palliative care principles and services. An interventional trial evaluating a decision-making interven-tion considering procedures that included information about health status and

1	the addition of palliative care principles and services. An interventional trial evaluating a decision-making interven-tion considering procedures that included information about health status and prognosis increased the likelihood of choosing less aggressive treatment options among patients with frailty (OR 3.41, 95% CI 1.39–8.39) or dementia (OR 1.66, 95% CI 1.06–2.64).60 In a study of preoperative care consultation in frail older adults, Ernst et al found that preoperative palliative care consultations were associated with reduced mortality.61 Several studies have found that postoperative palliative care improves symptoms, including uncertainly, symptom distress, and depres-sion, and improves quality of life.62-63Brunicardi_Ch47_p2045-p2060.indd 205328/02/19 2:08 PM 2054SPECIFIC CONSIDERATIONSPART IITable 47-5Core Competencies in surgical palliative careDOMAINCOMPETENCYPatient Care Possess the capacity to guide the transition from curative and palliative goals of treatment

1	47-5Core Competencies in surgical palliative careDOMAINCOMPETENCYPatient Care Possess the capacity to guide the transition from curative and palliative goals of treatment to palliative goals alone based on patient information and preferences, scientific and outcomes evidence, and sound clinical judgmentPerform an assessment and gather essential clinical information about symptoms, pain, and sufferingPerform palliative procedures competently and with sound judgment to meet patient goals of care at the end of lifeProvide management of pain and other symptoms to alleviate sufferingCommunicate effectively and compassionately bad news and poor prognosesConduct a patient and family meeting regarding advance directives and end-of-life decisionsExercise sound clinical judgment and skill in the withdrawal and withholding of life supportMedical knowledge Acute and chronic pain managementNon-pain symptom managementEthical and legal basis for advance directives, informed consent,

1	in the withdrawal and withholding of life supportMedical knowledge Acute and chronic pain managementNon-pain symptom managementEthical and legal basis for advance directives, informed consent, withdrawal and withholding of life support, and futilityGrief and bereavement in surgical illnessQuality of life outcomes and prognosticationRole of spirituality at the end of lifeInterpersonal and communication skillsSurgeons must be competent and compassionate communicators with patients, families, and other health care providers. They should be effective in communicating bad news and prognosis and in redefining hope in the context of cultural diversity. The interdisciplinary nature of palliative care requires that the surgeon is skilled as both a leader and a member of an interdisciplinary team and maintains collegial relationships with other health care providers.ProfessionalismSurgeons must maintain professional commitment to ethical and empathic care, which is patient focused, with

1	team and maintains collegial relationships with other health care providers.ProfessionalismSurgeons must maintain professional commitment to ethical and empathic care, which is patient focused, with equal attention to relief of suffering along with curative therapy. Respect and compassion for cultural diversity, gender, and disability is particularly important around rituals and bereavement at the end of life. Maintenance of ethical standards in the withholding and withdrawal of life support is essential.Systems-based practiceSurgeons must be aware and informed of the multiple components of the health care system that provide palliative and end-of-life care. Surgeons should be knowledgeable and willing to refer patients to hospice, palliative care consultation, pain management, pastoral care, social services, etc., and to understand resource utilization and reimbursement issues involved.Data from Mohanty S, Rosenthal RA, Russell MM, et al: Optimal Perioperative Management of the

1	care, social services, etc., and to understand resource utilization and reimbursement issues involved.Data from Mohanty S, Rosenthal RA, Russell MM, et al: Optimal Perioperative Management of the Geriatric Patient: A Best Practices Guideline from the American College of Surgeons NSQIP and the American Geriatrics Society, J Am Coll Surg. 2016 May;222(5):930-947.SPECIAL CONSIDERATIONSFunctional RecoveryIn the past decade, there has been increasing attention to the examination of functional outcomes after major surgery in the geriatric population. In a large prospective cohort study of patients age 60 years and older undergoing abdominal surgery, Lawrence et al found that older adults required several months to fully return to basic activities of daily living (ADLs) and up to 6 months to become independent in more complex instrumental activities of daily living (IADLs).65 Older adults are frequently discharged to postacute facilities even when they are functional dependent at baseline

1	independent in more complex instrumental activities of daily living (IADLs).65 Older adults are frequently discharged to postacute facilities even when they are functional dependent at baseline and have an uncomplicated postoperative course.66Among frail older adults, functional decline after surgery is often substantial and sustained. Studies examining functional outcomes after surgery among nursing home residents have demonstrated that the majority of nursing home residents who undergo surgery do not return to baseline levels of function post-operatively.67-68 Among residents who underwent colectomy for cancer, 53% were dead after 1 year and over half of 1-year survi-vors experienced functional decline.68 For residents who undergo lower extremity bypass, half die within a year of surgery.67 At 1 year, 13% of the initial vascular surgery cohort was ambulatory, and 18% had maintained or improved their baseline functional status—calling into question the efficacy of this procedure in

1	At 1 year, 13% of the initial vascular surgery cohort was ambulatory, and 18% had maintained or improved their baseline functional status—calling into question the efficacy of this procedure in the nursing home. A study of hip fracture repair in nursing home resi-dents found that over a third of residents died and over half of resi-dents had died or experienced functional decline within 180 days after fracture. Residents with multiple comorbidities and advanced cognitive impairment and those who did not undergo surgical cor-rection of the fracture experienced the worst outcomes.5Brunicardi_Ch47_p2045-p2060.indd 205428/02/19 2:08 PM 2055SURGICAL CONSIDERATIONS IN OLDER ADULTSCHAPTER 47Cancer SurgeryApproximately 50% of cancer diagnoses are currently made in patients age 70 years or older.69 It is predicted that the increase in the older adult population will account for up to a 50% increase in the number of patients undergoing oncologic procedures by the year 2020. The increased

1	It is predicted that the increase in the older adult population will account for up to a 50% increase in the number of patients undergoing oncologic procedures by the year 2020. The increased life expectancy of the geriatric patient coupled with the increasing incidence of cancer with advancing age will lead to an increased prevalence of malignant disease requiring surgical intervention. This is an area of great interest given that randomized clinical trials to determine the out-comes of older adult patients undergoing curative resections, as well as neoadjuvant and adjuvant therapy, are lacking. In addi-tion, older adult patients are rarely included in clinical trials; therefore, treatment decisions are often based on individual sur-geon experience and nongeriatric data, and they may be flawed by inherent biases regarding the outcome of complete oncologic resections in older adult patients. Surgeons may also be reluctant to expose older patients to the toxic effects of chemotherapy

1	flawed by inherent biases regarding the outcome of complete oncologic resections in older adult patients. Surgeons may also be reluctant to expose older patients to the toxic effects of chemotherapy and radiation without proven efficacy in this geriatric population. This highlights the need for research targeting the specific needs of older adult patients with malignancy to aid in the development of specific treatment guidelines for various cancers within this age cohort.Numerous studies have documented increased risk of post-operative morbidity and mortality in older adults with cancer. Evaluation of a national surgical registry found that older adults undergoing major gastrointestinal surgery have substan-tially higher risks of complications and death than individuals younger than 65 years.70 The impact of age on risk was present across all operations but had most impact in liver and rectal surgery. Surgeons are challenged to decide whether major sur-gery is justified in older adult

1	The impact of age on risk was present across all operations but had most impact in liver and rectal surgery. Surgeons are challenged to decide whether major sur-gery is justified in older adult patients, especially those with limited life expectancy. Effectiveness of oncologic surgery in older adult patients depends on whether a cure can be achieved safely without compromise to functional status or quality of life. Postoperative life expectancy should be improved by surgery, or, at the very least, not diminished.Emergency SurgeryEmergent surgery caries exceptionally high risk for older adult patients. In an analysis of patients age 90 years and older, 90-day mortality after emergency gastrointestinal procedures was 54%.71 In a large cohort of patients undergoing endovas-cular repair for ruptured aortic aneurysm, 30-day mortality was 35% after primary aortic repair and 52% after open conversion of endovascular aortic repair.72Frail institutionalized elders are at substantial risk for

1	ruptured aortic aneurysm, 30-day mortality was 35% after primary aortic repair and 52% after open conversion of endovascular aortic repair.72Frail institutionalized elders are at substantial risk for poor surgical outcomes after emergent surgery. In an analysis of over 70,000 nursing home residents who underwent emer-gent abdominal operations (surgery for bleeding ulcer, chole-cystectomy, appendectomy, and colectomy), operative mortality was twoto threefold higher than among matched community-dwelling elders.73 In addition, invasive life-sustaining interven-tions after surgery were significantly higher in the nursing home population than among noninstitutionalized Medicare enrollees, ranging from 18% vs. 5%, respectively, after cholecystectomy to 55% vs. 43%, respectively, after ulcer surgery. The combined effects of poor nutrition, decreased cogni-tion, and immune impairments due to nutritional or phar-macologic factors create a treacherous circumstance for older adult patients with

1	The combined effects of poor nutrition, decreased cogni-tion, and immune impairments due to nutritional or phar-macologic factors create a treacherous circumstance for older adult patients with poorly defined symptoms or who pres-ent with more advanced disease. In acute abdominal conditions, such as acute appendicitis and acute cholecystitis, one-third of older adult patients will lack an elevated white blood cell count, one-third will lack fever, and one-third will lack physical find-ings of localized peritonitis.74 These deficits contribute to a threefold higher rate of perforated appendicitis and of gangrene of the gallbladder in older adult patients compared to young patients. An “unimpressive” physical exam in an older adult patient with acute onset of abdominal symptoms should never be taken as a sign of the absence of surgical disease.Cardiovascular SurgeryWith advances in cardiopulmonary bypass technique, myocar-dial protection, and improved perioperative care, coronary artery

1	be taken as a sign of the absence of surgical disease.Cardiovascular SurgeryWith advances in cardiopulmonary bypass technique, myocar-dial protection, and improved perioperative care, coronary artery bypass grafting (CABG) and valve replacement operations have become safer in older patients. When considering cardiovascular surgery in elders, it is essential to consider that advanced age is not the strongest predictor of poorer outcomes or increased mor-tality compared in older patients. It has been demonstrated that emergency operations, preoperative New York Heart Association (NYHA) functional class 3 or greater, and chronic renal failure are the strongest independent predictors of increased operative mortality.75 In one study, preoperative renal dysfunction, cere-brovascular disease, valve surgery, and catastrophic state were independent predictors of increased mortality in older adult patients.76 Older adult patients with non–dialysis-dependent renal dysfunction had a 60% chance of

1	surgery, and catastrophic state were independent predictors of increased mortality in older adult patients.76 Older adult patients with non–dialysis-dependent renal dysfunction had a 60% chance of death during a 5-year follow-up period compared to 25% in older adult patients with-out a history of renal dysfunction. Similarly, the presence of cerebrovascular disease resulted in a two-fold increase in mor-tality among older adult patients.76 Even patients who were 80 years of age or more did not have any significant increase in surgical risk and within this population, and the 4-year actuarial survival was 70.5% with an event-free survival of 60.6%.There has been an increase in definitive operative interven-tion to older patients with operable coronary artery disease. The Society of Thoracic Surgeons reports that perioperative mortality rates range from 1.6% in patients 51 to 60 years of age to 7.7% in those 81 to 90 years of age.77 Older patients are more likely to have significant

1	Surgeons reports that perioperative mortality rates range from 1.6% in patients 51 to 60 years of age to 7.7% in those 81 to 90 years of age.77 Older patients are more likely to have significant three-vessel disease accompanied by poor ejec-tion fraction, left ventricular hypertrophy, significant valvular disease, and previous history of myocardial infarction than are younger patients.77 Older patients also are more likely to be clas-sified as NYHA functional class 3 or higher and are more likely to present on an emergent basis, in part because of reluctance to provide elective surgical intervention because of presumptive poorer outcome. Despite the increased risk of morbidity and mortality compared to younger patients, older adult patients, including those >80 years old, can undergo CABG with accept-able mortality risk. The overall mortality rate is approximately 7% to 12% for older adult patients, including those in whom CABG is performed under emergency conditions. The mortality

1	with accept-able mortality risk. The overall mortality rate is approximately 7% to 12% for older adult patients, including those in whom CABG is performed under emergency conditions. The mortality rate decreases to approximately 2.8% when CABG is performed electively with careful preoperative evaluation.78Valve ReplacementAs the population ages, the incidence of senile calcific aortic stenosis and referral for aortic valve replacement are increas-ing. The operative mortality from aortic valve replacement is estimated to be between 3% and 10%, with an average of approximately 7.7%.76 If aortic stenosis is allowed to progress without operative intervention, CHF will ensue. The average survival of these patients is approximately 1.5 to 2 years. If a patient is deemed fit for operative intervention, age should not 6Brunicardi_Ch47_p2045-p2060.indd 205528/02/19 2:08 PM 2056SPECIFIC CONSIDERATIONSPART IIbe a deterrent, especially considering the potential to increase life expectancy. It

1	age should not 6Brunicardi_Ch47_p2045-p2060.indd 205528/02/19 2:08 PM 2056SPECIFIC CONSIDERATIONSPART IIbe a deterrent, especially considering the potential to increase life expectancy. It has been recommended that the carefully selected, minimally symptomatic octogenarian with aortic ste-nosis should be considered a low-risk patient and be expected to experience an uneventful operative course and expedient recov-ery. More importantly, if elective procedures are delayed until symptoms or left ventricular dysfunction develop, patients may suffer from unnecessary increased operative risk and mortality.75 Early intervention results in a demonstrable improvement in quality of life in these patients, with many improving their NYHA functional classification.Older patients are candidates for mitral valve surgery when ischemic regurgitation is present. Surgery for mitral valve disease carries a higher morbidity and mortality risk than for aortic intervention, with an estimated mortality

1	mitral valve surgery when ischemic regurgitation is present. Surgery for mitral valve disease carries a higher morbidity and mortality risk than for aortic intervention, with an estimated mortality rate as high as 20%.77 Left ventricular function usually is compromised in patients requiring intervention, leading to a poorer outcome in these patients. The surgical outcome for mitral valve proce-dures depends on the extent of the disease, age of the patient, presence of pulmonary hypertension, and extent of coronary artery disease. The presence of comorbid conditions combined with the emergent nature of surgery in a large percentage of older patients further worsens the outcome. Therefore, a deci-sion regarding management of mitral valve disease should be individualized to each patient. Another concern regarding older patients who are candidates for valve disease surgery is the additional need for coronary revascularization—an important contributor to morbidity and mortality from

1	Another concern regarding older patients who are candidates for valve disease surgery is the additional need for coronary revascularization—an important contributor to morbidity and mortality from surgical interven-tion. To mitigate risk, an older patient with multiple comorbid conditions in need of a combined procedure should only have critically stenosed vessels bypassed.79 Neurologic complications from valve surgery are particularly common in older patients. It has been estimated that approximately 30% of patients >70 years old who undergo valve procedures develop either transient or permanent neurologic dysfunction.22 This often is a result of embolism from debris dislodged from the valve during the procedure or from a formed thrombus in the right atrium.An important consideration in valve replacement proce-dures in older patients is the type of prosthesis to be used. Older patients are at increased risk from bleeding-associated anticoagu-lation complications. This risk is

1	in valve replacement proce-dures in older patients is the type of prosthesis to be used. Older patients are at increased risk from bleeding-associated anticoagu-lation complications. This risk is especially significant in patients who have experienced falls and minor trauma that have resulted in intracranial hemorrhage. To avoid the lifelong requirement for anticoagulants, bioprosthetic valves should be used in place of mechanical valves whenever possible.79 Although the biopros-thetic valves are not as durable as mechanical valves, studies demonstrate excellent structural integrity 10 years post proce-dure, making it an appropriate choice in older patients.Transcatheter aortic valve implantation/replacement (TAVI/TAVR) is increasingly being used to treat aortic stenosis. Initially, this technique was reserved for individuals with high surgical risk. A systematic review of transcatheter aortic valve implantation versus surgical aortic valve replacement revealed that, compared to

1	this technique was reserved for individuals with high surgical risk. A systematic review of transcatheter aortic valve implantation versus surgical aortic valve replacement revealed that, compared to surgical repair, the transcatheter approach may have similar or better early and midterm outcomes, including among lowto intermediate-risk patients.80 Furthermore, there is increasing evidence that suggests TAVI results in acceptable long-term results in the older adult population.81,82Endovascular Aortic SurgeryWith increasing use of screening abdominal CT scans and ultrasounds for evaluation of various abdominal complaints, abdominal aortic aneurysms (AAA) are being identified with greater frequency, The percentage of AAA rises from about 1% at age 55 to 60 years to approximately 10% in patients 80 years of age or older.83 Historically, very old patients were deemed poor operative candidates for the traditional open repair given the frequent presence of comorbid conditions and limited

1	80 years of age or older.83 Historically, very old patients were deemed poor operative candidates for the traditional open repair given the frequent presence of comorbid conditions and limited car-diopulmonary reserve to tolerate a major operation or the many hours of required operative time and general anesthesia. The dissemination of endovascular techniques for repair of AAA, however, has shifted the risk-benefit ratio for operative inter-vention, allowing greater life expectancy for the elective repair of this potentially life-threatening condition with the benefits of a minimally invasive approach.Multiple studies have demonstrated that endovascular aortic repair (EVAR) is feasible and efficacious in older adult patients, including those previously considered unfit for open repair. EVAR is a minimally invasive technique in which a pros-thetic graft is introduced into the aortic lumen via the common femoral artery to exclude the aortic aneurysm sac. EVAR sig-nificantly reduces

1	EVAR is a minimally invasive technique in which a pros-thetic graft is introduced into the aortic lumen via the common femoral artery to exclude the aortic aneurysm sac. EVAR sig-nificantly reduces operative and anesthesia times, blood loss, intensive care needs, length of stays, and major postoperative morbidity associated with open AAA repair. This procedure also can be done using epidural anesthesia for high-risk candi-dates who may tolerate general anesthesia poorly (Fig. 47-2).Careful consideration of the life expectancy and the risk of rupture dictate the necessity for intervention. EVAR remains a viable option in older adult patients. Nonoperative management is justified in frail older adult patients with multiple comorbidi-ties and reduced life expectancy whose operative risks outweigh the risk of rupture and in those who are unlikely to survive long enough to benefit from the repair.Palliative SurgeryPalliative surgery is defined as surgical intervention targeted to alleviate

1	the risk of rupture and in those who are unlikely to survive long enough to benefit from the repair.Palliative SurgeryPalliative surgery is defined as surgical intervention targeted to alleviate a patient’s symptoms, thus improving the patient’s quality of life despite minimal impact on the patient’s survival.54 With an increasing number of older patients presenting with advanced disease, surgeons must be familiar with the concept of palliation to control symptoms. This concept focuses on pro-viding the maximal benefit to the patient using the least-invasive intervention. Ideally, this intervention leads to symptom relief and preservation of the quality of life in terminal disease states by alleviating symptoms such as intractable vomiting and severe pain. The success of palliative surgery is a careful bal-ance between achieving symptom relief without the develop-ment of new symptoms from the intervention itself. A recent meta-analysis of outcomes after palliative surgery for

1	surgery is a careful bal-ance between achieving symptom relief without the develop-ment of new symptoms from the intervention itself. A recent meta-analysis of outcomes after palliative surgery for malignant bowel obstruction from peritoneal carcinomatosis revealed that although palliative surgery can benefit some patients, many patients experience serious complications, incomplete resolu-tion of symptoms, and substantial hospitalization relative to the patient’s remaining survival time.84 It is essential to provide patients with realistic information about expected outcomes after palliative surgery to ensure that this surgical intervention is in line with their care preferences. The core competencies for surgical palliative care are shown in Table 47-5.SUMMARYMajor surgery in older adults requires careful consideration. In addition to chronic medical conditions, many elders have geriat-ric syndromes that put them at high risk for increased morbidity, Brunicardi_Ch47_p2045-p2060.indd

1	requires careful consideration. In addition to chronic medical conditions, many elders have geriat-ric syndromes that put them at high risk for increased morbidity, Brunicardi_Ch47_p2045-p2060.indd 205628/02/19 2:08 PM 2057SURGICAL CONSIDERATIONS IN OLDER ADULTSCHAPTER 47Figure 47-2. Endovascular repair of abdominal aortic aneurysms (AAAs) has gained favor for suitable older adult patients to prevent rupture. Through minimal groin incisions, this 82-year-old patient underwent repair of an AAA and right iliac artery aneurysm and was discharged on post-op day 2.mortality, and poor functional recovery after surgery. Screen-ing for and optimization of multiple domains of vulnerability is essential to improve outcomes in this vulnerable population. Furthermore, the incorporation of palliative care principles into the surgical care of frail elders will improve patient-centered decision-making, symptom management, and quality of life.REFERENCESEntries highlighted in bright blue are key

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1	Am J Psychiatry. 2003;160(6):1147-1156. 31. Li C, Friedman B, Conwell Y, Fiscella K. Validity of the Patient Health Questionnaire 2 (PHQ-2) in identifying major depres-sion in older people. J Am Geriatr Soc. 2007;55(4):596-602. 32. Blazer DG, Wu LT. The epidemiology of at-risk and binge drinking among middle-aged and older adult community adults: National Survey on Drug Use and Health. Am J Psychiatry. 2009;166(10):1162-1169. 33. Nath B, Li Y, Carroll JE, Szabo G, Tseng JF, Shah SA. Alcohol exposure as a risk factor for adverse outcomes in elective sur-gery. J Gastrointest Surg. 2010;14(11):1732-1741. 34. Tonnesen H, Kehlet H. Preoperative alcoholism and postopera-tive morbidity. Br J Surg. 1999;86(7):869-874. 35. National Institute on Alcohol Abuse and Alcoholism. CAGE Questionnaire. 2002. Available at: https://pubs.niaaa.nih.gov/publications/aa65/aa65.htm. Accessed August 21, 2018. 36. Gal J, Bogar L, Acsady G, Kertai MD. Cardiac risk reduction in non-cardiac surgery: the role of

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1	with the American College of Surgeons, American Society of Anesthesiologists, American Society of Echocardiography, American Society of Nuclear Cardiology, Heart Rhythm Society, Society for Cardiovascular Angiography and Interventions, Society of Cardiovascular Anesthesiologists, and Society of Vascular Medicine Endorsed by the Society of Hospital Medicine. J Nucl Cardiol. 2015;22(1):162-215. 39. Ansaloni L, Catena F, Chattat R, et al. Risk factors and inci-dence of postoperative delirium in older adult patients after elective and emergency surgery. Br J Surg. 2010;97(2):273-280. 40. Landesberg G, Beattie WS, Mosseri M, Jaffe AS, Alpert JS. Perioperative myocardial infarction. Circulation. 2009;119(22):2936-2944. 41. Smetana GW, Macpherson DS. The case against routine preop-erative laboratory testing. Med Clin North Am. 2003;87(1):7-40. 42. Smetana GW, Cohn SL, Lawrence VA. Update in perioperative medicine. Ann Intern Med. 2004;140(6):452-461. 43. Johnson RG, Arozullah AM, Neumayer

1	testing. Med Clin North Am. 2003;87(1):7-40. 42. Smetana GW, Cohn SL, Lawrence VA. Update in perioperative medicine. Ann Intern Med. 2004;140(6):452-461. 43. Johnson RG, Arozullah AM, Neumayer L, Henderson WG, Hosokawa P, Khuri SF. Multivariable predictors of postop-erative respiratory failure after general and vascular surgery: results from the patient safety in surgery study. J Am Coll Surg. 2007;204(6):1188-1198. 44. White JV, Guenter P, Jensen G, et al. Consensus statement: Academy of Nutrition and Dietetics and American Society for Parenteral and Enteral Nutrition: characteristics recom-mended for the identification and documentation of adult malnutrition (undernutrition). JPEN J Parenter Enteral Nutr. 2012;36(3):275-283. 45. Barnett SR. Polypharmacy and perioperative medications in the older adult. Anesthesiol Clin. 2009;27(3):377-389. 46. Qato DM, Alexander GC, Conti RM, Johnson M, Schumm P, Lindau ST. Use of prescription and over-the-counter medications and dietary supplements

1	older adult. Anesthesiol Clin. 2009;27(3):377-389. 46. Qato DM, Alexander GC, Conti RM, Johnson M, Schumm P, Lindau ST. Use of prescription and over-the-counter medications and dietary supplements among older adults in the United States. JAMA. 2008;300(24):2867-2878. 47. Hajjar ER, Cafiero AC, Hanlon JT. Polypharmacy in older adult patients. Am J Geriatr Pharmacother. 2007;5(4):345-351. 48. Seckler AB, Meier DE, Mulvihill M, Paris BE. Substituted judgment: how accurate are proxy predictions? Ann Intern Med. 1991;115(2):92-98. 49. Fried TR, Bradley EH, Towle VR, Allore H. Understanding the treatment preferences of seriously ill patients. N Engl J Med. 2002;346(14):1061-1066. 50. American Society of Anesthesiologists C. Practice guidelines for preoperative fasting and the use of pharmacologic agents to reduce the risk of pulmonary aspiration: application to healthy patients undergoing elective procedures: an updated report by the American Society of Anesthesiologists Com-mittee on

1	agents to reduce the risk of pulmonary aspiration: application to healthy patients undergoing elective procedures: an updated report by the American Society of Anesthesiologists Com-mittee on Standards and Practice Parameters. Anesthesiology. 2011;114(3):495-511. 51. Classen DC, Evans RS, Pestotnik SL, Horn SD, Menlove RL, Burke JP. The timing of prophylactic administration of antibi-otics and the risk of surgical-wound infection. N Engl J Med. 1992;326(5):281-286. 52. Silber JH, Rosenbaum PR, Trudeau ME, et al. Preopera-tive antibiotics and mortality in the older adult. Ann Surg. 2005;242(1):107-114. 53. Harari D, Hopper A, Dhesi J, Babic-Illman G, Lockwood L, Martin F. Proactive care of older people undergoing surgery (‘POPS’): designing, embedding, evaluating, and funding a comprehensive geriatric assessment service for older elective surgical patients. Age Ageing. 2007;36(2):190-196. 54. Englesbe MJ, Grenda DR, Sullivan JA, et al. The Michigan Surgical Home and Optimization

1	geriatric assessment service for older elective surgical patients. Age Ageing. 2007;36(2):190-196. 54. Englesbe MJ, Grenda DR, Sullivan JA, et al. The Michigan Surgical Home and Optimization Program is a scalable model to improve care and reduce costs. Surgery. 2017;161(6): 1659-1666. 55. Temel JS, Greer JA, Muzikansky A, et al. Early palliative care for patients with metastatic non-small-cell lung cancer. N Eng J Med. 2010;363(8):733-742.Brunicardi_Ch47_p2045-p2060.indd 205828/02/19 2:08 PM 2059SURGICAL CONSIDERATIONS IN OLDER ADULTSCHAPTER 47 56. Kavialieratos D, Corbelli J, Zhang D, et al. Association between palliative care and patient and caregiver outcomes. A systematic review and meta-analysis. JAMA. 2016;316(20):2104-2114. 57. Zimmermann C, Swami N, Krzyzanowska M, et al. Early palliative care for patients with advanced cancer: a clusterrandomised controlled trial. Lancet. 2014;383(9930): 1721-1730. 58. Olmsted CL, Johnson AM, Kaboli P, et al. Use of pal-liative care and

1	palliative care for patients with advanced cancer: a clusterrandomised controlled trial. Lancet. 2014;383(9930): 1721-1730. 58. Olmsted CL, Johnson AM, Kaboli P, et al. Use of pal-liative care and hospice among surgical and medical spe-cialties in the Veterans Health Administration. JAMA Surg. 2014;149(11):1169-1175. 59. Surgeons Palliative Care Workgroup. Office of Promoting Excellence in End-of-Life Care: Surgeon’s Palliative Care Work-group report from the field. J Am Coll Surg. 2003;197(4):661-686. 60. Moorhouse P, Mallery LH. Palliative and therapeutic harmoni-zation: a model for appropriate decision-making in frail older adults. J Am Geriatr Soc. 2012;60(12):2326-2332. 61. Ernst KF, Hall DE, Schmid KK, et al. Surgical palliative care consultations over time in relationship to systemwide frailty screening. JAMA Surg. 2014;149(11):1121-1126. 62. McCorkle R, Dowd M, Ercolano E, et al. Effects of a nursing intervention on quality of life outcomes in post-surgical women with

1	systemwide frailty screening. JAMA Surg. 2014;149(11):1121-1126. 62. McCorkle R, Dowd M, Ercolano E, et al. Effects of a nursing intervention on quality of life outcomes in post-surgical women with gynecological cancers. Psychooncology. 2009;18(1):62-70. 63. McCorkle R, Strumpf NE, Nuamah IF, et al. A specialized home care intervention improves survival among older post-surgical cancer patients. J Am Geriatr Soc. 2000;48(12):1707-1713. 64. Englesbe MJ, Grenda DR, Sullivan JA, et al. The Michigan Sur-gical Home and Optimization Program is a scalable model to improve care and reduce costs. Surgery. 2017;161(6):1659-1666. 65. Lawrence VA, Hazuda HP, Cornell JE, et al. Functional inde-pendence after major abdominal surgery in the older adult. J Am Coll Surg. 2004;199(5):762-772. 66. Balentine CJ, Naik AD, Berger DH, et al. Postacute care after major abdominal surgery in older adult patients: intersection of age, functional status, and postoperative complications. JAMA Surg.

1	CJ, Naik AD, Berger DH, et al. Postacute care after major abdominal surgery in older adult patients: intersection of age, functional status, and postoperative complications. JAMA Surg. 2016;151(8):759-766. 67. Oresanya L, Zhao S, Gan S, et al. Functional outcomes after lower extremity revascularization in nursing home residents: a national cohort study. JAMA Intern Med. 2015;175(6):951-957. 68. Finlayson E, Zhaao S, Boscardin WJ, et al. Functional status after colon cancer surgery in older adult nursing home residents. J Am Geriatr Soc. 2012;60(5):967-673. 69. Pasetto LM, Lise M, Monfardini S. Preoperative assessment of older adult cancer patient. Crit Rev Oncol Hematol. 2007;64:10. 70. Yeo HL, O’Mahoney PR, Lachs M, et al. Surgical oncol-ogy outcomes in the aging US population. J Surg Res. 2016;205(1):11-18. 71. Sudlow A, Tuffaha H, Strearns AT, et al. Outcomes of surgery in patients aged >=90 years in the general surgical setting. Ann R Coll Surg Engl. 2018;24:1-6. 72. Scali ST,

1	Res. 2016;205(1):11-18. 71. Sudlow A, Tuffaha H, Strearns AT, et al. Outcomes of surgery in patients aged >=90 years in the general surgical setting. Ann R Coll Surg Engl. 2018;24:1-6. 72. Scali ST, Runge SJ, Feezor RJ, et al. Outcomes after endovas-cular aneurysm repair conversion and primary aortic repair for urgent and emergency indications in the Society for Vascular Surgery Quality Initiative. J Vasc Surg. 2016;64(2):338-347. 73. Finlayson E, Wang L, Landefeld CS, et al. Major abdominal surgery in nursing home residents: a national study. Ann Surg. 2011;254(6):921-926. 74. Zenilman ME. Surgery in the older adult. Curr Probl Surg. 1998;35:99-179. 75. Cerillo AG, Kodami AA, Solinas M, et al. Aortic valve surgery in the older adult patient: a retrospective review. Interact Car-diovasc Thorac Surg. 2007;6:308-313. 76. Srinivasan AK, Oo AY, Grayson AD, et al. Mid-term survival after cardiac surgery in older adult patients: analysis of predic-tors for increased mortality. Interact

1	Thorac Surg. 2007;6:308-313. 76. Srinivasan AK, Oo AY, Grayson AD, et al. Mid-term survival after cardiac surgery in older adult patients: analysis of predic-tors for increased mortality. Interact Cardiovasc Thorac Surg. 2004;3:289-293. 77. Davis EA, Gardner TJ, Gillinov AM, et al. Valvular disease in the older adult: influence on surgical results. Ann Thorac Surg. 1993;55:333-337. 78. Richmond TS, Kaunder D, Strumpf N, et al. Characteristics and outcomes of serious traumatic injury in older adults. J Am Geri-atr Soc. 2002;50:215-222. 79. Aziz S, Grover FL. Cardiovascular surgery in the older adult. Cardiol Clin. 1999;17:213-231. 80. Garguilo G, Sannino A, Capodanno D, et al. Transcatheter aortic valve implantation versus surgical aortic valve replace-ment: a systematic review and meta-analysis. Ann Intern Med. 2016;165(50):334-344. 81. Chakos A, Wilson-Smith A, Arora S, et al. Long term outcomes of transcatheter aortic valve implantation (TAVI): a systematic review of 5-year survival

1	Ann Intern Med. 2016;165(50):334-344. 81. Chakos A, Wilson-Smith A, Arora S, et al. Long term outcomes of transcatheter aortic valve implantation (TAVI): a systematic review of 5-year survival and beyond. Ann Cardiothorac Surg. 2017;6(5):432-443. 82. D’Onofrio A, Facchin M, Besola L, et al. Intermediate clini-cal and hemodynamic outcomes after transcatheter aortic valve implantation. Ann Thorac Surg. 2016;101:881-8; discussion 888. 83. Biebl M, Lau LL, Hakaim AG, et al. Midterm outcomes of endo-vascular abdominal aortic aneurysm repair in octogenarians: a single institution’s experience. J Vasc Surg. 2004;40:435-442. 84. Olson P, Pinkerton C, Brasel KJ, et al. Palliative surgery for malignant bowel obstruction from carcinomatosis: a system-atic review. JAMA Surg. 2014;149(4):383-392.Brunicardi_Ch47_p2045-p2060.indd 205928/02/19 2:08 PM

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1	Ethics, Palliative Care, and Care at the End of LifeDaniel E. Hall, Eliza W. Beal, Peter A. Angelos, Geoffrey P. Dunn, Daniel B. Hinshaw, and Timothy M. Pawlik48chapterDedicated to the advancement of surgery along its scientific and moral side. June 10, 1926, dedication on the Murphy Auditorium, the first home of the American College of SurgeonsWHY ETHICS MATTEREthical concerns involve not only the interests of patients but also the interests of surgeons and society. Surgeons choose among the options available to them because they have particu-lar opinions regarding what would be good (or bad) for their patients. Aristotle described practical wisdom (Greek: phronesis) as the capacity to choose the best option from among several imperfect alternatives (Fig. 48-1).1 Frequently, surgeons are confronted with clinical or interpersonal situations in which there is incomplete information, uncertain outcomes, and/or complex personal and familial relationships. The capacity to choose

1	are confronted with clinical or interpersonal situations in which there is incomplete information, uncertain outcomes, and/or complex personal and familial relationships. The capacity to choose wisely in such circumstances is the challenge of surgi-cal practice.DEFINITIONS AND OVERVIEWBiomedical ethics is the system of analysis and deliberation dedicated to guiding surgeons toward the “good” in the prac-tice of surgery. One of the most influential ethical “systems” in the field of biomedical ethics is the principalist approach as articulated by Beauchamp and Childress.2 In this approach to ethical issues, moral dilemmas are deliberated using four guiding principles: autonomy, beneficence, nonmaleficence, and justice.2The principle of autonomy respects the capacity of indi-viduals to choose their own destiny, and it implies that indi-viduals have a right to make those choices. It also implies an obligation for physicians to permit patients to make autonomous choices about their medical

1	their own destiny, and it implies that indi-viduals have a right to make those choices. It also implies an obligation for physicians to permit patients to make autonomous choices about their medical care. Beneficence requires that proposed actions aim at and achieve something good whereas nonmaleficence aims at avoiding concrete harm: primum non nocere.* Justice requires fairness where both the benefits and burdens of a particular action are distributed equitably.The history of medical ethics has its origins in antiquity. The Hippocratic Oath along with other professional codes has guided the actions of physicians for thousands of years. However, the growing technical powers of modern medicine raise new questions that were inconceivable in previous gen-erations. Life support, dialysis, and modern drugs, as well as organ and cellular transplantation, have engendered new moral and ethical questions. As such, the ethical challenges faced by the surgeon have become more complex and

1	and modern drugs, as well as organ and cellular transplantation, have engendered new moral and ethical questions. As such, the ethical challenges faced by the surgeon have become more complex and require greater attention.The case-based paradigm for bioethics is used when the clinical team encounters a situation in which two or more val-ues or principles come into apparent conflict. The first step is to clarify the relevant principles (e.g., autonomy, beneficence, nonmaleficence, and justice) and values at stake (e.g., self-determination, quality of life). After identifying the principles and values that are affecting the situation, a proposed course of action is considered given the circumstances.Much of the discourse in bioethics adopts this “principal-ist” approach in which the relevant principles are identified, weighed, and balanced, and then applied to formulate a course of action. This approach to bioethics is a powerful technique for thinking through moral problems because the

1	principles are identified, weighed, and balanced, and then applied to formulate a course of action. This approach to bioethics is a powerful technique for thinking through moral problems because the four princi-ples help identify what is at stake in any proposed course of action. However, the principles themselves do not resolve ethi-cal dilemmas. Working together, patients and surgeons must use wise judgment to choose the best course of action for the specific case.Why Ethics Matter 2061Definitions and Overview 2061Specific Issues in Surgical Ethics 2062Informed Consent / 2062The Boundaries of Autonomy: Advance Directives and Powers of Attorney / 2064Withdrawing and Withholding Life-Sustaining Therapies / 2065Living Donor Liver Transplantation / 2066Palliative Care 2066General Principles of Palliative Care / 2066Concepts of Suffering, Pain, Health, and Healing / 2067Effective Communication and Negotiating the Goals of Care / 2067Care at the End of Life 2068The Syndrome of Imminent

1	of Palliative Care / 2066Concepts of Suffering, Pain, Health, and Healing / 2067Effective Communication and Negotiating the Goals of Care / 2067Care at the End of Life 2068The Syndrome of Imminent Demise / 2068Common Symptoms at the End of Life and Their Management / 2068Pronouncing Death / 2072Aid in Dying / 2072Professional Ethics: Conflict of Interest, Research, and Clinical Ethics 2072Conflict of Interest / 2072Research Ethics / 2072Special Concerns in Surgical Research / 2072Surgical Innovation / 2073The Ethics of Authorship / 2073Clinical Ethics: Disclosure of Errors / 2074*“First do no harm.”Brunicardi_Ch48_p2061-p2076.indd 206119/02/19 1:49 PM 2062Figure 48-1. Bust of Aristotle. Marble, Roman copy after a Greek bronze original by Lysippos from 330 b.c. (From http://en.wikipedia.org/wiki/File:Aristotle_Altemps_Inv8575.jpg: Ludovisi Collection, Accession number Inv. 8575, Palazzo Altemps, Location Ground Floor, Branch of the National Roman Museum. Photographer/-source

1	Ludovisi Collection, Accession number Inv. 8575, Palazzo Altemps, Location Ground Floor, Branch of the National Roman Museum. Photographer/-source Jastrow [2006] from Wikipedia.)Choosing wisely requires the virtue of practical wisdom first described by Aristotle (see Fig. 48-1). Along with the other cardinal virtues of courage, justice and temperance, practical wisdom is a central component of virtue ethics which comple-ment principalist ethics by guiding choices toward the best options for treatment. Practical wisdom cannot be learned from books and is developed only through experience. The appren-ticeship model of surgical residency fosters the development of practical wisdom through experience. More than teaching merely technical mastery, surgical residency is also moral training. In fact, the sociologist Charles Bosk argues that the “postgraduate training of surgeons is above all things an ethical training.”3SPECIFIC ISSUES IN SURGICAL ETHICSInformed ConsentAlthough a relatively

1	fact, the sociologist Charles Bosk argues that the “postgraduate training of surgeons is above all things an ethical training.”3SPECIFIC ISSUES IN SURGICAL ETHICSInformed ConsentAlthough a relatively recent development, the doctrine of informed consent is one of the most widely established tenets of modern biomedical ethics. During the nineteenth and early twentieth centuries, most physicians practiced a form of benign paternalism whereby patients were rarely involved in the deci-sion-making process regarding their medical care, relying instead on the beneficence of the physician. Consensus among the wider public eventually changed such that surgeons are now expected to have an open discussion about diagnosis and treatment with the patient to obtain informed consent. In the United States, the legal doctrine of simple consent dates from the 1914 decision in Schloendorff vs. The Society of New York Hospital regarding a case in which a surgeon removed a diseased uterus after the patient

1	the legal doctrine of simple consent dates from the 1914 decision in Schloendorff vs. The Society of New York Hospital regarding a case in which a surgeon removed a diseased uterus after the patient had consented to an examination under anesthesia, but with the express stipulation that no operative excision should be performed. The physician argued that his decision was justified by the beneficent obligation to avoid the risks of a second anes-thetic. However, Justice Benjamin Cardozo stated:Every human being of adult years and sound mind has a right to determine what shall be done with his body; and a surgeon who performs an operation without his patient’s consent commits an assault, for which he is liable in damages . . . except in cases of emergency, where the patient is unconscious, and where it is neces-sary to operate before consent can be obtained.4Having established that patients have the right to deter-mine what happens to their bodies, it took some time for the modern

1	and where it is neces-sary to operate before consent can be obtained.4Having established that patients have the right to deter-mine what happens to their bodies, it took some time for the modern concept of informed consent to emerge from the ini-tial doctrine of simple consent. The initial approach appealed to a professional practice standard whereby physicians were obligated to disclose to patients the kind of information that experienced surgeons customarily disclosed.5 However, this dis-closure was not always adequate for patient needs. In the 1972 Key Points1 The physician should document that the patient or surrogate has the capacity to make a medical decision.2 Sufficient details regarding diagnosis and treatment options should be disclosed to the patient so that the patient can pro-vide informed consent.3 Living wills are written to anticipate treatment options and choices in the event that a patient is rendered incompetent by a terminal illness.4 The durable power of attorney

1	informed consent.3 Living wills are written to anticipate treatment options and choices in the event that a patient is rendered incompetent by a terminal illness.4 The durable power of attorney for healthcare identifies sur-rogate decision makers and invests them with the authority to make healthcare decisions on behalf of patients in the event that they are unable to speak for themselves.5 Surgeons should encourage their patients to complete a liv-ing will and clearly identify their surrogates early in the course of treatment.6 Earlier referrals and wider use of palliative and hospice care may help more patients achieve their goals at the end of life.7 Seven requirements for the ethical conduct of clinical research studies have been articulated: value, scientific validity, fair subject selection, favorable risk-benefit ratio, independent review, informed consent, and respect for enrolled subjects.8 Individuals working together on research endeavors should have clear discussions early

1	favorable risk-benefit ratio, independent review, informed consent, and respect for enrolled subjects.8 Individuals working together on research endeavors should have clear discussions early in the planning process about authorship, and those discussions should be continued throughout the project or study.9 Disclosure of error is consistent with recent ethical advances in medicine toward more transparency, openness with patients, and the involvement of patients in their care.Brunicardi_Ch48_p2061-p2076.indd 206219/02/19 1:49 PM 2063ETHICS, PALLIATIVE CARE, AND CARE AT THE END OF LIFECHAPTER 48Patient establishes selfas decision-makerNoYesEngage patientdirectly in informedconsent process andin all aspects of careIdentify patient’s culturalidentity and, if possible,explicit preferences formaking decisions,including degree of familyinvolvementMaintain heightenedawareness of patient-family or patient-surrogate interactionsMake sure family orother surrogate is willingparticipant in

1	formaking decisions,including degree of familyinvolvementMaintain heightenedawareness of patient-family or patient-surrogate interactionsMake sure family orother surrogate is willingparticipant in informedconsent processContinuously reassessfor signs that patient isunhappy with current role ininformed consent processSecure private discussionwith patient and remindpatient of right toinformed consent processMake sure patient hasnot deferred decision-making involuntarilyFigure 48-2. Algorithm for navigating the process of informed consent. (Modified with permission from Childers R, Lipsett A, Pawlik T. Informed consent and the surgeon, J Am Coll Surg. 2009 Apr;208(4):627-634.)landmark case, Canterbury vs. Spence, the court rejected the professional practice standard in favor of the reasonable person standard whereby physicians are obliged to disclose to patients all information regarding diagnosis, treatment options, and risks that a “reasonable patient” would want to know in a similar

1	person standard whereby physicians are obliged to disclose to patients all information regarding diagnosis, treatment options, and risks that a “reasonable patient” would want to know in a similar situation. Rather than relying on the practices or consensus of the medical community, the reasonable person standard empow-ers the public (reasonable persons) to determine how much information should be disclosed by physicians to ensure that consent is truly informed. The court did recognize, however, that there are practical limits on the amount of information that can be communicated or assimilated.5 Subsequent litigation has revolved around what reasonable people expect to be disclosed in the consent process to include the nature and frequency of potential complications, the prognostic life expectancy,6 and the surgeon-specific success rates.4 Despite the litigious environ-ment of medical practice, it is difficult to prosecute a case of inadequate informed consent so long as the

1	life expectancy,6 and the surgeon-specific success rates.4 Despite the litigious environ-ment of medical practice, it is difficult to prosecute a case of inadequate informed consent so long as the clinician has made a concerted and documented effort to involve the patient in the decision-making process.Adequate informed consent entails at least four basic ele-ments: (a) the physician documents that the patient or surrogate has the capacity to make a medical decision; (b) the sur-geon discloses to the patient details regarding the diagno-sis and treatment options sufficiently for the patient to make an informed choice; (c) the patient demonstrates understanding of the disclosed information before (d) authoriz-ing freely a specific treatment plan without undue influence (Fig. 48-2). These goals are aimed at respecting each patient’s prerogative for autonomous self-determination. To accomplish these goals, the surgeon needs to engage in a discussion about the causes and nature of the

1	goals are aimed at respecting each patient’s prerogative for autonomous self-determination. To accomplish these goals, the surgeon needs to engage in a discussion about the causes and nature of the patient’s disease, the risks and ben-efits of available treatment options, as well as details regarding what patients can expect after an operative intervention includ-ing possible outcomes and complications.7-14Certain clinical settings make obtaining informed consent challenging. For example, obtaining consent for emergency surgery can be difficult, as the clinical team is forced to make decisions with incomplete information. Emergency consent requires the surgeon to consider if and how possible interven-tions might save a patient’s life, and if successful, what kind of disability might be anticipated. Surgical emergencies are one of the few instances where the limits of patient autonomy are freely acknowledged, and surgeons are empowered by law and ethics to act promptly in the best

1	anticipated. Surgical emergencies are one of the few instances where the limits of patient autonomy are freely acknowledged, and surgeons are empowered by law and ethics to act promptly in the best interests of their patients according to the surgeon’s judgment. Most applicable medi-cal laws require physicians to provide the standard of care to incapacitated patients, even if it entails invasive procedures without the explicit consent of the patient or surrogate. If at all possible, surgeons should seek the permission of their patients to provide treatment, but when emergency medical conditions render patients unable to grant that permission, and when delay is likely to have grave consequences, surgeons are legally and ethically justified in providing whatever surgical treatment the surgeon judges necessary to preserve life and restore health.4 This justification is based on the social consensus that most people would want their lives and health protected in this way, and this

1	surgeon judges necessary to preserve life and restore health.4 This justification is based on the social consensus that most people would want their lives and health protected in this way, and this consensus is manifest in the medical profession’s gen-eral orientation to preserve life. It may be that subsequent care may be withdrawn or withheld when the clinical prognosis is clearer, but in the context of initial resuscitation of injured patients, incomplete information makes clear judgments about the patient’s ultimate prognosis or outcome impossible.The pediatric population also presents unique challenges for the process of consent. For many reasons, children and ado-lescents cannot participate in the process of giving informed 1122Brunicardi_Ch48_p2061-p2076.indd 206319/02/19 1:49 PM 2064SPECIFIC CONSIDERATIONSPART IIconsent in the same way as adults. Depending on their age, children may lack the cognitive and emotional maturity to participate fully in the process. In addition,

1	2064SPECIFIC CONSIDERATIONSPART IIconsent in the same way as adults. Depending on their age, children may lack the cognitive and emotional maturity to participate fully in the process. In addition, depending on the child’s age, their specific circumstances, as well as the local jurisdiction, children may not have legal standing to fully par-ticipate on their own independent of their parents. The use of parents or guardians as surrogate decision makers only partially addresses the ethical responsibility of the surgeon to involve the child in the informed consent process. The surgeon should strive to augment the role of the decision makers by involving the child in the process. Specifically, children should receive age-appropriate information about their clinical situation and therapeutic options delivered in an appropriate setting and tone so that the surgeon can solicit the child’s “assent” for treatment. In this manner, while the parents or surrogate decision makers formally give the

1	delivered in an appropriate setting and tone so that the surgeon can solicit the child’s “assent” for treatment. In this manner, while the parents or surrogate decision makers formally give the informed consent, the child remains an inte-gral part of the process.Certain religious practices can present additional chal-lenges when treating minor children whose parents disallow medically indicated blood transfusions; however, case law has made clear the precedent that parents, regardless of their held beliefs, may not place their minor children at mortal risk. In such a circumstance, the physician should seek counsel from the hospital medicolegal team, as well as from the institutional ethics team. Legal precedent has, in general, established that the hospital or physician can proceed with providing all necessary care for the child.Obtaining “consent” for organ donation deserves spe-cific mention.15 Historically, discussion of organ donation with families of potential donors was

1	with providing all necessary care for the child.Obtaining “consent” for organ donation deserves spe-cific mention.15 Historically, discussion of organ donation with families of potential donors was performed by transplant professionals, who were introduced to families by intensivists after brain death had been confirmed and the family had been informed of the fact of death. In other instances, consent might be obtained by intensivists caring for the donor, as they were assumed to know the patient’s family and could facilitate the process. However, issues of moral “neutrality” as part of end-of-life care in the intensive care unit have caused a shift in how obtaining “consent” for organ donation is handled. Responsibility for obtaining consent from the donor family is now vested in trained “designated requestors” (or “organ procurement coordinators”)16 or by “independent” intensivists who do not have a therapeutic clinical relationship with the potential donor.17 In this way, the

1	“designated requestors” (or “organ procurement coordinators”)16 or by “independent” intensivists who do not have a therapeutic clinical relationship with the potential donor.17 In this way, the donor family can be allowed to make the decision regarding donation in a “neutral” environment without erosion of the therapeutic relationship with the treating physician or perceived undue pressure from the transplant team.The process of informed consent also can be limited by the capacity of patients to assimilate information in the context of their illness. For example, despite the best efforts of surgeons, evidence suggests that patients rarely retain much of what is dis-closed in the consent conversation, and they may not remember discussing details of the procedure that become relevant when postoperative complications arise.18 It is important to recognize that the doctrine of informed consent places the most emphasis on the principle of autonomy precisely in those clinical situa-tions

1	postoperative complications arise.18 It is important to recognize that the doctrine of informed consent places the most emphasis on the principle of autonomy precisely in those clinical situa-tions when, because of their severe illness or impending death, patients are often divested of their autonomy.The Boundaries of Autonomy: Advance Directives and Powers of AttorneySevere illness and impending death can often render patients incapable of exercising their autonomy regarding medical decisions. One approach to these difficult situations is to make decisions in the “best interests” of patients, but because such decisions require value judgments about which thoughtful peo-ple frequently disagree, ethicists, lawyers, and legislators have sought a more reliable solution. Advance directives of various forms have been developed to carry forward into the future the autonomous choices of competent adults regarding healthcare decisions. Furthermore, the courts often accept “informal” advance

1	various forms have been developed to carry forward into the future the autonomous choices of competent adults regarding healthcare decisions. Furthermore, the courts often accept “informal” advance directives in the form of sworn testimony about state-ments the patient made at some time previous to their illness. When a formal document expressing the patient’s advance directives fails to exist, surgeons should consider the comments patients and families make when asked about their wishes in the setting of debilitating illness.Living wills are written to anticipate treatment options and choices in the event that a patient is incapacitated by a terminal illness. In the living will, the patient indicates which treatments she wishes to permit or prohibit in the setting of terminal illness. The possible treatments addressed often include mechanical ventilation, cardiopulmonary resuscitation, artificial nutri-tion, dialysis, antibiotics, or transfusion of blood products. Unfor-tunately,

1	The possible treatments addressed often include mechanical ventilation, cardiopulmonary resuscitation, artificial nutri-tion, dialysis, antibiotics, or transfusion of blood products. Unfor-tunately, living wills are often too vague to offer concrete guidance in complex clinical situations, and the language (“termi-nal illness,” “artificial nutrition”) can be interpreted in many ways. Furthermore, by limiting the directive only to “terminal” conditions, it does not provide guidance for common clinical sce-narios like advanced dementia, delirium, or persistent vegetative states where the patient is unable to make decisions, but is not “terminally” ill. Perhaps even more problematic is the evidence that demonstrates that healthy patients cannot reliably predict their preferences when they are actually sick. This phenomenon is called “affective forecasting” and applies to many situations. For example, the general public estimates the health-related qual-ity of life (HRQoL) score of

1	are actually sick. This phenomenon is called “affective forecasting” and applies to many situations. For example, the general public estimates the health-related qual-ity of life (HRQoL) score of patients on dialysis at 0.39, although dialysis patients themselves rate their HRQoL at 0.56.19 Similarly, patients with colostomies rated their HRQoL at 0.92, compared to a score of 0.80 given by the general public for patients with colostomies.19 For these and other reasons, living wills are often unable to provide the extent of assistance they promise.20An alternative to living wills is the durable power of attor-ney for healthcare in which patients identify surrogate decision makers and invest them with the authority to make healthcare decisions on their behalf in the event that they are unable to speak for themselves. Proponents of this approach hope that the surrogate will be able to make decisions that reflect the choices that the patients themselves would make if they were able.

1	unable to speak for themselves. Proponents of this approach hope that the surrogate will be able to make decisions that reflect the choices that the patients themselves would make if they were able. Unfortunately, several studies demonstrate that surrogates are not much better than chance at predicting the choices patients make when the patient is able to state a preference. For example, a recent meta-analysis found that surrogates predicted patients’ treatment preferences with only 68% accuracy.21 These data reveal a flaw in the guiding principle of surrogate decision making: Surrogates do not necessarily have privileged insight into the autonomous preferences of patients. However, the dura-ble power of attorney at least allows patients to choose the person who will eventually make prudential decisions on their behalf and in their best interests; therefore, respecting the judgment of the surrogate is a way of respecting the self-determination of the incapacitated patient.22There is

1	decisions on their behalf and in their best interests; therefore, respecting the judgment of the surrogate is a way of respecting the self-determination of the incapacitated patient.22There is continuing enthusiasm for a wider use of advance directives. In fact, the 1991 Patient Self Determination Act requires all U.S. healthcare facilities to (a) inform patients of 334Brunicardi_Ch48_p2061-p2076.indd 206419/02/19 1:49 PM 2065ETHICS, PALLIATIVE CARE, AND CARE AT THE END OF LIFECHAPTER 48their rights to have advance directives, and (b) to document those advance directives in the chart at the time any patient is admitted to the healthcare facility.4 However, only a minority of patients in U.S. hospitals have advance directives despite concerted efforts to teach the public of their benefits and pro-vide resources to help patients prepare and maintain them. For example, the ambitious SUPPORT trial used specially trained nurses to promote communication between physicians, patients, and

1	and pro-vide resources to help patients prepare and maintain them. For example, the ambitious SUPPORT trial used specially trained nurses to promote communication between physicians, patients, and their surrogates to improve the care and decision making of critically ill patients. Despite this concerted effort, the interven-tion demonstrated “no significant change in the timing of do not resuscitate (DNR) orders, in physician-patient agreement about DNR orders, in the number of undesirable days (patients’ experiences), in the prevalence of pain, or in the resources consumed.”23Some of the reluctance around physician–patient agree-ment about DNR orders may reflect patient and family anxiety that DNR orders equate to “do not treat.” Patients and families should be assured, when appropriate, that declarations of DNR/do not intubate will not necessarily result in a change in ongoing routine clinical care. The issue of temporarily rescinding DNR/do not intubate orders around the time of an

1	that declarations of DNR/do not intubate will not necessarily result in a change in ongoing routine clinical care. The issue of temporarily rescinding DNR/do not intubate orders around the time of an operative procedure may also need to be addressed with the family.Patients should be encouraged to clearly identify their sur-rogates, both formally and informally, early in the course of treatment and before any major elective operation. Often, around the time of surgery or at the end of life, there are limits to patient autonomy in medical decision-making. Seeking an advance directive or surrogate decision maker requires time that is not always available when the clinical situation deterio-rates. As such, these issues should be clarified as early as pos-sible in the patient–physician relationship.Withdrawing and Withholding Life-Sustaining TherapiesThe implementation of various forms of life support technol-ogy raise a number of legal and ethical concerns about when it is permissible to

1	and Withholding Life-Sustaining TherapiesThe implementation of various forms of life support technol-ogy raise a number of legal and ethical concerns about when it is permissible to withdraw or withhold available therapeu-tic technology. There is general consensus among ethicists that there are no philosophic differences between withdrawing (stopping) or withholding (not starting) treatments that are no longer beneficial.24 However, the right to refuse, withdraw, and withhold beneficial treatments was not established before the landmark case of Karen Ann Quinlan. In 1975, Quinlan lapsed into a persistent vegetative state requiring ventilator support. After several months without clinical improvement, Quinlan’s parents asked the hospital to withdraw ventilator support. The hospital refused, fearing prosecution for euthanasia. The case was appealed to the New Jersey Supreme Court where the justices ruled that it was permissible to withdraw ventilator support.25 This case established a

1	fearing prosecution for euthanasia. The case was appealed to the New Jersey Supreme Court where the justices ruled that it was permissible to withdraw ventilator support.25 This case established a now commonly recognized right to with-draw “extraordinary” life-saving technology if it is no longer desired by the patient or the patient’s surrogate.The difference between “ordinary” and “extraordinary” care, and whether there is an ethical difference in withhold-ing or withdrawing “ordinary” vs. “extraordinary” care, has been an area of much contention. The 1983 Nancy Cruzan case highlighted this issue. In this case, Cruzan had suffered severe injuries in an automobile crash that rendered her in a persistent vegetative state. Cruzan’s family asked that her tube feeds be withheld, but the hospital refused. The case was appealed to the U.S. Supreme Court, which ruled that the tube feeding could be withheld if her parents demonstrated “clear and convincing evidence” that the incapacitated

1	refused. The case was appealed to the U.S. Supreme Court, which ruled that the tube feeding could be withheld if her parents demonstrated “clear and convincing evidence” that the incapacitated patient would have rejected the treatment.26 In this ruling, the court essentially ruled that there was no legal distinction between “ordinary” vs. “extraor-dinary” life-sustaining therapies.27 In allowing the feeding tube to be removed, the court accepted the principle that a competent person (even through a surrogate decision maker) has the right to decline treatment under the Fourteenth Amendment of the U.S. Constitution. The court noted, however, that there has to be clear and convincing evidence of the patient’s wishes (con-sistent with the principle of autonomy) and that the burdens of the medical intervention should outweigh its benefits (consistent with the principles of beneficence and nonmaleficence).In deliberating the issue of withdrawing vs. withholding life-sustaining therapies,

1	intervention should outweigh its benefits (consistent with the principles of beneficence and nonmaleficence).In deliberating the issue of withdrawing vs. withholding life-sustaining therapies, the principle of “double effect” is often mentioned. According to the principle of “double effect,” a treatment (e.g., opioid administration in the terminally ill) that is intended to help and not harm the patient (i.e., relieve pain) is ethically acceptable even if an unintended consequence (side effect) of its administration is to shorten the life of the patient (e.g., by respiratory depression). Under the principle of double effect, a physician may withhold or withdraw a life-sustaining therapy if the surgeon’s intent is to relieve suffering, not to has-ten death. The classic formulation of double effect has four ele-ments (Fig. 48-3).Withholding or withdrawing of life-sustaining therapy is ethically justified under the principle of double effect if the phy-sician’s intent is to relieve

1	double effect has four ele-ments (Fig. 48-3).Withholding or withdrawing of life-sustaining therapy is ethically justified under the principle of double effect if the phy-sician’s intent is to relieve suffering, not to kill the patient. Thus, in managing the distress of the dying, there is a fundamental eth-ical difference between titrating medications rapidly to achieve relief of distress and administering a very large bolus with the 55Double effectConditionsActionGood effectBad effectGood effectBad effectActIntrinsic moral wrongAgentIntendsGood effectBad effect1234ActBad effectGood effectFigure 48-3. The four elements of the double effect principle: (1) The good effect is produced directly by the action and not by the bad effect. (2) The person must intend only the good effect, even though the bad effect may be foreseen. (3) The act itself must not be intrinsically wrong, or needs to be at least neutral. (4) The good effect is sufficiently desirable to compensate for allowing the bad

1	the bad effect may be foreseen. (3) The act itself must not be intrinsically wrong, or needs to be at least neutral. (4) The good effect is sufficiently desirable to compensate for allowing the bad effect.Brunicardi_Ch48_p2061-p2076.indd 206519/02/19 1:49 PM 2066SPECIFIC CONSIDERATIONSPART IIintent of causing apnea. It is important to note, however, that although the use of opioids for pain relief in advanced illness is frequently cited as the classic example of the double effect rule, opioids can be used safely without significant risk. In fact, if administered appropriately, in the vast majority of instances the rule of double effect need not be invoked when administering opioids for symptom relief in advanced illness.28In accepting the ethical equivalence of withholding and withdrawing of life-sustaining therapy, surgeons can make dif-ficult treatment decisions in the face of prognostic uncertainty.24 In light of this, some important principles to consider when con-sidering

1	of life-sustaining therapy, surgeons can make dif-ficult treatment decisions in the face of prognostic uncertainty.24 In light of this, some important principles to consider when con-sidering withdrawal of life-sustaining therapy include: (a) Any and all treatments can be withdrawn. If circumstances justify withdrawal of one therapy (e.g., IV pressors, antibiotics), they may also justify withdrawal of others; (b) Be aware of the sym-bolic value of continuing some therapies (e.g., nutrition, hydra-tion) even though their role in palliation is questionable; (c) Before withdrawing life-sustaining therapy, ask the patient and family if a spiritual advisor (e.g., pastor, imam, rabbi, or priest) should be called; and (d) Consider requesting an ethics consult.Although the clinical setting may seem limited, a range of options usually exists with respect to withdrawing or with-holding treatment, allowing for an incremental approach, for example (a) continuing the current regimen without adding

1	limited, a range of options usually exists with respect to withdrawing or with-holding treatment, allowing for an incremental approach, for example (a) continuing the current regimen without adding new interventions or tests; (b) continuing the current regimen but withdrawing elements when they are no longer beneficial; and (c) withdrawing and withholding all treatments that are not tar-geted to relieve symptoms and maximize patient comfort.34The surgeon might consider discussing the clinical situ-ation with the patient or proxy decision maker, identify the various therapeutic options, and delineate the reasons why with-holding or withdrawing life-sustaining therapy would be in the patient’s best interest. If the patient (or designated proxy deci-sion maker) does not agree with withholding or withdrawing life-sustaining therapy, the surgeon should consider involving consultants who have participated in the patient’s care, experts in palliative or end-of-life care or recommend a second

1	or withdrawing life-sustaining therapy, the surgeon should consider involving consultants who have participated in the patient’s care, experts in palliative or end-of-life care or recommend a second medical opinion. If the second opinion corroborates that life-sustaining therapy should be withheld or withdrawn but the patient/family continues to disagree, the surgeon should consider assistance from institutional resources such as the ethics committee and hospital administration. Although the surgeon is not ethically obligated to provide treatment that he or she believes is futile, the surgeon is responsible for continued care of the patient, which may involve transferring the patient to a surgeon who is willing to provide the requested intervention.24Living Donor Liver TransplantationOne unique ethical issue that deserves special mention is that of living donor liver transplantation. Living donor kidney transplantation has been practiced for almost 50 years and has become a routine

1	unique ethical issue that deserves special mention is that of living donor liver transplantation. Living donor kidney transplantation has been practiced for almost 50 years and has become a routine part of clinical care, but living donor liver transplantation was first performed in the late 1980s with par-ent-to-child grafts and in the late 1990s for adult-to-adult grafts. These procedures are unique in that there are two patients, one with a diseased organ who requires intervention to be made well and one who is healthy and is being made unwell, albeit usu-ally temporarily, during the intervention. Performing an ethi-cal analysis of this situation requires considering both risks and benefits to each of the patients individually.For the recipient, the benefits of receiving a living donor organ as opposed to a deceased donor organ are many: first, there is reduced risk of death on the waitlist, and second, there is a potential for improved post-transplant outcomes due to improved

1	organ as opposed to a deceased donor organ are many: first, there is reduced risk of death on the waitlist, and second, there is a potential for improved post-transplant outcomes due to improved matching between relatives and the absence of hemo-dynamic instability often present before organ procurement in a deceased donor.30 Furthermore, the use of living donor organs is supported by the principal of utility, maximizing efficient use of organs.32The benefit to the organ donor is in fulfillment of an altru-istic ideal and satisfaction associated with having extended the recipient’s life, while the risks are those associated with partial hepatectomy, a procedure that is not without risks including postoperative complications and mortality, the risk of which is estimated to be 0.15%.29 The ethical concern in this case is hav-ing possibly violated the principle of nonmaleficence.This particular ethical issue emphasizes the importance of truly informed consent. The donor should be

1	The ethical concern in this case is hav-ing possibly violated the principle of nonmaleficence.This particular ethical issue emphasizes the importance of truly informed consent. The donor should be provided with information on local complication and mortality rates and allowed sufficient time to consider the risks and benefits with-out pressure from healthcare workers.30 Furthermore, experi-enced centers have recommended that living donors have access to sufficient resources and strong support from an institutions’ ethics committee, given substantial pressure exerted by the criti-cal illness of a family member.31PALLIATIVE CAREGeneral Principles of Palliative CarePalliative care is a coordinated, interdisciplinary effort that aims to relieve suffering and improve quality of life for patients and their families in the context of serious illness.33 It is offered simultaneously with all other appropriate medical treatment, and its indication is not limited to situations associated with a

1	and their families in the context of serious illness.33 It is offered simultaneously with all other appropriate medical treatment, and its indication is not limited to situations associated with a poor prognosis for survival. Palliative care strives to achieve more than symptom control, but it should not be confused with noncurative treatment.The World Health Organization defines palliative care as “an approach that improves the quality of life of patients and their families facing the problems associated with life-threat-ening illness, through the prevention and relief of suffering by means of early identification and impeccable assessment and treatment of pain and other problems, physical, psychosocial, and spiritual.”34 Palliative care is both a philosophy of care and an organized, highly structured system for delivering care.Palliative care includes the entire spectrum of intervention for the relief of symptoms and the promotion of quality of life. No specific therapy, including

1	structured system for delivering care.Palliative care includes the entire spectrum of intervention for the relief of symptoms and the promotion of quality of life. No specific therapy, including surgical intervention, is excluded from consideration. Therefore, surgeons have valuable contri-butions to make to palliative care. In fact, the term palliative care was coined in 1975 by Canadian surgeon, Balfour Mount. Furthermore, surgical palliative care can be defined as the treat-ment of suffering and the promotion of quality of life for seri-ously or terminally ill patients under the care of surgeons.36 The standard of palliative treatment lies in the agreement between patient and physician that the expected outcome is relief from distressing symptoms, lessening of pain, and improvement of quality of life. The decision to intervene is based on the treat-ment’s ability to meet the stated goals, rather than its impact on the underlying disease.The fundamental elements of palliative care

1	quality of life. The decision to intervene is based on the treat-ment’s ability to meet the stated goals, rather than its impact on the underlying disease.The fundamental elements of palliative care consist of pain and nonpain symptom management, communication among patients, their families, and care providers, and conti-nuity of care across health systems and through the trajectory Brunicardi_Ch48_p2061-p2076.indd 206619/02/19 1:49 PM 2067ETHICS, PALLIATIVE CARE, AND CARE AT THE END OF LIFECHAPTER 48of illness. Additional features of system-based palliative care are team-based planning that includes patient and family; close attention to spiritual matters; and psychosocial support for patients, their families, and care providers, including bereave-ment support.Indications for palliative care consultation in surgical practice include: (a) patients with conditions that are progres-sive and life-limiting, especially if characterized by burdensome symptoms, functional decline, and

1	care consultation in surgical practice include: (a) patients with conditions that are progres-sive and life-limiting, especially if characterized by burdensome symptoms, functional decline, and progressive cognitive defi-cits; (b) assistance in clarification or reorientation of patient/family goals of care; (c) assistance in resolution of ethical dilemmas; (d) situations in which a patient/surrogate declines further invasive or curative treatments with stated preference for comfort measures only; (e) patients who are expected to die imminently or shortly after hospital discharge; and (f) provision of bereavement support for patient care staff, particularly after loss of a colleague under care36 (Table 48-1). Although all patients, regardless of prognosis, may benefit from the services of a palliative care physician, hospice care is a specific form of palliative care intended for patients who have an estimated prognosis of 6 months or less to live. Hospice care is covered under

1	of a palliative care physician, hospice care is a specific form of palliative care intended for patients who have an estimated prognosis of 6 months or less to live. Hospice care is covered under Medicare Part A, and benefits may be continued beyond the original 6 months of estimated survival if physicians certify that life expectancy remains limited to 6 months or less. Although most Americans indicate a preference to die at home, nearly 75% die in an institutional setting. Earlier referral and wider use of the hospice benefit may help more patients achieve their goal of dying at home.Concepts of Suffering, Pain, Health, and HealingPalliative care specifically addresses the individual patient’s experience of suffering due to illness. Indeed, the philosophi-cal origins of palliative care began with attention to suffering and the existential questions suffering engenders. More than mere technologic evolution in the management of symptoms, the early proponents of palliative care sought

1	began with attention to suffering and the existential questions suffering engenders. More than mere technologic evolution in the management of symptoms, the early proponents of palliative care sought a revolution in the moral foundations of medicine that challenged the assumptions that so often seemed to result in futile invasive intervention, and identified many of the problems that were subsequently taken up by medical ethicists. This reorientation of the goals of medical care from a focus on disease and its management to the patient’s experience of illness focuses attention on the purpose of medicine and the meaning of health and healing.Over the past half century, several concepts and theo-ries about the nature of pain, suffering, and health have been proposed in service of the evolving conceptual framework of palliative care. For example, while considering the differences between disease-oriented and illness-oriented approaches to the care of seriously ill patients, psychiatrist

1	conceptual framework of palliative care. For example, while considering the differences between disease-oriented and illness-oriented approaches to the care of seriously ill patients, psychiatrist Arthur Kleinman wrote, “There is a moral core to healing in all societies. [Healing] is the central purpose of medicine . . . the purpose of medicine is both control of disease processes and care for the illness experience. Nowhere is this clearer than in the relationship of the chronically ill to their medical system: For them, the control of disease is by definition limited; care for the life problems created by the disorder is the chief issue.”33The relief of pain has been the clinical foundation for hospice and palliative care. Pain is defined by the International Association for the Study of Pain as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage.”38 For purposes of interdisciplinary palliative care,

1	Pain as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage.”38 For purposes of interdisciplinary palliative care, Saunders’s concept of “total pain”37 is a more useful definition and is frequently used as the basis for palliative assessments. Total pain is the sum total of four principal domains of pain: physical, psychologic, social or socioeconomic, and spiritual. Each of these contributes to, but is not synonymous with, suffering.Effective Communication and Negotiating the Goals of CareChanging the goals of care from cure to palliation near the end of life can be both emotionally and clinically challenging. It depends on determination of a clear prognosis and can be aided by effective communication. Unfortunately, prognostication can be notoriously difficult and inaccurate in advanced illness, and Christakis has argued that, to a large degree, physicians have abdicated their traditional

1	Unfortunately, prognostication can be notoriously difficult and inaccurate in advanced illness, and Christakis has argued that, to a large degree, physicians have abdicated their traditional responsibility to provide clear prognosis regarding incurable disease and approaching death.40 However, there are validated tools for prognosis in critical ill-ness (APACHE, MODS, etc.), and with most advanced diseases, functional status is the most powerful predictor of survival. For example, patients with advanced metastatic cancer who are rest-ing or sleeping for 50% or more of normal waking hours and require some assistance with activities of daily living (ADL) have a projected survival of weeks, and patients who are essen-tially bedfast and dependent for ADL have a projected survival of days to a week or two at best. Table 48-2 shows a simple prognostic tool to aid clinicians in recognizing patients nearing the end of life.Alternatively, the Karnofsky Performance Scale is a scale of

1	days to a week or two at best. Table 48-2 shows a simple prognostic tool to aid clinicians in recognizing patients nearing the end of life.Alternatively, the Karnofsky Performance Scale is a scale of functional status ranging from 100 (high level of function) to 0 (death). It is commonly used in palliative care to roughly assess a patient’s anticipated needs as well as prognosis. The Palliative Performance Scale41 is a validated42 expansion of the Karnofsky Performance Scale that includes five palliative-focused domains, including ambulation, activity level, self-care, intake, and level of consciousness, in addition to evidence of disease. The Missoula-Vitas Quality of Life Index is a 25-question scale specifically for palliative care and hospice patients that scores symptoms, function, interpersonal relationships, well-being, and spirituality. Updates and Spanish versions are available.39Regardless of the prognostic tool used, the prognosis should be conveyed to the patient and

1	interpersonal relationships, well-being, and spirituality. Updates and Spanish versions are available.39Regardless of the prognostic tool used, the prognosis should be conveyed to the patient and family. If done well, communication and negotiation with patients and families about advanced terminal illnesses can potentially avoid great 66Table 48-1Indications for palliative care consultationPatients with conditions that are progressive and life-limiting, especially if characterized by burdensome symptoms, functional decline, and progressive cognitive deficitsAssistance in clarification or reorientation of patient/family goals of careAssistance in resolution of ethical dilemmasSituations in which patient/surrogate declines further invasive or curative treatments with stated preference for comfort measures onlyPatients who are expected to die imminently or shortly after hospital dischargeProvision of bereavement support for patient care staff, particularly after loss of a colleague under

1	measures onlyPatients who are expected to die imminently or shortly after hospital dischargeProvision of bereavement support for patient care staff, particularly after loss of a colleague under careBrunicardi_Ch48_p2061-p2076.indd 206719/02/19 1:49 PM 2068SPECIFIC CONSIDERATIONSPART IITable 48-2Simple prognostication tool in advanced illness (especially cancer)FUNCTIONAL LEVELPERFORMANCE STATUS (ECOG)PROGNOSISAble to perform all basic ADLs independently and some IADLs2MonthsResting/sleeping up to 50% or more of waking hours and requiring some assistance with basic ADLs3Weeks to a few monthsDependent for basic ADLs and bed-to-chair existence4Days to a few weeks at mostThese observations apply to patients with advanced, progressive, incurable illnesses (e.g., metastatic cancer refractory to treatment).Basic ADL = activities of daily living (e.g., transferring, toileting, bathing, dressing, and feeding oneself); IADL = instrumental activities of daily living (e.g., more complex

1	to treatment).Basic ADL = activities of daily living (e.g., transferring, toileting, bathing, dressing, and feeding oneself); IADL = instrumental activities of daily living (e.g., more complex activities such as meal preparation, performing household chores, balancing a checkbook, shopping, etc.); ECOG = Eastern Cooperative Oncology Group functional (performance) status.Table 48-3Communicating unfavorable news: important principles• Setting: Find a quiet, private place to meet. Sit down close to the patient.• Listen: Clarify the patient’s and/or the family’s understanding of the situation.• “Warning shot”: Prepare patient and family and obtain their permission to communicate bad news (e.g., “I’m afraid I have bad news.”).• Silence: Pause after giving bad news. Allow patient/family to absorb/react to the news.• Encourage: Convey hope that is realistic and appropriate to the circumstances (e.g., patient will not be abandoned; symptoms will be controlled).psychologic harm and help make a

1	to the news.• Encourage: Convey hope that is realistic and appropriate to the circumstances (e.g., patient will not be abandoned; symptoms will be controlled).psychologic harm and help make a difficult transition easier. To communicate effectively and compassionately, it is helpful to pursue an organized process similar to the structured history and physical central to the evaluation of any patient. One such structured approach to delivering unfavorable news proposes six steps that can be easily learned by clinicians: (a) getting started by selection of the appropriate setting, introductions, and seating; (b) determining what the patient or family knows; (c) determining what the patient or family wants to know; (d) giving the information; (e) expressing empathy; and (f) establishing expectations, planning, and aftercare (Table 48-3).43 Success with this approach to breaking bad news is critically depen-dent upon the clinician’s ability to empathically respond to the patient’s (and

1	planning, and aftercare (Table 48-3).43 Success with this approach to breaking bad news is critically depen-dent upon the clinician’s ability to empathically respond to the patient’s (and family’s) reaction to the news.44 The empathic response does not require the surgeon to share the same emo-tions of the patient, but it does require the surgeon to identify the patient’s emotion and accurately reflect that awareness back to the patient. Such effective communication may be facilitated by involving other members of the healthcare team who have developed relationships with the patient and their family. Patient assessment in these conversations should give the highest prior-ity to identifying and responding to the most immediate source of distress. Relieving a pressing symptom is prerequisite for a more thorough search for other potential sources of suffering, and the assessment process itself can be therapeutic if conducted in a respectful and gentle manner.CARE AT THE END OF LIFEThe

1	for a more thorough search for other potential sources of suffering, and the assessment process itself can be therapeutic if conducted in a respectful and gentle manner.CARE AT THE END OF LIFEThe process of dying and the care of a patient at the time of death is a distinct clinical entity that demands specific skills from physicians. The issues specific to dying and the available tools for compassionate care at the end of life are addressed in this section.The Syndrome of Imminent Demise34,45In a patient who has progressed to the terminal stage of an advanced illness (e.g., cancer), a number of signs provide evi-dence of imminent death. As terminally ill patients progress toward death, they become increasingly bedbound, requiring assistance for all basic ADL. There is a steady decrease in desire and requests for food and fluids. More distressing to the dying patient is a progressively dry mouth that may be confused by the treating team as thirst. It is often exacerbated by

1	decrease in desire and requests for food and fluids. More distressing to the dying patient is a progressively dry mouth that may be confused by the treating team as thirst. It is often exacerbated by anticholinergic medications, mouth breathing, and supplemental oxygen (O2) administered without humidification.With progressive debility, fatigue, and weight loss, it is common for terminally ill patients to experience increasing dif-ficulty swallowing. This may result in aspiration episodes and an inability to swallow tablets, requiring alternative routes for medication administration (e.g., IV, SC, PR, sublingual, buccal, or transdermal). In addition to the increased risk of aspiration, patients near death develop great difficulty clearing oropharyn-geal and upper airway secretions, leading to noisy breathing or the so-called “death rattle.” As death approaches, the respiratory pattern may change to increasingly frequent periods of apnea often following a Cheyne-Stokes pattern of rapid,

1	to noisy breathing or the so-called “death rattle.” As death approaches, the respiratory pattern may change to increasingly frequent periods of apnea often following a Cheyne-Stokes pattern of rapid, progressively longer breaths leading up to an apneic period. As circulatory instability develops near death, patients may exhibit cool and mottled extremities. Periods of confusion are often accompanied by decreasing urine output and episodes of fecal and urinary incontinence.A number of cognitive changes occur as death approaches. Patients who are in the last days of life may demonstrate some signs of confusion or delirium. Agitated delirium is a promi-nent feature of a difficult death. Other cognitive changes that may be seen include a decreased interest in social interactions, increased somnolence, reduced attention span, disorientation to time (often with altered sleep-wake cycles), and an altered dream life, including vivid “waking dreams” or visual halluci-nations. Reduced hearing

1	reduced attention span, disorientation to time (often with altered sleep-wake cycles), and an altered dream life, including vivid “waking dreams” or visual halluci-nations. Reduced hearing and visual acuity may be an issue for some patients; however, patients who appear comatose may still be aware of their surroundings. Severely cachectic patients may lose the ability to keep their eyes closed during sleep because of loss of the retro-orbital fat pad.Common Symptoms at the End of Life and Their Management34,45,46The three most common, major symptoms that threaten the comfort of dying patients in their last days are respiratory Brunicardi_Ch48_p2061-p2076.indd 206819/02/19 1:49 PM 2069ETHICS, PALLIATIVE CARE, AND CARE AT THE END OF LIFECHAPTER 48Table 48-4Principles of pharmacotherapy in palliative care• Believe patient report of symptoms.• Modify pathologic process when possible and appropriate.• In terminally ill patients, avoid medications not directly linked to symptom

1	in palliative care• Believe patient report of symptoms.• Modify pathologic process when possible and appropriate.• In terminally ill patients, avoid medications not directly linked to symptom control.• Use a multidisciplinary approach.• Consider nonpharmacologic approaches whenever possible.• Engage participation of clinical pharmacist in treatment plan.• Select drugs that can multitask (i.e., use haloperidol for agitated delirium and nausea).• For pain, use adjuvant medications when possible (see Table 48-7).• When using opioids, spare when possible (adjuvant medication, local or regional anesthetics, surgical interventions, etc.).• Avoid fixed combination drugs.• Avoid excessive cost.• Select agents with minimum side effects.• Anticipate and prophylax against side effects.• For older adult patients, the hypoproteinemic, the azotemic: “Start low and go slow.”• Oral route whenever possible and practical.• No intramuscular injections.• Scheduled dosing, not as needed, for persistent

1	adult patients, the hypoproteinemic, the azotemic: “Start low and go slow.”• Oral route whenever possible and practical.• No intramuscular injections.• Scheduled dosing, not as needed, for persistent symptoms.• Stepwise approach. (See the World Health Organization Analgesic Ladder for pain, Table 48-5.)• Reassess continuously and titrate to effect.• Use equianalgesic doses when changing opioids (see Table 48-5).• Assess the patient’s and family’s comprehension of management plan.Table 48-5The World Health Organization’s three-step ladder for control of cancer pain30Step 1: mild pain (visual analogue scale, 1–3) Nonopioid ± adjuvant medicationStep 2: moderate pain (visual analogue scale, 4–6) Opioid for mild to moderate pain and nonopioid ± an adjuvantStep 3: severe pain (visual analogue scale, 7–10) Opioid for moderate to severe pain ± nonopioid ± an adjuvantThe primary treatment of dyspnea (air hunger) in the dying is opioids, which should be cautiously titrated to increase com-fort

1	7–10) Opioid for moderate to severe pain ± nonopioid ± an adjuvantThe primary treatment of dyspnea (air hunger) in the dying is opioids, which should be cautiously titrated to increase com-fort and reduce tachypnea to a range of 15 to 20 breaths per minute. Air movement across the face generated by a fan can sometimes be quite helpful. If this is not effective, empirical use of supplemental O2 by nasal cannula (2–3 L/min) may bring some subjective relief, independent of observable changes in pulse oximetry. Supplemental O2 should be humidified to avoid exacerbation of dry mouth. Typical starting doses of an immedi-ate release opioid for breathlessness should be one-half to two-thirds of a starting dose of the same agent for cancer pain. For patients already on opioids for pain, a 25% to 50% increment in the dose of the current immediate release agent for breakthrough pain often will be effective in relieving breathlessness in addi-tion to breakthrough pain.The availability and variety

1	increment in the dose of the current immediate release agent for breakthrough pain often will be effective in relieving breathlessness in addi-tion to breakthrough pain.The availability and variety of drugs should not prevent consideration of nonpharmacologic therapy. Massage therapy, music therapy, art therapy, guided imagery, hypnosis, physi-cal therapy, pet therapy, and others play a constructive role not only for the relief of symptoms but also for promoting a sense of hope through improving function, aesthetic pleasure, and social connectedness. Talents and capacities neglected during the treatment and progression of disease can be recovered even in the most advanced stages of illness.Pain is often less of a problem in the last days of life because the reduced activity level is associated with lower inci-dent pain. This, combined with lower renal clearance of opioids, may result in greater potency of the prescribed agents. Severe pain crises are fortunately rare, but when they

1	with lower inci-dent pain. This, combined with lower renal clearance of opioids, may result in greater potency of the prescribed agents. Severe pain crises are fortunately rare, but when they are inadequately addressed, can cause great and lasting distress (complicated grief) for loved ones who witness the final hours or days of agony. Such situations may require continuous administration of parenteral opioids. As death approaches and patients become less verbal, it is important to assess pain frequently, including the use of close observation for nonverbal signs of distress (e.g., grimacing, increased respiratory rate). Adequate dosing of opi-oid analgesics may require alternate route(s) of administration other than oral as patients become more somnolent or develop swallowing difficulties. Opioids should not be stopped abruptly, even if the patient becomes nonresponsive, because sudden withdrawal can cause severe distress.49,50Cognitive failure at the end of life is manifested in

1	Opioids should not be stopped abruptly, even if the patient becomes nonresponsive, because sudden withdrawal can cause severe distress.49,50Cognitive failure at the end of life is manifested in most patients by increasing somnolence and delirium. Gradually increasing somnolence can be accompanied by periods of dis-orientation and mild confusion, and it may respond to the reas-suring presence of loved ones and caregivers with minimal need for medications. A more distressing form of delirium also can distress, pain, and cognitive failure. General principles that are applicable to symptom management in the last days of life include (a) anticipating symptoms before they develop; (b) minimizing technologic interventions (usually manage symp-toms with medications); and (c) planning alternative routes for medications in case the oral route fails. It may be possible to cautiously reduce the dose of opioids and other medications as renal clearance decreases near the end of life, but it is

1	routes for medications in case the oral route fails. It may be possible to cautiously reduce the dose of opioids and other medications as renal clearance decreases near the end of life, but it is important to remember that increased somnolence and decreasing respira-tions are prominent features of the dying process independent of medication side effects. Sudden cessation of opioid analgesics near the end of life could precipitate withdrawal symptoms, and therefore medications should not be stopped for increasing som-nolence or slowed respirations.The principles of pharmacotherapy for pain and non-pain symptoms in the palliative care setting are outlined in Table 48-4. The World Health Organization,35 the United States Agency for Healthcare Policy and Research,47 the Academy of Hospice and Palliative Medicine,48 and many other agencies have endorsed a “step ladder” approach to cancer pain man-agement that can predictably result in satisfactory pain control in most patients (Table

1	and Palliative Medicine,48 and many other agencies have endorsed a “step ladder” approach to cancer pain man-agement that can predictably result in satisfactory pain control in most patients (Table 48-5). More refractory pain problems require additional expertise, and occasionally, more invasive approaches (Tables 48-6 and 48-7).Brunicardi_Ch48_p2061-p2076.indd 206919/02/19 1:49 PM 2070SPECIFIC CONSIDERATIONSPART IITable 48-6Analgesics for persistent painDRUGINITIAL DOSING (ADULT, >60 kg)COMMENTSMild persistent pain, visual analogue scale (VAS) 1–3 Acetaminophen (Tylenol)325–650 mg PO four times a day Maximum = 3200 mg/24 hUse <2400 mg if other potentially hepatotoxic drugs taken. Acetaminophen contained in concurrent nonprescription medications can easily exceed maximum daily allowable dose. Aspirin600–1500 mg PO four times a dayGastric bleeding, platelet dysfunction Choline magnesium trisalicylate (Trilisate)750–1500 mg PO twice a dayUseful for avoiding platelet

1	daily allowable dose. Aspirin600–1500 mg PO four times a dayGastric bleeding, platelet dysfunction Choline magnesium trisalicylate (Trilisate)750–1500 mg PO twice a dayUseful for avoiding platelet dysfunction Ibuprofen (Advil, Motrin)200–400 mg PO four times a day Maximum = 3200 mg/24 hGastropathy, nephropathy, decreased platelet aggregation Naproxen (Naprosyn)250 mg PO twice a day Maximum = 1300 mg/24 hAvailable as a transcutaneous gelModerate persistent pain, VAS 4–6 Hydrocodone (Vicodin, Lortab)5–7.5 mg PO every 4 hoursMost prescribed drug in the United StatesAcetaminophen in compounded drug limits use to moderate pain Oxycodone5 mg PO every 4 hoursSold as single agent or compounded with aspirin or acetaminophenSlow release form available (Oxycontin)Severe persistent pain, VAS 7–10 Morphine10 mg PO every 2–4 hours 2–4 mg IV, SC every 1–2 hoursStandard drug for comparison to alternative opioids. Avoid or caution when giving to older adults, patients with diminished glomerular

1	mg PO every 2–4 hours 2–4 mg IV, SC every 1–2 hoursStandard drug for comparison to alternative opioids. Avoid or caution when giving to older adults, patients with diminished glomerular filtration rate, or liver disease. Slow release PO form available (MS Contin). Hydromorphone1–3 mg PO, PR every 4 hours 1 mg IV, SC every 1–2 hoursSuppository form availableOral dose forms limited to 4 mg maximum Fentanyl, transdermal12 μg/h patch every 72 hoursNot for acute pain management. Do not use on opioid-naive patients. Absorption unpredictable in cachectic patients. MethadoneConsultation with pain management, clinical pharmacists, or palliative care/hospice services skilled in methadone use is recommended for those inexperienced in prescribing methadone.Not a first-line agent, although very effective, especially for pain with a neuropathic componentVery inexpensiveCan be given PO, IV, SC, PR, sublingually, and vaginallyIts long half-life makes dosing more difficult than alternative opioids and

1	especially for pain with a neuropathic componentVery inexpensiveCan be given PO, IV, SC, PR, sublingually, and vaginallyIts long half-life makes dosing more difficult than alternative opioids and close monitoring is required when initiating.Numerous medications, alcohol, and cigarette smoking can alter its serum levels.Physicians who write methadone prescriptions for pain should specify this indication. Methadone use for drug withdrawal treatment requires special licensure.Risk factors for NSAID-induced nephropathy include: advanced age, decreased glomerular filtration rate, congestive heart failure, hypovolemia, pressors, hepatic dysfunction, concomitant nephrotoxic agents. Dose reduction and hydration reduce risk.Opioids compounded with aspirin or acetaminophen are limited to treatment of moderate persistent pain because of dose-limiting toxicities of acetaminophen and aspirin.Slow-release preparations of morphine and oxycodone may be given rectally.Timed-release tablets or patches

1	of moderate persistent pain because of dose-limiting toxicities of acetaminophen and aspirin.Slow-release preparations of morphine and oxycodone may be given rectally.Timed-release tablets or patches should never be crushed or cut.Opioid analgesics are the agents of choice for severe cancer-related pain. Sedation is a common side effect when initiating opioid therapy. Tolerance to this usually develops within a few days. If sedation persists beyond a few days, a stimulant (methylphenidate 2.5–5 mg PO twice a day) can be given.Initiate bowel stimulant prophylaxis for constipation when prescribing opioids unless contraindicated.Adjuvant or coanalgesic agents are drugs that enhance analgesic efficacy of opioids, treat concurrent symptoms that exacerbate pain, or provide independent analgesia for specific types of pain (e.g., a tricyclic antidepressant for treatment of neuropathic pain). Coanalgesics can be initiated for persistent pain at any visual analogue scale level. Gabapentin is

1	for specific types of pain (e.g., a tricyclic antidepressant for treatment of neuropathic pain). Coanalgesics can be initiated for persistent pain at any visual analogue scale level. Gabapentin is commonly used as an initial agent for neuropathic pain.No place for meperidine (Demerol), propoxyphene (Darvon, Darvocet, or mixed agonist-antagonist agents [Stadol, Talwin]) in management of persistent pain.Always consider alternative approaches (axial analgesia, operative approaches, etc.) when managing severe persistent pain.Note: These are not recommendations for specific patients. The interand intraindividual variability to opioids requires individualizing dosing and titration to effect.Adapted with permission from Cameron JL: Current Surgical Therapy, 9th ed. Philadelphia, PA: Elsevier; 2008.Brunicardi_Ch48_p2061-p2076.indd 207019/02/19 1:49 PM 2071ETHICS, PALLIATIVE CARE, AND CARE AT THE END OF LIFECHAPTER 48Table 48-7Examples of adjuvant medications for treatment of neuropathic,

1	207019/02/19 1:49 PM 2071ETHICS, PALLIATIVE CARE, AND CARE AT THE END OF LIFECHAPTER 48Table 48-7Examples of adjuvant medications for treatment of neuropathic, visceral, and bone painaDRUG CLASSINITIAL DOSING (ADULT, >60 kg)COMMENTSTricyclic antidepressants Best for continuous burning or tingling pain and allodynia Efficacy for pain not due to antidepressant effectAmitriptyline 10–25 mg PO before bedNortriptyline 10–25 mg PO one per daySedating properties may be useful for relief of other concurrent symptoms. Side effects may precede benefit. Avoid in older adult patients due to anticholinergic side effects. Dose generally less than that required for antidepressant effectDoxepin 10–25 mg PO before bedLess anticholinergic effect Dose titrated up every few days until effect. Pain may respond to alternative antidepressants if no response to initial agent.Imipramine 10–25 mg PO one per day Anticonvulsants For shooting, stabbing painGabapentin 100–1200 mg PO three times a day.

1	may respond to alternative antidepressants if no response to initial agent.Imipramine 10–25 mg PO one per day Anticonvulsants For shooting, stabbing painGabapentin 100–1200 mg PO three times a day. Titrate up rapidly as needed. Max: 3600 mg daily in divided dosesCommonly used first-line agent. Generally well tolerated. Does not require blood level monitoring. Carbamazepine 200 mg PO every 12 hoursPregabalin starting dose 25–50 mg PO three times a dayEffective. Well studied. Requires blood monitoring.Does not require blood monitoring. Valproic acid 250 mg PO three times a day Local anesthetics Systemic use requires monitoring. Nebulized local anesthetics (lidocaine, bupivacaine) can be used for severe, refractory cough.Lidocaine transdermal patch 5%. Apply to painful areas. Max: 3 simultaneous patches over 12 hours (each patch contains 700 mg lidocaine).Lidocaine/prilocaine topical. Apply to painful areas.Systemic toxicity can result from applying more than recommended number per

1	patches over 12 hours (each patch contains 700 mg lidocaine).Lidocaine/prilocaine topical. Apply to painful areas.Systemic toxicity can result from applying more than recommended number per unit time and in patients with liver failure. Effective for postherpetic neuralgia.MiscellaneousBisphosphonates (pamidronate, zoledronic acid)For bone pain and reduced incidence of skeletal complications secondary to malignancy—best results in myeloma and breast cancer. Contraindicated in renal failure. Calcitonin nasal sprayRefractory bone pain DexamethasoneFor bone pain, acute nerve compression, visceral pain secondary to tumor infiltration or luminal obstruction by reducing inflammatory component of tumor Radionuclides (Sr-89)For malignant bone pain secondary to osteoclastic activity. 4–6 wk delay in benefit. Requires adequate bone marrow reserve. For prognosis of more than 3 mo. OctreotideReduces GI secretions that contribute to visceral painaRecommendations are based on experience of

1	delay in benefit. Requires adequate bone marrow reserve. For prognosis of more than 3 mo. OctreotideReduces GI secretions that contribute to visceral painaRecommendations are based on experience of practitioners of hospice and palliative medicine and in some instances do not reflect current clinical trials.Brunicardi_Ch48_p2061-p2076.indd 207119/02/19 1:49 PM 2072SPECIFIC CONSIDERATIONSPART IIdevelop, manifested by increasing agitation that may require the use of neuroleptic medications. Increasing amounts of opioids and/or benzodiazepines may exacerbate the delirium (especially in the elderly).Pronouncing Death51If the body is hypothermic or has been hypothermic, such as a drowning victim pulled from the water in the winter, the phy-sician should not declare death until warming attempts have been made. In the hospital, hospice, or home setting, the dec-laration of death becomes part of the medical or legal record of the event. There are a number of physical signs of death a

1	attempts have been made. In the hospital, hospice, or home setting, the dec-laration of death becomes part of the medical or legal record of the event. There are a number of physical signs of death a physician should look for in confirming the patient’s demise: complete lack of responsiveness to verbal or tactile stimuli, absence of heart beat and respirations, fixed pupils, skin color change to a waxen hue as blood settles, gradual poikilothermia, and sphincter relaxation with loss of urine and feces. For deaths in the home with patients who have been enrolled in hospice, the hospice nurse on call should be contacted immediately. In some states, deaths at home may require a brief police investiga-tion and report. For deaths in the hospital, the family must be notified (in person, if possible). A coroner or medical examiner may need to be contacted under specific circumstances (e.g., deaths in the operating room), but most deaths do not require their services. The pronouncing

1	if possible). A coroner or medical examiner may need to be contacted under specific circumstances (e.g., deaths in the operating room), but most deaths do not require their services. The pronouncing physician will need to complete a death certificate according to local regulations. Survivors may also be approached, if appropriate, regarding potential autopsy and organ donation. Finally, it is important to accommodate religious rituals that may be important to the dying patient or the family. Bereavement is the experience of loss by death of a person to whom one is attached. Mourning is the process of adapting to such a loss in the thoughts, feelings, and behaviors that one experiences after the loss.52 Although grief and mourn-ing are accentuated in the immediate period around death, it is important to note that patients and families may have begun the process of bereavement well before the time of death as patients and families grieve incremental losses of independence, vitality, and

1	important to note that patients and families may have begun the process of bereavement well before the time of death as patients and families grieve incremental losses of independence, vitality, and control. In addition to the surviving loved ones, it is impor-tant to acknowledge that caregivers also experience grief for the loss of their patients.53,54Aid in DyingFive European countries, Canada, and six U.S. states have legal-ized physician-assisted suicide, medical assistance in dying, or aid-in-dying, in some form, ranging from hospital-based pro-grams to provision of fatal doses of medications for home self-administration.55-57 Medical assistance in dying is a complex ethical and legal issues with divergent opinions among the pub-lic and healthcare providers.58,59 While aid-in-dying laws passed in the United States vary somewhat, these laws essentially all allow physicians to prescribe a lethal dose of medication to men-tally, competent, terminally ill adult patients for the

1	laws passed in the United States vary somewhat, these laws essentially all allow physicians to prescribe a lethal dose of medication to men-tally, competent, terminally ill adult patients for the purpose of achieving the end of life.60,61 Key areas of ethical consideration in this area include the benefit and harm of death; the relation-ship between passive euthanasia, active euthanasia, withholding treatment, and withdrawing treatment; the morality of physician and nursing participation in deliberately causing death; and the management of conscientious objection.60,62 Although surgeons outside of the critical care arena may only infrequently be asked to participate in aid-in-dying, it is important to be familiar with local legislation so that appropriate information can be provided to patients who request it.PROFESSIONAL ETHICS: CONFLICT OF INTEREST, RESEARCH, AND CLINICAL ETHICSConflict of InterestConflicts of interest for surgeons can arise in many situations in which the potential

1	who request it.PROFESSIONAL ETHICS: CONFLICT OF INTEREST, RESEARCH, AND CLINICAL ETHICSConflict of InterestConflicts of interest for surgeons can arise in many situations in which the potential benefits or gains to be realized by the surgeon are, or are perceived to be, in conflict with the respon-sibility to put the patient’s interests before the surgeon’s own. Conflicts of interest for the surgeon can involve actual or per-ceived situations in which the individual stands to gain mon-etarily by his or her role as a physician or investigator. In the academic community, monetary gain may not be the primary factor. Instead, motivators such as power, tenure, or authorship on a publication may serve as potential sources of conflict of interest. For example, the accrual of subjects in research studies or patients in surgical series may ensure surgeons better author-ship or more financial gains. The dual-role of the surgeon-scien-tist therefore needs to be considered because the duty as

1	studies or patients in surgical series may ensure surgeons better author-ship or more financial gains. The dual-role of the surgeon-scien-tist therefore needs to be considered because the duty as surgeon can conflict with the role of scientist or clinical researcher.Research EthicsOver the last three decades in the United States, the ethical requirements for the conduct of human subject research have been formalized and widely accepted. Although detailed informed consent is a necessary condition for the conduct of ethically good human subject research, other factors also deter-mine whether research is designed and conducted ethically. Emanuel and colleagues63 described seven requirements for all clinical research studies to be ethically sound: (a) value—enhancement(s) of health or knowledge must be derived from the research; (b) scientific validity—the research must be methodologically rigorous; (c) fair subject selection—scientific objectives, not vulnerability or privilege, and the

1	must be derived from the research; (b) scientific validity—the research must be methodologically rigorous; (c) fair subject selection—scientific objectives, not vulnerability or privilege, and the potential for and distribution of risks and benefits, should deter-mine communities selected as study sites and the inclusion cri-teria for individual subjects; (d) favorable risk-benefit ratio—within the context of standard clinical practice and the research protocol, risks must be minimized, potential benefits enhanced, and the potential benefits to individuals and knowl-edge gained for society must outweigh the risks; (e) independent review—unaffiliated individuals must review the research and approve, amend, or terminate it; (f) informed consent—individuals should be informed about the research and pro-vide their voluntary consent; and (g) respect for enrolled subjects—subjects should have their privacy protected, the opportunity to withdraw, and their well-being monitored.63Special

1	and pro-vide their voluntary consent; and (g) respect for enrolled subjects—subjects should have their privacy protected, the opportunity to withdraw, and their well-being monitored.63Special Concerns in Surgical ResearchA significant issue for clinical surgical research is that many surgical studies are retrospective in nature and are not com-monly undertaken in a prospective, double-blind, randomized fashion. For a randomized trial to be undertaken, the researchers should be in a state of equipoise—that is, there must be a state of genuine uncertainty on the part of the clinical investigator or the expert medical community regarding the comparative thera-peutic merits of each arm in a trial.64 To randomize subjects to receive two different treatments, a researcher must believe that the existing data are not sufficient to conclude that one treat-ment strategy is better than another. In designing surgical trials, surgeons usually have biases that one treatment is better than another

1	existing data are not sufficient to conclude that one treat-ment strategy is better than another. In designing surgical trials, surgeons usually have biases that one treatment is better than another and often have difficulty maintaining the state of equi-poise. As such, it is frequently difficult to demonstrate that a 77Brunicardi_Ch48_p2061-p2076.indd 207219/02/19 1:49 PM 2073ETHICS, PALLIATIVE CARE, AND CARE AT THE END OF LIFECHAPTER 48Table 48-8ICMJE criteria for authorshipAccording to ICMJE best practices recommendations, authors should fulfill each of the following four criteria67:1. Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work2. Drafting the work or revising it critically for important intellectual content3. Final approval of the version to be published4. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part

1	content3. Final approval of the version to be published4. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolvedContributors who do not fulfill all four criteria should be named in the manuscript in the acknowledgment section.Adapted with permission from Cameron JL: Current Surgical Therapy, 9th ed. Philadelphia, PA: Elsevier; 2008.randomized trial is necessary or feasible, and treatment options that question the validity of clinical tenets are difficult to accept. Meakins has suggested that a slightly different hierarchy of evi-dence applies to evidence-based surgery.65A second major issue for surgical trials is whether it is ethically acceptable to have a placebo-controlled surgical trial. Some commentators have argued that sham surgery is always wrong because, unlike a placebo medication that is harmless, every surgical procedure carries some risk.66

1	placebo-controlled surgical trial. Some commentators have argued that sham surgery is always wrong because, unlike a placebo medication that is harmless, every surgical procedure carries some risk.66 Others have argued that sham operations are essential to the design of a valid ran-domized clinical trial because, without a sham operation, it is not possible to know if the surgical intervention is the cause of improvement in patient symptoms or whether the improve-ment is due to the effect of having surgery.67,68 Most surgeons readily agree that designing an appropriately low-risk sham sur-gical procedure would create problems for the surgeon-patient relationship in that the surgeon would need to keep the sham a secret.69 In this sense, a sham surgical arm of a trial is very different from a placebo medication in that there cannot be blinding of the surgeon as to which procedure was undertaken. As a result, to have a sham surgery arm in a clinical trial, the interactions between the

1	a placebo medication in that there cannot be blinding of the surgeon as to which procedure was undertaken. As a result, to have a sham surgery arm in a clinical trial, the interactions between the surgeon and the subject must be lim-ited, and the surgeon performing the procedure should not be the researcher who follows the subject during the trial. Despite difficulties with designing a surgical trial in which the surgeon could ethically perform a sham operation, there are specific cir-cumstances that allow for placebo operations to be conducted, so long as certain criteria are met and are analyzed on a case by case basis.70,71Surgical InnovationAn important issue is whether surgical innovation should be treated as research or as standard of care. Throughout history, many advances in surgical techniques and technologies have resulted from innovations of individual surgeons crafted dur-ing the course of challenging operations—such innovations and technologies have served to move the

1	techniques and technologies have resulted from innovations of individual surgeons crafted dur-ing the course of challenging operations—such innovations and technologies have served to move the field of surgery forward.72 In the Korean and Vietnam wars, military guidelines for treat-ment of vascular injuries recommended ligation and amputation rather than interposition grafting of vascular injuries. Individual surgeons chose to ignore those guidelines and subsequently demonstrated the value of the reconstructive techniques that ultimately became the standard of care. It is debated whether modifications to an accepted surgical technique in an individual patient based on their circumstances and within the skill and judg-ment of an individual surgeon should require the same type of prior approval that enrollment in a clinical trial would warrant.73 However, if a surgeon decides to use a new technique on sev-eral occasions and to study the outcomes, Institutional Review Board approval and

1	that enrollment in a clinical trial would warrant.73 However, if a surgeon decides to use a new technique on sev-eral occasions and to study the outcomes, Institutional Review Board approval and all other ethical requirements for research are necessary. These situations require strict oversight as well as explicit consent by the patient.74 In particular, when developing new and innovative techniques, the surgeon should work in close consultation with his or her senior colleagues, including the chairperson of the department. Frequently, more senior individuals can provide sage ethical advice regarding what constitutes minor innovative changes in a technique vs. true novel research.Compared to the formalized process for new drug approval by the Food and Drug Administration, the process for a surgeon developing an innovative operation can be relatively unregu-lated and unsupervised.The Ethics of AuthorshipAuthorship specifies who is responsible for published research. It confers both

1	a surgeon developing an innovative operation can be relatively unregu-lated and unsupervised.The Ethics of AuthorshipAuthorship specifies who is responsible for published research. It confers both recognition for academic achievement as well as responsibility for the academic integrity of the published con-tent. Authorship is the stock in trade of productivity for aca-demic surgeons, and it plays a significant role in promotion and tenure. It can also be commodified in the form of intellectual property and patents in which the author and the author’s insti-tution have vested interests. Yet it can also become a liability if a given piece of work becomes embroiled in accusations of plagiarism, data fabrication, or other academic misconduct.In the past, criteria for authorship were unspecified: Those submitting manuscripts simply listed the authors with little or no need to substantiate their contribution to the work. Unfortu-nately, this informal process led to confusion and even abuse.

1	Those submitting manuscripts simply listed the authors with little or no need to substantiate their contribution to the work. Unfortu-nately, this informal process led to confusion and even abuse. For example, there has been a long tradition of awarding author-ship to the investigator who supervised or obtained funding for research, regardless of that person’s specific contribution to the manuscript. However, current recommendations specify that supervision and funding, by themselves, are insufficient criteria for authorship, and thus such individuals should only be included as authors if they make direct contributions to the work.75,76 A more disturbing example is the practice of “ghost writing” by which senior investigators publish industry-written research under their own name to bolster their productivity while providing a luster of academic integrity to industry.To address these conflicts of interest and to provide guid-ance to investigators, the International Committee of

1	bolster their productivity while providing a luster of academic integrity to industry.To address these conflicts of interest and to provide guid-ance to investigators, the International Committee of Medical Journal Editors (ICMJE) provides recommendations on criteria for authorship so that individuals who contributed to the intel-lectual content of a work get appropriate credit and that all those listed as authors take responsibility and are accountable for the published work. The ICMJE recommendations for authorship can be found in Table 48-8.75 Furthermore, the ICMJE recom-mends that each author should be able to identify the contribu-tion that each other author made to the work and be confident regarding the integrity of their co-authors. The ICMJE also recommends that individuals who do not meet these criteria be acknowledged in the manuscript, providing appropriate pro-cedures for such acknowledgement. Additionally, the ICMJE Brunicardi_Ch48_p2061-p2076.indd 207319/02/19 1:49

1	not meet these criteria be acknowledged in the manuscript, providing appropriate pro-cedures for such acknowledgement. Additionally, the ICMJE Brunicardi_Ch48_p2061-p2076.indd 207319/02/19 1:49 PM 2074SPECIFIC CONSIDERATIONSPART IIspecifically excludes certain types of contributions including acquisition of funding, general supervision of a research group, administrative support, writing assistance, technical editing, language editing, and proofreading.75Many journals have adopted these criteria, operational-izing them at the time of submission by having each author specify his or her contributions. These contributions are then disclosed in the published manuscript to further specify how credit and responsibility is shared.77 This approach has been shown to provide valuable information and has proved feasible in several journals, including The Lancet.78As research becomes increasingly interdisciplinary with ever-expanding teams of contributors, it can be difficult to determine

1	and has proved feasible in several journals, including The Lancet.78As research becomes increasingly interdisciplinary with ever-expanding teams of contributors, it can be difficult to determine which contributions warrant full authorship rather than simple acknowledgement. Individuals working together on research endeavors should have clear discussions early in the planning process about authorship, and those discus-sions should be continued throughout the project or study.Clinical Ethics: Disclosure of ErrorsDisclosure of error—either in medical or research matters—is important, but often difficult (see Chapter 12). Errors of judg-ment, errors in technique, and system errors are responsible for most errors that result in complications and deaths. Hospitals are evaluated based on the number of complications and deaths that occur in surgical patients, and surgeons traditionally review their complications and deaths in a formal exercise known as the mortality and morbidity conference,

1	of complications and deaths that occur in surgical patients, and surgeons traditionally review their complications and deaths in a formal exercise known as the mortality and morbidity conference, or M&M. The exercise places importance on the attending surgeon’s responsibility for errors made, whether he or she made them themselves, and the value of the exercise is related to the effect of “peer pressure”— the entire department knows about the case—on reducing repeated occurrences of such an error. Although a time-honored ritual in surgery, the M&M conference is nonetheless a poor method for analyzing causes of error and for developing methods to prevent them. Moreover, the proceedings of the M&M con-ference are protected from disclosure by the privilege of “peer review,” and the details are thus rarely shared with patients or those outside the department.A report from the United States Institute of Medicine titled “To Err Is Human” highlighted the large number of medi-cal errors that

1	thus rarely shared with patients or those outside the department.A report from the United States Institute of Medicine titled “To Err Is Human” highlighted the large number of medi-cal errors that occur and encouraged efforts to prevent patient harm.79 Medical errors are generally considered to be “prevent-able adverse medical events.”80 Medical errors occur with some frequency, and the question is what and how should patients be informed that a medical error has occurred.81Disclosure of error is consistent with the ethical virtue of candor (e.g., transparency and openness) and the ethical prin-ciple of respect for persons by involving patients in their care. In contrast, failing to disclose errors to patients under-mines public trust in medicine and potentially compro-mises adequate treatment of the consequences of errors and effective intervention to prevent future errors. In addition, fail-ure to self-disclose medical errors can be construed as a breach of professional ethics, as

1	of the consequences of errors and effective intervention to prevent future errors. In addition, fail-ure to self-disclose medical errors can be construed as a breach of professional ethics, as it is a failure to act in the patient’s best interests. Information regarding a medical error may be needed so that patients can make independent and well-informed deci-sions about future aspects of their care. The principles of auton-omy and justice dictate that surgeons need to respect individuals by being fair in providing accurate information about all aspects of their care—even when an error has occurred.Disclosing one’s own errors is therefore part of the ethi-cal standard of honesty and putting the patient’s interests above one’s own. Disclosing the errors of others is more complicated and may require careful consideration and consultation. Sur-geons sometimes discover that a prior operation has included an apparent error; an injured bile duct or a stenotic anastomosis may lead to the

1	may require careful consideration and consultation. Sur-geons sometimes discover that a prior operation has included an apparent error; an injured bile duct or a stenotic anastomosis may lead to the condition for which the surgeon is now treating the patient. Declaring a finding as an “error” may be inaccurate, however, and a nonjudgmental assessment of the situation is usually advisable. When clear evidence of a mistake is at hand, the surgeon’s responsibility is defined by his or her obligation to act as the patient’s agent.REFERENCESEntries highlighted in bright blue are key references. 1. Aristotle. Nichomachean Ethics, Book VI. In Ackrill J, ed. A New Aristotle Reader. Princeton, NJ: Princeton University Press; 1987:416. 2. Beauchamp TL, Childress JF. Principles of Biomedical Ethics, 3rd ed. New York: Oxford University Press; 1989. 3. Bosk C. Forgive and Remember, 2nd ed. Chicago, University of Chicago Press, 2003 (1979). 4. McCullough LB, Jones JW, Brody BA, eds. Surgical

1	3rd ed. New York: Oxford University Press; 1989. 3. Bosk C. Forgive and Remember, 2nd ed. Chicago, University of Chicago Press, 2003 (1979). 4. McCullough LB, Jones JW, Brody BA, eds. Surgical Ethics. New York: Oxford University Press; 1998. 5. Faden RR, Beauchamp TL. A History and Theory of Informed Consent. New York: Oxford University Press; 1986. 6. Bernat JL, Peterson LM. Patient-centered informed consent in surgical practice. Arch Surg. 2006;141:86-92. 7. Schneider CE. The Practice of Autonomy: Patients, Doctors, and Medical Decisions. New York: Oxford University Press; 1998. 8. Robb A, Etchells E, Cusimano MD, et al. A randomized trial of teaching bioethics to surgical residents. Am J Surg. 2005;189:453-457. 9. Steinemann S, Furoy D, Yost F, et al. Marriage of professional and technical tasks: a strategy to improve obtaining informed consent. Am J Surg. 2006;191:696-700. 10. Guadagnoli E, Soumerai SB, Gurwitz JH, et al. Improving dis-cussion of surgical treatment options for

1	tasks: a strategy to improve obtaining informed consent. Am J Surg. 2006;191:696-700. 10. Guadagnoli E, Soumerai SB, Gurwitz JH, et al. Improving dis-cussion of surgical treatment options for patients with breast cancer: local medical opinion leaders versus audit and perfor-mance feedback. Breast Cancer Res Treat. 2000;61:171-175. 11. Braddock CH III, Edwards KA, Hasenberg NM, et al. Informed decision making in outpatient practice: time to get back to basics. JAMA. 1999;282:2313-2320. 12. Leeper-Majors K, Veale JR, Westbrook TS, et al. The effect of standardized patient feedback in teaching surgical resi-dents informed consent: results of a pilot study. Curr Surg. 2003;60:615-622. 13. Courtney MJ. Information about surgery: what does the public want to know? ANZ J Surg. 2001;71:24-26. 14. Newton-Howes PA, Dobbs B, Frizelle F. Informed con-sent: what do patients want to know? N Z Med J. 1998;111: 340-342. 15. Streat S. Clinical review: moral assumptions and the pro-cess of organ

1	PA, Dobbs B, Frizelle F. Informed con-sent: what do patients want to know? N Z Med J. 1998;111: 340-342. 15. Streat S. Clinical review: moral assumptions and the pro-cess of organ donation in the intensive care unit. Crit Care. 2004;8:382-388. 16. Williams MA, Lipsett PA, Rushton CH, et al. The physician’s role in discussing organ donation with families. Crit Care Med. 2003;31:1568-1573. 17. Pearson IY, Zurynski Y. A survey of personal and professional attitudes of intensivists to organ donation and transplantation. Anaesth Intensive Care. 1995;23:68-74. 18. Sulmasy DP, Lehmann LS, Levine DM, et al. Patients’ percep-tions of the quality of informed consent for common medical procedures. J Clin Ethics. 1994;5:189-194. 19. Ubel PA, Loewenstein G, Jepson C. Whose quality of life? A commentary exploring discrepancies between health state 8899Brunicardi_Ch48_p2061-p2076.indd 207419/02/19 1:49 PM 2075ETHICS, PALLIATIVE CARE, AND CARE AT THE END OF LIFECHAPTER 48evaluations of patients

1	exploring discrepancies between health state 8899Brunicardi_Ch48_p2061-p2076.indd 207419/02/19 1:49 PM 2075ETHICS, PALLIATIVE CARE, AND CARE AT THE END OF LIFECHAPTER 48evaluations of patients and the general public. Qual Life Res. 2003;12:599-607. 20. Schneider CE. After autonomy. Wake Forest Law Review. 2006;41:411. 21. Shalowitz DI, Garrett-Mayer E, Wendler D. The accuracy of surrogate decision makers: a systematic review. Arch Intern Med. 2006;166(5): 493-497. 22. Sulmasy DP, Hughes MT, Thompson RE, et al. How would terminally ill patients have others make decisions for them in the event of decisional incapacity? A longitudinal study. J Am Geriatr Soc. 2007;55:1981-1988. 23. SUPPORT Principle Investigators. A controlled trial to improve care for seriously ill hospitalized patients. The study to under-stand prognoses and preferences for outcomes and risks of treatments (SUPPORT). The SUPPORT Principal Investigators. JAMA. 1995;274:1591-1598. 24. Pawlik TM. Withholding and

1	The study to under-stand prognoses and preferences for outcomes and risks of treatments (SUPPORT). The SUPPORT Principal Investigators. JAMA. 1995;274:1591-1598. 24. Pawlik TM. Withholding and withdrawing life-sustain-ing treatment: a surgeon’s perspective. J Am Coll Surg. 2006;202:990-994. 25. In re Quinlan. 355 A2d 647 (JN). Vol 429 US 9221976. 26. Cruzan vs. Director, Missouri Dept of Health, 497(1990). 27. Annas GJ. Nancy Cruzan and the right to die. N Engl J Med. 1990;323:670-673. 28. Sykes N, Thorns A. The use of opioids and sedatives at the end of life. Lancet Oncol. 2003;4:312-318. 29. Trotter JF, Adam R, Lo CM, Kenison J. Documented deaths of hepatic lobe donors for living donor liver transplantation. Liver Transpl. 2006;12(10):1485-1488. 30. Singer PA, Siegler M, Whitington PF, et al. Ethics of liver transplantation with living donors. N Engl J Med. 1989;321(9):620-622. 31. Fournier V, Foureur N, Rari E. The ethics of living donation for liver transplant: beyond donor

1	PF, et al. Ethics of liver transplantation with living donors. N Engl J Med. 1989;321(9):620-622. 31. Fournier V, Foureur N, Rari E. The ethics of living donation for liver transplant: beyond donor autonomy. Med Healthcare Philos. 2013;16(1):45-54. 32. Shapiro RS, Adams M. Ethical issues surrounding adult-to-adult living donor liver transplantation. Liver Transpl. 2000; 6(6 suppl 2):S77-S80. 33. Kleinman A. The Illness Narratives. Suffering, Healing & the Human Condition. New York: Basic Books; 1988. 34. Nelson KA, Walsh D, Behrens C, et al. The dying cancer patient. Semin Oncol. 2000;27:84. 35. WHO. Definition of palliative care, 2008. World Health Orga-nization. Available at: http://www.who.int/cancer/palliative/definition/en/. Accessed August 29, 2018. 36. Dunn G. Surgical palliative care. In: Mosby, ed. Current Surgical Therapy, 9th ed. Philadelphia: Elsevier; 2008. 37. Saunders C. The challenge of terminal care. In: Symington T, Carter R, eds. Scientific Foundations of Oncology.

1	In: Mosby, ed. Current Surgical Therapy, 9th ed. Philadelphia: Elsevier; 2008. 37. Saunders C. The challenge of terminal care. In: Symington T, Carter R, eds. Scientific Foundations of Oncology. London: Heineman; 1976:673. 38. International Association for the Study of Pain, Subcommittee on Taxonomy. Part II. Pain Terms: a current list with definitions and notes on usage. Pain. 1979;6:249. 39. Byock IR, Merriman MP. Measuring quality of life for patients with terminal illness: the Missoula-VITAS quality of life index. Palliat Med. 1998;12:231-244. 40. Christakis NA, Lamont EB. Extent and determinants of error in doctors’ prognoses in terminally ill patients: prospective cohort study. BMJ. 2000;320:469-472. 41. Anderson F, Downing GM, Hill J, et al. Palliative performance scale (PPS): a new tool. J Palliat Care. 1996;12:5-11. 42. Morita T, Tsunoda J, Inoue S, et al. Validity of the palliative performance scale from a survival perspective. J Pain Symptom Manage.

1	scale (PPS): a new tool. J Palliat Care. 1996;12:5-11. 42. Morita T, Tsunoda J, Inoue S, et al. Validity of the palliative performance scale from a survival perspective. J Pain Symptom Manage. 1999;18:2-3. 43. Buckman R. How to Break Bad News. A Guide for Healthcare Professionals. Baltimore: Johns Hopkins University Press; 1992. 44. Kubler-Ross E. On Death and Dying. London: Routledge; 1973. 45. Twycross R, Lichter I. The terminal phase. In: Doyle D, Hanks G, MacDonald N, eds. Oxford Textbook of Palliative Medicine. New York: Oxford University Press; 1998:977. 46. Hinshaw DB. Spiritual issues in surgical palliative care. Surg Clin North Am. 2005;85:257-272. 47. Jacox A, Carr D, Payne R, et al. Management of cancer pain. AHCPR Publication No. 94-052: Clinical Practice Guideline No. 9. Rockville: US Department of Health and Human Services, Public Health Service; 1994. 48. Storey P, Knight C. UNIPAC Three: Assessment and Treat-ment of Pain in the Terminally Ill. 2nd ed. New York: Mary

1	US Department of Health and Human Services, Public Health Service; 1994. 48. Storey P, Knight C. UNIPAC Three: Assessment and Treat-ment of Pain in the Terminally Ill. 2nd ed. New York: Mary Ann Liebert Inc; 2003. 49. Rubenfeld GD, Crawford SW. Principles and practice of with-drawing life-sustaining treatment in the ICU. In: Curtis JR, Rubenfeld GD, eds. Managing Death in the Intensive Care Unit. New York: Oxford University Press; 2001. 50. Rousseau P. Existential distress and palliative sedation. Anesth Analg. 2005;101:611-612, 51. The EPEC-O Project, Educating Physicians in End-of-Life Care-Oncology: Module 6: Last Hours of Living. Bethesda: National Cancer Institute; 2007. 52. Worden J. Bereavement Care. Philadelphia: Lippincott Williams and Wilkins; 2002. 53. Bishop JP, Rosemann PW, Schmidt FW. Fides ancilla medici-nae: on the ersatz liturgy of death in biopsychosociospiritual medicine. Heythrop J. 2008;49:20. 54. Schroeder-Sheker T. Transitus: A Blessed Death in the Modern World.

1	FW. Fides ancilla medici-nae: on the ersatz liturgy of death in biopsychosociospiritual medicine. Heythrop J. 2008;49:20. 54. Schroeder-Sheker T. Transitus: A Blessed Death in the Modern World. Mt. Angel: St. Dunstan’s Press; 2001. 55. Li M, Watt S, Escaf M, et al. Medical assistance in dying—implementing a hospital-based program in Canada. N Engl J Med. 2017;376(21):2082-2088. 56. Emanuel EJ, Onwuteaka-Philipsen BD, Urwin JW, Cohen J. Attitudes and practices of euthanasia and physician-assisted suicide in the United States, Canada, and Europe. JAMA. 2016;316:79-90. 57. Trice Loggers E, Starks H, Shannon-Dudley M, Back AL, Appelbaum FR, Stewart FM. Implementing a Death with Dignity program at a comprehensive cancer center. N Engl J Med. 2013;368:1417-1424. 58. Rhee JY, Callaghan KA, Stahl A, et al. Physician-assisted sui-cide and euthanasia is incompatible with medicine: a response from medical students. Crit Care Med. 2017;45(6):e626-e627. doi:

1	JY, Callaghan KA, Stahl A, et al. Physician-assisted sui-cide and euthanasia is incompatible with medicine: a response from medical students. Crit Care Med. 2017;45(6):e626-e627. doi: 10.1097/CCM.0000000000002354. 59. Vogelstein E. Evaluating the American Nurses Associa-tion’s arguments against nurse participation in assisted suicide. Nurs Ethics. 2017;969733017694619. doi: 10.1177/0969733017694619. 60. Sharpe JT. Is there a significant moral distinction between active and passive euthanasia? Critique. 2011;5:11-16. 61. Buchbinder M. Aid-in-dying laws and the physician’s duty to inform. J Med Ethics. 2017;43(10):666-669. doi: 10.1136/medethics-2016-103936. 62. Goligher EC, Ely EW, Sulmasy DP, et al. Physician-assisted suicide and euthanasia in the ICU: a dialogue on core ethi-cal issues. Crit Care Med. 2017;45(2):149-155. doi: 10.1097/CCM.0000000000001818. 63. Emmanuel EJ, Wendler D, Grady C. What makes clinical research ethical? JAMA. 2000;283:2701-2711. 64. Freedman B. Equipoise and

1	Care Med. 2017;45(2):149-155. doi: 10.1097/CCM.0000000000001818. 63. Emmanuel EJ, Wendler D, Grady C. What makes clinical research ethical? JAMA. 2000;283:2701-2711. 64. Freedman B. Equipoise and the ethics of clinical research. N Engl J Med. 1987;317:141-145. 65. Meakins J. Innovation in surgery. The rules of evidence. Am J Surg. 2002;183:399-405. 66. Lefering R, Neugebauer E. Problems of randomized controlled trials in surgery. Paper presented at: Nonrandomized Compara-tive Clinical Studies. Heidelberg, 1997. 67. Flum DR. Interpreting surgical trials with subjective out-comes: avoiding UnSPORTsmanlike conduct. JAMA. 2006;296:2483-2485.Brunicardi_Ch48_p2061-p2076.indd 207519/02/19 1:49 PM 2076SPECIFIC CONSIDERATIONSPART II 68. Moseley JB, O’Malley K, Petersen NJ, et al. A controlled trial of arthroscopic surgery for osteoarthritis of the knee. N Engl J Med. 2002;347:81. Summary for patients in: J Fam Pract. 2002;51:813. 69. Angelos PA. Sham surgery in research: a surgeon’s view.

1	trial of arthroscopic surgery for osteoarthritis of the knee. N Engl J Med. 2002;347:81. Summary for patients in: J Fam Pract. 2002;51:813. 69. Angelos PA. Sham surgery in research: a surgeon’s view. Am J Bioeth. 2003;3:65-66. 70. Miller FG. Sham surgery: an ethical analysis. Sci Eng Ethics. 2004;10:157-166. 71. Angelos P. Sham surgery in clinical trials. JAMA. 2007;297:1545-1546, author reply 1546. 72. Riskin DJ, Longaker MT, Gertner M, et al. Innovation in sur-gery: a historical perspective. Ann Surg. 2006;244:686-693. 73. Biffl WL, Spain DA, Reitsma AM, et al. Responsible develop-ment and application of surgical innovations: a position state-ment of the Society of University Surgeons. J Am Coll Surg. 2008;206(6):1204-1209. 74. McKneally MF, Daar AS. Introducing new technologies: pro-tecting subjects of surgical innovation and research. World J Surg. 2003;27:930-934. 75. International Committee of Medical Journal Editors. Defin-ing the role of authors and contributors. Available at:

1	subjects of surgical innovation and research. World J Surg. 2003;27:930-934. 75. International Committee of Medical Journal Editors. Defin-ing the role of authors and contributors. Available at: http://www.icmje.org/recommendations/browse/roles-and-respon-sibilities/defining-the-role-of-authors-and-contributors.html> Accessed August 29, 2018. 76. Eggert LD. Best practices for allocating appropriate credit and responsibility to authors of multi-authored articles. Front Psychol. 2011;2:196. 77. Rennie D, Yank V, Emanuel L. When authorship fails. A proposal to make contributors accountable. JAMA. 1997;278(7):579-585. 78. Yank V, Rennie D. Disclosure of researcher contributions: a study of original research articles in The Lancet. Ann Intern Med. 1999;130(8):661-670. 79. Kohn LT, Corrigan JM, Donaldson MS. To Err Is Human: Building a Safer Health System. Washington: National Academy Press; 2000. 80. Brennan TA, Leape LL, Laird NM, et al. Incidence of adverse events and negligence in

1	JM, Donaldson MS. To Err Is Human: Building a Safer Health System. Washington: National Academy Press; 2000. 80. Brennan TA, Leape LL, Laird NM, et al. Incidence of adverse events and negligence in hospitalized patients. Results of the Harvard Medical Practice Study I. N Engl J Med. 1991;324:370-376. 81. Hebert PC, Levin AV, Robertson G. Bioethics for clinicians: 23. Disclosure of medical error. CMAJ. 2001;164:509-513.Brunicardi_Ch48_p2061-p2076.indd 207619/02/19 1:49 PM

1	Global SurgeryKatherine E. Smiley, Haile T. Debas, Catherine R. deVries, and Raymond R. Price 49chapterINTRODUCTIONModern surgery can save lives, help expand economies, and offer hope to individuals and communities. Prior to the accep-tance and availability of aseptic technique to prevent or decrease infections, and improved anesthesia for controlling pain, sur-gery as a specialty was held in very low esteem by medical doc-tors and the general public. Over the last 100 years, surgery has developed into a highly regarded discipline that not only pro-vides opportunities for curing certain diseases but also fulfills a special role in preventing and mitigating disability.Yet, surgery is currently unavailable to most people world-wide. The vast majority—90%—of the world’s population receives only 10% of the surgical care delivered. Said another way, 90% of surgical resources are consumed by the most privi-leged 10% of the world’s population. More than 5 billion people lack access to safe,

1	10% of the surgical care delivered. Said another way, 90% of surgical resources are consumed by the most privi-leged 10% of the world’s population. More than 5 billion people lack access to safe, timely, and affordable surgical care.1 Very few surgical procedures occur in countries spending less than U.S. $100 per person on health care per year compared to coun-tries spending greater than U.S. $1000 per person (Fig. 49-1).2Examples of disparities abound. In many countries, including the wealthiest, islands of poverty coexist within cities replete with material resources. Tertiary level hospitals operate within eyesight of slums whose inhabitants have no access to even basic care. Most of the people without access—people in rural areas and in countries with poor infrastructure—are the very people most at risk for death or disability due to lack of surgical care. Often the poor accept and endure many painful and potentially correctable fatal conditions as a fact of life.3-6 Care for

1	people most at risk for death or disability due to lack of surgical care. Often the poor accept and endure many painful and potentially correctable fatal conditions as a fact of life.3-6 Care for trauma and obstetrical emergencies is considered a basic surgical need but is absent in many rural regions. Other chronic conditions—often equally debilitating—progress to death or serious disability due to lack of available, safe surgery and anesthesia.Many factors contribute to the disparity in access to surgi-cal care. Poverty, a primary risk factor for all types of diseases, is a major obstacle hindering access to surgery. Healthcare pro-fessionals, including surgeons, migrate from areas of need due to a lack of infrastructure (hospitals, roadways, and stable elec-trical sources), limited supplies and equipment, lack of human resources, few opportunities for professional development, and concerns for personal safety. Until recently, there has been a significant lack of information

1	supplies and equipment, lack of human resources, few opportunities for professional development, and concerns for personal safety. Until recently, there has been a significant lack of information regarding the burden of surgical disease and surgery’s positive impact on communities. Current research substantiates that investment in surgical care improves economies and is an integral and necessary component of global health.7,8Disparities in care and outcomes are multidimensional, and no simple solution exists to improve access to appro-priate and affordable surgical care. Yet, five major forces are reshaping priorities and strategies leading the charge for the globalization of surgical care.1. The epidemiologic transition of diseases from primarily in-fectious to more chronic conditions2. The mobile nature of the world’s populations, allowing peo-ple to move freely between more isolated areas of the world, leading to a more integrated global community3. Ubiquitous information access

1	mobile nature of the world’s populations, allowing peo-ple to move freely between more isolated areas of the world, leading to a more integrated global community3. Ubiquitous information access exponentially enabling wide-spread participation in understanding and designing innova-tive opportunities for high-quality surgical care4. A revolution for equity and human rights where the world’s poor are demanding benefits to surgical care similar to those found in high-income countries (HICs)5. Recognition of the cost-effectiveness of surgical care and its potential to build economies, demonstrating the value of in-cluding surgery in global health strategies9-12The greatest burden of disease occurs in areas where human resources—physicians, nurses, pharmacists, and other healthcare workers—are scarce (Fig. 49-2).13 The proportion of physicians is low both in high-population areas and in areas where the population is growing most rapidly (Fig. 49-3).14,15 Fully trained surgeons and

1	scarce (Fig. 49-2).13 The proportion of physicians is low both in high-population areas and in areas where the population is growing most rapidly (Fig. 49-3).14,15 Fully trained surgeons and anesthesiologists comprise only a small proportion of the total number of the Human Resources in Health (HRH), and efforts to meet the 12Introduction 2077Defining Global Surgery 2079Global Surgery Ecosystem / 2079Human Resources / 2081Burden of Surgical Disease / 2081Strategies for Development 2086Essential Surgery: Current and Evolving Concepts / 2086Outreach and Engagement / 2087International Organizations / 2088Global Surgery and Public Health / 2091Cancer Initiatives / 2098Integrating Value into Global Surgery / 2102Advanced Surgical Care for Resource-Poor Areas / 2102Academic Global Surgery / 2103Ethics / 2105Innovation in Global Surgery / 2106The Future for Global Surgery 2106Brunicardi_Ch49_p2077-p2112.indd 207713/02/19 5:53 PM 2078surgical needs are now embracing a multifaceted

1	/ 2103Ethics / 2105Innovation in Global Surgery / 2106The Future for Global Surgery 2106Brunicardi_Ch49_p2077-p2112.indd 207713/02/19 5:53 PM 2078surgical needs are now embracing a multifaceted approach, including advanced care practitioners. The Lancet Commission on Global Surgery estimates that an additional 143 million sur-gical procedures are needed each year in LMICs to prevent death and disability caused by lack of care.16The potential benefits of surgical care for economic pro-ductivity are astounding. Considering that the annual eco-nomic loss from road traffic injuries alone exceeds U.S. $500 billion globally, a panel of expert economists at the Copenhagen Consensus of 2012, including four Nobel prize lau-reates, prioritized strengthening surgical capacity as the eighth most cost-effective investment for addressing the world’s most pressing problems. The Consensus reconvened in 2015, syn-chronizing their recommendations to the United Nations’ Sus-tainable Development

1	cost-effective investment for addressing the world’s most pressing problems. The Consensus reconvened in 2015, syn-chronizing their recommendations to the United Nations’ Sus-tainable Development Goals, and reaffirmed that surgery-related initiatives (i.e., circumcision and skilled obstetrical support), offer the best “value-for-money” in terms of alleviating world poverty.17-19 The Lancet Commission on Global Surgery, a research and advisory working group with contributors from 110 nations, echoed these sentiments. Surgery should be viewed as an investment rather than a cost.16,20Much of the economic modeling and strengthening of political will related to surgical and anesthesia care has been carried out by collaborative groups and consortia including aca-demic, nongovernmental, and other organizations. The efforts of these groups truly coalesced in 2015, when several major consensus statements from governmental organizations, such as the World Bank and the World Health Assembly,

1	organizations. The efforts of these groups truly coalesced in 2015, when several major consensus statements from governmental organizations, such as the World Bank and the World Health Assembly, recognized the importance of surgery in public health for the first time. 3$100$101-400$401-1000> $100012,00010,00080006000400020000Number of procedures/100,000 populationUS dollars/person healthcare/yearFigure 49-1. Worldwide distribution of surgical procedures. (Data from Weiser TG, Regenbogen SE, Thompson KD, et al. An estima-tion of the global volume of surgery: a modelling strategy based on available data, Lancet. 2008 Jul 12;372(9633):139-144.)Key Points1 There are five major forces reshaping priorities and strate-gies for the globalization of surgical care:a. The epidemiologic transition of diseasesb. The mobile nature of the world’s populationsc. Ubiquitous information accessd. A revolution for equity and human rightse. Recognition of the cost-effectiveness of surgical care for

1	of diseasesb. The mobile nature of the world’s populationsc. Ubiquitous information accessd. A revolution for equity and human rightse. Recognition of the cost-effectiveness of surgical care for treatment and prevention of disease2 The burden of disease is greatest in areas where human resources—physicians, nurses, pharmacists, and other healthcare workers—are the least.3 Surgery should be viewed as an investment rather than a cost.4 The key components of the global surgery ecosystem include technology, education, community, healthcare, business, and multidisciplinary engagement between a variety of disciplines.5 Understanding and addressing the necessary communica-tion, energy, and transportation technologies along with the underlying cultural context represent the foundation critical to implementing sustainable infrastructure for appropriate surgical care.6 There has been a significant shift from communicable, maternal, neonatal, and nutritional causes of disease to noncommunicable

1	implementing sustainable infrastructure for appropriate surgical care.6 There has been a significant shift from communicable, maternal, neonatal, and nutritional causes of disease to noncommunicable causes, many of which require surgi-cal care.7 Patients and their communities in lowand middle-income countries (LMICs) bear a much greater share of the burden of cancer than high-income countries (HICs).8 Globally, trauma has become a leading cause of death and disability; 90% of trauma deaths occur in LMICs.9 Essential surgical services should be integrated into com-prehensive health care delivery, with the potential to avert 1.5 million deaths per year in LMICs.10 Surgery is gaining an increasingly recognized role for improving public health, having a role in prevention as well as treatment.11 The cost-effectiveness of surgical care has been demon-strated, and its value as a public health investment is increasingly understood by policymakers.12 Developing capabilities for surgical care

1	cost-effectiveness of surgical care has been demon-strated, and its value as a public health investment is increasingly understood by policymakers.12 Developing capabilities for surgical care has the ability to promote system-strengthening in resource-poor countries and to mitigate migration of health professionals at all levels.13 Academic global surgery provides a unique environment to study health systems, identify solutions and implement them collaboratively, fulfilling many institutions’ mis-sions to strengthen multidisciplinary training, advocacy, and research.14 Surgical innovations that bring value by balancing cost with quality designed for challenging energy environ-ments will foster equity in surgical care for LMICs.Brunicardi_Ch49_p2077-p2112.indd 207813/02/19 5:53 PM 2079GLOBAL SURGERYCHAPTER 49The third edition of World Bank’s Disease Control Priorities (DCP3) and the World Health Assembly’s Resolution 68.15 both specifically discuss the vital nature of surgical care

1	SURGERYCHAPTER 49The third edition of World Bank’s Disease Control Priorities (DCP3) and the World Health Assembly’s Resolution 68.15 both specifically discuss the vital nature of surgical care as part of any health system.7,21 Such resolutions represent a sea change in terms of how the global policy community views surgical care for LMICs and, indeed, the entire world.This chapter examines the ongoing need to expand and strengthen surgical care globally, explores some of the signifi-cant challenges of global surgery, and presents potential guid-ing concepts along with examples of successful strategies for sustainable surgical development.DEFINING GLOBAL SURGERYGlobal Surgery EcosystemTo understand how surgery fits into healthcare systems and to understand its unique needs, it is helpful to consider global surgery as an ecosystem. The emerging field of global surgery considers surgical care to be a fundamental component of global health. As a system with both local and international

1	to consider global surgery as an ecosystem. The emerging field of global surgery considers surgical care to be a fundamental component of global health. As a system with both local and international scope, global surgery encompasses not just the medical and technical aspects of surgical care, but also the societal and environmental context in which surgery is per-formed. Global surgery also refers to a worldwide lens through which we view challenges collaboratively; thus, global sur-geons may focus on resource-limited areas where needs are profound, but the ultimate goal should be to make surgical care equitable, accessible, and affordable for every human being.22 Surgery as an ecosystem considers the diverse but interrelated systems that must be functional for quality surgical care to be delivered. Only part of these systems falls within the tradi-tional training of surgeons. Yet, modern surgical care requires these systems to work in a coordinated fashion to support three priorities

1	delivered. Only part of these systems falls within the tradi-tional training of surgeons. Yet, modern surgical care requires these systems to work in a coordinated fashion to support three priorities critical for expanding surgery globally—accessibility, affordability, and innovation (Fig. 49-4). Global surgery is a way to consider a “systems-based practice” beyond a single hospital or community, for the benefit of people worldwide. Many interrelated components of this surgical ecosystem orig-inate outside the hospital.Disparities in surgical care have geographical, socioeco-nomic, and cultural components. Most people who live in major cities in the northern and western hemispheres take for granted a functioning energy grid. The development of energy beyond major cities has enabled wealthier communities to imagine, and indeed, to expect healthcare to be available at all times and affordable. Yet, a lack of reliable energy sources is a major limiting factor. Communication and

1	wealthier communities to imagine, and indeed, to expect healthcare to be available at all times and affordable. Yet, a lack of reliable energy sources is a major limiting factor. Communication and transportation technologies, for example, the mobile phone and air and ground travel, have dramatically progressed in highand middle-income countries but are still rudimentary in poor countries. Many of the current disparities in health care, particularly surgical care, are due to the lack of penetration of these technologies. Under-standing and addressing the necessary communication, energy, and transportation deficits as well as the underlying cultural nuances are necessary to support the sustainable development of surgical care.Electricity is necessary for all modern surgery. Anesthe-sia monitoring, operating room lighting, cautery, suction, and patient warming devices all require sources of electricity that are stable, without huge electrical surges. Only in the last 50 years or so could

1	operating room lighting, cautery, suction, and patient warming devices all require sources of electricity that are stable, without huge electrical surges. Only in the last 50 years or so could stable electricity be expected in most wealthy cities. However, in rural areas of even wealthy countries, electricity remains unpredictable (Fig. 49-5).23In poorer countries, the cost and availability of electric-ity is frequently the limiting factor for more advanced diagnos-tic and therapeutic technology—from laboratories that require refrigeration to radiology in all of its various branches. Modern design for surgical devices has, for the most part, not taken into account the wide range of energy environments where surgery is practiced. Fragile instruments and monitors that cannot survive the rigors of the real working environment limit the types of surgery that can be provided.4535302520151050Percent of global disease burdenPercent of global

1	and monitors that cannot survive the rigors of the real working environment limit the types of surgery that can be provided.4535302520151050Percent of global disease burdenPercent of global workforceAmericasEuropeWesternPacificSouth-EastAsiaAfricaEasternMediterranean* Even with grants and loans from abroad.054045303520251015Africa suffers from 24% of the global burden of disease but has access to only 3% of health workers and less than 1% of the world's financial resources.*Figure 49-2. Distribution of healthcare workers by burden of disease in WHO regions. (Reproduced with permission from Intermountain Healthcare.)Brunicardi_Ch49_p2077-p2112.indd 207913/02/19 5:53 PM 2080SPECIFIC CONSIDERATIONSPART IIComponents of the Global Surgery Ecosystem. Improve-ments in energy, transportation, and communication are criti-cal to support the growth of surgical care.9 Building capacity for surgical care requires interaction between the various com-ponents that create a functioning,

1	and communication are criti-cal to support the growth of surgical care.9 Building capacity for surgical care requires interaction between the various com-ponents that create a functioning, sustainable system. When surgeons think of surgery, they usually think in terms of science and hands-on technical expertise. However, global surgery requires a broader understanding of systems in other disciplines. Surgeons must work collaboratively with engineers and busi-ness leaders to develop technology that can function in lower 0–1717–3434–50Physicians (per 10,000 population)2005–201050–67200,000,000–600,000,0000–200,000,000600,000,000–1,400,000,000Population20120.48%–1.96%1.96%–3.45%3.45%–4.93%Population growth rate2012–1.01%– –0.48%Figure 49-3. Number of physicians, world populations, and world population growth rates. (Reproduced with permission from World Health Organization, 2018 (Density of Physicians [total number per 1000 population]); 2013 World Population Data Sheet Interactive Map;

1	population growth rates. (Reproduced with permission from World Health Organization, 2018 (Density of Physicians [total number per 1000 population]); 2013 World Population Data Sheet Interactive Map; World Bank, population growth (annual %) map.)Brunicardi_Ch49_p2077-p2112.indd 208013/02/19 5:53 PM 2081GLOBAL SURGERYCHAPTER 49resource environments. These innovations can provide a source of economic growth for the community, which in turn supports better health care (Fig. 49-6).No sustainable surgical system in the modern age can function without specialists in bioengineering, sterile process, supply chain, hospital safety, and waste management. These often unappreciated colleagues make possible the daily practice of surgery. Similarly, specialists in anesthesia, nursing, and the diagnostic specialties of radiology, pathology, and laboratory services are fundamental to a fully functional surgical service.Human ResourcesPrimary care physicians, nurses, midwives, or advanced care

1	specialties of radiology, pathology, and laboratory services are fundamental to a fully functional surgical service.Human ResourcesPrimary care physicians, nurses, midwives, or advanced care practitioners (ACPs) provide much of the basic surgical and anesthetic care in LMICs. Where regulations allow, “task sharing,” or training ACPs to deliver surgery and anesthesia services previously allowed only under the purview of fully trained specialists, can provide expanded access to care.24-26 Non-MD practitioners, known as assistant medical officers (AMOs) or “tecnicos de cirurgia” in Mozambique, often have extensive operative experience, including obstetrical care, and are the pri-mary surgical providers in some regions.27-29 Task sharing with ACPs also occurs in the United States and other countries where they fill a need otherwise unmet by specialists even in major tertiary care centers.30 However, concerns about the quality of care, lack of adequate supervision, and the effect on

1	countries where they fill a need otherwise unmet by specialists even in major tertiary care centers.30 However, concerns about the quality of care, lack of adequate supervision, and the effect on prestige and professional development for specialists and ACPs, continue to be topics for debate.31,32Migration of practitioners to economically and culturally favorable locales is universal and not restricted to low-resource countries.33,34 However, the net impact on poor countries is greater. In a 2004 study, more than 23% of U.S. physicians received their medical training from other countries; of these 64% were from low-income countries.35 Using 2013 data, another study showed annual emigration rates of sub-Saharan physicians to the United States are increasing, despite a World Health Organization Global Code of Practice in 2010 aimed at LMIC workforce retention.36 Investments in training greater numbers of doctors in these countries, including surgical spe-cialists, have been only

1	Global Code of Practice in 2010 aimed at LMIC workforce retention.36 Investments in training greater numbers of doctors in these countries, including surgical spe-cialists, have been only partially successful in meeting demand in poor countries. Until economic conditions improve or oppor-tunities for professional development increase, and incentives enticing migration of health care workers to high-income countries abate, it is unlikely that the most skilled practitio-ners will remain in resource-poor areas beyond their immediate obligations.37-41Burden of Surgical DiseaseEpidemiologic Transition of Disease. The population on Earth currently stands at more than 7 billion. While the rate of growth has slowed in recent years, projections estimate that AccessibilityAffordabilityInnovationGLOBALSURGERYFigure 49-4. Global surgery priorities. (Reproduced with permission from University of Utah Center for Global Surgery and Intermountain Healthcare.)Figure 49-5. Map of world electrification.

1	49-4. Global surgery priorities. (Reproduced with permission from University of Utah Center for Global Surgery and Intermountain Healthcare.)Figure 49-5. Map of world electrification. (Reproduced with permission from NASA, Visible Earth, Available at: http://visibleearth.nasa.gov/view.php?id=79765.)Brunicardi_Ch49_p2077-p2112.indd 208113/02/19 5:53 PM 2082SPECIFIC CONSIDERATIONSPART IIthe population will continue to grow to 9 billion by 2050.42 Population characteristics are changing rapidly. According to United Nations’ estimates, the entire world is aging even in low-income countries, and by 2050, 2 billion people will be over the age of 60. Currently, Asia is home to 55% of the world’s population over the age of 60.43 Just before the year 2020, the percentage of the world’s population over age 65 years is predicted to surpass the percentage of children under age 5 years, on an unprecedented reversal of trajectories for both age demographics. While this represents a victory for

1	over age 65 years is predicted to surpass the percentage of children under age 5 years, on an unprecedented reversal of trajectories for both age demographics. While this represents a victory for infectious disease control, the dramatic increase in longevity will present new challenges in terms of treating noncommu-nicable disease in the older adult population.44 At the same time, Sub-Saharan Africa’s population is experiencing a much different trend: a current “baby boom” will lead the region to quadruple its population, from 960 million to 4 billion, by the year 2100.45Until recently, infectious diseases dominated public health strategy. Now with major scourges like polio isolated to rela-tively small regions of the world, and HIV and malaria decreas-ing in their relative impact worldwide, chronic diseases and their complications, as well as the effects of aging, are gaining dominance in health care needs. Many of these chronic diseases are best approached by surgery.The lack of

1	chronic diseases and their complications, as well as the effects of aging, are gaining dominance in health care needs. Many of these chronic diseases are best approached by surgery.The lack of metrics and paucity of data identifying the unmet burden of surgical need in many countries have been obstacles facing global surgery initiatives. The 2010 Global Burden of Disease Study was the first worldwide comprehensive burden of disease evaluation since the initial 1990 epidemiologic study. Using the disability-adjusted life year (DALY), a metric that captures both premature mortality and the prevalence and severity of illnesses, disease burdens were calculated for 291 causes in 21 regions of the world (including 187 countries) for 1990, 2005, and 2010 to enable identification of significant trends over time.46 While the global DALYs remained stable from 1990 to 2010, the study identified a significant shift from communicable, maternal, neonatal, and nutritional causes of disease to

1	trends over time.46 While the global DALYs remained stable from 1990 to 2010, the study identified a significant shift from communicable, maternal, neonatal, and nutritional causes of disease to noncommuni-cable causes (Fig. 49-7).47In 2015, the previous estimate by the second edition of Disease Control Priorities in 2006 of an 11% global surgical dis-ease burden was updated to 30%, obtained from provider-based survey data from the Lancet Commission.1,10 Using country-wide population surveys (the Surgeons OverSeas Assessment of Surgical Need Survey [SOSAS]) in Sierra Leone, Rwanda, and Nepal, the overall presence of surgically treatable condi-tions was 11.2%, with 25.6% of deaths potentially avoidable had surgical care been available. Applying these percentages to the 48 low-income countries, as defined by the World Bank, suggests that there are 288.2 million people currently living with surgically treatable conditions; providing improved access to surgical care could prevent 5.6

1	as defined by the World Bank, suggests that there are 288.2 million people currently living with surgically treatable conditions; providing improved access to surgical care could prevent 5.6 million deaths per year.48-50 Untreated acute and chronic surgical conditions represent a sig-nificant unmet burden of disease that has major impact on the economies of these nations.7,16,51Cancer. Patients and their communities in LMICs bear a much greater share of the burden of cancer than HICs. The dramatic increase in the proportion of reported cancer cases in LMICs is a result of population growth, aging populations, and decreased mortality from infectious diseases. In 1970, only 15% of newly reported cancer cases worldwide were from the developing world; by 2008, this proportion rose dramatically to 58% and is expected to grow to 70% by 2030.52 Since 2013, the second leading cause of death world-wide has been cancer, and an estimated 20% of all global surgery is now cancer-related.53

1	to 58% and is expected to grow to 70% by 2030.52 Since 2013, the second leading cause of death world-wide has been cancer, and an estimated 20% of all global surgery is now cancer-related.53 Previously thought to be a 67ElectricityLaundrySupply chainKnowledgeCultureHealthcareFamily supportGeographyPatientRoad/travel infrastructurePre-hospital careSpiritualityExpectationsLaboratory servicesHospital safetyAdministrationBusinessHealthcarePublic healthInsurersRegulatory agenciesTechnologyPublic policyEducationSURGICALECOSYSTEMBioengineeringWaste managementSterile processingAnesthesiologyRadiologyNursingPharmacyPathologyRESOURCESCOMMUNITYMULTIDISCIPLINARY ENGAGEMENTSURGICAL CARE INFRASTRUCTUREValue-driven outcomesPatient-centered careFigure 49-6. The global surgery ecosystem requires broad integration of many fields in a multidisciplinary context. (Reproduced with permission from Intermountain Healthcare.)Brunicardi_Ch49_p2077-p2112.indd 208213/02/19 5:53 PM 2083GLOBAL SURGERYCHAPTER

1	of many fields in a multidisciplinary context. (Reproduced with permission from Intermountain Healthcare.)Brunicardi_Ch49_p2077-p2112.indd 208213/02/19 5:53 PM 2083GLOBAL SURGERYCHAPTER 49disease almost exclusive to high-income countries, nearly two-thirds of the 7.6 million cancer deaths worldwide occur in LMICs. Mortality from cancer correlates inversely with a country’s economy for certain treatable cancers, including breast, testicular, and cervical cancer—LMICs have higher case fatality rates than HICs (Fig. 49-8).52,54For example, breast cancer case fatality rates illustrate the great disparity in outcomes between regions. Case fatality rates in East Africa reach an unacceptable 59% compared to 19% in the United States.54 In LMICs, patients have very lim-ited access to screening. They present for care with much later stages of cancer. In Haiti, after the great earthquake in 2010, with its initial onslaught of orthopedic injuries, many aid orga-nizations found themselves

1	They present for care with much later stages of cancer. In Haiti, after the great earthquake in 2010, with its initial onslaught of orthopedic injuries, many aid orga-nizations found themselves faced with the unmet underlying burden of disease, including late-stage breast cancer and other tumors (Fig. 49-9). The DCP3 has devoted an entire chapter to cancer screening in LMICs, emphasizing the importance of proper infrastructure for screening and treatment, as well as considering cost-effectiveness and ethical concerns related to screening and subsequent treatment of detected cancers.55 The number and quality of training programs in surgical oncology is also inversely related to a country’s income, leaving LMICs with few adequately trained providers. Collaborative training programs between HIC and LMIC centers, as well as tele-teaching and mobile consultation, may address this shortage in a relatively low-cost, high-impact way.56Trauma. Trauma has become a leading cause of death (5.8

1	HIC and LMIC centers, as well as tele-teaching and mobile consultation, may address this shortage in a relatively low-cost, high-impact way.56Trauma. Trauma has become a leading cause of death (5.8 million people per year) and disability around the world; 90% of trauma deaths occur in LMICs.57 Approximately 32% more people die as a result of injuries than from malaria, tuberculosis, and HIV/AIDS combined, representing 10% of the world’s deaths (Fig. 49-10).58,59 The major causes of death from injuries are road traffic accidents (RTAs), suicides, homicides, falls, drownings, and burns; in every category except burns, almost twice as many men die compared to women.60847354354101119902010% Worldwide DALYsCommunicable, maternal, neonatal, nutritionalNoncommunicableInjuriesFigure 49-7. Shift in disease burden 1990–2010. (Reproduced with permission from Intermountain Healthcare.)ThyroidHighHigh-middleLow-middleLow0.80.60.40.20.0Ratio of mortality to incidenceIncome

1	49-7. Shift in disease burden 1990–2010. (Reproduced with permission from Intermountain Healthcare.)ThyroidHighHigh-middleLow-middleLow0.80.60.40.20.0Ratio of mortality to incidenceIncome classBreastCervicalTesticularProstateColorectalFigure 49-8. Ratio of mortality to incidence by solid tumor type and country income (2008). (Reproduced with permission from Intermountain Healthcare.)Brunicardi_Ch49_p2077-p2112.indd 208313/02/19 5:53 PM 2084SPECIFIC CONSIDERATIONSPART IIOver 1.25 million people die from RTAs, causing LMICs to lose 3% of their GDP; 50 million more people incur nonfatal injuries, many with resulting lifelong disabilities.61 Globally, RTAs are the main cause of death for young people between the ages of 15 and 29. Forty-nine percent of all traffic deaths are among pedestrians, cyclists, and motorcycles.62 In the United States, a patient presenting with an injury in a rural community has a higher mortality than those from an urban setting.63 This disparity is much more

1	cyclists, and motorcycles.62 In the United States, a patient presenting with an injury in a rural community has a higher mortality than those from an urban setting.63 This disparity is much more pronounced in eco-nomically disadvantaged societies, where seriously injured patients from road traffic accidents are twice as likely to die compared to similarly injured patients in a high-income set-ting (Fig. 49-11).58,64 Additionally, death is much more likely to occur in the prehospital settings for injured patients from low-income countries. The lack of integrated communication and emergency transportation systems contribute to prehospital risk, while the lack of infrastructure, supplies, and personnel contribute to inhospital mortality.The number of deaths from RTAs has remained the same between 2007 and 2013. The predicted increase in mortality from RTAs, expected from the increase in population and global motorization, did not materialize, suggesting that interventions to improve

1	between 2007 and 2013. The predicted increase in mortality from RTAs, expected from the increase in population and global motorization, did not materialize, suggesting that interventions to improve global road safety (i.e., The Decade of Action for Road Safety 2011–2020) may be having some success in pre-venting deaths from RTAs.62Burns. The World Health Organization estimates that 265,000 people die of burn injuries each year, mostly (95%) from LMICs; the vast majority never present for medical care.65 Scalds and electrical burns represent another significant source of death and disability. Women and children in LMICs are most likely to be burned in domestic kitchens; men are more likely to be burned in the workplace. The economic and social impact from long hospitalizations and from the resulting disfigurement provides a significant negative stigma causing ostracism and rejection.Of all the forms of trauma worldwide, burns are the only type that predominantly afflict women and

1	the resulting disfigurement provides a significant negative stigma causing ostracism and rejection.Of all the forms of trauma worldwide, burns are the only type that predominantly afflict women and children. Southeast Asia accounts for 27% of burn-related deaths worldwide; 70% of people dying from burns in this region are women.66 Cooking on wood, charcoal, or low kerosene stoves also puts children at risk, particularly from scalding (Fig. 49-12). Small children in the WHO African region have triple the number of burn deaths as children worldwide. Con-trast this with the United States, where more burns and burn deaths affect men.People living in rural areas suffer disproportionately because there are fewer facilities capable of managing the acute and chronic aspects of burns and because the population is gen-erally poorer. Surgical grafting and management of contractures is often best done in specialized burn centers, but these are rare in LMICs. Telemedicine has been shown to be

1	the population is gen-erally poorer. Surgical grafting and management of contractures is often best done in specialized burn centers, but these are rare in LMICs. Telemedicine has been shown to be effective in man-aging burns and preventing complications, and now, in the era of high resolution mobile phones, it can effectively diagnose and triage many burn patients appropriately.67 Telemedicine can also be useful in providing much-needed education of rural providers in basic burn care (Box: Telemedicine and Tele-education at the University of Utah).6801234567Deaths per year (millions)InjuryHIV/AIDS, TB, and malariaInjuries and violence:the scale of the problemFigure 49-10. Injuries and violence: the scale of the problem. (Reproduced with permission from World Health Organization, Geneva. Injuries and violence: the facts. http://www.who.int/ violence_injury_prevention/key_facts/VIP_key_fact_1.pdf.)Figure 49-9. Underlying unmet cancer burden in Haiti (2010). (Reproduced with permission

1	Injuries and violence: the facts. http://www.who.int/ violence_injury_prevention/key_facts/VIP_key_fact_1.pdf.)Figure 49-9. Underlying unmet cancer burden in Haiti (2010). (Reproduced with permission from Intermountain Healthcare. Photo contributor: R. Dirk Noyes, MD.)Brunicardi_Ch49_p2077-p2112.indd 208413/02/19 5:54 PM 2085GLOBAL SURGERYCHAPTER 49Figure 49-11. Change in traffic fatality risk (deaths per 10,000 persons, 1975–1998). (Reproduced with permission from Intermountain Healthcare.)Telemedicine and Tele-education at the University of UtahThe University of Utah’s Burn Center has utilized Project ECHO (Extension for Community Healthcare Outcomes), an HIPAA-compliant tele-education platform developed at the University of New Mexico. Project ECHO provides live, free, interactive educational materials to rural physicians, nurses, and EMTs in eight surrounding states.69 This model has been expanded internationally through the University of New Mexico to 21 countries, with the

1	educational materials to rural physicians, nurses, and EMTs in eight surrounding states.69 This model has been expanded internationally through the University of New Mexico to 21 countries, with the potential to drastically improve the knowledge base of providers in many resource-limited settings.70Direct patient care has also been achieved at the University of Utah’s Burn Center, through their Telemedicine outreach program. “TeleBurn” currently provides approximately 400 video consultations per year, serving 80 sites in five surrounding states that lack specialized burn care. For a region like the Intermountain West, where travel can be limited by inclement weather and long distances, the TeleBurn program provides better access to specialty care, at lower costs to patients.71Figure 49-12. Domestic kitchen: risk factor for burns in women and children in LMICs. (Used with permission from James H. Kenney, Jr.)CanadaAustriaFranceIndiaChinaUnited

1	at lower costs to patients.71Figure 49-12. Domestic kitchen: risk factor for burns in women and children in LMICs. (Used with permission from James H. Kenney, Jr.)CanadaAustriaFranceIndiaChinaUnited StatesColumbiaBotswana400350250200150100500–50–100High income countriesLow income countriesHigh income countriesLow income countriesPercentage changeBrunicardi_Ch49_p2077-p2112.indd 208513/02/19 5:54 PM 2086SPECIFIC CONSIDERATIONSPART IISTRATEGIES FOR DEVELOPMENTEssential Surgery: Current and Evolving ConceptsDr. Jim Yong Kim, President of the World Bank, aptly stated that surgery is an “indivisible, indispensable part of health care.”72 The wisdom of this statement has been supported by the findings of two landmark publications in 2015: Disease Control Priorities, third edition (DCP3), and the Lancet Com-mission on Global Surgery 2030 (LCGS).7,16 According to the DCP3, “the provision of essential surgical procedures would avert 1.5 million deaths a year or 6.7% of all avertable deaths

1	the Lancet Com-mission on Global Surgery 2030 (LCGS).7,16 According to the DCP3, “the provision of essential surgical procedures would avert 1.5 million deaths a year or 6.7% of all avertable deaths in LMICs,” and according to the Lancet Commission, 5 billion people do not have access to safe, affordable surgical and anes-thesia care when needed. Taken together, these and other find-ings suggest that without the provision of accessible, affordable essential surgical care in all LMICs, the lofty goal of another Lancet Commission report, Global Health 2035: A World Con-verging Within a Generation, would be unachievable.73 This earlier Lancet Commission believes that, with adequate invest-ment in global health, all countries could reduce their infectious, maternal, and childhood mortality rates down to those currently seen in the best-performing middle-income countries (e.g., the 4C countries: Chile, China, Costa Rica, and Cuba) within a gen-eration by 2035. They also make the

1	rates down to those currently seen in the best-performing middle-income countries (e.g., the 4C countries: Chile, China, Costa Rica, and Cuba) within a gen-eration by 2035. They also make the interesting observation that the LMICs can use their own resources for much of the funding needed.The critical role of essential surgical and anesthesia ser-vices in global health, in general, and in saving lives and disabil-ities in LMICs, in particular, has been established by fact-based evidence and analysis provided by the two landmark publica-tions of 2015, DCP3 and LCGS. Table 49-1 summarizes the key findings and recommendations of the two publications.The DCP3 adopted a working definition of essential sur-gical conditions as those that (a) are primarily or extensively treated by surgery; (b) have a large health burden; and (c) can be successfully treated by a surgical procedure that is cost-effective and feasible to promote globally. Using this def-inition, the DCP3 identified 44 essential

1	a large health burden; and (c) can be successfully treated by a surgical procedure that is cost-effective and feasible to promote globally. Using this def-inition, the DCP3 identified 44 essential procedures, most of which can be performed in first-level hospitals (Table 49-2).74 The first-level (district) hospital is the appropriate platform to provide essential surgical service. These procedures rank among the most cost-effective of all interventions and include those that treat injuries, obstetric complications (including fistulas), abdominal emergencies, cataracts, and congenital anomalies.The LCGS Report, based on extensive research and analy-sis of factual evidence, provides recommendations to improve access to safe, affordable anesthesia and surgical care in LMICs. Essential surgical services should be integrated into a compre-hensive platform of healthcare delivery. At the core of delivery of essential surgery is the first (district) hospital, which must be capable of

1	surgical services should be integrated into a compre-hensive platform of healthcare delivery. At the core of delivery of essential surgery is the first (district) hospital, which must be capable of delivering three bellwether essential surgery pro-cedures (hysterectomy, laparotomy, and treatment of an open fracture). A hospital that can provide these three procedures safely is presumed to have the necessary expertise in general and orthopedic surgery, obstetrics, and anesthesia to perform all essential surgical procedures.The cost of untreated surgical conditions is huge and, until now, not recognized. At the present time, some 33 million individuals face catastrophic health expenditure for surgical and anesthesia care in LMICs. The LCGS estimates that it would cost U.S. $420 billion to scale up the surgical workforce 9Table 49-1Key findings and recommendations from the Disease Control Priorities (DCP3) and the Lancet Commission for Global Surgery (LCGS)DCP3LCGSProvision of essential

1	up the surgical workforce 9Table 49-1Key findings and recommendations from the Disease Control Priorities (DCP3) and the Lancet Commission for Global Surgery (LCGS)DCP3LCGSProvision of essential surgery in LMICs would prevent 1.5 million deaths, or 6.7% of all avertable deaths5 billion people lack access to safe, affordable surgical and anesthesia care when neededEssential surgical procedures rank among the most cost-effective of all health interventions143 million more operations are needed in LMICs, where only 6% of all worldwide procedures are now doneEffective and affordable measures (such as task-sharing) increase access to surgical care33 million face catastrophic health expenditure from surgery and anesthesia care each yearInvestments must be made to expand capacity buildingWithout urgent investment, LMICs will lose US $12.3 trillion in economic productivity between 2015 and 2030Substantial disparities exist between countries in safety of surgical and anesthetic care. Feasible

1	urgent investment, LMICs will lose US $12.3 trillion in economic productivity between 2015 and 2030Substantial disparities exist between countries in safety of surgical and anesthetic care. Feasible and affordable measures (e.g., surgical safety checklist) improve safety and qualitySurgery is an indivisible, indispensable part of health care. Surgical care should be part of the National Health Care System, and should be “available, accessible, safe, timely, and affordable.”Universal coverage of essential surgery should be publicly financed early on the path to universal health coverage LMIC = Low and Middle Income CountryTable 49-2Essential surgical procedures that can be performed in first level (District) hospitals (DCP3)Obstetric Complications Severe postpartum hemorrhage, obstructed labor, prolonged labor, eclampsia, prolapsed cord, fetal distress, tubal pregnancy, postabortion endometritis/myometritis, postabortion sepsis, intrauterine fetal deathTrauma and Violence Major limb

1	labor, prolonged labor, eclampsia, prolapsed cord, fetal distress, tubal pregnancy, postabortion endometritis/myometritis, postabortion sepsis, intrauterine fetal deathTrauma and Violence Major limb fracture/injury, joint dislocation, major soft tissue injury, pneumo/hemothorax, ruptured spleenAcute Surgical Emergencies Strangulated hernia, intestinal obstruction, intestinal perforation, appendicitis, liver abscess, major wound infection, osteomyelitis/septic arthritisNonacute Surgical Conditions Congenital hernia, hernia, breast cancer, chronic osteomyelitis, hydrocele, urethral stricture, prostatic hypertrophy, cataract, eye injuryBrunicardi_Ch49_p2077-p2112.indd 208613/02/19 5:54 PM 2087GLOBAL SURGERYCHAPTER 49to have 20 surgical, anesthetic, and obstetric providers (SAOPs) per 100,000 population in LMICs by 2030. This figure must be compared to the U.S. $20.7 trillion loss in global economy that surgical conditions would be responsible for.Recent studies have shown that

1	100,000 population in LMICs by 2030. This figure must be compared to the U.S. $20.7 trillion loss in global economy that surgical conditions would be responsible for.Recent studies have shown that essential surgical condi-tions account for about 18% of the global burden of disease. Investment in essential surgical services is critical and should be done early in the path towards universal health coverage (UHC). The barriers to essential surgical services in LMICs are formi-dable. The shortage in surgical workforce is huge, and it is clear that the deficit cannot be satisfactorily addressed without task sharing. Infrastructure deficits (clinics, hospitals, equipment, drugs, blood banks, etc) are equally enormous. The first (district) hospital is the important platform for delivery of essential surgical services. The DCP3 estimates that it would cost U.S. $43 million annually of additional spending to provide universal coverage of essential surgery applicable to first-level hospitals

1	surgical services. The DCP3 estimates that it would cost U.S. $43 million annually of additional spending to provide universal coverage of essential surgery applicable to first-level hospitals worldwide.Outreach and EngagementMany models for outreach and engagement have had a positive impact on the accessibility of surgery. Organizations participat-ing in outreach are guided by a wide range of motivations and resources (Fig. 49-13). Some organizations are purely humani-tarian and service oriented; others are primarily educational. Some even use the promise of healthcare to advance political, religious, or personal agendas.Many patients have benefited from the multitude of service-oriented volunteer “missions” providing much needed surgical care that would otherwise have been unavailable. While volun-teerism and medical missions provide needed clinical surgical care for underserved populations, they may not be a sustainable solution to long-term manpower shortages for health.75

1	While volun-teerism and medical missions provide needed clinical surgical care for underserved populations, they may not be a sustainable solution to long-term manpower shortages for health.75 Com-prehensive initiatives are necessary to engage local healthcare professionals and organizations, governments, and academic institutions to build sustainable capacity.76Charitable Surgical Delivery Platforms. A significant bur-den of surgical disease is addressed through charitable organi-zations. The DCP3 divides these charitable surgical delivery platforms into two types: temporary delivery platforms and spe-cialty surgical hospitals (Table 49-3).77Short-term charitable surgical platforms bring entire surgi-cal teams along with equipment and supplies needed to operate in local facilities for a short period of time. Local physicians provide the majority of follow-up care.78-80 Self-contained plat-forms bring the entire surgical infrastructure (fully functional operating rooms, postoperative

1	short period of time. Local physicians provide the majority of follow-up care.78-80 Self-contained plat-forms bring the entire surgical infrastructure (fully functional operating rooms, postoperative recovery capability) through various modes of transportation: airplanes, ships, trucks, and buses. These self-contained platforms tend to stay in-country longer, may still provide short-term care, and tend not to leave behind any physical structure.78,80,81Little information exists on outcomes and cost-effectiveness of these temporary surgical platforms. Where no other services Politicians/AdministratorsResourcesFinancialHumanPoliticalMotivationsHumanitarianEducationalAcademicPoliticalOtherCataractHerniaVesico-vaginalfistulaLaparoscopicsurgeryEducatorsIndividualpeopleHealthcareprovidersCountryProvinceUrban vs.RuralDisease(technique specific)GeographicTargeted audienceGlobalsurgery initiativesPoliticians/administratorsFigure 49-13. Global surgery initiatives. (Reproduced with permission

1	vs.RuralDisease(technique specific)GeographicTargeted audienceGlobalsurgery initiativesPoliticians/administratorsFigure 49-13. Global surgery initiatives. (Reproduced with permission from Intermountain Healthcare.)Brunicardi_Ch49_p2077-p2112.indd 208713/02/19 5:54 PM 2088SPECIFIC CONSIDERATIONSPART IIexist, they may provide needed services. However, some of these charitable organizations report higher complications rates in lower-resource settings, which seem to increase even more with complex procedures. Some of these platforms include edu-cation for local care providers along with clinical care. While some question their ability to sustainably train local surgical teams, one charitable partnership with short-term, concentrated surgical training trips over 9 years documented a countrywide transition from open cholecystectomy to laparoscopic cholecys-tectomy in Mongolia.82Specialty surgical hospitals establish entire hospitals or facilities within existing hospitals. Some target

1	transition from open cholecystectomy to laparoscopic cholecys-tectomy in Mongolia.82Specialty surgical hospitals establish entire hospitals or facilities within existing hospitals. Some target specific diseases (Addis Ababa Fistula Hospital) while others provide a wide range of surgical and medical services (Pan-African Academy of Christian Surgeons [PAACS] mission hospitals); many are supported through partnerships with various charitable and gov-ernmental organizations.77International OrganizationsUnited Nations. Committed to maintaining international peace, developing friendly relations between nations, and promoting better standards of living (conquering hunger, dis-ease, and illiteracy) and human rights, representatives from 51 nations in 1945 signed the United Nations (UN) Charter at the United Nations Conference on International Organization in San Francisco, California.83 There are now 193 member states.84 The UN promotes a social justice agenda advocating for world-wide

1	at the United Nations Conference on International Organization in San Francisco, California.83 There are now 193 member states.84 The UN promotes a social justice agenda advocating for world-wide health, engagement of philanthropies, and civil society in global health initiatives, and it supports the sustainable develop-ment goals (SDGs).85Sustainable Development Goals. In September 2000, the UN led a worldwide, organized effort to set benchmarks for social, economic, and environmental development. Leaders from 189 countries agreed on eight specific “millennium devel-opment goals” (MDGs), spanning poverty, mortality, education, sustainability, and development.86The MDGs created a framework for improvement that some criticized as unattainable; nevertheless nearly 1 billion people were lifted out of extreme poverty, and primary education for girls made measurable improvements.87 Still, many challenges, including some related to lack of surgical care, remained. In 2015, the UN General

1	out of extreme poverty, and primary education for girls made measurable improvements.87 Still, many challenges, including some related to lack of surgical care, remained. In 2015, the UN General Assembly reconvened to raise the bar yet again, in what was declared a “supremely ambitious and transforma-tive vision.”88 Eight MDGs became 17 SDGs with 169 specific targets, to be achieved by 2030 (Table 49-4).85 Most relevant to the global surgeon is SDG #3, “good health and well-being,” which builds upon the MDGs’ primarily maternaland child-mortality focus, as well as communicable disease prevention. SDG #3 broadens the focus to nine health targets, including a one-third reduction in deaths by noncommunicable diseases, as well as halving the rate of deaths and injuries from road traffic accidents by 2020. In addition, a novel push to strengthen and retain the global health workforce and systems for protection and prevention of disease also falls squarely within the realm of the surgical

1	by 2020. In addition, a novel push to strengthen and retain the global health workforce and systems for protection and prevention of disease also falls squarely within the realm of the surgical provider. Finally, the SDGs have garnered praise for closely involving local stakeholders, versus the expert consensus that produced the MDGs. Funding to work towards achievement of the SDGs is also divided between wealthier and poorer nations, whereas the MDGs relied primarily on funding from HICs to sup-port their mission.89World Health Organization. The initial UN Conference in 1945 voted to establish a new international health organization. The Constitution of the World Health Organization (WHO) was approved and ratified in 1948.83 The first World Assembly in 1948 established malaria, tuberculosis, venereal diseases, maternal and child health, sanitary engineering, and nutrition as WHO priorities. One of the WHO’s greatest public health sto-ries is the worldwide eradication of smallpox that

1	venereal diseases, maternal and child health, sanitary engineering, and nutrition as WHO priorities. One of the WHO’s greatest public health sto-ries is the worldwide eradication of smallpox that began with the USSR proposal for the WHO-led program in 1958 culminating in the last identified case in Somalia in 1977.While the disease burden from communicable diseases has abated in large part from these successful international coopera-tive interventions, little has been done to address the growing global burden of surgical disease. Despite the laudable aims of the 1978 Declaration of Alma Alta, which expressed the need for urgent action for the world community to protect and pro-mote health for all people, the declaration did so by crowning primary health care as the key to achieving the goal of health for all—which was then accepted by the member countries in the World Health Organization.90 Although the Alma Ata slogan Table 49-3Examples of charitable surgical delivery

1	the goal of health for all—which was then accepted by the member countries in the World Health Organization.90 Although the Alma Ata slogan Table 49-3Examples of charitable surgical delivery platformsTemporary DeliveryShort-Term Trips World Surgical Foundation Kenya Orthopedic Program APRIDEC Medical Outreach Group Self-Contained Mobile Surgical Platforms Mercy Ships Cinterandes FoundationSpecialty Surgical Hospitals Addis Ababa Fistula Hospital Aravind Eye Hospital Mission Hospitals (PAACS*)*Pan-African Association of Christian hospitalsTable 49-4Sustainable development goals 1No poverty 2Zero hunger 3Good health and well-being 4Quality education 5Gender equality 6Clean water and sanitation 7Affordable and clean energy 8Decent work and economic growth 9Industry, innovation, and infrastructure10Reduced inequalities11Sustainable cities and communities12Responsible consumption and production13Climate action14Life below water15Life on land16Peace, justice, and strong

1	and infrastructure10Reduced inequalities11Sustainable cities and communities12Responsible consumption and production13Climate action14Life below water15Life on land16Peace, justice, and strong institutions17Partnerships for the goalsBrunicardi_Ch49_p2077-p2112.indd 208813/02/19 5:54 PM 2089GLOBAL SURGERYCHAPTER 49“health for all by 2000” did not materialize, it did galvanize efforts for global partnerships for healthcare improvements and poverty reduction. In 2015, the World Health Assembly (WHA) published resolution WHA 68.15, which proclaimed surgical and anesthesia care as a crucial component of primary care worldwide—for the first time in history. The resolution urged member states to complete nine actions, including prioritizing a core set of emergency and essential surgery and anesthesia services at the primary care level, ensuring access to essential medications and infection control techniques, and developing policies for providers’ minimum skills, among others.

1	and anesthesia services at the primary care level, ensuring access to essential medications and infection control techniques, and developing policies for providers’ minimum skills, among others. Addition-ally, the Director-General of the WHO was asked to complete ten actions related primarily to policyand advocacy-related endeavors at the international level. The resolution was voted in unanimously by 194 member states.91The Violence and Injury Prevention Program (VIP) and the Global Initiative for Emergency and Essentials Surgical Care (GIEESC) are two programs related to surgery within the WHO that began before 2008. But as a response to a growing recognition of the significant unmet surgical need, in 2008 the WHO for the first time included basic surgery as a component for community primary health care (Fig. 49-14).92The Global Initiative for Emergency and Essential Surgical Care. The Clinical Procedures (CPR) team in the WHO Department of Essential Health Technologies (EHT)

1	primary health care (Fig. 49-14).92The Global Initiative for Emergency and Essential Surgical Care. The Clinical Procedures (CPR) team in the WHO Department of Essential Health Technologies (EHT) convened a multidisciplinary group of experts from various surgical disci-plines, professionals, and civic leaders from national and inter-national organizations, as well as representatives from various WHO departments, in December 2005 in Geneva, Switzerland to formally organize the Global Initiative for Emergency and Essential Surgical Care (GIEESC).93 GIEESC’s main aim was to assist member states with capacity strengthening in the safe Waste disposalinspectionMammographyAlcoholismHerniaPlacentapraeviaTrafficaccidentConsultantsupportReferral formulti-drugresistanceReferral forcomplicationsGenderviolenceSurgeryMaternityEnvironmental health labTraining centreTrainingsupportCancerscreeningcentreWomen’sshelterAlcoholicsanonymousCommunitymentalhealth unitEmergencydepartmentHospitalSpecialized

1	health labTraining centreTrainingsupportCancerscreeningcentreWomen’sshelterAlcoholicsanonymousCommunitymentalhealth unitEmergencydepartmentHospitalSpecialized careDiagnosticservicesTB controlcentreDiabetes clinicCTscanCytologylabDiagnostic supportPap smearsSelf-helpgroupLiaisoncommunityhealth workerOtherOtherSocialservicesNGOsSpecialized prevention servicesCommunityPrimary-care team:continuous,comprehensive,person-centred careFigure 49-14. Emergency and essential surgery: an integral component of primary care. (Reproduced with permission from The World Health Report 2008—primary Health Care (Now More Than Ever). http://www.who.int/whr/2008/en/.)Mongolia GIEESCThe WHO situational analysis tool, developed in 2007 to assess the availability of emergency and essential surgical care (EESC) at individual health facilities, has been utilized to document limited infrastructure, human resources, procedures, equipment, and supplies available for even basic EESC in many countries.95 For example,

1	health facilities, has been utilized to document limited infrastructure, human resources, procedures, equipment, and supplies available for even basic EESC in many countries.95 For example, there were no trained surgeons or anesthetists at 44 first-referral hospitals in Mongolia.3 Only 66% of the facilities had electricity, and 45% had running water (Fig. 49-15).Most facilities lacked any policy for EESC, disaster preparedness, basic equipment to provide EESC, or any and appropriate use of emergency and essential surgical care (procedures, equipment) at resource-limited healthcare facilities through training and education programs. The training program was built around the WHO Integrated Management of Emer-gency and Essential Surgical Care (IMEESC) tool kit.94 The tool kit included best practice protocols, guidelines on policies, training curriculum, emergency equipment, teaching slides, and monitoring and evaluation instructions. Additionally, low-cost editions of the manual Surgical

1	protocols, guidelines on policies, training curriculum, emergency equipment, teaching slides, and monitoring and evaluation instructions. Additionally, low-cost editions of the manual Surgical Care at the District Hospital have been made available in local languages. As of 2015, GIEESC had over 2100 members in 140 countries.95 A Mongolian edition facilitated early expansion of GIEESC throughout the country. Mongolia has improved basic infrastructure, human resources, and capabilities; and the use of the tool kit system has led to its incorporation into the countrywide healthcare plan96 (Box: Mongolia GIEESC).Brunicardi_Ch49_p2077-p2112.indd 208913/02/19 5:54 PM 2090SPECIFIC CONSIDERATIONSPART IIFigure 49-15. First Level (Soum) Hospital (left); Suturing of lacerated tongue by natural light (right). (Reproduced with permission from Intermountain Healthcare. Photo contributor: Raymond R. Price,

1	49-15. First Level (Soum) Hospital (left); Suturing of lacerated tongue by natural light (right). (Reproduced with permission from Intermountain Healthcare. Photo contributor: Raymond R. Price, MD.)BayankhongorKhentiiBayan-UlgiiBulganTuvDundgobiKhovdAltaiUvurkhangaiDornodDarkhanUulSukhbaatarOrkhonGobi-Sumber187 Soum doctors from 14 Aimags (Soum doctors = Doctor of health care centers)657 Surgeons and anesthesia doctors from aimag and city hospitals 29 Midwife and feldshers from rural provinces50 Nurses from rural provincesFigure 49-16. EESC Project: Mongolia 2004–2010. (Reproduced with permission from Henry JA, Orgoi S, Govind S, et al: Strengthening surgical services at the soum (first-referral) hospital: the WHO emergency and essential surgical care (EESC) program in Mongolia, World J Surg. 2012 Oct;36(10):2359-2370.)access to training for EESC. Adopting a health systems strengthening approach to rectify these glaring deficiencies, Mongolia implemented a nationwide EESC program

1	J Surg. 2012 Oct;36(10):2359-2370.)access to training for EESC. Adopting a health systems strengthening approach to rectify these glaring deficiencies, Mongolia implemented a nationwide EESC program involving 14 of the 21 provinces (Aimags) from 2004 to 2010 (Fig. 49-16).96 In 6 years, dramatic improvements in short-term process measures were identified using the WHO Monitoring and Process form: 57.1% increase in availability of emergency rooms; 59.1% increase in the supply of emergency tool kits; and a 73.6% increase in the recording of emergency cases (Figs. 49-17 and 49-18).96 More importantly, countrywide morbidity and mortality dropped significantly (Fig. 49-19).97Violence and Injury Prevention. The Violence and Injury Prevention (VIP) program promotes numerous activities to assist countries to prevent and mitigate the consequences of violence and injury.98 While injury prevention is paramount, VIP provides guidance for strengthening trauma systems in countries of all economic

1	countries to prevent and mitigate the consequences of violence and injury.98 While injury prevention is paramount, VIP provides guidance for strengthening trauma systems in countries of all economic levels to improve emergency care and rehabilitation. VIP encourages development of systematic data collection and analysis to better guide appropriate interventions. Prevention programs include the WHO Helmet initiative, while the Essential Trauma Care Project (EsTC) creates standards for the care of injured patients and promotes systematic capacity building. VIP advocates support for the UN Decade of Action for Road Safety 2011–2020 and initiatives to achieve SDG 3.6, Brunicardi_Ch49_p2077-p2112.indd 209013/02/19 5:54 PM 2091GLOBAL SURGERYCHAPTER 49halving the number of global deaths and injuries from road traf-fic accidents by 2020, and SDG 11.2, which aims to provide access to safe, affordable, accessible, and sustainable transport systems for all by 2030.99WHO Safe Surgery Saves

1	injuries from road traf-fic accidents by 2020, and SDG 11.2, which aims to provide access to safe, affordable, accessible, and sustainable transport systems for all by 2030.99WHO Safe Surgery Saves Lives Initiative. Surgeons have always sought ways to prevent perioperative complications. Aseptic technique, one of the greatest forms of prevention in surgical care, requires vigilant reinforcement to prevent serious wound infections. In resource-limited areas inad-equate perioperative monitoring, lack of critical medications, and poor documentation place patients at increased risk for serious complications. The WHO Safe Surgery Saves Lives Initiative is a worldwide attempt to prevent perioperative complications.100Deaths from surgery occur at 0.4% to 0.8% globally; however, they may exceed 5% to 10% in developing countries. There are about 1 million deaths and 7 million disabling com-plications related to surgery worldwide, 50% of which are estimated to be preventable. The WHO Safe

1	5% to 10% in developing countries. There are about 1 million deaths and 7 million disabling com-plications related to surgery worldwide, 50% of which are estimated to be preventable. The WHO Safe Surgery Saves Lives initiative targets preventable surgical injuries.100 The initiative identified 10 basic and essential objectives that can help prevent perioperative injuries (Table 49-5).101 A simple, three-stage checklist (initiated as the patient enters the operat-ing room, just before the procedure, and just prior to the patient leaving the room) implemented in eight high-, middle-, and low-income countries found a 50% reduction in the failure to meet basic safety standards resulting in a 50% decrease in mor-tality (Fig. 49-20).102Global Surgery and Public HealthSurgical care is increasingly recognized as an integral compo-nent of public health. Traditional teaching portrays sur-gery as the antithesis of public health: treating the individual instead of the community, reactionary

1	recognized as an integral compo-nent of public health. Traditional teaching portrays sur-gery as the antithesis of public health: treating the individual instead of the community, reactionary instead of pre-ventative, and too expensive especially for countries with devel-oping economies. Yet in reality, surgery and public health share many priorities and would benefit from greater integration in many areas (Fig. 49-21). For example, providing access to obstetrical care or birth attendants for every delivery could 10BeforeAfter0102030405060Wound debridementWound suturing, dressing, suture removalIncision and drainage of abscessesResuscitationFracture managementPenetrating injuriesBlunt injuriesAmputationBurns managementSkin graftingContracture releaseChest tube insertionCricothyroidotomy/TracheostomyRemoval of foreign bodyVenous peripheral cutdownUterine rupture/ectopic pregnancySuprapubic puncture/cystostomyLocal anaesthestic inÿltrationFigure 49-17. Surgical procedures performed 1 to

1	of foreign bodyVenous peripheral cutdownUterine rupture/ectopic pregnancySuprapubic puncture/cystostomyLocal anaesthestic inÿltrationFigure 49-17. Surgical procedures performed 1 to 2 years post training (13 Soum hospitals evaluated). (Reproduced with permission from Henry JA, Orgoi S, Govind S, et al: Strengthening surgical services at the soum (first-referral) hospital: the WHO emergency and essential surgical care (EESC) program in Mongolia, World J Surg. 2012 Oct;36(10):2359-2370.)Brunicardi_Ch49_p2077-p2112.indd 209113/02/19 5:54 PM 2092SPECIFIC CONSIDERATIONSPART IIprevent the majority of vesicovaginal fistulas and markedly decrease the most common cause of maternal death—hemorrhage—for entire communities. Ninety percent of mortal-ity from injury occurs in LMICs, providing another area for surgical teams to lead preventative, population-based strategies to improve public health.103 Male circumcision is another exam-ple of a well-documented preventative, minor surgical

1	another area for surgical teams to lead preventative, population-based strategies to improve public health.103 Male circumcision is another exam-ple of a well-documented preventative, minor surgical proce-dure, capable of reducing the transmission of HIV.104,1050102030405060708090Establishmentof emergencyroomSupply ofemergency kitsRecording ofemergencycare casesInstructions onfacility andinstrumentusageBeforeAfterFigure 49-18. Pilot Soum hospitals’ evaluation 2 years post training. (Reproduced with permission from Henry JA, Orgoi S, Govind S, et al: Strengthening surgical services at the soum (first-referral) hospital: the WHO emergency and essential surgical care (EESC) program in Mongolia, World J Surg. 2012 Oct;36(10):2359-2370.).60.50.40.30.20.10.00200120022003200420052006200720082009Percentage of deathsPercentage of complicationsFigure 49-19. Surgical morbidity and mortality: Mongolia 2001–2009. (Reproduced with permission from Intermountain

1	of deathsPercentage of complicationsFigure 49-19. Surgical morbidity and mortality: Mongolia 2001–2009. (Reproduced with permission from Intermountain Healthcare.)Brunicardi_Ch49_p2077-p2112.indd 209213/02/19 5:54 PM 2093GLOBAL SURGERYCHAPTER 49devotes an entire volume to essential surgery, emphasizing its importance as a key part of health worldwide.7 There are three significant developments helping to accelerate the integration of surgery and public health:1. Improved understanding of the burden of surgical disease and its significant component of the overall burden of global disease2. Recognition that surgery has a primary, secondary, and ter-tiary preventative role (Table 49-6)3. Documentation that surgical care can be cost-effective for community-based healthcareTable 49-5Ten basic and essential objectives for safe surgery (WHO*)1. Operate on the correct patient at the correct site2. Use method known to prevent harm from anesthetic administration, while protecting the patient

1	and essential objectives for safe surgery (WHO*)1. Operate on the correct patient at the correct site2. Use method known to prevent harm from anesthetic administration, while protecting the patient from pain3. Recognize and effectively prepare for life-threatening loss of airway or respiratory function4. Recognize and effectively prepare for risk of high blood loss5. Avoid inducing any allergic or adverse drug reaction known to be a significant risk for the patient6. Consistently use method known to minimize risk of surgical site infection7. Prevent inadvertent retention of instruments or sponges in surgical wounds8. Secure and accurately identify all surgical specimens9. Effectively communicate and exchange critical patient information for the safe conduct of the operation10. Establish routine surveillance of surgical capacity, volume, and results*WHO: World Health Organization.Data from WHO Guidelines for Safe Surgery

1	information for the safe conduct of the operation10. Establish routine surveillance of surgical capacity, volume, and results*WHO: World Health Organization.Data from WHO Guidelines for Safe Surgery 2009.PreventiondiagnosistreatmentrehabilitationSurgeryPublichealthFigure 49-21. Overlapping priorities of surgery and public health. (Reproduced with permission from Intermountain Healthcare.)Even after Learmonth presented his landmark lecture in 1949 “The Contributions of Surgery to Preventive Medicine” at the University of London’s Heath Clark Lecture series, surgery has been neglected as a component of public health.106,107 DCP3 Before induction of anaesthesia Before skin incisionBefore patient leaves operating roomSurgical safety checklist(with at least nurse and anaesthetist)(with nurse, anaesthetist and surgeon)(with nurse, anaesthetist and surgeon)This checklist is not intended to be comprehensive. Additions and modifications to fit local practice are encouraged.Nurse verbally

1	anaesthetist and surgeon)(with nurse, anaesthetist and surgeon)This checklist is not intended to be comprehensive. Additions and modifications to fit local practice are encouraged.Nurse verbally confirms: The name of the procedure Completion of instrument, sponge and needle counts Specimen labeling (read specimen labels aloud, including patient name) Whether there are any equipment problems to be addressedTo surgeon, anaesthetist and nurse: What are the key concerns for recovery and management of this patient?Based on the WHO Surgical Safety Checklisthttp://whqlibdoc.who.int/publications/2009/9789241598590_eng_Checklist.pdf© World Health Organization 2009 All rights reservedHas the patient confirmed his/her identity,site, procedure, and consent? YesIs the site marked? Yes Not applicableIs the anaesthesia machine and medicationcheck complete? YesIs the pulse oximeter on the patient andfunctioning? YesDoes the patient have a:Known allergy? No YesDifficult airway or aspiration risk? No

1	the anaesthesia machine and medicationcheck complete? YesIs the pulse oximeter on the patient andfunctioning? YesDoes the patient have a:Known allergy? No YesDifficult airway or aspiration risk? No Yes, and equipment/assistance availableRisk of >500 ml blood loss (7 ml/kg in children)? No Yes, and two IVs/central access and fluids planned Confirm all team members have introduced themselves by name and role. Confirm the patient’s name, procedure, and where the incision will be made.Has antibiotic prophylaxis been given within the last 60 minutes? Yes Not applicableAnticipated critical eventsto Surgeon: What are the critical or non-routine steps? How long will the case take? What is the anticipated blood loss?To anaesthetist: Are there any patient-specific concerns?To nursing team: Has sterility (including indicator results) been confirmed? Are there equipment issues or any concerns?Is essential imaging displayed? Yes Not applicableFigure 49-20. Surgical safety checklist. (Reproduced

1	(including indicator results) been confirmed? Are there equipment issues or any concerns?Is essential imaging displayed? Yes Not applicableFigure 49-20. Surgical safety checklist. (Reproduced with permission from WHO surgical safety checklist, 2009, http://whqlibdoc.who.int/publications/2009/9789241598590_eng_Checklist.pdf. © World Health Organization 2009 All rights reserved.)Brunicardi_Ch49_p2077-p2112.indd 209313/02/19 5:54 PM 2094SPECIFIC CONSIDERATIONSPART IIStrategies for Integration of Global Surgery and Public Health. Three areas stand out as opportunities for integration of global surgery and public health: education, professional societies, and multinational health policy organizations. From an education standpoint, several universities in HICs have developed formal programs for the study of surgery and public health.108,109Diseases commonly present in very late stages in LMICs and in disadvantaged populations in developed countries. Many morbid conditions could have

1	for the study of surgery and public health.108,109Diseases commonly present in very late stages in LMICs and in disadvantaged populations in developed countries. Many morbid conditions could have been cured while localized in their earlier stages and likely eradicated by a local surgical pro-cedure. Early recognition and treatment of surgically correctable diseases is a critical preventive role for surgery. Many surgical procedures are not only a form of tertiary prevention, but are also forms of primary prevention (Table 49-7).110Assigning Disease Priorities. Global surgery interventions can be prioritized to identify those conditions in which clini-cians and public health professionals should collaborate most closely—targeting those diseases that impose the largest bur-den on a society and have a highly successful surgical outcome (Table 49-8).94,111 There are four broad, high-priority areas where surgery has an important role for public health interventions: trauma care;

1	a society and have a highly successful surgical outcome (Table 49-8).94,111 There are four broad, high-priority areas where surgery has an important role for public health interventions: trauma care; obstetrical emergencies; acute-surgical emergen-cies; and nonacute surgical conditions that significantly affect the quality of life (Table 49-9).10Trauma Care. The Essential Trauma Care Project (EsTC) begun in 2001 is a collaboration effort between the International Association for Trauma Surgery and Intensive Care, an inte-grated society within the International Society of Surgery-Societe-Internationale Chirurgie (ISS-SIC) and the World Health Organization (WHO), specifically the Violence and Injury Prevention unit. The project culminated in a document that identified 11 core essential trauma care services (“the rights of the injured patient”) that ought to be available at all levels of healthcare facilities (Table 49-10).112 In addition, the docu-ment delineated 260 human and physical

1	care services (“the rights of the injured patient”) that ought to be available at all levels of healthcare facilities (Table 49-10).112 In addition, the docu-ment delineated 260 human and physical resources that should be available based on the type of facility (Table 49-11).Table 49-6Prevention strategiesPREVENTION STRATEGYTARGETGOAL1. PrimaryRoot causes of diseaseEliminate or reduce risk of developing illness2. SecondaryIllness or disease at earliest stagesLimit progression of disease3. TertiaryDisease at later stagesCure or limit the effect of existing diseaseData from deVries C, RR Price: Global Surgery and Public Health: A New Paradigm, 1st ed. Sudbury, MA: Jones & Bartlett Learning, LLC; 2012.Table 49-7The role of surgery for primary prevention of cancerTERTIARY SURGICAL PROCEDUREPRIMARY CANCER PREVENTEDBreast lumpectomy for ductal carcinoma in situBreastColonoscopic polypectomyColonColposcopy and excisionCervicalResection of actinic keratosisSkinResection of leukoplakia and

1	CANCER PREVENTEDBreast lumpectomy for ductal carcinoma in situBreastColonoscopic polypectomyColonColposcopy and excisionCervicalResection of actinic keratosisSkinResection of leukoplakia and erythroplakiaOralData from Riviello R, Meara JG, Rogers SO. Comemntary: Cancer Care and Control–the role of surgery. Global Surgery and Anesthesia, 2010. http://www.ghdonline.org/surgery/discussion/cancer-care-and-control-the-role-of-surgery/.Table 49-8Prioritization of surgical conditionsPRIORITY*PUBLIC HEALTH BURDENSURGICAL PROCEDURE SUCCESSFULCOST-EFFECTIVE AND FEASIBLE TO PROMOTE GLOBALLY1HighHighlyHighly2ModerateModeratelyModerately3LowNeither highly or moderatelyLow*Priority one implies that all three conditions must be met. The priority should be shifted to 2 or 3 if any of the conditions are moderate or low.Data from Mock C, Cherian M, Juillard C, et al: Developing priorities for addressing surgical conditions globally: furthering the link between surgery and public health policy, World J

1	or low.Data from Mock C, Cherian M, Juillard C, et al: Developing priorities for addressing surgical conditions globally: furthering the link between surgery and public health policy, World J Surg. 2010 Mar;34(3):381-385.Table 49-9The role of surgery for public health strategiesTrauma carePrevention of death and chronic disability by the provision of timely, expert, and complete surgical careObstetrical emergenciesTimely surgical intervention in obstructed labor, in preand post-partum hemorrhage, and other obstetrical complicationsAcute surgical emergenciesProvision of competent surgery to treat a wide range of emergency abdominal and nonabdominal conditionsNonacute surgical conditionsSurgical care for several elective conditions that have a significant effect on the quality of life such as cataract, otitis media, clubfoot, and herniasData from Jamison DT, Breman JG, Measham AR et al: Disease Control Priorities in Developing Countries, 2nd ed. New York, NY: Oxford University Press for

1	cataract, otitis media, clubfoot, and herniasData from Jamison DT, Breman JG, Measham AR et al: Disease Control Priorities in Developing Countries, 2nd ed. New York, NY: Oxford University Press for the World Bank; 2006.Brunicardi_Ch49_p2077-p2112.indd 209413/02/19 5:54 PM 2095GLOBAL SURGERYCHAPTER 49The EsTC recommendations provide a cost-effective framework for LMICs to improve their trauma care. These recommendations have been used as a planning guide and as an advocacy statement. To catalyze strengthening trauma and emergency care in lowand middle-income countries, in 2007, the WHA adopted a resolution on emergency care systems (resolution WHA 60.22).113,114 This first-ever WHA resolution dedicated specifically to trauma care highlights the importance accorded by world governments in caring for their injured.Quality improvement programs provide inexpensive tools to strengthen trauma systems. National trauma registries, integral for trauma research, can be used to monitor and

1	in caring for their injured.Quality improvement programs provide inexpensive tools to strengthen trauma systems. National trauma registries, integral for trauma research, can be used to monitor and improve patient outcomes.115,116 Yet very few trauma regis-tries exist in LMICs.117,118 The World Bank stated, “It is criti-cal for LMICs to create or strengthen existing trauma systems to improve outcomes.”119,120 Trauma systems exist in varying states of development in different countries, and even within countries. Initiatives to strengthen trauma systems target the full spectrum of services: prevention, prehospital and defini-tive hospital care, rehabilitation, and process improvement and patient safety initiatives. Nearly 2 million lives could be saved each year if LMICs could design and implement simple trauma care initiatives that reduced the case fatality rates among seri-ously injured patients to equal those in HICs (Fig. 49-22).119,120 In one Canadian province, introducing simple

1	simple trauma care initiatives that reduced the case fatality rates among seri-ously injured patients to equal those in HICs (Fig. 49-22).119,120 In one Canadian province, introducing simple prehospital Table 49-10Essential trauma care services1. Obstructed airway appropriately maintained2. Impaired breathing supported3. Pneumothorax and hemothorax promptly diagnosed and treated4. Bleeding promptly stopped (internal or external)5. Shock recognized and treated appropriately (I.V. fluids)6. Timely decompression of space occupying lesions to prevent secondary brain injury7. Abdominal injuries diagnosed and promptly repaired (intestinal injuries and others)8. Disabling extremity injuries corrected9. Potentially unstable spine injuries identified and managed (early immobilization)10. Minimize consequences of injuries by appropriate rehabilitative services11. Medication to provide above services and relieve pain readily availableData from Mock C, Joshipura M, Goosen J, et al: Overview of

1	consequences of injuries by appropriate rehabilitative services11. Medication to provide above services and relieve pain readily availableData from Mock C, Joshipura M, Goosen J, et al: Overview of the Essential Trauma Care Project, World J Surg. 2006 Jun;30(6):919-929.Table 49-11Airway management recommendations for physical and human resources based on type of facility (sample from EsTC*) FACILITY LEVELKNOWLEDGE AND SKILLSBASICGENERAL PRACTITIONERSPECIALISTTERTIARYAssessment of airway compromiseEEEEManual maneuvers (chin lift, jaw thrust)EEEEInsertion of oral or nasal airwayDEEEEndotracheal IntubationDDEEEquipment and supplies Oral or nasal airwayDEEELaryngoscopeDDEEEndotracheal tubeDDEECapnographyIDDDE: essential; D: desirable; I: irrelevant (not usually to be considered as the level in question).*EsTC: Essential Trauma Care.Data from Mock C, Lromand JD, Goosen J, et al: Guidelines for essential trauma care. Geneva: World Health Organization; 2004.Kumasi,Ghana0LowMiddleCountry

1	in question).*EsTC: Essential Trauma Care.Data from Mock C, Lromand JD, Goosen J, et al: Guidelines for essential trauma care. Geneva: World Health Organization; 2004.Kumasi,Ghana0LowMiddleCountry incomeHigh20406080Fatalities (percent)(ISS ˜9)Monterrey,MexicoSeattle,WashingtonUnited StatesFigure 49-22. Case fatality rates for severely injured people, 1998. Note: ISS = injury severity score. Income classifications are based on status at the time of the study. Ghana is now a lower-middle income country, and Mexico is an upper-middle-income country. (Reproduced with permission from Intermountain Healthcare.)Brunicardi_Ch49_p2077-p2112.indd 209513/02/19 5:54 PM 2096SPECIFIC CONSIDERATIONSPART IIinterventions and improving the focus on trauma at the policy level demonstrated consistent improvement in trauma-related morbidity and mortality, over a relatively short time period (Fig. 49-23).121Obstetrical and Other Acute Surgical Emergencies. Reduction of maternal deaths and long-term

1	in trauma-related morbidity and mortality, over a relatively short time period (Fig. 49-23).121Obstetrical and Other Acute Surgical Emergencies. Reduction of maternal deaths and long-term disability are high priorities for the international community.122 Despite the 44% reduction in maternal deaths from 1990 to 2015, approxi-mately 830 women—mostly in LMICs—still die daily from preventable causes related to pregnancy and childbirth.123 For every maternal death, 30 women are incapacitated by chronic problems that reduce their quality of life and ability to care for their families. High priority surgical procedures to improve maternal health include cesarean section, hysterectomy for postpartum bleeding and uterine rupture, management of ectopic pregnancy, and dilatation and curettage.111 In 2015, the LCGS reported that maternal mortality was closely related to density of surgical, anesthetic, and obstetrical providers (SAOPs). They showed that maternal mortality throughout the world

1	In 2015, the LCGS reported that maternal mortality was closely related to density of surgical, anesthetic, and obstetrical providers (SAOPs). They showed that maternal mortality throughout the world appeared to decrease—by 13.1% on average—for every 10 unit increase in SAOPs per 100,000 persons, a strong argument for addressing maldistribution of providers related to surgical disease.16In terms of nonobstetrical acute surgical emergencies, about 90% could be addressed by developing the capability to care for the 10 most common acute surgical conditions in any local region. While a few types of disease processes vary by geo-graphical location, there are many that are universal, including appendicitis, strangulated hernia, small bowel obstruction, per-forated peptic ulcer, fractures, lacerations, and wounds.Nonacute Surgical Conditions. Even common nonacute conditions can have significant impact on the quality of life. Hernias can prevent otherwise healthy individuals from work-ing,

1	and wounds.Nonacute Surgical Conditions. Even common nonacute conditions can have significant impact on the quality of life. Hernias can prevent otherwise healthy individuals from work-ing, especially in societies where the economy relies heavily on manual labor. Cleft lip and cleft palate deformities interfere with the ability to speak or eat properly and predispose affected indi-viduals to chronic ear infections leading to hearing loss. Many live in isolation because social ostracism prevents them from attending school, marrying, or holding jobs.124 Plastic surgeons who pioneered global outreach for reconstructive procedures for cleft lip and palate opened the door for subsequent outreach by other specialties, including ophthalmology, orthopedics, general surgery, urology, and dentistry.125-127The most common form of blindness is caused by cata-racts. Cataracts decrease the quality of life and the socioeco-nomic status for both the blind person and his or her family. The fact that

1	most common form of blindness is caused by cata-racts. Cataracts decrease the quality of life and the socioeco-nomic status for both the blind person and his or her family. The fact that 90% of blind people no longer work reveals the extra burdens carried by the family members who care for them.128 The Himalayan Cataract Project (HCP) is a highly successful initiative focusing on cataracts in Asia and Africa. HCP pri-orities and measurable outcomes illustrate how combining key public health concepts with a comprehensive approach to sur-gical care creates a model for curing disease, building econo-mies, and delivering hope in resource-poor areas9 (Box: The Himalayan Cataract Project: A Sustainable Public Health Approach for Curing Blindness).1992–930%10%20%30%Trauma mortality40%50%60%1993–941994–951995–961996–971997–981998–991999–002000–012001–021990: Government ofQuebec deÿnes traumacare as a priority1993: Four Quebechospitals designatedlevel 1 trauma centers1993: Introduction

1	Government ofQuebec deÿnes traumacare as a priority1993: Four Quebechospitals designatedlevel 1 trauma centers1993: Introduction ofguidelines aimed atreducing prehospital times1995: Implementation oftriage and transfer protocolsbased on injury severity1995: Designation oflevel II and III centers1996–97: Specializedtrauma centersdesignated2002: Eliminationof MDs fromprehospital care1994–95: Traumabecomes a priority inlevel 1 centers2000: Guidelines todecrease MD dispatch toprehospital traumaFigure 49-23. Improvement in mortality among severely injured patients in Canada through evolving interventions. Note: Inclusion criteria specified death as a result of injury or an injury severity score (ISS) exceeding 12, a prehospital index exceeding 3, two or more injuries with an abbreviated injury scale score of 3 or higher, or a hospital stay exceeding 3 days. (Adapted with permission from Liberman M, Mulder D, Lavoie A et al. Implementation of a Trauma Care System: Evolution through

1	injury scale score of 3 or higher, or a hospital stay exceeding 3 days. (Adapted with permission from Liberman M, Mulder D, Lavoie A et al. Implementation of a Trauma Care System: Evolution through Evaluation, J Trauma. 2004 Jun;56(6):1330-1335.)Brunicardi_Ch49_p2077-p2112.indd 209613/02/19 5:54 PM 2097GLOBAL SURGERYCHAPTER 49The Himalayan Cataract Project (HCP): A Sustainable Public Health Approach for Curing BlindnessAccording to the WHO criteria, 285 million people worldwide are visually disabled. Of that population, 39 million are classified as bilaterally blind; 90% live in the developing world where poor water quality, lack of sanitation, malnutrition, and inadequate services cause a higher incidence of eye disease.128 The most common cause of avoidable blindness in LMICs is cataract (43%). Nepal has one of the highest incidences of cataracts partially due to increased exposure to ultraviolet sunlight encountered at its higher elevations; 62% of total blindness in Nepal is

1	(43%). Nepal has one of the highest incidences of cataracts partially due to increased exposure to ultraviolet sunlight encountered at its higher elevations; 62% of total blindness in Nepal is due to cataracts.129In 1995, Sanduk Ruit joined forces with Geoffrey Tabin to establish the Himalayan Cataract Project (HCP). In the early 1990s, difficult geography with inadequate transportation, the high cost of intraocular lenses, and a lack of trained ophthalmologists, assistants, and nurses limited access to cataract surgery for the poor.HCP developed and defined six priorities, each with an associated public health principle and outcome measurement that provided the basis for assessing success and for implementing change (Fig. 49-24). HCP’s care model targeted the entire population of blind people with cataracts regardless of the ability to pay. Since most of the potential patients lived in remote areas, HCP found it imperative to take cataract surgery to the local communities. The

1	people with cataracts regardless of the ability to pay. Since most of the potential patients lived in remote areas, HCP found it imperative to take cataract surgery to the local communities. The Tilganga Institute of Ophthalmology (TIO) in Katmandu, Nepal, has served as a base from which 493 doctors and over 19,000 ophthalmic personnel of all levels have received training since 1994. Through the TIO and its outreach programs, over 4,657,748 people have been screened, and more than 307,611 eye surgeries have been performed since 1994 (Fig. 49-25).130The TIO developed an ophthalmology residency training program implementing standards set forth by the American Academy of Ophthalmology. In addition to the formal residency program for ophthalmologists, HCP established training programs for community eye care workers in a three-year Ophthalmic Assistant Training Program.Ruit developed an innovative sutureless technique for cataract surgery yielding equivalent results to those in developed

1	eye care workers in a three-year Ophthalmic Assistant Training Program.Ruit developed an innovative sutureless technique for cataract surgery yielding equivalent results to those in developed countries but also reproducible in resource-constrained areas. By redesigning the intraocular lens implant and mass producing it locally in Nepal for U.S. $4, Ruit and Tabin provided a low-cost alternative to the higher-priced lens produced in developed countries. A local business—the Fred Hollows Intraocular Lens Factory—mass produces the lenses and supports the local economy by creating a new sustainable business.131HCP also designed a compassion-driven, culturally acceptable method for cost-recovery that involves a sliding scale for payment: 45% of patients pay U.S. $120; 20% pay a smaller amount based on their economic situation; and 35% receive cataract surgery for free.With the rapidity and scale of success experienced in Nepal, HCP and TIO began expanding their efforts globally. HCP is now

1	on their economic situation; and 35% receive cataract surgery for free.With the rapidity and scale of success experienced in Nepal, HCP and TIO began expanding their efforts globally. HCP is now actively working to replicate and proliferate their model in countries throughout South Asia and Africa by developing high-quality eye care systems, supporting local institutions, and training local doctors and ophthalmic personnel. Since 2005, HCP has trained over 300 ophthalmic personnel from 19 countries.HCPprioritiesPublic healthprinciplesImplementationAccessibleAppropriateDisruptive technologySustainable growthAffordableSustainabilityHumanitarianHigh qualityInnovationDirect impactAffordabilityReplicationEntire populationCare at local levelCare comparable to western standardsDisease with high incidence/prevalenceDesigned $4 lensLocal businessSkills transferBuilding infrastructureDelivery model$20 cost/cataractMeet needs of current populationCulturally and economically acceptableFigure

1	incidence/prevalenceDesigned $4 lensLocal businessSkills transferBuilding infrastructureDelivery model$20 cost/cataractMeet needs of current populationCulturally and economically acceptableFigure 49-24. Himalayan cataract project priorities, public health principles, and outcome measurements. (Redrawn from Himalayan Cataract Project and Tilganga Eye Center, Cureblindness.org, 129-131, by permission. Illustration reproduced with permission from Intermountain Healthcare.)Brunicardi_Ch49_p2077-p2112.indd 209713/02/19 5:54 PM 2098SPECIFIC CONSIDERATIONSPART IICancer InitiativesSurgery for cancer in public health plays a role not only for curative surgery, but also for early diagnosis, prevention, and palliation.52,110,132,133 Solid tumors, in their early stages, presents insidiously as a nonacute surgical problem. Due to cancer’s recent recognition as a leading cause of death, cancer has been identified as a health priority in LMICs. Most solid tumors are incurable without surgery and

1	surgical problem. Due to cancer’s recent recognition as a leading cause of death, cancer has been identified as a health priority in LMICs. Most solid tumors are incurable without surgery and at a minimum require surgical excision of the primary lesion.110It is often not appreciated that surgeons provide a signif-icant amount of primary care and are the principle providers involved in endoscopic screening and treatment of gastrointes-tinal tumors in LMICs. In countries without specialized ser-vices, low-cost and effective treatment options combining early prevention and treatment with off-patent drug use have led to coverage of cancer treatment in several middle-income coun-tries’ national health insurance plans.52 Cancer care provides significant opportunity for including surgery in community-wide public health programs as a high priority according to the prioritization model (see Table 49-6); cancer has a high public health burden, is treated with highly successful procedures, and

1	public health programs as a high priority according to the prioritization model (see Table 49-6); cancer has a high public health burden, is treated with highly successful procedures, and can be cost-effective and feasible globally. In 2009, a coalition of leaders in cancer care and public health organized the Global Task Force on Expanded Access to Cancer Care and Control in Developing Countries (GTFCCC).134 GTFCCC’s mission is to expand access to cancer prevention, detection, and care in LMICs. Successful partnerships have already been entered into Haiti, Rwanda, Mexico, Malawi, and Jordan.Cost-Effectiveness of Surgical Care. Funders in healthcare look for measurable return on their investments. While compari-son of outcomes and objective measures would be ideal, reality demonstrates that healthcare budgets more commonly are dic-tated by politics rather than actual need. Nevertheless, in a world of limited resources and tightening budgets for healthcare, cost-effective analyses of

1	healthcare budgets more commonly are dic-tated by politics rather than actual need. Nevertheless, in a world of limited resources and tightening budgets for healthcare, cost-effective analyses of various options for intervention are critical for policy makers. Comparing various options that have differ-ent outcomes is an approach called cost-utility analysis (CUA). Surgical interventions can be evaluated by specific diseases or conditions, or by systems or services required to support the delivery of surgical care. In 1990, the World Bank defined the Disability Adjusted Life Year (DALY) as the sum of Years of Life Lost (YLL) due to premature mortality in the population and the Years Lost due to Disability (YLD) for people living with the health condition or its consequences (DALY = YLL + YLD). Evaluating the cost per DALY averted is one approach for comparing the cost-utility between medical and surgical interventions. Recent surgical cost/DALY studies identifying the

1	(DALY = YLL + YLD). Evaluating the cost per DALY averted is one approach for comparing the cost-utility between medical and surgical interventions. Recent surgical cost/DALY studies identifying the cost-effectiveness of various types of surgical care have allowed surgical initiatives to be considered when prioritizing public health initiatives.The World Bank arbitrarily defined U.S. $100 per DALY averted per day in low-income countries as highly cost-effective. Compared to other public health initiatives, developing basic and emergency surgical care at the district level hospital is as cost-effective as, or more so, than typical public health programs such as retroviral treatments for HIV/AIDS or immunization for measles (Fig. 49-26).135-139Using the WHO’s cost-effectiveness standards, investing in emergency obstetrical systems, including timely caesarean delivery, can also be considered “highly cost-effective” for 48 of 49 countries in which there are currently inadequate numbers of

1	in emergency obstetrical systems, including timely caesarean delivery, can also be considered “highly cost-effective” for 48 of 49 countries in which there are currently inadequate numbers of cesarean deliveries.140,141 The median cost per DALY averted by cesarean-section was $304. In addition, the cost-benefit ratio in 46 of 49 countries was >1, suggesting that investment in cae-sarean delivery is a viable economic proposition.Inguinal hernia repair is one of the most common opera-tions performed worldwide. Tension-free inguinal hernia repairs performed with mosquito netting or polypropylene mesh were cost-effective in Western Ecuador and Western Ghana ($78.18 per DALY and $12.88 per DALY averted, respectively).136,142 Using mosquito netting in India was 3700 times cheaper than using traditional polypropylene mesh.143Using “value of lost output” (VLO) data representing 90% of the world’s population, it is estimated that U.S. $20.7 trillion would be lost between 2015 and 2030 due to

1	polypropylene mesh.143Using “value of lost output” (VLO) data representing 90% of the world’s population, it is estimated that U.S. $20.7 trillion would be lost between 2015 and 2030 due to unmet surgical needs and their inherent morbidity and mortality.144 Projected 1119941996199820002002200420062008201020122014201635,00030,00025,00020,00015,00010,00050000SurgeriesYearFigure 49-25. Eye surgeries at Tilganga Eye Center and outreach. Note: 2015 was the year of a devastating earthquake in Nepal. (Redrawn from Himalayan Cataract Project and Tilganga Eye Center, Cureblindness.org, 129-131, by permission. Illustration reproduced with permission from Intermountain Healthcare.)Brunicardi_Ch49_p2077-p2112.indd 209813/02/19 5:54 PM 2099GLOBAL SURGERYCHAPTER 49economic losses of such magnitude have underlined the impor-tance of prioritizing surgical infrastructure and the cost-effective nature of many interventions within the surgical realm.Factors Affecting Utilization and Outcome for

1	have underlined the impor-tance of prioritizing surgical infrastructure and the cost-effective nature of many interventions within the surgical realm.Factors Affecting Utilization and Outcome for Surgical Care. There are three major factors that severely limit utiliza-tion of surgical services:1. Socioeconomic and cultural factors2. Accessibility of facilities3. Quality of care (Fig. 49-27)145The decision to seek timely care is affected by the costs associated with time off from work and inability to support the family during the absence, transportation and lodging, and the surgical services themselves. Cultural and religious tradi-tions may define acceptability of various treatment options. For example, many people in Mongolia refuse to have surgery on Tuesdays as this is viewed as a “bad luck” day. Understanding local customs and cultural concerns can improve utilization of surgical services.At the intersection of cost and culture are “willingness to pay” (WTP) models, which predict

1	luck” day. Understanding local customs and cultural concerns can improve utilization of surgical services.At the intersection of cost and culture are “willingness to pay” (WTP) models, which predict how a society’s perceived costs of obtaining care versus tolerating a medical condition will lead to or prevent them from seeking care. Such calcula-tions can inform which policies are most likely to yield improved health for a country or region, and they rely heavily on per capita gross domestic product (GDP) and DALYs averted. It is vital to understand that these models, and the policies they inform, are context-dependent. What is perceived as socially valuable in Tanzania may be seen as overpriced or unnecessary in Haiti. As global surgical advocates work with public health experts to strengthen surgical systems, it will be important to remember that context, culture, and cost are indivisible from one another.146Austere environments, difficult terrain, and long distances from health

1	surgical systems, it will be important to remember that context, culture, and cost are indivisible from one another.146Austere environments, difficult terrain, and long distances from health care facilities significantly delay or prevent access to surgical care. Triage and transfer guidelines along with tele-medicine have the potential to mitigate the limitations of geog-raphy. However, without adequately trained care providers and support staff, the risk for poor outcomes is increased.Recognizing these three important factors for increasing utilization and outcomes, Mongolia initiated a public health approach for the management of gallbladder disease incorpo-rating minimally invasive surgery (Box: The Public Health Approach to Management of Gallbladder Disease in Mongolia).Figure 49-26. Cost-effectiveness of surgical interven-tions, compared to two key medical interventions. Note: DALY = disability-adjusted life year. (Reproduced with permission from Intermountain

1	49-26. Cost-effectiveness of surgical interven-tions, compared to two key medical interventions. Note: DALY = disability-adjusted life year. (Reproduced with permission from Intermountain Healthcare.)Orthopedic surgery tripTrauma centerCesarean deliveryHydrocephalus repairTrachoma surgeryCleft lip and palate repairHernia repairSurgical hospitalCataract surgeryObstetric hospitalMeasles vaccinationAntiviral therapy for HIV$1/DALY$10/DALY$100/DALY$1000/DALYRange Cost/DALY (2012 US$)Figure 49-27. Factors affecting utilization and outcome of surgical care. (Adapted with permission from UNFPA United Nations Population Fund (UNFPA); Setting stan-dards for emergency obstetric and new-born care; Available from: https://www.unfpa.org/resources/setting-standardsemergency-obstetric-and-newborn-care. Illustration reproduced with permission from Intermountain Healthcare.)Socioeconomic/cultural factorsAccessibility offacilitiesQuality ofcarePhase IDecision toseek carePhase IIIdentifying &

1	Illustration reproduced with permission from Intermountain Healthcare.)Socioeconomic/cultural factorsAccessibility offacilitiesQuality ofcarePhase IDecision toseek carePhase IIIdentifying & reachingmedical facilityPhase IIIReceipt of adequate &appropriate treatmentFactors affectingutilization & outcomePhases of delayBrunicardi_Ch49_p2077-p2112.indd 209913/02/19 5:54 PM 2100SPECIFIC CONSIDERATIONSPART IIThe Public Health Approach to Management of Gallbladder Disease in MongoliaMongolia, the most sparsely populated country in the world, covers a large geographic area nestled between China and Siberia.147 The austere environment with extremes of weather, dry deserts, and high mountains present significant obstacles for road building limiting transportation for patients in the vast rural areas (Fig. 49-28). Significant deficiencies in infrastructure, supplies, equipment, and human resources at primary healthcare facilities exist: sporadic electricity, no fully qualified surgeons or

1	areas (Fig. 49-28). Significant deficiencies in infrastructure, supplies, equipment, and human resources at primary healthcare facilities exist: sporadic electricity, no fully qualified surgeons or anesthesiologists, and less than half the facilities with running water.3 In 2006, Healthcare expenditures reached only U.S. $23.2 per capita.148,149The second most common cause of inpatient morbidity in Mongolia has transitioned to gastrointestinal diseases with liver disease, appendicitis, and gallbladder disease the top three causes.150 While laparoscopic cholecystectomy was introduced in Mongolia in 1994, by 2005 only 2% of gallbladders were removed laparoscopically, and then, only in the capital city.151 A cohort study in 2005 comparing open with laparoscopic cholecystectomy by Dr. Sergelen, the chief of surgery at the Health Sciences University of Mongolia (HSUM), found the wound infection rate to be significantly lower, hospital stays shorter, and hospital expenditures 50% less with

1	the chief of surgery at the Health Sciences University of Mongolia (HSUM), found the wound infection rate to be significantly lower, hospital stays shorter, and hospital expenditures 50% less with laparoscopy compared to open cholecystectomy.152Dr. Sergelen formulated a plan to expand access to laparoscopic surgery throughout Mongolia. This plan targeted the three main areas affecting utilization and outcome:1. Quality of Care:a. Develop a laparoscopic training didactic and practical course to train surgical teams and transfer skills safely.b. Improve the surgical infrastructure for each facility.c. Expand the surgical residency to include laparoscopic training.2. Accessibility of Quality Care:a. Begin training surgical teams in the capital city, but then expand them to four carefully selected regional diagnostic treatment and referral centers (RDTRCs) in all four quadrants of the country.b. Invite industry to offer cost-affordable supplies and replacement parts to sustain the

1	selected regional diagnostic treatment and referral centers (RDTRCs) in all four quadrants of the country.b. Invite industry to offer cost-affordable supplies and replacement parts to sustain the laparoscopic equipment in Mongolia.3. Socioeconomic/Cultural Factors:a. Educate the public on the increased benefits of laparoscopic surgery so they would initiate lobbying efforts demanding the government increase funding for these services.b. Educate government leaders about the need and benefit of laparoscopic cholecystectomy for the Mongolian people.The strategic initiative that began by expanding laparoscopic cholecystectomy within the capital city and then to the four key Regional Diagnostic and Treatment Referral Centers (RDTRCs) created the foundation for countrywide access to high-quality modern surgery for a regionally prevalent disease (Fig. 49-29).147,153In 2011, through a multinational partnership (HSUM, the Dr. WC Swanson Family Foundation (SFF), the Society of American

1	modern surgery for a regionally prevalent disease (Fig. 49-29).147,153In 2011, through a multinational partnership (HSUM, the Dr. WC Swanson Family Foundation (SFF), the Society of American Gastrointestinal and Endoscopic Surgeons (SAGES), and the University of Utah Department of Surgery), Mongolia experienced a Figure 49-28. Rural Ger. (Used with permission from Michelle K. Price.)Brunicardi_Ch49_p2077-p2112.indd 210013/02/19 5:54 PM 2101GLOBAL SURGERYCHAPTER 49dramatic transformation from open to laparoscopic cholecystectomy; laparoscopic replaced open cholecystectomy and became the primary method to remove the gallbladder in Mongolia (Fig. 49-30).154As people began to see their neighbors return to functional ability faster with the laparoscopic approach, the Mongolian people developed increased trust in their healthcare providers and the quality of care they could receive. The Ministry of Health committed increased funding for laparoscopic surgery and changed existing laws

1	increased trust in their healthcare providers and the quality of care they could receive. The Ministry of Health committed increased funding for laparoscopic surgery and changed existing laws making it easier for hospitals to purchase the needed equipment and supplies solidifying the needed financial and business models to support laparoscopic surgery in Mongolia.By 2016, with the introduction of laparoscopic training into the surgical residency program and development of laparoscopic fellowship training for surgical teams from outlying provinces, 17 of 21 provinces now provide laparoscopic cholecystectomy allowing patients the benefits of less pain, smaller incisions, fewer wound infections, and more rapid return to work (Fig. 49-31).KhovdChoybalsanUlaanbaatarErdenetArvaikheerMurunArvaikheer regionaldiagnostic and treatmentreferral centerHospital #1Hospital #2Cancer hospitalNomun hospitalMurungeneral hospitalErdenet regionaldiagnostic and treatmentreferral centerKhovd

1	regionaldiagnostic and treatmentreferral centerHospital #1Hospital #2Cancer hospitalNomun hospitalMurungeneral hospitalErdenet regionaldiagnostic and treatmentreferral centerKhovd regionaldiagnostic and treatmentreferral centerChoybalsan regionaldiagnostic and treatmentreferral centerRussiaChinaFigure 49-29. The public health approach to expanding laparoscopy to the regional diagnostic treatment and referral centers of Mongolia (RDTRCs). (Reproduced with permission from Intermountain Healthcare.)20050204060Percent of total cases801002006200720082009Year2010201120122013U1BAU1BR1BR1BR2R3BR3R4BR2R5BR4R5BR6BU2BU2BU1U3AU1Ap ˜ 0.001CROSSOVER FROM OPEN TO LAP CHOLE(SAGES)(SAGES)RURALURBANU1BAU1BR1BR1BR2R3BR3R4BR2R5BR4R5BR6BU2BU2BU1U3AU1A(SAGES)= Open= Laparoscopy= Plan for countrywide lap surgery expansion= Advanced course= Basic course= UrbanN= RuralN= Center numberABURNFigure 49-30. Transition from open to laparoscopic cholecystectomy in Mongolia. (Reproduced with permission from Wells

1	expansion= Advanced course= Basic course= UrbanN= RuralN= Center numberABURNFigure 49-30. Transition from open to laparoscopic cholecystectomy in Mongolia. (Reproduced with permission from Wells KM, Lee YJ, Erdene S, et al: Building operative care capacity in a resource limited setting: The Mongolian model of the expansion of sustainable laparo-scopic cholecystectomy, Surgery. 2016 Aug;160(2):509-517.)Brunicardi_Ch49_p2077-p2112.indd 210113/02/19 5:54 PM 2102SPECIFIC CONSIDERATIONSPART IIIntegrating Value into Global SurgeryAnother topic closely related to quality is the concept of value in healthcare, often described in terms of the value equation. The value equation states that value equals quality plus service, divided by cost. In this model, systems that generate high-quality care, at lower costs, produce greater value for stakeholders in that system (i.e., patients, physicians, insurers, and hospitals). This discussion is most pertinent in high-income countries such as the

1	at lower costs, produce greater value for stakeholders in that system (i.e., patients, physicians, insurers, and hospitals). This discussion is most pertinent in high-income countries such as the United States, where rising healthcare expenditures are cause for worry. It is also relevant in LMICs, where proposed interventions to improve quality must also control cost. A bidi-rectional exchange of information related to surgical systems between HICs and LMICs may provide one strategy for reduc-tion of cost in HICs and maximization of quality in LMICs.Advanced Surgical Care for Resource-Poor AreasLimited financial, physical, and human resources, political and social conflicts, and austere environments cause many to believe that advanced surgical care is inappropriate in resource poor countries.111,155-157 Misconception of the needs and abilities of people in LMICs cause some policymakers to discount the desire of people worldwide for advanced surgical care.144 Developing these

1	countries.111,155-157 Misconception of the needs and abilities of people in LMICs cause some policymakers to discount the desire of people worldwide for advanced surgical care.144 Developing these capabilities in resource-poor coun-tries has the potential to decrease overall cost and actually develop the infrastructure necessary to entice physicians and other healthcare workers to remain in their own countries. Establishing advanced surgical care requires expertise and ser-vices that symbiotically support and improve general medical care. Therefore, many developing countries are actively build-ing capacity and capability to provide the full spectrum of modern surgical care locally.158As economies improve and the benefits of laparoscopic surgery for resource-poor areas become better delineated, patients and doctors, surgical societies, ministries of health, and industries are demanding the benefits of minimally invasive surgery for patients and communities.150,159-165 The economic

1	patients and doctors, surgical societies, ministries of health, and industries are demanding the benefits of minimally invasive surgery for patients and communities.150,159-165 The economic impact of laparoscopy may be even greater in LMICs than in developed countries.166 Worldwide, surgeons have identified laparoscopic training as one of their greatest needs. In a 2010 survey, developing laparoscopic and endoscopic skills were identified as the most important skills desired by surgeons from the West Africa College of Surgeons (WACS) (Fig. 49-32).167Transplantation is another area of great interest to people in poor countries partly because of the high prevalence of kidney failure and because chronic dialysis facilities are limited. Hepatoma and liver failure are very common in countries with a strong prevalence of hepatitis B and C. Transplantation has become the treatment of choice for end-stage kidney disease in developed countries as it dramatically improves the qual-ity of life

1	with a strong prevalence of hepatitis B and C. Transplantation has become the treatment of choice for end-stage kidney disease in developed countries as it dramatically improves the qual-ity of life and increases survival rates compared to medical management.168 Yet, transplantation eludes most of the developing world. Initial attempts to transport critically ill patients from LMICs to developed countries for kidney transplantation were cost-prohibitive.169 With the alarming increase in the rate at which young people have been presenting with kidney disease in developing countries, the increased utilization placed on the few dialysis machines has been overwhelming.170 Dialysis units which previously were utilized three times a week, now oper-ate 24 hours a day, 7 days a week, and cannot begin to provide the needed services to the multitudes needing treatment. Even programs to develop peritoneal dialysis cannot fully ease the demand.12Remaining states without laparoscopic surgery

1	begin to provide the needed services to the multitudes needing treatment. Even programs to develop peritoneal dialysis cannot fully ease the demand.12Remaining states without laparoscopic surgery capabilityLaparoscopic Surgery expanded with internal Mongolian trainersLaparoscopic Surgery developed with HSUM/SFF/SAGES/U of U partnershipBayankhongorArhangaiKhovsgolZavkhanUvsKhentiiBayan-UlgiiBulganTuvDundgobiOmnogoviDomogoviKhovdGovi-altaiUvurkhangaiDornodSelengeDarkhanUulSukhbaatarOrkhonGobi-SumberFigure 49-31. Laparoscopic cholecystectomy expanded to 17 of 21 provinces in Mongolia by 2016. (Data from Raymond Price via personal communication with Dr. Erdene Sergelen. Illustration reproduced with permission from Intermountain Healthcare.)Brunicardi_Ch49_p2077-p2112.indd 210213/02/19 5:54 PM 2103GLOBAL SURGERYCHAPTER 49The majority of kidney transplants in developing countries are from living related donation because of cultural and legal prohibitions precluding cadaveric

1	5:54 PM 2103GLOBAL SURGERYCHAPTER 49The majority of kidney transplants in developing countries are from living related donation because of cultural and legal prohibitions precluding cadaveric transplantation. Laparoscopic living related donation has the potential to increase the volun-tary donor pool as patients have less postoperative pain, return to work and activities quicker, and have much better cosmesis than open surgery.171 Ethical concerns exist for nonrelated dona-tions, however, because of concern for coercion in some coun-tries. Adapting to the limited resources, surgeons have described various cost-saving techniques to facilitate the laparoscopic approach in resource poor areas, such as using endoclips instead of staplers for vascular control, modifications to the surgical approach, and suprapubic extraction of the kidney rather than endocatch removal.171-173Academic Global SurgeryThere has been a paradigm shift from traditional reliance on intermittent short-term

1	approach, and suprapubic extraction of the kidney rather than endocatch removal.171-173Academic Global SurgeryThere has been a paradigm shift from traditional reliance on intermittent short-term volunteerism toward a strengthening of the education and research pillars for surgical healthcare in developing regions, a role ideally suited for academic sur-gery. Global surgery is emerging as a new academic field of endeavor (Table 49-12). Academic institutions have histori-cally pioneered discovery in disease causation and treatment. As globalization expands, academic surgical programs are begin-ning to respond by broadening their vision and mission. This vision and mission includes interdisciplinary and collaborative approaches to designing innovative, affordable surgical care that is accessible to all through research, education, development, and advocacy.174,175Responding to the challenges of disparities, new genera-tions of students, faculty, philanthropists, private industry

1	accessible to all through research, education, development, and advocacy.174,175Responding to the challenges of disparities, new genera-tions of students, faculty, philanthropists, private industry lead-ers, and policymakers have demonstrated a growing passion to address global surgery as part of global health.176,177 Prior to 1984, only 0.32% of physicians and 0.12% of nurses were involved in international health (either paid or volunteer).178 Recently, interest in global health has exploded among medical students, residents, and faculty in the United States.179,180Figure 49-32. West African College of Surgeons: most desired skills. CT = computed tomography; MRI = magnetic resonance imag-ing. (Adapted with permission from Akporiaye L. Trigen survey: West African College of Surgeons. Trigen, Lagos, Nigeria. Unpublished data. 2010. Illustration reproduced with permission from Intermountain Healthcare.)706050403020100Open surgical skills(suturing, dissection)Minimal access

1	Trigen, Lagos, Nigeria. Unpublished data. 2010. Illustration reproduced with permission from Intermountain Healthcare.)706050403020100Open surgical skills(suturing, dissection)Minimal access skillsEndoscopic skillsSurgical critical careIntraoperative respiratory &hemodynamic monitoringRadiologic image interpretation(ultrasound, CT, MRI)Ultrasound skillsTable 49-12Examples of academic global surgery programsINSTITUTIONNAME OF GLOBAL SURGERY CENTERBrigham and Women’s HospitalCenter for Surgery and Public HealthEmory University School of MedicineGlobal Surgery ProgramHarvard Medical SchoolProgram in Global Surgery and Social ChangeKing’s College LondonKing’s Center for Global Health and Health PartnershipsMcGill UniversityCentre of Global SurgeryOregon Health and Sciences UniversityGlobal Health Advocacy Program in SurgeryUniversity of British ColumbiaBranch for International Surgical CareUniversity of California San FranciscoCenter for Global Surgical StudiesUniversity of ChicagoGlobal

1	Advocacy Program in SurgeryUniversity of British ColumbiaBranch for International Surgical CareUniversity of California San FranciscoCenter for Global Surgical StudiesUniversity of ChicagoGlobal Surgery ProgramUniversity of UtahCenter for Global SurgeryBrunicardi_Ch49_p2077-p2112.indd 210313/02/19 5:54 PM 2104SPECIFIC CONSIDERATIONSPART IIAcademic involvement in global surgery provides train-ing for the next generation of surgical leaders where they can learn the necessary skills to develop systems for quality and affordable surgical care, both locally and internationally. Leaders for the 21st century will need to know how to provide outstanding cost-effective clinical care for all environments.Global Surgery programs use a variety of methods to engage faculty, residents, and students to foster an envi-ronment of creativity and innovation necessary to generate new ideas for solving some of the most vexing problems in global health. For example, some academic collabora-tive

1	to foster an envi-ronment of creativity and innovation necessary to generate new ideas for solving some of the most vexing problems in global health. For example, some academic collabora-tive programs introduce medical, engineering, and business students to the fascinating world of medical device innova-tion focusing on a variety of communities with very limited resources.181,182 Other academic endeavors include global surgery resident rotations, fellowships, journal clubs, classes (locally and online), certificates, Masters of Public Health, bilateral exchange programs, and a variety of educational, research, and entrepreneurial meetings (local, national, and international). Many of these activities provide opportuni-ties for collaborative scholarly work (journal publications, books, book chapters, films, grants, research trials) that pres-ent new avenues for academic advancement supporting both colleagues from abroad as well as the home institution. More importantly, Ministries of

1	chapters, films, grants, research trials) that pres-ent new avenues for academic advancement supporting both colleagues from abroad as well as the home institution. More importantly, Ministries of Health are responding to results of these scholarly works by designing policies that include surgi-cal care in their countrywide health plans.Partnering academic programs from high-income coun-tries with LMICs and/or with NGOs provides opportuni-ties for collaboration183 (Box: Academic Global Surgery 13Global surgery engagementsLEVEL OF INTEGRATIONAssociationAllianceCollaborationPartnership+–Figure 49-33. Training outcomes from NGO/academic partner-ship. (Reproduced with permission from IVUmed and Intermoun-tain Healthcare.)Academic Global Surgery PartnershipsA. Rwanda Human Resources for Health (HRH) ProgramThe Rwanda Human Resources for Health (HRH) program is an ambitious 7-year long program of the Ministry of Health (MOH) of Rwanda, funded by the U.S. Government and the Global Fund to

1	(HRH) ProgramThe Rwanda Human Resources for Health (HRH) program is an ambitious 7-year long program of the Ministry of Health (MOH) of Rwanda, funded by the U.S. Government and the Global Fund to Fight AIDS, Tuberculosis, and Malaria. The HRH Program seeks to greatly expand and improve Rwanda’s health care workforce by strengthening national training programs of specialized physicians, nurses, oral health providers, and health managers. The HRH Program is also designed to strengthen the capacity of academic institutions in Rwanda to sustain the training programs initiated and supported by the HRH Program through (a) recruitment and retention of Rwandan faculty, (b) transfer of knowledge and skills to Rwandan faculty, (c) establishment of additional academic partnerships and collaborations between Rwandan academic institutions and U.S. academic institutions. Currently, 22 U.S. academic medical centers and universities are participating in this program, in collaboration with the

1	between Rwandan academic institutions and U.S. academic institutions. Currently, 22 U.S. academic medical centers and universities are participating in this program, in collaboration with the University of Rwanda—College of Medicine and Health Sciences (UR-CMHS) training faculty. Since the launch of the program in 2012, U.S. institutions have deployed about 100 faculty members per year across these four health-related professions. The recruited U.S. faculty are twinned with UR faculty and senior trainees, paired along common goals and interests, and together they engage in a diversity of activities—including teaching, training, research, clinical care, and care delivery improvement projects.The program is currently in its fifth year (August 2016–July 2017). Focusing our discussion on the surgical disciplines, the annual intake of postgraduate students (residents) has dramatically increased. The anesthesiology residency, whose annual matriculation ranged from zero to three residents,

1	surgical disciplines, the annual intake of postgraduate students (residents) has dramatically increased. The anesthesiology residency, whose annual matriculation ranged from zero to three residents, now routinely admits 10 new residents yearly. The surgery residency has divided into the four specialties of general surgery, orthopedics, urology, and neurosurgery. Annual matriculation across all for programs now ranges from 15 to 20 compared to 3 to 6, prior to the HRH program’s support of the surgery department. In July 2016, UR graduated eight new general surgeons and one new urologist, the largest output to date. Similar training output is noted across the other disciplines and specialties as well. As a result, Rwanda is now on track to achieve most of its targets for the health workforce. Most graduates are deployed across provincial hospitals to provide specialty level care in a decentralized fashion, while a portion are maintained at the teaching hospitals to be recruited as new

1	Most graduates are deployed across provincial hospitals to provide specialty level care in a decentralized fashion, while a portion are maintained at the teaching hospitals to be recruited as new faculty. The HRH Program also aims to strengthen the quality of the training programs through competency-based training and pedagogic innovation, improvements in infrastructure and equipment within the schools at the CMHS and the teaching hospitals, and stronger administration of the training programs. As the HRH program comes into its final years, efforts are underway towards faculty professional development program that will both ensure that the UR-CMHS is able to Partnerships). Global surgery engagements exist along a con-tinuum from simple associations, to alliances, collaborations, or formal partnerships (Fig. 49-33). A true partnership usually involves specified and joint rights and responsibilities. The other engagements vary depending on the amount of integra-tion between the

1	formal partnerships (Fig. 49-33). A true partnership usually involves specified and joint rights and responsibilities. The other engagements vary depending on the amount of integra-tion between the institutions and organizations. Successful sustainable progress for global surgery can occur within any of the different levels of engagement. Many partnerships begin with a simple association or alliance before growing into a formal partnership.Academic Global Surgery helped progress the founda-tional knowledge in defining the burden of surgical disease, clarifying the cost-effectiveness of surgical care, and estab-lishing baseline values of surgical capacity.184 Further work is necessary to move beyond data collection and to use this foun-dational knowledge to now develop interventional strategies and stimulate sustainable solutions for accessible, affordable, appropriate surgical care for all.185Brunicardi_Ch49_p2077-p2112.indd 210413/02/19 5:54 PM 2105GLOBAL SURGERYCHAPTER

1	strategies and stimulate sustainable solutions for accessible, affordable, appropriate surgical care for all.185Brunicardi_Ch49_p2077-p2112.indd 210413/02/19 5:54 PM 2105GLOBAL SURGERYCHAPTER 49continue making the aforementioned human resource gains independent of this large foreign aid grant, and that the relationships and collaborations forged between academic institutions may continue to grow and find new avenues for productive work together.186—Robert Riviello, MD, MPH, FACSB. Coordinating Nongovernmental Organizations (NGO) and Academic Organizations: IVUmedNonprofit organizations (NGO) have filled a niche in establishing surgical care in countries where training centers and healthcare systems are historically nonexistent or understaffed. More recently, professional organizations have developed a focus on specific diseases or patient groups and have become a resource for education and training in poor countries.For more than 20 years, the IVUmed NGO has focused on urological

1	have developed a focus on specific diseases or patient groups and have become a resource for education and training in poor countries.For more than 20 years, the IVUmed NGO has focused on urological education and hands-on training in Africa, Asia, and Latin America. IVUmed evolved from a need identified by plastic surgeons that had seen many children with hypospadias and other urological anomalies, such as exstrophy, when providing care for children with cleft lip and palate. Adult surgeons were not trained in the delicate reconstruction of pediatric genitourinary anomalies, and pediatric surgeons were not trained in endoscopic or reconstructive urological surgery. The program has expanded to support training in all aspects of urological care, including adult reconstruction, oncology, and endoscopic management of stones and prostatic disease.As a nonprofit organization, IVUmed is a partnership between surgeons, anesthesiologists and nurses, academic medical centers, urological

1	and endoscopic management of stones and prostatic disease.As a nonprofit organization, IVUmed is a partnership between surgeons, anesthesiologists and nurses, academic medical centers, urological professional associations, industry, and the public with urologic surgery training in more than 20 countries. It also provides North American trainees scholarships to travel to low-resource countries to learn and to share knowledge gained in their own programs. Many former scholars become mentors for other residents when they complete their training. The sites with the longest collaborations have developed their own educational programs in general urology or subspecialty areas and are now providing advanced training and care locally (see Fig. 49-33).C. Cancer Disparities Consortium in West AfricaNoncommunicable diseases, such as cancer, are a major public health problem in lowand middle-income countries (LMIC). In many LMIC, surgeons, due to the lack of medical oncologists, treat all stages

1	diseases, such as cancer, are a major public health problem in lowand middle-income countries (LMIC). In many LMIC, surgeons, due to the lack of medical oncologists, treat all stages of noninfectious related cancers, such as breast and colorectal cancer (CRC). In 2011, to address the disparity in outcomes for patients with cancer in West Africa compared to the United States, a research and training collaboration was formed between the Obafemi Awolowo University Teaching Hospital in Nigeria and Memorial Sloan Kettering Cancer Center (MSK) in New York. This relationship has now grown to become a consortium of five Nigerian hospitals and the Global Cancer Disparity Initiative Team at MSK.The consortium began by focusing on important questions regarding CRC: how can early stage patients be identified; what are the demographics of CRC patients in Nigeria; and is the biology of CRC different in Nigeria compared to the USA? These questions are being answered, with the support of two NIH

1	identified; what are the demographics of CRC patients in Nigeria; and is the biology of CRC different in Nigeria compared to the USA? These questions are being answered, with the support of two NIH grants, by creating a robust prospective database with a matching biobank. With over 250 patients, the consortium can now describe the metastatic patterns, stages of presentation, and risk factors for CRC in West Africa. Given that over 65% of patients present with stage IV disease, the development of a risk model to identify patients with early stage disease is a priority. This is being accomplished with a 400-patient prospective trial of colonoscopy in patients over 45 years of age with rectal bleeding in three Nigerian cities. Future projects include studying new technologies for CRC and breast cancer screening.—Peter Kingham, MD, FACSEthicsThe ethics involved in working outside one’s own country are complex. While a practitioner’s scope of practice is usually constrained by regulation

1	cancer screening.—Peter Kingham, MD, FACSEthicsThe ethics involved in working outside one’s own country are complex. While a practitioner’s scope of practice is usually constrained by regulation in America and Europe, in many countries the limits of what one can do are neither regulated nor enforced. Guidelines for what should be done, where, and under what circumstances are beyond the expertise of some ministries of health. Some problems are so episodic that they are not anticipated, and few guidelines exist. For example, in natural disasters and emergencies, should any willing provider from any country be granted permission to provide care? Should specific disaster-related training be encouraged or required?187,188 In the nonacute setting, should practitioners not licensed or credentialed in their home environments be allowed to perform volunteer surgery in other countries? What entity should oversee the flow of volunteer practitioners? Can a standard set of guidelines meet the

1	in their home environments be allowed to perform volunteer surgery in other countries? What entity should oversee the flow of volunteer practitioners? Can a standard set of guidelines meet the needs of most countries? Currently, there is little cross-national agreement between state entities, like ministries of health and independent organizations and individuals. While many countries require at least temporary licensure, some do not. In many cases enforce-ment is inconsistent.With respect to research, the poor historically have not received benefit from research performed on them. In inter-national studies, even local collaborators have been left out of study design and publication.189 As internet communications have improved, these lapses are no longer tolerated.190 Informed consent for surgical procedures, in the appropriate language and respectful of local customs, is becoming the norm. Few hospitals outside academic medical centers have institutional review boards (IRBs) to

1	for surgical procedures, in the appropriate language and respectful of local customs, is becoming the norm. Few hospitals outside academic medical centers have institutional review boards (IRBs) to oversee the implementation and review of clinical research. In recent years, peer reviewed journals have become more mindful of attribution of credit, and authors are strongly encouraged to design and report studies with local input at all levels.With regard to transplantation, many countries have laws against cadaveric transplants because of the very real concern for illegal marketing of organs. Even living-donor Brunicardi_Ch49_p2077-p2112.indd 210513/02/19 5:54 PM 2106SPECIFIC CONSIDERATIONSPART IItransplantation has seen effects of coercion in some regions and for some populations such as prisoners. Nevertheless, the need and popular desire for transplantation is accelerating acquisition of skills and technology to make transplantation available worldwide.191Finally, what is

1	such as prisoners. Nevertheless, the need and popular desire for transplantation is accelerating acquisition of skills and technology to make transplantation available worldwide.191Finally, what is considered ethical in one country or com-munity might be considered highly unethical in another. Con-sent for surgery may in one setting rest with the patient, but in another, with the community or family. Values about privacy vary markedly from region to region. Health information in many cultures is considered to be a community concern, not the personal property of an individual patient.Innovation in Global SurgeryThe pressing need for surgical care at all levels and the shortage of fully trained surgeons, anesthesiologists, and support personnel as well as equipment and supplies means that opportunities abound for innovation. Innovations in education, including simulation, can shorten the time necessary for learn-ing technical skills. Gaming technology can teach algorithms for

1	that opportunities abound for innovation. Innovations in education, including simulation, can shorten the time necessary for learn-ing technical skills. Gaming technology can teach algorithms for interpretation of X-rays and ultrasounds. Telemedicine/tele-health is transforming education through combinations of clini-cal case-based learning and massively open online courses (MOOC) (Box: Telemedicine). The potential for education innovation in surgery beyond the apprenticeship system cham-pioned by Halsted in 1904 is vast.14Sophisticatedtechnology thatsimplifiesLow-cost,innovativebusiness modelsEconomicallycoherent valuenetworkRegulations and standards thatfacilitatechangeFigure 49-34. Elements of disruptive innovation. (Reproduced with permission from Intermountain Healthcare.)Innovation that radically changes the way we do things and that changes a paradigm of a service or system is called “disruptive”; it abruptly changes an older and more expensive system in favor of a less

1	that radically changes the way we do things and that changes a paradigm of a service or system is called “disruptive”; it abruptly changes an older and more expensive system in favor of a less expensive, more widely available technology or process. The ability for disruptive innovations to transform products and services into affordable realities requires three main factors: a sophisticated technology that simplifies, a low-cost business model, and an economically coherent value network (Fig. 49-34).195Regulations and standards that vary between countries and locales can facilitate or impede disruptive change. While disrup-tions often are not qualitatively superior to the status quo, they make the process both less expensive and more accessible, and through multiple iterations, ultimately improve quality as they cycle through the transformative process.Decentralizing education, laboratory testing, and medical records have been made possible through free and open-source software, apps,

1	improve quality as they cycle through the transformative process.Decentralizing education, laboratory testing, and medical records have been made possible through free and open-source software, apps, and devices such as smart phones, tablets, and laptop computers. Monitoring and imaging devices and lapa-roscopic instruments designed for low resource environments have the potential to not only improve accessibility in poor countries but also to radically reduce surgical costs in wealthy ones.196THE FUTURE FOR GLOBAL SURGERYSurgeons of the future will need to educate themselves in areas that have not historically been taught in surgical curricula. Beyond the technical aspects of surgical practice, there is a complex ecosystem that supports surgical care. Surgeons must become more aware of the complexities of cost in order to be able to shape the environment in which they work. They must understand better what patients are seeking from the surgical experience, rather than focusing

1	of the complexities of cost in order to be able to shape the environment in which they work. They must understand better what patients are seeking from the surgical experience, rather than focusing primarily on a narrow view of what surgery might have to offer. Surgeons must engage in policy development and advocate for affordable and accessible surgical care without sacrificing quality. Thoughtful technology design can focus on improving quality and on decreasing cost, both in poor and wealthy countries. Building surgical capac-ity through a health systems-focused approach, with robust data collection, and establishment of global surgery centers of Telementoring in Global SurgeryOne excellent example of successful telementoring in surgery is a program started by Allan Okrainec, MD, a minimally invasive surgeon at the University of Toronto, and Georges Azzie, MD, a pediatric surgeon at Toronto’s Hospital for Sick Children. In the mid-2000s, the two imagined utilizing laparoscopic box

1	invasive surgeon at the University of Toronto, and Georges Azzie, MD, a pediatric surgeon at Toronto’s Hospital for Sick Children. In the mid-2000s, the two imagined utilizing laparoscopic box trainers and videoconferencing technology via Skype to teach minimally invasive techniques to surgeons in LMICs.192 As part of the “Go Global” Initiative of the Society of American Gastrointestinal and Endoscopic Surgeons (SAGES), Dr. Okrainec’s team traveled to Botswana in 2007 to teach a 3-day Fundamentals of Laparoscopic Surgery (FLS) course in person. Although only two of twenty surgeons achieved certification, average posttest scores showed promising improvement.Realizing that continued mentorship promotes success, Dr. Okrainec’s team reconfigured their teaching model to include telesimulation. In 2009, they carried out an 8-week course in FLS, with weekly meetings via videoconference and real-time simulation demonstration and feedback between Botswana and Toronto. This time, 100% of

1	In 2009, they carried out an 8-week course in FLS, with weekly meetings via videoconference and real-time simulation demonstration and feedback between Botswana and Toronto. This time, 100% of participants in the tele-simulation group attained certification.193 Subsequently, the team expanded its program to Colombia, with similar success in skill acquisition.194 Since 2009, the program has expanded to 15 countries, with satellite telesimulation sites in Colombia and Ukraine. They have trained more than 300 individuals in FLS skills around the world.Brunicardi_Ch49_p2077-p2112.indd 210613/02/19 5:54 PM 2107GLOBAL SURGERYCHAPTER 49excellence will stimulate improvements in the provision of sur-gical and anesthesia services.197 Further integration of surgical care into national health plans of governments should encour-age increased investments and political will necessary to create capacity, leading to timely, quality surgical care to all without risk of financial ruin.16,91 Our

1	plans of governments should encour-age increased investments and political will necessary to create capacity, leading to timely, quality surgical care to all without risk of financial ruin.16,91 Our colleagues in public health and the World Bank, Paul Farmer and Jim Kim, have challenged us: “We need our surgical colleagues to speak fluently about rebuilding infrastructure, training, personnel, and delivering high-quality care to the very poorest.”106REFERENCESEntries highlighted in bright blue are key references. 1. Shrime MG, Bickler SW, Alkire BC, Mock C. Global burden of surgical disease: an estimation from the provider perspec-tive. Lancet Glob Health. 2015;3(suppl 2):S8-S9. 2. Weiser TG, Regenbogen SE, Thompson KD, et al. An esti-mation of the global volume of surgery: a modelling strategy based on available data. Lancet. 2008;372(9633):139-144. 3. Chang L, Lacy BE, Spiegel BM. Quantifying surgical and anesthetic availability at primary health facilities in Mongolia. World J

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1	surgical diseases: a modelling study. Lancet Glob Health. 2015;3(suppl 2):S21-S27. 145. United Nations Population Fund (UNFPA); Providing emer-gency obstetric and newborn care to all in need. Available at: http://www.unfpa.org/public/mothers/pid/4385. Accessed August 22, 2018. 146. Marseille E, Larson B, Kazi DS, et al. Thresholds for the cost-effectiveness of interventions: alternative approaches. Bull World Health Organ. 2015;93:118-124. 147. Price R, Sergelen O, Unursaikhan C. Improving surgical care in Mongolia: a model for sustainable development. World J Surg. 2012;37:1492-1499. 148. Gunsentsoodol B, Nachin B, Dashzeveg T. Surgery in Mongo-lia. Arch Surg. 2006;141(12):1254-1257. 149. List of countries by total health expenditure per capita. Avail-able at: http://en.wikipedia.org/wiki/List_of_countries_by_total_health_expenditure_%28PPP%29_per_capita. Accessed August 22, 2018. 150. Health Indicators 2007, National Center for Health Development:Ulaanbataar. Ministry of Health,

1	Accessed August 22, 2018. 150. Health Indicators 2007, National Center for Health Development:Ulaanbataar. Ministry of Health, Mongolia. Available at: http://www.chd.mohs.mn/images/pdf/sma/uzuulelt/ENGLISH2007.pdf. Accessed October 30, 2018. 151. Rusher AH. Outreach surgery. Surgical technology arrives in Mongolia. Bull Am Coll Surg. 1999;84(3):21-25. 152. Sergelen O. Development of laparoscopic surgery in Mongolia. 2006. Available at: http://www.gfmer.ch/Medical_education_En/PGC_RH_2006/Reviews/pdf/Orgoi_laparoscopy_2006.pdf. Accessed August 22, 2018. 153. Straub CM, Price RR, Matthews D, Handrahan DL, Sergelen D. Expanding laparoscopic cholecystectomy to rural Mongo-lia. World J Surg. 2011;35(4):751-759. 154. Wells KM, Lee YJ, Erdene S, et al. Building operative care capacity in a resource limited setting: the Mongolian model of the expansion of sustainable laparoscopic cholecystectomy. Surgery. 2016;160(2):509-517. 155. Manning RG, Azziz AQ. Should laparoscopic cholecystec-tomy be

1	limited setting: the Mongolian model of the expansion of sustainable laparoscopic cholecystectomy. Surgery. 2016;160(2):509-517. 155. Manning RG, Azziz AQ. Should laparoscopic cholecystec-tomy be practiced in the developing world? The experience of the first training program in Afghanistan. Ann Surg. 2009; 249(5):794-798. 156. Contini S, Taqdeer A, Gosselin RA. Should laparoscopic cho-lecystectomy be practiced in the developing World? The expe-rience of the first training program in Afghanistan. Ann Surg. 2010;251(3):574; author reply 575. 157. Alfa-Wali M, Antoniou A. Should laparoscopic cholecys-tectomy be practiced in the developing world? Ann Surg. 2010;251(2):387; author reply 387-388. 158. Castadot RG, Magarick RH, Sheppard L, Burkman RT. A review of ten years’ experience with surgical equipment in international health programs. Int J Gynaecol Obstet. 1986;24(1):53-60.Brunicardi_Ch49_p2077-p2112.indd 211013/02/19 5:54 PM 2111GLOBAL SURGERYCHAPTER 49 159. Udwadia TE.

1	surgical equipment in international health programs. Int J Gynaecol Obstet. 1986;24(1):53-60.Brunicardi_Ch49_p2077-p2112.indd 211013/02/19 5:54 PM 2111GLOBAL SURGERYCHAPTER 49 159. Udwadia TE. Navigating laparoscopic surgery into the next decade in developing countries—a personal perspec-tive. Langenbecks Arch Surg. 2007;392(1):99-104. 160. Udwadia TE. One world, one people, one surgery. Surg Endosc. 2001;15(4):37-343. 161. Vargas G, Price RR, Sergelen O, Lkhagvabayar B, Batcholuun P, Enkhamagalan T. A successful model for laparoscopic train-ing in Mongolia. Int Surg. 2012;97(4):363-371. 162. Clegg-Lamptey JN, Amponsah G. Laparoscopic cholecystec-tomy at the Korle Bu Teaching Hospital, Accra, Ghana: an initial report. West Afr J Med. 2010;29(2):113-116. 163. Baigrie RJ, Stupart D. Introduction of laparoscopic colorectal cancer surgery in developing nations. Br J Surg. 2010;97(5):625-627. 164. Udwadia TE. Low-cost laparoscopic cholecystectomy. (Br J Surg. 2002; 89:1602-1607). Br J

1	of laparoscopic colorectal cancer surgery in developing nations. Br J Surg. 2010;97(5):625-627. 164. Udwadia TE. Low-cost laparoscopic cholecystectomy. (Br J Surg. 2002; 89:1602-1607). Br J Surg. 2003;90(6):761. 165. Piukala S. Laparoscopic cholecystectomy: complications and experiences in Tonga. Pac Health Dialog. 2006;13(2): 107-110. 166. Udwadia TE, Udwadia RT, Menon K, et al. Laparoscopic sur-gery in the developing world. An overview of the Indian scene. Int Surg. 1995;80(4):371-375. 167. Akporiaye L. Trigen survey: West African College of Sur-geons. Trigen, Lagos, Nigeria. Unpublished data. 2010. 168. Al-Bazzaz PH. Kidney transplantation in Erbil, Iraq: a single-center experience. Saudi J Kidney Dis Transpl. 2010;21(2):359-362. 169. Basinda SL, Maro EE, McLarty DG, Young AE, Wing AJ. Ten Tanzanian transplants: problems and perspectives. Postgrad Med J. 1988;64(756):778-782. 170. Korle-Bu to begin kidney transplant in last quarter. 2012. Available at:

1	DG, Young AE, Wing AJ. Ten Tanzanian transplants: problems and perspectives. Postgrad Med J. 1988;64(756):778-782. 170. Korle-Bu to begin kidney transplant in last quarter. 2012. Available at: http://www.ghanaweb.com/GhanaHomePage/NewsArchive/artikel.php?ID=238839. Accessed August 22, 2018. 171. Kumar A, Dubey D, Gogoi S, Arvind NK. Laparoscopy-assisted live donor nephrectomy: a modified cost-effective approach for developing countries. J Endourol. 2002;16(3):155-159. 172. Kumar A, Chaudhary H, Srivastava A, Raghavendran M. Laparoscopic live-donor nephrectomy: modifications for developing nations. BJU Int. 2004;93(9):1291-1295. 173. Simforoosh N, Basiri A, Tabibi A, Shakhssalim N. Laparo-scopic donor nephrectomy—an Iranian model for developing countries: a cost-effective no-rush approach. Exp Clin Trans-plant. 2004;2(2):249-253. 174. Wells KM, Price RR, Finlayson SG, deVries CR. Fundamen-tals for establishing and maintaining an academic centre for global surgery: the University of

1	Exp Clin Trans-plant. 2004;2(2):249-253. 174. Wells KM, Price RR, Finlayson SG, deVries CR. Fundamen-tals for establishing and maintaining an academic centre for global surgery: the University of Utah Experience. Lancet Glob Health. 2014;2(S47):47. 175. Price RR, Butler MW, deVries CR, Abdullah F. Promoting, developing, and sustaining academic global surgery programs. In: Swaroop M, Krishnaswami S, eds. Academic Global Surgery. New York: Springer International Publishing; 2016. 176. Banerjee A. Medical electives: a chance for international health. J R Soc Med. 2010;103(1):6-8. 177. Powell AC, Casey K, Liewehr DJ, Hayanga A, James TA, Cherr GS. Results of a national survey of surgical resident interest in international experience, electives, and volun-teerism. J Am Coll Surg. 2009;208(2):304-312. 178. Baker TD, Weisman C, Piwoz E. U.S. physicians in international health. Report of a current survey. JAMA. 1984;251(4):502-504. 179. Busnaina I. Medical school admissions doctor. 2012.

1	TD, Weisman C, Piwoz E. U.S. physicians in international health. Report of a current survey. JAMA. 1984;251(4):502-504. 179. Busnaina I. Medical school admissions doctor. 2012. Available at: https://www.usnews.com/education/blogs/medical-school-admissions-doctor/2012/04/09/medical-students-should-consider-overseas-clinical-experience. 180. Provenzano AM, Graber LK, Elansary M, Khoshnood K, Rastegar A, Barry M. Short-term global health research projects by U.S. medical students: ethical challenges for partnerships. Am J Trop Med Hyg. 2010;83(2):211-214. 181. University of Utah Health: Transforming Healthcare through Innovation: Center for Medical Innovation. Available from: http://healthsciences.utah.edu/center-for-medical-innovation/ 182. Stanford University: Extreme: Design for Extreme Affordabil-ity. Available at: https://extreme.stanford.edu.) 183. Busse H, Azazh A, Teklu S, et al. Creating change through collaboration: a twinning partnership to strengthen emergency medicine at

1	Affordabil-ity. Available at: https://extreme.stanford.edu.) 183. Busse H, Azazh A, Teklu S, et al. Creating change through collaboration: a twinning partnership to strengthen emergency medicine at Addis Ababa University/Tikur Anbessa Special-ized Hospital—a model for international medical education partnerships. Acad Emerg Med. 2013;20(12):1310-1318. 184. Blair KJ, Paladino L, Shaw PL, Shapiro MB, Nwomeh BC, Swaroop M. Surgical and trauma care in lowand middle-income countries: a review of capacity assessments. J Surg Res. 2017;210:139-151. 185. Carlson LC, Lin JA, Ameh EA, et al. Moving from data col-lection to application: a systematic literature review of surgi-cal capacity assessments and their applications. World J Surg. 2015;39(4):813-821. 186. Cancedda C, Riviello R, Wilson K, et al. Building workforce capacity abroad while strengthening global health programs at home: participation of seven Harvard-affiliated institutions in a health professional training initiative in

1	K, et al. Building workforce capacity abroad while strengthening global health programs at home: participation of seven Harvard-affiliated institutions in a health professional training initiative in Rwanda. Acad Med. 2017;92(5):649-658. 187. Sztajnkrycer MD, Madsen BE, Alejandro Báez A. Unstable ethical plateaus and disaster triage. Emerg Med Clin North Am. 2006;24(3):749-768. 188. Ramsey KM, Weijer CR. Ethics of surgical training in devel-oping countries. World J Surg. 2007;31(11):2067-2069; discussion 2070-2071. 189. Zumla A, Costello A. Ethics of healthcare research in develop-ing countries. J R Soc Med. 2002;95(6):275-276. 190. Kushner AL, Kyamanywa P, Adisa CA, et al. Editorial policy on co-authorship of articles from lowand middle-income countries. World J Surg, 2011;35:2367-2368. 191. Cameron JS, Hoffenberg R. The ethics of organ transplanta-tion reconsidered: paid organ donation and the use of executed prisoners as donors. Kidney Int. 1999;55(2):724-732. 192. International

1	JS, Hoffenberg R. The ethics of organ transplanta-tion reconsidered: paid organ donation and the use of executed prisoners as donors. Kidney Int. 1999;55(2):724-732. 192. International Surgery Canada. Telementoring in Botswana. Available at: http://internationalsurgerycanada.com/telemen-toring-in-botswana. Accessed August 22, 2018. 193. Okrainec A, Henao O, Azzie G. Telesimulation: an effec-tive method for teaching the fundamentals of laparoscopic surgery in resource-restricted countries. Surg Endosc. 2010;24(2):417-422. 194. Henao O, Escallon J, Green J, et al. Fundamentals of lapa-roscopic surgery in Colombia using telesimulation: an effective educational tool for distance learning. Biomedica. 2013;33(1):107-114. 195. Christensen CM, Grossman JH, Hwang J. The Innovator’s Prescription. New York: McGraw-Hill; 2009:441. 196. Beck M. New low-cost surgical tool could help patients in third world. The Wall Street Journal. September 23, 2016. 197. Ng-Kamstra JS, Greenberg SLM, Abdullah F,

1	York: McGraw-Hill; 2009:441. 196. Beck M. New low-cost surgical tool could help patients in third world. The Wall Street Journal. September 23, 2016. 197. Ng-Kamstra JS, Greenberg SLM, Abdullah F, et al. Global Surgery 2030: a Roadmap for high income country actors. BMJ Global Health. 2016;1(1):e000011.Brunicardi_Ch49_p2077-p2112.indd 211113/02/19 5:54 PM

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1	ENHANCED RECOVERY AFTER SURGERYHistory and Overview of Enhanced RecoveryAs anesthetic techniques, antibiotics, and minimally invasive surgery have improved surgical care over the centuries, fur-ther strategies to continue to improve patient outcomes have emerged. A novel perioperative regimen for patients following colon surgery using early oral nutrition, early mobilization, and epidural analgesia was first described in 1995 by Professor Hen-rik Kehlet from Copenhagen, Denmark in a small group of nine patients.1 He subsequently outlined a more detailed multimodal approach to perioperative care in 1997, reducing length of stay to a median of 2 days following sigmoid resection, and thus, has been described as the founder of enhanced recovery after surgery (ERAS).2,3 Soon thereafter, several others duplicated that applying standard perioperative care protocols could reduce length of stay following colon surgery.4The ERAS Study Group was founded in 2001 by Professor Ken Fearon and

1	several others duplicated that applying standard perioperative care protocols could reduce length of stay following colon surgery.4The ERAS Study Group was founded in 2001 by Professor Ken Fearon and Professor Olle Ljungqvist to further expand on the ideas proposed by Professor Kehlet. As there was a great discrepancy between actual practices and evidence-based best practices, as well as geographical and practitioner variations in care, the group desired to create a consensus on best practices with guidelines that could be employed in the clinical arena. The primary goal of ERAS is to treat the surgical patient in a multidisciplinary team approach throughout the perioperative course with the unified goal of accelerating functional recov-ery and optimizing patient outcomes based on evidence-based medicine (Fig. 50-1).In order to develop the key tenets of ERAS, the details of preoperative care, intraoperative surgical and anesthetic tech-nique, and postoperative care were scrutinized

1	medicine (Fig. 50-1).In order to develop the key tenets of ERAS, the details of preoperative care, intraoperative surgical and anesthetic tech-nique, and postoperative care were scrutinized and standards for each facet of care were developed (Fig. 50-2).ERAS and its associated principles truly represented a paradigm shift in perioperative care, breaking from the clas-sical teaching of prolonged fasting postoperatively until signs of bowel function, excessive fluid administration, and delayed postoperative mobilization. Instead, mitigating the metabolic and stress responses to surgery through multimodal care and uti-lizing evidenced based medicine allows for “fast-track” recov-ery and improved outcomes.Each facet of perioperative care may have modest benefit to the patient when applied alone, but when an integrated, mul-timodal enhanced recovery pathway (ERP) is used, the benefi-cial effects become synergistic. Patients return to presurgical functional states faster, spend less time in

1	but when an integrated, mul-timodal enhanced recovery pathway (ERP) is used, the benefi-cial effects become synergistic. Patients return to presurgical functional states faster, spend less time in the hospital, and expe-rience less morbidity.5 Furthermore, resource utilization is reduced, healthcare system cost is less, and societal cost is lower with faster return to work and reduced homecare needs.6 Though initial studies of ERP were primarily performed for colorectal surgery, ERP can be applied to a wide variety of specialties in the inpatient and outpatient setting including urology, orthopedics, and gynecology. Preoperative OptimizationFirst proposed in 1949 by the anesthesiologist, Dr. J. Albert Lee, a preanesthetic and presurgical evaluation by an anesthesiolo-gist is associated with improved outcomes for the efficiency of the operating room, the hospital, and most importantly, the patient.7 The use of a preoperative evaluation results in identify-ing patients at elevated

1	with improved outcomes for the efficiency of the operating room, the hospital, and most importantly, the patient.7 The use of a preoperative evaluation results in identify-ing patients at elevated respiratory risk, a 55% decrease in pre-operative testing, an 88% reduction in case cancellations, reduction in day of surgery delays, reduced total length of stay, a positive impact on hospital finances with cost reduction, and lower in-hospital mortality.8-13 Therefore, while it is very impor-tant for the surgeon to see the patient prior to a surgery, it is also 1Optimizing Perioperative Care: Enhanced Recovery and Chinese MedicineJennifer Holder-Murray, Stephen Esper, Zhiliang Wang, Zhigang Cui, and Xima Wang 50chapterEnhanced Recovery after Surgery 2113History and Overview of Enhanced Recovery / 2113Preoperative Optimization / 2113Metabolic Stress Response to Surgery / 2116Preoperative Fasting and Preoperative Carbohydrate Loading / 2117Intraoperative Considerations / 2117Perioperative

1	/ 2113Preoperative Optimization / 2113Metabolic Stress Response to Surgery / 2116Preoperative Fasting and Preoperative Carbohydrate Loading / 2117Intraoperative Considerations / 2117Perioperative Pain Management / 2119Postoperative Nausea and Vomiting Prevention / 2121Early Nutrition and Postoperative Ileus Prevention / 2121Mobilization / 2122ERAS in CRS / 2122ERAS in Hepatopancreaticobiliary Surgery / 2123ERAS in Gastrectomy and Esophagectomy / 2123ERAS in Bariatric Surgery / 2123ERAS in Other Surgical Specialties / 2123Setting Up an ERAS Program / 2124Traditional Chinese Medicine in Surgical Patients 2124History of Traditional Chinese Medicine / 2124Preoperative Nutritional Optimization / 2126Bowel Preparation for Surgery / 2126Preoperative Optimization During Sepsis and Infection / 2126Perioperative Pain Management / 2127Postoperative Nausea and Vomiting Prevention / 2127Early Nutrition and Postoperative Ileus Prevention / 2127Traditional Chinese Medicine in Common Surgical

1	/ 2126Perioperative Pain Management / 2127Postoperative Nausea and Vomiting Prevention / 2127Early Nutrition and Postoperative Ileus Prevention / 2127Traditional Chinese Medicine in Common Surgical Conditions / 2127Brunicardi_Ch50_p2113-p2136.indd 211301/03/19 9:39 AM 2114Key Points1 Enhanced recovery after surgery (ERAS) is a paradigm shift in the surgical care of patients. As a multimodal, integrated, evidence-based care pathway, ERAS optimizes patient care in the preoperative, intraoperative, and post-operative setting in order to achieve best patient outcomes. Patients recover faster, experience less physiological stress, enjoy shorter stays in the hospital, and have fewer complications.2 Setting appropriate expectations, optimizing nutritional and physical status through prehabilitation, and treating medical comorbidities optimizes patients before surgery.3 Achieving normovolemia both intraoperatively and post-operatively is important in order to maintain perfusion without

1	and treating medical comorbidities optimizes patients before surgery.3 Achieving normovolemia both intraoperatively and post-operatively is important in order to maintain perfusion without volume overload, as hypervolemia and hypovo-lemia are both associated with significant complications. Goal-directed therapy approaches maintain normovolemia with zero fluid balance.4 Intravenous normal saline administration results in hyper-chloremia, which has been associated with increased mor-tality and morbidity.5 As pain is a subjective response and cannot therefore be experienced while unconscious, the use of opioids intraop-eratively should generally be avoided in order to minimize the multiorgan system side effects of these medications. Additionally, avoiding intraoperative opioids actually improves postoperative pain scores and reduces the need for postoperative opioids.6 Multimodal analgesia, which includes oral or IV nonopioid analgesia and regional analgesic techniques, can reduce

1	improves postoperative pain scores and reduces the need for postoperative opioids.6 Multimodal analgesia, which includes oral or IV nonopioid analgesia and regional analgesic techniques, can reduce postoperative physiological stress and decrease complica-tions associated with surgery as part of a pain management regimen. Multimodal analgesia has been shown to reduce the number of opioids required for analgesia.7 The strategies for avoiding postoperative nausea and vom-iting include the avoidance of general anesthesia, the use of totally intravenous anesthesia, avoidance of nitrous oxide and volatile agents, minimizing intraoperative and postoperative opioids, and adequate hydration.8 Enhanced recovery after surgery care pathways can be applied to numerous types of surgery including colorectal, liver, pancreas, bariatric, gynecologic, and urologic sur-gery with success.9 Traditional Chinese medicine has been practiced for thou-sands of years and serves as a distinct cultural heritage

1	liver, pancreas, bariatric, gynecologic, and urologic sur-gery with success.9 Traditional Chinese medicine has been practiced for thou-sands of years and serves as a distinct cultural heritage of China. Its unique theories and methods are still applied widely in the practice of modern medicine, including dis-ease prevention, disease treatment, and perioperative management.10 Acupuncture and transcutaneous electroacupuncture can reduce the number of opioids utilized in the perioperative setting. Additionally, acupuncture, transcutaneous elec-troacupuncture, and some Chinese herb decoctions are effective in the prevention and treatment of postoperative nausea and vomiting.highly important for the patient to have an opportunity to dis-cuss the upcoming perisurgical and perianesthetic experience with a physician that is trained in the realm of periopera-tive optimization. Setting Expectations and Patient Education. Setting expectations in the preoperative clinic helps to orient patients

1	with a physician that is trained in the realm of periopera-tive optimization. Setting Expectations and Patient Education. Setting expectations in the preoperative clinic helps to orient patients regarding the entire surgical experience, from what they are expected to do at home before the surgery to the entire length of the recovery both in the hospital and at home. Information on the procedure and typical recovery should be clear, well defined, and consistently reinforced from all healthcare person-nel that interact with the patient. Clear expectations of goals prior to surgery, in the hospital, and after discharge should be communicated long before the surgery. Expected length of stay and disposition should also be clearly communicated in order to optimize timely discharge. The preoperative clinic helps in this role and to establish the patient as the leader in his or her own care. The patient must understand that his or her active partici-pation throughout the perioperative

1	clinic helps in this role and to establish the patient as the leader in his or her own care. The patient must understand that his or her active partici-pation throughout the perioperative experience will facilitate the recovery. A surgeon can do an operation. An anesthesiologist can keep a patient alive while the patient asleep. However, in reality, it is up to the patient to make his or her own care a prior-ity. If individuals train for a race, should they not also prepare for their procedure so that the recovery is swift? The optimiza-tion for success at surgery begins preoperatively with smoking cessation, exercise, and nutrition, but it also continues in the hospital and after discharge with pain control, physical activ-ity, discharge planning, and returning to daily activities. Clear expectations at each point in the perioperative continuum, which are communicated to the patient, will improve the perioperative experience for the patient and the provider.According to Costa,

1	Clear expectations at each point in the perioperative continuum, which are communicated to the patient, will improve the perioperative experience for the patient and the provider.According to Costa, “Evidence shows that patients suffer needlessly due to inadequate preoperative preparation and lack of information regarding their postoperative course as indicated by reports of unexpected pain, fatigue, and the inability to care for oneself.”14 Patients enter physician offices and procedures with a great deal of fear and anxiety related to the identification of a disease, the consequences of treatment of this disease, and/or the fear of death. Eliciting the patient’s concerns and provid-ing optimal communication and education can allay much of this fear and anxiety. There is a substantial perioperative cul-ture change that is brought about by the elements of ERAS, and many patients have personally had or have had a close relative that has experienced surgery that likely did not include

1	cul-ture change that is brought about by the elements of ERAS, and many patients have personally had or have had a close relative that has experienced surgery that likely did not include many 2ERAS: Team-centered approachPatientSurgical teamPreoperative nursing teamAnesthesiology teamPACU nursing teamInpatient nursing teamOffice/clinic teamERAS educatorPharmacyERAS project managerInformation technological teamFigure 50-1. Enhanced recovery multidisciplinary team. ERAS = enhanced recovery after surgery; PACU = postanesthesia care unit.Brunicardi_Ch50_p2113-p2136.indd 211401/03/19 9:39 AM 2115OPTIMIZING PERIOPERATIVE CARE: ENHANCED RECOVERY AND CHINESE MEDICINECHAPTER 50components of ERAS. Therefore, this culture change must be clearly disseminated to the patients and include new fasting guidelines, analgesic management, and patient participation in preoperative optimization. Patients can then anticipate and plan for certain events and sensations, such as what and when to eat and

1	guidelines, analgesic management, and patient participation in preoperative optimization. Patients can then anticipate and plan for certain events and sensations, such as what and when to eat and drink, how to exercise, what medications will be used, what tubes or lines will be present, and what criteria are used for dis-charge and return to daily activities. This kind of teaching has been defined as a therapeutic communication to help the patient face and cope with the surgical procedure in a calm manner.15Approximately 90 million people have difficulty under-standing and subsequently acting upon health information dis-tributed to them.16 Further, the ability of patients to process and understand basic information to make appropriate health decisions is directly related to socioeconomic status and to Caucasian race; furthermore, the readability of patient-directed healthcare material may be too advanced for comprehension by much of the surgical population.17,18 Thus, it is

1	status and to Caucasian race; furthermore, the readability of patient-directed healthcare material may be too advanced for comprehension by much of the surgical population.17,18 Thus, it is appropriate for patient information material to be at no higher than a sixth grade reading level, friendly, clear, concise, and simply designed. The employment of audiovisual aids may assist some patients as well.Nutrition. Surgery results in a significant catabolic stress response on the body, triggering inflammation and nutrient depletion. This stress response results in downstream effects on numerous organ systems and can lead to a higher risk of post-operative complications. Ensuring preoperative adequate nutri-tion is imperative before a large surgical procedure in order to mitigate adverse outcomes.While enteral or parenteral nutritional supplementation can be considered for the most nutritionally compromised patient, the enteral route is always preferred if clinically appropriate and can be

1	enteral or parenteral nutritional supplementation can be considered for the most nutritionally compromised patient, the enteral route is always preferred if clinically appropriate and can be adequately achieved in a timely fashion. Two main approaches to preoperative enteral nutrition include standard oral nutrition supplements and immunonutrition supplements, each providing extra protein and calories to supplement the diet. There is no statistical difference in infections, complications, and length of hospital stay between patients given standard oral versus immunonutrition supplements.19Standard oral nutrition products are high in protein, contain vitamins and minerals, and are widely available. Immunonutrition supplements usually similarly contain high protein, vitamins, and minerals, but they also have the addition of arginine to improve immunity and tissue repair and omega-3 fatty acids to mediate the inflammatory response.19 The exact dosage of arginine and omega-3 fatty acids

1	but they also have the addition of arginine to improve immunity and tissue repair and omega-3 fatty acids to mediate the inflammatory response.19 The exact dosage of arginine and omega-3 fatty acids that contribute to improved outcomes is not known. Standard oral nutrition prod-ucts may contain no or lesser quantities of arginine and omega-3 fatty acids when compared to immunonutrition supplements. The exact duration and frequency of supplementation have not been established; however, it is clear that the use of standard oral nutrition products has a positive impact on surgical out-comes by reducing postsurgical complications.19Exercise and Prehabilitation. Prehabilitation is defined as “the process of enhancing the functional capacity of the indi-vidual to enable him or her to withstand a stressful event.”20,21 Both exercise and prehabilitation, which have, heretofore, focused on cardiopulmonary rehabilitation prior to surgery, are very important to optimization of patient outcomes.

1	a stressful event.”20,21 Both exercise and prehabilitation, which have, heretofore, focused on cardiopulmonary rehabilitation prior to surgery, are very important to optimization of patient outcomes. Peri-operative cardiopulmonary exercise testing and prehabilitation in relation to ERAS programs around the world have been ana-lyzed, and it is noted that a reduction in fitness prior to sur-gery is associated with increased mortality and morbidity in the postoperative arena.22 Patients who actively exercise even when suffering from documented coronary artery disease, heart failure, hypertension, diabetes, chronic obstructive pulmonary disease, depression, dementia, cancer, and stroke have better outcomes.23-36 Furthermore, other literature supports the signifi-cant merits of exercise therapy and cardiopulmonary exercise therapy before and after major surgery, with the ability to reduce infection, hospital-associated complications, length of stay, and postoperative mortality.37There is a

1	and cardiopulmonary exercise therapy before and after major surgery, with the ability to reduce infection, hospital-associated complications, length of stay, and postoperative mortality.37There is a significant amount of evidence indicating that exercise training is feasible and safe in patients with a spec-trum of severe cardiac and pulmonary diseases as many of these patients require surgery to manage other disease processes. A randomized controlled trial involving 246 low-risk patients undergoing cardiac surgery reported a 1-day reduction in ICU stay and a reduced hospital length of stay in the intervention group.38 Cardiopulmonary fitness was found to be a strong independent predictor of survival after lung surgery, especially for non–small cell lung cancer.39 Preliminary nonrandomized Preoperative phasePatient education & set expectationsSmoking & alcohol cessationPrehabilitation with diet & exerciseShortened fastingAntimicrobial prophylaxisVTE prophylaxisMinimize bowel

1	Preoperative phasePatient education & set expectationsSmoking & alcohol cessationPrehabilitation with diet & exerciseShortened fastingAntimicrobial prophylaxisVTE prophylaxisMinimize bowel preparationIntraoperative phasePain blocksMinimally invasive surgeryGoal-directed fluid therapyMultimodal pain reliefPrevention of PONVAvoidance of tubes, drains, linesNormothermiaPostoperative phaseGoal-directed fluid therapyPrevention of PONVEarly ambulationEarly feedingMultimodal pain reliefEarly urinary catheter removalDefined discharge criteriaFigure 50-2. Phases and components of an enhanced recovery after surgery pathway. VTE = venous thromboembolism; PONV = postopera-tive nausea and vomiting.Brunicardi_Ch50_p2113-p2136.indd 211501/03/19 9:39 AM 2116SPECIFIC CONSIDERATIONSPART IIdata from patients undergoing elective rectal cancer surgery within an ERAS program have shown the feasibility of provid-ing a cardiopulmonary exercise interval training program that is delivered three times per

1	undergoing elective rectal cancer surgery within an ERAS program have shown the feasibility of provid-ing a cardiopulmonary exercise interval training program that is delivered three times per week for 6 weeks in a hospital setting after neoadjuvant chemoradiotherapy and before surgery.40 The interval following neoadjuvant therapy offers a unique window of time to improve the fitness and nutrition of many oncologic patients, which is especially important as these patients can often be some of the most debilitated.The addition of physical fitness and activity to a preopera-tive regimen for elderly patients undergoing major abdominal surgery significantly improved mortality, discharge to home versus a care facility, and length of stay.41 While this study showed that patients benefited from 60-minute sessions 2 to 4 weeks prior to surgery, even brief therapy before surgery, ranging from 1 day to 7 days before major abdominal surgery, have been associate with a significant decrease in

1	60-minute sessions 2 to 4 weeks prior to surgery, even brief therapy before surgery, ranging from 1 day to 7 days before major abdominal surgery, have been associate with a significant decrease in postopera-tive complications.42,43 Therefore, some oncologic or semiurgent patients may actually benefit from preoperative cardiopulmo-nary exercise programs.Smoking Cessation. Clearly tobacco use, especially smok-ing, has been well documented across all surgical specialties to increase postoperative mortality, as well as increase postopera-tive complications including prolonged ventilation, pneumonia, deep venous thrombosis, wound infection, delayed wound heal-ing, and reduced bone fusion.44-47 Physiologically, the carbon monoxide and nicotine from tobacco products increase heart rate and blood pressure and the body’s demand for oxygen. Nic-otine also causes vasoconstriction, reducing perfusion to many tissue beds. Obviously, it is advantageous for patients to cease smoking preoperatively.

1	pressure and the body’s demand for oxygen. Nic-otine also causes vasoconstriction, reducing perfusion to many tissue beds. Obviously, it is advantageous for patients to cease smoking preoperatively. There is debate about the duration of the nicotine free days needed preoperatively to offer best out-comes, though the literature suggests that the longer duration of smoking cessation prior to surgery portends better outcomes.48 This is likely to allow for bronchiolar and collagen remodeling and the several weeks following last exposure that are needed to achieve blood free of nicotine and its derivatives.Metabolic Stress Response to SurgeryMultiple organ systems interact in numerous metabolic and inflammatory cascades following the stress response to surgery leading to insulin resistance and protein catabolism (Fig. 50-3). Neuroendocrine responses, stress hormones cascades, activation of cytokine, and immune reactions all occur, leading to a cata-bolic state.49 Central to this metabolic

1	protein catabolism (Fig. 50-3). Neuroendocrine responses, stress hormones cascades, activation of cytokine, and immune reactions all occur, leading to a cata-bolic state.49 Central to this metabolic and inflammatory cas-cade is the development of insulin resistance, whereby a normal insulin concentration results in a subnormal biologic response. As insulin is the main anabolic hormone involved in glucose control, fat metabolism, and protein balance, insulin resistance disrupts many metabolic pathways.Hyperglycemia from insulin resistance results from an increase in glucose production and a decrease in glucose uptake by the periphery. In a fed state, insulin levels surge to 6 to 8 times basal levels, which stops glucose production and increases peripheral glucose uptake threeto fourfold.50 When fasting, insulin levels remain in a relative steady state with minimal effects on glucose and protein metabolism. Even when insulin levels increase to three times basal levels, there is no

1	When fasting, insulin levels remain in a relative steady state with minimal effects on glucose and protein metabolism. Even when insulin levels increase to three times basal levels, there is no increase in peripheral glucose uptake. Therefore, in the postoperative fast-ing state, without the assistance of exogenous insulin, peripheral Counterregulatory hormonescytokinesGlycogenolysisGlucoseLipolysisInsulinresistanceWhole-bodyprotein catabolismGluconeogenesisHypothalamusPituitaryGlycerolAmino acidsAcute-phaseproteinsHyperglycemiaPyruvateGlycolysisProteolysisLactateInjury siteAfferentsensorynervesSympathetic nervesHumoral agentsNeural impulsesFigure 50-3. Metabolic stress response to surgery results in insulin resistance. (Reproduced with permission from McGill University Health Center Patient Education Office, Montreal, Quebec, Canada.)Brunicardi_Ch50_p2113-p2136.indd 211601/03/19 9:39 AM 2117OPTIMIZING PERIOPERATIVE CARE: ENHANCED RECOVERY AND CHINESE MEDICINECHAPTER 50glucose

1	Education Office, Montreal, Quebec, Canada.)Brunicardi_Ch50_p2113-p2136.indd 211601/03/19 9:39 AM 2117OPTIMIZING PERIOPERATIVE CARE: ENHANCED RECOVERY AND CHINESE MEDICINECHAPTER 50glucose uptake is reduced. The resulting hyperglycemia can be corrected, however, with the use of exogenous insulin, and when normoglycemia is achieved in the perioperative period, the main components of metabolism also normalize.51The preoperative and postoperative fasting state triggers insulin resistance resulting in a catabolic state with gluconeo-genesis and protein breakdown. Following prolonged fasting states with stress such as following surgery, protein catabolism can be increased several fold over baseline. As less glycogen is stored in the muscle and loss of lean body mass occurs, there is less muscle function and therefore less capacity to mobilize.In addition to the typical metabolic effects of surgery, pain has been demonstrated to increase insulin resistance. In healthy male volunteers

1	muscle function and therefore less capacity to mobilize.In addition to the typical metabolic effects of surgery, pain has been demonstrated to increase insulin resistance. In healthy male volunteers undergoing painful stimulation, glucose uptake was reduced as a direct result of decreased insulin sensitivity.52 In addition, serum cortisol, epinephrine, and free fatty acids were all increased following painful stimulation.Elective surgery results in a state of insulin resistance, with the magnitude of surgery corresponding to a decrease in insulin sensitivity.53 For example, the difference in insulin sen-sitivity following laparoscopic cholecystectomy versus open cholecystectomy is 2.5-fold. More complex abdominal surgery such as an open colorectal resection results in a 3.5-fold increase in insulin resistance over laparoscopic cholecystectomy. As lev-els of insulin resistance increase, complications also increase.54 In addition to an association with complications, insulin resis-tance

1	insulin resistance over laparoscopic cholecystectomy. As lev-els of insulin resistance increase, complications also increase.54 In addition to an association with complications, insulin resis-tance has been shown to be an independent predictor of length of stay.53The increased postoperative complications associated with insulin resistance may be not only from the direct meta-bolic effects of insulin on glucose but also from free radical formation. In peripheral tissues that are independent of insulin metabolism, and therefore do not store glycogen, the increased plasma glucose levels result in greater glycolysis and oxygen free radical formation. This leads to alterations in gene expres-sion, which in turn propagates a cycle of increased inflamma-tion causing even more insulin resistance.55 Elective surgery has been implicated in increased inflammatory gene pathways and changes in insulin signaling genes in both adipose and skeletal muscle tissues.56,57Components of an ERP may offset

1	Elective surgery has been implicated in increased inflammatory gene pathways and changes in insulin signaling genes in both adipose and skeletal muscle tissues.56,57Components of an ERP may offset the metabolic and stress responses of surgery. Preoperative carbohydrate supple-mentation has been shown to counter the catabolic effects of the fasted state by stimulating glucose uptake and transition-ing metabolism to a more anabolic state with improved insulin sensitivity.58 Preoperative carbohydrate supplementation also reduces protein loss and improves muscle strength postopera-tively.59-61 When preoperative carbohydrate supplementation is added to epidural analgesia, there is even greater improvement in insulin resistance.62Preoperative Fasting and Preoperative Carbohydrate LoadingTraditionally, patients have been instructed to fast for 6 to 12 hours before surgery to reduce the risk of aspiration of gastric contents during the induction of anesthesia. This fasting state results in a

1	patients have been instructed to fast for 6 to 12 hours before surgery to reduce the risk of aspiration of gastric contents during the induction of anesthesia. This fasting state results in a prolonged period without nutrients or hydration prior to and during surgery, and it can lead to insulin resistance, hyper-glycemia, failure to achieve a postsurgical anabolic state, and sometimes, the need for insulin. Both European and American Societies of Anesthesiology guidelines have supported the use of clear liquid oral intake up to 2 hours prior to surgery with the exception of patients with gastroparesis, intestinal obstruction, or dysphagia.63-65 Carbohydrate oral intake up to 2 hours prior to surgery does not increase aspiration in healthy adults under-going elective surgery and in fact reduces preoperative hunger, thirst, anxiety, and nausea.66,67 In addition, a fasting time of 2 to 4 hours versus more than 4 hours actually results in smaller gastric volume and a higher gastric pH

1	reduces preoperative hunger, thirst, anxiety, and nausea.66,67 In addition, a fasting time of 2 to 4 hours versus more than 4 hours actually results in smaller gastric volume and a higher gastric pH value.68-75 Nevertheless, prior to the introduction of ERAS, the dogma of prolonged nothing-by-mouth status widely adopted many decades ago had little advocacy to change, despite evidence supporting more lib-eral fasting parameters. Current guidelines support fasting from clear liquids for 2 hours and solid food for 6 hours.Preoperative carbohydrate loading prior to surgery in the form of a carbohydrate rich clear liquid improves patient nausea and discomfort over preoperative water hydration or a prolonged fasting state.76 It may also have further benefits over low carbohydrate clear liquid beverages or fasting by changing the overnight fasting state to a fed state and thus altering glu-cose, protein, and fat metabolism by increasing postoperative insulin sensitivity.77-79 Furthermore, in

1	or fasting by changing the overnight fasting state to a fed state and thus altering glu-cose, protein, and fat metabolism by increasing postoperative insulin sensitivity.77-79 Furthermore, in patients whose expected length of stay is greater than 2 days, there is a significant length of stay reduction in patients that receive preoperative carbohy-drate loading.80 The best carbohydrate loading drink is unclear as studies are heterogeneous and the carbohydrate content is variable. Nevertheless, the carbohydrate drink should be hypo-osmolar for faster gastric emptying, result in a fed state with full glycogen stores, and reduce postoperative insulin resis-tance. The most commonly studied carbohydrate loading drink includes 100 g of carbohydrate the evening prior and 50 g of carbohydrate 2 to 3 hours prior to surgery.Intraoperative ConsiderationsSurgical Considerations. Prevention of surgical site infection consists of the use of mechanical, chemical, and/or antimicro-bial modalities.

1	to 3 hours prior to surgery.Intraoperative ConsiderationsSurgical Considerations. Prevention of surgical site infection consists of the use of mechanical, chemical, and/or antimicro-bial modalities. Mechanical and chemical methods include the use of patient bathing preoperatively and skin preparation with betadine, chlorhexidine, or similar chemical in order to limit the microbial content of the skin. Additionally, the appropriate use of antimicrobial prophylaxis should be employed and follow guidelines specific to the type of surgery and for duration of antibiotic prophylactic administration.Minimally invasive surgical approaches should be con-sidered as minimally invasive techniques have demonstrated improved outcomes across surgical specialties, including reduc-tions in length of stay and postoperative complications. Addi-tionally, the use of catheters or drains should be limited unless necessary, as these hinder the patient’s perceived ability for ambulation.81-84Hypothermia

1	stay and postoperative complications. Addi-tionally, the use of catheters or drains should be limited unless necessary, as these hinder the patient’s perceived ability for ambulation.81-84Hypothermia Prevention. Hypothermia is a common periop-erative problem. Up to 90% of all patients undergoing elective surgery suffer from inadvertent postoperative hypothermia.85,86 Those at highest risk include patients over the age of 60 years, and/or patients that have malnourishment, preexisting hypo-thermia, preexisting medical comorbidities that impair body temperature regulation (including advanced diabetes with neu-ropathy and hypothyroidism), who are undergoing general anes-thesia, and who are undergoing a major long surgery. Further, in patients who experience hypothermia, surgical complications are increased, including impaired wound healing, wound infec-tion, pressure ulcers, cardiac disorders including arrhythmia and infarction, as well as increased bleeding requiring blood transfu-sion

1	are increased, including impaired wound healing, wound infec-tion, pressure ulcers, cardiac disorders including arrhythmia and infarction, as well as increased bleeding requiring blood transfu-sion (Table 50-1).Brunicardi_Ch50_p2113-p2136.indd 211701/03/19 9:39 AM 2118SPECIFIC CONSIDERATIONSPART IITable 50-1Relative risk of elective surgical complications secondary to hypothermia86 RELATIVE RISK95% CONFIDENCE INTERVALSImpaired wound healing3.251.35–7.84Cardiac disorders4.491–20.16Blood transfusion1.331.06–1.66The reasons for hypothermia are multifactorial. Radiation, the transfer of heat by electromagnetic waves through space without a medium, accounts for 50% to 70% of heat loss. Con-vection, the loss of heat through ambient air stream, accounts for 15% to 25% of heat loss. Evaporation accounts for 5% to 20%, and conduction accounts for 3% to 5%.85 Temperature reduction can also be accelerated by cold intravenous fluids, low operating room temperatures, and a decreased

1	Evaporation accounts for 5% to 20%, and conduction accounts for 3% to 5%.85 Temperature reduction can also be accelerated by cold intravenous fluids, low operating room temperatures, and a decreased thermoregu-latory threshold, which occurs during the administration of gen-eral anesthesia. Further, the ability to compensate for reduction in body temperature is also compromised by muscle relaxation and anesthesia in general, as these processes impair shivering and thermoregulatory vasoconstriction.87 There are steps to take to prevent this hypothermia including active, convective heating using clean, filtered, forced-air warming blankets in patients in the preoperative area (prewarming) and also during anesthesia; thermal insulation; warmer ambient operating room tempera-tures, warmed irrigation solutions during surgery; and warmed infusions and blood products.88-99Venous Thromboembolism Prophylaxis. Venous throm-boembolism (VTE), which includes deep venous thrombosis (DVT) and

1	irrigation solutions during surgery; and warmed infusions and blood products.88-99Venous Thromboembolism Prophylaxis. Venous throm-boembolism (VTE), which includes deep venous thrombosis (DVT) and pulmonary embolism (PE), is the number one cause of potentially preventable death in common but preventable causes of morbidity and mortality in the perioperative patient. Several national quality improvement organizations have cited VTE prophylaxis for patients at risk as a priority for individual physicians and for hospitals because this intervention reduces adverse patient outcomes and hospital costs.Surgical patients have increased risk for VTE due to advanced age, multiple medical comorbidities, prolonged pro-cedure times, the inflammatory and hypercoagulable state of surgery, and immobility. Specific risk factors include major general, vascular, or orthopedic surgery; lower extremity paral-ysis due to spinal cord injury; fracture of the pelvis, hip, or long bones; multiple trauma;

1	Specific risk factors include major general, vascular, or orthopedic surgery; lower extremity paral-ysis due to spinal cord injury; fracture of the pelvis, hip, or long bones; multiple trauma; cancer; prior VTE; age 40 years and higher; obesity; immobility; oral contraceptive use; hypervis-cosity syndromes; and severe cardiopulmonary disease (prior myocardial infarction, congestive heart failure, chronic obstruc-tive pulmonary disease).Postoperative DVT is usually asymptomatic, and fatal PE can often be the first sign of VTE. DVT occurs after approxi-mately 25% of all major surgeries without prophylaxis, and PE occurs after 7%. Since screening modalities (such as venous duplex imaging) in asymptomatic patients have low sensitivity to detect clot, the best approach is to systematically apply pre-vention strategies to all patients undergoing surgery, with treat-ment choices based on patient-related and procedure-related risks.VTE prophylaxis is therefore an important component in

1	apply pre-vention strategies to all patients undergoing surgery, with treat-ment choices based on patient-related and procedure-related risks.VTE prophylaxis is therefore an important component in optimal perioperative care and current surgical practice and should be included in all practice guidelines. Appropriate VTE prophylaxis should be given preoperatively, intraopera-tively, and postoperatively based upon current guidelines for the surgery type.100 Examples of nonpharmacologic methods include early ambulation, graduated compression stockings, and intermittent pneumatic compression devices. Pharmaco-logic methods include the use of low dose unfractionated hepa-rin, low molecular weight heparin, and in some case, factor Xa inhibitors.Perioperative Fluid Management. Current and traditional fluid management strategies, which are based on a fixed fluid requirement per patient per case, have failed to improve out-comes. More modern goal-directed therapy (GDT) intravenous fluid

1	traditional fluid management strategies, which are based on a fixed fluid requirement per patient per case, have failed to improve out-comes. More modern goal-directed therapy (GDT) intravenous fluid approaches rely on the use of advanced medical devices, including esophageal Doppler monitors and other noninvasive cardiac output or bioimpedence models to determine whether or not patients are “fluid responsive” during surgery.101 In the setting of a normal ejection fraction, fluid is only administered when the expectation is that cardiac output will increase, and vasopressors are utilized if the aforementioned devices show fluid will not increase cardiac output. Excess fluid in certain general surgical cases can cause ileus and bowel edema, and in cardiac cases, it can cause hemodilution. Patients random-ized to restricted and liberal fluid resuscitation strategies found a clear linear relationship between total fluids administered (and weight gain) and complications following

1	Patients random-ized to restricted and liberal fluid resuscitation strategies found a clear linear relationship between total fluids administered (and weight gain) and complications following colorectal surgery including pulmonary edema and tissue-healing complications.102 Further multiple studies exist demonstrating fewer complica-tions with normovolemia than with liberal strategies of fluid resuscitation.102-108It must be understood that goal-directed therapy does, in no way, mean reduction in fluid administration. For some pro-cedures, it may be necessary to administer more than anticipated fluid volumes (orthopedics), while for others, the opposite may be true (abdominal). Normovolemia is important to maintain perfusion without volume overload. Thus, the idea behind goal-directed therapy is to maintain zero fluid balance coupled with minimal weight gain or loss. Hypovolemia is associated with reduced circulating blood volume, decreased renal perfusion, altered coagulation,

1	therapy is to maintain zero fluid balance coupled with minimal weight gain or loss. Hypovolemia is associated with reduced circulating blood volume, decreased renal perfusion, altered coagulation, microcirculation compromise, and endothe-lial dysfunction, among other processes. Hypervolemia is asso-ciated with splanchnic edema, decreased pulmonary gas exchange secondary to pulmonary edema, impaired wound heal-ing, anastomotic dehiscence, decreased mobility, altered coagu-lation, and endothelial dysfunction, amidst others processes109 (Fig. 50-4). Esophageal Doppler is a mode by which ultrasound is used to monitor and guide intraoperative fluid management has been used quite frequently. The use of this device for fluid optimi-zation has been studied in several randomized controlled trials or meta-analyses, all of which showed a significant reduction in length of stay of up to 4 days.110,111 Reductions in length of stay have been seen in gastrointestinal surgery, trauma surgery,

1	trials or meta-analyses, all of which showed a significant reduction in length of stay of up to 4 days.110,111 Reductions in length of stay have been seen in gastrointestinal surgery, trauma surgery, urologic surgery, and also the orthopedic population.112-116 Alter-native devices such as arterial waveform analyzers and pulse oximeter waveform analyzers have been studied and may be promising with the added advantage of lower cost over esopha-geal Doppler.Postoperatively, once the patient is adequately tolerat-ing at least a liquid diet and maintaining adequate hydration, 3Brunicardi_Ch50_p2113-p2136.indd 211801/03/19 9:39 AM 2119OPTIMIZING PERIOPERATIVE CARE: ENHANCED RECOVERY AND CHINESE MEDICINECHAPTER 50supplemental intravenous fluids should be minimized or ter-minated. The use of Dopplers or other volume status wave form analyzers have not been studied in the nonventilated postoperative patient and therefore cannot be used to reli-ably assess volume status. Clinical judgment

1	Dopplers or other volume status wave form analyzers have not been studied in the nonventilated postoperative patient and therefore cannot be used to reli-ably assess volume status. Clinical judgment based on patient factors, surgery type, and the clinical findings should be considered in the decision to continue intravenous flu-ids. However, once the patient is able to maintain adequate hydration, supplemental fluids should be used judiciously to limit fluid overload, tissue and lower extremity edema, and the constraints that the intravenous medication pole has upon patient-initiated ambulation.It is not enough to have normovolemia, but one must also consider the type of fluid that should be used for resuscitation. From a recent Cochrane review, there is no evidence that col-loids are superior to crystalloid for resuscitation in patients.117 Therefore, crystalloid fluids should generally be the primary intravenous fluid during the perioperative course. In cardiac sur-gery, the

1	are superior to crystalloid for resuscitation in patients.117 Therefore, crystalloid fluids should generally be the primary intravenous fluid during the perioperative course. In cardiac sur-gery, the utilization of 0.9% normal saline solution was associ-ated with hyperchloremia and poor postoperative outcomes, including higher length of stay and increased mortality.118 Fur-ther, a more balanced crystalloid, such as Plasma-Lyte, was associated with improved outcomes in 22,851 surgical patients.119 In this study, there was a 2.05 odds ratio predictor of mortality with normal saline. Other complications such as acute kidney injury, gastrointestinal complications, major hemor-rhage, and major infection were also increased in the group of patients that were hyperchloremic after normal saline adminis-tration. Based on such evidence, it would seem prudent to pro-ceed with a more balanced solution, such as PlasmaLyte, to reduce complications. Perioperative Pain ManagementAccording to the

1	adminis-tration. Based on such evidence, it would seem prudent to pro-ceed with a more balanced solution, such as PlasmaLyte, to reduce complications. Perioperative Pain ManagementAccording to the International Association for the Study of Pain (IASP) Taxonomy, the definition of pain is described as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage.”120 There are two important implications of this: pain is completely subjective in that it is whatever the patient says it is, and patients cannot experience pain while unconscious.121 This has some effects on how the anesthesiologist treats sympathetic stimulation in the operating room. Heretofore, elevations in heart rate and blood pressure were treated with opioid medica-tions, as these sympathetic markers were considered surrogates for patients experiencing pain while under general anesthesia. However, other medications can be used to treat the

1	with opioid medica-tions, as these sympathetic markers were considered surrogates for patients experiencing pain while under general anesthesia. However, other medications can be used to treat the sympa-thetic response to surgery such as β-blockers and deepening the anesthesia without administering opioid medications while the patient is unable to experience the pain.The mainstay of alleviating pain has historically relied almost exclusively on opioids, especially with the usage of patient controlled analgesia devices (PCAs). However, limiting opioids in the perioperative setting is of substantial benefit. Opi-oids, in fact, reduce pain immediately after administration. However, they also worsen pain scores after they wear off, increase postoperative opioid requirements, increase nausea and vomiting, cause respiratory depression, reduce gastrointestinal motility, worsen urinary retention, induce endocrine dysfunc-tion, and suppress the immune system.87 There have been a number of

1	and vomiting, cause respiratory depression, reduce gastrointestinal motility, worsen urinary retention, induce endocrine dysfunc-tion, and suppress the immune system.87 There have been a number of randomized controlled trials that have shown that as opioid administration increases, pain scores and postoperative nausea and vomiting increase.121-129 Exposure to any fentanyl or opioid in the operating room worsens postoperative pain scores and should therefore be limited or omitted.122,123,127,129 Opioid containing PCAs have been the standard for opioid administra-tion because of their safety and efficacy in patients to control the administration of opioids. However, because a PCA only offers opioid medication, there is the possibility that the desired analgesic effect will be associated with the aforemen-tioned complications of opioids. Despite their disadvantages, opioids are still quite useful in the treatment of pain. However, the ERAS protocols focus on opioids as a single component

1	the aforemen-tioned complications of opioids. Despite their disadvantages, opioids are still quite useful in the treatment of pain. However, the ERAS protocols focus on opioids as a single component of a comprehensive pain relief strategy, not as the mainstay for treatment. Instead, multimodal 45Complications andpostoperative morbidityNormovolemiaVolume statusHypervolemia consequences• Hyperchloremic acidosis• Pulmonary edema• Impaired wound healing• Anastamotic dehiscence• Decreased tissue perfusion• Altered coagulation• Multiple organ failureHypovolemia consequences• Reduced intravascular volume• Hypotension• Endothelial dysfunction• Altered coagulation• Decreased renal blood flow• HypoxiaFigure 50-4. Volume status affects postoperative morbidity. (Reproduced with permission from Francis N, Kennedy RH, Ljungqvist O, et al: Manual of Fast Track Recovery for Colorectal Surgery. London: Springer-Verlag; 2012.)Brunicardi_Ch50_p2113-p2136.indd 211901/03/19 9:39 AM 2120SPECIFIC

1	Francis N, Kennedy RH, Ljungqvist O, et al: Manual of Fast Track Recovery for Colorectal Surgery. London: Springer-Verlag; 2012.)Brunicardi_Ch50_p2113-p2136.indd 211901/03/19 9:39 AM 2120SPECIFIC CONSIDERATIONSPART IITable 50-2Analgesic medications ADVANTAGESDISADVANTAGESOpioids141Relieve pain immediately after administrationWorsen pain scores after opioids wear offIncrease postoperative opioid requirementsCentral Nervous System: Euphoria/dysphoria Sedation Respiratory depression Nausea/vomiting Cough suppressionCardiovascular: Bradycardia/tachycardia Arterial/venous dilationRenal: Antidiuretic Increased sodium resorption Urinary retentionGastrointestinal: Decreased motility Constipation Biliary colicEndocrine: Stimulates release of antidiuretic hormone Stimulates release of prolactin Stimulates release of somatotropin Decreases luteinizing hormoneImmunologic: Decreased immune system function Cancer growthNSAIDs142Reduce inflammationSynergistic effect with opioidsRenal

1	prolactin Stimulates release of somatotropin Decreases luteinizing hormoneImmunologic: Decreased immune system function Cancer growthNSAIDs142Reduce inflammationSynergistic effect with opioidsRenal insufficiencyIncreased bleedingDelay healingAdverse cardiovascular riskLocal anesthetics140Opioid-sparing effectDecrease PONVReduce ileusPossible anticancer effectCardiac toxicityCentral nervous system toxicityKetamine136,137,143Opioid-sparing effectMay prevent opioid-induced hyperalgesia and chronic pain syndromesDysphoriaHallucinationsGabapentinoids130-135Opioid-sparing effectReduce opioid side effectsReduce postoperative painVisual disturbancesanalgesia should be emphasized by utilizing multiple medi-cations to limit postoperative pain and therefore opioid use. Multimodal analgesia mitigates the side effects of opioids by opioid reduction and enhances pain management. Preoperative and postoperative administration of acetaminophen and cele-coxib or other nonsteroidal anti-inflammatory

1	the side effects of opioids by opioid reduction and enhances pain management. Preoperative and postoperative administration of acetaminophen and cele-coxib or other nonsteroidal anti-inflammatory drugs, as well as gabapentin have been shown to be efficacious.130-135 Intra-operatively, the utilization of ketamine, lidocaine, and magne-sium, act as adjunctive measures to limit pain and have been utilized to reduce the utilization of opioids in the postoperative period.136-140 Administration of lidocaine and ketamine can also be continued in the postoperative setting (Table 50-2).Neuraxial opioid analgesia, the administration of opioids through either the intrathecal or epidural route, can be accom-plished by either a single shot (both spinal and epidural) or catheter-based therapy (epidural). The use of opioids by this route was shown to have improved pain relief when compared to preoperative oral, IV, or intramuscular morphine.144 Further, neuraxial opioid analgesia is associated with

1	The use of opioids by this route was shown to have improved pain relief when compared to preoperative oral, IV, or intramuscular morphine.144 Further, neuraxial opioid analgesia is associated with lower postopera-tive pain scores in adults and children who undergo surgery.145 Neuraxial analgesia can also be performed with local anesthetic only. Finally, the American Pain Society (APS) recommends the utilization of such postoperative analgesic methods in patients who undergo major surgeries, including thoracic and abdominal Brunicardi_Ch50_p2113-p2136.indd 212001/03/19 9:39 AM 2121OPTIMIZING PERIOPERATIVE CARE: ENHANCED RECOVERY AND CHINESE MEDICINECHAPTER 50procedures, cesarean sections, and hip and lower-extremity sur-geries; this is especially recommended for patients at risk for cardiopulmonary complications or prolonged ileus.145Multimodal analgesia can also include regional analgesic techniques such as peripheral nerve blocks, paravertebral blocks, plexus blocks, and local

1	cardiopulmonary complications or prolonged ileus.145Multimodal analgesia can also include regional analgesic techniques such as peripheral nerve blocks, paravertebral blocks, plexus blocks, and local infiltration, which can reduce postoperative physiological stress and decrease complications associated with surgery as part of a regimen.146 These techniques have been shown to reduce the amount of opioids required for analgesia and also have been shown to reduce the adverse events seen with epidural local anesthetics (such as urinary reten-tion and hypotension) and/or opioid-containing PCAs. Postoperative Nausea and Vomiting PreventionPostoperative nausea and vomiting (PONV) is very common and can cause significant distress to patients, with the incidence of vomiting at approximately 30%, nausea at 50%, and the com-bination of PONV as high as 80%. All result in poor patient satisfaction, increased recovery room length of stay, and higher costs to the health-care system.147-152 This

1	nausea at 50%, and the com-bination of PONV as high as 80%. All result in poor patient satisfaction, increased recovery room length of stay, and higher costs to the health-care system.147-152 This could further increase the time to first feeding, which in turn may prolong ileus and/or hospital stay.Dr. Gan and colleagues developed a consensus guideline for the management of nausea and vomiting and details the risk and possible choices for the treatment of PONV.153 Risk factors include female sex, history of PONV or motion sick-ness, nonsmoking, younger age, general versus regional anes-thesia, use of volatile anesthetics and nitrous oxide, postoperative opioids, duration of anesthesia, and the type of surgery including cholecystectomy, laparoscopy, gynecologi-cal, and strabismus. The strategies for avoiding PONV include the avoidance of general anesthesia, the use of totally intravenous anesthesia, avoidance of nitrous oxide and volatile agents, minimizing intraoperative and

1	The strategies for avoiding PONV include the avoidance of general anesthesia, the use of totally intravenous anesthesia, avoidance of nitrous oxide and volatile agents, minimizing intraoperative and postoperative opioids, and adequate hydration.150,152,154-159 The medications to prevent, abort, and reduce PONV include perphenazine, aprepitant, dexamethasone, scopolamine, dolasetron, granisetron, and ondansetron, among others.160-164 PONV should be tar-geted before it occurs for optimal prevention (Fig. 50-5). Early Nutrition and Postoperative Ileus PreventionPostoperative ileus is the most common cause of prolonged hospital stay and readmissions following surgery on the diges-tive tract, occurring in up to 19% of cases.165 Not only is this adverse to the individual patient clinically, this also results in doubling of the total cost of the index hospital stay and thus carries a tremendous socioeconomic impact globally.166 Numer-ous risk factors contribute to postoperative ileus and

1	this also results in doubling of the total cost of the index hospital stay and thus carries a tremendous socioeconomic impact globally.166 Numer-ous risk factors contribute to postoperative ileus and include open surgery, increased surgery length of time, blood transfu-sion, fasting, fluid overload, opioids, postoperative nausea and vomiting, and other pharmacological agents. While some risk factors are unavoidable in certain patients, others are modifi-able, and therefore minimization of the risk of postoperative ileus is achievable.Nasogastric tubes (NGTs) were previously used prophy-lactically to prevent ileus, limit distension on the gastrointestinal anastomosis, as well as to prevent pulmonary complications. However, NGT use actually delays return of gastrointesti-nal activity and increases pulmonary complications without preventing anastomotic leaks in numerous types of surgery, including gastroduodenal, biliary, trauma, and esophageal.167-169 Therefore, the routine use of NGTs

1	pulmonary complications without preventing anastomotic leaks in numerous types of surgery, including gastroduodenal, biliary, trauma, and esophageal.167-169 Therefore, the routine use of NGTs for prophylaxis should be avoided.Addressing the numerous risk factors for postoperative ileus has a benefit on the reduction of the incidence of postoper-ative ileus. For example, mitigating the surgical trauma through 67Risk Factors:History of PONV/motion sicknessNonsmokerFemale sexPostoperative opioidsEmetogenic surgeryLow:Choose 1 treatmentMedium/High:Choose 2 treatmentsTotally intravenous anesthesiaRegional anesthesiaDexamethasoneAntidopaminergic5-HT3 antagonistDimenhydrinateScopolaminePatientPerphenazineFigure 50-5. Risk factors of and treatment options for postoperative nausea and vomiting. PONV = postoperative nausea and vomiting.Brunicardi_Ch50_p2113-p2136.indd 212101/03/19 9:39 AM 2122SPECIFIC CONSIDERATIONSPART IIminimally invasive surgery and meticulous surgery with mini-mal blood

1	= postoperative nausea and vomiting.Brunicardi_Ch50_p2113-p2136.indd 212101/03/19 9:39 AM 2122SPECIFIC CONSIDERATIONSPART IIminimally invasive surgery and meticulous surgery with mini-mal blood loss reduces postoperative ileus, either directly by limiting the inflammatory response with smaller incisions or indirectly through reduced opioid use.170-174 Anesthetic tech-nique can also aid in prevention of postoperative nausea and vomiting, which in turn reduces the need for parenteral opioids, a known risk factor for postoperative ileus. Multimodal pain strategies and neuraxial blocks reduce opioid use and therefore minimize nausea, improve early enteral nutrition, limit intra-venous fluid administration, and improve ambulation. Main-tenance of normovolemia in the perioperative setting should be achieved as fluid overload and dehydration both negatively affect return of bowel function, length of stay, and complica-tions.105,106,175 Clearly, each facet of the perioperative care

1	should be achieved as fluid overload and dehydration both negatively affect return of bowel function, length of stay, and complica-tions.105,106,175 Clearly, each facet of the perioperative care pro-cess is intricately intertwined to the next and has implications in total body homeostasis.Other measures may also assist in the prevention of post-operative ileus; however, the role of each in the setting of an ERAS pathway is unclear. Chewing gum is hypothesized to reduce postoperative ileus by stimulating the cephalovagal reflex and is considered a form of sham feeding. Prior to the introduction of ERAS, the use of chewing gum in multiple abdominal surgeries demonstrated faster intestinal recovery with variable impact on length of stay.176-178 Following the use of ERAS and associated early enteral feeding, the benefit of chewing gum is less clear.179,180 Alvimopan is a mu opioid recep-tor antagonist that is administered prior to surgery and twice daily postoperatively. Pooled analysis

1	enteral feeding, the benefit of chewing gum is less clear.179,180 Alvimopan is a mu opioid recep-tor antagonist that is administered prior to surgery and twice daily postoperatively. Pooled analysis of phase III trials dem-onstrated a reduction in postoperative NGT use, faster return of bowel function, and earlier discharge by 0.7 days.181 However, following ERAS implementation, small studies demonstrate a reduction in ileus and length of stay with the use of alvimopan in open surgeries without a benefit in laparoscopic surgery.182-184MobilizationThough no metric of mobilization has been clearly defined, early mobilization following surgery is an important compo-nent of ERAS that accelerates the return to baseline functional status. Prolonged postoperative bedrest leads to decondition-ing, increased deep venous thrombosis risk, and loss of muscle mass. Deterioration of mobility and activities of daily living can be seen in older patients after only 2 days of hospitalization.185

1	increased deep venous thrombosis risk, and loss of muscle mass. Deterioration of mobility and activities of daily living can be seen in older patients after only 2 days of hospitalization.185 Therefore, preoperative encouragement of an exercise program and perioperative mobilization can have dramatic impacts on not only the elderly but all patients undergoing surgery. Patients that begin a preoperative exercise program are more active post-operatively and have a faster return to baseline exercise capacity when compared to patients undergoing a postoperative exercise program.186 As patients in an ERAS program are directed to spend time out of bed and to ambulate, early mobilization is therefore encouraged. Many other facets of ERAS will assist in this early mobilization: postoperative nausea prevention, limit-ing drain use, and improved pain control. Setting preoperative expectations of mobility through patient education in the clinic setting and postoperative nursing unit engagement

1	prevention, limit-ing drain use, and improved pain control. Setting preoperative expectations of mobility through patient education in the clinic setting and postoperative nursing unit engagement in promot-ing mobility provide the proper setting for improved patient compliance with early and frequent mobilization. However, compliance with this is highly variable and difficult to track and may be hindered if pain in inadequately controlled or if the patient is tethered to devices such as drains, catheters, and IVs. As with other components of ERAS, engagement of all provid-ers of patient care from outpatient nursing, to inpatient nursing, physicians, and physical therapists, will improve compliance not only with mobility but often with other components as well. Reinforcement of expectations can be achieved with preopera-tive educational pamphlets, postoperative daily task lists, mobil-ity logs, and pedometers.ERAS in CRSAs the initial development of ERAS pathways occurred in colon

1	can be achieved with preopera-tive educational pamphlets, postoperative daily task lists, mobil-ity logs, and pedometers.ERAS in CRSAs the initial development of ERAS pathways occurred in colon surgery patients, the largest preponderance of data exists in this surgical specialty with the first ERAS guidelines developed in 2012 after many studies demonstrated positive outcomes apply-ing the basic tenets of ERAS surgery.81 In 1997, Henrik Kehlet published the initial series of patients, applying novel periopera-tive care strategies to colon resection patients.2,3 Following this, several groups also applied these principles to colon resection patients. These studies demonstrated that the principles of early mobilization, early feeding, and optimized intravenous fluid administration resulted in patients tolerating a diet sooner, faster return of bowel function, and earlier discharge.187-189 Further-more, these studies elucidated reduction in complications such as urinary tract infections,

1	patients tolerating a diet sooner, faster return of bowel function, and earlier discharge.187-189 Further-more, these studies elucidated reduction in complications such as urinary tract infections, ileus, and cardiopulmonary compli-cations. Nygren et al also demonstrated that muscle strength and lung function were less reduced after colon resection within an enhanced recovery protocol compared to traditional periopera-tive care.187 Additionally, as ERAS is expected to diminish the metabolic and hormonal stress response to surgery, attenuation of TNF-α, IL-1β, IL-6, and IFG-γ occurred after ERAS cases when compared to traditional perioperative care, and cortisol levels were not found to increase immediately postoperatively in ERAS, while those with traditional perioperative care experi-enced immediate and sustained cortisol elevation.190Larger series of patients followed with Delaney et al pub-lishing a single institutional experience of 1000 consecutive laparoscopic colectomy patients

1	immediate and sustained cortisol elevation.190Larger series of patients followed with Delaney et al pub-lishing a single institutional experience of 1000 consecutive laparoscopic colectomy patients with short length of stay and low readmission and mortality rates.191 The protocol was further applied to rectal surgery with success.187,192 Meta-analyses and systematic reviews demonstrate less opioid use, shorter length of stay, decreased morbidity, and no increase in readmission rates for laparoscopic or open colon or rectal resections when an ERAS protocol is utilized.193-197 Several groups have even discharged patients in as little as 24 hours following colon resec-tion, with Gignoux et al even discharging patients on the same day.198-200 ERAS can also be applied to octogenarian patients with compliance to the protocols and with no increased readmis-sion or mortality rates.201 Also, patients with diverting stomas can benefit from ERAS protocols, though diverting ileostomy may slightly

1	compliance to the protocols and with no increased readmis-sion or mortality rates.201 Also, patients with diverting stomas can benefit from ERAS protocols, though diverting ileostomy may slightly delay discharge over patients with no ileostomy.202Adherence to the numerous tenets of ERAS is inversely related to length of stay postoperatively in colorectal sur-gery.203,204 When compliance with ERAS measures is lower, length of stay is longer. The strongest predictors for shorter duration of stay include preoperative carbohydrate loading, no nasogastric tube, early mobilization, early oral nutrition, totally intravenous anesthesia, early removal of urinary catheter, and the use of nonopioid analgesia. Predictors for deviation from an ERAS program and thus resultant longer length of stay include pathologic diagnosis, intraoperative complications, high blood loss, surgery length, lack of mobilization, emesis, persistent use of intravenous fluids, reinsertion of urinary catheter, and poor

1	pathologic diagnosis, intraoperative complications, high blood loss, surgery length, lack of mobilization, emesis, persistent use of intravenous fluids, reinsertion of urinary catheter, and poor pain control.205-208 Though readmissions are no higher than traditional perioperative care, several factors have been impli-cated in readmission, including poor ERAS compliance and Brunicardi_Ch50_p2113-p2136.indd 212201/03/19 9:39 AM 2123OPTIMIZING PERIOPERATIVE CARE: ENHANCED RECOVERY AND CHINESE MEDICINECHAPTER 50preoperative neoadjuvant chemoradiation.209,210 Predictive tools using artificial neural networks may assist in clinical decision-making.209 The most common reasons for readmission include bowel obstruction and skin and soft tissue infection. While patient and quality outcomes are clearly important, the added benefit of cost savings has been demonstrated. Surgery using the tenets of ERAS is both less costly for direct and indirect costs to the hospital and effective.211

1	are clearly important, the added benefit of cost savings has been demonstrated. Surgery using the tenets of ERAS is both less costly for direct and indirect costs to the hospital and effective.211 Furthermore, patients in an ERAS program returned to work faster and had less caregiver burden, resulting in huge indirect savings as well.6ERAS in Hepatopancreaticobiliary SurgeryAn initial experience applying an ERAS protocol in 61 con-secutive patients undergoing liver resection demonstrated 92% of patients tolerating a diet on postoperative day 1, a reduction in length of stay from 8 to 6 days, and no increased readmis-sions or morbidity.212 A subsequent randomized trial in patients undergoing open major liver resection demonstrated a length of stay reduction from 7 to 4 days with a decrease in medically related postoperative complications and no increase in surgi-cal complications or readmission; ERAS patients also reported improved quality of life over controls.213 Several

1	a decrease in medically related postoperative complications and no increase in surgi-cal complications or readmission; ERAS patients also reported improved quality of life over controls.213 Several meta-analyses have similarly supported the use of ERAS protocols in liver surgery, citing reduced morbidity, hospital stays, cost, and time to recovery of bowel function without increasing mortality or readmission rates.214-216 When adherence to all elements of an ERAS protocol for liver resection was less, hospital length of stay was longer.217In 2012, the ERAS Society published recommendations for patients undergoing pancreaticoduodenectomy after several studies published early outcomes in this surgical population.82 Patients undergoing pancreaticoduodenectomy often have high rates of delayed gastric emptying; the use of ERAS has reduced the incidence of delayed gastric emptying by nearly half, thus allowing earlier feeding in this complex patient population.218,219 Additionally, multiple

1	gastric emptying; the use of ERAS has reduced the incidence of delayed gastric emptying by nearly half, thus allowing earlier feeding in this complex patient population.218,219 Additionally, multiple prospective cohort or retrospective stud-ies have shown that ERAS protocols offer significant benefit to patients undergoing both distal pancreatectomy and pancre-aticoduodenectomy with reduced hospital stay and complica-tions.220-226 Several meta-analyses or systematic reviews have confirmed shorter length of stays, decreased complications, and lower cost with ERAS protocols.227-230 Furthermore, the use of ERAS protocols in elderly patients undergoing pancreaticoduo-denectomy continue to show improved outcomes in length of stay and morbidity, showing that ERAS protocols allow this more fragile patient population to recover faster.231,232ERAS in Gastrectomy and EsophagectomyPatients undergoing foregut surgery have notoriously been sub-jected to prolonged periods of nasogastric tube

1	fragile patient population to recover faster.231,232ERAS in Gastrectomy and EsophagectomyPatients undergoing foregut surgery have notoriously been sub-jected to prolonged periods of nasogastric tube decompression and resultant starvation while surgical dogma dictated this fast-ing time diminished the risk of complications from anastomotic leak. Nevertheless, more liberal removal of the nasogastric tube and limited fasting, as components of ERAS protocols, have demonstrated improved recovery and outcomes. Randomized controlled trials demonstrate that removal of the nasogastric tube in the operating room and early feeding, as components in an ERAS program, result in shorter length of stay, fewer grade III or higher postoperative complications, and faster return to baseline weight and functional status.233,234 Multiple nonran-domized studies and meta-analyses verify reduction in length of stay and no increase in complications.235-238 In 2014, consen-sus guidelines for ERAS after

1	functional status.233,234 Multiple nonran-domized studies and meta-analyses verify reduction in length of stay and no increase in complications.235-238 In 2014, consen-sus guidelines for ERAS after gastrectomy were published, and these include no routine use of nasogastric decompression, early feeding within the first postoperative day, and early consider-ation for nutritional support if the patient is malnourished or unable to maintain at least 60% of caloric requirements.239Esophagectomy surgery is notoriously complicated and fraught with complications secondary to multiple factors includ-ing surgical complexity and medical comorbidity. Postoperative management is governed by the idiosyncrasies of the operating surgeon more so than many other specialties and therefore het-erogeneous. While studies of ERAS in gastrectomy suggest no routine use of nasogastric tubes and include early feeding, most ERAS programs for esophagectomy encompass all components not related to feeding, but

1	While studies of ERAS in gastrectomy suggest no routine use of nasogastric tubes and include early feeding, most ERAS programs for esophagectomy encompass all components not related to feeding, but prolonged nasogastric decompression remains.240 Many of these patients, however, do receive early enteral nutrition through the use of jejunostomy tubes com-monly placed at the time of resection. When defined protocols are followed in this cohort of patients, length of stay is reduced, and complications and readmissions are, at a minimum, not increased.241-243 Systematic reviews demonstrate a reduction in length of stay, anastomotic leak, and pulmonary complications without increased mortality or readmission.244,245ERAS in Bariatric SurgeryBariatric surgeons have applied clinical pathways to both the preoperative and postoperative periods for many years, which have resulted in improved outcomes. The adoption of ERAS protocols in these clinical pathways has offered further success for these

1	the preoperative and postoperative periods for many years, which have resulted in improved outcomes. The adoption of ERAS protocols in these clinical pathways has offered further success for these patients. A randomized trial for laparoscopic sleeve gastrectomy demonstrated a reduced length of stay to 1 day post-operatively in ERAS patients, and others have also discharged patients on postoperative day 1 following Roux-en-Y gastric bypass.246-248 Furthermore, earlier discharge of patients on post-operative day 1 has not been demonstrated to increase resource utilization, with no increase in patient phone calls, emergency department visits, or readmissions.249 A meta-analysis confirms success of ERAS in bariatric surgery with reduction of length of stay without increase in complication or complication sever-ity, while a second meta-analysis demonstrates an increase in minor complications without increasing patient morbidity.250,251 Following a thorough review of the literature

1	or complication sever-ity, while a second meta-analysis demonstrates an increase in minor complications without increasing patient morbidity.250,251 Following a thorough review of the literature supporting its use, the ERAS Society published guidelines for ERAS bariatric pro-tocols in 2016.84ERAS in Other Surgical SpecialtiesThough ERAS has been applied more broadly to complex abdominal surgery, there is surprising little data in its use in large ventral hernia repair and other abdominal wall reconstruc-tive techniques. Three studies report experience in open large ventral hernia repair with varying techniques of abdominal wall reconstruction including myofascial release.252-254 Each study cites faster return of gastrointestinal function and reduction in length of stay by up to 2.5 days. Furthermore, there were no increases in readmission, postoperative complications, or reop-eration. Though no long-term follow-up, there is no report that early feeding results in intestinal compromise

1	there were no increases in readmission, postoperative complications, or reop-eration. Though no long-term follow-up, there is no report that early feeding results in intestinal compromise from “tight” clo-sure or early hernia recurrence. In fact, as ERAS reduces the incidence of postoperative vomiting and ileus, it is likely very beneficial for this patient population.ERAS has been introduced to non–general surgery subspe-cialties as well. Complex urological procedures such as radical cystectomy have trialed ERAS over the last decade with favor-able results. In this patient population, length of stay was reduced, and complications were similar to or reduced when Brunicardi_Ch50_p2113-p2136.indd 212301/03/19 9:39 AM 2124SPECIFIC CONSIDERATIONSPART IIcompared to baseline controls.255-257 Similarly, ERAS has been applied to both minimally invasive and open complex cytore-ductive gynecological oncology surgery with favorable results for earlier discharge and decreased pain.258-263

1	Similarly, ERAS has been applied to both minimally invasive and open complex cytore-ductive gynecological oncology surgery with favorable results for earlier discharge and decreased pain.258-263 Total joint replacement surgery in orthopedics has also used ERAS with improved early mobility, a significant length of stay reduc-tion, and decreased morbidity.264,265 Setting Up an ERAS ProgramThe successful implementation of an enhanced recovery pro-gram depends heavily upon cultural change and excellent orga-nizational behavior. As the ERAS program encompasses so many facets of patient care, the implementation team should not only include surgeons and anesthesiologists but also inpa-tient and outpatient nurses, pharmacists, information technology specialists, compliance officers, and hospital administration.Initial strategy for ERAS implementation should define the scope of practice change by identifying the current state and the goal state of care. Protocol content can be discussed in a

1	administration.Initial strategy for ERAS implementation should define the scope of practice change by identifying the current state and the goal state of care. Protocol content can be discussed in a small group of engaged stakeholders. Once the protocol ele-ments are defined, all stakeholders should review and discuss the protocol in detail in order to identify barriers to implementa-tion, identify solutions to these barriers, and finalize the proto-col. This allows for all stakeholders to remain engaged and have ownership in the protocol. Appropriate informational resources for hospital and office staff education are created in order to have a thorough and successful educational campaign. Patient informational resources should also be developed in order to set clear expectations throughout the perioperative process. Addi-tionally, and importantly, standardized order sets are also devel-oped to ensure that all components of an ERAS program have little variation in order to improve

1	the perioperative process. Addi-tionally, and importantly, standardized order sets are also devel-oped to ensure that all components of an ERAS program have little variation in order to improve compliance. A final imple-mentation date is defined after coordinating that all stakeholders are indeed ready for launch. Appropriate educational campaigns are performed in a timely fashion prior to final implementation to outpatient office staff, inpatient units, preoperative and post-operative care units, operating room personnel and physicians, and midlevel providers and trainees (Fig. 50-6).Compliance and auditing should be done with relative frequency in the beginning, from weekly to biweekly and then monthly. Team meetings with all stakeholders present allow the team to address any issues in timely fashion with a mul-tidisciplinary approach and thus upholds accountability. The frequency of checkpoint meetings can be reduced over time. Sharing data of successes and failures keeps the team

1	timely fashion with a mul-tidisciplinary approach and thus upholds accountability. The frequency of checkpoint meetings can be reduced over time. Sharing data of successes and failures keeps the team engaged. It is also recommended that a financial team be employed. The financial savings after adoption of an ERAS protocol can be substantial from reduction in length of stay, medication use, and resource utilization. Ideally, a portion of the cost savings should be funneled back into the ERAS program in order to ensure program maintenance and compliance and also to assist with expansion to other service lines or patient care improvement projects.TRADITIONAL CHINESE MEDICINE IN SURGICAL PATIENTSHistory of Traditional Chinese MedicineTraditional Chinese medicine is one of China’s outstanding national cultural heritages and the quintessence of China, shar-ing a deep history and common homology with the Chinese culture.266 It is derived from the rich experience and theoretical knowledge

1	national cultural heritages and the quintessence of China, shar-ing a deep history and common homology with the Chinese culture.266 It is derived from the rich experience and theoretical knowledge that Chinese people have used to combat disease for thousands of years. The achievements of traditional Chinese medicine caught the attention of the world because it examines each function of the human organism and adjusts those func-tions to achieve ultimate balance.Traditional Chinese medicine is the oldest medicine in China. It is a medical system with unique theory, style, diagno-ses, and treatments, which were gradually formed throughout the historical medical practice of the Chinese nation. Its devel-opment not only depended on the practice but also resulted from the systemic mode of thinking and Chinese philosophy of protecting life shape. Yet, it is a traditional subject that still stands in the modern world of science.Traditional Chinese medicine theory mainly resulted from the

1	and Chinese philosophy of protecting life shape. Yet, it is a traditional subject that still stands in the modern world of science.Traditional Chinese medicine theory mainly resulted from the summary of practice and was continuously enriched and developed in practice. As early as 2000 years ago, Huang Di Nei Jing wrote the earliest existing theory of traditional Chinese medicine in China, and it summarized the treatment experience and medical theory to that time. Combining the achievements of other natural science branches and simple materialism and dialectical thinking of the Chinese culture, Huang Di Nei Jing comprehensively expounded the knowledge of human anatomy, physiology, and pathology and also stated the diagnosis, treat-ment, and prevention of disease, thus founding the preliminary theoretical basis of traditional Chinese medicine.Based on herbal remedies of primitive people, Shen Nong Ben Cao was the earliest existing monograph on herbal pharma-cology in China.267 It summed

1	theoretical basis of traditional Chinese medicine.Based on herbal remedies of primitive people, Shen Nong Ben Cao was the earliest existing monograph on herbal pharma-cology in China.267 It summed the study of herbology to date, including 365 kinds of drugs until the Han Dynasty. Long-term clinical practice and modern scientific research show that the effects of the drugs described in the book are mostly correct.In the third century, the famous physician Zhongjing Zhang of the Eastern Han Dynasty delved into the classical medical books such as Su Wen, Zhen Jing, and Nan Jing and extensively collected the effective prescriptions, combining this with his own clinical experience. He published a famous book about typhoid fever, Shang Han Bing Za Lun. This book established the theoretical system and treatment principles that applied dialectical therapy of Chinese medicine and influenced the future of traditional Chinese medicine.In 610 a.d., Yuanfang Chao et al wrote Treatise on the

1	system and treatment principles that applied dialectical therapy of Chinese medicine and influenced the future of traditional Chinese medicine.In 610 a.d., Yuanfang Chao et al wrote Treatise on the Etiology of Various Diseases, which is the earliest exiting mono-graph on causes of symptomatology in China. This book also recorded the intestinal anastomosis, abortion, tooth extraction, and other operations, indicating the breadth of surgery practiced at that time. Subsequently, the ancient Chinese government in 659 a.d. issued Tang Xiu Ben Cao; it was not only the first phar-macopoeia of ancient China but also the first national pharma-copoeia of the world. It was published 883 years earlier than the Nuremberg Pharmacopoeia, which was issued by the European Nuremberg government in 1542 a.d.The Tang Dynasty physician Simiao Sun authored Bei Ji Qian Jin Yao Fang and Qian Jin Yi Fang. In these two books, clinical subjects, acupuncture, dietary therapy, disease preven-tion, and life

1	a.d.The Tang Dynasty physician Simiao Sun authored Bei Ji Qian Jin Yao Fang and Qian Jin Yi Fang. In these two books, clinical subjects, acupuncture, dietary therapy, disease preven-tion, and life preservation were discussed. These were an out-standing achievement of the time, especially in the prevention and treatment of nutritional deficiency diseases.Between the 12th and 14th centuries, i.e., the Jin and Yuan eras of China, several new Chinese medicine theories emerged. There were four representative scholars. Wansu Liu (1120–1200 a.d.) thought the symptoms of shanghan (exogenous febrile disease) were related to “excessive internal heat,” so the herbal 8Brunicardi_Ch50_p2113-p2136.indd 212401/03/19 9:39 AM 2125OPTIMIZING PERIOPERATIVE CARE: ENHANCED RECOVERY AND CHINESE MEDICINECHAPTER 50Figure 50-6. Implementation process of an enhanced recovery after surgery pathway. ERAS = enhanced recovery after surgery; CRNA = certified registered nurse anesthetist; NP = nurse

1	MEDICINECHAPTER 50Figure 50-6. Implementation process of an enhanced recovery after surgery pathway. ERAS = enhanced recovery after surgery; CRNA = certified registered nurse anesthetist; NP = nurse practitioner; PA = physician assistant; IT = information technology; PACU = postanesthesia care unit; APP = advanced practice provider; LOS = length of stay; PCA = patient-controlled analgesia.Define ERAS teamSite workflow meetingsCommunication & trainingImplementation & initial monitoringDefine site-specific teamleaders:• Anesthesiologist• Surgeon• Quality nurse coordinatorDefine implementation teammembers, including:Anesthesiologists, surgeons,nurses, CRNAs, NPs, PAs,fellows, residents, ITIntro team meeting• General ERAS overview with core leadership teamDiscuss protocol logistics and workflow:• Site meetings to discuss logistics of implementing ERASEscalation of site issues tocore team• Site leadership to escalate issues to leadership teamImplementation planning:• Site leadership team

1	Site meetings to discuss logistics of implementing ERASEscalation of site issues tocore team• Site leadership to escalate issues to leadership teamImplementation planning:• Site leadership team to ensure all workflow issues have been addressed and implementation date plannedProtocol & order set training:• Clinic• Pre-Op nurses• PACU nurses & anesthesia• Floor nurses• APPsMonthly monitoring of keyoutcomes × 3 mos(minimum):Including: Overall LOS, Post-OpLOS, 30-day readmission andPCA utilization, and othersMonthly meeting × 3 mos(minimum):• Discussion of initialimplementation issues• Include all members of site-specific implementation teamImplement protocol &order set go-liveMonthly checkpointmeeting with ERASleadershipGo-live communication:ERAS site coordinator tocommunicate go-live date forprotocol & order set to siteteam and ERAS projectmanagerGo-live date must be givento ERAS PM at least 1month prior to go-liveERAS nurse educator toliaise with site championsERAS order set to

1	date forprotocol & order set to siteteam and ERAS projectmanagerGo-live date must be givento ERAS PM at least 1month prior to go-liveERAS nurse educator toliaise with site championsERAS order set to beimplemented up to twoweeks prior to theprotocol for order entryERAS leadership teamto provide outcomesto ERAS site teamBrunicardi_Ch50_p2113-p2136.indd 212501/03/19 9:39 AM 2126SPECIFIC CONSIDERATIONSPART IIcharacteristics of cold and cool were used in treatment, and this was known as the “cold and cool” treatment style. Congzheng Zhang (approximately 1156–1228 a.d.) thought the cause of disease was “exogenous evil” (exogenous pathogenic factor) invading the human body; therefore, the treatment focused on “eliminating evil,” through the diaphoresis, emetic, and/or purgative methods, which were known as the “offensive pre-cipitation” style. Dongyuan Li (1180–1251 a.d.) proposed that “internal injury of viscera causes various diseases,” and pyretic tonification of the spleen and

1	which were known as the “offensive pre-cipitation” style. Dongyuan Li (1180–1251 a.d.) proposed that “internal injury of viscera causes various diseases,” and pyretic tonification of the spleen and stomach was emphasized in the treat-ment, known as the “invigorating the spleen” style. Zhenheng Zhu (1281–1358 a.d.) thought “yang was always excessive, while yin was always insufficient” in the body, so the treatment was focused on nourishing yin and cutting down heat, which was known as the “nourishing yin” style.In approximately the 11th century, the Chinese began to use human pox vesicle exposure as vaccination to prevent smallpox and thus pioneered the field of medical immunology and vaccination. In the 17th to 19th centuries, due to the epi-demics of infectious diseases, the seasonal febrile disease theory developed. This theory broke the traditional Chinese medicine long-term conception that the pathogens invaded from the sur-face to the inside of the body. In the mid-17th century

1	disease theory developed. This theory broke the traditional Chinese medicine long-term conception that the pathogens invaded from the sur-face to the inside of the body. In the mid-17th century when bacteriology had not yet appeared, this was undoubtedly a great pioneering work and laid the groundwork for the epidemiology of the spread of disease.In the period from the Opium War (1838–1842 a.d.) to the founding of the People’s Republic of China in 1949 a.d., Western medicine was introduced to the continent of China. However, Western medicine and Chinese medicine theories were unique to each other. There was almost no interchange between them, either in theory or in practice; yet together, they formed a unique and relatively mature theoretical system. In the 1920s to 1930s, Western medicine proposed abolishing Chinese medicine. At the same time, Chinese medicine did not deny the merits of Western medicine but thought that Chinese medicine was superior to Western medicine. However,

1	proposed abolishing Chinese medicine. At the same time, Chinese medicine did not deny the merits of Western medicine but thought that Chinese medicine was superior to Western medicine. However, there were several advocates to combine the theories who published such works as “Chinese Medicine for Main, Western Medicine for Use,” “Using Their Respective Strengths, Reaching the Same Goal by Different Means,” and “Chinese Medicine Treat Internal Disease, Western Medicine Treat Surgical Disease.”268 In short, the pervasive opinion was to learn from the other’s strong points to make up for one’s deficiencies. This forged the trail for establishing modern Chinese integrative medicine.In the past decade, a series of significant progresses and breakthroughs have been made in the modern study of tradi-tional Chinese medicine theory and its application to clinical practice. For example, acupuncture anesthesia can be used for small splint fixation, and the treatment of acute abdomen inte-grates

1	Chinese medicine theory and its application to clinical practice. For example, acupuncture anesthesia can be used for small splint fixation, and the treatment of acute abdomen inte-grates both traditional Chinese and Western medicine.269 Most lately, Tu Yo Yo’s team found that artemisinin, derived from the wormwood plant, treats malaria, and the team was awarded the Nobel Prize in 2015.Clearly, integration of traditional Chinese medicine with modern science and technology will advance knowledge and treatment. Modern diagnostic instruments and techniques have become auxiliary methods of clinical diagnosis and treatment of traditional Chinese medicine, making up for the deficiency of the traditional “four ways of diagnosis” methods and improv-ing the accuracy and efficiency of traditional Chinese medicine diagnosis and treatment. Thus, modern science and technology’s infiltration, transformation, and integration in all aspects of Chi-nese medicine will be one of the distinctive

1	Chinese medicine diagnosis and treatment. Thus, modern science and technology’s infiltration, transformation, and integration in all aspects of Chi-nese medicine will be one of the distinctive characteristics of the future development of Chinese medicine.In summary, traditional Chinese medicine and pharmacy are an important part of the splendid culture of the Chinese nation, making outstanding contributions over thousands of years because of its systemic theory, distinctive treatment methods, sig-nificant efficacy, and abundant historical documentation. Modern Chinese medicine includes traditional Chinese medicine and thus offers integrated and superior health service. This integration of Eastern and Western medical ideas and philosophies are important for the future of this modern medical era.270 Preoperative Nutritional OptimizationMany surgical abdominal diseases have a long incubation period before clinical presentation, during which time the patients may develop malnutrition

1	era.270 Preoperative Nutritional OptimizationMany surgical abdominal diseases have a long incubation period before clinical presentation, during which time the patients may develop malnutrition including specific nutrient deficiencies and hypoproteinemia. These comorbidities directly influence the sur-gical treatment and postoperative effect of the patient undergoing an operation. In this setting, some experts advocate the use of parenteral nutrition and other adjunctive nutritional measures. These measures can often improve the patient’s nutritional sta-tus, but they are difficult to popularize because of the high cost of treatment and common complications with parenteral nutrition.In traditional Chinese medicine theory, it is thought that there are a variety of “asthenic symptoms” in patients who need surgical treatment and that applying the treatment principle of “treating deficiency with tonification” improves conditions throughout the body. On the basis of the traditional Chinese

1	who need surgical treatment and that applying the treatment principle of “treating deficiency with tonification” improves conditions throughout the body. On the basis of the traditional Chinese medicine theory of the “concept of holism” and “treatment according to syndrome differentiation,” the all nourishing decoction (Shiquan Dabu decoction) and Buzhong Yiqi decoc-tion are used in patients with the “deficiency of vital energy and blood syndrome” before surgery and have achieved good results.271-275 Similar success has been shown with the Shenmai injection and Astragalus injection.276,277 For example, when tra-ditional Chinese medicine is used effectively to treat patients with breast cancer before an operation, it enhances the general body status, improves the patient’s energy, and regulates the liver and kidney functions, which ultimately promotes the suc-cess of the operation and controls progression of the tumor.278,279Bowel Preparation for SurgeryIn gastrointestinal surgery, the

1	the liver and kidney functions, which ultimately promotes the suc-cess of the operation and controls progression of the tumor.278,279Bowel Preparation for SurgeryIn gastrointestinal surgery, the method of “purgation and offen-sive precipitation” is used in preparation for the operation. Either Large Chengqi decoction or Seasoning Chengqi decoc-tion significantly increases the gastrointestinal motility and washes the gastrointestinal stagnation to prepare the bowel for surgery. These decoctions also improve visceral blood flow and peritoneal absorption, promote early recovery of postoperative bowel function, and prevent superimposed infection of the intes-tine and the effect of endotoxin.280,281Preoperative Optimization During Sepsis and InfectionDue to infection, endotoxemia, blood loss, and other factors, many critically ill patients will deteriorate or progress to shock. Initial surgery during profound shock may be counterproductive until adequate resuscitation is achieved. In

1	and other factors, many critically ill patients will deteriorate or progress to shock. Initial surgery during profound shock may be counterproductive until adequate resuscitation is achieved. In addition to resuscita-tion, blood transfusion, and antimicrobial treatment if indicated for sepsis, the traditional Chinese medicine treatment based on dialectics can help to create a favorable condition for surgery, if indicated.9Brunicardi_Ch50_p2113-p2136.indd 212601/03/19 9:39 AM 2127OPTIMIZING PERIOPERATIVE CARE: ENHANCED RECOVERY AND CHINESE MEDICINECHAPTER 50For traumatic shock and anaphylactic shock patients, the use of flavored pure ginseng decoction supplemented by blood transfusion and other comprehensive antishock measures, can rapidly raise blood pressure and provide a more optimal set-ting for surgical treatment.282 Qingdan decoction and Qingyi decoction, which are composed of herbs to clear away heat and toxins, remove stasis, purge the bowel, and are used to treat acute

1	set-ting for surgical treatment.282 Qingdan decoction and Qingyi decoction, which are composed of herbs to clear away heat and toxins, remove stasis, purge the bowel, and are used to treat acute obstructive suppurative cholangitis and hemorrhagic and/or necrotizing pancreatitis.283-285Perioperative Pain ManagementThe application of traditional Chinese medicine in perioperative pain management is has become more prevalent in recent years. Research of acupuncture for analgesia began in the 1950s in China and has developed into a combined acupuncture and medicine anesthesia that is currently recognized by the medical field.286 During surgery, the combined anesthesia is composed of acupuncture and opioid drugs. Experimental data show that the combined acupuncture anesthesia could reduce the dose of opioid by 50%.287 Acupuncture also helps the management of postoperative pain by enhancing the level of the endogenous opioid, encephalin. Encephalin restrains the pain signal from being

1	the dose of opioid by 50%.287 Acupuncture also helps the management of postoperative pain by enhancing the level of the endogenous opioid, encephalin. Encephalin restrains the pain signal from being transmitted to the central nervous system, blocks the body’s reaction to pain, and increases the pain threshold accord-ingly.288 In several clinical trials, the needed doses of opioid at 8, 24, and 72 hours postoperatively were reduced in acupuncture groups compared to control groups.289 In addition, electroacu-puncture has been demonstrated to be effective at alleviating postoperative pain and assisting in recovery. One randomized controlled trial shows electroacupuncture significantly reduced the dose of fentanyl used, improved the quality of recovery and decreased the incidence of anesthesia related side effects for patients undergoing surgery.290 Postoperative Nausea and Vomiting PreventionPONV is a common complication after surgery. Acupuncture and herbs can be applied to prevent and

1	related side effects for patients undergoing surgery.290 Postoperative Nausea and Vomiting PreventionPONV is a common complication after surgery. Acupuncture and herbs can be applied to prevent and treat PONV. Acupunc-ture or transcutaneous electroacupuncture improves stress-induced impairment in gastric motility functions, significantly inhibits the frequency of transient lower esophageal sphincter relaxations in response to gastric distention, and suppresses retrograde peristaltic contraction.291-293 In clinical trials, intra-operative P6 acupuncture point (Neiguan acupoint) stimulation during surgery significantly reduces the incidence of PONV over 24 hours, and the efficacy of P6 stimulation is similar to that of commonly used antiemetic drugs in the prevention of PONV.294 Furthermore, electroacupuncture restrains the release of gastrointestinal peptides and consequently relieves PONV with an efficacy comparable to ondansetron.295 Moreover, a randomized, prospective,

1	Furthermore, electroacupuncture restrains the release of gastrointestinal peptides and consequently relieves PONV with an efficacy comparable to ondansetron.295 Moreover, a randomized, prospective, double-blinded clinical trial shows that auricular acupressure within 24 hours postoperatively has a similar effect to prevent PONV.296Additionally, some Chinese herb decoctions, such as Liu Jun Zi decoction and Cheng Qi decoction, administrated periop-eratively, show possible effectiveness to reduce the severity of PONV and to relieve abdominal distension.297,298Early Nutrition and Postoperative Ileus PreventionIn traditional Chinese medicine, acupuncture and decoctions have a role in the prevention of postoperative ileus. For example, early acupuncture on Zusanli, Shangjuxu, and Xijuxu acupoints, combined with early enteral nutrition, can effectively improve gastrointestinal function and shorten the length of stay after sur-gery.299 In addition, Dachengqi decoction applied to patients

1	combined with early enteral nutrition, can effectively improve gastrointestinal function and shorten the length of stay after sur-gery.299 In addition, Dachengqi decoction applied to patients after laparotomy improves gastric dysrhythmia, promotes intestinal peristalsis, and enhances gastrointestinal motility.300,301 Further-more, in one randomized trial, the combination of Simo decoc-tion and acupuncture reduces the incidence of postoperative ileus and shortens hospital stay for patient undergoing abdominal sur-gery when compared to the perioperative use of chewing gum.302 Other traditional Chinese medicine methods such as electroacu-puncture combined with Evodia hot compress, confers benefit in postoperative recovery of gastrointestinal function of patients who have undergone abdominal surgery.303Traditional Chinese Medicine in Common Surgical ConditionsColon Surgery. Several traditional Chinese medicine decoc-tions can assist in bowel preparation prior to surgery. For exam-ple,

1	surgery.303Traditional Chinese Medicine in Common Surgical ConditionsColon Surgery. Several traditional Chinese medicine decoc-tions can assist in bowel preparation prior to surgery. For exam-ple, during the bowel cleansing before surgery, Dachengqi decoction can be used to promote bowel peristalsis and evacua-tion, thus preventing contamination during surgery and reducing the risk of postoperative complications of infection. Addition-ally, after colon surgery, traditional Chinese medicine therapies such as acupuncture and decoctions prevent postoperative ileus, reduce the incidence of PONV, and promote the recovery of colon function. As a result, traditional Chinese medicine can shorten the hospital stay after colon surgery.Appendicitis. In general, patients suffering from acute appen-dicitis will undergo appendectomy. Exceptionally, when a case of acute simple appendicitis or a periappendiceal abscess is encountered, Chinese herbs together with antibiotics can serve as an

1	appen-dicitis will undergo appendectomy. Exceptionally, when a case of acute simple appendicitis or a periappendiceal abscess is encountered, Chinese herbs together with antibiotics can serve as an alternative treatment. In early acute simple appendicitis, oral Qinghua decoction can help the appendix infection resolve without surgical management.304 This nonsurgical treatment also can be applied to patients with periappendiceal abscess that is unsuitable for surgery. These Chinese medicine herbs activate blood flow, dissolve stasis, clear heat, and remove toxicity.Biliary Disease. Cholelithiasis is a common disease of the biliary tract that can result in cholecystitis and the possible need for cholecystectomy. Several traditional Chinese medicine herbs can relieve the symptoms of acute cholecystitis and delay the progression of the disease, possibly preventing the need for cholecystectomy. These Chinese medicine herbs are composed of herbs for clearing heat and secreting bile (Qing Re

1	and delay the progression of the disease, possibly preventing the need for cholecystectomy. These Chinese medicine herbs are composed of herbs for clearing heat and secreting bile (Qing Re Li Dan), herbs for promoting circulation of Qi and relieving pain (Xing Qi Zhi Tong), and herbs for clearing heat and promoting diure-sis (Qing Re Li Shi). According to different Chinese medicine therapies, the use of these herbs can also be applied to patients postoperatively from abdominal or biliary surgery to adjust bili-ary excretion and/or prevent cholestasis.Unfortunately, severe cholecystitis or cholangitis may progress to liver abscess. When a liver abscess develops, per-cutaneous catheter drainage and Chinese medicine herbs are applied in Chinese medicine practice.305 Generally, in the early stage of abscess, Chinese medicine herbs are used for clearing heat and removing toxicity (Qing Re Jie Du) and for promoting blood circulation and removing blood stasis (Huo Xue Hua Yu). When abscess

1	stage of abscess, Chinese medicine herbs are used for clearing heat and removing toxicity (Qing Re Jie Du) and for promoting blood circulation and removing blood stasis (Huo Xue Hua Yu). When abscess is evident, Chinese medicine herbs for clearing heat and cooling blood are added.305 The main function of these Chinese medicine herbs is to relieve infection, reduce inflam-mation, and activate intestinal motility in order to evacuate the 10Brunicardi_Ch50_p2113-p2136.indd 212701/03/19 9:39 AM 2128SPECIFIC CONSIDERATIONSPART IItoxicity, reduce the inflammatory response, and thus hasten recovery.Pancreas Surgery. In recent years, the early use of traditional Chinese medicine and enteral nutrition treatment in patients after pancreaticoduodenectomy, can help to hasten recovery in gastrointestinal function, improve nutritional status and immune function, and also reduce postoperative complications. The combined use of total parenteral nutrition and Astragalus injection can improve the

1	function, improve nutritional status and immune function, and also reduce postoperative complications. The combined use of total parenteral nutrition and Astragalus injection can improve the nutrition status of patients with obstructive jaundice and improve the immune function of these patients.306 All Nourishing (Shiquan Dabu) decoction has the role of increasing the level of plasma albumin and hemoglobin, which can be used as a recipe in surgical nutrition therapy.307Intestinal Obstruction. Adhesive ileus is the most com-mon type of small intestinal obstruction and also is the kind to which traditional Chinese medicine therapies apply widely. The methods of traditional Chinese medicine treatment include acupuncture, Chinese herbal enema, and gastrointestinal intuba-tion. Acupuncture or transcutaneous electroacupuncture applied to acupoints such as Zusanli point, Neiguan point, Zhongwan point, and Tianshu point has remarkable regulatory effects on gastrointestinal function and can

1	transcutaneous electroacupuncture applied to acupoints such as Zusanli point, Neiguan point, Zhongwan point, and Tianshu point has remarkable regulatory effects on gastrointestinal function and can promote relief of obstruction.308 Dachengqi decoction combined with the Chinese medicine herbs for clearing heat and removing toxicity (Qing Re Jie Du), and for promoting blood circulation and removing blood stasis (Huo Xue Hua Yu), can enhance gastrointestinal motility, improve blood circulation of the intestine, reduce intestinal capillary perme-ability, protect the barrier function of the intestinal mucosa, and help inflammatory edema to resolve.309 Additionally, acupunc-ture, Chinese herbal enema, and gastrointestinal intubation can shorten the time of the obstructive event and reduce the length of stay in patients suffering from adhesive intestinal obstruction.310 These methods of traditional Chinese medicine treatment can also be applied to the treatment of postoperative

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1	of Houpupaiqi Heji on the gastrintestinal function of patients after gastric cancer surgery. Mod Oncol. 2015;23:1268-1270. 299. Zhang X, Yuan H, Qin W. Study on early enteral nutrition combined with acupuncture to promote the recovery of gastro-intestinal function in patients after surgery. Chinese Journal of Surgery of Integrated Traditional and Western Medicine. 2016;22:542-545. 300. Jian W, Heng L, Hui QQ. Effect of Da-Cheng-Qi-Tang on gastrointestinal motility in patients undergoing laparotomy. Hepatogastroenterology. 2011;58:1887-1892. 301. You S, Wu X, Liu M. Effects of dachengqi decoction on gut hormones and intestinal movement after cholecystectomy. Zhongguo Zhong Xi Yi Jie He Za Zhi. 1994;14:522-524. 302. Yang Y, Zuo HQ, Li Z, et al. Comparison of efficacy of simo decoction and acupuncture or chewing gum alone on postop-erative ileus in colorectal cancer resection: a randomized trial. Sci Rep. 2017;7:37826. 303. Chen Z, Cao L, Wen Z, et al. Study of Evodia hot compress plus

1	acupuncture or chewing gum alone on postop-erative ileus in colorectal cancer resection: a randomized trial. Sci Rep. 2017;7:37826. 303. Chen Z, Cao L, Wen Z, et al. Study of Evodia hot compress plus electro-acupuncture in patients who have undergone abdominal surgery. Int J Clin Exp Med. 2015;8:16167-16174. 304. Wu X. Practice of Abdominal Surgery. 3rd ed. Tianjin Science and Technology Press; 2004. 305. Cui Z, Kong D. 74 cases of biiliary liver abscess treated with integrated traditional and western medicine. Chinese Journal of Surgery of Integrated Traditional and Western Medicine. 2014;20:167-169. 306. Sun S, Xu W. Effects of TPN and immune regulator radix astragali on postoperative patients of obstructive jaundice. J Parenter Enteral Nutr. 2002;9:151-157. 307. Ma X, Yang W, Zhang X. The study of effect of shiquan dabu decoction in surgeical nutrition therapy. Beijing Journal of Traditional Chinese Medicine. 1991;4:31-32. 308. Dong Y, Cui Z. Acupuncture research on adhesive

1	X. The study of effect of shiquan dabu decoction in surgeical nutrition therapy. Beijing Journal of Traditional Chinese Medicine. 1991;4:31-32. 308. Dong Y, Cui Z. Acupuncture research on adhesive intestinal obstruction. Clin J Chinese Med. 2001;3:86-88. 309. Li G, Ma J, Shang H, et al. Application of Dechengqi decoc-tion in abdominal surgery. World Chinese Journal of Diges-tion. 2008;16:1672-1676. 310. Li H, Hong Y, Huang M. Clinical effect observation of Chinese herbal enema, external application and acupuncture in adhe-sive ileus. Journal of Guangzhou University of Traditional Chinese Medicine. 2007;24:282-285.Brunicardi_Ch50_p2113-p2136.indd 213601/03/19 9:39 AM

1	Understanding, Evaluating, and Using Evidence for Surgical PracticeAndrew J. Benjamin, Andrew B. Schneider, Jeffrey B. Matthews, and Gary An 51chapterINTRODUCTIONThe singular importance of this chapter rests on the following chain of reasoning:1. The state of surgical science and knowledge is constantly changing.2. The education of a surgeon is a continuous process.3. Surgeons need to know how to evaluate new surgical knowl-edge to maintain their education in order to best serve their patients.4. This chapter provides guidance as to how surgeons might navigate, interpret and apply this new knowledge.Notably, this reasoning also applies to the process of acquisition of new knowledge itself, which explains why this inaugural chapter on evidence-based medicine is occurring in the 11th edition of this book. Recognizing the impermanence and fluidity of knowledge is a critical insight for the responsible surgeon, but so too is realizing that “good practice” cannot occur without reference

1	of this book. Recognizing the impermanence and fluidity of knowledge is a critical insight for the responsible surgeon, but so too is realizing that “good practice” cannot occur without reference points as to what should be done given the current imperfect state of knowledge. These dual recognitions inform the organization of this chapter, which introduces and describes the currently accepted approach to evidence-based medicine, and then follows by noting a series of current issues that anticipate the likelihood that what is meant by “evidence-based medicine” will evolve over the coming years. Also note that this chapter is not intended to be a primer on statistics and clinical trial design; there are entire textbooks devoted to those subjects. Rather, this chapter will focus on how those tools are aggregated and presented in order to inform a surgeon how to keep abreast with current developments in practice.WHAT IS EVIDENCE-BASED MEDICINE?For centuries, the practice of medicine was

1	tools are aggregated and presented in order to inform a surgeon how to keep abreast with current developments in practice.WHAT IS EVIDENCE-BASED MEDICINE?For centuries, the practice of medicine was guided primarily by anecdotal experience, often based on rationales that did not arise from a rigorous scientific process and sustained by the fundamental barriers associated with being able to learn from one’s experience (e.g., cognitive bias). For example, treatments such as bloodletting and purging were based on ostensible prin-ciples of bodily humors originating from the Ancient Greeks, and persisted well into the 18th century despite repeated disas-trous outcomes. To a great degree, the goal of the Scientific Method, through its emphasis on skepticism and falsifiability, is predicated upon overriding observational/experiential bias by the application of rigorous methodology statistical analysis. The dangers of bias were recognized at the dawn of the Scien-tific Era, and continue to

1	overriding observational/experiential bias by the application of rigorous methodology statistical analysis. The dangers of bias were recognized at the dawn of the Scien-tific Era, and continue to manifest today (Box: The History and Sources of Bias in Biomedical Literature).Introduction2137What is Evidence-Based Medicine?2137Searching for Information: Patient/Population, Intervention, Comparison, and Outcome / 2139Types of Studies / 2139Hierarchies of Evidence / 2140Tools to Evaluate a Body of Evidence / 2140Synthesis of Evidence—Clinical Guidelines / 2142The Challenges of Applying EBM to Surgery2144Analysis of a Surgical Randomized Control Trial / 2144Internal Validity / 2144External Validity / 2146Additional Challenges to Conducting a Surgical RCT / 2146Use and Misuse of Statistical Significance2147Type I and Type II Errors / 2147P Values / 2147Alternative to P Values / 2148How do the Tools of EBM Perform?2148External Consistency / 2148Internal Consistency / 2148System

1	Significance2147Type I and Type II Errors / 2147P Values / 2147Alternative to P Values / 2148How do the Tools of EBM Perform?2148External Consistency / 2148Internal Consistency / 2148System Issues / 2148Validity / 2149Implications of EBM / 2149The Alternatives to EBM2149What can Researchers do to Improve the Validity of Research Findings?2149The History and Sources of Bias in Biomedical LiteratureIn Sir Francis Bacon’s initial description of the scientific method in his Novum Organum1 he notes what he terms the “idols of the mind,” in essence recognizing and classifying the sources of cognitive bias that limit the reliability of sub-jective observation and interpretation. These “idols” are:• Idols of the tribe (Idola tribus): A humans’ tendency to per-ceive more order and regularity in systems than truly exists, and arises from their preconceived ideas about things• Idols of the cave (Idola specus): Arising from an individ-ual’s personal limitations in reasoning due to particular

1	systems than truly exists, and arises from their preconceived ideas about things• Idols of the cave (Idola specus): Arising from an individ-ual’s personal limitations in reasoning due to particular personalities, subjective likes and dislikesBrunicardi_Ch51_p2137-p2152.indd 213728/02/19 4:19 PM 2138• Idols of the marketplace (Idola fori): Arising from the con-fusion in the use of language and taking some words in sci-ence to have a different meaning than their common usage• Idols of the theatre (Idola theatri): Arising from the following of academic dogma and not asking questions about the worldThese descriptions still resonate today, illustrating just how long the dangers of cognitive bias have been recognized, and just how embedded those tendencies may be. For instance, the following are sources of bias in biomedical literature:• Publication bias: publishers incentivized to accept posi-tive results• Prevailing field bias: supporting entrenched opinions• Citation bias: tendency

1	are sources of bias in biomedical literature:• Publication bias: publishers incentivized to accept posi-tive results• Prevailing field bias: supporting entrenched opinions• Citation bias: tendency to cite positive studies• Time-lag bias: delay the reporting of negative results• Reporting bias: emphasizing positive over negative resultsKey Points1 Cognitive bias is inescapable, and limits the ability of both individual practitioners and the surgical field in general, to advance and improve in a scientific fashion. Evidence Based Medicine is an attempt to codify the process of interpret-ing experience, assessing the literature and translating it into practice.2 Dealing with and interpreting the vast amount of surgical literature available on the Internet can be daunting, and this task can be aided by the application of identified formats for executing online search. The PICO (Patient/Population, Intervention, Comparison and Outcome) format is a com-monly used method for codifying online

1	aided by the application of identified formats for executing online search. The PICO (Patient/Population, Intervention, Comparison and Outcome) format is a com-monly used method for codifying online search.3 Not all literature or evidence is created equal. There exist various approaches, such as the Oxford Center for Evidence Based Medicine (CEBM) Levels of Evidence or the GRADE (Grading and Recommendations, Assessment, Development and Evaluation) system, that have been developed to provide guidance in assessing and reifying scientific literature.4 The conversion of evidence into clinical practice often mani-fests in the creation of clinical guidelines. As with all things related to evidence based medicine, not all guidelines are created equal, and therefore there are certain characteristics that can be used to evaluate the quality of a particular clini-cal guideline.5 There are specific challenges in the application of evidence based medicine to surgery, not least of which is the

1	that can be used to evaluate the quality of a particular clini-cal guideline.5 There are specific challenges in the application of evidence based medicine to surgery, not least of which is the difficulty in performing a truly randomized clinical trial. The CONSORT (Consolidated Standard of Reporting Clinical Trials) guidelines were developed to serve as minimal rec-ommendations for reporting randomized clinical trials.6 The well-known saying “There are lies, damn lies and then statistics” points to the recognition that statistical tools can be prone to misuse, and emphasizes the need to understand the appropriate application, limits of and interpretation of reported statistics.7 Evidence based medicine has not thus far been held to its own standards of evidence. Recognizing that available “evidence” is a constantly shifting landscape should warn one against the dangers of epistemic certainty, and further emphasizes the fact that surgical education is an ongoing and perpetual

1	available “evidence” is a constantly shifting landscape should warn one against the dangers of epistemic certainty, and further emphasizes the fact that surgical education is an ongoing and perpetual process.In the medical field, the transition from accumulated anec-dote to true statistical analysis can be seen in the emergence of clinical epidemiology as a field in 1938, which began to shift the focus from descriptions of individual patients to trends affect-ing entire populations. This shift, however, was accompanied by new challenges, as different means of turning anecdotal experience into statistics (e.g., case series, observational stud-ies, retrospective studies, prospective studies) meant that now practitioners needed to be able to compare these “scientific” presentations against each other in order to best establish their practices. The processes and methods of aggregating, compar-ing, and translating these different types of data from the medi-cal literature into clinical

1	each other in order to best establish their practices. The processes and methods of aggregating, compar-ing, and translating these different types of data from the medi-cal literature into clinical practice were explicitly established in the latter part of the 20th century, particularly arising from efforts at McMaster University, which eventually led to a fun-damental framework for literature-informed medical decision-making known as evidence-based medicine (EBM).EBM is defined as the “conscientious, explicit and judi-cious use of current best evidence in making decisions about treating individual patients.”2,3 This term was coined by Gordon Guyatt in 1991, focusing on assessing the credibility of the medical literature, understanding the presented results, and applying the information to individual practice. EBM is defined by three epistemological principles4:• Principle 1: Not all evidence is created equal, and the practice of medicine should be based on the best available

1	to individual practice. EBM is defined by three epistemological principles4:• Principle 1: Not all evidence is created equal, and the practice of medicine should be based on the best available evidence• Principle 2: The pursuit of truth is best accomplished by evaluation of the totality of the evidence, and not selecting evidence that favors a particular claim• Principle 3: Clinical decision-making requires consideration of patients’ values and preferenceThe adoption of EBM in the discipline of surgery has lagged compared to nonsurgical specialties. To a great extent, this is due to the challenges of achieving the highest level of evidence noted in principle 1: definitive conclusions from a randomized controlled trial (RCT). A literature analysis of MEDLINE from 1966 to 2000 demonstrated that only 15.1% of the 134,689 RCTs evaluated a surgical topic.5 In the early days of EBM during the 1990s, surgical RCTs accounted for only 7% of published articles in surgical journals; most of the

1	that only 15.1% of the 134,689 RCTs evaluated a surgical topic.5 In the early days of EBM during the 1990s, surgical RCTs accounted for only 7% of published articles in surgical journals; most of the articles were retrospective studies and case series,6 which are essentially aggregated anecdotes. Over the next decade, the rela-tive frequency of RCTs in surgery further decreased, account-ing for 3.4% of all publications in 2003.5 As a result, most of the available evidence to guide surgical practice today remains based on retrospective reviews, nonrandomized trials, and expert opinion. The barriers to performing prospective RCTs in surgery remain substantial: standardization of clinical pre-sentation and, of course, accounting for variations in operative technique and the ability to blind studies to reduce experimental bias. The relative paucity in RCTs in surgery make it even more 1Brunicardi_Ch51_p2137-p2152.indd 213828/02/19 4:19 PM 2139UNDERSTANDING, EVALUATING, AND USING

1	studies to reduce experimental bias. The relative paucity in RCTs in surgery make it even more 1Brunicardi_Ch51_p2137-p2152.indd 213828/02/19 4:19 PM 2139UNDERSTANDING, EVALUATING, AND USING EVIDENCE FOR SURGICAL PRACTICECHAPTER 51important that surgeons understand the best-practice methods to critically appraise available evidence, while recognizing the limitations and potential pitfalls of those methods, in order to optimize their practice and decision-making regarding patient care when high quality evidence may not be available. Herein we present the steps of such a workflow, starting with an initial search for information, identification of the classes of informa-tion that such a search can return, and then guidelines by which that information is evaluated, compared, and aggregated.Searching for Information: Patient/Population, Intervention, Comparison, and OutcomeTechnology has substantively changed how information can be sought and retrieved. Online search engines such as

1	for Information: Patient/Population, Intervention, Comparison, and OutcomeTechnology has substantively changed how information can be sought and retrieved. Online search engines such as MEDLINE via PubMed, which contains over 26 million citations, have dramatically enhanced the ability to access bio-medical literature.7 However, there is a very real potential for such access to become overwhelming. Effective and efficient use of search engines can be enhanced by framing the clinical question in a format designed to improve the relevancy of search results. PICO is one such format, where the acronym stands for Patient/Population, Intervention, Comparison, and Outcome.8• Patient or population is the specific group of individuals for which the questions is being asked.• Intervention is the treatment or technique of interest for the defined patient or population. Intervention might be a proce-dure, such as “laparoscopic appendectomy” or be defined as an exposure of interest, such as

1	the treatment or technique of interest for the defined patient or population. Intervention might be a proce-dure, such as “laparoscopic appendectomy” or be defined as an exposure of interest, such as “smoking.”• Comparison is the alternative treatment or technique to which you are comparing the intervention. Terms might include, for example, “open appendectomy” or “observation.”• Outcome of interest is the final step of the PICO format. Examples include “mortality,” “operative time,” and “wound infection.”As with all online search strategies, there is a trade-off between the specificity of the search and the breadth of the returned items. When using PICO to inform clinical decision-making, it is generally advantageous to be as precise and specific as possible when initiating a search: this increases the likelihood the search will return information most germane to the particular clinical scenario. This is accomplished by the use of “AND” in the framing of the search to encompass the

1	increases the likelihood the search will return information most germane to the particular clinical scenario. This is accomplished by the use of “AND” in the framing of the search to encompass the set of questions of interest. For example, one could construct a query consisting of a particular procedure, with a particular method, with a particular outcome metric, such as “(distal pancreatectomy) AND splenectomy AND (splenic preservation) AND morbidity” to frame a PICO question.Types of StudiesPrinciple 1 of EBM states that not all evidence is created equal; therefore, evaluating the evidentiary quality of the results of an online search requires classifying the returned search items by type of study. As noted earlier, acknowledging that the “gold standard” level of evidence, RCTs, are rare in the surgical literature, the application of EBM to surgery requires increased familiarity with the types of alternative studies available, with their relative strengths and weaknesses. These

1	rare in the surgical literature, the application of EBM to surgery requires increased familiarity with the types of alternative studies available, with their relative strengths and weaknesses. These types are listed below:• Meta-analysis: A meta-analysis is a technique to combine similarly published data in order to increase the overall 2statistical power compared to each study individually. The amount of interstudy heterogeneity (methods, study popula-tion, endpoints, etc.) should be limited to allow for the gen-eration of informative conclusions. The pooling of similar studies enables researchers to generate a new statistical con-clusion based on a substantially larger sample size. These approaches, though useful, have their limitations: the inclu-sion of inappropriate studies and the mislabeling of a meta-analysis leading to inaccurate conclusions. Attention should be directed toward this type of evidence when clinical guide-lines do not exist.• Systematic Review: Like

1	and the mislabeling of a meta-analysis leading to inaccurate conclusions. Attention should be directed toward this type of evidence when clinical guide-lines do not exist.• Systematic Review: Like meta-analyses, systematic reviews use standardized methods to search for and appraise studies in order to attempt to reduce bias. However, systematic reviews do not utilize quantitative methods to summarize the results. For this reason, systematic reviews are often not considered to provide the same strength of evidence as a meta-analysis.• Cross-Sectional Studies: In a cross-sectional study, expo-sures and outcomes are measured at a single point in time. The prevalence of the outcome is then compared in patients who did and did not have the exposure. Multiple exposures and outcomes can be measured at the same time, which is an advantage; however, there are important limitations. One significant limitation is that a temporal relationship cannot be determined between exposure and outcome

1	at the same time, which is an advantage; however, there are important limitations. One significant limitation is that a temporal relationship cannot be determined between exposure and outcome because they are measured simultaneously. These studies will often form the foundation for more definitive studies.• Case Control Study: In a case-control study, cohorts are determined by the presence or absence of a particular out-come of interest. This is in contrast to a cross-sectional study where samples are determined by the presence or absence of an exposure. Once the samples have been identi-fied based upon outcome, then possible prior exposures are identified, and the odds of those exposures are compared between cohorts.• Case Series: A Case Series involves a report of a small group of patients that share specified clinical features; this gener-ally does not include description of a control group. Case series are prevalent in the field of surgery, and some of the most famous eponymous

1	that share specified clinical features; this gener-ally does not include description of a control group. Case series are prevalent in the field of surgery, and some of the most famous eponymous procedures originated from case series, including the Whipple procedure9 and Nissen fundo-plication.10 This type of study provides weak evidence due to issues with patient selection, biases, and confounding factors. However, the findings from a case series can be used to gen-erate hypotheses for a randomized control trial.• Expert Opinion: Expert opinions represent the lowest level of evidence and is representative of a clinician’s individual experience and anecdotes. Prior to evidence-based medicine, expert opinion was the primary means of teaching medicine and shaping the field. However, the opinions of clinicians can vary substantially leading to a wide range of potential unproven treatments for a medical issue. Thus, expert opinion should only be solicited in the complete absence of

1	opinions of clinicians can vary substantially leading to a wide range of potential unproven treatments for a medical issue. Thus, expert opinion should only be solicited in the complete absence of evidence in the literature.It should also be noted that irrespective of the type of study or recommendation, there are additional factors that can contribute to bias in publication. To a great degree these are extrapolations of the sources of individual cognitive bias, but writ large across an entire community (see Box: The History and Sources of Bias in Biomedical Literature).Brunicardi_Ch51_p2137-p2152.indd 213928/02/19 4:19 PM 2140SPECIFIC CONSIDERATIONSPART IIHierarchies of EvidenceThe original architects of EBM codified the notion that certain types of evidence are superior to others based on charac-teristics of study design, depicting this concept as a “pyra-mid,” with expert opinion comprising the base of the pyramid and randomized controlled trials at the peak (Fig. 51-1).

1	based on charac-teristics of study design, depicting this concept as a “pyra-mid,” with expert opinion comprising the base of the pyramid and randomized controlled trials at the peak (Fig. 51-1). Although conceptually appealing, this initial attempt to “rank” the evidence was relatively simplistic and rested on unproven assumptions that RCT were inherently superior to observational studies. While RCTs theoretically provide higher quality evi-dence compared to observational studies, RCTs can also have significant limitations and biases (see later section, “The Chal-lenges of Applying EBM to Surgery”). Furthermore, translating the results from well-crafted RCTs can be challenging, where the specific restrictive criteria for executing a high-quality RCT can inherently limit its applicability to clinical scenarios not specifically noted or tested in the RCT. Therefore, one could find oneself in the situation of trying to compare an RCT on a related but clearly distinct use case with a

1	to clinical scenarios not specifically noted or tested in the RCT. Therefore, one could find oneself in the situation of trying to compare an RCT on a related but clearly distinct use case with a well performed obser-vational study that more closely approximated the clinical sce-nario in question. This led to the subsequent development of more refined frameworks to assess the quality of evidence in order to try and address these issues, although there is currently no consensus on a single framework. The current situation is that while many newer systems have devised ways in which studies can move up and down the pyramid, for well-designed studies, the pyramid largely remains intact.The initial hierarchies of evidence were limited because they entangled the method of evidence collection with underly-ing study design. They failed to recognize principle 2 of EBM: “the pursuit of truth is best accomplished by evaluation of the totality of evidence” and the principle that “health claims be

1	underly-ing study design. They failed to recognize principle 2 of EBM: “the pursuit of truth is best accomplished by evaluation of the totality of evidence” and the principle that “health claims be based upon systematic reviews which summarize the best avail-able evidence.”4 The earliest hierarchies positioned systematic reviews at the top of the pyramid followed by RCTs; how-ever, this classification failed to acknowledge that systematic reviews can summarize any type of evidence. Cohort studies, case-control studies, and even case reports can be the subject of systematic review. The importance of systematic review in EBM cannot be understated: systematic reviews are the most 3cited type of study, and these studies are essential for the devel-opment of clinical guidelines and influencing the direction of future studies.2,11 When applied in a timely manner, systematic reviews have resulted in major practice changes, for example, encouraging early postoperative enteral feeding compared

1	the direction of future studies.2,11 When applied in a timely manner, systematic reviews have resulted in major practice changes, for example, encouraging early postoperative enteral feeding compared to parenteral nutrition to prevent sepsis.12Tools to Evaluate a Body of EvidenceBy 2002, over 100 unique evidence rating systems existed,2 and the differences among them may be nontrivial. Depending upon the specific criteria used, the “strength” of evidence might dif-fer widely from system to system. For example, the American Association of Orthopedic Surgeons (AAOS) published guide-lines in 2009 for prevention of venous thromboembolism (VTE) in patients undergoing hip or knee surgery that conflicted with the widely used American College of Chest Physician (ACCP) guidelines, despite having access to the same data. While the ACCP considered VTE prophylaxis to be a grade 1 recommen-dation with level A evidence, the AAOS recommendation var-ied based upon risk of pulmonary embolism and

1	access to the same data. While the ACCP considered VTE prophylaxis to be a grade 1 recommen-dation with level A evidence, the AAOS recommendation var-ied based upon risk of pulmonary embolism and bleeding, with no recommendation being greater than B and all recommenda-tions being based upon level III evidence.13 In the following section we present a few of the most widely accepted tools for assessing the quality of evidence.CEBM Levels of Evidence. One of the most widely adopted systems for grading evidence is the Oxford Center for Evi-dence Based Medicine (CEBM) Levels of Evidence. The origi-nal CEBM system was released in 2000 and was subsequently updated in 2011. Earlier systems of evidence ranking were criti-cized because they categorically placed randomized trials above observational studies, although observational studies and even anecdotes can occasionally give the “best” evidence in certain clinical situations. CEBM was therefore developed to not only improve the ranking of

1	studies, although observational studies and even anecdotes can occasionally give the “best” evidence in certain clinical situations. CEBM was therefore developed to not only improve the ranking of evidence but also to aid clinicians in quickly searching for the best evidence available for a given clinical question (Table 51-1). It is designed as both a tool for traditional critical appraisal as well as a pragmatic system that clinicians can use to answer clinical questions in real time. It can be used as a heuristic that clinicians and patients can utilize to answer clinical questions quickly and without resorting to preap-praised sources.14 The CEBM Levels of Evidence system begins with choosing a clinical question from the first column of the table provided by the creators (see Table 51-1) (for example, “How common is the problem?”, “Does this intervention help?”, or “Is this test worthwhile?”). Therefore, each row of the CEBM Levels of Evidence represents a series of steps one

1	51-1) (for example, “How common is the problem?”, “Does this intervention help?”, or “Is this test worthwhile?”). Therefore, each row of the CEBM Levels of Evidence represents a series of steps one should fol-low to find the best evidence for the question chosen. Strong evidence is likely to be found in columns to the left of the table, while weak evidence will be found in columns to the right. After completing a clinical query using the table, a final “level” of evidence is assigned on a scale from 1 to 5 based upon the types of studies found to answer the initial question (1 = highest rated evidence; 5 = lowest rated evidence). However, the levels are not intended to provide one with a definitive judgment regarding the quality of evidence. There may be cases where “lower level” evi-dence—for example, an observational study with a large treat-ment effect—provides stronger evidence than a “higher level” study, such as a systematic review with an inconclusive result.CEBM should be

1	example, an observational study with a large treat-ment effect—provides stronger evidence than a “higher level” study, such as a systematic review with an inconclusive result.CEBM should be thought of as a hierarchy of the likely best evidence. An advantage of CEBM is that it allows the potential of resorting to individual studies for the best evidence, while other systems generally assume that there is a systematic RCTCohort studyCase control studyCase seriesCase reportsAnimal researchIn-vitro researchExpert experience/opinionFigure 51-1. Evidence-based hierarchy.Brunicardi_Ch51_p2137-p2152.indd 214028/02/19 4:19 PM 2141UNDERSTANDING, EVALUATING, AND USING EVIDENCE FOR SURGICAL PRACTICECHAPTER 51Table 51-1Oxford center for evidence-based medicine 2011 levels of evidenceQUESTIONSTEP 1 (LEVEL 1*)STEP 2 (LEVEL 2*)STEP 3 (LEVEL 3*)STEP 4 (LEVEL 4*)STEP 5 (LEVEL 5)How common is the problem?Local and current random sample surveys (or censuses)Systematic review of surveys that allow

1	1 (LEVEL 1*)STEP 2 (LEVEL 2*)STEP 3 (LEVEL 3*)STEP 4 (LEVEL 4*)STEP 5 (LEVEL 5)How common is the problem?Local and current random sample surveys (or censuses)Systematic review of surveys that allow matching to local circumstances**Local non-random sample**Case-series**n/aIs this diagnostic or monitoring test accurate? (Diagnosis)Systematic review of cross-sectional studies with consistently applied reference standard and blindingIndividual cross-sectional studies with consistently applied reference standard and blindingNon-consecutive studies, or studies without consistently applied reference standards**Case-control studies, or “poor or non-independent reference standard**Mechanism-based reasoningWhat will happen if we do not add a therapy? (Prognosis)Systematic review of inception cohort studiesInception cohort studiesCohort study or control arm of randomized trial*Case-series or case-control studies, or poor quality prognostic cohort study**n/aDoes this intervention help? (Treatment

1	studiesInception cohort studiesCohort study or control arm of randomized trial*Case-series or case-control studies, or poor quality prognostic cohort study**n/aDoes this intervention help? (Treatment Benefits)Systematic review of randomized trials or n-of-1 trialsRandomized trial or observational study with dramatic effectNon-randomized controlled cohort/follow-up study**Case-series, case-control studies, or historically controlled studies**Mechanism-based reasoningWhat are the COMMON harms? (Treatment Harms)Systematic review of randomized trials, systematic review of nested case-control studies n-of-1 trial with the patient you are raising the question about, or observational study with dramatic effectIndividual randomized trial or (exceptionally) observational study with dramatic effectNon-randomized controlled cohort/follow-up study (post-marketing surveillance) provided there are sufficient numbers to rule out a common harm. (For long-term harms the duration of follow-up must be

1	controlled cohort/follow-up study (post-marketing surveillance) provided there are sufficient numbers to rule out a common harm. (For long-term harms the duration of follow-up must be sufficient.)**Case-series, case-control, or historically controlled studies**Mechanism-based reasoningWhat are the RARE harms? (Treatment Harms)Systematic review of randomized trials or n-of-1 trialRandomized trial or (exceptionally) observational study with dramatic effect Is this (early detection) test worthwhile? (Screening)Systematic review of randomized trialsRandomized trialNon-randomized controlled cohort/follow-up study**Case-series, case-control, or historically controlled studies**Mechanism-based reasoning*Level may be graded down on the basis of study quality, Imprecision, Indirectness (study PICO does not match questions PICO), because of inconsistency between studies, or because the absolute effect size is very small; Level may be graded up if there is a large or very large effect

1	PICO does not match questions PICO), because of inconsistency between studies, or because the absolute effect size is very small; Level may be graded up if there is a large or very large effect size.**As always, a systematic review is generally better than an individual study.How to cite the Levels of Evidence TableOCEBM Levels of Evidence Working Group*. “The Oxford 2011 Levels of Evidence”.Oxford Centre for Evidence-Based Medicine. http://www.cebm.net/index.aspx?o=5653*OCEBM Table of Evidence Working Group = Jeremy Howick, Iain Chalmers (James Lind Library), Paul Glasziou, Trish Greenhaigh, Carl Heneghan, Alessandro Liberati, Ivan Moschetti, Bob Phillips, Hazel Thornton, Olive Goddard, and Mary HodgkinsanBrunicardi_Ch51_p2137-p2152.indd 214128/02/19 4:19 PM 2142SPECIFIC CONSIDERATIONSPART IIDefinitions of GRADE Evidence QualityHigh quality – Further research is very unlikely to change confidence in the estimate of effect.Moderate quality – Further research is likely to have an

1	of GRADE Evidence QualityHigh quality – Further research is very unlikely to change confidence in the estimate of effect.Moderate quality – Further research is likely to have an important impact confidence in the estimate of effect and may change the estimate.Low quality – Further research is very likely to have an important impact on confidence in the estimate of effect and is likely to change the estimate.Very low quality – Any estimate of effect is very uncertain.review available. Additionally, other systems are built around considering the strength of evidence for therapeutic effects and harms, while CEBM allows appraisal of evidence for prevalence of disease, accuracy of diagnostic tests, prognosis, therapeutic effects, rare harms, common harms, and usefulness of screening.Grading and Recommendations, Assessment, Development, and Evaluation. Alternatively, the Grading and Recommendations, Assessment, Development and Evaluation (GRADE) system classifies the quality of evidence

1	and Recommendations, Assessment, Development, and Evaluation. Alternatively, the Grading and Recommendations, Assessment, Development and Evaluation (GRADE) system classifies the quality of evidence into one of four levels: high, moderate, low, and very low15 (Box: Definitions of GRADE Evidence Quality). Evidence quality in the GRADE system is not assigned solely on study design. For example, a randomized controlled trial begins at “high quality,” but may be demoted due to one or more of the following: study limitations, inconsistent results, indirectness of evidence, imprecision, or reporting bias. Alternatively, observational studies (cohort or case-control studies) start as “low quality” but may be upgraded if there is a large magnitude of the treatment effect, evidence of a dose-response relationship, or if all plausible biases would decrease the magnitude of a treatment effect. Thus, the GRADE system of evaluating the quality of evidence provides more granularity than the

1	relationship, or if all plausible biases would decrease the magnitude of a treatment effect. Thus, the GRADE system of evaluating the quality of evidence provides more granularity than the traditional hierarchy system, which assigns quality based upon study design alone. Although the GRADE system has significant advantages, it is more complex and has a steeper learning curve than traditional systems. Finally, GRADE is intended for appraising a body of evidence, such as in a systematic review.In addition to providing a transparent approach to grading evidence quality, the GRADE system outlines an approach to the development and assignment of strength to clinical recommendations. GRADE’s sophisticated hierarchy of evidence allows the system to protect against both superficial assessment and unwarranted confidence in all classes of study design. Since its development, the increasing use of GRADE has resulted in higher quality and rigor of systematic reviews due to standards outlined by

1	unwarranted confidence in all classes of study design. Since its development, the increasing use of GRADE has resulted in higher quality and rigor of systematic reviews due to standards outlined by the system.15 In creating a recommendation regarding a body of evidence, GRADE allows experts to account for limitations in bodies of evidence comprising of RCTs, while also allowing for the rating of observational studies as high quality in cases where RCTs are not feasible (i.e., an RCT cannot ethically be performed). GRADE therefore potentially allows for observational studies to provide definitive evidence of causal association (e.g., alcohol causing cirrhosis or asbestos causing mesothelioma) where RCTs may not be ethical or necessary.One of the major advantages of GRADE is that it specifi-cally addresses the process of moving from evidence to recom-mendations. The process begins with the creation of a summary of findings table. A summary of findings table consists of a presentation

1	addresses the process of moving from evidence to recom-mendations. The process begins with the creation of a summary of findings table. A summary of findings table consists of a presentation not only of evidence quality but also estimates of the relative and absolute effects of patient-centered outcomes (Fig. 51-2). The summary of findings format was created to min-imize framing effects, where different raters may come to varied conclusions based upon identical information due to the infor-mation having a contrasting presentation in terms of gain versus loss.16 GRADE and similar EBM systems specifically takes into consideration judgement of risk versus benefit, resource use, feasibility, and equity to attempt to make decision-making as consistent as possible across a range of reviewers.2 Despite all of the aforementioned considerations when constructing a guide-line, it is important to realize that patient values or preferences may immediately invalidate any recommendation. Evidence

1	Despite all of the aforementioned considerations when constructing a guide-line, it is important to realize that patient values or preferences may immediately invalidate any recommendation. Evidence is often constructed based upon measurement of outcomes such as morbidity, mortality, or survival; however, patients may be more concerned with quality of life or avoiding invasive inter-ventions. GRADE attempts to acknowledges this intrinsic vari-ability within its system of grading.In terms of the overall strength of a recommendation that GRADE can assign, two grades are possible: “strong” and “weak.” A strong recommendation is one where positive effects of an intervention clearly outweigh the negative effects or vice versa. A weak recommendation is one where the asso-ciation is less clear, either because of low quality evidence or because the evidence clearly suggests that the positive and negative effects are similar. However, quality of evidence is not the only factor that affects the

1	because of low quality evidence or because the evidence clearly suggests that the positive and negative effects are similar. However, quality of evidence is not the only factor that affects the strength of a recommenda-tion (Table 51-2). Factors such as uncertainty of patient values or whether an intervention is an appropriate use of resources can play a role in the strength of a recommendation as well. Therefore, it is important to note that a “strong” or “weak” rec-ommendation may be given regardless of the classification of the evidence. For example, there is a strong recommendation that patients with Zollinger-Ellison syndrome be treated with PPI. This recommendation is made despite weak evidence to support this practice because the potential benefits far out-weigh the potential risks.17Although the systems for grading evidence are well devel-oped, it is important to remember that the studies used for evi-dence are judged based on their internal validity, or the extent to which a

1	the systems for grading evidence are well devel-oped, it is important to remember that the studies used for evi-dence are judged based on their internal validity, or the extent to which a causal conclusion is warranted based upon applica-tion of the results to the study population. This means that care must be exercised when applying a recommendation to a given patient, as the external validity of a recommendation, or gener-alizability of a causal conclusion to populations outside of the scope of the original studies, may not be appropriate. Therefore, all evidence must be applied within the context of the patient in front of you.Synthesis of Evidence—Clinical GuidelinesThe Institute of Medicine defines a clinical guideline as “state-ments that include recommendations, intended to optimize patient care, that are informed by a systematic review of evidence and an assessment of the benefits and harms of alterna-tive care options.”18 Clinical guidelines may reflect previous published

1	patient care, that are informed by a systematic review of evidence and an assessment of the benefits and harms of alterna-tive care options.”18 Clinical guidelines may reflect previous published studies of varying design and quality, as well as expert opinion, and often represent the highest level of applied clinical evidence. Numerous guidelines have been published; however, like individual studies, even guidelines can vary in quality. The highest quality and most clinically useful guide-lines tend to have the following qualities:4Brunicardi_Ch51_p2137-p2152.indd 214228/02/19 4:19 PM 2143UNDERSTANDING, EVALUATING, AND USING EVIDENCE FOR SURGICAL PRACTICECHAPTER 51Summary of findings:Compression stockings compared with no compression stockings for people taking long flightsPatients or population: Anyone taking a long flight (lasting more than 6 hours)Settings: International air travelIntervention: Compression stockings1Comparison: Without stockingsOutcomesIllustrative comparative

1	population: Anyone taking a long flight (lasting more than 6 hours)Settings: International air travelIntervention: Compression stockings1Comparison: Without stockingsOutcomesIllustrative comparative risks* (95% CI)Relativeeffect(95% CI)Number ofparticipants(studies)Qualityof theevidence(GRADE)Comments Assumed riskCorresponding riskWithout stockingsWith stockingsSymptomaticdeep vein thrombosis (DVT)See comment See comment Not estimable2821(9 studies)See comment0 participants developed symptomatic DVT in these studies.Symptom-lessdeep vein thrombosis Low risk population2RR 0.10(0.04 to 0.26) 2637(9 studies) ++++High10 per 10001 per 1000 (0 to 3)High risk population230 per 10003 per 1000(1 to 8)Superficial vein thrombosis13 per 10006 per 1000(2 to 15)RR 0.45(0.18 to 1.13)1804(8 studies)+++OModerate3 OedemaPost-flight values measured on a scale from 0, no oedema, to 10, maximum oedema.The mean oedema score ranged across control groups from6 to 9.The mean oedema score in the

1	studies)+++OModerate3 OedemaPost-flight values measured on a scale from 0, no oedema, to 10, maximum oedema.The mean oedema score ranged across control groups from6 to 9.The mean oedema score in the intervention groups was on average4.7 lower(95% CI –4.9 to –4.5). 1246(6 studies)++OOLow4 Pulmonary embolusSee commentSee commentNot estimable2821(9 studies)See comment0 participants developed pulmonary embolus in these studies.5DeathSee commentSee commentNot estimable2821(9 studies)See comment0 participants died in these studies.Adverse effectsSee commentSee commentNot estimable1182(4 studies)See commentThe tolerability of the stockings was described as very good with no complaints of side effects in 4 studies.6*The basis for the assumed risk is provided in footnotes. The corresponding risk (and its 95% confidence interval) is based on the assumed risk in the intervention group and the relative effect of the intervention (and its 95% CI).CI: Confidence interval; RR: Risk ratio GRADE:

1	(and its 95% confidence interval) is based on the assumed risk in the intervention group and the relative effect of the intervention (and its 95% CI).CI: Confidence interval; RR: Risk ratio GRADE: GRADE Working Group grades of evidence (see explanations)11 All the stockings in the 9 trials included in this review were below-knee compression stockings. In four trials the compression strength was 20–30 mmHg at the ankle. It was 10–20 mmHg in the other four trials. Stockings come in different sizes. If a stocking is too tight around the knee it can prevent essential venous return causing the blood to pool around the knee. Compression stockings should be fitted properly. A stocking that is too tight could cut into the skin on a long flight and potentially cause ulceration and increased risk of DVT. Some stockings can be slightly thicker than normal leg covering and can be potentially restrictive with tight foot wear. It is a good idea to wear stockings around the house prior to travel to

1	DVT. Some stockings can be slightly thicker than normal leg covering and can be potentially restrictive with tight foot wear. It is a good idea to wear stockings around the house prior to travel to ensure a good, comfortable fitting. Stockings were put on 2 to 3 hours before the flight in most of the trials. The availability and cost of stockings can vary.2Two trials recruited high risk participants defined as those with previous episodes of DVT, coagulation disorders, severe obesity, limited mobility due to bone or joint problems, neoplastic disease within the previous two years, large varicose veins or, in one of the studies, participants taller than 190 cm and heavier than 90 kg. The incidence for 7 trials that excluded high risk participants was 1.45% and the incidence for the 2 trials that recruited high-risk participants (with at least one risk factor) was 2.43%. We have rounded these off to 10 and 30 per 1000 respectively.3The confidence interval crosses no difference and does

1	that recruited high-risk participants (with at least one risk factor) was 2.43%. We have rounded these off to 10 and 30 per 1000 respectively.3The confidence interval crosses no difference and does not rule out a small increase.4The measurement of oedema was not validated or blinded to the intervention. All of these studies were conducted by the same investigators.5If there are very few or no events and the number of participants is large, judgement about the quality of evidence (particularly judgements about precision) may be based on the absolute effect. Here the quality rating may be considered “high” if the outcome was appropriately assessed and the event, in fact, did not occur in 2821 studied participants.6None of the other trials reported adverse effects, apart from 4 cases of superficial vein thrombosis in varicose veins in the knee region that were compressed by the upper edge of the stocking in one trial.Figure 51-2. Example of a “summary of findings”

1	from 4 cases of superficial vein thrombosis in varicose veins in the knee region that were compressed by the upper edge of the stocking in one trial.Figure 51-2. Example of a “summary of findings” table.Brunicardi_Ch51_p2137-p2152.indd 214328/02/19 4:19 PM 2144SPECIFIC CONSIDERATIONSPART IITable 51-2Factors that affect the strength of a recommendationFACTOREXAMPLES OF STRONG RECOMMENDATIONSEXAMPLES OF WEAK RECOMMENDATIONSQuality of evidenceMany high quality randomized trials have shown the benefit of inhaled steroids in asthmaOnly case series have examined the utility of pleurodesis in pneumothoraxUncertainty about the balance between desirable and undesirable effectsAspirin in myocardial infarction reduces mortality with minimal toxicity, inconvenience, and costWarfarin in low risk patients with atrial fibrillation results in small stroke reduction but increased bleeding risk and substantial inconvenienceUncertainty or variability in values and preferencesYoung patients with

1	patients with atrial fibrillation results in small stroke reduction but increased bleeding risk and substantial inconvenienceUncertainty or variability in values and preferencesYoung patients with lymphoma will invariably place a highervalue on the life prolonging effects of chemotherapy than on treatment toxicityOlder patients with lymphoma may not place a higher value on the life prolonging effects of chemotherapy than on treatment toxicityUncertainty about whether the intervention represents a wise use of resourcesThe low cost of aspirin as prophylaxis against stroke in patients with transient ischemic attacksThe high cost of clopidogrel and of combination dipyridamole and aspirin as prophylaxis against stroke in patients with transient ischaemic attacks1. An explicit description of development and funding pro-cesses that is publicly available.2. A transparent process that minimizes bias, distortion, and conflicts of interest.3. Developed by a multidisciplinary panel composed of:

1	and funding pro-cesses that is publicly available.2. A transparent process that minimizes bias, distortion, and conflicts of interest.3. Developed by a multidisciplinary panel composed of: clini-cians, methodological experts, and representatives, includ-ing a patient or consumer, of populations expected to be affected by the guideline.4. Utilizes rigorous systematic evidence review and considers quality, quantity, and consistency of the aggregate of avail-able evidence.5. Summarizes evidence about potential benefits and harms relevant to each recommendation.6. Explains the parts that values, opinion, theory, and clinical experience play in deriving recommendations.7. Provides a rating of the level of confidence in the evidence underpinning each recommendation and a rating of the strength of each recommendation.8. Undergoes extensive external review that includes an open period for public comment.9. Has a mechanism for revision when new evidence becomes available.Depending upon the

1	of each recommendation.8. Undergoes extensive external review that includes an open period for public comment.9. Has a mechanism for revision when new evidence becomes available.Depending upon the clinical question, such guidelines are often interpreted as the standard of care. However, multiple clinical guidelines may be applicable with respect to various aspects of a given clinical situation and must not be followed blindly without considering specific situational issues through the lens of an experienced clinician. Moreover, guidelines do not (and probably cannot) exist for all clinical situations. Clini-cians often must resort to other resources to enrich the context in which decisions are made, and, as with all evidence, care must be taken not to extrapolate the application of a clinical guideline beyond its specific conditions.THE CHALLENGES OF APPLYING EBM TO SURGERYAs noted earlier, the application of EBM to surgery has lagged behind other fields of medicine, and this has been

1	guideline beyond its specific conditions.THE CHALLENGES OF APPLYING EBM TO SURGERYAs noted earlier, the application of EBM to surgery has lagged behind other fields of medicine, and this has been attributed to the difficulty in establishing a sufficient mass of evidence with the “gold standard” RCT. Here we describe the process of evaluating the quality of a RCT and note the challenges related to the execution of a high-quality RCT in a surgical context.Analysis of a Surgical Randomized Control TrialSufficient knowledge of the trial’s methodological accuracy and results are essential for critical appraisal. However, less than half of journal articles adequately report the study design.19 This deficiency led to the development of the Consoli-dated Standards of Reporting Trials (CONSORT) guidelines in 1992, which was subsequently revised in 2010.20 These guide-lines are a minimal set of recommendations for reporting RCTs (blinding, randomization, etc) to facilitate critical appraisal.

1	in 1992, which was subsequently revised in 2010.20 These guide-lines are a minimal set of recommendations for reporting RCTs (blinding, randomization, etc) to facilitate critical appraisal. Many of the surgical journals now require completion of a CONSORT checklist prior to submission of the RCT manu-script (Fig. 51-3). Establishing this requirement has standard-ized the way articles are presented and analyzed. The two key aspects to focus on when assessing a RCT are internal and external validity.Internal ValidityDetermining the degree that the results of the RCT are accurate and consistent for the sample patients is called internal validity. Without internal validity, a study cannot be properly appraised, as the study was not constructed properly to answer the hypoth-esis without avoiding bias or confounding factors.21 The internal validity of a RCT requires the evaluation of several properties: randomization, blinding, equivalence among groups, complete-ness of follow-up, and

1	bias or confounding factors.21 The internal validity of a RCT requires the evaluation of several properties: randomization, blinding, equivalence among groups, complete-ness of follow-up, and accuracy of analysis. These properties are discussed in the following section.Randomization. Randomization is the creation of participant groups with similar known and unknown prognostic factors to achieve the goal of eliminating selection bias. For example, if the investigator can decide which treatment the patient receives, he or she may assign a participant to a study arm that is more favorable for that specific patient. On outcomes analysis, certain groups may have an overestimated treatment effect due to patient selec-tion and not necessarily the intervention itself. The methodology 5Brunicardi_Ch51_p2137-p2152.indd 214428/02/19 4:19 PM 2145UNDERSTANDING, EVALUATING, AND USING EVIDENCE FOR SURGICAL PRACTICECHAPTER 51SectionItem NoChecklist itemTitle and Abstract1aIdentification as a

1	214428/02/19 4:19 PM 2145UNDERSTANDING, EVALUATING, AND USING EVIDENCE FOR SURGICAL PRACTICECHAPTER 51SectionItem NoChecklist itemTitle and Abstract1aIdentification as a randomized trial in the title1bStructured summary of trial design, methods, results, and conclusionsBackground and Objectives2aScientific background and explanation of rationale2bSpecific objectives or hypothesesTrial Design3aDescription of trial design (such as parallel, factorial) including allocation ratio3bImportant changes to methods after trial commencement with reasonsParticipants4aEligibility criteria for participants4bSettings and locations where the data were collectedInterventions5The interventions for each group with sufficient details to allow replication, including how and when they were administeredOutcomes6aCompletely defined pre-specified primary and secondary outcome measures, including how and when they were assessed6bAny changes to trial outcomes after the trial commenced, with reasonsSample

1	defined pre-specified primary and secondary outcome measures, including how and when they were assessed6bAny changes to trial outcomes after the trial commenced, with reasonsSample size7aHow sample size was determined7bWhen applicable, explanation of any interim analyses and stopping guidelinesRandomization: Sequence Generation8aMethod used to generate the random allocation sequence8bType of randomization; details of any restriction (such as blocking and block size)Allocation concealment mechanism9Mechanism used to implement the random allocation sequenceImplementation10Who generated the random allocation sequence, who enrolled participants, and who assigned interventionsBlinding11aIf done, who was blinded after assignment to interventions and how11bIf relevant, description of the similarity of interventionsResults Participant flow13aFor each group, the numbers of participants who were randomly assigned, received intended treatment, and were analyzed for the primary outcome13bFor each

1	of interventionsResults Participant flow13aFor each group, the numbers of participants who were randomly assigned, received intended treatment, and were analyzed for the primary outcome13bFor each group, losses and exclusions after randomization, together with reasonsRecruitment14aDates defining the periods of recruitment and follow-up14bWhy the trial ended or was stoppedBaseline data15A table showing baseline demographic and clinical characteristics for each groupNumbers analyzed16For each group, number of participants (denominator) included in each analysis and whether the analysis was by original assigned groupsOutcomes and estimation17aFor each primary and secondary outcome, results for each group, and the estimated effect size and its precision (such as 95% confidence interval)17bFor binary outcomes, presentation of both absolute and relative effect sizes is recommendedAncillary analyses18Results of any other analyses performed, including subgroup analyses and adjusted analyses,

1	binary outcomes, presentation of both absolute and relative effect sizes is recommendedAncillary analyses18Results of any other analyses performed, including subgroup analyses and adjusted analyses, distinguishing pre-specified from exploratoryHarms19All important harms or unintended effects in each groupDiscussion Limitations20Trial limitations, addressing sources of potential bias, imprecision, and, if relevant, multiplicity of analysesGeneralizability21Generalizability (external validity, applicability) of the trial findingsInterpretation22Interpretation consistent with results, balancing benefits and harms, and considering other relevant evidenceOther Information Registration23Registration number and name of trial registryProtocol24Where the full trial protocol can be accessed, if availableFunding25Sources of funding and other support (such as supply of drugs), role of fundersFigure 51-3. CONSORT checklist. (Reproduced with permission from Schulz KF, Altman DG, Moher D, et al:

1	availableFunding25Sources of funding and other support (such as supply of drugs), role of fundersFigure 51-3. CONSORT checklist. (Reproduced with permission from Schulz KF, Altman DG, Moher D, et al: CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials, Int J Surg. 2011;9(8):672-677.)Brunicardi_Ch51_p2137-p2152.indd 214528/02/19 4:19 PM 2146SPECIFIC CONSIDERATIONSPART IIof randomization should always be reported and carefully ana-lyzed by the reader. Certain approaches of randomization called quasi-random allocation (date of birth, day of week, participant number, etc.) are not truly random and cannot be fully concealed from study personnel. Additionally, the concept of randomization eliminating bias is only theoretical. To truly ensure the probability of confounders being equally balanced between groups, a trial must be repeated indefinitely. Understanding this impracticality, we accept that randomization will suffice.Blinding. Blinding aims

1	of confounders being equally balanced between groups, a trial must be repeated indefinitely. Understanding this impracticality, we accept that randomization will suffice.Blinding. Blinding aims to reduce certain biases that can affect the outcome of the study. A subject’s knowledge of the group that they were randomized can lead to a performance bias, which can influence subjective outcomes (placebo effect). Importantly, authors should be explicitly clear regarding which groups (sub-jects, clinicians, assessors) are blinded and avoid using non-specific phrases such as “double-blinded” or “triple-blinded.” Achieving blinding and minimizing bias is a major hurdle in the execution of surgical RCTs, where there are the ethical dilemmas surrounding “sham” or placebo surgery22 (though for a counter-argument, see reference no. 23). Moreover, blinding is impossible when comparing an operative versus a nonoperative intervention.Equivalence Among Groups. During accrual, randomiza-tion helps to

1	see reference no. 23). Moreover, blinding is impossible when comparing an operative versus a nonoperative intervention.Equivalence Among Groups. During accrual, randomiza-tion helps to ensure that each group in the study shares equiva-lent baseline demographics and unmeasured biases. However, throughout the study, each group should be treated equally (excluding the actual intervention) with respect to the number of clinical visits, diagnostic tests, etc. Enforcing the same pro-tocol to each study participant further decreases sources of bias and provides increased validity when performing final analysis.Completeness of Follow-Up. Attrition bias is the differences that occur between the groups when participants withdraw from the study. A pattern can usually be identified (the treatment, side effects of treatment, long follow-up time, or other factors) that leads to withdrawal from the study. These events can hinder the ability to interpret the results of the study, and researchers

1	side effects of treatment, long follow-up time, or other factors) that leads to withdrawal from the study. These events can hinder the ability to interpret the results of the study, and researchers should consider these implications during trial design. Furthermore, the mechanism of attrition may manifest in a bias; patients who elect to remain in a study may in fact select for characteristics that affect or determine efficacy (see the following section).Accuracy of Analysis. Analyzing the results of only partici-pants who completed all follow-up visits throughout the study can lead to skewed and inaccurate conclusions. Thus, most RCTs follow the principle of intention-to-treat (ITT) analysis. ITT analysis includes study participants who underwent initial randomization assignment regardless of events that transpired after randomization; thus ITT analysis is often described as “once randomized, always analyzed.” Removal of noncompli-ers from statistical analysis may overestimate the

1	of events that transpired after randomization; thus ITT analysis is often described as “once randomized, always analyzed.” Removal of noncompli-ers from statistical analysis may overestimate the effect size of the intervention. Furthermore, in clinical practice, a portion of patients will be noncompliant, and thus ITT analysis will more accurately represent the overall population.External ValidityThe goal of an RCT is to show a causative relationship between an intervention and an outcome. However, to change clinical practice, the results of the RCT must be both relevant and gen-eralizable to the clinical population; this assessment is called external validity.Number Needed to Treat. The number needed to treat (NNT) is defined as the number of patients that undergo the interven-tion before a single patient benefits compared to the control group in the trial. It is computed as the inverse of the risk dif-ference between two groups. The smaller the NNT, the more efficacious a treatment.

1	a single patient benefits compared to the control group in the trial. It is computed as the inverse of the risk dif-ference between two groups. The smaller the NNT, the more efficacious a treatment. For example, in an RCT comparing laparoscopic cholecystectomy to observation to prevent recur-rent idiopathic acute pancreatitis, the number needed to treat was five patients.24 The NNT should also be weighed against the adverse effects of the intervention.Number Needed to Harm. While NNT reports the number of patients who undergo the intervention before a single patient benefits, number needed to harm (NNH) describes how many patients undergo the intervention for one person to have an adverse event. The higher the NNH, the safer a treatment is. In general, interventions with a low NNT and high NNH are pre-ferred. However, NNT and NNH should not be used in isolation when determining the appropriateness of intervention as neither number takes into account the degree of benefit to

1	and high NNH are pre-ferred. However, NNT and NNH should not be used in isolation when determining the appropriateness of intervention as neither number takes into account the degree of benefit to harm.Generalizability of Results. RCTs have specific exclusion and inclusion criteria to recruit a study population that is homogenous with the goal of limiting sources of bias. While this method is appropriate for RCTs, the results may not directly translate to “real-world” situations with greater heterogeneity within the potential target population (see prior comment in “Hierarchies of Evidence”), leading to a potentially significant discrepancy between trial results and their implementation for day-to-day clinical decisions. In addition, RCTs often come to a conclusion that determines the best treatment for the “average” patient enrolled in the trial. However, most patients are not “average,” and therefore the proposed conclusion may not be relevant. Additional studies about the

1	the best treatment for the “average” patient enrolled in the trial. However, most patients are not “average,” and therefore the proposed conclusion may not be relevant. Additional studies about the intervention of interest in more heterogeneous populations can help convince physicians to change their clinical practice; these correlate to phase 4 pharmaceutical trials and point to the importance of continued postpractice change data collection and analysis. More importantly, principle 3 of EBM, which states that “clinical decisions should be influenced by patient values and preference,” needs to be accounted for, especially with the implementation of a new practice guideline or pattern.Additional Challenges to Conducting a Surgical RCTIn addition to methodological issues that might limit the reli-ability of a RCT, there are also considerable logistical barriers to performing a RCT. These are not trivial factors, and they contribute heavily to the number and size of RCTs that can be

1	the reli-ability of a RCT, there are also considerable logistical barriers to performing a RCT. These are not trivial factors, and they contribute heavily to the number and size of RCTs that can be done, particularly in surgical populations.Recruitment. One of the most challenging aspects of an RCT is recruiting an adequate number of patients to provide a high and sufficient degree of statistical power to demonstrate a measurable difference between interventions. This becomes exponentially more difficult with the prevalence of certain rare diseases. To help overcome low accrual, many trials expand their study to other hospitals and facilities at the expense of increased heterogeneity. While this may decrease internal valid-ity, the benefit is the increase in external validity.Learning Curves and Expertise-Based Design. Pharma-ceutical-based RCTs normally have higher internal validity compared to surgical trials because of the effect of surgeon experience and technique affecting

1	Curves and Expertise-Based Design. Pharma-ceutical-based RCTs normally have higher internal validity compared to surgical trials because of the effect of surgeon experience and technique affecting patient outcomes; this is especially impactful when new surgical procedures are intro-duced. While the administration of a drug is a straightforward process without measurable deviation, the same cannot be said Brunicardi_Ch51_p2137-p2152.indd 214628/02/19 4:19 PM 2147UNDERSTANDING, EVALUATING, AND USING EVIDENCE FOR SURGICAL PRACTICECHAPTER 51Table 51-3Decisions regarding the null hypothesisTABLE OF ERROR TYPES NULL HYPOTHESIS (H0) ISTRUEFALSEDecision about null hypothesis (H0) RejectType I error (false positive)Correct inference (true positive)Fail to rejectCorrect inference (true negative)Type II error (false negative)regarding surgery. Novel surgical procedures have defined learning curves even for the most experienced surgeons. During this learning process, surgeon inexperience,

1	II error (false negative)regarding surgery. Novel surgical procedures have defined learning curves even for the most experienced surgeons. During this learning process, surgeon inexperience, either in technical features of the procedure or procedure-related decision-making, can lead to adverse patient outcomes. Thus, neglecting the learn-ing curve can lead to an underestimation of the success of the experimental intervention; conversely, accounting for the learn-ing curve can be necessary in assessing how a new procedure can best be disseminated across the community. Furthermore, beyond the evaluation of new procedures, even with established procedures each individual surgeon is likely to have acquired throughout his/her career unique techniques and habits when operating on patients. This heterogeneity of surgeon experience and technique can limit standardization for a trial intervention.To help solve the issue of surgeon heterogeneity and inex-perience, RCTs can employ

1	patients. This heterogeneity of surgeon experience and technique can limit standardization for a trial intervention.To help solve the issue of surgeon heterogeneity and inex-perience, RCTs can employ “expertise-based design.” In this method, patients remain randomized to either the intervention or control, but the operating surgeons are experts in the surgery they are performing. This technique is already followed during cross-specialty RCTs, such as open gastrostomy tube placement versus interventional-radiology (IR) gastrostomy tube place-ment. However, this does not model every day clinical practice because not all surgeons are considered experts in the procedure described in a particular trial.All-or-None Situation. Despite continual pressure to prove treatment effect by using a RCT, there are situations when conducting a trial does not make ethical or common sense. A famous example is from the British Medical Journal in 2003 that questioned as to why there are no RCTs evaluating

1	there are situations when conducting a trial does not make ethical or common sense. A famous example is from the British Medical Journal in 2003 that questioned as to why there are no RCTs evaluating the use of parachutes during gravitational free-fall.25 The authors state that the evidence to support the use of parachutes is purely observational yet it is considered a “gold standard” practice. This demonstrates the concept of an all-or-none situation, where the study population exposed to a risk experiences the outcome and none of the population experiences the outcome with the intervention. Performing an RCT on this type of situation would be dangerous and unethical, and thus purely observational data can provide a high degree of sufficient evidence.Noninferiority Trials. As reviewed earlier, trialing a new therapy compared to a placebo or sham raises serious ethical issues, especially when an effective therapy has already been established. Moreover, a portion of randomized control

1	trialing a new therapy compared to a placebo or sham raises serious ethical issues, especially when an effective therapy has already been established. Moreover, a portion of randomized control trials today are evaluating secondary endpoints, such as quality of life, safety, and cost efficiency of a new therapy compared to the existing gold standard. These studies are called noninferiority trials, with the intent to prove efficacy that is not worse than the existing therapy. For example, a 2004 study compared open versus laparoscopic colectomy for colon cancer. The aim was to show similar oncologic endpoints with improved secondary outcomes (improved cosmesis, decreased postoperative pain, decreased hernia incidence).26 The prevalence of these trials have increased substantially from under 100 in 2005 to nearly 600 in 2015.27 The most important consideration when evaluating this type of trial is the prespecified margin of noninferiority, a value that is largely arbitrary in the

1	under 100 in 2005 to nearly 600 in 2015.27 The most important consideration when evaluating this type of trial is the prespecified margin of noninferiority, a value that is largely arbitrary in the literature.28USE AND MISUSE OF STATISTICAL SIGNIFICANCEThe use of statistical methods is central to the scientific process; it is only through statistics that the problem of induction29 can be addressed. While this chapter is not intended to be 6a comprehensive description of statistical methods, understand-ing the appropriate application of statistical tools is critical to being able to assess the conclusions presented in the literature, and therefore we present a summary of those statistical terms that are most germane to being able to interpret a clinical study.Type I and Type II ErrorsBy necessity, statistical testing requires declaration of a null hypothesis, usually corresponding to the “default” state (i.e., no difference or the patient is healthy). The alternative hypothesis would

1	statistical testing requires declaration of a null hypothesis, usually corresponding to the “default” state (i.e., no difference or the patient is healthy). The alternative hypothesis would then negate the stated null hypothesis (i.e., there is a dif-ference or the patient is unhealthy). The result of a statistical significance test may either reject or accept the null hypothesis, and this result can correspond with the true state (a correct deci-sion) or not correspond with the true state (an error). Two types of error are possible (Table 51-3).Type I Error. A type I error occurs when the null hypothesis is rejected but is actually true in the population. This may also be referred to as a false positive. The type I error rate, denoted by the Greek letter α (alpha), is the probability that the null hypothesis is rejected given that it is true. The error rate may also be referred to as the significance level, and often a value of 0.05, or 5%, is frequently used in the literature.Type

1	the null hypothesis is rejected given that it is true. The error rate may also be referred to as the significance level, and often a value of 0.05, or 5%, is frequently used in the literature.Type II Error. A type II error is the failure to reject the null hypothesis when the null hypothesis is false. This error may also be referred to as a false negative. The type II error rate is denoted by the Greek letter β (beta), and is related to the power of a study. Power can range from 0 to 1, and as power increases, there is decreasing probability of making a type II error. Power is related to three main factors: (a) the statistical significance criterion of the study, (b) the magnitude of the effect of interest, and (c) the sample size used to detect the effect. Power analysis can be used to calculate the minimum sample size required for a study so that one can be likely to detect an effect of a given size.P ValuesThe P value was an innovation most closely associated with Sir Ronald

1	to calculate the minimum sample size required for a study so that one can be likely to detect an effect of a given size.P ValuesThe P value was an innovation most closely associated with Sir Ronald Fisher, one of the founders of modern statistics. The definition of a P value is the probability of an observed result given the assumption that the null hypothesis is true. The arbi-trary value established for a result having statistical significance rather than “pure chance” is less than 1 in 20 defined as a P value less than 0.05.30 Put differently, the chance of making a false-positive conclusion is 5% at a P value of 0.05 (type I error). This risk of making a false-positive conclusion is called a “type I error.” Importantly, the P value reported in the study is specific for that study’s patient sample and may not be gen-eralizable to the overall population. The probability of a false positive report not actually having an association depends not Brunicardi_Ch51_p2137-p2152.indd

1	patient sample and may not be gen-eralizable to the overall population. The probability of a false positive report not actually having an association depends not Brunicardi_Ch51_p2137-p2152.indd 214728/02/19 4:19 PM 2148SPECIFIC CONSIDERATIONSPART IIonly on the associated P value, but also the prior probability that the association is real and the statistical power of the given study.31,32 The basis of this is due to perpetual undersampling of all possible relationships in a given scientific domain. This will inherently lead to type I errors with respect to all clinical pos-sibilities. Recently, statisticians have postulated that utilizing a P value of 0.05 will lead to wrong conclusions at least 30% of the time and may be even higher with underpowered studies.33The use of P values also categorizes statistical conclusions in a binary format. Should a P value of 0.049 be significant but a 0.051 not be significant? Furthermore, P values provide no insight into the effect size being

1	categorizes statistical conclusions in a binary format. Should a P value of 0.049 be significant but a 0.051 not be significant? Furthermore, P values provide no insight into the effect size being measured. Simply, an intervention may be statistically significant but lack any clinical significance. Purely utilizing a P value to determine the value of research findings without assessing the effect size, confidence interval, and power of the study can be misleading.Despite these flaws identified in P values, the frequency of their appearance in modern literature has continued to increase.34 Each reader should be carefully skeptical of P values and await replication with similar significance for confirmation. Fisher did not anticipate or endorse the use of the modern P <0.05 criteria. Rather, he envisioned that experiments would be repeated until the investigator was sure that he or she had learned how to use the experimental intervention to get a predictable result.Alternative to P

1	he envisioned that experiments would be repeated until the investigator was sure that he or she had learned how to use the experimental intervention to get a predictable result.Alternative to P ValuesOne potential alternative to Fisher’s approach and the limita-tion of P values is Bayesian statistics. The common element of Bayesian statistics is to provide a probability of a hypothesis being true by using prior knowledge or empirical data to esti-mate four probabilities:1. The probability that the hypothesis is true.2. The probability that the hypothesis is true given the observed data.3. The probability that the alternative hypothesis is true.4. The probability that the data would have been observed if the alternative hypothesis is true.These parameters are used to calculate a Bayes factor, or a ratio of the likelihood probability of two competing hypotheses. One difficulty for many studies is that there can be very little reli-able data that can be used to estimate these probability

1	or a ratio of the likelihood probability of two competing hypotheses. One difficulty for many studies is that there can be very little reli-able data that can be used to estimate these probability parameters.It is important to remember that both P values and Bayes factors are mathematically defined entities, and many of the issues that have arisen with P values are due to how they are interpreted by scientists and clinicians. A false interpretation of a Bayes fac-tor is just as troublesome as a false interpretation of a P value.HOW DO THE TOOLS OF EBM PERFORM?As mentioned previously, GRADE has been widely adopted by national and international medical societies, health-related branches of government, healthcare regulatory bodies, and online medical resources such as UpToDate.16 Widespread use of the system has emphasized consistency in the rating of guidelines and an easy to understand strength assessment based upon evi-dence quality. However, at the heart of any EBM system is a

1	use of the system has emphasized consistency in the rating of guidelines and an easy to understand strength assessment based upon evi-dence quality. However, at the heart of any EBM system is a central paradox: as systems have evolved during the EBM move-ment, there is no evidence that the systems themselves are reliable.7External ConsistencyGRADE is one of several EBM systems that aim to evaluate evidence and create recommendations, but it is unknown how it compares with other previously established systems.The GRADE Working Group attempted to address this question by comparing six different systems (The American College of Chest Physicians Evidence-Based Guidelines, Australian National Health and Medical Research Council Guidelines, Oxford Centre for Evidence-Based Medicine, Scottish Intercollegiate Guidelines Network, U.S. Preventive Services Task Force Recommendations, U.S. Task Force on Community Preventive Services Recommendations) on 12 criteria to assess the overall usefulness

1	Guidelines Network, U.S. Preventive Services Task Force Recommendations, U.S. Task Force on Community Preventive Services Recommendations) on 12 criteria to assess the overall usefulness of each approach. The authors found that there was poor agreement about the sensibility of the six systems.35 Given that there is no agreed upon or proven gold standard, one may be concerned about the lack of external consistency among different systems. GRADE was constructed to overcome these issues; however, the system’s ability to do so has never been formally assessed.The example of the Surviving Sepsis Campaign (SSC), an important attempt to produce guidelines to improve the care of patients with sepsis or septic shock, suggests that GRADE has not overcome these problems. The endorsement of the SSC by many influential organizations underscores its importance. Nonetheless, the SSC illustrates some of the important difficul-ties with grading in general and with the GRADE system (Box: Examples of

1	by many influential organizations underscores its importance. Nonetheless, the SSC illustrates some of the important difficul-ties with grading in general and with the GRADE system (Box: Examples of Inconsistent Use of EBM).Examples of Inconsistent Use of EBMSurviving Sepsis Campaign• The Surviving Sepsis Campaign recommended rapid use of intravenous antibiotics in their 2004 guidelines, which was given a grade of “E,”36 corresponding to a recommenda-tion based upon level IV or V evidence, or the lowest levels possible.• In the 2008 update, the same recommendation was given; however, it was given a grade of 1B/1D (depend-ing on if shock was present), corresponding to a “strong” recommendation.37• Between 2004 and 2008, three additional studies were published; however, none were randomized controlled trials or came to conclusions that were different than the numerous studies that were published prior to 2004.38-40 Internal ConsistencyIn 2005, the GRADE working group published a pilot

1	controlled trials or came to conclusions that were different than the numerous studies that were published prior to 2004.38-40 Internal ConsistencyIn 2005, the GRADE working group published a pilot study of the system which found varied levels of agreement on the qual-ity of evidence for the outcomes in question among 17 asses-sors (kappa values [Box: The Kappa Coefficient] for agreement beyond chance ranged from 0 to 0.82; mean k = 0.27; k <0 for four judgements). The authors concluded that “judgements about evidence and recommendations are complex” and stated that with discussion they could resolve most disagreements.41 No assessment of reliability or proof of usefulness has been presented regarding the GRADE system since these findings.42System IssuesThe GRADE group considers the “strength” of their recom-mendations to reflect “the degree of confidence that the desir-able effects of adherence to a recommendation outweigh the The Kappa CoefficientThe Kappa coefficient is a statistic

1	of their recom-mendations to reflect “the degree of confidence that the desir-able effects of adherence to a recommendation outweigh the The Kappa CoefficientThe Kappa coefficient is a statistic that measures inter-rater agreement for qualitative items. It is thought to be a more robust measure than simple percent agreement since κ takes into account the possibility of the agreement occurring by chance. In general, κ values < 0 indicate no agreement, 0 to 0.2 slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.80 substantial agreement, and 0.81 to 1 as almost perfect agreement.Brunicardi_Ch51_p2137-p2152.indd 214828/02/19 4:19 PM 2149UNDERSTANDING, EVALUATING, AND USING EVIDENCE FOR SURGICAL PRACTICECHAPTER 51Internal ConsistencyIn 2005, the GRADE working group published a pilot study of the system which found varied levels of agreement on the qual-ity of evidence for the outcomes in question among 17 asses-sors (kappa values [Box: The Kappa

1	working group published a pilot study of the system which found varied levels of agreement on the qual-ity of evidence for the outcomes in question among 17 asses-sors (kappa values [Box: The Kappa Coefficient] for agreement beyond chance ranged from 0 to 0.82; mean k = 0.27; k <0 for four judgements). The authors concluded that “judgements about evidence and recommendations are complex” and stated that with discussion they could resolve most disagreements.41 No assessment of reliability or proof of usefulness has been presented regarding the GRADE system since these findings.42System IssuesThe GRADE group considers the “strength” of their recom-mendations to reflect “the degree of confidence that the desir-able effects of adherence to a recommendation outweigh the The Kappa CoefficientThe Kappa coefficient is a statistic that measures inter-rater agreement for qualitative items. It is thought to be a more robust measure than simple percent agreement since κ takes into account the

1	Kappa coefficient is a statistic that measures inter-rater agreement for qualitative items. It is thought to be a more robust measure than simple percent agreement since κ takes into account the possibility of the agreement occurring by chance. In general, κ values < 0 indicate no agreement, 0 to 0.2 slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.80 substantial agreement, and 0.81 to 1 as almost perfect agreement.undesirable effects.”43 However, at the same time, the GRADE system allows the strength of a given recommendation to exist independent of the quality of evidence that underpins that recom-mendation. The GRADE Working Group states that “separating the judgements regarding the quality of evidence from judge-ments about the strength of recommendations is a critical and defining feature of this new grading system.”42 However, such a system allows for “high quality” evidence for small effects while “low quality” evidence with a strong

1	is a critical and defining feature of this new grading system.”42 However, such a system allows for “high quality” evidence for small effects while “low quality” evidence with a strong recommendation is highly implausible except for certain obvious observations.Finally, the touted advantage of the leveling process in determining the quality of evidence requires significant indi-vidual adjudication. A given study design begins at a level of quality and can be upgraded or downgraded based on several judgments regarding adequacy of blinding, follow-up, consis-tency, generalizability, and effect size. Graders are supposed to balance the level of quality using these factors, yet each is fun-damentally different and cannot be simply added or subtracted, and it is therefore up to individual judgment as to how to weigh each factor.ValidityThe GRADE system is well described in a series of publica-tions; however, none of the publications provide validation, data, or proof of the usefulness of

1	as to how to weigh each factor.ValidityThe GRADE system is well described in a series of publica-tions; however, none of the publications provide validation, data, or proof of the usefulness of the system. The only pub-lication with data is mentioned earlier, which showed a low kappa for interobserver agreement.41 Based upon the systematic tenets of EBM and lack of literature-based proof for the effec-tiveness of GRADE, there would not be a basis for its use in creating recommendations. For example, no RCT assessing the effect of using EBM on patient outcomes has been undertaken. Therefore, EBM does not satisfy its own requirements and is, ironically, a form of systematic expert opinion. There is no data to suggest that systematic EBM approaches are superior to the decision-making capabilities of competent physicians with knowledge of the recent medical literature.Implications of EBMThe GRADE Working Group suggests that “strong recommen-dations should require little debate and would

1	of competent physicians with knowledge of the recent medical literature.Implications of EBMThe GRADE Working Group suggests that “strong recommen-dations should require little debate and would be implemented in most circumstances.”42 Although most strong recommenda-tions are likely accurate, definitive recommendations may have unintended consequences. For example, a definitive recom-mendation may have the effect of limiting debate or further research on a topic where the recommendation is misguided, and there are numerous examples where “strong” recommenda-tions were later retracted. High-level EBM recommendations concluded that antibiotic prophylaxis should be used in necro-tizing pancreatitis based upon multiple prospective randomized controlled trials, meta-analyses, and systematic reviews.14,44,45 These recommendations were later reversed, as additional tri-als showed that there was no benefit to antibiotic use in these patients.46A valid concern regarding EBM is that established

1	These recommendations were later reversed, as additional tri-als showed that there was no benefit to antibiotic use in these patients.46A valid concern regarding EBM is that established systems may lead to “strong” recommendations that are hard to challenge. This may even lead to situations where life-saving prospective studies are deemed “unethical” due to the presence of high-level, strong recommendations. As such, some groups have even issued warnings about converting practice guidelines into law.47,48THE ALTERNATIVES TO EBMEBM is appealing due to its ability to reduce and cope with uncer-tainty; however, the ability to mitigate uncertainty is not without drawbacks. The various EBM systems that exist are not always consistent in their evaluation of evidence, and even a single sys-tem may assign varying grades based on several subjective fac-tors. Finally, the performance of EBM in improving patient care has never been validated. Therefore, while most certainly a useful tool, the

1	may assign varying grades based on several subjective fac-tors. Finally, the performance of EBM in improving patient care has never been validated. Therefore, while most certainly a useful tool, the limitations of EBM must be recognized to avoid blind adherence to guidelines and oversimplification of the complex clinical decision making that occurs in daily clinical care.Although striving for certainty is understandable, it is con-trary to the reality of medicine in which decisions regarding indi-vidual patients are inherently complex. In fact, as science strives for “precision” and “individualized” medicine, EBM’s focus of creating guidelines to care for the “average” patient will exist as a paradox. The best physicians function on a foundation of scien-tific theory expressed in a setting of practical knowledge gained in a local context, or tacit knowledge. This is how complex phys-iology and pathology are combined to make a specific decision for an individual patient. Therefore,

1	of practical knowledge gained in a local context, or tacit knowledge. This is how complex phys-iology and pathology are combined to make a specific decision for an individual patient. Therefore, although it is tempting to think that EBM makes surgery more scientific, one must remem-ber that EBM itself is not founded in scientific principal.So, what is the alternative? The alternative is a common-sense application of scientific principals and healthy skepticism for the ongoing use of EBM as a guideline for practice. This allows physicians to use published guidelines, applied within the context of their practice, until a grading system has defini-tively been shown to positively affect patient outcomes or more precise application of patient data is made possible. Recommen-dations certainly can be useful information; however, clinicians should also understand that there is a nuance with respect to adherence to guidelines and that much lies outside the reaches of EBM. As such,

1	certainly can be useful information; however, clinicians should also understand that there is a nuance with respect to adherence to guidelines and that much lies outside the reaches of EBM. As such, understanding that daily clinical practice involves hundreds of decisions that require varying proportions of explicit and tacit knowledge is important in devising a system where guidelines are flexible and receptive to continual feed-back based upon the experiences of practicing physicians.WHAT CAN RESEARCHERS DO TO IMPROVE THE VALIDITY OF RESEARCH FINDINGS?Although it is impossible to know the truth with absolute certainty, researchers can take steps to ensure that the posttest probability is maximized. First, researchers can attempt to obtain better-powered evidence. Although even high-powered, low-bias meta analyses are not perfect, they do approach a theoretical “gold standard” of research, and although increasing power is important in arriving at correct conclusions, even

1	high-powered, low-bias meta analyses are not perfect, they do approach a theoretical “gold standard” of research, and although increasing power is important in arriving at correct conclusions, even high-powered studies can have significant biases. Additionally, obtaining large-scale evidence may not be possible for many research questions.Brunicardi_Ch51_p2137-p2152.indd 214928/02/19 4:19 PM 2150SPECIFIC CONSIDERATIONSPART IICrisis of Reproducibility and Medical Reversal: Implications for EBM“You keep using that word. I do not think it means what you think it means.”—Inigo Montoya from The Princess BrideThis chapter started by noting that the landscape of scientific knowledge is constantly evolving and that this fact impacts how we use and evaluate evidence as well. This 11th edition of Schwartz’s Principles of Surgery is being produced at a particularly volatile period in biomedical research as basic assumptions as to how scientific literature determines what constitutes

1	edition of Schwartz’s Principles of Surgery is being produced at a particularly volatile period in biomedical research as basic assumptions as to how scientific literature determines what constitutes “evidence” are being reassessed in a critical fashion. We believe it does a disservice to our readers if we fail to note and describe these trends, as they directly affect the basis of this chapter. The reassessment of biomedical literature and clinical trials can be loosely grouped into two distinct, but related topics: the crisis of reproducibility and the issue of medical reversal.The Crisis of ReproducibilityOver the past decade it has become increasingly recognized that certain medical studies, held forth as index publications upon which were based either fundamental precepts of practice or to justify entire directions of drug discovery, could not be repro-duced independently. This failure strikes at a fundamental assumption of science: that well performed studies with sufficient

1	or to justify entire directions of drug discovery, could not be repro-duced independently. This failure strikes at a fundamental assumption of science: that well performed studies with sufficient statistical significance represented generalizable knowledge that could be built upon. However, estimates of irreproducibility range from 75% to 90% based on mathematical inference, and practical investigations have shown as few as 0 in 52 observa-tional study findings being confirmed by randomized controlled trials (RCTs).49 Methodological errors in study design, patient selection, or research practices have been proposed as major contributing factors in the debate over replication of scientific stud-ies. However, despite the importance of replicating research findings, there is increasing concern that in modern research there is an intrinsic bias towards positive results in publication. Biases in study design, data collection, data analysis, or presentation of findings can lead to research

1	that in modern research there is an intrinsic bias towards positive results in publication. Biases in study design, data collection, data analysis, or presentation of findings can lead to research findings when they do not truly exist. As bias increases, the positive predictive value (PPV) of a given finding being true decreases considerably. The overall effect of bias again depends on both the power and prestudy odds of a given study. In some fields, it may in fact be the case that research findings are simply a measure of the prevailing bias. Medical research operates in areas with low preand poststudy probability for true findings, meaning it may be quite common that observed effect sizes varying around the null hypothesis (what one would expect from chance alone) are simply measuring the prevailing bias of a given field.In addition to bias, the globalization of research means that at any given time it is almost a certainty that multiple research teams are investigating the same

1	the prevailing bias of a given field.In addition to bias, the globalization of research means that at any given time it is almost a certainty that multiple research teams are investigating the same question or topic. Despite this fact, research findings by single teams are often considered in isolation, and the first to report a finding receives significantly more attention than subsequent studies. Suppose multiple research teams are investigating a given question with the null hypothesis being that there is no difference in treatment two treatment strategies. The probability that at least one of the groups will claim a significant research finding increases, and the positive predictive value decreases as the number of research teams increases. Unfortunately, there is little way to control for this phenomenon other than increasing the power of each individual study.Due to the combination of the aforementioned factors, the current framework of research means it is quite difficult to

1	for this phenomenon other than increasing the power of each individual study.Due to the combination of the aforementioned factors, the current framework of research means it is quite difficult to end up with a PPV >50%. Based on mathematical principles, even a well-constructed, adequately powered RCT with a pretest probability of 50% will arrive at a true conclusion only about 85% of the time.31 These findings limit the available literature upon which evidence-based medicine (EBM) relies and place a greater burden on practitioners when they are attempting to analyze and draw conclusions from what they find.Medical ReversalA related topic that directly impacts how EBM is carried out is that of medical reversal. This term was introduced by Vinay Prasad and Adam Cifu in 201150,51 to describe the process and pitfalls by which a previously established practice or drug falls out of favor because it is subsequently identified not to work. As such, the issue of medical reversal is impacted by

1	the process and pitfalls by which a previously established practice or drug falls out of favor because it is subsequently identified not to work. As such, the issue of medical reversal is impacted by the decision for a particular therapy to become adopted in the first place (ostensibly based on the principles of EBM) and the barriers to how subsequent evi-dence (either acquired through studies, or, more importantly, upon a more critical reassessment of the basis of its initial adoption) can reverse a prior recommendation. The set of intersecting issues related to medical reversal are highly complex (interested readers are encouraged to delve into the growing list of reports on this topic), but in terms of EBM, central issues addressed in medical reversal pertain to the use of surrogate endpoints in clinical trials, the presentation/misrepresentation of clinical trial effects, the effect of bias (academic and economic) in trial reporting and dissemination, and the strength and

1	endpoints in clinical trials, the presentation/misrepresentation of clinical trial effects, the effect of bias (academic and economic) in trial reporting and dissemination, and the strength and reliability of alternatives to RCTs (for all their flaws). As with the crisis of reproducibility, understanding the factors of medical reversal directly impacts what is appropriately considered “evidence” when executing EBM, placing greater responsibility on the surgical practitioner when deter-mining what is appropriate or optimal care.It should come as no surprise to the attentive reader that many of the issues related to the crisis of reproducibility and medical reversal refer back to the sources of bias and potentially perverse incentives originally noted by Francis Bacon back in 1620 (Box: The History and Sources of Bias in Biomedical Literature).Brunicardi_Ch51_p2137-p2152.indd 215028/02/19 4:19 PM 2151UNDERSTANDING, EVALUATING, AND USING EVIDENCE FOR SURGICAL PRACTICECHAPTER

1	The History and Sources of Bias in Biomedical Literature).Brunicardi_Ch51_p2137-p2152.indd 215028/02/19 4:19 PM 2151UNDERSTANDING, EVALUATING, AND USING EVIDENCE FOR SURGICAL PRACTICECHAPTER 51Second, as was noted previously, multiple teams often simultaneously address a given research question, and it is not proper to focus on any one study in isolation. Instead, clinicians should focus on the body of evidence in its entirety. A poten-tial solution would be connecting groups through networking of data. This would allow for more accurate analysis and drawing of conclusions, although it would require a significant change in the culture of academic research practices.Today, clinicians rely on the statistics provided in a scien-tific study to provide a summary of the results. We place trust and confidence that the paper’s biostatistician accurately and truthfully calculated these statistics without incorporating con-scious bias. Each article should completely answer four ques-tions

1	and confidence that the paper’s biostatistician accurately and truthfully calculated these statistics without incorporating con-scious bias. Each article should completely answer four ques-tions regarding the results of the study:1. What is the statistical significance of the results?2. What is the effect size and is this clinical relevant?3. What is the confidence interval?4. What is the underlying power of the study to detect a mean-ingful difference?Significant progress has been made since the adoption of EBM; however, the current direction of EBM-based guidelines have focused on populations as opposed to the complex, nuanced interactions that occur on a case by case basis. Algorithmic protocols actually serve to steer the focus away from an individual patient, at times leading to a disconnect between patients and physicians when physicians propose treatment based upon guidelines that do not adhere to that patient’s goals and values. So what can surgeons do to combat this, and how

1	between patients and physicians when physicians propose treatment based upon guidelines that do not adhere to that patient’s goals and values. So what can surgeons do to combat this, and how should they practice? One must ask: “What is the best course of action for this patient, in these circumstances, at this point in their illness or condition?” Therefore, evidence must be synthesized and then individualized for each patient encounter by interconnecting it with the ethics, personality, and values associated with the case at hand. Tools such as risk calculators are useful in informing discussion, but they should by no means be definitive evidence to recommend for or against a particular treatment. Judgment remains necessary in the practice of medicine, and therefore guidelines should be thought of as “rules of thumb” that require context as opposed to “rules of law.”REFERENCESEntries highlighted in bright blue are key references. 1. Bacon F, Fowler T. Bacon’s Novum Organum. Oxford:

1	thought of as “rules of thumb” that require context as opposed to “rules of law.”REFERENCESEntries highlighted in bright blue are key references. 1. Bacon F, Fowler T. Bacon’s Novum Organum. Oxford: Clarendon Press; 1878. 2. Djulbegovic B, Guyatt GH. Progress in evidence-based medi-cine: a quarter century on. Lancet. 2017;390(10092):415-423. Available at: http://dx.doi.org/10.1016/S0140-6736(16) 31592-6. Accessed August 27, 2018. 3. Sackett DL, Rosenberg WM, Gray JA, Haynes RB, Richardson WS. Evidence based medicine: what it is and what it isn’t. BMJ. 1996;312(7023):71-72. 4. Djulbegovic B, Guyatt GH, Ashcroft RE. Epistemologic inquiries in evidence-based medicine. Cancer Control. 2009;16(2):158-168. 5. Wente MN, Seiler CM, Uhl W, Buchler MW. Perspectives of evidence-based surgery. Dig Surg. 2003;20(4):263-269. 6. Solomon MJ, McLeod RS. Clinical studies in surgical journals—have we improved? Dis Colon Rectum. 1993;36(1):43-48. 7. Pollock AV. Surgical evaluation at the crossroads. Br

1	2003;20(4):263-269. 6. Solomon MJ, McLeod RS. Clinical studies in surgical journals—have we improved? Dis Colon Rectum. 1993;36(1):43-48. 7. Pollock AV. Surgical evaluation at the crossroads. Br J Surg. 1993;80(8):964-966. 8. Richardson WS, Wilson MC, Nishikawa J, Hayward RS. The well-built clinical question: a key to evidence-based decisions. ACP J Club. 1995;123(3):A12-A13. 9. Whipple AO, Parsons WB, Mullins CR. Treatment of carcinoma of the ampulla of vater. Ann Surg. 1935;102(4):763-779. 10. Nissen R. A simple operation for control of reflux esophagitis (in German). Schweiz Med Wochenschr. 1956;86(suppl 20): 590-592. 11. Patsopoulos NA, Analatos AA, Ioannidis JP. Relative citation impact of various study designs in the health sciences. JAMA. 2005;293(19):2362-2366. 12. Moore FA, Feliciano DV, Andrassy RJ, et al. Early enteral feeding, compared with parenteral, reduces postoperative septic complications. The results of a meta-analysis. Ann Surg. 1992;216(2):172-183. 13. Eikelboom

1	DV, Andrassy RJ, et al. Early enteral feeding, compared with parenteral, reduces postoperative septic complications. The results of a meta-analysis. Ann Surg. 1992;216(2):172-183. 13. Eikelboom JW, Karthikeyan G, Fagel N, Hirsh J. American Association of Orthopedic Surgeons and American College of Chest Physicians guidelines for venous thromboembolism prevention in hip and knee arthroplasty differ: what are the implications for clinicians and patients? Chest. 2009;135(2): 513-520. 14. Bassi C, Larvin M, Villatoro E. Antibiotic therapy for prophylaxis against infection of pancreatic necrosis in acute pancreatitis. Cochrane Database Syst Rev. 2003;(4):CD002941. 15. Guyatt GH, Oxman AD, Vist GE, et al. GRADE: an emerg-ing consensus on rating quality of evidence and strength of recommendations. BMJ. 2008;336(7650):924-926. 16. Higgins JPT, Green S, eds. Cochrane Handbook for Systematic Reviews of Interventions. Version 5.1.0. The Cochrane Collabo-ration; 2011. Available at:

1	recommendations. BMJ. 2008;336(7650):924-926. 16. Higgins JPT, Green S, eds. Cochrane Handbook for Systematic Reviews of Interventions. Version 5.1.0. The Cochrane Collabo-ration; 2011. Available at: https://handbook-5-1.cochrane.org. Accessed August 27, 2018. 17. Delle Fave G, Kwekkeboom DJ, Van Cutsem E, et al. ENETS Consensus Guidelines for the management of patients with gastroduodenal neoplasms. Neuroendocrinology. 2012;95(2):74-87. 18. Graham R, Mancher M, Miller Wolman D, Greenfield S, Steinberg E, eds. Institute of Medicine (US) Committee on Standards for Developing Trustworthy Clinical Prac-tice Guidelines. Clinical Practice Guidelines We Can Trust. Washington DC: National Academies Press; 2011. 19. Adie S, Harris IA, Naylor JM, Mittal R. CONSORT compliance in surgical randomized trials: are we there yet? A systematic review. Ann Surg. 2013;258(6):872-878. 20. Schulz KF, Altman DG, Moher D; Consort Group. CON-SORT 2010 statement: updated guidelines for reporting par-allel

1	trials: are we there yet? A systematic review. Ann Surg. 2013;258(6):872-878. 20. Schulz KF, Altman DG, Moher D; Consort Group. CON-SORT 2010 statement: updated guidelines for reporting par-allel group randomised trials. Int J Surg. 2011;9(8):672-627. 21. Higgins JPT, Altman DG, Gøtzsche PC, et al. The Cochrane Collaboration’s tool for assessing risk of bias in randomised trials. BMJ. 2011;343:d5928. 22. Das AK. Randomised clinical trials in surgery: a look at the ethical and practical issues. Indian J Surg. 2011;73(4): 245-250. 23. Wartolowska K, Judge A, Hopewell S, et al. Use of placebo controls in the evaluation of surgery: systematic review. BMJ. 2014;348:g3253. 24. Raty S, Pulkkinen J, Nordback I, et al. Can laparoscopic cholecystectomy prevent recurrent idiopathic acute pancreatitis? A prospective randomized multicenter trial. Ann Surg. 2015; 262(5):736-741. 25. Smith GC, Pell JP. Parachute use to prevent death and major trauma related to gravitational challenge: systematic

1	A prospective randomized multicenter trial. Ann Surg. 2015; 262(5):736-741. 25. Smith GC, Pell JP. Parachute use to prevent death and major trauma related to gravitational challenge: systematic review of randomised controlled trials. BMJ. 2003;327(7429): 1459-1461. 26. Clinical Outcomes of Surgical Therapy Study Group, Nelson H, Sargent DJ, et al. A comparison of laparoscopically assisted and open colectomy for colon cancer. N Engl J Med. 2004;350(20):2050-2059.Brunicardi_Ch51_p2137-p2152.indd 215128/02/19 4:19 PM 2152SPECIFIC CONSIDERATIONSPART II 27. Mauri L, D’Agostino RB Sr. Challenges in the design and interpretation of noninferiority trials. N Engl J Med. 2017;377(14):1357-1367. 28. Ho PM, Peterson PN, Masoudi FA. Evaluating the evi-dence: is there a rigid hierarchy? Circulation. 2008; 118(16):1675-1684. 29. Hume D, Norton DF, Norton MJ. A Treatise of Human Nature. Oxford; New York: Oxford University Press; 2000. 30. Dahiru T. P-value, a true test of statistical

1	Circulation. 2008; 118(16):1675-1684. 29. Hume D, Norton DF, Norton MJ. A Treatise of Human Nature. Oxford; New York: Oxford University Press; 2000. 30. Dahiru T. P-value, a true test of statistical significance? A cautionary note. Ann Ib Postgrad Med. 2008;6(1):21-26. 31. Ioannidis JP. Why most published research findings are false. PLoS Med. 2005;2(8):e124. 32. Wacholder S, Chanock S, Garcia-Closas M, El Ghormli L, Rothman N. Assessing the probability that a positive report is false: an approach for molecular epidemiology studies. J Natl Cancer Inst. 2004;96(6):434-442. 33. Colquhoun D. An investigation of the false discovery rate and the misinterpretation of P values. R Soc Open Sci. 2014;1(3): 140216. 34. Chavalarias D, Wallach JD, Li AH, Ioannidis JP. Evolution of reporting P values in the biomedical literature, 1990-2015. JAMA. 2016;315(11):1141-1148. 35. Atkins D, Eccles M, Flottorp S, et al. Systems for grading the quality of evidence and the strength of recommendations I:

1	in the biomedical literature, 1990-2015. JAMA. 2016;315(11):1141-1148. 35. Atkins D, Eccles M, Flottorp S, et al. Systems for grading the quality of evidence and the strength of recommendations I: critical appraisal of existing approaches The GRADE Working Group. BMC Health Serv Res. 2004;4(1):38. 36. Dellinger RP, Carlet JM, Masur H, et al. Surviving Sepsis Campaign guidelines for management of severe sepsis and septic shock. Crit Care Med. 2004;32(3):858-873. 37. Dellinger RP, Levy MM, Carlet JM, et al. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008. Crit Care Med. 2008;36(1): 296-327. 38. Houck PM, Bratzler DW, Nsa W, Ma A, Bartlett JG. Timing of antibiotic administration and outcomes for Medicare patients hospitalized with community-acquired pneumonia. Arch Intern Med. 2004;164(6):637-644. 39. Kumar A, Haery C, Paladugu B, et al. The duration of hypotension before the initiation of antibiotic treatment is a critical

1	community-acquired pneumonia. Arch Intern Med. 2004;164(6):637-644. 39. Kumar A, Haery C, Paladugu B, et al. The duration of hypotension before the initiation of antibiotic treatment is a critical determinant of survival in a murine model of Escherichia coli septic shock: association with serum lactate and inflammatory cytokine levels. J Infect Dis. 2006;193(2):251-258. 40. Proulx N, Frechette D, Toye B, Chan J, Kravcik S. Delays in the administration of antibiotics are associated with mortality from adult acute bacterial meningitis. QJM. 2005;98(4):291-298. 41. Atkins D, Briss PA, Eccles M, et al. Systems for grading the quality of evidence and the strength of recommendations II: pilot study of a new system. BMC Health Serv Res. 2005;5(1):25. 42. Kavanagh BP. The GRADE system for rating clinical guidelines. PLoS Med. 2009;6(9):e1000094. 43. The GRADE Working Group. 2018. Available at: http://www .gradeworkinggroup.org. Accessed August 27, 2018. 44. Sharma VK, Howden CW. Prophylactic

1	clinical guidelines. PLoS Med. 2009;6(9):e1000094. 43. The GRADE Working Group. 2018. Available at: http://www .gradeworkinggroup.org. Accessed August 27, 2018. 44. Sharma VK, Howden CW. Prophylactic antibiotic administration reduces sepsis and mortality in acute necrotizing pancreatitis: a meta-analysis. Pancreas. 2001;22(1):28-31. 45. Bassi C, Mangiante G, Falconi M, Salvia R, Frigerio I, Pederzoli P. Prophylaxis for septic complications in acute necrotizing pancreatitis. J Hepatobiliary Pancreat Surg. 2001;8(3):211-215. 46. Villatoro E, Mulla M, Larvin M. Antibiotic therapy for prophylaxis against infection of pancreatic necrosis in acute pancreatitis. Cochrane Database Syst Rev. 2010;(5):CD002941. 47. Fein IA, Corrato RR. Clinical practice guidelines: culture eats strategy for breakfast, lunch, and dinner. Crit Care Med. 2008;36(4):1360-1361. 48. Jacobson PD. Transforming clinical practice guidelines into legislative mandates: proceed with abundant caution. JAMA.

1	for breakfast, lunch, and dinner. Crit Care Med. 2008;36(4):1360-1361. 48. Jacobson PD. Transforming clinical practice guidelines into legislative mandates: proceed with abundant caution. JAMA. 2008;299(2):208-210. 49. Begley CG, Ioannidis JP. Reproducibility in science: improv-ing the standard for basic and preclinical research. Circ Res. 2015;116(1):116-126. 50. Prasad V, Cifu A. Medical reversal: why we must raise the bar before adopting new technologies. Yale J Biol Med. 2011;84(4):471-478. 51. Prasad V, Gall V, Cifu A. The frequency of medical reversal. Arch Intern Med. 2011;171(18):1675-1676.Brunicardi_Ch51_p2137-p2152.indd 215228/02/19 4:19 PM

1	Ambulatory SurgeryMarcus Adair, Stephen Markowiak, Hollis Merrick, James R. Macho, Kara Richardson, Moriah Muscaro, Munier Nazzal, and F. Charles Brunicardi 52chapterINTRODUCTIONAmbulatory SurgeryAmbulatory Surgery is a multidisciplinary field in which surgi-cal procedures are performed on patients who are not expected to be admitted to the hospital. The field includes procedures performed on patients in the setting of hospital outpatient departments (HOPDs), freestanding ambulatory surgery cen-ters (ASCs), and those performed in doctor’s offices. The word ambulatory comes from the Latin ambulare, which means “to walk,” indicating that the patients arrived at the procedure on their own and departed after the procedure to their home environment.1Improved anesthesia techniques, the development of min-imally invasive procedures, and changes to healthcare policy (particularly healthcare funding) have been the driving fac-tors behind the increase in ambulatory surgery. Prior to these

1	of min-imally invasive procedures, and changes to healthcare policy (particularly healthcare funding) have been the driving fac-tors behind the increase in ambulatory surgery. Prior to these advances, almost all surgery was performed in an inpatient hos-pital setting. Any outpatient surgeries were minor, performed in physicians’ offices, and paid for by Medicare and insurers as part of the physician’s office visit reimbursement.2Since the early 1980s, the volume of ambulatory surgery has increased in the United States.2,3 Between 1981 and 2005, the number of outpatient surgeries nationwide grew almost 10-fold to over 32.0 million per year. Outpatient procedures grew from 19% to 60% of all surgical procedures, by volume, in the United States from 1981 to 2011.2,4,5 Strong financial incentives exist for hospi-tals to shift some surgeries to an outpatient setting. The number of Medicare-certified ASCs has also increased steadily, from fewer than 300 in the early 1980s2 to 5532 in 2016

1	exist for hospi-tals to shift some surgeries to an outpatient setting. The number of Medicare-certified ASCs has also increased steadily, from fewer than 300 in the early 1980s2 to 5532 in 2016 (Fig. 52-1).5By definition, procedures at ASCs and physician’s offices are performed without the full resources of a hospital. The press has sensationalized a few adverse patient outcomes in these set-tings and made claims about the overall safety based on these isolated events. However, much of the convenience, high patient satisfaction rates, and cost-efficiency of ambulatory surgery is lost when performed in a hospital setting. Thus, the challenge underlying ambulatory surgery is performing safe operations on carefully selected patients in a manner that is patient-family–centric and economical.A majority of large hospitals also have their own outpatient surgery departments that exist within the hospital. ASCs per-form procedures faster and more efficiently than HOPDs and at higher volume

1	majority of large hospitals also have their own outpatient surgery departments that exist within the hospital. ASCs per-form procedures faster and more efficiently than HOPDs and at higher volume than both HOPDs and physicians’ offices. Addi-tionally, the number of certified, freestanding ASCs nationwide has eclipsed the total number of hospitals by more than 1000 centers.5 For these reasons, a plurality of outpatient surgical vol-ume in the United States is now performed in ASCs. It is critical for the modern surgeon to have a grasp of the unique clinical challenges and economic impacts of ambulatory surgery.Ambulatory Surgery CentersASCs are independent healthcare that offer patients the conve-nience of having surgery performed safely without admission to a hospital. ASCs provide only elective surgical services rather than emergency care. According to the Centers for Medicare & Medicaid Services (CMS), effective May 18, 2009, “ASCs are any distinct entity that operates exclusively

1	elective surgical services rather than emergency care. According to the Centers for Medicare & Medicaid Services (CMS), effective May 18, 2009, “ASCs are any distinct entity that operates exclusively for the purpose of providing surgical services to patients not requiring hospi-talization and which the expected duration of services would not exceed 24 hours following an admission.”4 Ambulatory sur-gery centers should not be confused with office-based surgery practices or with other outpatient centers that provide diagnos-tic services or primary healthcare, such as urgent care centers, community health centers, mobile diagnostic units, or rural health clinics. ASCs are distinguished from these other health-care facilities by (a) their use of a referral system for accept-ing patients and (b) their maintenance of a dedicated operating room. The first feature means that any patient who wants to be treated in an ambulatory surgery center must first consult a Introduction2153Ambulatory

1	(b) their maintenance of a dedicated operating room. The first feature means that any patient who wants to be treated in an ambulatory surgery center must first consult a Introduction2153Ambulatory Surgery / 2153Ambulatory Surgery Centers / 2153Aspects Leading to Increased Utilization / 2154History of Ambulatory Surgery and Ambulatory Surgery Centers2154Procedures Performed2156Benefits of Ambulatory Surgery Centers2156Factors Contributing to Popularization of ASCs / 2157Regulation, Costs, and Quality2158Regulation / 2158Costs / 2158Quality / 2159Ownership / 2159Potential for Conflict of Interest / 2159Challenges2159Reimbursement / 2159Patient Selection / 2159ASCs vs. Hospital Outpatient Departments vs. Office-Based Surgical Suites / 2160Aging Population / 2160Conclusion2160Brunicardi_Ch52_p2153-p2162.indd 215328/02/19 4:17 PM 2154199660005000Number of ASCs4000300020001000019982000200220042006200820102012Figure 52-1. Number of Medicare-certified ambulatory surgery centers

1	215328/02/19 4:17 PM 2154199660005000Number of ASCs4000300020001000019982000200220042006200820102012Figure 52-1. Number of Medicare-certified ambulatory surgery centers from 1996 to 2013.Key Points1 Define ambulatory surgery and the unique aspects of ambu-latory surgery centers (ASCs).2 Understand the history of ambulatory surgery and ASCs in the United States.3 Review the most common procedures performed in ASCs compared to those performed in a hospital setting.4 Discuss the financial benefits for patients and physicians within the structure of ASCs as they pertain to physician ownership, healthcare systems partnerships, and insurance reimbursement.5 Understand the regulatory and accreditation processes that affect ASCs.6 Predict how anticipated medical advances, technological development, and an aging population will affect the field of ambulatory surgery.primary healthcare provider (PCP) and choose to have the con-dition treated by surgery rather than an alternative approach.

1	and an aging population will affect the field of ambulatory surgery.primary healthcare provider (PCP) and choose to have the con-dition treated by surgery rather than an alternative approach. The second characteristic means that all ASCs must have at least one dedicated operating room and the equipment needed to per-form surgery safely and ensure quality patient care.1 Patients who choose to have surgery in an ASC arrive on the day of their procedure, have their surgery in a fully equipped operating room, and recover under the care of a highly-skilled anesthesia team, all without hospital admission.Aspects Leading to Increased UtilizationTechnological Developments. Improvements in anesthe-sia have facilitated the safe practice of outpatient surgery by the use of new medications, improved techniques in regional anesthesia, and better management of postoperative pain. The development of minimally invasive surgical techniques such as fiberoptic endoscopy, arthroscopy, ophthalmologic

1	techniques in regional anesthesia, and better management of postoperative pain. The development of minimally invasive surgical techniques such as fiberoptic endoscopy, arthroscopy, ophthalmologic procedures, and laparoscopic and robotic surgery have made it possible for patients to be discharged the on the same day as the surgery.Reduced Cost for Patients Without Compromise in Quality of Care. Ambulatory surgery facilities are highly specialized centers originating from a service model rather than the tradition hospital model. This approach allows for streamlined processes and reduced costs. Staffing is the largest cost for most healthcare facilities including ASCs, thus same-day surgery eliminates the need for overnight nursing and support staff.ASCs Offer Reduced Cost for Healthcare Systems. A review of commercial medical claims data found that annual U.S. healthcare costs are reduced by approximately $3.8 billion due to the availability of ASCs. Patients save more than $1.5

1	Systems. A review of commercial medical claims data found that annual U.S. healthcare costs are reduced by approximately $3.8 billion due to the availability of ASCs. Patients save more than $1.5 bil-lion due to lower deductibles and coinsurance payments. Over the next decade, ASCs are expected to save the U.S. healthcare system between $32.5 and $57.6 billion. This cost reduction is driven by the fact that, in general, ASC prices are significantly lower than HOPD prices for the same procedure in all mar-kets, regardless of payer.6 Table 52-1 displays the cost savings compared to hospital outpatient departments for the most com-monly performed procedures at ASCs nationwide. While most hospitals offer outpatient surgery, ambulatory surgery centers are regarded as a superior choice for certain procedures because of facility efficiencies and price regulation under the outpatient prospective payment system.6HISTORY OF AMBULATORY SURGERY AND AMBULATORY SURGERY CENTERSAmbulatory surgical

1	procedures because of facility efficiencies and price regulation under the outpatient prospective payment system.6HISTORY OF AMBULATORY SURGERY AND AMBULATORY SURGERY CENTERSAmbulatory surgical practice traces its history from the early work of itinerant dental surgeons who traveled their circuits by horseback and trains. They frequently operated in hotel rooms and then moved on. In 1909, James Nicoll (Fig. 52-2), a pediatric surgeon in Scotland, wrote of his experiences with ambulatory anesthesia and surgery on nearly 9000 children as outpatients during a 10-year interval at Glasgow Royal Hospi-tal for Sick Children.8 Operations included cleft lip and palate repair, correction of pyloric stenosis, mastoidectomy, repair of inguinal and umbilical hernias, and management of spina bifida and depressed skull fractures. Nicoll pleaded with his fellow surgeons to perform more pediatric operations on an outpatient basis, stating that “a large number of the cases at present treated in-door

1	depressed skull fractures. Nicoll pleaded with his fellow surgeons to perform more pediatric operations on an outpatient basis, stating that “a large number of the cases at present treated in-door constitutes a waste of the resources of a children’s hos-pital . . . . The results obtained in the out-patient department at a tithe [small part] of the cost are equally good.”8Ralph Waters (Fig. 52-3) was a pioneer in the field of ambulatory surgery. He developed an office-based practice in Sioux City, Iowa in 1919. Waters used nitrous oxide, morphine, and scopolamine. He believed medical conditions had to be well controlled prior to surgery and that certain medical condi-tions precluded outpatient care. In these ways, Waters’ clinic became the prototype for the modern free-standing ASC. Waters subsequently went on to establish the first academic residency program for training anesthesiologists at the University of Wisconsin.9Prior to the advent of freestanding ASCs, the concept of

1	Waters subsequently went on to establish the first academic residency program for training anesthesiologists at the University of Wisconsin.9Prior to the advent of freestanding ASCs, the concept of ambulatory surgery first needed to gain acceptance in the form of HOPDs. In 1959, Eric Webb and Horace Graves advocated out-patient surgery because of a shortage of hospital beds in Vancou-ver. The first HOPD in the United States was established in 1962 at the University of California, Los Angeles by David Cohen and John Dillon, who also sought to address a shortage of hospital beds. These efforts proved to be safe and cost-effective.9Brunicardi_Ch52_p2153-p2162.indd 215428/02/19 4:17 PM 2155AMBULATORY SURGERYCHAPTER 52Figure 52-2. James Henderson Nicoll, pediatric surgeon. (Repro-duced with permission from University of Glasgow Archives & Special Collections, University collection, GB 248 PH/PR 2475.)Figure 52-3. Ralph Milton Waters, anesthesiologist. (Used with permission from the

1	with permission from University of Glasgow Archives & Special Collections, University collection, GB 248 PH/PR 2475.)Figure 52-3. Ralph Milton Waters, anesthesiologist. (Used with permission from the American Society of Anesthesiologists.)Table 52-1Comparison of top 10 procedures performed at ASCs vs. hospitals nationwideTOP 10 PROCEDURES PERFORMED AT AMBULATORY SURGICAL CENTERS BY VOLUME AND CPT CODENUMBER PERFORMEDAVG PAY PER CLAIMSAVINGS AT ASCTOP 10 PROCEDURES PERFORMED AT HOSPITAL OUTPATIENT DEPARTMENTS (HOPD) BY VOLUME AND CPT CODENUMBER PERFORMEDAVG PAY PER CLAIM 1. Cataract surgery with intraoccular lens(66984)1155, 283$959$219 1. Subcutaneous tissue debridement (11042)841,517$213 2. Esophagogastroduodenoscopy with biopsy (43239)524,082$301$110 2. Esophagogastroduodenoscopy with biopsy (43239)628,900$411 3. Colonoscopy and biopsy (45380)416,218$352$172 3. Aspiration/injection of joint (20610)578,407$141 4. Colonoscopy with lesion removal (45385)331,565$401$20 4. Cataract

1	biopsy (43239)628,900$411 3. Colonoscopy and biopsy (45380)416,218$352$172 3. Aspiration/injection of joint (20610)578,407$141 4. Colonoscopy with lesion removal (45385)331,565$401$20 4. Cataract surgery with IOL implant (66984)512,191$1,178 5. Spine epidural injection foraminal (64483)282,962$335 5. Colonoscopy and biopsy (45380)472,886$524 6. Postlaser cataract surgery capsulotomy (66821)275,760$227 6. Colonoscopy with lesion removal (45385)350,001$421 7. Spine epidural injection lumbar, sacral (62311)210,159$358$120 7. Spine epidural injection lumbar, sacral (62311)326,956$478 8. Injection, paravertebral facet joint (64493)174,450$306 8. Insertion of temporary bladder catheter (51702)308,614$69 9. Diagnostic colonoscopy (45378)157,951$401$100 9. Appl. of multilayer compression system (29581)303,026$9710. Colorectal screening, high-risk individual (G0105)128,181$33310. Diagnostic colonoscopy (45378)253,350$501The first truly freestanding ASC, “SurgiCenter,” was opened in

1	system (29581)303,026$9710. Colorectal screening, high-risk individual (G0105)128,181$33310. Diagnostic colonoscopy (45378)253,350$501The first truly freestanding ASC, “SurgiCenter,” was opened in Phoenix, Arizona by Wallace A. Reed and John L. Ford in 1970 (Fig. 52-4). Reed and Ford were committed to pro-viding timely, convenient, and comfortable surgical services to patients in their community, and therefore, avoiding more imper-sonal venues like regular hospitals. Prior to opening SurgiCen-ter, Reed and Ford were frustrated with having patients wait 6 weeks or more to get elective surgery, and in some cases, found surgeries canceled because the rooms booked were needed for emergencies.9Brunicardi_Ch52_p2153-p2162.indd 215528/02/19 4:17 PM 2156SPECIFIC CONSIDERATIONSPART IIFigure 52-4. Image of “SurgiCenter” in 1970. (Used with permission from Banner Health.)Figure 52-5. George Isaac Minimally Invasive Surgery Center at the University of Toledo Medical Center. (Used with

1	52-4. Image of “SurgiCenter” in 1970. (Used with permission from Banner Health.)Figure 52-5. George Isaac Minimally Invasive Surgery Center at the University of Toledo Medical Center. (Used with permission from University of Toledo.)In its first 6 months, Surgicenter performed more than 1200 operations by 153 surgeons. No adverse cardiac events occurred during the procedures, and only one patient was hos-pitalized following surgery (due to poorly controlled diabetes, not because of the operation itself).10 SurgiCenter’s incredible success resulted in more than 400 visitors touring the facility in the first year in order to learn about the new model for patient care.11 Reed and Ford realized that a tremendous need existed for freestanding, independent ASCs. According to Reed, this propelled the formation of what eventually became the Ambula-tory Surgery Center Association (ASCA), a major credentialing body within the field of ambulatory surgery.11The Orkand Report of 1976, a U.S.

1	the formation of what eventually became the Ambula-tory Surgery Center Association (ASCA), a major credentialing body within the field of ambulatory surgery.11The Orkand Report of 1976, a U.S. government–spon-sored study of outpatient surgery, concluded that ambulatory surgical facilities can significantly reduce costs while main-taining the same high quality of surgical and anesthetic care achieved in hospitals.12The Society for Ambulatory Anesthesia (SAMBA) was established in 1984 to further the development of ambulatory anes-thesiology as a subspecialty. The field continued to advance with the publication of Wetchler’s Anesthesia for Ambulatory Surgery in 1985, the introduction of the journal Ambulatory Surgery in 1993, and with the first state requiring accreditation for all outpatient facilities (California in 1996). Many other states have since adopted these high standards and require accreditation of ASCs. CMS now requires certification for all ASCs (Fig. 52-5 and

1	all outpatient facilities (California in 1996). Many other states have since adopted these high standards and require accreditation of ASCs. CMS now requires certification for all ASCs (Fig. 52-5 and 52-6).5PROCEDURES PERFORMEDBy 1982, CMS had approved payments to ASCs for more than 200 procedures. Steady growth in the number of ASCs (Fig. 52-7) and the number of surgical procedures performed in the outpatient setting, including HOPDs, has continued since. Each year physicians perform more than 23 million procedures in ASCs. This shift toward outpatient procedures has increased due to advancements in medical practice and technology that have reduced the need for overnight hospital stays. Most patients, except those with complicated health conditions, can be served in the outpatient setting. Common ASC procedures include colonoscopies, cataract surgeries, tonsillectomies, and arthroscopic orthopedic surgeries. CMS currently approves and reimburses more than 3500 procedures in the ASC

1	Common ASC procedures include colonoscopies, cataract surgeries, tonsillectomies, and arthroscopic orthopedic surgeries. CMS currently approves and reimburses more than 3500 procedures in the ASC setting.5 New developments continue to expand the scope of ASCs.ASCs may perform surgeries in several specialties or dedi-cate their services to one specialty, such as eye care or sports medicine. The procedure must not pose a significant safety risk and not require an overnight stay when performed in an ASC. The types of surgical procedures performed in ASCs have undergone significant changes in recent years. Many of the early ASCs were outpatient centers for plastic surgery. Advances in minimally invasive surgical techniques in other specialties, how-ever, led to the establishment of ASCs for orthopedic, dental, and ophthalmologic procedures. See Fig. 52-8 for a recent anal-ysis of specialty services provided by ASCs nationwide.BENEFITS OF AMBULATORY SURGERY CENTERSSince their founding over

1	dental, and ophthalmologic procedures. See Fig. 52-8 for a recent anal-ysis of specialty services provided by ASCs nationwide.BENEFITS OF AMBULATORY SURGERY CENTERSSince their founding over 40 years ago, ASCs have grown exponentially. These distinct entities have provided physi-cians an avenue to provide specialized, efficient, and quality Figure 52-6. ProMedica Parkway Surgery Center. (Used with permission from ProMedica Health Systems.)Brunicardi_Ch52_p2153-p2162.indd 215628/02/19 4:17 PM 2157AMBULATORY SURGERYCHAPTER 5219486171118694117415 (AK)2 (GU)21 (HI)12218366426347196924956081693533941724 (PR)1 (VI)67136104523318610125122779923413411626 (NH)56 (MA)10 (RI)48 (CT)269 (NJ)23 (DE)345 (MD)3 (DC)79452Figure 52-7. As of June 2017, California has 794 ASCs, making it the leading state in terms of number of ASCs. It is followed by Florida with 417 ASCs and Texas with 366 ASCs. Vermont and the U.S. Virgin Islands have the lowest number of ASCs with one

1	making it the leading state in terms of number of ASCs. It is followed by Florida with 417 ASCs and Texas with 366 ASCs. Vermont and the U.S. Virgin Islands have the lowest number of ASCs with one each.13Painmanagement10%Urology5%Orthopedics15%Plastic8%Gi14%Ophthalmology30%Other18%Figure 52-8. Specialties served in ASCs. GI = gastroenterology. (Reproduced with permission from Centers for Medicare & Medicaid Services, 2011.)care to patients who need surgical procedures. Patient satisfac-tion with same-day surgery has remained relatively high since ASCs started in 1970. It is important to recognize that patients undergoing generally nonemergent surgery that does not require a hospital stay are relatively satisfied overall. Historically, the field of ambulatory surgery has been associated with very high patient satisfaction.14-15 In the future, the CMS Consumer Assessment of Healthcare Providers and Systems (CAHPS) sur-vey will report on nationwide patient satisfaction with ASCs and

1	with very high patient satisfaction.14-15 In the future, the CMS Consumer Assessment of Healthcare Providers and Systems (CAHPS) sur-vey will report on nationwide patient satisfaction with ASCs and HOPDs.16Factors Contributing to Popularization of ASCsCost. In many cases an outpatient procedure performed in an ASC is between one-half to one-third the cost as the same pro-cedure performed in a hospital. In large part, ASCs affect cost savings by eliminating overnight hospitalizations and emer-gency procedures. ASCs perform fewer extensive diagnostic tests and dispense fewer medications. These facilities are not staffed around the clock and are not encumbered by the need for expensive and highly specialized equipment as are hospitals. For example, the Medicare Payment Advisory Commission found that a cataract operation cost only $942 at an ambulatory surgery center in 2001 as opposed to $1334 at a hospital. Figures for an endoscopy and biopsy of the upper digestive tract were $429 and

1	that a cataract operation cost only $942 at an ambulatory surgery center in 2001 as opposed to $1334 at a hospital. Figures for an endoscopy and biopsy of the upper digestive tract were $429 and $359; for a diagnostic colonoscopy, $429 and $401; and for epi-dural anesthesia, $320 and $183, respectively5,7 (see Table 52-1).Organization, staffing, and specialization may play a large role in the cost differences between ASCs and HOPDs. On aver-age, patients who were treated in ASCs spend 31.8 fewer min-utes undergoing procedures than patients who were treated in HOPDs. ASCs could generate savings of $363 to $1000 per outpatient case.4Comfort. Whereas most hospitals keep patients recovering from a surgical procedure in separate rooms, in an ASC the patient usually can spend the recovery period after surgery with their loved ones. Limiting the number of delays and disruption of emergent cases allows the surgeon to spend more time with the patient in the preoperative and postoperative

1	period after surgery with their loved ones. Limiting the number of delays and disruption of emergent cases allows the surgeon to spend more time with the patient in the preoperative and postoperative areas.17-19Convenience. Because ASCs usually schedule routine cases lasting no longer than 2 hours (average 30–45 minutes), and handle no emergency cases, scheduling is typically accurate. By avoiding the logjam, ASCs reduce the waiting time for elective procedures. A study by Hair et al reviewing Medicare patients again showed freestanding ASCs performed surgeries in less time than hospital-based ASCs overall and for procedures on various anatomic systems that resulted in reduced total time spent in facility with earlier discharge.19 These results corrobo-rate the notion that freestanding ASCs tend to be more efficient than HOPDs.20 One possible advantage for patients would be that they are able to leave an ASC relatively quickly after their surgery, resulting in less time away from work

1	tend to be more efficient than HOPDs.20 One possible advantage for patients would be that they are able to leave an ASC relatively quickly after their surgery, resulting in less time away from work and family. This may be particularly true for pediatric patients or parents.Efficiency. This advantage is particularly important to sur-geons. It takes much less time to prepare an operating room in a specialized ASC for the next patient than in a standard hospital. Improved efficiency allows the surgeon to treat more Brunicardi_Ch52_p2153-p2162.indd 215728/02/19 4:17 PM 2158SPECIFIC CONSIDERATIONSPART IIpatients in the same amount of time than he or she would be able to do in a hospital; some surgeons maintain that they can do three times the number of procedures in an ASC as they could in a hospital setting. Many doctors prefer working in an ASC because they can set the standards for staffing, safety precautions, and postoperative care, rather than having these things decided for them

1	a hospital setting. Many doctors prefer working in an ASC because they can set the standards for staffing, safety precautions, and postoperative care, rather than having these things decided for them by a hospital manager.1 Trentman and coauthors discuss several factors that affect patient flow and could result in differences in preoperative and recovery times for outpatient procedures between ASCs and hospitals.20 For example, compared to the situation in hospitals, in ASCs sur-geons are more likely to be assigned to a single operating room for all cases, which reduces delays; the operating room often is closer to the preoperative and recovery rooms because facili-ties are smaller; teams of staff have clearer and more consistent roles, with less personnel turnover; and staffing is not done by shifts—that is, staff members go home only after all cases are finished, which creates incentives to work quickly. In addition, hospitals may be more likely to have emergency add-on and

1	not done by shifts—that is, staff members go home only after all cases are finished, which creates incentives to work quickly. In addition, hospitals may be more likely to have emergency add-on and bring-back cases for more complex cases that compete with outpatient procedures for operating room time.3,5,19 These dif-ferences suggest that hospitals would have to adopt a substan-tially different and highly specialized organizational model to achieve the same efficiencies as ASCs.REGULATION, COSTS, AND QUALITYRegulationHealthcare facilities in the United States are highly regulated by federal and state entities. ASCs are included in this over-sight, with both federal and state laws and regulations govern-ing all aspects of them. Independent observers evaluate the safety and quality of care provided in ASCs through three pro-cesses: Medicare certification, state licensure, and voluntary accreditation.To obtain Medicare certification, ASCs must meet the Medicare certification

1	of care provided in ASCs through three pro-cesses: Medicare certification, state licensure, and voluntary accreditation.To obtain Medicare certification, ASCs must meet the Medicare certification requirements, known as the Conditions for Coverage. These conditions include specifying standards for administration of anesthesia, quality evaluation, operating and recovery rooms, medical staff, nursing services, and other aspects of care. An ASC must have an inspection conducted by a state official or a representative of an organization that the government has authorized to conduct that inspection. These inspectors visit the ASC to verify that it meets established stan-dards. Each state determines the specific requirements ASCs must meet for licensure. An ASC does not have to be certi-fied by Medicare in order to be accredited by JCAHO; how-ever, most ASCs provide care to Medicare beneficiaries, so it is important to meet their requirements in order to be reimbursed appropriately. Medicare

1	in order to be accredited by JCAHO; how-ever, most ASCs provide care to Medicare beneficiaries, so it is important to meet their requirements in order to be reimbursed appropriately. Medicare inspection and certification of ambu-latory surgery centers is a separate process from professional accreditation.To obtain state licensure, many states have independent rules and regulations as well as associated fees. These third-party bodies can include Accreditation Association for Ambulatory Healthcare (AAAHC), American Association for Accreditation of Ambulatory Surgery Facilities (AAAASF), and the Joint Commission on Accreditation of Healthcare Organizations (JCAHO). In states for which accreditation is not mandated, ASCs may undergo voluntary accreditation through these same associations. In 37 states, any party looking to open an ASC must demonstrate a need for the ASC to exist.21 State licensure requirements generally exist for both healthcare facilities and healthcare

1	associations. In 37 states, any party looking to open an ASC must demonstrate a need for the ASC to exist.21 State licensure requirements generally exist for both healthcare facilities and healthcare professionals.14There was previously controversy as to whether accredi-tation status affects safety outcomes. In multivariate analyses that controlled for facility volume and patient characteristics, patients at Joint Commission–accredited facilities were still significantly less likely to be hospitalized after colonoscopy. Specifically, compared with patients treated in nonaccredited ASCs regulated by the state agency, patients treated at accred-ited facilities were less likely to be hospitalized within 7 to 30 days after surgery.22 All accredited ASCs must meet spe-cific standards that are evaluated during on-site inspections. Patients who visit accredited ASCs can be assured that those medical facilities have rigorous checkpoints to ensure high standards.The Ambulatory Surgery Center

1	evaluated during on-site inspections. Patients who visit accredited ASCs can be assured that those medical facilities have rigorous checkpoints to ensure high standards.The Ambulatory Surgery Center Association (ASCA) or ASC Association has an integral role to ensure top-quality healthcare from the nation’s ASCs. The ASCA was established when the two leading national ASC associations—Federated Ambulatory Surgery Association (FASA) and the American Association of Ambulatory Surgery Centers (AAASC)—merged. The ASCA serves as the national membership orga-nization as well as the advocacy group for ASCs.21 The ASCA works with legislative and regulatory bodies, liaises with other organizations to improve access, reduce the costs of healthcare, encouraging insurance coverage of outpatient procedures, and works to establish standards for ASCs. ASCA requires all of its facility members to be accredited, licensed, or Medicare certi-fied. The ASCA was instrumental in forming the accrediting body

1	and works to establish standards for ASCs. ASCA requires all of its facility members to be accredited, licensed, or Medicare certi-fied. The ASCA was instrumental in forming the accrediting body that is now the largest accreditor of ambulatory surgery centers in the country.22CostsToday, more than 5300 Medicare-certified ASCs offer simi-lar services compared to those performed at HOPDs, and do so at a more efficient rate with lower costs. ASCs are able to accomplish this by decreasing administrative and overhead expenses.5 Expenses for an ASC include staff wages, insur-ance, utilities, rent, janitorial services, as well as resources necessary to handle patient records, including technology sys-tems. ASCs are able to schedule procedures without the risk of surgeries getting moved or cancelled due to emergencies. Additionally, ASCs enable patients to go home on the same day, therefore spending less time with staff in postoperative recovery rooms.Starting in 1982, Medicare has covered

1	due to emergencies. Additionally, ASCs enable patients to go home on the same day, therefore spending less time with staff in postoperative recovery rooms.Starting in 1982, Medicare has covered surgical proce-dures provided in ASCs.21 There are two primary elements of total cost in a surgical procedure: the cost of physicians’ pro-fessional services and cost of the facility. Typically, providers bill for professional service separately, whereas facility costs are paid to the ASCs. Currently Medicare provides separate payments for 3500 surgical procedures under the ASC pay-ment system.5 The payment system is maintained by CMS, which adjusts fees annually to maintain budget neutrality. Through the Medicare Prescription Drug, Improvement, and Modernization Act of 2003, payments to ASCs are based on the Outpatient Prospective Payment System (OPPS) and capped at no more than 59% of what is paid to hospitals for the same service.5During the period of 2008 to 2011, the growth in number of

1	based on the Outpatient Prospective Payment System (OPPS) and capped at no more than 59% of what is paid to hospitals for the same service.5During the period of 2008 to 2011, the growth in number of ASCs plateaued. The system saved an estimated $7.5 bil-lion for the Medicare program and its beneficiaries.5 However, Brunicardi_Ch52_p2153-p2162.indd 215828/02/19 4:17 PM 2159AMBULATORY SURGERYCHAPTER 52growth in procedure volume during this time was greater in ASCs than in hospitals.3 This suggests that physicians and patients still preferred using ASCs, possibly due to advantages in cost, convenience, comfort, or the inability for hospitals to meet demand for outpatient surgeries.QualityThe majority of ambulatory surgery centers ensure safety through maintaining certification and licensing of the facili-ties and associated staff. There are strict regulations in place at both the state and federal level that ensure quality in ASCs, which have been detailed earlier. ASCs are held to

1	of the facili-ties and associated staff. There are strict regulations in place at both the state and federal level that ensure quality in ASCs, which have been detailed earlier. ASCs are held to the same high standard of care as all U.S. medical facilities. They have comparable rates of use of perioperative antibiotics, patient falls, wrong-site surgery, and use of safety checklists.23 Nation-wide and among all procedures, from 2014 to 2016 fewer than 2% of all visits to ASCs resulted in an unplanned hospital visit within 7 days.5 Of these hospital visits, 1.6% were emergency department or observation stays, and 0.6% were unplanned inpatient admissions.24 CMS is working to develop a new qual-ity measure that will track unplanned hospitalizations after care at an ASC. This would help create increasing data on the quality of ASCs. Due to the isolated nature of ASCs, provid-ers are often unaware when a patient goes to the emergency department or is admitted to an unaffiliated hospital

1	increasing data on the quality of ASCs. Due to the isolated nature of ASCs, provid-ers are often unaware when a patient goes to the emergency department or is admitted to an unaffiliated hospital following a procedure. Thus, such a measure from the CMS will help educate providers and allow for continued quality improve-ment among ASCs.Looking at the general surgical population, cholecys-tectomy represents a key procedure to track because it is frequently performed, requires technical skill, and can result in serious complications. Provided appropriate patient selec-tion, outpatient cholecystectomy has been demonstrated to be safely performed in ASCs. ASCs charge significantly less for performing this procedure after controlling for the variety of indications (median of $6028 for ASCs compared to $10,876 for HOPDs).25OwnershipIn 2017, 90% of ASCs have partial or complete physician own-ership, while 25% to 30% of ASCs are at least partially owned by hospitals, as compared to HOPDs,

1	compared to $10,876 for HOPDs).25OwnershipIn 2017, 90% of ASCs have partial or complete physician own-ership, while 25% to 30% of ASCs are at least partially owned by hospitals, as compared to HOPDs, which are by definition owned and operated exclusively by the hospital. A trend is developing for hospitals to purchase ASC ownership stakes due to their cost efficiency and as a means of diversifying revenue streams.26 Physicians have been a driving force in the devel-opment of ASCs through their ownership and building of new facilities. Ownership of an ASC provides many clear advantages to the physician because of the increased control and autonomy over their practice. Some advantages include ease of scheduling, shorter waiting times to get patients in for elective surgery, and the ability to hire specially trained and highly skilled staff. Phy-sicians can also avoid the bureaucracies of a hospital, including having elective surgeries cancelled to make room for emergent surgeries and

1	to hire specially trained and highly skilled staff. Phy-sicians can also avoid the bureaucracies of a hospital, including having elective surgeries cancelled to make room for emergent surgeries and delays due to more complicated surgeries. They can also ensure that the facility has specialized equipment for their particular specialty and can design the facility to meet their specific needs.26 Furthermore, physicians who perform surgeries in their own ASCs receive a share of the ASC’s facility payment in addition to payment for their professional services. This could present a conflict of interest in terms of referring patients and lowering the threshold for surgery, as discussed further in the next section.Potential for Conflict of InterestApproximately 90% of ASCs nationwide have at least some physician ownership stake. Many are joint ventures between hospitals and physicians.27 Increasing investment in these cen-ters may be explained in a number of ways, including an attempt by

1	least some physician ownership stake. Many are joint ventures between hospitals and physicians.27 Increasing investment in these cen-ters may be explained in a number of ways, including an attempt by providers to assert greater control over their professional lives, such as by having greater authority in scheduling sur-geries and in purchasing equipment. Alternatively, this invest-ment trend may be explained by declining reimbursements for physician services and rising practice costs. These economic pressures have intensified providers’ interest in nontraditional revenue sources, such as ASC investment, as a means of gen-erating income.Ownership entitles physicians to collect a share of the facil-ity’s profits from referrals, in addition to their professional fees. One potential conflict of interest is that physician-owners might lower their thresholds for intervention, exposing the patient and healthcare system to the harm and cost associated with unneces-sary treatment. After

1	of interest is that physician-owners might lower their thresholds for intervention, exposing the patient and healthcare system to the harm and cost associated with unneces-sary treatment. After differences between patients and health-care markets are adjusted for, physicians with ownership in an ASC have been found to perform a higher number of procedures compared with nonowner physicians at the same facilities.27 It has been noted that the increase in outpatient surgery at ASCs was more than double the decline in similar procedures per-formed in the hospital setting.28-29The other potential conflict in physician ASC ownership is in regard to patient referral. There is some evidence that physi-cians with an ownership stake may refer well-insured patients to their own facilities while referring Medicare and Medicaid patients to hospital outpatient clinics.30-32Regardless of the reason for increasing investment in and utilization of ASCs, it is important to note that ASC owner-ship

1	referring Medicare and Medicaid patients to hospital outpatient clinics.30-32Regardless of the reason for increasing investment in and utilization of ASCs, it is important to note that ASC owner-ship creates a potential conflict of interest for physicians. In the United States, physician financial interests are heavily regulated by the Stark Law. This can be either a financial investment, employment, or compensation agreement. To avoid conflicts of interest and potential legal violations, physicians should consult with attorneys and advisors knowledgeable in medical law and ethics prior to entering a financial relationship.33CHALLENGESReimbursementSavings from use of ASCs are primarily to the patient and health-care system. Reimbursement to ASCs for services provided is, as a direct result, lower than reimbursement paid to hospitals for the same procedures. It is up to ASCs, which operate as private corporations, to find profit and viability by keeping their costs low through

1	result, lower than reimbursement paid to hospitals for the same procedures. It is up to ASCs, which operate as private corporations, to find profit and viability by keeping their costs low through efficiency and staffing. Some steps suggested to improve the financial viability of maintaining an ASC include increasing the variety of procedures offered and grouping sur-geries in such a way as to maximize staffing usage.34Patient SelectionSafe use of ASCs is based on identification of patients who are unlikely to require admission to a hospital after their procedure. Thus far, ASCs do very well in this aspect. Unplanned admis-sions after ambulatory surgery occur in approximately 0.5% to 2.0% of cases.5,35-37 In the future, ASCs will be challenged to reduce this unplanned admission rate even further.Patients with Medicaid insurance, lower median household income, and a greater preoperative comorbidity burden have the Brunicardi_Ch52_p2153-p2162.indd 215928/02/19 4:17 PM 2160SPECIFIC

1	further.Patients with Medicaid insurance, lower median household income, and a greater preoperative comorbidity burden have the Brunicardi_Ch52_p2153-p2162.indd 215928/02/19 4:17 PM 2160SPECIFIC CONSIDERATIONSPART IIhighest odds of unplanned acute care use. These patients may benefit from interventions that enhance and streamline postop-erative follow-up.38 Additionally, the potential costs associated with postoperative acute care following procedures performed in ASCs are not insignificant. Patient specific predictors of unplanned hospital admission include age 65 years or older, anticipated operating time longer than 120 minutes, cardiac comorbidities, peripheral vascular disease, cerebrovascular dis-ease, malignancy, positive for human immunodeficiency virus (HIV), and regional or general anesthesia use.39The strongest predictor for unplanned inpatient hospital admission was the individual patient’s own history of previ-ous hospitalizations, particularly among older adults.

1	or general anesthesia use.39The strongest predictor for unplanned inpatient hospital admission was the individual patient’s own history of previ-ous hospitalizations, particularly among older adults. African American and Hispanic individuals also have had a markedly elevated risk of inpatient hospital admission, possibly related to cultural or socioeconomic issues of access to care.40 These mea-sures may provide a valuable target for quality improvement, cost improvement, and innovation.ASCs vs. Hospital Outpatient Departments vs. Office-Based Surgical SuitesCompetition is increasing among ASCs, HOPDs, and office-based surgical practices. The same improvements in anesthe-sia and surgical equipment that made outpatient surgery in a freestanding ASCs safe to perform have also led to a growing number of office based surgical suites and HOPDs. Procedures such as dental, ophthalmologic, endoscopy, cosmetic surgery, and liposuction are increasingly being performed in office-based

1	to a growing number of office based surgical suites and HOPDs. Procedures such as dental, ophthalmologic, endoscopy, cosmetic surgery, and liposuction are increasingly being performed in office-based facilities.41Physicians’ offices are under lower regulatory oversight in comparison to ASCs and HOPDs. This has resulted in the phenomenon of “practice drift,” whereby physicians perform procedures outside of their typical scope of practice. Despite several high-profile adverse events in lay media, prospective studies have demonstrated office-based surgical suites to be of similar safety.41,42 A recent, large study in the area of cosmetic surgery compared hospital-based procedures with office-based surgical suites and with ASCs and found accredited office-based surgical suites to be a safe alternative to ASCs and hospitals.43Aging PopulationWith increased risk of complications from surgery, the older adult population present a unique challenge to ASCs. ASCs must be prepared for

1	a safe alternative to ASCs and hospitals.43Aging PopulationWith increased risk of complications from surgery, the older adult population present a unique challenge to ASCs. ASCs must be prepared for complications that may arise from operating on older patients, and they must do so without the same resources as a hospital. The potential benefit of outpatient surgery for elderly patients is substantial, however, as older adults often suffer from postoperative cognitive dysfunction in an inpatient setting, which may be minimized with early discharge.44 Addi-tional benefits include lower cost to patients on fixed incomes and increased time and comfort at home.CONCLUSIONASCs represent a large benefit to society because of their poten-tial to reduce the financial burden of the healthcare system on the economy. At the same time, ASCs also provide high-quality care which is patient and family centered and convenient for providers. ASCs reduce the length of stay and minimize surgi-cal delays

1	on the economy. At the same time, ASCs also provide high-quality care which is patient and family centered and convenient for providers. ASCs reduce the length of stay and minimize surgi-cal delays and cancellations. In the future, ASC quality will be rigorously tracked due to more regulatory oversight and data collection. The future of ambulatory surgical centers remains bright. Case numbers are increasing across many specialties, including general surgery, plastic surgery, vascular, urologic, and orthopedic practices. Surgeons should follow the matura-tion of ambulatory surgery closely.REFERENCESEntries highlighted in bright blue are key references. 1. Senagore EAJ. Ambulatory surgery centers. In: Encyclopedia of Surgery. 1st ed. Farmington Hills, MI: Gale Group; 2004:37-43. 2. Hall MJ, Schwartzman A, Zhang J, Liu X. Ambulatory surgery data from hospitals and ambulatory surgery centers: United States, 2010. Natl Health Stat Report. 2017;(102):1-15. 3. Cullen KA, Hall MJ, Golosinskiy

1	Schwartzman A, Zhang J, Liu X. Ambulatory surgery data from hospitals and ambulatory surgery centers: United States, 2010. Natl Health Stat Report. 2017;(102):1-15. 3. Cullen KA, Hall MJ, Golosinskiy A. Ambulatory surgery in the United States, 2006. Natl Health Stat Report. 2009;(11):1-25. 4. Munnich EL, Parente S. Procedures take less time at ambu-latory surgery centers, keeping costs down and ability to meet demand up. Health Affairs 2014;33(5):764-769. 5. Medicare Payment Advisory Commission. 2018. Report to the Congress: Medicare Payment Policy. Washington, DC: MedPAC. Available at: http://www.medpac.gov/-documents-/reports. Accessed August 27, 2016. 6. Advancing Surgical Care Association. Medicare cost savings tied to ASCs. 2013. Available at: http://www.advancingsurgicalcare.com/medicarecostsavings. Accessed August 27, 2018. 7. Definitive Healthcare. Top 10 outpatient procedures at ASCs and hospitals. 2014. Available at:

1	Available at: http://www.advancingsurgicalcare.com/medicarecostsavings. Accessed August 27, 2018. 7. Definitive Healthcare. Top 10 outpatient procedures at ASCs and hospitals. 2014. Available at: https://www.definitivehc.com/news/top-10-outpatient-procedures-at-ascs-and-hospitals. Accessed August 27, 2018. 8. Nicoll J. The surgery of infancy. Br Med J. 1909;18:753-754. 9. Eger EI, Saidman LJ, Westhorpe RN, eds. The Wondorous Story of Anesthesia. New York: Springer; 2014. 10. The report. Arizona Republic. Sept. 10, 1970. 11. Ambulatory Surgery Center Association. Looking back and looking forward where it all began. FASA Update. May/June 2004. 12. Orkand DS. Comparative Evaluation of Costs, Quality, and System Effects of Ambulatory Surgery Performed in Alterna-tive Settings: Final Report. Washington: U.S. Dept. of Health, Education, and Welfare, Health Care Financing Administra-tion, Office of Policy Planning and Research; 1977. 13. Ambulatory Surgery Center Association. Number of ASCs

1	U.S. Dept. of Health, Education, and Welfare, Health Care Financing Administra-tion, Office of Policy Planning and Research; 1977. 13. Ambulatory Surgery Center Association. Number of ASCs per State. ASCs: a positive trend in healthcare. Advancing Surgi-cal Care. June 2017. Available at: https://www.ascassociation.org/advancingsurgicalcare/asc/numberofascsperstate. Accessed August 27, 2018. 14. Koenig L, Doherty J, Dreyfus J, Xanthapoulos J. An Analysis of Recent Growth of Ambulatory Surgical Centers. KNG Con-sulting. June 5, 2009. Available at: http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.512.4498&rep=rep1&type= pdf 15. Colorado Ambulatory Surgery Center Association. Ambulatory surgery center history. Available at: https://www.coloradoasc.org/general-resources/asc-history/. Accessed August 27, 2018. 16. Centers for Medicare & Medicaid Services. Overview. www .cms.gov/Medicare/Medicare-Fee-for-Service-Payment/ASCPayment/index.html. 17. Manchikanti L. Ambulatory surgery

1	Accessed August 27, 2018. 16. Centers for Medicare & Medicaid Services. Overview. www .cms.gov/Medicare/Medicare-Fee-for-Service-Payment/ASCPayment/index.html. 17. Manchikanti L. Ambulatory surgery centers and interven-tional techniques: a look at long-term survival. Pain Physician. 2011;14(2):177-215. 18. Ambulatory Surgery Center Association. What is an ASC? Available at: https://www.advancingsurgicalcare.com/ advancingsurgicalcare/asc/whatisanasc, Accessed August 27, 2018. 19. Hair B, Hussey P, Wynn B. A comparison of ambulatory periop-erative times in hospitals and freestanding owners. Am J Surg. 2012;204(1):23-27. 20. Trentman TL, Mueller JT, Gray RJ, Pockaj BA, Simula DV. Outpatient surgery performed in an ambulatory surgery center Brunicardi_Ch52_p2153-p2162.indd 216028/02/19 4:17 PM 2161AMBULATORY SURGERYCHAPTER 52versus a hospital: comparison of perioperative time intervals. Am J Surg. 2010;200(1):64-67. 21. Rechtoris M. 51 things to know about the ASC industry. Becker’s

1	PM 2161AMBULATORY SURGERYCHAPTER 52versus a hospital: comparison of perioperative time intervals. Am J Surg. 2010;200(1):64-67. 21. Rechtoris M. 51 things to know about the ASC industry. Becker’s ASC Review. Available at: https://www.beckersasc.com/asc-turnarounds-ideas-to-improve-performance/50-things-to-know-about-the-asc-industry-2017.html. Accessed August 27, 2018. 22. Ambulatory Surgery Center Association. Medicare certifi-cation. Available at: www.ascassociation.org/federalregula-tions/medicarecertification. Accessed August 27, 2018. 23. Ambulatory Surgery Center Quality Collaboration. Quarterly Report, 2nd Quarter 2018. http://www.ascquality.org/qualityre-port.cfm. 24. Dickson V. CMS seeks closer look at quality of care at ambu-latory surgical centers. Modern Healthcare. 2017. Available at: http://www.modernhealthcare.com/article/20170711/NEWS/170719987. Accessed August 27, 2018. 25. Paquette IM, Smink D, Finlayson SR. Outpatient cholecys-tectomy at hospitals versus

1	2017. Available at: http://www.modernhealthcare.com/article/20170711/NEWS/170719987. Accessed August 27, 2018. 25. Paquette IM, Smink D, Finlayson SR. Outpatient cholecys-tectomy at hospitals versus freestanding ambulatory surgical centers. J Am Coll Surg. 2008;206(2):301-305. 26. Ambulatory Surgery Center Association. Benefits of physician ownership. 2011. Available at: https://www.advancingsurgicalcare. com/asc/benefitsofphysicianownership. Accessed August 27, 2018. 27. Becker’s Hospital Review. ASC ownership trends in 2017: more complex joint venture deals. Available at: www.beckershospitalreview.com/hospital-transactions-and-valuation/asc-ownership-trends-in-2017-more-complex-joint-venture-deals.html. Accessed August 27, 2018. 28. Conrad DA, Sales A, Liang SY, et al. Impact of financial incen-tives on physician productivity in medical groups. Health Serv Res. 2002;37(4):885-906. 29. Hollenbeck BK, Dunn RL, Suskind AM, Strope SA, Zhang Y, Hollingsworth JM. Ambulatory surgery

1	incen-tives on physician productivity in medical groups. Health Serv Res. 2002;37(4):885-906. 29. Hollenbeck BK, Dunn RL, Suskind AM, Strope SA, Zhang Y, Hollingsworth JM. Ambulatory surgery centers and their intended effects on outpatient surgery. Health Serv Res. 2015;50(5):1491-1507. 30. Plotzke MR, Courtmanche C. Does procedure profitability impact whether an outpatient surgery is performed at an ambu-latory surgery center or a hospital? Health Econ. 20(7):817-830. 31. Mitchell JM. Effect of physician ownership of specialty hos-pitals and ambulatory surgery centers on frequency of use of outpatient orthopedic surgery. Arch Surg. 2010;145(8):732-738. 32. Gable JR, Fahlman C, Kang R, Wozniak G, Kletke P, Hay JW. Where do I send thee? Does physician-ownership affect referral patterns to ambulatory surgery centers? Health Affairs. 2008;27(3):165-174. 33. U.S. Department of Health and Human Services. Office of the Inspector General. A Roadmap for New Physicians: Fraud & Abuse Laws.

1	to ambulatory surgery centers? Health Affairs. 2008;27(3):165-174. 33. U.S. Department of Health and Human Services. Office of the Inspector General. A Roadmap for New Physicians: Fraud & Abuse Laws. Available at: oig.hhs.gov/compliance/physician-education/01laws.asp. Accessed August 27, 2018. 34. American College of Surgeons. Should your health care system invest in an ambulatory surgery center? A decision-making framework [Internet]. Bulletin of the American College of Surgeons. 2017 [cited 2018 Nov 28]; Available from: http://bulletin.facs.org/2017/11/should-your-health-care-system-invest-in-an-ambulatory-surgery-center-a-decision-making-framework/. 35. Mezei G, Chung F. Return hospital visits and hospital readmis-sions after ambulatory surgery. Ann Surg. 1999;230(5):721-727. 36. Twersky R, Fishman D, Homel P. What happens after discharge? Return hospital visits after ambulatory surgery. Anesth Analg. 1997;84:319-324. 37. Fox JP, Vashi AA, Ross JS, Gross CP. Hospital based, acute

1	R, Fishman D, Homel P. What happens after discharge? Return hospital visits after ambulatory surgery. Anesth Analg. 1997;84:319-324. 37. Fox JP, Vashi AA, Ross JS, Gross CP. Hospital based, acute care after ambulatory center discharge. Surgery. 2014;155(5): 743-753. 38. Molina G, Neville BA, Lipsitz SR, Gibbons L, Childers AK, et al. Postoperative acute care use after freestanding ambulatory surgery. J Surg Res. 2016;205(2):331-340. 39. Fleisher LA, Pasternak LR, Lyles A. A novel index of ele-vated risk of inpatient hospital admission immediately fol-lowing outpatient surgery. Arch Surg. 2007;142(3):263-268. 40. Fleisher LA, Pasternak LR, Herbert R, Anderson GF. Inpa-tient hospital admission and death after outpatient surgery in elderly patients: importance of patient and system charac-teristics and location of care. Arch Surg. 2004;139(1):67-72. 41. Urman RD, Punwani N, Shapiro FE. Office-based surgical and medical procedures: educational gaps. Ochsner J 2012;

1	and system charac-teristics and location of care. Arch Surg. 2004;139(1):67-72. 41. Urman RD, Punwani N, Shapiro FE. Office-based surgical and medical procedures: educational gaps. Ochsner J 2012; 12(4):383-388. 42. Coldiron B, Shreve E, Balkrishnan R. Patient injuries from surgical procedures performed in medical offices: three years of Florida data. Dermatol Surg. 2004;30(12 pt 1):1435-1443, discussion 1443. 43. Gupta V, Parikh R, Nguyen L, et al. Is office-based surgery safe? Comparing outcomes of 183,914 aesthetic surgical procedures across different types of accredited facilities. Aesthet Surg J. 2017;37(2):226-235. 44. Aurini L, White P. Anesthesia for the elderly outpatient. Curr Opin Anesthesiol. 2014;27:563-575.Brunicardi_Ch52_p2153-p2162.indd 216128/02/19 4:17 PM

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1	BACKGROUNDIntroductionThe basic American surgical training model is patterned after that established by William Steward Halsted at the Johns Hopkins Hospital in the late 19th century. By tradition, it has consisted of a regulated apprenticeship system with incremental increases in clinical responsibility for resident surgeons over a specified period of time culminating with the expectation that independent surgical practice will be possible at its conclu-sion. While this approach has served well throughout the 20th century, the pace of changes in healthcare delivery and society itself has driven the need for new approaches to education and training. Among the important changes that the surgical training community has had to confront are an expanding array of medi-cal and surgical therapies, complex, new and sometimes disrup-tive technologies advancing at a dizzying pace, and increasing recognition of the opportunities for errors and adverse outcomes because of inadequate preparation

1	complex, new and sometimes disrup-tive technologies advancing at a dizzying pace, and increasing recognition of the opportunities for errors and adverse outcomes because of inadequate preparation for this new world of surgical practice.1 Appreciation of the scale and insidious consequences of medical errors began to take hold in the 1990s when the groundbreaking Institute of Medicine report “To Err is Human” presented to the public a dire picture of the frequency and impli-cations of medical errors.2 Among the proposed new approaches to deal with this problem was the use of simulation training.Simulation is the imitation of an actual or possible real-world condition or event. The degree to which a simulation resembles its real-world counterpart describes its fidelity. There are innumerable specific applications of simulation to train or assess human performance on anticipated real-world tasks. As such, it is a valuable tool with great potential to increase safety in high-stakes

1	specific applications of simulation to train or assess human performance on anticipated real-world tasks. As such, it is a valuable tool with great potential to increase safety in high-stakes undertakings such as commercial aviation, military training, and nuclear power generation. These are a few areas where such training has been implemented successfully. With the advent of widely available digital computing, the level of technology applied to simulation and simulators has made near-complete fidelity possible. Moreover, the enormous expense associated with full motion flight simulation, for example, has been made acceptable by safety gains that benefit entire popula-tions. In medicine, there has been gradual acceptance of the role of simulation to achieve these same goals, and this has led to an array of simulation methods and uses that specifically target surgical providers at all levels. The fundamental justification for commitment of resources to simulation is an ethical one: to

1	led to an array of simulation methods and uses that specifically target surgical providers at all levels. The fundamental justification for commitment of resources to simulation is an ethical one: to reduce patient risk associated with invasive surgical procedures or management of complex clinical problems. Simulation meth-ods provide opportunities for surgical learners to practice their skills under safe conditions in preparation for clinical experi-ences and to be assessed and deemed ready for those encounters.Skills Labs and Skills TrainingThe history of surgical skills training outside the clinical oper-ating room (OR) is a long one, involving practice of surgical skills and procedures using various models, including animals and cadavers. Newer training practices, including simulation use in a laboratory setting, have emerged that focus on objec-tive assessment of skill and establishment of specific, defined levels of proficiency. These assessment-based approaches are relatively

1	use in a laboratory setting, have emerged that focus on objec-tive assessment of skill and establishment of specific, defined levels of proficiency. These assessment-based approaches are relatively recent educational developments and are being imple-mented as a means to improve surgeon skill in a safe setting for both learners and patients.3 The use of inanimate benchtop mod-els to test surgical skills required a leap into the world of vali-dation of measurement methodologies. The most important of these pioneering efforts was the implementation of the objective Skills and SimulationNeal E. Seymour and Carla M. Pugh 53chapterBackground2163Introduction / 2163Skills Labs and Skills Training / 2163Scientific Underpinnings of Simulator Use / 2165Simulation in Graduate Medical Education / 2165“Bootcamps” / 2166Specific Simulation Training and Assessment Applications2166Training Basic Surgical Skills / 2166Fundamentals of Laparoscopic Surgery / 2168Bench Models for Training Specific

1	2165“Bootcamps” / 2166Specific Simulation Training and Assessment Applications2166Training Basic Surgical Skills / 2166Fundamentals of Laparoscopic Surgery / 2168Bench Models for Training Specific Procedural Skills / 2168Transfer of Training / 2169Virtual Reality / 2169Virtual Reality for Flexible Endoscopy / 2172Virtual Reality for Endovascular Interventions / 2173Patient-Specific VR Surgery Simulation / 2174Robotic Surgery Simulators / 2174Fundamentals of Robotic Surgery / 2174Nontechnical Skills2175High-Fidelity Patient Simulation and Team Skills / 2175Error Prevention2177Simulation and Patient Outcomes2178Simulation Training for the Practicing Surgeon and Maintenance of Skill2179Future Considerations2179Brunicardi_Ch53_p2163-p2186.indd 216322/02/19 4:39 PM 2164Key Points1 Learning basic skills at the point of care imposes inef-ficiencies that might very well endanger support for the education mission.2 In 2006, the Accreditation Council for Graduate Medical Education

1	basic skills at the point of care imposes inef-ficiencies that might very well endanger support for the education mission.2 In 2006, the Accreditation Council for Graduate Medical Education Residency Review Committee for Surgery instituted a formal requirement for simulation training in surgical residency.3 Procedural skills training in a simulated environment has been shown to transfer to the real-life clinical setting.4 Early studies of virtual reality training using both proficiency-based and non–proficiency-based training methods showed it to be an effective means of improving laparoscopic skill both in the lab and in the operating room compared to non–virtual reality trained controls.5 Use of proficiency-based training in the context of a larger curriculum appears to be the best way to achieve good training results irrespective of the training platform used.6 When assessing simulator validity, researchers have noted that the use of robotic surgery simulators does translate to

1	to achieve good training results irrespective of the training platform used.6 When assessing simulator validity, researchers have noted that the use of robotic surgery simulators does translate to the clinical environment and the learning curve for initial console training for surgeons is significantly decreased.7 Simulation training for communication and other teamwork-pertinent nontechnical skills requires learners to be embedded in realistic scenarios pertinent to a healthcare team’s actual clinical responsibilities.8 Simulation technology allows trainees the opportunity to execute a variety of tasks and procedures while also expe-riencing the cognitive demands of surgery, including error correction and surgical planning decisions.9 The immediate future of simulation in surgery will likely see expanded use of proficiency-based training given the consistent demonstrations of effectiveness in improving surgeon skills and improved educational outcomes as mea-sured in clinical

1	will likely see expanded use of proficiency-based training given the consistent demonstrations of effectiveness in improving surgeon skills and improved educational outcomes as mea-sured in clinical settings.10 Advances in wearables, motion tracking, and sensor tech-nologies allow for a wide variety of hybrid and augmented experiences in simulation as well as extensive opportuni-ties for the development of new performance metrics.structured assessment of technical skills (OSATS) program by educators at the University of Toronto.4,5 Using a series of repro-ducible physical models of surgical tasks (excision of a skin lesion, bowel anastomosis, insertion of a T tube, and abdominal wall closure) and carefully designed rating instruments, it was possible to show validity of these measurements when com-pared with skill manifested during surgery in animal models. This demonstration of practical measurement of skill in the lab, where observation for skills rating purposes can be more

1	when com-pared with skill manifested during surgery in animal models. This demonstration of practical measurement of skill in the lab, where observation for skills rating purposes can be more read-ily achieved, was seminal in sparking both additional interest in lab-based training and in simulation as a mainstream educa-tional method for surgeons.The advent of laparoscopic general surgery in the late 1980s and early 1990s, a disruptive technology at that time, was a major driver for the use of surgical simulation to gain unfa-miliar and nonintuitive skills needed to safely perform limited-access videoscopic surgery. The recognition that bile duct injury risk is increased by surgeon inexperience stimulated interest in simulation in order to separate a component of the psychomotor learning curve for laparoscopy from the clinical OR and patient by transfering it to the training lab.6-8 At the same time, interest in the science of skills acquisition and measurement made efforts to gain

1	curve for laparoscopy from the clinical OR and patient by transfering it to the training lab.6-8 At the same time, interest in the science of skills acquisition and measurement made efforts to gain surgical proficiency with simulation more meaningful. The measured performance characteristics of experienced sur-geons came to be appreciated as useful learning targets for less experienced surgical learners, and the concept of proficiency-based training began to be implemented successfully not only for research purposes but also for formative education.9As simulation began to be used more extensively and skills labs either evolved or merged with multidisciplinary education centers using simulation as a primary instructional method, recognition of such centers as focal points for surgical education also grew. The American College of Surgeons (ACS) Education Division recognized early on that simulation training was an important educational method that surgeons could take advantage of and

1	also grew. The American College of Surgeons (ACS) Education Division recognized early on that simulation training was an important educational method that surgeons could take advantage of and began to explore ways to facilitate growth and implement training centers. In 2006, the ACS began to accredit education centers engaged in simulation training as Level 1 (comprehensive) and Level 2 (focused) Education Institutes.10 The consortium of 95 institutes, as of early 2017, now spans the globe with centers across the United States and Canada as well as several institutions outside of North America.In 2007, the American College of Surgeons and the Asso-ciation of Program Directors in Surgery (APDS) initiated a project to provide a standardized skills curriculum for surgi-cal residents. These efforts produced the modular ACS-APDS skills curricula that represent the first comprehensive and widely available resource that prescribes simulation experi-ences as the principal means to achieve

1	efforts produced the modular ACS-APDS skills curricula that represent the first comprehensive and widely available resource that prescribes simulation experi-ences as the principal means to achieve educational objectives.11 The resources and curricula are readily available to residency programs to address surgical resident learning needs and to facilitate simulation lab use as mandated by the Accreditation Council for Graduate Medical Education (ACGME). The three components of the curriculum are basic technical skills, proce-dural skills, and team skills. Although these have been acknowl-edged to be valuable and have been utilized to meet resident training needs, implementation has been limited according to a recent survey of residency programs.12 Elements of the basic skills curriculum were reported to be used by 36% of respon-dents while procedural and team training modules were reported to be in use by about half as many programs. Nonetheless, the article notes that simulation

1	were reported to be used by 36% of respon-dents while procedural and team training modules were reported to be in use by about half as many programs. Nonetheless, the article notes that simulation methods had permeated surgical education and the various stakeholder organizations had taken steps to either endorse or implement programs for simulation use. The need to explore simulation-based education, as stated by the Surgical Council on Resident Education (SCORE), sug-gests a pathway by which simulation methods might become fully integrated into standardized cognitive elements of surgical residency curriculum.13 Based on current trends, a competency framework can be envisioned that combines advanced forms of Brunicardi_Ch53_p2163-p2186.indd 216422/02/19 4:39 PM 2165SKILLS AND SIMULATIONCHAPTER 53standards-based learning using simulation and measured per-formance in the OR for purposes of trainee advancement and certification.Scientific Underpinnings of Simulator UseThe assumption

1	53standards-based learning using simulation and measured per-formance in the OR for purposes of trainee advancement and certification.Scientific Underpinnings of Simulator UseThe assumption that surgical technical skills can be effec-tively learned and tested outside the OR serves as the basis for simulation based assessment of skills before, during, and following training. This capability permits the explicit descrip-tion of understandable surgeon performance characteristics including those that can be characterized as desirable training goals (e.g., “expert”). The effectiveness of specific models of training can also be ascertained by comparative studies examin-ing educational endpoints, such as rate of learning, or testable performance either in simulations or in clinical settings. Sim-ulation-based learning has been described using various peda-gogical models to help educators understand and leverage the most effective strategies to achieve educational goals. The most fundamental

1	learning has been described using various peda-gogical models to help educators understand and leverage the most effective strategies to achieve educational goals. The most fundamental concept used to justify time spent in skills training is the prospect of achieving progressively higher levels of skill pertinent to clinical care. For medical care providers, this pro-gression is commonly related to the learning model described by Dreyfus and Dreyfus.14 This model depicts changes in specific mental functions (recollection, recognition, decision, awareness) associated with incremental steps in the progres-sion from novice status to that of expert (Fig. 53-1). The rel-evance of Anders Ericsson’s description of deliberate practice to development of clinical skills is also widely accepted.15 This is predicated on the concept that “expert” performance is fun-damentally different than normal performance and results from behaviors that “reflect a life-long period of deliberate effort to

1	This is predicated on the concept that “expert” performance is fun-damentally different than normal performance and results from behaviors that “reflect a life-long period of deliberate effort to improve performance in a specific domain.”16 The drive to achieve mastery in surgery, provides an aspirational model for the surgeon’s use of simulation because the opportunity for deliberate, repetitive practice of numerous skills in the clinical setting alone is insufficient and in many cases nearly impossi-ble. For this concept to be translated into effective educational results, simulation-based education must be of high quality, and the curricular framework created for its use must be directed toward carefully crafted, attainable, and clinically relevant edu-cational goals.Simulation in Graduate Medical EducationSurgery residency has been a particular area of focus for simula-tion use in assessment and training. The need for safe and rapid skills development is especially important in

1	Medical EducationSurgery residency has been a particular area of focus for simula-tion use in assessment and training. The need for safe and rapid skills development is especially important in this group of learn-ers whose time in training is limited and for whom every hour spent in education must be prioritized for highest impact. The added direct costs of operative care in training institutions has also provides a strong imperative to conduct training that mini-mizes negative implications of resident involvement in surgical cases.17 Learning basic skills at the point of care imposes inef-ficiencies that might very well endanger support for the education mission.11. Transcends reliance on rules, guidelines, and maxims2. Intuitive grasp of situations based on deep understanding3. Has a vision of what is possible4. Uses an analytical approach in new situationsExpertProÿcient1. Holistic view of situation2. Prioritizes importance of aspects3. Perceives deviations from the normal

1	Has a vision of what is possible4. Uses an analytical approach in new situationsExpertProÿcient1. Holistic view of situation2. Prioritizes importance of aspects3. Perceives deviations from the normal pattern4. Employs maxims for guidance, with meanings that adapt to the situation at handNovice1. Rigid adherence to taught rules or plans2. No exercise of discretionary judgementAdvancedbeginner1. Limited situational perception2. All aspects of work treated separately with equal importanceCompetent1. Coping with crowdedness (multiple activities, information)2. Some perception of actions in relation to goals3. Deliberate planning4. Formulates routinesFigure 53-1. Dreyfus model describing stepwise skills development. In surgery, specific stages of expertise are achieved through cognitive learning, technical practice, and experience and are defined by specific cognitive and behavioral characteristics affecting how we perceive, process, and act in the task environment. (Reproduced with

1	learning, technical practice, and experience and are defined by specific cognitive and behavioral characteristics affecting how we perceive, process, and act in the task environment. (Reproduced with permission from University of South Australia; Teaching and Learning in Health Sciences: https://lo.unisa.edu.au/mod/book/view.php?id=611284&chapterid=104113.)Brunicardi_Ch53_p2163-p2186.indd 216522/02/19 4:39 PM 2166SPECIFIC CONSIDERATIONSPART IIDespite early examples of excellence in simulation lab use and fairly wide availability, a survey of residency programs in 2006 suggested the quality of usage for laparoscopic skills training was probably low and that more than half did not use a defined curriculum.18 Also in 2006, the ACGME Residency Review Committee (RRC) for Surgery instituted a formal requirement for simulation training in surgical residency.19 While the core program requirement did not define any specific educational objectives, performance outcomes, or specific methods

1	a formal requirement for simulation training in surgical residency.19 While the core program requirement did not define any specific educational objectives, performance outcomes, or specific methods to be used, there was a mandate that simulation in surgical education be objectives-based and that it be incorpo-rated into residency curricula within 2 years. As of 2016, the relevant core program requirement indicates that “resources must include simulation and skills laboratories” and that “these facilities must address acquisition and maintenance of skills with a competency-based method of evaluation.”20 Since the original notification of the need to conduct this training, man-dated activities have expanded with the pairing of ACGME statements and new American Board of Surgery (ABS) require-ments for certifications in Fundamentals of Laparoscopic Surgery (FLS; instituted for 2010 residency graduates) and in Fundamentals of Endoscopic Surgery (FES; instituted for 2018 residency

1	require-ments for certifications in Fundamentals of Laparoscopic Surgery (FLS; instituted for 2010 residency graduates) and in Fundamentals of Endoscopic Surgery (FES; instituted for 2018 residency graduates). Both of these certifications demand lab-based practice in order to successfully pass respective technical skill test components.Both high and low technology and fidelity, off-the-shelf, simulation training solutions have become available for sur-gery residents’ lab-based training. Curricular content such as the ACS-APDS skills modules are readily available to facilitate a sufficient level of implementation to meet the RRC require-ments, although preparation methods have not been specifically prescribed by either the ACGME or the ABS. In 2015, the ABS issued a new requirement for a comprehensive multilevel Flexible Endoscopy Curriculum (FEC) paired with FES certification. This curriculum states explicitly that experiential requirements at the lower levels can be met with

1	for a comprehensive multilevel Flexible Endoscopy Curriculum (FEC) paired with FES certification. This curriculum states explicitly that experiential requirements at the lower levels can be met with simulation methods but it is left to individual programs to decide what preparatory practice in simulation might consist of for their own residents.21Options for simulation use in surgical training are cur-rently numerous and can be implemented with basic facilities and equipment, but the determination to use these methods suc-cessfully requires considerable effort that is greatly aided by fully motivated and engaged faculty members with protected time. There are now numerous guides and recommendations for successful surgical simulation lab start-ups.18,22-24“Bootcamps”Preparatory training for surgery residency both before and immediately after the start of residency is now a widely used educational practice. The rationale for these programs includes early development of basic skills that

1	surgery residency both before and immediately after the start of residency is now a widely used educational practice. The rationale for these programs includes early development of basic skills that would be of obvious use to the new trainee as well as determination of the status of basic skills that would serve as a needs assessment to model curricu-lar efforts expected to be most appropriate for the individual resident.25-27 The suggestion that new interns will render safer care has been garnered from results of intensive, short-duration preparatory training that shows higher level of skills measured in simulation are feasible compared to the “control” situation of not using such training.28 However, none of the current reports address whether intensive preparatory training addresses the “July Effect” by improving clinical effectiveness during the earliest months of training.28Much of the training and assessment during these pro-grams is accomplished using simulation methods. No

1	the “July Effect” by improving clinical effectiveness during the earliest months of training.28Much of the training and assessment during these pro-grams is accomplished using simulation methods. No stan-dardized approach has yet been suggested, and at the present time the impact of specific simulation components is difficult to assess for senior students entering surgical training, or new interns. Sound recommendations on this await further study. A single meta-analysis of postgraduate “boot camp” programs both prior to or at the start of residency found that all programs utilized high and low technology simulation methods as “a key component.”29 Although the analysis included all medical spe-cialties, 93% of the studies were surgical in nature, underscor-ing the perceived value of this preparatory training for new surgeons. The examined studies were those where preand posttraining effects were measured in some way. The compiled data revealed that trainees who completed the programs

1	preparatory training for new surgeons. The examined studies were those where preand posttraining effects were measured in some way. The compiled data revealed that trainees who completed the programs had uni-formly strong increases in skills development, knowledge, and confidence. In 2014 the ABS, ACS, APDS, and Association for Surgical Education, citing the evidence of effectiveness of these now numerous preparatory courses, officially endorsed them as a useful method to position fourth-year students and interns for early success in residency.30 Whether preresidency exposure to surgical simulation can influence a medical student’s decision to pursue a surgical career remains to be determined.SPECIFIC SIMULATION TRAINING AND ASSESSMENT APPLICATIONSTraining Basic Surgical SkillsThe use of the simulation lab to train open, laparoscopic, and flexible endoscopic basic skills in preparation for care and prac-tice clinical constitutes the most accessible and widely adopted set of simulation

1	simulation lab to train open, laparoscopic, and flexible endoscopic basic skills in preparation for care and prac-tice clinical constitutes the most accessible and widely adopted set of simulation training practices. In connection with these, the term “proficiency-based” training (sometimes used synony-mously with competency-based or objectives-based) is often used, and can be defined as the use of meaningful performance standards as educational goals for training. Implicit in the term is the expectation that if proficiency standards are achieved, a learner will be better positioned to perform to a desirable stan-dard in the clinical setting. Even these most basic skills have been shown to improve with proficiency-based training.31 Addi-tional benefits of proficiency-based approaches to basic surgical skills include knowledge of a surgical learner’s specific educa-tional needs and design of a larger skills curriculum roadmap that envisions progression to more advanced skills

1	to basic surgical skills include knowledge of a surgical learner’s specific educa-tional needs and design of a larger skills curriculum roadmap that envisions progression to more advanced skills training.Educators at Southern Illinois University implemented a program of intensive lab-based practice of basic skills as a pre-liminary requirement to participate in operative cases. Termed “Verification of Proficiency,” this program targets junior train-ees for assessment of skill using OSATS-like rating instruments, but its unique feature is the definition of a minimal level of skill that would permit a resident to assume the role of operator for specific case types.32,33More recently, the concept of “proficiency-based progression” has been used to describe a formalized process of use of sequenced proficiency standards and a continuum of progres-sively more advanced and challenging simulation experiences as a potential future model of training.34 Such a model would span whatever period

1	proficiency standards and a continuum of progres-sively more advanced and challenging simulation experiences as a potential future model of training.34 Such a model would span whatever period of time was necessary for each learner to progress from basic surgical skills to more advanced ones.Open Surgical Skills. A variety of benchtop models for prac-tice and assessment of basic open skills are available, the most 2Brunicardi_Ch53_p2163-p2186.indd 216622/02/19 4:39 PM 2167SKILLS AND SIMULATIONCHAPTER 53Table 53-1ACS-APDS basic skills curriculum components• Asepsis and instrument identification• Knot tying• Suturing• Skin flaps• Skin grafts• Urethral catheterization• Airway management• Chest tube insertion• Central line insertion• Surgical biopsy• Laparotomy opening and closure• Basic laparoscopy skills• Advanced laparoscopy skills• Hand-sewn bowel anastomosis• Stapled bowel anastomosis• Arterial anastomosisFigure 53-2. Abdominal wall closure model made from commonly available

1	laparoscopy skills• Advanced laparoscopy skills• Hand-sewn bowel anastomosis• Stapled bowel anastomosis• Arterial anastomosisFigure 53-2. Abdominal wall closure model made from commonly available materials using instructions in the ACS-APDS Basic Skills Curriculum module for this task.well-known of which are the OSATS tasks that remain in use more than 20 years after their inception. The ACS-APDS Basic Skills Curriculum (Table 53-1) is a useful resource for this.Generally, lower-fidelity models are more cost effec-tive for repetitive training because durability with reuse may be better than high-fidelity physical models and replacement costs can be minimized for many tasks. Examples can include simple models for abdominal wall closure (Fig. 53-2). The use of low-fidelity models in well-formulated curricular train-ing can be very effective in increasing resident skill, although high-quality measurement can be labor intensive. Bowel and vascular anastomosis training has been an

1	well-formulated curricular train-ing can be very effective in increasing resident skill, although high-quality measurement can be labor intensive. Bowel and vascular anastomosis training has been an especially impor-tant point of focus given their prominence and highly technical nature in clinical practice. In a recent analysis of resident expe-rience with gastrointestinal anastomosis, Nemeth reported that although frequently performed (average 67 per resident), stapled anastomosis experience predominates during training (91% of laparoscopic procedures and 82% of open ones), suggesting a relatively small clinical experience with hand-sewn anastomotic methods, which remain critically relevant.35 Simulated bowel anastomosis models are widely available (Fig. 53-3), and there are numerous reports of successful curriculum-based devel-opment of this core technical skill generally utilizing OSATS rating instruments.36-38 Although further study is required to demonstrate a clinical effect of

1	of successful curriculum-based devel-opment of this core technical skill generally utilizing OSATS rating instruments.36-38 Although further study is required to demonstrate a clinical effect of this training, other experience strongly suggests that use of proficiency-based anastomosis training should be the next step. In an example of how this could be used for trainee benefit, Palter conducted a randomized trial of use of a proficiency-based technical skills and cognitive cur-riculum for abdominal wall closure.39 The technical skills com-ponent utilized a low-fidelity model for an OSATS assessments of both lab and OR performance. Residents in the intervention arm performed better on both clinical abdominal wall closure and a test of procedural knowledge than controls.Basic Laparoscopic Manipulative Skills. Current evidence supports the concept that basic laparoscopic skills should be developed in the training lab, avoiding safety concerns as well as the expenditure of time and

1	Manipulative Skills. Current evidence supports the concept that basic laparoscopic skills should be developed in the training lab, avoiding safety concerns as well as the expenditure of time and effort that would result from learning in clinical settings. Specific programs for basic laparo-scopic skills development coupled with skills assessment were set forth 20 years ago at a time when this need was newly articu-lated for surgeons at all levels.7,40,41 These utilized videoscopic training “boxes” for two-handed practice using laparoscopic instruments to manipulate box contents. Programs such as the Rosser drills employed various dexterity tasks emphasizing time for task completion as a measure of performance. Later, McGill investigators began to apply greater scientific rigor to the design Brunicardi_Ch53_p2163-p2186.indd 216722/02/19 4:39 PM 2168SPECIFIC CONSIDERATIONSPART IIABFigure 53-3. A. Hand-sewn GI anastomosis from the ACS-APDS Basic Skills Curriculum module for this

1	design Brunicardi_Ch53_p2163-p2186.indd 216722/02/19 4:39 PM 2168SPECIFIC CONSIDERATIONSPART IIABFigure 53-3. A. Hand-sewn GI anastomosis from the ACS-APDS Basic Skills Curriculum module for this task. A full instructional presen-tation is available with the curriculum along with an assessment instrument for the educator to use. B. OSATS stapled bowel anastomosis model. (Reproduced with permission from de Montbrun SL, Macrae H Simulation in surgical education, Clin Colon Rectal Surg. 2012 Sep;25(3):156-165.)and testing of basic skills curricula, with a particular focus on the ability to characterize the skills acquisition process and dif-ferences between learner groups based on careful repetitive measurement.42 Further study showed that performance, mea-sured using these bench training tasks, correlated with perfor-mance measured in vivo animal models.43 In addition to helping learners prepare for basic tissue manipulation, training on basic tasks were found to shorten the

1	training tasks, correlated with perfor-mance measured in vivo animal models.43 In addition to helping learners prepare for basic tissue manipulation, training on basic tasks were found to shorten the learning curves for more com-plex laparoscopic tasks such as suturing.44Fundamentals of Laparoscopic SurgeryFundamentals of Laparoscopic Surgery (FLS) was devised by minimally invasive surgical leaders in the Society of American Gastrointestinal and Endoscopic Surgeons as a means to assess laparoscopic surgical knowledge and skills for the purposes of certification of basic ability.45 This program represents the first broadly applied effort to demonstrate achievement of a specified level of basic surgical skill with such specific test-ing. Although most utilized by surgical residents, the resulting skills certification is applicable to a broad range of learners, including surgeons in practice as well as laparoscopic sur-geons in specialties other than general surgery. FLS consists of

1	resulting skills certification is applicable to a broad range of learners, including surgeons in practice as well as laparoscopic sur-geons in specialties other than general surgery. FLS consists of separate tests of knowledge and technical ability. The lat-ter component required integration of simulation tasks with a high degree of validation for both effectiveness in discerning skill and relevance to the respective clinical tasks. The McGill Inanimate System for Training and Evaluation of Laparoscopic Skills (MISTELS) tasks were selected as the technical skills assessment component.46 These were developed independently from the FLS program and adapted to the FLS based on the predictive value of a subset of the tasks for clinical skills mani-fested in the OR. These tasks have remained core features of the certifying examination and have been extensively studied both as training curriculum components and as predictors of clinical performance.47,48 Recent work has suggested that

1	core features of the certifying examination and have been extensively studied both as training curriculum components and as predictors of clinical performance.47,48 Recent work has suggested that spe-cific proficiency-based training can increase pass rates on FLS to 100%, supporting the general suppositions about the benefits of this model of training.49The more recently available FES serves a similar purpose to FLS for flexible endoscopy.50 This certification adds the important feature of use of a virtual reality flexible endoscopy simulator as one of the platforms for delivery of the technical skills test.Bench Models for Training Specific Procedural SkillsProcedure-specific simulation offers a unique opportunity to practice and evaluate wholesome surgical skill. This includes training and evaluating the relationship between basic and com-plex technical skills and surgical decision-making. While the majority of procedural simulation trainers focus on bedside procedures such as

1	and evaluating the relationship between basic and com-plex technical skills and surgical decision-making. While the majority of procedural simulation trainers focus on bedside procedures such as central venous catheter placement, blad-der catheterization, and intubation, a number of trainers also have been designed to simulate more complex procedures such as laparoscopic ventral hernia, laparoscopic colectomy, and robotic nephrectomy.51-53 Currently, both virtual reality and physical or hybrid models are used for procedure-specific simulations.54 While each technology has specific benefits, there is still a critical need for fabrication and design approaches that are efficient, cost effective, and produce anatomically accurate models with realistic tissue properties.55 A number of groups have explored the use of three-dimensional (3D) printing with varying degrees of success.56,57 As the printing substrate materi-als and 3D machines continue to decrease in price and increase in ease

1	have explored the use of three-dimensional (3D) printing with varying degrees of success.56,57 As the printing substrate materi-als and 3D machines continue to decrease in price and increase in ease of use, there will likely be a significant upsurge in the use of this approach to facilitate development of procedure spe-cific simulations. In addition to full immersion virtual reality, a number of groups are still exploring the benefits of computer-based learning for training procedure-specific surgical skills.58The training and assessment benefits of procedure-specific simulation are numerous. Not only does it allow an opportunity to assess technical skill in the context of a multistep procedure, forced errors and critical decisions can be combined with this technology, allowing for a more in-depth learning experience and skills assessment. By way of example, two multistep bed-side procedures were modified: bladder catheterization and central venous catheter insertion. Both simulations

1	in-depth learning experience and skills assessment. By way of example, two multistep bed-side procedures were modified: bladder catheterization and central venous catheter insertion. Both simulations had embed-ded clinical scenarios, which if executed incorrectly in real life might produce patient injury due to incorrect technique or equipment choices. During the implementation of these modi-fied, multistep procedures, it was possible to identify individual cognitive and technical errors that serve as opportunities for additional training.59,60 The ACS-APDS Procedural Skills Curriculum was developed to complement the Basic Skills Curriculum and offers detailed learning objectives for a vari-ety of surgical procedures. The goal of this curriculum was to address holistic surgical skills in a context where both technical Brunicardi_Ch53_p2163-p2186.indd 216822/02/19 4:39 PM 2169SKILLS AND SIMULATIONCHAPTER 53Table 53-2ACS-APDS procedural skills curriculum components• Laparoscopic

1	in a context where both technical Brunicardi_Ch53_p2163-p2186.indd 216822/02/19 4:39 PM 2169SKILLS AND SIMULATIONCHAPTER 53Table 53-2ACS-APDS procedural skills curriculum components• Laparoscopic ventral hernia repair• Open colon resection, lap right colon resection• Laparoscopic sigmoid resection• Open right colon resection• Laparoscopic/open bile duct exploration• Laparoscopic ventral/incisional hernia repair (porcine model)• Laparoscopic appendectomy• Laparoscopic Nissen fundoplication• Sentinal node biopsy and axillary lymph node dissection• Open inguinal/femoral hernia repair• Laparoscopic inguinal hernia repair• Laparoscopic/open splenectomy• Laparoscopic/open cholecystectomy• Gastric resection and peptic ulcer disease• Parathyroidectomy/thyroidectomyand cognitive performance could be assessed at the same time.11 The simulation scenarios defined for this curriculum are listed in Table 53-2.Similar to the literature on basic skills training and assess-ment, procedural skills

1	be assessed at the same time.11 The simulation scenarios defined for this curriculum are listed in Table 53-2.Similar to the literature on basic skills training and assess-ment, procedural skills training and assessment has been shown to benefit learners when delivered via a structured curriculum. In addition, procedural skills training in a simulated environ-ment has been shown to transfer to the real-life clinical setting. In 2005, Issenberg et al published a review of the features and uses of simulation training that lead to effective learning.61 After reviewing 109 articles that specifically used simulation as an educational intervention and not for assessment alone, Issenberg and colleagues noted that feedback, repetitive practice, and curricular intervention were among the top three features that lead to effective learning. Of note, in this same article, it was noted that simulator validity was at the bottom of the list of features that lead to effective learning. In essence,

1	features that lead to effective learning. Of note, in this same article, it was noted that simulator validity was at the bottom of the list of features that lead to effective learning. In essence, trainees need protected time to engage in simulation-based learning exercises that have a high-level focus on deliberate and repetitive practice based on personalized feedback. This model is very similar to that used to train professional athletes.62Transfer of TrainingThe determination that simulation-based education is effective requires assessment of training effect in either the clinical setting or one with a demonstrated relationship to a clinical setting. The latter might result from comparison to a validated “gold standard” training method. Establishing a relationship between lab-based training and performance in the OR requires validated measures of operative skills such as the widely used Global Assessment of Operative Laparoscopic Skills (GOALS) method described by Vassiliou.63 The

1	and performance in the OR requires validated measures of operative skills such as the widely used Global Assessment of Operative Laparoscopic Skills (GOALS) method described by Vassiliou.63 The majority of studies of laparoscopic simulation training that examine transfer of skills to the clinical OR make comparisons to control groups without highly structured and non-simulation based training. The results of these studies should eliminate any doubts that surgical learners, especially students and residents, can achieve training benefits from both proficiency-based and timeor repetitionor session number-based simulation practice. When the results of proficiency-based training are dissected out from the other models of training, consistently higher levels of clinical or 3animal OR performance is observed with such training compared to without.64-67 In one such review of skills transfer studies conducted between 2007 and 2013, Dawe reported results for general surgery and gynecology

1	is observed with such training compared to without.64-67 In one such review of skills transfer studies conducted between 2007 and 2013, Dawe reported results for general surgery and gynecology procedures using different virtual reality and non-VR laparoscopic simulation platforms (Table 53-3).48,67-77 For the 12 randomized controlled trials with surgical residents as study subjects, all but one showed significantly better clinical performance for intervention groups compared to non–simulation-trained controls.The comprehensive reviews of skills transfer data under-score the wide disparities in study design characteristics, metrics, simulator types, and the difficulties in comparing effectiveness of different simulation interventions. The “transfer effectiveness ratio” (TER) has been forwarded as a means of expressing the relative magnitude of the training effect and may provide a basis for comparison of cost or time efficiency of different training methods.78 To determine TER for a

1	as a means of expressing the relative magnitude of the training effect and may provide a basis for comparison of cost or time efficiency of different training methods.78 To determine TER for a simulation training effort, one would calculate the difference in clinical effort (time or some other measure such as number of cases) between simulator-trained and alternative-trained groups to achieve a desired level of clinical performance, divided by training time received by the simulator-trained group.Korndorffer raised concerns with studies of transfer of training when, irrespective of the improved results in lab performance with proficiency-based practice, residents studied by their group did not readily meet performance standards for more advanced skills set by expert surgeons in practice.79 Looking for opportunities to maximize the effectiveness of simulation training, Stefanidis proposed training to “automaticity” by adding a secondary visuospatial task to practice with laparoscopic

1	Looking for opportunities to maximize the effectiveness of simulation training, Stefanidis proposed training to “automaticity” by adding a secondary visuospatial task to practice with laparoscopic suturing, but only after proficiency levels were achieved with more standard laparoscopic suturing practice.80 It was suggested that the added attentional challenge likely prompted the achievement of automaticity, the end result of which was much greater performance than was seen for proficiency-based practice alone. It is not clear, however, that simply adding to task difficulty improves training results if learner capabilities are not taken into account. In a separate study, Stefanidis also reported that increased task difficulty during proficiency-based training causes measurable increases in cognitive workload and that when confronted with these challenges, novice learners did not perform better than those in proficiency-based training at a lower level of difficulty, suggesting a

1	in cognitive workload and that when confronted with these challenges, novice learners did not perform better than those in proficiency-based training at a lower level of difficulty, suggesting a possible mismatch between task challenges and capacity to learn.81 More work is required to characterize the proper balance of training difficulty and the capacity for learning in specific learners or learner groups.Virtual RealityThe use of virtual reality (VR) simulation as a way to deliver training experiences in surgery was proposed by Satava in 1993.82 Within a few years, practical applications of this technology led to the first commercially available laparoscopic simulators and studies to determine their value. The earliest and most functional VR platform was MIST-VR which permitted manipulation of abstract virtual objects using a realistic physical interface that transduced instrument motion into actions that could be observed in the virtual environment (Fig. 53-4).83 Even without a

1	of abstract virtual objects using a realistic physical interface that transduced instrument motion into actions that could be observed in the virtual environment (Fig. 53-4).83 Even without a force feedback apparatus and haptic, or “sense of touch,” cues expected with instrument–instrument or instrument–object interactions, learners could experience the psychomotor challenges of videolaparoscopy and iteratively train until Brunicardi_Ch53_p2163-p2186.indd 216922/02/19 4:39 PM 2170SPECIFIC CONSIDERATIONSPART IITable 53-3Randomized trials studying the effects of virtual reality training on surgical and OB-GYN resident operative performance vs. control trainees without virtual reality trainingAUTHOR (YEAR)PARTICIPANTS (N) AND SIMULATORPROCEDURE ASSESSEDCONCLUSIONAhlberg et al68 (2007)PGY 1–2 surgery residentsIG (7); Lap Sim VR simulatorCG (6) no simulationLaparoscopic cholecystectomyIG made fewer errors (P = 0.004), exposure (P = 0.040), clipping and tissue division (P <0.008), and

1	1–2 surgery residentsIG (7); Lap Sim VR simulatorCG (6) no simulationLaparoscopic cholecystectomyIG made fewer errors (P = 0.004), exposure (P = 0.040), clipping and tissue division (P <0.008), and dissection (P <0.031) compared with CGBanks et al69 (2007)PGY 1 GYN residentsIG (10); Limbs & Things box trainerCG (10) no simulationBilateral tubal ligationIG scored higher than CG with all three evaluation tools: task-specific checklist (P = 0.002), OSATS (P = 0.003), pass-fail grade (P = 0.003)Cosman et al70 (2007)Junior surgical traineesIG (5); Lap Sim VR simulatorCG (5) no simulationLaparoscopic cholecystectomy (clip application and cystic artery division)IG had fewer errors (P = 0.05), better bimanual coordination (P = 0.05), higher global score (P = 0.04) than CGGala et al71 (2013)GYN residentsIG (48); FLS box trainerCG (54) no simulationPomeroy bilateral tubal ligationIG had higher OSATS progression score than CG (P = 0.03)Hogle et al72 (2009)PGY 1 surgery residentsIG (6);

1	residentsIG (48); FLS box trainerCG (54) no simulationPomeroy bilateral tubal ligationIG had higher OSATS progression score than CG (P = 0.03)Hogle et al72 (2009)PGY 1 surgery residentsIG (6); Lap Sim VR simulatorCG (6) no simulationLaparoscopic cholecystectomyNo significant difference between IG and CG in GOALSDomain areas of depth perception, bimanual dexterity, efficiency, tissue handling, autonomyLarsen et al73 (2009)Firstand second-year OB-GYN registrarsIG (13); Lap Sim VR simulatorCG (11) no simulationSalpingectomyIG had higher score than CG in OSA-LS scale (P <0.001). IG completed procedure faster than CG (P <0.001)Sroka et al48 (2010)PGY 1–3 surgery residentsIG (8); FLS box trainerCG (8) no simulationLaparoscopic cholecystectomy (excision from liver bed)IG had higher total GOALS score than CG (P <0.001) and better GOALS domain scores for bimanual dexterity (P = 0.04), tissue handling (P = 0.04)Van Sickle et al74 (2008)PGY 3, 5–6 surgery residentsIG (11); MIST-VR

1	GOALS score than CG (P <0.001) and better GOALS domain scores for bimanual dexterity (P = 0.04), tissue handling (P = 0.04)Van Sickle et al74 (2008)PGY 3, 5–6 surgery residentsIG (11); MIST-VR simulatorCG no simulationNissen fundoplication (placement of intracorporeal sutures)IG completed task in less time (P <0.003), committed fewer errors (P <0.01), and had fewer needle manipulations (P <0.05) than CGZendejas et al75 (2011)Surgery residentsIG (26); Guildford MATTU TEP hernia task trainerCG (24) no simulationTEP inguinal hernia repairIG faster on first procedure (P <0.001) and had higher participation rates (P <0.001). For subsequent repairs IG remained faster than CG. GOALS score higher for IG (P = 0.001). Complications and overnight stay less likely for first TEP procedure in IG (P <0.05).Palter et al76 (2012)PGY 2–4 surgery residentsIG (9); curriculum including simulation training on LapSim VR simulatorCG (9) no simulationRight hemicolectomyIG had higher OSATS score (P = 0.030)

1	et al76 (2012)PGY 2–4 surgery residentsIG (9); curriculum including simulation training on LapSim VR simulatorCG (9) no simulationRight hemicolectomyIG had higher OSATS score (P = 0.030) than CG.IG able to perform more operative steps than CG (P = 0.021)Palter et al77 (2013)PGY 1–2 surgery residentsIG (9); curriculum including simulation training on LapSim VR simulator and on FLS box trainerCG (9) no simulationCholecystectomyIG had higher OSATS scores for first four procedures (P = 0.004, P = 0.036, P = 0.021, P = 0.023)PGY = postgraduate year; IG = intervention group; CG = control group; VR = virtual reality; OSATS = Objective Structured Assessment of Technical Skills; GOALS = Global Operative Assessment of Laparoscopic Skills; OSA-LS = Objective Structured Assessment of Laparoscopic Salpingectomy; TEP = totally extraperitoneal.Simulators: LapSim VR simulator (Surgical Science, Gothenburg, Sweden); laparoscopic stimulator and Minimal Access Therapy Unit (MATTU) (Limbs and Things,

1	TEP = totally extraperitoneal.Simulators: LapSim VR simulator (Surgical Science, Gothenburg, Sweden); laparoscopic stimulator and Minimal Access Therapy Unit (MATTU) (Limbs and Things, Bristol, UK); Fundamentals of Laparoscopic Surgery (FLS) Training Box simulator (SAGES, Los Angeles, California, USA); Minimally Invasive Surgical Trainer—Virtual Reality (MIST-VR; Mentice, Gothenburg, Sweden).Modified with permission from Dawe SR, Pena GN, Windsor JA, et al. Systematic review of skills transfer after surgical simulation-based training, Br J Surg. 2014 Aug;101(9):1063-1076.Brunicardi_Ch53_p2163-p2186.indd 217022/02/19 4:39 PM 2171SKILLS AND SIMULATIONCHAPTER 53ABFigure 53-4. The surgical interface for MIST VR (A), consisting of rightand left-handed laparoscopic instruments on a gimbaled mount that transduces instrument motions into digital spatial data, which determine the location and actions of instruments in the virtual space (shown in B) in the course of manipulating

1	a gimbaled mount that transduces instrument motions into digital spatial data, which determine the location and actions of instruments in the virtual space (shown in B) in the course of manipulating two-handed virtual objects. Although basic renderings of geometric shapes, these virtual objects could be grasped, positioned, and treated with electrosurgery instruments, and they were shown to be an effective means to transfer skills to clinical surgery despite a relatively low level of fidelity to human tissues and absence of haptic feedback in the instrument interface.Table 53-4Studies comparing laparoscopic operative performance after training on virtual reality simulators vs. videoscopic box trainersAUTHOR (YEAR)STUDY ARMS (N)SUBJECTSPROFICIENCY-BASED TRAINING?CONCLUSIONSHamilton et al92 (2002)VR (24)Box trainer (25)Firstand second-year surgical residentsNoImproved rater-blinded global assessments of laparoscopic cholecystectomy for VR but not box-trainer trained subjectsYoungblood

1	(2002)VR (24)Box trainer (25)Firstand second-year surgical residentsNoImproved rater-blinded global assessments of laparoscopic cholecystectomy for VR but not box-trainer trained subjectsYoungblood et al93 (2005)VR (16)Box trainer (17)No training (13)Medical studentsNoVR-trained students performed better than box trainer-trained on selected tasks in live porcine model. Both trained groups outperformed nontrained.Diesen et al94 (2011)VR (10)Box trainer (8)InternsMedical studentsNoNo difference in interval blinded video assessments of animal OR task performanceVR = virtual reality; OR = operating room.performance goals for precision, efficiency, and error avoidance were achieved. Performance measurement was automated and included time, instrument motion, and electrosurgery use metrics, as well as a tally of the occurrence of predefined errors. All metrics were free of human observer bias. Early studies of VR training using both proficiency-based and non–proficiency-based training

1	as well as a tally of the occurrence of predefined errors. All metrics were free of human observer bias. Early studies of VR training using both proficiency-based and non–proficiency-based training methods showed it to be an effective means of improving laparoscopic skill both in the lab and in the operating room compared to non-VR trained controls.68,84-87 Since the first studies of this type were performed almost 20 years ago, several comprehensive reviews of the growing body of literature on VR have continued to support the conclusion that skills acquired in VR transfer to the clinical setting (Table 53-3), not only for laparoscopy, but also for flexible endoscopy, sinuscopic surgery, and endovascular interventions.66,67,88-91 Largely due to small study sizes and some 4design limitations, the quality of evidence of these studies is consistently described as below level I.Based on available evidence, expanded use of VR for skills training could be justified, but few comparisons of

1	the quality of evidence of these studies is consistently described as below level I.Based on available evidence, expanded use of VR for skills training could be justified, but few comparisons of training effectiveness have been made between physical laparoscopic video trainer (“box” trainer) and laparoscopic VR simulator-based training. Crossover studies designed to determine if training in one environment improves performance in the other have not been especially helpful in defining the value of either. Only a few studies have compared the effects of the two training methods on OR performance (Table 53-4). Although some advantage has been suggested, the prevailing view is that both can be used for highly effective laparoscopic practice. Until better comparisons are made, use of proficiency-based training in the context of a larger curriculum appears to be the best way to achieve good training results irrespective of the training platform used.5Brunicardi_Ch53_p2163-p2186.indd

1	training in the context of a larger curriculum appears to be the best way to achieve good training results irrespective of the training platform used.5Brunicardi_Ch53_p2163-p2186.indd 217122/02/19 4:39 PM 2172SPECIFIC CONSIDERATIONSPART IIVR simulator systems are significant capital investments for simulation centers that have competing procurement priori-ties. In a prospective randomized trial, Orzech analyzed the cost impact of each type of training across Canadian residency pro-grams and found that the transfer of training effect was greater for VR as compared to the box trainer group. However, box trainer use was found to be more cost effective except in larger residencies.95 Although the reasons for the latter finding require further analysis, the high acquisition costs of VR systems can be offset by an economy of scale benefit not realized with physical models where staffing for purposes of task setup and assess-ment and consumable items related to many tasks represent

1	systems can be offset by an economy of scale benefit not realized with physical models where staffing for purposes of task setup and assess-ment and consumable items related to many tasks represent additive costs. Flexible endoscopy VR simulator device sharing was shown to work effectively for a skills acquisition program across a network of Texas institutions where procurement of numerous such simulators might not be practical.96The role of haptics has been debated for laparoscopic VR simulators since the inclusion of sophisticated force feedback hardware adds substantially to the cost of surgical VR as well as to system computing demands. In simulated endoscopic and laparoscopic procedures, effective haptic cues are important contributors to the fidelity of the experience. For basic skills acquisition these features have not been shown to offer significant advantages over nonhaptic VR systems, although for more advanced skills haptic cues may permit greater precision of instrument

1	acquisition these features have not been shown to offer significant advantages over nonhaptic VR systems, although for more advanced skills haptic cues may permit greater precision of instrument use.97,98 However, surgical VR has advanced to the point where inventories of procedure types offered on specific commercially available systems are quite extensive and these will inevitably increase and encompass entire procedures. The realism of the user experience, including the haptic experience, may prove essential to effective learning of advanced surgical skills in a new generation of VR devices. The current generation of laparoscopic simulators are highly capable devices with a high degree of graphical realism, full haptic features, and numerous tasks available for training basic and procedural skills for general surgical, urologic, and gynecologic procedures (Fig. 53-5).Despite encouraging progress made in VR simulation, two separate publications in 2007 and 2015 identified ongoing

1	skills for general surgical, urologic, and gynecologic procedures (Fig. 53-5).Despite encouraging progress made in VR simulation, two separate publications in 2007 and 2015 identified ongoing com-putational challenges in development of very high-fidelity simu-lations for surgery.99,100 These include the unique VR problems of modeling human tissues and the added demands of rendering the appropriate deformations when tissues are manipulated. One of the practical examples of this is that of guidewire behavior during VR as compared to real-life endovascular procedures, where variable elasticity characteristics of blood vessel walls were observed to result in real tissue or wire deformations that the VR software could not depict accurately.101 Also identified were disparities between levels of resourcing for surgical VR versus mainstream computer gaming, raising important ques-tions on how the full power of this technology can be realized to more fully simulate complex operations with a

1	of resourcing for surgical VR versus mainstream computer gaming, raising important ques-tions on how the full power of this technology can be realized to more fully simulate complex operations with a high degree of fidelity and realism.Virtual Reality for Flexible EndoscopyThe use of virtual reality simulation to train for flexible endos-copy procedures is now well established and supported by consistent evidence of both skills transfer. Sedlack compared colonoscopy skills between small groups of inexperienced VR-trained and nontrained gastroenterology fellows and found that VR training resulted in farther progression into the colon, better inspection skills, and a higher percentage of completed studies (those that reached the cecum).102 This performance advantage extended out to 30 posttraining procedures. In the years since that report, four randomized controlled trials of VR training with blinding to training status during posttraining clinical colonos-copy have been

1	to 30 posttraining procedures. In the years since that report, four randomized controlled trials of VR training with blinding to training status during posttraining clinical colonos-copy have been conducted.103-106 Despite methodological issues with each of these trials (no proficiency-based training, unsuper-vised VR practice on one, vaguely defined training characteris-tics of control groups), all but one showed training benefits for Figure 53-5. Three different current generation laparoscopic virtual reality simulators. A. LapVR VR Simulator, CAE Healthcare, Sarasota, FL. B. Simbionix LAP Mentor VR simulator, Littleton, CO. C. LapSim VR simulator, Surgical Science AB, Gothenburg, Sweden.ABCBrunicardi_Ch53_p2163-p2186.indd 217222/02/19 4:39 PM 2173SKILLS AND SIMULATIONCHAPTER 53ABFigure 53-6. Representation of patient-specific aortic vascular anatomy during simulation of endovascular aortic replacement (EVAR) for abdominal aortic aneurysm, with distal graft limbs in the

1	53ABFigure 53-6. Representation of patient-specific aortic vascular anatomy during simulation of endovascular aortic replacement (EVAR) for abdominal aortic aneurysm, with distal graft limbs in the ballerina (A. crossed) and standard (B. uncrossed) configurations. This example of patient-specific rehearsal requires preparation the virtual aorta from a DICOM (Digital Imaging and Communications in Medicine) format file of patient computed tomography (CT) or CT angiogram imaging data. Image is rendered on ANGIO Mentor VR simulator (3D Systems, Littleton, CO). (Reproduced with permission from Pakeliani D, Van Herzeele I, Lachat ML, et al: EVAR 2020: Training Future Aortic Spe-cialists. Emerging needs and the role of simulation. Endovascular Today 2017 March;16(3):95-100.)previously inexperienced VR-trained residents versus controls, especially in the earlier posttraining clinical cases based on either subjective determination of competency or measurement of procedure length. The study

1	VR-trained residents versus controls, especially in the earlier posttraining clinical cases based on either subjective determination of competency or measurement of procedure length. The study that did not show improved clini-cal performance compared to controls was a noninferiority com-parison to control subjects with undefined patient-based training where the two groups performed comparably well. Randomized trials of VR training for upper endoscopy have shown signifi-cant performance advantages in both procedure length and sub-jective assessment of competency compared to both nontrained and patient-based training control groups.107-109Based on the highly standardized test environment that can be experienced in VR and concurrent validity to clinical endos-copy, the SAGES FES program utilizes VR flexible endoscopy simulation for certification of skills. Systematic review of pub-lished literature on VR flexible endoscopy skills cite the need for better quality evidence in support of

1	VR flexible endoscopy simulation for certification of skills. Systematic review of pub-lished literature on VR flexible endoscopy skills cite the need for better quality evidence in support of best training practices and improved patient outcomes.110 Further study is required, especially in the area of proficiency-based VR flexible endos-copy training, which has been generally underutilized.Virtual Reality for Endovascular InterventionsVirtual reality simulation has been shown to be an effective alternative to training with animal or cadaver models for catheter-based vascular interventions. Current simulations allow development of basic guidewire and catheter handling skills, as well as practice in use of fluoroscopy, angioplasty, and stenting techniques. Procedural training for coronary, carotid, renal, neuro, peripheral vascular, and other interven-tions are feasible with measurement of learner performance in numerous areas such as procedure and fluoroscopy time, con-trast use, and

1	carotid, renal, neuro, peripheral vascular, and other interven-tions are feasible with measurement of learner performance in numerous areas such as procedure and fluoroscopy time, con-trast use, and intervention effectiveness. Simulators display fluoroscopy images and allow for the selection and insertion of virtual catheter, balloon, and stent types, which are pertinent to the procedure being performed (Fig. 53-6). In small randomized trials, VR training has been shown to increase residents’ periph-eral angioplasty skills with transfer of lab-acquired skills to the clinical OR.111,112 After having previously shown that VR simu-lator assessment can effectively discriminate the level of clini-cal experience with carotid artery stenting among experienced interventionalists, Van Herzeele demonstrated that experienced interventionalists could also significantly increase carotid artery stenting skills following a 2-day intensive course of didactic and VR training for this procedure.113,114

1	that experienced interventionalists could also significantly increase carotid artery stenting skills following a 2-day intensive course of didactic and VR training for this procedure.113,114 Following training, decreased procedure and fluoroscopy time and decreased time for placement and retrieval of the embolic protection device were observed. Although differing clinical outcomes were not seen in this small study, it was noted that internal carotid artery spasm frequency decreased after training.The incorporation of actual patient vascular anatomic information taken from computerized tomography data into a vascular interventional simulation as an aid to procedural plan-ning or technical procedure performance has been referred to as “mission rehearsal” or more commonly now, procedure specific rehearsal or procedure specific simulation. It has been described most extensively for carotid artery stenting procedures and high-lights how VR simulation can be directed toward the immedi-ate

1	rehearsal or procedure specific simulation. It has been described most extensively for carotid artery stenting procedures and high-lights how VR simulation can be directed toward the immedi-ate problems of clinical practice. Cates’ brief report of a single procedure was followed by small studies further demonstrat-ing the feasibility and general impressions of the value of this method.115-117 In a randomized comparison of trainees who per-formed either part-task rehearsal or rehearsal of the entire proce-dure, Willaert reported that a similar performance benefit could be achieved, suggesting a potentially more time-efficient way to train, although the embolic protection device was in place slightly longer in the part-task-trained group.118Brunicardi_Ch53_p2163-p2186.indd 217322/02/19 4:39 PM 2174SPECIFIC CONSIDERATIONSPART IIIn a recent multinational European study, Desender and colleagues randomized a series of 100 patients scheduled to undergo elective endovascular aneurysm

1	4:39 PM 2174SPECIFIC CONSIDERATIONSPART IIIn a recent multinational European study, Desender and colleagues randomized a series of 100 patients scheduled to undergo elective endovascular aneurysm repair (EVAR) for infrarenal aortic aneurysm to either have their procedure rehearsed with VR simulation preoperatively or to have the pro-cedure performed without rehearsal.119 There were 26% fewer minor errors, 76% fewer major errors, and a 27% fewer errors causing procedural delay in the VR rehearsal group. In addition, this group had significantly fewer angiograms performed to visualize proximal and distal graft landing zones. In a follow-up of this study, Desender reported that patient-specific rehearsal before EVAR resulted in alteration of the operative plan for proximal landing zone (54%), distal landing zone (76%), stent graft main body size (16%), contralateral limb size (34%) or orientation (16%), and iliac extension size (28%). Ninety-two percent of these changes were

1	zone (54%), distal landing zone (76%), stent graft main body size (16%), contralateral limb size (34%) or orientation (16%), and iliac extension size (28%). Ninety-two percent of these changes were implemented during the actual EVAR case.119Patient-Specific VR Surgery SimulationIn addition to patient specific rehearsal for endovascular inter-ventions, VR simulations for OR surgical procedures have begun to use patient imaging data to rehearse procedures preop-eratively. While many surgeons consciously and subconsciously mentally rehearse procedures before entering the operating room, this process does not allow for fully explicit informa-tion sharing between team members. Moreover, even when the surgeon verbalizes a plan for other members of the OR team after the mental rehearsal, it is not uncommon to unintentionally exclude important details that team members may value. The use of anatomically accurate VR simulations, based on patient-specific anatomy, may allow for team-based

1	is not uncommon to unintentionally exclude important details that team members may value. The use of anatomically accurate VR simulations, based on patient-specific anatomy, may allow for team-based rehearsals and reduce the risk of human error. In addition, VR-based rehearsals may also facilitate doctor-patient communication.120Patient-specific VR simulations have recently emerged for a variety of complex operations including pancreatectomies, hepatectomies, renal surgery, and hand surgery.120-123 For one of the renal surgery simulations, patient-specific computed tomog-raphy (CT) data was captured and used to create 3D imaging for incorporation into the simulation.122 The anatomical accuracy of various structures such as arteries, veins, ureters, and even tumors was reported to be high. Another group compared the appearance of individual vascular structures while performing several patient-specific virtual hepatectomies simultaneously with real-life hepatectomies and also noted a

1	Another group compared the appearance of individual vascular structures while performing several patient-specific virtual hepatectomies simultaneously with real-life hepatectomies and also noted a high degree of accuracy. In addition, similar to the way CT angiograms are used in the OR for surgical planning, this group was able to increase and decrease the transparency level of the patient spe-cific VR simulation and use it both as a real-time operative guide (minimal transparency mode) as well as an operative planning guide (high transparency–vessel only view). This enabled one system to be used seamlessly throughout the actual operation.123According to reports, the time needed to create patient-specific VR simulations is relatively short. On average, it took approximate 2.5 hours each for both the hepatectomy and pan-createctomy simulations.120 In addition, compared to the use of 3D printed simulations, patient-specific VR simulations are readily reusable and do not consume as many

1	both the hepatectomy and pan-createctomy simulations.120 In addition, compared to the use of 3D printed simulations, patient-specific VR simulations are readily reusable and do not consume as many resources. While these recent advances are quite promising, patient-specific VR simulators are a new technology, and thus additional studies are required to more fully understand the pros and cons of introduc-ing this technology into the patient care arena. As there contin-ues to be major improvements in patient-specific 3D rendering, including organ and tissue deformation in reaction to surgical manipulation, this increases the possibility of even more sophis-ticated and accurate VR simulations that can be used for pre-operative planning and rehearsing for complicated procedures.Robotic Surgery SimulatorsAfter the da Vinci surgical system was first introduced in the United States in 1999, a number of simulation systems for teaching robotic surgery emerged. While there have been sev-eral

1	SimulatorsAfter the da Vinci surgical system was first introduced in the United States in 1999, a number of simulation systems for teaching robotic surgery emerged. While there have been sev-eral versions of the da Vinci system deployed worldwide, the basic system components usually include dual hand controls, foot pedals, and a controllable 3D camera.124 Consistency in these system components allow for similar consistency in simu-lation design and delivery. Currently, there are four different simulators geared towards imparting some level of competency in using the da Vinci System: the SEP-Robot (SurgicalSim Educational Platform Robot; SimSurgery, Oslo, Norway); RoSS (Robotic Surgery Simulator-Simulated Surgical Systems, San Jose, CA); dV-Trainer (Mimic Simulation, Seattle, WA), and the da Vinci Skills Simulator (Intuitive Surgical, Santa Clara, CA).124,125 The SEP-Robot is a desktop-like system for training robotic skills in a VR graphical interface. The da Vinci Skills Simulator,

1	da Vinci Skills Simulator (Intuitive Surgical, Santa Clara, CA).124,125 The SEP-Robot is a desktop-like system for training robotic skills in a VR graphical interface. The da Vinci Skills Simulator, also called the “backpack,” is a hardware system that loads VR simulations into the actual da Vinci console.126 The RoSS and dV-Trainer systems are stand-alone devices with surgical controls resembling those of the da Vinci system.124 These simulators largely focus on hand-eye coordination, tissue manipulation, suturing and knot tying.127 The major benefit of VR simulators for training da Vinci robotic skills is that they produce performance metrics including time, error measures, and motion analysis.124 These simulators are increasingly being used for training novice surgeons in robotic skills for a variety of surgical specialties.When assessing simulator validity, researchers have noted that the use of robotic surgery simulators does translate to the clinical environment and the learning

1	for a variety of surgical specialties.When assessing simulator validity, researchers have noted that the use of robotic surgery simulators does translate to the clinical environment and the learning curve for initial console training for surgeons is significantly decreased.124,126 Unfortunately, the available robotic surgery simulators still come with a high sticker price and varying agreement on the level of fidelity that is currently present in these technologies.124 These deficiencies are likely due to the early stage of the robotic surgery approach, and it is likely that cheaper and more sophis-ticated systems will be available in the near future.128Fundamentals of Robotic SurgeryThe Fundamentals of Robotic Surgery (FRS) is a robotic surgi-cal skills training and assessment program designed to provide a proficiency-based curriculum of basic technical skills to prepare surgeons for performing robotic surgery procedures across a wide range of specialties. The FRS program was

1	designed to provide a proficiency-based curriculum of basic technical skills to prepare surgeons for performing robotic surgery procedures across a wide range of specialties. The FRS program was developed over a 2-year period by subject matter experts from multiple surgi-cal societies, surgical educational societies, surgical boards, and other governing organizations through a series of four consensus conferences, which included over 80 international robotic sur-gery experts, behavioral psychologists, medical educators, stat-isticians, and psychometricians.129 The multidisciplinary team of experts agreed upon the critical skills and tasks to be included in a comprehensive basic curriculum, and a task deconstruction was performed to identify the tasks, subtasks, and errors that needed to be measured. A modified Delphi methodology was then used to create a matrix of specific robotic surgery tasks, common errors, desired outcomes, and quantitative metrics to

1	and errors that needed to be measured. A modified Delphi methodology was then used to create a matrix of specific robotic surgery tasks, common errors, desired outcomes, and quantitative metrics to 6Brunicardi_Ch53_p2163-p2186.indd 217422/02/19 4:39 PM 2175SKILLS AND SIMULATIONCHAPTER 53Table 53-5The four online modules for the fundamentals of robotic surgery curriculumModule 1Introduction to Surgical Robotic Systems, includes an overview of minimally invasive surgery, advantages of robotic assisted surgery, components of robotic systems, and system functionalityModule 2Didactic Instructions for Robotic Surgery Systems, provides an overview of robotic surgery systems, as well as detailed information regarding the pre-, intra-, and postoperative phasesModule 3Psychomotor Skills Curriculum, consists of background and general principles of the psychomotor tasks, an introduction to the physical model on which the tasks are performed, and general scoring guidelines for all the tasks,

1	consists of background and general principles of the psychomotor tasks, an introduction to the physical model on which the tasks are performed, and general scoring guidelines for all the tasks, followed by detailed descriptions of each task, including the targeted primary and secondary skills and metricsModule 4Team Training and Communication Skills, includes background on the degradation of situation awareness and the TeamSTEPPS process followed by detailed content covering communication, situational awareness, mutual support, leadership, the preoperative phase, robotic docking, intraoperative phase, postoperative phase, and a review of five scenariossupport those outcomes. Finally, a second round classic Delphi anonymous rating was used to ensure concurrence, prioritize the task rankings, and eliminate low-scoring tasks.All trainees must first complete an online curriculum consisting of four modules. Each of the four online modules is followed by a short quiz, requiring a minimum of

1	and eliminate low-scoring tasks.All trainees must first complete an online curriculum consisting of four modules. Each of the four online modules is followed by a short quiz, requiring a minimum of 70% cor-rect to proceed in the training (Table 53-5). The curriculum also includes a cumulative, cognitive test following completion of all modules.ABFigure 53-7. A. The simulated abdominal cavity for training in the Fundamentals of Robotic Surgery. B. The 18-cm removable dome model featuring well-defined areas for executing each of the seven psychomotor exercises outlined in Table 53-6. (Used with permission from the Institute for Surgical Excellence.)Following online course completion, trainees must com-plete seven psychomotor exercises using a surgical robot, a simulated abdomen, and an 18-cm, removable dome model (Fig. 53-7).The psychomotor exercises consist of the following tasks: docking/instrument insertion, ring tower transfer, knot tying, railroad track, 3rd arm cutting, puzzle

1	removable dome model (Fig. 53-7).The psychomotor exercises consist of the following tasks: docking/instrument insertion, ring tower transfer, knot tying, railroad track, 3rd arm cutting, puzzle piece dissection, and ves-sel energy dissection (Table 53-6).NONTECHNICAL SKILLSFor surgeons, the term “nontechnical skills” refers to the cog-nitive knowledge and teamwork-related abilities that must be integrated with psychomotor skills and abilities. There are no sharp demarcations between these areas of skill, but different simulation methods are suitable for training each, and all should be addressed.High-Fidelity Patient Simulation and Team SkillsRecognition of the role of human factors in the occurrence of preventable errors has spawned various efforts to train behav-iors conducive to high-performing teams. Crew resource man-agement (CRM) training utilizing simulation has been credited with increased safety in aviation.130,131 Lessons learned from CRM have been adapted to medical

1	high-performing teams. Crew resource man-agement (CRM) training utilizing simulation has been credited with increased safety in aviation.130,131 Lessons learned from CRM have been adapted to medical training with simulators focusing on medical team performance in complex clinical situ-ations. This development came about in the 1990s driven pri-marily by anesthesiologists responsible for establishing the first high-fidelity simulation environments.132,133 These were devel-oped for simulation of crisis-level events where management could be practiced under realistic but safe conditions.134-136 Such training could be scaled to involve a single learner to focus on clinical management up to an entire care team able to practice team processes. Development of a program for such training requires an understanding of the principles underlying team effectiveness and the specific characteristics of an expert team. In a 2012 discussion paper on team-based health care emerging from the Best

1	an understanding of the principles underlying team effectiveness and the specific characteristics of an expert team. In a 2012 discussion paper on team-based health care emerging from the Best Practices Innovation Collaborative of the Institute of Medicine (IOM) Roundtable on Value & Science-Driven Health Care, such principles were clearly laid out following a careful analysis of effective medical teams across the country (Table 53-7).137 This document provides an excellent review of characteristics and values that surgical teams can aspire to. The necessity to train these skills has been widely accepted as Brunicardi_Ch53_p2163-p2186.indd 217522/02/19 4:39 PM 2176SPECIFIC CONSIDERATIONSPART IITable 53-6The seven psychomotor dome tasks for the fundamentals of robotic surgery curriculumTask 1: Docking/Instrument InsertionThe first psychomotor exercise provides training and assessment of proper docking and instrument insertion using the simulated abdomen.Task 2: Ring Tower

1	curriculumTask 1: Docking/Instrument InsertionThe first psychomotor exercise provides training and assessment of proper docking and instrument insertion using the simulated abdomen.Task 2: Ring Tower TransferThe trainee removes a ring from the right middle tower and places it on the lower left tower. Primary skills assessed include hand, eye, and instrument coordination, camera navigation, and use of the camera pedal.Task 3: Knot TyingThe trainee ties a surgeon’s knot to approximate two eyelets such that they touch each other. Primary skills assessed include appropriate handling of suture material and tying secure knots.Task 4: Railroad TrackThe trainee must perform horizontal mattress suturing through a series of target points to approximate the tissue. Primary skills assessed include holding and manipulation of the needle, following the curve of the needle, utilizing the full range of motion of the endowrist, and using graspers.Task 5: 3rd Arm CuttingThe trainee must switch control

1	and manipulation of the needle, following the curve of the needle, utilizing the full range of motion of the endowrist, and using graspers.Task 5: 3rd Arm CuttingThe trainee must switch control between different instruments to use the monopolar scissors to cut a simulated vein. Primary skills assessed include switching between and controlling multiple robot arms and cutting.Task 6: Puzzle Piece DissectionIn this task, the trainee must cut and remove a puzzle shape without incising the underlying tissue or cutting outside of the lines. Primary skills assessed include dissection, cutting, atraumatic tissue handling, sharp dissection, and blunt dissection.Task 7: Vessel Energy DissectionThe trainee must dissect through a fat layer to expose a vessel then coagulate the vessel at two points and finally cut the vessel between the two coagulated points. Primary skills assessed include accurate activation and use of energy sources, dissection of vessels and tissues, cutting and coagulation of

1	finally cut the vessel between the two coagulated points. Primary skills assessed include accurate activation and use of energy sources, dissection of vessels and tissues, cutting and coagulation of vessels, and multiple arm control.Table 53-7Principles of team-based health care• Shared goals: The team—including the patient and, where appropriate, family members or other support persons—works to establish shared goals that reflect patient and family priorities, and can be clearly articulated, understood, and supported by all team members.• Clear roles: There are clear expectations for each team member’s functions, responsibilities, and accountabilities, which optimize the team’s efficiency and often make it possible for the team to take advantage of division of labor, thereby accomplishing more than the sum of its parts.• Mutual trust: Team members earn each other’s trust, creating strong norms of reciprocity and greater opportunities for shared achievement.• Effective communication:

1	more than the sum of its parts.• Mutual trust: Team members earn each other’s trust, creating strong norms of reciprocity and greater opportunities for shared achievement.• Effective communication: The team prioritizes and continuously refines its communication skills. It has consistent channels for candid and complete communication, which are accessed and used by all team members across all settings.• Measurable processes and outcomes: The team agrees on and implements reliable and timely feedback on successes and failures in both the functioning of the team and achievement of the team’s goals. These are used to track and improve performance immediately and over time.Reproduced with permission from Mitchell P, Wynia M, Golden R, et al. Core Principles & Values of Effective Team-Based Health Care. Discussion Paper. Institute of Medicine, October 2012.a means of increasing the safety of healthcare. There are meth-ods to accomplish medical team training that do not involve simulation,

1	Care. Discussion Paper. Institute of Medicine, October 2012.a means of increasing the safety of healthcare. There are meth-ods to accomplish medical team training that do not involve simulation, but high-fidelity patient simulation has proven to be highly effective in increasing health care team competency, and systematic reviews have given evidence based endorsement of this approach.138Simulation training for communication and other teamwork-pertinent nontechnical skills requires learners to be embed-ded in realistic scenarios pertinent to a healthcare team’s 7actual clinical responsibilities where activities and interactions prompted by the simulated clinical circumstances can be prac-ticed and observed. The computer-driven high-fidelity manikin simulator serving as the “patient” at the center of these activities can be monitored and controlled to demonstrate realistic physi-ology consistent with the clinical condition needed for the scenario.139 Software-driven, physiologic

1	at the center of these activities can be monitored and controlled to demonstrate realistic physi-ology consistent with the clinical condition needed for the scenario.139 Software-driven, physiologic changes from the baseline state can occur in response to either manual commands or programmed adjustments to accurately depict, for example, new, ongoing and unexpected clinical developments with blood loss, sepsis, or myocardial ischemia. Basic interventions such as airway management, drug administration and wound care for moulaged body parts can also be performed. Hybrid simulations using both manikin and open abdominal or laparoscopic surgi-cal simulators have also been used to extend scenarios to an operating room setting, with all members of the surgical team engaged in their role-specific tasks.136,140,141 These events can be conducted in a dedicated simulation suite or in an actual clinical area where it would be termed in situ simulation.The postsimulation debriefing is an

1	tasks.136,140,141 These events can be conducted in a dedicated simulation suite or in an actual clinical area where it would be termed in situ simulation.The postsimulation debriefing is an essential compo-nent of simulation-based team training. This is where learning points are reinforced and progress towards desired knowledge, attitudes, and behavior can be developed.142,143 Participants are prompted to reflect on the events of the simulation and to openly discuss positive and negative aspects of the experience. The debriefing environment and discussion ought to be open, nonjudgmental, and directed at improvement in individual and overall team performance. A facilitator with strong content knowledge should ensure that the discussion includes identifica-tion of gaps between the observed and desired performance. An Brunicardi_Ch53_p2163-p2186.indd 217622/02/19 4:39 PM 2177SKILLS AND SIMULATIONCHAPTER 53effective facilitator is cognizant of the need to keep the debrief-ing

1	and desired performance. An Brunicardi_Ch53_p2163-p2186.indd 217622/02/19 4:39 PM 2177SKILLS AND SIMULATIONCHAPTER 53effective facilitator is cognizant of the need to keep the debrief-ing learner-centric and to keep discussion focused on oppor-tunities for improvement. Although debriefing is uniformly viewed as essential to health care team simulation effectiveness and structured debriefing models are frequently cited as highly effective, there is no consensus on which specific methods, including video review debriefings, represent best practices.144 Quality of implementation is consistently cited as the most important contributing factor to effectiveness of debriefing.The complexity of team-managed clinical events makes measurement of team performance challenging, but several assessment tools have been developed and used successfully in simulation settings. NOTECHS (Non-Technical Skills) and the NOTSS (Non-Technical Skills for Surgeons) instruments have been used to study

1	assessment tools have been developed and used successfully in simulation settings. NOTECHS (Non-Technical Skills) and the NOTSS (Non-Technical Skills for Surgeons) instruments have been used to study nontechnical abilities of individuals in surgi-cal teams.141,145-147 The principal focus of these rating scales is on the quality and effectiveness of situational awareness and com-munication. Instruments such as the Mayo High Performance Teamwork scale or the surgery-specific OTAS (Observational Team Assessment Scale) place focus on the team dynamics that extend beyond the single team member.148,149 These have been used to detect changes in team performance with training. Rosen suggested a framework for a best practices approach to team performance assessment in simulation that details specific appli-cations of measurement techniques in the simulation training environment.150Simulation training directed at nontechnical skills has been shown to improve clinical performance and improve

1	appli-cations of measurement techniques in the simulation training environment.150Simulation training directed at nontechnical skills has been shown to improve clinical performance and improve knowledge and attitudes about team functioning as measured in simulated surgical settings such as the trauma bay and the OR for interdis-ciplinary surgical teams and for surgical trainees.136,151-154 Two systematic reviews of simulation training to increase team skills in the operating room consistently bear out this result, but both also cited the small number of studies where simulation training effects were investigated in the clinical OR setting and lack of evidence of improved clinical outcomes with such training.155,156ERROR PREVENTIONError avoidance and prevention are the overarching goals of sur-gical care and the time-honored focus of surgical training.157 Fac-ulty who are responsible for training the next generation of highly qualified surgeons must facilitate the delicate balance

1	of sur-gical care and the time-honored focus of surgical training.157 Fac-ulty who are responsible for training the next generation of highly qualified surgeons must facilitate the delicate balance between resident autonomy and patient safety. From a training perspective, gradual increases in patient responsibility, autonomous decision-making, and operative action provide a critical opportunity for independent hands-on performance, critical thinking, and action-based skill assessment. However, resident autonomy must be bal-anced with the goal of delivering high-quality, error-free patient care. A key component to achieving residency training goals includes exposure to techniques and strategies for avoiding errors. The relationship between errors and patient safety is well estab-lished in the literature and is also the cornerstone of the case review process for surgical morbidity and mortality conferences.158 Simulation technology allows trainees the opportunity to execute a variety of

1	literature and is also the cornerstone of the case review process for surgical morbidity and mortality conferences.158 Simulation technology allows trainees the opportunity to execute a variety of tasks and procedures while also experiencing the cognitive demands of surgery, including error correction and surgical planning decisions. Fig. 53-8 presents a framework for categorizing surgical errors that may take place on the cognitive-motor continuum.159Simulation-based curricular approaches to exposing and training surgical errors include (a) the use of error-enabled 8CognitiveError preventionWhat will prevent an error?Knows the anatomy: right technique; natural hx of disease; how to avoid an errorSelect the right operation; the right stitch; the right instrumentWhat should you be concerned about?Cognitive-motorError recognitionEstimate errorrisk for: technicalapproach andpatient selectionWhich of thesemay result in anerror?Error rescueWhat areyour optionsfor

1	should you be concerned about?Cognitive-motorError recognitionEstimate errorrisk for: technicalapproach andpatient selectionWhich of thesemay result in anerror?Error rescueWhat areyour optionsfor errorrescue?Describewhat youwould do ifError preventionPre-error makesadjustments totechnical approachbased on errorpreventionError recognitionNear miss recognitionfollowed by correctiveactionError rescuePost-error knows andexecutes options forerror correctionIdentify propertechnique;potential error:error riskKnows -surgicaloptionsExample: Plansahead of time toadjust tensionduring knot tyingbased on tissuetypeExample: Recognizesthat the first knot wasa little tight then makesan adjustment to getthe second knot justrightExample: First knotpulled throughRescue: Freshenstissue edge, placesa new stitchError loopFigure 53-8. A framework for understanding error prevention, error recognition, and error rescue/correction during both the cognitive phase as well as the cognitive-motor phase where

1	stitchError loopFigure 53-8. A framework for understanding error prevention, error recognition, and error rescue/correction during both the cognitive phase as well as the cognitive-motor phase where there is fluid integration and updating of motor and cognitive decisions. This fluid integration allows for an error loop, as each decision is based on the results of each motor action in a dynamic fashion where an error can take place with each decision or action. (Reproduced with permission from Pugh CM, Santacaterina S, DaRosa DA, et al: Intra-operative decision making: more than meets the eye, J Biomed Inform. 2011 Jun;44(3):486-496.)Brunicardi_Ch53_p2163-p2186.indd 217722/02/19 4:39 PM 2178SPECIFIC CONSIDERATIONSPART IIsimulations, where the learner can make any of a variety of errors during the course of the task; and (b) the use of forced-error simulations, where the learner experiences a specific, usually unexpected error and demonstrates error management as well as options for

1	during the course of the task; and (b) the use of forced-error simulations, where the learner experiences a specific, usually unexpected error and demonstrates error management as well as options for correction. Error management is a human factors process that includes making, detecting, and correcting errors.160,161 Several studies show that there is wide variation in the timing and type of operative decisions that residents make when interacting with an error-enabled simulation.112,162-164 Specific error training simulations have been developed using a cognitive task analysis approach to guide the design of error scenarios. Cognitive task analysis involves the use of structured interviews to explore how experts approach the error management process, including intraoperative decision-making and technical approaches.165,166 The error-enabled approach allows for a broad assessment of learning needs. The forced-error approach allows instructors to engage in specific error management

1	and technical approaches.165,166 The error-enabled approach allows for a broad assessment of learning needs. The forced-error approach allows instructors to engage in specific error management skills and metrics. Error-enabled and forced-error surgical simulation trainers have been used in a variety of research and training modules to allow observation and assessment of surgical residents as they independently perform operative procedures and practice the error management process.112,166,167Another approach to error training and assessment is the use of assessments that focus on errors. Current assessment tools for surgical skills include task-specific and global rating scales, final product analysis, and documentation of critical fail-ures.160,168 These existing tools for assessing surgical residents largely focus on manual techniques and procedure time and do not capture the cause of resident performance failures.169 Use of error-related theories in human factors allows for the

1	residents largely focus on manual techniques and procedure time and do not capture the cause of resident performance failures.169 Use of error-related theories in human factors allows for the devel-opment of error-centric skills assessments.161,170,171 One study used previously developed human error classifications, includ-ing omission versus commission and cognitive versus technical as a means of assessing surgical residents.162 Omission errors were defined as failure to perform a step entirely. Commis-sion errors represented failure to perform a step correctly. For example, failure to measure the hernia defect was categorized as an omission error, whereas measuring the hernia defect with an inaccurate method was categorized as a commission error. Errors in information, diagnosis, and strategy were categorized as cognitive, and errors in action, procedure, or mechanics were classified as technical. Use of assessment surveys with this type of differentiation allows for development of

1	were categorized as cognitive, and errors in action, procedure, or mechanics were classified as technical. Use of assessment surveys with this type of differentiation allows for development of error metrics in sur-gery and focused error training and feedback.172SIMULATION AND PATIENT OUTCOMESSurgical simulation training is intended to make patient care safe and free of avoidable errors and to maximize opportunities for good clinical outcomes. The Kirkpatrick four-level scale (Table 53-8) characterizes educational intervention effects, including those that might improve surgeon performance in clinical settings (Kirkpatrick level 3) or those that might actually improve patient outcomes (Kirkpatrick level 4).173 Despite the expanding use of simulation in proficiency-based practice models over at least 15 years, there are surprisingly few studies of simulation-based surgical training that can be described as Kirkpatrick level 4. On the other hand, there are numerous studies showing that

1	at least 15 years, there are surprisingly few studies of simulation-based surgical training that can be described as Kirkpatrick level 4. On the other hand, there are numerous studies showing that clinician performance during the course of clinical care is improved after simulation training (Kirkpatrick level 3). As detection of changes in clinical Table 53-8Kirkpatrick level scale of educational outcomesKIRKPATRICK LEVELLEVEL DESCRIPTIONDESCRIPTION FOR SIMULATIONLevel 1ReactionDid the learner perceive value in using a simulator or participating in simulation training?Level 2LearningDid the learner’s knowledge, skill, or attitude improve as a result of the simulation training?Level 3Behavioral changeDid the knowledge, skills, and attitudes acquired during simulation transfer to the clinical environment?Level 4Institutional impactDid the simulation training program lead to improved patient outcomes?Data from Cox T, Seymour N, Stefanidis D: Moving the Needle: Simulation’s Impact on

1	environment?Level 4Institutional impactDid the simulation training program lead to improved patient outcomes?Data from Cox T, Seymour N, Stefanidis D: Moving the Needle: Simulation’s Impact on Patient Outcomes, Surg Clin North Am. 2015 Aug;95(4):827-838.outcomes can be quite challenging, it may be difficult to isolate simulation training effects from numerous other factors that can also affect patient outcomes. When looking specifically at a low-frequency complication event such as bile duct injury with laparoscopic cholecystectomy, the detection of a small, positive training effect is statistically improbable. However, the use of available Kirkpatrick level 3 data to support assumptions about training benefits is fully supportable given the preponderance of literature showing such benefit.The best current evidence for improved patient outcomes with simulation is that of technical and cognitive training for central venous catheter (CVC) insertion. In 2009, Barsuk et al monitored

1	best current evidence for improved patient outcomes with simulation is that of technical and cognitive training for central venous catheter (CVC) insertion. In 2009, Barsuk et al monitored catheter-related bloodstream infection incidence in an ICU setting over a 32-month period before and after institu-tion of proficiency-based simulation training. After simulation-trained medical residents began performing CVC insertion, an 85% reduction in these infections was observed (3.2 per 1000 catheter-days reduced to 0.50 infections per 1000 catheter-days).174 Subsequently, the same group reported that the finan-cial savings realized with these improved outcomes amounted to a 7:1 return of the investment for the training.175 In a similar sin-gle unit observational study, Burden et al reported a reduction in catheter-related bloodstream infection incidence from 6.47 per 1000 catheter days to 2.44 per 1000 catheter days after training intervention and comparable financial savings with shorter

1	reduction in catheter-related bloodstream infection incidence from 6.47 per 1000 catheter days to 2.44 per 1000 catheter days after training intervention and comparable financial savings with shorter ICU and hospital stays.176 Single cohort studies have inherent weak-nesses, but a somewhat smaller randomized trial of simulation training versus traditional apprenticeship model-trained controls also showed this reduced infection incidence (1.0 vs. 3.4 infec-tions per 1000 catheter-days, respectively).177Riley and colleagues conducted a study in three small community hospitals, administering TeamSTEPPS (Team Strategies and Tools to Enhance Performance and Patient Safety) didactic team training to perinatal care teams at one hospital, TeamSTEPPS with an accompanying program of in situ simula-tions at a second, and no intervention at the third hospital, which Brunicardi_Ch53_p2163-p2186.indd 217822/02/19 4:39 PM 2179SKILLS AND SIMULATIONCHAPTER 53served as the study control.178,179

1	at a second, and no intervention at the third hospital, which Brunicardi_Ch53_p2163-p2186.indd 217822/02/19 4:39 PM 2179SKILLS AND SIMULATIONCHAPTER 53served as the study control.178,179 The simulation interventions were designed to involve triage, labor and delivery, and OR components of care. Perinatal outcomes were assessed using the Weighted Adverse Outcomes Score (WAOS). Simulation train-ing resulted in improved clinical results with a 37.4% decrease in WAOS observed in the simulation intervention group, while similar benefit was not observed in either the didactic-trained or control groups.TeamSTEPPS with supplemental simulation training was also studied by Capella and colleagues for team performance and patient outcomes in trauma care.180 Trauma resuscitations for successive 2-month periods separated by didactic and scenario-based simulation training to subjectively-defined proficiency levels (33 before training, 40 after training) were assessed in multiple teamwork domains

1	periods separated by didactic and scenario-based simulation training to subjectively-defined proficiency levels (33 before training, 40 after training) were assessed in multiple teamwork domains including overall performance using the TPOT (Trauma Team Performance Observation Tool). Significant improvements were observed post training in leader-ship, situational monitoring, mutual support, communication, and overall scoring. In addition, time to OR was also observed to decrease. Steinemann and colleagues reported on a larger number of trauma resuscitations, 141 before and 103 after in situ trauma bay patient simulation training with accompanying didactic instruction.181 Although significant improvements in patient outcomes were not observed in this study, resuscitation time did decrease following training.In a comprehensive review of literature examining simula-tion impact on patient outcomes, Zendejas identified 50 stud-ies purporting to show patient outcomes.182 For the majority of

1	training.In a comprehensive review of literature examining simula-tion impact on patient outcomes, Zendejas identified 50 stud-ies purporting to show patient outcomes.182 For the majority of these, the quality of clinical outcomes evidence was considered low, and appropriate validity data were reported for results in only a small number of published reports. In a subsequent review of 1328 articles resulting from an exhaustive literature search, Cox identified 12 individual articles that reported suf-ficient patient outcomes data to be considered Kirkpatrick level 4.183 Concerns regarding the relatively few studies of this nature are primarily methodological and relate to the prepon-derance of use of the observational preand posttraining study model with its inherent bias risk, rather than randomized con-trolled trials. These studies have also tended to be quite limited in size. In surgery, the general areas of surgeon performance that have lent themselves to study of educational

1	than randomized con-trolled trials. These studies have also tended to be quite limited in size. In surgery, the general areas of surgeon performance that have lent themselves to study of educational outcomes in the clinical setting after simulation training include technical abilities and observable behaviors during team-based activi-ties. The use of decreased operative time or technical errors as clinical outcomes after simulation training might suggest, but do not clearly establish, a patient safety or other efficacy benefit. However, these metrics have frequently been used to make the case for the potential for such benefits in skills transfer studies for procedures such as laparoscopic cholecystectomy and lapa-roscopic hernia repair.56,63 There is a clear need to obtain high quality evidence of how proficiency-based simulation training impacts surgical patient outcomes.In a recent study examining intern response to pediatric codes, simulation training was shown to significantly

1	evidence of how proficiency-based simulation training impacts surgical patient outcomes.In a recent study examining intern response to pediatric codes, simulation training was shown to significantly reduce the time to request help, to initiate bag-mask ventilation, and to ini-tiate chest compressions.184 Citing the rarity of pediatric codes, investigators measured this effect in in mock code situations using patient simulators. This use of a high-fidelity simulation environment as a surrogate for clinical events, that might oth-erwise be difficult to observe, may be the only practical model available for the study of provider performance outcomes with educational interventions.SIMULATION TRAINING FOR THE PRACTICING SURGEON AND MAINTENANCE OF SKILLGiven the current requirement for FLS and FES certification in residency, some authors have posed the important question of whether such certification should be applied more broadly to surgeons in practice.185 In a 2012 press release, both

1	FES certification in residency, some authors have posed the important question of whether such certification should be applied more broadly to surgeons in practice.185 In a 2012 press release, both the ACS and SAGES made the recommendation that all surgeons per-forming laparoscopy obtain FLS certification. The potential to lower malpractice litigation risk under a self-insurance model was used as justification to certify 37 surgeons in FLS in the Harvard system in 2009.186 In a recent report, surgical oncolo-gists new to minimally invasive inguinal lymph node dissec-tion (MILND) were FLS tested prior to performance of their first clinical procedures, which were video-assessed using the GOALS scoring method.187 The FLS score was shown to corre-late with both GOALS results and operative time but not lymph node yield. However, evidence that a lab-based technical and cognitive skills test predicts observed operative technical skill in practicing surgeons is promising and warrants

1	time but not lymph node yield. However, evidence that a lab-based technical and cognitive skills test predicts observed operative technical skill in practicing surgeons is promising and warrants investigative follow-up.At the present time, the American Board of Surgery’s requirements for Maintenance of Certification (MOC) do not specifically include any certification of technical skills that might use surgical simulation.188 Nonetheless, simulation training and testing can be made available to surgeons in practice to provide an avenue for specific training. Although most investigations of VR use for laparoscopy have examined basic skills acquisition in the lab setting far in advance of any measured impact in the clinical OR, a recent innovative study of the use of a VR simulator for “warm-up” practice immediately before a procedure showed that this improved OR performance.189 As new surgical procedures and technologies are introduced to clinical practice, simulation training solutions

1	practice immediately before a procedure showed that this improved OR performance.189 As new surgical procedures and technologies are introduced to clinical practice, simulation training solutions could serve just as important a purpose to prepare for these as simulation training methods currently serve for laparoscopic surgery. In a recent publication relating to practicing surgeons, Sullivan et al. provide a framework for development of simulation-based certification models for both trainees and faculty surgeons.190FUTURE CONSIDERATIONSSimulation-based training and assessment is firmly established in surgical education, especially in graduate medical education where learners have been more extensively studied than any other simulation user group. The immediate future of simulation in surgery will likely see expanded use of proficiency-based training given the consistent demonstrations of effectiveness in improving surgeon skills and improved educa-tional outcomes as measured in

1	surgery will likely see expanded use of proficiency-based training given the consistent demonstrations of effectiveness in improving surgeon skills and improved educa-tional outcomes as measured in clinical settings.The question of what types of simulation-based assess-ments and training activities might be possible raises questions of where technological advances might open new opportunities.191 The advancement of virtual reality is inevitable, and much richer virtual experiences entering the mainstream in surgical educa-tion seems likely in the near future. Three-dimensional printing technology has been growing in its use and applications and has greatly facilitated the development of anatomically accurate bench top simulations for complex surgical procedures. Some of the materials allow for elegant instrument-based dissections, including the use of electrosurgery.192-1949Brunicardi_Ch53_p2163-p2186.indd 217922/02/19 4:39 PM 2180SPECIFIC CONSIDERATIONSPART IIAdvances in

1	allow for elegant instrument-based dissections, including the use of electrosurgery.192-1949Brunicardi_Ch53_p2163-p2186.indd 217922/02/19 4:39 PM 2180SPECIFIC CONSIDERATIONSPART IIAdvances in wearables, motion tracking, and sensor tech-nologies allow for a wide variety of hybrid and aug-mented experiences in simulation as well as extensive opportunities for the development of new performance metrics. One study using sensor technology for evaluating clinical breast examination skills noted key performance differences in expe-rienced physicians. Both the sensor data and sensor-guided video analysis allowed for skill quantifications that were previ-ously unknown but critical to performance excellence.195-196Opportunities for remote collaboration are now greatly improved with higher internet speeds, improvements in aug-mented reality technology, and ever-increasing camera reso-lution. VIPAR (Virtual Interactive Presence and Augmented Reality) allows for the visual field of a surgeon

1	speeds, improvements in aug-mented reality technology, and ever-increasing camera reso-lution. VIPAR (Virtual Interactive Presence and Augmented Reality) allows for the visual field of a surgeon to be converted to a simulation and projected in a remote location.197-198 As such, the system allows for intraoperative collaboration and telementoring.199Irrespective of what new simulation and engineering tech-nologies emerge, these technologies are here not just to stay but to grow as assessment and educational tools. This presents abundant opportunities for simulation leaders in surgery to improve the delivery of care by defining best practice in sim-ulation applications and keeping step with current and future changes in surgical practice.REFERENCESEntries highlighted in bright blue are key references. 1. Leape LL. Error in medicine. JAMA. 1994;272(23):1851-1857. 2. To err is human: building a safer health system. Washington, DC: National Academy Press, Institute of Medicine;

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1	trial of virtual reality simulator training: transfer to live patients. Am J Surg. 2007;194(2):205-211. 106. Haycock A, Koch AD, Familiari P, et al. Training and transfer of colonoscopy skills: a multinational, randomized, blinded, controlled trial of simulator versus bedside training. Gastrointest Endosc. 2010;71(2):298-307. 107. Ferlitsch A, Schoefl R, Puespoek A, et al. Effect of virtual endoscopy simulator training on performance of upper gastrointestinal endoscopy in patients: a randomized controlled trial. Endoscopy. 2010;42(12):1049-1056. 108. Ende A, Zopf Y, Konturek P, et al. Strategies for training in diagnostic upper endoscopy: a prospective, randomized trial. Gastrointest Endosc. 2012;75(2):254-260. 109. Shirai Y, Yoshida T, Shiraishi R, et al. Prospective randomized study on the use of a computer-based endoscopic simulator for training in esophagogastroduodenoscopy. J Gastroenterol Hepatol. 2008;23(7 pt 1):1046-1050.Brunicardi_Ch53_p2163-p2186.indd 218222/02/19 4:39

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1	Web-Based Education and Implications of Social MediaLillian S. Kao and Michael E. Zenilman 54chapterINTRODUCTIONSurgical education has changed significantly over the past two decades. Disruptive forces such as work hour restrictions and the advent of laparoscopy have forced educators to rethink how and where to teach residents. Technologies, including the inter-net and web-based applications, have further enabled educators to redesign surgical education (Fig. 54-1). The internet has become an integral tool not just in surgical education but also in Americans’ lives by changing the way that people communicate with each other, access information, and conduct their daily lives. Today, almost 9 in 10 American adults use the internet. Furthermore, the internet has revolutionized education by allowing for expanded reach, asynchronous learning whereby students and instructors do not have to be on the same time schedule, and multimedia materials.Like internet usage, social media has seen a

1	allowing for expanded reach, asynchronous learning whereby students and instructors do not have to be on the same time schedule, and multimedia materials.Like internet usage, social media has seen a rise in adop-tion over the past decade. Social media is a term that encom-passes multiple computer-mediated platforms that are used for creating and sharing information, ideas, and other content. Social media facilitates communication and interactions across virtual networks. Commonly used platforms include Facebook, Twitter, Snapchat, and Instagram. Social media can be used for multiple purposes including social and professional network-ing; however, this chapter will focus on its uses in surgical education.WEB-BASED EDUCATIONWeb-based educational resources include lectures and webi-nars, simulators, assessment tools, and interactive mentoring and coaching. Furthermore, entire web-based curricula have been developed that can link to online resources such as journal articles, interactive

1	assessment tools, and interactive mentoring and coaching. Furthermore, entire web-based curricula have been developed that can link to online resources such as journal articles, interactive anatomy modules, and videos of operations. There are multiple advantages to web-based education. For sur-gical trainees, web-based educational materials allow access regardless of time of day or night, provide interactive tools for learning (i.e., anatomy), and videos for viewing operations and procedures. Furthermore, for practicing surgeons, web-based educational resources include forums for sharing challenging cases and procuring advice, activities for obtaining continuing 12education, and rapid access to information about new technolo-gies and research. However, there may also be disadvantages in terms of costs and technical problems.Multiple studies have evaluated learning outcomes after implementation of web-based educational interventions. In sur-gical education, these interventions may be

1	terms of costs and technical problems.Multiple studies have evaluated learning outcomes after implementation of web-based educational interventions. In sur-gical education, these interventions may be used to teach patient care and decision-making via online case studies, convey knowledge using online didactic materials, or introduce surgical skills. However, studies evaluating these interventions tend to be nonrandomized, small, and single center. A 2008 systematic review and meta-analysis by Cook et al evaluated the effect of internet-based learning across healthcare in general. The review suggested that internet-based learning is better than no interven-tion but has similar effectiveness as traditional educational methods. A more recent 2015 systematic review by Jayakumar et al focused on web-based education in surgery. They reported a positive effect, but the majority of studies included in the review lacked a control. Based on the current literature, the internet should be

1	on web-based education in surgery. They reported a positive effect, but the majority of studies included in the review lacked a control. Based on the current literature, the internet should be considered one tool among many that can facilitate learning. However, further studies are necessary to identify the key elements that improve effectiveness. Web-based educational materials should be developed keeping adult learning theories and principles in mind.Web-based surgical curricula have been developed both at an institutional and at a national level. A widely-used curricu-lum is the Surgical Council on Resident Education (SCORE) curriculum, which is available via an online portal. Developed in 2006, SCORE is based on the six core competencies required of a graduating resident: patient care, medical knowledge, professionalism, communication, practice-based learning, and systems-based practice. SCORE is the result of an ongoing col-laborative effort of the American Board of Surgery,

1	care, medical knowledge, professionalism, communication, practice-based learning, and systems-based practice. SCORE is the result of an ongoing col-laborative effort of the American Board of Surgery, American College of Surgeons, American Surgical Association, Asso-ciation of Program Directors in Surgery, Association for Surgical Education, Residency Review Committee for Surgery of the Accreditation Council of Graduate Medical Education, and Society of American Gastrointestinal and Endoscopic Surgeons. The SCORE curriculum provides content for topics to be covered during a 5-year general surgery residency and is adding fellowship-level content as well. The SCORE Portal modules for each topic include learning objectives, discussion 3Introduction2187Web-Based Education2187Social Media–Based Education2188What is Social Media? / 2188Journal Clubs / 2190Live-Tweeting Conferences / 2190Interactive Forums and Communities / 2190Public Education / 2192Pitfalls in Web and Social Media–Based

1	Education2188What is Social Media? / 2188Journal Clubs / 2190Live-Tweeting Conferences / 2190Interactive Forums and Communities / 2190Public Education / 2192Pitfalls in Web and Social Media–Based Education2192Implications and Future Directions2194Brunicardi_Ch54_p2187-p2196.indd 218713/02/19 2:37 PM 2188Disruptive forces1995200020052010ImpactSurgical educationEnabling technologiesFigure 54-1. The relationship between disruptive forces, enabling technologies, and surgical education. (Reproduced with permission from Pugh CM, Watson A, Bell RH, Jr, et al. Surgical education in the internet era. J Surg Res. 2009 Oct;156(2):177-182.)Key Points1 The internet has become an integral tool not just in surgical education but also in Americans’ lives by changing the way that people communicate with each other, access informa-tion, and conduct their daily lives.2 The internet has revolutionized surgical education by allow-ing for expanded reach—asynchronous learning whereby students and

1	with each other, access informa-tion, and conduct their daily lives.2 The internet has revolutionized surgical education by allow-ing for expanded reach—asynchronous learning whereby students and instructors do not have to be on the same time schedule—and multimedia materials such as interactive les-sons and videos.3 Despite the appeal of web-based education, systematic reviews and meta-analyses have failed to identify high-quality studies demonstrating that it is superior to standard educational methods in improving learning outcomes.4 Web-based and virtual reality simulators can be used both to teach technical skills and to assess performance. Virtual reality simulators have been effective in training surgeons on technical skills that translate to operating room performance.5 Online and social media–based journal clubs can overcome barriers associated with traditional journal clubs such as lack of a convenient time and no local clinical or methodological experts. However, they may

1	social media–based journal clubs can overcome barriers associated with traditional journal clubs such as lack of a convenient time and no local clinical or methodological experts. However, they may require more time and commit-ment from the leaders, and they may not be as effective as traditional, in-person, faculty-moderated journal clubs.6 Social media rapidly and exponentially increases the spread of information.7 Multiple pitfalls exist with regards to web and social media–based education, including but not limited to: (a) need for more widespread adoption and use, (b) lack of accuracy and regulatory oversight over educational content, (c) issues regarding patient confidentiality and privacy, (d) nondisclo-sure of conflicts of interest, and (e) paucity of evidence for effectiveness of these materials for improving knowledge, attitudes, skills, and outcomes.8 Social media has become a necessary component of surgical practice.questions, text resources and videos, and self-assessment

1	these materials for improving knowledge, attitudes, skills, and outcomes.8 Social media has become a necessary component of surgical practice.questions, text resources and videos, and self-assessment quizzes. Although improvements in quality examination per-formance among residencies that subscribe to SCORE are prom-ising, no studies have definitely demonstrated that SCORE use improves resident knowledge, skills, or clinical performance.Web-based education can also be used for assessing and teaching surgical skills. In 2013, Birkmeyer et al performed a study that correlated surgical skills in bariatric surgery, based on blinded reviews of videotaped operations, to clini-cal outcomes. The ability to discriminate surgeons with good and poor technical skills using video-based assessments has signifi-cant implications for training surgeons and for evaluating their performance. With regards to training, multiple web-based and virtual reality simulators have been developed that allow

1	has signifi-cant implications for training surgeons and for evaluating their performance. With regards to training, multiple web-based and virtual reality simulators have been developed that allow resi-dents to practice tasks and skills repetitively at their own pace and on their own time. These simulators can quantify efficiency of motion and time to complete a task as well as provide 4real-time feedback. These metrics have been demonstrated to have construct validity (in that they measure what they are sup-posed to be measuring) and criterion validity (in that they cor-relate with operative performance). For example, randomized trials have demonstrated that surgical simulation training corre-lates with decreased operative time and improves subjectively rated performance on technical skills in the live setting. Web-based assessments have also been used in combination with physical simulators to provide similar metrics.With regards to surgeons in practice, there has been increased

1	skills in the live setting. Web-based assessments have also been used in combination with physical simulators to provide similar metrics.With regards to surgeons in practice, there has been increased enthusiasm for the use of video-based coaching to complement intraoperative teaching. In particular, postopera-tive review of videotaped procedures allows surgeons to receive individualized feedback about opportunities for improvement without the time constraints or pressures of the operating room. Randomized trials of surgical coaching in simulated settings suggests benefits over traditional simulator training, and larger trials in a live setting are ongoing. Although most coaching occurs face-to-face, there are opportunities to use web-based coaching. As an example, telementoring has been used to proc-tor surgeons in the operating room, even across the globe.Barriers to web-based education include the up-front costs for development and the need for technical expertise. As already noted,

1	to proc-tor surgeons in the operating room, even across the globe.Barriers to web-based education include the up-front costs for development and the need for technical expertise. As already noted, web-based education for teaching knowledge may not be more effective than traditional methods. On the other hand, simulation and video-based coaching hold significant prom-ise in improving training in and assessment of surgical skills. However, widespread implementation of video-based coaching will require a culture shift for surgeons to accept assistance and resources such as time, availability of coaches, and finances. Ongoing studies will provide data regarding the effectiveness of these educational strategies.SOCIAL MEDIA–BASED EDUCATIONWhat is Social Media?Social media is a term describing websites and web-based applications that enable users to share ideas, information, and Brunicardi_Ch54_p2187-p2196.indd 218813/02/19 2:37 PM 2189WEB-BASED EDUCATION AND IMPLICATIONS OF SOCIAL

1	websites and web-based applications that enable users to share ideas, information, and Brunicardi_Ch54_p2187-p2196.indd 218813/02/19 2:37 PM 2189WEB-BASED EDUCATION AND IMPLICATIONS OF SOCIAL MEDIACHAPTER 54@Twitteruser1@Twitteruser2A@Twitteruser1@Twitteruser2BFigure 54-2. Social media usage can be (a) unidirectional or (b) bidirectional. User names on Twitter are denoted by “@”. A. @Twitteruser2 is following @Twitteruser1. She is receiving all of his messages in her Twitter feed. However, @Twitteruser1 does not follow her back and therefore does not receive her messages in return. B. @Twitteruser1 and @Twitteruser2 follow each other. Therefore, they each receive each other’s messages in their Twitter feeds.content through virtual networking. Although social media is often used to interact with friends and family, social media can also be used for educational and professional purposes. Examples include Twitter-based journal clubs, Facebook-based discussion forums, and professional

1	with friends and family, social media can also be used for educational and professional purposes. Examples include Twitter-based journal clubs, Facebook-based discussion forums, and professional networking sites such as LinkedIn or ResearchGate. Social media platforms can serve different purposes including social networking, microblogging, blogging, photo sharing, video sharing, and crowdsourcing.Commonly used social media platforms in surgery include Facebook, Twitter, and YouTube. Facebook is the most popular social networking site; it can be accessed via desktops, laptops, and mobile phones. It allows users to exchange information, photos, and videos with specified contacts or “friends” with whom there is a two-way relationship. Twitter is another popu-lar social media platform. It is a microblogging site that, like Facebook, allows exchange of messages and photos but limits messages or tweets to 140 characters or less. Twitter users may have one or two-way relationships with other

1	microblogging site that, like Facebook, allows exchange of messages and photos but limits messages or tweets to 140 characters or less. Twitter users may have one or two-way relationships with other users. Followers of a user receive all of that person’s tweets in their Twitter feed. YouTube is a social media platform that allows users to share videos.Social media usage in surgery may be unidirectional or bidirectional (Fig. 54-2). For example, journals such as the New England Journal of Medicine may have a large number of followers but may be following very few users back. Given that the number of journal articles published daily has risen expo-nentially, particularly with the advent of open access journals, keeping up with the surgical literature can be overwhelming. Following journals on social media is one strategy for staying updated. Although conventional media outlets such as newspa-pers and news channels may draw attention to practice-changing studies, social media is another

1	on social media is one strategy for staying updated. Although conventional media outlets such as newspa-pers and news channels may draw attention to practice-changing studies, social media is another platform by which such infor-mation can be promoted and disseminated by journals. Many surgical journals have an online and social media presence, and many have social media editors who curate the posted materials. Popular social media platforms for journals include Facebook and Twitter. Both platforms allow journals to post text, figures, and links to abstracts or journal articles. Both platforms allow others to share information or comment on articles. However, Twitter restricts text to 140 characters. A recent innovation that may counteract the limited number of allowed characters is the visual abstract, which is a concise pictorial representation of an article’s key points (Fig. 54-3). Recently, a prospective, case-control crossover study was performed whereby tweets about articles

1	visual abstract, which is a concise pictorial representation of an article’s key points (Fig. 54-3). Recently, a prospective, case-control crossover study was performed whereby tweets about articles from Annals of Surgery were either accompanied by a visual abstract or tweeted with text alone. Accompaniment of a tweet with a visual abstract resulted in a threefold increase in article visits. Thus, the majority of journal followers may merely receive the information about new publications (uni-directional flow of information). However, users may also choose to respond to posts with comments (bidirectional flow of information).Surgicaltreatment ASurgicaltreatment BOutcomesComplications15%11%MortalityAuthor A et al. Journal. Date.Journal Logo8%4%Comparison of Surgical Treatment A VersusSurgical Treatment BPatients with disease XFigure 54-3. A visual abstract is a graphical summary of the main results of a journal article.Brunicardi_Ch54_p2187-p2196.indd 218913/02/19 2:37 PM

1	Treatment BPatients with disease XFigure 54-3. A visual abstract is a graphical summary of the main results of a journal article.Brunicardi_Ch54_p2187-p2196.indd 218913/02/19 2:37 PM 2190SPECIFIC CONSIDERATIONSPART IIJournal ClubsJournals may promote bidirectional flow of information by hosting social media–based journal clubs. From an educational standpoint, journal clubs have traditionally served not only as an adjunct to lectures but also as a forum to teach about critical appraisal of the literature. Furthermore, when facilitated by faculty with clinical expertise on the subject being discussed, surgical trainees can bet-ter evaluate how to incorporate the evidence into practice. How-ever, barriers to traditional journal clubs may include poor participation, lack of a convenient time, or absence of local exper-tise in either the clinical topic or research methodology. Social media–based journal clubs can help to overcome these barriers by allowing for asynchronous discussion

1	time, or absence of local exper-tise in either the clinical topic or research methodology. Social media–based journal clubs can help to overcome these barriers by allowing for asynchronous discussion and expert moderators. Online journal clubs can be carried out in real time, but they also allow respondents to comment hours or even days later to a conversation. Multiple specialties, including surgery, have developed social media–based journal clubs. Tips for suc-cessfully launching an online journal club can be garnered from the expanding experience with them (Table 54-1).Social media–based journal clubs in surgery have been conducted via Facebook, Twitter, or a combination of the two platforms. They have also taken the form of a blog. Although commonly associated with personal journals or diaries, blogs can also be found on professional websites that are updated frequently by a person or group (i.e., by a journal or surgical society). Conversations from journal club discussions can

1	or diaries, blogs can also be found on professional websites that are updated frequently by a person or group (i.e., by a journal or surgical society). Conversations from journal club discussions can also be compiled and summarized into a transcript either manually or using web-based applications such as Storify. These transcripts can be posted on the journal website or shared. Furthermore, 5aTable 54-1Ten tips for setting up an online journal club TIP 1Create an online home page that serves as a launching pad for your journal club discussions 2Develop and register a hashtag on Twitter 3Incorporate not only Twitter but other collaborative platforms as part of your online journal club 4Ensure that the time of the journal club is convenient for your target audience 5Help prepare participants by aggregating other online resources relevant to the article 6Consider inviting the authors of the featured article or other experts in the field 7Suggest journal club participants consider using

1	by aggregating other online resources relevant to the article 6Consider inviting the authors of the featured article or other experts in the field 7Suggest journal club participants consider using specific Twitter management applications during the Tweet chat 8Engage the participants to cultivate and incentivize more discussion 9Connect to the online community by following and engaging with other relevant social media accounts10Link back to the original paper by inserting a comment on PubMed CommonsData from Chan TM, Thoma B, Radecki R, et al. Ten steps for setting up an online journal club, J Contin Educ Health Prof. 2015 Spring;35(2):148-154.summaries from either in-person or online journal clubs can be added to the PubMed citation via PubMed Commons.Preliminary data suggests that online journal clubs increase discussion about articles, views of the abstract, and downloads (Fig. 54-4). For example, the International Gen-eral Surgery Journal Club held four moderated discussions of

1	journal clubs increase discussion about articles, views of the abstract, and downloads (Fig. 54-4). For example, the International Gen-eral Surgery Journal Club held four moderated discussions of journal articles on Twitter between March and June 2014. The reviewed articles covered topics relating to bariatric surgery (March), venous thromboembolism in trauma (April), diverticu-litis (May), and contralateral prophylactic mastectomy for breast cancer (June). Although the authors and invited experts only moderated discussions for 3 days, Twitter activity increased in the days preceding and following these discussions. Further-more, daily views of the article and downloads increased cor-respondingly. Thus, online journal clubs are a potential strategy for increasing surgeon education about seminal articles.While social media–based journal clubs hold much appeal, there is a paucity of data regarding their effectiveness in teach-ing participants about critical appraisal skills. A

1	about seminal articles.While social media–based journal clubs hold much appeal, there is a paucity of data regarding their effectiveness in teach-ing participants about critical appraisal skills. A multicenter randomized trial compared journal clubs moderated by a faculty member to online discussions. Surgical resi-dents in both arms utilized modules developed by the Evidence Based Reviews in Surgery Steering Committee; these modules include the relevant guide to critical appraisal and a method-ological and clinical review. Residents randomized to the mod-erated group scored higher on a validated test evaluating critical appraisal skills. Further study is required to assess the effective-ness of social media–based journal clubs in disseminating new knowledge as well as in teaching critical appraisal.Live-Tweeting ConferencesTraditionally, surgeons have attended regional and national conferences to network, learn new information relevant to their practice, and exchange ideas. However,

1	appraisal.Live-Tweeting ConferencesTraditionally, surgeons have attended regional and national conferences to network, learn new information relevant to their practice, and exchange ideas. However, with the advent of social media, surgeons no longer have to physically attend a conference to perform all those activities. Live-tweeting is a term used to describe the posting of comments on Twitter about an event while it is ongoing. Multiple surgical and nonsurgical societies have adopted Twitter to expand the reach of their conferences. By denoting tweets as emanating from a specific conference with a unique hashtag, the reach and number of impressions can actually be measured (Fig. 54-5). The reach refers to the number of unique recipients of messages from a specific group of Twitterers (or people posting on Twitter). Impressions refers to each time a message was delivered to a recipient; a recipient may receive the same message more than once. Neither reach nor impressions measure

1	(or people posting on Twitter). Impressions refers to each time a message was delivered to a recipient; a recipient may receive the same message more than once. Neither reach nor impressions measure whether the recipient read the tweet. As an example of how reach and impressions can be used to provide metrics for social media, the Healthcare Hashtag Project allows registered conference hashtags to track the latest tweets, the most prolific Twitterers of conference-related tweets, the most commonly mentioned Twitterers, and the number of impressions (Fig. 54-6) (https://www.symplur.com/healthcare-hashtags/). As noted in the figure, social media rapidly and exponentially increases the spread of information. Transcripts of conference-related tweets can also be assembled to allow a conversation thread to be organized into a cohesive discussion.Interactive Forums and CommunitiesInteractive forums and communities are another method by which both the internet and social media can be used for

1	thread to be organized into a cohesive discussion.Interactive Forums and CommunitiesInteractive forums and communities are another method by which both the internet and social media can be used for 5b6Brunicardi_Ch54_p2187-p2196.indd 219013/02/19 2:37 PM 2191WEB-BASED EDUCATION AND IMPLICATIONS OF SOCIAL MEDIACHAPTER 54ATimeDaily HTML Views & PDF Downloads of Featured Article During Each of 4 Monthly IGSJC Twitter Journal ClubsTweets025050075010001250Tweet Activity on #IGSJC10. Mar24. Mar7. Apr21. Apr5. May19. May2. JunB0100Number of HTML Views PDF Downloads200300400100Dates of Journal Club +/–5 DaysMarch 3-5June 4-50255075HTML ViewPDF DownloadFigure 54-4. Impact of a social media–based journal club on Twitter activity, hypertext markup language (HTML) views, and portable document format (PDF) downloads. A. Activity during a Twitter-based journal club such as the International General Surgery Journal Club (IGSJC) can be tracked by denoting the journal club related tweets with a

1	(PDF) downloads. A. Activity during a Twitter-based journal club such as the International General Surgery Journal Club (IGSJC) can be tracked by denoting the journal club related tweets with a hashtag (#IGSJC). Twitter activity increased during each of four journal clubs. B. Daily HTML views and PDF downloads of featured articles also increased around the time period of the four Twitter journal clubs. (Unpublished data from Sarah Bryczowski and Michael E. Zenilman.)@Twitteruser14 followers(3 unique, 1 shared)Reach: 7 unique usersImpressions: 8 impressionsEach user receivedtweet from@Twitteruser1:1 impression eachReceivedtweet fromboth users:2impressionsEach user receivedtweet from@Twitteruser2:1 impression each@Twitteruser24 followers(3 unique, 1 shared)Figure 54-5. Difference between reach and impressions in Twitter. If there are two Twitter users and each has three unique followers and one shared follower, then there are seven unique recipients of their combined tweets. Their total

1	and impressions in Twitter. If there are two Twitter users and each has three unique followers and one shared follower, then there are seven unique recipients of their combined tweets. Their total reach is seven unique users. If both users tweet the same message, then one user will have received the message twice. However, each time the message was delivered counts as an impression; thus, the followers will have a total of eight impressions.Brunicardi_Ch54_p2187-p2196.indd 219113/02/19 2:37 PM 2192SPECIFIC CONSIDERATIONSPART IIImpressionsTweetsParticipantsAvg Tweets/HourAvg Tweets/Participant4272298443,601The Numbers@User1 123@User2 65@User3 62@User4 46@User5 42@User6 32@User7 21@User8 19@User9 17@User10 16Top 10 by Mentions@User3 27@User11 25@User7 23@User4 23@User12 19@User13 13@User14 12@User15 11@User2 10@User1 9Top 10 by Tweets@User13 103,937@User24 80,956@User4 42,976@User3 40,911@User6 28,966@User41 22,534@User52 14,566@User72 13,514@User35 11,965@User68 9862Top 10 by

1	12@User15 11@User2 10@User1 9Top 10 by Tweets@User13 103,937@User24 80,956@User4 42,976@User3 40,911@User6 28,966@User41 22,534@User52 14,566@User72 13,514@User35 11,965@User68 9862Top 10 by ImpressionsThe #Surgery Conference In˜uencersFigure 54-6. Example of conference analytics from Healthcare Hashtags (https://www.symplur.com/healthcare-hashtags/). Sur-gery conferences can tag tweets by using a prespecified hashtag (i.e., #SurgeryConference) to denote conference-related messages. These can then be tracked. A mention occurs when a user includes another user’s name in the tweet. Note in the example that even if there are only a few users tweeting about a conference, the number of impressions can be large if several of those users have a large number of followers. For example, @User13 only tweeted 13 times but had 103,937 impressions.educational purposes. They are examples of crowdsourcing whereby information is gathered by enlisting the assistance of a large number of participants.

1	tweeted 13 times but had 103,937 impressions.educational purposes. They are examples of crowdsourcing whereby information is gathered by enlisting the assistance of a large number of participants. Crowdsourcing often involves the general public or a loosely defined group of individuals as opposed to outsourcing, which tasks a project to a specific company or group. For example, Wikipedia is an example of crowdsourcing. In surgery, an example of crowdsourcing are the web-based communities formed by the American College of Surgeons. Members can belong to one or more communities which are based on specialty (i.e., general surgery or endocrine surgery), geography (i.e., Florida Chapter), level of education (i.e., medical students or senior surgeons), special interest (i.e., surgeon writers), or issue (i.e., quality improvement and advo-cacy). Members can post and respond to discussion threads that cover a wide range of topics including advice for challenging cases, surgical history, and

1	or issue (i.e., quality improvement and advo-cacy). Members can post and respond to discussion threads that cover a wide range of topics including advice for challenging cases, surgical history, and political issues.An example of a social media–based interactive forum is the International Hernia Collaboration (IHC) which is housed on Facebook. Participants include not only surgeons but also healthcare providers and industry representatives interested in improving outcomes after hernia surgery. Membership in the IHC is limited to those who have been vetted and approved. The forum allows members to ask for advice on difficult cases, debate controversial topics, post step-by-step instructions on procedures or management strategies, and disseminate informa-tion rapidly to a wide audience. Collaborative forums such as the IHC allow for real-time, interactive, case-based, continuing education.Public EducationThe educational opportunities provided by the internet and social media are not

1	Collaborative forums such as the IHC allow for real-time, interactive, case-based, continuing education.Public EducationThe educational opportunities provided by the internet and social media are not limited to healthcare providers. The inter-net and social media are also being increasingly used to educate patients and their families. There are multiple media through which health education is spread, including, but not limited to, online or social media discussion forums run by patients, patient support groups, healthcare providers, or healthcare orga-nizations; blogs or newsrooms; or electronic and mobile health patient portals. Social media allows patients rapid access to information regardless of time or location. However, as noted in the following section, the accuracy of social media–based educational materials cannot be guaranteed, and patients should utilize caution in relying on the information to make important healthcare decisions.PITFALLS IN WEB AND SOCIAL MEDIA–BASED

1	media–based educational materials cannot be guaranteed, and patients should utilize caution in relying on the information to make important healthcare decisions.PITFALLS IN WEB AND SOCIAL MEDIA–BASED EDUCATIONDespite the many advantages of web and social media–based education, significant potential pitfalls still remain. First, widespread adoption and utilization is a challenge. Although internet and mobile phone usage is prevalent, effec-tiveness of web-based educational materials is dependent upon trainees’ and surgeons’ uptake of the technology and available resources. Despite increasing availability of the internet and mobile technology, not all surgeons use it for educational pur-poses. For example, a systematic review and meta-analysis by Guraya et al found that three out of four medical students use social networking sites, but only one out of five uses them for educational purposes. Furthermore, lack of utilization can impact educational effectiveness. In the previously

1	medical students use social networking sites, but only one out of five uses them for educational purposes. Furthermore, lack of utilization can impact educational effectiveness. In the previously mentioned multicenter trial comparing a moderated journal club to an online version, low participation in the Internet journal club was postulated to be a significant factor in the poorer perfor-mance on a critical appraisal test. More attention to instruc-tional design may improve uptake and effectiveness. A systematic review and meta-analysis by Cook et al determined that features such as interactivity, practice exercises, repeti-tion, and feedback are associated with improved learning out-comes. The authors also noted that the evidence base upon which to design internet-based learning programs is limited by poor study methodology, failure to use conceptual frame-works, and lack of adherence to reporting standards. Thus, while the aforementioned features should be considered in designing

1	is limited by poor study methodology, failure to use conceptual frame-works, and lack of adherence to reporting standards. Thus, while the aforementioned features should be considered in designing future web and social media–based educational pro-grams, further evidence-based guidance is needed.Second, the quality of information available online and via social media may not be accurate or reliable. Multiple reports have been published regarding the inaccuracies of web-based educational materials. Surgeons and surgical trainees should carefully evaluate the source of educational material, search for conflicts of interest that may result in biased information, assess how recently the information was updated, and cross-check references. The lay public may have more difficulty in identifying trustworthy surgical educational materials on the internet. Healthcare providers should guide patients to reputable websites and to encourage discussion regarding the accuracy of the content.Third,

1	trustworthy surgical educational materials on the internet. Healthcare providers should guide patients to reputable websites and to encourage discussion regarding the accuracy of the content.Third, useful dialog and advice about difficult cases must be balanced with ethical considerations surrounding patient con-fidentiality and privacy. Appropriate safeguards must be taken 7Brunicardi_Ch54_p2187-p2196.indd 219213/02/19 2:37 PM 2193WEB-BASED EDUCATION AND IMPLICATIONS OF SOCIAL MEDIACHAPTER 54Table 54-2The American College of Physicians Ethics, Professionalism and Human Rights Committee; the American College of Physicians Council of Associates; and the Federation of State Medical Boards Special Committee on Ethics and Professionalism published a position paper on online medical professionalismPosition 1Use of online media can bring significant educational benefits to patients and physicians, but it may also pose ethical challenges. Maintaining trust in the profession and in

1	professionalismPosition 1Use of online media can bring significant educational benefits to patients and physicians, but it may also pose ethical challenges. Maintaining trust in the profession and in patient–physician relationships requires that physicians consistently apply ethical principles for preserving the relationship, confidentiality, privacy, and respect for persons to online settings and communications.Position 2The boundaries between professional and social spheres can blur online. Physicians should keep the two spheres separate and comport themselves professionally in both.Position 3Email or other electronic communications should only be used by physicians in an established patient–physician relationship and with patient consent. Documentation about patient care communications should be included in the patient’s medical record.Position 4Physicians should consider periodically “self-auditing” to assess the accuracy of information available about them on physician-ranking

1	should be included in the patient’s medical record.Position 4Physicians should consider periodically “self-auditing” to assess the accuracy of information available about them on physician-ranking websites and other sources online.Position 5The reach of the internet and online communications is far and often permanent. Physicians, trainees, and medical students should be aware that online postings may have future implications for their professional lives.Data from Farnan JM1, Snyder Sulmasy L, Worster BK, et al: Online medical professionalism: patient and public relationships: policy statement from the American College of Physicians and the Federation of State Medical Boards, Ann Intern Med. 2013 Apr 16;158(8):620-627.to ensure that patients cannot be identified based on provided information, that patients have consented to have their informa-tion posted anonymously, and that all case-related comments are appropriate and professional. While common sense should be utilized in posting

1	that patients have consented to have their informa-tion posted anonymously, and that all case-related comments are appropriate and professional. While common sense should be utilized in posting about patient cases, only a few organizations have published guidelines for how to safeguard against potential pitfalls. In 2013, the American College of Physicians Ethics, Professionalism, and Human Rights Committee; the American College of Physicians Council of Associates; and the Federa-tion of State Medical Boards Special Committee on Ethics and Professionalism published a position statement about online medical education (Table 54-2). The paper stated that “Maintain-ing trust in the profession and in patient–physician relationships requires that physicians consistently apply ethical principles for preserving the relationship, confidentiality, privacy, and respect for persons to online settings and communications.”Fourth, conflicts of interest must be clearly stated. Jour-nals require

1	for preserving the relationship, confidentiality, privacy, and respect for persons to online settings and communications.”Fourth, conflicts of interest must be clearly stated. Jour-nals require authors to declare relevant conflicts of interest, but multiple studies suggest that these often go unreported. Simi-larly, conflicts of interest should be disclosed on social media. However, such disclosures may be more difficult on social media due to the limited number of allowable characters (i.e., 140 characters for Twitter) or to the way information is propa-gated. For example, a surgeon may disclose an industry relation-ship on an original tweet, but the disclosure may not appear in subsequent comments of a discussion thread. Surgeons post-ing on social media must make it their ethical and professional obligation to disclose their conflicts of interest. Furthermore, users of social media content must be aware of the potential for bias introduced by undisclosed conflicts of interest and

1	professional obligation to disclose their conflicts of interest. Furthermore, users of social media content must be aware of the potential for bias introduced by undisclosed conflicts of interest and per-form due diligence in assessing the reliability of the source. Lastly, regulatory bodies and professional organizations should publish standardized guidelines for disclosing on social media or develop mechanisms by which disclosure can be publicly accessed (such as the Open Payments database).Fifth, professionalism must always be maintained. Sur-geons posting content on the web or on social media must be aware that information will be widely disseminated and avail-able for posterity; messages posted on social media cannot be fully retracted. As already mentioned, surgeons must strive to maintain patient privacy, ensure accuracy of information, and disclose conflicts of interest. Furthermore, surgeons must be aware of unintentional interpretations of messages (i.e., as discriminatory

1	maintain patient privacy, ensure accuracy of information, and disclose conflicts of interest. Furthermore, surgeons must be aware of unintentional interpretations of messages (i.e., as discriminatory or unprofessional). Multiple studies of health-care providers’ social media sites have identified potentially and clearly unprofessional content; these studies have included medical students, residents, and practicing surgeons. Despite the prevalence of unprofessional content, few surgical residency programs have formal institutional social media policies. Fur-thermore, the American College of Physicians and Federation of State Medical Boards position statement only addresses a few of the issues surrounding web-based activities including for patient and physician education (Table 54-3).Table 54-3Online medical professionalism and educationPOSITIONS ON PROFESSIONALISM IN ONLINE PATIENT AND PHYSICIAN EDUCATION• The Internet can be a powerful tool for education.• Physicians should guide

1	54-3Online medical professionalism and educationPOSITIONS ON PROFESSIONALISM IN ONLINE PATIENT AND PHYSICIAN EDUCATION• The Internet can be a powerful tool for education.• Physicians should guide patients to high quality online resources that are accurate and objective. These sites should have peer-reviewed content or have verifiable mechanisms for quality control of information.• Online resources for learning can be used by patients and physicians.• The internet and social networking can be used to improve public health. Physicians engaged in online communities should ensure the security of the networks and restriction of participation to verified users. Clinical scenarios should not contain any personal identifying information, and patient consent should be obtained before sharing the vignette.• Discussion of frustrations online undermines trust and professionalism and should be avoided.Data from Farnan JM1, Snyder Sulmasy L, Worster BK, et al: Online medical professionalism:

1	the vignette.• Discussion of frustrations online undermines trust and professionalism and should be avoided.Data from Farnan JM1, Snyder Sulmasy L, Worster BK, et al: Online medical professionalism: patient and public relationships: policy statement from the American College of Physicians and the Federation of State Medical Boards, Ann Intern Med. 2013 Apr 16;158(8):620-627.Brunicardi_Ch54_p2187-p2196.indd 219313/02/19 2:37 PM 2194SPECIFIC CONSIDERATIONSPART IILastly, studies evaluating the effectiveness of web-based education have had varied results. A recent systematic review by Taveira-Gomes et al assessed 251 articles using computer-based learning methodologies in medical education; the num-ber of articles on this topic has increased over time. The most commonly used metrics for evaluating the effectiveness of these methodologies were assessments of knowledge, attitudes, and skills. The majority of studies reported positive effects on these outcomes, although the more rigorous

1	the effectiveness of these methodologies were assessments of knowledge, attitudes, and skills. The majority of studies reported positive effects on these outcomes, although the more rigorous studies (i.e., randomized trials) were less likely to find a positive effect. Online activ-ity (i.e., number of posts or views) was tracked in a few stud-ies, but results were conflicting regarding whether increased engagement correlated with improved performance. This review suggests that high-quality studies are needed of web-based edu-cational interventions and that these studies need to include measures of clinical performance and outcomes.IMPLICATIONS AND FUTURE DIRECTIONSThe Society of University Surgeons’ Social and Legislative Committee issued a position statement entitled: “Social media is a necessary component of surgery practice.” Given the rapid pace with which technology is advancing and the familiar-ity of the current generation (Generation Z) with the inter-net, surgeons have no

1	a necessary component of surgery practice.” Given the rapid pace with which technology is advancing and the familiar-ity of the current generation (Generation Z) with the inter-net, surgeons have no choice but to harness the power of the internet and social media or risk being left behind. Many jour-nals are phasing out print versions, and several journals are already online only. Furthermore, textbooks may also become a relic from the past as publishers move towards developing digi-tal versions that include interactive graphics, audio, and video.As surgical education continues to evolve, future directions may include broader indications for utilization of weband social media-based resources. For example, video-based coaching with face-to-face discussions have been used postoperatively to sup-plement intraoperative teaching. However, preoperative crowd-sourcing in planning a challenging case or intraoperative video telementoring are other applications of video-based coaching.Future

1	to sup-plement intraoperative teaching. However, preoperative crowd-sourcing in planning a challenging case or intraoperative video telementoring are other applications of video-based coaching.Future research efforts should focus on identifying the most effective formats and components of web and social media–based educational interventions, using rigorous methods to compare educational methods, and measuring clinical out-comes. Moreover, standardized guidelines should be instituted in order to safeguard against ethical and professional misconduct.BIBLIOGRAPHYBirkmeyer JD, Finks JF, O’Reilly A, Oerline M, et al; Michigan Bariatric Surgery Collaborative. Surgical skill and complica-tion rates after bariatric surgery. N Engl J Med. 2013;369(15): 1434-1442. This study correlated blinding reviewer ratings of videotaped laparoscopic gastric bypass procedures to clinical outcomes. Greater skill was associated with lower rates of com-plications, reoperations, readmissions, and emergency

1	reviewer ratings of videotaped laparoscopic gastric bypass procedures to clinical outcomes. Greater skill was associated with lower rates of com-plications, reoperations, readmissions, and emergency depart-ment visits.Bresnahan ER, Huynh DTK, Jacob B. Social media and education in hernia repair. In: Hope WW, Cobb WS, Adrales GL, eds. Textbook of Hernia. Cham: Springer International Publishing; 2017:373-379. This book chapter describes the International Hernia Collaboration, which is a Facebook-based community of practicing surgeons, trainees, and industry representatives. The goals of the collaboration include exchange of ideas and advice regarding patient care, discussion and debate of controversial topics in hernia management, and dissemination of information to a global audience.Chan TM, Thoma B, Radecki R, et al. Ten steps for setting up an online journal club. J Contin Educ Health Prof. 2015; 35(2):148-154. Written by a multidisciplinary group of authors, this article provides 10

1	Thoma B, Radecki R, et al. Ten steps for setting up an online journal club. J Contin Educ Health Prof. 2015; 35(2):148-154. Written by a multidisciplinary group of authors, this article provides 10 helpful hints for setting up an online journal club, using examples from existing journal clubs.Cook DA, Levinson AJ, Garside S, Dupras DM, Erwin PJ, Montori VM. Instructional design variations in internet-based learn-ing for health professions education: a systematic review and meta-analysis. Acad Med. 2010;85(5):909-922. This systematic review and meta-analysis of 51 studies, including 30 random-ized trials, identified several key features of internet-based learning interventions that were associated with improved learning outcomes: interactivity, practice exercises, repeti-tion, and feedback. However, the evidence base upon which to design internet-based learning programs is limited by poor study designs, failure to use conceptual frameworks, and lack of adherence to reporting

1	feedback. However, the evidence base upon which to design internet-based learning programs is limited by poor study designs, failure to use conceptual frameworks, and lack of adherence to reporting standards.Cook DA, Levinson AJ, Garside S, Dupras DM, Erwin PJ, Montori VM. Internet-based learning in the health professions: a meta-analysis. JAMA. 2008;300(10):1181-1196. This meta-analysis of 201 studies reported that internet-based instruction had a positive effect on educational outcomes as compared to no intervention, but that there was similar effectiveness between internet-based instruction and traditional educational methods.Farnan JM, Synder Sulmasy L, Worster BK, Chaudhry HJ, Rhyne JA, Arora VM; American College of Physicians Ethics, Profes-sionalism and Human Rights Committee; American College of Physicians Council of Associates; Federation of State Medical Boards Special Committee on Ethics and Professionalism. Online medical professionalism: patient and public rela-tionships:

1	College of Physicians Council of Associates; Federation of State Medical Boards Special Committee on Ethics and Professionalism. Online medical professionalism: patient and public rela-tionships: policy statement from the American College of Physicians and the Federation of State Medical Boards. Ann Intern Med. 2013;158(8):620-627. This position statement pro-vides recommendations on issues relating to professional use of the internet and social media including but not limited to: use of social media for nonclinical purposes, patient confidentiality in online discussions, and use of web-based patient education resources.Guraya SY. The usage of social networking sites by medical stu-dents for educational purposes: a meta-analysis and systematic review. N Am J Med Sci. 2016;8(7):268-278. This review of 10 articles published between 2004 and 2014 suggested that although 75% of medical students use social networking sites, only 20% use them for academic or educational purposes.

1	This review of 10 articles published between 2004 and 2014 suggested that although 75% of medical students use social networking sites, only 20% use them for academic or educational purposes. Fur-thermore, none of these studies evaluated whether use of social media improved academic performance.Ibrahim AM, Lillemoe KD, Klingensmith ME, Dimick JE. Visual abstracts to disseminate research on social media: a prospective, case-control crossover study. Ann Surg. 2017;266(6):e46-e48. This prospective, case-control crossover study compared the impact of tweets alone versus tweets accompanied by a visual abstract on article visits; there was a threefold increase when visual abstracts accompanied the tweets.Jayakumar N, Brunckhorts O, Dasgupta P, Khan MS, Ahmed K. e-Learning in surgical education: a systematic review. J Surg Educ. 2015;72(6):1145-1157. This systematic review of 38 studies suggested that while the majority of studies evaluating electronic learning in surgical education

1	a systematic review. J Surg Educ. 2015;72(6):1145-1157. This systematic review of 38 studies suggested that while the majority of studies evaluating electronic learning in surgical education demonstrate a positive effect, most lack a proper control.McLeod RS, MacRae HM, McKenzie ME, Victor JC, Brasel KJ; Evidence Based Reviews in Surgery Steering Committee. A moderated journal club is more effective than an Internet journal club in teaching critical appraisal skills: results of a multicenter randomized controlled trial. J Am Coll Surg. 8Brunicardi_Ch54_p2187-p2196.indd 219413/02/19 2:37 PM 2195WEB-BASED EDUCATION AND IMPLICATIONS OF SOCIAL MEDIACHAPTER 542010;211(6):769-776. This multicenter randomized trial reported that surgical residents who participated in a journal club moderated by a faculty member scored higher on a vali-dated test of critical appraisal than those who participated in an online journal club.Pugh CM, Watson A, Bell RH Jr, et al. Surgical education in the

1	by a faculty member scored higher on a vali-dated test of critical appraisal than those who participated in an online journal club.Pugh CM, Watson A, Bell RH Jr, et al. Surgical education in the internet era. J Surg Res. 2009;156(2):177-182. This article describes the factors that led to a change in surgical education over the last two decades.Taveira-Gomes T, Ferreira P, Taveira-Gomes I, Severo M, Ferreira MA. What are we looking for in computer-based learning inter-ventions in medical education? A systematic review. J Med Internet Res. 2016;18(8):e204. This systematic review assessed recent studies on computer-based learning (CBL) for types of software platforms and interventions and adherence to current recommendations for CBL research.Brunicardi_Ch54_p2187-p2196.indd 219513/02/19 2:37 PM